Structure–activity relationship of salicylic acid derivatives on inhibition of TNF-α dependent NFκB activity: Implication on anti-inflammatory effect of N-(5-chlorosalicyloyl)phenethylamine against experimental colitis

Abstract

To develop a more potent NFκB inhibitor from salicylic acid which is known to inhibit activity of NFκB, a transcription factor regulating genes involved in immunity, inflammation and tumorigenesis, derivatives of salicylic acid (SA) where the 5 position, carboxyl or hydroxyl group was modified were treated in HCT116 cells transfected with an NFκB dependent luciferase gene and LPS-stimulated RAW264.7 cells. Amidation of the carboxylic group or substitution of chlorine at the 5 position increased the ability of SA to suppress the expression of NFκB dependent luciferase and inducible nitric oxide synthase, a product of an NFκB target gene. Moreover, simultaneous amidation and chlorination of SA (5-chlorosalicylamide; 5-CSAM) conferred an additive NFκB inhibitory activity on SA. To further enhance the inhibitory activity, N-modification was imposed on 5-CSAM. N-(5-chlorosalicyloyl)phenethylamine (5-CSPA), N-(5-chlorosalicyloyl)3-phenylpropylamine (5-CSPPA) and N-(5-chlorosalicyloyl)4-hydroxyphenylethylamine (5-CSHPA) showed greater potencies for inhibiting NFκB activity than other derivatives. Their IC₅₀s’ in the luciferase assay measured 15 μM (5-CSPA), 17 μM (5-CSPPA) and 91 μM (5-CSHPA). Rectal administration of 5-CSPA ameliorated TNBS-induced rat colitis, which was more effective than a conventional drug, 5-aminosalicylic acid. These data may provide useful information for development of a therapeutic agent for treatment of diseases where NFκB plays a critical role in the pathogenic progresses.

Introduction

NFκB is an important transcription factor that regulates genes involved in immunity and inflammation [1]. Stimulation with pro-inflammatory cytokines such as TNF-α initiates an intracellular signaling cascade, resulting in the phosphorylation and subsequent degradation of IκB by the 26S-proteasome [2]. The degradation of IκBα releases NFκB allowing it to translocate into the nucleus and transactivates target genes such as cyclooxygenase-2 (COX-2), cytokines and chemokines that are pivotal mediators of the immune and inflammatory responses.

NFκB activity and levels of the pro-inflammatory cytokine TNF-α have been shown to be increased in the colon epithelial cells and mucosa of patients with inflammatory bowel disease (IBD) which comprises ulcerative colitis and Crohn’s disease [3]. It is known that expression of TNF-α, which strongly activates NFκB, is itself up-regulated by NFκB. This provides a positive autoregulatory loop that amplifies the inflammatory response and perpetuates chronic intestinal inflammation [3]. For this reason, therapeutic intervention against TNF-α or NFκB activation has been used for treatment of inflammatory bowel disease (IBD) [4]. In fact, inhibition of NFκB activity has been suggested to be a major component of the anti-inflammatory activity of glucocorticoid and 5-aminosalicylic acid (5-ASA) both of which are frequently used for treatment of chronic intestinal inflammation [5], [6]. Moreover, new strategies that specifically regulate NFκB activity using p65 (RelA) antisense oligonucleotides, proteasome inhibitors or adenoviral IκB expression vector show beneficial therapeutic effects in experimental colitis [7], [8].

Recent papers reported that NFκB inhibition is one of the anti-inflammatory mechanisms of salicylates such as sulfasalazine, salicylate, aspirin and 5-ASA [9], [10], [11]. Although the salicylates whose structures are different possess a common biological activity, there has been no prior study on structure–activity relationship on the NFκB inhibition activity. In the present work, derivatives of salicylic acid were purchased or prepared and structure–activity relationship of salicylic acid derivatives on inhibition of TNF-α dependent NFκB activity was investigated, which could provide valuable information for development of a new therapeutic agent for treatment of diseases where NFκB plays a critical role as a pathological mechanism. Based on the potential therapeutic application, the anti-inflammatory activity of one of the derivatives, 5-chlorosalicylic acid phenethylamide (5-CSPA), was evaluated in a TNBS-induced rat colitis model, which was compared to 5-ASA, the most widely used drug for the treatment of IBD.