A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

Article abstract of DOI:10.1016/j.bmcl.2012.10.089

A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5- carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [³H]5-HT uptake in COS-7 cells (K_i = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative to the transporters for dopamine and norepinephrine. Visualization of the SERT with compound 8 was demonstrated by confocal microscopy in HEK293 cells stably expressing EGFP-SERT.

Full text of DOI:10.1016/j.bmcl.2012.10.089

Bioorganic & Medicinal Chemistry Letters 23 (2013) 323–326
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe
for the serotonin transporter
Peng Zhang a, Trine Nygaard Jørgensen b, Claus J. Loland b, Amy Hauck Newman a,
⇑
a Molecular Targets and Medications Discovery Branch, Medicinal Chemistry Section, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health,
333 Cassell Drive, Baltimore, MD 21224, USA
b Molecular Neuropharmacology Laboratory and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Faculty of Health and Medical
Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the
serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution
on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)propyl)-1-
(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting
rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency
to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative to the transport-
ers for dopamine and norepinephrine. Visualization of the SERT with compound 8 was demonstrated by
confocal microscopy in HEK293 cells stably expressing EGFP–SERT.
Received 7 September 2012
Revised 15 October 2012
Accepted 19 October 2012
Available online 1 November 2012
Keywords:
Fluorescent ligand
Citalopram
SERT
Published by Elsevier Ltd.
Rhodamine
The serotonin transporter (SERT) is located in the membranes of
presynaptic serotonergic neurons, and functions to transport ex-
cess serotonin into the cell from the synaptic cleft to terminate
serotonergic signaling.1 Although selective serotonin reuptake
inhibitors (SSRIs) are used clinically for the treatment of anxiety
and depression, drug-protein interactions and long-term effects
of these drugs on SERT trafficking and function that affect clinical
efficacy remain largely unknown. Fluorescent probes can be pow-
erful tools for the detection of and structure–function studies on
membrane proteins.2–15
A fluorescently labeled ligand that selectively binds to a trans-
porter or receptor protein can be used to localize protein distribu-
tion in tissue and monitor its trafficking, including internalization
in live cells or tissue.3,4 In addition, these tools can be used to study
protein function, such as upstream and downstream molecular
regulation,2,10,11 protein oligomerization,5 or potentially, clinical
efficacy for certain pathological conditions.15 Combined with other
techniques, these structure–function studies can aid in under-
standing ligand–protein interactions, such as protein conforma-
tional changes caused by ligand binding.7,9 and also facilitate
protein structure analysis.13,14
Previously, fluorescently tagged 2b-carbomethoxy-3b-(3,4-
dichlorophenyl)tropane analogues (e.g., JHC 1-064, 1, Fig. 1) were
synthesized and used to visualize dopamine transporter (DAT) traf-
ficking in cultured live midbrain dopaminergic neurons.3,4,16 DAT,
along with the norepinephrine transporter (NET) and SERT are
three highly homologous transporters belonging to the neurotrans-
mitter: sodium symporter (NSS) family, which share a common
protein tertiary structure with twelve transmembrane (TM) do-
mains.1 Since the tropane-based analogues reported previously
bind to all three transporters with similar affinities,16 a fluorescent
molecule, based on a SERT-selective ligand, could be an important
addition to the armamentarium of tools to study the structure and
function of the SERT.
The antidepressants citalopram (2, Fig. 1) and its S-enantiomer
are selective serotonin reuptake inhibitors (SSRI) that bind with
high affinity to SERT (Ki = 1.94, 0.89 nM, respectively) and selec-
tively over NET (ratio = 3070, 11,800, respectively) and DAT (ra-
tio = 4780, 9160, respectively).17 Structure–activity relationship
(SAR) studies, based on a series of citalopram (2) analogues, have
been described wherein modifications at the N-, 4, and 5-positions
have identified points of attachment that maintain both high affin-
ity and SERT selectivity.17–20 Specifically, most modifications at the
5-position of the dihydroisobenzofuran ring were well tolerated at
the SERT (Ki = 1–40 nM), and none of these analogues demon-
strated high binding affinity at NET or DAT, which revealed that
Abbreviations: SERT, serotonin transporter; NET, norepinephrine transporter;
DAT, dopamine transporter; NSS, neurotransmitter/sodium (Na+/Clꢀ) symporter;
SSRI, selective serotonin reuptake inhibitor; NBD, nitrobenzoxadiazolealanine;
NMM, N-methylmorpholine; NHS, N-hydroxysuccinimide; EGFP, enhanced green
fluorescent protein.
⇑
Corresponding author. Tel.: +1 443 740 2887; fax: +1 443 740 2111.
E-mail address: anewman@intra.nida.nih.gov (A.H. Newman).
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.10.089

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P. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 323–326
Figure 1. JHC 1-064 (1) citalopram (2) and its 5-anilino analogue (3).
Scheme 1. Synthesis of SERT fluorescent compound 8. Reagents and conditions: (a) 150 °C, 2 h; (b) thionyl chloride, CH2Cl2, reflux, 5 h; (c) NEt3, CH2Cl2, rt, overnight; (d)
NH2NH2, rt, 5 h; (e) (i-pr)2ethylamine, DMF, rt, overnight.
steric bulk is tolerated at this position.17 This is important as the
fluorophore is a molecule of equal or greater molecular weight
compared to the pharmacophore and thus must be attached to a
position where steric bulk does not significantly diminish binding
affinity. Among these compounds, the 5-(30-NH2-Ph) analogue 3
(Ki = 10.4 nM)17 was chosen as the template for synthesis of the
first citalopram-based fluorescent ligand, as it contained a poten-
tially modifiable NH2 group.
In this study, we chose rhodamine as the fluorophore, as previ-
ous studies with rhodamine RedX-NHS ester established that it
was ideally suited for straightforward synthesis and visualization
studies.16 In addition, rhodamine is widely used for fluorescence
studies in biological systems due to its photostability and high
quantum yield. It also has a suitable excitation spectrum for the
use of relatively inexpensive lasers and is outside the intrinsic fluo-
rescence from protein tryptophans.

P. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 323–326
325
Table 1
Ki values of novel citalopram analogues to hSERT, hDAT and hNET determined by their inhibition potency of radiolabeled substratesa
Compound
hSERT
n
hDAT
n
hNET
n
[3H]5-HT inhib. Ki (nM)
[3H]DA inhib. Ki (nM)
[3H]DA inhib. Ki (nM)
5-HT (Km
1, JHC 1-064
2
3
8
)
491[416;579]
392[286;537]
16[12;22]
390[313;485]
225[208;241]
—
8
3
6
6
6
nd
nd
62[37;105]
>10,00,000
nd
10,900[9300;12900]
1010[909;1120]
3
2
194[153;244]
>10,00,000
nd
11,800[10,800;13,000]
396[369;425]
3
4
3
4
3
5
DA (Km
)
a
Uptake inhibition of [3H]5-HT at hSERT and [3H]dopamine ([3H]DA) at hDAT and hNET (Ki values) (mean [SE interval]). Ki values were determined from uptake inhibition
experiments of indicated radiolabeled in COS7 cells stably expressing hSERT, hDAT or hNET. The Ki values were calculated from IC50 values determined by non-linear
regression analysis of uptake data using the equation Ki = IC50/(1 + (L + Km)) where L is the concentration of [3H]5-HT or [3H]DA. The Km value for 5-HT and DA were
determined in parallel. nd, not determined.
4 100 5
×
The strategy shown in Scheme 1 wherein compound 3 was re-
acted with freshly prepared acyl chloride 5, made from the N-pro-
tected alanine 4, following a modified procedure23 was successful.
The resulting compound 6 was deprotected with hydrazine to give
the free amine 7, which was reacted with rhodamine red-X NHS es-
ter to give the desired fluorescent compound 8.
Excitation
Emission
3 100 5
×
2 100 5
×
The binding affinity and SERT selectivity of compound 8 was
determined measuring inhibition of [3H]5-HT uptake into COS-7
cells transiently expressing the hSERT and [3H]dopamine ([3H]DA)
uptake into COS-7 cells transiently expressing the hDAT and hNET.
These data were compared to the inhibition potency of 1 as deter-
mined previously16 (Table 1).
1 100 5
×
0
450
500
550
600
650
wavelength (nm)
The fluorescent compound 8 showed a similar potency to 1 for
inhibition of [3H]5-HT uptake and was selective for SERT (ꢁ50-
fold) over DAT and NET, although less selective than the parent
molecule 2.
The fluorescent properties of compound 8 were determined as
shown in Figure 2. The maximal excitation and emission wave-
lengths are at 570 and 592 nM, respectively, (IF, fluorescence inten-
sity. a.u., arbitrary units.)
To demonstrate specific labeling of SERT with compound 8, we
used a HEK293 cell line stably expressing hSERT with EGFP fused to
the N-terminus. Cells were incubated with compound 8 for 60 min
at 37 °C with or without the specific SERT inhibitor paroxetine. As
shown in Figure 3, live cell imaging using confocal microscopy re-
vealed a uniform distribution of compound 8 (red) at the cell sur-
face overlapping with the distribution of EGFP–SERT (green). In the
Figure 2. Excitation (black) and emission (red) wavelength spectra of compound 8.
The first synthetic strategy developed was to link compound 3
directly to rhodamine RedX-NHS ester. Unfortunately, the aniline
group was not sufficiently reactive, under basic reaction condi-
tions, either at room temperature or at 45 °C to couple to the rho-
damine reagent, hence, an ethylamino linker was introduced.16
However, no reaction was detected under basic conditions using
2-bromoethylphthalimide as a reagent, even when the tempera-
ture reached >100 °C. Another published method also failed to con-
dense the aniline group of compound 3 with N-protected alanine
under the condition of N-methylmorpholine and isobutylchlorofor-
mate.21 In addition, reaction between compound 3 and N-pro-
tected alanine anhydride22 failed and resulted in a salt form of 3.
Fig. 3. Visualizing 8 binding to hSERT in transfected HEK293 cells. HEK293 cells stably expressing EGFP–hSERT were preincubated in buffer with or without 1
for 30 min before one hour incubation with 20 nM 8 at 37 °C. Compound 8 labeling was visualized by confocal microscopy. Pictures are representatives of more than three
individual experiments. Scale bar, 10 m.
lM paroxetine
l

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P. Zhang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 323–326
presence of paroxetine, no fluorescence was observed at the cell
surface in the red channel. The same result was observed when
incubation of compound 8 was performed in the presence of
1 lM S-citalopram or 100 lM 5-HT (data not shown). These data
demonstrate that compound 8 labels SERT with high specificity
References and notes
1. Butler, S. G.; Meegan, M. J. Curr. Med. Chem. 2008, 15, 1737.
2. Colgan, L. A.; Putzier, I.; Levitan, E. S. J. Neurosci. 2009, 29, 15878.
3. Eriksen, J.; Bjorn-Yoshimoto, W. E.; Jorgensen, T. N.; Newman, A. H.; Gether, U.
J. Biol. Chem. 2010, 285, 27289.
4. Eriksen, J.; Rasmussen, S. G.; Rasmussen, T. N.; Vaegter, C. B.; Cha, J. H.; Zou, M.
F.; Newman, A. H.; Gether, U. J. Neurosci. 2009, 29, 6794.
5. Fjorback, A. W.; Pla, P.; Muller, H. K.; Wiborg, O.; Saudou, F.; Nyengaard, J. R.
Biochem. Biophys. Res. Commun. 2009, 380, 724.
6. Fowler, A.; Seifert, N.; Acker, V.; Woehrle, T.; Kilpert, C.; de Saizieu, A. J. Biomol.
Screen. 2006, 11, 1027.
7. Gether, U.; Lin, S.; Kobilka, B. K. J. Biol. Chem. 1995, 270, 28268.
8. Guignet, E. G.; Segura, J. M.; Hovius, R.; Vogel, H. ChemPhysChem 2007, 8, 1221.
9. Kobilka, B. K.; Gether, U. Methods Enzymol. 2002, 343, 170.
10. Martinat, C.; Bacci, J. J.; Leete, T.; Kim, J.; Vanti, W. B.; Newman, A. H.; Cha, J. H.;
Gether, U.; Wang, H.; Abeliovich, A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
2874.
and enables SERT visualization in live cells.
In summary, a novel fluorescent ligand selective for hSERT was
designed and synthesized using citalopram (2) as the SERT-selective
pharmacophore. The resulting rhodamine-labeled ligand 8 demon-
strated suitable serotonin uptake kinetics (Ki = 225 nM) and selec-
tivity over DAT and NET. Specific labeling of the SERT with
compound 8 was visualized by confocal microscopy in HEK293 cells
stably expressing EGFP–SERT. Further studies comparing compound
8 and 1 in labeling SERT in living neuronal cells are underway.
11. Oz, M.; Libby, T.; Kivell, B.; Jaligam, V.; Ramamoorthy, S.; Shippenberg, T. S. J.
Neurochem. 2010, 114, 1019.
12. Rasmussen, S. G.; Adkins, E. M.; Carroll, F. I.; Maresch, M. J.; Gether, U. Eur. J.
Pharmacol. 2003, 479, 13.
Funding sources
13. Rasmussen, S. G.; Gether, U. Biochemistry 2005, 44, 3494.
14. Sucic, S.; Dallinger, S.; Zdrazil, B.; Weissensteiner, R.; Jorgensen, T. N.; Holy, M.;
Kudlacek, O.; Seidel, S.; Cha, J. H.; Gether, U.; Newman, A. H.; Ecker, G. F.;
Freissmuth, M.; Sitte, H. H. J. Biol. Chem. 2010, 285, 10924.
15. Yuan, C. G.; Xue, X. L.; He, L. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao
(Shanghai) 2000, 32, 605.
16. Cha, J. H.; Zou, M. F.; Adkins, E. M.; Rasmussen, S. G.; Loland, C. J.;
Schoenenberger, B.; Gether, U.; Newman, A. H. J. Med. Chem. 2005, 48, 7513.
17. Zhang, P.; Cyriac, G.; Kopajtic, T.; Zhao, Y.; Javitch, J. A.; Katz, J. L.; Newman, A.
H. J. Med. Chem. 2010, 53, 6112.
18. Eildal, J. N.; Andersen, J.; Kristensen, A. S.; Jorgensen, A. M.; Bang-Andersen, B.;
Jorgensen, M.; Strømgaard, K. J. Med. Chem. 2008, 51, 3045.
19. Bigler, A. J.; Bogeso, K. P.; Toft, A.; Hansed, V. . Eur. J. Med. Chem – Chimaca
Therapeutica 1977, 12, 289.
20. Andersen, J.; Stuhr-Hansen, N.; Zachariassen, L.; Toubro, S.; Hansen, SM.; Eildal,
JN.; Bond, AD.; Bøgesø, KP.; Bang-Andersen, B.; Kristensen, AS.; Strømgaard, K.
Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 12137.
This work was funded by the NIDA-Intramural Research
Program (A.H.N.), the Center for Pharmacogenomics, University of
Copenhagen, NIH Grant P01 DA 12408, The Lundbeck Foundation
and University of Copenhagen Program of Excellence (C.J.L). The
Danish Council for Independent Research, Sapere Aude Program
(C.J.L.)
Acknowledgments
A special thanks to Erick Garcia-Gorbea for the HPLC analysis of
compound 8 and to Dr. Amina Woods for the MALDI-TOF analysis
of compound 8.
21. de Groot, F. M.; de Bart, A. C.; Verheijen, J. H.; Scheeren, H. W. J. Med. Chem.
1999, 42, 5277.
Supplementary data
22. Sakamoto, T.; Mahara, A.; Yamagata, K.; Iwase, R.; Yamaoka, T.; Murakami, A.
Biophys. J. 2005, 89, 4122.
23. Lelais, G.; Micuch, P.; Josien-Lefebvre, D.; Rossi, F.; Seebach, D. Helv. Chim. Acta
2004, 87, 3131.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.089.