Structure-based design of novel NSAID ester prodrugs: Dual targeting of cyclooxygenase-2 (COX-2) and α7 nicotinic receptors

Non-steroidal anti-inflammatory drugs (NSAIDs) are a common treatment for chronic inflammatory disorders but suffer from several unwanted side effects, particularly in the gastrointestinal (GI) tract. The GI-related side-effects can be reduced by masking of the carboxylate moiety of the NSAID, e.g., by chemical conversion into esters. Recently, it has been shown that activation of 7 nicotinic acetylcholine receptors (nAChRs) on macrophages and other immune cells, inhibits the release of proinflammatory mediators, thereby suppressing inflammatory processes. In the current work, we have used crystal structure data of acetylcholine-binding protein (AChBP), a ligand-binding domain (LBD) homolog to the α-7 nAChR, to design NSAID ester prodrugs that are also capable of activating 7 nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure-based design, chemical synthesis and the pharmacological evaluation of these novel dual action anti-inflammatory NSAID prodrugs. Chapter 6 Structure-based design of novel NSAID ester prodrugs: Dual targeting of cyclooxygenase-2 (COX-2) and α7 nicotinic receptors 130 Introduction Inflammation is the complex biological response of tissues to harmful stimuli, such as pathogens, damaged cells or irritants and is part of the body‟s natural defense system against injury and disease. However, in some cases the defensive response may be inappropriately deployed against tissues of the body itself. In this case, the inflammatory response can produce damage and may be part of the disease process itself, like for example in asthma, rheumatoid arthritis and atherosclerosis. 160 The most common treatment for these disorders are nonsteroidal anti-inflammatory drugs (NSAIDs). In general, NSAIDs exert their effects through inhibition of arachidonate cyclooxygenase type 2 (COX-2) enzymes, attenuating the production of proinflammatory mediators such as prostaglandins and thromboxanes. 161 However, the chronic use of NSAIDs may lead to side effects, particularly in the gastrointestinal (GI) tract but also in the liver, kidney, spleen, blood and bone marrow. 162 It has become clear that the GI-related side effects of NSAIDs are associated with inhibition of COX-1 and not COX-2. 163 As a consequence, several selective COX-2 inhibitors such as valdecoxib, celecoxib, etoricoxib and rofecoxib were approved and introduced to the market in the first few years of the new millennium. Unfortunately, shortly after their introduction, reports of cardiovascular side effects of the “coxibs” began to emerge and ultimately rofecoxib (Vioxx®) and valdecoxib (Bextra®) were withdrawn from the market in 2004 and 2005, respectively. 164,165 In an alternative approach to reduce or abolish GI toxicity, considerable efforts have been focused on prodrugs in which the carboxylic group of non-selective NSAIDs is temporarily masked by converting their carboxylate functionalities into esters. This strategy aims at chemical and/or enzymatic transformation of the prodrug, resulting in release of the active NSAID after passage of the GI tract. A large amount of studies exemplify the success of this approach as reduced toxicity and similar or improved anti-inflammatory activity have been reported. These studies have recently been reviewed by Halen and coworkers. 166 So far, these efforts have not translated into clinical utility and the ideal NSAID prodrug with a superior therapeutic advantage remains to be identified. 166 The current practice in drug discovery focuses mainly on a “one target, one disease” approach. However, the redundancy that exists in biological networks may mean that targeting single proteins will not be sufficient to tackle complex diseases. 167 As such, approaches in which compounds are being developed that modulate multiple targets are gaining interest. 167-171 These approaches are known as “polypharmacology”, “designed multiple ligands (DMLs)”, “multi-target drugs (MTDs)” or “hybrid drugs” and have been applied in inflammation-related disorders as well. Examples include, dual COX-2 and 5-lipoxygenase (5-LOX) inhibitors 172,173 , dual inhibitors of 5-LOX and TxA2 synthase 174 and nitric oxide (NO) donating NSAIDS 175 . Recently, studies have emerged that show that the nervous system controls peripheral inflammatory responses via efferent vagal nerves. 176-178 Vagal nerve stimulation has been shown to result in the release of peripheral acetylcholine. The released acetylcholine subsequently activates 7 nAChRs that are expressed on macrophages and other immune cells. 37,179 Activation of this “cholinergic antiinflammatory pathway” inhibits the production of TNF- and other pro-inflammatory Chapter 6 Structure-based design of novel NSAID ester prodrugs: Dual targeting of cyclooxygenase-2 (COX-2) and α7 nicotinic receptors 131 cytokines and as a consequence inflammatory processes are being suppressed. 37,145 The important role of the 7 nAChR in inflammatory processes is underlined by studies that show that the anti-inflammatory effects of nicotine on macrophages can be abolished by selective 7 antagonists such as bungarotoxin. 145,179 In contrast to wildtype mice, vagus nerve stimulation does not result in attenuation of TNF- release in 7-knockout mice. 37 Notably, treatment with 7 (partial) agonists such as nicotine and GTS-21 (DMXBA), has been shown to be effective in several animal models of inflammation. 180-182 These findings prompted us to examine if we could combine the advantages of NSAID ester prodrugs with α7 nAChR active compounds. Our strategy aims on derivatization of commercially available NSAIDs with moieties that would render the resulting prodrug capable of activating the 7 nAChR. In this manner, we expect to obtain dual action anti-inflammatory compounds with a reduced GI toxicity profile, see figure 1. Figure 1: Dual action approach to treat inflammatory disorders. I. Activation of α7 nAChRs on macrophages (or other immune cells) by the NSAID ester prodrug will suppress the release of proinflammatory cytokines. II. Subsequent hydrolysis releases the parent NSAID that will exert an additional anti-inflammatory effect by acting on COX-2, lowering the formation of proinflammatory prostaglandins. Nizri and coworkers have recently shown that dual targeting of 7 nAChRs and COX-2 may indeed increase therapy effectiveness as a dual action compound consisting of the 7 nicotinic agonist cytisine and the traditional NSAID ibuprofen was significantly more effective in a central nervous system (CNS) inflammatory model than when both compounds were dosed separately, or both unconjugated. 183 Their approach, however, differed significantly from the approach that is described in this work, as an octyl spacer was used to connect the NSAID and the nicotinic agonist. Very recently, we have disclosed 184 a fragment (VUF10663, Chapter 4) that exhibits good ligand efficiency 118 (LE = 0.43) for AChBP, as well as for the 7 Chapter 6 Structure-based design of novel NSAID ester prodrugs: Dual targeting of cyclooxygenase-2 (COX-2) and α7 nicotinic receptors 132 nicotinic receptor (LE = 0.44, Chapter 5). This fragment is an ester of benzoic acid and nortropinyl. Since the fragment VUF10663 displays affinity for the 7 receptor and the benzoic acid part exhibits high structural resemblance to certain NSAIDs or structural fragments of NSAIDs, we investigated if esterification of NSAIDs with tropine-like moieties would afford NSAID prodrugs with intrinsic 7 nAChR activity, see Figure 2. Figure 2: Fragment VUF10663 and the 5 NSAID derivatives that were selected in order to obtain NSAID prodrugs with an additional 7 nAChR mediated antiinflammatory effect. Upon analyzing the available literature on NSAID prodrugs, two publications by Yadav and co-workers were considered very interesting as they report on the antiinflammatory action of tropine esters of several NSAIDs including ibuprofen and ketoprofen. Their aim, however, was not to target 7 nAChRs but to construct ester prodrugs that display reduced GI toxicity by targeting muscarinic acetylcholine receptors in the gut 185 or to achieve selective localization of the ester prodrug in inflamed joints caused by rheumatoid arthritis. 186 As anticipated, the NSAID tropine esters exhibited reduced GI toxicity, as well as selective localization in inflamed joints when derivatized with a quaternary nitrogen atom and comparable antiinflammatory activity to their parent NSAIDs in a chronic arthritis model. To select which NSAIDs are most suitable for modification towards α7 nAChR activation we have used molecular docking in X-ray structures of acetylcholine-binding protein (AChBP) in complex with (partial) agonists for the α7 nAChRs. 63 AChBP is widely recognized as a water soluble structural homolog of the ligand binding domain (LBD) of nAChRs, and in particular of the α7 nAChR subtype. 116,117,187 The obtained structural information on AChBP, therefore enables structure-based design of the potential dual action anti-inflammatory agents. Based on the in silico results, a selection of NSAIDs was chemically modified with tropine moieties to obtain ester prodrugs with potential α7 nAChR activity. Here we describe the structure-based design, chemical synthesis and the pharmacological evaluation of these potential dual action anti-inflammatory NSAID prodrugs. Chapter 6 Structure-based design of novel NSAID ester prodrugs: Dual targeting of cyclooxygenase-2 (COX-2) and α7 nicotinic receptors 133 Results Structure-based Design Over 20 currently marketed NSAIDs that contain a carboxylate functionality (Table 1) were derivatized in silico with tropine moieties containing an unsubstituted (NH), methyl substituted (NMe) and dimethyl substituted nitrogen atom (N + (Me)2). Subsequently, the NSAID tropine esters were docked using Gold 4.0 125 in the X-ray structures of Lymnaea stagnalis AChBP (Ls-AChBP) in complex with nicotine (PDB: 1UW6) and in the structure of fragment VUF10663 in complex with AChBP from Aplysia californica (Ac-AChBP). 53,63 The obtained binding modes were visually inspected, and evaluated on complementarity towards the AChBP binding site and a conserved water molecule (that is involved in hydrogen bonds between the AChBP binding site and the pyridine nitrogen atom of nicotine) 63 in terms of polar, cation- and hydrophobic interactions while maintaining low energy conformations. In addition, our previously obtained co-crystal complex of VUF10663 with Ac-AChBP was particularly useful to evaluate the NSAIDs of the salicylate (e.g., salicylic acid) and fenamate class (e.g., flufenamic acid), since their respective tropine esters contain VUF10663 as a structural fragment (Figure 2). Assuming that these NSAID tropine esters would take similar binding modes as VUF10663, the VUF10663-Ac-AChBP co-crystal structure revealed that a hydroxyl moiety at the 2-position of VUF10663‟s phenyl moiety, as would be the case for the salicylates, is in close proximity to AcAChBP‟s Met114. Sequence alignments between AchBPs and human nAChR subunits (Chapter 1, Table 2) suggest that in the human α7 nAChR, a glutamine (Gln117) is located at this specific position. In silico mutation of Met114 for a glutamine, followed by an exploration of putative rotamers revealed that a glutamine is capable of forming hydrogen bonds with the 2-hydroxyl functionality of salicylate tropine esters (Figure 4). This finding was considered interesting as the formation of additional hydrogen-bonds with the α7 nAChR binding site is likely to be beneficial in terms of affinity and selectivity. Furthermore, inspection of the VUF10663-AChBP complex showed that between the tip of loop C (Cys188Cys189) and Gln55 on the complementary side, space is available that can accommodate larger NSAIDs such as flufenamic acid, ketoprofen and diflunisal. Moreover, the positioning of the ketone functionality of the ketoprofen tropinyl ester also indicated putative hydrogen bond formation with Gln117 in the α7 nAChR binding site. Based on these molecular modeling and docking results, and their commercial availability, 5 NSAIDs were selected for chemical derivatization, see Figure 2,3 and 4. Chapter 6 Structure-based design of novel NSAID ester prodrugs: Dual targeting of cyclooxygenase-2 (COX-2) and α7 nicotinic receptors 134 Table 1: Chemical structures of the NSAIDs that were evaluated for chemical derivatization to obtain α7 nAChR active NSAID ester prodrugs NSAID Chemical structure NSAID Chemical structure Salicylic acid Aspirin Diflunisal Flurbiprofen