Aryl-1,3,5-triazine derivatives as histamine H4 receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2014.06.032

A series of novel 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazine derivatives with different aryl substituents in the 6-position was designed, synthesized and evaluated for histamine H₄ receptor (H₄R) affinity in Sf9 cells expressing human H₄R co-expressed with G-protein subunits. Triazine derivative 8 with a 6-(p-chlorophenyl) substituent showed the highest affinity with hH₄R K_i value of 203 nM and was classified as an antagonist in cAMP accumulation assay. This compound, identified as a new lead structure, demonstrated also anti-inflammatory properties in preliminary studies in mice (carrageenan-induced edema test) and neither possessed significant antiproliferative activity, nor modulated CYP3A4 activity up to concentration of 25 μM. In order to discuss structure-activity relationships molecular modeling and docking studies were undertaken.

Full text of DOI:10.1016/j.ejmech.2014.06.032

Accepted Manuscript
Aryl-1,3,5-triazine derivatives as histamine H receptor ligands
4
Dorota Łażewska, Małgorzata Więcek, Joanna Ner, Katarzyna Kamińska, Tim
Kottke, J. Stephan Schwed, Małgorzata Zygmunt, Tadeusz Karcz, Agnieszka Olejarz,
Kamil Kuder, Gniewomir Latacz, Marek Grosicki, Jacek Sapa, Janina Karolak-
Wojciechowska, Holger Stark, Katarzyna Kieć-Kononowicz
PII:
S0223-5234(14)00551-0
10.1016/j.ejmech.2014.06.032
EJMECH 7075
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2014
Revised Date: 17 April 2014
Accepted Date: 16 June 2014
Please cite this article as: D. Łażewska, M. Więcek, J. Ner, K. Kamińska, T. Kottke, J.S. Schwed, M.
Zygmunt, T. Karcz, A. Olejarz, K. Kuder, G. Latacz, M. Grosicki, J. Sapa, J. Karolak-Wojciechowska, H.
Stark, K. Kieć-Kononowicz, Aryl-1,3,5-triazine derivatives as histamine H receptor ligands, European
4
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.032.
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ACCEPTED MANUSCRIPT
Aryl-1,3,5-triazine derivatives as histamine H receptor ligands
4
1
1
1
1
2
Dorota Łażewska , Małgorzata Więcek , Joanna Ner , Katarzyna Kamińska , Tim Kottke , J.
2
3
1
1
1
Stephan Schwed , Małgorzata Zygmunt , Tadeusz Karcz , Agnieszka Olejarz , Kamil Kuder ,
Gniewomir Latacz , Marek Grosicki , Jacek Sapa , Janina Karolak-Wojciechowska , Holger
1
1
3
4
2
,5
1*
Stark , Katarzyna Kieć-Kononowicz
1
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
2
College, Medyczna 9 str., 30-688 Kraków, Poland; Goethe University, Institute of
Pharmaceutical Chemistry, Biozentrum, ZAFES/CMP/ICNF, Max-von-Laue-Str. 9, 60438
3
Frankfurt am Main, Germany; Department of Pharmacodinamics, Jagiellonian University
4
Medical College, Medyczna 9 str., 30-688 Kraków, Poland; Institute of General and
Ecological Chemistry, Technical University of Łódź, Żeromskiego 116 str., 90-924 Łódź,
5
Poland; Heinrich-Heine-University, Institute of Pharmaceutical and Medicinal Chemistry,
Universitaetsstr. 1, 40225 Duesseldorf, Germany
*
Corresponding author: tel +48 12 6205580; fax + 48 12 620 55 96
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec-Kononowicz)
NH2
K = 203 nM
N
N
i
N
N
N
Cl
CH3
8

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Highlights
•
A series of 2,4,6-trisubstituted 1,3,5-triazines were obtained as potential histamine
H R ligands
4
•
•
Some compounds showed K values in the low submicromolar range
i
Some compounds exhibited in vivo anti-inflammatory activity

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Aryl-1,3,5-triazine derivatives as histamine H receptor
4
ligands
1
1
1
1
2
Dorota Łażewska , Małgorzata Więcek , Joanna Ner , Katarzyna Kamińska , Tim Kottke , J.
Stephan Schwed , Małgorzata Zygmunt , Tadeusz Karcz , Agnieszka Olejarz , Kamil Kuder ,
2
3
1
1
1
1
1
3
4
Gniewomir Latacz , Marek Grosicki , Jacek Sapa , Janina Karolak-Wojciechowska , Holger
2
,5
1*
Stark , Katarzyna Kieć-Kononowicz
1
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9
2
str., 30-688 Kraków, Poland; Goethe University, Institute of Pharmaceutical Chemistry, Biozentrum,
ZAFES/CMP/ICNF, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany; Department of
3
Pharmacodinamics, Jagiellonian University Medical College, Medyczna 9 str., 30-688 Kraków, Poland;
4
Institute of General and Ecological Chemistry, Technical University of Łódź, Żeromskiego 116 str., 90-924
5
Łódź, Poland; Heinrich-Heine-University, Institute of Pharmaceutical and Medicinal Chemistry,
Universitaetsstr. 1, 40225 Duesseldorf, Germany
*
Corresponding author: tel +48 12 6205580; fax + 48 12 620 55 96
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec-Kononowicz)
Abstract: A series of novel 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazine derivatives
with different aryl substituents in the 6-position was designed, synthesized and evaluated for
histamine H receptor (H R) affinity in Sf9 cells expressing human H R co-expressed with G-
4
4
4
protein subunits. Triazine derivative 8 with a 6-(p-chlorophenyl) substituent showed the
highest affinity with hH R K value of 203 nM and was classified as an antagonist in cAMP
4
i
accumulation assay. This compound, identified as a new lead structure, demonstrated also
anti-inflammatory properties in preliminary studies in mice (carrageenan-induced edema test)
and neither possessed significant antiproliferative activity, nor modulated CYP3A4 activity up
to concentration of 25 ꢀM. In order to discuss structure-activity relationships molecular
modeling and docking studies were undertaken.
Keywords: Histamine H receptor, 4-methylpiperazines, 2,4,6-trisubstituted 1,3,5-triazines,
4
anti-inflammatory properties
1. Introduction
The human histamine H receptor (hH R) was cloned in 2000 and has constituted an
4
4
interesting target for drug development thereafter. Pharmacological studies suggest utility of
H R antagonists/inverse agonists in the treatment of inflammatory and immunomodulatory
4
1
diseases, e.g., allergic rhinitis, asthma, atopic dermatitis, colitis and pruritus. Among
described H R antagonists/inverse agonists pyrimidine-2-amine derivatives consist important
4
2
-7
lead structures with many potent compounds [for review see ]. Some of early developments

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already made their way to clinical trials and promising results from the clinical studies for
8
,9
compounds UR-63325 and ZPL-38937887 were reported.
The 1,3,5-triazine (s-triazine) moiety is present in many biologically active structures
with the broad range of therapeutic utility. Such compounds have been proved to exhibit
1
0,11
12
13,14
antimicrobial,
antimalarial activity and chemotherapeutic potential.
Moreover, for
some triazine dimers the ability to mimic the protein A binding to murine and human IgG
1
5
16
antibody was described. The chemistry of 1,3,5-triazine was recently reviewed by Blotny.
A couple of 4-(4-methylpiperazin-1-yl)-1,3,5-triazines were disclosed in the patent
application from Johnson&Johnson and some of them showed high affinities (K < 20 nM) for
i
1
7
hH R (e.g. 1, Fig. 1).
4
1
8
Taking together the published results on pyrimidine derivatives (e.g. 2) as well as
information on triazines (e.g. 1) a search was initiated to find active compounds among 2-
1
7
amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazines. In this work, the synthesis, H R affinities,
4
functional properties, receptor selectivity and anti-inflammatory properties of these
compounds (exemplified by general structure on Fig. 2) are described. Also for selected
compounds antiproliferative effect and metabolic stability was checked.
2. Results and Discussion
2
.1 Chemistry
The general synthetic procedure for preparation of 2,4,6-trisubstituted 1,3,5-triazines is
outlined in Scheme 1. Triazine derivatives 3-23 were obtained as the result of the reaction of
esters 3a-23a with biguanidine dihydrochloride. 4-Methylpiperazin-1-yl biguanidine
dihydrochloride intermediate was obtained by the heating of cyanoguanide and 4-
1
9
methylpiperazine dihydrochloride in 1-butanol. All acid methyl esters were commercially
2
0
available (except 7a and 21a which were synthesized according to the known procedure ).
Cyclization of 4-methylpiperazin-1-yl biguanidine dihydrochloride with the appropriate
carboxylic acid methyl ester in the presence of sodium methoxide have been known to yield
2
1
1
3
,3,5-triazines. The reaction was led in the boiling point and proceeded in low yields (4-
5%).
2
.2 Histamine H receptor screening
4
The series of novel substituted phenyl derivatives of 1,3,5-triazine with one to three
3
substituents at the benzene ring was screened at hH R in [ H]histamine binding experiments
4
(Table 1). Investigated was the SAR of the 6 substituent of the 1,3,5-triazine derivatives on
the hH R activity. All compounds share the 4-methylpiperazinyl moiety (as basic center) and
4
the primary aromatic amino group as additional basic center as well as hydrogen
acceptor/donor functionality. These both elements are widely present in many published H R
4
2
-7
antagonists. Most of the obtained compounds showed micro/submicromolar affinities.
The most potent in these group was compound 8 with chlorine substituent in the para-
6
position with submicromolar affinity (K = 203 nM ). The change of chlorine position to meta
i
(
5, K = 408 nM) and especially to ortho (4, K = 1261 nM) position drastically reduced
i
i
affinity. Generally, the same trend was also observed for methyl and cyano substituents
compare 6 vs 12 and 7 vs 14). In addition, the change of chlorine in the para-position for
(

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different substituents (F, Br, I, CH , CF , CN, N(CH ) , compounds 9-15), led to decrease of
3
3
3 2
potency compared with that of 8. Interestingly, compound 3, the unsubstituted analogue, had
higher affinity than the majority of the substituted derivatives.
The introduction of the second substituent (chlorine 18 or methoxy group 16, 17) to
meta-position e.g. compound 5 did not improve the affinity. Two fluorine substituents in 2-
and 6-position (19) are less beneficial than two (20) or three atoms (21) in respectively the
3
,5-position and the 3,4,5-position.
Comparing the present results with the published data for pyrimidine analog of 8 (compound
1
8
2
, Fig. 1 ) a reduction in affinity of more than one log unit was noticed. Although, it seems
that the additional nitrogen in the central core (triazine ring instead of pyrimidine one) is
detrimental for H R affinity, some compounds display submicromolar affinities. So further
4
investigations should be done (e.g. blocking free amino group) as some related triazines with
1
7
high H R affinities have recently been described.
4
2
.3 Functional properties at histamine H receptor and selectivity over histamine H3
4
receptor for selected compounds
In order to better characterize the pharmacological profile of the investigated
compounds selected structures (8 and 10) were additionally tested for their functional
properties in cAMP accumulation assay at recombinant cellular model. Their selectivity over
histamine H receptor (H R) subtype, representing the highest degree of homology with H R,
3
3
4
was also evaluated.
Investigated compounds caused a blockade of the histamine-induced cAMP reduction in
CHO-hH R-cAMPzen cells, co-treated with forskolin and were therefore classified as
4
antagonists at H Rs. Antagonist potency (IC ) was determined by performing a dose response
4
50
curve in presence of H R agonist (histamine, 140 nM, corresponding to its EC ) and
4
80
forksolin (10 µM) (Fig. 3). Obtained IC values for both evaluated compounds were well in
5
0
accordance with their K values at hH Rs and the order of potency also was retained in
i
4
functional tests (8, K = 203 nM, IC = 512 nM; 10, K = 524 nM, IC = 1630 nM).
i
50
i
50
Since H R shows the highest sequence homology (~60% in the transmembrane
4
2
2
domains) to the H R subtype , considered structures were additionally tested for their
3
interaction with H R. For that purpose radioligand binding assays were performed with the
3
3
α
use of [ H]N -methylhistamine and membrane preparations from HEK-293 cells stably
expressing human H R.
3
Performed assays revealed that compounds 8 and 10 show low affinity for H R (8, K =
3
i
1
3.2 ꢀM; 10, K =15.0 ꢀM) and are therefore selective towards H R subtype (H R K /H R K :
i
4
3
i
4
i
8
, 29-fold selectivity; 10, 65-fold selectivity).
2
.4 Anti-inflammatory activities of selected compounds
Radioligand receptor binding assay showed that some of the investigated 1,3,5-triazine
derivatives have micro/submicromolar affinities for the hH R and act as antagonists at that
4
receptor. These results were inspiration to study the anti-inflammatory activity of the selected
2
3,24
1
,3,5-triazine derivatives in vivo in the carrageenan-induced paw edema test.
The tested

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compounds (5, 8, 9, 10, 12, 18 and 23) administered intraperitonally caused inhibition of
carrageenan-induced edema in mice, when compared to the control (ketoprofen; Fig. 4).
Compound 10, in all doses, throughout duration of the experiment, showed a
statistically significant anti-inflammatory activity. At a dose 50 mg/kg ip, compound 10,
decreased carrageenan-induced edema by 90.6%, 90.0% and 91.8% in 1, 2 and 3 hours of the
experiment, respectively, while at a dose 25 mg/kg, by 68.8%, 70.0% and 81.6%,
respectively. Compound 10 at a dose 10 mg/kg, suppress edema paw in the range 37.5% -
6
3.3%. Compound 8 showed statistically significant effects in this experimental model. Given
intraperitoneally at a dose 50 mg/kg, inhibited carrageenan-induced edema in 1, 2, 3 hours of
the experiment by 90.6%, 92.5%, 91.8%, respectively, while at a dose 25 mg/kg, by 56.2%,
6
5.0% and 71.5%, respectively. The half maximal the anti-inflammatory effect was observed
at the lowest dose, analogously by 46.9%, 50.0%, 59.2%. Compound 12 at a dose 50 mg/kg
and 25 mg/kg, reduced carrageenan-induced edema, in the first hour of measurement by
9
0.6% and 50.0%, in the second hour of measurement by 75.0% and 52.5%, and in the third
hour by 79.5% and 61,2%, respectively. Compound 9 given at a dose 50 mg/kg, suppress
carrageenan-induced edema by 81.3%, 77.5%, 81.6% in 1, 2 and 3 hours of the experiment,
respectively. The same compound, given at the lower dose 25 mg/kg, also significantly
significant inhibited edema by 75%, 57.5%, 69.4%, compared to the control group. However,
reduction of the dose lead to a decreased anti-inflammatory activity. Compound 5 showed
statistically significant anti-inflammatory effect, given in two doses. This compound (5)
administered at a dose 50 mg/kg and 25 mg/kg, decreased carrageenan- induced edema,
throughout duration of the observation, by 81.2% - 87.7% and 46.8% - 69.3%, respectively.
Compound 23 at a dose of 50 mg/kg inhibited paw edema, in the first, second and third hour,
after carrageenan injection, by 65.6%, 65%, 71.4%, respectively. In lower doses (25 mg/kg or
1
0 mg/kg) the decrease in activity was observed. Compound 18 given in the range 50 mg/kg -
1
0 mg/kg, showed anti-inflammatory effect. This effect was intensified, with an increase in
the duration of the experiment, reaching a maximum in the third hour, which was 51.0% (50
mg/kg), 42.8% (25 mg/kg) and 44.8% (10 mg/kg). Ketoprofen was used as the reference
compound in dosages from 50 to 10 mg/kg. The largest anti-inflammatory activity was
observed at the highest dose. Ketoprofen administered at a dose 50 mg/kg, reduced
carrageenan-induced edema, after the first, second and third hour after injection of
carrageenan, by 66.7%, 70.7%, 77.4%, respectively. Ketoprofen given intraperitoneally, at a
dose of 25 mg/kg, inhibited edema, in the third hour of the experiment by 20.0%, 55.0%,
6
7.3%, respectively, while at a dose 10 mg/kg, suppress edema by 26.7%, 55.1% and 62.7%.
Preliminary pharmacological studies showed, that all tested compounds had anti-
inflammatory effect. The strongest inhibition of paw edema was shown at a dose of 50 mg/kg
in three measurement times, in the range from 42.5% to 92.5%. The compounds administered
at a dose 25 mg/kg, reduced paw edema in the first, second and third hour of the experiment
in the range 27.5% - 81.6%, while at a dose 10 mg/kg in the range 14.7% -63.2%. In
comparison of the anti-inflammatory properties of tested compounds with the activity of the
reference compound, it has been shown, that compounds 8 and 10 at all doses, throughout
duration of the experiment, showed stronger anti-inflammatory activity than ketoprofen, while
compounds 5 and 9 showed apparently stronger anti-inflammatory activity than ketoprofen,
only in the two highest doses.

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2
.5 Antiproliferative assay
The most important properties in drug discovery which describe the ability of the
compound to be an ideal drug candidate are absorption, distribution, metabolism, elimination
2
5
and toxicity. Recently, the in vitro studies are increasingly applied for toxicity studies during
earlier stages of drug discovery. This strategy is a reliable alternative to in vivo methods and
allows for study of greater number of compounds. Additionally, the in vitro tests reduce the
2
6
usage of animals (3Rs approach). Moreover, the toxicity tests, for example antiproliferative
or cytotoxicity assays, are useful tools in conventional anticancer drug discovery and
2
7
development. During this study we used formazan dye based EZ4U test for the
determination of antiproliferative effect of 8 and 10 on HEK-293 and IMR-32 cell lines. The
incubation of HEK-293 cells in the presence of the tested compounds for 48 h showed weak
antiproliferative effect. The reduction of cell viability was observed only at very high
concentrations: 100 and 250 µM. However, it must to be pointed out, that in comparison to
the calculated IC value of the standard drug doxorubicin (DX) against HEK-293 (455 nM)
5
0
the significant antiproliferative effect 8 and 10 was observed in more than 200 fold higher
concentration (Fig. 5).
Measured antiproliferative activity of the examined compounds against neuroblastoma
cell line IMR-32 was similar to that observed in HEK-293 cells. Compound 8 did not show
any significant effect on cells viability up to the concentration of 10 µM and 10 up to the
concentration of 100 µM. However, compared to DX (calculated IC : 5.1 nM) the examined
5
0
compounds did not possess the significant antiproliferative activity (Fig. 6).
2
.6 Metabolic stability
The in silico prediction of the sites of metabolism in phase I reactions is commonly used
in the preliminary stage of study on the metabolic pathway prediction. MetaSite is an example
of the easy and inexpensive computational method for in silico prediction of metabolic
transformations related to CYP450 reactions (e.g. CYP3A4). MetaSite identifies the most
likely sites of metabolism in an examined compound and predicts structures of metabolites by
consideration of two factors: a thermodynamic factor (enzyme-substrate recognition) and a
2
8,29
kinetic factor describing the chemical transformations catalyzed by the enzymes.
Cytochrome P450 3A4 is known to metabolize the majority of therapeutics agents and
endogenous substrates and in the recent years, many drug interactions were documented as
3
0
associated with metabolic activity of this cytochrome. In our study, compounds 8 and 10
were examined in silico in two models of metabolism: liver and CYP3A4 exclusively. For
both compounds (8 and 10) in the used models, the highest probability of metabolism
occurred at N-methyl group of piperazine moiety and the suggested reaction was N-
demethylation. The locations of metabolic modifications are shown on Fig. 7. The darker
color of the marked functional group indicates higher probability of its involvement in the
metabolism pathway.
Moreover, we also used luminescent CYP3A4 P450-Glo™ Assay (Promega) for testing
the effects of these two H R ligands (8 and 10) on CYP3A4 activities. In vitro studies with the
4
recombinant human CYP3A4 isoforms are the reliable methods for evaluation of metabolic

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stability and drug-drug interaction. Luminescent CYP3A4 P450-Glo™ Assay is based on the
conversion of the Luciferin-PPXE, the beetle D-luciferin derivative, into D-luciferin by
recombinant human CYP3A4. After addition of the firefly luciferase the measured
luminescence is used to evaluate the potential inhibition/induction of CYP3A4 by tested
compounds. The measured amount of light produced in that reaction is proportional to the
3
1
CYP3A4 activity. As is shown on Fig. 8 compounds 8 and 10 did not modulate significantly
of CYP3A4 activity up to 25 µM, whereas the reference compound ketoconazole inhibited
completely the CYP3A4 activity already at 10 µM.
2
.7 X-ray study
From topological point of view, all studied derivatives can be expressed in terms of
triazine with the fastened phenyl and piperazine rings. The mutual position of all three rings
and suitable crystal architecture depends mainly on the phenyl ring substitution. At the same
time, in the frame of studied group of compounds, degree of affinity to the designed receptor
must be associated with rings arrangements as well as with ability to generate suitable
intermolecular interactions. Thus, crystallographic studies of compounds 8 (most potent with
para-Cl) and 17 (4-Cl-6-methoxy compound) seem to be sufficient for the description of
structural phenomena for studied triazines.
In both X-ray structures for 8 and 17 (Fig. 9) benzene and piperazine rings are slightly
inclined to triazine and the distances between them are almost identical (7.08 Å and 6.89 Å
respectively for 8 and 17). Hence the impact of substituents nature and position on molecule
conformation seems to be insignificant.
However this impact is evident in the pattern of molecules assembling in the crystal.
The main motif in both structures is based on N3-H…N strong H-bond involving free amine
group (N3 nitrogen) and one of three triazine nitrogens. Thus, in 8 the N3-H…N2 attaches
molecules into the dimers, while in 17 molecules are assembled in helix through N3-H…N6
hydrogen bond. The N4 triazine nitrogen does not contribute to the crystal net. In both
crystals hydrogen bonds from N3 to piperazines N4’ are connecting main motifs (for details
see respective Fig. 10 and Fig. 11). Therefore, it is evident that benzene ring substituents
nature and position are able to manipulate with the charge of N2 and N6 triazine nitrogens. In
consequence ability to create complexes with designed receptor depends on the benzene
substitution as was shown with the screening data.
2
.8. In silico molecular and docking studies
In order to determine protein-ligand interactions and its possible influence on in vitro activity
docking studies for all synthesized ligands, as well as for two reference compounds (1-2) were
performed. All of the docked ligands showed moderate to good docking score values in the
range of -4.185 (13) to -6.738 (1).
3
2
As it has been previously described by Jongejan the H R has two major anchoring points:
4
3.32
5.46
ASP94 and GLU182 . Two possible ligand orientations in the binding pocket of H R has
also been found, which is in agreement with the study of Feng and recent studies of
Schultes (Fig. 12). For all the obtained docked ligands (3-23), interactions with only
were found. However, for the most active ligands in this group (e.g. 5, 8-10, 12)
protonated methylpiperazine nitrogen is in very close proximity to GLU182
4
3
3
3
4
3.32
ASP94
5
.46
and might

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form weak hydrogen bonds. For most of the compounds, hydrogen bond formation between
6
.51
protonated nitrogen and TYR225
was found. Stabilization of the benzene ring via π- π
stacking interactions for compounds 3, 5, 6, and with pyrimidine ring of compound 2 was
observed.
3
.33
For compounds with triazine core, benzene ring of TYR95
stabilize receptor-ligand
complexes through π- π stacking interactions with triazine ring for all of tested ligands and
with substituted benzene ring for compounds 1, 3, 6, 17. For compounds 1, 3, 5, 6 and 17 also
additional ligand stabilization through π- π stacking interactions between substituted phenyl
6
.54
and PHE228 was observed. For most active compounds in this series 8-10, halogen atoms
2
.53
para substituted to benzene ring fits in the hydrophobic pocket formed by VAL64
,
2
.58
3.28
ILE69
and TRP90
(Fig. 13). It can be observed that for the structural isomers of 8,
3
5
compounds 4 and 5, what is in agreement with Surgand et.al ortho and meta substituted
chlorine of 4 and 5 might not fit in the above mentioned pocket. For the most active
compounds 1 and 2 protonated methylpiperazine nitrogen forms a hydrogen bond with
5
.42,
THR178
(Fig. 13), that seems to highly influence the stability of ligand in H R binding
4
pocket, as well as ligands’ in vitro affinity. Even though the distance between the atoms do not
meet the requirements of hydrogen bond with methylated piperazine nitrogen, for most active
compounds (only in binding manner A) in this series methylated piperazine nitrogen was in
5
.42
very close proximity to THR178 (3,2-3,6 Å). Moreover, paying attention to the activity of
reference compound 2 with free amine group and its position in H R homology model binding
4
pocket, it could be observed, that the amine group of compound 2 is in very close proximity to
6
.51
TYR225 (3,17 Å) and thus might influence higher affinity of compound 2 (vs e.g. 5) to
H R, by forming hydrogen bond of moderate strength with this moiety.
4
3. Conclusions
Generally, the change of the substituent in the phenyl moiety largely influenced the H R
4
affinity although it seems that this part of the structure did not decide on the activity but only
influenced its degree. Histamine H receptor antagonist - 8, with promising submicromolar
4
affinity (203 nM) and good selectivity over hH R, could be a good lead structure for our
3
further investigations. X-ray studies indicated the preferences of triazine nitrogen N2 to be the
potential H-bond acceptor in complexes with designed receptor. Molecular modelling and
docking studies allowed to discuss the mode of ligands interaction with elements of histamine
H R model. Additional preliminary pharmacological studies demonstrated the in vivo anti-
4
inflammatory properties of tested triazines. The selected compounds (8 and 10) did not show
the significant antiproliferative effect and did not modulate CYP3A4 activity.
4. Experimental
4
.1 Chemistry
Melting points were determined on MEL-TEMP II apparatus and are uncorrected. IR
spectra were measured as KBr pellets on FT Jasco IR spectrometer. Mass spectra (LC/MS)
1
13
were performed on Waters TQ Detector mass spectrometer. H-NMR and C NMR spectra
were recorded on Varian-Mercury 300 MHz spectrometer in DMSO-d . Chemical shifts in
6

ACCEPTED MANUSCRIPT
1
H-NMR spectra are expressed in ppm downfield from deuterated solvent signal treated as
reference. Data are reported in the following order: multiplicity (br, broad; def, deformed; s,
singlet; d, doublet; t, triplet; m, multiplet; Ph, phenyl; Pp, piperazine), approximate coupling
constants J in Hertz (Hz), number of protons. 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 determinated (%).
Elemental analyses (C, H, N) were measured on Elemental Vario-EL III instrument and are
within ± 0.5% of the theoretical values. TLC was carried out using silica gel F254 plates
(Merck). The spots were visualized with Dragendorff's reagent or by UV absorption at 254
nm.
4
.1.1. General procedure for synthesis of 1,3,5-triazines (3-22)
To 10 mL of absolute methanol was added 10 mmol of sodium and then 4-methylpiperazin-1-
19
yl biguanidine x 2HCl (5 mmol) and the appropriate carboxylic acid ester (5 mmol). The
reaction mixture was refluxed for 15-30 h and 10 mL of water was added and stirred in the
room temperature for 0.5h. The precipitate was separated and crystallized from methanol.
4
.1.2. 4-(4-methylpiperazin-1-yl)-6-phenyl-1,3,5-triazin-2-amine (3)
0
1
White solid. Mp. 171-174 C. Yield:21% (280 mg). C H N (MW 270.33). H-NMR
1
4
18
6
[
3
DMSO-d ]: δ=8.28 (m, 2H, Ph 2,6-H), 7.41-7.53 (m, 3H, Ph 3,4,5-H), 6.89 (br s, 2H, -NH ),
6 2
1
3
.78 (br s, 4H, Pp-2,6-H), 2.33 (t, 4H, J=4.87 Hz, Pp-3,5-H), 2.19 (s, 3H, CH ). C NMR
3
[
(
DMSO-d ]: δ (ppm) 170.1, 167.7, 165.2, 137.4, 131.7, 128.6, 128.2, 54.9, 46.3, 43.0. IR
6
-1
+/-
+
KBr) [cm ] ν= 3348 (br NH ). LC/MS : purity: 100%, t =2.40,(ESI) m/z [M+H] 271.19.
2
R
4
.1.3. 4-(4-methylpiperazin-1-yl)-6-(2-chloro-phenyl)-1,3,5-triazin-2-amine (4)
0
1
White solid. Mp. 214-216 C. Yield:5% (80 mg). C H N Cl (MW 304.79). H-NMR
1
4
17
6
[
DMSO-d ]: δ=7.62-7.57 (m, 1H, Ph-3-H), 7.50-7.34 (m, 3H, Ph-4,5,6-H), 6.97 (br s, 2H,
6
13
NH ), 3.73-3.65 (m, 4H, Pp-2,6-H), 2.30-2.20 (br s, 4H, Pp-3,5-H), 2.17 (s, 3H, CH ). C
2
3
NMR [DMSO-d ]: δ (ppm) 172.0, 167.3, 164.7, 138.0, 131.2, 130.4, 127.3, 54.8, 46.2. IR
6
-1
+/-
+
(
KBr) [cm ] ν= 3298, 3121 (br NH ). LC/MS : purity: 100%, t =2.54, (ESI) m/z [M+H]
2 R
3
05.16.
.1.4. 4-(4-methylpiperazin-1-yl)-6-(3-chloro-phenyl)-1,3,5-triazin-2-amine (5)
4
0
1
White solid. Mp 196-198 C. Yield:11% (160 mg). C H N Cl (MW 304.79). H-NMR
1
4
17
6
[
DMSO-d ]: δ=8.25-8.21 (m, 1H-Ph-2-H), 8.21-8.19 (m, 1H, Ph-6-H), 7.60-7.55 (m, 1H, Ph-
6
4
2
-H), 7.49 (t, 1H, J = 7.7 Hz, Ph-5-H), 6.98 (br s, 2H, NH ), 3.90-3.65 (br s, 4H, Pp-2,6-H),
2
-1
13
.40-2.30 (m, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). IR (KBr) [cm ] ν= 3384 (br NH ). C NMR
3
2
[
DMSO-d ]: δ (ppm) 168.8, 167.6, 165.2, 139.6, 133.6, 131.4, 130.7, 127.8, 126.8, 54.8, 46.2.
6
+
/-
+
LC/MS : purity: 99.7%, t =2.54, (ESI) m/z [M+H] 305.25. Anal. Calcd for C H N Cl:
R
14 17
6
C55.16, H5.62, N27.58; Found: C55.30, H5.69, N27.65.
4
.1.5. 4-(4-methylpiperazin-1-yl)-6-(3-methyl-phenyl)-1,3,5-triazin-2-amine (6)
0
1
White solid. Mp 187-190 C. Yield:11% (160 mg). C H N (MW 284.36). H-NMR
1
5
20
6
[
DMSO-d ]: δ=8,05-8,08 (m, 2H, Ph 4,5-H), 7,31-7,34 (m, 2H, Ph-2,6-H), 6,86 (br s, 2H, -
6
1
3
NH ), 3,78 (br.s, 4H, Pp 3,5-H), 2,33 (t.def, 7H, Pp-3,5-H + PhCH ), 2,19 (s, 3H, CH ). C
2
3
3

ACCEPTED MANUSCRIPT
NMR [DMSO-d ]: δ (ppm) 170.2, 167.6, 165.3, 137.7, 137.4, 132.3, 128.7, 128.5, 125.5,
6
-
1
+/-
5
4.9, 46.3, 43.0, 21.5. IR (KBr) [cm ] ν= 3323, 3140 (br NH ). LC/MS : purity: 98.8%,
2
+
t =2.93, (ESI) m/z [M+H] 285.29. Anal. Calcd for C H N : C63.36; H7.09; N29.55;
R
15 20
6
Found: C63.25; H7.20; N29.34.
4
.1.6. 4-(4-methylpiperazin-1-yl)-6-(3-cyano-phenyl)-1,3,5-triazin-2-amine (7)
0
1
White solid. Mp 242-243 C. Yield:13% (190 mg). C H N (MW 295.34). H-NMR [DMSO-
15
17
7
d ]: δ=8.61-8.53 (m, 2H, Ph-2,6-H), 7.99 (dt, J = 7.7 Hz, J = 1.5 Hz, 2H, Ph-4-H), 7.69 (t,
6
1
2
J =7.7 Hz, 1H, Ph-5-H), 7.04 (br s, 2H, NH ), 3.80 (br s, 4H, Pp-2,6-H), 2.34 (def t, 4H, Pp-
2
2
1
3
3
1
,5-H), 2.20 (s, 3H, CH ). C NMR [DMSO-d ]: δ (ppm) 168.3, 167.5, 165.08, 138.6, 135.0,
3 6
-1
32.7, 131.6, 130.2, 119.1, 111.9, 54.8, 39.90, 46.2, 43.0. IR (KBr) [cm ] ν= 3431, 3294 (br
NH ). LC/MS : purity: 99.4%, t =2.82, (ESI) m/z [M+H] 296.26. Anal. Calcd for C H N :
+
/-
+
2
R
15 17
7
C61.00, H5.80, N33.20; Found: C60.59, H5.84, N32.70.
4
.1.7. 4-(4-methylpiperazin-1-yl)-6-(4-chloro-phenyl)-1,3,5-triazin-2-amine (8)
0
1
White solid. Mp 229-231 C. Yield: 20% (300 mg). C H N Cl (MW 304.79). H-NMR
1
4
17
6
[
DMSO-d ]: δ=8.30-8.20 (m, 2H, Ph-2,6-H), 7.60-7.45 (m, 2H, Ph-3,5-H), 6.94 (br s, 2H,
6
13
NH ), 3.90-3.65 (br s, 4H, Pp-2,6-H), 2.40-2.30 (m, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C
2
3
NMR [DMSO-d ]: δ (ppm) 169.2, 167.6, 165.2, 136.4, 136.3, 130.0, 128.8, 54.8, 46.2. IR
6
-1
+/-
+
(
KBr) [cm ] ν= 3322, 3138 (br NH ). LC/MS : purity: 100%, t =3.60, (ESI) m/z [M+H]
2 R
3
05.23. Anal. Calcd for C H N Cl: C55.16, H5.62, N27.58; Found: C54.79, H5.75, N27.59.
14 17 6
4
.1.8. 4-(4-methylpiperazin-1-yl)-6-(4-fluoro-phenyl)-1,3,5-triazin-2-amine (9)
0
1
White solid. Mp 198-200 C. Yield:15% (210 mg). C H N F (MW 288.33). H-NMR
1
4
17
6
[
DMSO- d ]: δ= 8.36-8.29 (m, 2H, Ph-2,6-H), 7.29-7.24 (m, 2H, Ph-3,5-H), 6.91 (br s, 2H,
6
13
NH ), 3.90-3.65 (br s, 4H, Pp-2,6-H), 2.34-2.31 (m, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C
2
3
NMR [DMSO-d ]: δ (ppm) 169.2, 167.6, 165.2, 133.9, 130.7, 130.6, 115.7, 115.4, 54.8, 46.2,
6
-1
+/-
4
3.0. IR (KBr) [cm ] ν= 3318, 3138 (br NH ). LC/MS : purity: 96.3%, t =2.83, (ESI) m/z
2 R
+
[
M+H] 289.21.
4
.1.9. 4-(4-methylpiperazin-1-yl)-6-(4-bromo-phenyl)-1,3,5-triazin-2-amine (10)
0
1
White solid. Mp 243-245 C. Yield:17% (350 mg). C H N Br (MW 349.23). H-NMR
1
4
17
6
[
2
DMSO-d ]: 8.20 (d, J= 8.7 Hz, 2H, Ph-2,6-H), 7.66 (d, J= 8.7 Hz, 2H, Ph-3,5-H), 6.95 (br s,
6
1
3
H, NH ), 3.78 (br s, 4H, Pp-2,6-H), 2.33 (t, J=4.9 Hz, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C
2 3
NMR [DMSO-d ]: δ (ppm) 169.3, 167.6, 165.2, 136.7, 131.742.99, 46.22, 54.81, 125.44,
6
-1
+/-
1
30.23, , IR (KBr) [cm ] ν= 3325, 3141 (br NH ). LC/MS : purity: 100%, t =3.77, (ESI)
2 R
m/z [M+] 349.24. Anal. Calcd for C H N Br: C48.15, H4.91, N24.06; Found: C48.15,
1
4
17
6
H5.06, N24.09.
4
.1.10. 4-(4-methylpiperazin-1-yl)-6-(4-iodo-phenyl)-1,3,5-triazin-2-amine (11)
0
1
Light yellow solid. Mp 242-245 C. Yield:18% (360 mg). C H N I (MW 396.23). H-NMR
1
4
17
6
[
DMSO-d ]: δ= 8.04 (d, J=8.7 Hz, 2H, Ph-2,6-H), 7.84 (d, J=8.5 Hz, 2H, Ph-3,5-H), 6.91 (br
6
s, 2H, NH ), 3.78 (br s, 4H, Pp-2,6-H), 2.33 (t, J=5.1Hz, 4H, Pp-3,5-H), 2.20 (s, 3H, CH ).
2
3
1
3
C NMR [DMSO-d ]: δ (ppm) 169.5, 167.6, 165.1, 137.6, 137.0, 130.2, 99.3, 54.8, 46.2. IR
6

ACCEPTED MANUSCRIPT
-1
+/-
+
(
KBr) [cm ] ν= 3324, 3142 (br NH ). LC/MS : purity: 98.6%, t =3.95, (ESI) m/z [M+H]
2 R
3
97.23. Anal. Calcd for C H N I: C42.44, H4.32, N21.21; Found: C42.33, H4.37, N20.81.
14 17 6
4
.1.11. 4-(4-methylpiperazin-1-yl)-6-(4-methylphenyl)-1,3,5-triazin-2-amine (12)
0
1
White solid. Mp. 216-219 C. Yield:16% (223 mg). C H N (MW 284.36). H-NMR
1
5
20
6
[
2
DMSO-d ]: δ=8.19 (d, J=8,1 Hz, 2H, Ph 2,6-H), 7.27 (d, J=8.0 Hz, 2H, Ph 3,5-H), 6.84 (br s,
6
1
3
H, -NH ), 3.79 (br s, 4H, Pp 2,6-H), 2.35 (m, 7H, Pp-3,5-H +Ph-CH ), 2.21 (s, 3H, CH ). C
2 3 3
NMR [DMSO-d ]: δ (ppm) 170.1, 167.6, 165.2, 141.5, 134.7, 129.2, 128.2, 54.9, 46.3, 42.9,
6
-
1
+/-
+
2
2
1.5. IR (KBr) [cm ] ν= 3319 (br NH ). LC/MS : purity: 100%, t =2.92, (ESI) m/z [M+H]
2 R
85.29. Anal. Calcd for C H N : C63.36, H7.09, N29.55; Found: C63.92, H7.16, N29.43.
15
20
6
4
.1.12. 4-(4-methylpiperazin-1-yl)-6-(4-trifluoromethylphenyl)-1,3,5-triazin-2-amine (13)
0
1
White solid. Mp 204-205 C. Yield:6% (100 mg). C H N F (MW 338.34). H-NMR
1
5
17
6 3
[
DMSO-d ]: δ=8.47 (d, 2H, J= 8.79 Hz, Ph-2,6-H), 7.85 (d, 2H, J= 8.75 Hz, 7.03 (br s, 2H,
6
1
3
NH ), 3.81 (br s, 2H, Pp-2,6-H), 2.36 (t, 4H, J=4.9 Hz, 4H, Pp-3,5-H), 2.21 (s, 3H, CH ). C
2
3
NMR [DMSO-d ]: δ (ppm) 168.9, 167.6, 165.1, 141.4, 141.3, 131.7, 131.3, 128.9, 126.4,
6
-
1
1
25.7, 125.6, 122.8, 54.8, 46.2, 43.0, 43.0. IR (KBr) [cm ] ν= 3336, 3145 (br NH ).
2
+
/-
+
LC/MS : purity: 99.5%, t =4.10, (ESI) m/z [M+H] 339.26. Anal. Calcd for C H N F :
R
15 17
6 3
C53.25, H5.06, N24.84; Found: C53.03, H4.98, N25.11.
4
.1.13. 4-(4-methylpiperazin-1-yl)-6-(4-cyanophenyl)-1,3,5-triazin-2-amine (14)
0
1
White solid. Mp. 208-211 C. Yield 12% (170 mg). C H N (MW 295.35). H-NMR
1
5
17
7
[
DMSO-d ]: δ=8.40 (dd, J = 8.5 Hz; J = 4.9 Hz, 2H, Ph-2,6-H), 7.93 (dd, J = 8.2 Hz, J = 5.1
6 1 2 1 2
Hz, 2H, Ph-3,5-H), 7.05 (br s, 2H, NH ), 3.80 (br s, 4H, Pp-2,6-H), 2,33 (def t, 4H, Pp-3,5-H),
2
+
/-
+
2
.19 (s, 3H, CH ). LC/MS : (ESI) [M+H] 296. Anal. Calcd for C H N : C61.00, H5.80,
3 15 17 7
N33.20; Found: C60.62, H5.96, N32.96.
4
(
.1.14. 4-(4-methylpiperazin-1-yl)-6-(4-(dimethylamino)phenyl)-1,3,5-triazin-2-amine
15)
White solid. Mp. 253-256 C. Yield 4% (60 mg). C H N (MW 313.40). H-NMR [DMSO-
0
1
16
23
7
d6]: 8.11 (d, J= 8.98 Hz, 2H, Ph-2,6-H), 6.70 (d, J= 9.0 Hz, 2H, Ph-3,5-H), 6.66 (br s, 2H,
NH ), 3.76 (br s, 4H, Pp-2,6-H), 2.96 (s, 6H, N-(CH ) ), 2.31 (t, J=4.9 Hz, 4H, Pp-3,5-H),
2
3 2
1
3
2
1
9
.19 (s, 3H, CH ). C NMR [DMSO-d ]: δ (ppm) 170.2, 167.5, 165.2, 152.8, 129.7, 124.3,
3 6
-1
+/-
11.3, 54.9, 46.2, 42.8, 39.9. IR (KBr) [cm ] ν= 3277, 3079 (br NH ). LC/MS : purity:
9.6%, t =2.26, (ESI) m/z [M+H] 314.40. Anal. Calcd for C H N : C61.32, H7.40, N31.28;
R 16 23 7
2
+
Found: C61.47, H7.45, N31.19.
4
(
.1.15. 4-(4-methylpiperazin-1-yl)-6-(3-chloro-4-methoxyphenyl)-1,3,5-triazin-2-amine
16)
White solid. Mp 233-235 C. Yield:8% (130 mg). C H N OCl (MW 334.81). H-NMR
0
1
1
5
19
6
[
DMSO- d ]: δ=8.25 (s, 1H, Ph-2-H), 8.21 (d, 2H, J= 8.5 Hz, Ph-6-H), 7.21 (d, 1H, J = 8.7
6
Hz, Ph-5-H), 6.98 (br s, 2H, NH ), 3.91 (s, 3H, OCH ), 3.90-3.65 (br s, 4H, Pp-2,6-H), 2.40-
2
3
1
3
2
1
.30 (m, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C NMR [DMSO-d ]: δ (ppm) 168.7, 167.5,
3 6
-1
65.1, 157.3, 130.7, 129.5, 128.6, 121.3, 112.7, 56.8, 54.9, 46.3. IR (KBr) [cm ] ν= 3434 (br

ACCEPTED MANUSCRIPT
+
/-
+
NH ). LC/MS : purity: 97.4%, t =3.37, (ESI) m/z [M+H] 335.28. Anal. Calcd for
2
R
C H N OCl : C53.81, H5.72, N25.10; Found: C53.73, H5.84, N25.19.
15
19
6
4
(
.1.16. 4-(4-methylpiperazin-1-yl)-6-(3-chloro-6-methoxyphenyl)-1,3,5-triazin-2-amine
17)
White solid. Mp 198-200 C. Yield:31% (520 mg). C H N OCl (MW 334.81). H-NMR
0
1
1
5
19
6
[
DMSO- d ]: δ=7.45-7.39 (m, 2H, Ph-4,6-H), 7.08 (d, 1H, J = 9.5 Hz, Ph-3-H), 6.88 (br s, 2H,
6
NH ), 3.80-3.60 (m, 7H, Pp-2,6-H + OCH ), 2.35-2.20 (m, 4H, Pp-3,5-H), 2,17 (s, 3H, CH ).
2
3
3
1
3
C NMR [DMSO-d ]: δ (ppm) 171.3, 167.1, 164.85, 156.4, 130.5, 130.3, 129.8, 124.2,
6
-1
+/-
1
14.6, 56.6, 54.7, 46.2, 42.9. IR (KBr) [cm ] ν= 3358 (br NH ). LC/MS : purity: 100%,
2
+
t =2.35, (ESI) m/z [M+H] 335.28. Anal. Calcd for C H N OCl : C53.81, H5.72, N25.10;
R
15 19
6
Found: C53.46, H5.80, N24.85.
4
.1.17. 4-(4-methylpiperazin-1-yl)-6-(3,4-dichlorophenyl)-1,3,5-triazin-2-amine (18)
0
1
White solid. Mp. 214-216 C. Yield:3% (40 mg). C H N Cl (MW 339.22). H-NMR
1
4
16
6
2
[
DMSO-d ]: δ=8.39 (d, J=2.1 Hz, 1H, Ph-2-H), 8.21 (dd, J=10.1 Hz, J=2.1 Hz, 1H, Ph-6-H),
6
7
.73 (d, J=8.5 Hz, 1H, Ph-5-H), 7.01 (br s, 2H, -NH ), 3.78 (br s, 4H, Pp-3,5-H), 2.33 (t,
2
1
3
J=4.2 Hz, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C NMR [DMSO-d ]: δ (ppm) 168.1. 167.5,
1
NH ). LC/MS : purity: 100%, t =4.37, (ESI) m/z [M+H] 339.20.
3
6
-1
65.1, 138.1, 134.3, 131.6, 131.1, 129.7, 128.2, 54.8, 46.2, 43.0. IR (KBr) [cm ] ν= 3371 (br
+
/-
+
2
R
4
.1.18. 4-(4-methylpiperazin-1-yl)-6-(2,6-difluorophenyl)-1,3,5-triazin-2-amine (19)
0
1
White solid. Mp. 218-219 C. Yield 3% (40 mg). C H N F (MW 306.32). H-NMR
1
4
16
6 2
[
DMSO-d ]: δ=7.50 (m, 1H, Ph-4-H), 7.03 (m, 4H, Ph-3,5-H + NH ), 3.68 (br s, 4H, Pp-2,6-
6 2
13
H), 2.29 (br s, 4H, Pp-3,5-H), 2.18 (s, 3H, CH ). ). C NMR [DMSO-d ]: δ (ppm) 167.0,
1
3
3
6
-1
66.4, 164.6, 161.6, 161.5, 158.2, 131.5, 112.5, 112.1, 54.7, 46.2, 42.9. IR (KBr) [cm ] ν=
+/-
+
328, 3157 (br NH ). LC/MS : purity: 98.9%, t =2.28, (ESI) m/z [M+H] 307.29.
2
R
4
.1.19. 4-(4-methylpiperazin-1-yl)-6-(3,5-difluorophenyl)-1,3,5-triazin-2-amine (20)
0
1
White solid. Mp. 225-227 C. Yield:5% (72 mg). C H N F (MW 306.32). H-NMR
1
4
16
6 2
[
DMSO-d ]: δ=7.89-7.82 (m, 2H, Ph-2,6-H), 7-46-7.37 (m, 1H, Ph-4-H), 7.03 (br s, 2H,
6
1
3
NH ), 3.80 (br s, 4H, Pp-2,6-H), 2.33 (t, J = 4.87 Hz, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C
2
3
NMR [DMSO-d ]: δ (ppm) 167.9, 165.1, 164.4, 161.2, 141.6, 111.1, 110. 8, 107.0, 54.8,
6
-
1
+/-
4
[
6.2, 43.1. IR (KBr) [cm ] ν= 3386 (br NH ). LC/MS : purity: 100%, t =3.51, (ESI) m/z
2 R
+
M+H] 307.29. Anal. Calcd for C H N F : C54.89; H5.27; N27.42; Found: C54.62; H5.02;
14 16 6 2
N27.72.
4
.1.20. 4-(4-methylpiperazin-1-yl)-6-(3,4,5-trifluorophenyl)-1,3,5-triazin-2-amine (21)
0
1
White solid. Mp. 186-188 C. Yield 3% (25 mg). C H N F (MW 324.29). H-NMR
1
4
15
7 3
[
DMSO-d ]: δ=8.04 (m, 2H, Ph-2,6-H), 7.05 (br s, 2H, NH ), 3.78 (br s, 4H, Pp-2,6-H), 2.32
6 2
1
3
(t, J=4.9 Hz, 4H, Pp-3,5-H), 2.19 (s, 3H, CH ). C NMR [DMSO-d ]: δ (ppm) 167.4, 165.0,
3 6
+
/-
1
52.4, 149.1, 134.4, 112.6, 112.3, 112.3, 54.8, 46.1, 43.0. LC/MS : purity: 96.2%, t =3.94,
R
+
(ESI) m/z [M+H] 325.24.
4
.1.21. 4-(4-methylpiperazin-1-yl)-6-(3,5-dimethylphenyl)-1,3,5-triazin-2-amine (22)

ACCEPTED MANUSCRIPT
0
1
White solid. Mp.156-160 C. Yield 8% (129 mg). C H N (MW 299.39). H-NMR [DMSO-
16
22
6
d ]: δ=7.88 (s, 2H, Ph-2,6-H), 7.12 (s, 2H, Ph-4-H), 6.86 (br s, 2H, -NH ), 3.77 (br s, 4H, Pp
6
2
1
3
2
,6-H), 2.30 (m, 10H, Pp-3,5-H + Ph-3,5-CH ), 2.19 (s, 3H, CH ). C NMR [DMSO-d ]: δ
3 3 6
(
[
ppm) 175.1, 172.3, 167.0, 142.3, 142.1, 137.8, 130.8, 59.6, 51.0, 47.7, 44.7, 26.2. IR (KBr)
-1
+/-
+
cm ] ν= 3378 (br NH ). LC/MS : purity: 100%, t =3.47, (ESI) m/z [M+H] 299.31. Anal.
2
R
Calcd for C H N : C64.40, H7.43, N28.16; Found: C64.37, H7.76, N27.70.
1
6
22
6
4
.1.22. 4-(4-methylpiperazin-1-yl)-6-(3,4-dimethylphenyl)-1,3,5-triazin-2-amine (23)
0
1
White solid. Mp. 223-226 C. Yield 18% (259 mg). C H N (MW 299.39). H-NMR
1
6
22
6
[
4
DMSO-d ]: δ=7.88 (s, 2H, Ph-2,6-H), 7.12 (s, 2H, Ph-4-H), 6.86 (br s, 2H, -NH ), 3.77 (br s,
6 2
1
3
H, Pp 2,6-H), 2.30 (m, 10H, Pp-3,5-H + Ph-3,4-CH ), 2.19 (s, 3H, CH ). C NMR [DMSO-
3 3
d ]: δ (ppm) 170.2, 167.6, 165.3, 140.2, 136.3, 135.0, 129,8, 129.2, 125.9, 54.9, 46.3, 20.0. IR
6
-1
+/-
+
(
KBr) [cm ] ν= 3289 (br NH ). LC/MS : purity: 100%, t =3.27, (ESI) m/z [M] 299.31.
2 R
Anal. Calcd for C H N : C64.40, H7.43, N28.16; Found: C64.28, H7.46, N27.98.
1
6
22
6
4
4
.2 Pharmacology
3
.2.1. In vitro [ H]histamine binding assay on hH R
4
Before the experiments, cell membranes were sedimented by a 10-min centrifugation at
+
4°C and 16,000 × g and resuspended in binding buffer (12.5 mM MgCl , 1 mM EDTA and
2
7
5 mM Tris/HCl, pH 7.4). Competition binding experiments were carried out by incubating
membranes, 35-40 ꢀg/well (prepared from Sf9 cells transiently expressing hH R, co-
4
expressed with G- protein Gα and Gβ γ subunits) in a final volume of 200 ꢀL containing
i2
1 2
3
binding buffer and [ H]histamine (10 nM). Assays were run in triplicates with four to seven
appropriate concentrations between 0.1 nM and 100 ꢀM of the test compound. Incubations
were performed for 60 min at room temperature and shaking at 250 rpm. Non-specific binding
was determined in the presence of 10 ꢀM unlabeled JNJ7777120. Bound radioligand was
separated from free radioligand by rapid filtration through GF/B filters pretreated with 0.3%
(mass/vol) polyethylenimine (PEI) solution and washed three times with 0.3 mL of ice-cold
binding buffer (+4°C). The amount of radioactivity collected on the filter was determined by
liquid scintillation counting. Competition binding data were analyzed by the software
GraphPad Prism™ (version 3.02, San Diego, CA, USA) using non-linear least squares fit.
Affinity values (K ) were expressed as mean ± standard deviation (SD) and mean absolute
i
deviation (MD). K values were calculated from the IC values according to Cheng–Prusoff
i
50
3
6
equation .
4
.2.2. cAMP accumulation assay in cells expressing hH R
4
Intracellular cAMP accumulation was measured with homogenous TR-FRET
immunoassay, using LANCE Ultra cAMP kit (PerkinElmer). Frozen γ-irradiated recombinant
CHO cells, expressing the human histamine H receptor were thawed completely in a 37°C
4
water bath and resuspended in stimulation buffer (HBSS, 100ꢀM RO-201724, 5mM HEPES,
0
.1% BSA, pH 7.4). An antagonist dose-response experiments were performed in a total assay
volume of 20 ꢀl in white 384-well plates, using 600 cells/well. Cell stimulation was
performed for 30 min at room temperature, and histamine (140 nM), forskolin (10 ꢀM) and

ACCEPTED MANUSCRIPT
antagonists in appropriate concentrations (in range 3-100 ꢀM) were added simultaneously to
cell suspension. After incubation, five microliters of europium (Eu) chelate-labeled cAMP
tracer and 5ꢀl of ULight-labeled anti-cAMP mAb working solutions were added, mixed and
incubated for 1 h. TR-FRET signal was read on an EnSpire microplate reader (PerkinElmer).
Measured TR-FRET signal was translated into actual quantities of produced cAMP on the
basis of cAMP standard curve and obtained results were presented as % of maximal response.
Sigmoidal dose-response curve fitting was performed with use of GraphPad Prism™ software
(version 5.01, San Diego, CA, USA). Showed results represent the mean of three separate
experiments, each performed in triplicates.
3
α
4
.2.3. In vitro [ H]N -methylhistamine binding assay on hH R
3
Prior to the binding experiments, thawed cell membranes were homogenized by
sonication in ice-cold binding buffer (12.5 mM MgCl , 100 mM NaCl and 75 mM Tris/HCl,
2
pH 7.4). Competition binding experiments were carried out in total volume of 200ꢀl per
3
α
sample. [ H]N -methylhistamine (2 nM) was incubated with HEK293-hH R cells membranes
3
(20–25 ꢀg/well) and different concentrations of the tested compound. Assays were run at least
in duplicates with seven appropriate concentrations of investigated ligand, spanning from 0.01
nM to 100 ꢀM. Samples were incubated for 90 min at 25 °C with shaking (250 rpm). Non-
specific binding was determined in the presence of selective histamine H receptor inverse
3
agonist/antagonist - pitolisant (10 ꢀM). The bound radioligand was separated from free
radioligand by filtration through GF/B filters, pre-treated with 0.3% (m/v) PEI, followed by
three washing steps with 0.3 ml/well of ice-cold washing buffer (50 mM Tris/HCl, 120 mM
NaCl, pH 7.4). Competition binding data were analyzed similarly as for H R affinity studies,
4
utilizing GraphPad Prism™ software non-linear least squares fit (version 3.02, San Diego,
3
6
CA, USA) and Cheng–Prusoff equation .
4
.2.4. In vivo Anti-inflammatory activity
The activity of compounds was determined in vivo using the Carrageenan-Induced
Edema Model in mice. Male albino Swiss mice (18-25 g) were used for these studies. The
animals were housed and fed in a laboratory kept at constant temperature of 22°C under the
standard conditions (12:12 h L:D cycle, standard pellet diet, tap water). Each experimental
group consisted of 8 animals/dose and all the animals were used only once. Treatment of the
used laboratory animals in the present study was in full accordance with the respective Polish
and European regulations and was approved by the Local Ethics Committee at the
Jagiellonian University, Cracow, Poland.
4
.2.4.1. Determination of anti-inflammatory activities of investigated compounds
Male Swiss albino mice (18–25 g) were used in hind paw edema test. Mice were
divided into four groups, one of them being the control. In order to produce inflammation, 0.1
ml of 1% carrageenan solution in water was injected into hind paws subplantar tissue of mice,
2
3
24
according to the modified method of Winter and Lence . The development of paw edema
was measured with a plethysmometr (Plethysmometr 7140, Ugo Basile). Prior to this
administration, paw diameters were measured by dividers and recorded. The investigated

ACCEPTED MANUSCRIPT
compounds were administered at doses of 10, 25, 50 mg/kg, intraperitonally (as a suspension
in methylcellulose), prior to carrageenan injection. Methylcellulose was administered by the
same route, to the control group (methylcellulose had no effect on edema, data not shown).
After these administrations, paw diameters were measured at 1, 2, and 3 hours, % of edema
and edema inhibition was calculated according to the formulas given below.
Edema % = (N’×100)/N.
Edema inhibition % = (N–N’×100)/N.
N: paw diameters measured 1, 2 and 3 hours after injection of carrageenan to the control
group – paw diameters at the beginning.
N’: paw diameters measured 1, 2, and 3 hours after injection of carrageenan to the test groups
–
paw diameters at the beginning.
Data are expressed as mean ± SEM (Table 2; supplemental material). A value of P < 0.05 was
considered statistically significant. The obtained data were evaluated by the Student’s t-test
for grouped data. The software package GraphPad Prism™ (version 6.0, San Diego, CA,
USA) was used to process data.
4
4
.2.5. Antiproliferative assay
.2.5.1 Cell lines
HEK-293 and IMR-32 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium
DMEM (Gibco). Complete growth media contained 10 % fetal bovine serum (FBS), 100
mg·mL streptomycin and 100 U·mL penicillin. Cells were cultured at 37°C in an
-
-1
-1
atmosphere containing 5% CO . Neuroblastoma IMR-32 cell line was provided by
2
Department of Oncogenomics, Academisch Medisch Centrum, Amsterdam, Holland. Human
embryonic kidney HEK-293 cell line (ATCC CRL-1573) was kindly donated by Prof. Dr.
Christa Müller (Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn).
4
.2.5.2 In vitro antiproliferative assay
4
The cells were seeded in 96-well plates at a concentration of 2 × 10 cells/well (IMR-
4
3
2) or 1.5 × 10 cells/well (HEK-293) in 200 ꢀl culture medium and routinely cultured for 24
h to reach 60 % confluence. Next, the stock solutions of examined compounds in DMSO were
diluted into fresh growth medium and added into the microplates at the final concentrations
0
.01 ꢀM – 250 ꢀM. The maximal DMSO concentration did not exceed 1%. After 48 h of
incubation 20 ꢀl of EZ4U labeling mixture (EZ4U Non-radioactive cell proliferation and
cytotoxicity assay, Biomedica) 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 (PerkinElmer) at 492 nm. The activity of the standard drug doxorubicin (DX) was
estimated in the same way at the concentrations: 0.001-50 ꢀM for IMR-32 cells and at 0.005-
1
00 ꢀM for HEK-293 cells. GraphPad Prism™ software (version 5.01, San Diego, CA, USA)
was used to calculate the IC values.
5
0

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4
4
4
.3. Physicochemical studies
.3.1. Metabolic stability
.3.1.1. In silico study
The metabolic biotransformation of H R ligands 8 and 10 was studied in silico by using
4
computational procedure MetaSite 4.1.1 provided by Molecular Discovery Ltd. The potential
sites of metabolism were analyzed using either liver or cytochrome CYP3A4 computational
model exclusively.
4
.3.1.2. In vitro CYP3A4 P450-Glo™ Assay
The luminescent CYP3A4 P450-Glo™ Assay was purchased from Promega. The
CYP3A4 inhibitor ketoconazole was purchased from Sigma-Aldrich. The enzymatic reactions
were performed in white polystyrene, nontreated, flat-bottom multiwall ½ AreaPlate™ - 96
(PerkinElmer). The luminescence signal was measured with a microplate reader in
luminescence mode (PerkinElmer). The CYP3A4 P450-Glo™ assays were performed
according to the manufacturer’s procedure. The mixture consisting 0.5 pmol of CYP3A4
membranes and 4X Luciferin-PPXE in 100mM Tris-HCl was titrated in 96-well plate. Next,
an equal volume of the test compound or Luciferin-Free Water or ketoconazole (inhibition
control) was added to give one-half of the final reaction volume. After the 10-minute pre-
incubation one-half of the final volume of 2X NADPH regeneration system was added to
initiate CYP3A4 reaction. The final concentrations of TR7 and TR18 were 0.025 ꢀM – 25
ꢀ
M, the CYP3A4 inhibitor ketoconazole 0.025 ꢀM - 10 µM and Luciferin-PPXE 25 mM. The
total volume of DMSO did not exceed of recommended by manufacturer 0.2 %. The reaction
mixture with inactive control membranes (control) was prepared simultaneously as was
described above. The reaction mixture was incubated at 37 °C for 30 minutes. Afterwards, to
initiate the luminescence the reconstituted Luciferin Detection Reagent containing firefly
luciferase was added. The luminescence was measured after 20 minutes incubation of the
reaction mixture at room temperature. For calculation the total luminescence the average
luminescence of the control reaction containing inactive membranes was subtracted from the
luminescence of CYP3A4 containing reactions. The luminescence of the reactions containing
Luciferin-Free Water instead of the tested compound indicated the total (100%) CYP3A4
activity.
4
.3.2. X-ray crystal structures determination
The relevant crystals were obtained from methanol solutions. Pertinent crystallographic
parameters are summarized in Table 3 and selected metric parameters are presented in Table 4
supplemental material). The structures were solved by direct methods and refined with
(
3
7
SHELXTL . E-map provided positions for all non-H-atoms. The full-matrix least-squares
2
refinement was carried out on F ’s using anisotropic displacement factors for all non-H-
atoms. All C-bound H atoms were placed in idealized locations and were refined using a
riding model, with C–H = 0.93 Å and U (H) = 1.2U (C). The H-atoms attached to nitrogens
iso
eq
were located in difference Fourier map and refined with the restraints N-H = 0.86 Å and Uiso=
.2U (N).
1
eq

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Further details on the CIF files are available from the Cambridge Crystallographic Data
Center, 12 Union Road, Cambridge CB2 1EZ quoting the deposition numbers CCDC 913574
&
913575 (Fax: Int code + (1223)336-033; E-mail: deposit@ccdc.cam.ac.uk).
Crystal data for 8: C H N Cl , M = 304.79, monoclinic, space group P2 /c, a = 6.4519(4)
1
4
17
6
5
1
3
Ǻ, b = 16.5378(14) Ǻ, c = 13.9923(10) Ǻ, β= 91.056(7)°, V = 1492.73(19) Ǻ , Z = 4, D =
1
x
-3
-1
.356 g cm , T = 293 K, µ = 0.259 mm , λ = 0.71073 Ǻ, data/parameters = 2649/ 191; final
R = 0.0678.
1
Crystal data for 17: C15 H19 N O Cl S, M = 334.81, monoclinic, space group Cc, a =
6
3
1
4
2
2.9909(5) Ǻ, b = 18.2448(7) Ǻ, c = 7.3449(4) Ǻ, β = 113.968(5)°, V = 1590.75(14)Ǻ , Z =
-3
-1
, D = 1.398 g cm , T = 293 K, µ = 0.255 mm , λ = 0.71073 Ǻ, data/parameters = 2607/
x
11; final R = 0.0258.
1
4
4
.3.3. In silico molecular and docking studies
.3.3.1. Homology model
Histamine H receptor homology model used in this studies was built on the basis of
4
previously described and published crystal structure of histamine H receptor (PDB entry:
1
3
2
3
RZE), paying attention to mutagenesis data described by Jongejan et al. .
3
8
After the alignment of sequences, a total of 100 models was obtained using Modeller .
Models were then fitted to H R crystal structure using Schrödinger software . Missing side
3
9
1
chains were added using Prime module, H-bonds optimization and energy minimization
(using OPLS2005 forcefield) was then performed using Protein Prepararion Wizard
implemented in Schrödinger suite. Structures evaluation and stereochemical analyses were
4
0
performed using PROCHECK , and Ramachandran plots. Further docking studies allowed to
choose one model used for docking studies. Obtained model lacks the large intracellular loop,
due to its length difference in desired (H R) and template (H R) model.
4
1
4
.3.3.2. Ligands
For this study two crystal structures of ligands, 5 and 18 were used. For the tested set of
compounds energetically optimal conformers were found using ConfGen and prepared using
LigPrep module. For all of the structures 5 energetically best conformations were used for
docking. All of the tested compounds were used in their N-protonated form.
Receptor grid was generated by GlideGrid module and validated with known histamine H4
receptor ligands 4-methylhistamine and JNJ 7777120, according to the procedure described
3
3
by Feng . Docking studies were performed via GlideDock module. Results were interpreted
by the means of Docking Score functions. Receptor-ligand interactions were visualized using
4
1
PyMOL .
Acknowledgments
We grateful thank Prof. R. Seifert (Hannover, Germany) for providing Sf9 cells and baculovirus stock solutions
encoding for the human histamine H receptor and G-protein G and Gβ1γ2 subunits.
4
αi2

ACCEPTED MANUSCRIPT
The authors acknowledge the partial support of ESF COST Action BM0806 ‘Recent advances in histamine
receptor H R research’, financed by EU-FP7, of Grant No. 594/N-COST/2009/0, National Science Center,
4
granted on the basis of decision No DEC-2011/02/A/NZ4/00031, of the European COST Actions BM1007,
CM1103, CM1207, of Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational
Research Innovation – Pharma (TRIP), of Fraunhofer-Institut für Molekularbiologie und Angewandte Ökologie
IME, Projektgruppe Translationale Medizin und Pharmakologie TMP (Fh-TMP), and that of LOEWE OSF.
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Table 1. Histamine H R affinities of synthesized compounds 3-23.
4
NH2
N
N
R
N
N
N
CH3
(
m)
(m)
(2)
No
3
R
Histamine H
4
R
n
No
14
R
Histamine H
affinity
i
K [nM] ± SD (MD)
4
R
n
a
a
affinity
[nM] ± SD (MD)
K
i
(
3)
1099 ± 485 (345)
3
2
2358 ± 1277 (903)
1
NC
(
2)
(2)
(2)
4
5
Cl
1261 ±332 (235)
15
16
64490 ± 31396 (22200)
2
(
CH ) N
3
2
(
3)
Cl
408 ± 134 (99)
3
2
Cl
1472 ± 57 (41)
2
2
H C
3
O
(
2)
2)
(3)
6
7
H C
1747 ± 1500 (1061)
17
Cl
6007 ± 1538 (1088)
3
O
CH3
(
(3)
(2)
NC
5161 ± 2440 (1726)
203 ± 65 (47)
1
3
18
19
Cl
790 ± 320 (226)
5077 ± 445 (315)
3
1
Cl
b
(3)
8
F
Cl
F
F
(
3)
(2)
(2)
(2)
9
378 ± 96 (73)
524 ± 225 (159)
2391 ± 1477 (1044)
3
2
1
20
21
22
F
1191 ± 71 (50)
1787 ± 71 (50)
1
1
1
F
F
c
(2)
1
0
F
F
Br
I
(2)
1
1
2
H C
10567 ± 5110 (3614)
3
CH3
(
4)
2)
(3)
1
1
431 ± 99 (72)
2
1
23
H C
834 ± 494 (379)
2
3
H C
H C
3
3
(
3
4239 ± 673 (477)
F3C
a
3
[
H]Histamine displacement with membrane preparation of Sf9 cells expressing human histamine H
subunits. Each determination was performed with four or seven different concentration points (initial screening with four
concentration points with at least two independent measurements, for compounds with good affinity values, i.e. K < 1000 nM, seven
4
receptor, co-expressed with G protein
1 2
Gαi2 and Gβ γ
i
concentration points in at least three independent experiments were performed) ranging from 0.01 nM to 100 ꢀM test compound
concentration in triplicates.
SD: standard deviation; MD: mean absolute deviation; n: number of experiments in triplicates, m: number of measurements
b
K
i
value of 8 at human histamine H receptor: 13.2 ± 0.8 ꢀM
3
value of 10 at human histamine H receptor: 15.0 ± 2.2 ꢀM
3
c
K
i

ACCEPTED MANUSCRIPT
NH2
K = 10 nM
i
K = 4.8 nM
i
N
N
N
N
N
N
N
N
N
1
Cl
CH3 Cl
CH3
2
1
7
Figure 1. Structures and H R affinities of (4-methylpiperazin-1-yl)-1,3,5-triazine (1 ) and -
pyrimidine-2-amine derivatives (2 ).
4
1
8

ACCEPTED MANUSCRIPT
NH2
N
N
N
N
R
N
CH3
R= mono/di/trisubstituted with F, Cl, Br, I, CH , CF , CN, OCH , N(CH )
3 2
3
3
3
Figure 2. General structure of synthesized compounds.

ACCEPTED MANUSCRIPT
A
B
100
100
8
0
0
0
0
80
60
40
6
4
2
20
IC50 = 512 ± 62 nM
IC50 = 1630 ± 140 nM
0
0
1
0-8
10-7
10-6
10-5
10-4
10-8
10-7
10-6
10-5
10-4
[
8], M
[10], M
Figure 3. cAMP accumulation studies in CHO cells expressing the human histamine H4
receptor, co-treated with forskolin, histamine and tested compounds: 8 (A) and 10 (B).

ACCEPTED MANUSCRIPT
Figure 4. Anti-inflammatory effect of compounds 8, 10 and 18 (doses: 10 mg/kg, 25 mg/kg
and 50 mg/kg) on paw edema induced by subplantar injection of carrageenan.

ACCEPTED MANUSCRIPT
1
20
00
8
1
1
0
DX
80
60
40
20
0
1
0-9 10-8 10-7 10-6 10-5 10-4 10-3
[
compound], M
Figure 5. The antiproliferative effect of 8, 10 and DX (reference) against HEK-293 cell line.

ACCEPTED MANUSCRIPT
1
20
00
8
1
1
0
DX
8
6
4
2
0
0
0
0
0
1
0-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3
compound], M
[
Figure 6. The antiproliferative effect of 8, 10 and DX (reference) against IMR-32 cell line.

ACCEPTED MANUSCRIPT
Figure 7. Plots of MetSite predictions for sites of metabolism for 8 and 10 by liver and
CYP3A4.

ACCEPTED MANUSCRIPT
CYP3A4 P450-Glo™
150
8
1
0
100
ketoconazole
50
0
0.01
0.1
1
10
100
µM
Figure 8. The effect of 8, 10 and ketoconazole on CYP3A4 activity.