Antifungal Aromadendrane Sesquiterpenoids from the Leaves of Xylopia brasiliensis

Um novo sesquiterpeno, aromadendrano-4β,10α,15-triol, foi isolado das folhas de Xylopia brasiliensis Spreng (Annonaceae), juntamente com quatro derivados de esqueleto aromadendrano e três esteróides. As estruturas dos metabólitos foram definidas por meio da análise dos respectivos espectros de RMN, incluindo experimentos bidimensionais, além de espectrometria de massas. Os sesquiterpenos aromadendranos diidroxilados apresentaram atividade antifúngica frente a Cladosporium cladosporioides.


Introduction
The genus Xylopia (Annonaceae) comprises about 160 species with occurrence in South and Central America, Africa and Asia. 1,2 Aproximatelly 25 species of Xylopia were identified in Brazil, mainly in Amazonia. 2 These species produce a wide variety of metabolites including alkaloids, amides, lignoids, acetogenins and terpenoids. [3][4][5][6][7] X. brasiliensis, a very large tree distributed in Southeastern Brazil, is known as "pindaíba", "pindaubuna" and "bindaíba" and has been used in the folk medicine as a sedative and analgesic. Previous investigations of the chemical constituents of this plant resulted in the isolation of diterpenoids and aporphine alkaloids from the fruits and stem bark, respectively. 8,9 In addition of our studies on X. brasiliensis, we describe herein the isolation and identification of five aromadendrane sesquiterpenoids, including a new derivative, besides three known steroids. All the sesquiterpenoids were submitted to antifungal assay against Cladosporium cladosporioides and the dihydroxylated derivatives showed pronounced activity.
The sesquiterpenoids 1, 2, 3 and 4 and the steroids 6, 7 and 8 were identified by analysis of their 1 H and 13 C NMR spectroscopic data and comparison with literature data. [10][11][12][13][14] The 13 C NMR spectrum of 5 displayed fifteen signals, three of which are of carbinolic carbon atoms [δ 80.5 (C), 75.8 (C) and 62.1 (CH 2 )]. These data, associated to molecular ion peak at 255 Da [M+H] + in ESIMS, suggested the structure of a trihydroxysesquiterpenoid with molecular formula C 15 H 26 O 3 . The 1 H NMR spectrum of 5 showed absorptions of a cyclopropane ring at δ 0.41 (1H, 829 Antifungal Aromadendrane Sesquiterpenoids from the Leaves of Xylopia brasiliensis Vol. 14, No. 5, 2002 dd, J 10.8 and 9.6 Hz) and at δ 0.65 (1H, m), which are a characteristic feature of the aromadendrane skeleton. 11 This spectrum also showed one singlet at δ 1.02 (6H), assigned to a gem-dimethyl group linked to a quaternary carbon, and one singlet at δ 1.25 (3H) assigned to one methyl group joined to a tertiary carbinolic carbon. The data of 5, compared with those of related compounds aromadendrane-4α,10α-diol (3) and aromadendrane-4β,10α-diol (3a), 12 indicated that the hydroxyl groups at C-4 and C-10 have β and α orientation, respectively. 11 The presence of an AB system at δ 3.66 (1H, J 11.3 Hz) and at δ 3.73 (1H, J 11.3 Hz) suggested the presence of a primary alcohol in 5. Accordingly the signal at δ 62.1 in the 13 C NMR spectra (BBD and DEPT 135°) was assigned to the oxygenated methylenic group. The third hydroxyl group was thus positioned at C-15 shielding C-9 chemical shift by a γ-gauche effect of 5 ppm, when compared with aromadendrane-4β,10β-diol (3b). 11 This proposal was confirmed by HMBC spectrum, which showed a long range coupling between C-15/H-1, C-15/H-9 and C-10/H-15. The positioning of the hydroxyl group at C-15 was also confirmed by analysis of 13 C NMR data of the monoacetylated derivative (5-Ac), which showed a deshielding effect of 2.7 ppm of C-15 and a shielding effect of 0.8 ppm to C-10, due to β and γ effects respectively, when compared with those of 5. Therefore, the structure of 5 was defined as aromadendrane-4β,10α,15-triol.
The antifungal activity of sesquiterpenoids 2, 3, 4 and 5 was evaluated by means of direct bioautography on TLC plate. The aromadendrane derivatives 2, 3 and 4 showed antifungal activity against C. cladosporioides and their minimun amount required to inhibit growth of this fungus were detemined as 5 µg. Nystatin (5 µg) and miconazole (1 µg) were used as positive controls.

Experimental
General NMR spectra were recorded at 75 and 125 MHz to 13 C and 300 and 500 MHz for 1 H (Bruker DPX-300 and DRX-500 spectrometers, respectively). The spectra were recorded in CDCl 3 (Aldrich); δ values were expressed in ppm relative to the residual signal of CHCl 3 in CDCl 3 ; IR spectra were obtained using KBr pellets in a Perkin-Elmer Infrared Spectrometer model 1750; ESIMS were obtained in a Platform II-Micromass Spectrometer (quadrupole). EIMS were obtained with a 70 eV INCOS 50 Finnigan-Matquadrupole mass spectrometer; silica gel 60 (Merck) and Sephadex LH-20 (Pharmacia) were used for column chromatography (63-200 µm); pre-coated aluminium silica gel plates PF 254 (Merck) were used for TLC analysis.

Plant material
The leaves of X. brasiliensis were collected at the Instituto de Botânica on February, 1998, São Paulo, Brazil. A voucher specimen (YOUNG10) is deposited at the herbarium of Instituto de Botânica (SMA-SP).

Antifungal assay
Dilutions containing 100, 50, 25, 10, 5, 1 and 0.1 µg of pure compounds were applied on a pre-coated TLC plate. The plate was developed with hexane/EtOAc (7:3). The chromatogram was sprayed with a spore suspension of C. cladosporioides in glucose and salt solution and was incubated for 72 h in darkness in a moistened chamber at 25 °C. 15 Clear inhibition zones appeared against a dark blackground, indicating the minimal amount required for inhibition of fungal growth on TLC plate. Nystatin (5 µg) and miconazole (1 µg) were used as positive controls. The microorganism used in the antifungal assay, C. cladosporioides (Pemzig) SPC 491, has been maintained at Instituto de Botânica (SP).