Syntheses of 3-carbomethoxy-4-(aryl)piperidines and in vitro and in vivo pharmacological evaluation: Identification of inhibitors of the human dopamine transporter

Article abstract of DOI:10.1016/S0968-0896(02)00528-X

A series of 3-carbomethoxy-4-(aryl-substituted)piperidines with various aryl groups were synthesized and examined for binding and reuptake inhibition at the human dopamine transporter, the human serotonin transporter, and the human norepinephrine transpor

Full text of DOI:10.1016/S0968-0896(02)00528-X

Bioorganic & MedicinalChemistry 11 (2003) 775–780
Syntheses of 3-Carbomethoxy-4-(aryl)piperidines and In Vitro
and In Vivo Pharmacological Evaluation: Identification
of Inhibitors of the Human Dopamine Transporter
a
a
b
Xianqi Feng, Keith Fandrick, Robert Johnson,
b
a,
Aaron Janowsky and John R. Cashman *
aHuman BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA
bVeterans Administration Hospital, Portland, OR 97201, USA
Received 9 July 2002; accepted 16 September 2002
Abstract—A series of 3-carbomethoxy-4-(aryl-substituted)piperidines with various aryl groups were synthesized and examined for
binding and reuptake inhibition at the human dopamine transporter, the human serotonin transporter, and the human nor-
epinephrine transporter. The binding potency and reuptake inhibition efficacy was compared with that of (ꢀ)-cocaine to determine
the significance of removing the two-carbon bridge of the cocaine nucleus on the inhibition of transporter binding and reuptake. Of
the transporters examined, the substituted piperidines were relatively selective for the human dopamine transporter. In all cases
examined, the cis-diastereomer of the 3-carbomethoxy-4-(aryl-substituted)piperidine was observed to be a more potent inhibitor of
the human dopamine transporter than the trans diastereomer. Based on the K (binding) and IC (reuptake inhibition) values
i
50
0
obtained, the most potent inhibitor of the series was cis-3-carbomethoxy-4-(4 -chlorophenyl)piperidine, and this compound sup-
pressed spontaneous- and cocaine-induced stimulation in non-habituated male Swiss-Webster mice. The conclusion is that sub-
stantialportions of the cocaine structure can be dissected away to provide compounds with significant binding and reuptake
inhibition of the human dopamine transporter.
#
2002 Elsevier Science Ltd. All rights reserved.
Introduction
the pharmacological effects of cocaine, it is the dopa-
mine transporter (DAT), controlling the uptake of the
neurotransmitter dopamine (DA) that is most often
targeted in anti-cocaine abuse medications develop-
ment. One of the reinforcing (addictive) properties of
cocaine stems from the inhibition of binding to and the
re-uptake of DA, 5-HT and NE, into the pre-synaptic
neurons. In theory, development of compounds that
potently and selectively bind to the human DAT and do
not inhibit DA re-uptake or only modestly inhibit DA
re-uptake may be usefulas agents to antagonize or par-
tially antagonize, respectively, the pharmacological
properties of cocaine.
The plant alkaloid cocaine from the leaves of Erythroxylon
coca is a potent centralnervous stim ua lnt. Cocaine
abuse and addiction are serious medicalprob el ms in the
1
1
1
United States and toxicity due to cocaine overuse
includes cardiac arrest, stroke, grand-mal seizures and
2
drug-induced psychosis.
Development of therapies to combat cocaine addiction
and abuse is an important goal. While significant
3ꢀ6
advances in the use of enzymes
and catalytic anti-
ꢀ8
to protect against the toxicity of cocaine by
6
bodies
inactivation have been reported, small-molecule-based
approaches for medications development of cocaine
Using the structure of cocaine as a lead, molecular dis-
section of the aryltropane nucleus by removing the
2-carbon bridge showed that 3-carbomethoxy-4-(aryl-
substituted)piperidines possessed considerable human
DAT affinity and re-uptake inhibition potency. The
goalof our work was to examine the effect of aromatic
substitution on the binding and reuptake inhibition of
3-carbomethoxy-4-(aryl-substituted)piperidines at the
antagonists have dominated the field.9
ꢀ11
Although
compounds that interact with serotonin (5-HT) and
norepinephrine (NE) systems are involved in modulating
*
9
Corresponding author. Tel.: +1-858-458-9305; fax: +1-858-458-
311; e-mail: jcashman@hbri.org; URL: http://www.hbri.org
0
968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00528-X

776
X. Feng et al. / Bioorg. Med. Chem. 11 (2003) 775–780
Table 1. Inhibition of radioligand binding in HEK-hDAT, HEK-
a
hSERT and HEK-hNET cells by 3-carbomethoxy-4-(aryl)piperidines
HEK hDAT
(nM) SEM
HEK hSERT
(nM) SEM
HEK hNET
i
K (nM) SEM
Compd
K
i
K
i
Figure 1. Chemicalstructure of cocaine, compound 1 and chemical
structures of 3-carbomethoxy-4-(aryl)piperidines as cis and trans dia-
stereomers.
1
2
3
4
5
6
8
1
, Cocaine
272
98.88
356.21
339.14
2038.04
4615.01
>10 mM
ꢂ58
601
684.03
ꢂ132
830
2200.62 ꢂ544.54
5223.21 ꢂ2126.81 2279.26 ꢂ842.9
2247.74
ꢂ147
ꢂ6.42
ꢂ17.96
ꢂ26.13
ꢂ205.04
ꢂ330.72
2441.22 ꢂ140.85
ꢂ560.75 >10 mM
—
>10 mM
—
—
—
—
—
—
—
, 7
, 9
0
ꢂ894.44
1286.92
2518.3
ꢂ335.13 >10 mM
ꢂ688.62 >10 mM
human-DAT, human 5-HT transporter (hSERT) and
human NE transporter (hNET). In addition, the stereo-
selectivity of the process was examined. Highly active
compounds identified in vitro were also evaluated in
vivo for their ability to alter locomoter activity and
compete with the locomoter affects of a parallel dose of
cocaine.
—
5134.89 ꢂ1462.63 >10 mM
—
—
—
—
>10 mM
>10 mM
10 mM
11
1
1
>10 mM
2664.96
6830.92
—
ꢂ604.87
>10 mM
10 mM
2
3
ꢂ743.52 >10 mM
>10 mM
aDrug inhibition of [125I]-RTI-55 binding in HEK-hDAT, HEK-hSERT or
HEK-hNET cell membranes. Values represent the mean ꢂ SEM for three to
four experiments unless the mean of three experiments exceeded 10 mM.
Results and Discussion
Table 2. Inhibition of radiolabelled neurotransmitter uptake in
HEK-hDAT, HEK-hSERT and HEK-hNET cells by 3-carbo-
Molecular dissection of the 2-carbon bridge of cocaine,
compound 1, afforded 3-carbomethoxy-4-(benzoyloxy)-
piperidine (Fig. 1). Recognizing that replacement of the
benzoyloxy moiety with a phenyl group (as in the case
a
methoxy-4-(aryl)piperidines
HEK hDAT
HEK hSERT
HEK hNET
Compd
IC50 (nM) SEM
IC50 (nM) SEM
IC50 (nM) SEM
1
2
of aryltropanes ) in some cases significantly increased
the potency of binding to the hDAT and improved the
efficacy of inhibition of DA reuptake, a series of 3-carbo-
methoxy-4-(aryl-substituted)piperidines were stereo-
selectively synthesized (Fig. 1). The target compounds
were tested in binding and uptake inhibition assays in
the presence of hDAT, hSERT and hNET. The most
active compounds were also evaluated in vivo as inhibi-
tors of mouse locomoter activity.
1
2
3
4
5
6
, cocaine
271
151
325.2
339.9
446.2
4736.5
>10 mM
4657
>10 mM
1788.7
988.44
ꢂ56
243
544
3133
793
2868
687
437.6
2198
3891
3414
3025
ꢂ64
ꢂ208
ꢂ67
188
1841
2084
724
ꢂ48
ꢂ421
ꢂ75
ꢂ243
ꢂ387
—
ꢂ6.88
ꢂ63.9
ꢂ55.9
ꢂ125.7
ꢂ2048.7
—
ꢂ75
ꢂ931
ꢂ57
1127
, 7
>10 mM
>10 mM
1282
2706
4852
8, 9
ꢂ224.9
ꢂ758
ꢂ160
ꢂ591
ꢂ313
—
10
11
12
ꢂ2171
ꢂ262
ꢂ894
ꢂ1852
—
—
ꢂ580.7
ꢂ465.0
13
>10 mM
a
3
3
3
Inhibition of [ H]-DA, [ H]-5HT or [ H]-NE in the presence of HEK-hDAT,
HEK-hSERT or HEK-hNET cells, respectively. Values are the mean ꢂ SEM for
three to four experiments unless the mean of three experiments exceeded 10 mM.
The 3-carbomethoxy-4-(aryl) piperidines were prepared
by combining arecoline free base to the appropriate
ꢁ
Scheme 1). The addition of the aryl group followed by
arylmagnesium bromide in ether/CH Cl at ꢀ40
C
2
2
(
treatment of the resulting product with trifluoroacetic
greater binding affinity for the hDAT. For the hSERT
and hNET, inhibition of radiolabelled RTI-55 binding
was not significantly more potent than that of cocaine.
Inhibition of neurotransmitter uptake by compound 2
was significantly greater than inhibition by cocaine.
However, compounds 3–12 showed little or no
improved efficacy for inhibition of 5-HT or NE reup-
take in HEK cells transfected with their corresponding
human transporters, as compared with cocaine.
ꢁ
acid in ether at ꢀ78 C and extractive work up provided
a mixture of diastereomeric pairs. Using this procedure,
generally, a predominance of the cis-b-b-compound was
obtained. Flash silica gel chromatography readily
resolved most of the diastereomeric pairs. The struc-
tures of all the compounds were consistent with the
proton NMR and mass spectraldata.
With the exception of the p-methoxy and m-methoxy
aryl-substituted compounds, each pair of cis and trans
When the IC50 value for neurotransporter reuptake
inhibition (Table 2) is divided by the binding constant
for inhibition of radiolabelled RTI-55 binding (Table
1) to provide a ratio (Table 3) this correlation ratio
value can provide insight into the efficacy of a com-
pound as an antagonist of cocaine action. An IC /K
i
3
-carbomethoxy-4-(aryl-substituted)piperidine diaster-
eomers were examined for potency of inhibition of
binding of radiolabelled RTI-55 to the hDAT, hSERT
and hNET. As shown in Table 1, compared with
cocaine, compound 1, only compound 2 possessed
5
0
correlation ratio of 25 or greater suggests that the
Scheme 1. Synthesis of cis- and trans-3-carbomethoxy-4-(aryl)piperidines.

X. Feng et al. / Bioorg. Med. Chem. 11 (2003) 775–780
777
Figure 2. Chemicalstructure of cis- and trans-3-carbomethoxy-4-(aryl)piperidines and corresponding identification numbers.
compound possesses binding potency and reuptake
inhibition efficacy of sufficient magnitude to compete
with the pharmacological effects exerted by cocaine. As
shown in Table 3, however, none of the compounds
examined attained the desired level of efficacy.
in affinity at the rat brain DAT compared with
1
3ꢀ15
cocaine.
The fumarate salts of compounds 2 and 4 were prepared
and tested for suppression of spontaneous locomoter
activity in groups of non-habituated male Swiss-Web-
ster mice. Compounds 2 and 4 were individually tested
for suppression of spontaneous locomoter activity at
intra-peritonealdoses ranging from 3–100 mg/kg in the
period of 0–60 min after dosing. The period of 20–60 or
0–30 min was selected for analysis of dose-response data
because these were the time periods that maximalsup-
pression first appeared as a function of dose and this is
the time period in which cocaine produces its maximal
effects. The mean average horizontalactivity (counts/10
min) for these time periods was fit to a linear function of
log10 dose of the descending portion of the dose–effect
Compared with cocaine, compounds 2 and 3 were more
selective for the hDAT. Thus, the K (hSERT)/K (hDAT)
and K (hNET)/K (hDAT) values for compounds 2 and 3
i
i
i
i
were 7, 14.5 and 14.7 and 6.4, respectively. On the basis
of the limited number of compounds examined, there
does not appear to be a significant electronic sub-
stitutent effect on the inhibition of binding to the hDAT
or on the inhibition of reuptake of radiolabelled neuro-
transmitter by HEK cells transfected with hDAT,
hSERT or hNET. The para-position of the arylring
may be sensitive to lipophilicity or some other effect
because the isoelectronic cis compounds 2 and 4 (i.e.,
1
2
curve. The ID50 (dose producing maximaldepressant
para-Cland para-CH ) had among the highest binding
3
activity where maximaldepression is zero counts per 30
min) was calculated as 29.5 and 38.3 mg/kg, respec-
tively, for compounds 2 and 4, respectively.
and uptake inhibition values. However, there was a sig-
nificant dependence of pharmacological activity on the
stereochemistry of the compound (Fig. 2).
An interaction study was also conducted examining the
effect of various intra-peritonealdoses of compound 2
(3–100 mg/kg) on the stimulant activity of 20 mg/kg of
cocaine. The mean average horizontalcounts/10 min on
the descending portion of the dose-effect curve (10–100
mg/kg dose range) for the 30-min period were fit to a
linear function of log10 dose, and the AD50 (the dose
attenuating cocaine-induced stimulation by 50%) was
calculated to be 47.7 mg/kg. After receiving 100 mg/kg
of compound 2, the locomoter activity of 20 mg/kg
cocaine was completely suppressed. However, at a dose
of 100 mg/kg 2, the entire group of eight mice showed
tremors during the pretreatment period. It is possible
that other centralnervous system mechanisms con-
tribute to the pharmacological activity observed for 2.
In each example examined, the cis diastereomer was
more potent an inhibitor of binding and more effica-
cious an inhibitor of uptake than the corresponding
trans diastereomer. The preference for cis stereo-
selectivity was pronounced for binding and uptake
inhibition at the hDAT, but was less pronounced at the
hSERT. There was no apparent stereoselectivity
observed for binding at the hNET. The results are in
agreement with previous studies that showed that a ser-
ies of cis-3b-carbomethoxy-4b-(p-chlorophenyl)piperi-
dine analogues of cocaine exhibited a 2.5-fold increase
Table 3. Calculated correlation ratios of uptake inhibition to binding
affinity
HDAT
IC50/K
hSERT
HNET
IC50/K
i
a
Compd
i
IC50/K
i
Conclusion
2
3
4
5
1.53
0.9
1.0
0.22
1.03
1.0
0.91
1.0
0.79
0.6
0.84
0.91
0.296
0.11
1
In summary, a convenient synthetic procedure was used
for the procurement of cis and trans diastereomers of
0.35
0.29
0.83
0.17
0.22
0.39
0.34
0.3
6, 7
8, 9
10
1
1
1
3-carbomethoxy-4-(aryl)piperidines. This method was
13ꢀ15
1
similar to the one previously reported.
Retro-
0.13
0.27
0.48
1
synthetic analysis of the target compounds showed that
the structure was derived from molecular dissection of
1
2
3
0.67
0.14
(
ꢀ)-cocaine and related (ꢀ)-aryltropanes. The sig-
aThe IC50 value (Table 2) for potency of uptake inhibition was divided by the K
value (Table 1) for potency of binding to obtain the correlation ratio listed.
nificance of this approach is that it could lead to highly
potent inhibitors and highly efficacious uptake inhibitors
i

778
X. Feng et al. / Bioorg. Med. Chem. 11 (2003) 775–780
of hDAT that could be substantially free of the addic-
tion liability inherent to the parent compound, (ꢀ)-
cocaine. Of the 3-carbomethoxy-4-(aryl)piperidines
examined, the most active compounds possessed a para-
chloro aryl substituent or its isosteric equivalent, a para-
methylgroup. This resutl is in agreement with work
solution of the arylmagnesium bromide was added to
ꢁ
the flask drop-wise. The reaction was stirred at ꢀ30 C
ꢁ
10 mL of TFA in 10 mL of Et O was added to the
ꢁ
to ꢀ40 C for 3 h and then cooled to ꢀ78 C for 30 min.
2
reaction drop-wise. The reaction was allowed to warm
to 0 C and 30 mL of 1N HClwas added to the mixture
ꢁ
followed by enough saturated NH OH to raise the pH
1
3ꢀ15
published previously.
A notable degree of stereo-
4
selectivity was observed in the potency of binding or in
the efficacy of uptake inhibition of hDAT. The cis dia-
stereomer was considerably more active than the corre-
sponding trans isomer. This is in keeping with studies of
of the reaction to >12. The aqueous layer was extracted
three times with 40 mL of EtOAc. The combined
organic extracts were dried over Na SO and con-
2
4
centrated to a thick oil. A mixture of diastereomeric
products were crudely separated with flash silica
chromatography (R =0.3 and 0.2) using Et N/hexanes
(ꢀ)-cocaine or (ꢀ)-aryltropanes that also show sig-
nificantly greater pharmacological efficacy for the beta,
beta orientation of the C-2 and C-3 substituents of the
tropane nucleus. Molecular dissection as shown in Fig-
ure 1 afforded compounds with marked hDAT binding
and re-uptake inhibition that were considerably selec-
tive for the hDAT. However, examination of the most
active compounds in vivo showed that only modest
suppression of cocaine-induced locomoter activity was
observed following administration of compound 2.
Future studies will be directed at examining the mole-
cular basis for the action of 3-carbomethoxy-4-(aryl)
piperidines and developing other agents with greater
potency that possesses non-toxic and non-addicting
properties.
f
3
25/75, v:v) to afford in approximately 40% yield a mix-
ture of cis and trans compounds in a ratio of 2:1,
respectively. Each diastereomer was further purified by
silica chromatography (MeOH/CH Cl 5:95, v:v) to
2
2
afford highly purified cis and trans 3-carbomethoxy-4-
(aryl)piperidines.
General procedure for preparing the fumarate salts of
selective piperidines
To an ethereal solution of the 3-carbomethoxy-4-(aryl)-
piperidine (1g, 6.5 mmol) was added 1 equivalent of
fumaric acid (0.75 g, 6.5 mmol) in ether. After stirring for
30 min, the reaction mixture was evaporated to dryness
to afford a crystalline salt of the desired piperidine.
Experimental
Synthesis
0
1
cis 3-Carbomethoxy-4-(4 -chlorophenyl)piperidine, 2. H
NMR (CDCl ) d 1.80 (dd, 1H, J=2.8, 13.4 Hz), 2.08
3
(dt, 1H, J=2.4, 11.2 Hz), 2.29 (s, 3H), 2.35 (dd, 1H,
General. Chemicals, reagents, buffers and solvents used
in this study were of the highest purity available from
commercialsources. So vl ents used in the reaction (i.e.,
tetrahydro furan, ether, CH Cl ) were freshly distilled
J=3.2, 11.5 Hz), 2.67 (dq, 1H, J=3.5, 11.8 Hz), 2.78–
2.81 (m, 1H), 2.97–3.00 (m, 2H), 3.18 (dd, 1H, J=1.0,
11.4 Hz), 3.52 (s, 3H), 7.20–7.26 (m, 5H); EI–MS m/z
+
268/270 (M +1), 234/235, 202/203, 174/175, 70.
2
2
prior to use. Cocaine was provided by the National
Institute on Drug Abuse Drug Supply Program,
NationalInstitute of He at lh (Bethesda, MD, USA).
RTI-55 was a kind gift from Dr. Ivy Carroll (RTI,
Research Triangle Park, NC, USA). Other buffers,
reagents and solvents were obtained from VWR (San
Diego, CA, USA). Chromatography was done with
silica gel 60 (230–400 mesh) from Acros Chemical
0
trans 3-Carbomethoxy-4-(4 -chlorophenyl)piperidine, 3.
1
H NMR (CDCl ) d 1.78–1.82 (m, 2H), 2.06.2.11 (m,
3
1H), 2.16 (t, J=11.2 Hz), 2.34 (s, 3H), 2.74 (m, 1H),
2.88 (dt, 1H, J=3.8, 11.2 Hz), 2.93 (br d, 1H, J=11.7
Hz), 3.07 (dd, 1H, J=2.6, 7.6 Hz), 3.44 (s, 3H), 7.11–
+
7.26 (m, 5H); EI–MS m/z 268/270 (M +1), 208/210.
(
done with silica gel F plates from E. Merck (Darm-
Pittsburgh, PA), and thin layer chromatography was
cis 3-Carbomethoxy-4-(p-toluene) piperidine, 4. 1H
NMR (CDCl ) d 1.81–1.86 (m, 1H), 2.07–2.13 (m, 1H),
2.29 (s, 3H), 2.34 (s, 3H), 2.72–2.76 (m, 1H), 2.88-2.96
(m, 2H), 3.07-3.10 (m, 1H), 3.44 (s, 3H), 7.08 (s, 4H),
2
54
3
3
3
3
stadt, Germany). [ H]-DA, [ H]-5HT, [ H]-NE and
25
1
[ I]RTI-55 were purchased from DuPont—New Eng-
land Nuclear (Boston, MA, USA). The preparation of
+
EI–MS m/z 248 (M +1).
1
6
the hDAT used was described previously. The hSERT
cDNA and HEK cells transfected with hNET was sup-
plied by Dr. Randy Blakely (Vanderbilt University,
trans 3-Carbomethoxy-4-(p-toluene)piperidine, 5. 1H
NMR (CDCl ) d 1.82 (m, 1H), 2.15 (m, 1H), 2.29 (s,
3H), 2.30 (s, 3H), 2.40 (m, 1H), 2.68 (m, 1H), 2.92 (m,
1H), 3.00 (m, 2H), 3.14 (m, 1H), 3.51 (s, 3H), 7.10 (m,
2H), 7.17 (m, 2H), EI–MS m/z 248 (M +1).
3
1
Nashville, TN, USA). H NMR spectra were recorded
on a Bruker spectrometer operating at 500 MHz with
TMS as an internalstandard. Mass spectra were
obtained on a HP 1100 MSD (Palo Alto, CA, USA).
+
0
cis- And trans 3-carbomethoxy-4-(4 -methoxyphenyl)-
1
piperidine, 6 and 7. H NMR (CDCl ): d 2.29 (s, 3H),
3
General procedure for synthesis of cis and trans
3
3
.52 (s, 3H), 7.20-7.26 (m, 5H); EI–MS m/z 263
+
-carbomethoxy-4-(aryl) piperidines
(M +1), 232, 204, 98.
4
.99 g (1 equiv) of arecoline free base in 40 mL of Et O,
2
ꢁ
was treated with 64.4 mL (2 equiv) of a 1 M ethereal
0
and 50 mL of CH Cl was cooled to ꢀ30 to ꢀ40 C and
cis- And trans 3-carbomethoxy-4-(3 -methoxyphenyl)-
1
2
2
piperidine, 8 and 9. H NMR (CDCl ): d 2.29 (s, 3H),
3

X. Feng et al. / Bioorg. Med. Chem. 11 (2003) 775–780
779
3
.52 (s, 3H), 7.20–7.26 (m, 5H); EI–MS m/z 264
+
filters presoaked with 0.05% polyethylenimine using a
Tomtech call harvester and scintillation counting as
described above. Specific uptake was defined as the
difference in uptake in the presence and absence of 5
mM mazindol(hDAT and hNET) or 5 mM imipra-
mine (hSERT).
(
M +1), 232, 192, 77.
cis 3-Carbomethoxy-4-(4 -phenyl)piperidine, 10. 1H
NMR (CDCl ) d 1.84 (dd, 1H, J=3.0, 12.6 Hz), 2.10
0
3
(
dt, 1H, J=2.2, 11.2 Hz), 2.29 (s, 3H), 2.38 (dd, 1H,
J=3.1, 11.4 Hz), 2.70 (dq, 1H, J=2.9, 12.0 Hz), 2.86
m, 1H), 2.98–3.02 (m, 2H), 3.18 (dd, 1H, J=1.0, 6.5
Hz), 3.50 (s, 3H), 7.17–7.30 (m, 5H); EI–MS m/z 234
(
In vivo studies
+
(
M +1), 202, 173, 131, 70.
Separate non-habituated male Swiss-Webster mice were
used in the conduct of this work. Fully approved animal
protocols were used and the studies were done in keep-
ing with the NIH standards for use of experimental
animals.
trans 3-Carbomethoxy-4-(4 -phenyl)piperidine, (11). 1H
NMR (CDCl ) d 1.82–1.87 (m, 2H), 2.10 (dt, 1H,
J=5.6, 11.2 Hz), 2.19 (t, J=11.2 Hz), 2.34 (s, 3H), 2.75
(dt, 1H, J=5.6, 11.2 Hz), 2.91 (dd, 1H, J=3.9, 11.3
Hz), 2.95 (dd, 1H, J=4.8, 9.2 Hz), 3.10 (dd, 1H, J=2.4,
0
3
Data analysis
1
2
1.3 Hz), 3.42 (s, 3H), 7.16–7.28 (m, 5H); EI–MS m/z
+
34 (M +1), 202, 174, 131, 79.
GraphPad Prism (GraphPad Software, San Diego, CA,
USA) was used to determine the saturation and binding
kinetic data. IC50 values were converted to K values
0
cis 3-Carbomethoxy-4-(4 -fluorophenyl)piperidine, (12).
1
i
H NMR (CDCl ) d 1.79–1.82 (br d, 1H), 2.09 (br d,
using the Cheng–Prusoff equation.
3
1
1
3
H), 2.28 (s, 3H), 2.29 (s, 3H), 2.37 (m, 1H), 2.67 (dq,
H, J=2.0, 12.0 Hz), 2.80 (m, 1H), 2.95–3.00 (m, 2H),
.16 (m, 1H), 3.51 (s, 3H), 6.90 (m, 2H), 7.25 (m, 2H);
In vivo evaluation of selected piperidines. Dose–response
study. A dose–response study of compounds 2- and
4-induced locomotor activity was conducted according
to the locomotor activity studies standard operation
procedure of the IntramuralResearch Program of the
NationalInstitute on Drug Abuse (B at li more, MD,
USA). The study was conducted using 16 Digiscan
locomotor activity testing chambers (40.5ꢃ40.5ꢃ30.5
cm). Panels of infrared beams (16 beams) and corre-
sponding photodetectors were located in the horizontal
direction along the sides of each activity chamber.
Separate groups of eight non-habituated male Swiss-
Webster mice were injected via the intraperitoneal(IP)
route with either vehicle (saline) or the test compounds
(3, 10, 30, or 100 mg/kg), 20 min prior to locomotor
activity testing. Just prior to placement in the appara-
tus, all mice received a saline injection IP. In all studies,
horizontalactivity (interruption of one photocelbeam)
was measured for 1-h within 10-min periods. Testing
was conducted with one mouse per activity chamber.
+
EI–MS m/z 252 (M +1), 113.
Transporter binding assays: [125I]RTI-55 binding
HEK-hDAT,-hSERT, or-hNET cells were grown until
1
7
confluent as described previously. Cells were scraped
from plates and centrifuged for 20 min at 30,000g and
the pellet was re-suspended in 0.32 M sucrose with a
Polytron at a setting of 1 for 5 s. Assays contained 50
mL of membrane preparation, 25 mL of the test com-
pound and 25 mL of [ I]RTI-55 (40–80 pM finalcon-
centration) in a finalvo ul me of 250 mL Krebs HEPES
buffer (25 mM HEPES, 122 mM NaCl, 5 mM KCl, 1.2
mM MgSO , 2.5 mM CaCl , 1 mM pargyline, 100 mm tro-
polone, 2 mg glucose/mL and 0.2 mg ascorbic acid/mL at
pH 7.4). Membranes were pre-incubated with test com-
pounds for 10 min before addition of [ I]RTI-55. Specific
binding was determined as the difference in binding
observed in the presence and absence of 5 mM mazindol
1
25
4
2
1
25
(
HEK-hDAT and hNET) or 5 mM imipramine (HEK-
Interaction study
hSERT). The incubations were done in the dark and
terminated by filtration onto a Whatman GF/C filter
The interaction study was conducted using 16 Digiscan
locomotor activity testing chambers as described in the
previous section. Twenty minutes following IP vehicle
or compound 2 injections (3, 10, 30, 100 mg/kg), groups
of eight non-habituated male Swiss-Webster mice were
injected with either 0.9% saline or 20 mg/kg cocaine IP
and placed in the Digiscan apparatus for a 1-h session.
IC50 values were estimated from a linear regression
against log doses. A two-way analysis of variance was
conducted on horizontalactivity counts/10 min of
treatment, 10-min periods and the interaction of periods
and treatment.
(
Whatman, Inc., Clifton, NI, USA) using a 96-well Tom-
tech cell harvester (Tomtech, Orange, CT, USA). Scintil-
lation fluid was added to each filter spot and radioactivity
remaining on the filter was determined using a Wallace
b-plate reader (Wallace Labs, Cranbury, NJ, USA).
Inhibition of substrate uptake
HEK-hDAT,-hSERT or -hNET cells were grown as
above. Cells were scraped from the plates and sus-
pended cells were added to a 96-well plate containing
test compounds and Krebs-HEPES buffer in a final
assay volume of 0.5 mL. After a 10-min preincu-
bation in a 25 C water bath, [ H]-labeled neuro-
transmitter (50 mL, 20 nM finalconcentration) was
added and the assay was initiated. After 10 min, the
incubation was terminated by filtration onto GF/C
ꢁ
3
Acknowledgements
We acknowledge the generous gifts of authentic cocaine
and cocaine metabolites provided from the Drug Supply

780
X. Feng et al. / Bioorg. Med. Chem. 11 (2003) 775–780
Program of the NationalInstitute on Drug Abuse. We
thank Dr. Jonathan Katz of the IntramuralResearch
Program of the NationalInstitute on Drug Abuse (Ba-l
timore, MD, USA) for performing the in vivo studies.
The financialsupport of the NationalInstitute on Drug
Abuse, NationalInstitutes of Hea lt h, Grant DA08531 is
gratefully acknowledged.
8. Cashman, J. R.; Berkman, C. E.; Underiner, G. E.
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