Laboratoire de Chimie et Biochimie Pharmacologiques et
Toxicologiques (C.M., R.A.), URA 400 CNRS, Université René
Descartes; and the
Institut de Recherches Chimiques et Biologiques
Appliquées (P.H., B.D.)
This study was undertaken to validate the use of microbial
biotransformation systems for drug metabolism studies. Thymoxamine 1 was rapidly hydrolyzed to desacetylthymoxamine (DAT) 2 by
numerous fungi. Other known animal metabolites, such as
N-desmethyl-desacetylthymoxamine 3 and
desacetylthymoxamine-O-sulfate 6, were produced from
DAT by Mucor rouxii and Mortierella isabellina.
DAT-N-oxide 5, a putative animal microsomal
metabolite, was also produced by M. isabellina. In
addition, a few strains (such as Actinomucor elegans, Mucor hiemalis, and Mucor janssenii) produced a
glycosylated metabolite that was identified by high-resolution
1H- and 13C-NMR, MS, and enzymatic hydrolysis
as the corresponding
[4-(2-dimethylaminoethoxy)-5-isopropyl-2-methyl-phenyl]-1-
-D-glucopyranoside 7. A similar glucosylation reaction was observed when
thymohydroquinone 10 was incubated with A. elegans.
Several strains were able to produce transiently thymohydroquinone from
DAT-N-oxide 5, possibly through a -elimination
mechanism.
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Introduction |
Thymoxamine
1 {moxisylyt,
[4-(2-dimethylaminoethoxy)-5-isopropyl-2-methyl phenyl acetate]} is
a well-known and representative molecule in a family of 
-adrenergic
blocking agents (1-4) devoid of -blocking activity (5) that have
been used in the treatment of various vascular disorders. Recently,
this molecule, and differently substituted congeners (fig.
1), have been also demonstrated to be useful in the
urological field (6-10). The metabolism of thymoxamine itself in
humans and animals is now well established (4, 11-17); it has been
shown that the active drug is DAT1
2 derived from the prodrug 1 by early
deacetylation catalyzed by intestinal and plasma esterases (11).
Further hepatic biotransformations mainly involve
N-desmethylation to DMAT 3 and sulfoconjugation
and glucuronoconjugation of these metabolites. In addition, small
amounts (<1%) of 4-hydroxy-5-isopropyl-2-methyl phenol, in
equilibrium with thymoquinone 4, have been shown to be
present in the human urinary elimination metabolites (16).
Use of microbial models of mammalian metabolism, first established in
the early 1970s by Smith and Rosazza (18, 19), has now gained a large
audience, especially when it is necessary to obtain significant amounts
of xenobiotics phase I metabolites, sometimes unavailable by chemical
synthesis (20). More recently, it has been shown that phase II
metabolites of phenolic compounds may also be obtained
by the same microbial methods, including glucuronides (21, 22),
glucosides (21, 23-30) and sulfoconjugates (21, 31-35). It was thus
interesting to complete the survey of metabolites formed from compounds
1 and 2 by investigating those formed by
microorganisms (especially fungi), and to compare them with those
produced in mammalian species, including phase II-conjugated
derivatives.
Materials and Methods
Chemicals.
All solvents were analytical grade. Thymoxamine 1, DAT
2, DMAT (15, 16) 3, DAT-N-oxide
5, DAT-O-sulfate 6 (15), thymoquinone
4 (36), and thymohydroquinone 10 (37) were
provided by the Institut de Recherches Chimiques et Biologiques
Appliquées, Laboratoires Debat (Vicq, France). Silica gel
H60 (Merck, 230-400 mesh) was used for flash
chromatography. -Glucosidase (type I, from Baker's yeast),
-glucosidase (from almonds), -galactosidase (from
Aspergillus niger), phenyl--D- and
phenyl--D-glucopyranosides,
4-nitrophenyl--D- and
4-nitrophenyl--D-glucopyranosides,
2-nitrophenyl--D- and 4-nitrophenyl--D-galactopyranosides, salicin
([2-hydroxymethyl]phenyl--D-glycopyranoside), and
arbutin (hydroquinone--D-glucopyranoside) were purchased from Sigma Chemical Co. (St. Louis, MO). -Galactosidase (from Escherichia coli) was purchased from Boehringer Mannheim
(Meylan, France). D-Glucuronic acid (a sodium salt
monohydrate) was purchased from Janssen Chemica (Paris, France). The
specific colorimetric determination kit for D-glucose
(based on glucose oxidase activity) was obtained from Biotrol (Paris,
France).
Synthesis of 5-isopropyl-4-methoxy-2-methylphenol(11) (fig.
2). 4-Isopropyl-3-methoxy-toluene.
A solution of thymol (5 g, 33 mmol) in methanol (15 ml) and a 16 M
solution of potassium hydroxide (2 ml, 32 mmol) were refluxed, whereas
dimethyl sulfate (11 ml, 115 mmol) and a 16 M potassium hydroxide (8 ml, 128 mmol) in methanol were alternatively added. After completion of
the reaction, monitored by GC (capillary SE30 column, 120°-160°C,
4°C/min), the reaction mixture was filtered, and the filtrate and
washing concentrated in vacuo to a small volume. The residue
was taken up in ethyl ether, which was washed with a saturated sodium
hydrogen carbonate solution, dried on sodium sulfate, and then
evaporated under reduced pressure. The residual oil was purified by
filtration on silica gel H60 and eluted with pentane
(yield: 4.1 g, 75%).
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Fig. 2.
Synthesis of
5-isopropyl-4-methoxy-2-methylphenol (11).
(a) Dimethyl sulfate, KOH/MeOH; (b)
polyphosphoric acid/Ac2O, 60°C; (c) HCOOH,
H2O2; and (d) KCN/MeOH, 80°C.
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2-Acetyl-4-isopropyl-5-methoxy-toluene.
A mixture of 4-isopropyl-3-methoxy-toluene (3 g, 18.3 mmol),
polyphosphoric acid (12 g, 73 mmol), and water (1 ml, 73 mmol) were
heated to 60°C, and acetic anhydride (7.5 ml, 79 mmol) was added
dropwise. After 1 hr, the cold mixture was added with water and brought
to alkaline pH with sodium hydrogen carbonate. After repeated
extraction with dichloromethane and washing with brine, the organic
phase was dried on magnesium sulfate and evaporated under reduced
pressure. The oily residue was purified by flash chromatography with
pentane-ethyl acetate (98:2; yield: 3 g, 80%).
2-Acetoxy-4-isopropyl-5-methoxy-toluene.
To a solution of 2-acetyl-4-isopropyl-5-methoxy-toluene (1 g, 4.85 mmol) in formic acid (4.85 ml), 30% hydrogen peroxide (0.82 ml, 9 mmol) was added dropwise. The reaction mixture was left at 33°C for 1 hr, cooled to 15°C, and neutralized at this temperature with sodium
hydrogen carbonate, then extracted repeatedly with dichloromethane.
After usual workup and evaporation of the organic phase, the oily
residue was purified by flash chromatography with pentane:ethyl acetate
(95:5; yield: 0.87 g, 81%).
5-Isopropyl-4-methoxy-2-methyl Phenol.
A solution of 2-acetoxy-4-isopropyl-5-methoxy-toluene (100 mg, 0.45 mmol) and potassium cyanide (15 mg) (38) in methanol (3 ml) is refluxed
during 1 hr. After cooling and acidification with 1 N hydrochloric
acid, ethanol was evaporated under reduced pressure and the residue,
taken up in a small amount of water, was extracted with
dichloromethane. After usual workup of the organic phase, flash
chromatography (pentane:ethyl acetate, 98:2) and recrystallization in
ethanol yielded pure 11 (75 mg, 93%). M.p. 65°-67°C
[lit. (39) 67°C]. 1H-NMR (CD3OD), 
ppm:
1.11 (d, 6H, J = 6.8 Hz, CH3-9 and
CH3-10), 2.11 (s, 3H, CH3-7), 3.16 (m, 1H,
H-8), 3.69 (s, 3H, OCH3), 6.57 and 6.59 (2s, 2H, H-3, and
H-6). 13C-NMR. (CD3OD), ppm: 16.1 (CH3-7), 23.3 (CH3-9 and CH3-10), 27.6 (CH-8), 56.8 (OCH3), 113.8 and 115.0 (CH-3 and CH-6),
122.8 (quat. C-5), 136.3 (quat. C-2), 150.0 and 151.4 (quat. C-1 and C-4). MS (electron ionization) m/z (%): 180 (41)
M+; 165 (100) [M-15]+.
Instrumentation.
1H- and 13C-NMR (one-dimensional and
two-dimensional) spectra were performed at 250.13 and 62.9 MHz,
respectively, on a Bruker WM250-FT instrument, using standard pulse
sequences. Electron impact and chemical ionization (ammonia) mass
spectrometric analyses were performed on a R10-10 Nermag instrument at
the Laboratoire de Chimie, École Normale Supérieure
(Paris, France). UV spectra were recorded with a Uvikon 810 spectrophotometer (Kontron). Melting points were determined in
capillary tubes with a Büchi instrument and are uncorrected.
Optical rotations were measured using a Perkin-Elmer 241C
spectropolarimeter in a 1 dm cell.
Microorganisms.
All cultures were maintained on agar slants containing (per liter)
yeast extract (Difco, Detroit, MI) 5 g, malt extract (Difco) 5 g, glucose 20 g, and Bacto-agar (Difco) 20 g; stored
at 4°C; and subcultured before use. Fungi were purchased from the
American Type Culture Collection (ATCC strains; Rockville, MD) the
Northern Regional Research Laboratories (NRRL strains; Peoria, IL), the Centralbureau voor Schimmelcultures (CBS strains; Baarn, the
Netherlands), or the Mycothèque of the Museum d'Histoire
Naturelle (MMP strains; Paris, France). Some strains (no strain number)
are from local origin.
Analytical Chromatographic Procedures.
Reversed-phase HPLC was conducted using the following conditions:
column, Lichrospher 100 RP18 Merck (125 × 4 mm);
solvent, MeOH:water:28% NH4OH (65:34.5:0.5 or
60:39.5:0.5), 0.8-1 ml/min using a Beckmann 110A pump, a Gilson 231 sample injector equipped with a 20-µl loop, a Pye-Unicam LC-UV
detector set at 278 nm, and a Shimadzu C-R6A integrator recorder. TLC
was performed with the ascending method using silica gel
60F254 precoated aluminium sheets (Merck, Germany), and
CH2Cl2:MeOH:28% NH4OH (80:19:1) as solvent. Spots were detected under UV light and by spraying with a
2.5% ammonium molybdate:1% cerium sulfate solution in 10% aqueous H2SO4.
Microbial Biotransformations. Culture and Screening
Procedures.
All microorganisms were grown at 27°C in a liquid medium containing
(per liter) corn steep liquor (Roquette, France) 10 g, glucose
30 g, KH2PO4 1 g,
K2HPO4 2 g, NaNO3 2 g,
KC1 0.5 g, MgSO4 · 7H2O 0.5 g, and
FeSO4 · 7H2O 0.02 g (final pH: ~6.5).
For screening experiments, 250-ml conical flasks containing 100 ml of
sterile liquid medium were inoculated with a few drops of a spore
suspension obtained from a freshly grown agar slant, and orbitally
shaken (200 rpm) at 27°C for 48-72 hr in a Gallenkamp incubator.
Crystalline substrates were then added to yield a final concentration
of 0.5 g/liter. Samples (1-2 ml) were aseptically withdrawn every day, centrifuged, and the supernatants were microfiltered (0.45 µm). Control incubations in the sterile culture medium without inoculated microorganisms were systematically included in all biotransformation experiments. Aliquots of the filtrates were analyzed by reversed-phase HPLC, and the remaining fraction was adjusted at pH 9.0, saturated with
sodium chloride, and extracted with ethyl acetate for TLC analysis.
Most transformations were continued until no further increase of
metabolite(s) was observed (usually 7-10 days). In some experiments,
the grown biomass (66 hr) was separated from the culture medium by
filtration on a Whatman GF/A glass fiber paper, washed with distilled
water, and resuspended in an equal volume of 0.05 M potassium phosphate
buffer (pH 7.0); the substrate was then added and incubation performed
as previously described.
Transformation of DAT 2 by Actinomucor elegans.
Three 100-ml volumes of liquid medium were inoculated and grown as
described. After full growth of the mycelium (66 hr), DAT (50 mg/flask)
was added, and incubation was continued in the same conditions during 7 days. The flask contents were pooled, and the mycelium was separated
from broth by filtration. The filtrate was saturated with sodium
chloride, filtered again on Celite, adjusted to pH 9.0 with 2 N sodium
hydroxide, then extracted 3 times with 150 ml of ethyl acetate. The
pooled organic extracts were washed with brine, dried on sodium
sulfate, then evaporated under reduced pressure to yield 230 mg of
crude extract. This extract was deposited on a silica gel
H60 (Merck, 230-400 mesh) column (2 × 15 cm) that
was eluted with CH2Cl2:MeOH:28%
NH4OH (80:19:1). Collected fractions were pooled to give
unchanged substrate (40 mg) and metabolite 7 (66 mg) as a
glassy solid, which after solubilization in water and lyophilization
yielded a white hygroscopic powder used for structure elucidation. M.p.
40°-48°C (sealed tube). []D24 = 
22.8° (C 0.2, MeOH). MS (CI, NH3) m/z (%):
400 (100) [MH]+; 238 (28)
[(M162)H]+. HRMS: calc. for
C20H33NO7, 399.22569, found
399.2255.
Enzymic Hydrolysis of
[4-(2-Dimethylaminoethoxy)-5-isopropyl-2-methylphenyl]-1--D-glucopyranoside
(7).
A solution of metabolite 7 (0.5 mg, 1.25 µmol) in 0.1 M
sodium acetate buffer [pH 5.0 (1 ml)] was incubated with 0.25 units
of almond -glucosidase at 37°C. A control sample was incubated without enzyme. Aliquots (100 µl) were withdrawn periodically for 32 hr and used for HPLC determination of released DAT and colorimetric-enzymatic determination of D-glucose. Similar
experiments were conducted using yeast -glucosidase in 0.1 M sodium
phosphate buffer (pH 7.0). Control experiments in the same conditions
showed a total hydrolysis of
4-nitrophenyl-1--D-glucopyranoside or
4-nitrophenyl-1--D-glucopyranoside, respectively, in
~15 min. Similarly, metabolite 7 (2 µmol) in 0.1 M
sodium phosphate buffer pH 7.0 (1 ml) was incubated with 0.36 units of
-galactosidase or 0.15 units of -galactosidase (previously
activated with 0.3 M mercaptoethanol and 0.01 M magnesium chloride) at
37°C. In the same conditions,
4-nitrophenyl-1--D-galactopyranoside and
2-nitrophenyl-1--D-galactopyranoside were, respectively, hydrolyzed in 15 min and 10 min.
Transformation of DAT 2 by Mucor rouxii.
Two 100-ml volumes of liquid medium were inoculated and grown as
described. DAT (50 mg/flask) was added, and incubation was continued in
the same conditions during 7 days. Flask contents were pooled and
mycelium was separated from broth by filtration. The filtrate was
saturated with sodium chloride, filtered again on Celite, adjusted to
pH 9.0 with 2 N sodium hydroxide, then extracted 3 times with ethyl
acetate. Pooled organic extracts were washed with brine, dried on
sodium sulfate, then evaporated under reduced pressure to yield 135 mg
of crude extract. This extract was deposited on a silica gel
H60 column (2 × 12 cm) that was eluted with
CH2Cl2:MeOH:28% NH4OH
(90:9.5:0.5). Collected fractions were pooled to give unchanged
substrate (90 mg), and metabolite 3 (10 mg) was identical to
an authentic sample of DMAT.
Transformation of DAT 2 by Mortierella isabellina.
Two 100-ml volumes of liquid medium were inoculated and grown as
described. DAT (50 mg/flask) was added, and incubation was continued in
the same conditions for 7 days. Flask contents were pooled, and the
mycelium was separated from broth by filtration. The filtrate was
lyophilized to give a white-yellowish powder, which was taken up in
MeOH and deposited on a silica gel column (Merck H60
230-400 mesh, 2.5 × 13 cm) that was eluted with
CH2Cl2:MeOH:28% NH4OH (80:19:1).
Metabolite 5 was first eluted (17 mg). M.p. = 112°-113°C (authentic sample of DAT-N-oxide, 114°C).
1H-NMR (CD3OD), ppm: 1.12 (d, 6H,
J = 6.8 Hz, CH3-9 and CH3-10), 2.11 (s, 3H, CH3-7), 3.19 (m, 1H, H-8), 3.34 (s, 6H,
CH3-13 and CH3-14), 3.77 (t, 2H,
J = 4.8 Hz, H-12), 4.37 (t, 2H, J = 4.8 Hz, H-11), 6.61 and 6.69 (2s, 2H, H-3 and H-6). 13C-NMR
(CD3OD), ppm: 17.8 (CH3-7), 25.4 (CH3-9 and CH3-10), 29.3 (CH-8), 59.7 (CH3-13 and CH3-14), 65.6 (CH2-12),
72.1 (CH2-11), 115.6 and 118.1 (CH-3 and
CH3-6), 125.2 (quat. C-2), 138.4 (quat. C-5); 150.8 and
153.1 (quat. C-1 and C-4). MS (CI, NH3) m/z
(%): 254 (12) [MH]+; 238 (100) [(M 16)H]+; 224 (70) [(M 30)H]+.
Metabolite 6 (18 mg) was eluted later, with partial
hydrolysis on the silica gel column. M.p. = 260°C dec. (identical to
an authentic sample of DAT-O-sulfate). 1H-NMR
(D2O), ppm: 1.23 (d, 6H, J = 6 Hz,
CH3-9 and CH3-10), 2.33 (s, 3H,
CH3-7), 3.07 (s, 6H, CH3-13 and
CH3-14), 3.31 (m, 1H, H-8), 3.70 (t, 2H, J = 4.8 Hz, H-12), 4.42 (t, 2H, J = 4.8 Hz, H-11), 6.97 (s, 1H, H-3), 7.32 (s, 1H, H-6). 13C-NMR (D2O),
ppm: 25.0 (CH3-9 and CH3-10), 25.2 (CH3-7), 28.7 (CH-8), 46.2 (CH3-13 and
CH3-14), 59.7 (CH2-12), 66.0 (CH2-11), 117.8 (CH-3), 122.5 (CH-6), 132.5 (quat. C-2),
139.3 (quat. C-5), 147.0 and 147.6 (quat. C-1 and C-4). MS (CI,
NH3) m/z (%): 335 (14) [M + NH4]+, 300 (10) [(M-18)H]+, 238 (100) [(M-80)H]+.
Transformation of Thymohydroquinone (10) by
Actinomucor elegans.
Four 100-ml volumes of liquid medium were inoculated and grown as
described. Thymohydroquinone (30 mg/flask, solubilized in 0.5 ml
ethanol) was added, and incubation was continued in the same conditions
for 7 days. Flask contents were pooled, and the mycelium was separated
from broth by filtration. The filtrate was saturated with NaCl,
filtered on Celite, and extracted 3 times with 150 ml of ethyl acetate.
Pooled organic extracts were washed with brine, dried on
Na2SO4, and then evaporated under reduced pressure to yield 280 mg of crude extract. This extract was deposited on a silica gel column (Merck H60, 230-400 mesh, 2.5 × 15 cm) that was eluted with CH2Cl2:MeOH:28%
NH4OH (80:19:1). Collected fractions were pooled to give
unchanged substrate (12 mg, 10%), then 50 mg (25%) of a 6:4 mixture
of 1- and 4-thymohydroquinone--D-glucosides, unseparated
after crystallization in ethyl acetate. M.p. = 98°-100°C. MS (CI,
NH3) m/z (%): 346 (100) [M + NH4]+, 299 (22) [(M-30)H+], 282 (92) [(M-47)H]+, 166 (18) [(M-162)H]+.
Thymohydroquinone-1-O-glucoside (40).
1H-NMR (acetone-d6), ppm: 1.11 (d, 6H, J = 6.8 Hz, CH3-9 and
CH3-10), 2.11 (s, 3H, CH3-7), 3.23 (m, 1H,
H-8), 3.30-4.45 (m, 10H, 4 D2O-exchangeable OH + H-2
, H-3, H-4, H-5, and 2 H-6), 4.16 (t, 2H, J = 5.6 Hz, H-11), 4.72 (d, 1H, J = 7 Hz, H-1), 6.66 and
6.91 (2s, 2H, H-3 and H-6). 13C-NMR (CD3OD),
ppm: 16.0 (CH3-7), 23.1 (CH3-9 and
CH3-10), 27.0 (CH-8), 62.7 (CH2-6), 71.6 (CH-4), 75.1 (CH-2), 78.0 and 78.3 (CH-3 and CH-5), 104.4 (CH-1),
113.1 and 120.3 (CH-3 and CH-6), 123.2 (quat. C-2), 138.2 (quat. C-5),
149.2 and 151.8 (quat. C-1 and C-4).
Thymohydroquinone-4-O-glucoside (40).
1H-NMR (acetone-d6), ppm: 1.17 (d, 6H, J = 6 Hz, CH3-9 and
CH3-10), 2.13 (s, 3H, CH3-7), 3.23 (m, 1H,
H-8), 3.30-4.45 (m, 10H, 4 D2O-exchangeable OH + H-2, H-3, H-4, H-5, and 2 H-6), 4.16 (t, 2H, J = 5.6 Hz, H-11), 4.72 (d, 1H, J = 7 Hz, H-1), 6.59 and
6.98 (2s, 2H, H-3 and H-6). 13C-NMR (CD3OD),
ppm: 16.1 (CH3-7), 23.6 and 23.7 (CH3-9 and
CH3-10), 27.9 (CH-8), 62.7 (CH2-6), 71.6 (CH-4), 75.1 (CH-2), 78.0 and 78.3 (CH-3 and CH-5), 104.5 (CH-1),
116.1 and 117.9 (CH-3 and CH-6), 127.2 (quat. C-2), 134.2 (quat. C-5),
150.5 and 150.6 (quat. C-1 and C-4).
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Results |
Among the 40 fungi screened (table 1) for the
metabolization of thymoxamine 1 at 0.5 g/liter
concentration, only a small number of strains were relatively inactive,
producing slowly the desacetyl derivative 2 (sometimes >7
days to complete hydrolysis, a rate comparable with the spontaneous
hydrolysis of this phenol-acetate molecule in the conditions of the
culture medium). On the contrary, most strains showed the ability to
hydrolyze 1 quite rapidly (24 hr or less); some of
them
such as A. elegans MMP 2092, M. isabellina
NRRL 1757, Mucor hiemalis, Mucor janssenii NRRL
3628 (= Mucor circinelloides f. janssenii), or M. rouxii CBS 41677produced on longer incubation times
noticeable amounts of other metabolites, as detected by HPLC of the
incubation medium (figs.
3, 4, 5). Quite evidently,
such metabolites (fig. 6) were produced from DAT
(2), and further preparative investigations were conducted
using this compound as substrate.
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Fig. 3.
Reversed-phase HPLC profile of the filtrate
of 7 days incubation of DAT (0.5 g/liter) with a M. rouxii
culture.
Solvent: MeOH:water:28% NH4OH (60:39.5:0.5); flow rate:
0.8 ml/min. For other experimental details, see Materials and
Methods. A, DAT (2); B, DMAT
(3).
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Fig. 4.
Reversed-phase HPLC profile of the filtrate
of 4 days incubation of DAT (0.5 g/liter) with a M.
isabellina culture.
Solvent: MeOH:water:28% NH4OH (65:34.5:0.5); flow rate: 1 ml/min. For other experimental details, see Materials and
Methods. A, DAT (2); (B,
DAT-N-oxide (5) + DAT-O-sulfate (6).
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Fig. 5.
Reversed-phase HPLC profile of 24 hr
incubation of DAT (0.5 g/liter) with an A. elegans
culture.
For experimental details, see fig. 3. A, DAT
(2); B,
DAT-1-O--D-glucoside (7).
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Fig. 6.
Chemical structure of metabolites produced
from thymoxamine (1) and DAT (2) by M.
isabellina, M. hiemalis, M. janssenii, and A.
elegans.
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Fungal Metabolite Isolation and Identification.
When DAT was incubated with a 65-hr grown culture of M. rouxii, it was slowly metabolized (~50% in 5-7 days), and a
small amount (~10%) was recovered as a single product, identical
with an authentic sample of DMAT 3 (fig. 3).
After 7 days incubation with M. isabellina, DAT (100 mg) was
completely converted to a mixture of two main highly polar
water-soluble products that were not separated by HPLC (fig. 4), but
appeared as distinct spots on TLC plates. Both metabolites could be
recovered from the incubation filtrate after lyophilization and were
separated by silica gel chromatography using an aqueous ammonia-organic eluent mixture. The first eluted product (15 mg) was characterized by a
1H-NMR spectrum very similar to that of DAT; only a few
signals, corresponding to the ethoxyamino side chain, were shifted to
lower fields (
H-11, 0.1 ppm; H-12, 0.19 ppm; and H-13/H-14,
0.35 ppm). Similarly, in the 13C-NMR spectrum, only C-11,
C-12, and C-13/C-14 exhibited a slight low-field shift (6, 6, and 14 ppm, respectively), and no change in the multiplicity of any signal
could be observed. Because MS indicated a 16 mass unit incrementthus
suggesting the introduction of an extra oxygen atomthe formation of
an N-oxy-derivative 5 (fig. 6) was strongly
suggested, which was entirely confirmed by comparison with an authentic
specimen.
The second product (18 mg) obtained from the incubation of DAT with
M. isabellina was also characterized by 1H- and
13C-NMR. All signals for hydrogens were present, but low
field shifted, especially those corresponding to one of the aromatic
hydrogens (H-3 or H-6, = 0.45 ppm). Most of the
13C signals of aromatic carbons were similarly shifted,
together with the 7-CH3 group ( = 7.6 ppm), whereas
the ethoxyamino side-chain signals were nearly unchanged. MS (CI,
NH3) exhibited a significant
[MNH4]+ signal at m/z 335, corresponding to a molecular weight of 317 (i.e., 80 mass
units higher than DAT). The presumed structure of a sulfate ester
6 (fig. 6) was confirmed by comparison with an authentic
sample [obtained by treatment of DAT with chlorosulfonic acid (16) or
amidosulfonic acid in pyridine (15)]. The sulfate ester was probably
formed in a higher amount during the incubation, but it was slowly
hydrolyzed during the chromatographic purification step.
After 7 days incubation with A. elegans, DAT (150 mg) was
partially converted (fig. 5) to a major polar product (fig.
7) that was extracted from the incubation medium, at
alkaline pH, with organic solvent and separated from residual substrate
by silica gel chromatography, thus affording 66 mg of metabolite
7 (fig. 6), the structure of which was determined by
spectroscopic and enzymatic methods. The molecular formula was
suggested as C20H33NO7, from the
CI-mass spectral data (MH+ at m/z 400, 100%),
with the most important fragment at m/z 238 (DAT + H+, 28%). The increase in molecular weight was confirmed
by 1H- and 13C-NMR spectra and
1H-13C-heterocorrelation spectra, which
characteristically indicated the presence of an additional
C6-glycosyl unit. The assignments listed in table
2 were highly indicative of an aryl glycoside derivative
and strongly suggested a -hexopyranoside conjugation. The
1H-NMR signals (table 2) of the aromatic part of the DAT
moiety were slightly deshielded (by 0.1-0.2 ppm), whereas the signals of the ethoxyamino side chain were shifted (by 0.1-0.6 ppm) to lower
frequencies. Signals of H-6 (2 dd) and H-1 of the glycosyl moiety
were well individualized, whereas H-2 to H-5 protons were observed in
the expected 3.48-3.64 ppm range and poorly separated. The assignment
of sugar protons was facilitated by the corresponding two-dimensional
(1H-1H-correlated) spectrum (fig.
8). A set of 17 signals was observed in the
13C-NMR spectrum (table 2), all of which could be assigned
by comparison with DAT and
1H-13C-heterocorrelation spectra. The
13C chemical shifts observed for the sugar moiety were in
agreement with an aryl -glycopyranoside structure (21, 25-30).
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Fig. 8.
2.5-5 ppm region of the
two-dimensional-(1H-1H-correlated) NMR
spectrum (D2O, 500 MHz) of the glycosylated metabolite
7.
|
|
Current assignments of - or -aryl glucoside structures are
generally based on the chemical shift and the characteristic coupling
constant of the anomeric proton of the sugar, seen as a doublet at
5.0-5.7 ppm. A large (7-8 Hz) coupling constant with H-2 is
indicative of the ax-ax coupling of a
-glucopyranoside, whereas a small (3-4 Hz) constant corresponds to
the ax-eq coupling of an -glucopyranoside. The
anomeric proton of metabolite 7 was clearly identified at
4.96 ppm, corresponding to a -glucoside, butdepending on the
concentration and the solvent (D2O or
CD3OD)the H-1 signal was obviously more complicated than
a simple doublet (fig. 9).
Surprisingly, when authentic aryl--D-glucopyranosides
(phenyl--D-glucopyranoside,
4-nitrophenyl--D-glucopyranoside, salicine) were
analyzed in identical conditions, similar patterns of the H-1 signal
were observed, with symmetrical or unsymmetrical features. On the
contrary, the H-1 signal of arbutin (another
aryl--D-glucopyranoside) appeared in D2O as
a simple doublet at 5.04 ppm (J = 7.6 Hz). These
unusual spectral patterns can be readily interpreted and simulated as
the X part of XAA'A' or XAA'B signals, wherein the H-1'
anomeric proton (X), coupled with H-2' (A), is also weakly coupled
(4J coupling = 0.5 to 1 Hz) with H-3'
(A') and eventually with H-5' (A' or B), with a trans
diaxial large coupling constant (9-10 Hz) existing between H-2' and
H-3', situated at very close chemical shifts. Such features introduce
a second order appearance into the H-1' (X) signal with a composition
rays pattern strongly depending on very slight differences in chemical
shifts of protons H-2' and H-3', whereas the symmetry of the system
is itself depending on the chemical shift difference of the
5'-proton, explaining the concentration or solvent effects. The use
of pyridine or dimethylsulfoxide as solvent and/or higher resolution
(500 MHz) spectra did not introduce sufficient separation between
H-2' and H-3' signals to get the expected simplified doublet quoted
in the literature. In NMR simulations, best fittings were effectively
obtained for chemical shifts in a 0.01 ppm range, as observed for
protons H-2', H-3', and H-5', a [H-1'-H-2'] coupling
constant of 7.5 Hz, corresponding to the usual value admitted for
-glucopyranosides, and a [H-2-H-3] coupling constant of ~9 Hz
(fig. 9).
The nature and stereochemistry of the glycosyl residue were entirely
confirmed by enzymatic treatment with - or -glucosidases. Only
almond -glucosidase supported hydrolysis of the metabolite (fig.
10), thus releasing equivalent amounts of DAT
(2) and D-glucose, respectively, estimated by
reversed-phase HPLC and specific enzymic determination. Enzymatic
hydrolysis was slow, requiring ~30 hr, compared with 15 min for
4-nitrophenyl--D-glucopyranoside in similar conditions.
No significant hydrolysis could be detected using yeast -glucosidase
(fig. 10). -Galactosidase from A. niger was similarly
ineffective, and <10% hydrolysis was observed after 32 hr with
E. coli -galactosidase.
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Fig. 10.
D-Glucose (determined by
enzymic oxidation) and DAT (determined by HPLC) released by enzymic
hydrolysis of the glycosylated metabolite 7 (1.25 µmol),
obtained from A. elegans (using almond -glucosidase;  ,
D-glucose;  , DAT; and using Baker's yeast
-glucosidase:  , D-glucose).
|
|
Similar results were obtained for the metabolization of thymoxamine or
DAT by M. hiemalis or M. janssenii. The major
product (60% and 30% respectively, in 7 days) was identical to the
-D-glucopyranoside 7; but, a minor product
(~5%) was simultaneously produced and identified as DMAT
(3), by HPLC and TLC comparison with the authentic compound.
To increase the production of the glycoside, or to shift it to the
production of a glucuronide (one of the currently observed animal
metabolites), D-glucose or D-glucuronic acid
was fed, together with DAT, in the incubation media, either directly to
the culture (after resuming of growth) or to a suspension of the washed
grown biomass in a 0.05 M phosphate buffer (pH 7). The results (table 3) showed a marked difference in the behavior of
A. elegans and M. hiemalis. Formation of the
glucoside was suppressed in both strains when incubated in buffer, but
glucose was able to restore it at a nearly normal level only in
M. hiemalis. In both strains, any attempt to obtain a new
glucuronide-like metabolite by glucuronic acid addition was
unsuccessful: moreover, glucuronic acid suppressed the formation of the
glucoside in A. elegans, but was without effect in M. hiemalis.
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|
TABLE 3
Formation (expressed in % of substrate converted) of
DAT--D-glucopyranoside 7 by A.
elegans and M. hiemalis in various incubation
conditions (DAT 0.5 g/liter, 7 days, 27°C)
|
|
It was interesting to know whether this aryl glucoside formation was a
general reaction, and a series of simple phenolic compounds 8-11 structurally related to DAT (fig. 11)
were incubated in the usual conditions with A. elegans.
Among them, only thymohydroquinone 10 was partially
(~10%) converted to a 4:6 mixture of unseparated 1- and
4--D-glucopyranosides, identified in the mixture by
1H- and 13C-NMR, and comparison of their
13C-NMR data with those previously described for the same
glucosides extracted from Geum japonicum (40).
DAT-N-oxide 5, a putative animal metabolite that
was isolated in significant amounts from incubations of DAT with
M. isabellina (see previous data) was not detectable in
incubations with other strains. In a limited screening, when
DAT-N-oxide itself was incubated in the usual conditions
with 14 fungal strains, a slow reduction to DAT was essentially
observed. However, in a few cases (Curvularia lunata, Aspergillus
candidus, or M. isabellina) thymohydroquinone
10, without any trace of the corresponding quinone
4,2 was transiently formed (up
to 20% in 4 days; fig. 12).
| |
Discussion |
Fair similarities could be found in the metabolism of thymoxamine
by fungal microorganisms when compared with animal metabolism. First,
enzymic hydrolysis of the phenyl acetate ester group by most
microorganisms (but not all) is similar to that found in animals, thus
emphasizing again that DAT is probably the effective active drug.
Second, two among the major known animal metabolites, DMAT and a phase
II sulfoconjugated derivative of DAT, are also produced by some strains
(M. isabellina and M. rouxii), whereas a new
conjugation product (DAT--D-glucoside) is produced by
several other strains (A. elegans, M. janssenii, or M. hiemalis). This is another illustration of the variety and
versatility of metabolic detoxification pathways found in such
microorganisms that can be advantageously used in model metabolic
studies.
Glucoside Formation.
Although not a general one, the glucosylation of phenolic derivatives
by, inter alia, A. elegans, M. janssenii, or M. hiemalis seems to be relatively common (24-29), compared with the rare examples reported for the glycosylation of aliphatic alcohols or carboxylic acids (23, 30). However, with the same A. elegans strain, for example, such a phase II metabolism is not the rule for all phenolic compounds, because only a unique phenol related to thymoxamine (thymohydroquinone) can be glucosylated. Whereas the nature of the
sugar can differ slightly [e.g. in the 4-O-methylglucosides formed by
Beauveria bassiana (24, 25, 28)], glucuronides are found
rarely in mould metabolism (21, 22) and could not be found as
thymoxamine metabolites, even by using a M. rouxii strain
that is known to produce glucuronic acid-containing polymers (41).
Similarly, but not unexpectedly, the use of the glucuronic acid
addition did not allow shifting of the formation of glucoside to
glucuronide. This kind of detoxification metabolite thus seems to be
rather specific for the mammalian metabolism of xenobiotics. However,
several known chemical methods (23, 42, 43) may allow preparation of
the glucuronide derivatives by simple oxidation of the corresponding
microbially formed glucosides.
Thymoquinone-Thymohydroquinone Formation.
The formation of thymoquinone 4 and/or thymohydroquinone
10 from thymoxamine may be put forward as possibly responsible for a few recent examples of acute hepatotoxicity promoted
by higher doses of ingested thymoxamine (Uroalpha) (44-48). These
compounds have been detected in low amounts as human urinary metabolites (16). Quinones are generally considered to be able to exert
highly cytotoxic effects through two main mechanisms (49, 50):
i) their capacity (shared with hydroquinones) to produce
reactive semiquinone radicals that can subsequently enter redox cycles
with molecular oxygen, leading to the formation of reactive oxygen
radicals (51, 52); and ii) their electrophilicity, which
enables them to form adducts to cellular constituents (mainly thiols)
(53). These properties are shared by a number of quinonoid compounds
(benzo-, naphtho-, and anthraquinones) and the more complex quinone
derivatives eventually used are anticancer agents (51, 54-56).
Although it is possible to visualize an oxidative metabolic
dealkylating splitting of the ethoxyamino side chain of DAT by microsomal enzymes, such an oxidation has never been observed in our
microbial systems. The intermediate formation of an N-oxide, a classical microsomal metabolite (57), may allow a different explanation of the formation of the quinone (or hydroquinone), through
a facilitated -elimination process on DAT-N-oxide
5 (fig. 13). Such an
elimination reaction would either give the hydroquinone, which could be
then oxidized to the quinone (pathway a), or directly afford the
quinone if combined, in a concerted reaction, with an hydride
acceptor (pathway b).
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|
Fig. 13.
Mechanisms for the splitting of the
ethoxyamino side chain of DAT, through oxidative -elimination in
DAT-N-oxide and formation of thymohydroquinone
10 and/or thymoquinone 4.
|
|
DAT-N-oxide is more generally reduced by our microorganisms.
However, the transient formation of thymohydroquinone (not
thymoquinone) in a few cases (see fig. 12) substantiates a
-elimination mechanism through pathway a, which might also be
operative in animal microsomes, followed by oxidation to thymoquinone
and possibly explain some of the toxicity accidents.
The high potential of certain fungal strains to produce not only
primary ("phase I") metabolites, but also sulfo- and
glycoconjugated ("phase II") metabolites of drugs, is illustrated
again by the results obtained with thymoxamine. The more general
formation of glucoside derivatives, in place of the glucurono
derivatives usually formed in animals, characterizes fungal metabolism.
Nevertheless, formation of such products may be of great interest for
analytical purposes and for new biologically active compound
preparation. One of the phase I metabolites observed,
DAT-N-oxide, may be at the origin of the formation of small
amounts of thymoquinone and thus explain some toxicity accidents caused
by high doses of the drug.
This study was supported by the Institut de Recherches
Chimiques et Biologiques Appliquées (Vicq, France), Laboratoires
Fournier (Dijon, France), and partially by an European Communities
Programme "Human Capital and Mobility: Biooxygenations" (Contract
ERBCHRXCT930259). This work was completed by C. Moussa in partial
fulfillment of her Doctorate of Science degree.
| 1.
|
A. T. Birmingham and
J. Szolcsanyi:
Competitive blockade of adrenergic receptors and histamine receptors by thymoxamine.
J. Pharm. Pharmacol.
17,
449-458 (1965)[Medline].
|
| 2.
|
L. Fawke:
Is thymoxamine a specific alpha-adrenoceptor blocking agent?
Br. J. Pharmacol.
44,
362P-363P (1972)[Medline].
|
| 3.
|
G. M. Drew:
Effects of alpha-adrenoceptor agonists and antagonists on pre- and postsynaptically located alpha-adrenoceptors.
Eur. J. Pharmacol.
36,
313-320 (1976)[Medline].
|
| 4.
|
M. H. Creuzet,
G. Prat,
A. Malek,
F. Fauran, and
J. Roquebert:
In vitro and in vivo -blocking activity of thymoxamine and its two metabolites.
J. Pharm. Pharmacol.
32,
209-213 (1980)[Medline].
|
| 5.
|
R. W. Foster:
The nature of the adrenergic receptors of the trachea of the guinea pig.
J. Pharm. Pharmacol.
18,
1-12 (1966)[Medline].
|
| 6.
|
M. Poirier,
J. P. Riffaud,
J. Y. Lacolle, and
C. Dupont:
Effects of five alpha-blockers on the hypogastric nerve stimulation of the canine lower urinary tract.
J. Urol.
140,
165-167 (1988)[Medline].
|
| 7.
| J. P. Riffaud: Recherche biochimique et pharmacologique de
nouveaux alpha-bloquants urosélectifs. J.A.M.A. (Suppl.
French Ed.) 9-12 (Jan. 1992).
|
| 8.
|
K. Watanabe,
Y. Hayashi,
K. Ikeda, and
H. Ohnishi:
Effect of moxisylyte on the lower urinary tract. 1. Effect on the isolated rabbit urethra and bladder.
Folia Pharmacol. Jpn.
97,
145-151 (1991).
|
| 9.
|
K. Watanabe,
Y. Hayashi,
K. Ikeda, and
H. Ohnishi:
Effect of moxisylyte on the lower urinary tract. 2. Effect on the urethra in anesthetized dogs.
Folia Pharmacol. Jpn.
97,
153-165 (1991).
|
| 10.
|
A. Zaroli,
S. Bernasconi, and
P. Bossi:
Effet d'un medicament alpha-lytique (thymoxamine) sur le profil des pressions de l'urètre.
Arch. Ital. Neurol. Nefrol.
51,
281-289 (1979).
|
| 11.
|
C. Feniou,
B. Neau,
G. Prat,
C. Cheze,
F. Fauran, and
J. Roquebert:
Metabolism of thymoxamine: identification of metabolites in rat.
J. Pharm. Pharmacol.
32,
104-107 (1980)[Medline].
|
| 12.
|
F. Nielsen-Kudsk,
P. Jakobsen, and
I. Magnussen:
Plasma induced biotransformation of thymoxamine and its kinetics.
Acta Pharmacol. Toxicol.
47,
11-16 (1980)[Medline].
|
| 13.
|
K.-O. Vollmer,
B. Liedtke,
A. Poisson,
A. VonHodenberg, and
W. Steinbrecher:
Metabolism of thymoxamine. I. Studies with 14C-thymoxamine in rats.
Eur. J. Drug Metab. Pharmacokinet.
10,
61-69 (1985)[Medline].
|
| 14.
|
K.-O. Vollmer and
A. Poisson:
Metabolism of thymoxamine: II. Studies with 14C-thymoxamine in man.
Eur. J. Drug Metab. Pharmacokinet.
10,
71-76 (1985)[Medline].
|
| 15.
|
K.-O. Vollmer and
A. VonHodenberg:
Metabolism of thymoxamine. III. Structure elucidation of the metabolites and interspecies comparison.
Eur. J. Drug Metab. Pharmacokinet.
10,
139-145 (1985)[Medline].
|
| 16.
|
P. Duchene,
C. Bernouillet,
M. Bromet-Petit,
J. Mosser,
C. Feniou,
D. Gaudin, and
H. Virelizier:
Metabolism of 14C-thymoxamine in rat and man.
Xenobiotica
18,
919-928 (1988)[Medline].
|
| 17.
|
C. Marquer,
J. H. Trouvin,
J. Y. Lacolle,
C. Dupont, and
C. Jacquot:
Pharmacokinetics of a prodrug thymoxamine: dose-dependence of the metabolite ratio in healthy subjects.
Eur. J. Drug Metab. Pharmacokinet.
16,
183-188 (1991)[Medline].
|
| 18.
|
R. V. Smith and
J. P. Rosazza:
Microbial models of mammalian metabolism. Aromatic hydroxylation.
Arch. Biochem. Biophys.
161,
551-558 (1974)[Medline].
|
| 19.
|
R. V. Smith and
J. P. Rosazza:
Microbial models of mammalian metabolism.
J. Pharm. Sci.
11,
1737-1759 (1975).
|
| 20.
|
R. J. P. Cannell,
A. R. Knaggs,
M. J. Dawson,
G. R. Manchee,
P. J. Eddershaw,
I. Waterhouse,
D. R. Sutherland,
G. D. Bowers, and
P. J. Sidebottom:
Microbial biotransformation of the angiotensin II antagonist GR117289 by Streptomyces rimosus to identify a mammalian metabolite.
Drug Metab. Dispos.
23,
724-729 (1995)[Abstract].
|
| 21.
|
C. E. Cerniglia,
J. P. Freeman, and
R. K. Mitchum:
Glucuronide and sulfate conjugation in the fungal metabolism of aromatic hydrocarbons.
Appl. Environ. Microbiol.
43,
1070-1075 (1982)[Abstract/Free Full Text].
|
| 22.
|
T. S. Chen,
L. So,
R. White, and
R. L. Monaghan:
Microbial hydroxylation and glucuronidation of the angiotensin-II (AII) receptor antagonist MK-954.
J. Antibiot.
46,
131-134 (1993)[Medline].
|
| 23.
| K. Petzoldt, K. Kieslich, and H. Steinbeck (Schering AG):
Oestratriene 3-beta glucosides preparation by adding 3-hydroxy steroid
to a carbohydrate-containing fungal culture medium. German Patent
2,326,084 (19 May 1973), C.A. 82, 86566s (1975).
|
| 24.
|
K. Kieslich,
H.-J. Vidic,
K. Petzoldt, and
G. A. Hoyer:
Microbiologische Umwandlungen nichsteroider Strukturen. VII. Mikrobielle Glucosidierung einer phenolischen Hydroxylgruppe.
Chem. Ber.
109,
2259-2265 (1976).
|
| 25.
| G. Neef, U. Eder, K. Petzoldt, A. Seeger, and P. Wieglepp:
Microbial hydroxylations of -carboline derivatives. J. Chem. Soc. Chem. Commun. 366 (1982).
|
| 26.
|
M. S. Raju,
G. S. Wu,
A. Gard, and
J. P. Rosazza:
Microbial transformations of natural antitumor agents. 20. Glucosylation of viridicatum toxin.
J. Natl. Prod.
45,
321-327 (1982).
|
| 27.
|
H. Kamimura:
Conversion of zearalenone to zearalenone glycoside by Rhizopus sp.
Appl. Environ. Microbiol.
52,
515-519 (1986)[Abstract/Free Full Text].
|
| 28.
|
B. Vigne,
A. Archelas,
J.-D. Fourneron, and
R. Furstoss:
Microbial transformations. Part 4(1). Regioselective para hydroxylation of aromatic rings by the fungus Beauveria sulfurescens. The metabolism of isopropyl N-phenyl carbamate (Propham®).
Tetrahedron
42,
2451-2456 (1986).
|
| 29.
|
S. ElSharkawy and
Y. J. AbulHajj:
Microbial transformation of zearalenone. I. Formation of zearalenone-4-O--glucoside.
J. Natl. Prod.
50,
520-521 (1987).
|
| 30.
|
M. Hezari and
P. J. Davis:
Microbial models of mammalian metabolism. Furosemide glucoside formation using the fungus Cunninghamella elegans.
Drug Metab. Dispos.
21,
259-267 (1993)[Abstract].
|
| 31.
|
S. H. ElSharkawy and
Y. J. AbulHajj:
Microbial transformation of zearalenone. 2. Reduction, hydroxylation and methylation products.
J. Org. Chem.
53,
515-519 (1988).
|
| 32.
| S. H. ElSharkawy: Microbial transformation of zearalenone.
III. Formation of 2,4-O--diglucoside. Acta Pharm.
Jugosl. 39, 303-310 (1989).
|
| 33.
|
S. H. ElSharkawy,
M. I. Selim,
M. S. Afifi, and
F. T. Halaweish:
Microbial transformation of zearalenone to a zearalenone sulfate.
Appl. Environ. Microbiol.
57,
549-552 (1991)[Abstract/Free Full Text].
|
| 34.
|
A. Ibrahim and
Y. J. AbulHajj:
Microbial transformation of flavonoids: sulfation of 5-hydroxyflavone by Streptomyces fulvissimus.
Appl. Environ. Microbiol.
55,
3140-3142 (1989)[Abstract/Free Full Text].
|
| 35.
|
B. C. Foster,
B. H. Thomas,
J. Zamecnik,
B. A. Dawson,
D. L. Wilson,
R. Duhaime,
G. Solomonraj,
I. J. McGilveray, and
B. A. Lodge:
Aromatic hydroxylation and sulfation of phenazopyridine by Cunninghamella echinulata.
Can. J. Microbiol.
37,
504-508 (1991).
|
| 36.
|
D. Liotta,
J. Arbiser,
J. W. Short, and
M. Saindane:
A simple, inexpensive procedure for the large scale production of alkyl quinones.
J. Org. Chem.
48,
2932-2933 (1983).
|
| 37.
|
R. D. Stolow,
P. M. McDonagh, and
M. M. Bonaventura:
Conformational studies. VI. Intramolecular hydrogen bonding in non-chair conformations of cis,cis,cis-2,5-dialkyl-1,4-cyclohexanediols.
J. Am. Chem. Soc.
86,
2165-2170 (1964).
|
| 38.
| K. Mori, M. Tominaga, T. Takigawa, and M. Matsui: A mild
transesterification method. Synthesis 790-791 (1973).
|
| 39.
|
E. Zavarin and
A. B. Anderson:
Extractive components from incense-cedar heartwood (Libocedrus decurrens Torrey). II. Occurrence and synthesis of p-methoxythymol and p-methoxycarvacrol, two new phenolic compounds.
J. Org. Chem.
20,
443-447 (1955).
|
| 40.
|
S. Shigenaga,
I. Kouno, and
N. Kawano:
Triterpenoids and glycosides from Geum japonicum.
Phytochemistry
24,
115-118 (1985).
|
| 41.
|
A. Flores-Carreon and
S. Bartnicki-Garcia:
Polyuronide biosynthesis by cell free extracts of Mucor rouxii.
J. Gen. Microbiol.
128,
2023-2027 (1982)[Abstract/Free Full Text].
|
| 42.
|
C. A. Marsh and
G. A. Levvy:
The synthesis of aryl glycosiduronic acids.
Biochem. J.
68,
617-621 (1958).
|
| 43.
|
N. J. Davis and
S. L. Flitsch:
Selective oxidation of monosaccharide derivatives to uronic acids.
Tetrahedron Lett.
34,
1181-1184 (1993).
|
| 44.
|
T. Vial,
N. Stremdoerfer,
L. Ollivier,
B. Veyre,
G. Goujon,
M. Pelletier, and
T. Fontanges:
Un cas d'hépatite aiguë au chlorhydrate de moxisylyte.
Gastroenterol. Clin. Biol.
15,
95-96 (1991)[Medline].
|
| 45.
|
D. Larrey:
Découverte tardive de l'hépatotoxicité des médicaments.
Gastroenterol. Clin. Biol.
15,
875 (1991)[Medline].
|
| 46.
|
A. Levy-Bruhl,
J. Germanaud,
D. Gargot, and
J.-L. Legoux:
Une hépatite cytolytique au moxisylyte?
Gastroenterol. Clin. Biol.
15,
979 (1991)[Medline].
|
| 47.
|
G. Mignot,
G. Dumas,
R. M. Chichmanian, and
A. Spreux:
Moxisylyte: atteinte hépatique. Un cas avec réadministration positive.
Gastroenterol. Clin. Biol.
15,
979-980 (1991).
|
| 48.
|
V. Ratziu,
T. Poynard,
H. Sabah,
S. Naveau, and
J.-C. Chaput:
Hépatotoxicité du chlorhydrate de moxisylyte: un nouveau cas.
Gastroenterol. Clin. Biol.
15,
980 (1991)[Medline].
|
| 49.
|
K. Öllinger and
A. Brunmark:
Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes.
J. Biol. Chem.
266,
21496-21503 (1991)[Abstract/Free Full Text].
|
| 50.
|
P. J. O'Brien:
Molecular mechanisms of quinone cytotoxicity.
Chem.-Biol. Interact.
67,
129-138 (1991).
|
| 51.
|
S. A. Akman,
M. Dietrich,
R. Chlebowski,
P. Limberg, and
J. B. Block:
Modulation of cytotoxicity of menadione sodium bisulfite versus leukemia L1210 by the acid-soluble thiol pool.
Cancer Res.
45,
5257-5262 (1985)[Abstract/Free Full Text].
|
| 52.
|
C. R. Stubberfield and
G. M. Cohen:
Interconversion of NAD(H) to NADP(H). A cellular response to quinone-induced oxidative stress in isolated hepatocytes.
Biochem. Pharmacol.
38,
2631-2637 (1989)[Medline].
|
| 53.
|
L. Rossi,
G. A. Moore,
S. Orrenius, and
P. J. O'Brien:
Quinone toxicity in hepatocytes without oxidative stress.
Arch. Biochem. Biophys.
251,
25-35 (1986)[Medline].
|
| 54.
|
P. Workman:
Enzyme-directed bioreductive drug development revisited: a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase.
Oncol. Res.
6,
461-475 (1994)[Medline].
|
| 55.
|
H. D. Beall,
A. M. Murphy,
D. Siegel,
R. H. J. Hargreaves,
J. Butler, and
D. Ross:
Nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase (DT-diaphorase) as a target for bioreductive antitumor quinones: quinone cytotoxicity and selectivity in human lung and breast cancer cell lines.
Mol. Pharmacol.
48,
499-504 (1995)[Abstract].
|
| 56.
|
Z. Djuric,
T. H. Corbett,
F. A. Valeriote,
L. K. Heilbrun, and
L. H. Baker:
Detoxification ability and toxicity of quinones in mouse and human cell lines used for anticancer drug screening.
Cancer Chemother. Pharmacol.
36,
20-26 (1995)[Medline].
|
| 57.
|
J. W. Gorrod and
L. A. Damani (eds.):
Biological oxidation of nitrogen in organic molecules. In
"Ellis Horwood Health Science Series" (A. Wiseman, ed.), pp. 445. Ellis Horwood & VCH, Chichester, 1985.
|
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Abstract
Introduction
Abstract
-2
