Preparative Enantioseparation of (RS)‐Baclofen: Determination of Molecular Dissymmetry

Abstract

The present work reports preparative enantioseparation of (RS)-baclofen using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Diastereomers were synthesized using a new monochloro-s-triazine-based chiral derivatizing reagent (CDR), namely, N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-L-phenylalanine, under microwave irradiation. Acetonitrile-0.1% aq. triflouroacetic acid in gradient elution mode and CH3 OH-CH2 Cl2 (4:5; v/v) were successful as mobile phase in HPLC and TLC, respectively. The two diastereomers were isolated by preparative TLC. Molecular dissymmetry was established by developing the lowest energy optimized structures of the diastereomers based on Density Functional Theory and with the help of (1) H NMR showing anisotropic effect associated with aromatic ring of s-triazine (in the CDR). The configuration of diastereomers was established as [L-Phe-(R)-Bac] and [L-Phe-(S)-Bac], where the first notation refers to the configuration of chiral auxiliary (of the CDR) and the second to that of the analyte Bac. Limits of detection were found to be 0.056 and 0.061 ng mL(-1), respectively, for the two diastereomers. Determination of absolute configuration of the two diastereomers lent support to the elution order and separation mechanism.

Enantioresolution of (RS)‐baclofen by liquid chromatography: A review

“…An MCT reagent (CDR ‐1 ), namely, N‐ (4‐chloro‐6‐piperidinyl‐[1,3,5]‐triazine‐2‐yl)‐ l ‐phenylalanine, was synthesized (Vashistha & Bhushan, ) by nucleophilic substitution of one of the chlorine atoms by a piperidinyl group and the second with l ‐Phe (as the chiral auxiliary). The CDR‐ 1 so synthesized was reacted with ( RS )‐Bac by microwave irradiation (MWI) for 2 min in the presence of NaHCO 3 when the pair of diastereomeric derivatives was obtained as a substitution of the remaining Cl atom in the MCT reagent by the nucleophilic amino group of the Bac molecule.…”

“…The significant aspect of these studies (on enantioseparation using CDR‐ 1 ) is the determination of the configuration of diastereomers by 1 H NMR spectroscopy (showing an anisotropic effect associated with aromatic ring of s ‐triazine in the CDR) and by developing the lowest energy optimized structures of the diastereomers using the Gaussian software based on density functional theory (which showed that the aromatic ring of Bac and triazine moiety were situated in different planes). Since the configuration of diastereomers was confirmed by 1 H NMR spectra, the elution order (on TLC) was interpreted so that [ l ‐( R )]‐diastereomeric derivative eluted before [ l ‐( S )]‐diastereomer where l ‐ denotes the configuration of the l ‐Phe moiety introduced as chiral auxiliary (Vashistha & Bhushan, ). The literature reveals that all the previous reports on enantioseparation of ( RS )‐Bac by indirect approach are limited to synthesis of CDRs and the diastereomers followed by only HPLC separation.…”

“…Successful TLC separation of diastereomers of ( RS )‐Bac [prepared with CDR‐ 1 (MCT reagent having l ‐Phe as chiral auxiliary), as mentioned above in this paper] was obtained using MeOH–CH 2 Cl 2 (4:5, v /v) as the solvent system at 20°C (Vashistha & Bhushan, ). A photograph of an actual chromatogram showing separation of diastereomers is shown in Figure (a).…”

“…An MCT reagent (CDR-1), namely, N-(4-chloro-6-piperidinyl-[1,3,5]triazine-2-yl)-L-phenylalanine, was synthesized (Vashistha & Bhushan, 2015) by nucleophilic substitution of one of the chlorine atoms by a piperidinyl group and the second with L-Phe (as the chiral auxiliary).…”

“…Nap-Btz (CDR-25), being an amide, is more stable than CDR-26 and CDR-27, which are esters, owing to higher thermodynamic stability of amides over esters. Synthesizing CDR-27 (Nap-Phth) is easier than the other two, since H-Phth, is neither explosive in nature like benzotriazole nor hygroscopic like H-Suc (Bhushan & Nagar, 2014 6.1 | TLC enantioseparation of (RS)-Bac by indirect approach and complimentary to HPLC Successful TLC separation of diastereomers of (RS)-Bac [prepared with CDR-1 (MCT reagent having L-Phe as chiral auxiliary), as mentioned above in this paper] was obtained using MeOH-CH 2 Cl 2 (4:5, v/v) as the solvent system at 20°C (Vashistha & Bhushan, 2015). A The RP-TLC chromatogram is shown in Figure 11(b) and hR F values are given in Table 4; the spots were yellow and visible in ordinary light.…”