Bacillisporins D and E, New Oxyphenalenone Dimers from Talaromyces bacillisporus

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The oligophenalenone dimer duclauxin and two new analogues, bacillisporins D and E, were isolated from the fungus Talaromyces bacillisporus in addition to the previously reported bacillisporins A, B and C. Structures were established by spectroscopic studies. Duclauxin and bacillisporins A, B, C and E were evaluated for cytotoxicity against three human cancer cell lines. Bacillisporin A was strongly active against MCF-7 and NCI-H460 and moderately active against SF-268 while bacillisporins B, C and duclauxin were moderately active against all three cell lines.

An earlier article on the chemistry of the fungus Talaromyces bacillisporus [1] described three new oligophenalenone dimers [2], which because the authors misspelled the name of the organism were misnamed bacillosporins A, B and C (1, 2 and 3) instead of more properly bacillisporins A - C, a designation which we will use henceforth. A new xanthone pinselin was also reported in the same article. Other authors [3] have isolated a cytotoxic pyrrolizidinedione derivative from the same fungus. The effect of bacillisporin A on mitochondrial respiration has been studied [4]. We have examined T. bacillisporus from a location in Thailand and have isolated not only bacillisporins A - C, but also the oligophenalenone dimer duclauxin (4) [5], [6], [7], [8] and two new oligophenalenone dimers bacillisporins D (5) and E (6) (Fig. [1]). Duclauxin has been previously reported from three other Talaromyces species [9] and several articles have described various biological activities of this substance [10], [11], [12], [13].

¹H- and ¹³C-NMR spectra of the new oligophenalenone dimers 5, C₂₆H₁₈O₁₀, and 6, C₂₈H₂₀O₁₁, in DMSO at 500 and 125 MHz are listed in Tables [1] and [2] together with those of duclauxin (1) and bacillisporins A - C (2 - 4). The available literature on duclauxin [5], [6], [7], [8] contains only an ¹H-NMR spectrum at an unspecified but presumably much lower field strength, while those of 2, 3 and 4 were run earlier [2] in acetone or tetrahydrofuran on a 100 MHz spectrometer. Assignments are based on HSQC, COSY and HMBC measurements. The mass spectra and spectroscopic data showed that bacillisporin E (6) is formally related to duclauxin by elimination of the elements of methanol from the ring containing the ketone group and that bacillisporin D (5) is the deacetyl derivative of E; conversely, biogenesis of duclauxin might involve acetylation of bacillisporin E followed by addition of methanol.

Oxaphenalenones 1 - 4 and 6 were screened for in vitro cytotoxicity against three human tumor cell lines MCF-7, NCI-H-460 and SF-268. The results, given in concentrations causing 50 % cell growth inhibition (GI₅₀), are summarized in Table [3]. These show that bacillisporin A (1) exhibited strong growth inhibitory effects on MCF-7 and NCI-H460 and a moderate growth inhibitory effect on SR-268. Bacillisporins B (2), C (3) and duclauxin (4) exhibited moderate inhibitory effects against all three cell lines but bacillisporin E (6) showed little activity. Bacillisporin D (5) was not tested because of the small amount of material isolated from the fungus.

Materials and Methods

General experimental procedures: ¹H- and ¹³C-NMR spectra were recorded at ambient temperature in DMSO on a Bruker DRX instrument operating at 500 and 125 MHz respectively. EI mass spectra were measured on a Hitachi Perkin-Elmer RMV-GM instrument. HR mass spectra were measured on a Kratos Concept II 2 sector mass spectrometer. Melting points were recorded on a Bock Monoscope and are uncorrected. Rotations were determined on a Polax-2L instrument. Silica gel for chromatography was Silica gel 60 (0.2 - 0.5 mm Merck) for analytical work and for preparative TLC Silica gel 60 GF 254 Merck.

Biological material: Talaromyces bacillisporus R. Benjamin was isolated from a soil sample collected on the campus of Kasetsart University, Bangkok, Thailand in July 2003 and identified by L. M. on the basis of the description in [1] and standard tests. A sample with accession number KPFC 3350 has been deposited in the Department of Plant Pathology, Faculty of Agriculture, Kasetsart University.

Culture and isolation of constituents: Twenty-five 1000-mL Erlenmeyer flasks, each containing 200 g of rice and 100 mL of H₂O were autoclaved at 121 °C for 15 min, inoculated with three mycelium plugs from the T. bacillisporus T2O culture and incubated at 28 °C for 30 days. To each flask containing the moldy rice was added 400 mL of EtOAc, after which the contents were left to macerate for 3 days and then filtered using filter paper. Evaporation of the combined filtrates to a volume of 1000 mL at reduced pressure followed by addition of anhydrous sodium soleplate, filtration and evaporation of the filtrate at reduced pressure furnished 105 g of dark brown crude EtOAc extract which was extracted with CHCl₃ (3 × 500 mL). The CHCl₃ extracts were combined and concentrated at reduced pressure to afford 85 g of a brown viscous mass.

The crude CHCl₃ extract was applied to a silica gel column (200 g) and eluted with CHCl₃-petrol and CHCl₃-acetone, 300 mL frs being collected as follows: Frs 1 - 142 (CHCl₃-petrol, 1 : 1), 143 - 218 (CHCl₃-petrol, 7 : 3), 219 - 286 (CHCl₃-petrol, 9 : 1), 287 - 315 (CHCl₃-acetone, 9 : 1), 316 - 343 (CHCl₃-acetone, 4 : 1), 344 - 365 (CHCl₃-acetone, 7 : 3). Frs 23 - 28 (600 mg) were combined and recrystallized from CHCl₃-petrol to give bacillisporin A (1) as a pale yellow solid (300 mg). TLC of the mother liquor (silica gel, CHCl₃-acetone-HCO₂H, 95 : 5:1) gave 35 mg of duclauxin (4) as a yellow solid. Frs 29 - 111 (2 g) were combined, applied to a silica gel column (50 g) and eluted with CHCl₃-petrol and CHCl₃-acetone, 100 mL subfrs being collected as follows: Frs 1 - 22 (CHCl₃-petrol, 7 : 3), 23 - 64 (CHCl₃-petrol, 9 : 1), 65 - 98 (CHCl₃-acetone, 9 : 1). Subfrs 3 - 5 were combined (800 mg) and recrystallized from CHCl₃-petrol to give more bacillisporin A (1, 250 mg). Purification of the mother liquor by TLC (silica gel, CHCl₃-acetone-HCO₂H, 95 : 5:1) gave 27 mg of duclauxin (4) as a yellow solid. Subfrs 43 - 64 (30 mg) were combined and purified by TLC (silica gel, CHCl₃-acetone-HCO₂H, 85 : 15 : 1) to give 9 mg of bacillisporin D (5) as a yellow solid.

Frs 143 - 160 (2 g) were combined and recrystallized from CHCl₃-acetone to give 480 mg of bacillisporin E (6). The mother liquor was chromatographed over silica gel (20 g) and eluted with CHCl₃-petrol and CHCl₃-acetone as follows using 100 mL subfractions, subfrs 1 - 61 (CHCl₃-petrol, 7 : 3), 62 - 72 (CHCl₃-petrol, 9 : 1), 73 - 87 (CHCl₃-acetone, 9 : 1) and 88 - 100 (CHCl₃-acetone, 4 : 1). Subfrs 16 - 21 (200 mg) were combined and purified by TLC (silica gel, CHCl₃-acetone-HCO₂H, 85 : 15 : 1) to give 22 mg of bacillisporin E (6). Combination of frs 161 - 180 (1.5 g) and recrystallization from CHCl₃-acetone afforded 600 mg of bacillisporin C (3). Combination of frs 211 - 215 (180 mg) and recrystallization from CHCl₃-acetone afforded 200 mg of bacillisporin B (2).

Bacillisporin A (1): Pale yellow solid precipitated from a mixture of CHCl₃ and acetone; m. p. 244 - 246 °C; [α]_D ²⁰: + 21.7° (MeOH, c 0.023 g/100 mL); ¹H- and ¹³C-NMR see Tables [1] and [2].

Bacillisporin B (2): Pale yellow solid precipitated from a mixture of CHCl₃ and acetone; m. p. 263 - 265 °C; [α]_D ²⁰: + 31.8° (MeOH, c 0.022 g/100 mL); ¹H- and ¹³C-NMR see Tables [1] and [2].

Bacillisporin C (3): Pale yellow solid precipitated from a mixture of CHCl₃ and acetone; m. p. above 280 °C with decomposition; [α]_D ²⁰: + 23.4° (MeOH, c 0.063 g/100 mL); ¹H- and ¹³C-NMR see Tables [1] and [2].

Duclauxin (4): White solid precipitated from a mixture of CHCl₃ and petroleum ether; m. p. 224 - 226 °C; [α]_D ²⁰: + 11.2° (CHCl₃, c 0.018 g/100 mL); ¹H- and ¹³C-NMR see Tables [1] and [2].

Bacillisporin D (5): Yellow semisolid; [α]_D ²⁰: -19.4° (CHCl₃, c 0.031 g/100 mL); ¹H- and ¹³C-NMR see Tables [1] and [2]; FAB-HR-MS: m/z = 491.09789 [M + H]⁺; calcd. for C₂₆H₁₉O₁₀: 491.09782.

Bacillisporin E (6): Pale yellow semisolid; [α]_D ²⁰: -21.4° (CHCl₃, c 0.047 g/100 mL); ¹H- and ¹³C-NMR see Tables [1] and [2]; FAB-HR-MS: m/z = 533.10830 [M + H]⁺; calcd. for C₂₈H₂₁O₁₁: 533.10839.

Tumor cell growth assay: Stock solutions of compounds 1, 2, 3, 4 and 6 in DMSO were prepared at 400 times the maximum test concentrations and stored at -20 °C. The frozen samples were diluted with culture medium immediately prior to the assay. Control cultures were always mixed with an equivalent amount of DMSO. Effects of compounds 1, 2, 3, 4 and 6 on the growth of human tumor cell lines were evaluated by the procedure used by the National Cancer Institute for the in vitro anticancer drug discovery screen which uses the protein-binding dye sulforhodamine B (SRB) to assess cell growth inhibition [14]. Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium (Invitrogen; Carlsbad, CA, USA) supplemented with 5 % heat-inactivated fetal bovine serum, 2 mM glutamine and 50 μg/mL of gentamicin at 37 °C in a humidified atmosphere containing 5 % CO₂. The optimal plating density of each cell line ensuring exponential growth throughout the period of the experiment was identical with that published originally and was 1.5 × 10⁵ cells/mL for MCF-7 and SF-268 and 7.5 × 10⁴ cells/mL for NCI-N460. Cells were exposed for 48 hours to five concentrations of 1, 2, 3, 4 and 6 starting from a maximum concentration of 100 μM. For each compound tested and for each cell line a dose response curve was generated and GI₅₀ was calculated as described [15]. Doxorubin was used as positive control.

Acknowledgements

Work in Portugal was supported by Fundação para a Ciência e Tecnologia of Portugal (Unidade de I & D 226/96), POCTI (QAIII), FEDER, CIIMAR Pluriannual. Tida Dethoup thanks the Thailand Research fund (TRF) for a fellowship under the RGJ-PHD program.

References

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  Search in Google Scholar
• 2 Yamazaki M, Okuyama E. Isolation and structures of oxaphenalenone dimers from Talaromyces bacillosporus (sic!).  Chem Pharm Bull. 1980;  28 3649-55
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• 6 Ogihara Y, Iitaka Y, Shibata S. X-Ray study of monobromoduclauxin. Tetrahedron Lett 1965: 1289-90
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• 12 Kawai K, Nozwa Y, Ito T, Yamanaka N. Effects of xanthomegnin and duclauxin on culture cells of murine leukemia and Ehrlich ascites tumor.  Res Commun Chem Pathol Pharmacol. 1982;  3 29-38
  PubMedSearch in Google Scholar
• 13 Shiojiri H, Kawai K, Kato T, Ogihara Y, Nozawa Y. Cytotoxicities on culture cells and inhibitory effects on mitochondrial respiration by duclauxin and related compounds.  Mycotoxins. 1983;  18 38-41
  PubMedSearch in Google Scholar
• 14 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer drug screening.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 15 Monks A, Scudiero D, Skehan P, Shoemaker R, Paul K, Vistica D. et al . Feasibility of a high-flux anticancer drug screening using a diverse panel of cultured human tumor cell lines.  J Natl Cancer Inst. 1991;  83 757-76
  CrossrefPubMedSearch in Google Scholar

Prof. Werner Herz

Department of Chemistry and Biochemistry

Florida State University

Tallahassee

FL 32306-4390

USA

Fax: +1-850-644-8281

Email: jdulin@chem.fsu.edu

References

• 1 Stolk A C, Samson R A. The genus Talaromyces: Studies on Talaromyces and related genera II. Studies in mycology No 2. Baarn; Centralbureau voor Schimmelcultures (CBS) 1972: p 1-65
  Search in Google Scholar
• 2 Yamazaki M, Okuyama E. Isolation and structures of oxaphenalenone dimers from Talaromyces bacillosporus (sic!).  Chem Pharm Bull. 1980;  28 3649-55
  PubMedSearch in Google Scholar
• 3 Ishii K, Itoh T, Kobayashi K, Horie Y, Ueno Y. Isolation of a cytotoxic metabolite of Talaromyces bacillosporus (sic!).  Appl Envir Microbiol. 1995;  61 941-3
  PubMedSearch in Google Scholar
• 4 Shiojiri H, Nakamura T, Hissada K, Kawai K, Nozawa Y, Okuyama E. et al . The effect of bacillosporin A from Talaromyces bacillosporus (sic!) on mitochondrial respiration.  Mycotoxins. 1984;  20 17-9
  PubMedSearch in Google Scholar
• 5 Shibata S, Ogihara Y, Tokutake N, Tanaka O. Duclauxin, a metabolite of Penicillium duclauxii. Tetrahedron Lett 1965: 1287-8
  Search in Google Scholar
• 6 Ogihara Y, Iitaka Y, Shibata S. X-Ray study of monobromoduclauxin. Tetrahedron Lett 1965: 1289-90
  Search in Google Scholar
• 7 Kuhr I, Fuska J. Antitumor antibiotic produced by Penicillium stipitatum. Its identity with duclauxin.  J Antibiot. 1973;  26 535-6
  PubMedSearch in Google Scholar
• 8 Cooke R G, Edwards G M. Naturally occurring phenalenones and related compounds. In: Herz W, Grisebach H, Kirby GW, editors Progress in the chemistry of organic natural products. Wien; Springer Verlag 1981: p 153-90
  Search in Google Scholar
• 9 Frisvad J C, Filtenborg O, Samson R A, Stolk A C. Chemotaxonomy of the genus Talaromyces .  Antonie van Leuwenhoek. 1990;  57 179-89
  PubMedSearch in Google Scholar
• 10 Fuskova A, Proksa B, Fuska J. In vitro effect of duclauxin and derivatives of coumarin on nucleic acid and protein synthesis in Ehrlich’s ascites carcinoma cells.  Pharmazie. 1977;  32 291
  PubMedSearch in Google Scholar
• 11 Kovac L, Bohmerova E, Fuska J. Inhibition of mitochondrial functions by the antibiotics bikaverin and duclauxin.  J Antibiot. 1978;  31 616-20
  PubMedSearch in Google Scholar
• 12 Kawai K, Nozwa Y, Ito T, Yamanaka N. Effects of xanthomegnin and duclauxin on culture cells of murine leukemia and Ehrlich ascites tumor.  Res Commun Chem Pathol Pharmacol. 1982;  3 29-38
  PubMedSearch in Google Scholar
• 13 Shiojiri H, Kawai K, Kato T, Ogihara Y, Nozawa Y. Cytotoxicities on culture cells and inhibitory effects on mitochondrial respiration by duclauxin and related compounds.  Mycotoxins. 1983;  18 38-41
  PubMedSearch in Google Scholar
• 14 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer drug screening.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 15 Monks A, Scudiero D, Skehan P, Shoemaker R, Paul K, Vistica D. et al . Feasibility of a high-flux anticancer drug screening using a diverse panel of cultured human tumor cell lines.  J Natl Cancer Inst. 1991;  83 757-76
  CrossrefPubMedSearch in Google Scholar

Prof. Werner Herz

Department of Chemistry and Biochemistry

Florida State University

Tallahassee

FL 32306-4390

USA

Fax: +1-850-644-8281

Email: jdulin@chem.fsu.edu