Design, Synthesis And Biological Evaluation Of Anticancer Efficacy Of Multiple Substituted Indole Derivates

Along with gradually increased understanding of nosogennesis of tumor, developing anticancer drugs targeting signaling pathways which are highly active in cancer cells is becoming the hot spot. PI3K/Akt signaling pathway plays an important role in the regulation of cell survival, proliferation and growth in tumor cells. Akt belongs to Phosphatidylinositol3-kinase (PI3K)/Akt signaling pathway and recent studies indicate that unregulated activation of the PI3K/Akt pathway is a crucial feature in many human cancers. Akt is over-expressed or activated in all major cancers, such as ovarian, breast and pancreatic cancers. The activated Akt phosphorylates a series of substrates, which could block apoptosis pathway, promote cell proliferation, and maintain the survival of tumor cells. Inhibition of Akt proves to be an effective way to cancer intervention.Although there is no marketed drug targeting Akt, studies on Akt inhibitors has become more and more important. Akt inhibitors with different mechanisms have been reported, including ATP competitive inhibitors, allosteric inhibitors, phosphatidylinositol analogs inhibitors, pseudosubstrate inhibitors and other mechanism unknown inhibitors. MK-2206, an allosteric inhibitor of Akt, is in Phase I clinical trials. Inhibitors of phosphatidylinositol analogs and pseudosubstrates are currently in preclinical stages. Mechanism unknown inhibitors are reported to inhibite Akt with unknown mechanisms. API-2belongs to this kind of inhibitors which is currently in clinical trials. Most of the research on the Akt inhibitors focused on ATP competitive inhibitors. Several ATP competitive inhibitors have been into the clinical trials.Based on the three dimensional crystal structures of Akt and ATP competitive inhibitors complex, this study designed and synthesized three series of multiple substituted indole derivates:1) C-7phenyl substituted compounds;2) C-7methoxyl or halogen substituted compounds and3) oxdiazole compounds.The target compounds were synthesized using5-substituted salicylaldehyde as the starting material by benzyl chloride protecting reaction, aldol condensation, cyclization reaction, hydrogenation reduction, debenzylation by AlCl3, Suzuki reaction, substitution reaction with methoxyl group, methylation, etherification reaction, Mitsunobu reaction and deprotection reaction of Boc group.In this paper, we obtained40novel target compounds which are not reported previously. These compounds are identified by1H NMR and HRMS.We designed and synthesized10compounds in series1firstly,. The enzyme assay showed that several compounds exhibited Aktl inhibitory activities and the most potential compound is compound10c with coordinative activity to lead compound. The structure-activity relationship of this series compounds showed that: The introduction of methyl on N-1position is helpful to Aktl inhibition activities; The side chain optimization on C-4position suggested that compounds with two carbon atoms between the amino group and the oxygen atom gave better Akt pocency than that with three carbon atoms; The compounds with methyl ester group were proved to be more active than that with carboxyl, acid amide and hydroximic acid. However, phenyl substituted on C-7did not gave better Akt pocency as expected. Based on the results of series1, we optimized the structure of compounds with compound10c as lead compound to get series2. In this series, we obtained23target compounds and preliminary result showed that most of compounds displayed better Aktl inhibitory activities than the series1. Compounds29b,29f,33ac,33bc and33bf showed potent Aktl inhibitory activities with inhibition rate at72.5%,70.3%,73.0%,73.5%and76.9%respectively, which were higher than that of positive control GSK690693. The docking study of this series showed that the methoxyl group was headed into the back pocket as we designed. The glycine rich loop of the Akt was curved over the plane of the indole ring, maximizing hydrophobic interactions between the kinase and the inhibitor. The series3were designed based on the series2by replacing methyl ester group with oxdiazole using bioisosterism principle. Compound37b exhibited the most potent inhibitory activity with inhibition rate at78.6%. The docking study of37b and Akt showed that the comformation of37b was different with former compounds because of the oxdiazole ring. The oxdiazole ring went deep into the pocket A, forming a hydrogen bond with Ala232. The methoxyl group was headed into the back pocket with a hydrogen bond between oxygen atom and Asp293. The amino group on C-4side chain formed two hydrogen bolds with Glu236and a water molecule. The phenyl group on C-4side chain stretched out to the G-loop.The antiproliferative activities against human prostatic cancer cells PC-3were tested using MTT assay. The results showed that compounds29b,29f,33ac,33bc,33bf and37b, which showed good enzyme inhibitory activities against Aktl, also exhibited well potency against the proliferation of PC-3cell with IC50valus of3.83μM,3.10μM,4.98μM,3.24μM,10.18pM and5.34μM, respectively. These compounds were more active than the positive control GSK690693. We determined GSK-3phosphorylation in PC-3cells for up to4h with series concentration of compounds29b and37b. The results showed that compounds29b and37b could decrease GSK-3phosphorylation by directly inhibiting Akt in PC-3cells, which led to inhibition of cell proliferation. Meanwhile, both compounds showed dose dependent inhibition of GSK-3phosphorylation.