Discovery of quinazolines as a novel structural class of potent inhibitors of NF-κB activation
Compound 11q exhibited a highly inhibitory activity toward NF-κB activation and also showed an anti-inflammatory effect.
Introduction
Autoimmune diseases and allergies, such as rheumatoid arthritis, septic shock, transplant rejection, asthma, and psoriasis, are considered to be caused by abnormalities of T cell immune responses.1 In particular, the activation of T cells initiates a network of events that results in the overproduction of certain transcription factors and proinflammatory cytokines.2 Transcription factors are DNA-binding proteins that regulate the production of proinflammatory cytokines as well as that of a variety of other cellular regulators.
Nuclear factor-κB (NF-κB) is a pivotal transcription factor that functions to enhance the transcription of proinflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-8, which are thought to be important in the generation of acute inflammatory responses.3 Activation and regulation of NF-κB are tightly controlled by members of a family of inhibitory proteins referred to as IκB. Several IκB proteins have been identified, including IκB-α, IκB-β, IκB-γ, IκB-ε, and Bcl-3.4
In resting cells, NF-κB is sequestered in the cytoplasm through its interaction with its inhibitor, IκB-α, IκB-β or IκB-ε. However, a large variety of inflammatory conditions, such as inflammatory cytokines, bacterial and viral infections, and certain chemical agents, induce NF-κB activity.5 NF-κB activation involves signaled phosphorylation, ubiquitination, and proteolysis of IκB-α.4 Liberated NF-κB migrates to the nucleus, where it stimulates the transcription of its target gene. The activation of NF-κB initiates both extracellular and intracellular regulatory events that result in autoregulation of the inflammatory cascade through modulation of NF-κB activation. The inhibition of NF-κB activation would lead to suppression of proinflammatory cytokines levels and be beneficial for the treatment of the above-mentioned diseases.6
Several disclosures have reported some compounds with the inhibitory activities toward NF-κB-mediated transcriptional activation. Low-molecular-weight compounds, such as MG-132 (1),7 BAY 11-7085 (2),8 and an indan derivative (3),9 as well as natural products, such as caffeic acid phenylethyl ester (4)10 and the sesquiterpene lactone helenalin (5),11 have been shown to inhibit NF-κB activation (Fig. 1).
In order to find a new structural class of NF-κB activation inhibitors, we conducted a reporter gene-based screening featuring phorbol 12-myristate-13-acetate (PMA) plus phytohemagglutin (PHA) stimulation of human Jurkat T cells transfected with the reporter DNA having the binding sequence for NF-κB and the luciferase gene.12 This led to the identification of the quinazoline 6a as a lead compound (IC50=2381 nM). To date, no one has reported quinazoline derivatives as NF-κB activation inhibitors. This situation prompted us to investigate structure–activity relationship (SAR) of quinazoline derivatives as inhibitors of NF-κB activation. In this paper, we describe the results of our preliminary SAR studies on the quinazolines, conducted on the basis of the lead compound, 6a.
Section snippets
Chemistry
Procedures for the preparation of 6a–6h having the 6–6 condensed-ring systems are shown in Scheme 1. Chlorination of 4-quinazolone with phosphorus oxychloride followed by the reaction of the corresponding amines provided the quinazolines 6a–6e. The quinoline (6f), the isoquinoline (6g), and the phthalazine (6h) were also prepared by the same procedures used for the quinazolines.
The general synthetic pathway to the phenethylaminoquinazolines is shown in Scheme 2. Treatment of the appropriate
Results and discussion
To screen compounds for their inhibitory potency toward NF-κB transcriptional activation, we set up a luciferase reporter gene assay using human Jurkat cells. Compounds were also evaluated for their inhibitory activities toward TNF-α production by using murine splenocytes stimulated with LPS. Cytotoxicity toward both human Jurkat cells and murine splenocytes was measured by using the MTS assay. The results obtained by using these tools to drive the SAR efforts are shown in Table 1, Table 2.
Conclusions
Based on the lead compound 6a obtained from random screening, we identified and synthesized a novel structural class of NF-κB activation inhibitors. Our SAR investigation allowed us to identify the 6-amino-4-phenethylaminoquinazoline skeleton as the basic framework and led to the discovery of 11q, whose inhibitory activity toward NF-κB transcriptional activation represented a 2 order of magnitude increase relative to that of 6a. Compound 11q was also highly potent in the inhibition of TNF-α
General
All reagents and solvents were obtained from commercial suppliers and were used without further purification. Melting points were measured with a BÜCHI 535 melting point apparatus and were uncorrected. 1H NMR was recorded on a Jeol GSX270 FT NMR spectrometer. Chemical shifts were given in parts per million (ppm) using tetramethylsilane as the internal standard for spectra obtained in DMSO-d6 and CDCl3. TOFMS (time-of-flight mass spectrometry) was recorded on a Kompact MALDI 3 V 4.0.0
Acknowledgements
We are grateful for all of the help provided by Dr. Satoru Misawa. In addition, we would like to express our thanks to Mr. Toru Negishi, Mr. Toshiyuki Sato, and Mr. Hideo Omukai for their technical assistance in obtaining the biological data.
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