Using diverse arylmethyl groups to replace the benzyl moiety of the lead Hsp90 inhibitor 1 (N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-N-benzyl-1,2,3,4-tetrahydro-3-iso quinolinecarboxamide), thirty four derivatives (10-43) were developed, and exhibited improved Hsp90 inhibitory and antiproliferative activities. SAR analysis indicated that the southeastern aryl substitutions influenced their antiproliferative activities obviously, with the para-pyridyl group (41) outperforming all other substitution patterns. In this regard, compound 41 was selected for further evaluation. CETSA melt and ITDRF (isothermal dose-response fingerprint) curves for Hsp90α further proved that 41 interacted with intracellular Hsp90α powerfully. Compared with the lead compound 1, docking and MD refinement of the Hsp90α-41 complex revealed a favorable H-bonding interaction between the side-chain of Tyr139 and the pyridine moiety of 41, which is the first time to be used for resorcinol-based Hsp90 inhibitors. With broad-spectral antitumor activity, compound 41 induced time- and dose-dependent growth inhibition and G0/G1 cell cycle arrest on human breast cancer MDA-MB-453 cell line. In addition, flow cytometry and Western blot analyses confirmed that 41 induced apoptosis of human breast cancer MDA-MB-453 cell line. Via degradation of IKKs and suppression of IKKs activity, compound 41 inhibited TNF-α-induced NF-κB activation. The overall properties warrant compound 41 a promising Hsp90 inhibitor and further biological characterizations. This study provides insights into the chemical evolution of Hsp90 inhibitors, and may facilitate the design of next generation Hsp90 inhibitors for the antitumor drug development.