Synthesis and computer-aided analysis of the role of linker for novel ligands of the 5-HT6 serotonin receptor among substituted 1,3,5-triazinylpiperazines

Article abstract of DOI:10.1016/j.bioorg.2018.11.046

A series of 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazines was designed based on previously published 2-amino-4-benzyl-(4-methylpiperazin-1-yl)-1,3,5-triazines in order to evaluate the role of a linker between the triazine moiety and an aromatic substituent for the human serotonin 5-HT₆ receptor affinity. As new linkers two carbon atoms (ethyl or ethenyl) or an oxyalkyl chain (methoxy, 2-ethoxy, 2-propoxy) were introduced. Affinities of the compounds for the 5-HT₆R as the main target, and for the 5-HT_1AR, 5-HT₇R and D₂R as competitive ones, were determined in the radioligand binding assays. Docking to the 5-HT₆R homology model was performed to support SAR analysis. Results showed that the branching of the methoxyl linker increased affinity for the human 5-HT₆R whereas an unsaturated bond within the linker dramatically reduced desirable activity. Both experimental and theoretical studies confirmed the previously postulated beneficial role of the aromatic size for interaction with the 5-HT₆R. Thus, the largest naphthyl moiety yielded the highest activity. In particular, 4-(4-methylpiperazin-1-yl)-6-(1-(naphthalen-1-yloxy)ethyl)-1,3,5-triazin-2-amine (24), the most potent 5-HT₆R agent found (K_i = 23 nM), can be a new lead structure for further search and development.

Full text of DOI:10.1016/j.bioorg.2018.11.046

Accepted Manuscript
Synthesis and computer-aided analysis of the role of linker for novel ligands of
the 5-HT6 serotonin receptor among substituted 1,3,5-triazinylpiperazines
Dorota Łażewska, Rafał Kurczab, Małgorzata Więcek, Grzegorz Satała,
Katarzyna Kieć-Kononowicz, Jadwiga Handzlik
PII:
S0045-2068(18)30763-6
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.046
YBIOO 2654
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 July 2018
21 November 2018
24 November 2018
Please cite this article as: D. Łażewska, R. Kurczab, M. Więcek, G. Satała, K. Kieć-Kononowicz, J. Handzlik,
Synthesis and computer-aided analysis of the role of linker for novel ligands of the 5-HT6 serotonin receptor among
substituted 1,3,5-triazinylpiperazines, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.046
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Synthesis and computer-aided analysis of the role of linker for novel ligands of
the 5-HT6 serotonin receptor among substituted 1,3,5-triazinylpiperazines
Dorota Łażewska1*, Rafał Kurczab2, Małgorzata Więcek1, Grzegorz Satała2, Katarzyna Kieć-
Kononowicz1, Jadwiga Handzlik1**
1Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian
University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland.
2Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of
Sciences, 12 Smętna Street, 31-343 Kraków, Poland
*Corresponding author
**Corresponding author
Email adresses: dlazewska@cm-uj.krakow.pl (D. Łażewska); j.handzlik@uj.edu.pl (J.
Handzlik)
1

Abstract
A series of 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazines was designed based on
previously published 2-amino-4-benzyl-(4-methylpiperazin-1-yl)-1,3,5-triazines in order to
evaluate the role of a linker between the triazine moiety and an aromatic substituent for the
human serotonin 5-HT6 receptor affinity. As new linkers two carbon atoms (ethyl or ethenyl)
or an oxyalkyl chain (methoxy, 2-ethoxy, 2-propoxy) were introduced. Affinities of the
compounds for the 5-HT6R as the main target, and for the 5-HT1AR, 5-HT7R and D2R as
competitive ones, were determined in the radioligand binding assays. Docking to the 5-HT6R
homology model was performed to support SAR analysis. Results showed that the branching
of the methoxyl linker increased affinity for the human 5-HT6R whereas an unsaturated bond
within the linker dramatically reduced desirable activity. Both experimental and theoretical
studies confirmed the previously postulated beneficial role of the aromatic size for interaction
with the 5-HT6R. Thus, the largest naphthyl moiety yielded the highest activity. In particular,
4-(4-Methylpiperazin-1-yl)-6-(1-(naphthalen-1-yloxy)ethyl)-1,3,5-triazin-2-amine (24), the
most potent 5-HT6R agent found (Ki = 23 nM), can be a new lead structure for further search
and development.
2

1.Introduction
The serotonin 5-HT6 receptors (5-HT6Rs) are mainly distributed in the brain in the
areas relevant with cognition. Potential utility of the 5-HT6R ligands, especially antagonists,
could be in the treatment of cognition deficits in diseases such as Alzheimer’s disease (AD),
depression/anxiety and schizophrenia [1,2]. Most of compounds advanced to clinical trials
(phase III) were evaluated with therapeutic indications of cognitive impairments associated
with AD, e.g. idalopirdine or intepirdine 1 [3] (Fig. 1), and have shown no higher efficacy
versus placebo as adjuncts to donepezil [4]. However, new promising 5-HT6R ligands have
appeared and are now tested in phase I trials, e.g. AVN-492 (2; Fig. 1) [5]. Furthermore,
positive effects were also observed for the 5-HT6R antagonists in obesity and metabolic
related disorders [6,7]. Recently the 5-HT6R antagonists (e.g. 3; Fig. 1) showed also
neuropathic pain-alleviating effects in a rat spinal nerve ligation model [8].
Fig. 1. Structures of described 5-HT6R ligands. eeAChE – acetylcholinesterase from the electric eel; hAChE –
recombinant human acetylcholinesterase; eqBuChE – butyrylcholinesterase from equine serum.
Thus, 5-HT6R is an attractive target in the search for biologically active substances with
promising pharmacological utility. So far, intensive works done by academic and
pharmaceutical industry researchers led to obtain many potent compounds with different
3

chemical structures. In 2006 Holenz et al. [9] have suggested a classification of 5-HT6R
ligands into the following groups: indole derivatives, indole-like derivatives, aryl-piperazines,
bi- and tricyclic piperazines, and arylsulfonyl derivatives. Recently very interesting aryl-
piperazines,
designed as multi-target-directed ligands blocking 5-HT6Rs, inhibiting
acetyl/butyrylcholinesterase and amyloid β anti-aggregation, were described by Więckowska
et al. (4, Fig. 1) [10].
On the other hand, in our recent studies, we discovered a totally new chemical family
of potent 5-HT6R agents among 2,4,6-trisubstituted derivatives of 1,3,5-triazine, that goes
beyond the classification of Holenz, despite of a presence of methyl-piperazine fragment [11].
Benzyl derivatives of 4-piperazinyl-1,3,5-triazine were the most active members
demonstrating submicromolar 5-HT6R affinities (e.g. 5; Fig.1, Table 1).
Table 1. Structures of tested compounds (5-26).
R
X
-----
-CH2-
6
7
---
12
---
16
---
---
---
8
5
9
10
13
14
19
---
---
11
---
15
20
23
25
---
---
18
22
---
---
-CH2CH2-
-CH=CH-
-O-CH2-
-O-CH(CH3)-
-O-C(CH3)2-
---
17
21
24
26
The computer-aided structure-activity relationship (SAR) analysis, performed for that
group, has indicated an important role of a size and properties of aromatic fragments linked
by methylene spacer to the 1,3,5-triazine ring [11]. It was especially remarkable, that the
potent 5-HT6R affinity of arylmethyl compounds, containing methylene spacer (5 and 10-12;
Table 1), significantly decreased with the spacer deletion in the case of all corresponding
4

aryl-triazines (6 and 7, Table 1). This observation suggests that presence of linker between
the triazine and aromatic moieties is a crucial structural feature for the desired receptor action.
However, there were not enough data to fully analyze that hypothesis within our previous
studies due to only two linker-variants in account (Table 1). In this context, our present study
reports on deeper insight into the role of the linker between the triazine moiety and the
aromatic substituent. Apart from two linkers described earlier, two carbon atoms (ethyl or
ethenyl) or an oxyalkyl chain (methoxy, 2-ethoxy, 2-propoxy) were under consideration.
Herein, we describe synthesis of new compounds, molecular modeling docking studies and
receptors radioligand binding assays. Computer-aided SAR analysis has been performed on
the basis of a series of 22 compounds (5–26) that contains 5 corresponding aromatic moieties
with different linkers (Table 1).
2. Synthesis
Synthesis of compounds 5–7, 10–12, 14–18 and 20 was described earlier, as follows: 5, 10
and 20 were reported by Latacz et al. [12], compounds 6 and 7 by Łażewska et al. [13],
compounds 11 and 12 by Łażewska et al. [11], whereas compounds with vinyl linker (14–18)
by Kamińska et al. [14]. Compounds 8, 9, 13, 19, and 21–26 were prepared from the proper
esters (8a, 9a, 13a, 19a, 21a–26a) and 4-methylpiperazin-1-yl biguanide dihydrochloride in
the presence of sodium methoxide (Scheme 1).
Scheme 1. Synthesis of compounds 8, 9, 13, 19, 21–26. (i) Reagents and conditions: CH3ONa, rt or reflux: 24-
48h; 5-65%.
The reaction was carried out at reflux temperature as described previously [13] (for 9, 13, 19,
23 and 25) or at room temperature (for 8, 21, 22, 24 and 26). Commercially unavailable esters
(13a, 19a, and 21a–26a) were obtained by two methods (Scheme 2). The first method
comprised esterification of commercially available aromatic acids with methanol in the
5

presence of sulfuric acid or with iodomethane and DBU (Scheme 2a). In the second synthetic
procedure, proper phenols or naphthols were alkylated with suitable bromo-esters in the
presence of potassium carbonate in acetone (Scheme 2b). The structures and purity of the
final compounds 8, 9, 13, 19, and 21–26 were confirmed by means of spectral (1H-NMR, 13C
NMR, MS, IR) and chromatographic methods (TLC).
Scheme 2a. Synthesis of esters 13a and 19a. (i) Reagents and conditions: CH3OH, H2SO4, rt: 144h, 77% (ii)
CH3I, DBU, toluene, rt 25h, 80%.
Scheme 2b. Synthesis of esters 21a–26a. (i) Reagents and conditions: acetone:H2O (4:1), K2CO3, reflux: 4-15h,
38-83%.
3. In vitro pharmacologiacal studies
3.1. Affinity for human serotonin 5-HT6 receptor
All compounds were screened for human 5-HT6R affinity in the radioligand binding assay
with [3H]-LSD as a specific radioligand. Results from these studies are collected in Table 2.
For comparison, affinity data for our previous triazine compounds (5–7 and 10–12) were
included [11]. Nine (13, 18, 20–26) out of all new 5-HT6R ligands investigated (8, 9, 13–26)
had significant affinity for the 5-HT6R with Ki values below 250 nM, and compounds 24 and
26 displayed particularly potent affinities (Ki: 23 nM and 33 nM, respectively) in the range of
6

the most active compounds found previously (5, 11 and 12). In contrary, two compounds
described previously (6, 7) and three new compounds (14–16) showed very low affinities for
the target 5-HT6R.
Table 2. Affinity of investigated compounds for serotonin (5-HT6, 5-HT1A, 5-HT7) and dopamine D2 receptors.
Cpds
Ki (nM)
5-HT1A
a
b
c
d
5-HT6
20
D2
5-HT7
895
5060
2937
5372
5
4189
2708
3103
5188
95
10680
10840
6
e
e
e
-
-
-
7
15590
2221
68110
8
e
e
e
-
-
-
9
965
1359
1946
606
17560
2189
9453
2447
10470
1858
4337
574
9263
6665
16790
2114
319700
27690
13400
8015
3465
9531
5701
4943
5533
5880
9574
2411
2305
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
52
22
218
7077
11190
2524
7562
460
11190
10470
894
206
507
226
150
191
84
2336
1348
2729
1262
1548
1900
319
1782
8726
3020
225
4136
1395
2357
158
23
88
554
33
1036
3349
b 3
a[3H]-LSD binding assay in HEK293 cells stably expressing the human serotonin 5-HT6R. [ H]-
c 3
Raclopride binding assay in HEK293 cells stably expressing the human dopamine D2 receptor; [ H]-
Raclopride binding assay in HEK293 cells stably expressing the human serotonin 5-HT1A receptor;
d[3H]-5-Carboxyamidotryptamine binding assay in HEK293 cells stably expressing the human
serotonin 5-HT7 receptor; enot tested.
7

3.2. Intrinsic activity for human serotonin 5-HT6 receptor of compounds 5, 12, 24 and 26
Selected compounds (5, 12, 24 and 26) were additionally determined on their intrinsic activity
in functional tests. The cAMP accumulation assay in HEK293 cells expressing the human
recombinant 5-HT6R was used. Results, expressed as Kb values, are shown in Table 3.
Addition of considered compounds resulted in a blockade of 5-carboxyamidotryptamine (5-
HT3R agonist) activity, leading to dose-dependent increase of cAMP level in cells co-treated
with 5-CT and forskolin. On the basis of the obtained results, all the compounds can be
classified as potent 5-HT6R antagonists with Kb values corresponding to Ki ones in the
radioligand binding assays (5, 12, 24, Table 2) or a bit higher (26).
Table 3. Functional activity of investigated compounds (5, 12, 24 and 26) for serotonin 5-HT6 receptor.
Cpd
5-HT6R functional
antagonism
Kb [nM]
5
12
24
26
26
18
14
150
3.3. Selectivity in respect to other GPCRs (D2, 5HT1A, 5HT7)
Most of the compounds (excluding 7 and 9), tested for affinities towards serotonin receptors
5-HT1A and 5-HT7 as well as for dopamine D2R, predominantly demonstrated low affinities
(Ki > 1.2 µM; Table 2). It is notable that all active 5-HT6R agents displayed significantly
lower affinities for the competitive serotonin and dopamine receptors tested. However, some
compounds (5, 10, 13, 17, 21, 24 and 25) had a moderate affinity for dopamine D2 and 5-
HT1A receptors with Ki values < 1 µM. Compound 24 was the most active at dopamine D2
receptor, with Ki of 319 nM. The highest affinity for 5-HT1A showed compounds 21 and 25
(Ki: 225 nM and 158 nM, respectively). None of the tested compounds showed significant
affinity for the 5-HT7 receptor (Ki > 2.1 µM). Compounds 11, 12, 23 and 26 were the most
selective among the potent 5-HT6R ligands with Ki < 100 nM (Table 2).
4. Molecular docking studies
Molecular docking studies were performed to take an insight into the binding mode of the
tested compounds to the 5-HT6R model. For this purpose, the recently developed 5-HT6R
homology models [11] based on the β2 adrenergic receptor template [15–17] and trained on
1,3,5-tiazine derivatives were used. For each compound, five top-scored complexes were
8

considered, of which the best with a binding mode coherent to the previously described [11]
was analyzed. The molecular docking results indicated that all newly investigated compounds
(13–26), generally, showed very consistent binding mode to the previously reported 1,3,5-
triazine derivatives [11]. However, some influence of linker properties on both, strength and
manner of binding has been observed (Fig. 2), and was in good concordance with results of
the radioligand binding assay.
Fig. 2. The interactions of 1,3,5-triazinylpiperazine derivatives with different modification of linker with 5-
HT6R. (A) A comparison of the binding modes of analogs with a different carbon linker length between the
1,3,5-triazine and the aromatic ring (10 – green, 13 – yellow). (B) A comparison of the binding modes of
compound 13 (yellow) and its analog 19 (magenta) with oxygen atom in a linker. (C) Illustration of orientation
of analogs with branching in the methoxyl linker (20 – violet, 23 – brown, and 25 – cyan). (D) A superposition
of the poses with different aromatic ring, i.e.: phenyl (10 – green), 4-chlorophenyl (11 – blue), 3-indolyl (12 –
orange), and 1-naphtyl (5 – red).
9

5. Molecular modeling-aided SAR discussion
Results of both, radioligand binding assay and docking analysis, have provided an
interesting insight on SAR within this chemical group, accentuating a role of the linkers
between aromatic and triazine moieties.
The most potent compounds for 5-HT6R (Ki < 35 nM) found in this (24 and 26) and
previous (5 and 12) studies belong to the flexible linker-containing derivatives of 1,3,5-
triazines with fused (hetero)aromatic rings of the 1-naphthyl (5, 24, 26) or 3-indole (12). In
contrast, linker-free aryl derivatives of the 1,3,5-triazine (6–9; Ki > 2 µM) and the compounds
with vinylene linker (14–16, Ki > 7 µM) were very weak 5-HT6R agents, almost regardless
the kind of their aromatic substituent. However, significant impact of the aromatic substituent
is distinctly seen in the case of more active compounds (5, 10–13, 18–26). Generally, the
phenyl derivatives (6, 10, 14, 19) were less active than their corresponding naphthyl
analogues (8, 5, 17–18, 21–22). As impact of the linker, the following relationships can be
observed: (i) an elongation of the methylene chain to the ethylene one (13) slightly decreased
the 5-HT6R affinity, whereas the inflexible vinylene linker (14–17) caused much stronger
activity decrease; (ii) an introduction of oxygen instead of carbon within the ethylene linker
resulted in decrease of the affinity, e.g. 13 vs 19, while (iii) branching in the methoxyl linker
(-OCH(CH3)- or -OC(CH3)2-) increased the 5-HT6R affinity in comparison to the straight-
chain (e.g. 20 vs 23 vs 25 or 21 vs 24 vs 26), and let obtain compounds with comparable
affinity to the most active methylene-linked analogues (e.g. 11 vs 23 vs 25 or 5 vs 24 vs 26).
Results of the docking studies (Fig. 2) indicated that, in addition to the crucial salt bridge
interaction with D3.32 found previously [11], all of the docked compounds formed at least
one specific aromatic interaction (CH–π or π–π stacking) with F6.52 and partially with F6.51.
The NH2 group of the 1,3,5-triazine ring was additionally hydrogen bonded with carbonyl
oxygen of V3.33 (Fig. 2A). A comparison of the binding mode of analogs with a different
carbon linker length (ranged from 0 to 2) between 1,3,5-triazine and aromatic moieties (Fig.
2A), showed that the methylene is an optimal linker followed by an ethylene. The less active
analog in the series, without linker (6) was not docked, probably due to its conformational
rigidity. For the similar reason, stiffened vinylene-linked derivatives (14–16) were the least
active 5-HT6R ligands within the whole series. However, the low linker-flexibility seems to
be partly compensated by presence of profitable aromatic moieties of the naphthalene in the
10

case of 17 and 18. Both compounds (17 and 18) exhibited submicromolar 5-HT6R-affinities,
with predominant activity of the β-naphthyl derivative (18).
In contrast, a decrease of the affinity for the derivative with the ethylene linker (13) can be
attributed to its higher flexibility leading to the dispersion of the spatial position of the phenyl
ring in the cavity of the binding crevice formed by helices 4–6 and extracellular loop 2. An
introduction of oxygen instead of carbon into the ethylene linker led to the further decrease of
the affinity (from Ki = 218 nM to 507 nM). A comparison of the binding modes of compound
13 and its analog 19 with oxygen atom (Fig. 2B) indicated that the methoxyl linker induces a
larger rigidity of this fragment of molecule, causing a difficult adaptation to the binding
pocket compared to the more flexible analog with the ethylene linker. The branching of the
methoxyl linker by one or two methyl groups led to improve the affinity significantly.
Molecular docking indicated (Fig. 2C) that methyl substituents better filling the hydrophobic
binding cavity formed by helices 5 and 6. A comparison of the binding mode of analogs with
different aromatic substituents showed that an increase of the activity may result from an
enlargement of hydrophobic surface of a substituent that interacts stronger with the
hydrophobic crevice formed by helices 3–5 and extracellular loop 2. This is in line with our
previous study among arylmethyl 1,3,5-triazine derivatives [11].
SAR analysis indicted that a new promising group of alkylether-linked aryl triazine
derivatives was found in this study as an extension of the previous active group of arylmethyl
derivatives. The influence of both, a kind of the linker and a kind of the aromatic substituent,
are pivotal for the 5-HT6R interaction of the 1,3,5-triazine derivatives investigated and some
balance of linker-aromatic moiety can be observed. However, the population of representative
compounds is still not enough to find a regular principle guiding this relationship. Due to the
small number of representatives, further studies are necessary to well define an influence of
the ether-linker, in “collaboration” with a variety of aromatic substituents, on the 5-HT6R
affinity and selectivity. In this context, the most active α-naphthyl derivative with branched
ether linker (24) could be a lead structure for further chemical modifications.
5. Conclusions
A series of new compounds with a different linker between the triazine and aromatic moieties
were designed based on our previous 5-HT6R ligands found among 4-benzyl-1,3,5-triazines.
The evaluated linkers showed different influence on the 5-HT6R affinity. The observed
positive effect decreased in order: methylene ≥ branched methoxylene > methoxylene >
11

ethylene >> vinylene ~ lack of linker. Replacement of the phenyl (4-chlorophenyl) substituent
with the naphthyl ring resulted in an increase of the 5-HT6R affinity. Results of the studies
accent two following structural factors as beneficial in the search for active ligands: the
branched methoxylene linkers and the bulky aromatic system. The role of both factors is
worth to be further investigated in separate expanded studies. Summing up, this study allowed
to identify a new group of potent 5-HT6R agents among (branched) methoxylene derivatives
of 1,3,5-triazines. The most potent member, 4-(4-methylpiperazin-1-yl)-6-(1-(naphthalen-1-
yloxy)ethyl)-1,3,5-triazin-2-amine (24), seems to be a good lead structure for further
modifications.
6. Experimental
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 (mp) were determined using MEL-TEMP II apparatus and are
uncorrected. The 1H NMR and 13C NMR spectra were obtained on a Varian Mercury-VX 300
1
MHz spectrometer or 400 MHz Spectrometer in DMSO-d6. Chemical shifts in H NMR
spectra were reported in parts per million (ppm) on the  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 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 (%).
6.1. Chemistry
6.6.1. Synthesis of esters
Esters 8a and 9a were commercially available. Esters 13a, 19a, and 21a–26a were obtained
according the procedure described by Łażewska et al. [11] as seen in Scheme 2, and some of
them (13a, 19a, 21a, 22a, 23a, 25a) are known in Chemical Abstract Database:
Methyl 3-phenylpropanoate (13a) CAS: 103-25-3. Methyl 2-phenoxyacetate (19a) CAS:
2065-23-8. Ethyl (naphthalen-1-yloxy)acetate (21a) CAS: 41643-81-6. Ethyl (naphthalen-2-
12

yloxy)acetate (22a) CAS: 6036-14-2. Methyl 2-(4-chlorophenoxy)propanoate (23a) CAS:
18671-89-1. Ethyl 2-(4-chlorophenoxy)-2-methylpropanoate (25a) CAS: 55162-41-9
6.6.2. Synthesis of triazines (8, 9, 13, 19, 21–26)
Triazines (Table 1) were obtained as presented in Scheme 1 by cyclization of 4-
methylpiperazin-1-yl biguanidine dihydrochloride with the appropriate carboxylic acid methyl
ester in the presence of sodium methoxide according to the previously published procedure
[13] (at reflux: 9, 13, 19, 23, 25) or with modification (at room temperature; 9, 21, 22, 24, 26).
Crystallization from acetonitrile (21), acetonitrile/water (8), ethyl acetate (22), methanol (9,
13, 19, 23, 25, 26), and methanol/water (24) was performed.
4-(4-methylpiperazin-1-yl)-6-(naphthalen-1-yl)-1,3,5-triazin-2-amine (8)
Obtained as white solid (Yield 5%) from commercially available methyl 1-naphthoate, mp
1
173-176˚C. C18H20N6 (MW 320.39). H NMR (300 MHz, DMSO-d6) δ: 8.72 (dd, J = 3.52,
5.86 Hz, 1H, Naph-8-H), 7.87 - 8.11 (m, 3H, Naph-2,4,5-H), 7.45 - 7.63 (m, 3H, Naph-3,6,7-
H), 7.01 (br. s, 2H, NH2), 3.76 (br. s, 4H, Pp-2,6-H2), 2.33 (br. s, 4H, Pp-3,5-H2), 2.19 (s, 3H,
13
-CH3). C NMR (100 MHz, DMSO-d6) δ: 173.23, 167.42, 164.97, 135.98, 133.89, 130.63,
128.73, 128.44, 126.80, 126.55, 126.21, 125.48, 54.83, 46.27, 43.03. LC/MS+/-: purity: 100%,
tR = 3.21, (ESI) m/z [M+H]+ 321.38.
4-(4-Methylpiperazin-1-yl)-6-(naphthalen-2-yl)-1,3,5-triazin-2-amine (9)
Obtained as white solid (Yield 13%) from commercially available methyl 2-naphthoate, mp
230-231 ˚C. C18H20N6 (MW 320.39). 1H NMR (300 MHz, DMSO-d6): : 8.86 (s, 1H, Naph-1-
H), 8.39-8.36 (dd, J = 1.79 Hz, J = 8.71 Hz, Naph-4-H), 8.06-8.03 (m, 1H, Naph-3-H), 7.99-
7.94 (m, 2H, Naph-5,8-H), 7.61-7.53 (m, 2H, Naph- 6,7-H), 6.96 (br.s, 2H, NH2), 3.84 (br.s,
4H, Pp-2,6-H), 2.25 (s, 4H, Pp-3,5-H), 2.20 (s, 3H, Pp-CH3). 13C NMR (100 MHz, DMSO-d6)
δ: 170.13, 167.68, 165.30, 134.97, 132.92, 129.46, 128.46, 128.08, 128.06, 127.82, 126.89,
125.29, 54.93, 46.29, 43.06. LC/MS+/-: purity: 100 %, tR = 3.83, (ESI) m/z [M+H]+ 321.19.
4-(4-Methylpiperazin-1-yl)-6-phenethyl-1,3,5-triazin-2-amine (13)
Obtained as white solid (Yield 10%) from ester 13a, mp 137-140 ˚C. C16H22N6 (MW 298.37).
1H NMR (300 MHz, DMSO-d6): : 7.29-7.13 (m, 5H, Ph-2,3,4,5,6-H), 6.72 (br.s, 2H, NH2),
3.69 (br.s, 4H, Pp-2,6-H), 2.96 (def. t, 2H, Ph-CH2-CH2), 2.67 (def. t, 2H, Ph-CH2), 2.29 (t, J
13
= 5.04 Hz, 4H, Pp-3,5-H), 2.19 (s, 3H, Pp-CH3). C NMR (100 MHz, DMSO-d6) δ: 176.95,
13

167.22, 167.22, 164.91, 142.09, 128.73, 128.70, 126.23, 54.85, 46.28, 42.83, 33.00. LC/MS+/-
: purity: 99.22 %, tR = 2.30, (ESI) m/z [M+H]+ 299.26.
4-(4-Methylpiperazin-1-yl)-6-(phenoxymethyl)-1,3,5-triazin-2-amine (19)
Obtained as white solid (Yield 9%) from ester 19a, mp 128-130 ˚C. C15H20N6O (MW
300.36). 1H NMR (400 MHz, DMSO-d6): : 7.27 (t, J = 7.8 Hz, 2H, Ph-3,5-H), 7.06-6.90 (m,
5H, Ph-2,4,6-H + NH2), 4.77 (s, 2H, OCH2), 3.67 (t, J = 4.7 Hz, 4H, Pp-2,6-H), 2.28 (br.s,
4H, Pp-3,5-H), 2.18 (s, 3H, Pp-CH3). 13C NMR (100 MHz, DMSO-d6) : 173.41, 167.26,
164.77, 158.89, 129.83, 121.06, 115.08, 70.03, 54.76, 46.22, 42.87. LC/MS+/-: purity: 100 %,
tR = 2.50, (ESI) m/z [M+H]+ 301.25.
4-(4-methylpiperazin-1-yl)-6-((naphthalen-1-yloxy)methyl)-1,3,5-triazin-2-amine (21)
Obtained as white solid (Yield 50%) from ester 21a, mp 153-155˚C. C19H22N6O (MW
350.42). 1H NMR (300 MHz, DMSO-d6) δ: 8.31-8.17 (m, 1H, Naph-8-H), 7.91-7.78 (m, 1H,
Naph-5-H), 7.63 - 7.41 (m, 3H, Naph-4,6,7-H), 7.36 (t, J = 7.90 Hz, 1H, Naph-3-H), 6.89 (m,
3H, Naph-2-H + NH2), 4.98 (s, 2H, -O-CH2), 3.63 (br.s, 4H, Pp-2,6-H), 2.32 - 2.10 (m, 7H,
Pp-3,5-H + Pp-CH3). 13C NMR (75 MHz, DMSO-d6) δ: 173.39, 167.26, 164.77, 154.42,
134.49, 127.84, 126.86, 126.55, 125.62, 125.49, 122.30, 120.54, 106.24, 70.47, 54.70, 46.19,
42.86. LC/MS+/-: purity: 100%, tR=3.85,(ESI) m/z [M+H]+ 351.16.
4-(4-methylpiperazin-1-yl)-6-((naphthalen-2-yloxy)methyl)-1,3,5-triazin-2-amine (22)
Obtained as white solid (Yield 32%) from ester 22a, mp 119˚C. C19H22N6O (MW 350.42). 1H
NMR (300 MHz, DMSO-d6) δ: 7.80 (d, J = 8.79 Hz, 2H, Naph-5,8-H), 7.73 (d, J = 8.21 Hz,
1H, Naph-4-H), 7.42 (t, J = 7.03 Hz, 1H, Naph-7-H), 7.37-7.24 (m, 2H, Naph-3,6-H), 7.19
(dd, J = 2.34, 8.79 Hz, 1H, Naph-1-H), 6.95 (br.s, 2H, NH2), 4.88 (s, 2H, -O-CH2), 3.66 (br.s,
4H, Pp-2,6-H), 2.37-2.01 (m, 7H, Pp-3,5-H + Pp-CH3). 13C NMR (75 MHz, DMSO-d6) δ:
173.21, 167.27, 164.75, 156.77, 134.57, 129.68, 128.97, 127.93, 127.12, 126.81, 124.07,
119.12, 107.71, 70.19, 54.72, 46.19. LC/MS+/-: purity: 99.45%, tR=3.72,(ESI) m/z [M+H]+
351.16.
4-(1-(4-Chlorophenoxy)ethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (23)
Obtained as white solid (Yield 65%) from 23a, mp 132-133 ˚C. C16H21ClN6O (MW 348.83).
1H NMR (400 MHz, DMSO-d6): : 7.27 (d, J = 8.61 Hz, 2H, Ph-3,5-H), 6.96 (br.s, 2H, NH2),
14

6.87 (d, J = 9.0 Hz, Hz, 2H, Ph-2,6-H), 4.88 (q, J = 6.65 Hz, 1H, OCH-CH3), 3.66 (br.s, 4H,
Pp-2,6-H), 2.27 (br.s, 4H, Pp-3,5-H), 2.18 (s, 3H, Pp-CH3), 1.53 (d, J = 6.65 Hz, 3H, CH-
CH3). 13C NMR (100 MHz, DMSO-d6) : 176.80, 167.44, 164.92, 157.30, 129.30, 124.54,
117.30, 76.45, 54.69, 46.21, 42.91, 20.73. LC/MS+/-: purity: 100 %, tR = 3.58, (ESI) m/z
[M+H]+ 349.24.
4-(4-methylpiperazin-1-yl)-6-(1-(naphthalen-1-yloxy)ethyl)-1,3,5-triazin-2-amine (24)
Obtained as white solid (Yield 29%) from ester 24a, mp 134-136˚C. C20H24N6O (MW
364.44). 1H NMR (300 MHz, DMSO-d6) : 8.32 - 8.18 (m, 1H, Naph-8-H), 7.89-7.75 (m, 1H,
Naph-5-H), 7.56-7.43 (m, 2H, Naph-6,7-H), 7.43-7.36 (m, 1H, Naph-4-H), 7.35-7.23 (m, 1H,
Naph-3-H), 7.08-6.72 (m, 3H, Naph-2-H + -NH2), 5.15-5.00 (m, 1H, -CH(CH3)), 3.60 (br.s,
4H. Pp-2,6-H2), 2.33-2.02 (m, 7H, Pp-3,5-H2 + Pp-CH3), 1.65 (d, J = 6.45 Hz, 3H, CH3). 13C
NMR (100 MHz, DMSO-d6) : 177.23, 167.49, 164.97, 154.04, 134.55, 127.79, 126.82,
126.50, 125.68, 125.54, 122.50, 120.35, 106.98, 76.84, 54.66, 46.20, 20.94. LC/MS+/-: purity:
100%, tR = 4.17, (ESI) m/z [M+H]+ 365.25.
4-(2-(4-Chlorophenoxy)propan-2-yl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (25)
Obtained as white solid (Yield 62%) from ester 25a, mp 137-138 ˚C. C17H23ClN6O (MW
1
362.86). H NMR (400 MHz, DMSO-d6): : 7.22 (d, J = 8.99 Hz, 2H, Ph-3,5-H), 6.90 (br.s,
2H, NH2), 6.67 (d, J = 9.0 Hz, Hz, 2H, Ph-2,6-H), 3.66 (br.s, 4H, Pp-2,6-H), 2.26 (br.s, 4H,
Pp-3,5-H), 2.18 (s, 3H, Pp-CH3), 1.59 (s, 6H, C-(CH3)2). 13C NMR (100 MHz, DMSO-d6) :
178.66, 167.57, 165.05, 155.25, 129.14, 124.93, 120.71, 81.36, 54.70, 46.22, 42.93, 26.95.
LC/MS+/-: purity: 100 %, tR = 3.76, (ESI) m/z [M+H]+ 363.26.
4-(4-methylpiperazin-1-yl)-6-(2-(naphthalen-1-yloxy)propan-2-yl)-1,3,5-triazin-2-amine (26)
Obtained as creamy solid (Yield 10%) from ester 26a, mp 210-213˚C. C21H26N6O (MW
378.47). 1H NMR (300 MHz, DMSO-d6) : 8.30-8.15 (m, 1H, Naph-8-H), 7.90-7.73 (m, 1H,
Naph-5-H), 7.56-7.32 (m, 3H, Naph-4,6,7-H), 7.29-7.14 (m, 1H, Naph-3-H), 6.90 (br.s, 2H, -
NH2), 6.48 (d, J = 7.62 Hz, 1H, Naph-2-H), 3.63 (br.s, 4H, Pp-2,6-H2), 2.35-2.06 (m, 7H, Pp-
13
3,5-H2 + Pp-CH3), 1.70 (s, 6H, 2x CH3). C NMR (100 MHz, DMSO-d6) : 179.07, 167.63,
165.13, 151.76, 134.71, 127.83, 127.36, 126.55, 126.08, 125.47, 122.86, 120.40, 111.0, 81.54,
54.70, 46.22, 42.93, 26.93. LC/MS+/-: purity: 99.33%, tR = 4.32, (ESI) m/z [M+H]+ 379.21.
6.2. Radioligand binding studies
15

6.2.1. Radioligand binding assay for serotonin 5HT-6 receptor
The affinity of the synthesized compounds for human serotonin 5-HT6R stably expressed in
HEK293 cells in radioligand binding assays via the displacement of [3H]-LSD was performed
as described previously [11].
6.2.2. Intrinsic activity for human serotonin 5-HT6 receptor of compounds 5, 12, 24 and 26
The functional properties of compounds on 5-HT6R were evaluated using their ability to
inhibit cAMP production induced by 5-CT (1000 nM) – a 5-HT6R agonist. Compounds were
tested in triplicate at 8 concentrations (10-11 – 10-4 M). The level of adenylyl cyclase activity
was measured using frozen recombinant 1321N1 cells expressing the Human Serotonin 5-
HT6R (PerkinElmer). Total cAMP was measured using the LANCE cAMP detection kit
(PerkinElmer), according to the manufacture's directions. For quantification of cAMP levels,
cells (5 µL) were incubated with mixture of compounds (5 µL) for 30 min at room
temperature in 384-well white opaque microtiter plate. After incubation, the reaction was
stopped and cells were lysed by the addition of 10 µL working solution (5 µL Eu-cAMP and 5
µL ULight-anti-cAMP). The assay plate was incubated for 1h at room temperature. Time-
resolved fluorescence resonance energy transfer (TR-FRET) was detected by an Infinite
M1000 Pro (Tecan) using instrument settings from LANCE cAMP detection kit manual. Kb
values were calculated from Cheng–Prusoff equation [18] specific for the analysis of
functional inhibition curves: Kb = IC50/(1 + A/EC50) where A is an agonist concentration, IC50
is the concentration of antagonist producing a 50% reduction in the response to agonist, and
EC50 is the agonist concentration which causes a half of the maximal response.
6.2.2. Radioligand binding studies for serotonin receptors (5HT1A, 5HT7) and dopamine D2
Tests for human serotonin (5-HT2A and 5-HT7) and human dopamine D2 receptors were
conducted in the radioligand binding assays as described by Canale et al.[19].
6.3. Molecular modelling studies
The building of homology models of 5-HT6 receptor, and validation of these models on the
1,3,5-triazine derivatives were previously described [11]. Since 5-HT6R homology models
built on β2 adrenergic template showed coherent for the whole set of compounds, and
explained the main structure-activity relationships, they were used in this study.
16

3-Dimensional structures of the ligands were prepared using LigPrep v3.6 [20], and the
appropriate ionization states at pH = 7.4±1.0 were assigned using Epik v3.4 [21]. Compounds
with stereogenic centres obtained as racemates (23 and 24) or those with unknown Z/E
configurations (14–18) were docked in all possible, R and S or Z and E, configurations,
respectively. One low energy ring conformation per ligand was generated. 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
[22]) by centring the grid box with a size of 12 Å on D3.32 side chain. Automated flexible
docking was performed using Glide v6.9 at SP level [23].
Acknowledgements
This study was financially supported by Polish National Science Centre (NCN) grants: No
UMO-2015/17/B/NZ7/02973 and DEC-2011/02/A/NZ4/00031 (synthesis of 13, 19, 23 and
25). Thanks to students: Joanna Ner, Paulina Porwisz and Michał Stelmasiński for their
contribution in the synthesis works (compounds 13, 19, 23 and 25).
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19

Figures, Schemes and Tables captions
Fig. 1. Structures of described 5-HT6R ligands. eeAChE – acetylcholinesterase from the electric eel; hAChE –
recombinant human acetylcholinesterase; eqBuChE – butyrylcholinesterase from equine serum.
Fig. 2. The interactions of 1,3,5-triazinylpiperazine derivatives with different modification of linker with 5-
HT6R. (A) A comparison of the binding modes of analogs with a different carbon linker length between the
1,3,5-triazine and the aromatic ring (10 – green, 13 – yellow). (B) A comparison of the binding modes of
compound 13 (yellow) and its analog 19 (magenta) with oxygen atom in a linker. (C) Illustration of orientation
of analogs with branching in the methoxyl linker (20 – violet, 23 – brown, and 25 – cyan). (D) A superposition
of the poses with different aromatic ring, i.e.: phenyl (10 – green), 4-chlorophenyl (11 – blue), methylindole (12
– orange), and 1-naphtyl (5 – red).
Scheme 1. Synthesis of compounds 8, 9, 13, 19, 21–26. (i) Reagents and conditions: CH3ONa, rt or reflux: 24-
48h; 5-65%.
Scheme 2a. Synthesis of esters 13a and 19a. (i) Reagents and conditions: CH3OH, H2SO4, rt: 144h, 77% (ii)
CH3I, DBU, toluene, rt 25h, 80%.
Scheme 2b. Synthesis of esters 21a–26a. (i) Reagents and conditions: acetone:H2O (4:1), K2CO3, reflux: 4-15h,
38-83%.
Table 1. Structures of tested compounds (5-26).
Table 2. Affinity of investigated compounds for serotonin (5-HT6, 5-HT1A, 5-HT7) and dopamine D2 receptors.
Table 3. Functional activity of investigated compounds (5, 12, 24 and 26) for serotonin 5-HT6 receptor.
20

Research Highlights
1. Synthesis of a new series of 1,3,5-triazinylpiperazines
2. Affinities for receptors: 5-HT6, 5-HT1A and D2 in radioligand binding assays
3. Molecular modeling docking studies to 5-HT6 receptor homology model
4. SAR studies on the role of linkers for the 5-HT6 receptor affinity
5. New lead structure in search for potent 5-HT6 receptor ligands was found
21

Graphical abstract
22