5-HT2 receptor affinity, docking studies and pharmacological evaluation of a series of 1,3-disubstituted thiourea derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.03.073

A series of 10 thiourea derivatives have been synthesized by the reaction of aromatic amine with a substituted aryl (compounds 1-3, 6-8) and alkylphenyl (4, 5, 9, 10) isothiocyanates. Their in vitro and in vivo pharmacological properties were studied. Amo

Full text of DOI:10.1016/j.ejmech.2016.03.073

European Journal of Medicinal Chemistry 116 (2016) 173e186
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
5-HT2 receptor affinity, docking studies and pharmacological
evaluation of a series of 1,3-disubstituted thiourea derivatives
,
*
b
c
d
Anna Bielenica a , Ewa Ke˛dzierska , Michał Kolinski , Sebastian Kmiecik ,
ꢀ
d
e
e
e
ꢀ
Andrzej Kolinski , Ferdinando Fiorino , Beatrice Severino , Elisa Magli ,
Angela Corvino e, Ilaria Rossi f, Paola Massarelli f, Anna E. Kozioł g,
Aleksandra Sawczenko g, Marta Struga h
a Chair and Department of Biochemistry, Medical University of Warsaw, 02-097, Warszawa, Poland
b Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, 20-031, Lublin, Poland
c Bioinformatics Laboratory, Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106, Warszawa, Poland
d Faculty of Chemistry, University of Warsaw, 02-093, Warszawa, Poland
e
ꢁ
Dipartimento di Farmacia Universita di Napoli “Federico II”, Via D. Montesano, 49, 80131, Naples, Italy
f
ꢁ
Dipartimento di Scienze Mediche, Chirurgiche e Neuroscienze Strada delle Scotte, Universita di Siena, 6 53100, Siena, Italy
g Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031, Lublin, Poland
h Department of Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw, 02-097, Warszawa, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 10 thiourea derivatives have been synthesized by the reaction of aromatic amine with a
substituted aryl (compounds 1e3, 6e8) and alkylphenyl (4, 5, 9, 10) isothiocyanates. Their in vitro and
in vivo pharmacological properties were studied. Among the evaluated compounds, two displayed very
high affinity for the 5-HT2A receptor (1e0.043 nM and 5e0.6 nM), being selective over the 5-HT2C re-
ceptor. Derivatives 3, 5, 9, 10 by 70e89% diminished L-5-HTP-induced head twitch episodes. Compounds
1 and 5 as the 5-HT2A receptor antagonists produced a dose-dependent decrease in the number of DOI-
elicited HTR. Compounds 1e5 strongly reduced amphetamine-evoked hyperactivity in rodents. In
another test, 1 and 2 caused hyperthermia in mice, whereas 9 and 10 led to hypothermia. Antinociceptive
and anticonvulsant properties of selected derivatives were demonstrated. Molecular docking studies
using a homology model of 5-HT2A revealed a significant role of hydrogen bonds between both thiourea
Received 28 January 2016
Received in revised form
18 March 2016
Accepted 25 March 2016
Available online 30 March 2016
Keywords:
5-HT2A receptor ligands
5-HT2C receptor ligands
Docking studies
Thiourea
NH groups and Asp155/Tyr370 residues, as well as
pe
p
interaction with Phe339.
CNS activity
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
[5,6], depression, obsessive-compulsive disorders (5-HT2C agonists)
[1,7] and generalized anxiety disorders (5-HT2A/5-HT2C antago-
nists) [6]. Type 2 serotonin receptors (5-HT2) mediate the action of
large number of psychoactive drugs, including antipsychotics,
hallucinogens, anxiolytics and anti-depressants [2,8,9]. The key site
for hallucinogen action is the 5-HT2A receptor subtype, which was
developed by correlation of the high affinity for that receptor and
behavioral activity of hallucinogenic amphetamines [4]. It is known
that 5-HT2C rather than 5-HT2A blockade can prevent the extrapy-
ramidal side effects induced by atypical antipsychotics, such as
Haloperidol [10].
The 5-HT2 receptor family comprises of the 5-HT2A, 5-HT2B and
5-HT2C subreceptors, which are G-protein coupled receptors linked
to the inositol phosphate signal transduction system [1]. Since 5-
HT2A and 5-HT2C receptors exhibit 46e50% overall sequence iden-
tity [2,3], their molecular and pharmacological profiles are similar
[4]. Both are involved in a range of physiological functions, such as
motor behavior, mood, pain, appetite, sleep, thermoregulation, urea
excretion and hormone secretion [1,4]. They are implicated in
several psychiatric disorders, including schizophrenia (mixed D2/
D1/5-HT2 antagonists), psychosis (mixed D2/5-HT2A antagonists)
Several structurally different compounds are known to bind 5-
HT2A receptors. Among them, some urea and thiourea derivatives
have been extensively studied (Fig. 1). All of them display nano-
molar potency at 5-HT2A receptors, but only for Pimavanserin high
* Corresponding author.
E-mail address: abielenica@wum.edu.pl (A. Bielenica).
http://dx.doi.org/10.1016/j.ejmech.2016.03.073
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

174
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
N
N
NH
N
O
H
F
O
O
O
N
NH
N
F
N
H
N
H
S
O
Pimavanserin
Lisuride
Altanserin
Fig. 1. Some representative ligands for 5-HT2A receptors.
selectivity over 5-HT2C is observed [11]. What is more, it lacks other
serotonin, as well as dopamine receptors activity, in contrast to
Lisurde [12] and Altanserin [7,13], which effectively block dopa-
mine D2 receptors. The mode of action at 5-HT2A receptors among
mentioned above (thio)urea derivatives also varies: Pimavanserin is
an inverse agonist [14,15], the profile of Lisuride is described as a
partial agonism [12], and Altanserin acts as an antagonist [7].
It is supposed that 5-HT2A receptor blockade, without affecting
dopamine D2 receptors, represented e.g. by pimavanserin, could be
the proper target mechanism for non-motor disorders accompa-
nying Parkinson‘s disease, such as depression, sleep problems, de-
mentia and psychosis, known as Parkinson's disease psychosis
[11,16]. Another urea connection, Lisuride, is applied in an initial
anti-Parkinsonian therapy, aimed at alleviating movement
dysfunction. Additionally, it also lowers prolactin and its low doses
prevent migraine attacks [12]. On the other hand, 5-HT2A antago-
nists and inverse agonists are also being developed as potential
anti-insomnia drugs [17], without many of the less desirable side
effects of benzodiazepines [14].
The substituted amphetamine hallucinogen, 5-HT2A/2C receptor
agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI) has emerged as
the most popular pharmacological tool used in HTR studies of
hallucinogens. Numerous receptor systems are involved in the
behavioral response induced by DOI and an activation of the 5-HT2A
receptor is essential for this effect [18]. Some studies suggest
considering also the 5-HT2B receptor as a potential modulator of
HTRs. What is more, recent investigations have demonstrated that
5-HT2C and 5-HT1A agonists [17,19], chronic administration of se-
rotonin norepinephrine transporter inhibitors (SNRIs), selective
AMPA glutamate antagonists, GABAA agonists, adenosine A1 ago-
nists, D1 receptor antagonists [18], as well as indirect cannabinoid
agonists decrease the number of DOI-elicited HTRs [20]. It has also
been suggested that head twitch shakes may be useful as a model of
Tourette's syndrome [21].
[5,10,29e31].
This work reports the synthesis, docking studies and evaluation
of 1,3-disubstituted thiourea derivatives related with 5-HT2A and 5-
HT2C receptors, as well as determination of their in vivo properties
in rodent behavioral models. The outcome enabled us to identify
the key structural features responsible for the serotonergic activity,
therefore leading to novel agents with antipsychotic action.
2. Results and discussion
2.1. Chemistry
The synthetic route for the preparation of 1,3-disubstituted
thiourea derivatives is shown in Scheme 1. The amine compounds
were reacted with substituted isothiocyanates in dry acetonitrile
medium and gave some 3-(trifluoromethyl)phenyl)thiourea (1e5)
and (4-chloro-3-nitrophenyl)thiourea derivatives (6e10). The
structures of newly obtained compounds were elucidated by
spectral analyses, and molecular structure of 2 and 6 was confirmed
by an X-ray crystallography (Fig. 2).
The set of substituents at the thiourea nitrogen was selected on
the basis of our previous experiences with the substituent impact
on the pharmacological properties [25,26,32]. As a result, the
representative N-arylthiourea and N-alkylthiourea derivatives of 3-
(trifluoromethyl)aniline (1e5) and 4-chloro-3-nitroaniline (6e10)
were presented. From the first group, compounds with electron-
withdrawing functionalities 1 (3-chloro-4-fluorophenyl), 3 and 7
(2-chlorophenyl)
were
investigated,
as
well
as
4-
methoxyphenylthiourea 2, that contains an electron-donating
substituent on benzene ring. In the N-alkylthiourea set, the nitro-
gen atom of thiourea was linked to phenylethyl (4, 10) or benzyl (5,
9) fragment. Unsubstituted phenyl (1) and benzoyl (8) derivatives
were also evaluated. As a result, the tested collection of compounds
presented diverse and comparable group.
Some reports concerning a strong influence of urea [22e24] and
thiourea [24e27] derivatives on the central nervous system (CNS)
in rodents have been presented. Several N-arylthioureas [26],
similarly as atypical opioid analgesic (morphine, Tramadol) and
antipsychotics (Clozapine), inhibited L-5-HTP-induced head twitch
responses (HTR) in mice, an animal model for the activation of the
CNS 5-HT2A receptors [28]. It was demonstrated that also Pima-
vanserin shared a behavioral characteristics consistent with the
atypical antipsychotic drugs, such as potently inhibits head-
twitching produced by DOI [15,16]. Both urea and thiourea con-
nections are known of their anticonvulsant properties [24,27],
indicating the therapeutic potential in petit mal seizures. Moreover,
a sedative effect of thioureas was observed [22,23], making them
potentially useful for panic attacks, anxiety and sleep disorders.
There is no data concerning binding modes of (thio)urea de-
rivatives to 5-HT2 receptors so far; what is more, limited docking
studies are available for other class of 5-HT2A receptor ligands, as
2.2. In vitro tests
All the new compounds were tested for their affinity to 5-HT2A
and 5-HT2C receptors. Some of the synthesized derivatives showed
affinities in the subnanomolar range towards 5-HT2A receptors and
no affinity for other relevant 5-HT2C receptors (Table 1). Besides the
outstanding 5-HT2A receptor affinity and selectivity of compound 1
(Ki ¼ 0.043 0.009 nM), another interesting Ki value was that of
compound 5 (Ki ¼ 0.60 0.024 nM). Although the derivatives 1e10
showed different substituents R1 and R2 on the aromatic rings
linked to the thiourea scaffold, the R2 functionalities do not seem to
be decisive in determining a general trend towards 5-HT2 receptors.
The affinity/selectivity profiles towards 5-HT2A receptor are more
influenced by the presence of a m-CF3 group on the aromatic ring
than R1 substituent. In fact, the association of this structural feature
to a 3-chloro-4-fluorophenyl group and a phenylethyl fragment on
R2 position of the thioureidic scaffold (1 and 5), conferred the
tryptamines,
phenylisopropylamines
or
n-alkylpiperidines

A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
175
R1
R1
NH
NH
MeCN
+
NH2
R2
N
C
S
R
2
12-36 h
S
6 R1= 3-NO2,4-Cl, R2= Ph
1 R1= 3-CF3, R2= 3-Cl,4-F-Ph
2 R1= 3-CF3, R2= 4-OCH3-Ph
3 R1= 3-CF3, R2= 2-Cl-Ph
4 R1= 3-CF3, R2= CH2CH2-Ph
5 R1= 3-CF3, R2= CH2-Ph
7 R1= 3-NO2,4-Cl, R2= 2-Cl-Ph
8 R1= 3-NO2,4-Cl, R2= -(C=O)-Ph
9 R1= 3-NO2,4-Cl, R2= CH2-Ph
10 R1= 3-NO2,4-Cl, R2= -CH(CH3)-Ph
Scheme 1. Synthesis of thiourea derivatives 1e10.
Fig. 2. Perspective view of molecules 2 and 6 (conformers observed in solid).
Table 1
Affinities values of compounds 1e10 for 5-HT2A and 5-HT2C receptors.
Comp.
R1
R2
Receptor affinity Ki SD (nM)
ClogPa
5-HT2A [3H]Ketanserin
5-HT2C [3H]Mesulergine
1
2
3
4
5
6
7
8
3-CF3
3-CF3
3-CF3
3-CF3
3-Cl, 4-F-Ph
4-OCH3-Ph
2-Cl-Ph
eCH2CH2ePh
eCH2ePh
Ph
0.043 0.009
> 104
> 104
> 104
> 104
> 104
> 104
> 104
> 104
> 104
> 104
> 104
e
4.75
3.57
3.84
4.19
3.76
3.23
3.42
3.62
3.71
4.02
> 104
> 104
3-CF3
0.60 0.024
3-NO2,4-Cl
3-NO2,4-Cl
3-NO2,4-Cl
3-NO2,4-Cl
3-NO2,4-Cl
e
> 104
2-Cl-Ph
> 104
e(C]O)Ph
eCH2ePh
eCH(CH3)ePh
e
> 104
9
10
> 104
> 104
Ketanserine
Mesulergine
0.85
e
e
e
1.9
a
Calculated by ChemBioDraw Ultra 14.
highest affinity and selectivity values for the 5-HT2A receptor.
Moreover the outstanding affinities and selectivity of compounds 1
and 5 towards 5-HT2A receptors agree with data already reported in
literature by Parker et al. [33] and could be explained also in terms
of correlation between lipophilicity and binding affinity, as
confirmed by molecular docking studies.
Therefore we decided to use crystal structure of
inactive state of the receptor as a template. Moreover, b2AR was
b
2AR presenting
also used as a template for homology modeling of 5-HT2A receptor
and ligand docking in previous studies [36]. The molecular model of
the receptor was refined during 10 ns MD simulation, in the
membrane environment, with weak position restraints imposed on
the protein backbone. The conducted simulation allowed for
relaxation and optimization of the receptor side-chains (see Fig. S1,
Supplementary Materials). The obtained model was used in the
docking. For each of the 10 compounds, we have obtained a set of
1000 lowest energy conformers during docking procedure. The
estimated values of free binding energies were ranging
from ꢀ5.53 kcal/mol to ꢀ7.9 kcal/mol. Final docking conformation
was selected by means of structural clustering. Depending on a
compound case, the largest cluster consisted from 193 to 970
members (see Table 2 for detail description of docking results).
These numbers indicate a high degree of convergence towards a
single and favored binding mode for each of the ligands. Additional
2.3. Molecular docking studies
In this study binding modes of all synthesized compounds to 5-
HT2A receptor using molecular docking approach was analyzed. The
5-HT2A receptor model was created applying comparative
modeling methods based on the crystal structure of b2AR. In 2013,
two crystal structures of serotonin receptors subtypes 5-HT1B [34]
and 5-HT2B [35] were determined. However, in both structures
different conformational patterns are observed which show fea-
tures of an intermediate active-state for the 5-HT1B receptor and a
b
-arrestin-biased activation-state for the 5-HT2B receptor [35].

176
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
Table 2
5-HT2A binding data based on docking results for compounds 1e10.
Comp. Position LC/LBE
Ligand surrounding amino acids (d ꢁ 3 Å)
LC ¼ 327; LBE ¼ ꢀ6.05 kcal/ TRP151, ILE152, ASP155, VAL156, SER226, PHE222, LEU228, LEU229, VAL235, GLY283, SER239, SER242, PHE339,
1
LBM
UBM
LBM
UBM
LBM
UBM
UBM
UBM
UBM
mol PHE340, ASN234, VAL366
LC ¼ 384; LBE ¼ ꢀ5.53 kcal/ SER131, THR134, ILE135, GLY138, ARG140, TRP141, TRP151, ILE152, CYS227, LEU228, LEU229, PHE339, ASN363,
mol VAL366, TYR370
2
3
LC ¼ 311; LBE ¼ ꢀ6.18 kcal/ ILE152, ASP155, VAL156, SER226, PHE222, LEU228, LEU229, TRP336, PHE339, PHE340, ASN234, VAL366, TYR370
mol
4
LC ¼ 193; LBE ¼ ꢀ6.49 kcal/ SER131, THR134, ARG140, TRP141, TRP151, ILE152, CYS227, LEU228, ASN363, VAL366, TRP367, TYR370,
mol
5
LC ¼ 742; LBE ¼ ꢀ5.95 kcal/ TRP151, ILE152, ASP155, SER226, PHE222, CYS227, LEU228, LEU229, TRP336, PHE339, PHE340, ASN234, TYR370
mol
6
LC ¼ 970; LBE ¼ ꢀ6.69 kcal/ SER131, THR134, ILE135, ARG140, TRP141, TRP151, ILE152, ASP155, PHE222, CYS227, LEU228, LEU229, ASN363,
mol
TYR370
7
LC ¼ 291; LBE ¼ ꢀ7.48 kcal/ THR134, ILE135, ARG140, TRP141, TRP151, ILE152, PHE222, SER226, LEU228, LEU229, TYR370
mol
8
LC ¼ 277; LBE ¼ ꢀ7.6 kcal/ THR134, ILE135, ARG140, TRP141, TRP151, ILE152, ASP155, VAL156, PHE222, SER226, CYS227, LEU228, LEU229
mol
9
LC ¼ 268; LBE ¼ ꢀ7.42 kcal/ SER131, THR134, ILE135, ARG140, TRP141, TRP151, ILE152, CYS227, LEU228, LEU229, TYR370
mol
10a
(S0 isomer)
UBM
(R0 isomer)
LBM
LC ¼ 295; LBE ¼ ꢀ7.9 kcal/ THR134, ILE135, TRP151, ILE152, ASP155, CYS227, LEU228, LEU229, VAL366, TYR370
mol
ILE152, ASP155, VAL156, PHE222, LEU228, LEU229, VAL235, GLY283, SER239, SER242, TRP336, PHE339,ASN234,
LC ¼ 329; LBE ¼ ꢀ7.4 kcal/ VAL366, TYR370
mol
LBM ¼ lower binding mode.
UBM ¼ upper binding mode.
LC ¼ number of members of the largest cluster calculated for 1000 docking runs using RMSD tolerance ¼ 2 Å
LBE ¼ estimated lowest free energy of binding by AutoDock4 energy function.
a
Two isomers were investigated.
MD simulations of predicted complexes could be applied for
detailed investigation of protein-ligand interaction/interplay.
However, they would require an extensive long timescale simula-
tions [37], which is beyond the scope of this study.
HT2A receptor antagonists by other authors [38]. In models for
triangular arylpiperazine antagonists developed previously by
Mokrosz [39,40] or Klabunde and Evers [5], the distances between
groups crucial for activity are different than these described in this
study, mainly in the relative position of aromatic groups versus the
nitrogen atom. To reach maximum binding affinity, the thiourea NH
group has to be placed in the distance 4.3e4.9 Å from both aromatic
centers.
Analysis of resulting binding modes revealed that ligands were
grouped in two distinct regions of the receptor binding site. Com-
pounds 2, 4, 6, 7, 8, 9 and 10 (R0 isomer) preferred upper portion of
the receptor binding site, whereas compounds 1, 3, 5 and 10 (S0
isomer) were grouped at the bottom of the pocket. Graphical rep-
resentation of the two distinct binding modes is shown in Fig. 3.
Those two distinct binding modes correlate well with binding af-
finity experimental data. According to experiment, compounds
interacting at the lower portion of the binding site show high af-
finity to the receptor, in contrast to derivatives showing no affinity
for the receptor protein that are grouped near the extracellular side
of the protein. Two ligands 1 and 5 (exhibiting the highest exper-
imental affinity) bind at the lower portion of the binding site,
penetrating deeper into the receptor alpha helical core. The ligands
are stabilized mainly by hydrogen bonds with Asp155 residue. In
case of 5 additional hydrogen bond is created with Tyr370. That
The studied structural modifications were focused on the aro-
matic substituents at thiourea nitrogen atoms. Results of both af-
finity and molecular modeling studies confirmed the role of the
modified fragment for the biological activity. Within the group of
(3-trifluoromethyl)phenyl derivatives 1e5, compounds substituted
at N3 with 3-chloro-4-fluoro-phenyl (1) or benzyl (5) ring belong to
the class of the most potent ligands, deeply penetrating the binding
pocket of the 5-HT2A receptor. This bottom part of the active site
was also preferred by the derivative 3, possessing electron-
withdrawing chlorine substituent at the benzene ring (similarly
as 1) and S-enantiomer of phenylethyl compound 10 (close analog
of 5). In the group of weakly bounded ligands, gathered in the
upper place of the binding site, derivatives of 4-chloro-3-
nitroaniline can be found (6e9, R-enantiomer of 10). Within this
series, the presence of unsubstituted phenyl ring (6), benzoil (8) or
benzyl (9) rings at the end of the molecules critically decreased
affinity for the 5-HT2A receptor. Introduction of longer spacer at the
terminal phenyl ring (as for phenylethyl compound 4, homolog of
5) or an electron-donating functionality (p-methoxy in 2) was
definitely unprofitable for serotonin receptor affinity. Comparing
two pairs of corresponding thiourea derivatives (3e7 and especially
5e9) it can be concluded that the (3-trifluoromethyl)phenyl con-
taining molecules are more active and/or fit better to the receptor
pocket in each pair.
ligand is also engaged in
pep interaction with Phe339 (Fig. 4).
Interestingly, the thiourea 5 is the representative of the largest
cluster (LC ¼ 742, see Table 2) of the lowest energy conformers
localized at lower portion of the binding site. In contrast, two
compounds 6 and 8 (showing no experimental affinity) interact at
the upper portion of the receptor binding pocket. Both of them are
engaged in hydrogen bond network with backbone fragments of
the residues located in extracellular loops (ECLs), namely: Trp141
from ECL I and Cys227 from ECL II. In addition, the Trp151 residue
formed
pep interactions with the molecule 8 (Fig. 5). What is
more, the ClogP values for inactive compounds 6e8 are the lowest
among the series that indicate their higher hydrophilicity (Table 1).
Based on the docking results obtained in this study, a general
pharmacophore model for ligands showing high experimental af-
finity was proposed. The model consists of the two aromatics sites
and one hydrogen bond donor (HBD) site (Fig. 6). Similar three-
point pharmacophores models have been also generated for 5-
2.4. In vivo tests
On the basis of docking and affinity binding results, several
thioureas were chosen for in vivo experiments. Compounds with

A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
177
and drug-evoked locomotor potency, and anticonvulsant or anal-
gesic activities. Additionally, the involvement in alteration of body
temperature in mice was studied, as it shows the link with 5-HT1
receptors. The established ED50 of tested compounds were the
following: 286.2 mg/kg (1), 771.4 mg/kg (2), 2000 mg/kg (3),
709.6 mg/kg (4), 731.3 mg/kg (5), 909 mg/kg (9) and 1031.3 mg/kg
(10). The progressive doses, calculated as a part of ED50, were used
in behavioral experiments.
In the first instance, no observable neurotoxic effects were
detected in the rota-rod and chimney tests for any of investigated
compounds (data not shown). Thus, one can ascertain that tested
thiourea derivatives do not disturb motor coordination and do not
provoke myoreleaxation.
The head twitch responses (HTR) in rodents induced by 5-HTP, a
precursor of 5-HT, is considered as a specific behavioral model for
the activation of serotonergic neurons [28]. According to literature
data, the attenuating action on HTR reflects an antagonist activity of
a compound at 5-HT2A receptors [42]. Early behavioral studies
revealed that 5-HT2A receptor antagonists, such as ketanserin and
ritanserin, inhibit HTR by more than 80% [43]. However, this test is
not considered very specific, because other substances, like
adrenergic ligands, can influence HTR [44]. The effect of new
compounds (used at the dose of 0.1 ED50) on HTR evoked by L-5-
HTP (230 mg/kg) is presented in Fig. 7. Compounds 3, 5, 9 and 10
seem to be the strongest blocking agents of head twitch frequency
among the whole series, as compared to the basal activity after
administration of L-5-HTP. The head twitch shakes were decreased
in the presence of derivatives 3, 5, 9 and 10 by 72.4%, 73.3%, 70.5%
(p < 0.05) and 89.0%, respectively (p < 0.01). The results for 1, 2 and
4 did not reach statistical significance, though decrements of the
responses were also noticed.
Experimental evidence in animals proved that some hallucino-
gens like 2,5-dimethoxy-4-iodoamphetamine (DOI) induce hallu-
cinations by activating 5-HT2A receptors, and their properties are
blocked by mainly selective 5-HT2A receptor antagonists [6]. Ac-
cording to in vitro affinity results, synthesized derivatives 1 and 5
were found as very potent 5-HT2A ligands, therefore their antago-
nistic properties were assessed in the ( )DOI)-induced head twitch
responses in mice (Fig. 8). Studied compounds produced a dose-
dependent decrease in the number of ( )DOI-induced head
shakes (Table 3), that proved their 5-HT2A receptor antagonistic
activities. The thiourea 5 was stronger 5-HT2A antagonist than 1,
inhibiting the HTR by 72.2%, if administrated in the dose equivalent
to 0.1 ED50 (p < 0.001) and by 69.4%, when taken in its lower dose
(0.05 ED50). These findings are consistent with docking results,
since the ligand 5 has formed more interactions in the upper
binding site of the receptor protein. Although the binding constant
Ki of the derivative 1 is even higher than this estimated for both 5
and Ketanserine, its bioavailability in vivo seems to be lower, which
could explain weaker inhibitory effect against head twitch re-
sponses observed among laboratory animals.
The influence of a thiourea series on spontaneous motility and
amphetamine-induced hyperactivity was assessed (Fig. 9). It was
noticed that 3, administered at the dose of 0.1 ED50 inhibited the
locomotor activity in studied animals (p < 0.05), while the other
derivatives (1, 2, 4, 5, 7e10) did not produce any significant effects.
Nonetheless, it was observed that almost all 3-(trifluoromethyl)
phenyl derivatives, administrated at the same dose, generated a
significant decline of the amphetamine-induced hyperactivity of
animals (p < 0.05 for 2, 5, p < 0.01 for 1, 4, Fig. 10). Derivatives 1 and
4 reduced the amphetamine-induced motility by 47.30% and
42.48%, respectively. This effect was not observed for tested 4-
chloro-3-nitrophenylthioureas (9, 10). Literature survey shows
that in contrast to the atypical antipsychotic drugs, the urea de-
rivative such as pimavanserin lacks D2 antagonist activity and
Fig. 3. Structure of 5-HT2A receptor model. Colored surface indicates portion of the
binding site occupied by ligands: the color green represents lower binding mode for
compounds 1 and 5; the color red represents upper binding mode for compounds: 6
and 8. Residue Asp155 is shown in yellow. a) View parallel to the membrane plain, b)
View from the extracellular side. (For interpretation of the references to color in this
figure caption, the reader is referred to the web version of this article.)
the highest affinity for 5-HT2A receptor (1, 5), derivatives with
preferred lower localization in the active site (3, 10), as well as their
very close structural analogs (2, 4, 9) were investigated. The
intrinsic activity of structures 6e8 was not examined, because they
occupy upper, non-preferred place of the receptor pocket that
suggested no potential biological potency.
To determine the 5-HT2 binding profile precisely, the tendency
to diminish the head twitch responses (HTR), evoked by either 5-
hydroxy-L-tryptophan
(L-5-HTP)
and
2,5-dimethoxy-4-
iodoamphetamine (DOI) was assessed. Previously performed the
rota-rod and chimney tests allowed to check possible occurrence of
drug-induced changes in the muscle relaxant activity of mice,
which may have contributed to their behavior. Since already re-
ported structurally related thiourea derivatives strongly influenced
the central nervous system in mice [22e28], some common
behavioral tests were estimated, such as effects on the spontaneous

178
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
Fig. 4. Binding modes of compounds a) 5 and b) 1. The left panel presents receptoreligand interaction scheme generated using PoseView server [41]. Black dashed lines indicate
hydrogen bonds. Green solid lines show hydrophobic interactions and green dashed lines show interactions. The right panel shows ligand position inside the lower portion of
pep
binding the site of the receptor protein. Red dotted lines indicate hydrogen bonds. (For interpretation of the references to color in this figure caption, the reader is referred to the
web version of this article.)
therefore does not effectively reverse amphetamine-induced hy-
peractivity [16]. Comparing studied compounds with that drug we
could conclude that mechanism of action of compounds 1, 2, 4, 5 is
mediated by catecholamine system, since amphetamine exerts its
behavioral effects by modulating monoamine neurotransmission in
the brain [45].
after the administration of the compound 3 at the dose of 0.1 ED50
(p < 0.01) and after 4, 5 (p < 0.05; p < 0.01) and 9, 10 (p < 0.01 and
p < 0.001) at the dose of 0.05 ED50. Pretreatment with Naloxone did
not reverse these antinociceptive effects of tested compounds. It
leads to the conclusion, that mechanism of their activity is not
linked with endogenous opioid system and the compounds do not
behave as its agonists.
The antinociceptive activity was measured as the ability to
reduce the number of abdominal writhes induced by i.p. injection
of acetic acid in mice (Fig. 11). This test, known as “writhing test”, is
one of the most sensitive methods used to detect even very weak
antinociceptive agents. It is also considered as an experimental
model closest to the nature of clinical pain. Although it does not
allow to determine the length of analgesic action and it is not
specific, it allows to evaluate activity of both central and peripheral
origin [46,47]. It was observed that substances 3e5, 9, 10, used at a
dose equivalent to 0.1 ED50, were the most potent in this test
(p < 0.01). When given at half a dose (0.05 ED50), statistically sig-
nificant reduction in the number of writhing episodes was
observed for 4, 5, 9 and 10 (p < 0.05). These derivatives adminis-
trated at a dose equivalent to 0.025 ED50, still exhibited anti-
nociceptive activity, though without any statistical importance.
In order to check the correlation between the antinociceptive
effect of a compound and the opioid system, the writhing test in the
presence of nonselective opioid receptor antagonist, Naloxone, was
performed (Fig. 12). One-way ANOVA showed significant changes
in the number of writhing episodes of mice after the administration
of tested derivatives (F(10,96 ¼ 5443; p < 0.0001). Post hoc Dunnett's
test showed a significant reduction in the writhing episodes of mice
The anticonvulsant activity of new derivatives was determined
by using pentetrazol-induced seizure test (Table 4). This rodent
model is widely used as the standard method for predicting pro-
tection against tonic-clonic seizures. In PTZ-induced seizures
model compound 4 administrated at a dose equivalent to 0.1 ED50,
significantly protected from tonic seizures (by 46,66%) and death of
animals (by 48.33%, p < 0.05). Derivative 5 reduced clonic seizures
(by 40.0%) and was found effective in preventing from tonic con-
vulsions (by 46.66%, p < 0.05); the ability to decrease the mortality
by this compound was also observed. Among 4-chloro-3-
nitrophenyl compounds, derivative 9 considerably (in 50e43.75%)
reduced both tonic and clonic seizures, as well as the mice mor-
tality. Used in half of its dose (0.05 ED50), it still diminished death
episodes (p < 0.05). The obtained results have confirmed other
authors findings [24,48], since N-alkylphenylthiourea analogues
were found to possess higher anticonvulsant activity than N-
arylthioureas, because of the bulk and lipophilicity.
The results of measurement of the body temperature in
normothermic mice may also suggest the link between the sero-
tonergic system and mechanism of action of studied compounds
(Figs. 13 and 14). According to literature data, that system is

A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
179
Fig. 5. Binding modes of compounds a) 6 and b) 8. The left panel presents receptoreligand interaction scheme generated using PoseView server [12]. Black dashed lines indicate
hydrogen bonds. Green solid lines show hydrophobic interactions and green dashed lines show interactions. The right panel shows ligand position inside the upper portion of
pep
the binding site of the receptor protein. Red dotted lines indicate hydrogen bonds. (For interpretation of the references to color in this figure caption, the reader is referred to the
web version of this article.)
20
control
0.1 ED50
15
p < 0.01
**
p < 0.05
*
vs control
10
5
*
*
*
**
0
1
2
3
4
5
9
10
Fig. 6. The 5-HT2A receptor pharmacophore model: Ar - aromatic; HBDehydrogen
bond donor. For comparison the graphical representation of 5 in its receptor inter-
acting conformation is superimposed on the pharmacophore model.
Fig. 7. The influence of new derivatives on head-twitch responses (HTR) evoked by L-
5-HTP (230 mg/kg, i.p). Compounds were injected 60 min before the test, whereas L-5-
HTP immediately before the test. Data are expressed as mean SEM values. **p < 0.01,
*p < 0.05 vs. control group (Dunnett's test).
involved in the body temperature regulation in hypothalamus
[49,50]; 5-HT2 receptor agonists (e.g. DOI) and 5-HT1A antagonists
can induce hyperthermia, whereas 5-HT2 antagonists (e.g. 8-OH-
DPAT, Ketanserine) or 5-HT1A agonists cause hypothermia [51,52].
Bonferroni's post hoc test revealed a significant increase in the
body temperature of mice after the administration of the com-
pound 1 at the dose of 0.1 ED50 from 30 to 180 min (p < 0.001), and
after intake of the compound 2 in 30 (p < 0.01), 60 (p < 0.001) and
90 min (p < 0.05). The compound 9, given in a dose corresponding
to 0.1 ED50, provoked considerable decrease in the body tempera-
ture (in 60 (p < 0.01), 90 (p < 0.001) and 120 min (p < 0.01). The
derivative 10 was even more effective e it has decreased the body
temperature from 30 to 120 min (p < 0.001, 0.1 ED50). Other tested
compounds did not affect body temperature in a clear, statistically
significant manner, as compared to the control. The results of body
temperature measurement allow for conclusion that the action of 1,
2 and 9, 10 could be an effect of a stimulation of 5-HT1A receptors.
To sum up, derivatives of 3-(trifluoromethyl)aniline (1e5)
regardless of the type of the terminal substituent, have consider-
ably reduced amphetamine-evoked hyperactivity, thus the

180
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
involvement between the observed activity and the catecholamine
system is supposed. The common feature of behaviorally tested
derivatives was also the ability to diminish the number of L-5-HTP-
induced head twitch episodes in mice e for derivatives 1 and 5 it
was linked with their direct antagonistic impact on 5-HT2A re-
ceptors. However, N-alkylphenylthiourea derivatives (4, 5, 9, 10)
were most potent and multidirectional in performed behavioral
tests than their aryl analogues. The connection of the analgesic
potency of phenylethyl (4, 10) benzyl (5, 9) and also 2-chlorophenyl
(3) derivatives with endogenous opioid system was confirmed. The
presence of an alkylphenyl fragment attached to thiourea in 4, 5
and 9 was responsible for their anticonvulsant properties. In
contrast to other derivatives, the sedative effect of thiourea
analogue 3 on spontaneous activity was denoted. The administra-
tion of N-arylthioureas 1 and 2 induced hyperthermia, however
alkylphenyl connections of thiourea 9 and 10 caused decreasing
effect on the body temperature in animals. No compounds exerted
a depressant activity on the CNS in rodents.
Fig. 8. The effect of compounds 1 and 5 on ( )DOI-induced head twitches reaction in
mice. The investigated compounds (i.p.) were administrated 60 min before ( )DOI
(i.p.). ***p < 0.001 versus ( )DOI treated group (one-way ANOVA followed by Bon-
ferroni's post hoc test).
It is worth noting that the presented CNS-activity of derivatives
bearing 3-(trifluoromethyl)phenyl moiety is more broad than the
potency of 4-chloro-3-nitrophenylthioureas. The first group was
dominant by strong 5-HT2A receptor antagonists, as they reduced
head twitch evoked by both L-5-HTP and DOI. Compounds of these
groups expressed also comparable analgesic activity in writhing
test and strongly influenced the body temperature in mice. How-
ever, the tendency of 3-(trifluoromethyl)phenylthioureas to
diminish drug-induced hyperlocomotion tend to be more evident.
All observed effects seem to be connected primarily with seroto-
nergic and/or opioid systems.
Table 3
Effect of compounds 1 and 5 on ( )DOI-induced head twitches reaction (HTR) in
mice.
Treatment
Dose (mg/ Head twitches/20 min
% of HTR
reduction
kg)
(mean SEM)
(
)DOI
2.5
27.38 1.19
13.25 2.03
7.62 1.74
8.37 1.55
0.0 0.0
1
5
28.6
73.1
36.55
5
51.6
72.2
69.4
100
Ketanserin
(KET)
400
300
200
100
0
3. Conclusion
control
0.1 ED50
Our study performed for the series of 1,3-disubstituted thiourea
derivatives 1e10 provided new information in the field of structural
properties responsible for affinity and selectivity toward the 5-HT2A
p < 0.05
vs control
*
*
receptor, comparing to the 5-HT2C. The molecular modeling
demonstrated the role of three structural parameters: two terminal
aromatic fragments and at least one thiourea NH group. (3-
Trifluoromethyl)phenyl ring and 3-chloro-4-fluorophenyl (com-
pound 1) or benzyl (5) moieties were particularly profitable for
affinity and selectivity for the 5-HT2A receptor. 4-Chloro-3-
nitrophenyl part at the thiourea nitrogen was unfavorable for af-
finity for both 5-HT2 receptors. Hydrogen bond between NH group
and Asp155 residue of the receptor was the main stabilizing
interaction. For the derivative 5 also other hydrogen bonds and
1
2
3
4
5
9
10
Fig. 9. The influence of new derivatives on the spontaneous locomotor activity of mice.
Compounds tested were injected 60 min before the test. Locomotor activity was
measured for a period of 30 min. Data are expressed as mean SEM values. *p < 0.05
vs. control group (Dunnett's test).
pep interactions with the receptor were decisive. These two most
active compounds, along with 3 and 10 (S-isomer) occupied upper
portion of the receptor binding site. On the other hand, 2, 4, 6e9, 10
1500
###
control
amph 5mg/kg
0.1 ED50 + amph 5 mg/kg
1000
*
*
### p < 0.001
vs control
**
**
** p < 0.01
500
p < 0.05
*
vs control
0
1
2
3
4
5
9 10
Fig. 10. The influence of new derivatives on the amphetamine-induced hyperactivity of mice. Compounds tested were injected 60 min and amphetamine 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. **p < 0.01, *p < 0.05 vs. control group (Dunnett's test).

A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
181
30
20
10
0
control
0.1 ED50
0.05 ED50
0.025 ED50
p < 0.01
p < 0.05
vs control
*
**
*
*
*
**
**
** *
**
**
1 2
3
4
5
9
10
Fig. 11. The influence of the new derivatives on nociceptive reactions in the writhing test in mice. Compounds tested were injected 60 min and acetic acid (0.6% solution) 5 min
before the test. Data are expressed as mean SEM values. *p < 0.05; **p < 0.01 and ***p < 0.001 vs. control group (Dunnett's test).
Fig. 12. The effect of Naloxone on the antinociceptive effects of compounds 3, 4, 5, 9 and 10 assessed in the writhing test in mice. The results are expressed as mean SEM values.
Table 4
protein that was revealed by molecular modeling studies. The
in vivo evaluation exhibited their distinct antagonistic potency in
DOI-evoked HTR test. The accordance between theoretically
Effect of new derivatives on the pentylenetetrazole (PTZ)-induced clonic seizures,
tonic convulsions and death of mice.
Comp.
Clonic seizuresa Tonic convulsionsa Mortalityb
favorable position in the receptor pocket and head twitch inhibiting
potency in 5-HTP-induced HTR test was also proved for derivatives
3 and 10. Since ligands of the 5-HT1A receptor could also diminish
head twitch shakes [17,19]. That serotonergic affinity was
confirmed experimentally for 2 and 9 by their influence on the
body temperature in laboratory animals. In general, compounds
1e5, 9, 10 have exerted strong influence on the central nervous
system of laboratory animals.
The results of in vivo and in vitro investigations, along with the
molecular docking studies on the 5-HT2A receptor, have provided a
valuable basis for further structural design in the search for novel
potential antipsychotics based on the structure of 1,3-disubstituted
thiourea derivatives. Extended studies concerning affinity and
Control (PTZ 110 mg/kg) 12/12
8/12
8/10
5/10
8/10
2/10*
4/10
2/10*
8/10
15/16
5/10*
6/10
8/10
7/12
6/10
5/10
7/10
1/10*
4/10
2/10
8/10
15/16
5/10*
5/10*
8/10
1 (0.1 ED50
2 (0.1 ED50
3 (0.1 ED50
4 (0.1 ED50
4 (0.05 ED50
5 (0.1 ED50
5 (0.05 ED50
Control (PTZ 110 mg/kg) 16/16
9 (0.1 ED50
9 (0.05 ED50
10 (0.1 ED50
)
)
)
)
10/10
10/10
9/10
8/10
)
)
10/10
6/10*
8/10
)
)
5/10*
8/10
10/10
)
)
*p < 0.05 vs. control (Fisher's exact test).
a
Number of mice reacting/animals tested.
Number of dead mice/animals tested.
b
selectivity towards the 5-HT1A, a-adrenergic and dopaminergic
receptors develop their full pharmacological profile.
(R-isomer) were gathered at the bottom of the pocket, that resulted
in lack of affinity.
4. Experimental
A strict correlation between docking results, binding affinity
studies and in vivo tests was observed. Compounds 1 and 5 were
strong and selective ligands of the 5-HT2A receptor (according to
in vitro data), deeply penetrating the active site of the receptor
4.1. Chemistry
4.1.1. General procedure
The starting amines (3-(trifluoromethyl)aniline and 4-chloro-3-

182
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
Fig. 13. The influence of derivatives 1e5 (used at the dose of 0.1 ED50) on the mice body temperature. Body temperature was measured over a total period of 240 min (60 min before
and 180 min after the tested compound injection). Data are expressed as mean SEM values. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group (Bonferroni's test).
Fig. 14. The influence of derivatives 9 and 10 (used at the dose of 0.1 ED50) on the mice body temperature. Body temperature was measured over a total period of 180 min (60 min
before and 120 min after the tested compound injection). Data are expressed as mean SEM values. *p < 0.05 and **p < 0.01 vs. control group (Bonferroni's test).
nitroaniline) were commercially available (Alfa Aesar). Iso-
thiocyanates were supplied from Alfa Aesar or Sigma Aldrich.
Organic solvents (acetonitrile, chloroform and methanol) were
supplied from POCh (Polskie Odczynniki Chemiczne). All chemicals
were of analytical grade and were used without any further puri-
fication. Prior usage, dry acetonitrile was kept in crown cap bottles
over anhydrous phosphorus pentoxide (Carl Roth). The NMR
spectra were recorded on Varian VNMRS 300 Oxford NMR spec-
trometer, operating at 300 MHz (1H NMR) and 75.4 MHz (13C NMR).
equiped with the microfucus X-ray source (Cu K
a
,
l
¼ 1.54184 Å)
and CCD detector. The CRYSALIS program system [53] was used for
data collection, cell refinement and data reduction. The data were
corrected for Lorentz and polarization effects. A multi-scan ab-
sorption correction was applied. The structure was solved using
direct methods implemented in the SHELXS-97, and refined and
refined by the full-matrix least-squares on F2 with the SHELXL-97
program [54]. All non-H atoms were refined with anisotropic
displacement parameters. The H-atoms attached to carbon were
positioned geometrically and refined using the riding model with
Uiso(H) ¼ 1.2Ueq(C). The nitrogen bonded H-atoms were found in
the difference-Fourier map and refined with isotropic displacement
parameters.
Chemical shifts (d) were expressed in parts per million relative to
tetramethylsilane used as the internal reference. Mass spectral ESI
measurements were carried out on Waters ZQ Micro-mass in-
struments with quadruple mass analyzer. The spectra were per-
formed in the negative or positive ion mode at a declustering
potential of 40e60 V. The sample was previously separated on a
UPLC column (C18) using UPLC ACQUITYTM system by Waters
connected with DPA detector. Elemental analyses were carried out
on a Carlo Erba model 1106. Flash chromatography was performed
on Merck silica gel 60 (230e400 mesh) using chloroform eluent.
Analytical TLC was carried out on silica gel F254 (Merck) plates
(0.25 mm thickness). The diffraction data for 2 and 6 were collected
at 120(2) K on a SuperNova diffractometer (Oxford Diffraction)
4.1.2. General procedure for the synthesis of N-substituted 3-
(trifluoromethyl)phenyl)thiourea (1e5) and (4-chloro-3-
nitrophenyl)thiourea derivatives (6e10)
A
solution of commercially available amines: 3-(tri-
fluoromethyl)aniline (0.0031 mol, 0.50 g) or 4-chloro-3-
nitroaniline (0.0029 mol, 0.50 g) in anhydrous acetonitrile
(10 mL) was treated with appropriate isothiocyanate in 1:1 M ratio.
The mixture was stirred at room temperature for 12e36 h. After

A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
183
solvent evaporating, the residue was either crystallized from
acetonitrile (or chloroform) or purified by column chromatography
(chloroform).
pale yellow powder, m.p. 163e165 ꢂC. FT-IR (KBr, cmꢀ1): 3323.5,
3161.3 (
NeO); 1482.0 (
1298.8, 1187.6, 1143.4 (
695.2 (
C]S). 1H NMR (300 MHz, DMSO)
n
NeH); 3089.6, 2982.8 (
arCeC); 1407.5 (
arCeH); 1047.1 (
n
arCeH); 1592.0 (
d
NeH); 1527.6
(n
n
d
ar(1,4)CeC); 1362.7 (
n
NeO);
CeCl);
The
fluoromethyl)phenyl]thiourea (1), 1-(2-phenylethyl)-3-[3-(tri-
fluoromethyl)phenyl]thiourea (4) and 1-benzyl-3-[3-
synthesis
of
1-(3-chloro-4-fluorophenyl)-3-[3-(tri-
d
n
C]S); 863.7 (
d
n
d: 10.15 (s, 2H, NH), 8.35
(d, 1H, J ¼ 2.4 Hz, AreH), 7.81 (dd, 1H, J1 ¼ J2 ¼ 2.4 Hz, AreH), 7.70
(d, 1H, J ¼ 9.0 Hz, AreH), 7.47e7.44 (m, 2H, AreH), 7.37 (t, 2H,
J ¼ 7.8 Hz, AreH), 7.18 (t,1H, J ¼ 7.35 Hz, AreH). 13C NMR (75.4 MHz,
(trifluoromethyl)phenyl]thiourea (5) has been described recently
[55]. The method of preparation of derivatives 1-(4-chloro-3-
nitrophenyl)-3-phenylthiourea (6), 1-(4-chloro -3-nitrophenyl)-3-
(2-chlorophenyl)thiourea (7), 1-benzyl-3-(4-chloro-3-nitrophenyl)
thiourea (9) and 1-(4-chloro-3-nitrophenyl)-3-(1-phenylethyl)
thiourea (10) has also been presented [56].
DMSO)
d: 179.95, 147.09, 140.05, 139.08, 131.53, 128.92, 128.62,
125.31,124.20,119.98,119.65. ESI MS: m/z ¼ 330.0 [MþNa]þ (100%).
Anal. calcd for C13H10ClN3O2S: C 50.73, H 3.28, N 13.65, S 10.42;
found: C 50.92, H 3.28, N 13.67, S 10.44.
Crystal data for 6: C13H10Cl N3O2 S, Mw ¼ 307.75, crystal system
(1) Anal. calcd for C14H9ClF4N2S: C 48.22, H 2.60, N 8.03, S 9.19;
found: C C 48.31, H 2.60, N 8.04, S 9.20.
(4) Anal. calcd for C16H15F3N2S: C 59.25, H 4.66, N 8.64, S 9.89;
found: C 59.47, H 4.67, N 8.66, S 9.90.
(5) Anal. calcd for C15H13F3N2S: C 58.05, H 4.22, N 9.03, S 10.33;
found: C 58.26, H 4.21, N 9.04, S 10.35.
(7) Anal. calcd for C13H9Cl2N3O2S: C 45.63, H 2.65, N 12.28, S
9.37; found: C 45.53, H 2.63, N 12.24, S 9.36.
triclinic, space group P-1; unit cell dimensions a ¼ 6.836(2) Å,
b ¼ 8.012(3) Å, c ¼ 12.542(4) Å,
a
¼ 76.88(2)ꢂ,
b
¼ 88.62(2)ꢂ,
g
m
¼ 89.54(2)ꢂ, V ¼ 668.8(4) Å3; Z ¼ 2, Dcalc ¼ 1.528 g/cm3,
¼ 4.041 mmꢀ1, F(000) ¼ 316;
q
range 3.62e73.22ꢂ, reflections
collected/independent/observed 4269/2589/2363; Goodness-of-fit
on F2 1.071, final R indices [I > 2
s
(I)] R1 ¼ 0.0335, wR2 ¼ 0.0864,
residual electron density max/min 0.30/-0.33 e Åꢀ3. CCDC No.
1457556.
(9) Anal. calcd for C14H12ClN3O2S: C 52.26, H 3.76, N 13.06, S
9.96; found: C 52.20, H 3.76, N 13.08, S 9.93.
(10) Anal. calcd for C15H14ClN3O2S: C 53.65, H 4.20, N 12.51, S
9.55; found: C 53.48, H 4.19, N 12.48, S 9.56.
4.1.2.4. N-[(4-chloro-3-nitrophenyl)carbamothioyl]benzamide
(8).
The synthesis of 8 was described previously [57], however without
spectral data presentation.
Yield 72%, cream powder, m.p. 168e170 ꢂC. FT-IR (KBr, cmꢀ1):
3333.7 (
NeH); 1524.4 (
1187.7, 1143.1 (
C]S). 1H NMR (300 MHz, DMSO)
n
NeH); 2998.9 (
NeO); 1483.4 (
arCeH); 1042.5 (
n
arCeH); 1672.8 (
arCeC); 1327.5 (
C]S); 881.5 (
: 12.67 (s, 1H, NH), 11.79 (s, 1H,
n
C]O); 1587.7 (
NeO); 1275.6,
CeCl); 683.4 (n
d
4.1.2.1. 1-(4-Methoxyphenyl)-3-[3-(trifluoromethyl)phenyl]thiourea
(2). Yield 70%, white crystals, m.p. 143e144.5 ꢂC. FT-IR (KBr, cmꢀ1):
n
d
n
n
n
d
d
3185.5 (
C N); 1449.8 (
1160.6, 1115.4 ( arCeH); 1069.4 (
C]S). 1H NMR (300 MHz, DMSO)
d
n
NeH); 3025.7, 3004.9 (
arCeC); 1328.9 (
C]S); 1039.5 (
(ppm): 9.83 (m, 2H, NH),
n
arCeH); 1596.9 (
arCeH); 1279.4 (
CeOeC); 724.6
d
NeH); 1548.2
(
n
n
d
n
CeOeC);
NH), 8.60 (d,1H, J ¼ 2.1 Hz, AreH), 8.00e7.96 (m, 3H, AreH), 7.81 (d,
d
n
n
1H, J ¼ 9.0 Hz, AreH), 7.67 (t, 1H, J ¼ 7.35 Hz, AreH), 7.55 (t, 2H,
(n
J ¼ 7.5 Hz, AreH). 13C NMR (75.4 MHz, DMSO)
d: 179.64, 167.74,
7.96e7.93 (m, 1H, AreH), 7.75 (d, 1H, J ¼ 8.4 Hz, AreH), 7.54 (t, 1H,
J ¼ 7.9 Hz, AreH), 7.44 (d, 1H, J ¼ 7.8 Hz, AreH), 7.35e7.30 (m, 2H,
AreH), 6.95e6.88 (m, 2H, AreH), 3.75 (s, 3H, OCH3). 13C NMR
146.66, 137.75, 132.97, 131.66, 131.31, 129.72, 128.44 (2C), 128.18
(2C),121.58,121.12. ESI MS: m/z ¼ 334.0 [M ‒ H]ꢀ (100%). Anal. calcd
for C14H10ClN3O3S: C 50.08, H 3.00, N 12.51, S 9.55; found: C 49.92,
H 3.00, N 12.47, S 9.56.
(75.4 MHz, DMSO)
d (ppm): 179.98, 156.79, 140.59, 131.67, 129.33,
128.64 (q), 127.26, 126.08 (2C), 124.08 (q), 120.37 (q), 119.76 (q),
113.83 (2C), 55.81. ESI MS: m/z ¼ 349.0 [MþNa]þ (100%). Anal. calcd
for C15H13F3N2OS: C 55.21, H 4.02, N 8.58, S 9.83; found: C 55.15, H
4.03, N 8.59, S 9.82.
4.2. In vitro tests
Crystal data for 2: C15H13F3N2OS, Mw ¼ 326.33; crystal system
orthorhombic, space group Pna21, unit cell dimensions
a ¼ 13.538(3) Å, b ¼ 14.789(2) Å, c ¼ 7.293(1) Å, V ¼ 1460.2(4) Å3,
4.2.1. Binding affinity studies
4.2.1.1. General procedures. The synthesized compounds were
tested for in vitro affinity for serotonin 5-HT2A and 5-HT2C receptors
by radioligand binding assays. All the compounds were dissolved in
5% DMSO. The following specific radioligands and tissue sources
were used: (a) serotonin 5-HT2A receptor, [3H]ketanserin, rat brain
cortex; (b) serotonin 5-HT2C receptor, [3H]mesulergine, rat brain
cortex.
Z ¼ 4; Dcalc ¼ 1.484 g/cm3,
m
¼ 2.321 mmꢀ1, F(000) ¼ 672;
q range
4.43e73.73ꢂ, reflections collected/independent/observed 20306/
2934/, Goodness-of-fit on F2 0.889, final R indices [I > 2
s(I)]
R1 ¼ 0.0285, wR2 ¼ 0.0764, residual electron density max/min
0.25/ꢀ0.23 e Åꢀ3. CCDC No. 1457555.
4.1.2.2. 1-(2-Chlorophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea
(3). Yield 85%, white powder, m.p. 162e163 ꢂC. FT-IR (KBr, cmꢀ1):
Non-specific binding was determined as described below, and
specific binding as the difference between total and non-specific
binding. Blank experiments were carried out to determine the ef-
fect of 5% DMSO on the binding and no effects were observed.
Competition experiments were analyzed by PRISM 5 (Graph-
PadPrism®, 1992e2007, GraphPad Software, Inc., La Jolla, CA, USA)
to obtain the concentration of unlabeled drug that caused 50% in-
hibition of ligand binding (IC50), with six concentrations of test
compounds, each performed in triplicate. The IC50 values obtained
were used to calculate apparent inhibition constants (Ki) by the
method of Cheng and Prussoff [58], from the following equation:
Ki ¼ IC50/(1 þ S/KD) where S represents the concentration of the hot
ligand used and KD its receptor dissociation constant (KD values,
obtained by Scatchard analysis [59], were calculated for each
labeled ligand).
3296.1, 3189.6 (
1546.3 ( C N); 1509.9, 1450.1 (
arCeH); 1069.3 ( C]S); 870.1 (
(300 MHz, DMSO) (ppm): 10.17 (s, 1H, NH), 9.69 (s, 1H, NH), 8.02
n
NeH); 3030.8, 3004.7 (
arCeC); 1334.5, 1167.6, 1125.3 (
CeCl); 721.6 (
C]S). 1H NMR
n arCeH); 1595.2 (d NeH);
n
n
d
n
d
n
d
(m, 1H, AreH), 7.80e7.77 (m, 1H, AreH), 7.60e7.46 (m, 4H, AreH),
7.39e7.34 (m, 1H, AreH), 7.32e7.26 (m, 1H, AreH). 13C NMR
(75.4 MHz, DMSO)
d (ppm): 179.69, 140.19, 132.58, 130.06, 129.63,
129.33 (q), 128.41, 126.98, 125.19, 124.22, 123.09 (q), 122.01, 120.79
(q), 119.84 (q). ESI MS: m/z ¼ 353.2 [MþNa]þ (100%). Anal. calcd for
C
14H10ClF3N2S: C 50.84, H 3.05, N 8.47, S 9.69; found: C 50.89, H
3.04, N 8.49, S 9.66.
4.1.2.3. 1-(4-Chloro-3-nitrophenyl)-3-phenylthiourea (6). Yield 56%,

184
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
4.2.2. 5-HT2A and 5-HT2C binding assays
1000 lowest energy conformers obtained during docking proce-
dure. The central structure of the largest observed cluster was
selected as the final ligand binding conformation. Visual inspection
and analysis of resulting complexes and ligand-receptor in-
teractions were performed using VMD program [67].
Radioligand binding assays were performed as previously re-
ported by Herndon et al. [60]. Briefly, frontal cortical regions of
male SpragueeDawley rats (180e220 g) were dissected on ice and
homogenized (1:10 w/v) in ice-cold buffer solution (50 mM Tris
HCl, 0.5 mM EDTA, and 10 mM MgCl2 at pH 7.4) with a Polytron
PT10 (setting 5 for 15 s) and centrifuged at 3000 g for 15 min. The
pellet was resuspended in buffer (1:30 w/v), incubated at 37 ꢂC for
15 min and then centrifuged twice more at 3000 g for 10 min (with
resuspension between centrifugations). The final pellet was
resuspended in buffer that also contained 0.1% ascorbate and
10ꢀ5 M pargyline.
4.4. In vivo tests
The experiments were carried out on male Albino Swiss mice
(18e30 g). The animals were kept 8e10 to a cage under standard
laboratory conditions (at a temperature of 20
1
ꢂC and a 12 h
light/dark cycle) with free access to food (LSM, Motycz, Poland) and
water. All experiments were performed between 9:00 a.m. and
4:00 p.m., 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 (51/2013 i 73/2015).
The investigated substances in all tests were administered
intraperitoneally (i.p.), as a suspensions in aqueous solution of 0.5%
methylcellulose (tylose) and were injected 60 min before the tests.
All substances were administered in a volume of 10 mL/kg. The
control animals received an equivalent volume of the solvent at the
respective time before the test. All tests performed, suggested by
Vogel and Vogel [47], are generally accepted as basic in investiga-
tion of the central activity by behavioral methods. The acute
toxicity of the compound was assessed in mice acc. to Litchfield and
Wilcoxon method [68] as the ED50 calculated as “the loss of righting
reflex” within 48 h: after an i.p. administration of tested com-
pounds, an observation of mice was carried out in the following
time intervals: in 15-, 30-, 60-, 180-min and 24, 48 h. Each com-
pound in behavioral experiments was injected in doses equivalent
to 0.1, 0.05, 0.025, 0.0125 ED50. In addition, the activity of com-
pounds was assessed in the following tests:
Assays were performed in triplicate in a 2.0 mL volume con-
taining 5 mg wet weight of tissue and 0.4 nM [3H]ketanserin hy-
drochloride (47.3 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA,
USA) for 5-HT2A receptor assays, and 10 mg wet weight of tissue
and 1 nM [3H]mesulergine (83.1 Ci/mmol; Perkin Elmer Life Sci-
ences, Boston, MA, USA) for 5-HT2C receptor assays. Cinanserin
(1.0
mM) was used to define nonspecific binding in the 5-HT2A assay.
In the 5-HT2C assays, mianserin (1.0
mM) 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 ꢂC, filtered on Schliecher and Schuell (Keene, NH, USA)
glass fiber filters presoaked in polyethylene imine, and washed
with 10 mL of ice-cold buffer. Filters were counted at an efficiency
of 50%.
4.3. Molecular docking studies
The sequences of human 5-HT2A receptor (UniProt entry:
P28223) and the sequence of beta2-adrenergic receptor (b2AR)
were aligned using ClustalW program [61] and manually adjusted
in order to obtain correct position of corresponding trans-
membrane regions (see Supplementary material, Fig. S2, Supple-
mentary Materials). Molecular model of 5-HT2A receptor was con-
structed applying MODELLER program [62] using (PDB ID: 2RH1) as
a template. The protein fragments including: N-terminal domain
(residues Met1 to His70), second intracellular loop (residues Val269
to Thr311) and C terminal domain (residues Gln398 to Val471) have
been omitted during structure prediction, because they have no
impact on predicted receptor binding site. In the next step, mo-
lecular dynamics simulations (MD) were performed to optimize the
geometry and packing of amino acids side chains. The receptor
model was inserted into POPC membrane and solvated with water
molecules. The simulation box consisted of 115 POPC lipids, 11424
water molecules and 5 Cl- ions. During the MD simulation lasting
10 ns the weak position restraints were applied on protein back-
bone atoms in order to preserve its initial conformation. The
modified version of GROMOS force field [63] (parameter set 53A6)
was used and simulation time step was equal to 2 fs The final step of
model refinement included energy minimization using 2000 steps
of steepest descent algorithm. All simulation were performed using
GROMACS v. 4.6.5 program [64].
Molecular structures for 10 analyzed compounds were con-
structed using Automated Topology Builder server (ATB Version
2.2) [65]. Ligand docking to receptor model and analysis of resulting
ligand-receptor binding modes were conducted using AutoDock4
(v. 4.2) and AutoDockTools4 [66]. For each of 10 ligands 1000 in-
dependent docking calculations were performed using Lamarckian
Genetic Algorithm (LGA), (see Supplementary materials for detailed
description of docking parameters, Fig. S3, Supplementary Mate-
rials). Ligand molecules were fully flexible during molecular dock-
ing procedure whereas receptor remained rigid. For each of 10
compounds the most favorable binding mode was identified by
means of structural clustering (with RMSD 2 Å cutoff) of the set of
- locomotor activity was measured for single mice in photo-
resistor actometers (circular cages, diameter 25 cm, two light
beams, Multiserv, Poland; the number of crossed light beams by
the mice was recorded) for 30 min as:
a) spontaneous activity
b) amphetamine-induced hyperactivity: mice received sub-
cutaneously (s.c.) 5 mg/kg of amphetamine 30 min before the
test;
- nociceptive reactions were studied in the acetic acid (0.6%)-
induced ‘writhing’ test [26,69] The number of writhing episodes
was measured for 10 min starting 5 min after i.p. administration
of acid solution;
- motor coordination was evaluated in rota-rod test [70]; and
chimney test [71].
-
body temperaturdarmanie in normothermic mice was
measured in the rectum by thermistor thermometer during a
total period of 180 min (60 min before and 120 min after tested
compound injection). The mean value from the first two mea-
surements (60 and 30 min before drug administration) was
assumed as initial temperature (ti). The final temperature (tf)
was measured 30, 60, 90 and 120 min after the injection of
tested compounds at a dose of 0.1 ED50. Body temperature
changes (Dt) were calculated according to the formula:
Dt ¼ tf ꢀ ti
- pentylenetetrazole (110 mg/kg, s.c.)-induced convulsions were
evaluated as the number of mice with clonic seizures, tonic
convulsions and dead animals;

A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
Res. 39 (2014) 2008e2017.
185
- HTR after 5-hydroxytryptophan (5-HTP), acc. to Corne et al.
[17] C.E. Canal, R.G. Booth, D. Morgan, Neuropharmacology 70 (2013) 112e121.
[72]. Mice received 5-HTP (230 mg/kg, i.p.) and the number of
HTR was recorded in 6 2-min intervals (4e6, 14e16, 24e26,
34e36, 44e46, 54e56 min).
- head twitches induced by (( )-DOI (2.5 mg/kg). Immediately
after treatment, the head twitches were counted throughout
20 min [73]. The investigated compounds were administered
60 min before (( )-DOI.
[18] C.E. Canal, D. Morgan, Drug Test. Anal. 4 (2012) 556e576.
[19] C.E. Canal, U.B. Olaghere da Silva, P.J. Gresch, E.E. Watt, E. Sanders-Bush,
D.C. Airey, Psychopharmacol. Berl. 209 (2010) 163e174.
[20] N.A. Darmani, Pharmacol. Biochem. Behav. 68 (2001) 311e317.
[21] R.L. Hayslett, Y. Tizabi, Pharmacol. Biochem. Behav. 76 (2003) 409e415.
[22] B.T. Purwanto, World J. Pharm. Sci. 3 (2015) 58e61.
ꢀ
[23] E. Szacon, M. Rza˛dkowska, A.A. Kaczor, E. Ke˛dzierska, S. Fidecka, D. Matosiuk,
Molecules 20 (2015) 3821e3840.
ꢂ
€
ꢂ
ꢂ
[24] B.K. Kaymakçioglu, S. Rollas, E. Korcegez, F. Aricioglu, Eur. J. Pharm. Sci. 26
(2005) 97e103.
[25] J. Stefanska, D. Szulczyk, A.E. Koziol, B. Miroslaw, E. Kedzierska, S. Fidecka,
B. Busonera, G. Sanna, G. Giliberti, P. La Colla, M. Struga, Eur. J. Med. Chem. 55
(2012) 205e213.
4.5. Statistics
[26] A. Bielenica, E. Kedzierska, S. Fidecka, H. Maluszynska, B. Miroslaw, A.E. Koziol,
J. Stefanska, S. Madeddu, G. Giliberti, G. Sanna, M. Struga, Lett. Drug Discov.
Dev. 12 (2015) 263e276.
[27] S. Karakus, B. Koçyigit-Kaymakcioglu, H.Z. Toklu, F. Aricioglu, S. Rollas, Arch.
Pharm. Weinh. 342 (2009) 48e53.
[28] H.L. Sun, J.W. Zheng, K. Wang, R.K. Liu, J.H. Liang, Life Sci. 72 (2003)
1221e1230.
[29] R.B. Westkaemper, R.A. Glennon, Curr. Top. Med. Chem. 2 (2002) 575e598.
[30] D.A. Shapiro, K. Kristiansen, W.K. Kroeze, B.L. Roth, Mol. Pharmacol. 258
(2000) 877e886.
[31] B.J. Ebersole, I. Visiers, H. Weinstein, S.C. Sealfon, Mol. Pharmacol. 63 (2003)
36e43.
[32] M. Struga, J. Kossakowski, A.E. Koziol, E. Kedzierska, S. Fidecka, P. La Colla,
Ch Ibba, G. Collu, G. Sanna, B. Secci, R. Loddo, Eur. J. Med. Chem. 44 (2009)
4960e4969.
Obtained data were calculated by Fisher exact test (PTZ-induced
seizures), two-way analysis of variance (ANOVA) followed by
Bonferroni post hoc test (body temperature) and one-way ANOVA
followed by Dunnett's post hoc test (other tests). Each group of
animals consisted of 8e12 mice. All results are presented in the
figures as mean SEM. A level of p < 0.05 was considered as sta-
tistically significant. All the figures were prepared by the GraphPad
Prism version 5.00 for Windows, GraphPad Software (San Diego,
California, USA), www.graphpad.com.
ꢂ
ꢂ
Aknowledgments
[33] M.A. Parker, D.M. Kurrasch, D.E. Nichols, Bioorg. Med. Chem. 16 (2008)
4661e4669.
The work was supported by Polish Ministry of Science and
Higher Education grant ‘Iuventus plus’ no. IP2012 016372. Research
subject was carried out with the use of CePT infrastructure financed
by the European Union e the European Regional Development
Fund within the Operational Programme “Innovative economy for
2007e2013”.
The research was also carried out with the facilities purchased
thanks to the financial support of the European Regional Devel-
opment Fund in the framework of the Operational Program
Development of Eastern Poland 2007e2013 (Contract No.
POPW.01.03.00-06-009/11-00, Equipping the laboratories of the
Faculties of Biology and Biotechnology, Mathematics, Physics and
Informatics, and Chemistry for studies of biologically active sub-
stances and environmental samples).
[34] C. Wang, Y. Jiang, J. Ma, H. Wu, D. Wacker, V. Katritch, G.W. Han, W. Liu,
X.P. Huang, E. Vardy, et al., Science 340 (2013) 610e614.
[35] D. Wacker, C. Wang, V. Katritch, G.W. Han, X.P. Huang, E. Vardy, J.D. McCorvy,
Y. Jiang, M. Chu, F.Y. Siu, et al., Science 340 (2013) 615e619.
[36] A. Gandhimathi, R. Sowdhamini, J. Biomol. Struct. Dyn. (2015) 1e19.
[37] S. Yuan, K. Palczewski, Q. Peng, M. Kolinski, H. Vogel, S. Filipek, Angew. Chem.
Int. Ed. Engl. 54 (2015) 7560e7563.
[38] A. Bielenica, A.E. Koziol, M. Struga, Mini Rev. Med. Chem. 13 (2013) 933e951.
ꢀ
[39] J.L. Mokrosz, L. Strekowski, B. Duszynska, D.B. Harden, M.J. Mokrosz,
A.J. Bojarski, Pharmazie 49 (1994) 801e806.
[40] K. Stierand, P.C. Maass, M. Rarey, Bioinformatics 22 (2006) 1710e1716.
ꢀ
[41] J.L. Mokrosz, L. Strekowski, W.X. Kozak, B. Duszynska, A.J. Bojarski,
ꢀ
ꢀ
A. Kłodzinska, A. Czarny, M.T. Cegła, A. Deren-Wesołek, E. Chojnacka-Wojcik,
et al., Arch. Pharm. Weinh. 328 (1995) 659e666.
[42] F.C. Colpaert, P.A. Janssen, Neuropharmacology 22 (1983) 993e1000.
[43] Y. Morishima, T. Shibano, Pharmacol. Biochem. Behav. 52 (1995) 755e758.
[44] S.L. Handley, J. Brown, Neuropharmacology 21 (1982) 507e510.
[45] G.M. Miller, J. Neurochem. 116 (2011) 164e176.
[46] D. Le Bars, M. Gozariu, S. Cadden, Pharmacol. Rev. 53 (2001) 597e652.
[47] G.H. Vogel, W.H. Vogel, Drug discovery and evaluation, in: Pharmacological
Assays, Springer-Verlag, Berlin, Germany, 1997, pp. 204e207.
[48] S.N. Pandeya, V. Mishra, P.N. Singh, D.C. Rupainwar, Pharmacol. Res. 37 (1998)
17e22.
[49] K.E. Cooper, W.I. Cranston, A.J. Honour, J. Physiol. 181 (1965) 852e864.
[50] W. Feldberg, R.D. Myers, Nature 200 (1963) 1325e1328.
[51] F.-S. Chueh, C.-P. Chang, C.-C. Chio, M.-T. Lin, J. Pharmacol. Sci. 96 (2004)
420e427.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.073.
References
[52] S. Hjorth, J. Neural Transm. Gen. Sect. 61 (1985) 131e135.
[53] CrysAlisPro, Agilent Technologies, Version 1.171.37.34, 2014.
[54] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112e122.
[1] S.L. Kitson, Curr. Pharm. Des. 13 (2007) 2621e2637.
[2] D. Hoyer, J.P. Hannon, G.R. Martin, Pharmacol. Biochem. Behav. 71 (2002)
533e554.
[3] P.J. Pauwels, 5-HT Receptors and Their Ligands. Toric Reviews No.25, Toric
Cookson, Bristol, UK, 2003, pp. 1e12.
[4] D.E. Nichols, C.D. Nichols, Chem. Rev. 108 (2008) 1614e1641.
[5] T. Klabunde, A. Evers, Chembiochem 6 (2005) 876e889.
[6] B.J. Jones, T.P. Blackburn, Pharmacol. Biochem. Behav. 71 (2002) 555e568.
[7] H. Kristiansen, B. Elfving, P. Plenge, L.H. Pinborg, N. Gillings, G.M. Knudsen,
Synapse 58 (2005) 249e257.
ꢀ
ꢀ
ꢀ
[55] A. Bielenica, J. Stefanska, K. Ste˛pien, A. Napiorkowska, E. Augustynowicz-
ꢀ
Kopec, G. Sanna, S. Madeddu, S. Boi, G. Giliberti, M. Wrzosek, M. Struga, Eur. J.
Med. Chem. 101 (2015) 111e125.
ꢀ
ꢀ
ꢀ
[56] A. Bielenica, K. Ste˛pien, A. Napiorkowska, E. Augustynowicz-Kopec,
S. Krukowski, M. Włodarczyk, M. Struga, Chem. Biol. Drug Des. (2016), http://
dx.doi.org/10.1111/cbdd.12723 (in press).
[57] A.K. Saxena, S.K. Pandey, S. Seth, C. Dikshit, Bioorg. Med. Chem. 9 (2001)
2025e2034.
[58] Y.C. Cheng, W.H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099e3108.
[59] G. Scatchard, The attraction of proteins for small molecules and ions, Ann. N.Y.
Acad. Sci. 51 (1949) 660e672.
[60] J.L. Herndon, A. Ismaiel, S.P. Ingher, M. Teitler, R.A. Glennon, J. Med. Chem. 35
(1992) 4903e4910.
[8] D. Van Oekelen, W.H. Luyten, J.E. Leysen, Life Sci. 72 (2003) 2429e2449.
[9] M.E. Silva, R. Heim, A. Strasser, S. Elz, S. Dove, J. Comput. Aided Mol. Des. 25
(2011) 51e66.
ꢀ
[10] J. Brea, J. Rodrigo, A. Carrieri, F. Sanz, M.I. Cadavid, M.J. Enguix, M. Villazon,
~
G. Mengod, Y. Caro, C.F. Masaguer, E. Ravina, N.B. Centeno, A. Carotti, M.I. Loza,
[61] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan,
H. McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson,
T.J. Gibson, D.G. Higgins, Bioinformatics 23 (2007) 2947e2948.
[62] B. Webb, A. Sali, Curr. Protoc. Bioinforma. 47 (2014) 5.6.1e5.6.32.
[63] C. Oostenbrink, A. Villa, A.E. Mark, W.F. van Gunsteren, J. Comput. Chem. 25
(2004) 1656e1676.
[64] B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J. Chem. Theory Comput. 4
(2008) 435e447.
[65] K.B. Koziara, M. Stroet, A.K. Malde, A.E. Mark, J. Comput. Aided Mol. Des. 28
(2014) 221e233.
J. Med. Chem. 45 (2002) 54e71.
[11] T. Friedman, Expert Opin. Pharmacother. 14 (2013) 1970e1975.
[12] C.T. Egan, K. Herrick-Davis, K. Miller, R.A. Glennon, M. Teitler, Psycho-
pharmacol. Berl. 136 (1998) 409e414.
[13] A.J. Bojarski, Curr. Top. Med. Chem. 6 (2006) 2005e2026.
[14] A. Abbas, B.L. Roth, Expert Opin. Pharmacother. 9 (2008) 3252e3259.
[15] L.R. Gardell, K.E. Vanover, L. Pounds, R.W. Johnson, R. Barido, G.T. Anderson,
I. Veinbergs, A. Dyssegaard, P. Brunmark, A. Tabatabaei, R.E. Davis, M.R. Brann,
U. Hacksell, D.W. Bonhaus, J. Pharmacol. Exp. Ther. 322 (2007) 862e870.
[16] U. Hacksell, E.S. Burstein, K. McFarland, R.G. Mills, H. Williams, Neurochem.

186
A. Bielenica et al. / European Journal of Medicinal Chemistry 116 (2016) 173e186
[66] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, J. Comput. Chem. 30 (2009) 2785e2791.
[67] W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph 14 (1996), 33e38, 27e38.
[68] L.T. Litchfield, F. Wilcoxon, J. Pharm. Exp. Ther. 96 (1949) 99e113.
[69] R. Koster, M. Anderson, E.J. de Beer, Fed. Proc. 18 (1959) 412.
[70] F. Gross, J. Tripod, R. Meir, Schweiz Med. Wochschr. 85 (1955) 305e309.
[71] J.R. Boissier, J. Tardy, J.C. Diverres, Med. Exp. Basel 3 (1960) 81e84.
[72] S.J. Corne, R.W. Pickering, Psychopharmacologia 11 (1967) 65e78.
[73] N.A. Darmani, B.R. Martin, U. Pandey, R.A. Glennon, Pharmacol. Biochem.
Behav. 36 (1990) 901e906.