Antifungal Piperolides from Piper malacophyllum ( Prels ) C . DC .

O estudo fitoquímico biomonitorado do extrato em CH 2 Cl 2 das folhas de Piper malacophyllum através da técnica de bioautografia frente a fungos, resultou no isolamento de dois piperolidos bioativos: 4,6-dimetóxi-5-E-fenilbutenolido (1) e 4,6-dimetóxi-5-Z-fenilbutenolido (2). Essas substâncias foram identificadas com base na análise dos espectros de IV, EM e RMN, incluindo técnicas bidimensionais, e comparação com dados descritos na literatura. A configuração E da ligação dupla confere maior atividade para 1 considerando-se que as quantidades mínimas de 1 e 2 necessária para inibir o crescimento dos fungos Cladosporium cladosporioides e C. sphaerospermum foram de 1,0/1,0 μg (1) e de 5,0/10,0 μg (2), respectivamente.


Introduction
2][3] In the course of our search aiming to unravel new antifungal metabolites from plants in Brazilian Atlantic Forest and Cerrado, 4,5 the crude CH 2 Cl 2 extract from leaves of Piper malacophyllum was selected for bioactivity-guided phytochemical investigation due to the strong activity observed against Cladosporium cladosporioides and C. sphaerospermum.After several chromatographic steps, this extract yielded two structurally related piperolides, which had their structures identified by analysis of spectrometric data.

Results and Discussion
Bioactivity-guided fractionation of the CH 2 Cl 2 extract from leaves of P. malacophyllum by silica gel and Sephadex LH-20 chromatography yielded two butenolides of the piperolide series.
Compound 1 had its molecular formula indicated as C 13 H 12 O 4 by analysis of LREIMS (70 eV, [M] +. at m/z 232), and 13 C NMR (BBD and DEPT 135°).The IR spectrum exhibited bands at 1748 (conjugated lactone carbonyl group) and at 1592, 1493, and 1446 (aromatic) cm -1 .The 1 H NMR (Table 1) spectrum of 1 showed multiplets at δ 7.43-7.45(2H) and δ 7.73 (3H), a pattern of a monosubstituted aromatic ring.This spectrum also exhibited two methoxyl signals at δ 3.99 (s) and δ 3.65 (s) besides one singlet at δ 5.27 (1H).These information, associated to the 13 C NMR resonances at δ 171.7 (C), 168.1 ) gave a positive nOe for the hydrogens at δ 3.65 (6-OCH 3 ) and δ 5.27 (H-3), indicating the E configuration to double bond.Therefore, the compound 1 was identified as 4,6-dimethoxy-5-E-phenylbutenolide (5,6-E-fadyenolide), previously isolated from roots of Piper fadyenii. 6,7he LREIMS spectrum of 1 and 2 showed the same molecular ion-peak and fragment ions, suggesting similar structures.The IR spectrum with bands at 1745, 1592, 1493, and 1443 cm -1 confirming the presence of conjugated lactone group and aromatic ring in the structure of compound 2. The 13 C NMR spectra (Table 1) exhibited signals of two methoxyl groups (δ 58.9 / 58.8), two olefinic carbons [δ 144.0 (C) and 130.The 1 H NMR spectrum showed a multiplet at δ 7.37-7.42(5H), indicative of a monosubstituted aromatic ring, and three singlets at δ 5.11 (1H), 3.59 (3H) and 3.74 (3H), which confirmed the structural similarity between 1 and 2. The cross-peaks between δ 3.59 (OCH 3 ) / 170.8 (C-4) and δ 3.74 (OCH 3 ) / 144.0 (C-6) in its HMBC spectrum determined the position of the methoxyl groups at C-4 and C-6, as depicted to compound 1.This spectrum showed also cross peaks between H-3 (δ 5.11) and C-2 (δ 167.8) / C-5 (δ 129.0) and between H-8/H-12 (δ 7.37-7.42)and C-6 (δ 144.0).Thus the connectivity between the butenolide and aromatic rings and the same planar structure of 1 was confirmed.However, irradiation of signal at δ 3.59 (4-OCH 3 ) gave a nOe enhancement for the hydrogens at δ 5.11 (H-3) and δ 7.42 (H-8/H-12) while the signal at δ 3.74 (6-OCH 3 ) were not affected, indicating the Z configuration for the double bound.Therefore, the compound 2 was identified as 4,6dimethoxy-5-Z-phenylbutenolide, as previously defined to 5,6-Z-fadyenolide, also isolated from roots and aerial parts of Piper fadyenii. 6,7here are only few report of piperolides in Piperaceae species and to date they have been described from Piper fadyenii 6,7 and P. sanctum 8,9 which occur from Jamaica and Mexico, respectively.In such reports 8 the NMR data of compounds 1 and 2 were assigned only to the butenolide moiety.Thus herein we present an unambiguous assignment of their NMR data based on several 2D spectra including HMBC experiment (Table 1).Both compounds were isolated in a bioactivity-guided fractionation procedure.The antifungal activity of compounds 1 and 2 was determined by means of direct bioautography on TLC plate 11 .The detection limits of these compounds required to inhibit growth of the fungus C. cladosporioides and C. sphaerospermum (Table 2) were obtained according to methodology described elsewhere. 5,11,12Compound 1 presented stronger antifungal activity than that observed for compound 2, suggesting that the configuration of the double bond in the butenolide ring could be associated to their fungitoxic potential.There was no previous report describing the fungitoxic potential of these compounds, mainly 1 which showed a similar activity of the positive control (nystatin and miconazole).

Instrumental and chromatography materials
Silica gel (Merck 230-400 mesh) and Sephadex LH-20 (Pharmacia) were used for column chromatographic separation.Silica gel PF 254 (Merck) was used to TLC preparative purification.The NMR spectra were recorded on Bruker DRX-500 operating at 500 MHz to 1 H and at 125 MHz to 13 C, in CDCl 3 with TMS as internal standard.IR spectra were obtained on a FT-IT 510 Nicolet spectrometer.LREIMS were measured at 70 eV on a HP 5990/5988A spectrometer.UV spectra were recorded on a UV/Visible Hitachi U-3000 spectrophotometer.

Plant material
The leaves of Piper malacophyllum (Prels) C. DC. was collected in December 2000 in the Parque Estadual Intervales, São Paulo State, Brazil and was identified by Dr. Elsie F. Guimarães (Jardim Botânico -Rio de Janeiro).

Antifungal assays
The microorganisms used in the antifungal assays C. sphaerospermum (Penzig) SPC 491 and C. cladosporioides (Fresen) de Vries SPC 140 have been maintained at the Instituto de Botânica, São Paulo, SP, Brazil.Ten microliters of the solutions of the crude extracts, fractions and pure compounds were prepared, in different concentrations, corresponding to 20, 10, 5 and 1 µg for pure compounds and 100 µg for the crude extracts or fractions.The samples were applied to TLC plates, these being eluted with CHCl 3 -MeOH 99:1 followed by complete removal of the solvent at room temperature.The chromatographic plates were sprayed with spores suspension of C. sphaerospermum and C. cladosporioides in a nutritive medium (glucose and salt solution 11 ) and incubated for 48 h and 37 °C.After incubation, clear inhibition zones appeared against a dark background chromatogram.Nystatin and miconazole were used as positive controls whereas ampicillin and chloramphenicol were used as negative controls. 5,11
Positive control: nystatin (1.0 µg) and miconazole (1.0 µg); a Minimum amount required for the inhibition of fungal growth on thin-layer chromatographic plates (TLC).

Table 2 .
Antifungal activity of compounds 1 and 2 against Cladosporium cladosporioides and C. sphaerospermum