Triterpenes from the frond exudate of the fern Notholaena greggii

Phytochemistry, Vol. 31,No. 3, pp.923-927,1992 Printedin GreatBritain.

003l-9422/92 $5.00 + 0.00 0 1992Pergamon Press plc

TRITERPENES FROM THE FROND EXUDATE OF THE FERN

NOTHOLAENA GREGGII GIOVANNI APPENDINO,*

PIERLUIGI GARIBOLDI,~ ECKHARD WOLLENWEBER,$ ANGELO SIRONI~ and HENRIETTE MOLINARI\(

Dipartimento di Scienza e Tecnologiadel Farmaco, Cso Raffaello 31,10125 Torino, Italy; t Dipartimentodi Scienze Chimiche, Via S. Agostino 1,62032 Camerino (MC), Italy; $Institut fiir Botanik der Technischen Hochschule, SchnittspahnstraBe 3,610O Darmstadt, Germany; $Istituto di Chimica Strutturistica Inorganica, via Venezian 21,20133 Milano, Italy; IIDipartimento di Chimica Organica e Industriale, Via C. Golgi 19, 20133 Milano, Italy (Received 24 June 1991) Key Word Index-Notholaena

greggii; Pteridaceae; frond exudate; dammarane triterpenoids.

Almtract-Along with known flavonoid aglycones, the frond exudate of N. greggii yielded two ocotillol-type dammarane triterpenoids, whose structure was established by chemical reactions, spectral data and X-ray analysis.

INTRODUCTION

In several genera of ferns, belonging to the Pteridaceae, the lower frond surface of certain species bears a farinose

or waxy coating. This exudate material often consists of various flavonoid aglycones [ 1,2], sometimes along with considerable amounts of terpenoids. Kaurenoic acid has been reported as a major exudate constituent in Cheilanthes kaul&ssi, Notholaena incana (with its dihydroxy derivative, abbeokutone) and N. pallens [S]. A labdadienoic acid has been found to be the major constituent of the C. argentea exudate [4], while an acylated triterpene acid is excreted by N. candida var. candida [S]. We now report the isolation of two new triterpenoids from the exudate of N. greggii (Mett.) Maxon. RESULTS AND DISCUSSION

Apigenin, its 7- and 4’-monomethyl ethers and its 7,4’dimethyl ether have been reported earlier as characteristic flavonoid aglycones in the farinose exudate of N. greggii [6]. However, the crystalline dammaranes 1 and 2 were the major constituents (9.1 and 23%, respectively) of this material. Inspection of the ‘H and 13C NMR spectra showed that 1 and 2 are triterpene derivatives having five oxygenated carbons, one of them being a keto group in 2. Oxidation of 1 and 2 afforded the same diketone 3, showing that 2 differed from 1 only in the presence of a keto group in place of a hydroxyl. The formation of the y-lactone 4 (vcxo 1775 cm-‘) as the minor product of the oxidation suggested the presence. of a tetracyclic skeleton bearing an ether bridge between C-20 and C-24 and a hydroxyl at C-25 in the side-chain. This was further confirmed by the presence of a prominent ion at m/z 143 (A) in the mass spectra of 1 and 2. The two remaining oxygenated functions were tentatively placed at C-3 and C-12 on the basis of the following considerations: in 1 a strong intramolecular hydrogen bond was present (6,:

*Author to whom correspondence should be addressed. PRY31:3-A

6.66, unaffected by dilution.) This bond was still present in the ketol 2, but disappeared upon oxidation to the diketone 3. As no cross-peak was present between the sets of protons adjacent to the two carbonyls in the COSY spectrum of 3, the two hydroxyls are on different rings, and the intramolecular hydrogen bond most probably involves one of the secondary hydroxyls and the ethereal oxygen. Both keto groups are on a six-membered ring (8216.93 and 210.51, a cyclopentanone carbonyl would resonate at lower fields [7]), showing that the D-ring was not oxygenated. Furthermore, of the two hydroxyls, one was adjacent to a methylene and a quaternary centre (aproton: dd at 63.45, b-carbons: 639.75 s and 28.54 t), and the other to a methylene and a methine (cr-proton: td at 6 3.77, p-carbons: 648.73 d and 32.22 t). These data, along with comparison with literature data [8] suggested the presence of a 3,12-dioxygenated dammarane skeleton for 1 and 2. The structure of the diketone 3 was firmly assessed by a single-crystal X-ray analsyis, that also established the relative stereochemistry of the side-chain. An ORTEP drawing of the molecule is given in Fig. 1, and the atomic coordinates in Table 1. The relative configuration of the secondary hydroxyl(s) in the natural products was then deduced from the splitting pattern of the cc-protons, thus leading to stereostructures 1 and 2. Inspection of models shows the possibility of an intramolecular hydrogen bond between the hydroxyl at C-12 and the ethereal oxygen. Compounds 1 and 2 are structurally related to known sapogenins; indeed oxygenated triterpenes like 1 and 2 are widespread in seed plants [S], but they have very rarely been encountered in ferns, whose triterpenoids generally lack the C-3 oxygen [9]. The occurrence of 1 and 2 in N. greggii is, therefore, taxonomically relevant. In spite of the pharmacological importance of dammarane triterpenoids [S] and the wealth of NMR data available on this class of compounds [8], relatively few modem NMR studies have been conducted [lo]. For the complete assignment of the spectra, a thorough NMR study was thus undertaken. The aim was to assign all the 13C and ‘H resonances. This study was prompted

923

924

G.

APPENDINO

et

al.

OH

2-S

ie R,

4

RZ

1

P -OH;a-H

B-OH:

a-H

2

=o

P-OH:

a-H

3

=o

=o

Fig. 1. ORTEP drawing of compound

by the presence in the exudate of several minor triterpenoids differing from 1 and 2 by the oxidation of one of the methyls to a hydroxymethyl group. A set ofconsistent 13C NMR assignments could, in fact, allow a tentative location of the primary hydroxyl. A preliminary analysis of the spectra of l-4 showed that the diketone 3 was the best candidate for this study, on account of the better distribution of ‘H chemical shifts induced by the anisotropy ofthe two carbonyls. However, the extensive signal overlapping of the aliphatic protons discouraged the use of traditional assignment techniques. Even a homonuclear COSY experiment in the DQF version [11] could localize only few ‘H connections (e.g. H-13-H-17; H-9-H-l 1; H-l-H-2). The following experiments were thus performed: (i). ‘H-broad band decoupled r3C NMR spectrum to measure chemical shifts, followed by a DEPT-experiment, to assign multiplicities. (ii). 13C-lH COSY via one-bond couplings, which allowed assignment of a few carbons directly bonded to the protons already revealed by the homonuclear DQF-COSY. Well resolved 1D traces taken along F2 (’ 3C frequency) in the phased mode, allowed extraction of

3.

the single ‘H subspectra, taking advantage of the complete chemical shift resolution in the 13C frequency domain. This made it possible to assign all ‘H chemical shifts after complete 13C assignments were made based upon two further experiments described below. In several instances the coupling pattern of the protons could also be fully described (Table 2). (iii). rH-r3C COSY via twoand three bond couplings. The recently published FLOCK sequence [12] was used. This turned out to be very efficient in suppressing one-bond correlation peaks and in giving rise to several long-range cross-peaks with excellent sensitivity. This experiment allowed assignment of most 13C and ‘H resonances, starting from those previously assigned and $rmping’ by two or three bonds along the carbon skeleton. The fully classified long-range correlation peaks are reported in Table 2. After this analysis only a few 13C resonances could not be unambiguously assigned (C-6, C-7, C-15, C-16, C-22 and C-23), due to their close chemical shifts, the lack of indicative long-range r3C-‘H correlation peaks and the extensive overlapping of the corresponding proton signals. (iv). 2DINADEQUATE experiment. In spite of its well-known

Triterpenes from Notholaena greggii Table 1. Positional parameters and their estimated standard deviations Atom

x

o-1 o-2 o-3 O-4 C-l c-2 c-3 c-4 c-5 C-6 c-7 c-s c-9 c-10 c-11 c-12 c-13 c-14 c-15 C-16 c-17 C-18 c-19 c-20 c-21 c-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 c-30

0.6695 (4) 0.8586 (4) 0.6419 (6) 0.1310(7) 0.4109 (7) 0.2946 (8) 0.1723 (8) 0.0964 (7) 0.2208 (6) 0.1470(7) 0.2830(7) 0.3927 (6) 0.4550(6) 0.3192(6) 0.5753 (6) 0.7135 (7) 0.6540 (7) 0.5531(7) 0.5260(S) 0.6956 (8) 0.7797 (7) 0.2855 (7) 0.2110(7) 0.8236 (7) 0.8848 (8) 0.9424 (8) 0.8825 (8) 0.6930(8) 0.5902 (9) 0.623(l) 0.406 (1) 0.0532 (8) -0.0676(7) 0.6673 (8)

z

Y -0.4684 (3) - 0.0866 (3) -0.551 l(4) 0.3930(4) 0.2046 (5) 0.3137(5) 0.3027 (5) 0.1774(5) 0.0718(5) - 0.0547 (5) -0.1519(5) -0.1552(4) -0.0219(5) 0.0814(4) -0.0190(4) -0.1147(5) - 0.2407 (4) -0.2344(5) -0.3724 (5) -0.4313(5) -0.3468 (5) -0.2067 (5) 0.0756(5) -0.4119(5) -0.3266(5) -0.5200(6) -0.6138(5) -0.5954(5) -0.6288 (5) -0.7598 (6) -0.6078 (7) 0.1743 (6) 0.1726 (6) -0.1854(5)

0.1807(l) 0.1432(l) 0.2613 (1) 0.0418 (2) 0.1030 (2) 0.0964 (2) 0.0613(2) 0.0512(l) 0.0612(l) 0.0549 (2) 0.0533 (2) 0.0932(l) 0.1025(l) 0.1035(l) 0.1403 (2) 0.1381(l) 0.1279(l) 0.0862 (1) 0.0777 (2) 0.0899 (2) 0.1230(2) 0.1295(2) 0.1438 (2) 0.1643(2) 0.1983 (2) 0.1589(2) 0.1905 (2) 0.1882(2) 0.2275 (2) 0.2411(2) 0.2192(2) 0.0041(2) 0.0761(2) 0.0501(2)

B(A2) 3.91(8) 4.94 (9) 6.0(l) 8.5 (1) 3.7(l) 4.7(l) 4.6(l) 3.8(l) 3.2(l) 4.0(l) 3.9(l) 2.8(l) 2.8(l) 2.8(l) 3.0(l) 3.3(l) 3.0(l) 3.0(l) 4.3(l) 4.6(l) 3.7(l) 3.6(l) 3.4(l) 3.5(l) 4.8(l) 5.1(l) 5.3(l) 4.3(l) 4.9(l) 7.7 (2) 6.6(2) 5.5 (2) 5.1(l) 4.7(l)

Starred atoms were refined isotropically. Anisotropically relined atoms are given in the form of the isotropic equivalent thermal parameter &fined as: 1 (4/3) * [a2*B(l, l)+b2*B(2, 2)+c2*B(3, 3)+ab(cosy)*B(l, 2)+ ac(cos jl) * B (1, 3) + bc(cos a) + B(2, 3)].

low sensitivity, this technique seemed the only experiment that could allow the assignment of the remaining frequencies. The spectrum displayed all the cross-peaks due to one-bond 13C-‘3C couplings necessary to assign the remaining resonances, and also allowed a check of the correctness of the previous assignments. In spite of the high field used (125.77 MHz) and the intrinsically high resolution power of this experiment [spreading of crosspeaks along the ‘double quantum frequency’ (F,)], a number of signals still remained overlapping. Thus, only the concomitant use of different NMR techniques allowed a complete spectral analysis of compound 3. Based on the results of this study, many ambiguities in the spectral analysis of dammaranes, including the assignment of the angular methyls [8], can now be solved. EXPERIMENTAL Plant material. NotkoLaenn greggii was collected in Dec. 1981

in the Canyon of Rio Nazas in Durango/Mexico,

in a locality

925

where it is abundant on south-facing limestone slopes. Vouchers (T. Reeves, L. Reeves and E. Wollenweber, 7513) are kept at ASU and in E.W.‘s private herbarium. Fronds were clipped in the field and dried in a paper bag. Isolation of the constituents. Dried fronds (138 g) were rinsed with MezCO, giving 16.3 g of extract. Further washings with toluene afforded 2.1 g of extract. The overall yield was thus 13.3%. The extract (10.13 g) was dissolved in CHzCI, and 10 g silica gel 40 (35-70 mesh) added. After removal of the solvent (water pump), the residue was charged on to a 150 g silica gel column and eluted with hexane containing increasing amounts of EtOAc. Frs of 25 ml were taken. Frs 18-46 and 47-62 (hexane_EtOAc 4: 1) gave a solid residue. After crystalliition from EtzO, apigenin 7.4’~dimethyl ether (42 mg) and a mixture of apigenin 7 and 4’-monomethyl ethers (132 mg) were obtained. Frs 63-102 (hexane-EtOAc, 1: 1) upon crystallization from hexane-Et,0 at -5” gave 2.4Og of ketal 2, and frs 103-118 (hexane-EtOAc, 3 : 7) 2.15 g of crude trio1 1. Crystallization from hexane-Et,0 gave 920 mg of pure 1. (38,12B,20S,24S)20,24-Epoxydammarane-3,12,25-trioI (I). Crystals (hexane-EtzO), mp 147”, [a]h5 -24.5” (CHzCI,; c O.Sk IR vz;crn-‘: 3380, 1380, 1125, 1080, 1045, 880; ‘H NMR (300 MHz, pyridine-d,: 66.06 (brs, OH), 4.18(dd, J= 10.6 and 5.5 Hz, H-24), 3.77 (td, J = 10.0,lO.Oand 5.0 Hz, H-12), 3.45 (dd, J = 10.4 and 5.6 Hz, H-3), 1.45,1.31,1.31, 1.23, 1.06, 1.05,0.93,0.91 (s, 8 methyls); EIMS (70 eV), m/z (rel. int.): no molecular ion (C3cH5z04), 458 [M - IS] + (lo), 4; 7 (30), 400 (15), 399 (60), 381 (35), 143 (100). (12/?,20S,24S)20,24-Epoxydammarane-12,25-dioI-3-one

(2).

Crystals (hexane-EtzO), mp 173”, [a]:: +22” (CHzClz; ~0.7); IR ~2: cm-‘: 3270,1695,1460,1380,1040,1015,880, ‘H NMR (300 MHz, pyridinad,): 66.36 (br s, OH), 4.13 (dd, .I= 10.2 and 5.2 Hz, H-24), 3.68 (td, J= 10.4,10.4 and 4.8 Hz, H-12), 1.25,1.21, 1.08, 1.06, 1.02, 1.01,0.96,0.90 (s, 8 methyls) EIMS (70 eV), m/z (rel. int.): no molecular ion (C,,H,,O,), 456 [M- 18]+ (2), 400 (lo), 399 (lo), 376 (S), 192 (20), 143 (100). Oxidation of ketol 2. Compound 2 (920 mg) was dissolved in 60 ml dry CHzCl, and an excess of PDC (17.5 g) was added. After stirring for 11 days at room temp., Et,0 was added and the reaction mixture was filtered through a pad of celite. The filtrate was evapd and purified by CC (20 g silica gel). Frs eluted with hexane_EtOAc (1: 1) gave 142 mg of a mixture of y-&tone 4 and diketone 3, and 432 mg of pure 3. The mixture of 3 and 4 was further separated by HPLC (hexane-EtOAc, 3 :2, Water Microporasil column) to give 41 mg of 4 and 22 mg of 3. The overall yield was 50% for 3 and 4.9% for 4. Oxidation oftriol 1. Compound 1 was oxidized as described for 2. From 300 mg 1, 120 mg 3 (40%) and 16 mg 4 (5.8%) were obtained. (20S,24S)20,24-Epoxydammarane-25-o1-3,12-dione (3). Crystals (diisopropyl ether), mp 149”,IR vz= cm-‘: 1700,1460,1380, 1180, 1050, 900. EIMS (70 ev), m/z (rel. int.): no molecular ion (C3cH4s04), 454 [M - 18]+ (5), 399 (lo), 398 (lo), 190 (20), 143 (100). Lactone 4. Crystals (EtzO), IR v&t: cm-‘: 1775, 1710, 1390, 1260,1225,1170,945; ‘H NMR (300 MHz, CDCI,): 6 1.24,1.24, 1.04, 1.03, 1.00, 0.76 (s, 6 methyls). NMR experiments. ‘H and i3C NMR spectra were run on a 300 MHz spectrometer. A 0.05 M soln of 3 was used. lH-iaC correlation spectra were run on the same instrument using a 0.2 M CDCl, soln of 3. The sequence HETCOR available in the VXR-4000 software was used for one-bond correlations; a spectral window of 3500 Hz with a digital resolution of 3.4 Hz/point was chosen in F2. 256 traces of 54 transients each were collected. 2024 data points were used for the Fourier transformation. The FLOCK sequence [12] was used for long-

G. APPENDINOet al.

926

Table 2. NMR spectral data for compounds l-4 (CJXI,, ‘H NMR: 500 MHz. J in Hz; 13C NMR: 125.77 MHz for 3 and 75.04 MHz for 1, 2 and 4) H

3

la lb 2a+b 5 6a, b 7a. b 9 lla lib 13 15a 15b 16a 16b 17 18 19 21 22a 22b 23a 23b 24 26 27 28 29 30

1.74 br I (11.5) 1.46 dd (11.5, 3.2) ca 2.48 M 1.40 dd (11.9, 3.2) ca 1.63 m ca 1.53 m 1.83 dd (10.0, 3.6) 2.40 t (13.1) 2.32 dd (13.1, 4.5) 3.00 d (9.4) 1.73 m 1.18 m 1.74 m 1.58 m 2.56 dt (10.2, 4.0, 4.0) 1.25 s 1.05 s 1.05 s 1.88 m 1.65 m 1.78 m 3.73 dd (10.6, 5.6) 1.11 sa 1.20 sa 1.11 s 1.08 s 0.78 s

C

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

38.92 t 28.54 t 78.31 d 39.75 s 55.95 d 18.30 t 34.77 t 39.74 s 50.22 d 37.17 s 32.22 t 70.45 d 48.73 d 52.17 s 31.61 t 25.10 t 48.93 d 17.78 q 15.46 q 87.15 s 27.93 q 31.61 t 27.37 t 87.40 d 70.00 s 24.10 q 28.00 q 28.87 q 15.32 q 16.29 q

2

3

39.72 t 32.19 ta 217.83 s 47.32 s 55.26 d 19.69 t 34.05 t 39.61 s 49.55 d 36.84 s 32.06 ta 70.36 d 48.87 d 52.15 s 31.64 t 25.05 t 48.93 d 17.69 q 15.13 q 87.11 s 27.96 q 31.63 t 28.51 t 87.43 d 70.03 s 24.29 q 28.87 q 26.74 q 20.93 q 16.09 q

39.19 t 33.80 t 216.93 s 47.35 s 55.13 d 19.74 t 33.61 t 40.30 s 53.71 d 37.32 s 39.81 t 210.51 s 57.37 d 55.98 s 31.97 t 24.81 t 43.03 d 15.24 q 15.76 q 85.24 s 26.72 q 36.55 t 26.21 t 87.63 d 70.30 s 23.96 q 27.71 q 26.65 q 21.02 q 16.53 q

Long range corr.

C

4

H-2, H-19 H-la, b H-2, H-28, H-29 H-28, H-29 H-19, H-28, H-29 H-5 H-18 H-9, H-lla,b, H-18, H-30 H-lla,b, H-18, H-19 H-5, H-lla, b, H-19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

39.18 t 33.78 r 216.78 s 47.37 s 56.07 d 19.67 t 33.78 t 40.31 s 53.61 d 37.30 s 39.72 t 210.09 s 56.93 d 55.12 s 31.58 t 24.97 t 42.74 d 15.42 q 15.75 q 88.64 s 24.26 q 28.93 ta 32.43 t’ 76.92 s

H-11, H-13 H-30 H-13, H-18, H-30 H-30 H-17 H-13, H-21 H-9 H-9, H-5 H-13, H-21 H-21 H-26, H-27 H-26, H-27 H-27 H-26 H-29 H-13

26.60 q 21.02 q 16.37 q

“Interchangeable assignments. range correlations. Three different spectra were run; one for aliphatic carbons (14-58 ppm), one for oxygenated carbons (68-88 ppm) and one for carbonyl carbons (205-218 ppm); a digital resolution of 3.6 Hz/point was always used in F2. A 1024 Hz spectral window was chosen in Fl for the three spectra; 512,256 and 256 traces of 128 transient each were collected in the corresponding spectra. 68 and 35 msec were chosen for delays A1 and AZ, respectively [12]. For the DQFCOSY experiment [ll], a spectral window of 940 Hz was used in both dimensions; 256 traces of 16 transients each were collected and 1026 data points were used for both Fourier transformations. The INADEQUATE experunent was carried out on a 5OOMHz spectrometer, operating at 125.77 MHz for C, at 309 K, on a sample 0.4 M in CDCI,, to which 10 mg bi(Acac), had been added, in a 5 mm tube without spinning. A 125” read pulse was used to provide quadrature detection in the double quantum frequency domain, with an improved sensitivity of the double quantum signal; the 90” carbon pulse was 9.6 psec, and the delay of the echo period necessary to excite the double quantum coherence was set to l/45,_,= 6 msec. The proton decoupling was achieved through standard composite schemes, with a 90” pulse set to 59 psec. The sweep width in both dimensions was 11904.762 Hz, the acquisition time was 0.806 set in F2 and 0.0054 set in Fl. 2048 transients were collected for each FID. Sine bell window was applied in both dimensions prior to Fourier transformation. X-ray analysis. A transparent colourless crystal of dimensions 0.22 x 0.10 x 0.009 mm was mounted on a Enraf-Nonius CAD-4

diffractometer, and 25 intense reflections having a t? value in the range 10&14.0” were centred using graphite-monochromated MoK, radiation (1.=0.71073 A). Least-squares refinements of their setting angles resulted in the unit-cell parameters reported in Table 3, together with an orientation matrix relating the crystal axes to the diffractometer axes. A total of 2548 chffracted intensities 11427 with I> 30(l)] were collected at room temp. with constant scan speed (scan time for each reflection: 90 set), by exploring the octant of the reciprocal lattice with O
Triterpenes from Notholaena greggii REFERENCES

Table 3. Crystallographic data Formula M, Crystal system Space group a (A) b(A) c (A) V(A3) Z DEBled (g cm- ? p(MoK,) (cm- ‘) Scan mode o-scan width (” ) O-range ( “) Octants of reciprocal space explored Measured reflections Unique observed reflections with I > 30(I) Final R and R, indices* No. of variables GOFt *R=[Z(F,-klF,l)/ZF,],

921

GoH,sO, 472.71 orthorhombic P2,&2, 7.989 (2) 10.920(l) 31.580(l) 2755(l) 4 1.14 0.68 0 1.0+0.35 tans l-25 +h, +k, +I 2548 1427 0.046, 0.053 307 1.421

R,=[~w(F,-k(F,I)‘/ZwF,*1”*.

tGOF=[Zw(F,-kJF,J)‘/(N obser”.tio”% “arhblcs)11’2. w= l/(o(F,))‘, o(F,)= [u’(I )+(0.041)‘,“2;F,L,. SDP-Plus Structure Determination Package [15]. Maximum residual in the final difference Fourier synthesis: 0.18 eA_“. A list of computed hydrogen atoms positions, anisotropic temperature factor coefficients and observed and calculated structure factor moduli has been deposited at the Cambridge file. Acknowledgements-E.W. thanks the Deutsche Forschungsgemeinschaft for financial support. This work was also supported by the Minister0 della Pubblica Istruzione (Fondi 40 and 60%). Thanks are due to T. and L. Reeves (Tsaile, AZ) for help with plant collection. Note added in proof A triterpene closely related to compounds 1 and 2 has been found in Notholaena rigida [Arriaga-Giner, F. J., Rullkiitter, J., Peakman, T. M. and Wollenweber, E. (1992) 2. Naturforsch., in press].

1. Wollenweber, E. (1978) Am. Fern. J. 68, 13. 2. Wollenweber, E. (1984) Rev. Latinoam. Quim. 15, 3. 3. Ruedi, P., Wollenweber, E., Marx, D. and Scheele, C. (1989) Z. Naturforsch. Uc, 901. Wollenweber, E., Ruedi, P. and Seigler, D. (1982) Z. Naturforsch. 37c, 1283. Arriaga-Giner, F.-J. and Wollenweber, E. (1986) Phytochemistry 25, 735.

Wollenwebcr, E. (1982) in Z%ePlant Cuticle (Cutler, D. F., Alvin, K. L. and Price, C. E., eds) @inn. Sot. Symp. Ser. 10). Academic Press, London. 7. Pretsch, E., Clerc, T., Seibl, J. and Simon, W. (1989) in

8. 9. 10. 11.

Spectral Data for Structure Determination of Organic Compounds, p. C190. Springer. Tanaka, 0. and Kasai, R. (1984) Progr. Chem. Org. Nat. Prod. 46, 1. Murakami, T. and Tanaka, N. (1988) Progr. Chem. Org. Nat. Prod. 54, 1. Wilkins, A. L., Elix, J. A. and Whitton, A. A. (1990) Aust. J. Chem. 43,411. Piantini, V., Sorensen, D. W. and Ernst, R. R. (1982) J. Am. Chem. Sot. 104, 6800.

12. Reynolds, W. F., McLean, S., Perpick-Dumont, M. and Enriques, R. G. (1989) Magn. Res. Chem. 27, 162. 13. Cromer, D. T. (1974) Znternational Tables for X-Ray Crystallography, Vol. 4, Table 2.3.1, The Kynoch Press, Birmingham (present distributor: Kluwer Academic Publishers, Dordrecht). 14. Cromer, D. T. and Waber, J. T. (1974) International Tables for X-Ray Crystallography, Vol. 4, Table 2.2.b. The Kynoch Press, Birmingham (present distributor: Kluwer Academic Publishers, Dordrecht). 15. Frenz, B. A. and Associates (1980) SDP Plus Version 1.0. Enraf-Nonius, Delft, The Netherlands. 16. Sheldrick, G. M. (1985) SHELXS86, in Crystallographic Computing, Vol. 3 (Sheldrick, G. M., Kruger, C. and Goddard, R., eds), pp. 175-189. Clarendon Press, Oxford.