Echinulin, a novel cyclic dipeptide carrying a triprenylated indole moiety from an Anacardiaceae, a Cucurbitaceae and two Orchidaceae plants : detailed high resolution 2D-NMR and mass spectral studies

A levorotatory compound, C 11 N 3 0 2 was isolated from the edible fruits of Rhus parviflora (Anacardiaceae) along with citric acid 2-methyl ester. A detailed 1 H and 13 C NMR spectral studies including 1 11- 1 H correlation, long range \3C- 1 H cur relation (HMBC and IIMQC) and also NOESY confirmed its structure as JS,6S,3-112-(I,I-dimethylallyl)-5,7-bis(3,3-dimethylallyl-l H-indu-3-yllmethyiJ-6-mcthyl-2,5-pipcrazinedione (I) and allowed the unambiguous assignments of each car bon and proton signals. This biogenetically interesting novel cyclic dipeptide was designated echinulin, being reported first in 1943 from the moulds of Aspergillus eclrinulatus; later on it was reported from the moulds of the A. glaucus group. Echinulin has since been isolated in this laburator·y from three other plants, viz. two Orchidaceae plants, Dendrobium fimbriatum and Cymbidium uloifolium and the peels of an edible vegetable (fruits, Bengali name Potu/) of Triclzosunthes dioica (Cucurbitaceae). To our knowledge, this is the first report on the occurrence of this mould metabolite, echinulin in four· higher plants belonging to three different families. The absolute configuration (S) at each of its two chiral centres and its mass frag mentation have also been discussed.

l 1 when at work or travelling along the hill track to quench thirst and get energy. A citric acid derivative was isolated from these fruits in this laboratory which may be responsible for such relief. This compound 3 identified as 2-hydroxy-1 ,2,3-propanetricarboxylic acid 2-methyl ester serves as an interesting example of a simple achiral molecule having three adjacent prochiral centres and two enantiotopic pairs of diastereotopic geminal hydrogens as demonstrated from its 1 H NMR spectral data.
The chI oro form extract of the dried fruits on si I ica ge I chromatography yielded an amorphous material forming colourless crystals, m.p. 238-239° (from ethanol), [a] 0 -36.3° (CHCI 3 ), M+ at rn!z 461 (C 29 H 39 N 3 0 2 ). Its UV spectrum established the presence of an indole chromophore in the molecule, while its IR spectrum showed sharp absorption peaks for aromatic NH (3665), two amide NH groups (3190 and 3030) and two amide carbonyls ( 1677 and 1667 cm-1 ).
Its 300 MHz 1 H NMR as well as 75 MHz 13 C NMR spectra showed a large number of peaks which could be mostly assigned in structures 2 and 3, but necessitated further organization for correct informational pool. The unambiguous assignments of all peaks and the unequivocal elucidation of its structure have been possible through extensive studies at much stronger magnetic fields of its 1 H NMR  Table I   H-12 HA-3' Hr4" (m) and methyl proton resonance at 151.81 ( d, J 1.0) as well as at 151.87 (br s). The presence of a monosubstituted olefin unit was also discernible from the COSY spectrum. Further, the 1 H NMR spectrum also displayed two methyl signals at 151.~08 and 1.511 as singlets implying the presence of a l, 1-dimethylallyl part in the molecule.
The molecule also had two aromatic proton resonances at 156.81 (br s) and 7.13 (br s) showing mutual correlation among themselves in COSY spectrum and another broad singlet at r5 8.05 (br s) for indole NH proton. Their relative dispositions were ascertained from NOESY experiment The aliphatic methylene proton signal at r5 3.39 (d) showed NOESY correlation with both the aromatic proton resonances at 156.81 (br s) and 7.31 (br s) while the NOESY correlation peaks were discernible between the other aliphatic methylene proton resonance at r5 3.53 (d) and the aromatic proton resonance at r5 6.81 as well as the indole NH resonance at r5 8    and 145.7 were for C-2"', C-2" and C-2', respectively, as they had 1 Jc-H correlation with H-4, H-6, H-2"', 1-1-2" and H-2' resonances. Similarly, the resonance positions for various methyl carbons as summarized in Table 2 were identified. The resonance positions for non-protonated sp 2 carbons C-2, C-3, C-3a, C-5, C-7, C-7a, C-10, C-13, C-3" and C-3'" were conclusively ascertained from the analysis of HMBC spectrum of echinulin (I). It was observed that

Mass ji-agmentation :
The mass fragmentation pattern ofechinulin is well consistent with its structure. The genesis of the major peaks is delineated in Scheme I. Biogenesis : Echinulin 4 ' 5 (I) has been isolated earlier from the myce-1 ium of the moulds of the Aspergillus echinulatus and Aspergillus glauc:a groups as an important constituent. It has been shown to be 2-( l, 1-dimethylallyl)-5,7-bis(3,3dimethylallyl)-3-(6-methyl-2,5-dioxopiperizinyl)methylindole on the basis of its degradative and spectroscopic studies as well as from some model experiments related to the synthesis of echinulin 6 . This mould metabolite being bio- H ~ ~~ -¥~V genetically interesting as it has probable origin from tryptophan, alanine and three isoprene units, has evolved much interest of a number of research groups involved in the biosynthetic studies of natural products with special reference to indole alkaloids 7 · 8 . This conjecture has been supported by the incorporation of L-tryptophan into echinulin in the mould Aspergillus glauca 9 , Aspergillus echinulatus 9 as well as Aspergillus amstelodarin 10 . The homochirality being the hallmark in living systems, from biogenetic point of view the cyclic peptides are formed from L-amino acids and this acceptance settles the stereochemistry of two asymmetric centres as S. However, in the review by Grundon eta!. the structure of echinul in has been drawn in such a way that the configuration of asymmetric centres appeared to be R.
The echinulin has also been isolated from the chloroform extracts ofboth Dendrobiumjimbriatum (Orchidaceae) and Cimbidium aloifolium (Orchidaceae) and also from the peels of the fruits of Trichosanthes dioica (Cucurbitaceae) widely used as vegetables.
Since the occurrence of echinulin was not known in higher plants prior to our report, initially we took it as a new cyclic peptide. However, literature survey 4 · 5 settles its identity with a mould metabolite isolated from mycelium of the moulds of Aspergillus glauca groups. Experimental M.ps. measured in open capillary tubes are uncorrected. IR spectra (KBr) were recorded on a Perkin-Elmer spectrophotometer. 1 H NMR (600 MHz) and 13 C NMR (150 MHz) spectra were recorded on a Varian-600 spectrometer in CDCI 3 (unless mentioned otherwise); the TMS (50.00) or solvent (CHCI 3 57.26 and CDCI 3 577.0) was used as the internal standard and J values are expressed in Hz. The mass spectra were recorded at 70 eV. The silica gel60-120 mesh (Qualigens) and silica gel 230--400 mesh (SRL) were used for column chromatography and flash chromatography. Petrol refers to b.p. 60-80° fraction. The eluate fractions were monitored using microscopic slides with silica gel G layer put on them by dipping in its chloroform slurry, taking out and drying in ambient temperature; simi Jar fractions were combined.
Extraction: Air-dried and powdered (450 g) fruits of R. parvijlora was extracted exhaustively in a soxhlet apparatus with chloroform and methanol successively for 40 h each. The extracts were evaporated in vacuo to afford residues A (I 5 g) from chloroform and B (30 g) from methanol, re-spectively, which were chromatographed over silica gel (60-120 mesh) using solvents and solvent mixtures of increasing polarity. Fractions of a similar composition, as indicated by TLC, were combined. Air-dried orchids, Dendrobium fimbriatum and Cimbidium aloifolium as well as the dried peels of the fruits of Trichosanthes dioica (Cucurbitaceae) were separately extracted with petrol and chloroform respectively in a soxhlet apparatus. Each chloroform extract was chromatographed as above and yielded echinulin in 0.0015, 0.00 I 0 and 0.0005%, yield, respectively.