Development of molecular probes for the human 5-HT6 receptor

Article abstract of DOI:10.1021/jm1007177

In this work we report the synthesis of a set of labeled ligands targeting the human 5-HT₆ receptor (h5-HT₆R). Among the synthesized compounds, fluorescent probe 10 (K_i = 175 nM and -_f = 0.21) and biotinylated derivative 15 (K_i = 90 nM) deserve special attention because they enable direct observation of the h5-HT₆R in cells. Thus, they represent the first molecular probes for 5-HT₆R visualization. These results are the starting point for introducing a variety of tags in these or other 5-HT₆R ligand scaffolds aimed at the development of optimized probes with tailored profiles in terms of fluorescence, affinity, or selectivity.

Full text of DOI:10.1021/jm1007177

J. Med. Chem. 2010, 53, 7095–7106 7095
DOI: 10.1021/jm1007177
Development of Molecular Probes for the Human 5-HT6 Receptor
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§
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ꢀ
´
ꢀ †
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Henar Vazquez-Villa, Juan A. Gonzalez-Vera, Bellinda Benhamu, Alma Viso, Roberto Fernandez de la Pradilla,
Elena Junquera,‡ Emilio Aicart,‡ Marı
guez,*,† and Silvia Ortega-Gutierrez*
ꢀ
a L. Lopez-Rodrı
´
,†
ꢀ
†
‡
Departamento de Quımica Organica I, and Departamento de Quımica Fısica, Facultad de Ciencias Quımicas, Universidad Complutense de
´
ꢀ
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Madrid, E-28040 Madrid, Spain, and Instituto de Quımica Organica General, Consejo Superior de Investigaciones Cientıficas,
§
´
ꢀ
´
Juan de la Cierva 3, E-28006 Madrid, Spain
Received June 15, 2010
In this work we report the synthesis of a set of labeled ligands targeting the human 5-HT6 receptor
(h5-HT6R). Among the synthesized compounds, fluorescent probe 10 (Ki = 175 nM and Φf = 0.21) and
biotinylated derivative 15 (Ki = 90 nM) deserve special attention because they enable direct observation
of the h5-HT6R in cells. Thus, they represent the first molecular probes for 5-HT6R visualization. These
results are the starting point for introducing a variety of tags in these or other 5-HT6R ligand scaffolds
aimed at the development of optimized probes with tailored profiles in terms of fluorescence, affinity, or
selectivity.
Introduction
With this broad objective in mind, we have initiated a
project aimed at the search of fluorescent probes that enable
direct visualization of GPCRs in complex biological systems.
Although some GPCR fluorescent ligands have been recently
described, their suitability for application in cell systems has
proved challenging.11 Ideally, excellent probes would be
ligands with high affinity (i.e., Ki<100 nM) and with fluo-
rescence emission intensity as high as possible.
In this work, we have focused our efforts on the 5-HT6
receptor (5-HT6R), one of the most recent additions to the
serotonin family of GPCRs. This receptor has been involved
in the pathogenesis of CNS diseases related to cognitive or
eating disorders,12,13 and several 5-HT6R ligands have entered
clinical trials although none of them is on the market yet. In
this regard, availability of agents able to directly monitor the
5-HT6R, which to our knowledge has not been described so
far, would aid the therapeutic validation of this receptor for
the proposed or even new indications.
In this work we report the synthesis of a set of probes 1-15
based on the potent 5-HT6R antagonist developed by
SmithKlineBeecham SB-27104614,15 (16, pKi = 8.9; pA2 =
8.7, Figure 1). Among the synthesized compounds, fluores-
cent probe 10 (Ki = 175 nM and fluorescence quantum yield,
Φf, of 0.21) and biotinylated derivative 15 (Ki = 90 nM)
deserve special attention, as they enable direct observation
of the h5-HT6R in cells. These results support the validity of
our approach and represent the starting point for further probe
optimization with tailored profiles in terms of tag incorpora-
tion, affinity, and selectivity, experiments that are underway in
our laboratory.
Fluorescence-based molecular imaging facilitates visualiza-
tion of specific biomolecules in their native environments.
Current advances in this field include the development of
fluorescent small molecules that allow detection of metabo-
lites and ions,1,2 imaging of glycan- and lipid-containing pro-
teins,3-5 and profiling of enzyme activities.6 However, extension
of these strategies to G-protein-coupled receptors (GPCRsa),
which are the target of about the 50% of current marketed
drugs, has proved challenging. Changes in levels and expression
pattern of GPCRs have been associated with a variety of disease
states including central nervous system (CNS) related patholo-
gies and cancer.7-9 Therefore, the ability to directly monitor
GPCRs in the context of cells, tissues, and organisms would
undoubtedly augment our understanding of their physiological
significance and provide new clinical tools boosting drug devel-
opment. Although antibodies against GPCRs enable their
visualization in vitro or ex vivo, they have in vivo limited use.
In addition, generation of GPCR antibodies is difficult be-
cause of their structure, poor immunogenicity, and low recep-
tor density.10 These technical challenges have hampered the
general availability of GPCR antibodies and are associated
with the restricted utility in many experimental formats of
some of the available ones. Thus, the development of com-
plementary methods for GPCR imaging is of great impor-
tance. Moreover, the introduction of alternative tags other
than fluorophores to obtain additional information in a high-
throughput manner comparable to activity-based protein
profiling approaches in the field of enzymes is still lacking.6
*For M.L.L.-R.: phone, þ34 913944239; fax, þ34 913944103; e-mail,
mluzlr@quim.ucm.es. For S.O.-G.: phone, þ34 913944894; fax, þ34
913944103; e-mail, siortega@quim.ucm.es.
Results and Discussion
Initially we based our structural exploration on the scaffold
of 16, in which we have studied different structural possibi-
lities for the introduction of the fluorescent tag aiming to have
a minimal influence on the biological activity of the parent
compound. Accordingly, we considered small sets of probes
where the tag was introduced (a) in the piperazine ring, (b) in
a Abbreviations: 5-HT, serotonin; ACN, acetonitrile; a.u., arbitrary
units; Ds, dansyl; DIPEA, N,N-diisopropylethylamine; Phth, phthali-
mide; BINAP, (()-2,20-bis(diphenylphosphino)-1,10-binaphthalene;
Em, emission; Ex, excitation; Φf, fluorescence quantum yield; GPCR,
G-protein-coupled receptor; h5-HT6R, type 6 of the human 5-HT
receptor; HOBt, 1-hydroxybenzotriazole; I, intensity; SAR, structure-
activity relationship.
r
2010 American Chemical Society
Published on Web 09/16/2010
pubs.acs.org/jmc

7096 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Vaꢀzquez-Villa et al.
Figure 1. Labeled derivatives of the h5-HT6R antagonist 16.
Scheme 1. Synthesis of Dansylated Derivatives 1-3a
a Reagents and conditions: (a) DsCl (1.5 equiv), Et3N (2 equiv), CH2Cl2, room temp, 12 h, 66-74%; (b) H2 (40 psi), Pd/C (8% wt), EtOH/H2O
(200:1), room temp, 16 h, 94-96%; (c) 5-chloro-3-methylbenzo[b]thiophene-2-sulfonyl chloride (1.5 equiv), pyridine (2 equiv), CH2Cl2, room temp,
12 h, 62-66%; (d) NaI (2 equiv), ACN, reflux, 32 h, 79-89%; (e) TFA (20 equiv), CH2Cl2, room temp, 10 h, 99%.
the sulfonamide moiety, or (c) in the methoxy group
(Figure 1). As tag, a dansyl ((5-dimethylamino)naphthalene-
1-sulfonyl, Ds) group was originally selected by virtue of its
syntheticversatility, smallsize, relatively large Stokes shift, the
high degree of sensitivity of its emission wavelength and
quantum yield to environmental changes, and its suitability
for use in cellular environments.16,17 In all cases, the tag was
attached either directly or through a variable length aliphatic
spacer that may provide some flexibility to the fluorophore in
order to minimally affect probe-receptor affinity.
Synthesis of Probes Based on the Scaffold of 16. We
prepared the first series of labeled compounds by attaching
a dansyl group to the nitrogen of the piperazine ring of 16
either directly or through a spacer (Figure 1). The reaction

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7097
Scheme 2. Synthesis of Dansylated Derivatives 4-6a
a Reagents and conditions: (a) DsCl (1.5 equiv), DIPEA (2 equiv), DMAP cat., CH2Cl2, room temp, 12 h, 85%; (b) TFA (20 equiv), CH2Cl2, room
temp, 6 h, 81-97%; (c) NaH (1.8 equiv), DMF, 90 °C, 16 h, 95-96%; (d) N2H4 H2O (15 equiv), EtOH, 50 °C, 5 h, 86-98%; (e) DsCl (1.5 equiv), Et3N
3
(3 equiv), CH2Cl2, room temp, 16 h, 73-76%.
Scheme 3. Synthesis of Dansylated Derivative 7a
a Reagents and conditions: (a) DEAD (2 equiv), PPh3 (2 equiv), THF, reflux, 34 h, 60%; (b) 1-Boc-piperazine (3 equiv), Pd(OAc)2 (10 mol %), BINAP
(15 mol %), Cs2CO3 (1.5 equiv), toluene, 100 °C, 24 h, 21%; (c) H2 (40 psi), Pd/C (8% wt), EtOH/H2O (100:1), room temp, 16 h, 99%; (d) 5-chloro-3-
methylbenzo[b]thiophene-2-sulfonyl chloride (1.5 equiv), pyridine (2 equiv), CH2Cl2, room temp, 12 h, 80%; (e) TFA (20 equiv), CH2Cl2, room temp,
10 h, 88%.
of 1-(2-methoxy-5-nitrophenyl)piperazine 17 with dansyl
chloride yielded intermediate 18. Hydrogenation of the nitro
group in 18 afforded the aniline derivative 19 that was then
reacted with 5-chloro-3-methylbenzo[b]thiophene-2-sulfonyl
chloride to afford the target compound 1. Compound 2, in
which the dansyl group is attached to the piperazine through a
hexamethylene chain, was prepared following a similar proce-
dure starting with alkylation of 17 with N-Boc-6-bromohexy-
lamine. Further increase of the spacer length (compound 3)
was achieved by direct alkylation of 16 with the dansylated
bromooctylamine 24 (Scheme 1).
A second series of labeled ligands derived from 16 was
synthesized by linking the dansyl group to the nitrogen atom
of the sulfonamide group through alkyl spacers of variable
length (Figure 1). Compound 4, where the dansyl group is
directly attached to the core structure, was prepared by reaction
of the derivative 25 with dansyl chloride in the presence of
DIPEA. Compounds 5 and 6, in which the fluorophore is
linked to the sulfonamide nitrogen through four and eight
carbon alkyl chains, respectively, were prepared following
an alkylation sequence (Scheme 2). Thus, reaction of 25 with
the corresponding N-(bromoalkyl)phthalimide afforded the
alkylated products 27 and 28. Subsequent removal of the
phthalimide followed by reaction with dansyl chloride led to
the protected intermediates 31 and 32 that were finally trans-
formed into the target compounds 5 and 6 by cleavage of the
Boc group with TFA in dichloromethane.
Finally, we also explored whether it was possible to sub-
stitute the methoxy group of 16 by a dansyl tag attached
through a spacer (see Figure 1). This compound was pre-
pared following the synthetic route shown in Scheme 3.
Mitsunobu reaction of 2-bromo-4-nitrophenol with the dansyl
tagged alcohol 33 yielded compound 34. Buchwald-Hartwig
coupling of 34 with 1-Boc-piperazine led to 35 that was then
transformed to the corresponding aniline derivative 36. Its
reaction with the sulfonyl chloride derivative followed by

7098 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 1. 5-HT6R Affinity and Fluorescence Values for Probes 1-7
Vaꢀzquez-Villa et al.
Scheme 4. Synthesis of Compounds 8 and 9a
a Reagents and conditions: (a) DsCl (1 equiv), pyridine (2 equiv),
CH2Cl2, room temp, 15 h, 82%; (b) TFA (20 equiv), CH2Cl2, room
temp, 6 h, 83-93%; (c) DsCl, DIPEA (2 equiv), DMAP cat., CH2Cl2,
room temp, 12 h, 50%.
Table 2. 5-HT6R Affinity and Fluorescence Values for Probes 8-12
a The values are the mean ( SEM from two to four independent
experiments performed in triplicate. b Fluorescence values were deter-
mined at 10 μM in binding assay buffer (50 mM Tris-HCl, 0.5 mM
MgSO4, pH 7.4) at 25 °C using an excitation slit of 2.5 nm. Excitation
wavelength (λex) was 350 nm for all the compounds except for 3 (λex
=
338 nm). c Emission slit of 5 nm.
cleavage of the Boc protecting group rendered the final
compound 7.
Biological Data and Fluorescent Properties of Probes 1-7.
Table 1 shows the affinity values for the h5-HT6R and the
fluorescent properties of the probes 1-7. Affinity values
were determined by competition binding assays on mem-
branes from h5-HT6R stably expressing HEK-293 EBNA
cells, using [3H]LSD as radioligand. The fluorescent proper-
ties of compounds 1-7 were determined in 10 μM solutions
of the corresponding compound in the buffer solution used
for the binding assays. In general, the introduction of a
dansyl group at any of the positions of 16 was detrimental
for the h5-HT6R affinity, which remained between moderate
(compounds 1, 2, and 6) and low (compounds 3, 5, and 7).
Only derivative 4 conserved high affinity (Ki = 27 nM),
though in this case with a negligible fluorescence. The highest
fluorescence values were shown by derivatives 2, 3, and 6.
Taken together, all these data indicate that modification of
16 by attachment of a fluorophore is not likely to yield a
fluorescent high-affinity 5-HT6R ligand.
a The values are the mean ( SEM from two to four independent
experiments performed in triplicate. b Fluorescence values were deter-
mined at 10 μM in binding assay buffer (50 mM Tris-HCl, 0.5 mM
MgSO4, pH 7.4 at 25 °C) using an excitation slit of 2.5 nm. λex was
350 nm for 8 and 9, 340 nm for 10 and 11, and 338 nm for 12. c Emission
slit of 5 nm.
was likely to keep high 5-HT6R affinity and we were con-
fident in that this new scaffold would be fluorescent.
Dansyl-Based Probes. Initially, we synthesized an analo-
gue of 16 in which the dansyl group replaces the benzothio-
phenesulfonyl group (Scheme 4). Thus, reaction of N-Boc-
1-(5-amino-2-methoxyphenyl)piperazine 38 with dansyl
chloride in the presence of pyridine yielded the expected
compound 8 after deprotection of the piperazine ring by
treatment with TFA. Although this compound showed a
high 5-HT6R affinity (Ki = 8 nM), it was not fluorescent
(Table 2). Not even the introduction of a second dansyl
moiety in the sulfonamide group (derivative 9), obtained
Therefore, we envisioned the possibility of replacing the
benzothiophenesulfonyl moiety of 16 for the dansyl group.
According to our previous pharmacophore model and struc-
ture-affinity relationship (SAR) studies,18 this modification

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7099
Scheme 5. Synthesis of Compounds 10-12a
a Reagents and conditions: (a) Br(CH2)nNHBoc, NaI (2 equiv), ACN, reflux, 32 h, 38-89%; (b) TFA (20 equiv), CH2Cl2, room temp, 10 h, 99%;
(c) DsCl (1.5 equiv), Et3N (3 equiv), CH2Cl2, room temp, 12 h, 74-79%; (d) H2 (40 psi), Pd/C (8% wt), EtOH/H2O (200:1), room temp, 16 h, 94%;
(e) DsCl (1.5 equiv), pyridine (2 equiv), CH2Cl2, room temp, 16 h, 65-71%; (f) 24, NaI (2 equiv), ACN, reflux, 32 h, 45%.
Scheme 6. Synthesis of Compounds 13 and 14a
a Reagents and conditions: (a) dapoxyl chloride (1.1 equiv), pyridine (2 equiv), CH2Cl2, room temp, 16 h, 84%; (b) TFA (20 equiv), CH2Cl2, room
temp, 6 h, 91-99%; (c) Lissamine rhodamine B sulfonyl chloride (1 equiv), Et3N (5 equiv), THF, 80 °C, 24 h, 52%.
Table 3. 5-HT6R Affinity and Fluorescence Values for Compounds 13
under the same conditions as 8 when diisopropylethylamine
and 14
was used as base, allowed for obtaining significant fluores-
cence. Therefore, we focused our efforts on establishing
whether the introduction of a second dansyl group attached
to the piperazine by a methylene spacer of variable length
would render simultaneous high 5-HT6R affinity and fluor-
escence. Compounds 10 and 11 were synthesized following a
route analogous to that used for compound 2 (see Scheme 1),
attaching the second dansyl group to the aromatic ring in the
last step of the sequence, whereas derivative 12, with the
longest spacer (n = 8), was synthesized by direct alkylation
of the piperazine nitrogen of the parent compound 8 with the
tagged chain 24 (Scheme 5).
fluorescenceb
compd
R1
R2 R3 Ki a (nM) λex c (nm) λem d (nm) Iem (a.u.)
13
14
dapoxyl
lissamine
H
H
H
H
523 ( 22
>1000
370
570
e
550
588
a The values are the mean ( SEM from two to four independent
experiments performed in triplicate. b Fluorescence values were deter-
mined at 10 μM in binding assay buffer (50 mM Tris-HCl, 0.5 mM
MgSO4, pH 7.4 at 25 °C). Wavelengths for the maximal absorption
and emission. c Excitation slit of 2.5 nm. d Emission slit of 5 nm. e Non-
fluorescent compound.
Determination of the 5-HT6R affinity and fluorescence
(Table 2) of the synthesized compounds showed that 10, with
a Ki value of 175 nM, a fluorescence emission intensity (Iem
)
above 1000 a.u. for excitation and emission slits of 2.5 and
5 nm and a high Stokes shift, exhibited the best profile in

7100 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Scheme 7. Synthesis of Biotin-Tagged Compound 15a
Vaꢀzquez-Villa et al.
a Reagents and conditions: (a) NaI (2 equiv), ACN, reflux, 36 h, 81%; (b) TFA (20 equiv), CH2Cl2, room temp, 6 h, 83%; (c) D-(þ)-biotin (0.83 equiv),
HOBt (0.2 equiv), DCC (1.1 equiv), DMAP (1 mol %), DMF, room temp, 24 h, 82%.
Figure 2. Labeling of COS7 cells transiently transfected with h5-HT6R with fluorescent compound 10. Cells were incubated in the presence of
10 (5 μM) for 10 min, washed, fixed, mounted, and then observed by confocal microscopy (A). To assess specificity, nontransfected cells were
labeled under the same conditions (B) or transfected cells were incubated with 10 in the presence of an excess (50 μM) of 16 (C). Scale bar
represents 25 μm. Preparations were visualized under a SP2 Leica confocal microscope with a 63ꢀ objective with constant laser beam and
photodetector sensitivity and are representative of two or three independent experiments.
Figure 3. Labeling of COS7 cells transiently transfected with h5-HT6R with biotinylated compound 15. Cells were fixed, incubated in
the presence of 15 (5 μM) for 10 min followed by streptavidin-Alexa Fluor 488 conjugate, mounted, and then observed by fluorescence
microscopy. Nuclei were counterstained with Hoechst 33258 (A). To assess specificity, nontransfected cells were labeled under the same
conditions (B) or in the absence of probe (C). Scale bar represents 25 μm. Preparations were visualized under a Zeiss Axioplan2 fluorescence
microscope. Images are representative of two independent experiments.
terms of affinity and fluorescence and therefore could be a
candidate to assess its potential as a probe for 5-HT6R
visualization in cellular systems. In spite of this encouraging
data, we notice that even the best values obtained for simul-
taneous fluorescence and affinity (compound 10, Ki=175 nM;
Iem > 1000 a.u., Table 2) are only moderate, so it was con-
ceivable that further optimization of the probes would be
highly desirable for their use in complex biological settings.
However, the extensive structural variations addressed in
derivatives 1-12 suggest that dansyl labeling of 16 does not
allow for high affinity and fluorescent 5-HT6R ligands. There-
fore, we envisioned the possibility of incorporating other
fluorophores or a versatile biotin subunit. Considering that
substitution of the benzothiophenesulfonyl group in 16 for a
dansyl moiety (compound 8) kept 5-HT6R affinity [Ki (8)=
8 nM], we tried to replace it for dapoxyl (4-{5-[4-(dimethylamino)-
phenyl]-1,3-oxazol-2-yl}benzenesulfonyl) or lissamine rhodamine
B (2-(chlorosulfonyl)-5-[6-(diethylamino)-3-(diethyliminio)-3H-
xanthen-9-yl]benzenesulfonate), fluorescent moieties with higher
extinction coefficients than dansyl group. Dapoxyl-labeled
derivative 13 was synthesized by treatment of N-Boc-1-(5-
amino-2-methoxyphenyl)piperazine 38 with dapoxyl chlo-
ride followed by cleavage of the Boc protecting group under
standard conditions. On the other hand, reaction of 38 with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7101
lissamine rhodamine B sulfonyl chloride (mixture of isomers)
and subsequent deprotection of the piperazine ring yielded
probe 14 as a mixture of isomers (Scheme 6).
physiological states. In addition to visualization, implementa-
tion of other chemical reporters with different tags that enable
covalent binding or affinity pull-downs should provide new
tools to interrogate GPCR states. These strategies should be
extendable even to complex biological settings, a final goal
worthy of pursuit given the enormous importance of the
members of the GPCR superfamily in normal and disease
states. Increase of the arsenal of tools for probing GPCRs
belonging to different families and assessment of their bio-
logical potential are currently underway in our laboratory.
Unfortunately, both compounds exhibited poor 5-HT6R
affinity and low fluorescence (Table 3). These results made us
consider the piperazine ring as an attachment point of the
biotin moiety. Thus, N-alkylation of 16 with N-Boc-6-bro-
mohexylamine led to compound 43. Cleavage of the Boc
protecting group followed by coupling reaction with biotin
afforded the tagged compound 15 (Scheme 7) which was
characterized as 5-HT6R ligand (Ki = 90 nM).
Cell Visualization. On the basis of all these results, we have
selected two probes to carry out visualization experiments,
dansyl-based derivative 10, with the best balance between
5-HT6R affinity and fluorescence (Ki=175 nM; Iem>1000 a.u.
for excitation and emission slits of 2.5 and 5 nm; λex = 340 nm;
Experimental Section
Chemistry. Melting points (uncorrected) were determined on
a Stuart Scientific electrothermal apparatus. Infrared (IR) spectra
were measured on a Bruker Tensor 27 instrument equipped with a
Specac ATR accessory of 5200-650 cm-1 transmission range.
Frequencies (ν) are expressed in cm-1. Nuclear magnetic reso-
nance (NMR) spectra were recorded on a Bruker Avance DPX
300 spectrometer (1H, 300 MHz; 13C, 75 MHz) or Bruker Avance
500 spectrometer (1H, 500 MHz; 13C, 125 MHz) at the UCM’s
NMR facilities or on a Varian INOVA-300 (1H, 300 MHz; 13C,
75 MHz) or Varian INOVA-400 (1H, 400 MHz; 13C, 100 MHz) at
the CSIC’s NMR facilities. Chemical shifts (δ) are expressed in
parts per million relative to internal tetramethylsilane. Coupling
constants (J) are in hertz (Hz). The following abbreviations are
used to describe peak patterns when appropriate: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), br (broad), app
(apparent). Two-dimensional NMR experiments (HMQC and
HBQC) of representative compounds were carried out to assign
protons and carbons of the new structures. High resolution mass
spectrometry (HRMS) was carried out on a FTMS Bruker APEX
Q IV (UCM) or Agilent QTOF 6520 (CSIC) spectrometer in
electrospray ionization (ESI) mode. High pressure liquid chroma-
tography-mass spectrometry (HPLC-MS) analysis was per-
formed using an Agilent 1200LC-MSD VL instrument. LC
separation was achieved with an Eclipse XDB-C18 column
(5 μm, 4.6 mm ꢀ 15 mm) together with a guard column (5 μm,
4.6 mm ꢀ 12.5 mm). The gradient mobile phases consisted of A
(95:5 water/acetonitrile) and B (5:95 water/acetonitrile) with 0.1%
ammonium hydroxide and 0.1% formic acid as the solvent
modifiers. The gradient started at 0% B (for 5 min) and increased
linearly to 100% B over the course of 20 min, with a flow rate of
0.5 mL/min, and it was followed by an isocratic gradient of 100%
B for 5 min before equilibrating for 5 min at 0% B. MS analysis
was performedwithanESIsource. Thecapillary voltage wassetto
3.0 kV, and the fragmentor voltage was set at 70 eV. The drying
gas temperature was 350 °C, the drying gas flow was 10 L/min,
and the nebulizer pressure was 20 psi. Elemental analyses (C, H,
N, S) were obtained on a LECO CHNS-932 apparatus at the
UCM’s or CSIC’s analysis services and were within 0.5% of the
theoretical values, confirming a purity of at least 95% for all tested
compounds. Analytical thin-layer chromatography (TLC) was
run on Merck silica gel plates (Kieselgel 60 F-254) with detection
by UV light, ninhydrin solution, or 10% phosphomolybdic acid
solution in ethanol. Flash chromatography was performed on
glass column using silica gel type 60 (Merck, particle 230-400
mesh). Unless stated otherwise, starting materials, reagents, and
solvents were purchased as high-grade commercial products from
Sigma-Aldrich, Fluka, Lancaster, ABCR, Acros, Scharlab, or
Panreac and were used without further purification. In general,
solvents were obtained via elution through a Pure Solv column
drying system from Innovative Technology, Inc. THF was dis-
tilled from sodium benzophenone ketyl and used immediately.
Dichloromethane was distilled from calcium hydride.
λem = 495 nm; Φf = 0.21), and the biotinylated probe 15 (Ki =
90 nM), which can be visualized using fluorophore-conjugated
streptavidin. To assess whether these probes kept the binding
profile of the parent compound 16, we selected some representa-
tive metabotropic serotonin receptors (5-HT1A, 5-HT5A, and
5-HT7) for which 16 displayed moderate affinity.15 Both com-
pounds 10 and 15 (at 1 μM) did not significantly bind (radio-
ligand displacement <15%) to any of the analyzed receptors,
matching the binding affinities described for 16.
In order to assess the full potential of these probes for
visualization of the 5-HT6R in cells, 10 and 15 were used in in
vitro cell labeling experiments. Incubation of cells with probe 10
(5 μM) allowed direct observation of the h5-HT6R in transiently
transfected COS7 cells using conventional confocal microscopy
(λex = 405 nm) (Figure 2A). In order to assess specificity of the
labeling, nontransfected cells were incubated with compound 10
under the same conditions (Figure 2B). In this case a diffuse
fluorescence pattern that did not allow the clear distinction of the
cells was obtained. In addition, incubation of transfected cells
with probe 10 in the presence of an excess of 16 essentially
eliminated fluorescent labeling (Figure 2C).
Although these results support that this probe enables
5-HT6R visualization, biotinylated probe 15 might exhibit
better properties considering its higher receptor affinity
(Ki(15) = 90 nM vs Ki(10) = 175 nM) but especially its
versatility. In this regard, there exists an array of easily
available streptavidin-fluorophore conjugates optimized
for fluorescence microscopy. Incubation of cells with probe
15 in similar conditions as above also allowed direct obser-
vation of the h5-HT6R using fluorescence microscopy
(Figure 3A). Supporting specificity, labeling is essentially
absent in nontransfected cells (Figure 3B). Figure 3C shows
cellular autofluorescence in the absence of probe. Consid-
ered together, all these results confirm the suitability of 10
and 15 as probes for cellular visualization of the h5-HT6R.
Conclusions
In summary, the synthesized compounds 10 and 15 described
herein represent the first fluorescent probes for the specific
labeling of the h5-HT6R in cells. These results are the starting
point in order to introduce a variety of tags in the scaffold of
other 5-HT6R ligands different from 16, aimed at the develop-
ment of optimized probes with tailored profiles in terms of
fluorescence, affinity, or selectivity. Availability of these probes
should help boost the molecular imaging field focused on the
GPCR superfamily by providing tools that facilitate direct
monitoring of spatiotemporal changes associated with (patho)-
Collected data for compounds 1-15 refer to free bases, and
then hydrochloride salts were prepared prior to elemental
analyses and biological assays. Spectroscopic data of all de-
scribed compounds were consistent with the proposed struc-
tures. Satisfactory HPLC chromatograms were also obtained

7102 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Vaꢀzquez-Villa et al.
for the final compounds 1-15 and for 16. The data of final
compounds 1-15 are described.
5-Chloro-N-(3-{4-[8-({[5-(dimethylamino)-1-naphthyl]sulfonyl}-
amino)octyl]piperazin-1-yl}-4-methoxyphenyl)-3-methyl-1-benzo-
thiophene-2-sulfonamide (3). Sodium iodide (59 mg, 0.39 mmol)
and 24 (100 mg, 0.23 mmol) were added to a solution of 16
(89 mg, 0.20 mmol) in anhydrous acetonitrile (10 mL) under an
argon atmosphere, and the reaction mixture was refluxed for
34 h. Solvent was removed under reduced pressure and the
residue was purified by flash chromatography on silica gel
(elution with 150:1 to 20:1 CH2Cl2/MeOH) to afford pure 3
(126mg, 79%yield) asa beigesolid(mp=158-160 °C). Rf =0.45
(CH2Cl2/MeOH, 10:1). IR (KBr) ν = 3271, 2931, 2853, 1588,
1506, 1322, 1158 cm-1. 1H NMR (CDCl3, 300 MHz) δ=1.08-
1.26 (m, 8H, CH2), 1.31-1.44 (m, 2H, CH2), 1.46-1.58 (m, 2H,
CH2), 2.24 (s, 3H, CH3), 2.35-2.48 (m, 2H, CH2), 2.64 (m, 4H,
CH2), 2.79-2.98 (m, 12H, CH3, CH2), 3.81 (s, 3H, CH3), 4.73
(t, J = 6.1 Hz, 1H, NH), 6.60 (d, J = 2.2 Hz, 1H, Ar), 6.67 (d,
J = 8.6 Hz, 1H, Ar), 6.73 (dd, J = 8.6, 2.2 Hz, 1H, Ar), 7.19 (d,
J = 7.2 Hz, 1H, Ar), 7.40 (dd, J=8.6, 2.0 Hz, 1H, Ar), 7.50-7.62
(m, 2H, Ar), 7.65 (dd, J = 1.8, 0.5 Hz, 1H, Ar), 7.70 (dd, J = 8.6,
0.5 Hz, 1H, Ar), 8.25 (dd, J = 7.3, 1.2 Hz, 1H, Ar), 8.29 (d, J =
8.7 Hz, 1H, Ar), 8.54 (d, J = 8.5 Hz, 1H, Ar) ppm. 13C NMR
(CDCl3, 75 MHz) δ = 12.2 (CH3), 26.4 (CH2), 27.3 (CH2), 28.9
(CH2), 29.3 (CH2), 29.6 (CH2), 29.8 (CH2), 43.4 (CH2), 45.6
(2CH3), 49.9 (2CH2), 53.0 (2CH2), 55.7 (CH3), 58.6 (CH2), 111.3
(CH), 115.3 (CH), 116.1 (CH), 118.8 (CH), 119.9 (CH), 123.3
(CH), 123.5 (CH), 123.9 (CH), 127.9 (CH), 128.4 (C), 128.5
(CH), 129.7 (C), 129.8 (CH), 130.0 (C), 130.5 (CH), 131.5 (C),
134.8 (C), 136.0 (C), 137.3 (C), 137.7 (C), 140.6 (C), 141.4 (C),
151.3 (C), 152.1 (C) ppm. HRMS (ESI) calcd for C40H51ClN5-
O5S3 (M þ H)þ: 812.2582. Found: 812.2589. Elemental analysis
calcd (%) for C40H50ClN5O5S3 2.5HCl 3H2O: C 50.16, H 6.16,
5-Chloro-N-[3-(4-{[5-(dimethylamino)-1-naphthyl]sulfonyl}-
piperazin-1-yl)-4-methoxyphenyl]-3-methyl-1-benzothiophene-2-
sulfonamide (1). Pyridine (42 μL, 0.52 mmol) was added to a
solution of 19 (114 mg, 0.26 mmol) in anhydrous CH2Cl2 (2 mL)
under an argon atmosphere at room temperature. A solution of
5-chloro-3-methylbenzo[b]thiophene-2-sulfonyl chloride (81 mg,
0.29 mmol) in anhydrous CH2Cl2 (1 mL) was added dropwise,
and the mixture was stirred for 12 h at room temperature. The
reaction was then quenched with a 1 M aqueous solution of HCl
(5 mL), and the aqueous phase was extracted with CH2Cl2 (2 ꢀ
10 mL). The combined organic layers were washed with water
(1 ꢀ 10 mL) and brine (1 ꢀ 10 mL), dried over Na2SO4, filtered,
and evaporated under reduced pressure. Flash chromatography
of the residue on silica gel (elution with CH2Cl2 to 500:1 CH2Cl2/
MeOH) gave 210 mg (66% yield) of 1 as a pale yellow solid
(mp =161-163 °C). Rf = 0.40 (hexane/ethyl acetate, 1:1). IR
(KBr) ν = 3439, 2924, 2855, 1597, 1508, 1453, 1393, 1334, 1242,
1154 cm-1. 1H NMR (CDCl3, 300 MHz) δ = 2.26 (s, 3H, CH3),
2.88 (m, 4H, CH2), 2.97 (s, 6H, CH3), 3.27 (m, 4H, CH2), 3.73 (s,
3H, CH3), 6.60 (s, 1H, Ar), 6.64 (d, J = 8.6 Hz, 1H, Ar), 6.70 (dd,
J = 8.6, 2.3 Hz, 1H, Ar), 6.87 (s, 1H, NH), 7.20 (br d, J = 7.2 Hz,
1H, Ar), 7.41 (dd, J = 8.6, 2.0 Hz, 1H, Ar), 7.58 (app t, J = 7.8 Hz,
2H, Ar), 7.64 (d, J = 2.0 Hz, 1H, Ar), 7.68 (d, J = 8.6 Hz, 1H, Ar),
8.21 (dd, J =7.3, 1.0 Hz, 1H, Ar), 8.53 (d, J = 8.7 Hz, 1H, Ar), 8.67
(d, J =7.3Hz, 1H, Ar) ppm. 13CNMR (CDCl3, 75 MHz) δ= 12.2
(CH3), 45.8 (2CH3), 45.9 (2CH2), 50.1 (2CH2), 55.7 (CH3), 111.5
(CH), 115.6 (CH), 115.8 (CH), 119.5 (2CH), 123.5 (2CH), 123.9
(CH), 128.1 (CH), 128.2 (CH, C), 128.6 (C), 130.6 (CH), 130.8 (C),
130.9 (CH), 131.6 (C), 132.6 (C), 135.7 (C), 137.5 (C), 137.8 (C),
140.6 (2C), 151.1 (2C) ppm. HRMS (ESI) calcd for C32H32ClN4-
O5S3 ([M - H]þ): 683.1223. Found: 683.1229. Elemental analysis
calcd (%) for C32H33ClN4O5S3 2HCl 2.5H2O: C 47.85, H 5.02, N
3
3
N 7.31, S 10.04. Found: C 50.16, H 6.12, N 6.89, S 9.73.
5-Chloro-N-{[5-(dimethylamino)-1-naphthyl]sulfonyl}-N-(4-me-
thoxy-3-piperazin-1-ylphenyl)-3-methyl-1-benzothiophene-2-sulfonamide
(4). TFA (0.21 mL, 2.8 mmol) was added to a solution of 26 (110 mg,
0.14 mmol) in anhydrous CH2Cl2 (5 mL) under an argon atmo-
sphere, and the mixture was stirred for 6 h at room temperature. The
mixture was then neutralized with a 10% aqueous solution of
NaHCO3, and the aqueous phase was extracted with CH2Cl2 (2 ꢀ
10 mL). The combined organic layers were washed with water (1 ꢀ
10 mL) and brine (1 ꢀ 10 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. Flash chromatography of the
residue on silica gel (elution with 50:1 to 10:1 CH2Cl2/MeOH) gave
75 mg (81% yield) of 4 as a pale yellow solid (mp =135-137 °C).
Rf =0.14(CH2Cl2/MeOH, 9:1). IR (KBr) ν= 3435, 2939, 2833, 1588,
3
3
6.98, S 11.98. Found: C 47.80, H 4.63, N 6.80, S 11.69.
5-Chloro-N-(3-{4-[6-({[5-(dimethylamino)-1-naphthyl]sulfonyl}-
amino)hexyl]piperazin-1-yl}-4-methoxyphenyl)-3-methyl-1-benzothio-
phene-2-sulfonamide (2). Pyridine (32 μL, 0.40 mmol) was added to a
solution of 23 (110 mg, 0.20 mmol) in anhydrous CH2Cl2 (2 mL)
under an argon atmosphere at room temperature. A solution of
5-chloro-3-methylbenzo[b]thiophene-2-sulfonyl chloride (62 mg,
0.22 mmol) in anhydrous CH2Cl2 (1 mL) was added dropwise,
and the reaction mixture was stirred for 12 h at room temperature.
The reaction was then quenched with a 10% aqueous solution of
NaHCO3 (3 mL), and the aqueous phase was extracted with CH2Cl2
(2 ꢀ 10 mL). The combined organic layers were washed with water
(1 ꢀ 10 mL) and brine (1 ꢀ 10 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. Flash chromatography of the
residue on silica gel (elution with 100:1 to 10:1 CH2Cl2/MeOH) gave
98 mg (62% yield) of 2 as a pale yellow solid (mp = 149-151 °C).
Rf = 0.54 (CH2Cl2/MeOH, 9:1). IR (KBr) ν = 3290, 2929, 1508,
1322, 1158 cm-1. 1H NMR (CDCl3, 500 MHz) δ = 1.10-1.28 (m,
4H, CH2), 1.32-1.50 (m, 4H, CH2), 2.22 (s, 3H, CH3), 2.30-2.40
(m, 2H, CH2), 2.54 (m, 4H, CH2), 2.84 (m, 4H, CH2), 2.80-2.98 (m,
8H, CH3,CH2),3.80(s,3H, CH3),4.78(t, J= 6.1 Hz, 1H, NH), 6.55
(d, J = 2.2 Hz, 1H, Ar), 6.67 (d, J = 8.6 Hz, 1H, Ar), 6.72 (dd, J =
8.6, 2.2 Hz, 1H, Ar), 7.18 (d, J = 7.6, 1H, Ar), 7.41 (dd, J = 8.6, 2.0
Hz, 1H, Ar), 7.52 (dd, J= 8.5, 7.3 Hz, 1H, Ar), 7.55 (dd, J= 8.6, 7.6
Hz, 1H, Ar), 7.65 (d, J = 1.7 Hz, 1H, Ar), 7.70 (d, J = 8.6 Hz, 1H,
Ar), 8.25 (dd, J = 7.3, 1.2 Hz, 1H, Ar), 8.29 (d, J = 8.7 Hz, 1H, Ar),
8.54(d,J= 8.5 Hz, 1H, Ar) ppm. 13CNMR(CDCl3,125MHz) δ=
12.0 (CH3), 25.8 (CH2), 26.1 (CH2), 26.7 (CH2), 29.3 (CH2), 43.2
(CH2), 45.4 (2CH3), 49.8 (2CH2), 52.7 (2CH2), 55.5 (CH3), 58.0
(CH2), 111.1 (CH), 115.1 (CH), 116.5 (CH), 118.7 (CH), 120.6 (CH),
123.1 (CH), 123.2 (CH), 123.7 (CH), 127.6 (CH), 128.1 (C), 128.3
(CH), 129.5 (CH), 129.6 (C), 129.8 (C), 130.3 (CH), 131.3 (C), 134.7
(C), 136.1 (C), 136.9 (C), 137.4 (C), 140.4 (C), 141.3 (C), 151.2 (C),
151.9 (C) ppm. MS (ESI) 784.5 (M þ H)þ. Elemental analysis calcd
(%) for C38H46ClN5O5S3 2.5HCl 2.5H2O: C 49.57, H 5.86, N 7.61,
1
1504, 1372, 1355, 1250, 1166 cm-1. H NMR (CDCl3, 500 MHz)
δ = 2.29 (s, 3H, CH3), 2.59-2.63 (m, 2H, CH2), 2.67-2.70 (m, 2H,
CH2), 2.89 (s, 6H, CH3), 2.91 (m, 4H, CH2), 3.86 (s, 3H, CH3),6.49(d,
J = 2.5 Hz, 1H, Ar), 6.75 (d, J = 8.5 Hz, 1H, Ar), 6.92 (dd, J =
8.5, 2.5 Hz, 1H, Ar), 7.16 (d, J = 7.5 Hz, 1H, Ar), 7.37 (dd,
J = 8.5, 7.5 Hz, 1H, Ar), 7.48 (dd, J = 8.5, 2.0 Hz, 1H, Ar), 7.52
(dd,J=8.0,7.5Hz,1H,Ar),7.74(d,J=7.0Hz,1H,Ar),7.76(s,1H,
Ar), 8.05 (d, J = 9.0 Hz, 1H, Ar), 8.36 (dd, J = 7.5, 1.5 Hz, 1H, Ar),
8.59 (d, J = 8.5 Hz, 1H, Ar) ppm. 13C NMR (CDCl3, 125 MHz) δ=
12.4 (CH3), 45.4 (2CH3), 45.9 (2CH2), 51.5 (2CH2), 55.6 (CH3), 110.7
(CH), 115.3 (CH), 119.2 (CH), 122.1 (CH), 123.2 (CH), 123.7 (CH),
123.8(CH),125.5(C),126.9(CH),128.4(CH),128.6(CH),129.7(C),
129.8 (C), 131.5 (C), 131.9 (CH), 132.7 (CH), 134.6 (C), 134.8 (C),
138.7 (C), 139.8 (C), 140.4 (C), 141.8 (C), 151.9 (C), 153.7 (C) ppm.
MS (ESI) 685.3 (M þ H)þ. Elemental analysis calcd (%) for
C32H33ClN4O5S3 2.5HCl H2O: C 48.38, H 4.76, N 7.05, S 12.11.
3
3
Found: C 48.31, H 5.01, N 6.80, S 11.73.
General Procedure for the Synthesis of Final Compounds 5 and
6. TFA (20 equiv) was added to a solution of the corresponding
N-Boc protected compound (31 and 32) in anhydrous CH2Cl2
(20 mL/mmol) under an argon atmosphere, and the mixture was
stirred for 6 h at room temperature. The mixture was then
neutralized with a 10% aqueous solution of NaHCO3, and the
aqueous phase was extracted with CH2Cl2 (2ꢀ). The combined
organic layers were washed with water (1ꢀ) and brine (1ꢀ),
3
3
S 10.45. Found: C 49.70, H 5.92, N 7.66, S 10.90.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7103
dried over Na2SO4, filtered, and evaporated under reduced
pressure. Flash chromatography of the residue on silica
gel (elution with 50:1 to 10:1 CH2Cl2/MeOH) afforded pure
5 and 6.
1H NMR (CDCl3, 500 MHz) δ = 1.11-1.30 (m, 6H, CH2),
1.31-1.44 (m, 4H, CH2), 1.66-1.78 (m, 2H, CH2), 2.29 (s, 3H,
CH3), 2.78-2.94 (m, 12H, CH3, CH2), 3.07 (m, 4H, CH2), 3.87
(t, J = 6.2 Hz, 2H, CH2), 4.36 (br s, 1H, NH), 6.51 (d, J = 2.3
Hz, 1H, Ar), 6.65 (d, J = 8.7 Hz, 1H, Ar), 6.75 (dd, J = 8.6, 2.4
Hz, 1H, Ar), 7.16 (d, J = 7.4 Hz, 1H, Ar), 7.40 (dd, J = 8.6, 2.0
Hz, 1H, Ar), 7.49 (dd, J = 8.4, 7.4 Hz, 1H, Ar), 7.52-7.57 (m,
1H, Ar), 7.66 (d, J = 1.8 Hz, 1H, Ar), 7.69 (d, J = 8.6 Hz, 1H,
Ar), 8.21 (dd, J = 7.3, 1.1 Hz, 1H, Ar), 8.31 (d, J = 8.6 Hz, 1H,
Ar), 8.52 (d, J = 8.5 Hz, 1H, Ar) ppm. 13C NMR (CDCl3, 125
MHz) δ = 12.3 (CH3), 26.2 (CH2), 26.4 (CH2), 29.0 (CH2), 29.2
(CH2), 29.4 (CH2), 29.8 (CH2), 43.5 (CH2), 45.4 (2CH2), 45.6
(2CH3), 50.4 (2CH2), 68.5 (CH2), 112.5 (CH), 115.3 (CH), 115.4
(CH), 119.0 (CH), 119.2 (CH), 123.4 (CH), 123.5 (CH), 123.9
(CH), 127.9 (CH), 128.5 (CH), 128.7 (C), 129.5 (CH), 129.8 (C),
130.0 (C), 130.4 (CH), 131.6 (C), 135.2 (C), 136.2 (C), 137.2 (C),
137.8 (C), 140.7 (C), 141.7 (C), 150.6 (C), 152.0 (C) ppm. HRMS
(ESI) calcd for C39H47ClN5O5S3 (M - H)þ: 796.2428. Found:
5-Chloro-N-[4-({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)-
butyl]-N-(4-methoxy-3-piperazin-1-ylphenyl)-3-methyl-1-benzothio-
phene-2-sulfonamide (5). General procedure was followed using 31
(200 mg, 0.23 mmol) to afford 170 mg of final compound 5 (96%
yield) (mp = 145-147 °C). Rf = 0.29 (CH2Cl2/MeOH, 10:1). IR
(KBr) ν = 3317, 2927, 2851, 1586, 1506, 1452, 1346, 1320, 1240,
1153 cm-1. 1H NMR (CDCl3, 300 MHz) δ = 1.34-1.56 (m, 4H,
CH2), 2.04 (s, 3H, CH3), 2.73 (m, 4H, CH2), 2.82-2.88 (m, 8H,
CH3, CH2), 2.94 (m, 4H, CH2), 3.54 (t, J = 6.6 Hz, 2H, CH2), 3.83
(s, 3H, CH3), 5.19 (br s, 1H, NH), 6.40 (d, J= 2.2 Hz, 1H, Ar), 6.62
(dd, J = 8.6, 2.2 Hz, 1H, Ar), 6.69 (d, J = 8.6 Hz, 1H, Ar), 7.16 (d,
J = 7.5 Hz, 1H, Ar), 7.41 (dd, J = 8.6, 2.0 Hz, 1H, Ar), 7.47-7.55
(m, 2H, Ar), 7.65 (d, J = 1.8 Hz, 1H, Ar), 7.71 (d, J = 8.6 Hz, 1H,
Ar), 8.20 (dd, J = 7.3, 1.0 Hz, 1H, Ar), 8.26 (d, J = 8.6 Hz, 1H,
Ar), 8.51 (d, J = 8.5 Hz, 1H, Ar) ppm. 13C NMR (CDCl3, 75
MHz) δ = 12.1 (CH3), 25.4 (CH2), 26.4 (CH2), 42.7 (CH2), 45.5
(2CH3), 46.0 (2CH2), 50.9 (CH2), 51.6 (2CH2), 55.7 (CH3), 111.1
(CH), 115.2 (CH), 118.7 (CH), 118.8 (CH), 123.3 (CH), 123.4
(2CH), 123.8 (CH), 127.8 (CH), 128.4 (CH), 129.6 (CH), 129.7 (C),
129.9 (C), 130.4 (CH), 130.8 (C), 131.5 (C), 134.8 (2C), 137.1 (C),
137.6 (C), 140.6 (C), 142.1 (C), 152.0 (C), 152.3 (C) ppm. HRMS
(ESI) calcd for C36H43ClN5O5S3 (M þ H)þ: 756.2106. Found:
796.2433. Elemental analysis calcd (%) for C39H48ClN5O5S3
3
3.5HCl 4H2O: C 46.93, H 6.01, N 7.02, S 9.64. Found: C 46.63,
3
H 6.19, N 7.28, S 9.91.
5-(Dimethylamino)-N-(4-methoxy-3-piperazin-1-ylphenyl)naph-
thalene-1-sulfonamide (8). TFA (0.31 mL, 4 mmol) was added to a
solution of 39 (109 mg, 0.20 mmol) in anhydrous CH2Cl2 (4 mL)
under an argon atmosphere, and the mixture was stirred for 6 h at
room temperature. The mixture was then neutralized with a 10%
aqueous solution of NaHCO3, and the aqueous phase was
extracted with CH2Cl2 (2 ꢀ 10 mL). The combined organic layers
were washed with water (1 ꢀ 10 mL) and brine (1 ꢀ 10 mL), dried
over Na2SO4, filtered, and concentrated. Flash chromatography
of the residue on silica gel (elution with 25:1 to 5:1 CH2Cl2/
MeOH) afforded final compound 8 (82 mg, 93% yield) as a pale
yellow solid (mp = 160-161 °C). Rf = 0.10 (CH2Cl2/MeOH,
9:1). IR (KBr) ν = 3435, 2942, 2832, 1588, 1506, 1454, 1316, 1234,
756.2109. Elemental analysis calcd (%) for C36H42ClN5O5S3
3
4.5HCl 5H2O: C 42.79, H 5.64, N 6.93, S 9.52. Found: C 42.65,
H 5.41, N 6.70, S 9.86.
3
5-Chloro-N-[8-({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)-
octyl]-N-(4-methoxy-3-piperazin-1-ylphenyl)-3-methyl-1-benzothio-
phene-2-sulfonamide (6). General procedure was followed using 32
(140 mg, 0.15 mmol) to afford 120 mg of final compound 6 (97%
yield) (mp =165-167 °C). Rf = 0.10 (CH2Cl2/MeOH, 10:1). IR
(KBr) ν = 3320, 2925, 2855, 1587, 1506, 1455, 1241, 1154 cm-1. 1H
NMR (CDCl3, 300 MHz; mixture of conformers) δ = 1.11-1.42
(m, 12H, CH2), 2.14 (s, 1.3H, CH3), 2.18 (s, 1.6H, CH3), 2.80-2.85
(m, 6H, CH2), 2.93 (s, 2.8H, CH3), 2.95 (s, 3.2H, CH3), 3.18 (m,
2H, CH2), 3.34 (m, 2H, CH2), 3.62 (t, J = 6.7 Hz, 2H, CH2), 3.84
(s, 1.6H, CH3), 3.86 (s, 1.4H, CH3), 5.74 (br s, 1H, NH), 6.48-6.62
(m, 1H, Ar), 6.75 (d, J = 9.6 Hz, 2H, Ar), 7.22 (m, 1H, Ar), 7.43
(dd, J = 8.5, 1.9 Hz, 1H, Ar), 7.50-7.61 (m, 2H, Ar), 7.67-7.76
(m, 2H, Ar), 8.23 (dd, J = 7.3, 1.0 Hz, 1H, Ar), 8.37 (app t, J = 7.8
Hz, 0.4H, Ar), 8.45 (d, J = 8.5 Hz, 0.6H, Ar), 8.62 (app t, J = 7.8
Hz, 1H, Ar) ppm. 13C NMR (CDCl3, 75 MHz) δ = 12.2 (CH3),
26.0 (CH2), 26.2 (CH2), 28.6 (CH2), 28.7 (CH2), 28.8 (CH2), 29.4
(CH2), 43.3 (CH2), 43.8 (2CH2), 45.7 (2CH3), 50.4 (CH2), 51.5
(2CH2), 55.8 (CH3), 111.2 (CH), 115.5 (CH), 119.4 (CH), 119.9
(CH), 123.4 (CH), 123.8 (CH), 124.0 (CH), 124.1 (CH), 127.8
(CH), 128.2 (CH), 129.5 (CH), 129.7 (CH), 129.9 (C), 130.1 (C),
131.2 (C), 131.5 (C), 135.0 (C), 135.1 (C), 136.8 (C), 137.6 (C),
139.9 (C), 140.6 (C), 152.1 (2C) ppm. HRMS (ESI) calcd for
C40H51ClN5O5S3 (M þ H)þ: 812.2735. Found: 812.2729. Elemen-
tal analysis calcd (%) for C40H50ClN5O5S3 3HCl 2H2O: C 50.15,
1
1144 cm-1. H NMR (CD3OD, 400 MHz) δ = 2.70 (m, 4H,
CH2), 2.84 (s, 6H, CH3), 2.88 (m, 4H, CH2), 3.35 (s, 1H, NH), 3.71
(s, 3H, CH3), 6.41 (d, J = 2.4 Hz, 1H, Ar), 6.62 (dd, J = 8.6, 2.5
Hz, 1H, Ar), 6.67 (d, J = 8.7 Hz, 1H, Ar), 7.25 (d, J = 7.5 Hz, 1H,
Ar), 7.43 (dd, J = 8.5, 7.4 Hz, 1H, Ar), 7.57 (dd, J = 8.7, 7.6 Hz,
1H, Ar), 8.06 (dd, J = 7.3, 1.2 Hz, 1H, Ar), 8.39 (d, J = 8.7 Hz,
1H, Ar), 8.49 (d, J = 8.5 Hz, 1H, Ar) ppm. 13C NMR (CD3OD,
100 MHz) δ = 45.8 (2CH3), 46.3 (2CH2), 52.0 (2CH2), 56.2
(CH3), 113.1 (CH), 115.3 (CH), 116.4 (CH), 119.2 (CH), 120.6
(CH), 124.2 (CH), 129.2 (CH), 131.1 (C), 131.3 (C), 131.4 (2CH),
131.6 (C), 136.2(C), 142.8 (C), 151.8(C), 153.3 (C) ppm. MS(ESI)
441.2 (M þ H)þ. Elemental analysis calcd (%) for C23H28N4O3S
3
2.5HCl H2O: C 50.25, H 5.96, N 10.19, S 5.83. Found: C 50.16, H
6.19, N 10.04, S 5.44.
3
5-(Dimethylamino)-N-(5-(dimethylamino)naphthalen-1-ylsulfo-
nyl)-N-(4-methoxy-3-(piperazin-1-yl)phenyl)naphthalene-1-sulfon-
amide (9). To a solution of 40 (155 mg, 0.20 mmol) in anhydrous
CH2Cl2 (4 mL) under an argon atmosphere, TFA (0.31 mL,
4 mmol) was added, and the mixture was stirred for 6 h at room
temperature. The reaction mixture was then neutralized with a
10% aqueous solution of NaHCO3, and the aqueous phase was
extracted with CH2Cl2 (2 ꢀ 10 mL). The combined organic layers
were washed with water (1 ꢀ 10 mL) and brine (1 ꢀ 10 mL), dried
over Na2SO4, filtered, and evaporated under reduced pressure.
Flash chromatography of the residue on silica gel (elution with
50:1 to 10:1 CH2Cl2/MeOH) gave 111 mg (83% yield) of 9 as a
solid (mp = 110-112 °C). Rf = 0.10 (CH2Cl2/MeOH, 9:1). IR
(KBr) ν = 3435, 2941, 2832, 1587, 1572, 1504, 1350, 1250, 1176
3
3
H 6.00, N 7.31, S 10.04. Found: C 50.13, H 5.92, N 6.96, S 9.94.
5-Chloro-N-(4-{[8-({[5-(dimethylamino)-1-naphthyl]sulfonyl}-
amino)octyl]oxy}-3-piperazin-1-ylphenyl)-3-methyl-1-benzothio-
phene-2-sulfonamide (7). To a solution of 37 (30 mg, 0.033 mmol)
in anhydrous CH2Cl2 (1 mL) under an argon atmosphere, TFA
(51 μL, 0.66 mmol) was added, and the mixture was stirred for
6 h at room temperature. Then the reaction mixture was
neutralized with a 10% aqueous solution of NaHCO3 and the
aqueous phase was extracted with CH2Cl2 (2 ꢀ 10 mL). The
combined organic layers were washed with water (1 ꢀ 10 mL)
and brine (1 ꢀ 10 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. Flash chromatography of
the residue on silica gel (elution with 50:1 to 10:1 CH2Cl2/
MeOH) gave 23 mg (88% yield) of the final compound 7
(mp=139-141 °C). Rf = 0.28 (CH2Cl2/ethyl acetate, 1:1). IR
1
cm-1. H NMR (CDCl3, 400 MHz) δ = 2.40 (m, 4H, CH2),
2.78-2.86 (m, 16H, CH3, CH2), 3.81 (s, 3H, CH3), 6.15 (d, J = 2.4
Hz, 1H, Ar), 6.68 (d, J = 8.7 Hz, 1H, Ar), 6.99 (dd, J = 8.6, 2.2
Hz, 1H, Ar), 7.06-7.08 (m, 4H, Ar), 7.47 (app t, J = 8.0 Hz, 2H,
Ar), 7.75-7.81 (m, 2H, Ar), 8.30 (d, J = 7.5 Hz, 2H, Ar), 8.56 (d,
J = 8.5 Hz, 2H, Ar) ppm. 13C NMR (CDCl3, 100 MHz) δ = 45.4
(4CH3), 45.9 (2CH2), 51.4 (2CH2), 55.6 (CH3), 110.6 (CH), 115.1
(2CH), 119.3 (2CH), 122.0 (CH), 123.2 (2CH), 125.4 (C), 127.3
(KBr) ν = 3315, 2925, 2857, 1584, 1505, 1422, 1241, 1154 cm-1
.

7104 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Vaꢀzquez-Villa et al.
(CH), 128.0 (2CH), 129.4 (2C), 130.0 (2C), 131.7 (2CH), 132.8
(2CH), 134.0(2C), 141.3(C), 151.6 (2C), 153.4 (C) ppm. MS (ESI)
674.5 (M þ H)þ. Elemental analysis calcd (%) for C35H39N5-
O5S2 2.5HCl 5H2O: C 49.16, H 6.07, N 8.19, S 7.50. Found: C
(CH), 130.7 (CH), 134.3 (C), 135.1 (C), 140.6 (C), 150.4 (C), 152.1
(C), 152.2 (C) ppm. MS (ESI) 773.2 (M þ H)þ. Elemental analysis
calcd (%) for C41H52N6O5S2 3.5HCl 3.5H2O: C 51.10, H 6.54, N
3
3
8.72, S 6.65. Found: C 51.11, H 6.23, N 9.01, S 6.36.
3
3
49.56, H 6.39, N 7.89, S 7.67.
5-(Dimethylamino)-N-(8-{4-[5-({[5-(dimethylamino)-1-naphthyl]-
sulfonyl}amino)-2-methoxyphenyl]piperazin-1-yl}octyl)naphthalene-
1-sulfonamide (12). Sodium iodide (16 mg, 0.11 mmol) and 24
(24 mg, 0.055 mmol) were added to a solution of 8 (22 mg, 0.05
mmol) in anhydrous acetonitrile (3 mL) under an argon atmo-
sphere, and the reaction mixture was refluxed for 32 h. Solvent was
removed under reduced pressure and the residue was purified by
flash chromatography on silica gel (elution with 150:1 to 20:1
CH2Cl2/MeOH) to afford pure 12 (20 mg, 45% yield) as a pale
yellow solid (mp =151-153 °C). Rf = 0.50 (CH2Cl2/MeOH, 9:1).
IR (KBr) ν = 3438, 2926, 2854, 1625, 1506, 1456, 1404, 1314, 1237,
1146 cm-1. 1H NMR (CDCl3, 300 MHz) δ = 1.00-1.26 (m, 8H,
CH2), 1.28-1.39 (m, 2H, CH2), 1.40-1.56 (m, 2H, CH2), 2.30-
2.46 (m, 2H, CH2), 2.59 (m, 4H, CH2), 2.79 (m, 4H, CH2),
2.85-2.88 (m, 14H, CH3, CH2), 3.71 (s, 3H, CH3), 4.80 (t, J =
5.9 Hz, 1H, NH), 6.30 (d, J = 2.2 Hz, 1H, Ar), 6.55 (d, J = 8.7 Hz,
1H, Ar), 6.60 (dd, J = 8.6, 2.2 Hz, 1H, Ar), 7.17 (dd, J = 7.2, 5.3
Hz, 2H, Ar), 7.36 (dd, J = 8.4, 7.4 Hz, 1H, Ar), 7.46-7.60 (m, 3H,
Ar), 8.05 (dd, J = 7.3, 1.1 Hz, 1H, Ar), 8.24 (dd, J = 7.3, 1.2 Hz,
1H, Ar), 8.30 (d, J = 8.7 Hz, 1H, Ar), 8.34 (d, J = 8.7 Hz, 1H, Ar),
5-(Dimethylamino)-N-(3-{4-[4-({[5-(dimethylamino)-1-naphthyl]-
sulfonyl}amino)butyl]piperazin-1-yl}-4-methoxyphenyl)naphthalene-
1-sulfonamide (10). Pyridine (23 μL, 0.28 mmol) was added to a
stirred solution of 50 (111 mg, 0.21 mmol) in anhydrous CH2Cl2
(1 mL) under an argon atmosphere at room temperature. A solution
of dansyl chloride (117 mg, 0.43 mmol) in anhydrous CH2Cl2
(1 mL) was added dropwise, and the reaction mixture was stirred
at room temperature for 16 h. Then the reaction was quenched with
a 10% aqueous solution of NaHCO3 (3 mL) and the aqueous phase
was extracted with CH2Cl2 (2 ꢀ 10 mL). The combined organic
layers were washed with water (1 ꢀ 10 mL) and brine (1 ꢀ 10 mL),
dried over Na2SO4, filtered, and evaporated under reduced pres-
sure. Flash chromatography of the residue on silica gel (elution with
150:1 to 20:1 CH2Cl2/MeOH) gave 105 mg (65% yield) of 10 as a
yellow solid (mp = 169-171 °C). Rf = 0.40 (CH2Cl2/MeOH,
10:1). IR (KBr) ν = 3441, 2929, 2842, 1616, 1581, 1506, 1456, 1314,
1237, 1145 cm-1. 1H NMR (CDCl3, 300 MHz) δ = 1.43-1.56 (m,
4H, CH2), 2.24-2.35 (m, 2H, CH2), 2.49 (m, 4H, CH2), 2.75 (m,
4H, CH2), 2.84 (s, 6H, CH3), 2.88 (s, 6H, CH3), 2.90-2.99 (m, 2H,
CH2), 3.72 (s, 3H, CH3), 6.29 (d, J = 2.2 Hz, 1H, Ar), 6.55 (d, J =
8.7 Hz, 1H, Ar), 6.60 (dd, J = 8.6, 2.2 Hz, 1H, Ar), 7.16 (dd, J =
7.3, 3.6 Hz, 2H, Ar), 7.37 (dd, J = 8.4, 7.5 Hz, 1H, Ar), 7.45 (dd,
J = 7.1, 6.1 Hz, 1H, Ar), 7.52 (dd, J = 15.2, 7.5 Hz, 2H, Ar), 8.08
(dd, J = 7.3, 1.1 Hz, 1H, Ar), 8.23 (dd, J = 7.3, 1.1 Hz, 1H, Ar),
8.35 (app t, J = 8.3 Hz, 2H, Ar), 8.45 (d, J = 8.5 Hz, 1H, Ar), 8.50
(d, J= 8.5 Hz, 1H, Ar) ppm. 13CNMR(CDCl3, 75MHz) δ=24.4
(CH2), 28.5 (CH2), 43.0 (CH2), 45.5 (2CH3), 45.6 (2CH3), 49.8
(2CH2), 53.0 (2CH2), 55.6 (CH3), 57.8 (CH2), 111.3 (CH), 114.8
(CH), 115.2 (CH), 115.3 (CH), 118.4 (CH), 118.9 (CH), 119.4
(CH), 123.3 (2CH), 128.2 (CH), 128.6 (CH), 129.4 (C), 129.5 (CH),
129.8 (C), 129.9 (C), 130.0 (C), 130.1 (C), 130.2 (CH), 130.6 (CH),
130.7 (CH), 134.4 (C), 135.7 (C), 141.3 (C), 150.6 (C), 151.9 (C),
152.1 (C) ppm. MS (ESI) 745.3 (M þ H)þ. Elemental analysis calcd
(%) for C39H48N6O5S2 3HCl 3H2O: C 51.57, H 6.32, N 9.25, S
8.45 (d, J = 8.5 Hz, 1H, Ar), 8.53 (d, J = 8.5 Hz, 1H, Ar) ppm. 13
C
NMR (CDCl3, 75 MHz) δ = 26.2 (CH2), 26.4 (CH2), 27.3 (CH2),
28.9 (CH2), 29.2 (CH2), 29.6 (CH2), 43.4 (CH2), 45.6 (4CH3), 49.7
(2CH2), 53.1 (2CH2), 55.6 (CH3), 58.6 (CH2), 111.2 (CH), 114.9
(CH), 115.3 (2CH), 118.7 (CH), 118.8 (CH), 118.9 (CH), 123.3
(CH), 123.4 (CH), 128.5 (CH), 128.6 (CH), 129.2 (C), 129.7 (2CH),
129.8 (C), 130.0 (2C), 130.5 (CH), 130.6 (CH), 130.7 (C), 134.2 (C),
134.9 (C), 141.1 (C), 150.5 (C), 152.0 (C), 152.1 (C) ppm. MS (ESI)
801.2 (M þ H)þ. Elemental analysis calcd (%) for C43H56N6O5S2
3
4HCl 3H2O: C 51.60, H 6.65, N 8.40, S 6.41. Found: C 51.71, H
6.92, N 8.02, S 6.16.
3
4-{5-[4-(Dimethylamino)phenyl]-1,3-oxazol-2-yl}-N-(4-methoxy-
3-piperazin-1-ylphenyl)benzenesulfonamide (13). TFA (66 μL, 0.86
mmol) was added to a solution of 41 (27 mg, 0.043 mmol) in
anhydrous CH2Cl2 (1 mL) under an argon atmosphere, and the
mixture was stirred for 6 h at room temperature. Then the mixture
was neutralized with a 10% aqueous solution of NaHCO3, and
the aqueous phase was extracted with CH2Cl2 (2 ꢀ 10 mL). The
combined organic layers were washed with water (1 ꢀ 10 mL) and
brine (1 ꢀ 10 mL), dried over Na2SO4, filtered, and evaporated
under reduced pressure. Flash chromatography of the residue on
silica gel (elution with 25:1 to 5:1 CH2Cl2/MeOH) gave compound
13 (21 mg, 91% yield) as a yellow solid (mp = 189 °C, dec.). Rf =
0.24 (CH2Cl2/MeOH, 1:1). IR (KBr) ν = 3315, 2925, 2855, 1671,
1605, 1510, 1456, 1281, 1164 cm-1. 1H NMR (CD3CO, 500 MHz)
δ = 2.84-2.97 (m, 8H, CH2), 3.02 (s, 6H, CH3), 3.76 (s, 3H, CH3),
6.72-6.81 (m, 3H, Ar), 6.83 (d, J = 9.0 Hz, 2H, Ar), 7.47 (s, 1H,
Ar), 7.68 (d, J = 9.0 Hz, 2H, Ar), 7.85 (d, J = 8.7 Hz, 2H, Ar), 8.18
(d, J = 8.6 Hz, 2H, Ar) ppm. 13C NMR (CD3CO, 125 MHz) δ =
40.3 (2CH3), 46.5 (2CH2), 51.8 (2CH2), 55.9 (CH3), 113.0 (CH),
113.1 (2CH), 114.3 (CH), 116.2 (C), 117.3 (CH), 122.1 (CH), 126.5
(2CH), 126.7 (2CH), 128.8 (2CH), 131.3 (C), 132.1 (C), 141.5 (C),
143.4 (C), 151.3 (C), 151.9 (C), 154.3 (C), 158.8 (C) ppm. HRMS
(ESI) calcd for C28H32N5O4S (M þ H)þ: 534.2169. Found:
534.2163. Elemental analysis calcd (%) for C28H31N5O4S 3HCl
3
3
7.06. Found: C 51.39, H 6.35, N 9.04, S 7.46.
5-(Dimethylamino)-N-(3-{4-[6-({[5-(dimethylamino)-1-naphthyl]-
sulfonyl}amino)hexyl]piperazin-1-yl}-4-methoxyphenyl)naphthalene-
1-sulfonamide (11). Pyridine (97 μL, 1.2 mmol) was added to a
solution of 23 (216 mg, 0.4 mmol) in anhydrous CH2Cl2 (4 mL)
under an argon atmosphere at room temperature. A solution of
dansyl chloride (129 mg, 0.48 mmol) in anhydrous CH2Cl2 (2 mL)
was added over 5 min, and the reaction mixture was stirred at room
temperature for 16 h. Then the reaction was quenched with a 10%
aqueous solution of NaHCO3 (3 mL) and the aqueous phase was
extracted with CH2Cl2 (2 ꢀ 10 mL). The combined organic layers
were washed with water (1 ꢀ 10 mL) and brine (1 ꢀ 10 mL), dried
over Na2SO4, filtered, and evaporated under reduced pressure.
Flash chromatography of the residue on silica gel (elution with
150:1 to 20:1 CH2Cl2/MeOH) gave 220 mg (71% yield) of 11 as pale
yellow solid (mp = 155-157 °C). Rf = 0.48 (CH2Cl2/MeOH, 9:1).
IR (KBr) ν = 3277, 2926, 2855, 1579, 1506, 1457, 1314, 1145 cm-1
.
1H NMR (CDCl3, 300 MHz) δ = 1.14-1.32 (m, 6H, CH2), 1.36-
1.47 (m, 2H, CH2), 2.38-2.48 (m, 2H, CH2), 2.65 (m, 4H, CH2),
2.78-3.05 (m, 18H, CH3, CH2), 3.74 (s, 3H, CH3), 4.83 (br s, 1H,
NH), 6.30 (s, 1H, Ar), 6.55-6.65 (m, 2H, Ar), 7.19 (dd, J = 7.4, 4.7
Hz, 2H, Ar), 7.38 (dd, J= 8.4, 7.5 Hz, 1H, Ar), 7.52 (dd, J= 8.4, 7.4
Hz, 1H, Ar), 7.57 (app t, J = 8.1 Hz, 2H, Ar), 8.05 (d, J = 7.3 Hz,
1H, Ar), 8.24 (dd, J = 7.3, 1.1 Hz, 1H, Ar), 8.32 (d, J = 8.7 Hz, 1H,
Ar), 8.36 (d, J = 8.7 Hz, 1H, Ar), 8.46 (d, J = 8.5 Hz, 1H, Ar), 8.53
(d, J =8.5Hz, 1H, Ar) ppm. 13CNMR (CDCl3, 75 MHz) δ= 25.9
(CH2), 26.4 (CH2), 29.2 (CH2), 29.3 (CH2), 43.3 (CH2), 45.5
(4CH3), 49.2 (br, 2CH2), 52.8 (2CH2), 55.7 (CH3), 57.9 (CH2),
111.4 (CH), 114.9 (CH), 115.3 (2CH), 118.5 (CH), 118.9 (CH),
119.0 (CH), 123.3 (CH), 123.4 (CH), 128.5 (CH), 128.7 (CH), 129.5
(C), 129.6 (CH), 129.8 (2C), 129.9 (C), 130.0 (C), 130.4 (CH), 130.6
3
3
7H2O: C 43.72, H 6.29, N 9.11, S 4.17. Found: C 43.42, H 5.95, N
8.77, S 4.06.
Mixture of 2-[6-(Diethylamino)-3-(diethyliminio)-3H-xanthen-9-
yl]-5-{[(4-methoxy-3-piperazin-1-ylphenyl)amino]sulfonyl}benzene-
sulfonate and 5-[6-(Diethylamino)-3-(diethyliminio)-3H-xanthen-9-
yl]-2-{[(4-methoxy-3-piperazin-1-ylphenyl)amino]sulfonyl}benzene-
sulfonate (14). TFA (65 μL, 0.85 mmol) was added to a solution of
42 (36 mg, 0.042 mmol) in anhydrous CH2Cl2 (1 mL) under an
argon atmosphere, and the mixture was stirred for 6 h at room
temperature. Afterward, the solvent was evaporated to afford the
trifluoroacetate salt of compound 14 (mixed isomers; 36 mg, 100%

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7105
yield) as a red solid. IR (KBr) ν = 3402, 2928, 2851, 1592, 1456,
suspension were incubated at 37 °C for 120 min with 2 nM [3H]-
8-hydroxy-DPAT (170.2 Ci/mmol, Perkin-Elmer) in the pre-
sence or absence of the competing drug in a final volume of
200 μL of assay buffer. Nonspecific binding was determined by
radioligand binding in the presence of a saturating concentra-
tion of 10 μM serotonin and represented less than 10% of total
binding.
1339, 1151 cm-1 1H NMR (CD3OD, 500 MHz; mixture of
.
isomers) δ = 1.27-1.35 (m, 12H, CH3), 3.25 (m, 4H, CH2), 3.29
(m, 4H, CH2), 3.50-3.77 (m, 8H, CH2), 3.83 (s, 3H, CH3),
6.79-6.82 (m, 1H, Ar), 6.87-7.16 (m, 8H, Ar), 7.39-7.46 (m,
1H, Ar), 7.97-8.00 (m, 1H, Ar), 8.57-8.58 (m, 1H, Ar) ppm. 13
C
NMR (CD3OD, 125 MHz) δ = 12.9 (2CH3), 13.9 (CH3), 14.9
(CH3), 39.3 (CH2), 45.1 (2CH2), 46.9 (CH2), 47.2 (CH2), 47.4
(CH2), 47.9 (CH2), 48.7 (2CH2), 56.4 (CH3), 97.1 (CH), 97.3 (CH),
104.1 (C), 108.5 (C), 112.4 (CH), 113.6 (CH), 114.6 (CH), 115.1
(CH), 115.2 (CH), 115.6 (C), 115.7 (C), 116.1 (CH), 117.0 (CH),
117.1 (CH), 118.9 (CH), 119.2 (CH), 128.4 (CH), 129.8 (CH),
130.2 (CH), 131.8 (C), 132.4 (CH), 132.5 (CH), 133.5 (CH), 134.1
(CH), 134.2 (CH), 135.1 (C), 135.3 (C), 135.7 (C), 141.7 (C), 142.7
(C), 142.9 (C), 146.9 (C), 151.6 (C), 153.4 (C), 154.0 (C), 154.3 (C),
156.6 (C), 157.2 (C), 157.5 (C), 157.9 (C), 158.1 (C), 158.2 (C),
158.6 (C), 159.4 (C), 159.5 (C), 160.0 (C) ppm. HRMS (ESI) calcd
for C38H46N5O7S2 (M þ H)þ: 748.2833. Found: 748.2827. Ele-
5-HT5A Receptor. Cell membranes (6.0 mg/mL) were homo-
genized in 199 volumes of assay buffer (50 mM Tris-HCl, 0.3%
BSA, pH 7.4 at 25 °C). Fractions of 500 μL of the membrane
suspension were incubated at 27 °C for 60 min with 1 nM
[3H]LSD (82.7 Ci/mmol, Perkin-Elmer) in the presence or absence
of the competing drug in a final volume of 550 μL of assay buffer.
Nonspecific binding was determined by radioligand binding in the
presence of a saturating concentration of 10 μM 5-carboxamido-
triptamine and represented less than 10% of total binding.
5-HT6 Receptor. Cell membranes (6.0 mg/mL) were homo-
genized in 7 volumes of assay buffer (50 mM Tris-HCl, 10 mM
MgCl2, 0.5 mM EDTA, pH 7.4 at 25 °C). Fractions of 20 μL of
the membrane suspension were incubated at 37 °C for 60 min
with 2.5 nM [3H]LSD (79.2 Ci/mmol, Perkin-Elmer) in the
presence or absence of the competing drug (ranging from 10-5
to 10-10 M) in a final volume of 200 μL of assay buffer. Non-
specific binding was determined by radioligand binding in the
presence of a saturating concentration of 100 μM serotonin and
represented less than 10% of total binding.
5-HT7 Receptor. Cell membranes (6.8 mg/mL) were homo-
genized in 200 volumes of assay buffer (50 mM Tris-HCl, 10 mM
MgSO4, 0.5 mM EDTA, pH 7.4 at 25 °C). Fractions of 500 μL of
the membranes suspension were incubated at 27 °C for 120 min
with 3 nM [3H]LSD (79.2 Ci/mmol, Perkin-Elmer) in the presence
or absence of the competing drug in a final volume of 540 μL of
assay buffer. Nonspecific binding was determined by radioligand
binding in the presence of a saturating concentration of 25 μM
clozapine and represented less than 15% of total binding.
For all binding assays, competing drug and nonspecific, total,
and radioligand bindings were defined in triplicate. Incubation
was terminated by rapid vacuum filtration through Wallac
Filtermat A filters, presoaked in polyethylenimine (0.5% for
5-HT1A and 5-HT6 receptors and 0.3% for the 5-HT7 receptor)
or 0.5% BSA (for 5-HT5A), using a FilterMate Unifilter 96-
Harvester. The filters were then washed 9 times with 500 μL of
ice-cold 50 mM Tris-HCl buffer (pH 7.4 at 25 °C) and dried. The
radioactivity bound to the filters was measured by scintillation
spectrometry, using a Microbeta TopCount instrument. The
data were analyzed by an iterative curve-fitting procedure using
GraphPad Prism program. Ki values were calculated from the
Cheng-Prusoff equation19 and are the mean values of two to
four experiments performed in triplicate.
Fluorescence Spectroscopy. Absorption spectra of compounds
1-14 were determined in 10 μM solutions of the corresponding
compound in buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM
EDTA, pH 7.4) at 25 °C on a Shimadzu UV-2550 UV-vis
spectrophotometer in 1 cm path length quartz cells. Spectra were
recorded between 250 and 700 nm (0.5 nm increments and 0.1 s
integration time), and they were corrected for background absor-
bance by subtracting a blank scan of the buffer solution.
Emission spectra of compounds 1-14 were determined for
the same solutions on a PerkinElmer LS50B luminescence
spectrometer in 1 cm path length quartz cells. The spectra were
recorded between 340 and 690 nm (0.5 nm increments and 0.1 s
integration time) with excitation set at the appropriate excita-
tion wavelength. Slit widths were set to 2.5 nm for excitation and
to 2.5, 5, or 10 nm for emission, depending on the observed
emission intensity. All the spectra were corrected for back-
ground fluorescence by subtracting a blank scan of the buffer
solution.
mental analysis calcd (%) for C38H45N5O7S2 CF3CO2H H2O: C
3
3
54.72, H 5.28, N 7.98, S 7.30. Found: C 54.35, H 5.74, N 8.12, S 7.57.
N-{6-[4-(5-{[(5-Chloro-3-methyl-1-benzothien-2-yl)sulfonyl]-
amino}-2-methoxyphenyl)piperazin-1-yl]hexyl}-5-[(3aS,4S,6aR)-
2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide (15).
D-(þ)-Biotin (49 mg, 0.20 mmol) and N-hydroxybenzotriazole
(5.4 mg, 0.04 mmol) were suspended in anhydrous DMF (2 mL)
with 4 A molecular sieves under an argon atmosphere, and the
mixture was heated until a clear solution was obtained (∼45 °C).
When the mixture was cooled, a solution of dicyclohexylcarbo-
diimide in CH2Cl2 (0.22 mL of a 1 M solution in CH2Cl2, 0.22
mmol) was added dropwise and the mixture was stirred at room
temperature for 3 h. 44 (133 mg, 0.24 mmol) and DMAP (0.3 mg,
0.0024 mmol) were added, and themixture was stirred at 60 °C for
4 h and then at room temperature for 24 h. The mixture was
filtered and washed with CH2Cl2/MeOH (1/1 v/v, 5 mL), and the
filtrate was concentrated and purified by flash chromatography
on silica gel (elution with 100:1 to 10:1 CH2Cl2/MeOH) to afford
pure 15 (128 mg, 82% yield) as an off-white solid (mp =196-
198 °C). [R]25D þ23 (c, 36 mg/mL, ethanol). Rf = 0.17 (CH2Cl2/
MeOH, 10:1). IR (KBr) ν = 3400, 3078, 2932, 2856, 1693, 1641,
1506, 1458, 1334, 1232, 1156 cm-1. 1H NMR (CDCl3, 300 MHz)
δ = 1.24-1.36 (m, 4H, CH2), 1.38-1.57 (m, 6H, CH2), 1.58-
1.80 (m, 4H, CH2), 2.21 (t, J = 7.2 Hz, 2H, CH2), 2.32 (s, 3H,
CH3), 2.38 (dd, J = 12.4, 5.4 Hz, 2H, CH2), 2.56 (m, 4H, CH2),
2.72 (d, J = 12.8 Hz, 1H, CH2), 2.79-2.97 (m, 5H, CH2), 3.15
(dd, J = 11.7, 7.1 Hz, 1H, CH), 3.23 (dd, J = 12.5, 6.4 Hz, 2H,
CH2), 3.79 (s, 3H, CH3), 4.34 (dd, J = 7.2, 5.0 Hz, 1H, CH), 4.52
(dd, J = 7.2, 5.0 Hz, 1H, CH), 5.27 (br s, 1H, NH), 5.84 (br s, 2H,
NH), 6.62-6.68 (m, 2H, Ar), 6.78 (dd, J = 8.6, 2.1 Hz, 1H, Ar),
7.39 (dd, J = 8.7, 1.9 Hz, 1H, Ar), 7.66 (d, J = 1.9 Hz, 1H, Ar),
7.69 (d, J = 8.6 Hz, 1H, Ar) ppm. 13C NMR (CDCl3, 75 MHz)
δ = 12.2 (CH3), 25.8 (CH2), 26.3 (CH2), 26.8 (CH2), 27.1 (CH2),
28.1 (CH2), 28.2 (CH2), 29.5 (CH2), 36.1 (CH2), 39.5 (CH2), 40.7
(CH2), 50.1 (2CH2), 53.6 (2CH2), 55.69 (CH), 55.7 (CH3), 58.2
(CH2), 60.4 (CH), 61.9 (CH), 111.3 (CH), 115.1 (CH), 118.6
(CH), 123.4 (CH), 123.9 (CH), 127.8 (CH), 129.1 (C), 131.4 (C),
136.3 (C), 137.0 (C), 137.7 (C), 140.7 (C), 141.6 (C), 150.8 (C),
164.1 (CdO), 173.4 (CdO) ppm. HRMS (ESI) calcd for
C36H50ClN6O5S3 (M þ H)þ: 777.2687. Found: 777.2687. Ele-
mental analysis calcd for C36H49ClN6O5S3 2HCl 3H2O: C
3
3
47.81, H 6.35, N 9.29, S 10.64. Found: C 47.80, H 6.08, N 8.96,
S 10.87.
Binding Assays. Membranes from HEK-293-EBNA (5-HT1A
)
CHO-K1 (5-HT5A), and HEK-293 (5-HT6 and 5-HT7) cells
expressing the indicated human serotonin receptors were pur-
chased from Perkin-Elmer and conserved at -80 °C in packaging
buffer for subsequent use. Competitive inhibition assays were
performed according to standard procedures detailed below.
5-HT1A Receptor. Cell membranes (6.4 mg/mL) were homo-
genized in 7 volumes of assay buffer (50 mM Tris-HCl, 0.5 mM
MgSO4, pH 7.4 at 25 °C). Fractions of 20 μL of the membrane
Fluorescent quantum yield (Φf) of compound 10 was calcu-
lated with respect to quinine sulfate (Aldrich) in 0.1 M H2SO4 as
a standard (Φf = 0.54).20 Solutions of both the sample and the

7106 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Vaꢀzquez-Villa et al.
ꢀ
Social Fund for Juan de la Cierva and Ramon y Cajal grants
to J.A.G.-V. and S.O.-G., respectively. The authors thank
´
Prof. I. Rodrıguez-Crespo (Universidad Complutense de
Madrid, Spain) for the gift of COS7 cells and thank the
Microscopy and Cytometry and NMR Core Facilities from
Universidad Complutense de Madrid.
reference were prepared by dilution of stock solutions whose
absorbance was below 0.1 at the same excitation wavelength
(350 nm). Fluorescence measurements were taken for each
solution with the same instrument parameters, and the fluores-
cence spectra were corrected for instrumental response before
integration. The integrated corrected emission intensities were
plotted against the absorbance, and data were least-squares-
fitted to a straight line. The slopes of the lines are proportional to
the quantum yield of the different samples. Absolute values are
calculated using standard samples of known Φf, according the
following equation:21
Supporting Information Available: Full synthetic procedures
and analytical and spectral characterization data of intermedi-
ates 18-24, 26-37, 39-44. This material is available free of
charge via the Internet at http://pubs.acs.org.
ꢀ
ꢁ
ꢀ
ꢁ
2
2
Fx As ηx
slopex ηx
Φf,x ¼ Φf,s
¼ Φf,s
References
Ax Fs ηs
slopes ηs
(1) Terai, T.; Nagano, T. Fluorescent probes for bioimaging applica-
tions. Curr. Opin. Chem. Biol. 2008, 12, 515–521.
where A is the absorbance at the excitation wavelength, F is the
area under the corrected emission curve, η is the refractive index
of the solvent, and the slopes refer to the integrated corrected
emission vs absorbance plots for the sample (x) and the standard
(s). The correction for the refractive index (ηx/ηs)2 was found21
to be very close to 1 (0.9997), as expected for diluted aqueous
solutions. Therefore, it was considered to be of no significance.
Cell Visualization. COS-7 cells, kindly donated by Prof. I.
(2) Lee, J.-S.; Kang, N.-Y.; Kim, Y. K.; Samanta, A.; Feng, S.; Kim,
H. K.; Vendrell, M.; Park, J. H.; Chang, Y.-T. Synthesis of a BODIPY
library and its application to the development of live cell glucagon
imaging probe. J. Am. Chem. Soc. 2009, 131, 10077–10082.
(3) Laughlin, S. T.; Bertozzi, C. R. Imaging the glycome. Proc. Natl.
Acad. Sci. U.S.A. 2009, 106, 12–17.
(4) Neef, A. B.; Schultz, C. Selective fluorescence labeling of lipids in
living cells. Angew. Chem., Int. Ed. 2009, 48, 1498–1500.
(5) Nguyen, U. T. T.; Guo, Z.; Delon, C.; Wu, Y.; Deraeve, C.;
Rodrıguez-Crespo (Universidad Complutense de Madrid, Spain),
´
€
Franzel, B.; Bon, R. S.; Blankenfeldt, W.; Goody, R. S.;
were routinely grown in DMEM media (Invitrogen) supplemented
with 10% fetal bovine serum (FBS, Gibco), 1% penicillin-strepto-
mycin solution (Gibco), 1% sodium pyruvate (Gibco), and 1%
nonessential amino acids (Gibco) in a humidified atmosphere with
5% CO2 at 37 °C. For labeling studies, cells were treated with
0.125% trypsin (Invitrogen) and plated onto gelatin-coated glass
coverslips in 24-well tissue culture dishes at a density of 15 000 cells/
well and cultured under the same conditions as above for an
additional 24 h. Cells were transiently transfected with the HA-
tagged-5-HT6 receptor cDNA18 using Escort IV transfection re-
agent (Sigma) and following the manufacturer’s instructions.
Stock solutions of the compounds were prepared in DMSO
and then diluted to the final concentration with PBS buffer so
that DMSO content was <1%.
For cell labeling with fluorescent derivative 10, culture media
were aspirated off and cells were washed with PBS and incu-
bated in the presence or absence of the compound in PBS buffer
for 10 min at ∼20 °C. Afterward, buffer was removed and cells
were washed with PBS, fixed with 2% paraformaldehyde,
washed again, and mounted on glass slides with Immu-mount
(Thermo Scientific). Preparations were observed with a SP2
Leica confocal microscope with a 63ꢀ, 1.4 NA, oil immersion
objective (excitation at 405 nm and emission window at 461-
568 nm).
For cell labeling with biotinylated derivative 15, culture
media were aspirated off, cells were washed with PBS, fixed
with 2% paraformaldehyde, and washed again, and endogenous
biotin was blocked with the Molecular Probes kit. Cells were
incubated in the presence or absence of the compound in PBS
buffer for 10 min at ∼20 °C. Afterward, buffer was removed,
cells were washed with PBS, incubated with the streptavi-
din-Alexa Fluor 488 conjugate (Invitrogen Molecular Probes)
at 1:1000 for 1 h at room temperature, and washed again, and
nuclei were counterstained with Hoechst-33258 (1:5000 of a
10 mg/mL solution, 10 min, room temperature). After removal
of Hoechst solution, cells were washed with PBS and mounted
on glass slides with Immu-mount (Thermo Scientific). Fluores-
cence images were acquired using Metamorph-Offline 6.2 soft-
ware (Universal Imaging) and a Zeiss Axioplan 2 microscope.
Waldmann, H.; Wolters, D.; Alexandrov, K. Analysis of the
eukaryotic prenylome by isoprenoid affinity tagging. Nat. Chem.
Biol. 2009, 5, 227–235.
(6) Cravatt, B. F.; Wright, A. T.; Kozarich, W. Activity-based protein
profiling: from enzyme chemistry to proteomic chemistry. Annu.
Rev. Biochem. 2008, 77, 383–414.
(7) Eisenstein, M. GPCRs: insane in the membrane. Nat. Methods
2009, 6, 929–933.
(8) Li, S.; Huang, S.; Peng, S. B. Overexpression of G protein-coupled
receptors in cancer cells: involvement in tumor progression. Int. J.
Oncol. 2005, 27, 1329–1339.
(9) Catapano, L. A.; Manji, H. K. G protein-coupled receptors in
major psychiatric disorders. Biochim. Biophys. Acta 2007, 1768,
976–993.
(10) Bahouth, S. W. Development of Antibodies to Adrenergic Recep-
tors. In Adrenergic Receptor Protocols, 1st ed.; Machida, C. A., Ed.;
Methods in Molecular Biology, Vol. 126; Humana Press: Totowa, NJ,
2000; pp 281-299.
(11) Kuder, K.; Kiec-Kononowicz, K. Fluorescent GPCR ligands as
new tools in pharmacology. Curr. Med. Chem. 2008, 15, 2132–2143.
(12) Heal, D. J.; Smith, S. L.; Fisas, A.; Codony, X.; Buschmann, H.
Selective 5-HT6 receptor ligands: progress in the development of a
novel pharmacological approach to the treatment of obesity and
related metabolic disorders. Pharmacol. Ther. 2008, 117, 207–231.
(13) Liu, K. G.; Robichaud, A. J. 5-HT6 antagonists as potential treatment
for cognitive dysfunction. Drug Dev. Res. 2009, 70, 145–168.
(14) Bromidge, S. M.; King, F. D.; Wyman, P. A. Sulphonamide
Derivatives, Process for Their Preparation, and Their Use as
Medicaments. WO9827081, 1998.
(15) Bromidge, S. M.; Brown, A. M.; Clarke, S. E.; Dodgson, K.;
Gager, T.; Grassam, H. L.; Jeffrey, P. M.; Joiner, G. F.; King,
F. D.; Middlemiss, D. N.; Moss, S. F.; Newman, H.; Riley, G.;
Routledge, C.; Wyman, P. 5-Chloro-N-(4-methoxy-3-piperazin-1-
yl- phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046):
a potent, selective, and orally bioavailable 5-HT6 receptor antago-
nist. J. Med. Chem. 1999, 42, 202–205.
(16) Lavis, L. D.; Raines, R. T. Bright ideas for chemical biology. ACS
Chem. Biol. 2008, 3, 142–155.
(17) Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz,
P. G. A genetically encoded fluorescent amino acid. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 9785–9789.
´
(18) de la Fuente, T.; Martın-Fontecha, M.; Sallander, J.; Benhamu, B.;
ꢀ
Campillo, M.; Medina, R. A.; Pellissier, L. P.; Claeysen, S.; Dumuis,
ꢀ
A.; Pardo, L.; Lopez-Rodrıguez, M. L. Benzimidazole derivatives as
´
new serotonin 5-HT6 receptor antagonists. Molecular mechanisms of
receptor inactivation. J. Med. Chem. 2010, 53, 1357–1369.
(19) Cheng, Y. W.; Prusoff, H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes 50%
inhibition (IC50) of an enzymatic reaction. Biochem. Pharmacol.
1973, 22, 3099–3108.
(20) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic/Plenum Press: New York, 1999.
(21) Handbook of Organic Chemistry; Scaiano, J. C., Ed.; CRC Press:
Boca Raton, FL, 1989.
Acknowledgment. This work was supported by grants
ꢀ
from the Comunidad Autonoma de Madrid (CAM, Grant
S-SAL-249-2006) and the Spanish Ministerio de Ciencia e
ꢀ
Innovacion (MICINN, Grants SAF-2007/67008-C02-01,
CTQ2009-07752, FIS2008-06197-C02-01, and SAF-2010/
22198-C02-01). The authors thank MICINN and European