Research paper
Design, synthesis and pharmacological evaluation of N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-N-arylmethyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamides as potent Hsp90 inhibitors

https://doi.org/10.1016/j.ejmech.2017.11.013Get rights and content

Highlights

  • A structure-based optimization was conducted on the lead Hsp90 inhibitor 1.

  • CETSA and WB showed that 41 with the para-pyridyl group bound to cellular Hsp90α.

  • Compound 41 formed an H-bonding interaction with the side-chain of Tyr139.

  • Compound 41 inhibited tumor cells growth, induces cell cycle arrest and apoptosis.

  • Compound 41 inhibited TNF-α-induced NF-κB activation through IKKs.

Abstract

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 (1043) 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 ITDRFCETSA (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.

Introduction

Heat shock protein 90 (Hsp90) is a ubiquitous and abundant ATP-dependent molecular chaperone, which is documented to interact with more than 200 different “client” proteins involved in signal transduction, protein trafficking, receptor maturation, and innate and adaptive immunity [1], [2], [3]. Impairment of Hsp90 ATPase activity disrupts the dynamic ATP-coupled chaperone cycle and alters its interaction with client proteins, resulting in client protein destabilization and eventual degradation via the ubiquitin−proteasome pathway [4], [5], [6]. Therefore, Hsp90 inhibitors allow the simultaneous inhibition of multiple oncoproteins and signaling pathways that are essential in maintaining the malignant phenotype of tumors [4]. This is a particularly attractive feature given the extraordinary ability of tumor cells to rapidly evolve resistance through mutations or activation of alternative signaling pathways to therapeutics targeting a single oncogene [7], [8], [9], [10].

One of the most characteristic and striking features of Hsp90 protein is its extreme flexibility, which allows efficient Hsp90 chaperone cycles, in concert with cochaperones and adaptor proteins [11]. Among documented movements that have been observed in the Hsp90 protein, conformational changes observed in the N-ter ATP site are critical for the inhibition of ATP hydrolysis and client processing.

As previously reported for 8-benzyladenine series of inhibitor (purine-scaffold), a conformational change in the flexible lid of Hsp90 occurs upon ligand binding and creates a new binding channel [12], [13]. In this class, the 8-aryl moiety rearranges the flexible Hsp90 lid, exposing a lipophilic pocket whereby the phenyl ring becomes stacked among Leu107 and Phe138, and forms further favorable interactions with Met98 and Leu103. This motif (Leu107 and Phe138) is referred to the “aryl-binding pocket” [13], [14].

Recently, we have embarked on the development of N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamides as novel Hsp90 inhibitors [15]. Among them, compound 1 (B7 in ref. [15], N-(5-chloro-2,4-dihydroxybenzoyl)-(R)-N-benzyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide) was selected as lead compound, in which the resorcinol moiety is linked to a benzyl group via a (R)-Tic bridge. Previous research indicated that the benzyl group of 1 was buried in the aryl-binding pocket (Fig. 1), which enabled us to rationalize chemical modifications for this compound to improve Hsp90 binding affinity. In order to explore the properties of this newly developed pocket, we now report the optimization of the southeastern aryl substitutions of 1, which provides a series of new and potent Hsp90 inhibitors.

Section snippets

Chemistry

A series of tetrahydroisoquinoline derivatives with the general structure were designed, based on the structure of lead compound 1. The target compounds were synthesized by following the procedures described (Scheme 1). Compound 4 was synthesized from methyl 2,4-dihydroxy-5-methyl benzoate (2) by chlorination of the aromatic ring followed by benzyl protection of the phenols, which was hydrolyzed to furnish acid 5 in good yield. Esterification of chiral monomer 6 gave (R)-methyl

Conclusions

Focusing on the southeastern aryl moiety of lead compound 1, 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

Cell culture

The SHSY5Y cells, U251 cells, HCT116 cells, H1299 cells, A549 cells, HepG2 cells, PC3 cells, MDA-MB-231 cells, HeLa cells, and 293T cells were maintained in DMEM containing 10% fetal bovine serum (FBS). The SW480 cells, MCF7 cells, SKOV3 cells and MDA-MB-453 cells were maintained in RPMI 1640 media containing 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. All cells were obtained from American Type Culture Collection (ATCC).

In vitro antiproliferative evaluations

Growth

Acknowledgments

This work was supported by 973 Program (2010CB833802), NSFC Projects (81273384, 91313303, U1405223), Natural Science Foundation of Shandong Province, China (ZR2013HQ048), Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R68) and Distinguished Young Scholars Grant to Y. S. (30325044).

References (26)

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