Synthesis and computer-aided SAR studies for derivatives of phenoxyalkyl-1,3,5-triazine as the new potent ligands for serotonin receptors 5-HT6

Article abstract of DOI:10.1016/j.ejmech.2019.06.022

This research has provided the most active 5-HT₆R agents among 1,3,5-triazine derivatives investigated to date and has also identified the world's first selenium-containing 5-HT₆R ligands. The studies are focused on design, synthesis, biological evaluation and docking-supported SAR analysis for novel 5-HT₆R agents as derivatives of lead structure 4-(4-methylpiperazin-1-yl)-6-(phenoxymethyl)-1,3,5-triazin-2-amine (7). The lead modifications included an introduction of: (i) various small substituents at benzene ring, (ii) a branched ether linker or (iii) the ether oxygen replacement with other chalcogen (S, Se) or sulfonyl moiety. Hence, a series of new compounds (7–24) was synthesized and examined on their affinities for 5-HT₆R and selectivity, in respect to the 5-HT_1AR, 5-HT_2AR, 5-HT₇R and dopamine D₂ receptor, in the radioligand binding assays. For representative most active compounds functional bioassays and toxicity profile in vitro and antidepressant-like activity in vivo were examined. The 2-isopropyl-5-methylphenyl derivative (10) was found as the most active triazine 5-HT₆R antagonist (K_i = 11 nM). SAR analysis indicated, that an exchange of oxygen to selenium (7 vs. 22), and especially, to sulfur (7 vs. 19) was beneficial to increase both affinity and antagonistic action for 5-HT₆R. Surprisingly, an introduction of SO₂ caused a drastic decrease of the 5-HT₆R affinity, which was explained at a molecular level based on docking studies. All in vivo tested compounds (10, 18 and 21) did not show any risk of toxicity in the safety studies in vitro.

Full text of DOI:10.1016/j.ejmech.2019.06.022

Accepted Manuscript
Synthesis and computer-aided SAR studies for derivatives of phenoxyalkyl-1,3,5-
triazine as the new potent ligands for serotonin receptors 5-HT
6
Wesam Ali, Małgorzata Więcek, Dorota Łażewska, Rafał Kurczab, Magdalena
Jastrzębska-Więsek, Grzegorz Satała, Katarzyna Kucwaj-Brysz, Annamaria
Lubelska, Monika Głuch-Lutwin, Barbara Mordyl, Agata Siwek, Muhammad Jawad
Nasim, Anna Partyka, Sylwia Sudoł, Gniewomir Latacz, Anna Wesołowska,
Katarzyna Kieć-Kononowicz, Jadwiga Handzlik
PII:
S0223-5234(19)30547-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.022
EJMECH 11425
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 February 2019
Revised Date: 31 May 2019
Accepted Date: 7 June 2019
Please cite this article as: W. Ali, Mał. Więcek, D. Łażewska, Rafał. Kurczab, M. Jastrzębska-Więsek,
G. Satała, K. Kucwaj-Brysz, A. Lubelska, M. Głuch-Lutwin, B. Mordyl, A. Siwek, M.J. Nasim, A. Partyka,
S. Sudoł, G. Latacz, A. Wesołowska, K. Kieć-Kononowicz, J. Handzlik, Synthesis and computer-aided
SAR studies for derivatives of phenoxyalkyl-1,3,5-triazine as the new potent ligands for serotonin
receptors 5-HT , European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
6
j.ejmech.2019.06.022.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis and computer-aided SAR studies for derivatives of phenoxyalkyl-
1,3,5-triazine as the new potent ligands for serotonin receptors 5-HT6.
Wesam Alia,b, Małgorzata Więceka, Dorota Łażewskaa, Rafał Kurczabc, Magdalena
Jastrzębska-Więsekd, Grzegorz Satałac, Katarzyna Kucwaj-Brysza, Annamaria Lubelskaa,
Monika Głuch-Lutwine, Barbara Mordyle, Agata Siweke, Muhammad Jawad Nasima,b,
Anna Partykad, Sylwia Sudoła, Gniewomir Latacza, Anna Wesołowskad, Katarzyna Kieć-
Kononowicza, and Jadwiga Handzlika*
a Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy,
Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
b Division of Bioorganic Chemistry, School of Pharmacy, University of Saarland,
Campus B2 1, D-66123 Saarbruecken, Germany
c Department of Medicinal Chemistry Institute of Pharmacology, Polish Academy
of Sciences, Smętna 12, PL 31-343 Kraków, Poland
dDepartment of Clinical Pharmacy, Faculty of Pharmacy, Jagiellonian
University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
eDepartment of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University,
Medical College, Medyczna 9, PL 30-688 Kraków, Poland
* Corresponding authors:
(J. Handzlik) E-mail address: j.handzlik@uj.edu.pl
1

ACCEPTED MANUSCRIPT
Abstract
This research has provided the most active 5-HT6R agents among 1,3,5-triazine
derivatives investigated to date and has also identified the world’s first selenium-
containing 5-HT6R ligands. The studies are focused on design, synthesis, biological
evaluation and docking-supported SAR analysis for novel 5-HT6R agents as
derivatives of lead structure 4-(4-methylpiperazin-1-yl)-6-(phenoxymethyl)-1,3,5-
triazin-2-amine (7). The lead modifications included an introduction of: (i) various
small substituents at benzene ring, (ii) a branched ether linker or (iii) the ether oxygen
replacement with other chalcogen (S, Se) or sulfonyl moiety. Hence, a series of new
compounds (7-24) was synthesized and examined on their affinities for 5-HT6R and
selectivity, in respect to the 5-HT1AR, 5-HT2AR, 5-HT7R and dopamine D2 receptor,
in the radioligand binding assays. For representative most active compounds
functional bioassays and toxicity profile in vitro and antidepressant-like activity in
vivo were examined. The 2-isopropyl-5-methylphenyl derivative (10) was found as
the most active triazine 5-HT6R antagonist (Ki = 11 nM). SAR analysis indicated, that
an exchange of oxygen to selenium (7 vs. 22), and especially, to sulphur (7 vs. 19)
was beneficial to increase both affinity and antagonistic action for 5-HT6R.
Surprisingly, an introduction of SO2 caused a drastic decrease of the 5-HT6R affinity,
which was explained at a molecular level based on docking studies. All in vivo tested
compounds (10, 18 and 21) did not show any risk of toxicity in the safety studies in
vitro.
Keywords:
Serotonin receptors; 5-HT6R ligands; 1,3,5-triazine; thioether; selenoether; antidepressive
2

ACCEPTED MANUSCRIPT
1. Introduction
The 5-hydroxytryptamine (5-HT: serotonin) subtype 6 receptor (5-HT6R) has gained a
great interest as an attractive target for drug discovery [1]. The growing appeal of this
receptor stems, at least in part, from its almost exclusive expression in the central nervous
system (CNS), and the potential therapeutical indications of the 5-HT6R ligands which
encompass a spectrum of diseases; including depression, cognitive dysfunction associated
with Alzheimer's disease, anxiety, schizophrenia, and obesity [2-8]. Intriguingly, both 5-
HT6R agonists and antagonists may paradoxically evoke pro-cognitive, antidepressant-like,
or anti-anxiety-like effects [8]. Lines of evidence indicated several selective 5-HT6R
agonists (e.g. WAY-181187), partial agonists (e.g. EMDT 386088) and antagonists (e.g.
SB-271046, SB-399885) [9-11], intensively explored in preclinical and clinical trials but
none of them has reached the pharmaceutical market as an accepted drug (Fig. 1a). Recent
years have provided new groups of 5-HT6R antagonists, based on pyrrolo[2,3-b]pyridine-
(1) [12], pyrrolo[3,2-c]quinoline- (2) [13] or non-sulfonyl imidazo[4,5-b]pyridine (3) [14]
derivatives (Fig. 1b). The compounds (1-3) displayed potent nanomolar affinities,
nonetheless their structures resemble known ligands (Fig. 1a vs. Fig. 1b) due to the
presence of an indole-like core and/or sulfonyl moieties. Thus, more efforts of medicinal
chemists are needed to search for 5-HT6R agents in more diverse chemical groups which
could provide both potent and selective action on 5-HT6R and satisfying CNS-drugability
properties. In this context, we started to explore a new group of piperazine 1,3,5-triazine
derivatives as potent and linker-dependent 5-HT6R agents [15]. Hence, compounds with
methylene linker (4 and 5, Fig. 1c) displayed significant affinities, whereas those with
direct aromatic substituents (6) were very weak 5-HT6-agents. The decrease in activity was
also observed for triazine compounds with stiffened linker, i.e. incorporating vinyl- [16] or
hydantoin [17] moieties. Wider investigations of the role of linkers have provided an ether
compound (7) with a moderate activity towards 5-HT6R. The unsubstituted phenyl ring of
7 provides various possibilities for substitutions in order to improve pharmacophoric
properties of the hydrophobic feature. Thus, compound 7 was selected as a new lead
structure for further modifications which were performed within this study.
3

ACCEPTED MANUSCRIPT
a)
S
Cl
NH
NH
Cl
O
S
N
H
N
Cl
N
b)
N
O
O
S
Cl
N
S
O
N
NH
S
O
O
O
O
O
N
H
N
NH2
HN
EMDT 386088
SB-399885
Cl
SB-271046
WAY-181187
CF3
NH
HN
N
N
N
N
O
O
Cl
S
N
N
NH
N
S
N
N
Cl
O
3
2
O
1
Ki (5-HT6R) = 20 nM
Ki (5-HT6R) = 6 nM
Ki (5-HT6R) = 3 nM
c)
Cl
CH3
H2N
N
H2N
H2N
N
N
N
N
N
N
Cl
O
N
N
N
N
N
N
N
N
N
N
N
NH2
N
H3C
H3C
4
N
5
H3C
6
7
Ki (5-HT6R) = 507 nM
Ki (5-HT6R) = 29 nM
Ki (5-HT6R) = 2707 nM
Ki (5-HT6R) = 52 nM
Fig. 1. Structures of the 5-HT6R ligands found within previous studies; a) explored in preclinical or
clinical trials [9-11]; b) found by other research teams in 2016-2017 [12-14]; c) 1,3,5-triazine
derivatives [15, 16].
Apart from pharmacomodulation focused on benzene substituents, the lead
structure allows for variety of linker modifications, such as branching the ether linker or a
heteroatom exchange. Based on interesting pro- or/and antioxidant properties of chalcogen
compounds [18, 19], the significance of sulfur in previous 5-HT6R ligands [12, 13] and
growing interest of selenium-compounds as neuroprotective agents with therapeutic
potential [20-22], especially confirmed for selenides [23, 24], we sought to explore the
potency of S and Se-containing 1,3,5-triazine derivatives of the lead 7 as 5-HT6R ligands
with pharmacological activity in vivo.
In this context, the design and synthesis of new derivatives of lead 7 with
(un)branched linker and a different substitution at phenyl ring as well as their respective S-
4

ACCEPTED MANUSCRIPT
or Se analogues (8-24, Table 1), including one sulfone analogue (24), were performed. The
5-HT6R affinity and selectivity for the series were tested in the radioligand binding assay.
For selected compounds intrinsic activity in functional bioassays, toxicity profile in vitro
and antidepressant-like activity in vivo were examined.
Table 1. Structure of the compounds investigated.
NH2
N
N
X
N
N
N
R2
H3C
R1
7-24
Cpd
R1
R2
X
Cpd
R1
R2
X
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
3-Me, 4-Cl
3-Cl
2-Cl
H
H
Me
Me
H
O
O
H
2-Cl
7
16
17
18
19
20
21
22
23
24
8
3-Cl
O
9
2-i-Pr, 5-Me
2-Me
S
10
11
12
13
14
15
2-Cl
2-Cl
H
H
S
3-Me
Me
H
S
4-Me
Se
Se
SO2
2-Me, 4-Cl
2,4-diCl
H
Me
H
2-Cl
2. Results
2.1. Synthesis
The synthesis of the compounds (8-24) investigated followed several chemical approaches
due to different availability of starting materials (Scheme 1). The common step for
compounds 8-23 (Scheme 1a) was the formation of the triazine ring via cyclic
condensation of 4-methyl piperazine-1-yl biguanide dihydrochloride (25) with an
appropriate ester (26-41), according to a method already described [15-17, 25, 26]. In the
case of synthesis of sulfone 24, selective oxidation of the thioether sulfur atom of 20 was
performed according to Scheme 1b. The ester intermediates necessary for synthesis of 8-28
were obtained via five different routes. For preparation of compounds 11, 12, 16-18 and
21, commercially available (thio)ether derivatives of methyl esters (26-31) were purchased.
For the compounds 8, 9, 13, 19 and 20, related (thio)ether derivatives of methyl- or ethyl
esters (32-36, Scheme 1c) were obtained during O- or S-alkylation of adequate
5

ACCEPTED MANUSCRIPT
(thio)phenols (42-46) with suitable alkyl bromoacetates (47, 48). Moreover, methyl ester
37 was obtained via acid-catalyzed esterification of corresponding commercial
phenylacetic acid (49), whereas reactions of suitable carboxylic acids (50, 51) with
iodomethane in the presence of DBU were carried out for esters 38 and 39 [15] (Scheme
1d). The synthesis of Se-containing esters 40 and 41 was performed starting from diphenyl
diselenides, and commercial bromoesters (48, 52) were employed as alkylation agents
(Scheme 1e).
R1
NH2
a)
H2N
HN
NH
NH
N
N
(i)
X
N
N
+
X
R2
N
N
R2
H3C
R1
x 2HCl
O
O
R3
N
8-23
CH3
Compounds 11,12, 16-18 and 21
synthesized from commercial esters 26-31
25
26-41
R1
b)
c)
NH2
N
Cl
(ii)
N
O
R1
O
(iii)
R2
S
X
20
XH
+
N
N
Br
R3
O
O
N
24
H3C
O
O
47, 48
42-46
R3
47: R3=Et
48: R3=Me
32-36
d)
e)
37
(iv)
O
R3
Br
(vi)
Se
R1
+
Se
O
OH
(v)
40, 41
O
R2
O
38, 39
48, 52
49-51
8, 32, 42:
9, 33, 43:
10, 37, 49:
11, 26:
X=O; R1=2-Cl;
X=O; R1=3-Cl;
R2=H; R3=Et
R2=H; R3=Et
16, 28:
17, 29:
18, 30:
19, 35, 45:
20, 36, 46:
21, 31:
X=O; R1=3-Me,4-Cl; R2=H; R3=Me
X=O; R1=3-Cl,
X=O; R1=2-Cl;
X=S; R1=H;
X=S; R1=2-Cl;
X=S; R1=H;
X=Se; R1=H;
X=Se; R1=H;
R2=Me; R3=Me
R2=Me; R3=Me
R2=H; R3=Me
R2=H; R3=Me
R2=Me; R3=Me
R2=H; R3=Me
R2=Me; R3=Me
X=O, R1=2-i Pr, 5-Me; R2=H; R3=Me
X=O; R1=2-Me;
X=O; R1=3-Me
X=O; R1=4-Me;
R2=H; R3=Me
R2=H; R3=Me
R2=H; R3=Et
12, 27:
13, 34, 44:
14, 38, 50:
15, 39, 51:
X=O, R1=2-Me, 4-Cl; R2=H; R3=Me
22, 40:
23, 41, 52:
X=O, R1=2,4-diCl;
R2=H; R3=Me
Scheme 1. Synthesis route for final compounds 8-24 (a, b) and intermediates 32-36, 40 and 41 (c-
e). a) The cyclic condensation for 8-23; (i) absolute methanol, Na, reflux 15-30 h. b) The selective
.
oxidation for 24; (ii) ethanol, Na2WO4 2H2O, H2O2, rt. c) O- or S-alkylation for 32-36; (iii)
acetonitrile, K2CO3, reflux. d) Esterification for 37-39; (iv) dried methanol, concentrated H2SO4,
reflux; (v) dried toluene, DBU, iodomethane, rt. e) Synthesis of Se-intermediates 40 and 41; (vi)
THF/water, NaBH4, N2, rt.
6

ACCEPTED MANUSCRIPT
2.2. Molecular Modelling
Molecular docking was employed to study the mechanism of action of a newly synthesized
library of compounds with the 5-HT6R. Since this receptor has not been crystallized so far,
its homology models used for studying the interaction of other 1,3,5-triazine derivatives
[15, 16] have been applied. In general, results revealed that newly synthesized compounds
(7–24) had a very consistent binding mode with the previously reported 1,3,5-triazine
derivatives [15, 16]. Small structural modifications introduced in the obtained library, and
their influence on 5-HT6R activity was reflected in the ligand-receptor complexes obtained.
Emphasis was placed on: (i) the influence of the oxygen replacement in the linker on the
sulfur and sulfone group, (ii) the influence of different substituents in the phenyl ring, and
(iii) the role of the branching in the linker (Fig. 2).
Fig. 2. The interactions of selected 1,3,5-triazine analogs with 5-HT6R. (A) A difference of the
binding modes of derivatives with O (8–green), S (20–cyan), and sulfonyl (24–red) in the linker.
(B) A comparison of the binding modes of selected analogs with different substituents in the
phenyl ring (10–orange, 14–magenta, 11–brown). (C) Illustration of the binding modes for analogs
with hydrogen (20–cyan) and methyl (21–yellow) linker substituent.
2.3. Radioligand binding assays
Radioligand binding assays were applied to determine the affinity and selectivity profiles
of the newly synthesized compounds for human serotonin 5-HT6R in comparison to 5-HT7,
5-HT1A, 5-HT2A and dopaminergic D2 receptors. All derivatives tested, except 14 and 24,
displayed higher affinity towards 5-HT6R (Ki < 400 nM) than that of lead 7 (Table 2).
Compounds 10, 11, 18, 19, 21 demonstrated high affinities for 5-HT6R (Ki < 100 nM),
particularly potent in the case of 10, 18, 19 and 21 (Ki ≤ 30 nM). Each of the active
7

ACCEPTED MANUSCRIPT
compounds featured predominant affinity toward 5-HT6R in comparison to both 5-HT1AR
and D2R. Compounds 10, 11, 18, 19 and 21 exhibited the highest selectivity to 5-HT6R
over D2, 5-HT1A, 5-HT2A and 5-HT7 receptors. Compound 10 was identified as the most
active one with an affinity for 5-HT6R in the range of the standard 5-HT6R antagonist
olanzapine. In parallel, compound 10 was much more selective with respect to the D2-
dopamine receptor when compared to the dual target reference olanzapine.
Table 2. Radioligand binding assays results for the investigated compounds (7-24).
Ki [nM]a
Cpd
5-HT6R
507
129
148
11
D2R
1348
2362
674
1094
4247
740
2916
4369
1234
1224
566
1001
734
329
520
506
5-HT1AR
8726
5467
7505
12530
14160
15070
28510
7688
1610
3584
3438
4540
4389
1680
3384
3176
5311
>106
5-HT2AR
2628
nt
5-HT7R
9531
9247
5818
11950
514
10660
16050
15180
3804
5840
8361
38730
2871
614
7
8
9
nt
430
17170
2274
nt
nt
nt
197
590
1830
197
nt
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Refb-d
87
207
235
911
128
355
118
23
26
126
19
407
406
376
4570
2329
4247
98430
18d
242
111
3574
7b
656
50140
9b
11590
-
20c
aTested experimentally in the radioligand binding assay; binding affinity, Ki, expressed as the
average of at least two independent experiments. Radioligands used: [3H]-8-OH-DPAT (5-
HT1A), [3H]-Ketanserin (5-HT2A), [3H]-LSD (5-HT6), [3H]-5-CT (5-HT7) and [3H]-Raclopride
b-d
b
c
(D2). Results for 5-HT6R bolded. Reference ligands for GPCRs investigated: olanzapine,
buspirone, d clozapine.
2.4. Functional assays
For selected active 5-HT6R ligands (10, 18, 21), their intrinsic activity was investigated in
vitro by the cellular aequorin-based functional assay with recombinant CHO-K1 cells
expressing mitochondrially targeted aequorin, human GPCR and the promiscuous G
protein α16 for 5-HT6R. Compound SB-742457 served as the highly potent antagonist,
mianserin as moderate one, and serotonin as reference agonist (Table 3). The results
obtained indicated a lack of agonistic activity, and rather pointed towards a potent
8

ACCEPTED MANUSCRIPT
antagonist action for the triazines tested, with Kb values in the nanomolar range (Kb = 2.9–
4.6 nM).
Table 3. Functional- vs. Radioligand binding assays results for 10, 18 and 21.
5-HT6R
Compound Binding affinity (RBA)
Antagonist mode*
Agonist mode**
Ki [nM]
Kb [nM]
4.60
3.00
2.90
nc
Emax [%]
11
23
19
-
-
-
1
1
1
100
2
1
10
18
21
Serotonin
SB-742457
Mianserin
0.67
30.0
*Results were normalized as percentage of maximal response in the absence of antagonist.
**Results were normalized as percentage of maximal agonist response (serotonin 10-6 M). Emax is
the maximum possible effect; nc - not calculable.
2.5. In vivo studies
2.5.1. Antidepressant-like activity of compounds investigated
Compounds 10, 18 and 21 were selected for behavioral studies in vivo in male Wistar rats.
Their potential antidepressant-like properties were assessed in the forced swim test (FST)
[27]. These compounds exhibited different activity in the FST (Fig. 3). Compound 10, for
instance, decreased about 20% immobility at the doses of 0.3–3 mg/kg (but not at 0.1
mg/kg) compared to the vehicle group (one-way ANOVA, F(4,35) = 4.5546; p < 0.01).
Compound 18 showed antidepressant-like activity only in one medium (1 mg/kg) dose; it
decreased the immobility time about 22 % vs the respective vehicle treated group (one-way
ANOVA F(3,24)=4.0751; p < 0.05) (Fig. 3). Compound 21 was not active in the FST (one-
way ANOVA F(3,24)=2.4252; NS; Fig. 3). The reference 5-HT6 receptor antagonist,
compound SB-399885, was active in the FST only at the dose of 10 mg/kg
(F(3,28)=9.7153; p < 0.0001; Fig. 3) [28]. An U-shaped dose-response in the FST test for
compounds 18 and SB-399885 was observed. This effect seems to be typical for some
antidepressants with different mechanisms of action also producing U-shaped dose-
response effects in some animal models and tests for antidepressant-like activity [29, 30].
Moreover this effect was also observed for other 5-HT6 ligands, such as standard potent 5-
HT6R antagonist SB-258585 [31]
.
9

ACCEPTED MANUSCRIPT
Fig. 3. Effects of compounds 10, 18, 21 and SB-399885 on the immobility time of rats in the forced
swim test. Compounds 10, 18, and 21 were administered i.p. 60 min, whilst SB-399885 was given
30 min before the test. The animals were observed for 5 min. The data are presented as the mean ±
SEM of 6-8 rats. The data were statistically evaluated by one-way ANOVA followed by
Bonferroni’s post-hoc test, * p < 0.05, vs. respective vehicle group.
2.5.2. Locomotor activity
For compounds 10 and 18, locomotor activity assays in rats were also performed. These
triazine derivatives, especially compound 10 at the doses active in the FST, slightly
increased locomotor activity measured in the open field (OF) test (Table 4).
Table 4. Effects of compounds 10 and 18 on the rat locomotor activity measured in the OF
test.
Dose
(mg/kg)
0
Total distance
(cm)
2165 ± 119
Ambulation
X
190 ± 12
Ambulation
Y
187 ± 15
Treatment
Vehicle
Rearings
57 ± 6
2833 ± 157 ***
307 ± 19 ***
290 ± 19 **
56 ± 3
0.3
0
F(1,10)=11.502; F(1,10)=27.956; F(1,10)=18.645; F(1,10)=0.0157;
10
p<0.01
929 ± 36
1005 ± 68
p<0.001
213 ± 13
269 ± 8 **
p<0.01
187 ± 14
227 ± 14
NS
68 ± 7
46 ± 4 *
Vehicle
18
1
F(1,10)=0.9711; F(1,10)=14.308; F(1,10)=4.5275; F(1,10)=6.4150;
NS p<0.01 NS p<0.05
Compounds were administered i.p. 60 min before the test. The animals were observed for 5 min.
The data are presented as the mean ± SEM of 5-6 rats. The data were statistically evaluated by one-
way ANOVA followed by Bonferroni’s post-hoc test, * p<0.05, **p<0.01, ***p<0.001 vs vehicle
group, NS=not significant.
10

ACCEPTED MANUSCRIPT
These results indicate that the antidepressant-like effects in the FST observed may not be
specific ones. Compound SB-399885 did not change the rats’ locomotor activity under
identical experimental settings [31].
2.6. Drug-likeness
The safety profile of the most active triazine 5-HT6R agents (10, 18 and 21) was estimated
in, both neurotoxicity and mutagenicity assays in vitro.
Neurotoxicity was examined in the SH-SY5Y eukaryotic cell line. The colorimetric MTS
assay allowed the determination of SH-SY5Y cell viability after 72 h of incubation with
10, 18 and 21, respectively. A statistically significant decrease in cell viability was
observed only for the highest dose for each of the 5-HT6R agents tested (100 µM, Fig. 4).
Fig. 4. The impact of doxorubicin (DX), 10, 18 and 21 on the viability of the neuroblastoma SH-
SY5Y cell line after 72 h of incubation. Statistical significance was evaluated by one-way
ANOVA, followed by Bonferroni’s comparison test (∗∗∗p < 0.001 compared with the negative
control).
The mutagenicity of 10, 18 and 21 was determined following the Ames microplate
fluctuation protocol (MPF) with Salmonella typhimurium strain TA100, which enabled the
detection of frame shift mutations [32]. The results were compared to the reference
mutagen nonyl-4-hydroxyquinoline-N-oxide (NQNO) at 0.5 µM. The numbers of
revertants were estimated colourimetrically and the mutagenicity potential was evaluated
according to the procedure provided by the manufacturer [32]. The medium control
baseline (MCB) was calculated first, as a number of revertants observed in the growth
medium with 1% DMSO as negative control plus standard deviation. A compound was
considered as mutagenic one if data point with fold increase ≥ 2 of MCB, and Binomial B-
value ≥ 0.99 (Table 5). The results of the study did not indicate any risk of mutagenicity
for the triazine derivatives 10, 18 and 21 tested.
11

ACCEPTED MANUSCRIPT
Table 5. The mutagenicity potential of 10, 18 and 21.
Conc.
[µM]
0.5
Fold increase Binomial B-
Cpd
Number of revertants ± SD
over MCB
value*
1.0000
0.0570
5.41
46.00 ± 2.29
4.67 ± 1.53
NQNO
10
0.55
1
18
0.73
0.1755
8.00 ± 2.65
21
0.94
0.47
0.7035
0.0172
10.33 ± 1.53
4.00±1.00
10
10
18
1.00
0.76
0.8259
0.2366
11.00 ± 2.65
8.33 ± 3.06
21
* Binomial B-value indicates the probability that spontaneous mutation events alone. The result
≥0.99 indicates that chances of spontaneous mutation are ≤1 %.
In summary, compounds 10, 18 and 21 displayed statistically significant neurotoxicity only
at > 4000-fold higher concentrations than the one required for 5-HT6R activity (Ki= 11–23
nM, Table 1) and 100–fold higher concentrations than the one for the positive control
doxorubicin. Furthermore, none of the compounds investigated caused any mutagenic
effects. Thus, results of the study using models in vitro indicated high pharmaceutical
safety for these active 5-HT6R antagonists.
3. Discussion
The modifications performed on the lead 7 were addressed in order to analyse an influence
on the affinity for 5-HT6R of three following structural factors: (i) the substituents at
phenyl ring, including kind, number and position; (ii) the methyl (un)branched linker and
(iii) different chalcogen-ether linker with respect to the S-oxidized form (SO2) that is an
indispensable structural feature of more than 80% 5-HT6R agents found within the 20-year
global studies on this receptor [33]. The results obtained within this study allow to perform
comprehensive structure-activity relationship discussion.
The first subseries of 11 unbranched ether derivatives (7–16, Table 1) enabled to estimate
SAR for substituents at the phenyl ring. On the basis of previous results, we took into
consideration a variety of alkyl- and chlorine substituents only, since they were found as
beneficial, in contrast to alkoxyl moieties [15]. Thus, the following order of the 5-HT6R
affinity decrease can be observed: 2-iPr-5-Me>>2-Me>2-Cl~2,4-diCl>3-Cl>3-Me>4-
Cl>4-Me>3-Me-4-Cl>H>2-Me-4-Cl (Table 2) [16]. This order confirmed the general
12

ACCEPTED MANUSCRIPT
preference of electrodonating and hydrophobic substituents at the phenyl ring that was also
observed for previous methylene- [15] and hydantoin-linked aromatic triazine 5-HT6R
agents [17]. The isopropyl derivative (10) seems to be a special example of optimal
electrodonating-hydrophobic balance for the substituent that, as more hydrophobic than
methyl- (10 vs. 11, Table 1), and stronger electro-donating than the hydrophobic chlorine
substituent (10 vs. 8), provided an outstanding 5-HT6 affinity, not only in the considered
series but also among all 1,3,5-triazines 5-HT6R agents investigated so far [15-17]. As
position of substituents at phenyl ring, the previous studies indicated the preferable meta-
position in the case of benzyltriazine derivatives [15], while para one was emphatically
beneficial for the 5-HT6R affinity of the hydantoin-linked series [17]. In contrast, the
presently considered ether-linked 1,3,5-triazines distinctly favour ortho-position in the
binding with 5-HT6R in comparison to both meta and para. Furthermore, the following
trend of the 5-HT6R affinity increase: para < meta < ortho, can be observed, either for Cl-
(5-HT6R Ki: 226nM>148nM>129nM) [16] or for Me- (5-HT6R Ki: 235nM>207nM>87nM)
substituents. Otherwise, an impact of di-substitution is not so clear and seems to be rather
unprofitable (16, 14) or at best indifferent (15), and also responsible for selectivity
decrease (16 vs. 12).
In concordance to previous studies [15-17], results of this work underline the linker-
dependent influence of Ph-substituents on the activity towards 5-HT6R. In the case of S-
and Se-ether linked triazines (19-23), the impact of substituents at the phenyl ring seems to
be less meaningful than that observed for O-ether compounds (7-16), and significantly
dominated by the impact of methyl-branching. It is particularly evident during analysis of
results for S-ether compounds 19–21, where the methyl-branched o-Cl-phenyl- (21) and
the unbranched phenyl-unsubstituted (19) derivatives displayed comparable affinities for
5-HT6R, and significantly more potent than that of the o-Cl-phenyl-, but linker-
unbranched, derivative 20 (Table 1). The similar trend is also observed for selenoethers (22
and 23) demonstrating in 2-fold increase of the 5-HT6R affinity with the appearance of
methyl-branching within the linker.
Two parallel comparative analyses of the linker-activity relationship, performed for o-Cl-
(8, 18, 20, 21 and 24) and (un)substituted phenyl (7, 19, 22 and 23) derivatives,
respectively, allow to assess the role of hetero-atom within the linker, connecting phenyl
moiety with 1,3,5-triazine. In the case of o-chlorophenyl derivatives, the following activity
order was found: S (Me-branched) ≥ O (Me-branched) > S (unbranched) ≥ O (unbranched)
>> SO2, while the 5-HT6R affinity decrease for the phenyl derivatives was as follows: S
13

ACCEPTED MANUSCRIPT
(unbranched) > Se (Me-branched) > Se (unbranched) > O (unbranched). Both sequences
distinctly indicate the beneficial impact of the sulphide linker, whereas the selenoether
seems to be slightly more profitable than the simple O-ether. It is worth to emphasize that
presence of SO2 drastically decreased the desirable pharmacological activity. This is a
breakthrough observation against the majority of world's lines of evidence [33]. Thus, we
have carried out computer-aided analysis to explain this and other SAR-observations in the
molecular level.
The docked compounds showed very consistent binding mode, i.e. they formed the salt
bridge interaction with D3.32, an aromatic interaction (CH–π or π–π stacking) with F6.52,
and a hydrogen bond between NH2 group of 1,3,5-triazine ring and carbonyl oxygen of
V3.33 and/or A5.42 (Fig. 2). These contacts were crucial for recognition the active
scaffolds by the 5-HT6 receptor, however, the substituted aromatic moiety linked with the
triazine ring was found to have a significant influence on the activity modulation [15, 16].
This fragment interacted with the hydrophobic cavity formed by transmembrane domains
(TM) 3–5 and extracellular loop 2 (ECL2), and the positioned into this cavity was resulted
by the tetrahedral conformation of the linker.
Two used modifications of the linker, namely: (1) changing the oxygen on the
chalcogen atoms (S and Se) or a sulfone group and (2) branching the linker by adding the
methyl group, were also explored within this simulation in silico. The derivatives with
oxygen, sulfur and selenium atom in the linker showed high-to-medium activity to 5-HT6R
(11–911 nM). Analysis of the docking results (Fig. 2A) indicated that these compounds
had a very similar binding mode, and the potential role of the atoms mentioned above is to
bend the conformation of the molecule and further directing the aryl fragment into the
hydrophobic pocket formed by TM3–5 and ECL2. However, a significant decrease in the
activity was found for derivative with sulfonyl group 24 (Ki = 3574 nM). The analysis of
binding mode revealed that it may be caused by forcing a straightened conformation (in
which the phenyl and triazine rings are in one plane and do not form an angle of 90 ), and
thus no interaction with the hydrophobic pocket is probable. In order to confirm this
hypothesis, the geometry optimization for compound 24 was additionally performed using
quantum-mechanical methods (DFT: B3LYP/6-31++G**, PCM model with water as a
solvent). The calculations confirmed that the most energetically stable conformation is
straightened and the repulsion interaction between free electron pairs of one of the triazine
nitrogen atoms and oxygen of the sulfone group was responsible for inducing such
conformation.
14

ACCEPTED MANUSCRIPT
A comparison of the binding modes for analogs having different substituents at phenyl
fragment (Fig. 2B) showed that modulation of the activity can be related with the position
and the size of the substituents. The most active compound 10 (Ki = 11 nM) was very well
fitted to the hydrophobic sub-pocket formed by helices 3–5 and extracellular loop 2.
However, the less active 14 (Ki = 911 nM) showed that improper configuration of
substituents at the phenyl ring may even lead to destabilization of the complex and
breaking some key interactions (the NH2 group does not form hydrogen bonds, Fig. 2B).
The branching of the linker by a methyl group increased the affinity for each of the
compared pairs, i.e.: 8 vs. 18; 20 vs. 21; 22 vs. 23. Molecular docking indicated (Fig. 2C)
that in each case the methyl group was located in a small binding cavity formed by helices
5 and 6 which increased the stability of the resulting ligand–receptor complexes.
Our comprehensive studies allowed also to examine the selectivity of the series (7–
24) towards the target 5-HT6R as well as the intrinsic activity, the primary antidepressive-
like action in vivo and safety properties in vitro for the most active compounds elected (10,
18 and 21). Although some of the assays were performed for a few members only,
different results are noteworthy for an initial qualitative SAR analysis. Results of the
receptor screening have indicated a favourable 5-HT6R-selectivity, in respect to 5-HT7-, 5-
HT1A, 5-HT2A- and dopaminergic D2L receptors, for almost whole investigated ether 1,3,5-
triazine derivatives (7–24), with one exception of the 3-methyl-4-chlorophenyl derivative
16 that acted almost 2-fold stronger towards 5-HT2A (Table 1). However, the affinity of 16
for either 5-HT6R- or 5-HT2AR was rather moderate (Ki > 100 nM). Hence, the results for
the whole series indicate that this chemical family, consisting the skeleton of 2-amine-
1,3,5-traizine core with methyl piperazine at position 4 and ether-linked phenyl at position
6, tend to selective action towards desirable 5-HT6R over other GPCR-competitors.
The functional assays have confirmed a strong antagonistic action for the tested
compounds (10, 18 and 21), the most potent in the case of the thioether 21 (Kb = 2.8 nM).
However, this potent antagonist did not demonstrate antidepressant-like activity in the
behavioral tests in vivo, while both the 2-isopropylphenyl (10) and 2-chlorophenyl (18) O-
ether derivatives were active in the FST assay with probable non-specific action, estimated
on the basis of the open field test results. Taking into account, still not enough recognized
mechanisms of physiological actions mediated by 5-HT6R receptor and lines of evidence
indicating wider pharmacological abilities of its ligands, including: pro-cognitive,
anxiolytic and anti-obesity properties, the obtained lack of anti-depressive potency in vivo
15

ACCEPTED MANUSCRIPT
for the potent sulfide antagonist 21 does not exclude other possible pharmacological
directions for this agent, and even suggests some selectivity in comparison to the O-
analogs (10 and 18). Furthermore, results of both, the neurotoxicity- (MTT-test) and the
mutagenicity AMES screening, confirmed profitable CNS-drug-likeness properties for all
the investigated compounds (10, 18 and 21).
Results obtained for the two “pilot” selenoethers (22 and 23) are worth emphasizing
since both 22 and 23 displayed significant affinities, although they do not possess any
beneficial substituents at the phenyl moiety. The unbranched Se-ether 22 was two-fold
more active than corresponding O-ether derivative 7.
4. Conclusions
The studies reported here are concerned with the design, synthesis, molecular
modelling and biological screening, including: (i) RBA-evaluated affinity and selectivity
towards 5-HT6R in respect to 5-HT1AR, 5-HT2AR, 5-HT7R and dopamine D2 receptor, (ii)
functional assays, (iii) behavioural assays in vivo and (iv) safety screening in vitro for a
new series of phenoxyalkyl derivatives of 1,3,5-triazine. On the basis of the data obtained,
an insightful SAR-analysis was performed. In the course of the study, a new family of
active and selective 5-HT6R antagonists was obtained, which may serve as a tool for
deeper insight into the mechanisms of action of this most intriguing member of the
serotonin GPCRs. Although this series was not such SAR-regular as the arylmethyl-1,3,5-
triazines described previously, it has provided the most active 1,3,5-triazine 5-HT6R agents
to date, i.e. 4-((2-isopropyl-5-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-
triazin-2-amine (10) and 4-(1-((2-chlorophenyl)thio)ethyl)-6-(4-methylpiperazin-1-yl)-
1,3,5-triazin-2-amine (21), with Ki < 20 nM. Furthermore, the world’s first selenium-
containing 5-HT6R agents (22 and 23, Ki < 250 nM) were identified within these studies.
SAR-analysis indicated that presence of sulphide is especially beneficial, while the SO2-
containing derivative (24) investigated proved to be distinctly less active than the other
members of the series (Ki > 3 µM). This result, contrary to a majority of lines of evidence,
was justified in the molecular level by our docking studies. Taking into account lines of
evidence indicating antidepressive, procognitive and neuroprotective roles of selenides, in
addition to a wide range of pharmacomodulation possibilities for these unsubstituted-
phenyl derivatives, the selenocompounds 22 and 23 seem to represent an excellent starting
16

ACCEPTED MANUSCRIPT
point for new chemical modifications in search for successful CNS-selenoagents acting via
5-HT6 receptor.
The most active agents 10 and 21 require wider pharmacological evaluation with a
prospect to produce new CNS-drug candidate. Compounds 10 and 21 and the most active
selenocompound 23 can be considered as suitable new lead structures for further chemical
modifications in search for highly potent and selective 5-HT6R antagonists.
The series of phenoxyalkyl-1,3,5-triazine derivatives presented seems to be a new
promising alternative in search for 5-HT6R agents which, as distinctly structurally differing
from the world’s dominating derivatives of sulfone and indole, may form a basis for a
breakthrough in the search for new therapies of the civilization CNS diseases related to this
exceptional serotonin GPCR.
5. Experimental
5.1. Chemical synthesis
Melting points (mp) were determined using MEL-TEMP II apparatus and are uncorrected.
1H-NMR and 13C-NMR spectra were recorded on a Varian Mercury-VX 300 MHz PFG
instrument or FT-NMR JEOL (JNM-ECZR500 RS1 v. ECZR) 500 MHz instrument (13C-
NMR for 8-10, 12-16 and 21) in DMSO-d6 (for most compounds) or MeOD (13C-NMR for
19 and 24) at ambient temperature using the solvent signal as an internal standard: the
values of the chemical shifts expressed as δ values in (ppm) and the coupling constants (J)
in Hz. Data are reported as follows: chemical shift, multiplicity (s, singlet; br. s, broad
singlet; d, doublet; t, triplet; dd, doublet of doublet, q, quintet, td triplet of doublet, m,
multiplet), coupling constant J, number of protons, proton’s position (Ph-phenyl, Pp-
piperazine). Mass spectra recorded on a UPLC–MS/MS system consisted of a Waters
ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) coupled to a Waters TQD
mass spectrometer (electrospray ionization mode ESI-tandem quadrupole). The UPLC/MS
purity of all the final compounds was confirmed to be higher than 95% (except 24).
Retention time values (tR) are given in minutes. Elemental analyses (C, H, N) were
performed on an Elemental Analyser Vario El III (Hanau, Germany) and agreed with
theoretical values within ±0.4%.Thin-layer chromatography (TLC) was performed on pre-
coated Merck silica gel 60 F254 aluminum sheets. The reactions at fixed temperature were
carried out using a magnetic stirrer with a contact thermometer Heidolph MR 2001. Esters
32-41 have been synthesized due to their high commercial costs. However, all of them are
17

ACCEPTED MANUSCRIPT
1
already described in Chemical Abstract Database, thus, their CAS numbers and H-NMR
or LC/MS+ are indicated in Supplementary Information.
5.1.1. General procedure for the synthesis of final 1,3,5-triazine derivatives (8–23)
Sodium (10 mmol) was dissolved in 10 ml of absolute methanol, then 4-methylpiperazine-
1-yl biguanide HCl (5 mmol) and a suitable carboxylic acid ester containing O- S- or Se-
ether, respectively, (26–41, 5 mmol) was added. The reaction mixture was refluxed for 15-
30 hours. After cooling to room temperature, the solvent was evaporated, and the residue
was dissolved in water (10 ml), stirred for 30 minutes at room temperature and kept
overnight at the fridge. The precipitated triazine product was isolated by filtration and
crystallized from methanol to give the desire final products as solids in basic form (method
A). In case of lack of desirable precipitate, the methanol solution was saturated with
gaseous HCl to give pure final product in hydrochloric salt form (method B)
5.1.1.1. 4-((2-Chlorophenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (8)
Method A. White solid, Yield 23%, mp 141-142˚C. C15H19ClN6O (MW 334.80). LC/MS+:
1
purity: 100%, tR=3.25, (ESI) m/z [M+H]+ 335.31. H-NMR
δ
: 7.41-7.37 (dd, J1=7.8 Hz,
J2=1.67 Hz, 1H, Ph), 7.27-7.24 (m, 1H, Ph), 7.05-6.98 (m, 4H, Ph-2,6-H, NH2), 4.88 (s,
2H, CH2), 3.62 (s, 4H, Pp-2,6-H), 2.23 (s, 4H, Pp-3,5-H), 2.15 (s, 3H, CH3). 13C-NMR
(125 MHz, DMSO-d6) δ: 173.01; 167.28; 164.75; 154.46; 130.44; 128.61; 122.04; 121.78;
114.73; 70.53; 54.79; 46.27. Anal. calcd. for C15H19ClN6O: C 53.81%, H 5.72%, N
25.10%. Found: C 53.86%, H 5.86%, N 25.19%
5.1.1.2. 4-((3-Chlorophenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (9)
Method A. Pale yellow solid. Yield 30%, mp 140-141˚C. C15H19ClN6O (MW 334.80).
1
LC/MS+: purity: 100%, tR=3.45, (ESI) m/z [M+H]+ 335.31. H-NMR
δ
: 7.26 (t, J=8.1 Hz,
1H-Ph), 7.00-6.86 (m, 2H, NH2), 7.00 (m, 1H, Ph), 6.98-6.94 (m, 1H, Ph), 4.80 (s, 2H,
CH2), 3.65-3.62 (m, 4H, Pp-2,6-H), 2.24 (s, 4H, Pp-3,5-H), 2.15 (s, 3H, CH3).
13C-NMR (125 MHz, DMSO-d6) δ: 173.03; 167.29; 164.76; 159.95; 134.06; 131.27;
121.12; 115.38; 114.33; 70.25; 54.80; 46.28. Anal. calcd. for C15H19ClN6O: C 53.81%, H
5.72%, N 25.10%. Found: C 53.80%, H 5.72%, N 25.20%
5.1.1.3. 4-((2-Isopropyl-5-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-
2-amine (10)
Method A. White solid. Yield 23%, mp 92-93˚C. C19H28N6O (MW 356.47). LC/MS+:
1
purity: 100%, tR=4.57, (ESI) m/z [M+H]+ 357.38. H-NMR
δ
: 7.02 (d, J=7.7 Hz, 1H, Ph),
18

ACCEPTED MANUSCRIPT
6.87 (br. s, 2H, NH2), 6.71 (s,1H, Ph), 6.67 (d, J=7.9 Hz, 1H, Ph), 4.74 (s, 2H, CH2), 3.65
(t, J=4.7 Hz, 4H, Pp-2,6-H), 3.30-3.22 (m, 1H, CH), 2.25 (s, 4H, Pp-3,5-H), 2.20 (s, 3H,
CH3), 2.15 (s, 3H, CH3), 1.14 (d, J=6.9 Hz, 6H, 2x-CH3). 13C-NMR (125 MHz, DMSO-d6)
δ: 173.70; 167.31; 164.93; 156.03; 136.14; 133.82; 126.09; 121.67; 113.31; 70.30; 54.80;
46.27; 26.76; 23.12; 21.52. Anal. calcd. for C19H28N6OxH2O C 60.88%, H 8.01%, N
22.43%. Found: C 60.60%, H 7.90%, N 22.39%.
5.1.1.4. 4-(4-Methylpiperazin-1-yl)-6-(o-tolyloxymethyl)-1,3,5-triazin-2-amine (11)
Method A. White solid. Yield 45%, mp 252-254˚C. C16H22N6O (MW 314.39). LC/MS+:
1
purity: 100%, tR=3.19, (ESI) m/z [M+H]+ 315.14. H-NMR
δ
: 7.12-7.04 (m, 2H, Ph-3,5-
H), 6.83-6.77 (m, 4H, 2H, Ph-2,6-H, NH2), 4.78 (s, 2H-CH2), 3.65 (s, 4H, Pp-2,6-H), 2.25
13
(s, 4H, Pp-3,5-H), 2.18 (s, 3H, CH3), 2.15 (s, 3H, CH3). C-NMR (DMSO-d6) δ: 173.62,
167.23, 164.78, 157.06, 130.83, 127.21, 126.33, 120.75, 112.18, 70.22, 54.72, 46.20,
16.60.
5.1.1.5. 4-(4-Methylpiperazin-1-yl)-6-(m-tolyloxymethyl)-1,3,5-triazin-2-amine (12)
Method A. White solid. Yield 10%, mp 132-135˚C. C16H22N6O (MW 314.39). LC/MS+:
1
purity: 100%, tR=3.11, (ESI) m/z [M+H]+ 315.21. H-NMR
δ
: 7.11 (t, J=7.6 Hz, 1H, Ph),
7.05-6.78 (br. s, 2H, NH2), 6.78-6.61 (m, 3H, Ph), 4.72 (s, 2H, CH2), 3.65 (s, 4H, Pp-2,6-
H), 2.34-2.19 (m, 7H, 4H, Pp-3,5-H, Pp-CH3), 2.16 (s, 3H, CH3). 13C-NMR (125 MHz,
DMSO-d6) δ: 173.52, 167.34, 164.84, 159.00, 139.34, 129.64, 121.91, 115.91, 112.09,
70.12, 54.83, 46.29, 42.99, 21.65
5.1.1.6. 4-(4-Methylpiperazin-1-yl)-6-(p-tolyloxymethyl)-1,3,5-triazin-2-amine (13)
Method A. White solid. Yield 21%, mp 144-145˚C. C16H22N6O (MW 314.39). LC/MS+:
purity: 96.95%, tR=3.18, (ESI) m/z [M+H]+ 315.30. 1H-NMR
δ: 7.03 (d, J=8.7 Hz, 2H, Ph),
6.96-6.84 (m, 2H, NH2), 6.78 (dd, J1=6.41 Hz, J2=2.05 Hz, 2H, Ph), 4.69 (s, 2H, CH2),
13
3.64 (s, 4H, Pp-2,6-H), 2.25 (s, 4H, Pp-3,5-H), 2.20 (s, 3H, CH3), 2.16 (s, 3H, CH3). C
NMR (125 MHz, DMSO-d6) δ: 173.60; 167.34; 164.85; 156.87; 130.23; 129.75; 114.98;
70.25; 54.83; 46.29; 20.59. Anal. calcd. for C16H22N6O C 61.13%, H 7.05%, N 26.73%.
Found: C 61.10%, H 7.09%, N 26.69%
19

ACCEPTED MANUSCRIPT
5.1.1.7. 4-((4-Chloro-2-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (14)
Method A. White solid. Yield 44%, mp 132-133˚C. C16H21ClN6O (MW 348.83). LC/MS+/-
1
: purity: 99.71%, tR=4.05, (ESI) m/z [M+H]+ 349.33. H-NMR
δ
: 7.19 (d, J=2.6 Hz, 1 H,
Ph), 7.11 (dd, J1=8.71 Hz, J2=2.82 Hz, 1H, Ph), 6.90 (br. s, 2H, NH2), 6.83 (d, J=8.7 Hz,
1H, Ph), 4.79 (s, 2H, CH2), 3.63 (s, 4H, Pp-2,6-H), 2.24 (br. s, 4H, Pp-3,5-H), 2.17 (s, 3H,
13
Ph-CH3), 2.15 (s, 3H, CH3). C-NMR (125 MHz, DMSO-d6) δ: 173.46; 167.33; 164.84;
154.42; 148.32; 141.70; 108.46; 106.48; 101.51; 98.51; 71.06; 54.83; 46.29. Anal. calcd.
for C16H21ClN6O: C 55.09%, H 6.07%, N 24.09%. Found: C 55.31%, H 6.10%, N 24.19%
5.1.1.8. 4-((2,4-Dichlorophenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(15)
Method A. White solid. Yield 35%, mp 104-105˚C. C15H18Cl2N6O (MW 369.25). LC/MS+:
1
purity: 100%, tR= 4.06, (ESI) m/z [M]+ 369.28. H-NMR
δ: 7.53 (d, J=2.6 Hz, 1H, Ph),
7.29 (dd, J1=8.86 Hz, J2=2.56 Hz, 1H, Ph), 7.02 (d, J=8.9 Hz, 1H-Ph), 6.95-6.88 (br s, 2H,
NH2), 4.91 (s, 2H, CH2), 3.61 (s, 4H, Pp-2,6-H), 2.22 (br s, 4H, Pp-3,5-H), 2.15 (s, 3H,
CH3). 13C NMR (125 MHz, DMSO-d6) δ: 172.69; 167.23; 164.68; 153.64; 129.72; 128.39;
124.94; 116.02; 70.58; 54.77; 46.27. Anal. calcd. for C15H18Cl2N6O: C 48.79%, H 4.91%,
N 22.76. Found: C 49.1%, H 5.08%, N 22.63%
5.1.1.9. 4-((4-Chloro-3-methylphenoxy)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (16)
Method A. White solid. Yield 19%, mp 131-134˚C. C16H21N6OCl (MW 348.83). LC/MS+:
purity: 100%, tR=3.85, (ESI) m/z [M+H]+ 349.10. 1H-NMR
δ: 7.24 (d, J = 8.7 Hz, 1H, Ph),
7.03-6.79 (m, 3H, Ph-5-H, NH2), 6.74 (dd, J1 = 3.1 Hz, J2=9.0 Hz, 1H, Ph), 4.74 (s, 2H,
CH2), 3.64 (t, J = 4.5 Hz, 4H, Pp-2,6-H), 2.30-2.20 (m, 7H, Pp-3,5-H, CH3), 2.16 (s, 3 H,
CH3). 13C-NMR (125 MHz, DMSO-d6) δ: 173.20, 167.30, 164.78, 157.75, 136.82, 129.88,
125.17, 118.08, 117.65, 114.28, 70.27, 68.56, 54.81, 46.29, 42.95, 20.33.
5.1.1.10. 4-(1-(3-Chlorophenoxy)ethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(17)
Method A. Brown solid. Yield 16%, mp 138˚C. C16H21N6OCl (MW 348.83). LC/MS+:
1
purity: 100%, tR=3.60, (ESI) m/z [M+H]+ 349.17. H-NMR δ: 7.25 (t, J=8.1 Hz, 1H, Ph),
7.07–6.92 (m, 3H, Ph), 6.91–6.77 (m, 2H, NH2), 4.95 (q, J =6.5 Hz, 1H, CH), 3.66 (s, 4H,
Pp-2,6-H), 2.27 (s, 4H, Pp-3,5-H), 2.18 (s, 3H, CH3), 1.53 (d, J=6.5 Hz, 3H, CH3).13C-
20

ACCEPTED MANUSCRIPT
NMR (DMSO-d6) δ: 176.59, 167.46, 164.89, 159.41, 133.90, 131.17, 120.85, 115.72,
114.61, 76.56, 54.69, 46.20, 20.59.
5.1.1.11. 4-(1-(2-Chlorophenoxy)ethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(18)
Method A. White solid. Yield 30%, mp 156˚C. C16H21N6OCl (MW 348.83). LC/MS+:
1
purity: 100%, tR=3.57,(ESI) m/z [M+H]+ 349.17. H-NMR δ: 7.40 (dd, J1=7.9 Hz, J2=1.6
Hz, 1H, Ph), 7.30–6.93 (m, 3H-Ph), 7.00-6.80 (m, 2H, NH2,), 4.98 (q, J=6.5 Hz, 1H, CH,),
3.67 (s, 4H, Pp-2,6-H), 2.38–2.19 (m, 4H, Pp-3,5-H), 2.17 (s, 3H, CH3), 1.57 (d, J=6.5 Hz,
3H, CH3). 13C-NMR (DMSO-d6) δ: 176.56, 167.44, 164.88, 154.01, 130.41, 128.46,
122.03, 121.87, 115.37, 77.20, 54.68, 46.21, 20.74.
5.1.1.12. 4-(4-Methylpiperazin-1-yl)-6-((phenylthio)methyl)-1,3,5-triazin-2-amine
hydrochloride (19)
Method B. White solid. Yield 33%; mp 250˚C. C15H21ClN6S (MW 352.89, MW 316.15-
basic form). LC/MS+: purity: 100%, tR=2.92,(ESI) m/z [M+H]+ 317.14. 1H-NMR δ: 11.95
(br. s, 1H, NH+), 8.40–7.87 (br, 2H, NH2), 7.49–7.46 (m, 2H, Ph), 7.36–7.33 (m, 2H, Ph),
7.27–7.23 (m, 1H, Ph), 4.13 (s, 2H, CH2), 3.45 (s, 4H, Pp-2,6-H), 3.16–2.72 (m, 7H, Pp-
3,5-H, CH3). 13C-NMR (MeOD-d4) δ: 166.58, 161.54, 157.07, 132.88, 131.30, 128.97,
127.72, 52.30, 52.03, 42.08, 40.73, 40.55, 36.62
5.1.1.13. ((2-Chlorophenyl)thio)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(20)
Method A. White solid. Yield 37%, mp 170˚C. C15H19ClN6S (MW 350.11). LC/MS+:
purity: 98.94%, tR=3.40,(ESI) m/z [M+H]+ 351.36. 1H-NMR δ: 7.58 (dd, J1=8.0 Hz, J2=1.4
Hz, 1H, Ph-3-H), 7.41 (dd, J1=7.9 Hz, J2=1.4 Hz, 1H, Ph-6H), 7.27 (td, J1=7.7 Hz, J2=1.4
Hz, 1H, Ph-5-H), 7.18–7.11 (m, 1H, Ph-4-H), 6.97-6.87 (br, 2H, NH2,), 3.94 (s, 2H, CH2),
3.67–3.60 (m, 4H, Pp-2,6-H), 2.24 (s, 4H, Pp-3,5-H,), 2.15 (s, 3H-CH3). 13C-NMR
(DMSO-d6) δ: 173.78, 167.24, 164.67, 136.51, 131.00, 129.67, 128.15, 128.01, 126.76,
54.72, 49.00, 46.20
5.1.1.14. 4-(1-((2-Chlorophenyl)thio)ethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine hydrochloride (21)
Method B. White solid. Yield 65%, mp 271˚C. C16H22Cl2N6S (MW 401.36, MW 364.12 –
1
basic form). LC/MS+: purity: 99.49%, tR=3.90, (ESI) m/z [M+H]+ 365.12. H-NMR δ:
11.87 (br. s, 1H, NH+), 8.05 (br, 2H, NH2), 7.66 (dd, J1=7.5 Hz, J2=1.7 Hz, 1H, Ph), 7.53
21

ACCEPTED MANUSCRIPT
(m, 1H, Ph), 7.39–7.29 (m, 2H, Ph), 4.39 (q, J=7.0 Hz, 1H, CH), 3.46 (d, J=10.5 Hz, 4H,
Pp-2,6-H), 2.92 (m, 7H, Pp-3,5-H, Pp-CH3), 1.60 (d, J=7.0 Hz, 3H, CH3). 13C-NMR
(DMSO-d6) δ: 171.21, 162.27, 160.33, 135.37, 133.83, 132.24, 130.34, 130.32, 128.40,
51.92, 45.03, 42.42, 18.04.
5.1.1.15. 4-(4-Methylpiperazin-1-yl)-6-((phenylselenyl)methyl)-1,3,5-triazin-2-amine
hydrochloride (22)
Method A. White solid. Yield 39%; mp 249˚C. C15H20N6Se (MW 364.09). LC/MS+:
purity: 100%, tR=2.82,(ESI) m/z [M+H]+ 365.12. 1H-NMR δ: 7.60–7.47 (m, 2H, Ph), 7.31–
7.15 (m, 3H, Ph), 6.85 (s, 2H, NH2), 3.82 (d, J=5.4 Hz, 2H, CH2), 3.60 (s, 4H, Pp-2,6-H),
2.23 (s, 4H, Pp-3,5-H), 2.15 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ: 175.48, 171.16,
167.35, 164.82, 134.10, 131.78, 131.40, 130.45, 129.49, 129.25, 127.03, 125.59, 54.77,
46.23, 42.84, 34.62, 33.19.
5.1.1.16. 4-(4-Methylpiperazin-1-yl)-6-(1-(phenylselenyl)ethyl)-1,3,5-triazin-2-amine
hydrochloride (23)
White solid. Yield 12%; mp 238˚C. C16H23ClN6Se (MW 413.81, MW 378.11- basic form).
1
LC/MS+/: purity: 100%, tR=3.23, (ESI) m/z [M+H]+ 379.08. H-NMR δ: 11.88 (br. S, 1H,
NH+), 8.16 (br, ,2H, NH2), 7.56 (d, J= 6.7 Hz, 2H, Ph ), 7.38 (m, 3H, Ph), 4.32 (q, J=7.1
Hz, 1H, CH), 3.45 (s, 4H, Pp-2,6-H), 2.75 (s, 7H, Pp-3,5-H, Pp-CH3), 1.61 (s, 3H, CH3).
13C-NMR δ: 162.14, 136.07, 131.31, 129.97, 129.62, 129.20, 128.32, 51.75, 42.45, 42.37,
22.49, 18.24.
5.1.2. Synthesis of 4-(((2-chlorophenyl)sulfonyl)methyl)-6-(4-methylpiperazin-1-yl)-1,3,5-
triazin-2-amine (24)
A solution of 20 (2 mmol, 701 mg) in ethanol (4 ml) was cooled to 0° C and sodium
.
tungstate dehydrate (Na2WO4 2H2O) (0.23 mmol, 303 mg) was added. After 5 minutes
at 0°C, 35% aqueous solution of H2O2 (4.5 ml) was added dropwise. The resulting mixture
was stirred at 0°C for 30 minutes. Subsequently, the reaction mixture was stirred at room
temperature for 24 hours. Saturated aqueous sodium thiosulfate anhydrous Na2S2O3
(15mL) was added and resulting layers were separated. The aqueous layer was extracted
with ethyl acetate three times and the combined organic layers was washed with brine,
dried over MgSO4 anhydrous, filtered and the solvents was removed under reduced
pressure. The pure product was obtained by recrystallization from methanol to give beige
crystals of desired product.
22

ACCEPTED MANUSCRIPT
White solid. Yield 37%, mp 170˚C. C15H19ClN6O2S (MW 382.10). LC/MS+: purity:
1
93.49%, tR=2.77,(ESI), m/z [M+H]+ 383.20. H-NMR δ: 7.80 (d, J=7.1 Hz, 1H, Ph-3-H),
7.66–7.55 (m, 3H, Ph-4,5,6-H), 7.13-7.00 (m, 2H, NH2), 4.06 (m, 2H, CH2), 3.68 (m, 8H,
Pp), 1.11 (s, 3H, CH3). 13C-NMR (125 MHz, MeOD-d4) δ: 170.24, 168.40, 165.75,
134.02, 131.79, 131.14, 129.45, 127.22, 65.82, 59.93, 38.69.
5.2. Molecular modeling procedures
The procedure of 5-HT6R homology models generation based on the β2 adrenergic
receptor template and these further validation for the support of structure-activity
relationship analyses of our earlier 1,3,5-triazine derivatives was described previously [15,
16]. In order to select the best subset of the receptor conformations, all the newly
synthesized derivatives (7–24) were docked. Only models showing coherent binding mode
to the previously described triazine derivatives and explained the main structure-activity
relationships were used.
The LigPrep [34] and Epik [35] were used to prepare the 3-dimensional structures and to
assigned appropriate ionization states at pH=7.4±1.0 of the ligands, respectively. If not
specified, the all possible R/S or Z/E isomers were generated and used in the docking. 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 [36]) 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 [37]. All of the used
programs are a part of Schrödinger Suite.
5.3. Radioligand binding assay
Radioligand binding assays to determine affinities of the tested compounds towards 5-
HT6R and competitive GPCRs were carried out, using [3H]-8-OH-DPAT (135.2 Ci/mmol),
[3H]-Ketanserin (53.4 Ci/mmol), [3H]-LSD (83.6 Ci/mmol), [3H]-5-CT (80.1 Ci/mmol) and
[3H]-Raclopride (76.0 Ci/mmol) for 5- HT1A, 5-HT2A, 5-HT6, 5-HT7 and D2, respectively.
Non-specific binding is defined with 10 mM of 5-HT in 5-HT1AR and 5-HT7R binding
experiments, whereas 10 mM of chlorpromazine, 10 mM of methiothepine or 10 mM of
haloperidol were used in 5-HT2AR, 5- HT6R and D2L assays, respectively. Each compound
was tested in triplicate at 7–8 concentrations (10-11–10-4 M). The inhibition constants (Ki)
were calculated from the Cheng-Prusoff equation [38]. HEK293 cells with stable
23

ACCEPTED MANUSCRIPT
expression of human serotonin 5-HT1AR, 5-HT6R, 5-HT7bR or dopamine D2LR as well as
CHO-K1 cells with stable expression of human serotonin 5-HT2AR purchased from
PerkinElmer BioSignal Inc were used in the assays, accroding to the procedures described
previously [14-17].
5.4. Functional assays for 5-HT6 receptor
Test and reference compounds were dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 1 mM. Serial dilutions were prepared in 96-well microplate in assay
buffer and 8 to 10 concentrations were tested. A cellular aequorin-based functional assay
was performed with recombinant CHO-K1 cells expressing mitochondrially targeted
aequorin, human GPCR and the promiscuous G protein α16 for 5-HT6R. Assay was
executed according to previously described protocol [39]. After thawing, cells were
transferred to assay buffer (DMEM/HAM’s F12 with 0.1% protease free BSA) and
centrifuged. The cell pellet was resuspended in assay buffer and coelenterazine h was
added at final concentrations of 5 µM. The cells suspension was incubated at 16 °C,
protected from light with constant agitation for 16 h and then diluted with assay buffer to
the concentration of 100,000 cells/ml. After 1 h of incubation, 50 µl of the cell’s
suspension was dispensed using automatic injectors built into the radiometric and
luminescence plate counter MicroBeta2 LumiJET (PerkinElmer, USA) into white opaque
96-well microplates preloaded with test compounds. Immediate light emission generated
following calcium mobilization was recorded for 60 s. In antagonist mode, after 30 min of
incubation the reference agonist was added to the above assay mix and light emission was
recorded again. Final concentration of the reference agonist was equal to EC80 (40 nM
serotonin).
5.5. Safety in vitro
SH-SY5Y (ATCC® CRL-2266™) cell line was purchased from American Type Culture
Collection ATCC (Manassas, VA, USA). The CellTiter 96® AQueous Non-Radioactive
Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI, USA). The
cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) with 10% fetal
bovine serum (FBS) (Gibco, Carlsbad, CA, USA) at 37°C in an atmosphere containing 5%
of CO2. The cells were seeded in 96-well plates at a concentration of 1.5 × 104 cells/well in
100 µl culture medium and cultured for 24 h to reach 60% confluence. The 10 mM stock
solutions of 5-HT6R ligands in DMSO were diluted into fresh growth medium and added
24

ACCEPTED MANUSCRIPT
into the microplates at the final concentrations 0.1—100 µM (DMSO concentration did not
exceed 1%). After 72h of incubation, the MTS labeling mixture was added to the each
well, and the cells were incubated under the same conditions for 5 h. The absorbance of the
samples was measured using a microplate reader EnSpire (PerkinElmer, Waltham, MA
USA) at 490 nm. Each compound was tested in at least three repetitions. GraphPad
Prism™ software (version 5.01, San Diego, CA, USA) was used to calculate statistical
significances.
The Salmonella typhimurium TA100 strain with base pair substitution (hisG46 mutation,
which target is GGG) was purchased from Xenometrix, Allschwil, Switzerland. The
reference mutagen nonyl-4-hydroxyquinoline-N-oxide (NQNO) was purchased from
Sigma-Aldrich, (St. Louis, MO, USA). All experiments were performed as described
before [40,41]. The mutagenic potential was calculated according to the procedure
provided by Xenometrix [32]. Each compound was tested in triplicate.
5.6. In vivo studies
5.6.1. Animals
The experiments were performed on male Wistar rats (200-250 g) obtained from an
accredited animal facility at the Jagiellonian University Medical College, Poland. The
animals were housed in group of four in controlled environment (ambient temperature
21±20C; 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. 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. Procedures involving animals and their care were
conducted in accordance with current European Community and Polish legislation on
animal experimentation. Additionally, all efforts were made to minimize animal suffering
and to use only the number of animals necessary to produce reliable scientific data. Animal
testing was limited to the study only for compounds that have previously demonstrated
enough interest in in vitro experiments. 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 EU Directive 2010/63/EU for animal experiments,
and were in accordance with the National Institutes of Health guide for the care and use of
Laboratory animals (NIH Publications No. 8023, revised 1978).
25

ACCEPTED MANUSCRIPT
5.6.2. Drugs
The following drugs were administered: SB-399885 (N-[3,5-dichloro-2-(methoxy)-
phenyl]-4-(methoxy)-1-(piperazinyl)benzenesulfonamide; GlaxoSimthKline, UK).
All the compounds were suspended in 1% Tween 80 immediately before
administration in a volume of 2 ml/kg. Compounds investigated were administered
intraperitoneally (i.p.) 60 minutes, while SB-399885 i.p. 30 minutes before testing. Control
animals received vehicle (1% Tween 80) according to the same schedule.
5.6.3. Behavioral procedures in rats
5.6.3.1. Forced swim test (FST test)
The experiment was carried out according to the method of Borsini and Meli [30].
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 re-placed 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.
5.6.3.2. Open field (OF) test
The experiment was performed employing a Motor Monitor System (Campden
Instruments, Ltd., UK) consisting of two SmartFrame Open Field stations (40 x 40 x 38
cm) with 16 x 16 beams, located in sound attenuating chambers and connected to PC
software by control chassis. Individual vehicle- or drug-injected animals were gently
placed in the center of the station. An automated Motor Monitor System recorded
ambulation (in X and Y axis), the number of rearing episodes, and total distance covered
by a rat for 5 min.
5.6.4. Statistical analysis
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).
26

ACCEPTED MANUSCRIPT
Acknowledgements
Authors thank Prof. Andrzej J. Bojarski for the opportunity to conduct RBA and MM
studies in his Department. Authors thank Prof. Claus Jacob, the tutor of PhD-students
Wesam Ali and Jawad Nasim, for his contribution in chemical, not biological, part of this
study (supervision of Se-intermediates synthesis). This study was financially supported by
National Science Centre, Poland grants: No UMO-2015/17/B/NZ7/02973, UMO-
2016/21/N/NZ7/03265 (Safety studies in vitro) and DEC-2011/02/A/NZ4/00031 (synthesis
of
7-11).
Wesam
Ali
was
financed
by
Saarland
University,
“Landesforschungsförderungsprogramm” (Grant No. WT/2 – LFFP 16/01).
References
[1]. M. L. Woolley, C. A. Marsden, K. C. Fone, 5-HT6 Receptors, Curr. Drug Targets CNS
Neurol. Disord. 3 (2004) 59-79.
[2]. K. Wicke, A. Haupt, A. Bespalov, Investigational drugs targeting 5-HT6 receptors for
the treatment of Alzheimer’s disease, Expert. Opin. Investig. Drugs. 24 (2015) 1515-
1528.
[3]. M. Kołaczkowski, M. Marcinkowska, A. Bucki, J. Śniecikowska, M. Pawłowski, G.
Kazek, A. Siwek, M. Jastrzębska-Więsek, A. Partyka, A. Wasik, A. Wesołowska, P.
Mierzejewski, P. Bienkowski, Novel 5-HT6 receptor antagonists/D2 receptor partial
agonists targeting behavioral and psychological symptoms of dementia, Eur. J. Med.
Chem. 92 (2015) 221-235.
[4]. C. Mattsson, P. Svensson, H. Boettcher, C. Sonesson, Structure activity relationship of
5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole analogues as 5-HT6
receptor agonists, Eur. J. Med. Chem. 63 (2013) 578-588.
[5]. M. Kotańska, K. Lustyk, A. Bucki, M. Marcinkowska, J. Śniecikowska, M.
Kołaczkowski, Idalopirdine, a selective 5-HT6 receptor antagonist, reduces food intake
and body weight in a model of excessive eating, Metab. Brain. Dis. 33 (2018) 733-740.
[6]. S. E. Shortall, O. Negm, M. Fowler, L. C. Fairclough, P.J. Tighe, P. M. Wigmore, M.
V. King, Characterization of Behavioral, Signaling and Cytokine Alterations in a Rat
Neurodevelopmental Model for Schizophrenia, and Their Reversal by the 5-HT6
Receptor Antagonist SB-399885, Mol. Neurobiol. 55 (2018) 7413-7430.
[7]. A. Więckowska, T. Wichur, J. Godyń, A. Bucki, M. Marcinkowska, A. Siwek, K.
Więckowski, P. Zaręba, D. Knez, M. Głuch-Lutwin, G. Kazek, G. Latacz, K. Mika, M.
27

ACCEPTED MANUSCRIPT
Kołaczkowski, J. Korabecny, O. Soukup, M. Benkova, K. Kieć-Kononowicz, S.
Gobec, B. Malawska, Novel Multitarget-Directed Ligands Aiming at Symptoms and
Causes of Alzheimer's Disease, ACS. Chem. Neurosci. 9 (2018) 1195-1214.
[8]. J. R. Hong, H. Choo, G. Nam, Neuropathic pain-alleviating effects of pyrazole-
conjugated arylsulfonamides as 5-HT6 receptor antagonists, Bioorg. Med. Chem. Lett.
27 (2017) 4146-4149.
[9]. A. Wesołowska, Potential role of the 5-HT6 receptor in depression and anxiety: an
overview of preclinical data, Pharmacol. Rep. 62 (2010) 564-577.
[10]. H. M. Yun, H. Rhim, The Serotonin-6 Receptor as a Novel Therapeutic Target, Exp.
Neurobiol. 20 (2011) 159-168.
[11]. M. Jastrzębska-Więsek, A. Siwek, A. Partyka, M. Kołaczkowski, M. Walczak, M.
Smolik, G. Latacz, K. Kieć-Kononowicz, A. Wesołowska, Study on the effect of
EMD386088, a 5-HT6 receptor partial agonist, in enhancing the anti-immobility action
of some antidepressants in rats, Naunyn Schmiedebergs Arch. Pharmacol. 391 (2018)
37-49.
[12]. C. H. Fabritius, U. Pesonen, J. Messinger, R. Horvath, H. Salo, M. Gałęzowski, M.
Galek, K. Stefanska, J. Szeremeta-Spisak, M. Olszak-Płachta, A. Buda, J. Adamczyk,
M. Król, P. Prusis, M. Sieprawska-Lupa, M. Mikulski, K. Kuokkanen, H. Chapman, R.
Obuchowicz, T. Korjamo, N. Jalava, M. Nowak, 1-Sulfonyl-6-Piperazinyl-7-
Azaindoles as potent and pseudoselective 5-HT6 receptor antagonists, Bioorg. Med.
Chem. Lett. 26 (2016) 2610-2615.
[13]. K. Grychowska, G. Satała, T. Kos, A. Partyka, E. Colacino, S. Chaumont-Dubel, X.
Bantreil, A. Wesołowska, M. Pawłowski, J. Martinez, P. Marin, G. Subra, A.J.
Bojarski, F. Lamaty, P. Popik, P. Zajdel, Novel 1H-Pyrrolo[3,2-c]quinoline Based 5-
HT6 Receptor Antagonists with Potential Application for the Treatment of Cognitive
Disorders Associated with Alzheimer’s Disease, ACS. Chem. Neurosci. 7 (2016) 972-
983.
[14]. D. Vanda, M. Soural, V. Canale, S. Chaumont-Dubel, G. Satała, T. Kos, P. Funk, V.
Fülöpová, B. Lemrová, P. Koczurkiewicz, E. Pękala, A. J. Bojarski, P. Popik, P. Marin,
P. Zajdel, Novel non-sulfonamide 5-HT6 receptor partial inverse agonist in a group of
imidazo[4,5-b]pyridines with cognition enhancing properties, Eur. J. Med. Chem. 144
(2017) 716–729.
[15]. D. Łażewska, R. Kurczab, M. Więcek, K. Kamińska, G. Satała, M. Jastrzębska-
Więsek, A. Partyka, AJ. Bojarski, A. Wesołowska, K. Kieć-Kononowicz, J. Handzlik,
28