Bioactivation of benzylamine to reactive intermediates in rodents: Formation of glutathione, glutamate, and peptide conjugates

Article abstract of DOI:10.1021/tx020063q

The in vivo and in vitro disposition of benzylamine was investigated in rats. Benzylamine was metabolized to only a small extent by rat liver subcellular fractions. In contrast, it was extensively metabolized in vivo in rats. In vivo studies performed wit

Full text of DOI:10.1021/tx020063q

1
190
Chem. Res. Toxicol. 2002, 15, 1190-1207
Bioa ctiva tion of Ben zyla m in e to Rea ctive In ter m ed ia tes
in Rod en ts: F or m a tion of Glu ta th ion e, Glu ta m a te, a n d
P ep tid e Con ju ga tes
,
†
‡
‡
‡
Abdul E. Mutlib,* Patricia Dickenson, Shiang-Yuan Chen, Robert J . Espina,
†
§
J . Scott Daniels, and Liang-Shang Gan
Drug Metabolism and Pharmacokinetics Section, Bristol-Myers Squibb Pharma Company,
Wilmington, Delaware 19880
Received J uly 8, 2002
The in vivo and in vitro disposition of benzylamine was investigated in rats. Benzylamine
was metabolized to only a small extent by rat liver subcellular fractions. In contrast, it was
extensively metabolized in vivo in rats. In vivo studies performed with stable isotope-labeled
benzylamine enabled rapid mass spectrometric identification of metabolites present in rat bile
and urine. The major metabolite of benzylamine was the hippuric acid formed by glycine
conjugation of benzoic acid. LC/MS analysis of bile and urine obtained from rats dosed with
1
0 7 0 2
:1 equimolar mixture of either d :d - or d :d -benzylamine showed the presence of several
glutathione adducts in addition to the hippuric acid metabolite. The presence of various
glutathione adducts indicated that benzylamine was metabolized to a number of reactive
intermediates. Various metabolic pathways, including those independent of P450, were found
to produce these intermediates. A previously undocumented pathway included the formation
of a new carbon-nitrogen bond that led to a potentially reactive intermediate, Ar-CH
CO)-X, capable of interacting with various nucleophiles. The origin of this reactive intermediate
is postulated to occur via the formation of either a formamide or carbamic acid metabolites.
Metabolites which were produced by the reaction of this intermediate, Ar-CH -NH(CO)-X with
2
-NH-
(
2
nucleophiles included S-[benzylcarbamoyl] glutathione, N-acetyl-S-[benzylcarbamoyl]cysteine,
S-[benzylcarbamoyl] cysteinylglycine, S-[benzylcarbamoyl] cysteinylglutamate, N-[benzylcar-
bamoyl] glutamate, and an oxidized glutathione adduct. Bioactivation of amines via this
pathway has not been previously described. The oxidative deamination of benzylamine yielding
the benzaldehyde was demonstrated to be a precursor to the hippuric acid metabolite and
S-benzyl-L-glutathione. The formation of the S-benzyl-L-glutathione conjugate showed that a
net displacement of amine from benzylamine had taken place with a subsequent addition of
glutathione at the benzylic position. In addition to these novel pathways, a number of other
glutathione-derived adducts formed as a result of epoxide formation was characterized. It was
demonstrated that benzylamine was converted by rat P450 2A1 and 2E1 to benzamide that
was rapidly metabolized to an epoxide. Mechanisms are proposed for the formation of various
GSH adducts of benzylamine.
In tr od u ction
The ability to characterize unusual and minor metabo-
yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC
23) and its analogues, using various spectroscopic
4
techniques (2, 3). The metabolism studies with DPC 423
and its structural analogues revealed that the benzy-
lamine moiety was a metabolic soft spot that was being
metabolized extensively to several products, including a
number of novel metabolites. The benzylamine moiety
was converted by phase I and phase II enzymes produc-
ing an aldehyde, a carboxylic acid, acyl glucuronides, a
carbamyl glucuronide, oxime, and a glutathione adduct
lites has been greatly accelerated with the introduction
of versatile analytical techniques such as liquid chroma-
tography/mass spectrometry (LC/MS) and liquid chro-
matography/nuclear magnetic resonance (LC/NMR) spec-
troscopy. Characterization of such minor metabolites has
demonstrated the existence of previously undocumented
metabolic pathways, such as the formation of glutamic
acid conjugates of acetaminophen glutathione-derived
adducts (1). Recently we reported the characterization
of unique glutamate conjugates of 1-[3-(aminomethyl)
phenyl]-N-[3-fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4-
(2-5). The presence of these metabolites implicated
bioactivation of these compounds. The interesting dispo-
sition pathways of DPC 423 (see Figure 1) prompted us
to continue with a more detailed investigation into the
rodent metabolism of benzylamine.
*
To whom correspondence should be addressed. Telephone: (734)
Benzylamines differ from the more widely studied
arylamines by virtue of a having a methylene bridge
between the aromatic system and the amino moiety.
Several reports have appeared in the literature describ-
ing the metabolism of arylamines. The reactivities as-
6
22-2198. Fax: (734) 622-1459. E-mail: abdul.mutlib@pfizer.com.
†
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., 2800
Plymouth Road, Ann Arbor, MI 48105.
‡
Bristol-Myers Squibb Pharma Company.
Department of Drug Metabolism and Pharmacokinetics, Millenium
§
Pharmaceuticals Inc., Cambridge, MA 02139.
1
0.1021/tx020063q CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/17/2002

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1191
derived. Importantly, data from our DPC 423 work
implicated the involvement of hydroxylamine species as
a reactive precursor to the formation of oxime-derived
GSH adducts in rats. Therefore, the in vivo metabolism
of benzylhydroxylamine in rodents was also investigated
in order to determine any possible links of this species
to the bioactivation of benzylamine.
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d Su p p lies. Benzylamine, benzaldehyde, syn-
benzaldehyde oxime, benzamide, benzohydroxamic acid, benzoic
acid, benzyl alcohol, N-benzohydroxamic acid, N-benzylhydroxy-
lamine, benzyl isocyanate, N-acetylcysteine, cysteinylglycine,
cysteinylglutamate, hippuric acid, L-glutamic acid, GSH (re-
duced and oxidized), and various chemical reagents were
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).
0 2 7
F igu r e 1. Structures of DPC 423 and d -, d -, d -benzylamine.
Benzyl-d
7
-amine hydrochloride was obtained from CDN Isotopes
(
Quebec, Canada). S-Benzyl-L-cysteine was purchased from
sociated with the formation of hydroxylamines, nitroso,
and other metabolic intermediates from arylamines have
been very well documented (6-9). In contrast, the
metabolism of benzylamines has largely been overlooked.
A more comprehensive investigation into the metabolic
disposition of benzylamine is therefore required. The
literature reveals that several metabolism studies have
been performed with benzylamine itself. However, most
of these studies were limited to the description of
oxidative deamination of benzylamine, mediated by vari-
ous amine oxidases (10-15). The oxidative deamination
of benzylamine by the amine oxidases affords benzalde-
hyde. The biotransformation of benzylamine to benzal-
dehyde has been attributed to mitochondrial monoamine
oxidase (MAO)-B (10-12) and to semicarbazide-sensitive
amine oxidases (SSAO) present in the plasma of various
animal species (13-15).
Benzylamine has been shown to be extensively deami-
nated to benzaldehyde by liver subcellular fractions from
various species (11). However, due to the unavailability
of LC/MS and LC/NMR techniques at that time, the
structure elucidation of polar conjugates was not possible.
We therefore re-visited the metabolism of benzylamine
with an anticipation that it might be metabolized to
similar metabolites as DPC 423 (2-5). One objective of
our study was to thoroughly investigate the metabolic
conversion of benzylamine in rodents. We therefore
employed LC/MS and NMR to evaluate if reactive
intermediates were formed during its biotransformation.
Deuterium-labeled benzylamine was employed to im-
prove the detection of metabolites in complex biological
matrixes as well as to aid in structure elucidation. During
the course of this study, synthetic standards were
prepared and the LC/MS retention times and MS/MS1
spectra were obtained for comparison with the metabo-
lites. We also attempted to isolate polar metabolites from
biological samples for which no synthetic standards were
available. NMR studies were performed with these
samples to unambiguously assign the structures. The
GSH adducts, in particular, were fully characterized in
order to provide valuable insight into the possible identity
of the reactive intermediates from which they were
Fluka Chemicals (Bassle, Switzerland). Bond Elut C18 car-
tridges (10 g/60 cm ) were obtained from Varian Sample
3
Preparation Products (Harbor City, CA). All general solvents
and reagents were of the highest grade available commercially.
Syn th esis of Meta bolite Sta n d a r d s. Synthetic standards
of potential metabolites of benzylamine were either obtained
commercially or synthesized in-house. The purity of each
standard was confirmed by LC/MS and NMR analysis to be
greater than 95% as determined by the integration of peak
signals. All of the standards showed the presence of a single
peak [detected both by UV and total ion current (TIC)] during
the LC/MS analysis.
Syn th esis of Ben zyl-d
2
-a m in e Hyd r och lor id e. To a sus-
pension of LiAlD (4.2 g, 52 mmol) in THF (100 mL) was added
4
a solution of benzonitrile (5.2 g, 50 mmol) in THF (20 mL) at
50 °C. The mixture was stirred at 50 °C for 4 h. After cooling in
an ice bath, 6 mL of 1 N NaOH was added to the mixture slowly.
The mixture was filtered, and the filtrate evaporated to dryness.
The residue was dissolved in CH
column chromatography (silica gel), eluting with a solution of
CH Cl /methanol/NH OH (80:15:5 v/v). The fraction correspond-
ing to benzylamine was collected and evaporated to dryness.
The residue was dissolved in 1 N HCl (20 mL), and the solution
was lyophilized to yield the desired product d
hydrochloride (2 g, 18 mmol). 1H NMR in DMSO-d
2 2
Cl and purified using flash
2
2
4
2
-benzylamine
showed
6
aromatic proton signals at δ7.2-7.5 (5H, m). LC-ESI/MS showed
MH at m/z 110.
+
Syn th esis of S-Ben zyl-L-glu ta th ion e. To a round-bottom
flask containing 5 mL of methanol:water (1:1 v/v) were added
reduced GSH (1.6 mmol) and benzyl bromide (1.5 mmol).
Triethylamine (1.5 mmol) was added, and the reaction mixture
stirred for 1 h at room temperature, after which the solvent was
removed under a stream of nitrogen. The dried powder was
dissolved in a methanol:water mixture (1:9 v/v) and chromato-
graphed on a Bond Elut C18 cartridge that was previously
preconditioned with methanol and water. After the samples
eluted under gravity, the cartridge was washed with 30 mL of
water followed by elution with 30 mL aliquots of different
percentages of methanol in water. It was found that most of
the GSH conjugate had eluted with 10-15% methanol in water.
The solvents were removed, and the dried powder was subse-
1
quently analyzed by LC/MS and NMR. H NMR: δ 8.42 (1H,
bs, cys NH), 8.10 (1H, bs, gly NH), 7.30 (4H, m, Ar-H), 7.20 (1H,
m, Ar-H), 4.48 (1H, m, cys R), 3.75 (1H, s, Ar-CH -S), 3.60 (2H,
m, gly R), 3.35 (1H, m, glu R), 2.83 (1H, m, cys â), 2.62 (1H, m,
cys â′), 2.35 (1H, m, glu γ), and 1.95 (1H, m, glu â′). LC-ESI/MS
2
+
showed MH at m/z 398.
1
Syn th esis of S-[Ben zylca r ba m oyl]glu ta th ion e. GSH (1.7
mmol) was stirred in 5 mL of methanol in a 10 mL round-bottom
flask. Benzyl isocyanate (1.5 mmol) was added to the flask and
the reaction mixture stirred. To the reaction mixture was added
Abbreviations: LC-ESI/MS, liquid chromatography electrospray
ionization mass spectrometry; LC-APCI/MS, liquid chromatography
1
atmospheric pressure chemical ionization mass spectrometry; H NMR,
proton nuclear magnetic resonance; MS/MS, mass spectrometry/mass
spectrometry; TOCSY, total correlated spectroscopy; HMBC, hetero-
nuclear multiple bond correlation; HSQC, heteronuclear single quan-
tum coherence.
1
0 µL of concentrated sodium hydroxide (6 M) and the solution
stirred for 30 min at room temperature. The solvents were

1
192 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
removed under vacuum and the powder purified on a C18
cartridge as described above. The fraction containing the GSH
adduct was dried and re-purified on a semipreparative column
to a methanolic solution of benzyl isocyanate (0.2 mmol) and
the reaction stirred for 30 min at room temperature. At the end
of the reaction, the solvent was removed under a stream of
nitrogen and the dried extract analyzed by LC/MS without any
(
Supelco, C18, 10 × 250 mm). The column was eluted with an
isocratic mobile phase consisting of acetonitrile:0.05% TFA (1:5
v/v) delivered at 3.5 mL/min. The GSH conjugate eluting at t
4.0 min was collected, dried and subsequently analyzed by
+
further purification. LC-ESI/MS showed MH at m/z 746. Due
R
to the low yield of M11, further characterization by NMR was
not performed. The structure of metabolite M11 was assigned
tentatively.
)
1
LC/MS and NMR. H NMR: δ 7.31 (2H, m, Ar-H), 7.23 (3H, m,
Ar-H), 4.30 (2H, m, Ar-CH -NH-), 4.25 (1H, m, cys R), 3.50 (1H,
2
Liqu id Ch r om a togr a p h y/Ma ss Sp ectr om etr y. The me-
tabolites were separated on a HPLC column (Phenomenex, Aqua
C18, 4.6 × 150 mm, 5 µm) by a gradient solvent system
consisting of two components, A (100% methanol with 0.1%
formic acid) and B (ammonium formate, 10 mM, pH 3.6). The
percentage of A was increased from 0 to 35 over 15 min with
the solvent flow rate set at 1.0 mL/min. After maintaining the
organic solvent at 35% for an additional 3 min, the proportion
of A was ramped linearly to 75% over the next 7 min. After 25
min, the column was washed with 90% A for 5 min before re-
equilibrating with the initial mobile phase. To avoid contami-
nating the source of the mass spectrometer, the solvent was
diverted to waste at 25 min. Aliquots of bile (20 µL) and urine
samples (80 µL) were injected directly onto the HPLC column,
and the eluent was introduced into the source of the mass
spectrometer. Subsequent studies also utilized a smaller diam-
eter column (Phenomenex, Aqua C18, 2.1 × 100 mm, 3 µm)
using the same mobile phase eluted at a flow rate of 0.3 mL/
min. To detect the metabolites in the fractions from C18
cartridges and from semipreparative HPLC column, aliquots
(20-50 µL) were introduced to the mass spectrometer using the
flow injection analysis method. The mobile phase consisted of a
mixture of A and B (1:1 v/v) delivered at a rate of 0.35 mL/min.
LC/MS was carried out by coupling a Hewlett-Packard HPLC
system (HP1100) to a Finnigan LCQ ion trap mass spectrom-
eter. Liquid chromatography atmospheric pressure chemical
ionization-mass spectrometry (LC-APCI/MS) or liquid chroma-
tography electrospray ionization mass spectrometry (LC-ESI/
MS) was performed in the positive ion mode. The metabolites
were detected by operating the mass spectrometer either in the
full scan mode or by selected ion monitoring of the pseudomo-
lecular ions of the conjugate. MS/MS spectra of fragment ions
on the LCQ mass spectrometer were obtained with 25-30%
relative collision energy.
m, glu R), 3.40 (2H, m, gly R), 3.35 (1H, m, cys â), 2.95 (1H, m,
cys â′), 2.38 (1H, m, glu γ), 2.30 (1H, m, glu γ′), 2.00 (1H, m, glu
â), and 1.90 (1H, m, glu â′). LC-ESI/MS showed MH at m/z
+
4
41.
Syn th esis of N-Acetyl-S-[ben zylca r ba m oyl]cystein e. N-
Acetylcysteine (1.7 mmol) was dissolved in 5 mL of methanol
in a 10 mL round-bottom flask. Benzyl isocyanate (1.5 mmol)
was added to the flask, and the reaction performed as described
above. At the end of the reaction, the solvents were removed
under vacuum, and the residue was purified on a C18 cartridge
as described above. The N-acetylcysteine conjugate eluted with
4
0% methanol in water. The solvents were removed under
vacuum, and the dried sample was analyzed by LC/MS and
1
NMR without any further purification. H NMR: δ 8.70 (1H,
m, Ar-CH
2
-NH-), 8.20 (1H, d, NH-CO(CH
3
), 7.32 (2H, m, Ar-
H), 7.25 (3H, m, Ar-H), 4.32 (2H, m, Ar-CH
2
-NH-), 4.30 (1H, m,
cys R), 3.35 (1H, m, cys â), 3.02 (1H, m, cys â′), and 1.85 (3H, s,
+
CO(CH
3
). LC-ESI/MS showed MH at m/z 297.
Syn th esis of S-[Ben zylca r ba m oyl]cystein ylglycin e. Cys-
teinylglycine (0.1 mmol) was dissolved in 5 mL of methanol in
a 10 mL round-bottom flask. Benzyl isocyanate (0.1 mmol) was
added to the flask, and the reaction performed as described
above. At the end of the reaction, the solvents were removed
under vacuum and the residue was purified on a C18 cartridge
as described above. No further purification was required for this
1
standard. H NMR showed δ at 8.75 (1H, m, Ar-CH
1H, m, CO-NH-CH -), 7.31 (2H, m, Ar-H), 7.23 (3H, m, Ar-H),
.30 (2H, Ar-CH -NH-), 4.30 (1H, m, cys R), 3.45 (2H, d, CH-
NH ) 3.30 (1H, m, cys â), 3.18 (2H, m, gly R), 2.90 (1H, m, cys
2
-NH-), 7.82
(
4
2
2
2
+
â′). LC-ESI/MS showed MH at m/z 312.
Syn th esis of S-[Ben zylca r ba m oyl]cystein ylglu ta m a te.
Cys-Glu (0.8 µmol) was added to 1.1 M equiv of benzyl isocy-
anate in methanol, and the reaction performed at room tem-
perature as described above. At the end of the reaction, the
solvents were removed and the dried extract was purified on a
semipreparative column (Supelco, 10 × 250 mm). The column
was eluted with an isocratic mobile phase consisting of a mixture
of acetonitrile and 0.05% TFA (3:7 v/v) delivered at 3.5 mL/min.
Selected ion monitoring (SIM) of metabolites present in bile
and urine was performed, and the peak area response for each
metabolite obtained. Known amounts of synthetic standards of
metabolites were also injected onto the LC/MS, and the peak
area response was obtained for each compound. An estimate of
the levels of each metabolite was obtained by comparing the
peak areas determined from SIM experiments. The volumes of
urine and bile were taken into consideration when obtaining
an estimate of the total amount of metabolite excreted. The ions
monitored for M1, M2, M3, M5, M6, M8, M9, M10, M12, and
M13 were at m/z 180, 445, 316, 441, 312, 297, 281, 384, 398,
and 212, respectively.
The conjugate eluted at t
to this compound was collected from several injections, pooled,
dried under vacuum, and analyzed by LC/MS and NMR. 1
NMR: δ 8.70 (1H, m, Ar-CH -NH-), 8.35 (1H, d, CH-NH-CO-)
2
.32 (2H, m, Ar-H), 7.23 (3H, m, Ar-H), 4.30 (2H, m, Ar-CH -
R
) 5.0 min. The peak corresponding
H
2
7
NH-), 4.25 (1H, m, cys R), 3.60 (1H, m, glu R), 3.35 (1H, m, cys
â), 3.00 (1H, m, cys â′), 2.30 (2H, m, glu γ), 2.00 (1H, m, glu â),
+
and 1.90 (1H, m, glu â′). LC-ESI/MS showed MH at m/z 384.
High -F ield NMR. Spectra were obtained on a Bruker
1
Syn th esis of N-[Ben zylcar bam oyl]glu tam ate. L-Glutamate
Avance 600 MHz NMR spectrometer equipped with 2.5 mm H/
1
3
(0.3 mmol) was reacted with benzyl isocyanate (0.3 mmol) in
C inverse conventional NMR probe. Suppression of the re-
methanol as described above. At the end of the reaction, the
solvents were removed under vacuum and the residue subse-
quently purified on a semipreparative column (Supelco, 10 ×
sidual water and acetonitrile signals was carried out using the
WET solvent suppression method in all the NMR experiments.
Chemical shifts were referenced to DMSO at δ 2.49 ppm and to
acetonitrile at δ 2.0 ppm. The structures of metabolites were
determined from proton and carbon one-dimensional NMR as
well as proton-proton total correlated spectroscopy (TOCSY),
proton-carbon heteronuclear single-quantum coherence (HSQC),
and long-range proton-carbon heteronuclear multiple-bond cor-
relation (HMBC) two-dimensional NMR.
2
50 mm). The column was eluted with an isocratic mobile phase
consisting of a mixture of acetonitrile and 0.05% TFA (3:7 v/v)
delivered at 3.5 mL/min. The glutamate conjugate eluted at t
6.0 min. The peak corresponding to this conjugate was
R
)
collected from several injections, pooled, dried under vacuum,
and analyzed by LC/MS and NMR. 1H NMR: δ 7.28 (2H, m,
2
Ar-H), 7.24 (3H, m, Ar-H), 4.20 (2H, s, Ar-CH -NH-), 4.12 (1H,
In Vivo Stu d ies. Male Sprague-Dawley rats with cannu-
lated bile ducts (weighing between 250 and 350 g) were
administered oral doses of benzylamine hydrochloride, N-
benzylhydroxylamine, benzamide or benzaldehyde at 300 mg/
kg, and urine and bile collected over ice. Benzylamine hydro-
chloride and N-benzylhydroxylamine were dissolved in normal
m, glu R), 2.30 (1H, m, glu γ), 2.25 (1H, m, glu γ′), 1.95 (1H, m,
+
glu â), and 1.72 (1H, m, glu â′). LC-ESI/MS showed MH at
m/z 281.
Syn th esis of Meta bolite M11 (a d d u ct of oxid ized glu -
ta th ion e). Oxidized glutathione (GSSG, 0.2 mmol) was added

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1193
saline while benazamide and benzaldehyde were prepared as a
suspension in 0.5% methocel. These were administered orally
at 10 mL/kg. In another study, groups of three rats were
v/v) methanol:10 mM ammonium formate, pH 3.7, before further
purification on a semipreparative HPLC column (Beckman C18,
10 × 250 mm). The mobile phase consisted of an isocratic solvent
mixture of A (5% methanol in 10 mM ammonium formate, pH
3.7) and B (0.1% formic acid in methanol). Two peaks at
approximate retention times of 5.4 and 4.8 min were collected,
corresponding to metabolites M2 and M3, respectively. A Waters
Fraction Collector II was used to collect the fractions from the
HPLC. The fractions corresponding to each metabolite were
dried under vacuum and subsequently analyzed by NMR
spectroscopy.
0 7 0 2
administered 1:1 equimolar mixtures of either d :d or d :d
benzylamine hydrochloride (in normal saline) at 300 mg/kg and
bile/urine collected. The rats were housed individually in
suspended stainless steel wire-mesh cages equipped with an
automatic watering system. The study room was environmen-
tally controlled for temperature (72 ( 4 °F), relative humidity
(40-70%), and light (a 12 h light/dark cycle). Rats had free
access to water and were given a specific amount of certified
Purina rodent chow each day. The samples were collected at
0
-7 and 7-24 h time intervals and stored at -20 °C until
analyzed.
Stu d y w ith Am in oben zotr ia zole (ABT). Three bile duct-
Resu lts
In Vitr o Stu d ies. Rat liver S9 and microsomal frac-
tions did not produce detectable levels of any of the
benzylamine metabolites that were observed in vivo
cannulated male Sprague-Dawley rats were orally adminis-
tered ABT (100 mg/kg) for 3 days. On the second and third days,
the rats were administered benzylamine hydrochloride (150 mg/
kg) 1 h after the ABT dose. The bile and urine samples were
collected over ice and stored as described above.
(
Scheme 1). The human cDNA expressed FMO1, FMO3,
or FMO5 did not metabolize benzylamine. The lack of in
vitro benzylamine metabolism suggested a minor role for
P450 and FMO in the metabolic disposition of benzy-
lamine. It appears that the MAO activities responsible
for the formation of benzaldehyde from benzylamine were
either absent or present at low levels in the S9 fractions.
Experiments performed with rat cDNA-expressed P450
enzymes, however, showed conversion of benzylamine by
P450 2A1 and 2E1 to 3,4-dihydroxybenzamide (Scheme
Micr osom a l, S9, a n d F MO Meta bolism of Ben zyla m in e.
0 7
Benzylamine hydrochloride or a 1:1 w/w mixture of d :d
benzylamine hydrochloride or N-benzylhydroxylamine (all dis-
solved in normal saline) were incubated with rat liver mi-
crosomes (phenobarbital or dexamethasone induced or nonpre-
treated, 1 mg/mL) or S9 fraction (4 mg/mL) using the following
protocol: NADPH (2 mM), substrate (10 or 100 µM), +/- GSH
2
(3 mM), MgCl (3 mM), and 0.1 M phosphate buffer (pH 7.4) to
2
). The tentative structure of this metabolite was estab-
a final volume of 1 mL. The mixtures were incubated for 1 h
after which 2 mL of cold acetonitrile was added and the proteins
precipitated. After centrifuging the samples at 3500g for 5 min,
the supernatants were transferred to clean culture tubes and
dried under a stream of nitrogen at 25 °C. The dried extracts
were reconstituted in the HPLC mobile phase and analyzed by
LC/MS as described earlier. Studies were also conducted with
human FM01, FM03, and FM05 using the following protocol:
NADPH (2 mM), substrate (10 or 100 µM), +/- GSH (3 mM),
lished by mass spectral analysis of the incubations
employing deuterium labeled benzylamine (Figure 1).
The metabolite showed MH at m/z 154, 154, and 157
+
0 2 7
from incubations performed with d -, d - and d -benzy-
lamine, respectively. This amounted to a net addition of
46 amu to the nonlabeled benzylamine. An addition of
three oxygen atoms with the possibility of further oxida-
tion of an alcohol functional group to the corresponding
ketone appeared likely. The LC/MS analysis of extract
in which CYP2A1 was incubated with d :d - or d :d -
0 7 0 2
benzylamine showed that four and two deuteriums were
lost from the labeled compounds, respectively. The total
0
.5 mg of cDNA expressed human FMO1, FMO3 and FMO5,
diethylenetriaminepentaacetic acid (1.2 mM), and 0.05 M
phosphate buffer (pH 8.4) to a final volume of 1 mL. The
incubations, extractions of samples, and LC/MS analysis of
samples were done as described above.
loss of two deuteriums from d
2 0
-benzylamine in the d :
In Vit r o Met a b olism b y cDNA E xp r essed R a t P 450
En zym es. To a buffered solution (either sodium phosphate or
d
2
-benzylamine incubation implicates an oxidation had
occurred at the benzylic position with the formation of
benzamide. This would account for an addition of 14 amu
to the mass of benzylamine. Further oxidation on the
aromatic ring with an insertion of two oxygen atoms
TRIS, 100 mM, pH 7.4) of d
20 µM), MgCl (3 mM), and rat P450 enzyme (50 pmol) was
added NADPH (2 mM), and the solution was incubated at 37
C for 45 min. The incubations were performed in duplicate with
one set of samples fortified with GSH. The reactions were
terminated by the addition of 2 mL of acetonitrile. The super-
natant was dried and analyzed by LC/MS. The following is a
list of rat cDNA-expressed enzymes employed in the study: 1A1,
0 7 0 2
:d -, d :d -benzylamine or benzamide
(
2
°
(resulting in a loss of two deuteriums from the aromatic
ring) would account for the other 32 amu; hence, a net
addition of 46 amu was observed. In vitro metabolism
studies with benzamide (nonlabeled) incubated with
either CYP 2E1 or 2A1 showed the formation of the same
1
3
A2, 2A1, 2A2, 2B1, 2C6, 2C11, 2C12, 2D1, 2D2, 2E1, 3A1, and
A2.
+
metabolite (MH at m/z 154), as was observed with
P la sm a Sta bility of Ben zyla m in e in Ra ts. The in vitro
benzylamine, providing evidence in support of benzamide
as the likely precursor.
plasma stability of benzylamine was determined by incubating
the compound (50 µM) in rat plasma (1 mL) at 37 °C for 4 h.
After precipitation of proteins with acetonitrile (4 mL), the
supernatants were dried under nitrogen and the residue recon-
stituted in the HPLC mobile phase (200 µL) and analyzed for
benzylamine metabolite(s).
Benzylamine was fairly stable in rat plasma. This is
in contrast to the results we had obtained earlier with
DPC 423 (Figure 1), a compound possessing a benzy-
lamine moiety, which was susceptible to degradation by
semicarbazide sensitive amine oxidase (SSAO) present
in rat plasma (4). In the present study we observed that
Isola tion of Meta bolites M2 a n d M3 fr om Ra t Bile. Bile
duct-cannulated male Sprague-Dawley rats were dosed with
benzylamine and bile collected as described above. The bile
samples were diluted 1:1 with distilled water and loaded onto
C18 cartridges preconditioned with methanol and water. The
samples were allowed to elute under gravity. The cartridges
were subsequently washed with 30 mL of water followed by
elution with 30 mL aliquots of different percentages of methanol
in water. Each of the fractions was checked by LC/MS for the
presence of metabolites. The fractions containing the metabolites
were pooled and dried, and the residues reconstituted in (5:95
benzylamine was slowly metabolized to the glucoside
+
conjugate (MH at m/z 270 and 277 for d
0
- and d
7
-
benzylamine, respectively) in the presence of rat plasma.
Most of benzylamine had remained unchanged over the
course of 4 h with the glucoside conjugates accounting
for less than 5% conversion of the substrate. A similar
conversion was observed from incubations with plasma
from both dog and human.

1
194 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Sch em e 1. P r op osed Meta bolic P a th w a ys of Ben zyla m in e in Ra ts
Mutlib et al.
Sch em e 2. P 450 2A1 a n d 2E1 Con ver sion of Ben zyla m in e to Ben za m id e a n d 3,4-Dih yd r oxyben za m id ea
a
The lack of specific GST in the cDNA expressed system leads to the catechol metabolite rather than to the GSH adduct, M2.
In Vivo Stu d ies. Benzylamine was extensively me-
phase I metabolites such as the aldehyde and carboxylic
acid (Scheme 1) were not detected in the bile or urine
samples. The hippurate (M1) was the major metabolite
detected in the bile and urine samples of rats dosed with
tabolized by rats to a number of metabolites including
hippuric acid and several GSH adducts. Little or no
glucuronide or sulfate conjugates were detected. The

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1195
F igu r e 2. (A) LC/MS/MS total ion current (TIC); (B) UV trace; and (C) MS/MS spectrum of the protonated parent ion MH+ at m/z
80 of hippuric acid metabolite (glycine conjugate) present in urine of a rat dosed with 300 mg/kg of benzylamine hydrochloride.
1
benzylamine hydrochloride. The LC/MS total ion current
TIC) and LC/UV traces of urine samples (Figure 2)
as the base peak. The MS/MS spectrum of this metabolite
showed ions at m/z 427, 370 (-glycine), 316 (-pyro-
glutamate), 306 (GSH), 298, 288, 281, and 177. Selected
ion monitoring (SIM) studies suggested that this me-
tabolite accounted for less than 2% of the administered
dose. The structure of M2 was confirmed by isolating and
(
indicated that greater than 95% of the drug-related
material was excreted as the hippuric acid. Likewise, the
LC/MS and LC/UV traces of bile samples suggested that
hippuric acid was the major component. The GSH ad-
ducts accounted for less than 20% of total dose excreted
in bile as determined by selected ion monitoring studies.
However, in the absence of a radiolabeled compound, it
was difficult to obtain the absolute levels of each me-
tabolite in the biological samples, most notably in the
presence of endogenous components.
1
characterizing by NMR experiments. The H NMR showed
signals at δ 7.85 (1H, bs, gly NH), 7.70 (1H, bs, CONH
7.10 (1H, bs, CONH
(1H, m), 4.20 (1H, d, CH(OH)), 4.00 (1H, d, CHSCH
2
),
2
), 6.80 (1H, d), 6.18 (1H, m), 6.05
2
),
3.50 (1H, glu R, m), 3.35 (1H, m, cys R), 3.30 (2H, m, gly
R), 2.72 (1H, m, cys â), and 2.60 (1H, m, cys â′), 2.35 (2H,
m, glu γ), 2.00 (1H, m, glu â), and 1.90 (1H, m, glu â′).
Ch a r a cter iza tion of Meta bolites in Ben zyla m in e
Dosed Ra ts. Metabolites M1-M4 showed a character-
istic retention of five deuteriums as indicated by the
1
The H NMR of this metabolite was identical to that of
M3 except for the presence of glutamate proton signals
in the spectrum of GSH conjugate (M2).
+
presence of pairs of MH ions, separated by 5 amu, in
the mass spectra of bile samples obtained from rats dosed
Metabolite M3: The cysteinylglycine conjugate derived
from M2 produced MH at m/z 316. The SIM studies
+
with 1:1 w/w mixture of d
spectra of metabolites from rats dosed with d
0
:d
7
-benzylamine. The mass
:d -benzy-
0
2
suggested that this metabolite accounted for less than
5% of the administered dose. This metabolite was isolated
from rat bile, and its structure determined by NMR. The
MS/MS spectrum of the protonated parent ion produced
lamine showed a total loss of both deuteriums, and hence,
+
MH of these compounds appeared as a single peak.
Metabolite M1: This was the major metabolite found
+
1
in rat bile and urine. Metabolite M1 produced MH at
ions at m/z 298, 281, 177 (cys-gly), and 152. The H NMR
+
m/z 180 with the ammonium adduct [M + NH
4
] at m/z
(see Figure 3) showed signals at δ 7.85 (1H, bs, gly NH),
1
97 in the mass spectrum. The structure of M1 was
7.70 (1H, bs, CONH
d), 6.18 (1H, m), 6.05 (1H, m), 4.20 (1H, d, CH(OH)), 4.00
(1H, d, CHSCH ), 3.35 (1H, m, cys R), 3.30 (2H, m, gly
2 2
), 7.10 (1H, bs, CONH ), 6.80 (1H,
confirmed by comparing the LC/MS retention time and
mass spectral data with those produced by hippuric acid.
The formation of M1 as a metabolite of benzylamine was
also confirmed by studies done with labeled compounds.
The LC/MS spectrum of bile and urine from rats dosed
2
R), 2.72 (1H, m, cys â), and 2.60 (1H, m, cys â′). The
structure of M3, shown in Figure 3 and Scheme 1, was
determined by TOCSY and HMBC experiments.
Metabolites M4: The cysteine conjugate formed from
0 7
with 1:1 mixture of d :d -benzylamine hydrochloride
+
+
showed MH at m/z 180 and 185, respectively (Figure
the catabolism of M2 produced MH at m/z 259. An
2
1
). The MS/MS spectrum of MH+ ion showed ions at m/z
05 and 162 corresponding to characteristic losses of
estimate of M2 could not be obtained due to the unavail-
ability of synthetic standard. This metabolite found in
rat bile was not further characterized.
glycine (-75 amu) and water, respectively.
+
Metabolite M2: Metabolite M2 showed MH at m/z
Metabolites M5-M11 showed a characteristic reten-
tion of seven deuteriums as indicated by the presence of
4
45 and was found in rat bile. The mass spectrum showed
+
a loss of water from the protonated parent ion (m/z 427)
pairs of MH ions, separated by 7 amu, in the mass

1
196 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
F igu r e 3. 1H NMR of the cysteinylglycine conjugate (M3) isolated from bile of rats dosed with 300 mg/kg of benzylamine hydrochloride.
spectra of bile samples from rats dosed with 1:1 w/w
mixture of d :d -benzylamine. Similarly these metabolites
leading to a carbon-nitrogen bond had to be ruled out.
This was demonstrated by HMBC experiments whereby
it was shown that the methylene of the cysteine moiety
was coupled to the carbonyl group (data not shown). The
methylene from the aminomethyl group of benzylamine
was also coupled to the same carbonyl group. The
structure of this metabolite was determined to be S-
[benzylcarbamoyl] glutathione (Scheme 1).
Metabolite M6: The cysteinylglycine conjugates pro-
duced from M5 showed the protonated molecular ions at
m/z 312 and 319, corresponding to nonlabeled and labeled
metabolites, respectively. The SIM studies suggested that
this metabolite accounted for less than 2% of the admin-
istered dose. The LC-MS/MS spectrum of this conjugate,
0
7
showed retention of both deuteriums as observed by the
+
presence of pairs of MH ions, separated by 2 amu, in
the spectra of samples from rats dosed with 1:1 w/w
mixture of d :d -benzylamine. LC/MS/MS analyses of the
0 2
metabolites suggested that the aminomethyl functional
group was intact; however an increment of 28 amu to
molecular mass of benzylamine, corresponding to carbon
monoxide addition, suggested the possibility of an isocy-
anate intermediate. The existence of this additional CO
group was confirmed by NMR studies with synthetic
standards of metabolites produced by reacting benzyl
isocyanate with the appropriate nucleophiles.
Metabolite M5: This GSH conjugate was found in rat
bile. The SIM studies suggested that this metabolite
accounted for less than 7% of the administered dose. The
protonated molecular ions for the nondeuterated and
deuterated GSH adducts appeared at m/z 441 and 448,
respectively. The MS/MS spectrum of m/z 441 showed ion
fragments at m/z 366 (-glycine), 312 (-pyroglutamate),
and 179. A similar mass spectrum of ions produced from
loss of glycine and pyroglutamate was produced from MS/
2
found in rat bile, showed ions at m/z 179 (loss of Ar-CH -
NHCO), 162, and 144. Further MS/MS studies with the
fragment ion at m/z 179 showed the fragmentation
pattern was identical to that produced by an authentic
standard of cysteinylglycine. The LC/MS retention time
and MS/MS fragmentation pattern of synthetic standard
was obtained and found to be identical to those produced
by the metabolite. The structure of M6, shown in Scheme
1, was confirmed as S-[benzylcarbamoyl]cysteinylglycine.
+
MS of the deuterated analogue (MH at m/z 448). The
Metabolite M7: The cysteine conjugates derived from
+
LC/MS retention time and MS/MS fragmentation pattern
of synthetic standard was obtained and found to be
identical to those produced by the metabolite. The
structure of the synthetic standard was confirmed by
NMR experiments. It was important to demonstrate that
during the chemical synthesis of this standard, isocyan-
ate had reacted with the sulfhydryl of GSH forming a
carbon-sulfur bond. The possibility of benzyl isocyanate
reacting with the free amine of GSH (glutamate moiety)
M6 produced MH at m/ z 255 and 262 for d
0
- and d
7
-
labeled compounds, respectively. An estimate of M7
levels in bile could not be obtained due to the unavail-
ability of synthetic standard. No further characterization
of this metabolite was done.
Metabolite M8: This metabolite was detected in the
urine of rats dosed with benzylamine. The SIM studies
suggested that this metabolite accounted for less than
3% of the administered dose. The mass spectrum showed

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1197
F igu r e 4. 1H NMR of the synthetic N-acetyl-S-[benzylcarbamoyl]cysteine, M8.
MH+ at m/z 297 with ion fragments at m/z 164 (N-
glutamate was coupled to the same carbonyl group as
was the methylene of the benzylamine. The LC/MS
retention time and MS/MS fragmentation pattern of
synthetic standard was obtained and found to be identical
to those produced by the metabolite. The structure of M9,
shown in Scheme 1, was confirmed as N-[benzylcarbam-
oyl]glutamate.
acetylcysteine) and 122 (cysteine). The deuterium-labeled
+
cysteine conjugate showed MH at m/z 304 as expected
with the retention of all seven deuteriums. The LC/MS
retention time and MS/MS fragmentation pattern of
synthetic standard was obtained and found to be identical
1
to those produced by the metabolite. The H NMR of the
N-acetylcysteine conjugate is shown in Figure 4. The
isocyanate linkage to the sulfhydryl of N-acetylcysteine
was clearly demonstrated by HMBC experiments whereby
it was shown that the methylene of the cysteine moiety
was coupled to the carbonyl group (Figure 5). The
methylene from the aminomethyl group of benzylamine
was also found to be coupled to the same carbonyl group.
The existence of this carbonyl group, added to the
benzylamine during its metabolism was adequately
demonstrated by these NMR experiments. The structure
of M8 was confirmed as N-acetyl-S-[benzylcarbamoyl]-
cysteine.
Metabolite M10: The cysteinylglutamate conjugate
found in rat bile displayed a MH at m/z 384. The SIM
+
studies suggested that this metabolite accounted for less
than 2% of the administered dose. The MS/MS of pseudo-
molecular ion at m/z 384 produced major fragment ion
at m/z 255 (-pyroglutamate). The LC/MS retention time
and MS/MS fragmentation pattern of synthetic standard
was obtained and found to be identical to those produced
by the metabolite. The structure of M10, shown in
Scheme 1, was confirmed as S-[benzylcarbamoyl]cys-
teinylglutamate.
Metabolite M11: Rats dosed with benzylamine ex-
creted a unique oxidized GSH conjugate of benzyl isocy-
anate. The SIM studies suggested that this metabolite
accounted for less than 1% of the administered dose. The
Metabolite M9: The glutamate conjugate of benzyl
isocyanate was found in rat bile. The SIM studies
suggested that this metabolite accounted for less than
+
1
% of the administered dose. The metabolite produced
protonated molecular ions (MH ) were observed at m/z
+
MH at m/z 281 with the major fragment ion at m/z 148
corresponding to the protonated glutamic acid). The urea
0 7
746 and 753, for d - and d -labeled metabolites, respec-
+
(
tively. The MS/MS spectra of these metabolites (MH at
m/z 746 and 753) are shown in Figure 7. The ion
fragments at m/z 639, 613, 595, 538, 510, 484, 466, 409,
and 355 were found to be common in the mass spectra of
both labeled and nonlabeled adduct. However, the MS/
linkage formed between the amine of the glutamic acid
and the carbonyl group of the benzyl isocyanate was
demonstrated by HMBC results (Figure 6). It was shown
that the R carbon bearing the carboxylic acid of the

1
198 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
F igu r e 5. HMBC spectrum of the N-acetylcysteine conjugate (M8) showing the correlation between the carbonyl group and the
methylene groups (Ar-CH -NH-CO-S-CH -). The carbonyl group of the carboxylic acid of cysteine was found to be coupled to the cys
2
2
R proton only.
F igu r e 6. HMBC spectrum of the glutamic acid conjugate (M9) showing the correlation between the carbonyl group and the methylene
groups (Ar-CH -NH-CO-NH-CH(COOH)-CH -).
2
2
MS spectra also showed important differences between
the ion fragments produced by these conjugates. Specific
ions at m/z 728, 671, 617, and 542 were only seen in the
mass spectrum of the nonlabeled adduct (Figure 7).

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1199
F igu r e 7. LC/MS/MS of protonated parent ions of (A) nonlabeled (m/z 746) and (B) labeled (m/z 753) glutathione conjugate (M11)
present in bile of rats dosed with d :d -benzylamine hydrochloride.
0
7
Similarly ions unique to the d
7
-labeled adduct were
observed in the mass spectrum. The analysis of bile
samples from rats dosed with d - and d -benzylamine
observed and included fragments at m/z 735, 678, 624,
and 549 which were 7 amu higher than those produced
by the nonlabeled adduct. Further MS/MS studies of the
common fragments (found in spectra of both labeled and
nonlabeled adducts) indicated losses of pyroglutamate
0
7
+
showed MH ions at m/z 398 and 404, respectively. The
LC/MS retention time and MS/MS fragmentation pattern
of synthetic standard was obtained and found to be
1
identical to those produced by the metabolite. The H
(
-129 amu) and glycine (-75 amu). The fragmentation
NMR of the adduct M12 is shown in Figure 8. The
structure of M12, shown in Scheme 1, was confirmed as
S-benzyl-L-glutathione.
patterns suggested an existence of a peptide capable of
losing two glycine and one glutamate moiety. It was
postulated that the adduct was formed by an interaction
of reactive benzyl isocyanate (or its precursor) with
oxidized GSH. The MS/MS studies showed a loss of only
one glutamate, suggesting that the amine functional
group of one of the glutamic acids in GSSG reacted with
the benzyl isocyanate. A synthetic standard was pre-
pared, and its LC/MS and MS/MS fragmentation pattern
studied. The MS/MS of the MH+ (at m/z 746) of the
synthetic standard matched that of the metabolite de-
tected in the bile sample. However, due to the incomplete
structural elucidation of the synthetic standard, the
structure of M11 was tentatively assigned as the oxidized
GSH conjugate of benzyl isocyanate.
Metabolite M13: This metabolite was formed via
catabolism of the M12. The SIM studies suggested that
this metabolite accounted for less than 3% of the admin-
istered dose. The mass spectrum of metabolite showed
+
MH at m/z 212. The loss of one deuterium atom from
the aminomethyl side chain was confirmed by observing
+
MH at m/z 212, 213, and 218 from bile samples of rats
dosed with d -, d -, and d -benzylamine (see Scheme 1).
0
2
7
The MS/MS spectrum of this conjugate showed ions at
m/z 195 (base peak) and 91. The LC/MS retention time
and MS/MS fragmentation pattern of synthetic standard
was obtained and found to be identical to those produced
by the metabolite. The structure of M13, shown in
Scheme 1, was confirmed as S-benzyl-L-cysteine.
Metabolites M12-M13 showed a characteristic reten-
tion of six deuteriums as evidenced by the presence of
pairs of MH ions, separated by 6 amu, in the mass
Metabolites M14-M15: Metabolites M14 and M15
+
+
were confirmed as GSH conjugates with MH at m/z 473
spectra of bile samples obtained from rats dosed with 1:1
and 455, respectively. The structures of these metabolites
were tentatively based on MS/MS data from labeled and
nonlabeled compounds. Synthetic standards were not
available to confirm the structures of these metabolites.
Metabolites M14 and M15, based on the SIM experi-
ments done with other GSH conjugates, each appeared
to account for less than 1% of the administered dose.
Ch a r a cter iza tion of Meta bolites in N-Hyd r oxyl-
ben zyla m in e Dosed Ra ts. Rats dosed with N-hydroxy-
lbenzylamine excreted a GSH conjugate, which produced
w/w mixture of d
w/w mixture of d
0
:d
:d
7
-benzylamine. The studies with 1:1
2
-benzylamine showed retention of
0
+
only one deuterium in the MH of these two metabolites.
Metabolite M12: This was a GSH conjugate with MH+
at m/z 398. The SIM studies suggested that this metabo-
lite accounted for less than 5% of the administered dose.
The MS/MS data revealed a characteristic loss of 129
amu (-pyroglutamate) to give the base peak at m/z 269.
An ion at m/z 323 formed by loss of glycine was also

1
200 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
F igu r e 8. 1H NMR of S-benzyl-L-glutathione, M12.
Sch em e 3. P r op osed P a th w a ys Lea d in g to th e F or m a tion of M5-M11 fr om Ben zyla m in ea
a
A reactive intermediate, Ar-CH2-NH(CO)-X such as carbamyl glucuronide (pathway 1 with X as glucuronic acid) or benzyl isocyanate
(
pathway 2) formed from a formamide (X ) H) are postulated to react with nucleophiles such as GSH, GSSG and glutamate to form the
metabolites M5-M11.
MH+ at m/z 427. The SIM studies suggested that this
metabolite accounted for less than 10% of the adminis-
tered dose. The MS/MS spectrum of this conjugate
showed ion fragments at m/z 352, 298, 265 (base peak),
and 177. The formation of this conjugate has previously
been postulated to occur via the hydroxylamine and the
nitroso intermediates (Scheme 5) (5). The structure of this
metabolite was confirmed by comparing the chromato-
graphic and mass spectral behavior of the metabolite
with an authentic standard that was previously synthe-
sized (5). It was shown that the metabolite found in rat
bile was identical to the standard and hence was con-
firmed as having the structure as shown in Figure 9. In
addition to the GSH adduct, rats excreted both M1 and
benzylamine. These were the major metabolites account-
ing for greater than 90% of the administered dose as
determined by SIM. None of the GSH adducts previously
identified in the benzylamine-dosed rats were found in
the bile of animals dosed with N-hydroxyl benzylamine.
Ch a r a cter iza tion of Meta bolites in Ben za m id e
Dosed Ra ts. Rats dosed with benzamide produced large
quantities of metabolites M2, M3, and M4. The LC/UV

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1201
Sch em e 4. P r op osed P a th w a y for th e F or m a tion of M2 fr om Ben zyla m in e via Ben za ld eh yd e In ter m ed ia te
Sch em e 5. Oxid a tion of N-Ben zylh yd r oxyla m in e to Rea ctive In ter m ed ia tes a n d Su bsequ en t Con ju ga tion s
profiles indicated that the GSH-derived adducts (M2, M3,
and M4) were the major drug-related components in all
of the bile samples. Trace quantities of benzamide were
found in bile and urine samples. A representative LC/
UV profile of bile from a rat dosed with benzamide is
shown in Figure 10. Interestingly, the hippuric acid (M1)
was also found in the bile and urine samples. M1 was
the major metabolite in urine of benzamide dosed rats.
In addition to these metabolites, at least two glutamate
conjugates, present as minor metabolites, were found.
These were the glutamate adducts of M2 and M4, giving
derived from M12 was also detected in the bile. This
metabolite, with MH at m/z 269, produced ion fragments
at m/z 252, 166, and 149. The levels of this metabolite
could not be estimated due to the unavailability of the
synthetic standard.
+
Discu ssion
We had previously shown that DPC 423 (Figure 1), a
compound possessing a benzylamine moiety, was con-
verted to several novel metabolites (3-5). Studies were
performed to compare and contrast the metabolic disposi-
tion of benzylamine with DPC 423 and its analogues. The
in vivo metabolism of benzylamine was investigated in
rats, and the structures of metabolites were identified
by LC/MS, high-field NMR, and by comparison with
synthetic standards. Furthermore, the metabolism of
primary metabolites (benzaldehyde and benzamide) of
benzylamine was studied to confirm the proposed meta-
bolic pathways (Scheme 1). Benzylamine was converted
primarily to hippuric acid by rats, although a number of
other minor metabolites were detected and characterized
(Scheme 1). These included novel metabolites produced
by pathways not previously described. Some of the
+
MH at m/z 574 and 388, respectively. These adducts
were not characterized further.
Ch a r a cter iza tion of Meta bolites in Ben za ld eh yd e
Dosed Ra ts. Benzaldehyde dosed rats excreted hippuric
M1 as the major metabolite (greater than 90% of the
administered dose) in both bile and urine. However, it
was found that metabolites M12 and M13 were also
present in bile samples. Our SIM studies suggested that
these metabolites accounted for less than 10% of the
administered dose. The identities of M12 and M13 were
confirmed by comparing the MS/MS data previously
obtained from synthetic standards. A metabolite corre-
sponding to the cysteinylglycine conjugate intermediate

1
202 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
F igu r e 9. LC/MS/MS TIC (top) and MS/MS spectrum (middle) of the protonated parent ion MH+ at m/z 427 of the GSH conjugate
of benzaldehyde oxime present in rat bile dosed with benzylhydroxylamine HCl. The UV trace of the sample is shown at the bottom
[the asterisk (*) representing the GSH adduct in the bile].
F igu r e 10. LC/MS/UV profile of bile from a rat dosed with 300 mg/kg of benzamide. The UV chromatogram (A) shows the presence
of M2, M3, and M4. The mass spectrum of the GSH adduct (M2) is shown in the bottom pane (B).
metabolites were formed through P450 oxidation while
others were formed independent of P450. The metabolite
profile of benzylamine in rats showed the presence of
various GSH adducts which were formed via different
metabolic pathways. In addition to the known metabolic
pathways that led to bioactivation of benzylamines, at
least two previously unreported metabolic routes were
discovered during these studies.
(M1). The hippurate was the major metabolite excreted
in the bile and urine of rats dosed with benzylamine,
benzaldehyde, or benzamide. Studies with benzaldehyde
confirmed that it was being formed from benzylamine as
a precursor to the carboxylic acid. Dosing rats with an
equimolar mixture of d - and d -benzylamine confirmed
0
7
the oxidative deamination of benzylamine. The LC-APCI/
MS spectrum showed that the d - and d -benzylamine
0
7
+
Benzylamine was metabolized to benzaldehyde via
oxidative deamination. The aldehyde was rapidly con-
verted to the carboxylic acid, which was subsequently
conjugated with glycine and excreted as hippuric acid
produced protonated (MH ) ions at m/z 180 and 185 of
nonlabeled and labeled hippuric acid metabolites, respec-
tively. However, the relative intensities of these ions
suggested a significant intramolecular deuterium isotope

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1203
effect in vivo. The deuterium isotope effect in forming a
carboxylic acid from a benzylamine has been previously
demonstrated (4).
product. On the other hand, hydroxylation at the benzylic
position of benzylamine leads to a product capable of
being further oxidized. The remaining proton at the
benzylic position becomes very acidic (after the initial
hydroxylation) and is amenable to an abstraction by
P450. An abstraction of the benzylic proton (one electron
abstraction) would produce a radical that is resonance-
stabilized over the aromatic ring. A subsequent P450-
catalyzed electron abstraction would yield the corre-
sponding benzamide. The existence of M2 is indirect
evidence for the existence of a putative epoxide interme-
diate (Scheme 1). The epoxide intermediate is cleaved by
GSH to form a product that yields a characteristic NMR
pattern for a trisubstituted hexadiene ring (see Figure 3
The use of deuterium-labeled benzylamine also pro-
vided evidence that the hippuric acid was indeed a
metabolite and not entirely an endogenous product.
Analysis of control bile samples showed that hippuric acid
was excreted in the bile and urine of rats as an endog-
enous product, albeit at a very low level. Hippuric acids
are normal constituents in urine of mammals, and its
elevated levels in urine of animals are sometimes used
as a biomarker of tissue damage (16, 17). On dosing the
animals with benzylamine, benzaldehyde or benzamide,
the levels of hippuric acid in urine increased by greater
than 1000-fold. The deuterium-labeled benzylamine dem-
onstrated that the hippuric acid was indeed a metabolite,
and its high levels in the urine/bile were not due to an
injury to a tissue. The in vivo studies performed with the
primary metabolites, benzaldehyde (formed by MAO),
and benzamide (formed by P450) showed that both of
these compounds were capable of forming the hippuric
acid.
The aldehydes produced by oxidative deamination of
xenobiotics are generally metabolized either to alcohols
by aldehyde reductase or alcohol dehydrogenase or to
acids by aldehyde dehydrogenase or aldehyde oxidase.
In this instance it appears that rats preferentially convert
the aldehyde to carboxylic acid. The aldehyde and car-
boxylic acid metabolic intermediates were not detected
in the bile/urine samples probably due to the rapid
conjugation of the benzoic acid with glycine. The mito-
chondrial metabolism of benzylamine to the benzalde-
hyde, mediated by amine oxidases, has been previously
described (10-15). Benzaldehydes are converted to ben-
zoic acid by the aldehyde dehydrogenase present in the
hepatic mitochondria. The benzoic acid has been found
to be conjugated with glycine in the mitochondria where
all the cofactorssATP, CoA, and glycinesexist (18). The
extremely low turnover of benzylamine by rat microsomes
suggests that P450 may not play a significant role in
converting it to the benzoic acid. This was supported by
coadministering benzylamine with ABT, a potent P450
inhibitor (19, 20), to rats. The analysis of bile/urine from
these rats showed that the levels of M1 were essentially
unchanged while the formation of other metabolites such
as M2 and M12 was reduced but not totally abolished.
This is consistent with the previous observations that
benzylamine is a good substrate for MAO present in
mitochondria (10-15).
1
for H NMR of cysteinylglycine conjugate, M3). The
identification of M2 and M3 was achieved by NMR
analysis of the isolated metabolites. Rats that were dosed
with 1:1 w/w mixture of d
0
:d
that showed MH at m/z 316 only; i.e., both the deute-
7
riums were lost. Studies with 1:1 w/w mixture of d :d -
2
-benzylamine produced M3
+
0
benzylamine showed loss of two deuteriums and retention
of five labels, presumably on the aromatic ring of M3 (see
Figure 11). This study clearly demonstrated that oxida-
tion occurred at the labeled aminomethyl side chain.
Additional in vivo studies with benzamide confirmed the
hypothesis that benzylamine was converted to benzamide
prior to the formation of M2. Metabolites M3 (cysteinylg-
lycine) and M4 (cysteine) were found as catabolites of M2.
In addition to these metabolites, glutamate conjugates
of M2 and M4 were also found in rat bile. This is
consistent with our previous observations that glu-
tathione-derived adducts are capable of being conjugated
with glutamic acid via γ-glutamyltranspeptidase (1, 3).
The results from the in vivo studies were consistent with
the data obtained from in vitro studies. It was demon-
strated that benzylamine was metabolized to the benza-
mide and 3,4-dihydroxybenzamide by rat CYP2A1 and
2E1 enzymes (data not shown). The presence of 3,4-
dihydroxybenzamide from incubations with either ben-
zylamine or benzamide suggests that CYP2E1 and 2A1
are responsible for both of the metabolic steps (see
Scheme 2). It is postulated that the benzamide is
converted to an epoxide that reacts with GSH in the
presence of a specific GST to form M2. In contrast, in
the absence of GST, as is the case with cDNA expressed
microsomal system, this expoxide is further catabolized
to the catechol, 3,4-dihydroxybenzamide.
The structures of M5-M11 show an unusual addition
of nucleophiles such as GSH and glutamate to benzy-
lamine. A prerequisite modification of benzylamine may
be required prior to the addition of these nucleophiles.
The structures of the metabolites suggest that benzy-
lamine is converted to a reactive metabolite via a novel
metabolic pathway. It is postulated that the reactive
intermediate is either the benzyl isocyanate or an un-
The structures of the GSH adducts suggest that
benzylamine is metabolized to several chemically reactive
intermediates capable of interacting with a nucleophile
such as GSH. The formation of M2 appears to be via an
initial oxidation of benzylamine to benzamide, which is
subsequently converted to an epoxide intermediate. The
formation of amide from amines has been very well
documented, especially for cyclic compounds (21, 22). The
formation of cotinine from nicotine is a typical example
of such a metabolic pathway (21). Nicotine is R-hydroxy-
lated in the pyrrolidine ring to give a carbinolamine that
is oxidized further to the major metabolite, cotinine.
However, the formation of amides from alkylamines is
usually precluded by the rapid hydrolysis of the unstable
carbinolamine that is formed during the P450-catalyzed
N-dealkylation. This usually leads to an N-dealkylated
product and an aldehyde instead of a further oxidized
known precursor, Ar-CH
capable of undergoing a nucleophilic displacement. In-
teraction of benzyl isocyanate or Ar-CH -NH(CO)-X with
2
-NH(CO)-X (see Scheme 3),
2
GSH, glutamic acid, cysteinyl glutamate, or oxidized GSH
leads to the formation of M5, M9, M10, and M11,
respectively. Further catabolism of M5 via the mercap-
turic acid pathway produces metabolites M6, M7, and
M8. The formation of GSH adducts from isocyanates and
isothiocyanates have been described in the literature
(23-26). The partial positive charge residing on the
carbonyl of isocyanates makes it susceptible to nucleo-

1
204 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
+
F igu r e 11. LC/MS spectra of cysteinylglycine conjugate (showing the MH ) present in bile samples of rats dosed with 300 mg/kg
of (1:1 w/w) mixture of d :d -benzylamine hydrochloride (A) and d :d -benzylamine hydrochloride (B).
0
7
0
2
philic attack (e.g., GSH). However, the mechanism for
the formation of the benzyl isocyanate from benzylamine
has not been fully elucidated. In vitro studies with
various rat P450 enzymes (fortified with GSH) suggested
that M5 could not be produced by any of the isoenzymes.
This was expected since P450 would not add an element
of CO to the benzylamine. The possible pathways leading
to the formation of these unusual conjugates (M5-M11)
are shown in Scheme 3. These pathways suggest a
formation of new carbon-nitrogen bond in benzylamine.
The formation of new carbon-nitrogen bonds via N-
methylation, N-acetylation, and N-glucuronidation has
been described previously (27-29). All of these metabolic
processes are non-P450 dependent and are considered
phase II metabolism. However, none of these metabolic
pathways are expected to yield an intermediate that
could ultimately lead to the formation of metabolites
M5-M11. It is postulated that a formamide of benzy-
lamine could be formed where X ) H (in Scheme 3). The
existence of formamides as metabolites of a number of
amines has been described previously (30-34). The
formamide metabolites of these compounds have been
attributed to both enzymatic formylation mechanisms
involving enzymes such as kynurenine formamidase (35)
in the case of some arylamines or have been demon-
strated to arise from alpha carbon oxidation in the case
of aminopyrine (36) and recipavrin (34). Kynurenine
formamidase is capable of transferring the formyl group
administration of ABT (P450 inhibitor) to rats dosed with
benzylamine perhaps did not produce a total elimination
of P450 activity, hence the conversion of formamide to
isocyanate was not totally blocked.
It is also proposed that the amine nitrogen can react
with an endogenous compound to form an acyl conjugate
(such as the carbamyl glucuronide), which has the
potential to interact with other endogenous components
or decompose to an isocyanate (Scheme 3). The formation
of carbamic acid from amines is a metabolic pathway that
leads to the creation of a new carbon-nitrogen bond. Due
to its inherent instability, the existence of carbamic acid
metabolites is usually demonstrated by characterizing
their glucuronide conjugates, often referred to as car-
bamyl glucuronides. The formation of carbamic acids
from amines trapped as carbamyl O-â-D-glucuronides has
been described previously (42-44). The pathway leading
to carbamic acid intermediates from amines is believed
be due to the nonenzymatic reaction of the amine with
dissolved carbon dioxide (44). Spontaneous formation of
carbamates has been reported for a number of amino
acids (45) and other amines (46). Compared to our
previously studied benzylamine, DPC 423 (4, 5), which
formed the carbamyl glucuronide as the major metabo-
lite, benzylamine itself did not excrete a similar carbamyl
glucuronide conjugate. One could postulate that if the
carbamyl glucuronide of benzylamine was formed, it
2
could have acted as the reactive intermediate Ar-CH -
(
from N-formyl-L-kynurenine) to aromatic amines such
NH(CO)-X, with glucuronic acid as a good leaving group
(X in Scheme 3). The high reactivity of this carbamyl
glucuronide could explain its absence in the biological
fluids of rats dosed with benzylamine. Furthermore, since
the formation of carbamyl glucuronide is not dependent
on P450 enzymes, administration of ABT would not have
affected the levels of metabolites M5-M11. Indeed, rats
dosed with ABT showed only a slight decrease in the
levels of M5-M11. Experiments will be conducted in the
future to determine if the carbamyl glucuronide or the
formamide of benzylamine are potential precursors to
metabolites M5-M11.
as aniline (35). Usually formylation catalyzed by this
enzyme is regarded as a detoxification pathway, espe-
cially for aromatic amines. However, in this case the
formation of formamide and its subsequent metabolism
could have led to a more reactive isocyanate intermediate
as shown previously (25, 37, 38). The toxicities of forma-
mides have been attributed to the formation of these
reactive isocyante intermediates via P450-catalyzed reac-
tions (38-41). Sequential one-electron oxidations of me-
thyformamide by P450 2E1 have been suggested to lead
to the reactive methyl isocyanate metabolite (40). The

Bioactivation of Benzylamine
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1205
The reactivity of benzyl isocyanate or its precursor was
also demonstrated by isolating and characterizing other
peptide adducts such as the glutamate (M9) and oxidized
GSH adducts (M11). The formation of isocyanate-derived
peptide adducts was interesting since it showed that the
reactive intermediate was not only capable of interacting
with sulfhydryl but also reacted with other nucleophiles
such as the amino groups of the glutamate and GSSG.
Adducts of GSSG are seldom, if ever, described for
compounds that form reactive intermediates. Typically,
the GSH adduct, formed by an interaction of sulfhydryl
of GSH with an electrophile, is identified. An interaction
of amino group of GSSG with an electrophilic metabolite
such as the benzyl isocyanate has not been previously
described. LC/MS/MS capability of an ion trap instru-
ment as well as the availability of selectively labeled
benzylamine facilitated the identification of this conju-
gate. The LC/MS/MS spectra of labeled and nonlabeled
benzylamine. These included metabolites such as M14
and M15 whose structures were tentatively assigned
based on mass spectral data.
Because these studies were performed at appreciable
doses of benzylamine, it is difficult to predict the relative
contribution of each pathway at trace doses of the
compound. It is quite possible that, at the high doses of
benzylamine, metabolic switching toward the formation
of hippuric acid predominated. Alternatively, the monoam-
ine oxidases responsible for the deamination to benzal-
dehyde are present in abundant quantities in vivo and
are capable of easily metabolizing benzylamine at all dose
levels. Further studies with low doses of benzylamine
were not performed. However, such experiments may
elucidate the relative contributions of other metabolic
pathways (as compared to hippuric acid pathway) at low
doses of benzylamine.
The metabolic disposition of benzylamine was also
investigated to provide mechanistic insight into the
biotransformation of the previously described benzy-
lamine, DPC 423. Although there were clear similarities
observed in the metabolites generated from the respective
compound, e.g., oxidative deamination to the aldehyde,
we found there to be several diverging pathways from
that observed previously from DPC 423 metabolism.
Previous work in our laboratory resulted in a proposed
scheme for DPC 423 involving the oxidation of the
benzylamine moiety to a hydroxylamine intermediate,
which, after further oxidation to the nitroso species,
tautomerizes to its corresponding oxime. The oxime of
DPC 423 was consequently conjugated by GSH (4), the
mechanism of which is not fully elucidated. Although the
current data support the involvement of a similar oxida-
tion pathway for the metabolism of the aminomethyl side
chain of N-benzylhydroxylamine (see Scheme 5), the
metabolism of benzylamine diverges, mediated predomi-
nantly by MAO, to yield the benzaldehyde intermediate.
Subsequent oxidation of benzaldehyde and conjugation
by glycine leads to the formation of the hippurate M1
and represents a divergent pathway from that observed
with DPC 423. Importantly, one can also imagine a
scheme in which the oxime of N-benzylhydroxylamine is
hydrolyzed and conjugated to form the hippurate M1
(Scheme 5). However, it appears that benzylamine does
not form the N-hydroxylbenzylamine as readily as DPC
423. It is noteworthy that N-hydroxylbenzylamine is
disposed of in a manner similar to DPC 423, i.e., further
oxidation to a nitroso and forms the GSH conjugate of
the oxime. The GSH conjugation of the putative benzal-
dehyde metabolite, leading to M12 and M13, represents
the second divergent metabolic pathway between benzy-
lamine and DPC 423. While there was no apparent
intermediacy of an amide implicated in the disposition
of DPC 423, such an intermediate appears to represent
the third divergent pathway for benzylamine from DPC
423, leading to the GSH-derived metabolites M2, M3, and
M4. The formation of the benzamide as a metabolite of
benzylamine is perhaps due to the stability conferred on
a P450-generated radical intermediate capable of under-
going a second electron abstraction. Likewise, the in-
+
benzylamine-GSSG adduct with MH at m/z 746 and 753,
respectively, are shown in Figure 7. MS/MS studies were
done to demonstrate that the benzylamine moiety was
linked through a urea bond to the glutamate amino
functional group of the oxidized GSH. A synthetic stan-
dard of this hexapeptide adduct was made, and LC/MS/
MS studies were performed to show that it was identical
to the biological product. The postulated presence of a
2
reactive isocyanate or its precursor Ar-CH -NH(CO)-X is
indeed a novel pathway for metabolism of a nitrogen
containing compound. A review of the literature revealed
that such a metabolic reaction has not been described
before. Importantly, the reactivity of benzylamine via
formation of isocyanate or an isocyanate-derived meta-
bolic intermediate may possibly extend to other amines.
Toxicities associated with the bioactivation of amines
may be attributed in part to existence of such reactive
intermediates. With the application of versatile analytical
techniques, it is now possible to identify some of these
elusive metabolites that were once difficult to character-
ize.
The formation of M12-M13 showed a net displace-
ment of an amino group by GSH. A nucleophilic displace-
ment of halogen, such as bromide, with GSH has been
previously reported for haloalkanes (47-49). Halogens
are good leaving groups and their displacement by a
nucleophile such as GSH is expected to occur with or
without enzyme catalysis. However, a direct displace-
ment of amino functional group with GSH is unprec-
edented and is not expected to occur without any bioac-
tivation of the amine. Studies done with deuterium-
labeled benzylamine showed that one of the deuteriums
on the aminomethyl side chain was lost on formation of
M12. This implied that at least one electron oxidation
must have taken place prior to the formation of M12. The
metabolic conversion of benzylamine to benzaldehyde was
proposed and a study was subsequently conducted in vivo
to demonstrate that benzaldehyde was the precursor to
M12. A mechanism is proposed whereby the aldehyde is
further reduced to an alcohol, which is subsequently
converted by phase II enzymes to a good leaving group.
Displacement of this leaving group with GSH would
produce M12 (Scheme 4). Metabolite M13 is a cysteine
adduct derived from M12 via the mercapturic acid
pathway. The identities of M12 and M13 were confirmed
by comparisons with synthetic standards. In addition to
these metabolites, a number of other GSH adducts were
also detected and characterized in bile of rats dosed with
volvement of the proposed reactive intermediate, Ar-CH
2
-
NH(CO)-X, which leads to the formation of the conjugates
M5, M9, and M10, represents another major distinguish-
ing fate during the metabolism of benzylamine. The
determinants that orchestrate the final outcomes of
benzylamine and DPC 423 metabolism seem to be

1
206 Chem. Res. Toxicol., Vol. 15, No. 9, 2002
Mutlib et al.
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P450 oxidation, respectively. However, the obvious struc-
tural differences between DPC 423 and benzylamine may
alone influence metabolic fates due to substrate specifica-
tions of the various phase I and II enzymes involved in
disposition of these compounds.
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