Deuterium isotope effects for the oxidation of 1-methyl-3-phenyl-3-pyrrolinyl analogues by monoamine oxidase B

Article abstract of DOI:10.1016/j.bmc.2008.09.001

The parkinsonian inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a cyclic tertiary allylamine exhibiting good monoamine oxidase B (MAO-B) substrate properties. MAO-B catalyzes the ring α-carbon 2-electron bioactivation of MPTP to yield the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP⁺). The corresponding 5-membered ring MPTP analogue, 1-methyl-3-phenyl-3-pyrroline, also undergoes MAO-B-catalyzed oxidation to give the 2-electron oxidation product, 1-methyl-3-phenylpyrrole. Here we report the kinetic deuterium isotope effects on V_max and V_max/K_m for the steady-state oxidation of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-fluorophenyl)-3-pyrroline by baboon liver MAO-B, using the corresponding pyrroline-2,2,4,5,5-d₅ analogues as the deuterated substrates. The apparent isotope effects for the two substrates were 4.29 and 3.98 on V_max, while the isotope effects on V_max/K_m were found to be 5.71 and 3.37, respectively. The values reported for the oxidation of MPTP by bovine liver MAO-B with MPTP-6,6-d₂, as deuterated substrate, are ^D(V_max) = 3.55; ^D(V_max/K_m) = 8.01. We conclude that the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl substrates is similar to that of the tetrahydropyridinyl substrates and that a carbon-hydrogen bond cleavage step is, at least partially, rate determining.

Full text of DOI:10.1016/j.bmc.2008.09.001

Bioorganic & Medicinal Chemistry 16 (2008) 8813–8817
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Deuterium isotope effects for the oxidation of 1-methyl-3-phenyl-3-pyrrolinyl
analogues by monoamine oxidase B
Anél Pretorius a, Modupe O. Ogunrombi a, Gisella Terre’Blanche a, 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 and Edward Via College of Osteopathic Medicine, Blacksburg, VA 24061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 June 2008
Accepted 2 September 2008
Available online 5 September 2008
The parkinsonian inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a cyclic ter-
tiary allylamine exhibiting good monoamine oxidase B (MAO-B) substrate properties. MAO-B catalyzes
the ring a-carbon 2-electron bioactivation of MPTP to yield the 1-methyl-4-phenyl-2,3-dihydropyridini-
um species (MPDP+). The corresponding 5-membered ring MPTP analogue, 1-methyl-3-phenyl-3-pyrro-
line, also undergoes MAO-B-catalyzed oxidation to give the 2-electron oxidation product, 1-methyl-3-
phenylpyrrole. Here we report the kinetic deuterium isotope effects on Vmax and Vmax/Km for the
steady-state oxidation of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-fluorophenyl)-3-pyrroline
by baboon liver MAO-B, using the corresponding pyrroline-2,2,4,5,5-d5 analogues as the deuterated sub-
strates. The apparent isotope effects for the two substrates were 4.29 and 3.98 on Vmax, while the isotope
effects on Vmax/Km were found to be 5.71 and 3.37, respectively. The values reported for the oxidation
of MPTP by bovine liver MAO-B with MPTP-6,6-d2, as deuterated substrate, are D(Vmax) = 3.55;
D(Vmax/Km) = 8.01. We conclude that the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl sub-
strates is similar to that of the tetrahydropyridinyl substrates and that a carbon–hydrogen bond cleavage
step is, at least partially, rate determining.
Keywords:
Monoamine oxidase B
MPTP
1-Methyl-3-phenyl-3-pyrroline
Kinetic isotope effect
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
C6H5
C6H5
The metabolic activation of the proneurotoxin, 1-methyl-4-phe-
nyl-1,2,3,6-tetrahydropyridine [MPTP (1)], is catalyzed by the fla-
voenzyme, monoamine oxidase B (MAO-B) (Scheme 1),1,2 to yield
MAO-B
N
N
the
a-carbon 2-electron oxidation product, the corresponding 1-
methyl-4-phenyl-2,3-dihydropyridinium species, MPDP+ (2H+).
Following a second 2-electron oxidation, presumably via the corre-
sponding free base 2, the ultimate neurotoxic metabolite, 1-
methyl-4-phenylpyridinium [MPP+ (3+)] is generated.1,3,4 MPP+ is
a mitochondrial toxin which selectively damages nigrostriatal neu-
rons and induces a parkinsonian syndrome in humans5,6 and other
susceptible mammals.7–9 MPTP is the first reported cyclic tertiary
allylamine that acts as a good MAO-B substrate.1 Several other ter-
tiary aminyl substrates of MAO-B have since been described.10,11
Among these, 1-methyl-3-phenyl-3-pyrroline (4a), the 5-mem-
bered ring analogue of MPTP, has MAO-B substrate properties com-
CH3
CH3
2H+
1
C6H5
C6H5
-H+
-2e-, H+
?
N
N
CH3
CH3
3+
2
parable to those of MPTP.12,13 The ring
a-carbon oxidation of 4a
Scheme 1. The MAO-B-catalyzed
a-carbon oxidation of MPTP (1) to yield the
results in 1-methyl-3-phenylpyrrole (5a), an overall 2-electron
process that most likely arises via 6Ha+, the short-lived conjugate
acid of 5a (Scheme 2).
corresponding 2,3-dihydropyridinium product MPDP+ (2H+). Further oxidation via
an unknown pathway results in the pyridinium species MPP+ (3+).
In the present study, we report the steady-state kinetic
deuterium isotope effects on Vmax and Vmax/Km for the oxidation
of 1-methyl-3-phenyl-3-pyrroline (4a) and 1-methyl-3-(4-fluoro-
* Corresponding author. Tel.: +27 18 2992206; fax: +27 18 2994243.
E-mail address: jacques.petzer@nwu.ac.za (J.P. Petzer).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.001

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A. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8813–8817
removed via fractional recrystallization of the oxalate salt (4aÁoxa-
C6H4X
MAO-B
C6H4X
late) as reported.13
In order to replace the methylene protons of the ring a-carbons
(C-2 and C-5) of the pyrrolinyl analogues with deuterons, a similar
synthetic approach was initially followed (Scheme 4). Because of
the relative ease of purification of the 4-fluorophenyl substituted
pyrrolinyl analogue 4b compared to the unsubstituted analogue
4a (see above), compound 4b-d8 was chosen as the target 3-pyrro-
line. The key starting material, 7b-d14 was prepared by reaction of
4-fluorophenylacetic acid with DMF-d7 in the presence of POCl3.18
1H NMR analysis indicated that deuterium incorporation was >99%
at the anticipated methyl and methinyl carbons (Scheme 4). Treat-
ment of 7b-d14 with a sodium methoxide solution (prepared with
methanol-d1) in dry pyridine yielded the pyrrolyl analogue 5b-
d6.19,20 While the deuterium incorporation at the N–CH3 carbon
was high (97.8%), proton–deuteron exchange occurred at the C-2,
C-4, and C-5 of the pyrrolyl heterocyclic system resulting in deu-
teron loss to the extent of 6–27%. However, upon reduction with
zinc/DCl (to yield 4b-d8),13,15,21 deuterium incorporation at C-2,
C-4, and C-5 of the pyrrolinyl ring were again relatively high
(>96%). This result indicated that the zinc/DCl reduction procedure
could be accompanied by the exchange of pyrrolinyl protons for
deuterons. It was possible to take advantage of this type of reaction
to prepare both 4b-d5 and the more difficult to purify 4a-d5, by
simply reducing the corresponding pyrrolyl analogue with zinc/
DCl. Accordingly, zinc/DCl reduction of 5a and 5b yielded 4a-d5
and 4b-d5, respectively, with a relatively high degree (>98%) of
deuterium incorporation at C-2, C-4, and C-5 of the pyrrolinyl ring
(Scheme 5). No deuterium incorporation was observed at N–CH3.
N
N
CH3
CH3
4a: X = H
4b: X = 4-F
6Ha+: X = H
6Hb+: X = 4-F
C6H4X
-H+
N
CH3
5a: X = H
5b: X = 4-F
Scheme 2. The MAO-B-catalyzed
a
-carbon oxidation of 1-methyl-3-phenyl-3-
pyrrolinyl analogues (4a and 4b) to yield intermediates 6Ha+ and 6Hb+. Deproto-
nation of 6Ha+/6Hb+ results in the corresponding 1-methyl-3-phenylpyrrolyl
species 5a and 5b.
phenyl)-3-pyrroline (4b) by baboon liver mitochondrial MAO-B.
For this purpose we have employed the corresponding 1-methyl-
3-phenyl-3-pyrrolinyl-2,2,4,5,5-d5 (4a-d5) and 1-methyl-3-(4-
fluorophenyl)-3-pyrrolinyl-2,2,4,5,5-d5 (4b-d5) analogues as
deuterated substrates (Scheme 3). The apparent isotope effects
for the two substrates were compared to the isotope effects previ-
ously reported for the oxidation of MPTP by bovine liver mitochon-
drial MAO-B using MPTP-6,6-d2 (1-d2) as deuterated substrate.14
The results of this study are discussed with reference to the extent
to which carbon–hydrogen bond cleavage is rate limiting during
MAO-B-catalyzed oxidation of pyrrolinyl substrates.
2.2. General enzymology
In order to measure the rates of oxidation of the pyrrolinyl ana-
logues 4a–4b and 4a-d5–4b-d5, the substrates were incubated with
2. Results
D
D
2.1. Chemistry
CD3
CD3
i
N
N
Ar
The 1-methyl-3-phenyl-3-pyrrolinyl analogues (4a–4b) exam-
ined here, were prepared by partial reduction of the corresponding
pyrrolyl analogues 5a–b as previously reported.13,15,16 TLC and 1H
NMR analysis indicated that the 3-pyrrolines prepared in this man-
ner were contaminated with the corresponding pyrrolidinyl deriv-
atives formed by the over-reduction of the pyrrolyl substrates.17
Also, HPLC analysis revealed trace amount of the pyrrolyl starting
material present in samples of the free bases of 4a and 4b. In order
to remove the volatile pyrrolidinyl contaminant from 4b, the free
base was subjected to high vacuum (see Section 4) while the pyrr-
olyl contaminant was removed by converting 4b to the corre-
sponding oxalate salt (4bÁoxalate). For the purification of 4a, both
the corresponding pyrrolidinyl and pyrrolyl contaminants were
CO2H
CD3 Ar
CD3
7b-d14: Ar = 4-FC6H4
D
Ar
D
D
Ar
iii
ii
D
D
N
D
D
D
N
CD3
CD3
5b-d6: Ar = 4-FC6H4
4b-d8: Ar = 4-FC6H4
Scheme 4. Synthetic pathway to the deuterated pyrrolinyl substrate, 4b-d8.
Reagents and conditions: (i) DMF-d7, POCl3, 80 °C; (ii) NaOCH3 (from methanol-
d1), pyridine, reflux; (iii) Zn/DCl.
C6H5
D
C6H4X
C6H4X
D
C6H4X
D
D
D
i
D
D
D
D
N
N
D
D
N
N
D
CH3
CH3
CH3
CH3
4a-d5: X = H
4b-d5: X = 4-F
1-d2
5a: X = H
5b: X = 4-F
4a-d5: X = H
4b-d5: X = 4-F
Scheme 3. The structures of 1-methyl-3-phenyl-3-pyrroline-2,2,4,5,5-d5 (4a-d5),
1-methyl-3-(4-fluorophenyl)-3-pyrroline-2,2,4,5,5-d5 (4b-d5), and MPTP-6,6-d2
(1-d2).
Scheme 5. Synthetic pathway to the deuterated pyrrolinyl substrates 4a-d5 and 4b-
d5. Reagent: (i) Zn/DCl.

A. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8813–8817
8815
the enzyme for a fixed period of time at 37 °C and the correspond-
ing pyrrolyl oxidation products were quantified by HPLC-UV anal-
ysis (Fig. 1) as described previously for 4a.13 HPLC-UV analysis was
preferred to spectrophotometry since background interference in
the near-UV wavelength range by the mitochondrial fractions used
here as enzyme source, prevented accurate spectrophotometric
measurements at 270 nm, the approximate wavelength of maxi-
mal absorption of the pyrrolyl products. The incubation time of
the enzyme-catalyzed reactions were chosen to be 10 min since
the oxidation of all the substrates evaluated here (4a–4b and 4a-
d5–4b-d5) was found to be linear (Fig. 2) for at least 15 min at sub-
with the inactivated mitochondrial preparation was only 8.4% of
that formed with the fully active preparation (6.42 0.18 M).
Similarly, when the deuterium labeled analogue 4a-d5 was incu-
l
bated with (R)-deprenyl pre-inactivated mitochondria only
0.037 0.001
l
M (about 2.6%) of the pyrrolyl oxidation product
M)
was observed compared to the amount formed (1.42 0.05
l
with the fully active preparation. These results document that oxi-
dation of the pyrrolinyl substrates by the baboon liver mitochon-
drial fraction is MAO-B dependent. The residual activity not
suppressed by (R)-deprenyl has been observed previously in exper-
iments where tetrahydropyridines served as the MAO-B sub-
strates.22 The factors contributing to this residual activity remain
to be identified. At least for baboon liver mitochondria the residual
activity is not related to the action of the MAO-A isoform since the
baboon liver mitochondria are devoid of MAO-A activity.22 Fur-
thermore, 1-methyl-3-phenyl-3-pyrroline (4a) is reported to be
an MAO-B selective substrate.12
strate concentrations of 100 lM. Control incubations of the sub-
strates carried out with no enzyme source added were used to
verify the absence of the pyrrolyl products in the incubation
samples.
To estimate the extent on non-MAO-B mediated oxidation of
the pyrrolinyl substrates 4a and 4a-d5, the baboon liver mitochon-
drial fractions that served as enzyme source were preincubated
with the MAO-B selective inactivator (R)-deprenyl.22 Following
2.3. Steady-state kinetic deuterium isotope effects on Vmax and
incubation of the test substrates (50
lM) with the mitochondria
Vmax/Km
inactivated with (R)-deprenyl, only low concentrations of the pyrr-
olyl oxidation products were observed. For example, for substrate
In order to estimate the values of the steady-state kinetic
4a the yield of pyrrolyl oxidation product 5a (0.54 0.052
l
M)
parameters (Km and Vmax) for the oxidation of the oxalate salts of
the pyrrolinyl analogues (4a–4b and 4a-d5–4b-d5) by MAO-B, ini-
tial rates were measured at eight substrate concentrations span-
ning at least two orders of magnitude. As illustrated by example
with Figure 3, the steady-state oxidation of the substrates (4a–4b
and 4a-d5–4b-d5) by MAO-B, followed Michaelis–Menten behavior.
Also, large kinetic isotope effects on Vmax were apparent from the
graphs. The Km and Vmax values as well as the steady-state kinetic
deuterium isotope effects obtained for the oxidation of the pyrroli-
nyl substrates by baboon liver mitochondrial MAO-B are summa-
rized in Table 1. The apparent isotope effects on Vmax for the
oxidation of 4a and 4b were found to be 4.29 and 3.98 using 4a-
d5 and 4b-d5, respectively, as the deuterated substrates. These val-
ues are comparable to the D(Vmax) value of 3.55 previously reported
for the oxidation of MPTP by bovine liver mitochondrial MAO-B
with MPTP-6,6-d2 as the deuterated substrate.14 We have observed
a smaller difference between the Km value for 4a and its deuterated
analogue 4a-d5 [D(Km = 0.80)] than the reported difference be-
tween the Km values of MPTP and MPTP-6,6-d2 [D(Km = 0.45)].14
The Km value of substrate 4b, was found to be, within the range
100
80
60
40
20
0
1
2
3
4
min
Figure 1. An HPLC-UV tracing showing the presence of 1-methyl-3-phenylpyrrole
(5a) (retention time of 2.96 min) in an incubation of 1-methyl-3-phenyl-3-
pyrroline (4a) with baboon liver MAO-B (0.15 mg protein/mL of the mitochondrial
preparation). Following 10 min incubation, the reaction was terminated by the
addition of trichloroacetic acid. After centrifugation, 50 lL of the supernatant was
injected into the HPLC and the effluent was monitored at a wavelength of 270 nm.
10
8
8
4a
6
4
6
4
4a-d5
2
2
0
0
0
500
1000
1500
2000
[S], µM
0
2
4
6
8
10
12
14
16
Figure 3. Determination of the Km and Vmax values for the oxidation of 4a (filled
circles) and 4a-d5 (open circles) by baboon liver MAO-B (0.15 mg protein/mL of the
mitochondrial preparation). The concentrations of the pyrrolyl oxidation products
were measured by HPLC analysis following a 10 min incubation at 37 °C. The rate
data were fitted to the Michaelis–Menten equation. All measurements were
conducted in triplicate and the concentration of the test substrates in the
Incubation time (min)
Figure 2. Linearity in the oxidation of pyrrolinyl analogues 4a (filled circles), 4b
(open circles), 4a-d5 (filled triangles) and 4b-d5 (open triangles) by baboon liver
mitochondrial MAO-B (0.15 mg protein/mL of the mitochondrial preparation). The
concentration of pyrrolyl products was quantified via HPLC-UV analysis following
termination of the enzyme-catalyzed reaction at time points of 2.5, 5.0, 10.0 or
incubations ranged from 12.5 to 2000
lM. The initial rates are expressed as
15.0 min. The concentration of the substrates used in this study was 100 lM.
nanomoles product formed/min-mg protein.

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A. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8813–8817
Table 1
4.2.1. 2-(4-Fluorophenyl)-3-(dimethylamino)allylidene(dimethyl)-
Steady-state kinetic constants and deuterium isotope effects for the MAO-B-catalyzed
oxidation of 1-methyl-3-phenyl-3-pyrrolinyl analogues
ammonium perchlorate-d14 (7b-d14
)
Yield 82.5%; mp 128–133 °C (from ethanol); 1H NMR (DMSO-d6)
d 7.22–7.29 (m, 2H), 7.31–7.38 (m, 2H); 13C NMR (DMSO-d6) d
38.45 (m), 47.57 (sep), 103.55, 115.27 (d), 128.74, 134.11 (d),
160.47, 162.66 (t), 163.74; FAB-MS m/z: 236 (MH+).
D(Vmax
)
D(Km
)
D(Vmax/Km
)
a
Compound
Vmax
Km
(
l
M)
4a
4b
4a-d5
4b-d5
9.07 0.41
15.7 1.73
2.11 0.05
3.96 0.52
118 7.27
87.2 1.87
157 26.1
73.8 4.46
4.29 0.08
3.98 0.09
0.80 0.15
1.19 0.10
5.71 0.88
3.37 0.20
4.3. Synthesis of 1-methyl-3-phenylpyrroles 5a–b and 5b-d6
a
Values are expresses in nmol/min-mg mitochondria.
1-Methyl-3-phenylpyrroles 5a–b were synthesized according to
a modification20 of the method described in the literature.19 Com-
pound5b-d6 was synthesizedfrom 7b-d14 accordingto thefollowing
procedure19,20: sodium pieces (13 mmol) was reacted with metha-
nol-d1 (4.5 mL) and the resulting solution was added under an atmo-
sphere of argon to a solution of 7b-d14 (6 mmol) in 24 mL dry
pyridine (distilled over CaH2 and stored over 4 Å molecular sieves).
The reaction was heated under reflux for 24 h and most of the pyri-
dine was removed via vacuum distillation to obtain a yellow pasty
residue to which 25 mL D2O was added. The resulting suspension
was stirred for 30 min on an ice bath and filtered. The crude solid
so obtained was dissolved in a minimum amount of ethylacetate
and purified on a short column (30 Â 60 mm) by neutral aluminum
oxide chromatography (Fluka) with petroleum ether/ethylacetate,
90:10 as mobile phase to give a white solid. The melting points of
previously reported 5a and 5b were as follows: 5a mp 46–48 °C
(from petroleum ether), lit. mp 46–47 °C19; 5b mp 100–101 °C (from
methanol), lit. mp 98–100 °C20; The characterization of previously
unreported 5b-d6 is summarized below.
expected for experimental error, equal to that of its deuterated
analogue, 4b-d5 [D(Km = 1.19)].
3. Discussion
In this study we have measured the kinetic deuterium isotope
effects on Vmax and Vmax/Km for the steady-state oxidation of 1-
methyl-3-phenyl-3-pyrroline (4a) and 1-methyl-3-(4-fluoro-
phenyl)-3-pyrroline (4b) by baboon liver mitochondrial MAO-B.
For this purpose the corresponding pyrroline-2,2,4,5,5-d5 ana-
logues (4a-d5 and 4b-d5) were employed as deuterated substrates.
Moderately large apparent isotope effects of 4.29 and 3.98 on Vmax
were observed for the two substrates, respectively. The isotope ef-
fects on Vmax/Km were found to be 5.71 and 3.37 for the two sub-
strates, respectively. Although these values may not reflect the
intrinsic isotope effects, the Vmax isotope effects suggest that a car-
bon–hydrogen bond cleavage step is at least partially rate deter-
mining.23 The isotope effects on Vmax for the two pyrrolinyl
substrates are comparable to those reported for the oxidation of
MPTP by bovine liver MAO-B [D(Vmax) = 3.55]. We conclude that
the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl
substrates is similar to that of the tetrahydropyridinyl substrates.
4.3.1. 1-Methyl-3-(4-fluorophenyl)pyrrole-d6 (5b-d6)
The title compound was synthesized from 7b-d14 in a yield of
46.8%: mp 102–103 °C; 1H NMR (CDCl3) d 6.37 (0.06H), 6.61
(0.0+1H), 6.82 (s, 0.27H), 6.97–7.04 (m, 2H), 7.39–7.46 (m, 2H);
13C NMR (CDCl3) d 35.67 (m), 106.11 (d), 115.25 (d), 118.19 (m),
122.67 (d), 124.09, 126.28 (d), 132.16 (d), 159.45, 162.68; EI-HRMS
calcd. 181.11739, found 181.11819 (MÅ+).
4. Experimental
4.1. Chemicals and instrumentation
4.4. Synthesis of 1-methyl-3-phenyl-3-pyrrolinyl analogues
(4a–4b, 4a-d5–4b-d5 and 4b-d8) and their respective oxalate salts
All starting materials, unless otherwise stated, were obtained
from Sigma–Aldrich and were used without purification. Deuter-
ated solvents and reagents were obtained from Cambridge Isotope
Laboratories. Proton and carbon NMR spectra were recorded on a
Varian Gemini 300 spectrometer. 1H NMR spectra were recorded
at a frequency of 300 MHz and 13C NMR spectra at 75 MHz. Chem-
ical shifts are reported in parts per million (d) downfield from the
signal of tetramethylsilane added to the NMR solvents, CDCl3,
DMSO-d6, and CD3OD. Spin multiplicities are given as s (singlet),
d (doublet), t (triplet), sep (septet) or m (multiplet) and the cou-
pling constants (J) are given in hertz (Hz). Fast atom bombardment
mass spectra (FAB-MS) were recorded with a VG 7070E mass spec-
trometer. Direct insertion electron impact ionization mass spectra
(EI-HRMS) were obtained with a AutoSpec ETOF (Micromass).
Melting points (mp) were determined with a Stuart SMP10 melting
point apparatus; all melting points are uncorrected. HPLC analyses
were performed with an Agilent 1200 HPLC system equipped with
an Agilent 1200 series variable wavelength detector. Thin layer
chromatography (TLC) was carried out using neutral aluminum
oxide 60 (Merck) with UV254 fluorescent indicator.
Compounds 4a–b were synthesized from the corresponding
pyrrolyl analogues, 5a–5b, according to a modification13 of the zinc
reduction procedure reported in the literature.15,16 Compounds 4a-
d5, 4b-d5, and 4b-d8 were synthesized from 5a, 5b, and 5b-d6,
respectively, according to the following procedure: DCl (15 mL,
20%) was cooled on an ice bath (0 °C) and zinc dust (20 mmol)
was carefully added followed by the appropriate pyrrolyl analogue
(4 mmol) dissolved in 34 mL ethanol-d1. The resulting suspension
was stirred for 1 h at 0 °C and DCl (10 mL, 35%) was added. The
reaction was allowed to return to room temperature and stirred
for another 1 h. Another 2 portions of zinc dust (40 mmol) were
added and the reaction was stirred for 2 h during which the reac-
tion cleared. The remaining zinc was removed via filtration and
washed with 5 mL D2O. The residual ethanol-d1 was removed from
the filtrate under reduced pressure, the pH was adjusted to ꢀ12
with NaOH (8 N) and the reaction was extracted to diethylether
(3Â 30 mL). The organic phase was dried over MgSO4, removed un-
der reduced pressure to yield the product as a light yellow oil. The
pyrrolinyl analogues were converted to their respective oxalic acid
salts in diethyl ether. Compounds 4a and 4a-d5 were purified by
recrystallizing their oxalate salts twice from methanol. Compounds
4b, 4b-d5 and 4b-d8 were purified by placing the free bases in a
vacuum oven at 40 °C for 24 h. For previously described 4aÁoxalate
the melting point was recorded as 151–153 °C (from methanol), lit.
mp 152–154 °C.13 The characterizations of compounds that were
previously unreported are summarized below.
4.2. Synthesis of 2-(4-fluorophenyl)-3-(dimethylamino)-
allylidene(dimethyl)ammonium perchlorate-d14 (7b-d14
)
Compound 7b-d14 was prepared in high yield from 4-fluorophe-
nylacetic acid, N,N-dimethylformamide-d7 (DMF-d7), and phos-
phoryl chloride according the method described in the literature.18

A. Pretorius et al. / Bioorg. Med. Chem. 16 (2008) 8813–8817
8817
4.4.1. 1-Methyl-3-phenyl-3-pyrroline-d5 (4a-d5)
incubated with 0.15 mg protein/mL of the baboon liver mitochon-
drial fraction. At time points 2.5, 5, 10, and 15 min, the reactions
were terminated with the addition of 20 lL trichloroacetic acid
(1 g/mL, w/v) and the MAO-B-generated pyrrolyl products were
measured by HPLC analysis as described above. All measurements
were conducted in triplicate and the concentrations of the pyrrolyl
products were expressed as means SEM.
The title compound was synthesized from 5a in a yield of 16.5%
(oxalate salt): mp 152–155 °C; 1H NMR (CD3OD) d 3.10 (s, 3H),
7.35–7.43 (m, 3H), 7.47–7.50 (m, 2H); 13C NMR (CD3OD) d 41.68,
60.93 (m), 62.00 (m), 117.87 (m), 126.43, 129.37, 129.58, 132.21,
137.59, 166.28; EI-HRMS calcd. 164.13618, found 164.13641 (MÅ+).
4.4.2. 1-Methyl-3-(4-fluorophenyl)-3-pyrroline (4b)
The title compound was synthesized from 5b in a yield of 29.5%
(free base): mp 64–66 °C; 1H NMR (CDCl3) d 2.53 (s, 3H), 3.60–3.65
(m, 2H), 3.77–3.80 (m, 2H), 6.02 (m, 1H), 6.95–7.01 (m, 2H), 7.27–
7.31 (m, 2H); 13C NMR (CDCl3) d 42.67, 62.38 (d), 115.30 (d),
121.93 (d), 127.00 (d), 130.67 (d), 138.97, 160.52, 163.79; EI-HRMS
calcd. 177.09538, found 177.09629 (MÅ+).
4.7. (R)-Deprenyl studies
In order to estimate the degree to which the oxidation of the pyrr-
olinyl substrates (4a and 4a-d5) was dependent upon MAO-B, ba-
boon liver mitochondria (0.3 mg protein/mL) were preincubated
with (R)-deprenylÁHCl (3 Â 10À6 M) at 37 °C for 30 min in 100 mM
sodium phosphate buffer, pH 7.4. The pre-inactivated mitochondrial
fractionswere thenadded to thetestsubstrates to yieldfinalconcen-
trations of 0.15 mg mitochondrial protein/mL. The volume of these
4.4.3. 1-Methyl-3-(4-fluorophenyl)-3-pyrroline-d5 (4b-d5)
The title compound was synthesized from 5b in a yield of 36.3%
(free base): mp 63–66 °C; 1H NMR (CDCl3) d 2.53 (s, 3H), 6.94–7.00
(m, 2H), 7.26–7.30 (m, 2H); 13C NMR (CDCl3) d 42.66, 61.95 (m),
115.28 (d), 121.90 (m), 126.99 (d), 130.65 (d), 138.79, 160.50,
163.77; EI-HRMS calcd. 182.12676, found 182.12585 (MÅ+).
incubation mixtures were 500
theoxalatesalts of thetestsubstrates was50
incubated for 10 min at 37 °C and terminated with the addition of
20 L trichloroacetic acid (1 g/mL, w/v). Control incubation reac-
l
L and the final concentration of
lM. Thereactionswere
l
tions were carried out following the same procedure with the excep-
tion that the preincubations were conducted in the absence of (R)-
deprenyl. The concentrations of the pyrrolyl products in the enzy-
matic reactions were measured by HPLC analysis as describedabove.
All measurements were conducted in triplicate and the concentra-
tions of the pyrrolyl products were expressed as means SEM.
4.4.4. 1-Methyl-3-(4-fluorophenyl)-3-pyrroline-d8 (4b-d8)
The title was synthesized from 5b-d6 in a yield of 48.0% (free
base): mp 64–68 °C; 1H NMR (CDCl3) d 6.94–7.02 (m, 2H), 7.24–
7.32 (m, 2H); 13C NMR (CDCl3) d 41.90 (m), 61.95 (m), 115.30
(d), 121.90 (m), 127.01 (d), 130.69 (d), 138.83, 160.52, 163.79;
EI-HRMS calcd. 185.14559, found 185.14566 (MÅ+).
Acknowledgments
4.5. Steady-state MAO-B activity measurements
We are grateful to Jan du Preez and the staff of the Analytical
Technology Laboratory, North-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 Coun-
cil, South Africa.
Baboon liver mitochondria were isolated as described in the lit-
erature24 and stored at À70 °C. The mitochondrial isolates were
suspended in 1 volume sodium phosphate buffer (100 mM, pH
7.4) containing 50% glycerol (w/v) and the protein concentrations
were determined by the method of Bradford.25 Since baboon liver
is devoid of MAO-A activity, inactivation of MAO-A was deemed
unnecessary.22 In order to estimate the Km and Vmax values for
the oxidation of the oxalate salts of the pyrrolinyl analogues 4a–
4b and 4a-d5–4b-d5 by MAO-B, initial rates were measured at eight
substrate concentrations spanning at least two orders of magni-
References and notes
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In order to determine the time interval for which MAO-B-cata-
lyzed substrate oxidation remains linear, 100
lM of the oxalate
salts of the test substrates (4a–4b and 4a-d5–4b-d5) were