Synthesis of NF-κB activation inhibitors derived from epoxyquinomicin C

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Abstract

In order to develop new inhibitors of NF-κB activation, we designed and synthesized dehydroxymethyl derivatives of epoxyquinomicin C, namely, DHM2EQ and its regioisomer DHM3EQ. These derivatives were synthesized from 2,5-dimethoxyaniline in 5 steps. Since DHM2EQ was more active and less toxic than DHM3EQ, its stereochemical configuration was determined by X-ray crystallographic analysis. Each enantiomer of the protected DHM2EQ was separated by a chiral column and deprotected. DHM2EQ inhibited TNF-α-induced activation of NF-κB in human T cell leukemia cells, and also inhibited collagen-induced arthritis in a rheumatoid model in mice.

Introduction

NF-κB is a transcription factor that mediates the expression of a variety of cellular genes regulating the inflammatory response. For example, the expression of various cytokines such as IL-1, IL-2, IL-8 and TNF-α is regulated by NF-κB. NF-κB is located in the cytoplasm along with its endogenous inhibitor, I-κB. Stimulatory signals such as TNF-α and phorbol esters induce degradation of I-κB, and NF-κB consequently activated enters the nucleus to bind to the κB site of DNA. Inhibitors of this process of NF-κB activation are likely to become new anti-inflammatory and anti-rheumatoid agents.1 Recently, panepoxydone2 and cycloepoxydon3 were reported to inhibit NF-κB activation, both of which have the 4-hydroxy-5,6-epoxycyclohexenone structure. We previously isolated four novel 5,6-epoxycyclohexenone compounds named epoxyquinomicins from Amycolatopsis sp. MK299-95F4 as antibiotics and anti-inflammatory agents.4, 5 Epoxyquinomicin C was the simplest among them, having a 4-hydroxy-5,6-epoxycyclohexenone structure, however, it did not inhibit activation of NF-κB. Epoxyquinomicin C has an additional hydroxymethyl group compared with panepoxydone that inhibits NF-κB. Therefore, we designed and synthesized 5-dehydroxymethyl derivatives of epoxyquinomicin C.

Section snippets

Synthesis

The synthetic route for DHM2EQ and DHM3EQ is outlined in Scheme 1. We employed the Wipf method6 with modifications for the preparation of the quinone monoketal structure. Commercially available 2,5-dimethoxyaniline, 3, and acetylsalicyloyl chloride were coupled in pyridine to give salicylamide 4, which was subsequently oxidized by iodobenzenediacetate in methanol to yield quinone monoketal 5, in 50% yield. Epoxidation of 5 with alkaline hydrogen peroxide in aqueous THF gave epoxide 6 in 53%

Biological activity

Human T cell leukemia Jurkat cells were used for the assay of NF-κB activity. The cells were transfected with 2μg of DNA by the DEAE-Dextran method. The transfected cells were seeded into 12-well plates at 1×106/well. Chemicals dissolved in DMSO and TNF-α were added at 14 and 16 h, respectively. Six hours after this TNF-α addition, the cells were harvested and lysed; and the lysate was used for the luciferase assay with luciferin and ATP (Promega Luciferase Assay Systems). Luminescence was

Acknowledgements

The authors wish to thank Ms. K. Kameo and Ms. M. Endoh, Mercian Corporation, for measuring the physico-chemical data. This work was financially supported in part by the Special Coordination Funds for Promotion of Science and Technology from the Science and Technology Agency, and by a grant from the Ministry of Education, Science, Culture, and Sports of Japan (Academic Frontier Promotion Project).

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