Vesicular monoamine transporter substrate/inhibitor activity of MPTP/MPP+ derivatives: A structure-activity study

Article abstract of DOI:10.1021/jm070875p

The active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), N-methyl-4-phenylpyridinium (MPP⁺), selectively destroys the dopaminergic neurons and induces the symptoms of Parkinson's disease. Inhibition of mitochondrial complex

Full text of DOI:10.1021/jm070875p

760
J. Med. Chem. 2008, 51, 760–768
Vesicular Monoamine Transporter Substrate/Inhibitor Activity of MPTP/MPP+ Derivatives: A
Structure–Activity Study
D. Shyamali Wimalasena,† Rohan P. Perera,† Bruce J. Heyen,‡ Inoka S. Balasooriya,† and Kandatege Wimalasena*,†
Department of Chemistry, Wichita State UniVersity, Wichita, Kansas 67260-0051, and Department of Chemistry, Tabor College,
Hillsboro, Kansas 67063
ReceiVed July 19, 2007
The active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), N-methyl-4-phenylpyridinium
(MPP+), selectively destroys the dopaminergic neurons and induces the symptoms of Parkinson’s disease.
Inhibition of mitochondrial complex I and/or the perturbation of dopamine metabolism through cellular and
granular accumulation have been proposed as some of the major causes of neurotoxicity. In the present
study we have synthesized and characterized a number of MPTP and MPP+ derivatives that are suitable for
the comparative neurotoxicity and complex I inhibition versus dopamine metabolism perturbation studies.
Structure-activity studies with bovine chromaffin granule ghosts show that 3′-hydroxy-MPP+ is one of the
best known substrates for the vesicular monoamine transporter (VMAT). A series of compounds that combine
the structural features of MPP+ and a previously characterized VMAT inhibitor, 3-amino-2-phenyl-propene,
have been identified as the most effective VMAT inhibitors. These derivatives have been used to define the
structural requirements of the VMAT substrate and inhibitor activities.
Introduction
Although comparative structure–activity studies with respect
to complex I inhibition versus DA metabolism perturbation by
MPP+ could provide valuable information to distinguish
between the above two mechanisms, such detailed studies have
not been carried out to our knowledge. In addition, accumulating
experimental evidence indicates that the neurotoxicity of a large
number of illicit drugs as well as other physicopharmacologi-
cally active agents may be due to the perturbation of catechola-
mine metabolism in the central nervous system (CNS).5,6 Thus,
the specific uptake of MPP+ through DAT and its granular
accumulation through VMAT also make it a convenient probe
to study the in vivo effects of the perturbation of catecholamine
metabolism. Similarly, properly designed MPP+ derivatives
could also be useful probes for the structure–activity studies of
DAT and VMAT in vitro, since MPP+ is a good substrate for
the catecholamine uptake systems in plasma membrane and
storage vesicles.7,8 The structural rigidity of these derivatives
in comparison to that of the regular phenylethylamine type
substrates makes them ideal candidates for such studies. Based
on these rationales, we have synthesized and characterized a
number of MPTP and MPP+ derivatives that are suitable for
the structure–activity studies of DAT and VMAT as well as
the comparative neurotoxicity and complex I inhibition versus
DA metabolism perturbation studies. In the present paper we
describe the synthesis and characterization of these derivatives
and their use to define the structural requirements of the VMAT
substrate and inhibitor activities using resealed chromaffin
granule ghosts as a model.
The neurotoxin, N-methyl-4-phenyl-tetrahydropyridinium
(MPTPa), selectively destroys the dopaminergic neurons of the
subatantia nigra and induces the symptoms of Parkinson’s
disease.1–4 Numerous studies have shown that MPTP ac-
cumulates in the brain by crossing the blood brain barrier, and
it is readily converted to the fully aromatized product, the
N-methyl-4-phenylpyridinium salt (MPP+), by monoamine
oxidase. It has also been well accepted that the MPTP
neurotoxicity is solely due to the metabolite MPP+. In agreement
with this proposal, direct administration of MPP+ to the rat brain
has been shown to specifically destroy the dopaminergic
neurons. In addition, MPP+ is shown to be taken up into
dopaminergic neurons through the plasma membrane dopamine
transporter (DAT) as well as into catecholamine storage vesicles
through the granule vesicular monoamine transporter (VMAT).1–4
Furthermore, MPP+ inhibits oxidative phosphorylation by
inhibiting complex I of the electron transport chain.3 Although
the exact mechanism of the specific toxicity of MPP+ to
dopaminergic neurons has not been fully understood, ac-
cumulating evidence suggests that at least two important
characteristics of MPP+ could be responsible for its toxic effects.
While some studies suggest that the inhibition of complex I is
the main cause, other studies appear to indicate that the
perturbation of dopamine (DA) metabolism through cellular and
granular accumulation may also play a role in the specific
dopaminergic neurotoxicity of MPP+.4
Results and Discussion
* To whom correspondence should be addressed. Phone: (316)-978-7386.
Fax: (316)-978-3431. E-mail: kandatege.wimalasena@wichita.edu.
Chemistry. MPP+ (1), MPTP (15), 2′-Me and 2′-NH2 MPTP
derivatives (16–17), and MPDP+ClO4- (22) were obtained from
Sigma Aldrich. The 4′- and 3′-halo- (7–9), 4′-methyl- (12), 4′-
methoxy- (5), and 4′- and 3′-trifluoromethyl- (10-11) MPP+
derivatives were synthesized by the copper catalyzed coupling
reaction between the appropriate ArMgBr derivative and an
N-protected pyridine derivative followed by rearomatization of
the pyridine ring by KMnO4 and methylation of the pyridine
nitrogen using methyl iodide (Scheme 1).9,10 The hydroxy MPP+
†
Wichita State University.
Tabor College.
‡
a Abbreviations: APP, 3-amino-2-phenylpropene; ATP, adenosine triph-
osphate; CNS, central nervous system; DAT, plasma membrane dopamine
transporter; DA, dopamine; HEPES, 4-(2-hydroxyethyl)-1-piperazine sul-
fonic acid; MPP+, N-methyl-4-phenylpyridinium; MPTP, N-methyl-4-
phenyl-1,2,5,6-tetrahydropyridine; MPDP, 1-methyl-4-phenyl-5,6-dihydro-
pyridine; NE, norepinephrine; RES, reserpine; TBZ, tetrabenazine,VMAT,
granule vesicular monoamine transporter(s); VMAT2, granule vesicular
monoamine transporter-2.
10.1021/jm070875p CCC: $40.75
2008 American Chemical Society
Published on Web 01/26/2008

VMAT Substrate/Inhibitor ActiVity of MPP+ DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 761
Scheme 1. Synthesis of 1-Methyl-4-phenylpyridinium Saltsa
a (i) CuI/THF; (ii) KMnO4/acetone, 0 °C, 15 min; (iii) CH3I, THF, 0 °C.
Scheme 2. Synthesis of Hydroxy Derivatives of 1-Methyl-4-phenylpyridinium Saltsa
a (ii) Pyridine/HCl, 200 °C, 2-3 h; (ii) 1 M NaOH, 5 h; (iii) HCl, NaHCO3; (iv) CH3I, THF, 0 °C, 5 h.
Scheme 3. Synthesis of Hydroxy Derivatives of 1-Methyl-3-phenylpyridinium Salts (N3PP+)a
a (i) Dichlorobis(triphenyl-phosphine)nickel(II)/THF, 12 h; (ii) pyridine/HCl; (iii) CH3I, THF, 0 °C, 5 h.
Scheme 4. Synthesis of 1-Methyl-4-phenyl-1,2,5,6
Scheme 5. Synthesis of MPP+-APP Conjugates
tetrahydropyridine (MPTP) Derivativesa
a (i) THF, room temperature, 12 h; (ii) 20% HAc in HBr, reflux 5 h.
All new compounds were fully characterized and found to
be highly stable in purified form. Under the biological assay
(pH 7–7.5) or storage conditions, these compounds showed no
tendency toward oxidation, polymerization, or hydrolytic cleav-
age of the N-substituents. Even under strong basic conditions
(pH 12–14), compound 23 and its analogues showed no sign
of hydrolysis of the pyridinium ring from the R-methyl styrene
group. Compounds 23–28 were sparingly soluble in water and
were freely soluble in polar organic solvents such as ethanol or
DMSO. The concentrated stock solutions of those compounds
were prepared in ethanol and used in all kinetic studies.
However, the highest ethanol concentrations in the inhibition
studies were kept to a minimum, usually less than 1%.
derivatives (3, 4, and 6) were prepared by the demethylation of
the corresponding methoxy derivatives by using pyridine/HCl
according to the procedure of Das et al.,11 followed by
methylation of the pyridine nitrogen by methyl iodide (Scheme
2). The 1-methyl-3-phenyl pyridinium salts (13 and 14) were
synthesized from 3-bromo pyridine as shown in Scheme 3. The
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) deriva-
tives (18–21) were synthesized by the Grignard reaction of
1-methyl-4-piperidone and the desired bromobenzene derivative,
followed by a dehydration using 20% HAc in HBr under
refluxing conditions (Scheme 4). The MPP+-3-amino-2-phenyl-
propene conjugates (23–31) were prepared by nucleophilic
displacement of Br- from the appropriate 3-bromo-2-phenyl-
propene derivative (Scheme 5).12
Biological Studies. Bovine adrenal chromaffin cells and their
granules have been previously used in our laboratory to study
DA uptake and conversion to norepinephrine (NE) and are
proven to be effective models for the study of the granular

762 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Wimalasena et al.
Table 1. Uptake and DA-Uptake Inhibition Kinetic Parameters of MPTP and MPP+ Derivatives for Bovine Chromaffin Granule VMATa
d
inhibitor/substrate
Ki (µM)b
Km (µM)c
73 ( 11
Vmax (nmols/mg·min)c
1.4 ( 0.1
Km,DA/Ki,I
MPP+ Derivatives
MPP+I- (1)
92 ( 14
95 ( 10
0.3
0.3
9.7
0.7
0.2
0.8
1.0
5.8
1.1
1.3
1.8
1.0
0.1
1.2
2′-MeMPP+I- (2)
3′-OHMPP+I- (3)
4′-OHMPP+I- (4)
4′-OMeMPP+I- (5)
3-Me-4′-OHMPP+I- (6)
4′-FMPP+I-(7)
2.4 ( 0.1
82 ( 11
8.4 ( 2.1
107 ( 16
135 ( 9
1.9 ( 0.1
1.6 ( 0.1
1.6 ( 0.1
106 ( 14e
65 ( 19e
51.3 ( 6.3e
7.6 ( 0.7
46.3 ( 5.4e
33.8 ( 4.2e
24.6 ( 3.0e
51.8 ( 8.7
338 ( 142
36.6 ( 4.3e
4′-ClMPP+I- (8)
3′-ClMPP+I- (9)
4′-CF3MPP+I- (10)
3′-CF3MPP+I- (11)
4′-CH3MPP+I- (12)
4′-OHN3PP+I- (13)
3′-OHN3PP+I- (14)
41.3 ( 6.6
409 ( 88
0.9 ( 0.1
1.7 ( 0.3
MPTP Derivatives
MPTPHCl (15)
52.5 ( 6.3
38.4 ( 5.2
193 ( 39e
25.8 ( 3.1e
134 ( 24e
27.7 ( 3.4
49.7 ( 6.3e
53.0 ( 13.6e
0.5
0.7
0.1
1.7
0.3
1.6
0.9
0.7
2′-MeMPTPHCl (16)
2′-NH2MPTPHCl (17)
3′-OHMPTP HCl (18)
4′-OHMPTP HCl (19)
3′-ClMPTP HCl (20)
4′-F MPTP HCl (21)
-
MPDP+ClO4 (22)
a Uptake and DA-uptake inhibition kinetics were determined using resealed chromaffin granule ghosts under standard uptake conditions as previously
described.12–14 Resealed granules ghosts were prepared from frozen membrane stores prior to the kinetic experiment and used immediately. The kinetic
parameters were determined for each derivative using the same ghost preparation, but different preparations were used for different derivatives. b The Ki
values were obtained by fitting the experimental data (typically four to five different inhibitor and six different DA concentrations) to the Cleland’s COMP
program.23 All inhibitions were competitive with respect to DA. The ( values were standard errors obtained from fitting routines. c The Km and Vmax values
for various substrates were determined by fitting the initial uptake rates at 12 different concentrations of the substrate to the hyperbolic form of the
Michaelis-Menten equation. d The Km,DA/Ki,I ratios were calculated from the respective Ki,I and Km,DA parameters determined for each compound in order
to normalize the variation of Km for DA for difference ghost preparations. e Ki values were estimated by fitting the experimental data (obtained at 0 and 200
µM inhibitor and 6 different DA concentrations) to the standard competitive inhibition equation using Sigmaplot (Cambridge Software Corp.).
Table 2. Structures and VMAT Inhibition Kinetic Parameters of MPP+-APP Conjugates
b
b
inhibitor
Kia (µM)
Km,DA/Ki,I
inhibitor
Kia (µM)
Km,DA/Ki,I
23
24
25
26
27
38.4 ( 4.2
53.0 ( 13.6
16.4 ( 2.7
13.4 ( 1.7
1.4 ( 0.2
0.9
0.7
1.8
2.7
24.1
28
29
30
31
2.8 ( 0.1
0.4 ( 0.1
0.5 ( 0.1
4.5 ( 0.5
8.7
55
41.5
11.3
a Ki, inhibition kinetics was determined in resealed chromaffin granule ghosts under standard uptake conditions as detailed in Table 1. All inhibitions
were competitive with respect to DA. The ( values were standard errors obtained from fitting routines. b The Km,DA/Ki,I ratios were calculated from the
respective Ki,I and Km,DA parameters determined for each compound in order to normalize the variation of Km for DA for difference ghost preparations as
in Table 1.
metabolism of catecholamines.12–14 The relevance of chromaffin
granule model studies to the central nervous system catechola-
mine storage vesicles is substantiated by the finding that bovine
adrenal chromaffin granules express the same brain specific
VMAT2 as the major transmembrane monoamine transporter.15
We have previously reported the experimental protocols to
examine the kinetics of uptake and conversion of intragranular
DA to NE by using resealed bovine chromaffin granule ghosts
without using radiolabeled substrates, and we have also shown
that the resealed chromaffin granules are a good model system
to study the structure–activity of VMAT2.12–14 As previously
reported,12 minor variations of uptake kinetics between day to
day ghost preparations could be normalized using the kinetic
parameters determined for the DA uptake with the same ghost
preparations. Therefore, the Km,DA/Ki,I ratio is a more accurate
parameter in estimating the relative inhibition potencies of
VMAT inhibitors for more quantitative purposes. However, as
evident from the data in Tables 1 and 2, these variations are
relatively small, and the intrinsic Ki parameters could still
be used in qualitative analysis of the kinetic data. Thus, we
have used bovine chromaffin granule ghosts as a model to
characterize a number of novel MPTP and MPP+ derivatives

VMAT Substrate/Inhibitor ActiVity of MPP+ DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 763
with respect to their interactions with the VMAT to determine
their suitability as DA perturbation probes.
porter-3 complex is significantly slower than that of the transport
(k–1 < k2). These findings suggest that the 3′-OH group of 3
may play a critical role in the interaction with the VMAT, which
parallels the effect of 3′-OH substitution on 3-amino-2-phenyl-
propene (APP) derivatives.12 In contrast to the high inhibition
potency of 3, the 4′-OH derivative (4) does not compete
effectively for VMAT and showed a weaker inhibition potency
(Ki ) 82 ( 11 µM), which is consistent with its weak substrate
activity. However, this behavior sharply contrasts the effects
of the 4′-OH substitution in APP inhibitors.12 Strikingly, 4′-Cl
substitution (8) also increased the inhibition potency of the
parent compound significantly. Although not experimentally
verified, the efficient competition of this derivative (8) with DA
for uptake suggests that it could probably be a good substrate
for VMAT as well.
Proper granular storage of catecholamines is highly critical
for the functioning of the catecholaminergic nervous system.
Perturbation of granular accumulation of catecholamines has
been recognized as a major factor in the physicopharmacological
and neurotoxic toxic effects of a large number of illicit drugs.5,6
In spite of this convincing evidence, little attention has been
paid to understand the molecular details of the accumulation
and storage of catecholamines in storage vesicles through the
VMAT. For example, in contrast to the earlier proposal that
the unprotonated form of amines is the only form transported
through the VMAT, the finding that the quaternary ammonium
salt MPP+ is also effectively transported under physiological
conditions led to the proposal that protonated amine may be
the substrate for the VMAT.7,8 Apart from this information very
little is known about the specificity, the molecular nature of
the interaction of the substrates or inhibitors, or the mechanism
of the amine transfer through the VMAT. Although highly
potent well-known inhibitors such as reserpine, tetrabenazine,
and ketanserin have been extensively used in biochemical and
pharmacological studies of the VMAT,16–18 their application
in structure–activity studies has been limited due to the
following: (a) nonspecific interaction with the membrane lipids
altering the integrity of the membrane, (b) very tight binding
to the transporters, (c) low solubility in the experimental
medium, (d) structural complexity, and so forth. Therefore,
development of specific inhibitors and/or alternate substrates
that may be conveniently used in the structure–function and
mechanistic studies of VMATs is a significant goal. We have
previously characterized a series of 3-amino-2-phenyl-propene
(APP) derivatives as novel and simple reversible inhibitors for
the bovine chromaffin granule VMAT.12 In the present study,
we have taken advantage of the VMAT substrate and inhibitor
activities of MPP+ and 3-amino-2-phenyl-propene (APP) de-
rivatives, respectively, to develop and characterize a series of
probes, and we have used them to further define the structure–
activity relationships of the substrates and inhibitors of the
VMAT.
Among the MPTP derivatives tested, the 3′-OH derivative
(18) appeared to be the best competitive inhibitor for VMAT
with respect to DA uptake, which further confirmed the
importance of the 3′-OH group in the inhibition as well as the
VMAT substrate activities of both series of compounds.
However, the inhibition potency of 18 is about 10 times weaker
than that of the corresponding MPP+ derivative (3). Interest-
ingly, the inhibition potencies of MPTP as well as some of its
derivatives (for example 16 and 20) are higher than those of
the corresponding MPP+ derivatives, suggesting that they
interact well with the transporter in spite of poor substrate
activities. Thus, the transport through the VMAT appears to
require more stringent molecular interactions than the inhibition.
The above results also suggest that the 3′-OH groups of both
MPP+ and MPTP play an important role in the interaction with
the VMAT. A similar trend is also apparent with the 3′-CF3 in
the MPP+ series. Therefore, the 3′-substituents of these two
classes of compounds may play a critical role in the interaction
with the VMAT through H-bonding with an amino acid residue
in the binding site. However, in contrast to the regular
phenylethylamine VMAT substrates, the 4′-OH groups of MPP+
or MPTP do not contribute significantly to the substrate activity
or the inhibition potencies. On the other hand, bulky hydro-
phobic substituents at the 4′-position, specifically halogen,
positively contribute to the binding, similar to the trend that
was observed with previously characterized APP inhibitors.12
Structure–Activity Studies. Previous studies have suggested
that 1-methyl-4-phenyl pyridinium (1, MPP+) is a good substrate
for resealed chromaffin granular ghost VMAT.7,8 However, the
data presented in Table 1 clearly show that it is a relatively
weak substrate, in comparison to DA. Similarly, most MPP+
derivatives tested were also weak substrates for the VMAT,
except the 3′-OH derivative (3). While this derivative is an
excellent substrate with a Km of 8.4 ( 2.1 µM and a Vmax of
1.9 ( 0.1 nmols/mg·min, the 4′-OH derivative (4) was a very
weak substrate (Table 1), which is unexpected, since both 4′-
and 3′-tyramines are almost equally good substrates for VMAT
(D. S. Wimalasena and K. Wimalasena, unpublished observa-
tions). In contrast to MPP+ derivatives, all of the MPTP
derivatives tested were found to be very weak or not substrates
for the VMAT.
The inhibition kinetics of substituted MPP+ derivatives show
that 3 very effectively competes with DA for uptake under the
standard conditions (Ki ) 2.4 ( 0.1 µM). It interacts with the
VMAT about 10 times better than DA and about 35 times better
than the parent compound, MPP+ (1, Ki ) 92 ( 14 µM), under
similar experimental conditions. In addition, the Km determined
for 3 under standard uptake conditions was about 4 times greater
than the Ki determined by DA competition experiments (Table
1), suggesting that it is a kinetically sticky substrate for the
VMAT; that is, the rate constant for the dissociation of the trans-
To further exploit the interaction of these derivatives with
the VMAT, we have designed, synthesized, and characterized
a series of probes that mimic the structural features of both APP
(a previously characterized inhibitor12) and MPP+ (substrate).
The uptake studies with granule ghosts have shown that the
prototypical parent derivative, the MPP+-APP conjugate (23),
was not detectably taken up even at the 200–500 µM concentra-
tions, suggesting that the bulky substituent (2-phenylpropene)
on nitrogen completely abolishes the VMAT substrate activity
of MPP+. However, 23 was found to be a reasonably good
competitive inhibitor for the VMAT with a Ki of 38 ( 4 µM
(Table 2). Comparison of the inhibition potency of 23 with that
of 1 indicates that the bulky hydrophobic substituent on the
nitrogen significantly enhances the inhibition potency of MPP+.
Based on these findings, a number of structurally distinct
APP-MPP+ conjugates were synthesized and characterized, and
their VMAT inhibition potencies were determined. These studies
have revealed that the replacement of the 4-phenyl group of
the parent compound (23) with a cyclohexyl group (24)
decreases the inhibition potency (Ki ) 53.0 ( 13.6 µM),
suggesting that the planar aromatic ring at the 4-position of the
pyridine ring of these conjugates plays a favorable role in the
interaction with the transporter. However, the replacement of

764 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Wimalasena et al.
the pyridine ring with a piperidine or pyrimidine ring (25, 26)
increased the inhibition potency noticeably (Ki ) 16.5 ( 2.7
and 13.4 ( 1.7 µM, respectively). More importantly, the
replacement of the pyridine ring with a piperazine ring (27)
increased the inhibition potency even more significantly. Thus,
piperazine derivative 27 is approximately 25-fold more potent
than the parent compound 23 and about 10–11-fold more potent
than the piperidine- or pyrimidine-based inhibitors 25 and 26.
These findings appear to indicate that the piperazine ring binds
more favorably to the transporter than the planar pyridinium
ring of conjugate inhibitors. We believe these interesting
differences could be due to the relatively high flexibility of the
piperazine ring in comparison to the pyrimidine ring of 26 [the
presence of an additional nitrogen atom on the ring may also
positively contribute to the inhibition potency of this derivative
(see below)]. In addition, these findings appear to indicate that
the permanently charged quaternary ammonium functionality
of MPP+ derivatives is not mandatory and/or optimal for the
effective interaction with the transporter, which is consistent
with our finding that MPTP is a better inhibitor than MPP+ for
VMAT (see below).
Figure 2. Overlay of energy minimized structures of tetrabenazine
with 29 (top) and reserpine with 29 (bottom).
conjugate inhibitors may interact with the transporter in a
slightly altered fashion in comparison to MPP+ itself.
The above results suggest that the MPP+ portion of the above
conjugates may largely determine their inhibition properties and
the APP portion could play an auxiliary role, presumably
through nonspecific hydrophobic interactions. This proposal is
supported by the finding that the addition of an extra phenyl
group to the 2-phenyl propene moiety of the parent conjugate
(31) significantly enhanced the VMAT inhibition potency (Ki
) 4.5 ( 0.5 µM). It is also consistent with our previous
observation that the inhibition potency of the 4′-halogen
substituted APP derivative increases with the increasing hy-
drophobicity of the halogen (I > Br > Cl > F).12 Furthermore,
the crystallographic analysis of the structures of these derivatives
(the structure of 28 is shown in Figure 1; this derivative gave
X-ray quality crystals readily, in comparison to the most potent
inhibitor, 29) shows that these molecules possess a well-defined
“L” shape architecture, and both the APP and MPP+ portions
may interact with the VMAT as two separate entities, leading
to higher inhibition potencies in comparison to those of either
of the two derivatives.
Figure 1. Crystal structure of 28.
To determine the distinct roles of the MPP+ and APP portions
of the above conjugates in the interaction with the VMAT, a
series of APP-MPP+ conjugates with different substituents on
the MPP+ phenyl ring have been synthesized and examined.
These studies revealed that substitution of a 4′-OMe on the
phenyl ring of the conjugate (28) increases the inhibition potency
by about 14-fold compared to the parent compound (23). This
result is quite opposite of the effects observed in 4′-OMe
substitution on the substrate/inhibitor activities of MPP+ itself
(Table 1). Introduction of a 3′-OH on the MPP+ phenyl and a
4′-I on the 2-phenyl propene moieties of the parent conjugate
(29) (previous studies have shown that bulky hydrophobic
halogens on the 4′-position of APP derivatives significantly
enhance their inhibition potencies12) increases the inhibition
potency significantly, and this was the most potent conjugate
inhibitor (Ki ) 0.44 ( 0.05 µM) discovered in the present study.
However, again in contrast to the trend observed in substrate
activities, 4′-OH substitution also increases the inhibition
potency of the conjugates to a level very similar to that of the
3′-OH substitution (30). These drastic differences in substituent
effects between the inhibition potencies of MPP+ and the
conjugates appear to suggest that the MPP+ portion of the
Computer modeling studies show that the MPP+-APP
conjugates structurally resemble the known potent VMAT
inhibitors reserpine (RES) and tetrabenazine (TBZ) (Figure 2).
Overlay of the optimized structures of 29 and TBZ shows that
the two molecules are structurally similar with respect to steric
constraints and positioning of the 4′- and 3′-phenyl ring oxygen
substituents. However, the ring nitrogens could not be fully
overlaid keeping the above constraints intact, indicating that
the nitrogens of the VMAT bound 29 and TBZ may not occupy
the same positions of the active site. Similar modeling studies
with RES show that the nonaromatic nitrogen of RES and the
pyridine nitrogen of 29 superimpose well (Figure 2), suggesting
that they could occupy similar positions in the binding site.
These proposals are consistent with the finding that the
replacement of the pyridine ring of the MPP+ portion of 23

VMAT Substrate/Inhibitor ActiVity of MPP+ DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 765
with nonaromatic piperidine (25) or piperazine (27) increases
the inhibition potency significantly. Thus, the high affinity of
TBZ (Ki ∼ 4 nM) in comparison the MPP+-APP conjugates
could be due to the optimal positioning of the nitrogen and the
carbonyl oxygen (which is not present in MPP+-APP conju-
gates or RES) in the binding site. We note however that the
large trimethoxyphenyl tail of RES could not be mimicked by
TBZ or any of the above MPP+-APP conjugate inhibitors.
Thus, since the carbonyl oxygen is not present and the nitrogens
of RES may not contribute toward optimal binding, its extended
hydrophobic tail must provide a significant contribution toward
its high binding affinity, especially since the binding site of the
transporter is shown to be highly hydrophobic. This proposal
is further supported by the previous report that the trimethoxy-
phenyl acetate-cleaved RES derivative, reserpic acid, is at least
a 2000-fold weaker inhibitor than RES (the Ki’s of RES and
reserpic acid are 1–7 nM19 and 10 µM,20 respectively). Since
the above conjugates may not have optimal positioning of the
nitrogen and also may lack an extended hydrophobic tail, they
are substantially weaker inhibitors for VMAT in comparison
to TBZ and RES, but they are comparable to reserpic acid. The
excellent substrate activity of 3′-OH MPP+ (probably the best
known substrate for the VMAT) in comparison to the 4′-OH
MPP+ and DA shows that the relative positioning of 3′-OH
with respect to the pyridine nitrogen in MPP+ derivatives is
optimal for the VMAT substrate activity. Thus, the similar
inhibition potencies observed for the MPP+-APP conjugates
with respect to 3′- and 4′-OH further indicate that the mode of
interaction of these as well as RES and TBZ inhibitors with
the transporter could be distinct from that of regular substrates.
Based on the above findings, a hypothetical working model
is proposed for the interaction of substrates and inhibitors of
the VMAT. The salient features of the model are as follows:
(a) substrate transport through the VMAT requires much more
stringent interactions than the inhibition, (b) the orientation of
the 3′-OH with respect to the pyridine nitrogen of MPP+ (3) is
optimal for substrate activity in contrast to regular phenylethyl-
amine substrates, (c) the interaction of MPP+-APP conjugates
as well as TBZ and RES may not precisely mimic the mode of
substrate interaction, (d) the MPP+-APP conjugates, TBZ, and
RES may interact with the transporter in a similar mode, and
(e) the nitrogen and carbonyl functionalities of TBZ and the
trimethoxyphenyl hydrophobic tail of RES may contribute
significantly to their high inhibition potencies.
wide variability of the VMAT substrate and/or the inhibitory
activities of these simple derivatives make them highly desirable
probes to examine the molecular mechanisms pertinent to the
neurotoxicity of MPP+. The most effective inhibitors identified
in the present study were a series of compounds that integrate
the structural features of MPP+ and APP, a previously char-
acterized VMAT inhibitor.12 The overall structures of these
MPP+-APP conjugates were remarkably similar to the well-
known classical VMAT inhibitors RES and TBZ. The struc-
ture–activity analysis of these three series of inhibitors supports
a proposal that the high inhibition potency of TBZ could be
due to the optimal placement of the ring nitrogen and carbonyl
oxygen in the active site, and in the case of RES, nonoptimal
nitrogen interactions and the lack of a corresponding carbonyl
oxygen may be compensated by the contributions from the
hydrophobic trimethoxyphenyl tail. We believe this information
would enable the design and synthesis of potent and structurally
simple inhibitors for monoamine transporter(s) for further
structure–activity studies. In addition, the comparative toxico-
logical studies of the MPP+ derivatives and conjugates char-
acterized in the present studies should also be useful in
determining the underlying mechanisms responsible for the
specific dopaminergic neurotoxicity of MPP+. These studies are
currently underway in our laboratory.
Experimental Section
A. Materials. All reagents and solvents were obtained from
various commercial sources with the highest purity available and
used without further purification. Tetramethylsilane (for deuterated
organic solvents) or 3-(trimethylsilyl)propionic acid sodium salt (for
D2O) was used as internal standard for 1H NMR. Beef liver catalase
(65 000 units/mg protein) was obtained from Boehringer-Mannheim.
Protein assay reagents were obtained from Bio-Rad laboratories.
The compounds MPP+I- (1), MPTPHCl (15), 2′Me-MPTPHCl
(16), 2′NH2-MPTPHCl (17), and MPDP+ClO4– (22) were obtained
from Sigma Aldrich.
B. General Methods. All 1H and 13C NMR spectra were
recorded on a Varian Inova 400 or 300 MHz spectrometer, and
mass spectra were obtained from a Finnigan LCQ-Deca ion trap
mass spectrometer (Thermoquest Corporation, San Jose, CA). All
melting points were uncorrected and determined using MEL-TEMP
II (Laboratory Devices, USA). All of the elemental analyses were
carried out by Desert Analytics (Tucson, AZ). Compounds 5 and
7 have been previously synthesized and characterized.10 Compounds
2, 8-12, 18-20, 25-27, and 30 are structurally simple and related
to the analytically fully characterized parent compounds. Therefore,
their structural conformations and purities were assessed by “clean”
1H and 13C NMR spectra, mass spectrometry, and C18-reversed
phase HPLC.
As mentioned above, the conjugate inhibitors characterized
in the present study are significantly weaker than the commonly
employed classical inhibitors such as RES and TBZ. However,
structural simplicity and well-defined structural rigidity together
with the easy synthetic accessibility of these derivatives make
them more appealing for systematic structure–activity studies.
On the other hand, they share some common limitations that
are associated with those classical inhibitors such as low
solubility and nonspecific membrane interactions. Further
structural refinements of these inhibitors for better inhibition
potencies and increased solubilities are currently underway.
C. Computational Calculations. Ab initio calculations were
carried out using the Gaussian 98-ReVision A.9 suite21 of programs.
The overlay of minimized structures was carried out by using the
built-in overlay function of Chem 3D Ultra (CambridgeSoft,
Cambridge, MA).
D. Chromaffin Granule Isolation. Chromaffin granules were
prepared as previously described with minor modifications.12–14
Fresh bovine adrenal glands were dissected, and medullary tissue
was collected in ice cold 0.3 M sucrose, 10 mM HEPES buffer,
pH 7.0. The tissues were homogenized with a Biospec biohomo-
genizer, and the homogenate was centrifuged at 3600 rpm (1200g)
for 10 min at 4 °C. The resulting supernatant was centrifuged at
18 000 rpm (27 000g) at 4 °C for 25 min; the supernatants were
discarded. The pellets were washed carefully by swirling with ∼1–2
mL of 0.3 M sucrose, 10 mM HEPES buffer, pH 7.0 to remove
the white fluffy upper layer. The washed pellets were resuspended
in the above buffer and homogenized using a Potter-Elvejhem
homogenizer, and the above centrifugation and washing steps were
repeated. The pellets were resuspended in 0.3 M sucrose, 10 mM
Conclusions
A series of MPTP and MPP+ derivatives have been designed,
synthesized, and characterized as novel substrates/inhibitors for
the VMAT. The 3′-OH derivative of MPP+ has been identified
as one of the best known substrates for the VMAT. Comparative
structure–activity analysis of these inhibitors and substrates
suggests that the VMAT substrate activity must satisfy much
more stringent structural requirements than the inhibition. The

766 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Wimalasena et al.
HEPES buffer, pH 7.0, and homogenized, and the homogenate was
layered on 1.6 M sucrose, 10 mM HEPES buffer, pH 7.0, and then
centrifuged at 27 500 rpm (58 700g) for 90 min at 4 °C. The pellets
were washed and resuspended in lysing buffer containing 0.2 M
tris phosphate buffer, 100 µg/mL catalase, pH 7.0, and glycerol
(7:1 v/v) and homogenized. The lysed granules were stored at
-70 °C.
c. 1-Methyl-4-(4′-fluorophenyl)pyridinium Iodide (7). Yield
1
38%; mp 231–234 °C; H NMR (D2O) δ 4.31 (s, 3H), 7.19 (d,
2H), 7.45 (t, 2H), 8.19 (q, 2H), 8.58 (d, 2H); 13C NMR (D2O) δ
50.0, 119.2, 119.4, 127, 132.2, 132.9, 133, 144.4, 157.1, 166.2,
168.7; MS (ESI) m/z 188.09 (M+).
d. 1-Methyl-4-(4′-chlorophenyl)pyridinium Iodide (8). Yield
1
32%; mp 225 °C; H NMR (D2O) δ 4.32 (s, 3H), 7.38 (d, 2H),
7.30 (d, 2H), 8.09 (d, 2H), 8.61 (d, 2H); 13C NMR (D2O) δ 50.1,
130.3, 132.9, 146.2, 147.2, 157.9; MS (ESI) m/z 207.12 (M+).
e. 1-Methyl-4-(3′-chlorophenyl)pyridinium Iodide (9). Yield
34%; mp 226–228 °C; 1H NMR (D2O) δ 4.34 (s, 3H), 7.54-7.64
(m, 4H), 8.19 (d, 2H), 8.63 (d, 2H); 13C NMR (D2O) δ 50.0, 127.5,
128.9, 130.3, 133.6, 134.2, 137.5, 138.2, 147.6, 157.3; MS (ESI)
m/z 207.12 (M+).
E. Biological Evaluation. All kinetic experiments were carried
out using properly characterized resealed bovine chromaffin granule
ghosts as previously described.12–14 Briefly, the washed granule
membranes isolated from the above stores were resealed to contain
20 mM Tris phosphate, 100 mM KCl, 150 mM sucrose, 10 mM
fumarate, and 100 µg/mL catalase (pH 7.0) and were purified by a
discontinuous Ficoll/sucrose density gradient. The resealed granule
ghosts were incubated in a medium (0.5 mL total volume)
containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5
mM MgSO4, and 100 µg/mL catalase, (pH 7.0) at 30 °C for 10
min (uptake conditions). Then, the desired concentrations of the
inhibitor were added to the mixture, which was incubated further
for 2 min. Following the incubation period, the uptake reaction
was initiated by the addition of the desired concentration of DA,
and 400 µL aliquots of the incubate were withdrawn at 6 min time
intervals and diluted into 5.0 mL of ice cold 0.4 M sucrose, 10
mM HEPES, pH 7.0. These samples were then centrifuged at
36 000g for 25 min at 4 °C, the supernatants removed, the pellets
gently washed three times with 0.4 M sucrose, 10 mM HEPES,
pH 7.0, and the tubes swabbed dry. Then, 500 µL of 0.1 M HClO4
was added, the pellets were homogenized, and the extraction was
allowed to proceed for 20 min at room temperature. After low speed
centrifugation to remove coagulated protein, the catecholamines
in the acidic extracts (intragranular catecholamines) were separated
and quantified by reversed phase HPLC-EC. In uptake experiments,
the same procedure was used, except that DA was excluded from
the incubations and the intragranular levels of various substrates
were quantified by HPLC-UV.
F. Synthesis. 1. Technical Statement. Due to potential health
hazards of MPP+ and MPTP and related derivatives, extreme
caution should be taken in their synthesis and use. The guidelines
for proper handling of MPTP derivatives have been reported.22
2. Synthesis of 1-Methyl-4-phenyl Pyridinium Salts. These
compounds were synthesized according to the procedure described
by Das et al. with minor modifications (Scheme 1).9 To a suspension
of anhydrous CuI (0.3 g, 1.5 mM) and dry pyridine (0.04 mol) in
dry THF (100 mL) was added ethyl chloroformate (0.03 mol) under
N2, at -30 °C, and the reaction mixture was stirred for 30 min.
Then, a solution of Grignard reagent (0.04 mol) prepared from the
respective phenyl bromide and Mg turnings in THF (100 mL) was
added dropwise over 15 min. The mixture was stirred for 15 min
at -30 °C and then at room temperature until the solution became
clear (in some cases the solution was refluxed for 10 h under N2 to
obtain a clear solution). Then, the reaction mixture was treated with
saturated NH4Cl (50 mL), extracted with ether, dried with Na2SO4,
and concentrated, and the resultant dihydropyridine intermediate
was oxidized by dropwise addition of KMnO4 in acetone at 0 °C
(until the pink permanganate color persisted for several minutes).
After evaporation of acetone, the brown solid was suspended in
ether and filtered. The ether solution was washed several times with
5% NaHCO3, dried over Na2SO4, and concentrated under vacuum.
The free base was dissolved in anhydrous THF and treated with
MeI at 0 °C to give the corresponding quaternary ammonium salt,
which was crystallized using ethanol/ether.
f. 1-Methyl-4-(4′-trifluorophenyl)pyridinium Iodide (10). Yield
1
38%; mp 236–237 °C; H NMR (D2O) δ 4.31 (s, 3H), 7.84 (d,
2H), 8.12 (d, 2H), 8.28 (d, 2H), 8.32 (d, 2H); 13C NMR (D2O) δ
50.0, 126.53, 131.2, 133.9, 134.1, 148.1, 148.9, 157.4, 162.4; MS
(ESI) m/z 238.09 (M+).
g. 1-Methyl-4-(3′-trifluorophenyl)pyridinium Iodide (11). Yield
23%; mp 218–221 °C; 1H NMR (D2O) δ 4.31 (s, 3H), 7.34-7.51
(m, 4H), 7.82 (d, 2H), 8.51 (d, 2H); 13C NMR (D2O) δ 50.0, 119.1,
129.4, 126, 128.2, 131.3, 134.3, 143.4, 155.1, 159.2, 164.7; MS
(ESI) m/z 238.09 (M+).
h. 1-Methyl-4-(4′-methylphenyl)pyridinium Iodide (12). Yield
46%; mp 216–219 °C; 1H NMR (D2O) δ 2.43 (s, 3H), 4.32 (s,
3H), 7.38 (d, 2H), 7.70 (d, 2H), 8.09 (d, 2H), 8.61 (d, 2H); 13C
NMR (D2O) δ 22.3, 49.8, 126.3, 130.9, 132.2, 132.8, 146.1, 147.2,
157.9; MS (ESI) m/z 184.11 (M+).
3. Synthesis of Hydroxy Derivatives of MPP+. All hydroxyl
derivatives of MPP+ were obtained by O-demethylation of the
corresponding -OMe derivatives of 4-phenylpyridine according to
the procedure of Das et al.11 followed by N-methylation of pyridine
nitrogen with CH3I (Scheme 2). Briefly, a mixture of 10 mL of
pyridine and excess concentrated HCl in a three necked flask fitted
with a thermometer was heated up to 180 °C until complete removal
of water. Then the salt solution was cooled to 140 °C, and 1.0 g of
the corresponding methoxy-MPP derivative was added, and the
mixture was heated to 200 °C under N2 while stirring for 3 h. The
resulting brown solution was cooled to room temperature, and 1
M NaOH was added dropwise until the formation of an orange
yellow precipitate was complete. The mixture was stirred for 1 h,
and the precipitate was filtered, dissolved in absolute ethanol, and
acidified with concentrated HCl. Ethanol was evaporated under
vacuum, and the solid was crystallized with ethanol/ether. The
resulting solid was filtered, basified with 1% NaHCO3, and extracted
into ether. The ether layer was dried over Na2SO4 and evaporated
under vacuum to obtain a white solid. The resulting solid was
dissolved in anhydrous THF and cooled to 0 °C, and a 10–20%
molar excess of CH3I was added and stirred for 1 h at 0 °C and for
4 h at room temperature. The resulting light yellow precipitate was
filtered and crystallized with ethanol/ether.
a. 1-Methyl-4-(3′-hydroxyphenyl)pyridinium Iodide (3′-OHM-
1
PP+) (3). Yield 26%; mp 228–229 °C; H NMR (D2O) δ 4.36 (s,
3H), 7.10–7.12 (m, 1H), 7.26 (m, 1H), 7.40–7.49 (m, 2H), 8.16 (d,
2H), 8.71 (d, 2H); 13C NMR (D2O) δ 49.7, 117.1, 121.9, 123.1,
127.6, 134.2, 147.5, 158.3, 159.1; MS (ESI) m/z 186.6 (M+).
b. 1-Methyl-4-(4′-hydroxyphenyl)pyridinium Iodide (4′-OHM-
1
PP+) (4). Yield 23%; mp 222–224 °C; H NMR (D2O) δ 4.29 (s,
3H), 7.06 (d, 2H), 7.87 (d, 2H), 8.16 (d, 2H), 8.59 (d, 2H); 13C
NMR (D2O) δ 49.5, 119.3, 125.8, 127.7, 132.7, 147.08, 157.8,
163.03; MS (ESI) m/z 186.6 (M+); Anal. (C12H12ONI) C, H, N.
c. 1,3-Dimethyl-4-(4′-hydroxyphenyl)pyridinium Iodide (6).
Yield 12%; mp 224–226 °C; 1H NMR (D2O) δ 2.48 (s, 3H), 4.31
(s, 3H), 7.03 (d, 2H), 7.46 (d, 2H), 7.83 (d, 1H), 8.25 (d, 1H), 8.61
(s, 1H); 13C NMR (D2O) δ 20.3, 49.4, 127.3, 130.3, 132.9, 136.5,
158.2, 162.9; MS (ESI) m/z 200.4 (M+); Anal. (C13H14ONI) C,
H, N.
a. 1-Methyl-4-(2′-methylphenyl)pyridinium Iodide (2). Yield
41%; mp 226–228 °C; 1H NMR (D2O) δ 2.17 (s, 3H), 4.24 (s,
3H), 7.24–7.26 (m, 4H), 7.86 (d, 2H), 8.60 (d, 2H); 13C NMR (D2O)
δ 20.4, 48.5, 127.3, 130.3, 132.9, 1343.9, 135.9, 136.5, 147.2, 148.2,
158.9; MS (ESI) m/z 184.11 (M+).
b. 1-Methyl-4-(4′-methoxyphenyl)pyridinium Iodide (5). Yield
42%; mp 226–228 °C; 1H NMR (D2O) δ 2.42 (s, 3H), 4.32 (s,
3H), 7.38 (d, 2H), 7.70 (d, 2H), 8.09 (d, 2H), 8.61 (d, 2H); 13C
NMR (D2O) δ 41.3, 49.5, 126.3, 130.3, 132.9, 133.9, 146.2, 147.2,
157.9; MS (ESI) m/z 200.1 (M+).
4. Synthesis of Hydroxy Derivatives of 1-Methyl-3-phenyl
Pyridinium Salts. A solution of 4-bromoanisole (0.03 mol) in THF
was stirred with Mg turnings (0.09 mol) and for 5 h under N2 at

VMAT Substrate/Inhibitor ActiVity of MPP+ DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 767
room temperature. The resulting solution was added dropwise to a
solution of dichlorobis(triphenylphosphine)nickel(II) and 3-bro-
mopyridine in anhydrous THF and stirred for 12 h under N2. The
reaction was quenched by adding diluted HCl, and unreacted starting
materials were removed by extracting into CH2Cl2. The aqueous
phase was basified with NaHCO3 and extracted into ether. The ether
layer was dried and concentrated under vacuum to obtain a white
solid. The solid was dissolved in ethanol, acidified with concentrated
HCl, and purified by crystallization with ethanol/ether. The product
was O-demethylated and then N-methylated as described for
compound 3, above (Scheme 3).
57, 123.8, 127.6, 129.2, 130.6, 131.8, 132.3, 135, 138.8, 143.7,
146.7, 159.6; MS (ESI) m/z 272.24 (M+); Anal. (C20H18BrN) C,
H, N.
b. 4-Cyclohexyl-1-(2-phenyl-allyl)pyridinium Bromide (24).
Yield 58%; mp 231–232 °C; 1H-NMR (D2O) δ 1.25 (m, 1H),
1.35–1.42 (m, 3H), 1.64–1.83 (m, 4H), 2.80 (t, 1H), 5.56 (s, 1H),
5.63 (s, 2H), 5.81 (s, 1H), 7.35–7.51 (m, 5H), 7.80 (d, 2H), 8.67
(d, 2H); 13C NMR (D2O) δ 27.7, 28.3, 34.9, 46.6, 57.0, 66.3, 123.5,
129.1, 129.2, 131.7, 131.8, 138.8, 143.8, 146.1; MS (ESI) m/z
348.19 (M+); Anal. (C26H22BrN) C, H, N.
c. 4-(4′-methoxyphenyl)-1-(2-phenyl-allyl)pyridinium Bromide
(28). Yield 74%; mp 241–243 °C; 1H NMR (D2O) δ 3.78 (s, 3H),
5.50 (s, 1H), 5.52 (s, 2H), 5.72 (s, 1H), 6.97 (d, 2H), 7.28 (m, 3H),
7.41 (d, 2H), 7.64 (d, 2H), 7.89 (d, 2H), 8.53 (d, 2H); 13C NMR
(D2O) δ 38.3, 56.6, 123.9, 128.6, 129.8, 131.6, 132.8, 135.8, 138.8,
143.8, 145.4, 1.58.2, 161.6; MS (ESI) m/z 302.15 (M+); Anal.
(C21H20BrNO) C, H, N.
d. 4-(3′-hydroxyphenyl)-1-[2-(4-iodophenyl)-allyl]pyridinium
Bromide (29). Yield 46%; mp 245–248 °C; 1H NMR (CD3OD) δ
5.54 (s, 1H), 5.76 (s, 2H), 5.84 (s, 2H), 7.04 (m, 1H), 7.31 (m,
1H), 7.39–7.45 (m, 6H), 8.29 (d, 2H), 8.90 (d, 2H); 13C NMR (D2O)
δ 64.3, 115.6, 120.2, 120.7, 121.6, 124.2, 126.1, 129.1, 132.1, 133.3,
136.3, 136.8, 142.5, 145.8, 158.8, 160.1; MS (ESI) m/z 414.06
(M+); Anal. (C20H17BrINO) C, H, N.
a. 1-Methyl-3-(4′-hydroxyphenyl)pyridinium Iodide (13). Yield
1
16%; mp 236 °C; H NMR (D2O) δ 4.42 (s, 3H), 7.01 (d, 2H),
7.62 (d, 2H), 8.00 (q, 1H), 8.61 (t, 2H), 8.94 (s, 1H); 13C NMR
(D2O) δ 49.3, 117.1, 121.9, 123.1, 127.6, 134.2, 147.5, 158.3, 162.1;
MS (ESI) m/z 186.6 (M+); Anal. (C12H12ONI) C, H, N.
b. 1-Methyl-3-(3′-hydroxyphenyl)pyridinium Iodide (14). Yield
1
28%; mp 227–229 °C; H NMR (D2O) δ 4.26 (s, 3H), 7.10–7.12
(m, 1H), 7.26 (m, 1H), 7.40–7.49 (m, 2H), 8.11 (q, 1H), 8.64 (t,
2H), 8.91 (s, 1H); 13C NMR (D2O) δ 49.7, 117.1, 121.9, 122.4,
123.1, 127.6, 129.5, 134.2, 147.5, 158.3, 159.1; MS (ESI) m/z 186.6
(M+); Anal. (C12H12ONI) C, H, N.
5. Synthesis of 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine
(MPTP) Derivatives. A solution of 1-methyl-4-piperidone (typically
3 mmol scale) in dry THF was mixed with the desired phenyl
magnesium bromide and stirred for 12 h at room temperature. The
reaction mixture was treated with saturated NH4Cl, and unreacted
starting materials were removed by washing with ethyl acetate. The
aqueous layer was basified with saturated NaHCO3 and extracted
into ether. The ether layer was dried with Na2SO4 and evaporated
to dryness; the residue was dissolved in ethanol, acidified with
concentrated HCl, and evaporated to dryness. The product hydro-
chloride was crystallized with ethanol/ether (Scheme 4).
e. 4-(4′-hydroxyphenyl)-1-[2-(4-bromophenyl)-allyl]pyridin-
1
ium Bromide (30). Yield 74%; mp 239–240 °C; H NMR (D2O)
δ 5.48 (s, 1H), 5.72 (s, 2H), 5.74 (s, 1H), 7.24 (d, 2H), 7.26 (d,
2H), 7.44 (d, 2H), 7.87 (d, 2H), 8.21 (d, 2H), 8.76 (d, 2H); 13C
NMR (D2O) δ 55.7, 63.9, 115.1, 119.3, 122.4, 125.8, 127, 129,
130.7, 134.8, 135.2, 141.3, 144.2, 158.5, 166.4; MS (ESI) m/z
366.04 (M+).
f. 4-Phenyl-1-(2-biphenyl-allyl)pyridinium Bromide (31). Yield
46%; mp 241–243 °C; 1H NMR (CD3OD) δ 4.95 (s, 1H), 4.98 (s,
2H), 5.15 (s, 1H), 6.64–6.71 (m, 4H), 6.78–6.81 (m, 6H), 6.83–6.85
(m, 4H), 7.68 (d, 2H), 8.32 (d, 2H); 13C NMR (CD3OD) δ 57.0,
123.5, 129.1, 129.2, 131.7, 131.8, 132.4, 133.5, 133.8, 138.8, 143.8,
156.1; MS (ESI) m/z 348.19 (M+); Anal. (C26H22BrN) C, H, N.
g. 4-Phenyl-1-(2-phenyl-allyl)piperidine Chloride (25). Yield
a. 1-Methyl-4-(3-hydroxyphenyl)-1,2,3,6-tetrahydropyridine HCl
1
(18). Yield 23%; H NMR (D2O) δ 2.83–3.01 (m, 2H), 3.12 (s,
3H), 3.25–3.32 (m, 1H), 3.66–3.76 (m, 1H), 4.02 (dd, 1H), 4.07
(dd, 1H), 6.13 (dd, 1H), 6.92 (dd, 1H), 6.97 (t, 1H) 7.06–7.08 (m,
1H), 7.35 (t, 1H); 13C NMR δ 25.8, 44.3, 50.1, 52.2, 118.7, 125.4,
127.5, 130.3, 132.2, 136.0, 148.7, 159.9; MS (ESI) m/z 190.12
(M+).
1
31%; mp 224–226 °C; H NMR (D2O) δ 3.53 (br s, 9H), 4.41 (s,
2H), 5.72 (s, 1H), 5.87 (s, 1H), 7.17–7.20 (m, 3H), 7.41–7.59 (m,
7H); 13C NMR (D2O) δ 47.7, 50.9, 60.2, 118.3, 124.3, 125.2, 126.7,
129.3, 130, 137.3, 137.4, 146.9; MS (ESI) m/z 278.19 (M+).
h. 4-Phenyl-1-(2-phenyl-allyl)pyrimidine Hydrochloride (26).
b. 1-Methyl-4-(4-hydroxylphenyl)-1,2,3,6-tetrahydropyridine HCl
1
(19). Yield 32%; H NMR (D2O) δ 2.82–2.85 (m, 2H), 3.01 (s,
3H), 3.34 (m, 1H), 3.68–3.74 (m, 1H), 3.78 (dd, 1H), 4.00 (dd,
1H), 6.02 (dd, 1H), 6.91 (d, 2H), 7.41 (d, 2H); 13C NMR (D2O) δ
25.5, 44.3, 50.1, 52.5, 117.2, 128.5, 146.3, 148.8, 161.1; MS (ESI)
m/z 190.12 (M+).
1
Yield 34%; H NMR (D2O) δ 5.71 (s, 1H), 5.72 (s, 2H), 5.91 (s,
1H), 7.39–7.54 (m, 4H), 7.54–7.61 (m, 5H), 7.64–7.73 (m, 1H),
8.21 (d, 2H), 8.41 (d, 1H), 9.07 (dd, 1H), 9.56 (s, 1H); 13C NMR
(D2O) δ 56.8, 57.9, 119.3, 124.3, 125.2, 126.7, 129.3, 130, 137.3,
137.4, 156.9; MS (ESI) m/z 273.19 (M+).
c. 1-Methyl-4-(3-chlorophenyl)-1,2,3,6-tetrahydropyridine HCl
1
(20). Yield 41%; H NMR (D2O) δ 2.83–3.01 (m, 2H), 3.12 (s,
i. 4-Phenyl-1-(2-phenyl-allyl)piperazine Hydrochloride (27).
3H), 3.25–3.32 (m, 1H), 3.66–3.76 (m, 1H), 4.02 (dd, 1H), 4.07
(dd, 1H), 6.13 (dd, 1H), 6.92 (dd, 1H), 6.97 (t, 1H), 7.06–7.08 (m,
1H), 7.35 (t, 1H); 13C NMR (D2O) δ 26.4, 44.0, 52.8, 54.2, 118.7,
125.4, 127.5, 130.3, 132.2, 135.6, 136.0, 142.7; MS (ESI) m/z
208.19 (M+).
1
Yields 34%; mp 212–214 °C; H NMR (D2O) δ 4.24 (br s, 8H),
5.12 (s, 2H), 5.62 (s, 1H), 5.89 (s, 1H), 7.17–7.20 (m, 3H),
7.41–7.59 (m, 7H); 13C NMR (D2O) δ 49.7, 52.7, 60.8, 119.6,
125.8, 126.4, 126.7, 129.8, 133.5, 137.8, 137.2, 146.9; MS (ESI)
m/z 279.19 (M+); Anal. (C19H23ClN2) C, H, N.
d. 1-Methyl-4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine HCl
1
(21). Yield 36%; H NMR (D2O) δ 2.81–2.86 (m, 2H), 3.01 (s,
Acknowledgment. This work was supported by the National
Institutes of Health (NS 39423).
3H), 3.34 (m, 1H), 3.68–3.74 (m, 1H), 3.78 (dd, 1H), 4.05 (dd,
1H), 6.09 (dd, 1H), 7.18 (t, 2H), 7.51 (q, 2H); 13C NMR (D2O) δ
26.4, 44.9, 53.0, 55.2, 117.7, 129.5, 135.3, 136.9, 163.6, 166.1;
MS (ESI) m/z 192.12 (M+).
Supporting Information Available: Elemental analysis data of
compounds 4, 6, 13, 14, 23–24, 27–29, and 31. This material is
available free of charge via the Internet at http://pubs.acs.org.
6. Synthesis of MPP+-APP Conjugates. A mixture of the
desired 4-phenylpyridine derivative (6.5 mmol) and excess NaHCO3
in THF was treated with 6.5 mmol of the desired 3-bromo-2-
phenylpropene12 and stirred for 5 h at room temperature (Scheme
5). The solvent was removed under reduced pressure, and the
resultant solid was dissolved in absolute ethanol, and insoluble
NaHCO3 was removed by filtration. Filtrate was evaporated, and
the residue was crystallized with EtOH/ether.
References
(1) Gerlach, M.; Riederer, P.; Przuntek, H.; Youdim, M. B. MPTP
mechanisms of neurotoxicity and their implications for Parkinson’s
disease. Eur. J. Pharmacol. 1991, 208, 273–286.
(2) Tipton, K. F.; Singer, T. P. Advances in our understanding of the
mechanisms of the neurotoxicity of MPTP and related compounds.
J. Neurochem. 1993, 61, 1191–1206.
(3) Fiskum, G.; Starkov, A.; Polster, B. M.; Chinopoulos, C. Mitochondrial
mechanisms of neural cell death and neuroprotective interventions in
Parkinson’s disease. Ann. N.Y. Acad. Sci. 2003, 991, 111–119.
a. 4-Phenyl-1-(2-phenyl-allyl)pyridinium Bromide (23). Yield
67%; mp 237–238 °C; 1H NMR (D2O) δ 5.64 (s, 1H), 5.70 (s,
2H), 5.86 (s, 1H), 7.39–7.42 (m, 3H), 7.51–7.55 (m, 4H), 7.80–7.83
(m, 2H), 8.14–8.17 (d, 2H), 8.79–8.82 (d, 2H); 13C NMR (D2O) δ

768 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Wimalasena et al.
(4) Obata, T. Dopamine efflux by MPTP and hydroxyl radical generation.
J. Neural Transm. 2002, 109 (9), 1159–1180.
(5) Kahlig, K. M.′; Galli, A. Regulation of dopamine transporter function
and plasma membrane expression by dopamine, amphetamine, and
cocaine. Eur. J. Pharmacol. 2003, 479, 153–8.
(6) Torres, G. E. The dopamine transporter proteome. J. Neurochem. 2006,
97, 3–10.
(7) Daniels, A. J.; Reinhard, J. F., Jr. Energy-driven uptake of the
neurotoxin 1-methyl-4-phenylpyridinium into chromaffin granules.
J. Biol. Chem. 1988, 263, 5034–5036.
(8) Darchen, F.; Scherman, D.; Henry, J.-P. Characteristics of the transport
of quaternary ammonium 1-methyl-4-phenylpyridinium by chromaffin
granules. Biochem. Pharmacol. 1988, 37, 4381–4387.
(9) Das, A.; Jeffery, J. C.; Maher, J. P.; McCleverty, J. A.; Schatz, E.;
Ward, M. D.; Wollermann, G. Mono- and binuclear molybdenum and
tungsten complexes containing asymmetric bridging ligands: effects
of ligand conjugation and conformation on metal-metal interactions.
Inorg. Chem. 1993, 32, 2145–2155.
transporter from chromaffin granules. J. Exp. Biol. 1994, 196, 251–
262.
(16) Darchen, F.; Scherman, D.; Henry, J.-P. Reserpine binding the
chromaffin granules suggests the existence of two conformations of
the monoamine transporter. Biochemistry 1989, 28, 1692–1697.
(17) Scherman, D.; Henry, J.-P. Effect of drugs on the ATP-induced and
pH-gradient-driven monoamine transport by bovine chromaffin gran-
ules. Biochim. Biophys. Acta 1980, 601, 664–677.
(18) Darchen, F.; Scherman, D.; Laduron, P. M.; Henry, J.-P. Ketanserin
binds to the monoamine transporter of chromaffin granules and of
synaptic vesicles. Mol. Pharmacol. 1988, 33, 672–677.
(19) Parti, R.; Ozkan, E. D.; Harnadek, G. J.; Njus, D. Inhibition of
norepinephrine transport and reserpine binding by reserpine derivatives.
J. Neurochem. 1987, 48, 949–953.
(20) Chaplin, L.; Cohen, A. H.; Huettl, P.; Kennedy, M.; Njus, D.;
Temperley, S. J. Reserpic acid as an inhibitor of norepinephrine
transport into chromaffin vesicle ghosts. J. Biol. Chem. 1985, 260,
10981–10985.
(10) Arora, P. K.; Riachi, N. J.; Fiedler, G. C.; Singh, M. P.; Abdallah, F.;
Harik, S. I.; Sayre, L. M. Structure-neurotoxicity trends of analogues
of 1-methyl-4-phenylpyridinium (MPP+), the cytotoxic metabolite of
the dopaminergic neurotoxin MPTP. Life Sci. 1990, 46, 379–390.
(11) Das, A.; Jeffery, J. C.; Maher, J. P.; McCleverty, J. A.; Schatz, E.;
Ward, M. D.; Wollermann, G. Angew. Chem., Int. Ed. Engl. 1992,
31, 1515–1518.
(12) Perera, R. P.; Wimalasena, D. S.; Wimalasena, K. Characterization
of a series of 3-amino-2-phenylpropene derivatives as novel bovine
chromaffin vesicular monoamine transporter inhibitors. J. Med. Chem.
2003, 46, 2599–2605.
(13) Wimalasena, D. S.; Wimalasena, K. Kinetic evidence for the chan-
neling of dopamine between monoamine transporter and membranous
dopamine ꢀ-monooxygenase in chromaffin granule ghosts. J. Biol.
Chem. 2004, 279, 15298–15304.
(14) Wimalasena, K.; Wimalasena, D. S. The reduction of membrane-bound
dopamine ꢀ-monooxygenase in resealed chromaffin granule ghosts:
is intra-granular ascorbic acid a mediator for the extra-granular
reducing equivalents. J. Biol. Chem. 1995, 270, 27516–27524.
(15) Henry, J. P.; Botton, D.; Sagne, C.; Isambert, M. F.; Desnos, C.;
Blanchard, V.; Raisman Vozari, R.; Krejci, E.; Massoulie, J.; Gasnier,
B. Biochemistry and molecular biology of the vesicular monoamine
(21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.;
Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels,
A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone,
V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.;
Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.;
Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Baboul, A. G.; Stefanov,
B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts,
R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen,
W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.;
Replogle, E. S.; Pople, J. A. Gaussian 98, Revision-A.9; Gaussian,
Inc.: Pittsburgh, PA, 1998.
(22) Przedborski, S.; Jackson-Lewis, V.; Naini, A. B.; Jakowec, M.;
Petzinger, G.; Miller, R.; Akram, M. The parkinsonian toxin 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): A technical review of
its utility and safety. J. Neurochem. 2001, 76, 1265–1274.
(23) Cleland, W. W. Statistical analysis of enzyme kinetic data. Methods
Enzymol. 1979, 63, 103–138.
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