Talaroxanthone , a Novel Xanthone Dimer from the Endophytic Fungus Talaromyces sp . Associated with Duguetia stelechantha ( Diels ) R . E . Fries

Departamento de Química, Universidade Federal do Amazonas, 69077-000 Manaus-AM, Brazil Instituto de Química, Universidade Estadual de Campinas, 13084-971 Campinas-SP, Brazil Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, 69065-001 Manaus-AM, Brazil Departamento de Química, Universidade Federal do Paraná, 81531-990 Curitiba-PR, Brazil Departamento de Genética e Evolução, Universidade Federal de São Carlos, 13565-905 São Carlos-SP, Brazil


Introduction
Fungi from the genus Talaromyces are known to be a rich and reliable source of biologically active and/ or chemically novel compounds. 1][4] Endophytic organisms have been fructuous producers of new xanthones, 5,6 as illustrated by the antibacterial and antifungal microsphaeropsones from Microsphaeropsis spp., 7 the antimalarial phomoxanthones A and B from Phomopsis sp., 8 the cytotoxic tajixanthone from Emericella variecolor 9 and the antibacterial blennolides from Blennoria sp. 10 Xanthones found in fungi are always found as tetrahydroxanthone group of dimers and heterodimers. 5Many of these xanthone dimers from fungi displayed several biological activities such as antibacterial, antifungal and anticancer. 7Therefore, endophytic fungi are considered to be a promising resource for new lead compounds in drug development.Our research group focuses on the discovery of new structural and bioactive secondary metabolites from endophytic fungi that have been isolated from Amazonian endemic plants. 11This work describes the chemical investigation of Talaromyces sp., an endophytic fungus strain from the Amazonian Rainforest plant Duguetia stelechantha (Annonaceae), which resulted in the identification of a new xanthone dimer, talaroxanthone (Figure 1).

Results and Discussion
Talaroxanthone (Figure 1) was isolated as a yellow powder from the extract of mycelia from flask cultures of Talaromyces sp.DgCr22.1b.The molecular formula C 32 H 30 O 14 was established by high-resolution mass spectrometry (HRMS).The 13 2) between the carbinolic hydrogen at H-5 (d H 3.75) along with the methine hydrogen at H-6 (d H 2.42) was observed by the coupling constant J 5,6 12 Hz previously reported for the eumitrin A 2 . 12The comparison of the 13 10 These comparisons led to the establishment of the orientations of the hydroxyl group at C-5 and the methyl connected to C-6 as pseudoequatorial (Figure 2).
The aromatic hydrogens at d H 6.60 (H-2) and d H 8.04 ppm (H-3) were assigned as being ortho to each other due to their coupling constants (J 2,3 8.7 Hz) and COSY (correlation spectroscopy) data.The observed chemical shift for H-3 is different than the observed for phomoxanthone A, 8 but any other connection between the aromatic rings was dismissed by the observed data.The symmetry observed by 1 H and 13 C NMR and confirmed by HRMS/MS indicates that this molecule is a homodimer connected at C4-C4', which is rare in fungal metabolites. 8This connectivity was also confirmed by HMBC correlations of the hydroxyl group at C-1. Talaroxanthone differs from the phomoxanthones at positions C-10a, C-10a' and C-5, presenting as substituents methylacetate at C-10a and C-10a' and a hydroxyl at C-5.The Phomopsis xanthones, on the other hand, contain -CH 2 OCCH 3 groups at C-10a and C-10a' and an acetoxy group at C-5a.Talaroxanthone is related to the secalonic acids, also called ergochromes, 5 which are also tetrahydroxanthones, but with a C2-C2' linkage pattern, 14 usually found in compounds from the Claviceps, 15 Penicillium, 16 Pyrenochaeta and Aspergillus genera. 17

Conclusions
The potential of endophytic fungi as sources of novel substances was demonstrated by the isolation of a new xanthone dimer named talaroxanthone from the mycelial mass of the endophytic fungus Talaromyces DgCr22.1b.This compound is responsible for the yellow pigmentation observed for this strain.To the best of our knowledge, compound 1 is the first example of a xanthone homodimer related to the secalonic acids with C4-C4' linkage along with the presence of methylacetate groups at the positions C10a and C10a' differently from the previous isolated phomoxanthones.

Experimental
General procedures 1D and 2D NMR experiments were recorded on a Bruker AVANCE 400 spectrometer operating at 9.4 T, equipped with a 5 mm multinuclear direct detection probe with z-gradient operating at 400 MHz for 1 H and 100 MHz for 13 C.The chemical shifts were referenced to the solvent peak CDCl 3 at d 7.27 and 77.0 ppm, respectively.High resolution electrospray ionization mass spectrometry (HRESIMS) measurements were recorded on a Waters Synapt HDMS instrument with quadrupole time-of-flight (QTOF) geometry.Optical rotations were recorded on a Jasco P-1020 polarimeter.Fourier transform infrared  Talaroxanthone, a Novel Xanthone Dimer from the Endophytic Fungus Talaromyces sp.J. Braz.Chem.Soc.882 (FTIR) spectra were recorded on a Bomem MB102 spectrophotometer.A Shimadzu LC-6AD pump equipped with a Shimadzu SPD-10AV UV detector and a Rheodyne injector was used for high pressure liquid chromatography (HPLC).Silica gel 60 (70-230 mesh) was used for column chromatography, while silica gel 60 F 254 was used for analytical (0.25 mm) thin layer chromatography (TLC).A Phenomenex Phenyl Hexyl 5 µm preparative column (10 mm × 250 mm) was used for the HPLC separations.All solvents used for chromatography and mass spectrometry were from Tedia (HPLC grade), and H 2 O was ultrapure (Milli-Q, Millipore).All microbiological culture media were purchased from Biosystems.

Fungal material
The roots of Duguetia stelechantha (Diels) R. E. Fries were collected in the experimental farm of the Universidade Federal do Amazonas, Amazonas State, Brazil.The plant material was washed with detergent and sterile water for external cleaning, then fragments were immersed in 70% alcohol, later in 3% hypochlorite solution and finally in sterile water. 18After this process, plant fragments were inoculated in Petri dishes containing ISP2-agar medium and incubated for five days.The fragments containing hyphae of fungi were subsequently transferred to test tubes with the same medium and incubated at 26 °C for 30 days.After this period, the mitosporic strain was purified by the Tween technique. 19The isolated strain was identified according to traditional morphological criteria and sequencing of the fungus ITS-1 to ITS-2 rDNA and compared with sequences from the GenBank. 18A voucher was deposited in the fungal collection of the GEMMA group of the Universidade Federal do Amazonas under the code DgCr22.1b.

Production and isolation
An isolated culture of the strain DgCr22.1b was grown on ISP 2 broth medium (International Streptomyces Project 2) at 25 °C for 19 days, into 39 × 1 L Erlenmeyer flasks each containing 300 mL of medium.The mycelia were separated from the broth and extracted with EtOAc 1:1 MeOH (1 L, 2 days), the solvent was evaporated under reduced pressure and provided an red solid gum (30.2 g).The crude extract was subjected to column chromatography (silica gel, 4 × 20 cm) with EtOAc as eluent.A yellow pigment was eluted after ca.500 mL elution with EtOAc.This process was repeated twice to yield a yellow gum (2.1 g).This yellow gum was subjected to another column chromatography (silica gel, 2.5 × 30 cm) eluted with a gradient of hexane and EtOAc (9:1, 0:1) to give 17 main fractions.The fractions 11-13 (1:9, 104 mg) were subjected to semi-preparative HPLC using isocratic MeOH as the eluent at a flow rate of 8.7 mL min -1 to obtain 1 (70.6 mg, t R 5 min).

Figure 2 .
Figure 2. Key long-range correlations observed in the HMBC and NOESY of compound 1.

Table 1 .
13 and13C NMR spectral data a for compound 1 Experiments were carried out at 400 MHz for 1 H and 100 MHz for a