| Aim:1) To design, synthesize the analogues of natural product corynantheine.2) Test their vasodilatory property and analysis the structure activity relationship.3) Investigate the vasodilatory mechanisms of compound 8.4) Test the inhibitory activity of compounds 2-6 in vitro on human tumor cells.Methods:1) We start out synthesis route with tryptamine, and obtained the products by Pictet-Spengler condensation, Boc-protected, Cbz-protected reaction in the N-position, then selectively reduction reaction with DIBA1H, Knoevenagel reaction and catalytic hydrogenation in hydrogen atmosphere with Pd/C. And we also synthesized a series of derivatives of compounds 5 and 8.2) Isolated SD rats’thoracic aortic artery rings were prepared to test the vasodilatory effect of the synthesized compounds ex vivo. Compounds were added cumulatively after the aortic rings’contraction with PE reached stable condition, and the tension of the rings was recorded. The potency and the activity concentration curve of the new synthesized compounds were calculated and analyzed, as well as the structure activity relationship.3) Compound 8, which owns the best vasodilatory potency among all the compounds, was chose to investigate the mechanisms of the vasodilatory effect. The function of endothelium cells, L-type calcium channel, KATp+channel, the intracellular calcium release pathway, as well as the al-adrenaline receptor were tested in vitro.4) Compound 2-6 own inhibitory properties via MTT test on human tumor cells in vitro.Result:1) Fifteen corynantheine analogues were synthesized, including eleven novel compounds. The structures of all the compounds were confirmed with MS, HRMS, ’H-NMR,13C-NMR.2) The pharmacological results showed that fourteen among these fifteen compounds have vasodilatory effect ex vitro on isolated rats’thoracic aortic artery rings in concentration dependent manner. Eight of them exhibited better activity compared to natural product rhynchophylline (IC50< 128.5μM). The structure activity relationship analysis revealed that the structure of compounds 5 and 8, the best two compounds both have a hydroxyl residue. Once other substitutions replaced the hydrogen of the hydroxyl function, the pharmacological potency decreased (IC50 of 9-11> 5; IC50 of 12-16> 8). On condition that the substitutions were acyl groups we found that the longer of the carbon chain, the weaker of the vasodilatory effect (IC50 of 13> 12; IC50 of 10> 9). Besides, large group with benzene ring substitutions replaced the hydrogen atom will lead to further decrease of the potency (1C50 of 14～16> 100μM). Besides, compared 15 and 16 in pairs, except for their weak activities we found that compound 16 with a fluorine in the residue have better activity than compound 15 with a chloride in the residue. Still, from the data of the compounds which exhibited vasodilatory properties indicated that their common structure -- carboline with Boc in 1-N position may be an essential factor for their pharmacological effect.3) The previous work showed that compound 8 have the best vasodilatory effect (IC5o=1.293μM), and it was chosen to investigate the pharmacological mechanisms. The endothelium cells played an important role during the vasodilatory process of compound 8. In endothelium-denuded group (IC5o=15.23μM), and endothelium-intact group which were pretreated with 10-5M L-NAME or 10-5M atropine, the vasodilatory effect of compound 8 decreased but still existed. Compound 8 and nifedipine exhibited similar potency in endothelium-denuded artery rings contracted by PE (IC50=134.6μM and 15.23 μM respectively), however, the potency of the two agents was hugely different in endothelium-denuded artery rings contracted by KC1 (60mM): the potency of compounds 8 decreased to 1/10 while the potency of nifedipine increased quite a lot. In calcium free medium, compound 8 could inhibit the contracture of the aortic artery rings in concentration dependent manner. Once pretreated with 10-5M glibenclamide, the vasodilatory effect of compound 8 was inhibited (IC50=66.20 μM). Compound 8 didn’t affect resting artery rings, and pre-treat with compound 8 could inhibit the contraction of artery rings by PE ex vitro.4) The result of the MTT test indicates that compounds 2-6 own varied inhibitory properties on human tumor cells in vitro.Conclusion:we designed and synthesized fifteen novel indole corynantheine analogues, and verified their vasodilatory activities in vitro. The results revealed that fourteen of the fifteen compounds have vasodilatory properties, and eight of them own better potency than natural product rhynchophylline (IC50< 128.5μM). According to the structure activity relationship analysis, we found that the hydroxyl plays an essential role for the compounds to exert considerable activities. The mechanisms investigation of compound 8 demonstrated that compound 8 hardly affect the physiological function of artery rings in resting condition and the pre-treat of compound 8 may inhibit a-adrenaline receptor. Compound 8 exhibited vasodilatory effect in both endothelium dependent and independent manner, and the eNOS and muscarinic receptor were involved in the process. Besides, compound 8 reduced the intracellular concentration of calcium ion via the inhibition of intracellular calcium release pathway rather than the inhibition of L-type calcium channel. Still, KATp+ channel was involved in the process and compound 8 may act as a potassium channel opener. Besides, compounds 2-6 exhibited inhibitory activity in vitro on human tumor cells, indicated that compounds could affect the growth of the cell lines in vitro. |
