Suraia Said a and Ivone Carvalho *,a a

-1 ). The biotrasformation of the tetrahydrofuran lignan, (-)-grandisin, by the endophitic fungus Phomopsis sp, obtained from Viguiera arenaria, led to the formation of a new compound determined as 3,4-dimethyl-2-(4'-hydroxy-3',5'-dimethoxyphenyl)-5-methoxy-tetrahydrofuran. The metabolite was evaluated against the parasite Trypanosoma cruzi, the causative agent of Chagas's disease, and showed a trypanocidal activity (IC 50 9.8 µmol L -1 ) similar to the natural precursor (IC 50 3.7 µmol L -1 ).


Experimental
General procedures GC-MS were measure at 70 eV on a Shimadzu Model: QP2010, 120 ºC (oven temp.), using a DB-5MS (30 m × The spectral data from the detector were collected over de 200-400 nm range of absorption chromatograms, and the chromatograms were plotted at 225 nm.Grandisin was obtained from dichloromethane extracts from twigs of Virola surinamensis, which yielded two steroids, two lignans (compounds 6 and 8), five flavonoids and one polyketide. 4

Microorganisms and culture
The endophytic fungi have been isolated and identified by their rDNA sequences as previously described. 29The strains are maintained on PDA slants at 4 ºC.Initially the fungi were screened for their ability to transform grandisin.Fungi were cultivated on PDA in Petri dish for 7 days at 30 ºC and agar pieces (1 cm 2 ) containing fungal mycelia was used as inocullum.Strain SS42 was also identified by "Micoteca URM/ Depto.de Microbiologia/ CCB/ UFPE" (Av.Prof. Nelson Chaves, s/n, 50670-420 Recife-PE, Brazil).
A two-step culture was used for each fungus.Initially, the fungi were inoculated into 20 mL of Czapek medium, supplemented with 3% of sucrose and the cultures were incubated at 30 ºC for 3 days, with shaking at 120 ºC rpm.After cultivation the resulting mycelia were harvested by filtration and transferred to 20 mL of Czapek medium, containing 1% of sucrose and supplemented with a solution of grandisin in DMSO, resulting 0.4 mg mL -1 as final concentration in the media.The cultures were reincubated at the same conditions for 8 days.

Extraction of the metabolites
The culture broths were separated from mycelia by vacuum filtration, followed by 3 times partition sequentially with ethyl ether, ethyl acetate and n-butanol (10 mL).The solvents were evaporated and the crude extracts were analyzed by TLC and HPLC (condition A).

Isolation of the metabolites
After the preliminary screening, Phomopsis sp (VA35) culture was scaled up to 150 mL using the same conditions already described, including the proportion of grandisin (6) (60 mg), but changing the fermentation time to 9 days.The ethyl ether and ethyl acetate extracts were joined because they showed similar profiles.The crude extract obtained was purified by Silica-gel column chromatography using hexane/ethyl acetate 1:1, yielding 4 fractions.Fraction 3 was further purified through HPLC (Condition B) to yield compound 10 (1.0 mg).

In vitro trypanocidal assay
The bioassays were carried out using the blood of infected Swiss albino mice, collected by cardiac puncture on the parasitemy peak (7 th day) after infection with Y strains of T. cruzi.The infected blood was diluted with the blood health mice to achieve a concentration of 10 6 trypomastigote per mL.The assays were performed on titration microplates (96 wells) in duplicate.The compound 10 was dissolved in dimethyl sulfoxide (DMSO) and was added into the infected mouse to provide concentration of 0.5, 2.0, 8 and 32 µmol L -1 .The plates were incubated at 4 ºC and the numbers of parasites were counted after 24 h.Negative and positive controls containing either DMSO or crystal violet at 250 µg mL -1 , respectively were run in parallel. 30

Results and Discussion
The microbial transformations of (-)-grandisin ( 6) by endophytic fungi were investigated in a small scale screening test (8 mg) monitored by TLC and HPLC analyses (condition A).Among the 20 endophytic fungi, Curvularia lunata (SS-42), isolated from Smallanthus sonchifolius, and Phomopsis sp.(VA-35), obtained from Viguiera arenaria, showed better results in the biotransformation of 6. Phomopsis sp. was selected to be cultivated in large scale experiments because of its great ability to generate metabolites in the preliminary tests.
The metabolization of compound 6 (60 mg), with a retention time of 40.23 min as indicated by HPLC analysis (condition B), led to the generation of a major metabolite (10)  with a distinct retention time of 10.35 min.Compound 10 was purified by column chromatography, followed by HPLC (condition B).Condition B was established by exploring its different interaction properties with the reverse phase column (C-18), in a manner similar to that previously reported for butyrolactones isolated from Virola surinamensis. 31,32C-MS analysis showed that compound 10 has a molecular weight lower than grandisin with a [M] +. at m/z 282 and fragments (m/z 68, 85, 100, 167 and 182) suggesting the molecular formula C 15 H 22 O 5 .In fact, the High Resolution Mass Spectrometry confirms the proposed molecular formula with a detectable [M + Na] + 305.1368 and a corresponding theoretic mass of 305.1365.
The structure of compound 10 was established by 1D and 2D NMR data.For instance, the 1 H NMR spectrum of compound 10 showed two methoxy proton signals at d 3.37 and 3.83 with a relative integration of 3:6, which were characteristic of groups attached to aliphatic and aromatic skeleton, respectively.Moreover, the ortho-aromatic proton signals were assigned at d 6.52 (s, 2H), while the methyl groups at C-13 and C-12 were observed at d 1.10 and d 0.87 as two doublets (J 6.9 Hz).
Nearly all relevant spectral details of the isolated metabolites were closely comparable to the corresponding 13 C and 1 H features of grandisin, 4,33 but no data for the second aromatic ring were observed by the integration of signals.In addition, a hemiacetal proton was observed at d 4.71 (d, 1H), attached to a carbon at d 111.1 as observed in the HMQC experiment.Also, the signal at d 3.83 was assigned to the methoxy groups at meta positions of the remaining aromatic ring linked to the tetrahydrofuran nucleus, indicating the presence of a free hydroxyl group at para position.Methoxy aliphatic protons at d 3.37 and methyl protons at d 1.10 showed correlation in the HMBC with carbon at d 111.1, unequivocally establishing the methoxy group at C-5.These data confirmed the presence of a single aromatic ring in compound 10, and an extra methoxy group at C-5.The main HMBC correlations for compound 10 are shown in Figure 1 and data described in Table 2.
We propose that the peculiar aliphatic methoxy groups of compound 10 should be generated firstly by a microbial oxidative metabolism at position C-5 followed by rearrangement to a lactone displacing the aromatic ring.Further reduction to the hemiketal function and O-methylation of the nearly formed hydroxyl group might explain how the fungus converted the 3,4,5 trimethoxyphenyl group of compound 6 to methoxy group of the metabolite 10.
The HPLC separation (condition B) provided a second metabolite with a retention time of 12.50 min.GC-MS analysis showed that this compound has the same molecular weight and fragments presented by compound 10.The High Resolution Mass Spectrometry detected [M + Na] + 305.1368. 1 H NMR spectrum revealed the same integrals, but insufficient data could be obtained from 2D analysis because of the small isolated quantity.
The results from 1 H NMR spectrum, GC-MS and high resolution mass spectrometry suggested that this secondary compound might be an epimer of compound 10.The percentage of lyses observed for the trypomastigote form of T. cruzi treated with compound 10, as outlined in Figure 2, was similar to the corresponding natural product 6. 4 Based on experiments involving the Y strains of T. cruzi compounds 6 and 8 were able to cause lyses of parasites with IC 50 3.7 and 2.3 µmol L -1 respectively, 33,34 while the biotransformation product 10 displayed IC 50 9.8 µmol L -1 .

Conclusions
The product 3,4-dimethyl-2-(4'-hydroxy-3',5'dimethoxyphenyl)-5-methoxy-tetrahydrofuran (10) was successfully obtained from the biotransformation of grandisin (6) by the endophytic fungus Phomopsis sp.The evaluation of the trypanocidal activity of compound 10 and its corresponding natural precursor 6 gave similar results, revealing that a unique methoxylated aromatic ring bound to the central tetrahydrofuran scaffold might preserve the trypanocidal activity, this represents an interesting molecular simplification for synthetic purposes.

Figure 1 .
Figure 1.Selected HMBC correlations for metabolite 10 obtained in the biotrasformation process.