Selective antagonism activity of alkaloids from bulbs Fritillariae at muscarinic receptors: Functional studies

15 alkaloids were isolated from five Fritillariae species and 6 derivatives were synthesized. Alkaloids having anticholinergic effect on guinea-pig tracheal smooth muscle were screened out and their mechanism was further studied on the cAMP formation in Chinese hamster ovary cells stably expressing human muscarinic M2 receptor (CHO-hM2 cells) and intracellular calcium ([Ca2+]i) transient in Chinese hamster ovary cells stably expressing human muscarinic M3 receptor (CHO-hM3 cells). In normal Krebs–Henseleit (KH) solution, imperialine (15), 3β-acetylimperialine (16) and sinpeinine A (17) concentration-dependently relaxed 1 μM carbachol-induced contraction of guinea-pig tracheal rings with EC50 of 4.19, 1.71 and 4.67 μM, respectively. In Ca2+-free KH solution, 10 μM 3β-acetylimperialine (16), imperialine (15) and sinpeinnine A (17) caused 97.42%, 5.45% and 6.55% inhibition, respectively, which indicated that the three components might inhibit muscarinic receptor in different mechanism. Results of muscarinic M2 receptor-inhibited cAMP formation in CHO-hM2 cells showed that imperialine (15) and sinpeinine A (17) could potently elevate the cAMP formation whereas 3β-acetylimperialine (16) only had weak effect on antagonism of cAMP inhibition. Furthermore, the investigations of muscarinic M3 receptor-stimulated [Ca2+]i transient in CHO-hM3 cells revealed that imperialine (15) and sinpeinine A (17) could not antagonize [Ca2+]i transient, but 3β-acetylimperialine (16) significantly inhibited [Ca2+]i peak elevation with an IC50 of 5.26 μM. The functional studies suggest that the mechanism of relaxant action of imperialine (15) and sinpeinine A (17) is due to their selective inhibitory effects on muscarinic M2 receptors and the mechanism of 3β-acetylimperialine (16) originates from its selective muscarinic M3 receptors antagonism.