Synthesis of new serotonin 5-HT7 receptor ligands. Determinants of 5-ht7/5-ht1a receptor selectivity

Article abstract of DOI:10.1021/jm8014553

We report the synthesis of a new set of compounds of general structure I (1-20) with structural modifications in the pharmacophoric elements of the previously reported lead UCM-5600. The new derivatives have been evaluated for binding affinity at 5-HT

Full text of DOI:10.1021/jm8014553

2384
J. Med. Chem. 2009, 52, 2384–2392
Synthesis of New Serotonin 5-HT7 Receptor Ligands. Determinants of 5-HT7/5-HT1A Receptor
Selectivity
Roc´ıo A. Medina,†,‡ Jessica Sallander,‡,§ Bellinda Benhamu´,† Esther Porras,†,| Mercedes Campillo,§ Leonardo Pardo,*,§ and
Mar´ıa L. Lo´pez-Rodr´ıguez*,†
Departamento de Qu´ımica Orga´nica I, Facultad de Ciencias Qu´ımicas, UniVersidad Complutense de Madrid, E-28040 Madrid, Spain, and
Laboratori de Medicina Computacional, Unitat de Bioestad´ıstica, Facultat de Medicina, UniVersitat Auto`noma de Barcelona, E-08193
Bellaterra, Barcelona, Spain
ReceiVed NoVember 18, 2008
We report the synthesis of a new set of compounds of general structure I (1-20) with structural modifications
in the pharmacophoric elements of the previously reported lead UCM-5600. The new derivatives have been
evaluated for binding affinity at 5-HT7 and 5-HT1A receptors. The influence of the different structural features
in terms of 5-HT7/5-HT1A receptor affinity and selectivity was analyzed by computational simulations of
the complexes between compounds I and ꢀ2-based 3-D models of these receptors. Compound 18 (HYD1 )
1,3-dihydro-2H-indol-2-one; spacer ) -(CH2)4-; HYD2 + HYD3 ) 3,4-dihydroisoquinolin-2(1H)-yl) exhibits
high 5-HT7R affinity (Ki ) 7 nM) and selectivity over the 5-HT1AR (31-fold), and has been characterized
as a partial agonist of the human 5-HT7R.
Chart 1. Structures of 5-HT7R Agents
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) is an important
neurotransmitter that interacts with 14 serotoninergic receptor
subtypes, classified into seven families (5-HT1-7a),1 to elicit
numerous physiological processes.2 Many of these receptors
(e.g., 5-HT1A, 5-HT1D, 5-HT3, 5-HT4) are tractable targets for
drug discovery, with several compounds in advanced clinical
development or marketed.3-5 The more recently discovered
5-HT7 receptor (5-HT7R),6,7 first cloned in 1993,8,9 has been
identified in several species, including human, and is positively
coupled to adenylyl cyclase via Gs proteins.10 The 5-HT7R
displays a low degree of homology (40%) with other serotonin
G protein-coupled receptors (GPCRs). Recent distribution
studies in brain have revealed a high abundance of the 5-HT7R
protein in hippocampus, thalamus, hypothalamus and cerebral
cortex.11 In the hypothalamus, it is mainly expressed in the
suprachiasmatic nucleus, which functions as the circadian clock.
It is thus a potential target for sleep disturbances12 such as jet
lag, and for related psychiatric disorders such as mental fatigue
or depression.13 The presence in the hypothalamus also cor-
relates with its involvement in thermoregulation and endocrine
function.6 In addition, the significant density of the 5-HT7R in
the hippocampus accounts for its role in learning and memory.14
The 5-HT7R subtype has also been found in smooth muscle
cells and in blood vessels of the skull and other peripheral
tissues,15,16 so it is suggested as a putative target for migraine17
and irritable bowel syndrome18 treatments. All together, many
important functional roles for the 5-HT7R have been reported
in various pathophysiological processes. Therefore, this receptor
has become an attractive target for drug discovery and in the
past years intense efforts have led to the identification of
different structural classes of 5-HT7R agents, mainly by high
throughput screening of pharmaceutical companies (Chart
1).19-22 Some of them have been the subject of extensive
preclinical research. Nevertheless, to date there have not been
many 5-HT7R ligands in clinical development programs, and
current and future research should determine whether a ligand
of this receptor is suitable as a therapeutic agent.
In the course of a program aimed at the discovery of new
potent and selective serotonin 5-HT7R ligands, we postulated a
ligand-based pharmacophore model for 5-HT7R antagonism.23,24
The model consists of five features: a positive ionizable atom
(PI), a H-bonding acceptor group (HBA), and three hydrophobic
regions (HYD1-HYD3) (Figure 1). Other pharmacophore
models of 5-HT7R agonism25 and antagonism/inverse agonism26,27
were reported. These models of antagonism have added to our
previous proposal either a fourth hydrophobic region (HYD4)26
or a second H-bonding acceptor group (HBA2).27 Our pharma-
cophore model was validated through the design and synthesis
of a series of naphtholactam and naphthosultam derivatives that
led us to confirm the essential structural requirements for ligand
* To whom correspondence should be addressed. For M.L.L.-R.: phone,
34-91-3944239; fax, 34-91-3944103; e-mail, mluzlr@quim.ucm.es. For L.P.:
phone, 34-93-5812797; fax, 34-93-5812344; e-mail, Leonardo.Pardo@
uab.es.
†
Universidad Complutense de Madrid.
Both authors contributed equally.
Universitat Auto`noma de Barcelona.
Present address: GlaxoSmithKline, Severo Ochoa 2, E-28760 Tres
‡
§
|
Cantos, Madrid, Spain.
a Abbreviations: GPCR, G protein-coupled receptor; 5-HT7R, 5-HT7
receptor; 5-HT1AR, 5-HT1A receptor; PI, positive ionizable atom; HBA,
H-bonding acceptor group; HYD, hydrophobic; TM, transmembrane helix;
EL, extracellular loop; IL, intracellular loop.
10.1021/jm8014553 CCC: $40.75
2009 American Chemical Society
Published on Web 03/27/2009

New Serotonin 5-HT7 Receptor Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2385
Table 1. Binding Affinity of Synthesized Compounds I (1-20) at
5-HT7 and 5-HT1A Receptors
Figure 1. (a) Pharmacophore model proposed for 5-HT7R antagonism.
(b) Lead compound UCM-5600 and compounds of general structure I.
recognition.24 Analogue UCM-5600 (Ki ) 89 nM) was identified
as a lead for the search of new 5-HT7R ligands. Here, we have
studied a new set of compounds with structural modifications
at the different pharmacophoric elements present in UCM-5600
(Figure 1). We report the synthesis of new compounds of general
structure I and their binding affinities at 5-HT7 and 5-HT1A
receptors. Computational simulations of the complexes between
compounds I and ꢀ2-based 3-D models of both 5-HT receptors
have permitted us to study the influence of the different
structural features in terms of 5-HT7R affinity and selectivity
over the 5-HT1AR. This study has provided valuable information
about the molecular details of the ligand-receptor interaction
in this family of compounds and has allowed us to propose a
hypothesis for 5-HT7/5-HT1A receptor selectivity. This selectivity
has been achieved in ligand 18 (HYD1 ) 1,3-dihydro-2H-indol-
2-one; spacer ) -(CH2)4-; HYD2 + HYD3 ) 3,4-dihydroiso-
quinolin-2(1H)-yl), which was pharmacologically characterized
as a partial agonist of the h5-HT7R.
Results and Discussion
a Values are the mean of two to four experiments performed in triplicate.
Synthesis. Target compounds of general structure I (Table
1) were obtained following the synthetic routes described in
Scheme 1. In general, appropriate commercially available or
previously synthesized arylpiperazine or arylpiperidine was
alkylated with halides 21-29 using triethylamine and acetoni-
trile solvent to afford final compounds 1-18 (see Experimental
Section for details). Compound 10 (Ar ) 4-(benzimidazol-4-
yl)) was obtained by subsequent treatment with THF, acetic acid
and water that was required for selective removal of the trityl
protecting group in the benzimidazole ring. Bromoalkyl deriva-
tives 21, 22 were prepared by reduction of the corresponding
1-(ω-bromoalkyl)phthalimide with tin in hydrobromic and acetic
acids, as previously reported28 (Scheme 1). Intermediates 23-29
were obtained by reaction of 1,3-dihydro-2H-indol-2-one with
the appropriate dihalide in the presence of K2CO3 (23-27) or
Cs2CO3 (28, 29) in acetonitrile solution (Scheme 1). 1-(Naphth-
1-yl)piperazine (32) and 1-(1-tritylbenzimidazol-4-yl)piperazine
(33) were synthesized via Pd(0)-catalyzed coupling of the
corresponding aromatic halide with piperazine, following previ-
ously described procedures.29,30 In the case of final compounds
19, 20 the alkylation reaction was performed between readily
available 1,3-dihydro-2H-indol-2-one and corresponding 2-(ω-
haloalkyl)-1,2,3,4-tetrahydroisoquinoline 30, 31 (Scheme 1),
which were prepared by treatment of commercial 1,2,3,4-
tetrahydroisoquinoline with 1,3-dibromopropane or with 2-chlo-
roethanol followed by thionyl chloride,31 respectively.
b Value from ref 24. c Displacement of radioligand at 1 µM concentration.
by radioligand binding assays, using [3H]LSD in transfected
CHO-K1 cells and [3H]-8-OH-DPAT in transfected HEK-293
cells, respectively (see Experimental Section for details). All
compounds were assayed as hydrochloride salts. The competitive
inhibition assays were first performed at a fixed dose of 10-6
M, and the complete dose-response curve, at six different
concentrations of the ligand, was determined for those com-
pounds that presented a displacement of the radioligand over
55%. The inhibition constant Ki was calculated from the IC50
value using the Cheng-Prusoff equation,32 and the values in
Table 1 are the mean of two to four experiments. The following
structure-affinity relationships can be drawn from the binding
affinity data presented in Table 1. (a) The isoindolin-1-one
moiety (ring A) is a less favorable HYD1 region than the 1,3-
dihydro-2H-indol-2-one system (ring B) for 5-HT7R affinity,
since compounds 1-3 (Ki(1) > 1000 nM, Ki(2) ) 158 nM, Ki(3)
) 203 nM) display much lower affinity than related derivatives
4, 5, and 7 (Ki(4) ) 74 nM, Ki(5) ) 32 nM, Ki(7) ) 63 nM),
respectively. (b) In agreement with the optimum length (5.2-6.2
Å) between HYD1 and the basic center PI proposed in our
pharmacophore model (see Figure 1), a spacer containing at
least four methylene units is necessary for high 5-HT7R affinity.
Notably, binding affinity is decreased when the length is
progressively shortened from four to three and two methylenes
in compounds 19 and 20 (Ki(19) ) 105 nM, Ki(20) ) 350 nM
Vs Ki(18) ) 7 nM). However, these ligands become selective
over the 5-HT1AR (Ki >1000 nM). It is also observed that a
Binding Affinities and Structure-Affinity Relationships.
New synthesized compounds I (1-20) were assessed for in Vitro
affinity at human serotoninergic 5-HT7 and 5-HT1A receptors

2386 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Medina et al.
Scheme 1a
a Reagents and conditions: (a) Sn, HBr, acetic acid, reflux, 6 h; (b) K2CO3 or Cs2CO3, acetonitrile, reflux, 3-5 h; (c) (1) Et3N, acetonitrile, 60 °C, 24 h;
(2) 10: THF/acetic acid/H2O, reflux, 3 h.
saturated alkyl chain is preferred for binding the 5-HT7R. Indeed,
unsaturated analogues 11, 13-15 are poorly active or inactive
(Ki(11) ) 250 nM, Ki(13-15) >1000 nM) in contrast with
related saturated derivatives 6 and 9 (Ki(6) ) 47 nM, Ki(9) )
69 nM). Only unsaturated compound 12, with a trans double
bond in the spacer, conserves 5-HT7R affinity (Ki(12) ) 69 nM).
(c) HYD2 and HYD3 pharmacophoric regions seem to play an
important role in 5-HT7/5-HT1A receptor selectivity. Thus,
compounds 5, 7, 8, and 18, containing a monocyclic system as
HYD3, exhibit affinity for the 5-HT7R and are inactive or poorly
active at the 5-HT1AR; whereas compounds 6, 9, 10, and 12,
containing bicyclic moieties, exhibit affinity for both receptors.
Computational Models of 5-HT7 and 5-HT1A Receptors
in Complex with Ligands I. In an attempt to get a better
understanding of the ligand-receptor interactions involved in
5-HT7/5-HT1A receptor affinity and selectivity, we have per-
formed computational simulations of the complexes between
compounds of general structure I and both receptors (see
Experimental Section for details). The amino acid residues of
aminergic GPCRs involved in ligand binding have been
identified by site-directed mutagenesis within transmembrane
helices (TMs) 3, 5, 6, and 7; and extracellular loop (EL) 2.33
This has been confirmed by the recent crystal structures of the
ꢀ1- and ꢀ2-adrenergic receptors bound to the antagonist cyan-
opindolol34 and the partial inverse agonist carazolol,35,36
respectively. In addition, we have recently proposed that the
aromatic moiety of antagonists of the GPCR family occupy a
small cavity between TMs 5 and 6, and act by blocking the
proposed conformational transition of W6.48 of the highly
conserved CWxPF motif in TM 6 and the subsequent steps in
the activation process.37 Thus, in the complex of compound 6
with the 5-HT7R (Figure 2a) the aromatic ring B (HYD1
pharmacophoric feature) enters this cavity to interact with the
aromatic side chains of F5.47 and F6.52; the carbonyl group
(HBA) hydrogen bonds S6.55; the protonated amine (PI) forms
an ionic interaction with D3.32; and the naphthalene ring (HYD2
+ HYD3) expands toward TMs 2 and 3 to interact with F3.28.
Figure 2b shows the binding of compound 6 to the 5-HT1AR.
Despite compound 6 shows similar binding affinity for 5-HT1A
and 5-HT7 receptors, the proposed binding mode is different.
The carbonyl group (HBA) of compound 6 hydrogen bonds
S5.42 of the 5-HT1AR rather than S6.55, as in the 5-HT7R.
Figure 3 shows the sequence alignment of TMs 2, 3, 5-7 of
5-HT7 and 5-HT1A receptors. The absence of S6.55 in the
5-HT1AR, which contains Ala at this position, causes compound
6 to hydrogen bond S5.42. Notably, the environment of TM 5
is identical in both receptors, containing S5.42, T5.43, and
A5.46; and the bulky V3.33 in TM 3, which points toward TM
5, defining the binding site crevice. Thus, it is important to
remark that compound 6 could also bind the 5-HT7R in a similar
manner to the 5-HT1AR, i.e. via S5.42. Compounds of general
formula I containing structurally different spacers were designed
to experimentally probe the proposed different binding modes
to 5-HT1A and 5-HT7 receptors. First, we designed unsaturated
derivatives 11, with a cis double bond in the spacer (Figure 2c,
pink compound), and 12, with a trans double bond (Figure 2c,
blue compound), which superimpose with the different confor-
mations of compound 6 in the binding to the 5-HT1AR (via
S5.42, Figure 2b) and in the binding to the 5-HT7R (via S6.55,
Figure 2a), respectively. Notably, constrained derivative 11
exhibits similar binding affinity for the 5-HT1AR (Ki(6) ) 22
nM Vs Ki(11) ) 11.5 nM) and decreases the binding affinity
for the 5-HT7R (Ki(6) ) 47 nM Vs Ki(11) ) 250 nM), supporting
the proposed binding modes. Trans analogue 12 probably retains
enough flexibility to bind both the 5-HT1AR (Ki(6) ) 22 nM Vs
Ki(12) ) 39 nM) and the 5-HT7R (Ki(6) ) 47 nM Vs Ki(12) )
69 nM). This influence of the cis/trans conformation of the
spacer in the 5-HT7/5-HT1A receptor selectivity is in agreement
with previous results.38 The triple bond in unsaturated compound
13 directs the carbonyl group (HBA feature) to S5.42, maintain-
ing the affinity for the 5-HT1AR (Ki ) 26 nM). However, 13
becomes inactive at the 5-HT7R (Ki >1000 nM), probably due
to the fact that the rigidity of the triple bond does not permit
the naphthalene ring to accommodate into the small cavity
between TMs 2 and 7 and ELs 1 and 2 in the 5-HT7R, but it
does into the larger 5-HT1AR cavity (see below). Second, we
designed derivatives 19 and 20 in which the number of
methylene units in the spacer was decreased to three and two,
respectively, to impede binding to the more distant S5.42 and
force binding to the closer S6.55 (see Figure 2h). In agreement,
compounds 19 and 20 keep moderate 5-HT7R binding affinity
(Ki(18) ) 7 nM Vs Ki(19) ) 105 nM, Ki(20) ) 350 nM), and
are unable to bind the 5-HT1AR (Ki(18) ) 219 nM Vs Ki(19)
>1000 nM, Ki(20) >1000 nM). In compound 9, the spacer of

New Serotonin 5-HT7 Receptor Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2387
Figure 2. (a, b) Computational models of the complexes between compound 6 and (a) the 5-HT7 and (b) 5-HT1A receptors, constructed from the
crystal structure of the ꢀ2-adrenergic receptor. The protonated amine of compound 6 is predicted to form an ionic interaction with D3.32; ring B,
located in a small hydrophobic cavity between TMs 5 and 6, performs aromatic-aromatic interactions with the side chains of F5.47 and F6.52; the
carbonyl group hydrogen bonds to S6.55 in the 5-HT7R, and to S5.42 in the 5-HT1AR; and the naphthalene aromatic ring interacts with F3.28 in
the 5-HT7R, and with both F3.28 and Y2.64 in the 5-HT1AR. (c) Molecular models of the complexes between unsaturated compounds 11, with a
cis double bond in the spacer (pink); and 12, with a trans double bond (blue), and the 5-HT7R. (d, e) The binding cavity between TMs 2, 3 and
7 and ELs 1 and 2 is larger for the (e) 5-HT1AR than for the (d) 5-HT7R. (f, g) Computational model of the complexes between compounds 5 (f),
with a methoxyphenyl moiety; and 18 (g), with a benzofused aromatic ring, and the 5-HT7R. (h, i) Molecular models of the complexes between
compounds 19 (h), with three methylene units in the spacer; and 9 (i), with five methylene units, and the 5-HT7R. The CR traces of TMs 2 (gray),
3 (yellow), 4 (gray), 5 (red), 6 (blue), and 7 (purple) are shown.
Figure 3. Sequence alignment of TMs 2, 3, 5-7 and ELs 1 and 2 of 5-HT1A and 5-HT7 receptors. Residues referenced in the manuscript are
boxed. “Hot-spots”, residues with an important contribution to the selective binding of ligands to their receptors, are in black; identical residues in
dark gray; and dissimilar residues in white. The highly conserved CWxPF motif in TM6 is shown in light gray.
five methylene units favors binding to the more distant S5.42
(Figure 2i); accordingly, 9 maintains binding affinity for both
5-HT7 and 5-HT1A receptors. Thus, these data point to S6.55 as
a “hot spot”, i.e. residues with an important contribution to the
selective binding of ligands to their receptors.
acid longer in the 5-HT7R than in the 5-HT1AR, also restricting
the size of the binding site; and the 5-HT7R possesses the bulky
L232 instead of T188 in the 5-HT1AR (Figure 3). Moreover,
near this recognition cavity D2.65 in the 5-HT7R is interacting
with R7.36, absent in the 5-HT1AR, which provides additional
rigidity to this hydrophobic pocket in the 5-HT7R. Therefore,
the cavity between TMs 2, 3 and 7 to accommodate the ligand
is smaller in the 5-HT7R than in the 5-HT1AR (Figures 2d and
2e). Accordingly, the smaller the hydrophobic moiety of the
ligand placed into this small cavity, the better the binding affinity
for the 5-HT7R should be. In this way compound 18 (Ki ) 7
nM), with a benzofused aromatic system in the HYD2 + HYD3
region, possesses higher binding affinity than compounds with
larger HYD2 + HYD3 moieties [e.g., 4 (Ki ) 74 nM), 6 (Ki )
47 nM), and 16 (Ki ) 160 nM)]. Figure 2g shows compound
18 into the 5-HT7R. Indeed, the tetrahydroisoquinoline ring of
18 is optimally placed into this cavity, without clashing with
V2.61, and interacting with F3.28. The 5-HT1AR, in addition
of possessing a larger cavity, incorporates Y2.64 toward this
binding cavity that is Thr in the 5-HT7R (Figure 3). Thus, the
presence of both Y2.64 and F3.28 in the 5-HT1AR allows the
large naphthalene ring of compound 6 (Ki ) 22 nM) to interact
with these aromatic residues (Figure 2e) more properly than
the tetrahydroisoquinoline ring of 18 (Ki ) 219 nM). Thus,
Figure 2f shows the model of the complex between the
5-HT7R and compound 5 that contains an o-methoxyphenyl
moiety in the HYD3 region instead of the naphthalene ring of
compound 6 (Figure 2a). The polar methoxy group of compound
5 hydrogen bonds R7.36 of the 5-HT7R, which is also forming
an ionic interaction with D2.65 (Figure 2f). The absence of both
residues in the 5-HT1AR (Figure 3) explains the observed
selectivity of compound 5 (5-HT7: Ki ) 32 nM, 5-HT1A: Ki >
1000 nM), in contrast with compound 6 that exhibits high
affinity for both receptors (5-HT7: Ki ) 47 nM, 5-HT1A: Ki )
22 nM). This data points to R7.36 as another important element
for 5-HT7/5-HT1A receptor selectivity, as has been previously
proposed.27
Other “hot spots” in the selectivity of 5-HT7R ligands over
the 5-HT1AR are located within TMs 2 and 7 and ELs 1 and 2.
The 5-HT7R contains, in front of D3.32, the bulky V2.61 and
L7.39 residues that are Ala and Asn, respectively, in the
5-HT1AR (Figure 3) and, thus, defining the size of the binding
site between TMs 2 and 7 (Figures 2d and 2e). EL1 is one amino

2388 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Medina et al.
compounds 5, 7, 8, 16-20 with a monocyclic system as HYD3
moiety are inactive or poorly active at the 5-HT1AR whereas
those with a bicyclic moiety exhibit affinity for both receptors
(6, 9, 10, and 12).
provided valuable information about the molecular determinants
of 5-HT7/5-HT1A receptor selectivity. The major differences
between both receptors are as follows: S6.55 is present only in
the 5-HT7R whereas the 5-HT1AR contains Ala at this position;
the 5-HT7R possesses the ionic D2.65 · · ·R7.36 interaction that
is absent in the 5-HT1AR; the 5-HT7R contains the bulky V2.61
and L7.39 residues that are Ala and Asn, respectively, in the
5-HT1AR; and Tyr replaces T2.64 of the 5-HT7R (Figures 2 and
3). Thus, a decrease of the distance between PI and HBA
features forces the ligand to bind S6.55, increasing selectivity;
polar substitutions at the HYD2 + HYD3 region might interact
with R7.36, increasing selectivity; and an increase of the size
of HYD2 + HYD3 region clashes with V2.61 in the 5-HT7R
and favors the interaction with Y2.64 in the 5-HT1AR, decreasing
selectivity.
Aimed 5-HT7/5-HT1A receptor selectivity (31-fold) has been
achieved in ligand 18 (HYD1 ) 1,3-dihydro-2H-indol-2-one;
spacer ) -(CH2)4-; HYD2 + HYD3 ) 3,4-dihydroisoquinolin-
2(1H)-yl), which was pharmacologically characterized as a
partial agonist. These hypotheses provide the basis for the design
and development of new specific 5-HT7R ligands with prede-
termined pharmacological properties.
Functional Activity of Ligand 18 on the 5-HT7R. The
agonist and antagonist effects of selective ligand 18 were
assessed by determining the adenylate cyclase activity in CHO
cells expressing the human 5-HT7R.39 Cells were treated with
the ligand for 45 min at 37 °C, in the absence (agonist effect)
or the presence (antagonist effect) of serotonin (100 nM), and
the cAMP content was then measured by homogeneous time-
resolved fluorescence (HTRF) (see Experimental Section for
details). Adenylate cyclase activity is expressed as the percent-
age of the maximal effect obtained with serotonin. Compound
18 produced an increase of cAMP levels in a dose-dependent
manner. The concentration that produced the half-maximal effect
(EC50) was 0.31 ( 0.04 µM, and the maximal activation effect
was 40 ( 1%. After pretreatment with serotonin, ligand 18
induced a decrease of cAMP concentration in a dose-dependent
manner. The IC50 value was above the highest tested concentra-
tion of 10 µM, and the maximal inhibitory effect was 43 (
4%. Serotonin (EC50 ) 15 nM) and mesulergine (IC50 ) 120
nM) were assayed as agonist and antagonist reference com-
pounds, respectively. These results indicate that compound 18
behaves as a partial agonist at the h5-HT7R.
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 resonance (NMR)
spectra were recorded on a Bruker Avance 300 AM (1H, 300 MHz;
13C, 75 MHz) or Bruker 200-AC spectrometer (1H, 200 MHz; 13C,
50 MHz) at the UCM’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), qt (quintet), m (multiplet). Mass spectrometry
(MS) was carried out on a Bruker LC-Esquire or a single quadrupole
Agilent (1100 series) instrument in electrospray mode (ESI).
Elemental analyses (C, H, N) were obtained on a LECO CHNS-
932 apparatus at the UCM’s analysis services and were within 0.4%
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, iodine, ninhydrin solution, or 10% phos-
phomolybdic acid solution in ethanol. Flash chromatography was
performed on glass column using silica gel type 60 (Merck, particle
230-400 mesh) or on a Supelco VersaFlash station using silica
gel cartridges (Supelco, particle size 20-45 µM). Unless stated
otherwise, starting materials, reagents and solvents were purchased
as high-grade commercial products from Sigma, Aldrich, Fluka,
Lancaster, Scharlab or Panreac, and were used without further
purification.
The following compounds were synthesized according to de-
scribed procedures: 2-(4-bromobutyl)isoindolin-1-one (21),28 1-(4-
bromobutyl)-1,3-dihydro-2H-indol-2-one (23),43 1-(5-bromopentyl)-
1,3-dihydro-2H-indol-2-one (24),43 2-(2-chloroethyl)-1,2,3,4-
tetrahydroisoquinoline (31),31 1-(naphth-1-yl)piperazine (32),29
1-(1-tritylbenzimidazol-4-yl)piperazine (33).30 Collected data for
compounds 1-20 refer to free bases, and then hydrochloride salts
were prepared prior to melting point determination, elemental
analyses, and biological assays. Spectroscopic data of all described
compounds were consistent with the proposed structures. For series
25-29 and 1-18 we include the data of compounds 25, 28, 2, 3,
6, 10, 16-18.
The molecular mechanism underlying GPCR agonism is a
key subject in medicinal chemistry. To date, there is not a clear
idea as to the rearrangements occurring at the extracellular
domain of serotonin receptors upon 5-HT stimulation. However,
it has been proposed for other GPCRs that TM 6 performs an
inward movement of the extracellular part toward TM 3 (the
global toggle switch of Schwartz et al.) that is stabilized by the
interaction with agonists.40 The hydrogen bond interaction
between compound 18 and S6.55 (Figure 2g) probably induces
this proposed active conformation of TM 6. In addition, agonist
binding also triggers the rotamer toggle switch of W6.48
(conserved in 71% of rhodopsin-like GPCRs) of the CWxP(F/
Y) motif in TM 6, that undergoes a conformational transition
from the inactive gauche+ (pointing toward TM 7) to the active
trans (toward TM 5) conformation,37,41 which can be directly
stabilized by agonist binding.42 Notably, the aromatic ring B
(HYD1 pharmacophoric feature) of compound 18 (Figure 2g)
blocks this conformational transition of W6.48 by occupying
the small cavity between TMs 5 and 6 that accommodate the
trans conformation of the W6.48 side chain in the active state
of the receptor. This dual function of compound 18 might
explain its pharmacological profile of partial agonist.
Conclusions
Herein we report the synthesis of a new set of compounds of
general structure I with structural modifications in the pharma-
cophoric elements of the previously reported lead UCM-560024
(Figure 1), and their binding affinities at 5-HT7 and 5-HT1A
receptors. Using computational models of the complexes
between new ligands I and both receptors, we have analyzed
the influence of the different structural features in 5-HT7/5-HT1A
receptor affinity and selectivity. In the interaction model with
the 5-HT7R, the protonated amine (PI pharmacophoric element)
of the ligand forms an ionic interaction with D3.32; the aromatic
ring (HYD2 + HYD3) expands toward TM 7 to interact with
F3.28; the carbonyl group (HBA) hydrogen bonds S6.55; and
the dihydroindolone ring (HYD1) enters into a small cavity
between TMs 5 and 6, to interact with the aromatic side chains
of F5.47 and F6.52 (Figures 2a and 2g). This study has also
General Procedure for the Synthesis of Intermediate
Compounds 25-29. To a stirred solution of 1,3-dihydro-2H-indol-
2-one (0.80 g, 6 mmol) and of the appropriate commercial dihalide
(8 mmol) in anhydrous acetonitrile (90 mL), K2CO3 (1.66 g, 12

New Serotonin 5-HT7 Receptor Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2389
mmol) or Cs2CO3 (2.90 g, 9 mmol) was added. The reaction mixture
was heated at reflux under an argon atmosphere for 3-5 h, then
cooled to room temperature and filtered. The resulting solution was
evaporated under vacuum, and the crude material was resuspended
in water and extracted with dichloromethane (3 × 30 mL). The
organic layers were dried (Na2SO4), filtered and evaporated under
reduced pressure. The obtained residue was purified by column
chromatography using the appropriate eluent, to afford pure 25-29.
2-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}isoindolin-1-
one (2). Obtained from 21 and 1-(2-methoxyphenyl)piperazine, in
80% yield. Chromatography: from ethyl acetate/ethanol, 9:1 to 8:2;
mp 230-232 °C (dec). IR (CHCl3): 1678, 1622, 1595, 1500, 1472,
1456 cm-1. 1H NMR (CDCl3): δ 1.51-1.79 (m, 4H), 2.45 (t, J )
7.3, 2H), 2.63 (m, 4H), 3.07 (m, 4H), 3.65 (t, J ) 6.8, 2H), 3.84
(s, 3H), 4.38 (s, 2H), 6.82-7.03 (m, 4H), 7.39-7.56 (m, 3H), 7.83
(dd, J ) 7.3, 1.7, 1H). 13C NMR (CDCl3): δ 24.2, 26.4, 42.2, 49.9,
50.6, 53.5, 55.4, 58.2, 111.2, 118.2, 121.0, 122.7, 122.9, 123.7,
128.0, 131.2, 133.1, 141.1, 141.4, 152.3, 168.5. MS (ESI) 380.2
(M + H)+. Anal. (C23H29N3O2 ·2HCl·1/2H2O) C, H, N.
2-[5-(4-Phenylpiperazin-1-yl)pentyl]isoindolin-1-one (3). Ob-
tained from 22 and 1-phenylpiperazine, in 68% yield. Chromatog-
raphy: ethyl acetate; mp 188-190 °C. IR (CHCl3): 1680, 1620,
1599, 1580, 1502, 1472, 1456 cm-1. 1H NMR (CDCl3): δ 1.39 (qt,
J ) 6.8, 2H), 1.52-1.78 (m, 4H), 2.38 (t, J ) 7.3, 2H), 2.58 (t, J
) 5.1, 4H), 3.18 (t, J ) 5.1, 4H), 3.63 (t, J ) 7.3, 2H), 4.38 (s,
2H), 6.80-6.94 (m, 3H), 7.25 (t, J ) 7.1, 2H), 7.40-7.56 (m, 3H),
7.84 (dd, J ) 8.1, 1.2, 1H). 13C NMR (CDCl3): δ 24.6, 26.1, 28.2,
42.1, 48.8, 49.8, 53.0, 58.2, 116.0, 119.6, 122.5, 123.6, 127.9, 129.0,
131.1, 132.9, 141.0, 151.1, 168.4. MS (ESI) 364.2 (M + H)+. Anal.
(C23H29N3O·2HCl·1/2H2O) C, H, N.
1-[(2Z)-4-Chlorobut-2-enyl]-1,3-dihydro-2H-indol-2-one (25).
Obtained from 1,3-dihydro-2H-indol-2-one and (2Z)-1,4-dichlo-
robut-2-ene, in 27% yield. Chromatography: hexane/ethyl acetate,
from 8.5:1.5 to 8:2; mp 76-78 °C. IR (CHCl3): 1708, 1616, 1488,
1
1465 cm-1. H NMR (CDCl3): δ 3.48 (s, 2H), 4.19 (d, J ) 7.8,
2H), 4.39 (d, J ) 6.7, 2H), 5.51-5.63 (m, 1H), 5.76-5.89 (m,
1H), 6.79 (d, J ) 7.6, 1H), 6.98 (t, J ) 7.4, 1H), 7.17-7.24 (m,
2H). 13C NMR (CDCl3): δ 35.7, 36.5, 38.6, 108.5, 122.5, 124.5,
127.9, 128.4, 129.2, 144.4, 175.0. MS (ESI) 222.0 (M + H)+.
1-[(2E)-4-Chlorobut-2-enyl]-1,3-dihydro-2H-indol-2-one (26).
Obtained from 1,3-dihydro-2H-indol-2-one and (2E)-1,4-dichlo-
robut-2-ene, in 25% yield. Chromatography: hexane/ethyl acetate,
9.5:0.5; mp 72-74 °C.
1-(4-Chlorobut-2-ynyl)-1,3-dihydro-2H-indol-2-one (27). Ob-
tained from 1,3-dihydro-2H-indol-2-one and 1,4-dichlorobut-2-yne,
in 25% yield. Chromatography: hexane/ethyl acetate, 8:2; mp
69-72 °C.
1-[4-(4-Phenylpiperazin-1-yl)butyl]-1,3-dihydro-2H-indol-2-
one (4). Obtained from 23 and 1-phenylpiperazine, in 79% yield.
Chromatography: from ethyl acetate to ethyl acetate/ethanol, 8:2;
mp 218-221 °C (dec). Anal. (C22H27N3O·HCl·4/3H2O) C, H, N.
1-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-1,3-dihydro-
2H-indol-2-one (5). Obtained from 23 and 1-(2-methoxyphe-
nyl)piperazine, in 91% yield. Chromatography: ethyl acetate/ethanol,
8:2; mp 198-201 °C. Anal. (C23H29N3O2 ·2HCl·1/2H2O) C, H, N.
1-{4-[4-(1-Naphthyl)piperazin-1-yl]butyl}-1,3-dihydro-2H-in-
dol-2-one (6). Obtained from 23 and 32, in 65% yield. Chroma-
tography: ethyl acetate/hexane, 8:2; mp 226-228 °C (dec). IR
1-[3-(Bromomethyl)benzyl]-1,3-dihydro-2H-indol-2-one (28).
Obtained from 1,3-dihydro-2H-indol-2-one and 1,3-bis(bromom-
ethyl)benzene, in 20% yield. Chromatography: hexane/ethyl acetate,
8:2; mp 111-113 °C. IR (CHCl3): 1712, 1614, 1487, 1465 cm-1
.
1H NMR (CDCl3): δ 3.56 (s, 2H), 4.38 (s, 2H), 4.84 (s, 2H), 6.64
(d, J ) 7.7, 1H), 6.95 (t, J ) 7.5, 1H), 7.09-7.27 (m, 6H). 13C
NMR (CDCl3): δ 33.2, 35.8, 43.6, 109.0, 122.6, 124.6, 127.5, 128.0,
128.1, 128.5, 129.4, 136.7, 138.5, 144.3, 175.2. MS (ESI) 316.0
(M + H)+.
(CHCl3): 1701, 1614, 1491, 1468 cm-1 1H NMR (CDCl3): δ
.
1.64-1.70 (m, 4H), 2.45 (t, J ) 7.0, 2H), 2.72 (m, 4H), 3.07 (m,
4H), 3.47 (s, 2H), 3.71 (t, J ) 7.0, 2H), 6.82 (d, J ) 7.6, 1H),
6.93-7.03 (m, 2H), 7.17-7.33 (m, 2H), 7.38-7.42 (m, 3H), 7.48
(d, J ) 8.3, 1H), 7.73-7.78 (m, 1H), 8.10-8.15 (m, 1H). 13C NMR
(CDCl3): δ 24.3, 25.4, 35.9, 39.9, 53.0, 53.8, 58.1, 108.5, 114.7,
122.2, 123.7, 124.6, 125.4, 125.8, 125.9, 127.7, 128.4, 129.3, 135.1,
144.9,150.0,175.5.MS(ESI)400.1(M+H)+.Anal.(C26H29N3O·2HCl·7/
2H2O) C, H, N.
1-[4-(Bromomethyl)benzyl]-1,3-dihydro-2H-indol-2-one (29).
Obtained from 1,3-dihydro-2H-indol-2-one and 1,4-bis(bromom-
ethyl)benzene, in 20% yield. Chromatography: dichloromethane;
mp 115-117 °C.
Synthesis of 2-(3-Bromopropyl)-1,2,3,4-tetrahydroisoquino-
line (30). To a stirred solution of 1,2,3,4-tetrahydroisoquinoline
(1.87 mL, 15 mmol) in acetone (20 mL)/NaOH (3 mL), 1,3-
dibromopropane (2.03 mL, 20 mmol) was added, and the reaction
was stirred at room temperature for 7 h. The solvent was evaporated
to dryness, and the residue was resuspended in water and extracted
with dichloromethane. The organic layers were dried (Na2SO4), the
solvent was evaporated under reduced pressure, and the crude
material was purified by column chromatography using hexane/
ethyl acetate, 9.5:0.5, to afford pure 30 in 25% yield. IR (CHCl3):
1-[5-(4-Phenylpiperazin-1-yl)pentyl]-1,3-dihydro-2H-indol-2-
one (7). Obtained from 24 and 1-phenylpiperazine, in 98% yield.
Chromatography:ethylacetate;mp212-214°C.Anal.(C23H29N3O·2HCl ·1/
2H2O) C, H, N.
1-{5-[4-(2-Methoxyphenyl)piperazin-1-yl]pentyl}-1,3-dihydro-
2H-indol-2-one (8). Obtained from 24 and 1-(2-methoxyphe-
nyl)piperazine, in 86% yield. Chromatography: ethyl acetate; mp
195-197 °C. Anal. (C24H31N3O2 ·2HCl·H2O) C, H, N.
1-{5-[4-(1-Naphthyl)piperazin-1-yl]pentyl}-1,3-dihydro-2H-in-
dol-2-one (9). Obtained from 24 and 32, in 56% yield. Chroma-
tography: ethyl acetate/hexane, 9:1; mp 234-235 °C (dec). Anal.
(C27H31N3O·HCl·H2O) C, H, N.
1
1649, 1498, 1453 cm-1. H NMR (CDCl3): δ 2.16 (qt, J ) 6.6,
2H), 2.66 (t, J ) 6.9, 2H), 2.71 (t, J ) 5.9, 2H), 2.90 (t, J ) 5.9,
2H), 3.51 (t, J ) 6.6, 2H), 3.63 (s, 2H), 6.93-7.62 (m, 4H). 13C
NMR (CDCl3): δ 29.1, 30.3, 31.9, 51.0, 56.2, 56.3, 125.6, 126.2,
126.5, 128.7, 132.4, 135.4. MS (ESI) 254.0 (M + H)+.
1-{5-[4-(1H)-benzimidazol-4-yl)piperazin-1-yl]pentyl}-1,3-di-
hydro-2H-indol-2-one (10). 1-{5-[4-(1-trityl(1H)-benzimidazol-4-
yl)piperazin-1-yl]pentyl}-1,3-dihydro-2H-indol-2-one was obtained
from 24 and 33, in 42% yield. Chromatography: ethyl acetate/
General Procedure for the Synthesis of Final Compounds
1-18. To a suspension of corresponding intermediate 21-29 (0.9
mmol) and commercially available or synthesized cyclic amine (1.5
mmol) in anhydrous acetonitrile (4 mL), triethylamine was added
(0.2 mL, 1.5 mmol). The reaction mixture was heated at 60 °C
under an argon atmosphere for 24 h. Upon cooling to room
temperature, the solvent was evaporated under reduced pressure
and the crude material was resuspended in water and extracted with
dichloromethane (3 × 10 mL). The organic layers were dried
(Na2SO4), filtered and evaporated, and the resulting oil was purified
by column chromatography using the appropriate eluent, to provide
pure 1-18.
ethanol, 9.5:0.5 (oil). IR (CHCl3): 1710, 1495, 1445, 1430 cm-1
.
1H NMR (CDCl3): δ 1.36-1.48 (m, 2H), 1.67-1.83 (m, 4H), 2.58
(t, J ) 7.8, 2H), 3.17 (m, 4H), 3.47 (m, 4H), 3.51 (s, 2H), 3.70 (t,
J ) 7.1, 2H), 6.10 (d, J ) 8.4, 1H), 6.54 (d, J ) 7.8, 1H), 6.75-6.87
(m, 2H), 6.98-7.23 (m, 6H), 7.25-7.37 (m, 12H), 7.81 (s, 1H).
13C NMR (CDCl3): δ 24.7, 27.1, 35.6, 39.7, 49.3, 53.0, 58.2, 75.1,
107.3, 108.1, 108.7, 121.9, 122.6, 124.2, 124.5, 127.6, 127.8, 129.8,
135.8, 136.6, 141.2, 141.5, 141.7, 142.8, 144.5, 174.8. MS (ESI)
646.2 (M + H)+.
2-[4-(4-Phenylpiperazin-1-yl)butyl]isoindolin-1-one (1). Ob-
tained from 21 and 1-phenylpiperazine, in 62% yield. Chromatog-
raphy: ethyl acetate; mp 232-234 °C (dec). Anal. (C22H27N3O·
2HCl·3H2O) C, H, N.
A solution of above synthesized trityl derivative (110 mg, 0.2
mmol) in THF (1.4 mL), acetic acid (1.4 mL) and water (1.4 mL)
was heated at reflux for 3 h. The mixture was cooled to room
temperature, acidified with 1 M HCl until pH 1-2, and then

2390 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Medina et al.
extracted with ethyl acetate. Anhydrous K2CO3 was added to the
aqueous layer until pH 9-10, and the basic solution was extracted
with dichloromethane. The organic layers were dried (Na2SO4),
filtered and evaporated to provide final compound 10, in 58% yield.
to afford pure 19 in 44% yield. IR (CHCl3): 1716, 1612, 1489,
1467 cm-1. 1H NMR (CDCl3): δ 1.90 (qt, J ) 7.0, 2H), 2.51 (t, J
) 7.0, 2H), 2.64 (t, J ) 6.0, 2H), 2.82 (t, J ) 5.9, 2H), 3.42 (s,
2H), 3.54 (s, 2H), 3.75 (t, J ) 7.0, 2H), 6.85 (d, J ) 7.6, 1H),
7.00-7.09 (m, 2H), 7.10-7.12 (m, 3H), 7.17-7.20 (m, 2H). 13C
NMR (CDCl3): δ 25.1, 29.1, 35.8, 38.2, 50.9, 55.3, 56.1, 108.4,
122.1, 124.4, 124.6, 125.6, 126.1, 126.5, 127.8, 128.6, 134.3, 134.7,
144.8, 175.1. MS (ESI): 307.2 (M + 1). Anal. (C20H22N2O·HCl·
3H2O) C, H, N.
Synthesis of 1-[2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethyl]-1,3-
dihydro-2H-indol-2-one (20). To a suspension of 31 (310 mg, 1.6
mmol) and 1,3-dihydro-2H-indol-2-one (319 mg, 2.4 mmol) in
anhydrous acetonitrile (7 mL), KI (26.6 mg, 0.16 mmol) was added,
and the mixture was heated at 60 °C for 20 h under an argon
atmosphere. The reaction was cooled to room temperature, the
solvent was evaporated under reduced pressure, and the residue
was then resuspended in water and extracted with dichloromethane
(3 × 10 mL). The organic layers were dried (Na2SO4) and
evaporated, and the crude material was purified by column
chromatography eluting with dichloromethane/ethyl acetate, 9.5:
0.5, to give pure 20 in 55% yield. IR (CHCl3): 1711, 1614, 1490,
1467 cm-1. 1H NMR (CDCl3): δ 2.71-2.83 (m, 6H), 3.46 (s, 2H),
3.70 (s, 2H), 3.92 (t, J ) 7.2, 2H), 6.87 (d, J ) 7.7, 1H), 7.00-7.09
(m, 2H), 7.10-7.12 (m, 3H), 7.17-7.20 (m, 2H). 13C NMR
(CDCl3): δ 29.1, 35.9, 38.1, 51.0, 54.7, 56.4, 108.4, 122.3, 124.6,
124.7, 125.8, 126.3, 126.6, 127.9, 128.8, 134.3, 134.8, 146.4, 174.9.
MS (ESI): 293.2 (M + 1). Anal. (C19H20N2O·HCl·5/2H2O) C, H,
N.
1
IR (CHCl3): 1710, 1495, 1445, 1430 cm-1. H NMR (CDCl3): δ
1.31-1.34 (m, 2H), 1.56-1.67 (m, 4H), 2.37 (t, J ) 7.5, 2H), 2.66
(m, 4H), 3.46 (m, 6H), 3.64 (t, J ) 7.2, 2H), 6.59 (t, J ) 4.2, 1H),
6.77 (d, J ) 7.7, 1H), 6.97 (t, J ) 7.2, 1H), 7.08-7.30 (m, 4H),
7.92 (s, 1H). 13C NMR (CDCl3): δ 24.6, 25.5, 27.1, 35.8, 39.7,
49.4, 53.0, 58.1, 107.0, 108.2, 108.3, 122.2, 123.6, 124.4, 124.5,
127.8, 135.7, 136.5, 138.7, 142.4, 144.4, 175.1. MS (ESI) 404.3
(M + H)+. Anal. (C24H29N5O·3HCl·3H2O) C, H, N.
1-{(2Z)-4-[4-(1-Naphthyl)piperazin-1-yl]but-2-enyl}-1,3-dihy-
dro-2H-indol-2-one (11). Obtained from 25 and 32, in 42% yield.
Chromatography: hexane/ethyl acetate, 3:7; mp 226-228 °C (dec).
Anal. (C26H27N3O·2HCl·3H2O) C, H, N.
1-{(2E)-4-[4-(1-Naphthyl)piperazin-1-yl]but-2-enyl}-1,3-dihy-
dro-2H-indol-2-one (12). Obtained from 26 and 32, in 44% yield.
Chromatography: hexane/ethanol, 8:2; mp 173-175 °C (dec). Anal.
(C26H27N3O·2HCl·7/2H2O) C, H, N.
1-{4-[4-(1-Naphthyl)piperazin-1-yl]but-2-ynyl}-1,3-dihydro-
2H-indol-2-one (13). Obtained from 27 and 32, in 25% yield.
Chromatography: hexane/ethanol, 9:1; mp 171-173 °C (dec). Anal.
(C26H25N3O·2H2O·9/2HCl) C, H, N.
1-(3-{[4-(1-Naphthyl)piperazin-1-yl]methyl}benzyl)-1,3-dihy-
dro-2H-indol-2-one (14). Obtained from 28 and 32, in 30% yield.
Chromatography: hexane/ethyl acetate, 7:3; mp 209-211 °C (dec).
Anal. (C30H29N3O·2HCl·7/2H2O) C, H, N.
1-(4-{[4-(1-Naphthyl)piperazin-1-yl]methyl}benzyl)-1,3-dihy-
dro-2H-indol-2-one (15). Obtained from 29 and 32, in 25% yield.
Chromatography: hexane/ethyl acetate, 7:3; mp 210-212 °C (dec).
Anal. (C30H29N3O·2HCl·H2O) C, H, N.
Pharmacology. Radioligand Binding Assays. Membranes from
HEK-293 and CHO-K1 cells expressing human 5-HT7 and 5-HT1A
receptors, respectively, were purchased from Perkin-Elmer and
conserved at -80 °C in packaging buffer for subsequent use.
Specific radioligands [3H]LSD (79.2 Ci/mmol) and [3H]-8-OH-
DPAT (135 Ci/mmol) were from NEN (Perkin-Elmer). Competitive
inhibition assays were performed according to standard procedures,
briefly detailed below.
5-HT7 Receptor. Cell paste (6.8 mg/mL) was homogenized 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, 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.
5-HT1A Receptor. Cell paste (36.8 mg/mL) was homogenized
in 29.1 volumes of assay buffer (50 mM Tris-HCl, 5 mM MgSO4,
pH 7.4 at 25 °C). Fractions of 20 µL of the membranes suspension
were incubated in the dark at 37 °C for 120 min with 4.9 nM [3H]-
8-OH-DPAT, in the presence 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
concentration of 10 µM 5-HT, and represented less than 20% of
total binding.
For all binding assays, competing drug, nonspecific, total, and
radioligand bindings were defined in triplicate. Incubation was
terminated by rapid vacuum filtration through Wallac Filtermat A
filters, presoaked in poly(ethylenimine) (0.3% for the 5-HT7R and
0.5% for the 5-HT1AR), 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 heated at 80 °C. 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 (program
Prism, Graph Pad), which provided IC50, Ki, and r2 values for test
compounds, Ki values being calculated from the Cheng-Prusoff
equation.32
1-[4-(4-Phenylpiperidin-1-yl)butyl]-1,3-dihydro-2H-indol-2-
one (16). Obtained from 23 and 4-phenylpiperidine, in 40% yield.
1
Chromatography: hexane/ethyl acetate/ammonia, 9:1:0.1 (oil). H
NMR (CDCl3): δ 1.52-1.84 (m, 4H), 1.81-1.84 (m, 4H), 2.05 (t,
J ) 11.2, 2H), 2.42 (t, J ) 7.1, 2H), 2.47-2.50 (m, 1H), 3.04 (d,
J ) 10.9, 2H), 3.54 (s, 2H), 3.76 (t, J ) 6.9, 2H), 6.88 (d, J ) 7.8,
1H), 7.04 (t, J ) 7.5, 1H), 7.21-7.27 (m, 7H). 13C NMR (CDCl3):
δ 24.2, 25.3, 33.3, 35.7, 40.0, 43.6, 54.2, 58.7, 108.3, 122.0, 124.3,
126.0, 126.7, 127.7, 128.3, 143.3. 147.5, 175.2. MS (ESI) 349.0
(M + H)+. Anal. (C23H28N2O·HCl·5H2O) C, H, N.
1-[4-(3-Phenylpiperidin-1-yl)butyl]-1,3-dihydro-2H-indol-2-
one (17). Obtained from 23 and 3-phenylpiperidine, in 42% yield.
Chromatography: hexane/ethanol/ammonia, 9:1:0.1 (oil). 1H NMR
(CDCl3): δ 1.36-1.90 (m, 8H), 1.96 (t, J ) 11.2, 2H), 2.39 (t, J )
7.1, 2H), 2.79-2.81 (m, 1H), 2.97 (d, J ) 10.8, 2H), 3.44 (s, 2H),
3.67 (t, J ) 7.0, 2H), 6.78 (d, J ) 7.6, 1H), 6.96 (t, J ) 7.5, 1H),
7.10-7.27 (m, 7H). 13C NMR (CDCl3): δ 22.7, 24.2, 31.2, 35.8,
40.1, 43.9, 53.8, 58.9, 108.4, 122.2, 124.5, 127.3, 127.8, 128.5,
144.3,147.5,175.2.MS(ESI)349.0(M+H)+.Anal.(C23H28N2O·HCl·9/
2H2O) C, H, N.
1-[4-(3,4-Dihydroisoquinolin-2(1H)-yl)butyl]-1,3-dihydro-2H-
indol-2-one (18). Obtained from 23 and 1,2,3,4-tetrahydroisoquino-
line, in 45% yield. Chromatography: hexane/ethyl acetate, 3:7 (oil).
IR (CHCl3): 1701, 1614, 1558, 1489, 1468 cm-1. 1H NMR (CDCl3):
δ 1.69-1.78 (m, 4H), 2.56 (t, J ) 6.0, 2H), 2.73 (t, J ) 6.0, 2H),
2.90 (t, J ) 6.0, 2H), 3.50 (s, 2H), 3.63 (s, 2H), 3.77 (t, J ) 6.0,
2H), 6.87 (d, J ) 7.8, 1H), 7.00-7.10 (m, 2H), 7.11-7.13 (m,
3H), 7.22-7.27 (m, 2H). 13C NMR (CDCl3): δ 24.4, 25.3, 29.1,
35.8, 39.7, 50.9, 56.2, 57.6, 108.4, 122.1, 124.4, 124.6, 125.6, 127.8,
128.6, 134.3, 134.8, 144.6, 174.9. MS (ESI) 321.1 (M + H)+. Anal.
(C21H24N2O·HCl·5/2H2O) C, H, N.
Synthesis of 1-[3-(3,4-Dihydroisoquinolin-2(1H)-yl)propyl]-
1,3-dihydro-2H-indol-2-one (19). A mixture of 30 (76.3 mg, 0.3
mmol), 1,3-dihydro-2H-indol-2-one (39.9 mg, 0.3 mmol), and
anhydrous K2CO3 (148 mg, 1.1 mmol) in acetone (8.9 mL) was
heated at reflux for 9 h. Upon cooling the reaction to room
temperature, the solid was filtered off, and the resulting solution
was evaporated to dryness. The residue was purified by column
chromatography eluting with hexane/ethyl acetate, from 9:1 to 8:2,
5-HT7R Adenylate Cyclase Assay. Effects on adenylate cyclase
activity were determined at CEREP (Le Bois l’Eveque, 86600 Celle
L’Evescault, France) according to previously published methods.39
Briefly, CHO cells expressing the h5-HT7R were incubated at 37

New Serotonin 5-HT7 Receptor Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2391
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°C for 45 min with the ligand, in the absence or presence of 100
nM serotonin, and the cAMP concentration was measured by HTRF.
Adenylate cyclase activity is expressed as the percentage of the
maximal effect obtained with 10 µM serotonin. EC50 and IC50 values
were calculated by nonlinear regression analysis of the concentra-
tion-response curves using Hill software developed at CEREP.
Computational Methods. Models of the Ligand-Receptor
Complexes. Models of the TM domains 1-7 of the 5-HT7 and
5-HT1A receptors were constructed by homology modeling tech-
niques using the crystal structure of the ꢀ2-adrenergic receptor (PDB
code 2RH1)35,36 as template. SCWRL-3.044 was employed to add
the side chains of the nonconserved residues, using a backbone-
dependent rotamer library. Modeler 9v145 was used to add
intracellular loops IL1-2 and extracellular loops EL1-3 using the
structure of the ꢀ2-adrenergic receptor as template. Internal water
molecules 506, 519, 528, 529, 532, 534, 537, 543, 546, and 548,
that mediate a number of interhelical interactions36 and seem
conserved in the rhodopsin-like family of GPCRs,46 were also
included in the model. The Duan et al. (2003) force field47 was
used for peptides, and the general Amber force field (GAFF)48 and
HF/6-31G*-derived RESP atomic charges were used for the ligands.
Molecular dynamics simulations of the ligand-receptor complexes
were performed with the Sander module of AMBER 949 using the
protocol previously described.41
Acknowledgment. This work has been supported by grants
from the Spanish Ministerio de Educacio´n (MEC, SAF-2007/
67008), Comunidad Auto´noma de Madrid (CAM, S-SAL-249-
2006), Instituto de Salud Carlos III (RD07/0067/0008), and
AGAUR (SGR2005-00390). The authors thank MEC for
predoctoral grants to R.A.M. and J.S.
Supporting Information Available: Synthesis of compound 22,
spectral characterization data of compounds 22, 26, 27, 29, 1, 4, 5,
7-9, 11-15, and combustion analysis data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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