Towards new 5-HT7 antagonists among arylsulfonamide derivatives of (aryloxy)ethyl-alkyl amines: Multiobjective based design, synthesis, and antidepressant and anxiolytic properties

Article abstract of DOI:10.1016/j.ejmech.2015.11.040

A series of 39 arylsulfonamide/amide derivatives of (aryloxy)ethyl alkyl amines, was designed with the support of the Virtual Combinatorial Library-Virtual Screening protocol, and synthesized using solidphase methodologies. Representative compounds were biologically evaluated for their affinity for 5-HT₇Rs and for their selectivity over related 5-HTRs (5-HT_1ARs, 5-HT_2ARs, 5-HT₆Rs), dopamine D₂Rs and adrenergic a1Rs. The study identified the derivatives 27 (3-fluoro-N-{1-[2-(2-cyclopentylphenoxy) ethyl]piperidin-4-yl}-benzenesulfonamide; PZ-1417) and 35 (4-fluoro-N-(1-{2-[(propan-2-yl)phenoxy] ethyl}-8-azabicyclo[3.2.1]octan-3-yl)-benzenesulfonamide; PZ-1150) as being potent 5-HT₇R antagonists with antidepressant and anxiolytic properties in the forced swim test (0.625e5 mg/kg and 0.625 mg/kg, respectively), the tail suspension test (0.625 mg/kg and 0.625 mg/kg, respectively), and in four plate test (0.625 mg/kg and 1.25e2.5 mg/kg, respectively) in mice. It has to be stressed that new compounds displayed higher activity than that of SB-269970, a reference 5-HT₇R antagonist. Finally, the study provided valuable insight into the development of potential therapeutic agents for the treatment of CNS disorders.

Full text of DOI:10.1016/j.ejmech.2015.11.040

European Journal of Medicinal Chemistry 108 (2016) 334e346
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Towards new 5-HT7 antagonists among arylsulfonamide derivatives of
(aryloxy)ethyl-alkyl amines: Multiobjective based design, synthesis,
and antidepressant and anxiolytic properties
Vittorio Canale a, Rafał Kurczab b, Anna Partyka c, Grzegorz Satała b, Tomasz Lenda d,
Magdalena Jastrze˛bska-Wie˛sek c, Anna Wesołowska c, Andrzej J. Bojarski b,
,
*
Paweł Zajdel a
a
ꢀ
Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krakow, Poland
Department of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
Department of Neuro- and Psychopharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krakow, Poland
b
c
ꢀ
ꢀ
d
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 39 arylsulfonamide/amide derivatives of (aryloxy)ethyl alkyl amines, was designed with the
support of the Virtual Combinatorial Library-Virtual Screening protocol, and synthesized using solid-
phase methodologies. Representative compounds were biologically evaluated for their affinity for 5-
HT7Rs and for their selectivity over related 5-HTRs (5-HT1ARs, 5-HT2ARs, 5-HT6Rs), dopamine D2Rs
and adrenergic a1Rs. The study identified the derivatives 27 (3-fluoro-N-{1-[2-(2-cyclopentylphenoxy)
ethyl]piperidin-4-yl}-benzenesulfonamide; PZ-1417) and 35 (4-fluoro-N-(1-{2-[(propan-2-yl)phenoxy]
ethyl}-8-azabicyclo[3.2.1]octan-3-yl)-benzenesulfonamide; PZ-1150) as being potent 5-HT7R antagonists
with antidepressant and anxiolytic properties in the forced swim test (0.625e5 mg/kg and 0.625 mg/kg,
respectively), the tail suspension test (0.625 mg/kg and 0.625 mg/kg, respectively), and in four plate test
(0.625 mg/kg and 1.25e2.5 mg/kg, respectively) in mice. It has to be stressed that new compounds
displayed higher activity than that of SB-269970, a reference 5-HT7R antagonist. Finally, the study
provided valuable insight into the development of potential therapeutic agents for the treatment of CNS
disorders.
Received 29 July 2015
Received in revised form
23 November 2015
Accepted 24 November 2015
Available online 30 November 2015
Keywords:
Screening of combinatorial library
Molecular docking
5-HT7 antagonist
SB-269970
Forced swim test
Tail suspension test
Four plate test
© 2015 Elsevier Masson SAS. All rights reserved.
Depression
Anxiety
1. Introduction
nervous system (CNS), have suggested their involvement in phys-
iological functions including the circadian rhythm, learning and
The 5-HT7 receptors (5-HT7Rs) are the most recently described
members of the serotonin family [1]. These receptors are typical
metabotropic receptors (GPCRs) positively coupled with adenylyl
cyclase through the stimulatory Ga12 proteins [2]. Correlations
between the distribution of 5-HT7Rs (e.g. thalamus, suprachias-
matic nuclei, and hippocampus) and their function in the central
memory as well as in mood disorders, schizophrenia, anxiety or in
cognitive decline processes [3]. Cognitive impairment observed in
CNS disorders such as schizophrenia have in several studies shown
to be addressed by 5-HT7Rs antagonists [4]. Moreover, many piece
of evidences have confirmed that the pharmacological blockade of
5-HT7Rs produced an antidepressant-like behavior in animal
models [5], suggesting 5-HT7R antagonists may be clinically rele-
vant for the treatment of depression [6,7]. Finally, the antidepres-
Abbreviations: BAL, Backbone amide linker; Boc, Di-tert-butyl-dicarbonate; TEA,
Trimethylamine; DBU, 1,8-diazabicyclo [5,4,0]udec-7-ene; DMF, Dimethylforma-
mide; TFA, Trifluoroacetic acid; AcOH, Acetic acid; TMSOtf, Trimethylsilyl tri-
fluoromethanesulfonate; CHO, Chinese Hamster Ovary; HEK, Human Embryonic
Kidney; FST, forced swim test; TST, tail suspension test; FPT, four-plates test.
* Corresponding author.
sant effects of atypical antipsychotics
e amisulpride and
lurasidone, could be related to the antagonism at the 5-HT7Rs [8,9].
Initially, the quest for selective 5-HT7R ligands led to arylsulfo-
namide derivatives of alkylamines (e.g., SB-269970 and SB-656104)
[10,11]. Soon after long-chain arylpiperazines/piperidines
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
http://dx.doi.org/10.1016/j.ejmech.2015.11.040
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
335
functionalized with different terminal fragments (e.g., tetrahy-
drobenzindoles, oxindoles, benzothiophenes, biphenyl, benzoimi-
dazolones) [12e21] and their bioisosteres (i.e.,
tetrahydroisoquinoline) [22] have emerged as an important class of
5-HT7R agents. Further efforts resulted in the identification of JNJ-
18038683 with efficacy confirmed in preclinical models, which has
been recently evaluated in a clinical trial (phase II) for the treat-
ment of major depression [23]. These findings indicated the sig-
nificant interest in the development of 5-HT7R antagonists with
appropriate drug-like properties for pre-clinical and clinical
validation.
Our research group has recently reported the design of a new
class of 5-HT7R antagonists, namely arylsulfonamide derivatives of
(aryloxy)ethyl alicyclic amines, and identified several compounds
(e.g., PZ-766 and PZ-1404) that displayed distinct antidepressant-
like and anxiolytic-like properties in the forced swim test (FST)
and the four plate test (FPT) in mice, respectively, and pro-cognitive
activity in the novel object recognition (NOR) test in rats (Fig. 1)
[24,25].
Encouraged by those promising results, we decided to further
explore the group of 5-HT7Rs antagonists with PZ-766 and PZ-1404
lead structures, expanding chemical diversity of central amine part
to identify other high affinity scaffolds, and to improve their overall
pharmacological profile by modifications in both aryloxy and
arylsulfonamide fragments. For this purpose we adapted the
approach of Virtual Combinatorial Library-Virtual Screening (VCL-
VS), consisting of construction of VCL and its subsequent filtration
by physicochemical descriptors, pharmacophore model, and mo-
lecular docking with SVM-SIFt based post-docking ranking scheme,
to prioritize the synthesis [24]. Structural modifications of the
finally synthesized compounds comprised the introduction of new
alicyclic amine scaffolds (3-amino-azetidine, 3-amino-8-azabicyclo
[3.2.1]octane and 2,5-diazabicyclo[2.2.1]heptane), small and steri-
cally encumbered substituents in position-2 of aryloxy fragment,
and replacement of the arylsulfonamide group with arylamide and
arylurea moieties.
Scheme 1. Synthesis of (aryloxy)ethyl bromides 16{1e9}. Reagents and conditions: (i)
1,2-dibromoethane, K2CO3, KI, (CH3)2CO, 60 ꢂC, 48e72 h.
of potassium carbonate in refluxing acetone, yielding (aryloxy)
ethyl bromides 16{1e9} (Scheme 1). These building blocks were
subsequently used for a solid-phase approach.
Next, a solid-supported synthesis protocol was performed on a
commercially available BAL-functionalized polystyrene resin 10
(Scheme 2). The initial step consisted of one-pot reductive amina-
tion to anchor the Boc-protected secondary amine 11{1e3}
(Table 1) onto the solid support.
The obtained secondary amines were further treated with
different arylsulfonyl chlorides 13{1e8}, arylacyl chlorides 13
{9e10} and arylisocyanates 13{11e12} (Table 2) in the presence of
TEA and CH2Cl2.
After Boc removal, the obtained resin-bound compounds 15
were further alkylated with different (aryloxy)ethyl bromides 16
{1e9} (Table 3) under the DBU promoted reaction in DMF. The final
step involved cleavage from the resin of the final compounds 18e52
under acidic conditions.
Compounds containing the 2,5-diazabicyclo[2.2.1]heptane core
11{4} were synthesized in alternative way (Scheme 3). After
alkylation of the Boc-protected secondary amine with (aryloxy)
ethyl bromides 16{6} and 16{9}, final arylsulfonamides 55e58 were
obtained upon treatment of the deprotected intermediates 53e54
with the selected arylsulfonyl chlorides.
2.2. Molecular modeling
To explore available chemical space around central amine part
of PZ-766 a set of 680 cyclic secondary amine reagents of different
ring size and additional primary or secondary amine group was
selected from MolPort database. Next, Virtual Combinatorial Li-
brary based on the elaborated synthetic protocol (Scheme 2) was
generated by the in-house script for iterative combination of the
above mentioned set of central amine scaffolds and small sets of
acylating and alkylating agents (Tables 2 and 3). Acylating and
alkylating agents were selected on a basis of our previous reports
[24,25]; to further explore an impact of the sp2 configuration, a set
of acylating agents was extended to isocyanates (Table 2). The ob-
tained VCL of PZ-766 derivatives (680 cores ꢁ 9 alkylating
agents ꢁ 12 acylating agents ¼ 73,440 cmpds) was processed by the
hierarchical multistep virtual screening protocol including:
physico-chemical filter (the strongest basic pKa < 10, logP < 6,
logD < 5), ADMET filter (QPPCaco e gut-blood barrier): <25 poor,
>500 great, solubility in water at 25 ꢂC (QPlogS: e6.5 e 0.5) and the
We further report on the extensive structureꢀactivity relation-
ships among the synthesized analogs regarding their activity for 5-
HT7 and 5-HT1ARs, an extended evaluation of the selected com-
pounds for serotoninergic, dopaminergic and adrenergic receptors
(5-HT2A, 5-HT6, D2 and a1Rs), as well as for off-targets, i.e. adren-
ergic b1, histamine H1, muscarinic M1 receptors, in vitro functional
signaling, and in vivo behavioral evaluation in rodents models of
depression and anxiety.
2. Results and discussion
2.1. Chemistry
The designed compounds 18e52 were synthesized according to
a multistep procedure. In the first step, commercially available
phenols 1e9 were treated with 1,2-dibromoethane in the presence
Fig. 1. 5-HT7 receptor antagonists e SB-269970 [10] and arylsulfonamide derivative of (2-isopropylphenoxy)ethyl piperidine.

336
V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
Scheme 2. The solid-phase synthesis of compounds 18e52. Reagents and conditions: (i) NaBH3CN, 1% AcOH/DMF, 60 ꢂC, 24 h; (ii) TEA, CH2Cl2, RT, 2 ꢁ 2 h; (iii) TMSOTf, 2,6-lutidine,
CH2Cl2, RT, 2 ꢁ 30’; (iv) DBU, KI, DMF, 60 ꢂC, 24 h; (v) TFA/CH2Cl2 (90/10; v/v), RT, 2 h.
Table 1
blood brain barrier penetration (QPlogBB: e3.0 e 1.2), 3D phar-
macophore model based on long-chain arylpiperazines and dock-
ing to 5-HT7R homology models. The resultant ranking of amine
cores (Table 1-S.I.) was generated by application of SUM method of
data fusion for all scores calculated by filters used in virtual
screening protocol [24].
The diversity of the selected alicyclic amine cores 11{1e4} and their relative ranking
based on VCL-VS analysis.
ID
Structure
Rankinga ID
Structure
Rankinga
11{1}
1
11{3}
3
4
11{2}
2
11{4}
2.3. Pharmacology
2.3.1. In vitro evaluation
a
For details see Table 1-S.I.
Radioligand binding assays were employed to determine the
affinity and selectivity profiles of the synthesized compounds in
competition binding experiments for human serotonin 5-HT7B, 5-
HT1A, 5-HT2A, 5-HT6 and D2L receptors, which were all stably
expressed in HEK293 cells. According to the previously published
procedures [24,26e28], the experiments were carried out using
[3H]-5-CT (39.2 Ci/mmol), [3H]-8-OH-DPAT (187 Ci/mmol), [3H]-
Ketanserin (66.9 Ci/mmol), [3H]-LSD (85.2 Ci/mmol), [3H]-
Raclopride (74.4 Ci/mmol) and [3H]-prazosin (22 Ci/mmol) for 5-
HT7B, 5-HT1A, 5-HT2A, D2 and a1 receptors, respectively.
Table 2
The diversity of selected arylsulfonyl chlorides 13{1e8}, arylacyl chlorides 13{9e10},
and arylisocyanates 13{11e12}.
ID
Structure
ID
Structure
13{1}
13{7}
13{2}
13{8}
2.3.2. Functional in vitro evaluation and extended in vitro profile
The functional properties of compounds PZ-766, PZ-1404, 27,
34, 35 and 39 on 5-HT7R were evaluated using their ability to
inhibit cAMP production induced by 5-CT (10 nM) e a 5-HT7R
agonist, in a HEK293 cells overexpressing 5-HT7R. Each compound
was tested in triplicate at 8 concentrations (10ꢀ11e10ꢀ4 M). The
level of adenylyl cyclase activity was measured using recombinant
HEK293 cells stably expressing the human 5-HT7b receptor. Kb
values were calculated from Cheng-Prusoff equation [29] specific
for the analysis of functional inhibition curves: Kb ¼ IC50/(1 þ A/
EC50) where A is an agonist concentration, IC50 is the concentration
of antagonist producing a 50% reduction in the response to agonist,
and EC50 is the agonist concentration which causes a half of the
maximal response. The antagonist properties of the selected com-
pounds were evaluated in functional cAMP cellular assays, per-
formed at Eurofins Cerep using CHO cells, which stably expressed
the human 5-HT7Rs. The results were obtained as % inhibition of
control agonist response (serotonin 300 nM) at 10ꢀ6 M and 10ꢀ7 M.
Additionally, the affinity for off targets b1, H1 and M1Rs of selected
13{3}
13{4}
13{9}
13{10}
13{5}
13{6}
13{11}
13{12}

V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
337
Table 3
The diversity of N-alkylating agents (aryloxyl)ethyl bromides 16{1e9}.
ID
Structure
ID
Structure
ID
Structure
16{1}
16{4}
16{7}
16{2}
16{3}
16{5}
16{6}
16{8}
16{9}
Scheme 3. Synthesis of arylsulfonamides 55e58. Reagents and conditions: (i) 16{6} or 16{9}, K2CO3, KI, (CH3)2CO, 60 ꢂC, 48e72 h; (ii) TFA/CH2Cl2 (90/10; v/v), RT, 2 h; (iii) 13{1} or
13{3}, CH2Cl2, 0 ꢂC, 6e12 h.
Table 4
compounds 27 and 35 were tested at Eurofins Cerep. The results
The binding data of structural analogs of PZ-766 for 5-HT7 and 5-HT1ARs.
were obtained as % inhibition of control agonist response according
to experimental protocols described online at www.cerep.fr.
2.3.3. Behavioral evaluation
The antidepressant activity of the selected 5-HT7R antagonists
27, 34, 35 and 39 was evaluated in the forced swim test (FST) in
mice [30] and their activity was compared with that of the selective
5-HT7R antagonist SB-269970 and selective serotonin reuptake
inhibitors, citalopram. The influence of the effective doses recorded
in the FSTon spontaneous locomotor activity in mice was studied in
order to exclude the possibility of competing behaviors such as
general locomotor activity. Furthermore, the antidepressant prop-
erties of the most active in the FST compounds 27 and 35 were
confirmed in tail suspension test (TST) in C57BL/6J mice [31] and in
the FST in Swiss Albin rats [32]. Finally, the anxiolytic properties of
selected compounds 27 and 35 were evaluate in the mouse four
plate test (FPT), an unconditioned fear model of anxiety [33], using
diazepam as active control.
Compd
R
R0
Ki [nM]a
5-HT7
5-HT1A
PZ-766b
18
19
20
21
Isopropyl
H
Methyl
Methoxyl
Isopropoxyl
Isopropyl
H
H
H
H
0.3
855
163
344
27
436
1764
745
463
197
H
22
Isopropyl
1918
6147
a
Ki values are means of three independent binding experiments (SEM ꢃ 21%).
b
Ref. [24].
donating properties) maintained
a high affinity for 5-HT7Rs
2.4. Structure-activity relationship studies
(Ki ¼ 27 nM), this modification decreased the selectivity over the 5-
HT1A sites (21 vs PZ-766). It thus seems, that substituents with
strong electron donating properties decreases the selectivity, while
the symmetry of substituent increases the affinity and selectivity.
Furthermore, compound 22, which had two isopropyl groups in
positions 2 and 6, was 3000-fold less potent compared with PZ-
766. These data are consistent with those of molecular modeling,
indicating that the introduction of small but branched hydrophobic
substituent in position-2 of the (aryloxy)ethyl fragment improved
the docking score (Fig. 2), but due to limited volume of hydrophobic
pocket formed by the TM3, TM5 and TM6 helices, 2,6-disubstituted
derivatives showed strongly decreased affinity for 5-HT7Rs.
We have recently conducted extensive SAR and pharmacological
studies in a group of alkyl/arylsulfonamide derivatives of (aryloxy)
ethyl alicyclic amines and found that encumbered substituents in
position-2 of the aryloxy fragment were preferred for interaction
with 5-HT7Rs [24,25]. To finally confirm this preferential interac-
tion assumption, our first efforts centered on exploring the pilot set
with hydrogen, small alkyl, and alkoxyl substituents as close ana-
logs of PZ-766 (Table 4).
Compound 18 with no substituent or compounds 19 and 20 with
methyl, and methoxyl, respectively, displayed low-to-moderate
affinity for 5-HT7Rs. Although the replacement of the isopropyl
fragment (symmetric with weak electron donating properties) with
the isopropoxyl fragment (symmetric with strong electron
The position of the (aryloxy)ethyl fragment in the binding cavity
is largely fixed by several aromatic interactions with Phe6.51and

338
V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
Fig. 2. A e Binding mode of structural analogs of PZ-766 with different substituents in position-2 of aryloxy fragment (PZ-766 e green, compounds 18, 19, 20, 21 and 22 e in blue,
pink, orange, cyan and yellow, respectively). B e the shape of the binding cavity formed by TM3, TM5 and TM6. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
Phe6.52 but less by that with Phe5.47. Moreover, the Cys3.36,
Thr3.37 and Ser5.42 amino acids limit the size and shape of the
cavity, which can explain the highest affinity of the analog with the
isopropyl substituent. The remaining substituents, indeed, fit into
the cavity; however, probably due to poorer filling, the hydrophobic
interactions they formed are weaker. The docking score of the 2,6-
diisopropyl analog was the worst within this series, because the
second isopropyl group did not fit into the cavity. Note that all VCL
members containing the isopropyl groups in position-2 and
position-6 exhibited lower scores compared to their analogs with
no substituents in position-6. Finally, following the previous re-
ports disclosing the influence of a type of substituent in position-3
at the aryloxy fragment for the interaction with 5-HT7Rs (acet-
amide, fluoro, phenyl; as well as 2-phenylthiazole) [24,34] the
current study confirmed our rationale for the substitution pattern
in the (aryloxy)ethyl fragment to adopt the biologically active
conformation.
In the next step, in an attempt to identify potent 5-HT7R an-
tagonists and to further extend the structureeactivity relationship
studies, our strategy focused on the sterically hindered substituent
in position-2 at the aryloxy fragment. Most of the compounds with
isopropyl, cyclopentyl, and phenyl substituents displayed high af-
finity for 5-HT7Rs (Ki < 50 nM), except 25, 38 and 43 (the reason is
discussed below). In contrast, the replacement of isopropyl with a
tert-butyl fragment decreased the affinity for 5-HT7Rs yet main-
tained high selectivity over 5-HT1ARs (e.g., PZ-766 vs 24) (Table 5).
Subsequently, we evaluated the replacement of the piperidine
scaffold (present in PZ-766 and PZ-1404) with four-membered
azetidine or sterically hindered azabicyclo[3.2.1]octane (tropane).
The amine cores used in the synthetic protocol were selected based
on adapted virtual screening protocol, combining: MolPort reagent
filtering, VCL construction and multivariable analysis (Table 1-S.I.).
Finally, in selection of the definitive set of amine cores (Table 1)
synthetic accessibility and financial issues were also decisive. A
replacement of the piperidine with the azetidine scaffold provided
compounds 29e32, which displayed comparable affinities for 5-
HT7Rs but were not selective over the 5-HT1A sites. Following the
amine ranking generated from VCL-VS, the tropane moiety pro-
vided active 5-HT7 ligands and were selective over the 5-HT1ARs.
We also explored the replacement of piperidine with the
diazabicyclo-heptane moiety (55e58). In line with in silico exper-
iments, this modification significantly reduced the affinity for the
5-HT7Rs (Ki > 162 nM) (Table 6).
(44e48) for interaction with Phe3.28 in the 5-HT7 binding site.
Although these derivatives interacted with amino acids in a very
similar manner, differences in the orientation of the terminal aro-
matic ring against Phe3.28 were observed (Fig. 3C). The amide and
urea groups, due to their conformational rigidity, caused retraction
of the aromatic fragment from Phe3.28 of approximately 1e2 Å
further (depending on the receptor conformation and type of an-
alogs) than in the case of the sulfonamide group. The sp3 hybridi-
zation of the sulfonyl group forced the bending of the terminal
aromatic ring towards Phe3.28 (average distance of approximately
4 Å) to form an aromatic face-to-face interaction. This interaction
caused an additional stabilization of the L-R complex, which is also
confirmed by the better average scoring function for the com-
pounds with the sulfonamide group than their amide and urea
counterparts (Table 2).
To further analyze SAR around PZ-766 in the arylsulfonyl moi-
ety, several sulfonylating agents were chosen (Table 2). Mono-
halogenated derivatives exhibited some preference for interaction
with 5-HT7Rs over their disubstituted analogs (34 vs. both 36 and
37 and 41 vs. 42).
Surprisingly, one of the most highly ranked building blocks, i.e.,
the 1-N-methyl-4-pyrazole moiety was less favorable for the
interaction with 5-HT7Rs (e.g., 38 and 43 with Ki ¼ 70 and 50 nM,
respectively).
Regarding the lower lipophilicity of compounds as a positive
attribute in view of their undesired promiscuity and ADME issues,
we subsequently evaluated derivatives containing 4-aminophenyl
and 3-pyridinyl fragments. This modification decreased the affin-
ity for 5-HT7Rs and the selectivity over 5-HT1A sites (PZ-766 vs. 38,
49 and 50). The binding mode of these analogs are consistent and
demonstrated that differently substituted arylsulfonyl moiety
occupied the same binding-site crevice. However, the significantly
higher affinity of the fluorine analog (PZ-766) could be explained by
the ability of fluorine to form several interactions. The close insight
revealed that fluorine forms polar contacts with the side chain of
Glu7.35 and the guanidinium group of Arg7.36 and has hydropho-
bic interaction with the lipophilic side chain of Leu7.39. These in-
teractions increased stabilization of R-L complex of the fluorine
analog in comparison to the arylsulfonyl moieties used (Fig. 3D).
2.5. Extended in vitro and in vivo pharmacology
Having identified 27, 34, 35 and 39 as promising compounds
with the highest affinity for 5-HT7Rs and selectivity over 5-HT1ARs,
we next investigated their in vitro functional activities in cAMP
assays (Table 7). Subsequently, these compounds were evaluated in
extended biological assays for other related serotonin (5-HT2A, 5-
HT6), dopamine D2 and adrenergic a1 receptors revealing high-to-
Recently, we investigated the effect of bioisosteric replacement
of the amide bond with sulfonamide, which was beneficial for the
interaction with 5-HT7Rs [24]. Herein, we finally confirmed that the
tetrahedral configuration of the sulfonamide fragment is prefer-
ential to the planar one presented in the amide and urea derivatives

V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
339
Table 5
The binding data of the solid-supported synthesized compounds 23e52 for 5-HT7 and 5-HT1ARs.
Compd
Ar
X
Aminea
R
Ki [nM]b
c
5-HT7
5-HT1A
S1A/
7
PZ-1404d
PZ-1129d
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
3-F-phenyl
4-F-phenyl
3-Cl-phenyl
4-F-phenyl
1-N-methyl-4-pyrazyl
3-Cl-phenyl
3-F-phenyl
4-F-phenyl
3-F-phenyl
4-F-phenyl
3-Cl-phenyl
1-N-methyl-4-pyrazyl
3-Cl-phenyl
3-F-phenyl
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
CO
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Azetidine
Azetidine
Azetidine
Azetidine
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Azabicyclo-octane
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Piperidine
Isopropyl
Phenyl
t-Butyl
t-Butyl
t-Butyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Isopropyl
Isopropyl
Phenyl
9
356
159
1847
4570
5555
556
545
373
28
45
23
46
206
142
98
99
51
431
254
123
170
74
947
199
148
591
190
718
502
371
407
536
40
23
25
85
66
33
31
23
2
7
74
54
84
17
19
16
14
7
20
40
4
2
1
6
1
1
Phenyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Isopropyl
Phenyl
Isopropyl
Isopropyl
Phenyl
Isopropyl
Isopropyl
Phenyl
52
71
98
11
13
6
18
10
12
4
19
1
1
2
1
4-F-phenyl
3,4-diF-phenyl
2,5-diF-phenyl
1-N-methyl-4-pyrazyl
3-Cl-phenyl
3-F-phenyl
4-F-phenyl
3,4-diF-phenyl
1-N-methyl-4-pyrazyl
4-F-phenyl
3-Cl-phenyl
3-Cl-phenyl
4-F-phenyl
3-Cl-phenyl
4-NH2-phenyl
3-pyridinyl
4-NH2-phenyl
3-pyridinyl
9
4
70
14
12
14
20
50
285
197
264
201
198
39
72
26
53
CO
NHCO
NHCO
NHCO
SO2
SO2
SO2
SO2
4
13
5
16
10
Phenyl
a
Amine cores are decoded as follows: azabicyclo-octane (m ¼ 1, n ¼ 1, with dashed bonds), azetidine (m ¼ 0, n ¼ 0, no dashed bonds), piperidine (m ¼ 1, n ¼ 1, no dashed
bonds).
b
c
Ki values are means of three independent binding experiments (SEM ꢃ 38%).
Ratio of affinity for 5-HT1A vs 5-HT7Rs.
Ref. [24,25].
d
Table 6
The binding data of synthesized compounds 55e58 for 5-HT7 and 5-HT1ARs.
Compd
Ar
Amine
R
Ki [nM]a
b
5-HT7
5-HT1A
S1A/
7
55
56
57
58
3-Cl-phenyl
4-F-phenyl
3-Cl-phenyl
4-F-phenyl
Diazabicyclo-heptane
Diazabicyclo-heptane
Diazabicyclo-heptane
Diazabicyclo-heptane
Isopropyl
Isopropyl
Phenyl
168
162
319
292
393
260
2667
1068
2
2
8
4
Phenyl
a
Ki values are means of three independent binding experiments (SEM ꢃ 27%).
Ratio of affinity for 5-HT1A vs 5-HT7Rs.
b
moderate selectivity (Table 7).
antidepressant drug belonging to the group of selective serotonin
reuptake inhibitors, as references [35]. All the tested compounds
werefoundtosignificantly reduce the immobility time of mice in the
range of 18e43% at the minimum effective doses (MED) of 0.625 mg/
The antidepressantactivityof theselected5-HT7R antagonists 27,
34, 35 and 39 were further evaluated in vivo in the FST in mice using
the selective 5-HT7R antagonist SB-269970 and citalopram, the

340
V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
Fig. 3. Panel demonstrating molecular docking results of the library members for the 5-HT7 receptor. (A) The comparison of the binding modes for PZ-766 (isopropyl, green) and its
analogs with different 2-substituted aryloxy fragment PZ-1129 (phenyl, cyan), 24 (t-butyl, pink) and 28 (cyclopentyl, yellow). (B) The highly scored complexes of PZ-766 (green) and
its analogs with different amine cores (azetidine e cyan, azabicyclo octane e pink and diazabicyclo-heptane e yellow). (C) The perturbation of the binding mode caused by
bioisosteric replacement of sulfonamide (PZ-766 e green) with amide (44 e orange) and urea (47 e blue) bonds. (D) The comparison of PZ-766 (green) with its derivatives
containing 4-aminophenyl (49 e pink) and 3-pyridinyl fragments (50 e cyan). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Table 7
produced their effect at MED ¼ 2.5 mg/kg, being similar to the
The antagonist activity for 5-HT7Rs of selected compounds and their binding data for
5-HT7, 5-HT1A, 5-HT2A, 5-HT6, D2 and a1Rs.
active dose of SB-269970 and concurrently eight times lower than
MED of citalopram (20 mg/kg). Finally, compound 27
Compd
Ki [nM]a Kb[nM] % inhb
Ki [nM]a
(MED ¼ 1.25 mg/kg) exerted effects characteristic of antidepres-
sants in the FST in rats at the same dose as the reference 5-HT7R
antagonist e SB-269970 (Table 8) [36].
5-HT7
5-HT1A 5-HT2A 5-HT6 D2
a1
979
PZ-1404
9
19
2
26 nM
85 nM
96 nM
74 nM
99%/70% 356
90%/61% 545
86%/46% 142
178
303
1297
1295
1146
471
281
539
559
325
102
322 1525
199
60
In contrast to the well-established role for 5-HT7Rs in depres-
sion, their involvement in the regulation of anxiety-like behaviors
is less consistent. The literature data demonstrated both no dif-
ferences between 5-HT7R knock-out mice and wild-type controls in
the elevated plus maze or light/dark transfer test [37,38] and spe-
cific anxiolytic-like effects of 5-HT7 antagonists as well as agonists
in rats and mice preclinical tasks [39e41]. These pharmacological
data prompted us to evaluate the compounds with the most
antidepressant-like activity, 27 and 35, in the mouse FPT, an un-
conditioned fear model of anxiety (Table 8). Compounds 27
(MED ¼ 1.25 mg/kg) and 35 (MED ¼ 0.625 mg/kg) as well as SB-
269970 (MED ¼ 1 mg/kg) and diazepam (MED ¼ 1.25 mg/kg)
exerted anxiolytic-like activity via increase in the number of shocks
accepted by mice (Fig. 1-S.I.). The effects of compounds 27 and 35
were similar to those produced by diazepam.
Active doses of the investigated compounds, SB-269970 and
reference drugs had no influence on the spontaneous locomotor ac-
tivity measured over a period of time equal to the observation period
in the FST (i.e., from 2 to 6 min), in the TST (i.e., 6 min) and in the FPT
(i.e., 1 min 15 s); thus, the antidepressant and anxiolytic effects of
those compounds appeared to be specific (data not shown).
Finally, the most in vivo potent compounds 27 and 35 were also
evaluated for their affinity for off-targets, i.e. a1 (>300 nM) b1, H1
and M1Rs [42]. In line with our previous study [25], selected
compounds displayed weak affinity for a1 (>300 nM) and H1
27
34
35
39
250
339
742
1
98%/68%
98
14
178 nM 93%/65% 254
94
a
Ki values are means of three independent binding experiments (SEM ꢃ 28%).
b
% inhibition of control agonist response (serotonin 300 nM) at 10ꢀ6 M/10ꢀ7 M;
performed at Eurofins Cerep.
kg (27), 2.5 mg/kg (34), 0.625 mg/kg (35) and 10 mg/kg (39).
The potency of the antidepressant-like activity of the investi-
gated compounds was similar to that of the selective 5-HT7R
antagonist SB-269970, which exerted its characteristic antide-
pressant effect at one medium dose (10 mg/kg), corresponding to a
reduction in the mouse immobility time by 31%. Note that all of the
tested compounds, except for 39, displayed their antidepressant-
like activity at doses 4e16 times lower than the active dose of the
reference compound and that only compound 27 was active over a
wide range of doses (0.625e5 mg/kg) (Fig. 4). Citalopram
(1.25e5 mg/kg), significantly and dose-dependently shortened the
immobility time in mice. The antidepressant-like activity of the
tested compounds was similar or even higher (compounds 27 and
35) to that of citalopram administered in the lowest dose.
Subsequently, the antidepressant properties of the most active
in the FST compounds 27 and 35 were confirmed in tail suspension
test (TST) in C57BL/6J mice (Table 2-S.I.). Two selected compounds

V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
341
Fig. 4. Effects of the active compounds 27, 34, 35, 39, SB-269970 and citalopram on the immobility time of mice in the forced swim test. Data are presented as the mean standard
error of the mean of N ¼ 7e12 animals per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the respective vehicle group, followed by the Bonferroni's
comparison test after a significant analysis of variance.
Table 8
In vivo antidepressant and anxiolytic activity of 27, 35 and reference compounds.
Compound
TST in mice MED (mg/kg) i.p.
FST in rats MED (mg/kg) i.p.
FPT in mice MED (mg/kg) i.p.
27
35
2.5
2.5
2.5
20
1.25
NTa
1.25
0.625
1
NT
1.25
SB-269970
Citalopram
Diazepam
1.25c
NAb,d
NT
NT
a
NT e not tested.
NA e not active.
Ref. [39].
Ref. [5].
b
c
d
receptors (<78% at a concentration 1
and M1 receptors (<4% at a concentration 1
may result in low propensity of tested compounds to produce hy-
potension, sedation or other peripheral and CNS side effects related
to these sites.
m
M), and did not bind to b1
models (FST, TST and FPT) in doses lower or comparable to SB-
269970, citalopram, and diazepam, used as active controls.
Additional studies on this group of 5-HT7R antagonists, which
may improve the available therapies, will be useful to further
confirm their potential application in the treatment of CNS
disorders.
mM). These findings
3. Conclusions
4. Experimental section
Using VCL-VS protocol integrated with solid-supported meth-
odologies, we designed and synthesized a series of 39 arylsulfo-
namide/amide/urea derivatives of (aryloxy)ethyl alicyclic amines as
5-HT7R antagonists. The work expanded chemical diversity of
central amine part of PZ-766 and PZ-1404, as well as arylsulfona-
mide/amide and aryloxy moieties, to identify other high affinity
analogs.
Structure-activity relationship studies supported by molecular
modeling analysis confirmed that sterically encumbered sub-
stituents in position-2 of aryloxy fragment (isopropyl, tert-butyl,
cyclopentyl and phenyl) preferentially interacted with 5-HT7Rs.
Moreover, replacement of the piperidine moiety with the 8-
azabicyclo[3.2.1]octane (tropane) scaffold led to active 5-HT7 li-
gands and selectivity over the 5-HT1A receptors. Finally, we
confirmed that sulfonamide group was more preferable for inter-
action with 5-HT7 receptors than amide and urea bonds.
4.1. Chemistry
Solution and solid-phase organic transformations and resin
washes were carried out at ambient temperature, unless indicated
otherwise. Organic solvents (from Aldrich and Chempur) were of
reagent grade and were used without purification. BAL linker
MBHA-resin (loading 1.1 mmol/g) was purchased from Iris Chem-
icals. All other commercially available reagents were of the highest
purity (from Aldrich, Fluorochem).
All workup and purification procedures were carried out with
reagent-grade solvents under ambient atmosphere. Column chro-
matography was performed using silica gel Merck 60
(70e230 mesh ASTM).
Mass spectra were recorded on a UPLC-MS/MS system consisted
of a Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA,
USA) coupled to a Waters TQD mass spectrometer (electrospray
ionization mode ESI-tandem quadrupole). Chromatographic sepa-
rations were carried out using the Acquity UPLC BEH (bridged ethyl
The study allowed for the identification of the potent 5-HT7R
antagonists 27 (PZ-1417) and 35 (PZ-1150), which displayed sig-
nificant antidepressant-like and anxiolytic properties in rodents

342
V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
hybrid) C18 column; 2.1 ꢁ 100 mm, and 1.7
m
m particle size,
chromatographic purification over silica gel with CH2Cl2/MeOH (9/
0.7); UPLC/MS purity 96%, tR ¼ 4.65. C20H25FN2O4S, MW 408.49,
Monoisotopic Mass 408.15, [MþH]þ 409.3 1H NMR (300 MHz,
equipped with Acquity UPLC BEH C18 VanGuard pre-column;
2.1 ꢁ 5 mm, and 1.7
mm particle size. The column was maintained
at 40 ꢂC, and eluted under gradient conditions from 95% to 0% of
eluent A over 10 min, at a flow rate of 0.3 mL minꢀ1. Eluent A:
water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid
CDCl3) d 1.46e1.55 (m, 2H), 1.69e1.72 (m, 2H), 2.08e2.16 (m, 2H),
2.73e2.77 (m, 4H), 2.79e2.84 (m, 1H), 3.84 (s, 3H), 4.01e4.03 (m,
2H), 6.83e6.99 (m, 4H), 7.06e7.15 (m, 2H), 7.80e7.88 (m, 2H).
(0.1%, v/v). Chromatograms were made using Waters el PDA de-
tector. Spectra were analyzed in 200e700 nm range with 1.2 nm
resolution and sampling rate 20 points/s. MS detection settings of
Waters TQD mass spectrometer were as follows: source tempera-
ture 150 ꢂC, desolvation temperature 350 ꢂC, desolvation gas flow
rate 600 L hꢀ1, cone gas flow 100 L hꢀ1, capillary potential 3.00 kV,
cone potential 40 V. Nitrogen was used for both nebulizing and
drying gas. The data were obtained in a scan mode ranging from 50
to 2000 m/z in time 1.0 s intervals. Data acquisition software was
MassLynx V 4.1 (Waters). The UPLC/MS purity of all the final
compounds was confirmed to be 95% or higher.
4.1.4. 4-Fluoro-N-{1-[2-(2-isopropoxyphenoxy)ethyl]piperidin-4-
yl}benzenesulfonamide (21)
Yellow oil, 31 mg (36% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 95%, tR ¼ 4.77. C22H29FN2O4S, MW 436.54, Mono-
isotopic Mass 436.18, [MþH]þ 437.3 1H NMR (300 MHz, CDCl3)
d
1.30 (d, J ¼ 1.00 Hz, 6H), 1.41e1.56 (m, 2H), 1.71e1.81 (m, 2H),
2.16e2.28 (m, 2H), 2.78 (t, J ¼ 5.69 Hz, 2H), 2.90 (d, J ¼ 12.31 Hz,
2H), 3.11e3.25 (m, 1H), 4.06 (t, J ¼ 5.69 Hz, 2H), 4.46 (spt,
J ¼ 6.09 Hz, 1H), 6.85e6.91 (m, 4H), 7.14e7.22 (m, 2H), 7.86e7.93
(m, 2H).
1H NMR and 13C NMR spectra were obtained in Varian BB 200
spectrometer using TMS (0.00 ppm) as an internal standard in
CDCl3, and were recorded at 300 and 75 MHz, respectively. The J
values are reported in Hertz (Hz), and the splitting patterns are
designated as follows: s (singlet), d (doublet), t (triplet), dd (doublet
of doublets), td (triplet of doublets), ddd (doublet of doublet of
doublets), m (multiplet), br s (broad signal).
Elemental analyses for C, H, N and S were carried out using the
elemental Vario EI III Elemental Analyzer (Hanau, Germany). All
values are given as percentages, and were within 0.4% of the
calculated values.
4.1.5. 4-Fluoro-N-{1-[2-(2,6-diisopropylphenoxy)ethyl]piperidin-4-
yl}-benzenesulfonamide (22)
Yellow oil, 30 mg (32% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 95%, tR ¼ 6.05. C25H35FN2O3S, MW 462.62, Mon-
oisotopic Mass 462.24, [MþH]þ 463.2. 1H NMR (300 MHz, CDCl3)
d
1.19 (d, J ¼ 6.92 Hz, 12H), 1.47e1.61 (m, 2H), 1.80 (dd, J ¼ 13.45,
3.03 Hz, 2H), 2.11e2.21 (m, 2H), 2.74 (t, J ¼ 5.82 Hz, 2H), 2.83e2.90
(m, 2H), 3.12e3.23 (m, 1H), 3.30 (quin, J ¼ 6.91 Hz, 2H), 3.80 (t,
J ¼ 5.82 Hz, 2H), 4.84 (br s, 1H), 7.07 (s, 3H), 7.15e7.22 (m, 2H),
7.88e7.96 (m, 2H).
Melting point (Mp) were determine with a Büchi apparatus and
are uncorrected.
The synthesis of final compounds 18e52 and 55e58 were per-
formed according to the previously published procedures [24,25].
Compounds selected for behavioral evaluation (27, 34, 35 and 39)
were converted into the hydrochloride salts.
Spectroscopic data for representative compounds are summa-
rized below, while all the synthesis methods and characterization
data for the intermediate and remaining final compounds are re-
ported in the Supporting Information.
4.1.6. 1-Methyl-N-(1-{2-[(2-tert-butyl)phenoxy]ethyl}piperidin-4-
yl}-1H-pyrazole-4-sulfonamide (25)
Yellow oil, 45 mg (35% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 98%, tR ¼ 4.72, C21H32N4O3S, MW 420.57, Mono-
isotopic Mass 420.22, [MþH]þ 421.3. 1H NMR (300 MHz, CDCl3)
d
1.37 (s, 9H), 1.45e1.59 (m, 2H), 1.82e1.92 (m, 2H), 2.17e2.27 (m,
2H), 2.79e2.84 (m, 2H), 2.85e2.91 (m, 2H), 3.14e3.26 (m, 1H), 3.95
(s, 3H), 4.07 (t, J ¼ 6.03 Hz, 2H), 4.50 (s, 1H), 6.83 (dd, J ¼ 8.13,
1.14 Hz, 1H), 6.88 (td, J ¼ 7.53, 1.25 Hz, 1H), 7.12e7.19 (m, 1H), 7.29
(d, J ¼ 1.67 Hz, 1H), 7.75e7.81 (m, 1H).
4.1.1. 4-Fluoro-N-[1-(2-phenoxyethyl)piperidin-4-yl]-
benzenesulfonamide (18)
Yellow oil, 32 mg (33% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 95%, tR ¼ 4.10. C19H23FN2O3S, MW 378.46, Mono-
isotopic Mass 378.14, [MþH]þ 379.1. 1H NMR (300 MHz, CDCl3)
4.1.7. 3-Fluoro-N-{1-[2-(2-cyclopentylphenoxy)ethyl]piperidin-4-
yl}-benzenesulfonamide (27)
d
1.45e1.56 (m, 2H), 1.73e1.83 (m, 2H), 2.12e2.23 (m, 2H), 2.75 (t,
Yellow oil, 38 mg (38% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.5);
UPLC/MS purity 99%, tR ¼ 5.87, C24H31FN2O3S, MW 446.58, Mono-
isotopic Mass 446.20, [MþH]þ 447.1. 1H NMR (300 MHz, CDCl3)
J ¼ 5.75 Hz, 2H), 2.80e2.88 (m, 2H), 3.14e3.23 (m, 1H), 4.04 (t,
J ¼ 5.75 Hz, 2H), 4.54 (br s, 1H), 6.87 (dd, J ¼ 8.74, 0.98 Hz, 2H),
6.90e6.97 (m, 1H), 7.15e7.25 (m, 4H), 7.85e7.93 (m, 2H).
d
1.39e1.62 (m, 6H), 1.66e1.93 (m, 6H), 2.00e2.28 (m, 2H),
4.1.2. 4-Fluoro-N-{1-[2-(o-tolyloxy)ethy]piperidin-4-yl}-
benzenesulfonamide (19)
3.21e3.36 (m, 6H), 3.52e3.72 (m, 2H), 4.37 (br. s., 1H), 6.73e6.82
(m, 1H), 6.85e6.93 (m, 1H), 7.02e7.19 (m, 4H), 7.79e7.89 (m, 2H).
Yellow oil, 37 mg (32% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 95%, tR ¼ 4.53. C20H25FN2O3S, MW 392.49, Mon-
oisotopic Mass 392.16, [MþH]þ 393.1. 1H NMR (300 MHz, CDCl3)
13C NMR (75 MHz, CDCl3)
d 25.28, 29.60, 29.63, 33.17, 38.44,
52.66, 62.69, 111.38, 111.50, 116.19, 116.48, 121.95, 126.77, 126.80,
126.86, 129.29, 129.41, 134.34, 154.76. Hydrochloride: white pow-
der, MW 483.08, Anal. calcd for C24H31FN2O3S$HCl: C: 59.68, H:
6.68, N: 5.80, S: 6.64; Found C: 59.94, H: 6.58, N: 5.64, S: 6.60. Mp
for C24H31FN2O3S$HCl: 208.5e209.1 ꢂC.
d
1.42e1.55 (m, 2H), 1.74e1.83 (m, 2H), 2.18 (s, 3H), 2.21e2.27 (m,
2H), 2.79 (t, J ¼ 5.66 Hz, 2H), 2.83e2.90 (m, 2H), 3.15e3.23 (m, 1H),
4.05 (t, J ¼ 5.66 Hz, 2H), 4.52 (br s, 1H), 6.77 (d, J ¼ 9.23 Hz, 1H),
6.82e6.88 (m,1H), 7.12 (dd, J ¼ 6.49,1.49 Hz, 2H), 7.15e7.23 (m, 2H),
7.86e7.93 (m, 2H).
4.1.8. 3-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}azetidin-3-
yl)-benzenesulfonamide (29)
Yellow oil, 35 mg (33% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.5);
UPLC/MS purity 98%, tR ¼ 5.09. C20H25FN2O3S, MW 392.49, Mon-
oisotopic Mass 392.16, [MþH]þ 393.1. 1H NMR (300 MHz, CDCl3)
4.1.3. 4-Fluoro-N-{1-[2-(2-methoxyphenoxy)ethyl]piperidin-4-yl}
benzenesulfonamide (20)
Yellow oil, 33 mg (30% isolated yield) following

V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
343
d
1.16 (d, J ¼ 6.95 Hz, 6H), 2.80 (t, J ¼ 5.27 Hz, 2H), 2.98 (t,
4.1.12. 1-Methyl-N-{8-[2-(2-(biphenyl-2-yloxy)ethyl]-8-azabicyclo
[3.2.1]octan-3yl}-1H-pyrazole-4-sulfonamide (43)
J ¼ 6.99 Hz, 2H), 3.20 (dt, J ¼ 13.81, 6.90 Hz, 1H), 3.54e3.61 (m, 2H),
3.91 (t, J ¼ 5.29 Hz, 2H), 4.05 (t, J ¼ 6.22 Hz, 1H), 6.73 (dd, J ¼ 8.13,
0.75 Hz, 1H), 6.89e6.95 (m, 1H), 7.12 (td, J ¼ 7.76, 1.73 Hz, 1H),
7.16e7.22 (m, 2H), 7.51 (td, J ¼ 8.04, 5.27 Hz, 1H), 7.61 (dt, J ¼ 8.08,
1.91 Hz, 1H), 7.69 (d, J ¼ 7.82 Hz, 1H). 13C NMR (75 MHz, CDCl3)
Yellow oil, 33 mg (30% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 100%, tR ¼ 4.69, C25H30N4O3S, MW 466.60, Mon-
oisotopic Mass 466.20, [MþH]þ 467.5. 1H NMR (300 MHz, CDCl3)
d
22.73, 26.43, 44.67, 57.88, 62.79, 67.24, 111.10, 116.31, 116.61,
d
1.24e1.35 (m, 2H), 1.52 (d, J ¼ 14.71 Hz, 2H), 1.68e1.92 (m, 5H),
120.91, 126.01, 126.55, 129.59, 129.66, 129.71, 136.85, 155.69, 163.42,
166.81.
2.56e2.64 (m, 2H), 2.99e3.11 (m, 2H), 3.79 (s, 3H), 3.98 (t,
J ¼ 4.89 Hz, 2H), 4.09 (br. s., 1H), 6.80e6.91 (m, 2H), 7.10e7.32 (m,
7H), 7.58 (s, 1H), 7.71 (s, 1H).
4.1.9. 3-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}-8-azabicyclo
[3.2.1]octan-3-yl)-benzenesulfonamide (34)
Yellow oil, 36 mg (32% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 99%, tR ¼ 5.53, C24H31FN2O3S, MW 446.58, Mono-
4.1.13. (1-{2-[(propan-2-yl)phenoxy]ethyl}piperidin-4-yl)-3-(4-
fluorophenyl)urea (47)
Yellow oil, 29 mg (31% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 100%, tR ¼ 5.10, C23H30FN3O2, MW 399.50, Mono-
isotopic Mass 399.23, [MþH]þ 400.4. 1H NMR (300 MHz, CDCl3)
isotopic Mass 446.2, [MþH]þ 447.1. 1H NMR (300 MHz, CDCl3)
d 1.19
(d, J ¼ 6.92 Hz, 6H), 1.62e1.71 (m, 2H), 1.91e2.06 (m, 4H), 2.10e2.21
(m, 2H), 2.78 (t, J ¼ 5.24 Hz, 2H), 3.21e3.31 (m, 1H), 3.32e3.42 (m,
3H), 4.08 (t, J ¼ 5.58 Hz, 2H), 5.40 (br. s., 1H), 6.81 (dd, J ¼ 8.14,
0.87 Hz, 1H), 6.89e6.95 (m, 1H), 7.12 (td, J ¼ 7.76, 1.73 Hz, 1H), 7.20
(dd, J ¼ 7.52, 1.59 Hz, 1H), 7.25e7.32 (m, 1H), 7.51 (td, J ¼ 8.04,
5.27 Hz, 1H), 7.61 (dt, J ¼ 8.08, 1.91 Hz, 1H), 7.69 (d, J ¼ 7.82 Hz, 1H).
d
1.19 (d, J ¼ 6.92 Hz, 6H), 1.42e1.54 (m, 2H), 1.96 (d, J ¼ 12.63 Hz,
2H), 2.23e2.35 (m, 2H), 2.83 (t, J ¼ 5.67 Hz, 2H), 2.97 (d,
J ¼ 12.05 Hz, 2H), 3.23e3.34 (m, 1H), 3.63e3.74 (m, 1H), 4.09 (t,
J ¼ 5.69 Hz, 2H), 4.97 (br. s, 2H), 6.78e6.88 (m, 2H), 6.90e7.01 (m,
3H), 7.10e7.17 (m, 1H), 7.19e7.24 (m, 2H).
13C NMR (75 MHz, CDCl3)
d 22.72, 25.61, 26.70, 35.91, 45.80,
51.36, 59.25, 67.50, 111.22, 120.87, 125.29, 126.01, 126.60, 127.32,
130.57, 132.94, 135.41, 136.81, 141.24, 155.61. Hydrochloride: white
powder, MW 483.08. Anal. calcd for C24H31FN2O3S$HCl: C: 59.68, H:
6.68, N: 5.80, S: 6.64; Found C: 59.88, H: 6.51, N: 5.59, S: 6.40. Mp
for C24H31FN2O3S$HCl: 226.8e227.2 ꢂC.
4.1.14. N-(1-{2-[(propan-2-yl)phenoxy]ethyl}piperidin-4-yl-
pyridine-3-sulfonamide (50)
Yellow oil, 38 mg (30% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 99%, tR ¼ 4.55, C21H29N3O3S, MW 403.54, Mono-
isotopic Mass 403.19, [MþH]þ 404.3. 1H NMR (300 MHz, CDCl3)
4.1.10. 4-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}-8-
azabicyclo[3.2.1]octan-3-yl)-benzenesulfonamide (35)
Yellow oil, 38 mg (37% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 100%, tR ¼ 5.28, C24H31FN2O3S, MW 446.58, Mon-
oisotopic Mass 446.2, [MþH]þ 447.4. 1H NMR (300 MHz, CDCl3)
d
1.18 (d, J ¼ 6.92 Hz, 6H), 1.45e1.59 (m, 2H), 1.75e1.85 (m, 2H),
2.16e2.27 (m, 2H), 2.78 (t, J ¼ 5.66 Hz, 2H), 2.84e2.94 (m, 2H),
3.20e3.32 (m, 2H), 4.04 (t, J ¼ 5.64 Hz, 2H), 6.78 (d, J ¼ 8.11 Hz, 1H),
6.88e6.95 (m, 1H), 7.08e7.15 (m, 1H), 7.19 (dd, J ¼ 7.52, 1.52 Hz, 1H),
7.46 (dd, J ¼ 8.00, 4.86 Hz, 1H), 8.17 (dt, J ¼ 8.06, 1.93 Hz, 1H), 8.80
(dd, J ¼ 4.82, 1.52 Hz, 1H), 9.10 (d, J ¼ 1.99 Hz, 1H).
d
1.19 (d, J ¼ 6.92 Hz, 6H), 1.42e1.56 (m, 2H), 1.79 (dd, J ¼ 13.62,
3.08 Hz, 2H), 2.17e2.25 (m, 2H), 2.78 (t, J ¼ 5.72 Hz, 2H), 2.82e2.90
(m, 2H), 3.20e3.34 (m, 3H), 4.04 (t, J ¼ 5.71 Hz, 2H), 4.72 (br s, 1H),
6.78e6.82 (m, 1H), 6.92e6.97 (m, 1H), 7.12e7.21 (m, 4H), 7.88e7.95
(m, 2H). 13C NMR (75 MHz, CDCl3)
d
22.69, 25.80, 26.73, 36.26,
4.1.15. 3-Chloro-N-{7-[2-(2-(biphenyl-2-yloxy))ethyl]-2,7-
diazabicyclo[2.2.1]heptan-2-yl}-benzenesulfonamide (57)
45.99, 51.47, 59.06, 67.92, 111.25, 114.43, 114.75, 119.81, 120.10,
120.76,122.91,122.94, 122.96,126.00,126.55, 130.98,136.88,155.77,
160.82, 164.15.
Yellow oil, 90 mg (70% yield) following chromatographic puri-
fication over silica gel with CH2Cl2/MeOH (9/0.7); UPLC/MS purity
100%, tR ¼ 5.75, C25H25ClN2O3S, MW 469.00, Monoisotopic Mass
Hydrochloride: white powder, MW 483.08. Anal. calcd for
C
24H31FN2O3S$HCl: C: 59.68, H: 6.68, N: 5.80, S: 6.64; Found C:
468.13, [MþH]þ 469.2. 1H NMR (300 MHz, CDCl3)
d 1.25e1.29 (m,
59.72, H: 6.67, N: 5.81, S: 6.58. Mp for
C
24H31FN2O3S$HCl:
2H), 1.53e1.59 (m, 1H), 2.58 (ddd, J ¼ 9.91, 3.30, 1.60 Hz, 2H),
2.81e2.92 (m, 2H), 3.32 (s, 2H), 3.38 (d, J ¼ 9.97 Hz, 2H), 3.97e4.17
(s, 1H), 6.90e6.95 (m, 1H), 7.00e7.06 (m, 1H), 7.27e7.31 (m, 1H),
7.32e7.43 (m, 3H), 7.44e7.49 (m, 3H), 7.54e7.58 (m, 1H), 7.68 (dt,
J ¼ 7.73, 1.36 Hz, 1H), 7.80 (t, J ¼ 1.81 Hz, 1H).
225.6e226.1 ꢂC.
4.1.11. 3-Chloro-N-{8-[2-(biphenyl-2-yloxy)ethyl]-8-azabicyclo
[3.2.1]octan-3yl}benzenesulfonamide (39)
Yellow oil, 32 mg (28% isolated yield) following chromato-
graphic purification over silica gel with CH2Cl2/MeOH (9/0.7);
UPLC/MS purity 99%, tR ¼ 5.75, C27H29ClN2O3S, MW 497.05, Mon-
oisotopic Mass 496.16, [MþH]þ 497.3. 1H NMR (300 MHz, CDCl3)
4.2. Molecular modeling
d
1.42e1.52 (m, 2H), 1.70e1.90 (m, 3H), 1.95e2.06 (m, 2H), 2.63 (t,
4.2.1. Software
J ¼ 5.47 Hz, 2H), 3.13e3.15 (m, 2H), 3.33 (q, J ¼ 5.94 Hz, 1H), 4.05 (t,
J ¼ 5.45 Hz, 2H), 5.07 (d, J ¼ 5.01 Hz, 1H), 6.91e7.05 (m, 2H),
7.24e7.38 (m, 5H), 7.43e7.51 (m, 3H), 7.54e7.59 (m, 1H), 7.74 (dt,
J ¼ 7.75, 1.38 Hz, 1H), 7.85 (t, J ¼ 1.83 Hz, 1H). 13C NMR (75 MHz,
The in-house script was generated by using CombiGlide [43].
The Instant JChem version 15.3.16.0 was used to management of
chemical structures, to search and to predict a simple molecular
properties [44]. The LigPrep [45] was used to prepare a high quality
3D ligand structures with assignment of proper protonation states
and stereoisomers, calculation of simple ADME descriptors was
performed by QikProp [46], and Glide with standard precision (SP)
mode was used to automated ligand-receptor docking [47]. The
Discovery Studio 3.5 was used to perform 3D pharmacophore
model generation and screening [48].
CDCl3)
d 25.75, 36.26, 46.00, 51.14, 58.81, 68.60, 112.19, 120.95,
125.22, 126.80, 127.25, 127.80, 128.61, 129.55, 130.50, 130.82, 130.94,
132.85, 135.38, 138.58, 141.64, 155.63. Hydrochloride: white pow-
der, MW 533.55. Anal. calcd for C27H29ClN2O3S$HCl: C: 60.78, H:
5.67, N: 5.25, S: 6.01; Found C: 60.77, H: 5.64, N: 5.32, S: 6.17. Mp for
C
27H29ClN2O3S$HCl: 242.9e243.5 ꢂC.

344
V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
4.3. In vitro pharmacology
solution (5 mL Eu-cAMP and 5 mL ULight-anti-cAMP). The assay plate
was incubated for 1 h at room temperature. Time-resolved fluores-
cence resonance energy transfer (TR-FRET) was detected by an
Infinite M1000 Pro (Tecan) using instrument settings from LANCE
cAMP detection kit manual.
4.3.1. Cell culture and preparation of cell membranes
Cell pellets were thawed and homogenized in 20 volumes of
assay buffer using an Ultra Turrax tissue homogenizer and centri-
fuged twice at 35,000 g for 20 min at 4 ꢂC, with incubation for
15 min at 37 ꢂC in between. The composition of the assay buffers
was as follows: for 5-HT1AR: 50 mM TriseHCl, 0.1 mM EDTA, 4 mM
MgCl2, 10 mM pargyline and 0.1% ascorbate; for 5-HT6R: 50 mM
TriseHCl, 0.5 mM EDTA and 4 mM MgCl2, for 5-HT7bR: 50 mM
TriseHCl, 4 mM MgCl2, 10 mM pargyline and 0.1% ascorbate; for
dopamine D2LR: 50 mM TriseHCl, 1 mM EDTA, 4 mM MgCl2,
120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate. All as-
4.4. In vivo pharmacology
The experiments were performed on male Albino Swiss
(22e28 g, FST and FPT), C57BL/6J mice (20e24 g, TST) and on male
Wistar rats (220e250 g, FST). The animals were kept at a room
temperature of 20
1
ꢂC, and had free access to food (standard
laboratory pellets) and tap water before the experiment. All the
experiments were conducted in the light phase between 9 a.m. and
2 p.m. All the experimental procedures were approved by the Local
Ethics Commission for Animal Experiments of Jagiellonian Uni-
versity in Cracow. Compounds 34, 39, SB-269970 and diazepam
were suspended in a 1% aqueous solution of Tween 80, while
compounds 27, 35 and citalopram were dissolved in distilled water.
All the tested compounds were injected intraperitoneally (ip)
60 min (FST, TST and FPT in mice) or 30 min (FST in rats) before the
test. Diazepam were injected intraperitoneally (ip) 60 min, while
SB-269970 and citalopram 30 min before the test. Each experi-
mental group consisted of six to ten animals, and all the animals
were used only once.
says were incubated in a total volume of 200 ml in 96-well micro-
titre plates for 1 h at 37 ꢂC, except for 5-HT1AR that was incubated
for 1 h at room temperature. The process of equilibration is
terminated by rapid filtration through Unifilter plates with a 96-
well cell harvester and radioactivity retained on the filters was
quantified on a Microbeta plate reader. Non-specific binding is
defined with10
mM of 5-HT in 5-HT1AR and 5-HT7R binding ex-
periments, whereas10
mM of methiothepine or 1
m
M of (þ)buta-
clamol were used in 5-HT6R and D2L assays, respectively. Each
compound was tested in triplicate at 7e8 concentrations
(10ꢀ11e10ꢀ4 M). The inhibition constants (Ki) were calculated from
the Cheng-Prusoff equation [28]. Results were expressed as means
of at least three separate experiments. Membrane preparation and
general assay procedures for cloned receptors were adjusted to 96-
microwell format based on described protocols (Perkin Elmer).
Membrane preparation and general assay procedures for cloned
receptors were adjusted to 96-microwell format based on
described protocols.
4.4.1. Forced swim test in mice protocol
The experiment was carried out according to the method of
Porsolt et al. [30]. Mice were individually placed in a glass cylinder
(25 cm high; 10 cm in diameter) containing 10 cm of water main-
tained at 23e25 ꢂC, and were left there for 6 min. A mouse was
regarded as immobile when it remained floating on the water,
making only small movements to keep its head above it. The total
duration of immobility was recorded during the last 4 min of a 6-
min test session.
a1-Adrenoceptor binding assays were carried out on the rat
cerebral cortex. [3H]-Prazosin (22 Ci/mmol) was used as ligand. The
incubation mixture (final volume of 550
membrane suspension, 50
l of a [3H]-Prazosin (0.6 nM) solution
and 50 l of buffer containing seven or eight concentrations
(1 nMe100 M) of the investigated compounds. For measuring
ml) consisted of 450 ml of
m
m
m
4.4.2. Forced swim test in rats protocol
unspecific binding, phentolamine at a final concentration of 10
was present.
m
M
The experiment was carried out according to the method of
Porsolt et al. [31]. On the first experimental day, the animals were
gently individually placed in Plexiglas cylinders (40 cm height,
18 cm in diameter) containing 15 cm of water maintained at 25 ꢂC
for 15 min. On removal from water, the rats were placed for 30 min
in a Plexiglas box under a 60-W bulb to dry. On the following day,
the rats were re-placed in the cylinder and the total duration of
immobility was recorded during the whole 5-min test period. Fresh
water was used for each animal.
4.3.2. Effects on adenylate cyclase activity
The functional properties of compounds on 5-HT7R were evalu-
ated using their ability to inhibit cAMP production induced by 5-CT
(10 nM) e a 5-HT7R agonist, in a HEK293 cells over expressing the
human 5-HT7bR. Each compound was tested in triplicate at 8 con-
centrations(10ꢀ11e10ꢀ4 M). Thelevelofadenylylcyclaseactivitywas
measured using recombinant HEK293 cells which stably expressing
the human 5-HT7b receptor. Cells (prepared with the use of Lip-
ofectamine 2000) were maintained at 37 ꢂC in a humidified atmo-
sphere with 5% CO2 and were grown in Dulbeco's Modifier Eagle
Medium containing 10% dialyzed fetal bovine serum and 500 mg/mL
G418 sulfate. For functional experiments, cells were subcultured in
25 cm diameter dishes, grown to 90% confluence, washed twice with
prewarmed to 37 ꢂC phosphate buffered saline (PBS) and were
centrifuged for 5 min (160 ꢁ g). The supernatant was aspirated, the
cell pellet was resuspended in stimulation buffer (1 ꢁ HBSS, 5 mM
HEPES, 0.5 mM IBMX, 0.1% BSA). The cAMP level was measured using
the LANCE cAMP detection kit (PerkinElmer), according to the
manufacturer's directions. For the investigation of antagonist effect
on 5-HT7R, the agonist, 5-carboxyamidotryptamine (5-CT;
EC50 ¼ 1 nM) was used in submaximal concentration (6.2 nM) to
stimulate cAMP production and cells (450 per well) were incubated
4.4.3. Tail suspension test in mice protocol
The testing procedure was based on the method of Steru et al.
[32]. An automated device (Campden Instruments Ltd., United
Kingdom) was used. The mice were suspended by the tail with tape
to an aluminum hook connected to a strain gage, and were posi-
tioned such that the base of their tail was aligned with the bottom
of the hook. This positioning was found to decrease the propensity
for the mice to climb their tail during the test. A strain gage con-
nected to computer software detected any movements made by the
mouse, to record the number of times (events) each subject entered
into an escape behavior (struggling episodes), the duration of the
events, and the average strength of each event during a 6-min test
session. The total duration of immobility was calculated as the time
in which the force of the movements of the mice was below a preset
threshold. An optimum threshold was determined by comparing
manually scored videotapes with automated scores. The following
settings were used in all experiments: threshold 0.20 N, off delay
30 ms
with compound (1
white opaque microtiter plate. After incubation, the reaction was
stopped and cells were lysed by the addition of 10 L working
mM) for 30 min at room temperature in 384-well
m

V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
345
T. Ishiyama, M. Taiji, The role of 5-HT7 receptor antagonism in the amelio-
ration of MK-801-induced learning and memory deficits by the novel atypical
antipsychotic drug lurasidone, Behav. Brain Res. 244 (2013) 66e69.
[10] I.T. Forbes, S. Dabbs, D.M. Duckworth, A.J. Jennings, F.D. King, P.J. Lovell,
A.M. Brown, L. Collin, J.J. Hagan, D.N. Middlemiss, G.J. Riley, D.R. Thomas,
N. Upton, (R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl) propyl]
benzenesulfonamide: the first selective 5-HT7 receptor antagonist, J. Med.
Chem. 41 (1998) 655e657.
[11] D.R. Thomas, S. Melotto, M. Massagrande, A.D. Gribble, P. Jeffrey, A.J. Stevens,
N.J. Deeks, P.J. Eddershaw, S.H. Fenwick, G. Riley, T. Stean, C.M. Scott, M.J. Hill,
D.N. Middlemiss, J.J. Hagan, G.W. Price, I.T. Forbes, SB-656104-A, a novel se-
lective 5-HT7 receptor antagonist, modulates REM sleep in rats, Br. J. Phar-
macol. 4 (2003) 705e714.
4.4.4. Four-plates test in mice protocol
The four-plate apparatus (BIOSEB) consists of a plastic cage
(25 ꢁ 18 ꢁ 16 cm) floored by four identical rectangular metal plates
(8 ꢁ 11 cm) separated from one another by a gap of 4 mm. The top
of the cage is covered by a transparent Perspex lid that prevents
escape behavior. The plates are connected to a device that can
generate electric shocks. Following a 15-s habituation period, the
animal's motivation to explore a novel environment was sup-
pressed by an electric foot shock (0.8 mA, 0.5 s) every time it moves
from one plate to another during a 1-min test session. This action is
referred to as a ‘punished crossing’, and was followed by a 3 s shock
interval, during which the animal can move across plates without
receiving a shock [33]. The number of punished crossings received
by an animal was recorded during the 60 s period.
[12] C. Kikuchi, T. Hiranuma, M. Koyama, Tetrahydrothienopyridylbutyltetrahy-
drobenzindoles: new selective ligands of the 5-HT7 receptor, Bioorg. Med.
Chem. Lett. 12 (2002) 2549e2552.
ꢀ
ꢀ
[13] B. Volk, J. Barkoczy, E. Hegedus, S. Udvari, I. Gacsalyi, T. Mezei, K. Pallagi,
H. Kompagne, G. Levay, A. Egyed, L.G.J. Harsing, M. Spedding, G. Simiq,
(Phenylpiperazinyl-butyl)oxindoles as selective 5-HT7 receptor antagonists,
J. Med. Chem. 51 (2008) 2522e2532.
4.4.5. Spontaneous locomotor activity assay protocol
[14] L. Berrade, B. Aisa, M.J. Ramirez, S. Galiano, S. Guccione, L.R. Moltzau, F.O. Levy,
F. Nicoletti, G. Battaglia, G. Molinaro, I. Aldana, A. Monge, P. Perez-Silanes,
Novel benzo[b]thiophene derivatives as new potential antidepressants with
rapid onset of action, J. Med. Chem. 54 (2011) 3086e3090.
Locomotor activity was recorded with an Opto M3 multi-
channel activity monitor (MultiDevice Software v.1.3, Columbus
Instruments). Swiss albino or C57BL/6J mice were individually
placed in plastic cages (22 ꢁ 12 ꢁ 13 cm) for 30 min habituation
period, and then the ambulation were counted from 2 to 6 min,
6 min or 1 min 15 s that is the time equal to the observation period
respectively in the forced swim, tail suspension and four plate tests.
The cages were cleaned up with 70% ethanol after each mouse.
[15] Y. Kim, J. Tae, K. Lee, H. Rhim, I.H. Choo, H. Cho, W.K. Park, G. Keum, H. Choo,
Novel N-biphenyl-2-ylmethyl 2-methoxyphenylpiperazinylalkanamides as 5-
HT7R antagonists for the treatment of depression, Bioorg. Med. Chem. 22
(2014) 4587e4596.
ꢀ
[16] P. Zajdel, G. Subra, P. Verdie, E. Gabzdyl, A.J. Bojarski, B. Duszynska,
J. Martinez, M. Pawłowski, Sulfonamides with the N-alkyl-N0-dialkylguanidine
moiety as 5-HT7 receptor ligands, Bioorg. Med. Chem. Lett. 19 (2009)
4827e4831.
[17] V. Canale, R. Kurczab, A. Partyka, G. Satała, J. Witek, M. Jastrze˛bska-Wie˛sek,
M. Pawłowski, A.J. Bojarski, A. Wesołowska, P. Zajdel, Towards novel 5-HT7
versus 5-HT1A receptor ligands among LCAPs with cyclic amino acid amide
fragments: design, synthesis, and antidepressant properties, Part II. Eur. J.
Med. Chem. 92 (2015) 202e211.
4.4.6. Statistics
All the data are presented as the mean SEM. The statistical
significance of results was evaluated by a one-way analysis of
variance (ANOVA) followed by Bonferroni's Comparison Test.
p < 0.05 was considered statistically significant.
[18] E. Badarau, F. Suzenet, A.J. Bojarski, A.L. Fînaru, G. Guillaumet, Benzimidazo-
lone-based serotonin 5-HT1A or 5-HT7R ligands: synthesis and biological
evaluation, Bioorg. Med. Chem. Lett. 19 (2009) 1600e1603.
ꢁ
[19] L. Salerno, V. Pittala, M.N. Modica, M.A. Siracusa, S. Intagliata, A. Cagnotto,
M. Salmona, R. Kurczab, A.J. Bojarski, G. Romeo, Structure-activity relation-
ships and molecular modeling studies of novel arylpiperazinylalkyl 2-
benzoxazolones and 2-benzothiazolones as 5-HT7 and 5-HT1A receptor li-
gands, Eur. J. Med. Chem. 85 (2014) 716e726.
Acknowledgments
This study was supported by the National Science Center Grant
No DEC-2012/05/B/NZ7/03076.
[20] G. Spadoni, A. Bedini, S. Bartolucci, D. Pala, M. Mor, T. Riccioni, F. Borsini,
W. Cabri, D. Celona, M. Marzi, G. Tarzia, S. Rivara, P. Minetti, Towards the
development of 5-HT7 ligands combining serotonin-like and arylpiperazine
moieties, Eur. J. Med. Chem. 80 (2014) 8e35.
[21] H.D. Hansen, E. Lacivita, P. Di Pilato, M.M. Herth, S. Lehel, A. Ettrup,
V.L. Andersen, A. Dyssegaard, P. De Giorgio, R. Perrone, F. Berardi,
N.A. Colabufo, M. Niso, G.M. Knudsen, M. Leopoldo, Synthesis, radiolabeling
and in vivo evaluation of [11C](R)-1-[4-[2-(4-methoxyphenyl)phenyl]piper-
azin-1-yl]-3-(2-pyrazinyloxy)-2-propanol, a potential PET radioligand for the
5-HT7 receptor, Eur. J. Med. Chem. 79 (2014) 152e163.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.11.040.
References
ꢀ
ꢀ
[22] R.A. Medina, H. Vazquez-Villa, J.C. Gomez-Tamayo, B. Benhamú, M. Martín-
ꢀ
Fontecha, T. de la Fuente, G. Caltabiano, P.B. Hedlund, L. Pardo, M.L. Lopez-
Rodríguez, The extracellular entrance provides selectivity to serotonin 5-HT7
receptor antagonists with antidepressant-like behavior in vivo, J. Med. Chem.
57 (2014) 6879e6884.
[1] T.W. Lovenberg, B.M. Baron, L. De Lecea, J.D. Miller, R.A. Prosser, M.A. Rea,
P.E. Foye, M. Racke, A.L. Slone, B.W. Siegel, A novel adenylyl cyclase-activating
serotonin receptor (5-HT7) implicated in the regulation of mammalian
circadian rhythms, Neuron 11 (1993) 449e458.
[2] E. Kvachnina, G. Liu, A. Dityatev, U. Renner, A. Dumuis, D.W. Richter,
G. Dityateva, M. Schachner, T.A. Voyno-Yasenetskaya, E.G. Ponimaskin, 5-HT7
receptor is coupled to G alpha subunits of heterotrimeric G12-protein to
regulate gene transcription and neuronal morphology, J. Neurosci. 25 (2005)
7821e7830.
[23] P. Bonaventure, C. Dugovic, M. Kramer, P. De Boer, J. Singh, S. Wilson,
K. Bertelsen, J.N. Di, J. Shelton, L. Aluisio, L. Dvorak, I. Fraser, B. Lord,
D. Nepomuceno, A. Ahnaou, W. Drinkenburg, W.Y. Chai, C. Dvorak, S. Sands,
N. Carruthers, T.W. Lovenberg, Translational evaluation of JNJ-18038683, a 5-
hydroxytryptamine type 7 receptor antagonist, on rapid eye movement sleep
and in major depressive disorder, J. Pharmacol. Exp. Ther. 342 (2012)
429e440.
[24] P. Zajdel, R. Kurczab, K. Grychowska, G. Satała, M. Pawłowski, A.J. Bojarski, The
multiobjective based design, synthesis and evaluation of the arylsulfonamide/
amide derivatives of aryloxyethyl- and arylthioethyl- piperidines and pyrro-
lidines as a novel class of potent 5-HT₇ receptor antagonists, Eur. J. Med.
Chem. 56 (2012) 348e360.
[3] A. Matthys, G. Haegeman, K. Van Craenenbroeck, P. Vanhoenacker, The se-
rotonin 5-HT7 receptors: two decades of research, Mol. Neurobiol. 230 (2011)
555e568.
[4] A. Nikiforuk, CNS Drugs 29 (2015) 265e275.
[5] A. Wesolowska, E. Tatarczynska, A. Nikiforuk, E. Chojnacka-Wojcik,
Enhancement of the anti-immobility action of antidepressants by a selective
5-HT7 receptor antagonist in the forced swimming test in mice, Eur. J. Phar-
macol. 555 (2007) 43e47.
[25] P. Zajdel, V. Canale, A. Partyka, K. Marciniec, G. Satała, R. Kurczab, A. Siwek,
M. Jastrze˛bska-Wie˛sek, A. Wesołowska, T. Kos, P. Popik, A.J. Bojarski, Aryl-
sulfonamide derivatives of (aryloxy)ethyl-piperidines as selective 5-HT7 re-
ceptor antagonists and their psychotropic properties, Med. Chem. Comm. 6
(2015) 1272e1277.
[6] G. Sarkisyan, A.J. Roberts, P.B. Hedlund, The 5-HT(7) receptor as a mediator
and modulator of antidepressant-like behavior, Behav. Brain Res. 209 (2010)
99e108.
ꢀ
[26] A.J. Bojarski, M.T. Cegła, S. Charakchieva-Minol, M.J. Mokrosz, M. Mackowiak,
ꢀ
[7] O. Mnie-Filali, C. Faure, L. Lambas-Senas, M.M. El, H. Belblidia, E. Gondard,
S. Misztal, J.L. Mokrosz, Structure-activity relationship studies of CNS agents.
Part 9: 5-HT1A and 5-HT2 receptor affinity of some 2-and 3-substituted 1, 2,
3, 4-tetrahydro-beta-carbolines, Pharmazie 4 (1993) 89e94.
A. Etievant, H. Scarna, A. Didier, A. Berod, P. Blier, N. Haddjeri, Pharmacological
blockade of 5-HT7 receptors as a putative fast acting antidepressant strategy,
Neuropsychopharmacology 36 (2011) 1275e1288.
ꢀ
ꢀ
[27] M.H. Paluchowska, R. Bugno, B. Duszynska, E. Tatarczynska, A. Nikiforuk,
[8] A.I. Abbas, P.B. Hedlund, X.P. Huang, T.B. Tran, H.Y. Meltzer, B.L. Roth, Ami-
sulpride is a potent 5-HT7 antagonist: relevance for antidepressant actions
in vivo, Psychopharmacology 205 (2009) 119e128.
ꢀ
T. Lenda, E. Chojnacka-Wojcik, The influence of modifications in imide frag-
ment structure on 5-HT(1A) and 5-HT(7) receptor affinity and in vivo phar-
macological properties of some new 1-(m-trifluoromethylphenyl)piperazines,
[9] T. Horisawa, H. Nishikawa, S. Toma, A. Ikeda, M. Horiguchi, M. Ono,

346
V. Canale et al. / European Journal of Medicinal Chemistry 108 (2016) 334e346
blockade on the behavioral and neurochemical effects of imipramine in rats,
Bioorg. Med. Chem. 15 (2007) 7116e7125.
ꢀ
ꢀ
[28] P. Zajdel, K. Marciniec, A. Maslankiewicz, G. Satała, B. Duszynska, A.J. Bojarski,
Pharmacol. Rep. 60 (2008) 464e474.
ꢀ
A. Partyka, M. Jastrze˛bska-Wie˛sek, D. Wrobel, A. Wesołowska, M. Pawłowski,
[37] S. Pellow, A.L. Johnston, S.E. File, Selective agonists and antagonists for 5-
hydroxytryptamine receptor subtypes, and interactions with yohimbine and
FG 7142 using the elevated plus-maze test in the rat, J. Pharm. Pharmacol. 39
(1987) 917e928.
Quinoline- and isoquinoline-sulfonamide derivatives of LCAP as potent CNS
multi-receptor-5-HT1A/5-HT2A/5-HT7 and D2/D3/D4-agents: the synthesis
and pharmacological evaluation, Bioorg. Med. Chem. 20 (2012) 1545e1556.
[29] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and
the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
[38] M.J. Millan, The neurobiology and control of anxious states, Prog. Neurobiol.
70 (2003) 83e244.
ꢀ
[39] A. Wesołowska, A. Nikiforuk, K. Stachowicz, E. Tatarczynska, Effect of the
[30] R.D. Porsolt, A. Bertin, M. Jalfre, Behavioral despair in mice:
a
primary
selective 5-HT7 receptor antagonist SB 269970 in animal models of anxiety
and depression, Neuropharmacology 51 (2006) 578e586.
[40] W. Adriani, D. Travaglini, E. Lacivita, L. Saso, M. Leopoldo, G. Laviola, Modu-
latory effects of two novel agonists for serotonin receptor 7 on emotion,
motivation and circadian rhythm profiles in mice, Neuropharmacology 62
(2012) 833e842.
screening test for antidepressants, Arch. Int. Pharmacodyn. Ther. 229 (1977)
327e336.
[31] L. Steru, R. Chermat, B. Thierry, P. Simon, The tail suspension test: a new
method for screening antidepressants in mice, Psychopharmacology 85
(1985) 367e370.
[32] R.D. Porsolt, G. Anton, N. Blavet, M. Jalfre, Behavioural despair in rats: a new
model sensitive to antidepressant treatments, Eur. J. Pharmacol. 47 (1978)
379e391.
[41] R. Canese, F. Zoratto, L. Altabella, P. Porcari, L. Mercurio, F. De Pasquale,
E. Butti, G. Martino, E. Lacivita, M. Leopoldo, G. Laviola, W. Adriani, Persistent
modification of forebrain networks and metabolism in rats following
adolescent exposure to a 5-HT7 receptor agonist, Psychopharmacology 23
(2015) 275e289.
[33] C. Aron, P. Simon, C. Larousse, J.R. Boissier, Evaluation of a rapid technique for
detecting minor tranquilizers, Neuropharmacology 10 (1971) 459e469.
[34] K. Grychowska, K. Marciniec, V. Canale, M. Szymiec, G. Glanowski, G. Satała,
[42] The percentage of inhibition for b1, H1 and M1 receptors were evaluated at
Eurofins Cerep. Experimental conditions for these assays are described online
at www.cerep.fr.
ꢀ
A. Maslankiewicz, M. Pawłowski, A.J. Bojarski, P. Zajdel, Quinolinesulfona-
mides of aryloxy-/arylthio-ethyl piperidines: influence of an arylether frag-
ment on 5-HT1A/5-HT7 receptor selectivity, Arch. Pharm. 346 (2013)
180e188.
€
[43] CombiGlide, version 3.5, Schrodinger, LLC, New York, NY, 2014.
[44] Instant JChem 15.3.16.0, 2015, ChemAxon (http://www.chemaxon.com).
€
[35] J.J. Hagan, G.W. Price, P. Jeffrey, N.J. Deeks, T. Stean, D. Piper, M.I. Smith,
N. Upton, A.D. Medhurst, D.N. Middlemiss, G.J. Riley, P.J. Lovell, S.M. Bromidge,
D.R. Thomas, Characterization of SB-269970-A, a selective 5-HT(7) receptor
antagonist, Br. J. Pharmacol. 130 (2000) 539e548.
[45] LigPrep, version 3.2, Schrodinger, LLC, New York, NY, 2014.
€
[46] QikProp, version 4.2, Schrodinger, LLC, New York, NY, 2014.
€
[47] Glide, version 6.5, Schrodinger, LLC, New York, NY, 2014.
[48] Discovery Studio 3.5, BIOVIA Inc., San Diego, CA, USA.
[36] A. Wesołowska, M. Kowalska, Influence of serotonin 5-HT(7) receptor