Twelve chiral derivatizing reagents (CDRs) were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene (DFDNB) with three optically pure amines [(R)-(-)-1-cyclohexylethylamine, (+)-dehydroabietylamine and (S)-(-)- ,4-dimethylbenzylamine], six amino acid amides [L-Ala-NH2, L-Phe-NH2, L-Val-NH2, L-Leu-NH2, L-Met-NH2 and D-Phg-NH2] and three amino acids [L-Ala, L-Val and L-Leu]. The work was led by Professor Ravi Bhushan from the Department of Chemistry at the Indian Institute of Technology Roorkee in India and documented in Biomedical Chromatography [23 (12) 1291 – 1299 (2009)].
β-blockers are commonly used in the treatment of hypertension, angina pectoris, cardiac arrhythmias and glaucoma. Most of them are marketed as racemic mixtures and used in therapy as such. Their enantiomers show significant differences in the pharmacological effects and activities. “Generally, the S-(-)-enantiomer of β-blockers is pharmacologically effective showing about 50-500 fold higher activities. Considering the two enantiomers as different drugs for the differences in their stereo selective mechanism the current prescription of racemic β-adrenergic blockers thus needs a review from the clinical, medical and health point of views,” explained Professor Bhushan.
In the study, 14 CDRs were synthesized; twelve of them were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene (DFDNB) with three optically pure amines, six amino acid amides and three amino acids (CDR 1-12). One was synthesised by the reaction of DNP- L-proline with N-hydroxysuccinimide in presence of dicyclohexyl carbodiimide (CDR 13) and the last CDR is N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate (SINP; CDR 14). “This is the first report on synthesis of diastereomers of the six β-blockers with these 14 CDRs particularly the ones having optically pure amines, as the chiral auxiliary, followed by high-performance liquid-chromatographic resolution of the diastereomers,” Bhushan continued.
According to Bhushan, of the total 84 pairs of diastereomers there were resolved 64 pairs showing sharp peaks with base line separation. In the mobile phase, MeCN was found to be a better organic solvent compared with methanol as larger retention times and broader peaks were obtained with the latter. “The results indicate that this method can be applied for the detection of (S)-propranolol in (R)-propranolol up to 0.002 %. The recoveries of (R)- and (S)-propranolol were found to be between 95 to 99.5%. The reagents, dinitrophenyl- L-Pro-N-hydroxysuccinimide ester and the N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate were capable of producing diastereomers of Salbutamol where the secondary N is sterically hindered and many other CDRs do not produce the corresponding diastereomers,” he said.
The derivatization time required in the present study is either 50 seconds under microwave irradiation for reaction with CDRs 1-12, and 10-15 minutes at room temperature for reaction with CDR 13 and 14. The LODs are in the range of 10-15 pg/mL which are much lower than the literature reports.
The derivatization time for atenolol (or some other β-blocker) is reported to be 30 minutes at room temperature using different CDRs; e,g., FLEC, t-BOC- L-leu anhydride, 2,3,4,6-tetra-O-acetyl-β-D-glycopyranosyl isothiocyanate, and 2,3,4-tri-O-acetyl-β-D-arabinopyrsnosyl isothiocyanate; the limit of detection (LOD) in these literature reports varies from 0.2 ng/ mL to 3 ng/mL.
“There is a growing need for marketing single enantiomers,” he continued. This could either be through enantioselective synthesis or via enantiomeric resolution. The chromatographic conditions for indirect mode of enantioseparation can be optimized with greater ease because CDRs can provide high sensitive detector response for the analysis of biological samples and thus often result in better resolution than use of a direct method. Marfey’s reagent (1-fluoro-2,4-dinitrobenzene-5- L-alaninamide, FDAA, FDNP- L-Ala-NH2, MR) can safely be considered as a chiral variant of 2,4-di-nitrofluorobenzene (DNFB, Sanger´s reagent).
“CDRs based on Sanger’s reagent have been used, in this laboratory, for indirect enantiomeric resolution of compounds containing amino group such as atenolol including that from rat plasma, protein and non-protein amino acids, penicillamine, and baclofen. The present results offer a fast, reliable and superior method. The application of Sanger’s reagent and its chiral variants in the present work further establishes their utility and importance and suggest their future application in analysis and control of enantiomeric purity in pharmaceutical industry,” he concluded.
For more information, contact the author at rbushfcy@gmail.com