Chlorine substituents and linker topology as factors of 5-HT6R activity for novel highly active 1,3,5-triazine derivatives with procognitive properties in vivo

Article abstract of DOI:10.1016/j.ejmech.2020.112529

In the light of recent lines of evidence, 5-HT₆R ligands are a promising tool for future treatment of memory impairment. Hence, this study has supplied highly potent 5-HT₆R agents with procognitive effects, which represent an origina

Full text of DOI:10.1016/j.ejmech.2020.112529

Journal Pre-proof
Chlorine substituents and linker topology as factors of 5-HT R activity for novel highly
6
active 1,3,5-triazine derivatives with procognitive properties in vivo
Sylwia Sudoł, Katarzyna Kucwaj-Brysz, Rafał Kurczab, Natalia Wilczyńska,
Magdalena Jastrzębska-Więsek, Grzegorz Satała, Gniewomir Latacz, Monika Głuch-
Lutwin, Barbara Mordyl, Ewa Żesławska, Wojciech Nitek, Anna Partyka, Kamila
Buzun, Agata Doroz-Płonka, Anna Wesołowska, Anna Bielawska, Jadwiga Handzlik
PII:
S0223-5234(20)30501-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112529
EJMECH 112529
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 April 2020
Revised Date: 14 May 2020
Accepted Date: 1 June 2020
Please cite this article as: S. Sudoł, K. Kucwaj-Brysz, R. Kurczab, N. Wilczyńska, M. Jastrzębska-
Więsek, G. Satała, G. Latacz, M. Głuch-Lutwin, B. Mordyl, E. Żesławska, W. Nitek, A. Partyka,
K. Buzun, A. Doroz-Płonka, A. Wesołowska, A. Bielawska, J. Handzlik, Chlorine substituents and
linker topology as factors of 5-HT R activity for novel highly active 1,3,5-triazine derivatives with
6
procognitive properties in vivo, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112529.
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Graphical Abstract

Chlorine substituents and linker topology as factors of 5-HT R activity for novel highly
6
active 1,3,5-triazine derivatives with procognitive properties in vivo
a
a,b
b
c
Sylwia Sudoł , Katarzyna Kucwaj-Brysz , Rafał Kurczab , Natalia Wilczyńska , Magdalena
c
b
a
d
Jastrzębska-Więsek , Grzegorz Satała , Gniewomir Latacz , Monika Głuch-Lutwin , Barbara
d
e
f
c
a,g
Mordyl , Ewa Żesławska , Wojciech Nitek , Anna Partyka , Kamila Buzun , Agata Doroz-
a
c
g
a
Płonka , Anna Wesołowska , Anna Bielawska , Jadwiga Handzlik *
a
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy,
Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
b
Department of Medicinal Chemistry, Maj Institute of Pharmacology, Polish Academy of
Sciences, Smętna 12, PL 31-343 Kraków, Poland
c
Department of Clinical Pharmacy, Faculty of Pharmacy, Jagiellonian University,
Medical College, Medyczna 9, PL 30-688 Kraków, Poland
d
Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University, Medical
College, Medyczna 9, PL 30-688 Kraków, Poland
e
Institute of Biology, Pedagogical University of Cracow, Podchorążych 2, PL 30-084 Kraków, Poland
f
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, PL 30-387 Kraków, Poland
g
Deparmtent of Biotechnology, Medical University of Białystok, PL 15-222 Białystok, Poland
*
Corresponding author: j.handzlik@uj.edu.pl
1

Abstract
In the light of recent lines of evidence, 5-HT R ligands are a promising tool for future
6
treatment of memory impairment. Hence, this study has supplied highly potent 5-HT R agents
6
with procognitive effects, which represent an original chemical class of 1,3,5-triazines,
different from widely studied sulfone and indole-like 5-HT R ligands. The new compounds
6
were rationally designed as modifications of lead, 4-(1-(2-chlorophenoxy)ethyl)-6-(4-
methylpiperazin-1-yl)-1,3,5-triazin-2-amine (1), involving an introduction of: (i) two
chlorines at benzene ring and (ii) varied linkers joining the triazine ring to aromatic ethers.
Synthesis, in vitro and in vivo biological tests and computer-aided SAR analysis for 19 new
compounds were carried. Most of the new triazines displayed high affinity (K < 100 nM) and
i
selectivity towards 5-HT R, with respect to 5-HT R, 5-HT R and D R. The crystallography-
6
2A
7
2
supported docking studies, including quantum-polarized ligand docking (QPLD), indicated
that chlorine atoms may be involved in different type of halogen bonding, however, the linker
properties seem to predominately affect the 5-HT R affinity. 4-[1-(2,5-
6
Dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(9),
which
displayed: the highest affinity (K = 6 nM), very strong 5-HT R antagonistic action (K = 27
i
6
B
pM), procognitive effects in vivo in novel object recognition (NOR) test in rats, a very good
permeability in PAMPA model and satisfying safety in vitro, was identified as the most potent
1
,3,5-triazine agent so far, useful as a new lead for further research.
Keywords
Serotonin receptors, 5-HT antagonist, 1,3,5-triazine, docking QPLD, procognitive,
6
halogen bonds
2

1
. Introduction
The serotoninergic 5-HT receptor was discovered in 1993 as one of the latest members of
6
serotoninergic system [1], being structurally and pharmacologically different from other
subtypes [2]. As typical G-protein coupled receptor, it stimulates activity of adenylyl cyclase,
which results with cyclic AMP increase. The attractiveness of this receptor as therapeutic
target has been growing up since many preclinical studies confirmed an efficacy of the 5-
HT R ligands in serious central nervous system (CNS) dysfunctions, such as: depression [3],
6
Alzheimer’s disease (AD) [4,5], schizophrenia [6] and obesity [7,8]. Additionally, very recent
research indicated that 5-HT R antagonism may be a useful tool in treatment of irritable
6
bowel syndrome (IBS) [9]. Importantly, both the 5-HT R antagonist and agonist, were able to
6
improve memory impairment in novel object recognition- (NOR), social recognition- (SRT),
Y-maze continuous spontaneous alternation- (Y-CAT) and Morris water maze (MWM) tests
in rats [10,11]. This makes 5-HT receptor an intriguing protein target, demanding in-depth
6
research that leads to elucidation of its mechanism of action.
Despite of a number of already synthesized and pharmacologically evaluated 5-HT R ligands,
6
none has been approved as new drug in pharmaceutical market. Several 5-HT R agents have
6
reached clinical trials (Fig.1) [12–17] but, for the time being, the clinical experiments have
not confirmed clearly either significant procognitive effects in patients with mild to severe
AD or anxiolytic ones in patients with schizophrenia, thus highlighting a need of further
studies on 5-HT R-mediated mechanisms of action. Worth noting, the majority of previously
6
found 5-HT R ligands, belongs mainly to two chemical classes, i.e.: indole-derivatives or
6
sulfones [18]. Given all the mentioned-above facts, a further search for chemically original 5-
HT R agents, which could be translated into efficacy in clinical trials, seems to be relevant in
6
terms of rational strategy for new potential treatment of CNS disorders.
On the other hand, halogen atoms are substituents very widely used in medicinal chemistry as
their introduction into molecules often results in an improvement of metabolic stability [19],
blood-brain barrier (BBB) penetration [20] or other biological membrane permeability [21].
Moreover, their presence may provide higher activity thanks to ability to form non-covalent
interactions, so-called halogen bonding (XB), thus stabilizing protein-ligand complex [22].
The importance of this phenomena has been already confirmed in search for 5-HT R
6
antagonists [23], but also for ligands of other GPCRs (e.g. 5-HT1A [24], 5-HT R [25], 5-
2B
HT R [26], D R, D R [27]). Taking into account aforementioned facts, halogen substituents
7
2
4
3

can be considered as those of the first order in pharmacomodulation of novel biologically
active small molecules.
Fig. 1. The 5-HT R antagonists, which reached clinical trials
.
6
Intending to explore a new chemical space for 5-HT R ligands, our recent research was
6
concentrated on design, synthesis and pharmacological characteristics of novel, non-indole
and non-sulfone, family of 1,3,5-triazine derivatives to give a group of new 5-HT R agents
6
with high affinity (K < 100 nM) and selectivity for 5-HT receptor [28–31]. The structure-
i
6
activity relationship (SAR) analysis for those 1,3,5-triazine 5-HT R ligands showed a
6
significant role of their linker and a linker-dependent substitution in the aromatic system.
Thus, the lipophilic substituent in meta position was conducive to activity in the series of
1
,3,5-triazines with the methylene linker [31]. In the case of the cyclic moiety in the linker,
para substitution was the most advantageous [29], while compounds containing an ether
linker were found as the most interesting group, giving broad possibilities for further
modifications [28,30,32]. In that last group, compound MST4, containing alkyl substituents
at both, 2- and 5-positions of benzene, was the most active derivative found so far (K = 11
i
nM, Fig. 2) [32]. It is worth to underline, that alkyl mono-substituted (at the benzene ring)
analogues of MST4 were significantly less active (K = 87-235 nM), while better results were
i
observed for some chlorine mono-substituted derivatives. Among them, compound WA-13
(1, Fig. 2), containing o-chlorobenzene and the methyl branching linker took our special
attention, as displaying both potent (K = 23 nM) and selective action on 5-HT R as well as
i
6
4

satisfying CNS-druglike properties. Furthermore, antidepressant effect of 1 was confirmed in
the forced swim test (FST) in mice [28]. In this context, the compound 1 was selected as an
appropriate lead structure for further chemical modifications.
Therefore, this study was focused on design, synthesis and biological evaluation of novel
derivatives of the lead 1, where the main chemical modifications concerned a variety of
1
chlorine di-substitutions at the phenyl aromatic ring (R ). Furthermore, modifications of the
2
linker, i.e. length (n) and branching (R ), were carried out (Fig. 2). In term to complete SAR
analysis, one compound with the unsubstituted phenyl ring, but branched linker, was under
consideration.
Fig. 2. The previously reported hit compound (MST4), the lead structure (WA-13, 1) and its
chemical modifications investigated within this study.
For this purpose, synthesis of 19 new derivatives of WA-13 (2–20, Fig. 2) has been
performed. Whole the series (2–20) was tested for the affinity and selectivity towards 5-HT R
6
in the radioligand binding assay (RBA). Selected active compounds have been evaluated in
extended screening, including: (i) their intrinsic activity in functional assays, (ii) ADMETox
properties in vitro as well as (iii) behavioral tests in vivo. In order to perform comprehensive
SAR analysis, crystal structures for two representative members with different length of
5

linkers have been carried out, followed by molecular modelling, including quantum-polarized
ligand docking (QPLD), with special emphasize on role of halogen bond in ligand-receptor
complexes.
2
. Results
2
.1. Synthesis
The compounds (2–20) were obtained within 2-step synthesis pathways, elaborated on the
basis of procedures described for previous 1,3,5-triazines [13]. In the first step, O-alkylation
of commercial (dichloro)phenols with appropriate bromoesters was used to give aromatic
ethers (22–40). Then, the cyclic condensation of 4-methylpiperazin-1-yl biguanide
dihydrochloride (21) with the adequate ester (22–40) was carried out to form a 1,3,5-triazine
ring resulting in final products 2–20 (Scheme 1). As the racemic mixtures of bromoesters
were applied for the synthesis of the aromatic ethers (22–26, 28–31, 33, 34, 36–39),
consequently racemic mixtures of the final compounds (2–6, 8–11, 13, 14, 16–19) were
obtained.
Scheme 1. Synthetic route for compounds for 2–20. Reagents and conditions: (i) BuOH, reflux, yield:
8
1
6%; (ii) acetonitrile, K CO , reflux, 2–16 hours, yield: 36-90%; (iii) absolute methanol, Na, reflux
5–30 hours, yield: 13-52%.
2
3
6

2
2
.2. Pharmacology in vitro
.2.1. Radioligand binding assays
The binding affinities for all the newly synthesized 1,3,5-triazine derivatives 2–20 towards the
-HT receptor and competitive ones, i.e. serotoninergic receptors 5-HT , 5-HT and
5
6
2A
7
dopaminergic D receptor, have been assessed in radioligand binding assays (Table 1).
2
Table 1. Radioligand binding assays results for the newly synthesized compounds 2–20.
a
K [nM]
i
5-HT2A
1830
5047
268
1
2
Compound
R
R
n
5
-HT6
23
21
16
6
5-HT7
38730
19940
12550
5202
D2
1001
1506
432
421
495
506
206
375
320
229
103
413
754
580
356
157
368
377
270
Lead 1
2-Cl
H
Me
Et
0
0
0
0
0
0
2
0
0
0
0
2
0
0
2
0
0
0
0
2
-
2
3
4
5
6
7
8
9
2,3-diCl
2,3-diCl
2,3-diCl
Me
Et
209
Pr
23
73
274
13
6
310
13420
5265
2,3-diCl n-Bu
470
2,3-diCl
2,5-diCl
2,5-diCl
2,5-diCl
H
Me
Et
649
8592
355
15050
5706
484
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
Pr
12
17
470
95
86
382
12470
6263
2,5-diCl n-Bu
431
2,5-diCl
3,4-diCl
3,4-diCl
3,4-diCl
3,5-diCl
3,5-diCl
3,5-diCl
H
Me
Et
1457
576
7924
5928
696
5098
H
1061
27
906
4879
Me
Et
412
9398
11
463
9483
Pr
26
488
19850
10880
1904
3,5-diCl n-Bu
51
839
c
c
2,4-diCl
-
H
-
999
7
nt
nt
b
Ref
9
a
the standard deviation values are reported in Supplementary materials, Table S2;
olanzapine; nt – not tested
b
c
7

Serotonin 5-HT R and dopaminergic D R were chosen as protein off-targets in order to
2
A
2
control interactions which might be responsible for undesired side effects. According to
literature, activation of 5-HT R leads to hallucinations [33], whereas interaction with D R is
2
A
2
associated with extrapyramidal symptoms (e.g. acute dystonia and parkinsonian symptoms)
34]. Additionally, studies on animal models showed that 5-HT R ligands may cause
[
7
procognitive effects [35], similarly as 5-HT R agents. In order to clarify, if herein investigated
6
procognitive effects are a result of interaction with 5-HT R, the 5-HT R was also selected as
6
7
competitive protein target.
According to results obtained, most of the triazine compounds (2–6, 8–11, 13, 14 and 16–19)
showed high affinity for 5-HT R (K < 100 nM) and significant selectivity, with respect to
6
i
other GPCR’s. Ten compounds were more potent than lead structure WA-13 (K < 23 nM).
i
Compounds 4 and 9 turned out to have the highest 5-HT R affinity (K = 6 nM), in 2-fold
6
i
more potent than the most active triazine 5-HT R ligand described so far (MST4, K = 11 nM)
6
i
[
18]. Compound 2 demonstrated the most significant selectivity towards 5-HT R, with respect
6
to the rest of considered receptors (5-HT R: 240-fold, 5-HT R: 950-fold, D R: 72-fold).
2
A
7
2
2
.2.2. Functional assays towards 5-HT receptor
6
Selected active compounds (3, 8, 9, 11 and 16) were tested in the functional assays, in order to
study their intrinsic activity towards 5-HT R. During the experiments, the level of cAMP was
6
measured (Table 2).
Table 2. The results from functional assays for compounds 3, 8, 9, 11 and 16.
Binding affinity Antagonist mode
Agonist mode
Emax [%] ± SEM
Compound
a
b
c
pKi
pKB ± SEM
d
SEROTONIN
-
-
-
N.C.
100 ± 2
4 ± 0.5
4 ± 0.0
4 ± 0.0
6 ± 0.5
4 ± 0.0
4 ± 0.0
11 ± 0.5
SB258585
8.68 ± 0.055
6.28 ± 0.023
8.24 ± 0.317
8.44 ± 0.138
10.57 ± 0.065
6.36 ± 0.031
8.78 ± 0.332
MIANSERIN
3
8
9
7.80
7.89
8.22
7.77
7.57
1
1
1
6
a
b
Calculated according to the data from Table 1. Results were normalized as percentage of reference
-5
c
antagonist (SB258585 10 M). Results were normalized as percentage of maximal agonist response
(
-5
d
serotonin 10 M); E is the maximum possible effect. N.C. - not calculable. The full data with
max
standard deviation values are presented in Supplementary materials, Table S3
8

None of the tested derivatives showed agonistic mechanism, while four out of them showed
strong antagonistic action in the low nanomolar- (3, 8, 16) or even picomolar range in the case
of 9 (pK = 10.57, i.e. K = 27 pM, Table S3, Supplementary).
B
B
2
2
.3. Drug-likeness in vitro
.3.1. Permeability
For active representatives of the novel triazine derivatives, 8 and 9, permeability was tested
using Parallel Artificial Membrane Permeability Assay (PAMPA). The test is based on
compounds’ passive penetration through the bilayer artificial membranes. This membrane
may imitate barriers for compound absorption from the intestines with a good correlation to in
vivo conditions. The assay was performed in accordance to previously described methods
[
36–40]. Both tested ligands (8 and 9) had a high permeability coefficient (Pe), higher than
that for MST4 [32] and compared to Pe estimated for caffeine, the reference high-permeable
compound (Table 3).
Table 3. Permeability coefficient of compounds 8 and 9.
-6
Compound
Pe* [10 cm/s] ± SD
8
9
18.0 ± 1.2
18.9 ± 0.9
MST4
Reference
12.3 ± 1.98
Caffeine 15.1 ± 0.4
*
tested in triplicate
2
.3.2. Metabolic stability
The metabolic stability was investigated in vitro by using rat liver microsomes (RLMs) and
supported with MetaSite 6.01 software. The most probable structures of metabolites were
determined. In silico predicted sites of metabolism for both compounds 8 and 9 were specified
for N-methylpiperazine moiety and para position of the chlorine di-substituted aromatic ring
(Fig. 3).
9

8
9
Fig. 3. In silico prediction of the sites of metabolism by MetaSite 8.0.1 for 8 and 9. Blue circle marked
on the functional group structures indicates the highest biotransformation probability. The fading red
color shows the decreasing of the metabolism probability.
The incubation with RLMs for 120 min resulted in the formation of six (9) or seven (8)
metabolites, and more than 90% of each compound was biotransformed (Table S1; Fig. 4A,
4
B). Obtained results indicated rather low metabolic stability for both compounds, lower than
that for MST4 [32,41], while 9 was almost in 3-fold more stable than 8 (8.93% of 9 vs.3.17%
of 8, remained in the reaction mixture).
1
0

Fig. 4. The UPLC spectra of 8 (A) and 9 (B) and metabolites obtained after 120 min reaction with
RLMs.
These studies allowed us to predict the most probable metabolic pathway, i.e. the
hydroxylation at the phenyl ring (the metabolite M1), while other probable metabolic
pathways included demethylation and hydroxylation of piperazine ring, which were found for
both the 5-HT R ligands (8, 9) by MS analyses (Table S1; Fig. S1A-G, Fig. S2A-F,
6
Supplementary).
1
1

2
.3.3. Toxicity
The safety profile of the 1,3,5-triazine derivatives (8, 9) was estimated in the hepatotoxicity
assay in vitro using hepatoma HepG2 cell line, according to the protocol reported earlier [37–
4
0]. Both tested compounds 8 and 9 demonstrated a weak hepatotoxicity effect compared to
the used reference toxins (doxorubicin and 3-chlorophenyl-hydrazone). The statistically
significant decrease in cells viability was observed only at the highest applied concentration
of compound 8 (100 µM, Fig. 5). Compound 9, due to partial precipitation at 50 and 100 µM,
could be correctly tested only at lower concentrations, showing no toxicity up to 10 µM,
inclusively. Hence, both compounds (8, 9) displayed a satisfied safety in the level
corresponding to the best derivatives of 1,3,5-triazines tested previously [28, 31, 32, 38].
Fig. 5. The effect of tested compounds 8, 9 and references: doxorubicin (DX, 1 µM), mitochondrial
toxin carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP, 10 µM) on hepatoma HepG2 cell line
viability. Compound 9 partly precipitated at 50 and 100 under assay conditions. 1% DMSO in cell
growth media was used as a negative control. GraphPad Prism 8.0.1 was used to calculate the
statistical significances by one-way ANOVA, followed by Bonferroni's comparison test (****p <
0
.0001, **p<0.01).
2
.4. Behavioral studies in vivo
Taking into account that various previously studied 5-HT receptor ligands, especially
6
antagonists, exhibited beneficial effects on cognition in animal models [39], the most active 5-
HT R antagonist in this study, compound 9, has been investigated in two models of the
6
memory impairment tests, i.e. NOR and NOL (Novel Object Location) tests. Compound SB-
1
2

7
42457, the potent 5-HT R antagonist with the confirmed ability to reverse MK-801-induced
6
memory impairment in NOR test after both, acute and chronic administrations [10] was used
as reference (Fig. 6). The compound 9 dose-depend ameliorated the memory impairment
induced by MK-801 (0.1 mg/kg), in the statistically significant manner at the dose of 3 mg/kg
(Fig. 6a). The obtained data of discrimination index for the triazine derivative 9 at the dose of
3
6
mg/kg are comparable with those for the reference SB-742457 at the same dose used (Fig.
a). Despite, we did not observe any activity of this compound (9) in the NOL test (Fig. 6b).
Fig. 6. Effects of compound 9 on the memory impairment induced by MK-801 in NOR (a) and NOL
b) tests. Compound 9 and SB-742457 were administered i.p. 60 min, while MK-801 30 min before
the T1 session. The animals were observed for 5 min. The data are presented as the mean ± SEM of 6–
rats. The data were statistically evaluated by one-way ANOVA followed by Bonferroni’s post-hoc
test,*p< 0.05,** p<0.01 vs respective vehicle-treated group, and #p<0.05, ##p<0.01 vs respective MK-
01-treated group. (one-way ANOVA for discrimination index for NOR test: F(4,30)=3.99, p<0.01
for compound 9) and F(3,40)=6.78, p<0.001 (for SB-742457); and for NOL test: F(4,29)=2.82,
(
8
8
(
p<0.05)
Thus, the improvement of memory was observed only in the case of the memory for objects
(what) in the NOR but not in the spatial location (where) in the NOL test. A deficit in episodic
memory is one of the well-established cognitive deficits in schizophrenic patients and it is the
most profound and earliest cognitive deficit in AD [42]. The improved novel object
recognition task, but not the novel location place object preference task, demonstrates
dissociation between the effects of the investigated compound in a spatial location (NOL test)
and the object recognition (NOR test) version of animal model of episodic memory task. As
1
3

this dissociation was obtained using identical stimulus types, arena, and identical retention
delays of 1 h, it cannot be accounted for by differences in the response requirements of the
procedures or attentional or motivational processes. At this stage of our research, it is difficult
to explain this loss of activity of compound 9 in NOL test. We can only suppose that this
compound at the range of doses used, may possesses ability rather to reverse memory
impairment in recognition than in spatial memory components of episodic memory, but it
needs extended investigations in the future.
2
.5. The computer-aided structure-activity relationship analysis
In order to support structure-activity relationship discussion, in-depth inside into 3D-structure
of the investigated compounds (2–20) and their interactions with target 5-HT R protein in the
6
molecular level were performed, including both experimental crystallography and molecular
modelling.
2
.5.1. Crystallographic studies
Crystallographic analyses for compounds, representing either the shortest (9) or the longest
20) linker structural category, were carried out giving a starting point to find active
(
conformations for the whole series (2–10) within molecular modelling studies.
The overall shapes of the 9 and 20 molecules with the atom-numbering schemes are presented
in Fig. 7. Both molecules are isomers possessing the 2-amine-4-(4'-methylpiperazin-1'-yl)-
1
,3,5-triazine moiety, as well as a di-substituted aromatic ring (2,5-dichloro for 9 and 2,4-
dichloro for 20) connected with the triazine ring by the ethylmetoxylene linker for 9 and the
propoxylene linker for 20. In both molecules the piperazine ring adopts chair conformation
with equatorial position of the methyl group. However, the substituent at the N2 atom is not in
a typical equatorial position with a torsion angle C4-N2-C7-C8 of about 180°. The values of
this angle are 111.1(1)° and 133.8(1)° for 9 and 20, respectively.
1
4

(a)
(b)
Fig. 7. The molecular structures of (a) 9 and (b) 20, showing the atom-numbering schemes.
Displacement ellipsoids are drawn at the 50% probability level.
These values are lower than in other three crystal structures containing the 2-amine-4-(4'-
methylpiperazin-1'-yl)-1,3,5-triazine moiety [43,44] but similar to one observed in the crystal
structure of the 2-amine-6-(5-chloro-2-methoxyphenyl)-4-(4'-methylpiperazin-1'-yl)-1,3,5-
triazine [43], as well as similar to these observed in the crystal structures of 2-(4-
methylpiperazin-1-yl)-1H-imidazol-5(4H)-one derivatives [45]. The mutual orientation of
three rings differs in presented crystal structures. The interplanar angles between triazine and
piperazine rings are 43.30(5)° and 28.65(5)°, while between triazine and aromatic rings are
8
1.50(4)° and 76.62(3)° for 9 and 20, respectively.
The intermolecular interactions are similar in both structures. The main motif of interactions
is based on the N-H···N hydrogen bonds (Fig. 8). These interactions lead to formation of the
dimers. In addition, the crystal structures are stabilized by N–H···Cl and C-H···π contacts.
1
5

(a)
(b)
Fig. 8. The dimers in the crystal structure of 9 (a) and 20 (b). Dashed lines indicates the hydrogen
bonds.
2
.5.2. Molecular modelling
The molecular docking study for the newly synthesized derivatives 2–20 to 5-HT receptor
6
homology models showed coherent binding mode with the previously reported 1,3,5-triazine
derivatives [28–31,41]. The crucial interactions are the salt bridge between protonated
methylpiperazine nitrogen and negatively charged D3.32 side chain, an aromatic CH–π or π–π
interaction with F6.52 and/or F6.51, and hydrogen bond between NH group of 1,3,5-triazine
2
ring and carbonyl oxygen of V3.33 and/or A5.42. Substituted aromatic fragment linked with
the 1,3,5-triazine ring interacted with the hydrophobic cavity formed by transmembrane
domains (TM) 3–5 and extracellular loop 2 (ECL2), moreover, its positioned into this cavity
is caused by the tetrahedral conformation of the linker. In the light of the molecular docking
1
6

results, the comparison of the most active derivative 9 with the lead structure 1 (Fig. 9A)
showed a retaining of a common binding mode. However, elongation of the linker caused a
decrease of activity, which is in line with the molecular docking study. For instance, a
comparison of binding modes (Fig. 9A) of compound 9 (K = 6 nM) vs. 12 (K = 470 nM)
i
i
showed that too long linker may lead to destabilization of the complex and breaking some key
interactions (e.g. with A5.42/V3.33).
The branching of the linker showed increase of the affinity in order methyl < ethyl, but
decrease in order propyl > butyl for each of the compared series. Molecular docking indicated
(Fig. 9B) that the branching alkyl group was placed into a small binding cavity formed by
helices 5 and 6, which increased the stability of the resulting ligand–receptor complexes for
shorter, and destabilized the complexes (perhaps because of the overfilling of this pocket) for
the longer branching group.
In term to investigate the role of different substitution of chlorine atoms in the phenyl
fragment, the docking procedure involves quantum-polarized ligand docking (QPLD) and the
MM/GBSA algorithm was used. This procedure showed better performance in reproducing
the X-ray geometries of protein–ligand complexes with halogen bonding than classical
docking approach [46], and was also used to the study of the halogen bonding with privileged
amino acids as an important factor modulating the activity to 5-HT receptor [29,47]. The
6
disubstituted series, including: 2,3-diCl (4), 3,5-diCl (17), 2,5-diCl (9), and 3,4-diCl (14)
derivatives, and also the unsubstituted analog (2) in the phenyl fragment (Fig. 9C), was
selected.
1
7

Fig. 9. Illustration of the binding modes of selected 1,3,5-triazine derivatives with 5-HT receptor. (A)
6
Comparison of the docking poses for compounds 9 (green), 12 (yellow), and the lead structure 1
(
(
cyan). (B) Binding modes of analogs with methyl (3–pink), ethyl (4–blue), propyl (5–red), and butyl
6–brown) linker branch. (C) Comparison of the binding modes of unsubstituted 2 (orange) with its
differently disubstituted of chlorine atoms derivatives, namely: 2,3-diCl (4–blue), 3,5-diCl (17–violet),
,5-diCl (9–green), and 3,4-diCl (14–limon). Amino acids that are crucial for the binding of the
2
presented compounds are shown as thick dark-grey sticks.
It was possible to calculate the changes in the L–R binding free energy resulting from the
substitution of halogen atoms in this fragment (ΔΔG). Analysis of the binding modes showed
that chlorine atoms were involved in the formation of different types of halogen bonds (all
with backbone carbonyl oxygen), which were also visible in the change of binding free energy
and activity of a given derivative. The highest increase of the binding free energy was noted
for the most active derivatives 4 and 9 (K = 6 nM). The first analogue (2,3-diCl) showed
i
ΔΔG = –2.89 kcal/mol and two halogen bonds with P4.60 (XB distance = 4.1Å, σ-hole angle
=
165°) and A4.56 (XB distance = 3.0Å, σ-hole angle = 141°, with chlorine in position 2).
1
8

The second derivative (2,5-diCl) displayed ΔΔG = –1.96 kcal/mol and halogen bonds with
A4.56 (XB distance = 2.8Å, σ-hole angle = 148°) formed by chlorine in position 2, and
surprisingly, with L162 from extracellular loop 2 (XB distance = 2.7Å, σ-hole angle = 162°).
The 3,5-diCl analogue 17 showed only slightly lower increase of binding free energy
compared to 2,5-diCl (ΔΔG = –1.78 kcal/mol), for which two halogen bonds were found as
well – with S4.57 (XB distance = 3.8Å, σ-hole angle = 161°), and with L162 (XB distance =
2
.9Å, σ-hole angle = 152°) formed by chlorine in position 5 (as in the 2,5-diCl derivative).
The last derivative 14 (3,4-diCl) showed the lowest increase of binding free energy equal –
.73 kcal/mol, and only one halogen bond was formed between chlorine in position 3 with
0
S4.57 (XB distance = 3.0Å, σ-hole angle = 172°), whereas the chlorine at position 4 did not
show any specific interaction with the binding site. This analysis demonstrated that chlorine
atoms may be involved in the formation of halogen bonding, additionally stabilizing the L–R
complex, but their contribution is rather moderate (the highest improvement in activity after
chlorine substitution is approximately 4-fold).
2
.5.3 General SAR discussion
The chemical modifications of the lead 1 were introduced to analyze an influence on the
affinity towards 5-HT R of two following structural factors: (i) dichloro-substitution at phenyl
6
ring and (ii) the diverse (un)branched linker. The results obtained within this study allow us to
perform comprehensive structure-activity relationship discussion.
Sixteen out of nineteen newly synthesized compounds demonstrated a high affinity for 5-
HT R, with K values lower than 100 nM. The best pharmacological activity profile was
6
i
observed for 2,5- and 2,3-dichlorophenoxy 1,3,5-triazine derivatives with a short but branched
alkoxy linker, i.e. compounds 4 (K = 6 nM) and 9 (K = 6 nM). Both compounds had higher
i
i
affinity for 5-HT R than reference olanzapine as well as the strong picomolar antagonistic
6
action, found for the 2,5-dichlorophenyl derivative 9 (K = 0.027 nM), is worth emphasizing.
B
Among the series of analogues with chlorine atoms at the same positions of phenyl ring, a
change of branching in linker, from methyl to ethyl group, increased the 5-HT R affinity,
6
simultaneously maintaining significant selectivity. However, further elongation of branching
did not improve activity, giving the least beneficial effects for the butyl group, which is
consistent with molecular docking results. The mentioned-above results, together with the
high 5-HT R affinity (K = 21 nM) of the compound unsubstituted at the aromatic ring (2),
6
i
indicate that the type of linker is the predominant structural factor for the 5-HT R activity of
6
1
9

the compounds investigated within this study. This conclusion may be also supported by
previous results, in which the unbranched analogue of 2 displayed significantly lower affinity
for the 5-HT R target (K = 507 nM) [29]. Additionally, molecular modelling studies
6
i
confirmed that chlorine atoms might be involved in stabilization of L–R complex through the
different type of halogen bonds, however, their effect on the activity improvement is
moderate. These results allow to fully explain the high and little-varied activity of branched
derivatives ortho-, meta- or unsubstituted at the phenyl ring.
On the other hand, the presence of two chlorine substituents at the phenyl ring was found as
profitable also for “drug-like” properties of the explored 1,3,5-triazines due to an increase of
lipophilicity. The higher lipophilicity seems to be desirable to penetrate membrane barriers,
including BBB, which is especially important in the case of compounds destined to act on
CNS. The representative 2,5-dichlorophenyl derivatives tested (8, 9) demonstrated high
-6
permeability (Pe about 18 x 10 cm/s) and rather low risk of hepatotoxicity in the studies in
vitro.
Despite the low metabolic stability in vitro, the significant procognitive effect of the 2,5-
dichlorophenyl derivative 9 in vivo was observed in the NOR test in rats. Furthermore, the
active dose of 9 was corresponding to that of compounds MST4 tested previously, although
the metabolic stability of MST4 was in 6-fold higher (59.25% vs. 8.93%), and the 5-HT R
6
affinity only slightly weaker (11 nM vs. 6 nM) [32]. The observed procognitive action of
compound 9 in vivo seems to be a result of combined factors, i.e. (i) the beneficial effects of
both the highly potent 5-HT R-antagonistic action and a putative very good blood-brain
6
barrier penetration (high permeability confirmed in PAMPA test) on one hand, but (ii) an
unfavorable low metabolic stability effect, on the other. In this context, the resultant effect
indicates a distinctly greater impact of those favorable factors. Otherwise, the significant
activity in the behavioral test (NOR) could be also caused by synergistic procognitive action
of either the compound 9 or the metabolites formed. However, these hypotheses need wider
pharmacological and ADMET considerations to be enough confirmed.
3
. Conclusions
The described herein novel series of 1,3,5-triazine derivatives enlarged the original family of
highly potent 5-HT R antagonists, structurally different from widely investigated sulfone and
6
indole-like compounds. In particular, the systematic modifications of o-chloro substituted lead
(1), via the addition of one more chlorine substituent and changes within the ether linker,
2
0

resulted in two the strongest 5-HT R agents (4 and 9), in 2-fold more active than MST4, the
6
most potent triazine 5-HT R ligand described so far. Furthermore, the performed elimination
6
of the chlorine-substituent from the lead 1 gave a potent and highly selective ligand (2),
displaying selectivity for 5-HT R in the range of 71-867, with respect to 5-HT , 5-HT and
6
2A
7
D dopamine receptors. SAR studies supported by crystallography and extended molecular
2
modelling indicated that (i) the short linker branched with ethyl chain and (ii) the dichloro-
substituted phenyl ring at positions ortho and meta are the most favorable structural factors
for strong interaction with the target 5-HT R, but the role of the first one is predominant.
6
The performed study allows to find the new “hit’, i.e. 4-[1-(2,5-dichlorophenoxy)propyl]-6-
(
4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (9), which displayed extremely high 5-HT R
6
antagonistic action in the functional assays, significant procognitive effects in vivo in rats,
very good permeability in PAMPA model and a satisfying safety in vitro. However, the
disadvantageous weak metabolic stability of 9 and its methyl-branched analogue (8) was
indicated in vitro, which requires a further pharmacomodulation.
Apart from compound 9, its 2,3-dichlorophenyl analogue (4), found as the equally active 5-
HT R agent, as well as the most selective phenyl-unsubstituted compound 2 seem to be worth
6
of wider considerations, too. Thus, the presented results set the new lead structures for further
studies concerning search for 5-HT R agents, which may be a useful tool for future treatment
6
of cognitive impairment associated with Alzheimer Disease.
4
. Experimental
4
.1. Chemical synthesis
1
Melting points (mp) were determined using MEL-TEMP II apparatus and are uncorrected. H
1
3
NMR and C NMR spectra were recorded on a Varian Mercury-VX 400 MHz PFG
1
3
instrument or FT-NMR JEOL (JNM-ECZR500 RS1 v. ECZR) 500 MHz instrument ( C
NMR for 3-20) in DMSO‑ d (for almost all compounds, except from compound 2 – 400
6
MHz) 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, m, multiplet), coupling constant J, number of
protons, protons position (Pip-piperazine, Ph-phenyl). Mass spectra recorded on a UPLC–
MS/MS system consisted of a Waters ACQUITY® UPLC® (Waters Corporation, Milford,
2
1

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 14). Retention time values (t ) are given in minutes. Thin-layer
R
chromatography (TLC) was performed on pre-coated Merck silica gel 60 F254 aluminum
sheets (Hex/EtOAc 4:1, DCM/MeOH 95:5). The reactions at fixed temperature were carried
out using a magnetic stirrer with a contact thermometer Heidolph MR 2001. Intermediates
(
22-40) were synthesized based on previous methods [28-31], and used in a crude form
purity 54-99%) for synthesis of the final compounds (2-20). Details of their synthesis and
(
chemical characterization are provided in Supplementary.
4
.1.1. General procedure for the synthesis of final compounds (2-20)
Sodium (10 mmol) was dissolved in 10 ml of absolute methanol, then 4-methylpiperazine-1-
yl biguanide hydrochloride (21) (5mmol) and a proper ester (22–40, 5mmol) was added. The
reaction mixture was refluxed for 15–30 h. After cooling to room temperature, the residue
was washed with water (10 ml), stirred for 30 min. at room temperature. The precipitated
product was isolated by filtration and crystallized from methanol to give the desire final
products as solids (method A). In case of lack of desirable precipitate, final product was
converted into hydrochloric salt form using solution of HCl in diethyl ether (method B).
4
.1.1.1. (RS)-4-(4-methylpiperazin-1-yl)-6-(1-phenoxypropyl)-1,3,5-triazin-2-amine (2)
+
Ester 22, reaction time: 16 h. Method A. White solid. Yield 38%, LC/MS purity: 96,4%,
o
1
t =3.29, mp=112–114 C, C H N O (MW= 328.42). H NMR (400 MHz, DMSO-d ) δ
R
17 24
6
6
[
ppm]: 0.97 (t, J = 7.4 Hz, 3H, CH ), 1.84 – 1.93 (m, 2H, CH ), 2.17 (s, 3H, N-CH ), 2.27 (br.
3 2 3
s, 4H, Pip-3,5-H), 3.66 (br. s, 4H, Pip-2,6-H), 4.63 (t, J = 6.5 Hz, 1H, O-CH), 6.77 – 6.89 (m,
1
3
4
H, Ph-2,3,5,6-H), 7.00 (br. s, 1H, Ph-4-H), 7.18 – 7.24 (m, 2H, NH ). C NMR (101 MHz,
2
DMSO-d ) δ [ppm]: 10.50, 27.90, 42.86, 46.22, 54.70, 81.40, 115.56, 120.84, 129.78, 158.82,
6
1
64.94, 167.41, 176.51.
4
.1.1.2. (RS)-4-[1-(2,3-dichlorophenoxy)ethyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (3)
Ester 23, reaction time: 15 h. Method A. White solid. Yield 24%, LC/MS purity: 100%,
+
o
1
t =4.15, mp=187–189 C, C H Cl N O (MW= 383.28). H NMR (500 MHz, DMSO-d ) δ
R
16 20
2
6
6
[
ppm]: 1.54 (d, J=5.73 Hz, 3H, CH ) 2.13 (s, 3H, N-CH ), 2.21 (br. s, 4H, Pip-3,5-H), 3.60
3 3
2
2

(
br. s, 4H, Pip-2,6-H), 5.00 (d, J=6.30 Hz, 1H, O-CH), 6.68-6.99 (m, 2H, NH ), 6.87-6.89 (d
2
1
3
def., 1H, Ph-6-H), 7.09-7.10 (m, 1H, Ph-5-H), 7.16 (t, J=7.20 Hz, 1H, Ph-4-H). C NMR
126 MHz, DMSO-d ) δ [ppm]: 20.72, 43.02, 46.25, 54.72, 77.63, 113.86, 120.93, 122.40,
(
6
1
28.67, 132.83, 155.71, 164.90, 167.49, 176.25.
4
.1.1.3. (RS)-4-[1-(2,3-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (4)
+
Ester 24, reaction time: 21 h. Method A. White solid. Yield 40%, LC/MS purity: 100%,
o
1
t =4.73, mp=174–176 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
ppm]: 1.02 (t, J = 7.4 Hz, 3H, CH ), 1.97 (p, J = 7.3 Hz, 2H, CH ), 2.16 (s, 3H, N-CH ), 2.25
3 2 3
(br. s, 4H, Pip-3,5-H), 3.63 (br. s, 4H, Pip-2,6-H), 4.80 (t, J = 6.3 Hz, 1H, O-CH), 6.86 – 6.89
(
m, 1H, Ph-6-H), 7.01 (br. s, 2H, NH ), 7.13 – 7.16 (m, 1H, Ph-5-H), 7.19 – 7.23 (m, 1H, Ph-
2
1
3
4
1
-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 10.38, 27.90, 46.26, 54.73, 82.57, 113.76,
6
20.91, 122.35, 128.75, 132.79, 155.93, 164.83, 167.41, 175.53.
4
.1.1.4. (RS)-4-[1-(2,3-dichlorophenoxy)butyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (5)
+
Ester 25, reaction time: 18 h. Method A. White solid. Yield 18%, LC/MS purity: 100%,
o
1
t =5.20, mp=186–188 C, C H Cl N O (MW= 411.33). H NMR (500 MHz, DMSO-d ) δ
R
18 24
2
6
6
[
ppm]: 0.89 (t, J = 6.8 Hz, 3H,CH ), 1.36 – 1.57 (m, 2H, CH ), 1.78 – 1.98 (m, 2H, CH ),
3 2 2
2
4
.12 (s, 3H, N-CH ), 2.21 (s, 2H, Pip-3,5-H), 2.46 (s, 2H, Pip-3,5-H), 3.59 (s, 4H, Pip-2,6-H),
3
.76 – 4.88 (m, 1H, O-CH), 6.77 – 6.91 (m, 2H, NH ), 6.98 (br. s, 1H, Ph-5-H), 7.07 – 7.23
2
1
3
(
m, 2H, Ph-4,6-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 14.23, 18.81, 36.69, 46.26,
6
5
4.71, 81.17, 113.67, 120.86, 122.35, 128.76, 132.80, 155.91, 164.83, 167.41, 175.75.
4
.1.1.5. (RS)-4-[1-(2,3-dichlorophenoxy)penthyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (6)
+
Ester 26, reaction time: 19 h. Method A. White solid. Yield 52%, LC/MS purity: 98,5%,
o
1
t =5.41, mp=150–152 C, C H Cl N O (MW= 425.36). H NMR (500 MHz, DMSO-d ) δ
R
19 26
2
6
6
[
ppm]: 0.81 (t, J=7.16 Hz, 3H, CH ), 1.26-1.31 (q def., 2H, CH ), 1.37-1.48 (m, 2H, CH ),
3 2 2
1
4
7
.85-1.95 (m, 2H, CH ), 2.11 (s, 3H, N-CH ) 2.20 (s, 4H, Pip-3,5-H), 3.31 (s, 4H, Pip-2,6-H),
2 3
.78-4.81 (d def., 1H, O-CH), 6.82 (d, J=7.45 Hz, 1H, Ph-6-H), 6.99-7.01 (m, 2H, NH2),
1
3
.09-7.10 (m, 1H, Ph-5-H), 7.14-7.15 (m, 1H, Ph-4-H). C NMR (126 MHz, DMSO-d ) δ
6
2
3

[
ppm]: 14.42, 22.40, 27.68, 34.30, 43.20, 46.26, 54.72, 81.45, 113.68, 120.89, 122.35, 128.74,
1
32.81, 155.92, 164.84, 167.42, 175.73.
4
.1.1.6. 4-[3-(2,3-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(7)
+
Ester 27, reaction time: 20 h. Method A. White solid. Yield 42%, LC/MS purity: 100%,
o
1
t =4.18, mp=84–86 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
ppm]: 2.08 (d, J=6.30 Hz, 2H, CH ), 2.12 (br. s, 3H, N-CH ), 2.21 (br. s, 4H, Pip-3,5-H),
2 3
2
.53 (t, J=6.59 Hz, 2H, CH ), 3.61 (br. s, 4H, Pip-2,6-H), 4.09 (br. s, 2H, O-CH ), 6.74 (br. s,
2 2
2
H, NH ), 7.05 (d, J=8.02 Hz, 1H, Ph-4-H), 7.14 (d, J= 8.02 Hz, 1H, Ph-6-H), 7.23 (t, J= 7.73
2
1
3
Hz, 1H, Ph-5-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 26.58, 34.89, 42.85, 46.30, 54.85,
6
6
9.24, 112.69, 120.61, 122.34, 128.98, 132.77, 155.99, 164.96, 167.30, 177.16.
4
.1.1.7. (RS)-4-[1-(2,5-dichlorophenoxy)ethyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (8)
+
Ester 28, reaction time: 22 h. Method A. White solid. Yield 43%, LC/MS purity: 96,4%,
o
1
t =4.02, mp=153–155 C, C H Cl N O (MW= 383.28). H NMR (500 MHz, DMSO-d ) δ
R
16 20
2
6
6
[
(
ppm]: 1.53 (d, J=6.87 Hz, 3H, CH ), 2.12 (s, 3H, N-CH ), 2.21 (br. s, 4H, Pip-3,5-H), 3.61
3 3
br. s, 4H, Pip-2,6-H), 5.01 (q, J=6.68 Hz, 1H, O-CH), 6.70-6.94 (m., 2H, NH ) 6.92-6.94 (d
2
1
3
def., 1H, Ph-4-H), 7.06 (s, 1H, Ph-6-H), 7.37 (d, J=8.02 Hz, 1H, Ph-3-H). C NMR (126
MHz, DMSO-d ) δ [ppm]: 20.43, 43.09, 46.25, 54.75, 77.65, 115.77, 121.05, 121.74, 131.46,
6
1
32.38, 154.76, 164.82, 167.53, 175.91.
4
.1.1.8. (RS)-4-[1-(2,5-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (9)
+
Ester 29, reaction time: 20 h. Method A. White solid. Yield 47%, LC/MS purity: 100%,
o
1
t =4.60, mp=163–165 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
(
(
ppm]: 0.94 (t, J=7.45 Hz, 3H, CH3), 1.91 (q, J=7.16 Hz, 2H, CH ), 2.11 (s, 3H, N-CH ), 2.21
2 3
br. s, 4H, Pip-3,5-H), 3.61 (br. s, 4H, Pip-2,6-H), 4.77 (t, J=6.30 Hz, 1H, O-CH), 6.84-6.90
m, 2H, NH ), 6.90-6.93 (d def., 1H, Ph-6-H), 7.02-7.05 (d def., 1H, Ph-4-H), 7.36 (d, J=8.59
2
1
3
Hz, 1H, Ph-3-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 10.25, 27.60, 43.11, 46.23,
6
5
4.74, 82.69, 115.66, 121.11, 121.66, 131.41, 132.39, 155.01, 164.79, 167.49, 175.23.
2
4

4
.1.1.9. (RS)-4-[1-(2,5-dichlorophenoxy)butyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (10)
+
Ester 30, reaction time: 22 h. Method A. White solid. Yield 54%, LC/MS purity: 96,7%,
o
1
t =5.13, mp=143–145 C, C H Cl N O (MW= 411.33). H NMR (500 MHz, DMSO-d ) δ
R
18 24
2
6
6
[
ppm]: 0.92 (t, J = 7.4 Hz, 3H, CH ), 1.37 – 1.55 (m, 2H, CH ), 1.83 – 2.00 (m, 2H, CH ),
3 2 2
2
.16 (s, 3H, N-CH ), 2.25 (br. s, 4H, Pip-3,5-H), 3.57 – 3.74 (m, 4H, Pip-2,6-H), 4.88 (dd, J1
3
=
8.2 Hz, J = 4.9 Hz, 1H, O-CH), 6.90 – 6.94 (m, 1H, NH ), 6.96-6.98 (m, 1H, Ph-6-H), 7.05
2 2
1
3
(
d, J = 2.3 Hz, 1H, Ph-4-H), 7.06 – 7.09 (m, 1H, NH ), 7.41-7.44 (m, 1H, Ph-3-H). C NMR
2
(
126 MHz, DMSO-d ) δ [ppm]: 175.43, 167.46, 164.77, 154.97, 132.37, 131.47, 121.70 ,
6
1
21.05, 115.60, 81.24, 54.74, 46.27, 36.38, 18.70, 14.25.
4
.1.1.10. (RS)-4-[1-(2,5-dichlorophenoxy)penthyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (11)
+
Ester 31, reaction time: 25h. Method A. White solid. Yield 33%, LC/MS purity: 98,8%,
o
1
t =5.34, mp=143–145 C, C H Cl N O (MW= 425.36). H NMR (500 MHz, DMSO-d ) δ
R
19 26
2
6
6
[
ppm]: 0.80 (t, J=7.16 Hz, 3H, CH ), 1.24-1.32 (m, 2H, CH ), 1.35-148 (m, 2H, CH ), 1.85-
3 2 2
1
.96 (m, 2H, CH ), 2.11 (s, 3H, N-CH ), 2.20 (br. s, 4H, Pip-3,5-H), 3.61 (br. s, 4H, Pip-2,6-
2 3
H), 4.80-4.83 (m, 1H, O-CH), 6.84-6.92 (m, 2H, NH ), 6.90-6.92 (m, 1H, Ph-6-H), 7.01 (d,
2
1
3
J=2.29 Hz, 1H, Ph-4-H), 7.36 (d, J=8.02 Hz, 1H, Ph-3-H). C NMR (126 MHz, DMSO-d ) δ
6
[
ppm]: 14.39, 22.42, 27.58, 34.02, 43.02, 46.23, 54.75, 81.56, 115.60, 121.08, 121.65, 131.41,
1
32.40, 154.98, 164.79, 167.50, 175.41.
4
.1.1.11. 4-[3-(2,5-dichlorophenoxy)propyl]-6-(4-methylopiperazin-1-yl)-1,3,5-triazin-2-
amine (12)
+
Ester 32, reaction time: 16 h. Method A. White solid. Yield 28%, LC/MS purity: 98,8%,
o
1
t =3.71, mp=126–128 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
ppm]: 2.05-2.06 (m, 2H, CH ), 2.12 (br. s, 3H, N-CH ), 2.21 (br. s, 4H, Pip-3,5-H), 2.50 (t,
2 3
J=7.16 Hz, 2H, CH ), 3.62 (br. s, 4H, Pip-2,6-H), 4.09 (t, J=6.01 Hz, 2H, O-CH ), 6.70 (br. s,
2
2
2
H, NH ), 6.94-6.96 (m, 1H, Ph-4-H), 7.16 (s, 1H, Ph-6-H), 7.37 (d, J= 8.59 Hz, 1H, Ph-3-H).
2
1
3
C NMR (126 MHz, DMSO-d ) δ [ppm]: 26.56, 34.81, 42.87, 46.31, 54.86, 69.14, 114.50,
6
1
20.80, 121.58, 131.32, 132.94, 155.14, 164.97, 167.30, 177.14.
4
.1.1.12. (RS)-4-[1-(3,4-dichlorophenoxy)ethyl]-6-(4-methylopiperazin-1-yl)-1,3,5-triazin-2-
amine hydrochloride (13)
2
5

+
Ester 33, reaction time: 30 h. Method B. White solid. Yield 13%, LC/MS purity: 100%,
o
1
t =4.28, mp=258–260 C, C H Cl N O (MW= 383.11). H NMR (500 MHz, DMSO-d ) δ
R
16 20
2
6
6
[
ppm]: 1.55 (d, J=6.87 Hz, 3H, CH ), 2.70 (br. s, 3H, N-CH ), 3.03 (br. s, 4H, Pip-3,5-H),
3 3
4
7
1
1
.55-4.63 (m, 4H, Pip-2,6-H), 5.31 (d, J=6.30 Hz, 1H, O-CH), 7.06-7.08 (m, 1H, Ph-6-H),
.38-7.39 (m, 1H, Ph-2-H), 7.48-7.49 (m, 1H, Ph-5-H), 8.18-8.36 (m, 2H, NH ), 11.85 (br. s,
2
+
13
H, NH ). C NMR (126 MHz, DMSO-d ) δ [ppm]: 19.67, 42.44, 51.69, 51.91, 74.69,
6
16.98, 118.55, 123.87, 131.49, 132.01, 156.82, 162.80.
4
.1.1.13. (RS)-4-[1-(3,4-dichlorophenoxy)propyl]-6-(4-methylopiperazin-1-yl)-1,3,5-triazin-
2
-amine (14)
+
Ester 34, reaction time: 20 h. Method A. White solid. Yield 50%, LC/MS purity: 91,4%,
o
1
t =4.61, mp=126–128 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
ppm]: 0.90 (t, J=7.45 Hz, 3H, CH ), 1.84 (q, J=6.70 Hz, 2H, CH ), 2.11 (s, 3H, N-CH ), 2.21
3 2 3
(br. s, 4H, Pip-3,5-H), 3.61 (br. s, 4H, Pip-2,6-H), 4.67 (t, J=6.30 Hz, 1H, O-CH), 6.82-6.84
(
m, 1H, Ph-6-H), 6.82-7.00 (m, 2H, NH ), 7.12 (s, 1H, Ph-2-H), 7.40 (d, J=9.16 Hz, 1H, Ph-
2
1
3
3
-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 10.34, 27.64, 43.11, 46.26, 54.73, 81.99,
6
1
16.52, 117.74, 122.80, 131.36, 131.81, 158.35, 164.85, 167.42, 175.57.
4
.1.1.14. 4-[3-(3,4-dichlorophenoxy)propyl]-6-(4-methylopiperazin-1-yl)-1,3,5-triazin-2-
amine (15)
+
Ester 35, reaction time: 18 h. Method A. White solid. Yield 45%, LC/MS purity: 99,2%,
o
1
t =3.91, mp=90–92 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
(
ppm]: 2.01-2.03 (m, 2H, CH ), 2.12 (s, 3H, N-CH ), 2.20 (br. s, 4H, Pip-3,5-H), 2.46-2.47
2 3
m, 2H, CH ), 3.61 (br. s, 4H, Pip-2,6-H), 3.98 (t, J=6.01 Hz, 2H, O-CH ), 6.74 (br. s, 2H,
2
2
NH ), 6.86-6.89 (m, 1H, Ph-6-H), 7.12 (d, J=2.29 Hz, 1H, Ph-2-H), 7.42 (d, J=9.16 Hz, 1H,
2
1
3
Ph-3-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 26.60, 34.85, 42.85, 46.29, 54.84, 68.40,
6
1
15.89, 116.80, 122.72, 131.40, 132.09, 158.62, 164.94, 167.29, 177.19.
4
.1.1.15. (RS)-4-[1-(3,5-dichlorophenoxy)ethyl]-6-(4-methylopiperazin-1-yl)-1,3,5-triazin-2-
amine (16)
+
Ester 36, reaction time: 20 h. Method A. White solid. Yield 29%, LC/MS purity: 100%,
o
1
t =4.33, mp=180–182 C, C H Cl N O (MW= 383.28). H NMR (500 MHz, DMSO-d ) δ
R
16 20
2
6
6
[
ppm]: 1.48 (d, J=6.30 Hz, 3H, CH ), 2.12 (s, 3H, N-CH ), 2.21 (s, 4H, Pip-3,5-H), 3.31 (s,
3 3
4
H, Pip-2,6-H), 4.96 (q, J=6.30 Hz, 1H, O-CH), 6.88 (br. s, 2H, NH ), 6.93 (d, J=1.72 Hz,
2
2
6

1
3
2
H, Ph-2,6-H), 7.03 (s, 1H, Ph-4-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 20.40, 42.90,
6
4
6.25, 54.74, 77.03, 115.13, 120.74, 134.86, 160.00, 164.86, 167.50, 176.09.
4
.1.1.16. (RS)-4-[1-(3,5-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (17)
+
Ester 37, reaction time: 16 h. Method A. White solid. Yield 48%, LC/MS purity: 100%,
o
1
t =4.90, mp=124–126 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d ) δ
R
17 22
2
6
6
[
ppm]: 0.95 (t, J = 7.4 Hz, 3H, CH ), 1.88 – 1.95 (m, 2H, CH ), 2.16 (s, 3H, N-CH ), 2.25 (br.
3 2 3
s, 4H, Pip-3,5-H), 3.54-3.76 (br. s, 4H, Pip-2,6-H), 4.77 (t, J = 6.5 Hz, 1H, O-CH), 6.91 (br. s,
H, NH ), 6.97 (d, J = 1.8 Hz, 2H, Ph-2,6-H), 7.03 (br. s, 1H, NH ), 7.10 (t, J = 1.8 Hz, 1H,
1
2
2
1
3
Ph-4-H). C NMR (126 MHz, DMSO-d ) δ [ppm]: 10.29, 27.48, 46.27, 54.76, 82.09,
6
1
15.16, 120.74, 134.85, 160.33, 164.81, 167.42, 175.35.
4
.1.1.17. (RS)-4-[1-(3,5-dichlorophenoxy)butyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (18)
+
Ester 38, reaction time: 21 h. Method A. White solid. Yield 46%, LC/MS purity: 98,3%,
o
1
t =5.26, mp=164–166 C, C H Cl N O (MW= 411.33). H NMR (500 MHz, DMSO-d ) δ
R
18 24
2
6
6
[
ppm]: 0.90 (t, J = 7.3 Hz, 3H, CH ), 1.31 – 1.49 (m, 2H, CH ), 1.79 – 1.91 (m, 2H, CH ),
3 2 2
2
.16 (s, 3H, N-CH ), 2.25 (br. s, 4H, Pip-3,5-H), 3.63 (br. s, 4H, Pip-2,6-H), 4.83 (dd, J = 7.8
3 1
Hz, J =5.2 Hz, 1H, O-CH), 6.91 (br. s, 1H, NH ), 6.94 – 6.97 (m, 2H, Ph-2,6-H), 7.03 (br. s,
2
2
1
3
1
1
H, NH ), 7.07 – 7.10 (m, 1H, Ph-4-H). C NMR (126 MHz, DMSO-d6 ) δ [ppm]: 175.57,
2
67.42, 164.81, 160.30, 134.86, 120.75, 115.12, 80.65, 54.73, 46.26, 36.34, 18.72, 14.21.
4
.1.1.18. (RS)-4-[1-(3,5-dichlorophenoxy)pentyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-
amine (19)
+
Ester: 39, reaction time: 22 h. Method A. White solid. Yield 44%, LC/MS purity: 99,5%,
o
1
t =5.70, mp=154–156 C, C H Cl N O (MW= 425.36). H NMR (500 MHz, DMSO-d ) δ
R
19 26
2
6
6
[
ppm]: 0.86 (t, J = 5.4 Hz, 3H, CH ), 1.25 – 1.46 (m, 4H, CH -CH ), 1.84 – 1.93 (m, 2H,
3 2 2
CH ), 2.16 (s, 3H, N-CH ), 2.25 (s, 4H, Pip-3,5-H), 3.64 (s, 4H, Pip-2,6-H), 4.79 – 4.84 (m,
2
3
1
–
3
H, O-CH), 6.91 (br. s, 1H, NH ), 6.95 – 6.98 (m, 2H, Ph-2,6-H), 7.04 (br. s, 1H, NH ), 7.08
2
2
1
3
7.11 (m, 1H, Ph-4-H). C NMR (126 MHz, DMSO-d6 ) δ [ppm]: 14.40, 22.42, 27.55,
3.98, 46.21, 54.74, 80.90, 115.12, 120.75, 134.86, 160.29, 164.80, 167.43, 175.55.
4
.1.1.19. 4-[3-(2,4-dichlorophenoxy)propyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine
(20)
2
7

+
Ester 40, reaction time: 17 h. Method A. White solid. Yield 43%, LC/MS purity: 100%,
o
1
t =4.09, mp=150–152 C, C H Cl N O (MW= 397.30). H NMR (500 MHz, DMSO-d6 ) δ
R
17 22
2
6
[
ppm]: 2.06-2.07 (m, 2H, CH ), 2.12 (br. s, 3H, N-CH ), 2.21 (br. s, 4H, Pip-3,5-H), 2.52 (br.
2 3
s, 2H, CH ), 3.62 (br. s, 4H, Pip-2,6-H), 4.06 (br. s, 2H, O-CH ), 6.73 (br. s, 2H, NH ), 7.10
2
2
2
1
3
(
br. s, 1H, Ph-6-H), 7.29 (br. s, 1H, Ph-5-H), 7.49 (br. s, 1H, Ph-3-H). C NMR (126 MHz,
DMSO-d6 ) δ [ppm]: 26.56, 34.84, 42.85, 46.30, 54.86, 69.04, 115.46, 122.91, 124.75,
1
28.58, 129.73, 153.57, 164.97, 167.30, 177.15.
4
.2. X-ray crystallographic studies
Crystals suitable for an X-ray structure analysis for both compounds were obtained from
methanol by slow evaporation of the solvent at room temperature.
Data for single crystals were collected using the XtaLAB Synergy-S diffractometer, equipped
with the Cu (1.54184 Å) Kα radiation source and graphite monochromator. The phase
problem was solved by direct methods using SIR-2014[48] and all non-hydrogen atoms were
2
refined anisotropically using weighted full-matrix least-squares on F . Refinement and further
calculations were carried out using SHELXL[49] . The hydrogen atoms bonded to carbons
were included in the structure at idealized positions and were refined using a riding model
with U (H) fixed at 1.5 U (C) for methyl groups and 1.2 U (C) for the other hydrogen
iso
eq
eq
atoms. Hydrogen atoms attached to nitrogen atoms were found from the difference Fourier
map and refined without any restraints. For molecular graphics MERCURY[50] program was
used.
Crystallographic data for 9: C H Cl N O, M = 397.30, wavelength 1.54184 Å, crystal
17
22
2
6
r
3
size = 0.13 x 0.18 x 0.60 mm , triclinic, space group Pī, a = 8.6624(3) Å, b = 9.8661(4) Å, c =
3
1
2.2609(5) Å, α = 70.529(4)°, β = 77.394(3)°, γ = 86.601(3)°, V = 964.02(7) Å , Z = 2, T =
1
00(2) K, 25842 reflections collected, 3867 unique reflections (R = 0.0301), R1 = 0.0314,
int
wR2 = 0.0777 [I > 2σ(I)], R1 = 0.0325, wR2 = 0.0786 [all data].
Crystallographic data for 20: C H Cl N O, M = 397.30, wavelength 1.54184 Å, crystal
17
22
2
6
r
3
size = 0.08 x 0.15 x 0.39 mm , monoclinic, space group P2 /n, a = 15.5045(1) Å, b =
1
3
6
5
0
.4311(1) Å, c = 18.8146(5) Å, β = 94.185(1)°, V = 1871.02(3) Å , Z = 4, T = 100(2) K,
6835 reflections collected, 3876 unique reflections (Rint = 0.0391), R1 = 0.0276, wR2 =
.0727 [I > 2σ(I)], R1 = 0.0285, wR2 = 0.0733 [all data].
2
8