A potent and orally active NK1 antagonist, trans-N-[3, 5-bis(trifluoromethyl)benzyl]-7,8-dihydro-N, 7-dimethyl-5-(4-methylphenyl)-8-oxo-1,7-naphthyridine-6-carboxamide (1t), was shown to exist as a mixture of separable and stable (R)- and (S)-atropisomers (1t-A and 1t-B) originating from the restricted rotation around the -C(6)-C(=O)- bond; the antagonistic activities of 1t-A were ca. 6-13-fold higher than those of 1t-B. Analogues of 1t (3), which have (S)- and (R)-methyl groups at the benzylic methylene portion of 1t, were prepared and separated into the diastereomeric atropisomers, 3a-A, 3a-B and 3b-A, 3b-B, in enantiomerically pure forms. Among the four isomers of 3, the (aR, S)-enantiomer (3a-A) exhibited the most potent antagonistic activities with an IC50 value of 0.80 nM (in vitro inhibition of [125I]BH-SP binding in human IM-9 cells) and ED50 values of 9.3 micrograms/kg (iv) and 67.7 micrograms/kg (po) (in vivo inhibition of capsaicin-induced plasma extravasation in guinea pig trachea), while the activity of the (aS,R)-enantiomer (3b-B) was the weakest with an IC50 value of 620 nM. The structure-activity relationships in this series of antagonists indicate that the (R)-configuration at the axial bond and the stacking (or stacking-like) conformation between the two phenyl rings as shown in 1t-A and 3a-A are essential for high-affinity binding and suggest that the amide moiety functions as a hydrogen bond acceptor in the interaction with the receptor.
Quantitative NMR (qNMR) is applied to determine the absolute quantitative value of analytical standards for HPLC-based quantification. We have previously reported the optimal and reproducible sample preparation method for qNMR of hygroscopic reagents, such as saikosaponin a, which is used as an analytical standard in the assay of crude drug section of Japanese Pharmacopoeia (JP). In this study, we examined the absolute purity determination of a hygroscopic substance, indocyanine green (ICG), listed in the Japanese Pharmaceutical Codex 2002, using qNMR for standardization by focusing on the adaptation of ICG to JP. The purity of ICG, as an official non-Pharmacopoeial reference standard (non-PRS), had high variation (86.12 ± 2.70%) when preparing qNMR samples under non-controlled humidity (a conventional method). Additionally, residual ethanol (0.26 ± 0.11%) was observed in the non-PRS ICG. Next, the purity of non-PRS ICG was determined via qNMR when preparing samples under controlled humidity using a saturated sodium bromide solution. The purity was 84.19 ± 0.47% with a lower variation than that under non-controlled humidity. Moreover, ethanol signal almost disappeared. We estimated that residual ethanol in non-PRS ICG was replaced with water under controlled humidity. Subsequently, qNMR analysis was performed when preparing samples under controlled humidity in a constant temperature and humidity box. It showed excellent results with the lowest variation (82.26 ± 0.19%). As the use of a constant temperature and humidity box resulted in the lowest variability, it is recommended to use the control box if the reference ICG standard is needed for JP assays.
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