Br. J. Pharmacol. (1990), 101, 501-503

SPECIAL REPORT

.-:) MacmiUan Press Ltd, 1990

Selective chiral inhibitors of 5-lipoxygenase with anti-inflammatory activity 'R.M. McMillan, 29*J-M. Girodeau & S.J. Foster Bioscience 1, ICI Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK1O 4TG and *ICI Pharma, Centre de Recherches, Zone Industrielle Sud-Est, 51064 Reims, France The studies described here, using enantiomers of an optically-active methoxy alkyl thiazole IC1216800 (1a specific, chiral interaction with 5-lipoxygenase. In accordance with their biochemical efficacy these compounds also demonstrate enantio-specific anti-inflammatory activity in a leukotriene-mediated model of inflammation. This is the first class of compounds for which 5-lipoxygenase inhibition and anti-inflammatory activity have been shown to be mediated via a specific chiral interaction.

methoxy-6{naphth-2-yl-methoxy)-1-(thiazol-2-yl)indane), provide unequivocal evidence for

Introduction Discovery of 5-lipoxygenase inhibitors which are orally active and selective compared to a related enzyme, cyclo-oxygenase, has proved difflicult. Most of the previously described inhibitors have the potential to participate in redox reactions or to chelate iron and may have effects on other biological systems that involve iron or redox processes. Although we and others have identified orally-active and selective inhibitors based on these mechanisms (Tateson et al., 1988; Carter et al., 1989; Foster et al., 1990) there has been little evidence for a specific interaction with 5-lipoxygenase. In particular there is no difference in biological potency between enantiomers of optically-active compounds (Salmon et al., 1989). The studies described here, using enantiomers of an

optically-active methoxy alkyl thiazole, IC1216800, provide unequivocal evidence for a specific, chiral interaction with 5lipoxygenase. Moreover, these compounds also demonstrate enantio-specific anti-inflammatory activity in a leukotrienemediated model of inflammation. Methods Enantiomer synthesis and resolution Each of the two enantiomers of ICI216800 [1-methoxy-6-(naphth-2yl-methoxy)-l-(thiazol-2-yl)indane] were obtained pure (as confirmed by analytical high performance liquid chromatography (h.p.l.c.) and nuclear magnetic resonance spectroscopy) by preparative h.p.l.c. using a Pirkle ionic preparative chiral column (Pirkle & Finn, 1982). For the (+)-enantiomer the chiral phase of the column was prepared from Nucleosil 10 NH2 (lO,um aminopropyl) silica (Machery-Nagel GMBH, Duren, Germany) and S(+}3,5-dinitrobenzoylphenylglycine and was eluted with hexane/dioxane (97:3). For the (--enantiomer a similar column was prepared but with R(-)-3,5dinitrobenzoylphenylglycine as the chiral component to the phase.

Spectrophotometric analysis of 5-lipoxygenase Inhibition of cell-free 5-lipoxygenase activity was determined spectrophotometrically by modification of the procedure of Aharony & Stein (1986), using the high speed (150,000g) supernatant from rat basophilic leukaemia (RBL-1) cells as the enzyme source. Eicosanoid generation Eicosanoid generation in heparinised rat and human blood and protein-free mouse peritoneal macrophage cultures was determined as previously described (Foster et al., 1990). ' Author for correspondence. 2 Present address: Roussel Uclaf, Division Agroveterinaire Recherches Chimiques, 102 Route de Noisy, 93230 Romainville,

France.

Arachidonic acid-induced inflammation in rabbit skin Antiinflammatory efficacy of 5-lipoxygenase inhibitors was determined by a modification of the procedure previously described (Aked & Foster, 1987). Fur from the back of New Zealand White rabbits (3.0-4.0kg: Ranch Rabbits, Crawley Down, Sussex) was removed with clippers 24 h before an intravenous injection of "251-labelled human serum albumin, in a 0.5% solution of Evans blue in physiological saline. Five min later inflammation was induced by the topical application of arachidonic acid alone (300,ug, in 10,ul acetone; 6 sites) or together with the appropriate concentrations of lipoxygenase inhibitor (3 sites). Sites treated with 10pl of acetone were used to assess background 1251-counts. Animals were killed 2h later with a lethal dose of pentobarbitone sodium (Euthatal; May and Baker, Dagenham). The skin was removed and the injection sites were separated with a 15 mm diameter metal punch. 125I-counts were determined in the skin sites and aliquots of plasma with an LKB ultra-gamma counter and the plasma volume at each inflammation site was calculated in Mul after subtracting the mean background count. Statistical analysis Differences between means were assessed either by Student's paired t test or by the use of a one-tailed Student's t test with P < 0.05 regarded as significant in both cases. Materials Materials used were as previously described (Foster et al., 1990 and references therein). REV5901 and all ICI compounds were synthesized in Chemistry Department 1, ICI Pharmaceuticals or at ICI Pharma, Centre de Recherches, Reims.

Results The optically-active compound IC1216800 (Table 1), is a potent and selective inhibitor of 5-lipoxygenase. In plasma-free peritoneal macrophage cultures, the compound produced potent inhibition of leukotriene C4 (LTC4) synthesis (mean IC50 = 0.014pM; n = 2) but did not inhibit synthesis of prostaglandin E2 (PGE2) at doses up to 30M which indicates a selectivity ratio (cyclo-oxygenase: lipoxygenase) of more than 2000. Inhibition of LTB4 synthesis by IC1216800 in human blood (IC50 = 0.69 + 0.18 pM; n = 4) was also selective with no significant inhibition of synthesis of the cyclooxygenase product, thromboxane B2 (TxB2), at doses up to

100pM.

In order to investigate the chiral specificity of the inhibition, the enantiomers of ICI216800 were resolved and their effects compared to the parent compound (Table 1). The racemic mixture produced potent inhibition of 5-lipoxygenase in a cell-free enzyme assay and in human blood. Evaluation of the resolved enantiomers, demonstrated that the lipoxygenase

502

SPECIAL REPORT

Table I Structure and chiral inhibition of 5-lipoxygenase by ICI216800

IC50 (.M) (+ )-Enantiomer

Racemate

(--Enantiomer

>40 0.54 + 0.17 LTB4 synthesis 0.69 + 0.18 in human blood >20 0.13 0.77 Cell-free 5-lipoxygenase LTB4 = leukotriene B4. Results in human blood are mean IC50 values ± s.d. (n = 4). IC50 values for cell-free data are derived from mean values of duplicate determinations.

inhibitory activity resided predominantly in the (+ -enantiomer: (+)-IC1216800 was at least 70 times more potent than (-)-IC1216800 in both assays (Table 1). Figure 1 compares the anti-inflammatory activity of the enantiomers against arachidonic acid-induced inflammation in rabbit skin, which is mediated in part by LTB4 (Aked & Foster, 1987). Topical administration of (+)-IC1216800 produced dose-dependent inhibition of plasma extravasation with ED30 (n = 4) of 15.2 nmol per site. In accord with the in vitro data described above, (-)-IC1216800 was inactive at doses up to at least 78 nmol per site. 50-

40C

20.0

30101 0

1

10

100

nmol per site Figure 1 Topical anti-inflammatory activity of enantiomers of IC1216800: (-), (+)-ICI216800; (-), (-)IC1216800. Values are the mean percentage inhibition of plasma extravasation in 4 rabbits; vertical bars show s.e.mean. The amount of plasma extravasation induced by arachidonic acid alone was 88.0 + 10.5jp1. * P

Selective chiral inhibitors of 5-lipoxygenase with anti-inflammatory activity.

The studies described here, using enantiomers of an optically-active methoxy alkyl thiazole ICI216800 (1-methoxy-6-(naphth-2-yl-methoxyl)-1- (thiazol-...
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