Novel 5-HT7R antagonists, arylsulfonamide derivatives of (aryloxy)propyl piperidines: Add-on effect to the antidepressant activity of SSRI and DRI, and pro-cognitive profile

Article abstract of DOI:10.1016/j.bmc.2017.03.057

A novel series of arylsulfonamide derivatives of (aryloxy)propyl piperidines was designed to obtain potent 5-HT₇R antagonists. Among the compounds evaluated herein, 3-chloro-N-{1-[3-(1,1-biphenyl-2-yloxy)2-hydroxypropyl]piperidin-4-yl}benzenesulfonamide (25) exhibited antagonistic properties at 5-HT₇R and showed selectivity over selected serotoninergic and dopaminergic receptors, as well as over serotonin, noradrenaline and dopamine transporters. Compound 25 demonstrated significant antidepressant-like activity in the forced swim test (0.625–2.5?mg/kg, i.p.) and in the tail suspension test (1.25?mg/kg, i.p.), augmented the antidepressant effect of inactive doses of escitalopram (selective serotonin reuptake inhibitor) and bupropion (dopamine reuptake inhibitor) in the FST in mice, and similarly to SB-269970, exerted pro-cognitive properties in the novel object recognition task in cognitively unimpaired conditions in rats (0.3?mg/kg, i.p.). Such an extended pharmacological profile, especially the augmentation effect of the identified 5-HT₇R antagonist on SSRI activity, seems promising regarding the complexity of affective disorders and potentially improved outcomes, including mnemonic performance.

Full text of DOI:10.1016/j.bmc.2017.03.057

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 5-HT R antagonists, arylsulfonamide derivatives of (aryloxy)
7
propyl piperidines: Add-on effect to the antidepressant activity of SSRI
and DRI, and pro-cognitive profile
a
b
c
d
d
c
Vittorio Canale , Anna Partyka , Rafał Kurczab , Martyna Krawczyk , Tomasz Kos , Grzegorz Satała ,
a
b
b
c
Bartłomiej Kubica , Magdalena Jastrz e˛ bska-Wi e˛ sek , Anna Wesołowska , Andrzej J. Bojarski ,
Piotr Popik , Paweł Zajdel
Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
Department of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sm e˛ tna Street, 31-343 Krakow, Poland
Department of Behavioral Neuroscience and Drug Development, Institute of Pharmacology, Polish Academy of Sciences, 12 Sm e˛ tna Street, 31-343 Krakow, Poland
Faculty of Health Sciences, Jagiellonian University Medical College, 20 Michałowskiego Street, 31-126 Kraków, Poland
d,e
a,
⇑
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of arylsulfonamide derivatives of (aryloxy)propyl piperidines was designed to obtain
Received 21 February 2017
Revised 23 March 2017
Accepted 26 March 2017
Available online xxxx
potent 5-HT
yloxy)2-hydroxypropyl]piperidin-4-yl}benzenesulfonamide (25) exhibited antagonistic properties at 5-
HT R and showed selectivity over selected serotoninergic and dopaminergic receptors, as well as over
serotonin, noradrenaline and dopamine transporters. Compound 25 demonstrated significant antidepres-
sant-like activity in the forced swim test (0.625–2.5 mg/kg, i.p.) and in the tail suspension test (1.25 mg/
kg, i.p.), augmented the antidepressant effect of inactive doses of escitalopram (selective serotonin reup-
take inhibitor) and bupropion (dopamine reuptake inhibitor) in the FST in mice, and similarly to SB-
7
R antagonists. Among the compounds evaluated herein, 3-chloro-N-{1-[3-(1,1-biphenyl-2-
7
Keywords:
Selectivity prediction
5
7
-HT antagonist
2
69970, exerted pro-cognitive properties in the novel object recognition task in cognitively unimpaired
conditions in rats (0.3 mg/kg, i.p.). Such an extended pharmacological profile, especially the augmenta-
tion effect of the identified 5-HT R antagonist on SSRI activity, seems promising regarding the complexity
of affective disorders and potentially improved outcomes, including mnemonic performance.
Ó 2017 Elsevier Ltd. All rights reserved.
Serotonin reuptake inhibitor
Dopamine reuptake inhibitor
Depression
7
Cognitive deficit
1
. Introduction
7 7
Recently, targeting the 5-HT receptors (5-HT Rs), has been pro-
posed as an alternative strategy for the treatment of mood disor-
ders including depression and may have beneficial effects in
accelerating/enhancing the therapeutic effects of clinically used
Depression is a common, recurrent and debilitating mental dis-
order characterized by a complex pattern of genetic, epigenetic,
1
7,8
developmental, and environmental factors. Despite the progress
antidepressant drugs. A large body of evidence demonstrated
in pharmacotherapy, currently available antidepressants require
long-term administration (3–6 weeks) before the full efficacy is
7
that genetic inactivation or pharmacological blockade of 5-HT Rs
by the administration of the selective antagonists SB-269970 or
JNJ-18038683 produced a significant antidepressant effect in pre-
2
,3
attained, often followed by tolerance, the recurrence of disease,
and the appearance of several side effects such as sexual dysfunc-
9
–11
clinical studies.
Additionally, it was found that the co-adminis-
4
tion and/or weight gain. The addition of thyroid hormone (T3) or
tration of individually ineffective doses of SB-269970 and citalo-
pram (a representative of selective serotonin reuptake inhibitors,
SSRI) enhanced the behavioral effects decreasing the immobility
time of mice in the forced swim test (FST) and the tail suspension
test (TST), and reducing the delayed onset and the REM sleep sup-
lithium represents a classic augmentation strategy often used in
the treatment of resistant depression. Soon later, it was found that
the response to antidepressants could be augmented by joint
administration with second-generation antipsychotics, e.g., queti-
5
,6
12–14
apine and aripiprazole, via antagonism at serotonin receptors.
pression induced by SSRIs.
Moreover, several studies have revealed that antagonism at
5
-HT
Rs may affect hippocampal-dependent cognitive processes15
7
⇑
Corresponding author.
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
and may improve the acquisition of recognition memory in the
http://dx.doi.org/10.1016/j.bmc.2017.03.057
968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
0
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2
V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
O
O
O
N
O
S
O
N
O
S
O
N
O O
F
S
Cl
N
N
N
H
H
H
F
PZ-766
PZ-1404
PZ-1130
K
i
(5-HT
7
) = 0.3 nM K
(5-HT ) = 1 nM
FST (MED = 5 mg/kg),
NORT (MED = 1 mg/kg)
i
(5-HT1A) = 436 nM
K
i
(5-HT
7
) = 9 nM K
(5-HT
i
(5-HT1A) = 356 nM
) = 26 nM
K
i
(5-HT
7
) = 7 nM K (5-HT1A) = 234 nM
7
(5-HT ) = 6 nM
i
K
b
7
K
b
7
K
b
FST (MED = 2.5 mg/kg), FPT (MED = 1.25 mg/kg)
NORT (MED = 1 mg/kg)
FST (MED = 5 mg/kg)
NORT (MED = 1 mg/kg)
7
Fig. 1. Chemical structures along with the in vitro and in vivo pharmacological activity of selected 5-HT R antagonists in a group of arylsulfonamide derivatives of (aryloxy)
ethyl alicyclic amines.1
9,20
novel object recognition task (NORT) in rats,16,17 suggesting an
additional therapeutic benefit in the treatment of the cognitive
impairment associated with depression.18
ethyl spacer with a branched or linear propylene linker. Next, in
an attempt to increase the stabilization of the ligand-receptor com-
plex via the formation of a net of hydrogen bonds, a secondary
hydroxyl group was introduced into the propylene spacer (Fig. 2).
7
Continuing our effort in the development of potent 5-HT R
antagonists, we have recently reported on a series of arylsulfon-
amide derivatives of (aryloxy)ethyl alicyclic amines (i.e., PZ-766,
PZ-1404 and PZ-1130, Fig. 1) with significant in vivo behavioral
activities in rodent models of depression (FST), anxiety (the four-
plate test, FPT) and cognitive decline (NORT).1
Extensive SAR studies toward this group of derivatives showed
that sterically hindered substituents in an ortho position at the ary-
loxy fragment (i.e., isopropyl and phenyl) guaranteed high affinity
7
In this study we further report on the in vitro activity for 5-HT -
Rs, and selected monoaminergic receptors, functional cellular
activity, and in vivo behavioral effects of selected compounds in
animal models of depression. A special interest was focused on
the interaction between tested compounds and selected antide-
pressants with different mechanism of action (i.e., escitalopram,
reboxetine and bupropion), followed by determination of the
pro-cognitive properties of the most promising compounds.
9,20
7
for 5-HT R and selectivity over other related GPCRs, e.g., serotonin-
ergic, dopaminergic, adrenergic, muscarinic and histamine recep-
2
. Results and discussion
1
9,21
tors.
sulfonamide moiety (i.e., 3-chloro, 3-fluoro and 4-fluoro) preferen-
tially interacted with 5-HT Rs over their di-substituted
Based on these findings, we designed and synthesized
Moreover, compounds mono-halogenated at the phenyl
2.1. Chemistry
7
analogs.2
0,22
The synthesis of the designed series of compounds, grouped in
a limited series of arylsulfonamide derivatives of (aryloxy)alkyl
piperidines. Chemical modifications included replacement of an
the three sets 15–18 (Set I), 19–22 (Set II) and 23–26 (Set III),was
carried out by a multi-step procedure according to Scheme 1. In the
first step, alkylation of the commercially available phenols 1 and 2
under biphasic conditions using different alkylating agents, i.e., 1-
chloropropan-2-one, 1,3-dibromoethane, and epichlorohydrin,
yielded intermediates 3 and 4; 5 and 6; and 7 and 8, respectively.
The ketone derivatives 3 and 4 were subsequently reductively ami-
nated in the presence of sodium cyanoborohydride to give the sec-
ondary amines 9 and 10. Alternatively, compounds 5–8 reacted
with the Boc-protected 4-aminopiperidine to give intermediates
R
R
2
O
S
O
N
O
n
R
1
N
R
3
H
3 2 3 1
R = 3-F, 3-Cl, 4-F; R = H, CH ; R = H, OH; n = 0, 1; R = isopropyl, phenyl
1
1–14.
After the removal of the protecting group, the final arylsulfon-
amides 15–26, were synthesized by the treatment of the primary
Fig. 2. Structural modification of arylsulfonamide derivatives of (aryloxy)propyl
piperidines.
O
R
R
R
O
ii
O
iv, v
iv, v
O
N
O O
S
N
Boc
i
N
H
N
Set I
R
1
H
3
-4
9-10
15-18
R
HO
R
R
O
R
O
i
iii
Br
O
N
N
N
O
O
1
-2
Boc
S
i
N
H
N
5
-6
11-12
R
Set II
1
H
1
9-22
R
O
R
O
iv, v
O
R
iii
O
O
O
N
S
OH
Boc
OH
N
H
N
R
1
H
Set III
7
-8
13-14
2
3-26
R = isopropyl, phenyl; R
1
= 3-F, 3-Cl, 4-F
Scheme 1. The synthesis of three series of arylsulfonamide derivatives of (aryloxy)propyl piperidines 15–18 (Set I), 19–22 (Set II) and 23–26 (Set III). Reagent and
conditions: i) alkylating agent, K CO , KI, (CH CO, 60 °C, 24–48 h; ii) 4-(Boc-N-amino)piperidine, NaBH CN, CH Cl , r.t., 4 h; iii) 4-(Boc-N-amino)piperidine, K CO , KI,
CH CO, 60 °C, 24 h; iv) TFA/CH Cl (80/20; v/v), r.t., 1 h; v) arylsulfonyl chloride, TEA, CH Cl , 0 °C, 2–6 h.
2
3
)
3 2
3
2
2
2
3
(
)
3 2
2
2
2
2
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V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
amine derivatives with the selected arylsulfonyl chloride in the
presence of triethylamine (yields 70–75%).
To further support the design, molecular docking was per-
formed with the use of recently developed 5-HT homology models
based on dopaminergic D receptor template (PDB ID: 3PBL).
7
21–23
3
The results showed the fully coherent binding mode of the titled
compounds with that of previously synthesized analogs (i.e., PZ-
2.2. In silico selectivity prediction and molecular docking
7
66, and PZ-1130). The main L-R interaction contacts involved a
salt-bridge with Asp3.32, a cation– interaction of the arylsulfon-
amide moiety with Arg7.36, and CH– interactions between
A recently developed machine learning-based algorithm,23
build on three molecular fingerprints (i.e. MACCS, CDK and Kle-
p
p/p–p
2
4–26
kota-Roth),
focused library-members 15–26 for 5-HT
tionally, the model was updated using the recently published aryl-
was employed to access the selectivity of the
the (aryloxy)alkyl fragment and the side chains of Phe6.51 and
Phe6.52 (Fig. 4).
7
Rs over 5-HT1ARs. Addi-
Moreover, to analyze the influence of each structural modifica-
2
0–22
sulfonamide derivatives of (aryloxy)ethyl alicyclic amines.
Scores were averaged and ranged from 0 (low selectivity) to 4
high selectivity) (Table 1-SI). Regarding the selectivity threshold
7
tion on the 5-HT R affinity, the analysis of the binding modes was
enriched by the calculation of binding free energy changes
reported as DDG values (Table 1-SI).
(
23
assumed in the model generation (>5), the performance of the
binary prediction was calculated by means of Recall (fraction of
correctly identified positive examples), BEDROC (to address the
problem of ‘‘early recognition” in the virtual screening of com-
pounds library) and AUC (area under the receiver operator charac-
teristic curve). The updated models, compared to these developed
previously, showed equal BEDROC value (1.00) and improved
Recall (0.90 compared to 0.70) and AUC (0.95 compared to 0.85)
values (Fig. 3).
2
2
.3. Pharmacology
.3.1. In vitro evaluation
Radioligand competition binding assays for human serotonin, 5-
6
HT1A, 5-HT2A, 5-HT , 5-HT7B and D2L receptors, which were all sta-
bly expressed in HEK293 or CHO-K1 cells, were employed to deter-
mine the affinity and selectivity profiles of the synthesized
27–
compounds. According to the previously published procedures,
2
9
3
the experiments were carried out using [ H]-8-OH-DPAT
3
3
(
(
(
135.2 Ci/mmol), [ H]-Ketanserin (53.4 Ci/mmol), [ H]-LSD
83.6 Ci/mmol), [ H]-5-CT (80.1 Ci/mmol) and [ H]-Raclopride
76.0 Ci/mmol) for 5-HT1A, 5-HT2A, 5-HT , 5-HT7B and D , respec-
3
3
6
2
tively. Additionally, the affinity of compounds 20 and 25 for sero-
tonin, noradrenaline and dopamine transporters (SERT, NET and
DAT, respectively), and
at Eurofins Cerep. The results were expressed as the % inhibition
of the control binding for SERT, NET, DAT, and -adrenergic recep-
1
a -adrenergic receptors, were evaluated
a
1
tors according to experimental protocols described online at www.
cerep.fr.
2.3.2. Functional in vitro evaluation
The functional properties of selected compounds 20, 21 and 25
on 5-HT
7
R were evaluated using their ability to inhibit cAMP pro-
R agonist, in HEK293
duction induced by 5-CT (10 nM), a 5-HT
cells over expressing 5-HT
triplicate at 8 concentrations (10 –10 M). The level of adenylyl
cyclase activity was measured using a LANCE cAMP detection kit.
7
2
1,22
7
R.
Each compound was tested in
ꢀ
11
ꢀ4
Fig. 3. The ROC curves illustrating the real performance of recently developed (red
line) and updated (green line) models in the prediction of selectivity for the library
of synthesized compounds 15–26.
2.3.3. Behavioral evaluation
The antidepressant activities of selected compounds 20 and 25
were evaluated in FST and TST in Swiss albino and C57BL/6J mice,
Fig. 4. Molecular docking results of the representative library members for the 5-HT
7
receptor. A) Comparison of the binding modes for compounds 16, 20, 24 and PZ-766
(
yellow, cyan, pink and green, respectively). B) Comparison of the binding modes for compounds PZ-1130, 17, 21 and 25 (yellow, cyan, pink and green, respectively).
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4
V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
respectively, and their effects were compared with those of SB-
69970, a selective 5-HT R antagonist, and escitalopram, represen-
the ligand-receptor complex. This effect was pronounced for iso-
propyl derivative 16 in comparison to its phenyl analog 17
2
7
tative of SSRI drugs. In the next stage, to determine synergic inter-
actions (add-on effects) between the tested compounds and
currently used antidepressants, inactive doses of 20 and 25 were
co-administered with inactive doses of the selective serotonin,
noradrenaline and mixed noradrenaline and dopamine reuptake
inhibitors (i.e., escitalopram, reboxetine and bupropion, respec-
tively). 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. Moreover, the pro-cognitive properties of com-
pounds 20 and 25 were further determined in the NORT in Sprague
Dawley rats.
(
DDG = 5.93 and 1.12 kcal/mol respectively).
Subsequently, elongation of the alkylene spacer from 2 to 3
7
methylene groups slightly decreased the affinity for 5-HT Rs (PZ-
766 vs 20 and PZ-1130 vs 25) yet maintaining high selectivity over
5-HT1A sites (S1A/7 > 30) (Table 1). According to the results obtained
from binding free energy calculations, the introduction of a hydro-
xyl group in the propylene linker of ortho-isopropyl derivatives
decreased the stability of the ligand-receptor complex
(
DDG = 11.23 kcal/mol) reducing the affinity for 5-HT
fold (19 vs 23 and 20 vs 24). In contrast, this modification retained
the high affinity of ortho-phenyl analogs for 5-HT R and selectivity
7
R up to 3-
7
over 5-HT1AR when compared to their unsubstituted congeners (25
vs 21 and 26 vs 22). This discrepancy might be partially explained
by molecular docking analysis. It was found that despite the possi-
bility for the OH group of both subseries (ortho-isopropyl deriva-
tives 23 and 24, ortho-phenyl analogs 25 and 26) to create a
hydrogen bond with Asp3.32 or the SH group of Cys3.36, only in
case of ortho-isopropyl derivatives could the orientations of the
hydrogen atom in the OH group of ligands and the SH group of
Cys3.36 produce a repulsion between the free electron pairs of
oxygen and sulfur (Fig. 4A–B).
2.4. Structure-activity relationship studies
All the newly synthesized compounds 15–26 were evaluated in
in vitro binding experiments for their affinity for 5-HT
Rs. In general, binding assays revealed that compounds displayed
high-to-moderate affinity for 5-HT R (K = 32–213 nM) and moder-
ate-to-low affinity for 5-HT1ARs (K = 297–2081 nM) (Table 1).
7
and 5-HT1A-
7
i
i
The radioligand binding assays revealed that the introduction of
a methyl group at the alpha carbon of the ethylene spacer (proxi-
mal to the nitrogen atom of piperidine) differently influenced
2.5. Extended in vitro and in vivo pharmacology
interaction with 5-HT
the affinity of compounds containing an ortho-isopropyl sub-
stituent at the aryloxy fragment for 5-HT Rs (PZ-766 vs both 15
and 16, K = 0.3, 170 and 74 nM, respectively). On the other hand,
it has not so significantly impacted the affinity of ortho-phenyl
substituted analogs (PZ-1130 vs 17 and 18, K = 7, 32 and 34 nM,
respectively). These results are in line with molecular modeling
indicating that although the possibility to form a CH- interaction
7
R. This modification significantly decreased
Next, the most interesting derivatives with the highest affinity
for 5-HT Rs (K < 50 nM) and selectivity over 5-HT1ARs (S1A/7 > 40)
7 i
were evaluated in in vitro functional activity studies in cAMP
assays (Table 2).
7
i
i
Having identified compounds 20 and 25 as potent 5-HT
antagonists, we further investigated their affinity for other related
serotoninergic 5-HT2A, 5-HT and dopaminergic D Rs, and finding
7
R
p
6
2
with Phe6.52 exists, the presence of the methyl group destabilized
high-to-moderate selectivity for those sites (Table 2). Furthermore,
Table 1
The binding data of the synthesized compounds 15–26 for 5-HT
7
and 5-HT1ARs.
R
R
2
O
S
O
N
O
n
R
1
N
H
R
3
1
5-26
Cmpd
R
n
R
1
R
2
R
3
K
i
[nM]a
b
5-HT
7
5-HT1A
S
1A/7
PZ-766c
PZ-1130
Isopropyl
Phenyl
Isopropyl
Isopropyl
Phenyl
0
0
0
0
0
0
1
1
1
1
1
1
1
1
H
H
H
H
H
H
H
H
H
H
H
4-F
3-Cl
3-F
4-F
3-Cl
4-F
3-F
4-F
3-Cl
4-F
3-F
4-F
3-Cl
4-F
0.3
7
170
74
32
34
52
32
52
58
436
234
962
1169
696
297
1792
2081
1630
1839
742
506
1329
1322
1453
33
6
16
22
9
34
65
31
32
4
d
15
16
17
18
19
20
21
22
23
24
25
26
CH
CH
CH
CH
H
H
H
H
H
3
3
3
3
Phenyl
Isopropyl
Isopropyl
Phenyl
Phenyl
H
Isopropyl
Isopropyl
Phenyl
OH
OH
OH
OH
213
104
33
H
H
H
5
40
22
Phenyl
59
a
b
c
K
i
values are means of three independent binding experiments (SEM ꢁ17%).
Ratio of affinity for 5-HT1Avs 5-HT
Data taken from Ref. 19.
Data taken from Ref. 20.
7
Rs.
d
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V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 2
The antagonist activity for 5-HT
7
Rs of selected compounds 20 and 25 and their binding data for 5-HT1A, 5-HT2A, 5-HT
6
and D
2
Rs.
[nM]
Compd
K
i
[nM]
K
b
[nM]
K
i
5
-HT
7
5-HT1A
5-HT2A
5-HT
6
D
2
2
2
0
5
32
33
1
25
2081
1329
1352
2115
268
295
328
343
compound 20 displayed a moderate affinity for SERT (79% @ 1
and did not bind to NET and DAT (<10% @ 1 M). On the other
hand, compound 25 showed a low affinity for all of the trans-
porters (<40% @ 1 M). Both compounds displayed no affinity for
-adrenergic receptors (<10% @ 1 M).
The antidepressant activities of the selected 5-HT
l
M)
10 mg/kg, corresponding to a reduction in the mouse immobility
time by 31%. Escitalopram given at doses of 1.25 and 2.5, but not
0.625 mg/kg, shortened the immobility time by 27 and 34%,
respectively.
l
l
a
1
l
The antidepressant-like properties of 20 and 25 were further
confirmed in the TST. Both 20 and 25 increased the average force
of fights and decreased the immobility time when given at a dose
of 2.5 mg/kg. The significant increase in number of fight episodes
was visible after administration of 20 at a dose of 1.25 mg/kg
and 25 at doses of 0.625 and 1.25 mg/kg. Moreover, at a dose of
1.25 mg/kg, 25 significantly shortened the duration of immobility.
For comparison, after the administration of a single dose of 2.5 mg/
kg, SB-269970 produced antidepressant-like effects in the TST,
while escitalopram given at doses of 5, 10 and 20 mg/kg elicited
an antidepressant-like effect, significantly reducing the immobility
7
R antagonists
2
0 and 25 were then evaluated in vivo in the FST in mice using SB-
2
69970 and escitalopram as active compounds for comparison.
Tested compounds displayed antidepressant-like activities at
doses of 0.625–2.5 mg/kg, significantly reducing the duration of
the immobility time by 19–53% for 20 and 28–46% for 25 (Fig. 5).
Thus, compounds 20 and 25 produced higher antidepressant-like
effect than compounds PZ-766 and PZ-1130, and the observed
effects were found at up to 8-fold lower doses. SB-269970 exerted
its characteristic antidepressant effect at one medium dose of
Fig. 5. Effects of the active compounds 20, 25, SB-269970, and escitalopram on the immobility time of mice in the forced swim test. Data are presented as the
⁄
⁄⁄⁄⁄
p < 0.0001 compared with the respective vehicle group, followed by the Bonferroni’s
mean ± standard error of the mean of N = 7–10 animals per group. p < 0.05,
comparison test after a significant analysis of variance.
Fig. 6. Effects of the active compounds 20, 25, SB-269970, and escitalopram on the average force of fights, number of fight episodes and immobility time of mice in the tail
⁄
⁄⁄
⁄⁄⁄
⁄⁄⁄⁄
p < 0.0001 compared with the
suspension test. Data are presented as the mean ± standard error of the mean of N = 7–9 animals per group. p < 0.05, p < 0.01, p < 0.001,
respective vehicle group, followed by the Bonferroni’s comparison test after a significant analysis of variance.
Please cite this article in press as: Canale V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.057

6
V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
time and additionally increasing the average force of fights at the
highest dose only (Fig. 6). The discrepancies in antidepressant-
active doses of the tested compounds in the FST and TST may lie
in the different mice strains used in those tests (Swiss albino in
FST) and (C57BL/6J in TST).
The antidepressant-like activities of 20 and 25 cannot be attrib-
uted to changes in the general activity, as these compounds used at
doses producing anti-immobility effects in the FST and TST do not
change the locomotor activity of mice measured over a period of
time equal to the observation time in both tests (Table 2-SI).
An augmentation strategy is often implemented to improve an
efficacy and limit side effects of antidepressant treatment. Specifi-
Fig. 8. Effects of the tested compounds 20, 25 and SB-269970 in the novel object
cally, previous data showed that 5-HT
antidepressant effect of SSRI in the FST and TST.
other hand, the interaction between 5-HT R antagonists and nora-
7
R antagonists increased the
recognition test in rats. The data are presented as the mean ± standard error of the
12,13,30,31
⁄
⁄
⁄⁄⁄
On the
mean of N = 8–10 animals per group. p < 0.01 vs. vehicle, p < 0.001 vs. vehicle.
7
drenaline reuptake inhibitors (NRI) or dopamine reuptake inhibi-
tors (DRI) have been much less studied and are ambiguous. A
combination of SB-269970 and reboxetine (NRI) reduced immobil-
ity in the FST but not in the TST.31 Additionally, the co-administra-
tion of SB-269970 and GBR-12909 (selective DRI) did not influence
the immobility time either in the FST or in the TST. Compounds 20
and 25 (0.312 mg/kg) co-administered with escitalopram
(Fig. 8). It is worth noting, that compound 25 displayed pro-cogni-
tive properties in the same dose as it improved the efficacy of inac-
tive doses of escitalopram and bupropion in the FST.
3. Conclusions
(
0.625 mg/kg) or, to a lesser extent, bupropion (1.25 mg/kg), but
not reboxetine (0.312 mg/kg), produced significant anti-immobil-
ity effects in the FST (Fig. 7). It was also found, that compound
As part of ongoing efforts to develop potent 5-HT R antagonist a
focused library of arylsulfonamide derivatives of (aryloxy)propyl
7
2
0 co-administrated with escitalopram produced slightly higher
piperidines was designed, and an algorithm to predict 5-HT /5-
7
effect than 25 (32.6% vs 25.4%, respectively). This might partly
result from a higher affinity of compound 20 for the serotonin
transporter. All of the positive interactions described above seem
to be specific, since these drugs (given alone or jointly) do not
increase the spontaneous locomotor activity of mice (data not
shown). The data of the interaction studies suggest that antide-
pressant-like effects of the tested compounds observed in the FST
in mice are mediated via serotoninergic and dopaminergic rather
than noradrenergic systems.
HT1A receptor selectivity was fine-tuned. Structure-activity rela-
tionship studies supported by molecular modeling analysis
demonstrated that the elongation of the alkylene spacer from 2
to 3 methylene units maintained the high affinity for 5-HT R and
7
selectivity over 5-HT1AR. Moreover, the introduction of a secondary
alcohol functional group in the propylene spacer was preferential
for interaction with 5-HT R only in compounds with an ortho-phe-
7
nyl substituent at the aryloxy fragment. The study allowed identi-
fication
of
3-chloro-N-{1-[3-(1,1-biphenyl-2-yloxy)-2-
Cognitive decline constitutes an integral part of the clinical pic-
ture of depressed patients, thus the improvement of cognitive pro-
cesses such as executive functions and working memory is an
important target for pharmacological intervention in the treatment
of depression. Several pre-clinical studies have demonstrated that
hydroxypropyl]piperidin-4-yl}benzene sulfonamide (25), as
a
potent 5-HT R antagonist. Compound 25 produced antidepres-
7
sant-like effects at lower doses than SB-269970 and escitalopram
in the FST and TST (MED = 0.625 and 1.25 mg/kg, respectively),
potentiated the antidepressant properties of inactive doses of esc-
italopram and bupropion in the FST in mice (0.325 mg/kg), and dis-
played pro-cognitive properties in rat’s NORT (0.3 mg/kg) in not
disturbed conditions. These findings further support the data that
5
7
-HT Rs modulate cognitive processes involved in memory consol-
idation, further suggesting that the pharmacological blockade of
these receptors may lead to pro-cognitive effects.1
6,32
Thus, in
the next step, compounds 20 and 25 were evaluated in the NORT
associating antagonism at 5-HT R with the inhibition of sero-
7
3
3
in rats to determine their effects on visual episodic memory.
tonin/dopamine reuptake might be beneficial regarding the com-
The acute administration of 20 and 25 reversed in a dose-
dependent manner, the natural forgetting impairment in rats (in
plexity of CNS disorders, specifically in increasing the response
rate of available drugs to obtain remission, overcome treatment-
resistant depression and improve cognition performance.
2
4 h interval) producing similar effects to those of SB-269970
Fig. 7. Effects of the co-administration of inactive doses of compounds 20 (A) and 25 (B) and the antidepressants escitalopram, reboxetine and bupropion on the immobility
⁄
⁄⁄⁄⁄
p < 0.0001 versus the vehicle
time of mice in the forced swim test. Data are presented as the mean ± standard error of the mean of N = 7–10 animals per group. p < 0.05,
treated group, #p < 0.05, p < 0.01 versus the respective antidepressant treated group, followed by the Bonferroni’s comparison test after a significant analysis of variance.
##
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V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
4
. Experimental section
designated as follows: br.s. (broad singlet), d (doublet), t (triplet),
q (quartet), quin (quintet), sept (septet), dd (doublet of doublets),
dt (doublet of triplets), dq (doublet of quartets), td (triplet of dou-
blets), ddd (doublet of doublet of doublets), tdd (triplet 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 cal-
culated values.
Melting points (Mp) were determined with a Büchi apparatus
and are uncorrected.
Compounds selected for behavioral evaluation (20 and 25) were
converted into the hydrochloride salts.
4.1. Molecular docking
The 3-dimensional structures of the ligands were prepared
3
4
using LigPrep v3.6, and the appropriate ionization states at
pH = 7.4 were assigned using Epik v3.4.35 The Protein Preparation
Wizard was used to assign the bond orders, appropriate amino acid
ionization states and to check for steric clashes. The receptor grid
was generated (OPLS_2005 force field) by centring the grid box
with a size of 12 Å on Asp3.32. Automated docking was performed
using Glide v6.9 at the SP level with the flexible docking option
3
6
turned on.
Spectroscopic data for all of the final compounds and all of the
synthesis methods are summarized below, while characterization
data for the intermediates are reported in the Supporting
Information.
4
.2. Binding free energy calculation
MM-GBSA (Generalized-Born/Surface Area) was used to calcu-
late the binding free energy for all ligand-receptor complexes
obtained from molecular docking using Prime software from
Schrodinger. Firstly, the complexes were minimized using the local
optimization feature in Prime, whereas the energies were calcu-
lated with the OPLS-2005 force field and Generalized-Born/Surface
Area continuum solvent model. Based on the MM-GBSA binding
free energies, a difference between binding free energy (DDG) of
a parent compound PZ-766 and PZ-1130 and their structural ana-
logs was calculated to assess the influence of a given modification
on the binding energy.
4
.3.1. General procedure for the synthesis of intermediates 3–8
A solution of the proper alkylating agent, chloropropa-2-one,
,3-dibromopropane or epichlorohydrin (0.04 mol) in 40 mL of
1
acetone was added dropwise into the mixture of respective phenol
0.02 mol) and K CO (0.04 mol) in 30 ml of acetone. Subsequently,
(
2
3
a catalytic amount of KI (0.3 mmol) was added and the resulting
mixture was stirred at 60 °C for 24–48 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.
4.3. Chemistry
Organic synthesis were carried out at ambient temperature,
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, Fluorochem, TCI).
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 (70–230
mesh ASTM).
4
.3.2. General procedure for the synthesis of intermediates 9 and 10
Amine (1.5 mmol) and ketone (1.5 mmol) were stirred in anhy-
(
drous THF (5 mL) for 5 h at room temperature, and then were trea-
ted with sodium cyanoborohydride (3 mmol). The mixture was
further stirred until the reagents were consumed as determined
by HPLC analysis. Subsequently, the reaction was quenched by
adding a saturated solution of NaHCO
3
, washed with H
SO , filtered and concentrated. The
2
O (2ꢂ),
brine (1ꢂ), dried over Na
2
4
obtained crude product was purified using silica gel with CH
MeOH as an eluting system.
2
Cl
2
/
Mass spectra were recorded on an UPLC-MS/MS system con-
sisted of a Waters ACQUITYÒ UPLC (Waters Corporation, Milford,
MA, USA) coupled to a Waters TQD mass spectrometer (electro-
spray ionization mode ESI-tandem quadrupole). Chromatographic
separations were carried out using the Acquity UPLC BEH (bridged
ethyl hybrid) C18 column; 2.1 ꢂ 100 mm, and 1.7 mm particle size,
equipped with Acquity UPLC BEH C18 Van Guard pre-column;
Ò
4
.3.3. General procedure for the synthesis of intermediates 11–14
-(Boc-N-amino)piperidine (0.015 mol) was dissolved in ace-
tone (15 mL), followed by addition of K CO (0.045 mol), catalytic
amount of KI. Subsequently, selected alkylating agent 5–8
0.018 mol) was added dropwise in 20 min and the mixture was
4
2
3
2
.1 ꢂ 5 mm, and 1.7 mm particle size. The column was maintained
(
at 40 °C, and eluted under gradient conditions from 95% to 0% of
stirred under reflux for 24 h. Inorganic residues were filtered off
and organic mixture was concentrated under reduced pressure.
The obtained crude product was purified using silica gel with CH
Cl /MeOH as an eluting system.
ꢀ1
eluent A over 10 min, at a flow rate of 0.3 mL min . Eluent A:
water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid
2
-
(
0.1%, v/v). Chromatograms were made using Waters ek PDA detec-
2
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
4.3.4. General procedure for the synthesis of final compounds 15–26
Removal of Boc function of the intermediates 9–14 was accom-
plished by treatment with a mixture of TFA/CH Cl (80/20 v/v).
2 2
ꢀ
1
ꢀ1
rate 600 L h
,
cone gas flow 100 L h
,
capillary potential
3
.00 kV, cone potential 40 V. Nitrogen was used for both nebulizing
Upon removal of organic solvents under reduced pressure, TFA
salts of deprotected intermediates 9–14 were left under vacuum
overnight. Then, a mixture of the appropriate (aryloxy)propyl
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.
H NMR and C NMR spectra were obtained in Varian BB 200
spectrometer using TMS (0.00 ppm) as an internal standard in
CDCl and were recorded at 300 and 75 MHz, respectively. The J
values are reported in Hertz (Hz), and the splitting patterns are
2 2
piperidine (0.38 mmol) in CH Cl (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
1
13
3
sulfonamides were purified using silica gel column with CH
MeOH as an eluting system.
2 2
Cl /
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8
V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4
.4. Characterization data for final compounds 15–26
.4.1. 3-Fluoro-N-(1-{1-[(propan-2-yl)phenoxy]propan-2-yl}
(m, 2H), 3.21–3.25 (m, 1H), 3.24–3.34 (m, 1H), 3.97 (t, J = 5.9 Hz,
H), 5.02 (br.s., 1H), 6.81 (dd, J = 8.2, 1.2 Hz, 1H), 6.87–6.94 (m,
1H), 7.09–7.16 (m, 1H), 7.20 (dd, J = 7.6, 1.8 Hz, 1H), 7.23–7.31
(m, 1H), 7.50 (td, J = 8.2, 5.3 Hz, 1H), 7.61 (dt, J = 8.2, 2.1 Hz, 1H),
7.68–7.72 (m, 1H).
2
4
piperidin-4-yl)benzenesulfonamide) (15)
Yellow oil, 80 mg following chromatographic purification over
silica gel with CH
2
Cl
S, MW 434.57, Monoisotopic Mass 434.20,
3
M+H] 435.2. H NMR (300 MHz, CDCl ) d 1.15 (d, J = 6.8 Hz,
2
/MeOH (9/0.7); UPLC/MS purity 98%,
t
R
= 5.57. C23
H
31FN
2
O
3
4.4.6. 4-Fluoro-N-(1-{3-[(2-isopropylphenoxy)]propyl}piperidin-4-yl)
benzenesulfonamide (20)
+
1
[
3
2
3
5
H), 1.20 (d, J = 6.9 Hz, 6H), 1.45–1.52 (m, 2H), 1.74–1.84 (m,
Yellow oil, 150 mg following chromatographic purification over
H), 2.33–2.48 (m, 2H), 2.76–2.88 (m, 2H), 2.99–3.08 (m, 1H),
.12–3.21 (m, 1H), 3.28 (quin, J = 6.9 Hz, 1H), 3.83 (dd, J = 9.4,
.9 Hz, 1H), 3.98 (dd, J = 9.4, 5.4 Hz, 1H), 4.53 (br.s., 1H), 6.79 (dd,
silica gel with CH
2
Cl
S, MW 434.57, Monoisotopic Mass 434.20,
) d 1.19 (d, J = 6.9 Hz,
2
/MeOH (9/0.7); UPLC/MS purity 100%,
t
R
= 5.79. C23
[M+H] 435.1. H NMR (300 MHz, CDCl
H
31FN
2 3
O
+
1
3
J = 8.2, 1.1 Hz, 1H), 6.89–6.95 (m, 1H), 7.09–7.16 (m, 1H), 7.21
6H), 1.45–1.57 (m, 2H), 1.78 (dd, J = 13.2, 3.4 Hz, 2H), 1.90–1.97
(m, 2H), 2.06 (t, J = 10.4 Hz, 2H), 2.47–2.56 (m, 2H), 2.79 (d,
J = 11.9 Hz, 2H), 3.17 (d, J = 6.6 Hz, 1H), 3.28 (sept, J = 6.9 Hz, 1H),
3.97 (t, J = 6.1 Hz, 2H), 6.80 (dd, J = 8.1, 1.0 Hz, 1H), 6.87–6.94 (m,
(
(
dd, J = 7.4, 1.7 Hz, 1H), 7.27 (tdd, J = 8.3, 2.6, 0.9 Hz, 1H), 7.49
td, J = 8.0, 5.3 Hz, 1H), 7.59 (ddd, J = 8.2, 2.5, 1.7 Hz, 1H), 7.65–
7
.69 (m, 1H).
1
3
1
H), 7.08–7.21 (m, 4H), 7.86–7.95 (m, 2H). C NMR (75 MHz,
CDCl ) d 22.6, 26.8, 27.0, 32.9, 52.0, 55.2, 65.9, 111.1, 116.2,
116.5, 120.5, 126.0, 126.5, 129.5, 129.7, 136.9, 156.0. Anal. calcd
for C23
4.4.2. 4-Fluoro-N-(1-{1-[(propan-2-yl)phenoxy]propan-2-yl}
3
piperidin-4-yl)benzenesulfonamide (16)
Yellow oil, 100 mg following chromatographic purification over
H31FN
2
O
3
SꢃHCl: C: 58.65, H: 6.85, N: 5.95, S: 6.81; Found
silica gel with CH
2
Cl
S, MW 434.57, Monoisotopic Mass 434.20,
3
M+H] 435.2. H NMR (300 MHz, CDCl ) d 1.15 (d, J = 6.8 Hz,
2
/MeOH (9/0.7); UPLC/MS purity 100%,
C: 58.89, H: 6.81, N: 6.05, S: 6.51. M.p. for C23
185.3–188.7 °C.
2
H31FN O
3
SꢃHCl:
t
R
= 5.52. C23
H
31FN
2
O
3
+
1
[
0
3
2
3
5
H), 1.20 (d, J = 6.9 Hz, 6H), 1.41–1.52 (m, 2H), 1.73–1.83 (m,
4.4.7. 3-Chloro-N-{1-[3-(1,1 -biphenyl-2-yloxy)propyl]piperidin-4-yl}
benzenesulfonamide (21)
H), 2.37–2.40 (m, 2H), 2.77–2.87 (m, 2H), 2.98–3.10 (m, 1H),
.12–3.14 (m, 1H), 3.28 (quin, J = 6.9 Hz, 1H), 3.83 (dd, J = 9.4,
.8 Hz, 1H), 3.98 (dd, J = 9.4, 5.4 Hz, 1H), 4.69 (br.s., 1H), 6.79 (dd,
Yellow oil, 220 mg following chromatographic purification over
silica gel with CH
2
Cl
S, MW 485.04, Monoisotopic Mass 484.16,
3
[M+H] 485.3. H NMR (300 MHz, CDCl ) d 1.37–1.53 (m, 2H),
2
/MeOH (9/0.7); UPLC/MS purity 100%,
J = 8.2, 1.0 Hz, 1H), 6.89–6.95 (m, 1H), 7.11 (dd, J = 8.0, 1.7 Hz,
H), 7.14–7.23 (m, 3H), 7.86–7.93 (m, 2H). 13C NMR (75 MHz,
CDCl ) d 13.3, 22.7, 26.7, 33.6, 47.9, 51.2, 58.4, 69.7, 111.0, 116.2,
t
R
= 5.89. C26
H
29ClN
2 3
O
+
1
1
3
1.69–1.80 (m, 2H), 1.80–1.89 (m, 2H), 1.91–2.03 (m, 2H), 2.32–
2.40 (m, 2H), 2.63–2.72 (m, 2H), 3.18 (t, J = 9.1 Hz, 1H), 3.96 (t,
J = 6.2 Hz, 2H), 4.65 (br. s., 1H),6.95 (dd, J = 8.2, 0.8 Hz, 1H), 7.02
1
1
16.5, 120.6, 120.7, 126.0, 126.1, 126.5, 129.6, 129.7, 137.0,
37.4, 155.8, 163.3, 166.7.
(
td, J = 7.4, 1.0 Hz, 1H), 7.27–7.33 (m, 3H), 7.34–7.40 (m, 2H),
0
4
4
.4.3. 3-Chloro-N-{1-[1-(1,1 -biphenyl-2-yloxy)propan-2yl]piperidin-
7.42–7.46 (m, 1H), 7.47–7.52 (m, 2H), 7.52–7.57 (m, 1H), 7.77
(dq, J = 7.4, 1.0 Hz, 1H), 7.88 (t, J = 1.8 Hz, 1H). C NMR (75 MHz,
CDCl ) d 26.7, 29.7, 32.8, 33.4, 33.5, 50.8, 51.9, 55.0, 66.6, 112.6,
1
3
-yl}benzenesulfonamide (17)
Yellow oil, 120 mg following chromatographic purification over
3
silica gel with CH
2
Cl
S, MW 485.04, Monoisotopic Mass 484.16, [M+H]
) d 1.01 (d, J = 6.9 Hz, 3H), 1.28–
2
/MeOH (9/0.7); UPLC/MS purity 97%, t
R
= 5.82.
116.2, 116.5, 121.0, 126.8, 127.8, 128.6, 129.6, 129.7, 130.8,
131.0, 137.4, 137.4, 138.5, 155.8, 163.3, 166.6.
+
C
4
1
26
H
29ClN
2 3
O
1
85.3. H NMR (300 MHz, CDCl
.42 (m, 2H), 1.60–1.72 (m, 2H), 2.14–2.25 (m, 2H), 2.54–2.66
3
0
4.4.8. 4-Fluoro-N-{1-[3-(1,1 -biphenyl-2-yloxy)propyl]piperidin-4-yl}
(
m, 2H), 2.87–2.99 (m, 1H), 3.08–3.10 (m, 1H), 3.80–3.86 (m,
benzenesulfonamide (22)
1H), 3.91–3.97 (m, 1H), 4.57 (br.s., 1H), 6.93 (d, J = 8.2 Hz, 1H),
7.02 (td, J = 7.5, 1.2 Hz, 1H), 7.26–7.39 (m, 5H), 7.42–7.45 (m,
1H), 7.45–7.51 (m, 2H), 7.52–7.57 (m, 1H), 7.75 (dt, J = 7.8,
1.4 Hz, 1H), 7.87 (t, J = 1.8 Hz, 1H).
Yellow oil, 180 mg following chromatographic purification over
silica gel with CH
2
Cl
S, MW 468.58, Monoisotopic Mass 468.19, [M+H]
) d 1.36–1.51 (m, 2H), 1.70–1.79
2 R
/MeOH (9/0.7); UPLC/MS purity 99%, t = 5.52.
+
C
26 2 3
H29FN O
1
469.4. H NMR (300 MHz, CDCl
3
(
m, 2H), 1.80–1.88 (m, 2H), 1.91–2.00 (m, 2H), 2.33–2.39 (m,
0
4
4
.4.4. 4-Fluoro-N-{1-[1-(1,1 -biphenyl-2-yloxy)propan-2-yl]piperidin-
2H), 2.63–2.71 (m, 2H), 3.11–3.20 (m, 1H), 3.96 (t, J = 6.2 Hz, 2H),
4.63 (br.s., 1H), 6.95 (dd, J = 8.2, 0.8 Hz, 1H), 7.02 (td, J = 7.6,
1.0 Hz, 1H), 7.14–7.23 (m, 2H), 7.25–7.32 (m, 3H), 7.32–7.40 (m,
2H), 7.47–7.52 (m, 2H), 7.86–7.93 (m, 2H).
-yl}benzenesulfonamide) (18)
Yellow oil, 100 mg following chromatographic purification over
silica gel with CH
2
Cl
S, MW 468.58, Monoisotopic Mass 468.19, [M+H]
) d 1.00 (d, J = 6.7 Hz, 3H), 1.27–
2 R
/MeOH (9/0.7); UPLC/MS purity 97%, t = 5.45.
+
C
4
1
26
H
29FN
2 3
O
1
69.4. H NMR (300 MHz, CDCl
3
4.4.9. 3-Fluoro-N-(1-{2-hydroxy-3-[(propan-2-yl)phenoxy]propyl}
piperidin-4-yl)benzenesulfonamide (23)
.41 (m, 2H), 1.57–1.72 (m, 2H), 2.13–2.25 (m, 2H), 2.60 (t,
J = 12.3 Hz, 2H), 2.89–2.98 (m, 1H), 2.99–3.12 (m, 1H), 3.79–3.86
m, 1H), 3.90–3.98 (m, 1H), 4.56 (br. s, 1H), 6.93 (d, J = 8.2 Hz,
Yellow oil, 150 mg following chromatographic purification over
(
silica gel with CH
2
Cl
S, MW 450.57, Monoisotopic Mass 450.20, [M+H]
3
451.1. H NMR (300 MHz, CDCl ) d 1.20 (d, J = 6.9 Hz, 6H), 1.41–
2 R
/MeOH (9/0.7); UPLC/MS purity 99%, t = 5.24.
+
1
5
H), 7.03 (td, J = 7.4, 1.0 Hz, 1H) 7.12–7.21 (m, 2H), 7.24–7.39 (m,
H), 7.46–7.51 (m, 2H), 7.84–7.93 (m, 2H).
C
23 2 4
H31FN O
1
1
.60 (m, 2H), 1.75–1.86 (m, 2H), 2.04–2.16 (m, 1H), 2.30–2.41
4
.4.5. 3-Fluoro-N-(1-{3-[(2-isopropylphenoxy)]propyl}piperidin-4-yl)
(m, 1H), 2.51–2.56 (m, 2H), 2.72–2.76 (m, 1H), 2.88–2.91 (m,
1H), 3.19–3.34 (m, 2H), 3.48 (q, J = 7.0 Hz, 1H), 3.89–3.99 (m,
2H), 4.01–4.11 (m, 1H), 6.79–6.84 (m, 1H), 6.89–6.97 (m, 1H),
7.13 (td, J = 7.75, 1.68 Hz, 1H), 7.20 (dd, J = 7.5, 1.5 Hz, 1H), 7.26–
benzenesulfonamide (19)
Yellow oil, 130 mg following chromatographic purification over
silica gel with CH
2
Cl
S, MW 434.57, Monoisotopic Mass 434.20,
) d 1.20 (d, J = 7.0 Hz,
2
/MeOH (9/0.7); UPLC/MS purity 100%,
t
R
= 5.43. C23
H
31FN
2
O
3
7.32 (m, 1H), 7.50 (td, J = 8.01, 5.3 Hz, 1H), 7.60 (dt, J = 8.1,
+
1
13
[
M+H] 435.4. H NMR (300 MHz, CDCl
3
3
2.1 Hz, 1H), 7.69 (dt, J = 7.9, 1.1 Hz, 1H). C NMR (75 MHz, CDCl )
6
H), 1.44–1.56 (m, 2H), 1.78 (dd, J = 13.2, 3.2 Hz, 2H), 1.89–1.98
d 22.6, 26.9, 32.9, 33.1, 51.2, 60.7, 65.9, 70.2, 111.3, 114.1, 114.5,
(
m, 2H), 2.05 (t, J = 10.3 Hz, 2H), 2.45–2.53 (m, 2H), 2.73–2.82
119.7, 120.0, 122.6, 126.1, 126.6, 130.9, 131.0, 137.0, 143.3, 155.7.
Please cite this article in press as: Canale V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.057

V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
4.4.10. 4-Fluoro-N-(1-{2-hydroxy-3-[(propan-2-yl)phenoxy]propyl}
thepine or 10 mM of haloperidol were used in 5-HT2AR, 5-HT
6
R and
piperidin-4-yl)-benzenesulfonamide (24)
D2L assays, respectively. Each compound was tested in triplicate at
ꢀ11
ꢀ4
Yellow oil, 160 mg following chromatographic purification over
i
7–8 concentrations (10 –10 M). The inhibition constants (K )
3
7
silica gel with CH
2
Cl
S, MW 450.57, Monoisotopic Mass 450.20, [M+H]
) d 1.19 (d, J = 6.9 Hz, 6H), 1.47–
2
/MeOH (9/0.7); UPLC/MS purity 99%, t
R
= 5.03.
were calculated from the Cheng-Prusoff equation. Results were
expressed as means of at least three separate experiments.
+
C
4
1
23
H
31FN
2 4
O
1
51.1. H NMR (300 MHz, CDCl
.62 (m, 2H), 1.75–1.84 (m, 2H), 2.07–2.18 (m, 1H), 2.36 (t,
3
4.5.2. Functional in vitro evaluation
J = 10.1 Hz, 1H), 2.53–2.58 (m, 2H), 2.77 (d, J = 11.7 Hz, 1H), 2.93
d, J = 12.0 Hz, 1H), 3.16–3.22 (m, 1H), 3.24–3.33 (m, 1H), 3.89–
Cells (prepared with the use of Lipofectamine 2000) were main-
tained at 37 °C in a humidified atmosphere with 5% CO and were
2
(
4
6
.02 (m, 2H), 4.04–4.12 (m, 1H), 6.81 (dd, J = 8.1, 0.8 Hz, 1H),
.90–6.96 (m, 1H), 7.10–7.22 (m, 4H), 7.87–7.94 (m, 2H).
grown in Dulbeco’s Modifier Eagle Medium containing 10% dial-
ysed foetal bovine serum and 500 mg/mL G418 sulphate. For func-
tional experiments, cells were subcultured in 25 cm diameter
dishes, grown to 90% confluence, washed twice with pre warmed
to 37 °C phosphate buffered saline (PBS) and were centrifuged for
5 min (160g). The supernatant was aspirated, the cell pellet was
resuspended in stimulation buffer (1 ꢂ 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
4.4.11. 3-Chloro-N-{1-[3-(1,1-biphenyl-2-yloxy)2-hydroxypropyl]
piperidin-4-yl}benzenesulfonamide (25)
Yellow oil, 200 mg following chromatographic purification over
silica gel with CH
2
Cl
S, MW 501.04, Monoisotopic Mass 500.15,
M+H]+ 501.3. H NMR (300 MHz, CDCl
) d 1.39–1.54 (m, 2H),
3
2
/MeOH (9/0.7); UPLC/MS purity 100%,
t
[
R
= 5.38. C26
H
29ClN
2 4
O
1
1
1
3
.71–1.80 (m, 2H), 1.99 (td, J = 11.4, 2.4 Hz, 2H), 2.17–2.30 (m,
H), 2.32–2.43 (m, 2H), 2.57–2.65 (m, 1H), 2.72–2.81 (m, 1H),
.13–3.25 (m, 1H), 3.86–3.96 (m, 3H), 4.81 (br.s., 1H), 6.97 (dd,
7
on 5-HT R, the agonist, 5-carboxyamidotryptamine (5-CT;
EC50 = 1 nM) was used in submaximal concentration (10 nM) to
stimulate cAMP production. Cells (5 ml) were incubated with com-
pounds (5 ml)for 30 min at room temperature in 384-well white
opaque microtiter plate. After incubation, the reaction was stopped
and cells were lysed by the addition of 10 mL working solution (5 mL
Eu-cAMP and 5 mL ULight-anti-cAMP). The assay plate was incu-
bated for 1 h at room temperature. Time-resolved fluorescence res-
onance energy transfer (TR-FRET) was detected by an Infinite
M1000 Pro (Tecan) using instrument settings from LANCE cAMP
J = 8.2, 0.8 Hz, 1H), 7.04 (td, J = 7.4, 1.0 Hz, 1H), 7.26–7.34 (m,
3
7
1
6
1
H), 7.34–7.41 (m, 2H), 7.42–7.46 (m, 1H), 7.47–7.52 (m, 2H),
.52–7.57 (m, 1H), 7.76 (dq, J = 7.7, 1.0 Hz, 1H), 7.88 (t, J = 1.7 Hz,
H). 1 C NMR (75 MHz, CDCl
3
) d 29.7, 32.8, 33.0, 50.7, 51.2, 53.1,
3
0.4, 66.0, 70.9, 113.1, 121.5, 124.9, 126.9, 127.0, 127.9, 128.7,
29.6, 130.5, 130.8, 131.2, 132.7, 135.3, 138.4, 143.1, 155.5. Anal.
calcd for C26
2
H29ClN O
4
SꢃHCl: C: 58.10, H: 5.63, N: 5.21, S: 5.97;
2 4
Found C: 58.29, H: 5.36, N: 5.42, S: 5.77. M.p. for C26H29ClN O -
SꢃHCl: 198.1–200.3 °C.
detection kit manual.K
b
values were calculated from Cheng-Prusoff
equation specific for the analysis of functional inhibition curves:
b
K = IC50/(1 + A/EC50) where A is an agonist concentration, IC50 is
3
7
0
4.4.12. 4-Fluoro-N-{1-[3-(1,1 -biphenyl-2-yloxy)2-hydroxypropyl]
piperidin-4-yl}benzenesulfonamide (26)
Yellow oil, 170 mg following chromatographic purification over
silica gel with CH Cl /MeOH (9/0.7); UPLC/MS purity 99%, t = 5.38.
S, MW 484.58, Monoisotopic Mass 484.18, [M+H]
) d 1.38–1.54 (m, 2H), 1.69–1.79
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.
2
2
R
+
C
4
26
H
29FN
2 4
O
1
85.4. H NMR (300 MHz, CDCl
3
4.6. In vivo pharmacology
(
2
(
m, 2H), 1.98 (td, J = 11.4, 2.3 Hz, 1H), 2.16–2.30 (m, 2H), 2.31–
.43 (m, 2H), 2.56–2.65 (m, 1H), 2.73–2.81 (m, 1H), 3.11–3.21
m, 1H), 3.86–3.96 (m, 3H), 4.76 (br.s., 1H), 6.97 (dd, J = 8.1,
4.6.1. Forced swim test and tail suspension test in mice
4.6.1.1. Subject. The experiments were performed on male Swiss
albino (FST and respective locomotor activity test) or C57BL/6J
(TST and respective locomotor activity test) mice (22–26 g) pur-
chased from a licensed breeder Staniszewska (Ilkowice, Poland).
Mice were kept in groups of ten to Makrolon type 3 cages (dimen-
sions 26.5 ꢂ 15 ꢂ 42 cm). The animals were kept in an environ-
mentally 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 environment
for one week before commencement of the experiments. Standard
laboratory food (Ssniff M-Z) and filtered water were freely avail-
able. All the experimental procedures were approved by the I Local
Ethics Commission at the Jagiellonian University in Krakow.
0
7
.9 Hz, 1H), 7.04 (td, J = 7.5, 1.2 Hz, 1H), 7.14–7.23 (m, 2H), 7.26–
.34 (m, 3H), 7.34–7.40 (m, 2H), 7.46–7.52 (m, 2H), 7.85–7.93
(
m, 2H).
4
4
.5. In vitro evaluation
.5.1. Radioligand binding assays
Cell pellets were thawed and homogenized in 20 volumes of
assay buffer using an Ultra Turrax tissue homogenizer and cen-
trifuged twice at 35,000g for 20 min at 4 °C, with incubation for
1
5 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
5
mM MgCl
0 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl
R: 50 mM Tris–HCl, 0.5 mM EDTA and 4 mM MgCl
-HT7BR: 50 mM Tris–HCl, 4 mM MgCl , 10 mM pargyline and
.1% ascorbate; for dopamine D2LR: 50 mM Tris–HCl, 1 mM EDTA,
mM MgCl , 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl and 0.1%
2
, 10 mM pargyline and 0.1% ascorbate; for 5-HT2AR:
and 0.1% ascorbate;
, for
2
4.6.1.2. Forced swim test experimental procedure. All the experi-
ments were conducted in the light phase between 09.00 and
14.00 h. Each experimental group consisted of 7–10 animals/dose.
The animals were used only once. The experiments were per-
formed by an observer unaware of the treatment administered.
The experiment was carried out according to the method of Porsolt
et al. Swiss albino mice were individually placed in a glass cylin-
der (25 cm high; 10 cm in diameter) containing 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 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.
for 5-HT
6
2
5
0
4
2
2
2
ascorbate. All assays were incubated in a total volume of 200 ml
in 96-well microtitre plates for 1 h at 37 °C, except for 5-HT1AR that
was incubated for 1 h at room temperature. The process of equili-
bration is terminated by rapid filtration through Unifilter plates
with a 96-well cell harvester and radioactivity retained on the fil-
ters was quantified on a Microbeta plate reader. Non-specific bind-
3
8
ing was defined with 10 mM of 5-HT in 5-HT1AR and5-HT
7
R binding
experiments, whereas 10 mM of chlorpromazine, 10 mM of methio-
Please cite this article in press as: Canale V., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.057

1
0
V. Canale et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4
.6.1.3. Tail suspension test experimental procedure. The testing pro-
4.7.2. Experimental procedure
39
cedure was based on the method of Steru et al. An automated
device (Campden Instruments Ltd., United Kingdom) was used.
The mice were suspended by the tail with tape to an aluminium
hook connected to a strain gage, and were positioned 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 connected to com-
puter 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
The experiments were conducted based on the previously
4
0
reported procedures. At least 1 h before the start of the experi-
ment, rats were transferred to the experimental room for acclima-
tion. Rats were tested in a dimly lit (25 lx) ‘‘open field” apparatus
made of a dull gray plastic (66 ꢂ 56 ꢂ 30 cm). After each measure-
ment, the floor was cleaned and dried. Procedure consisted of
habituation to the arena (without any objects) for 5 min, 24 h
before the test and test session comprised of two trials separated
by an inter trial interval (ITI). For memory improvement paradigm,
24 h inter-trail interval was chosen. During the first trial (familiar-
ization, 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 pre-
sented 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 used were the glass beakers filled with the gravel
and the plastic bottles filled with the sand. The heights of the
objects were comparable (ꢄ12 cm) and the objects were heavy
enough not to be displaced by the animals. The sequence of pre-
sentations 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 sys-
tem. Based on exploration time (E) of two objects during T2, dis-
crimination index (DI) was calculated according to the formula:
3
0 ms.
4
.6.2. Spontaneous locomotor activity assay protocol
Locomotor activity was recorded with an Opto M3 multi-chan-
nel activity monitor (MultiDevice Software v.1.3, Columbus Instru-
ments). Swiss albino or C57BL/6J mice were individually placed in
plastic cages (22 ꢂ 12 ꢂ 13 cm) for 30 min habituation period, and
then the ambulation were counted from 2 to 6 min or 6 min, that is
the time equal to the observation period respectively in the forced
swim and tail suspension. The cages were cleaned up with 70%
ethanol after each mouse.
4.6.3. Drugs
DI = (E
B
ꢀ E
A A B
)/(E + A ). Tested compounds 20 and 25 as well as
The following drugs were used: escitalopram (hydrochloride;
the reference SB-269970 were administrated i.p. 30 min before T1.
Adamed, Pienkow, Poland), reboxetine (mesylate; Tocris Bio-
science, Bristol, UK), bupropion (hydrochloride; Tocris, Bristol,
UK) ant the tested compounds 20 and 25. All the compounds were
dissolved in distilled water immediately before administration and
were injected intraperitoneally (i.p.) in a volume of 10 ml/kg.
Reboxetine, bupropion, 20 and 25 were given 60 min while esci-
talopram 30 min before the test. In an interaction study the drugs
4.7.3. Statistics
Data on exploratory preference and distance travelled were
analyzed using two-way repeated measures ANOVAs with trial as
repeated factor; DI data were analyzed by one-way ANOVA fol-
lowed by Newman-Keuls post hoc test.
–
when administered at the same pre-treatment time – were given
in two separate injections (20 or 25 first, and immediately after-
wards an antidepressant). Control mice received vehicle according
to the same schedule. The doses of drugs refer to their salt forms.
Acknowledgments
This study was supported by the Polish National Science Center
Grant No DEC-2012/05/B/NZ7/03076.
4.6.4. Statistics
All the data are presented as the mean ± SEM. The data were
evaluated by an analysis of variance (ANOVA): one-way ANOVA
when one drug was given) or two-way ANOVA (when two drugs
were used) followed by Bonferroni’s multiple comparison test;
p < 0.05 was considered significant.
A. Supplementary data
(
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.03.057.
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