Limonoids from Australian members of the meliaceae

P~~~oc~e~~s~r~, Vol. 31, No. 12, pp. 4163-4166,1992 Printed in Great Britain.

LIMONOIDS

0031-9422/92$5.~+0.~ 0 I992 Pergamon Press Ltd

FROM AUSTRALIAN MELIACEAE

MEMBERS OF THE

DULCIE A. MULHOLLANDand DAVID A. H. TAYLOR Department of Chemistry, University of NataI, King George V Avenue, Durban 4001, South Africa (Received27 April 1992)

Key Word Index- -Owe&asp., Toona austra’atis; Xylocarpusmoluccensis;Meliaceae;glabretal; cedrelone; Iimonoids; chemotaxonomy.

Abstract-The seeds of Owenia acidula and of Oweniu uenosa were found to contain a simple limonoid and a derivative of the cyciopropane protolimonoid giabretal, which has also been found in the timber of Aglain ferruginea. The timber of lbona australis was found to contain cedrelone, and the timber and seed of X~l~~rp~ rn~l~ccens~ (sensu Mab~rley) have been found to contain a rich mixture of limonoids. The chemotaxonomic significance of these results is discussed.

INTRODUCTION

We have reported in a series of papers on the chemistry and chemotaxonomy of African members of the family Meliaceae, and more recently on the Madagascan members. We have now examined some of the Australian members of this family. Eleven genera and 33 species of the Meliaceae occur in Australia, of which the genera Owenia and Synoum do not occur elsewhere. We have collected and examined species of all genera except Anthocarapa and Chisocheton. Herbarium specimens are preserved in the Forest Herbarium, Oxford. An Indian species of Chisocheton has been examined before Cl], and was a rich source of limonoids. Previously, of the Australian species of the family, only representatives of Dysoxylon and Toona had been examined. All three species of the genus Xylocurpus are found in Australia, and in view of the taxonomic problems associated with this genus, these were made the object of a special study. The genus Xylocu~~~s consists of trees growing around the littoral of the tropical Indian Ocean, and extending to the Pacific Islands. It has a rather confused history, and misidentification of specimens, which are not always easy to identify in the herbarium, especially in the absence of adequate collector’s notes, has been common. According to a recent revision by Mabberley [Z], there are three rather well defined species, and it was the purpose of this investigation to see how far the chemistry of the genus followed this classification. All three species are familiar to the senior author, in East Africa, in Australia, orboth.

of the characteristic mahogany type. It occurs in mangrove swamps on the ocean coast, and in the estuaries of tidal rivers, and ranges from East Africa to the Pacific Islands, appearing quite the same in East Africa and in Queensland, where it extends as far south as Cairns. The fruit is grapefruit sized, hard and heavy, leading to the common name of the ‘cannon ball tree’. This is identified as X. greats Koenig. Species B is a smaller, less branched mangrove, with pointed leaves, deeply serrated bark, and an undistinguished timber. It is confined to the Eastern part of the area, and extends further south than either of the other species, reaching south of Townsville in Queensland. The fruit is the size of a manda~n orange. This has been known as X. australiacus in Australia, but according to Mabberley is correctly named as X. ~l~c~ensis Lam. ex Roehm. Species C is very similar to this, and not easily distinguished in the herbarium. Ecologically, it is quite distinct, as it is not a mangrove, growing instead on sandy beaches, or in estuaries above the high tide mark. It extends from East Africa to the Pacific, but has a more tropical distribution than the other species, being found in Australia only on the north coast or on the off-shore islands. It has, confusingly, usually been called X. mollucensis Lam. ex Roehm, and its chemistry has been described under that name [3]. According to Mabberley, it is correctly X. ~mphii (Kostel.) Mabb. This view has more recently been challenged [4].

Chemistry

RESULTSANDDISCUSSION morphology

Species A is a large spreading mangrove, with rounded coriaceous leaves, smooth thin bark, and an abundant red heartwood, which furnishes a useful, if rather hard, timber

From the biosynthetic point of view, X~lo~~r~s is an interesting genus, the most characteristic products being the xyloccensins, isolated from X. ~rn~hii [3, 5-j. These are mexicanolide derivatives with a hemi-ketal bridge, not yet found elsewhere, which are the only compounds so far obtained intermediate in complexity between the common mexicanolide and phra~alin groups. [Sj.

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D. A. MULHOLLANDand D. A. H. TAYLOR

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Extraction of the timber of X. ~~~~ufu~ has given gedunin (I), [6], the seed of this species gave mainly d~ti~oyl-6”d~oxyswiete~ne acetate (2) and xylocarpin (3), with small amounts of gedunin and an unidentified compound [7]. Extraction of timber or seed of X. ra~@i [3, 51 has given the xyloccensins, phragmahn, and methyl angolensate in varying proportions. The xyloccensins possess the hemi-ketal structure (4), they differ in the presence or absence of a 2-hydroxy group and a 14,15 double bond, and in the este~fying acids. As usual with limonoids, these are most often isobutyric, 2-methylbutyric or acetic, these mixtures can be difficult and unrewarding to separate. Further work [5] has given additional, retated compounds of possible biosynthetic interest, but present in too small quantity to be usefuf taxonomic markers.

Xylocarpus molluccensis (sensu Mabberly) has not previously been examined. Samples were collected at Townsv~le, at Cairns, and north of Cairns; specimens are deposited in Oxford. Extraction of the timber gave destigloyl-6-deoxyswietenine acetate (2) as a crystalline solid in good yield, the main additional constituent was angustidienolide (5), [8], isolated by prep. TLC. Chromatography of the residue gave phragmalin 3,30_diacetate, phra~a~in 2,3,3~~tria~tate and minor unidentified compounds. The seed extract contained a complex mixture of limonoids, the main constituent, isolated by prep. TLC was the 2-hydroxy derivative (6) of 2. This has been isolated several times before, e.g. from Khaya nyasica (D. A. H. Taylor, unpublished results) and from Swietenia humilis [9], but has not previously been described. Chromatography of the residue gave 7-0x0-7-

Limonoids of Meliaceae

deacetoxygedunin f?), humilin B (g), [9, lo]? a new limonoid, and some unidentified fractions. These were not xylo~~nsins, as they did not show the ~hara~te~sti~ ketat structure in the r 3C NMR spectrum. The new hmonoid, compound B3, C,sH,,O, r, showed ‘HNMR signals due to the usual furan ring, a methyl ester, an acetate, and an isobutyrate. It also showed a carbonyl group at the unusual position of S, 198.2, su~~ting a fury1 carbonyl group, as in pseudrelone B [lt]. However, there was not a second carbomethoxy group, as in pseudreione B, and a lactone was still present, suggesting that the normal C-16, C-17 lactone had opened and recyclised on to C-30. A ditertiary double bond was present, the location of this at C-14, C-15 was confirmed by the coupling of C-15 (53.9) with H-9, (62.8). There was no normaI, saturated carbonyl resonance, hence C-l had reacted in some way. The only known ~mbinations that C-1 enters into are to form hemiketals, which was not the case here as there was no hemiketal carbon resonance, or to take part in the formation of a bridged ring A as in phragmalln, in which case C-l bears a tertiary hydroxyl group. That this had happened was suggested by the fact that the spectrum of B3 showed only three nuclear methyl groups, instead of the four in mex~~nolide, and confirmed by the presence of an isotated methylene group (8, 41.6, 5n2.22, 1.96, J= 18 Hz), representing the ring A bridge, a tertiary carbon at 6o90.4 then represented C-l. Use of COSY and HETCOR enabled the assignment of all the hydrogen and most of the carbon resonances, these are listed in the Experimentat section. We therefore propose the structure 9 for compound B3, the ester groups being assigned by analogy with known, related compounds. This structure is a very interesting one, as it is the first mexi~nolide derivative to have a bridged A ring without hydroxyl substitution at C-8, C-9; its existence would seem to effectively demohsh the theory of phra~alin biosynthesis proposed in ref. [S]. These results suggest that although the three species are very similar, they can be distinguished chemically by examination of the timber, X. gra~t~rn and X. mo~~ccensis (sensu Mabberley) giving crystalline gedunin or destigloyl-6-deoxyswiet~ine acetate, if in sufBcient amount, and X. ~rn~~i~ giving a non~ryst~line mixture of xyi~ns~s, idenii~able by TLC with reference samples, or by the appearance of a ketaf carbon in the 13CNMR spectrum of purified fractions. However, a recent report [t2] describes the isolation of xyloccensins from the seed of Fijian samples of X. grumztum and X. moluccensis tpresumabiy X. ~rn~~i~ (Kostel.) Mabb.], suggesting that the range of variation ofX. ~ru~uturn may be wider than suspected. Further examina~on of this point seems to be desirable, Toona austrulis (F. Muell.) Harms is a valuabie Australian timber tree, it is considered to be conspecitic with T. ciiriuta Roxburgh. The timber of T. ciliata is well known chemically as the source of cedrelone [13], while the leaves of T. austrulb are the source of toona~~in [f4], but corresponding parts of the two trees have never been examined. Examination of a timber specimen provided by the Forestry Department at Atherton gave 0.03% of cedrelone, thus supporting the identity of the two taxa. Specimens of Owenia acid&a F. Muell. and 0. uenosa F. Muell. were collected near Rockhampton. Extraction of the timber of both gave a series of compounds. The mass spectrum of one fraction suggested that it was the tride-

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canoic acid ester of an un~dent~~ed monohydroxy triterpene alcohol, Investigation of these extracts was not pursued. The seeds of Owenia are notoriously difficult to remove from the fruit, and also difficult to germinate. Fruit was collected with the assistance of Mr Neil Hoy of Rockhampton, who specialises in growing the plant, and minced in a powerful machine. Extracts of the two species were so similar on TLC that they were combined, prep. TLC then gave 3-isobut~~~-7-dea~tylglabre~~ (IO), (0.4%), mp 195”, identified by comparison of the ‘H NMR and r3C NMR spectra with those of the corresponding acetate (D. A. Mulholland, unpublished results) and a much smaller amount of a new limonoid, 6x-acetoxydeoxyhavanensin (Il)r as an oil. The spectra of this mater&d showed four acetate groups in a limonoid nucleus with a Eve-membered unsaturated ring D. Two of the acetates were at C-l and C-3, since the adjacent protons were both coupled to the same two protons, and to nothing else, the couplings showed both were in the c+configuration. The two remaining acetates were vicinal, since the adjacent protons were directly coupled. One was only coupled to the other, the second was also coupled to one single, otherwise uncoupled proton. These could represent H-7 and H-6, or possibly H-12 and H-11, however not only in the latter case woutd ring B be unsubstituted, which is highly unlikely, but also the chemical shift of the vinyl H1.5is in agreement with the presence of a 7a-acetate. The stereoch~jstry at C-6 and C-7 follows from the coupling constants [15]. 6~-Substituted derivatives of havanensin are very common, but usually with the &oxygen atom cyclised on to the 4a-methyl group, the simple acetate has not previously been reported. Timber samples of Synoum ghdulosum (Sm.) A. Juss. and of V@uaeaamicomm Benth. gave no terpenoid material; seed was not obtained. Seeds of various Aglaia species examined previously have given no limonoids, however the timber of A.Sernrginea White and Francis has recently been found to contain 3-acetyl-7-deacetylglabretal [16]. Only the timber of Dysoxy~on was available; this has so far given no interesting extractives, although the seeds have previously furnished limonoids [ 17J.

The new evidence provided by this work is of the occurrenCe of glabretaI derivatives in Agiaia and in Owenia, and of 6u-acetoxydeoxyhavanensin acetate in Uwenia. It has also been discovered recently that giabretat derivatives occur in Turraea o~t~s~~j~ Hochst. [lg], while the original isolation was from Gtlarea &bra [19].

D.

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A. M~ILH~LLAND and D. A. H. TAYLOR

As pointed out by the original authors, glabretal is a curious and unexpected by-product of limonoid metabolism, and these new occurrences therefore suggest either that giabretal derivatives are widespread and have previously escaped notice, or that Aglain, &area, Owe& and Turraea are closely related. In the classification of Pennington and Styles [203, these four genera are placed in four different tribes of the subfamily Meliodeae. However, in the recent intensive search for limonoids in Melia and Azadirachta it seems unlikely that a substantial amount of glabretal, if present, would have been overlooked, whereas in the fruit of Uwenia it is present in considerable amount. Further work is required before any conclusions can be drawn. 6~-A~toxyhavanensin, on the other hand, is of a very common type and oxidation pattern, related compounds are well known in Melia and other genera. However, these are rarely found alone, but are further metabolised into more complex limonoids. The nearest known analogues in which oxidation stops at a similar stage are found in Turraea and Trichilia, of which the latter alone is placed in the same tribe as ~~~en~~, EXPERIMENTAL

Timber of X. moluccensia (sensu Mabberley) (2 kg) was ground and extracted with refluxing hexane. The extract deposited a powder (5 g) which crystallised from MeOH to give destigloyl-6deoxyswjetenine actetate (2) (2 g), identical with previous samples. The mother liquor was coned and a portion (0.36 g) sepd by prep. TLC giving further 2, angustidienolide (S), and phragmalin di- and triacetates, a11identical with previous samples. The seed, collected at Townsville, (1 I4 8) was minced and extracted with refluxing hexane, giving a powder (4 g) which did not crystal&se. A sample (0.4 g) sepd by prep. TLC gave 2hydroxydestigloyi-6-deoxyswietenine acetate (6) as a crystalline solid, mp 250-252” (MeOH).[a]p - 10.5: “CNMR: 6215.0 s, 173.8s, 171.0s, 169.5.9, 143.1 d, 141.9d, 136.2s, 129Sd, 120.3s, llO.Od, 85.1 d, 77.4d, 56Sd, 52.4q, 49.5 s, 45.0d.41.8d, 39.1 s, 36.6 s, 34.3 t, 32.8 t: 30.1 t, 29.8 t, 22.1 q, 22.Oq, 20.6 q. 20.3 q, 19.8 q, 15.8 q. ‘H NMR: 6 7.8 m, 7.43 m, 6.48 m, 5.71 s, 5.38 m, 4.88 s, 3.72, 2.14, 1.25, 1.09, 0.82, 0.75. (all 3Hs). Chromatography of the remainder also gave 7-0x0-7deacetylgedunin (7), 2-hydroxyfissinolide, and a new limonoid, B3, not obtained crystalline (9). 13C NMR: 6198.2 (C-17), 176.6 (iPrCO), 173.4 (C-7). 169.5 (MeCO). 168.9 (C-16), 145.8 CC-8 (14)3, 143.3 (C-23), 141.7 (C-21), 130.7 cc-14 (811, 120.0 (C-20), 109.7 (C-22), 90.4 [C-l (2)], 89.5 (C-3), 80.2 (C-30), 80.0 [C-2(1)], 52.0 (OMe), 47.7 s, 44.5 (C-9), 43.4 s, 41.6 (C-29),39.0 (C-5), 38.9 s,35.2(C-15),33.9(hife,~CH~CO),33.8(C-6),30.3(C-12),20.8q, 19.7(C-11). 19.Oq,18.9q, 18.8q,17.8q, 14.6q. ‘HNMR:S7.5(H21), 7.44 (H-23), 6.45 (H-22), 5.5 (H-30), 5.09 (H-3), 4.49 (OH), 3.9 (Hz-15), 3.68 (OMe), 2.92 (H-5), 2.74 (H-9), 2.5 m (J =6.9X Hz, Me,CH.CO), 2.22 (H-6), 2.12 (J= 18 Hz, H-29a), 1.96 (OAc), 1.92(H-29b), 1,75(H,-II), 1.25(H,-12), 1.20, 1.10,0.87,3 xMe; 1.13, 1.12 (2d, J=6.98 Hz, Me,CH.CO). Fruit of Qwenia acidula (67 g) and of 0. ~enosa (50 g) were minced and extracted separately with refluxing hexane. The greenish extracts were partitioned between hexane and 90% aq. MeOH, the MeOH layer strongly diluted with H,O and extracted with CH,Cl,, Evaporation gave greenish gums (1.0,0.95 g, respectively). Each of these was subjected to prep. TLC; the fractions which seemed to be identical by TLC were then combined and further processed together. Further prep. TLC

gave 3-isobutyry~-7-deacetylg~abretal (460 mg), mp 195” (EtOAc). [a];’ -41” (N.B. in the spectra some peaks are doubled, due to the presence of both anomers at the ketal carbon). “C NMR: 6176.4 fiPrCO), 102.0(98.1) (C-21), 78.8 (77.2) (C-23), 78.8 (C-3); 74.2 (C-7); 67.7 (65.5) (C-24); 57.75 (57.0) (C-25); 49.3, (C-17), 44.8 (C-20), 44.2 (C-9), 41.2 (C-5), 39.0 (C-8), 37.3 (C-10); 37.3 (C-13); 36.9 (C-14k 36.2 (C-4); 33.9 (Me,CH.CO), 33.7 (Cl), 30.8 (29.6) (C-22), 29.1 (C-16), 27.6 q. 27.5 (C-15X 25.6 (C-12), 24.9 q, 24.3 (C-6), 22.7 (C-Z), 21.8 (I, 19.4 q, 19.1 q, 18.9 q, 18.8 q, 16.2 (C-11), 15.6 q, 13.8 (C-18). ‘HNMR: 6 5.46 (H-21), 4.65 (H3~,3.9(H-23),3.7S(H-7),2.86(2.71)(d,J=7.3Hz,H-24),2.55(m, 5=7 Hz, Me,CH,CO), 1.33, 1.31, 1.07, 0.91, 0.91, 0.88 (6 xCMe), 1.20 (d, J=7 Hz, Me, .CH.CO). A faster moving fraction gave 6a-acetoxyhavanensin acetate (11) as an oil. 13CNMR: 6170.3, 170.1, 170.1, 169.9 (4 x MeCO), 158.5 (C-14), 142.5 (C-23), 139.6 (C-21), 124.5 (C-20), 119.6 (C15), lll.O(C-22).77.3d,75.5d,72.3d,69.9d,51.2(C-17),47.1(C13), 42.5 (C-IO), 41.8 (C-8). 41.1 (C-5). 36.2 (C-4), 35.2 (C-9), 34.3 (C-16), 32.7 (C-12), 30.5 q, 27.2 q. 25.7 (C-2), 22.3 q, 21.6, 21.5, 21.2, 21.1, (4xMeCO), 20.1 q. 17.0 q, 15.8 (C-11). ‘HNMR: 67.37 (H-23), 7.23 (H-21), 6.26 (H-22), 5.53 (dd, J=2.5, 12.2 Hz, H-6), 5.35 (H-7, H-15), 4.68.4.58 (H-l, H-31, 2.61 (d, J= 12.2 Hz, H-5), 2.26 (H-2a), 2.05, 2.04, 2.03, 2.02 (4xOAc), 1.98 (H-2b), 1.28, 1.25, 1.10, 0.98, 0.78 (5 x Me). REFERENCFS

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