Department of Toxicology, University of Tuebingen (U.M., U.B.-P.),
Tuebingen, Germany; and Department of Analytics, Boehringer Ingelheim
Pharma, Biberach, Germany (H.W.)
The antiallergic drug ketotifen is chiral due to a nonplanar
seven-membered ring containing a keto group. Earlier studies have
revealed glucuronidation at the tertiary amino group as a major
metabolic pathway in humans. Chemical synthesis of glucuronides from
racemic ketotifen now led to four isomers separable by HPLC of which
two each could be ascribed to (R)-(+)- and
(S)-(
)-ketotifen by synthesis from the enantiomers.
According to 1H NMR analysis of the
(S)-ketotifen N-glucuronides, the
conformation of the piperidylidene ring differs between the two
isomers. Enzymatic hydrolysis with Escherichia coli
-glucuronidase proceeded at a lower rate with the slower eluting
(S)-ketotifen glucuronide than with the three other
isomers. On incubation of the ketotifen enantiomers (0.5-200 µM)
with human liver microsomes in the presence of UDP-glucuronic acid and
Triton X-100, the N-glucuronides of (R)-ketotifen were produced with an apparent
KM 15 µM and
Vmax 470 pmol/min/mg protein. The two
(S)-ketotifen glucuronides were formed by two-enzyme
kinetics with KM1 1.3 µM and
KM2 92 µM and Vmax values of 60 and 440 pmol/min/mg
protein. After ingestion of 1 mg of racemic ketotifen, 10 healthy
subjects excreted in urine 17 ± 5% of the dose in the form of
N-glucuronides. The (R)-ketotifen glucuronide isomers contributed one-sixth only, whereas the remainder consisted primarily of the (S)-ketotifen glucuronide
isomer, which eluted last. Differential hydrolysis or membrane
transport may be responsible for the discrepancy between
N-glucuronide isomer ratios in vitro and in vivo.
 |
Introduction |
Ketotifen
[(R,S)-4-(1-methyl-4-piperidylidene)-9,10-dihydro-4H-benzo[4,5]cyclohepta[1,2-b]thiophene-10-one]
is an oral H1 antihistamine used in the control
of asthma and other allergic conditions (Grant et al., 1990
). Its
central seven-membered ring is nonplanar, giving rise to chirality, and
enantiomers that differ in pharmacological potency have been separated
by formation of diastereomeric salts (Polívka et al., 1989).
Absorption of orally administered ketotifen was fast with maximal
plasma concentrations of about 1 to 2 nmol/liter 2 to 4 h after a
2-mg dose (Julien-Larose et al., 1983; Grahnén et al., 1992). It
was supposedly complete, but bioavailability was reported to be only
about 50% due to first-pass metabolism (Grant et al., 1990). Terminal
elimination half-life varied between 7 and 27 h (mean 12 h)
in the largest and most careful study (Grahnén et al., 1992). The
metabolic fate in humans of racemic ketotifen has been studied by
measuring metabolites produced in hepatocytes (Le Bigot et al., 1987),
occurring in plasma (Julien-Larose et al., 1983), and excreted in urine
(Guerret et al., 1981). Major biotransformation pathways were reduction
of the carbonyl group and glucuronidation at the tertiary amino group.
The formation of a quaternary ammonium glucuronide (Fig.
1) in human liver microsomes has been
studied in detail by Le Bigot et al. (1983). Using native microsomes,
they measured production rates conforming to two-enzyme kinetics,
whereas activation by Triton X-100 led to one-enzyme kinetics.
N-Glucuronidation of tertiary amines is a biotransformation
pathway largely restricted to humans and nonhuman primates. In a recent
review, Hawes (1998) listed 23 antihistamines, tricyclic antidepressants, neuroleptics, and related drugs for which quaternary ammonium glucuronides were measured in human urine. The drugs with the
highest percentages of the dose represented by
N-glucuronides were ketotifen (24%) and doxepin (23%). The
enzymatic basis of quaternary ammonium glucuronide formation has been
elucidated by the use of UDP-glucuronosyl transferases
(UGTs)1 expressed
in cell lines. Of the more than 30 UGTs studied, only human UGT1A3 and
1A4 proved able to catalyze the N-glucuronidation of
aliphatic tertiary amines (review by Green and Tephly, 1998). Although
the substrate spectrum was similar with the two enzymes, homogenates of
cells expressing UGT1A4 usually achieved higher conjugation rates
(Green et al., 1995, 1998; Green and Tephly, 1996); this also applied
to ketotifen glucuronidation. Kinetic studies resulted in apparent
KM values around or above 100 µM with
amitriptyline, chlorpromazine, and clozapine for UGT1A4 (Green et al.,
1995, 1998; Green and Tephly, 1996), whereas a 1.8-fold higher value
was obtained with amitriptyline for UGT1A3 (Green et al., 1998).
However, the conjugation of amitriptyline in human liver microsomes
exhibited a concentration dependence compatible with two-enzyme
kinetics, with a high-affinity apparent KM
around 1 µM and a low-affinity component around 300 µM
(Breyer-Pfaff et al., 1997). Whereas the low-affinity
KM would be in agreement with that measured
for UGT1A3, the biphasic character of the kinetics can not be well
explained by the additional activity of UGT1A4, because a
KM ratio of 1.8 would not be discernible in
kinetic analysis. Thus, the data would argue in favor of UGT1A3 and 1A4 being enzymes that conjugate amitriptyline with a high
KM value, whereas no UGT has as yet been
expressed with a KM as low as it became
apparent in liver microsomes. The kinetics of diphenhydramine N-glucuronidation in liver microsomes also appeared to be
biphasic, both KM values being about 3-fold
those measured with amitriptyline (Breyer-Pfaff et al., 1997).
For the present investigation, ketotifen was chosen as the substrate
because of the relatively high percentage of the dose recovered as
N-glucuronide (see above) and because of the possibility to
compare the kinetic behavior of two enantiomers. It was expected that
two diastereomeric glucuronides would be produced from the racemate and
that their ratio in vivo would mirror the kinetics of their production
in vitro. Actually, two N-glucuronides originated from each
one of the enantiomers, the conjugation kinetics of the enantiomers
differed distinctly, and one of the isomers by far exceeded the three
others in quantity as urinary metabolites.
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Experimental Procedures |
Materials.
Human liver samples were kindly supplied by Dr. W. Lauchart, Department
of Surgery, University of Tuebingen. These were either samples from
livers excluded from transplantation for medical reasons or excess
normal tissue obtained on partial hepatectomy for tumor metastases.
They were cut into pieces of 5 to 10 g and stored at 80°C.
The pamoate salt of racemic ketotifen and (R)-(+)- and
(S)-()-ketotifen (free bases) were generously donated by
Novartis Pharma AG (Basel, Switzerland).
-Glucuronidase/arylsulfatase from Helix pomatia,
-glucuronidase from E. coli, and UDP-glucuronate were
purchased from Boehringer (Mannheim, Germany),
D-glucuronolactone from Sigma-Aldrich
(Deisenhofen, Germany), Isolute SPE (solid-phase extraction)
columns filled with 500 mg of the strongly acidic cation exchanger SCX
from ict (Bad Homburg, Germany), and HPLC grade acetonitrile
from E. Merck (Darmstadt, Germany). Rat -glucuronidase was prepared
from preputial glands of female rats (Tulsiani and Touster, 1978).
Synthesis of Ketotifen Glucuronides.
The procedure of Luo et al. (1992) was modified in the following way.
Racemic ketotifen (1 g) was liberated from the pamoate by extracting an
alkalinized suspension in water with 10 ml of toluene. In the toluene
solution, 1 g of freshly prepared
methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-
-D-glucopyranosuronate was dissolved and the solution was added to 20 ml of 0.5 M
NaHCO3. After 2 days of continuous stirring in an
atmosphere of nitrogen, the aqueous phase was replaced by a fresh
NaHCO3 solution and 1 g of glucuronic acid
derivative was added. This procedure was repeated after another 2 days,
and the third aqueous phase was sampled after 2 days. The combined
aqueous phases contained conjugate in which the carboxyl group had been
liberated by methyl ester hydrolysis. They were extracted three times
with 10 ml of diisopropyl ether, adjusted to pH 12.5 to remove
the protecting acetyl groups, extracted again with 3 × 10 ml of
diisopropyl ether, and extracted once more after adjustment to pH 7 (Luo et al., 1992). The aqueous solution was passed through a column
with 5 ml of C18-silica gel (Polygoprep 60-50
C18; Macherey-Nagel, Düren, Germany), which was washed with 8 ml of water/methanol (9:1, v/v) and eluted with 10 ml
of methanol/water (9:1). The residue of the eluate was purified by
thin-layer chromatography on two 20 × 20 cm sheets coated
with silica gel (Alugram Sil G/UV254;
Macherey-Nagel) in 1-butanol/acetic acid/water (4:1:1, v/v/v).
The UV-absorbing band at RF 0.35 was extracted with methanol and the extract evaporated under a stream of
nitrogen. Glucuronides of (R)- and (S)-ketotifen
were synthesized in the same way on a smaller scale, starting from 20 mg of drug and 0.1 g of glucuronic acid derivative in 1 ml of
toluene stirred with 2 ml of 0.5 M NaHCO3. The
molar quantities and synthesis yields were determined from
E300 E330 of
an aliquot in methanol, assuming that the molar absorption
difference was the same as that of ketotifen (10,500 cm
1M1). From
racemic ketotifen, 0.21% was recovered as N-glucuronides.
Microsome Preparation.
Liver homogenates were prepared with four volumes of buffer (250 mM
sucrose, 20 mM Tris-HCl, 5 mM EDTA, adjusted to pH 7.4 at 37°C) and
microsomes were obtained by fractionated centrifugation in the last
step at 85,000g for 1 h. They were washed by
resuspension in the same buffer followed by centrifugation. Pellets
were suspended in buffer (0.6 ml per gram of liver, resulting in about
20 mg protein/ml) and aliquots of 0.2 ml were frozen in liquid nitrogen and stored at 80°C. Protein was measured according to Lowry et al.
(1951) with BSA as standard.
Binding Experiments.
Binding of (R)- and (S)-ketotifen to microsomal
protein was determined by equilibrium dialysis (Brinkschulte and
Breyer-Pfaff, 1979) at 37°C in 57 mM Tris-HCl pH 8.0 containing 5 mM
MgCl2. The enantiomers (50 µM) were added to
one 1-ml half-cell, and the concentrations in both cells were measured
by HPLC after 2, 4, and 6 h. In experiments without microsomes,
equal concentrations in the two cells were achieved after 4 and 6 h. Therefore, dialysis time was fixed at 5 h. In experiments with
variable ketotifen concentrations (2-100 µM), the concentration of
microsomal protein was 0.5 mg/ml, and when the microsome quantity was
varied between 0.2 and 1 mg of protein/ml, the ketotifen concentration
was constant at 50 µM. Ketotifen was added to the microsome
suspension, and after dialysis was measured in both half-cells by HPLC
(see below). Samples were adjusted to 0.2 N
HClO4, after those with ketotifen added to 50 or
100 µM had been diluted with water 3- and 5-fold, respectively.
Glucuronidation Assay.
Standard incubation mixtures of 1 ml containing 57 mM Tris-HCl pH 8.0, 5 mM MgCl2, 2 mM UDP-glucuronate, 0.02% Triton
X-100, 0.5 to 200 µM (R)- or (S)-ketotifen, and
0.5 mg/ml microsomal protein were shaken for 25 min at 37°C. The
reaction was stopped by three extractions with 1 ml of
tert.-butyl methyl ether and one extraction with 1 ml of
n-hexane, care being taken to remove the protein interphase
with the organic layer. A weighed aliquot of the aqueous phase was
mixed with 10% of its volume of 2 N perchloric acid, centrifuged at
10,000 rpm after cooling on ice, and 0.5 ml was injected for HPLC. When
N-glucuronide mixtures were incubated with microsomes in the
absence of UDP-glucuronate, extracted, and analyzed by HPLC after
HClO4 addition, mean recoveries were 101 and
97%, respectively, in duplicate experiments with the glucuronides of
(R)- and (S)-ketotifen. Therefore, the quantities
measured were not corrected for recoveries. In kinetic measurements,
all incubations were carried out in duplicate and means were used for
calculations. The coefficients of variation within series with 10 to 11 different substrate concentrations varied between 4 and 11% (mean
7%).
HPLC Analyses.
For N-glucuronide analyses, samples of 0.5 ml were applied
to a 20 × 4.6 mm clean-up column with
C18-silica gel (Grom-Sil 120 ODS-4 HE, 11 µm;
Grom, Herrenberg, Germany) by pumping water (1 ml/min) for 2 min.
Samples were transferred to the analytical column (Prodigy 5 µm ODS
(3) 100 Å, 250 × 4.6 mm; Phenomenex, Hösbach, Germany) by
running the eluent (10 mM sodium phosphate buffer pH 6/acetonitrile,
78:22, v/v, 1 ml/min) for 3 min in the reverse direction. The clean-up
column was conditioned with methanol for 0.5 min and with water for 2 min. The eluate was monitored at 300 nm and data were registered by the
MT2 integration program (Kontron Instruments, München, Germany).
Quantities of individual glucuronides were calculated from peak areas
relative to external standards with known total concentrations. It was
assumed that the contribution of each glucuronide to total peak area
corresponded to its relative quantity.
For recording spectra during the HPLC run, the pump was stopped close
to the peak maximum and the absorption was measured between 240 and 340 nm with a UVIS-205 detector (Linear Instruments, Reno, NV).
For 1H NMR and mass spectrometric
investigations, about 0.4 µmol each of the two isomeric
(S)-ketotifen glucuronides were obtained from the
glucuronide mixture synthesized from racemic ketotifen. Quantities of
about 0.1 µmol were injected for HPLC, and eluates corresponding to
the first and fourth peaks were sampled separately. Eluates from 18 consecutive injections were combined and concentrated on
C18-silica gel (cartridges Bond Elut C18/OH; ict,
Frankfurt, Germany), from which the individual glucuronides were eluted
with methanol.
Free ketotifen was analyzed in a system consisting of a 6 × 5 mm
clean-up column (Polygoprep 60-50 C18; Macherey-Nagel) and a 250 × 4.6 mm analytical column filled with
C18-silica gel (Nucleosil 5 C18, Macherey-Nagel). Samples of 0.2 ml were
applied to the clean-up column with 30 mM perchloric acid adjusted to
pH 2.5 with NaOH, flow 1.5 ml/min, for 2 min. The eluent (10 mM
perchloric acid buffered to pH 2.5/acetonitrile 70:30, v/v, 1.2 ml/min)
was run in reverse direction for 1 min. The clean-up column was
conditioned with 30 mM perchloric acid, pH 2.5, for 2.5 min. Ketotifen
(RT 14 min) was detected at 300 nm and its
quantity was derived from the peak height relative to those of external standards.
Enzymatic Hydrolysis of Ketotifen N-Glucuronides.
Incubations with -glucuronidase/arylsulfatase from Helix
pomatia or -glucuronidase from rat were carried out in 0.1 N
sodium acetate/acetic acid pH 5, those with -glucuronidase from
E. coli in 75 mM potassium phosphate buffer pH 6.8. Rat and
E. coli glucuronidase activities were standardized with
phenolphthalein glucuronide (Stahl and Fishman, 1984) and specific
activities were found to be 1.4 and 0.16 U/mg protein, respectively, at
37°C (producer information on the E. coli enzyme: 20 U/mg
with 4-nitrophenyl--D-glucuronide as
substrate). Quantities used for measuring the rate of ketotifen N-glucuronide hydrolysis were 0.1 to 1 mU/ml of the E. coli enzyme and 5 to 10 mU/ml of that from rat. Samples were drawn
after 1 to 8 h at 37°C and analyzed by HPLC for unhydrolyzed glucuronides.
For confirmation of structure, an excess of the Helix
pomatia enzyme (0.1 ml) was incubated with 3.5 nmol
(S)- or (R,S)-ketotifen glucuronides for 12 h at 56°C and the liberated ketotifen was extracted from the
alkalinized incubate and measured by HPLC.
Ketotifen N-Glucuronidation In Vivo.
Five female and five male healthy volunteers took 1 capsule of
ketotifen (Zaditen; Sandoz) containing 1.38 mg of
(R,S)-ketotifen hydrogen fumarate (corresponding to 1 mg
free base) with 50 ml of water at bedtime. They collected urine in
three 8-h fractions that were analyzed for N-glucuronides by
a modification of the procedure of Fischer and Breyer-Pfaff (1995).
Urine (3-5 ml) was diluted with three volumes of 10 mM sodium
phosphate pH 4.0, the pH was adjusted to 4.0 and the solution was
passed within 13 min through an SCX column, which was subsequently
washed with 2 ml 10 mM sodium phosphate pH 4.0, 2 ml of water, and 4 ml
of methanol, and slowly eluted with 6 ml of the following mixture:
methanol/0.5 M ammonium acetate adjusted to pH 8.0 with ammonia and
containing 0.15% triethylamine (4:1, v/v). The eluate was concentrated
to about 60% under a stream of nitrogen and evaporated to dryness under reduced pressure at 35°C. The residue was dissolved in 1 ml of
0.2 N HClO4, of which 0.5 ml was analyzed by
HPLC. Recovery experiments were carried out with synthetic
(R,S)-ketotifen N-glucuronide at a concentration
of 1.5 nmol/ml urine. With fresh columns, the N-glucuronide
recovery was 88 ± 5%. Before use, columns had to be conditioned
by washing with 3 ml each of methanol, water, and 10 mM sodium
phosphate pH 4.0.
Mass Spectrometry.
Mass spectra of (R)- and (S)-ketotifen and of the
two glucuronides synthesized from (S)-ketotifen were
recorded with a TSQ 700 triple quadrupole mass spectrometer (Finnigan
MAT, San Jose, CA) and Finnigan acquisition software in the
electrospray ionization (ESI) and collision-induced dissociation (CID)
modes. For ESI and CID mass spectra the samples were dissolved in
methanol/water (9:1) to concentrations of 10 to 20 ng/µl. These
solutions were infused via a syringe pump at a flow rate of 1.5 µl/min into the ion source. The positive and negative ion
electrospray needle voltages were +4500 and 3500 V, respectively. The
temperature of the heated transfer capillary was set to 120°C. Sheath
gas was nitrogen. Spectra were acquired over the mass range 100 to 700 amu in 2 s. The acquisition time was 1 min and the recorded spectra were averaged.
In the CID mass spectrometry mode, argon was used as collision
gas. The collision cell pressure was 1.9 mtorr and the collision offset
voltage was 24 eV for the N-glucuronides and 28 eV for ketotifen. The scan range was between 20 and 495 amu, the scan time
1.5 s, and the acquisition time 2 min. The recorded spectra were averaged.
NMR Spectroscopy.
The 600-MHz 1H NMR spectra were recorded on a
Bruker DRX 600 at 303°K using Bruker standard software and pulse
programs. The 1H data were referenced to a trace
of tetramethylsilane (0.00 ppm). Samples were dissolved in
dimethyl sulfoxide-d6 to concentrations of 4 and
2 mg/0.5 ml for (R)- and (S)-ketotifen and about
0.05 mg/0.5 ml for the N-glucuronides. Hartman-Hahn
correlation spectroscopy (HH COSY) and Hartman-Hahn rotating-frame
Overhauser effect spectroscopy (HH ROESY) experiments were
included as two-dimensional measurements. Data acquisition was
performed with sweep widths of 4167 to 6250 (COSY) or 6250 to 11363 Hz
(ROESY) and relaxation delays of 5 to 6 or 7.5 to 11 s, respectively.
Calculations.
Kinetic parameters were calculated according to the Michaelis-Menten
equation for one or two enzymes by nonlinear least-squares regression
analysis (Fig. P; Biosoft, Cambridge, UK). Intrinsic clearance
(Clint) was calculated as
Vmax/KM.
Variations given are S.D.
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Results |
Synthesis and Characterization of Ketotifen
N-Glucuronides.
Under the conditions used for other antihistamines (Luo et al., 1992),
ketotifen could be derivatized at the tertiary amino group with the
production of quaternary ammonium salts, although at a poor yield.
Although two diastereomeric glucuronides had been expected to be formed
from the racemic drug, four reaction products were separated under
optimal HPLC conditions. Records of their UV spectra run during HPLC
were identical with each other and with that of ketotifen exhibiting
maxima at 300 nm. Incubation with -glucuronidase/arylsulfatase led
to the disappearance of all four peaks and the formation of ketotifen,
indicating that the peaks contained glucuronides. Syntheses starting
from (R)- or (S)-ketotifen resulted in the
glucuronides with RT 13.5 and 13.85 min
[designated (R)-GlucA and
(R)-GlucB] or those with
RT 12.4 and 14.5 min
[(S)-GlucA and
(S)-GlucB], respectively. Syntheses from racemic ketotifen yielded the N-glucuronides of the two
enantiomers in nearly equal quantities. On the other hand, the ratios
between the two isomers derived from either (R)- or
(S)-ketotifen were variable, but never exceeded 2 whether
the racemic drug or one of the enantiomers was derivatized.
To elucidate the difference between
(S)-GlucA and
(S)-GlucB, quantities sufficient for
instrumental analysis were collected from HPLC eluates. Their
1H NMR and mass spectra were compared with those
of (R)- and (S)-ketotifen, the latter two giving
identical spectra throughout. In the ESI positive ion mode, the mass
spectrum of ketotifen shows the expected [M+H]+
ion at m/z 310 as the base peak and in the
negative ion mode at m/z 308 as
[MH] (in accordance with the sum formula
C19H19NOS). The CID mass spectrum of the precursor ion m/z 310 shows
besides the base peak at m/z 96 [C6H10N]+
only two noticeable ions of low abundance, m/z
213 [M+H97(C6H11N)]+
and m/z 82 [C5H8N]+.
In the ESI mode, the two N-glucuronides exhibit nearly
identical mass spectra with molecular ions
[M+H]+ at m/z 486, sodium
adduct ions [M+Na]+ at
m/z 508, and [MH]
ions at m/z 484. These are compatible with the
sum formula
C25H27NO7S. The CID spectra of the two N-glucuronides show identical
fragmentation patterns. The precursor ions m/z
486 eliminate 176 amu [glucuronic acidH2O]
with formation of the base peak at m/z 310 [ketotifen+H]+. Additional fragment ions of low
abundance are m/z 159 [glucuronic acid2H2O+H]+,
m/z 131 [159CO]+,
m/z 113 [159HCOOH]+,
and m/z 96 [C6H10N]+.
1H NMR data are presented in Table
1. The proton signals of the
piperidylidene ring of ketotifen form two groups because the protons on the side directed toward the benzene ring (H-13 and H-14,
see Fig. 1) are more shielded and may underlie the magnetic field
produced by the ring current. The signals of axial (ax) and equatorial
(eq) protons of both groups exhibit identical coupling patterns and can
be assigned on the basis of their coupling constants and cross signals
in COSY. The eq protons at C-11 and C-14 are influenced by the magnetic
anisotropy of the exocyclic double bond and give signals upfield to
those of the axial protons. ROESY experiments demonstrate the close
steric proximity of H-3 on the thiophene ring to the two hydrogens at
C-11 by nearly equally intensive cross-peaks. On the other hand, those
between H-5 (and H-6) on the benzene ring and H-14ax and H-14eq are
weak (or very weak) and hardly exceed the H-3 to H-12eq signal in
intensity. This leads to the conclusion that the axes C-11 to C-12 and
C-14 to C-13 are not aligned parallel to the theoretical axis C-4a to
C-10, but rather at a right angle. The piperidylidene ring is
apparently present in a rigid chair form.
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TABLE 1
1H NMR assignment for (S)-ketotifen and its isomeric
N-glucuronides (S)-GlucA and (S)-GlucB
Spectra were recorded in d6-dimethyl sulfoxide. Chemical shifts
are in ppm relative to tetramethylsilane and coupling constants (in
parentheses) in Hz; coupling patterns: s, singlet; d, doublet; dd,
double doublet; ddd, double-double doublet; t, triplet; dt, double
triplet; m, multiplet.
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Very intense ROESY cross-peaks indicate a close proximity of H-8 and
H-9a, which should be in the plane of the benzene ring, whereas H-9b is
on the convex side of the tricyclic system. The keto group is oriented
in the thiophene ring plane.
(S)-GlucA exhibits small differences
from ketotifen with regard to the signals of the aromatic protons and
those of H-9a and H-9b. In the piperidylidene ring, the methylene
groups in -position to the quaternary nitrogen atom (H-12 and H-13)
are deshielded by 1.17 to 1.55 ppm and those in -position (H-11 and
H-14) by 0.20 to 0.71 ppm. Geminal (2J) and
vicinal ax-ax (3J) coupling constants of the
methylene groups exceed those of ketotifen, whereas the ax-eq and eq-eq
(3J) coupling constants are nearly the same. A
symmetrical chair or boat form can be excluded because according to the
equal intensity of the ROESY cross-peaks between H-3 and H-11eq on the
one hand and H-5 and H-14ax on the other, these distances should be
similar (Fig. 2A). A strong cross-peak is
also registered between H-1' and H-14ax, indicating a pseudoaxial or
isoclinal position of the glucuronosyl C-1' on the quaternary ring
nitrogen. A close proximity between H-1' and H-13eq is indicated by a
weak cross-peak; therefore, the plane of the glucuronosyl ring should
be oriented vertically to the benzene ring. The most probable
conformation of the piperidylidene ring is a twist form analogous to
that of cis-1-tert.-butyl-4-phthalimidocyclohexane
(Kellie and Riddell, 1974). In the present case, a twist form may be
further favored by the presence of a
sp2-hybridized carbon atom in the piperidylidene
ring.
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Fig. 2.
Structural formulae showing the
conformations of the (S)-ketotifen N-glucuronides
(S)-GlucA (A) and
(S)-GlucB (B).
Close steric proximities giving rise to cross signals in ROESY
experiments are indicated by dotted lines.
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In (S)-GlucB the deshielding effect of
the quaternary nitrogen amounts to 1.05 to 1.65 ppm in H-12 and H-13
and to 0.28 to 0.47 ppm in H-11 and H-14. An unusual finding is the
identity of the shifts of H-13ax and H-13eq, probably caused by the
ring torsion that removes H-13ax from the extra magnetic field produced by the ring current of the benzene ring. In comparison with
(S)-GlucA, H-1' and H-5' show upfield
shifts of 0.47 and 0.24 ppm, respectively (Table 1), and indicate the
influence of anisotropic magnetic fields in their neighborhood. As in
ketotifen, the ROESY experiment indicates nearly identical H-3 to
H-11ax and H-3 to H-11eq distances. In addition, the small internuclear
distance of H-1' and H-13(ax or eq) becomes apparent from a strong
cross-peak, and a weaker peak points to the proximity of H-1' and
H-14eq (Fig. 3). Therefore, C-1' should
be attached axially to the quaternary nitrogen and the piperidylidene
ring should be present in a distorted boat form. This is in accordance
with the 3J coupling constants that are increased
relative to those of ketotifen in ax-eq and eq-eq interactions. The
H-13ax to H-14ax constant is smaller, but 3J for
H-11ax to H12ax is larger and the geminal (2J)
values for H-11 and H-14 are distinctly larger. The glucuronosyl residue is assumed to be placed parallel to the plane of the
seven-membered ring on the convex side of the tricyclus (Fig. 2B).
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Fig. 3.
Two-dimensional NMR ROESY spectrum of the
ketotifen N-glucuronide (S)-GlucB.
Arrows point to cross-peaks providing decisive structural
information.
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Chemical Stability and Enzymatic Hydrolysis of Ketotifen
N-Glucuronides.
The synthetic glucuronides (S)-GlucA
and (S)-GlucB were not hydrolyzed on
heating to 56°C in 1 N sulfuric acid for 30 min nor did an
interconversion or a conversion to (R)-ketotifen
glucuronides take place. Also in neutral medium, the compounds were
sterically stable during 8 h at 37°C, and samples illuminated by
a neon tube for 24 h were not converted to isomers. No
racemization of the ketotifen moiety was observed on storage of the
glucuronides at 4°C in the dark for several months.
Partial hydrolysis by limiting quantities of -glucuronidase
from E. coli or rat usually was not linear with time. In
most cases, the rate was maximal within the first 1 or 2 h with
progressive slowing in the following 3 or 6 h; on the other hand,
hydrolysis rates increased linearly with enzyme concentrations.
Differential hydrolysis took place when the two
(S)-ketotifen glucuronides were incubated with E. coli -glucuronidase. As an example, Fig. 4B demonstrates the 26% hydrolysis of
(S)-GlucA within 1 h that is
increased to 42% up to 4 h, whereas the small fraction of
(S)-GlucB hydrolyzed became measurable
only after 4 h (7%). On the other hand, the two
(R)-ketotifen glucuronides were split at comparable rates
(Fig. 4A). -Glucuronidase from rat was about 40-fold less active
than the bacterial enzyme in N-glucuronide hydrolysis
relative to its activity toward phenolphthalein. Incubation of 5.8 µM
(S)-ketotifen glucuronides with 5.1 mU/ml for 2 h led
to 25 and 18% hydrolysis, respectively, of
(S)-GlucA and
(S)-GlucB, and little more reaction up
to 8 h. Under the same conditions,
(R)-GlucA and
(R)-GlucB were hydrolyzed by 9 and
21%, respectively, within 2 h.
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Fig. 4.
Time course of the hydrolysis of (R)- and
(S)-ketotifen N-glucuronides with -glucuronidase from E. coli.
A, (R)-ketotifen N-glucuronide (6.6 µM)
was incubated with 0.16 mU/ml of the enzyme (assayed with
phenolphthalein glucuronide); B, (S)-ketotifen
N-glucuronide (25 µM) was incubated with 0.32 mU/ml
enzyme.
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Ketotifen N-Glucuronidation in Liver Microsomes.
Because only substrate not bound nonspecifically to a microsomal
suspension is available for metabolism (Obach, 1997), the free
fractions of the ketotifen enantiomers were determined under the
conditions of microsomal incubations, but with omission of UDP-glucuronate and Triton X-100. A low degree of binding that was
independent of the protein concentration in the range 0.2 to 1 mg/ml
and of the drug concentration in the range 2 to 100 µM was
observed. The main free fraction of (R)-ketotifen was
91 ± 5% and that of the (S)-enantiomer 94 ± 6%
(n = 9). These fractions were used for correcting the
substrate concentrations added to microsomal incubates.
For in vitro glucuronidation experiments, optimal conditions were
determined as shown in Table 2.
Glucuronidation rates increased with pH, but values above 8.0 were not
tested. Small enhancements were observed on increasing the
UDP-glucuronic acid concentration above 2 mM. Addition of 0.01 or
0.02% Triton X-100 stimulated the reaction distinctly, but at 0.03%
the rates declined. Reaction rates increased linearly with protein
concentrations between 0.25 and 0.75 mg/ml, and they were constant with
time up to 40 min.
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TABLE 2
Influence of reaction conditions on the rates of (R)- and (S)-ketotifen
glucuronidation in human liver microsomes
Reaction rates are given relative to those under standard conditions
(pH 8.0, 2 mM UDP-glucuronic acid, and 0.02% Triton X-100) set to
1.00.
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On incubation of about 5 or 100 µM racemic ketotifen, all four
isomeric N-glucuronides were produced (Fig.
5). Their identities were confirmed by
recording their UV spectra and by enzymatic hydrolysis, which resulted
in the disappearance of the glucuronides and a recovery of 78% as
ketotifen. No peaks interfering with N-glucuronide analysis
were detected in control samples incubated without UDP-glucuronate.
(S)-Ketotifen glucuronides represented about two-thirds of
the total quantity with (S)-GlucB
exceeding (S)-GlucA 2- to 3-fold.
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Fig. 5.
HPLC record of the N-glucuronide mixture
produced on incubation of 4.8 µM racemic ketotifen with human liver
microsomes and UDP-glucuronic acid in the presence of Triton
X-100.
1: (S)-GlucA, 2:
(R)-GlucA, 3:
(R)-GlucB, 4:
(S)-GlucB. Total
N-glucuronide quantity was 1 µmol/ml and the ratio of
the individual conjugates 20:9:26:45.
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For kinetic measurements, (R)- and (S)-ketotifen
were incubated separately in the concentration range 0.5 to 200 µM.
No higher concentrations were used because in preliminary experiments
substrate inhibition had become apparent. N-Glucuronide
quantities produced from 400 µM (R)- or
(S)-ketotifen amounted to about two-thirds of those measured
with 200 µM substrate. In each sample obtained from one of the
enantiomers, two isomeric N-glucuronides were measured with
mean
(R)-GlucB/(R)-GlucA
ratios around 2 and
(S)-GlucB/(S)-GlucA ratios that increased with increasing substrate concentrations from
1.5-2 to 2.3-3.5. Kinetic calculations were based on the sums of the
two isomers.
The concentration dependence of production of N-glucuronides
from (S)-ketotifen in microsomes from four livers could be
depicted by Michaelis-Menten kinetics with two enzymes, one of them
exhibiting a very high affinity (KM1 1.3 µM, Table 3). This can be visualized by
Eadie-Hofstee plots (Fig. 6). Omission of
Triton X-100 from the incubation medium reduced
KM1, Vmax1, and
Vmax2 values in a comparative experiment
with HL 23 microsomes (Table 3), but kinetics remained biphasic (Fig.
6). Nonlinear regression coefficients were higher in all cases when
parameters were fitted to two-enzyme kinetics (Table 3) than to
one-enzyme kinetics. Apparent intrinsic clearances
(Vmax/KM) of
the high-affinity enzyme exceeded those of the low-affinity component,
on average 9-fold, in experiments with detergent.
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TABLE 3
Apparent kinetic parameters for the N-glucuronidation of (S)-ketotifen
in human liver microsomes
KM values are given in µM, Vmax
in pmol/min/mg protein, Vmax/KM
in µl/min/mg protein.
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Fig. 6.
Eadie-Hofstee plots of the kinetics of
N-glucuronide formation from (S)-ketotifen with microsomes from three
human liver samples.
HL 23 microsomes were incubated in the presence and absence of Triton
X-100, the others in its presence only. The lines were calculated from
the parameters for two-enzyme Michaelis-Menten kinetics listed in Table
3.
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(R)-Ketotifen N-glucuronidation kinetics were
more complex, because in incubations without Triton X-100 a
contribution of a high-affinity enzyme became apparent that in the
presence of Triton was not discernible in two of three microsomal
preparations (Table 4, Fig.
7). Parameters for two-enzyme kinetics
have to be regarded as estimates, because the differences between
KM1 and KM2
were too small for exact evaluations of the individual components. As
with (S)-ketotifen, the contribution of the high-affinity enzyme to total intrinsic clearance in the absence of Triton X-100 was
higher than that of the low-affinity enzyme. In experiments with
Triton, one-enzyme kinetics were applicable, resulting in a mean
KM value of 15 µM and, thus, intermediate
between those of the two components without detergent, and a very
similar intrinsic clearance in spite of a higher
Vmax value (Table 4). When intrinsic clearances in the presence of Triton were compared, their sum with
(S)-ketotifen as the substrate was on average about 50%
higher than the value for (R)-ketotifen.
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TABLE 4
Apparent kinetic parameters for the N-glucuronidation of (R)-ketotifen
in human liver microsomes with and without addition of Triton X-100
KM values are given in µM, Vmax
in pmol/min/mg protein, Vmax/KM
in µl/min/mg protein.
Parameters were calculated according to one- and two-enzyme
Michaelis-Menten kinetics.
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Fig. 7.
Eadie-Hofstee plots of the kinetics of
N-glucuronide formation from (R)-ketotifen with microsomes from two
human liver samples in the presence and absence of Triton
X-100.
Solid lines were calculated from the parameters for two-enzyme
Michaelis-Menten kinetics and dashed lines with those for one-enzyme
kinetics listed in Table 4. In the case of HL 18 with detergent,
one-enzyme kinetics only were applicable and are depicted by a solid
line.
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Ketotifen N-Glucuronidation in vivo.
After ingestion of 1 mg of racemic ketotifen, 10 healthy volunteers
excreted in urine all four N-glucuronide isomers, but (S)-GlucB exceeded the others by far
in quantity (Fig. 8, Table 5). Within 24 h, urinary
glucuronides corresponded to 17.3% of the dose on average, with about
7.6, 6.4, and 3.3% being excreted in the 0- to 8-, 8- to 16-, and 16- to 24-h intervals. This indicates that excretion was not complete after
24 h. The same conclusion can be drawn from the observation that
subject WG excreted a higher percentage (29.9%) under continuous
dosing with 1 mg ketotifen/day than in the acute experiment (22.9%,
Table 5). No significant correlation existed between the percentage of
the dose excreted as N-glucuronides and the urine volume.
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Fig. 8.
HPLC record of ketotifen N-glucuronides in
urine of volunteer BP collected 8 to 16 h after ingestion of 1 mg
of racemic ketotifen.
Of the extract from 4 ml of urine, an aliquot of 50% was injected.
Quantities found were (S)-GlucA (1) 0.11 nmol, (R)-GlucA + (R)-GlucB (2 + 3) 0.07 nmol, and
(S)-GlucB (4) 0.56 nmol.
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TABLE 5
Urinary excretion of ketotifen glucuronides by healthy subjects after
ingestion of 1 mg of racemic ketotifen
Percentages refer to the dose of 1 mg, not to those of the enantiomers.
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To probe chiral stability of ketotifen and its glucuronides, two
subjects (BP and MU) took 1 mg (R)-ketotifen as the free base. Their 24-h excretion of (R)-ketotifen glucuronides
amounted to 6 and 0.9% of the dose, and in addition, 3.3 and 0.8%,
respectively, were found as (S)-ketotifen glucuronides.
Thus, quaternary ammonium glucuronides excreted in urine were
mainly derived from (S)-ketotifen, of which a mean fraction of 26% was found as (S)-GlucB and 3%
as (S)-GlucA, whereas on average less
than 6% of the (R)-ketotifen dose was glucuronidated in
experiments with 1 mg of the racemic drug as well as with 1 mg of the
(R)-enantiomer alone.
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Discussion |
Diastereomeric quaternary ammonium glucuronides were
obtained by chemical synthesis or in vitro biosynthesis in human liver microsomes from racemic ketotifen. Unexpectedly, each one of the diastereomers occurred in two conformations that, according to experiments with the (S)-ketotifen derivatives, were not
interconverted at temperatures up to 37°C in neutral media nor at
56°C in acid. The same four isomeric N-glucuronides were
excreted by humans after ingestion of the racemic drug. Detailed
1H NMR analysis led to the conclusion that
differences in the conformation of the piperidylidene ring are the
basis for different arrangements of the glucuronosyl group relative to
the tricyclic system. As a six-membered saturated ring, the
piperidylidene ring may adopt chair as well as nonchair conformations
depending on the substituents (Kellie and Riddell, 1974). In ketotifen,
1H NMR data were in favor of a rigid chair
conformation, whereas in the N-glucuronide
(S)-GlucB the data pointed to a
distorted boat form, and in (S)-GlucA
to a twist form. As a consequence, the glucuronosyl moiety can be
assumed to be directed parallel to the tricyclus in
(S)-GlucB (Fig. 2B), and this may be
the reason for the relatively lipophilic character manifested by a
longer retention time in reversed phase HPLC than for
(S)-GlucA. In the latter, the
hydrophilic glucuronosyl group is less protected (Fig. 2A); this may
lead to faster elution (Fig. 5). Because of the poorer resolution
between the (R)-ketotifen N-glucuronide isomers, separate investigation was not possible. An alternative possibility for
the occurrence of isomers is the chiral character of the quaternary nitrogen in the N-glucuronides. However, the NMR data
allowed us to exclude that (S)-GlucA
and (S)-GlucB are diastereomers, because in this case one would expect nearly identical chemical shifts
and coupling constants for the piperidylidene ring protons.
An isomerism as it was revealed now has to our knowledge not been
described for other quaternary N-glucuronides. Two isomers were separated on HPLC of the tertiary 10-N-glucuronide of
the tricyclic neuroleptic olanzapine (Kassahun et al., 1997, 1998). The
nature of the isomerism has apparently not been elucidated.
The ketotifen N-glucuronides proved resistant toward acid
hydrolysis in accordance with the behavior of other drug-derived quaternary ammonium glucuronides (Breyer-Pfaff et al., 1990; Hawes, 1998). The rate of their enzymatic hydrolysis relative to that of
phenolphthalein glucuronide was highest with -glucuronidase from
E. coli. This has been observed with the glucuronides of amitriptyline and diphenhydramine (Fischer and Breyer-Pfaff, 1995) and
confirmed for those of chlorpromazine, doxepin, and cyclizine (Hawes,
1998). The bacterial enzyme also proved considerably more active than
one from rat toward the ketotifen N-glucuronides, although
differences were apparent among the isomers. The hydrolysis of
(S)-GlucB proceeded much slower than
that of (S)-GlucA (Fig. 4B), possibly
due to a poorer accessibility of the glycosidic linkage. Enzymes
produced by E. coli and other bacteria can be assumed to
hydrolyze N-glucuronides synthesized in liver and secreted via bile into the intestine, thus leading to enterohepatic drug cycling. Whether this plays a role for ketotifen kinetics in vivo is
not clear, but several observations argue in favor of such a process.
First, ketotifen plasma concentration profiles sometimes exhibited
secondary peaks or shoulders 5 to 8 h after oral ingestion (Julien-Larose et al., 1983; Grahnén et al., 1992), a phenomenon usually interpreted as indicative of enterohepatic cycling. Second, human feces contained the 10-N-glucuronide after oral
administration of olanzapine (Kassahun et al., 1997) and a glucuronide
of unidentified structure after ingestion of clozapine (Dain et al.,
1997). These two tricyclic drugs bear a structural similarity to
ketotifen, and in all three drugs the molecular masses of their
glucuronides approach 500 Da, a value regarded as the threshold for
efficient biliary excretion of amphiphilic substances into human bile
(Klaassen and Watkins, 1984). Third, after i.v. administration of the
quaternary amitriptyline N-glucuronide, deconjugation could
be demonstrated in vivo (Breyer-Pfaff et al., 1990). It can, however,
not be decided whether this took place in the intestine.
The N-glucuronidation kinetics of (S)-ketotifen
in human liver microsomes were clearly biphasic, suggesting the
participation of at least two enzymes with distinctly different
affinities (Table 3, Fig. 6). In the presence of Triton X-100, the
apparent KM value of the high-affinity
component was very similar to that found with amitriptyline as the
substrate (Breyer-Pfaff et al., 1997), whereas that of the low-affinity
component was lower. Two-enzyme kinetics could also be applied to an
experiment without Triton addition. Under these conditions,
(R)-ketotifen was N-glucuronidated biphasically,
although the two components differed less in
KM (Table 4) and thus could not be as well
evaluated as with (S)-ketotifen. Thus, it has to be
concluded that as in the conjugation of amitriptyline, another member
of the UGT enzyme family besides UGT1A4 is catalyzing the
reaction. On Triton addition, (R)-ketotifen
conjugation became monophasic in two of three microsomal preparations.
A qualitatively similar behavior was described by Le Bigot et al.
(1983), who studied the conjugation of racemic ketotifen and apparently
measured the sum of all four N-glucuronides. In the absence
of Triton, they found KM values of 12.5 and
100 µM, the first one clearly exceeding
KM1 of 2.4 and 1 µM measured with
(R)- and (S)-ketotifen, respectively, under the
same conditions. Moreover, the Vmax values of the previous authors were 2- to 4-fold lower. When Triton was present at 0.06% (3-fold the concentration used now), they observed monophasic glucuronidation with KM 42 µM,
a value that does not agree with any of the constants measured now. The
detection of biphasic kinetics required the use of lowest drug
concentrations in the low or submicromolar range in the present
investigation and in a previous one (Breyer-Pfaff et al., 1997). The
minimal concentration of racemic ketotifen used by Le Bigot et al.
(1983) was 2 µM and those of tricyclic psychoactive drugs were 10 to 20 µM (Green et al., 1995, 1998; Green and Tephly, 1996). Still, the
lowest ketotifen concentration of 0.5 µM was about 1000-fold higher
than the maximal plasma level expected after a 1-mg dose (Grahnén
et al., 1992). Therefore, the two enzymes catalyzing N-glucuronidation should contribute to total reaction rate
according to their intrinsic clearances. If those measured with
detergent in vitro correctly reflect the ratios in vivo (see below),
the high-affinity enzyme is responsible for about 90% of
(S)-ketotifen N-glucuronidation in liver, whereas
no valid estimate can be made with regard to (R)-ketotifen.
Some of the present experiments have been published in abstract form
(Mey and Breyer-Pfaff, 1999), but the Vmax
values reported are erroneous.
There were conspicuous differences between the contributions of
individual ketotifen N-glucuronides to total
N-glucuronide production in vitro and to total excretion in
human urine (Table 5). Intrinsic clearances derived from the ratios of
apparent Vmax and
KM values (Tables 3 and 4) should reflect
the efficiencies of individual metabolic pathways. The majority of in
vitro data was obtained in the presence of detergent because the
latency of UGT enzymes that is overcome by the detergent is assumed not to prevail in vivo (Bossuyt and Blanckaert, 1995). On this basis, (R)-ketotifen glucuronides would be expected to account for
about 40% of N-glucuronides formed from racemic ketotifen
in human liver, whereas its contribution to N-glucuronides
in urine was only 16%. In addition, the
(S)-GlucB/(S)-GlucA
ratio was 1.5 to 2 at low substrate concentrations in vitro, but
on average 11 in urine. Several reasons are conceivable:
N-Glucuronidation is possibly not confined to liver, but may
be carried out in other organs with different stereoselectivity. For an
O-glucuronidation this has been demonstrated, inasmuch as
(+)-E-10-hydroxynortriptyline was conjugated in human liver
microsomes and kidney homogenate, whereas the ()-enantiomer was a UGT
substrate in intestinal homogenate (Dahl-Puustinen et al., 1989).
Alternatively, renal tubular secretion can be stereoselective, in this
case with preferential transport of
(S)-GlucB, whereas the
N-glucuronides with lower renal clearances are more likely
to undergo rehydrolysis. Tubular secretion of xenobiotic glucuronides
has been claimed repeatedly (Møller and Sheikh, 1982), but firm
experimental evidence from animal experiments is available only for
acyl glucuronides (Meffin et al., 1983) and 1-naphthol glucuronide
(Redegeld et al., 1988). Interestingly, morphine 6-glucuronide seemed
to be reabsorbed in the rat kidney (van Crugten et al., 1991). If such
a process occurs with ketotifen N-glucuronides, another
possibility for stereoselectivity would be given. The
N-glucuronides may undergo enzymatic rehydrolysis either
systemically or after biliary excretion (Sperker et al., 1997) to
varying extents, as indicated by the experiments with rat and E. coli -glucuronidase (Fig. 4).
(S)-GlucB proved relatively resistant
to the action of the bacterial enzyme, which should predominate in
intestinal contents, but according to present knowledge reabsorption of
unhydrolyzed glucuronide is rather improbable. Finally, competing
routes of metabolism can affect the (R)/(S) ratio
of N-glucuronides. A major reaction, reduction of the
carbonyl group to the secondary alcohol, could be shown to be catalyzed by aldo-keto reductases in human liver cytosol, and these prefer (S)- over (R)-ketotifen as substrate
(Breyer-Pfaff and Nill, 1999). Other reactions, namely
N-demethylation and N-oxidation, are of minor
quantitative importance (Le Bigot et al., 1987) and their stereoselectivity is not known. The possibility of racemization in vivo
has been checked by administering pure (R)-ketotifen to two
volunteers. The low percentage of (S)-ketotifen
N-glucuronides recovered from their urine would argue
against a significant contribution of racemization to the observed
discrepancy of (R)/(S) ratios of the
N-glucuronides in vitro and in vivo.
In conclusion, conjugation of racemic ketotifen or one of its
enantiomers at the tertiary amino group either chemically or enzymatically produced two quaternary ammonium glucuronides from each
enantiomer. These are conformers differing in piperidylidene ring
folding. The four isomers showed differential sensitivity toward
enzymatic hydrolysis. The kinetics of their production in human liver
microsomes were biphasic in the absence of a detergent and with
(S)-ketotifen also in its presence, indicating the
involvement of at least two UGT isozymes. Of an oral ketotifen dose, a
mean of 17% was detected as N-glucuronides in urine with
preferential excretion of one of the (S)-ketotifen
glucuronides. Discrepancies between isomer ratios in microsomal
incubates and in human urine can be due to extrahepatic
N-glucuronidation, to differential rehydrolysis, and/or to
selective transport of individual glucuronides.
We thank the persons and institutions who provided materials, K. Nill
for expert advice concerning HPLC technique, Dr. W. Zimmermann
(Department of Pharmaceutics, University of Tuebingen) for support in
chemical syntheses, M. Cavegn, E. Endris, and H. Gorcica (Boehringer
Ingelheim Pharma, Biberach) for measuring NMR and mass spectra, and Dr.
K. Wagner for the opportunity for using the analytical instruments.
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Abstract
Introduction
ínsky M,
Holubek J,
Schneider B,
edivy Z,
Svátek E,
Matou
