Antimicrobial Azaphilones from the Fungus Hypoxylon multiforme

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Fungal material
• Extraction and isolation
• Antimicrobial activity
• Results and Discussion
• Acknowledgements
• References

Abstract

Four new azaphilones named multiformins A - D together with a known compound 4 : 5:4′:5′-tetrahydroxy-1 : 1′-binaphthyl were isolated from the methanolic stromatal extract of the xylariaceous ascomycete Hypoxylon multiforme. Their absolute structures were characterised by 2D-NMR, UV, IR, mass and CD spectroscopy. Multiformins A - D showed strong and apparently non-selective antimicrobial activity.

Introduction

Azaphilones have been shown to possess various bioactivities such as telomerase inhibitory activity [1], inhibition of cholesteryl ester transfer protein [2], inhibition of gp120-CD4 binding [3], and inhibition of monoamine oxidase [4], as well as antimicrobial and nematicidal effects [5], [6], [7]. In the course of our investigation of biologically active compounds from xylariaceous fungi, we previously reported the isolation of several cytotoxic cytochalasins from Daldinia sp. [8], cohaerins A and B from H. cohaerens [9] and four antimicrobial active azaphilones from Creosphaeria sassafras [10]. In continuation, we report here on the isolation and structural characterisation of four new azaphilones and their antimicrobial activity.

Materials and Methods

General experimental procedures

Optical rotations were measured on a JASCO DIP-1000 polarimeter in MeOH. UV spectra were obtained on a Shimadzu UV-1650PC instrument in EtOH. IR spectra were measured on a Perkin Elmer Spectrum One FT-IR spectrometer. The ¹H- and ¹³C-NMR spectra were recorded on a Varian Unity 600 NMR spectrometer (600 MHz for ¹H and 150 MHz for ¹³C), using CDCl₃ as solvent. Mass spectra were recorded on a JEOL JMS AX-500 spectrometer. Preparative HPLC was performed using two different systems: System 1 comprised a Gilson Abimed (Ratingen, Germany) HPLC with UV detector, an MZ (Mainz, Germany) Kromasil C18 column (7 μm; 250 × 40 mm; 0.1 % trifluoroacetic acid:acetonitrile; gradient elution at 7 mL/min as specified below). System 2 comprised a Shimadzu liquid chromatograph LC-10AS with RID-6A and SPD-10A detectors using a 5 SL-II column (10 × 250 mm, Nacalai Tesque, Japan). Conventional column chromatography was carried out on silica gel 60 (0.2 - 0.5 mm, 0.04 - 0.063 mm, Merck) and gel permeation chromatography on Sephadex LH-20 (Amersham BioSciences, Sweden) columns, respectively.

Fungal material

Stromata of Hypoxylon multiforme (Fr.) Fr. (Ascomycota, Pezizomycotina, Sordariomycetes, Xylariomycetidae, Xylariales, Xylariaceae) were collected and identified on October 11, 2003 in the mixed forest ”Osterholz”, growing on a dead trunk of Betula pendula in the vicinity of Wuppertal-Schöller, Germany, by M. Stadler. This fungus is very common on Betulaceae and Salicaceae in the Northern hemisphere [10], [11]. A voucher specimen (No. STMA 03 047) is deposited at the mycological herbarium of the Fuhlrott-Museum, Wuppertal, Germany, as well as in the personal herbarium of M. Stadler. Only young stromata that had just begun to form ascospores were used for extraction.

Extraction and isolation

Dried stromata of H. multiforme (23 g) were carefully detached from the woody substrate, pulverised in a mortar and extracted with methanol (3 × 500 mL, each for 30 min) in an ultrasonic bath. The resulting extracts were filtered, combined and evaporated under vacuum (40 °C) to give an oily residue (1125 mg), which was separated by HPLC on MZ40, using the following gradient: linear from t = 0 min (20 % acetonitrile) to t = 30 min (50 % acetonitrile), isocratic from t = 30 min to t = 50 min, linear from t = 50 min to t = 70 min to 100 % acetonitrile, thereafter isocratic at 100 % acetonitrile. The fractionation was followed by UV adsorption at 210 nm, and the crude extract was divided into eight fractions. While fractions 1 and 2 and 7 and 8 contained a heterogeneous mixture of as yet unidentified components, which were not separated further for lack of sufficient material (and to some extent, due to apparent instability), the prevailing metabolites were located in fractions 3 - 6 and were further processed by complementary chromatographic methods. Fraction 3 (81.6 mg) was further chromatographed on an SiO₂ column (20 × 800 mm) using CHCl₃/MeOH/H₂O (25 : 2.5 : 0.5, 450 mL) and then by preoperative HPLC on 5 SL-II, EtOAc (400 mL), flow rate 1 mL/min and UV detection at 254 nm, Rt = 26 min to give 1 (2.2 mg). Fraction 4 (96.2 mg) was subjected to SiO₂ column (20 × 800 mm) chromatography, CHCl₃/MeOH/H₂O (25 : 2.5 : 0.5, 500 mL) to afford 2 (18.5 mg), 3 (6.5 mg) and 4 (4.9 mg). Fraction 5 (42.6 mg) was purified by gel permeation chromatography on a Sephadex LH-20 column (20 × 800 mm), CH₃OH/CHCl₃ (1 : 1, 200 mL) to yield 5 (12.2 mg). Fraction 6 (32.5 mg) was also chromatographed on an SiO₂ column (500 × 15 mm), CHCl₃/MeOH/H₂O (25 : 2.5 : 0.5, 300 mL), followed by gel permeation chromatography on Sephadex LH-20 column (500 × 15 mm), CH₃OH/CHCl₃ (1 : 1, 200 mL) to afford 2 (2.0 mg) and 3 (6.7 mg).

Multiformin A (1): [α]_D ²⁰: -69.3° (MeOH, c 0.81); UV (EtOH): λ_max (log ε) = 356 (4.0), 223 (4.0), 203 nm (4.0); CD (MeOH): λ_ext nm (Δε) = 376 (+ 20.0), 332 (-16.4), 261 (+ 8.2), 239 (-27.8); IR (KBr): ν_max = 1774, 1718, 1683, 1626, 1551, 1246, 1087, 1046, 914 cm^-1; FAB-MS: m/z = 411 [M + H]⁺; HR-FAB-MS: m/z = 411.1830 (calcd. for C₂₄H₂₇O₆ : 411.1808); ¹H- and ¹³C-NMR (CDCl₃), see Tables [1] and [2].

Multiformin B (2): [α]_D ²⁰: + 221.5° (MeOH, c 1.05); UV (EtOH): λ_max (log ε) = 340 (4.1), 264 (4.2), 221 (4.2), 203 nm (4.2); CD (MeOH): λ_ext nm (Δε) = 354 (-108.0), 277 (+ 80.0), 210 (-129.8); IR (KBr): ν_max = 1755, 1682, 1634, 1590, 1542, 1245, 1173, 915 cm^-1; FAB-MS: m/z = 409 [M + H]⁺; HR-FAB-MS: m/z = 409.1626 (calcd. for C₂₄H₂₅O₆ : 409.1651); ¹H- and ¹³C-NMR (CDCl₃), see Tables [1] and [2].

Multiformin C (3): [α]_D ²⁰: + 86.0° (MeOH, c 0.87); UV (EtOH): λ_max (log ε) = 343 (4.0), 265 (4.0), 205 nm (4.2); CD (MeOH): λ_ext nm (Δε) = 362 (-80.7), 290 (+ 46.2), 255 (+ 50.1), 210 (-84.0); IR (KBr): ν_max = 3233, 1763, 1635, 1455, 1372, 1087, 913 cm^-1; FAB-MS: m/z = 407 [M + H]⁺; HR-FAB-MS: m/z = 407.1482 (calcd. for C₂₄H₂₃O₆ : 407.1495); ¹H- and ¹³C-NMR (CDCl₃), see Tables [1] and [2].

Multiformin D (4): [α]_D ²⁰: + 79.2° (MeOH, c 0.94); UV (EtOH): λ_max (log ε) = 340 (3.8), 265 (3.8), 202 nm (4.1); CD (MeOH): λ_ext nm (Δε) = 354 (-39.7), 275 (+ 29.4), 212 (-37.8); IR (KBr): ν_max = 3338, 1745, 1714, 1683, 1634, 1541, 1424, 1372, 1255, 1033, 914 cm^-1; FAB-MS: m/z = 427 [M + H]⁺; HR-FAB-MS: m/z = 427.1770 (calcd. for C₂₄H₂₇O₇ : 427.1757); ¹H- and ¹³C-NMR (CDCl₃), see Tables [1] and [2].

Antimicrobial activity

The antimicrobial activity of multiformins A - D (1 - 4) was assayed in analogy to a conventional plate diffusion assay [13] as described in Reference [10].

Results and Discussion

The methanol extract of H. multiforme was purified by reverse-phase HPLC, SiO₂ column chromatography and finally by preparative HPLC to obtain five compounds. Four of them are new azaphilones named multiformins A - D (1 - 4); one compound is the known 4 : 5:4′:5′-tetrahydroxy-1 : 1′-binaphthyl (5) (Fig. [1]) [11].

Multiformin A (1) was characterised as C₂₄H₂₆O₆ by HR-FAB-MS. The ¹H-NMR spectrum (Table [1]) showed the presence of three singlet olefinic protons, two vicinal-coupling olefinic protons (δ_H = 6.15 and 7.05), one primary methyl, two secondary methyls and one tertiary methyl group. Its ¹³C-NMR spectrum (Table [2]) revealed the resonances of 24 carbon signals, including two conjugated ketones (δ_C = 191.6 and 194.6), one saturated ketone (δ_C = 206.1) and one lactone (δ_C = 168.5). Its spectral data are closely related to the reported known azaphilone, trichoflectin [5] except for the substitution groups at C-3, C-18 and the presence of two methine protons at C-8 and C-18 (δ_H = 3.89 and 3.97). The first unit was determined to be 6-methyl-2-oxocyclohex-3-ene by 2D NMR (Figs. [2] and [3]). This unit was attached at C-3 due to an HMBC correlation between H-10 and C-3 (Fig. [3]). The second unit, which was characterised to be 2-methylbutan-1-one, was located at C-18 by the HMBC correlation (Fig. [3]) from H-18 to C-19. The relative stereochemistries of C-7, C-8, C-10, C-15 and C-18 were determined to be 7S*, 8S*, 10S*, 15S* and 18R*, respectively, by the NOE correlations between (1) H-8 and H-9; (2) H-8 and H-18; (3) H10 and H-16 in the NOESY spectrum (Fig. [3]), and by the coupling constant of H-10 (d, J = 12.3 Hz) indicating that H-10 is axial. Therefore, multiformin A (1) was established as (6aS*,9R*,9aS*)-6a-methyl-9-(2-methylbutyryl)-3-[(1S*,6S*)-6-methyl-2-oxocyclohex-3-enyl]-9,9a-dihydro-6aH-furo[2,3-h]isochromene-6,8-dione. The configuration of C-20 has not been established. The relative stereochemical relationship between the furanone and cyclohexenone ring systems could not be determined.

The spectral data of multiformin B (2) resembled those of multiformin A (1) except for the disappearance of two hydrogen atoms at C-8 and C-18 to form an unsaturated lactone (δ_C = 167.8). The absolute configuration at C-7 was established to be S by the CD spectrum, which showed negative (-354 and -210 nm) and positive (+ 277 nm) Cotton effects [12]. From these spectral findings, multiformin B (2) was found to be (6aS)-6a-methyl-9-(2-methylbutyryl)-3-[(1S*6S*)-6-methyl-2-oxocyclohex-3-enyl]-6aH-furo [2,3-h]isochromene-6,8-dione as shown in Fig. [1]. The configuration of C-20 as well as the relative stereochemical relationship between C-7 and the cyclohexenone ring system could not be determined.

The FAB-MS of multiformin C (3) showed a molecular ion peak at 407 [M + H]⁺ corresponding to the molecular formula of C₂₄H₂₂O₆. The ¹H- and ¹³C-NMR spectra (Tables [1] and [2]) were similar to those of 2 except for the signals of the substitution group at C-3. Integration of its ¹H-¹H-COSY spectrum revealed that the aromatic proton H-13 (δ_H = 7.24) correlated with two aromatic protons H-12 (δ_H = 6.80), H-14 (δ_H = 6.86). In addition, the methyl group (H-16) coupled to C-10, C-14 and C-15 in the HMBC spectrum suggesting that the methyl group be located at C-15. Furthermore, H-12 and H-13 correlated with the phenolic carbon C-11 in the HMBC spectrum. Thus, the substitution group was identified as 3-methylphenol, which was connected to C-3 due to the HMBC correlation between H-4 and C-10. The absolute configuration of 3 at C-7 was determined to be S by comparing its CD spectrum with that of 2, which showed negative (-362 and -210 nm) and positive (+ 290 and + 255 nm) Cotton effects. Consequently, multiformin C (3) was deduced to be (6aS)-3-(2-hydroxy-6-methyl-phenyl)-6a-methyl-9-(2-methylbutyryl)-6aH-furo[2,3-h]isochromene-6,8-dione. The configuration of C-20 could not be determined.

The spectral data of multiformin D (4) were also highly similar to those of 2 and 3 aside from the notably different signals for the substituent at C-3. The structure of this moiety was found to be 4-hydroxy-2-methyl-6-oxocyclohexyl by 2D NMR. Its relative configuration was established by the NOESY spectrum, in which H-10, H-13 and H-15 were all axial due to the NOE correlations between (1) H-10 and H-14, H-16; (2) H-13 and H-15, and coupling constants of H-10 (d, J = 12.0 Hz), H-13 (tt, J = 4.7, 11.0 Hz). Furthermore, the absolute configuration at C-7 was also established to be S by comparing its CD spectrum, which showed negative (-354 and -212 nm) and positive (+ 275 nm) Cotton effects, with those of 2 and 3. From the above discussion, multiformin D (4) was deduced to be (6aS)-3-[(1S*,2S*,4R*)-4-hydroxy-2-methyl-6-oxocyclohexyl]-6aH-furo[2,3-h]isochromene-6,8-dione. The configuration of C-20 as well as the relative stereochemical relationship between C-7 and the cyclohexanone ring system could not be determined.

In vitro antimicrobial activity against a panel of human pathogenic microorganisms was measured using the established disc diffusion assay [13] at a dose of 50 μg per paper disc. Table [3] summarises the antimicrobial properties of multiformins A - D (1 - 4). Moderate to strong activity was observed against all tested strains. The highest inhibition zone values against the medically important pathogens were observed for 2 being closest to the activity of standard antibiotics. Gram-negative and Gram-positive strains were equally susceptible to the tested compounds. This is in contrast to the frequently reported resistance of Gram-negative strains that is attributed to a higher impermeability due to a more complex three-dimensional lipopolysaccharide structure of their cell walls [14], as compared to Gram-positive bacteria. The yeast, Candida albicans, and the fungal filamentous organism A. niger were the most resistant organisms to 1 - 4 in our assay; however, compounds 2 and 3 showed considerable activity against Candida albicans and Aspergillus niger. Mainly bacteriostatic or fungistatic, but weak or no cidal activity was observed for some strains (S. enteriditis and E. coli) in the cases of compounds 1 and 4. Compound 4 has shown specific activity against Klebsiella pneumoniae, while compound 1 is active against K. pneumoniae and S. aureus only.

The inhibitory effects of structurally related compounds, the deflectins [7], could be reversed by the addition of serum or serum albumin. This implies that the well known strong affinity of azaphilones to primary amines, i. e., the pyran moiety of the molecules, may at least in part be responsible for the observed activity. It is worthwhile mentioning that the noticeable difference in activity between 1 and 2 can be regarded as a result of the reduction of the C-8/C-18 double bond, the unsaturated lactone moiety, which deprives the molecule of a structural feature reminiscent of many antimicrobial sesquiterpene lactones, the α-methylene-γ-lactone unit, considered to play the role of a Michael acceptor of biological nucleophiles, e. g., with thiol groups of proteins [15]. Likewise, the rather dramatic decline in activity of 4 compared to 2 can be explained as a consequence of the loss of another unsaturated carbonyl at C-11 as well as by the presence of a hydroxy group at C-13 of 4.

The results therefore suggest a broad spectrum of strong antimicrobial activity for compounds 2 and 3. Since many fungal azaphilones such as the related deflectins [7] have been previously been reported to be strongly cytotoxic, and for structural properties of the multiformins discussed above, further selectivity tests, including determinations of their effects against mammalian cell lines should be carried out before a judgement as to their possible benefits can be made. On the other hand, this paper only focuses on the antimicrobial activity of multiformins.

It is noteworthy that the multiformins also appear to be structurally related to the cohaerins, which we concurrently reported from Hypoxylon cohaerens, a fungus that is known to be closely related to H. multiforme and which also belongs to Hypoxylon sect. Annulata [9]. In the latter study, a large number of representatives of H. multiforme had been included, and retrospective analyses of the HPLC data generated in this study were carried out. It was found that, like in H. cohaerens, the azaphilones appear to be present in particular high concentrations in young, growing stromata, while they are not easily detected in fully mature and overmature specimens. Hence, the relatively high concentrations of these metabolites observed in the stromatal extracts (and their location in granules directly beneath the stromatal surface) suggest that these broad-spectrum antibiotics may act as a means of natural chemical defence against other micro-organisms, or even other natural enemies such as insects and nematodes that may attempt to feed on the growing stromata [9], [16].

Acknowledgements

We gratefully acknowledge the financial support of JSPS (Japan Society for the Promotion of Science) for granting a postdoctoral fellowship to D. N. Quang, No. P04162. Thanks are also due to Dr. M. Tanaka and Miss. Y. Okamoto (TBU, Japan) for recording NMR and mass spectra, respectively.

References

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Yoshinori Asakawa

Faculty of Pharmaceutical Sciences

Tokushima Bunri University

Yamashiro-cho

Tokushima 770-8514

Japan

Fax: +88-655-3051

Email: asakawa@ph.bunri-u.ac.jp

References

• 1 Tabata Y, Ikegami S, Yaguchi T, Sasaki T, Hoshiko S, Sakuma S, Shin-Ya K, Seto H. Diazaphilonic acid, a new azaphilone with telomerase inhibitory activity.  J Antibiot. 1999;  52 412-4
  PubMedSearch in Google Scholar
• 2 Tomoda H, Matsushima C, Tabata N, Namatame I, Tanaka H, Bamberger M J. et al . Structure-specific inhibition of cholesteryl ester transfer protein by azaphilones.  J Antibiot. 1999;  52 160-70
  PubMedSearch in Google Scholar
• 3 Matsuzaki K, Tahara H, Inokoshi J, Tanaka H, Masuma R, Omura S. New brominated and halogen-less derivatives and structure-activity relationship of azaphilones inhibiting gp120-CD4 binding.  J Antibiot. 1998;  51 1004-11
  PubMedSearch in Google Scholar
• 4 Yoshida E, Fujimoto H, Baba M, Yamazaki M. New azaphilone metabolites isolated from ascomycetous fungi, Talaromyces luteus and T. helicus .  Tennen Yuki Kagobutsu Toronkai Koen Yoshishu. 1993;  35 290-7
  PubMedSearch in Google Scholar
• 5 Thines E, Anke H, Sterner O. Trichoflectin, a bioactive azaphilone from the ascomycete Trichopezizella nidulus .  J Nat Prod. 1998;  61 306-8
  CrossrefPubMedSearch in Google Scholar
• 6 Stadler M, Anke H, Dekermendjian K, Reiss R, Sterner O, Witt R. Novel bioactive azaphilones from fruit bodies and mycelial cultures of the Ascomycete Bulgaria inquinans (Fr.)  Nat Prod Lett. 1995;  7 7-14
  PubMedSearch in Google Scholar
• 7 Anke H, Kemmer T, Hoefle G. Deflectins, new antimicrobial azaphilones from Aspergillus deflectus .  J Antibiot. 1981;  34 923-8
  CrossrefPubMedSearch in Google Scholar
• 8 Hashimoto T, Asakawa Y. Biologically active substances of Japanese inedible mushrooms.  Heterocycles. 1998;  47 1067-110
  PubMedSearch in Google Scholar
• 9 Quang D N, Hashimoto T, Nomura Y, Wollweber H, Hellwig V, Fournier J. et al . Cohaerins A and B, azaphilones from the fungus Hypoxylon cohaerens, and comparison of HPLC-based metabolite profiles in Hypoxylon sect.  Annulata Phytochemistry. 2005;  66 797-809
  PubMedSearch in Google Scholar
• 10 Quang D N, Hashimoto T, RadulovicŽ N, Fournier J, Stadler M, Asakawa Y. Sassafrins A - D, new antimicrobial azaphilones from the fungus Creosphaeria sassafras .  Tetrahedron. 2005;  61 1743-8
  CrossrefPubMedSearch in Google Scholar
• 11 Stadler M, Wollweber H, Mühlbauer A, Henkel T, Asakawa Y, Hashimoto T. et al . Secondary metabolite profiles, genetic fingerprints and taxonomy of Daldinia and allies.  Mycotaxon. 2001;  77 379-429
  PubMedSearch in Google Scholar
• 12 Takahashi M, Koyama K, Natori S. Four new azaphilones from Chaetomium globosum var. flavo-viridae .  Chem Pharm Bull. 1990;  38 625-8
  CrossrefPubMedSearch in Google Scholar
• 13 NCCLS (National Committee for Clinical Laboratory S tandards). Performance standards for antimicrobial disk susceptibility testing, 6^th International Supplement. Fort Wayne; Wayne Publishing 1997: pp M2-A6
  Search in Google Scholar
• 14 Smith-Palmer A, Stewart J, Fyfe L. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens.  Lett Appl Microbiol. 1998;  26 118-22
  CrossrefPubMedSearch in Google Scholar
• 15 Rodriguez E, Towers G HN, Mitchell J C. Biological activities of sesquiterpene lactones.  Phytochemistry. 1976;  15 1573-80
  CrossrefPubMedSearch in Google Scholar
• 16 Stadler M, Wollweber H, Fournier J. A new species of Hypoxylon in France, and notes on the chemotaxonomy of the ”Hypoxylon rubiginosum complex”.  Mycotaxon. 2004;  90 187-211
  PubMedSearch in Google Scholar

Yoshinori Asakawa

Faculty of Pharmaceutical Sciences

Tokushima Bunri University

Yamashiro-cho

Tokushima 770-8514

Japan

Fax: +88-655-3051

Email: asakawa@ph.bunri-u.ac.jp