C-Methyl Flavones from the Leaf Wax of ...

C-Methyl Flavones from the Leaf Wax of Leptospermum laevigatum (Myrtaceae) E ck h ard W ollenw eber, K arin M anna and James N. R oitm anh a Institut für Botanik der Technischen Hochschule, Schnittspahnstrasse 3, D-64287 Darmstadt, Bundesrepublik Deutschland b Western Regional Research Center, U SD A -A R S, 800 Buchanan St., Albany, CA^94710, U.S.A. Z. Naturforsch. 51c, 8 - 1 0 (1996); received November 10/D ecem ber 11, 1995 Leptosperm um laevigatum, Myrtaceae, Leaf Wax, Flavonoid Aglycones, C-M ethyl Flavones Flavonoid aglycones were detected as minor constituents o f the leaf wax o f the Myrtaceae Leptosperm um laevigatum. They belong to the rarely encountered C-methyl flavonoids. One of them is a new natural product, 5-hydroxy-3,7,3',4'-tetramethoxy-6-C-methyl flavone.

Introduction Several m em bers of the M yrtaceae have p re ­ viously been found to exhibit the rare C-m ethylated flavones as m inor com ponents of epicuticular wax layers (W ollenw eber and K ohorst, 1981). We now have studied the leaf wax of L eptosperm um laevigatum (G a e rtn e r) F. Müll, for the presence of such com pounds. The genus Leptosperm um en ­ com passes som e 50 species, grow ing in M alaysia, A ustralia and N ew Z ealan d (Willis, 1973). From the epicuticular m aterial of L. laevigatum we have isolated four C -m ethyl flavonoid aglycones, one of which is a novel n atu ral product. Materia! and Methods Leaves of L eptosperm um laevigatum w ere col­ lected from a p lan t cultivated for m any years in the B otanical G ard en of the Technische H ochschule D arm stad t. (A voucher specim en is k ep t in the h erb ariu m of the B otanical G arden of the T H D arm stad t). Fresh leaves w ere rinsed with acetone to dissolve the waxy epicuticular m aterial. The gum m y residue obtained upon ev aporation of th e solvent was d efatted (M eO H , -10°) and passed over Sephadex LH-20, eluted with M eO H , to sep­ arate the flavonoids from the prevailing te rp e ­ noids. Flavonoid fractions w ere fu rth er ch ro m ato­ g raphed over Polyam id SC- 6 , eluted with toluene and increasing am ounts of m ethylethyl ketone and

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m ethanol. TLC was perform ed on silica with to lu ­ ene - M eC O E t 9:1 o r with toluene - dioxane H O A c 1 8 :5:1, and on polyam ide DC-11 with petrol ioo_ i4 o° - to luene - M eC O E t - M eO H 1 2 :6 :1 :1 , with toluene - petrol 100_ 1 4 0 ° - M eC ­ O E t - M eO H 1 2 :6 :2 :1 , and with toluene - M eC ­ O E t - M eO H 1 2 :5 :3 . C hrom atogram s w ere viewed under U V 3 6 6 before and after spraying with “N aturstoffreagenz A”. Terpenoids w ere vis­ ualized by spraying silica plates with M nC l 2 re a­ gent, followed by heating (Jork et al., 1989). Flavo­ noid aglycones w ere identified by direct com parison with m ark ers and/ or by th e ir spectral data. N M R spectra w ere recorded in D M SO -d 6 at 400 M Hz ( !H ) and 100 M Hz ( 1 3 C) on a B ruker A RX -400 instrum ent. Two flavonoids w ere isolated in crystalline form , although in very small am ounts. H ow ever, spectra of good quality w ere obtained and the data are presented below. Two additional flavonoids w ere identified by direct com parisons with m arkers available in E. W .'s lab (W ollenw eber and Ko­ horst, 1984). C om pound 1. M p 195-196° C (uncorrrected). U V lmax (E tO H ) nm: 348, 274, 259; + A1C13 367, 268, AICI 3 + HC1 366, 282, 264; N aO H 410, 267; + N aO A c 413, 270; N aO A c + H 3 B 0 3 354, 273, 259. MS m/z (rel. int.): 358 (100, M+), 357 (57), 343 (47, M - C H 3), 327 (10), 315 (34), 181 ( 8 ), 165 (20), 151 (13). 'H -N M R 6 (ppm ): 12.82 (s, O H -5); 7.68 (d, J = 2 Hz; H -2'); 7.63 (dd, J = 2, 8 Hz; H - 6 '); 6.97 (d, J = 8 Hz; H -5 '); 6.80, s (H - 8 ); 3.92, 3.90, 3.83 (s, 3 x O M e); 2.00 (s, C-M e). 1 3 C -N M R 6 (ppm ): 178.0 (C-4), 163.1 (C-7), 157.2 (C-5), 155.5 (C-2), 154.5 (C-9), 149.9 (C -4'), 147.5 (C -3'), 138.0

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E. W ollenweber et al. • C-Methyl Flavones from Leptosperm um

(C-3), 122.3 (C - 6 '), 120.8 ( C - l') , 115.7 (C -5'), 112.1 (C -2 '), 107.2 (C - 6 ), 104.8 (C-10), 90.1 (C- 8 ), 59.7 (3-O M e), 56.3 (7-O M e), 55.8 (3 '-O M e), 7.3 ( 6 -M e). The MS fragm entation is in very good agreem ent w ith (R ab esa and Voirin, 1985), while the U V d a ta differ som ew hat. C om pound 2. U V Xmax (E tO H ) nm: 343, 271, 253; + A1C13 357, 262; A1C13 + HC1 362, 280, 261; N aO H 344, 274, 254; no reaction w ith N aO A c. MS m/z (rel. int.) 372 (100, M +), 371 (56), 357 (41, M C H 3), 341 (12), 329 (39). 'H -N M R Ö (ppm ); 12.80 (s, O H -5); 7.74 (dd, J = 2, 8 Hz; H - 6 '); 7.67 (d, J = 2 Hz; H -2 '); 7.17 (d, J = 8 Hz; H -5 '); 6.87 (s, H - 8 ); 3.93, 3.87, 3.87, 3.83 (s, 4 x O M e); 2.02 (s, C-M e). , 3 C -N M R 6 (ppm ): 178.0 (C-4), 163.2 (C-7), 157.2 (C-5), 155.3 (C-2), 154.6 (C-9), 151.3 (C -4'), 148.5 (C -3'), 138.4 (C-3), 122.2 (C - 6 '), 122.1 (C -l'), 111.6* (C -5 '), 111.3* (C -2 '), 107.3 (C - 6 ), 104.9 (C10), 90.3 (C - 8 ), 59.8 (3-O M e), 56.4 (7-O M e), 55.72, 55.70 (3'-O M e, 4 '-O M e), 7.3 ( 6 -M e). C om pound 1 was dem eth y lated with pyridinium hydrobrom ide (H ow ard and M abry, 1970) to yield com pound 3 (pinoquercetin, M + 316); U V A.max (E tO H ) nm: 375, 260; + A1C13 463, 275; A1C13 + HC1 440, 270; + N aO H 425 (dec.), 338, 246; + N aO A c 402, 340, (275); N aO A c + H3BO3 385, 345, 300, 260. O n polyam ide T L C 3 app ears as a dark spot th at exhibits orange fluorescence after spray­ ing with N A . Its R f is ab o u t the sam e as th at of quercetin. Results and Discussion The m ajor p art of the leaf wax of Leoptosperm u m laevigatum consists of terpenoids, the p re ­ dom inant constituent being oleanolic acid. Four Cm ethylated flavonoid aglycones are m inor constit­ uents of this epicuticular m aterial. 5,4'-D ihydroxy3,7,3'-trim ethoxy-6-C -m ethyl flavone (com pound 1) and 5-hydroxy-3,7,3',4'-tetram ethoxy-6-C m ethyl flavone (com pound 2 ) w ere isolated as crystalline p roducts and identified by exam ining their spectroscopic data. 5,4'-D ihydroxy-7-m ethoxy- 6 -C -m ethyl flavone ( 8 -desm ethyl-sideroxylin) and 5,4'-dihydroxy-3,7-dim ethoxy-6-C -m ethyl flavone ( 8 -desm ethyl-latifolin) w ere identified by direct com parisons w ith m arkers.

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In an earlier p aper (W ollenw eber and K ohorst, 1984) we explained how parallels in R ,-differences and in colour reactions with N aturstoffreagenz helped to find the structures of four new C-m ethyl flavonols. Sim ilar considerations led us to po stu ­ late that com pound 1 is a C-m ethyl flavonol with two O H groups, three O M e groups and one C-Me and that com pound 2 is its 4'-m ethyl e th e r (corre­ sponding to quercetin-3,7,4'-triM e and quercetin3 ,7,3',4'-tetraM e). These assum ptions w ere con­ firm ed by the spectral data. C om pound 1 has been found once previously, nam ely in the bark and spines of the D idieraceae Alliiaudia hum bertii (R abesa and Voirin, 1985). C om pound 2 , its 4'-m ethyl ether, is a novel natural com pound. The dem ethylation product of com ­ pound 1 has been rep o rte d as a n atu ral product from the bark of Pinus ponderosa (K urth et al., 1956). 8 -D esm ethyl-sideroxylin was re p o rted for the first tim e as a m inor constituent of the leaf wax of several Eucalyptus species (W ollenw eber and K ohorst, 1981) and found later in the leaf wax of som e E ricaceae (W ollenw eber and K ohorst, 1984). 8 -D esm ethyl-latifolin is thus far know n only from K alm ia latifolia and from Gaultheria procum bens leaf wax (W ollenw eber and K ohorst, 1984). A ccording to W ollenw eber (1994) and Wollenw eber and Jay (1988) som e 12 flavones with Cm ethylation have been found thus far, 1 0 of which were discovered as free aglycones, m ostly in Myrtaceae. T hree tim es as m any C -M ethyl-flavonols have been rep o rted and are d istributed in a num ber of families. The reasons th at plants elabo­ rate C -m ethyl flavonoids are presently unknow n, although one can speculate that they play a d efen­ sive role reducing attack by specific pred ato ry in­ sects or m icroorganism s (Proksch and R odriguez, 1985). A cknow ledgem ent Thanks are due to Lyn C raven at the A ustralian N ational H erbarium (C S IR O D ivision of Plant In­ dustry) in C anberra, A ustralia, for d eterm ination of the plant studied. E.W. also wishes to thank Dr. K. Siems, B erlin, for N M R analysis of the te rp e ­ noid fraction.

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E. Wollenweber et al. ■C-Methyl Flavones from L eptospennum

Howard G. and Mabry T. J. (1970), M yricetin 3 -0 methyl ether 3'-0-b-D -glucoside, the major flavonoid of O enothera speciosa (O nagraceae). Phyto­ chemistry 9, 2 4 1 3 -2 4 1 4 . Jork H., Funk W., Fischer W. and W immer H. (1989), D iinnschicht-chrom atographie, Vol. la , Verlag Chem ie, W einheim. Kurth E. F., Ram anathan V. and Venkataraman K. (1956), The colouring matters o f ponderosa pine bark: Part I. J. Sei. Ind. Res. 1 5 B, 1 3 9 -1 4 6 . Proksch P. and R odriguez E. (1985), Baja California. B iologie in unserer Z eit 15, 7 5 -8 0 . Rabesa Z .-A . and Voirin B. (1985), Sur la presence de trois C-methyl flavonols nouveaux, derives de la quercetine, chez A lluaudia hum bertii. C. R. Acad. Sc. Paris 301, 3 5 1 -3 5 4 .

Willis, J. C. (1973), A Dictionary o f the Flowering Plants and Ferns. 8. Ed. Cambridge University Press, Cambridge etc. W ollenweber E. and Kohorst G .(1981), Epicuticular leaf flavonoids from Eucalyptus species and from Kalm ia latifolia Z. Naturforsch. 36 c, 9 1 3 -9 1 5 . W ollenweber, E. and G. Kohorst (1984), N ovel epicu­ ticular leaf flavonoids from K alm ia and G aultheria (Ericaceae). Z. Naturforsch. 39c, 710 -713. W ollenweber E. (1993), Flavones and Flavonols. In: The Flavonoids - A dvances in Research since 1986, (Harborne, J. B., ed.). Chapman and Hall, London, pp. 2 5 9 -3 3 5 . W ollenweber E. and Jay M. (1988), Flavones and Fla­ vonols. In: The Flavonoids - A dvances in R esearch since 1980 (Harborne, J. B., ed.). Chapman and Hall, London-N ew York, pp. 2 3 3 -3 0 2 .