Characterization of a series of 3-amino-2-phenylpropene derivatives as novel bovine chromaffin vesicular monoamine transporter inhibitors

Article abstract of DOI:10.1021/jm030004p

A series of 3-amino-2-phenylpropene (APP) derivatives have been synthesized and characterized as novel competitive inhibitors, with K_i values in the μM range, for the bovine chromaffin granule membrane monoamine transporter(s) (bVMAT). Although, these inhibitors are structurally similar to the bVMAT substrate tyramine, none of them were measurably transported into the granule. Structure - activity studies have revealed that, while the 3′- or 4′-OH groups on the aromatic ring enhance the inhibition potency, Me or OMe groups in these positions reduce the inhibition potency. Halogen substitution on the 4′-position of the aromatic ring causes gradual increase of the inhibition potency parallel to the electron donor ability of the halogen. Substituents on the NH₂ as well as on the 3-position of the alkyl chain reduce the inhibition potency. Comparative structure - activity analyses of APP derivatives with tyramine and the neurotoxin 1-methyl-4-phenylpyridinium suggest that the flexibility of the side chain and the relative orientation of the NH₂ group may be critical for the efficient transport of the substrate through the bVMAT. Comparable bVMAT affinities of these inhibitors to that of DA and other pharmacologically active amines suggest that they are suitable for the structure activity and mechanistic studies of monoamine transporters and may also be useful in modeling the mechanism of action of amphetamine-related derivatives.

Full text of DOI:10.1021/jm030004p

J . Med. Chem. 2003, 46, 2599-2605
2599
Ch a r a cter iza tion of a Ser ies of 3-Am in o-2-p h en ylp r op en e Der iva tives a s Novel
Bovin e Ch r om a ffin Vesicu la r Mon oa m in e Tr a n sp or ter In h ibitor s
Rohan P. Perera, D. Shyamali Wimalasena, and Kandatege Wimalasena*
Department of Chemistry, Wichita State University, Wichita, Kansas 67260-0051
Received J anuary 11, 2003
A series of 3-amino-2-phenylpropene (APP) derivatives have been synthesized and characterized
as novel competitive inhibitors, with Ki values in the µM range, for the bovine chromaffin
granule membrane monoamine transporter(s) (bVMAT). Although, these inhibitors are
structurally similar to the bVMAT substrate tyramine, none of them were measurably
transported into the granule. Structure-activity studies have revealed that, while the 3′- or
4′-OH groups on the aromatic ring enhance the inhibition potency, Me or OMe groups in these
positions reduce the inhibition potency. Halogen substitution on the 4′-position of the aromatic
ring causes gradual increase of the inhibition potency parallel to the electron donor ability of
the halogen. Substituents on the NH2 as well as on the 3-position of the alkyl chain reduce the
inhibition potency. Comparative structure-activity analyses of APP derivatives with tyramine
and the neurotoxin 1-methyl-4-phenylpyridinium suggest that the flexibility of the side chain
and the relative orientation of the NH2 group may be critical for the efficient transport of the
substrate through the bVMAT. Comparable bVMAT affinities of these inhibitors to that of DA
and other pharmacologically active amines suggest that they are suitable for the structure-
activity and mechanistic studies of monoamine transporters and may also be useful in modeling
the mechanism of action of amphetamine-related derivatives.
All monoamine storage vesicles (catecholaminergic,
serotonergic, or histaminergic) utilize two, closely re-
lated, relatively nonspecific transporters for the vesicu-
lar uptake of monoamines.1-4 These transporters are
responsible for maintaining high catecholamine con-
centration gradients (>105) in catecholamine storage
vesicles. They are acidic glycoproteins with an apparent
molecular weight of 70 000 Da with 12 transmembrane
domains.5,6 A functional vesicular monoamine trans-
porter from a pheochromocytoma cDNA library (VMAT1)
has been cloned and characterized.6 A second form of
the vesicular monoamine transporter (VMAT2) has been
cloned from a cDNA library of the brain.5 The central,
peripheral, and enteric neurons express only VMAT2,
while neuroendocrine, including chromaffin and entero-
chromaffin cells exclusively express VMAT1.7-9 How-
ever, VMAT2 appears to be the major transporter in
chromaffin granules of the bovine adrenal medulla.7-10
Catecholamines and histamines are shown to exhibit
3- and 30-fold more affinity, respectively, toward VMAT2
in comparison to VMAT1.7 Reserpine and ketanserin are
slightly more potent inhibitors of VMAT2, while tetra-
benazine is a specific inhibitor for VMAT2.1-4 In addi-
tion, amphetamine, methylenedioxyamphetamine, and
phenylethylamines are better inhibitors for VMAT2
than VMAT1.7,11
least partly, through interference with the physiological
functions of VMATs.6-7,10,12-19 Despite the physiological
significance, the molecular mechanisms related to the
biochemical functions of VMATs are still poorly under-
stood.20 Therefore, it is a significant goal to develop
specific probes, which could be used in the structure-
function and mechanistic studies of VMATs. In the
present study, we have characterized a series of 3-amino-
2-phenylpropene (APP) derivatives as novel reversible
competitive inhibitors for the bovine chromaffin granule
monoamine transporter(s) (bVMAT) with potencies in
the low µM range. Although, the affinities of these
inhibitors are significantly less than that of the classical
inhibitors such as reserpine, ketanserine, and tetra-
benazine,21-23 they are comparable to the affinities of
the most pharamacologically active monoamines, mak-
ing them highly suitable for pharmacological studies of
VMATs. In addition, their high water solubility, com-
petitive kinetics with respect to DA, and structural
simplicity make them suitable for structure-activity
studies of VMATs as well.
Ch em istr y. The syntheses of 1-3 have been previ-
ously reported.24-26 The parent compound, 3-amino-2-
phenylpropene (1; APP; previously incorrectly named
and abbreviated as PAME; see ref 26), could be con-
veniently prepared by the allylic bromination of R-
methylstyrene, with N-bromosuccinimide (NBS), fol-
lowed by Gabriel phthalimide synthesis. The starting
materials for the synthesis of 2 and 3 (the corresponding
R-methylstyrene derivatives) could be easily obtained
by the treatment of the corresponding ring-hydroxy
acetophenones with methylmagnesium bromide fol-
lowed by simultaneous dehydration/acetylation with
acetic anhydride.24-27 The R-methylstyrene derivatives
Vesicular monoamine transporters play a critical role
not only in sorting out, storing, and releasing of neuro-
transmitters, but also in fine-tuning the neuronal and
endocrine informational output.1-4 In addition, a large
number of illicit drugs and neurotoxins are proposed to
exert their neuropharmacological and toxic effects, at
* To whom correspondence should be addressed, Tel: (316)-978-
7386, Fax (316)-978-3431, e-mail:kandatege.wimalasena@wichita.edu.
10.1021/jm030004p CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/08/2003

2600 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Perera et al.
Sch em e 1a
a
(I) a. Ph3PCH2Br, DMSO; b. n-BuLi; (II) NBS, CHCl3; (III)
potassium phthalimide, DMF (IV) a. NH2NH2, EtOH; b. NaOH.
for the synthesis of other para-substituted derivatives
5-10 were obtained from the corresponding aceto-
phenones using the general Wittig reaction (Scheme 1)
which gave a better yield than the two-step Grignard
procedure (compounds 4, 5, 7, and 11 have also been
previously reported.28,29) Since N-bromosuccinimide (NBS)
bromination of 4-methoxyphenyl R-methylstyrene ex-
clusively produced the vinyllic isomer, 4 was obtained
from the corresponding 4-OH phthalimide derivative by
methylation of the ring hydroxyl group with CH3I
followed by hydrazinolysis to remove the phthalimide
group.
Biologica l Eva lu a tion . Bovine adrenal chromaffin
cells and their granules have been extensively used as
models in the study of pharmacologically active amines
and related compounds.1-4 Resealed bovine adrenal
chromaffin granule ghosts have also been commonly
used as a model for the catecholamine neurotransmitter
storage vesicles in the study of the DA uptake and
norepinephrine biosynthesis.1-4,27,30 We have previ-
ously developed experimental protocols to examine the
kinetics of dopamine (DA) uptake and conversion to
norepinephrine (NE) inside resealed bovine chromaffin
granule ghosts without using radiolabeled substrates.27,30
In the present study we have extended these protocols
to examine the kinetics of DA uptake into resealed
bovine chromaffin granule ghosts through the vesicular
monoamine transporter (bVMAT) (see Experimental
Section; details of these methods will be published
elsewhere). The results presented below clearly dem-
onstrate that the resealed chromaffin granule ghosts are
an ideal model system for the detailed kinetic charac-
terization of bVMAT inhibitors and substrates. While
the kinetic parameters determined for DA uptake in the
present study are in excellent agreement with the
previously reported values for intact bovine chromaffin
granules and granule ghosts,15,18,27,30 they are about
6-8 times higher than the kinetic parameters reported
for both VMAT1 and VMAT2 expressed in digitonin-
permeabilized fibroblast cells.7
F igu r e 1. The effect of external 4′OH APP on the time courses
of NE production in resealed chromaffin granule ghosts under
turnover conditions: Ghosts were resealed to contain 20 mM
Tris phosphate, pH 7.0, 100 mM KCl, 150 mM sucrose, 10 mM
fumarate, and 100 µg/mL catalase with 20 mM ascorbic acid
(pH 7.0) and incubated in a medium (2.5 mL total volume)
containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5
mM MgSO4, 20 mM ascorbic acid, and 100 µg/mL catalase (pH
7.0) at 30 °C for 10 min. Then, 0.5 mM 4′OH APP was added
to one of the mixtures, and both were allowed to incubate
further for 2 min. At zero time, the conversion reaction was
initiated by the addition of DA to a final concentration of 200
µM, and, at 5 min time intervals, 400 µL aliquots of the
incubate were withdrawn and intragranular DA and NE levels
were quantified by HPLC-EC as detailed in Experimental
Section. (a), b, NE level in the control, 9, NE level in the
presence of 500 µM external 4′OH APP; O, DA level in the
control; 0, DA level in the presence of 500 µM external 4′OH
APP.
our experiments revealed that externally applied 2 does
not accumulate in resealed granule ghosts even at 200-
500 µM concentrations, under standard uptake condi-
tions (see Experimental Section). On the other hand,
the incubation of resealed chromaffin granule ghosts in
a medium containing 500 µM 2 and 200 µM DA under
standard turnover conditions resulted in a marked
decrease in both DA accumulation and NE production
inside the granule ghosts (Figure 1). Similarly, the
incubations of resealed granule ghosts with equimolar
concentrations of DA + 2 (200 µM) under uptake
(nonturnover) conditions revealed that DA uptake is
markedly inhibited by 2, suggesting that 2 could be an
efficient inhibitor for bVMAT (data not shown).
To further confirm the above results, the kinetics of
the inhibition of bVMAT by 2 was studied using resealed
chromaffin granule ghosts under standard uptake con-
ditions. As shown in Figure 2A and B, DA uptake
inhibition by 2 was competitive with respect to DA with
a Ki of 15.5 ( 0.9 µM. Under similar experimental
conditions, Km for DA was determined to be 23.6 ( 1.4
µM, suggesting that 2 interacts with bVMAT slightly
more efficiently than DA (Km,DA/Ki ∼ 1.5). Furthermore,
the parent compound, APP (1), was also found to be a
weaker competitive inhibitor for bVMAT with a Ki of
40.3 ( 5.3 µM (Km,DA/Ki ∼ 0.6), demonstrating that the
4′-OH group of 2 plays a significant role in the inhibition
of bVMAT which is consistent with the known specificity
of bVMAT.1-4 On the other hand, the transposition of
the ring OH group from the 4′- to the 3′-position (3) did
Resu lts a n d Discu ssion
Although DA is the physiological substrate for
bVMAT, previous studies have shown that tyramine is
also a good substrate for bVMAT with kinetic param-
eters similar to that of DA.30 The structural similarity
between tyramine and 4′OH APP (2) suggests that 2
should also be a good substrate for bVMAT. However,

Chromaffin Vesicular Monoamine Transporter Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2601
F igu r e 2. The kinetics of inhibition of DA uptake into resealed chromaffin granule ghosts by 4′OH-APP: Ghosts were resealed
to contain 20 mM Tris phosphate, pH 7.0, 100 mM KCl, 150 mM sucrose, 10 mM fumarate, and 100 µg/mL catalase and incubated
in a medium containing 0.3 M sucrose, 10 mM HEPES, 5 mM Mg-ATP, 5 mM MgSO4, and 100 µg/mL catalase at 30 °C for 10 min
(0.5 mL total volume; final pH 7.0). Then, the appropriate concentration of 4′OH APP (50 µM-400 µM) was added to the incubation
mixtures, and they were allowed to incubate further for 2 min. The uptake reaction was initiated by the addition of DA to the
respective incubation mixture to give a final DA concentration of 10, 15, 25, 50, 100, and 200 µΜ. After 6 min, 400 µL aliquots
of each incubate were withdrawn, and internal DA levels were quantified by HPLC-EC as detailed in the Experimental Section.
(A) double reciprocal plot of DA uptake inhibition by (0) 0 µM; (4) 50 µM; (2) 100 µM; (O) 200 µM, and (b) 400 µM external 4′OH
APP; (B) re-plot of the slope versus [4′OH APP].
Ta ble 1. Inhibition Kinetic Parameters of APP Derivatives for
these derivatives were actively taken up into the
resealed granule ghosts.
bVMATa
The kinetic data presented in Table 1 clearly dem-
onstrate that the APP derivatives tested behave as
competitive inhibitors for bVMAT with respect to DA.
These data (Table 1) also demonstrate that the inhibi-
tion potencies of APP derivatives are dependent on the
APP derivatives
Ki (µM)
Km,DA/Kib
ring, side chain, and N-substituents. As mentioned
above, while both 3′ and 4′-OH groups contribute
positively toward the inhibition potency, 4′- and 3′-OMe
substitutions (4 and 5) did not improve the inhibition
potency with respect to the parent compound 1 signifi-
cantly. In fact, while 4′-OMe substitution had a negative
effect on the inhibition, relative to the parent compound
(1), 3′-OMe substitution only slightly improved the
inhibition potency, again suggesting the distinct nature
of the effects of 3′- and 4′-substituents. More notably,
the substitution of halogens on the 4′-position of the
phenyl ring exhibited an interesting effect on the
inhibition potency. While F substitution on this position
had a minimal effect on the inhibition potency, Cl, Br,
and I substitutions gradually increased the inhibition
potency, yielding 4′-iodo derivative 9 as one of the most
potent inhibitors of the series with a Ki of 12.9 ( 2.3
µM (Km,DA/Ki ∼ 3). Substitution of a Me-group on the
4′-position of the aromatic ring (10) had only a small
effect on the inhibition potency. Substitution of a Me
group on the primary amine functionality of 4′OH APP
(11) reduced the inhibition potency by a factor of ∼6.
Similarly, the substitution of the primary amine group
with N,N-diethyl (12) or N,N,N-triethyl (13) groups
decreased the inhibition potency similar to that of
N-methyl substitution. The weak inhibition potency of
the N,N,N-triethyl (13) and pyridine (14) derivatives
suggest that a permanent positive charge at nitrogen
does not improve the inhibition potency significantly.
In addition, substitution of a Me group in the R-carbon
to yield a derivative structurally similar to amphet-
amines (15, 16) decreased the inhibition potency sig-
nificantly. Finally, substitution of a bulky Br-group (17)
on the double bond had only a small effect on the
inhibition potency.
1
2
3
4
5
6
7
8
APP
4′OH APP
3′OH APP
4′OMe APP
3′OMe APP
4′F APP
4′Cl APP
4′Br APP
40.3 ( 5.3
15.5 ( 0.9
16.7 ( 1.1
102.5 ( 8.1c
30.2 ( 1.5
42.3 ( 3.1c
18.0 ( 0.9c
17.7 ( 2.0
12.9 ( 2.3
55.9 ( 4.4c
93.7 ( 11.8
202 ( 42c
d
0.57
1.52
1.40
0.33
0.76
0.80
1.76
1.79
2.96
0.59
0.39
0.15
-
9
4′I APP
4′ Me APP
10
11
12
13
14
15
16
17
18
19
N-Me 4′OH APP
N,N-diethyl 4′OH APP
N,N,N-trimethyl APP
phenylpyridyl methyl ethane
3-Me APP
N,N-diethyl 3-Me 4′OH APP
4′OH 1-Br(E) APP
4′OMe 1-Br (E) APP
2′-Me, 4′OH APP
241 ( 84c
78.4 ( 24.6c
207 ( 37c
50.0 ( 6.1c
d
0.12
0.41
0.15
0.46
d
a
Ki, inhibition constants for DA uptake were determined in
resealed chromaffin granule ghosts under standard uptake condi-
tions as detailed in the Experimental Section. The Ki values were
obtained by fitting the experimental data (typically, obtained using
four to five different inhibitor, and six different DA concentrations;
for further details see Figure 2 legend) to the Cleland’s COMP
program.33 All inhibitions were competitive with respect to DA.
b
The Km,DA/Ki ratios were calculated from the respective Ki and
K
m,DA parameters determined for each compound to normalize the
variation of Km for DA for difference ghost preparations. c Deter-
mined by fitting the experimental data (obtained with 0 and 200
µM inhibitor and six different DA concentrations) to the standard
competitive inhibition equation using Sigmaplot Cambridge Soft-
d
ware Corp.). Screening experiments show that the inhibition
potencies are significantly less than those reported in the table.
not alter the inhibition potency significantly (Table 1),
suggesting that both 3′- and 4′- ring hydroxyl groups of
the inhibitor may play a similar but distinct role in the
interaction with the transporter. In addition, the ap-
propriate control experiments revealed that none of

2602 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Perera et al.
inhibition of bVMAT. As shown in Figure 3, the dihedral
angles C7-C3-C2-N1 of the optimized structures
(RHF/6-31G*) of 4′OH APP (note that numbering of
atoms is different from that of Table 1) and tyramine,
in protonated forms, are similar and are 54.17° and
54.62°, respectively. However, the close inspection of the
torsional barriers calculated for the dihedral angle C7-
C3-C2-N1 of these two compounds show that the in
plane configuration of C10, C7, and N1 (i.e., C7-C3-
C2-N1 dihedral angle is 180°) in 4′OH APP is about
1.9 kcal/mol more unfavorable in comparison to tyramine.
Therefore, if such a planer conformation is critical at a
high energy state of the transport process then, APP
may not be efficiently transported due to the relatively
high rotational barrier. Further studies are currently
underway in our laboratory to test the above model for
the transport of biogenic amines through bVMAT.
F igu r e 3. Energy-optimized structures of [4′OH APP],
tyramine, MPP+: ab initio calculations were carried out using
the Gaussian 98-Revision A.9 suite of programs.31 The molec-
ular geometries were optimized using restricted Hartree-Fock
method employing 6-31G* basic set. Energy-optimized struc-
tures of (A) 4′OH APP; (B) tyramine; (C) MPP+. Color code:
C: gray; H: light blue; O: red; N: dark blue.
Con clu sion s
The above results demonstrate that although the
bVMAT inhibition potencies of the APP derivatives are
weaker than the classical inhibitors such as reserpine,
tetrabenazine, and ketanserin, they are kinetically fully
characterizable and possess affinities similar to that of
DA and most other pharmacologically active mono-
amines. Therefore, they are good candidates for the
structure-activity, mechanistic, and pharmacological
studies of monoamine transporters. Based on the struc-
ture-activity relationships of a number of APP deriva-
tives, a testable molecular model for the transport vs
inhibition of the transporter is proposed. In addition,
due to the remarkable structural similarities between
APP and amphetamine derivatives, we believe that they
may also possess interesting pharmacological properties
and may be useful in modeling the mechanism of action
of amphetamine-related derivatives.
The structure-activity profile of the APP derivatives
tested (Table 1) shows that the hydroxyl and large
halogens, especially Br and I at the 4′ position of the
aromatic ring, enhanced the inhibition potency signifi-
cantly. Although, the effect of the 4′-OH could be due
to H-bonding with a critical residue in the cytosolic
phase of the transporter, as previously proposed for
bVMAT substrates, the gradual increase of the inhibi-
tion potency from F to I in the 4′-halogen-substituted
series indicates that the inhibition potency may increase
with the electron donor ability of the halogen. Clearly,
the effect of halogen substitution could not be due to
the increase in the hydrophobicity, since 4′-methyl
substitution decreased the inhibition potency noticeably.
The experimental data also suggest that the NH2 group
makes an important contribution to the interaction with
the transporter probably through H-bonding and/or ionic
interactions, and the bulky substituents on nitrogen or
at the R-C are not tolerated.
Apart from the monoamines, the only other known
substrate for VMAT is the neurotoxin MPP+.18,19 Since
MPP+ is structurally rigid and significantly different
from the common monoamines, several key structural
parameters that are critical for bVMAT substrates could
be derived from its structure (Figure 3C). For example,
(a) the permanent positive charge on the nitrogen of
MPP+ indicates that the protonated form of the mono-
amine may be the substrate for bVMAT; (b) the rela-
tively bulky pyridine ring in place of the ethylamine side
chain of the monoamines suggests that the steric bulk
at the benzylic carbon does not affect the substrate
activity significantly; and (c) since 3-phenyl derivative
of MPP+ is not a substrate for bVMAT [Wimalasena,
D. S.; Perera, R. P., Wimalasena, K. unpublished results
(manuscript in preparation)], the planer arrangement
of the C10, C7 of the aromatic ring with the N1 nitrogen
atom of the pyridine ring (Figure 3C) may be critical
for the transport through bVMAT. On the basis of this
evidence, we propose that the conformationally re-
stricted mobility of the side chain of APP derivatives,
in comparison to the corresponding phenyethylamines
such as tyramine, may play a critical role in the
Exp er im en ta l Section
VMAT In h ibition Kin etics. All inhibition kinetic experi-
ments were carried out using properly characterized resealed
bovine chromaffin granule ghosts as previously described
under standard uptake conditions30 (further details of these
procedures will be published elsewhere). Briefly, the washed
granule membranes were resealed to contain 20 mM Tris
phosphate, 100 mM KCl, 150 mM sucrose, 10 mM fumarate,
and 100 µg/mL catalase, and with no ascorbate (pH 7.0).
Resealed granule ghosts were purified by a discontinued 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
was added to the mixture and allowed to incubate further for
2 min. Following the incubation period, the uptake reaction
was initiated by the addition of the desired concentration of
DA and, at 6 min time intervals, 400 µL aliquots of the
incubate were withdrawn and diluted into 5.0 mL of ice-cold
0.4 M sucrose, 10 mM HEPES, pH 7.0, and stored at 0 °C until
the incubation period was completed. 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, 20 µL of the acidic extracts were
analyzed for catecholamines by reversed phase HPLC-EC. In

Chromaffin Vesicular Monoamine Transporter Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2603
standard turnover experiments, the granules were resealed
to contain 20 mM ascorbic acid and incubated as above except
that 20 mM ascorbic acid was included in the external
incubation medium.
Syn th esis. All 1H and 13C NMR spectra were recorded with
a Varian Inova 400 MHz spectrometer (Varian), and mass
spectra were obtained from a Finnigan LCQ-Deca ion trap
mass spectrometer (Thermoquest Corporation, San J ose, CA).
All reagents and solvents were obtained from various com-
mercial sources with the highest purity available and used
without further purification. Trimethylsilane (for organic
solvents) or 3-(trimethylsilyl)propionic acid sodium salt (for
D2O) were used as internal standards for NMR.
Com p u ta tion a l Ca lcu la tion s. Ab initio calculations were
carried out using the Gaussian 98-Revision A.9 suite of
programs.31 The rotational barriers were calculated by using
the built-in scan mode. The dihedral angles C7-C3-C2-N1
(see Figure 3A,B) were changed by 5° increments, the geom-
etries were completely optimized, and the energies were
calculated.
3-Am in o-2-(4′-flu or op h en yl)p r op en e‚HCl (6). Mp 168-
169 °C; 1H NMR (D2O) δ 3.97 (s, 2H), 5.32 (s, 1H), 5.52 (s,
1H), 7.07 (t, 2H), 7.40 (d, d, 2H); 13C NMR (D2O) δ 42.9, 115.7,
116.0, 117.5, 128.3, 128.4, 133.4, 139.7, 161.3, 164.5; MS (ESI),
m/z 152.3 (M+). Anal. (C9H11ClFN) C, H, N.
3-Am in o-2-(4′-ch lor op h en yl)p r op en e‚HCl (7). Mp 178-
178 °C; 1H NMR (D2O) δ 3.98 (s, 2H) 5.36 (s, 1H), 5.58 (s, 1H),
7.37 (s, 4H); 13C NMR (D2O) d 45.2, 120.5, 130.4, 131.6, 136.7,
138.30, 142.1; MS (ESI), m/z 168.3 (M+). Anal. (C9H11Cl2N) C,
H, N.
3-Am in o-2-(4′-br om op h en yl)p r op en e‚HCl (8). Mp 183-
185 °C; 1H NMR (D2O) δ3.98 (s, 2H), 5.37 (s, 1H), 5.60 (s, 1H),
7.30 (d, 2H), 7.52 (d, 2H); 13C NMR (D2O) d 45.0, 120.4, 124.7,
130.5, 134.4, 138.7, 142.0; MS (ESI), m/z 214.2 (M+). Anal.
(C9H11BrClN) C, H, N.
3-Am in o-2-(4′-iod op h en yl)p r op en e‚HCl (9). Mp 208-
210 °C; 1H NMR (D2O) δ 4.08 (s, 2H), 5.46 (s, 1H), 5.70 (s,
1H), 7.28 (d, 2H), 7.82 (d, 2H);13C NMR (D2O) d 45.5, 97.2,
121.0, 131.2, 139.8, 141.1, 142.8; MS (ESI), m/z 260.0 (M+).
Anal. (C9H11ClIN) C, H, N.
3-Am in o-2-p h en ylp r op en e‚HCl (1). Mp 178-179 °C (lit.
178-179 °C);26 1H NMR (D2O) δ 7.47-6.95 (m, 5 H), 5.73 (s, 1
H), 5.54 (s, 1 H), 4.13 (s, 2 H); 13C NMR (D2O) δ 44.8, 119.5,
128.4, 131.1, 131.2, 139.2, 142.6; MS (ESI) m/z 134.2 (M+).
3-Am in o-2-(4′-h yd r oxyp h en yl)p r op en e‚HCl (2). This
was synthesized by the method of Padgette et al.26 Yield 17.9%;
mp 173-175 °C (lit. 175 °C);26 1H NMR (D2O) δ 7.3 (d, 2 H),
6.81 (d, 2 H), 5.41 (s, 1 H), 5.18 (s, 1 H), 3.86 (s, 2 H); 13C
NMR (D2O) δ 45.4, 118.3, 118.4, 130.5, 132.0, 142.5, 158.8;
MS (ESI) m/z 149 (M+).
3-Am in o-2-(3′-h yd r oxyp h en yl)p r op en e‚HCl (3). This
was also synthesized by using the same procedure as (2). Yield
31%; mp 144-145 °C (lit. 143-145 °C);26 1H NMR (D2O) δ
6.86-7.61 (m, 4 H), 5.73 (s, 1 H), 5.54 (s, 1 H), 4.13 (s, 2 H);
13C NMR (D2O) δ 45.3, 115.8, 118.5, 120.2, 121.1, 133.1, 141.5,
142.8, 158.6; MS (ESI) m/z 149 (M+).
3-Am in o-2-(4′-m eth oxyp h en yl)p r op en e‚HCl (4). An eth-
anol solution of N-[2-(4′-acetoxyphenyl)-2-propenyl]phthal-
imide was treated with 1 M NaOH until the pH of the solution
was about 10 and stirred for 15 min. The resultant solution
was acidified to pH 8 using concentrated HCl, and ethanol was
removed under reduced pressure. The resultant product was
extracted into CHCl3 and concentrated under reduced pressure
to yield a white solid which was dissolved in DMF and treated
with CH3I in the presence of K2CO3 to give N-[2-(4′-methoxy)-
2-propenyl]phthalimide. This was purified by column chro-
matography using 10% ethyl acetate/hexane, and the phthal-
imide group was removed as described by McDonald et al.24
to yield 3-amino-2-(4′-methoxy)propene. Yield: 18.6%; mp
162-164 °C; 1H NMR (D2O) δ 7.31 (d, 2H), 7.01 (d, 2H), 5.68
(s, 1H), 5.45 (s, 1H), 4.13 (s, 2H), 3.86 (s, 3H); 13C NMR (D2O)
δ 45.3, 58.1, 114.8, 117.4, 131.4, 131.7, 140.0, 162.7; MS (ESI)
m/z 149 (M+). Anal. (C10H14ClNO) C, H, N.
2-(3′-Meth oxyp h en yl)p r op en e. A solution of n-BuLi (18
mL, 0.05 mol in hexane) was added dropwise to a solution of
triphenylphosphonium bromide (14.3 g, 0.04 mol) in 25 mL of
DMSO. The solution was stirred for 1 h at RT, and 3-methoxy-
acetophenone (6 g, 0.04 mol) in 10 mL of DMSO was added
dropwise. The mixture was stirred overnight and quenched
with water, and the product was extracted with hexane. The
concentrated hexane extract was purified by column chroma-
tography using hexane as a solvent. Yield 89%; 1H NMR
(CDCl3) δ 7.60 (m, 2 H), 7.27(d, d 1 H), 6.98 (d, 1 H) 5.35(s, 1
H), 5.09 (s, 1 H), 2.21 (s, 3 H), 2.12 (s, 3 H).
3-Am in o-2-(4′-m eth ylph en yl)pr open e‚HCl (10). Mp 170-
173 °C (lit. 170-173 °C);28 1H NMR (D2O) δ 2.25 (s, 3H) 3.97
(s, 2H), 5.28 (s, 1H), 5.53 (s, 1H), 7.18 (d, 2H), 7.29 (d, 2H);
13C NMR (D2O) d 22.9, 45.3, 119.1, 128.8, 132.3, 136.8, 142.1,
142.9; MS (ESI), m/z 148.1 (M+). Anal. (C10H14NCl) C, H, N.
3-(N -Me t h yla m in e )-2-(4′-h yd r oxyp h e n yl)p r op e n e ‚
HCl (11). A solution of 4′-acetoxy-phenyl R- (bromomethyl)-
styrene (1 g, 3.9 mmol) in THF (10 mL) was added dropwise
to a solution of methylamine (2 M solution, 5 mL, 0.001 mol)
in THF in the presence of NaHCO3 (0.2 g) and stirred for 5 h
under nitrogen. The resulting solution was filtered and
concentrated under reduced pressure. The HCl salt of the
product was recrystalized from ethanol/ether. Yield 35.9%; mp
1
149-150 °C; H NMR (D2O) δ 7.3 (d, 2 H), 6.81 (d, 2 H), 5.41
(s, 1 H), 5.18 (s, 1 H), 3.86 (s, 2 H); 13C NMR (D2O) δ 48.4,
52.1, 116.7, 121.2, 122.4, 130.5, 132.2, 143.1, 158.2; MS (ESI)
m/z 164.4 (M+). Anal. (C10H14ClNO) C, H, N.
3-(N,N-Diet h yla m in o)-2-(4′-h yd r oxyp h en yl)p r op en e‚
HCl (12). This was synthesized by the same procedure
described for 11, except that diethylamine was used in place
of methylamine. Yield 32.4%; mp 182-185 °C; 1H NMR (D2O)
δ 7.41 (d, 2H), 6.95(d, 2H), 5.66 (s, 1H), 5.58 (s, 1H), 4.68 s,
2H), 3.27 (q, 4H), 1.21 (t, 6H); 13C NMR (D2O) δ 15.0, 59.2,
66.9, 119.1, 123.7, 131.6, 133.8, 146.1, 159. 7; MS (ESI) m/z
206.3 (M+). Anal. (C13H20ClNO) C, H, N.
3 -(N ,N ,N -T r i m e t h y l a m i n o )-2 -p h e n y l -p r o p e n e ‚
HBr (13). This was synthesized by the same procedure used
for 11, except that triethylamine was used in place of methyl-
amine. Yield 60%; mp 123-124 °C; 1H NMR (D2O) δ 8.46-
8.48 (m, 2 H), 8.37-8.39 (m, 3 H), 6.85 (s, 1 H), 6.67 (s, 1 H),
5.37 (s, 2 H), 3.90 (s, 9H); 13C NMR (D2O) δ 56.0, 71.42, 129.2,
131.5, 131.9, 139.9, 141.6; MS (ESI) m/z 176.1 (M+). Anal.
(C12H18BrN) C, H, N.
1-(2-P h en yl-2-p r op en yl)p yr id in iu m Br om id e (14). This
was synthesized by the same procedure used for 11, except
that pyridine was used in place of methylamine. Mp 115-116
°C (lit. 115-116);28 1H NMR (D2O) δ 5.68 (s, 1H), 5.81 (s, 2H),
5.86 (s, 1H), 7.42-7.48 (m, 3H), 7.54-7.58 (m, 2H), 8.04 (t,
2H), 8.51 (t, 1H), 8.95 (d, 2H); 13C NMR (D2O) δ 66.1, 123.9,
128.9, 130.7, 131.6, 131.6, 138.3, 143.0, 146.6, 148.9; MS (ESI),
m/z 196.1(M+).
2-Br om o-1-p h en yl-1-p r op a n on e. This was prepared by
the method of King and Ostrum29 in 82% yield. 1H NMR
(CDCl3) δ 1.88 (d, 2H), 5.29 (q, 1H), 7.44 (m, 3H), 8.11 (d, 2H).
The following compounds 5-10 were synthesized from the
corresponding R-methylstyrene derivatives which were ob-
tained by the general Wittig reaction as described for 2-(3′-
methoxyphenyl)propene using the standard procedure.
3-Am in o-2-(3′-m eth oxyph en yl)pr open e‚HCl (5). Mp 158-
160 °C; 1H NMR (D2O) δ 6.86-7.61 (m, 4 H), 5.73 (s, 1 H),
5.54 (s, 1 H), 4.13 (s, 2 H); 13C NMR (D2O) δ 45.3, 58.1, 114.8,
117, 120.6, 121.8, 133.0, 141.5, 142.9, 161.9; MS (ESI) m/z 149
(M+). Anal. (C10H14ClNO) C, H, N.
2-P h th alim ido-1-ph en ylpr opan on e. A solution of 2-bromo-
1-phenyl-1-propanone (3 g, 0.01 mol) in 10 mL of DMF,
potassium phthalimide (1.85 g, 0.01 mol) and K2CO3 (0.3 g)
were mixed and heated to 90 °C under N2 for 1 h and an
additional 1 h at RT and filtered. The filtrate was concentrated
under vacuum. The solid product was recrystalized from
CHCl3/hexane. Yield 56%; 1H NMR (CDCl3) δ 1.74 (d, 3H), 5.62
(q, 1H), 7.41 (m, 3H), 7.71 (dd, 2H), 7.96 (m, 4H).

2604 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Perera et al.
3-P h th a lim id o 2-p h en ylbu ten e. This was synthesized
using the same procedure as for 1-(3′-methoxyphenyl)propene.
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1
Yield 75%; H NMR (CDCl3) δ 1.24 (d, 3H), 5.01 (s, 1H), 5.05
(q, 1H), 5.12 (s, 1H), 7.25 (m, 3H), 7.5 (dd, 2H), 7.61-7.72 (m,
4H).
3-Am in o-2-p h en ylb u t en e‚HCl (15). Hydrazinolysis of
3-phthalimido-2-phenylbutene gave the desired product. Yield
59%; mp 177-179 °C; 1H NMR (D2O) δ 1.45 (d, 3H), 4.548 (q,
1H), 5.39 (s, 1H), 5.54 (s, 1H), 7.48 (s, 5H); 13C NMR (D2O) δ
20. 7, 52.4, 117.3, 129.8, 131.6, 131.8, 141.3, 149.4; MS (ESI)
m/z (%) 148.1 (M+). Anal. (C10H14ClN) H, N, C: calc, 65.39;
found, 64.03.
2-Br om o-1-(4′-h yd r oxyp h en yl)p r op a n on e. This was pre-
pared by the method of King and Ostrum.32 Yield 66%; 1H
NMR (DMSO-d6) δ 10.12 (broad, 1H), 7.89 (d, 2H), 6.85 (d,
2H), 5.65 (q, 2H), 1.66 d, 3H).
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2-(N,N-Diet h yla m in e)-1-(4′-h yd r oxyp h en yl)p r op a n -
on e. 2-Bromo-1-(4-hydroxyphenyl)propanone (2 g, 8.6 mmol)
was dissolved in 20 mL of THF and diethylamine (1.2 mL, 8.6
mmol) was added dropwise. The solution was stirred for 2 h,
and the unreacted amine was removed under reduced pressure
to yield a light brown liquid. Yield 86%; H NMR (DMSO-d6)
δ 10.32 (broad, 1H), 7.88 (d, 2H), 6.88 (d, 2H), 5.69 (q, 1H),
2.94 (q, 4H), 1.79 (d, 2H) 1.08 (t, 6H).
3-(N,N-Diet h yla m in e)(4′-h yd r oxyp h en yl)b u t en e‚H Cl
(16). This was synthesized from 2-(N,N-diethylamine)-1-(4-
hydroxyphenyl)propanone using the general Wittig reaction
as described for 1-(3-methoxyphenyl)propene. Yield 16.8%; mp
186 °C; 1H NMR (D2O) δ 7.28 (d, 2H), 6.82 (d, 2H), 5.49 (s,
1H), 5.41 (s, 1H), 4.52 (q, 1H), 3.06 (q, 4H), 1.41 (d, 3H), 1.07
(t, 6H); 13C NMR (D2O) δ 57.2, 62.1, 118.9, 122.7, 131.5, 133.7,
146.1, 159.2; MS ESI m/z 220.2 (M+). Anal. (C14H22NOCl) C,
H, N.
1
(E)-2-(4′-Hyd r oxyp h en yl)-3-br om oa llyla m in e‚HCl (17).
This was prepared by the method of McDonald et al.24 starting
from 2-(4′-acetoxyphenyl)propene. Yield 16.3%; mp 187-189
1
°C; H NMR (D2O) δ 7.41 (d, 2H), 6.95 (d, 2H), 6.82 (s, 1H),
4.08 (s, 2H); 13C NMR (D2O) δ 45.39, 113.72, 119.21, 119.43,
130.54, 132.23, 143.12, 159.15; MS (ESI) m/z 228 (M+). Anal.
(C9H11BrClNO) C, H,N.
(E)-2-(4-Meth oxyp h en yl)-3-br om oa llyla m in e‚HCl (18).
The NBS bromination of 4-methoxy-R-methylstyrene exclu-
sively produced the corresponding dibromide in 81% yield. This
was converted to the corresponding phthalimide, and the
desired amine was obtained by usual hydrazinolysis. Yield
67%; mp 190-192; °C 1H NMR (D2O) δ 7.42 (d, 2H), 7.03 (d,
2H), 6.82 (s, 1H), 4.28 (s, 2H), 3.86 (s, 3H); 13C NMR (D2O) δ
43.2, 58.1, 113.7, 117.3, 131.0, 131.5, 139.9, 162.30; MS (ESI)
m/z 242.1 (M+).
3-Am in o-2-(2′-m eth yl-4′-h yd r oxyp h en yl)p r op en e‚HCl
(19). This was synthesized by the same procedure as (3)
starting from 4′-hydroxy-2′-methyl acetophenone. Yield 32%;
1
mp 190-192 °C H NMR (D2O) δ 7.13 (d, 2H), 6.83-6.78 (m,
3H), 5.58 (s, 1H), 5.24 (s, 1H), 3.89 (s, 2H), 2.27 (s, 3H); 13C
NMR (D2O) δ 21.7, 47.1, 115.5, 119.9, 121.6, 132.8, 133.1,
140.5, 143.2, 158.0; MS (ESI) m/z 164.2 (M+).
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (NS 39423).
Ap p en d ix
Abbreviations: APP, 3-amino-2-phenylpropene; ATP,
adenosine triphosphate; bVMAT, bovine chromaffin
granule vesicular monoamine transporter(s); DA, dopam-
ine; HEPES, 4-(2-hydroxyethyl)-1-piperazinesulfonic
acid; m-DâM, membranous dopamine â-monooxygenase;
MPP+, 1-methyl-4-phenylpyridinium; NBS, N-bromo-
succinimide; NE, norepinephrine; s-DâM, soluble dopam-
ine â-monooxygenase; VMAT1, human vesicular mono-
amine transporter-1; VMAT2, human vesicular mono-
amine transporter-2.
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