Novel and high affinity fluorescent ligands for the serotonin transporter based on (S)-citalopram

Article abstract of DOI:10.1021/ml5000806

Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently

Full text of DOI:10.1021/ml5000806

Letter
pubs.acs.org/acsmedchemlett
Novel and High Affinity Fluorescent Ligands for the Serotonin
Transporter Based on (S)‑Citalopram
Vivek Kumar,† Troels Rahbek-Clemmensen,‡ Christian B. Billesbølle,‡ Trine Nygaard Jorgensen,‡
Ulrik Gether,‡ and Amy Hauck Newman*,†
†Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural
Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States
‡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
S
* Supporting Information
ABSTRACT: Novel rhodamine-labeled ligands, based on (S)-
citalopram, were synthesized and evaluated for uptake
inhibition at the human serotonin, dopamine, and norepi-
nephrine transporters (hSERT, hDAT, and hNET, respec-
tively) and for binding at SERT, in transiently transfected
COS7 cells. Compound 14 demonstrated high affinity binding
and selectivity for SERT (Ki = 3 nM). Visualization of SERT,
using confocal laser scanning microscopy, validated compound
14 as a novel tool for studying SERT expression and
distribution in living cells.
KEYWORDS: Fluorescent ligand, SERT, citalopram, rhodamine
heterologous cell systems as well as monitoring endogenous
DAT in cultured dopaminergic neurons.5−8 However, the lack
of selectivity across monoamine transporters and relatively low
affinity for SERT decreases the utility of this fluorescent ligand
for SERT trafficking studies. Recently, we reported our first
attempt at a SERT selective fluorescent ligand ZP 455, using
( )-citalopram as the parent compound (Figure 1).9 While ZP
455 demonstrated moderately high affinity and selectivity for
SERT (Ki = 225 nM) over DAT and NET in HEK293 cells,
labeling of SERT in neurons was weak and characterized by
he serotonin transporter (SERT) is a member of the
TNeurotransmitter:Sodium Symporter (NSS) family of
transporters and regulates serotonin signaling and homeostasis
by mediating reuptake of extracellular serotonin.1 The
important classes of selective serotonin reuptake inhibitors
(SSRIs) and tricyclic antidepressants (TCAs) that are
prescribed for the treatment of anxiety, and major depressive
disorders work through inhibition of serotonin reuptake via
SERT. Nevertheless, only a rudimentary understanding of the
molecular and cellular mechanisms governing native SERT
expression, distribution, and trafficking exists currently. Hence,
further investigation is critical for understanding the
pathophysiology of these disorders, the effects of chronic
drug use, and how to improve long-term treatment.2
One approach to studying native protein function in live cells
or brain tissue is to use fluorescently labeled small molecules.3,4
Fluorescent ligands that have high affinity and selectivity for
their target protein can retain pharmacological properties of the
parent molecule and allow concomitant localization and real
time monitoring of cellular processes, including distribution,
trafficking, sequestering, and postendocytotic sorting.4,5 Thus,
with the technological advances in fluorescent microscopy,
these small molecules can serve as powerful tools for
investigating CNS target−ligand interactions and the systems
in which they reside and function.
Figure 1. JHC1-64 and ZP 455 (compound 8 in Zhang et al.9).
The tropane-based fluorescent ligand, JHC1-64, as well as
related analogues that have fluorophores other than rhodamine,
have proven highly useful in studying DAT in transfected
Received: February 21, 2014
Accepted: March 21, 2014
Published: March 27, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
rapid dissociation of the ligand, possibly due to its relatively low
affinity for SERT.
aluminum−nickel catalyst in formic acid to its aldehyde
intermediate 9 (Scheme 2).10 Compound 9 underwent
In the present study, using recently developed SAR10 and
chiral separation of ( )-citalopram to give the pure eutomer,
(S)-citalopram, we developed novel C-1 and C-5 substituted
rhodamine labeled ligands with high binding affinity and SERT
selectivity.
Scheme 2. Synthesis of SERT Fluorescent Compounds 12
a
and 14
Scheme 1 outlines the synthetic strategy used for the
synthesis of C-1 based fluorescent citalopram analogues 7 and
Scheme 1. Synthesis of SERT Fluorescent Compounds 7 and
a
8
a
Reagents and conditions: (a) Ni-AI alloy, formic acid, reflux, 5 h; (b)
cyanoacetic acid, morpholine, DMF, reflux, 5 h; (c) NaBH4, CoCI2,
MeOH, 0 °C to RT, 16 h; (d) rhodamine-NHS ester, (i-
pr)2ethylamine, DMF, 40−45 °C, 24 h; (e) LAH, THF, 5 h.
nucleophilic substitution with cyanoacetic acid to afford
intermediate 10, which upon reduction provided compound
11.14,15 Rhodamine-NHS ester was coupled to 11 under basic
conditions to afford compound 12. To synthesize compound
14, (S)-citalopram was reduced to intermediate 1310 and
coupled with rhodamine-NHS ester.9
a
Reagents and conditions: (a) (i) NH4OH (ii) ACE-CI, DCE, 18 h;
All four novel rhodamine analogues (7, 8, 12, and 14) were
evaluated for inhibition of serotonin or dopamine uptake at
their respective transporters (e.g., hSERT, hDAT, and hNET)
and compared to JHC1-64 in transiently transfected COS7
cells using [3H]5-HT for SERT and [3H]dopamine for NET
and DAT. On the basis of these assays, compounds 12 and 14
showed the highest apparent affinities at SERT suggesting that
substitution at the C-5 position of (S)-citalopram resulted in
the most well-tolerated analogues at SERT, and the affinities
were not significantly influenced by the difference in the linker
length between the fluorophore and the parent compound
(Table 1). None of the four new fluorescent analogues had any
inhibitory effect on the uptake of [3H]dopamine at DAT or
NET, at a concentration of 10 μM, in contrast to JHC1-64.
Next, we determined the affinities of the (S)-citalopram-
based analogues in competition binding assays using [3H](S)-
citalopram as the radioligand. Because the compounds were
highly SERT specific according to the uptake inhibition assay
data, only hSERT binding affinities were assessed (Table 2). All
four compounds showed moderate to high affinities for SERT;
however, compound 14 displayed an affinity similar to that of
(b) (+)-DPTTA, MeOH; (c) formaldehyde, formic acid, H2O, reflux,
5 h; (d) NaBH(OAc)3, AcOH, DCE, RT, 12 h; (e) hydrazine, EtOH,
reflux, 3 h; (f) Rhodamine red-X NHS ester, (i-pr)2ethylamine, DMF,
40−45 °C, 24 h.
8. The HBr salt of ( )-citalopram was neutralized and
demethylated to afford desmethylcitalopram, as previously
described.10 The (S)-enantiomer was separated using
(+)-DPTTA ((+)-O,O′-di-p-toluoyl-D-tartaric acid) as the
chiral resolving agent with modifications of a previously
reported procedure.11 (S)-Desmethylcitalopram was further
N-alkylated and reduced in the presence of NaBH(OAc)3 to
give intermediates 3 and 4.12 Deprotection of compounds 3
and 4 using hydrazine gave the free amines 5 and 6,
respectively. Rhodamine-NHS ester was coupled to the free
amine intermediate 5 and 6 under basic conditions to afford the
final products 7 and 8. (S)-Desmethylcitalopram was N-
alkylated to afford (S)-citalopram according to the published
procedure.13
To carry out the synthesis of the C-5 based fluorescent ligand
12, (S)-citalopram was oxidized in the presence of an
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ACS Medicinal Chemistry Letters
Letter
Table 1. Uptake Inhibition at hSERT, hDAT, and hNET
a
Expressed in COS7 Cells
SERT Ki (nM)
[SE interval]
DAT Ki (nM)
[SE interval]
NET Ki (nM)
[SE interval]
compd
(S)-citalopram
7.4 [3.0−19]
140 [110−180]
250 [130−450]
37 [22−61]
IA
IA
7
IA
IA
8
IA
IA
12
IA
IA
14
34 [19−69]
IA
IA
JHC1-64
94 [72−120]
20 [8−51]
91 [73−110]
a
For determination of Ki values, IC50 values were determined as
described in the Supporting Information from nonlinear regression
analysis of uptake inhibition assays on transfected COS7 cells using
2,5,6-[3H]-dopamine for DAT and NET or 5-hydroxy[3H]tryptamine
for SERT. The KM values for 2,5,6-[3H]-dopamine or 5-hydroxy[3H]-
tryptamine were determined in parallel and used for calculating
indicated Ki values. The SE interval for each Ki value is indicated and
was calculated from pKI SE. Data are means [SE interval] of three to
four repeated measurements performed in triplicate. IA = inactive at a
conc. of 10 μM.
Figure 2. Excitation and emission data for compounds 7, 8, 12, and
14. Data are shown as percent of maximum fluorescence and obtained
as described in the Supporting Information.
enhanced green fluorescent protein (EGFP-SERT). The cells
were incubated with 20 nM 14 and imaged by confocal laser
scanning microscopy after 0, 1, 5, 10, or 20 min. As seen in
Figure 3, we observed a gradual increase in the fluorescent
Table 2. hSERT Affinities of the Fluorescent Analogues
a
Assessed by Competition Binding
compd
SERT Ki or Kd (nM) [SE interval]
(S)-citalopram
2.6 [1.8−3.8]
11 [10−13]
180 [160−190]
11 [6−19]
7
8
12
14
3.0 [2.9−3.1]
a
For determination of Ki values, IC50 values were determined as
Figure 3. Compound 14 binds specifically to EGFP-SERT in a time-
dependent manner in HEK293 cells. Visualization of SERT with
compound 14 using confocal live imaging of HEK293 cells transiently
expressing EGFP-SERT. Cells were incubated with 20 nM 14 for 0, 1,
described in the Supporting Information from nonlinear regression
analysis of competition binding assays using [3H]-S-citalopram (2 nM)
as the radioligand. The Kd value for [3H]-S-citalopram was determined
in parallel and used for calculating indicated Ki values. The SE interval
for each Ki value is indicated and was calculated from pKI SE. Data
are means [SE interval] of three to four repeated measurements
performed in triplicate.
5, 10, or 20 min
100 μM paroxetine at room temperature. Left
panels show fluorescent EGFP signal (EGFP-SERT). Remaining
panels show the fluorescent rhodamine signal. Lower panels show
imaging of cells coincubated with an excess of paroxetine (100 μM).
The imaging was performed as described in the Supporting
Information. The fluorescent rhodamine images are merged with a
transmission image in all panels. The experiment shown is
representative of three identical experiments.
the parent compound, (S)-citalopram; ∼4-fold higher affinity
than compounds 7 and 12 and 60-fold higher affinity than
compound 8 (Table 2). The finding that compound 14 binds
with low nanomolar affinity to SERT, and thus with an affinity
comparable to the parent compound, is remarkable considering
the size of the rhodamine appended to the C-5 position and
suggests that it does not interfere with critical binding
interactions at the SERT binding site. Our previous
fluorescently tagged citalopram ligand, ZP 455, also had
rhodamine appended in the C-5 position; however, its SERT
binding affinity was ∼75-fold lower than for compound 14. In
addition to being racemic, ZP 455 possessed an aniline moiety
attached to the C-5 position, and this may have contributed to
the poorer solubility and binding kinetics of ZP 455 compared
to the present set of compounds.
The fluorescent properties for the four compounds 7, 8, 12,
and 14 were determined on a fluorescence spectrometer
(Figure 2). The compounds had an excitation maximum of 571,
570, 572, and 572 nm, respectively, and all four analogues had
an emission maximum of 589 nm. Thus, the different linker
lengths or position of substitution had no effect on their
fluorescent properties.
rhodamine signal that reached maximum after ∼10 min and
overlapped with the fluorescent EGFP signal in the plasma
membrane of EGFP-SERT expressing cells. Importantly, we did
not see any labeling of cells without EGFP signal, and
moreover, coincubation with an excess of the high affinity
SERT inhibitor paroxetine completely blocked labeling of cells
with compound 14 (Figure 3).
In summary, we report herein novel fluorescent ligands based
on (S)-citalopram with modifications at the C-1 and C-5
positions. We discovered that unlike the tropane-based JHC1-
64 that binds to DAT, NET, and SERT, these (S)-citalopram-
based fluorescent ligands are highly specific for SERT without
any binding to DAT or NET. Moreover, modification at the C-
5 position was particularly well tolerated at SERT, and binding
affinity was not adversely influenced by the change in linker
length between the fluorophore and (S)-citalopram. Com-
pound 14 demonstrated the highest affinity and selectivity for
SERT with Ki = 3.0 nM, which is comparable to the parent
compound (Ki =2.6 nM), indicating that the rhodamine does
not interfere with binding of the pharmacophore to the SERT
binding site. Application of confocal microscopy demonstrated
To assess whether it was possible to selectively label and
visualize SERT in live cells with compound 14, we used
HEK293 cells expressing SERT tagged at the N-terminus with
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ACS Medicinal Chemistry Letters
Letter
Rhodamine-labeled 2b-carbomethoxy-3b-(3,4-dichlorophenyl)-tropane
analogues as high-affinity fluorescent probes for the dopamine
transporter. J. Med. Chem. 2005, 48, 7513−7516.
(7) Eriksen, J.; Rasmussen, S. G. F.; Rasmussen, T. N.; Vaegter, C. B.;
Cha, J. H.; Zou, M. F.; Newman, A. H.; Gether, U. Visualization of
dopamine transporter trafficking in live neurons by use of fluorescent
cocaine analogs. J. Neurosci. 2009, 29, 6794−6808.
(8) Rickhag, M.; Hansen, F. H.; Sørensen, G.; Strandfelt, K. N.;
Andresen, B.; Gotfryd, K.; Madsen, K. L.; Klewe, I. V.; Ammendrup-
furthermore that compound 14 can be used to specifically
visualize SERT in the plasma membrane of live cells. Taken
together, the combination of compound 14’s high affinity and
selectivity for SERT and the strong signal-to-noise ratio seen in
confocal imaging illustrates how well-suited this unique
fluorescent tool should be in further studies of SERT
expression, localization, and trafficking directly in living cells.
ASSOCIATED CONTENT
* Supporting Information
Johnsen, I.; Eriksen, J.; Newman, A. H.; Fuchtbauer, E. M.; Gomeza, J.;
̈
■
Woldbye, D. P.; Wortwein, G.; Gether, U. Nat. Commun. 2013, 4,
1580.
̈
S
Experimental details for the synthesis and purification of the
compounds and their in vitro pharmacological characterization.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(9) Zhang, P.; Jørgensen, T. N.; Loland, C. J.; Newman, A. H. A
rhodamine-labeled citalopram analogue as a high-affinity fluorescent
probe for the serotonin transporter. Bioorg. Med. Chem. Lett. 2013, 23,
323−326.
(10) Banala, A. K.; Zhang, P.; Plenge, P.; Cyriac, G.; Kopajtic, T.;
Katz, J. L.; Loland, C. J.; Newman, A. H. Design and synthesis of 1-(3-
(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofur-
an-5-carbonitrile (citalopram) analogues as novel probes for the
serotonin transporter S1 and S2 binding sites. J. Med. Chem. 2013, 56,
9709−9724.
AUTHOR INFORMATION
Corresponding Author
*(A.H.N.) Tel: +1-443-740-2887. Fax: +1-443-740-2111. E-
mail: anewman@intra.nida.nih.gov.
■
Author Contributions
(11) Kaushik, V. K.; Mohammed, U. K.; Bobbali, N. R.; Srinivasan, R.
K.; Dandala, R.; Meenakshisunderam, S. Process for the preparation of
Escitalopram. Eur. Pat. Appl. 2017271A1, 2009.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(12) Xiao, X.; Antony, A.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Design, synthesis, and biological evaluation of cytotoxic 11-amino-
alkenylindenoisoquinoline and 11- diaminoalkenylindenoisoquinoline
topoisomerase I inhibitors. Bioorg. Med. Chem. 2004, 12, 5147−5160.
(13) Elati, C. R.; Kolla, N.; Vankawala, P. J.; Gangula, S.; Chalamala,
S.; Sundaram, V.; Bhattacharya, A.; Vurimidi, H.; Mathad, V. T.
Substrate modification approach to achieve efficient resolution:
didesmethylcitalopram: a key intermediate for escitalopram. Org.
Process Res. Dev. 2007, 11, 289−292.
Funding
This research was funded by the NIDA Intramural Research
Program and by the National Institute of Health Grants P01
DA 12408 (to U.G.), the Danish Medical Research Council (to
U.G.), and the Lundbeck Foundation Center for Biomem-
branes in Nanomedicine (to U.G.). V.K. was supported by an
NIH Visiting Fellowship.
(14) Masuda, K.; Ito, Y.; Horiguchi, M.; Fujita, H. Studies on the
solvent dependence of the carbamic acid formation from -(1-
naphthyl)alkylamines and carbon dioxide. Tetrahedron 2005, 61,
213−229.
(15) Gautier, F.-M.; Jones, S.; Li, X.; Martin, S. J. Scope of the
organocatalysed asymmetric reductive amination of ketones with
trichlorosilane. Org. Biomol. Chem. 2011, 9, 7860−7868.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A special thanks is given to Dr. Amina Woods for the MALDI-
TOF analysis of all the final compounds. We thank Anette
Dencker Kaas for excellent technical assistance.
ABBREVIATIONS
■
SERT, serotonin transporter; SSRI, selective serotonin reuptake
inhibitors; DAT, dopamine transporter; NET, norepinephrine
transporter; LAH, lithium aluminum hydride; EGFP, enhanced
green fluorescent protein; RT, room temperature
REFERENCES
■
(1) Kristensen, A. S.; Andersen, J.; Jørgensen, T. N.; Sørensen, L.;
Eriksen, J.; Loland, C. J.; Strømgaard, K.; Gether, U. SLC6
Neurotransmitter transporters: structure, function, and regulation.
Pharmacol. Rev. 2011, 63, 585−640.
(2) Benmansour, S.; Cecchi, M.; Morilak, D. A.; Gerhardt, G. A.;
Javors, M. A.; Gould, G. G.; Frazer, A. Effects of chronic
antidepressant treatments on serotonin transporter function, density,
and mRNA level. J. Neurosci. 1999, 19, 10494−10501.
(3) Baindur, N.; Triggle, D. J. Concepts and progress in the
development and utilization of receptor-specific fluorescent ligands.
Med. Res. Rev. 1994, 14, 591−664.
(4) Joubert, J.; Dyk, S. V.; Malan, S. F. Small molecule fluorescent
ligands as central nervous system imaging probes. Joubert et al. Mini-
Rev Med. Chem. 2013, 13, 682−696.
(5) Eriksen, J.; Bjørn-Yoshimoto, W. E.; Jørgensen, T. N.; Newman,
A. H.; Gether, U. J. Biol. Chem. 2010, 28, 27289−27301.
(6) Cha, J. H.; Zou, M. F.; Adkins, E. M.; Rasmussen, S. G. F.;
Loland, C. J.; Schoenenberger, B.; Gether, U.; Newman, A. H.
699
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