Computer-aided studies for novel arylhydantoin 1,3,5-triazine derivatives as 5-HT6 serotonin receptor ligands with antidepressive-like, anxiolytic and antiobesity action in vivo

Article abstract of DOI:10.3390/molecules23102529

This study focuses on the design, synthesis, biological evaluation, and computer-aided structure-activity relationship (SAR) analysis for a novel group of aromatic triazine-methylpiperazines,with an hydantoin spacer between 1,3,5-traizine and the aromatic fragment. New compounds were synthesized and their affinities for serotonin 5-HT₆, 5-HT_1A, 5-HT_2A, 5-HT₇, and dopamineD2 receptorswere evaluated. The induced-fit docking (IFD) procedure was performed to explore the 5-HT₆ receptor conformation space employing two lead structures. It resulted in a consistent binding mode with the activity data. For the most active compounds found in each modification line, anti-obesity and anti-depressive-like activity in vivo, as well as "druglikeness" in vitro, were examined. Two 2-naphthyl compounds (18 and 26) were identified as themost active 5-HT₆R agents within each leadmodification line, respectively. The 5-(2-naphthyl)hydantoin derivative 26, themost active one in the series (5-HT₆R: K_i = 87 nM), displayed also significant selectivity towards competitive G-protein coupled receptors (6-197-fold). Docking studies indicated that the hydantoin ring is stabilized by hydrogen bonding, but due to its different orientation, the hydrogen bonds formwith S5.44 and N6.55 or Q6.58 for 18 and 26, respectively. Compound 26 exerted anxiolytic-like and antidepressant-like activities. Importantly, it demonstrated anti-obesity properties in animals fed palatable feed, and did not show toxic effects in vitro.

Full text of DOI:10.3390/molecules23102529

molecules
Article
Computer-Aided Studies for Novel Arylhydantoin
1,3,5-Triazine Derivatives as 5-HT6 Serotonin Receptor
Ligands with Antidepressive-Like, Anxiolytic and
Antiobesity Action In Vivo
2
4
Rafał Kurczab 1,†, Wesam Ali 2,3,†, Dorota Łazewska , Magdalena Kotan´ska ,
˙
Magdalena Jastrze˛bska-Wie˛sek 5, Grzegorz Satała 1, Małgorzata Wie˛cek 2, Annamaria Lubelska 2,
Gniewomir Latacz 2, Anna Partyka 5, Małgorzata Starek 6, Monika Da˛browska 6, Anna Wesołowska 5,
Claus Jacob 3, Katarzyna Kiec´-Kononowicz 2 and Jadwiga Handzlik 2,*
1
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna 12,
PL 31-343 Cracow, Poland; kurczab@if-pan.krakow.pl (R.K.); satala@if-pan.krakow.pl (G.S.)
Department of Technology and Biotechnology of Drugs, Jagiellonian University, Medical College, Medyczna 9,
2
PL 30-688 Cracow, Poland; s8wealii@stud.uni-saarland.de (W.A.); dlazewska@cm-uj.krakow.pl (D.Ł.);
mwiecek@cm-uj.krakow.pl (M.W.); annamaria.lubelska@doctoral.uj.edu.pl (A.L.);
glatacz@cm-uj.krakow.pl (G.L.); mfkonono@cyf-kr.edu.pl (K.K.-K.)
3
Division of Bioorganic Chemistry, School of Pharmacy, University of Saarland, Campus B2 1,
D-66123 Saarbruecken, D-66123 Saarland, Germany; c.jacob@mx.uni-saarland.de
Department of Pharmacodynamics, Jagiellonian University, Medical College, Medyczna 9,
4
PL 30-688 Cracow, Poland; magda.dudek@uj.edu.pl
Department of Clinical Pharmacy, Jagiellonian University, Medical College, Medyczna 9,
5
PL 30-688 Cracow, Poland; mj.wiesek@gmail.com (M.J.-W.); annairena.partyka@uj.edu.pl (A.P.);
awesolowska@cm-uj.krakow.pl (A.W.)
Department of Inorganic Chemistry, Jagiellonian University, Medical College, Medyczna 9,
6
PL 30-688 Cracow, Poland; m.starek@uj.edu.pl (M.S.); monika.1.dabrowska@uj.edu.pl (M.D.)
*
†
Correspondence: j.handzlik@uj.edu.pl; Tel.: +48-12-620-55-84
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 September 2018; Accepted: 24 September 2018; Published: 3 October 2018
Abstract: This study focuses on the design, synthesis, biological evaluation, and computer-aided
structure-activity relationship (SAR) analysis for a novel group of aromatic triazine-methylpiperazines, with
an hydantoin spacer between 1,3,5-traizine and the aromatic fragment. New compounds were synthesized
and their affinities for serotonin 5-HT6, 5-HT1A, 5-HT2A, 5-HT7, and dopamine D2 receptors were evaluated.
The induced-fit docking (IFD) procedure was performed to explore the 5-HT6 receptor conformation
space employing two lead structures. It resulted in a consistent binding mode with the activity data.
For the most active compounds found in each modification line, anti-obesity and anti-depressive-like
activity in vivo, as well as “druglikeness” in vitro, were examined. Two 2-naphthyl compounds (18
and 26) were identified as the most active 5-HT6R agents within each lead modification line, respectively.
The 5-(2-naphthyl)hydantoin derivative 26, the most active one in the series (5-HT6R: Ki = 87 nM), displayed
also significant selectivity towards competitive G-protein coupled receptors (6–197-fold). Docking studies
indicated that the hydantoin ring is stabilized by hydrogen bonding, but due to its different orientation,
the hydrogen bonds form with S5.44 and N6.55 or Q6.58 for 18 and 26, respectively. Compound 26 exerted
anxiolytic-like and antidepressant-like activities. Importantly, it demonstrated anti-obesity properties in
animals fed palatable feed, and did not show toxic effects in vitro.
Keywords: serotonin receptors; 5-HT6 ligands; 1,3,5-triazine; hydantoin; docking; obesity;
antidepressive; ADMET in vitro
Molecules 2018, 23, 2529; doi:10.3390/molecules23102529
www.mdpi.com/journal/molecules

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1. Introduction
The serotonin receptor 5-HT6 is one of the recently discovered members of the 5-HTRs family [
It is quite unique, since it includes a short third cytoplasmatic loop in parallel, with a long C-terminal
tail, and one introne located in the middle of the third cytoplasmatic loop [ ]. The localization
1].
2,3
of 5-HT6R is limited to the central nervous system, especially as it is located in the brain areas
involved in learning and memory processes. Relatively limited, but intensive, research efforts in this
area led to several families of potent 5-HT6R ligands [
treatment in the cognitive dysfunction associated with Alzheimer’s disease, anxiety or depression [
Over 20 compounds have qualified for clinical trials, most of which in studies following a cure for
Alzheimer’s disease [ ]. Nonetheless, none has reached the pharmaceutical market yet. Recent lines
4
,5], which have been proposed for potential
5–7
].
5
of evidence also point towards promising properties of some 5-HT6R ligands in the battle against
obesity [8,9]. Therefore, the search for new ligands for the 5-HT6R, in particular, those with an action
directed against obesity, seems to be a challenge for current medicinal chemistry.
Lines of evidence have identified several groups of chemical compounds, which displayed
significant affinities for the serotonin receptors 5-HT6 [4,10,11]. The structure-activity relationship
(SAR) analysis performed for the 5-HT6 ligands obtained previously enabled to identify
pharmacophore features, including a triangle topology with tops of: A bulky hydrophobic area
(HYD), positive ionizable nitrogen (PI), and hydrogen bond acceptor (HBA), as well as a central
aromatic fragment (AR) (Figure 1) [12].
Figure 1. Modified pharmacophore model based of the 1,3,5-triazine with mapped compound 2,
including four key pharmacophore features, namely: A positive ionizable group (PI, red), an aromatic
ring (AR, orange), a hydrogen bond acceptor group (HBA, green) and hydrophobic group (HYD, blue)
(
A
). The lead compound
2 mapped onto the binding site of 5-HT6R (B). Examples of the most active
1,3,5-traizne 5-HT6 ligands found recently 1–3 (C).
Taking into consideration the pharmacophore features, our previous studies have successfully
found a new group of potent 5-HT6R agents among methylpiperazine derivatives of benzyl
2-amino-1,3,5-triazines which fit 3 out of 4 pharmacophore features. Two closed HBAs were the missing
feature [13]. The computer-aided SAR analysis allowed us to modify the previous pharmacophore
features and it has underlined the notable importance of aromatic substituents separated by a short
linker from the 1,3,5-triazine. Thus, a bulky aromatic moiety, represented by the naphthyl (1) or

Molecules 2018, 23, 2529
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m-chloro- (
2) or m-methyl- (3) substituted phenyl ring (Figure 1) was beneficial for the kind of 5-HT6R
affinity evaluated experimentally [13].
Taking into account both, the need to search for the 5-HT6R agents with new pharmacological
possibilities and the results of previous SAR studies, we decided to explore a novel group of
triazine-methylpiperazine derivatives (Figure 2), in which the spacer between 1,3,5-traizine and
aromatic fragment is represented by a differently positioned hydantoin system containing both,
two closed HBAs and HYD/AR features. In the first step, five pilot compounds (
and evaluated with regard to their affinity for the 5-HT6R. These initial studies resulted in two parallel
lead structures and (Figure 2). Chemical modifications of the lead structures subsequently provided
a series of compounds 27 (Table 1). Therefore, these studies are focused on the design, synthesis and
biological evaluation, including affinity and selectivity for the 5-HT6R, as well as computer-aided SAR
discussion for an entire series of hydantoin-triazines ( 27). For representative compounds, anti-obesity
4–8) were synthesized
5
7
9–
4
–
and anti-depressive activity in vivo, as well as their “drugability” properties, were also examined.
2. Results
2.1. Identification of Lead Structures
In the first step, a nonaromatic 5,5-dimethylhydantoin 1,3,5-triazine derivative
4 was found as a
moderate 5-HT6R ligand after the radioligand binding assay and was therefore selected as an initial
lead structure (Figure 2). Then, four different hydantoin moieties were taken into consideration as lead
modifications, including: two N-substituted p-chlorobenzyl derivatives
3-N ( ) and 1-N ( ), the 5-(4-chlorophenyl)-5-methyl derivative and 5,5-bis(4-chlorophenyl)hydantoin
8 (Figure 2).
5 and 6, with respect to position
5
6
7
Figure 2. The rationale for the design of novel lead structures of the 5-HT6R ligand among hydantoin
1,3,5-triazine derivatives.

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Table 1. Structures and affinities for serotonin/dopamine receptors of hydantoin-triazine compounds 4–26.
Ki (nM) a
5-HT1A [3H]-8-OH-DPAT
Cpd
Gr
R
5-HT6 [3H]-LSD
D2 [3H]-Raclopride
5-HT2A [3H]-Ketanserin
5-HT7 [3H]-5-CT
4
5
6
7
8
B
-
A
B
-
Methyl
-
4-Chlorophenyl
4-Chlorophenyl
-
427
4275
127
176
1058
592
271
597
205
663
447
570
692
895
182
726
418
403
457
195
667
121
87
22,330
5007
4098
31,240
6855
8783
4631
2867
681
6557
6577
6677
15,490
5207
888
41,040
16,780
11,550
15,090
4738
23,300
7811
4247
9 b
9.99 × 106
15,430
23,300
28,070
3052
42,630
13,570
4155
nt
nt
nt
nt
nt
1596
328
809
3589
3548
239
1327
2036
817
1907
12,150
7396
11,050
10,770
3433
24,410
8080
17,170
-
35,790
1620
3711
938
594
350
159
279
1116
953
88
571
3587
420
1525
1096
791
1565
10,130
8905
564
9
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
Phenyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Ref.
3-Chlorophenyl
2,5-dichlorophenyl
2,4-dichlorophenyl
4-Fluorophenyl
3-Fluorophenyl
3-Bromophenyl
3-Methoxyphenyl
3-Methylphenyl
2-Naphthyl
7693
48,360
37,090
12,930
23,910
16,990
5718
31,620
4101
26,190
13,980
6028
Phenyl
3-Chlorophenyl
2,5-dichlorophenyl
2,4-dichlorophenyl
2,3,4-trichlorophenyl
4-Methylphenyl
1-Naphthyl
22,320
7146
3367
514
2-Naphthyl
14,160
20 c
7 b
18 d
a Tested experimentally in the radioligand binding assay, binding affinity, Ki, expressed as the average of at least two
b–d
independent experiments; nt—not tested.
clozapine.
Reference ligands for GPCRs investigated, b olanzapine, c buspirone,
d
Compounds
radioligand binding assays. Results displayed an increase of the 5-HT6R affinity in the case
of compounds and , whereas both and were much less active than the initial lead 4.
5–8 were synthesized and their affinity for the 5-HT6R was evaluated in the
6
7
5
8
Interestingly, the structure-activity relationship indicated a crucial role for the position of the
1,3,5-triazine substitution at hydantoin ring. Thus, 1,3,5-triazine linked by a methylene group
at position 3 of hydantoin seems to be profitable (
1 of hydantoin caused a huge decrease in activity (
1-(4-chlorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-hydantoin
), and the 3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(4-chlorophenyl)-5
-methylhydantoin ( ) were selected in parallel as two particularly promising lead structures (Lead
1 and Lead 2, Figure 2). These two leads were subsequently modified within their aromatic area to
6
), whilst that fragment situated at position
5). Taking into account these results, both
(6
7
generate a library of two distinct series of derivatives, i.e., compounds
respectively (Table 1).
9–18 and compounds 19–26,

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2.2. Chemical Synthesis of Compounds 5–26
The final compounds
The synthesis of 5,5-dimethylhydantoin triazine derivative
the syntheses of the 3-benzyl derivative ( ) and the 1-arylmethyl ones (6, 9–
5
–
26 were obtained using four different synthesis pathways (Scheme 1a–d).
was described earlier [14]. As for
18), the commercial
4
5
5,5-dimethylhydantoin (DMH) was a starting material, which was substituted with an appropriate
arylmethyl halide and methyl bromoacetate in a different order, respectively (a and b, Scheme 1).
The reactions were performed as basic two-phase alkylations in acetone in the presence of a catalytic
amount of benzyltriethylammonium chloride (TEBA). In case of 5, an alkylation with 4-chlorobenzyl
at position 3 was the first step (27) [15], followed by the introduction of the methyl acetate fragment at
position 1 (28, Scheme 1a). In contrast, the methyl ester 29a for the synthesis of
29b, i.e., the common first intermediate for the synthesis of 18, were obtained by an alkylation of
5,5-dimethylhydantoin with a suitable alkyl bromoacetate at position 3. The ester 29 was substituted
using commercial arylmethyl halides to give compounds 30 40. Although a variety of purification
methods was applied, including extraction, crystallization with charcoal and chromatography, only
compounds 33 34 and 40 gave precipitates (purity > 90%). Compounds 30 32 and 35 39 were obtained
as glue-like residues with varying contents of the target product (44–81%) and were used for final
cyclization in the crude form. In the case of 5-aryl-5-methylhydantoin derivatives ( 19 26), standard
Bucherer-Bergs cyclic condensation of suitable commercial ketones (41 49) was the first synthetic step
providing 5-aryl-5-methylhydantoins 50 58. Next, esters 59 67 were synthesized via N-alkylation of
50 58 in the hydantoin position 3 with methyl bromoacetate, under similar conditions to those for 29
and purification methods as those for 30 40 (Scheme 1c). Among the esters (59 67), compounds 60
and 66 were obtained in glue forms (purity 76–85%) with the remaining compounds (59 61 65 and
67) yielding white crystals (purity 95–100%). The ester intermediate for compound was obtained
within a 3-step synthesis, starting from the acyloin condensation of commercial 4-chlorobenzaldehyde
68), followed by oxidation to benzyl compound 69. The benzyl intermediate was used to perform
6 and the ethyl ester
9–
–
,
–
–
7
,
–
–
–
–
–
–
–
,
–
8
(
a cyclic condensation with urea providing 5,5-bis(4-chlorophenyl)hydantoin (70), followed by an
alkylation with methyl bromoacetate in position 3 of the hydantoin, in the aforementioned conditions
of two-phase alkylation, to produce ester 71 (Scheme 1d). All ester hydantoin intermediates (28
59 67, and 71) were converted into the 1,3,5-triazine final products ( 26) via cyclic condensation with
1-(imino(4-methylpiperazin-1-yl)methyl)guanidine, which proceeded in sodium methanolate under
the conditions corresponding to those for a series of benzyl-1,3,5-triazines described previously [13 14].
, 30–40,
–
5–
,

Molecules 2018, 23, 2529
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Cl
a)
Cl
O
O
O
O
i
ii
iii
HN
NH
HN
N
N
N
H3CO
5
O
O
O
DMH
27
28
b)
H2N
ii
O
O
N
O
N
i
iii
N
N
N
CH2COOR1
N
N
N
N
HN
N
CH2COOR1
R
R
O
O
O
29
30-40
6, 9-18
29a, 30 R1: CH3
29b, 31-40 R1: C2H5
30, 6 R: 4-Cl-Ph
31, 9 R: Ph
32, 10 R: 3-Cl-Ph
33, 11 R: 2,5-diCl-Ph
34, 12 R: 2,4-diCl-Ph
35, 13 R: 4-F-Ph
36, 14 R: 3-F-Ph
37, 15 R: 3-Br-Ph
38, 16 R: 3-MeO-Ph
39, 17 R: 3-Me-Ph
40, 18 R: 2-Naphthyl
c)
H2N
O
N
N
O
O
iv
O
R
ii
R
iii
R
N
N
HN
N
N
HN
NH
HN
N
R
CH2COOCH3
O
O
O
7, 19-26
41-49
50-58
59-67
22, 45, 54, 63 R: 2,4-diCl-Ph
7, 41, 50, 59 R: 4-Cl-Ph
19, 42, 51, 60 R: Ph
20, 43, 52, 61 R: 3-Cl-Ph
21, 44, 53, 62 R: 2,5-diCl-Ph
23, 46, 55, 64 R: 2,3,4-triCl-Ph
24, 47, 56, 65 R: 4-Me-Ph
25, 48, 57, 66 R: 1-Naphthyl
26, 49, 58, 67 R: 2-Naphthyl
d)
Cl
Cl
Cl
Cl
Cl
O
H
O
v, vi
vii
ii
iii
O
O
HN
O
8
HN
O
N
NH
CH2COOCH3
Cl
O
Cl
69
68
70
71
Scheme 1.
) 1-arylmethylhydantoin derivatives (
) 5,5-diarylhydantoin derivative and triazines (8
Synthesis of compounds
5
–
26 (Table 1);
18); ( ) 5-aryl-5-methylhydantoin derivatives (
). Reagents and conditions: (i) RCH2Cl, K2CO3,
(
a
) 3-arylmethylhydantoin product
5;
(
b
6,
9–
c
7
,
19–26);
(d
TEBA, acetone, 3–5 h reflux; (ii) BrCH2COOMe, K2CO3, TEBA, acetone, 5–7 h reflux; (iii)
1-(imino(4-methylpiperazin-1-yl)methyl)guanidine hydrochloride, MeONa, reflux for 3–5 h, then rt, 10–20 h;
(iv) KCN, (NH4)2CO3, 50% EtOH, 55 ◦C; (v) KCN, EtOH, 1 h reflux; (vi) HNO3 conc., 3 h reflux; (vii)
EtONa, urea, 2 h reflux, HCl.
2.3. Molecular Modeling
Molecular docking was performed to gain an insight into the binding mode of the library of
compounds synthetized (5–26) to the recently developed 5-HT6R homology models [13] built on the β2
adrenergic receptor template and optimized for the structures of Lead 1 and 2. The molecular docking
indicated that newly synthesized compounds, generally, exhibited a very consistent binding mode with
recently reported 5-HT6 ligands [16–18]. An influence of the topology of aromatic substituents and
the position of the 1,3,5-triazine substitution at the hydantoin ring on the binding has been observed
(Figure 2) and was in good agreement with results of the radioligand binding assay (see below).

Molecules 2018, 23, 2529
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2.4. Pharmacology
2.4.1. Radioligand Binding Assay
Compounds
5–26 were evaluated with respect to their affinity and selectivity for the target
5-HT6R in radioligand binding assays based on the human serotonin receptors 5-HT6, 5-HT1A, 5-HT2A
,
5-HT7bR and dopaminergic D2LR, all of whom were expressed stably in HEK-293 cells. Five selective
radioligands were employed (Table 1). Results expressed as Ki indicate that all derivatives of both leads
displayed submicromolar affinities for the target 5-HT6R. More active agents (6, 7, 18, 23, 25 and 26)
exerted Ki(5-HT6R) values lower than 200 nM. Compound 26 was the most active one (Ki = 87 nM) and
also highly selective over 5-HT1AR, 5-HT2AR, and D2LR, with some selectivity over 5-HT7R (6-fold).
Furthermore, a slightly less active 5-HT6R agent, compound 25, was highly selective with respect to
all competitive GPCRs tested (Table 1). Most of the compounds displayed much weaker affinities for
5-HT1A, 5-HT2A and D2LR receptors than for 5-HT6R. Notably, compounds 8–11, 14, 17 and 24 were
more potent for 5-HT7R than for the target 5-HT6R, in particular, compound 14 (5-HT7R; Ki = 88 nM),
which was also selective toward 5-HT7R (Table 1).
2.4.2. Behavioral Tests In Vivo
The most potent 5-HT6R agent identified in the radioligand binding assays, i.e., compound 26
,
was selected for behavioral assays to determine its antidepressant- and anxiolytic-like properties
in vivo in male Wistar rats.
Antidepressant-Like Activity of Compound 26
In the forced swim test (FST), compound 26 significantly decreased immobility time about 20%
(ANOVA: F(3,28) = 3.7674, p < 0.05) vs. vehicle treated group. A U-shaped dose-response in the FST
test for compound 26 was observed (Figure 3).
Figure 3. Influence of compound 26 on the immobility time of rats in the forced swim test.
Compound 26 was administered i.p. 60 min before the test. The animals were observed for 5 min.
The data are presented as the mean
ANOVA followed by Bonferroni’s post-hoc test, * p < 0.05 vs. vehicle group.
±
SEM of 6–8 rats. The data were statistically evaluated by one-way

Molecules 2018, 23, 2529
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This effect seems to be typical for some antidepressants with different mechanisms of
action also producing U-shaped dose-response curves in some animal models and evaluated for
antidepressant-like activity [19
(i.e., SB258585) [21].
,20]. Moreover, this effect was also observed for other 5-HT6 ligands
Anxiolytic-Like Activity of Compound 26
Anxiolytic-like activity was assessed using a Vogel conflict drinking test. As demonstrated in
Figure 4, compound 26, administered at a dose of just 3 mg/kg, has significantly increased (by 76%) the
number of accepted shocks (ANOVA: F(3,22) = 3.4967, p < 0.05) during the 5 min experimental session.
Thus, compound 26 (at a dose of 3 mg/kg) exerted anxiolytic-like activity in this behavioral test.
Figure 4. Compound 26 increases the number of shocks accepted in the Vogel conflict test in rats.
Compound 26 was administered i.p. 60 min before the test. The animals were monitored for 5 min.
The data are presented as the mean
ANOVA followed by Bonferroni’s post-hoc test, * p < 0.05 vs. vehicle group.
±
SEM of 6–8 rats. The data were statistically evaluated by one-way
2.4.3. Metabolic Assays In Vivo
Compounds
B (26), were evaluated with regard to their influence on the body weight of male Wistar rats within
metabolic assays in vivo (Figure 5). Animals that were fed palatable feed, and treated with , exhibited
6 and 26, representing the most potent 5-HT6R agents of both groups, A (6) and
6
significantly less weight gain when compared to animals in the control group consuming a standard
feed. From day 16 of the experiment, there was a significant difference between the groups. The body
weight of rats treated with
6 having access to preferential feed differed significantly from the weight of
control animals fed standard feed. Results are shown in Figure 3A,B.
Similarly, animals fed palatable feed and treated with 26 showed significantly less weight gain
than animals in the control group consuming a preferential feed (Figure 5C,D), but there was a
significant difference between the groups from day 12 of the experiment,. Additionally, the body
weight of rats treated with 26 and having access to preferential feed did not differ significantly from
the weight of control animals fed standard feed. No effects on body weight were noted in animals
treated either with 6 or with 26 and consuming standard feed.

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Figure 5. Influence of long-term administration of
The change in body weight in Wistar rats fed palatable diet or standard diet and in Wistar rats fed
palatable diet or standard diet treated for 21 days with ) or 26 ). Results are means SEM,
n = 6. Multiple comparisons were performed by two-way ANOVA, Sidak post-hoc ( ) or by one-way
ANOVA, Sidak post-hoc ( ); * p < 0.05, ** p < 0.01, *** p < 0.001 significant vs. control rats fed
standard diet; ˆ p < 0.05, ˆˆ p < 0.01 significant vs. control rats fed palatable diet.
6 or 26 on the body weight in male Wistar rats.
6
(A
,
B
(C
,
D
±
A,C
B,D

Molecules 2018, 23, 2529
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2.5. “Drug-Like” Properties
2.5.1. Lipophilicity
The lipophilicity for compounds considered in the search for lead structures (
5
–
8) in particular
for the most active compounds found as derivatives of both lead modifications (18
determined with the standard RP-TLC method [22]. For comparison, the active arylmethyl-triazine
compounds previously found ( ) were also examined. Stationary phase RP-18 and mixtures of
, 23, 25 and 26) was
1–3
water/methanol (1:9, v/v) as a mobile phase were employed. RM values were calculated from the RF
data obtained. It was found that the RM parameters decreased linearly with increasing amounts of the
organic modifier (methanol) in the mobile phases tested. The regression coefficients (r2) determined
for all compounds were higher than 0.95. On the basis of the linear relationship between RM values
and the volume fraction of methanol, values of RM0 in the systems analyzed corresponding to 100% of
water were obtained by extrapolation. RM0 values received for the compounds tested ranged from
3.14 to 4.49 (Figure 6).
5
4.49
4.46
4.16
4.04
3.76
4
3
2
1
0
3.58
3.51
3.39
3.25
3.22
3.14
1
2
3
5
6
7
8
18
23
25
26
Figure 6. Lipophilicity parameters evaluated using R-TLC: RM0 values for compounds
25 and 26.
1–3, 5–8, 18, 23,
The most lipophilic character is associated with compound 23 (RM0 = 4.49), while the least
lipophilic hydantoin compound is a weak 5-HT6R agent (RM0 = 3.22). The arylmethyl derivatives
previously investigated ( ) displayed slightly less lipophilic properties than those of the most active
hydantoin agents (18 26). Overall, these results indicate a good “drug-like” lipophilicity for the entire
5
1–3
–
series of compounds investigated, taking into consideration either classical rules (Rules of Five, Ghose
or Pfizer) or the latest CNS MPO approach [23].
2.5.2. Toxicity In Vitro
The preliminary evaluation of the safety profile for the most promising 5-HT6R ligands
6 and
26 was performed in vitro with the HEK-293 eukaryotic cell line. The compounds examined were
dissolved in culture growth media and incubated with cells for 72 h. Next, the MTS test was applied to
determine the viability of the cells. As shown in Figure 7, a statistically significant decrease of viability
(*** p < 0.001) was observed only for compound
6
and only at the highest concentration used, i.e.,
M was also
100 M. The reference cytostatic drug doxorubicin (DX) at the very low concentration 1
µ
µ
cytotoxic. Thus, the results obtained confirm a low or no significant toxicity for compounds
6 and
26, respectively.

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Figure 7. The influence of doxorubicin (DX) and 5-HT6R ligands on the viability of cultured
HEK-293 cells. Statistical significance was evaluated by one-way ANOVA, followed by Bonferroni’s
comparison test (*** p < 0.001 compared with the negative control).
2.5.3. Blood-Brain Barrier Permeability
In order to estimate an ability of the library synthetized (4–26) to penetrate the blood-brain
barrier (BBB), the permeability QPlogBB parameter was calculated (Table 2) using QikProp from
Schrodinger Suite 2017 [24]. QikProp predictions are for orally delivered drugs, and thus some natural
neurotransmitters, e.g., dopamine and serotonin, are CNS negative, because they are extremely polar
to cross the blood-brain barrier. Recommended values range for compounds that penetrate BBB is from
−
3.0 to 1.2. All the hydantoin-triazines investigated (
with QPlogBB values from −0.98 (25) to −0.33 (12).
4–26) are predicted as BBB-permeable (Table 2)
Table 2. The blood-brain barrier permeability parameter (QPlogBB) for compounds 4–26.
Cpd
QPlogBB
Cpd
QPlogBB
Cpd
QPlogBB
4
5
6
7
8
9
10
11
−0.97
−0.60
−0.60
−0.89
−0.62
−0.84
−0.40
−0.37
12
13
14
15
16
17
18
19
−0.33
−0.73
−0.46
−0.65
−0.92
−0.54
−0.63
−0.75
20
21
22
23
24
25
26
−0.63
−0.46
−0.47
−0.38
−0.84
−0.98
−0.92
3. Computer-Aided SAR Analysis
In order to expand the current knowledge about structural properties responsible for 5-HT6R
activity in the group of recently discovered aryl derivatives of 1,3,5-triazine, this study took into account
the crucial role of the linker, which was found by comparative SAR analysis for the previously active
arylmethyl- and inactive aryl-triazine molecules [13]. The new series of hydantoin-triazines (5–26)
were designed as an extension of the methylene linker of the most active 2-arylmethyl-1,3,5-triaines,
via an introduction of the inflexible, cyclic hydantoin insert, containing also hydrogen bond acceptors,
and occurring in two topological variants found to be profitable in the preliminary lead identification
(4–8). Both leads (6 and 7) and their derivatives (9–18 and 19–26, respectively) displayed significant
submicromolar affinities for the target 5-HT6 receptors, much more potent than those of previously
investigated linker-free aryl-triazines [13], but slightly less potent if compared to the arylmethyl
ones (1–3, Figure 1). Interestingly, the influence of the topology of the aromatic moieties in the
group of 1,3,5-triazine derivatives seems to be distinctly linker-dependent. Although the bulky
aromatic area of the naphthalene was beneficial for all the groups considered, it was distinctly
predominant in the case of the 5-arylhydantoin (25, 26 vs. 7 and 19–24; Table 1), comparable with

Molecules 2018, 23, 2529
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the 3-methylphenyl and 3-chlorophenyl compounds in the group arylmethyl triazines (
1–
3, Figure 1),
and slightly less favourable than the 4-chlorophenyl substituent within the 5,5-dimethylhydantoin
group (18 vs. , Table 1). Furthermore, the preferable m-substitution within the phenyl ring, found for
the benzyl 1,3,5-triazine 5-HT6R agents earlier [13], has not been confirmed in this study, either for the
5,5-dimethyl-1-benzylhydantoin- ( vs. 10) or for the 5-phenyl-5-methylhydantoin triazine compounds
vs. 20). Consequently, an influence of both variants of the hydantoin linkers (1,3,5-substitution,
6
6
(7
group A or 3,5-substitution, group B) on the 5-HT6R activity was dependent on the properties of the
aromatic moiety. Thus, both variants appear to be comparable, although the most active compound
(26) is a member of the 5-aryl-5-methylhydantoin derivatives (group B).
Results of our docking studies have provided data useful to explain SAR on the molecular level
(Figure 8).
Figure 8. Molecular docking results of the representative library members for the 5-HT6 receptor.
(
A
) Comparison of the binding mode of compounds
6
(cyan) and
7
(gray). (
B) Binding mode of 18
(orange) and 26 (blue). ( ) A superposition of the poses of compounds
C
9
(magenta), 17 (yellow), 6
(cyan), and 10 (violet) against the putative halogen binding pocket interaction spheres for the 5-HT6
receptor. The chlorine-oxygen theoretical interaction spheres illustrate the projected qualities of the
formed ligand-receptor halogen bonds. Key residues in the binding site are presented as thick dark-grey
sticks. The dotted yellow lines represent hydrogen bonds with polar residues.
In general, all of the newly synthesized derivatives of Lead 1 and Lead 2 exhibited a binding
mode to 5-HT6R, which was very consistent with derivatives of 1,3,5-tiazines studied previously [13].
In addition to the crucial salt bridge interaction with D3.32, all of the docked ligands formed CH–
π
aromatic interactions with F6.52, and partially with F6.51. Additionally, the –NH2 group of the
1,3,5-triazine fragment was, usually, hydrogen-bonded with the carbonyl oxygen of A5.42 and V3.33
(Figure 8A). The methylene linker connecting 1,3,5-triazine and hydantoin rings, due to its tetrahedral
conformation, forced the terminal aromatic group into a hydrophobic cavity formed by transmembrane
helices 3–5 and extracellular loop 2. Thus, the structure-activity relationship in this series is determined
by the different substituents at the terminal aromatic ring, and by the different orientation of hydantoin
ring induced by its different positioning within the compound’s structure. A comparison of the
analogues with different positions of the 1,3,5-triazine substitution at the hydantoin ring indicated that
linking by the methylene group at position 3 of hydantoin is profitable (
significant decrease of activity was noted for the derivative where the 1,3,5-triazine was linked with
hydantoin via position 1 ( , Ki = 4275 nM). The analysis of the binding mode showed that a differently
positioned hydantoin ring induces its different spatial orientation in the binding site (Figure 8A),
6, Ki = 127 nM), whereas a
5
and only the complex of
Q6.58 side chain.
6 with the receptor is stabilized additionally by a hydrogen bond with a
Interestingly, within the group A and B, the best activity showed 2-naphthyl derivatives (18 and 26
,
respectively; Figure 8B). It should be noted, that apart from the length of the linker and the positioning
of the hydantoin ring, the difference of activity between 18 and 26 is negligible—a comparison of its
binding modes indicated that the most significant difference is noted for the hydantoin ring interaction.

Molecules 2018, 23, 2529
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In both derivatives, the hydantoin ring is stabilized by hydrogen bonding, but due to its different
orientation, the hydrogen bonds form with S5.44 and N6.55 or Q6.58 for 18 and 26, respectively. It is
worth to note that the shift of the chlorine atom from 3- to 4-position resulted in a 4- (for modification B)
to 5-fold (for modification A) increase of affinity for 5-HT6R compared to its unsubstituted analogues.
This relationship was confirmed by molecular docking (Figure 8C) revealing that only the presence of
chlorine atom in 4-position stabilized the L–R complex through the formation of halogen bond with
◦
the carbonyl oxygen of P4.60 residue (Cl···O distance = 3.31 Å,
σ
-hole angle = 151.47 ). As the halogen
bond appeared to possess a highly directional nature, to explain the decrease in activity by shifting the
chlorine atom from position 3 to 4 in the phenyl ring, the interaction sphere [25 26] was plotted onto
,
the relevant backbone carbonyl oxygens (Figure 8C). The 4-chloro substituent was positioned within
the energetically favorable areas of the sphere, whereas the 3-chloro substituent pointed outside of the
sphere, indicating no ability to form halogen bond.
4. Experimental Section
4.1. Chemistry
Reagents were purchased from Alfa Aesar (Karlsruhe, Germany) or Sigma Aldrich (Darmstadt,
Germany). Methanol was dried over calcium oxide Reaction progress was verified using thin layer
chromatography (TLC), which was carried out on 0.2 mm Merck silica gel 60 F254 plates. Spots were
visualized by UV light or treatment with Dragendorff reagent. Melting points (m.p.) were determined
using MEL-TEMP II apparatus (LD Inc., Long Beach, CA, USA) and are uncorrected. The 1H-NMR
and 13C-NMR spectra were obtained on a Mercury-VX 300 Mz spectrometer (Varian, Palo Alto, CA,
USA) in DMSO-d6 or CDCl3. Chemical shifts in 1H-NMR spectra were reported in parts per million
(ppm) on the
δ scale using the solvent signal as an internal standard. Data are reported as follows:
Chemical shift, multiplicity (s, singlet; br.s., broad singlet; d, doublet; t, triplet; m, multiplet), coupling
constant J in Hertz (Hz), number of protons, proton’s position (Ind–indole, Naph—naphthalene,
Ph—phenyl, Pp—piperazine). LC-MS were carried out on a system Water TQ Detector (Water
Corporation, Milford, CT, USA) consisting of a Waters Acquity UPLC, coupled to a Waters TQD
mass spectrometer. Retention times (tR) are given in minutes. The UPLC/MS purity of all final
compounds was determined (%).
Synthesis of compounds 27, 29, 50–58, 63, 65 and 68–70 was obtained by our [15,27] or other research
groups [28–32] earlier and are reported in Chemical Abstract Database (see Supplementary Information).
4.1.1. Synthesis of Methyl 2-(3-(4-chlorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)acetate (28
)
A mixture of 3-(4-chlorobenzyl)-5,5-dimethylimidazolidine-2,4-dione 27 (20 mmol, 5.05 g), TEBA
(2.60 mmol, 0.60 g), K2CO3 (58 mmol, 8 g) in acetone (100 mL) was heated under reflux for 30 min.
Methyl bromoacetate (20 mmol, 3.06 g) in acetone (20 mL) was added. The mixture was heated under
reflux for 5 h and stirred at room temperature overnight. The inorganic precipitate was separated by
filtration. The filtrate was evaporated, and the residue was crystallized with methanol. White solid,
◦
1
m.p. 78–80 C, yield: 58% (11.60 mmol, 3.77 g), C15H17ClN2O4 (MW 324.76). H-NMR (300 MHz,
DMSO-d6) = 7.38–7.41 (d def., 2H, Ar-3,5-H), 7.22–7.25 (d def., 2H, Ar-2,6-H), 4.57 (s, 2H, Ar-CH2),
δ
4.18 (s, 2H, CH2COO), 3.68 (s, 3H, OCH3), 1.30 (s, 6H, 2
tR = 6.14, (ESI) m/z [M + H]+ 325.11.
×
5-CH3) ppm. LC/MS+: Purity: 98.40%,
4.1.2. General Procedure for the Synthesis of Alkyl
2-(1-arylmethyl-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (30–40)
A mixture of alkyl 2-(5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate 29 (12 mmol, 29a: 2.40 g
or 29b: 2.58 g), TEBA (1.20 mmol, 0.36 g), K2CO3 (12 mmol, 4.80 g) in acetone (60 mL) was heated
under reflux for 30 min. The appropriate arylmethyl halide (12 mmol) in acetone (15 mL) was added.
The mixture was heated under reflux for 5–7 h and stirred at room temperature for further 24–48 h.

Molecules 2018, 23, 2529
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The inorganic precipitate was separated by filtration. The filtrate was collected, the solvent then
evaporated and the residue was dissolved in 30 mL of DCM and washed with 1% NaOH (2 30 mL)
and H2O (2 30 mL). The organic phase was dried on anhydrous Na2SO4 overnight, separated from
×
×
the drying agent and the solvent was evaporated. Then, Methods A or B were applied for purification.
Method A: The residue was crystallized with EtOH and charcoal to give crystals of the intermediates.
Method B: If Method A was applied, but no crystal appeared, the ethanolic solution was concentrated
to give crude ester intermediates in glue form. No ester intermediate (30–40) was isolated in pure
form (purity < 80%). The purity and identity of crude intermediates were evaluated using LC-MS.
Taking into consideration the results of LC-MS a desirable ester was used for the synthesis of the final
product in the amount suitable to its concentration in the crude product obtained (40–80%)
Ethyl 2-(1-(4-chlorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (30). 1-Chloro-4-(chloromethyl)
benzene (1.93 g) and 29a were used. Method A. White crystals, m.p. 88–90 ◦C, yield: 68% (8.16 mmol,
1
2.65 g), C15H17ClN2O4 (MW 324.76). H-NMR (300 MHz, DMSO-d6)
δ = 7.36–7.37 (d def., 4H, Ar),
4.52 (s, 2H, Ar-CH2), 4.25 (s, 2H, CH2COO), 3.70 (s, 3H, OCH3), 1.25 (s, 6H, 2
Purity: 81%, tR = 6.31, (ESI) m/z [M + H]+ 325.18.
×
CH3) ppm. LC/MS+:
Ethyl 2-(1-benzyl-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (31). Benzyl chloride (1.52 g) and 29a
were used. Method B. Glue mixture containing 44% of 31, yield: 61.37%, C15H18N2O4 (MW 304.15).
LC/MS+: Purity: 44%, tR = 6.09, (ESI) m/z [M + H]+ 304.14.
Ethyl 2-(1-(3-chlorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (32). 1-Chloro-3-(chloromethyl)
benzene (1.93 g) and 29a were used. Method B. Sticky liquid, yield: 85.22%, C15H19ClN2O4 (MW
1
338.59). H-NMR (300 MHz, DMSO-d6)
δ
= 7.41–7.33 (m, 1H, Ph-5-H), 7.31–7.28 (m, 3H, Ph-2,4,6-H),
CH3),
4.54 (s, 2H, Ph-CH2), 4.23 (s, 2H, CH2COO), 4.10 (q, J = 7.05 Hz, 2H, CH3–CH2), 1.26 (s, 6H, 2
×
1.16 (t, J = 7.35 Hz, 3H, CH3–CH2) ppm. LC/MS+: Purity: 67.03%, tR = 6.73, (ESI) m/z [M + H]+ 339.59.
Ethyl 2-(1-(2,5-dichlorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl) acetate (33). 1-Bromo-2,5-(di-chloro
◦
methyl) benzene (3.60 g) and 29a were used. Method A. yellowish Solid, m.p. 98 C, yield: 98.21%,
C16H18Cl2N2O4 (MW 373.04). 1H-NMR (300 MHz, DMSO-d6)
δ
= 7.52–7.41 (m, 1H, Ph-6-H), 7.39–7.40 (m,
2H, Ph-3,4-H), 4.58 (s, 2H, Ph-CH2), 4.24 (s, 2H, CH2COO), 4.10 (q, J = 7.00 Hz, 2H,CH3–CH2), 1.29 (s, 6H,
2
×
CH3), 1.17 (d, J = 7.00 Hz, 3H, CH3–CH2) ppm. LC/MS+: Purity: 93.90%, tR = 7.28, (ESI) m/z [M +
H]+ 373.03.
Ethyl 2-(1-(2,4-dichlorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (34). 1-Chloro-2,4-(di-chloro
◦
methyl) benzene (2.34 g) and 29a were used. Method B. White Solid, m.p. 105 C, yield: 91.52%,
C16H18Cl2N2O4 (MW 373.04). 1H-NMR (300 MHz, DMSO-d6)
δ
= 7.66 (d, J = 2.0 Hz, 1H, Ph-3-H), 7.47–7.37
(m, 2H-Ph-5,6-H), 4.60 (s, 2H-Ph-CH2), 4.26 (s, 2H, CH2COO), 4.16 (q, J = 7.1 Hz, 2H–CH3–CH2), 1.29
(s, 6H, 2
×
CH3), 1.21 (t, J = 7.1 Hz, 3H, CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
δ = 175.83, 167.53,
154.84, 134.60, 133.22, 133.18, 130.71, 129.32, 127.98, 62.51, 61.83, 22.68, 14.38 ppm. LC/MS+: Purity: 91.74%,
tR = 7.52, (ESI) m/z [M + H]+ 374.04.
Ethyl 2-(1-(4-fluorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (35). 4-Flouro benzyl chloride
(2.17 g) and 29a were used. Method B. Sticky liquid, yield: 90.72%, C16H19FN2O4 (MW 322.99).
LC/MS+: Purity: 69.51%, tR = 7.52, (ESI) m/z [M + H]+ 323.99.
Ethyl 2-(1-(3-fluorobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (36). 3-Flouro benzyl chloride
(1.74 g) and 29a were used. Method A. Sticky liquid, yield: 77.06%, C16H19FN2O4 (MW 322.99).
1H-NMR (300 MHz, DMSO-d6)
δ = 7.41–7.36 (m, 1H, Ph-5-H), 7.19–7.13 (m, 3H, Ph-2,4,6-H), 4.55 (s,
2H, Ph-CH2), 4.23 (s, 2H, CH2COO), 4.10 (q, J = 7.05 Hz, 2H, CH3–CH2), 1.26 (s, 6H, 2× CH3), 1.16 (t,
J = 7.05 Hz, 3H, CH3–CH2) ppm. LC/MS+: Purity: 69.43%, tR = 6.23, (ESI) m/z [M + H]+ 323.99.
Ethyl 2-(1-(3-bromobenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (37). 3-Bromo benzyl chloride
(3.75 g) and 29a were used. Method B. Sticky liquid, yield: 60%, C16H19BrN2O4 (MW 383.24). LC/MS+:
Purity: 78.26%, tR = 6.85, (ESI) m/z [M + H]+ 384.24.

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Ethyl 2-(1-(3-methoxybenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (38). 3-Methoxy benzyl
chloride (1.88 g) and 29a were used. Method B. Sticky liquid, yield: 68.94%, C17H22N2O5 (MW
334.38). LC/MS+: Purity: 88.03%, tR = 6.65, (ESI) m/z [M + H]+ 335.38.
Ethyl 2-(1-(3-methylbenzyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (39). 3-Methyl benzyl chloride
(1.69 g) and 29a were used. Method B. Sticky liquid, yield: 61.05%, C17H22N2O4 (MW 318.38). LC/MS+:
Purity: 81.94%, tR = 6.12, (ESI) m/z [M + H]+ 319.38.
Ethyl 2-(1-(2-naphthyl)-5,5-dimethyl-2,4-dioxoimidazolidin-3-yl)acetate (40). 2-(Chloromethyl)naphthalene
◦
(2.65 g) and 29a were used. Method B. White solid, m.p. 95 C, yield: 81.47%, C20H22N2O4 (MW
354.19). 1H-NMR (300 MHz, DMSO-d6)
δ
= 7.90–7.84 (m, 4H, naft-1,4,5,8-H), 7.52–7.45 (m, 3H, naft-3,6,7-H),
4.71 (s, 2H, Ph-CH2), 4.26 (s, 2H, CH2COO), 4.16–4.11 (q, J = 7.35 Hz, 2H, CH3–CH2), 1.26 (s, 6H, 2 CH3),
1.17(d, J = 7.00 Hz, 3H, CH3–CH2) ppm. LC/MS+: Purity: 90.56%, tR = 7.08, (ESI) m/z [M + H]+ 355.15.
×
4.1.3. General Procedures for the Synthesis of Methyl 2-(5-arylhydantoin-3-yl)acetates (60
–67 and 71)
A mixture of a suitable arylhydantoin 50 58 (Scheme 1) or 70 (15–20 mmol) with K2CO3 (6–8 g),
–
TEBA (0.45–0.60 g) in acetone (75–100 mL) was stirred and refluxed for 30 min. Then, the solution
of methyl bromoacetate (15–20 mmol, 2.30–3.06 g) in acetone (15–20 mL) was added. The mixture
was stirred and refluxed for 3–6 h, then, stirred at room temperature overnight. The inorganic
components were separated by filtration. The filtrate was concentrated, and the residue was purified
using the similar methods as those for compounds 30–40, but MeOH in place of EtOH was used
for crystallization.
Methyl 2-(5-methyl-2,4-dioxo-5-phenylimidazolidin-3-yl)acetate (60). Compound 51 (2.85 g) and methyl
bromoacetate (2.30 g) were used. Sticky liquid, yield: 48.6%, C13H14N2O4 (MW 262.26).
1H-NMR (300 MHz, DMSO-d6)
δ = 9.06 (s, 1H, N1–H), 7.47–7.50 (d, J = 7.00 Hz, 2H, Ph-2,6-H),
7.33–7.42 (m, 3H Ph-3,4,5-H), 4.19 (s, 2H, N3–CH2), 3.67 (s, 3H, O-CH3), 1.70 (s, 3H, CH3) ppm.
LC/MS+: Purity: 85.92%, tR = 4.43, (ESI) m/z [M + H]+ 263.11.
Methyl 2-(5-(3-chlorophenyl)-5-methyl-2,4-dioxoimidazolidin-3-yl)acetate (61). Compound 52 (4.49g) and
methyl bromoacetate (3.06 g) were used. White Chrystal, m.p. 102 ◦C, yield: 51.10%, C13H13ClN2O4
1
(MW 296.71). H-NMR (300 MHz, DMSO-d6)
δ = 9.13 (s, 1H, N1–H), 7.51 (s, 1H, Ph-6-H), 7.43–7.47 (m,
3H, Ph-2,4,5-H), 4.20 (s, 2H, N3-CH2), 3.66 (s, 3H, O–CH3), 1.70 (s, 3H, CH3) ppm. LC/MS+: Purity:
100%, tR = 5.25, (ESI) m/z [M + H]+ 297.07.
Methyl 2-(5-(2,5-dichlorophenyl)-5-methyl-2,4-dioxoimidazolidin-3-yl)acetate (62). Compound 53 (4.14 g)
and methyl bromoacetate (2.45 g) were used. White solid, m.p. 214 ◦C, yield: 83.19%, C13H12Cl2N2O4
1
(MW 331.15). H-NMR (300 MHz, DMSO-d6)
δ
= 8.75 (s, 1H, N1-H), 7.69 (s, 2H, Ph-4-H), 7.52 (s, 2H,
Ph-3,6-H), 4.25 (s, 2H, N3–CH2), 3.68 (s, 3H, O–CH3), 1.80 (s, 3H, CH3) ppm. LC/MS+: Purity: 99.10%,
tR = 5.41, (ESI) m/z [M + H]+ 330.69.
Methyl 2-(5-(2,3,4-trichlorophenyl)-5-methyl-2,4-dioxoimidazolidin-3-yl)acetate (64). Compound 54 (5.18 g)
and methyl bromoacetate (3.06 g) were used. White solid, m.p. 172 ◦C, yield: 37.76%, C13H12Cl2N2O4
(MW 331.15). 1H-NMR (300 MHz, DMSO-d6)
δ = 8.75 (s, 1H, N1–H), 7.68–7.71 (d, J = 8.8 Hz, 1H,
Ph-3-H), 7.65–7.66 (s, 1H, Ph-5-H), 7.48–7.52 (m, 1H, Ph-6-H), 4.25 (s, 2H, N3–CH2), 3.68 (s, 3H, O–CH3),
1.78 (s, 3H, CH3) ppm. LC/MS+: Purity: 96.70%, tR = 5.49, (ESI) m/z [M + H]+ 330.76.
Methyl 2-(5-methyl-5-(naphthalen-1-yl)-2,4-dioxoimidazolidin-3-yl)acetate (66). 5-Methyl-5-(naphthalen-1-
yl)imidazolidine-2,4-dione 57 (20 mmol, 4.80 g), K2CO3 (8 g), TEBA (0.60 g) in acetone (100 mL) and
methyl bromoacetate (20 mmol, 3.06 g) in acetone (20 mL) were stirred and refluxed for 6 h. Method B.
◦
White glue-crystals, m.p. 105–109 C, yield: 78% (15.60 mmol, 4.86 g), C17H16N2O4 (MW 312.11).
1H-NMR (300 MHz, DMSO-d6)
δ = 8.98 (s, 1H, NH), 7.94–8.01 (m, 3H, Ar-4,5,8), 7.75 (d, J = 7.44 Hz,
1H, Ar-2), 7.48–7.57 (m, 3H, Ar-3,6,7), 4.33–4.39 (m, 2H, –CH2CO), 3.73 (s, 3H, –OCH3), 1.94 (s, 3H,
5-CH3) ppm. LC/MS+: Purity: 65.46%, tR = 5.47, (ESI) m/z [M + H]+ 312.95.

Molecules 2018, 23, 2529
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Methyl 2-(5-methyl-5-(naphthalen-2-yl)-2,4-dioxoimidazolidin-3-yl)acetate (67). 5-Methyl-5-(naphthalen-2-
yl)imidazolidine-2,4-dione 58 (15 mmol, 3.60 g), K2CO3 (6 g), TEBA (0.45 g) in acetone (75 mL) and
methyl bromoacetate (15 mmol, 2.30 g) in acetone (15 mL) were stirred and refluxed for 5 h. Method A.
1
White crystals, m.p. 134–138 ◦C, yield: 58% (8.7 mmol, 2.72 g), C17H16N2O4 (MW 312.11). H-NMR
(300 MHz, DMSO-d6)
δ = 9.15 (s, 1H, NH), 8.02 (s, 1H, Ar-1-H), 7.60 (dd, J1 = 8.72 Hz, J2 = 1.80 Hz, 2H,
Ar-6,7-H), 7.87–7.99 (m, 3H, Ar-4,5,8-H), 7.51–7.57 (m, 1H, Ar-3), 4.23 (s, 2H, –CH2CO), 3.66 (s, 3H,
–OCH3), 1.81 (s, 3H, 5-CH3) ppm. LC/MS+: Purity: 76.61%, tR = 5.63, (ESI) m/z [M + H]+ 312.82.
Methyl 2-(5,5-bis(4-chlorophenyl)-2,4-dioxoimidazolidin-3-yl)acetate (71).
5,5-bis(4-Chlorophenyl)-
imidazolidin-2,4-dione 70 (15 mmol, 4.80 g), K2CO3 (6 g), TEBA (0.45 g) in acetone (75 mL) and
methyl bromoacetate (15 mmol, 2.30 g) in acetone (15 mL) were stirred and refluxed for 4 h. Method A.
◦
Creamy crystals, m.p. 120–121 C, yield: 62%, C18H14Cl2N2O4 (MW 392.03). 1H-NMR (300 MHz,
DMSO-d6) δ = 7.53 (d, J = 8.7 Hz, 4H-ph), 7.37 (d, J = 8.7 Hz, 4H-ph), 4.31 (s, 2H–CH2), 3.70 (s, 3H-CH3),
1.04 (d, J = 6.1 Hz, 1H-NH) ppm. LC/MS+: Purity: 91.91%, tR = 7.26, (ESI) m/z [M + H]+ 393.10.
4.1.4. General Procedure for the Synthesis of 1,3,5-Triazine Final Products (5–26)
Sodium (8–10 mmol) was dissolved in 10 mL of absolute methanol, then 4-methylpiperazin-1-yl
biguanidine
added. The reaction mixture was refluxed for 15–30 h. After cooling to room temperature, water
(10 mL) was added and the mixture was stirred for 0.5 h. The triazine product precipitated ( 26) was
×
2HCl (4–5 mmol) and an appropriate ester 28, 30–40, 59–67 or 71 (4–5 mmol) were
5
–
separated and purified using Methods C, D or E. Method C: Crystallization with methanol. Method D:
crystallization with methanol and charcoal. Method E: crystallization with ethanol.
3-(4-Chlorobenzyl)-1-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (
5
). Ester 28 (4.30 mmol, 1.40 g) and 4-methylpiperazin-1-yl biguanidine
×
2HCl (4.30 mmol,
◦
1.11 g) were used (48 h refluxed). Method C. White solid, m.p. 166–169 C, yield: 34% (0.67 g),
C21H27N8O2Cl (MW 458.95). H-NMR (300 MHz, DMSO-d6)
1
δ 7.34–7.52 (m, 2H, Ph-3,5-H), 7.21–7.32
(m, 2H, Ph-2,6-H), 6.82 (br. s., 2H, NH2), 4.55 (s, 2H, Ph-CH2), 4.19 (s, 2H, TR-CH2), 3.72–3.37 (br s, 4H,
Pp-2,6-H), 2.15 (s, 7H, Pp-3,5-H + Pp-CH3), 1.31 (s, 6H, 2
×
CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
δ
= 176.83, 173.77, 167.04, 164.65, 154.86, 136.23, 132.52, 129.73, 129.03, 62.11, 54.67, 46.19, 44.19, 42.75, 41.08,
22.84 ppm. LC/MS+: Purity: 99.46%, tR = 3.75, (ESI) m/z [M + H]+ 459.23.
1-(4-Chlorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (
6
). Ester 30 (4.30 mmol, 1.40 g) and 4-methylpiperazin-1-yl biguanidine
×
2HCl (4.30 mmol,
◦
1.11 g) were used (50 h refluxed). Method C. White solid, m.p. 204–205 C, yield: 19% (0.38 g),
C21H27N8O2Cl (MW 458.95). H-NMR (300 MHz, DMSO-d6)
1
δ = 7.38 (s, 4H, Ph-2,3,5,6-H), 6.87 (br. s.,
2H, NH2), 4.51 (s, 2H, Ph-CH2), 4.27 (s, 2H, TR-CH2), 3.55 (br. s., 4H, Pp-2,6-H), 2.15 (s, 7H, Pp-3,5-H +
Pp-CH3), 1.26 (s, 6H, 2× CH3) ppm. LC/MS+: Purity: 100%, tR = 3.94, (ESI) m/z [M + H]+ 459.23.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(4-chlorophenyl)-5-methylimidazolidine-2,4-
dione (
were used (13.5 h refluxed). Method C. White solid, m.p. 218–220 ◦C, yield: 23% (0.42 g), C19H23N8O2Cl
(MW 430.89). 1H-NMR (300 MHz, DMSO-d6)
= 9.02 (s, 1H, Hyd-1-H), 7.51–7.69 (m, 2H, Ph-3,5-H),
7). Ester 59 (5 mmol, 1.48 g) and 4-methylpiperazin-1-yl biguanidine
×
2HCl (4.30 mmol, 1.11 g)
δ
7.36–7.51 (m, 2H, Ph-2,6-H), 6.83 (br. s., 2H, NH2), 4.21 (s, 2H, TR-CH2), 3.53 (br. s., 2H, Pp-2,6-He),
3.27–3.32 (m, 2H, Pp-2,6-Ha), 2.13 (s, 7H, Pp-3,5-H + Pp-CH3), 1.68 (s, 3H, Hyd-CH3) ppm. 13C-NMR
(75 MHz, DMSO-d6) δ = 175.25, 171.94, 166.94, 164.54, 155.78, 139.22, 133.15, 128.87, 127.93, 62.99, 54.57,
46.16, 42.63, 42.45, 25.92 ppm. LC/MS+: Purity: 100%, tR = 3.23, (ESI) m/z [M + H]+ 431.19.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-bis(4-chlorophenyl)imidazolidine-2,4-dione
(
8
). Ester 71 (4.80 mmol, 1.90 g) and 4-methylpiperazin-1-yl biguanidine
used (30 h refluxed). Method C. White solid, m.p. 266–268 ◦C, yield: 40% (0.83 g), C24H24N8O2Cl2 (MW
526.14). 1H-NMR (300 MHz, DMSO-d6)
= 9.79 (s, 1H, Hyd-1-H), 7.34–7.59 (m, 8H, 2 Ph-2,3,5,6-H), 6.85
(br. s, 2H, NH2), 4.30 (s, 2H, TR-CH2), 3.55 (br. s, 2H, Pp-2,6-He), 3.09 (br. s, 2H, Pp-2,6-Ha), 1.76–2.35 (m,
×
2HCl (4 mmol, 1.03 g) were
δ
×

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7H, Pp-3,5-H + Pp-CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
δ
= 173.01, 171.74, 166.92, 164.52, 155.38,
138.75, 133.57, 129.12, 129.01, 68.59, 54.58, 46.16, 42.77, 42.53 ppm. LC/MS+: Purity: 100%, tR = 3.23, (ESI)
m/z [M]+ 527.16.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-1-benzyl-5,5-dimethylimidazolidine-2,4-dione (
Ester 31 (3 mmol, 2.03 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (3 mmol, 0.77 g) were used
(36 h refluxed). Method D. White solid, m.p. 206 ◦C, yield: 3.94%, C21H28N8O2 (MW 424.23). 1H-NMR
9).
×
(300 MHz, DMSO-d6) δ = 7.36–7.25 (m, 5H-Ph), 6.87 (s, 2H, NH2), 4.52 (s, 2H, CH2), 4.28 (s, 2H, TR-CH2),
3.62 (s, 4H, Pp-2,6-H), 2.15–2.23 (m, 7H, Pp-3,5-H, Pp-CH3), 1.25 (m, 6H, 2× CH3) ppm. LC/MS+: Purity:
98.30%, tR = 3.50, (ESI) m/z [M]+ 425.20.
1-(3-Chlorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (10). Ester 32 (5 mmol, 2.52 g) and 4-methylpiperazin-1-yl biguanidine
were used (6 h refluxed), and stirred for another 24 h in a room temperature. Method D. White solid, m.p.
168 ◦C, yield: 24.81%, C21H28N8O2 (MW 458.19). 1H-NMR (300 MHz, DMSO-d6)
= 7.42 (s, 1H, Ph-6-H),
7.33 (m, 3H, Ph-2,4,5-H), 6.86 (s, 2H, NH2), 4.52 (s, 2H–CH2), 4.27(s, 2H, TR-CH2), 3.58 (s, 4H, Pp-2,6-H),
×
2HCl (5 mmol, 0.92 g)
δ
2.13 (m, 7H, Pp-3,5-H, Pp-CH3), 1.27 (s, 6H, 2
×
CH3) ppm. LC/MS+: Purity: 96.12%, tR = 5.34, (ESI) m/z
[M]+ 459.23.
1-(2,5-Dichlorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (11). Ester 33 (5 mmol, 2.01 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (5 mmol, 0.92 g)
were used (6 h refluxed), and stirred for another 144 h in room a temperature. Method D. White solid, m.p.
163 ◦C, yield: 27.32%, C21H26Cl2N8O (MW 492.16). 1H-NMR (300 MHz, DMSO-d6)
= 7.53–7.50 (s, 1H,
Ph-4-H), 7.41–7.38 (m, 2H, Ph-3,6-H), 6.84 (s, 2H, NH2), 4.58 (s, 2H, CH2), 4.29 (s, 2H, TR-CH2), 3.63 (s,
×
δ
4H, Pp-2,6-H), 2.23–2.15 (m, 7H, Pp-3,5-H, Pp-CH3), 1.30 (s, 6H, 2
×
CH3) ppm. LC/MS+: Purity: 100%,
tR = 4.40, (ESI) m/z [M]+ 493.13.
1-(2,4-Dichlorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (12). Ester 34 (5 mmol, 2.03 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (5 mmol, 0.92 g)
were used (3 h refluxed), and stirred for another 24 h in room a temperature. Method D. White solid, m.p.
123 ◦C, yield: 23.98%, C21H26Cl2N8O (MW 492.16). 1H-NMR (300 MHz, DMSO-d6)
= 7.65–7.64 (s, 1H,
Ph-6-H), 7.40–7.41 (m, 2H, Ph-3,5-H), 6.88 (s, 2H, NH2), 4.58 (s, 2H, CH2), 4.28 (s, 2H, TR-CH2), 3.62 (s,
×
δ
4H, Pp-2,6-H), 2.52–2.26 (m, 7H, Pp-3,5-H, Pp-CH3), 1.29 (s, 6H, 2
×
CH3) ppm. LC/MS+: Purity: 100%,
tR = 4.56, (ESI) m/z [M]+ 493.13.
1-(4-Fluorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (13). Ester 35 (5 mmol, 2.32 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (5 mmol, 0.92 g)
×
were used (10 h refluxed), and stirred for another 24 h in room a temperature. Method D. White solid, m.p.
1
180 ◦C, yield: 9.23%, C21H27FN8O2 (MW 442.22). H-NMR (300 MHz, DMSO-d6)
δ
= 7.38–7.43 (m, 2H,
Ph-2,6-H), 7.11–7.17 (m, 2H, Ph-3,5-H), 6.86 (s, 2H, NH2), 4.51 (s, 2H, CH2), 4.27 (s, 2H, TR-CH2), 3.57 (s,
4H, Pp-2,6-H), 2.14–2.23 (m, 7H, Pp-3,5-H, Pp-CH3), 1.26 (m, 6H, 2
×
CH3) ppm. LC/MS+: Purity: 100%,
tR = 3.68, (ESI) m/z [M]+ 443.28.
1-(3-Fluorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (14 Ester 36 (5 mmol, 2.34 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (5 mmol, 0.92 g)
were used (10 h refluxed), and stirred for another 24 h in room a temperature. Method D. White solid, m.p.
).
×
◦
207 C, yield: 9.32%, C21H27FN8O2 (MW 442.22). 1H-NMR (300 MHz, DMSO-d6)
δ = 7.34 (m, 1H, Ph-6-H),
7.13 (m, 3H, Ph-2,4,5-H), 6.86 (s, 2H, NH2), 4.53 (s, 2H, CH2), 4.27 (s, 2H, TR-CH2), 3.58 (s, 4H, Pp-2,6-H),
2.14 (m, 7H, Pp-3,5-H, Pp-CH3), 1.27 (s, 6H, 2
×
CH3) ppm. LC/MS+: Purity: 100%, tR = 3.59, (ESI) m/z
[M]+ 443.22.
1-(3-Bromobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (15). Ester 37 (5 mmol, 2.51 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (5 mmol, 0.92 g)
were used (4 h refluxed), and stirred for another 24 h in room a temperature. Method D. White powder,
×

Molecules 2018, 23, 2529
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1
m.p. 125 ◦C, yield: 56%, C21H27BrN8O2 (MW 502.14). H-NMR (300 MHz, DMSO-d6)
δ
= 7.59 (s, 1H,
Ph-4-H), 7.51–7.44 (m, 1H, Ph-2-H), 7.38 (d, J = 7.8 Hz, 1H, Ph-6-H), 7.30 (t, J = 7.7 Hz, 1H, Ph-5-H), 6.88
(s, 2H, NH2), 4.54 (s, 2H, CH2), 4.29 (s, 2H, TR-CH2), 3.77–3.44 (m, 4H, Pp-2,6-H), 2.16 (m, 7H, Pp-3,5-H,
Pp-CH3), 1.29 (s, 6H, 2× CH3) ppm. LC/MS+: Purity: 97.66%, tR = 4.07, (ESI) m/z [M]+ 505.16.
1-(3-Methoxybenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (16). Ester 38 (5 mmol, 2.42 g) and 4-methylpiperazin-1-yl biguanidine
were used (10 h refluxed), and stirred for another 24 h in room a temperature. Method D. White solid,
m.p. 111 ◦C, yield: 13.79%, C22H30N8O3 (MW 454.24). 1H-NMR (300 MHz, DMSO-d6)
= 7.25–7.20 (m,
1H, Ph-3-H), 6.93 (m, 2H, Ph-2,4-H), 6.84–6.82 (m, 1H, Ph-6-H, 2H, NH2), 4.49 (s, 2H, CH2), 4.28 (s, 2H,
×
2HCl (5 mmol, 0.92 g)
δ
TR-CH2), 3.71 (s, 3H, CH3), 3.59 (s, 4H, Pp-2,6-H), 2.15(m, 7H, Pp-3,5-H, Pp-CH3), 1.25 (m, 6H, 2
×
CH3)
ppm. LC/MS+: Purity: 97.60%, tR = 3.55, (ESI) m/z [M]+ 455.24.
1-(3-Methylbenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione (17). Ester 39 (5 mmol, 2.61 g) and 4-methylpiperazin-1-yl biguanidine 2HCl (5 mmol, 0.92 g)
were used (5 h refluxed) and stirred for another 24 h in room a temperature. Method D. White solid, m.p.
×
◦
154 C, yield: 13.79%, C22H30N8O (MW 438.25). 1H-NMR (300 MHz, DMSO-d6)
δ = 7.22–7.07 (m, 4H, Ph),
6.86 (s, 2H, NH2), 4.48 (s, 2H, CH2), 4.27 (s, 2H, TR-CH2), 3.59 (s, 4H, Pp-2,6-H), 2.27–2.15(m, 10H, Pp-3,5-H,
Pp-CH3, Ph-CH3), 1.25 (s, 6H, 2× CH3) ppm. LC/MS+: Purity: 98.88%, tR = 3.94, (ESI) m/z [M]+ 439.23.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethyl-1-(naphthalen-1-ylmethyl)
imidazolidine-2,4-dione (18). Ester 40 (5 mmol, 1.96 g) and 4-methylpiperazin-1-yl biguanidine
×
2HCl (5 mmol, 0.92 g) were used (5 h refluxed) and stirred for another 24 h in room a temperature.
1
Method D. White powder, m.p. 116 ◦C, yield: 13.79%, C25H30N8O2 (MW 474.25). H-NMR (300 MHz,
DMSO-d6)
δ = 7.88–7.84 (m, 4H, naft-1,4,5,8-H), 7.50–7.48 (m, 3H, naft-3,6,7-H), 6.86 (s, 2H, NH2), 4.70
(s, 2H, CH2), 4.30 (s, 2H, TR-CH2), 3.55 (s, 4H, Pp-2,6-H), 2.22–2.02 (m, 7H, Pp-3,5-H, Pp-CH3), 1.29 (s,
6H, 2× CH3) ppm. LC/MS+: Purity: 100%, tR = 4.31, (ESI) m/z [M]+ 475.25.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-methyl-5-phenylimidazolidine-2,4-dione (19).
Ester 60 (5 mmol, 1.31g) and 4-methylpiperazin-1-yl biguanidine
(4.5 h refluxed) and stirred for another 24 h in room a temperature. Method C. White solid, m.p. 270 ◦C,
yield: 50%, C19H24N8O2 (MW 396.45). 1H-NMR (300 MHz, DMSO-d6)
= 9.00 (s, 1H, N1-H), 7.54–7.56
×
2HCl (5 mmol, 0.92 g) were used
δ
(d, J = 7.5 Hz, 2H, Ph-2,6-H), 7.39–7.43 (t, J = 7.25 Hz, Ph-3,5-H), 7.33–7.36 (d, J = 7.5 Hz,1H, Ph-4-H), 6.85
(s, 2H, NH2), 4.24 (s, 2H, N3-CH2), 3.61 (s, 2H, Pp-2,6-Hb), 3.40–3.50 (m, 2H, Pp-2,6-Ha), 2.16–2.24 (s, 4H,
Pp-3,5-H), 2.16 (s, 3H, N–CH3), 1.73 (s, 3H, 5-CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
δ = 175.62, 172.08,
166.96, 164.61, 155.92, 140.14, 128.88, 128.31, 125.98, 63.40, 54.64, 46.20, 42.72, 42.47, 25.82 ppm. LC/MS+:
Purity: 96.38%, tR = 2.62, (ESI) m/z [M]+ 397.22.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(3-chlorophenyl)-5-methylimidazolidine-2,4-
dione (20). Ester 61 (5 mmol, 1.49 g) and 4-methylpiperazin-1-yl biguanidine
used (5 h refluxed). Method C. White solid, m.p. 234 ◦C, yield: 56.80%, C19H23ClN8O2 (MW 430.89).
1H-NMR (300 MHz, DMSO-d6)
= 9.00 (s, 1H, N–H), 7.54–7.58 (m, 2H, Ph-2,6-H), 7.45–7.49 (m, 2H,
×
2HCl (5 mmol, 0.92 g) were
δ
Ph-4,5-H), 6.85 (s, 2H, NH2), 4.25 (s, 2H, N3–CH2), 3.60 (s, 2H, Pp-2,6-Hb), 3.30 (s, 2H, Pp-2,6-Ha), 2.26 (s,
2H, Pp-3,5-Hb), 2.16 (s, 3H, N–CH3), 2.09 (s, 2H, Pp-3,5-Ha), 1.72 (s, 3H, 5-CH3) ppm. 13C-NMR (75 MHz,
DMSO-d6) δ = 175.08, 171.94, 166.96, 164.55, 155.74, 142.65, 133.63, 130.92, 128.38, 125.80, 124.85, 63.11, 54.60,
46.19, 42.67, 42.50, 26.21 ppm. LC/MS+: Purity: 100%, tR = 3.23, (ESI) m/z [M]+ 431.12.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(2,5-dichlorophenyl)-5-methylimidazolidine-
2,4-dione (21). Ester 62 (5 mmol, 1.65 g) and 4-methylpiperazin-1-yl biguanidine
were used (3.5 h refluxed). Method C. White solid, m.p. 166 ◦C, yield: 79.10%, C19H22Cl2N8O2 (MW
465.34). 1H-NMR (300 MHz, DMSO-d6)
= 8.66 (s, 1H, N1–H), 7.72 (s, 1H, Ph-4-H), 7.54 (s, 2H, Ph-3,6-H),
6.89 (s, 2H, NH2), 4.30 (s, 2H, N3–CH2), 3.67 (m, 4H, Pp-2,6-H), 2.29 (s, 4H, Pp-3,5-H), 2.19 (s, 3H, N–CH3),
1.87 (s, 3H, 5-CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
= 174.90, 172.31, 167.02, 164.70, 156.23, 137.56,
×
2HCl (5 mmol, 0.92 g)
δ
δ

Molecules 2018, 23, 2529
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133.13, 132.49, 132.26, 130.68, 130.05, 62.65, 54.71, 46.21, 42.90, 42.49, 25.18 ppm. LC/MS+: Purity: 99.11%,
tR = 3.53, (ESI) m/z [M]+ 465.15.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(2,4-dichlorophenyl)-5-methylimidazolidine-
2,4-dione (22). Ester 63 (5 mmol, 1.66 g) and 4-methylpiperazin-1-yl biguanidine
were used (4.5 h refluxed). Method C. White solid, m.p. 228 ◦C, yield: 76.56%, C19H22Cl2N8O2 (MW
465.34). 1H-NMR (300 MHz, DMSO-d6)
= 8.65 (s, 1H, N1–H), 7.71–7.73 (d, 1H, Ph-3-H), 7.67–7.68 (s, 1H,
Ph-5-H), 7.50–7.53 (d, 1H, Ph-6-H), 6.89 (s, 2H, NH2), 4.30 (s, 2H, N3–CH2), 3.67 (s, 4H, Pp-2,6-H), 2.29 (s,
4H, Pp-3,5-H), 2.19 (s, 3H, N–CH3), 1.86 (s, 3H, 5-CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
= 175.12,
×
2HCl (5 mmol, 0.92 g)
δ
δ
172.34, 167.02, 164.70, 156.25, 134.70, 134.66, 134.57, 131.70, 130.83, 127.80, 62.49, 54.71, 46.21, 42.88, 42.47,
25.29 ppm. LC/MS+: Purity: 100%, tR = 3.66, (ESI) m/z [M]+ 465.08.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(2,3,4-trichlorophenyl)-5-methylimidazolidine-
2,4-dione (23). Ester 64 (5 mmol, 1.83 g) and 4-methylpiperazin-1-yl biguanidine
were used (4 h refluxed). Method C. White crystal, m.p. 256 ◦C, yield: 83.68%, C19H21Cl3N8O2 (MW
499.78). 1H-NMR (300 MHz, DMSO-d6)
= 7.74 (s, 2H, Ph-5,6-H), 6.89 (s, 2H, NH2), 4.32 (s, 2H, N3–CH2),
3.67 (s, 4H, Pp-2,6-H), 2.29 (s, 4H, Pp-3,5-H), 2.19 (s, 3H, N–CH3), 1.88 (s, 3H, 5-CH3) ppm. 13C-NMR
×
2HCl (5 mmol, 0.92 g)
δ
(75 MHz, DMSO-d6) δ = 175.94, 172.32, 167.01, 164.70, 156.33, 136.85, 133.94, 133.58, 132.24, 129.48, 129.09,
63.11, 54.71, 46.21, 42.90, 42.59, 25.56 ppm. LC/MS+: Purity: 98.93%, tR = 4.07, (ESI) m/z [M]+ 499.05.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-(4-methylphenyl)-5-methylimidazolidine-
2,4-dione (24). Ester 65 (5 mmol, 1.38 g) and 4-methylpiperazin-1-yl biguanidine
×
2HCl (5 mmol,
0.92 g) were used (4.5 h refluxed). Method C. White solid, m.p. 240 ◦C, yield: 49.27%, C20H26N8O2
1
(MW 410.47). H-NMR (300 MHz, DMSO-d6)
δ = 7.41–7.43 (d, J = 8.3 Hz, 2H, Ph-2,6), 7.20–7.22 (d,
J = 7.9 Hz, 2H, Ph-3,5-H), 6.84 (s, 2H, NH2), 4.23 (s, 2H, N3–CH2), 3.59 (s, 4H, Pp-2,6-H), 2.30 (s, 3H,
Ph-CH3), 2.16 (s, 4H, Pp-3,5-H), 2.10 (s, 3H, N–CH3), 1.69 (s, 3H, 5-CH3) ppm. 13C-NMR (75 MHz,
DMSO-d6) δ = 175.74, 172.10, 166.96, 164.61, 155.91, 137.49, 137.29, 129.39, 125.87, 63.21, 54.65, 46.20,
42.70, 42.44, 25.83, 21.03 ppm. LC/MS+: Purity: 98.31%, tR = 3.10, (ESI) m/z [M]+ 411.18.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-methyl-5-(naphthalen-1-yl)imidazolidine-
2,4-dione (25). Ester 66 (5 mmol, 1.56 g) and 4-methylpiperazin-1-yl biguanidine
were used (5 h refluxed). Method E. White solid, m.p. 208 ◦C, yield: 32.87%, C23H26N8O2 (MW 446.50).
1H-NMR (300 MHz, DMSO-d6)
= 8.95 (s, 1H, N1-H), 8.05–8.07 (m, 1H, Naft-8-H), 7.96–8.00 (t, 2H,
×
2HCl (5 mmol, 0.92 g)
δ
Naft-4,5-H), 7.76–7.78 (d, J = 7.5 Hz; 1H, Naft-2-H), 7.51–7.55 (t, J = 7.7 Hz, 3H, Naft-3,6,7-H), 6.90 (s, 2H,
NH2), 4.40 (s, 2H, N3–CH2), 3.63 (s, 4H, Pp-2,6-H), 2.17 (s, 4H, Pp-3,5-H), 2.07 (s, 3H, N–CH3), 2.02 (s, 3H,
5-CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
δ = 176.31, 172.32, 167.07, 164.73, 156.03, 134.61, 134.21, 130.87,
130.22, 129.62, 126.92, 126.43, 126.10, 125.43, 125.11, 64.18, 54.66, 46.20, 42.88, 42.71, 26.74 ppm. LC/MS+:
Purity: 99.06%, tR = 3.55, (ESI) m/z [M]+ 447.20.
3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5-methyl-5-(naphthalen-2-yl)imidazolidine-
2,4-dione (26). Ester 67 (5 mmol, 1.56 g) and 4-methylpiperazin-1-yl biguanidine
×
2HCl (5 mmol,
0.92 g) were used (3 h refluxed). Method E. White solid, m.p. 144 ◦C, yield: 34.57%, C23H26N8O2 (MW
1
446.50). H-NMR (300 MHz, DMSO-d6)
δ
= 9.12 (s, 1H, N1–H), 8.07 (s, 1H, Naft-1-H), 7.93–7.99 (m, 3H,
Naft-4,5,8-H), 7.72–7.74 (d def., 1H, Naft-3-H), 7.54–7.57 (m, 2H, Naft-6,7-H), 6.85 (s, 2H, NH2), 4.27 (s, 2H,
N3–CH2), 3.40–3.60 (m, 2H, Pp-2,6-Hb), 3.05–3.20 (m, 2H, Pp-2,6-Ha), 2.09 (s, 4H, Pp-3,5-H), 2.00 (s, 3H,
N–CH3), 1.83 (s, 3H, 5-CH3) ppm. 13C-NMR (75 MHz, DMSO-d6)
δ = 175.56, 172.01, 166.95, 164.53, 155.92,
137.75, 132.96, 132.81, 128.62, 128.57, 127.89, 126.95, 126.90, 124.51, 124.33, 63.56, 53.90, 46.00, 42.56, 42.47,
25.94 ppm. LC/MS+: Purity: 99.11%, tR = 3.49, (ESI) m/z [M]+ 447.20.

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4.2. Molecular Modeling
4.2.1. Optimization of the 5-HT6R Binding Site Using Induced-Fit Docking Procedure
The 5-HT6R homology model (build on β2 adrenergic template) was optimized using the
induced-fit docking (IFD) [33] protocol from Schrödinger. The IFD combines flexible ligand docking,
using the Glide algorithm [34], with receptor structure prediction and side chain refinement in Prime.
The structures of Lead 1 and Lead 2 were used as inputs to IFD. In each case, the centroid of the grid
box was anchored on D3.32 and allowed on residues refinement within 12 Å of ligand poses. The ten
top-scored L–R complexes were inspected visually to select those showing the closest compliance
with the common binding mode for monoaminergic receptor ligands [35]. The final validation of the
receptor conformations selected was performed by docking (Glide SP mode) the synthetized library,
retaining only those with a coherent binding mode for the whole set.
4.2.2. Molecular Docking
The 5-HT6R models selected in IFD procedure were used to study the binding mode of the library
synthesized. 3-dimensional structures of the ligands were prepared using LigPrep v3.6 (Schrödinger,
New York, NY, USA) [36], and the appropriate ionization states at pH = 7.4
±
1.0 were assigned using
Epik v3.4 (Schrödinger, New York, NY, USA) [37]. Compounds with unknown absolute configuration
were docked in all configurations. The Protein Preparation Wizard was used to assign the bond
orders, appropriate amino acid ionization states and to check for steric clashes. The receptor grid was
generated (OPLS3 force field [38]) by centering the grid box with a size of 12 Å on the D3.32 side chain.
Automated flexible docking was performed using Glide at SP level [39].
4.2.3. Plotting Interaction Spheres for Halogen Bonding
The halogen bonding web server was used (accessed on 22 February 2018, http://www.
halogenbonding.com/) to visualize (plotting interaction spheres) the potential contribution of halogen
bonding to ligand–receptor complexes.
4.3. Lipophilicity Study
4.3.1. Thin-Layer Chromatography
The mobile phases were prepared by mixing the respective amounts of water and organic modifier
(methanol) in a range from 40 to 90% (v/v) in 5% increments. TLC was performed on Silica gel 60RP-18 F254
plates (7
the substances. Solutions (10
10 mm apart and 10 mm from the lower edge and sides of the plates, by using Linomat V applicator
(Camag, Basel, Switzerland). The vertical chamber (Sigma–Aldrich, St. Louis, MI, USA), 20 10 18 cm
×
10 cm) plates (Merck, Darmstadt, Germany). Methanol was used to prepare the solutions of
µL) of the analyzed compounds were applied to the plates as 5 mm bands,
×
×
in size, was saturated with mobile phase for 20 min. Development was carried out over 9 cm from the
◦
starting line at a temperature of 20 C. Next, the plates were dried at room temperature, and the spots were
observed under ultraviolet light at 254 nm and/or 366 nm (UV lamp, Camag, Basel, Switzerland). In each
case, sharp and symmetric spots without a tendency for tailing were obtained. Each experiment was run
in triplicate and mean RF (retardation factor) values were calculated.
Starting from the RF values, the RM parameters were computed as described in the formula:
RM = log(1/RF − 1)
The linear correlations between the RM values of the substances and the concentration of methanol
in mobile phases were calculated for each compound with the Soczewin´ski-Wachtmeister equation [22]:
RM = RM0 + aC

Molecules 2018, 23, 2529
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where C is the concentration of the organic modifier (in %) in the mobile phase, a is the slope, and RM0
is the concentration of organic modifier extrapolated to zero.
4.3.2. Statistical Methods
Statistical analysis was performed using Statistica v10 software (StatSoft, Tulsa, OK, USA).
The correlation coefficients (r, r2), and the standard errors of the slope, interception, and estimate (Sa,
Sb, Se) were used as the basis for testing the linearity of regression plots.
4.4. Studies In Vitro
4.4.1. Radioligand Binding Assay
Radioligand binding assays were used to determine the affinity of the synthesized compounds
for human serotonin 5-HT6R, which were stably expressed in HEK-293 cells. This procedure was
accomplished via the displacement of [3H]-LSD (85.2 Ci mmol−1) for 5-HT6R. Each compound was
tested in triplicate at 7 to 8 different concentrations (10−11 to 10−4 M). The inhibition constants (Ki)
were calculated using the Cheng–Prusoff equation [40], and the results are expressed as the mean of at
least two independent experiments.
4.4.2. Toxicity In Vitro Test
The ATCC CRL 1573 HEK-293 cells were seeded in 96-well plates (NuncTM) at a concentration of
1
×
104 cells/well in 200 µ
L in Dulbecco’s Modified Eagle’s Medium (Gibco®) supplemented with 10%
fetal bovine serum (Gibco®), and cultured at 37 ◦C in an atmosphere containing 5% of CO2 for 24 h to
reach 60% confluence. The 10 mM stock solutions of 5-HT6R ligands 6 and 26 in DMSO were diluted
into fresh growth medium and added into the microplates at the final concentrations 0.1
µM –100
µM. Total DMSO concentration did not exceed 1% in growth media. The cytostatic drug doxorubicin
(Sigma-Aldrich) at the concentration 1 µM was used as a reference. After 72 h of incubation 20 µL
of CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) (Promega®) was added
to each well and the cells were incubated under the same conditions for 2 h. The absorbance of the
samples was measured using a microplate reader EnSpire (PerkinElmer, Waltham, MA, USA) at 490 nm.
Statistical significance was analyzed by GraphPad Prism
™ software v. 5.01 (GraphPad Software, La
Jolla, CA, USA) using One-way ANOVA and Bonferroni’s Multiple Comparison Post Test.
4.5. Studies In Vivo
4.5.1. Animals
The experiments were performed on male Wistar rats (200–220 g—behavioural test or
170–190 g—metabolic assay) obtained from an accredited animal facility at the Jagiellonian University
Medical College, Poland. The animals were housed in a group of four in a controlled environment
◦
(ambient temperature 21
±
2 C; relative humidity 50–60%; 12 h light/dark cycles (lights on at
8:00). Standard laboratory food (LSM-B) and filtered water were freely available (behavioral tests).
Animals were assigned randomly to treatment groups. All the experiments were performed by two
observers unaware of the treatment applied between 9:00 and 14:00 on separate groups of animals.
All animals were used only once.
The experimental protocols and procedures described in this manuscript were approved by the I
Local Ethics Commission in Cracow (no 293/2015) and complied with the European Communities
Council Directive of 24 November 1986 (86/609/EEC) and were in accordance with the 1996 NIH
Guide for the Care and Use of Laboratory Animals.

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4.5.2. Drugs
Compounds were suspended in 1% Tween 80 immediately before administration in a volume
of 2 mL/kg—behavioral tests or 1 mL/kg—body weight measures. Compounds were administered
intraperitoneally (i.p.) 60 min before testing—behavioral tests. Control animals received vehicle (1%
Tween 80) according to the same schedule.
4.5.3. Behavioral Procedures in Rats
Forced Swim Test (FST Test)
The experiment was carried out according to the method of Porsolt et al. [41]. On the first day of
an experiment, the animals were gently individually placed in Plexiglas cylinders (40 cm high, 18 cm
in diameter) containing 15 cm of water maintained at 23–25 ◦C for 15 min. On removal from water,
the rats were placed for 30 min in a Plexiglas box under 60-W bulb to dry. On the following day (24 h
later), the rats were replaced in the cylinder and the total duration of immobility was recorded during
the whole 5-min test period. The immobility was assigned when no additional activity was observed
other than that necessary to keep the rat’s head above the water. Fresh water was used for each animal.
Vogel Conflict Drinking Test
The testing procedure was based on a method of Vogel et al. [42] and used the Anxiety Monitoring
System “Vogel test” produced by TSE Systems (Germany). It was consisted of polycarbonate cages
(dimensions 26.5
×
15
×
42 cm), equipped with a grid floor made from stainless steel bars and drinking
bottles containing tap water. Experimental chambers were connected to PC software by control chassis
and on electric shocks’ generator. On the first day of the experiment, the rats were adapted to the
test chambers and drink water from the bottle spout for 10 min. Afterward, the rats were returned to
their home cages and were given 30 min free access to water followed by a 24 h water deprivation
period. The adaptation session and water deprivation protocols were repeated on the second day of the
experiment. On the third day, the rats were placed again in the test chambers 60 min after compound
26 and diazepam administration and were given free access to the drinking tube. Recording data
started immediately after the first lick and rats were punished with an electric shock (0.5 mA, lasting
1 s) delivered to the metal drinking tube every 20 licks. The number of licks and the number of shocks
received during a 5 min experimental session were recorded automatically.
4.5.4. Metabolic Assays In Vivo
The Effect of compound 6 or 26 on Body Weight by Non-Obese Rats Fed Palatable Diet (Model of
Excessive Eating)
In order to determine the anorectic activity of
6 or 26, the model of excessive eating was
used [43,44]. Male Wistar rats (170–190 g) were housed in a pair in plastic cages in constant temperature
facilities exposed to a light-dark cycle. Water and food were available ad libitum. Two groups of 6 rats
were fed with diets consisting of milk chocolate with nuts, cheese, salted peanuts, and 7% condensed
milk and also had access to standard feed (Labofeed B, Morawski Manufacturer Feed, Kcynia, Poland)
and water ad libitum for 3 weeks. Palatable control group (palatable diet + vehicle) received vehicle
(1% Tween 80, intraperitoneally), while palatable test group (palatable diet + 6 or palatable + 26)
was injected (intraperitoneally) with 6 or 26 at the dose of 5 mg/kg b.w. dissolved in 1% Tween 80
(1 mL/kg). Body weights were measured daily, immediately prior to administration of drugs.
The palatable diet contained: 100 g peanuts—614 kcal; 100 mL condensed milk—131 kcal; 100 g
milk chocolate—529 kcal; 100g cheese (Greek type)—270 kcal. The standard diet contained 100 g
feed—280 kcal.

Molecules 2018, 23, 2529
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The Effect of compound 6 or 26 on Body Weight by Non-Obese Rats Fed Only with Standard Diet
Male Wistar rats (170–190 g) were housed in a pair in plastic cages in constant temperature
facilities exposed to a light-dark cycle. Control group (standard diet + vehicle) received vehicle (1%
Tween 80, intraperitoneally), while the test group (standard diet + 6 or standard diet + 26) was injected
(intraperitoneally) with
6 or 26 at the dose of 5 mg/kg b.w. dissolved in 1% Tween 80 (1 mL/kg).
Body weights were measured daily immediately prior to administration of drugs.
4.6. Statistical Analysis
4.6.1. Lipophilicity
Statistical analysis was performed using Statistica v10 software (StatSoft, Tulsa, OK, USA).
The correlation coefficients (r, r2), and the standard errors of the slope, interception, and estimate (Sa,
Sb, Se) served as the basis for testing the linearity of regression plots.
4.6.2. Studies In Vitro and In Vivo
Statistical calculations were performed using GraphPad Prism 6 software (GraphPad Software,
La Jolla, CA, USA). Results are provided as arithmetic means with a standard error of the mean.
The data of behavioral studies were evaluated by an analysis of variance one-way ANOVA followed by
Bonferroni’s post hoc test (statistical significance set at p < 0.05). Statistical significance of body weight
was calculated using one-way ANOVA with Sidak’s multiple comparison test post-hoc or two-way
ANOVA with Sidak’s multiple comparison test post-hoc. Differences were considered statistically
significant at: p ≤ 0.05, p ≤ 0.01, p ≤ 0.001.
5. Conclusions
These comprehensive studies, including: Computer-aided design, synthesis, pharmacological
screening in vitro and in vivo, “drugability” experiments and an extensive docking-based SAR analysis,
have provided an insight into chemical properties, ligand–receptor interactions and potential therapeutic
perspectives for the new group of 1,3,5-triazine molecules. Lead structures with two topological variants
of the hydantoin linker have been identified, followed by the synthesis of numerous lead modifications.
As result, a new series of 18 active 5-HT6 agents, with submicromolar affinities confirmed in the
radioligand binding assays, has been obtained (groups A and B). Computer-aided SAR analysis indicates
the significance of both, linker and aromatic moiety properties, and their mutual dependences, for the
5-HT6R affinity of the 1,3,5-triazine derived compounds. Thus, the combination of 1-unsubstituted
hydantoin (group B) with the
followed by either the corresponding
1-(4-chlorobenzyl)-3-((4-amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)methyl)-5,5-dimethylimidazolidine-
2,4-dione ( , group A). The most active agent (26) displayed also antidepressant-like and anxiolytic-like
activities in vivo, whereas the best members of both groups ( and 26) demonstrated anti-obesity properties
β-naphthyl moiety at position 5 (26) was found as the most profitable,
α-naphthyl analogue (25) and the tri-substituted combination of
6
6
in animals fed palatable feed, as well as lipophilicity favorable for CNS drugs with a low risk of toxicity
in vitro.
The interesting pharmacological properties together with a promising “drugability” designate this
group of chemical molecules as a good starting point for further consideration in order to explore
molecular mechanisms of the 5-HT6R–ligand interaction, and even to search of new drug candidates for the
treatment of depression and obesity. In this context, 3-((4-Amino-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
yl)methyl)-5-methyl-5-(naphthalen-2-yl)imidazolidine-2,4-dione (26) can be selected as a new lead structure.

Molecules 2018, 23, 2529
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Supplementary Materials: The supplementary materials are available online.
Author Contributions: R.K. and J.H. conceived and designed the article; W.A., D.Ł., M.W. and J.H. performed
syntheses; J.H. and K.K.-K. supervised synthesis works; W.A. elaborated synthesis description (paragraph 4.1); R.K.
performed molecular modeling studies, G.S. performed radioligand binding assays; M.K. performed metabolic tests
in vivo; M.J.W., A.P., and A.W. performed behavioral tests in vivo, A.L. and, G.L. performed toxicity in vitro test; M.S.
and M.D. performed lipophilicity experiments, R.K., J.H., W.A., and C.J. wrote the paper.
Funding: This study was financially supported by Polish National Science Centre (NCN) grants: No
UMO-2015/17/B/NZ7/02973, UMO-2016/21/N/NZ7/03265 (Toxicity in vitro) and UMO-2014/15/D/NZ7/01782.
Authors thank also to the University of Saarland and the Erasmus Plus scheme for financial support of Wesam Ali.
Acknowledgments: Authors thank Andrzej J. Bojarski for the opportunity to conduct RBA and MM studies in
his Department. Thanks to our excellent technician, Maria Kaleta and to students: Kinga Półchłopek, Angelika
Nowakowska and Raluca Mihaela Sisu, for their contribution in the synthesis work.
,
,
Conflicts of Interest: The authors declare no conflict of interest.
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