Biotin tethered homotryptamine derivatives: High affinity probes of the human serotonin transporter (hSERT)

Article abstract of DOI:10.1016/j.bmcl.2011.01.102

Quantum dot conjugates of compounds capable of inhibiting the serotonin transporter (SERT) could form the basis of fluorescent probes for live cell imaging of membrane bound SERT. Additionally, quantum dot-SERT antagonist conjugates may be amenable to flu

Full text of DOI:10.1016/j.bmcl.2011.01.102

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1678–1682
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biotin tethered homotryptamine derivatives: High affinity probes
of the human serotonin transporter (hSERT)
Ian D. Tomlinson a, Hideki Iwamoto b, Randy D. Blakely b,c,f, Sandra J. Rosenthal a,b,c,d,e,
⇑
a The Department of Chemistry, Vanderbilt University, Station B, 351822, Nashville, TN 37235-1822, USA
b The Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 27232-8548, USA
c Vanderbilt Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA
d The Department of Physics and Astronomy, Vanderbilt University, 1807, Station B, Nashville, TN 37235, USA
e The Department of Chemical and Bimolecular Engineering, Vanderbilt University, Station B, 351631, 5824 Stevenson Center, Nashville, TN 37235-1631, USA
f The Department of Psychiatry, Suite 3105, 1601, 23rd Avenue South, Nashville, TN, 37212, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Quantum dot conjugates of compounds capable of inhibiting the serotonin transporter (SERT) could form
the basis of fluorescent probes for live cell imaging of membrane bound SERT. Additionally, quantum
dot-SERT antagonist conjugates may be amenable to fluorescence-based, high-throughput assays for this
transporter. This Letter describes the synthesis of SERT-selective ligands amenable to conjugation to
quantum dots via a biotin–streptavidin binding interaction. SERT selectivity and affinity were incorpo-
rated into the ligand via a tetrahydropyridine or cyclohexylamine derivative and the affinity of these
compounds for SERT was measured by their ability to produce SERT-dependent currents in Xenopus laveis
oocytes.
Received 12 November 2010
Revised 20 January 2011
Accepted 21 January 2011
Available online 2 February 2011
Keywords:
Quantum dot
Serotonin
SERT antagonist
Homotryptamine
Leak current
Ó 2011 Elsevier Ltd. All rights reserved.
Neurotransmitters such as dopamine, norepinephrine and
5-hydroxytryptamine (serotonin, 5-HT) modulate neuronal signal-
ing in response to a variety of sensory and physiological stimuli.
Synaptic concentrations of these neurotransmitters are regulated
by presynaptic transporters, specifically the dopamine transporter
(DAT), the norepinephrine transporter (NET) and the serotonin
transporter (SERT).1,2 Methodologies used to study the location
and temporal dynamics of these transporters within the mem-
brane may provide important insights to their dysfunction, which
is suspected to contribute to disorders such as autism, OCD,
depression, ADHD and addiction. Our efforts currently center on
the development of fluorescent probes based upon DAT and SERT
antagonists that can be conjugated to quantum dots.3–6 Ultimately,
these probes will be used to study the expression, location and
dynamics of these transporters within the presynaptic mem-
brane7–9 and to identify abnormalities and therapeutics pertinent
to transporter-related illnesses.
R
N
(2) R = H
(3) R = CH CH NH-Biotin
2
2
(4) R = (CH ) NHCO-PEG3400NH-Biotin
2 2
(5) R = CH CH NH
2
2
2
(6) R= (CH ) NHCO-PEG3400-NH
N
H
2 11
2
(7) R = (CH ) NH
2 11
2
Figure 1. High affinity SERT ligands for conjugation to quantum dots.
Table 1
Biological activity of free and conjugated ligands against hSERT
Compound no.
IC50 (nM)
Conjucate IC50 (nM)
2
3
4
5
6
7
80a
2
1
2000
30
25
Not applicable
30
100
Not conjucated
10
Not conjucated
In earlier studies, we identified 3-(1,2,3,6-tetrahydropyrindin-
4-yl)-1H-indole (2) as a high affinity SERT antagonist that may be
conjugated to quantum dots.8,9 A linker arm may be attached to
this tetrahydropyridyl nitrogen atom, this biological activity is
retained (Fig. 1, Table 1). The ability of these derivatives to inhibit
the uptake of tritiated serotonin (5-HT) in human SERT (hSERT)
Literature value.11
a
expressing HEK cells was measured. After conjugation to quantum
dots, the uptake inhibition by these conjugates was measured and
IC50 values were calculated following titration of conjugated dot
concentrations (Table 1). Studies of nonspecific binding of
quantum dots to cellular membranes suggested a long polyethyl-
⇑
Corresponding author.
E-mail address: sandra.j.rosenthal@vanderbilt.edu (S.J. Rosenthal).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.102

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1678–1682
1679
R
R
HN
HN
O
S
O
H
H
N
X
N
N
N
N
H
N
H
X
n
O
O
n
H
N
NH
HN
O
S
HN
O
(8) IDT357, R = H, n = 10, X = PEG5000
(13) IDT374, R = H, n =10, X = PEG5000
(9) IDT318, R = OMe, n = 10, X = PEG5000
(10) IDT366, R = H, n = 5, X = PEG5000
(11) IDT317, R = OMe, n = 1, X = PEG5000
(12) IDT361, R = CN, n = 10, X = PEG5000
O
N
PEG5000 NH
R
H
O
HN
H
N
NH2
O
N
S
HN
(15)
n
(14) IDT373, R = H, n = 10
Figure 2. Ligands used in this study.
ene glycol chain on the surface of the dot was necessary to
reduce nonspecific adsorption of the quantum dots to cellular
membranes.10 Such compounds were shown to retain antagonist
activity.
Although we have demonstrated our ligands have a high affinity
for hSERT in uptake inhibition assays, we sought further evidence
that ligand-conjugated dots bind tightly to hSERT in intact cells
in real-time physiological assays. Using hSERT expressing oocytes,
it is possible to detect antagonist suppressed hSERT leak currents
(which appear as outward currents above a pretreatment baseline),
from these measurements it is possible to gain insights regarding
the relative potency of hSERT ligands. In this Letter, the synthesis
of a series of high-affinity hSERT ligands (Fig. 3.) and their ability
to suppress hSERT leak currents in hSERT expressing oocytes was
studied.
Figure 3. The ratio of antagonist revealed leak current to 5-HT induced current for
Derivatives of both (2) and cyclohexenyl amine were synthe-
sized with an alkyl spacer attached to biotin-PEG5000 (Fig. 2).
The alkyl spacer length was changed to study the effect on binding
(compounds (8), (10) and (11)). The effect of substituting on the in-
dole ring was studied by synthesizing compounds (8), (9) and (12).
Compound (13) and the intermediate (14) were synthesized
extending the basic nitrogen from the tetrahydropyridine ring to
study the effects of this modification on binding. Finally, a methyl
caped PEG ligand (15) was synthesized and used as a negative
control.
Compounds (8)–(12) were synthesized as shown in Scheme 1.
Initially (2), the methoxy, and the cyano analogs were synthesized
using the method described by Guillaume et al.11; these were then
attached to 2-(bromoalkyl)isoindoline-1,3-dione (15a), (15b) or
(15c), resulting in (16a)–(16f) and were converted to the amines
(17a)–(17f)9, after coupling to Biotin-PEG5000-NHS compounds
(8)–(12) were obtained.12 The synthesis of (16a) and (16b) are pre-
viously described by Tomlinson et al.9
the analogs used in this study.
was converted to N-methyl-1,4-dioxaspiro[4.5]decan-8-amine
(19) by a reductive amination with sodium triacetoxy borohydride
and methylamine. The ketal was hydrolysed to yield 4-(methyl-
amino)cyclohexanone (20) and this was reacted with indole to
yield 4-(1H-indol-3-yl)-N-methylcyclohex-3-enamine (21) in a
20% overall yield.13 The 11 carbon spacer was attached to this
using the same methodology as previously described to give (22)
in a 64% yield.14 The phthalimide protecting group was removed
by stirring in ethanol in the presence of hydrazine mono hydrate
using the method previously described9 to give (14) in a 93% yield.
This was coupled to Biotin-PEG5000-NHS to give compound (13).15
Compound (15) was obtained by reacting Biotin-PEG5000-NHS
with methylamine.16
The ability of compounds (8)–(15) to suppress hSERT-depen-
dent leak currents in Xenopus oocytes was measured by perfusing
Compound (13) was synthesized as outlined in Scheme 2. The
mono ethyl ketal of commercially available cyclohexane dione
oocytes with a 1
logical recordings for each ligand were obtained using a low-pass
lM solution of (8)–(14) in buffer. Electrophysio-

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I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1678–1682
O
N
O
n
Br
H
N
N
n
O
N
O
R
R
(i)
+
N
N
H
H
(17a) R = H, n = 10
(17b) R = OMe, n =10
(17c) R = H, n = 5
(17d) R = OMe, n = 1
(17e) R = CN, n = 10
R = H, OMe, CN
(16a) n = 5
(16b) n = 10
(16c) n =1
(ii)
R
NH2
n
HN
N
O
N
N
PEG5000 NH
n
H
(iii)
R
O
N
H
H
(8) IDT357, R = H, n = 10
(9) IDT318, R = OMe, n = 10
(10) IDT366, R = H, n = 5
(11) IDT317, R = OMe, n = 1
(12) IDT361, R = CN, n = 10
(18a) R = H, n = 10
(18b) R = OMe, n = 10
(18c) R = H, n = 5
(18d) R = OMe, n =1
(18e) R = CN, n = 10
N
O
S
HN
Scheme 1. Reagents: (i) Et3N; (ii) hydrazine monohydrate; (iii) Biotin-PEG5000-NHS.
filter at 10 Hz and digitized at 20 Hz. All analyses were performed
using Origin 7 (OriginLab, Northampton, MA). The magnitude of
the leak currents for (8)–(14) was obtained using five replicates
and the magnitudes of the leak currents for compounds (8)–(13)
are shown in Figure 3. An example raw trace of leak suppression
and 5-HT induced currents is shown in Figure 4. Compound (15)
was a control compound and induced no suppression of leak cur-
rent indicating that the indole derivative is necessary for biological
activity.
creased in length from two carbon atoms in IDT317 (11) to six car-
bon atoms in IDT366 (10), an increase in leak current suppression
can be observed. This trend continues when the alkyl spacer is in-
creased to 11 carbon atoms (IDT357 (8)), indicating that the alkyl
spacer in these molecules participates in binding. One hypothesis
is that hydrophobic interactions within SERT itself support ligand
interactions, or another possibility may be that hydrophobic inter-
actions between the alkyl spacer and the lipid membrane increase
the tightness of binding. Substitution on the indole ring has a less
significant effect on the magnitude of the leak current. Derivatives
including electron withdrawing substituents on the 5-position of
the indole ring, such as the cyano derivative (IDT361 (12)), have
a slightly larger leak current suppression than derivatives where
an electron donating substituent, such as a methoxy derivative is
on the ring (IDT318 (9)). However these differences do not seem
to correlate well with IC50 values since it has been reported that
the cyano substituted parent drug has a potency that is approxi-
mately 20-fold greater than the methoxy substituted parent
drug.17 A large increase in the magnitude of the leak current sup-
pression was obtained by moving the basic nitrogen out of the tet-
rahydropyridyl ring to give IDT374 (13), suggesting this basic
nitrogen may be interacting with the binding site in hSERT. The ba-
sic nitrogen may be acting as a hydrogen bond acceptor and the
binding strength is likely dependent upon the orientation of the
lone pair of electrons on the hydrogen bond acceptor. Conse-
quently, it is not surprising any modification in the structure of
the homotryptamine, which changes the orientation of this nitro-
gen atom and its lone pair of electrons, will have a significant im-
pact upon the tightness of binding to hSERT if it is acting as a
hydrogen bond acceptor.
Figure 4 shows a raw data trace obtained for the intermediate
IDT373 (14).
In addition to measuring leak current suppression, the ability of
the ligands to inhibit the uptake of tritiated 5-HT in hSERT express-
ing Xenopus oocytes was also measured and IC50 values were ob-
tained using five replicates. Figure 5 shows a correlation of the
IC50 value obtained using this oocyte system with the observed
leak current suppression.
Compounds (8)–(14) showed leak currents that are typical of
SERT antagonists. It is crucial to understand how tightly our li-
gands are binding to the SERT since we intend to utilize them in
a variety of biological assays. Ligands that bind tightly and do
not diffuse into the surrounding media during an assay may be re-
quired for assays to track the location and temporal dynamics of
SERT within live cell membranes. On the other hand, ligands that
do not bind as tightly may be more suitable for displacement
assays which could be the basis of a quantum dot based high-
throughput (HTS) assay. The magnitude of the measured leak
currents and IC50 value will enable the selection of the appropriate
ligand.
The magnitude of the leak current is proportional to the potency
of the ligand for hSERT interactions. IDT374 (13) exhibited the
greatest activity while IDT317 (11) exhibited the lowest leak cur-
rent suppression, and consequently, the weakest binding to hSERT.
These data were mirrored by the measured IC50 values for SERT up-
take inhibition. The relative potencies of our SERT ligands appears
to be determined by three factors. The 1st of these is the length of
the alkyl spacer in the linker arm; when the alkyl spacer is in-
In conclusion, the relative potency for interactions with
hSERT for our ligands follows the order: IDT374 (13) ꢀ IDT373
(14) > IDT361 (12) ꢀ IDT357 (8) = IDT318 (9) > IDT366 (10) >
IDT317 (11). In addition to these leak current experiments, we also
demonstrated that IDT317 (11) binds relatively weakly to hSERT
since it can be easily washed off, restoring a normal 5-HT induced
influx current (data not shown). The leak current revealed by

I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1678–1682
1681
NH
O
O
NH
NH
O
Br
N
10
(iii)
(i)
(ii)
+
O
N
H
O
O
O
O
(20)
(21)
(19)
(iv)
O
N
N
O
(22)
HN
(v)
N
NH2
(14)
HN
(vi)
H
N
N
PEG5000
NH
O
O
(13)
HN
H
N
O
S
HN
Scheme 2. Reagents: (i) Methylamine, Na(AcO)3BH; (ii) TFA; (iii) indole, NaOMe; (iv) Et3N; (v) Hydrazine monohydrate; (vi) Biotin-PEG5000-NHS.
Figure 4. A raw data trace showing a representative leak current obtained for the
intermediate IDT373 (14).
Figure 5. Leak current correlations with 5-HT uptake inhibition IC50 in oocytes
obtained for compounds (8)–(14).
incubating IDT317 with hSERT expressing oocytes was similar in
magnitude to the leak current obtained with fluoxetine, while the
antagonists with longer alkyl spacers had leak currents of similar
magnitude to the higher affinity hSERT antagonist paroxetine.
These data suggest that the other ligands have a higher affinity
for hSERT, bind tighter than IDT317, and one or more of these
compounds may be amenable to the development of quantum
dot-based fluorescent assays using hSERT-expressing mammalian
cells (preliminary data not shown).
Acknowledgments
This work was supported by grants from the National Institutes
of Health (RO1EB003728-02 and GM72048-02). We would also like
to thank Professor Louis De Felice for providing assistance with
measuring the leak currents and IC50 values in this study.

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I. D. Tomlinson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1678–1682
yield crude 4-(methylamino)cyclohexanone
(20).
The
crude
4-
References and notes
(methylamino)cyclohexanone (20) was dissolved in methanolic KOH solution
(2M, 200 ml) and indol (4.1 g, 45 mmol) was added. The mixture was heated at
reflux for 8 h cooled to ambient temperature and evaporated. The product
was purified by column chromatography on silica gel eluted with ethyl
acetate (98%): triethyl amine (2%) to give 1.4 g of 4-(1H-indol-3-yl)-N-
methylcyclohex-3-enamine (21) as a colorless solid in a 20% yield. 1H NMR
(CDCl3) d 8.55 (s, 1H), 7.90 (d, 1H), 7.34 (d, 1H), 7.13 (m, 3H), 6.21 (s, 1H), 2.77
(m, 1H), 2.55 (m, 4H), 1.88 (m, 2H), 1.64 (m, 4H). 13C NMR (CDCl3) d 136.78,
131.08, 125.22, 122.00, 121.14, 120.72, 119.86, 119.65, 118.47, 111.27, 54.70,
33.63, 32.57, 28.99, 27.53
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1,3-dione
(22).
4-(1H-indol-3-yl)-N-methylcyclohex-3-enamine
(1.2 g,
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12. IDT357 (8) was synthesized by dissolving Biotin-PEG5000-NHS (0.2 g) in
methylene chloride (100 ml) and 11-(4-(1H-indol-3-yl)-5,6-dihydrpyridin-
1(2H)-yl)undecylamine (17a) 0.015 g was added. This solution was stirred
for 18 h then evaporated under reduced pressure. The product was repeatedly
washed with diethylether (5 ꢁ 100 ml) to yield 0.14 g of (8) as a pale yellow
solid. Compounds (9)–(11) were synthesized using the same protocol.
13. 4-(1H-Indol-3-yl)-N-methylcyclohex-3-enamine (21). 1,4-cyclohexanedione
monoethyl ketal (5 g, 32 mmol), methylamine 33% in ethanol (10 ml) and
triacetoxy sodium borohydride (9 g, 42 mol) were mixed in methylene chloride
(100 ml) and stirred at room temperature under nitrogen for 18 h. Sodium
hydroxide (2 M, 100 ml) was added and the organic solution was separated
and dried over magnesium sulfate. This was filtered and evaporated to yield
5.7 g of N-methyl-1,4-dioxaspiro[4.5]decan-8-amine (19) which was used
without further purification. The N-methyl-1,4-dioxaspiro[4.5]decan-8-amine
(19) was dissolved in tetrahydrofuran (50 ml) and trifluoroacetic acid (50 ml)
was added. This mixture was heated at reflux for 18 h cooled and evaporated to
5.3 mmol) and 11-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-undecanyl bromide
(2 g, 5.3 mmol) were mixed in acetonitrile (100 ml) and triethyl amine (5 ml)
was added. The mixture was heated at reflux for 18 h cooled and evaporated.
The product was recrystalized from acetonitrile to yield 1.8 g (64%) of 2-(11-
((4-(1H-indol-3-yl)cyclohex-3-enyl)(methyl)aminoundecyl)isoindoline-1,3-
dione (22) as a pale brown solid. 1H NMR (DMSO-d6) d 11.18 (s, 1H), 7.85 (m,
4H), 7.42 (m, 2H), 7.10 (m, 2H), 6.11 (s, 1H), 3.55 (t, 2H), 3.36 (s, 6H), 3.11 (m,
4H), 2.77 (s, 2H), 1.70 (m, 4H), 1.29 (m, 14H). 13C NMR (DMSO-d6) d 167.95,
136.89, 134.44, 134.40, 131.56, 126.00, 124.54, 123.10, 122.99, 119.98, 119.33,
116.80, 115.53, 111.81, 45.61, 37.35, 29.36, 28.80, 28.50, 27.84, 26.20, 26.08,
23.70
15. IDT374 (13) was synthesized by dissolving Biotin-PEG5000-NHS (0.2 g) in dry
dimethyl formamide (10 ml) and 2-(11-((4-(1H-indol-3-yl)cyclohex-3-
enyl)(methyl)aminoundecyl)isoindoline-1,3-dione (0.016 g) was added. The
solution was stirred for 18 h then tenta gel derivatized with NHS was added
(1 g) and stirred for 6 h the solution was filtered and evaporated and the
resulting material was dissolved in deionised water then filtered and
evaporated to yield IDT374 (0.16 g) as a pale brown solid.
16. Compound (14). Using the same synthetic protocol as was used to synthesize
(8); 120 mg of IDT364 (12) was obtained from 150 mg of Biotin-PEG5000-NHS
and methylamine (33%, 5 ml) dissolved in methanol.
17. Deskus, J. A.; Epperson, J. R.; Sloan, C. P.; Cipollina, J. A.; Dextraze, P.; Qian-
Cutrone, J.; Gao, Q.; Ma, B.; Beno, B. R.; Mattson, G. K.; Molski, T. F.; Krause, R.
G.; Taber, M. T.; Lodge, N. J.; Mattson, R. J. Bioorg. Med. Chem. Lett. 2007, 17,
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