Total Synthesis And Structure-activity Relationship Study Of 7-hydroxyneolamellarin A

Tumor cells are often in a hypoxic microenvironment due to the abnormal structure and function of their blood vessels,leading to overexpression of hypoxia-inducible factor-1?HIF-1?,which is associated with tumor angiogenesis,proliferation and metabolism,invasion and metastasis and chemotherapy resistance.In recent years,extensive experimental and clinical data have validated HIF-1 as a therapeutic target for cancer,many small molecule inhibitors targeting HIF-1 signaling pathway were explored to seek excellent clinical candidates.A novel marine pyrrole alkaloid 7-hydroxyneolamellarin A,derived from sponge Dendrilla nigra,has been proven to possess excellent hypoxia-induced HIF-1 inhibitory activity in T47D cells(IC₅₀=1.9?M),but there is no more report on the total synthesis of this compound and the mechanism of action remains unknown.In this paper,we designed and synthesized 7-hydroxyneolamellarin A and its derivatives,and evaluated their HIF-1inhibitory activity by dual-luciferase reporter gene assay.The structure-activity relationship was established to lay the foundation for further study of mechanism of action.Total synthesis of 7-hydroxyneolamellarin A was achieved in 12 steps by a convergent synthesis strategy,with an over-all yield of 0.4%.The route was:separately preparing3,4-bis?4-?benzyloxy?phenyl?-1H-pyrrole and benzyl?1-?4-?benzyloxy?phenyl?-2-chloro-2-Oxyethyl?carbonate,followed by N-acylation under n-butyllithium to synthesize hydroxyl-protected intermediates 1-2,finally debenzylated by Pd/C,the target compound was obtained.HeLa cells expressing HRE-dependent firefly luciferase reporter and CMV-driven renilla luciferase reporter were transfected with Lentiviral and selected with puromycin.The test conditions were explored and a dual-luciferase reporter gene evaluation system was successfully constructed.Using a dua-luciferase reporter gene evaluation system,HIF-1 inhibitory activities of 137-hydroxyneolamellarin A derivatives?3 of which were from this work and 10 were pre-synthesized?were evaluated and the cytotoxicities were tested by MTT assay.Structure-activity relationship showed that the length of aliphatic carbon chain had no significant effects on the activity.Compounds with p-hydroxyl group exhibited more potent HIF-1 inhibitory activity than the corresponding compounds with p-methoxyl group,whereas their cytotoxic effects were also higher than those of methoxyl-substituted derivatives.C-7hydroxy-substituted analogue 2b(IC₅₀=11.9±3.6?M)showed comparable potency with neolamellarin A 2a(IC₅₀=10.8±1.0?M),demonstrating that the 7-hydroxyl group is critical for the HIF-1 inhibitory activity of this skeleton.All the compounds we synthesized had no significant cytotoxicity at the concentrations they effectively inhibited HIF-1 activation.