1-Heteroaryl-3-phenoxypropan-2-ones as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: Effect of the replacement of the ether oxygen with sulfur and nitrogen moieties on enzyme inhibition and metabolic stability

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Abstract

Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that certain 3-phenoxy-substituted 1-heteroarylpropan-2-ones are inhibitors of cPLA2α and/or FAAH. Starting from 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylic acid (3) and 1-(1H-benzotriazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (4), the effect of the replacement of the oxygen in position 3 of the propan-2-one scaffold by sulfur and nitrogen containing moieties on inhibition of cPLA2α and fatty acid amide hydrolase as well as on metabolic stability in rat liver S9 fractions was investigated. As a result of these structure–activity relationship studies it was found that the ether oxygen is of great importance for enzyme inhibitory potency. Replacement by sulfur led to an about 100-fold decrease of enzyme inhibition, nitrogen and substituted nitrogen atoms at this position even resulted in inactivity of the compounds. The effect of the structural variations performed on metabolic stability of the important ketone pharmacophore was partly different in the two series of compounds. While introduction of SO and SO2 significantly increased stability of the ketone against reduction in case of the indole-5-carboxylic acid 3, it had no effect in case of the benzotriazole 4. Further analysis of the metabolism of 3 and 4 in rat liver S9 fractions revealed that the major metabolite of 3 was the alcohol 53 formed by reduction of the keto group. In contrast, in case of 4 beside keto reduction an excessive hydroxylation of the terminal phenoxy group occurred leading to the dihydroxy compound 50. Experiments with enzyme inhibitors showed that the phenylhydroxylation of 4 was catalyzed by tranylcypromine sensitive cytochrome P450 isoforms, while the reduction of the ketone function of 3 and 4 was mainly caused by cytosolic short chain dehydrogenases/reductases (cSDR).

Introduction

In mammalian organism, derivatives of arachidonic acid play important roles as algesic and pro-inflammatory as well as analgesic and anti-inflammatory mediators. On the one hand, oxidation products of arachidonic acid such as prostaglandin E2 and leukotriene B4 formed via the arachidonic acid cascade are involved in the pathophysiology of pain and inflammation.1 On the other hand, the arachidonic acid amide anandamide generated via the endocannabinoid pathway has analgesic and anti-inflammatory properties.2

The key enzyme in the formation of oxidized derivatives of arachidonic acid is cytosolic phospholipase A2 α (cPLA2α).3, 4 This enzyme, which contains a catalytic serine in its active site, provides arachidonic acid for prostaglandin and leukotriene synthesis by cleaving membrane phospholipids at the sn-2 position. Therefore, cPLA2α is considered as a target for treatment of pain and inflammatory diseases.5, 6, 7 First-generation cPLA2α inhibitors were analogues of arachidonic acid with the COOH group replaced by COCF3 (AACOCF3)8, 9 or CH2PO(OCH3)F (MAFP).10 Compounds with high in vitro cPLA2α inhibitory potency reported later are benzhydrylindoles from Wyeth,11 thiazolidinedione compounds from Shionogi,12 propan-2-ones from AstraZeneca13 and α-ketoamides of the Kokotos group.14 We have developed several heteroaryl-substituted propan-2-ones, which also exhibit a strong inhibition of cPLA2α such as compound 1 (Fig. 1).15

An important enzyme in the endocannabinoid metabolism is fatty acid amide hydrolase (FAAH). This enzyme, which is also a serine hydrolase, rapidly cleaves the analgesic and anti-inflammatory lipid mediator anandamide into arachidonic acid and ethanolamine.16, 17, 18 Inhibition of FAAH is supposed to enhance the action of anandamide. Therefore, like cPLA2α inhibitors, inhibitors of FAAH may represent new agents against pain and inflammation.19 In the past years many potent inhibitors of FAAH have been described.20, 21, 22, 23 Recently, we have found that several of our cPLA2α inhibitors with heteroaryl-substituted propan-2-one structure also inhibit FAAH. One of the most active inhibitors of this enzyme was the benzotriazole derivative 2 (Fig. 1).24, 25

An important pharmacophoric element of compounds 1 and 2 is the central ketone group, which possesses an enhanced reactivity due to the electron withdrawing effects of the neighbouring heterocycle and ether oxygen, respectively. This highly electrophilic ketone is supposed to form covalent bondings with the serine residue of the active sites of cPLA2α and FAAH, respectively.13, 15, 24 Consistently, metabolic reduction of the ketone to an alcohol results in a loss of activity against both enzymes. Starting from the phenoxy-substituted derivatives 3 and 4 of the initial lead compounds 1 and 2, we have now investigated the effect of the replacement of the ether oxygen by sulfur and nitrogen containing moieties on enzyme inhibitory potency and metabolic stability.

Section snippets

Chemistry

Scheme 1 outlines the synthesis of the thio analogue of compound 3. Opening of the epoxide ring of the allyl ester of 1-(oxiran-2-ylmethyl)indole-5-carboxylic acid (6) with 4-phenoxythiophenol under solvent free conditions afforded the hydroxy intermediate 7. Subsequent oxidation of this alcohol with Dess–Martin periodinane gave the keto ester 8. Cleavage of the allyl ester moiety of 8 by reaction with tetrakis(triphenylphosphine)palladium(0) led to the target compound 9.

The synthesis of the

Results and discussion

One aim of this study was to investigate how the replacement of the ether oxygen of 3 and 4 by sulfur and nitrogen containing moieties affect inhibition of cPLA2α and FAAH. Because it has been found that many of our heteroarylpropan-2-one inhibitors are readily inactivated by metabolic reduction of the ketone group, it should also be investigated, whether these structural modifications lead to an improvement of metabolic ketone stability.

Starting compounds were the indole-5-carboxylic acid

General

Column chromatography was performed on silica gel 60, particle size 0.040–0.063 mm, from Merck or Macherey & Nagel. For preparative HPLC a RP18 column was applied (Kromasil 100, 5 μm, 10 mm (I.D.) × 250 mm protected with an analogously filled guard column 10 mm (I.D.) × 50 mm). Melting points were determined on a Büchi B-540 apparatus and are uncorrected. 1H NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer (400 MHz) or a Bruker AV300 spectrometer (300 MHz). 13C NMR spectra (100 MHz) were

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