Triterpenoids from gum mastic, the resin ofPistacia lentiscus

Phytochemistry, Vol. 30, No. 11, pp. 3709-3712,1991

0031-9422/91 $3.00+0.00 © 1991PergamonPress plc

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TRITERPENOIDS F R O M G U M MASTIC, THE RESIN OF PISTACIA LENTISCUS FRANZ-JOSEF MARNER, ANTJE FREYER and JOHANN LEX* Institut fiir Biochemie, Universit/it zu K61n Z/ilpicher StraBe 47, D-5000 K61n 1, Germany; *Institut f/Jr Organische Chemic, Universit/it zu K61n Greinstral3e 4, D-5000 K61n 41, Germany (Received 25 February 1991)

Key Word Index--Pistacia lentiscus; Anacardiaceae; gum mastic; triterpenes; norlupane derivatives; noroleanane derivatives; malabaricane derivatives; polypodane derivatives; epoxysqualene cyclization. Abstract--Two novel nortriterpenoids and two, hitherto unknown, representatives of the rare malabaricane and polypodane type, respectively, were found in the neutral fraction of gum mastic, the resin of Pistacia lentiscus. In addition, several tri-, tetra- and pentacyclic triterpenes were identified. The significance of these results for the geometry of the epoxyqualene cyclase/substrate interaction in Pistacia lentiscus is discussed.

INTRODUCTION Mastic, the bleed resin of Pistacia lentiscus L. (Anacardiaceae), is abundantly available and has widely been used for several purposes. Nevertheless, very little is known about its composition. First attempts to analyse the resin led to the identification of three triterpenoid keto-acids and the tetracyclic triterpenol tirucallol [1, 2]. Probably due ro the extreme complexity of the mixture no further efforts on its separation and analysis have been made until recently, when Boar et al. [3,1 succeeded in isolating the new bicyclic triterpenoid diol 1 from the neutral fraction of the gum. This compound turned our attention to mastic as it (resp. its diastereoisomer at C-8) may be an intermediate in the formation of the iridals and cycloiridals, a family of mono- or bicyclic triterpenoids with completely new structures (e.g. 2) which we have isolated from various Iris species [4,1.

RESULTSAND DISCUSSION The neutral fraction of the resin [2] was carefully fractionated on silica gel and five main fractions obtained. Analysis by GC and GC-MS led to the identification of the known tetra- or pentacyclic triterpenoids tirucallol, dipterocarpol, lupeol, fl-amyrin, fl-amyrone, oleanonic aldehyde and germanicol, the identity of which was confirmed by comparison with authentic standards. Although not reported previously as constituents of mastic (except for tirucallot), several of these compounds have been found in galls produced by insects on the leaves of various Pistacia species and in the bleed resin of Pistacia vera [5-8,1. The components, not identified by GC-MS, were purified to homogeneity by reversed phase- or argentation-chromatography, if necessary after derivatization of functional groups. Structure elucidation of the compounds isolated was achieved by spectroscopic methods.

From their mass spectra a noroleanane skeleton was assigned to a ketone (3) and its corresponding 3r-alcohol (3a) as they gave rise to a molecular ion at m/z 410 and 412, respectively, and a prominent retro-Diels-Alder fragment at m/z 204 which indicated the absence of a methyl group at ring D or E. The 13C NMR spectrum of 3 clearly showed that the molecule lacked C-28, because in comparison to fl-amyrin [91 the methyl resonance at 6 27.3 was missing. This was confirmed by X-ray analysis of the ketone (3) (Fig. 1) which crystallized from methanol. Compound 3 has been shown to be present in sediments found offthe east coast of Borneo [10,1 and recently has been reported as a constituent of a drug used in Taiwan [11]. The nortriterpenol (3a), however, has not been found before and for both compounds spectroscopic data had not yet been published. The most plausible pathway for the formation of these compounds from fl-amyrin or fl-amyrone is by stepwise oxidation of C-28 to a carboxylic acid and subsequent decarboxylation. All intermediates in this reaction sequence are present in mastic. Thus, besides fl-amyrone, oleanonic aldehyde and oleanonic acid [2"1, 28-hydroxyfl-amyrone (3b) is a constituent of the resin, since we isolated its diacetate (3c) which was prepared by reduction of 3b and acetylation of the resulting diol. Compound 3c was identified readily by its typical mass spectrum [12] and its 1H and 13CNMR spectra. The structure was further confirmed by preparation of 3e from oleanonic aldehyde. Although the intermediates are still missing, the same demethylation sequence presumably takes place in the lupane group, as, besides lupeol, the novel 3-oxo-28norlup-20(29)-ene (4) is present in the extracts. The N M R and mass spectra made the pentacyclic nature of the compound apparent, with a norlupane type skeleton and only one olefinic double bond in the exocyclic C-20(29)position. The absence of the C-28 methyl was deduced from H - H - and H-C~COSY experiments and com-

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parison of the 1H and l a C N M R data with the values reported for lupeol and lupenone [13]. In addition to dipterocarpol, two more tetracyclic triterpenoids of the dammarane group were isolated, namely 20(S)-3fl-acetoxy-20-hydroxydammar-24-ene (5) and 3-oxo-dammara-20(21),24-diene (Sa). The E1 mass spectrum of the latter is almost undistinguishable from the one of dipterocarpol which, after spontaneous loss of water, shows the same fragmentation pattern. Differences are of course obvious, however, in the NMR spectra. Thus, the exomethylene double bond is responsible for typical resonances as shown previously for the appropriate 3fl-alcohol [14]. Similarly, the physical properties of the acetate (5) are consistent with the values known for the underivatized diol [ 15]. Additionally, the identity of 5 was confirmed by its preparation from dipterocarpol. The less common triterpenoids are not restricted to the bicyclic diol (1). Thus, we isolated the hitherto unknown corresponding ketone (la). Its structure was deduced easily by comparison with 1. Following Boar's procedure [3] the absolute configuration was confirmed by degradation of the compound to (+)-ambreinolide. Two tricyclic triterpenoids with the rare malabaricane skeleton are constituents of the extract. From spectral evidence, they could be assigned the 3fl-hydroxy- and the

3-oxo-malabaricatriene structures (6) and (6a), respectively. Ozonolysis of 6 and esterification of the reaction mixture yielded the diketoester (7). This established the tricyclic nature of the compounds. The structure of the side chain was proven by the GC-MS identification of levulinic acid as a degradation product. The ketone (6a) and the acetate of the triterpenol (6) have earlier been found only in the roots of Pyrethrum santolinoides [16]. The optical rotation of 6a, and the 13C NMR and NOE difference spectra of both compounds found in mastic, are in full agreement with the literature values reported for these compounds. Therefore, the configurations shown is assigned to these components. It is interesting to note that P. lentiscus is the first example of a plant producing triterpenoids with bi-, tri-, tetra- and pentacyclic skeletons from epoxysqualene. So far only the fern Lemmaphyllum microphyllum var. obovatum is known to contain the corresponding hydrocarbons which are formed by proton-triggered cyclization of squalene [17]. These results give rise to the assumption that the enzyme-substrate interactions of the epoxysqualene cyclase from P. lentiscus obey the following rules: Squalene epoxide is affixed in the chair-chair-chairboat form to the active site of the enzyme. Apparently, the

Triterpenoids from Pistacia lentiscus

3711

Fig. 1. Three-dimensional drawing of the noroleanone (3) obtained by X-ray analysis.

C-14/C-15 double bond of the substrate, is not firmly held in place, as its tertiary or quaternary carbon can participate in the formation of the third ring, the former leading to the malabaricanes and the latter to the tetra- or pentacyclic structures in the usual way. O n the other hand, this double bond and the carbocation at C-8 may be so far apart that the d o n o r of a hydroxy group can push its way to the carbenium ion, which results in the formation of the bicyclic polypodanes. EXPERIMENTAL

Optical rotations were measured in C H 2 C I 2 o r C H C | 3 at 20°. Mps are uncorr. NMR spectra were recorded in CDCI 3 (1H at 300 or 400 MHz and 13C at 75 or 100 MHz, respectively). Chemical shifts are reported in 6-values relative to TMS. If available, resonances were assigned on the basis of H-H and H-C COSY experiments. ElMS were recorded at 70 eV,. CH 4 served as reagent gas for CIMS. GC separations were carried out on a 10 m WCOT fused silica capillary column coated with SE30 (CB-phase). Commercial gum mastic was obtained from Carl Roth GmbH, Karlsruhe, F.R.G. The neutral fr. of the material was obtained as described previously I2] and was chromatographed on silica gel using a petrol-Et20 gradient affording frs containing ketones fir. 1:petrol-Et2 O, 9:1), dicarbonyl compounds fir. 2: petrol-Et20, 17:3), alcohols (fr. 3: petrol-Et20, 4:1), ketoalcohols fir. 4: petrol-Et20, 7:3) and diols fir. 5: petrol-Et20, 3: 2), respectively. fl-Amyrone (fr. 1), oleanonic aldehyde (fr. 2), fl-amyrin, germanicol, lupeol, tirucallol (all fr. 3) and dipterocarpol fir. 4) were identified by their mass spectra and their GC retention behaviPHYTO30:11-0

our as compared to authentic samples. Fr. 5 consisted of almost pure (8R)-3fl,8-dihydroxy-polypoda-13E,17E,21-triene (1). The 3-oxo-28-norolean-12-ene (3) crystallized from a MeOH soln of fr. 1 (0.74% of the resin). After acetylation of fr. 3 the acetate of the corresponding 3//-hydroxy derivative (3a) was identified by GC-MS. Confirmation of this result was acbeived by conversion of 3 into 3a. Further chromatography of several frs on silica gel impregnated with AgNO3 (10%) led to the purification of the following triterpenoids: from fr. 1: 3-oxo-28-norlup-20(29)-ene (4) (0.01%), 3-oxo-dammara-20(21),24-diene (Sa) (0.01%) and 3-oxomalabarica-14(26),17E,21-triene (ra) (0.015%). From fr. 4: (8R)3-oxo-8-hydroxypolypoda-13E,17E,21-triene (la) (1.6%) and from the same fr. after reduction with NaBH4 and acetylation the diacetate (3c), thus proving the presence of 28-hydroxy-fl-amyrone (3b) (0.21%). By low pressure LC of ft. 3 on RP 18 (MeOH) the (20S)-3fl-acetoxy-20-hydroxydammar-24-ene (5) (0.31%) and 3fl-hydroxymalabarica-14(26),17E,21-triene (6) (0.26%) were isolated. Except for the two noroleanes 3 and 3a and the novel ketones la and 4 the physical data of all compounds (or their homologues at C-3, e.g. 3-hydroxy- instead of 3-oxo-derivatives) have been reported earlier. (8R)-3-Oxo-8-hydroxypolypoda-13E,17E,21-triene (la). Oil [~]57s: + 11° (CH2C12; c4.7); ElMS m/z (rel. int,): 424 [M - H 2 0 ] + (0.5), 355 (0.5), 287 (0.1), 189 (1.5), 137 (17), 94 (32), 81 (85), 69 (100); CIMS (pos.) m/z 442 [M]+; CIMS (neg.) m/z 441 [ M - 1]-; IH NMR: 60.88 (3H, s), 0.96 (3H, s), 1.03 (3H, s), 1.13 (3H, s), 1.53 (9H, s), 1.61 (3H, s), 1.8-2.1 (10n, m), 2.3-2.6 (3H, m), 5.0-5.15 (3H, m); laC NMR: 6217.9 (s, C-3), 135.3 (s, C-14), 134.9 (s, C-18), 131.1 (s, C-22), 124.6 (d, C-13), 124.2 (d, C-21), 124.0 (d, C-17), 73.5 (s, C-8), 60.2 (d, C-9), 55.0 (d, C-5), 47.4 (s, C-4), 43.6 (t,

3712

F.-J. MARNERet al.

C-7), 39.6 (t, C-19), 39.6 (t, C-15), 38.4 (t, C-l), 38.2 (s, C-10), 33.8 (t, C-2), 31.1 (t, C-11), 26.6 (t, C-20), 26.5 (t, C-16), 26.2 (q, C-23), 25.7 (t, C-12), 25.6 (q, C-30), 23.4 (q, C-26), 22.5 (t, C-6), 21.2 (q, C24), 17.6 (q, C-29), 16.1 (q, C-27), 15.9 (q, C-28), 14.7 (q, C-25). 3-Oxo-28-norolean-12-ene (3). Mp: 191-193 ° (MeOH); [~t]STS: +184.5 (CHC13; c 1.7); EIMS m/z (rel. int.): 410 [M] + (1), 395 (0.5), 271 (0.2), 257 (0.3), 243 (0.5), 217 (0.8), 204 (100), 189 (35); 1H NMR: 60.84 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 1.02 (3H, s), L03 (3H, s), 1.07 (3H, s), 1.09 (3H, s), 1.1-2.0 (m), 2.3-2.6 (2H, m), 5.2 (1H, t, J=3.6 Hz, H-12); laCNMR: 6217.9 (s, C-3), 146.0 (s, C13), 120.8 (d, C-12), 55.3 (d, C-5), 47.4 (s, C-4), 47.0 (d, C-9), 44.9 (t), 42.5 (s, C-14), 41.1 (t0, 39.1 (t, C-1), 39.1 (t, C-8), 36.8 (s, C-10), 35.7 (d), 34.1 (t, C-2), 33.6 (q, C-29), 33.6 (t), 32.5 (t, C-7), 31.2 (s, C-20), 31.0 (t), 27.9 (t), 26.5 (q, C-23), 24.9 (q, C-27), 23.8 (q, C-30), 23.4 (t, C-11), 22.2 (t), 21.5 (q, C-24), 19.7 (t, C-6), 17.4 (q, C-26), 15.1 (q, C25); X-ray analysis: Crystal data: C29H460 ([M] + 410.69), orthorhombic, a=6.695 (2), b= 11.886 (2), c=30.919 (6).~, V =2460.518 .~3 (cell constants were determined by least square technique involvingdiffractometer 20 angles for 25 automatically centred reflections, 2 =0.71069 A); space group P212121 (No. 19), Z=4, Dca~c=1.109 g cm-3; needles, sealed in Lindemann capillaries; crystal dimensions: 0.28 x 0.30 x 0.32 mm 3, #(MoK~) = 0.599 cm-1. Data collection: Data for unit cell determination and for the structural studies were collected utilizing an EnrafNonius CAD-4 diffractometer. A total of 2927 reflections was measured at room temp. with graphite-monochromated MoK~ radiation up to ® =27 °, using an co- 20 scan mode with co- scan width=0.70+0.35tan® and co scan specd=l.ldegmin -1. After data reduction, 1870 reflections with I > 23 (I) were taken as observed. Structure analysis and refinement: The structure was solved by direct methods (MULTAN 11/82) [18], hydrogen atoms were obtained from a difference Fourier map. The subsequent full-matrix least-squares refinement with anisotropic thermal parameters for all non-hydrogen atoms and isotropic thermal parameters for the hydrogen atoms led to R = 0.045 and R,, = 0.052 for 455 parameters. The final difference Fourier map showed no significant features. Scattering factors are taken from ref. [19]. All crystallographic calculations were performed using the Enraf-Nonius SDP package [20]*. Acetate of 3fl-hydroxy-28-norolean*l 2-ene (3a). EIMS m/z (rel. int.): 454 [M] ÷ (2.8),439 (0.6), 379 (0.3), 354 (0.3), 257 (0.5),243 (1), 204 (100), 189 (25), 175 (8), 161 (3), 148 (4), 133 (5), 119 (7), I05 (9), 95 (9), 81 (12), 69 (17), 55 (12), 43 (58). 3-Oxo-28-norlup-20(29)-ene (4). Mp: 160-161 ° (Et20); [ct]57s: +69.7 ° (CHC13; c0.8); EIMS m/z (rel. int.): 410 [M] ÷ (4), 395 (0.1), 367 (4), 218 (6), 205 (18), 191 (28), 175 (26), 161 (17), 147 (23), 135 (51), 121 (46), 107 (66), 55 (100). IH NMR: 60.89 (3H, s, H-27), 0.93 (3H, s, H-25), 0.98 (3H, s, H-26), 1.02 (3H, s, H-24), 1.07 (3H, s, H-23), 1.68 (3H, s, H-30), 1.2-2.0 (m),2.37-2.55 (3H, m, H-19, H2), 4.6/4.67 (2H, d, H-29); laCNMR: 6218.4 (s, C-3), 150.8 (s, C20), 108.1 (t, C-29), 54.9 (d, C-5), 52.5 (d, C-19), 50.6 (d, C-9), 47.3 (s, C-4), 44.9 (d, C- 17), 41.0 (s, C-14), 40.5 (s, C-8), 39.7 (t, C-l), 39.7 (d, C-13), 37.7 (d, C-18), 36.9 (s, C-10), 34.1 (t, C-2), 33.0 (t, C-7), 30.2 (t, C-21), 30.1 (t, C-16), 26.8 (q, C-23), 26.7 (t, C-12), 26.4 (t, C15), 23.2 (t, C-22), 21.9 (t, C-11), 20.9 (q, C-24), 20.7 (q, C-30), 19.6 (t, C-6), 16.3 (q, C-25), 15.4 (q, C-26), 14.2 (q, C-27).

*Tables of final positional and thermal parameters, bond distances and angles, torsional angles and observed and calculated structure factors are available from the Cambridge Crystallographic Centre, University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, U.K.

Ozonolysis of the 3fl-hydroxymalabarica-14(26),17E,21-triene (6) gave after oxidative work-up and esterification the dioxoester (7) in 4.3% yield: oil; [ct]sTs: +62.5 ° (CHCI3; c0.3); EIMS m/z (rel. int.): 344 [M - H20 ] + (0.1), 331 (0.2), 220 (0.3), 205 (10), 187 (1), 143 (100), 111 (83), 55 (40); CIMS (pos.) m/z 363 [ M + I ] + ; 1H NMR: 60.91 (3H, s, H-20), 1.0 (3H, s, H-19), 1.03 (3H, s,H-18), 1.04 (3H, s, H-21), 3.65 (3H, s, H-22), 2.32-2.66 (4H, m), 2.66-2.76 (2H, at); 13C NMR: 6 217.4 (s, C-3), 212.8 (s, C-14), 173.4 (s, C-17), 61.2 (d, C-13), 55.9 (d, C-9), 54.5 (d, C-5), 51.8 (q, C-22), 47.2 (s, C4), 46.3 (s, C-8), 40.3 (t, C-15), 38.7 (t, C-l), 36.2 (s, C-10), 35.9 (t, C7), 33.9 (t, C-2), 27.7 (t, C-16), 26.7 (q, C-18), 24.0 (t, C-12), 23.8 (q, C-21), 21.2 (t, C-11), 20.9 (q, C-19), 20.4 (t, C-6), 15.4 (q, C-20). Acknowledgements--The authors wish to thank Drs H. R6ttele (Karlsruhe) and H. Schmickler (Cologne) for running the NMR spectra and Dr P. G. Gfilz (Cologne) for the generous supply of triterpenoid standards. Financial support by the Deutsche Forschungsgemeinschaft, Bad-Godesherg, and the Fonds der Chemischen Industrie, Frankfurt, is gratefully acknowledged.

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