Studies on the monoamine oxidase-B-catalyzed biotransformation of 4- azaaryl-1-methyl-1,2,3,6-tetrahydropyridine derivatives

Article abstract of DOI:10.1021/jm9900319

The substrate properties of a series of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridinyl (MPTP) analogues in which the C-4 phenyl group has been replaced with various 4-azaaryl moieties have been examined in an effort to evaluate the contribution of electro

Full text of DOI:10.1021/jm9900319

1828
J . Med. Chem. 1999, 42, 1828-1835
Stu d ies on th e Mon oa m in e Oxid a se-B-Ca ta lyzed Biotr a n sfor m a tion of
4-Aza a r yl-1-m eth yl-1,2,3,6-tetr a h yd r op yr id in e Der iva tives
Sandeep K. Nimkar, Ste´phane Mabic, Andrea H. Anderson, Sonya L. Palmer, Thomas H. Graham,
Milly de J onge, Lisa Hazelwood, Sean J . Hislop, and Neal Castagnoli, J r.*
Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061
Received J anuary 21, 1999
The substrate properties of a series of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinyl (MPTP)
analogues in which the C-4 phenyl group has been replaced with various 4-azaaryl moieties
have been examined in an effort to evaluate the contribution of electronic, polar, and steric
parameters to the MAO-B-catalyzed oxidation of this type of cyclic tertiary allylamine to the
corresponding dihydropyridinium metabolite. No significant correlation could be found with
the calculated energy of the C-H bond undergoing cleavage. A general trend, however, was
observed between the magnitude of the log P value with the magnitude of kcat/Km. The results
indicate that the placement of a polar nitrogen atom in the space occupied by the phenyl group
of MPTP leads to a dramatic decrease in substrate properties. Enhanced substrate properties,
however, were observed when benzoazaarenes replaced the corresponding five-membered
azaarenes. These results are consistent with our previously published molecular model of the
active site of MAO-B.
In tr od u ction
efficient inactivator of MAO-B.12 The 4-benzyl analogue
12, however, proved to be both a good substrate and an
inactivator of this enzyme.5 Furthermore, the 4-thio-
phenoxy (13), 4-phenoxy (14), and 4-(2-methylphenyl)
(15) analogues14 are devoid of inactivator properties but
are good to excellent MAO-B substrates with kcat/Km
values for conversion to the dihydropyridinium metabo-
The flavoenzymes monoamine oxidase-A and -B (MAO-
A and -B) catalyze the oxidative deamination of endog-
enous neurotransmitters such as dopamine and sero-
tonin1-3 and a variety of xenobiotics. A novel structural
type displaying MAO substrate properties is the 1-meth-
yl-4-substituted-1,2,3,6-tetrahydropyridinyl system A
shown in Scheme 1. (The only classes of cyclic tertiary
amines reported to be substrates for MAO are the six
(1,2,3,6-tetrahydropyridinyl) and five (pyrrolinyl) sys-
tems; both R-carbon oxidations occur at allylic centers.)
These compounds undergo allylic, R-carbon oxidation to
form, via the C-6 radicals B, the generally unstable
dihydropyridinium intermediates C that are oxidized
further to the pyridinium metabolites D. An important
member of this series of compounds is the parkinsonian
inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine (MPTP, 1) which is biotransformed to the
dihydropyridinium species MPDP+ (2) and subsequently
to the neurotoxic pyridinium metabolite MPP+ (3)
(Scheme 1).
lites ranging from 635 (15) to 1980 (13) min-1 mM-1
.
When subjected to model SET reaction conditions, all
of the cyclopropyltetrahydropyridinyl derivatives un-
derwent ring-opening reactions.16 No evidence of R-car-
bon oxidation was observed. These results plus the
report that cyclopropylaminyl radical cations undergo
ring opening at rates too fast to measure17 suggested
that the MAO-B-mediated R-carbon oxidation of these
compounds did not proceed via cyclopropylaminyl radi-
cal cations. On the other hand, the alignment of the half-
filled p-orbitals of the aminyl radical cations with the
p-type orbitals of the cyclopropyl carbon-carbon bonds
required for efficient ring opening of these systems could
be prevented by steric constraints imposed by the active
site.
The MAO catalytic pathway has been a topic of
debate. Arguments in support of a hydrogen atom-
transfer (HAT) pathway (4 f 6 f 7, Scheme 2)5-7 and
a single-electron-transfer (SET) pathway (4 f 5 f 6 f
7)8-11 have been advanced. Particularly persuasive
evidence supporting the SET pathway is the mecha-
nism-based inactivation properties of cyclopropylaminyl
derivatives such as N-benzylcyclopropylamine (8). MAO-
B-catalyzed bioactivation of 8 proceeds through the
cyclopropylaminyl radical cation 9 and the ring-opened
primary carbon radical 10 that bioalkylates an active
site functionality of MAO-B.10
The structural features of the active sites of MAO-A
and -B are not well-defined. In the absence of X-ray
crystallographic data,18-20 SAR studies have been pur-
sued to gain an appreciation of how steric, electronic,
and polar properties influence the interactions of sub-
strates and inhibitors with these enzymes.13,15,21-27 We
have focused our SAR work on analogues of MPTP
(Chart 1). The poor MAO-B substrate properties of the
4-pyridinyl analogues 16-18 (kcat/Km ) 6-60 min-1
mM-1) compared to MPTP (kcat/Km ) 1471 min-1 mM-1
)
point to the possible importance of electronic and/or
polar factors.15 The poor substrate properties of the
4-(pyrrol-2-yl) analogue 19 (kcat/Km ) 155 min-1 mM-1
versus the 4-(1-methylpyrrol-2-yl) analogue 20 (kcat/Km
) 1800 min-1 mM-1) may reflect an influence of steric
Studies with a variety of MPTP analogues have
provided evidence both supporting and challenging the
SET pathway.5,12-15 Consistent with the SET pathway,
the 4-phenyl-1-cyclopropyl analogue 11 (Chart 1) is an
)
10.1021/jm9900319 CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/30/1999

MAO-B-Catalyzed Biotransformation of MPTPs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1829
Sch em e 1. MAO-B-Catalyzed Oxidation of MPTP
Ch a r t 1. Structures of 1,2,3,6-Tetrahydropyridinyl Derivatives Discussed in the Text
Sch em e 2. Proposed HAT and SET Pathways for the
MAO-Catalyzed Oxidation of Amines
sufficiently nucleophilic to undergo reaction with 38 at
room temperature to generate the pyridinium interme-
diates 30, 32, 34, and 36 in good yield. These compounds
proved to be hygroscopic and were reduced without
purification to the desired tetrahydropyridinyl target
compounds 22, 23, 26, and 27. The final products were
converted to their stable oxalate salts.
The success achieved with the chloromethylpyri-
dinium substrate led us to examine 4-chloropyridine 39
as a possible electrophile for these condensation reac-
tions. As expected, 39 did not react with 1H-[1,2,3]-
triazole 31, even at elevated temperatures. The corre-
sponding triazolylsodium salt 40, however, did undergo
reaction at 100 °C. GC-EIMS analysis of the reaction
mixture indicated the presence in low yield of two
isomeric triazolylpyridinyl products (M•+ 146 Da) in a
ratio of 2/1 (Scheme 4). The major isomer gave a
fragment ion at m/z ) 118, corresponding to a loss of
N2 (28 amu), which is characteristic of 1H-triazolyl
derivatives.29 The second isomer gave a fragment ion
at m/z ) 119, corresponding to a loss of CHN (27 amu),
as expected for a 2H-triazolyl isomer. These compounds
were separated by SiO2 column chromatography, and
factors.15 In the present paper, results from studies with
two series (21-24 and 25-28) of 4-azaaryl MPTP
analogues are summarized. The geometry within each
series is constant, while the polar and electronic features
of members of each series differ. We have determined
the MAO-B substrate properties of these compounds
using MAO-B purified from beef liver and have exam-
ined the relationships between these substrate proper-
ties and the corresponding log P values. Additionally
we have calculated the heats of formation of the putative
C-6 radical intermediates B in an attempt to identify
possible electronic contributions of the azaaryl groups
on the catalytic pathway.
1
the structures were established by H NMR spectros-
copy. The major isomer, 4-1H-[1,2,3-triazolyl]pyridine
41, was not processed further since the required tet-
rahydropyridinyl product 23 had already been prepared.
The minor isomer proved to be the symmetrical 2H-
[1,2,3]triazolyl product 42 which displayed the expected
three sets of signals for the aromatic protons. Methy-
lation of 42 afforded the pyridinium intermediate 43
which, upon reduction with NaBH4, gave the tetrahy-
dropyridinyl product 24.
Resu lts a n d Discu ssion
Ch em istr y. All of the 1-methyl-4-azaaryl-1,2,3,6-
tetrahydropyridinyl derivatives C were prepared by
NaBH4 reduction of the corresponding 1-methylpyri-
dinium intermediates B (Scheme 3).28 Various ap-
proaches, principally involving nucleophilic aromatic
substitution chemistry, were employed to obtain the
required methylpyridinium intermediates.
The basic properties of imidazole 29, 1H-[1,2,3]tria-
zole 31, benzimidazole 33, benzotriazole 35, and inda-
zole 37 suggested the possibility that these azaarenes
might be adequately nucleophilic to participate in
nucleophilic aromatic substitution chemistry with the
4-chloro-1-methylpyridinium species 38 serving as the
electrophilic substrate. The reaction with indazole 37
failed. The remaining basic azaarenes, however, were
The marginal success obtained in this reaction indi-
cates that 4-chloropyridine is not adequately reactive
to be of general utility in these types of substitution
reactions. A literature report on the coupling reaction
between 4-fluoropyridine 44 and indole 4530 prompted
us to examine this method to prepare the indolylpyri-
dinyl (46) and 4-(1-indazolyl)pyridinyl (47) derivatives
(Scheme 5). The reactions were carried out with the
sodium salt of the azaarenes 45 and 48 in DMF at high

1830 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Nimkar et al.
Sch em e 3. Synthesis of 4-Azaaryl-1-methyl-1,2,3,6-tetrahydropyridinyl Derivatives 22, 23, 26, and 27
Sch em e 4. Synthesis of 4-2H-[1,2,3]Triazolyl Derivative 24a
a
Key: (a) DMF, 100 °C, 31% yield of 41 + 42; (b) CH3I, acetone, 25 °C, 90% yield; (c) NaBH4, CH3OH; (d) oxalic acid, CH3OH, Et2O,
70% yield.
Sch em e 5. Synthetic Routes to the Indazolyl (28) and
Indolyl (25) Tetrahydropyridinyl Derivativesa
product 53 was treated with CH3I to give the pyridinium
intermediate 54. Compound 54 was reduced with NaBH4
to yield the tetrahydropyridinyl product 21 that was
purified as its oxalate salt (Scheme 6).
Quantitative estimations of the rates of substrate
turnover (A f C, Scheme 1) required molar extinction
coefficients for the corresponding 1,2-dihydropyridinium
metabolites C. For the five-membered heterocyclic
system we used the ꢀ value (16 000 M-1 15
of the
)
dihydropyridinium metabolite (C: R ) 1-methylpyrrol-
2-yl) derived from 1-methyl-4-(1-methylpyrroyl-2-yl)-
1,2,3,6-tetrahydropyridine (20, see Chart 1). The dihy-
dropyridinium metabolite 55 of the indazolyl analogue
27 was prepared, and its ꢀ value was used for all of the
benzo-fused ring structures. The synthetic approach was
based on an established route to this system32 which
proceeds via the corresponding N-oxide 56 (Scheme 7).
Treatment of 56 with trifluoroacetic anhydride gave the
dihydropyridinium product 55, presumably via the
corresponding trifluoroacetyloxy intermediate 57. Di-
hydropyridinium derivatives such as 55 are unstable
unless fully protonated, and therefore the final product
was purified as its perchlorate salt. The ꢀ value for 55
a
Key: (a) DMF, 100 °C, 90% yield for 46, 60% yield for 47; (b)
CH3I, acetone, 25 °C, 90% yield for 49, 91% yield for 50; (c) NaBH4,
CH3OH; (d) oxalic acid, CH3OH, Et2O, 70% yield for 25, 64% yield
for 28.
temperature. Subsequent reaction with iodomethane
followed by NaBH4 reduction of the resulting pyridinium
species 49 and 50 gave the desired tetrahydropyridinyl
products 25 and 28.
The remaining target substrate was 4-(1-pyrrolyl)-
1,2,3,6-tetrahydropyridine (21). The synthesis of 21 was
achieved by the condensation of 4-aminopyridine (51)
with 2,5-dimethoxytetrahydrofuran (52) under acidic
conditions (Scheme 6).31 The resulting pyrrolylpyridinyl
was estimated to be 21 000 M-1
.
En zym ology. UV scans of 10 mM solutions of the
1-methyl-4-imidazolyl analogue 20 and 4-triazolyl ana-
logues 23 and 24 in the presence of 0.16 µM MAO-B
showed that the substrate properties of these com-
pounds were too poor to generate useful kinetic data.
The 1-methyl-4-pyrrol-1-yl (21), 4-benzimidazolyl (26),
4-benzotriazolyl (27), and 4-indazolyl (28) analogues

MAO-B-Catalyzed Biotransformation of MPTPs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1831
Sch em e 6. Synthesis of 4-(Pyrrol-1-yl) Derivative 21a
a
Key: (a) AcOH, reflux, 90% yield; (b) CH3I, acetone, 25 °C, 90% yield; (c) NaBH4, CH3OH, 99% yield; (d) oxalic acid, CH3OH, Et2O,
70% yield.
Sch em e 7. Synthesis of the Indazolyldihydropyridinium Species 55a
a
Key: (a) m-CPBA, CHCl3, 0 °C, 96% yield; (b) (CF3CO)2O, CH2Cl2; (c) HClO4, 73% yield.
Ta ble 1. MAO-B Kinetic Data and Calculated (ACD LogP) log
P Values for Various 1-Methyl-4-azaaryl-1,2,3,6-
tetrahydropyridinyl Analogues
Sch em e 8. Fate of 26 and 27 upon Incubation with
MAO-B
kcat
Km
kcat/Km
compd C-4 substituent (min-1) (mM) (min‚mM)-1 log P
21
22
23
24
25
26
27
28
1
16
17
18
19
20
1-pyrrolyl
268
0.65
410
2.17
1.31
1.13
1.13
3.61
2.54
2.36
2.85
2.74
1.24
1.44
1.24
1.27
1.73
1-imidazolyl
1H-triazolyl
2H-triazolyl
1-indolyl
1-benzimidazolyl
1-benzotriazolyl
1-indazolyl
phenyl
2-pyridinyl
3-pyridinyl
4-pyridinyl
2-pyrrolyl
414
200
54
120
271
56
67
14
85
360
0.11
0.90
0.08
0.1
0.2
0.9
1.9
2.4
1.80
0.2
3350
220
675
850
1470
60
35
6
46
1800
proved to be better substrates. UV scans of 0.5 mM
solutions of these tetrahydropyridines in the presence
of 0.08 µM MAO-B showed the time-dependent forma-
tion of chromophores near 360 nm as expected for the
corresponding dihydropyridinium metabolites. Linear
initial velocities versus substrate concentration plots
and linear double-reciprocal plots were obtained for all
four compounds. Closer examination of the UV scans
of incubation mixtures containing 26 or 27 and MAO-B
revealed that the spectra corresponding to the dihydro-
pyridinium metabolites 46 and 47 (Scheme 8) shifted
with time from a λmax of 360 nm toward a λmax of 325
nm, suggesting their further oxidation to the pyridinium
species 34 and 36, respectively. In the absence of pure
synthetic standards, the possible formation of pyri-
dinium products in these reactions was examined by
GC-EIMS following treatment of the incubation mix-
tures with NaBD4.33 The detection of 26-2,6-d 2 con-
firmed the suspected formation of the benzimidazolyl-
pyridinium species 34. The absence of 27-2,6-d 2 in the
corresponding experiment with the triazolyl substrate
indicated that the dihydropyridinium metabolite 47
derived from 27 had experienced an alternative fate.
The possibility that 47 and (possibly) 46 had undergone
hydrolytic cleavage to yield benzotriazole 35 and benz-
imidazole 33, respectively, and the aminoenone 58 (λmax
) 324 nm), analogous to 4-aryloxydihydropyridinium
metabolites,34 was examined with the aid of an HPLC-
diode array assay. The detection of 58 in these assays
established that both dihydropyridinium metabolites 46
and 47 spontaneously hydrolyze to 58 and the corre-
NCH3-2-pyrrolyl
sponding azaarenes. The rates of hydrolysis, however,
were slow, and therefore estimations of the initial rates
of conversion of the tetrahydropyridinyl substrates to
their dihydropyridinium metabolites could be obtained
during the first 120 s of the incubation.
Table 1 presents the MAO-B substrate properties and
log P values of MPTP and the 4-azaaryl analogues that
have been studied to date. In the discussion that follows,
we have taken kcat/Km as the measure of the overall
efficiency of enzyme catalysis. The electron-rich pyrrolyl
analogue 19 is a good substrate, while the activities of
the more electron-deficient imidazolyl (20) and triazolyl
(21 and 22) analogues are too slow to measure. This is
somewhat reminiscent of the poor substrate properties
of the pyridinyl analogues 16-18 compared to those of
MPTP,15 suggestive of electronic and/or polar effects.
Since the cleavage of the C6-H bond in this class of
biotransformation (A f B, Scheme 1) is accompanied
by a primary isotope effect,6 we speculated that stabi-
lization of the C-6 radical B, generated by either C-H
bond homolysis (HAT pathway) or deprotonation of the
aminyl radical cation (SET pathway), by electron-
donating substituents at C-6 could lead to a decrease
in ∆Gq and an increase in kcat. The calculated C6-H
bond energies [∆∆Hf(C6H-C6 radical)] for all the

1832 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Nimkar et al.
substituents at C-2 and C-3 on the phenyl ring24 and
by the excellent substrate properties of 4-aryloxy34 and
4-benzyl35 analogues of MPTP. Substituents in this area
might enhance the interactions with the active site and
“lock” the nitrogen of the tetrahydropyridine in a more
suitable position for catalysis. (The structure shown in
Figure 1 places the off-axis groupings on the same side
as the tetrahydropyridinyl double bond. This represen-
tation is arbitrary since these results provide no infor-
mation on the preferred orientation of the molecule in
the active site. Calculations show that the syn structure
(off-axis substituent on the side of the double bond as
in Figure 1) and the anti structure differ in energy by
0.4 kcal/mol or less.)
Studies currently in progress focus on the region
identified as area B. We are particularly interested in
studying MPTP analogues bearing polar functionalities
that may occupy the same regions of the active site that
accommodate the phenolic OH groups of the catechola-
mines and serotonin.
F igu r e 1. Areas of substrate interactions defined by azaaryl
analogues of MPTP.
N-linked analogues, including the benzoazaarenes, how-
ever, show no clear relationship to substrate turnover
and vary by less than 0.75 kcal/mol. (Calculations are
not reported for the pyridinyl series 16-18 nor for the
pyrrolyl derivatives 19 and 20, because they are not
N-linked. In addition, the six-membered ring in the
pyridinyl series induces a new steric parameter which
may influence the stabilization of the C-6 radical
intermediate by the C-4 substituent. There is very little
steric interaction between five-membered rings and the
hydrogen atoms at positions C-3 and C-5 of the tetra-
hydropyridinyl ring. The six-membered rings, on the
other hand, do interact with these C-3 and C-5 hydrogen
atoms. Comparison of stabilization energy then would
become hazardous, as different factors operate for the
various series of compounds.) Consequently, it appears
unlikely that electronic factors contribute significantly
to the substrate properties of these systems. The fact
that only cyclic tertiary allylamines are substrates for
MAO suggests that electronic factors still may be
important in determining the substrate behavior of
these systems.
Exp er im en ta l Section
Ca u tion ! MPTP is a known nigrostriatal neurotoxin, and
therefore compounds of this class should be handled using
disposable gloves in a properly ventilated hood. Detailed
procedures for the safe handling of MPTP have been re-
ported.27
Gen er a l Meth od s. All nonaqueous reactions were carried
out using glassware that had been flame-dried under an inert
atmosphere of dry nitrogen. All common reagents were pur-
chased from Aldrich Chemical Co. (Milwaukee, WI) or Lan-
caster Synthesis Inc. (Windham, NH) and were used without
further purification. The following compounds, 4-fluoropyridine
(32),36,37 4-(1-indolyl)pyridine (33),30 and 4-chloro-1-methylpy-
ridinium iodide (37),38 were synthesized as described previ-
ously. The synthesis of compound 41 will be described sepa-
rately.39 THF was distilled from sodium/benzophenone ketyl
and acetone from K2CO3. UV absorption spectra were recorded
on a Beckman DU-7400 spectrophotometer. Proton NMR
spectra were recorded on a Brukers WP 270 or AM-360 or a
Varian 400-MHz spectrometer. Chemical shifts (δ) are reported
relative to tetramethylsilane as an internal standard. Spin
multiplicities are given as s (singlet), bs (broad singlet), d
(doublet), t (triplet), q (quartet), or m (multiplet). Coupling
values (J ) are given in hertz (Hz). Gas chromatography-
electron ionization mass spectrometry (GC-EIMS) was per-
formed on a Hewlett-Packard 5890 GC fitted with an HP-1
capillary column which was coupled to a Hewlett-Packard 5870
mass selective detector. Data were acquired using an HP 5970
ChemStation. Unless otherwise stated, the temperature pro-
gram employed was as follows: 125 °C for 1 min, then 25 °C/
min to 275 °C. Normalized peak heights are reported as a
percentage of the base peak. Melting points were performed
on a Thomas-Hoover melting point apparatus and are uncor-
rected. Microanalyses were performed by Atlantic Microlab,
Inc., Norcross, GA. The lipophilicity parameter log P was
calculated using the ACD/LogP software (ACD/Labs). Expe-
rimetal and calculated log P values were essentially identical
for 1 (2.71 vs 2.74) and 18 (1.25 vs 1.24).25 Calculations were
performed on a Macintosh G3, using the semiempirical method
AM1 embedded in MacSpartan Plus software. Unrestricted
Hartree-Fock method was used for calculations on radical
species.
In contrast to the five-membered azaarenes, all of the
benzoazaarenes are MAO-B substrates. As shown in
Table 1, all of these fused ring systems also are more
lipophilic than the corresponding monocyclic analogues.
In fact, a good trend exists between the log P and kcat
/
Km values with the most lipophilic compound, the
1-indolyl analogue 23, being the most lipophilic and the
best substrate. Compounds with kcat/Km values of less
than 60 (min‚mM)-1 have log P values below 1.5. On
the basis of this analysis, lipophilicity is judged to be
an important factor contributing to the efficiency of
substrate turnover in this series of compounds.
The direct correlation between the MAO-B substrate
properties of azaaryltetrahydropyridinyl derivatives and
their lipophilic character parallels those effects observed
with phenyl-substituted MPTP derivatives.25 On the
other hand, some polar compounds, such as the dopam-
ine (log P 0.12), are excellent MAO-B substrates.
Therefore the poor substrate properties observed with
some of these azaaryl derivatives may describe regions
of unfavored polar interactions. Results with the five-
membered azaaryl and pyridinyl analogues suggest that
nitrogen atoms with localized nonbonding lone pairs of
electrons located in area A of the model shown in Figure
1 may form hydrogen bonds with active site proton
donors that disfavor effective binding of the substrate.
The improved substrate properties of the benzoazaaryl
analogues suggest a region of favorable interactions
indicated by area B in the model. This depiction is
consistent with the improved substrate properties of
MPTP analogues bearing a variety of polar and nonpolar
4-(P yr r ol-1-yl)p yr id in e (53). Under anhydrous conditions,
a mixture of 4-aminopyridine (51; 9.4 g, 100 mmol) and 2,5-
dimethoxytetrahydrofuran (52; 16.5 g, 125 mmol) in 100 mL
of glacial AcOH (100 mL) was heated to reflux for 4.5 h. An
EtOAc (199 mL) solution of the residue obtained after remov-
ing the AcOH was washed extensively with aqueous 10%
NaOH (8 × 50 mL) followed by H2O and brine. The organic
phase was dried (MgSO4), the solvent was removed, and the

MAO-B-Catalyzed Biotransformation of MPTPs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1833
residue was filtered through a column of Al2O3 using CH2Cl2
as an eluent to give 13.7 g (90.4 mmol, 90% yield) of 53 as a
white solid: mp 78 °C. Anal. (C9H8N2) C, H, N.
1-Meth yl-4-(p yr r ol-1-yl)p yr id in iu m Iod id e (54). A mix-
ture of 53 (1.73 g, 12 mmol) and CH3I (5.07 g, 36 mmol) in
120 mL of anhydrous acetone was stirred in the dark for 24
h. The solid iodide salt was collected and washed with Et2O
to give pure 54 (3.1 g, 10.8 mmol, 90% yield) as a white solid:
mp 197-198 °C. Anal. (C10H11IN2) C, H, N.
Oxa la te Sa lt of 1-Meth yl-4-(p yr r ol-1-yl)-1,2,3,6-tetr a h y-
d r op yr id in e (21). Reduction of 54 with an excess of NaBH4
in MeOH for 1 h followed by removal of the solvent and
extraction with CH2Cl2/H2O gave a crude residue of 21 free
base, which under treatment in Et2O with an excess of oxalic
acid gave the corresponding oxalate that was recrystallized
from MeOH to yield 21 (600 mg, 2.7 mmol, 70% yield) as a
white crystalline solid: mp 185-186 °C; 1H NMR (DMSO-d6)
δ 7.13 (dd, J ) 2.4 and 2.0 Hz, 2H), 6.16 (dd, J ) 2.4 and 2.0
Hz, 2H), 5.82 (m, 1H), 3.72 (dd, J ) 2.4 and 5.6 Hz, 2H), 3.46
(t, J ) 6.4 Hz, 2H), 2.85 (m, 2H), 2.78 (s, 3H); 13C NMR
(DMSO-d6) δ 165.1, 133.6, 118.3, 110.2, 50.5, 49.6, 42.2, 24.0;
MS (m/z, rel int) 162 (27), 161 (19), 118 (47), 96 (100), 94 (20),
42 (47); UV (nm, MeOH) 207, 246. Anal. (C12H16N2O4) C, H,
N.
1-Meth yl-4-(in d ol-1-yl)p yr id in iu m Iod id e (49). A mix-
ture of 46 (1.94 g, 10 mmol) and CH3I (4.2 g, 30 mmol) in 100
mL of anhydrous acetone was stirred in the dark for 28 h. The
solid iodide salt was collected and washed with Et2O to give
pure 49 (3.05 g, 9.1 mmol, 91% yield) as a white solid: mp
210-212 °C. Anal. (C12H13IN2) C, H, N.
Oxa la te Sa lt of 1-Meth yl-4-(in d ol-1-yl)-1,2,3,6-tetr a h y-
d r op yr id in e (25). Same procedure as described for the
synthesis of 21 was followed which yielded a white solid from
MeOH/Et2O in 91% yield: mp 209-210 °C; 1H NMR (CD3OD)
δ 7.59 (2H, t, J ) 8.0 Hz), 7.35 (1H, d, J ) 3.3 Hz), 7.20 (1H,
t, J ) 8.0 Hz), 7.09 (1H, t, J ) 8.0 Hz), 6.59 (1H, dd, J ) 3.35
Hz, J ) 0.8 Hz), 5.97 (1H, bs), 4.03 (2H, bs), 3.64 (2H, t, J )
4.0 Hz), 3.05 (3H, s), 2.99 (2H, bs); 13C NMR (CD3OD) δ 160.5,
135.6, 131.8, 128.9, 128.6, 127.2, 123.7, 123.6, 121.8, 121.6,
115.8, 52.4, 50.1, 41.5, 24.4; GC (tR 5.57 min)-EIMS m/z (rel
int) 212 (22%), 180 (2), 168 (25), 143 (2), 130 (7), 96 (100), 89
(9), 53 (9); UV (nm, MeOH) 337. Anal. (C16H18N2O4) C, H, N.
1-Meth yl-4-(in d a zol-1-yl)p yr id in iu m Iod id e (50). The
procedure described for 54 yielded a yellow solid in 90%
yield: mp 261-263 °C. Anal. (C13H12IN3) C, H, N.
Oxa la te Sa lt of 1-Meth yl-4-(in d a zol-1-yl)-1,2,3,6-tet-
r a h yd r op yr id in e (28). Reduction of 50 as described for 21
yielded a white crystalline solid in 68% yield after recrystal-
lization from MeOH: mp 174-175 °C; 1H NMR (DMSO-d6) δ
8.28 (1H, d, J ) 0.9 Hz), 7.85 (m, 2H), 7.49 (1H, ddd, J ) 1.2
Hz, 6.9 Hz, 8.0 Hz), 7.25 (1H, ddd J ) 0.6 Hz, 6.9 Hz, 7.9 Hz),
6.12 (1H, m), 3.87 (2H, m), 3.42 (2H, d, J ) 6.1 Hz), 3.03 (2H,
m), 2.84 (3H, s); 13C NMR (DMSO-d6) δ 164.1, 137.8, 134.9,
134.0, 127.3, 124.8, 121.7, 121.4, 111.3, 108.8, 50.3, 49.5, 41.9;
MS (m/z, rel int) 213 (40), 185 (24), 157 (36), 144 (18), 130 (9),
96 (27), 70 (100); UV (nm, MeOH) 208, 244, 301. Anal.
(C15H17N3O4) C, H, N.
equiv). The precipitated oxalate salt was recrystallized from
CH3CN/MeOH.
Oxa la te Sa lt of 4-(1-Im id a zolyl)-1-m eth yl-1,2,3,6-tet-
r a h yd r op yr id in e (22). This compound was obtained in 30%
1
yield: mp 153-154 °C; H NMR (DMSO-d6) δ 9.88 (bs, 2H),
8.14 (bs, 1H), 7.62 (bs, 1H), 7.09 (bs, 1H), 6.07 (bs, 1H), 3.82
(bs, 2H), 3.43 (bs, 2H), 2.90 (bs, 2H), 2.84 (s, 3H); GC (tR
)
4.26 min)-EIMS m/z (%) 163 (M.+, 27), 119 (26), 96 (100), 94
(28); UV (0.1 M sodium phosphate buffer, pH 7.4) λmax ) 257
nm (ꢀ ) 490 M-1). Anal. (C9H13N3‚1.1C2H2O4) C, H, N.
Bisoxalate Salt of 4-(1-Ben zim idazolyl)-1-m eth yl-1,2,3,6-
tetr a h yd r op yr id in e (26). This compound was obtained in
1
22% yield: mp 146 °C; H NMR (DMSO-d6) δ 8.43 (bs, 1H),
7.69-7.72 (m, 2H), 7.25-7.33 (m, 2H), 6.13 (bs, 1H), 3.94 (bs,
2H), 3.49-3.52 (m, 2H), 2.98 (bs, 2H), 2.90 (s, 3H); 13C NMR
(DMSO-d6) δ 163.5, 143.9, 142.5, 132.9, 131.3, 123.8, 123.0,
120.3, 113.5, 112.2, 50.6, 49.6, 41.9, 25.2; GC (tR ) 7.06 min)-
EIMS m/z (%) 213 (M.+, 22), 169 (26), 96 (100). Anal.
(C17H19N3O8) C, H, N.
Oxa la te Sa lt of 4-(1-Ben zotr ia zolyl)-1-m eth yl-1,2,3,6-
tetr a h yd r op yr id in e (27). This compound was obtained in
45% yield: mp 190-191.5 °C dec; 1H NMR of free base (CDCl3)
δ 8.07-8.10 (d, J ) 6.5 Hz, 1H), 7.69-7.71 (d, J ) 6.5 Hz,
1H), 7.48-7.53 (m, 1H), 7.37-7.41 (m, 1H), 6.17-6.18 (m, 1H),
3.28-3.30 (m, 2H), 3.02-3.06 (m, 2H), 2.84 (m, 2H), 2.50 (s,
3H); 13C NMR of free base (CDCl3) δ 146.0, 134.0, 132.0, 127.8,
124.2, 120.1, 117.1, 111.0, 53.3, 51.7, 45.4, 28.4; GC (tR ) 6.72
min)-EIMS m/z (%) 214 (M.+, 2), 186 (20), 157 (20), 143 (70),
130 (75), 94 (25), 77 (100), 53 (95); UV (0.1 M sodium
phosphate buffer, pH 7.4) λmax ) 266 nm (ꢀ ) 7000 M-1) and
293 nm (ꢀ ) 6200 M-1). Anal. (C14H16N4O4) C, H, N.
Oxa la te Sa lt of 1-Meth yl-4-(1H-[1,2,3]tr ia zolyl)-1,2,3,6-
tetr a h yd r op yr id in e (23). This compound was obtained in
27% yield: mp 238-239 °C; 1H NMR (DMSO-d6) δ 8.0 (s, 2H),
6.43-6.45 (m, 1H) 3.72 (bs, 2H), 3.29 (m, 2H), 2.98 (bs, 2H),
2.74 (s, 3H); 13C NMR (DMSO-d6) δ 164.3, 135.9, 133.8, 108.3,
50.1, 49.2, 41.9, 22.8; GC (tR ) 2.38 min)-EIMS m/z (%) 164
(M.+, 67), 163 (35), 110 (52), 96 (52), 94 (100), 70 (55); UV (0.1
M sodium phosphate buffer, pH 7.4) λmax ) 254 nm (ꢀ ) 12 000
M-1). Anal. (C10H14N4O4) C, H, N.
4-(1H-[1,2,3]Tr ia zolyl)p yr id in e (41) a n d 4-(2H-[1,2,3]-
Tr ia zolyl)p yr id in e (42). Under anhydrous conditions, 1,2,3-
triazole (7.7 mmol, 0.53 g) in DMF (35 mL) was treated
portionwise with NaH (8.05 mmol, 0.19 g). The freshly
prepared free base of 4-chloropyridine (39; 8 mmol, 1.0 g) in
solution in DMF (15 mL) was then added, and the reaction
mixture was heated under reflux for 14 h. After cooling to 25
°C, a saturated aqueous solution of NH4Cl (10 mL) was added.
The mixture was washed with hexane (2 × 40 mL), neutralized
with a saturated aqueous solution of K2CO3 (15 mL), and
extracted with Et2O (4 × 50 mL). The combined organic phase
was dried over MgSO4 and evaporated under reduced pressure.
Column chromatography (SiO2, EtOAc) of the resulting mix-
ture gave 41 as a pale-yellow solid in 9% yield: mp 125.5-
127 °C. Anal. (C7H6N4) C, H, N. A second fraction from the
column yielded 42 as a pale-yellow solid in 22% yield: mp:
128-130 °C. Anal. (C7H6N4) C, H, N.
1-Meth yl-4-(2H-[1,2,3]tr ia zolyl)p yr id in iu m Iod id e (43).
Methylation of 42 as described for 53 gave 43 as a yellow solid
in 82% yield: mp 255-256 °C dec. Anal. (C8H9IN4) C, H, N.
Oxa la te Sa lt of 1-Meth yl-4-(2H-[1,2,3]tr ia zolyl)-1,2,3,6-
tetr a h yd r op yr id in e (24). Treatment of 43 as described for
the synthesis of 21 gave 24 as a white solid in 25% yield: mp
189-190 °C; 1H NMR (CD3OD, 360 MHz) δ 7.82 (2H, s), 6.02
(1H, bs), 3.97 (2H, bs), 3.63 (2H, t, J ) 4.0 Hz), 3.05 (3H, s),
2.98 (2H, bs); 13C NMR (CD3OD, 90 MHz) δ 121.3, 114.2, 52.4,
50.1, 41.5, 24.4; GC-EIMS m/z (rel int) 164 (MH.+, 22%), 136
(100), 115 (12), 96 (42); UV (MeOH) λmax 290, 265 nm. Anal.
(C10H14N4O4) C, H, N.
1-Meth yl-4-(1H-in d a zolyl)-2,3-d ih yd r op yr id in iu m P er -
ch lor a te (55). To a solution of 1-methyl-4-(1H-indazolyl)-
1,2,3,6-tetrahydropyridine (28; 250 mg, 1.2 mmol) in 15 mL
of anhydrous CHCl3 at 0 °C was added m-chloroperoxybenzoic
acid (m-CPBA; 50%, 384 mg, 1.1 mmol) in one portion. After
Gen er a l P r oced u r e for th e Syn th esis of th e 4-Aza a r yl-
1,2,3,6-tetr a h yd r op yr id in yl Der iva tives 22, 23, 26, a n d
27. A mixture of 4-chloro-1-methylpyridinium iodide (38) (2
equiv), the appropriate azaarene (2 equiv), and freshly distilled
triethylamine (3 equiv) was stirred in CH3CN at room tem-
perature under nitrogen for 14 h. The solvent was evaporated,
and the residue was treated with a saturated solution of K2-
CO3. This mixture was extracted with CH2Cl2 (3 × 60 mL),
and the combined organic extracts were washed with brine
and dried over MgSO4. Evaporation of the solvent gave the
crude, hygroscopic pyridinium product which was treated
directly with NaBH4 (2.5 equiv) in 25 mL of MeOH at 0 °C.
After stirring for 15 min, the solvent was removed under
reduced pressure and the residue in 20 mL of H2O was
extracted with CH2Cl2 (2 × 25 mL). The combined organic
layer was dried over MgSO4, filtered, and evaporated, and the
residue in 25 mL of dry Et2O was treated with oxalic acid (3

1834 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Nimkar et al.
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effect studies on the MAO-B catalyzed oxidation of 4-benzyl-1-
cyclopropyl-1,2,3,6-tetrahydropyridine. Biochemistry 1996, 35,
3335-3340.
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monoamine oxidase B. J . Biol. Chem. 1989, 264, 13684-13688.
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stirring for 15 min, the reaction mixture was concentrated in
vacuo. The residue was purified by column chromatography
on basic alumina, eluting with CH3Cl/MeOH (9.5:0.5) to give
246 mg (1.1 mmol, 96%) of the N-oxide 56 as a yellow oil: 1H
NMR (DMSO-d6, 360 MHz) δ 8.27 (d, J ) 0.8 Hz, 1H), 7.84
(m, 2H), 7.47 (ddd, J ) 1.2 Hz, J ) 6.9 Hz, J ) 8.2 Hz, 1H),
7.23 (ddd, J ) 1.0 Hz, J ) 6.9 Hz, J ) 8.0 Hz, 1H), 5.97 (d, J
) 8.0 Hz, 1H), 4.26 (m, 1H), 3.81 (m, 1H), 3.58 (m, 1H), 3.44
(m, 1H), 3.34 (m, 1H), 3.21 (s, 3H), 2.88 (m, 1H); 13C NMR
(DMSO-d6, 90 MHz) δ 137.8, 134.6, 133.7, 127.1, 124.7, 121.5,
121.3, 111.3, 108.7, 79.2, 65.6, 62.2, 59.0; UV (MeOH) λmax 208,
244, 299 nm. HR-CIMS Calcd for C15H15N3O: 229.1215123.
Found: 229.12247.
A mixture of 56 (105 mg, 0.46 mmol) and trifluoroacetic
anhydride (115 mg, 0.55 mmol) in 2 mL of CH2Cl2 was stirred
for 15 min at 0 °C. Addition of a solution of perchloric acid
(134 mg, 0.92 mmol) in 4 mL of MeOH followed by stirring for
1 h gave 105 mg of a yellow crystalline product (0.34 mmol,
73%): mp 174-175 °C; 1H NMR (DMSO-d6, 360 MHz) δ 8.74
(d, J ) 0.8 Hz, 1H), 8.55 (dddd, J ) 1.0 Hz, J ) 1.0 Hz, J )
1.0, J ) 5.6 Hz, 1H), 8.18 (dddd, J ) 0.8 Hz, J ) 0.8 Hz, J )
0.8 Hz, J ) 8.6 Hz, 1H), 8.01 (ddd, J ) 1.0 Hz, J ) 1.0 Hz, J
) 8.0, 1H), 7.72 (ddd, J ) 1.2 Hz, J ) 7.1 Hz, J ) 8.4 Hz, 1H),
7.51 (ddd, J ) 0.6 Hz, J ) 7.2 Hz, J ) 7.9, 1H), 6.89 (d, J )
5.5 Hz, 1H), 4.05 (m, 2H), 3.83 (m, 2H), 3.63 (s, 3H); 13C NMR
(DMSO-d6, 90 MHz) δ 163.4, 157.9, 154.5, 142.2, 138.4, 129.8,
127.4, 125.0, 122.9, 114.2, 98.5, 47.3, 45.4; UV (MeOH) λmax
382 nm (ꢀ ) 21 000 M-1). Anal. (C13H14N3O4Cl‚0.7H2O) C, H,
(10) Zhong, B. Y.; Silverman, R. B. Identification of the active site
cysteine in bovine liver monoamine oxidase B. J . Am. Chem. Soc.
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Anderson, A. H.; Burch, H.; Castagnoli, N., J r. Probing the
mechanism of bioactivation of MPTP type analogues by mono-
amine oxidase B: structure-activity studies on substituted
4-phenoxy-, 4-phenyl, and 4-thiophenoxy-1-cyclopropyl-1,2,3,6-
tetrahydropyridines. Chem. Res. Toxicol. 1995, 8, 703-710.
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N. HR-CIMS Calcd for
212.118454.
C13H14N3: 212.1187726. Found:
En zym e Stu d ies. MAO-B was isolated from bovine liver
mitochondria according to the method of Salach and Weyler40
with minor modifications as described previously.41 The puri-
fied enzyme concentration was calculated to be 8 nmol/mL.
All enzyme assays were performed at 37 °C on a Beckman DU-
7400 spectrophotometer. Stock solutions (2-10 mM) of the
tetrahydropyridinyl derivatives were prepared in 100 mM
sodium phosphate buffer, pH 7.4. In preliminary experiments,
the potential MAO-B substrate properties of each test com-
pound (0.25-10 mM) were examined by recording repeated
scans (500-250 nm) in the presence of 0.08-0.16 µM MAO-
B. For kinetic analyses, initial rates of oxidation of the
tetrahydropyridinyl derivatives were determined at four sub-
strate concentrations, which bracketed the Km value. A
mixture of each test compound (50-500 µM) and MAO-B
(0.08-0.16 µM) was added to a sample cuvette, and the initial
rates of oxidation were estimated by monitoring the increase
in absorbance of the corresponding dihydropyridinium me-
tabolite every 5 s for 2 min. The Km and kcat values were
calculated from Lineweaver-Burke plots. Duplicate analyses
gave kcat/Km values that differed by 10% or less.
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and functional properties of the monoamine oxidases. Hetero-
cycles 1994, 39, 933-955.
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features of human monoamine oxidase A elucidated from cDNA
and peptide sequences. J . Neurochem. 1988, 51, 1321-1324.
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Ack n ow led gm en t. This work was supported by the
National Institute of Neurological and Communicative
Disorders and Stroke Grant NS 28792 and the Harvey
W. Peters Center.
Su p p or tin g In for m a tion Ava ila ble: Spectroscopic data
on all synthetic intermediates. This material is available free
of charge via the Internet at http://pubs.acs.org.
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