Xanthones and Oxepino[2, 3- b ]chromones from Three Endophytic Fungi

DOI: 10.1002/chem.200900749

Xanthones and Oxepino ACHTUNGTRENNUNG[2, 3-b]chromones from Three Endophytic Fungi**

Karsten Krohn,*[a] Simeon F. Kouam,[a, b] Guy M. Kuigoua,[a, b] Hidayat Hussain,[a]

Stephan Cludius-Brandt,[a] Ulrich Flçrke,[a] Tibor Kurt�n,[c] Gennaro Pescitelli,[d]

Lorenzo Di Bari,[d] Siegfried Draeger,[e] and Barbara Schulz[e]

Dedicated to Professor Dr. S�ndor Antus on the occasion of his 65th birthday

Introduction

Xanthones and partially hydrogenated di- or tetrahydroxan-thones are widespread classes of natural products[2] withconsiderable bioactivity[3–11] that occur in fungi,[9,12–14]

plants,[15–18] ferns,[19] and lichens.[20,21] Due to their pro-nounced toxicity, they are classified as mycotoxins.[12,22] Thetetrahydroxanthones occur as monomers, for example, as a-and b-diversonolic esters,[23,24] monodictysins,[25] or blenno-lides, the recently isolated monomeric units of secalonicacids.[26] Other examples of highly active anticancer dihy-droxanthones are globosuxanthone A (11),[27] nidulalin A,xanthoquinodins, and parnafungins.[28] Symmetrical or asym-metrical coupling of identical or slightly different monomer-ic units is very common and often increases the bioactivityand toxicity. Some examples of these highly toxic mycotox-ins include secalonic acids,[29,30] eumetrins,[31,32] xanthonols,[33]

rugulotrosins,[34] hirtusneanoside,[35] phomoxanthone,[36, 37]

and dicerandrols.[38] Herein, we report the discovery of sev-eral new aromatic, hydrogenated, and structurally uniquering-extended xanthones from different endophytic fungi.The different structures of these metabolites, together withrelated compounds from our previous work and examples

[a] Prof. K. Krohn, Prof. S. F. Kouam, G. M. Kuigoua, Dr. H. Hussain,S. Cludius-Brandt, Dr. U. FlçrkeDepartment Chemie, Universit�t PaderbornWarburger Strasse 100, 33098 Paderborn (Germany)Fax: (+49) 5251-603245E-mail : [email protected]

[b] Prof. S. F. Kouam, G. M. KuigouaDepartment of ChemistryHigher Teachers� Training CollegeUniversity of Yaounde I, BP 47, Yaounde (Cameroon)

[c] Dr. T. Kurt�nDepartment of Organic ChemistryUniversity of Debrecen, POB 204010 Debrecen (Hungary)

[d] Dr. G. Pescitelli, Prof. L. Di BariDipartimento di Chimica e Chimica IndustrialeUniversit� di Pisavia Risorgimento 35, 56126 Pisa (Italy)

[e] Dr. S. Draeger, Dr. B. SchulzInstitut f�r MikrobiologieTechnische Universit�t BraunschweigSpielmannstrasse 7, 31806 Braunschweig (Germany)

[**] Biologically Active Secondary Metabolites from Fungi, Part 43; forPart 42, see [1]: J. Dai, K. Krohn, S. Draeger, B. Schulz, Eur. J. Org.Chem. 2009, 1564–1569.

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/chem.200900749.

Abstract: Three new metabolites, mi-crosphaeropsones A–C (1–3) with aunique oxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-one(ring-enlarged xanthone) skeleton,were isolated from the endophyticfungus Microsphaeropsis species, co-oc-curring with their putative biogeneticanthraquinoide precursors citreorosein(4) and emodin (5). From another Mi-crosphaeropsis species, large amountsof fusidienol A (8 a), smaller amountsof emodin (5), the known aromaticxanthones 9 a and 9 b, the new 3,4-dihy-drofusidienol A (8 b), and the new aro-matic xanthone 9 c were isolated. The

endophyte Seimatosporium species pro-duced a new aromatic xanthone, seima-toxanthone A (10), and 3,4-dihydroglo-bosuxanthone A (12), closely related toa-diversolonic ester (13) from Microdi-plodia sp.. The structures were deter-mined mainly by extensive 1D and 2DNMR experiments and supported byX-ray single-crystal analysis of 1 and

the oxidation product 7. The absoluteconfigurations of the microsphaerop-sones A–C (1–3) were established bycomparison of the electronic and vibra-tional circular dichroism (ECD andVCD) spectra of 1 with time-depen-dent DFT (TDDFT) and DFT calcula-tions by using either the solid-statestructures or DFT-optimized geome-tries as inputs. Preliminary studies indi-cated that 1, 2, and enone 7 showed an-tibacterial, fungicidal, and algicidalproperties.

Keywords: anthraquinones · biolog-ical activity · circular dichroism ·density functional calculations ·xanthones

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taken from the literature, enable classification and discus-sion of the typical structural features of naturally occurringxanthones.

Results and Discussion

We recently reported the isolation of a number of aromaticand partially hydrogenated xanthones from Microdiplo-dia[27b] and Blennoria species.[26] In continuation of oursearch for bioactive metabolites from endophytic fungi,[39]

we investigated three different new species: two Micro-sphaeropsis sp. and one Seimatosporium sp. We now reporton the isolation of bioactive metabolites from the culturesof these productive fungi. Three new oxepinoACHTUNGTRENNUNG[2,3-b]chromen-6-ones (a very rare class of ring-enlarged xan-thones), namely, microsphaeropsones A–C (1–3); a knownoxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-one, fusidienol A (8 a);[40,41] thenew 3,4-dihydrofusidienol A (8 b); the two known anthra-quinones citreorosein (4)[42] and emodin (5);[42] the knownpalmarumycine C11 (6)[43] (bipendensin);[44] two known aro-matic xanthones 9 a and 9 b ;[45, 46] and two new aromatic xan-thones, namely, microxanthone (9 c) and seimatoxanthone A(10) were isolated in addition to the common fungal metab-olites ergosterol and 5a,8a-epidioxyergosterol (Scheme 1

and 2).[47, 48] A semisynthetic dihydrooxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-one 7 was prepared by oxidation of the allylic alcohol 1with manganese dioxide. In a second fermentation of thepreviously investigated Microdiplodia sp.,[27b] we discovered3,4-dihydroglobosuxanthone A (12), a bridge to the closelyrelated a-diversolonic ester (13) (revised structure).[24]

The first endophytic fungus Microsphaeropsis sp. (internalstrain no. 8875) was isolated from the shoots of Lycium intri-catum, a thorny, succulent bush from Playa del Ingles(Gomera, Spain). It was cultivated at room temperature ona biomalt solid agar media for 21 days. The cultures, bothfungus and medium, were extracted with ethyl acetate toafford a crude extract that showed antibacterial and antifun-

gal properties and was separated into two fractions by flashcolumn chromatography on silica gel. The less polar fractionwas eluted with petroleum ether and contained mainly fattyacids and lipids and it was not further investigated. Thesecond fraction was further purified by column chromatog-raphy on silica gel and preparative TLC to afford three newcompounds (i.e., 1–3), five known compounds (i.e., 4–6 ; seeScheme 1), and ergosterol and 5a,8a-epidioxyergosterol.

The optically active new compound 1 ([a]D =++280) wasobtained as a yellow gum that crystallized on standing(m.p. 139 8C) with the molecular formula C16H14O8, as de-duced from HRMS (EI) and NMR spectroscopic data. TheIR spectrum showed strong absorptions for the hydroxy andester carbonyl groups at 3400, 1740, and 1650 cm�1, whereasthe 1H NMR spectrum (see the Experimental Section) ex-hibited the presence of one allylic methyl group (dH =

1.70 ppm) and a chelated phenolic hydroxy group (dH =

11.92 ppm). The 1H NMR spectra also showed signals atdH = 5.19 and 3.49 ppm, which were assigned to two hydroxygroups as evidenced by analysis of the HMQC and HMBCspectra (Figure 1). The 13C NMR spectrum of 1 (see the Ex-

perimental section) displayed signals for sixteen carbonatoms, and the DEPT spectrum indicated the presence ofone allylic methyl (dC =14.2 ppm), one methoxy (dC =

53.3 ppm), five methines (dC =137.0, 135.6, 111.9, 106.6,71.7 ppm), and nine quaternary carbon atoms, including twocarbonyl groups (dC =185.1, 172.0 ppm). According to theHMQC experiment, the signals at dC =76.4 and 71.7 ppmcan be assigned to an oxygenated quaternary sp3 carbon andan oxymethine group. In addition, analysis of the couplingpattern of the 1H NMR spectrum showed the presence ofthree vicinal protons on a benzene nucleus at dH =6.80 (d,J=8.3 Hz), 7.46 (t, J=8.3 Hz), and 6.75 ppm (d, J=8.3 Hz).In the HMBC experiment (Figure 1), one of the hydroxyresonances (dH = 5.19 ppm) displayed correlations withcarbon atoms that resonate at dC =103.6 (C9a), 76.4 (C1),71.7 (C2), and 172.0 ppm (ester carbonyl group). The reso-nance at dC =14.2 ppm (C12) showed 3J correlations withthe olefinic carbon at dC =137.0 ppm (C4) and the oxyme-thine carbon at dC = 71.7 ppm (C2). Thus, the ester carbonylgroup was placed at C1. The a position of this group wasunambiguously established on the basis of its NOESY spec-trum. A correlation was observed between the hydroxygroup at dH = 5.19 ppm and the proton signal at dH =

Scheme 1. Compounds 1–6 isolated from the endophytic fungus Micro-sphaeropsis sp. (8875) and 7 obtained by the oxidation of 1.

Figure 1. Selected 1H–1H COSY and HMBC correlations for 1 and 3.

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4.94 ppm (H2). These data, and in particular the additionaloxygen atom shown in the mass spectrum in comparison tonormal xanthones, suggested a ring-enlarged xanthone,namely an oxepino ACHTUNGTRENNUNG[2, 3-b]chromen-6-one system, as presentin fusidienol A (8 a).[40,41] The assignment was further con-firmed by 1H–1H COSY, HMQC, and HMBC correlations(Figure 1) and definitely established by X-ray crystallo-graphic analysis of a single crystal of 1 (Figure 2). Enone 7

(Scheme 1) was prepared by oxidation of the allylic hydroxygroup at C2 of microsphaeropsone A (1) with manganese di-oxide; furthermore, single crystals suitable for X-ray crystal-lographic analysis could also be obtained (Figure 3). On the

basis of the X-ray crystallographic data, the compound wasassigned structure 1 and named microsphaeropsone A, afterthe producing fungus. To the best of our knowledge, this ex-ample is the first dihydrooxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-onesystem to be found in nature. Fusidienol A (8 a)[40,41] has acorresponding but completely unsaturated system(Scheme 2). Interestingly, isomeric chromone-3-oxepines,isofusidienols, were recently isolated from the endophyticfungus Chalara sp.[49a]

To assign the absolute configuration of microsphaerop-sone A (1) and related compounds, their electronic and vi-brational circular dichroism (ECD and VCD) spectra wererecorded and interpreted through TDDFT and DFT calcula-tions,[50–53] respectively. Because the X-ray crystal structureof 1 was available, two possibilities were accessible for theapplication of ECD TDDFT calculations by using either thecalculated solution structure or the solid-state X-ray crystalgeometry. UV/Vis absorption and ECD spectra of 1 were,therefore, recorded in solutions of acetonitrile and chloro-form and in a glassy KCl matrix (Figure 4). The complex

chromophoric system gives rise to several transitions in therange 185–400 nm. The first absorption band at 323 nm(CH3CN) is allied with a weak CD signal, that is, a Cottoneffect (CE), in solution, with a low g factor De/e and signal-to-noise ratio, whereas a weak negative exciton couplet isobserved in the solid state. Other CD bands (a weak nega-tive CE at 295 nm, and a structured overlap of various posi-tive CEs in the 225–286-nm region) are instead more pre-served under the various conditions, at least down to225 nm. Below that wavelength, a moderate negative CE at215 nm followed by a stronger positive CE are detected insolution, which appear as a trough and a positive CE at198 nm in the solid state.

The solid-state CD spectrum may be compared with aTDDFT-calculated CD spectrum by using the X-ray crystalgeometry as the input structure. This approach, known assolid-state CD/TDDFT,[26,27b,54–61] was recently developed bysome of the authors and has the advantage of making con-formational searches superfluous. The unit cell of singlecrystals of 1 contains two independent molecules, molA andmolB, which differ slightly from each other (root meansquare (RMS) deviation between heavy atoms=0.13 �). In

Figure 2. Molecular structure of molecule A of 1 in the crystal. Displace-ment ellipsoids are drawn at the 50% probability level.

Figure 3. Molecular structure of the semisynthetic enone 7 obtained fromcrystallographic studies. Displacement ellipsoids are drawn at the 50 %probability level.

Figure 4. Experimental ECD spectra of 1 in solutions of CH3CN andCDCl3 and in the solid state as a KCl disc.

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both structures (molA is shown in Figure 2), ring A lies in adistorted-boat conformation,[62] in which atoms C2, C1, C9a,C4, and O11 are almost coplanar, as is also true for atomsC2, C3, C4, and O11, with the two planes that define a dihe-dral angle of about 608. The two OH groups attached at C1and C2 are in a gauche-diequatorial arrangement, whereasthe axial carboxylic group has its C=O bond almost eclipsedwith C1�O. Upon reoptimization of the hydrogen atomswith the DFT method (B3LYP/6-31G(d)), three intramolec-ular hydrogen bonds are obtained (C8-OH···O=C9, C1-OH···O=C13, and C2-OH···OC1). The ECD/TDDFT calcu-lations on X-ray crystal structures with a 1R,2R absoluteconfiguration led to moderately different results dependingon the functional employed (BP86, B3LYP, andBH&HLYP; in all cases, the TZVP basis set was used). Thebest match between the calculated and experimental solid-state ECD spectrum was observed with the BH&HLYP/TZVP method. Different spectra were obtained for the twostructures molA and molB, which were then averaged(Figure 5). Apart from a wavelength shift of about Dl=

50 nm to the left, the calculated average spectrum reprodu-ces well the relative position and intensity of CD bandsbelow 300 nm in the experimental solid-state spectrum(Figure 4, dotted line). At longer wavelengths, a small nega-tive band is predicted instead of the negative couplet foundexperimentally. This latter couplet may conceivably be dueto the exciton coupling between distinct molecules closelypacked in the solid state. In each unit cell, molA and molB

lie with aromatic rings almost parallel to each other at a dis-tance of �3.3 � in a skewed, chiral fashion with a twistangle between long axes (that pass through the centers of

rings B and C) of about �1108. Because the first p–p* tran-sition responsible for the absorption band at 323 nm is ap-proximately polarized along the same long axis in each chro-mophore, the geometrical arrangement between molA andmolB is suitable for an efficient exciton coupling. It is clearthat any feature that appears in the solid-state ECD spec-trum that is of intermolecular origin[63,64] cannot be predict-ed by single-molecule ECD calculations, therefore the pooragreement between the solid-state experimental and calcu-lated ECD spectra above 300 nm is understandable.

To check the consistency between the solution and solid-state chiroptical properties of 1, we decided to reproduce so-lution ECD spectra as well. This process required a full con-formational analysis run by means of a molecular-mechanicsconformational search followed by DFT geometry optimiza-tions of all minima thus found. The results were thenchecked against NMR spectroscopic data, such as 3JC�H cou-plings between C2�H and its relative b carbon atoms. Final-ly, 13C NMR spectra were calculated on DFT-optimized geo-metries and compared with the experimental set[65] (see theSupporting Information for details of the whole procedure).The ECD/TDDFT calculations on DFT-optimized geome-tries with a 1R,2R configuration were again in agreementwith the experimental ECD solution spectra below 300 nm(see Figure S1 in the Supporting Information). However,several factors that affect the calculated spectra made itseem advisable for us to check for a further independent as-signment. These included 1) the above-mentioned depend-ence on the functional employed, with B3LYP and BP86leading to poorer agreement with the experiment; 2) the ap-parent overlap between several CEs in the significant regionof the spectrum; 3) the high-lying nature of the virtual states(with positive eigenvalues)[66] involved in all computed tran-sitions from the third state onward.

Then, we resorted to measuring the VCD spectrum andcomparing it with the DFT-calculated spectrum, an ap-proach known to allow for reliable absolute configurationalassignments at low computational cost, at least for mole-cules with restricted flexibility.[52,53] Figure 6 shows the VCDspectra measured for a solution of 1 in CDCl3 at 900–1800 cm�1 (see also Figure S3 in the Supporting Informationfor the IR spectrum). VCD calculations were run on DFT-optimized geometries of 1, with the solvent effect taken intoaccount with the SCRF-PCM model[67,68] (see the SupportingInformation for the calculation details). Boltzmann-aver-aged IR and VCD spectra calculated for (1R,2R)-1 were innice agreement with experimental spectra for a large majori-ty of bands (Figures 6 and S3 in the Supporting Informa-tion). As a result of VCD and two independent ECD calcu-lations, which came to the same conclusion, the absoluteconfiguration of microsphaeropsone A could be safely as-signed as (1R,2R)-(1).

The second new compound, namely, microsphaeropsoneB (2), was isolated as an optically active amorphous gum([a]D =++ 11). Its molecular formula was established asC16H14O7 by HRMS (ESI) at m/z 319.0812 [M+ H]+ (calcd:319.0818), 16 mass units less than that observed for 1. The

Figure 5. TDDFT-calculated ECD spectra of the solid-state structure of(1R,2R)-1 (BH&HLYP/TZVP). The two independent molecules A and Bfound in the unit cell were considered and their average spectrum was es-timated.

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absorption bands in the IR and UV spectra (see the Experi-mental section) were similar to those of 1. The presence oftwo carbonyl groups was confirmed by the 13C NMR spec-trum with resonances at dC = 182.8 and 172.6 ppm. The1H NMR spectrum of 2 (see the Experimental Section) wasvery similar to that of 1, except for the missing singlet atdH = 5.19 ppm assigned to C1�OH of microsphaeropsone A(1). In addition, the spectrum of 2 showed a spin system(dH =4.74 ppm, d, J= 3.4 Hz; dH =4.53 ppm, dd, J= 3.4,11.0 Hz; and dH = 3.94 ppm, d, J= 11.0 Hz) that suggestedthe presence of the fragment (�CHCHOH), which was fur-ther confirmed by the COSY spectrum. The proposed struc-ture was further supported by 13C NMR spectroscopic data(see the Experimental Section) with assignments based onthe DEPT, HMQC, and HMBC spectra. On the basis of theco-occurrence of 1 and 2 in the same fungus and the cou-pling constant of J1,2 = 3.4 Hz,we propose the a configurationfor the methoxycarbonyl andhydroxy group in microsphaer-opsone B (2) and a 1S,2S con-figuration (the inversion withrespect to 1 is only formal;Scheme 1). The cis-relativeconfiguration of 1-CO2CH3

and C2-OH was further sup-ported by the very sluggish b-elimination under thermal-,acid-, or base-catalyzed reac-tions, because the trans-diaxialorientation of H1 and 2-OHrequired for the elimination in

the CO2CH3/OH cis-relative configurations has a relativelyhigh energy. The microsphaeropsones A (1) and B (2) repre-sent the first dihydrooxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-ones to befound in nature.

Compound 3 was obtained as an amorphous gum with themolecular formula C16H12O7 as deduced from HRMS (EI)spectral and 13C NMR spectroscopic data. The 1H NMRspectrum (see the Experimental Section) exhibited resonan-ces that were similar to those of microsphaeropsones A (1).A comparison of the 1H and 13C NMR spectra (see the Ex-perimental Section) of microsphaeropsone C (3) with thoseof 1 and 2 showed that ring A was fully unsaturated, con-firmed by the low-field resonance at dH = 7.37 ppm that cor-responds to H4 in the 1H NMR spectrum for the vinylic con-jugated proton. This finding was further supported by13C NMR spectroscopic data (see the Experimental Section)and by comparison with the reported values for fusidienol A(8 a).[40, 41] Thus, the structure of microsphaeropsone C (3)was established as (2Z,4E,5aZ)-methyl 7-hydroxy-9-methyl-6-oxo-6H-oxepinoACHTUNGTRENNUNG[2,3-b]chromene-5-carboxylate.

The two known anthraquinones citreorosein (4)[42] andemodin (5),[42] the known palmarumycine C11

[43] (bipende-nin)[44] (6), ergosterol,[47] and 5a,8a-epidioxyergosterol[48]

were identified by comparison of their spectra with reporteddata. The co-occurrence of the ring-extended xanthones 1–3with anthraquinones 4 and 5 is very interesting from a bio-synthetic point of view; the latter compounds are putativeprecursors of xanthones.[69] In addition, the co-occurrence ofoxepino ACHTUNGTRENNUNG[2,3-b]chromenes and anthraquinones with palmaru-mycines, all putative polyketide-derived secondary metabo-lites, sheds light on the diversity of the Polyketide Synthe-tase II enzyme system of this Microsphaeropsis sp.

Another endophytic Microsphaeropsis sp. (internal strainno. 7177) was isolated from the shoots of the plant Zygo-phyllum fortanesii, a halotolerant succulent from Valle GranRey (Gomera, Spain). From the crude extract of the culturein ethyl acetate, chromatography on silica gel afforded theknown fusidienol A (8 a)[40,41] and the known aromatic xan-thone 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylicacid methyl ester (9 a)[45,46] (Scheme 2). The co-occurrenceof the ring-extended and aromatic xanthones 8 a and 9 a in

Figure 6. Experimental (0.3 m CDCl3 solution, 50-mm cell) and calculatedVCD spectrum of (1R,2R)-1. Calculated spectrum is Boltzmann-averagedover three lowest-energy DFT structures (computed with DFT, B3LYP/6-31G(d), including the solvent, with the self-consistent reaction field–po-larizable continuum model (SCRF-PCM); see the Supporting Informa-tion for details).

Scheme 2. Anthraquinone derivative 5 and xanthone derivatives 8 a–9c isolated from Microsphaeropsis sp.(7177) and 10 from Seimatosporium sp. (8883).

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the same fungus indicates their biogenetic relationship. Thisconnection is also supported by the fact that monocyclic ox-epins are in equilibrium with their 1,2-epoxybenzene deriva-tives and that acid catalysis can convert them into phenolsby a 1,2-hydride shift.[49] In addition, the very raredihydrooxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-ones occurred in both Mi-crosphaeropsis isolates. However, the position of the methyland hydroxy groups in the metabolite fusidienol A (8 a) dif-fers from that of the microsphaeropsones 1–3, thus suggest-ing a somewhat different biosynthetic pathway in the twofungi of the same genus. This interesting change in themethyl group position also occurs in xanthones from otherfungal genera (see Scheme 4).

Four additional metabolites were isolated as minor com-ponents from Microsphaeropsis sp. (7177). The putative pre-cursor emodin (5) was isolated in trace amounts in additionto one known xanthone, that is, methyl 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (9 b),[70] and a newxanthone 9 c, named microxanthone after the producingfungus obtained as a yellow solid from the extract of Micro-sphaeropsis sp in ethyl acetate. In addition to these aromaticxanthones, another new non-aromatic ring-enlarged 3,4-di-hydrofusidienol A (8 b) was isolated as a minor component.The structure was mainly elucidated by comparison of itsNMR spectra with that of fusidienol A (8 a).[41] A noticeabledifference was the occurrence of 1H and 13C NMR signalsfor two methylene groups at dH =2.65 (dt, J= 6.5, 5.4 Hz;dC =28.7 ppm) and 4.65 ppm (t, J=6.5 Hz; dC = 78.4 ppm) ofC3 and C4, respectively. The mass spectrum of 3,4-dihydro-fusidienol A (8 b) showed [M+] at 302.0790 in HRMS (EI),two mass units more than that of fusidienol A (8 a),[41] thusindicating the two additional protons. These differenceswere supported by 1H–1H COSY correlations of H2 to H3and H3 to H2 and H4; furthermore, the HMBC correlationsof H2 to CO2CH3, C1, C3, C4; H3 to C1, C2, C4; and H4 toC2, C3, C4, C4a provided further evidence. Thus, 3,4-dihy-drofusidienol A (8 b) was determined to be (4E,5aZ)-meth-yl 7-hydroxy-9-methyl-6-oxo-3,6-dihydro-2H-oxepinoACHTUNGTRENNUNG[2,3-b]-chromene-5-carboxylate. It is worth noting that the dihydrocompounds usually co-occur with the fully desaturatedparent compounds (see 3,4-dihydroglobosuxanthone A (12);Scheme 3).[49]

The molecular formula of microxanthone (9 c) was deter-mined to be C16H12O6 by HRMS (EI). The 1H and 13C NMRspectra exhibited the presence of 11 proton and 16 carbonsignals, respectively. Fourteen of the carbon signals were inthe region dC = 107.0–180.3 ppm, which is indicative ofhighly substituted aromatic and heteroaromatic ring systems.The 1H NMR spectrum of 9 c in CDCl3 displays the presenceof two 3H singlets for an aromatic methyl proton (dH =

2.43 ppm), meta-coupled aromatic protons (dH = 6.78,6.65 ppm, d, J= 1.0 Hz), and a chelated hydroxy group (dH =

12.13 ppm), whereas a 13C NMR signal at dC = 180.3 ppmwas assigned to a chelated carbonyl carbon (see the Experi-mental section). The HMBC correlations of 1-OH to C2,C9a; H2 to C1, C3, C4; and H4 to C2, C3, C4a suggestedthat the left-side ring A of 9 c is identical to the known xan-

thones 9 a and 9 b.[70] Two ortho-coupling doublets at dH =

7.31 and 7.23 ppm for H6 and H7 and both the 1H NMR(dH =3.99 ppm, s, 3 H) and 13C NMR spectra (dC = 169.6 and53.0 ppm) show the presence of a methyl ester. The substitu-tion pattern of 9 c on the side ring was based on the HMBCcorrelations of H6 to C4b, C5, C7, C8 and H7 to CO2CH3,C5, C6, C8, C8a. Thus, microxanthone (9 c) was determinedto be methyl 4,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate. It is interesting to note that all three aromaticxanthones 9 a–c can plausibly be generated by eliminationor allylic rearrangement from the diversolonic ester type(13) non-aromatic xanthones (Scheme 2 and 3).

Finally, the extract of the endophytic fungus Seimatospori-um sp. (internal strain no. 8883) in ethyl acetate that hadbeen isolated from the halotolerant herbaceous plant Salsolaoppositifolia from Playa del Ingles (Gomera, Spain) was in-vestigated for bioactive metabolites. Seimatoxanthone A(10) was obtained as a yellow solid and was assigned themolecular formula C16H14O6 by HRMS (EI). The IR spec-trum showed absorptions at 3400 and 1610 cm�1 for the OHgroup and a conjugated carbonyl functionality, respectively,which also appeared at dC = 178.0 ppm (C9) in the 13C NMRspectrum of 10. Inspection of the 1H NMR spectrum of sei-matoxanthone A (10) showed two double doublets and onetriplet for AMX-type protons at dH =7.07 (t, J=8.0 Hz),7.65 (dd, J= 8.0, 1.5 Hz), and 7.58 ppm (dd, J=8.0, 1.5 Hz);one singlet proton at dH = 6.45 (s); and three methoxygroups at dH = 3.95 (s), 3.91 (s), and 3.85 ppm (s). The13C NMR spectrum showed 16 signals that, according to theDEPT spectrum, could be attributed to three CH3 units,four CH units, and nine quaternary carbon atoms, includingsix oxygenated carbon atoms. These results indicated that 10has a xanthone skeleton.[71] Irradiation of the aromatic sin-glet at dH = 7.58 ppm caused NOE enhancements of the che-lated hydroxy group at dH =12.17 ppm, thus suggesting thatthe latter must be attached to C8 with a system of AMX-type with three aromatic protons in one aromatic ring. Thisconclusion was further supported by the HMBC spectrumwith cross-peaks between the chelated hydroxy group andC7, C8, C8a and important HMBC and NOE correlations ofthe three AMX-type aromatic protons (Figure 7). The loca-tion of the methoxy groups at C1, C2, and C4 was also de-duced unambiguously from NOE correlations (Figure 7).The structures of ergosterol[47] and 5a,8a-epidioxyergoster-ol,[48] which were also isolated from this fungus, were con-firmed by comparison of their reported spectroscopic data.

Figure 7. Important 1H–13C HMBC and 1H–1H NOE correlations in theNMR spectrum of 10.

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The reinvestigation of a Microdiplodia sp.[27b] resulted inthe isolation of 3,4-dihydroglobosuxanthone A (12 ;Scheme 3), in addition to the previously isolated metabolites

2-hydroxyvertixanthone,[27, 72] vertixanthone,[27,72] globosuxan-thone A (11),[27] and the usual fungal metabolite ergoster-ol.[47] The NMR spectra of the new 3,4-dihydroglobosuxan-tone 12 (see the Experimental Section) had similarities withthe relevant data of globosuxanthone A (11), except thatthe two signals for vinylic protons were replaced by signalsfor methylene protons that showed the expected dddd multi-plicity for the H3 protons and the ddd pattern for the H4protons. This outcome was confirmed by the 1H–1H COSYcorrelations and the mass spectrum with two more massunits than recorded for 11 (m/z 306 [M+]). The couplingconstant of J2,3a =10.3 Hz for diaxial protons showed theequatorial position of 2-OH,similar to that of the parentcompound 11,[27] thus suggest-ing the same stereochemistry.This deduction was furtherconfirmed by hydrogenation ofa small sample of 11 to the cor-responding 3,4-dihydro com-pound 12.

Having these metabolites ofdifferent xanthones of fungalorigin and those elucidated inour previous investigations inhand,[26,27b] a critical compari-son of their structures and thereported structure is possible,which is interesting with re-spect to biosynthesis, taxono-

my, drug development,[73] and the generation of the chemicaldiversity of the tetrahydroxanthones. The a- and b-diverso-lonic esters, isolated in 1983 from Penicillium diversum,have been known for a long time.[23] Their structures wererecently shown to be incorrect[26] and were revised to 13, ana-diversolonic ester (Scheme 3), by total synthesis.[24] Withthe knowledge of their recently corrected structures (the b-diversolonic ester is the 2-epimer of 13), the structural simi-larity to 3,4-dihydroglobosuxanthone A (12) produced byMicrodiplodia sp. is evident. However, the presence of amethyl group at C6 in the diversolonic esters is worthnoting. In contrast to the globosuxanthones and diversolonicesters (which can be considered to belong to the “type-B”tetrahydroxanthone family), most other hydrogenated xan-thones have the ester group at the angular C4a position, asshown in the selected structures 17 and 18 (“type-A” tetra-hydroxanthones). In fact, this outcome points to the biosyn-thetic origin from anthraquinones by oxidative cleavage tobenzophenone intermediates, as already discussed byHolker et al.[23] and shown in Scheme 4 with chrysophanolas one possible precursor (20). The oxidative opening ofchysophanol leads to type-A xanthones with a C6 methylgroup (pathway a; methyl and ester groups on differentsides, e.g., 16) or to the respective type-B xanthones with a3-methyl group (pathway b; methyl and ester groups on thesame side, e.g., 17) through the benzophenone intermediates19 or 21, respectively (Scheme 4).

Both pathways initially lead to tetrahydroxanthones withan ester group at the angular position C4a (see 17 and 18 inScheme 3). The ester group in type-A xanthones can furtherbe reduced at the hydroxymethyl level, as present in pho-moxanthone[36,74] and xanthonol monomers,[9] or to themethyl stage, as in diversonol (14)[75] or monodictyns (15).[25]

A very interesting question is whether one fungus can useboth pathways a and b in the synthesis of tetrahydroxan-thones. From an endophytic Blennoria sp., we recently iden-tified seven new xanthone derivatives of type A, namely,blennolides.[26] Interestingly, blennolide C (16) is a 6-methylderivative, produced by pathway a, whereas, from the same

Scheme 3. Structure of 12 from Microdiplodia sp. and comparison withrelated xanthones from other fungi.

Scheme 4. Putative biosynthesis of tetrahydroxanthones with a methyl group at C3 or C6.[23]

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FULL PAPERIsolation and Characterization of Xanthone Derivatives from Endophytic Fungi

fungus, bennolide A (17) has a C3 methyl group producedby pathway b. Thus, from this evidence, both pathways canco-occur in the same fungus. Moreover, rearranged g-lac-tones, such as blennolide E (18), can also be produced bythe same microorganism to increase the diversity of xan-thone derivatives even further. The different placement ofthe methyl group also occurs within the ring-enlarged xan-thones with the dihydrooxepino ACHTUNGTRENNUNG[2,3-b]chromen-6-one skele-ton. In 1 and 2 from cultures of the endophytic Micro-sphaeropsis sp. (8875), the methyl group is placed on theseven-membered ring A, whereas in fusidienol A (8 a)[40,41]

from another Microsphaeropsis sp. (7177) the methyl groupis located at the aromatic ring C.

Bioactivity : The antibacterial, fungicidal, and algicidal prop-erties of seven selected compounds are compiled in Table 1in comparison to a number of standard antibiotics and the

solvent acetone. All the compounds have excellent antibac-terial activities, of particular note is the antibacterial activityagainst the gram-negative bacterium Escherichia coli. Thesecompounds are also algicidal, with the exception of aromaticxanthone 9 a and 3,4-dihydroglobosuxanthone A (12). It isremarkable that the oxidation product of microsphaerop-sone A (1) shows significant antifungal properties in con-trast to the desaturated microsphaeropsones C (3).

In summary, structurally unique ring-expanded (i.e., 1–3),aromatic (i.e., 9 c and 10), and hydrogenated xanthone de-rivatives (i.e., 8 b and 12) have been isolated from differentendophytic fungi. Analysis of these hydrogenated xanthonestructures and those reported previously revealed an aston-ishing ability of the fungal enzyme system in the generationof a diversity of monomeric and dimeric xanthones. Thechemical principles are surprisingly simple and some of thefeatures are aromatic versus hydrogenated xanthones, ringextension, rearrangements, different placement of themethyl group(s), reduction of the ester group, and, very im-

portantly, creation of stereoisomers. In addition, the dimeri-zation of these different monomers (even heterodimers oflinear (e.g., 16) and rearranged (e.g., 18) xanthones areknown)[26,76] further extends the chemical diversity of hydro-genated xanthones and creates metabolites of potentiatedbioactivity relative to the monomers.

Experimental Section

General : For general methods and instrumentation, see ref. [74]; for mi-crobiological methods and conditions of culture, see ref. [77]; and fortests for biological activity, see ref. [78]. The melting points were deter-mined on a Gallenkamp micromelting point apparatus and are uncorrect-ed. The NMR spectra were recorded on a Bruker-500 NMR avance spec-trometer and a Varian VXR 600 spectrometer. The 3JC,H couplings weremeasured by means of pulsed field gradient HMBC spectra recorded byvarying the J-refocusing time between t=0.04 and 0.14 s (interval:10 ms), which corresponds to J= 1/(2t)=3.6–12.5 Hz. The 3JC,H valueswere estimated with least-squares sinusoidal fits of the experimentallydetermined cross-peak intensities as a function of J. Mass spectra wereobtained on a MAT 8200 mass spectrometer (EI). The ECD spectra wererecorded on a J-810 spectropolarimeter. The VCD spectra were obtainedon a Jasco FVS-4000 spectrometer using default acquisition parameters(resolution: 4 cm�1) and 4000 accumulations (total scan time: �1 h). Thecompounds were detected on TLC plates (Merck AG; silica gel 60 F254)by spraying with cerium–molybdenum spray reagent followed by heating.

Extraction and isolation : The endophytic fungus Microsphaeropsis sp.(strain 8875) was isolated from the plant Lycium intricatum fromGomera (Spain). It was cultured for 21 days on biomalt solid agar (12 L,5% w/v) at room temperature. The culture and medium were extractedwith ethyl acetate to give a crude extract (5.6 g). A portion of this extract(5 g) was dissolved in a minimum amount of ethyl acetate. The solutionwas kept at room temperature and the mixture of compounds precipitat-ed by the addition of petroleum ether. After filtration, a solid mixturewas obtained (350 mg) and purified by column chromatography on silicagel with CH2Cl2/MeOH (98:2) as the eluant to afford 4, (7 mg), 5(15 mg), and 6 (23 mg). The filtrate was concentrated and the residue(4.5 g) was subjected to flash column chromatography on silica gel usingpetroleum ether with a gradient of ethyl acetate as the eluent. The elu-ates were monitored by TLC analysis, and similar fractions were com-bined to give three main fractions (A–C). Fraction A (2.8 g), which con-tained mainly fatty acids and lipids, was not further investigated. FractionB (1.6 g) was purified by column chromatography on silica gel with pe-troleum ether/ethyl acetate (80:20) as the eluant and the third fraction ofthe chromatography (0.9 g) was further purified on column chromatogra-phy on silica gel to afford 1 (50 mg), 2 (5 mg), and ergosterol (246 mg).Fraction C (1.2 g) was purified again by column chromatography on silicagel with varying proportions of petroleum ether and ethyl acetate (15–45%) as the eluent to afford xanthone 3 (3 mg).

Another Microsphaeropsis sp. (internal strain no. 7177) was isolated fromthe plant Zygophyllum fortanesii, also from Gomera (Spain). From theextract of the culture in ethyl acetate (cultivated on biomalt solid agarmedium (12 L, 5% w/v) at room temperature for 21 days), the crude ex-tract was isolated (20.6 g) and column chromatography on silica gel withgradients of ethyl acetate/petroleum ether afforded the known fusidienolA (8a ; 3.56 g) and the known aromatic xanthone (9 a ; 0.57 g) as themajor compounds. The mother liquors from crystallization of 8a and 9aand the intermediate fractions were combined and rechromatographedon silica gel with gradients of ethyl acetate/petroleum ether to afford,with increasing polarity, emodin (5 ; 6.7 mg), 3,4-dihydrofusidienol A (8b ;5.2 mg), and xanthones 9b (6.3 mg) and 9c (7.2 mg).

The endophytic fungus Seimatosporium sp. (internal strain no. 8883) wasisolated from Salsola oppositifolia, from Gomera (Spain), and was culti-vated on biomalt solid agar medium (12 L, 5% w/v) at room temperaturefor 21 days. The cultures were then extracted with ethyl acetate to afford

Table 1. Biological activities of pure metabolites against microbial testorganisms in an agar diffusion assay.[a]

Metabolites/ Radius of the zone of inhibition [mm]standards Escherichia

coliBacillusmegaterium

Microbotryumviolaceum

Chlorellafusca

1 8 10 0 123 6 10 0 94 9 10 10 107 11 6 10 6

8 a 8 10 9 149 a 7 13 0 1012 7 8 0 10

penicillin n.t. 18 0 0tetracycline n.t. 18 0 10 gi

nystatin n.t. 0 20 0actidione n.t. 0 50 35

acetone n.t. 0 0 50

[a] Conditions: A 50-mL aliquot (concentration: 1 mgmL�1, which equals0.05 mg of substance/test filter disc) of the pure and control substanceswere tested in an agar diffusion assay. n.t.=not tested, gi =partial growthinhibition, that is, there was some growth within the zone of inhibition.

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K. Krohn et al.

a residue (3.5 g). The extract was separated into two fractions by columnchromatography on silica gel with a gradient of n-hexane/ethyl acetate(90:10, 50:50, 0:100) as the eluent. The least polar fraction 1 (2.1 g) con-tained mainly fatty acids and lipids. The next polar fraction 2 (1.1 g) wasseparated by column chromatography on silica gel with n-hexane/ethylacetate (10:1, 5:1) to give two subfractions A and B. Fraction B was sepa-rated by column chromatography on silica gel with n-hexane/ethyl ace-tate (7:3) to give seimatoxanthone A (10 ; 10.2 mg) and 5a,8a-epidioxyer-gosterol (7.3 mg). Similarly, fraction A was separated by column chroma-tography on silica gel with n-hexane/ethyl acetate (8.5:1.5) to give ergo-sterol (13.2 mg).

The endophytic fungus Microdiplodia sp. (internal strain no. 7092) wasisolated from Erica arborea, from Gomera (Spain),[27b] and cultivated in asecond fermentation on biomalt solid agar medium (2 L, 5 % w/v) atroom temperature for 28 days. The culture medium was then extractedwith ethyl acetate to afford a residue (1.4 g) after removal of the solventunder reduced pressure. The extract was separated by column chroma-tography on silica gel as described previously[27b] to afford 2-hydroxyver-tixanthone (11 mg), vertixanthone (10 mg), globosuxanthone A (11;5 mg), ergosterol (25 mg), and the new 3,4-dihydroglobosuxanthone A(12 ; 15 mg).

Microsphaeropsone A (1): Yellow crystals; m.p. 139 8C; [a]D =++280 (c=

1.1 in CH2Cl2), [a]D =++249 (c =0.059 in CH2Cl2); CD (MeCN, c =3.04 10�4): lmax (De)=331 (0.16), 295 (�0.71), 269 (4.12), 237 (5.54), 215(�2.11), 197 sh (7.38), 187 nm (13.34 mol�1 dm3 cm�1); CD (CDCl3, c=

6.2 10�3) lmax (De)= 324 (0.56), 294 (�0.42), 272 (4.88), 253 (5.53),241 nm (5.95 mol�1 dm3 cm�1); CD (KCl, 37 mg in 258 mg of KCl): lmax

(mdeg)=359 (�0.76), 325 (1.24), 296 (�0.72), 269 (4.56), 235 (4.59),198 nm (8.81); 1H NMR (500 MHz, CDCl3): d= 11.92 (s, 1H; 8-OH), 7.46(t, J =8.3 Hz, 1H; H6), 6.80 (d, J =8.3 Hz, 1H; H5), 6.75 (d, J =8.3 Hz,1H; H7), 6.52 (s, 1 H; H4), 5.19 (s, 1H; 1-OH), 4.94 (s, 1H; H2), 3.77 (s,3H; OMe), 3.49 (s, 1H; 2-OH), 1.70 ppm (s, 3 H; 3-Me); 13C NMR(125 MHz, CDCl3): d=185.1 (s; C9), 172.0 (s; C13), 162.9 (s; C4a), 160.5(s; C8), 152.9 (s; C10a), 137.0 (d; C4), 135.6 (d; C6), 129.5 (s; C3), 111.9(d; C7), 108.6 (s; C8a), 106.6 (d; C5), 103.6 (s; C9a), 76.4 (s; C1), 71.7 (d;C2), 53.3 (q; OMe), 14.2 ppm (q; 3-Me); IR (KBr): nmax =3426, 1733,1652, 1436, 812 cm�1; (CHCl3 + MeOH): 3400 (OH), 1740 (COO),1650 cm�1 (CO); UV (MeOH): lmax (log e)= 322 (3.83), 272 sh (3.81),242 nm (4.21 mol�1 dm3 cm�1); MS (EI, 70 eV): m/z (%): 334 [M+] (23),287 (18), 284 (49), 275 (62), 257 (42), 229 (100), 205 (80), 137 (61), 97(20); HRMS (ESI, 70 eV): m/z : calcd for C16H15O8: 335.0767; found:335.0761 [M+H]+ .

Microsphaeropsone B (2): Amorphous gum; [a]D =++11 (c =0.04 inCH2Cl2); 1H NMR (500 MHz, CDCl3): d= 12.34 (s, 1H; 8-OH), 7.50 (t,J =8.3 Hz, 1 H; H6), 6.83 (dd, J=8.3, 0.8 Hz, 1 H; H5), 6.81 (dd, J =8.3,0.8 Hz, 1H; H7), 6.29 (s, 1H; H4), 4.74 (d, J =3.4 Hz, 1H; H1), 4.53 (dd,J =11.0, 3.4 Hz, 1 H; H2), 3.94 (d, J=11.0 Hz, 1 H; 2-OH), 3.77 (s, 3H;OMe), 1.82 ppm (s, 3H; 3-Me); 13C NMR (125 MHz, CDCl3): d=182.8(s; C9), 172.6 (s; C13), 163.2 (s; C4a), 160.5 (s; C8), 153.4 (s; C10a),135.2 (d; C6), 132.1 (d; C4), 125.1 (s; C3), 111.9 (d; C7), 108.3 (s; C8a),106.5 (d; C5), 98.1 (s; C9a), 68.4 (d; C2), 43.8 (s; C1), 52.7 (q; OMe),16.1 ppm (q; 3-Me); IR (KBr, film): nmax =3515, 1745, 1650, 1234, 1093,1031, 815 cm�1; UV (MeOH): lmax (log e)=322 (3.85), 278 sh (3.85),243 nm (4.23 mol�1 dm3 cm�1); MS (EI, 70 eV): m/z (%): 318 (100), 287(10), 259 (22), 230 (56), 217 (32), 189 (13), 149 (83), 137 (67), 121 (37), 83(27), 69 (51), 57 (59); HRMS (ESI, 70 eV): m/z : calcd for C16H15O7:319.0818; found: 319. 0812 [M+H]+.

Microsphaeropsone C (3): Amorphous gum; 1H NMR (500 MHz,CDCl3): d=12.36 (s, 1 H; 8-OH), 7.55 (t, J =8.4 Hz, 1 H; H6), 7.37 (s,1H; H4), 6.90 (d, J =8.4 Hz, 1H; H5), 6.78 (d, J =8.4 Hz, 1 H; H7), 3.93(s, 3H; OMe), 2.42 ppm (s, 3 H; 3-Me); 13C NMR (125 MHz, CDCl3): d=

180.4 (s; C9), 169.6 (s; C13), 161.4 (s; C8), 155.6 (s; C10a), 151.8 (s; C2),150.6 (s; C4a), 137.1 (s; C3), 136.2 (d; C6), 122.3 (d; C4), 117.2 (s; C9a),113.2 (s; C1), 110.3 (d; C7), 109.2 (s; C8a), 106.6 (d; C5), 53.0 (q; OMe),16.9 ppm (q; 3-Me); IR (KBr): nmax =3420, 2930, 1720, 1660, 1636,687 cm�1; UV (MeOH): lmax (log e) =327 (4.30), 230 nm(3.54 mol�1 dm3 cm�1); MS (EI, 70 eV): m/z (%): 316 (50) [M+], 284 (33),

252 (54), 243 (100), 163 (72), 137 (66); HRMS (EI, 70 eV): m/z : calcd forC16H12O7: 316.0582; found: 316.0588 [M]+ .

Oxidation of microsphaeropsone A (1) to enone 7: A solution of micro-sphaeropsone (1; 15 mg, 0.045 mmol) in dry benzene (1 mL) was treatedwith MnO2 (5.8 mg, 0.067 mmol). The mixture was stirred for 48 h at21 8C, filtered, and the MnO2 thoroughly washed with diethyl ether. Thefiltrate was concentrated and the residue purified by preparative TLCwith petroleum ether/ethyl acetate (3:1) to afford 7 as yellow crystals(11 mg, 74 % yield). M.p. 130–131 8C; [a]D =�42 (c= 0.25 in CH2Cl2);CD (MeCN, c =3.11 10�4): lmax (De)=350 (1.47), 314 (�2.68), 279 sh(�3.81), 263 (�11.68), 240 (6.58), 225 (�3.99), 213 (�4.18), 205 nm(�4.16 mol�1 dm3 cm�1); CD (DMSO, c =3.08 10�4): lmax (De)=345(3.88), 293 sh (�2.03), 269 nm (�11.26 mol�1 dm3 cm�1), negative CEbelow l =257 nm; CD (MeOH, c=3.34 10�4): lmax (De)=347 (1.81), 313(�0.98), 289 (�1.82), 265 (�8.84), 244 (3.69), 227 (�9.44), 213 nm(�8.54 mol�1 dm3 cm�1); CD (KCl, 71 mg in 250 mg of KCl): lmax (mdeg)=

343 (5.57), 308 (1.07), 267 (�11.74), 230 (�16.73), 211 nm(�11.48 mol�1 dm3 cm�1); 1H NMR (500 MHz, CDCl3: d= 11.71 (s, 1H; 8-OH), 7.57 (t, J= 8.2 Hz, 1H; H6), 6.99 (s, 1H; H4), 6.86 (d, J =8.2 Hz,1H; H5), 6.86 (d, J=8.2 Hz, 1H; H7), 6.05 (s, 1H; 1-OH), 3.84 (s, 3 H;OMe), 1.96 ppm (s, 3H; 3-Me); 13C NMR (125 MHz, CDCl3: d= 188.6 (s;C2), 184.6 (s; C9), 169.7 (s; C13), 162.3 (s; C4a), 160.8 (s; C8), 152.9 (s;C10a), 142.4 (d; C4), 136.3 (d; C6), 121.1 (s; C3), 112.8 (d; C7), 108.6 (s;C8a), 106.5 (d; C5), 98.2 (s; C9a), 80.2 (s; C1), 53.7 (q; OMe), 15.1 ppm(q; 3-Me); IR (KBr, pellet): nmax =2953, 2925, 2853, 1750, 1650, 1604,1469, 1240, 1164 cm�1; UV (MeOH): lmax (log e)=328 (3.85), 288 (3.80),246 nm (4.20 mol�1 dm3 cm�1); ESI-TOF-MS: m/z : calcd for C16H12O8Na:355.042; found: 355.041 [M+Na]+ .

3,4-Dihydrofusidienol A (8 b): Yellow powder; m.p. 195–197 8C; 1H NMR(500 MHz, CDCl3): d=6.78 (t, J =6.5 Hz, 1 H; H2), 6.56 (d, J =1.0 Hz,1H; H5), 6.51 (d, J=1.0 Hz, 1H; H7), 4.65 (t, J= 6.5 Hz, 2 H; H4), 3.68(s, 3 H;1-CO2CH3,), 2.65 (dt, J =6.5, 5.4 Hz, 2 H; H3), 2.43 ppm (s, 3 H; 6-CH3); 13C NMR (125 MHz, CDCl3): d= 182.4 (s; C9), 168.4 (3; CO2CH3),165.2 (s; C4a), 159.8 (s; C8), 153.4 (s; C10a), 147.0 (s; C6), 136.7 (d; C2),129.0 (s; C1), 112.4 (d; C5), 107.0 (d; C7), 106.2 (s; C8a), 99.2 (s; C9a),78.4 (t; C4), 52.2 (q; 1-CO2CH3), 28.7 (t; C3), 22.0 ppm (q; 6-CH3); IR(CH2Cl2 + CH3OH): nmax =3440, 1725, 1650, 1590, 710 cm�1; UV (CHCl3)lmax (log e)=240 (3.11), 304 nm (4.10 mol�1 dm3 cm�1); MS (EI, 70 eV):m/z (%): 302.1 [M]+ (40), 284 [M�H2O]+ (18), 274 [M�CO]+ (10), 243[M�CO2CH3]

+ (80), 215 (18), 171 (10), 137 (11), 63 (5); HRMS (EI,70 eV): m/z : calcd for C16H14O6: 302.0790; found: 302.0781 [M]+ .

Microsxanthone (9 c): Yellow powder; m.p. 218 8C; 1H NMR (500 MHz,CDCl3): d=12.13 (s, 1 H; 1-OH), 7.31 (d, J =6.0 Hz, 1 H; H6), 7.23 (d,J =6.0 Hz, 1H; H7), 6.78 (d, J= 1.0 Hz, 1H; H4), 6.65 (d, J=1.0 Hz, 1 H;H2), 3.99 (s, 3H; 8-CO2CH3), 2.43 ppm (s, 3H; 3-CH3); 13C NMR(125 MHz, CDCl3): d= 180.3 (s; C9), 169.6 (s; CO2CH3), 161.7 (s; C1),154.9 (s; C4b), 149.4 (s; C3), 145.8 (s; C5), 144.2 (s; C4b), 124.4 (s; C8),123.1 (d; C7), 119.4 (d; C6), 118.3 (s; C8a), 112.3 (d; C2), 107.1 (d; C4),107.0 (s; C9a), 53.0 (q; 8-CO2CH3), 24.7 ppm (q; 3-CH3); IR (CH2Cl2):nmax =3430, 1725, 1656, 1590, 710 cm�1; UV (CHCl3) lmax (log e) =242(3.10), 309 nm (4.09 mol�1 dm3 cm�1); MS (EI, 70 eV): m/z (%): 300.1[M]+ (30), 282 [M�H2O]+ (18), 272 [M�CO]+ (10), 241 [M�CO2CH3]

+

(80), 227 (57), 215 (18), 171 (10), 137 (10), 79 (10), 63 (5), 39 (10);HRMS (EI, 70 eV): m/z : calcd for C16H12O6: 300.0626; found: 300.0634[M]+ .

Seimatoxanthone A (10): Yellow powder; m.p. 238 8C; 1H NMR(500 MHz, CDCl3): d=12.17 (s, 1H; 8-OH), 7.65 (dd, J =8.0, 1.5 Hz, 1 H;H5), 7.58 (dd, J= 8.0, 1.5 Hz, 1 H; H7), 7.07 (t, J=8.0 Hz, 1H; H6), 6.45(s, 1 H; H3), 3.95 (s, 3H; 4-OMe), 3.85 (s, 3H; 1-OMe), 3.91 ppm (s, 3 H;2-OMe); 13C NMR (125 MHz, CDCl3): d =178.0 (s; C9), 158.9 (s; C8),157.5 (s; C4b), 154.7 (s; C4), 150.9 (s; C1), 149.0 (s; C2), 138.0 (s; C4a),133.0 (d; C6), 128.6 (d; C7), 121.2 (d; C5), 119.6 (s; C9a), 105.3 (s; C8a),95.9 (d; C3), 62.1 (q; 4-OMe), 61.7 (q; 2-OMe), 56.4 ppm (q; 1-OMe);IR (CHCl3): nmax = 3400, 1610, 1590, 710 cm�1; UV (CHCl3) lmax (log e)=

253 (3.10), 285 (2.95), 350 nm (4.10 mol�1 dm3 cm�1); MS (EI, 70 eV): m/z(%): 302.1 [M+] (55); HRMS (EI, 70 eV): m/z : calcd for C16H14O6:302.0789; found: 302.0775 [M]+ .

Chem. Eur. J. 2009, 15, 12121 – 12132 � 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 12129

FULL PAPERIsolation and Characterization of Xanthone Derivatives from Endophytic Fungi

3,4-Dihydroglobosuxanthon A (12; (1R,2R)-methyl 1,2,8-trihydroxy-9-oxo-2,3,4,9-tetrahydro-1H-xanthene-1-carboxylate): Yellow crystals, m.p.210 8C (dec.); [a]D

25 =++ 38 (c =0.07 in CHCl3); 1H NMR (500 MHz,CDCl3): d=11.98 (s, 1 H; 8-OH), 7.51 (t, J =8.3 Hz, 1 H; H6), 6.86 (dd,J =8.3, 0.8 Hz, 1 H; H5), 6.78 (dd J =8.3, 0.8 Hz, 1 H; H7), 4.68 (s, 1H;OH), 4.08 (dd, J =10.3, 3.6 Hz, 2H; H2), 3.84 (s, 3H; OCH3), 2.93 (ddd,J =18.7, 6.1, 4.6 Hz, 1 H; H4e), 2.84 (ddd, J =18.7, 8.9, 6.2 Hz, 1H; H4a),2.79 (s, 1 H; 2-OH), 2.30 (dddd, J=19.4, 10.3, 8.9, 6.1 Hz, 1 H; H3a),2.19 ppm (dddd, J =19.4, 10.3, 8.0, 6.1 Hz, 1H; H3e); 13C NMR(125 MHz, CDCl3): d=182.3 (s; C9), 172.7 (s; C10), 167.2 (s; C8), 160.6(s; C4a), 156.1 (s; C10a), 135.7 (d; C6), 116.9 (s; C9a), 111.5 (d; C7),110.2 (s; C8a), 106.9 (d; C5), 76.1 (s; C1), 72.6 (d; C2) , 53.3 (s; 1-CO2CH3), 26.0 (t; C4), 24.2 ppm (t; C3); IR (CHCl3 +MeOH): nmax =

3504, 3400 (OH), 1739 (COO), 1647 (CO), 1473, 1274, 1232, 1093,817 cm�1; UV (MeOH): lmax (log e) =340 (3.50), 267 nm(4.24 mol�1 dm3 cm�1); MS (EI, 70 eV): m/z (%): 306 [M]+ (30), 270 (3),262 (12), 247 [M�COOCH3]

+ (100), 229 (57), 201 (90), 189 (49), 147(25), 137 (87), 108 (77), 91 (40), 65 (37); HRMS (EI, 70 eV): m/z : calcdfor C15H14O7: 306.0740; found: 306.0737 [M]+ .

Crystal structure determination of microsphaeropsone A (1):[79] C16H14O8,Mr =334.3, monoclinic, space group C2, a =26.064(6), b=7.6193(16), c=

17.138(4) �, b=122.628(4)8, V= 2866.3(11) �3, Z=8, 1calcd =1.549 gcm�3,F ACHTUNGTRENNUNG(000) =1392, T =120(2) K. Bruker-AXS SMART APEX CCD,[80] graph-ite monochromator, l ACHTUNGTRENNUNG(MoKa)=0.71073 �, m=0.126 mm�1, colorless crys-tal, size: 0.51 0.23 0.18 mm3, 12606 intensities collected, 1.4<q<27.98,�34<h<34, �9<k<10, �22< l<22. Structure solved by direct meth-ods,[80] full-matrix least-squares refinement[81] with 3661 independent re-flections based on F2 and 443 parameters, all but H atoms refined aniso-tropically, H atoms from difference Fourier maps refined with ridingmodel on idealized positions with U =1.5Uiso(O and methyl C atom) or1.2Uiso(C). Compound 1 crystallizes with two independent but similarmolecules A and B with numbering schemes 1xx and 2xx per asymmetricunit in the non-centrosymmetric space group C2; however, in the absenceof significant anomalous scattering effects, the Flack[81] parameter is es-sentially meaningless. Accordingly, Friedel pairs were merged. Refine-ment converged at R1 ACHTUNGTRENNUNG(I>2s(I))=0.038, wR2 ACHTUNGTRENNUNG(all data) = 0.091, S =1.04,max ACHTUNGTRENNUNG(d/s)<0.001, min/max height in final DF map �0.22/0.37 e�3.Figure 2 shows the molecular structure.

Crystal structure determination of enone (7):[79] C16H12O8, Mr =332.3,monoclinic, space group P21, a =7.8871(12), b=10.7528(16), c=

8.7598(13) �, b=105.035(3)8, V= 717.47(19) �3, Z= 2, 1calcd =

1.538 gcm�3, F ACHTUNGTRENNUNG(000) =344, T =120(2) K. Bruker-AXS SMART APEXCCD,[80] graphite monochromator, l ACHTUNGTRENNUNG(MoKa)= 0.71073 �, m =0.126 mm�1,colorless crystal, size: 0.43 0.37 0.33 mm3, 6265 intensities collected2.4<q<27.98, �10<h<8, �13<k<14, �11< l<11. Structure solved bydirect methods,[80] full-matrix least-squares refinement[80] with 1812 inde-pendent reflections based on F2 and 221 parameters, all but H atoms re-fined anisotropically, H atoms from difference Fourier maps refined withriding model on idealized positions with U =1.5Uiso(O and methyl Catoms) or 1.2Uiso(C). Compound 7 crystallizes in the non-centrosymmet-ric space group P21; however, in the absence of significant anomalousscattering effects, the Flack[81] parameter is essentially meaningless. Ac-cordingly, Friedel pairs were merged. Refinement converged at R1-ACHTUNGTRENNUNG(I>2s(I))=0.035, wR2 ACHTUNGTRENNUNG(all data) =0.088, S=1.10, max ACHTUNGTRENNUNG(d/s)<0.001, min/max height in final DF map �0.15/0.35 e�3. Figure 3 shows the molecularstructure.

Computational section : Conformational searches (MMFF method) andpreliminary geometry optimizations (DFT method, B3LYP/6–31G(d)level) were run with Spartan’06 (Wavefunction, Inc., Irvine CA), with de-fault parameters and convergence criteria. Refined DFT geometry-opti-mizations, TDDFT, frequency (IR and VCD), and magnetic-shielding(NMR) calculations were run with Gaussian’03W (Revision D.01, Gaussi-an, Inc., Wallingford, CT).[82, 83] An integral equation formalism polariza-ble continuum model (IEF-PCM) was employed in self-consistent reac-tion field (SCRF) calculations with solvent parameters for chloroformand spheres put on all the solute atoms with universal force field (UFF)-generated radii. The TDDFT calculations (ECD) used various function-als (B3LYP, BH&HLYP, BP86) and the TZVP basis set; the frequency

(VCD) and magnetic shielding (NMR) calculations used the B3LYPfunctional and 6–31G(d) basis set. The ECD spectra were generated assum of Gaussians with a half-height width of 2000 cm�1 (ca. 16 nm at280 nm) using dipole-velocity computed rotational strengths. All the ex-cited states above 180 nm had transition energies below the estimatedionization potential (7.7 eV, BH&HLYP/TZVP).[66] The IR and VCDspectra were generated as sum of Gaussians with a half-height width of8 cm�1, and their frequencies scaled by a factor of 0.97.[52] The magneticshielding s was computed with the gauge-independent atomic orbital(GIAO) method and converted into chemical shifts d (in ppm) by the re-gression d =200.65�1.0715s, which is appropriate for the B3LYP/6–31G(d) level of calculation.[65]

Acknowledgements

We thank BASF AG and the Bundesministerium f�r Bildung und For-schung (BMBF; project no. 03F0360A) for financial support. G.P. andL.D.B. thank MIUR (FIRB project no. RBPR05NWWC) for financialsupport. T.K. thanks the Hungarian Scientific Research Fund (OTKA),the National Office for Research and Technology (NKTH), and theBolyai J�nos Foundation for financial support (K-68429).

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Chem. Eur. J. 2009, 15, 12121 – 12132 � 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org 12131

FULL PAPERIsolation and Characterization of Xanthone Derivatives from Endophytic Fungi

B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L.Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Na-nayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen,M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian, Inc., WallingfordCT, 2004.

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Received: March 23, 2009Revised: July 8, 2009

Published online: September 23, 2009

www.chemeurj.org � 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2009, 15, 12121 – 1213212132

K. Krohn et al.