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Introduction
Benproperine, 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-piperidine (BPP,
1, Figure 1) is widely used as a cough
suppressant for non-productive coughs. It has a peripheral
and central action and can be given to humans
po in forms of embonate or
phosphate[1]. The antitussive activity is
comparable to that of codeine, but is devoid of the undesirable
codeine¡¯s side effects[2].
In general, drugs are metabolized to more polar,
hydrophilic entities, which can be excreted from the body more
easily. At the same time, drugs can be inactivated, be
activated or become a toxicant. There is a growing interest in
identifying metabolites and in establishing their
pharma-cokinetic, pharmacological, and toxicological
properties. Activated metabolites are sometimes drug candidates for
the treatment of a variety of diseases.
In our previous research, 5 mono-hydroxylated
metabolites of benproperine and their conjugates were detected in
human urine and 2 of them that were hydroxylated in phenyl
rings have been identified. Mass spectra indicated that the
other 2 mono-hydroxylates were probably hydroxylated in
the piperidyl ring[3]. To identify metabolites, liquid
chromatography-tandem mass spectrometry is an efficient approach,
which is used widely[4-6]. A comparison of the high
performance liquid chromatography (HPLC) retention times, as well
as MS/MS spectra of putative metabolite and authentic
standard, might be sufficient to make a more definitive
identification[4].
The purposes of the present study was to synthesize
4 putative benproperine metabolites:
1-[1-methyl-2-[2-(phenyl-methyl)phenoxy]ethyl]-4-piperidinol (2),
1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3), as well as
their glucuronides 1-O-[1-[1-methyl-2-
[2-(phenylmethyl)phenoxy]ethyl]-piperidin-4-yl]-b-
D-glucopyranosiduronic acid (4) and 1-O-[1-
[1-methyl-2-[2-(phenylmethyl)phenoxy]-ethyl]-piperidin-3-yl]-
b-D-glucopyranosiduronic acid (5) (Figure 1), to identify the chemical structures of the
metabolites and to evaluate the antitussive effects of 2 and 3
phos-phates.
Materials and methods
Instruments and chemicals Melting points were
determined in open capillary tubes and the thermometer was
uncorrected. MS/MS spectra were recorded on a Finnigan
LCQ ion trap mass spectrometer via an electrospray
ionization (ESI) source in positive ion detection mode.
1H and 13C NMR spectra were recorded on a Bruker ARX-600
instrument with tetramethylsilane as an internal standard.
Ultraviolet (UV) spectra were recorded on a Shimadzu UV-2201
instrument.
3-Piperidinol hydrochloride and 4-piperidinol were
obtained from the Aldrich Chemical Co. We prepared
1-[2-(phenylmethyl)phenoxy]-2-propanol p-toluensulfonate (6)
ourselves, according to published
procedures[7,8], from o-benzylphenol, which was kindly supplied by Aosen
Pharmaceutical Co. Methyl
(2,3,4-tri-O-acetyl-1-O-trichloroaceti-midoyl)-a-D-glucopyranuronate (7) was prepared by us,
according to Soliman
et al[9] from D(+)-glucuronolactone, which
was obtained from Wako Chemical Co. The structures of 6
and 7 were identified using
1H and 13C NMR. Column
chromatography was carried out on silica gel BW-820MH, which
was obtained from Fuji Silysia Chemical Co. Benproperine
phosphate capsule was obtained from Shenyang
Pharmaceutical Co. Benproperine phosphate was kindly supplied
by Aosen Pharmaceutical Co and was recrystallized by us;
the purity was 98.5% by HPLC. Citric acid was obtained
from Shenyang Dongxing Reagent Factory. Test compounds
and citric acid used to investigate antitussive were all
dissolved in physiological saline. Methanol was of HPLC grade,
and the other chemicals used were of analytical grade.
Distilled water, prepared from demineralized water, was used in
LC/MS/MS.
The putative metabolites were synthesized with the
procedure shown in Figure 2.
1-[1-Methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol (2) A total of 3.64 g (36.0 mmol) of 4-piperidinol
and 3.6 g (9.0 mmol) of
1-[2-(phenylmethyl)phenoxy]-2-propanol
p-toluensulfonate (6)[7,
8] were fused on an oil bath at 100°C for 3 h, and stirred. After cooling to an ambient
temperature, 15 mL of water was introduced. The resultant
was extracted with dichloromethane (20 mL×3). The
combined dichloromethane extracts were washed with water and
brine, and dried over anhydrous sodium sulfate. The
solvent was removed under reduced pressure, leaving a brown
oil. The residue was purified using silica gel column
chroma-tography, eluting with CHCl3/MeOH to leave the titled
compound 2 (2.49 g, 85.3%) as a pale-yellow oil. The oil 2 (2.49 g,
0.77 mmol) was dissolved in an aqueous solution of 0.3 mol/L
H3PO4 (25 mL) and the water was removed under reduced
pressure. The residue was recrystallized from ethanol,
leaving phosphate of 2 (2.60 g, 80.2%) as a colorless needle.
1-[1-Methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-
piperidinol (3) 3-Piperidinol was treated as in the procedure
described for 4-piperidinol, leaving 3 as a pale yellow oil
(82.1%) and its phosphate of 3 as a white powder.
1-O-[1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-
piperidin-4-yl]-b-D-glucopyrano-siduronic acid
(4) Phosphate of 2 (85 mg, 0.20 mmol) was suspended and stirred in
CH2Cl2 (4.0 mL) at -15°C under
N2 atmosphere.
BF3 Et2O (10 mL) was added to in one portion. After 10 min of stirring,
methyl (2,3,4-tri-O-acetyl-1-O-trichloroacetimidoyl)-
a-D-glucopyranuronate (7)
[9] (95 mg, 0.20 mmol) was added.
Stirring was continued for 48 h at an ambient temperature. EtOAc
(50 mL) was added, and the mixture was washed with
saturated Na2CO3 aqueous solution (10 mL×2) and brine (10 mL),
and dried over Na2SO4. The solution was evaporated and
purified on silica gel column chromatography, eluted with
n-C6H14-EtOAc-MeOH in gradient.
1-O-[1-[1-Methyl- 2-[2-(phenylmethyl)phenoxy]ethyl]-piperidin-4-yl]-2,3,4-tri-
O-acetyl-b-D-glu-copyranosiduronic acid methyl ester (8, 65
mg, 50.4%) was obtained as a colorless syrup. Compound 8
was dissolved in MeOH (2 mL), and
Na2CO3 aqueous solution (0.1 mol/L, 1 mL) was added at an ambient temperature
and stirred. After 2.5 h of stirring, the mixture was desalted
and concentrated. The titled compound 4 was obtained as a
colorless syrup.
1-O-[1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-
piperidin-3-yl]-b-D-glucopyranosiduronic acid (3-OH-BPP
glucuronide, 5) Phosphate of 3 was treated as in the
procedure described above for phosphate of 2 to give the
intermediate 9. Then, the titled compound 5 was obtained as a
pale-yellow syrup after hydrolysis.
Urine sample Seven healthy male volunteers, whose
mean (SD) age and weight were 22.7 (0.6) years and 62.2 (5.6)
kg, gave written informed consent to take part in the study
and local ethics approval was obtained. Each of the
volunteers swallowed 3 benproperine phosphate capsules (60 mg
BPP) with 200 mL of water. Urine samples were collected at
0 h-24 h. Blank urine was collected from the same volunteer
directly before being given the capsules. The urine samples
were stored at -20°C until analysis.
A 1.0 mL urine sample was filtered through
0.45 mm of precut membrane. The filter was applied to a 3.0-mL
Bond-Elute C18 cartridge (0.5 g silica gel) preconditioned with 2 mL
aliquots of methanol and water. After loading the sample,
the column was washed with 1 mL of water. Metabolites
were eluted with 1 mL of methanol. The eluate was
evaporated to dryness at 40°C under a gentle stream of
N2. The residue was dissolved in
100 mL of the mobile phase. A 20-mL aliquot of the resulting solution was injected onto the
LC/MS/MS system for analysis.
A 1.0 mL portion of blank urine sample was treated as in
the above procedure, in which the solutions of putative
metabolites (2, 3, 4, or 5) in mobile phase were used instead of
mobile phase to obtain the spiked urine sample for
LC/MS/MS analysis.
LC/MS/MS analysis and identification of metabolites
A Shimadzu LC-10AD pump was used in the LC/MS/MS system.
Chromatography was performed on a Diamonsil
C18 column (150×4.6 mm inner diameter,
5 mm, Dikma), which was coupled with a Security Guard
C18 guard column (4×3.0 mm inner diameter, Phenomenex). The components were eluted with
an isocratic mobile phase of methanol-water-formic acid
(50:50:1, v:v:v), and the flow rate was 0.5 mL/min. The
column temperature was maintained at
25 °C. A Finnigan LCQ ion trap mass spectrometer interfaced with liquid
chromatography via an electrospray ionization (ESI)
source was used for mass analysis in positive ion detection mode. The
capillary voltage was fixed at 16 V, and its temperature was
maintained at 200 °C. The spray voltage was set at 4.25 kV. The
HPLC fluid was nebulized using N2 as both the sheath gas at
a flow rate of 0.75 L/min, and the auxiliary gas at a flow
rate of 0.15 L/min. The MS/MS spectra were produced by
collision-induced dissociation (CID) of the selected
precursor ions with He present in the mass analyzer, and the
relative collision energy was set at 30%-40%. Data were
collected and analyzed using the Navigator software (version
1.2).
Animals Dunkin Hartley guinea pigs of both sexes,
weighing 200-300 g (Experimental Animal Center of
Shen-yang Pharmaceutical University, Shenyang, China) were
used. All animal studies were in accordance with the
Regulations for the Administration of Affairs Concerning
Experimental Animals (China, 1988) and Implementing Regulations
of the Administration on Medical Experiments on Animals
(China, 1989). The guinea pigs were maintained in standard
animal rooms, with food and water freely available, and on a
natural light-dark cycle. They were allowed to adapt to the
conditions for at least 1 week before being used in
experi-ments.
Investigation of antitussive effect The antitussive effect
of BPP, 2 and 3 phosphates was evaluated in conscious
guinea-pigs against citric acid-induced
coughs[10,11]. Guinea pigs were placed individually in a transparent plexiglass
cylinder chamber (10 cm×10 cm×21 cm) and exposed to a
nebulized solution of 7.5% citric acid for 3 min. An ultrasonic
nebulizer (402 AI, Shanghai Yuyue Medical Facilities) was
used to produce an aerosol with particles with an
aerodynamic mass median diameter of 1 mm; the volume of solution
aerosolized was approximately 0.6 mL/min. Animals were
selected for the study according to the number of coughs
observed 24 h before the test, and animals with more than 20
or fewer than 6 coughs during the 3 min test were not used.
The compounds and the vehicle (physiological) saline were
given ip (2 mL/kg) for 1.5 h (for
BPP×H3PO4) or for 40 min (for
2×H3PO4 and
3×H3PO4) before the test. The number of coughs
during the 3 min test and during the 5 min immediately after
the test was determined. The animals with different doses
(3 mg/kg, 9 mg/kg, 27 mg/kg for
2×H3PO4 and
3×H3PO4, 27 mg/kg for
BPP×H3PO4) of drugs and the vehicle were grouped
according to a random table. Animals were used only once
because of tachyphylaxis of the cough response. Coughing
sounds were recorded and amplified using a microphone and
loudspeaker. During the experiment, the animals were
continuously watched by a trained observer who was unaware
of the treatment. Sneezes and coughs were differentiated by
visual observation of the animals.
Statistical analysis Pharmacological results are
represented as mean±SEM. Data were analyzed using one-way
analysis of variance (ANOVA). Whenever anova was significant, further comparisons between vehicle- and
drug-treatment groups were performed using Dunnett¡¯s
t-test. The above analysis was performed using the software SPSS
V11.0 for Windows.
Results
Chemical synthesis Compound 6, which was
synthesized from 2-benzylphenol[8], was fused with 4-piperidinol to give 2 in 85% of yield. 3-Piperidinol was treated according to
the procedure for 4-piperidinol to give 3 in 82% of yield.
Glucuronides were synthesized from mono-hydroxylates (2
and 3) treated with trichloroacetimidate donor (7) followed
by basic hydrolysis (Figure 2). The structures of products
were identified using ESI-MS, 1H NMR, and
13C NMR (Table 1).
Metabolites of benproperine Compared with
corresponding blank samples, the urine after being given of BPP showed
5 peaks corresponding to hydroxylated metabolites and 5
peaks corresponding to their glucuronides in LC/MS/MS
analysis (Figure 3).
Metabolites M1 and M2 M1 and M2 gave the same
pseudo-molecular ions [M+H]+ at m/z
326, similar MS/MS spectra (Figure 4), but different retention times (Figure 3),
indicating that they were isomers. Their pseudo-molecular
ions were 16 u higher than that of the parent drug, indicating
the addition of a hydroxyl group to the parent drug.
The MS2 spectra of M1 and M2 displayed the same
fragment ions at m/z 308 and m/z 142 (Figure 4). The presence of
the prominent ion at m/z 308 was 18 u lower than precursor
ions, indicating the loss of a water molecule from
pseudo-molecular ions and further proving the presence of hydroxyl
group. The presence of the ion at m/z 142 indicates that the
hydroxyl group was in the piperidylpropanyl moiety.
However, the MS/MS data did not provide useful information for assigning the exact site of hydroxylation. Therefore,
M1 and M2 were definitely confirmed by comparing their
retention time and mass spectra with synthesized 2 and
3 (Figures 3 and 4). M1 possessed the same retention time
and mass spectra with 2, and was confirmed as
1-[1-methyl-2-[2-(phenylmethyl)-phenoxy]ethyl]-4-piperidinol and, in
the same way, M2 was identified as 1-[1-methyl-2-[2-
(phenylmethyl)phenoxy]-ethyl]-3-piperidinol.
Metabolites M3 and M4 M3 and M4 gave the same
pseudo-molecular ions
[M + H]+ at m/z 502, which were 176 u
higher than monohydroxylate metabolites, indicating
conjugation of a glucuronic acid. The
MS2 spectra of M3 and M4 displayed same fragment ions at
m/z 326, which possessed the same mass to charge ratio as the pseudo-molecular ions
of M1 and M2. The MS3 spectra of M3 and M4 displayed
the fragment ions at m/z 308 and m/z 142, which were
consistent with MS2 spectra of M1 and M2, respectively (Figure 4).
It indicated that M3 and M4 were the glucuronides of M1
and M2, respectively. The further confirmation was obtained
by comparing their retention times and MS/MS spectra with
the synthesized glucuronides 4 and 5 (Figure 3 and Figure 4).
M3 was identified as
1-O-[1-[1-methyl-2-[2-(phenylmethyl)phenoxy]-ethyl]-piperidin-4-yl]-
b-D-glucopyranosiduronic acid. In the same way, M4 was identified as
1-O-[1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-piperidin-3-yl]-
b-D-glucopyranosiduronic acid.
The peaks at 4.6 min and 6.7 min (Figure 3) corresponded
to the two identified monohydroxylate
metabolites[3]. Two peaks gave the same pseudo-molecular ions
[M + H]+ at m/z 326. The MS/MS spectra showed the fragment ion at
m/z 163 and 126 for the peak at 4.6 min and
m/z 126 for the peak at 6.7 min, respectively.
Pharmacology Exposure to a nebulized solution of 7.5%
citric acid aerosol caused coughing in control animals within
88.1±5.9 s (n=10), and both test compounds prolonged the
latency of cough without dose-dependency as shown in
Table 2. Immediately after exposure to citric acid, there is a
hypersensitive period and the animals tend to cough
continuously. For the control group, the number of coughs
during the 3 min test was 8.2±1.2; all three compounds
significantly decreased the number of coughs during the 3 min
test, but 2 and 3 failed to decrease the number of coughs
during the 5 min immediately after the test (Table 2).
Discussion
In synthesis of putative metabolites 4 and 5, the form of
basic acceptor, reaction promoter, and the order of addition
are all important variables in glucuronidation. The strong
electron-withdrawing character of the 6b-methoxycarbonyl
group in any activated glucuronate makes such species
notoriously poor donors[12], so that glucuronidation is difficult.
The phosphate of 2 was treated with a trichloroacetimidate
donor in the presence of
BF3×Et2O to give protected
glucuronide 4 in 52% of yield. However, in free form of 2,
no desired product was obtained. The salt form of the basic
acceptor was better than its free form for glucuronidation,
with Lewis acid used as the promoter in the present study.
When trimethysilyl trifluoromethane sulfonate
(TMSOTf)[9] was used as a promoter instead of
BF3×Et2O, more of the
by-product, 2 acetylate, was obtained and the yield of the
desired product was increased. An inverse addition, which
means trichloroacetimidate 7 was added into the mixed
solution of 2 phosphate and
BF3×Et2O, improved the yield to
52% from 21%. This result is consistent with the prior report
for glucuronidation of other
compounds[13].
In positive-ion mode, compounds 2 and 3 formed
pseudo-molecular ions [M+H] + at m/z
326. They are two isomers; each of them contains a hydroxyl group in the piperidyl ring
and an ether bond. The same product ions were observed in
the full scan of MS/MS spectra at m/z 308 and 142, with
different relative abundance for 2 compounds. The
fragment ion at m/z 142 was formed by cleavage of the ether
bond (Figure 4). The fragment ion at
m/z 308 was formed by dehydration, which was easier going in 3 than in
2 because p- p conjugated system was formed after
dehydra tion in the former. Therefore,
m/z 308 was the base peak for 3 and m/z 142 was the base peak
for 2.
Hydroxylation and glucuronidation are 2 general
pathways of drug metabolism, by which drug can be metabolized
to more polar, hydrophilic entities, which can be excreted
from the body more easily. The presence of M1
(2), M2 (3) and their
glucuronides M3 (4) and M4 (5) is consistent with
the general rule of metabolism. In human urine, some
dihydroxylate metabolites besides monohydroxylate were
detected; no dealkylation metabolite was detected. In the
present study, 4 monohydroxylate metabolites and 2
glucuronide metabolites were identified. The chromatogram
(Figure 3) indicates that the hydroxylated metabolites of
benproperine were recovered in urine mainly as glucuronides,
and in very low concentrations as free forms. M1 and M2
were also found in human plasma.
The free forms of compound 2 (M1) and
3 (M2) are oils, which are difficult to be weighed, and the water-solubility of
the free forms are poor. They are not suitable for use in the
study of antitussive activity. Benproperine is generally used
in clinical treatments as dihydrogenphosphate. Therefore,
we converted the free form of compounds 2 and
3 to their phosphates for pharmacological study.
The experimental model to induce coughing using citric
acid is the model most frequently adopted and extensively
studied in animals and in humans[14]. Citric acid can induce
coughing in guinea pigs by increasing the volume of
bronchial secretion[10], by acting on capsaicin sensitive sensory
neurons[15,16] or by disturbing the pH of the airway surface
liquid[17]. However, it induces the airway hyper-reactivity in
guinea pigs[18]. Although phosphates of
2 and 3 produced an increase in the latency of the first cough and decreased
the number of coughs during the 3 min test using citric acid,
they did not decrease the number of coughs during the 5 min
immediately after the test. The results showed that
phosphates 2 and 3 did not inhibit the coughing induced by citric
acid in guinea pigs.
In conclusion, 8 novel compounds, 2-5, 8, 9, and
phosphates of 2 and 3 were synthesized successfully for the first
time.
1-[1-Methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol
(2),
1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3),
1-O-[1-[1-methyl-2-[2-(phenyl
methyl)-phenoxy]ethyl]-piperidin-4-yl]-b-D-glucopyrano-
siduronic acid (4) and 1-O-[1-[1-methyl-
2-[2-(phenylmethyl)phenoxy]ethyl]-piperidin-3-yl]-b
-D-glucopyranosiduronic acid (5) were identified to be metabolites of benproperine in
human urine. Compounds 2 and 3 are inactive metabolites of
benproperine.
Acknowledgments
The authors thank Dr Osamu HARA from Meijo
University for his helpful discussions. We also thank Ms Wen LI, Mr Yi
SHA and Ms Ai-hua SONG in Shenyang Pharmaceutical
University for obtaining part of the NMR data and UV data.
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