Synthesis And Antitumor Mechanisms Of Novel Fascaplysin Derivatives As CDK4 Inhibitors

Fascaplysin with broad bioactivity is a CDK4-specific inhibitors,whereas its planar structure enable bind and intercalate DNA leading to quite toxic effects.Therefore,we decided to synthesis non-planar ?-carboline derivatives through cyclization,oxidation,and quaternary amine ways.And the bis-indole target compounds were obtained only by one step of amidation.All compounds have been confirmed by 13CNMR,1HNMR,and HRMS.MTT assay indicated that ?-carboline derivatives are more potent with the IC50 in the range of 1-100 ?M than indoles(IC50>200?M)in inhibiting the growth of A549,HeGp2,Hela,and MCF cells.The target compounds A1,B1 which are most active inhibit A549 cell growth with the IC50 of 7.8 ?M and 10.5 ?M respectively.The inhibition of the compounds against CDK4 enzyme were at the cellular level,and A1 with the IC50 of 5.2 ?W showed approximately equivalent inhibition with fascaplysin(IC50=0.6 ?M).Molecular docking analysis suggested that A1 was docked into the pocket between Phe 93 and Phe 159 through the hydrogen bond with Val 96 and the conjugation with His 95.Flow cytometry assay indicate that A1 provoke G1 phase arrest and B1 can induce significantly apoptosis in A549 cells.Western blot further confirmed the inhibition against CDK4/cyclin D of A1 through measuring the downregulation expression of CDK4 and cyclin D.After treatment of A549 with B1,the level of actived caspase-3 was markedly upregulated.This facts indicated that A1 exhibits anti-proliferation effect through arresting cell cycle progression at G1 phase,whereas through induction of apoptosis related with caspase-3 pathway for B1.In ultraviolet and fluorescence spectrum,the significantly red shift and subtractive effect similar with fascaplysin were not found,which proved Aland B1 could not interacted with DNA.All together,these data sustain our contention that A1 and B1 not only reserve the antitumor activity but also reduce the ability of intercalation with DNA,and provide much guidance to develop novel CDK4 inhibitor.