Synthesis And Pharmacological Activities Studies Of Hirsutine And Its Analogues

Objective:Natural product Hirsutine and its analogues are synthesized and evaluated for vasorelexant effects, and the indole analogues were designed for evaluating for antitumor activity. The structure activity relationship of Hirsutine and its analogues are developed and analyzed. Investigation was undertaken to elucidate the Hirsutine analogue Y-342, designed and synthesized from Hirsutine, as a vasorelaxant agent on ex vitro functional rat aorta test and to research its mechanism.Method:1) All the Hirsutine analogues are evaluated on in vitro rat thoracic aorta test for vasodilation activity. Cell survival rate is assessed by MTT experiment for antitumor activity.2) Isolated SD rat thoracic aortic artery rings were prepared to test the vasodilatory effects of the synthesized compounds ex vivo. Rats (250-350 g body weight) were sacrificed by cervical dislocation. And the thoracic aortas were removed and immersed in Krebs solution at room temperature, and than the thoracic aorta was cleaned of adhering connective tissue and was cut into 3-5 mm length rings. In some rings, the endothelium was removed. Then, tissue segments were mounted in stainless steel hooks, under an optimal tension of 2 g, in 10 mL organ baths containing warmed (37℃) and oxygenated (O2/CO2,19:1) Krebs solution (composition, mM:NaCI, 120.6; KC1,5.9; CaCl2,2.5; MgSO4,1.2; KH2PO4,1.2; NaHCO3,15.4; and glucose, 11.5, pH 7.4). Compounds were added cumulatively after the aortic rings contraction with PE reached stable condition. Changes in tension were measured and recorded using JH-2 force transducers (Jialong Instruments(?), Shanghai, China) connected to a Sump-PC analyzer (Jialong Instruments, Shanghai, China). 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 Y-342, which owns the best vasodilatory effect among all the Hirsutine analogues, was chose to investigate the mechanisms of the vasodilatory effect. The function of endothelium cells, voltage-gated potassium channel, Nonselective calcium sensitive potassium channels, NOS, cGMP, L-type calcium channel, the intracellular calcium release pathway, as well as the M-adrenaline receptor were tested in vitro.4) Indole analogues own inhibitory properties via MTT test on human tumor cells in vitro.Result:1.8 new Hirsutine analogues and 24 intermediate indole analogues are synthesized.2. All the Hirsutine analogues are involved in vasorelexant effects and five in this series are more potent than the natural product Rhynchophylline. IC50 of the most potent compounds are Y-482 (IC50=3.684×10-3μM,1.78×10-3 mg/L), Y-326 (IC50=0.09832μM,0.0321 mg/L) Y-342 (IC5o=5.891μM,2.015 mg/L, which is much stranger than Rhynchophylline (IC5o(Rhy)=174.2μM,66.893 mg/L).3. Compound Y-342 which was as a vasorelaxant agent on in vitro functional rat thoracic aorta test was the same as natural Hirsutine. Compound Y-342 (0.001-100 μM) induced significant relaxation in a concentration-and endothelium-dependent and-independent manners in the thoracic aortic rings pre-contracted with noradrenaline (NA,1 μM), and phenylephrine (PE,1μM). When endothelium was removed (-E) the vasorelaxant effect was not more significant (P> 0.05) than in endothelium-intact (+E) aorta rings. Pre-treatment with N-nitro-L-arginine methyl ester (L-NAME; 10μM), l-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-l-one (ODQ,1 μM) or atropine (1μM) produced a significant change of the relaxant response and activity was markedly inhibited, but not by tetraethyl ammonium (TEA,5 μM) and glibenclamide (1 μM). The experimental results show that the compound of Y-342 vasodilation activity may be through the NO/cGMP signal pathways and calcium channel blockade, and has nothing to do with the potassium channels.4. The result of the MTT test indicates that the 24 intermediate indole analogues express considerable antitumor activity on hepatoma carcinoma cells, grastric cells and colon cancer cells.Conclusion:This research accomplished total synthesis of Hirsutine and its analogues. Besides these,24 intermediate indole analogues are also synthesized. The rat thoracic aorta tests show that Hirsutine analogues had vasodilation activities, and the results of MTT cell survival experiments demonstrate that the intermediate indole analogues had antitumor effects.The mechanism research of compound Y-342 shows that the compound was mediated by NO/cGMP pathway and through calcium chanel blockade.