Synthesis, inhibitory activity and in silico docking of dual COX/5-LOX inhibitors with quinone and resorcinol core

Highlights

• Series of novel 1,4-benzoquinones, hydroquinones, and resorcinols were synthesized.

• Compounds 5b and 6b possessed effective dual inhibition of COX-1, COX-2 and 5-LOX.

• Most important modification was the elongation of alkyl chain from 5 to 11 carbons.

• SAR study and docking revealed other modifications important for bioactivity.

Abstract

Based on the significant anti-inflammatory activity of natural quinone primin (5a), series of 1,4-benzoquinones, hydroquinones, and related resorcinols were designed, synthesized, characterized and tested for their ability to inhibit the activity of cyclooxygenase (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) enzymes. Structural modifications resulted in the identification of two compounds 5b (2-methoxy-6-undecyl-1,4-benzoquinone) and 6b (2-methoxy-6-undecyl-1,4-hydroquinone) as potent dual COX/5-LOX inhibitors. The IC₅₀ values evaluated in vitro using enzymatic assay were for compound 5b IC₅₀ = 1.07, 0.57, and 0.34 μM and for compound 6b IC₅₀ = 1.07, 0.55, and 0.28 μM for COX-1, COX-2, and 5-LOX enzyme, respectively. In addition, compound 6d was identified as the most potent 5-LOX inhibitor (IC₅₀ = 0.14 μM; reference inhibitor zileuton IC₅₀ = 0.66 μM) from the tested compounds while its inhibitory potential against COX enzymes (IC₅₀ = 2.65 and 2.71 μM for COX-1 and COX-2, respectively) was comparable with the reference inhibitor ibuprofen (IC₅₀ = 4.50 and 2.46 μM, respectively). The most important structural modification leading to increased inhibitory activity towards both COXs and 5-LOX was the elongation of alkyl chain in position 6 from 5 to 11 carbons. Moreover, the monoacetylation in ortho position of bromo-hydroquinone 13 led to the discovery of potent (IC₅₀ = 0.17 μM) 5-LOX inhibitor 17 (2-bromo-6-methoxy-1,4-benzoquinone) while bromination stabilized the hydroquinone form. Docking analysis revealed the interaction of compounds with Tyr355 and Arg120 in the catalytic site of COX enzymes, while the hydrophobic parts of the molecules filled the hydrophobic substrate channel leading up to Tyr385. In the allosteric catalytic site of 5-LOX, compounds bound to Tyr142 and formed aromatic interactions with Arg138. Taken together, we identified optimal alkyl chain length for dual COX/5-LOX inhibition and investigated other structural modifications influencing COX and 5-LOX inhibitory activity.

Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of common pain as well as for chronic inflammatory diseases such as rheumatoid arthritis. Some of them are over-the-counter drugs and belong to the most widely used pharmaceuticals worldwide. NSAIDs reduce the production of prostaglandins via the inhibition of cyclooxygenase (COX) enzymes. However, chronic intake of non-selective COX inhibitors could result in severe side effects such as gastrointestinal bleeding [1,2]. Besides the inhibition of inducible COX-2 isoform responsible for inflammatory processes, non-selective inhibitors also suppress the activity of COX-1 that plays cytoprotective role in gastrointestinal system. Actually, the role of COX-1 (generally considered as homeostatic) and COX-2 (generally considered as inducible) is complex and depends on many factors such as organ where it actions, developmental stage, stage of inflammatory process etc. The application of selective COX-2 inhibitors (called Coxibs) avoided gastrointestinal complications but their usage resulted in the increased risk of cardiovascular events [3]. One of the reasons is that selective COX-2 inhibitors change homeostasis in the production of vasoactive prostanoids. Coxibs do not affect the production of thromboxane X₂ (TXA₂) promoting vasoconstriction and platelet aggregation but reduce biosynthesis of prostaglandin I₂ (PGI₂) that is vasodilator and potent inhibitor of platelet aggregation. Moreover, COX-2 mediates cardioprotective effects of the late phase of ischemic preconditioning and may also play role in angiogenesis [4]. Enzyme 5-lipoxygenase (5-LOX) catalyzes biosynthesis of leukotrienes (LTs) and lipoxins. Leukotriene B₄ (LTB₄) is important chemotactic and chemokinetic mediator regulating the immune response and inflammatory processes such as arthritis, asthma, and atherosclerosis. The cysteinyl leukotrienes are involved in asthma, allergic rhinitis, and chronic inflammation [5]. Until now, zileuton is only one approved 5-LOX inhibitor for asthma treatment. Moreover, zileuton is associated with adverse effects such as hepatotoxicity and adverse pharmacokinetic profile derived from a short half-life. The inhibition of COXs leads to the increased production of LTs, which could increase asthma. The reason is that both COXs and 5-LOX use as a substrate arachidonic acid (AA) and when the activity of COXs is inhibited AA availability is increased for 5-LOX pathway. Another reason is that the production of vasodilators PGI₂ and PGE₂ is reduced. Therefore, dual inhibition of COXs and 5-LOX is suggested as a promising approach that should diminish negative effects connected with the inhibition of COXs. Moreover, dual COX/5-LOX inhibition may exhibit better anti-inflammatory activity than NSAIDs because of the influence on inflammatory mediators not affected by COX inhibitors [1]. Previously, we found that some natural benzo- and naphthoquinones are able to inhibit COX as well as 5-LOX catalytic activity in vitro. As the most promising compound was identified benzoquinone primin [6,7]. Benzoquinones were found as potent 5-LOX inhibitors also in other studies. Hydroquinone miconidin acetate isolated from Eugenia hiemalis inhibited human recombinant 5-LOX in vitro [8]. Natural benzoquinone embelin was revealed as potent 5-LOX and microsomal prostaglandin E2 synthase-1 inhibitor [9]. Tests of series of embelin derivatives revealed compounds such as 4,5-dimethoxy-3-dodecyl-1,2-benzoquinone and 5-[(2-naphthyl)methyl]-2-hydroxy-2,5-cyclohexadiene-1,4-dione with significantly higher 5-LOX inhibitory activity than that of the parent compound [10,11]. Further, Peduto et al. optimized alkyl chain pattern of quinones and corresponding hydroquinones that provided the identification of highly potent 5-LOX inhibitors with in vivo anti-inflammatory effect [12]. In our study, we used different synthetic approach and prepared series of derivatives of natural quinone, primin. Obtained compounds were assayed for their ability to inhibit COX-1, COX-2 and 5-LOX catalytic activity as potential dual inhibitors. In addition, in silico docking experiments were employed to elucidate the binding of compounds into catalytic sites of COX as well as 5-LOX enzymes.