Introduction

Since the discovery of penicillin by Sir Alexander Fleming in 1928, several fungal-derived natural products have been discovered and developed as new drugs, such as the antibacterial agent cephalosporin C from Cephalosporium acremonium,1, 2, 3 the immunosuppressive drug cyclosporine from Tolypocladium inflatum4, 5 or the cholesterol-lowering agent lovastatin from Aspergillus terreus.6 However, the frequent re-isolation of known metabolites from fungi has turned the interest of natural product chemists to hitherto less investigated ecological niches, such as arctic glaciers, deep-sea hydrothermal vents or hypersaline lakes.7, 8 Fungi that live at elevated temperatures, at an acidic or alkaline pH, high pressure, high-salt concentration and/or low-nutrient concentration are called extremophiles, and have developed unique metabolic mechanisms to produce bioactive secondary metabolites as a response to environmental stress.9, 10, 11 Thus, extremophilic fungi are attracting considerable attention in recent years as new promising sources for biologically active compounds with potential pharmaceutical applications.12, 13

During our ongoing search for new bioactive compounds from fungi,14, 15, 16, 17 the extract of the fungus Cladosporium cladosporioides, isolated from the sediment of the hypersaline lake El Hamra located in Wadi el Natrun (Egypt), caused complete growth inhibition of the murine lymphoma cell line L5178Y when assayed at a dose of 10 μg ml−1. Bioassay-guided fractionation of the extract afforded a new viriditoxin derivative (1), together with viriditoxin (2) and two previously reported viriditoxin congeners (3 and 4).18, 19 Viriditoxin (2) was first isolated from Aspergillus viridinutans as a mycotoxin, whereas it was later also obtained from Aspergillus brevipes, Aspergillus fumigatus, Paecilomyces variotii and Spicaria divaricata.18, 19, 20 Details on the isolation and structure elucidation of 1, its biological activity and the plausible biogenesis of the isolated compounds are reported herein.

Results and Discussion

The ethyl acetate (EtOAc) extract of the sediment-derived fungus C. cladosporioides following cultivation on solid rice medium was partitioned between n-hexane and 90% aqueous methyl alcohol (MeOH). The resulting MeOH phase was fractionated by vacuum liquid chromatography on silica gel, followed by size exclusion chromatography over Sephadex LH-20 and separation on diol-functionalized silica. Final purification was achieved by semipreparative reversed-phase HPLC to yield a new viriditoxin derivative (1), along with three known compounds including viriditoxin (2) and its derivatives (3) and (4) (Figure 1).

Figure 1
figure 1

Structures of cladosporinone (1), viriditoxin (2) and viriditoxin derivatives (3 and 4).

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Compound 1 was isolated as a red, amorphous powder. Its molecular formula was established as C33H30O14 based on the prominent ion peak observed at m/z=651.1706 [M+H]+ in the high-resolution ESIMS spectrum. The 1H and 13C NMR data of 1 (Table 1) were similar to those of the co-isolated viriditoxin (2). Accordingly, the 1H NMR spectrum of 1 revealed signals corresponding to four methylene groups (H-4, δH 2.98 and 2.89; H-11, δH 2.81; H-4′, δH 2.36 and 2.11 and H-11′, δH 2.32 and 2.13), four methoxy groups (12-OCH3, δH 3.63; 7-OCH3, δH 3.77; 12′-OCH3, δH 3.49 and 7′-OCH3, δH 3.72), two oxymethine protons (H-3, δH 4.94 and H-3′, δH 3.84) and four singlets in the aromatic region (H-8, δH 6.84; H-8′, δH 6.70, H-5′, δH 6.58 and H-5, δH 6.41) (Table 1). In addition, one exchangeable proton detected at δH 12.81 was attributable to a chelated hydroxy group. The 13C and DEPT spectra revealed the presence of 19 sp2 quaternary carbons, including five carbonyl groups (δC 169.90, C-1; δC 169.87, C-12; δC 179.9, C-10′; δC 180.8, C-10a′ and δC 171.5, C-12′).

Table 1 NMR spectroscopic data of 1 [DMSO-d6] (1H NMR in 600 MHz, 13C NMR in 150 MHz, δ in p.p.m.)
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Further inspection of the 2D NMR (COSY, HSQC and HMBC) data allowed us to establish the planar structure of 1. The HMBC correlations from H-8 to C-9, C-9a and C-6, from H-5 to C-4, C-9a, C-10a, C-5a and C-6, and from H2-4 to C-10a, C-5, C-11 and C-3 indicated the presence of a 3,4-dihydro-α-napthopyrone structure. Further correlations from 7-OCH3 to C-7, from 12-OCH3 to C-12 and from H2-11 to C-4, C-3 and C-12 fully supported the semi-viriditoxin moiety in the structure of 1 (Figure 2). Analysis of the 1D and 2D NMR data revealed that 1 also contained a 1,2-naphthoquinone moiety as confirmed by the HMBC correlations from H-8′ to C-6′, C-9′ and C-9a′ and from H-5′ to C-9a′, C-6′, C-10a′, C-4′ and C-4a′. In addition, a 3′-OH-methyl butyrate side chain was located at C-4a′ of the 1,2-benzoquinone ring, as supported by the COSY correlations from H-3′ to H2-4′ and H2-11′, and by the HMBC correlations from H-4′ to C-3′, C-4a′, C-10a′, C-5′ and C-11′, from H-11′ to C-3′, C-4′ and C-12′, and from 12′-OCH3 to C-12′ (Figure 2). In a similar manner to viriditoxin, the HMBC correlations from 7′-OCH3 to C-7′ and from the chelated proton 9′-OH to C-8′, C-9′ and C-9a′, indicated the presence of 7′-methoxy and 9′-hydroxy substituents in the aromatic ring, leaving only C-6′ for the connection of the two monomers through a 6′,6-biaryl linkage. Thus, the planar structure of 1 was elucidated as shown in Figure 2.

Figure 2
figure 2

Selected HMBC and COSY correlations of 1.

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In an attempt to determine the absolute configuration of C-3′, the modified Mosher’s method was applied, however, this reaction failed to give the corresponding Mosher’s ester, probably, due to the chelated hydroxy group. The axial chirality of 1 was determined by comparing its electronic circular dichroism (ECD) spectrum (Figure 3) with that of the related viriditoxin (2), which had a negative exciton couplet (275 nm −194.7, 255 nm +174.0) in chloroform.18 The negative couplet of viriditoxin derives from coupling of the two 1Bb transitions of the interacting 6,6′-linked naphthopyrone chromophores having a counter-clockwise twist with a dihedral angle of about 90°. Although one of the naphthalene ring is replaced by a 1,2-naphthoquinone residue in 1, the alignment of the interacting electric transition moments are not expected to alter significantly, and thus similarly to viriditoxin, the negative exciton couplet of 1 derives from (aR) axial chirality of the biaryl system. As the ECD spectrum is governed predominantly by the axial chirality element, the central chirality of the fused α-pyrone ring of 1 could not be determined from ECD. However, this configuration was determined earlier for semi-viriditoxin, the naphthopyrone monomer of viriditoxin, as (S)21 and it was also confirmed by stereoselective synthesis.22 Thus, 1 was identified as a new natural heterodimer for which the name cladosporinone is proposed.

Figure 3
figure 3

ECD spectrum of 1 in acetonitrile. Δɛ values were multiplied by 5 above 314 nm for better visualization.

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The known compounds viriditoxin (2) and its analogs (3 and 4) were identified on the basis of NMR, mass spectrometry and optical rotation data analysis, as well as by comparison with the literature.18, 19 The axial chirality of 3 and 4 had been deduced earlier as (aR) by comparison of their ECD curves with those of 2.18, 19, 20 Several attempts were undertaken to influence the pattern of metabolites produced by C. cladosporioides either by changing the medium (OSMAC approach23) or by co-culturing the fungus in the presence of several bacteria, such as Bacillus subtilis 168 trpC2, Bacillus cereus T, Streptomyces lividans TK24 and Streptomyces coelicolor A2(3). Mixed fermentation of fungi and bacteria has repeatedly been shown to activate silent fungal biogenetic gene clusters, thus causing the accumulation of compounds not detected in axenic fungal cultures.24, 25 However, none of these attempts caused a significant change in the metabolite pattern on C. cladosporioides as shown by HPLC analysis of the respective extracts and by comparison to extracts of the fungus grown only on rice.

A plausible biogenetic pathway of compounds 1–4 includes a head-to-tail combination of one acetyl-CoA (starter unit) and seven malonyl-CoA (extender units) moieties, a process that is catalyzed by multidomain enzymes called polyketide synthases. Then, the octaketide chain would undergo regiospecific aromatization, reduction or cyclization reactions to form precursor B. Dehydration and lactonization of the latter (B) and subsequent methoxylation would afford the monomeric precursors C and D, respectively. Oxidative coupling of the different monomers at C-6/C-6′ would result in the formation of 2–4, as shown in Figure 4. An alternative biosynthetic route would include oxidative decarboxylation of precursor B to form the 1,2-naphthoquinone monomer, followed by oxidative coupling with monomer D to afford the new metabolite 1 (Figure 4).26

Figure 4
figure 4

Proposed biosynthetic pathway of 1–4.

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Compounds 1–4 were assayed for their cytotoxicity toward L5178Y (murine lymphoma) cells. Viriditoxin (2) exhibited the most potent cytotoxicity against the tested cell line, with an IC50 of 0.1 μm, followed by compound 3 with an IC50 of 0.25 μm and the new compound 1 with an IC50 of 0.88 μm (Table 2). Compound 4 was inactive in this bioassay. The loss of one or both methylated carboxyl groups at C-12 and C-12′ as in 3 and 4 weakens the cytotoxicity of the respective derivatives when compared to 2. Hydrolysis of one of the two lactone rings followed by oxidative decarboxylation as in 1 likewise attenuates the cytotoxicity as evident when compared to 2. In addition, compounds 1–4 were evaluated for their antimicrobial activities against Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25213, and Pseudomonas aeruginosa ATCC 27853 (Table 2). Interestingly, all compounds showed selective activity against S. aureus ATCC 29213, with viriditoxin (2) being by far the most active compound exhibiting a MIC of 0.015 μg ml−1 (0.023 μm), thus confirming the results of earlier studies.27, 28, 29

Table 2 Bioactivities of tested compounds 1–4
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Experimental Procedure

General experimental procedures

Optical rotations were determined on a Perkin-Elmer-241 MC polarimeter (Perkin-Elmer, Waltham, MA, USA). 1H, 13C, and 2D NMR spectra were recorded at 25 °C in DMSO-d6 on Bruker AVANCE DMX 600 NMR spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany). Chemical shifts were referenced to the solvent residual peaks, δH=2.50 p.p.m. for 1H and δc=39.51 p.p.m. for 13C. Mass spectra (ESI) were recorded with a Finnigan LCQ Deca mass spectrometer (Thermo Fisher Scientific GmbH, Bremen, Germany), and HRMS (ESI) spectra were obtained with a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer (Thermo Fisher Scientific GmbH). Solvents were distilled before use and spectral grade solvents were used for spectroscopic measurements. HPLC analysis was performed with a Dionex UltiMate3400 SD (Dionex Softron GmbH, Munich, Germany) with a LPG-3400 s.d. Pump (Dionex Softron GmbH) coupled to a photodiode array detector (DAD3000RS); routine detection was at 235, 254, 280 and 340 nm. The separation column (125 × 4 mm) was prefilled with Eurosphere-10 C18 (Knauer, Germany), and the following gradient was used (MeOH, 0.02% HCOOH in H2O): 0 min (10% MeOH); 5 min (10% MeOH); 35 min (100% MeOH); 45 min (100% MeOH). Semipreparative HPLC was performed using a Merck Hitachi HPLC System (Merck KGaA, Darmstadt, Germany; UV detector L-7400; Pump L-7100; Eurosphere-100 C18, 300 × 8 mm, Knauer, Germany). Column chromatography included LH-20 Sephadex (0.25–0.1 mm mesh size, GE Healthcare Europe GmbH, Freiburg, Germany), Merck MN Silica gel 60 M (0.04–0.063 mm, Merck KGaA) and Diol-functionalized silica gel spherical (40–75 μm, GE Healthcare Europe GmbH). TLC plates with silica gel F254 (Merck KGaA) were used to monitor fractions; detection was under UV at 254 and 366 nm or by spraying the plates with anisaldehyde reagent followed by heating.

Fungal material

The fungus C. cladosporioides was isolated from the sediment of the hypersaline lake El Hamra located in Wadi el Natrun of Egypt, which was collected in November 2012. The isolation was performed as described before.30

Identification of fungal cultures

The fungus was identified as C. cladosporioides according to a molecular biological protocol by DNA amplification and sequencing of the internal transcribed spacer (ITS) region as described previously.31 The sequence data was submitted to the GenBank, with accession number KT899331. The fungal strain is kept in one of the author’s laboratory (PP).

Fermentation

The fungus was cultivated on solid rice medium in six Erlenmeyer flasks. Solid rice medium was prepared by adding demineralized water (110 ml) to rice (100 g) in an Erlenmeyer flask, followed by autoclaving (121 °C, 20 min). The fungus, which nearly covered the whole surface of a petri dish, was inoculated onto this sterile rice medium under the clean bench and was allowed to grow (20 °C) for 35 days. Attempts to optimize the fermentation conditions were performed by adding 3.5% sea salt to the rice medium or by adjusting the pH of the medium to 9 or 11 by adding sodium hydroxide. In addition, co-cultivation experiments of C. cladosporioides with B. subtilis 168 trpC2, B. cereus T, S. lividans TK24 and S. coelicolor A2(3) were performed on rice medium as described before.24, 25

Extraction and isolation

Fungal cultures were extracted with EtOAc. The crude extract was evaporated to dryness, and then subjected to liquid–liquid partitioning between n-hexane and 90% aqueous MeOH. The 90% MeOH fraction amounted to 4.9 g, and was further separated by vacuum liquid chromatography (4.5 × 30 cm) on silica gel, using solvents in a gradient of increasing polarity—n-hexane–ethyl acetate–dichloromethane–methanol—to obtain a total of 11 fractions. The fractions eluted with n-hexane–ethyl acetate (30:70—100% EtOAc, 126 mg) and DCM–MeOH (90:10, 153 mg) were then subjected to column chromatography (3.5 × 60 cm) using Sephadex LH-20 as stationary phase and DCM–MeOH (50:50v/v) as mobile phase to remove pigments. The bioactive fraction (87 mg) was further subjected to column chromatography (2.5 × 50 cm) over diol-functionalized silica using DCM–MeOH (99:1v/v) as eluting solvent. Fractions were combined according to TLC monitoring (53 mg) and purified by reversed-phase HPLC (silica-based, semipreparative column), with gradient elution MeOH–H2O from 50:50 to 60:40 in 30 min, to yield the four compounds 1 (5.3 mg), 2 (21 mg), 3 (4.4 mg) and 4 (3.9 mg).

Cladosporinone (1): red, amorphous powder; [α]22D=−154° (c 0.35, MeOH); UV (MeCN) λmax log ɛ: 440sh (2.8), 383 (3.1), 334sh (2.9), 264 (3.9), 222sh (3.7). ECD (MeCN, λ (nm) (Δɛ), c=2.56 × 10−4 m): 471 (−1.8), 386 (1.5), 359 (−0.4), 333 (3.8), 289sh (−9.1), 268 (−91.6), 246 (64.3), 200 (−6.2).; 1H (600 MHz) and 13C (150 MHz) NMR, see Table 1; ESI-MS m/z 651.0 [M+H]+, 649.4 [M−H]−; HRESIMS m/z 651.1706 [M+H]+ (calcd for C33H31O14, 651.1708).