N-Alkylated arylsulfonamides of (aryloxy)ethyl piperidines: 5-HT7 receptor selectivity versus multireceptor profile

Article abstract of DOI:10.1016/j.bmc.2015.11.041

The N-alkylation of the sulfonamide moiety, in a group of arylsulfonamide derivatives of (aryloxy)ethyl piperidines, may be considered as a strategy for the design of selective 5-HT₇ receptor ligands or multifunctional agents to extend a polyph

Full text of DOI:10.1016/j.bmc.2015.11.041

Bioorganic & Medicinal Chemistry 24 (2016) 130–139
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Alkylated arylsulfonamides of (aryloxy)ethyl piperidines: 5-HT7
receptor selectivity versus multireceptor profile
a
b
c
b
d
e
´
Vittorio Canale , Rafał Kurczab , Anna Partyka , Grzegorz Satała , Karolina Słoczynska , Tomasz Kos ,
c
f
d
b
c
_
Magdalena Jastrze˛bska-Wie˛sek , Agata Siwek , Elzbieta Pe˛kala , Andrzej J. Bojarski , Anna Wesołowska ,
Piotr Popik e, Paweł Zajdel a,
⇑
a Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
b Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krakow, Poland
c Department of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
d Department of Pharmaceutical Biochemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
e Department of Drug Development, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krakow, Poland
f Department of Pharmacobiology, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The N-alkylation of the sulfonamide moiety, in a group of arylsulfonamide derivatives of (aryloxy)ethyl
piperidines, may be considered as a strategy for the design of selective 5-HT7 receptor ligands or multi-
functional agents to extend a polypharmacological approach to the treatment of complex diseases. The
study allowed for the identification of 31 (1-methyl-N-{1-[2-(2-(t-butyl)phenoxy)ethyl]piperidin-4-yl}-
N-cyclopropylmethyl-1H-pyrazole-4-sulfonamide), a potent and selective 5-HT7 receptor antagonist
and 33 (1-methyl-N-{1-[2-(biphenyl-2-yloxy)ethyl]piperidin-4-yl}-N-cyclopropylmethyl-1H-pyrazole-
4-sulfonamide), as multimodal 5-HT/dopamine receptor ligand, as 5-HT2A/5-HT7/D2 receptor antagonists.
Both selected compounds were evaluated in vivo in a forced swim test (FST) in mice and in a novel object
recognition (NOR) task in rats, demonstrating distinct antidepressant-like and pro-cognitive properties
(MED = 1.25 mg/kg and 1 mg/kg, ip, respectively). These findings warrant further studies to explore the
therapeutic potential of N-alkylated arylsulfonamides for the treatment of CNS disorders.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 30 September 2015
Revised 25 November 2015
Accepted 28 November 2015
Available online 30 November 2015
Keywords:
N-Alkylated sulfonamides
5-HT7 receptor antagonist
Multimodal 5-HT/dopamine ligands
Depression
Cognitive deficits
1. Introduction
development of further 5-HT7R antagonists is still required to
extend the knowledge of their therapeutic potentials.
Recent advances in neuropharmacology have demonstrated
new strategies for the development of antidepressant therapies.
Among them pharmacological blockade of 5-HT7 receptors,
enhancing REM sleep suppression and learning and memory pro-
cesses,1,2 might offer advantages over currently available drugs. It
has been found that several antidepressant and antipsychotic
drugs displayed high affinity for 5-HT7Rs. Furthermore, the antide-
pressant properties of certain antipsychotic drugs—amisulpride
and aripiprazole—could be related to the 5-HT7Rs.3,4 Finally, sev-
eral preclinical studies revealed that 5-HT7R antagonists may pro-
duce beneficial pro-cognitive effects in animal models of cognitive
decline.5 In view of the aforementioned findings, as well as the fact
that compound JNJ-1803883 has been recently evaluated in a
phase II clinical trial for the treatment of major depression,6 the
Although, primary sulfonamides are more present in marketed
drugs (i.e., anticonvulsant, antidiabetic, diuretics, chemotherapeu-
tics), a growing number of reports supports the fact that substitu-
tion at the sulfonamide group may be regarded as an important
approach in drug design.7 Since the introduction of the secondary
or tertiary sulfonamide group is chemically straightforward and
pharmacologically safe, substituted sulfonamides represent a suit-
able scaffold for the design and the development of new lead struc-
tures.8–12 An array of highly potent and selective 5-HT7R ligands
was reported in the arylsulfonamide and arylsulfone derivatives
of alkylamines, and led to early lead structures and pharmacolog-
ical tools (Fig. 1).13,14 It was further found that N-alkylation at
the sulfonamide in a group of arylpiperazine and perhydroiso-
quinoline derivatives was a successful approach for developing
selective 5-HT7R antagonists (Fig. 1).15–18
Herein we report for the first time N-alkylated arylsulfonamides
of (aryloxy)ethyl piperidines to establish the influence of the
N-alkylation of sulfonamide on 5-HT7R affinity and selectivity over
⇑
Corresponding author. Tel.: +48 126205450; fax: +48 126205405.
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
http://dx.doi.org/10.1016/j.bmc.2015.11.041
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
131
3. Chemistry
O
S
N
O
N
The designed compounds were synthesized according to a
multistep procedure (Scheme 1). Treating phenols 1–3 with
1,2-dibromoethane under biphasic conditions yielded (aryloxy)
ethyl bromides 4–6. These intermediates were then used for
the alkylation of the Boc-protected N-methyl-4-amino-piperi-
dine or 4-amino-piperidine to give compounds 7–9 and
21–23, respectively. The latter were subsequently reductively
aminated with cyclopropanecarbaldehyde, in the presence of
sodium cyanoborohydride, followed by removal of the Boc
group. The final arylsulfonamides 10–20 and 27–33 were
synthesized by the treatment of deprotected intermediates
7–9 and 22–24 with the selected arylsulfonyl chlorides. The
physicochemical data for the synthesized compounds are
reported in SI.
N
O
N
S
O
OH
SB-269970
Ki 5-HT7 = 1.2 nM
SB-258719
5-HT7 = 31 nM
Ki
N
N
N
O
S
O
N
O
N
S
N
N
N
O
F
A
PZ-376
5-HT7 = 13 nM
Ki
F
Ki 5-HT7 = 33 nM
Figure 1. Representatives of the arylsulfonamide/N-alkylated arylsulfonamide
class of 5-HT7 receptor ligands.
R
O
O
4. In vitro pharmacology
N
N
O
O
O
S
O
F
S
N
Ar
N
R1
4.1. In vitro radioligand binding evaluation
H
10-20; 27-33
PZ-1404
R = isopropyl, t-butyl, phenyl;
Ki (5-HT7) = 9 nM Kb (5-HT7) = 26 nM
Radioligand binding assays were employed to determine the
affinity and selectivity profiles of the synthesized compounds in
competition binding experiments for human serotonin 5-HT7, 5-
HT1A, 5-HT2A and D2 receptors, which were all stably expressed
in HEK293 cells, according to the previously published proce-
R1 = methyl,cyclopropylmethyl
Figure 2. Chemical structure of arylsulfonamide and N-alkylated sulfonamide
derivatives of (aryloxy)ethyl piperidines.
dures.20,23–25
a1-Adrenoceptor binding assays were carried out on
related monoaminergic receptors (i.e., 5-HT1A, 5-HT2A, D2). Struc-
tural modification comprised the introduction of an N-methyl
and N-cyclopropylmethyl moiety at the sulfonamide, as well as
the diversification of sulfonylating and alkylating agents resulting
from our previous reports (Fig. 2).19,20
We also evaluated the in vitro functional activity and metabolic
stability of selected compounds, as well as their in vivo pharmaco-
logical properties in behavioral models of depression and cognitive
decline test in rodents.
the rat cerebral cortex. The inhibition constants (Ki) were calcu-
lated from the Cheng–Prusoff equation.26 Results were expressed
as means of at least three separate experiments.
The percentage of inhibition for selected compounds 17, 20, 31
and 33 for SERT was determined according to the previously
reported method,27 whereas for D3, H1, 5-HT2C and M1 receptors
were evaluated at Eurofins Cerep. Experimental conditions for
these assays are described online at www.cerep.fr.
2. Molecular modeling
4.2. In vitro functional evaluation
Homology models of 5-HT7 receptor was built using crystal
structures of 5-HT1B (PDB ID: 4IAR), b2 (PDB ID: 3P0G), D3 (PDB
ID: 3PBL) and H1 (PDB ID: 3RZE) receptors,21 retrieved from the
Protein Data Bank.22 Based on the docking of a structurally diverse
set of ligands it was found that their fully coherent binding mode
was obtained only in models generated on the 5-HT1B template.
These models were next used for the study of the binding mode
of N-alkylated arylsulfonamides of (aryloxy)ethyl piperidines for
5-HT7R.
The functional properties of compounds 17, 20, 31 and 33 on 5-
HT7Rs were evaluated using their ability to inhibit cAMP produc-
tion induced by 5-CT (10 nM)—a 5-HT7R agonist, in HEK293 cells.
Each compound was tested in triplicate at 8 concentrations
(10À11 to 10À4 M). Additionally the antagonist properties of com-
pounds 20 and 33 against the human 5-HT2A and D2Rs were eval-
uated in functional cAMP cellular assays, performed at Eurofins
Cerep using HEK-293 cells, according to procedures described
online at www.cerep.fr.
R
R
R
R
O
O
O
HO
iv,v
ii
i
N
N
Br
O
S
O
Boc
Ar
N
N
4-6
7-9
10 20
-
1-3
ii
R
R
R
O
O
O
N
v
iii,iv
N
N
O
S
O
Boc
N
H
Ar
N
HN
24 26
21 23
-
27-33
-
R = isopropyl, t-butyl, phenyl
Ar = phenyl, 3-F-phenyl, 4-F-phenyl, 1-N-methyl-pyrazol-4-yl
Scheme 1. Synthesis of N-methyl/N-cyclopropylmethyl arylsulfonamide derivatives of (aryloxy)ethyl piperidines 10–20 and 27–33. Reagents and conditions: (i) 1,2-
dibromoethane, K2CO3, KI, (CH3)2CO, 60 °C, 48–72 h; (ii) 4-(Boc-N-methyl-amino)-piperidine or 4-(Boc-N-amino)-piperidine, K2CO3, KI, (CH3)2CO, 60 °C, 48–72 h; (iii)
cyclopropanecarbaldehyde, NaBH3CN, CH2Cl2, rt, 4 h; (iv) TFA/CH2Cl2 (90:10; v/v), 2 h; (v) arylsulfonyl chloride, CH2Cl2, 0 °C, 2–6 h.

132
V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
between the (aryloxy)ethyl fragment and the side chains of
5. In vitro metabolic stability
Phe6.51 and Phe6.52 (Fig. 3A).
Structure–activity relationship studies confirmed that N-alky-
lated sulfonamides generally displayed high affinity for 5-HT7Rs
yet, this modification had different influences on selectivity
towards other tested receptors compared with their N-unsubsti-
tuted analogs (Table 1). In the case of compounds containing iso-
propyl and phenyl substituents in the (aryloxy)ethyl fragment
this modification decreased 5-HT7/5-HT1A selectivity (i.e., PZ-
1404 vs both 11 and 27, and PZ-1345 vs both 20 and 33), while
compounds with a tert-butyl moiety showed increased selectivity
(i.e., PZ-1315 vs both 17 and 31). In line with the general trend
in selectivity, replacement of the methyl group with sterically hin-
dered N-cyclopropylmethyl, slightly decreased the affinity for 5-
HT7 sites (i.e., 11 vs 27), except for compounds 30 and 31 with a
tert-butyl substituent. It is worth noting, that the type of N-alky-
lated moiety at the nitrogen atom of sulfonamide did not impact
the position of the aryloxy fragment in the binding pocket (Fig. 3B).
Among tested sterically hindered substituents in the ortho posi-
tion of the (aryloxy)ethyl fragment, only tert-butyl guaranteed
acceptable selectivity and provided selective 5-HT7Rs ligands
(i.e., 17, 31). Compounds with an isopropyl moiety were classified
as dual 5-HT7/D2Rs ligands (i.e., 12, 28), while derivatives contain-
ing a phenyl ring were classified as 5-HT7/5-HT2A/D2 multireceptor
ligands (i.e., 20, 33).
It thus seems, that the N-alkylation of sulfonamide followed by
the selection of the type of substituent in the ortho position of the
(aryloxy)ethyl fragment may extend a strategy of designing either
selective 5-HT7Rs ligands or multireceptor ligands, the latter to
meet the criteria of polypharmacology for CNS disorder.
To further optimize the physicochemical parameters, the sub-
stituted phenylsulfonyl moiety was replaced with the less lipophi-
lic 1-N-methyl-pyrazol-4-yl. This fragment maintained high
affinity with no change in the selectivity profile (for selected
physicochemical parameters, see Table 1-S.I.).20
In vitro biotransformation assays of selected compounds 17, 20,
31 and 33 were performed using mouse liver microsomes (MLM),
potassium phosphate buffer, NADPH-regenerating system (NADP,
glucose-6-phosphate, glucose-6-phosphate dehydrogenase and
levallorphan as internal standard, according to the previously pub-
lished procedures.28,29 UPLC/MS analysis were performed to deter-
mine the quantity of starting material left in solution. The in vitro
half-time (t1/2) for test compounds was determined from the slope
of the linear regression of ln % parent compound remaining versus
incubation time. The calculated t1/2 was incorporated into the fol-
lowing equation to obtain intrinsic clearance (Clint) = (volume of
incubation [
l
l]/protein in the incubation [mg]) Â 0.693/t1/2
.
6. In vivo pharmacology
In the successive phase of our investigations, compounds 17, 20,
31 and 33 were tested in vivo in the forced swim test (FST) in mice,
to predict their antidepressant-like activity.30 The influence of the
effective doses recorded in the FST was studied in spontaneous
locomotor activity in mice in order to exclude the possibility of
competing behaviors such as general locomotor activity. Addition-
ally, pro-cognitive properties of the most interesting compounds
31 and 33 were further tested in the novel object recognition
(NOR) task in rats.
7. Results and discussion
We have recently reported on a class of 5-HT7R ligands, namely
arylsulfonamide derivatives of (aryloxy)ethyl piperidines which
behaved as antagonists at these sites and displayed antidepres-
sant-like, anxiolytic and pro-cognitive properties in rodent mod-
els.19,20 In the attempt to further extend the SAR studies towards
this group of 5-HT7R antagonists, we designed and synthetized a
series of N-alkylated sulfonamides, exploring the influence of this
modification on receptor profile and behavioral effects.
The concept of N-alkylation of the sulfonamide was confirmed
by results from molecular docking to 5-HT7R homology models,
indicating that the binding mode of N-methylsulfonamides 12,
16 and 19 was in line with SB-258719, involving a salt-bridge with
In the next step, selected compounds 17 and 31 with the high-
est 5-HT7R selectivity and with the most favorable CNS physico-
chemical properties (i.e. logP and logD values) were investigated
in vitro for their functional activity in cAMP assays, behaving as
potent antagonists at human 5-HT7Rs (Table 2). On the other hand,
multimodal ligands 20 and 33 were classified as antagonists of 5-
HT7, 5-HT2A and D2Rs and displayed high affinity for dopamine
Asp3.32, cation–
Arg7.36 and Thr2.64, additional hydrophobic interaction with
Cys146 and Leu147 from EL2,21 and the CH–
interactions
p interaction of arylsulfonamide moiety with
D3Rs (>91% at @ 1
and 33 showed no affinity for serotonin transporter (SERT) (<20%
l
M).31 Furthermore, compounds 17, 20, 31
p/p–p
Figure 3. Molecular docking results of the representative library members for the 5-HT7 receptors. (A) Comparison of the binding modes for compounds 12, 16, 19 and SB-
258719 (yellow, blue, violet, red, respectively); (B) comparison of the binding modes for compounds PZ-1315, 17 and 31 (cyan, pink, green, respectively).

V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
133
Table 1
The binding data of the synthesized compounds 10–20 and 27–33 for 5-HT7, 5-HT1A, 5-HT2A and D2Rs
R
O
N
O
S
O
Ar
N
R1
10-20; 27-33
Compd
Ar
R1
R
Kia (nM)
5-HT7
5-HT1A
5-HT2A
D2
PZ-1404b
3-F-phenyl
H
H
H
Isopropyl
t-Butyl
Phenyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
Phenyl
Phenyl
Phenyl
Isopropyl
Isopropyl
Isopropyl
t-Butyl
9
84
19
9
18
13
10
70
68
58
58
100
5
356
5555
321
140
190
178
576
179
264
230
382
121
1254
1928
1070
557
42
102
253
119
60
45
50
PZ-1315b
PZ-1345b
10
1-N-Methyl-pyrazol-4-yl
1-N-Methyl-pyrazol-4-yl
Phenyl
3-F-phenyl
4-F-phenyl
1-N-Methyl-pyrazol-4-yl
Phenyl
3-F-phenyl
4-F-phenyl
1-N-Methyl-pyrazol-4-yl
Phenyl
4-F-phenyl
1-N-Methyl-pyrazol-4-yl
3-F-phenyl
4-F-phenyl
1-N-Methyl-pyrazol-4-yl
4-F-phenyl
1-N-Methyl-pyrazol-4-yl
4-F-phenyl
1-N-Methyl-pyrazol-4-yl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
11
12
13
14
15
16
17
18
19
20
27
28
29
30
31
32
180
346
46
2346
4511
9054
9626
62
30
80
158
249
360
290
324
280
52
54
35
15
20
69
58
7
56
28
56
53
49
18
10
429
413
378
1328
1479
70
214
18
14,030
17,770
342
160
230
37
t-Butyl
Phenyl
Phenyl
33
159
30
16
a
Ki values are means of three independent binding experiments (SEM 6 21%).
Data taken from Ref. 20.
b
Table 2
20, 33) were further evaluated in the FST in mice. The obtained
results indicate that all of the tested compounds, 17 (0.312–
1.25 mg/kg), 20 (1.25–5 mg/kg), 31 (0.625–2.5 mg/kg) and 33
(0.312–1.25 mg/kg) as well as the selective 5-HT7R antagonist
SB-269970 in low doses, revealed antidepressant-like properties,
shortening the immobility time of mice (Fig. 4). The potency of
the antidepressant-like activity of the more stable compounds 31
and 33 administered at a dose of 1.25 mg/kg was similar to the
potency of SB-269970, which exerted its characteristic antidepres-
sant effect at one medium dose (10 mg/kg), but occurred at doses
eight times lower, similar to the effect of citalopram given at the
same dose of 1.25 mg/kg. Moreover, it is worth noting that all
investigated compounds as well as SB-269970 showed U-shaped
dose–response curves which may occur when ligands interact with
just one 5-HT receptor subtype.32–34 Active doses of the investi-
gated compounds, SB-269970 and citalopram had no influence
on spontaneous locomotor activity measured during the time
equal to the observation period in FST, that is, from 2 to 6 min,
(data not shown), thus the antidepressant effects of those
compounds appeared to be specific.
The antagonist activity of selected compounds for 5-HT7, 5-HT2A, and D2Rs
Compd
Kia (nM)
Kb (nM)
% inhb
% inhc
D2
5-HT7
5-HT2A
17
20
31
33
58
7
49
10
82
41
64
79
NTd
85
NTd
93
NTd
94
NTd
94
a
Ki values are means of three independent binding experiments (SEM 6 21%).
b
%
inhibition of control agonist response (serotonin 100 nM) at 10À6 M;
performed at Eurofins Cerep.
c
%
inhibition of control agonist response (dopamine 30 nM) at 10À6 M;
performed at Eurofins Cerep.
d
NT—not tested.
at @ 1 lM). Subsequently, above selected compounds were evalu-
ated for their affinity for ‘off-target’ receptors, displaying weak
affinity for adrenergic a1 (>300 nM) and histamine H1 receptors
(<64% at @ 1
5-HT2C receptors (<17% at @ 1
risk that the tested compounds would evoke undesirable cardio-
l
M), and did not bind to muscarinic M1 and serotonin
l
M).31 These results suggested a low
vascular or CNS side effects.
Impaired cognitive processes (including slowing, impairment of
executive functions and working memory) constitute an integral
part of clinical picture of depression, and cognitive decline is one
of the main causes of depressive patient’s poor functioning. Thus,
the improvement of cognitive deficits is considered as an impor-
tant therapeutic target in the treatment of depression. As many
pieces of evidences suggest that 5-HT7Rs antagonists modulate
cognitive processes involved in memory consolidation, and that
pro-cognitive properties may have some benefit in regard to the
treatment of depression,35 compounds 31 and 33 were tested in
a NOR task. Evaluated compounds were found to dose-dependently
(1 mg/kg, ip) ameliorate PCP-induced memory deficits in rats
(Fig. 5), with no influence on exploratory activity in NOR test. In
line with the recent finding by Nikiforuk et al.,5 these results
It was further found by an in vitro biotransformation mouse
microsomal assay that N-cyclopropylmethylated sulfonamides 31
and 33 showed acceptable metabolic stability with microsomal
clearance of 77.9 and 55.9 ll/mg/min, while their N-methylated
analogs (17, 20) as well as unsubstituted counterparts were more
susceptible to metabolism (see Table 2-S.I.). Interestingly, in the
case of the tert-butyl derivatives (17 and 31), the mono-hydroxy-
lated product at the (aryloxy)ethyl fragment represented the major
metabolite, while compounds containing the biphenyl moiety
(20 and 33) were mostly metabolized by N-dealkylation at the
sulfonamide moiety.
Finally, the antidepressant activities of selected 5-HT7 antago-
nists (i.e., 17, 31) and multimodal 5-HT7/5-HT2A/D2 ligands (i.e.,

134
V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
Figure 4. Effects of the compounds 17, 20, 31, 33, 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 = 7–12 animals per group. *p <0.05, **p <0.01, ***p <0.01 compared with the respective vehicle group, followed by the Bonferroni’s
comparison test after a significant analysis of variance.
9. Experimental
9.1. Molecular modeling
Sequences of the modeled receptor and selected templates were
aligned manually using Accelrys Discovery Studio,36 making sure
that the most conserved amino acids in each helix, and motifs
characteristic for class A GPCRs are on equivalent positions. Ranges
of helices were determined on the basis of crystal structures; loop
regions were modeled, but not refined.
For each template, 20 models were generated using MODELLER
9v8 software,37 and underwent validation by docking of active and
inactive compounds, and enrichment calculation. The active
Figure 5. Effects of the active compounds 31 and 33 in the novel-object recognition
(Ki < 100 nM) and inactive (Ki > 1000 nM) sets of compounds, were
retrieved from ChEMBL v11 database.38 The whole set of inactive
compounds (1594 for 5-HT7R) was used for docking, while actives
were hierarchically clustered using Moldprint2D, Tanimoto metric
and Complete Cluster Linking Method implemented in Canvas.39
From each cluster containing more than two members, the cen-
troid was selected. Finally, all centroids of active (118 for 5-
HT7R) and all sets of inactive compounds constituted an input for
docking. The 3-dimensional structures of the synthesized com-
pounds were prepared using LigPrep ver. 3.240 and the appropriate
ionization states at pH = 7.4 were assigned using Epik version 3.1.41
The Protein Preparation Wizard was used to assign the bond
orders, appropriate amino acid ionization states and check the
steric clashes. The receptor grids were generated (the OPLS_2005
force field) by centered the grid box of the size of 15 Å on the
Asp3.32. Automated docking was performed by using Glide version
6.6 at SP level with the flexible docking option turned on.42 The
docking procedure was validated by calculating the ROC curves,
based on the Glide Score values of docked compounds. Here, the
undocked actives and inactives were assigned as false negatives
and true negatives, respectively. The final model quality was deter-
mined by the Area Under ROC curve (AUROC). Three models per
template with the highest AUROC value were selected for further
studies. The final validation of the selected receptor models was
performed by docking (Glide SP mode) the all set of studied ana-
logs. The spatial constrain was imposed on creation of an ionic
interaction between the protonated amine group of the ligand
and Asp3.32 side chain. Only those models were kept for which
coherent, for the whole set of compounds, and closest compliance
test in rats. Data are presented as the mean standard error of the mean of N = 7–12
animals per group. **p <0.01 versus vehicle, ***p <0.001 versus vehicle, ##p <0.01
versus PCP, ###p <0.001 versus PCP.
may suggest that compounds 31 and 33, with 5-HT7 antagonistic
properties, may additionally represent a useful tools in the treat-
ment of cognitive deficits and some negative symptoms of
schizophrenia.
8. Conclusions
To summarize, in an attempt to establish the impact of N-alky-
lation of the sulfonamide on 5-HT7R affinity and selectivity over
the 5-HT1A, 5-HT2A and D2 sites, we designed and synthesized a
series of N-methyl and N-cyclopropylmethyl arylsulfonamide
derivatives of (aryloxy)ethyl piperidines. This structural modifica-
tion in combination with the kind of substituent in the (aryloxy)
ethyl fragment may be regarded as a strategy to design either
selective 5-HT7R agents (i.e., 17 and 31) or multimodal
5-HT/dopamine receptor ligands (i.e., 20 and 33). Compounds 31
and 33, produced an antidepressant-like effect in FST in mice
(MED = 1.25 mg/kg), in doses eight times as low as the active
comparator SB-269970, and additionally displayed distinct pro-
cognitive properties in NOR tasks in rats (MED = 1 mg/kg). Further
studies in the area of selective 5-HT7 receptor ligands or mixed
5-HT/dopamine compounds might be beneficial to confirm their
potential applications in the treatment of CNS disorders, especially
depression and cognitive decline.

V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
135
with common binding mode for monoaminergic receptor ligands
were observed.43
were filtered off and organic mixture was concentrated under
reduced pressure. The obtained crude product was purified using
silica gel with CH2Cl2/MeOH as an eluting system.
9.2. Chemistry
9.2.3. Boc-deprotection protocol
Organic transformations were carried out at ambient tempera-
ture, unless indicated otherwise. Organic solvents used in this
study (Sigma–Aldrich, Chempur) were of reagent grade and were
used without purification. All other commercially available
reagents were of the highest purity (from Sigma–Aldrich, Fluo-
rochem). All workup and purification procedures were carried
out with reagent-grade solvents under ambient atmosphere. Mass
spectra were recorded on a UPLC–MS/MS system consisted of a
Waters ACQUITYÒ UPLCÒ (Waters Corporation, Milford, MA, USA)
coupled to a Waters TQD mass spectrometer (electrospray ioniza-
tion mode ESI-tandem quadrupole). Chromatographic separations
were carried out using the Acquity UPLC BEH (bridged ethyl
TFA (4 ml) was added to the solution of Boc-protected alicyclic
amine in CH2Cl2 (2 ml) and stirred for 2 h at room temperature.
The excess of reagent and solvent were removed under reduced
pressure and left under vacuum overnight.
9.2.4. Reductive amination protocol
Amine (1.5 mmol) and cyclopropanecarbaldehyde (1.5 mmol)
were mixed in anhydrous CH2Cl2 (5 ml), for 5 h at room tempera-
ture and then treated with sodium cyanoborohydride (3 mmol).
The mixture was stirred until the reagents were consumed as
determined by HPLC analysis. subsequently, the reaction was
quenched by adding a saturated solution of NaHCO3, washed with
H2O (2Â), brine (1Â), dried over Na2SO4, filtered and concentrated.
The obtained crude product was purified using silica gel with
CH2Cl2/MeOH as an eluting system.
hybrid) C18 column; 2.1 Â 100 mm, and 1.7
equipped with Acquity UPLC BEH C18 VanGuard pre-column;
2.1 Â 5 mm, and 1.7 m particle size. The column was maintained
lm particle size,
l
at 40 °C, and eluted under gradient conditions from 95% to 0% of
eluent A over 10 min, at a flow rate of 0.3 mL minÀ1. Eluent A:
water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid
(0.1%, v/v). Chromatograms were made using Waters ek PDA detec-
tor. Spectra were analyzed in 200–700 nm range with 1.2 nm res-
olution and sampling rate 20 points/s. MS detection settings of
Waters TQD mass spectrometer were as follows: source tempera-
ture 150 °C, desolvation temperature 350 °C, desolvation gas flow
9.2.5. General procedure for preparation of final compounds
10–20 and 27–33
A
mixture of the appropriate (aryloxy)ethyl piperidine
(0.38 mmol) in CH2Cl2 (3 mL), and TEA (1.14 mmol) was cooled
down (ice bath), and arylsulfonyl chloride (0.46 mmol) was added
at 0 °C in one portion. The reaction mixture was stirred for 2–6 h
under cooling. Then, the solvent was evaporated and the sulfon-
amides were purified using silica gel column with CH2Cl2/MeOH
as an eluting system. Free bases were then converted into the
hydrochloride salts by treatment of their solution in anhydrous
ethanol with 1.25 M HCl in MeOH.
, , capillary potential
rate 600 L hÀ1 cone gas flow 100 L hÀ1
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 soft-
ware was MassLynx V 4.1 (Waters). The UPLC/MS purity of all
the final compounds was confirmed to be 95% or higher. 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), quin (quintet), dd (doublet of dou-
blets), td (triplet of doublets), qd (quartet of double), dt (double of
triplets), tt (triplet of triplets), ddd (doublet of doublet of doublets),
m (multiplet). Elemental analyses for C, H, N and S were carried out
using the elemental Vario EI III Elemental Analyser (Hanau,
Germany). All values are given as percentages, and were within
0.4% of the calculated values. Melting points (Mp) were
determined with a Büchi apparatus and are uncorrected.
9.2.6. Characterization data for final compounds
9.2.6.1. N-(1-{2-[(Propan-2-yl)phenoxy]ethyl}piperidin-4-yl)-N-
methyl-benzenesulfonamide (10).
Yellow oil, 90 mg follow-
ing chromatographic purification over silica gel with CH2Cl2/MeOH
(9:0.5); LC/MS purity 98%, tR = 5.39, C23H32N2O3S, MW 416.58,
Monoisotopic Mass 416.21, [M+H]+ 417.2. 1H NMR (300 MHz,
CDCl3) d 1.19 (d, J = 6.92 Hz, 6H), 1.40–1.46 (m, 2H), 1.68–1.72
(m, 2H), 2.07–2.11 (m, 1H), 2.80 (s, 3H), 3.33–3.39 (m, 1H), 3.59–
3.62 (m., 2H), 3.70 (d, J = 11.76 Hz, 2H), 4.12–4.24 (m, 1H), 4.34–
4.39 (t, J = 5.70 Hz, 2H), 6.93–7.00 (m, 2H), 7.12–7.19 (m, 1H),
7.24 (dd, J = 7.47, 1.47 Hz, 1H), 7.56–7.70 (m, 3H), 7.88 (d,
J = 6.89 Hz, 2H).
9.2.6.2. 3-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}piperi-
9.2.1. General procedure for the synthesis of (aryloxyl)ethyl
bromides (4–6)
din-4-yl)-N-methyl-benzenesulfonamide (11).
Yellow oil,
80 mg following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.5); LC/MS purity 97%, tR = 5.60, C23H31FN2O3S,
MW 434.57, Monoisotopic Mass 434.2, [M+H]+ 435.2. 1H NMR
(300 MHz, CDCl3) d 1.20 (d, J = 6.92 Hz, 6H), 1.41–1.49 (m, 2H),
1.68–1.73 (m, 2H), 2.02–2.18 (m, 2H), 2.78 (s, 3H), 2.81 (t,
J = 5.79 Hz, 2H), 2.98–3.06 (m, 2H), 3.29 (quin, J = 6.92 Hz, 1H),
3.76–3.85 (m, 1H), 4.06 (t, J = 5.72 Hz, 2H), 6.80 (d, J = 8.14 Hz,
1H), 6.89–6.95 (m, 1H), 7.13 (td, J = 7.76, 1.70 Hz, 1H), 7.19–7.24
(m, 1H), 7.26–7.30 (m, 1H), 7.45–7.54 (m, 2H), 7.59–7.63 (m, 1H).
A solution of 1,2-dibromoethane (0.08 mol) in 40 ml of acetone
was added dropwise into the mixture of respective phenol
(0.02 mol) and K2CO3 (0.04 mol) in 30 ml of acetone. Subsequently,
a catalytic amount of KI (0.3 mmol) was added and the resulting
mixture was stirred at 60 °C for 24–72 h. After the completion of
the reaction the inorganic residues were filtrated off and organic
mixture was concentrated under vacuum. The obtained crude pro-
duct was purified on silica gel with AcOEt/hexane as eluting
system.
9.2.6.3. 4-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}piperi-
9.2.2. General procedure for the alkylation of 4-(N-methyl-Boc-
amino)piperidine (7–9) and 4-(N-Boc-amino)piperidine (21–23)
Commercial 4-(N-methyl-Boc-amino)piperidine (0.015 mol) or
4-(Boc-N-amino)piperidine (0.015 mol) was dissolved in acetone
(15 ml). Then K2CO3 (0.045 mol) and catalytic amount of KI were
added followed by dropwise addition of (aryloxy)ethyl bromide
(0.018 mol). The reaction was refluxed for 48 h. Inorganic residues
din-4-yl}-N-methyl-benzenesulfonamide (12).
Yellow oil,
100 mg following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7); LC/MS purity 96%, tR = 5.61, C23H31FN2O3S,
MW 434.57, Monoisotopic Mass 434.20, [M+H]+ 435.3 1H NMR
(300 MHz, CDCl3) d 1.20 (d, J = 6.92 Hz, 6H), 1.40–1.49 (m, 2H),
1.71 (qd, J = 12.27, 3.86 Hz, 2H), 2.22 (td, J = 11.93, 2.00 Hz, 2H),
2.76 (s, 3H), 2.82 (t, J = 5.71 Hz, 2H), 2.98–3.07 (m, 2H), 3.29 (quin,

136
V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
J = 6.92 Hz, 1H), 3.80 (tt, J = 12.04, 4.10 Hz, 1H), 4.06 (t, J = 5.71 Hz,
2H), 6.80 (dd, J = 8.16, 1.01 Hz, 1H), 6.89–6.96 (m, 1H), 7.10–7.16
(m, 2H), 7.16–7.22 (m, 2H), 7.79–7.86 (m, 2H). 13C NMR (75 MHz,
CDCl3) d 22.69, 26.73, 28.81, 29.34, 29.68, 53.42, 55.20, 57.09,
66.39, 111.26, 114.40, 119.70, 120.80, 122.58, 122.63, 126.53,
130.85, 137.05, 142.20, 142.28, 155.78, 160.79, 164.13.
MW 434.60, Monoisotopic Mass 434.24, [M+H]+ 435.5. 1H NMR
(300 MHz, CDCl3) d 1.37 (s, 9H), 1.50–1.58 (m, 2H), 1.70 (qd,
J = 12.17, 3.89 Hz, 2H) 2.19 (td, J = 11.80, 2.23 Hz, 2H), 2.73 (s,
3H), 2.82 (t, J = 6.07 Hz, 2H), 2.97–3.04 (m, 2H), 3.76–3.88 (m,
1H), 3.93 (s, 3H), 4.06 (t, J = 6.06 Hz, 1H), 6.83 (dd, J = 8.21,
1.03 Hz, 1H), 6.87–6.91 (m, 1H), 7.15 (td, J = 7.71, 1.70 Hz, 1H),
7.27 (dd, J = 7.92, 1.44 Hz, 1H), 7.70 (s, 1H), 7.74 (s, 1H). 13C NMR
(75 MHz, CDCl3) d 28.58, 29.28, 29.68, 29.81, 34.81, 39.55, 53.48,
55.09, 57.13, 65.94, 112.03, 120.39, 122.24, 126.64, 126.97,
131.35, 138.10, 138.15, 157.49. Anal. calcd for C22H34N4O3SÁ2HCl:
C: 52.06, H: 7.15, N: 11.04, S: 6.32; Found C: 51.95, H: 6.95, N:
10.82, S: 6.11. Mp for C22H34N4O3SÁ2HCl: 181.7–182.3 °C.
9.2.6.4. 1-Methyl-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}piperi-
din-4-yl}-N-methyl-1H-pyrazole-4-sulfonamide (13). Yellow
oil, 90 mg following chromatographic purification over silica
gel with CH2Cl2/MeOH (9:0.7); LC/MS purity 96%, tR = 4.47,
C
21H32N4O3S, MW 420.57, Monoisotopic Mass 420.22, [M+H]+
421.3 1H NMR (300 MHz, CDCl3) d 1.19 (d, J = 6.92 Hz, 6H), 1.49–
1.58 (m, 2H), 1.71 (qd, J = 12.21, 3.88 Hz, 2H), 2.22 (td, J = 11.89,
2.24 Hz, 2H), 2.73 (s, 3H), 2.81 (t, J = 5.72 Hz, 2H), 2.99–3.07 (m,
2H), 3.29 (quin, J = 6.92 Hz, 1H), 3.81 (tt, J = 11.97, 4.22 Hz, 1H),
3.92 (s, 3H), 4.06 (t, J = 5.72 Hz, 2H), 6.80 (dd, J = 8.14, 0.99 Hz,
1H), 6.88–6.94 (m, 1H), 7.12 (td, J = 7.76, 1.73 Hz, 1H), 7.20 (dd,
J = 7.53, 1.67 Hz, 1H), 7.70 (d, J = 0.61 Hz, 1H), 7.74 (s, 1H). 13C
NMR (75 MHz, CDCl3) d 22.70, 26.73, 28.60, 29.27, 39.55, 53.47,
55.03, 57.13, 66.40, 111.26, 120.78, 122.21, 126.06, 126.53,
131.37, 137.03, 138.15, 155.79.
9.2.6.9. N-{1-[2-(Biphenyl-2-yloxy)ethyl]piperidin-4-yl}-N-me-
thyl-benzenesulfonamide (18).
Yellow oil, 80 mg following
chromatographic purification over silica gel with CH2Cl2/MeOH
(9:0.7); LC/MS purity 96%, tR = 5.33 C26H30N2O3S, MW 450.59,
Monoisotopic Mass 450.20, [M+H]+ 451.2. 1H NMR (300 MHz,
CDCl3) d 1.37–1.42 (m, 2H), 1.79–1.82 (m, 2H), 2.73 (s, 3H), 2.93
(t, J = 12.01 Hz, 2H), 3.33–3–35 (m, 2H), 3.40–3.45 (m, 2H), 3.92–
4.03 (m, 1H), 4.30–4.35 (m, 2H), 7.10 (d, J = 7.66 Hz, 2H), 7.23–
7.29 (m, 2H), 7.32–7.42 (m, 5H), 7.61–7.68 (m, 2H), 7.69–7.76
(m, 1H), 7.83–7.88 (m, 2H).
9.2.6.5. N-(1-{[2-(t-Butyl-2-yl)phenoxy]ethyl}piperidin-4-yl)-N-
methyl-benzenesulfonamide (14). Yellow oil, 70 mg following
chromatographic purification over silica gel with CH2Cl2/MeOH
(9:0.7); LC/MS purity 97%, tR = 5.76 C24H34N2O3S, MW 430.60,
Monoisotopic Mass 430.23, [M+H]+ 431.3. 1H NMR (300 MHz,
CDCl3) d 1.38 (s, 9H), 1.68–1.73 (m, 2H), 2.08–2.11 (m, 2H), 2.80
(s, 3H), 2.96–3.03 (m, 2H), 3.62 (t, J = 4.89 Hz, 2H), 3.71–3.75 (m,
2H), 4.19 (m, 1H), 4.41 (t, J = 5.32 Hz, 2H), 6.93 (td, J = 7.53,
1.19 Hz, 1H), 7.02 (dd, J = 8.19, 1.04 Hz, 1H), 7.15–7.21 (m, 1H),
7.30 (dd, J = 7.79, 1.63 Hz, 1H), 7.57–7.67 (m, 3H), 7.88 (dd,
J = 8.22, 1.36 Hz, 2H).
9.2.6.10. 4-Fluoro-N-{1-[2-(biphenyl-2-yloxy)ethyl]piperidin-4-
yl}-N-methyl-benzenesulfonamide (19).
Yellow oil, 60 mg
following chromatographic purification over silica gel with CH2Cl2/
MeOH (9:0.7); LC/MS purity 97%, tR = 5.36 C26H29FN2O3S, MW
468.58, Monoisotopic Mass 468.19, [M+H]+ 469.5. 1H NMR
(300 MHz, CDCl3) d 1.33–1.44 (m, 2H), 1.74–1.91 (m, 2H), 2.73 (s,
3H), 2.94 (t, J = 12.55 Hz, 2H), 3.33–3.37 (m, 2H), 3.40–3.46 (m,
2H), 3.96 (tt, J = 12.08, 3.95 Hz, 1H), 4.29–4.38 (m, 2H), 7.10 (d,
J = 7.47 Hz, 2H), 7.24–7.31 (m, 2H), 7.32–7.42 (m, 7H), 7.87–7.95
(m, 2H).
9.2.6.6. 3-Fluoro-N-(1-{[2-(t-butyl-2-yl)phenoxy]ethyl}piperi-
9.2.6.11. 1-Methyl-N-{1-[2-(biphenyl-2-yloxy)ethyl]piperidin-4-
din-4-yl)-N-methyl-benzenesulfonamide (15).
Yellow oil,
yl}-N-methyl-1H-pyrazole-4-sulfonamide (20).
Yellow oil,
60 mg following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7); LC/MS purity 98%, tR = 5.84 C24H33FN2O3S,
MW 448.59, Monoisotopic Mass 448.22, [M+H]+ 449.5. 1H NMR
(300 MHz, CDCl3) d 1.37 (s, 9H), 1.41–1.49 (m, 2H), 1.70 (qd,
J = 12.23, 3.85 Hz, 2H), 2.19 (td, J = 11.89, 2.15 Hz, 2H), 2.78 (s,
3H), 2.82 (t, J = 6.06 Hz, 2H), 2.96–3.03 (m, 2H), 3.75–3.87 (m,
1H), 4.06 (t, J = 6.07 Hz, 2H), 6.83 (dd, J = 8.14, 1.12 Hz, 1H), 6.88
(td, J = 7.53, 1.25 Hz, 1H), 7.12–7.18 (m, 1H), 7.26–7.30 (m, 1H),
7.45–7.49 (m, 1H), 7.49–7.54 (m, 1H), 7.59–7.63 (m, 1H).
100 mg following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7); LC/MS purity 100%, tR = 4.65, C24H30N4O3S,
MW 454.58, Monoisotopic Mass 454.20, [M+H]+ 455.5. 1H NMR
(300 MHz, CDCl3) d 1.40–1.48 (m, 2H), 1.62–1.65 (m, 2H), 2.05
(td, J = 11.90, 2.20 Hz, 2H), 2.66–2.70 (m, 2H), 2.71 (s, 3H), 2.83–
2.90 (m, 2H), 3.68–3.80 (m, 1H), 3.94 (s, 3H), 4.04 (t, J = 5.63 Hz,
2H), 6.93–6.97 (m, 1H), 7.03 (td, J = 7.45, 1.06 Hz, 1H), 7.28–7.33
(m, 3H), 7.34–7.40 (m, 2H), 7.49–7.51 (m, 1H), 7.51–7.53 (m,
1H), 7.69 (d, J = 0.54 Hz, 1H), 7.73 (s, 1H). 13C NMR (75 MHz, CDCl3)
d 28.59, 29.19, 39.57, 53.35, 55.00, 56.78, 66.94, 112.70, 121.18,
122.22, 126.83, 127.79, 128.58, 129.61, 130.86, 131.13, 131.35,
138.17, 138.48, 155.63. Anal. calcd for C24H30N4O3SÁHCl: C: 58.70,
H: 6.36, N: 11.41, S: 6.53; Found C: 58.96, H: 6.49, N: 11.42, S:
6.88. Mp for C24H30N4O3SÁHCl: 189.5–189.9 °C.
9.2.6.7. 4-Fluoro-N-(1-{[2-(tert-butyl-2-yl)phenoxy]ethyl}piper-
idin-4-yl)-N-methyl-benzenesulfonamide (16).
Yellow oil,
60 mg following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7); LC/MS purity 99%, tR = 5.54 C24H33FN2O3S,
MW 448.59, Monoisotopic Mass 448.22, [M+H]+ 449.3. 1H NMR
(300 MHz, CDCl3) d 1.37 (s, 9H), 1.44 (dd, J = 11.64, 2.18 Hz, 2H),
1.68 (qd, J = 12.25, 3.91 Hz, 2H), 2.17 (td, J = 11.88, 2.15 Hz, 2H),
2.75 (s, 3H), 2.82 (t, J = 6.09 Hz, 2H), 2.95–3.03 (m, 2H), 3.80 (tt,
J = 12.03, 4.14 Hz, 1H), 4.06 (t, J = 6.09 Hz, 2H), 6.83 (dd, J = 8.16,
1.11 Hz, 1H), 6.88 (td, J = 7.54, 1.23 Hz, 1H), 7.14–7.22 (m, 3H),
7.26–7.29 (m, 1H), 7.79–7.86 (m, 2H). 13C NMR (75 MHz, CDCl3)
d 28.72, 29.33, 29.81, 34.80, 53.45, 55.11, 57.11, 65.91, 112.05,
116.14, 116.44, 120.42, 126.64, 126.96, 129.46, 129.51, 129.58,
136.30, 136.34, 138.14, 157.50, 163.19, 166.56.
9.2.6.12. 4-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}piperi-
din-4-yl)-N-cyclopropylmethyl-benzenesulfonamide
(27).
Yellow oil, 80 mg following chromatographic purifica-
tion over silica gel with CH2Cl2/MeOH (9:0.5); LC/MS purity 98%,
tR = 6.24, C26H35FN2O3S, MW 474.63, Monoisotopic Mass 474.24,
[M+H]+ 475.2. 1H NMR (300 MHz, CDCl3) d 0.26–0.33 (m, 2H),
0.49–0.57 (m, 2H), 1.20 (d, J = 6.92 Hz, 6H), 1.55–1.64 (m, 2H),
1.75–1.82 (m, 2H), 2.13–2.23 (m, 2H), 2.80 (t, J = 5.71 Hz, 2H),
2.87–2.95 (m, 1H), 3.01–3.03 (m, 2H), 3.12 (d, J = 6.76 Hz, 2H),
3.29 (quin, J = 6.91 Hz, 1H), 3.61–3.64 (m, 1H), 4.06 (t, J = 5.69 Hz,
2H), 6.80 (dd, J = 8.11, 0.80 Hz, 1H), 6.92 (td, J = 7.43, 0.91 Hz,
1H), 7.10–7.16 (m, 1H), 7.18–7.25 (m, 2H), 7.47 (td, J = 8.03,
5.32 Hz, 1H), 7.56 (dt, J = 8.38, 2.04 Hz, 1H), 7.65 (dt, J = 7.84,
1.21 Hz, 1H). 13C NMR (75 MHz, CDCl3) d 5.10, 8.34, 12.48, 22.68,
9.2.6.8. 1-Methyl-N-(1-{[2-(t-butyl-2-yl)phenoxy]ethyl}piperi-
din-4-yl)-N-methyl-1H-pyrazole-sulfonamide (17). Yellow oil,
80 mg following chromatographic purification over silica gel with
CH2Cl2/MeOH (9:0.7); LC/MS purity 100%, tR = 4.81, C22H34N4O3S,

V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
137
26.74, 30.92, 48.40, 51.63, 53.75, 56.09, 57.07, 66.40, 111.24,
120.77, 122.57, 122.60, 122.62, 126.07, 126.52, 130.65, 130.75,
143.81, 143.90, 155.80.
(300 MHz, CDCl3) d 0.25–0.31 (m, 2H), 0.51–0.57 (m, 2H), 1.44–
1.52 (m, 2H), 1.92–2.06 (m, 2H), 2.25 (t, J = 11.70 Hz, 2H), 2.94–
3.02 (m, 2H), 3.05 (d, J = 6.80 Hz, 2H), 3.10–3.25 (m, 2H), 3.60 (tt,
J = 12.01, 3.85 Hz, 1H), 3.93–4.05 (m, 1H), 4.19 (t, J = 4.89 Hz, 2H),
6.92 (dd, J = 8.61, 0.95 Hz, 1H), 6.95–7.02 (m, 1H), 7.05 (td,
J = 7.49, 1.03 Hz, 1H), 7.14–7.20 (m, 2H), 7.26–7.33 (m, 4H), 7.34–
7.38 (m, 2H), 7.41–7.46 (m, 2H).
9.2.6.13. 4-Fluoro-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}piperidin-
4-yl}-N-cyclopropylmethyl-benzenesulfonamide (28). Yellow
oil, 80 mg following chromatographic purification over silica gel
with CH2Cl2/MeOH (9:0.5); LC/MS purity 97%, tR = 6.07,
C
26H35FN2O3S, MW 474.63, Monoisotopic Mass 474.24, [M+H]+
9.2.6.18. 1-Methyl-N-{1-[2-(biphenyl-2-yloxy)ethyl]piperidin-4-yl}-
475.3. 1H NMR (300 MHz, CDCl3) d 0.26–0.33 (m, 2H), 0.49–0.57
(m, 2H), 1.20 (d, J = 6.90 Hz, 6H), 1.54–1.62 (m, 2H), 1.79 (qd,
J = 12.11, 3.51 Hz, 2H), 2.13–2.23 (m, 2H), 2.81 (t, J = 5.67 Hz, 2H),
2.99–3.06 (m, 3H), 3.10 (d, J = 6.76 Hz, 2H), 3.29 (quin,
J = 6.92 Hz, 1H), 3.61 (tt, J = 12.00, 3.98 Hz, 1H), 4.06 (t,
J = 5.67 Hz, 2H), 6.80 (dd, J = 8.14, 0.83 Hz, 1H), 6.89–6.95 (m,
1H), 7.10–7.16 (m, 2H), 7.17–7.23 (m, 2H), 7.83–7.89 (m, 2H).
N-cyclopropylmethyl-1H-pyrazole-4-sulfonamide (33).
oil, 70 mg following chromatographic purification over silica gel
Yellow
with CH2Cl2/MeOH (9:0.7); LC/MS purity 100%, tR = 5.37,
C
27H34N4O3S, MW 494.65, Monoisotopic Mass 494.24, [M+H]+
495.4 1H NMR (300 MHz, CDCl3) d 0.25–0.31 (m, 2H), 0.47–0.55
(m, 2H), 1.52–1.58 (m, 2H), 2.32–2.59 (m, 4H), 3.00 (d,
J = 6.73 Hz, 2H), 3.22–3.37 (m, 4H), 3.66–3.76 (m, 2H), 3.92 (s,
3H), 4.37–4.41 (m, 2H), 6.93 (d, J = 8.17 Hz, 1H), 7.02 –7.09 (m,
1H), 7.22–7.38 (m, 7H), 7.65 (s, 1H), 7.78 (s, 1H). 13C NMR
(75 MHz, CDCl3) d 5.16, 12.14, 27.56, 39.52, 48.23, 52.22, 52.73,
52.76, 52.79, 55.72, 63.25, 112.65, 122.28, 123.23, 127.20, 128.00,
128.99, 129.55, 130.81, 131.41, 131.59, 137.84, 138.35, 154.12.
Anal. calcd for C27H34N4O3SÁ2HCl: C, 57.14; H, 6.39; N, 9.87; S,
5.65; Found: C, 56.94; H, 6.69’ N, 10.12; S, 5.36. Mp for
9.2.6.14. 1-Methyl-N-(1-{2-[(propan-2-yl)phenoxy]ethyl}pipe-
ridin-4-yl)-N-cyclopropylmethyl-1H-pyrazole-4-sulfonamide
(29). Yellow oil, 70 mg following chromatographic purification
over silica gel with CH2Cl2/MeOH (9:0.5); LC/MS purity 98%,
tR = 5.36, C24H36N4O3S, MW 460.63, Monoisotopic Mass 460.25,
[M+H]+ 461.4. 1H NMR (300 MHz, CDCl3) d 0.26–0.33 (m, 2H),
0.49–0.57 (m, 2H), 1.20 (d, J = 6.92 Hz, 6H), 1.62–1.71 (m, 2H),
1.72–1.87 (m, 2H), 2.20 (td, J = 11.79, 2.23 Hz, 2H), 2.81 (t,
J = 5.72 Hz, 2H), 3.03 (d, J = 1.83 Hz, 2H), 3.07–3.11 (m, 3H), 3.29
(quin, J = 6.93 Hz, 1H), 3.65 (tt, J = 11.89, 4.17 Hz, 1H), 3.93 (s,
3H), 4.07 (t, J = 5.71 Hz, 2H), 6.80 (dd, J = 8.16, 1.01 Hz, 1H), 6.89–
6.95 (m, 1H), 7.10–7.16 (m, 1H), 7.21 (dd, J = 7.53, 1.70 Hz, 1H),
7.71–7.76 (m, 2H).
C27H34N4O3SÁ2HCl: 198.3–198.8 °C.
9.3. In vitro pharmacology
9.3.1. Radioligand binding assay protocol
Radioligand binding assays were carried out using [3H]-5-CT
(39.2 Ci/mmol), [3H]-8-OH-DPAT (187 Ci/mmol), [3H]-Ketanserin
(66.9 Ci/mmol) and [3H]-Raclopride (74.4 Ci/mmol) for 5-HT7B, 5-
HT1A, 5-HT2A and D2 receptors, respectively. Cell pellets were
thawed and homogenized in 20 volumes of assay buffer using an
Ultra Turrax tissue homogenizer and centrifuged twice at
35 000g for 20 min at 4 °C, with incubation for 15 min at 37 °C in
between. The composition of the assay buffers was as follows:
for 5-HT1AR: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl2,
10 mM pargyline and 0.1% ascorbate; for 5-HT6R: 50 mM Tris–HCl,
0.5 mM EDTA and 4 mM MgCl2, for 5-HT7bR: 50 mM Tris–HCl,
4 mM MgCl2, 10 mM pargyline and 0.1% ascorbate; for dopamine
D2LR: 50 mM Tris–HCl, 1 mM EDTA, 4 mM MgCl2, 120 mM NaCl,
5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate. All assays were incu-
9.2.6.15. 4-Fluoro-N-{1-[2-(2-(t-butyl)phenoxy)ethyl]piperidin-
4-yl}-N-cyclopropylmethyl-benzenesulfonamide (30). Yellow
oil, 110 mg following chromatographic purification over silica gel
with CH2Cl2/MeOH (9:0.5); LC/MS purity 97%, tR = 6.52,
C
27H37FN2O3S, MW 488.66, Monoisotopic Mass 488.25, [M+H]+
489.3 1H NMR (300 MHz, CDCl3) d 0.24–0.31 (m, 2H), 0.49–0.56
(m, 2H), 1.37 (s, 9H), 1.55–1.64 (m, 2H), 1.74–1.89 (m, 2H), 2.17–
2.19 (m, 3H), 2.84 (t, J = 5.90 Hz, 2H), 3.03 (d, J = 11.57 Hz, 2H),
3.10 (d, J = 6.76 Hz, 2H), 3.61–3.64 (m, 1H), 4.08 (t, J = 5.95 Hz,
2H), 6.87 (ddd, J = 18.62, 7.85, 1.15 Hz, 2H), 7.12–7.20 (m, 3H),
7.28 (dd, J = 8.14, 2.08 Hz, 1H), 7.82–7.90 (m, 2H).
bated in a total volume of 200 ll in 96-well microtitre plates for
9.2.6.16. 1-Methyl-N-{1-[2-(2-(t-butyl)phenoxy)ethyl]piper-
idin-4-yl}-N-cyclopropylmethyl-1H-pyrazole-4-sulfonamide
(31). Yellow oil, 110 mg following chromatographic purification
over silica gel with CH2Cl2/MeOH (9:0.5); LC/MS purity 100%,
tR = 6.24, C25H38N4O3S, MW 474.66, Monoisotopic Mass 474.27,
[M+H]+ 475.2 1H NMR (300 MHz, CDCl3) d 0.25–0.31 (m, 2H),
0.51–0.57 (m, 2H), 1.38 (s, 9H), 1.56–1.63 (m, 2H), 1.74–1.89 (m,
2H), 2.18–2.21 (m, 3H), 2.84 (t, J = 5.92 Hz, 2H), 3.03 (d,
J = 11.55 Hz, 2H), 3.10 (d, J = 6.76 Hz, 2H), 3.61–3.64 (m, 1H), 3.91
(s, 3H), 4.12 (t, J = 5.95 Hz, 2H), 6.88 (qd, J = 7.73, 0.98 Hz, 2H),
7.14 (td, J = 7.72, 1.68 Hz, 1H), 7.24–7.29 (m, 1H), 7.66–7.70 (m,
1H), 7.78 (s, 1H). 13C NMR (75 MHz, CDCl3) d 5.12, 12.47, 29.69,
29.80, 30.01, 30.77, 34.81, 48.31, 53.79, 55.84, 57.08, 65.81,
112.03, 115.99, 116.28, 120.44, 126.66, 126.99, 129.42, 129.54,
138.09, 157.46, 163.05, 166.41. Anal. calcd for C25H38N4O3SÁHCl:
C, 58.75; H, 7.69; N, 10.96; S, 6.27; Found: C, 58.94; H, 7.79; N,
10.72; S, 6.16. Mp for C25H38N4O3SÁHCl: 193.7–194.5 °C.
1 h at 37 °C, 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 Micro-
beta plate reader. Non-specific binding is defined with10
5-HT in 5-HT1AR and 5-HT7R binding experiments, whereas10
of methiothepine or 1 M of (+)butaclamol were used in 5-HT6R
lM of
lM
l
and D2L assays, respectively. Each compound was tested in triplicate
at 7–8 concentrations (10–11 to 10–4 M). 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.
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
consisted of 450 of membrane suspension, 50 of
[3H]-Prazosin (0.6 nM) solution and 50
seven or eight concentrations (1 nM to 100
compounds. For measuring unspecific binding, phentolamine at a
ll)
l
l
ll
a
ll of buffer containing
9.2.6.17. 4-Fluoro-N-{1-[2-(biphenyl-2-yloxy)ethyl]piperidin-4-
lM) of the investigated
yl}-N-cyclopropylmethyl-benzenesulfonamide (32).
Yellow
oil, 100 mg following chromatographic purification over silica gel
with CH2Cl2/MeOH (9:0.7); LC/MS purity 96%, tR = 6.28, C29H33FN2
O3S, MW 508.65, Monoisotopic Mass 508.22, [M+H]+ 509.4 1H NMR
final concentration of 10 lM was present. SERT binding experi-
ments were conducted using tissue (rat cortex) and [3H]-citalo-
pram as radioligand according to previously published

138
V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
methods.27 Percent of specific binding was calculated in screening
between 09.00 and 14.00 h. Each experimental group consisted
of: 7–12 animals/dose. The animals were used only once. The
experiments were performed by an observer unaware of the
treatment administered. The following drugs were used: (2R)-1-
[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]
pyrrolidine (hydrochloride, SB-269970; synthesized by the team of
Ass. Prof. P. Zajdel, Department of Medicinal Chemistry, Jagiel-
lonian University Medical College, Krakow, Poland), citalopram
(hydrochloride Adamed Pharmaceuticals, Pienkow, Poland), Citalo-
pram and tested compounds were dissolved in distilled water, SB-
269970 was suspended in a 1% aqueous solution of Tween 80
(Sigma–Aldrich, UK) immediately before administration. Com-
pounds 17, 20, 31 and 33 was administered intraperitoneally (ip)
60 min, before the test, citalopram and SB-269970 was injected
ip 30 min before testing. Swiss albino mice were individually
placed in a glass cylinder (25 cm high; 10 cm in diameter) contain-
ing 10 cm of water maintained at 23–25 °C, and were left there for
6 min. A mouse was regarded as immobile when it remained float-
ing 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.
test. Each compound was tested at final concentration of 10À6
and 10À7 M run in duplicate.
M
9.3.2. Functional cAMP assay protocol
The functional properties of compounds on 5-HT7R were evalu-
ated using their ability to inhibit cAMP production induced by 5-CT
(10 nM)—a 5-HT7R agonist, in HEK293 cells overexpressing the
human 5-HT7bR. Each compound was tested in triplicate at 8 con-
centrations (10À11 to 10À4 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 equation20 specific for the analysis of functional
inhibition curves: Kb = IC50/(1 + A/EC50) where A is an agonist con-
centration, IC50 is the concentration of antagonist producing a 50%
reduction in the response to agonist, and EC50 is the agonist con-
centration which causes a half of the maximal response. Addition-
ally the antagonist properties of the selected compounds were
evaluated in functional cAMP cellular assays, performed at Eurofins
Cerep using HEK293 cells, which stably expressed the human 5-
HT2ARs. Experimental conditions for these assays are described
online at www.cerep.fr.
9.5.1.1. Statistics.
All the data are presented as the mean of
9.4. In vitro metabolic stability
SEM. The statistical significance of the results was evaluated by a
one-way ANOVA, followed by Bonferroni’s Comparison Test.
Test compounds incubation (20 lM) with mouse liver micro-
somes (MLM 0.2 mg/ml) was performed in 100 mM potassium
9.5.2. Spontaneous locomotor activity protocol
phosphate buffer (PB), pH 7.4, at 37 °C for 15 min in a final reaction
mixture volume of 150 ll. The reaction was initiated by the addi-
The locomotor activity was recorded with an Opto M3 multi-
channel activity monitor (MultiDevice Software v.1.3, Columbus
Instruments). The CD-1 mice were individually placed in plastic
cages (22 Â 12 Â 13 cm) for 30 min habituation period, and then
ambulation were counted during 1 h with data recording every
5 min. The Swiss albino mice were individually placed in plastic
cages (22 Â 12 Â 13 cm), and then the crossings of each channel
(ambulation) were counted from 2 to 6 min, that is, the time equal
to the observation period in the forced swim test. The cages were
cleaned up with 70% ethanol after each mouse.
tion of an NADPH-regenerating system (NADP, glucose-6-phos-
phate, glucose-6-phosphate dehydrogenase in 100 mM PB, pH
7.4) to the incubation mixture. The blank probe without the
NADPH—regenerating system was performed in parallel. Subse-
quently, the samples were incubated at 37 °C for 5, 15, 30 and
60 min (PZ-1315, PZ-1345, 17, 20 and 31) or additionally for 90
and 120 min (33) with gentle shaking. Next, an internal standard
(levallorphan, 20 lM) was added. The reaction was terminated at
different time points with perchloric acid (69–72%, by volume).
Thereafter, all samples were centrifuged and the supernatant was
analyzed using UPLC/MS in order to determine the quantity of
starting material left in solution. All samples were prepared in
duplicate.
The in vitro half-time (t1/2) for test compounds was determined
from the slope of the linear regression of ln % parent compound
remaining versus incubation time. The calculated t1/2 was incorpo-
rated into the following equation to obtain intrinsic clearance
9.5.3. Novel object recognition protocol
The experiments were conducted in accordance with the NIH
Guide for the Care and Use of Laboratory Animals and were
approved by the Ethics Committee for Animal Experiments, Insti-
tute of Pharmacology. Male Sprague–Dawley rats (Charles River,
Germany) weighing ꢀ250 g at the arrival were housed in the stan-
dard laboratory cages, under standard colony A/C controlled condi-
tions: room temperature 21 2 °C, humidity (40–50%), 12-h
light/dark cycle (lights on: 06:00) with ad libitum access to food
and water. Rats were allowed to acclimatize for at least 7 days
before the start of the experimental procedure. During this week
animals were handled for at least 3 times. Behavioral testing was
carried out during the light phase of the light/dark cycle. At least
1 h before the start of the experiment, rats were transferred to
the experimental room for acclimation.
(Clint) = (volume of incubation
[ll]/protein in the incubation
[mg]) Â 0.693/t1/2. MLM, glucose-6-phosphate, NADP, glucose-6-
phosphate dehydrogenase and levallorphan were obtained from
´
Sigma Aldrich (Poznan, Poland).
9.5. In vivo pharmacology
9.5.1. Forced swim test protocol
Rats were tested in a dimly lit (25 lÂ) ‘open field’ apparatus
made of a dull gray plastic (66 Â 56 Â 30 cm). After each measure-
ment, the floor was cleaned and dried. The procedure lasting for
2 days consisted of the habituation to the test arena (without any
objects) for 5 min. The test session comprising of two trials sepa-
rated by an inter-trial interval (ITI) of 1 h was carried out on the
next day. During the first trial (familiarization, T1) two identical
objects (A1 and A2) were presented in the opposite corners of
the open field, approximately 10 cm from the walls. During the
second trial (recognition, T2) one of the A objects was replaced
by a novel object B, so that the animals were presented with the
A = familiar and B = novel objects. Both trials lasted for 3 min and
the animals were returned to their home cages after T1. The objects
The experiments were performed on male Swiss albino mice
(22–26 g) purchased from a licensed breeder Staniszewska (Ilkow-
ice, Poland). Mice were kept in groups of ten to Makrolon type 3
cages (dimensions 26.5 Â 15 Â 42 cm). The animals were kept in
an environmentally controlled rooms (ambient temperature
22 2 °C; relative humidity 50–60%; 12:12 light/dark cycle, lights
on at 8:00). They were allowed to acclimatize with the environ-
ment for one week before commencement of the experiments.
Standard laboratory food (Ssniff M-Z) and filtered water were
freely available. All the experimental procedures were approved
by the I Local Ethics Commission at the Jagiellonian University in
Krakow. All the experiments were conducted in the light phase

V. Canale et al. / Bioorg. Med. Chem. 24 (2016) 130–139
10. Ivachtchenko, A. V.; Ivanenkov, Y. A. Expert Opin. Ther. Pat. 2012, 22, 917.
139
used were the glass beakers filled with the gravel and the plastic
bottles filled with the sand. The heights of the objects were compa-
rable (ꢀ12 cm) and the objects were heavy enough not to be dis-
placed by the animals. The sequence of presentations and the
location of the objects was randomly assigned to each rat.
The animals explored the objects by looking, licking, sniffing or
touching the object while sniffing, but not when leaning against,
standing or sitting on the object. Any rat exploring the two objects
for less than 5 s within 3 min of T1 or T2 was eliminated from the
study. Exploration time of the objects and the distance traveled
were measured using the Any-mazeÒ video tracking system. Based
on exploration time (E) of two objects during T2, discrimination
index (DI) was calculated according to the formula: DI = (EB À EA)/
(EA + AB).
11. Monti, S. M.; Supuran, C. T.; De Simone, G. Expert Opin. Ther. Pat. 2013, 23, 737.
12. Zajdel, P.; Partyka, A.; Marciniec, K.; Bojarski, A. J.; Pawlowski, M.; Wesołowska,
A. Future Med. Chem. 2014, 6, 57.
13. Thomas, D. R.; Melotto, S.; Massagrande, M.; Gribble, A. D.; Jeffrey, P.; Stevens,
A. J.; Deeks, N. J.; Eddershaw, P. J.; Fenwick, S. H.; Riley, G.; Stean, T.; Scott, C.
M.; Hill, M. J.; Middlemiss, D. N.; Hagan, J. J.; Price, G. W.; Forbes, I. T. Br. J.
Pharmacol. 2003, 139, 705.
14. Raubo, P.; Beer, M. S.; Hunt, P. A.; Huscroft, I. T.; London, C.; Stanton, J. A.;
Kulagowski, J. J. Bioorg. Med. Chem. Lett. 2006, 16, 1255.
15. Vermeulen, E. S.; Van Smeden, M.; Schmidt, A. W.; Sprouse, J. S.; Wikström, H.
V.; Grol, C. J. J. Med. Chem. 2004, 47, 5451.
16. Pat., WO 2,009,029,439, 2010.
´
17. Zajdel, P.; Marciniec, K.; Maslankiewicz, A.; Paluchowska, M. H.; Satała, G.;
Partyka, A.; Jastrze˛bska-Wie˛sek, M.; Wróbel, D.; Wesołowska, A.; Duszyn´ ska,
B.; Bojarski, A. J.; Pawłowski, M. Bioorg. Med. Chem. 2011, 19, 6750.
18. Zajdel, P.; Subra, G.; Verdie, P.; Gabzdyl, E.; Bojarski, A. J.; Duszyn´ ska, B.;
Martinez, J.; Pawłowski, M. Bioorg. Med. Chem. Lett. 2009, 19, 4827.
19. Zajdel, P.; Kurczab, R.; Grychowska, K.; Satała, G.; Pawłowski, M.; Bojarski, A. J.
Eur. J. Med. Chem. 2012, 56, 348.
20. Zajdel, P.; Canale, V.; Partyka, A.; Marciniec, K.; Satała, G.; Kurczab, R.; Siwek,
A.; Jastrze˛bska-Wie˛sek, M.; Wesołowska, A.; Kos, T.; Popik, P.; Bojarski, A. J.
MedChemComm 2015, 6, 1272.
21. Canale, V.; Kurczab, R.; Partyka, A.; Satała, G.; Witek, J.; Jastrze˛bska-Wie˛sek, M.;
Pawłowski, M.; Bojarski, A. J.; Wesołowska, A.; Zajdel, P. Eur. J. Med. Chem.
2015, 92, 202.
22. Bernstein, F. C.; Koetzle, T. F.; Williams, G. J.; Meyer, E. F., Jr.; Brice, M. D.;
Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977, 112,
535.
Phencyclidine (PCP), used to attenuate learning, was adminis-
tered at the dose of 5 mg/kg (ip) 45 min before familiarization
phase (T1). The compounds were administrated ip 1 h and
15 min before T1.
9.5.3.1. Statistics.
Data on exploratory preference and dis-
tance traveled were analyzed using two-way repeated measures
ANOVAs with trial as repeated factor; DI data were analyzed by
one-way ANOVA followed by Newman–Keuls post hoc test.
23. Bojarski, A. J.; Cegla, M. T.; Charakchieva-Minol, S.; Mokrosz, M. J.; Mackowiak,
M.; Misztal, S.; Mokrosz, J. L. Pharmazie 1993, 48, 289.
24. Paluchowska, M. H.; Bugno, R.; Duszyn´ ska, B.; Tatarczynska, E.; Nikiforuk, A.;
Lenda, T.; Chojnacka-Wójcik, E. Bioorg. Med. Chem. 2007, 15, 7116.
25. Zajdel, P.; Marciniec, K.; Mas´lankiewicz, A.; Satała, G.; Duszyn´ ska, B.; Bojarski,
A. J.; Partyka, A.; Jastrze˛bska-Wie˛sek, M.; Wróbel, D.; Wesołowska, A.;
Pawłowski, M. Bioorg. Med. Chem. 2012, 20, 1545.
Acknowledgement
This study was supported by the National Science Center Grant
No DEC-2012/05/B/NZ7/03076.
26. Cheng, Y.; Prusoff, W. H. Biochemistry 1973, 12, 2612.
´
27. Zajdel, P.; Marciniec, K.; Maslankiewicz, A.; Grychowska, K.; Satała, G.;
Duszyn´ ska, B.; Lenda, T.; Siwek, A.; Nowak, G.; Partyka, A.; Wróbel, D.;
Jastrze˛bska-Wie˛sek, M.; Bojarski, A. J.; Wesołowska, A.; Pawłowski, M. Eur. J.
Med. Chem. 2013, 60, 42.
Supplementary data
28. Di, L.; Kerns, E. H.; Hong, Y.; Kleintop, T. A.; McConnell, O. J.; Huryn, D. M. J.
Biomol. Screening 2003, 8, 453.
29. Huang, J.; Si, L.; Fan, Z.; Hu, L.; Qiu, J.; Li, G. J. Chromatogr., B: Analyt. Technol.
Biomed. Life Sci. 2011, 879, 3386.
30. Porsolt, R. D.; Bertin, A.; Jalfre, M. Arch. Int. Pharmacodyn. Ther. 1977, 229, 327.
31. The percentage of inhibition for D3, H1, 5-HT2C and M1 receptors were
evaluated at Eurofins Cerep. Experimental conditions for these assays are
described online at www.cerep.fr.
Supplementary data (characterization data for the intermediate
compounds, MS, 1H NMR and 13C NMR spectra for key final
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2015.11.041.
References and notes
32. Pellow, S.; Johnston, A. L.; File, S. E. J. Pharm. Pharmacol. 1987, 39, 917.
33. Millan, M. J. Prog. Neurobiol. 2003, 70, 83.
1. Matthys, A.; Haegeman, G.; Van Craenenbroeck, K.; Vanhoenacker, P. Mol.
Neurobiol. 2011, 43, 228.
2. Nikiforuk, A. CNS Drugs 2015, 29, 265.
3. Abbas, A. I.; Hedlund, P. B.; Huang, X.; Tran, T. B.; Meltzer, H. Y.; Roth, B. L.
Psychopharmacology 2009, 205, 119.
4. Cates, L. N.; Roberts, A. J.; Huitron-Resendiz, S.; Hedlund, P. B.
Neuropharmacology 2013, 70, 211.
34. Wesołowska, A.; Nikiforuk, A.; Stachowicz, K. Eur. J. Pharmacol. 2006, 553, 185.
35. Freret, T.; Paizanis, E.; Beaudet, G.; Gusmao-Montaigne, A.; Nee, G.; Dauphin,
F.; Bouet, V.; Boulouard, M. Psychopharmacology 2014, 231, 393.
36. Discovery Studio v. 3.5, Accelrys Software, San Diego, CA.
37. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
38. Gaulton, A.; Bellis, L. J.; Bento, A. P.; Chambers, J.; Davies, M.; Hersey, A.; Light,
Y.; McGlinchey, S.; Michalovich, D.; Al-Lazikani, B.; Overington, J. P. Nucleic
Acids Res. 2012, 40, 1100.
5. Nikiforuk, A.; Kos, T.; Fijal, K.; Holuj, M.; Rafa, D.; Popik, P. PLoS One 2013, 8,
e66695.
6. Bonaventure, P.; Dugovic, C.; Kramer, M.; De Boer, P.; Singh, J.; Wilson, S.;
Bertelsen, K.; Di, D. J.; Shelton, J.; Aluisio, L.; Dvorak, L.; Fraser, I.; Lord, B.;
Nepomuceno, D.; Ahnaou, A.; Drinkenburg, W.; Chai, W. J.; Dvorak, C.; Sands,
S.; Carruthers, N.; Lovenberg, T. W. J. Pharmacol. Exp. Ther. 2012, 342, 429.
7. Scozzafava, A.; Carta, F.; Supuran, C. T. Expert Opin. Ther. Pat. 2013, 23, 203.
8. Abulwerdi, F. A.; Liao, C.; Mady, A. S.; Gavin, J.; Shen, C.; Cierpicki, T.; Stuckey, J.
A.; Showalter, H. D. H.; Nikolovska-Coleska, Z. J. Med. Chem. 2014, 57, 4111.
9. Borhade, S. R.; Rosenström, U.; Sävmarker, J.; Lundbäck, T.; Jenmalm-Jensen, A.;
Sigmundsson, K.; Axelsson, H.; Svensson, F.; Konda, V.; Sköld, C.; Larhed, M.;
Hallberg, M. ChemistryOpen 2014, 3, 256.
39. Canvas, Version 2.0; Schrodinger, LLC: New York, NY, 2014.
40. LigPrep, Version 3.2; Schrodinger, LLC: New York, NY, 2014.
41. Epik, Version 3.1; Schrodinger, LLC: New York, NY, 2015.
42. Glide, Version 6.6; Schrodinger, LLC: New York, NY, 2015.
43. Lovell, S. C.; Davis, I. W.; Arendall, W. B., 3rd; de Bakker, P. I. W.; Word, J. M.;
Prisant, M. G.; Richardson, J. S.; Richardson, D. C. Proteins: Struct., Funct., Genet.
2002, 50, 437.