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Applied and Environmental Microbiology, June 2004, p . 3521-3527, Vol . 70, No . 6

Specific 12ß-Hydroxylation of Cinobufagin by Filamentous Fungi

Min Ye, Guiqin Qu, Hongzhu Guo, and Dean Guo*

The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, People's Republic of China

Received 30 May 2003/ Accepted 1 March 2004


   ABSTRACT

 
Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism . Microbial transformation of the cytotoxic steroid cinobufagin was investigated . Cinobufagin could be specifically hydroxylated at the 12ß-position by the fungus Alternaria alternata . Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12ß-hydroxyl cinobufagin, 12ß-hydroxyl desacetylcinobufagin, 3-oxo-12ß-hydroxyl cinobufagin, 3-oxo-12ß-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12{alpha}-hydroxyl cinobufagin . The last five products are new compounds . 12ß-Hydroxylation of cinobufagin by A . alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate . This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16 . Hydroxylation at C-12ß also was the first step in the metabolism of cinobufagin by a variety of fungal strains . In vitro cytotoxicity assays suggest that 12ß-hydroxyl cinobufagin and 3-oxo-12{alpha}-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities . The 12ß-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis .


   INTRODUCTION

 
Bufadienolides are steroids with a characteristic {alpha}-pyrone ring at the C-17 position; they have cardiotonic, blood pressure-stimulating, antiviral, and local anesthetic activities (20) . More than 300 bufadienolides have been isolated from natural sources including plants and animals (15, 23) . These compounds have been reported to have significant antitumor activities (9, 19, 31, 33) . Cinobufagin (compound I) is a bufadienolide with a 14ß,15ß-epoxy ring, originally isolated as a major component of the traditional Chinese drug, Chan'Su (also called toad venom or toad poison), which is prepared from the skin secretions of giant toads (10) . Cinobufagin can induce apoptosis and elevate intracellular Ca2+ levels (13, 14) . Against cancer cells it has a 50% inhibitory concentration (IC50) of approximately 10–8 mol/liter (30) . Unfortunately, it is poorly soluble in water and is toxic to humans; therefore, analogs with improved pharmaceutical properties, such as those obtained from plant cell suspension cultures (27, 29), are needed before this compound can be utilized effectively in a clinical setting .

Microbial transformation is an important tool for structural modification of organic compounds, especially natural products with complicated structures (17, 22) . It can be used to synthesize chemical structures that are difficult to obtain by other means (24) and as a model of mammalian metabolism due to the similarity between mammalian and microbial cytochrome P450 enzyme systems (1, 2, 4, 5, 12) . The metabolism of cinobufagin in rat liver microsomes produced at least six metabolites (32), resulting primarily from deacetylation at C-16 and epimerization of 3-OH via a 3-keto intermediate . The exact structures of these metabolites are still unknown since only small amounts of samples were recovered .

The objective of this study was to utilize Alternaria alternata as an in vitro model to prepare cinobufagin derivatives that are potential mammalian metabolites . Five of the six metabolites obtained are new compounds, and two significantly inhibit the growth of cultured human cancer cells .


   MATERIALS AND METHODS

 
General.
Melting points were determined with an XT4A apparatus (Dianguang Corp., Shanghai, China) and are uncorrected . Optical rotations were measured with a 243B polarimeter (Perkin-Elmer, Rotkreuz, Switzerland) . UV spectra were detected with a TU-1901 UV-Vis spectrophotometer (GenTech Inc., Arlington, Va.) . Infrared (IR) spectra were recorded in KBr with an Avatar 360 FT-IR spectrophotometer (Nicolet Inc., Madison, Wis.) . 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded on a DRX-500 spectrometer (Bruker, Karlsruhe, Germany) in dimethyl sulfoxide (DMSO-d6) at ambient temperature with tetramethylsilane (TMS) as the internal standard . The chemical shifts ({delta} values) are given in parts per million relative to TMS at 0 ppm . The coupling constants (J values) are reported in Hertz . Standard pulse sequences were used for distortionless enhancement by polarization transfer (DEPT), nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) experiments . High-resolution mass spectra (HR-MS) were obtained with an APEX II Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker) in the positive mode with a secondary ion mass spectrometry ion source .

Chemicals.
Cinobufagin was isolated from the Chinese medicine Chan'Su and identified unambiguously by NMR spectroscopy and MS and by comparison with authentic samples from the China Institute for Control of Pharmaceutical and Biological Products (Beijing, China) . The purity was >99% based on high-performance liquid chromatography-diode array detection (HPLC/DAD) and thin-layer chromatography analyses (10) . Silica gel (200 to 300 mesh) for column chromatography was purchased from Qingdao Marine Chemical Corp . (Qingdao, China) . Sephadex LH-20 was from Pharmacia (Piscataway, N.J.) . All chemical solvents used for product isolation were of analytical grade or higher .

Microorganisms and culture medium.
The microorganisms used were purchased from China General Microbiological Culture Collection Center (Beijing, China) (Table 1) . Fermentations were carried out in a potato medium consisting of 20 g of potato extract (10% [wt/wt], prepared from 200 g of potato slices extracted with boiling water for 30 min), 20 g of glucose, and 1,000 ml of distilled H2O . The media were sterilized at 121°C and 1.06 kg/cm2 for 30 min (8) . Strains were maintained on potato slants solidified with 30 g of agar per liter at 4°C .


TABLE 1 . Screening of fungal strains for the metabolism of cinobufagina

 
Preliminary screening tests.
Mycelia from the agar slants (1 cm3) were transferred to liquid medium and incubated at 25°C with rotary shaking at 180 rpm in the dark in 250-ml Erlenmeyer flasks containing 80 ml of medium . After 48 h of incubation, 2 mg of cinobufagin (10 mg/ml in ethanol) was added to the culture and the incubation was continued for 3 days . Mycelia were harvested by filtration through Whatman no . 1 filter paper, and the filtrate was extracted with an approximately equal volume of ethyl acetate . The organic extract was evaporated to dryness in a rotary evaporate under reduced pressure at 60°C, and the residue was dissolved in 1 ml of methanol . Thin-layer chromatography analyses were carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Corp.) developed with petroleum ether-acetone (1:1, vol/vol) . Compounds were detected as a spot on the plates after they were sprayed with 10% H2SO4 (in ethanol) and heated at 120°C for 10 min . There were two controls, one being a fermentation without cinobufagin and the other being sterile medium with cinobufagin but with no fungal inoculum .

HPLC analysis.
A 1100 HPLC apparatus (Agilent, Waldbronn, Germany) equipped with DAD and a quaternary pump system was used . The column contained Agilent Zorbax Extend-C18 packing (5 µm in diameter) and was 4.6 by 250 mm . The mobile phase was methanol-water (52:48, vol/vol) for the first 10 min and then a linear gradient to methanol-water (80:20, vol/vol) over 15 min, where it was held for 5 min . The flow rate was 1.0 ml/min, the detection wavelength was 296 nm, and the column temperature was 25°C . Samples were filtered through 0.45-µm-pore-size membranes, and 10 µl of each sample was injected into the HPLC instrument .

Preparative HPLC conditions.
A SpectraSeries HPLC apparatus (Thermo Quest, San Jose, Calif.) with a PEGASIL ODS column (pore size, 5 µm; 10 by 250 mm) (Senshu Pak, Tokyo, Japan) was used to isolate biotransformation products . The mobile phase flow rate was 2.0 ml/min, and the detection wavelength was 296 nm . Samples were eluted with mixtures (65:35 and 60:40, vol/vol) of methanol and water .

Scaled-up biotransformation of cinobufagin by A . alternata AS 3.4578.
Liquid cultures were prepared and incubated as described above for 36 h to make a stock inoculum . A 5-ml volume of the inoculum was added to a 1-liter flask containing 350 ml of potato medium and incubated for 48 h, at which time cinobufagin in ethanol (20 mg/ml) was added to a final concentration of 60 µg/ml . After an additional 4 days of incubation, cultures from 25 flasks were pooled and filtered through Whatman no . 1 filter paper . The filtrate was extracted three times with 5 liters of ethyl acetate . The organic extract was concentrated and evaporated to dryness in a rotary evaporator under reduced pressure at 60°C to yield 1.6 g of a brownish oily residue .

Isolation and purification of biotransformation products.
The 1.6 g of residue was dissolved in 1 ml of methanol . The sample was applied to a Sephadex LH-20 column and eluted with methanol to give 1 g of a yellowish solid that was subjected to silica gel column chromatography (200 to 300 mesh; 2 by 30 cm, with 40 g of silica gel), and eluted in 100-ml fractions with gradient mixtures of petroleum ether (60 to 90°C) and acetone (4:1 for 1,500 ml, 2:1 for 1,000 ml, 3:2 for 800 ml, and 1:1 for 1,000 ml, vol/vol) . Fractions 6 to 8 were subjected to preparative reversed-phase liquid chromatography and eluted with methanol-water (65:35, vol/vol) to obtain compounds VI (3.6 mg, 0.7% yield) and VII (4.8 mg, 1.0% yield); fractions 10 to 16 and fractions 19 to 26 were purified over a Sephadex LH-20 column and eluted with methanol to yield compounds IV (55 mg, 11% yield) and II (141 mg, 28% yield), respectively; fractions 36 to 38 were combined, subjected to preparative liquid chromatography, and eluted with methanol-water (60:40, vol/vol) to give compounds III (6.5 mg, 1.3% yield) and V (3.6 mg, 0.7% yield) .

Time course investigation of the biotransformation of cinobufagin by A . alternata.
The fungal incubation conditions were identical to those for preparative-scale biotransformation, with cinobufagin added after 48 h of incubation and flasks harvested at 2-h intervals for the first 24 h and then daily for 9 days . Mycelia were removed as above by filtration, and the filtrate was diluted to 300 ml with distilled water . Half of the sample was extracted with 150 ml of ethyl acetate . The organic extract was evaporated to dryness, and the residue was dissolved in 1 ml of methanol . Samples were filtered through 0.45-µm-pore-size membranes just prior to HPLC analysis . An aliquot of 10 µl was used for each injection .

12ß-Hydroxyl cinobufagin (II) was a white powder; C26H34O7; molecular weight (MW) = 458; melting point (mp) 261 to 263°C; [{alpha}]D25 –20.7° (c 0.24, CH3OH); UV {lambda}max(log {varepsilon})(CH3OH): 203.0 (3.59), 295.0 (3.50) nm; IR {nu}max(KBr): 3,523, 3,402, 2,942, 2,877, 1,722, 1,637, 1,539, 1,241, 1,127, 1,028, 957, and 829 cm–1; HR-FT-ICR MS m/z calculated for C26H35O7 [M + H]+, 459.2377; found, 459.2382; 1H-and 13C-NMR data, see Tables 2 and 3 .


TABLE 2 . 13C-NMR spectral data for compounds I to VIIa

 

TABLE 3 . 1H-NMR spectral data of compounds II, III, IV, VI, and VIIa

 
12ß-Hydroxyl desacetylcinobufagin (III) was a white crystalline powder (acetone); C24H32O6; MW 416; mp 275 to 276 °C; [{alpha}]D25 +5.0 ° (c 0.20, CH3OH); UV {lambda}max(log {varepsilon})(CH3OH): 204.0 (3.65), 295.0 (3.55) nm; IR {nu}max(KBr): 3,375, 3,308, 2,933, 2,858, 1,699, 1,623, 1,534, 1,148, 1,024, 962, 825 cm–1; HR-FT-ICRMS m/z calculated for C24H33O6 [M + H]+, 417.2271; found, 417.2274; 1H- and 13C-NMR data, see Tables 2 and 3 .

3-oxo-12ß-Hydroxyl cinobufagin (IV) was a white powder; C26H32O7; MW 456; mp 220 to 222°C; [{alpha}]D25 –5.8° (c 0.34, CH3OH); UV {lambda}max(log {varepsilon})(CH3OH): 205.0 (3.68), 295.0 (3.58) nm; IR {nu}max(KBr): 3,404, 2,953, 2,876, 1,713, 1,633, 1,539, 1,249, 1,142, 1,031, 958, 831 cm–1; HR-FT-ICRMS m/z calculated for C26H33O7 [M + H]+, 457.2220; found, 457.2227; 1H- and 13C-NMR data, see Tables 2 and 3 .

3-oxo-12ß-Hydroxyl desacetylcinobufagin (V) was a white powder; C24H30O6; MW 414; mp 231 to 233°C; [{alpha}]D25 +9.6° (c 0.21, CH3OH); UV {lambda}max(log {varepsilon})(CH3OH): 203.0 (3.65), 295.0 (3.44) nm; IR {nu}max(KBr): 3,462, 2,943, 2,860, 1,685, 1,619, 1,537, 1,454, 1,427, 1,284, 1,226, 1,030, 935, 833 cm–1; HR-FT-ICRMS m/z calculated for C24H31O6 [M + H]+, 415.2115; found, 415.2111; 1H-NMR (DMSO-d6, 300 MHz): 7.86 (1H, d, J = 9.6 Hz, H-22), 7.37 (1H, s, H-21), 6.16 (1H, d, J = 9.6 Hz, H-23), 5.07 (1H, d, J = 4.5 Hz, 12-OH), 4.82 (1H, d, J = 4.2 Hz, 16-OH), 4.57 (1H, dd, J = 9.0 Hz, 4.2 Hz, H-16), 3.52 (1H, s, H-15), 3.40 (1H, m, overlapped, H-12), 3.08 (1H, d, J = 9.0 Hz, H-17), 2.73 (1H, t, J = 14.1 Hz, H-4{alpha}), 2.42 (1H, dt, J = 5.4 Hz, 14.1Hz, H-2{alpha}), 0.94 (3H, s, 19-CH3), 0.58 (3H, s, 18-CH3); 13C-NMR data, see Table 2 .

12-oxo-Cinobufagin (VI) was a white powder; C26H32O7; MW 456; mp 223 to 225°C; [{alpha}]D25 –46.2° (c 0.13, CH3OH); UV {lambda}max(log{varepsilon})(CH3OH): 206.0 (3.66), 293.0 (3.54) nm; IR {nu}max(KBr): 3,460, 2,929, 2,871, 1,742, 1,706, 1,537, 1,248, 1,139, 1,037 cm–1; HR-FT-ICRMS m/z calculated for C26H33O7 [M + H]+, 457.2220; found, 457.2222; 1H- and 13C-NMR data, see Tables 2 and 3 .

3-oxo-12{alpha}-Hydroxyl cinobufagin (VII) was a white powder; C26H32O7; MW 456; mp 110 to 112°C; [{alpha}]D25 +22.2 ° (c 0.18, CH3OH); UV {lambda}max(log{varepsilon})(CH3OH): 206.0 (3.62), 294.0 (3.52) nm; IR {nu}max(KBr): 3,381, 2,939, 2,872, 1,715, 1,539, 1,377, 1,246, 1,132, 1,026, 972, 880, 802 cm–1; HR-FT-ICRMS m/z calculated for C26H33O7 [M + H]+, 457.2220; found, 457.2223; 1H- and 13C-NMR data, see Tables 2 and 3 .

Bioassay.
Human hepatoma Bel-7402 cells, human gastric cancer BGC-823 cells, human cervical carcinoma HeLa cells, and human leukemia HL-60 cells were maintained in RPMI 1640 medium (GIBCO/BRL, Gaithersburg, Md.) supplemented with 10% (vol/vol) fetal bovine serum and cultured in 96-well microtiter plates . Test compounds (10–3 to 102 µmol/liter) were added to the cultures, and the cells were incubated at 37°C under 5% CO2 for 72 h . Cell survival was evaluated by the MTT method (3) and is reported as IC50, i.e., the concentration (micromoles per liter) of the test compound that inhibits 50% of cell growth . Results presented are the means of triplicate determinations .


   RESULTS

 
Of the 20 strains examined, 15 could metabolize cinobufagin and 9 could transform it readily and convert most of the substrates into a number of products (Table 1) . A . alternata AS 3.4578 was one of the most active strains and was selected for scaled-up biotransformation . After 3 days of transformation, nearly all the substrates were exhausted and several new peaks appeared in the HPLC chromatogram with DAD on-line UV absorption maximum at 296 nm, characteristic of the {alpha}-pyrone ring of bufadienolides (Fig . 1) . Since neither the no-fungus nor the no-cinobufagin control contained compounds with similar peaks, these new peaks presumably represented biotransformation products .


FIG . 1 . HPLC profile of the biotransformed products (II to VII) from cinobufagin (I, tR = 21.9 min [not detected]) by A . alternata AS 3.4578.

 
Six pure products, II to VII (Fig . 2) were isolated from the culture supernatant of the preparative-scale biotransformation by repeated chromatography, and their structures were identified by extensive spectroscopical analyses (28) .


FIG . 2 . Proposed biotransformation pathway of cinobufagin (I) by A . alternata AS 3.4578 . Bold arrows indicated major transformation reactions . The structure in brackets is a presumed intermediate.

 
The high-resolution mass spectrum of metabolite II had the [M + H]+ ion peak at m/z 459.2382, suggesting a molecular formula of C26H34O7 . The 13C-NMR spectrum showed an additional oxygenated methine signal at {delta}73.4 . Compared to the corresponding data for cinobufagin, the C-11 ({delta}29.4) and C-13 ({delta}50.8) signals shifted downfield by 8.9 and 6.2 ppm, respectively, whereas C-18 ({delta}11.7) shifted upfield by 5.2 ppm, suggesting the hydroxylation of C-12 . This conclusion was confirmed by the correlations of H-12 ({delta}3.40, brd, J = 11.0 Hz) with C-18 and C-17 ({delta}44.7) in the HMBC spectrum . The enhancement between H-12 and H-16 ({delta}5.44, d, J = 9.5 Hz) in the NOESY spectrum suggested that 12-OH was located in the ß-configuration . When deuterated water was added, the H-12 signal appeared as a typical double doublet, resulting from the couplings of H-12 with H-11ß (3Jaa = 11.0 Hz) and H-11{alpha} (3Jae = 4.5 Hz) . This split pattern is consistent with the ß-configuration of H-12 . Thus, metabolite II was identified as 12ß-hydroxyl cinobufagin . This compound was first isolated from toad venom in 1972, but no NMR data were reported (11) .

The high-resolution mass spectrum of metabolite III had the [M + H]+ ion peak at m/z 417.2274, suggesting a molecular formula of C24H32O6 . As with metabolite II, the 13C-NMR spectrum of III also had an additional oxygen-bearing tertiary-carbon signal at {delta}73.6 . In addition, C-18 shifted upfield to {delta}11.8 while C-13 shifted downfield to {delta}50.6 . These data are consistent with metabolite III being a 12ß-hydroxylated product of cinobufagin . The NMR signals corresponding to the acetyl group in cinobufagin disappeared, and C-16 shifted upfield to {delta}70.6, both suggesting the loss of the 16-acetyl group in III . Thus, metabolite III was identified as 12ß-hydroxyl desacetylcinobufagin .

The high-resolution mass spectrum of metabolite IV had the [M + H]+ ion peak at m/z 457.2227, suggesting a molecular formula of C26H32O7 . The 13C-NMR spectrum showed an additional oxygen-bearing CH signal at {delta}73.3 . Its corresponding proton signal at {delta}3.47 had long-range couplings with C-17 ({delta}44.7) and C-18 ({delta}11.7), suggesting that C-12 in metabolite IV was hydroxylated . When deuterated water was added, H-12 appeared as a double doublet (J = 11.0 Hz, 4.5 Hz), indicating the ß-configuration of 12-OH . The 13C-NMR spectrum at very low field showed a new carbonyl signal at {delta}211.6, while the signal at {delta}64.5 for C-3 in cinobufagin disappeared, indicating that C-3 of metabolite IV was dehydrogenated to a ketone group (16) . The HMBC spectrum also showed that C-3 had long-range couplings with H-2 and H-4 . Thus, metabolite IV was characterized as 3-oxo-12ß-hydroxyl cinobufagin .

The high-resolution mass spectrum of product V had the [M + H]+ ion peak at m/z 415.2111, suggesting a molecular formula of C24H30O6 . The 13C-NMR spectrum was very similar to that of product IV, except that signals corresponding to the acetyl group in IV disappeared, suggesting that V is a deacetylated product of cinobufagin . The carbon signal at {delta}211.7 indicated that C-3 was oxidized to a carbonyl group . The CH signal at {delta}73.3 and the upfield shift to {delta}11.7 of C-18 both suggest the introduction of a 12ß-hydroxyl group in metabolite V . Thus metabolite V was identified as 3-oxo-12ß-hydroxyl desacetylcinobufagin .

The high-resolution mass spectrum of metabolite VI had the [M + H]+ ion peak at m/z 457.2222, suggesting a molecular formula of C26H32O7 . The 13C-NMR spectrum had a carbonyl signal at {delta}209.7, while signals for carbons of the A and B rings were almost identical to those of cinobufagin . The proton signal of 18-CH3 shifted downfield to {delta}0.93 and had a long-range correlation with the {delta}209.7 signal, which led to its assignment to C-12 . Accordingly, C-11 ({delta}37.0) and C-13 ({delta}59.1) shifted downfield by 16.0 and 14.5 ppm, respectively . The HMBC spectrum also showed long-range coupling between H-17 ({delta}4.05) and C-12 . Thus, metabolite VI was identified as 12-oxo-cinobufagin .

The high-resolution mass spectrum of product VII had the [M + H]+ ion peak at m/z 457.2223, suggesting a molecular formula of C26H32O7 . The 1H- and 13C-NMR spectra of metabolite VII were very similar to those of metabolite IV, except that C-18 ({delta}17.2) resonated at a much lower field . An additional methine signal at {delta}73.2 in the 13C-NMR spectrum suggested the hydroxylation of C-12 in metabolite VII, because of its long-range coupling with H-18 ({delta}0.68) . In the NOESY spectrum, the enhancements between H-12 ({delta}3.61) and 18-CH3, and between H-9 ({delta}2.35) and 12-OH ({delta}3.61) strongly suggested the {alpha}-configuration of 12-OH . Thus, product VII was identified as 3-oxo-12{alpha}-hydroxyl cinobufagin .

Metabolites III to VII are described here for the first time . All products are hydroxylated at C-12ß except for the minor products VI and VII, which could result from dehydrogenation or epimerization of 12ß-hydroxylated precursors (Fig . 2) .

Time course of biotransformation.
12ß-Hydroxyl cinobufagin (metabolite II) was the first metabolite formed and was detected 2 h following addition of the substrate . The cinobufagin added was quantitatively converted to metabolite II after 8 h of incubation . The concentration of metabolite II then decreased gradually until it could no longer be detected on day 7 . Meanwhile, 3-oxo-12ß-hydroxyl cinobufagin accumulated in the cultures, peaking on day 5 before slowly decreasing . The concentration of a third metabolite, 3-oxo-12ß-hydroxyl desacetylcinobufagin, increased gradually for the entire period (Fig . 3) . We concluded that cinobufagin metabolism by A . alternata was initiated by 12ß-hydroxylation followed by 3-OH dehydrogenation and then C-16 deacetylation . The last two steps are relatively slow compared to the 12ß-hydroxylation . Interestingly, the concentration of 12ß-hydroxyl desacetylcinobufagin (product III) did not increase concomitantly with 3-oxo-12ß-hydroxyl desacetylcinobufagin (product V) in the later stages of the biotransformation . This pattern suggests the importance of the C-3 carbonyl group for the deacetylation of cinobufagin derivatives and allowed us to propose a possible biotransformation pathway for the metabolism of cinobufagin (Fig . 2) .


FIG . 3 . Kinetics of the biotransformed products from cinobufagin in culture supernatant of A . alternata AS 3.4578 . Each point represents the mean and standard deviation from three replicate experiments.

 
Metabolites II and/or IV were the major products for most of the 20 strains screened, indicating that 12ß-hydroxylation might be a common step in the metabolism of cinobufagin by filamentous fungi (Table 1) . However, the biotransformation products produced by Fusarium avenaceum AS 3.4594 are very different from those produced by A . alternata AS 3.4578, suggesting that this Fusarium strain may use a different pathway to transform cinobufagin . Some new HPLC peaks also were observed in Mucor and Aspergillus fermentations .

The in vitro cytotoxicity of cinobufagin and metabolites II to VII was evaluated with four human cancer cell lines (Table 4) . All the metabolites were less cytotoxic than cinobufagin . The deacetylated products III and V were almost inactive against cancer cells, which is in good agreement with the results obtained by Kamano et al . (13, 14) . However, products II and VII had IC50 of approximately 10–7 mol/liter, and their potential as antitumor compounds warrants further evaluation .


TABLE 4 . Cytotoxic activities of the biotransformation products against human cancer cell linesa

 

   DISCUSSION

 
Bufadienolides are a promising class of antineoplastic agents, and cinobufagin is among the most widely studied due to its natural abundance and cytotoxicity . By obtaining a series of cinobufagin derivatives via biotransformation, our goal was to identity new antitumor lead compounds and to study their structure-activity relationship . Some of the fungal biotransformation products could be the same as those in humans due to the similarity between these two metabolic systems .

In the present study, a scaled-up fermentation of A . alternata AS 3.4578 with cinobufagin yielded six more polar products, each hydroxylated at the C-12 position . A screening test with 20 fungal strains suggested that 12ß-hydroxylation of cinobufagin is a common metabolic pathway . Although steroids are common exogenous substrates for filamentous fungi, hydroxylated at the 11{alpha}-, 11ß-, and 16{alpha}-positions in most cases, 12ß-hydroxylation is relatively rare (18) . Examples include hydroxylation of 24-epi-brassinolide and 24-epi-castasterone by Cunninghamella, of pregn-4-ene-3,20-dione by Cephalosporium, of mexrenone by Mortierella isabellina, and of scillarenin by Rhizopus arrhizus (6, 7, 21, 25) . These reactions are not specific, however, and are coupled with hydroxylation at other sites . This is the first report of specific 12ß-hydroxylation of steroids by filamentous fungi, which also is difficult to achieve by chemical means (17) . The 12ß-hydroxylated bufadienolides are relatively rare in the nature (15), and our result provides a simple and efficient approach to prepare this type of compounds .

We studied the substrate specificity of 12ß-hydroxylation of bufadienolides by examining the biotransformation of two cinobufagin analogs by A . alternata (15) . After incubation with resibufogenin, an analog with a 14ß,15ß-epoxy ring but without the C-16 substituent, 12ß-hydroxyl resibufogenin was detected as a major product . In the fermentation of A . alternata with bufalin, a 14ß-OH analog, 7ß-hydroxyl bufalin, was detected as a major product, together with the expected 12ß-hydroxyl bufalin . Thus, A . alternata could catalyze the 12ß-hydroxylation of different subtypes of bufadienolides . The characteristic {alpha}-pyrone ring of bufadienolides might be a structural requirement for this reaction . The 14ß,15ß-epoxy ring appears to favor the hydroxylation at C-12ß while inhibiting that at C-7ß . We think that this results from the adjacent spatial locations of C-7 and the epoxy ring . To prove this hypothesis, further study of the specific structure of the target enzyme that facilitates its binding with bufadienolides is needed .

Hydroxylation is a common way to increase the water solubility of less polar compounds (17, 24) . Preliminary results of the structure-activity relationship studies with bufadienolides show that introduction of a hydroxyl group at C-16{alpha} or C-11ß greatly reduces cytotoxicity, while bufadienolides with a 12ß- or 1ß-hydroxyl group have significant inhibitory effects . Thus, our discovery of an efficient means of 12ß-hydroxylation should speed the development of new cytotoxic bufadienolides .

The major transformation reactions of cinobufagin by Alternaria include 12ß-hydroxylation, 3-OH dehydrogenation, and C-16 deacetylation . The deacetylation was shared by rat liver microsomes, human intestinal bacteria, and plant cell suspension cultures (26, 27, 29, 32) and may well be the metabolic pathway for cinobufagin in humans . The 12ß-hydroxylation reaction is exclusive for filamentous fungi and is unknown in humans .

In conclusion, the specific 12ß-hydroxylation of cinobufagin by filamentous fungi provides a simple and efficient method of producing 12ß-hydroxylated bufadienolides . The compounds identified in this study will serve as references for the identification of cinobufagin metabolites in humans .

 


   ACKNOWLEDGMENTS

 
We thank Guangzhong Tu, Beijing Institute of Microchemistry, for recording NMR spectra . M . Ye thanks Yuxin Sheng and Peng Han for their technical assistance .


   FOOTNOTES

 
* Corresponding author . Mailing address: The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road #38, Beijing 100083, People's Republic of China . Phone: 86-10-8280-1516 . Fax: 86-10-8280-2700 . E-mail: gda{at}bjmu.edu.cn .


   REFERENCES

 

  1. Abourashed, E . A., A . M . Clark, and C . D . Hufford. 1999 . Microbial models of mammalian metabolism of xenobiotics: an updated review . Curr . Med . Chem . 6:359-374.
  2. Azerad, R. 1999 . Microbial models for drug metabolism, p . 169-218 . In T . Scheper (ed.), Advances in biochemical engineering/biotechnology, vol . 63 . Biotransformations . Springer-Verlag, Berlin, Germany . Biotechnol . 63:169-218.
  3. Carmichel, J., W . G . DeGraff, A . F . Gazdar, J . D . Minna, and J . B . Mitchell. 1987 . Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing . Cancer Res . 47:936-942.
  4. Cirino, P . C., and F . H . Arnold. 2002 . Protein engineering of oxygenases for biocatalysis . Curr . Opin . Chem . Biol . 6:130-135.
  5. Clark, A . M., and C . D . Hufford. 1991 . Use of microorganisms for the study of drug metabolism: an update . Med . Res . Rev . 11:473-501.
  6. Farooq, A . H., R . James, and Z . Iqbal. 1994 . Hydroxylation of progesterone by Cephalosporium aphidicola . Phytochemistry 37:723-726.
  7. Goerlich, B. 1971 . Transformation of scillarenin with Rhizopus arrhizus I . Structural determination of the Rhizopus arrhizus-induced transformation products of scillarenin II . Planta Med . Suppl . 5:113-126 . (In German.)
  8. Goodhue, C . T. 1982 . The methodology of microbial transformation of organic compounds, p . 9-44 . In J . P . Rosazza (ed.), Microbial transformations of bioactive compounds, vol . I . CRC Press, Boca Raton, Fla.
  9. Hashimoto, S., Y . Jing, N . Kawazoe, Y . Masuda, S . Nakajo, T . Yoshida, Y . Kuroiwa, and K . Nakaya. 1997 . Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs . Leuk . Res . 21:875-883.
  10. Hong, Z., K . Chan, and H . W . Yeung. 1992 . Simultaneous determination of bufadienolides in the traditional Chinese medicine preparation, Liu-Shen-Wan, by liquid chromatography . J . Pharm . Pharmacol . 44:1023-1026.
  11. Horiger, N., D . Zivanov, H . H . A . Linde, and K . Meyer. 1972 . Weitere bufadienolide aus Ch'an Su . Helv . Chim . Acta 55:2549-2562 . (In German.)
  12. Jezequel, S . G. 1998 . Microbial models of mammalian metabolism: uses and misuses (clarification of some misconceptions) . J . Mol . Catal . B Enzym . 5:371-377.
  13. Kamano, Y., A . Kotake, H . Hashima, M . Inoue, H . Morita, K . Takeya, H . Itokawa, N . Nandachi, T . Segawa, A . Yukita, K . Saitou, M . Katsuyama, and G . R . Pettit. 1998 . Structure-cytotoxic activity relationship for the toad poison bufadienolides . Bioorg . Med . Chem . 6:1103-1115.
  14. Kamano, Y., A . Yamashita, T . Nogawa, H . Morita, K . Takeya, H . Itokawa, T . Segawa, A . Yukita, K . Saito, M . Katsuyama, and G . R . Pettit. 2002 . QSAR evaluation of the Ch'an Su and related bufadienolides against the colchicine-resistant primary liver carcinoma cell line PLC/PRF/5 . J . Med . Chem . 45:5440-5447.
  15. Krenn, L., and B . Kopp. 1998 . Bufadienolides from animal and plant sources . Phytochemistry 48:1-29.
  16. Linde, H . H . A., and F . Lohrer. 1991 . Strukturaufklarung von Gamabufotaliniol (1) . Pharm . Acta Helv . 66:98-101.
  17. Loughlin, W . A. 2000 . Biotransformations in organic synthesis . Bioresour . Technol . 74:49-62.
  18. Mahato, S . B., and S . Garai. 1997 . Advances in microbial steroid biotransformation . Steroids 62:332-345.
  19. Masuda, Y., N . Kawazoe, S . Nakajo, T . Yoshida, Y . Kuroiwa, and K . Nakaya. 1995 . Bufalin induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells . Leuk . Res . 19:549-556.
  20. Nogawa, T., Y . Kamano, A . Yamashita, and G . R . Pettit. 2001 . Isolation and structure of five new cancer cell growth inhibitory bufadienolides from the Chinese traditional drug Ch'an Su . J . Nat . Prod . 64:1148-1152.
  21. Preisig, C . L., J . A . Laakso, U . M . Mocek, P . T . Wang, J . Baez, and G . Byng. 2003 . Biotransformations of the cardiovascular drugs mexrenone and canrenone . J . Nat . Prod . 66:350-356.
  22. Riva, S. 2001 . Biocatalytic modification of natural products . Curr . Opin . Chem . Biol . 5:106-111.
  23. Steyn, P . S., and F . R . van Heerden. 1998 . Bufadienolides of plant and animal origin . Nat . Prod . Rep . 15:397-413.
  24. Urlacher, V., and R . D . Schmid. 2002 . Biotransformations using prokaryotic P450 monooxygenases . Curr . Opin . Biotechnol . 13:557-564.
  25. Voigt, B., A . Porzel, H . Naumann, S . C . Hoerhold, and G . Adam. 1993 . Hydroxylation of the native brassinosteroids 24-epicastasterone and 24-epibrassinolide by the fungus Cunninghamella echinulata . Steroids 58:320-323.
  26. Yang, X . W., Z . T . Xing, J . R . Cui, Y . L . Fan, R . Zhao, and X . G . Li. 2001 . Studies on the metabolism of cinobufagin and cinobufotalin by human intestinal bacteria . J . Peking Univ . (Health Sci.) 33:199-204 . (In Chinese.)
  27. Ye, M., J . Dai, H . Guo, Y . Cui, and D . Guo. 2002 . Glucosylation of cinobufagin by cultured suspension cells of Catharanthus roseus . Tetrahedron Lett . 43:8535-8538.
  28. Ye, M., J . Han, H . Guo, and D . Guo. 2002 . Structural determination and complete NMR spectral assignments of a new bufadienolide glycoside . Magn . Reson . Chem . 40:786-788.
  29. Ye, M., L . Ning, J . Zhan, H . Guo, and D . Guo. 2003 . Biotransformation of cinobufagin by cell suspension cultures of Catharanthus roseus and Platycodon grandiflorum . J . Mol . Catal . B Enzymol . 22:89-95.
  30. Yeh, J . Y., W . J . Huang, S . F . Kan, and P . S . Wang. 2003 . Effects of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells . Prostate 54:112-124.
  31. Zhang, L . S., K . Nakaya, T . Yoshida, and Y . Kuroiwa. 1991 . Bufalin as a potent inducer of differentiation of human myeloid leukemia cells . Biochem . Biophys . Res . Commun . 178:686-693.
  32. Zhang, L . S., T . Yoshida, K . Aoki, and Y . Kuroiwa. 1991 . Metabolism of cinobufagin in rat liver microsomes . Identification of epimerized and deacetylated metabolites by liquid chromatography/mass spectrometry . Drug Metab . Dispos . 19:917-919.
  33. Zhang, L . S., K . Nakaya, T . Yoshida, and Y . Kuroiwa. 1992 . Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers . Cancer Res . 52:4634-4641.

 

 

 

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