Biologically active alkamide from the lichen Stereocaulon alpinum

Ph ytomedicine Vol. 4 (4 ), pp . 33 1-3 34, 199 7 © 1997 by Gustav Fischer Verlag

Biolog ically active alkamide from the lichen Stereocaulon alpinum K. INGOLFSOOnIR 1, S. R. GISSURARSON1, A. NENNINGER2, A. NESZMELYI3, B. WIEOEMANN2, H. WAGNER2 Dept. of Pharmacy, University of Iceland, Reykjavik, Iceland Institut fur Pharmazeutische Biologie, Ludwi g-M aximilians-Un iversitat, Munchen , Germany 3 Central Research Institute for Chemistry, Hungarian Academy of Sciences, Budapest, Hungary 1

2

Summary The pr esence of an alka mide in lichens is reported for the first time . Th e alkamide was iso lated from Stereocaulon alpinum Laur. and ident ified th rou gh spectrosc opic analysis as 9-cis-octadecenamide (1). With reference to previously known activity of alka mides fro m both the plant and animal king doms on inflamma tory processes, 1 wa s teste d for in vitro inh ibitory activity against th e key enzymes o f the major pathways invol ved in arach idonate metabolism. Th e compound was show n to exhibit moderate dose-dependent inhibitory act ivity aga inst cycloox ygenase from sheep seminal vesicle micro som es (IC so = 64 .3 11M) whil st showing onl y slight activity aga inst 5-lipoxygena se from porcine leucocytes . Key words: Stereocaulon alpinum, lichen, alkamide, 9-cis-octadecenam ide, cyclo ox ygena se, arachid on at e metabolism .

Introduction We have recently reported on the isolati on and in vitro testing for inh ibitory effects o n a rachido na te metabolism of two ch ar act erist ic secon dar y meta bolites fro m th e lichen Stereocaulon alpinum Laur. (Stereocaulaceae); the ~-orci­ nol depsid e arra norin and th e orcinol depsidone lobaric acid. The latter exhibited dose-dependent inhibito ry acti vity against 5-lipoxygena se from porcine leucocytes with an IC so value of 7.3 11M (Ing6 lfsd6ttir et aI., 1996 ) and comparable inhibitory effects agains t cysteinyl leuk otriene forma tion in guinea pig smooth muscle (Gissur arson et aI., 1997 ). Further studies on th e chemical co mposition of the lichen have led to the isolation of a lipid (1) belonging to the class of alkamide s. Sever al classes of alkamides are kn own in the animal kingdom, and progress co ntinues to me mad e in reco gn izing their biological importan ce. Anandam ide (arachidony leth anolamide ), for example, has been identified as an endogenous ligand for CNS -type cannabinoid receptors (Devane et aI., 1992; Felder et a1. , 19 93 ) and N -(2-hydroxy-

endogenous ligand for the peripheral cannabinoid receptor (Facci et a l., 1995 ), acting as a local autacoid , down-regu lating mast cell activat ion an d infla mm ation (M azzari et aI., 1996 ). Congeners of th e latter alkamide are currentl y being eva luat ed for their potential as ant i-inflammato ry agents. Th e distribution of alkamides in th e plant kingdom appears to be limite d to several higher plant families such as Asteraceae, Pipe raceae, Rutaceae and Aristo lochiaceae. The pre sence of alkamides in lichens has no t previously been rep orted to our kn owledge. Reported biological ef-

cr hyljhexadccanamidc ha s rec ently bee n sugge sted t o b e t h e

lic h en Ste roo ca u io n alpin u m ,

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1 Fig. 1. The structure of 9-cis-octadecenamide (1) isolated from the

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K. Ingolfsdottir et al.

fects of fatty acid amides from plants include a study in which palmitylethanolamide from soybeans and peanuts exhibited anti-inflammatory activity in guinea pigs (Kuehl et al., 1957). More recently, alkamides from Echinacea and Achillea species were shown to exhibit in vitro inhibitory activity against 5-lipoxygenase and cyclooxygenase (Wagner et al., 1989; Muller-jakic et al., 1994). Furthermore, synthetic arachidonamide and related amides have been found to inhibit 5-lipoxygenase from rat basophilic leukemic cells (Corey et al., 1984). In view of the previously reported effects of alkamides from both the plant and animal kingdoms on inflammatory processes, it was considered worthwhile to evaluate the effects of 1 on arachidonate metabolism. The compound was thus tested for in vitro inhibitory activity against cyclooxygenase as well as 5-lipoxygenase by using preparations from porcine leucocytes and sheep seminal vesicle microsomes respectively (Wagner and Fessler, 1986; Wagner et al., 1986).

Materials and Methods Plant material Stereocaulon alpinum Laur. (Stereocaulaceae) was collected in Holmsheidi, southwest Iceland. The plant was identified by Dr. Hordur Kristinsson, Director of the Icelandic Institute of Natural History, Akureyri, Iceland. Materials

Solvents and chemicals were of analytical grade. TLC was performed on silica gel 60 F2s4 coated glass plates (Merck) with toluene:glacial acetic acid (100:15), detection with 10% H 2S0 4 followed by heating at 100 0 • Silica gel, 0.063-0.100 11m (Merck 15101), was used for medium pressure LC. Labelled [14C]-arachidonic acid was purchased from Amersham Buchler GmbH, Braunschweig, Germany. Other chemicals and solvents were purchased from Merck, Darmstadt, Germany and Sigma, Deisenhofen, Germany and Sigma, U.S.A. Instruments

Column chromatography was performed on Buchi B-681 medium pressure liquid chromatography (MPLC). Melting point was determined on a Buchi 510 melting point apparatus and is uncorrected. IR spectra were recorded with a Perkin Elmer IR 1310 spectrophotometer and FABMS by using positive ion mode and 3-NBA as matrix with a MS 80 RFA (KRATOS) instrument (7 kV, 10 W, 5 x 10-sT, 4 KeV). NMR spectra were recorded in CDCI 3 (7 mg/O.8 ml) at room temperature with a Bruker AC 250 spectrometer at 250.1 MHz for lH and at 62.9 MHz for 13C (0, TMS as internal standard).

Isolation of 9-cis-octadecenamide (1)

Air-dried S. alpinum (530 g) was extracted in a Soxhlet apparatus with diethyl ether. After removal of precipitate A (4.4 g) which formed on cooling and precipitate B (4.5 g) which formed after partial concentration of the extract in vacuo and subsequent cooling, the extract was further concentrated to ca. 30 ml. On standing, two phases formed, the lower containing chlorophyll, the upper bearing a light yellow colour. After being taken to dryness, the latter (6.4 g) was chromatographed by medium pressure LC (column 46 cmx35 mm i.d.) with the following eluents: hexane (600 ml; fractions 1-6), hexane:CH 2Cl2 95:5 (300 ml; fractions 7-9), hexane:CH 2Cl2 90:10 (300 ml; fractions 10-12), hexane: CH 2Cl2 80:20 (300 ml; fractions 13-15), hexane: CH 2Cl2 50:50 (500 ml; fractions 16-20), hexane:CH 2Cl2 40:60 (500 ml; fractions 21-25), hexane: CH 2Cl2 25:75 (500 ml; fractions 26-30), CH 2Cl2 (1200 ml; fractions 31-54), CH 2CI2:MeOH 95:5 (650 ml; fractions 55-67), CH 2CI2 : MeOH 90:10 (600 ml; fractions 68-80), CH 2Cl2 : MeOH 80:20 (500 ml; fractions 81-90), CH 2CI2:MeOH 50:50 (600 ml; fractions 91-102), MeOH (600 ml; fractions 103-114). Fractions 50-74 were combined, reduced to dryness and the residue recrystallised twice from methanol to afford a white, waxy solid (105 mg), m.p. 60-62°. IR(KBr) u em:': 3370,3200,2920,2840, 1660, 1630, 1470, 1420, 1410. Positive FABMS, mlz (%, rel. int.): 282 (100) [M+H]+, 281 (23) [M]+, 256 (28), 228 (20), 154 (22),137 (23),136 (18), 128 (11), 107 (11), 95 (18), 69 (36). lH NMR (CDCI3 ) 0: 0.86 (3H, t,] = 6.6 Hz, H 3-18), 1.24-1.29 (20H, m, H 2-4 ~ H 2-7 and H 2-12 ~ H 2-17), 1.62 (2H, quintet,] = 7.25 Hz, H 2-3), 1.99 (4H, m, H 2-8 and H 2-11), 2.21 (2H, t,] = 7.6 Hz, H 2-2), 5.33 (2H, m, H-9 and H-10), 5.6 (lH, br s, N-H), 5.9 (1H, br s, N-H). 13C NMR (CDCI3 ) 0: 175.96, 130.01,129.73,35.89,31.87,29.72,29.66,29.49,29.29, 29.21,29.16,29.08,28.81,27.17,27.12,25.49,22.66, 14.11. Cyclooxygenase Assay

Inhibitory effects on cyclooxygenase activity were measured by the reduced production of prostaglandin E2 from labelled arachidonic acid according to Wagner et al. (1986) and as described by Ing6lfsd6ttir et al. (1994). ICso values are expressed as a mean of 3 determinations at six different concentrations by log regression analysis.

5-Lipoxygenase Assay

Inhibitory effects on 5-lipoxygenase activity were measured by the reduced production of 5-HETE (5-hydroxy6,8,11,14-eicosatetraenoic acid) from labelled arachidonic acid according to Wagner and Fessler (1986) and as described by Ing6lfsd6ttir et al. (1994).

Biologically active alkamide from the lichen Stereocaulon alpinum

Results and Discussion The assignment of the unknown lipid isolated from S. alpinum to the class of fatty acid amides was indicated by results from spectroscopic analysis. The presence of a long alkyl chain with one double bond was evident from IH and l3C NMR data. Bands typical for primary amides were observed in the IR spectrum. These data along with mass spectroscopic measurements indicated an octadecenamide. The position and configuration of the double bond were established on the basis of IH- and I3C NMR data. It has previously been shown that the difference in chemical shift between olefinic signals in the I3C NMR spectra of octadecenoic acids reveals valuable information about the position of the double bond (Gunstone, 1993a). The difference of 0.28 ppm observed in the present study is indicative of a 9,10-positioned double bond. The cis stereochemistry of the double bond was unambiguously established on the basis of the proton-proton coupling constant at the double bond (9 Hz) and corroborated by the positions of allylic signals in the IH and I3C NMR spectra. The proton multiplet at 5.33 ppm (of intensity 2H) was analyzed by spin-simulation using the LAOCOON-III program of Bothner-By (kindly submitted by the author). A six-spin system was calculated consisting of the protons on carbon atoms C-8 ~ C-ll. The best fit was obtained with ]HH (9, 10) = 9 Hz and a chemical shift difference of 0.0148 ppm for H-l 0 and H-9. It has been shown that the signals for the 4H multiplet for allylic protons in 9-cis and 9-trans octadecenamide lie in the region of 81.94-1.91 and 8 1.88-1.86 respectively (Cravatt, 1996). Allylic carbon signals in octadecenoic acids have been reported to lie in the range 27.3-27.4 ppm for cis isomers and 32.6-32.7 ppm for trans isomers (Cunstone, 1993a). The structure of the lichen lipid has thus been assigned as 9-cis-octadecenamide (olearnide; 1). BC NMR and IH NMR data of 1 are in accordance with that reported respectively for oleamide (Gunstone, 1993b; Kaneshiro et al., 1994). The alkamide (1) exhibited moderate in vitro inhibitory activity against the formation of cyclooxygenase products in sheep seminal vesicle microsomes. The effects were dosedependent and the ICIO was calculated as 64.3 J.tM. When tested for activity against 5-lipoxygenase in ionophorestimulated porcine leucocytes, 9-cis-octadecenamide showed only slight activity (lCIO = 252 J.tM). The preferential inhibition of cyclooxygenase vs. lipoxygenase exhibited by 1 is comparable to selective activity shown by pentadeca- and hexadeca-isoburylarnides from Echinacea angustifolia (Muller-jakic et al., 1994). The marked differences in the side chains of these compounds and 1, however, precludes further comparison of structure/activity. Interestingly, oleic acid has previously been shown to inhibit cyclooxygenase In vitro with an IClo of 14 J.tM (Bauer et al., 1996) and is thus more potent in this re-

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spect than the corresponding amide. The results presented do not merit further study on oleamide (1) as an inhibitor of arachidonate metabolism. The similar structural elements of 1 to those of known biologically active compounds from the animal kingdom, however, might render it a candidate for assays aimed at detecting other mechanisms of anti-inflammatory activity. Acknowledgements The authors wish to thank Dr. Hor our Kristinsson (Icelandic Institute of Natural History, Akureyri) for identification of the plant material, Dr. Sigridur jonsdottir (Science Institute, University of Iceland) for recording IH NMR and I3C NMR spectra and Mrs. M. Greuter (Institut fur Pharrnazeutische Biologic, LMU, Miinchen) for recording MS spectra. The financial support of the Hungarian National Science foundation (aTKA # Tl7833) is appreciated. The work was supported in part by the Icelandic Council of Science and The University of Iceland Research Fund.

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Bagby, M. 0.: 9(Z)-Octadecenamide and fatty amides by Bacillus megaterium (B-3437) conversion of oleic acid. j. Am. Oil Chem. Soc. 71 (5): 491-494,1994. Kuehl, E A., Jacob, T. A., Ganley, O. H., Ormond, R. E., Meisinger, M. A. P.: The identification of N-(2-hydroxyethyl)-palmitamide as a naturally occurring anti-inflammatory agent. j. Am. Chem. Soc. 79: 5577-5578, 1957. Mazzari, S., Canella, R., Petrelli, L., Marcolongo, G., Leon, A.: N(2-hydroxyethyl)hexadecanamide is orally active in reducing edema formation and inflammatory hyperalgesia by down-modulating mast cell activation. Eur. j. Pharm. 1996. Muller-jakic, B., Breu, W., Probstle, A., Redl, K., Greger, H., Bauer, R.: In vitro inhibition of cyclooxygenase and 5-lipoxygenase by alkamides from Echinacea and Achillea species. Planta A1ed. 60:37-40, 1994. Wagner, H., Fessler, B.: In vitro 5-Lipoxygenase Hemmung durch Ecliptaalba Extracte und das Coumestanderivat Wedelolacton. Planta A1ed. 52: 374-377,1986.

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Address K. Ing6lfd6ttir, Dept. of Pharmacy, University of Iceland, Hagi/Hofsvallagata, IS-107 Reykjavik, Iceland Tel.: 354-525-4000; Fax: 354-525-407; e-mail: [email protected].