- Email: [email protected]
Biotransformation of quinazoline and phthalazine by Aspergillus niger
Journal of Bioscience and Bioengineering VOL. 111 No. 3, 333 – 335, 2011 www.elsevier.com/locate/jbiosc
NOTE
Biotransformation of quinazoline and phthalazine by Aspergillus niger John B. Sutherland,⁎ Thomas M. Heinze, Laura K. Schnackenberg, James P. Freeman, and Anna J. Williams National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA Received 14 June 2010; accepted 22 November 2010 Available online 18 December 2010
Cultures of Aspergillus niger NRRL-599 in fluid Sabouraud medium were grown with quinazoline and phthalazine for 7 days. Metabolites were purified by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Quinazoline was oxidized to 4-quinazolinone and 2,4-quinazolinedione, and phthalazine was oxidized to 1-phthalazinone. @ 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aspergillus niger; Azaarene; Benzodiazine; Biotransformation; Phthalazine; Quinazoline]
The azaarene ring structures of quinazoline and phthalazine (Fig. 1) are found in various pharmaceuticals and dyes. Benzodiazines are persistent and toxic to aquatic organisms (1,2), and phthalazine also is positive for genotoxicity by the SOS-Chromotest (3). The benzodiazines, which may react with oxygen radicals in water, also are biotransformed by microorganisms. Quinazoline is metabolized to 4-quinazolinone, quinazoline cis-5,6-dihydrodiol, and quinazoline 5,6,7,8-tetrahydro-cis-5,6-dihydrodiol by Pseudomonas putida (4,5) and to 2,4-quinazolinedione by Streptomyces viridosporus (6). Phthalazine is metabolized to 1-phthalazinone by the bacteria Pseudomonas diminuta, Arthrobacter sp., and Streptomyces viridosporus and the fungus Fusarium verticillioides (6–9), and it is metabolized to phthalazine N-oxide by the fungus Cunninghamella elegans (9). Aspergillus niger is a common saprobic fungus that frequently has been proposed for use in the bioremediation of sites containing polycyclic aromatic hydrocarbons, heavy metals, pesticides, and textile dye effluents. It oxidizes indole to 3-hydroxyindole, later cleaving both benzo and pyrrole rings (10), and it transforms the diazine ring of cinnoline to produce two N-oxides (11). We dosed cultures of A. niger with quinazoline and phthalazine to find out whether it was able to metabolize these azaarenes; chemical analysis of the metabolites showed that it oxidized only the diazine ring of each. Triplicate 125-ml flasks, each containing 30 ml of fluid Sabouraud medium with 25 μM naphthalene (Aldrich Chemical Co., Milwaukee, WI) to induce oxidative enzymes (12), were inoculated with spores of A. niger NRRL-599 (U.S. Department of Agriculture, Peoria, IL) from slants of potato dextrose agar and incubated at 25 °C with shaking at 125 rpm. After 3 days, quinazoline or phthalazine (Aldrich) was dissolved in water, filter-sterilized, and used to dose each of the cultures (except for non-dosed controls) and the noninoculated controls directly for a final concentration of 0.44 mM. The cultures were harvested 7 days after dosing and both the mycelium and the ⁎ Corresponding author. Tel.: +1 870 543 7059; fax: +1 870 543 7307. E-mail address: [email protected] (J.B. Sutherland).
culture fluid were extracted three times with ethyl acetate. The extracts were combined, dried over anhydrous Na2SO4, and evaporated in vacuo. The residues were redissolved in methanol for analysis. The extracts were analyzed with an Agilent Technologies (Palo Alto, CA) 1100 series high-performance liquid chromatograph (HPLC). A 5-μm Waters (Milford, MA) Spherisorb ODS-2 column (250 × 2 mm) was used with a 20-min gradient of 20–60% methanol in buffer (50 mM ammonium formate, pH 5.5) with a 10-min hold at 60% methanol. The flow rate was 1 ml/min and the UV detector was at 254 nm. Peaks representing possible metabolites were collected and then the buffer was removed by solid-phase extraction. Metabolites were identified by gas chromatography/electron ionization mass spectrometry (GC/EI-MS) (9) and by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESIMSMS) (13) using a Phenomenex (Torrance, CA) Gemini 3-μm C18 column (150 × 2 mm); the spectra were compared with those of authentic compounds and published data. 2,4-Quinazolinedione (benzoyleneurea) was from Sigma Chemical Co. (St. Louis, MO), and 4-quinazolinone (4-hydroxyquinazoline) and 1-phthalazinone (phthalazone) were from Aldrich. Samples dissolved in methanol-d4 (Cambridge Isotope Laboratories, Andover, MA) were analyzed at 600 MHz by proton nuclear magnetic resonance (NMR) spectroscopy (14). Extracts from cultures of A. niger NRRL-599 dosed with quinazoline, analyzed by HPLC using ammonium formate buffer, showed peaks at 10.5 min (metabolite I), 13.2 min (metabolite II), and 19.0 min (quinazoline) (Fig. 1A). Extracts from cultures dosed with phthalazine showed peaks at 10.8 min (metabolite III) and 15.9 min (phthalazine) (Fig. 1B). Quinazoline metabolite I had the same retention time and UV spectrum (λmax = 219 and 310 nm) as authentic 2,4-quinazolinedione. GC/MS of both metabolite I and 2,4-quinazolinedione showed significant ions at m/z 162 [M+·] (100), 119 (66), and 92 (28). The product– ion spectra (LC/ESI-MSMS) of both metabolite I (Table 1) and 2,4-
1389-1723/$ - see front matter @ 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.11.017
334
SUTHERLAND ET AL.
J. BIOSCI. BIOENG.,
I
A
225
II
5
200
4
6
3
N
175 150
7
125
8
Absorbance at 254 nm (mAU)
100
2
N 1
75 50
Q
25 0 0
5
10
15
20
B
225
25
5
4
6
200
3
N
III
175 150
N 2
7
125
8
P
100
1
75 50 25 0 0
5
10
15
20
25
Time (min) FIG. 1. HPLC elution profiles at 254 nm of the ethyl acetate extracts from cultures of Aspergillus niger NRRL-599 dosed with (A) quinazoline and (B) phthalazine (I = 2,4-quinazolinedione; II= 4-quinazolinone; III = 1-phthalazinone; Q = quinazoline; P = phthalazine).
quinazolinedione showed ions at m/z 163 [M+ H]+ and 146, among others. The 1H NMR chemical shifts of metabolite I were 8.04 (H5, d, 7.9 Hz), 7.68 (H7, td, 8.4, 1.5 Hz), 7.26 (H6, td, 7.5, 1.1 Hz), and 7.21 (H8, d, 8.3 Hz). Metabolite I was confirmed, by comparison of the NMR spectrum with that of the authentic compound, as 2,4-quinazolinedione. Quinazoline metabolite II had the same retention time and UV spectrum (λmax = 200, 226, 264, 302, and 313 nm) as authentic 4-quinazolinone. The product–ion spectra (LC/ESI-MSMS) of metabolite II (Table 1) and 4-quinazolinone showed ions at m/z 147 [M + H]+, 130, and others, indicating that metabolite II was a quinazolinone. The 1H NMR chemical shifts of metabolite II were 8.26 (H5, d, 8.8 Hz), 8.13 (H2, s), 7.87 (H7, td, 7.9, 1.1 Hz), 7.74 (H8, d, 7.2 Hz), and 7.59 (H6, td, 7.2, 1.3 Hz). Metabolite II was confirmed, by comparison of the NMR spectrum with that of the authentic compound, as 4-quinazolinone. Phthalazine metabolite III had the same retention time, UV spectrum (λmax = 208, 222, 249, 277, 298, and 310 nm), GC/MS mass spectrum, and LC/ESI-MSMS product–ion spectrum (Table 1) as authentic 1-phthalazinone. The 1H NMR chemical shifts of metabolite III were 8.35 (H8, d, 7.5 Hz), 8.34 (H4, s), 7.92 (H5, H6, m), and 7.88 (H7, td, 7.7, 1.1 Hz). Metabolite III was confirmed, by comparison
of the NMR spectrum with that of the authentic compound, as 1-phthalazinone. Fig. 2 shows the structures proposed for the metabolites produced from benzodiazines by A. niger: 2,4-quinazolinedione (I), 4-quinazolinone (II), and 1-phthalazinone (III). Of the two metabolites produced from quinazoline by A. niger, 4-quinazolinone is also produced by P. putida (4,5) and 2,4quinazolinedione by S. viridosporus (6). Both are also produced by mammalian aldehyde oxidase (15,16). 4-Quinazolinone, 2-quinazolinone, and 2,4-quinazolinedione may also be produced from quinazoline by hydroxyl radicals. Hydroxyl radicals may be produced from H2O2 and Fe2+ by Fenton's reaction; and the glucose oxidase of A. niger may produce H2O2 (17). Both 4-quinazolinone and 2,4-quinazolinedione are strong inhibitors of mammalian poly(ADP-ribose) synthetase and mono(ADP-ribosyl) transferase (18). 4-Quinazolinone is known to regulate kinase cascades and transcription factors involved in septic shock in mice (19) and also to reduce serum cholesterol in rats (20). The metabolite produced from phthalazine by A. niger, 1phthalazinone, is also produced by several bacteria (6–8), by the fungus F. verticillioides (9), and by mammalian aldehyde oxidase (16). 1-Phthalazinone, which may also arise from a photochemical reaction
TABLE 1. Electrospray product–ion spectra of the metabolites produced by Aspergillus niger NRRL-599 from quinazoline and phthalazine. Metabolite 2,4-Quinazolinedione (I) 4-Quinazolinone (II) 1-Phthalazinone (III) a
Parent ion [M + H]+ (m/z)
Collision energy (eV)
Product–ion spectrum (m/z, with % relative abundance) a
163 147 147
20 22 22
163 (11) 146 (100) 145 (9) 120 (1) 118 (2) 90 (19) 147 (100) 130 (37) 129 (13) 120 (26) 104 (19) 102 (11) 92 (30) 77 (14) 65 (9) 147 (100) 130 (18) 129 (25) 120 (19) 118 (73) 104 (65) 102 (40) 90 (49) 77 (36)
Product–ion spectra were obtained by LC/ESI-MSMS.
VOL. 111, 2011
NOTE
5.
Quinazoline
4-Quinazolinone (II)
2,4-Quinazolinedione (I)
6.
7.
8.
Phthalazine 1-Phthalazinone (III) FIG. 2. Pathways proposed for the metabolism of quinazoline and phthalazine by cultures of Aspergillus niger NRRL-599.
9.
10.
between phthalazine and H2O2, inhibits mammalian poly(ADPribose) synthetase and mono(ADP-ribosyl) transferase (18). 1,4Phthalazinedione was not found. Aspergillus niger produced two metabolites from quinazoline (2-quinazolinone and 2,4-quinazolinedione) and one from phthalazine (1-phthalazinone) that are known to be produced by bacteria. The metabolite produced from phthalazine was found previously in cultures of a different fungus. Such metabolites have the potential to induce harmful effects in mammals. We thank C. E. Cerniglia, G. Gamboa da Costa, and D. L. Mendrick for helpful comments and advice. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.
11.
12.
13.
14.
15.
16.
References 17. 1. Schultz, T. W. and Cajina-Quezada, M.: Structure-toxicity relationships of selected nitrogenous heterocyclic compounds. II. Dinitrogen molecules, Arch. Environ. Contam. Toxicol., 11, 353–361 (1982). 2. Bleeker, E. A. J., Wiegman, S., de Voogt, P., Kraak, M., Leslie, H. A., de Haas, E., and Admiraal, W.: Toxicity of azaarenes, Rev. Environ. Contam. Toxicol., 173, 39–83 (2002). 3. Bartoš, T., Letzsch, S., Škarek, M., Flegrová, Z., Čupr, P., and Holoubek, I.: GFP assay as a sensitive eukaryotic screening model to detect toxic and genotoxic activity of azaarenes, Environ. Toxicol., 21, 343–348 (2006). 4. Boyd, D. R., McMordie, R. A. S., Porter, H. P., Dalton, H., Jenkins, R. O., and Howarth, O. W.: Metabolism of bicyclic aza-arenes by Pseudomonas putida to yield
18.
19.
20.
335
vicinal cis-dihydrodiols and phenols, J. Chem. Soc. Chem. Commun., i, 1722–1724 (1987). Boyd, D. R., Sharma, N. D., Dorrity, M. R. J., Hand, M. V., McMordie, R. A. S., Malone, J. F., Porter, H. P., Dalton, H., Chima, J., and Sheldrake, G. N.: Structure and stereochemistry of cis-dihydro diol and phenol metabolites of bicyclic azaarenes from Pseudomonas putida UV4, J. Chem. Soc. Perkin Trans., I i, 1065–1071 (1993). Sutherland, J. B., Freeman, J. P., and Williams, A. J.: Biotransformation of isoquinoline, phenanthridine, phthalazine, quinazoline, and quinoxaline by Streptomyces viridosporus, Appl. Microbiol. Biotechnol., 49, 445–449 (1998). Lehmann, M., Tshisuaka, B., Fetzner, S., Röger, P., and Lingens, F.: Purification and characterization of isoquinoline 1-oxidoreductase from Pseudomonas diminuta 7, a novel molybdenum-containing hydroxylase, J. Biol. Chem., 269, 11254–11260 (1994). Stephan, I., Tshisuaka, B., Fetzner, S., and Lingens, F.: Quinaldine 4-oxidase from Arthrobacter sp. Rü61a, a versatile procaryotic molybdenum-containing hydroxylase active towards N-containing heterocyclic compounds and aromatic aldehydes, Eur. J. Biochem., 236, 155–162 (1996). Sutherland, J. B., Freeman, J. P., Williams, A. J., and Deck, J.: Biotransformation of phthalazine by Fusarium moniliforme and Cunninghamella elegans, Mycologia, 91, 114–116 (1999). Kamath, A. V. and Vaidyanathan, C. S.: New pathway for the biodegradation of indole in Aspergillus niger, Appl. Environ. Microbiol., 56, 275–280 (1990). Sutherland, J. B., Freeman, J. P., Williams, A. J., and Deck, J.: Metabolism of cinnoline to N-oxidation products by Cunninghamella elegans and Aspergillus niger, J. Ind. Microbiol. Biotechnol., 21, 225–227 (1998). Baillie, G. S., Hitchcock, C. A., and Burnet, F. R.: Increased cytochrome P-450 activity in Aspergillus fumigatus after xenobiotic exposure, J. Med. Vet. Mycol., 34, 341–347 (1996). Adjei, M. D., Heinze, T. M., Deck, J., Freeman, J. P., Williams, A. J., and Sutherland, J. B.: Transformation of the antibacterial agent norfloxacin by environmental mycobacteria, Appl. Environ. Microbiol., 72, 5790–5793 (2006). Jung, C. M., Heinze, T. M., Schnackenberg, L. K., Mullis, L. B., Elkins, S. A., Elkins, C. A., Steele, R. S., and Sutherland, J. B.: Interaction of dietary resveratrol with animal-associated bacteria, FEMS Microbiol. Lett., 297, 266–273 (2009). McCormack, J. J., Allen, B. A., and Hodnett, C. N.: Oxidation of quinazoline and quinoxaline by xanthine oxidase and aldehyde oxidase, J. Heterocycl. Chem., 15, 1249–1254 (1978). Stubley, C., Stell, J. G. P., and Mathieson, D. W.: The oxidation of azaheterocycles with mammalian liver aldehyde oxidase, Xenobiotica, 9, 475–484 (1979). Pazur, J. H. and Kleppe, K.: The oxidation of glucose and related compounds by glucose oxidase from Aspergillus niger, Biochemistry, 3, 578–583 (1964). Banasik, M., Komura, H., Shimoyama, M., and Ueda, K.: Specific inhibitors of poly (ADP-ribose) synthetase and mono(ADP-ribosyl)transferase, J. Biol. Chem., 267, 1569–1575 (1992). Veres, B., Radnai, B., Gallyas, F., Varbiro, G., Berente, Z., Osz, E., and Sumegi, B.: Regulation of kinase cascades and transcription factors by a poly(ADP-ribose) polymerase-1 inhibitor, 4-hydroxyquinazoline, in lipopolysaccharide-induced inflammation in mice, J. Pharmacol. Exp. Ther., 310, 247–255 (2004). Refaie, F. M., Esmat, A. Y., Abdel Gawad, S. M., Ibrahim, A. M., and Mohamed, M. A.: The antihyperlipidemic activities of 4(3 H)quinazolinone and two halogenated derivatives in rats, Lipids Health Dis., 4, 22 (2005).
NOTE
Biotransformation of quinazoline and phthalazine by Aspergillus niger John B. Sutherland,⁎ Thomas M. Heinze, Laura K. Schnackenberg, James P. Freeman, and Anna J. Williams National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA Received 14 June 2010; accepted 22 November 2010 Available online 18 December 2010
Cultures of Aspergillus niger NRRL-599 in fluid Sabouraud medium were grown with quinazoline and phthalazine for 7 days. Metabolites were purified by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Quinazoline was oxidized to 4-quinazolinone and 2,4-quinazolinedione, and phthalazine was oxidized to 1-phthalazinone. @ 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aspergillus niger; Azaarene; Benzodiazine; Biotransformation; Phthalazine; Quinazoline]
The azaarene ring structures of quinazoline and phthalazine (Fig. 1) are found in various pharmaceuticals and dyes. Benzodiazines are persistent and toxic to aquatic organisms (1,2), and phthalazine also is positive for genotoxicity by the SOS-Chromotest (3). The benzodiazines, which may react with oxygen radicals in water, also are biotransformed by microorganisms. Quinazoline is metabolized to 4-quinazolinone, quinazoline cis-5,6-dihydrodiol, and quinazoline 5,6,7,8-tetrahydro-cis-5,6-dihydrodiol by Pseudomonas putida (4,5) and to 2,4-quinazolinedione by Streptomyces viridosporus (6). Phthalazine is metabolized to 1-phthalazinone by the bacteria Pseudomonas diminuta, Arthrobacter sp., and Streptomyces viridosporus and the fungus Fusarium verticillioides (6–9), and it is metabolized to phthalazine N-oxide by the fungus Cunninghamella elegans (9). Aspergillus niger is a common saprobic fungus that frequently has been proposed for use in the bioremediation of sites containing polycyclic aromatic hydrocarbons, heavy metals, pesticides, and textile dye effluents. It oxidizes indole to 3-hydroxyindole, later cleaving both benzo and pyrrole rings (10), and it transforms the diazine ring of cinnoline to produce two N-oxides (11). We dosed cultures of A. niger with quinazoline and phthalazine to find out whether it was able to metabolize these azaarenes; chemical analysis of the metabolites showed that it oxidized only the diazine ring of each. Triplicate 125-ml flasks, each containing 30 ml of fluid Sabouraud medium with 25 μM naphthalene (Aldrich Chemical Co., Milwaukee, WI) to induce oxidative enzymes (12), were inoculated with spores of A. niger NRRL-599 (U.S. Department of Agriculture, Peoria, IL) from slants of potato dextrose agar and incubated at 25 °C with shaking at 125 rpm. After 3 days, quinazoline or phthalazine (Aldrich) was dissolved in water, filter-sterilized, and used to dose each of the cultures (except for non-dosed controls) and the noninoculated controls directly for a final concentration of 0.44 mM. The cultures were harvested 7 days after dosing and both the mycelium and the ⁎ Corresponding author. Tel.: +1 870 543 7059; fax: +1 870 543 7307. E-mail address: [email protected] (J.B. Sutherland).
culture fluid were extracted three times with ethyl acetate. The extracts were combined, dried over anhydrous Na2SO4, and evaporated in vacuo. The residues were redissolved in methanol for analysis. The extracts were analyzed with an Agilent Technologies (Palo Alto, CA) 1100 series high-performance liquid chromatograph (HPLC). A 5-μm Waters (Milford, MA) Spherisorb ODS-2 column (250 × 2 mm) was used with a 20-min gradient of 20–60% methanol in buffer (50 mM ammonium formate, pH 5.5) with a 10-min hold at 60% methanol. The flow rate was 1 ml/min and the UV detector was at 254 nm. Peaks representing possible metabolites were collected and then the buffer was removed by solid-phase extraction. Metabolites were identified by gas chromatography/electron ionization mass spectrometry (GC/EI-MS) (9) and by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESIMSMS) (13) using a Phenomenex (Torrance, CA) Gemini 3-μm C18 column (150 × 2 mm); the spectra were compared with those of authentic compounds and published data. 2,4-Quinazolinedione (benzoyleneurea) was from Sigma Chemical Co. (St. Louis, MO), and 4-quinazolinone (4-hydroxyquinazoline) and 1-phthalazinone (phthalazone) were from Aldrich. Samples dissolved in methanol-d4 (Cambridge Isotope Laboratories, Andover, MA) were analyzed at 600 MHz by proton nuclear magnetic resonance (NMR) spectroscopy (14). Extracts from cultures of A. niger NRRL-599 dosed with quinazoline, analyzed by HPLC using ammonium formate buffer, showed peaks at 10.5 min (metabolite I), 13.2 min (metabolite II), and 19.0 min (quinazoline) (Fig. 1A). Extracts from cultures dosed with phthalazine showed peaks at 10.8 min (metabolite III) and 15.9 min (phthalazine) (Fig. 1B). Quinazoline metabolite I had the same retention time and UV spectrum (λmax = 219 and 310 nm) as authentic 2,4-quinazolinedione. GC/MS of both metabolite I and 2,4-quinazolinedione showed significant ions at m/z 162 [M+·] (100), 119 (66), and 92 (28). The product– ion spectra (LC/ESI-MSMS) of both metabolite I (Table 1) and 2,4-
1389-1723/$ - see front matter @ 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.11.017
334
SUTHERLAND ET AL.
J. BIOSCI. BIOENG.,
I
A
225
II
5
200
4
6
3
N
175 150
7
125
8
Absorbance at 254 nm (mAU)
100
2
N 1
75 50
Q
25 0 0
5
10
15
20
B
225
25
5
4
6
200
3
N
III
175 150
N 2
7
125
8
P
100
1
75 50 25 0 0
5
10
15
20
25
Time (min) FIG. 1. HPLC elution profiles at 254 nm of the ethyl acetate extracts from cultures of Aspergillus niger NRRL-599 dosed with (A) quinazoline and (B) phthalazine (I = 2,4-quinazolinedione; II= 4-quinazolinone; III = 1-phthalazinone; Q = quinazoline; P = phthalazine).
quinazolinedione showed ions at m/z 163 [M+ H]+ and 146, among others. The 1H NMR chemical shifts of metabolite I were 8.04 (H5, d, 7.9 Hz), 7.68 (H7, td, 8.4, 1.5 Hz), 7.26 (H6, td, 7.5, 1.1 Hz), and 7.21 (H8, d, 8.3 Hz). Metabolite I was confirmed, by comparison of the NMR spectrum with that of the authentic compound, as 2,4-quinazolinedione. Quinazoline metabolite II had the same retention time and UV spectrum (λmax = 200, 226, 264, 302, and 313 nm) as authentic 4-quinazolinone. The product–ion spectra (LC/ESI-MSMS) of metabolite II (Table 1) and 4-quinazolinone showed ions at m/z 147 [M + H]+, 130, and others, indicating that metabolite II was a quinazolinone. The 1H NMR chemical shifts of metabolite II were 8.26 (H5, d, 8.8 Hz), 8.13 (H2, s), 7.87 (H7, td, 7.9, 1.1 Hz), 7.74 (H8, d, 7.2 Hz), and 7.59 (H6, td, 7.2, 1.3 Hz). Metabolite II was confirmed, by comparison of the NMR spectrum with that of the authentic compound, as 4-quinazolinone. Phthalazine metabolite III had the same retention time, UV spectrum (λmax = 208, 222, 249, 277, 298, and 310 nm), GC/MS mass spectrum, and LC/ESI-MSMS product–ion spectrum (Table 1) as authentic 1-phthalazinone. The 1H NMR chemical shifts of metabolite III were 8.35 (H8, d, 7.5 Hz), 8.34 (H4, s), 7.92 (H5, H6, m), and 7.88 (H7, td, 7.7, 1.1 Hz). Metabolite III was confirmed, by comparison
of the NMR spectrum with that of the authentic compound, as 1-phthalazinone. Fig. 2 shows the structures proposed for the metabolites produced from benzodiazines by A. niger: 2,4-quinazolinedione (I), 4-quinazolinone (II), and 1-phthalazinone (III). Of the two metabolites produced from quinazoline by A. niger, 4-quinazolinone is also produced by P. putida (4,5) and 2,4quinazolinedione by S. viridosporus (6). Both are also produced by mammalian aldehyde oxidase (15,16). 4-Quinazolinone, 2-quinazolinone, and 2,4-quinazolinedione may also be produced from quinazoline by hydroxyl radicals. Hydroxyl radicals may be produced from H2O2 and Fe2+ by Fenton's reaction; and the glucose oxidase of A. niger may produce H2O2 (17). Both 4-quinazolinone and 2,4-quinazolinedione are strong inhibitors of mammalian poly(ADP-ribose) synthetase and mono(ADP-ribosyl) transferase (18). 4-Quinazolinone is known to regulate kinase cascades and transcription factors involved in septic shock in mice (19) and also to reduce serum cholesterol in rats (20). The metabolite produced from phthalazine by A. niger, 1phthalazinone, is also produced by several bacteria (6–8), by the fungus F. verticillioides (9), and by mammalian aldehyde oxidase (16). 1-Phthalazinone, which may also arise from a photochemical reaction
TABLE 1. Electrospray product–ion spectra of the metabolites produced by Aspergillus niger NRRL-599 from quinazoline and phthalazine. Metabolite 2,4-Quinazolinedione (I) 4-Quinazolinone (II) 1-Phthalazinone (III) a
Parent ion [M + H]+ (m/z)
Collision energy (eV)
Product–ion spectrum (m/z, with % relative abundance) a
163 147 147
20 22 22
163 (11) 146 (100) 145 (9) 120 (1) 118 (2) 90 (19) 147 (100) 130 (37) 129 (13) 120 (26) 104 (19) 102 (11) 92 (30) 77 (14) 65 (9) 147 (100) 130 (18) 129 (25) 120 (19) 118 (73) 104 (65) 102 (40) 90 (49) 77 (36)
Product–ion spectra were obtained by LC/ESI-MSMS.
VOL. 111, 2011
NOTE
5.
Quinazoline
4-Quinazolinone (II)
2,4-Quinazolinedione (I)
6.
7.
8.
Phthalazine 1-Phthalazinone (III) FIG. 2. Pathways proposed for the metabolism of quinazoline and phthalazine by cultures of Aspergillus niger NRRL-599.
9.
10.
between phthalazine and H2O2, inhibits mammalian poly(ADPribose) synthetase and mono(ADP-ribosyl) transferase (18). 1,4Phthalazinedione was not found. Aspergillus niger produced two metabolites from quinazoline (2-quinazolinone and 2,4-quinazolinedione) and one from phthalazine (1-phthalazinone) that are known to be produced by bacteria. The metabolite produced from phthalazine was found previously in cultures of a different fungus. Such metabolites have the potential to induce harmful effects in mammals. We thank C. E. Cerniglia, G. Gamboa da Costa, and D. L. Mendrick for helpful comments and advice. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.
11.
12.
13.
14.
15.
16.
References 17. 1. Schultz, T. W. and Cajina-Quezada, M.: Structure-toxicity relationships of selected nitrogenous heterocyclic compounds. II. Dinitrogen molecules, Arch. Environ. Contam. Toxicol., 11, 353–361 (1982). 2. Bleeker, E. A. J., Wiegman, S., de Voogt, P., Kraak, M., Leslie, H. A., de Haas, E., and Admiraal, W.: Toxicity of azaarenes, Rev. Environ. Contam. Toxicol., 173, 39–83 (2002). 3. Bartoš, T., Letzsch, S., Škarek, M., Flegrová, Z., Čupr, P., and Holoubek, I.: GFP assay as a sensitive eukaryotic screening model to detect toxic and genotoxic activity of azaarenes, Environ. Toxicol., 21, 343–348 (2006). 4. Boyd, D. R., McMordie, R. A. S., Porter, H. P., Dalton, H., Jenkins, R. O., and Howarth, O. W.: Metabolism of bicyclic aza-arenes by Pseudomonas putida to yield
18.
19.
20.
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