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Microsphaerol and Seimatorone: Two New Compounds Isolated from the Endophytic Fungi, Microsphaeropsis sp. and Seimatosporium sp. by Hidayat Hussain* a ) b ), Natalia Root a ), Farah Jabeen b ) c ), Ahmed Al-Harrasi b ), Manzoor Ahmad c ), Fazal Mabood b ), Zahid Hassan b ), Afzal Shah d ), Ivan R. Green e ), Barbara Schulz f ), and Karsten Krohn a ) a
) Department of Chemistry, University of Paderborn, Warburger Strasse 100, DE-33098 Paderborn (phone: þ 49-5251-602172; fax: þ 49-5251-603245; e-mail: [email protected]) b ) UoN Chair of Omans Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Postal Code 616, Birkat Al Mauz, Nizwa, Sultanate of Oman c ) Department of Chemistry, University of Malakand, Chakdara, Dir (L), Pakistan d ) Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan e ) Department of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1 Matieland 7602, South Africa f ) Institute of Microbiology, University of Braunschweig, Spielmannstraße 7, DE-38106 Braunschweig
A new polychlorinated triphenyl diether named microsphaerol (1), has been isolated from the endophtic fungus Microsphaeropsis sp. An intensive phytochemical investigation of the endophytic fungus Seimatosporium sp., led to the isolation of a new naphthalene derivative named seimatorone (2) and eight known compounds, i.e., 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (3), 1-(2,6dihydroxyphenyl)butan-1-one (4), 1-(2-hydroxy-6-methoxyphenyl)butan-1-one (5), 5-hydroxy-2-methyl-4H-chromen-4-one (6), 2,3-dihydro-5-hydroxy-2-methyl-4H-chromen-4-one (7), 8-methoxynaphthalen-1-ol (8), nodulisporins A and B (9 and 10, resp.), and daldinol (11). The structures of 1 and 2 were elucidated by detailed spectroscopic analysis including 1H- and 13C-NMR, COSY, HMQC, HMBC, and HR-EI-MS, while the structures of the known compounds were deduced from comparison of their spectral data with those in the literature. Preliminary studies revealed that microsphaerol (1) showed good antibacterial activities against B. Megaterium and E. coli, and good antilagal and antifungal activities against C. fusca, M. violaceum, respectively. On the other hand, seimatorone (2) exhibited moderate antibacterial, antialgal, and antifungal activities.
Introduction. – Fungi are among the important sources of biologically active natural products. For many years, they have provided metabolites used for a variety of applications. Most importantly, currently most of the new natural products reported in the literature were isolated from fungi [1]. In this regard, diverse environments ranging from sponges in the oceans to fungi in cohabitation with mammals have been the target for these investigations, rendering them as an infinite platform for production of secondary metabolites, and current estimations indicate that only a few percent of the worlds fungi are known today [1]. Fermentation processes additionally provide large quantities of fungal metabolites which have been developed for biological and chemical evaluation [1]. In continuation of our program on the characterization of structurally novel and/or biologically active metabolites derived from fungal endophyte cultures [1 – 5], we investigated the endophytic fungi Microsphaeropsis sp. (internal strain 2015 Verlag Helvetica Chimica Acta AG, Zrich
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No. 7820) and Seimatosporium sp. (internal strain No. 8883), isolated from the halotolerant herbaceous plant Salsola oppositifolia from Playa del Ingles (Gomera, Spain), which led to the isolation and structural determination of two new compounds named microsphaerol (1) and seimatorone (2), along with eight known compounds, i.e., 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (3), 1-(2,6-dihydroxyphenyl)butan-1one (4), 1-(2-hydroxy-6-methoxyphenyl)butan-1-one (5), 5-hydroxy-2-methyl-4Hchromen-4-one (6), 2,3-dihydro-5-hydroxy-2-methyl-4H-chromen-4-one (7), 8-methoxynaphthalen-1-ol (8), nodulisporins A and B (9 and 10, resp.), and daldinol (11). Results and Discussion. – The two endophytic fungi, Microsphaeropsis sp. and Seimatosporium sp., were cultivated on biomalt agar medium [6] [7], and phytochemical investigation of these two fungi provided compounds 1 – 11 (Fig. 1). Compound 1 was isolated as white powder, with a molecular-ion peak at m/z 482.0445 (M þ , for C23H21Cl3O þ5 ; calc. 482.0455) in its HR-EI-MS. The EI-MS spectrum of 1 showed the pseudo-molecular-ion peaks at m/z 482, 484, and 486, indicating the presence of the three Cl-atoms in the molecule. The IR data evidenced the presence of OH groups (3380 cm 1) and phenyl rings (1560 and 1460 cm 1). This was also confirmed by OH H-atom signals observed at (d(H) 9.64 and 10.53 in 1H-NMR spectrum ((D6 )DMSO). Signals of four Me groups attached to aromatic rings were detected at d(H) 2.57 (s, MeC(6)), 2.41 (s, MeC(2’)), 2.39 (s, MeC(6’)), and 1.87 (s, MeC(5’)) 1), and this finding was further supported by four peaks in the 13C-NMR
Fig. 1. Structures of compounds 1 – 11 isolated from Microsphaeropsis sp. and Seimatosporium sp.
1)
Arbitray atom numbering as indicated in Fig. 1. For the systematic name, see the Exper. Part.
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spectrum (d(C) 17.1 (MeC(6)), 10.6 (MeC(2’)), 11.9 (MeC(6’)), and 21.4 (MeC(5’))). A triplet at d(H) 7.33 (J ¼ 7.0) was assigned to HC(4), a doublet at d(H) 6.72 (J ¼ 7.0) to HC(3), and a second doublet at d(H) 6.62 (J ¼ 7.0) to HC(5), representing the ABC system of a 2-chloro-6-methyl substituted ring A, and suggesting that one Cl, one Me, and one O-bearing substituent were present in this ring. In addition, the 1H-NMR spectrum showed one aromatic H-atom singlet at d(H) 6.23 assigned to HC(2’’) and one MeO signal at d(H) 3.90 for the MeOC(5’’) group. The 13C-NMR spectrum displayed 23 signals including those of one MeO group (d(C) 56.0), four aromatic Me groups (d(C) 10.6, 11.9, 17.1, and 21.4), four aromatic CH groups (d(C) 131.3, 122.8, 108.6, and 94.5), and 14 quaternary C-atoms (d(C) 157.1, 153.6, 152.8, 147.4, 146.4, 144.7, 139.1, 127.7, 123.6, 123.7, 123.5, 116.9, 110.3, and 109.5), based on an APT experiment. 1D- and 2D-NMR analysis, including 8 Hz-optimized HMBC, allowed assignment of all the C-atoms in the three phenyl rings (Figs. 1 and 2), i.e., a 2-chloro-6-methyl-1-oxygenated phenyl ring, a 3-chloro-2,5,6-trimethyl-1,4dioxygenated phenyl ring, and a 6-chloro-1,3,4,5-tetraoxygenated phenyl ring. The signals of the remaining two H-atoms, suggested in the molecular formula, were not observed in the 1H-NMR spectrum (in CDCl3 ), and these two H-atoms may arise from exchangeable OH H-atoms. This was confirmed by the 1H-NMR spectrum recorded in (D6 )DMSO which exhibited the signals of these OH H-atoms at d(H) 9.64 and 10.53, and by an IR absorption band at 3380 cm 1. Moreover, there were no HMBCs (8 Hzoptimized HMBC experiment) between H- and C-atoms in adjacent rings (Fig. 2). Efforts were unsuccessful to grow crystals in different solvents suitable for X-ray diffraction analysis. The connectivities of the three rings have been established by determining a mixture of crucial four- and five-bond J(C,H) correlations by a modified HMBC experiment, optimized for nJ(H,C) of 2 Hz [8]. It was interesting to note that this latter HMBC experiment (Fig. 2) provided a series of important four- and fivebond correlations which confirmed the ether linkages between the three phenyl rings. On the other hand, the optical rotation of compound 1 was zero. Consequently, the structure was established to be 4-chloro-5-[2-chloro-4-(2-chloro-6-methylphenoxy)3,5,6-trimethylphenoxy]-3-methoxybenzene-1,2-diol (1), named microsphaerol, after the producing organism, Microsphaeropsis sp. The molecular formula of seimatorone 2 was deduced as C12H12O5 from the HR-EIMS with a molecular peak at m/z 236.0680, and 1H- and 13C-NMR data (see Exper.
Fig. 2. Key COSY correlations and HMBCs of microsphaerol (1) and seimatorone (2)
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Part). The IR spectrum showed absorption peaks at 1635 cm 1, for a chelated C¼O, and at 3200 – 3350 cm 1, which indicated the presence of a chelated OH group. The 1 H-NMR spectrum of 2 displayed one aromatic H-atom singlet at d(H) 6.54 assigned to HC(5) and a one H-atom singlet at d(H) 10.52 for an aldehyde. Moreover, this was further confirmed by the 13C-NMR and DEPT spectra that exhibited signals of one CH group at d(C) 101.7 (C(5)) and aldehyde C-atom at d(C) 188.4 (CHO). In addition, the 1 H-NMR spectrum also exhibited two non-equivalent and separate groups of CH2 Hatom multiplets at d(C) 3.03 – 2.68, assigned to HC(2), and 2.47 – 2.12, assigned to HC(3), and a CH H-atom multiplet at d(H) 4.75, assigned to HC(4). These two CH2 groups and the CH are interconnected, as confirmed from COSY and HMBCs (Fig. 2). The NMR spectrum of 2 furthermore showed signals for one MeO group (d(H) 3.75 assigned to the MeOC(6); d(C) 56.8 (MeOC(6))) and a chelated OH group (d(H) 13.83; with a corresponding (d(C) 167.0 (C(8)))). Moreover, the 13C-NMR spectrum showed a signal at d(C) 201.8 assigned to the C(1)¼O group. This was further confirmed by the IR absorption at 1635 cm 1 for the H-bonded CO group. Based on this information, it was evident that compound 2 had a tetralone skeleton. The positions of the MeO, aldehyde, and chelated OH groups (at C(6), C(7), and C(8), resp.) were confirmed by the HMBCs, i.e., MeO/C(6); CHO/C(6), C(7), and C(8); and OH/C(7), C(8), and C(8a). The 1H,1H and 1H,13C connectivities were determined from the 1 H,1H-COSY (Fig. 2) and HMQC spectra. The 13C-NMR spectrum and DEPT experiments displayed twelve signals attributed to one MeO group, and two CH2 and three CH groups (which includes one aldehyde signal), and six quaternary C-atoms. The positions and connectivities of the C(1) – C(4) part in compound 2 were confirmed from the HMBCs: HC(2)/C(1), C(3), C(4), and C(8a); HC(3)/C(2), C(4), and C(4a); and HC(4)/C(2), C(3), C(4a), C(5), C(8a). The configuration at C(4) could not be established unambiguously by NMR spectroscopy, and since the amount of compound 2 available was extremely limited, further transformations in order to confirm the stereochemical assignment were not further persued. Consequently, the structure was established to be 5,6,7,8-tetrahydro1,5-dihydroxy-3-methoxy-8-oxonaphthalene-2-carbaldehyde 2, named seimatorone, after the producing organism, Seimatosporium sp. 1-(2,6-Dihydroxyphenyl)-3-hydroxybutan-1-one (3) [9], 1-(2,6-dihydroxyphenyl)butan-1-one (4) [10] [11], 1-(2-hydroxy-6-methoxyphenyl)butan-1-one (5) [9], 5hydroxy-2-methyl-4H-chromen-4-one (6) [10], 2,3-dihydro-5-hydroxy-2-methyl-4Hchromen-4-one (7) [12], 8-methoxynaphthalen-1-ol (8) [13], nodulisporins A [9] and B (9 and 10, resp.) [9], and daldinol (11) [14] were all identified based on the comparison of their spectral data with those in the literature. Microsphaerol (1) and seimatorone (2) were evaluated in an agar diffusion assay for their antifungal, antibacterial, and algicidal activities against Escherichia coli, Bacillus megaterium, Chlorella fusca, and Microbotryum violaceum (Table). Compound 1 showed good antibacterial activities against B. megaterium and E. coli; particularly noteworthy is the activity against the Gram-negative bacterium E. coli. Moreover, 1 also exhibited good antilagal and antifungal activities against C. fusca, M. violaceum, respectively. On the other hand, seimatorone (2) demonstrated moderate antibacterial activities against B. Megaterium and E. coli, and antialgal and antifungal activities against C. fusca, M. violaceum, respectively.
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Table. Biological Activities of Pure Metabolites 1 and 2 against Microbial Test Organisms in Agar Diffusion Assay a ) b ) Compound
1 2 Penicillin Tetracycline Nystatin Actidione Acetone
Antialgal
Antifungal
Escherichia coli
Antibacterial Bacillus megaterium
Chlorella fusca
Microbotryum violaceum
8 3 14 18 0 0 0
9 PI 7 18 18 0 0 0
8.5 5 0 PI 10 0 35 0
PI c ) 9 PI 5 0 0 20 50 0
a ) Application of pure substances at a concentration of 0.05 mg (50 ml of 1 mg/ml). b ) The radius of zone of inhibition was measured in mm. c ) PI; Partial inhibition, i.e., there was some growth within the zone of inhibition.
Experimental Part 1. General. Column chromatography (CC): silica gel (SiO2 , 70 – 230 and 230 – 400 mesh; E-Merck, DE-Darmstadt). TLC: Aluminum sheets precoated with silica gel 60 F254 (0.2 mm thick; E-Merck) to check the purity of the compounds; visualization under UV light (254 and 366 nm), followed by spraying with ceric sulfate. The UV (MeOH) and IR (KBr) spectra: Hitachi U-3200 and Shimadzu-8900 spectrophotometers, resp. 1H- and 13C-NMR spectra: Bruker AMX-500 instruments at 500 (1H) and 125 MHz (13C), resp.; with TMS as an internal reference; chemical shifts in ppm (d); the coupling constants (J) in Hz. EI-MS and HR-EI-MS: MAT 8200 and Micromass LCT mass spectrometers; in m/z. Microbiological methods and culture conditions: as described in [6] [7]. 3.2. Culture, Extraction, and Isolation. The marine endophytic fungus Microsphaeropsis sp. (internal strain No. 7820) was cultivated at r.t. for 28 d [6] [7] on biomalt solid agar medium. The culture media were then extracted with AcOEt to afford 6.7 g of a residue after removal of the solvent under reduced pressure. The extract was separated into six fractions by CC (SiO2 ; CH2Cl2/AcOEt 85 : 15, 50 : 50, 0 : 100). The less polar Fr. 1 (3 g) contained mainly fatty acids and lipids. Fr. 3 was further purified by CC (SiO2 ; hexane/AcOEt 10 : 1 (1000 ml), 5 : 1 (1000 ml)) to give pure compound 1 (10 mg). The endophytic fungus Seimatosporium sp. (internal strain No. 8883) was isolated from Salsola oppositifolia (Gomera, Spain), and was cultivated on biomalt solid agar medium (12 l, 5% (w/v)) at r.t. for 21 d. The cultures were then extracted with AcOEt to afford a residue (3.5 g). The extract was separated into two fractions by CC (SiO2 ; hexane/AcOEt 90 : 10, 50 : 50, 0 : 100 as the eluent). The least polar Fr. 1 (2.1 g) contained mainly fatty acids and lipids. The next polar Fr. 2 (1.1 g) was separated by CC (SiO2 ; hexane/AcOEt 10 : 1, 5 : 1) to give five subfractions, Fr. F1 – F6 . Frs. F3 and F4 were further purified by CC (SiO2 ) and prep. TLC (CH2Cl2/AcOEt 85 : 15, 50 : 50, 0 : 100) to give pure compounds 2 (2.1 mg), 3 (4.6 mg), 4 (3.7 mg), 5 (4.0 mg), 6 (3.5 mg), 7 (4.1 mg), 8 (5.6 mg), 9 (2.9 mg), 10 (4.4 mg), and 11 (3.1 mg). 2.1. Microsphaerol ( ¼ 4-Chloro-5-[2-chloro-4-(2-chloro-6-methylphenoxy)-3,5,6-trimethylphenoxy]3-methoxybenzene-1,2-diol; 1). Pale-yellow amorphous solid. UV (MeOH): 205 (4.52); 290 (4.20). IR (KBr): 3380, 1630, 1560, 1460, 1250. 1H-NMR (500 MHz, CDCl3 ): 7.33 (t, J ¼ 7.0, HC(4)); 6.72 (d, J ¼ 7.0, HC(3)); 6.62 (d, J ¼ 7.0, HC(5)); 6.23 (s, HC(2’’)); 3.90 (s, MeOC(5’’)); 2.57 (s, MeC(6)); 2.41 (s, MeC(2’)); 2.39 (s, MeC(6’)); 1.87 (s, MeC(5’)). 13C-NMR (125 MHz, CD3OD): 157.1 (C(1)); 153.6 (C(5)); 152.8 (C(4’)); 147.4 (C(1’’)); 146.4 (C(3’’)); 144.7 (C(1’)); 139.1 (C(4’’)); 131.3 (C(4)); 127.7 (C(6)); 123.6 (C(6’)); 123.7 (C(5’)); 123.5 (C(2)); 122.8 (C(3)); 116.9 (C(3’)); 110.3 (C(2’)); 109.5 (C(6’’)); 108.6 (C(5)); 94.5 (C(2’’)); 56.0 (MeOC(5’’)); 17.1 (MeC(6)); 10.6 (Me(C(2’)); 11.9 (MeC(6’)); 21.4 (MeC(5’)). HR-EI-MS: 482.0445 (C23H21Cl3O þ5 ; calc. 482.0455).
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2.2. Seimatorone ( ¼ 5,6,7,8-Tetrahydro-1,5-dihydroxy-3-methoxy-8-oxonaphthalene-2-carbaldehyde; 2). Orange-red needles. M.p. 1698. UV (MeOH): 216 (4.30), 259.0 (4.7), 333 (3.40); IR (KBr): 3200 – 3350, 2930, 1635, 1580, 1420, 1250, 745. 1H-NMR (500 MHz, CDCl3 ): 13.83 (s, chealted OH); 10.52 (s, CHO); 6.54 (s, HC(5)); 4.72 – 476 (m, HC(4)); 4.01 (s, MeOC(6)); 3.03 – 2.68 (m, CH2(2)); 2.47 – 2.12 (m, CH2(3)). 13C-NMR (125 MHz, CDCl3 ): 201.8 (C(1)); 188.4 (CHO); 167.5 (C(6)); 167.0 (C(8)); 150.5 (C(4a)); 112.9 (C(8a)); 111.2 (C(7)); 101.7 (C(5)); 68.4 (C(4)); 56.8 (MeOC(6)); 34.7 (C(2)); 28.14 (C(3)). EI-MS (2308): 236.0 (16.7, M þ ). HR-EI-MS: 236.0680 (C12H12O þ5 ; calc. 236.0685). 3. Agar Diffusion Test. The tested compounds, 1 and 2, and extracts were dissolved in acetone at a concentration of 1 mg/ml. Fifty ml of the soln. were pipetted onto a sterile filter disc, which was placed onto an appropriate agar growth medium [6] for the respective test organism and subsequently sprayed with a suspension of the test organism. The test organisms were Chlorella fusca, Microbotryum violaceum, Escherichia coli, and Bacillus megaterium. The radius of the zone of inhibition was measured in mm. We thank BASF AG and the BMBF (Bundesministerium fr Bildung und Forschung; Project No. 03F0360A).
REFERENCES [1] H. Hussain, K. Krohn, S. Draeger, B. Schulz, Heterocycles 2007, 74, 331. [2] K. Krohn, S. F. Kouam, G. M. Kuigoua, H. Hussain, S. Cludius-Brand, U. Flçrke, T. Kurtn, G. Pescitelli, L. Di Bari, S. Draeger, B. Schulz, Chem. – Eur. J. 2009, 15, 12121. [3] H. Hussain, N. Akhtar, S. Draeger, B. Schulz, G. Pescitelli, P. Salvadori, S. Antus, T. Kurtn, K. Krohn, Eur. J. Org. Chem. 2009, 749. [4] H. Hussain, K. Krohn, S. Draeger, K. Meier, B. Schulz, Rec. Nat. Prod. 2009, 3, 114. [5] S. Qin, H. Hussain, B. Schulz, S. Draeger, K. Krohn, Helv. Chim. Acta 2010, 93, 169. [6] U. Hçller, A. D. Wright, G. F. Matthe, G. M. Kçnig, S. Draeger, H.-J. Aust, B. Schulz, Mycol. Res. 2000, 104, 1354. [7] B. Schulz, J. Sucker, H.-J. Aust, K. Krohn, K. Ludewig, P. G. Jones, D. Dçring, Mycol. Res. 1995, 99, 1007. [8] X.-C. Li, H. N. Elsohly, C. D. Hufford, A. M. Clark, Magn. Reson. Chem. 1999, 37, 856. [9] J. Dai, K. Krohn, U. Flçrke, S. Draeger, B. Schulz, A. Kiss-Szikszai, S. Antus, T. Kurtn, T. van Ree, Eur. J. Org. Chem. 2006, 3498. [10] A. Arnoldi, Synthesis 1984, 856. [11] H.-R. Huang, X.-L. Feng, Z.-G. She, Y.-C. Lin, L. L. P. Vrijmoed, E. B. G. Jones, Acta Crystallogr., Sect. E 2005, 61, 282. [12] L. E. Gray, H. W. Gardner, D. Weisleder, M. Leib, Phytochemistry 1999, 50, 1337. [13] H. Anke, V. Stadler, A. Mayer, O. Sterner, Can. J. Botany 1995, 73 (Suppl. 1), 932. [14] T. Hashimoto, S. Tahara, S. Takaoka, M. Tori, Y. Asakawa, Chem. Pharm. Bull. 1994, 42, 1528. Received February 28, 2014
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Microsphaerol and Seimatorone: Two New Compounds Isolated from the Endophytic Fungi, Microsphaeropsis sp. and Seimatosporium sp. by Hidayat Hussain* a ) b ), Natalia Root a ), Farah Jabeen b ) c ), Ahmed Al-Harrasi b ), Manzoor Ahmad c ), Fazal Mabood b ), Zahid Hassan b ), Afzal Shah d ), Ivan R. Green e ), Barbara Schulz f ), and Karsten Krohn a ) a
) Department of Chemistry, University of Paderborn, Warburger Strasse 100, DE-33098 Paderborn (phone: þ 49-5251-602172; fax: þ 49-5251-603245; e-mail: [email protected]) b ) UoN Chair of Omans Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Postal Code 616, Birkat Al Mauz, Nizwa, Sultanate of Oman c ) Department of Chemistry, University of Malakand, Chakdara, Dir (L), Pakistan d ) Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan e ) Department of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1 Matieland 7602, South Africa f ) Institute of Microbiology, University of Braunschweig, Spielmannstraße 7, DE-38106 Braunschweig
A new polychlorinated triphenyl diether named microsphaerol (1), has been isolated from the endophtic fungus Microsphaeropsis sp. An intensive phytochemical investigation of the endophytic fungus Seimatosporium sp., led to the isolation of a new naphthalene derivative named seimatorone (2) and eight known compounds, i.e., 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (3), 1-(2,6dihydroxyphenyl)butan-1-one (4), 1-(2-hydroxy-6-methoxyphenyl)butan-1-one (5), 5-hydroxy-2-methyl-4H-chromen-4-one (6), 2,3-dihydro-5-hydroxy-2-methyl-4H-chromen-4-one (7), 8-methoxynaphthalen-1-ol (8), nodulisporins A and B (9 and 10, resp.), and daldinol (11). The structures of 1 and 2 were elucidated by detailed spectroscopic analysis including 1H- and 13C-NMR, COSY, HMQC, HMBC, and HR-EI-MS, while the structures of the known compounds were deduced from comparison of their spectral data with those in the literature. Preliminary studies revealed that microsphaerol (1) showed good antibacterial activities against B. Megaterium and E. coli, and good antilagal and antifungal activities against C. fusca, M. violaceum, respectively. On the other hand, seimatorone (2) exhibited moderate antibacterial, antialgal, and antifungal activities.
Introduction. – Fungi are among the important sources of biologically active natural products. For many years, they have provided metabolites used for a variety of applications. Most importantly, currently most of the new natural products reported in the literature were isolated from fungi [1]. In this regard, diverse environments ranging from sponges in the oceans to fungi in cohabitation with mammals have been the target for these investigations, rendering them as an infinite platform for production of secondary metabolites, and current estimations indicate that only a few percent of the worlds fungi are known today [1]. Fermentation processes additionally provide large quantities of fungal metabolites which have been developed for biological and chemical evaluation [1]. In continuation of our program on the characterization of structurally novel and/or biologically active metabolites derived from fungal endophyte cultures [1 – 5], we investigated the endophytic fungi Microsphaeropsis sp. (internal strain 2015 Verlag Helvetica Chimica Acta AG, Zrich
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No. 7820) and Seimatosporium sp. (internal strain No. 8883), isolated from the halotolerant herbaceous plant Salsola oppositifolia from Playa del Ingles (Gomera, Spain), which led to the isolation and structural determination of two new compounds named microsphaerol (1) and seimatorone (2), along with eight known compounds, i.e., 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (3), 1-(2,6-dihydroxyphenyl)butan-1one (4), 1-(2-hydroxy-6-methoxyphenyl)butan-1-one (5), 5-hydroxy-2-methyl-4Hchromen-4-one (6), 2,3-dihydro-5-hydroxy-2-methyl-4H-chromen-4-one (7), 8-methoxynaphthalen-1-ol (8), nodulisporins A and B (9 and 10, resp.), and daldinol (11). Results and Discussion. – The two endophytic fungi, Microsphaeropsis sp. and Seimatosporium sp., were cultivated on biomalt agar medium [6] [7], and phytochemical investigation of these two fungi provided compounds 1 – 11 (Fig. 1). Compound 1 was isolated as white powder, with a molecular-ion peak at m/z 482.0445 (M þ , for C23H21Cl3O þ5 ; calc. 482.0455) in its HR-EI-MS. The EI-MS spectrum of 1 showed the pseudo-molecular-ion peaks at m/z 482, 484, and 486, indicating the presence of the three Cl-atoms in the molecule. The IR data evidenced the presence of OH groups (3380 cm 1) and phenyl rings (1560 and 1460 cm 1). This was also confirmed by OH H-atom signals observed at (d(H) 9.64 and 10.53 in 1H-NMR spectrum ((D6 )DMSO). Signals of four Me groups attached to aromatic rings were detected at d(H) 2.57 (s, MeC(6)), 2.41 (s, MeC(2’)), 2.39 (s, MeC(6’)), and 1.87 (s, MeC(5’)) 1), and this finding was further supported by four peaks in the 13C-NMR
Fig. 1. Structures of compounds 1 – 11 isolated from Microsphaeropsis sp. and Seimatosporium sp.
1)
Arbitray atom numbering as indicated in Fig. 1. For the systematic name, see the Exper. Part.
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spectrum (d(C) 17.1 (MeC(6)), 10.6 (MeC(2’)), 11.9 (MeC(6’)), and 21.4 (MeC(5’))). A triplet at d(H) 7.33 (J ¼ 7.0) was assigned to HC(4), a doublet at d(H) 6.72 (J ¼ 7.0) to HC(3), and a second doublet at d(H) 6.62 (J ¼ 7.0) to HC(5), representing the ABC system of a 2-chloro-6-methyl substituted ring A, and suggesting that one Cl, one Me, and one O-bearing substituent were present in this ring. In addition, the 1H-NMR spectrum showed one aromatic H-atom singlet at d(H) 6.23 assigned to HC(2’’) and one MeO signal at d(H) 3.90 for the MeOC(5’’) group. The 13C-NMR spectrum displayed 23 signals including those of one MeO group (d(C) 56.0), four aromatic Me groups (d(C) 10.6, 11.9, 17.1, and 21.4), four aromatic CH groups (d(C) 131.3, 122.8, 108.6, and 94.5), and 14 quaternary C-atoms (d(C) 157.1, 153.6, 152.8, 147.4, 146.4, 144.7, 139.1, 127.7, 123.6, 123.7, 123.5, 116.9, 110.3, and 109.5), based on an APT experiment. 1D- and 2D-NMR analysis, including 8 Hz-optimized HMBC, allowed assignment of all the C-atoms in the three phenyl rings (Figs. 1 and 2), i.e., a 2-chloro-6-methyl-1-oxygenated phenyl ring, a 3-chloro-2,5,6-trimethyl-1,4dioxygenated phenyl ring, and a 6-chloro-1,3,4,5-tetraoxygenated phenyl ring. The signals of the remaining two H-atoms, suggested in the molecular formula, were not observed in the 1H-NMR spectrum (in CDCl3 ), and these two H-atoms may arise from exchangeable OH H-atoms. This was confirmed by the 1H-NMR spectrum recorded in (D6 )DMSO which exhibited the signals of these OH H-atoms at d(H) 9.64 and 10.53, and by an IR absorption band at 3380 cm 1. Moreover, there were no HMBCs (8 Hzoptimized HMBC experiment) between H- and C-atoms in adjacent rings (Fig. 2). Efforts were unsuccessful to grow crystals in different solvents suitable for X-ray diffraction analysis. The connectivities of the three rings have been established by determining a mixture of crucial four- and five-bond J(C,H) correlations by a modified HMBC experiment, optimized for nJ(H,C) of 2 Hz [8]. It was interesting to note that this latter HMBC experiment (Fig. 2) provided a series of important four- and fivebond correlations which confirmed the ether linkages between the three phenyl rings. On the other hand, the optical rotation of compound 1 was zero. Consequently, the structure was established to be 4-chloro-5-[2-chloro-4-(2-chloro-6-methylphenoxy)3,5,6-trimethylphenoxy]-3-methoxybenzene-1,2-diol (1), named microsphaerol, after the producing organism, Microsphaeropsis sp. The molecular formula of seimatorone 2 was deduced as C12H12O5 from the HR-EIMS with a molecular peak at m/z 236.0680, and 1H- and 13C-NMR data (see Exper.
Fig. 2. Key COSY correlations and HMBCs of microsphaerol (1) and seimatorone (2)
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Part). The IR spectrum showed absorption peaks at 1635 cm 1, for a chelated C¼O, and at 3200 – 3350 cm 1, which indicated the presence of a chelated OH group. The 1 H-NMR spectrum of 2 displayed one aromatic H-atom singlet at d(H) 6.54 assigned to HC(5) and a one H-atom singlet at d(H) 10.52 for an aldehyde. Moreover, this was further confirmed by the 13C-NMR and DEPT spectra that exhibited signals of one CH group at d(C) 101.7 (C(5)) and aldehyde C-atom at d(C) 188.4 (CHO). In addition, the 1 H-NMR spectrum also exhibited two non-equivalent and separate groups of CH2 Hatom multiplets at d(C) 3.03 – 2.68, assigned to HC(2), and 2.47 – 2.12, assigned to HC(3), and a CH H-atom multiplet at d(H) 4.75, assigned to HC(4). These two CH2 groups and the CH are interconnected, as confirmed from COSY and HMBCs (Fig. 2). The NMR spectrum of 2 furthermore showed signals for one MeO group (d(H) 3.75 assigned to the MeOC(6); d(C) 56.8 (MeOC(6))) and a chelated OH group (d(H) 13.83; with a corresponding (d(C) 167.0 (C(8)))). Moreover, the 13C-NMR spectrum showed a signal at d(C) 201.8 assigned to the C(1)¼O group. This was further confirmed by the IR absorption at 1635 cm 1 for the H-bonded CO group. Based on this information, it was evident that compound 2 had a tetralone skeleton. The positions of the MeO, aldehyde, and chelated OH groups (at C(6), C(7), and C(8), resp.) were confirmed by the HMBCs, i.e., MeO/C(6); CHO/C(6), C(7), and C(8); and OH/C(7), C(8), and C(8a). The 1H,1H and 1H,13C connectivities were determined from the 1 H,1H-COSY (Fig. 2) and HMQC spectra. The 13C-NMR spectrum and DEPT experiments displayed twelve signals attributed to one MeO group, and two CH2 and three CH groups (which includes one aldehyde signal), and six quaternary C-atoms. The positions and connectivities of the C(1) – C(4) part in compound 2 were confirmed from the HMBCs: HC(2)/C(1), C(3), C(4), and C(8a); HC(3)/C(2), C(4), and C(4a); and HC(4)/C(2), C(3), C(4a), C(5), C(8a). The configuration at C(4) could not be established unambiguously by NMR spectroscopy, and since the amount of compound 2 available was extremely limited, further transformations in order to confirm the stereochemical assignment were not further persued. Consequently, the structure was established to be 5,6,7,8-tetrahydro1,5-dihydroxy-3-methoxy-8-oxonaphthalene-2-carbaldehyde 2, named seimatorone, after the producing organism, Seimatosporium sp. 1-(2,6-Dihydroxyphenyl)-3-hydroxybutan-1-one (3) [9], 1-(2,6-dihydroxyphenyl)butan-1-one (4) [10] [11], 1-(2-hydroxy-6-methoxyphenyl)butan-1-one (5) [9], 5hydroxy-2-methyl-4H-chromen-4-one (6) [10], 2,3-dihydro-5-hydroxy-2-methyl-4Hchromen-4-one (7) [12], 8-methoxynaphthalen-1-ol (8) [13], nodulisporins A [9] and B (9 and 10, resp.) [9], and daldinol (11) [14] were all identified based on the comparison of their spectral data with those in the literature. Microsphaerol (1) and seimatorone (2) were evaluated in an agar diffusion assay for their antifungal, antibacterial, and algicidal activities against Escherichia coli, Bacillus megaterium, Chlorella fusca, and Microbotryum violaceum (Table). Compound 1 showed good antibacterial activities against B. megaterium and E. coli; particularly noteworthy is the activity against the Gram-negative bacterium E. coli. Moreover, 1 also exhibited good antilagal and antifungal activities against C. fusca, M. violaceum, respectively. On the other hand, seimatorone (2) demonstrated moderate antibacterial activities against B. Megaterium and E. coli, and antialgal and antifungal activities against C. fusca, M. violaceum, respectively.
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Table. Biological Activities of Pure Metabolites 1 and 2 against Microbial Test Organisms in Agar Diffusion Assay a ) b ) Compound
1 2 Penicillin Tetracycline Nystatin Actidione Acetone
Antialgal
Antifungal
Escherichia coli
Antibacterial Bacillus megaterium
Chlorella fusca
Microbotryum violaceum
8 3 14 18 0 0 0
9 PI 7 18 18 0 0 0
8.5 5 0 PI 10 0 35 0
PI c ) 9 PI 5 0 0 20 50 0
a ) Application of pure substances at a concentration of 0.05 mg (50 ml of 1 mg/ml). b ) The radius of zone of inhibition was measured in mm. c ) PI; Partial inhibition, i.e., there was some growth within the zone of inhibition.
Experimental Part 1. General. Column chromatography (CC): silica gel (SiO2 , 70 – 230 and 230 – 400 mesh; E-Merck, DE-Darmstadt). TLC: Aluminum sheets precoated with silica gel 60 F254 (0.2 mm thick; E-Merck) to check the purity of the compounds; visualization under UV light (254 and 366 nm), followed by spraying with ceric sulfate. The UV (MeOH) and IR (KBr) spectra: Hitachi U-3200 and Shimadzu-8900 spectrophotometers, resp. 1H- and 13C-NMR spectra: Bruker AMX-500 instruments at 500 (1H) and 125 MHz (13C), resp.; with TMS as an internal reference; chemical shifts in ppm (d); the coupling constants (J) in Hz. EI-MS and HR-EI-MS: MAT 8200 and Micromass LCT mass spectrometers; in m/z. Microbiological methods and culture conditions: as described in [6] [7]. 3.2. Culture, Extraction, and Isolation. The marine endophytic fungus Microsphaeropsis sp. (internal strain No. 7820) was cultivated at r.t. for 28 d [6] [7] on biomalt solid agar medium. The culture media were then extracted with AcOEt to afford 6.7 g of a residue after removal of the solvent under reduced pressure. The extract was separated into six fractions by CC (SiO2 ; CH2Cl2/AcOEt 85 : 15, 50 : 50, 0 : 100). The less polar Fr. 1 (3 g) contained mainly fatty acids and lipids. Fr. 3 was further purified by CC (SiO2 ; hexane/AcOEt 10 : 1 (1000 ml), 5 : 1 (1000 ml)) to give pure compound 1 (10 mg). The endophytic fungus Seimatosporium sp. (internal strain No. 8883) was isolated from Salsola oppositifolia (Gomera, Spain), and was cultivated on biomalt solid agar medium (12 l, 5% (w/v)) at r.t. for 21 d. The cultures were then extracted with AcOEt to afford a residue (3.5 g). The extract was separated into two fractions by CC (SiO2 ; hexane/AcOEt 90 : 10, 50 : 50, 0 : 100 as the eluent). The least polar Fr. 1 (2.1 g) contained mainly fatty acids and lipids. The next polar Fr. 2 (1.1 g) was separated by CC (SiO2 ; hexane/AcOEt 10 : 1, 5 : 1) to give five subfractions, Fr. F1 – F6 . Frs. F3 and F4 were further purified by CC (SiO2 ) and prep. TLC (CH2Cl2/AcOEt 85 : 15, 50 : 50, 0 : 100) to give pure compounds 2 (2.1 mg), 3 (4.6 mg), 4 (3.7 mg), 5 (4.0 mg), 6 (3.5 mg), 7 (4.1 mg), 8 (5.6 mg), 9 (2.9 mg), 10 (4.4 mg), and 11 (3.1 mg). 2.1. Microsphaerol ( ¼ 4-Chloro-5-[2-chloro-4-(2-chloro-6-methylphenoxy)-3,5,6-trimethylphenoxy]3-methoxybenzene-1,2-diol; 1). Pale-yellow amorphous solid. UV (MeOH): 205 (4.52); 290 (4.20). IR (KBr): 3380, 1630, 1560, 1460, 1250. 1H-NMR (500 MHz, CDCl3 ): 7.33 (t, J ¼ 7.0, HC(4)); 6.72 (d, J ¼ 7.0, HC(3)); 6.62 (d, J ¼ 7.0, HC(5)); 6.23 (s, HC(2’’)); 3.90 (s, MeOC(5’’)); 2.57 (s, MeC(6)); 2.41 (s, MeC(2’)); 2.39 (s, MeC(6’)); 1.87 (s, MeC(5’)). 13C-NMR (125 MHz, CD3OD): 157.1 (C(1)); 153.6 (C(5)); 152.8 (C(4’)); 147.4 (C(1’’)); 146.4 (C(3’’)); 144.7 (C(1’)); 139.1 (C(4’’)); 131.3 (C(4)); 127.7 (C(6)); 123.6 (C(6’)); 123.7 (C(5’)); 123.5 (C(2)); 122.8 (C(3)); 116.9 (C(3’)); 110.3 (C(2’)); 109.5 (C(6’’)); 108.6 (C(5)); 94.5 (C(2’’)); 56.0 (MeOC(5’’)); 17.1 (MeC(6)); 10.6 (Me(C(2’)); 11.9 (MeC(6’)); 21.4 (MeC(5’)). HR-EI-MS: 482.0445 (C23H21Cl3O þ5 ; calc. 482.0455).
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2.2. Seimatorone ( ¼ 5,6,7,8-Tetrahydro-1,5-dihydroxy-3-methoxy-8-oxonaphthalene-2-carbaldehyde; 2). Orange-red needles. M.p. 1698. UV (MeOH): 216 (4.30), 259.0 (4.7), 333 (3.40); IR (KBr): 3200 – 3350, 2930, 1635, 1580, 1420, 1250, 745. 1H-NMR (500 MHz, CDCl3 ): 13.83 (s, chealted OH); 10.52 (s, CHO); 6.54 (s, HC(5)); 4.72 – 476 (m, HC(4)); 4.01 (s, MeOC(6)); 3.03 – 2.68 (m, CH2(2)); 2.47 – 2.12 (m, CH2(3)). 13C-NMR (125 MHz, CDCl3 ): 201.8 (C(1)); 188.4 (CHO); 167.5 (C(6)); 167.0 (C(8)); 150.5 (C(4a)); 112.9 (C(8a)); 111.2 (C(7)); 101.7 (C(5)); 68.4 (C(4)); 56.8 (MeOC(6)); 34.7 (C(2)); 28.14 (C(3)). EI-MS (2308): 236.0 (16.7, M þ ). HR-EI-MS: 236.0680 (C12H12O þ5 ; calc. 236.0685). 3. Agar Diffusion Test. The tested compounds, 1 and 2, and extracts were dissolved in acetone at a concentration of 1 mg/ml. Fifty ml of the soln. were pipetted onto a sterile filter disc, which was placed onto an appropriate agar growth medium [6] for the respective test organism and subsequently sprayed with a suspension of the test organism. The test organisms were Chlorella fusca, Microbotryum violaceum, Escherichia coli, and Bacillus megaterium. The radius of the zone of inhibition was measured in mm. We thank BASF AG and the BMBF (Bundesministerium fr Bildung und Forschung; Project No. 03F0360A).
REFERENCES [1] H. Hussain, K. Krohn, S. Draeger, B. Schulz, Heterocycles 2007, 74, 331. [2] K. Krohn, S. F. Kouam, G. M. Kuigoua, H. Hussain, S. Cludius-Brand, U. Flçrke, T. Kurtn, G. Pescitelli, L. Di Bari, S. Draeger, B. Schulz, Chem. – Eur. J. 2009, 15, 12121. [3] H. Hussain, N. Akhtar, S. Draeger, B. Schulz, G. Pescitelli, P. Salvadori, S. Antus, T. Kurtn, K. Krohn, Eur. J. Org. Chem. 2009, 749. [4] H. Hussain, K. Krohn, S. Draeger, K. Meier, B. Schulz, Rec. Nat. Prod. 2009, 3, 114. [5] S. Qin, H. Hussain, B. Schulz, S. Draeger, K. Krohn, Helv. Chim. Acta 2010, 93, 169. [6] U. Hçller, A. D. Wright, G. F. Matthe, G. M. Kçnig, S. Draeger, H.-J. Aust, B. Schulz, Mycol. Res. 2000, 104, 1354. [7] B. Schulz, J. Sucker, H.-J. Aust, K. Krohn, K. Ludewig, P. G. Jones, D. Dçring, Mycol. Res. 1995, 99, 1007. [8] X.-C. Li, H. N. Elsohly, C. D. Hufford, A. M. Clark, Magn. Reson. Chem. 1999, 37, 856. [9] J. Dai, K. Krohn, U. Flçrke, S. Draeger, B. Schulz, A. Kiss-Szikszai, S. Antus, T. Kurtn, T. van Ree, Eur. J. Org. Chem. 2006, 3498. [10] A. Arnoldi, Synthesis 1984, 856. [11] H.-R. Huang, X.-L. Feng, Z.-G. She, Y.-C. Lin, L. L. P. Vrijmoed, E. B. G. Jones, Acta Crystallogr., Sect. E 2005, 61, 282. [12] L. E. Gray, H. W. Gardner, D. Weisleder, M. Leib, Phytochemistry 1999, 50, 1337. [13] H. Anke, V. Stadler, A. Mayer, O. Sterner, Can. J. Botany 1995, 73 (Suppl. 1), 932. [14] T. Hashimoto, S. Tahara, S. Takaoka, M. Tori, Y. Asakawa, Chem. Pharm. Bull. 1994, 42, 1528. Received February 28, 2014
