3β-(4-ethyl-3-iodophenyl)nortropane-2β-carboxylic acid methyl ester as a high-affinity selective ligand for the serotonin transporter

Full text of DOI:10.1021/jm970492z

© Copyright 1997 by the American Chemical Society
Volume 40, Number 24
November 21, 1997
Communications to the Editor
3â-(4-Eth yl-3-iod op h en yl)n or tr op a n e-2â-
ca r boxylic Acid Meth yl Ester a s a
High -Affin ity Selective Liga n d for th e
Ser oton in Tr a n sp or ter
Bruce E. Blough,† Philip Abraham,†
Andrew C. Mills,† Anita H. Lewin,† J ohn W. Boja,‡,§
Ursula Scheffel,| Michael J . Kuhar,‡, and
F. Ivy Carroll*,†
Chemistry and Life Sciences,
Research Triangle Institute, P.O. Box 12194, Research
Triangle Park, North Carolina 27709, Neuroscience Branch,
National Institute on Drug Abuse (NIDA) Addiction
Research Center, P.O. Box 5180, Baltimore, Maryland
21224, and Department of Radiology, Division of Nuclear
Research, J ohns Hopkins University School of Medicine,
Baltimore, Maryland 21205
6b, respectively. Compound 6b was reported to be
about 22 times more active than cocaine in inhibiting
the uptake of [3H]norepinephrine (NE) in rat brain.7
Studies reported in the late 1980s showed that, like
cocaine, 6a and 6b inhibited the uptake of 5-HT and
dopamine (DA) as well as NE. In fact, the potency for
inhibition of DA uptake was greater than that for
inhibition of 5-HT and NE uptake.8,9 During the same
time frame, [3H]WIN 35,428 ([3H]6b) was developed as
a radioligand for assaying the inhibition of binding at
the dopamine transporter (DAT).10 We have utilized
this ligand for structure activity relationship (SAR)
studies to characterize the cocaine binding site on the
DAT.11 Early studies led to the identification of 6c as
a ligand with high affinity for the DAT and low
selectivity relative to inhibition of radioligand binding
to the serotonin transporter (5-HTT) and norepine-
phrine transporter (NET). Later studies showed that
the phenyl and isopropyl esters 6d (RTI-113) and 6e
(RTI-114), as well as the 2â-isoxazole analogue 6f (RTI-
177), possess high affinity for the DAT and very low
affinity for the 5-HTT and NET.12,13
Received J uly 29, 1997
Serotonin-selective reuptake inhibitors (SSRIs) such
as fluoxetine (1), fluvoxamine (2), paroxetine (3), and
sertraline (4) have proved to be important agents in the
treatment of depression.1,2 Even though this class of
antidepressants is burdened with fewer side effects than
other classes, they still cause gastrointestinal, sleep
disturbance, and sexual dysfunction side effects.3 Thus,
the development of new structural classes of clinically
useful SSRIs is a subject of continuing interest. More-
over, reports in the literature suggest that inhibition
of serotonin (5-HT) uptake modulates the reinforcing
properties of cocaine (5).4-6
In attempts to separate the stimulant (or antidepres-
sant) and local anesthetic actions of cocaine from its
toxicity and dependence liability, Clarke and co-workers
synthesized several 3â-(substituted phenyl)tropane-2â-
carboxylic acid methyl esters (6).7 These compounds are
exemplified by WIN 35,065-2 and WIN 35,428, 6a and
In this study, we report the synthesis of 3â-(4-ethyl-
3-iodophenyl)nortropane-2â-carboxylic acid methyl ester
(7), a cocaine/WIN 35,065-2 (6a ) analogue which has
high affinity for 5-HTT and relatively low affinity for
DAT and NET. We also report in vivo competition
binding studies in mice which show that the regional
distribution and ED50 values of 7 are very similar to
† Research Triangle Institute.
‡ NIDA Addiction Research Center.
§ Current address: Department of Pharmacology, Northeastern Ohio
University, College of Medicine, 4209 State Route 44, Rootstown, OH
44272.
| J ohn Hopkins University School of Medicine.
Current address: Yerkes Regional Primate Research Center,
Emory University, 954 Gatewood NE, Atlanta, GA 30329.
S0022-2623(97)00492-5 CCC: $14.00 © 1997 American Chemical Society

3862 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Communications to the Editor
Sch em e 1
those of 1 and 4. These results suggest that 7 may be
useful as an antidepressant and that an iodine-123
analogue would have potential as a single photon
emission computed tomography (SPECT) ligand for
imaging 5-HT neurons in living brain.
Ch em istr y. The route used to synthesize 7 is shown
in Scheme 1. Subjection of N-[(2,2,2-trichloroethyl)-
carbamoyl]-3â-(4-iodophenyl)nortropane-2â-carboxylic
acid methyl ester (8)14 to a Stille-type coupling using
vinylzinc and bis(triphenylphosphine)palladium(II) chlo-
ride in tetrahydrofuran gave 73% of the 4-vinyl com-
for the 5-HTT, and 0.5 nM [3H]nisoxetine for the NET.
The results are listed in Table 1.
In Vivo Bin d in g. Male CD-1 mice (Charles-River,
Wilmington, MA), weighing 28-32 g, were pretreated
with increasing doses of 7 (0.1-10 mg/kg, i.p., 0.2 mL
of saline) 60 min before intravenous injection of 2 µCi
(in 0.2 mL of saline) of [125I]RTI-55 ([125I]-3â-(4-iodo-
phenyl)tropane-2â-carboxylic acid methyl ester) into the
tail vein. The animals were sacrificed 2 h later as
previously described.17 The brains were immediately
dissected on ice, the individual tissues were weighed and
placed in plastic tubes, and the radioactivity was
assayed in an automatic gamma counter. The counting
error was kept below 3%. Aliquots of the injectate were
prepared as standards and counted along with the tissue
samples. The percent injected dose per gram tissue
(%ID/g) was calculated.
Resu lts a n d Discu ssion . In our SAR studies, we
had noticed that removing the N-methyl group from 6c
to give 3â-(4-chlorophenyl)nortropane-2â-carboxylic acid
methyl ester 12a increased affinity at both the DAT and
5-HTT; however, the effect on the 5-HTT was much
larger18 (see Table 1). Since our SAR studies also had
shown that the addition of a 4-ethyl group to the phenyl
moiety of WIN 35,065-2 to give 3â-(4-ethylphenyl)-
tropane-2â-carboxylic acid methyl ester 12b reduced
affinity at the DAT and increased affinity at the 5-HTT,
we expected that 3â-(4-ethylphenyl)nortropane-2â-car-
boxylic acid methyl ester 12c would have selectivity for
5-HTT.18 This compound, 12c, with IC50 values of 8.13
and 49.9 nM for the 5-HTT and DAT, was the first WIN
35,065-2 analogue whose affinity at the 5-HTT was
greater than at the DAT.18
1
pound 9. The H NMR spectrum of 9 was complicated
by the existence of rotamers caused by the N-(2,2,2-
trichloroethyl)carbamoyl group; however, the expected
vinyl resonances at 6.65 (1H, dd, J ) 10.9 and 17.6 Hz),
5.70 (1H, d, J ) 17.6 Hz), and 5.20 ppm (1H, d, J )
10.9 Hz) were clearly delineated. Catalytic reduction
of 9 using 10% palladium on carbon catalyst in methanol
afforded 4-ethyl compound 10 in 84% yield. The 1H
NMR spectrum showed the expected resonances for the
ethyl group and the absence of the vinyl resonances.
Iodination of 10 using iodine in a mixture of acetic acid
and perchloric acid containing mercuric oxide provided
N-[(2,2,2-trichloroethyl)carbamoyl]-3â-(4-ethyl-3-iodo-
phenyl)nortropane-2â-carboxylic acid methyl ester (11)
in 68% yield. The 1H NMR spectrum of 11 showed
broad resonances at 7.58 (1H) and 7.08 ppm (2H) for
the three aromatic protons. The N-[(2,2,2-trichloroeth-
yl)carbamoyl group was removed under the experimen-
tally mild conditions of 10% lead oxide-cadmium15 in
a mixture of tetrahydrofuran and 1 M aqueous am-
monium acetate at 25 °C to give 7 in 53% yield. The
substitution pattern of the phenyl ring was confirmed
by heteronuclear multiple bond correlation which showed
a three-bond interaction between the ethyl methylene
protons (quartet at 2.68 ppm) and the carbon bearing
the iodine (unique at 100 ppm). The chemical shift and
coupling pattern of the C-2 and C-3 protons are consis-
tent with previously reported compounds that possess
the 2â,3â-stereochemistry.14 The compound was further
characterized as the tartrate salt: mp 160-161 °C;
We also had reported that adding a 3-iodo group to
WIN 35,065-2 to give 3â-(3-iodophenyl)tropane-2â-car-
boxylic acid methyl ester 12d had little effect on affinity
at the DAT.19 Since present data demonstrated sub-
stantial enhancement of affinity for 12d at 5-HTT
relative to WIN 35,065-2 (Table 1), we reasoned that
the addition of a 3-iodo group to 12c might lead to a
compound with increased affinity and selectivity for the
[R]25 -56.9° (c 0.26, CH3OH).
D
In Vitr o Bin d in g. The binding affinities of the
compounds for DAT, 5-HTT, and NET were determined
using competitive binding assays following previously
reported procedures.16 The radioligands used were 0.5
nM [3H]WIN 35,428 for the DAT, 0.2 nM [3H]paroxetine

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3863
Ta ble 1. Comparison of Inhibitors of Radioligand Binding to Monoamine Transporters
IC50(nM)a
ratiob
5-HT/DA
DA
NE
5-HT
compd
X
Y
R
[3H]WIN 35,428
[3H]nisoxetine
[3H]paroxetine
NE/DA
cocainec
89.1
23
3300
920
1050
2000
37
40
60
33
0.5
7
71
2.5
61
12
87
50
40
0.002
9
0.52
0.2
1.5
6a , WIN 35, 065-2c
6b, WIN 35,428c
6c (RTI-31)c
H
F
Cl
C2H5
Cl
C2H5
C2H5
H
H
H
H
I
H
H
H
I
CH3
CH3
CH3
H
H
CH3
H
13.9 ( 1.7
1.12 ( 0.1
329 ( ( 13
0.62 ( 0.09
55.0 ( 2.1
49.9 ( 7.3
26 ( ( 1.7
835 ( 45
37 ( ( 2.1
148 ( ( 9.2
4.13 ( 0.62
39.0 ( 381
122 ( ( 12
1590 ( ( 120
692 ( 71
44.5 ( 1.3
0.69 ( 0.07
5.45 ( 0.21
28.4 ( 3.8
8.13 ( 0.3
38.8 ( 3.8
7 (EINT)
12a (RTI-110)c
12b (RTI-83)c
12c (RTI-173)c
12d (RTI-92)d
CH3
a
b
Data are mean standard error of three or four experiments performed in tripliate. Ratios of IC50values. c IC50 values taken from ref
d
18. IC50 value for DA taken from ref 19.
5-HTT. This compound, 3â-(4-ethyl-3-iodophenyl)nor-
tropane-2â-carboxylic acid methyl ester (7), was pre-
pared and was found to have an IC50 value of 0.69 nM
at the 5-HTT and IC50 values of 329 and 148 nM at the
DAT and NET, respectively. To our knowledge, 7 is the
most potent and selective WIN 35,065-2 analogue thus
far prepared for the 5-HTT.
The efficacy of the antidepressant drugs 1-4 as well
as the modulation of cocaine self-administration has
been associated with serotonin transporters. Since 7
is a potent and selective compound for the 5-HTT and
also contains an iodine that could be replaced by an
iodine-123, this compound could be useful as an SPECT
ligand to measure serotonin transporters in living
humans if it provided high levels of specific binding. To
examine the potential of 7 as a SPECT imaging agent,
and to compare its brain levels to those of the antide-
pressant drugs, sertraline and fluoxetine, the inhibition
of the binding of [125I]RTI-55, which binds to both DATs
(especially in striatum) and 5-HTTs, in mouse brain in
vivo was investigated; the results are shown in Figure
1. The regional distribution of [125I]RTI-55 binding in
mouse brain varied, with the striatum having the
highest, and the cerebellum having the lowest, radio-
activity. Consistent with the observed in vitro selectiv-
ity of 7 for 5-HTT relative to DAT, the analogue 7
inhibited [125I]RTI-55 binding in the hypothalamus and
thalamus, regions with high concentrations of 5-HTTs.
This effect was dose-dependent with [125I]RTI-55 bind-
ing decreasing with increasing doses of 7. In contrast,
in the striatum and olfactory tubercle, regions with high
concentrations of DATs relative to 5-HTTs, binding of
F igu r e 1. Inhibition of [125I]RTI-55 binding by 7 in mouse
brain. Compound 7 (1.0 or 10 mg/kg in 0.2 mL of saline) or
saline (0.2 mL) (controls) was injected intraperitoneally 60 min
before intravenous injection of the tracer via tail vein. All
animals were sacrificed 120 min after tracer injection. The
number of animals is given in parentheses. CB ) cerebellum;
OLF ) olfactory tubercle; HYP ) hypothalamus; HIP )
hippocampus; STR ) striatum; FTCTX ) prefrontal cortex;
CTX ) parietal cortex; THAL ) thalamus; SCOL ) superior
colliculus. Data are means ( SEM, n ) 3, *P < 0.05; **P <
0.01.
7, a compound possessing high affinity and selectivity
for the 5-HTT. In vivo competition binding studies in
mouse suggest that the iodine-123 analogue of 7 may
be a useful SPECT ligand for studying the 5-HTT in
living brain and that 7 may also be useful as an
antidepressant.
Ack n ow led gm en t. This research was supported by
the National Institute on Drug Abuse, Grant DA05477
and Contract DA-4-8308. We thank Theresa A. Kopajtic
for assisting in obtaining the monoamine transporter
binding data and Dr. J ason P. Burgess for assistance
[
125I]RTI-55 increased, in a dose-dependent manner,
with administration of 7 (Figure 1). The effectiveness
of 7 in decreasing the binding of [125I]RTI-55 in 5-HTT-
rich regions (hypothalamus and thalamus) supports the
notion that 7 is selective for 5-HTT. The increase of
1
in obtaining and interpreting the H NMR data.
125I]RTI-55 binding in DAT-rich regions is likely due
Su p p or tin g In for m a tion Ava ila ble: Experimental data
for the syntheses of 7 (4 pages). Ordering information is given
on any current masthead page.
[
to increased availability of [125I]RTI-55 following its
displacement from 5-HTTs by 7. The calculated ED50
of 7 in hypothalamus, 3.5 µmol/kg, is in the range of
ED50 values reported for sertraline (2.6 µmol/kg) and
fluoxetine (6.8 µmol/kg).17,20
Refer en ces
(1) Fuller, R. W. Pharmacologic modification of serotonergic func-
tion: Drugs for the study and treatment of psychiatric and other
disorders. J . Clin. Psychiatry 1986, 47 (4), 4-8.
(2) Thakore, J . H.; Berti, C.; Dinan, T. G. Treating depression with
specific serotonergic acting agents. J . Serotonin Res. 1996, 3,
145-160.
In summary, the removal of the N-methyl group
combined with the addition of 4-ethyl and 3-iodo sub-
stituents to the 3â-phenyl group of WIN 35,065-2 affords

3864 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Communications to the Editor
(3) Goodwin, G. M. How do antidepressants affect serotonin recep-
tors? The role of serotonin receptors in the therapeutic and side
effect profile of the SSRIs. J . Clin. Psychiatry 1996, 57 (Suppl.
4), 9-13.
(4) Spealman, R. D. Modification of behavioral effects of cocaine by
selective serotonin and dopamine uptake inhibitors in squirrel
monkeys. Psychopharmacology 1993, 112, 93-99.
(5) Walsh, S. L.; Cunningham, K. A. Serotonergic mechanisms
involved in the discriminative stimulus, reinforcing and subjec-
tive effects of cocaine. Psychopharmacology 1997, 130 (1), 41-
58.
(6) Howell, L. L.; Byrd, L. D. Serotonergic modulation of the
behavioral effects of cocaine in the squirrel monkey. J . Phar-
macol. Exp. Ther. 1995, 275 (3), 1551-1559.
(7) Clarke, R. L.; Daum, S. J .; Gambino, A. J .; Aceto, M. D.; Pearl,
J .; Levitt, M.; Cumiskey, W. R.; Bogado, E. F. Compounds
affecting the central nervous system. 4. 3â-Phenyltropane-2-
carboxylic esters and analogs. J . Med. Chem. 1973, 16, 1260-
1267.
(13) Kotian, P.; Mascarella, S. W.; Abraham, P.; Lewin, A. H.; Boja,
J . W.; Kuhar, M. J .; Carroll, F. I. Synthesis, ligand binding, and
quantitative structure-activity relationship study of 3â-(4′-
substituted phenyl)-2â-(heterocyclic)tropanes: Evidence for an
electrostatic interaction at the 2â-position. J . Med. Chem. 1996,
39, 2753-2763.
(14) Blough, B. E.; Abraham, P.; Lewin, A. H.; Kuhar, M. J .; Boja, J .
W.; Carroll, F. I. Synthesis and Transporter Binding Properties
of 3â-(4′-Alkyl-, 4′-alkenyl-, and 4′-alkynylphenyl)nortropane-
2â-carboxylic Acid Methyl Esters: Serotonin Transporter Selec-
tive Analogs. J . Med. Chem. 1996, 39, 4027-4035.
(15) Dong, Q.; Anderson, C. E.; Ciufolini, M. A. Reductive cleavage
of TROC groups under neutral conditions with cadmium-lead
couple. Tetrahedron Lett. 1995, 36, 5681-5682.
(16) Carroll, F. I.; Gray, J . L.; Abraham, P.; Kuzemko, M. A.; Lewin,
A. H.; Boja, J . W.; Kuhar, M. J . 3-Aryl-2-(3′-substituted-1′,2′,4′-
oxadiazol-5′-yl)tropane analogues of cocaine: Affinities at the
cocaine binding site at the dopamine, serotonin, and norepi-
nephrine transporters. J . Med. Chem. 1993, 36(20), 2886-2890.
(17) Scheffel, U.; Kim, S.; Cline, E. J .; Kuhar, M. J . Occupancy of
the serotonin transporter by fluoxetine, paroxetine, and sertra-
line: In vivo studies with [125I]RTI-55. Synapse 1994, 16, 263-
268.
(8) Reith, M. E. A.; Meisler, B. E.; Sershen, H.; Lajtha, A. Structural
requirements for cocaine congeners to interact with dopamine
and serotonin uptake sites in mouse brain and to induce
stereotyped behavior. Biochem. Pharmacol. 1986, 35 (7), 1123-
1129.
(18) Boja, J . W.; Kuhar, M. J .; Kopajtic, T.; Yang, E.; Abraham, P.;
Lewin, A. H.; Carroll, F. I. Secondary amine analogues of 3â-
(4′-substituted phenyl)tropane-2â-carboxylic acid esters and
N-norcocaine exhibit enhanced affinity for serotonin and nore-
pinephrine transporters. J . Med. Chem. 1994, 37, 1220-1223.
(19) Carroll, F. I.; Mascarella, S. W.; Kuzemko, M. A.; Gao, Y.;
Abraham, P.; Lewin, A. H.; Boja, J . W.; Kuhar, M. J . Synthesis,
ligand binding, and QSAR (CoMFA and classical) study of 3â-
(3′-substituted phenyl)-, 3â-(4′-substituted phenyl)-, and 3â-(3′,4′-
disubstituted phenyl)tropane-2â-carboxylic acid methyl esters.
J . Med. Chem. 1994, 37, 2865-2873.
(20) Cline, E. J .; Scheffel, U.; Boja, J . W.; Mitchell, W. M.; Carroll,
F. I.; Abraham, P.; Lewin, A. H.; Kuhar, M. J . In vivo binding
of [125I]RTI-55 to dopamine transporters: Pharmacology and
regional distribution with autoradiography. Synapse 1992, 12,
37-46.
(9) Ritz, M. C.; Cone, E. J .; Kuhar, M. J . Cocaine inhibition of ligand
binding at dopamine, norepinephrine and serotonin transport-
ers: A structure-activity study. Life Sci. 1990, 46, 635-645.
(10) Madras, B. K.; Spealman, R. D.; Fahey, M. A.; Neumeyer, J . L.;
Saha, J . K.; Milius, R. A. Cocaine receptors labeled by [3H]2â-
carbomethoxy-3â-(4-fluorophenyl)tropane. Mol. Pharmacol. 1989,
36, 518-524.
(11) Carroll, F. I.; Lewin, A. H.; Kuhar, M. J . Dopamine Transporter
Uptake Blockers: Structure-Activity Relationships. In Neu-
rotransmitter Transporters: Structure, Function, and Regula-
tion; Reith, M. E. A., Ed.; Humana Press Inc.: Totowa, NJ , 1997;
pp 263-295.
(12) Carroll, F. I.; Kotian, P.; Dehghani, A.; Gray, J . L.; Kuzemko,
M. A.; Parham, K. A.; Abraham, P.; Lewin, A. H.; Boja, J . W.;
Kuhar, M. J . Cocaine and 3â-(4′-substituted phenyl)tropane-2â-
carboxylic acid ester and amide analogues. New high-affinity
and selective compounds for the dopamine transporter. J . Med.
Chem. 1995, 38, 379-388.
J M970492Z