Chemical model studies on the monoamine oxidase-B catalyzed oxidation of 4-substituted 1-cyclopropyl-1,2,3,6-tetrahydropyridines

Article abstract of DOI:10.1016/S0968-0896(97)10033-5

Two catalytic pathways have been proposed for the flavoenzyme monoamine oxidase B (MAO-B-one based on an initial single electron transfer (SET) step from the nitrogen lone pair and the second based on an initial α-carbon hydrogen atom transfer (HAT) step. The SET pathway is consistent with the mechanism based inactivation properties of various cyclopropylamines. The observation that MAO-B catalyzes the efficient oxidation of certain 1- cyclopropyl-4-substituted-1,2,3,6-tetrahydropyridines to the corresponding dihydropyridinium metabolites suggests that the catalytic pathway for these cyclic tertiary allylamines may not proceed via the putative SET generated aminyl radical cations. The present paper describes the chemical fate of a series of N-cyclopropyltetrahydropyridines examined under reaction conditions that model the SET and the HAT pathways. All of the test compounds were rapidly converted under HAT reaction conditions to their dihydropyridinium products. Although the test compounds also were oxidized rapidly under SET conditions, no evidence for dihydropyridinium product formation was observed. The products that were identified most likely were formed after cyclopropyl ring opening of the initially formed cyclopropylaminyl radical cation. The results are discussed in terms of the mechanism of MAO-B catalysis.

Full text of DOI:10.1016/S0968-0896(97)10033-5

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 283±291
Chemical Model Studies on the Monoamine Oxidase-B
Catalyzed Oxidation of 4-Substituted
1-Cyclopropyl-1,2,3,6-tetrahydropyridines
Christelle Franot, Stephane Mabic and Neal Castagnoli, Jr.*
Department of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212, U.S.A.
Received 13 August 1997; accepted 17 October 1997
AbstractÐTwo catalytic pathways have been proposed for the ¯avoenzyme monoamine oxidase B (MAO-B)Ðone
based on an initial single electron transfer (SET) step from the nitrogen lone pair and the second based on an initial a-
carbon hydrogen atom transfer (HAT) step. The SET pathway is consistent with the mechanism based inactivation
properties of various cyclopropylamines. The observation that MAO-B catalyzes the ecient oxidation of certain 1-
cyclopropyl-4-substituted-1,2,3,6-tetrahydropyridines to the corresponding dihydropyridinium metabolites suggests
that the catalytic pathway for these cyclic tertiary allylamines may not proceed via the putative SET generated aminyl
radical cations. The present paper describes the chemical fate of a series of N-cyclopropyltetrahydropyridines examined
under reaction conditions that model the SET and the HAT pathways. All of the test compounds were rapidly con-
verted under HAT reaction conditions to their dihydropyridinium products. Although the test compounds also were
oxidized rapidly under SET conditions, no evidence for dihydropyridinium product formation was observed. The
products that were identi®ed most likely were formed after cyclopropyl ring opening of the initially formed cyclopro-
pylaminyl radical cation. The results are discussed in terms of the mechanism of MAO-B catalysis. # 1998 Published
by Elsevier Science Ltd. All rights reserved.
Introduction
and the observation that these are the only cyclic amines
which show MAO-B substrate properties.
The ¯avoproteins monoamine oxidase (MAO) A and B
catalyze the a-carbon oxidation of a variety of endo-
genous and exogenous amines.1,2 Two distinct pathways
have been proposed to account for MAO's catalytic
activity (see Scheme 1). One is based on a single electron
transfer (SET) step to form an aminyl radical cation.3±6
The second pathway involves a-hydrogen atom transfer
(HAT) as the initial step.7±9 We have focused our MAO-
B mechanistic studies on 1-methyl and 1-cyclopropyl-
1,2,3,6-tetrahydropyridinyl derivatives bearing a variety
of C-4 substituents.10±21 These studies have been driven
in part by interest in the MAO-B dependent tox-
icological properties of the parkinsonian inducing agent
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1)
MPTP is converted in an MAO-B catalyzed reaction to
the unstable dihydropyridinium species 4 (Scheme 1)22
which subsequently is oxidized further to the ultimate
neurotoxin, the 1-methyl-4-phenylpyridinium species
5.23 According to the SET pathway, product formation
proceeds via the aminyl radical cation 2 and a-carbon
radical 3 intermediates. According to the HAT pathway
the substrate molecule is converted directly to the a-
carbon radical 3. In an attempt to distinguish between
these pathways, we examined the behavior of various 4-
substituted 1-methyltetrahydropyridinyl derivatives
under reaction conditions that model the SET and HAT
pathways.
The
reactants
were
oxidized
to
the corresponding dihydropyridinium and pyridinium
species under both reaction conditions. However, we
were unable to make useful correlations between how
the compounds behaved in these chemical models with
their MAO-B substrate behavior.24
Key words: Monoamine oxidase; chemical model; SET (single
electron transfer); cyclopropylamine; tetrahydropyridine.
*Corresponding author. Tel: (540) 231-8202; fax: (540) 231-8890;
e-mail: ncastagnoli@chemserver.chem.vt.edu
0968-0896/98/$19.00 # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(97)10033-5

284
C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
Ph
Ph
Ph
- H
e
(HAT)
Ph
Ph
Ph
Ph
Ph
N
N
N
e
H
e
2e
CH2
(SET)
2H
N
N
N
N
N
CH3
CH3
CH3
CH3
CH3
6
8
3
7
1
2
4
5
Scheme 1. Proposed pathways for the MAO catalyzed oxida-
tion of MPTP.
Scheme 2. Proposed SET pathway mediating the inactivation
properties of 6.
It is well known that cyclopropylamines, presumably via
an SET generated cyclopropylaminyl radical cation
(Scheme 2), can be ecient time and concentration
dependent (mechanism based) inactivators of MAO-B.25
For example, although MPTP is an excellent MAO-B
substrate,13 the corresponding N-cyclopropyl analog 6 is
an ecient MAO-B mechanism based inactivator with
such poor substrate properties that metabolite forma-
tion could not be detected spectrophotometrically.16
The inactivation pathway presumably proceeds via the
cyclopropylaminyl radical cation 7 followed by ring
opening to form the distonic radical cation 8 that alkyl-
ates the enzyme active site (Scheme 2). The recent char-
acterization of a three carbon adduct, derived from the
cyclopropyl group of an N-benzylcyclopropylaminyl
inactivator, to a cysteinyl residue of beef liver MAO-B
provides strong evidence in support of the SET path-
way.26
MAO-B14 while the 4-phenoxy analogue 11 is an excel-
lent substrate that displays no inactivator properties
(Table 1)19. In an e€ort to characterize more completely
the pathway(s) by which MAO-B processes these types
of cyclopropyltetrahydropyridinyl derivatives, we have
examined the chemical fate of the N-cyclopropyl-
tetrahydropyridinyl derivatives 6 and 9±11 when treated
under SET and HAT oxidizing conditions. The results
are discussed in terms of the behavior of these com-
pounds in the presence of MAO-B.
Results and Discussion
Treatment of 6 and 9±11 under HAT conditions (t-butyl
peroxybenzoate in the presence of catalytic amounts of
CuI) followed by reduction of the reaction mixtures with
NaBD4 resulted in the corresponding tetrahydro-
pyridine-2,6-d2 species 6-d2 and 9-d2-11-d2, respectively
(Scheme 3).27,28 The reactions were rapid in all cases
since gas chromatography-electron ionization mass
spectral (GC-EIMS) analyses of reaction mixtures trea-
ted with NaBD4 showed that none of the starting d0
materials were present after a 10 min reaction time.
This behavior is similar to that observed when a series
of 1-methyltetrahydropyridinyl analogues was exam-
ined under HAT reaction conditions.24 We conclude,
The 4-(4-pyridinyl) analogue 9 and several structurally
related heteroaryl analogues also display MAO-B
mechanism based inactivation properties.14 Other
experimental evidence, however, suggests that aminyl
radical cations may not be obligatory intermediates
in the MAO-B catalyzed oxidation of tetrahydro-
pyridines.17,18 For example, the 4-(1-methyl)pyrrol-2-yl
analogue 10 is a good substrate and poor inactivator of
Table 1. MAO-B substrate and inactivator properties of various N-cyclopropyl MPTP analogs
R
N
N
R =
O
N
11
6
9
10
1
1
1
1
Compound
kcat (min
)
Km (mM)
kcat/KM (min mM
)
kinact (min
)
KI (mM)
kinact/KI (min mM )
6
9
Turnover too slow to detect
Turnover too slow to detect
0.2
0.7
0.1
0.18
1.4
0.1
0.1
10
11
58
215
290
1650
Rate of inactivation too slow to measure
Rate of inactivation too slow to detect
0.13

C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
285
t-BuOOOCPh
CuI
CuII + PhCOO
R
N
R
N
R
R
R
N
t-BuOH
t-BuO
H(D)
CuII
CuI
+
+
H
H
(D)H
N
N
R
C6H5
4-C5H4N
2-C4H3NCH3
6 (6-d2)
9 (9-d2)
10 (10-d2)
11 (11-d2)
24
25
26
27
12
13
14
15
16
17
18
19
20
21
22
23
OC6H5
Scheme 3. Proposed HAT pathway for the t-butoxy radical initiated oxidation of the 4-substituted 1-cyclopropyl-1,2,3,6-tetra-
hydropyridines 6 and 9±11
therefore, that the t-butoxyl radical converts these tetra-
hydropyridinyl derivatives to the corresponding allylic
radicals 12±15 which, in turn, are oxidized by the CuII
species, formed in the reaction between t-butyl peroxy-
benzoate and CuI, to the dihydropyridinium inter-
mediates 16±19 (Scheme 3). Deprotonation of 16±19
generates the corresponding free bases 20±23 that are
converted to the pyridinium products 24±27 by an analo-
gous oxidative sequence. Reduction of 24±27 with
NaBD4 provides the d2 products 6-d2 and 9-d2-11-d2
that were identi®ed by GC-EIMS analysis.
min) now dominated the tracing (>90% of the total ion
+
.
current). The mass spectrum (M
155) led to the
assignment of 4-phenylpyridine (28) for the structure of
the compound giving rise to the peak at 6.0 min. This
assignment was con®rmed by comparison of the corre-
sponding spectrum obtained with an authentic sample
of 28. Treatment of reaction mixtures with NaBD4 or
with water alone gave the same product with no deu-
terium incorporation suggesting that 28 was formed
during the course of the reaction, presumably from early
arising intermediates that were converted with NaBH4
to the two major products. The minor peak at 6.3 min
has not been identi®ed but may be derived from the
tetrahydropyridinyl starting material since it is not pres-
ent in the absence of this reactant. The minor peak at
7.2 min is derived from the phenanthroline reagent and
was observed at all time points examined.
The SET studies were performed using the FeIII phen-
anthroline complex Fe3+(Phen)3(PF6 )3 as the electron
acceptor29 in the presence of pyridine, the base used to
deprotonate the intermediate cyclopropylaminyl radical
cations. The reactions were run in CH3CN at room
temperature, conditions that worked well in the corre-
sponding study with a series of 4-substituted 1-methyl-
1,2,3,6-tetrahydropyridines.24 GC-EIMS analysis of the
reaction with 6 and the Fe+3 complex established that
no starting material remained after 10 min. Further-
more, none of the starting material was observed in the
GC-EIMS total ion current (TIC) chromatogram
(Figure 1) of a 10 min reaction mixture that had been
treated with NaBH4. Consequently, unlike the behavior
of the N-methyltetrahydropridines, 6 was not converted
to its dihydropyridinium or pyridinium oxidation pro-
ducts under SET conditions.
The GC-EIMS total ion current (TIC) chromatogram
(Figure 1) showed two major peaks [tR ˆ 6:7 min (73%)
and tR ˆ 7:3 min (13%)] and three minor peaks. The
appearance of the major peaks was dependent on
NaBH4 treatment. With time (16 h), neither of the major
peaks was present and one of the minor peaks (tR ˆ 6:0
Figure 1. GC-EIMS total ion current chromatogram of a
NaBH4 treated 10 min SET reaction mixture containing 6.

286
C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
Figure 3. GC-EI mass spectrum of 1-propyl-4-phenyl-1,2,3,6-
tetrahydropyridine (30) isolated from a NaBH4 treated 10 min
SET reaction mixture containing 6.
Figure 2. GC-EI mass spectrum of 1-ethyl-4-phenyl-1,2,3,6-
tetrahydropyridine (29) isolated from a NaBH4 treated 10 min
SET reaction mixture containing 6.
The mass spectrum of the major peak in the TIC tra-
cing with tR ˆ 6:7 min is shown in Figure 2. The
parent ion at m/z 187 is consistent with N-ethyltetra-
hydropyridinyl derivative 29. As expected, a major
fragment ion (i) at m/z 172 can be assigned to loss of a
CH3 radical. A synthetic standard of 29 gave an iden-
tical GC-EI mass spectrum. The structure of this com-
pound was fully con®rmed by 1H NMR analysis of
material isolated from a preparative scale SET reaction
mixture.
Similar experiments were carried out with the 4-(4-pyr-
idinyl)-, 4-(1-methylpyrrol-2-yl)-, and 4-phenoxy-1-
cyclopropyl-1,2,3,6-tetrahydropyridines (9, 10, and 11,
respectively). In all cases, the starting materials were
consumed rapidly and no evidence of ring a-carbon
oxidation was obtained. Quantitatively, the fate of each
molecule was dependent on the C4 substituent (see
Table 2). Reaction of the pyridinyl analogue 9 led to the
N-ethyl product 31 but the levels of the N-propyl prod-
uct 34 were too low to detect. As with the phenyl case,
however, the only product detected in the GC-EIMS
TIC chromatogram after a 16 h reaction period was the
pyridinyl product, in this case 4,40-bipyridine (37). The
intermediates leading to the N-ethyl (32) and N-propyl
(35) products derived from the pyrrolyl analogue 10
appeared to be relatively stable since 32 and 35 repre-
sented the major species at all time points analyzed with
only 5% of the pyridinyl species 38 being detected at
16 h. Finally, the behavior of the 4-phenoxy analogue 11
was intermediate in that about 50% of the starting
The mass spectrum (Figure 3) of the second major peak
with tR ˆ 7:3 min is similar to the spectrum of com-
pound 29 except that the parent ion (m/z 201) appears at
15 amu higher than the parent ion of 29. This informa-
tion plus the presence of the same fragment ion i at m/z
172 (loss of a CH3CH2 radical) led to the assignment of
the N-propyl derivative 30 as the structure of this prod-
uct. The identical spectrum was observed with a syn-
thetic sample of 30.
Table 2. GC-EI TIC yieldsa of various products from the cyclopropyltetrahydropyridine 6 and 9±11 observed following SET initiated
oxidation and NaBH4 treatment
R
N
R
N
R
N
R
N
10 min
90 min
16 h
10 min
90 min
16 h
10 min
890 min 16 h
R
C6H5
6
29
31
32
33
70%
74
50%
2
0%
30
34
35
36
20%
0
6%
0
0%
28
37
38
39
10%
26
0
44% 100%
4-C5H4N
2-C4H3NCH3
OC6H5
9
10
11
0
90
40
0
5
98
5
100
5
55
76
65
70
45
22
30
25
10
2
5
50
a
The actual yields are somewhat less since these estimates do not take into account the contributions of the minor peaks (10% or less)
to the integrated total ion current.

C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
287
Ph
N
Ph
Ph
Ph
?
NaBR4
SET
H
H
H
H
H
H
H
H
H
H
H
H
(R = H or D)
H
N
H
N
R'
N
CR'2
R3C
NH
R3C
6-d0
7-d0
40: R = H
40a-d3: R = D
29: R = R' = H
29a-d3: R = H; R' = D
29c-d3: R = D; R' = H
Ph
N
Ph
Ph
N
Ph
?
NaBH4
SET
D
D
D
D
D
D
D
D
D
D
D
N
D
D
D
N
H
CH2
H3C
NH
H3C
7-d4
40b-d3
6-d4
29b-d3
Scheme 4. Proposed pathway leading to various N-ethyl (29) products in the SET reaction.
material could be accounted for as the N-ethyl (36) and
N-propyl (39) species after the 16 h reaction period with
the remaining 50% recovered as the 4-pyridinyl species 39.
The di€erences in the life times of the intermediates
leading to the N-ethyl and N-propyl products (see
Schemes 4 and 5) presumably re¯ect stereoelectronic
e€ects of the C4 substituents on the stability of these
intermediates.
ethyl group. Based on these data, this d3 pro-
duct has been assigned the structure 29a±d3
(Scheme 4).
2. The N-propyl product incorporated two deuter-
+
.
ium atoms (M m/z 203). Once again, the frag-
ment ions observed at m/z 202 and 201 suggested
the presence of both a proton and a deuteron at
.
C6. The fragment ion resulting from loss of Et
+
.
(m/z 174, M
29) retained the two deuterium
Additional information regarding the origins of the
three products formed from 6 was sought with the aid of
deuterium labeling (Table 3). GC-EIMS analysis of a
10 min reaction mixture treated with NaBD4 established
the following:
atoms establishing that no deuterium atom had
been incorporated into the N-ethyl group. A
structure consistent with these data is 30±d2.
Ph
Ph
Ph
SET
R
R
R
R
R
R
R
R
R
R
R
1. The N-ethyl product incorporated three deuter-
+
N
R
N
N
.
ium atoms (M
fragment ions at m/z 189 and 188, corresponding
at m/z 190). The presence of
CH2
+
+
.
.
to (M
1)+ and (M
2)+, indicated the
8: R = H
8-d4: R = D
6: R = H
7: R = H
6-d4: R = D
7-d4: R = D
presence of one deuterium atom at the C6 posi-
tion. A weak fragment ion at m/z 161 (observed at
m/z 158 with 29 as the reactant), corresponding
Ph
Ph
Ph
NaBR4
R = H or D
+
.
to (M
29)+, is typical of C4-phenyl substituted
H
R
R
R
tetrahydropyridines.30 This fragment is formed
by loss of the C2/C3 ethylene group plus one
hydrogen atom from the phenyl ring. Conse-
quently, no deuterium is present at the C2 or
R
N
R"
R
N
R
R
N
R
R
CH
CH2R
CH2R
R'
42: R = H
42-d4: R = D
43: R = H
43-d4: R = D
30-d2: R = H; R' = R" = D
30-d4: R = R" = D; R' = H
C3 positions. Finally, the loss of the terminal
+
15)+ led to the
.
methyl group as CH3 (M
.
Scheme 5. Proposed SET pathway for the formation of 30-d2
from 6 (following NaBD4 treatment) and 30-d4 from 6-d4
(following NaBH4 treatment).
conclusion that the remaining 2 deuterium atoms
were located on the methylene carbon of the

288
C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
3. As mentioned earlier, the 4-phenylpyridine (28)
recovered from this reaction contained no deuter-
The N-propyl product isolated in the reaction with 6-d4
as substrate retained all four of the deuterium atoms
present in the d4 starting material. A relatively strong
fragment ion at m/z 204 indicated the presence of a
proton at C6. Furthermore, the base peak at m/z 175
ium (M + m/z 155).
.
A third experiment was conducted with 1-cyclopropyl-4-
phenyl-1,2,3,6-tetrahydropyridine-2,2,6,6-d4 (6-d4) as
.
resulting from loss of Et , appeared at 30 amu lower
.
than the mass of the parent ion. Consequently, Et must
substrate. As expected, the 4-phenylpyridine formed
+
.
from this reactant contained two deuterium atoms (M
157), most likely at the 2- and 6-positions. The N-ethyl
have contained one deuterium atom and that deuterium
atom must have been derived from the C6 position of
the starting material. These spectral features are well
accommodated by 30-d4 that would result from deuter-
ium migration as depicted in Scheme 5. SET initiated
oxidation of 6-d4 generates the aminyl radical cation 7-
d4 which, following ring opening, leads to the distonic
radical cation 8-d4. Rearrangement of 8-d4 yields the
more stable allylic radical cation 42-d4. Deprotonation
of 42-d4 followed by a second electron transfer yields 43-
d4 which, following NaBH4 reduction, gives 30-d4. Ear-
lier studies on the mass spectral characteristics of the
electron ionization generated radical cation 7-d4 from 6-
d4 have established that an analogous migration takes
place in the gas phase.31 This pathway also is consistent
with the results obtained with the NaBD4 treated reac-
tion mixture starting from 6. The sequence 6 ! 7 ! 8
! 42 would lead to 43 which, upon treatment with
NaBD4, would yield 30-d2 the mass spectrum of which is
consistent with that observed (Table 3).
+
product contained three deuterium atoms (M 190)+.
.
Since ions were observed at m/z 189, assigned to
+
+
+
+
.
.
H ) , and m/z 188, assigned to (M
.
.
D ) , we
have concluded that one hydrogen and one deuterium
(M
.
atom are attached to C6. The loss of Me from this
product gives the base peak at m/z 175 and therefore all
three deuterium atoms were retained in the fragment.
These data are consistent with 29b-2,2,6-d3 in which the
proton at C6 is derived from NaBH4 (Scheme 4).
Since it was dicult to rationalize the conversion of an
N-cyclopropyl group to an N-ethyl group, we considered
CH3CN as a potential source of the N-ethyl group.
When the reaction was run with 6-d0 in CD3CN and
worked-up with NaBH4, the product again contained
three deuterium atoms. Their location on the terminal
methyl position of the N-ethyl group was readily
demonstrated by mass spectral analysis since an intense
ion was present at m/z 172 due to the loss of the terminal
CD3 radical. These data led to the assignment of 29c-d3
for this product. We conclude, therefore, that aceto-
nitrile is the source of the N-ethyl group of 29. In addi-
tion, the corresponding reaction of 6 in proprionitrile
does not lead to the formation of the N-ethyl derivative
29 but, as expected, only to the N-propyl derivative 30.
In view of the deuterium labeling pattern, the most likely
structures of the intermediates leading to the various
N-ethyl products are the corresponding iminodihydro-
pyridinium species 40 shown in Scheme 4. The pathway
leading to 40, however, remains to be determined.
The available data from these SET experiments are
summarized in Scheme 6. N-Methyltetrahydropyridines
(A) undergo allylic ring a-carbon oxidations to yield B
in both model reactions. Compounds B also are formed
in the MAO-B catalyzed oxidation of A. N-Cyclo-
propyltetrahydropyridines (C) also undergo allylic ring
a-carbon oxidation to yield tetrahydropyridine products
(D) under HAT but not under SET reaction conditions.
Somewhat indirect evidence suggests that the products
obtained in these reactions that are trapped following
hydride (or deuteride) reduction are formed from the
Table 3. Key GC-EIMS ions of products obtained form compound 6 and 6-d4 following a 10 min reaction under the SET reaction
and workup conditions indicated
Substrate
Reaction conditions
Product
N-Ethyl product
N-Propyl product
+
.
+
+
.
+
+
+
+
+
.
Et )
.
.
Me )
.
M
.
.
H )+ or
.
D ]+
.
M
(M
or [M
H.)+
D.]+
(M
(M
(M
+
+
.
.
[M
6
NaBH4
(CH3CN)
NaBD4
187 (100)
190 (100)
190 (100)
190 (100)
186 (65)
[185 (0)]
189 (65)
[188 (15)]
189 (65)
[188 (15)]
189 (65)
[188 (0)]
172 (70)
175 (70)
[188 (15)]
172 (70)
201 (70)
203 (70)
205 (70)
201 (70)
200 (13)
[199 (0)]
202 (35)
172 (100)
174 (100)
175 (100)
172 (100)
6
(CH3CN)
NaBH4
[201 (15)]
204 (14)
[z203 (5)]
200 (13)
[199 (0)]
6-d4
64
(CH3CN)
NaBH
(CD3CN)

C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
289
hydropyridine (29)33 and 1-propyl-4-phenyl-1,2,3,6-tetra-
hydropyridine (30)12 were prepared as reported
previously. Free bases of the tetrahydropyridines were
extracted with ethyl acetate from saturated aqueous
potassium carbonate of the corresponding protonated
salts. The combined organic extracts were dried over
MgSO4 and the solvent was evaporated under reduced
pressure. All reactions were conducted using ¯ame dried
glassware under an atmosphere of dry nitrogen. Chro-
matography refers to ¯ash column chromatography on
silica gel unless otherwise noted. Proton NMR spectra
were recorded on a Bruker WP 270-MHz spectrometer.
Chemical shifts are expressed in ppm down®eld from
internal tetramethylsilane. Spin multiplicities are given
as s (singlet), brs (broad singlet), d (doublet), t (triplet)
or m (multiplet). Coupling constant values (J ) are given
in hertz (Hz). GC-EIMS analyses were performed at an
ionization voltage of 70 eV on a Hewlett Packard (HP)
5890 GC ®tted with an HP-1 methyl silicone capillary
column (12.5 mÂ0.2 mmÂ0.33 mm ®lm thickness)
employing helium as the carrier gas (40 mL/min). The
ion currents were detected with an HP 5870 mass-selec-
tive detector. Data were acquired using an HP 5970
Chemstation. Normalized peak heights are reported as a
percentage of the base peak.
Fe+3(Phen)3 (SET)
R
N
R
(HAT)
O
N
MAO-B
R
A
B
R
R
N
Fe+3(Phen)3
SET
O
N
N
HAT
E
D
C
MAO-B
Scheme 6. Fate of N-methyl- and N-cyclopropyltetrahydro-
pyridines in SET, HAT, and MAO-B oxidation conditions.
ring opened intermediate E, the same intermediate
postulated to account for the inactivation of MAO-B. It
may be reasonable to speculate, therefore, that the
MAO-B catalyzed ring allylic a-carbon oxidations of
certain N-cyclopropyltetrahydropyridines do not proceed
via the SET pathway but rather via a pathway that does
not include the cyclopropylaminyl radical cation inter-
mediate. An alternative explanation would invoke a
restricted conformation of the cyclopropylaminyl radi-
cal cation in the enzyme active site such that overlap of
the half-®lled p-orbital on the nitrogen atom and the p-
type orbitals of the cyclopropyl group required for ring
opening would be prohibited. We hope to explore this
possibility by examining the chemical fate under SET and
HAT reaction conditions of rigid N-cyclopropyltetra-
hydropyridinyl analogues that impose such constraints.
The HAT model. Solutions of the tetrahydropyridine
free bases 6 and 9±11 (0.114 mmol) and CuCl (1.1 mg,
10%) in acetonitrile (5 mL) were prepared. Nitrogen
was bubbled gently through the solution and t-butyl
peroxybenzoate (43 mL, 0.228 mmol) was added at room
temperature. Timed aliquots (0.1 mL) (1, 2, 3, 5 min) of
the reaction mixtures were treated with an excess of
NaBH4 or NaBD4 in CH3OH and 5 min later the solvent
was evaporated under reduced pressure. A saturated
solution of potassium carbonate was added and organic
compounds were extracted with ethyl acetate. The
organic phase was dried over MgSO4 and analyzed by
GC-EIMS. Ethyl acetate extracts of potassium carbonate
only treated aliquots also were examined by GC-EIMS.
Experimental
Important notice: 1-Methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine (1) and related 1,4-disubstituted 1,2,3,6-tetra-
hydropyridines are known nigrostriatal neurotoxin sand
should be handled using disposable gloves in a properly
ventilated hood. Detailed procedures for the safe handling
of MPTP have been reported.32
The SET model. To a solution of the tetrahydropyridine
free bases 6, 6-d4 and 9±11 (0.0125 mmol) and pyridine
(5 mL, 0.07 mmol) in 10 mL of acetonitrile was added at
room
temperature
Fe3+(Phen)3(PF6
)
(51 mg,
3
0.05 mmol). Aliquots (0.5 ml) of the reaction mixture
were treated with an excess of NaBH4 or NaBD4 in
CH3OH following which the solvents were removed
under reduced pressure. A saturated solution of aqu-
eous potassium carbonate was added and the organic
compounds were extracted into diethyl ether. The
extract was dried over MgSO4 and analyzed by GC-
EIMS. Alternatively, a 0.5 mL aliquot (5 min) was trea-
ted with saturated potassium carbonate and the organic
compounds directly extracted with diethyl ether and
analyzed by GC-EIMS.
General methods
Reagents and starting materials were obtained from
commercial suppliers and were used without further
puri®cation. Tris(1,10-phenanthroline)iron (III) tris(hexa-
¯uorophosphate) was prepared by a literature method.29
The preparations of the tetrahydropyridines 6,16 9,14
10,14 11,19 6-2,2,6,6-d4,27 1-ethyl-4-phenyl-1,2,3,6-tetra-

290
C. Franot et al./Bioorg. Med. Chem. 6 (1998) 283±291
Characterization of 1-ethyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine (29) formed from 6 in the SET model study.
To a solution of 6 free base [from 144.6 mg of the
corresponding oxalate salt (144.6 mg, 0.5 mmol)] and
pyridine (0.2 mL, 2.5 mmol) in 200 ml of acetonitrile was
added Fe3+(Phen)3(PF6 )3 (2 g, 2 mmol) at room tem-
perature. After 20 min, the mixture was added to a
solution of NaBH4 (189 mg, 5 mmol) in CH3OH
(100 mL) at 0 ꢀC. After stirring for 1 h, the solvent was
evaporated under reduced pressure, the solid residue
was stirred with diethyl ether (100 mL) and the red
reduced iron complex was removed by ®ltration. A
solution of saturated K2CO3 (100 mL) was added to the
ether ®ltrate. The aqueous layer was extracted an addi-
tional three times with diethyl ether (80 mL each) and
the combined organic extracts were dried over MgSO4
and solvent evaporated under reduced pressure. The
residue was chromatographed with CH2Cl2 employing a
CH3OH gradient from 0 to 5%. The 1-ethyl-4-phenyl-
tetrahydropyridine 29 was obtained as a yellow oil. The
1H NMR spectrum (CD3Cl) d 1.18 (t, 3H, CH3, J ˆ 7:2
4. Silverman, R. B.; Yamasaki, R. B. Biochemistry 1984, 23,
1322±1332.
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H. B.; Edmondson, D. E.; Stack, D. E.; Cremonesi, P.; Han-
son, A.; Doerge, D. R.; Divi, R. L.; Sayre, L. M.; Engelhart, D.
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0
Hz), 2.55±2.63 (m, 4H, C1 , C3), 2.74±2.78 (m, 2H, C2),
3.19±3.22 (m, 2H, C6), 6.05±6.07 (m, 1H, C5) and GC-
EIMS spectrum (free base, m/z, %) 187 (M +, 100), 186
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N. J. Med. Chem. 1994, 37, 944±949.
.
(46), 172 (76), 158(15), 130 (26), 129(43), 128(26), 115 (43),
110 (37), 91 (26), 77 (15) were identical to the corre-
sponding spectra of an authentic synthetic standard.33
14. Nimkar, S. K.; Anderson, A.; Rimoldi, J. M.; Stanton, M.;
Castagnoli, K. P.; Mabic, S.; Wang, X-Y.; Castagnoli, Jr. N.
Chem. Res. Toxicol. 1996, 9, 1013±1022.
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1997, 40, 1982±1989.
Characterization of the 1-ethyl-d2-4-phenyl-1,2,3,6-
tetrahydropyridine-6-d1 (29a-d3) formed from 6 in the
SET model study. The same procedure just described
was followed except that NaBD4 was used instead of
NaBH4. Compound 29a-d3 was obtained as a yellow oil:
1H NMR (CD3Cl) d 1.18 (s, 3H, CH3), 2.60±2.62 (m,
2H, C3), 2.73±2.76 (m, 2H, C2); 3.16±3.19 (m, 1H, C6),
6.06±6.07 (m, 1H, C5); GC-EIMS analysis (free base, m/
16. Hall, L.; Murray, S.; Castagnoli, K.; Castagnoli, Jr. N.
Chem. Res. Toxicol. 1992, 5, 625±633.
17. Kuttab, S.; Kalgutkar, A.; Castagnoli, Jr. N. Chem. Res.
Toxicol. 1994, 7, 740±744.
18. Anderson, A.; Kuttab, S.; Castagnoli, Jr. N. Biochemistry
1996, 35, 3335±3340.
19. Rimoldi, J. M.; Wang, Y-X.; Nimkar, S. K.; Kuttab, S. H.;
Anderson, A. H.; Burch, H.; Castagnoli, Jr. N. Chem. Res.
Toxicol. 1995, 8, 703±710.
z, %) 190 (M +, 100), 189 (70), 188 (28), 175 (76), 174
.
(28), 161 (9), 131 (33), 130 (48), 129 (30), 116 (33), 115
(22), 113 (39), 92 (13), 91 (20), 77 (15).
20. Ottoboni, S.; Caldera, P.; Trevor, A.; Castagnoli, Jr. N. J.
Biol. Chem. 1989, 264, 13684±13688.
21. Wang, Y-X.; Castagnoli, Jr. N. J. Med. Chem. 1995, 38,
1904±1910.
Acknowledgements
22. Chiba, K.; Trevor, A.; Castagnoli, Jr. N. Biochem. Biophys.
Res. Commun. 1984, 120, 574±578.
This work was supported by the National Institute of
Neurological and Communicative Disorders and
Strokes (NS 28792) and by the Harvey W. Peters Center
for the Study of Parkinson's Disease.
23. Singer, T. P.; Ramsay, R. R.; Sonsalla, P. K.; Nicklas, W.
J.; Heikkila, R. E. In Biochemical mechanism underlying
MPTP-induced and idiopathic parkinsonism; Singer, T. P.;
Ramsay, R. R.; Sonsalla, P. K.; Nicklas, W. J.; Heikkila, R. E.
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