APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1979, p. 311-313 0099-2240/79/08-0311/03$02.00/0
Vol. 38, No. 2
Microbial Transformations of Natural Antitumor Agents: Conversion of Lapachol to Dehydro-a-Lapachone by Curvularia lunata SHAREE OTTEN AND JOHN P. ROSAZZA* Division of Medicinal Chemistry and Natural Products, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242 Received for publication 22 May 1979
Microbial transformation of lapachol, a naturally occurring naphthoquinone, carried out by Curvularia lunata (NRRL 2178). The fungus brings about oxidative cyclization of the substrate to dehydro-a-lapachone, which was isolated and characterized by nuclear magnetic resonance and mass spectral analyses; its structure was verified by chemical synthesis. The metabolite is a naturally occurring chromene possessing antibacterial and antitumor activities. was
Microorganisms are known to transform a at 4°C in sealed screw-capped tubes on Sabouraudwide variety of naturally occurring antitumor maltose agar slants and grown according to the precompounds (15). These transformations are con- viously described two-stage fermentation procedure (1, 14) at 27°C in a soybean meal-glucose medium of ducted in order to prepare sufficient quantities the composition: soybean meal, 5 g; glucose, of metabolites for structure elucidation and bi- 20 g;following extract, 5 g; NaCl, 5 g; K2HPO4, 5 g; distilled ological testing and to identify new metabolic water,yeast 1,000 ml. The medium was adjusted to pH 7.0 pathways which may also occur in mammalian with 6 N HCl and autoclaved at 121°C (15 lb/in2) for metabolic systems (16, 17). 15 min. Lapachol (1) is a naturally occurring naphthControls consisted of fermentations without la' oquinone derivative found in the heartwood of pachol, and solutions of lapachol in buffers, including several species of Bignoniaceae and Verbena- 0.1 M citric acid (pH 3.1), 0.1 M sodium phosphate ceae and has antimalarial (6), antibiotic (7), and (pH 6.5), and 0.1 M tris(hydroxymethyl)aminomethane (pH 8.7), all of which were incubated with antitumor activities (5, 7). In a previous report, shaking for the duration of normal fermentations (72 we documented a novel oxidative ring fission of h). Although lapachol decomposed slightly (less than lapachol by Penicillium notatum (14). This re- 1% as estimated by TLC) to colorless products under port describes the oxidative conversion of la- all pH conditions, none of the decomposition products pachol (1) to dehydro-a-lapachone (2), a natu- was chromatographically comparable to the observed rally occurring chromene derivative (Fig. 1). microbial metabolites. Sampling and TLC. Samples (4 ml) of substrate MATERIALS AND METHODS containing stage II fermentations were withdrawn at NMR and mass spectra, melting points, and various time intervals, acidified to pH 2.0 with 6 N TLC. Nuclear magnetic resonance (NMR) spectra HCl, and extracted with 1 ml of ethyl acetate. Approxwere obtained with a Varian T-60 spectrometer, using imately 30 ,I of the ethyl acetate extract was spotted tetramethylsilane as an internal standard. Low-reso- on TLC plates, which were developed in benzenelution mass spectra were obtained with a Finnigan model 3200 mass spectrometer. Melting points were determined in open-ended capillary tubes in a Thomas-Hoover melting-point apparatus and were corrected. Thin-layer chromatography was performed on 0.25- and 1.0-mm-thick layers of Silica Gel GF254 (Merck) prepared on glass plates with a Quickfit Industries Spreader. The plates were activated at 120°C before use. Lapachol. Lapachol was purchased from Aldrich Chemical Co., and its characteristics were published (13, 14). Fermentation procedures. Preliminary tests identified several cultures capable of metabolizing lapachol, and Curvularia lunata (NRRL 2178) was selected for more detailed work. This culture was stored
acetic acid (95:5) or chloroform-ethanol-acetic acid (88:7:5). Chromatograms were visualized by fluorescence quenching under 254-nm ultraviolet light or by spraying developed plates with ceric ammonium sulfate [1%; Ce(NH4)4 (SO4)4] in 50% H3PO4 and warming the sprayed plates with a heat gun. Transformation of lapachol to dehydro-a-lapachone by C. lunata NRRL 2178. C. lunata reproducibly formed a single, major, bright orange metabolite (Rf 0.40 in benzene-acetic acid [95:5]) which was not formed by other cultures tested, or in controls. It was selected for a preparative-scale incubation for the purpose of obtaining sufficient quantities of the metabolite for structure elucidation. Stage II fermentations were conducted in 30 1-liter Erlenmeyer flasks each containing 200 ml of culture. Lapachol was dis-
311
312
OTTEN AND ROSAZZA
1 ~~~~~~~~~~~2 FIG. 1. Conversion of lapachol (1) to dehydro-a-
lapachone (2). solved in dimethylformamide (3 g/30 ml) and distributed evenly among the 24-h-old stage II culture flasks (500-ug/ml final concentration) which were incubated with shaking. TLC analysis indicated that approximately 50% of the lapachol was converted to the product 72 h after substrate addition and that no further transformation was taking place with prolonged incubation. The fermentation was harvested by filtration, and the metabolite was isolated from culture filtrates. The combined filtrate was acidified to pH 2.0 with 6 N HCI and exhaustively extracted with ethyl acetate. The combined extracts were dried over anhydrous Na2SO4 and evaporated to dryness (3.0 g). The mixture was adsorbed onto 5 g of silica gel and applied as a dried powder to the top of a silica gel column (50 g, Baker 3405, 60 to 200 mesh; column dimensions, 33 by 2.8 cm) which was eluted with chloroform-ethanol (99: 1) at a rapid flow rate while four 50-ml fractions were collected. The first fraction containing primarily the metabolite and lapachol was evaporated to dryness to yield 300 mg of solids. It was further purified by adsorption onto silica gel (0.75 g), addition to the top of a second silica gel column (60 g, Baker 3405; 33 by 2.8 cm), and elution with benzene-hexane-acetic acid (74:20:1) at a flow rate of 1 ml/min while 180 2-ml fractions were collected. Fractions 121 through 165 containing the metabolite were combined, partitioned with 5% NaHCO3 to remove remaining acetic acid, and evaporated to dryness. The crude metabolite (45 mg) thus obtained was further purified by preparative TLC (benzene-hexaneacetic acid [74:20:1]) to yield pure metabolite, which was crystallized from ethanol as orange needles, mp 141 to 145°C. The metabolite possessed the same melting point (mixture melting point undepressed) and NMR and mass spectral properties as the synthetic (2). Synthesis of dehydro-a-lapachone (8). A solution of 1.0 g of lapachol (0.004 M) in 25 ml of pyridine was heated at reflux for 4 h. The reaction mixture was allowed to cool to room temperature and was diluted with 25 ml of petroleum ether (bp 36 to 56°C) before extraction with two 50-ml portions of water. The aqueous extracts were combined and exhaustively reextracted with benzene. The combined benzene extracts were washed with equal volumes of 0.1 N HCI and water, dried over anhydrous Na2SO4, and concentrated to dryness. The crude dehydro-a-lapachone (0.78 g) crystallized from ethanol as orange needles (0.39 g): mp 142 to 144°C (literature [3], 143°C); mixture melting point with the metabolite, undepressed at 142 to 144.5°C. NMR (3): CDCl3-ppm 1.53 (6H, s,
APPL. ENVIRON. MICROBIOL.
Me2C), 5.60 (1H, d, J = 10, -CH=), 6.52 (1H, d, J = 10, -CH=), 7.50 (2H, m, ArH), and 7.92 (2H, m, ArH). Mass spectrum (11): mle (percent relative abundance) 240 (14), 225(100), 212(5), 196(36), 184(12), 183(38), 169(6), 155(4), 127(3), 104(19), 101(18), 76(22), and 50(11). Analysis-calculated for CI5HI203: C, 74.99; H, 5.03; found: C, 74.81; H, 4.71.
RESULTS AND DISCUSSION The NMR spectral comparisons between lapachol (13, 14) and the metabolite indicated that the aromatic ring and the gem-dimethyl groups were still present but that the phenolic hydroxyl group was absent. A pair of doublet signals at 5.60 and 6.53 ppm in the NMR spectrum of the metabolite suggested that a shift in the position of the side-chain double bond occurred. The mass spectrum revealed a molecular weight of 240 for the metabolite with a molecular formula of C,5H1203 and exhibited a fragmentation pattern consistent with a 1,4-naphthoquinone structure in which cleavage occurred at the ketonic oxygen atom. Peaks at m/e 212 and 184 can be explained by the elimination of 2 mol of carbon monoxide from the molecular ion. Fragmentation may also occur by initial loss of a methyl radical followed by elimination of carbon monoxide or ketene, or both, which results in peaks at m/e 225, 197, 183, and 169. These data are consistent with the structure of dehydro-a-lapachone (2). Synthesis of dehydro-a-lapachone from lapachol by refluxing in pyridine gave a compound with spectral and physical properties identical to those of the metabolite. Dehydro-a-lapachone (2) is a naturally occurring chromene substance which has been isolated from several plant species of Bignoniaceae (2, 19). It may be formed as an artifact from lapachol under certain conditions (3, 9), but in our case the control experiments showed that C. lunata produced it as a bona fide metabolite. The mechanism by which (2) is formed from lapachol by C. lunata is uncertain, but at least three pathways for its formation are possible. (i) Ionic or radical mechanisms may be involved. (ii) Dehydrogenation of lapachol could form an intermediate quinone methide, which in turn may cyclize to (2). This pathway is analogous to the mechanism proposed by Turner for the biogenesis of naturally occurring chromenes (18). (iii) An intramolecular attack of an intermediate side-chain epoxide followed by dehydration could occur to provide (2). This would be similar to the mechanism suggested for the formation of a chromene ring with mycophenolic acid (4, 10). Dehydro-a-lapachone has been shown to possess antibacterial (12) and antitumor activities
VOL. 38, 1979
CONVERSION OF LAPACHOL TO DEHYDRO-a-LAPACHONE
(5, 12). It is also marginally active against the L1210 leukemia test system, whereas lapachol itself is inactive.
8.
ACKNOWLEDGMENTS We acknowledge financial support through Public Health Service grant CA-13786 from the National Cancer Institute. S.O. thanks the American Foundation for Pharmaceutical Education for fellowship support.
9.
LITERATURE CITED
10.
1. Betts, R. E., D. E. Walters, and J. P. Rosazza. 1974. Microbial transformations of antitumor compounds. 1.
Conversion of acronycine to 9-hydroxyacronycine by Cunninghamella echinulata. J. Med. Chem. 17:599602. 2. Burnett, A. R., and R. H. Thomson. 1967. Naturally occurring quinones. X. The quinonoid constituents of Tabebuia avellanedae (Bignoniaceae). J. Chem. Soc. C 1967:2100-2104. 3. Burnett, A. R., and R. H. Thomson. 1967. Quinones. VIII. Dehydro-a- and -18-lapachone. J. Chem. Soc. C 1967:1261-1264. 4. Campbell, I. M., C. H. Calzadilla, and N. J. McCorkindale. 1966. Some new metabolites related to mycophenolic acid. Tetrahedron Lett. 1966:5107-5111. 5. Driscoll, J. S., G. F. Hazard, Jr., H. B. Wood, Jr., and A. Goldin. 1974. Structure-antitumor activity relationships among quinone derivatives. Cancer Chemother. Rep. Part 2 4:1-362. 6. Fieser, L. F., E. Berliner, F. J. Bondhus, F. C. Chang, W. G. Dauben, M. G. Ettlinger, G. Fawaz, M. Fields, M. Fieser, C. Heidelberger, H. Heymann, A. M. Seligman, W. R. Vaughan, A. C. Wilson, E. Wilson, M. I. Wu, M. T. Leffler, K. E. Hamlin, R. J. Hathaway, E. J. Matson, E. E. Moore, M. B. Moore, R. T. Rapala, and H. E. Zaugg. 1948. Naphthoquinone antimalarials. I. General survey. J. Am. Chem. Soc. 70: 3151-3155. 7. Goncalves de Lima, O., J. S. de B. Coelho, I. Leoncio d'Albuquerque, J. Francisco de Mello, D. G. Martins, A. L. Lacerda, and M. A. De Moraes e Souza. 1971. Antimicrobial compounds from higher plants. XXXV. Antimicrobial and antitumor activity of lawsone (2-hydroxy-1,4-naphthoquinone) compared with
11.
12.
13.
14.
15.
16. 17. 18. 19.
313
that of lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4naphthoquinone]. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife 11:21-26. Goncalves de Lima, O., L. Leoncio d'Albuquerque, M. A. P. Borba, and J. Francisco de Meilo. 1966. Antibiotic substances from higher plants. XXV. Isolation of xiloidone (dehydrolapachone) by the conversion of lapachol in the presence of pyridine. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife 6:23-34. Hooker, S. C. 1936. The constitution of lapachol and its derivatives. V. The structure of Paterno's "isolapachone." J. Am. Chem. Soc. 58:1190-1197. Jones, D. F., R. H. Moore, and G. C. Crawley. 1970. Microbial modification of mycophenolic acid. J. Chem. Soc. C 1970:1725-1737. Joshi, K. C., L Prakash, R. K. Bansal, and P. Singh. 1973. Mass spectrometric studies of dehydro-a-lapachone and dehydro-iso-a-lapachone. Z. Naturforsch. Teil C 28:646-649. Leoncio d'Albuquerque, I., M. do C. M. De Araujo, M. C. N. Maciel, G. M. Maciel, M. A. De Moraes e Souza, A. L Lacerda, and D. G. Martins. 1972. Microbial substances from higher plants. XL. New method for preparing xyloidone (dehydrolapachone) from lapachol. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife 12:25-30. Linardi, M. da C. F., M. M. de Oliveira, and M. R. P. Sampaio. 1975. A lapachol derivative active against mouse lymphocytic leukemia P-388. J. Med. Chem. 18: 1159-1161. Otten, S., and J. P. Rosazza. 1978. Microbial transformations of natural antitumor agents: oxidation of lapachol by Penicillium notatum. Appl. Environ. Microbiol. 35:554-557. Rosazza, J. P. 1978. Microbial transformations of natural antitumor agents. Lloydia 41:297-311. Rosazza, J. P., and R. V. Smith. 1979. Microbial models for drug metabolism. Adv. Appl. Microbiol. vol. 25, in press. Smith, R. V., and J. P. Rosazza. 1975. Microbial models of mammalian metabolism. J. Pharm. Sci. 64:17371759. Turner, A. B. 1964. Quinone methides. Quarterly Rev. 18:347-360. Weinberg, M. de L. D., 0. R. Gottlieb, and G. G. De Oliveira. 1976. Naphthoquinones from Zeyhera tuberculosa. Phytochemistry 15:570.
Vol. 38, No. 2
Microbial Transformations of Natural Antitumor Agents: Conversion of Lapachol to Dehydro-a-Lapachone by Curvularia lunata SHAREE OTTEN AND JOHN P. ROSAZZA* Division of Medicinal Chemistry and Natural Products, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242 Received for publication 22 May 1979
Microbial transformation of lapachol, a naturally occurring naphthoquinone, carried out by Curvularia lunata (NRRL 2178). The fungus brings about oxidative cyclization of the substrate to dehydro-a-lapachone, which was isolated and characterized by nuclear magnetic resonance and mass spectral analyses; its structure was verified by chemical synthesis. The metabolite is a naturally occurring chromene possessing antibacterial and antitumor activities. was
Microorganisms are known to transform a at 4°C in sealed screw-capped tubes on Sabouraudwide variety of naturally occurring antitumor maltose agar slants and grown according to the precompounds (15). These transformations are con- viously described two-stage fermentation procedure (1, 14) at 27°C in a soybean meal-glucose medium of ducted in order to prepare sufficient quantities the composition: soybean meal, 5 g; glucose, of metabolites for structure elucidation and bi- 20 g;following extract, 5 g; NaCl, 5 g; K2HPO4, 5 g; distilled ological testing and to identify new metabolic water,yeast 1,000 ml. The medium was adjusted to pH 7.0 pathways which may also occur in mammalian with 6 N HCl and autoclaved at 121°C (15 lb/in2) for metabolic systems (16, 17). 15 min. Lapachol (1) is a naturally occurring naphthControls consisted of fermentations without la' oquinone derivative found in the heartwood of pachol, and solutions of lapachol in buffers, including several species of Bignoniaceae and Verbena- 0.1 M citric acid (pH 3.1), 0.1 M sodium phosphate ceae and has antimalarial (6), antibiotic (7), and (pH 6.5), and 0.1 M tris(hydroxymethyl)aminomethane (pH 8.7), all of which were incubated with antitumor activities (5, 7). In a previous report, shaking for the duration of normal fermentations (72 we documented a novel oxidative ring fission of h). Although lapachol decomposed slightly (less than lapachol by Penicillium notatum (14). This re- 1% as estimated by TLC) to colorless products under port describes the oxidative conversion of la- all pH conditions, none of the decomposition products pachol (1) to dehydro-a-lapachone (2), a natu- was chromatographically comparable to the observed rally occurring chromene derivative (Fig. 1). microbial metabolites. Sampling and TLC. Samples (4 ml) of substrate MATERIALS AND METHODS containing stage II fermentations were withdrawn at NMR and mass spectra, melting points, and various time intervals, acidified to pH 2.0 with 6 N TLC. Nuclear magnetic resonance (NMR) spectra HCl, and extracted with 1 ml of ethyl acetate. Approxwere obtained with a Varian T-60 spectrometer, using imately 30 ,I of the ethyl acetate extract was spotted tetramethylsilane as an internal standard. Low-reso- on TLC plates, which were developed in benzenelution mass spectra were obtained with a Finnigan model 3200 mass spectrometer. Melting points were determined in open-ended capillary tubes in a Thomas-Hoover melting-point apparatus and were corrected. Thin-layer chromatography was performed on 0.25- and 1.0-mm-thick layers of Silica Gel GF254 (Merck) prepared on glass plates with a Quickfit Industries Spreader. The plates were activated at 120°C before use. Lapachol. Lapachol was purchased from Aldrich Chemical Co., and its characteristics were published (13, 14). Fermentation procedures. Preliminary tests identified several cultures capable of metabolizing lapachol, and Curvularia lunata (NRRL 2178) was selected for more detailed work. This culture was stored
acetic acid (95:5) or chloroform-ethanol-acetic acid (88:7:5). Chromatograms were visualized by fluorescence quenching under 254-nm ultraviolet light or by spraying developed plates with ceric ammonium sulfate [1%; Ce(NH4)4 (SO4)4] in 50% H3PO4 and warming the sprayed plates with a heat gun. Transformation of lapachol to dehydro-a-lapachone by C. lunata NRRL 2178. C. lunata reproducibly formed a single, major, bright orange metabolite (Rf 0.40 in benzene-acetic acid [95:5]) which was not formed by other cultures tested, or in controls. It was selected for a preparative-scale incubation for the purpose of obtaining sufficient quantities of the metabolite for structure elucidation. Stage II fermentations were conducted in 30 1-liter Erlenmeyer flasks each containing 200 ml of culture. Lapachol was dis-
311
312
OTTEN AND ROSAZZA
1 ~~~~~~~~~~~2 FIG. 1. Conversion of lapachol (1) to dehydro-a-
lapachone (2). solved in dimethylformamide (3 g/30 ml) and distributed evenly among the 24-h-old stage II culture flasks (500-ug/ml final concentration) which were incubated with shaking. TLC analysis indicated that approximately 50% of the lapachol was converted to the product 72 h after substrate addition and that no further transformation was taking place with prolonged incubation. The fermentation was harvested by filtration, and the metabolite was isolated from culture filtrates. The combined filtrate was acidified to pH 2.0 with 6 N HCI and exhaustively extracted with ethyl acetate. The combined extracts were dried over anhydrous Na2SO4 and evaporated to dryness (3.0 g). The mixture was adsorbed onto 5 g of silica gel and applied as a dried powder to the top of a silica gel column (50 g, Baker 3405, 60 to 200 mesh; column dimensions, 33 by 2.8 cm) which was eluted with chloroform-ethanol (99: 1) at a rapid flow rate while four 50-ml fractions were collected. The first fraction containing primarily the metabolite and lapachol was evaporated to dryness to yield 300 mg of solids. It was further purified by adsorption onto silica gel (0.75 g), addition to the top of a second silica gel column (60 g, Baker 3405; 33 by 2.8 cm), and elution with benzene-hexane-acetic acid (74:20:1) at a flow rate of 1 ml/min while 180 2-ml fractions were collected. Fractions 121 through 165 containing the metabolite were combined, partitioned with 5% NaHCO3 to remove remaining acetic acid, and evaporated to dryness. The crude metabolite (45 mg) thus obtained was further purified by preparative TLC (benzene-hexaneacetic acid [74:20:1]) to yield pure metabolite, which was crystallized from ethanol as orange needles, mp 141 to 145°C. The metabolite possessed the same melting point (mixture melting point undepressed) and NMR and mass spectral properties as the synthetic (2). Synthesis of dehydro-a-lapachone (8). A solution of 1.0 g of lapachol (0.004 M) in 25 ml of pyridine was heated at reflux for 4 h. The reaction mixture was allowed to cool to room temperature and was diluted with 25 ml of petroleum ether (bp 36 to 56°C) before extraction with two 50-ml portions of water. The aqueous extracts were combined and exhaustively reextracted with benzene. The combined benzene extracts were washed with equal volumes of 0.1 N HCI and water, dried over anhydrous Na2SO4, and concentrated to dryness. The crude dehydro-a-lapachone (0.78 g) crystallized from ethanol as orange needles (0.39 g): mp 142 to 144°C (literature [3], 143°C); mixture melting point with the metabolite, undepressed at 142 to 144.5°C. NMR (3): CDCl3-ppm 1.53 (6H, s,
APPL. ENVIRON. MICROBIOL.
Me2C), 5.60 (1H, d, J = 10, -CH=), 6.52 (1H, d, J = 10, -CH=), 7.50 (2H, m, ArH), and 7.92 (2H, m, ArH). Mass spectrum (11): mle (percent relative abundance) 240 (14), 225(100), 212(5), 196(36), 184(12), 183(38), 169(6), 155(4), 127(3), 104(19), 101(18), 76(22), and 50(11). Analysis-calculated for CI5HI203: C, 74.99; H, 5.03; found: C, 74.81; H, 4.71.
RESULTS AND DISCUSSION The NMR spectral comparisons between lapachol (13, 14) and the metabolite indicated that the aromatic ring and the gem-dimethyl groups were still present but that the phenolic hydroxyl group was absent. A pair of doublet signals at 5.60 and 6.53 ppm in the NMR spectrum of the metabolite suggested that a shift in the position of the side-chain double bond occurred. The mass spectrum revealed a molecular weight of 240 for the metabolite with a molecular formula of C,5H1203 and exhibited a fragmentation pattern consistent with a 1,4-naphthoquinone structure in which cleavage occurred at the ketonic oxygen atom. Peaks at m/e 212 and 184 can be explained by the elimination of 2 mol of carbon monoxide from the molecular ion. Fragmentation may also occur by initial loss of a methyl radical followed by elimination of carbon monoxide or ketene, or both, which results in peaks at m/e 225, 197, 183, and 169. These data are consistent with the structure of dehydro-a-lapachone (2). Synthesis of dehydro-a-lapachone from lapachol by refluxing in pyridine gave a compound with spectral and physical properties identical to those of the metabolite. Dehydro-a-lapachone (2) is a naturally occurring chromene substance which has been isolated from several plant species of Bignoniaceae (2, 19). It may be formed as an artifact from lapachol under certain conditions (3, 9), but in our case the control experiments showed that C. lunata produced it as a bona fide metabolite. The mechanism by which (2) is formed from lapachol by C. lunata is uncertain, but at least three pathways for its formation are possible. (i) Ionic or radical mechanisms may be involved. (ii) Dehydrogenation of lapachol could form an intermediate quinone methide, which in turn may cyclize to (2). This pathway is analogous to the mechanism proposed by Turner for the biogenesis of naturally occurring chromenes (18). (iii) An intramolecular attack of an intermediate side-chain epoxide followed by dehydration could occur to provide (2). This would be similar to the mechanism suggested for the formation of a chromene ring with mycophenolic acid (4, 10). Dehydro-a-lapachone has been shown to possess antibacterial (12) and antitumor activities
VOL. 38, 1979
CONVERSION OF LAPACHOL TO DEHYDRO-a-LAPACHONE
(5, 12). It is also marginally active against the L1210 leukemia test system, whereas lapachol itself is inactive.
8.
ACKNOWLEDGMENTS We acknowledge financial support through Public Health Service grant CA-13786 from the National Cancer Institute. S.O. thanks the American Foundation for Pharmaceutical Education for fellowship support.
9.
LITERATURE CITED
10.
1. Betts, R. E., D. E. Walters, and J. P. Rosazza. 1974. Microbial transformations of antitumor compounds. 1.
Conversion of acronycine to 9-hydroxyacronycine by Cunninghamella echinulata. J. Med. Chem. 17:599602. 2. Burnett, A. R., and R. H. Thomson. 1967. Naturally occurring quinones. X. The quinonoid constituents of Tabebuia avellanedae (Bignoniaceae). J. Chem. Soc. C 1967:2100-2104. 3. Burnett, A. R., and R. H. Thomson. 1967. Quinones. VIII. Dehydro-a- and -18-lapachone. J. Chem. Soc. C 1967:1261-1264. 4. Campbell, I. M., C. H. Calzadilla, and N. J. McCorkindale. 1966. Some new metabolites related to mycophenolic acid. Tetrahedron Lett. 1966:5107-5111. 5. Driscoll, J. S., G. F. Hazard, Jr., H. B. Wood, Jr., and A. Goldin. 1974. Structure-antitumor activity relationships among quinone derivatives. Cancer Chemother. Rep. Part 2 4:1-362. 6. Fieser, L. F., E. Berliner, F. J. Bondhus, F. C. Chang, W. G. Dauben, M. G. Ettlinger, G. Fawaz, M. Fields, M. Fieser, C. Heidelberger, H. Heymann, A. M. Seligman, W. R. Vaughan, A. C. Wilson, E. Wilson, M. I. Wu, M. T. Leffler, K. E. Hamlin, R. J. Hathaway, E. J. Matson, E. E. Moore, M. B. Moore, R. T. Rapala, and H. E. Zaugg. 1948. Naphthoquinone antimalarials. I. General survey. J. Am. Chem. Soc. 70: 3151-3155. 7. Goncalves de Lima, O., J. S. de B. Coelho, I. Leoncio d'Albuquerque, J. Francisco de Mello, D. G. Martins, A. L. Lacerda, and M. A. De Moraes e Souza. 1971. Antimicrobial compounds from higher plants. XXXV. Antimicrobial and antitumor activity of lawsone (2-hydroxy-1,4-naphthoquinone) compared with
11.
12.
13.
14.
15.
16. 17. 18. 19.
313
that of lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4naphthoquinone]. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife 11:21-26. Goncalves de Lima, O., L. Leoncio d'Albuquerque, M. A. P. Borba, and J. Francisco de Meilo. 1966. Antibiotic substances from higher plants. XXV. Isolation of xiloidone (dehydrolapachone) by the conversion of lapachol in the presence of pyridine. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife 6:23-34. Hooker, S. C. 1936. The constitution of lapachol and its derivatives. V. The structure of Paterno's "isolapachone." J. Am. Chem. Soc. 58:1190-1197. Jones, D. F., R. H. Moore, and G. C. Crawley. 1970. Microbial modification of mycophenolic acid. J. Chem. Soc. C 1970:1725-1737. Joshi, K. C., L Prakash, R. K. Bansal, and P. Singh. 1973. Mass spectrometric studies of dehydro-a-lapachone and dehydro-iso-a-lapachone. Z. Naturforsch. Teil C 28:646-649. Leoncio d'Albuquerque, I., M. do C. M. De Araujo, M. C. N. Maciel, G. M. Maciel, M. A. De Moraes e Souza, A. L Lacerda, and D. G. Martins. 1972. Microbial substances from higher plants. XL. New method for preparing xyloidone (dehydrolapachone) from lapachol. Rev. Inst. Antibiot. Univ. Fed. Pernambuco Recife 12:25-30. Linardi, M. da C. F., M. M. de Oliveira, and M. R. P. Sampaio. 1975. A lapachol derivative active against mouse lymphocytic leukemia P-388. J. Med. Chem. 18: 1159-1161. Otten, S., and J. P. Rosazza. 1978. Microbial transformations of natural antitumor agents: oxidation of lapachol by Penicillium notatum. Appl. Environ. Microbiol. 35:554-557. Rosazza, J. P. 1978. Microbial transformations of natural antitumor agents. Lloydia 41:297-311. Rosazza, J. P., and R. V. Smith. 1979. Microbial models for drug metabolism. Adv. Appl. Microbiol. vol. 25, in press. Smith, R. V., and J. P. Rosazza. 1975. Microbial models of mammalian metabolism. J. Pharm. Sci. 64:17371759. Turner, A. B. 1964. Quinone methides. Quarterly Rev. 18:347-360. Weinberg, M. de L. D., 0. R. Gottlieb, and G. G. De Oliveira. 1976. Naphthoquinones from Zeyhera tuberculosa. Phytochemistry 15:570.
