Novel multi-target azinesulfonamides of cyclic amine derivatives as potential antipsychotics with pro-social and pro-cognitive effects

Article abstract of DOI:10.1016/j.ejmech.2018.01.002

Currently used antipsychotics are characterized by multireceptor mode of action. While antagonism of dopamine D₂ receptors is responsible for the alleviation of “positive” symptoms of schizophrenia and the effects at other, particularly serotonergic receptors are necessary for their additional therapeutic effects, there is no consensus regarding an “ideal” target engagement. Here, a detailed SAR analysis in a series of 45 novel azinesulfonamides of cyclic amine derivatives, involving the aryl-piperazine/piperidine pharmacophore, central alicyclic amine and azinesulfonamide groups has led to the selection of (S)-4-((2-(2-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)pyrrolidin-1-yl)sulfonyl)isoquinoline (62). The polypharmacology profile of 62, characterized by partial 5-HT_1AR agonism, 5-HT_2A/5-HT₇/D₂/D₃R antagonism, and blockade of SERT, reduced the “positive”-like, and “negative”-like symptoms of psychoses. Compound 62 produced no catalepsy, demonstrated a low hyperprolactinemia liability and displayed pro-cognitive effects in the novel object recognition task and attentional set-shifting test. While association of in vitro features with the promising in vivo profile of 62 is still not fully established, its clinical efficacy should be verified in further stages of development.

Full text of DOI:10.1016/j.ejmech.2018.01.002

Accepted Manuscript
Novel multi-target azinesulfonamides of cyclic amine derivatives as potential
antipsychotics with pro-social and pro-cognitive effects
Paweł Zajdel, Tomasz Kos, Krzysztof Marciniec, Grzegorz Satała, Vittorio Canale,
Krzysztof Kamiński, Małgorzata Hołuj, Tomasz Lenda, Robert Koralewski, Marek
Bednarski, Leszek Nowiński, Jacek Wójcikowski, Władysława A. Daniel, Agnieszka
Nikiforuk, Irena Nalepa, Piotr Chmielarz, Justyna Kuśmierczyk, Andrzej J. Bojarski,
Piotr Popik
PII:
S0223-5234(18)30002-3
10.1016/j.ejmech.2018.01.002
EJMECH 10079
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 August 2017
Revised Date: 9 December 2017
Accepted Date: 1 January 2018
Please cite this article as: Paweł. Zajdel, T. Kos, K. Marciniec, G. Satała, V. Canale, K. Kamiński, Mał.
Hołuj, T. Lenda, R. Koralewski, M. Bednarski, L. Nowiński, J. Wójcikowski, Włł.A. Daniel, A. Nikiforuk, I.
Nalepa, P. Chmielarz, J. Kuśmierczyk, A.J. Bojarski, P. Popik, Novel multi-target azinesulfonamides of
cyclic amine derivatives as potential antipsychotics with pro-social and pro-cognitive effects, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.002.
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ACCEPTED MANUSCRIPT
Novel multi-target azinesulfonamides of cyclic amine derivatives as potential
antipsychotics with pro-social and pro-cognitive effects
a,*
b
c
f
a
Paweł Zajdel, Tomasz Kos, Krzysztof Marciniec, Grzegorz Satała, Vittorio Canale,
a
b
g
d
Krzysztof Kamiński, Małgorzata Hołuj, Tomasz Lenda, Robert Koralewski, Marek
e
e
i
i
Bednarski, Leszek Nowiński, Jacek Wójcikowski, Władysława A. Daniel, Agnieszka
b
h
h
h
f
Nikiforuk, Irena Nalepa, Piotr Chmielarz, Justyna Kuśmierczyk, Andrzej J Bojarski, Piotr
b,*
Popik
a
Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna
Street, 30-688 Kraków, Poland
b
Department of Behavioral Neuroscience and Drug Development, Institute of Pharmacology,
Polish Academy of Sciences, 12 Smętna Street, 31-343 Kraków, Poland
c
Department of Organic Chemistry, Medical University of Silesia, 4 Jagiellońska Street, 41-
2
00 Sosnowiec, Poland
d
TriMen Chemicals, 141 Piłsudskiego Avenue, 92-318 Łódź
e
Department of Pharmacological Screening, Jagiellonian University Medical College, 9
Medyczna Street, 30-688 Kraków, Poland
f
g
Department of Medicinal Chemistry, Department of Neuro and Psychopharmacology
h
i
Department of Brain Biochemistry, Department of Pharmacokinetics and Drug
Metabolism, Institute of Pharmacology, Polish Academy of Sciences,12 Smętna Street, 31-
43 Kraków, Poland
3
*
Corresponding authors:
Piotr Popik, Institute of Pharmacology, Polish Academy of Sciences, e-mail: nfpopik@cyf-
kr.edu.pl
Paweł Zajdel, Jagiellonian University Medical College, e-mail: pawel.zajdel@uj.edu.pl
1

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Table of Contents graphic
2

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Abstract
Currently used antipsychotics are characterized by multireceptor mode of action. While
antagonism of dopamine D receptors is responsible for the alleviation of “positive”
2
symptoms of schizophrenia and the effects at other, particularly serotonergic receptors are
necessary for their additional therapeutic effects, there is no consensus regarding an “ideal”
target engagement. Here, a detailed SAR analysis in a series of 45 novel azinesulfonamides
of cyclic amine derivatives, involving the aryl-piperazine/piperidine pharmacophore, central
alicyclic amine and azinesulfonamide groups has led to the selection of (S)-4-((2-(2-(4-
(benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)pyrrolidin-1-yl)sulfonyl)isoquinoline (62). The
polypharmacology profile of 62, characterized by partial 5-HT R agonism, 5-HT /5-
1
A
2A
HT /D /D R antagonism, and blockade of SERT, reduced the “positive”-like, and “negative”-
7
2
3
like symptoms of psychoses. Compound 62 produced no catalepsy, demonstrated a low
hyperprolactinemia liability and displayed pro-cognitive effects in the novel object
recognition task and attentional set-shifting test. While association of in vitro features with
the promising in vivo profile of 62 is still not fully established, its clinical efficacy should be
verified in further stages of development.
Keywords: multitarget directed ligands, polypharmacology, designed multiple ligands,
5
-HT1A partial agonist, 5-HT antagonist, dopamine D receptors, schizophrenia, cognitive
7 2
flexibility
3

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1
. Introduction
The key action of typical antipsychotic medications is attributed to the blockade of
dopamine D receptors (D Rs) in the mesolimbic and nigrostriatal brain areas [1]. However,
2
2
apart from the reduction of the most disturbing “positive” symptoms (hallucinations and
delusions) [2], this mechanism is also responsible for the undesired side-effects, including the
induction of extrapyramidal symptoms (EPS) [3] and for hyperprolactinemia [4].
Second generation antipsychotics exhibit significant affinity for various serotonin
receptors. Their prominent feature is an additional antagonism of 5-HT Rs (or, more
2
A
precisely, a high 5-HT /D receptor affinity ratio) [5], which contributes to the side-effects
2
A
2
reduction. For instance, an inverse agonist of 5-HT Rs, pimavanserin alleviates symptoms
2
A
of psychosis without causing EPS [6]. The stimulation of 5-HT Rs also contributes to the
1
A
alleviation of the negative and extrapyramidal symptoms and improves cognitive impairment
7]. Clinical studies have demonstrated that the 5-HT R partial agonist tandospirone
[
1
A
ameliorates cognitive decline in schizophrenic patients. In addition, 5-HT R activation
1
A
contributes also to the antidepressant and anti-anxiety properties of antipsychotics [8,9].
Next, the blockade of 5-HT Rs has emerged as a novel mechanism to treat cognitive
6
impairment in schizophrenia [10]. In this regard, antagonists and inverse agonists of 5-HT R
6
display pro-cognitive actions in preclinical and clinical settings [11]. Finally, recent
preclinical data demonstrate that the 5-HT R antagonist SB-269970 exerts pro-social effects
7
[
12], most likely contributing to an alleviation of the “negative” symptoms of schizophrenia
including, emotional flattening and social withdrawal) as well as to the pro-social and pro-
(
cognitive effects of the modern antipsychotics [13].
Although the introduction and development of second generation antipsychotics have
revolutionized the pharmacotherapy of schizophrenia, there is no clear consensus regarding
the precise mechanism of action of an “ideal” antipsychotic medication being effective
4

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against all three core symptoms and devoid of the undesired side-effects [14]. Because the
etiology and pathophysiology of psychoses remain elusive, a multi-target approach remains
the conventional strategy for the development of schizophrenia pharmacotherapies [15].
We designed compounds with a multi-receptor profile by combining privileged
structures (i.e., molecular frameworks that confer biological activity within a class of targets)
present in selected antipsychotics and compounds evaluated in preclinical and clinical trials
(Figure 1).
Figure 1.
Specifically, we used an alicyclic amine fragment modified with an arylsulfonamide moiety
as a privileged structure for 5-HT R, the azinesulfonyl fragment present in potent 5-HT R
7
6
and 5-HT R antagonists [16,17], different privileged amine cores stimulating 5-HT Rs (like
7
1A
eltoprazine and ziprasidone) and acting as antagonists/partial agonists at dopamine D Rs (like
2
ziprasidone, risperidone, bifeprunox and brexpiprazole), integrating these pharmacophores
into one molecule (Figure 1).
We report the chemical synthesis and structure-activity relationship results, followed
by neurochemical, functional, pharmacokinetic and toxicological evaluation. Present study
revealed compound 62 as a putative novel atypical antipsychotic addressing the “positive”-
like symptoms, reversing the “negative”-like symptoms, and cognitive deficits associated
with schizophrenia without causing catalepsy with a minimal side-effects profile.
5

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2
. Results and Discussion
2
.1. Chemistry
The synthesis of final compounds 38–82 was performed according to the multistep
procedure shown in Scheme 1. Boc-protected β-homo-amino acids 1–5 were reduced to the
corresponding primary alcohols 6–10, which were subsequently oxidized using
2
-iodoxybenzoic acid (IBX) [18] to yield aldehyde derivatives 11–15. These were further
used for reductive amination of selected arylpiperazines/arylpiperidines/tetrahydropyridines
in the presence of sodium triacetoxyborohydride, giving intermediates 16–37. After the
removal of the protecting group, the final quinoline- and isoquinoline-sulfonamides of cyclic
amine derivatives 38–82 were obtained while treating the secondary amines with the selected
azinesulfonyl chloride in the basic condition. The azinylsulfonyl chlorides were synthesized
from respective haloquinolines or isoquinoline via their S-methyl analogs according to the
previously reported method [19].
Scheme 1.
(
S)-(Boc-azepan-2-yl)acetic acid (5) used for the preparation of compounds 75, 76 and 81,
2 was synthesized following the routes presented in Scheme 2. A starting 2-acetamido-5-
hexenoic acid was submitted to enzymatic treatment giving (S)-2-amino-5-hexenoic acid
83). Upon the introduction of the Boc protection, this was further alkylated with allyl
8
(
bromide to give 84. The latter compound was submitted to ring closing metathesis, using
Grubbs second-generation catalyst [20], in refluxing dichloromethane for 3 days, to yield
cyclized Boc-tetrahydro-1H-azepine-2-carboxylic acid (85).
Scheme 2.
6

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After the double bond reduction, using palladium on activated charcoal, in a mixture of
EtOH/THF/H O at 36°C under a hydrogen atmosphere, the resulting intermediate Boc-
2
hexahydro-1H-azepine-2-carboxylic acid (86) was then submitted to Arndt-Eistert β-
homologation reaction. The reaction involved the creation of a diazoketone intermediate (87)
and its further conversion to acetic acid analog 5 in the presence of CF COOAg as a catalyst
3
[
21].
2
.2. Structure-affinity relationships studies
Our approach focused on the development of a putative novel antipsychotic, which, by
extending the idea of multitarget ligands fit the classical pharmacophore for monoaminergic
receptor (mainly serotonin and dopamine) ligands consisting of the protonated amine
anchored by Asp3.32 (amino acid numbering according to [22]) and specific hydrophobic
recognition involving aromatic interaction (CH–π or π–π) with the Phe6.51/Phe6.52/Trp6.48
aromatic cluster [23]. Moreover, in the case of compounds optionally containing the terminal
fragment connected with the basic center via alkylene linker, the additional aromatic and H-
bond interactions stabilize ligand binding in the second transmembrane pocket created
between TMHs 7–3 (involving specific amino acids characteristic for serotonin and
dopamine receptors) [23].
2
.2.1. The effect of the type of amine
We first focused on the amine pharmacophore moieties present in bifeprunox, which
behaves as 5-HT R agonist and D R partial agonist, and eltoprazine (classified as 5-HT R
1
A
2
1A
7

ACCEPTED MANUSCRIPT
partial agonist). Our results indicate that compounds bearing 1,3-benzoxazol-2-on-4-yl-
piperazine (I) were classified as dual 5-HT /D R ligands (Table 1), while 1,4-benzodioxin-
1
A
2
5
-yl-piperazine derivatives (II, eltoprazine) displayed a high affinity for 5-HT /D Rs and
1A 2
high-to-moderate affinity for the 5-HT sites. It was further found that a 5-chloroindol-3-yl-
7
tetrahydropyridine (III) core did not result in a high affinity for 5-HT Rs, which are crucial
2
A
for atypical antipsychotic activity.
Table 1.
Our amine scaffold replacement strategy involving introduction of 6-fluorobenzo[d]isoxazol-
-yl-piperidine (IV, present in risperidone) did not improve affinity for 5-HT1A receptors.
Compounds 49–51 displayed a mixed 5-HT /5-HT /D R profile, with a moderate affinity for
3
2
A
7
2
5
-HT1A and 5-HT Rs. These compounds showed a higher affinity for 5-HT R in comparison
6 6
to risperidone. It was further found, that replacement of 6-fluorobenzo[d]isoxazol-3-yl-
piperidine with benzo[d]isothiazol-3-yl-piperazine (V), present in ziprasidone, resulted in
compounds 52–57 with a high affinity for the four receptors, except for a moderate-to-high
affinity for 5-HT R.
6
The replacement of benzo[d]isothiazol-3-yl-piperazine (V) with benzo[d]isoxazol-3-yl-
piperazine (VI) generally had no influence on the affinity for 5-HT , 5-HT or D Rs, while
2
A
7
2
it decreased the affinity for 5-HT R. This receptor pattern is in line with the one observed
1
A
for 6-fluorobenzo[d]isoxazol-3-yl-piperidine. This result indicates that the benzo[d]isoxazole
moiety determines the affinity for 5-HT , 5-HT and D Rs regardless of the type of
2
A
7
2
connected alicyclic amine (i.e. piperidine or piperazine). It thus seems, that piperidine and
piperazine are equipotent regarding their interaction with the studied receptors.
8

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Indeed, the introduction of benzothiophen-4-yl-piperazine (VII), present in brexpiprazole
and responsible for its partial agonism of D receptors, conferred multimodality of all the
2
compounds for the five studied receptors. The representative compounds 61–66 with
benzothiophen-4-yl-piperazine were classified as high affinity ligands for 5-HT /5-HT /
1
A
2A
5
-HT /5-HT /D Rs. Interestingly, the replacement of the benzothiophen-4-yl-piperazine
6 7 2
fragment with indol-4-yl-piperazine (VIII) decreased the affinity of compounds 67–70 for
5
-HT R.
6
2
.2.2. The effect of the azine moiety
Our previous reports on azinesulfonamides of long-chain arylpiperazine derivatives with
multimodal mechanism [17,24] and selective 5-HT R antagonists [16] disclosed the influence
6
of the type of azinesulfonamide moiety (the position of the sulfonamide group in the α- or β-
position of the azine moiety and the localization of the sulfonamide group in a pyridine or
benzene ring) on receptor affinity. Thus, for the present study only beneficial α-position
azinesulfonamides were selected. Unfortunately, the quantitative influence of 5-quinolyl, and
4
- and 5-isoquinolyl moieties toward the tested receptor panel was difficult to unequivocally
establish.
2
.2.3. The effects of stereochemistry and size of alicyclic amine
As stereochemistry plays a role in ligand recognition by different serotonin and dopamine
receptors [25–28], we focused on the relevance of chirality to receptor activity profile. It was
found that for some amine scaffolds an interaction with 5-HT and to less extent 5-HT Rs
6
7
was dependent on stereochemistry. This observation was pronounced in the case of 5-HT Rs,
6
9

ACCEPTED MANUSCRIPT
where enantiomers (R) displayed 5–to–19-fold higher affinity than their (S) counterparts (e.g.,
4
5, 49, 53, 61 vs 47, 51, 56, 64, Table 1).
Encouraged by these data we then applied a scaffold hopping approach to investigate any
influence of the size of the alicyclic amine on the receptor profile (Table 2).
Table 2.
For this purpose, we synthesized analogs with (S) azetidine, six-member ring (R)
piperidine and its even higher seven-member analog – (S) azepane. The replacement of
pyrrolidine with azetidine generally decreased the binding, even though affinity for 5-HT ,
1
A
5
-HT , 5-HT , and D Rs stayed at the same level. Increasing the ring size from a pyrrolidine
2A 7 2
to piperidine and azepane also decreased the affinity for all receptors with the most
pronounced decrease or even loss of affinity of the evaluated compounds for 5-HT R (Table
6
2
2
5
).
.3. Preliminary pharmacokinetic study
Next, based on the binding data (Table 2), a series of 6 selected compounds, 53, 54, 56,
7, 62, 65 exhibiting a high affinity for 5-HT , 5-HT , 5-HT , and D Rs, and a high-to-
1
A
2A
7
2
moderate affinity for 5-HT R, were submitted to a preliminary pharmacokinetic study in male
6
Wistar rats. The results indicate that the three 4-isoquinolyl derivatives better penetrate the
blood−brain barrier and are taken up more by the brain than the two 5-isoquinolyl analogs
(Table 1. SI).
2
.4. In vitro intrinsic activity evaluation
10

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Subsequently, the selected 4-isoquinolyl derivatives (53, 62 and 65) were submitted for a
detailed evaluation of their functional profile using in vitro methods (Table 3). Among them,
compound 53 with benzo[d]isothiazol-3-yl-piperazine behaved as an antagonist at 5-HT ,
1
A
5
-HT , 5-HT , D , and D Rs. In contrast, benzothiophen-4-yl-piperazine analogs 62 and 65
2A 7 2 3
showed stimulatory activity in the agonist assays at 5-HT Rs. The latter two compounds
1
A
behaved as antagonists of 5-HT , 5-HT , D and D Rs (Table 3).
2
A
7
2
3
Table 3.
Interestingly, only compound 65 behaved as a potent 5-HT R antagonist. The functional
6
activity of 62 and 65 at 5-HT R was similar to that observed for clozapine, ziprasidone or
1
A
brexpiprazole (behaving as 5-HT R partial agonists).
1
A
2
.5. Preliminary in vivo behavioral evaluation
Uncompetitive NMDA receptor antagonists (e.g., ketamine or PCP,) induce
schizophrenia-like symptoms in healthy volunteers, thus administration of these
psychotomimetics to rodents serves as a model of psychoses [29]. Consequently, compounds
normalizing hyperactivity (distance traveled) display antipsychotic-like properties [30]. The
preliminary experiments (Table 4) allowed the selection of compounds 53 and 62, which
produced an antipsychotic effect on PCP-induced locomotor hyperactivity in rats at doses
lower than those evoking sedation (see Figure 1-SI and Figure 2-SI for details, respectively).
In contrast compound 56 and 65 (both R enantiomer) did not reverse the PCP-induced
hyperactivity and induced sedation. Importantly, clozapine (Figure 3-SI), and ziprasidone
(Figure 4-SI) displayed a favorable profile by reducing PCP-induced hyperactivity at doses
11

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lower than those inducing sedation, while haloperidol (Figure 5-SI) and olanzapine (Figure
6
-SI) inhibited the PCP-induced hyperactivity at doses higher than those producing sedation.
Impaired cognitive processes constitute an integral part of the clinical picture of
schizophrenia, and the improvement of cognitive deficits has become one of the priorities in
the development of antipsychotics. Thus, the novel object recognition task (NORT) was used
to study the influence of the investigated compounds on visual episodic memory [31].
This test is based on the spontaneous exploration of novel and familiar objects. The
administration of ketamine or PCP reduces the ability to discriminate a novel object from a
familiar one [13], and any compounds that have a protective effect against this deficit are
regarded as potentially useful for treating cognitive deficits that are characteristic of
psychoses [32].
Table 4.
Table 4 shows that compounds 53 (Figure 7-SI) and 62 (Figure 8-SI) (minimum effective
dose; MED = 1 mg/kg, i.p.) ameliorated PCP-induced learning deficits in the NORT. In the
same experimental settings, clozapine (Figure 9-SI), ziprasidone (Figure 10-SI) and
olanzapine (Figure 11-SI) but not haloperidol (Figure 12-SI) displayed similar pro-cognitive
effects.
2
.6. Cardiac safety, interaction with cytochrome P450, and metabolic stability of compounds
3 and 62
5
Since a cardiac toxicity is a common reason for increased cardiac death, ability of
compound 53 (Figure 13-SI) and 62 (Figure 14-SI) to block human hERG potassium
channels was investigated in a whole cell patch clamp assay in CHO-K1 cells with expression
12

ACCEPTED MANUSCRIPT
of hERG channels. Compound 53 tested at 10 µM and 50 µM concentrations showed a
propensity to block hERG, while 62 tested at the same concentrations displayed no
statistically significant changes in ion currents. Additionally, 62 displayed no agonist
-6
properties for 5-HT R (agonist effect @ 10 M counted 3 and 14 % for 53 and 62,
2
B
respectively) which are involved in valvulopathy and cardiac impairment [33].
As the QT interval prolongation is one of the safety issue often noted for atypical
antipsychotics, cardiac safety of compounds 53 and 62 was further tested in vivo in a guinea
pig model showing no QT prolongation or any cardiac liability at a dose of 5 mg/kg, and 10
mg/kg (i.p.), respectively. Compounds 53 (Table 4-SI) and 62 (Table 5-SI) at doses 5- and
1
0-fold higher than those used in the behavioral tests did not significantly change the normal
ECG or frequency of heart rate in guinea pigs. The lack of changes in the ECG may suggest
the safety of 53 and 62 regarding pro-arrhythmic activity while using behaviorally effective
doses.
We also investigated whether compound 53 (2 and 5 mg/kg; Figure 15-SI) and compound
6
2 (3 and 10 mg/kg, Figure 16-SI) affected blood pressure in rats. The results demonstrate
compounds 53 and 62 did not affect diastolic or systolic blood pressure in rats at any of the
doses tested.
In vitro biotransformation studies using rat liver microsomes indicate that compounds 53
and 62 showed metabolic stability with intrinsic clearance values of 1.73 and 2.81
µL/min/mg, respectively. Additionally, to extend the potential drug-drug interaction at the
level of the liver cytochrome P450, the influence of compounds 53 and 62 on five different
cytochrome P450 isozymes – CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, was
determined [34]. Compounds 53 and 62 only moderately inhibited CYP3A4 activity with
IC counting 23 and 10 µM, respectively (Table 6-SI). The obtained values indicate that
5
0
compounds 53 and 62 should not inhibit the CYP3A4 isoform in vivo since their
13

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pharmacological concentrations in the rat brain and plasma are below 1 µM. Hence, the new
compounds studied here are safe with respect to drug interaction at the level of cytochrome
P450 as other atypical neuroleptics (e.g., clozapine or risperidone) which is not true for
typical neuroleptics (e.g., phenothiazines or haloperidol), which are strong CYP2D6
inhibitors [35].
2
.7. Advanced in vivo pharmacological evaluation of 53 and 62
To further extend the evaluation of the antipsychotic properties of compounds 53 and 62,
the conditioned avoidance response (CAR) test was employed [36]. The CAR test is
recognized as a reliable tool with high predictive validity for “positive” symptoms in
antipsychotic development. Compounds 53 and 62, at a dose of 3 mg/kg, suppressed the
avoidance behavior without affecting failures, suggesting a “specific” antipsychotic-like
action in the CAR test (Table 5).
Table 5.
Similar specific effects were apparent for risperidone and clozapine; however, haloperidol,
olanzapine and ziprasidone significantly increased the failures, suggesting a sedative effect
under these experimental conditions (Table 7-SI).
In the ketamine-disrupted social interaction test summarized in Table 5, treatment with 53
(Figure 17-SI) and 62 (Figure 18-SI) ameliorated social deficits at doses of 3 and 1 mg/kg,
respectively. Similar effects were observed for 2 mg/kg aripiprazole (Figure 19-SI) and 0.1
mg/kg risperidone (Figure 20-SI). In contrast, clozapine (1 mg/kg; Figure 21-SI),
14

ACCEPTED MANUSCRIPT
haloperidol (0.1 mg/kg, Figure 22-SI), and ziprasidone (1 mg/kg, Figure 23-SI) were
inactive in the same experimental setting.
One of the major obstacles to the treatment with antipsychotics is their propensity to
produce extrapyramidal side-effects [3]. To examine the tendency of the novel compounds to
produce EPS-like side effects rats were scored for catalepsy for 240 min following i.p.
administration. Haloperidol produced dose-dependent cataleptogenic effects with an MED ~
0
.2 mg/kg, (Figure 24-SI). Compound 53 at 30 mg/kg and olanzapine at 40 mg/kg shows the
cataleptogenic effects (Figure 25-SI) In contrast, ziprasidone (30 mg/kg) produced
insignificant cataleptogenic effects, and compound 62 (30 mg/kg) was devoid of this action
(Table 5, Figure 25-SI).
The potential side effect profile of 62 was also assessed in terms of its ability to induce
hyperprolactinemia. In contrast to olanzapine and ziprasidone (which significantly increased
prolactin levels at a dose of 3 mg/kg), compound 62 did not affect plasma prolactin level in
rats up to a dose of 3 mg/kg (Figure 26-SI).
Apart from the aforementioned functional profile (Table 3), extended pharmacological in
vitro profiling was conducted for 62 to investigate its potential off-target activity (Table 6).
-6
The results show that 62 at 10 M inhibited the binding of the control to adrenergic α , α ,
1
2C
D , D , D , and 5-HT , but not M and 5-HT by at least 50%. It also inhibited binding to
1
3
4
2C
1
3
SERT (K = 76 nM). It was found that 62 behaved as an antagonist of adrenergic α , H , 5-
i
1A
1
HT , 5-HT Rs and showed modest antagonistic properties at α , D and 5-HT Rs.
1
D
2C
2C
3
1B
Consequently, like other atypical antipsychotics, 62 displays a complex pharmacology with
the desired profile, and appears to produce some potentially unwanted off-target effects.
These effects, which result from the antagonistic action of 62 on α1A and H receptors, could
1
suggest factors related to hypotension and sedation; however, these states were not found in
this study.
15

ACCEPTED MANUSCRIPT
Table 6.
The pharmacokinetic properties of 62 were then explored after administration of a single
dose of 9 mg/kg per os indicating that compound 62 is quickly absorbed from the
gastrointestinal tract (Tmax = 10 min) and reaches a maximum concentration of Cmax = 54 nM.
A similar Cmax value is reached in the brain after 60 min (Tmax).
Schizophrenic patients develop cognitive dysfunctions, including inflexibility in
modifying behavior in response to altering the relevance of stimuli. This aspect of executive
function, which is controlled by the prefrontal cortex, is commonly assessed in humans using
the WCST [37]. In fact, a poor WCST performance is a key behavioral symptom of
schizophrenia [38]. We thus examined the cognitive effects of compound 62 in the attentional
set-shifting test (ASST) [13,39]. While ketamine disturbed cognitive flexibility by
significantly and specifically impairing rats’ performance at the extradimensional (ED) stage,
compound 62 (1 mg/kg) reversed this deficit (Figure 27-SI).
3
. Conclusions
Currently used medications for schizophrenia can effectively control “positive” symptoms
but with some exceptions they display limited impact on cognitive deficits and social
withdrawal. These unmet medical needs were addressed in present study aimed at developing
novel antipsychotic. By combining the concept of multimodal ligands with privileged
structural elements favorable for interactions with selected monoaminergic receptors, we
designed, synthesized, and characterized a new series of azinesulfonamides of alicyclic amine
derivatives with arylpiperazine/piperidine moieties. SAR exploration of this library revealed
that the (isoquinolin-4-ylsulfonyl)-(S)-pyrrolidinyl fragment and the 1,2-benzothiazol-3-yl-
16

ACCEPTED MANUSCRIPT
and benzothiophen-4-yl-piperazine fragments were favorable for binding the 5-HT ,
1
A
5
-HT , 5-HT , 5-HT , D , and D receptors. Additionally, the interaction of the designed
2A 6 7 2 3
series with 5-HT R was highly dependent on the stereochemistry of the alicyclic amine.
6
From
a
diverse library, (S)-4-((2-(2-(4-(benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)
pyrrolidin-1-yl)sulfonyl)isoquinoline (62) was identified as a potential novel antipsychotic.
Taking advantage of the concept of D R blockade, which is the only clinically validated
2
approach, 62 combines a multi-receptor mechanism with prominent 5-HT R partial
1
A
agonism, 5-HT /5-HT /D R antagonism, and blockade to SERT. Because it reverses PCP-
2
A
7
3
induced hyperactivity and avoidance behavior in the CAR test, 62 appears to be useful for
addressing the positive symptoms of schizophrenia. Furthermore, its ability to reverse the
social interaction deficit in a ketamine model and memory decline in PCP- and ketamine-
disrupted conditions reveals that 62 can alleviate the negative symptoms and displays pro-
cognitive properties. Additionally, 62 displayed no cardiac toxicity, and no tendency of
inducing catalepsy. Further mechanistic studies are necessary to better understand the distinct
behavioral attribute profiles observed. Additionally, the receptor multimodality of 62 could
potentially provide opportunities for the treatment of various other CNS disorders, such as
anxiety, depression, psychoses associated with dementia, and stress and impulse control
disorders. Finally, since social withdrawal accompanies autism spectrum disorders and
because only two atypical antipsychotics – risperidone and aripiprazole – are registered for
treatment of autism, further studies should reveal the activity of novel antipsychotics with a
5
-HT R component against the core symptoms and communication impairments of autism.
7
17

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4
. Experimental Section
4
.1. Chemistry
Organic synthesis were carried out at ambient temperature, unless indicated otherwise.
Organic solvents used in this study (Sigma-Aldrich, Chempur) were of reagent grade and
were used without purification. All other commercially available reagents were of the highest
purity (from Sigma-Aldrich, Fluorochem, TCI). All workup and purification procedures were
carried out with reagent-grade solvents under ambient atmosphere.
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
4
0°C, and eluted under gradient conditions from 95% to 0% of eluent A over 10 min, at a
-1
flow rate of 0.3 mL min . 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
-
1
1
1
50°C, desolvation temperature 350°C, desolvation gas flow rate 600 L h , cone gas flow
-1
00 L h , 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.
18

ACCEPTED MANUSCRIPT
1
13
H NMR and C NMR spectra were obtained in Varian BB 200 or AV-III-500-HD
spectrometer using TMS (0.00 ppm) as an internal standard in CDCl and were recorded at
3
3
00 or 500 and 75 MHz, respectively. The J values are reported in Hertz (Hz), and the
splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd
(
doublet of doublets), dt (doublet of triplets), td (triplet of doublets), tt (triplet of triplets), qd
quartet of doublet), ddd (doublet of doublet of doublets), m (multiplet), br.s (broad singlet).
(
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.
Characterization data for representative compounds are summarized below, while all
characterization data for all of intermediate and remaining final compounds are reported in
the Supporting Information.
4
.1.1 General procedure for the synthesis of intermediates 6–10
To a mixture of 2-((2S)-1-tert-butoxycarbonylpyrrolidin-2-yl)acetic acid (2.0 g, 8.7 mmol)
in anhydrous CH Cl (20 mL) lithium aluminum hydride 2.0 M in THF (7.23 mL, 13.05
2
2
o
mmol) was added dropwise and the reaction mixture was heated to 55 C in inert atmosphere
under argon). After stirring for 4 hours, the reaction mixture was cooled to room
(
temperature, diluted with AcOEt (20 mL) and treated with saturated NaHCO (3 mL). Then
3
the solution was concentrated under reduce pressure. To the residue were added CH Cl (20
2
2
mL) and the organic layer was washed with water and brine, and then dried over anhydrous
Na SO , and concentrated under reduced pressure to give intermediate 6 (1.72 g, 8.0 mmol)
2
4
as light oil (yield 91.9%). The same procedure was used for the synthesis of intermediates 7–
0.
1
19

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4
.1.2. General procedure for the synthesis of intermediates 11–15
2
–Iodoxybenzoic acid (IBX, 6.72 g, 24 mmol) was dissolved in DMSO (50 mL), stirred
for 10 min. at room temperature and treated with a solution of intermediate 6 (1.72 g, 8.0
mmol) in DMSO (50 mL). After stirring overnight at room temperature the reaction mixture
o
was diluted with Et O (100 mL), cooled to 0 C and quenched by addition of water (100 mL).
2
The inorganic layer was diluted with brine (50 mL) and extracted with AcOEt (3 × 20 mL).
The combined organic layers were washed with saturated NaHCO , water and brine, and then
3
dried over anhydrous Na SO . After concentration under reduced pressure the crude product
2
4
was purified by column chromatography on silica gel, using a mixture of AcOEt/Hex (30/70
v/v) as eluting system yielding intermediate 11 (1.12 g, 5.24 mmol) as light oil (yield 65.5%).
The same procedure was used for the synthesis of intermediates 12–15.
4
.1.3. General procedure for the synthesis of intermediates 16–37
To a solution of intermediate 11 (1 g, 4.7 mmol), 1-(benzothiophen-4-yl)piperazine (1.5 g,
7
mmol) in THF (30 mL) was added sodium triaceoxyborohydride (1.98 g, 9.4 mmol) and
anhydrous AcOH (1 mL). After stirring for 4 hours at room temperature, saturated NaHCO3
was added to the mixture, and the aqueous layer was extracted with AcOEt (3 × 20 mL). The
organic layer was washed with water and brine, dried over anhydrous Na SO , and
2
4
concentrated under reduced pressure. The residue obtained was purified by column
chromatography on silica gel, using a mixture of CH Cl /MeOH (9/0.5 v/v), to give the Boc-
2
2
protected derivative 24 (1.70 g, 4.14 mmol) as light oil (yield 88%). The same procedure was
used for the synthesis of intermediates 16–37.
4
.1.4. General procedure for the synthesis of final compounds 38–82
Removal of Boc function of the intermediates 16–37 was accomplished by treatment with
a mixture of TFA/CH Cl (80/20 v/v). Upon removal of organic solvents under reduced
2
2
20

ACCEPTED MANUSCRIPT
pressure, TFA salts of deprotected intermediates 16–37 were left under vacuum overnight.
Then, a mixture of the proper secondary amine (0.8 mmol) in CH Cl (5 mL), and Et N (2.4
2
2
3
mmol) was cooled down (ice bath), and the selected azinesulfonyl chloride (0.96 mmol) was
added in one portion. The reaction mixture was stirred for 2-6 hours on ice bath. Then, the
solvent was evaporated and the final sulfonamides 38–82 were purified by column
chromatography using CH Cl /MeOH as eluting system.
2
2
4
4
.2. Characterization data for representative final compounds
.2.1. (S)-7-(4-(2-(1-(Isoquinolin-4-ylsulfonyl)pyrrolidin-2-yl)ethyl)piperazin-1-yl)benzo[d]
oxazol-2(3H)-one (39)
Yellow oil (150 mg, 78% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 100%, R = 3.94; C H N O S, MW 507.6,
2
2
t
26 29
5
4
+
1
Monoisotopic Mass 507.19, [M+H] 508.5. H NMR (300 MHz, CDCl ) δ (ppm) 1.60–1.72
3
(m, 4H), 1.73–1.81 (m, 1H), 1.85–1.94 (m, 1H), 2.34–2.46 (m, 2H), 2.51–2.59 (m, 2H), 2.60–
2.69 (m, 2H), 3.32 (t, J = 4.6 Hz, 4H) 3.44–3.52 (m, 2H), 4.07 (tt, J = 8.0, 3.9 Hz, 1H), 6.59–
6.71 (m, 2H), 7.02–7.10 (m, 1H), 7.77 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.92 (ddd, J = 8.6, 7.1,
1.4 Hz, 1H), 8.05 (br.s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.86 (dd, J = 8.7, 0.8 Hz, 1H), 9.13 (s,
1H), 9.43 (d, J = 0.5 Hz, 1H).
4
.2.2. (S)-4-((2-(2-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-5-yl)piperazin-1-yl)ethyl)pyrrolidin-
1
-yl)sulfonyl)isoquinoline (42)
White solid (140 mg, 69% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 97%, R = 4.42; C H N O S, MW 508.63,
2
2
t
27 32
4
4
+
1
Monoisotopic Mass 508.21, [M+H] 509.4. H NMR (300 MHz, CDCl ) δ (ppm) 1.63–1.82
3
(m, 4H), 1.89–2.00 (m, 1H), 2.05–2.18 (m, 1H), 2.24–2.36 (m, 1H), 3.34 (td, J = 3.1, 1.3 Hz,
4
H), 3.62–3.81 (m, 4H), 3.90–4.01 (m, 2H), 4.29 (t, J = 3.9 Hz, 2H), 4.42 (t, J = 2.9 Hz, 2H),
21

ACCEPTED MANUSCRIPT
4
.49–4.57 (m, 2H), 6.79–6.94 (m, 2H), 7.32 (dd, J = 6.9, 1.6 Hz, 1H), 7.95–8.08 (m, 1H),
8
.30 (t, J = 7.6 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 9.00 (d, J = 8.6 Hz, 1H), 9.26 (s, 1H), 9.96
1
3
(
s, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 24.2, 29.7, 30.5, 30.7, 51.7, 55.1, 58.4, 64.7,
3
1
04.8, 108.9, 122.6, 126.5, 128.5, 128.6, 128.9, 131.7, 132.7, 147.4, 155.7.
4
.2.3. (R)-5-((2-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)
pyrrolidin-1-yl)sulfonyl)quinoline (47)
Brown oil (140 mg, 71% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 99%, R = 5.03; C H ClN O S, MW 521.07,
2
2
t
28 29
4
2
+
1
Monoisotopic Mass 520.17, [M+H] 521.5. H NMR (300 MHz, CDCl ) δ (ppm) 1.58–1.75
3
(m, 4H), 1.76–1.87 (m, 1H), 2.03–2.16 (m, 1H), 2.44–2.55 (m, 4H), 2.58–2.78 (m, 2H), 3.18
(dd, J = 10.8, 2.1 Hz, 2H), 3.31–3.39 (m, 2H), 3.95–4.05 (m, 1H), 6.06–6.08 (m, 1H), 7.07–
7
.14 (m, 2H), 7.29 (s, 1H), 7.53 (dd, J = 8.8, 4.7 Hz, 1H), 7.73–7.80 (m, 2H), 8.26 (dd,
J = 7.0, 1.2 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.96 (dd, J = 4.1, 1.2 Hz, 1H), 9.07 (br. s., 1H),
1
3
9
4
1
.27 (dd, J = 8.8, 1.2 Hz, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 24.2, 28.5, 31.1, 33.0,
3
8.4, 50.1, 52.9, 54.8, 58.4, 112.5, 117.1, 118.3, 120.0, 122.3, 122.7, 123.0, 125.0, 125.6,
26.1, 127.7, 129.5, 129.9, 133.8, 134.4, 135.2, 135.5, 148.5, 151.1.
4
.2.4. (R)-6-Fluoro-3-(1-(2-(1-(quinolin-5-ylsulfonyl-pyrrolidin-2-yl)ethyl)piperidin-4-yl)-
1
,2-benzoxazole (51)
Brown oil (120 mg, 65% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 100%, R = 4.5; C H FN O S, MW 508.61,
2
2
t
27 29
4
3
+
1
Monoisotopic Mass 508.2, [M+H] 509.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.61–1.69
3
(
m, 6H), 1.72–1.89 (m, 3H), 1.99–2.11 (m, 8H), 3.37 (t, J = 6.3 Hz, 2H), 3.95–4.00 (m, 1H),
.06 (td, J = 8.89, 2.1 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 7.58 (dd, J = 8.8, 4.2 Hz, 1H), 7.69
dd, J = 8.7, 5.1 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 8.26–8.37 (m, 2H), 9.01 (dd, J = 4.1, 1.5
7
(
22

ACCEPTED MANUSCRIPT
1
3
Hz, 1H), 9.26 (d, J = 8.9 Hz, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 24.2, 29.7, 30.9, 30.9,
3
3
1
1.1, 48.5, 55.1, 58.4, 97.6, 117.4, 122.6, 124.5, 128.5, 128.6, 128.9, 131.7, 132.7, 147.4,
49.9, 157.7, 164.2, 166.3.
4
.2.5. (S)-3-(4-(2-(1-(Isoquinolin-4-ylsulfonyl)pyrrolidin-2-yl)ethyl)piperazin-1-yl)-1,2-
benzothiazole (53)
Yellow oil (250 mg, 78% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 100%, R = 4.85; C H N O S , MW 507.67,
2
2
t
26 29
5
2 2
+
1
Monoisotopic Mass 507.18, [M+H] 508.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.56–1.66
3
(m, 2H), 1.69–1.78 (m, 2H), 1.87–1.99 (m, 2H), 2.11–2.20 (m, 2H), 2.26–2.37 (m, 2H), 3.37–
3
7
.48 (m, 4H), 3.52–3.64 (m, 2H), 3.69–3.79 (m, 2H), 4.10–4.22 (m, 1H), 7.40–7.48 (m, 1H),
.51–7.58 (m, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.98–8.09 (m, 2H), 8.28 (t, J = 7.8 Hz, 1H), 8.51
1
3
(
d, J = 8.1 Hz, 1H), 8.98 (d, J = 8.6 Hz, 1H), 9.17 (s, 1H), 9.81 (s, 1H). C NMR (75 MHz,
CDCl ) δ (ppm) 23.7, 29.4, 30.7, 46.5, 48.6, 51.0, 51.6, 54.2, 58.0, 120.4, 123.1, 124.4,
3
1
24.9, 127.0, 127.9, 128.3, 131.2, 131.7, 132.8, 133.8, 135.6, 138.3, 152.5, 161.6. Anal. calcd
for C H N O S ·3HCl·2H O: C: 47.82, H: 5.56, N: 10.72, S: 9.82; Found C: 47.91, H: 5.47,
2
6
29
5
2
2
2
N: 10.75, S: 9.84. C H N O S ·3HCl·2H O: 201.1–202.3°C.
2
6
29
5
2
2
2
4
.2.6. (S)-3-(4-(2-(1-(Isoquinolin-5-ylsulfonyl)pyrrolidin-2-yl)ethyl)piperazin-1-yl)-1,2-
benzothiazole (54)
Yellow oil (220 mg, 71% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 98%, R = 4.35; C H N O S , MW 507.67,
2
2
t
26 29
5
2 2
+
1
Monoisotopic Mass 507.18, [M+H] 508.3. UPLC H NMR (300 MHz, CDCl ) δ (ppm)
3
1
2
7
.58–1.66 (m, 2H), 1.69–1.76 (m, 2H), 1.83–1.94 (m, 2H), 2.11–2.20 (m, 2H), 2.28–2.37 (m,
H), 3.35–3.48 (m, 4H), 3.52–3.67 (m, 2H), 3.72–3.81 (m, 2H), 4.12–4.22 (m, 1H), 7.19–
.24 (m, 1H), 7.51–7.58 (m, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.98–8.09 (m, 2H), 8.20 (d,
23

ACCEPTED MANUSCRIPT
13
J = 8.2 Hz, 1H), 8.42 (s, 1H), 8.62–8.72 (m, 2H), 9.33 (s, 1H). C NMR (75 MHz, CDCl ) δ
3
(
ppm) 24.2, 31.0, 32.8, 38.7, 48.3, 50.0, 53.0, 55.1, 58.4, 68.1, 120.5, 122.6, 123.9, 124.9,
27.6, 127.7, 128., 129.9, 133.7, 134.6, 135.5, 148.6, 151.1, 152.7, 163.9. Anal. calcd for
C H N O S ·2HCl·H O: C: 52.17, H: 5.56, N: 11.70, S: 10.71; Found C: 52.24, H: 5.45, N:
1
26
29
5
2
2
2
1
1.82, S: 11.01. Mp for C H N O S ·2HCl·H O: 201.5–202.4°C.
26 29 5 2 2 2
4
.2.7. (R)-3-(4-(2-(1-(Isoquinolin-4-ylsulfonyl)pyrrolidin-2-yl)ethyl)piperazin-1-yl)-1,2-
benzothiazole (56)
Brown oil (200 mg, 75% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 100%, R = 4.81; C H N O S , MW 507.67,
2
2
t
26 29
5
2 2
+
1
Monoisotopic Mass 507.18, [M+H] 508.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.59–1.75
3
(m, 4H), 1.81–1.91 (m, 2H), 2.03–2.16 (m, 2H), 2.38–2.47 (m, 2H), 3.40–3.46 (m, 4H), 3.55
(t, J = 4.7 Hz, 2H), 3.69–3.79 (m, 2H), 4.05 (dt, J = 8.1, 3.9 Hz, 1H), 7.31–7.38 (m, 1H), 7.45
(t, J = 7.52 Hz, 1H), 7.70–7.83 (m, 2H), 7.85–7.94 (m, 2H), 8.07 (d, J = 8.1 Hz, 1H), 8.84 (d,
1
3
J = 8.7 Hz, 1H), 9.10 (s, 1H), 9.39 (s, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 24.3, 31.0,
3
3
2.9, 48.5, 50.0, 53.0, 55.2, 58.5, 120.5, 123.9, 124.6, 127.5, 128.5, 128.6, 128.8, 129.0,
1
31.7, 132.6, 145.0, 152.7, 157.6, 163.9. Anal. calcd for C H N O S ·2HCl·H O: C: 52.17,
2
6
29
5
2
2
2
H: 5.56, N: 11.70, S: 10.71; Found C: 51.95, H: 5.95, N: 11.82, S: 10.11. Mp for
C H N O S ·2HCl·H O: 201.5–202.4°C.
26
29
5
2
2
2
4
.2.8. (R)-3-(4-(2-(1-(Isoquinolin-5-ylsulfonyl)pyrrolidin-2-yl)ethyl)piperazin-1-yl)-1,2-
benzothiazole (57)
Yellow oil (240 mg, 76% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 100%, R = 4.31; C H N O S , MW 507.67,
2
2
t
26 29
5
2 2
+
1
Monoisotopic Mass 507.18, [M+H] 508.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.56–1.73
3
(m, 4H), 1.80–1.89 (m, 1H), 2.00–2.12 (m, 1H), 2.36–2.44 (m, 2H), 2.51–2.60 (m, 2H), 2.63–
24

ACCEPTED MANUSCRIPT
2
.70 (m, 2H), 3.37–3.43 (m, 2H), 3.54 (t, J = 4.9 Hz, 4H), 3.96–4.04 (m, 1H), 7.32–7.38 (m,
1
H), 7.46 (td, J = 7.6, 1.0 Hz, 1H), 7.68–7.75 (m, 1H), 7.79–7.93 (m, 2H), 8.20 (d, J = 8.2
1
3
Hz, 1H), 8.41 (dd, J = 7.4, 1.2 Hz, 1H), 8.64–8.70 (m, 2H), 9.34 (s, 1H). C NMR (75 MHz,
CDCl ) δ (ppm) 24.2, 31.0, 32.8, 48.4, 50.1, 53.00, 55.1, 58.5, 120.6, 123.9, 125.9, 127.5,
3
1
28.0, 129.1, 132.1, 133.5, 133.8, 145.1, 152.7, 153.2, 163.9. Anal. calcd for
C H N O S ·2HCl·H O: C: 52.17, H: 5.56, N: 11.70, S: 10.71; Found C: 52.45, H: 5.26, N:
26
29
5
2
2
2
1
1.42, S: 10.81. Mp for Anal. calcd for C H N O S ·2HCl·H O: 201.5–202.4°C.
26 29 5 2 2 2
4
.2.9. (S)-3-(4-(2-(1-(Quinolin-5-ylsulfonyl)pyrrolidin-2-yl)ethyl)piperazin-1-yl)-1,2-
benzoxazole (58)
Yellow oil (150 mg, 70% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 100%, R = 4.25; C H N O S, MW 491.61,
2
2
t
26 29
5
3
+
1
Monoisotopic Mass 491.2, [M+H] 492.4. H NMR (300 MHz, CDCl ) δ (ppm) 1.58–1.70
3
(m, 4H), 1.79–1.89 (m, 1H), 1.96–2.08 (m, 1H), 2.33–2.40 (m, 2H), 2.48–2.56 (m, 2H), 2.59–
2
.67 (m, 2H), 3.38 (d, J = 5.9 Hz, 2H), 3.56 (t, J = 5.0 Hz, 4H), 4.00 (tt, J = 7.9, 3.9 Hz, 1H),
.22 (ddd, J = 7.9, 6.3, 1.4 Hz, 1H), 7.43–7.48 (m, 2H), 7.54–7.58 (m, 1H), 7.69 (d, J = 8.5
7
Hz, 1H), 7.79 (t, J = 7.9 Hz, 1H), 8.24–8.36 (m, 2H), 9.00 (dd, J = 4.2, 1.3 Hz, 1H), 9.26 (dt,
J = 8.8, 0.7 Hz, 1H).
4
.2.10. (S)-4-((2-(2-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)pyrrolidin-1-yl)sulfonyl)
isoquinoline (62)
Brown oil (230 mg, 75% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 100%, R = 5.06; C H N O S , MW 506.68,
2
2
t
27 30
4
2 2
+
1
Monoisotopic Mass 506.18, [M+H] 507.4. H NMR (300 MHz, CDCl ) δ (ppm) 1.61–1.73
3
(m, 4H), 1.73–1.80 (m, 1H), 1.86–1.95 (m, 1H), 2.39–2.50 (m, 2H), 2.60–2.75 (m, 4H), 3.20
(t, J = 4.0 Hz, 4H), 3.47 (dd, J = 7.2, 5.9 Hz, 2H), 4.07–4.12 (m, 1H), 6.92 (dd, J = 7.7, 0.7
25

ACCEPTED MANUSCRIPT
Hz, 1H), 7.27–7.32 (m, 1H), 7.39–7.44 (m, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.76 (ddd, J = 8.1,
7
.0, 0.9 Hz, 1H), 7.91 (ddd, J = 8.6, 7.1, 1.4 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 8.88 (dd,
13
J = 8.7, 0.8 Hz, 1H), 9.14 (s, 1H), 9.42 (s, 1H). C NMR (75 MHz, CDCl ) δ (ppm) 24.3,
3
3
1.1, 33.0, 48.5, 52.1, 53.5, 53.6, 55.2, 58.6, 112.2, 117.0, 121.9, 124.7, 124.9, 125.0, 128.4,
28.4, 128.6, 129.0, 129.1, 131.8, 132.5, 134.1, 141.1, 145.1, 148.4, 157.6. Anal. calcd for
1
C H N O S ·3HCl: C: 52.64, H: 5.40, N: 9.09, S: 10.41; Found C: 52.61, H: 5.72, N: 8.76,
27
30
4
2 2
S: 10.58. Mp for C H N O S ·3HCl: 202.5–203.8°C.
27
30
4
2 2
4
.2.11. (R)-4-((2-(2-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)pyrrolidin-1-yl)sulfonyl)
isoquinoline (65)
Brown oil (260 mg, 74% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 100%, R = 5.27; C H N O S , MW 506.68,
2
2
t
27 30
4
2 2
+
1
Monoisotopic Mass 506.18, [M+H] 507.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.60–1.74
3
(m, 4H), 1.85–1.96 (m, 2H), 2.08–2.18 (m, 2H), 2.39–2.53 (m, 2H), 2.58–2.77 (m, 2H), 3.21
(t, J = 6.0 Hz, 4H). 3.47 (dd, J = 7.2, 5.9 Hz, 2H), 4.09 (tt, J = 8.12, 3.9 Hz, 1H), 6.92 (dd,
J = 7.6, 0.6 Hz, 1H), 7.25–7.34 (m, 1H), 7.38–7.46 (m, 2H), 7.57 (d, J = 8.1 Hz, 1H), 7.76
(ddd, J = 8.1, 7.1, 0.9 Hz, 1H), 7.92 (ddd, J = 8.1, 7.1, 0.9 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H),
1
3
8
2
1
.88 (dd, J = 8.7, 0.7 Hz, 1H), 9.14 (s, 1H), 9.42 (s, 1H). C NMR (75 MHz, CDCl ) δ (ppm)
3
4.3, 31.0, 48.4, 52.1, 53.6, 55.2, 58.5, 112.2, 117.1, 117.9, 121.9, 125.0, 125.9, 133.5, 133.6,
45.1, 153.2. Anal. calcd for C H N O S ·3HCl: C: 52.64, H: 5.40, N: 9.09, S: 10.41;
2
7
30
4
2 2
Found C: 52.68, H: 5.67, N: 8.89, S: 10.34. Mp for C H N O S ·3HCl: 202.2–203.5°C.
2
7
30
4
2 2
4
.2.12. (S)-4-((2-(2-(4-(1H-Indol-4-yl)piperazin-1-yl)ethyl)pyrrolidin-1-yl)sulfonyl)
isoquinoline (68)
Yellow oil (120 mg, 67% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 97%, R = 4.38; C H N O S, MW 489.63,
2
2
t
27 31
5
2
26

ACCEPTED MANUSCRIPT
+
1
Monoisotopic Mass 489.22, [M+H] 490.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.63–1.76
3
(m, 6H), 1.86–1.93 (m, 1H), 2.38–2.49 (m, 2H), 2.58–2.65 (m, 2H), 2.68–2.75 (m, 2H), 3.26–
3
7
.31 (m, 4H), 3.41–3.47 (m, 2H), 4.02–4.11 (m, 1H), 6.51–6.62 (m, 2H), 7.04–7.18 (m, 3H),
13
.71–7.94 (m, 2H), 8.09 (s, 1H), 8.86 (d, J = 8.6 Hz, 1H), 9.11 (s, 1H), 9.40 (s, 1H). C
NMR (75 MHz, CDCl ) δ (ppm) 24.3, 30.1, 31.0, 48.4, 51.2, 53.2, 55.2, 58.6, 101.2, 105.8,
3
1
2
06.6, 121.2, 122.6, 122.7, 124.6, 128.4, 128.6, 128.9, 131.8, 132.6, 137.0, 145.0, 157.6,
07.0.
4
.2.13. (S)-5-((2-(2-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)azetidin-1-yl)sulfonyl)
quinoline (77)
Brown oil (150 mg, 66% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 99%, R = 5.06; C H N O S , MW 493.64,
2
2
t
25 27
5
2 2
+
1
Monoisotopic Mass 493.16, [M+H] 493.4. H NMR (300 MHz, CDCl ) δ (ppm) 1.74–1.89
3
(m, 2H), 1.97–2.08 (m, 2H), 2.09–2.22 (m, 2H), 2.59–2.62 (m, 4H), 3.13–3.17 (m, 4H), 3.57–
3.66 (m, 1H), 3.83–3.93 (m, 1H), 4.41 (qd, J = 8.2, 4.7 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H),
7.24–7.31 (m, 2H), 7.53–7.62 (m, 2H), 7.82 (dd, J = 8.5, 7.3 Hz, 1H), 8.29–8.41 (m, 2H),
9.03 (dd, J = 4.4, 1.5 Hz, 1H), 9.17 (d, J = 8.8 Hz, 1H).
4
.2.14. (R)-5-((2-(2-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)piperidin-1-yl)sulfonyl)
quinoline (79)
Brown oil (160 mg, 69% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.7); UPLC purity = 99%, R = 5.14; C H N O S , MW 520.71,
2
2
t
28 32
4
2 2
+
1
Monoisotopic Mass 520.2, [M+H] 521.4. H NMR (300 MHz, CDCl ) δ (ppm) 1.57–1.62
3
(m, 6H), 1.69–1.81 (m, 2H), 1.87–1.99 (m, 2H), 2.22–2.32 (m, 2H), 2.47–2.56 (m, 4H), 3.07–
3
.18 (m, 4H), 3.85–3.93 (m, 1H), 6.89 (dd, J = 7.8, 0.7 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H),
27

ACCEPTED MANUSCRIPT
7
.37–7.44 (m, 2H), 7.54–7.63 (m, 2H), 7.81 (dd, J = 8.5, 7.4 Hz, 1H), 8.31–8.40 (m, 2H),
8
.96–9.02 (m, 1H), 9.04 (dd, J = 4.2, 1.6 Hz, 1H).
4
.2.15. (S)-4-((2-(2-(4-(Benzo[b]thiophen-4-yl)piperazin-1-yl)ethyl)azepan-1-yl)sulfonyl)
isoquinoline (82)
Brown oil (180 mg, 73% yield) following chromatographic purification over silica gel with
CH Cl /MeOH (9/0.5); UPLC purity = 98%, R = 5.21; C H N O S , MW 534.74,
2
2
t
29 34
4
2 2
+
1
Monoisotopic Mass 534.21, [M+H] 535.3. H NMR (300 MHz, CDCl ) δ (ppm) 1.28–1.40
3
(m, 2H), 1.43–1.56 (m, 4H), 1.61–1.75 (m, 3H), 2.02–2.23 (m, 4H), 2.27–2.38 (m, 4H), 3.00–
3
.11 (m, 4H), 3.94–4.13 (m, 2H), 6.86 (d, J= 7.03 Hz, 1H), 7.26 (d, J = 15.8 Hz, 1H), 7.33–
.40 (m, 2H), 7.54 (d, J = 8.2 Hz, 1H), 7.71–7.77 (m, 1H), 7.88–7.94 (m, 1H), 8.08 (d,
7
J = 8.2 Hz, 1H), 8.62 (d, J = 7.6 Hz, 1H), 9.19 (s, 1H), 9.39 (s, 1H).
4
4
.3. In vitro pharmacology
.3.1. Radioligand binding assays
Radioligand binding assays were employed for determining the affinity and the selectivity
profile of the synthesized compounds for cloned serotonin: 5-HT , 5-HT , 5-HT , 5-HT
7
1
A
2A
6
and dopamine D receptors which were all stably expressed in HEK293 or CHO-K1 cells.
2
L
According to the previously published procedures [24,26,40,41], the experiments were
3
3
carried out using 2.5 nM [ H]-8 OH DPAT (135.2 Ci/mmol) for 5-HT R; 1 nM [ H]-
1
A
3
Ketanserin (53.4 Ci/mmol) for 5-HT R; 2 nM [ H]-LSD (83.6 Ci/mmol) for 5-HT R; 0.8
2
A
6
3
3
nM [ H]-5-CT (80.1 Ci/mmol) for 5-HT R or 2.5 nM [ H]-Raclopride (76.0 Ci/mmol) for
7
D R.
2
L
Non-specific binding is defined with 10 µM of 5-HT in 5-HT R and 5-HT R binding
1
A
7
experiments, whereas 10 µM of chlorpromazine, 10 µM of methiothepine or 10 µM of
28

ACCEPTED MANUSCRIPT
haloperidol were used in 5-HT R, 5-HT R and D assays, respectively. Each compound
2
A
6
2L
–
11
–4
was tested in triplicate at 7–8 concentrations (10 –10 M). The inhibition constants (K )
i
were calculated from the Cheng-Prusoff equation [42].
HEK293 cells with stable expression of human serotonin 5-HT R, 5-HT R, 5-HT R or
1
A
6
7b
dopamine D R (all prepared with the use of Lipofectamine 2000) were maintained at 37 °C
2
L
in a humidified atmosphere with 5% CO and were grown in Dulbeco’s Modifier Eagle
2
Medium containing 10% dialysed foetal bovine serum and 500 mg/ml G418 sulphate. For
membranes preparations, cells were subcultured in 10 cm diameter dishes, grown to 90%
confluence, 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.
Prior to membrane preparations pellets were stored at –80°C. CHO-K1 cells with stable
expression of human serotonin 5-HT R were purchased from PerkinElmer BioSignal Inc
2
A
and were maintained according to manufacturer’s protocol.
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-HT R: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl , 10 µM pargyline and
1
A
2
0
.1% ascorbate; for 5-HT R: 50 mM Tris–HCl, 0.1 mM EDTA, 4 mM MgCl and 0.1%
2A 2
ascorbate; for 5-HT R: 50 mM Tris–HCl, 0.5 mM EDTA and 4 mM MgCl , for 5-HT R: 50
6
2
7b
mM Tris–HCl, 4 mM MgCl , 10 µM pargyline and 0.1% ascorbate; for dopamine D R: 50
2
2L
mM Tris–HCl, 1 mM EDTA, 4 mM MgCl , 120 mM NaCl, 5 mM KCl, 1.5 mM CaCl and
2
2
0
.1% ascorbate.
All assays were incubated in a total volume of 200 ml in 96-well microtitre plates for 1 h
at 37°C, except for 5-HT R and 5-HT R which were incubated at room temperature for 1 h
1
A
2A
and 1.5 h, respectively. The process of equilibration is terminated by rapid filtration through
29