Rhodamine-labeled 2β-carbomethoxy-3β-(3,4-dichlorophenyl)tropane analogues as high-affinity fluorescent probes for the dopamine transporter

Article abstract of DOI:10.1021/jm050431y

Novel fluorescent ligands were synthesized to identify a high-affinity probe that would enable visualization of the dopamine transporter (DAT) in living cells. Fluorescent tags were extended from the N- or 2-position of 2β-carbomethoxy-3β-(3,4-dichlorophenyl)tropane, using an ethylamino linker. The resulting 2-substituted (5) and N-substituted (9) rhodamine-labeled ligands provided the highest DAT binding affinities expressed in COS-7 cells (K_i = 27 and 18 nM, respectively) in the series. Visualization of the DAT with 5 and 9 was demonstrated by confocal fluorescence laser scanning microscopy in stably transfected HEK293 cells.

Full text of DOI:10.1021/jm050431y

J. Med. Chem. 2005, 48, 7513-7516
7513
Letters
Rhodamine-Labeled
2â-Carbomethoxy-3â-(3,4-dichlorophenyl)-
tropane Analogues as High-Affinity
Fluorescent Probes for the Dopamine
Transporter
Joo Hwan Cha,§ Mu-Fa Zou,§ Erika M. Adkins,†
Søren G. F. Rasmussen,† Claus Juul Loland,†
Bernhard Schoenenberger,‡ Ulrik Gether,† and
Amy Hauck Newman*,§
Medicinal Chemistry Section, National Institute on Drug
AbusesIntramural Research Program,
National Institutes of Health, 5500 Nathan Shock Drive,
Baltimore, Maryland 21224, Molecular Pharmacology
Group, Department of Pharmacology, The Panum Institute,
University of Copenhagen, Blegdamsvej 3, DK-2200,
Copenhagen, Denmark, and
Figure 1. Fluorescent tags.
Fluka GmbH, CH-9471 Buchs, Switzerland
signal-to-noise ratio at the cell surface when using
saturating concentrations of this ligand (Rasmussen and
Gether, unpublished observation). More recently, co-
caine-fluorophore conjugates have been reported that
reproduce affinity constants of [3H]cocaine and thus
represent nonradioactive bioimaging tools with particu-
lar application toward the development of therapeutic
antibodies against cocaine.15
Received May 6, 2005
Abstract: Novel fluorescent ligands were synthesized to
identify a high-affinity probe that would enable visualization
of the dopamine transporter (DAT) in living cells. Fluorescent
tags were extended from the N- or 2-position of 2â-car-
bomethoxy-3â-(3,4-dichlorophenyl)tropane, using an ethylami-
no linker. The resulting 2-substituted (5) and N-substituted
(9) rhodamine-labeled ligands provided the highest DAT
binding affinities expressed in COS-7 cells (Ki ) 27 and 18
nM, respectively) in the series. Visualization of the DAT with
5 and 9 was demonstrated by confocal fluorescence laser
scanning microscopy in stably transfected HEK293 cells.
In this study, we set out to develop new fluorescent
analogues of cocaine displaying high binding affinity for
DAT and lower background staining than previously
prepared ligands. Moreover, we wished to use fluores-
cent moieties with higher photostability and quantum
yield than NBD, such as rhodamine derivatives that dis-
play quantum yields of 0.95-1.00.16 Ideally, we would
also like to obtain selectivity between SERT, DAT, and
the norepinephrine transporter (NET) that also display
high affinity for cocaine; however, for many purposes this
might be of less importance, e.g., when studying systems
where only one of the transporters is expressed. Struc-
ture-activity relationships derived from the 3-phenyltro-
pane class of DAT inhibitors have shown that the 3â-
3,4-diCl-phenyl ring substitution on the tropane ring
gives optimal DAT affinity and that large functional
groups attached to the tropane N- or the 2-position, via
an extended linking chain, are predicted to be well toler-
ated.17 Hence, we designed and synthesized compounds
in which rhodamine red-X NHS ester or the novel fluor-
escent tag N-[trans-4(succinimidyloxycarbonyl)cyclohex-
ylmethyl]sulforhodamine B acid amide, a rhodamine
red-X NHS ester analogue, or ATTO 610-NHS ester ({1-
[3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propyl]-11,11-di-
methyl-2,3,4,11-tetrahydro-1H-naphtho[2,3g]quinolin-
9-ylidene}dimethylammonium, Figure 1) was extended
from either the N- or 2-position of 2â-carbomethoxy-3â-
(3,4-dichlorophenyl)tropane (1), using an ethylamino
linker and the amine-reactive fluorescent molecule
derivatives.
The use of fluorescence techniques to probe the
structure and function of membrane proteins has proven
to be extremely powerful. Numerous applications exist
ranging from biophysical characterization to fluorescent
imaging of membrane proteins in living cells.1-4 Fluo-
rescently labeled high-affinity ligands represent a class
of widely applicable molecular tools. For example,
fluorescent ligands can be used to visualize their target
protein directly in living cells and to characterize the
biophysical microenvironment of a binding site and its
accessibility to the aqueous milieu upon which the
location of the binding site in the protein structure may
be inferred.5-13 Previous studies using a cocaine ana-
logue, which contained the fluorescent moiety nitroben-
zoxadiazol (NBD), were conducted to explore the bio-
physical properties of the cocaine binding site in the rat
serotonin transporter (rSERT).6 This analogue could not
be used for visualization of the SERT or the homologous
DAT transporter in living cells most likely because of
the low brightness of NBD (quantum yields for NBD
derivatives in water are <0.1).14 Thus, the low bright-
ness of the analogue resulted in an unacceptably poor
* To whom correspondence should be addresed. Phone: 410-550-
6568. Fax: 410-550-6855. E-mail: anewman@intra.nida.nih.gov.
§ National Institute on Drug Abuse.
In Scheme 1, 1 was prepared as previously reported18
and de-esterified. Formation of the acid chloride fol-
† University of Copenhagen.
‡ Fluka GmbH.
10.1021/jm050431y CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/05/2005

7514 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Letters
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) 1,4-dioxane/H2O, reflux, 17 h,
73%; (b) (i) POCl3, room temp, 4 h, (ii) hydroxyethylphthalimide,
CH2Cl2, Et3N, room temp, 17 h, 74%, two steps; (c) hydrazine,
ethanol, reflux, 1.5 h, 76%; (d) rhodamine red-X NHS ester,
(iPr)2NEt, DMF, 40-45 °C, 5 days, 43%.
a Reagents and conditions: (a) 2-bromoethylphthalimide, K2CO3,
DMF, 60 °C, 15 h, 93%; (b) hydrazine, ethanol, reflux, 1.5 h, 82%;
(c) rhodamine red-X NHS ester, (iPr)2NEt, DMF, 40-45 °C, 18 h,
87% 9 or the novel analogue of rhodamine red X-NHS ester, DMF,
room temp, 24 h, 68% 10 or ATTO 610-NHS ester, (iPr)2NEt, DMF,
room temp, 24 h, 58% 11.
Table 1. Uptake Inhibition Data at hDAT, hNET, and hSERTa
DAT Ki (nM)
NET Ki (nM)
SERT Ki (nM)
Table 2. hDAT Affinities of the Fluorescent Analogues
compd
[SE interval]
[SE interval]
[SE interval]
Assessed by Competition Bindinga
1
2.3 [1.3-3.9]
72 [45-116]
62 [37-105]
436 [382-497] 627 [594-661]
250 [198-318] 308 [268-354]
5.0 [4.3-5.7]
12 [8-18]
compd
DAT Ki (nM) [SE interval]
5
177 [157-199] 1490 [1340-1660]
9
194 [153-244]
392 [286-537]
705 [450-1440]
227 [197-261]
1
0.2 [0.1-0.5]
27 [23-31]
18 [15-22]
50 [39-65]
10
11
5
9
11
a Ki values (mean of n ) 3 with SE interval) determined
from uptake inhibition experiments in transfected COS-7 cells
([3H]dopamine uptake for hDAT and hNET; [3H]5-HT uptake for
hSERT). The IC50 values used in the estimation of Ki values were
determined from nonlinear regression analysis of uptake data and
calculated from the mean of pIC50 values and the SE interval from
the pIC50 ( SE. For determination of Ki values we used a KM for
[3H]dopamine in DAT (1.1 µM [0.9-1.3]), NET (0.18 µM [0.16-
0.2]), and [3H]5-HT in SERT (1.1 µM [0.8-1.5]) determined in
parallel. Data are the mean with SE intervals of three to six
experiments performed in triplicate.
a Ki values determined from competition binding experiments
on intact COS-7 cells using [3H]CFT (2â-carbomethoxy-3â-(4-
fluorophenyl)tropane) as the radioligand. Ki values were calculated
from nonlinear regression analysis of binding assays performed
as described in Supporting Information. For determination of Ki
values, we used a KD for [3H]CFT determined in parallel. Data
are the mean of two to three experiments performed in triplicate.
To assess the binding affinities of 5 and 9-11 for the
human dopamine transporter (hDAT) in comparison to
the corresponding norepinephrine (hNET) and serotonin
transporters (hSERT), we determined the potency by
which they inhibited [3H]dopamine uptake into COS-7
cells transiently expressing the hDAT or hNET and
[3H]5-HT uptake into COS-7 cells transiently expressing
the hSERT (Table 1). Compounds 5 and 9 demonstrated
the highest apparent binding affinities for hDAT (72 and
62 nM, respectively), whereas 10 and 11 displayed 4-
to 6-fold lower apparent affinities than 5 and 9. During
the course of these studies, we detected that 10 and 11
appeared to be less stable than 5 and 9 and their lower
binding affinities may have reflected some decomposi-
tion in solution. The affinities of all the fluorescent
analogues were lower than those observed for the parent
compound (1) having an apparent affinity of 2.3 nM. To
lowed by esterification with hydroxyethylphthalimide
gave the amine-protected intermediate 3. Deprotection
with hydrazine was followed by reaction with the amine-
reactive rhodamine red-X NHS ester (Molecular Probes).
This reaction was very slow, and the yield of the final
product 5 was not optimal (43%). In Scheme 2, 618 was
reacted with 2-bromoethylphthalimide to give interme-
diate 7. Deprotection was followed by reaction of 8 with
the rhodamine red-X NHS ester, which proceeded in 18
h and gave higher yields (87%) of the final product 9.
As the synthesis of the N-substituted product was more
satisfactory, addition of the two novel fluorescent tags
(Figure 1) was conducted likewise to give 10 and 11.
Isolation of the pure products was achieved by prepara-
tive thin-layer chromatography producing the conju-
gates as red (5, 9, and 10) or blue (11) solids.

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7515
labels. Compounds 5 and 9 also showed relatively high
apparent affinities for the hNET (∼2-fold lower than for
the hDAT), whereas the affinity of 5 for the hSERT was
∼20-fold lower and for 9 ∼4-fold lower than that
observed for hDAT.
The fluorescent properties of 5 and 9-11 were deter-
mined as shown in Figure 2. For compounds 5, 9, and
10 the excitation maxima were 567, 570, and 570 nm,
respectively. The emission maxima for 5, 9, and 10 were
586, 585, and 587 nm, respectively. For 11 the excitation
and emission maxima were 618 and 631 nm, respec-
tively.
On the basis of their chemical stability, binding, and
fluorescent profiles, 5 and 9 were further evaluated for
their capability to label the hDAT by use of confocal
laser scanning microscopy. HEK293 cells stably express-
ing the hDAT, or nontransfected controls cells, were
incubated with 25 nM 5 or 10 nM 9 for the indicated
times in Figure 3. With both fluorescent reagents, DAT
on the cell surface could be visualized within minutes
and with increasing intensity over time, reaching maxi-
mum staining after approximately 20 min. This was not
observed in HEK293 cells devoid of DAT. An additional
experiment was conducted in which pretreatment with
10 µM cocaine of the HEK293 cells expressing hDAT
protected the DAT from binding 5 because only non-
specific fluorescence was observed. Importantly, parallel
experiments with COS7 transiently expressing hDAT
showed similar results, with 5 and 9 displaying labeling
that could be blocked with cocaine and dopamine (data
not shown).
Figure 2. Excitation and emission spectra of (A) 5, (B) 9, (C)
10, and (D) 11. The excitation (dashed lines) and emission
(solid lines) spectra were obtained as described in Supporting
Information.
In summary, novel fluorescent ligands for the DAT
have been discovered with high DAT binding affinity
and appropriate fluorescence for visualization using
confocal microscopy. Compound 9 demonstrated the best
DAT fluoroprobe profile overall and has thus been
selected for future studies. The possibility of using this
ligand for the direct labeling of the DAT in living
neuronal cells represents a new and important approach
for understanding cellular targeting and trafficking.
Acknowledgment. J.H.C. was supported by an NIH
visiting fellowship and Post-doctoral Program of Korea
Science & Engineering Foundation (KOSEF). This
research was supported by NIDAsIRP, NIH, and in
part by the National Institutes of Health Grant P01 DA
12408 (U.G.), the Lundbeck Foundation (U.G.), the
Danish Health Science Research Council (U.G.), and the
Novo Nordic Foundation (U.G.). The MALDI-TOF spec-
tra were obtained by Dr. Amina Woods, NIDAsIRP.
HPLC chromatograms were carried out by Hansjoerg
Tinner, Fluka Production GmbH.
Figure 3. Visualization of DAT in HEK293 cells expressing
DAT by confocal laser scanning microscopy. Cells expressing
hDAT, or nontransfected controls cells, were incubated with
25 nM 5 (A) or 10 nM 9 (B) for the indicated times. All
experiments were performed at room temperature in PBS
buffer containing 5 mM D-glucose.
Supporting Information Available: Experimental meth-
ods. This material is available free of charge via the Internet
at http://pubs.acs.org.
further evaluate the affinities of the compounds for
hDAT, we also performed a competition binding assay
using the cocaine analogue [3H]CFT (2â-carbomethoxy-
3â-(4-fluorophenyl)tropane) as the radioligand (Table 2).
This showed the same rank order of potencies as that
observed in the uptake assay; however, the estimated
affinities were somewhat higher (Table 2). Altogether
the uptake and binding data demonstrated that al-
though addition of the fluorescent moieties decreased
binding affinities at the DAT, compared to the parent
ligand 1, compounds 5 and 9 still demonstrated affini-
ties for the DAT that made them useful as fluorescent
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JM050431Y