Design,Synthesis And Antitumor Activity Of Tetrahydroisoquinoline-3-carboxylic Acid-based Hsp90 Inhibitors

Heat shock protein 90(Hsp90)is an ATP-dependent molecular chaperone that is involved in the folding,activation,and stabilization of numerous oncogenic proteins.It has become an attractive therapeutic target,especially for eradicating malignant cancers and overcoming chemotherapy resistance.By means of FBDD,we rationally designed and synthesized two series of novel N-(5-chloro-2,4-dihydroxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide derivatives as Hsp90 inhibitors using(S)-Tic(A1-13)and(R)-Tic(B1-13)as scaffolds,respectively.The compounds of B-series showed evident anti-proliferative activities against MDA-MB-231 and HeLa cell lines,which reveals not only clear structure-activity relationships but also that the(R)-Tic scaffold is suitable for the design and synthesis of Hsp90 inhibitors.Target identifications by CETSA indicated that the cytotoxic compounds B1-13 bound to Hsp90α,which showed the same structure-activity relationships as that of SRB assays.The most potent compound B7 having the(R)-Tic scaffold showed not only the strongest cytotoxicity but also the most ability to induce thermal stabilization of Hsp90a.CETSA melt and ITDRFcETSA curves for Hsp90a further proved that B7 interacted with intracellular Hsp90α.Moreover,B7 was verified to be a novel inhibitor of Hsp90α by exhibiting the well-established cellular biomarkers as the known Hsp90 inhibitor GA at the IC50 concentration.In addition,docking and MD refinement of the Hsp90a-B7 complex gave a theoretical binding model and revealed a novel Π-stacking interaction between the residue Phe138 of Hsp90α and the benzylamino moiety of B7.The overall properties warrant compound B7 a promising lead of Hsp90 inhibitors and further biological characterizations.Focusing on the southeastern aryl moiety of lead compound B7,a series of derivatives were developed and exhibited improved Hsp90 inhibition and antiproliferative activities.However,the ring substitution requirements were very specific,with the para-pyridyl group outperforming all other substitution patterns,which extended our understanding into the structural elements that allowed for high affinity binding to Hsp90.In this regard,it highlighted the importance of substituents on the aryl moiety.Compound D4 were selected for further evaluation.CETSA melt and ITDRFCETSA curves for Hsp90a further proved that D4 interacted with intracellular Hsp90a.Docking and MD refinement of the Hsp90a-D4 complex gave a theoretical binding model and revealed a favorable H-bonding interaction between the side-chain of Tyr139 and the pyridine moiety of D4.With broad-spectrum antitumor activity,compound D4 induced time-and dose-dependent growth inhibition and G0/G1 cell cycle arrest.In addition,flow cytometry and Western blot analyses confirmed that D4 induced apoptosis.IKKs are well recognized as key regulators in the NF-κB pathway.Via degradation of IKKs and inhibition of IKKs activity,compound D4 inhibited the activity of NF-κB in response to TNF-a.The overall properties warrant compound D4 a promising Hsp90 inhibitor and further biological characterizations.Molecular modeling analysis of Hsp90-D4 complex indicated that tetrahydroisoquinoline fragment of D4 was closed to Lys58 and faced the solvent-exposed area.Based on this analysis,structure optimization of tetrahydroisoquinoline fragment created E and F series compounds,which was expected to improve pharmacology activity and drug safety.Synthesis and activity screening of these two series compounds have been completed at present.The study of antitumor mechanism is ongoing.In conclusion,based on the crystal structure of Hsp90 and the binding mode of the known Hsp90 inhibitors with the enzyme,we rationally designed and synthesized a series of Tic-based Hsp90 inhibitors via integrated application of medicinal chemistry,biochemistry and computational chemistry.This study introduced CETSA to identify target engagement and further researched anti-tumor mechanism of the representative compound.Moreover,this study provided insights into the chemical evolution of Hsp90 inhibitors and may facilitate the design of next generation Hsp90 inhibitors for the antitumor drug development.