Stress metabolites of Solanum mel~ngena:'?~ biosynthetic studies and isolation of auberganol and a- and P-eudesmol

ALBERT STOESSL AND J.B. STOTHERS London Research Centre, Agriculture Canada and the Department of Chemistry, University of Western Ontario,

London, Ont. Canada N6A 5B7

Received May 8, 1985

ALBERT STOESSL and J . B. STOTHERS. Can. J . Chem. 64, 1 (1986). Attempts to clarify the biosynthetic origin of the eggplant stress metabolite aubergenone (1) by incorporation of label from

sodium [2-'H3, I-13C]acetate were frustrated by low yields and enrichment levels. However, the presence of deuterium in the biosynthetically significant 5-position was demonstrated by 'Hmr for the closely related auberganol(5) and a- and P-eudesmol (10 and l l ) , suggesting that these compounds, and hence also 1 , are normal eudesmanes and not the products of a possible double rearrangement. Deuterium was also incorporated into the expected positions of 9-oxonerolidol(12), 9-oxynerolidol (13), and lubirnin (4). Incorporation of label from [ l ,2-13C2]acetate could be determined satisfactorily only for 13 and its allylic isomer 14, corresponding with expectations. This is the first report of 5 as a natural product and of 10,11, and the 10-epimer of 4 as eggplant stress metabolites.

ALBERT STOESSL et J . B. STOTHERS. Can. J . Chem. 64, 1 (1986). Dans le but de determiner l'origine biosynthetique de l'auberginone (I), un metabolite de stress de I'aubergine, on a effectue

des essais d'incorporation de l ' ac~ ta te - [ '~~-2 , '~C- l ] de sodium qui n'ont pas reussi i cause des faibles rendements et des faibles niveaux d'enrichissement. Toutefois, on a pu demontrer la presence de deuterium dans la position 5, qui est significative d'un point de vue biosynthetique, en faisant appel a la rmn du 'H de l'auberganol(5) et des a- et P-eudesmol(10 et 11) qui lui sont trks apparentes; ces resultats sugg6rent que ces composts et, par consequent, le compost 1 sont des eudesmanes normaux et non pas des produits provenant d'une double transposition. I1 y a aussi incorporation de deuterium dans les positions prCvues du 0x0-9 nCrolidol(12), du 0x0-9 nCrolidol(13) et de la lubimine (4). Ce n'est qu'avec le compose 13 et son isomhe allylique 14 que I'on a pu determiner d'une f a ~ o n satisfaisante I'incorporation d'un marqueur de I'a~Ctate-['~C~-l,2] qui corresponde aux expectatives. Les rksultats obtenus dans ce travail correspondent i une preiniere concernant l'existence du compose 5 comme produit nature1 et des produits 10, 11 et de 1'Cpimkre en position 10 du compost 4 comme metabolites de stress de l'aubergine.

[Traduit par le journal]

Aubergenone ( I ) , a metabolite of Solanum melongena, has until now been unique among the carbocyclic stress metabolites of the Solanaceae in that its structure is that of a normal eudesmane whereas the many other compounds of the group are derived from hypothetical eudesmane precursors by carbon and hydrogen migrations (1, 2). However, it has been shown recently (3) that the biosynthesis of rishitin (2), a member of the group, involves a remarkable second rearrangement of the vetispirane, hydroxylubimin (3) (Scheme 1). The possibility therefore arises that 1 may also be formed through a similar sequence. In principle, this question might be answered through the biosynthesis of 1 with [2-2H3,1-'3C]acetate incorporation. If a vetispirane is an intermediate, its formation may be accom- panied by hydrogen (deuterium) migration from C-5 to C-4, as has been unambiguously demonstrated for 2 , 3 , and lubimin (4) (4). In the present case this could give rise to an observable signal in an uncluttered region of the 2 ~ r n r spectrum provided, of course, that the incorporation level is sufficiently high and that the C-4 hydrogen (deuterium) does not itself participate in the hypothetical second rearrangement. On the other hand, if 1 arises from an acyclic precursor without carbon-carbon rear- rangement, the salient deuterium atom will remain bonded to C-5 and, therefore, be observable either directly or through the P-isotope shift in the C-6 absorption pattern.

To test these notions, eggplant fruit was treated with a sus- pension of Monolinia fructicola, as stress compound inducer, and, after 18h, with [2-2~3,1- '3~]aceta te (1.86 X 1 O p 3 ~ effective concentration). A small amount of 1 was isolated after

'part 121 of 13Cmr studies; for Part 120 see ref. 12. 'Communication No. 1010 from the Research Centre; Part 47 of

"Postinfectional inhibitors from plants"; for Part 46 see ref. 13; for Part 45 see ref. 14; for Part 44 see ref. 15.

2 days but the incorporation level was so low that only the deuterium signals for the methyl groups were observed in the 2 ~ m r spectrum. The 13C spectrum was similarly uninformative, revealing neither signal enhancements nor P-shifts for C-2, -4, -6, -8, -10, or -1 1. Fortunately, the situation was saved in part by the isolation of a new compound that was identified as a tetrahydroaubergenone, auberganol (S), by its 'H, 13Cmr, and mass spectra. Although this compound was not previously

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2 CAN. 1. CHEM.

known from eggplant or other natural sources, it has been described as one of the intermediates in the laboratory synthesis of 1 from a-cyperone (5). The ' ~ m r and ms data for the eggplant metabolite are in excellent agreement with the litera- ture values and the mp is satisfactorily close. To confirm the identity of 5, however, the compound was oxidized to 6, which was completely transformed to 7 on refluxing with ethanolic potassium hydroxide. Ketol 7 was obtained earlier as an inter- mediate in the synthesis of 4-epi-aubergenone (6). A sample of the latter compound3 was hydrogenated to furnish authentic 7, which was found to be identical to that obtained from 5. As an aid for the assignment of the 13Cmr spectrum of 5, a portion of ketol 7 was reduced with sodium borohydride to furnish a mixture of two diols 8 and 9. The major product 8 was isolated by fractional crystallization and exhibited the properties reported earlier for 8 as a precursor of 7 in the aforementioned synthesis of 4-epi-aubergenone (6). The minor diol9 was therefore the 3 a isomer. The 13c data for 5-9 are collected in Table 1.

As in the case of 1, the incorporation levels in 5 were too low to be detected by 13cmr. On the other hand, however, the 'Hmr spectrum clearly showed the presence of deuterium in the methyl groups, with well-defined signals at 6 0.84, 0.86, and 1.18 for the 4-, lo-, and 1 1-methyl groups, respectively ('H enrichment ca. 0.5%), and for some of the skeletal positions (broad absorptions ca. 6 1.05, 1.25, 1.4, 1.6). Most signifi- cantly, a sharp signal at 6 3.70 established the presence of deuterium at C-3 (enrichment ca. 0.15%) and the absence of absorption near 6 1.88 showed there was little or no deuterium at C-4. The absorption positions for the 3- and 4-deuterons were known from analysis of the proton spectrum in which irradiation at 6 1.88 collapsed the 4-methyl doublet, 6 0.84, to a singlet and eliminated a 5.5-Hz coupling in the carbinyl pattern at 6 3.70, thereby reducing the latter to a doublet of doublets, J = 5.5, 10.5 Hz. Some alteration in the spectrum near 6 1.25 indicated this to be the position of the roton, on, thereby tentatively identifying an additional 'H signal. From these observations it can be concluded that 5 was probably formed without deuterium migration from C-5 to C-4.4 A definitive conclusion follows from the presence of deuterium at C-3, since this establishes that 5 is not fomed by reduction of 1 but, rather, is either in a precursor or shunt relationship to 1.

Another crystalline substance, m.p. 81-83"C, was isolated from the product and from its I3C spectrum, found to be a 2:3 mixture of a- and P-eudesmols, 10 and 11, respectively. These could not be separated by either column or preparative thin- layer chromatography but corresponded in all respects to the like mixture of the two compounds obtained by Cordano et al. (7) from Balanites roxburghii. The 13cmr spectrum of our product once again showed no discernible enrichment or P- shifts; the data are listed in Table 1. The 'Hmr spectrum contained distinctive methyl signals at 6 0.71, 0.75, and 1.18 arising from the 4-, lo-, and 1 1-methyl groups, respectively, with broadened skeletal deuterium signals centred at 6 1.36,

3~rofessor R. B. Kelly kindly provided a generous sample of 4-epl- aubergenone.

4An unambiguous conclusion on this point is elusive because of the possibility of two consecutive deuterium migrations, i.e. an eudes- mane -+ a vetispirane -+ an eudesmane with C-5 + C-4 -+ C-5 deuterium migration, although it is known that the second migration does not occur in the rearrangement of 3 -+ 2 (4). It would appear that the only approach to a solution of this question would involve testing of suitable vetispiranes for incorporation into 1, experiments which are beyond the scope of the present study.

1.44,1.50,1.60, and 2.30. The 6 2.30 signal corresponded to a multiplet in the proton spectrum containing one large coupling, -12 Hz, which could be attributed to the 5-proton.

To evaluate the situation more fully, the major eggplant phytoalexins lubimin (4), 9-oxonerolidol (12), and 9-oxynero- lido1 (13) were also isolated for examination of their spectra. The 'Hmr spectrum of the lubimin sample revealed the presence of ca. 10% of the 10-epi isomer which had been found previously in potato (8) but not eggplant. The 'H spectrum of this sample was essentially the same as that recorded earlier for 4 isolated in the course of analogous incorporation experiments with potatoes (4), exhibiting readily assigned signals for deute- rium at the aldehydic (6 9.78), exo-methylene (6 4.65), allylic (6 1.60), and secondary rnethyl(6 0.93) positions. In addition, broad signals centred at 6 1 .O, 1.4, 1.8, and 2.3 arising from skeletal deuterons were evident, that at 1.4 corresponding to I - ~ H and (or) 4-'H. The 'Hrnr spectrum of 12 contained prominent signals for each of the four methyl groups, 6 1.20, 1.50, 1.80, and 2.05, with that at 6 1.50 superimposed on absorption for skeletal methylene deuterons. In addition, a signal at 6 3.00 arising from the 8-methylene deuterons and weaker signals at 6 5.25,5.95, and 6.10 confirmed the presence of deuterium at C-2, -6, and -10. These assignments followed from the proton spectra obtained in an earlier study (9) and reported previously by Hiroi and Takaoka (10). The major metabolite isolated in the experiment was 9-oxynerolidol (13) for which the 'H spectrum readily revealed incorporation at the four methyl sites, 6 1.20 and 1.54, the latter signal arising from the three unresolved allylic methyl groups and more than three times as intense as the 6 1.20 signal. In addition, methylene absorption at 6 2.05 and olefinic signals at 6 5.95 and 5.22 were apparent. The lack of absorption in the range 6 2.1-5.1 confirmed the absence of incorporation at C-9. The 13c spec- trum of 13 revealed the presence of two diastereomers, as found previously (9, 10) in a ratio of ca. 2: 1. In contrast to our earlier report, slightly different shieldings were found for 12 of the 15 carbons owing to the increased shift dispersion produced by the 3-fold increase of the observing frequency (300 vs. 100 MHz). The data are listed in Table 1. It may be noted that the original assignments for C-6 and -10 have been reversed (see below). ~hekffects of deuterium incorporation were just apparent in the patterns observed for each of the enriched centres, C- 1, -3, -5, -7, -9, and -1 1, with distinctive P-isotope shifts for C-1 (A =

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STOESSL AND STOTHERS

137 ppb), C-5 (1 10 ppb), C-7 (55 ppb), and C-9 (64 ppb) while the C-3 and - 1 1 signals were broadened.

In a preliminary experiment, similarly inoculated aubergines were treated with sodium [I ,2-13C2]acetate. Although auberge- none (1) was present in trace amounts, insufficient material could be isolated for spectroscopic examination. However, 13 and its allylic isomer 14 were isolated from tissue extracts by chromatography and their I3C spectra recorded to determine the pattern of intact acetate units in each compound as indicated by the bold linkages in 13 and 14. For each unit, the I3C-l3c coupling constants could be measured and these data are in- cluded in Table 1. The relative intensities of the 13c satellites and the central signal for the coupled carbons indicated that the 13C enrichments were low, 0.25 and 0.15 ? 0.05% for 13 and 14, respectively. The very low incorporation levels and the minimal yield of 1 dictated a change in tactics, leading to the experiment with [ 2 - ' ~ ~ , 1-13c]acetate described above.

Auberganol(5) and the eudesmols 8 and 9 were not present in readily detectable amounts in extracts from uninoculated auber- g ine~ . The experiments described above suggest that these, together with aubergenone (I), are normal, i.e. non-rearranged eudesmanoids. No eudesmanes have as yet been reported as stress metabolites of other species of the Solanaceae. It is at present unknown whether auberganol is antifungally active but at least one report describes (+)-P-eudesmol, isolated from Hymenoclea monogyra, as active against Alternaria spp. (1 1).

Experimental General experimental procedures and instrumentation utilized were

as described in earlier papers (8,9) unless otherwise indicated. The 13C and ' ~ m r spectra were recorded with a Varian XL-300 system using CDC13 (13C) and CHCl, ('H) solutions in 5-mm tubes.

Aubergines (imported from Florida) were purchased in the local market in November and were of unknown storage age and variety. The fruits (25) were cut longitudinally into equal halves, seeds and pith were scooped out, and the resulting shells were charged with a suspension of Monilinia fructicola (2 x lo5 spores/mL, 40 mL/shell) and, after 22 h, with sodium [2- '~~, l - '~C]acetate (Merck Isotopes Inc., 98% 'H, 90% I3C, 8 mg (0.093 mmol in 10 mL of H20/shell). Two days after inoculation, the liquid diffusate was collected, com- bined with water rinsings, and extracted with ether. Necrotic tissue was scraped from the shells and extracted by steeping overnight in MeOH/EtOAc (1:1), concentrating the filtered extract until mainly aqueous, and reextracting with ether. On column chromatography over Si02 (Camag DFO; 300 g) in sec-BuOHIEtOAc (5:95), the diffusate extract afforded fractions (20 mL each) that consisted predominantly of 9-oxonerolidol(l2) together with 10 and 11 (fractions 26-28,53 mg), 9-oxynerolidol (13) and its ally1 isomer 14 (fractions 29-34, 140 mg), a mixture of 4 and 1 (fractions 38-44, 41 mg), and crude crystalline 5 (fractions 50-61; 14.9 mg).

Rechromatography of fractions 26-28 gave 9-oxonerolidol (12) (18 mg, no discernible enhancement or shifts in the I3c spectrum) and compounds 10 and 11 as a crystalline mixture (13.5 mg); mp 81-83°C after two recrystallizations (light petrol, - 10°C); 'Hmr (CDC13) 6 (10): 0.75 (s, 3H, 10-Me), 1.18 (s, 6H, isopropyl Me's), 1.60 (bs, 3H, 4-Me), 5.30 (m, lH, H-3); 6 (11): 0.66 (s, 3H, 10-Me), 1.18 (s, 6H, isopropyl Me's), 4.42 (m, lH), and 4.70 (m, 1H) exo-methylene protons; I3Cm (see Table 1); ' ~ m r indicated low enrichment of ca. 0.016 atoms D/molecule; m/e: 222, 204, 189, 164, 161, 149, 93, and 59 (100%). Exact Mass calcd. for CI5Hz60: 222.19835; found: 222.19805.

9-Oxynerolidol(l3) was obtained nearly pure (97 mg) by rechroma- tography of fractions 29-34 (MeOH/CHC13 5:95). The small amount of its allylic isomer that was also obtained was not further examined.

Aubergenone (1) (5 mg) was obtained from fractions 38-44 by column chromatography (MeOH/CHC13 1:99) followed by multiple

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4 CAN. J. CHEM. VOL. 64, 1986

preparative tlc (high performance precoated plates, Whatman Multi-K type CS5KC18F/K5F; EtOAc/toluene/HOAc 30:60: 1) and was com- bined for spectroscopy with another 3.2 mg similarly obtained from the tissue extract.

Lubimin (15 mg) was obtained in the above purification of 1. It consisted of 4 accompanied by a small amount of its 10-epimer as judged from the 'Hmr spectrum.

Auberganol(5) was purified by recrystallization from EtOAc (5 mg), mp 155-158°C; after a further recrystallization (EtOAc/hexane) it had mp 173.0-173.5"C (lit. (5) mp 166-166S°C); ir (CHC13): 3605 (s), 3460 (br), 2935,2870,2855,1450 (d), 1382,1370,1130,1080,1008, 903 cm-'; too little sample was available for determination of the small rotation (lit. (5). [&ID -2.7'); l ~ m r (CDC13) 6: 0.86 (s, 3H, 10-Me), 0.84 (d, 3H, J = 7.4 Hz, 4-Me), 1.18 (s, 6H, isopropyl Me's), 1.92 (m, lH, H-4), 3.71 (dt, J = 10.4 and 5.5 Hz, lH, H-3); I3Cmr (see Table 1). Exact Mass calcd for CISH280: 240.20891 ; found: 240.20879.

The very complex tissue extract was used only for the isolation of 1 by similar chromatographic techniques.

In another experiment, similarly inoculated aubergines (20) were treated with sodium [ l ,2-I3C2]acetate (1.68 mg) in 1 mL of H20 added to 10 mL of spore suspension/shell. Aubergenone (1) was present only in trace amounts and could not be studied. 9-Oxynerolidol(13) (15 mg) and its isomer 14 (16 mg) were isolated from the tissue extract by chromatography over Si02 in EtOAc/CH2C12 1:4.

Reduction-epirnerization of 5 Auberganol (2.5 mg) in acetone ( I d ) at 0°C was treated at

increasingly long intervals with small drops of the Jones' reagent (59 mg Cr03, 2 mL H2SO4, H20 to 15 mL) until a yellow colour persisted for 30 min. Excess reagent was destroyed with MeOH (few drops) and the solution extracted with Et20 (2 X 10 mL); the extract was washed with 5% Na2C03 (2 mL) and water (2 x 4mL) and evaporated to give 6 (1.9 rng), homogeneous by tlc and 'H and I3crnr spectra. This product was refluxed in 0.1 M KOH in EtOH (2 mL) at 74°C for 1 h, poured into H20 (10 mL), acidified (2 M HCl, 0.11 mL), and extracted into CHC13 (2 x 5 mL) to give 7 as a syrup (-1 mg) identical (except for the presence of trace amounts of different impuri- ties) with the sample prepared from synthetic 4-epi-aubergenone by tlc, ir, 'H, and 13Cmr spectra.

Reduction of 4-epi-aubergenone The synthetic ketol (6) (15 mg) was hydrogenated over 5% Pd/C

(25 mg, prereduced) in EtOH (15 mL) for 6 h. Essentially pure trans- dihydrocarissone (7) was obtained as a syrup on evaporation of the filtered solution.

4-epi -Auberganols Synthetic 7 (12 mg) in MeOH (2 mL) was reduced with NaBH4

(few grains) at 0°C. Extraction (CHC13) from ice-water gave a mixture of diols 8 and 9 (I3Cmr, see Table 1) as a syrup (1 1.5 mg). Crystalliza- tion from CH2C12 at - 10°C gave 8 (3.2 mg), mp 136-140°C, raised to 138-140°C by a further crystallization; I ~ m r (CDC13) 6: 0.83 (s, 3H, 10-Me), 0.97 (d, 3H, J = 6.8Hz, 4-Me), 1.19 (s, 6H, (CH3)2C(OH)-), 3.14 (ddd, lH, J = 5.0, 9.8, 11 .O HZ, H-3).

Acknowledgements Thanks are due to G. Rock for general experimental assis-

tance, S. Wilson and D. Hairsine for recording 'Hmr and mass spectra, respectively; and to the Natural Sciences and Engineer- ing Research Council of Canada for financial support. We thank Professor R. B. Kelly for the sample of 4-epi-aubergenone.

1. J. B. STOTHERS. Pure Appl. Chem. 53, 1241 (1981). 2. A. STOESSL. In Phytoalexins. Edited by J. A. Bailey and J. W.

Mansfield, Blackie, Glasgow and London. 1982. p. 133. 3. A. MURAI, S. SATO, A. OSADA, N. KATSUI, and T. MASAMUNE.

J. Chem. Soc. Chem. Commun. 32 (1982). 4. A. STOESSL and J. B. STOTHERS. Can. J. Chem. 61,1766 (1983).

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