Application Of NMR In Chinese Medicine Quality Control And Drug Stereostructure Study

Nuclear magnetic resonance (NMR) technique has become an indispensable tool for structure characterization, dynamic process tracking and metabonomics analysis, widely used in drug development at all levels. This work studies the application of NMR in Chinese medicine quality control and drug stereostructure study. Studies include the establishment of quality evaluation and control system of Cimicifuga by1H NMR fingerprint combined with pattern recognition (PR) method, and the investigation of conformational behavior of antihypertensive agent trandolapril and anti-cancer drug capecitabine in solution by using dynamic NMR experiment.It is well known, at least eight species of Cimicifuga are distributed in China, and most are used in traditional Chinese medicine. Three species, C. foetida, C. heracleifolia and C. dahurica, all known by the same common Chinese name "shengma" and have been record in Chinese Pharmacopoeia. However, according to the pharmacopoeia method can not achieve the authentification of Cimicifuga species. In order to identify their species and quality, the metabolomic analysis of the three Cimicifuga species was performed using1H NMR spectroscopy and PR techniques in the first part.Dried plant material of38C. foetida,15C. heracleifolia and15C. dahurica were extracted by methanol to give extracts, which were detected by1H NMR spectroscopy. The reproducibility and precision of the all samples and the testing process were investigated, and the main peaks were assigned, thereby establishing the1H NMR fingerprint of Cimicifuga. The analysis using principal component analysis (PCA) and discriminant partial least square (DPLS) of the1H NMR spectra showed a clear discrimination between C. foetida and the other two species. The major metabolites responsible for the discrimination were triterpenoid saponins and saccharides. A coupled artificial neural network (ANN) and DPLS model was applied to the classification of C. heracleifolia and C. dahurica.In the second part, the conformational behaviors of two synthetic drugs in solution were investigated by dynamic NMR technique. To the best of our knowledge, the structure and conformational behavior of drug molecule is essential in obtaining information about the active site of the enzyme. In this study, the stereostructure of capecitabine and trandolapril were investigated by NMR spectroscopy combined with quantum chemical calculations. Trandopril was studied by NMR in various solvents and at various temperatures. The results showed that the two conformers interchange via rotation about the amide bond, and the cis conformation was the preferred conformation. The free energy of activation for the rotation about the amide bond was16.2kcal/mol. Quantum chemical calculations for trandolapril and capecitabine have been performed, and the results verified the NMR experiment conclusion.In addition, two concurrent exchanges arising due to the restricted rotation around the carbamate C-N bond and alkyl C-N bond were investigated in capecitabine. A total of four low energy conformers were evaluated in the molecule, two were coexisted with a ratio of1:0.7in CDCl3solution at265K. Dynamic NMR experiment indicate that the major conformer is actually a mixture of three conformers with an extremely low barrier for them to interconvert, and the minor conformer form intramolecular hydrogen bond because of the NH proton migration.This work established the quality evaluation and control system of Cimicifuga by1H NMR fingerprint combined with pattern recognition (PR) method for the first time. It provided an important method for authenticating C. foetida and distinguishing it from other species of Cimicifuga. This should facilitate the continued development of high-quality, unadulterated C. foetida products. Furthermore, we also found the presence of a variety conformers in solution of capecitabine and trandolapril, and then elucidated its stereostructure by NMR spectroscopy and quantum chemistry calculation for the first time. These results are not only a reasonable explanation of their conformational behavior from the level of molecular structure, also laid the foundation for further study about the drug efficacy and the mode of action of the drug molecule with the target enzyme molecule.