The impact of the halogen bonding on D2 and 5-HT1A/5-HT7 receptor activity of azinesulfonamides of 4-[(2-ethyl)piperidinyl-1-yl]phenylpiperazines with antipsychotic and antidepressant properties

Article abstract of DOI:10.1016/j.bmc.2017.04.046

A series of azinesulfonamides of long-chain arylpiperazine derivatives with semi-rigid alkylene spacer was designed, synthesized, and biologically evaluated using in vitro methods for their affinity for dopaminergic D₂ and serotoninergic 5-HT

Full text of DOI:10.1016/j.bmc.2017.04.046

Accepted Manuscript
The impact of the halogen bonding on D2 and 5-HT1A/5-HT7 receptor activity
of azinesulfonamides of 4-[(2-ethyl)piperidinyl-1-yl]phenylpiperazines with
antipsychotic and antidepressant properties
Anna Partyka, Rafał Kurczab, Vittorio Canale, Grzegorz Satała, Krzysztof
Marciniec, Agnieszka Pasierb, Magdalena Jastrzębska-Więsek, Maciej
Pawłowski, Anna Wesołowska, Andrzej J Bojarski, Paweł Zajdel
PII:
S0968-0896(16)30902-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.046
BMC 13716
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 October 2016
30 March 2017
20 April 2017
Please cite this article as: Partyka, A., Kurczab, R., Canale, V., Satała, G., Marciniec, K., Pasierb, A., Jastrzębska-
Więsek, M., Pawłowski, M., Wesołowska, A., Bojarski, A.J., Zajdel, P., The impact of the halogen bonding on
D2 and 5-HT1A/5-HT7 receptor activity of azinesulfonamides of 4-[(2-ethyl)piperidinyl-1-yl]phenylpiperazines with
antipsychotic and antidepressant properties, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
10.1016/j.bmc.2017.04.046
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The impact of the halogen bonding on D2 and 5-HT1A/5-HT7 receptor activity
of azinesulfonamides of 4-[(2-ethyl)piperidinyl-1-yl]phenylpiperazines with
antipsychotic and antidepressant properties
Anna Partyka,a Rafał Kurczab,b Vittorio Canale,c Grzegorz Satała,b Krzysztof Marciniec,d Agnieszka
Pasierb,c Magdalena Jastrzębska-Więsek,a Maciej Pawłowski,c Anna Wesołowska,a Andrzej J
Bojarski,b Paweł Zajdelc,*
a Department of Clinical Pharmacy, c Department of Medicinal Chemistry,
Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
b Institute of Pharmacology, Polish Academy of Sciences, Department of Medicinal Chemistry,
12 Smętna Street, 31-343 Krakow, Poland
d Department of Organic Chemistry, Medical University of Silesia,
4 Jagiellońska Street, 41-200 Sosnowiec, Poland
*Corresponding author:
Department of Medicinal Chemistry
Jagiellonian University Medical College
e-mail: pawel.zajdel@uj.edu.pl
Tel.: +48 12 620 54 50; fax: +48 12 620 54 05
1

Abstract
A series of azinesulfonamides of long-chain arylpiperazine derivatives with semi-rigid alkylene
spacer was designed, synthesized, and biologically evaluated using in vitro methods for their
affinity for dopaminergic D2 and serotoninergic 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptors.
Docking to homology models revealed a possible halogen bond formation in complexes of the most
potent ligands and the target receptors. The study allowed for the identification of compound 5-({4-
(2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(21),
which
behaved as D2, 5-HT1A and 5-HT7 receptor antagonist. In preliminary in vivo studies, compound 21
displayed distinct antipsychotic properties in the MK-801-evoked hyperactivity test in mice at a
dose of 10 mg/kg, and exerted antidepressant-like effect in a forced swim test in mice
(MED = 0.625 mg/kg, i.p.).
Keyword:
azinesulfonamides,
semi-rigid
long-chain
arylpiperazines,
multimodal
dopamine/serotonin ligands, multi-target ligands, halogen bonding, schizophrenia, depression.
2

1. Introduction
Despite recent advances in the pharmacology and neurophysiology which allowed for the
development of new therapeutic strategies, the outcome of pharmacological treatments of
schizophrenia as well as affective disorders e.g., depression, bipolar disorder still remains
unsatisfactory. Indeed, in the last decades a large amount of efforts were aimed to improve the
therapeutic efficiency of clinically used antipsychotics and antidepressants avoiding tolerance, long
time of onset of action or remission of a disease and to reduce the unwanted side effects (sexual
dysfunction, extrapyramidal symptoms, nausea/emesis, weight gain, endocrinopathies and
cardiovascular effects).1,2
Since schizophrenia displays high comorbidity with other serious CNS disorders (depression,
cognitive impairment and anxiety) a multi-target strategy might show a broad-based efficacy.3–5 The
essential characteristic of all currently used antipsychotics is their ability to block D2 receptors to
reverse positive symptoms of schizophrenia. It has been further found, that antagonism at 5-HT7
receptors (5-HT7Rs) may contribute to alleviation of negative symptoms (e.g., social withdrawal),6
while both antagonism at 5-HT7Rs and partial agonism at 5-HT1ARs may contribute to pro-
cognitive effects. Furthermore, there is strong evidence supporting the notation that antagonism at
5-HT7Rs as well as partial agonism at 5-HT1ARs may be involved in the antidepressant and/or
anxiolytic activity of second generation antipsychotics such as amisulpride, lurasidone and
cariprazine (Figure 1).7–9
Figure 1. Chemical structures of atypical antipsychotics lurasidone, aripiprazole and cariprazine.
3

Our research group has previously reported on the series of flexible quinoline- and isoquinoline-
sulfonamides of long-chain arylpiperazines (LCAPs) with halogen substituent at the
phenylpiperazine moiety which showed high affinity for 5-HT7 receptor and a mixed
5-HT2A/5-HT7/D2R antagonist profile, and displayed antidepressant and anxiolytic properties
(Figure 2).10–12
Figure 2. The chemical structure of compounds I–III as multimodal D2/5-HT1A/5-HT2A/5-HT7
receptor ligands.
Moreover, inspired by previous findings on a group of N-alkylated azinesulfonamides,13 we
identified
compound
III
(4-(4-{2-[4-(4-chlorophenyl)-piperazin-1-yl]-ethyl}-piperidine-1-
sulfonyl)-isoquinoline) with partially rigidified polimethylene spacer,10 which behaved as D2/
5-HT2A/5-HT7 receptor antagonist. Compound III showed antipsychotic-like properties in MK-801-
induced hyperlocomotor activity in mice (MED = 5 mg/kg, i.p), significant antidepressant-like
activity in the forced swim test (FST) in mice (MED = 30 mg/kg, i.p.), and anxiolytic-like effect in
the plus-maze test in rats (MED = 10 mg/kg, i.p.).
4

Figure 3. Structural modifications for the designed series of azinesulfonamide derivatives of 4-[(2-
ethyl)piperidin-1yl]phenylpiperazines.
Continuing study in this group of LCAP derivatives, we designed a new series of close analogs
of compound III with semi-rigid alkylene spacer (Figure 3). Structural modifications aimed to
verify an influence of the type of azinesulfonamide fragment as well as an impact of the halogen
substitution pattern at the phenylpiperazine moiety on the receptor affinity through the directional
interaction called halogen bonds. Herein, we report on the synthesis, in vitro evaluation for D2,
5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptors, followed by determination of in vitro functional
receptor profile for selected compounds. Finally, we present results of preliminary pharmacological
evaluation of the most in vitro active agents in mouse models of schizophrenia and depression.
2.
Chemistry
The synthesis of final compounds 10–24 was performed according to the Scheme 1. In the first
step, commercially available 1-Boc-protected-4-(2-hydroxyethyl)piperidine (1) was treated with
para-toluenesulfonyl chloride followed by nucleophilic substitution with the substituted
phenylpiperazines to obtain intermediates 2–9. After removal of Boc-protecting group, the resulted
secondary amines reacted with the selected azinesulfonyl chloride in basic condition yielding final
compounds 10–24.
5

Scheme 1. Synthesis of azinesulfonamides of LCAP with semi-rigid alkylene spacer 10–24.
Reagents and conditions: i) p-toluenesulfonyl chloride, 4-(dimethylamino)aniline, triethylamine,
CH2Cl2, 0°C, 12 h; ii) substituted phenylpiperazines, THF/Toluene (30/70, v/v), 70°C, 12 h;
iii) TFA/CH2Cl2 (80/20, v/v), rt, 2 h; iv) azinesulfonyl chloride, TEA, CH2Cl2, 0°C, 2–6 h.
The azinylsulfonyl chlorides were obtained from their respective bromo derivatives according to the
previously reported method.14 All of the final compounds were converted into their water-soluble
hydrochloride salts.
3. Molecular Modeling
Homology models built on D3 receptor crystal structure template developed and tested in our
previous studies,15–17 were used to investigate the molecular mechanism of binding of the
synthesized library of compounds for the interaction with D2, 5-HT1A, 5-HT7Rs and to support
results obtained from in vitro radioligand binding assays. The recently studied procedure involving
Quantum Mechanics/Molecular Mechanics-driven molecular docking with binding free energy
calculations was applied to assess the contribution of halogen bonding to the quality of binding
mode.18 To visualize (plotting interaction spheres) the possible contribution of halogen bonding for
resulting ligand-receptor complexes, the Halogen Bonding Webserver was used (access 19.03.2017,
http://www.halogenbonding.com/).19
6

4. In vitro pharmacology
4.1. In vitro radioligand binding evaluation
Radioligand binding assays were performed to determine the affinity and selectivity profiles of
the synthesized compounds in competition binding experiments for human dopaminergic D2 and
serotonin 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptors, which were all stably expressed in HEK293
cells, according to the previously published procedures.10,20,21 The inhibition constants (Ki) were
calculated from the Cheng-Prusoff equation.22 Results were expressed as means of at least three
separate experiments.
4.2. In vitro functional evaluation
The functional properties of selected compounds 16, 21 and 22 against the human D2, 5-HT7
and 5-HT1ARs were determined in functional cAMP cellular assays, performed at Eurofins Cerep
using CHO and HEK-293 cells, according to procedures described online at www.cerep.fr.
5.
In vivo pharmacology
The central activities of the most potent D2/5-HT1A/5-HT7 receptor antagonist (i.e. 16, 21 and
22) were tested in in vivo behavioral test commonly used to predict antipsychotic- and
antidepressant-like activity. MK-801-induced hyperactivity test in CD-1 mice was used to
determine the antipsychotic-like activity of the tested compounds while the potential antidepressant
properties were evaluated in the forced swim test (FST) in Swiss albino mice.23,24 Moreover, the
influence of the antipsychotic- and/or antidepressant-effective doses on the spontaneous locomotor
activity in mice was investigated in order to exclude the possibility of competing behaviors such as
general locomotor activity. Citalopram, aripiprazole and compound III were used as references.
7

6.
Results and Discussion
6.1. Structure-activity relationship studies
The newly synthesized azinesulfonamides 10–24 were evaluated in in vitro binding
experiments for their affinity for D2, 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7Rs. In general, binding
assays revealed that new compounds displayed high-to-low affinity for D2Rs (30–1821 nM) and
5-HT1A (Ki = 29–975 nM), high-to-moderate affinity for 5-HT7R (Ki = 14–209 nM), and finally low
affinity for 5-HT6Rs (Ki = 309–8279 nM) (Table 1). It is worth noting, that all the newly
synthesized compounds showed moderate affinity for 5-HT2AR (Ki = 139–691 nM) and displayed
lower affinity for these sites in comparison to compound III (Ki = 59 nM) (Table 1).
Following our previous studies,10,25 suggesting a preferential position of sulfonamide group
in α-position of the azinyl moiety, regardless localization of the sulfonamide group in pyridine or
benzene rings, 4-isoquinolyl and 5-quinolyl moieties were selected for design of analogs of
compound III. It was observed that a kind of azinyl moiety only slightly influenced affinity of
synthesized compounds for tested receptors.
Then, regarding the preliminary studies on the engagement of halogens in interaction of
LCAP derivatives with 5-HT1A and 5-HT7 receptors,26 a special interest was focused on impact of
halogen atoms (an influence of the kind of halogen and/or its position at the phenylpiperazine
moiety) in the binding of compounds 10–24 with D2, 5-HT1A, and 5-HT7 receptors. Although
halogen atoms have been routinely used in drug development processes to improve biological
activity for a given target(s) and/or pharmacokinetic properties (e.g., membrane permeability, oral
absorption), their role has been recently assigned to stabilize the ligand–receptor complexes via
formation of specific interactions called halogen bond.27,28
8

Table 1. The binding data of compound III and the synthesized compounds 10–24 for D2, 5-HT1A,
5-HT2A, 5-HT6 and 5-HT7Rs
Ki [nM]a
Cmpd
Q
R
D2
60
5-HT1A
3210
749
543
740
759
975
54
5-HT2A
59
5-HT6
16650
6712
2412
1020
1459
1863
445
5-HT7
47
IIIb
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4-isoquinolyl
5-quinolyl
4-Cl
H
1237
1218
141
223
418
76
646
410
152
139
156
197
157
665
691
530
161
286
371
336
179
209
174
37
4-isoquinolyl
5-quinolyl
H
4-Cl
5-quinolyl
4-F
29
4-isoquinolyl
5-quinolyl
4-F
14
3-Cl
37
4-isoquinolyl
5-quinolyl
3-Cl
37
32
1185
8279
3802
640
18
3-F
1821
1011
149
148
36
609
370
40
165
142
56
4-isoquinolyl
5-quinolyl
3-F
3-CF3
3-CF3
2,3-diCl
2,3-diCl
3,4-diCl
3,4-diCl
4-isoquinolyl
5-quinolyl
30
1016
401
52
45
55
4-isoquinolyl
5-quinolyl
30
29
309
39
121
145
257
324
791
52
4-isoquinolyl
1041
36
a Ki values are means of three independent binding experiments (SEM ≤ 21%);
b Data taken from Ref. 10
Binding data revealed that unsubstituted phenylpiperazine compounds 10 and 11 displayed low
affinity for all the tested receptors. On the other hand, the introduction of a chlorine atom in 4-
position increased the affinity for D2 and 5-HT7Rs up to 8- and 5-fold, respectively (10 vs 12).
9

Then, the replacement of the 4-chloro atom with the 4-fluoro was not preferred for the interaction
with D2R yet maintaining high affinity for 5-HT7Rs (13 vs 12). The higher affinity of compounds
III and 12 (with chlorine atom in 4-position) for D2Rs than unsubstituted and 4-fluoro analogs
might be explained by the ability of chlorine to stabilize the ligand-receptor (L-R) complex by the
formation of a halogen bond (X-bond distance = 2.95 Å, σ-hole angle = 171.87°) with the backbone
carbonyl group of Ser5.42 (Figure 4A). On the contrary, hydrophobic interactions (i.e., with
Ala5.46 and Ile3.40, Figure 4B) of compounds III and 14, seem to be more crucial for the
interaction with 5-HT7R (Ki = 47 and 14 nM, respectively). In line with in silico experiments
indicating a perturbation of L-R complex stability (lack of interaction with Asp3.32), both 4-chloro
(III) and 4-fluoro (14) derivatives displayed low affinity for 5-HT1ARs (Figure 4C).
Figure 4. A–C: Binding mode of compounds 11 (green), III (violet) and 14 (yellow) for D2, 5-HT7
and 5-HT1ARs, respectively; D–F: Binding mode of compounds 11 (green), 16 (cyan) and 18
(magenta) for D2, 5-HT7 and 5-HT1ARs respectively. The chlorine-oxygen theoretical interaction
spheres illustrate the projected qualities of the formed L-R halogen bonds.
10

Interestingly, the shift of the chlorine from 4- to 3-position increased the affinity for 5-HT1A and D2
sites (12 vs 15 and III vs 16). At the same time, this modification decreased the affinity for
5-HT2ARs by 3–4 fold. In consequence, compounds 15 and 16 were classified as D2/5-HT1A/5-HT7
receptor ligands. In contrast, an introduction of 3-fluorine atom at phenylpiperazine decreased
affinity for all of the tested receptors (17 vs 15). These findings were confirmed by molecular
modeling experiments revealing that only the presence of chlorine atom in 3-position stabilized the
L-R complex through the formation of halogen bond with Val5.39 residue of D2Rs (X-bond
distance = 2.95 Å, σ-hole angle = 171.87°) and with Thr5.39 residues of 5-HT7 and 5-HT1ARs (X-
bond distance = 2.88 and 3.83 Å, σ-hole angle = 155.80° and 137.46°, respectively) (Figure 4D–F).
It was found that increasing the electron withdrawing effect of the substituent in 3-position at the
phenylpiperazine moiety by the replacement of chlorine atom with the CF3 group decreased affinity
up to 3 fold for D2 and 5-HT7Rs (15 vs 19). Although introduction of a second chlorine atom in
2-position at the phenylpiperazine, yielding compound 21 and 22, created an additional halogen
bond with His6.55 (X-bond distance = 2.81 Å, σ-hole angle = 149.45°, Figure 5A), this
modification did not affect the receptor binding profile in comparison to 3-chloro counterpart (21 vs
15 or 22 vs 16). Thus, compounds 21 and 22 were classified as D2/5-HT1A/5-HT7 multi-receptor
ligands.
Figure 5. A–C: A superposition of the poses of compounds 15 (blue), 21 (red) and 23 (orange)
against putative halogen binding pocket interaction spheres for D2, 5-HT7 and 5-HT1ARs,
11

respectively. The chlorine-oxygen theoretical interaction spheres illustrate the projected qualities of
the formed L-R halogen bonds.
Further explanation results from the fact, that chlorine atom in 2-position of compound 21 was not
directly engaged in stabilization of ligand – 5-HT1A and 5-HT7 complexes. Consequently, chlorine
atom in 3-position stabilized the L–5-HT1A and 5-HT7R complexes through the formation of
halogen bonds with Thr5.39 (X-bond distance = 3.20 Å, σ-hole angle = 157.84° for 5-HT1AR, and
X-bond distance = 3.15 Å, σ-hole angle = 169.93° for 5-HT7R, respectively) (Figure 5B-C).
In contrary, introduction of the second chlorine atom in 4-position, giving 3-4-dichlorosubstituted
analogs 23 and 24, drastically decreased the affinity for 5-HT1A and D2Rs, yet maintaining a high
affinity for 5-HT7 site (21 vs 23 and 22 vs 24). This was confirmed by stabilization of the L-5-HT7R
complex by compound 23 through the formation of halogen bond with Thr5.39 (X-bond
distance = 3.31 Å; σ-hole angle = 172.12°) and Ser5.42 (X-bond distance = 2.84 Å, σ-hole
angle = 147.15°, Figure 5C).
Table 2. The antagonist activity of selected compounds for D2, 5-HT1A and 5-HT7Rs.
% inha
% inhb
% inhc
Compd
D2
85
88
5-HT1A
69
5-HT7
73
16
21
63
69
79
64
75
22
a inhibition of control agonist response (dopamine 30 nM) at 10-6M; performed at Eurofins Cerep
b inhibition of control agonist response (8-OH-DPAT 100 nM) at 10-6M; performed at Eurofins Cerep
c inhibition of control agonist response (serotonin 300 nM) at 10-6M; performed at Eurofins Cerep
In the next step, compounds displaying a mixed dopamine/5-HT activity were selected for
extended in vitro functional evaluation. It was found that tested compounds 16, 21 and 22 behaved
as moderate antagonist at the human 5-HT1A and 5-HT7Rs and were classified as potent antagonist
12

at the D2Rs (Table 2). Interestingly, in contrast to flexible azinesulfonamides of LCAPs with 2,3-
dichlorophenylpiperazine moiety (e.g., compound II, Figure 2), which behaved as 5-HT1AR partial
agonists, compounds 21 and 22 were classified as 5-HT1AR antagonists.
6.2. Behavioral evaluation
In view of the receptor profile and functional activity of the selected derivatives their potential
antipsychotic- and/or antidepressant-like properties were evaluated in behavioral tests in mice.
Acute administration of NMDA receptor antagonists, such as dizocilpine (MK-801), results in
behavioral syndrome which includes hyperlocomotion, stereotypic behaviors, and social and
cognitive deficits. These symptoms may be analogous to symptoms of schizophrenia in humans.29
Behavioral abnormalities induced in rodents by MK-801 are prevented by antipsychotic drugs.30
The binding data revealed that compounds 16, 21 and 22 displayed significant D2 receptor affinity,
thus their antipsychotic activity was studied in the MK-801-induced locomotor hyperactivity test in
CD-1 mice. Compounds 16 (30 mg/kg) and 21 (10 and 20 mg/kg) significantly attenuated the MK-
801-evoked increase of mice locomotor activity and the effect was similar to that produced by
reference compound III. On the other hand, compound 22 administered at doses of 5–20 mg/kg
produced no effects on the hyperactivity induced by MK-801. By comparison, the reference
antipsychotic drug, aripiprazole, at a dose of 0.5 mg/kg remarkably suppressed the stimulating
effect of dizocilpine (Figure 6). It is noteworthy that 16, 21 and aripiprazole given in antipsychotic
active doses remained without significant influence on the spontaneous locomotor activity of
animals, thus their effects in the MK-801-induced hyperactivity test seem to be specific (Table 1-
SI).
13

Figure 6. Effects of compounds 16, 21, 22, reference compound III and aripiprazole on the
hyperlocomotor activity induced by MK-801 in CD-1 mice. Data are presented as the mean
standard error of the mean of N = 8–10 animals per group.*p < 0.05, **p < 0.01, ***p < 0.001
compared with the respective vehicle group, followed by the Bonferroni’s comparison test after a
significant analysis of variance.
Next, we used the forced swim test to evaluate antidepressant-like activity of the tested derivatives.
The obtained results indicate that compounds 16 and 21 exhibit characteristic of antidepressant
drugs. In fact, 16 given at doses of 1.25 and 2.5 mg/kg (F(4,46) = 8.3915, p < 0.0001), and 21 at a
dose of 0.625 mg/kg (F(3,39) = 3.7577, p < 0.05) produced distinct antidepressant-like effects in
that test, significantly reducing by 36%, 32% and 23% the immobility time of animals (Figure 7).
14

Figure 7. Effects of the compounds 16, 21, citalopram and compound III 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 < 0.001 compared with the respective vehicle
group, followed by the Bonferroni’s comparison test after a significant analysis of variance.
Importantly, all of the tested compounds displayed their antidepressant-like activity at doses 12–48
times lower than the active dose of the reference compound III. It is worth to note that their potency
was similar to that of the clinically-used antidepressant citalopram, that administered at dose of 1.25
mg/kg shortened the immobility time of mice by 28% (F(3, 28) = 19.238, p < 0.0001). Moreover,
the antidepressant-like effects produced by all studied compounds seem to be specific, since the
effective doses of 16, 21 as well as citalopram had no influence on the spontaneous locomotor
activity of mice during initial 2–6 min session (i.e. at the time identical to the observation period in
the forced swim test) (data not shown).
It thus seems, that starting from compound III, classified as mixed D2/5-HT2A/5-HT7
antagonist, which showed antipsychotic-like properties in MK-801-induced hyperlocomotor activity
(MED = 5 mg/kg, i.p), and antidepressant-like activity in FST (MED = 30 mg/kg, i.p.), structural
modification in halogen substitution pattern led to compound 21 (D2/5-HT1A/5-HT7 antagonist),
which apart of its antipsychotic properties (MED = 10 mg/kg, i.p.) produced an antidepressant-like
activity at lower doses in FST (MED = 0.625 mg/kg).
15

7.
Conclusions
A small series of 15 azinesulfonamides of phenylpiperazine derivatives, based on compound
III with semi-rigid alkylene spacer, was synthesized and evaluated as multimodal
dopamine/serotonin receptor ligands. Structure-activity relationship studies supported with
molecular modeling approach revealed that the affinity of evaluated compounds for D2, 5-HT1A and
5-HT7 receptors was highly dependent upon formation of halogen interactions. The study allowed to
identify compound 21 which behaved as mixed D2/5-HT1A/5-HT7 receptor antagonist. Preliminary
pharmacological in vivo evaluation showed that compound 21 was active in MK-801-evoked
hyperactivity test in mice, and produced antidepressant-like activity in a mouse model of
depression. Further studies in the area of CNS agents with multiple mode of action might confirm
their
broad-based
efficacy
in
the
treatment
of
comorbid
symptoms
of
schizophrenia/depression/anxiety.
16

8.
8.1. Chemistry
Organic synthesis were carried out at ambient temperature, unless indicated otherwise. Organic
Experimental
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.
Mass spectra were recorded on a UPLC-MS/MS system consisted of a Waters ACQUITY®
UPLC® (Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer
(electrospray ionization mode ESI-tandem quadrupole). Chromatographic separations were carried
out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm, and 1.7 µm
particle size, equipped with Acquity UPLC BEH C18 VanGuard pre-column; 2.1 × 5 mm, and 1.7
µm particle size. The column was maintained 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 min-1. Eluent A: water/formic acid
(0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v). Chromatograms were made using Waters
eλ PDA detector. Spectra were analyzed in 200–700 nm range with 1.2 nm resolution and sampling
rate 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source
temperature 150 °C, desolvation temperature 350°C, desolvation gas flow rate 600 L h-1, cone gas
flow 100 L h-1, capillary potential 3.00 kV, cone potential 40 V. Nitrogen was used for both
nebulizing and drying gas. The data were obtained in a scan mode ranging from 50 to 2000 m/z in
time 1.0 s intervals. Data acquisition software was MassLynx V 4.1 (Waters). The UPLC/MS purity
of all the final compounds was confirmed to be 95% or higher.
13
1H NMR, C NMR and 19F NMR spectra were obtained in Varian BB 200 spectrometer using
TMS (0.00 ppm) as an internal standard in CDCl3 or DMSO-d6 and were recorded at 300, 75 and
282 MHz, respectively. The J values are reported in Hertz (Hz), and the splitting patterns are
17

designated as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td
(triplet of doublets), 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.
8.1.1.General procedure for the synthesis of intermediates 2–9
Boc-piperidineethanol 1 (23 mmol) was dissolved in pyridine (50 mL) and cooled down under
nitrogen. Then a tosyl chloride (27 mmol) was added portion-wise. The reaction was carried out for
3 h on bath-ice, then warm to room temperature and stirred for additional 10 h. The reaction
mixture was next extracted with CH2Cl2 (150 mL) and the organic phase was washed with 1M
KHSO4 (4 x 50 mL), water, brine, dried over Na2SO4, filtered and evaporated under pressure. The
obtained tosyl derivative were subsequently used without any further purification. Next, to a
solution of the differently substituted 4-phenylpiperazine (2 mmol) and TEA (20 mmol,) in a
mixture of THF/toluene (5 mL/10 mL), tosyl derivatives (2.5 mmol,) was added and the reaction
was stirred under reflux for 20 h. After solvent evaporation the crude product was purified on silica
gel column chromatography (CH2Cl2/MeOH) to yield Boc-protected piperidines 2–9.
8.1.2.Characterization data for intermediates 2–9
8.1.2.1. Tert-butyl-4-[2-(4-phenylpiperazin-1-yl)ethyl]piperidine-1-carboxylate (2)
White solid, 450 mg (yield 58%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.7); UPLC/MS purity 98%, tR = 4.86, C22H35N3O2, MW 373.53, Monoisotopic
Mass 373.27, [M+H]+ 374.4. 1H NMR (300 MHz, CDCl3) δ 1.06–1.21 (m, 2H), 1.45 (s, 9H), 1.46–
1.55–1.59 (m, 3H), 1.67–1.72 (m, 2H), 2.39–2.46 (m, 2H), 2.56–2.62 (m, 4H), 2.69 (t, J = 12.0 Hz,
2H), 3.17–3.24 (m, 4H), 4.00–4.10 (m, 2H), 6.85 (t, J = 7.3 Hz, 1H), 6.93 (d, J = 8.2 Hz, 2H), 7.22–
7.30 (m, 2H). Mp for C22H34ClN3O2: 148.3–149.8°C.
18

8.1.2.2. Tert-butyl-4-{2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl}piperidine-1-carboxylate (3)
White solid, 420 mg (yield 52%) following cchromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 98%, tR = 5.49, C22H34ClN3O2, MW 407.98,
1
Monoisotopic Mass 407.23, [M+H]+ 408.6. H NMR (300 MHz, CDCl3) δ 1.12–1.18 (m, 2H),
1.20–1.32 (m, 3H), 1.45 (s, 9H), 1.64–1.68 (m, 2H), 2.41–2.45 (m, 2H), 2.60–2.72 (m, 6H), 3.15–
3.18 (m, 4H), 4.05–4.23 (m, 2H), 6.81–6.85 (m, 2H), 7.18–7.26 (m, 2H). Mp for C22H34ClN3O2:
155.2–156.8°C.
7.1.2.3. Tert-butyl-4-{2-[4-(4-fluorophenyl)piperazin-1-yl]ethyl}piperidine-1-carboxylate (4)
White solid, 440 mg (yield 57%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 100%, tR = 4.78, C22H34FN3O2, MW 391.52,
1
Monoisotopic Mass 391.26, [M+H]+ 392.3. H NMR (300 MHz, CDCl3) δ 1.12–1.20 (m, 2H),
1.37–1.50 (m, 12H), 1.64–1.69 (m, 2H), 2.45 (t, J = 7.44 Hz, 2H), 2.58–2.72 (m, 6H), 3.13 (t,
J = 4.88 Hz, 4H), 4.05–4.08 (m, 2H), 6.84–6.88 (m, 2H), 6.92–6.98 (m, 2H). Mp for C22H34FN3O2:
179.1–180.2°C.
7.1.2.4. Tert-butyl-4-{2-[4-(3-chlorophenyl)piperazin-1-yl]ethyl}piperidine-1-carboxylate (5)
Yellow oil, 420 mg (yield 52%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 98%, tR = 5.17, C22H34ClN3O2, MW 407.98,
1
Monoisotopic Mass 407.23, [M+H]+ 408.3. H NMR (300 MHz, CDCl3) δ 1.10–1.25 (m, 2H),
1.45–1.49 (m, 12H), 1.64–1.69 (m, 2H), 2.40–2.44(m, 2H), 2.58–2.61 (m, 4H), 2.63–2.72 (m, 2H),
3.23–3.26 (m, 4H), 4.05–4.08 (m, 2H), 7.04–7.10 (m, 2H), 7.31–7.35 (m, 2H). Mp for
C22H34ClN3O2·HCl: 187.2–189.8°C.
7.1.2.5. Tert-butyl-4-{2-[4-(3-fluorophenyl)piperazin-1-yl]ethyl}piperidine-1-carboxylate (6)
White solid, 480 mg (yield 59%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 99%, tR = 4.63, C22H34FN3O2, MW 391.52, Monoisotopic
Mass 391.26, [M+H]+ 392.2. 1H NMR (300 MHz, CDCl3) δ 1.07–1.21 (m, 2H), 1.46 (s, 9H), 1.47–
19

1.52 (m, 2H), 1.63–1.72 (m, 3H), 2.38–2.45 (m, 2H), 2.55–2.60 (m, 4H), 2.69 (t, J = 12.0 Hz, 2H),
3.17–3.23 (m, 4H), 4.03–4.12 (m, 2H), 6.48–6.52 (m, 1H), 6.55–6.62 (m, 1H), 6.67 (dd, J = 8.2, 2.3
Hz, 1H), 7.14–7.22 (m, 1H). Mp for C22H34FN3O2: 182.4–183.2°C.
7.1.2.6. Tert-butyl-4-(2-{4-[3-(trifluoro)phenyl]piperazin-1-yl}ethyl)piperidine-1-carboxylate
(7)
White solid, 430 mg (yield 52%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.3); UPLC/MS purity 99%, tR = 5.49, C22H34F3N3O2, MW 441.53,
1
Monoisotopic Mass 441.26, [M+H]+ 442.3. H NMR (300 MHz, CDCl3) δ 1.08–1.25 (m, 2H),
1.45–1.48 (m, 12H), 1.65–1.69 (m, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.58–2.61 (m, 4H), 2.68 (t,
J = 12.0 Hz, 2H), 3.24 (t, J = 4.8 Hz, 4H), 4.05–4.07 (m, 2H), 7.04–7.10 (m, 3H), 7.35 (t, J = 7.9
Hz, 1H). Mp for C22H34F3N3O2: 178.2–179.8°C.
7.1.2.7. Tert-butyl-4-{2-[4-(2,3-dichlorophenyl)piperazin-1-yl]ethyl}piperidine-1-carboxylate
(8)
White solid, 500 mg (yield 59%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 99%, tR = 5.78, C22H33Cl2N3O2, MW 442.42,
1
Monoisotopic Mass 441.19, [M+H]+ 442.2. H NMR (300 MHz, CDCl3) δ 1.12–1.20 (m, 2H),
1.45–1.50 (m, 12H), 1.65–1.69 (m, 2H), 2.44 (t, J = 7.4 Hz, 2H), 2.63–2.73 (m, 6H), 3.07–3.11 (m,
4H), 4.03–4.07 (m, 2H), 6.92–6.98 (m, 1H), 7.10–7.17 (m, 2H). Mp for C22H33Cl2N3O2: 202.5–
204.3°C.
7.1.2.8. Tert-butyl-4-{2-[4-(3,4-dichlorophenyl)piperazin-1-yl]ethyl}piperidine-1-carboxylate
(9)
White solid, 480 mg (yield 57%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.3); UPLC/MS purity 100%, tR = 5.67, C22H33Cl2N3O2, MW 442.42,
20

1
Monoisotopic Mass 441.19, [M+H]+ 442.3. H NMR (300 MHz, CDCl3) δ 1.12–1.18 (m, 2H),
1.45–1.49 (m, 12H), 1.64–1.68 (m, 2H), 2.42 (t, J = 7.69 Hz, 2H), 2.55–2.58 (m, 4H), 2.68 (t,
J = 12.5 Hz, 2H), 3.15 (t, J = 5.1 Hz, 4H), 4.02–4.06 (m, 2H), 6.73 (dd, J = 8.9, 2.8 Hz, 1H), 6.94
(d, J = 2.8 Hz, 1H), 7.24–7.28 (m, 1H). Mp for C22H33Cl2N3O2: 205.5–206.9°C.
7.1.3.General procedure for the synthesis of final compounds 10–24
In the next stage intermediates 2–9 were converted into their TFA salts by treatment with a mixture
of TFA/CH2Cl2 (4 mL /1 mL). The excess of reagent and solvent were removed under reduced
pressure and left under vacuum overnight. Then, a mixture of the proper secondary amine (0.76
mmol) in CH2Cl2 (8 mL), and TEA (3.04 mmol) was cooled down (ice bath), and the selected
azinesulfonyl chloride (0.91 mmol) was added in one portion. The reaction mixture was stirred for
2-6 hours under cooling. Then, the solvent was evaporated and the sulfonamides were purified by
column chromatography using CH2Cl2/MeOH as eluting system. Final compounds were obtained as
hydrochloride salts by treatment with a solution of 1.25 M HCl in MeOH.
7.1.4.Characterization data for final compounds 10–24
7.1.4.1. 5-({4-[2-(4-phenylpiperazin-1-yl)ethyl]piperidin-1-yl}sulfonyl)quinoline (10)
White solid, 300 mg (yield 87%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.7); UPLC/MS purity 100%, tR = 4.19, C26H32N4O2S, MW 464.62,
Monoisotopic Mass 464.22, [M+H]+ 465.1. 1H NMR (300 MHz, CDCl3) δ 1.26–1.47 (m, 5H), 1.74
(d, J = 10.6 Hz, 2H), 2.30–2.37 (m, 2H), 2.51–2.55 (m, 4H), 3.12–3.19 (m, 4H), 3.85 (t, J = 11.7
Hz, 2H), 6.81–6.93 (m, 3H), 7.21–7.28 (m, 2H), 7.55 (q, J = 2.8 Hz, 1H), 7.77–7.82 (m, 1H), 8.22
(dd, J = 7.4, 1.2 Hz, 1H), 8.32 (dd, J = 8.46, 1.2 Hz, 1H), 9.00 (dd, J = 4.1, 1.5 Hz, 1H), 9.13 (ddd,
J = 8.7, 1.0, 0.7 Hz, 1H). Mp for C26H32N4O2S·2HCl: 186.4–188.0°C.
7.1.4.1. 4-({4-[2-(4-phenylpiperazin-1-yl)ethyl]piperidin-1-yl}sulfonyl)isoquinoline (11)
White solid, 280 mg (yield 84%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.7); UPLC/MS purity 100%, tR = 4.48, C26H32N4O2S, MW 464.62,
21

Monoisotopic Mass 464.22, [M+H]+ 465.5. 1H NMR (300 MHz, CDCl3) δ 1.28–1.48 (m, 5H), 1.75
(d, J = 9.9 Hz, 2H), 2.31–2.38 (m, 2H), 2.51–2.55 (m, 4H), 2.56–2.62 (m, 2H), 3.12–3.18 (m, 4H),
3.90 (d, J = 12.3 Hz, 2H), 6.81–6.92 (m, 3H), 7.21–7.28 (m, 2H), 7.71–7.78 (m, 1H), 7.84–7.91 (m,
1H), 8.09 (d, J = 7.6 Hz, 1H), 8.72 (d, J = 8.8 Hz, 1H), 9.08 (s, 1H), 9.40 (s, 1H). Mp for
C26H32N4O2S·2HCl: 187.0–188.1°C.
7.1.4.2. 5-({4-(2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(12)
White solid, 310 mg (yield 88%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.7); UPLC/MS purity 100%, tR = 4.77, C26H31ClN4O2S, MW 499.07,
1
Monoisotopic Mass 498.19, [M+H]+ 499.3. H NMR (300 MHz, CDCl3) δ 1.25–1.30 (m, 3H),
1.39–1.45 (m, 2H), 1.71–1.75 (m, 2H), 2.33 (t, J = 7.4 Hz, 2H), 2.46–2.53 (m, 6H), 3.11 (t, J = 4.8
Hz, 4H), 3.81–3.85 (m, 2H), 6.78–6.83 (m, 2H), 7.15–7.20 (m, 2H), 7.55 (q, J = 2.8 Hz, 1H), 7.77–
7.82 (m, 1H), 8.22 (dd, J = 7.4, 1.2 Hz, 1H), 8.32 (dd, J = 8.46, 1.2 Hz, 1H), 9.00 (dd, J = 4.1, 1.5
Hz, 1H), 9.13 (ddd, J = 8.7, 1.0, 0.7 Hz, 1H). Mp for C26H31ClN4O2S·2HCl: 197.1–199.2°C.
7.1.4.3. 5-({4-(2-[4-(4-Fluorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(13)
White solid, 270 mg (yield 77%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/1); UPLC/MS purity 100%, tR = 4.38, C26H31FN4O2S, MW 482.61, Monoisotopic
Mass 482.22, [M+H]+ 483.4. 1H NMR (300 MHz, CDCl3) δ 1.25–1.30 (m, 3H), 1.42–1.52 (m, 2H),
1.72–1.75 (m, 2H), 2.35 (t, J = 7.7 Hz, 2H), 2.42–2.53 (m, 6H), 3.09 (t, J = 4.6 Hz, 4H), 3.83 (d,
J = 12.3 Hz, 2H), 6.84–6.87 (m, 2H), 6.92–6.97 (m, 2H), 7.55 (q, J = 4.3 Hz, 1H), 7.77–7.82 (m,
1H), 8.23 (dd, J = 7.4, 1.2 Hz, 1H), 8.35 (dd, J = 8.4, 1.2 Hz, 1H), 9.01 (dd, J = 4.3, 2.2 Hz, 1H),
9.12 (dd, J = 8.7, 2.2 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ 29.2, 31.3, 32.4, 45.7, 46.5, 50.8,
53.4, 115.4, 115.8, 116.1, 118.4 (d, J (C–F) = 8.3 Hz), 124.2, 124.8, 131.0, 131.4, 131.8, 134.0,
138.7, 143.3, 146.6, 148.8, 155.5. 19F NMR (282 MHz, DMSO-d6) δ -123.9. Anal. calcd for
22

C26H31FN4O2S·2HCl: C: 56.21, H: 5.99, N: 10.09, S: 5.77; Found C: 56.23, H: 6.23, N: 9.85, S:
5.80. Mp for C26H31FN4O2S·2HCl: 187.6–189.8°C.
7.1.4.4. 4-({4-(2-[4-(4-Fluorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)isoquinoline
(14)
White solid, 310 mg (yield 88%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 98%, tR = 4.62, C26H31FN4O2S, MW 482.61,
1
Monoisotopic Mass 482.22, [M+H]+ 483.4. H NMR (300 MHz, CDCl3) δ 1.21–1.39 (m, 3H),
1.42–1.47 (m, 2H), 1.66–1.76 (m, 2H), 2.32–2.37 (m, 2H), 2.32–2.60 (m, 6H), 3.06–3.10 (m, 4H),
3.90 (d, J = 12.3 Hz, 2H), 6.82–6.88 (m, 2H), 6.90–6.99 (m, 2H), 7.74 (td, J = 6.9, 1.2 Hz, 1H),
7.88 (td, J = 7.4, 1.5 Hz, 1H), 8.08 (dd, J = 8.2, 0.7 Hz, 1H), 8.71 (dd, J = 8.4, 0.7 Hz, 1H), 9.07 (s,
1H), 9.40 (s, 1H). Mp for C26H31FN4O2S·2HCl: 189.5–191.6°C.
7.1.4.5. 5-({4-(2-[4-(3-Chlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(15)
White solid, 230 mg (yield 65%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.3); UPLC/MS purity 98%, tR = 5.27, C26H31ClN4O2S, MW 499.07,
1
Monoisotopic Mass 498.19, [M+H]+ 499.3. H NMR (300 MHz, CDCl3) δ 1.25–1.38 (m, 3H),
1.43–1.46 (m, 2H), 1.72–1.76 (m, 2H), 2.41 (t, J = 7.1 Hz, 2H), 2.54–2.68 (m, 4H), 2.74 (t, J = 12.0
Hz, 2H), 3.17 (t, J = 4.6 Hz, 4H), 3.96–4.00 (m, 2H), 6.65–6.79 (m, 1H), 6.82 (t, J = 2.0 Hz, 1H),
7.17 (t, J = 8.2 Hz, 2H), 7.54 (q, J = 8.7 Hz, 1H), 7.77–7.83 (m, 1H), 8.23 (dd, J = 7.4, 1.2 Hz, 1H),
8.34 (dd, J = 8.4, 1.2 Hz, 1H), 9.01 (dd, J = 4.1, 1.5 Hz, 1H), 9.13 (dd, J = 8.9, 1.2 Hz, 1H). Mp for
C26H31ClN4O2S·2HCl: 167.9–170.1°C.
7.1.4.6. 4-({4-(2-[4-(3-Chlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)isoquinoline
(16)
23

White solid, 210 mg (yield 60%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 99%, tR = 5.38, C26H31ClN4O2S, MW 499.07,
1
Monoisotopic Mass 498.19, [M+H]+ 499.3. H NMR (300 MHz, CDCl3) δ 1.23–1.30 (m, 3H),
1.42–1.45 (m, 2H), 1.73–1.76 (m, 2H), 2.35 (t, J = 7.1 Hz, 2H), 2.50–2.59 (m, 6H), 3.16 (t, J = 5.1
Hz, 4H), 3.91 (d, J = 12.3 Hz, 2H), 6.73–6.80 (m, 2H), 6.84 (t, J = 2.3 Hz, 1H), 7.18 (t, J = 8.2 Hz,
1H), 7.77 (td, J = 8.2, 1.0 Hz, 1H), 7.88 (td, J = 8.4, 1.5 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 8.72 (dd,
13
J = 7.6, 1.0 Hz, 1H), 9.07 (s, 1H), 9.41 (s, 1H). C NMR (75 MHz, DMSO-d6) δ 29.4, 31.3, 32.7,
45.2, 46.7, 50.6, 53.5, 114.6, 115.7, 119.6, 121.7, 128.4, 128.5, 129.4, 130.7, 131.1, 132.6, 134.4,
135.4, 149.4, 151.2, 153.9. Anal. calcd for C26H31ClN4O2S·2HCl·H2O:C: 52.93, H: 5.98, N: 9.50,
S: 5.43; Found C: 52.57, H: 5.93, N: 9.36, S: 5.36. Mp for C26H31ClN4O2S·2HCl·H2O: 187.8–
190.2°C.
7.1.4.7. 5-({4-(2-[4-(3-Fluorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(17)
White solid, 350 mg (yield 89%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 99%, tR = 4.25, C26H31FN4O2S, MW 482.61,
1
Monoisotopic Mass 482.22, [M+H]+ 483.2. H NMR (300 MHz, CDCl3) δ 1.22–1.34 (m, 3H),
1.38– 1.47 (m, 2H), 1.73 (d, J = 9.9 Hz, 2H), 2.29–2.37 (m, 2H), 2.46–2.57 (m, 6H), 3.11–3.19 (m,
4H), 3.85 (d, J = 12.3 Hz, 2H), 6.47–6.58 (m, 2H), 6.64 (dd, J = 8.5, 2.1 Hz, 1H), 7.12–7.21 (m,
1H), 7.54 (q, J = 8.7 Hz, 1H), 7.77–7.83 (m, 1H), 8.23 (dd, J = 7.4, 1.2 Hz, 1H), 8.34 (dd, J = 8.4,
1.2 Hz, 1H), 9.01 (dd, J = 4.1, 1.5 Hz, 1H), 9.13 (dd, J = 8.9, 1.2 Hz, 1H). Mp for
C26H31FN4O2S·2HCl: 188.5–190.3°C.
7.1.4.8. 4-({4-(2-[4-(3-Fluorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)isoquinoline
(18)
24

White solid, 300 mg (yield 86%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 99%, tR = 4.72, C26H31FN4O2S, MW 482.61,
1
Monoisotopic Mass 482.22, [M+H]+ 483.3. H NMR (300 MHz, CDCl3) δ 1.27–1.34 (m, 3H),
1.39–1.47 (m, 2H), 1.70–1.80 (m, 2H), 2.30–2.37 (m, 2H), 2.49–2.54 (m, 4H), 2.54–2.61 (m, 2H),
3.13–3.18 (m, 4H), 3.86–3.95 (m, 2H), 6.47–6.59 (m, 2H), 6.64 (dd, J = 7.9, 2.1 Hz, 1H), 7.17 (td,
J = 8.2, 7.0 Hz, 1H), 7.75 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.88 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H), 8.09
(d, J = 8.2 Hz, 1H), 8.69–8.74 (m, 1H), 9.07 (s, 1H), 9.41 (s, 1H). Mp for C26H31FN4O2S·2HCl:
188.2–190.0°C.
7.1.4.9. 5-({4-(2-(4-[3-(Trifluoromethyl)phenyl]piperazin-1-yl)ethyl)piperidin-1-yl}sulfonyl)
quinoline (19)
White solid, 230 mg (yield 65%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 100%, tR = 5.11, C27H31F3N4O2S, MW 532.62,
1
Monoisotopic Mass 532.21, [M+H]+ 533.3. H NMR (300 MHz, CDCl3) δ 1.25–1.31 (m, 3H),
1.46–1.49 (m, 2H), 1.72–1.75 (m, 2H), 2.30–2.36 (m, 2H), 2.38–2.54 (m, 6H), 3.22–3.26 (m, 4H),
3.81–3.86 (m, 2H), 7.02–7.05 (m, 3H), 7.33 (t, J = 7.4 Hz, 1H), 7.54 (q, J = 8.7 Hz, 1H), 7.77–7.83
(m, 1H), 8.23 (dd, J = 7.4, 1.2 Hz, 1H), 8.34 (dd, J = 8.4, 1.2 Hz, 1H), 9.01 (dd, J = 4.1, 1.5 Hz,
13
1H), 9.13 (dd, J = 8.9, 1.2 Hz, 1H). C NMR (75 MHz, DMSO-d6) δ 29.4, 31.3, 32.4, 45.1, 45.7,
50.5, 53.8, 116.3, 117.6, 121.2, 124.5, 128.6, 129.2, 129.7, 130.1, 131.2, 131.6, 132.1, 134.3, 144.0,
149.7, 158.1. Anal. calcd for C27H31F3N4O2S·2HCl·H2O: C: 50.55, H: 5.81, N: 8.73, S: 5.00; Found
C: 50.90, H: 5.78, N: 8.79, S: 5.28. Mp for C27H31F3N4O2S 2HCl·H2O: 179.4–181.2°C.
7.1.4.10.4-({4-(2-(4-[3-(Trifluoromethyl)phenyl]piperazin-1-yl)ethyl)piperidin-1-yl}sulfonyl)
isoquinoline (20)
White solid, 290 mg (yield 82%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.7); UPLC/MS purity 98%, tR = 5.42, C27H31F3N4O2S, MW 532.62,
25

1
Monoisotopic Mass 532.21, [M+H]+ 533.3. H NMR (300 MHz, CDCl3) δ 1.23–1.30 (m, 3H),
1.42–1.44 (m, 2H), 1.72–1.76 (m, 2H), 2.30–2.36 (m, 2H), 2.49–2.60 (m, 6H), 3.11–3.14 (m, 4H),
3.88–3.93(m, 2H), 6.69 (dd, J = 8.9, 3.0 Hz, 2H), 6.91 (d, J = 2.8 Hz, 1H), 7.23–7.27 (m, 1H), 7.74
(td, J = 6.9, 1.0 Hz, 1H), 7.88 (td, J = 6.9, 1.2 Hz, 1H), 8.09 (dd, J = 8.4, 0.7 Hz, 1H), 8.71 (dd,
J = 8.7, 0.7 Hz, 1H), 9.07 (s, 1H), 9.41 (s, 1H). Mp for C27H31F3N4O2S·2HCl: 174.8–176.8°C.
7.1.4.11.5-({4-(2-[4-(2,3-Dichlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(21)
White solid, 310 mg (yield 88%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.7); UPLC/MS purity 100%, tR = 5.23, C26H30Cl2N4O2S, MW 533.51,
1
Monoisotopic Mass 532.15, [M+H]+ 533.3. H NMR (300 MHz, CDCl3) δ 1.23–1.30 (m, 3H),
1.43–1.49 (m, 2H), 1.72–1.75 (m, 2H), 2.37–2.41 (m, 2H), 2.50–2.57 (m, 6H), 3.03–3.10 (m, 4H),
3.81–3.85 (m, 2H), 6.91–6.94 (dd, J = 6.6, 2.8 Hz, 1H), 7.10–7.25 (m, 2H), 7.55 (q, J = 4.1 Hz,
1H), 7.77–7.83 (m, 1H), 8.24 (dd, J = 7.1, 1.2 Hz, 1H), 8.33 (dd, J = 8.4, 1.2 Hz, 1H), 9.01 (dd,
J = 4.3, 1.8 Hz, 1H), 9.15 (dd, J = 8.7, 1.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 31.7, 33.0, 33.6,
45.9, 51.2, 53.3, 55.8, 118.5, 122.5, 124.6, 124.8, 127.4, 127.6, 130.4, 133.4, 133.6, 134.0, 135.6,
148.6, 151.1, 151.2. Anal. calcd for C26H30Cl2N4O2S·2HCl·2H2O: C: 48.61, H: 5.65, N: 8.72, S:
4.99; Found C: 48.85, H: 5.59, N: 8.42, S: 5.24.Mp for C26H30Cl2N4O2S·2HCl·2H2O: 173.9–
175.2°C.
7.1.4.12.4-({4-(2-[4-(2,3-Dichlorophenyl)iperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)
isoquinoline (22)
White solid, 310 mg (yield 88%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 100%, tR = 5.44, C26H30Cl2N4O2S, MW 533.51,
1
Monoisotopic Mass 532.15, [M+H]+ 533.3. H NMR (300 MHz, CDCl3) δ 1.23–1.26 (m, 3H),
1.40–1.45 (m, 2H), 1.73–1.77 (m, 2H), 2.35–2.40 (m, 2H), 2.53–2.57 (m, 6H), 3.03–3.10 (m, 4H),
26

3.89–3.93 (m, 2H), 6.91–6.96 (dd, J = 6.6, 3.0 Hz, 2H), 7.12–7.24 (m, 2H), 7.73 (td, J = 8.2, 1.2
Hz, 1H), 7.87 (td, J = 8.4, 1.5 Hz, 1H), 8.08 (dd, J = 8.2, 1.0 Hz, 1H), 8.72 (dd, J = 8.7, 1.0 Hz,
1H), 9.07 (s, 1H), 9.40 (s, 1H). Mp for C26H30Cl2N4O2S·2HCl: 198.9–201.9°C.
7.1.4.13.5-({4-(2-[4-(3,4-Dichlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)quinoline
(23)
White solid, 220 mg (yield 63%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); UPLC/MS purity 99%, tR = 5.23, C26H30Cl2N4O2S, MW 533.51,
1
Monoisotopic Mass 532.15, [M+H]+ 533.3. H NMR (300 MHz, DMSO-d6) δ 1.23–1.30 (m, 3H),
1.41–1.47 (m, 2H), 1.71–1.75 (m, 2H), 2.31–2.36 (m, 2H), 2.46–2.50 (m, 6H), 3.13–3.19 (m, 4H),
3.81–3.85 (m, 2H), 6.70 (dd, J = 8.9, 2.8 Hz, 1H), 6.92 (d, J = 2.8 Hz, 1H), 7.23–7.26 (m, 1H), 7.55
(q, J = 4.1 Hz 1H), 7.77–7.82 (m, 1H), 8.22 (dd, J = 8.4, 1.2 Hz, 1H), 9.34 (dd, J = 8.4, 1.2 Hz, 1H),
13
8.99 (dd, J = 4.1, 1.54 Hz, 1H), 9.13 (dd, J = 8.9, 1.0 Hz, 1H). C NMR (75 MHz, DMSO-d6) δ
29.2, 31.3, 32.4, 45.1, 45.7, 50.4, 53.5, 116.2, 117.4, 121.2, 124.0, 124.7, 130.7, 131.1, 131.5,
1321.1, 132.3, 133.8, 137.3, 144.8, 149.7, 149.7. Anal. calcd for C26H30Cl2N4O2S·2HCl·2H2O: C:
48.61, H: 5.65, N: 8.72, S: 4.99; Found C: 48.92, H: 5.61, N: 8.37, S: 4.89. Mp for
C26H30Cl2N4O2S·2HCl·2H2O: 172.5–174.1°C.
7.1.4.14.4-({4-(2-[4-(3,4-Dichlorophenyl)piperazin-1-yl]ethyl)piperidin-1-yl}sulfonyl)
isoquinoline (24)
White solid, 270 mg (yield 77%) following chromatographic purification over silica gel with
CH2Cl2/MeOH (9/0.5); LC/MS purity 98%, tR = 5.43, C26H30Cl2N4O2S, MW 533.51, Monoisotopic
Mass 532.15, [M+H]+ 533.3. 1H NMR (300 MHz, CDCl3) δ 1.23–1.30 (m, 3H), 1.40–1.44 (m, 2H),
1.72–1.76 (m, 2H), 2.34 (t, J = 7.1 Hz, 2H), 2.49–2.59 (m, 6H), 3.12 (t, J = 5.1 Hz, 4H), 3.88–
3.92(m, 2H), 6.70 (dd, J = 8.9, 2.8 Hz, 1H), 6.91 (d, J = 2.8 Hz, 1H), 7.73 (s, 1H), 7.74 (td, J = 8.2,
27

1.02 Hz, 1H), 7.89 (td, J = 8.4, 1.2 Hz, 1H), 8.09 (dd, J = 8.2 ,1.2 Hz, 1H), 8.71 (dd, J = 8.4, 0.7 Hz,
1H), 9.07 (s, 1H), 9.40 (s, 1H). Mp for C26H30Cl2N4O2S 2HCl: 163.3–165.4°C.
7.2. Molecular modeling
The 3-dimensional structures of the ligands were prepared using LigPrep v3.6,31 and the appropriate
ionization states at pH = 7.4 were assigned using Epik v3.4.32 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 centering the grid box with a
size of 12 Å on Asp3.32. Docking was performed by procedure involves quantum-polarized ligand
docking (QPLD) to obtain the QM-derived ligand atomic charges in the protein environment at the
B3PW91/cc-pVTZ level and the MM/GBSA algorithm to calculate the binding free energies, which
performance was recently evaluated for docking of halogenated ligands.18
7.3. In vitro pharmacology
7.3.1.Radioligand binding assay protocol
Radioligand binding assays were employed to determine the affinity and selectivity profiles of
the synthesized compounds in competition binding experiments for human serotonin, 5-HT7b,
5-HT1A, 5-HT2A, 5-HT6 and D2L receptors, which were all stably expressed in HEK293 cells.
According to the previously published procedures, the experiments were carried out using [3H]-5-
CT (39.2 Ci/mmol), [3H]-8-OH-DPAT (187 Ci/mmol), [3H]-Ketanserin (66.9 Ci/mmol), [3H]-LSD
(85.2 Ci/mmol) and [3H]-Raclopride (74.4 Ci/mmol) for 5-HT7, 5-HT1A, 5-HT2A, 5-HT6 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 35 000 g 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 µM 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 µM pargyline and 0.1% ascorbate; for dopamine D2LR: 50 mM Tris–HCl, 1 mM
28

EDTA, 4 mM MgCl2, 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate. All assays
were incubated in a total volume of 200 µl 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 equilibration is terminated
by rapid filtration through Unifilter plates with a 96-well cell harvester and radioactivity retained on
the filters was quantified on a Microbeta plate reader. Non-specific binding is defined with10 µM of
5-HT in 5-HT1AR and 5-HT7R binding experiments, whereas10 µM of methiothepine or 1 µM of
(+)butaclamol were used in 5-HT6R and D2L assays, respectively. Each compound was tested in
triplicate at 7–8 concentrations (10–11–10–4 M). The inhibition constants (Ki) were calculated from
the Cheng-Prusoff equation. Results were expressed as means of at least three separate experiments.
Membrane preparation and general assay procedures for cloned receptors were adjusted to 96-
microwell format based on described protocols (Perkin Elmer). Membrane preparation and general
assay procedures for cloned receptors were adjusted to 96-microwell format based on described
protocols.
7.3.2 Functional cAMP assay protocol
The functional properties of the selected compounds were evaluated in functional cAMP
cellular assays, performed at Eurofins Cerep using CHO cells which stably the human 5-HT7Rs and
HEK293 cells which stably expressed the human 5-HT1A and D2Rs. Experimental conditions for
these assays are described online at www.cerep.fr.
7.4. In vivo pharmacology
7.4.1. Subject
The experiments were performed on male Swiss albino mice (22–26 g) purchased from a
licensed breeder Staniszewska (Ilkowice, Poland) or male CD-1 mice from accredited animal
facility Jagiellonian University Medical College Kraków, Poland. Mice were kept in groups of ten
to Makrolon type 3 cages (dimensions 26.5 × 15 × 42 cm). The animals were kept in an
environmentally controlled rooms (ambient temperature 22 2°C; relative humidity 50–60%; 12:12
29