Isothiocyanate derivatives of 9-[3-(cis-3,5-dimethyl-1- piperazinyl)propyl]-carbazole (rimcazole): Irreversible ligands for the dopamine transporter

Article abstract of DOI:10.1021/jm9705519

Cocaine has been reported to bind to the dopamine transporter in a biphasic fashion, and it has been hypothesized that the low-affinity component may play a modulatory role in cocaine's psychomotor stimulant effects. In an effort to gain further insight i

Full text of DOI:10.1021/jm9705519

4340
J . Med. Chem. 1997, 40, 4340-4346
Isoth iocya n a te Der iva tives of 9-[3-(cis-3,5-Dim eth yl-1-p ip er a zin yl)p r op yl]-
ca r ba zole (Rim ca zole): Ir r ever sible Liga n d s for th e Dop a m in e Tr a n sp or ter
Stephen M. Husbands, Sari Izenwasser, Richard J . Loeloff, J onathan L. Katz, Wayne D. Bowen,†
Bertold J . Vilner,† and Amy Hauck Newman*
Psychobiology Section, National Institute on Drug Abuse - Intramural Research Program, 5500 Nathan Shock Drive,
Baltimore, Maryland 21224, and Laboratory of Medicinal Chemistry, NIDDK, Bethesda, Maryland 20892
Received August 15, 1997X
Cocaine has been reported to bind to the dopamine transporter in a biphasic fashion, and it
has been hypothesized that the low-affinity component may play a modulatory role in cocaine’s
psychomotor stimulant effects. In an effort to gain further insight into the roles of the two
sites, we have prepared a series of irreversible ligands based on rimcazole (9-[3-(cis-3,5-dimethyl-
1-piperazinyl)propyl]carbazole, 2), a compound that has been postulated to bind only to the
low-affinity site. The alkylating moiety (isothiocyanate) is attached to the distal nitrogen of
the piperazine ring via alkyl chains of varying lengths or directly attached to one of the aromatic
groups. It was found that substitution on the piperazine nitrogen caused an initial decrease
in affinity that was recovered as the alkyl chain length increased. Importantly, the analogue
16, with the highest affinity for the dopamine transporter (DAT), binds in a monophasic and
irreversible manner, as evidenced by the greatly diminished binding of [3H]WIN 35,428 in
tissue that had been preincubated with the ligand and then thoroughly washed using
centrifugation. The dose-dependent reduction in Bmax was accompanied by a concentration-
related decrease in KD values. This shift in KD to a lower value suggests that the preincubation
with 16 produced a preferential irreversible binding to the low-affinity [3H]WIN 35,428 site on
the dopamine transporter. These ligands may prove to be important tools with which to study
the significance of the low-affinity site on the DAT. Since rimcazole does not share the
behavioral profile of cocaine, and in fact appears to play a modulatory role, these compounds
may provide leads for a novel cocaine-abuse treatment.
The social and economic consequences of increasing
illicit drug use, and in particular cocaine (1) abuse,
emphasize the need for pharmacological intervention.
A wide range of potential medications have been studied
in clinical settings as possible pharmacotherapeutics,
from a number of diverse therapeutic classes, including
dopamine agonists and antagonists, dopamine reuptake
inhibitors, anticonvulsants, antidepressants, and opioid
agonists, antagonists, and partial agonists.1 The results
of trials with these compounds have often proved
inconclusive, with no single class appearing to be fully
effective across the range of cocaine addict profiles.
Consequently, there has been significant interest in the
design of novel compounds and, in particular, com-
pounds that could act as substitution therapies or
cocaine antagonists.2
have suggested a relationship between σ-receptors and
the dopamine transporter (DAT). For example, the low-
affinity binding of cocaine to σ-receptors labeled with
[3H]haloperidol in rats has been demonstrated,5 and the
photoaffinity label [125I]iodoazidococaine, that recognizes
both the high- and low-affinity cocaine binding sites on
the DAT, appears to label a σ-receptor when photo-
activated.6 From these studies, a relationship between
σ-sites and cocaine binding sites is apparent. In addi-
tion, many of the GBR series of DAT ligands bind with
high affinity to both the DAT and also to σ-receptors,
suggesting an overlap of structural requirements for
binding to the DAT and σ-sites.7,8 Recently, GBR-like
ligands have been prepared with greater selectivity for
the DAT; however, it is clear that there is still signifi-
cant overlap in the binding requirements.8
Although there are as yet no compounds that have
been proven to act as cocaine antagonists, there are
several compounds known that appear to attenuate,
rather than potentiate, cocaine’s effects in animal
models of psychomotor stimulant abuse. These include
compounds from the GBR3 series and various sigma (σ)
ligands.4 Interestingly, certain σ-ligands such as rim-
cazole (9-[3-(cis-3,5-dimethyl-1-piperazinyl)propyl]car-
bazole, 2) and BMY 14802 (4-(fluorophenyl)-4-(5-fluoro-
2-pyrimidinyl)-1-piperazinebutanol) have been shown to
attenuate the locomotor stimulant effects of cocaine at
doses that do not have marked behavioral effects of their
own.4 Results from research in a number of laboratories
A number of σ-ligands were previously tested for their
ability to inhibit dopamine uptake and [3H]WIN 35,428
binding to the DAT.9 Many of these ligands inhibited
dopamine uptake and displaced [3H]WIN 35,428 binding
† NIDDK.
X Abstract published in Advance ACS Abstracts, December 1, 1997.
S0022-2623(97)00551-7 This article not subject to U.S. Copyright. Published 1997 by the American Chemical Society

Irreversible Ligands for the Dopamine Transporter
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4341
in a dose-dependent manner. They also displayed
monophasic binding, in contrast to cocaine and many
cocaine analogues, which recognize both high- and low-
affinity binding sites. Further the σ-ligands also inhib-
ited dopamine uptake in a monophasic manner, again
in contrast to cocaine. These differences in actions at
the DAT, in comparison to cocaine, may suggest alter-
native interactions at the molecular level. As these
compounds do not produce cocaine-like psychomotor
stimulant effects and some attenuate locomotor stimu-
lant effects of cocaine, it is possible that a novel action
at the DAT may be responsible for the lack of cocaine-
like pharmacology.10
group on the carbazole ring system or attaching, via an
alkyl chain, to the secondary nitrogen of the piperazine
ring.
Ch em istr y
Initially our work centered on the synthesis of the
carbazole-substituted isothiocyanate. By the selective
mononitration of rimcazole, using conditions developed
for the nitration of imipramine,14 the nitro derivative
4a was formed. Reduction using H2 over a Pd/C catalyst
resulted in the p-amino analogue 5a in good yield. Due
to the presence of the secondary amine, and its interfer-
ence in the reaction with thiophosgene, a mixture of
products was formed, from which the isothiocyanate
derivative could not be isolated. By reductive meth-
ylation, using the standard conditions of paraform-
One approach to further characterize these actions
is to prepare an irreversible ligand that might bind
covalently to the low-affinity component of the trans-
porter and therefore eliminate those sites. Affinity
labels have proved to be immensely valuable pharma-
cological tools to study receptor and transporter phar-
macology.11,12 An irreversible ligand first binds in a
reversible manner to the protein; irreversibility is the
result of subsequent covalent bond formation between
the ligand and protein. To be of use as a pharmacologi-
cal tool, the proposed irreversible ligand must bind with
high affinity and selectivity to the target protein. On
binding, the reactive group (typically an electrophile)
present on the ligand must be located close to a suitable
nucleophile on the protein. In this particular case, little
is known about the relative positions of suitable nu-
cleophiles on the DAT. It is therefore necessary to
synthesize a number of potential irreversible ligands in
the hope that one will meet the above criteria.
15
aldehyde and NaCNBH3 to give 3, this secondary
amine was converted to a tertiary amine and the
possibility of interference in the reaction was removed.
Nitration and reduction were carried out as before, but
now the reaction of the amine with thiophosgene16
proceeded smoothly to yield the isothiocyanate 6 (Scheme
1).
For the alkyl-substituted isothiocyanates, two proce-
dures were employed depending on the length of the
alkyl chain. For chain lengths of two or three carbons,
simple alkylation of rimcazole with the carbobenzoxy-
protected aminobromoalkane gave the best results.
Yields were optimal when potassium carbonate was
used as the base, with dimethylformamide as the
solvent. Deprotection was then carried out with TMSI16
to give the primary amines 9 and 10, and the isothio-
cyanates 11 and 12 were then formed by reaction with
thiophosgene (Scheme 2). For the six-carbon-chain
analogue, simple alkylation, as just described in the
synthesis of 11 and 12, resulted in little or no reaction.
Better yields were obtained by first acylating to give
the amide 13. This intermediate was deprotected with
TMSI and then reduced using LiAlH4 to 15. Treatment
with thiophosgene again gave the desired isothiocyanate
16 (Scheme 3).
The isothiocyanate is the most widely employed
moiety in the design of irreversible ligands for receptors
or transporters. This group is fairly small and so has
minimum impact on the binding affinity and selectivity
of the parent ligand, though examples are known where
a change in selectivity has occurred.13 The isothio-
cyanate is also readily introduced into a molecule, being
synthesized from the corresponding primary amine.
This group is highly reactive toward amino and sulfhy-
dryl groups, while displaying low reactivity toward
water and other hydroxyl functions. Thus there is less
opportunity for nonspecific binding with the isothio-
cyanate group compared to other, more reactive elec-
trophiles.
Resu lts a n d Discu ssion
Binding affinities of the synthesized compounds for
the DAT in rat caudate-putamen and the σ1- and σ2-
receptors in guinea pig brain and rat liver, respectively,
were determined and are shown in Table 1. [3H]WIN
35,425 was used to label the DAT, while [3H]pentazocine
and [3H]DTG in the presence of dextrallorphan were
used for σ1- and σ2-sites, respectively. Additionally, one
compound (16) was tested for irreversible binding to the
DAT.
Methylation of rimcazole to 3 caused a decrease in
affinity for the DAT of approximately 4-fold. This could
be attributed either to the loss of hydrogen bond donor
capabilities or as a direct result of the increase in steric
bulk at this position. The carbazole-substituted isothio-
cyanate 6 was a further 5-fold lower in affinity at the
DAT, resulting in a compound with too low affinity (Ki
) 2374 nM) to be of further interest as a pharmacologi-
cal probe.
Of the σ-ligands studied, rimcazole had the highest
affinity for the DAT (Ki ) 103 nM)9 and displayed
significant activity in behavioral tests, antagonizing the
locomotor stimulant effects of cocaine at a dose that
displayed no significant activity of its own.4 Further-
more, rimcazole demonstrated a significantly lower
potency for inhibiting dopamine uptake (IC50 ) 4.22
µM)9 as compared to its binding affinity than any of the
other ligands evaluated, or any other dopamine trans-
porter ligands reported to date. This novel neurochemi-
cal and behavioral profile provided interest in choosing
rimcazole as a suitable candidate for further chemical
modification, particularly with the aim of developing
potential irreversible ligands that might allow the
functional significance of the high- and low-affinity sites
to be more clearly defined.
With the three alkylisothiocyanates prepared (11, 12,
16), affinity for the transporter proved to be directly
proportional to the length of the alkyl chain used. There
is a >2-fold increase in affinity on going from the two-
Examining the rimcazole structure, it is clear that
there is the possibility of placing the isothiocyanate

4342 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Husbands et al.
Sch em e 1a
Sch em e 2a
a
(i) Br(CH2)nNHCBz; (ii) TMSI; (iii) Cl2CS.
As previously discussed, rimcazole binds with moder-
ate affinity to σ-receptors. The new analogues, with the
exception of 6, were evaluated for their binding affinity
to σ-receptors (Table 1). There appeared to be a good
correlation between σ2-affinity and affinity at the DAT.
Thus, increasing chain length caused an initial drop in
affinity followed by an increase in affinity as the chain
length increased further. The significant exception to
this appears to be rimcazole itself, it having relatively
low affinity for σ2-sites and about 14-fold selectivity for
the DAT. With σ1-sites there was less of a variation in
affinity, with only 12 (three-carbon chain) showing a
drop in affinity. The approximately 3-fold selectivity
of 16 for the DAT over σ1-sites is similar to the
selectivity displayed by various GBR analogues, includ-
ing GBR 12935 (∼4-fold).8
a
(i) (CH2O)n, NaBH3CN; (ii) AcOH, HNO3; (iii) H2, Pd/C; (iv)
Cl2CS.
carbon chain (11: Ki ) 2241 nM) to the three-carbon
chain (12: Ki ) 935 nM) and a further 6-fold improve-
ment on going from three to six carbons (16: Ki ) 155
nM) in length (Table 1).
Because 16 displayed the highest affinity for the DAT,
it was chosen for further evaluation to determine its
potential for irreversible binding. As shown in Table
2, there was a concentration-dependent decrease in Bmax
values for [3H]WIN 35,428 binding following incubation
with 100 nM or 10 µM 16, as compared to buffer, despite
two washes with centrifugation. This result is indica-
tive of an irreversible interaction of 16 with the dopam-
ine transporter. Figure 1 shows representative Scat-
chard analyses of the binding of [3H]WIN 35,428 under
the different preincubation conditions. In contrast to
what typically has been reported, the binding of [3H]-
WIN 35,428 in tissue preincubated in buffer did not
model better for two sites than for one binding site. It
has previously been shown that the conditions used in
this assay can critically affect the ability to delineate
two binding sites.18 It is likely that the added prein-
It thus appears that additional bulk (or loss of
H-bond-donating capability) at the secondary nitrogen
causes an initial drop in affinity for the DAT. It seems
most likely that this negative interaction has at least a
large steric component, as there is a further significant
drop (5-fold) in affinity on going from NMe (3) to
N(CH2)2NCS (11). This could not be explained by
exclusively H-bonding interactions. As the chain length
increases further (to three and then six carbons) the
affinity begins to increase, presumably due to interac-
tions at another binding domain. The initial decrease,
followed by an increase in affinity on lengthening of the
tether between the functional group and the pharma-
cophore, has previously been observed in other series
of receptor ligands.17

Irreversible Ligands for the Dopamine Transporter
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4343
Sch em e 3a
a
(i) ClCO(CH2)5NHCBz; (ii) TMSI; (iii) LaAlH4; (iv) Cl2CS.
Ta ble 1. Binding Affinities at the DAT and σ1- and σ2-Receptors
IC50 (nM ( SEM)a
compound
substitution
[3H]WIN 35,428 binding (DAT)
[3H]pentazocine binding (σ1)
[3H]DTG binding (σ2)b
2, rimcazole
3
6
11
12
16
H
NMe
NMe, 4-NCS
N(CH2)2NCS
N(CH2)3NCS
N(CH2)6NCS
57.6 ( 14
244 ( 24
1480 ( 160
897 ( 180
ndc
949 ( 220
1830 ( 320
707 ( 29
386 ( 48
294 ( 26
ndc
1440 ( 44
877 ( 120
211 ( 15
1330 ( 40
1250 ( 100
523 ( 52
86.7 ( 14
a
b
IC50’s were determined as described in the Experimental Section. [3H]DTG was used in the presence of dextrallorphan. c nd ) not
determined.
Ta ble 2. Binding Constants (Bmax and KD) for Studies of
[3H]WIN 35,428 Saturation in Rat Caudate-Putamen Tissue
Preincubated in Either Buffer or Buffer with Different
Concentrations of 16
Bmax
KD
condition fmol/mg of prot % control
nM
% control
buffer
100 nM
10 µM
537 ( 19.6
167 ( 69.3
6.65 ( 1.66
100
31
1.2
16.6 ( 1.09
7.61 ( 4.60
5.60 ( 2.52
100
45
34
cubation period of the present study altered the condi-
tions sufficiently to affect this outcome. Despite not
fitting a two-site model significantly better than a one-
site model, the Scatchard plot showed a significant
deviation from linearity as assessed by a Runs test (p
) 0.0367). This result was evidence of heterogeneity
of binding which was not true for the tissues that had
been pretreated with 16.
In addition to changes in Bmax values, preincubation
with 16 also produced a concentration-related decrease
in KD values (Table 2). These decreases in KD values
can be seen in the altered slopes of the Scatchard plots
in Figure 1. This shift in KD to a lower value is evidence
of a loss of low-affinity binding. The decrease in KD
F igu r e 1. Representative Scatchard analyses of the binding
of [3H]WIN 35,428 after preincubation with buffer or buffer
with 16.
values indicates that the preincubation with 16 pro-
duced a preferential irreversible binding to the low-
affinity [3H]WIN 35,428-labeled site on the dopamine
transporter.
Con clu sion s
A series of potential irreversible ligands were syn-
thesized and evaluated for their DAT and σ-site affini-

4344 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Husbands et al.
layer was collected and the aqueous layer extracted with
CHCl3 (2 × 20 mL/mmol). The organic layers were combined,
dried (Na2SO4), and evaporated to yield crude isothiocyanate
product. The product was then filtered through silica gel using
EtOAc:CHCl3 (4:1) as the eluent. This gave products that were
pure by TLC.
ties. From the structural modifications made, it appears
that rimcazole is very sensitive to alterations in its
structure, with fairly large changes in affinity occurring
with each of the substitutions made. Although σ1-
affinity did not vary significantly within this series of
compounds, there seemed to be a good correlation
between σ2-affinity and affinity at the DAT for the
N-substituted analogues. In each case there was an
initial lowering of affinity on going from NMe to
N(CH2)2NCS; then affinity rose as the length of the side
chain was increased. This is further evidence of the
very similar structural requirements for binding to the
DAT and σ-sites.
One of the potential irreversible ligands, 16, was
found to have similar affinity to rimcazole at the DAT
and was thus deemed a suitable candidate for further
characterization aimed at determining its ability to bind
in an irreversible manner. Binding studies have shown
that 16 is binding irreversibly, and additionally, it
appears that 16 may be labeling only one binding site
on the DAT. This result is important, in that it may
allow the functional significance of the low- and high-
affinity binding sites on the DAT to be separated.
Experiments are being designed for further character-
ization in both in vitro and behavioral assays.
9-[3-(cis-3,4,5-Tr im et h yl-1-p ip er a zin yl)p r op yl]ca r b a -
zole (3). Rimcazole (1.41 g, 4.4 mmol) was dissolved in a
mixture of CH3CN (9 mL) and EtOH (9 mL) before adding
formaldehyde (0.72 mL of 37%). NaBH3CN (0.28 g, 4.4 mmol)
was then added slowly and stirring continued for 2 h. Water
was added to quench the reaction and the mixture evaporated
to dryness. The product was extracted into CHCl3 and washed
with water (2 × 20 mL). Drying (Na2SO4) and evaporation
yielded an oil. Column chromatography (7% CMA) gave a
white foam, 1.25 g (85%). The HCl salt was formed and
recrystallized from EtOH:2-PrOH: mp 214-215 °C; Rf 0.5
1
(10% CMA); IR (CHCl3) 742, 719 (Ph) cm-1; H NMR δ 1.03
(6H, d, J ) 6.1 Hz, 2 × Me), 2.19 (2H, t, J ) 6.8 Hz, 2H of
piperazine), 2.26 (5H, t, s, NMe, 2H of piperazine), 2.63 (2H,
d, J ) 9.6 Hz, 2H of piperazine), 4.32 (2H, t, J ) 6.6 Hz, CH2N);
13C NMR δ 18.21, 25.78, 37.92, 40.35, 54.55, 57.96, 60.93,
108.81, 118.69, 120.19, 122.76, 125.43, 140.43. Anal. (C22H31N3‚
2HCl‚2-PrOH) C, H, N.
4-Nitr o-9-[3-(cis-3,4,5-tr im eth yl-1-p ip er a zin yl)p r op yl]-
ca r ba zole (4b). Compound 3 (2.30 g, 6.9 mmol) was taken
up in glacial acetic acid (52 mL) and then nitric acid (1.04 mL
of 70%) slowly added at room temperature. Stirring was
continued for 2.5 h before diluting the reaction with water (100
mL) and HCl (4 mL of 37%). The solution was washed with
ether (2 × 40 mL) and basified with NH4OH, and the organics
were extracted into CHCl3. The organic layer was dried (Na2-
SO4) and evaporated to give a red oil: 2.3 g (88%); IR (CHCl3)
1515 (NO2, asymm), 1330 (NO2, symm) cm-1; 1H NMR δ 1.03
(6H, d, 2 × Me), 2.25 (3H, s, NMe), 2.64 (2H, d, 2H of
piperazine), 4.45 (2H, t, CH2N), 7.29 (1H, m, Ph), 7.48 (3H,
m, Ph), 8.12 (1H, d, Ph m- to NO2), 8.36 (1H, d, Ph o- to NO2),
8.99 (1H, s, Ph o- to NO2).
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Thomas-
Hoover melting point apparatus and are uncorrected. 1H and
13C NMR spectra were recorded using a Bruker (Billerica, MA)
AC-300 spectrometer. Samples were dissolved in CDCl3, and
the chemical shifts are reported as parts per million (δ) relative
to tetramethylsilane (Me4Si, 0.00 ppm) as internal standard.
Chemical shifts for 13C NMR spectra are reported as δ relative
to deuterated chloroform (CDCl3, 77.0 ppm). Infrared spectra
were recorded with a Perkin-Elmer 1600 series FTIR spectro-
photometer with KBr disks. Thin-layer chromatography was
performed on silica gel plates (Uniplates, Analtech, Inc.) with
10% CMA (89% CHCl3, 10% MeOH, 1% NH4OH) as solvent
unless noted otherwise. Compounds for in vitro testing were
converted to their HCl salts by dissolving in a minimum
volume of MeOH and slowly adding a saturated solution of
HCl in MeOH until acidic. Elemental analysis performed by
Atlantic Microlab, Inc. (Norcross, GA) agreed to within 0.4%
of the calculated values. All chemicals and reagents were
purchased from Lancaster Synthesis, Inc. or Aldrich Chemical
Co.
Gen er a l P r oced u r es. Meth od A: Nitr ogen Alk yla tion .
Rimcazole, the alkyl bromide (1.1 equiv), and K2CO3 (2 equiv)
were heated to 110 °C in DMF (3 mL/mmol of rimcazole) for 6
days. On days 2 and 4 an extra 0.5 equiv of the alkyl bromide
was added. The mixture was cooled to room temperature and
H2O added. The organics were extracted with EtOAc:Et2O (1:
1), dried (Na2SO4), and evaporated to yield the crude product.
Meth od B: CBz Dep r otection .17 The CBz-protected
amine was dissolved in CH3CN (5 mL/mmol) and the iodotri-
methylsilane then added (3.6 equiv). The solution was stirred
at room temperature for 1 h before quenching with MeOH
(0.5× CH3CN vol). The solution was reduced to dryness in
vacuo and the solid then taken up in ether and 1 N HCl. After
separation, the aqueous layer was washed with further Et2O
(6 × 10 mL/mmol), before then basifying the aqueous layer
with NH4OH and extracting the product into CHCl3:2-PrOH
(7:3). This solution was dried (Na2SO4) and evaporated to yield
crude product.
4-Am in o-9-[3-(cis-3,4,5-tr im eth yl-1-piper azin yl)pr opyl]-
ca r ba zole (5b). Compound 4b (380 mg, 1.0 mmol) was
dissolved in MeOH (25 mL) and hydrogenated under an
atmosphere of H2 at 30 psi, for 3 h, over a 10% Pd/C catalyst
(60 mg). Filtration through Celite followed by column chro-
matography (5-10% CMA) yielded a red oil: 190 mg (54%);
Rf 0.38 (10% CMA); IR (CHCl3) 3413, 3346 (NH2) cm-1 1H
;
NMR δ 1.07 (6H, d, 2 × CH3), 2.30 (3H, s, NMe), 2.64 (2H, d,
2H of piperazine), 4.32 (2H, t, CH2N), 6.89 (1H, m, Ph), 7.17
(1H, m, Ph), 7.28 (1H, s, Ph), 7.38 (3H, m, Ph), 7.94 (1H, d,
Ph).
4-Isoth iocyan ato-9-[3-(cis-3,4,5-tr im eth yl-1-piper azin yl)-
p r op yl]ca r ba zole (6). Using general procedure C with 5b
(180 mg, 0.51 mmol), but with EtOH as the eluent for
purification, the isothiocyanate 6 was obtained as an oily red
solid,, 160 mg (79%). The HCl salt was formed and recrystal-
lized in 2-PrOH:THF: mp 188-190 °C dec; Rf 0.05 (10% EtOH
in EtOAc); IR (CHCl3) 2108 (NCS) cm-1; 1H NMR δ 1.03 (6H,
d, J ) 6.4 Hz, 2 × Me), 2.22 (3H, s, NMe), 2.67 (2H, d, J )
10.5 Hz, 2H of piperazine), 4.37 (2H, t, J ) 6.5 Hz, CH2N),
7.28 (2H, m, aryl), 7.48 (3H, m, aryl), 7.92 (1H, d, J ) 2.0 Hz,
aryl-H o- to NCS), 8.04 (1H, d, J ) 7.5 Hz, aryl-H o- to NCS);
13C NMR δ 16.26, 25.78, 37.98, 41.12, 54.44, 58.04, 60.95,
109.36, 109.69, 117.73, 119.59, 120.60, 121.95, 123.31, 126.58,
139.50, 141.82. Anal. (C23H28N4S‚2HCl‚0.5H2O) C, H, N.
9-[3-[cis-3,5-Dim eth yl-4-[2-(ca r boben zoxya m in o)eth yl]-
1-p ip er a zin yl]p r op yl]ca r ba zole (7). Using general proce-
dure A with benzyl 2-bromoethylcarbamate.18 Column chro-
matography (7% CMA) of the crude product yielded an off-
white solid: 83%; Rf 0.55 (10% CMA); IR (CHCl3) 1715 (CO of
1
CBz) cm-1; H NMR δ 1.06 (6H, d, 2 × Me), 2.6-2.8 (6H, m),
3.21 (2H, t, CH2NHCBz), 4.38 (2H, t, CH2N), 5.09 (2H, s,
PhCH2).
Meth od C: Isoth iocya n a te (NCS) F or m a tion . The
amine was dissolved in a mixture of CHCl3 (60 mL/mmol) and
water (25 mL/mmol) with NaHCO3 (4.5 equiv). This mixture
was cooled in an ice bath before adding freshly distilled
thiophosgene (1.3 equiv). Stirring was continued for 2 h before
pouring the mixture into a separatory funnel. The organic
9-[3-[cis-3,5-Dim eth yl-4-[3-(car boben zoxyam in o)pr opyl]-
1-p ip er a zin yl]p r op yl]ca r ba zole (8). Using general proce-
dure A with benzyl 3-bromopropylcarbamate.18 Column chro-
matography (7% CMA) yielded an oil: 35%; Rf 0.5 (10% CMA);
1
IR (CHCl3) 1713 (CO of CBz) cm-1; H NMR δ 1.06 (6H, d, 2

Irreversible Ligands for the Dopamine Transporter
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4345
× Me), 2.59 (4H, m), 2.78 (2H, m), 4.37 (2H, t, CH2N), 5.13
9-[3-[cis-3,5-Dim eth yl-4-(3-isoth iocyan atopr opyl)-1-pip-
er a zin yl]p r op yl]ca r ba zole (12). Using general procedure
C with the amine 10 yielded the product as a white solid, 81%.
The HCl salt was formed and recrystallized in 2-PrOH:THF:
mp 230-232 °C; IR (CHCl3) 2110 (NCS) cm-1; 1H NMR δ 1.05
(6H, d, J ) 6.2 Hz, 2 × Me), 2.63 (4H, d, J ) 8.7 Hz, 2H of
piperazine, CH2N of side chain), 2.83 (2H, dd, J ) 7.9 Hz, 2H
of piperazine), 3.49 (2H, t, J ) 6.4 Hz, CH2NCS), 4.41 (2H, t,
J ) 6.5 Hz, CH2N at carbazole); 13C NMR δ 18.29, 25.69, 25.84,
40.53, 43.49, 45.77, 54.51, 54.74, 61.09, 108.82, 118.75, 120.26,
122.81, 125.49, 140.48. Anal. (C25H32N4S‚2HCl) C, H, N.
Bin d in g Meth od s. 1. Ch em ica ls. Chemicals and re-
agents were obtained from the following sources: [3H]WIN 35,-
428 (2â-carbomethoxy-3â-(4-fluorophenyl)tropane 1,5-naph-
thalene disulfonate; specific activity 83.5 Ci/mmol) from New
England Nuclear (Boston, MA); cocaine hydrochloride from
Sigma Chemical Co. (St. Louis, MO). [3H]-(+)-Pentazocine was
synthesized as described previously.19,20 [3H]DTG (39.1 Ci/
mmol) was purchased from DuPont/New England Nuclear
(Boston, MA). Dextrallorphan was provided by Dr. F. I.
Carroll (Research Triangle Institute, Research Triangle Park,
NC). Haloperidol and poly(ethylenimine) were purchased from
Sigma Chemicals (St. Louis, MO).
(2H, s, CH2Ph).
9-[3-[cis-3,5-Dim eth yl-4-[6-(ca r boben zoxya m in o)-1-ox-
oh exyl]-1-p ip er a zin yl]p r op yl]ca r ba zole (13). Rimcazole
(3.53 g, 11 mmol) was dissolved in toluene (90 mL) before
adding N-CBz-6-aminohexanoyl chloride (5.10 g, 18 mmol) and
triethylamine (2.6 mL, 18.7 mmol) and warming to 60 °C
overnight. The solution was cooled, and the organics were
extracted in EtOAc (3 × 50 mL) from a Na2CO3 solution.
Drying (Na2SO4) and evaporation gave a brown foam. Column
chromatography (2% CMA) yielded a cream-colored solid: 2.8
g (45%); Rf 0.83 (10% CMA); IR (CHCl3) 3317 (NH), 1713 (CO
of carbamate), 1625 (CO of amide) cm-1; 1H NMR δ 1.43 (6H,
m, 2 × Me), 2.76 (2H, d, 2H of piperazine), 3.20 (2H, t), 4.43
(2H, t, CH2N), 5.11 (2H, s, CH2Ph), 7.2-7.5 (11H, m, CBz,
carbazole H’s), 8.09 (2H, m, 2H of carbazole).
9-[3-[cis-3,5-Dim eth yl-4-(6-a m in o-1-oxoh exyl)-1-p ip er -
a zin yl]p r op yl]ca r ba zole (14). Using general procedure B
with the CBz-protected amine 13. Column chromatography
(5-10% CMA) of the crude product gave an oil: 75%; Rf 0.15
(10% CMA); IR (CHCl3) 3373 (NH), 3297 (NH), 1632 (CO of
amide) cm-1; 1H NMR δ 1.42 (6H, m, 2 6 × Me), 2.78 (4H, m,
2H of piperazine plus CH2N of side chain), 4.47 (2H, t, CH2N).
2. Dop a m in e Tr a n sp or ter Bin d in g Assa y. Male Spra-
gue-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 homog-
enized in 30 vol 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 D-glucose, pH adjusted
to 7.4) using a Brinkman polytron and centrifuged at 20000g
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 20000g for 10 min at 4 °C. Fresh homogenates were
used in all experiments.
Binding assays were conducted in modified Krebs-HEPES
buffer on ice. The total volume in each tube was 0.5 mL, and
the final concentration of membrane after all additions was
0.5% (w/v) corresponding to 200-300 mg of protein/sample.
[3H]WIN 35,428 (specific activity 82.4 Ci/mmol) 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 (Gaithers-
burg, MD). The filters were washed with three additional
3-mL washes and transferred to scintillation 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%. For determination
of IC50 values, triplicate samples of membrane suspension were
preincubated for 5 min in the presence or absence of the
compound being tested. Each compound was tested with
concentrations ranging from 0.01 nM to 100 µM for competition
against binding of [3H]WIN 35,428 (final concentration 1.5
nM), in three independent experiments. Under these assay
conditions, an average experiment yielded approximately 6000
dpm total binding per sample and approximately 250 dpm
nonspecific binding, defined as binding in the presence of 100
µM cocaine.
9-[3-[cis-3,5-Dim eth yl-4-(2-a m in oeth yl)-1-p ip er a zin yl]-
p r op yl]ca r ba zole (9). Using general procedure B with the
CBz-protected amine 7. The gummy white foam product was
purified by column chromatography (5-10% CMA) to give a
white solid: 75%; Rf 0.1 (10% CMA); IR (CHCl3) 3404, 3179
(NH) cm-1; 1H NMR δ 1.07 (6H, d, 2 × Me), 1.75 (4H, m), 2.08
(2H, t), 2.24 (2H, t), 2.6-2.8 (6H, m), 4.39 (2H, t, CH2N).
9-[3-[cis-3,5-Dim eth yl-4-(3-am in opr opyl)-1-piper azin yl]-
p r op yl]ca r ba zole (10). Using general procedure B with the
CBz-protected amine 8. The off-white, foam product was
purified by column chromatography (10% CMA) to yield a
white foam: 62%; Rf 0.1 (10% CMA); IR (CHCl3) 3400, 3200
(NH) cm-1; 1H NMR δ 1.01 (6H, d, 2 × Me), 2.69 (4H, m), 2.85
(2H, m), 4.39 (2H, t, CH2N).
9-[3-[cis-3,5-Dim eth yl-4-(6-a m in oh exyl)-1-p ip er a zin yl]-
p r op yl]ca r ba zole (15). The amide 14 was dissolved in dry
THF (1 mL/mmol) and added to a cooled mixture of LiAlH4
(1.3 equiv) in dry THF (1 mL/mmol). The mixture was allowed
to warm to room temperature and stirring continued overnight.
The reaction was quenched with solid Rochelle’s salt before
adding water (1 mL/2 mmol). The mixture was evaporated to
dryness, the product was taken up in 7:3 CHCl3:2-PrOH, and
the inorganic salts were filtered off. Drying (Na2SO4) and
evaporation gave the crude product as a thick oil; crude yield,
95%. Column chromatography gave the pure amine as a sticky
solid: 70%; Rf 0.3 (10% CMA); IR (CHCl3) 3356, 3282 (NH)
1
cm-1; H NMR δ 1.07 (6H, d, 2 × Me), 1.3-1.5 (6H, m), 2.70
(6H, m), 4.38 (2H, t, CH2N).
9-[3-[cis-3,5-Dim eth yl-4-(6-isoth iocya n a toh exyl)-1-p ip -
er a zin yl]p r op yl]ca r ba zole (16). Using general procedure
C with amine 15 yielded the product as an oily solid, 50%.
The HCl salt was formed and recrystallized from 2-PrOH:
1
Et2O: mp 210-212 °C dec; IR (CHCl3) 2102 (NCS) cm-1; H
NMR δ 1.05 (6H, d, J ) 6 Hz, 2 × Me), 2.68 (4H, d, J ) 9.8
Hz, 2H of piperazine; t, J ) 7.8 Hz, CH2N of side chain), 3.52
(2H, t, J ) 6.5 Hz, CH2NCS), 4.38 (2H, t, J ) 6.6 Hz, CH2N at
carbazole); 13C NMR δ 17.98, 23.21, 25.76, 26.48, 26.78, 29.88,
40.45, 44.85, 47.88, 53.62, 54.60, 61.13, 108.72, 118.62, 120.14,
122.90, 125.37, 140.37. Anal. (C28H38N4S‚2HCl‚0.25H2O) C,
H, N.
3. Ir r ever sible Bin d in g Assa y. Caudate-putamen tissue
from rat brain was homogenized in 30 vol of ice-cold modified
Krebs-HEPES buffer (see above) and centrifuged at 20000g
for 10 min at 4 °C. The resulting pellet was then washed one
additional time by resuspension in ice-cold buffer and cen-
trifugation. The tissue was then incubated on ice for 1 h in
the presence of drug-free buffer, buffer with 100 nM 16, or
buffer containing 10 µM 16. After this, the tissue was
resuspended in cold buffer and subjected to two more cen-
trifugations, each in 30 vol of buffer, as above. Twelve-point
[3H]WIN 35,428 saturation curves were performed over a
concentration range of 0.1-25 nM [3H]WIN 35,428, which was
added at the beginning of a 1-h incubation on ice, as described
above.
9-[3-[cis-3,5-Dim eth yl-4-(2-isoth iocya n a toeth yl)-1-p ip -
er a zin yl]p r op yl]ca r ba zole (11). Using general procedure
C with amine 9 yielded the product as an off-white solid, 55%.
The HCl salt was formed and recrystallized in EtOH:2-PrOH:
mp 203-204 °C dec; IR (CHCl3) 2108 (NCS) cm-1; 1H NMR δ
1.07 (6H, d, J ) 6.2 Hz, 2 × Me), 2.66 (4H, d, J ) 9 Hz, 2H of
piperazine, CH2N of side chain), 2.95 (2H, dd, J ) 7.3 Hz, 2H
of piperazine), 3.52 (2H, t, J ) 7.7 Hz, CH2NCS), 4.39 (2H, t,
J ) 6.5 Hz, NCH2 at carbazole); 13C NMR δ 18.37, 25.64, 40.37,
43.10, 48.78, 54.55, 55.24, 60.69, 108.72, 118.68, 120.18,
122.71, 125.41, 140.36. Anal. (C24H30N4S‚2HCl‚0.5H2O) C, H,
N.
4. Da ta An a lysis. Saturation data were analyzed by the
use of the nonlinear least-squares curve-fitting computer

4346 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Husbands et al.
program LIGAND.21 Saturation data from replicate experi-
ments were modeled together to produce a set of parameter
estimates (KD and Bmax values) and the associated standard
errors of these estimates. Protein values were determined
using a modification of the Lowry procedure.22
Sigma Receptors in Rat Brain. Life Sci. Pharmacol. Lett. 1990,
47, PL133-137.
(8) Matecka, D.; Rothman, R. B.; Radesca, L.; de Costa, B. R.;
Dersch, C. M.; Partilla, J . S.; Pert, A.; Glowa, J . R.; Wojnicki, F.
H. E.; Rice, K. C. Development of Novel, Potent, and Selective
Dopamine Reuptake Inhibitors through Alteration of the Pip-
erazine Ring of 1-[2-(Diphenylmethoxy)ethyl]- and 1-[2-(Bis(4-
fluorophenyl)methoxy)ethyl]-4-(3-phenylpropyl)piperazines
(GBR12935 and GBR12909). J . Med. Chem. 1996, 39, 4704-
4716.
5. σ-Bin d in g Assa ys. σ1-Receptors were labeled as de-
scribed previously, using the σ1-selective probe [3H]-(+)-
pentazocine and guinea pig brain membranes.20 Guinea pig
membranes (350-500 µg of membrane protein) were incubated
with 3 nM [3H]-(+)-pentazocine in a total volume of 0.5 mL of
50 mM Tris-HCl, pH 8.0. Incubations were carried out for 120
min at 25 °C. Nonspecific binding was determined in the
presence of 10 µM unlabeled haloperidol. Assays were termi-
nated by dilution with 5 mL of ice-cold Tris-HCl, pH 8.0, and
vacuum filtration through glass fiber filters using a Brandel
cell harvester. Filters were then washed twice with 5 mL of
ice-cold 10 mM Tris-HCl, pH 8.0. Filters were soaked in 0.5%
poly(ethylenimine) for at least 30 min at 25 °C prior to use.
Filters were counted in CytoScint cocktail (ICN, Costa Mesa,
CA) after an overnight extraction of counts. Membranes were
prepared from frozen guinea pig brains (minus cerebella) as
previously described.23
σ2-Receptors were labeled as previously described using rat
liver membranes, a rich source of σ2-sites, and [3H]-1,3-di-o-
tolylguanidine ([3H]DTG) in the presence of 1 µM dextrallor-
phan to mask σ1-receptors.23 Assays were performed in 50 mM
Tris-HCl, pH 8.0, for 120 min at 25 °C in a volume of 0.5 mL
with 160 µg of membrane protein and 5 nM radioligand.
Nonspecific binding was determined in the presence of 10 µM
haloperidol. All other manipulations were as described for the
σ1-receptor assay. Rat liver membranes were prepared from
the livers of male Sprague-Dawley rats as previously de-
scribed.23
(9) Izenwasser, S.; Newman, A. H.; Katz, J . L. Cocaine and Several
σ-Receptor Ligands Inhibit Dopamine Uptake in Rat Caudate-
Putamen. Eur. J . Pharmacol. 1993, 243, 201-205.
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Heterogeneity of Dopamine Transporter Binding and Function
and the Behavioral Pharmacology of Cocaine. Pharmacol. Bio-
chem. Behav. 1997, 57, 1-8.
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(13) Burke, T. R., J r.; J acobson, A. E.; Rice, K. C.; Silverton, J . V.;
Simonds, W. F.; Streaty, R. A.; Klee, W. A. Probes for Narcotic
Receptor Mediated Phenomena. 12. cis-(+)-3-Methylfentanyl
Isothiocyanate, a Potent Site Directed Acylating Agent for δ
Opioid Receptors - Synthesis, Absolute Configuration, and
Receptor Enantioselectivity. J . Med. Chem. 1986, 29, 1087-
1093.
(14) Rehavi, M.; Ittah, Y.; Skolnick, P.; Rice, K. C.; Paul, S. M.
Nitroimipramines - Synthesis and Pharmacological Effects of
Potent Long Acting Inhibitors of [3H] Serotonin Uptake and [3H]
Imipramine Binding. Naunyn-Schmeideberg’s Arch. Pharmacol.
1982, 320, 45-49.
(15) Gareau, Y.; Zamboni, R.; Wong, A. W. Total Synthesis of N-Me
LTC0: A Novel Methodology for the Monomethylation of Amines.
J . Org. Chem. 1993, 58, 1582.
(16) Newman, A. H.; Lueddens, H. W. M.; Skolnick, P.; Rice, K. C.
Novel Irreversible Ligands Specific for “Peripheral” Type Ben-
zodiazepine Receptors: (()-, (+)-, and (-)-1-(2-Chlorophenyl)-
N-(2-isothiocyanatoethyl)-3-isoquinolinecarboxamide and 1-(2-
Isothiocyanatoethyl)-7-chloro-1,3-dihydro-5-(4-chlorophenyl)-2H-
1,4-benzodiazepin-2-one. J . Med. Chem. 1987, 30, 1901.
(17) Shah, J . H.; Izenwasser, S.; Geter-Douglass, B.; Witkin, J . M.;
Newman, A. H. (()-(N-Alkylamino)benzazepine Analogues: Novel
Dopamine D1 Receptor Antagonists. J . Med. Chem. 1995, 38,
4284-4293.
(18) Izenwasser, S.; Rosenberger, J . G.; Cox, B. M. The Cocaine
Analogue WIN 35,428 Binds to Two Sites in Fresh Rat Caudate-
putamen under the Correct Assay Conditions. Life Sci./ Phar-
macol. Lett. 1993, 52, PL141-145.
(19) de Costa, B. R.; Bowen, W. D.; Hellewell, S. B.; Walker, J . M.;
Thurkauf, A.; J acobson, A. E.; Rice, K. C. Synthesis and
Evaluation of Optically Pure [3H](+)-Pentazocine, a Highly
Potent and Selective Radioligand for Sigma Receptors. FEBS
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(20) Bowen, W. D.; de Costa, B. R.; Hellewell, S. B.; Walker, J . M.;
Rice, K. C. [3H](+)-Pentazocine: A Potent and Highly Selective
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ropharmacol. 1993, 3, 117.
Ack n ow led gm en t. We would like to thank Phyllis
Terry for her expert technical assistance. Animals were
cared for according to the PHS guidelines and the NIH
guide for the care and use of laboratory animals.
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