Antidepressant and antipsychotic activity of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole targeting serotonin 5-HT 1A/5-HT2A/5-HT7 and dopamine D 2/D3 receptors

Article abstract of DOI:10.1016/j.ejmech.2012.11.042

A series of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole was synthesized to explore the influence of two structural features-replacement of ether/amide moiety with sulfonamide one, and localization of a sulfonamide group in the azine moiety. In contrast to aripiprazole, compound 33 (N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl) quinoline-7-sulfonamide) and 39 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl) butyl)isoquinoline-3-sulfonamide) displaying multireceptor 5-HT _1A/5-HT_2A/5-HT₇/D₂/D₃ profile, and behaving as 5-HT_1A agonists, D₂ partial agonists, and 5-HT_2A/5-HT₇ antagonists, produced significant antidepressant activity in FST in mice. On the other hand, their 4-isoquinolinyl analog 40 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl) isoquinoline-4-sulfonamide), with similar receptor binding and functional profile, additionally displayed remarkable antipsychotic properties in the MK-801-induced hyperlocomotor activity in mice.

Full text of DOI:10.1016/j.ejmech.2012.11.042

European Journal of Medicinal Chemistry 60 (2013) 42e50
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antidepressant and antipsychotic activity of new quinoline- and isoquinoline-
sulfonamide analogs of aripiprazole targeting serotonin 5-HT1A/5-HT2A/5-HT7
and dopamine D2/D3 receptors
,
b
b
a
Pawe1 Zajdel a , Krzysztof Marciniec , Andrzej Maslankiewicz , Katarzyna Grychowska ,
*
ꢀ
c
c
c
d
d
e
ꢀ
Grzegorz Sata1a , Beata Duszynska , Tomasz Lenda , Agata Siwek , Gabriel Nowak , Anna Partyka ,
Dagmara Wróbel e, Magdalena Jastrze˛bska-Wie˛sek e, Andrzej J. Bojarski c, Anna Weso1owska e,
Maciej Paw1owski a
a Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
b
ꢀ
Department of Organic Chemistry, Medical University of Silesia, 4 Jagiellonska Street, 41-200 Sosnowiec, Poland
c Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Kraków, Poland
d Department of Pharmacobiology, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
e Department of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole was synthesized to
explore the influence of two structural features e replacement of ether/amide moiety with sulfonamide
one, and localization of a sulfonamide group in the azine moiety. In contrast to aripiprazole, compound
33 (N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)quinoline-7-sulfonamide) and 39 (N-(4-(4-(2,3-
dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-3-sulfonamide) displaying multireceptor 5-HT1A/5-
HT2A/5-HT7/D2/D3 profile, and behaving as 5-HT1A agonists, D2 partial agonists, and 5-HT2A/5-HT7
antagonists, produced significant antidepressant activity in FST in mice. On the other hand, their 4-
isoquinolinyl analog 40 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-4-sulfonamide),
with similar receptor binding and functional profile, additionally displayed remarkable antipsychotic
properties in the MK-801-induced hyperlocomotor activity in mice.
Received 15 October 2012
Received in revised form
23 November 2012
Accepted 29 November 2012
Available online 5 December 2012
Keywords:
Quinoline-sulfonamides
Isoquinoline-sulfonamides
Long-chain arylpiperazines
2,3-Dichlorophenylpiperazine
Serotonin
Ó 2012 Elsevier Masson SAS. All rights reserved.
Dopamine
5-HT2A antagonist
D2 partial agonist
5-HT7 antagonist
Aripiprazole
Depression
Schizophrenia
1. Introduction
currently available antidepressants. Additional unmet clinical
needs include long time of onset of action, sexual dysfunction,
Affective disorders, e.g. major depressive disorder (MDD),
bipolar disorder and schizophrenia, are highly debilitating diseases
whose treatment, despite development of novel therapy strategies,
is still unsatisfactory because of poor efficiency and unwanted
effects of antidepressant/antipsychotic drugs. Furthermore, the
majority of patients with MDD fail to achieve full remission with
nausea/emesis, weight gain, and/or cardiovascular effects.
In a development of CNS active agents, the multitarget strategy
seems to be one of the most valuable approaches. Accordingly,
several recently launched antidepressant/antipsychotic drugs e.g.
amisulpride and aripiprazole, or compounds investigated in clinical
trials (vortioxetine) [1] display mixed serotonin (5-HT) and dopa-
mine (D) receptor profile. It was recently found, that these drugs
behave as 5-HT7 receptor antagonists, and this mechanism is
responsible for their antidepressant properties [2]. Moreover, the
multimodal receptor profile (5-HT1A, 5-HT2A, 5-HT7, D2, D3) of
* Corresponding author. Tel.: þ48 12 620 54 50; fax: þ48 12 620 54 05.
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.042

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
43
aripiprazole, and its functional profile e partial agonist of D2 and 5-
HT1A receptors and antagonist of 5-HT2A and 5-HT7 sites, might
underline its board efficacy e antipsychotic, antidepressant, and
anxiolytic effects.
(22e28) were prepared from respective bromoquinolines and
bromoisoquinolines, according to the previously reported method
[6]. For the pharmacological evaluation compounds 31e40 were
converted into their water-soluble hydrochloride salts.
Extending a concept of mixed 5-HT/D receptor agents as novel
antipsychotics, Butini et al. [3] discovered a series of aripiprazole
analogs combining high affinity for 5-HT1A, 5-HT2A receptors, and
low affinity for D2 sites, at the same time displaying high affinity
for D3 receptors and antagonistic properties at these sites.
Structures of compounds were based, among others, around 2,3-
dichlorophenylpiperazine core structure, which was functional-
ized with isoquinoline-amide and quinoline- and isoquinoline-
ether moieties (I, II; Fig. 1).
We have previously explored multireceptor strategy and re-
ported on quinoline- and isoquinoline-sulfonamide derivatives of
long-chain arylpiperazines (LCAP) with 3- or 4-chloro-phenyl-
piperazine moieties, as potential antidepressant, anxiolytic, and
antipsychotic agents. Among them, compound III (Fig. 2) with mixed
5-HTand D2, D3 receptor profile, behaved as antagonist of 5-HT1A, 5-
HT2A, 5-HT7, D2 postsynaptic sites, and produced antidepressant-
like effect in the forced-swim test in mice. On the other hand,
a semi-rigid compound IV containing4-isoquinolinylmoiety (Fig. 2),
that behaved as 5-HT2A, 5-HT7, and D2 antagonist, and showed
affinity for D3, and D4 receptors, produced antidepressant-like effect
and additionally displayed anxiolytic activity in plus-maze test and
antipsychotic properties in the MK-801 induced hyperlocomotor
activity in mice, a model related to negative symptoms and cognitive
impairments observed in schizophrenia [4].
Continuing our study in a group of azinesulfonamide derivatives
of LCAP [4,5], and to extend the studies aimed at verification of the
impact of localization of sulfonamide group in the azine moiety and
the length of the linker, we designed a series of quinoline- and
isoquinoline-sulfonamide analogs of aripiprazole. Herein, we
report on their synthesis, and evaluation for selected serotoninergic
(5-HT1A, 5-HT2A, 5-HT6, 5-HT7), dopaminergic (D2, D3), adrenergic
a1 receptors, and serotonin transporter (SERT), as well as deter-
mination of their intrinsic activity. We also present results of
preliminary evaluation of selected compounds in animal models of
depression and schizophrenia aimed at validation of the multi-
receptor approach and identification of structural elements
responsible for in vivo activity.
2.2. Pharmacology
2.2.1. In vitro evaluation
Radioligand binding assays were employed for determining the
affinity and the selectivity profile of the synthesized compounds for
cloned 5-HT1A, 5-HT2A, 5-HT6, 5-HT7 and dopamine D2 receptors.
This was accomplished by displacement of respective radioligands
from cloned human receptors, all stably expressed in HEK293 cells:
[3H]-8-OH-DPAT for 5-HT1AR, [3H]-ketanserin for 5-HT2AR, and
[3H]-LSD for 5-HT6R, [3H]-5-CT for 5-HT7R and [3H]-raclopride for
D2R. Displacement of [3H]-methylspiperone from the cloned
human receptor stably expressed in HEK293 cells was used in D3R
binding experiments (Cerep, Le Bois l’Eveque, 86600 Celle L’Ev-
escault, France) [7].
Compounds affinity for a1-adrenergic receptors and serotonin
transporter (SERT) was accomplished by determining their ability
to displace [3H]-prazosin and [3H]-citalopram, respectively, from
rat cerebral cortex.
2.2.2. Functional in vitro and in vivo evaluation
The functional activity of the selected compounds (33, 34, 37, 39,
40) on 5-HT1A and 5-HT7 receptors, on intracellular cAMP levels,
was determined at Cerep according to the previously published
methods [8,9]. Assays were carried out in CHO and HEK-293 cells,
which stably expressed the human 5-HT1A and 5-HT7 receptors,
respectively, and using 8-OH-DPAT and methiothepin as reference
compounds.
The functional activity of the selected compounds (33, 34, 37, 39,
40) at D2 and 5-HT2A receptors was tested in the commonly used
in vivo models. To determinate D2 receptor antagonistic effect of
the tested compounds, their ability to abolish the apomorphine-
induced climbing test in mice was tested. The antagonistic 5-HT2A
properties were assessed in the (ꢀ)DOI-induced head twitch
responses in mice [10].
2.2.3. Behavioral evaluation
In the successive phase of our investigations the most in vitro
active derivatives, i.e. 33, 34, 37, 39 and 40 were tested in vivo in
behavioral tests commonly used to predict antidepressant- and
antipsychotic-like activity. To evaluate the potential antidepressant
properties of the investigated compounds, the forced swim test
(FST) in mice was employed [11]. 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. Furthermore,
2. Results and discussion
2.1. Chemistry
The synthesis of target compounds (31e40) was carried out as
shown in Scheme 1, in reaction of the primary amines (29, 30) with
appropriate azinesulfonyl chloride (22e28) in the presence of
Hunig’s base. The quinoline- and isoquinoline-sulfonyl chlorides
H
O
N
O
N
Cl
N
N
Cl
Aripiprazole
H
N
N
O
N
Cl
N
Cl
O
N
Cl
N
Cl
I
II
Fig. 1. Aripiprazole and its quinoline- and isoquinoline amide and ether analogs.

44
P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
O
O
S
N
Cl
N
O
O
S
N
N
N
N
H
N
N
III
IV
Cl
Fig. 2. The structure of III, a mixed 5-HT1A/5-HT2A/5-HT7/D2/D3 ligand and IV, a mixed 5-HT2A/5-HT7/D2/D3/D4 agent.
spontaneous locomotor activity and the hyperactivity induced by
MK-801 were used to evaluate specific antipsychotic-like activity in
mice. Citalopram and aripiprazole were used as reference drugs.
unchanged within both the propylene and butylene derivatives of
2,3-diClePhPs, the 7-quinolinyl fragment, revealed to be the most
favorable for interaction with 5-HT1ARs. It thus confirmed the
structural analogy for dihydroquinolin-2-one moiety of parent
aripiprazole. As observed previously, introduction of halogen atom
to the 3-quinolinyl moiety led to decreased affinity for 5-HT1AR,
and had little effect on the 5-HT2A, and 5-HT7R affinity; this was
exemplified by 35, whose binding constant for 5-HT1AR decreased
up to 4 folds.
Furthermore, compounds 33 and 36 containing 7-quinolinyl
fragment displayed the highest affinity for 5-HT2A and 5-HT6Rs in
the quinoline set. This quinolinyl fragment was also the most
preferable, among propylene analogs 31e33, for binding to 5-HT7R.
On the other hand, such preference was not observed within
butylene derivatives; compounds 34, 37, and 38, with 3-quinolinyl,
7-quinolinyl, and 8-quinolinyl fragments, respectively, displayed
comparable affinity for 5-HT7Rs.
2.3. Structureeactivity relationship studies
Development of CNS active compounds was often abandoned by
low aqueous solubility, which greatly hindered in vivo evaluation of
arene derivatives [12,13]. Starting from a series of arene- and
quinoline-sulfonamides of LCAP derivatives [5] we have recently
reported on the design, synthesis and pharmacological evaluation
of a series of quinoline- and isoquinoline-sulfonamides of flexible
or semi-rigid LCAP derivatives [4]. In contrast to limited water
solubility of parent arene derivatives, a series of azinesulfonamides
displayed improved solubility, still exerting high therapeutic
potential.
In view of the literature data presenting four receptor systems e
5-HT1A, 5-HT2A, 5-HT6, and 5-HT7 e as targets for effective treat-
ment of affective disorders, all the synthesized compounds 31e40
were evaluated for these sites (Table 1). Quinoline- and
isoquinoline-sulfonamides displayed high affinity for 5-HT1A, 5-
HT2A, and 5-HT7Rs (14e112 nM, 22e115 nM and 12e124 nM,
respectively), and moderate-to-low for 5-HT6R (220e1331 nM).
Generally, in the evaluated quinoline derivatives of 2,3-
dichlorophenylpiperazines, elongation of propylene spacer to
butylene one, decreased affinity for 5-HT1A receptor, simulta-
neously increased affinity for 5-HT2A sites, and either retained or
decreased affinity for 5-HT7R.
Replacement of the 8-quinolinyl moiety 38 with its a-position
counterpart e 4-isoquinolinyl, resulted in compound 40, and up to
2-folds increased affinity for 5-HT1A and 5-HT6Rs, and up to 3-folds
decreased affinity for 5-HT2ARs.
Interestingly, 3-isoquinolinyl derivative 39 and 4-isoquinolinyl
one 40 displayed opposite preference for 5-HT1A and 5-HT7
receptors. This may suggest, that additional hydrogen bonds
formed between sulfonamide moiety in position 3 of isoquinoline
and an endocyclic nitrogen atom, which stabilizes compounds
structure in the terminal fragment, may account for higher affinity
of 39 for 5-HT1AR.
In line with the previous studies revealing new insights into
therapeutic role of 5-HT1AR in treating of various CNS disorders
[15], and extending our aim to discover compounds combining
multiple pharmacological action, in the next move of in vitro
evaluation, compounds with high affinity for 5-HT1A sites
(Ki < 60 nM), were investigated for D2, D3 receptors. For these
It was previously found, in a series of 2-methoxy-phenyl-
piperazine (PhP) and 3-ClePhP derivatives of quinoline- and
isoquinoline-sulfonamides, that localization of the sulfonamide
bond in the pyridine ring of quinoline was preferential for inter-
action with 5-HT1AR. Although the previously established rank
order (3-quinolinyl
>
6-quinolinyl
>
8-quinolinyl) remained
O
z
O
SNa
Br
SH
S
i
ii
iii
z
z
z
Cl
X
X
X
X
X
X
X
X
8 - 14
1 - 7
15 - 21
22 - 28
iv
H N
N
Cl
n
N
Cl
O
z
O
29, 30
X = N, CH
n = 1, 2
S
N
H
N
Cl
X
n
z = 3-Q, 6-Cl-3-Q, 6-Q, 7-Q
8-Q, 3-isoQ, 4-isoQ
X
N
Cl
31 - 40
Scheme 1. Synthesis of azinesulfonamide derivatives of LCAP 31e40. Reagents and conditions: (i) MeSNa (5 mol equiv.), DMF boiling temp., 4 h (ii) conc. HCl, 0 ꢁC (iii) NaOCl, 5 ꢁC,
30 min (iv) DIEA, CH2Cl2, 0 ꢁC / rt.

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
45
Table 1
Table 2
The binding data of the quinoline- and isoquinoline-sulfonamides (31e40) for 5-HT
receptors.
The binding data of the selected quinoline- and isoquinoline-sulfonamides for
dopamine receptors.
Compd Azinyl
n
Ki ꢀ SEM [nM]
Compd Azinyl
n
Ki ꢀ SEM [nM]
a
D2
D3
SERT
a1
5-HT1A
5-HT2A
5-HT6
5-HT7
33
34
37
39
40
7-Quinolinyl
3-Quinolinyl
7-Quinolinyl
3-Isoquinolinyl
4-Isoquinolinyl
1
2
2
2
2
16 ꢀ 2
9 ꢀ 1
90/99
94/99
99/101
95/98
93/95
224 ꢀ 6.2
186 ꢀ 0.5
170 ꢀ 1.6
136 ꢀ 2.6
255 ꢀ 21.5
160 ꢀ 3.9
31
32
33
34
35
36
37
38
39
40
6-Cle3-Quinolinyl
1
1
1
2
2
2
2
2
2
2
e
23 ꢀ 2
25 ꢀ 3
14 ꢀ 2
23 ꢀ 4
88 ꢀ 6
79 ꢀ 9
17 ꢀ 3
112 ꢀ 16
34 ꢀ 1
59 ꢀ 2
5.6 ꢀ 0.8a
115 ꢀ 18
65 ꢀ 7
47 ꢀ 1
34 ꢀ 3
42 ꢀ 2
23 ꢀ 2
22 ꢀ 1
29 ꢀ 4
35 ꢀ 2
90 ꢀ 14
21 ꢀ 1
901 ꢀ 8
326 ꢀ 27
257 ꢀ 19
1331 ꢀ 94
555 ꢀ 42
46 ꢀ 3
31 ꢀ 2
12 ꢀ 1
30 ꢀ 3
41 ꢀ 2
1200 ꢀ 12
788 ꢀ 25
335 ꢀ 6.2
783 ꢀ 24
6-Quinolinyl
7-Quinolinyl
3-Quinolinyl
6-Cle3-Quinolinyl
6-Quinolinyl
7-Quinolinyl
8-Quinolinyl
3-Isoquinolinyl
4-Isoquinolinyl
11 ꢀ 1
17 ꢀ 1
8 ꢀ 1
Aripiprazole
0.8 ꢀ 0.1 9.7 ꢀ 5.4b 1080 ꢀ 180b 25.7 ꢀ 5.0b
484 ꢀ 30 124 ꢀ 7
301 ꢀ 16
598 ꢀ 76
454 ꢀ 37
220 ꢀ 12
90 ꢀ 1
31 ꢀ 2
27 ꢀ 4
56 ꢀ 8
21 ꢀ 1
26 ꢀ 2
Screening procedure e displacement % at 10ꢂ7/10ꢂ6
Data taken from Ref. [14].
.
a
b
Aripiprazole
All tested derivatives 33 (5e30 mg/kg), 34 (10e30 mg/kg), 37
a
Data for 5-HT1A taken from Ref. [14].
(5e20 mg/kg), 39 (5e20 mg/kg) and 40 (1.25e20 mg/kg) as well as
aripiprazole (2.5e10 mg/kg) dose-dependently inhibited the (ꢀ)
DOI-induced head twitches in mice (Table 4) [10]. The highest
activity in apomorphine-induced climbing as well as in DOI-
induced head twitches tests displayed compound 40
(ID50 ¼ 11.29 mg/kg and 5.3 mg/kg, respectively).
assays one representative containing propylene spacer (33) and
four compounds with butylene spacer (34, 37, 39, 40) were selected
(Table 2). The binding data revealed that all compounds displayed
high affinity for D2 and D3 receptors. Furthermore, compounds
showed moderate-to-low affinity for SERT. Among them, the
highest affinity still in moderate range (224e335 nM), displayed
compound 33 and 39. Since a1 receptors may be considered as
potential source of undesirable cardiovascular effects, and addi-
tionally blockade of these receptors may decrease effectiveness of
antidepressant drugs, selected compounds were also tested for
these sites (Table 2).
Summarizing the results of our in vitro and in vivo functional
study it was found that the investigated compounds behaved as 5-
HT1A receptor agonists, 5-HT2A, 5-HT7 receptor antagonists and D2
receptor partial agonists.
2.5. Behavioral evaluation
Regarding many classes of 5-HT receptors described in CNS,
much attention has been devoted to the role of 5-HT1A, 5-HT2A, 5-
HT6 and 5-HT7 receptor subtypes in many psychiatric disorders, e.g.
depression. Behavioral effects similar to those of SSRIs have been
produced by agonists of 5-HT1A and 5-HT6 receptors. Also, antag-
onists of 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptors have been
reported to display antidepressant-like responses [16].
Generally, the compounds selected for extended in vitro eval-
uation may be classified as multimodal serotonin and dopamine
receptor ligands with high affinity for 5-HT1A/5-HT2A/5-HT7 and
D2/D3 receptors, and moderate affinity for SERT and a1. In
comparison to the previously reported by Butini et al. [3], ether or
amide analogs of aripiprazole, an introduction of azinesulfonamide
group remarkably increased affinity for D2 receptors.
The obtained results indicate that all of the tested compounds
33 (2.5e10 mg/kg), 34 (5e20 mg/kg), 37 (5e20 mg/kg), 39 (2.5e
20 mg/kg), 40 (10e30 mg/kg) and citalopram (1.25e5 mg/kg),
used as a reference drug, revealed antidepressant-like properties,
shortening to a various extent the immobility time of mice. On the
other hand, aripiprazole (0.01e0.1 mg/kg), used as a reference
antipsychotic drug, with structural similarity to the investigated
compounds was inactive in that test (Fig. 3). The doses of aripi-
prazole used in our investigations are based on the active doses in
FST and TST (tail suspension test) reported by Sarkisyan et al., who
2.4. Functional evaluation
Among compounds selected for functional evaluation 34, 37, 39,
40 behaved as weak 5-HT1A receptor agonist, while 33 displaying
58% of maximal effect of 5-HT, was classified as partial agonist
(Table 3A) [8]. Evaluated compounds inhibited cAMP accumulation
induced by 5-HT, and thus were classified as weak antagonists at
the h5-HT7Rs (Table 3B) [9].
The same compounds and aripiprazole revealed some agonistic
properties for D2 receptor as well, evoking climbing behavior in
mice. Assuming, that the effect produced by apomorphine (3 mg/
kg) is 100%, aripiprazole (1 mg/kg), 33 (5 mg/kg), 34 (5 mg/kg), 37
(5 mg/kg), 39 (1.25 mg/kg) and 40 (5 mg/kg) displayed
a maximum agonist effect of about 50, 50.5, 49.3, 43.6, 49.1 and
44.6%, respectively. Moreover, dose-dependent and considerable
reduction of the apomorphine-induced climbing behavior of mice
were observed for agents 34 (10e40 mg/kg), 37 (10e30 mg/kg),
39 (10e30 mg/kg), 40 (5e20 mg/kg) and aripiprazole (0.03e
3 mg/kg). Compound 33 (1.25e30 mg/kg) decreased that effect
in statistically significant manner only in a dose of 2.5 mg/kg
(Table 4).
Table 3
Effects of 33, 34, 37, 39, 40 on (A) cAMP accumulation (agonistic effect) and (B) 5-HT-
induced cAMP accumulation in vitro (antagonistic effect).
Compound
5-HT1A EC50 (M)
5-HT7 IC50 (M)
33
34
37
39
7.4 ꢃ 10ꢂ6
1.65 ꢃ 10ꢂ6
8.65 ꢃ 10ꢂ7
1.55 ꢃ 10ꢂ5
1.5 ꢃ 10ꢂ5
1.5 ꢃ 10ꢂ8
e
2.8 ꢃ 10ꢂ6
7.8 ꢃ 10ꢂ6
3.8 ꢃ 10ꢂ6
5.6 ꢃ 10ꢂ6
8.2 ꢃ 10ꢂ7
e
40
8-OHeDPAT
Methiothepine
2.2 ꢃ 10ꢂ9

46
P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
Table 4
Effects of the tested compounds and aripiprazole on (A) the apomorphine-induced
climbing behavior and (B) on the (ꢀ)DOI-induced head-twitches in Swiss albino
mice.
a
Compound
A (ID50a mg/kg i.p.)
B (ID50 mg/kg i.p.)
33
34
37
39
e
10.22
20.21
4.58
9.03
5.30
9.89
(9.30e11.13)
20.74
19.94
19.90
11.29
1.58
(13.9e27.57)
(16.99e23.43)
(4.07e5.09)
(3.94e14.12)
(3.75e6.74)
(Wide range)
(18.18e21.71)
(15.11e24.70)
(5.53e17.05)
(0.38e3.54)
40
Aripiprazole
a
ID50 e the dose inhibiting climbing behavior or head twitches in mice by 50%;
confidence limits (95%) given in parentheses (Graph Pad Prism 5 Software).
emphasized 5-HT7 receptor antagonistic properties as a potential
mechanism of antidepressant efficacy [2]. Similarly to our study,
the lack of aripiprazole’s antidepressant-like activity in FST was
reported by Bourin et al. [17], who indicated that at the dose of
0.125 mg/kg aripiprazole decreased the locomotor activity of mice.
These discrepancies may be caused by different strains of mice used
in the laboratories.
Fig. 4. Effects of the tested compounds and aripiprazole on the MK-801-induced
hyperlocomotor activity in CD-1 mice.
or adjunctive treatment of bipolar disorder, major depressive
disorder and/or treatment-resistant depression [18e20]. In view of
these facts, and considered that compounds 33, 34, 37, 39 and 40
display high affinityfordopamineD2 and/or D3 receptors(Table 3), at
the successive stage their antipsychotic potential was studied in the
MK-801-induced locomotor hyperactivity test, a model of the
negative and cognitive symptoms of schizophrenia [21,22].
Among the evaluated compounds, only 40, given at doses of 10
and 20 mg/kg, considerably attenuated the MK-801-evoked
hyperactivity in mice (Fig. 4). Its effect seems to be specific, since
40 administered at effective doses had no influence on the spon-
taneous locomotor activity in mice. Derivatives 33 in a dose of
20 mg/kg and 37 in a dose of 10 mg/kg significantly increased
hyperactivity induced by MK-801, while compounds 34 and 39
produced no effects on the hyperactivity evoked by MK-801. It is
noteworthy that 33 and 37 in above-mentioned doses visibly
augmented the spontaneous locomotor activity of mice, whereas
derivatives 34 and 39 remained without significant effect (Table 1-
SI). By comparison, aripiprazole in a dose of 0.5 mg/kg remarkably
attenuated the hyperactivity evoked by MK-801, and its effect
seems to be specific, since a dose of 1 mg/kg per se was the
minimum sedative one in the spontaneous locomotor activity
model (Fig. 4).
Compounds 34 and 37 administered at higher doses of 10 and
20 mg/kg reduced by 31% and 30% (34) and by 48% and 41% (37), the
immobility time of mice in FST. Compound 40 was active only in
a medium dose of 20 mg/kg, having significantly decreased this
parameter by 44%. The most active compounds were 33 and 39.
Derivative 33 administered at doses of 5 and 10 mg/kg considerably
reduced the immobility time of animals by 57% and 79%, respec-
tively, and compound 39 at doses of 5, 10 and 20 mg/kg decreased
this parameter in statistically significant manner by 43%, 53% and
83%, respectively. It is worth noting, that antidepressant-like
activity of 33 and 39 given in the highest doses (10 mg/kg and
20 mg/kg, respectively) was stronger in terms of its potency than
that of citalopram. In fact, citalopram in doses of 1.25, 2.5 and 5 mg/
kg substantially shortened (by 32%, 51% and 61%, respectively) the
immobility time of mice in FST (Fig. 3).
Antidepressant doses of derivatives 33, 34, 39 and 40 did not
influence the spontaneous locomotor activity of mice during initial
2e6-min session (i.e., at the time identical to the observation
period in the FST). Merely the antidepressant-like effect of
compound 37 given at the highest dose of 20 mg/kg was not
specific, since it increased the locomotor activity of animals
(Table 1-SM).
Both clinical and preclinical studies demonstrate the role of the
dopaminergic system in the pathophysiologyof depression and in its
treatment. Moreover, some newer atypical antipsychotics (olanza-
pine, quetiapine, aripiprazole, risperidone) exert antidepressant
effects and have been recently launched into the monotherapy and/
3. Conclusions
Extending our studies in a group of azinesulfonamide analogs of
aripiprazole allowed identifying some potent, multireceptor 5-
HT1A/5-HT2A/5-HT7/D2/D3 ligands, which behaved as 5-HT1A
receptor agonists/5-HT2A and 5-HT7 receptor antagonists/D2
receptor partial agonists. The results of pharmacological in vivo
studies showed that all of the selected compounds (33, 34, 37, 39,
40), but not aripiprazole, displayed some antidepressant-like
activity in a mouse model of depression (FST). On the other hand,
only compound 40, similarly to aripiprazole, was active in MK-801-
evoked hyperactivity test in mice.
Regardless receptor binding profile, we found the behavioral
activity dependent upon localization of sulfonamide group in the
azine moiety; the highest antidepressant response displayed
compounds containing 7-quinolinyl (33, 37) and 3-isoquinolinyl
(39) moieties in sulfonamide fragment. This peculiar relationship
may be attributed to localization of sulfonamide group in b-posi-
tion of azine moiety. The same substitution pattern was observed in
the structure of aripiprazole. On the other hand, still in line with
our previous findings [4], antipsychotic effect was detected in
Fig. 3. Effects of the tested compounds, aripiprazole and citalopram on the immobility
time in the forced swim test in Swiss albino mice.

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
47
compound containing sulfonamide group in
moiety (4-isoquinolinyl analog, 40).
a
-position of azine
anhydrous sodium sulfate. CHCl3 was evaporated to leave solid
residue. The residue was recrystallized from benzene to give
chlorosulfonylazines (22e28) [6].
Receptor binding and functional activity data, followed by
behavioral assays results with these compounds indicate a complex
relationship between structure and these properties. Further
studies in the area of multifunctional compounds might be bene-
ficial to both depression and schizophrenia, and will address
receptor selectivity and pharmacokinetic.
4.2.1. Isoquinoline-3-sulfonyl chloride (20)
Creamy solid, 755 mg (83% yield) following crystallization.
C9H6ClNO2S,
MW
227.67,
monoisotopic
mass
226.98,
[M þ H]þ ¼ 228.0. 1H NMR (CDCl3)
d
(ppm) 7.91e7.93 (m,1H), 7.94e
7.96 (m,1H), 8.10e8.11 (dd,1H), 8.16e8.19 (dd,1H), 8.55 (s,1H), 9.38
(s, 1H). Mp ¼ 133e134 ꢁC. Anal. Calc for C9H6ClNO2S (277.67): C,
47.48; H, 2.66; N, 6.15; found C, 47.52; H, 2.65; N, 6.14.
4. Experimental protocols
4.1. Chemistry
4.3. General procedure for the preparation of compounds 31e40
Organic transformations were carried out at ambient tempera-
ture, unless indicated otherwise. Organic solvents (from Sigmae
Aldrich and Chempur) were of reagent grade and were used
without purification. All other reagents were from SigmaeAldrich
and Alfa Aesar.
Purity of the synthesized compounds was confirmed by TLC
performed on Merck silica gel 60 F254 aluminum sheets (Merck,
Darmstadt, Germany). Spots were detected by their absorption
The starting N-(
were synthesized according to the Gabriel method. A mixture of
the appropriate N-( -aminoalkyl)-2,3-dichlorophenylpiperazine
u-aminoalkyl)-2,3-dichlorophenylpiperazines
u
(1.0 mmol) in CH2Cl2 (7 mL) and DIEA (2.4 mmol) was cooled
down (ice bath), and azinesulfonyl (22e28) (1.2 mmol) was added
at 0 ꢁC in one portion. The reaction mixture was stirred for 2e6 h
under cooling. Then, the solvent was evaporated and the
sulfonamides were separated by column chromatography using
SiO2 and a mixture of CH2Cl2/MeOH ¼ 9/0.7 or 9/0.5, as an
eluting system. Free bases were then converted into the
hydrochloride salts by treatment of their solution in anhydrous
ethanol with 1 M HCl in dioxane. The LC/MS of the identified
compounds 31e40 exceeded purity of 98%.
under UV light (
l
¼ 254 nm).
Analytical HPLC were run on a Waters Alliance HPLC instrument,
equipped with a Chromolith SpeedROD column (4.6 ꢃ 50 mm).
Standard conditions were eluent system A (water/0.1% TFA), system
B (acetonitrile/0.1% TFA). A flow rate of 5 mL/min and a gradient of
(0e100)% B over 3 min were used. Detection was performed on
a PDA detector. Retention times (tR) are given in minutes.
1H NMR spectra were recorded at 300 MHz (Varian BB 200
spectrometer) using TMS (0.00 ppm) and chloroform-d1, J values
are in hertz (Hz), and splitting patterns are designated as follows: s
(singlet), d (doublet), t (triplet), m (multiplet).
Mass spectrometry analyses: Samples were prepared in aceto-
nitrile/water (10/90 v/v) mixture. The LC/MS system consisted of
a Waters Acquity UPLC, coupled to Waters TQD mass spectrometer
(electrospray ionization mode ESI-tandem quadrupole). All the
4.3.1. 6-Chloro-N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)
quinoline-3-sulfonamide (31)
White solid, 307 mg (60% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.32.
C22H23Cl3N4O2S, MW 513.87, monoisotopic mass 512.06,
[M þ H]þ ¼ 512.8. 1H NMR (CDCl3)
d (ppm) 1.89e1.98 (m, 2H), 2.95e
3.15 (m, 6H), 3.18e3.31 (m, 4H), 3.45e3.50 (m, 2H), 6.84e6.88 (d,1H,
J ¼ 7.4 Hz, J ¼ 2.1 Hz), 6.99e7.08 (m, 3H), 7.78e7.82 (dd,1H, J ¼ 9.1 Hz,
J ¼ 2.2 Hz), 8.05e8.08 (m, 2H), 8.90e8.91 (d, 1H, J ¼ 2.3 Hz), 9.19e
9.20 (dd, 1H, J ¼ 4.6 Hz, J ¼ 1.5 Hz). C22H23Cl3N4O2S$HCl,
Mp ¼ 212.5e214.1. Anal. Calc for C22H23Cl3N4O2S HCl (550.33): C,
48.01; H, 4.40; N, 10.18; found C, 47.89; H, 4.42; N, 10.20.
analyses were carried out using an Acquity UPLC BEH C18
,
50 ꢃ 2.1 mm reversed-phase column. A flow rate of 0.3 mL/min and
a gradient of (5e95)% B over 5 min was used. Eluent A: water/0.1%
HCO2H; eluent B: acetonitrile/0.1% HCO2H. Nitrogen was used for
both nebulizing and drying gas. LC/MS data were obtained by
scanning the first quadrupole in 0.5 s in a mass range from 100 to
700 m/z; 8 scans were summed up to produce the final spectrum.
Elemental analyses were found within ꢀ0.4% of the theoretical
values. Melting points (mp) were determined with a Büchi appa-
ratus and are uncorrected. Column chromatography separations
were carried out on column with Merck Kieselgel 60.
4.3.2. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-
6-sulfonamide (32)
White solid, 320 mg (67% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.14.
C22H24Cl2N4O2S, MW 479.42, monoisotopic mass 478.10,
[M þ H]þ ¼ 479.0. 1H NMR (DMSO)
d (ppm) 1.86e1.91 (m, 2H),
2.86e2.92 (m, 2H), 3.08e3.23 (m, 6H), 3.35e3.39 (m, 2H), 3.46e
3.54 (m, 2H), 7.15e7.18 (dd, 1H, J ¼ 6.8 Hz, J ¼ 2.5 Hz), 7.33e7.34
(m, 2H), 7.84e7.88 (q, 1H, J ¼ 4.4 Hz), 8.14e8.16 (m, 1H), 8.18e
8.21 (dd, 1H, J ¼ 9.00 Hz, J ¼ 2.1 Hz), 8.34e8.37 (dd, 1H), 8.66e
8.67 (d, 1H, J ¼ 2.1 Hz), 8.86e8.89 (d, 1H, J ¼ 7.7 Hz), 9.17e9.19
(dd, 1H, J ¼ 4.6 Hz, J ¼ 1.5 Hz). C22H24Cl2N4O2S$HCl, Mp ¼ 144.2e
145.9 ꢁC. Anal. Calc for C22H24Cl2N4O2S HCl (515.88): C, 51.22; H,
4.88; N, 10.86; found C, 51.18; H, 4.86; N, 10.89.
4.2. Synthesis of azinesulfonyl chloride 22e28
A mixture of bromoazine (1e7) (4 mmol), sodium meth-
anethiolate (20 mmol) and dry DMF (12 mL) was boiled with stir-
ring under argon atmosphere for 4 h. It was then cooled to 70 ꢁC
and the volatile components were evaporated under vacuum from
water bath. The remaining crude sodium azinethiolates (8e14),
were cooled down on an ice-water bath (under argon atmo-
sphere), and carefully acidified with conc. hydrochloric acid
(12 mL). Then, CHCl3 (12 mL) was added and 6% aqueous solution of
sodium hypochlorite (19 mL, 13.3 mmol) was dropped within
30 min to the well-stirred (cold 5 ꢁC) mixture of hydrochloric acid
solution of mercaptoazines (15e21) and CHCl3 at such a rate that
temperature was maintained below 5 ꢁC. The mixture was poured
into 30 g of ice. The chloroform layer was separated, and aqueous
layer was extracted with CHCl3 (3 ꢃ 10 mL). The chloroform
extracts were combined, washed with water and dried over
4.3.3. N-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)quinoline-
7-sulfonamide (33)
Yellow solid, 301 mg (63% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.21.
C22H24Cl2N4O2S, MW 479.42, monoisotopic mass 478.10,
[M þ H]þ ¼ 479.3. 1H NMR (DMSO)
d (ppm) 1.87e1.96 (m, 2H),
2.86e2.92 (m, 2H), 3.05e3.23 (m, 6H), 3.35e3.39 (m, 2H), 3.47e
3.51 (m, 2H), 5.92 (br s, 1H), 7.15e7.18 (dd, 1H J ¼ 6.8 Hz,
J ¼ 2.6 Hz), 7.30e7.38 (m, 2H), 7.79e7.83 (q, 1H, J ¼ 4.4 Hz), 8.00e

48
P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
8.03 (dd, 1H, J ¼ 8.7 Hz, J ¼ 1.8 Hz), 8.17e8.21 (t, 1H, J ¼ 5.9 Hz), 8.50
(br s,1H), 8.65e8.68 (d,1H, J ¼ 8.0 Hz), 9.12e9.15 (dd,1H, J ¼ 4.5 Hz,
J ¼ 1.5 Hz). C22H24Cl2N4O2S$HCl, Mp ¼ 211.1e212.9 ꢁC. Anal. Calc for
C22H24Cl2N4O2S HCl (515.88): C, 51.22; H, 4.88; N, 10.86; found C,
51.36; H, 4.89; N, 10.83.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
[M þ H]þ ¼ 493.2. 1H NMR (DMSO)
d (ppm) 1.29 (br s, 4H), 2.06e
2.10 (m, 2H), 2.33 (br s, 4H), 2.82e2.88 (m, 6H), 7.09e7.13 (m,
1H), 7.21e7.33 (m, 3H), 7.69e7.73 (m, 2H), 8.27e8.32 (m, 2H), 8.53e
8.56 (dd, 1H, J ¼ 8.5 Hz, J ¼ 1.8 Hz), 9.06e9.08 (m, 1H).
C23H26Cl2N4O2S$HCl, Mp
C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,
¼
131.1e131.9 ꢁC. Anal. Calc for
4.3.4. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
3-sulfonamide (34)
52.29; H, 5.16; N, 10.53.
Yellow solid, 344 mg (70% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.38.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
4.3.9. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)
isoquinoline-3-sulfonamide (39)
White solid, 334 mg (68% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.33.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
[M þ H]þ ¼ 493.1. 1H NMR (DMSO)
d
(ppm) 1.35e1.37 (m, 4H), 2.17e
2.25 (m, 2H), 2.34 (br s, 4H), 2.84e2.87 (m, 6H), 7.04e7.09 (m, 1H),
7.26e7.32 (m, 2H), 7.72e7.77 (m, 1H), 7.90e7.95 (m, 2H), 8.11e8.13
(m, 1H), 8.22e8.25 (m, 1H), 8.86 (s, 1H), 9.17 (s, 1H).
[M þ H]þ ¼ 492.7. 1H NMR (CDCl3)
d (ppm) 1.58e1.60 (m, 4H),
C23H26Cl2N4O2S$HCl, Mp
C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,
¼
148.0e149.7 ꢁC. Anal. Calc for
2.42e2.44 (m, 2H), 2.72 (br s, 4H), 3.03 (br s, 2H), 3.18e3.21 (m, 4H),
6.99e7.04 (m, 1H), 7.08e7.16 (m, 2H), 7.74e7.85 (m, 2H), 7.94e7.97
(d, 1H, J ¼ 8.0 Hz), 8.04e8.07 (dd, 1H, J ¼ 7.82 Hz, J ¼ 0.8 Hz), 8.43 (s,
1H), 8.65 (br s, 1H), 9.26 (s, 1H). C23H26Cl2N4O2S$HCl, Mp ¼ 185.3e
186.7 ꢁC. Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H,
5.14; N, 10.57; found C, 52.03; H, 5.12; N, 10.59.
52.23; H, 5.15; N, 10.53.
4.3.5. 6-Chloro-N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)
quinoline-4-sulfonamide (35)
Yellow solid, 305 mg (58% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.54.
C23H25Cl3N4O2S, MW 527.89, monoisotopic mass 526.08,
4.3.10. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)
isoquinoline-4-sulfonamide (40)
Yellow oil, 344 mg (70% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.28.
C23H26Cl2N4O2S, MW 493.45, Monoisotopic Mass 492.12,
[M þ H]þ ¼ 527.2. 1H NMR (DMSO)
d
(ppm) 1.43e1.50 (m, 2H), 1.72
(m, 2H), 2.84e2.90 (m, 2H), 3.03e3.24 (m, 6H), 3.35e3.50 (dd, 4H,
J ¼ 31.0 Hz, J ¼ 12.0 Hz), 7.16e7.19 (m, 1H), 7.33e7.35 (m, 2H), 7.93e
7.97 (dd, 1H, J ¼ 9.0 Hz, J ¼ 2.3 Hz), 8.13e8.16 (d, 1H, J ¼ 9.23 Hz),
8.18e8.20 (m, 1H), 8.41e8.42 (d, 1H, J ¼ 2.3 Hz), 8.88e8.89 (d, 1H,
J ¼ 2.0 Hz), 9.21e9.22 (d, 1H, J ¼ 2.3 Hz). C23H25Cl3N4O2S$HCl,
Mp ¼ 191.2e192.7 ꢁC. Anal. Calc for C23H25Cl3N4O2S$HCl (564.35):
C, 48.95; H, 4.64; N, 9.93; found C, 48.81; H, 4.62; N, 9.95.
[M þ H]þ ¼ 493.0. 1H NMR (CDCl3)
d (ppm) 1.61 (br s, 4H), 2.42e
2.46 (t, 2H, J ¼ 5.1 Hz), 2.72 (br s, 4H), 3.03e3.06 (m, 2H), 3.19e
3.22 (t, 4H, J ¼ 4.5 Hz), 7.02e7.05 (q, 1H, J ¼ 3.2 Hz), 7.15e7.19 (m,
2H), 7.70e7.77 (m, 1H), 7.80e7.86 (m, 2H), 8.08e8.10 (d, 1H,
J ¼ 7.7 Hz), 8.70e8.73 (d, 1H, J ¼ 9.2 Hz), 9.11 (s, 1H), 9.39 (s, 1H).
C23H26Cl2N4O2S$HCl, Mp
¼
145.1e147.4 ꢁC. Anal. Calc for
4.3.6. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
6-sulfonamide (36)
C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N, 10.57; found C,
52.16; H, 5.13; N, 10.53.
Yellow solid, 305 mg (62% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5); tR ¼ 1.20.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
4.4. In vitro pharmacology
[M þ H]þ ¼ 493.2. 1H NMR (DMSO)
d
(ppm) 1.38e1.46 (m, 2H),
4.4.1. Cell culture
1.64e1.75 (br s, 2H), 2.79e2.84 (q, 2H, J ¼ 6.4 Hz), 3.04e3.16 (m,
6H), 3.37e3.49 (m, 4H), 7.17e7.20 (m, 1H), 7.33e7.36 (m, 2H), 7.81e
7.86 (m, 1H), 8.02e8.08 (m, 1H), 8.16e8.20 (dd, 1H, J ¼ 8.9 Hz,
J ¼ 2.1 Hz), 8.32e8.38 (d, 1H, J ¼ 9.0 Hz), 8.63e8.64 (d, 1H,
J ¼ 1.8 Hz), 8.82e8.88 (d, 1H, J ¼ 7.7 Hz), 9.15e9.18 (dd, 1H,
J ¼ 4.6 Hz, J ¼ 1.5 Hz). C23H26Cl2N4O2S$HCl, Mp ¼ 147.1e148.8 ꢁC.
Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N,
10.57; found C, 52.31; H, 5.14; N, 10.60.
All receptor cDNAs were obtained from the Missouri S&T cDNA
Resource Center (www.cdna.org). Human serotonin 5-HT2AR cDNA
was cloned into the pcDNA5/FRT/TO vector (Invitrogen) and
transfected into the HEK293 cells. Cell line with tetracycline-
dependent expression of 5-HT2AR was developed using the FLP-
IN T-REx system according to manufacturers protocols (Invi-
trogen). HEK293 cells with stable expression of human serotonin 5-
HT1AR, 5-HT6, 5-HT7bR or dopamine D2LR were obtained with the
use of Lipofectamine 2000 (Invitrogen). Cells were maintained at
37 ꢁC in a humidified atmosphere with 5% CO2 and were grown in
Dulbecco’s Modified Eagle Medium containing 10% dialyzed fetal
4.3.7. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
7-sulfonamide (37)
Yellow solid, 319 mg (65% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7); tR ¼ 1.27.
C23H26Cl2N4O2S, MW 493.45, monoisotopic mass 492.12,
bovine serum under selective conditions (75
m
g/ml hygromycin and
10 g/ml blasticidin for the 5-HT2AR cell line and 500
m
mg/ml G418
sulfate for the other cells). 48 h prior to cell culture harvest the
expression of the 5-HT2A receptor was induced by addition of the
2 mg/ml tetracycline. For membranes preparations, cells were
[M þ H]þ ¼ 493.0. 1H NMR (CDCl3)
d (ppm) 1.62 (br s, 4H), 2.42 (br s,
2H), 2.69 (br s, 4H), 3.06 (br s, 2H), 3.16e3.19 (t, 4H, J ¼ 4.6 Hz),
7.03e7.06 (m, 1H), 7.16e7.18 (m, 2H), 7.51e7.56 (q, 1H, J ¼ 4.1 Hz),
7.95 (m, 3H), 8.20e8.23 (m, 1H), 8.63 (s, 1H), 9.02e9.04 (dd, 1H,
J ¼ 4.4 Hz J ¼ 1.8 Hz). C23H26Cl2N4O2S$HCl, Mp ¼ 142.8e144.1 ꢁC.
Anal. Calc for C23H26Cl2N4O2S$HCl (529.91): C, 52.13; H, 5.14; N,
10.57; found C, 51.94; H, 5.13; N, 10.60.
subcultured into 150 cm2 cell culture flasks, grown to 90% conflu-
ence, washed twice with prewarmed to 37 ꢁC phosphate buffered
saline (PBS) and were pelleted by centrifugation (200 g) in PBS
containing 0.1 mM EDTA and 1 mM dithiothreitol, and stored
at ꢂ80 ꢁC.
4.3.8. N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)quinoline-
8-sulfonamide (38)
White solid, 255 mg (52% yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.5);tR ¼ 1.37.
4.4.2. 5-HT1A/5-HT2A/5-HT6/5-HT7/D2L radioligand binding assays
Membrane preparation and general assay procedures for cloned
receptors were adjusted to 96-microwell format based on protocols
was described by us previously [4,23,24].

P. Zajdel et al. / European Journal of Medicinal Chemistry 60 (2013) 42e50
49
Cell pellets were thawed and homogenized in 20 volumes of
assay buffer using an Ultra Turrax tissue homogenizer and centri-
fuged twice at 35,000 g for 20 min at 4 ꢁ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 TriseHCl, 0.1 mM EDTA, 4 mM
MgCl2, 10 mM pargyline and 0.1% ascorbate; for 5-HT2AR: 50 mM
TriseHCl, 0.1 mM EDTA, 4 mM MgCl2 and 0.1% ascorbate; for 5-
HT6R: 50 mM TriseHCl, 0.5 mM EDTA and 4 mM MgCl2, for 5-
HT7bR: 50 mM TriseHCl, 4 mM MgCl2, 10 mM pargyline and 0.1%
ascorbate; for dopamine D2LR: 50 mM TriseHCl, 1 mM EDTA, 4 mM
MgCl2, 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 0.1% ascorbate.
All assays were incubated in a total volume of 200 mL in 96-well
resuspended in 30 volumes of buffer and centrifuged again. The
resulting pellet was resuspended in the same quantity of the buffer
and centrifuged third time in the same conditions. [3H]-Citalopram
(spec. act. 50 Ci/mmol, NEN Chemicals) was used for labeling 5-HT-
transporter. 240
ml of the tissue suspension, 30
m
l of 1 nM [3H]-
l of 1 mM
Citalopram and 30
m
l of the analyzed compound or 30
m
imipramine (displacer) were incubated at 22 ꢁC for 1 h. The
concentrations of analyzed compounds ranged from 10ꢂ10 to
10ꢂ5 M. Incubations were terminated by vacuum filtration over GF/
B filters and washed 5 times with 200 ml of ice-cold buffer. Radio-
activity was measured in a MicroBeta TriLux e liquid scintillation
counter (PerkinElmer, USA). The inhibition constants (Ki) were
calculated from the ChengePrushoff equation [25]. Results were
expressed as means of at least two separate experiments.
microtitre plates for 1 h at 37 ꢁC, except for 5-HT1AR and 5-HT2A
R
which were incubated at room temperature for 1 h and 1.5 h,
respectively. The process of equilibration was 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 (PerkinElmer, USA).
4.5. Pharmacology
4.5.1. Subjects
For displacement studies the assay samples contained as radi-
oligands (PerkinElmer, USA): 1.5 nM [3H]-8-OH-DPAT (135.2 Ci/
mmol) for 5-HT1AR; 2 nM [3H]-ketanserin (53.4 Ci/mmol) for 5-
HT2AR; 2 nM [3H]-LSD (83.6 Ci/mmol) for 5-HT6R; 0.6 nM [3H]-5-
CT (39.2 Ci/mmol) for 5-HT7R or [3H]-raclopride (76.0 Ci/mmol)
for D2LR.
The experiments were performed on male Swiss albino mice (22e
26 g) purchased from a licensed breeder Staniszewska (Ilkowice,
Poland) or male CD-1 mice (accredited animal facility Jagiellonian
University Medical College Kraków, Poland) and mice were kept in
groups of ten to Makrolon type
3
cages (dimensions
26.5 ꢃ 15 ꢃ 42 cm). Male Wistar rats weighing 205e225 g obtained
fromaccreditedanimalfacilityJagiellonianUniversityMedicalCollege
(Kraków, Poland) were housed in polycarbonate Makrolon type 3
cages (dimensions 26.5 ꢃ 15 ꢃ 42 cm) in groups of four. All animals
were kept in an environmentally controlled rooms (ambient
temperature 22 ꢀ 2 ꢁC; relative humidity 50e60%; 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 experi-
ments. Standard laboratory food (Ssniff M-Z) and filtered water were
freely available. All the experimental procedures were approved by
theILocalEthicsCommissionattheJagiellonianUniversityinKraków.
Non-specific binding was defined with 10
HT1AR and 5-HT7R binding experiments, whereas 10
promazine, 10 M of methiothepine or 1
used in 5-HT2AR, 5-HT6R and D2L assays, respectively.
Each compound was tested in triplicate at 7e8 concentrations
(10ꢂ11e10ꢂ4 M). The inhibition constants (Ki) were calculated from
the ChengePrushoff equation [25]. Results were expressed as
means of at least two separate experiments.
m
M of 5-HT in 5-
M of chlor-
M of (þ)butaclamol were
m
m
m
4.4.3. Adrenergic a1 competition binding assay
Tissue (rat cortex) was homogenized in 20 volumes of ice-cold
50 mM TriseHCl buffer, pH 7.6 using an Ultra Turrax T25B (IKA)
homogenizer. The homogenate was centrifuged at 20,000ꢃ g for
20 min. The resulting supernatant was decanted and pellet was
resuspended in the same buffer and centrifuged again in the same
conditions. The final pellet was resuspended in appropriate volume
of buffer (10 mg/1 mL). [3H]-Prazosin (spec. act. 85 Ci/mmol, Per-
4.5.2. Experimental procedures
All the experiments were conducted in the light phase between
09.00 and 14.00 h. Each experimental group consisted of 5e10
animals/dose, and the animals were used only once in each test.
The experiments were performed by an observer unaware of the
treatment administered.
kinElmer, USA) was used for labeling a1-receptor. 240
tissue suspension, 30 l of 10 M phentolamine (displacer), 30
0.2 nM [3H]-Prazosin and 30
m
l of the
m
m
m
ml of
4.5.3. Head twitch response in Swiss albino mice
l of the analyzed compound were
In order to habituate mice to the experimental environment,
each animal was randomly transferred to a 12 (diameter ꢃ 20 cm)
(height) glass cage, lined with sawdust 20 min before the treat-
ment. Head twitches in mice were induced by (ꢀ)DOI (2.5 mg/kg).
Immediately after treatment, the number of head twitches was
counted during 20 min [10]. ID50 (the dose inhibiting the head
twitches in mice by 50%) was calculated using Graph Pad Prism 5
Software.
incubated at 30 ꢁC for 30 min. The concentrations of analyzed
compounds ranged from 10ꢂ10 to 10ꢂ5 M.
The incubation was terminated by rapid filtration over glass
fiber filters FilterMate B (PerkinElmer, USA) using 96-well Filter-
Mate harvester (PerkinElmer, USA). Five rapid washes were per-
formed with ice-cold 50 mM TriseHCl buffer, pH 7.6. Filter mate
was dried in microwave and placed in plastic bag (PerkinElmer,
USA) and soaked in 10 mL of liquid scintillation cocktail Ultima Gold
MV (PerkinElmer, USA). After even distribution of scintillation
cocktail filter bag was sealed. The radioactivity on the filter was
measured in MicroBeta TriLux 1450 scintillation counter (Perki-
nElmer, USA). The inhibition constants (Ki) were calculated from
the ChengePrushoff equation [25]. Results were expressed as
means of at least two separate experiments.
4.5.4. Apomorphine-induced climbing behavior in Swiss albino
mice
For observation, mice were placed in separate cages with walls
made of metal bars. Apomorphine (3 mg/kg) was injected 10 min
after the drugs. Twenty minutes after injection of apomorphine,
time of climbing was determined for 2 min. Climbing time was
defined as the period during which the animal held the 2, 3 or 4
paws on the wall. ID50 (the dose inhibiting climbing behavior in
mice by 50%) was calculated using Graph Pad Prism 5 Software.
4.4.4. SERT competition binding assay
The assay was performed according to the method of Owens
et al. [26] with slight modifications. Rat cerebral cortex was
homogenized in 30 volumes of ice-cold 50 mM TriseHCl containing
150 mM NaCl and 5 mM KCl, pH ¼ 7.7 at 25 ꢁC and centrifuged at
20,000ꢃ g for 20 min. The supernatant was decanted and pellet was
4.5.5. Forced swim test in Swiss albino mice
The experiment was carried out according to the method of
Porsolt et al. [11]. Briefly, mice were individually placed in a glass

50
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4.5.7. MK-801-induced hyperlocomotor in CD-1 mice
The locomotor activity was recorded according to the method
described above.
4.5.8. Drugs
The following drugs were used: imipramine (hydrochloride,
SigmaeAldrich), MK-801 (hydrogen maleate, SigmaeAldrich), ari-
piprazole (Adamed Pharmaceuticals), apomorphine (hydrochlo-
ride,
Tocris),
2,5-dimethoxy-4-iodoamphetamine
(DOI,
hydrochloride, Adamed Pharmaceuticals) and tested compounds:
33, 34, 37, 39, 40. Imipramine, apomorphine, DOI and MK-801 were
dissolved in distilled water; remaining compounds were sus-
pended in a 1% aqueous solution of Tween 80 immediately before
administration. All the compounds were administered intraperi-
toneally at a volume of 10 ml/kg 30 min before the test, excluding
MK-801 and apomorphine which were injected 15 min and 20 min,
respectively, before testing. Control animals received a vehicle
injection according to the same schedule.
4.5.9. Statistics
All the data are presented as the mean ꢀ SEM. The statistical
significance of the results was evaluated by a one-way ANOVA,
followed by Bonferroni Comparison Test.
Acknowledgments
This study was partly supported by the Polish Ministry of
Science and Higher Education (MNiSW), Grant No. N N405 378437.
Radioligand binding experiments were financially supported by the
Norwegian Financial Mechanism as part of the Polish-Norwegian
Research Fund, Grant No. PNRFe103eAI-1/07.
Appendix A. Supplementary material
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Part 9: 5-HT1A and 5-HT2 receptor affinity of some 2- and 3-substituted
1,2,3,4-tetrahydro-beta-carbolines, Pharmazie 48 (1993) 289e294.
Supplementary material related to this article can be found at
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