Novel Anthraquinone Derivatives Produced by Phoma sorghina , an Endophyte Found in Association with the Medicinal Plant Tithonia diversifolia ( Asteraceae )

Três antraquinonas conhecidas (1,7-diidroxi-3-metil-9,10-antraquinona, 1,6-diidroxi-3-metil9,10-antraquinona e 1-hidroxi-3-metil-9,10-antraquinona), uma nova antraquinona (1,7-diidroxi3-hidroximetil-9,10-antraquinona), e dois novos derivados hexaidroantraquinônicos, dendrióis E e F, foram isolados da cultura do fungo endofítico Phoma sorghina, associado a Tithonia diversifolia (Asteraceae). Suas estruturas foram identificadas com base em dados espectroscópicos, principalmente RMN 1D e 2D.


Introduction
Endophytes are considered outstanding and under explored sources of novel chemical diversity and bioactive compounds. 1,2These microorganisms can be detected at a particular moment within the tissues of apparently healthy plant hosts, 3 and they have been found in all plant species examined to date. 4 As they occupy unique biological niches, 5,6 the complex web of interactions with other endophytes and with the host might give rise to new chemical diversity and bioactive compounds. 1In fact, this prolific biosynthetic capability is illustrated by a number of new and/or bioactive metabolites isolated from endophytes. 2,6ost of the researches on the chemistry of endophytes have been done in the northern hemisphere.However, results have shown that tropical plants present greater diversity of endophytes species than those from temperate zones. 3[9][10][11][12][13] We have been interested in endophytes found in association with Tithonia diversifolia (Asteraceae), also known as Mexican sunflower.5][16] Anti-inflammatory, amoebicidal, antispasmodic, antifungal, antibacterial and antiviral activities have also been described for T. diversifolia extracts. 15,16Moreover, there are no previous reports on the isolation and cultivation of endophytes from T. diversifolia.
In this work Phoma sorghina was isolated as an endophytic fungus from the leaves of T. diversifolia.After cultivation on solid rice medium, three known and three novel anthraquinone derivatives were isolated and identified.

General experimental procedures
Optical rotations were measured on a PERKIN ELMER 241 polarimeter.UV spectra were obtained in MeOH solution on a SHIMADZU PC 1520 diode array spectrophotometer and IR spectra were measured with a Nicole Protégé 460 spectrophotometer.High-resolution ESI-MS were measured with an UltrO-TOF (Bruker-daltonics, Billarica, USA).Lowresolution ESI-MS data were acquired in the negative ion mode, using a MICROMASS QUATTRO-LC instrument equipped with an ESI/APCI "Z-spray" ion source.Semi preparative HPLC separations were carried out in a Shimadzu (LC-6AD apparatus, Japan) multisolvent delivery system, Shimadzu SPD-M10Avp Photodiode Array Detector, and an Intel Celeron computer for analytical system control, data collection and processing (software Class-VP), using VP 250/ 10 NUCLEOSIL 120-5 C18 or VP 250/10 NUCLEOSIL 100-5 C18 columns. 1 H and 13 C NMR spectroscopic experiments were recorded on BRUKER DRX-400 and BRUKER DRX-500 spectrometers with CD 3 OD and CDCl 3 as solvents and TMS as internal standard.

Microorganism
The general procedures adopted for isolation of the microorganism followed the methodology described by Kongsaeree et al. 17 After collected, healthy leaves of Tithonia diversifolia were washed with water and surface sterilized by immersion in 70% aqueous ethanol (2 minutes), followed by 5% aqueous sodium hypochlorite (90 seconds), and finally with 70% aqueous ethanol (1 minute).After these procedures, the leaves were rinsed with sterilized water.This latter water was incubated in Petri dishes to guarantee the elimination of all epiphytic microorganisms.Small pieces of the leaves were excised and placed on agar in Petri dishes containing PDA medium at 30 ºC.Individual hyphal tips of the emerging fungi were removed and replaced on PDA.
The molecular formula of compound 4 was established as C 15 H 10 O 5 by HRESIMS, as well as 1 H and 13 C NMR data.The IR spectrum of the compound 4 showed characteristic absorption bands from OH (broad, 3442 cm -1 ) and α,β-unsaturated ketone (1638 cm -1 ).The 13 C NMR spectrum of 4 (Table 2) showed 13 carbon signals (two carbons were not observed): 2 carbonyls (δ 190.6 and δ 182.6), five quaternary sp 2 carbons, five methine aromatic carbons, and one sp 3 methylene group.The 1 H NMR spectrum (Table 1) showed a singlet at δ 8.54, assigned to a hydroxyl group H-bonded to a carbonyl, and two metacoupled aromatic hydrogens at δ 7.21 (d, J 0.6 Hz, H-2) and δ 7.69 (d, J 0.6 Hz, H-4), suggesting a 1,2,3,5tetrasubstituted aromatic ring.The presence of a 1,2,4trisubstituted aromatic ring was evident from the signals at δ 6.90 (dd, J 8.6 and 2.5 Hz, H-6), δ 7.43 (d, J 2.5 Hz, H-8) and δ 8.02 (d, J 8.6 Hz, H-5).The position of hydroxyl group at C-7 was unequivocally ascribed by HMBC correlations and splitting patterns of the 1 H NMR signals.Both hydrogens at δ 7.69 (H-4) and δ 8.02 (H-5) showed long range correlations with the carbon at δ 182.6, establishing this ketone at C-10.H-5 was found to be orto coupled to H-6, suggesting a substitution at C-7.The remaining ketone group was attributed to C-9, which presented long range correlation in the HMBC experiment with the hydrogen at δ 7.43 (d, J 2.5 Hz, H-8).The meta coupling observed for H-8 is also only possible if the hydroxyl group is located at C-7.The typical signal for the methyl group attached to C-3 in anthraquinones was not observed.However, the cross peak in the HMQC between the hydrogens at δ 4.68 (s, 2H) and the carbon at δ 64.4 suggested a hydroxymethyl group at C-3.The HMBC and HMQC data confirmed the location of the hydroxymethyl group at C-3 through the observed correlations amongst H-11 and C-2, C-3 and C-4.
The 1 H and 13 C NMR spectra of compounds 5 and 6 also showed the signals related to the aromatic ring bearing hydroxyl and methyl groups, typical of the anthraquinones (Tables 2 and 3).However, they did reveal only one signal of ketone carbonyl, and no signals of the additional aromatic ring.The 1 H NMR spectra revealed signals at δ 1.19-2.48and δ 3.40-5.35,suggesting the presence of hexahydroanthraquinone frameworks.Similar compounds were previously isolated from the pathogenic fungus Dendryphiella sp., and were named dendryols. 23ompound 5 was obtained as a pale yellow amorphous solid.From its HRESIMS, as well as by the observed signals in the NMR spectra, it was possible to deduce its molecular formula as C 15 H 18 O 4 .The 1 H NMR, DEPT, HMBC and HMQC spectra revealed the presence of one methyl, three methylene, six methine, five quaternary carbons and three hydroxyl groups.COSY, HMBC and NOE experiments, and also J values, allowed us to unequivocally ascribe all the protons and carbons of the molecule, as well as the relative stereochemistries of the stereogenic carbons.Hydroxyl methine protons were observed at δ 3.63 (m) and δ 4.80 (d, J 9.8 Hz).In contrast with 9,10-anthraquinones 1-4, only one ketone carbonyl signal (δ 200.0) was observed in the HMBC experiment.The cross peak between the ketone carbon and the proton at δ 7.26 (br s, H-4), observed in the HMBC experiment, led us to locate the ketone at C-10.So, it was suggested that one hydroxyl group should be placed at C-9.Moreover, both deshielded chemical shifts for H-9 (δ 4.80) and C-9 (δ 73.8) suggested a benzylic position.Hydrogens H-9 (δ 4.80) and H-8a (δ 1.75) are coupled in a trans relationship, as suggested by the J value of 9.8 Hz.The stereochemistry of an analogue structure, dendryol A, has been established through X-ray diffraction analysis. 23So, we assumed the same relative configuration at C-9 for compound 5.In addition, of H-9 resulted in NOE with H-5a and H-8β, confirming an α-oriented hydroxyl group at C-9.The remaining hydroxyl methine hydrogen at δ 3.63 (H-6) was coupled to both methylene hydrogens at δ 1.19 (m) and δ 2.03 (m), as well as to additional methylene hydrogens at δ 1.24 (m) and δ 2.48 (m), as observed in the COSY experiment.COSY also showed the couplings of the methylene hydrogens at δ 1.35 (dddd) and δ 2.42 (dt) with both methylene hydrogens at δ 1.19 (m) and δ 2.03 (m) and with the methine at δ 1.75 (m).HMBC experiment revealed long range correlations between H-5α (δ 1.24) and the carbons at δ 34.7 (C-7), 70.7 (C-6), 48.4 (C-5a) and 200.0 (C-10).These data allowed to locate the hydroxyl at C-6. Irradiation of H-6 (δ 3.63, m) showed NOE with four hydrogen signals at δ 1.35 (H-8β), 2.03 (H-7β), 2.35 (H-5a), and 2.48 (H-5β), which are only possible with H-6 in the axial configuration (Figure 2).Therefore, the hydroxyl group at C-6 should be α-oriented.
Compound 6 presented similar spectroscopic data compared to 5. The HRESIMS and NMR data suggested C 15 H 18 O 5 as the molecular formula of compound 6.The additional hydroxyl group was evident from the MS data and also from the three hydroxyl methine protons at δ 5.35, 3.69 and 3.40, respectively attached to the carbons at δ 74.1, 81.7 and 74.0, as observed by the cross peaks in the HMQC experiment.These preliminary data also indicated a hexahydroanthraquinone framework for compound 6.The long range correlations of H-4 (δ 7.24, br s) with carbons at δ 20.7 and δ 199.0 led us to place the ketone group at C-10.Therefore, one hydroxyl group was also α-oriented at C-9. Proton H-9 (δ 5.35, d) was coupled to H-8a in trans stereochemistry, as suggested by the J value of 9.2 Hz.The HMBC experiment revealed a long range correlation between H-9 and the carbon at δ 81.7, allowing us to locate another hydroxyl group at C-8.The splitting pattern and J values of H-8 signal (t, J 9.2 Hz) suggested it should be axially oriented.Therefore, the hydroxyl group at C-8 had to be αoriented.COSY experiment showed cross peaks of H-8 signal with protons at δ 1.98 (ddd, H-8a) and δ 3.40 (ddd), so the remaining hydroxyl group should be β-oriented at C-7.In addition, NOE experiments are in agreement with the proposed stereochemistries.Irradiation of H-8 (δ 3.69) showed NOE with H-9 (δ 5.35), and irradiation of H-7 (δ 3.40) led to NOE on both H-8a (δ 1.98) and H-6α (δ 1.41) signals.Dendryol C was previously isolated from Dendryphiella sp. 20and has the same structure proposed for compound 6.However, in dendryol C the 7-OH and 8-OH groups are αand β-oriented, respectively, as evidenced by the splitting patterns and J values of H-7 (δ 3.92, dt, J 3.2 and 2.8 Hz) and H-8 (δ 4.23, dd, J 2.8 and 3.2 Hz).Therefore, compound 6 and dendryol C were assumed as diastereomers.
Compounds 5 and 6 were named dendryol E and dendryol F, respectively, in analogy to the analogue structures of dendryols A-D, previously isolated from the phytopatogenic fungus Dendryphiella sp., and reported as phytotoxic against barnyardgras. 20ovel anthraquinone derivatives have recently been reported from the endophytic fungi Penicillium janthinellum 11 and Pleospora sp. 24P. janthinellum produced known antimicrobial anthraquinones and also a new modified anthraquinone, janthinone, containing a lactone between C-10 and C-4a. 11In addition, deoxybostrycin, altersolanol B and dactylariol (1,2,3,4tetrahydro-9,10-anthraquinones) and a new 1,2,3,4,4a,9ahexahydro-9,10-anthraquinone (pleospdione) were isolated from Pleospora sp.Deoxybostrycin, altersolanol B and dactylariol exhibited significant cytotoxic activity against colon cancer and leukemia cell lines. 24he access to new biological diversity has often afforded new natural products. 1Few chemical investigations were previously carried out only with pathogenic strains of Phoma sorghina, leading to the isolation of phytotoxins. 25,26In this work we described the identification of compounds 4, 5 and 6 as novel anthraquinone derivatives.In addition, this is the first report of Phoma sorghina as an endophyte and its production of anthraquinones, although the production of anthraquinone derivatives by other Phoma species has already been reported. 20,27