Synthesis, docking studies, and pharmacological evaluation of 5HT2C ligands containing the N′-cyanoisonicotinamidine or N′-cyanopicolinamidine nucleus

Article abstract of DOI:10.1002/ardp.201800373

N′-Cyanoisonicotinamidine and N′-cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinities to the 5-HT_1A, 5-HT_2A, and 5-HT_2C receptors were evaluated. Several of the newly synthesized compounds, tested by binding studies, showed nanomolar affinity at the 5-HT_1A and 5-HT_2C receptors and moderate or no affinity for other relevant receptors (D₁, D₂, α₁, and α₂). Compound 8e (K_i = 21.4 nM) was the most affine for the 5-HT_2C receptor, showing, at the same time, a high selectivity with respect to the other receptors analyzed. Compounds 4a and 4c, instead, showed an interesting mixed 5-HT_1A/5-HT_2C activity with K_i values of 21.3/11.5 and 23.2/6.48 nM, respectively. The compounds with better affinity and selectivity binding profiles toward 5-HT_2C (4a, 4c, 8b, and 8e) were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that compounds 4a, 8b, and 8e exerted antidepressant-like effects and 4a and 8e revealed also significant anxiolytic properties. Among the developed derivatives, the most promising compound seems to be 4a, which displayed antipsychotic-, antidepressant- and anxiolytic-like properties. No side effects, like catalepsy, motor-impairment or ethanol-potentiating effects, were observed after the injection of the tested compounds.

Full text of DOI:10.1002/ardp.201800373

Received: 18 December 2018
DOI: 10.1002/ardp.201800373
Revised: 7 February 2019
Accepted: 9 February 2019
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F U L L P A P E R
Synthesis, docking studies, and pharmacological evaluation
of 5HT2C ligands containing the N′‐cyanoisonicotinamidine
or N′‐cyanopicolinamidine nucleus
Elisa Magli1
Angela Corvino1
Paola Massarelli5
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Ewa Kędzierska2
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Agnieszka A. Kaczor3,4
Francesco Frecentese1
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Beatrice Severino1
Irene Saccone1
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Elisa Perissutti1
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Ewa Gibuła‐Tarłowska2
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Jolanta H. Kotlińska2
Ferdinando Fiorino1
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Vincenzo Santagada1
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Giuseppe Caliendo1
1Dipartimento di Farmacia, Università di
Abstract
Napoli “Federico II”, Naples, Italy
2Department of Pharmacology and
N′‐Cyanoisonicotinamidine and N′‐cyanopicolinamidine derivatives, linked to an
arylpiperazine moiety, were prepared and their affinities to the 5‐HT1A, 5‐HT2A
Pharmacodynamics, Faculty of Pharmacy with
Division of Medical Analytics, Medical
University of Lublin, Lublin, Poland
,
and 5‐HT2C receptors were evaluated. Several of the newly synthesized compounds,
tested by binding studies, showed nanomolar affinity at the 5‐HT1A and 5‐HT2C
receptors and moderate or no affinity for other relevant receptors (D1, D2, α1, and
α2). Compound 8e (Ki = 21.4 nM) was the most affine for the 5‐HT2C receptor,
showing, at the same time, a high selectivity with respect to the other receptors
analyzed. Compounds 4a and 4c, instead, showed an interesting mixed 5‐HT1A/5‐
HT2C activity with Ki values of 21.3/11.5 and 23.2/6.48 nM, respectively. The
compounds with better affinity and selectivity binding profiles toward 5‐HT2C (4a, 4c,
8b, and 8e) were selected for further in vivo assays to determine their functional
activity. Finally, to rationalize the obtained results, molecular docking studies were
performed. The results of the pharmacological studies showed that compounds 4a,
8b, and 8e exerted antidepressant‐like effects and 4a and 8e revealed also significant
anxiolytic properties. Among the developed derivatives, the most promising
compound seems to be 4a, which displayed antipsychotic‐, antidepressant‐ and
anxiolytic‐like properties. No side effects, like catalepsy, motor‐impairment or
ethanol‐potentiating effects, were observed after the injection of the tested
compounds.
3Department of Synthesis and Chemical
Technology of Pharmaceutical Substances
with Computer Modeling Laboratory, Faculty
of Pharmacy with Division of Medical
Analytics, Medical University of Lublin, Lublin,
Poland
4School of Pharmacy, University of Eastern
Finland, Kuopio, Finland
5Dipartimento di Scienze Mediche,
Chirurgiche e Neuroscienze, Università di
Siena, Siena, Italy
Correspondence
Ferdinando Fiorino, Dipartimento di Farmacia,
Università di Napoli “Federico II”, Via D.
Montesano, 49, 80131 Naples, NA, Italy.
Email: fefiorin@unina.it
Funding information
Interdisciplinary Center for Mathematical and
Computational Modeling, Warsaw, Poland,
Grant/Award Number: G30‐18
K E Y W O R D S
5‐HT1A, 5‐HT2A, and 5‐HT2C ligands, arylpiperazine derivatives, behavioral tests, binding
assays, in vitro assay
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1
INTRODUCTION
human's mood, memory and learning, response to stress, sexual
behaviors, anxiety, schizophrenia, depression, and other disorders.[5]
Serotonin (5‐hydroxytryptamine, 5‐HT), a well‐known neurotransmitter,
regulates several physiological and behavioral functions in the central
nervous system (CNS), involved in sleep regulation, emotion and
Serotonin is also present in numerous other tissues including the
gastrointestinal tract, the cardiovascular, and immune systems. Pharma-
cological regulation of the 5‐HT system is believed to have therapeutic
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MAGLI ET AL.
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potential, and therefore it is the subject of intense studies.[4] As already
known the actions of 5‐HT are mediated by G‐protein coupled receptors
(GPCRs) except for the 5‐HT3 receptor, which is a cation channel. The
GPCR 5‐HT receptor family has been identified as a class A rhodopsin‐
like receptor. Six types of GPCR 5‐HT receptors are known: 5‐HT1,
5‐HT2, 5‐HT4, 5‐HT5, 5‐HT6, and 5‐HT7 with additional subclasses,
amounting to 15 receptors. Among these, one of the most interesting
and the first pharmacologically well‐characterized is the 5‐HT1A receptor
that mediates effects on a wide range of psychiatric disorders, and it is
involved in the proliferation of human tumor cells (PC3) and in human
hormone refractory prostate cancer tissue.[6,7] The 5‐HT2 receptor
gical evaluation of a set of arylpiperazine derivatives containing a
N′‐cyanoisonicotinamidine or N′‐cyanopicolinamidine nucleus; the bind-
ing data reported in these studies identified this original scaffold as an
optimal structural element to enhance 5‐HT1A and 5‐HT2A receptor
affinity respectively.[21,22] Successively, we designed a new set of
derivatives where the piperazine‐N‐alkyl moiety has been linked to an
isonicotinic or picolinic fragment as terminal part of LCAPs; this choice
was made considering that it represents a molecular simplification of
previously synthesized derivatives.[23,24] Consequently, we have verified
that for both the series the substitution of a cyanoamidine group with a
simple amide bond does not determine a general trend towards
5‐HT1A and 5‐HT2A receptor, but provide compounds characterized by
family comprises three subtypes, namely, 5‐HT2A, 5‐HT2B, and 5‐HT2C
.
Among these, 5‐HT2A receptor is the most abundant: it stimulates the
secretion of various hormones and influences neuronal plasticity;
peripheral 5‐HT2A receptors mediate several processes such as
vasoconstriction, platelet aggregation,[4] and intestinal motility and
secretions, by means of the conspicuous presence of serotonin‐contain-
ing enterochromaffin cells; 5‐HT causes diarrhea if present in excess, and
constipation if any at fault.[4,8–11] The 5‐HT2C receptor is probably the
one with the most widespread distribution in the CNS and is able to
mediate the action of 5‐HT in virtually all areas of the brain. It is
suspected to mediate tonic/constitutive and phasic controls in the
activity of central neurobiological networks.[8,9,12] Its molecular proper-
ties and its functional organization in the brain imply that the 5‐HT2C
receptor might be a target of survey. Changes of 5‐HT2C receptor
function have been evidenced in numerous psychiatric diseases.
Selective 5‐HT2C ligands have been synthesized for the treatment of
anxiety, depression, food intake, and schizophrenia, and could be
valuable in treating other psychiatric and neurological disorders.[12] It
is intriguing that both agonists and antagonists at 5‐HT2C receptor are
proposed in the treatment of anxiety/depression and can also be utilized
in the treatment of schizophrenia. The involvement of 5‐HT2C receptor
in various CNS pathologies and the identification of their function in
numerous cerebral networks continue to stimulate the search for new
ligands that are either selective, such as lorcaserin, (1R,3S)‐(−)‐trans‐
PAT, vabicaserin, (+)‐((1S,2S)‐2‐(2‐(allyloxy)‐5‐fluorophenyl)‐cyclopropyl)‐
methanamine, or multitarget compounds such as CP‐809,101, S32006,
S32212, or (−)‐trans‐(2S,4R)‐4‐(3′[meta]‐bromophenyl)‐N,N‐dimethyl‐1,2,
3,4‐tetrahydronaphthalen‐2‐amine [−]‐MBP.[12] One of the most‐studied
chemical classes, already known for their high affinity toward these
receptors is the long‐chain N‐1‐substituted N‐4‐arylpiperazines
(LCAPs).[10] The significance of the respective parts of the LCAP
structures on the serotonin receptor affinity, intrinsic activity, and
selectivity has been the subject of many structure–activity relationship
studies. In particular, much effort has been devoted to understand both
the role of the heterocyclic nucleus and of the substituent on the
piperazine scaffold in the ligand–receptor interaction and, consequently,
5‐HT1A, 5‐HT2A, and 5‐HT2C affinity and selectivity. Just some
interesting binding results towards 5‐HT2C receptors obtained for some
derivatives pushed us, in continuation of our research program, to
complete the N′‐cyanoisonicotinamidine and N′‐cyanopicolinamidine
series with some arylpiperazines which had not previously been
considered. All the new compounds were tested for their affinity to
5‐HT1A, 5‐HT2A, and 5‐HT2C receptors and the multireceptor profiles of
promising derivatives were also evaluated in terms of binding affinities
for dopaminergic (D1 and D2) and adrenergic (α1 and α2) receptors.
Moreover, the compounds showing better affinity and selectivity binding
profile towards 5‐HT2C receptors have been evaluated by in vivo assay
(i.e., behavioral tests), with a view to designing new drugs useful in
treating psychiatric and neurological disorders, such as schizophrenia,
depression, and anxiety. Therefore, we evaluated the antipsychotic
activity of the compounds in amphetamine‐induced hyperactivity test,
antidepressant‐like activity in the forced swim test (FST), and anxiolytic‐
like effects in the elevated plus‐maze test (EPM). Moreover, we used
additional tests—spontaneous locomotor activity, rotarod and chimney
test, ethanol and bar test—to assess potential adverse effects of the
compounds. Effects of tested compounds in the FST were compared
with commonly known antidepressant, fluoxetine, and those in EPM,
with clinically useful anxiolytic, buspirone.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The synthetic strategy used for the preparation of the N′‐cyanoisonico-
tinamidine derivatives (Table 1) is summarized in Scheme 1. The general
procedure is as follows: Isopropyl‐N‐cyano‐4‐pyridinecarboximidate (2)
was prepared starting from 4‐cyanopyridine (1) by base‐promoted
reaction with isopropanol followed by treatment with cyanamide in
aqueous phosphate buffer (NaH2PO4•2H2O/Na2HPO4 = 4:1). Compound
2 was converted to N‐(3‐bromopropyl)‐N′‐cyanoisonicotinamidine (3) by
reaction with 3‐bromopropylamine HBr and NaOMe in methanol.
a
great number of different fragments were used.[13,14] In our
Subsequent condensation of compound 3 with the desired 4‐X‐
laboratories, there has been an ongoing research work to develop more
selective serotoninergic ligands[15–24] to have novel pharmacological
tools that could improve our knowledge of the signal transduction
mechanism leading to compounds with high affinity and selectivity.
Previously described studies focused on the synthesis and pharmacolo-
substituted‐piperazine, performed in CH3CN in the presence of K2CO3
and NaI, under reflux, provided the final compounds 4a–e. Purification of
each final product was achieved by chromatography on silica gel column
and further by crystallization from the appropriate solvent. Instead, the
synthetic strategy used for the preparation of the N’‐cyanopicolinamidine

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TABLE 1 Affinities of compounds 4a–e for 5‐HT1A, 5‐HT2A, and 5‐HT2C receptors
Receptor affinity Ki SD (nM)
5‐HT1A
5‐HT2A
5‐HT2C
Compd
4a
X
[3H]‐8‐OH‐DPAT
[3H]ketanserin
[3H]mesulergine
21.3 0.8
1900 100
11.5 3.0
4b
4c
4d
No affinity
23.2 1.1
>104
>104
No affinity
6.48 0.219
>104
264 37.5
918 8.5
4e
No affinity
3000 152
No affinity
Note. For purpose of comparison, 8‐OH‐DPAT, ketanserine and mesulergine bind 5‐HT1A, 5‐HT2A, and 5‐HT2C receptors with values of 0.80, 0.85, and
1.90 nM, respectively, under these assay conditions.
5‐HT: 5‐hydroxytryptamine; SD: standard deviation.
derivatives (Table 2) is summarized in Scheme 2. The general procedure is
as follows: Methyl‐N‐cyano‐2‐pyridinecarboximidate (2) was prepared
starting from 2‐cyanopyridine (5) by base‐catalyzed conversion in
anhydrous methanol followed by treatment with cyanamide in aqueous
phosphate buffer (NaH2PO4•2H2O/Na2HPO4 = 4:1). The conversion of
methyl‐N‐cyano‐2‐pyridinecarboximidate (6) to N‐(3‐bromopropyl)‐N′‐
with the proposed structures. As already reported in literature, due to
tautomeric equilibrium about sp2 carbon, cyanoamidine derivatives may
exist as a mixture of two tautomers (I and II in Figure 1). The NMR
spectra of the final compounds 4a–e and 8a–e, confirmed that all final
cyanopicolinamidine (7) was obtained by reaction of
bromopropylamine∙HBr and triethylamine in anhydrous methanol.
Subsequent condensation of compound with the desired 4‐X‐
6 with 3‐
6
substituted‐piperazine, performed in CH3CN in the presence of K2CO3
and NaI, under reflux, provided the final compounds 8a–e. Purification of
each final product was achieved by chromatography on silica gel column
and further by crystallization from the appropriate solvent. All new
compounds gave satisfactory elemental analyses and were characterized
by 1H‐NMR and mass spectrometry (API 2000 Applied Biosystem, Foster
City, CA). 1H‐NMR and MS data for all final compounds were consistent
SCHEME 1 Synthesis of compounds 4a–e. Reagents and
conditions: (i) CH3ONa, H2NCN, iPrOH; (ii) Br(CH2)3NH2∙HBr,
CH3ONa, anhydrous MeOH; (iii) 4‐X‐substituted‐piperazine, K2CO3,
NaI, CH3CN, 70°C, 4 hr

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TABLE 2 Affinities of compounds 8a–e for 5‐HT1A, 5‐HT2A, and 5‐HT2C receptors
Receptor affinity Ki SD (nM)
5‐HT1A
5‐HT2A
5‐HT2C
Compd
8a
X
[3H]‐8‐OH‐DPAT
[3H]ketanserin
[3H]mesulergine
17.2 1.36
2730 143
904 10.2
93.3 2.8
No affinity
8b
8c
8d
>104
>104
>104
28.8 2.67
45.1 4.8
No affinity
>104
8e
>104
504 39.5
21.4 1.22
Note. For purpose of comparison, 8‐OH‐DPAT, ketanserine and mesulergine bind 5‐HT1A, 5‐HT2A, and 5‐HT2C receptors with values of 0.80, 0.85, and
1.90 nM, respectively, under these assay conditions.
5‐HT: 5‐hydroxytryptamine; SD: standard deviation.
compounds exist predominantly as the cyanoimino form (I). Moreover,
about the configurational determination of geometrical isomers (E/Z) on
the amidine bond C=N, the N‐cyanoamidine derivatives 4a–e and 8a–e
exhibited only one set of relevant 1H‐NMR signals, implying the existence
of either a single geometric form or, more probably, a fast equilibrium of
two isomeric forms as already described in the literature.[25]
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2.2
In vitro receptor binding
All the new compounds were tested for their affinity to 5‐HT1A
,
5‐HT2A, and 5‐HT2C receptors. Some of the synthesized derivatives
showed affinities in the nanomolar range towards 5‐HT2C and 5HT1A
receptors and lower affinity for 5‐HT2A receptors (Tables 1 and 2).
Besides the outstanding 5‐HT2C receptor affinity and selectivity of
compounds 8e (Ki = 21.4 nM) and 8b (Ki = 28.8 nM), other interesting Ki
values were those of compounds 4a (11.5 nM), 4c (6.4 nM), and 8c (45.1
SCHEME 2 Synthesis of compounds 8a–e. Reagents and
conditions: (i) CH3ONa, H2NCN, anhydrous MeOH; (ii) Br
(CH2)3NH2∙HBr, TEA, anhydrous MeOH; (iii) 4‐X‐substituted‐
piperazine, K2CO3, NaI, CH3CN, 70°C, 4 hr

MAGLI ET AL.
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N'
N'
FIGURE 1 Tautomeric equilibrium about sp2 carbon of the cyanoamidine derivatives
nM). Moreover compounds 4a and 4c showed an interesting mixed
5‐HT1A/5‐HT2C activity with Ki values of 21.3/11.5 nM and 23.2/6.48
nM, whereas compound 8c presented an attractive 5‐HT2A/5‐HT2C
activity with Ki value of 93.3/45.1 nM. The two series, 4a–e and 8a–e,
differ in the presence of a N′‐cyanoisonicotinamidine or N′‐cyanopico-
linamidine nucleus connected through an alkyl chain (three units) to
4‐substituted piperazines. Some derivatives (4a, 4b, 8a, and 8b) have
been already reported in two studies concerning N′‐cyanoisonicotina-
midine and N′‐cyanopicolinamidine derivatives,[21,22] whereas general
binding affinities of new derivatives, molecular docking studies were
carried out.
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2.3
Molecular docking studies
Recent studies by Peng et al.[26] and Podlewska et al.[27] revealed some
structural aspects of serotonin receptor polypharmacology. In particu-
lar, important aspects of 5‐HT2C receptor recognition have been
uncovered by a novel X‐ray structure of this receptor in complex with
the inverse agonist, ritanserin (Protein Data Bank [PDB] ID: 6BQH[26]
;
trend
a N′‐cyanoisonicotinamidine scaffold represents an optimal
Figure 2a), a ligand which shares some structural similarity with the
compounds reported here, in particular 8e (Figure 2b). As for most
other ligands of aminergic GPCRs the main contact with receptors is the
electrostatic interactions between the protonatable nitrogen atom of
the ligand and the conserved aspartate from the third transmembrane
helix, (Asp(3.32); see Figures 2, 3, and 4). The deep binding pose of
ritanserin in the 5‐HT2C receptor is characterized by one of the 4‐
structural element to enhance 5‐HT1A receptor binding, whereas the N
′‐cyanopicolinamidine nucleus drives the binding more toward 5‐HT2A
receptor. The cyanoamidine group as well as the different position of
nitrogen into the aromatic system of the considered nuclei do not seem
to be decisive in determining a specific profile towards 5‐HT2C receptors
subtype but the affinity/selectivity profile was more influenced by the
particular substituent on the piperazine moiety. Additionally, the affinity
of the most active compounds (4a, 4c, 8b, and 8e) on several other
receptors (α1 and α2 adrenergic and D1 and D2 dopaminergic receptors)
was examined to verify the selectivity of these compounds. Results are
summarized in Table 3. In particular, the compounds have moderate or
no affinity to the studied dopaminergic receptors. Regarding α1 and α2
adrenergic receptors, only compounds 8b and 8e showed quite
moderate affinity towards α1 (30 and 72 nM, respectively); these data
are very interesting considering the high degree of homology existing
between the considered receptors and demonstrate that these
compounds possess a very good binding profile. These results further
support the choice of N′‐cyanoisonicotinamidine or N′‐cyanopicolinami-
dine nucleus not only for the preparation of serotoninergic ligands
endowed with high 5‐HT1A and 5‐HT2A affinity, but also with a
interesting 5‐HT2C activity. Anyway, to rationalize the differential
fluorophenyl groups encased in
a hydrophobic pocket between
transmembrane helices V and VI, where it interacts via halogen‐
aromatic interactions with Phe(5.47) and Phe(6.44) and aromatic edge‐
to‐face π‐π stacking interactions with residues Phe(6.52) and the Trp
(6.48) “toggle switch” important for GPCR activation (see Figure 2a).[26]
This binding pose was additionally validated by mutation of Phe(5.47),
Phe(6.44), and Trp(6.48). The binding pose of compound 8e in the
human 5‐HT2C receptor is in accordance with that of ritanserin. In
particular, one 4‐fluorophenyl group interacts via aromatic edge‐to‐face
π‐π stacking interactions with residues Phe(6.52) and the Trp(6.48).
Moreover, pyrid‐2‐yl group of the ligand is also involved in π‐π stacking
interactions with Tyr118 from the first extracellular loop and forms
hydrogen bond with the side chain of Asn(7.35). Other ligands, that is,
compounds 4a (Figure 2c), 4c (Figure 2d), 8c (Figure 2e), and 8b (Figure
TABLE 3 Affinities of compounds 4a, 4c, 8b, and 8e for D1, D2, α1, and α2 receptors
Receptor affinity Ki SD (nM)
D1
D2
α1
α2
Compd
[3H]SCH‐23390
[3H]spiperone
[3H]prazosin
[3H]yohimbine
4a
>104
>104
4000
3400
>104
>104
120
190
348
30
516
4c
1200
3000
1800
8b
8e
1200
72
Note. SD: standard deviation.

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FIGURE 2 The studied compounds in the binding pocket of the human serotonin 5‐HT2C receptor. (a) The reference compound ritanserin
(X‐ray structure, PDB ID: 6BQH); (b) compound 8e, most similar to the reference compound ritanserin; (c) compound 4a; (d) compound 4c;
(e) compound 8c; (f) compound 8b. Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with gray carbon
atoms, most important residues represented as sticks. Interacting residues labeled according to Ballesteros–Weinstein nomenclature. Hydrogen
bonds shown as yellow dashed lines. Nonpolar hydrogen atoms not shown for clarity. 5‐HT: 5‐hydroxytryptamine; PDB: Protein Data Bank
2f) adopt corresponding binding poses with arylopiperazine moiety
penetrating deep in the receptor and pyrid‐2‐yl or pyrid‐4‐yl scaffold
reaching to extracellular loops of the receptor. Compound 4a (Figure
2c) interacts additionally with Trp(3.28) via π‐π stacking of pyrid‐4‐yl
group and with the main chain of Leu(7.34) via hydrogen bond with the
cyano group. Compound 4c (Figure 2d) has its pyrid‐4‐yl group also
involved in the hydrogen bond with the side chain of Ser(2.60). In case
of compound 8c (Figure 2e) pyrid‐2‐yl moiety forms a hydrogen bond
with the side chain of Asn(7.35) and for the compound 8b (Figure 2f) no
additional interactions have been found.
The lack of affinity of compounds 4d, 8d, and probably also 4e to
the human serotonin 5‐HT2C receptor might be attributed to too
bulky aryl substituent which resulted either in no docking pose
interacting with Asp(3.32) or
a pose 180 degrees rotated in
comparison to poses shown in Figure 2. No correct docking pose
was also obtained for 4b and 8a.
FIGURE 3 The studied compounds in the binding pocket of the human serotonin 5‐HT1A receptor. (a) Compound 8a; (b) compound 4a;
(c) compound 4c. Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with gray carbon atoms, most
important residues represented as sticks. Interacting residues labeled according to Ballesteros–Weinstein nomenclature. Hydrogen bonds
shown as yellow dashed lines. Nonpolar hydrogen atoms not shown for clarity. 5‐HT: 5‐hydroxytryptamine

MAGLI ET AL.
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FIGURE 4 The studied compounds in the binding pocket of the human serotonin 5‐HT2A receptor. (a) Ligand 8c; (b) ligand 4c; (c) ligand 8e.
Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with gray carbon atoms, most important residues
represented as sticks. Interacting residues labeled according to Ballesteros–Weinstein nomenclature. Hydrogen bonds shown as yellow dashed
lines. Nonpolar hydrogen atoms not shown for clarity. 5‐HT: 5‐hydroxytryptamine
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In vivo behavioral tests
Some of the studied compounds, that is, ligand 8a, 4a, and 4c are
also characterized by nanomolar affinity to the human 5‐HT1A
receptor (Figure 3).
2.4
Compounds 4a, 4c, 8e, and 8b were selected for further functional in
vivo studies. First part of experiments included: locomotor activity
and motor coordination tests, generally accepted as basic tests in
central activity investigations of new agents.[28] First, it should be
noted that none of the compounds at the doses of 30 and 60 mg/kg
changed the behavior of mice in the chimney test (Figure 5a), did not
impair motor coordination assessed in the rotarod test (Figure 5b). In
the locomotor activity test, the compound 4a at doses of 15, 30, and
60 mg/kg and other compounds at the doses of 30 and 60 mg/kg
decreased spontaneous motility after 6 min (Figure 6a), 30 min
(Figure 6b) of observation.
The docking poses of these compounds in the human 5‐HT1A
receptor (Figure 3) correspond to their docking poses to the
human 5‐HT2C receptor (Figure 1), with Asp(3.32) and Phe(6.52) as
the key residues for interactions. Moreover, π‐π stacking interac-
tions of pyrid‐2‐yl or pyrid‐4‐yl moiety of the ligand with residue
Trp(7.39) is important for binding of compounds 8a (Figure 3a) and
4a (Figure 3b). Hydrogen bond with Cys187 from the extracellular
loop 2 is also crucial for binding of 8a (Figure 3a). No correct
binding poses, that is, with the interaction with Asp(3.32) were
obtained for compounds 4b, 4d, and 8e. Only 180‐degree‐rotated
binding poses were obtained for compounds 4e, 8b, 8c, and 8d.
Similar pattern of interactions was found for compounds 8c, 4c,
and 8e and 5‐HT2A receptor, involving Asp(3.32) and Phe(6.51) or
Phe(6.52) (Figure 4). In case of ligand 8e only a binding pose 180
degrees rotated in comparison with those of other compounds was
found. Similar rotated binding poses were found for less active and
inactive compounds.
Taking into account that selective 5‐HT2C ligands may have the
therapeutic potential for the treatment of schizophrenia in humans,
in the second stage of our research, we intended to evaluate the
antipsychotic ability of the new compounds. The preclinical studies
utilize mainly the amphetamine model to estimate antipsychotic
properties of drugs.[29] As it is based on manipulation of the
dopaminergic pathways, it may primarily respond to drugs that
affect this neurotransmitter system and many neuroleptics acting as
dopaminergic antagonists reverse this effect.[30] However, it should
be noted that the hyperlocomotion following amphetamine admin-
istration is sensitive to other classes of drugs, including mGluR2/3
agonists.[31] On the other hand, several preclinical tests have pointed
the role for 5‐HT2C ligands in modulation of monoaminergic systems,
including dopaminergic. Indeed, dysfunction in serotoninergic activity
could contribute to the alteration of dopaminergic function seen in
schizophrenia.[32] In amphetamine model, only compound 4a at dose
of 7.5 mg/kg reduced amphetamine‐induced hyperactivity of mice
(Figure 7). The observed positive antipsychotic effect could be
related rather to its interaction with other than dopaminergic
receptors, because it is their weak ligand; it may be that 5‐HT2C
and/or 5‐HT1A receptors, through modulation of dopaminergic
system, may play a significant role in this activity. Furthermore,
Regarding serotonin receptor subtype selectivity and polyphar-
macology of the studied compounds, the binding of the privileged
arylpiperazine moiety to the serotonin 5‐HT2 receptor subtypes
requires Asp(3.32), Trp(6.48), and at least one aromatic residue at
positions 5.47 and/or 6.52 as reported for ritanserin.[26] In case of
5‐HT2A receptor Trp(7.39) can also be crucial as found by Podlewska
et al.[27] Our docking results indicate that in case of the interactions
of the studied ligands with 5‐HT2C receptor, the ligands form
additional hydrogen bonds between their pyridyl groups and Ser
(2.60) or Asn(7.35), which is not observed for interactions with
5‐HT1A and 5‐HT2A receptors. The number of π‐π stacking interac-
tions is also higher in case of interactions with 5‐HT2C receptor. In
summary, the presented molecular docking results can help in
designing even more potent serotonin receptor ligands with the
desired polypharmacology.

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due to modulation of the central serotonin neurotransmission, the
new compounds may also show anxiolytic and/or antidepressant
activity.[33,34]
EPM. This test is based on the hypothesis that exposure to an
elevated and open maze alley leads to an approach–avoidance
conflict that is considerably stronger in comparison to that evoked by
exposure to a closed maze alley.[35] Fear‐induced inhibition of
exploratory activity affects entries into open arms in this task. As a
significant increase in the percentage of time spent on the open arms
and the number of entries into open arms is observed only with drugs
Considering this premise, as well as in vitro data obtained for the
new compounds (5‐HT2C affinity profile for 8e and 8b, and mixed
5‐HT1A/5‐HT2C affinity profile for 4a and 4c), we undertook to
examine its antidepressant and anxiolytic potential in behavioral
models commonly used in mice, that is FST and EPM. The obtained
results indicated that compounds 4a, 8b, and 8e (15 and 30 mg/kg)
revealed antidepressant‐like properties, observed as a shortening of
the immobility time of mice to various extents in the FST (Figure 8).
In addition, fluoxetine at the dose 15 mg/kg, used as a reference drug,
caused statistically significant decrease in immobility time. The
antidepressant‐like effect of the compounds appeared, despite their
reducing effects on the spontaneous activity of mice. Additionally,
the two compounds active in the FST test (4a and 8e) exhibited
characteristics of anxiolytic drugs. In fact, these compounds showed
anxiolytic‐like activity, increasing, in a statistically significant manner,
the percentage of entries and the time spent in the open arms of the
FIGURE 6 The influence of the tested compounds 4a, 4c, 8b, and
8e (7.5–60 mg/kg) on the spontaneous locomotor activity of mice.
Investigated compounds were injected intraperitoneally 60 min before
the test. Locomotor activity was measured from two to six and after
30 min. Data are expressed as mean SEM values of the one
independent experiment. *p < 0.05, **p < 0.01, and ***p < 0.001 versus
control (Dunnett’s test). One‐way ANOVA showed significant changes
in locomotor activity of mice during 4 min observation period for the
compound 4a, F(4,25) = 15.62, p < 0.001; 4c, F(3,38) = 9.005, p < 0.001;
8e, F(3,32) = 5.422, p < 0.01; and 8b, F(3,40) = 10.33, p < 0.001.
Dunnett’s post hoc test confirmed a significant decrease in locomotor
activity of mice after the administration of compound 4a at the doses
of 15 (p < 0.05), 30 (p < 0.001), and 60 mg/kg (p < 0.001); 4c at the
doses of 30 (p < 0.01) and 60 mg/kg (p < 0.05); 8e at the dose of 30 and
60 mg/kg (p < 0.05); 8b at the doses 30 and 60 mg/kg (p < 0.001 and p
< 0.01, respectively) after 6 min of observation. One‐way ANOVA
revealed significant changes in locomotor activity of mice also in 30
min of observation for the compound 4a, F(4,25) = 21.59, p < 0.001; 4c,
F(3,38) = 3.481, p < 0.05; 8e, F(3,32) = 10.95, p < 0.001; and 8b,
F(3,40) = 5.246, p < 0.01. Dunnett’s post hoc test confirmed a
significant decrease in locomotor activity of mice after the
FIGURE 5 The influence of the tested compounds 4a, 4c, 8b, and
8e (30 and 60 mg/kg) on motor coordination in mice evaluated in
chimney (a) and rotarod (b) tests. Investigated compounds were
injected intraperitoneally 60 min before the test. Data are expressed
as mean SEM values of the one independent experiments. One‐way
analysis of variance did not show significant changes in the in
chimney, F(8,39) = 0.4529; p = 0.8812; and the time spent on the
rotarod, F(8,39) = 0.1174; p = 0.1174. SEM: standard error of means
administration of compound 4a at the doses of 15 (p < 0.01), 30, and
60 mg/kg (p < 0.001); 4c at the doses of 30 and 60 mg/kg (p < 0.05), 8e
at the doses of 30 (p < 0.05) and 60 mg/kg (p < 0.01), 8b at the doses of
30 and 60 mg/kg (p < 0.05) after 30 min of observation. ANOVA:
analysis of variance; SEM: standard error of means

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FIGURE 7 The influence of the tested compounds 4a, 4c, 8b, and 8e (7.5–15 mg/kg) on the amphetamine‐induced hyperactivity of mice.
Compounds tested were injected 60 min and amph 5 mg/kg 30 min before the test. Locomotor activity was measured for a period of 30 min.
Data are expressed as mean SEM values of the one independent experiment; **p < 0.01 versus control, ##p < 0.01 versus amph. One‐way
analysis of variance showed significant changes in the locomotor activity of mice, F(9,57) = 4.782, p < 0.001. Dunnett’s post hoc test confirmed a
significant increase in locomotor activity of mice after the administration of amph (5 mg/kg; p < 0.01). Moreover, compound 4a at the dose of
7.5 mg/kg decreased amphetamine‐induced hyperactivity of mice (p < 0.01). SEM: standard error of means
that are clinically effective anxiolytics,[36] this model has been used to
assess anxiolytic‐like activity of new putative anxiolytic com-
pounds.[37] Buspirone (5 mg/kg) used as a reference anxiolytic drug
also prolonged time spent in the open arms of the EPM and increased
percentage of entries into the open arms (Figure 9). In this test the
new compounds 4a and 8e were almost as efficacious as buspirone.
In addition, we evaluated the compounds in the tests used to
investigate their potential adverse effects, that is, the risk of sedation
and catalepsy, including ethanol and bar test. All tested compounds
completely lacked augmentative effects on ethanol‐induced anesthe-
sia up to 100 mg/kg.
8e revealed significant anxiolytic properties and, in the EPM test,
they were almost as efficacious as buspirone. Among the developed
derivatives, the most promising compound seems to be 4a, which
displayed antipsychotic‐like activity in amphetamine‐induced hyper-
activity test, that is believed to reflect the potential efficacy of drugs
in treating psychotic symptoms. Additionally, 4a was effective in
mouse models of depression and anxiety. Finally no side effects, like
catalepsy, motor‐impairment, or ethanol‐potentiating effects were
observed after the injection of this compound, as well as the other:
4c, 8b, and 8e compounds. The effectiveness and expected safety
margin implies therapeutic‐like potential of the tested compounds,
especially 4a and also 8b and 8e, however, further pharmacological
studies are necessary to determine their detailed mechanism of
action and prospective clinical usefulness.
The motor side effects of antipsychotic drugs are a main concern
in clinics. Various behavioral tests have been developed to determine
whether new antipsychotic agents or strategies generate motor side
effects in comparison to classical antipsychotics. Catalepsy in the bar
test is a frequently used model to predict liability of drugs to produce
extrapyramidal side effects (EPS) in humans.[38] By themselves,
5‐HT2C ligands do not induce catalepsy.[12] Also in our study tested
compounds 4a, 4c, 8e, and 8b did not exert any cataleptogenic
effects in the bar test.
In conclusion, data presented in this study demonstrate that,
surprisingly, the novel synthesized compounds did not display a
general trend of affinity towards 5‐HT1A or 5‐HT2A receptors, as
instead obtained with the series previously synthesized.[21,22] On the
other hand, the here reported compounds are characterized by an
important 5‐HT2C affinity/activity profile and molecular docking
studies supported these results highlighting some selective and
additional interactions of the identified ligands with the 5HT2C
receptor subtype.
|
3
CONCLUSIONS
We have described the synthesis of a new series of arylpiperazines as
serotoninergic ligands (4a–e and 8a–e). The N′‐cyanoisonicotinami-
dine derivatives 4a (Ki = 11.5 nM) and 4c (Ki = 6.4 nM), and the
N′‐cyanopicolinamidine derivatives 8b (Ki = 28.8 nM) and 8e (Ki =
21.4 nM), afforded a favorable affinity profile for 5‐HT2C receptors.
Due to their high 5‐HT2C receptor affinity and selectivity, these
compounds were selected for further in vivo studies to investigate
their functional activity.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General procedures
All reagents and substituted piperazines were commercial products
purchased from Sigma Aldrich (Milan, Italy). Melting points were
determined using a Buchi Melting Point B‐540 instrument (Flawil,
Switzerland) and are uncorrected. 1H‐NMR and 13C‐NMR spectra
The obtained results showed that compounds 4a, 8b, and 8e
exerted antidepressant‐like effects; additionally compounds 4a and

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were recorded on Varian Mercury Plus 400 MHz instrument (Varian,
Palo Alto, CA). Unless otherwise stated, all spectra were recorded in
CDCl3. Chemical shifts are reported in ppm using Me4Si as internal
standard. The following abbreviations are used to describe peak
patterns when appropriate: s (singlet), d (doublet), t (triplet), m
(multiplet), q (quartet), qt (quintet), dd (double doublet), ddd (double
dd), and bs (broad singlet). Mass spectra of the final products were
performed on API 2000 Applied Biosystem mass spectrometer.
Where analyses are indicated only by the symbols of the elements,
results obtained are within 0.4% of the theoretical values. All
reactions were followed by thin‐layer chromatography, carried out
on Merck silica gel 60 F254 plates (Merck, Darmstadt, Germany) with
fluorescent indicator and the plates were visualized with UV light
(254 nm). Preparative chromatographic purifications were performed
using silica gel column (Kieselgel 60, Machery‐Nagel, Duren,
Germany). Solutions were dried over Na2SO4 and concentrated with
Buchi (Flawil, Switzerland) rotary evaporator at low pressure.
Original spectra of the investigated compounds are provided as
Supporting Information. The InChI codes of the investigated
compounds together with some biological activity data are also
given in the Supporting Information.
|
4.1.2
Synthesis of isopropyl
N‐cyano‐4‐pyridinecarboximidate (2)
A mixture of 4‐cyanopyridine (1) (15 g, 0,144 mol) and NaOMe
(0.24 g, 0.0044 mol) in anhydrous isopropanol (180 ml) was stirred
FIGURE 9 The influence of the investigated compounds 4a, 4c,
8b, and 8e (7.5–30 mg/kg) on elevated plus‐maze performance in
mice: percentage of time spent in open arms (a), the percentage of
the open arm entries (b) and total arm entries (c). Investigated
compounds were injected intraperitoneally 60 min before the test.
The results are expressed as mean SEM of the one independent
experiment. ***p < 0.001, **p < 0.01, and *p < 0.05 versus control
(Dunnett’s test). One‐way analysis of variance showed significant
changes in percentage of time spent in open arms of EPM,
F(10,91) = 3.244, p < 0.01. (a) and in the percentage of open arm
entries, F(10,91) = 3.827, p < 0.001). (b) There were no significant
changes in the total arm entries, F(10,91) = 1.762, p > 0.05). (c)
Dunnett’s post-hoc test confirmed a significant increase in time
spent in open arms after the administration of compounds 4a and 8e.
Compound 4a significantly increased percentage of time spent in
open arms and percentage of open arms entries at the dose of 7.5
(p < 0.05) and 15 mg/kg (p < 0.01), whereas compound 8e was active
at doses 7.5 and 15 mg/kg (p < 0.01 and p < 0.001 for percentage of
time spent in open arms, respectively, and p < 0.05 and p < 0.01
for percentage of open arms entries, respectively). Also buspirone
(5 mg/kg) induced a significant increase in percentage of time spent
in open arms and in percentage of open arms entries (p < 0.01 and
p < 0.001, respectively). EPM: elevated plus‐maze test; SEM:
standard error of means
FIGURE 8 The influence of the investigated compounds 4a, 4c,
8b, and 8e (7.5–30 mg/kg) and fluoxetine (15 mg/kg) on the total
duration in the forced swim test in mice (FST). The investigated
compounds were administered intraperitoneally 60 min before the
test. The values represent means SEM of the one independent
experiment. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control
(Dunnett’s test). One‐way analysis of variance showed significant
changes in immobility time after administration of the compound 4a,
F(3,28) = 4.962, p < 0.01; 8e, (F(2,21) = 18.97, p < 0.001; and 8b,
F(2,20) = 16.64, p < 0.001. Dunnett’s post hoc test confirmed a
significant reduction in immobility time after the administration of
compounds 4a applied at the doses of 15 (p < 0.05) and 30 mg/kg
(p < 0.01), 8e at the doses of 15 and 30 mg/kg (p < 0.001), and 8b
applied at the doses of 15 and 30 mg/kg (p < 0.001). Also fluoxetine
(15 mg/kg) induced a significant reduction in the immobility time
(p < 0.001). SEM: standard error of means

MAGLI ET AL.
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overnight at 0°C. AcOH (0.25 g, 0.0042 mol) was then added with
stirring and the solution was evaporated in vacuo. Hexane (150 ml)
was added to the residue and the resultant precipitate was filtered
off. The filtrate was evaporated to give crude isopropyl‐4‐
pyridinecarboximidate (18.6 g) as an oil. Crude compound was then
added to a mixture of NH2CN (8.83 g, 0.21 mol), NaH2PO4·2H2O
(65.55 g) and Na2HPO4 (14.9 g) in water (112.5 ml). After vigorous
stirring for 6 hr at room temperature, the reaction mixture was
extracted several times with CH2Cl2. The combined organic layers
were dried on anhydrous Na2SO4 and concentrated in vacuo to
yield crude isopropyl‐N‐cyano‐4‐pyridinecarboximidate (2) (22 g),
which was used directly in the following reaction. 1H‐NMR (400
MHz, CDCl3) δ: 1.50 (d, 6H J = 6.1); 5.42 (m, 1H); 8.0–7.8 (m, 2H);
8.9–8.7 (m, 2H).
4H, 2CH2 pip.); 2.67 (t, 2H, N1‐CH2, J = 5.1); 2.84 (bs, 4H, 2CH2 pip.);
3.64 (bs, 2H, N‐CH2); 4.01 (q, 2H, OCH2, J = 6.9); 6.64 (d, 1H, J = 7.7);
6.81 (d, 1H, J = 7.7); 6.87 (t, 1H, J = 7.7); 6.95 (t, 1H, J = 7.7); 7.52 (d, 2H,
J = 5.4); 8.76 (d, 2H, J = 5.4); 9.72 (s, 1H). ESI‐MS: 393.5 [M+H]+; 415.3
[M+Na]+; 431.1 [M+K]+. Anal. (C22H28N6O), C, H, N.
N′‐Cyano‐N‐(3‐(4‐(2‐cyanophenyl)piperazin‐1‐yl)propyl)isoni-
cotinamidine (4b)
From 3 and 2‐(piperazin‐1‐yl)benzonitrile. Yield: 20%; mp 169–171°C.
1H‐NMR (500 MHz, CDCl3) δ: 1.85 (m, 2H); 2.22 (bs, 4H, 2CH2 pip.);
2.69 (t, 2H, N1‐CH2, J = 5.1); 2.93 (bs, 4H, 2CH2 pip.); 3.64 (bs, 2H,
N‐CH2); 6.75 (d, 1H, J = 7.1); 7.02 (t, 1H, J = 7.1); 7.47 (d, 1H, J = 7.1);
7.51 (d, 2H, J = 5.4); 7.54 (t, 1H, J = 7.1); 8.77 (d, 2H, J = 5.4); 9.42 (s,
1H). ESI‐MS: 374.4 [M+H]+. Anal. (C21H23N7), C, H, N.
N′‐Cyano‐N‐(3‐(4‐(3,4‐dichlorophenyl)piperazin‐1‐yl)propyl)‐
isonicotinamidine (4c)
|
4.1.3
Synthesis of N‐(3‐bromopropyl)‐
N′‐cyanoisonicotinamidine (3)
From 3 and 1‐(3,4‐dichlorophenyl)piperazine. Yield: 32%; mp 152–
154°C. 1H‐NMR (500 MHz, CDCl3) δ: 1.86 (m, 2H); 2.59 (bs, 4H,
2CH2 pip.); 2.64 (t, 2H, N1‐CH2, J = 5.1); 2.91 (bs, 4H, 2CH2 pip.); 3.63
(bs, 2H, N‐CH2); 6.59 (d, 1H, J = 8.0); 6.84 (s, 1H); 7.26 (d, 1H, J = 8.0);
7.13 (t, 1H, J = 8.0); 7.47 (d, 2H, J = 5.4); 8.73 (d, 2H, J = 5.4); 9.08 (s,
1H). ESI‐MS: 417.2 [M+H]+. Anal. (C20H22Cl2N6), C, H, N.
To a solution of isopropyl N‐cyano‐4‐pyridinecarboximidate (21.89
g, 0.116 mol) in anhydrous MeOH (120 ml), 3‐bromopropylami-
ne·HBr (28.24 g, 0.129 mol), and NaOMe (6.77 g, 0.125 mol) were
successively added and the reaction mixture was stirred at room
temperature for 18 hr. After evaporation, the residue was dissolved
in CHCl3. The solution was washed with water, dried on anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
column chromatography (diethyl ether/ethanol 9:1 [v/v]). The
combined and evaporated product fractions were crystallized from
diethyl ether/hexane, yielding 12.5 g (40%) of the desired product
as a white solid: mp: 201–203°C; 1H‐NMR (400 MHz, CDCl3) δ: 2.30
(qt, 2H, J = 6.2); 3.51 (t, 2H J = 6.2); 3.72 (q, 2H, J = 6.2); 7.49 (d, 2H,
J = 5.5); 8.83 (d, 2H, J = 5.5); 9.60 (s, 1H).
N′‐Cyano‐N‐(3‐(4‐(4‐chlorobenzhydryl)piperazin‐1‐yl)propyl)‐
isonicotinamidine (4d)
From 3 and 1‐(4‐chlorobenzhydryl)piperazine. Yield: 24%; mp 95–97°C.
1H‐NMR (500 MHz, CDCl3) δ: 1.78 (m, 2H); 2.48 (bs, 8H, 4CH2 pip.);
2.61 (t, 2H, N1‐CH2, J = 5.1); 3.59 (bs, 2H, N‐CH2); 3.88 (s, 1H, NCH);
7.18 (m, 9H); 7.46 (d, 2H, J = 5.4); 8.8 (d, 2H, J = 5.4); 9.75 (s, 1H). ESI‐
MS: 473.5 [M+H]+. Anal. (C27H29ClN6), C, H, N.
N′‐Cyano‐N‐(3‐(4‐(bis(4‐fluorophenyl)methyl)piperazin‐1‐yl)‐
propyl)isonicotinamidine (4e)
|
4.1.4
General procedure for the condensation of
4‐substituted arylpiperazines with derivative 3
From 3 and 1‐bis(4‐fluorophenyl)methyl)piperazine. Yield: 35%; mp
117–119°C. 1H‐NMR (500 MHz, CDCl3) δ: 1.78 (m, 2H); 2.47 (bs, 8H,
4CH2 pip.); 2.60 (t, 2H, N1‐CH2, J = 5.1); 3.60 (bs, 2H, N‐CH2); 3.87
(s, 1H, NCH); 6.92 (m, 4H); 7.22 (m, 4H); 7.47 (d, 2H, J = 5.4); 8.82
(d, 2H, J = 5.4); 9.70 (s, 1H). ESI‐MS: 475.3 [M+H]+. Anal.
(C27H28F2N6), C, H, N.
A
mixture of N‐(3‐bromopropyl)‐N′‐cyanoisonicotinamidine (3)
(0.006 mol) and NaI (0.009 mol) in acetonitrile was stirred under
reflux for 30 min. Then, the appropriate 4‐substituted arylpiperazine
(0.03 mol) and anhydrous K2CO3 (0.009 mol) were added. The
reaction mixture was stirred under reflux for 4 hr. After cooling,
the mixture was filtered, concentrated to dryness and the residue
was dissolved in water (50 ml). The solution was extracted several
times with CH2Cl2. The combined organic layers were dried on
anhydrous Na2SO4 and the solvent removed under vacuum. The
crude mixtures were purified by silica gel column chromatography
using diethyl ether/methanol 8:2 (v/v) as eluent. The crude products
were recrystallized from diethyl ether.
|
4.1.5
Synthesis of ethyl N‐cyano‐
2‐pyridinecarboximidate (6)
A mixture of 2‐cyanopyridine (5) (10 g, 0,096 mol) and NaOMe (0.26
g, 0.0048 mol) in anhydrous methanol (180 ml) was stirred overnight
at room temperature. AcOH (0.32 g, 0.0053 mol) was then added
with stirring to neutralize the reaction solution and the solution was
evaporated in vacuum. Diethyl ether (150 ml) was added to the
residue and the resultant precipitate was filtered off. The filtrate was
evaporated to give crude methyl‐2‐pyridinecarboximidate (7.0 g) as
an oil. Crude compound was then added to a mixture of NH2CN
N'‐Cyano‐N‐(3‐(4‐(2‐ethoxyphenyl)piperazin‐
1‐yl)propyl)isonicotinamidine (4a)
From 3 and 1‐(2‐ethoxyphenyl)piperazine. Yield: 25%; mp 117–119°C.
1H‐NMR (500 MHz, CDCl3) δ: 1.40 (t, 3Η, J = 6.9); 1.85 (m, 2H); 2.22 (bs,

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MAGLI ET AL.
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(4.33 g, 0.103 mol), NaH2PO4·2H2O (32.14 g), and Na2HPO4 (7.30 g)
in water (55 ml). After vigorous stirring for 4 hr at room temperature,
the reaction mixture was extracted several times with CH2Cl2. The
combined organic layers were dried on anhydrous Na2SO4 and
concentrated in vacuo to yield crude methyl‐N‐cyano‐2‐pyridinecar-
boximidate (6) (8.4 g), which was used directly in the following
reaction. 1H‐NMR (400 MHz, CDCl3) δ: 4.16 (s, 3H); 7.63 (dd, 1H,
J = 9.4, 7.3); 7.94 (d, 1H, J = 3.4); 7.98 (dd, 1H, J = 7.3, 2.4); 8.83 (ddd,
1H, J = 9.4, 3.4, 2.4). 13C‐NMR (400 MHz, CDCl3) δ: 46.35; 116.90;
123.64; 126.80; 136.25; 148.13; 149.15, 153.10.
(d, 1H, J = 7.7); 6.91 (t, 1H, J = 7.7); 6.96 (t, 1H, J = 7.7); 7.41 (t, 1H, J = 7.3);
7.85 (t, 1H, J = 7.7); 8.50 (d, 1H, J = 8.0); 8.55 (d, 1H, J = 4.4); 10.10 (s, 1H).
ESI‐MS: 393.5 [M+H]+; 415.2 [M+Na]+. Anal. (C22H28N6O), C, H, N.
N′‐Cyano‐N‐(3‐(4‐(2‐cyanophenyl)piperazin‐1‐yl)‐
propyl)picolinamidine (8b)
From 7 and 2‐(piperazin‐1‐yl)benzonitrile. Yield: 20%; mp 118–
120°C. 1H‐NMR (500 MHz, CDCl3) δ: 1.88 (m, 2H); 2.65 (t, 2H, N1‐
CH2, J = 5.1); 2.73 (bs, 4H, 2CH2 pip.); 3.30 (bs, 4H, 2CH2 pip.); 3.87
(bs, 2H, N‐CH2); 6.98 (d, 1H, J = 7.1); 7.02 (t, 1H, J = 7.1); 7.44–7.56
(m, 3H,); 7.58 (d, 1H, J = 7.3); 7.86 (t, 1H, J = 7.7); 8.53 (bs, 1H); 9.78
(s, 1H). ESI‐MS: 374.4 [M+H]+; 396.0 [M+Na]+; 412.0 [M+K]+. Anal.
(C21H23N7), C, H, N.
|
4.1.6
Synthesis of N‐(3‐bromopropyl)‐
N′‐cyanopicolinamidine
N′‐Cyano‐N‐(3‐(4‐(3,4‐dichlorophenyl)piperazin‐1‐yl)‐
propyl)picolinamidine (8c)
To a solution of methyl N‐cyano‐2‐pyridinecarboximidate (6) (8 g,
0.050 mol) in anhydrous MeOH (120 ml), 3‐bromopropylamine·HBr
(11.93 g, 0.055 mol), and triethylamine (5.56 g 0.055 mol) were
successively added and the reaction mixture was stirred at room
temperature for 18 hr. After evaporation, the residue was dissolved
in CHCl3. The solution was washed with water, dried on anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by
column chromatography (diethyl ether/ethanol 9:1 [v/v]). The
combined and evaporated product fractions were crystallized from
diethyl ether/hexane, yielding 8.4 g (63%) of the desired product as a
brown solid: mp: 73–74°C; 1H‐NMR (400 MHz, CDCl3) δ: 2.15 (qt,
2H, J = 6.2); 2.75 (t, 2H, J = 6.2); 3.83 (q, 2H, J = 6.2); 7.47 (t, 1H,
J = 7.3); 7.86 (t, 1H, J = 7.3); 8.5 (d, 1H, J = 7.3); 8.64 (d, 1H, J = 7.3);
9.62 (s, 1 H). 13C‐NMR (400 MHz, CDCl3) δ: 27.33, 38.70, 53.91,
117.20, 122.54, 125.66, 135.10, 148.23, 150.70, 154.20.
From
7 and 1‐(3,4‐dichlorophenyl)piperazine. Yield: 40%; mp
126–128°C. 1H‐NMR (500 MHz, CDCl3) δ: 1.89 (m, 2H); 2.64 (bs,
4H, 2CH2 pip.); 2.66 (t, 2H, N1‐CH2, J = 5.1); 3.24 (br s, 4H, 2CH2 pip.);
3.88 (br s, 2H, N‐CH2); 6.73 (d, 1H, J = 7.1); 6.94 (t, 1H, J = 7.1); 7.28
(d, 1H, J = 7.1); 7.40 (t, 1H, J = 5.4); 7.84 (t, 1H, J = 7.1); 8.39 (d, 1H,
J = 5.4); 8.52 (d, 1H, J = 5.4); 9.85 (s, 1H). ESI‐MS: 417.0 [M+H]+;
438.9 [M+Na]+; 454.9 [M+K]+. Anal. (C20H22Cl2N6), C, H, N.
N′‐Cyano‐N‐(3‐(4‐(4‐chlorobenzhydryl)piperazin‐1‐yl)‐
propyl)picolinamidine (8d)
From 7 and 1‐(4‐chlorobenzhydryl)piperazine. Yield: 35%; mp 93–95°C.
1H‐NMR (500 MHz, CDCl3) δ: 1.80 (q, 2H); 2.46 (bs, 8H, 4CH2 pip.); 2.55
(t, 2H, N1‐CH2, J = 5.1); 3.79 (bs, 2H, N‐CH2); 4.29 (s, 1H, NCH); 7.18 (m,
9H); 7.46 (t, 1H, J = 5.4); 7.85 (t, 1H, J = 7.1); 8.34 (d, 1H, J = 5.4); 8.48 (d,
1H, J = 5.4); 9.71 (s, 1H). ESI‐MS: 473.4 [M+H]+. Anal. (C27H29ClN6), C,
H, N.
|
4.1.7
General procedure for the condensation of
4‐substituted arylpiperazines with derivative (7)
N′‐Cyano‐N‐(3‐(4‐(bis(4‐fluorophenyl)methyl)piperazin‐1‐yl)-
propyl)picolinamidine (8e)
A mixture of N‐(3‐bromopropyl)‐N′‐cyanopicolinamidine (7) (0.006
mol) and NaI (0.009 mol) in acetonitrile was stirred under reflux for
30 min. Then, the appropriate 4‐substituted arylpiperazine (0.06 mol)
and anhydrous K2CO3 (0.009 mol) were added. The reaction mixture
was stirred under reflux for 4 hr. After cooling, the mixture was
filtered, concentrated to dryness and the residue was dissolved in
water (50 ml). The solution was extracted several times with CH2Cl2.
The combined organic layers were dried on anhydrous Na2SO4 and
the solvent removed under vacuum. The crude mixture was purified
by silica gel column chromatography using diethyl ether/methanol
8:2 (v/v) as eluent. The crude product were recrystallized from
diethyl ether.
From 7 and 1‐bis(4‐fluorophenyl)methyl)piperazine. Yield: 34%; mp
132–134°C. 1H‐NMR (500 MHz, CDCl3) δ: 1.83 (q, 2H); 2.46 (bs, 8H,
4CH2 pip.); 2.54 (t, 2H, N1‐CH2, J = 5.1); 3.79 (bs, 2H, N‐CH2); 4.28 (s,
1H, NCH); 6.95 (d, 4H); 7.30 (d, 4H); 7.46 (t, 1H, J = 5.4); 7.85 (t, 1H,
J = 7.1); 8.33 (d, 1H, J = 5.4); 8.48 (d, 1H, J = 5.4); 9.66 (s, 1H). ESI‐MS:
476.0 [M+H]+; 497.1 [M+Na]+; 513.0 [M+K]+. Anal. (C27H28F2N6), C,
H, N.
|
4.2
In vitro receptor binding
General procedures
|
4.2.1
The compounds dissolved in ethanol or in 5% dimethyl sulfoxide
(DMSO) were tested for in vitro affinity for 5‐HT1A, 5‐HT2A, and
5‐HT2C receptors by radioligand‐binding assays. The compounds
showing the highest affinity towards serotonin receptors were
selected and evaluated for their affinity for dopaminergic (D1 and
D2) and adrenergic (α1 and α2) receptors. All the following specific
radioligands and tissue sources were used: (a) serotonin 5‐HT1A
N′‐Cyano‐N‐(3‐(4‐(2‐ethoxyphenyl)piperazin‐1-yl)‐
propyl)picolinamidine (8a)
From 7 and 1‐(2‐ethoxyphenyl)piperazine. Yield: 14%; mp 122–125°C.
1H‐NMR (500 MHz, CDCl3) δ: 1.43 (t, 3H, J = 6.9); 1.88 (m, 2H); 2.64 (t,
2H, N1‐CH2, J = 5.1); 2.71 (bs, 4H, 2CH2 pip.); 3.17 (bs, 4H, 2CH2 pip.);
3.86 (bs, 2H, N‐CH2); 4.04 (q, 2H, OCH2, J = 6.9); 6.85 (d, 1H, J = 7.7); 6.89

MAGLI ET AL.
13 of 16
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receptor, [3H]‐8‐OH‐DPAT, rat brain cortex; (b) serotonin
5‐HT2A receptor, [3H]ketanserin, rat brain cortex; (c) serotonin
5‐HT2C receptor, [3H]mesulergine, rat brain cortex; (d) dopamine
D1 receptor, [3H]SCH‐23390, rat striatum; (e) dopamine D2
receptor, [3H]spiperone, rat striatum; (f) α1 adrenergic receptor,
[3H]prazosin, rat brain cortex; and (g) α2 adrenergic receptor, [3H]
yo-himbine, rat brain cortex.
4.2.3
5‐HT2A and 5‐HT2C binding assays
|
Radioligand‐binding assays were performed as previously reported by
Herndon et al.[46] Briefly, frontal cortical regions of male Sprague–Dawley
rats (180–220 g) were dissected on ice and homogenized (1:10 w/v) in
ice‐cold buffer solution (50 mM Tris‐HCl, 0.5 mM ethylenediaminete-
traacetic acid [EDTA], and 10 mM MgCl2 at pH 7.4) with a Polytron PT10
(setting 5 for 15 s) and centrifuged at 3000g for 15 min. The pellet was
resuspended in buffer (1:30 w/v), incubated at 37°C for 15 min and then
centrifuged twice more at 3000g for 10 min (with resuspension between
centrifugations). The final pellet was resuspended in buffer that also
contained 0.1% ascorbate and 10–5 M pargyline.
Nonspecific binding was determined as described in the supple-
mentary material, and specific binding as the difference between total
and nonspecific binding. Blank experiments were carried out to
determine the effect of 5% DMSO on the binding and no effects were
observed. Competition experiments were analyzed by PRISM
5
Assays were performed in triplicate in a 2.0 ml volume containing
5 mg wet weight of tissue and 0.4 nM [3H]ketanserin hydrochloride
(88.0 Ci/mmol; Perkin Elmer Life Sciences) for 5‐HT2A receptor
assays, and 10 mg wet weight of tissue and 1 nM [3H]mesulergine
(87.0 Ci/mmol; Amersham Biosciences Europe GmbH, Friburg,
Germany) for 5‐HT2C receptor assays. Cinanserin (1.0 µM) was used
to define nonspecific binding in the 5‐HT2A assay. In the 5‐HT2C
assays, mianserin (1.0 µM) was used to define nonspecific binding,
and 100 nM spiperone was added to all tubes to block binding to 5‐
HT2A receptors. Tubes were incubated for 15 min at 37°C, filtered on
Schleicher and Schuell (Keene, NH) glass fiber filters presoaked in
polyethylene imine, and washed with 10 ml of ice‐cold buffer. Filters
were counted at an efficiency of 50%.
(GraphPad Prism, 1992–2007, GraphPad Software, Inc., La Jolla, CA)
program to obtain the concentration of unlabeled drug that caused 50%
inhibition of ligand binding (IC50), with six concentrations of tested
compounds, each performed in triplicate. The IC50 values obtained were
used to calculate apparent inhibition constants (Ki) by the method of
Cheng and Prusoff,[39] from the following equation: Ki = IC50/(1 + S/KD)
where S represents the concentration of the hot ligand used and KD its
receptor dissociation constant; KD values, obtained by Scatchard
analysis,[40] were calculated for each labeled ligand.
The experiments only for compounds 8b and 8e on receptors D1,
D2, α1 and α2 were performed at Eurofins‐Cerep SA, Celle
L’Evescault, France (Study Number: 100045303) according to the
Standardized Operational Protocols in place, following the experi-
mental protocols already described for D1,[41] D2,[42] α1,[43] and α2.[44]
|
4.2.4
D1 dopaminergic binding assay
|
4.2.2
5‐HT1A binding assay
The binding assay for D1 dopaminergic receptors was that described by
Billard et al.[47] Corpora striata were homogenized in 30 vol. (w/v) ice‐
cold 50 mM Tris‐HCl buffer (pH 7.7 at 25°C), using a Polytron PT10
(setting 5 for 20 s). Homogenates were centrifuged twice for 10 min at
50,000g with resuspension of the pellet in fresh buffer. The final pellet
was resuspended in 50 mM ice‐cold Tris‐HCl containing 120 mM NaCl,
5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid, and 10 µM
pargyline (pH 7.1 at 37°C). Each assay tube contained 50 µl [3H]SCH‐
23390 (85.0 Ci/mmol, Perkin Elmer Life Sciences) to achieve a final
concentration of 0.4 nM, and 900 µl resuspended membranes (3 mg fresh
tissue). The tubes were incubated for 15 min at 37°C and the incubation
was terminated by rapid filtration under vacuum through Whatman GF/B
glass fiber filters. The filters were washed three times with 5 ml ice‐cold
50 mM Tris‐HCl buffer (pH 7.7 at 25°C). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific [3H]SCH‐
23390 binding was defined as the difference between binding in the
absence or in the presence of 0.1 µM piflutixol.
Radioligand‐binding assays were performed following a published
procedure.[45] Cerebral cortex from male Sprague–Dawley rats (180–
220 g) was homogenized in 20 volumes of ice‐cold Tris‐HCl buffer (50
mM, pH 7.7 at 22°C) with a Polytron PT10, Brinkmann Instruments,
Westbury, New York (setting 5 for 15 s), and the homogenate was
centrifuged at 50,000g for 10 min at 0°C. The resulting pellet was then
resuspended in the same buffer, incubated for 10 min at 37°C, and
centrifuged at 50,000g for 10 min. The final pellet was resuspended in
80 volumes of the Tris‐HCl buffer containing 10 µM pargyline, 4 mM
CaCl2, and 0.1% ascorbate. To each assay tube was added the following:
0.1 ml of the drug dilution (0.1 ml of distilled water if no competing drug
was added), 0.1 ml of [3H]‐8‐hydroxy‐2‐(di‐n‐propyl-amino)tetralin ([3H]‐
8‐OH‐DPAT; 170.0 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA) in
the same buffer as above to achieve a final assay concentration of 1 nM,
and 0.8 ml of resuspended membranes. The tubes were incubated for
30 min at 37°C, and the incubations were terminated by vacuum
filtration through Whatman GF/B filters (Brandel Biomedical Research
and Laboratories Inc., Gaithersburg, MD). The filters were washed twice
with 5 ml of ice‐cold Tris‐HCl buffer, and the radioactivity bound to the
filters was measured by liquid scintillation spectrometer (Packard TRI‐
CARB® 2000CA; Packard BioScience s.r.l., Pero, Milan, Italy). Specific
[3H]‐8‐OH‐DPAT binding was defined as the difference between
binding in the absence and presence of 5‐HT (10 µM).
|
4.2.5
D2 dopaminergic binding assay
The procedure used in the radioligand‐binding assay was reported in
detail by Creese et al.[48] Corpora striata were homogenized in
30 vol. (w/v) ice‐cold 50 mM Tris‐HCl buffer (pH 7.7 at 25°C) using a
Polytron PT10 (setting 5 for 20 s). Homogenates were centrifuged

14 of 16
MAGLI ET AL.
|
|
twice for 10 min at 50,000g with resuspension of the pellet in fresh
buffer. The final pellet was resuspended in 50 mM ice‐cold Tris‐HCl
containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1%
ascorbic acid, and 10 µM pargyline (pH 7.1 at 37°C). Each assay tube
contained 50 µl [3H]spiperone (15.7 Ci/mmol, Perkin Elmer Life
Sciences) to achieve a final concentration of 0.4 nM and 900 µl
resuspended membranes (3 mg fresh tissue). The tubes were
incubated for 15 min at 37°C and the incubation was terminated
by rapid filtration under vacuum through Whatman GF/B glass fiber
filters. The filters were washed three times with 5 ml ice‐cold 50 mM
Tris‐HCl buffer (pH 7.7 at 25°C). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific [3H]-
spiperone binding was defined as the difference between binding in
the absence or in the presence of 1 µM (+)‐butaclamol.
4.3
Computational methods
Protein models
|
4.3.1
The following protein models were used for molecular docking: X‐ray
structure of the human 5‐HT2C receptor in complex with the inverse
agonist ritanserin (PDB ID: 6BQH)[26] and previously reported
homology models of human 5‐HT1A and 5‐HT2A receptors con-
structed in inactive conformation.[50]
|
4.3.2
Ligand models
Compounds 4a–e and 8a–e were prepared with LigPrep[51] module
from Schrödinger suite of software. To sample different protonation
states of ligands at physiological pH, the Epik[52] module of
Schrödinger suite of software was used.
|
4.2.6
α1 adrenergic binding assay
|
4.3.3
Molecular docking
The procedure used in the radioligand‐binding assay has been reported in
detail by Greengrass and Bremner.[43] The brain cortex was homogenized
in 30 vol. (w/v) ice‐cold 50 mM Tris‐HCl buffer (pH 7.2 at 25°C), using a
Polytron PT10 (setting 5 for 20 s). Homogenates were centrifuged twice
for 10 min at 50,000g with resuspension of the pellet in fresh buffer. The
final pellet was resuspended in 50 mM ice‐cold Tris‐HCl, (pH 7.4 at 25 °
C). Each assay tube contained 50 µl drug solution, 50 µl [3H]prazosin
(80.5 Ci/mmol, Perkin Elmer Life Sciences) to achieve a final concentra-
tion of 0.4 nM, and 900 µl resuspended membranes (10 mg fresh tissue).
The tubes were incubated for 30 min at 25°C and the incubation was
terminated by rapid filtration under vacuum through Whatman GF/B
glass fiber filters. The filters were washed three times with 5 ml ice‐cold
50 mM Tris‐HCl buffer (pH 7.2 at 25°C). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific [3H]-
prazosin binding was defined as the difference between binding in the
absence or in the presence of 10 µM phentolamine.
Molecular docking was performed using Standard Precision (SP)
approach of Glide[53] incorporated in Schrödinger suite of software.
For 5‐HT2C receptor the grid file was constructed indicating
ritanserin as the orthosteric ligand and for 5‐HT1A and 5‐HT2A
receptors the grid file was based on the docked chlorprothixene as
previously reported.[50] The following residues were flexible for
molecular docking: Thr196 (5.40), Ser199 (5.43), Thr200 (5.44) for 5‐
HT1A receptor, Ser159 (3.36), Ser239 (5.44), Ser242 (5.461) for 5‐
HT2A receptor, and Ser110 (2.60), Ser138 (3.36), Thr139 (3.37),
Ser219 (5.44) for 5‐HT2C receptor (numbers in brackets according to
the Ballesteros–Weinstein nomenclature).[54] Fifty poses were
generated for each ligand and each receptor.
|
4.4
In vivo behavioral tests
General procedures
|
4.4.1
The studies were conducted on male Albino Swiss mice (18–23 g).
The mice were housed in cages, five individuals per cage in an
environmentally controlled rooms (ambient temperature 22 1°C;
relative humidity 50–60%; 12 hr light/dark cycle, lights on at 8:00).
Standard laboratory food (LSM, Agropol‐Motycz, Poland) and filtered
water were available ad libitum. All the experimental procedures
were carried out, according to the National Institute of Health
Guidelines for the Care and Use of Laboratory Animals and to the
European Community Directive for the Care and Use of Laboratory
of 24 November 1986 (86/609/EEC), and approved by the Local
Ethics Committee for Animal Experimentation (License No. 42/2018).
The investigated compounds (4a, 4c, 8b, and 8e) in all tests were
administered intraperitoneally, dissolved in DMSO (its final concentra-
tion of 0.1%) and then diluted by aqueous solution of 0.5%
methylcellulose and injected 60 min before the tests. Reference drugs
(buspirone and fluoxetine) were administered intraperitoneally, diluted
in saline, and injected 60 min before the tests. All compounds were
given in a volume of 10 ml/kg to mice. The control animals received an
equivalent volume of the solvent at the respective time before the
|
4.2.7
α2 adrenergic binding assay
The procedure used in the radioligand‐binding assay was reported in
detail by Perry and U’Prichard.[49] Brain cortex was homogenized in
30 vol. (w/v) ice‐cold 5 mM Tris‐HCl, 5 mM EDTA buffer (pH 7.3 at 25°C)
using a Polytron PT10 (setting 5 for 20 s). Homogenates were centrifuged
three times for 10 min at 50,000g with resuspension of the pellet in fresh
buffer. The final pellet was resuspended in 50 mM ice‐cold Tris‐HCl, 0.5
mM EDTA (pH 7.5 at 25°C). Each assay tube contained 50 µL drug
solution, 50 µL [3H]yohimbine (80.5 Ci/mmol, Perkin Elmer Life Sciences)
to achieve a final concentration of 1 nM, and 900 µl resuspended
membranes (10 mg fresh tissue). The tubes were incubated for 30 min at
25°C and the incubation was terminated by rapid filtration under vacuum
through Whatman GF/B glass fiber filters. The filters were washed three
times with 5 ml ice‐cold 50 mM Tris‐HCl, 0.5 mM EDTA buffer (pH 7.5 at
25°C). The radioactivity bound to the filters was measured by a liquid
scintillation counter. Specific [3H]yohimbine binding was defined as the
difference between binding in the absence or in the presence of 10 µM
phentolamine.

MAGLI ET AL.
15 of 16
|
|
EPM test
tests. All the experiments were conducted in the light phase between
09.00 a.m. and 14.00 p.m. The experiments were performed by an
observer unaware of the treatment administered.
4.4.6
The EPM studies were carried out on mice according to the method
of Lister.[59] The EPM apparatus was made of plexiglass and consisted
of two open (30 × 5 cm) and two enclosed (30 × 5 × 15 cm) arms. The
arms extended from a central platform of 5 × 5 cm. The apparatus
was mounted on a plexiglass base, raising it 38.5 cm above the floor,
and illuminated by a red light. The test consisted of placing a mouse
in the center of the apparatus (facing an open arm) and allowing it to
freely explore. The number of entries into the open arms and the
time spent in these arms were scored for a 5 min test period. An
entry was defined as placing all four paws within the boundaries of
the arm. The following measures were obtained from the test: the
total number of arm entries; the percentage of arm entries into the
open arms; and the time spent in the open arms expressed as a
percentage of the time spent in both the open and closed arms.
Anxiolytic activity was indicated by increases in the time spent in
open arms and in the number of open arm entries. The total number
of entries into either type of arm was used additionally as a measure
of overall motor activity.[24,58]
|
4.4.2
Statistics
The results were calculated by the one‐way analysis of variance,
followed by Dunnett's post-hoc test. The results are presented as
mean standard errors (SEM). The level of p < 0.05 was considered
as statistically significant. All the figures were prepared by the
GraphPad Prism version 5.00 for Windows, GraphPad Software (San
Diego, CA; www.graphpad.com).
|
4.4.3
Spontaneous locomotor activity and
amphetamine‐induced hyperactivity
The spontaneous motility of mice was measured using photocell
apparatus (Multiserv, Poland). The apparatus consists of round cages
(diameter 25 cm) made of plastics. The measuring element is made of
infrared motion sensors. Motility is interpreted as the number of crossed
light beams by freely moving mice. The animals were placed in the cage
individually, 50 min after the administration of the tested compounds, for
a period of 10 min for acclimatization. After this time, their activity was
noted within 2 and 6 min (corresponded with the time duration of the
FST and near (5 min) to the EPM test, respectively) and after 30 min, to
observe the dynamics of changes.[23] The cages were cleaned up with
70% ethanol after each mouse. In another set of experiments, the
influence of tested compound on amphetamine‐induced hyperactivity in
mice was evaluated. The study was conducted in the same apparatus, but
each mouse received amphetamine (5 mg/kg, s.c.) 30 min after injection
of vehicle or tested compounds. Motility was measured during 30 min.
|
4.4.7
Ethanol test
The ability of the drugs to potentiate the action of subnarcotic dose
of ethanol was examined using the appearance of loss of the righting
reflex as a behavioral index.[37] One hour after dosing of drugs or
vehicle, 30% ethanol was intraperitoneally administered to mice at
the volume of 10 ml/kg. The loss of the righting reflex was tested
10 and 20 min later. The number of mice which lost the righting
reflex for more than 5 s at either time point was determined.
|
4.4.8
Bar test
|
4.4.4
Motor coordination
The catalepsy was measured using the bar test 60 min after drug
administration: the front paws of each subject were placed on a
cylindrical metal bar (0.75‐cm diameter) that was elevated 4.5 cm
above the table. The time during which both forelimbs remained on
the bar was recorded up to a maximum of 30 s. The test was repeated
three times (intertrial interval: 1 min). Animals were put back in their
home cage after each measurement of catalepsy. Mice that remained
motionless with their paws on the bar for 10 s (with the exception of
respiratory movements) were scored as cataleptic.[60,61]
The effects of investigated compounds on motor coordination were
evaluated in the rotarod[23,24,55] and chimney tests.[23,24,56] In the first
test, motor impairments were measured, defined as the inability to keep
balance on
a rotating rod (at constant speed of 18 rpm) for
1 min. In the second test, motor impairments were assessed by mouse
inability to climb up the tube backwards (3 cm in inner diameter, 25 cm
long) within 60 s. Before the tests, the animals were trained once a day
for 3 days. The animals, able to stay on the rotating rod or to leave the
chimney for 60 s, were approved for experiments.
ACKNOWLEDGMENTS
|
4.4.5
FST (Porsolt's test) in mice
The NMR spectral data were provided by Centro di Ricerca Inter-
dipartimentale di Analisi Strumentale, Università degli Studi di Napoli
“Federico II”. The assistance of the staff is gratefully appreciated.
Calculations were partially performed under a computational grant by
Interdisciplinary Center for Mathematical and Computational Modeling
(ICM), Warsaw, Poland (Grant Number, G30‐18), under resources and
licenses from CSC, Finland.
The experiment was carried out according to the method of Porsolt
et al.[57] Mice were individually placed in a glass cylinder (25 cm high;
10 cm in diameter) containing water maintained at 23–25°C, and
were left there for 6 min. The total duration of immobility was
recorded during the last 4 min of a 6‐min test session. A mouse was
regarded as immobile when it remained floating on the water, making
only small movements to keep its head above the water.[58]

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[31] J. H. Kim, P. Vezina, Pharmacol. Biochem. Behav. 2002, 73(2), 333.
[32] H. Meltzer, Neuropsychopharmacology 1999, 21, 106S.
[33] M. Tomić, D. Ignjatović, G. Tovilović, D. Andrić, G. Roglić, S. Kostić‐
Rajačić, Bioorg. Med. Chem. Lett. 2007, 17, 5749.
ORCID
Ferdinando Fiorino
http://orcid.org/0000-0001-7357-5751
[34] S. Leucht, C. Corves, D. Arbter, R. R. Engel, C. Li, J. M. Davis, Lancet
2009, 373, 31.
REFERENCES
[35] P. Chopin, M. Briley, Psychopharmacology 1993, 110(4), 409.
[36] S. Pellow, P. Chopin, S. E. File, M. Briley, J. Neurosci. Methods 1985,
14(3), 149.
[1] H. G. Baumgarten, M. Gother, Handb. Exp Pharm, Vol. 129. Berlin:
Springer‐Verlag, 1997.
[37] K. Harada, M. Aota, T. Inoue, R. Matsuda, T. Mihara, T. Yamaji, et al.,
[2] G. R. Martin, R. M. Eglen, D. Hoyer, M. W. Hamblin, F. Yocca, Ann.
Eur. J. Pharmacol. 2006, 553(1‐3), 171.
N.Y. Acad. Sci. 1998, 861(1), xiii.
[38] D. C. Hoffman, H. Donovan, Psychopharmacology 1995, 120, 128.
[39] C. Yung‐Chi, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099.
[40] G. Scatchard, Ann. N. Y. Acad. 1949, 51, 660.
[41] Q. Y. Zhou, D. K. Grandy, L. Thambi, J. A. Kushner, H. H. Van Tol,
R. Cone, et al., Nature 1990, 347, 76.
[3] N. M. Barnes, T. Sharp, Neuropharmacology 1999, 38, 1083.
[4] D. Hoyer, J. P. Hannon, G. R. Martin, Pharmacol. Biochem. Behav.
2002, 71, 533.
[5] R. Fredriksson, M. C. Lagerström, L. G. Lundin, H. B. Schiöth, Mol.
Pharmacol. 2003, 63(6), 1256.
[42] D. A. Hall, P. G. Strange, Br. J. Pharmacol. 1997, 121, 731.
[43] P. Greengrass, R. Bremmer, Eur. J. Pharmacol. 1979, 55(3), 323.
[44] S. Uhlén, Wikberg JES, Pharmacol. Toxicol. 1991, 69, 341.
[45] J. R. Schlegel, S. J. Peroutka, Biochem. Pharmacol. 1986, 35, 1943.
[46] J. L. Herndon, A. Ismaiel, S. P. Ingher, M. Teitler, R. A. Glennon, J.
Med. Chem. 1992, 35, 4903.
[6] E. J. Siddiqui, M. Shabbir, D. P. Mikhailidis, C. S. Thompson, F. H.
Mumtaz, J. Urol. 2006, 176, 1648.
[7] A. Corvino, F. Fiorino, B. Severino, I. Saccone, F. Frecentese, E.
Perissutti, et al., Curr. Med. Chem. 2018, 25, 3214.
[8] M. Bancila, D. Verge, O. Rampin, J. R. Backstrom, E. Sanders‐Bush, K.
E. McKenna, et al., Neuroscience 1999, 92(4), 1523.
[47] W. Billard, V. Ruperto, G. Crosby, L. C. Iorio, A. Barnett, Life Sci.
1984, 35(18), 1885.
[9] Y. Kimura, K. Hatanaka, Y. Naitou, K. Maeno, I. Shimada, A.
Koakutsu, et al., Eur. J. Pharmacol. 2004, 483(1), 37.
[48] I. Creese, R. Schneider, S. H. Snyder, Eur. J. Pharmacol. 1977, 46(4), 377.
[49] B. D. Perry, D. C. U’Prichard, Eur. J. Pharmacol. 1981, 76(4), 461.
[50] A. A. Kaczor, K. M. Targowska‐Duda, B. Budzyńska, G. Biała, A. G.
Silva, M. Castro, Neurochem. Int. 2016, 96, 84.
[10] M. Lopez‐Rodriguez, D. Ayala, B. Benhamu, M. Morcillo, A. Viso, Curr.
Med. Chem. 2002, 9, 443.
[11] D. M. Kendig, J. R. Grider, Neurogastroenterol. Motil. 2015, 27, 899.
[12] G. Di Giovanni, De P. Deurwaerdère, Pharmacol. Ther. 2016,
157, 125.
[51] Schrödinger Release 2018‐2. Glide. New York, NY: Schrödinger,
LLC; 2018.
[13] H. Pessoa‐Mahana, R. Araya‐Maturana, B. Saitz, C. Pessoa‐Mahana,
Mini Rev. Med. Chem. 2003, 3, 77.
[52] Schrödinger Release 2018‐2. LigPrep. New York, NY: Schrödinger,
LLC; 2018.
[14] M. L. Lopez‐Rodriguez, M. J. Morcillo, T. K. Rovat, E. Fernandez, B.
Vicente, A. M. Sanz, et al., J. Med. Chem. 1999, 42, 36.
[15] G. Caliendo, F. Fiorino, P. Grieco, E. Perissutti, V. Santagada, B.
Severino, et al., Bioorg. Med. Chem. 2000, 8, 533.
[53] Schrödinger Release 2018‐2. Epik. New York, NY: Schrödinger,
LLC; 2018.
[54] J. A. Ballesteros, H. Weinstein, Methods Neurosci. 1995, 25, 366.
[55] F. Gross, J. Tripod, R. Meir, Schweiz. Med. Wochenschr. 1955, 85, 305.
[56] J. R. Boissier, J. Tardy, J. C. Diverres, Med. Exp. (Basel) 1960, 3, 81.
[57] R. D. Porsolt, G. Anton, N. Blavet, M. Jalfre, Eur. J. Pharmacol. 1978,
47, 379.
[16] G. Caliendo, F. Fiorino, E. Perissutti, B. Severino, S. Gessi, E.
Cattabriga, et al., Eur. J. Med. Chem. 2001, 36(11‐12), 873.
[17] G. Caliendo, F. Fiorino, E. Perissutti, B. Severino, D. Scolaro, S. Gessi,
et al., Eur. J. Pharm. Sci. 2002, 16, 15.
[58] E. Kędzierska, J. Dudka, E. Poleszak, J. H. Kotlińska, Pharmacol. Rep.
2017, 69(2), 276.
[18] F. Fiorino, E. Perissutti, B. Severino, V. Santagada, D. Cirillo, S.
Terracciano, et al., J. Med. Chem. 2005, 48, 5495.
[59] R. G. Lister, Psychopharmacology 1987, 92, 180.
[60] P. J. Little, D. R. Compton, M. R. Johnson, L. S. Melvin, B. R. Martin, J.
Pharmacol. Exp. Ther. 1988, 247(3), 1046.
[19] F. Fiorino, B. Severino, Angelis FDe, E. Perissutti, F. Frecentese, P.
Massarelli, et al., Arch. Pharm. (Weinheim) 2008, 341, 20.
[20] F. Fiorino, B. Severino, De F. Angelis, E. Perissutti, E. Magli, F.
Frecentese, et al., Pharmazie 2009, 64(9), 555.
[61] N. Aaltonen, E. Kedzierska, J. Orzelska‐Gorka, M. Lehtonen, D.
Navia‐Paldanius, H. Jakupovic, et al., J. Pharmacol. Exp. Ther. 2016,
359(1), 62.
[21] F. Fiorino, B. Severino, De F. Angelis, E. Perissutti, E. Magli, F.
Frecentese, et al., Bioorg. Med. Chem. Lett. 2010, 20(9), 2978.
[22] F. Fiorino, B. Severino, E. Magli, E. Perissutti, F. Frecentese, A.
Esposito, et al., Eur. J. Med. Chem. 2012, 47, 520.
SUPPORTING INFORMATION
[23] F. Fiorino, A. Ciano, E. Magli, B. Severino, A. Corvino, E. Perissutti, et
al., Eur. J. Med. Chem. 2016, 110, 133.
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
[24] F. Fiorino, E. Magli, E. Kedzierska, A. Ciano, A. Corvino, B. Severino,
et al., Bioorg. Med. Chem. 2017, 25(20), 5820.
[25] G. Sauvé, V. S. Rao, G. Lajoie, B. Belleau, Can. J. Chem. 1985,
63(11), 3089.
[26] Y. Peng, J. D. McCorvy, K. Harpsøe, K. Lansu, S. Yuan, P. Popov, et al.,
Polypharmacol. Cell. 2018, 172, 719.
How to cite this article: Magli E, Kędzierska E, Kaczor AA,
et al. Synthesis, docking studies, and pharmacological
evaluation of 5HT2C ligands containing the
[27] S. Podlewska, R. Kafel, E. Lacivita, G. Satała, A. J. Kooistra, M. Vass,
et al., Eur. J. Med. Chem. 2018, 151, 797.
[28] H. G. Vogel, In: H. Vogel, editor. Drug Discovery and Evaluation:
Pharmacological Assays. Berlin Heidelberg: Springer‐Verlag; 2008. 565
[29] P. H. Gobira, J. Ropke, D. C. Aguiar, Crippa JAS, F. A. Moreira, Rev.
Bras. Psiquiatr. 2013, 35(2), 132.
N′‐cyanoisonicotinamidine or N′‐cyanopicolinamidine nucleus.
Arch Pharm Chem Life Sci. 2019;e1800373.
https://doi.org/10.1002/ardp.201800373
[30] C. Jones, D. Watson, K. Fone, Br. J. Pharmacol. 2011, 164, 1162.