Neurotoxicity studies with the monoamine oxidase B substrate 1-methyl-3-phenyl-3-pyrroline

Article abstract of DOI:10.1016/j.lfs.2007.06.014

The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately l

Full text of DOI:10.1016/j.lfs.2007.06.014

Life Sciences 81 (2007) 458–467
www.elsevier.com/locate/lifescie
Neurotoxicity studies with the monoamine oxidase B substrate
1-methyl-3-phenyl-3-pyrroline
Modupe O. Ogunrombi a, Sarel F. Malan a, Gisella Terre'Blanche a, Kay Castagnoli b,
Neal Castagnoli Jr. b, Jacobus J. Bergh a, Jacobus P. Petzer a,
⁎
a
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa
b
Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA
Received 24 March 2007; accepted 8 June 2007
Abstract
The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its
metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately leads to the
permanently charged 1-methyl-4-phenylpyridinium species MPP+, a 4-electron oxidation product of MPTP and a potent mitochondrial toxin. The
corresponding 5-membered analogue, 1-methyl-3-phenyl-3-pyrroline, is also a selective MAO-B substrate. Unlike MPTP, the MAO-B-catalyzed
oxidation of 1-methyl-3-phenyl-3-pyrroline is a 2-electron process that leads to the neutral 1-methyl-3-phenylpyrrole. MPP+ is thought to exert its
toxic effects only after accumulating in the mitochondria, a process driven by the transmembrane electrochemical gradient. Since this energy-
dependent accumulation of MPP+ relies upon its permanent charge, 1-methyl-3-phenyl-3-pyrrolines and their pyrrolyl oxidation products should
not be neurotoxic. We have tested this hypothesis by examining the neurotoxic potential of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-
chlorophenyl)-3-pyrroline in the C57BL/6 mouse model. These pyrrolines did not deplete striatal dopamine while analogous treatment with MPTP
resulted in 65–73% depletion. Kinetic studies revealed that both 1-methyl-3-phenyl-3-pyrroline and its pyrrolyl oxidation product were present in
the brain in relatively high concentrations. Unlike MPP+, however, 1-methyl-3-phenylpyrrole was cleared from the brain quickly. These results
suggest that the brain MAO-B-catalyzed oxidation of xenobiotic amines is not, in itself, sufficient to account for the neurodegenerative properties
of a compound like MPTP. The rapid clearance of 1-methyl-3-phenylpyrroles from the brain may contribute to their lack of neurotoxicity.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Monoamine oxidase B; Neurotoxicity; MPTP; 1-Methyl-3-phenyl-3-pyrroline; 1-Methyl-3-phenylpyrrole; Dopamine; Striata
Introduction
ring α-carbon 2-electron oxidation of the parent compound to
yield the corresponding 1-methyl-4-phenyl-2,3-dihydropyridi-
nium species MPDP+ (2H+). This metabolic intermediate,
presumably via the corresponding free base 2, undergoes a
second 2-electron oxidation to generate the 1-methyl-4-
phenylpyridinium metabolite MPP+ (3+), the ultimate neuro-
toxin (Chiba et al., 1984; Ramsay et al., 1991; Markey et al.,
1984). This process appears to take place mainly in MAO-B
rich glial cells (Takada et al., 1990). MPP+ is thought to
accumulate via the plasma membrane dopamine transporter
(DAT) (Chiba et al., 1985; Javitch and Snyder, 1984) in
nigrostriatal nerve terminals where it localizes within the inner
mitochondrial membrane (Sayre et al., 1990). Inhibition of
complex I of the mitochondrial respiratory chain by MPP+ then
leads to downstream events such as ATP depletion and
The flavoenzyme monoamine oxidase B (MAO-B) has been
identified as the principal catalyst responsible for the metabolic
activation of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine [MPTP (1)] in the brains of mammals
including humans (Fig. 1) (Chiba et al., 1984; Heikkila et al.,
1984b). The molecular mechanisms by which MPTP selectively
damages nigrostriatal neurons and induces a parkinsonian
syndrome has been the subject of extensive research (Heikkila
et al., 1984a; Nicklas et al., 1985; Smeyne and Jackson-Lewis,
2005). Critical to its mode of action is the MAO-B-catalyzed
⁎
Corresponding author. Tel.: +27 18 299 2206; fax: +27 18 299 4243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.06.014

M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
459
respiratory chain than is MPP+ but since it is not concentrated
inside the mitochondria, this compound is not a physiological
inhibitor of mitochondrial respiration. Accordingly, chronic
exposure of mice to 4-phenylpyridine and 4-phenyl-1,2,3,6-
tetrahydropyridine was found not to cause any reduction in
striatal dopamine (Perry et al., 1987).
Based on these observations, 1-methyl-3-phenyl-3-pyrrolines
and their MAO-B-catalyzed pyrrolyl oxidation products should
not be neurotoxic. In this study we have tested this hypothesis by
measuring ex vivo striatal dopamine concentrations in C57BL/6
mice 7 days after administration of 1-methyl-3-phenyl-3-pyrroline
(4a) and 1-methyl-3-(4-chlorophenyl)-3-pyrroline (4b). Com-
pound 4b is also reported to be a MAO-B substrate (Williams
and Lawson, 1998). The findings of this study are discussed with
reference to the brain concentrations attained by both 1-methyl-3-
phenyl-3-pyrroline and its pyrrolyl oxidation product compared to
the brain concentrations attained by MPTP, MPDP+ and MPP+
following systemic treatment of MPTP.
Materials and methods
Caution: MPTP (1) is a known nigrostriatal neurotoxin and
should be handled using disposable gloves and protective
eyewear. Procedures for the safe handling of MPTP have been
described previously (Pitts et al., 1986).
Fig. 1. The MAO-B-catalyzed oxidation of MPTP (1) to the corresponding 2,3-
dihydropyridinium product MPDP+ (2H+) and the pyridinium species MPP+
(3+).
Chemicals and instrumentation
oxidative stress which eventually result in degeneration of
nigrostriatal dopaminergic neurons (Singer et al., 1988).
Experimental animals treated with MPTP have become useful
models for studying neurodegenerative processes. In a fre-
quently used protocol striatal dopamine concentrations of
C57BL/6 mice are measured 7 days after systemic injection
(multiple or single doses) of MPTP (Schmidt and Ferger, 2001).
The depletion of striatal dopamine is indicative of the
permanent loss of nigrostriatal dopaminergic cell bodies in the
substantia nigra.
All starting materials not described elsewhere were obtained
from Sigma-Aldrich and were used without purification.
MPTP·HCl, dopamine·HCl and isoprenaline·HCl were pur-
chased from Sigma-Aldrich. Petroleum ether used in this study
had a distillation range of 40–60 °C. Melting points (mp) were
determined with a Gallenkamp melting point apparatus and all
melting points are uncorrected. Proton and carbon NMR spectra
were recorded on a Varian Gemini 300 spectrometer. Proton (1H)
spectra were recorded at a frequency of 300 MHz and carbon
Over the years various tetrahydropyridinyl analogues of
MPTP have been found to be MAO-B substrates (Kalgutkar and
Castagnoli, 1992). One compound of particular interest is 1-
methyl-3-phenyl-3-pyrroline (4a), a 5-membered ring analogue
that has been shown to be a good and selective substrate of
MAO-B (Wang et al., 1998). Unlike MPTP, the MAO-B-
generated final metabolite of 4a is the neutral 1-methyl-3-
phenylpyrrole (5a). This overall 2-electron oxidation most
likely arises via 6H+, the short-lived conjugate acid of the
pyrrolyl product 5a (Fig. 2).
MPP+ is thought to exert its toxic actions only after
accumulating in the inner mitochondrial membrane of the
nigrostriatal nerve terminals, a process driven by the transmem-
brane electrochemical gradient (Sayre et al., 1990). This energy-
dependant accumulation of MPP+ relies upon its permanent
charge. Intrastriatal microdialysis studies have established that
only permanently charged MPP+ analogues exhibit dopaminer-
gic neurotoxic properties (Rollema et al., 1990). Interestingly,
the neutral N-desmethylated form of MPP+, 4-phenylpyridine, is
a much more potent inhibitor of complex I of the isolated
Fig. 2. The MAO-B-catalyzed oxidation of 1-methyl-3-phenyl-3-pyrrolines (4)
to 1-methyl-3-phenylpyrroles (5).

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M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
(13C) spectra at 75 MHz. Chemical shifts are reported in parts per
million (δ) downfield from the signal of tetramethylsilane added
to CD3OD. Spin multiplicities are given as s (singlet), d (doublet),
dd (doublet of doublets), t (triplet) or m (multiplet) and the
coupling constants (J) are given in hertz (Hz). Fast atom
bombardment mass spectra (FAB-MS) and high resolution mass
spectra (HRMS) were obtained on a VG 7070E mass spectrom-
eter. HPLC analyses were performed with an Agilent 1100 HPLC
system equipped with an Agilent 1100 series variable wavelength
detector. Thin layer chromatography (TLC) was carried out using
neutral alumina 60 (Merck) with UV254 fluorescent indicator. UV/
vis spectral measurements were recorded on a Milton-Roy
Spectronic 1201 spectrophotometer.
modifications. The oily product obtained from the diethyl ether
extract solidified at 0 °C. This solid was placed in a vacuum oven
at 40 °C for 24 h. The material was converted into its oxalic acid
salt in diethyl ether. Following recrystallization from methanol a
colorless solid was obtained in 26% yield: mp 150–152 °C. 1H
NMR (300 MHz, CD3OD): δ 7.48–7.34 (m, 4H), 6.32 (t, 1H.
J=2.19 Hz), 4.55 (d, 2H, J=1.65 Hz), 4.35 (d, 2H, J=1.51 Hz),
3.09 (s, 3H). 13C NMR (75 MHz, CD3OD): δ 166.63, 137.14,
135.91, 131.50, 130.06, 128.60, 119.85, 63.60, 62.38, 42.97.
FAB-MS m/z: 194 (MH+). HRMS calcd. 194.07365, found
194.07371 (MH+).
Synthesis of 1,1-dimethyl-3-phenyl-3-pyrrolinium iodide (8·I)
Synthesis of 3-phenyl-3-pyrroline hydrochloride (7·HCl)
Compound 8·I served as internal standard for the HPLC
analysis of 4a. The hydrochloric acid salt of 3-phenyl-3-
pyrroline (7·HCl) was converted to the corresponding free base
as described above for the synthesis of 4a. This free base
(1.43 mmol) in 10 ml tetrahydrofurane (THF) was treated with 3
equivalents of iodomethane (4.3 mmol). The reaction mixture
was stirred for 1 h at room temperature at which time another
10 ml THF was added followed by an additional 3 equivalents
of iodomethane. The suspension was heated under reflux for
30 min, cooled to 0 °C and filtered to collect the light yellow
precipitate in a yield of 88%. This product was recrystallized
from methanol to give the pure product in a yield of 29%: mp
3-Phenyl-3-pyrroline hydrochloride (7·HCl) was prepared
from 3-phenyl-3-pyrrolidinol according the method described
by the literature (Lee et al., 2002) in a yield of 45%: mp 189–
191 °C (ethanol), lit. mp 184 °C (Lee et al., 2002).
Synthesis of 1-methyl-3-phenyl-3-pyrroline oxalate (4a·C2H2O4)
The hydrochloric acid salt of 3-phenyl-3-pyrroline (7·HCl)
was converted to the corresponding free base with sodium
carbonate (2 M). The thick white precipitate that formed was
extracted into chloroform and the extract was dried over
anhydrous magnesium sulfate. After removal of the chloroform
phase, the residue appeared as a light yellow oil which solidified
upon cooling to room temperature. The residue (0.55 mmol)
was cooled to 0 °C while 98% formic acid (3.3 mmol) was
added slowly followed by 40% formaldehyde (3.3 mmol) (Pine
and Sanchez, 1971). The reaction mixture was stirred at 80 °C
for 3 h and the reaction progress was monitored by TLC on
neutral alumina (100% ethyl acetate). During this time,
bubbling in the reaction was observed and the reaction turned
dark red. Water (5 ml) was added to the reaction followed by an
aqueous solution of 5 ml sodium carbonate (2 M). The resulting
white precipitate was extracted to diethyl ether and the extract
was dried over anhydrous magnesium sulfate. Upon removal of
the solvent a light yellow oily residue remained which was
converted into its oxalic acid salt by the addition of diethyl ether
saturated with oxalic acid. The product was recrystallized from
methanol at −20 °C and light yellow colored crystals were
obtained in a yield of 30%: mp 152–154 °C. 1H NMR
(300 MHz, CD3OD): δ 7.49–7.35 (m, 5H), 6.28 (d, 1H,
J=1.76 Hz), 4.56 (s, 2H), 4.34 (s, 2H), 3.09 (s, 3H). 13C NMR
(75 MHz, CD3OD): δ 166.71, 139.28, 132.78, 130.18, 129.95,
127.03, 18.83, 63.59, 62.53, 42.95. FAB-MS m/z: 160 (MH+).
HRMS calcd. 160.11263, found 160.11260 (MH+).
1
224–226 °C. H NMR (300 MHz, CD3OD): δ 7.52–7.38 (m,
5H), 6.36 (m, 1H), 4.82 (dd, 2H, J=2.06, 2.03 Hz), 4.58 (dd,
2H, J=2.20, 2.09 Hz), 3.43 (s, 6H). 13C NMR (75 MHz,
CD3OD): δ 137.97, 132.43, 130.47, 130.03, 127.11, 118.27,
74.11 (t, J=3.4–3.2 Hz), 73.03 (t, J=3.8–3.7 Hz), 54.61 (t,
J=4.0–3.9 Hz). FAB-MS m/z: 174 (M+), 175 (MH+). HRMS
calcd. 175.13610, found 175.13610 (MH+).
Synthesis of 2-aryl-3-(dimethylamino)allylidene(dimethyl)am-
monium perchlorates (9a–c)
Compounds 9a–c were prepared in relatively high yields from
the corresponding phenylacetic acid derivatives, DMF and
phosphoryl chloride according to the method described in the
literature (Jutz et al., 1969). Compound 9c served as starting
material for the preparation of the HPLC internal standard 5c (see
below). The melting points of compounds were as follows: 9a mp
203–205 °C (from ethanol), lit. mp 193–194 °C (Jutz et al., 1969);
9b mp 149–151 °C (from ethanol), lit. mp 142–144 °C (Jutz et al.,
1969); 9c mp 134–136 °C (from ethanol), lit. mp 130–131 °C (Jutz
et al., 1969).
Synthesis of 1-methyl-3-phenylpyrroles (5a–c)
1-Methyl-3-phenylpyrroles (5a–c) were synthesized from
compounds 9a–c according to the method described in the
literature (Gallagher et al., 1990). The crude products obtained
were dissolved in a minimal amount of ethyl acetate and purified
on a short column of neutral alumina (Fluka 507C) with 100%
petroleum ether (5a, b) or petroleum ether/ethyl acetate, 90:10
(5c) as mobile phase. The fractions containing the product were
Synthesis of 1-methyl-3-(4-chlorophenyl)-3-pyrroline oxalate
(4b·C2H2O4)
Compound 4b was synthesized from 1-methyl-3-(4-chlor-
ophenyl)pyrrole (5b) according to the procedure reported in the
literature (Williams and Lawson, 1998, 1999) with the following

M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
461
recrystallized from an appropriate solvent as indicated below.
Compound 5c served as internal standard for the HPLC analysis
of 5a. The melting points were as follows: 5a mp 44–46 °C
(from petroleum ether), lit. mp 46–47 °C (Gallagher et al.,
1990); 5b mp 112–114 °C (from methanol), lit. mp 117.5–
119.5 °C (Gallagher et al., 1990); 5c mp 120–122 °C (from
methanol), lit. mp 126–128 °C (Gallagher et al., 1990).
package (Systat Software Inc.). This determination was carried
out in triplicate and the Km and Vmax values were expressed as
means standard error of the mean (S.E.M.).
Steady-state MAO-B activity measurements with MPTP as
substrate
In order to estimate Km and Vmax values for the oxidation of
MPTP by MAO-B, the same incubation procedures were
followed as that described for the 1-methyl-3-phenyl-3-pyrroline
substrates with the exception that the incubation time chosen was
12.5 min. The rates of oxidation of MPTP by MAO-B present in
the mitochondrial fractions of beef liver, baboon liver, mouse liver
and mouse brain were found to remain linear over this time period
(results not shown). The concentrations of MPDP+ in the
incubations were measured spectrophotometrically at a wave-
length of 343 nm (ɛ=16,000 M−1) (Castagnoli et al., 1997).
During the time needed to complete a typical experiment it was
found, by HPLC analysis of incubations of 30–90 μM of MPTP
with mouse brain mitochondria, that the concentrations of MPP+
present in the incubations ranged from approximately 4.5% to
10.5% of the corresponding MPDP+ concentrations. These HPLC
analyses were carried out as described previously (Petzer et al.,
2003).
Steady-state MAO-B activity measurements with 4a and 4b as
substrates
Mitochondria were isolated from the tissues of beef liver,
baboon liver, mouse liver and mouse brain as described in the
literature (Salach and Weyler, 1987) and stored at −70 °C. The
mitochondrial isolates were suspended in sodium phosphate
buffer (100 mM, pH 7.4, containing 50% glycerol, w/v) and the
protein concentrations were determined by the method of
Bradford (1976). Since beef liver, baboon liver and mouse liver
mitochondria are devoid of MAO-A activity, inactivation of
MAO-A was unnecessary where the steady-state kinetic
parameters of the oxidation of 1-methyl-3-phenyl-3-pyrrolines
by MAO-B were measured (Inoue et al., 1999). The
mitochondrial fraction obtained from mouse brain tissue is
reported to contain 15% MAO-A activity (Inoue et al., 1999)
but since the 1-methyl-3-phenyl-3-pyrrolines (4a and 4b)
examined in this study are reported to be MAO-B selective
substrates (Wang et al., 1998) inactivation of MAO-Awas again
not deemed necessary. In order to estimate the Km and Vmax
values for the oxidation of the 1-methyl-3-phenyl-3-pyrrolines
by MAO-B, initial rates were measured at eight substrate
concentrations spanning at least two orders of magnitude (6.25–
2000 μM). The reactions were carried out in a final volume of
500 μl (in 100 mM sodium phosphate buffer, pH 7.4) and the
enzyme concentration used was 0.15 mg mitochondrial protein/
ml. All reactions were incubated at 37 °C for 10 min. For this
time period the MAO-B-catalyzed production of the 1-methyl-
3-phenylpyrrole products was found to be linear. The reactions
were terminated by the addition of 70% perchloric acid (10 μl)
and the samples were centrifuged at 16,000 g for 10 min. The
supernatant fractions were removed and the concentrations of
the MAO-B-generated 1-methyl-3-phenylpyrrolyl products
were measured by HPLC analysis with UV detection (see
Chemicals and instrumentation). A Phenomenex Luna C18
column (4.60×250 mm, 5 μm) was used and the mobile phase
consisted of 25% distilled water [containing 0.6% (v/v) glacial
acetic acid and 1% (v/v) triethylamine] and 75% acetonitrile at a
flow rate of 1 ml/min. A volume of 50 μl of the supernatant was
injected into the HPLC system and the elution of 1-methyl-3-
phenylpyrrole (5a) or 1-methyl-3-(4-chlorophenyl)pyrrole (5b)
was monitored at wavelengths of 270 and 280 nm, respectively.
Quantitative estimations of 5a and 5b were made by means of
linear calibration curves ranging from 1.5 to 25 μM and the
initial rates were expressed as nmol of product formed per mg
mitochondrial protein per min. The steady-state kinetic data
(initial rates as a function of substrate concentration) were fitted
to the Michaelis–Menten equation using the nonlinear least-
squares fitting routine incorporated into the SigmaPlot software
Animal studies
Animal trials were conducted with retired breeder male
C57BL/6 mice (30–35 g, 9–11 months of age) which were
housed 5 animals per cage in a temperature (21 0.5 °C) and
humidity (50 5% relative humidity) controlled room on a 12 h
light–12 h dark cycle with free access to food and water. The
animals were provided by the Laboratory animal center of the
Potchefstroom campus and protocols for all animal experiments
were reviewed and approved by the Research Ethics Committee
of the North-West University. All injections were intraperito-
neal (i.p.) in a volume of 0.2 ml per 30 g mouse. Sterile saline
was used as vehicle for all of the test compounds. Mice were
sacrificed by rapid cervical dislocation.
Striatal dopamine measurements
The concentrations of dopamine in dissected mouse striata
were determined as described previously, (Harvey et al., 2006)
and were expressed as means S.E.M.
Brain MPTP, MPDP+ and MPP+ measurements
Immediately after sacrifice, the mouse brains were removed,
placed in microcentrifuge tubes and frozen in liquid nitrogen. A
portion (∼30 mg) of the brain tissue of each mouse was
weighed and the concentrations of MPTP, MPDP+ and MPP+
were determined via HPLC analysis as described previously
(Castagnoli et al., 1997). The only modification made to the
published procedure was that the internal standard used was
changed to 1-methyl-4-(1-methylpyrrol-2-yl)pyridinium iodide
(MMP+) at a final concentration of 1.33 μM. MPTP, MPDP+

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M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
and MPP+ concentrations were expressed as pmol/mg wet
weight of tissue (mean S.E.M.).
Results
Chemistry
Brain measurements of 4a and 5a
1-Methyl-3-phenyl-3-pyrroline (4a) was prepared from 3-
Four groups (n=5/group) of mice were treated with
238 μmol/kg 1-methyl-3-phenyl-3-pyrroline oxalate
(4a·C2H2O4) (see Animal studies above) and sacrificed at 10,
20, 40 and 60 min post-treatment, respectively. Their brains
were removed and placed in microcentrifuge tubes. The brain
tissues were frozen in liquid nitrogen until further sample
preparation. For the measurement of 1-methyl-3-phenylpyrrole
(5a) concentrations, a portion (∼30 mg) of the brain tissue of
each mouse was weighed and 10 μl/mg of the homogenizing
solution (an aqueous solution of 50% acetonitrile, 0.305 M
perchloric acid and 12.5 μM of compound 5c as internal
standard) was added. Following sonication (2×14 s, 14 μm) the
samples were placed on ice for 60 min, centrifuged at 16,000 g
for 15 min and 50 μl of the resulting supernatants were analyzed
for 1-methyl-3-phenylpyrrole content by reverse phase HPLC
equipped with a UV detection and a Phenomenex Luna C18
analytical column (see Chemicals and instrumentation). The
mobile phase consisted of 35% aqueous phase (0.6% glacial
acetic acid and 1% triethylamine in distilled water, pH 4.7) and
65% acetonitrile; the solvent was delivered at a flow rate of
1 ml/min. The elutions of 5a (4.93 min) and internal standard 5c
(4.41 min) were monitored at a wavelength of 270 nm. A linear
standard curve was constructed using four calibration standards
(3.125–25 μM) prepared in the homogenizing buffer. In order to
measure brain concentrations of 1-methyl-3-phenyl-3-pyrroline
(4a), a portion of brain tissue (∼30 mg) of each mouse was
weighed and 10 μl/mg of the homogenizing solution (an
aqueous solution of 10% acetonitrile, 0.305 M perchloric acid
and 12.5 μM of compound 8·I as internal standard) was added.
Following sonication (2×14 s, 14 μm) the samples were placed
on ice for 60 min, centrifuged at 16,000 g for 15 min and 50 μl
of the resulting supernatants were analyzed for 1-methyl-3-
phenyl-3-pyrroline content by reverse phase HPLC as described
for 1-methyl-3-phenylpyrrole above. The mobile phase con-
sisted of 90% aqueous phase (0.6% glacial acetic acid and 1%
triethylamine in distilled water, pH 4.7) and 10% acetonitrile
and solvent was delivered at a flow rate of 1 ml/min. The
elutions of 4a (9.66 min) and internal standard 8 (7.88 min)
were monitored at a wavelength of 250 nm. A linear standard
curve was constructed using four calibration standards (3.125–
25 μM) prepared in the homogenizing buffer. The concentra-
tions of 4a and 5a were expressed as pmol/mg wet weight of
tissue (mean S.E.M.).
phenyl-3-pyrroline (7) by the formic acid–formaldehyde
methylation procedure (Eschweiler–Clarke reaction) described
in the literature (Pine and Sanchez, 1971) (Fig. 3). As expected
this method of methylation yielded exclusively the tertiary
amine with no trace of the N,N-dimethylated pyrrolinium
species (8) as judged by TLC. In contrast, treatment of 3-
phenyl-3-pyrroline with one equivalent of iodomethane yielded
a mixture of the tertiary and quaternary amines. Treatment with
an excess of iodomethane (6 equiv.) yielded exclusively the
pyrrolinium iodide salt (8·I) which readily separated from the
reaction solvent (THF). The starting material 3-phenyl-3-
pyrroline (7) was prepared from glycine ethyl ester and ethyl
chloroformate in a synthetic route involving six steps (Wu et al.,
1962; Kuhn and Osswald, 1956; Lee et al., 2002) with an
overall yield of less than 3%. For this reason the preparation of
1-methyl-3-(4-chlorophenyl)-3-pyrroline (4b) was achieved via
an alternative route (Fig. 4) (Williams and Lawson, 1998). The
key starting material was 2-(4-chlorophenyl)-3-(dimethyla-
mino)allylidene(dimethyl)ammonium perchlorate (9b) which
was prepared in high yield from 4-chlorophenylacetic acid and
DMF (Jutz et al., 1969). Cyclization of 9b was achieved by
treatment with sodium methoxide in anhydrous pyridine to
yield 1-methyl-3-(4-chlorophenyl)pyrrole (5b) in fair yield
(52.8%) (Gallagher et al., 1990). The preparations of 1-methyl-
3-phenylpyrrole (5a) and 1-methyl-3-(4-methoxyphenyl)pyr-
role (5c) were achieved in a similar manner starting from
phenylacetic acid and 4-methoxyphenylacetic acid, respective-
ly. The 1-methyl-3-(4-chlorophenyl)pyrrole intermediate was
converted to the corresponding 1-methyl-3-(4-chlorophenyl)-3-
pyrroline (4b) by partial reduction with zinc and hydrochloric
acid (Williams and Lawson, 1998; Andrews and McElvain,
1929) in an overall yield of 31.9%. In our experience 3-
pyrrolines prepared in this manner are often contaminated with
what we believe is the corresponding pyrrolidinyl derivatives
(Hudson and Robertson, 1967) in amounts ranging from
1
approximately 10–25% as judged by H NMR. We found that
in selected instances (for example in the preparation of 4b, the
pyrrolidinyl contaminant may be removed from the 3-pyrrolinyl
free base under reduced pressure with slight heating (see
Statistical analysis
Striatal dopamine levels were analyzed using an one-way
analysis of variance (ANOVA), followed by multiple compar-
isons using the Dunnett's t-test to compare the experimental
groups to the control group. The Statistica software package
(StatSoft Inc.) was used for all data analysis and data are
expressed as the mean S.E.M.
Fig. 3. Synthetic pathways to 1-methyl-3-phenyl-3-pyrroline (4a) and 1,1-
dimethyl-3-phenyl-3-pyrrolinium iodide (8·I). Key: (i) formic acid, formalde-
hyde, 80 °C; (ii) CH3I (6 equiv.), K2CO3, THF, rt.

M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
463
measured at wavelengths of 270 and 280 nm, respectively.
HPLC-UV was chosen as the analytical technique since
background interference in the near-UV wavelength range by
the mitochondrial fractions used here as enzyme source was too
high to measure the pyrrolyl product concentrations accurately
by spectrophotometry. The incubation time of the enzyme-
catalyzed reactions were chosen to be 10 min since the
oxidation of both substrates (4a and 4b) was found to be linear
(results not shown) for at least 12 min at a substrate
concentration of 50 μM. Since the mitochondrial fraction
obtained from mouse brain tissue is reported to contain 15%
MAO-A activity (Inoue et al., 1999) we measured the
contribution of MAO-A towards the oxidation of 1-methyl-3-
phenyl-3-pyrroline (4a) by the mitochondria obtained from this
source. The MAO-B isoform was inactivated by pre-incubating
the mitochondria with the MAO-B selective inactivator (R)-
deprenyl (Inoue et al., 1999). Following incubation of 4a
(250 μM) with the MAO-B inactivated mitochondria only trace
amounts (0.28 0.03 μM) of the pyrrolyl oxidation product
were detected (results not shown). This was less than 5% of the
product concentration (8.58 0.31 μM) detected in experiments
with mitochondria not previously inactivated. In contrast, when
the MAO-A isoform was inactivated by pre-incubating the
mitochondria with the MAO-A selective inactivator clorgyline
(Inoue et al., 1999) relatively larger amounts (7.18 0.43 μM)
of the pyrrolyl product were detected which was approximately
84% of the product concentration detected in experiments with
mitochondria not previously inactivated. These results docu-
ment that MAO-A does not contribute significantly to the
oxidation of 4a by mouse brain mitochondrial isolates and that
the steady-state kinetic parameters measured for 4a are
representative of its oxidation by the MAO-B isoform. Since
mitochondria obtained from beef liver, baboon liver and mouse
liver tissues are devoid of MAO-A activity (Inoue et al., 1999),
the oxidation of 4a and 4b by these enzyme sources can be
exclusively attributed to the action of the MAO-B isoform.
Fig. 4. Synthetic pathway to 1-methyl-3-phenylpyrroles (5a–c) and 1-methyl-3-
(4-chlorophenyl)-3-pyrroline (4b). Key: (i) DMF, POCl3, 80 °C; (ii) NaOCH3,
pyridine, reflux; (iii) Zn, HCl, 60 °C.
Materials and methods). Alternatively the impurity can also be
removed by fractional crystallization of the oxalate salts.
General enzymology
Both 1-methyl-3-phenyl-3-pyrroline (4a) and 1-methyl-3-(4-
chlorophenyl)-3-pyrroline (4b) have previously been shown to
be substrates for MAO-B (Wang et al., 1998; Williams and
Lawson, 1998). The 1-methyl-3-phenyl analogue is reported to
have a Km value of 193 μM and a Vmax value of 397 min−1 for
oxidation by beef liver MAO-B (Wang et al., 1998) while the
Km and Vmax values for the oxidation by rat liver MAO-B are
reported to be 79 μM and 8.1 nmol/min mg, respectively
(Williams and Lawson, 1998). The Km and Vmax values for the
oxidation of 1-methyl-3-(4-chlorophenyl)-3-pyrroline by rat
liver MAO-B are reported to be 67 μM and 10.3 nmol/min mg,
respectively (Williams and Lawson, 1998). In the present study
we have confirmed both 4a and 4b to be good substrates of beef
liver, baboon liver, mouse liver and mouse brain MAO-B, and
also compared their substrate properties with that of MPTP. In
order to compare the substrate properties of 4a, 4b and MPTP, it
was necessary to determine the steady-state kinetic parameters
(Km and Vmax values) for all three substrates in a single species
since the MAO-B substrate properties often differs between
species and even between different tissues in the same species
(Inoue et al., 1999). Since the Vmax values are dependent upon
the MAO-B concentration in the mitochondrial preparation
used in the assays, and the enzyme concentrations differ from
preparation to preparation, cited values could not be used for
direct comparison of the substrates. All the substrates were
therefore re-examined. In order to measure the steady-state
kinetic parameters we chose to measure the extent of oxidation
of 4a and 4b by HPLC-UV analysis. The concentrations of the
MAO-B-catalyzed pyrrole products 5a and 5b could be readily
MAO-B substrate properties of 4a and 4b
As illustrated by example in Fig. 5 the steady-state oxidation
of the substrates by MAO-B followed Michaelis–Menten
behavior. The Km and Vmax values obtained for the oxidation
of the two pyrroline substrates and MPTP by beef liver, baboon
liver, mouse liver and mouse brain MAO-B are summarized in
Table 1. Except for the kinetic data generated with mouse liver
mitochondria no significantly large interspecies differences of
the Km values were apparent. The Km values of 4a, 4b and
MPTP were significantly larger with mouse liver mitochondria
than with the other mitochondrial sources examined here. For
example, a Km value of 461 26.9 μM was observed for the
oxidation of 4a by mouse liver MAO-B while the
corresponding value with mouse brain mitochondria was
found to be 125 7.04 μM. Similarly, for the oxidation of
MPTP, a Km value of 797 11.5 μM was recorded with mouse
liver MAO-B compared to a value of 52.1 3.89 μM observed
with mitochondria from mouse brain. These observations are in
agreement with the literature which reports a Km value for the

464
M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
two compounds (4a and 4b) were approximately equally good
substrates for MAO-B as judged by the similarity of their Vmax
/
Km values. These Vmax/Km values were found to be consistently
higher than for MPTP which was confirmed to be also a
relatively good substrate of MAO-B. These results are in
accordance with literature reports that MPTP and 1-methyl-3-
phenyl-3-pyrroline act as good substrates of beef liver (Wang
et al., 1998) as well as rat liver (Williams and Lawson, 1998)
MAO-B. We have previously shown that the well-known
MAO-B substrate benzylamine is oxidized by baboon liver
MAO-B with Km and Vmax values of 616 23 μM and 63
2.2 nmol/min mg, respectively (Vlok et al., 2006). This yields a
Fig. 5. Determination of the Km and Vmax values for the oxidation of 4a by
baboon liver MAO-B. The concentration of 5a produced was measured by
HPLC analysis following a 10 min incubation with 0.15 mg/ml baboon liver
mitochondria at 37 °C. The rate data were fitted to the Michaelis–Menten
equation using a nonlinear least-squares fitting routine. All measurements were
conducted in triplicate and the concentration of 4a in the incubations ranged
from 6.25 to 1000 μM. The initial rates are expressed as nmol/min mg protein of
5a formed.
V
max/Km value of 0.10 (min mg protein)−1 which compares with
the corresponding values obtained for the pyrroline substrates
4a and 4b [0.17 and 0.10 (min mg protein)−1]. Therefore,
consistent with expectation, it can be concluded that 4a and 4b
are good substrates of MAO-B isolated from a variety of species
and therefore these compounds should be oxidized efficiently in
vivo to the corresponding pyrrolyl products (5a and 5b).
oxidation of MPTP by mouse liver MAO-B of 520 μM while a
Km value of 96.8 μM was measured with mouse brain
mitochondria (Inoue et al., 1999). The factors contributing to
these tissue dependent differences in MAO-B activity remain to
be identified. Also of note, the Vmax values for the oxidation of
4a and 4b by mouse brain mitochondrial MAO-B were
significantly lower than the corresponding values obtained
with beef, baboon and mouse liver mitochondria as enzyme
sources. Assuming that there are no large interspecies
differences of the turnover numbers (kcat) for the oxidation of
4a and 4b by MAO-B, this difference in Vmax is possibly due to
the lower density of MAO-B in brain mitochondrial fractions
compared to the liver. The trend has been previously observed
in various species where consistently lower Vmax values have
been measured with brain mitochondria as MAO-B source
compared to the liver (Inoue et al., 1999). For example baboon
liver mitochondria oxidize MPTP with a Vmax value of
6.3 nmol/min mg protein while with brain mitochondria a
Vmax value of 2.4 nmol/min mg protein was observed. These
Neurotoxicity studies
It is well documented that a single intraperitoneal injection of
MPTP (95–238 μmol/kg) causes depletion of striatal dopamine
in aged C57BL/6 mice while still being sub-lethal (Di Monte
et al., 1997; Castagnoli et al., 2001). This depletion measured
7–10 days following treatment is frequently used as marker for
the nigrostriatal degeneration resulting from the neurotoxic
action of MPP+. In this study we investigated whether similar
Table 1
Steady-state kinetic parameters for the oxidation of 4a, 4b and MPTP by MAO-
B present in the mitochondrial fractions of beef liver, baboon liver, mouse brain
and mouse liver tissue
Km (μM)
Vma ax
9.0 0.5
9.1 0.8
Vmax/Kmb
Beef liver
4a
4b
56.4 2.82; (193)c
44.8 5.97
0.16
0.20
MPTP 138 2.90; (191)c
3.1 0.04 0.023
Baboon liver 4a
54.6 3.21
46.1 10.6
9.2 0.7
4.7 0.4
6.7 0.1
0.17
0.10
0.039
4b
MPTP 173 6.93 (87.5)d
Mouse brain
Mouse liver
4a
4b
125 7.04
21.0 3.54
0.8 0.02 0.007
0.4 0.02 0.019
MPTP 52.1 3.89; (40)e; (96.8)d
0.9 0.1
5.9 0.2
2.2 0.3
10.6 0.1
0.017
0.013
0.034
0.013
4a
4b
461 26.9
63.5 6.57
Fig. 6. Striatal dopamine levels of C57BL/6 male mice (n=10 mice/group)
treated i.p. with saline, MPTP·HCl (167 μmol/kg), 4a·C2H2O4 (238 μmol/kg)
(top) or 4b·C2H2O4 (238 μmol/kg) (bottom). Dopamine levels were measured
7 days after treatment and are expressed as pmol/mg tissue. ⁎Significantly
different (pb0.01) from the saline treated group.
MPTP 797 11.5; (520)d
The values are expressed in anmol/min mg mitochondria and b(min mg
protein)−1. cWang et al. (1998). dInoue et al. (1999). eCastagnoli et al. (1997).

M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
465
Table 2
Brain concentrations of 4a and 5a in C57BL/6 mice (n=5 mice/group) treated
with 238 μmol/kg of 4a·C2H2O4 and sacrificed at the indicated times (min)
Time
Brain concentrations (pmol/mg of wet tissue)
4a
5a
10
20
40
60
65.5 11.2
34.1 2.71
6.92 0.86
2.20 0.40
53.0 9.89
37.0 5.29
6.39 1.60
3.11 2.54
lower brain concentrations of the pyrrolyl products compared to
a concentration of MPP+ that leads to neuronal injury. Another
explanation for the lack of neurotoxicity of 4a and 4b is that the
bioavailability of 1-methyl-3-phenyl-3-pyrrolines to the brain
may be lower than that of MPTP or that the clearance of 1-
methyl-3-phenyl-3-pyrrolines and/or their pyrrolyl oxidation
products from the brain may be faster than MPTP or MPP+. To
test these theories we injected mice (n=5 mice/group) with
238 μmol/kg of 4a·C2H2O4 and measured the brain concentra-
tions of 4a and its MAO-B-catalyzed oxidation product 5a at
various time points (10, 20, 40 and 60 min) following treatment
(Fig. 7). In a second experiment, mice (n=6 mice/group)
similarly received 167 μmol/kg MPTP·HCl and the concentra-
tions of MPTP, MPDP+ and MPP+ in the brain tissue were
measured at 10 min and 90 min following treatment. It is
reported that MPTP concentrations reach a maximum at 10 min
following i.p. injection while MPP+ concentrations reach a
maximum at approximately 90 min following MPTP injection
(Castagnoli et al., 1997). Results in Tables 2 and 3 show that,
like MPTP, 4a reaches maximum brain concentrations relatively
early with a measured concentration of 65.5 11.2 pmol/mg
tissue at 10 min following treatment. This concentration is
similar to the MPTP concentration in the brain of 80.17
4.99 pmol/mg at 10 min following treatment with a neurotoxic
dose of MPTP. The MAO-B-generated pyrrole product 5a was
also found to reach maximal brain concentrations relatively
early with a measured concentration of 53.0 9.89 pmol/mg
tissue at 10 min following treatment with 4a. This relatively
high concentration of 5a is in contrast to the MPP+
concentration in the brain of only 11.17 2.15 pmol/mg tissue
at 10 min following MPTP treatment. The pyrrolyl product 5a,
however, was found to clear relatively quickly from the brain; a
concentration of only 3.11 2.54 pmol/mg tissue was detected
at 60 min post-treatment. This is again in contrast to MPP+
which was found to be present in the brain in a relatively high
concentration of 77.67 4.87 pmol/mg tissue even at 90 min
Fig. 7. HPLC-UV tracings showing the presence of 1-methyl-3-phenylpyrrole
(5a) (top; 4.93 min; λ=270 nm) and 1-methyl-3-phenyl-3-pyrroline (4a)
(bottom; 9.66 min; λ=250 nm) in the brain tissue of mice sacrificed 10 min
following treatment with 238 μmol/kg 1-methyl-3-phenyl-3-pyrroline oxalate
(4a·C2H2O4). The internal standards used were 1-methyl-3-(4-methoxyphenyl)
pyrrole (5c) (top; 4.41 min) and 1,1-dimethyl-3-phenyl-3-pyrrolinium iodide
(8·I) (bottom; 7.88 min).
treatment of mice with 4a and 4b also results in a loss of
dopamine in the striatum and, by association, nigrostriatal
injury. Two studies were carried out each containing a control
group (n=10 mice) which received saline (0.20 ml/30 g mouse)
and a MPTP group (n=10 mice) which received a relatively
lower dose of MPTP·HCl (167 μmol/kg). The third group
(n=10 mice) received 4a·C2H2O4 or 4b·C2H2O4 at a relatively
high dose of 238 μmol/kg. The animals were sacrificed 7 days
later, the striata were dissected and the dopamine concentrations
were determined by HPLC-ECD analysis as described in the
Materials and methods. The results (Fig. 6) show that neither 4a
nor 4b induced depletion of dopamine with the measured
dopamine concentrations at 97% (Fig. 6, top) and 110% (Fig. 6,
bottom) of the control values, respectively. In contrast, MPTP
treatment significantly (pb0.001) reduced the dopamine levels
to 35% (Fig. 6, top) and 27% (Fig. 6, bottom) of the control
values.
In vivo kinetic studies
Table 3
Brain concentrations of MPTP, MPDP+ and MPP+ in C57BL/6 mice (n=6 mice/
group) treated with 167 μmol/kg of MPTP·HCl and sacrificed at the indicated
times (min)
The finding that high doses of 4a and 4b do not mimic the
characteristic striatal dopamine depletion effect of MPTP is in
accordance with the idea that the permanent positive charge of
MPP+ is a key structural feature contributing to its neurotoxic
action. While the lack of neurotoxicity by 4a and 4b may be
explained by this hypothesis, it is also possible that the 1-
methyl-3-phenyl-3-pyrrolines are not metabolized as efficiently
as MPTP by MAO-B in vivo. This could result in relatively
Time
Brain concentrations (pmol/mg of wet tissue)
MPTP
MPDP+
MPP+
10
90
a
80.17 4.99
4.44 1.60
25.67 1.23
4.60 1.37
11.17 2.15
77.67 4.87a
MPP+ concentrations reach a maximum at ∼90 min following MPTP
injection (Castagnoli et al., 1997).

466
M.O. Ogunrombi et al. / Life Sciences 81 (2007) 458–467
post-MPTP treatment. The literature reports that MPP+
concentrations remain relatively high even at a time point of
240 min post-treatment (Di Monte et al., 1997; Castagnoli et al.,
1997).
West University for their support. The NMR and MS spectra
were recorded by André Joubert, Johan Jordaan and Louis
Fourie of the SASOL Centre for Chemistry, North-West
University. This work was supported by grants from the
National Research Foundation and the Medical Research
Council, South Africa.
Discussion
In this investigation the nigrostriatal neurotoxic potentials of
the 1-methyl-3-phenyl-3-pyrrolines 4a and 4b were compared
with that of MPTP. It is generally accepted that the
neurotoxicity of MPTP is a consequence of its MAO-B-
catalyzed metabolism in the brain that ultimately yields the
mitochondrial toxin MPP+ (Chiba et al., 1984; Heikkila et al.,
1984b). Evidence suggests that the mitochondrial toxicity and
subsequent neurotoxic action of MPP+ relies on it being
permanently charged (Sayre et al., 1990; Rollema et al., 1990).
Since the pyrrolyl oxidation products of 1-methyl-3-phenyl-3-
pyrrolines are neutral species, 4a and 4b are not expected to be
neurotoxic.
Here both 4a and 4b were shown to act as good substrates for
beef liver, baboon liver, mouse liver and mouse brain MAO-B.
Judging by the steady-state kinetic parameters, the in vitro
MAO-B-catalyzed oxidation of 4a and 4b was at least as
efficient as that of MPTP and, in some instances, even superior.
The results of the neurotoxicity studies, however, show that
even at high doses, 4a and 4b do not result in depletion of
striatal dopamine as observed with MPTP. We conclude,
therefore, that these pyrrolines, and probably also other 1-
methyl-3-phenyl-3-pyrrolines, are not MPTP-type dopaminer-
gic neurotoxins.
From the in vivo kinetic studies we conclude that 4a is
sufficiently bioavailable to reach brain concentrations similar to
those of MPTP following a neurotoxic dose. The pyrrolyl
product 5a also reaches the brain in relative high concentrations
which are comparable to the maximum concentration measured
for MPP+. The concentrations measured for 5a in the brain are
probably representative of 5a generated centrally by the action
of MAO-B on 4a as well as 5a partitioning from the periphery
where it is produced by peripherally located MAO-B and
possibly by the cytochrome P450 isozymes. Unlike 5a, MPP+
carries a permanent positive charge and is not expected to cross
the blood–brain barrier (BBB). Therefore MPP+ concentrations
measured in the brain are representative of centrally generated
MPP+ only. The slow clearance of MPP+ from the brain may
possibly be explained by its low BBB permeability. In contrast
pyrrole 5a is cleared from the brain relatively quickly since it
probably crosses the BBB freely. The relatively fast clearance
from the brain may be another factor that contributes to the
observed lack of neurotoxicity of the 1-methyl-3-phenylpyr-
roles 5a and 5b.
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