Journal of Chromatography A, 1363 (2014) 162–168
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: 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 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 b
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)-6trifluoromethyl-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); RS = 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) studies 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) elution 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 structurally novel class of large-conductance calcium-activated potassium (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 potentials, 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: [email protected] (B. Natalini). http://dx.doi.org/10.1016/j.chroma.2014.06.020 0021-9673/© 2014 Elsevier B.V. All rights reserved.
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 considerable 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 channel openers, decreasing cell excitability and causing smooth muscle relaxation, could offer a novel therapeutic approach to several diseases 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 BMS204352 (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
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
Fig. 1. Chemical structure of BK openers.
the biological activity of the 2-aryl-1,4-benzothiazine BK opener class, using compound 1 as the reference compound. The column screening procedure, the preparative enantioresolution of 1, the stereochemical characterization of the absolute configuration at the C-2 position for the enantiomeric fractions, and the evaluation of the biological activity for the racemic mixture and the pure enantiomers are discussed in the following sections. The “racemic approach” [15] was employed to obtain the enantiomers of 1: the synthesis of the racemate, followed by the application of a chromatographic preparative enantioresolution method, is now largely recognized as the most convenient way for a rapid access to small amounts of highly pure enantiomers [15]. Among the benefits provided by the racemic approach, the reduced complexity of non-enantioselective synthesis protocols, and the possibility to simultaneously obtain all the stereoisomers are worth highlighting. The latter advantage is especially convenient in the case of preliminary comparative biological assays. Moreover, chromatographic preparative enantioresolutions can be profitably performed on analytical columns, when only limited amounts of each enantiomer are required. In most cases, the direct chromatographic preparative enantioresolution of a given racemate is carried out after a preliminary screening of the available chiral stationary phases (CSPs) which are suitable for the compound of interest [15]. Unfortunately, operational guidelines for the selection of the most appropriate CSP for a given application are rarely effective in practice, with some notable exceptions [16]. 2. Experimental 2.1. Materials Racemic 1 was synthesized and characterized according to a previously reported procedure [1]. Analytical grade 2-propanol (IPA), n-hexane, chloroform, ethyl acetate (EtOAc), and 1,3,5-tri-tertbutylbenzene (used as the unretained marker for the calculation of the chromatographic performance) were purchased from SigmaAldrich (Milano, Italy). For biological studies, dimethyl sulfoxide (DMSO), tyrode salt solution, acetylcholine chloride, and potassium chloride (KCl) were purchased from Sigma-Aldrich. HPLC-grade water was obtained from a New Human Power I Scholar water purification system (Human Corporation, Seoul, Korea). All the employed mobile phases were degassed by sonication for 10 min before use. Samples for HPLC analysis were dissolved in the selected mobile phase and injected at the approximate concentration of 0.5–1.0 mg mL−1 . Samples for electronic circular dichroism (ECD) and UV spectroscopic analysis were prepared at a 100 M concentration in IPA. 2.2. Instrumentations For the quantitative enantioresolution analyses, the following six columns (Fig. 2) were preliminary screened with the same eluent system: Lux Amylose-2 (CSP 1; amylose tris(5-chloro2-methylphenylcarbamate)), Chiralpak AD-H (CSP 2; amylose tris(3,5-dimethylphenylcarbamate)), Lux Cellulose-2 (CSP 3;
163
cellulose tris(3-chloro-4-methylphenylcarbamate)), Sepapak-4 (CSP 4; cellulose tris(4-chloro-3-methylphenylcarbamate)), Chiralcel OD-H (CSP 5; cellulose tris(3,5-dimethylphenylcarbamate)), and Chiralpak IB (CSP 6; cellulose tris(3,5-dimethylphenylcarbamate)). CSP 1 and CSP 3 were purchased from Phenomenex (Torrance, CA, USA); CSP 2, CSP 5 and CSP 6 were purchased from Chiral Technologies (West Chester, PA, USA); CSP 4 was kindly provided by Sepaserve GmbH (Münster, Germany). In CSPs 1–5, the chiral selector is adsorbed onto a 5 m silica gel. In CSP 6, the chiral selector is immobilized onto a 5 m silica gel. All the columns were characterized by the same 250 mm × 4.6 mm I.D. dimensions. Columns were conditioned with the selected mobile phase at a 1.0 mL min−1 flow rate for at least 40 min before use. All the analyses were carried out at a 1.0 mL min−1 flow rate and with a 25 ◦ C column temperature. The HPLC analyses were performed on a Shimadzu (Kyoto, Japan) LC-20A Prominence, equipped with a CBM-20A communication bus module, two LC-20AD dual piston pumps, a SPD-M20A photodiode array detector, and a Rheodyne 7725i injector (Rheodyne Inc., Cotati, CA, USA) with a 100 L stainless steel loop. Column temperature was controlled through a Grace (Sedriano, Italy) heater/chiller (Model 7956R) thermostat. The same HPLC system was used for the preparative enantioresolution of 1 with CSP 4. Electronic circular dichroism (ECD) and UV spectroscopic analysis was carried out at 25 ◦ C on a Jasco (Tokyo, Japan) J-810 spectropolarimeter equipped with a PTC-423S Peltier-type temperature control system, using a 2 nm spectral bandwidth, a 50 nm min−1 scanning speed and a 2 s data integration time; spectra were averaged over 3 accumulation cycles. Quartz cells (Hellma, Milan, Italy) with a 1 mm path length were used to measure spectra in the 350–200 nm spectral range.
2.3. Theoretical chiroptical spectroscopy The theoretical chiroptical properties of (R)-1 were determined according to the standard protocol for stereochemical characterization by time-dependent density functional theory (TD-DFT) calculations [17,18]. A preliminary conformational search was performed by molecular mechanics (MM) calculations using the MMFF94s force field [19] and the Spartan’02 [20] software. DFT geometry optimization and frequency calculations were carried out using the Gaussian 09 software [21] (for the full citation, see the Supporting information), the B97D functional [22] with the resolution of identity (RI) approximation [23,24], the TZVP Ahlrichs-type triple- valence plus polarization basis set [25] and the IEFPCM solvation model [26,27] for 2-propanol. Conformational clustering was performed with a RMSD threshold value of 0.01 A˚ for heavy atoms. TD-DFT calculations were also carried out using the Gaussian 09 software. The PBE0 functional [28,29] was used in combination with the TZVP basis set and the IEFPCM solvation model for 2-propanol; calculations were performed on all optimized conformers. Theoretical values of oscillator strength (fj ), rotational strength in dipole velocity formalism (Rj ) and excitation energy (expressed as wavelength, j ) were calculated for the 50 lowestenergy electronic transitions of each optimized conformer. The theoretical spectra of optimized conformers were then derived by approximation of fj and Rj values to Gaussian bands with a value of 0.25 eV [30]. The theoretical UV and ECD spectra of (R)-1 were finally derived as the weighted average of the contribution of all conformers according to their Boltzmann equilibrium populations at 298.15 K and 1 atm, based on free energy values (G ), and compared to the experimental spectra.
164
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
Fig. 2. Structure of the polysaccharide-based selectors characterizing the selected CSPs.
2.4. Vasorelaxing activity All the experimental procedures were carried out following the guidelines of the European Community Council Directive 86609. To determine a possible vasodilator mechanism of action, the compounds were tested on isolated thoracic aortic rings of male normotensive Wistar rats (250–350 g). The rats were sacrificed by cervical dislocation under light ether anesthesia and bled. The aortae were immediately excised and freed of extraneous tissues. The endothelial layer was removed by gently rubbing the intimae surface of the vessels with a hypodermic needle. Five mm wide aortic rings were suspended, under a preload of 2 g, in 20 mL organ baths, containing tyrode salt solution (composition of saline in mM: NaCl 136.8; KCl 2.95; CaCl2 1.80; MgSO4 ·7H2 O 1.05; NaH2 PO4 0.41; NaHCO3 11.9; glucose 5.5), thermostated at 37 ◦ C and continuously gassed with a mixture of O2 (95%) and CO2 (5%). Changes in tension were recorded by means of an isometric transducer (Basile mod. 7005), connected to a unirecord microdynamometer (Basile mod. 7050). After an equilibration period of 60 min, the endothelial removal was confirmed by administering acetylcholine (ACh) (10 M) to KCl (20 mM)-precontracted vascular rings. A < 10% relaxation of the KCl-induced contraction was indicative of an acceptable lack of the endothelial layer, while the organs, showing a relaxation ≥10% (i.e., significant presence of the endothelium), were discarded. Thirty to
forty minutes after the confirmation of the endothelium removal, the aortic preparations were contracted by treatment with a single concentration of KCl (25 mM). When the contraction reached a stable plateau, 3-fold increased concentrations of the tested compounds were added cumulatively. Preliminary experiments showed that the KCl-induced contractions remained in a stable tonic state for at least 40 min. The tested compounds were dissolved (10 mM) in DMSO and further diluted in HPLC-grade water. All the solutions were freshly prepared immediately before use. Previous experiments showed a complete ineffectiveness of the administration of the vehicle. The vasorelaxing efficacy was evaluated as the maximal vasorelaxing response, expressed as the percentage (%) of the contractile tone induced by 25 mM KCl. The potency parameter was expressed as pIC50 , which was calculated as the negative logarithm of the molar concentration of the test compounds, evoking a half reduction of the contractile tone induced by 25 mM KCl. The pIC50 could not be calculated for compounds having an efficacy parameter close to or less than 50%. The efficacy and potency parameters were expressed as the mean ± standard error, for 5–10 experiments. The Student’s t-test was selected for the statistical analysis; P < 0.05 was considered a significant statistical difference. Experimental data were analyzed by a computer fitting procedure (software Graph Pad Prism 3.0).
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
165
Table 1 Chromatographic performance achieved with the six employed polysaccharidebased CSPs 1–6. CSP no.
1 2 3 4 5 6
Selected chromatographic parameters kI
kII
˛
RS
NI
NII
2.90 1.80 1.87 2.62 2.29 1.27
3.77 1.91 4.08 5.32 3.64 1.65
1.30 1.06 2.18 2.03 1.59 1.30
1.95 – 4.49 8.36 5.03 3.90
2013 – 1106 4570 3561 9348
1486 – 916 2978 3120 9754
I: First eluted enantiomer; II: second eluted enantiomer.
3. Results and discussion 3.1. Chromatographic preparative enantioresolution of 1 In the development of chiral separation methods, the majority of companies and research laboratories prefer to rely upon automated column screening procedures over the optimization of the mobile phase composition with a single column. Generally, the number of tested columns is chosen to provide the widest range of specificity possible for a given compound, according to a peculiar enantiorecognition mechanism; in most cases, few different types of CSPs are sufficient to ensure the baseline resolution of the enantiomers with a limited number of mobile phase compositions. For the present study, six polysaccharide-based CSPs (Fig. 2, CSPs 1–6) were selected as screening columns, owing to their recognized extreme versatility [31]. The screening procedure of the six CSPs was carried out with the same eluent system (n-hexane/IPA 90:10, v/v) and chromatographic conditions (see section 2.2 for details). CSPs 1–6 were chosen with the aim of evaluating: (i) the impact of the polymer winding (CSP 2 vs CSP 5), since cellulose- and amylose-based polymers are characterized by a different winding of the enantioresolving polymer, which affects the morphology of the “chiral grooves” lying along the main chain and hence the access by the analyte to the stereoselective binding sites; (ii) the type and position of the phenyl substituents (CSP 1 vs CSP 2; CSP 3 vs CSP 4 vs CSP 5), since the enantiorecognition ability of the polysaccharide-type CSPs is sensitive to inductive effects deriving from the substituents of the incorporated phenyl groups; (iii) the coated or immobilized nature of the modified polymer chain, on the overall chromatographic performance (CSP 5 vs CSP 6), since changes in the polymer supramolecular structure upon immobilization can account for the different enantiorecognition mechanism often observed between coated and immobilized polysaccharide-based CSPs [32–35]. With the aforementioned standard mobile phase composition, the comparative analyses revealed that cellulose-based CSPs are generally more suited for the enantioseparation of 1 than their amylose variants (Table 1). In particular, CSP 4 turned out to be the best performing chiral medium (Table 1 and Fig. 3), and hence it was considered as the elective choice to carry out the following preparative enantioresolution. Due to the excellent chromatographic results with the selected alkane/alcohol mobile phase system, an optimization of the eluent composition was not required for the purpose of the present study. Often, comparative results are speculatively rationalized on the basis of structural differences in the chiral selector chemistry, when differences in the production processes of these CSPs (quality of packing, chromatographic matrix employed, end-capping of the matrix, amount of chiral selector on the matrix, etc.) can be responsible for most of the observed differences.
Fig. 3. Chromatographic traces of 1: column screening. Experimental conditions: column, (a) CSP 1; (b) CSP 2; (c) CSP 3; (d) CSP 4; (e) CSP 5; (f) CSP 6; flow rate, 1.0 mL min−1 ; column temperature, 25 ◦ C; detection wavelength, 220 nm; mobile phase, n-hexane/IPA 90:10, v/v. For each trace, the y-axis is in mAU units.
166
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
Fig. 4. Chromatographic trace of a run of six consecutive injections of a 1 mg mL−1 solution of 1, repeated at 1-min intervals. Experimental conditions: column, CSP 4; flow rate, 1.0 mL min−1 ; column temperature, 25 ◦ C; detection wavelength, 220 nm; mobile phase, n-hexane/IPA 90:10, v/v. The y-axis is in mAU units.
Nevertheless, the excellent performance provided by the chloromethylated CSP 4 can be reasonably explained by the simultaneous presence of an electron donating (CH3 ) and an electron withdrawing (Cl) substituent on the carbamate moiety [35,36]. Indeed, IR studies demonstrated that the simultaneous presence of a methyl group and a chlorine atom on the phenyl ring of the carbamate moiety increases the number of NH and C O groups available on the stationary phase for stereoselective intermolecular binding contacts with analytes, at expenses of intramolecular interactions between adjacent carbamate motifs along the polymer chain [35,36]. However, it needs to be pointed out that the short time of analysis and the high quality of the overall chromatographic performance (Table 1 and Fig. 3) provided by CSP 6 should be preferred for analytical-scale assays on compound 1. Runs of six consecutive injections repeated at 1-min intervals with CSP 4 produced two clearly distinguishable groups of peaks, one for the first-eluted enantiomer and one for the second-eluted enantiomer, without band contamination (Fig. 4). This procedure was carried out considering the low concentration of injected samples for the preparative collection of enantiomers, due to the limited sample solubility in the selected eluent system and the decreased enantioselectivity with higher IPA amounts in the mobile phase. Repeated consecutive injections allowed to isolate approximately 20 mg of each enantiomer (about 0.5 mg of each enantiomer in a single run), with enantiomeric excess (e.e.) values higher than 97% and 98% for the first- and second-eluted enantiomer, respectively (Fig. 5a and b). Very profitably, the high volatility of the eluent system facilitated the solvent evaporation after the separation runs. Before being submitted to stereochemical characterization and biological evaluation, both isolated enantiomers were carefully purified by flash chromatography performed on silica gel with an eluent mixture based on n-hexane/EtOAc 80:20, v/v. This procedure assured to obtain enantiomeric fractions with purities higher than 98%, as revealed from RP-HPLC/MS analysis (data not shown). 3.2. Stereochemical characterization of the enantiomers of 1 A detailed report on the conformational analysis and theoretical chiroptical properties of 1 is given in the Supporting information. The comparison between the experimental UV and ECD spectra of the two enantiomeric fractions of 1 and the TD-DFT theoretical spectra of (R)-1 (Fig. 6) allows a clear assignment of the (R) absolute
Fig. 5. Chromatographic traces of (a) first-eluted enantiomer of 1; (b) secondeluted enantiomer of 1; (c) 1. Experimental conditions: column, CSP 4; flow rate, 1.0 mL min−1 ; column temperature, 25 ◦ C; detection wavelength, 220 nm; mobile phase, n-hexane/IPA 90:10, v/v. For each trace, the y-axis is in mAU units.
configuration to the first-eluted fraction of 1 on CSP 4. The profiles of both the UV and ECD spectra are reproduced with good accuracy, with the strong Cotton effect at 303 nm being positive and the two Cotton effects at 250 nm and 243 nm being negative. Consequently, the (S) absolute configuration can be assigned to the second-eluted fraction and the elution order on CSP 4 with the employed eluent system can be assessed as (R), (S). 3.3. Vasorelaxing potency and efficacy of 1 vs the single enantiomers The BK-opening activity was evaluated in vitro as the vasorelaxing effect on endothelium-denuded rat aortic rings precontracted with KCl (25 mM) according to the protocol described in section 2.4. The vasorelaxing activities for the racemate and the single
Table 2 Vasorelaxing potency and efficacy of 1 and its enantiomersa . Compound
Emax b ± SEMc (%)
pIC50 d ± SEMc
1 (R)-1 (S)-1
96 ± 2 90 ± 10 92 ± 2
6.70 ± 0.022 6.67 ± 0.083 6.69 ± 0.028
a The compounds were administered to isolated endothelium-denuded rat aortic rings, precontracted by 20 mM KCl. b Maximal vasorelaxing effect expressed as the % of contractile tension. c Standard error of a mean 5–10 separate experiments. d Vasorelaxing potency expressed as the negative log of the concentration evoking a half-reduction of the contractile tone.
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
167
allowed to assign the absolute configuration to the enantiomeric fraction and assess a (R), (S) elution order in the selected chromatographic conditions. Finally, experiments aimed at evaluating the vasorelaxing potency and efficacy of 1 in comparison with its enantiomers clearly indicated that the absolute configuration at the C-2 position does not play a role in the definition of the biological activity of 1, at least at a vascular level. Acknowledgment DFT and TD-DFT calculations for the stereochemical characterization of 1 were carried out on the computing cluster of Prof. Riccardo Zanasi’s research group (Department of Chemistry and Biology–University of Salerno, Italy). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.06.020. References
Fig. 6. Experimental UV and ECD spectra of the first- (dashed) and second-eluted (dotted) enantiomeric fractions of 1, compared to the theoretical spectra of (R)-1 (solid).
Fig. 7. Vasorelaxing activity of the racemic mixture and pure enantiomers of 1.
enantiomers, expressed as efficacy (%) and potency (pIC50 ), are reported in Table 2 and Fig. 7. The results indicate that 1 and its isolated enantiomers are endowed with equivalent levels of vasorelaxing potency. 4. Conclusions The “racemic approach” allowed to obtain highly pure enantiomers of the BK channel opener 1, which was already studied as racemate. Approximately 20 mg of each enantiomer were successfully isolated with the chloromethylated cellulose-based CSP 4 (RS = 8.36; ˛ = 2.03), by running repeated elutions with a standard normal-phase eluent system. The individual enantiomers were collected with sufficient yield to carry out stereochemical and biological investigations. ECD spectroscopy and TD-DFT calculations
[1] V. Calderone, R. Spogli, A. Martelli, G. Manfroni, L. Testai, S. Sabatini, O. Tabarrini, V. Cecchetti, J. Med. Chem. 51 (2008) 5085. [2] S. Ghatta, D. Nimmagadda, X. Xu, S.T. O’Rourke, Pharmacol. Therapeut. 110 (2006) 103. [3] A.A. Fodor, R.W. Aldrich, Annu. Rev. Physiol. 71 (2009) 19. [4] N.S. Atkinson, G.A. Robertson, B. Ganetzky, Science 253 (1991) 551. [5] P. Orio, P. Rojas, G. Ferreira, R. Latorre, News Physiol. Sci. 17 (2002) 156. [6] R. Lu, A. Alioua, Y. Kumar, M. Eghbali, E. Stefani, L. Toro, J. Physiol. 570 (2006) 65. [7] V. Calderone, Curr. Med. Chem. 9 (2002) 1385. [8] A. Nardi, S.-P. Olesen, Curr. Med. Chem. 15 (2008) 1126. [9] K. Lawson, N.G. McKay, Curr. Pharm. Des. 12 (2006) 459. [10] S.C. Turner, C.-C. Shieh, in: G.J. Triggle, M. Gopalakrishnan, D. Rampe, W. Zheng (Eds.), Methods and Principles in Medicinal Chemistry, Wiley-VCH, Weinheim, 2006, p. 310. [11] A. Nardi, V. Calderone, S.-P. Olesen, Lett. Drug Des. Discovery 3 (2006) 210. [12] P. Hewawasam, N.A. Meanwell, V.K. Gribkoff, S.I. Dworetzky, C.G. Boissard, Bioorg. Med. Chem. Lett. 7 (1997) 1255. [13] P. Hewawasam, M. Erway, S.L. Moon, J. Knipe, H. Weiner, C.G. Boissard, D.J. Post-Munson, Q. Gao, S. Huang, V.K. Gribkoff, N.A. Meanwell, J. Med. Chem. 45 (2002) 1487. [14] P. Hewawasam, V.K. Gribkoff, Y. Pendri, S.I. Dworetzky, N.A. Meanwell, E. Martinez, C.G. Boissard, D.J. Post-Munson, J.T. Trojnacki, K. Yeleswaram, L.M. Pajor, J. Knipe, Q. Gao, R. Perrone, J.E. Starrett Jr., Bioorg. Med. Chem. Lett. 12 (2002) 1023. [15] E. Francotte, in: E. Francotte, W. Lindner (Eds.), Chirality in Drug Research, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006, p. 155. [16] W.H. Pirkle, M.E. Koscho, J. Chromatogr. A 761 (1997) 65. [17] J. Autschbach, Chirality 21 (2009) E116. [18] L. Goerigk, H. Kruse, S. Grimme, in: N. Berova, P.L. Polavarapu, K. Nakanishi, R.W. Woody (Eds.), Comprehensive Chiroptical Spectroscopy, Wiley & Sons, Hoboken, NJ, 2012, p. 643. [19] T.A. Halgren, J. Comput. Chem. 20 (1999) 720. [20] Wavefunction, Inc., Spartan’02, Wavefunction, Inc., Irvine, CA, 2002. [21] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, Ö. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09 (Revision C.01), Gaussian, Inc., Wallingford, CT, 2010. [22] S. Grimme, J. Comp. Chem. 27 (2006) 1787. [23] B.I. Dunlap, J. Chem. Phys. 78 (1983) 3140. [24] B.I. Dunlap, J. Mol. Struct. THEOCHEM 529 (2000) 37. [25] A. Schäfer, C. Huber, R. Ahlrichs, J. Chem. Phys. 100 (1994) 5829. [26] J. Tomasi, B. Mennucci, E. Cancès, J. Mol. Struct. THEOCHEM 464 (1999) 211. [27] J. Tomasi, B. Mennucci, R. Cammi, Chem. Rev. 105 (2005) 2999. [28] J.P. Perdew, K. Burke, M. Ernzerhof, Phys. Rev. Lett. 77 (1996) 3865. [29] C. Adamo, V. Barone, J. Chem. Phys. 110 (1999) 6158. [30] P.J. Stephens, N. Harada, Chirality 22 (2010) 229.
168
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
[31] B. Chankvetadze, J. Chromatogr. A 1269 (2012) 26. [32] B. Natalini, R. Sardella, S. Massari, F. Ianni, O. Tabarrini, V. Cecchetti, Talanta 85 (2011) 1392. [33] R. Sardella, F. Ianni, A. Lisanti, M. Marinozzi, S. Scorzoni, B. Natalini, Biomed. Chromatogr. 28 (2014) 159.
˜ A. Conejo-García, M.C. [34] B. Natalini, R. Sardella, F. Ianni, M.E. García-Rubino, ˜ M.A. Gallo, J.M. Campos, Chromatographia 76 (2013) 475. Núnez, [35] B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39. [36] B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 694 (1995) 101.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: 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 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 b
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)-6trifluoromethyl-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); RS = 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) studies 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) elution 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 structurally novel class of large-conductance calcium-activated potassium (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 potentials, 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: [email protected] (B. Natalini). http://dx.doi.org/10.1016/j.chroma.2014.06.020 0021-9673/© 2014 Elsevier B.V. All rights reserved.
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 considerable 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 channel openers, decreasing cell excitability and causing smooth muscle relaxation, could offer a novel therapeutic approach to several diseases 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 BMS204352 (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
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
Fig. 1. Chemical structure of BK openers.
the biological activity of the 2-aryl-1,4-benzothiazine BK opener class, using compound 1 as the reference compound. The column screening procedure, the preparative enantioresolution of 1, the stereochemical characterization of the absolute configuration at the C-2 position for the enantiomeric fractions, and the evaluation of the biological activity for the racemic mixture and the pure enantiomers are discussed in the following sections. The “racemic approach” [15] was employed to obtain the enantiomers of 1: the synthesis of the racemate, followed by the application of a chromatographic preparative enantioresolution method, is now largely recognized as the most convenient way for a rapid access to small amounts of highly pure enantiomers [15]. Among the benefits provided by the racemic approach, the reduced complexity of non-enantioselective synthesis protocols, and the possibility to simultaneously obtain all the stereoisomers are worth highlighting. The latter advantage is especially convenient in the case of preliminary comparative biological assays. Moreover, chromatographic preparative enantioresolutions can be profitably performed on analytical columns, when only limited amounts of each enantiomer are required. In most cases, the direct chromatographic preparative enantioresolution of a given racemate is carried out after a preliminary screening of the available chiral stationary phases (CSPs) which are suitable for the compound of interest [15]. Unfortunately, operational guidelines for the selection of the most appropriate CSP for a given application are rarely effective in practice, with some notable exceptions [16]. 2. Experimental 2.1. Materials Racemic 1 was synthesized and characterized according to a previously reported procedure [1]. Analytical grade 2-propanol (IPA), n-hexane, chloroform, ethyl acetate (EtOAc), and 1,3,5-tri-tertbutylbenzene (used as the unretained marker for the calculation of the chromatographic performance) were purchased from SigmaAldrich (Milano, Italy). For biological studies, dimethyl sulfoxide (DMSO), tyrode salt solution, acetylcholine chloride, and potassium chloride (KCl) were purchased from Sigma-Aldrich. HPLC-grade water was obtained from a New Human Power I Scholar water purification system (Human Corporation, Seoul, Korea). All the employed mobile phases were degassed by sonication for 10 min before use. Samples for HPLC analysis were dissolved in the selected mobile phase and injected at the approximate concentration of 0.5–1.0 mg mL−1 . Samples for electronic circular dichroism (ECD) and UV spectroscopic analysis were prepared at a 100 M concentration in IPA. 2.2. Instrumentations For the quantitative enantioresolution analyses, the following six columns (Fig. 2) were preliminary screened with the same eluent system: Lux Amylose-2 (CSP 1; amylose tris(5-chloro2-methylphenylcarbamate)), Chiralpak AD-H (CSP 2; amylose tris(3,5-dimethylphenylcarbamate)), Lux Cellulose-2 (CSP 3;
163
cellulose tris(3-chloro-4-methylphenylcarbamate)), Sepapak-4 (CSP 4; cellulose tris(4-chloro-3-methylphenylcarbamate)), Chiralcel OD-H (CSP 5; cellulose tris(3,5-dimethylphenylcarbamate)), and Chiralpak IB (CSP 6; cellulose tris(3,5-dimethylphenylcarbamate)). CSP 1 and CSP 3 were purchased from Phenomenex (Torrance, CA, USA); CSP 2, CSP 5 and CSP 6 were purchased from Chiral Technologies (West Chester, PA, USA); CSP 4 was kindly provided by Sepaserve GmbH (Münster, Germany). In CSPs 1–5, the chiral selector is adsorbed onto a 5 m silica gel. In CSP 6, the chiral selector is immobilized onto a 5 m silica gel. All the columns were characterized by the same 250 mm × 4.6 mm I.D. dimensions. Columns were conditioned with the selected mobile phase at a 1.0 mL min−1 flow rate for at least 40 min before use. All the analyses were carried out at a 1.0 mL min−1 flow rate and with a 25 ◦ C column temperature. The HPLC analyses were performed on a Shimadzu (Kyoto, Japan) LC-20A Prominence, equipped with a CBM-20A communication bus module, two LC-20AD dual piston pumps, a SPD-M20A photodiode array detector, and a Rheodyne 7725i injector (Rheodyne Inc., Cotati, CA, USA) with a 100 L stainless steel loop. Column temperature was controlled through a Grace (Sedriano, Italy) heater/chiller (Model 7956R) thermostat. The same HPLC system was used for the preparative enantioresolution of 1 with CSP 4. Electronic circular dichroism (ECD) and UV spectroscopic analysis was carried out at 25 ◦ C on a Jasco (Tokyo, Japan) J-810 spectropolarimeter equipped with a PTC-423S Peltier-type temperature control system, using a 2 nm spectral bandwidth, a 50 nm min−1 scanning speed and a 2 s data integration time; spectra were averaged over 3 accumulation cycles. Quartz cells (Hellma, Milan, Italy) with a 1 mm path length were used to measure spectra in the 350–200 nm spectral range.
2.3. Theoretical chiroptical spectroscopy The theoretical chiroptical properties of (R)-1 were determined according to the standard protocol for stereochemical characterization by time-dependent density functional theory (TD-DFT) calculations [17,18]. A preliminary conformational search was performed by molecular mechanics (MM) calculations using the MMFF94s force field [19] and the Spartan’02 [20] software. DFT geometry optimization and frequency calculations were carried out using the Gaussian 09 software [21] (for the full citation, see the Supporting information), the B97D functional [22] with the resolution of identity (RI) approximation [23,24], the TZVP Ahlrichs-type triple- valence plus polarization basis set [25] and the IEFPCM solvation model [26,27] for 2-propanol. Conformational clustering was performed with a RMSD threshold value of 0.01 A˚ for heavy atoms. TD-DFT calculations were also carried out using the Gaussian 09 software. The PBE0 functional [28,29] was used in combination with the TZVP basis set and the IEFPCM solvation model for 2-propanol; calculations were performed on all optimized conformers. Theoretical values of oscillator strength (fj ), rotational strength in dipole velocity formalism (Rj ) and excitation energy (expressed as wavelength, j ) were calculated for the 50 lowestenergy electronic transitions of each optimized conformer. The theoretical spectra of optimized conformers were then derived by approximation of fj and Rj values to Gaussian bands with a value of 0.25 eV [30]. The theoretical UV and ECD spectra of (R)-1 were finally derived as the weighted average of the contribution of all conformers according to their Boltzmann equilibrium populations at 298.15 K and 1 atm, based on free energy values (G ), and compared to the experimental spectra.
164
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
Fig. 2. Structure of the polysaccharide-based selectors characterizing the selected CSPs.
2.4. Vasorelaxing activity All the experimental procedures were carried out following the guidelines of the European Community Council Directive 86609. To determine a possible vasodilator mechanism of action, the compounds were tested on isolated thoracic aortic rings of male normotensive Wistar rats (250–350 g). The rats were sacrificed by cervical dislocation under light ether anesthesia and bled. The aortae were immediately excised and freed of extraneous tissues. The endothelial layer was removed by gently rubbing the intimae surface of the vessels with a hypodermic needle. Five mm wide aortic rings were suspended, under a preload of 2 g, in 20 mL organ baths, containing tyrode salt solution (composition of saline in mM: NaCl 136.8; KCl 2.95; CaCl2 1.80; MgSO4 ·7H2 O 1.05; NaH2 PO4 0.41; NaHCO3 11.9; glucose 5.5), thermostated at 37 ◦ C and continuously gassed with a mixture of O2 (95%) and CO2 (5%). Changes in tension were recorded by means of an isometric transducer (Basile mod. 7005), connected to a unirecord microdynamometer (Basile mod. 7050). After an equilibration period of 60 min, the endothelial removal was confirmed by administering acetylcholine (ACh) (10 M) to KCl (20 mM)-precontracted vascular rings. A < 10% relaxation of the KCl-induced contraction was indicative of an acceptable lack of the endothelial layer, while the organs, showing a relaxation ≥10% (i.e., significant presence of the endothelium), were discarded. Thirty to
forty minutes after the confirmation of the endothelium removal, the aortic preparations were contracted by treatment with a single concentration of KCl (25 mM). When the contraction reached a stable plateau, 3-fold increased concentrations of the tested compounds were added cumulatively. Preliminary experiments showed that the KCl-induced contractions remained in a stable tonic state for at least 40 min. The tested compounds were dissolved (10 mM) in DMSO and further diluted in HPLC-grade water. All the solutions were freshly prepared immediately before use. Previous experiments showed a complete ineffectiveness of the administration of the vehicle. The vasorelaxing efficacy was evaluated as the maximal vasorelaxing response, expressed as the percentage (%) of the contractile tone induced by 25 mM KCl. The potency parameter was expressed as pIC50 , which was calculated as the negative logarithm of the molar concentration of the test compounds, evoking a half reduction of the contractile tone induced by 25 mM KCl. The pIC50 could not be calculated for compounds having an efficacy parameter close to or less than 50%. The efficacy and potency parameters were expressed as the mean ± standard error, for 5–10 experiments. The Student’s t-test was selected for the statistical analysis; P < 0.05 was considered a significant statistical difference. Experimental data were analyzed by a computer fitting procedure (software Graph Pad Prism 3.0).
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
165
Table 1 Chromatographic performance achieved with the six employed polysaccharidebased CSPs 1–6. CSP no.
1 2 3 4 5 6
Selected chromatographic parameters kI
kII
˛
RS
NI
NII
2.90 1.80 1.87 2.62 2.29 1.27
3.77 1.91 4.08 5.32 3.64 1.65
1.30 1.06 2.18 2.03 1.59 1.30
1.95 – 4.49 8.36 5.03 3.90
2013 – 1106 4570 3561 9348
1486 – 916 2978 3120 9754
I: First eluted enantiomer; II: second eluted enantiomer.
3. Results and discussion 3.1. Chromatographic preparative enantioresolution of 1 In the development of chiral separation methods, the majority of companies and research laboratories prefer to rely upon automated column screening procedures over the optimization of the mobile phase composition with a single column. Generally, the number of tested columns is chosen to provide the widest range of specificity possible for a given compound, according to a peculiar enantiorecognition mechanism; in most cases, few different types of CSPs are sufficient to ensure the baseline resolution of the enantiomers with a limited number of mobile phase compositions. For the present study, six polysaccharide-based CSPs (Fig. 2, CSPs 1–6) were selected as screening columns, owing to their recognized extreme versatility [31]. The screening procedure of the six CSPs was carried out with the same eluent system (n-hexane/IPA 90:10, v/v) and chromatographic conditions (see section 2.2 for details). CSPs 1–6 were chosen with the aim of evaluating: (i) the impact of the polymer winding (CSP 2 vs CSP 5), since cellulose- and amylose-based polymers are characterized by a different winding of the enantioresolving polymer, which affects the morphology of the “chiral grooves” lying along the main chain and hence the access by the analyte to the stereoselective binding sites; (ii) the type and position of the phenyl substituents (CSP 1 vs CSP 2; CSP 3 vs CSP 4 vs CSP 5), since the enantiorecognition ability of the polysaccharide-type CSPs is sensitive to inductive effects deriving from the substituents of the incorporated phenyl groups; (iii) the coated or immobilized nature of the modified polymer chain, on the overall chromatographic performance (CSP 5 vs CSP 6), since changes in the polymer supramolecular structure upon immobilization can account for the different enantiorecognition mechanism often observed between coated and immobilized polysaccharide-based CSPs [32–35]. With the aforementioned standard mobile phase composition, the comparative analyses revealed that cellulose-based CSPs are generally more suited for the enantioseparation of 1 than their amylose variants (Table 1). In particular, CSP 4 turned out to be the best performing chiral medium (Table 1 and Fig. 3), and hence it was considered as the elective choice to carry out the following preparative enantioresolution. Due to the excellent chromatographic results with the selected alkane/alcohol mobile phase system, an optimization of the eluent composition was not required for the purpose of the present study. Often, comparative results are speculatively rationalized on the basis of structural differences in the chiral selector chemistry, when differences in the production processes of these CSPs (quality of packing, chromatographic matrix employed, end-capping of the matrix, amount of chiral selector on the matrix, etc.) can be responsible for most of the observed differences.
Fig. 3. Chromatographic traces of 1: column screening. Experimental conditions: column, (a) CSP 1; (b) CSP 2; (c) CSP 3; (d) CSP 4; (e) CSP 5; (f) CSP 6; flow rate, 1.0 mL min−1 ; column temperature, 25 ◦ C; detection wavelength, 220 nm; mobile phase, n-hexane/IPA 90:10, v/v. For each trace, the y-axis is in mAU units.
166
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
Fig. 4. Chromatographic trace of a run of six consecutive injections of a 1 mg mL−1 solution of 1, repeated at 1-min intervals. Experimental conditions: column, CSP 4; flow rate, 1.0 mL min−1 ; column temperature, 25 ◦ C; detection wavelength, 220 nm; mobile phase, n-hexane/IPA 90:10, v/v. The y-axis is in mAU units.
Nevertheless, the excellent performance provided by the chloromethylated CSP 4 can be reasonably explained by the simultaneous presence of an electron donating (CH3 ) and an electron withdrawing (Cl) substituent on the carbamate moiety [35,36]. Indeed, IR studies demonstrated that the simultaneous presence of a methyl group and a chlorine atom on the phenyl ring of the carbamate moiety increases the number of NH and C O groups available on the stationary phase for stereoselective intermolecular binding contacts with analytes, at expenses of intramolecular interactions between adjacent carbamate motifs along the polymer chain [35,36]. However, it needs to be pointed out that the short time of analysis and the high quality of the overall chromatographic performance (Table 1 and Fig. 3) provided by CSP 6 should be preferred for analytical-scale assays on compound 1. Runs of six consecutive injections repeated at 1-min intervals with CSP 4 produced two clearly distinguishable groups of peaks, one for the first-eluted enantiomer and one for the second-eluted enantiomer, without band contamination (Fig. 4). This procedure was carried out considering the low concentration of injected samples for the preparative collection of enantiomers, due to the limited sample solubility in the selected eluent system and the decreased enantioselectivity with higher IPA amounts in the mobile phase. Repeated consecutive injections allowed to isolate approximately 20 mg of each enantiomer (about 0.5 mg of each enantiomer in a single run), with enantiomeric excess (e.e.) values higher than 97% and 98% for the first- and second-eluted enantiomer, respectively (Fig. 5a and b). Very profitably, the high volatility of the eluent system facilitated the solvent evaporation after the separation runs. Before being submitted to stereochemical characterization and biological evaluation, both isolated enantiomers were carefully purified by flash chromatography performed on silica gel with an eluent mixture based on n-hexane/EtOAc 80:20, v/v. This procedure assured to obtain enantiomeric fractions with purities higher than 98%, as revealed from RP-HPLC/MS analysis (data not shown). 3.2. Stereochemical characterization of the enantiomers of 1 A detailed report on the conformational analysis and theoretical chiroptical properties of 1 is given in the Supporting information. The comparison between the experimental UV and ECD spectra of the two enantiomeric fractions of 1 and the TD-DFT theoretical spectra of (R)-1 (Fig. 6) allows a clear assignment of the (R) absolute
Fig. 5. Chromatographic traces of (a) first-eluted enantiomer of 1; (b) secondeluted enantiomer of 1; (c) 1. Experimental conditions: column, CSP 4; flow rate, 1.0 mL min−1 ; column temperature, 25 ◦ C; detection wavelength, 220 nm; mobile phase, n-hexane/IPA 90:10, v/v. For each trace, the y-axis is in mAU units.
configuration to the first-eluted fraction of 1 on CSP 4. The profiles of both the UV and ECD spectra are reproduced with good accuracy, with the strong Cotton effect at 303 nm being positive and the two Cotton effects at 250 nm and 243 nm being negative. Consequently, the (S) absolute configuration can be assigned to the second-eluted fraction and the elution order on CSP 4 with the employed eluent system can be assessed as (R), (S). 3.3. Vasorelaxing potency and efficacy of 1 vs the single enantiomers The BK-opening activity was evaluated in vitro as the vasorelaxing effect on endothelium-denuded rat aortic rings precontracted with KCl (25 mM) according to the protocol described in section 2.4. The vasorelaxing activities for the racemate and the single
Table 2 Vasorelaxing potency and efficacy of 1 and its enantiomersa . Compound
Emax b ± SEMc (%)
pIC50 d ± SEMc
1 (R)-1 (S)-1
96 ± 2 90 ± 10 92 ± 2
6.70 ± 0.022 6.67 ± 0.083 6.69 ± 0.028
a The compounds were administered to isolated endothelium-denuded rat aortic rings, precontracted by 20 mM KCl. b Maximal vasorelaxing effect expressed as the % of contractile tension. c Standard error of a mean 5–10 separate experiments. d Vasorelaxing potency expressed as the negative log of the concentration evoking a half-reduction of the contractile tone.
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
167
allowed to assign the absolute configuration to the enantiomeric fraction and assess a (R), (S) elution order in the selected chromatographic conditions. Finally, experiments aimed at evaluating the vasorelaxing potency and efficacy of 1 in comparison with its enantiomers clearly indicated that the absolute configuration at the C-2 position does not play a role in the definition of the biological activity of 1, at least at a vascular level. Acknowledgment DFT and TD-DFT calculations for the stereochemical characterization of 1 were carried out on the computing cluster of Prof. Riccardo Zanasi’s research group (Department of Chemistry and Biology–University of Salerno, Italy). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2014.06.020. References
Fig. 6. Experimental UV and ECD spectra of the first- (dashed) and second-eluted (dotted) enantiomeric fractions of 1, compared to the theoretical spectra of (R)-1 (solid).
Fig. 7. Vasorelaxing activity of the racemic mixture and pure enantiomers of 1.
enantiomers, expressed as efficacy (%) and potency (pIC50 ), are reported in Table 2 and Fig. 7. The results indicate that 1 and its isolated enantiomers are endowed with equivalent levels of vasorelaxing potency. 4. Conclusions The “racemic approach” allowed to obtain highly pure enantiomers of the BK channel opener 1, which was already studied as racemate. Approximately 20 mg of each enantiomer were successfully isolated with the chloromethylated cellulose-based CSP 4 (RS = 8.36; ˛ = 2.03), by running repeated elutions with a standard normal-phase eluent system. The individual enantiomers were collected with sufficient yield to carry out stereochemical and biological investigations. ECD spectroscopy and TD-DFT calculations
[1] V. Calderone, R. Spogli, A. Martelli, G. Manfroni, L. Testai, S. Sabatini, O. Tabarrini, V. Cecchetti, J. Med. Chem. 51 (2008) 5085. [2] S. Ghatta, D. Nimmagadda, X. Xu, S.T. O’Rourke, Pharmacol. Therapeut. 110 (2006) 103. [3] A.A. Fodor, R.W. Aldrich, Annu. Rev. Physiol. 71 (2009) 19. [4] N.S. Atkinson, G.A. Robertson, B. Ganetzky, Science 253 (1991) 551. [5] P. Orio, P. Rojas, G. Ferreira, R. Latorre, News Physiol. Sci. 17 (2002) 156. [6] R. Lu, A. Alioua, Y. Kumar, M. Eghbali, E. Stefani, L. Toro, J. Physiol. 570 (2006) 65. [7] V. Calderone, Curr. Med. Chem. 9 (2002) 1385. [8] A. Nardi, S.-P. Olesen, Curr. Med. Chem. 15 (2008) 1126. [9] K. Lawson, N.G. McKay, Curr. Pharm. Des. 12 (2006) 459. [10] S.C. Turner, C.-C. Shieh, in: G.J. Triggle, M. Gopalakrishnan, D. Rampe, W. Zheng (Eds.), Methods and Principles in Medicinal Chemistry, Wiley-VCH, Weinheim, 2006, p. 310. [11] A. Nardi, V. Calderone, S.-P. Olesen, Lett. Drug Des. Discovery 3 (2006) 210. [12] P. Hewawasam, N.A. Meanwell, V.K. Gribkoff, S.I. Dworetzky, C.G. Boissard, Bioorg. Med. Chem. Lett. 7 (1997) 1255. [13] P. Hewawasam, M. Erway, S.L. Moon, J. Knipe, H. Weiner, C.G. Boissard, D.J. Post-Munson, Q. Gao, S. Huang, V.K. Gribkoff, N.A. Meanwell, J. Med. Chem. 45 (2002) 1487. [14] P. Hewawasam, V.K. Gribkoff, Y. Pendri, S.I. Dworetzky, N.A. Meanwell, E. Martinez, C.G. Boissard, D.J. Post-Munson, J.T. Trojnacki, K. Yeleswaram, L.M. Pajor, J. Knipe, Q. Gao, R. Perrone, J.E. Starrett Jr., Bioorg. Med. Chem. Lett. 12 (2002) 1023. [15] E. Francotte, in: E. Francotte, W. Lindner (Eds.), Chirality in Drug Research, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006, p. 155. [16] W.H. Pirkle, M.E. Koscho, J. Chromatogr. A 761 (1997) 65. [17] J. Autschbach, Chirality 21 (2009) E116. [18] L. Goerigk, H. Kruse, S. Grimme, in: N. Berova, P.L. Polavarapu, K. Nakanishi, R.W. Woody (Eds.), Comprehensive Chiroptical Spectroscopy, Wiley & Sons, Hoboken, NJ, 2012, p. 643. [19] T.A. Halgren, J. Comput. Chem. 20 (1999) 720. [20] Wavefunction, Inc., Spartan’02, Wavefunction, Inc., Irvine, CA, 2002. [21] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, Ö. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09 (Revision C.01), Gaussian, Inc., Wallingford, CT, 2010. [22] S. Grimme, J. Comp. Chem. 27 (2006) 1787. [23] B.I. Dunlap, J. Chem. Phys. 78 (1983) 3140. [24] B.I. Dunlap, J. Mol. Struct. THEOCHEM 529 (2000) 37. [25] A. Schäfer, C. Huber, R. Ahlrichs, J. Chem. Phys. 100 (1994) 5829. [26] J. Tomasi, B. Mennucci, E. Cancès, J. Mol. Struct. THEOCHEM 464 (1999) 211. [27] J. Tomasi, B. Mennucci, R. Cammi, Chem. Rev. 105 (2005) 2999. [28] J.P. Perdew, K. Burke, M. Ernzerhof, Phys. Rev. Lett. 77 (1996) 3865. [29] C. Adamo, V. Barone, J. Chem. Phys. 110 (1999) 6158. [30] P.J. Stephens, N. Harada, Chirality 22 (2010) 229.
168
R. Sardella et al. / J. Chromatogr. A 1363 (2014) 162–168
[31] B. Chankvetadze, J. Chromatogr. A 1269 (2012) 26. [32] B. Natalini, R. Sardella, S. Massari, F. Ianni, O. Tabarrini, V. Cecchetti, Talanta 85 (2011) 1392. [33] R. Sardella, F. Ianni, A. Lisanti, M. Marinozzi, S. Scorzoni, B. Natalini, Biomed. Chromatogr. 28 (2014) 159.
˜ A. Conejo-García, M.C. [34] B. Natalini, R. Sardella, F. Ianni, M.E. García-Rubino, ˜ M.A. Gallo, J.M. Campos, Chromatographia 76 (2013) 475. Núnez, [35] B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39. [36] B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 694 (1995) 101.
