Novel azido and isothiocyanato analogues of [3-(4-phenylalkylpiperazin-1- yl)propyl]bis(4-fluorophenyl)amines as potential irreversible ligands for the dopamine transporter

Article abstract of DOI:10.1021/jm049670w

Potential irreversible ligands were prepared, based on a series of 3-(1-piperazinyl)propyl-N/N-bis(4-fluorophenyl)amines, as molecular probes for the dopamine transporter (DAT). Both azidoand isothiocyanato-substituted phenylalkyl analogues were synthesiz

Full text of DOI:10.1021/jm049670w

6128
J. Med. Chem. 2004, 47, 6128-6136
Articles
Novel Azido and Isothiocyanato Analogues of
[3-(4-Phenylalkylpiperazin-1-yl)propyl]bis(4-fluorophenyl)amines as Potential
Irreversible Ligands for the Dopamine Transporter
Jianjing Cao,† John R. Lever,‡ Theresa Kopajtic,§ Jonathan L. Katz,§ Anh T. Pham,| Muhsinah L. Holmes,|
Joseph B. Justice,| and Amy Hauck Newman*,†
Medicinal Chemistry and Psychobiology Sections, Intramural Research Program, National Institute on Drug Abuse,
5500 Nathan Shock Drive, Baltimore, Maryland 21224, Department of Radiology, University of MissourisColumbia, and
Harry S. Truman Veterans Hospital, 800 Hospital Drive, Columbia, Missouri, 65201, and Department of Chemistry,
Emory University, Atlanta, Georgia 30322
Received May 3, 2004
Potential irreversible ligands were prepared, based on a series of 3-(1-piperazinyl)propyl-N,N-
bis(4-fluorophenyl)amines, as molecular probes for the dopamine transporter (DAT). Both azido-
and isothiocyanato-substituted phenylalkyl analogues were synthesized and evaluated for
displacement of [3H]WIN 35 428 in rat caudate putamen tissue. All of the analogues showed
moderate binding potencies at the DAT. The azido analogue, 16b, was radioiodinated and used
to photolabel human DAT-transfected HEK 293 cell membranes. [125I]16b irreversibly labeled
an ∼80 kDa band corresponding to the DAT detected by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. This radioligand provides a novel addition to the growing arsenal of
structurally diverse irreversible ligands that are being used to identify binding domains on
the DAT. Characterizing points of attachment of these irreversible probes to the DAT protein
will ultimately help elucidate the three-dimensional arrangement of the transmembrane
domains, identify individual binding sites of the DAT inhibitors, and direct future drug design.
The development of irreversible ligands for the char-
acterization of drug molecule-receptor protein interac-
tions has been a successful strategy for more than three
decades.1 With the technological advances in molecular
pharmacology, the application of these ligands for the
identification of binding sites, at the molecular level,
has resulted in a resurgence of interest in designing and
implementing these highly selective molecular probes.2
The dopamine transporter (DAT) is a primary target
of psychomotor stimulant drugs of abuse, such as
cocaine. Significant and rapid occupation of mesolimbic
dopamine transporters that results in the inhibition of
dopamine reuptake by cocaine is directly related to its
psychostimulant and reinforcing effects.3,4 Considerable
investigation of numerous chemical classes of dopamine
uptake inhibitors has provided ligands with both high
affinity and selectivity for the DAT.5 Many of these
ligands have provided important molecular tools with
which to further elucidate the role of the DAT in cocaine
abuse and to provide leads for potential medications for
its treatment.2,5-7 In addition, structure-activity rela-
tionships have provided direction toward the design of
specific molecular tools such as irreversible ligands for
the DAT.
An early report of the photolabel DEEP (1, Figure 1),
based on the potent dopamine uptake inhibitor GBR
12909, described its covalent attachment to a 58 kDa
protein in rat striatum, which was characterized as the
DAT.8 Additional reports of both photolabels and other
electrophile-substituted (e.g., NCS) analogues of dopam-
ine uptake inhibitors were subsequently described.9-13
Initial studies that used epitope-specific immunopre-
cipitation of proteolytic fragments of the DAT protein
showed [125I]DEEP was incorporated near transmem-
brane (TM) domains 1 and 2, whereas the 3-phenyltro-
pane photolabel [125I]RTI 82 (2) was incorporated in the
4-7 TM region of the DAT.14,15 These studies were the
first to provide experimental evidence of differences in
the binding domains of these structurally divergent
dopamine uptake inhibitors. In a follow-up study and
as a result of discovering structure-activity relationship
divergence between the two tropane-based classes of
dopamine uptake inhibitors (3-aryltropanes vs benz-
tropines), [125I]GA 2-34 (3) was discovered to photolabel
an 80 kDa protein in rat caudate putamen that was
identified as the DAT.16 Thus, [125I]3 labeled the 1-2
transmembrane region of the DAT protein, as did the
GBR 12909-based photolabel [125I]DEEP. This binding
domain for [125I]3 contrasts to the 4-7 transmembrane
region, identified as the binding domain of the tropane-
based ligand [125I]RTI-82.17 Subsequent studies resulted
in the design of an N-substituted 3-phenyltropane
photolabel, MFZ 2-24 (4),18 which has recently been
* Corresponding author: e-mail anewman@intra.nida.nih.gov.
† Medicinal Chemistry Section, National Institute on Drug Abuse.
‡ University of MissourisColumbia and Harry S. Truman Veterans
Hospital.
§ Psychobiology Section, National Institute on Drug Abuse.
| Emory University.
10.1021/jm049670w CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/30/2004

Potential Irreversible Ligands for Dopamine Transporter
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6129
Figure 1. Chemical structures of selected DAT photolabels.
radioiodinated. [125I]4 is currently being used to identify
amino acid residue(s) involved in covalent attachment
of this ligand to DAT.
More recently, the dual incorporation of another DAT
photoaffinity label, AD-96-129 (5) based on a piperidine
series of GBR 12909 analogues was reported.19,20 Pro-
teolysis and immunoprecipitation studies demonstrated
that [125I]5 photolabeled DAT fragments of 45 and 14
kDa, corresponding to those labeled with [125I]1, and 32
and 16 kDa fragments, corresponding to those labeled
with [125I]2.20,21
These studies encouraged our interest in another class
of dopamine uptake inhibitors, based on rimcazole.22-24
SAR studies have revealed that the rimcazole analogues
show similar trends to those of the GBR 12909 ana-
logues at the DAT. However, divergence in pharmaco-
logical and behavioral activity profiles, in animal models
of cocaine abuse,26 prompted us to further investigate
binding site interaction at the molecular level. Herein
we report the preparation of a series of potential
irreversible ligands based on this class of dopamine
uptake inhibitors and selection of the most promising
analogue for radioiodination and subsequent investiga-
tion of the DAT. Previous attempts at an irreversible
ligand, based on rimcazole, resulted in SH 1-12 (6),
which we reported to bind wash-resistantly to the
DAT.22 In that report, addition of an isothiocyanato
group extended away from the terminal piperazine
nitrogen by an n-hexyl chain resulted in the irreversible
DAT ligand. Subsequent SAR studies showed that, by
replacing the carbazole ring system of rimcazole with a
4,4′-difluorophenylaniline and eliminating the 2,6-di-
methyl substitution on the piperazine ring, ligands with
higher affinity and selectivity for DAT resulted.24,25 In
addition, substitution of the terminal piperazine ring
with alkylaryl groups further improved DAT affinity
and provided a template for both isothiocyanato or azido
groups, with or without iodo substitution. Hence, a

6130 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Cao et al.
Scheme 1a
a Reagents and conditions: (a) K2CO3, DMF/H2O, rt, overnight; (b) H2 (40 psi), Pd/C (10%), MeOH/EtOAc; (c) (1) 4-nitrocinnamic acid,
SOCl2, reflux, 3 h; (2) pentene-stabilized CHCl3, NaHCO3/H2O, rt, 1 h; (d) AlH3, THF.
series of potential irreversible ligands based on this
design (Figure 1) was prepared.
and 67% yield, respectively. All products were purified
by flash column chromatography and converted to the
oxalate salts.
As shown in Scheme 3, [125I]16b was prepared from
11b in a three-step, one-flask sequence involving elec-
trophilic radioiodination under no-carrier-added condi-
tions, followed by diazotization and substitution of the
diazo moiety by azide. Reverse-phase, ion-pair HPLC
was used for purification, and [125I]16b (tR ) 45.2 min,
k′ ) 77) was well resolved from radioactive and nonra-
dioactive side products that might interfere during
photoaffinity labeling trials (Figure 2). Based upon route
of synthesis, chromatographic lipophilicity, and previous
radiochemical studies of [125I]DEEP28 and [125I]RTI 82,29
the major nonradioactive materials are assigned as the
corresponding azide (tR ) 18.1 min, k′ ) 30) and
chloroazide (tR ) 31.4 min, k′ ) 53). In support of this
hypothesis, the putative chloroazide was not observed
during model studies in the absence of the oxidant
chloramine-T. For four consecutive preparations, iso-
lated yields of pure [125I]16b ranged from 22% to 30%.
Specific radioactivities were within 20% of theoretical
and ranged from 1811 to 1942 mCi/µmol.
Chemistry
The syntheses of novel analogues 14a-c, 16a-c, and
17b,c are depicted in Schemes 1 and 2. Previously, we
reported the synthesis of 4,4-difluorphenylamine (7)
using literature methods.25 These methods proved to be
unreliable on a larger scale and we report herein a
significantly improved procedure using the aryl halide
amination catalyst 1,1′-bis(diphenylphosphino)ferrocene
(DPPH).27 Under these conditions, the desired product
was obtained in >90% yield and this reaction could be
reliably used on a multigram scale. In Scheme 1,
compound 8 was prepared as previously reported25 but
with the new method for compound 7. Coupling of 8 with
9a followed by catalytic hydrogenation gave the inter-
mediate aniline 11a in 77% yield. Compound 11b was
prepared in a similar manner with its respective alky-
lating agent 9b. Compound 11c was prepared by the
coupling of 8 with 4-nitrocinnamoyl chloride, followed
by hydrogenation to 13 and reduction of the amide
carbonyl with AlH3 to give aniline 11c. In Scheme 2,
compounds 11a-c reacted with CSCl2, under classical
biphasic conditions, to give products 14a-c in 55-61%
yield. Amines 11a-c reacted with ICl in glacial acetic
acid to give iodo-substituted products 15a-c in 39-63%
yield, which were then treated with NaNO2 followed by
NaN3 to give the azido-iodo products 16a-c in 83-100%
yield. Iodo-substituted compounds 15b and 15c were
treated with CSCl2 to give products 17a and 17b in 62%
Results and Discussion
Compounds 14a-c, 16a-c, and 17b,c were evaluated
for binding at DAT by displacement of [3H]WIN 35 428
in rat caudate putamen (Table 1). The IC50 values show
that addition of the 3′-iodo, 4′-azido groups on the
alkylphenyl side chains decreased binding affinities as
compared to the corresponding unsubstituted arylalkyl

Potential Irreversible Ligands for Dopamine Transporter
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6131
Scheme 2a
a Reagents and conditions: (a) CSCl2, NaHCO3, CHCl3/H2O, 0 °C, 3 h; (b) ICl, HOAc (glacial), rt, 3 h; (c) (1) NaNO2, HOAc/H2O (1:1),
0 °C, 30 min; (2) NaN3, 0 °C, 30 min.
Scheme 3a
a Reagents and conditions: (a) [125I]NaI, chloramine-T, NaOAc (0.3 M, pH 5.0)/MeOH, rt, 30 min; (b) HOAc (3.0 M), NaNO2, (0.5 M),
-10 °C; (c) NaN3, rt, 20 min, Na2S2O5 (50 mM).
substituted analogues, for example, 18 vs 16a and 19
vs 16c.25 This is consistent with other series of com-
pounds such as the photolabel based on N-phenyl-n-
butyl-4′,4′′-difluorobenztropine, 2,16 and the piperidine-
based photolabel 3,19 and may reflect steric bulk and/
or electronic properties of these substituents that are
not as well tolerated at the DAT. There was not a
significant difference in binding affinities between the
N-benzyl, N-ethylphenyl, and N-propylphenyl analogues
in either the azido- or isothiocyanato-substituted groups.
One observation in this series was that the isothio-
cyanato analogues consistently showed a 2.5-3-fold
higher binding potency for DAT than their correspond-
ing iodo-azido analogues (e.g., 14a vs 16a, 14b vs 16b,
and 14c vs 16c). This increase in potency could not be
attributed to the detrimental effect of the 3′-iodo group
in the azides, as when the 3′-iodo group was added to
the isothiocyanates binding affinities were not reduced
(e.g., 17b and 17c). The binding assay procedure did
not include photoactivation, so presumably the azides
are not forming a reactive electrophile to covalently
attach to an amino acid residue on or near the binding
site. However, the isothiocyanates need no photoacti-
vation and are fully reactive as electrophiles under the

6132 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Cao et al.
Table 1. Binding Data at DAT for Azido and Isothiocyanato Analogues of
3-(4-[3-Phenylalkyl]-1-piperazinyl)Propyl-N,N-bis(4-fluorophenyl)amines
compd
R
DAT, [3H]WIN 35 428 IC50 ( SEMa (nM)
Hill coefficient
16a
CH2-3-I, 4-N3-Ph
242 ( 30
1.84b ( 0.40
1.54b ( 0.29
1.61b ( 0.23
1.06 ( 0.13
1.22b ( 0.14
1.32b ( 0.15
1.05 ( 0.13
1.70b ( 0.25
1.19 ( 0.15
1.44b ( 0.12
1.63b ( 0.16
1.56b ( 0.12
16b
CH2CH2-3-I, 4-N3-Ph
CH2CH2CH2-3-I, 4-N3-Ph
CH2-4-NCS-Ph
163 ( 25
16c
282 ( 28
14a
75.4 ( 3.5
68.3 ( 6.6
99.8 ( 9.3
66.0 ( 9.2
95.5 ( 12
29.7 (4.1
19.5 ( 2.9
21.1 ( 2.18
5.61 ( 0.82d
14b
CH2CH2-4-NCS-Ph
CH2CH2CH2-4-NCS-Ph
CH2CH2-3-I, 4-NCS-Ph
CH2CH2CH2-3-I, 4-NCS-Ph
CH2-Ph
14c
17b
17c
18c
19c
CH2CH2CH2-Ph
GBR 12909
1; DEEP
a The binding methods used are as reported in ref 33 and described in the Experimental Methods section. b This coefficient is significantly
different from 1.0 (p < 0.05). c Synthesis and Ki values for these compounds were previously reported in ref 25. d Sucrose PO4 buffer.
possible. Additional characterization of these covalent
interactions is ongoing.
In general, Hill coefficients of these ligands are >1,
suggesting that the actions of these drugs at the site
labeled by [3H]WIN 35 428 does not strictly follow the
law of mass action. As these compounds are potentially
irreversible, covalent attachment and lack of equilibria
in binding might account for these values. In fact, Dutta
et al.21 reported that the DAT photolabel 5 had a Hill
coefficient of 2. Subsequent studies using immunopre-
cipitation and protease cleavage of the photolabeled
DAT showed dual incorporation of this ligand, suggest-
ing attachment to two separate or overlapping sites on
DAT. Further discussion of Hill coefficients of >1 for
potential irreversible ligands, based on GBR 12935, has
been reported.10
On the basis of these studies and our interest in
further characterizing the binding domains of irrevers-
ible DAT ligands with structural diversity,17 we selected
the azido analogue 16b to radioiodinate for further
investigation. Using procedures similar to those em-
Figure 2. HPLC chromatogram for purification of [125I]16b.
A 50% incorporation of radioiodine into a peak at 45.2 min,
well resolved from side products and appropriate for [125I]16b,
was observed. The mass of the UV peak associated with the
radioactivity was 380 pmol.
ployed for [125I]DEEP,28 125I]RTI 82,29 125I]3,16 and [125I]-
[ [
5,20 we prepared [125I]16b in moderate yield, with high
purity and high specific radioactivity. The radioligand
was formulated as a concentrated ethanol solution
buffered with pH 7.4 Tris-HCl and was stable when
stored at -20 °C in the dark.
conditions of the binding assay. Thus, we speculate that
the lower IC50 values may be due to covalent attachment
to the DAT protein, which results in an apparent higher
binding affinity for the isothiocyanates. Indeed, a 3-fold
decrease might not be considered very significant when
IC50 values are compared. However, these small differ-
ences have been reported in DAT site-directed mu-
tagenesis studies, wherein a 3-fold change in cocaine’s
binding affinity has been attributed to a single amino
acid residue change that reduces either direct or indirect
interaction of the ligand with the mutated protein.30,31
Others have reported potential covalent interactions
with cysteine sulfhydryl groups on the DAT with ir-
reversible and dopamine sparing antagonist.32 Although
the chemical structures of those ligands and these
reported herein are significantly different, it suggests
that covalent interaction with a cysteine SH on or near
the binding domain of these DAT ligands is certainly
Membranes were prepared as described in the Ex-
perimental Methods section from HEK-293 cells stably
transfected with the human DAT engineered with a 6×-
histidine and Flag epitope tags on the N-terminus
(F6×H-hDAT). Photolabeling with [125I]16b was per-
formed and the solubilized [125I]16b-labeled protein was
applied directly to SDS-7.5% PAGE. A Western blot of
the gel, probed with antibody directed to the Flag
epitope incorporated into the hDAT, shows the F6×H-
hDAT at approximately 80 kDa (Figure 3). A 24 h
exposure of film overlaid onto the developed Western
blot generated an autoradiograph in which a dominant
band was also observed at approximately 80 kDa
(Figure 3), supporting the interpretation that [125I]16b

Potential Irreversible Ligands for Dopamine Transporter
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6133
compounds in rat caudate putamen by displacement of
[3H]WIN 35 428. Reduction in binding potency of ∼10-
fold for the iodo-azido analogues, as compared to their
unsubstituted parent compounds, was observed. How-
ever, this reduction in potency was reduced to ∼3-fold
for the analogous isothiocyanates. As photolabeling
experiments are still preferable for further character-
ization of DAT binding domains, using immunoprecipi-
tation and proteolytic techniques, compound 16b was
selected for radioiodination. [125I]16b labeled an ∼80
kDa peptide in rat striatum that has been identified as
the DAT. Further investigation by use of this and other
irreversible probes will ultimately provide structural
characterization of the DAT that has remained elusive
due in part to the lack of X-ray data on the crystalline
form of the functional DAT protein.
Figure 3. Autoradiograph of a western blot of [125I]16b-
labeled hDAT. Solubilized N3×Flag6×his-hDAT membranes
labeled with [125I]16b were separated on a 7.5% Tris glycine
minigel and transferred onto a PVDF membrane, as described
in the Experimental Methods section. On the right (b) is the
Western blot after being probed with the anti-Flag antibody
and visualized. A major band is observed at approximately 80
kDa. On the left (a) is an autoradiograph of the western blot.
The radioactive signal obtained from [125I]16b on the film
aligns with the 80 kDa signal obtained from hDAT on the
western blot.
Experimental Methods
All melting points were determined on a Thomas-Hoover
melting point apparatus and are uncorrected. The 1H and 13
C
NMR spectra were recorded on a Varian AS400 instrument.
Samples were dissolved in an appropriate deuterated solvent
(CDCl3 or D2O). Proton and carbon chemical shifts are reported
as parts per million (δ) relative to tetramethylsilane (Me4Si;
0.00 ppm), which was used as an internal standard. Mass
spectra were recorded on a Hewlett-Packard (Palo Alto, CA)
HP 6890 series. Infrared spectra were recorded as a neat film
on KBr plates with a Perkin-Elmer RX FT-IR. Microanalyses
were performed by Atlantic Microlab, Inc. (Norcross, GA) and
agree within (0.4% of calculated values. All flash column
chromatography was performed with flash-grade silica gel
(Aldrich, 230-400 mesh, 60 Å). All chemicals and reagents
were purchased from Aldrich Chemical Co. or Lancaster
Synthesis, Inc. unless otherwise indicated and were used
without further purification. Radioactivity was measured with
a dose calibrator (Capintec CRC-15W) employing similar
counting geometries, coupled with attenuation correction
factors as necessary, for each reading.
Chemistry: 4,4′-Difluorophenylamine (7). 4-Fluorophen-
ylamine (278 mg, 2.5 mmol), DPPF (166 mg, 0.3 mmol),
(DPPF)PCl2‚CH2Cl2 (81.7 mg, 0.01 mmol), and sodium tert-
butoxide (240 mg, 2.5 mmol) were added to 1-fluoro-4-iodo-
benzene (444 mg, 2 mmol) in dried tetrahydrofuran (THF) (4.5
mL) in a pressure tube under argon.27 The reaction mixture
was heated at 100 °C for 3 h and then cooled to room
temperature. HCl (1 M; 4 mL) was added; the mixture was
basified with NaHCO3 to pH 9 and extracted with EtOAc (3
× 15 mL); and the combined organic layer was dried over
MgSO4 and concentrated. The residue was purified by flash
column chromatography (10:1 to 4:1 hexane/EtOAc) to give the
product (380 mg, 93%). MS (EI) 205 (M).
3-[4-(4-Nitrobenzyl)-1-piperazinyl]propyl-N,N-bis(4-
fluorophenyl)amine (10a). To the mixture of DMF (20 mL)
and H2O (1 mL) was added compound 8,25 4-nitrobenzylbro-
mide (432 mg, 2 mmol), and K2CO3 (1.14 g, 8.26 mmol). The
mixture was stirred overnight at room temperature and
filtered. The filtrate was concentrated in vacuo. The residue
was diluted with H2O (20 mL) and extracted with CH2Cl2 (3
× 15 mL). The combined organic layer was dried over MgSO4
and evaporated to give the crude product, which was purified
by flash column chromatography (2% CH2Cl2/MeOH/NH4OH)
to give the pure product (0.83 g, 89%) as an oil. 1H NMR δ
1.74-1.79 (2H, m, J ) 7.24 Hz, CH2), 2.35-2.47 (10H, m), 3.59
(2H, s, N-CH2-aryl), 3.64-3.69 [2H, t, J ) 7.25 Hz, CH2N-
(PhF)2], 6.88-6.98 (10H, m, aryl-Hs), 7.49-7.52 (2H, d, J )
8.75 Hz, aryl-Hs).
3-[4-(4-Aminobenzyl)-1-piperazinyl]propyl-N,N-bis(4-
fluorophenyl)amine (11a). Compound 10a (1.25 g, 2.68
mmol) was dissolved in a mixture of MeOH (30 mL) and EtOAc
(30 mL), and a catalytic amount of Pd/C (10%) was added. The
mixture was placed on a Parr hydrogenator at 40 psi for 0.5 h
and then filtered over Celite. The filtrate was concentrated.
labels hDAT. The theoretical weight of the unglycosy-
lated form of the histidine-tagged hDAT is 76 kDa. The
80 kDa band is believed to be the mature glycosylated
Flag/histidine-tagged DAT. The immunoblot signal
obtained is consistent with western blot analysis rou-
tinely performed on hDAT.
Under the assay conditions of this procedure, if
covalent attachment of [125I]16b had not occurred, the
radiolabeled band would not be present. Solubilization
of the proteins from the membrane would free any
reversibly bound ligand and any other noncovalently
bound ligand in the membrane. The protein is also
denatured on the gel and any reversibly bound ligand,
if not already washed out in solubilizing the protein,
would migrate to the bottom of the gel. Together these
experiments demonstrate that [125I]16b photolabels the
DAT protein and can now be used for additional
investigation of its binding domain(s).
Summary
Significant indirect support for differing binding
domains on the DAT of structurally diverse DAT inhibi-
tors has provided a direction toward the design of novel
ligands that may have different pharmacological profiles
from cocaine.2 One approach to further characterizing
the DAT inhibitor binding sites is to prepare ligands
that can potentially form a covalent linkage with an
amino acid residue to ultimately identify points of
attachment on the DAT protein. To this end, photoaf-
finity ligands based on several classes of DAT inhibitors
have been designed and have demonstrated early suc-
cess.1,17,20 The DAT inhibitors, based on rimcazole, have
provided a pharmacologically distinct class of ligands
that do not demonstrate a cocaine-like behavioral
profile.26 SAR comparisons to other DAT inhibitors have
shown distinctions that might be explained by a differ-
ing binding mode that could be exploited toward medi-
cation development.23-25 Thus, we reasoned that further
characterization of the binding domain on the DAT, by
this class of compounds, might be achieved by use of
irreversible ligands, and hence a series of eight 4′-
isothiocyanato-, 3′-iodo-4′-isothiocyanato-, and 3′-iodo-
4′-azido-substituted phenylalkylpiperazines were pre-
pared. DAT binding data were obtained for these

6134 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Cao et al.
The residue was purified by flash column chromatography (2%
CH2Cl2/MeOH/NH4OH) to give the pure product (0.90 g, 86%)
as a yellow oil. 1H NMR δ 1.67-1.80 (2H, m, CH2), 2.23-2.44
(10H, m), 3.40 (2H, s, N-CH2-aryl), 3.62-3.67 [4H, m, CH2N-
(PhF)2, aryl-NH2], 6.63-6.65 (2H, m, aryl-Hs), 6.87-6.97 (8H,
m, aryl-Hs), 7.07-7.10 (2H, m, aryl-Hs).
3-[4-(4-Aminophenylethyl)-1-piperazinyl]propyl-N,N-
bis(4-fluorophenyl)amine (11b) was prepared as described
for 11a from 8 (660 mg, 2.0 mmol) by use of 2-(4-nitrophenyl)-
ethyl bromide (552 mg, 2.4 mmol). After coupling and hydro-
genation, the product (820 mg, 91%) was obtained as an oil.
1H NMR δ 1.73-1.88 (2H, m, CH2), 2.35-2.72 (14H, m), 3.64-
3.69 [2H, t, CH2N(PhF)2], 6.60-6.65 (2H, m, aryl-Hs), 6.88-
7.00 (10H, m, aryl-Hs).
3-[4-(3-Iodo-4-aminobenzyl)-1-piperazinyl]propyl-N,N-
bis(4-fluorophenyl)amine (15a). ICl (267 mg, 1.64 mmol)
in glacial acetic acid (5.6 mL) was added very slowly (over 3
h) to 11a (650 mg, 1.49 mmol) in glacial acetic acid (34.6 mL)
at room temperature. Acetic acid was then removed in vacuo.
The residue was diluted with water (20 mL) and then basified
with NH4OH and extracted with CH2Cl2 (3 × 15 mL). The
combined organic layer was dried over MgSO4 and concen-
trated. The crude product was purified by flash column
chromatography (1% CH2Cl2/MeOH/ NH4OH) to give the pure
product (0.53 g, 63%) as a brown oil.
3-[4-(3-Iodo-4-aminophenylethyl)-1-piperazinyl]propyl-
N,N-bis(4-fluorophenyl)amine (15b) was prepared as de-
scribed for 15a from 11b (650 mg, 1.44 mmol) to give the
product (440 mg, 53%) as a brown oil.
3-[4-(3-Iodo-4-aminophenylpropyl)-1-piperazinyl]-
propyl-N,N-bis(4-fluorophenyl)amine (15c) was prepared
as described for 15a from 11c (600 mg, 1.29 mmol) to give the
product (300 mg, 39%) as a brown oil.
3-[4-(3-Iodo-4-azidobenzyl)-1-piperazinyl]propyl-N,N-
bis(4-fluorophenyl)amine (16a). Compound 15a (258 mg,
0.46 mmol) was dissolved in glacial acetic acid (1.5 mL) and
H2O (1.5 mL). To the solution was added NaNO2 (44.6 mg,
0.65 mmol). The mixture was stirred at 0 °C for 30 min. Then
NaN3 (43.7 mg, 0.67 mmol) was added and the mixture was
stirred at 0 °C for another 30 min. The reaction mixture was
diluted with water (10 mL), basified with NaHCO3 to pH 9,
and extracted with CH2Cl2 (3 × 7 mL). The combined organic
layer was dried over MgSO4 and concentrated. The residue
was purified by flash column chromatography (2% CH2Cl2/
MeOH/NH4OH) to give the pure product (270 mg, 100%),
which was converted to the oxalate salt and recrystallized from
MeOH, mp 211 °C. 1H NMR δ 1.74-1.79 (2H, m, J ) 7.30 Hz,
CH2), 2.34-2.44 (10H, m), 3.43 (2H, s, N-CH2-aryl), 3.63-3.68
[2H, t, J ) 7.18 Hz, CH2N(PhF)2], 6.90-7.09 (9H, m, aryl-Hs),
7.26-7.35 (1H, m, aryl-H), 7.75 (1H, m, aryl-H); 13C NMR δ
25.1, 51.0, 53.4, 53.5, 55.9, 61.9, 88.0, 116.1, 116.4, 118.5, 122.6,
122.7, 130.6, 137.3, 140.7, 140.8, 145.0, 159.9, 168.2; Anal.
[C26H27F2IN6‚2C2H2O4‚0.5(2-PrOH)] C, H, N.
3-[4-(4-Aminophenylpropyl)-1-piperazinyl]propyl-N,N-
bis(4-fluorophenyl)amine (11c). A solution of 4-nitrocin-
namic acid (676 mg, 3.5 mmol) in SOCl2 (3.5 mL) was stirred
at reflux for 3 h. The excess thionyl chloride was removed in
vacuo. The pale yellow acid chloride was taken up in 1 mL of
pentene-stabilized CHCl3 and added dropwise to the two-phase
reaction mixture of 8 (1.16 g, 3.5 mmol), H2O (17.5 mL),
pentene-stablized CHCl3 (56 mL), and NaHCO3 (1.75 g, 21
mmol) at 0 °C. The reaction mixture was allowed to warm to
room temperature, stirred for 1 h, washed with H2O (50 mL),
and extracted with CH2Cl2 (3 × 40 mL). The combined organic
layer was dried over MgSO4 and concentrated to give an
orange solid (1.73 g, 98%). The intermediate amide (1.1 g, 68%)
was prepared as described for 11a (the reaction time was 7 h)
and then reduced by AlH3 in THF to give the product 11c as
1
an orange oil (652 mg, 61%). H NMR δ 1.71-1.88 (4H, m),
2.32-2.82 (14H, m), 3.63-3.68 [2H, t, J ) 7.30 Hz, CH2N-
(PhF)2], 6.60-6.64 (2H, m, aryl-Hs), 6.87-6.98 (10H, m, aryl-
Hs).
3-[4-(4-Isothiocyanatobenzyl)-1-piperazinyl]propyl-
N,N-bis(4-fluorophenyl)amine (14a). Compound 11a (240
mg, 0.55 mmol) was dissolved in a mixture of pentene-
stabilized CHCl3 (12 mL), H2O (6 mL), and NaHCO3 (184 mg).
To the vigorously stirring mixture was added freshly distilled
thiophosgene (54.6 l, 0.71 mmol) at 0 °C, and stirring was
continued for 3 h. The two layers were separated, and the
aqueous phase was extracted with CH2Cl2 (3 × 10 mL). The
combined organic layer was dried over MgSO4 and concen-
trated. The residue was purified by flash column chromatog-
raphy (1% CH2Cl2/MeOH/NH4OH) to give the pure product
(160 mg, 61%), which was converted to the oxalate salt and
3-[4-(3-Iodo-4-azidophenylethyl)-1-piperazinyl]propyl-
N,N-bis(4-fluorophenyl)amine (16b) was prepared as de-
scribed for 16a from 15b (220 mg, 0.381 mmol) to get the
product (190 mg, 83%), which was converted to the oxalate
1
salt and recrystallized from MeOH, mp 217 °C (decomp). H
1
NMR δ 1.73-1.82 (2H, m, CH2), 2.35-2.58 (12H, m), 2.75-
2.76 (2H, m), 3.64-3.69 [2H, t, J ) 7.25 Hz, CH2N(PhF)2],
6.88-7.06 (9H, m, aryl-Hs), 7.21-7.24 (1H, m, aryl-H), 7.63-
7.64 (1H, m, aryl-H); 13C NMR δ 25.1, 30.1, 32.8, 51.0, 53.6,
55.9, 60.3, 88.0, 116.1, 116.4, 118.6, 122.6, 122.7, 130.3, 139.3,
139.9, 140.4, 144.9, 145.0, 156.7, 159.9; Anal. [C27H29F2IN6‚
2C2H2O4‚0.5(2-PrOH)] C, H, N.
recrystallized from MeOH, mp 192-194 C. H NMR δ 1.74-
1.80 (2H, m, CH2), 2.34-2.53 (10H, m), 3.48 (2H, s, N-CH2-
aryl), 3.63-3.68 [2H, t, J ) 7.17 Hz, CH2N(PhF)2], 6.88-7.32
(12H, m, aryl-Hs); 13C NMR δ 25.1, 51.0, 53.5, 53.6, 55.9, 62.7,
116.1, 116.4, 122.6, 122.7, 125.9, 130.3, 130.5, 135.4, 138.3,
144.9, 156.7, 159.9, 167.5; Anal. (C27H28F2N4S2C2H2O4), C, H,
N.
3-[4-(3-Iodo-4-azidophenylpropyl)-1-piperazinyl]prop-
yl-N,N-bis(4-fluorophenyl)amine (16c) was prepared as
described for 16a from 15c (130 mg, 0.22 mmol) to give the
product (120 mg, 90%), which was converted to the oxalate
3-[4-(4-Isothiocyanatophenylethyl)-1-piperazinyl]-
propyl-N,N-bis(4-fluorophenyl)amine (14b) was prepared
as described for 14a from 11b (100 mg, 0.22 mmol) to give the
product (60 mg, 55%), which was converted to the oxalate salt
and recrystallized from MeOH, mp 198-199 °C. 1H NMR δ
1.73-1.82 (2H, m, CH2), 2.35-2.60 (12H, m), 2.76-2.81 (2H,
m), 3.64-3.69 [2H, t, J ) 7.21 Hz, CH2N(PhF)2], 6.90-6.97
(8H, m, aryl-Hs), 7.15-7.17 (4H, m, aryl-Hs); 13C NMR δ 25.1,
33.6, 51.0, 53.6, 55.9, 60.4, 116.1, 116.4, 122.6, 122.7, 126.1,
129.5, 130.2, 135.2, 140.4, 144.9, 145.0, 156.7, 159.9, 167.1,
173.8; Anal. (C28H30F2N4S‚2C2H2O4) C, H, N.
3-[4-(4-Isothiocyanatophenylpropyl)-1-piperazinyl]-
propyl-N,N-bis(4-fluorophenyl)amine (14c) was prepared
as described for 14a from 11c (120 mg, 0.26 mmol) to give the
product (80 mg, 61%), which was converted to the oxalate salt
and recrystallized from MeOH, mp 191 °C (decomp); 1H NMR
δ 1.72-1.84 (4H, m), 2.31-2.65 (14H, m), 3.63-3.68 [2H, t, J
) 7.20 Hz, CH2N(PhF)2], 6.88-7.05 (9H, m, aryl-Hs), 6.90-
6.97 (8H, m, aryl-Hs), 7.14-7.18 (4H, m, aryl-Hs); 13C NMR δ
25.1, 28.7, 33.7, 51.0, 53.6, 55.9, 58.1, 116.1, 116.4, 122.6, 122.7,
126.0, 129.2, 129.9, 142.2, 144.9, 145.0, 156.7, 159.9, 167.9;
Anal. (C29H32F2N4S‚2C2H2O4) C, H, N.
1
salt and recrystallized from MeOH, mp 212 °C (decomp). H
NMR δ 1.75-1.82 (4H, m), 2.30-2.60 (14H, m), 3.64-3.68 [2H,
t, J ) 7.23 Hz, CH2N(PhF)2], 6.88-7.05 (9H, m, aryl-Hs), 7.18-
7.21 (1H, m, aryl-H), 7.62-7.63 (1H, m, aryl-H); 13C NMR δ
23.8, 25.1, 26.8, 28.7, 30.1, 32.6, 32.8, 51.0, 53.6, 55.9, 57.9,
88.1, 116.1, 116.4, 118.6, 122.6, 122.7, 125.4, 130.0, 135.6,
139.6, 140.2, 141.1, 144.9, 145.0, 156.7, 159.9; Anal. [C28H31F2-
IN6‚2C2H2O4‚0.5(2-PrOH)] C, H, N.
3-[4-(3-Iodo-4-(Isothiocyanato)phenylethyl)-1-piper-
azinyl]propyl-N,N-bis(4-fluorophenyl)amine (17b) was
prepared as described for 14a from 15b (210 mg, 0.35 mmol)
to give the product (150 mg, 67%), which was converted to the
oxalate salt and recrystallized from MeOH, mp 206 °C (de-
1
comp). H NMR δ 1.73-1.82 (2H, m, CH2), 2.34-2.58 (12H,
m), 2.70-2.81 (2H, m), 3.64-3.69 [2H, t, J ) 7.15 Hz, CH2N-
(PhF)2], 6.91-6.98 (8H, m, aryl-Hs), 7.16 (2H, m, aryl-Hs), 7.66
(1H, m, aryl-H); 13C NMR δ 25.1, 33.0, 51.0, 53.6, 55.9, 60.0,
94.4, 116.1, 116.4, 122.6, 122.7, 127.0, 130.0, 133.3, 139.9,

Potential Irreversible Ligands for Dopamine Transporter
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6135
141.8, 144.9, 145.0, 156.7, 159.9, 167.9; Anal. (C28H29F2IN4S‚
2C2H2O4‚CH3COCH3) C, H, N.
through Whatman GF/B filters, presoaked in 0.1% BSA, by
use of a Brandel R48 filtering manifold (Brandel Instruments,
Gaithersburg, MD). The filters were washed twice with 5 mL
of cold buffer and transferred to scintillation vials. Beckman
Ready Safe (3.0 mL) was added and the vials were counted
the next day in a Beckman 6000 liquid scintillation counter
(Beckman Coulter Instruments, Fullerton, CA). Data were
analyzed by use of GraphPad Prism software (San Diego, CA).
3-[4-(3-Iodo-4-(Isothiocyanato)phenylpropyl)-1-piper-
azinyl]propyl-N,N-bis(4-fluorophenyl)amine (17c) was
prepared as described for 14a from 15c (120 mg, 0.20 mmol)
to give the product (80 mg, 62%), which was converted to the
oxalate salt and recrystallized from MeOH, mp 215 °C (de-
comp). 1H NMR δ 1.73-1.79 (4H, m), 2.29-2.60 (14H, m),
3.63-3.68 [2H, t, J ) 7.50 Hz, CH2N(PhF)2], 6.91-6.14 (9H,
m, aryl-Hs), 7.16-7.23 (1H, m, aryl-H), 7.62-7.64 (1H, m, aryl-
H); 13C NMR δ 25.1, 28.5, 30.1, 33.1, 51.0, 53.6, 55.9, 57.8,
94.4, 116.1, 116.4, 122.6, 122.7, 127.0, 129.7, 133.0, 139.7,
143.6, 144.9, 145.0, 156.7, 159.9, 167.9; Anal. [C29H31F2IN4S‚
2C2H2O4‚0.5(2-PrOH)] C, H, N.
Labeling hDAT. HEK 293 cells stably expressing DAT
were obtained as previously described.34 HEK-DAT cells were
subsequently modified with an N-terminal 3× Flag and an
N-terminal 6× histidine affinity tag.35 Cells were washed with
Krebs-Ringer-HEPES (KRH) buffer (120 mM NaCl, 4.7 mM
KCl, 2.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 10
mM glucose, pH 7.4), followed by lysis in 2 mM HEPES and 1
mM EDTA for 10 min at 4 °C. Lysed cells were scraped and
centrifuged (31000g, 4 °C, 20 min). Pellets and membranes
were resuspended twice in incubation buffer (100 mM NaCl
and 50 mM Tris base, pH 7.0), sonicated (Heat Systems-
Ultrasonics, model W-185F) for 10 s, and centrifuged as above.
Each pellet was then resuspended by sonicating in 150 mL of
incubation buffer, to which [125I]16b was added to a final
concentration of 30 nM, and the mixture was incubated for 1
h on ice. The membrane/ligand suspension was transferred to
a 60 mm × 15 mm culture dish (Corning) and irradiated for
45 s three times with a shortwave (254 nm) UVG-11 Miner-
alight lamp (UVP). To wash away unreacted [125I]16b, the
preparations were diluted 1:2 in incubation buffer and cen-
trifuged (20000g, 4 °C, 20 min). Pellets were resuspended in
1 mL of incubation buffer and centrifuged again. The resulting
pellets were brought up in 400 µL of Flag solubilization buffer
(1% Triton X-100, 1 mM EDTA, 150 mM NaCl, and 50 mM
Tris-HCl, pH 7.4) and thoroughly resuspended by repeatedly
drawing the mixture through a pipet tip. Solubilization was
carried out overnight at 4 °C. The preparation was then
centrifuged for 1 h at 31000g, 4 °C. The supernatant, contain-
ing the solubilized radiolabeled membrane proteins, was
removed and saved at 4 °C for SDS-PAGE.
Gel Electrophoresis. Precast Tris-HC1 minigels, 7.5%
acrylamide (10 cm × 10 cm × 0.8 mm), were from Bio-Rad.
Samples were prepared by addition of 5 volumes of samples
to 1 volume of SDS-PAGE sample buffer (20% glycerol, 1%
SDS, and 50 mM Tris, pH 6.8, with bromphenol blue). ProSieve
Color Protein Markers (Cambrex) were used for calibration.
Gels were run at 200 V for 30-35 min, on a Bio-Rad Protean
II apparatus with a Bio-Rad PowerPac 1000. The mini gel was
transferred onto a poly(vinylidene difluoride) (PVDF) mem-
brane and probed with primary antibody directed to the Flag
epitope (Sigma).
Immunoblotting. Precast gels were soaked in transfer
buffer consisting of 39 mM glycine, 40 mM Tris, and 10%
MeOH for 15 min. A Fisher Scientific or Bio-Rad semidry
blotting apparatus was used for electrotransfer of proteins to
PVDF membranes. Whatman 3 M filter paper, 2 × 6 layers,
was soaked with transfer buffer, as was the PVDF membrane
following wetting with methanol. Electrotransfer was carried
out for 90 min at 60 mA constant current per gel. After transfer
is complete, the PVDF membrane was allowed to air-dry.
Methanol was used to rehydrate the PVDF membrane. The
membranes were washed twice for 5 min with TBS buffer (150
mM NaCl and 10 mM Tris, pH 7.6). The membranes are then
blocked with blocking buffer (5% nonfat dry milk, 0.1% Tween-
20, 150 mM NaCl, and 10 mM Tris, pH 7.6) for 1 h, with
shaking, at room temperature. The blocked membrane was
then washed three times for 10 min with TTBS (0.1% Tween-
20, 150 mM NaCl, and 10 mM Tris, pH 7.6). Primary antibody
directed against Flag epitope was used according to the
manufacturer’s suggestion. The antibody was prepared in
blocking buffer, applied to the membrane, and incubated
overnight at 4 °C on a shaker. Next, the membranes were
washed three times for 10 min each with TTBS at room
temperature. A secondary antibody directed toward the pri-
mary antibody containing a phosphatase-conjugated IgG
(Pierce) was diluted 1:10 000 in blocking buffer and applied
[
125I]-3-[4-(3-Iodo-4-azidophenylethyl)-1-piperazinyl]-
propyl-N,N-bis(4-fluorophenyl)amine ([125I]16b) was pre-
pared by treating aniline 11b (75 µL, 3.0 mM) in aqueous
NaOAc buffer (pH 5.0; 0.3 M) containing MeOH (50% v/v) at
ambient temperature with no-carrier-added [125I]NaI (20 µL,
2.21 mCi; ca. 1.0 nmol) followed by N-chloro-4-toluenesulfona-
mide (chloramine-T) trihydrate (15 µL, 3.5 mM) for 30 min.
The mixture was chilled at -10 °C, treated with cold HOAc
(50 µL, 3.0 M) followed by NaNO2 (25 µL, 0.5 M), and allowed
to stand for 15 min. Sodium azide (25 µL, 0.5 M) was added,
and the mixture was allowed to warm to ambient temperature
over 20 min. The reaction was quenched with Na2S2O5 (5 µL,
50 mM), and taken up in a syringe along with rinses (2 × 100
µL) of the vessel with the ternary HPLC mobile phase: MeOH
(22.5%), CH3CN (22.5%), and an aqueous solution (55%) of
Et3N (2.1% v/v) and HOAc (2.8% v/v). The Waters HPLC
system was equipped with a UV absorbance detector (254 nm),
a flow-through radioactivity detector, and a Waters C-18 Nova-
Pak column (radial compression module, 8 × 100 mm, 6 µm).
By use of a flow rate of 5 mL/min, radioactive material (tR
)
45.2 min, k′ ) 77) that corresponded to 16b was resolved from
both nonradioactive and radioactive side products. The [125I]-
16b was collected (25 mL), diluted with an equal volume of
distilled water, and passed through an activated (EtOH/water)
solid-phase extraction cartridge (Waters Sep-Pak Light t-C-
18) that was flushed with water (2.5 mL), to remove residual
salts, and then with air. Elution with EtOH (1.0 mL) gave [125I]-
16b (0.66 mCi; 30%) as a concentrated solution. This material
coeluted with 16b (tR ) 12.8 min, k′ ) 17), and displayed g99%
radiochemical purity by HPLC [4 mL/min; 45% aqueous phase
as above, 55% MeOH/CH3CN (1:1)]. Overall recovery of radio-
ligand in the concentration step was 98%. The specific radio-
activity of [125I]16b was calculated as 1942 mCi/µmol by use
of HPLC to determine the mass associated with the UV
absorbance peak area of the carrier in a sample of known
radioactivity. The UV response for nonradioactive 16b was
linear (r2 ) 1.0) by HPLC for a six-point standard curve over
the region of interest (30-450 pmol). Formulations were
supplemented (1% v/v) with Tris-HCl buffer (5 mM, pH 7.4)
prior to storage at -20 °C in the dark.
DAT Binding Assay in Rat Caudate Putamen. Brains
from male Sprague-Dawley rats weighing 200-225 g (Taconic
Labs) were removed, and the striatum was dissected and
placed on ice. Membranes were prepared by homogenizing
tissues in 20 volumes (w/v) 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, and 10 mM
D-glucose, pH adjusted to 7.4) using a Brinkman Polytron
(setting 6 for 20 s) and centrifuged at 20000g for 10 min at 4
°C. The resulting pellet was resuspended in buffer, recentri-
fuged, and resuspended in buffer to a concentration of 25 mg/
mL. Ligand binding experiments were conducted in assay
tubes containing 0.5 mL of modified Krebs-HEPES buffer for
60 min on ice. Each tube contained 1.5 nM [3H]WIN 35 428
(specific activity 84 Ci/mmol) and 2.5 mg striatal tissue
(original wet weight). Nonspecific binding was determined by
use of 0.1 mM cocaine hydrochloride. For determination of
binding affinity, triplicate samples of membranes were pre-
incubated for 5 min in the presence or absence of the compound
being tested. Incubations were terminated by rapid filtration

6136 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
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Acknowledgment. This work was funded by the
NIDA-IRP, DA 15175, DA11176, and DA015805.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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