Two Novel Eremophilane Sesquiterpenes from an Endophytic Xylariaceous Fungus Isolated from Leaves of Cupressus lusitanica

Dois novos sesquiterpenos eremofilanos, cupressolideo A e cupressolideo B, além de dois outros conhecidos, foram isolados a partir do extrato AcOEt do meio de cultura de uma espécie de Xylaria, isolada como fungo endofítico dos tecidos sadios das folhas de Cupressus lusitanica. Estudos espectroscópicos, usando EM e RMN, levaram às estruturas dos dois sesquiterpenos de esqueleto eremofilanos, novos na literatura.


Introduction
Cupressus lusitanica, commonly known as a Mexican Cypress and Portuguese Cypress, belongs to the family Cupressaceae and is usually cultivated as an ornamental tree and in commercial forestry plantation. 1,2Due to its economic importance, this plant was included in our continuous program established to study the chemistry and biochemistry aspects of plant microorganisms interactions, with emphasis on those apparently symbiotic associations.
Among the endophytic fungi isolated from healthy tissues of C. lusitanica leaves, we obtained some strains with macro and micro morphology characteristics of those microorganisms belonging to the genus Xylaria.Besides these morphologic characteristics, Xylaria species are producers of secondary metabolites, including isocoumarin, 3,4 cytochalasins, [4][5][6] xanthones, 7 xyloketals, [8][9][10] sesquiterpenes, 11 that contribute to their classification in this genus.In our study we detected isocoumarins and cytochalasins in the fungus extracts using mass spectrometry, which reinforce the hypothesis of its classification as a Xylareaceous fungus.Among the compounds isolated from the fungus extract, two novel (1 and 2) and two known (3 and 4) eremophilane sesquiterpenes were identified.
The production of these terpenoids corroborated to classify the fungus within Xylariaceae family.[14][15]

Results and Discussion
The EtOAc extract of liquid medium from endophytic fungi was chromatographed on silica gel columns to give four compounds (1-4).Compounds 3 and 4 were previously reported in the literature. 16,17ompound 1 was obtained as a colorless crystal.The IR spectrum displayed a broad band at 3487 cm -1 characteristic of a hydroxyl group and a band at 1745 cm -1 attributed to a conjugated γ-lactone.The 13   The relative stereochemistry of 1 was elucidated using nOe and COSY spectroscopy.The β orientation of H-3 was inferred from the nOe correlation with CH 3 -14 and CH 3 -15.H-8 (d 4.90) should be α oriented as indicated by the homoallylic coupling with the methyl (CH 3 -13, d 1.85) bounded to C-11 observed in the COSY experiment.This homoallylic coupling requires a 90º dihedral angle of the methyl group at C-11 with H-6α and H-8, witch was in agreement with the nOe experiment.Furthermore, the nOe spectrum showed correlations of H-6α with H-4 (d 1.81) and of H-6β with CH 3 -13, CH 3 -14 and CH 3 -15.All NMR data are shown in the Table 1.
Compound 2 showed spectroscopic data very similar to those of 1, indicating the presence of an eremophilane skeleton.The IR spectrum displayed a broad band at 3508 cm -1 characteristic of a hydroxyl group.The The data were acquired at 400 and 100 MHz for 1 H and 13 C respectively in CDCl 3 .TMS was used as internal reference.2).The relative stereochemistry was based on those determined to compound 2. All NMR data can be observed in Table 2.

Conclusions
The genus Xylaria is known for being a rich source of structurally diverse natural products including isocoumarin, 3,4 cytochalsins, [4][5][6] xyloketals, 8-10 sesquiterpenes 11 and others.3][14][15] One member of this genus reported in the present study showed the notable ability to produce eremophilane sesquiterpenes, including two new in the literature, cupressolide A and cupressolide B. Due to the many biological activities shown  The data were acquired at 400 and 100 MHz for 1 H and 13 C respectively in CDCl 3 .TMS was used as internal reference.
by this class of compounds, this Xylaria deserves a careful study aiming to sesquiterpene production enhancement.

Experimental
Equipment IR spectra were run on a Bomen MB102 -IR spectrometer using KBr pellets.Optical rotation was measured on a Perkin-Elmer 241 polarimeter.GC/MS analyses were performed using GC 8000 series Fisons and VG Platform mass spectrometer detector.1D and 2D NMR spectra were obtained in CDCl 3 (Aldrich) on DRX 400 Bruker spectrometer operating at 400 MHz for hydrogen and 100 MHz for 13 C and TMS was used as internal standard.MS were acquired in positive ion mode on a triple quadrupole Micromass Quattro LC spectrometer, equipped with an ESI ion source.

Plant material
Health leaves of Cupressus lusitanica were collected in São Carlos, São Paulo State, Brazil.A voucher specimen (No. 7281) has been deposited in the Herbarium of the Botanic Department of Universidade Federal de São Carlos, Brazil.

Fungal material
The method of surface sterilization employed in this work was similar to that used by Petrini et al. 18 After the collection, the leaves were washed in abundant water (domestic use grade) and then in distilled water.The leaves were surface sterilization by consecutive immersion in 70% ethanol (2 s), sterile distilled water (2 s), 11% aqueous sodium hypochloride for 1-5 min and 70% ethanol (2 s), and then in sterile distilled water.The material was placed in Petri dishes containing PDA medium (potato, dextrose and agar) supplemented with 100 μg mL -1 terramicin and incubated at room temperature.Endophytic fungi growing from the plant tissues, were picked and recultured on PDA to determine culture purity.It was deposited at LaBioMMi -Laboratório de Bioquímica Micromolecular de Microorganismos -of the Departamento de Química at Universidade Federal de São Carlos, Brazil.Working stocks were prepared on potato dextrose agar.

GC/MS analysis
The extract was submitted to clean-up procedures using solid phase extraction (SPE).The SPE cartridge was activated with 100% Hexane and conditioned with 3 mL of CHCl 3 .The extract (10 mg) was dissolved in 3 mL of CHCl 3 and loaded to the SPE cartridge.Elution of SPE cartridge with CHCl 3 produced an apolar fraction.For the GC/MS analysis the injector temperature was kept at 180 ºC and the GC oven temperature was maintained at 70 ºC during 6 min and then increased to 250 ºC at a rate of 6 ºC min -1 and finally increased to 325 ºC at 3 ºC min -1 .The sample volume injected was 2 mL.

MS data collection
ESI-MS data were colleted from direct introduction of the sample solution 5 mL of compound 1 (5 mg mL -1 ).The optimal voltages found for the probe and ion source components to produce maximum intensity of the ions [M+H] + were 3.3 kV for the capillary, 19 V for the sample cone, and 4 V for the extractor cone.
12 and  5.26) in the1 H NMR spectrum were in agreement with the profile of an eremophilane with a double bound at C-11 and C-12.The epoxide group at C-1 and C-10 was deduced from the chemical shifts of H-1 (d 2.97), C-1 (d 59.7), and C-10 (d 65.8).The COSY analysis of 2 led to a partial structure as shown by bold-faced lines in Figure2, which were supported by HMBC correlations (Table
C NMR spectrum exhibited 15 signals which were assigned, by DEPT 135 and HSQC experiments to three methyls, three methylenes, four methines and three sp 2 carbons, one of this being a carbonyl group.Its ESI-MS spectrum contains an ion peak of [M+H] + at m/z 265, consistent with the molecular formula C 15 H 20 O 4 which also was in accordance with the NMR data.The 1 H NMR spectrum of 1 showed three signals d H 1.85, 0.83 and 0.96 attributed to CH 3 -13, Vol.21, No. 8, 2010 H 2.23 (1H, br d, J 13.6 Hz, H-6b) and d