Synthesis and biological evaluation of meperidine analogues at monoamine transporters

Article abstract of DOI:10.1021/jm0401614

A series of aryl-substituted meperidine analogues was synthesized, and the binding affinities were determined at the DAT, SERT, and NET as well as at μ-opioid receptors. Generally the analogues exhibited increased affinity for the DAT and SERT relative to

Full text of DOI:10.1021/jm0401614

1336
J. Med. Chem. 2005, 48, 1336-1343
Synthesis and Biological Evaluation of Meperidine Analogues at Monoamine
Transporters
Stacey A. Lomenzo,† Jill B. Rhoden,† Sari Izenwasser,‡ Dean Wade,‡ Theresa Kopajtic,§ Jonathan L. Katz,§ and
Mark L. Trudell†,*
Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148, Department of Psychiatry and
Behavioral Sciences, University of Miami School of Medicine, Miami, Florida 33136, and Psychobiology Section, National
Institute on Drug Abuse, Intramural Research Program, 5500 Nathan Shock Drive, Baltimore, Maryland 21224
Received September 2, 2004
A series of aryl-substituted meperidine analogues was synthesized, and the binding affinities
were determined at the DAT, SERT, and NET as well as at µ-opioid receptors. Generally the
analogues exhibited increased affinity for the DAT and SERT relative to meperidine but
exhibited low binding affinity for the NET. The 2-naphthyl derivative 7f was the most potent
ligand at the SERT (Ki ) 0.0072 µM) and was the most selective ligand for the SERT over the
DAT (DAT/SERT ) 158) and µ-opioid receptors (µ/SERT ) 281). The 3,4-dichlorophenyl
derivative 7e was the most potent ligand at the DAT (Ki ) 0.125 µM) and was the most selective
ligand for the DAT over µ-opioid receptors (µ/DAT ) 16.3) but remained slightly more selective
for the SERT over the DAT(DAT/SERT ) 6.68). Three compounds, the 3,4-dichlorophenyl
derivative 7e and the 2-naphthyl analogues 6f and 7f, were identified that were more potent
at the DAT than meperidine and that exhibited well-defined biphasic dopamine uptake
inhibition similar to meperidine. However, none of the analogues tested produced locomotor
effects or substituted for cocaine in drug discrimination studies, suggesting that the µ-opioid
effects of these analogues may contribute to the poor efficacy observed in vivo.
Introduction
Cocaine (1) binding at the dopamine transporter
(DAT) has been well established.1-11 Initially, specific
binding sites were identified in rodent brain that bind
[3H]cocaine at a single saturable site.1,2 Subsequent
studies using rodent,3 human,4 and nonhuman pri-
mate5,6 brain tissue preparations (striatum and caudate
putamen) later provided evidence that suggests that
cocaine binding at the dopamine transporter is hetero-
geneous. Two distinct binding sites on the dopamine
transporter have been proposedsa high affinity and a
low affinity site.3-6,10,13 These sites are termed high and
low based upon the relative affinity of cocaine for these
sites. The phenyltropane analogues of cocaine, specifi-
cally WIN 35,428 (2a), WIN 35,065-2 (2b), and WIN 35,-
981 (2c), in addition to several other cocaine-like
compounds, also have binding curves that are better fit
with a two-site than a one-site model.10,13 However, it
is uncertain if these compounds interact at the high and
low affinity site in a fashion similar to that of cocaine.
It may be possible for compounds other than cocaine to
have low affinity for the high affinity site and vice versa.
Alternatively, DAT inhibitors such as mazindol13,14 and
GBR 1290915,16 exhibit only single site affinity for the
DAT.
logues, the correlation between affinity for the dopamine
transporter and potency can depend on whether high-
or low-affinity sites are examined. For example, in
comparison of DAT binding affinity with the cocaine-
like discriminative-stimulus (subjective) effects, the
correlation between substitution potencies for cocaine
analogues and DAT affinity is greater for the high-
affinity constants than for the corresponding low-affinity
constants.19
Currently there are no known dopamine uptake
inhibitors that discriminate between high-affinity and
low-affinity sites on the dopamine transporter. However,
it has been reported that meperidine (3, an atypical
µ-opioid agonist with some stimulant effects) is a potent
inhibitor of [3H]dopamine uptake when examined in a
chopped tissue rather than synaptosomal preparation.20
Further, the concentration response curve exhibited a
plateau at approximately 20% inhibition over a broad
range of low concentrations of meperidine. This high-
affinity component to the curve resembled the high-
affinity component of the inhibition of dopamine uptake
produced by cocaine. The maximal inhibition of dop-
amine uptake produced by meperidine at low concentra-
tions (20%) was also consistent with the amount of total
binding of [3H]WIN 35,428 (2a) that is attributable to
high-affinity sites (18%).6,10 In addition, when the opioid-
A strong correlation between binding affinity at the
dopamine transporter (DAT) and potency for reinforcing
effects among cocaine (1) and its analogues has been
reported.17,18 For other effects of cocaine and its ana-
* To whom correspondence should be addressed. Phone: (504)-280-
7337. Fax: (504) 280-6860. E-mail: mtrudell@uno.edu.
† University of New Orleans.
‡ University of Miami School of Medicine.
§ National Institute on Drug Abuse.
10.1021/jm0401614 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/10/2005

Meperidine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1337
Scheme 1a
[3H]paroxetine, and at NET by inhibition of [3H]nisoxe-
tine binding as previously described.24,25 In addition, the
inhibition of [3H]dopamine uptake was determined for
each compound in the same manner as that originally
described for meperidine.20 Moreover, µ-opioid receptor
binding affinities were also determined using a [3H]-
DAMGO binding assay.26 The results of these in vitro
binding studies are summarized in Table 1.
a Reagents and conditions: (a) BrCH2CH(OCH3)2, NaNH2,
toluene. (b) 3 N HCl, 50 °C, 4.5 h. (c) CH3NH2‚HCl, CH3OH,
NaBH3CN. (d) H2SO4/H2O 120 °C; then EtOH, azeotropic distil-
lation.
In general, the esters 7 were significantly more potent
than the corresponding nitriles 6 at the DAT, SERT,
and NET. Like meperidine, all of the analogues exhib-
ited a greater affinity for the SERT than for the DAT.
The 2′-naphthyl analogue 7f (Ki ) 0.0072 µM) was the
most potent ligand of the series at the SERT and was
59-fold more potent than meperidine (3). The 4-iodo-
phenyl derivative 7c (Ki ) 0.021 µM) and the 3,4-
dichlorophenyl derivative 7e (Ki ) 0.019 µM) also
exhibited low nanomolar affinities for the SERT. The
increased SERT affinity of the 4-iodophenyl- 7c (20-fold)
and 2′-naphthyl- 7f (60-fold) derivatives relative to 3
followed a similar trend observed for the SAR of the
phenyltropane analogue, WIN 35,065-2 (2b). The 3â-
(4-iodophenyl) derivative 2d27 and 3â-(2′-naphthyl)
analogue 828 exhibited a 390-fold and a 2500-fold,
respectively, increase in potency at SERT relative to 2b.
The DAT/SERT ratios (Table 1) illustrate that the
meperidine analogues exhibited 3- to 158-times greater
affinity for SERT than for DAT. The high selectivity of
the 4-iodophenyl analogue 7c (DAT/SERT ) 155) was
unexpected since DAT/SERT selectivity is modest for
the corresponding 3â-(4-iodophenyl)tropane 2d DAT/
SERT ) 3.0).27 In addition, the high selectivity of the
2′-naphthyl analogue 7f (DAT/SERT ) 158) was quite
surprising since the SAR previously described for the
3â-(2-naphthyl)tropane derivative 8 and the correspond-
ing 2â-acyl analogue 9 have been reported to exhibit
moderate DAT selectivity.28,29 Moreover, the 3â-car-
bomethoxy-1-methyl-4â-(2′-naphthyl)piperidine (10) has
been reported to exhibit only slight SERT selectivity
(DAT/SERT ) 2.8).30
agonist actions of meperidine were blocked by naltrex-
one, meperidine substituted for cocaine in squirrel
monkeys trained to discriminate cocaine from saline.20
These results suggested that the high-affinity inhibition
of dopamine uptake by cocaine may underlie its subjec-
tive effects.
Upon the basis of these previous studies, it was of
interest to explore the structure-activity relationships
of a series of meperidine derivatives to develop a more
potent dopamine transporter ligand with selectivity for
the high-affinity cocaine binding site. Compounds that
allow a differentiation of high- and low-affinity actions
of cocaine would undoubtedly be significant pharmaco-
logical probes to explore the relationship between those
actions and the abuse liability of cocaine. In our
preliminary studies, a series of meperidine analogues
were prepared that exhibited increased affinity for
dopamine and serotonin transporters over that observed
for meperidine.21 Herein we report the synthesis and
biological evaluation of a series of meperidine analogues
at dopamine, serotonin, and norepinephrine transport-
ers as well as at µ-opioid receptors.
Results and Discussion
Chemistry. A general synthetic approach was devel-
oped that could be easily modified to explore a broad
spectrum of aryl-substituted derivatives of meperidine.
As illustrated in Scheme 1, the dialkylation of readily
available22 and commercially available 2-aryl acetoni-
triles 4 with bromoacetaldehyde dimethyl acetal af-
forded the corresponding diacetals 5 in good yields (75-
90%). Hydrolysis of the acetals in 3 N hydrochloric acid
generated the corresponding dialdehydes which upon
concomitant reductive amination with methylamine
hydrochloride and sodium cyanoborohydride in metha-
nol gave the N-methyl-4-aryl-4-piperidine-4-carboni-
triles 6 in moderate overall yields (26-45%) for the two-
step process. Careful control of the reaction temperature
of the acetal hydrolysis proved to be critical for reason-
able yields of the desired piperidines. Hydrolysis reac-
tion temperatures that exceeded 50 °C typically gave
intractable mixtures of pyran derivatives. The nitriles
6 were then converted into the corresponding ethyl
esters 7 via a two-step sequence that furnished the ethyl
esters 7 in good overall yields (52-76%).
The SAR of the meperidine analogues at the DAT
exhibited a similar trend to that of the 3â-phenyltro-
panes, albeit the meperidine derivatives were 160-4000
times less potent. Nevertheless, substitution of the
phenyl ring with halogen or a methyl group affected the
DAT binding affinity of the meperidine derivatives in a
fashion similar to substitution on the phenyl ring of 2b
(Ki or IC50: H > F > CH3, > Cl ≈ I > 3,4-Cl2).31,32 The
3,4-dichlorophenyl derivative 7e (DAT Ki ) 0.125 µM)
was found to be the most potent ligand of the series for
the DAT and was over 100-fold more potent than
meperidine (3). In addition, 7e was the least selective
of the ester analogues for the SERT (DAT/SERT ) 6.68).
From the SAR of the meperidine derivatives, it is clear
the 3,4-dichlorophenyl group is an important moiety for
molecular recognition at the DAT. Similar effects of the
3,4-dichlorophenyl group on DAT affinity have been
Biology. The transporter binding affinities were
determined for the meperidine analogues 6 and 7 by
their ability to displace bound radiolabeled ligands from
rat caudate-putamen tissue.23 The Ki values that are
reported in Table 1 are inhibition constants derived for
the unlabeled ligands. The binding affinities of the
meperidine analogues were determined at DAT by
inhibition of [3H]WIN 35,428, at SERT by inhibition of

1338 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Lomenzo et al.
Table 1. In Vitro Data for Meperidine Analogues
[3H]paroxetine [3H]nisoxetine
[3H]DA uptake
inhibition
[3H]WIN 35,428
(SERT)
(NET)
[3H]DAMGO
DAT/
SERT µ/DAT µ/SERT
compd
Ara
(DAT) Ki (µM)b
Ki (µM)b
Ki (µM)b
Ki (µM)b
IC50 (µM)b,c
1
32 ( 5d
405 ( 91d
388 ( 221d
17.8 ( 2.7
3
Ph
0.413 ( 0.044
0.92
0.61 ( 2.2 nM (22%)f 43.1
0.052
2.13
12.6 ( 1.2
6a
6b
6c
6d
6e
6f
4-F-Ph
4-Cl-Ph
4-I-Ph
45% @ 100 µMc 10.1 ( 0.4
22.0 ( 10.1
8.34 ( 0.67
41.8 ( 6.1
(101.4)e
(15)c
(8)c
5.11 ( 0.59
0.430 ( 0.034
13.7 ( 0.4
0.805 ( 0.12
0.125 ( 0.022
(106.2)e
(93.6)e
(102.9)e
(94.1)e
(88.1)e
36.8 (51)c
17.3 (61)c
40.6 (46)c
40.0 (46)c
15.4 (61)c
(36)c
4.31
19.4
3.05
3.32
1.67
2.07
0.97
15.0
6.53
0.18
40.2
2.96
49.7
123
36.7 ( 1.3
4-Me-Ph
(22)c
3,4-Cl2-Ph 2.67 ( 0.24
2-naphthyl 2.36 ( 0.66
11.1 ( 1.2
0.37 ( 2.0 nM (11%)f 18.9
21.8 ( 1.2
7a
7b
7c
7d
7e
4-F-Ph
4-Cl-Ph
4-I-Ph
10.7 ( 2.3
4.10 ( 1.27
3.25 ( 0.20
12.4 ( 5.2
0.308 ( 0.026
0.277 ( 0.040
(71.9)e
601.2 ( 45.9
1.47
4.41
2.35
2.67
2.04
(47)c
34.7
14.8
155
0.14
1.08
0.72
0.22
16.3
4.77
15.9
111
1.66
109
26.9 ( 1.2
11.1 ( 1.2
76.2 ( 1.2
0.0211 ( 0.0024 519.3 ( 51.1
4-Me-Ph
1.61 ( 0.11
(57.6)e
7.69
3,4-Cl2-Ph 0.125 ( 0.015
0.0187 ( 0.0026 74.5 ( 5.1
6.01 ( 6.26 nM (22%)f 6.68
1.40 ( 1.25
7f
2-naphthyl 1.14 ( 0.38
0.0072 ( 0.0001 71.1 ( 9.7
2.03
3.19 ( 7.41 nM (16%)f 158
11.6 ( 1.3
1.78
282
a All compounds were tested as the HCl salt. b All values are the mean ( SEM of three experiments performed in triplicate. c Percent
inhibition at highest dose tested (100 µM). d The Ki and IC50 values are reproduced from ref 39 and were collected under identical conditions.
e Percent specific binding at highest dose tested (10 µM). f Percent of total uptake inhibition.
a nonlinear model of the data. The graphs do not show
this model, but show the actual data points connected
by lines. Each of the compounds that modeled better
for two sites than for one site had a high-affinity uptake
plateau component of approximately 20% of the total
inhibition, which is similar to both meperidine and
cocaine. This is consistent with the percentage (18%) of
the total binding that corresponds to the high-affinity
component of WIN 35 428 (2a).6,10 This suggests that
modification of the phenyl group of 3 can increase DAT
binding affinity with the retention of an ability to
differentiate between the high-affinity and low-affinity
components of the inhibition of DA uptake. Of the
phenyl substitutions examined, the 3,4-dichlorophenyl
(7e) and the 2-naphthyl (7f) groups were identified as
important substructures for differentiating between the
high- and low-affinity components, while significantly
increasing binding affinity relative to meperidine. Al-
ternatively, the 4-chlorophenyl and 4-iodophenyl deriva-
tives 7b and 7c exhibited increased DAT binding
affinity relative to 3, but were unable to discriminate
the between the high- and low-affinity components.
Figure 1. Meperidine-like biphasic dopamine uptake inhibi-
tion of meperidine derivatives 7e and 7f.
observed for other tropane derivatives,32-34 non-nitrogen
tropane analogues,35,36 and piperidine derivatives.37
Although, the 3,4-dichlorophenyl group also increased
the binding affinity of 7e for the SERT, the overall effect
was only 15% of that observed for the DAT.
All of the meperidine nitrile analogues 6a-f prepared
in this study exhibited significantly diminished µ-opioid
receptor affinity relative to meperidine (3). The ester
analogues 7a-f exhibited only slightly reduced affinity
for µ-opioid receptors relative to 3, but all of the
analogues exhibited a marked increase in DAT and
SERT selectivity over µ-opioid receptors compared to
meperidine. The 3,4-dichlorophenyl analogue 7e (µ/DAT
) 16) was found to exhibit the greatest selectivity for
the DAT over that of µ-opioid receptors (Table 1) while
the 2-naphthyl analogue 7f (µ/SERT ) 282) exhibited
the greatest selectivity for the SERT over the µ-opioid
receptors (Table 1).
Selected compounds 7c, 7e, and 7f were evaluated for
their locomotor-stimulant activity in mice (Figure 2)
using experimental conditions previously reported.24
Upon the basis of the high selectivity of these com-
pounds for the dopamine and serotonin transporters, it
was believed initially that any behavioral effects medi-
None of the meperidine analogues 7a-f exhibited
significant affinity for the NET. The meperidine ester
analogues 7b, 7c, 7e, and 7f exhibited only low micro-
molar affinity for the NET, while all the other analogues
exhibited only partial, if any, inhibition of [3H]nisoxetine
at the highest dose tested (10 µM).
All of the meperidine derivatives exhibited weak
inhibition of dopamine uptake (Table 1). The dopamine
uptake inhibition produced by most of the meperidine
compounds modeled better for a single site (e.g. 7c,
Figure 1). However, several of the compounds (6f, 7e,
7f) modeled better for two sites, a high-affinity site and
a low-affinity site, similar to that observed for meperi-
dine. As illustrated in Figure 1, the esters, 7e and 7f
exhibited biphasic dopamine uptake inhibition with a
high-affinity uptake plateau similar to that for meperi-
dine (3) of approximately 20% of the total inhibition.20
The percent of total inhibition of uptake that is ac-
counted for by the high affinity component is based upon

Meperidine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1339
Figure 2. Dose-dependent effects of 7c, 7e, and 7f on
locomotor activity in mice. Ordinates: horizontal locomotor
activity counts after drug administration in counts per second.
Abscissae: dose of drug in µmol/kg, log scale. Each point
represents the average effect determined in six mice. The data
are from a 30-min period starting 30 min after drug admin-
istration. Note that none of the compounds produced a
stimulation of activity.
ated by µ-opioid receptors would be diminished relative
to the cocaine-like effects mediated by the transporters.
Naive mice were injected ip and locomotor activity
(horizontal activity counts) was assessed immediately
after injection for a 1-h period. Each data point is a
mean of eight subjects. None of the compounds tested
produced locomotor stimulant effects similar to cocaine.
Instead each of the compounds decreased locomotor
activity across a range of doses from those having no
effects to those that virtually eliminated activity.
The compounds 7c (SERT selective) and 7e (DAT
selective) also were tested in rats trained to discriminate
injections of cocaine from saline. Rats were trained to
discriminate ip injections of cocaine (10 mg/kg) from
saline injections until reliable performances were main-
tained according to procedures previously outlined.24
Compounds were then tested for their effectiveness in
substituting for cocaine immediately after injection
during sessions that lasted for a maximum of 20 min.
Each data point is typically a mean of six subjects;
however, lever selection data was excluded if more than
half of the subjects tested did not respond. This only
occurred at the highest doses tested. Neither compound
7c nor 7e substituted for cocaine in rats trained to
discriminate between cocaine and saline (Figure 3). In
addition, efficacy observed for these compounds closely
resembled that of meperidne (3). The low efficacy
observed for 7c and 7e may be due to their µ-opioid
activity, which may not be as diminished as originally
envisaged. It is unlikely that actions at the SERT
interfered with the discriminative stimulus effect as
these effects do not do so for cocaine and other drugs
that have affinity for both the DAT and SERT.38
Figure 3. Effects of 7c and 7e in rats trained to discriminate
injections of cocaine from saline. Ordinates for top panels:
percentage of responses on the cocaine-appropriate key. Or-
dinates for the bottom panels: rates at which responses were
emitted (as a percentage of response rate after saline admin-
istration). Abscissa: drug dose in µmol/kg (log scale). Each
point represents the effect in six to nine rats. The percentage
of responses emitted on the cocaine-appropriate key was
considered unreliable, and not plotted, if fewer than half of
the subjects responded at that dose. Note that neither com-
pound substituted for cocaine.
3,4-dichlorophenyl derivative 7e was the most potent
ligand at the DAT and was the most selective ligand
for the DAT over µ-opioid receptors but remained
slightly more selective for the SERT over the DAT.
Three compounds, the 3,4-dichlorophenyl derivative 7e
and the 2-naphthyl analogues 6f and 7f, were identified
that were more potent at the DAT than meperidine and
that exhibited dopamine uptake inhibition curves that
resembled the biphasic dopamine uptake inhibition of
meperidine. However, none of the analogues tested
produced locomotor effects or substituted for cocaine in
drug discrimination studies, suggesting that the µ-opioid
effects may still be significant. While the monoamine
transporter affinity SAR of these meperidine analogues
is somewhat reminiscent of the 3â-phenyltropanes, it
is apparent from the dopamine uptake inhibition that
the 3,4-dichlorophenyl and 2-naphthyl moieties are
important in the meperidine system for differentiating
between the high- and low-affinity components of this
effect as well as between the DAT and the SERT.
Additional studies directed toward the development of
DAT and SERT selective meperidine derivatives with
diminished µ-opioid receptor affinity are underway and
will be reported in due course.
In summary, a series of aryl-substituted meperidine
analogues were synthesized and the binding affinities
determined at the DAT, SERT, and NET as well as at
µ-opioid receptors. Generally the analogues exhibited
increased affinity for the DAT and SERT relative to
meperidine but exhibited low binding affinity for the
NET. The 2-naphthyl derivative 7f was the most potent
ligand at the SERT and was the most selective ligand
for the SERT over the DAT and µ-opioid receptors. The
Experimental Section
All chemicals were purchased from Aldrich Chemical Co.,
Milwaukee, WI, unless otherwise noted. THF, CH2Cl2, and
MeOH were dried under the argon system. Toluene and Et2O

1340 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Lomenzo et al.
were dried by distillation over Na/benzophenone. Chromatog-
raphy refers to column chromatography on silica gel (Silica
Gel 60, 230-400 mesh). Petroleum ether refers to pentanes
with a boiling point range of 30-60 C. Reported melting points
are uncorrected. NMR spectra were recorded on a Varian-
Gemini 400 MHz spectrometer. Chemical shifts are reported
as δ values with tetramethylsilane (TMS), employed as the
internal standard. Elemental analyses were obtained from
Atlantic Microlabs, Inc., Norcross, GA.
General Method A. Preparation of Hydrochloride
Salts. Some of the compounds were converted into the
hydrochloride salts for biological testing as well as for storage
and handling purposes. The base (100 mg) was dissolved in
the minimum amount of Et2O (1-2 mL) and added to a
saturated ethereal solution (10 mL) of anhydrous hydrogen
chloride. The hydrochloride salts crystallized and were washed
with Et2O (3 × 2 mL) and purified by tritration with Et2O and
ethyl acetate. Fractional moles of water could not be prevented
despite vigorous drying (110 °C, 1h) under vacuum (0.01
mmHg). All compounds were homogeneous by thin-layer
chromatography (CHCl3:MeOH:NH4OH, 90:9:1).
NMR: δ 7.74 (d, J ) 8.8 Hz, 2H), 7.22 (d, J ) 8.4 Hz, 2H),
4.27 (m, J ) 4.0 Hz, 2H), 3.32 (s, 6H), 3.17 (s, 6H), 2.40 (dd, J
) 7.2, 14.4 Hz, 2H), 2.13 (dd, J ) 4.4, 14.6 Hz, 2H). 13C NMR:
δ 137.8, 137.6, 127.8, 120.7, 101.7, 93.3, 53.5, 53.1, 43.2, 41.3.
Anal. (C16H22NO4I) C, H, N.
2-(4-Methylphenyl)-2-(2,2-dimethoxyethyl)-4,4-
dimethoxybutyronitrile (5d). General Method B. This
compound was obtained as an orange oil (1.3 g, 80%). 1H
NMR: δ 7.34 (d, J ) 8.4 Hz, 2H), 7.20 (d, J ) 8.4 Hz, 2H),
4.26 (m, 2H), 3.31 (s, 6H), 3.17 (s, 6H), 2.40 (dd, J ) 6.8, 14.4
Hz, 2H), 2.36 (s, 3H), 2.14 (dd, J ) 3.6, 14.4 Hz, 2H). 13C
NMR: δ 137.7, 134.8, 129.6, 125.8, 121.4, 102.1, 53.7, 53.0,
43.6, 41.6, 20.9. Anal. (C17H25NO4) C, H, N.
2-(3,4-Dichlorophenyl)-2-(2,2-dimethoxyethyl)-4,4-
dimethoxybutyronitrile (5e). General Method B. This com-
pound was obtained as an orange oil (1.2 g, 81%). 1H NMR: δ
7.56 (d, J ) 2.4 Hz, 1H), 7.49 (d, J ) 8.4 Hz, 1H), 7.31 (dd, J
) 2.4, 8.4 Hz, 1H), 4.30 (m, J ) 4.4 Hz, 2H), 3.33 (s, 6H), 3.19
(s, 6H), 2.40 (dd, J ) 6.4, 14.4 Hz, 2H), 2.13 (dd, J ) 4.4, 14.4
Hz, 2H). 13C NMR: δ 138.3, 133.0, 132.1, 130.6, 128.1, 125.4,
120.4, 101.8, 53.6, 53.3, 43.3, 41.5. Anal. (C16H21NO4Cl2) C,
H, N.
2-(2,2-Dimethoxyethyl)-4,4-dimethoxy-2-naphthalen-2-
yl-butyronitrile (5f). General Method B. This compound was
obtained as an orange oil (1.5 g, 82%). 1H NMR: δ 8.05 (s,
1H), 7.87 (m, 3H), 7.52 (m, 3H), 4.28 (dd, J ) 4.0, 7.0 Hz, 2H),
3.31 (s, 6H), 3.13 (s, 6H), 2.50 (dd, J ) 7.2, 14.2 Hz, 2H), 2.26
(dd, J ) 4.0, 14.6 Hz, 2H). 13C NMR: δ 135.0, 133.1, 132.6,
128.9, 128.2, 127.5, 126.7, 126.6, 125.6, 122.9, 121.3, 102.1,
53.7, 53.2, 43.5, 42.1. Anal. (C20H25NO4) C, H, N.
General Method C: 4-Aryl-1-methylpiperidine-4-car-
bonitrile (6). To a 250 mL three-neck round-bottom flask of
3 N HCl (120 mL) at 50 °C was added the diacetal 5 (7.5
mmol), and the mixture was allowed to stir overnight. The acid
mixture was allowed to cool to room temperature and then
extracted with Et2O (300 mL). The ether layer was washed
with NaHCO3 (100 mL) and dried (Na2SO4). The Et2O was
removed under reduced pressure. The resulting residue was
dissolved in dry MeOH (37 mL), the N-methylamine hydro-
chloride (7.5 mmol) was added followed by NaBH3CN (6.7
mmol), and the mixture was allowed to stir 48 h under an
atmosphere of nitrogen. The solvent was removed under
reduced pressure. The resulting residue was treated with
saturated NaHCO3 (120 mL) and extracted with Et2O (3 ×
100 mL). The combined organic fractions were dried (Na2SO4),
and the solvent was removed under reduced pressure. The
resulting crude product was purified by chromatography
(CHCl3:MeOH:NH4OH, 90:9:1) to afford the 4-arylpiperidine-
4-carbonitriles 6.
4-(4-Fluorophenyl)-1-methylpiperidine-4-carboni-
trile (6a). General Method C. This compound was obtained
as an orange solid (0.63 g, 39%) and converted into a
hydrochloride salt (General Method A), which was obtained
as a white solid, mp 215-216 °C (HCl salt). 1H NMR (free
base): δ 7.47 (m, 2H), 7.09 (m, 2H), 2.96 (m, 2H), 2.45 (m,
2H), 2.39 (s, 3H), 2.09 (m, 4H). 13C NMR (free base): δ 162.2
(JC-F ) 246 Hz), 135.9, 127.3, 121.7, 115.8, 52.6, 45.9, 41.6,
36.7. Anal. (C13H15N2F‚HCl) C, H, N.
4-(4-Chlorophenyl)-1-methylpiperidine-4-carboni-
trile (6b). General Method C. This compound was obtained
as an orange solid (0.46 g, 26%) and converted into a
hydrochloride salt (General Method A), which was obtained
as a white solid, mp 211-212 °C. 1H NMR (free base): δ 7.44
(d, J ) 7.6 Hz, 2H), 7.37 (d, J ) 9.2 Hz, 2H), 2.96 (m, 2H),
2.48 (m, 2H), 2.38 (s, 3H), 2.09 (m, 4H). 13C NMR (free base):
δ 138.6, 133.9, 129.1, 126.9, 121.4, 52.5, 45.9, 41.7, 36.4. Anal.
(C13H15N2Cl‚HCl) C, H, N.
4-(4-Iodophenyl)-1-methylpiperidine-4-carbonitrile (6c).
General Method C. This compound was obtained as a yellow
oil (0.76 g, 31%). The compound was converted into a hydro-
chloride salt (General Method A), which was obtained as a
white solid, mp 267-270 °C. 1H NMR (free base): δ 7.73 (d, J
) 8.8 Hz, 2H), 7.25 (d, J ) 8.4 Hz, 2H), 2.97 (d, J ) 14.8 Hz,
2H), 2.48 (m, 2H), 2.39 (s, 3H), 2.09 (m, 4H). 13C NMR (free
(4-Iodophenyl)acetonitrile (4c).22 A mixture of KCN (2.7
g, 41 mmol) and deionized water (4.0 mL) was heated in an
oil bath to dissolve the KCN. A solution of the 4-iodobenzyl
bromide (9.8 g, 33 mmol) in MeOH (190 mL) was added to the
reaction flask via addition funnel and allowed to stir at reflux
for 4 h. The reaction mixture was allowed to cool to room
temperature. The salts were filtered, and the filtrate was
partially concentrated under reduced pressure. The newly
formed salts were then filtered, and the remaining filtrate was
concentrated under reduced pressure. The resulting residue
was purified by bulb-to-bulb distillation. The impurities were
collected from 80 to 100 °C, and the product was collected from
100-120 °C. This compound was obtained as a white solid (4.8
1
g, 60% yield). mp 56-58 °C [lit.:22 mp 56-57C]. H NMR: δ
7.71 (d, J ) 8.4 Hz, 2H), 7.09 (d, J ) 8.4 Hz, 2H), 3.70 (s, 2H).
13C NMR: δ 138.1, 129.7, 129.5, 117.2, 93.5, 23.2. 1H NMR: δ
7.73-7.68 (m, 2H), 7.10-7.05 (m, 2H), 3.70 (s, 2H). 13C NMR:
δ 138.2, 129.7, 129.5, 117.2, 93.5, 23.2.
General Method B: 2-Aryl-2-(2,2-dimethoxyethyl)-4,4-
dimethoxybutyronitrile (5). To a round-bottom flask of
arylacetonitrile 4 (5.4 mmol) and bromoacetaldehyde dimethyl
acetal (1.6 mL, 13 mmol) in dry toluene (7 mL) under an
atmosphere of nitrogen was added portionwise (2 to 4 × 1 mL)
a suspension of NaNH2 (50wt % in toluene). The reaction was
allowed to stir at reflux for 15 min before each portion of
NaNH2 was added. This was repeated until the monosubsti-
tuted product was no longer visible by TLC (Et2O/hexanes,
1:1). The reaction mixture was cooled to room temperature and
quenched with water (20 mL). The aqueous layer was ex-
tracted with ether (4 × 25 mL). The combined organic fractions
were dried (Na2SO4) and filtered. The solvent was removed
under reduced pressure, and the crude product was purified
by flash column chromatography using an elution gradient of
25% Et2O/hexanes to 50% Et2O/hexanes to afford the diacetals
5.
2-(4-Fluorophenyl)-2-(2,2-dimethoxyethyl)-4,4-
dimethoxybutyronitrile (5a). General Method B. This
compound was obtained as an orange oil (1.2 g, 66%). 1H
NMR: δ 7.44 (m, 2H), 7.10 (m, 2H), 4.27 (dd, J ) 4.8, 4.2 Hz,
2H), 3.23 (s, 6H), 3.17 (s, 6H), 2.41 (dd, J ) 14.2, 6.6, 2H),
2.14 (dd, J ) 14.4, 4.0, 2H). 13C NMR: δ 162.0 (JC-F ) 246
Hz), 133.6, 127.7, 121.1, 115.7, 101.9, 53.6, 53.1, 43.7, 41.4.
Anal. (C16H22NO4F) C, H, N.
2-(4-Chlorophenyl)-2-(2,2-dimethoxy-ethyl)-4,4-
dimethoxybutyronitrile (5b). General Method B. This
compound was obtained as an orange solid (1.2 g, 66%). 1H
NMR: δ 7.40 (m, 4H), 3.32 (s, 6H), 3.17 (s, 6H), 2.40 (dd, J )
6.8, 14.4 Hz, 2H), 2.13 (dd, J ) 4.0, 14.4 Hz, 2H). 13C NMR:
δ 136.4, 133.7, 128.9, 127.4, 120.8, 101.9, 53.5, 53.1, 43.4, 41.5.
Anal. (C16H22NO4Cl) C, H, N.
2-(4-Iodophenyl)-2-(2,2-dimethoxyethyl)-4,4-dimethoxy-
butyronitrile (5c). General Method B. The compound was
obtained as an orange solid (1.7 g, 77%), mp 73-75 °C. 1H

Meperidine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1341
base): δ 139.8, 138.1, 127.5, 121.4, 93.7, 52.6, 46.0, 42.0, 36.4.
Anal. (C13H15N2I‚HCl) C, H, N.
as a white solid, mp 247-249 °C. 1H NMR (free base): δ 7.65
(d, J ) 8.8 Hz, 2H), 7.13 (d, J ) 8.4 Hz, 2H), 4.12 (m, J ) 7.2
Hz, 2H), 2.78 (d, J ) 8.4 Hz, 2H), 2.54 (d, J ) 13.2 Hz, 2H),
2.27 (s, 3H), 2.14 (t, J ) 10.8 Hz, 2H), 1.94 (t, J ) 10.4 Hz,
2H), 1.18 (t, J ) 7.6 Hz, 3H). 13C NMR (free base): δ 173.7,
137.5, 127.8, 92.7, 60.9, 53.2, 48.3, 46.1, 33.6, 14.0. Anal.
(C15H20NO2I‚HCl) C, H, N.
4-(4-Methylphenyl)-1-methylpiperidine-4-carboxylic
Acid Ethyl Ester (7d). General Method D. This compound
was obtained as a white solid (0.75.g, 52%) and converted into
a hydrochloride salt (General Method A), which was obtained
as a white solid, mp 196-197 °C (HCl salt). 1H NMR (free
base): δ 7.27 (d, J ) 8.0 Hz, 2H), 7.12 (d, J ) 8.0 Hz, 2H),
4.11 (q, J ) 7.2 Hz, 2H), 2.78 (br s, 2H), 2.57 (d, J ) 13.2 Hz,
2H), 2.30 (s, 3H), 2.26 (s, 3H), 2.13 (t, J ) 11.2 Hz, 2H), 1.96
(t, J ) 11.2 Hz, 2H), 1.17 (t, J ) 7.2 Hz, 3H). 13C NMR (free
base): δ 174.3, 136.4, 129.1, 125.5, 60.6, 53.4, 48.2, 46.1, 33.8,
20.7, 13.9. Anal. (C16H23NO2‚HCl) C, H, N.
4-(3,4-Dichlorophenyl)-1-methylpiperidine-4-carboxy-
lic Acid Ethyl Ester (7e). General Method D. This compound
was obtained as a white solid (1.3 g, 76%) and converted into
a hydrochloride salt (General Method A), which was obtained
as a white solid, mp 195-196 °C. 1H NMR (free base): δ 7.47
(d, J ) 2.0 Hz, 1H), 7.40 (d, J ) 8.8 Hz, 1H), 7.23 (dd, J ) 2.4,
8.4 Hz, 1H), 4.14 (m, J ) 6.8 Hz, 2H), 2.80 (d, J ) 8.4 Hz,
2H), 2.55 (d, J ) 12.8 Hz, 2H), 2.28 (s, 3H), 2.16 (t, J ) 11.6
Hz, 2H), 1.94 (m, J ) 11.2 Hz, 2H), 1.20 (t, J ) 6.8 Hz, 3H).
13C NMR (free base): δ 173.4, 132.6, 131.2, 130.4, 128.2, 125.4,
61.2, 53.2, 48.2, 46.0, 33.6, 14.0. Anal. (C15H19NO2Cl2‚HCl) C,
H, N.
1-Methyl-4-naphthalen-2-yl-piperidine-4-carboxylic
Acid Ethyl Ester (7f). General Method D. This compound was
obtained as a white solid (1.1 g, 73%) and converted into a
hydrochloride salt (General Method A), which was obtained
as a white solid, mp 191-193 °C. 1H NMR (free base): δ 7.80
(m, 4H), 7.55 (dd, J ) 2.0, 8.6 Hz, 1H), 7.44 (m, 2H), 4.12 (m,
J ) 6.8 Hz, 2H), 2.83 (d, J ) 9.2 Hz, 2H), 2.70 (d, J ) 12.4 Hz,
2H), 2.28 (s, 3H), 2.21 (t, J ) 11.6 Hz, 2H), 2.10 (m, 2H), 1.16
(t, J ) 6.8 Hz, 3H). 13C NMR (free base): δ 174.3, 133.3, 132.3,
128.1,128.0, 127.3, 126.0, 125.9, 124.6, 124.0, 60.8, 53.5, 48.7,
46.2, 33.9, 14.0. Anal. (C19H23NO2‚HCl) C, H, N.
[3H]WIN 35,428 Binding Assay. Male Sprague-Dawley
rats (200-250 g, Taconic, Germantown, NY) were decapitated
and their brains removed to an ice-cooled dish for dissection
of the caudate-putamen. The tissue was homogenized in 30
volumes of ice-cold modified Krebs-HEPES buffer (15 mM
HEPES, 127 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2.5 mM
CaCl2, 1.3 mM NaH2PO4, 10 mM glucose, pH adjusted to 7.4)
using a Teflon/glass homogenizer and centrifuged at 20 000g
for 10 min at 4 °C. The resulting pellet was then washed two
more times by resuspension in ice-cold buffer and centrifuga-
tion at 20,000g for 10 min at 4 °C. Fresh homogenates were
used in all experiments. Binding assays were conducted in
modified Krebs-HEPES buffer on ice, essentially as previously
described.23 The total volume in each tube was 0.5 mL, and
the final concentration of membrane after all additions was
approximately 0.3% (w/v) corresponding to 150-300 g of
protein/sample. Increasing concentrations of the drug being
tested were added to triplicate samples of membrane suspen-
sion. Five minutes later, [3H]WIN 35 428 (final concentration
1.5 nM) was added and the incubation was continued for 1 h
on ice. The incubation was terminated by the addition of 3
mL of ice-cold buffer and rapid filtration through Whatman
GF/B glass fiber filter paper (presoaked in 0.1% BSA in water
to reduce nonspecific binding) using a Brandel Cell Harvester
(Gaithersburg, MD). After filtration, the filters were washed
with three additional 3 mL washes and transferred to scintil-
lation vials. Absolute ethanol (0.5 mL) and Beckman Ready
Value Scintillation Cocktail (2.75 mL) were added to the vials
which were counted the next day at an efficiency of about 36%.
Under these assay conditions, an average experiment yielded
approximately 6000 dpm total binding per sample and ap-
proximately 250 dpm nonspecific binding. Nonspecific binding
was defined as binding in the presence of 100 µM cocaine. Ki
4-(4-Methylphenyl)-1-methylpiperidine-4-carboni-
trile (6d). General Method C. This compound was obtained
as an oil (0.63 g, 39%) and converted into a hydrochloride salt
(General Method A), which was obtained as a white solid, mp
198-200 °C (HCl salt). 1H NMR (free base): δ 7.38 (d, J ) 8.4
Hz, 2H), 7.20 (d, J ) 8.4 Hz, 2H), 2.96 (m, 2H), 2.48 (m, 2H),
2.38 (s, 3H), 2.35 (s, 3H), 2.10 (m, 4H). 13C NMR (free base):
δ 137.8, 137.2, 129.6, 125.4, 122.0, 52.7, 46.0, 41.7, 36.6, 20.8.
Anal. (C14H18N2‚HCl‚1/3H2O) C, H, N.
4-(3,4-Dichlorophenyl)-1-methylpiperidine-4-carboni-
trile (6e). General Method C. This compound was obtained
as an orange oil (0.79 g, 39%) and converted into a hydro-
chloride salt (General Method A), which was obtained as a
white solid, mp 266-268 °C. 1H NMR (free base): δ 7.56 (d, J
) 2.0 Hz, 1H), 7.45 (d, J ) 8.4 Hz, 1H), 7.31 (dd, J ) 2.4, 8.4
Hz, 1H), 2.95 (m, 2H), 2.46 (m, 2H), 2.36 (s, 3H), 2.07 (m, 4H).
13C NMR (free base): δ 140.2, 133.2, 132.4, 130.9, 127.9, 125.0,
121.0, 52.4, 45.8, 41.6, 36.3. Anal. (C13H14N2Cl2‚HCl‚1/4 H2O)
C, H, N.
1-Methyl-4-naphthalen-2-yl-piperidine-4-carboni-
trile (6f). General Method C. This compound was obtained
as an orange oil (1.0 g, 45%). The compound was converted
into a hydrochloride salt (General Method A), which was
obtained as a white solid, mp 256-258 °C. 1H NMR (free
base): δ 7.97 (d, J ) 1.6 Hz, 1H), 7.85 (m, 3H), 7.57 (dd, J )
2.0, 8.4 Hz, 1H), 7.50 (m, 2H), 2.99 (d, J ) 12.0, 2H), 2.53 (m,
2H), 2.40 (s, 3H), 2.22 (m, 4H). 13C NMR (free base): δ 137.3,
133.1, 132.7, 128.9, 128.1, 127.5, 126.6, 126.5, 124.6, 123.3,
121.9, 52.7, 46.0, 42.3, 36.5. Anal. (C17H18N2‚HCl‚1/3H2O) C,
H, N.
General Method D. 4-Aryl-1-methylpiperidine-4-car-
boxylic Acid Ethyl Ester (7). The nitriles 6 (5.2 mmol) in
an aqueous solution of sulfuric acid (6.5 mL H2SO4:H2O, 1:1)
was heated in an oil bath at 120 °C for 1.5 h. The flask was
then equipped with a Dean-Stark trap with a stopcock. The
water was azeotropically removed by distillation with con-
tinual drainage of the solvent collected in the trap over 4.5 h,
and alcohol was added as needed. The reaction was then
heated at reflux overnight. The reaction was allowed to cool
to room temperature. The solvent was removed under reduced
pressure. The residue was then cooled in an ice bath, and the
acid was neutralized to a pH of 10 with 1 N NaOH. The
aqueous layer was extracted with Et2O (3 × 75 mL). The
combined organic fractions were dried (Na2SO4), and the
solvent was removed under reduced pressure. The crude
product was purified by chromatography (MeOH:CHCl3, 2.5:
97.5) to afford the esters 7.
4-(4-Fluorophenyl)-1-methylpiperidine-4-carboxylic
Acid Ethyl Ester (7a). General Method D. This compound
was obtained as a white solid (1.0 g, 75%) and converted into
a hydrochloride salt (General Method A), which was obtained
as a white solid, mp 137-138 °C (HCl salt). 1H NMR (free
base): δ 7.36 (m, 2H), 7.00 (m, 2H), 4.12 (q, J ) 6.8 Hz, 2H),
2.78 (br s, 2H), 2.57 (d, J ) 10.2 Hz, 2H), 2.26 (s, 3H), 2.13 (t,
J ) 10.8 Hz, 2H), 1.94 (m, 2H), 1.18 (t, J ) 7.2 Hz, 3H). 13C
NMR (free base): δ 174.1, 161.7 (JC-F ) 245 Hz), 138.7, 127.5,
115.3, 60.8, 53.4, 48.1, 46.2, 34.0, 14.0. Anal. (C15H20NO2F‚
HCl‚1/3H2O) C, H, N.
4-(4-Chlorophenyl)-1-methylpiperidine-4-carboxylic
Acid Ethyl Ester (7b). General Method D. This compound
was obtained as a white solid (1.0 g, 69%) and converted into
a hydrochloride salt (General Method A), which was obtained
as a white solid, mp 168-169 °C (HCl salt). 1H NMR (free
base): δ 7.31 (m, 4H), 4.12 (m, J ) 6.8 Hz, 2H), 2.77 (br s,
2H), 2.56 (d, J ) 12.8 Hz, 2H), 2.27 (s, 3H), 2.13 (t, J ) 11.2
Hz, 2H), 1.93 (m, J ) 10.4 Hz, 2H), 1.18 (t, J ) 7.2 Hz, 3H).
13C NMR (free base): δ 174.0, 141.5, 132.9, 128.6, 127.3, 60.9,
53.4, 48.3, 46.2, 33.9, 14.0. Anal. (C15H20NO2Cl‚HCl) C, H, N.
4-(4-Iodophenyl)-1-methylpiperidine-4-carboxylic Acid
Ethyl ester (7c). General Method D. This compound was
obtained as a white solid (1.3 g, 68%) and converted into a
hydrochloride salt (General Method A), which was obtained

1342 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5
Lomenzo et al.
values were derived from 14 point competition assays using
increasing concentrations of unlabeled compounds (0.05 nM
to 100 µM) against 1.5 nM [3H]WIN 35 428. Data were
analyzed with GraphPad Prism software (San Diego, Califor-
nia).
ip injections were given and the subjects were placed in
experimental chambers. The chambers contained two response
levers, a device for delivering food pellets, and stimulus lights.
Session start was indicated by illumination of the lights.
Subjects were trained to press the response levers, with
responses on one lever reinforced with food presentation after
a presession injection of cocaine and responses on the other
lever reinforced after an injection of saline. Over the course
of several sessions, the number of responses required for each
food pellet was gradually increased to 20. “Cocaine” and
“saline” sessions alternated in a sequence of double alterna-
tions. Sessions ended after 20 food presentations or 15 min,
whichever occurred first. Test sessions began once perfor-
mances met a criterion for stability (greater than 85% correct
responses prior to the first food presentation and throughout
the entire session). Test sessions were identical to training
sessions with the exception that 20 or 30 consecutive responses
on either lever were reinforced.
Locomotor Activity Studies. Subjects were experimen-
tally naive group-housed male Swiss Webster mice approxi-
mately 14 weeks old. They were studied in 40 cm3 clear acrylic
chambers which counted ambulatory activity with photoelec-
tric detectors placed 2.56 cm apart along the walls. One
activity count was registered at each instance in which the
subject crossed a beam. Multiple interruptions of the same
beam (e.g. grooming, head bobbing) were not counted. Mice
were injected and placed into the apparatus for 60 min. Each
dose was studied in six mice.
[3H]Dopamine Uptake Inhibition Studies: Rats were
sacrificed by decapitation and their brains removed to an ice-
cooled dish for dissection of the caudate-putamen. [3H]Dopam-
ine uptake was measured in a chopped tissue preparation as
described previously.39 Briefly, the tissue was chopped into 225
µm slices on a McIllwain tissue slicer with two successive cuts
at an angle of 90°. The strips of tissue were suspended in
oxygenated modified Krebs-HEPES buffer (see above), which
was pregassed with 95% O2/5% CO2 and warmed to 37 °C.
After rinsing, aliquots of tissue slice suspensions were incu-
bated in buffer in glass test tubes at 37 °C to which either the
drug being tested or no drug was added, as appropriate. After
a 5 min incubation period in the presence of drug, [3H]-
dopamine (final concentration 15 nM) was added to each tube.
After 5 min the incubation was terminated by the addition of
2 mL of ice-cold buffer to each tube and filtration under
reduced pressure over glass fiber filters (presoaked in 0.1%
polyethylenimine in water). The filters were rinsed and placed
in scintillation vials to which 1 mL methanol and 2 mL 0.2 M
HCl were added to extract the accumulated [3H]dopamine.
Radioactivity was determined by liquid scintillation spectrom-
etry at an efficiency of approximately 30%. The reported values
represent specific uptake from which nonspecific binding to
filters was subtracted. Data were analyzed with GraphPad
Prism software (San Diego, CA).
[3H]Paroxeting Binding Assay. Brains from male Spra-
gue-Dawley rats weighing 200-225 g (Taconic Labs) were
removed, and midbrain was dissected and rapidly frozen.
Membranes were prepared by homogenizing tissues in 20
volumes (w/v) of 50 mM Tris containing 120 mM NaCl and 5
mM KCl (pH 7.4 at 25 °C), using a Brinkman Polytron (setting
6 for 20 s) and centrifuged at 50,000g for 10 min at 4 °C. The
resulting pellet was resuspended in buffer, recentrifuged, and
resuspended in buffer to a concentration of 15 mg/mL. Ligand
binding experiments were conducted in assay tubes containing
4.0 mL buffer for 90 min at room temperature. Each tube
contained 0.2 nM [3H]paroxetine (NEN) and 1.5 mg midbrain
tissue (original wet weight). Nonspecific binding was deter-
mined using 1 µM citalopram. Incubations were terminated
by rapid filtration through Whatman GF/B filters, presoaked
in 0.05% polyethylenimine, using a Brandel R48 filtering
manifold (Brandel Instruments Gaithersburg, MD). The filters
were washed twice with 5 mL cold buffer and transferred to
scintillation vials. Beckman Ready Safe (3.0 mL) was added,
and the vials were counted the next day using a Beckman 6000
liquid scintillation counter (Beckman Coulter Instruments,
Fullerton, CA). Data were analyzed by using GraphPad Prism
software (San Diego, CA).
Acknowledgment. We are grateful to the National
Institute on Drug Abuse (DA11528) and NIDA-IRP for
the financial support of this research. The authors
would like to thank Bettye Campbell and Dawn French
for technical support, Patty Ballerstadt for administra-
tive and clerical support, and Dawn French for expert
data analysis.
Supporting Information Available: Microanalysis data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Meperidine Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 5 1343
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