(2-Arylethenyl)-1,3,5-triazin-2-amines as a novel histamine H4 receptor ligands

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(2-Arylethenyl)-1,3,5-triazin-2-amines as a novel histamine

H receptor ligands

4

a

b

Katarzyna Kamińska , Julia Ziemba , Joanna Ner , Johannes Stephan Schwed , Dorota

a

Łażewska , Małgorzata Więcek , Tadeusz Karcz , Agnieszka Olejarz , Gniewomir Latacz ,

a

b

c

Kamil Kuder , Tim Kottke , Małgorzata Zygmunt , Jacek Sapa , Janina Karolak-

d

b

a

Wojciechowska , Holger Stark , Katarzyna Kieć-Kononowicz

a

Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9,

0-688 Kraków, Poland

3

b

c

d

Heinrich-Heine-University, Institute of Pharmaceutical and Medicinal Chemistry, Universitaetsstr. 1, 40225

Duesseldorf, Germany

Department of Pharmacodynamics, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków,

Poland

Institute of General and Ecological Chemistry, Technical University of Łódź, Żeromskiego 116, 90-924 Łódź,

Poland

Cl

N

H N

N

2

N

6

K = 253 nM

i

ACCEPTED MANUSCRIPT

(2-Arylethenyl)-1,3,5-triazin-2-amines as a novel histamine

H receptor ligands

4

a

b

Katarzyna Kamińska , Julia Ziemba , Joanna Ner , Johannes Stephan Schwed , Dorota

a

Łażewska , Małgorzata Więcek , Tadeusz Karcz , Agnieszka Olejarz , Gniewomir Latacz ,

a

b

c

Kamil Kuder , Tim Kottke , Małgorzata Zygmunt , Jacek Sapa , Janina Karolak-

d

b

a*

Wojciechowska , Holger Stark , Katarzyna Kieć-Kononowicz

a

Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9,

0-688 Kraków, Poland

3

b

c

d

Heinrich-Heine-University, Institute of Pharmaceutical and Medicinal Chemistry, Universitaetsstr. 1, 40225

Duesseldorf, Germany

Department of Pharmacodynamics, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków,

Poland

Institute of General and Ecological Chemistry, Technical University of Łódź, Żeromskiego 116, 90-924 Łódź,

Poland

*

Corresponding author: tel +48 12 6205580; fax + 48 12 620 55 96

E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec-Kononowicz)

Abstract: Within the constantly growing number of histamine H (H R) receptor ligands

4

there is a large group of azine derivatives. A series of novel compounds in the group of

-methylpiperazine-1,3,5-triazine-2-amines were designed and obtained. Considered

4

structures were modified at the triazine 6-position by introduction of variously substituted

arylethenyl moieties. Their affinities to histamine H receptors were evaluated in radioligand

4

binding assays with use of Sf9 cells, transiently expressing human H R. Pharmacological

4

studies results allowed to identify 4-[(E)-2-(3-chlorophenyl)ethenyl]-6-(4-methylpiperazin-1-

yl)-1,3,5-triazin-2-amine (K = 253 nM) as the most potent compound in the present series.

i

Keywords: Histamine H receptor; 1,3,5-Triazin-2-amines; 4-Methylpiperazines; Anti-

4

inflammatory properties

1. Introduction

The activity of histamine was known since 1927, when its role in pathophysiology was

described for the first time. Histamine plays many important roles in the human body,

regulating numerous vital processes, such as glandular secretion, allergic and inflammation

reactions, gastric acid secretion in the stomach, modulation of neurotransmission and smooth

muscle activity. It is also an important stimulant of the intracellular signal transduction in

blood platelets. Moreover, recent studies suggest a key role of histamine in control of cell

proliferation, regulation of multiple cytokines secretion (including those involved in

cancerogenic processes) and modulation of the immune system [1, 2]. It is known nowadays

that histamine can interact with four histamine receptor subtypes: H , H , H , and H . They all

1

2

3

4

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belong to the family A of G Protein coupled receptors (GPCR), but differ in functions and

distribution in the body [3].

Our interest was focused on the histamine H receptor (H R). It was discovered and

4

cloned by several independent research teams around the year 2000 and 2001 [4-8]. H R is

4

located primarily in immune system cells, suggesting its participation in the immune

responses and allergic or inflammatory conditions [9]. High histamine content and

biosynthesis rate, together with H R overexpression, have been reported in different human

4

neoplasias. These included melanoma, colon and breast cancers, as well as experimental

tumors, in which histamine has been postulated to behave as an important paracrine and

autocrine regulator of proliferation [10]. The positive therapeutic effect of H R

4

antagonists/inverse agonists was observed in vivo in such conditions as asthma, hay fever,

pruritus or bowel inflammation [11, 12].

The first orally available, potent and selective H R antagonist was, described in 2003,

4

non-imidazole derivative - JNJ 7777120 [13] (Fig. 1). Several studies revealed the anti-

inflammatory activity of JNJ 7777120. Its role in reducing allergic and inflammatory

processes, including chemotaxis of eosinophils and mast cells, was also confirmed [14, 15].

After a decade of research, since the discovery of histamine H R, there are many well-

4

characterized compounds which are potent and selective histamine H R ligands. The azine

4

derivatives constitute a large, constantly growing and promising group of such structures [16-

2

0] (Fig. 2). Accordingly, we decided to restrict our research to 4-methylpiperazin-1,3,5-

triazine derivatives. In our previous studies we have investigated compounds with (un-

substituted phenyl in the position 6 of triazine fragment [21]. As a continuation of our

research, the aim of these presented studies was to investigate the group of (2-arylethenyl)-

,3,5-triazin-2-amine derivatives (Fig. 2) in order to find new potent histamine H R ligands.

)

1

4

For these compounds, radioligand binding assays were carried out to assess the affinity to

human H R (hH R) and selectivity over closely related histamine H receptor. Affinity

4

3

towards mouse H R was also determined for most active compound in current series.

4

Additionally, intrinsic activity of the most active compounds at hH R were evaluated in

4

cAMP accumulation assay. In vitro toxicity assays, in vivo anti-inflammatory activity studies

and crystallographic investigations were also performed for chosen compounds.

2. Results and discussion

2

.1. Chemistry

Syntheses of 1,3,5-triazin-2-amines were conducted by using appropriate

acid derivatives as substrates: 1-(imino-(4-methylpiperazin-1-yl)methyl)guanidine

dihydrochlorides and corresponding cinnamic acid esters [22] (Scheme 1d). The guanidine

derivative was obtained by the reaction of cyanoguanidine and 1-methylpiperazine

dihydrochloride (Scheme 1c). Cinnamic acid esters which were not commercially available

were prepared using two methods. The first method was the esterification reaction carried out

in acidic solution. The second method was the reaction of properly substituted cinnamic acids

with iodomethane in the presence of DBU [23] (Scheme 1b). Non-commercially available

cinnamic acids used as a substrates were obtained by the reaction of properly substituted

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benzaldehydes with malonic acid, where piperidine was used in catalytic amount [24]

(Scheme 1a).

2

.2. Biological studies

.2.1. Structure-activity relationship analysis at histamine H receptor

4

Within the present studies, eighteen new compounds were synthesized and their

affinities to human histamine H receptor were determined in radioligand binding

4

experiments. Obtained results are presented in Table 1, whereas structural formulas of

considered compounds can be found in Fig. 3.

Analysis of pharmacological data confirmed that nearly all compounds (excluding 4 and

1

5) show affinity to H R in (sub-)micromolar range (K < 10 µM). Almost every

4 i

monosubstitution of hydrogen atom in benzene ring (except 4) was more advantageous

compared to the unsubstituted ring (1). In case of monosubstitution with methyl group or

chlorine atoms, the best K values were observed for meta-position (3 and 6), whereas

i

compounds substituted at ortho- (2 and 5) and para- (4 and 7) position showed lower activity.

For that reason meta-position of monosubstitution was chosen for further developed structures

(

10-14). For halogen atoms (6, 10, 11) no direct relationship between hH R affinity and

4

elements atomic number was observed. For the larger groups of: trifluoromethyl-, cyano- and

nitro- (12, 13, 14) moieties as meta- substituents, the K values were all in a very similar range

i

(

equal around 2,500 nM). The influence of the benzene ring disubstitution on affinity to hH R

4

was examined by introduction of chlorine atoms. Among disubstituted structures, containing

respectively 2,4-dichloro- (8) and 3,4-dichlorophenyl (9) fragments, one (9) was almost ten

times more potent than the other (8). The change from phenyl to naphthyl group (15, 16)

resulted in significant decrease of the H R affinity only for compound with 1-naphthylethenyl

4

substituent (15), whereas the K value for 16 (2-naphthylethenyl substituent) remained almost

i

unchanged, when compared to the result obtained for 1 (unsubstituted phenyl). The attempt to

introduce a bicyclic-heterocyclic ring (17) led to the structure showing higher affinity to hH R

4

than naphthalene-derivatives, however, minimally differing in K values from several phenyl-

i

derivatives described herein (2, 12, 13, 14).

Generally, it could be concluded, that the substituent at the 6-position of 1,3,5-triazine

ring did not have as crucial influence on the affinity to hH R as it was previously expected. In

4

most cases, the observed diversity of K values was relatively low and therefore it is quite hard

i

to definitely determine the impact of relative substituents positions at the phenylethenyl group

at the position 6 of 1,3,5-triazine on the affinity to H R. On other hand, it is important to note

4

that the rest of substituents of 1,3,5-triazine ring, like 2-amino group and 4-(4-

methylpiperazinyl) group, can also have a noticeable impact on affinity values and more

extensive modifications could be scheduled for the future studies in order to better

characterize the discussed group of compounds.

In order to evaluate if the affinities determined at human H R may be the subject to

4

significant variations due to interspecies differences in receptor pharmacology, radioligand

binding studies at mouse H R were additionally performed for most active structure (6). The

4

obtained results revealed a slight decrease in affinity of 6 towards mouse H R, when

4

compared with data for human receptor (6, hH R: K = 253 nM, mH R: K = 854 nM).

4

i

4

i

However, the K value of 6 at mouse receptor was still in submicromolar concentration range.

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2

.2.2. Intrinsic activity at histamine H receptor and selectivity over

4

histamine H receptor

3

Selected structures (3 and 6) were further investigated in functional tests in order to

better characterize their pharmacological profile. Intrinsic activity of most active compounds

was determined in cAMP accumulation assay at HEK293 cells expressing recombinant

human H R.

4

Investigated compounds caused a blockade of the histamine-induced cAMP level

reduction, in cells co-treated with forskolin (H R is a G -coupled receptor) and were therefore

4

i

classified as antagonists at histamine H Rs. Antagonist potency (IC ) was evaluated by

4

50

performing a dose-response curve experiment in presence of H R agonist (histamine, 140 nM,

4

corresponding to its EC ) and forksolin (10 µM) (Fig. 4). Obtained IC values for both

8

0

50

tested compounds were comparable to their K values at human H Rs. Moreover, order of

i

4

compounds activity, determined in radioligand binding assay, could also be confirmed in

functional tests (3, K = 703 nM, IC = 606 nM; 6, K = 253 nM, IC = 439 nM).

i

50

i

50

Since human H R shows the highest sequence homology (~60% in the transmembrane

4

domains) to the histamine H receptor subtype [25], considered structures were additionally

3

tested for their interaction with H R. For that purpose radioligand binding assay were

3

α

performed with use of [ H]N -methylhistamine and membrane preparations from HEK-293

cells stably expressing human H R.

3

Performed assays revealed that compounds 3 and 6 show low affinity to hH R. Both

3

α

compounds caused a significant decrease of [ H]N -methylhistamine binding only in

concentrations equal or higher than 30 ꢀM. Due to limited solubility of compounds, lower

plateau of competition binding curves could not be reached in performed experiments (data

not shown) and therefore precise determination of K values was unavailable. Nevertheless, it

i

could be assumed that the affinity of 3 and 6 to hH R is lower than 30 ꢀM. Basing on that

3

statement it could be concluded that both compounds are selective towards histamine H R

4

subtype, representing at least 30-fold selectivity.

2

.2.3. Toxicity and metabolic stability

The acceptable toxicity as well as preliminary ADME properties defines the compound

as “drug-like” which means an ideal clinical candidate [26]. During this study we estimated

some of important ADME/Toxicity parameters for 6, the most potent H R histamine ligand in

4

current series. Antiproliferative activity at human cells, genotoxicity, metabolic stability and

influence on cytochrome P450 3A4 activity were evaluated using approved in vitro and in

silico methods.

The effect of 6 on the proliferation of two cell lines: HEK-293 (human embryonic

kidney) and IMR-32 (neuroblastoma) was examined under various concentrations and

compared to doxorubicin (DX) as a reference drug. Obtained data showed no antiproliferative

activity of 6 against HEK-293 (Fig. 5) and weak activity against neuroblastoma cell line IMR-

3

1

2 (Fig. 6). However, significant effects of 6 against IMR-32 was observed under more than

000-fold higher concentration in comparison to IC value of DX, which was found to be 5.1

50

nM. In summary, 6 can be considered as non-cytotoxic compound as judged by used cellular

models.

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To evaluate the risk of genotoxic properties of 6, the Ames microplate fluctuation

protocol (MPF) assay was applied. Salmonella typhimurium strain TA98 was used in the

experiments, enabling the detection of frameshift-type mutations. Strain TA98 is incapable of

producing histidine, but mutagenic events may lead to reverse mutation which allow reverted

bacteria to grow in histidine-deficient media. The criteria used to evaluate Ames MPF test

results is the fold increase in number of revertants after the exposure to tested chemicals. Fold

increase of revertants is calculated relative to solvent control baseline (FIB) and the threshold

value of FIB equal 2.0 is considered as a cut-off parameter for identification of potential

mutagens.

In our experiment compound 6 was tested in two concentrations: 1 ꢀM and 100 ꢀM and

2

-nitrofluorene at 10 ꢀM was used as a positive control. As depicted in Fig. 7 compound 6

showed no genotoxic effect in Ames MPF test at both used concentrations.

The MetaSite computational procedure was used to predict the most likely sites of 6

metabolism and the structures of its metabolites [27, 28]. The plot of MetaSite in silico

predictions for sites of metabolism for compound 6 by liver model is shown below (Fig. 8).

The highest probability of metabolism occurred at N-methyl group of piperazine moiety,

with suggested reaction of demethylation (100% scores) and at the primary amine, as well as

at the 4-position of benzene moiety (both 30% scores), with suggested reaction of

hydroxylation.

The obtained in silico metabolite structures were further compared with in vitro results.

A full scan chromatogram of the reaction mixture after 48 h incubation of compound 6 with

human liver microsomes (HLMs) showed the presence of unreacted 6 (t = 3.63) and its two

metabolites: M1 (t = 3.92) and M2 (t = 3.55) (Fig. 9A). The LC/MS analysis determined the

molecular masses of obtained metabolites: M1 with m/z = 347.24, which corresponds to the

metabolite obtained in silico after reaction of hydroxylation and M2 with m/z = 317.33 ,

which corresponds to the metabolite obtained in silico after reaction of demethylation

(Fig. 9B).

The luminescent cytochrome P450 3A4 (CYP3A4) assay, based on the conversion of

the cytochrome substrate - luciferin-PPXE into D-luciferin by recombinant human CYP3A4,

was used to determine the influence of 6 on cytochrome CYP3A4 activity. The light signal

produced in the reaction, after the addition of firefly luciferase, was proportional to

D-luciferin concentration in sample and therefore was used for indirect assessment of

CYP3A4 activity [29]. As it can be seen at Fig. 10, compound 6 substantially inhibited the

cytochrome activity only in higher concentrations. The CYP3A4 activity, measured at the

presence of 25 µM 6, was inhibited by 58% as compared to non-treated control. However, it

can be concluded, that in comparison to the effect of ketoconazole (reference CYP3A4

inhibitor), which blocked the CYP3A4 activity completely at 10 µM concentration (calculated

IC = 0.14 ꢀM), 6 exhibit only weak CYP3A4 inhibitory properties.

5

0

2

.2.4. In vivo pharmacology

Taking into consideration the increasing number of scientific proofs for histamine H₄

receptor involvement in inflammatory and allergic responses [12, 30, 31], as well as clear

evidence for histamine H antagonists utility in reducing inflammatory processes in vivo [32,

4

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3

3], we decided to investigate the most potent ligands of current series (3 and 6) in the

carrageenan-induced edema test in mice [34, 35].

In performed studies, two considered 1,3,5-triazin-2 amine derivatives, administered

intraperitoneally (i.p.), showed anti-inflammatory activity, when compared to the control

(ketoprofen; Fig. 11). Compound 6 showed a statistically significant anti-inflammatory

activity in all doses, throughout duration of the experiment. At a dose of 50 mg/kg i.p., 6

decreased carrageenan-induced edema by 69%, 62% and 67% after one, two and three hours

of the experiment, respectively, while at a dose of 25 mg/kg i.p., by 59%, 58%, 65%,

respectively. At a dose of 10 mg/kg i.p., 6 suppressed edema paw in the range 50% - 61%.

Compound 3 showed statistically significant anti-inflammatory effect, administered in two

doses. At a dose of 50 mg/kg i.p. and 25 mg/kg i.p., 3 decreased carrageenan- induced edema,

throughout duration of observation, by 88% - 94% and 53% - 69%, respectively. Ketoprofen

was used as the reference compound. The largest anti-inflammatory activity was observed at

the highest dose. Ketoprofen administered at a dose of 50 mg/kg i.p., reduced carrageenan-

induced edema, after the first, second and third hour after injection of carrageenan, by 67%,

7

1%, 77%, respectively. Ketoprofen given intraperitoneally, at a dose of 25 mg/kg, inhibited

edema by 20%, 54%, 67%, after first, second and third hour of experiment, respectively,

while at a dose 10 mg/kg, suppressed carrageenan-induced edema by 27%, 55% and 63%.

Preliminary pharmacological studies showed, that two tested compounds 3 and 6

exhibit an anti-inflammatory properties. The highest activity was observed for both

compounds at a dose of 50 mg/kg i.p., with the inhibition of paw edema in the range from

6

3% to 94% in three measurement times. The compounds administered at a dose of 25 mg/kg

i.p., reduced paw edema in the first, second and third hour of the experiment in the range from

3% to 69%, while at a dose 10 mg/kg i.p. in the range from 13% to 61%. By comparison of

5

the anti-inflammatory activity of novel histamine H receptor antagonists and the activity of

4

the reference compound - ketoprofen, it can be said that compound 3 showed the strongest

anti-inflammatory activity among these compounds and also partially higher than ketoprofen

(only in the two highest doses) throughout the duration of the experiment.

2

.3. Crystallographic studies

The availability of real 3-D structures is very useful for the prospective 3-D QSAR

studies (calculation of electronic values as similarity descriptors, molecular modelling of the

remaining studied molecules, H-bonding main patterns in the crystals, docking to the receptor

active site). Required data can be easily obtained from X-ray structure analysis for selected

number of derivatives from the set. In these studies, structural investigations were intended to

examine the crystal and molecular structures of the molecules with various aromatic-ethenyl

substituents at triazine ring. Thus, two derivatives: 5 and 17 (first with 2-chlorophenyl and the

second with indole ring) were re-crystallised from methanol solution and obtained

monocrystals were used as the subject of crystallographic studies. The ORTEP drawings of

both molecular structures are presented in Fig. 12.

From topological point of view, both studied derivatives can be expressed in terms of

triazine with fastened aromatic-ethenyl and piperazine moieties. In both molecules ethenyl

linker and triazine ring are in one plane. Phenyl moiety in 5 is also in this plane (Fig. 12). But

in 17 heteroaromatic indole ring is significantly out of triazine plane (Fig. 12). The mutual

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positions of all rings with respect to ethenyl linker double bond in both structures are

identical.

Hence, the impact of aromatic ring nature on molecule conformation seems to be

insignificant. However, this impact is evident in the pattern of molecules assembling in the

crystal. The ORTEP drawings of the details for both molecules H-bonding pattern are

presented in Fig. S1 (supplemental material). The main motive in 5 is based on N3-H…N2

strong H-bond attached molecules in dimers. The stacked dimers form a column down the

crystal. In 17 the shortest N10-H…N2 bond involves indole nitrogen. But the main motive is

based on two bonds: N3-H…N2 and N3-H…N4. Therefore, it is evident that aromatic ring

nature is important for the H-bonding pattern in the crystal.

2

.4. Docking studies

In order to determine protein-ligand interactions and its possible influence on in vitro

activity, docking studies for chosen ligands (2, 3, 5, 6, 7 and 17) were performed. For the

studies, previously described histamine H R model, was used [21] built on the basis of crystal

4

structure of histamine H receptor (PDB entry: 3RZE), paying attention to mutagenesis data

1

described by Jongejan et al. [36]. Histamine H R models based on the crystal structure of

4

histamine H R could be more reliable than rhodopsin-based H R models and could help to

1

4

identify potential H R ligands through virtual screening [37, 38].

4

All of the docked ligands showed moderate to good docking score values in the range of

-

2.701 to -5.807. As it has been previously described by Jongejan [36] histamine H R has two

4

3.32

5.46

major anchoring points: ASP94 and GLU182 . For all of the docked ligands, interactions

with these two amino acids were found. Moreover, two possible ligand orientations in the

binding pocket of human H R has also been found, which is in agreement with the study of

4

Feng [37]. For one orientation we observed the formation of the salt bridges between

5

.46

GLU182 and amine group connected with triazine ring for compounds of relatively higher

3

.33

(

eg. 3 vs. 2, 6 vs. 5) in vitro affinity to H R. Benzene ring of TYR95

stabilize receptor-

4

ligand complexes for both orientations through π- π stacking interactions with triazine ring for

all of tested ligands and with: phenyl (compounds 2, 3, 5) or indole (compound 18) ring.

6

.51

Moreover, triazine ring is stabilized through π- π stacking with TYR225 , that additionally

forms hydrogen bond with one of triazine nitrogens for compounds 3, 6 or amine group in

compound 5.

Paying attention to the structure-activity relationship for isomer pairs: 2 and 3, 5 and 6,

it could be observed, that for the less active compounds (2 and 5), methyl or chlorine

substituent in ortho- position of the benzene ring, might be too close to the benzene ring of

3

.33

TYR95 , therefore meta- (3 and 6; Fig. 13) position seems to be more favorable for this

group of histamine H R ligands.

4

3. Conclusions

A series of novel 1,3,5-triazine-2-amine derivatives as a potent H R ligands was

4

synthesized. Among them compounds: 3 (K = 703 nM) and 6 (K = 253 nM) showed the

i

highest affinity to histamine H R, with a good selectivity over histamine H R and were

4

3

classified as antagonists on the basis of cAMP accumulation assay results. Additionally,

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compound 6 was found to be devoid of any significant cytotoxic activity against human

embryonic kidney cells (HEK-293) and neuroblastoma cell line (IMR-32). Its safety profile

was also supplemented with good results in the Ames MPF mutagenicity assay and CYP3A4

inhibition test.

Compounds 3 and 6 were emerged from docking studies as the most suitable to the H R

4

receptor model, because of the meta- position of methyl group and chlorine atom in the

phenyl substituent. Crystallographic data have suggested that the impact of aromatic ring

nature on molecule conformation seems to be insignificant, but they confirmed important role

of the aromatic ring nature for the H-bonding pattern in the crystal.

Preliminary pharmacological in vivo studies showed, that compounds 3 and 6 exhibit

statistically significant anti-inflammatory effect in carrageenan-induced hind paw edema test

in mice: for compound 3 in two doses (25, 50 mg/kg i.p.) and for compound 6 in three doses

(10, 25 and 50 mg/kg i.p.). In conclusion, with a view to the results of presented studies,

compound 6 can be highlighted to be the most promising H R ligand and a good lead

4

structure for the future investigations among the (2-arylethenyl)-1,3,5-triazin-2-amines.

4. Experimental section

4

.1. Chemistry

Reaction progress was verified using thin layer chromatography (TLC), which was

carried out on 0.2 mm Merck silica gel 60 F₂₅₄plates. Developed chromatograms were

visualized by UV absorbance or treatment with Dragendorff reagent. When necessary,

solvents were dried over calcium hydride (toluene) or calcium oxide (methanol). Organic

syntheses with use of microwave system were performed on a CEM Discover LabMate

apparatus. Melting points are uncorrected and were measured on Mel-Temp melting point

1

apparatus. H NMR spectra were recorded in CDCl or DMSO d on a Varian Mercury-VX

3

6

PFG spectrometer at the frequency of 300 MHz. Chemical shifts were reported in parts per

million on the δ scale using the solvent signal as an internal standard. Data are reported as

follows: chemical shifts, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet

and br=broad, def=deformed), coupling constant, integration and assignment (Ph-phenyl, Pp-

piperazine, Ind-indole, Nph-naphthyl). Infrared spectra were recorded in KBr pellets using

Jasco Corp. Furier Transform Infrared Spectrometer FT/IR-410. Elemental analysis was

performed using Elementar Vario EI III apparatus.

4

.1.1. General procedure of synthesis of cinnamic acid esters.

According to method I [2a, 3a, 6a, 9a, 10a, 12a, 13a]: 15 mmols of cinnamic acid were

dissolved in 20 ml of dried methanol and 0.5 ml of concentrated sulfuric acid was added.

Mixture was refluxed from 7 to 17 h. Next, the solvent was evaporated, residue was extracted

with 40 ml of ethyl acetate and washed with 0.5 % NaOH and brine. Organic layer was dried

with anhydrous sodium sulphate and solvent was evaporated again. When necessary, product

was crystallized from ethanol with addition of active carbon.

According to method II [23] [4a, 5a, 7a, 8a, 14a, 15a, 16a, 17a]: 15 mmols of cinnamic

acid were dissolved in 45 ml of dried toluene. Next, 15 mmols of DBU were added to the

mixture and stirred in room temperature for 1 h. After that time 15 mmols of iodomethane

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were added and stirring was continued for at least 20 h. Then, 20 ml of toluene was added and

mixture was washed twice with 70 ml of 0.5 % NaOH and 70 ml of water. Organic layer was

dried with anhydrous sodium sulphate. Finally, the solvent was evaporated.

4

.1.2. General procedure of synthesis 1,3,5-triazin-2-amines.

According to method I: 5 mmols of cinnamic acid ester and 5 mmols of guanidine

derivative were added to 10 ml sodium methoxide, fresh-prepared from 10 mmols of sodium

and 10 ml of dried methanol. The mixture was refluxed from 15 to 25 h. After that time, 15

ml of water was added to the mixture and stirred from 0.5 to 3 h. The obtained precipitate was

filtered and crystallized from methanol.

According to method II: 2.5 mmols of cinnamic acid ester and 2.5 mmols of guanidine

derivative were added to 3 ml of sodium methoxide, fresh-prepared from 5 mmols of sodium

and 3 ml of dried methanol. The mixture was refluxed using microwave irradiation from 2 to

8

h in conditions: T = 70-80°C; power = 120 W; t = 10 min; t = 60 min; p = 5.0-6.0 bar. After

0

the set time 15 ml of water was added to the mixture and stirred from 0.5 to 3 h. The obtained

precipitate was filtered and crystallized from methanol.

4

.1.3. 4-(4-Methylpiperazin-1-yl)-6-[(E)-2-phenyletenyl]-1,3,5-triazin-2-

amine (1)

Obtained according to method I. White solid; C H N (MW 296.37); Mp. 176-180°C; Yield

1

6

20

6

1

5

2

%. H NMR (DMSO-d ) δ : 7.81 (d, J=15.90Hz, 1H; Ph-CH=CH); 7.63-7.66 (m, 2H, Ph-

6

,5-H); 7.32-7.42 (m, 3H, Ph-3,4,5-H); 6.74-6.79 (br s, 2H, NH ; d, J=15.90Hz, 1H, Ph-

2

CH=CH); 3.73 (br s, 4H, Pp-2,6-H); 2.31 (t def, J =4.87Hz, J =4.62Hz, 4H, Pp-3,5-H); 2.19

1

2

1

3

(

1

s, 3H, Pp-CH ). C NMR (DMSO-d ) δ : 170.48, 167.38, 165.06, 138.34, 135.84, 129.72,

29.35, 128.11, 128.04, 54.87, 46.25, 42.91. IR (KBr) [cm ] ν

3 6

-1

= 3326.61 (NH ); 3143.40

N-H 2

(

NH ). Anal. Calcd for C H N : C64.84, H6.80, N28.36; Found: C65.13, H6.53, N28.45.

2 16 20 6

+

/-

+

LC/MS : purity: 100%, t =2.77, (ESI) m/z [M+H] 297.32.

R

4

.1.4. 4-[(E)-2-(2-Methylphenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (2)

Obtained according to method I. White solid; C H N (MW 310.40); Mp. 170-173°C; Yield

1

7

22

6

1

7%. H NMR (DMSO d ) δ : 8.08 (d, J=15.90Hz, 1H; Ph-CH=CH); 7.68 (d, J=6.15Hz, 1H,

6

Ph-6-H); 7.18-7.25 (m, 3H, Ph-3,4,5-H); 6.79 (br s, 2H, NH ) 6.66 (d, J=15.90Hz, 1H, Ph-

2

CH=CH); 3.73 (br s, 4H, Pp-2,6-H); 2.38 (s, 3H, Ph-CH ); 2.31 (t, J=4.87Hz, 4H, Pp-3,5-H);

3

13

2

1

.19 (s, 3H, Pp-CH₃). C NMR (DMSO-d ) δ : 170.52, 167.38, 165.08, 136.93, 135.60,

6

34.63, 131.05, 129.46, 129.11, 126.86, 126.53, 54.89, 46.27, 42.93, 19.94. IR (KBr) [cm ] ν

-1

=

3310.21 (br. NH ). Anal. Calcd for C H N : C65.78, H7.14, N27.08; Found: C66.19,

2 17 22 6

N-H

+

/-

+

H7.48, N27.05. LC/MS : purity: 100%, t =3.12, (ESI) m/z [M+H] 311.35.

R

4

.1.5. 4-[(E)-2-(3-Methylphenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (3)

Obtained according to method I. White solid; C H N (MW 310.40); Mp. 175-177°C; Yield

1

7

22

6

1

5%. H NMR (DMSO d ) δ : 7.78 (d, J=16.16 Hz, 1H; Ph-CH=CH); 7.44 (t, J =8.21Hz,

6 1

ACCEPTED MANUSCRIPT

J =7.69Hz, 2H, Ph-2,6-H); 7.27 (t def, J =7.69Hz, J =7.44Hz, 1H, Ph-5-H); 7.16 (d,

2

1

2

J=7.44Hz, 1H, Ph-4-H); 6.77 (br s, 2H, NH ; d, J=15.90Hz, 1H, Ph-CH=CH); 3.73 (br s, 4H,

2

1

3

Pp-2,6-H); 2.29 (s, 4H, Pp-3,5-H; 3H, Ph-CH ); 2.19 (s, 3H, Pp-CH ). C NMR (DMSO-d )

3

6

δ : 170.51, 167.39, 165.05, 138.53, 138.45, 135.78, 130.43, 129.22, 128.64, 127.85, 125.31,

-

1

5

4.89, 46.28, 42.93, 21.36. IR (KBr) [cm ] ν

= 3383.50 (br. NH ). Anal. Calcd for

N-H 2

+

/-

C H N : C65.78, H7.14, N27.08; Found: C65.67, H7.44, N26.87. LC/MS : purity: 100%,

17

22

6

+

t =3.25, (ESI) m/z [M+H] 311.35.

R

4

.1.6. 4-[(E)-2-(4-Methylphenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (4)

Obtained according to method I. White solid; C H N (MW 310.40); Mp. 190-193°C; Yield

1

7

22

6

1

3%. H NMR (DMSO d ) δ : 7.77 (d, J=15.90Hz, 1H; Ph-CH=CH); 7.36 (d, J=7.95Hz; 2H,

6

Ph-2,6-H); 7.19 (d, J=8.20Hz; 2H, Ph-3,5-H); 6.76 (br s, 2H, NH ); 6.70 (d, J=15.90Hz, 1H,

2

Ph-CH=CH); 3.73 (br s, 4H, Pp-2,6-H); 2.31 (s, 4H, Pp-3,5-H; 3H, Ph-CH ); 2.19 (s, 3H, Pp-

3

1

3

CH₃). C NMR (DMSO-d ) δ : 170.61, 167.39, 165.08, 139.44, 138.34, 133.12, 129.96,

6

-1

1

28.08, 127.00, 54.90, 46.27, 42.93, 21.41. IR (KBr) [cm ] ν

= 3376.75 (br. NH₂);

N-H

3

320.82 (NH ). Anal. Calcd for C H N : C65.78, H7.14, N27.08; Found: C65.88, H7.03,

2

17 22

6

+

/-

+

N26.93. LC/MS : purity: 100%, t =3.23, (ESI) m/z [M+H] 311.35.

R

4

.1.7. 4-[(E)-2-(2-Chlorophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (5)

Obtained according to method II. White solid; C H N Cl (MW 330.82); Mp. 172-174°C;

1

6

19

6

1

Yield 4%. H NMR (DMSO d ) δ : 8.15 (d, J=15.90Hz, 1H; Ph-CH=CH); 7.92 (m, 1H, Ph-3-

6

H); 7.50 (m, 1H, Ph-6-H); 7.37 (m, 2H, Ph-4,5-H); 6.86 (br s, 2H, NH ); 6.83 (d, 1H,

2

J=15.90Hz, Ph-CH=CH); 3.70 (br s, 4H, Pp-2,6-H); 2.33 (t, 4H, J=4.90Hz, Pp-3,5-H); 2.20

1

3

(

1

s, 3H, Pp-CH ). C NMR (DMSO-d ) δ : 170.14, 167.37, 165.07, 133.63, 133.58, 133.34,

3 6

-1

31.13, 130.32, 128.28, 128.17, 54.85, 46.26, 42.94. IR (KBr) [cm ] ν

= 3453.88 (br.

N-H

NH ). Anal. Calcd for C H N Cl: C58.09, H5.79, N25.40; Found: C57.98, H5.82, N25.10.

2

16 19

6

+

/-

+

LC/MS : purity: 93.60%, t =3.48, (ESI) m/z [M+H] 331.29.

R

4

.1.8. 4-[(E)-2-(3-Chlorophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (6)

Obtained according to method II. White solid; C H N Cl (MW 330.82); Mp. 163-165°C;

1

6

19

6

1

Yield 3%. H NMR (DMSO d ) δ : 7.80 (d, J=16.18Hz, 1H; Ph-CH=CH); 7.76 (s, 1H, Ph-6-

6

H); 7.62-7.66 (m, 1H, Ph-2-H); 7.41-7.43 (m, 2H, Ph-4,5-H); 6.86 (d, J=16.18Hz, 1H, Ph-

CH=CH); 6.80 (br s, 2H, NH ); 3.76 (br s, 4H, Pp-2,6-H); 2.33 (t, J=4.92Hz, 4H, Pp-3,5-H);

2

13

2

.21 (s, 3H, Pp-CH₃). C NMR (DMSO-d ) δ : 170.23, 167.40, 165.02, 138.18, 136.79,

6

-1

1

34.17, 131.09, 129.78, 129.31, 127.75, 126.68, 54.89, 49.06, 46.27, 42.93. IR (KBr) [cm ] ν

=

3087.48 (br. NH ). Anal. Calcd for C H N Cl: C58.09, H5.79, N25.40; Found:

2 16 19 6

N-H

+

/-

+

C58.24, H5.82, N25.29. LC/MS : purity: 100%, t =3.55, (ESI) m/z [M+H] 331.22.

R

ACCEPTED MANUSCRIPT

4

.1.9. 4-[(E)-2-(4-Chlorophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (7)

Obtained according to method I. White solid; C H N Cl (MW 330.82); Mp. 208-211°C;

1

6

19

6

1

Yield 14%. H NMR (DMSO d ) δ : 7.80 (d, J=16.16Hz, 1H; Ph-CH=CH); 7.70 (d, 2H, Ph-

6

2

3

,6-H); 7.45 (d,2H, Ph-3,5-H); 6.81 (d, J=16.16Hz, 1H, Ph-CH=CH); 6.79 (br s, 2H, NH );

2

13

.76 (br s, 4H, Pp-2,6-H); 2.33 (t, J=4 .92Hz, 4H, Pp-3,5-H); 2.10 (s, 3H, Pp-CH ). C NMR

3

(

5

DMSO-d ) δ : 170.32, 167.39, 165.03, 136.94, 134.83, 134.12, 129.84, 129.33, 128.94,

6

-1

4.89, 46.27, 42.94. IR (KBr) [cm ] ν

= 3371.92 (NH ); 3322.75 (NH ). Anal. Calcd for

N-H 2 2

+

/-

C H N Cl: C58.09, H5.79, N25.40; Found: C58.03, H5.93, N25.43. LC/MS : purity:

1

16

19

6

+

00%, t =3.55, (ESI) m/z [M+H] 331.29.

R

4

1

.1.10. 4-[(E)-2-(2,4-Dichlorophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-

,3,5-triazin-2-amine (8)

Obtained according to method I. White solid; C H N Cl (MW 365.26); Mp. 160-162°C;

1

6

18

6

2

1

Yield 27%. H NMR (DMSO d ) δ : 8.08 (d, 1H, J=15.90Hz, Ph-CH=CH); 7.96 (d, 1H,

6

J=8.80Hz, Ph-6-H); 7.68 (s, 1H, Ph-3-H); 7.44 (d, 1H, J=8.20Hz, Ph-5-H); 6.85 (d, 1H,

J=15.90Hz, Ph-CH=CH); 6.85 (br s, 2H, NH ); 3.70 (br s, 4H, Pp-2,6-H); 2.30 (t, 4H,

2

1

3

J=4.87Hz, Pp-3,5-H); 2.20 (s, 3H, Pp-CH ). C NMR (DMSO-d ) δ [ppm]: 169.99, 167.36,

3

6

1

65.05, 134.67, 134.32, 132.72, 132.15, 131.86, 129.75, 129.59, 128.39, 54.84, 46.26, 42.94.

-1

IR (KBr) [cm ] ν

= 3455.81 (br. NH ). Anal. Calcd for C H N Cl : C52.61, H4.97,

2 16 18 6 2

N-H

+

/-

N23.01; Found: C52.47, H5.14, N22.81. LC/MS : purity: 93.80%, t =4.35, (ESI) m/z

R

+

[

M+H] 365.26.

4

1

.1.11. 4-[(E)-2-(3,4-Dichlorophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-

,3,5-triazin-2-amine (9)

Obtained according to method I. White solid; C H N Cl (MW 365.26); Mp. 201-204°C;

1

6

18

6

2

1

Yield 6%. H NMR (DMSO d ) δ : 7.99 (d, J=1.89Hz, 1H; Ph-2-H); 7.79 (d, J=15.97Hz, 1H,

6

Ph-CH=CH); 7.62-7.71 (m, 2H, Ph-5,6-H); 6.89 (d, J=15.97Hz, 1H, Ph-CH=CH); 6.80 (br s,

2

H, NH ); 3.76 (br s, 4H, Pp-2,6-H); 2.33 (t, J=4.92Hz, 4H, Pp-3,5-H); 2.21 (s, 3H, Pp-CH ).

2 3

1

3

C NMR (DMSO-d ) δ : 170.13, 167.41, 164.99, 136.82, 135.77, 132.15, 131.80, 131.35,

6

-1

1

30.43, 129.95, 128.05, 54.88, 46.27, 42.93. IR (KBr) [cm ] ν

= 3326.61 (NH ); 3174.26

N-H 2

(

NH ). Anal. Calcd for C H N Cl : C52.61, H4.97, N23.01; Found: C52.62, H5.32, N22.89.

2 16 18 6 2

+

/-

+

LC/MS : purity: 100%, t =4.31, (ESI) m/z [M+H] 365.26.

R

4

.1.12. 4-[(E)-2-(3-Bromophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (10)

Obtained according to method II. White solid; C H N Br (MW 375.27); Mp. 148-152°C;

1

6

19

6

1

Yield 64%. H NMR (DMSO d ) δ : 7.88 (s, 1H, Ph-2-H); 7.76 (d, 1H, J=15.90Hz, Ph-

6

CH=CH); 7.66 (d, 1H, J=7.7Hz, Ph-4-H); 7.52 (d, 1H, J=9.0Hz, Ph-6-H); 7.34 (t, 1H,

J=8.0Hz, Ph-5-H); 6.86 (d, 1H, J=15.90Hz, Ph-CH=CH); 6.80 (br s, 2H, NH ); 3.70 (br s, 4H,

2

13

Pp-2,6-H); 2.35 (t, 4H, J=4.6Hz, Pp-3,5-H); 2.20 (s, 3H, Pp-CH ). C NMR (DMSO-d ) δ :

3

6

1

70.21, 167.40, 165.01, 138.45, 136.73, 132.21, 131.37, 130.66, 129.76, 126.99, 122.76,

ACCEPTED MANUSCRIPT

-

1

5

4.89, 46.27, 42.93. IR (KBr) [cm ] ν

= 3461.60 (br. NH ). Anal. Calcd for C H N Br:

N-H 2 6

16 19

+

C51.21, H5.10, N22.39; Found: C51.51, H5.12, N22.92. MS: m/z (%) = 375.21 ([M] , 20)

4

.1.13. 4-[(E)-2-(3-Fluorophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (11)

Obtained according to method I. White solid; C H N F; (MW 314.36); Mp. 172-174°C;

1

6

19

6

1

Yield 15%. H NMR (DMSO d ) δ : 7.79 (d, 1H, J=15.90Hz, Ph-CH=CH); 7.46 (m, 3H, Ph-

6

2

,4,6-H); 7.17 (t, 1H, J=7.95Hz, Ph-5-H); 6.84 (d, 1H, J=15.90Hz, Ph-CH=CH); 6.84 (br s,

H, NH ); 3.73 (br s, 4H, Pp-2,6-H); 2.31 (t, 4H, J=4.9Hz, Pp-3,5-H); 2.20 (s, 3H, Pp-CH ).

2

3

1

3

C NMR (DMSO-d ) δ : 170.25, 167.39, 165.03, 164.21, 161.79, 138.53, 138.45, 137.05,

6

1

4

37.02, 131.26, 131.17, 129.66, 124.51, 124.49, 116.45, 116.124, 114.40, 114.19, 54.88,

-1

6.27, 42.94. IR (KBr) [cm ] ν

= 3500.17 (br. NH ). Anal. Calcd for C H N F: C61.13,

2 16 19 6

N-H

+

/-

H6.09, N26.73; Found: C60.92, H6.36, N26.38. LC/MS : purity: 99.42%, t =3.09, (ESI) m/z

R

+

[

M+H] 315.27.

4

1

.1.14. 4-{(E)-2-[3-(Trifluoromethyl)penyl]etenyl}-6-(4-methylpiperazin-

-yl)-1,3,5-triazin-2-amine (12)

Obtained according to method II. Cream solid; C H N F (MW 364.38); Mp. 170-172°C;

1

7

19

6 3

1

Yield 10%. H NMR (DMSO d ) δ : 8.00 (m, 2H, Ph-2,4-H); 7.88 (d, 1H, J=16.20Hz, Ph-

6

CH=CH); 7.64 (m, 2H, Ph-5,6-H); 6.92 (d, 1H, J=16.0Hz, Ph-CH=CH); 6.80 (s, 2H, NH₂);

4

.08 (q, 1H, CH -OH-solvent); 3.74 (br s, 4H, Pp-2,6-H); 3.14 (d, 2H, J=5.12Hz, CH -OH-

3 2

13

solvent); 2.32 (t, 4H, J=4.9Hz, Pp-3,5-H); 2.20 (s, 3H, Pp-CH ). C NMR (DMSO-d ) δ :

3

6

1

70.19, 167.42, 165.02, 137.07, 136.70, 131.65, 130.36, 130.20, 54.89, 46.27, 42.93. IR

-1

+

(KBr) [cm ] ν

= 3342.03 (br. NH ). MS: m/z (%) = 364.381 ([M] , 18)

N-H

2

4

.1.15. 4-[(E)-2-(3-Cyanophenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (13)

Obtained according to method II. White solid; C H N (MW 321.39); Mp. 196-200°C;

1

7

19

7

1

Yield 6%. H NMR (DMSO d ) δ : 8.18 (s,1H, Ph-2-H); 8.10 (d, 1H, J=8.2Hz, Ph-4-H); 7.82

6

(d, 1H, J=16.2Hz, Ph-CH=CH); 7.79 (d, 1H, J=7.70Hz, Ph-6-H); 7.58 (t, 1H, J=8.0Hz, Ph-5-

H); 6.90 (d, 1H, J=15.90Hz, Ph-CH=CH); 6.80 (s, 2H, NH ); 3.75 (br s, 4H, Pp-2,6-H); 2.32

2

1

3

(

t, 4H, J=4.8Hz, Pp-3,5-H); 2.20 (s, 3H, Pp-CH ). C NMR (DMSO-d ) δ : 170.13, 167.40,

3 6

1

4

65.00, 137.21, 136.17, 132.87, 132.53, 131.71, 130.56, 130.48, 119.01, 112.54, 54.83, 46.21,

-1

+

2.90. IR (KBr) [cm ] ν

= 3468.35 (br. NH ). MS: m/z (%) = 321.388 ([M] , 100)

N-H

2

4

.1.16. 4-[(E)-2-(3-Nitropenyl)etenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-

triazin-2-amine (14)

Obtained according to method I. White solid; C H N O x 0.25H O (MW 345.87); Mp. 204-

1

6

19

7

2

1

2

08°C; Yield 26%. H NMR (DMSO d ) δ : 8.46 (t, 1H, J=3.8Hz, Ph-2-H); 8.17 (m, 2H, Ph-

6

4

,6-H); 7.93 (d, 1H, J=16.2Hz, Ph-CH=CH); 7.68 (d, 1H, J=16.0Hz, Ph-5-H); 6.94 (d, 1H,

J=16.2Hz, Ph-CH=CH); 6.82 (br s, 2H, NH ); 3.75 (br s, 4H, Pp-2,6-H); 2.33 (t, 4H, J=4.6Hz,

2

13

Pp-3,5-H); 2.20 (s, 3H, Pp-CH ). C NMR (DMSO-d ) δ : 170.04, 167.44, 164.99, 148.83,

3

6

-1

1

37.79, 136.07, 133.98, 131.01, 130.78, 123.94, 122.71, 54.90, 46.27, 42.95. IR (KBr) [cm ]

ACCEPTED MANUSCRIPT

ν _N_-_H= 3368.07 (br. NH ). Anal. Calcd for C H N O x 0,25H O: C55.56, H5.68, N28.35;

2

16 19

7

2

+

/-

+

Found: C55.64, H5,99, N28,10. LC/MS : purity: 99.00%, t =3.16, (ESI) m/z [M+H]

R

3

42.26.

4

1

.1.17. 4-(4-Methylpiperazin-1-yl)-6[(E)-2-(naphthalene-1-yl)ethenyl)]-

,3,5-triazin-2-amine (15)

Obtained according to method I. White solid; C H N (MW 346.43); Mp. 173-176 °C; Yield

2

0

22

6

1

9

%. H NMR (DMSO d ) δ : 8.64 (d, J=15.65 Hz, 1H, Ph-CH=CH); 8.18 (d, J= 8.21 Hz, 1H,

6

Nph-5-H); 7.99-7.92 (m, 3H, Nph-6,7,8-H); 7.66-7.52 (m, 3H, Nph-2,3,4-H); 6.84 (br s, 2H,

NH ; d, J=15.64Hz; 1H, Ph-CH=CH); 3.77 (br s, 4H, Pp- 2,6-H); 2.33 (t def, J=4.87Hz;

2

1

3

J=4.62Hz; 4H, Pp-3,5-H); 2.20 (s, 3H, Pp-CH ). C NMR (DMSO-d ) δ : 170.41, 167.43,

3

6

1

65.13, 134.57, 133.82, 132.78, 131.24, 130.73, 129.89, 129.19, 127.33, 126.63, 126.32,

-1

25.05, 123.44, 54.91, 46.27, 42.98. IR (KBr) [cm ] ν

= 3322.75 (br. NH ). Anal. Calcd

2

N-H

+

/-

for C H N : C69.34, H6.40, N24.26; Found: C69.22, H6.40, N24,14. LC/MS : purity:

2

0

22

6

+

1

00%, t =3.88, (ESI) m/z [M+H] 347.31.

R

4

1

.1.18. 4-(4-Methylpiperazin-1-yl)-6[(E)-2-(naphthalene-2-yl)ethenyl)]-

,3,5-triazin-2-amine (16)

Obtained according to method I. White solid; C H N (MW 346.43); Mp. 197-200 °C; Yield

2

0

22

6

1

4

3

2

6%. H NMR (DMSO d ) δ : 8.02 (d, J=15.9 Hz, 1H, Ph-CH=CH); 7.97-7.86 (m, 4H, Nph-

6

,4,5,8-H); 7.57-7.51 (m, 2H, Nph-6,7-H); 6.93 (d, J=15.64Hz; 1H, Ph-CH=CH); 6.80 (br s,

H, NH ) 3.78 (br s, 4H, Pp- 2,6-H); 2.34 (t, J=4.90Hz; 4H, Pp-3,5-H); 2.21 (s, 3H, Pp-CH ).

2

3

1

3

C NMR (DMSO-d ) δ : 170.54, 167.44, 165.08, 138.42, 133.76, 133.58, 133.47, 129.08,

6

-1

1

28.90, 128.76, 128.47, 128.08, 127.25, 127.05, 124.36, 54.90, 46.29, 42.96. IR (KBr) [cm ]

+

/-

+

ν _N_-_H= 3329.50 (br. NH ). LC/MS : purity: 100%, t =3.95, (ESI) m/z [M+H] 347.31.

2

R

4

.1.19. 4-([E]-2-(1H-Indol-3-yl)ethenyl)-6-(4-methylpiperazin-1yl)-1,3,5-

triazin-2-amine (17)

Obtained according to method I. Yellow solid; C H N (MW 335.41); Mp. 256-258 °C;

1

8

21

7

1

Yield 11%. H NMR (DMSO d ) δ : 11.85 (s, 1H, NH); 8.01 (d, J=16.2 Hz, 1H, Ind-

6

CH=CH); 7.85-7.82 (m, 2H, Ind-4,7-H ); 7.45-7.42 (m, 1H, Ind-2-H); 7.21-7.14 (m, 2H, Ind-

5

4

1

4

,6-H); 6.65-6.60 (m, 3H, NH + Ind-CH=CH); 3.74 (br s, 4H, Pp- 2,6-H); 2.48 (t, J=4.87Hz;

2

13

H, Pp-3,5-H); 2.33 (s, 3H, Pp-CH₃). C NMR (DMSO-d ) δ : 171.51, 167.33, 165.09,

6

37.79, 133.03, 130.12, 125.59, 122.64, 121.93, 120.96, 120.13, 113.06, 112.71, 54.95, 46.29,

2.91. Anal. Calcd for C H N : C64.46, H6.31, N29.23; Found: C63.86, H6.30, N28.82. IR

1

8

21

7

-

1

+/-

(KBr) [cm ] ν

= 3210.73 (NH ); 3308.23 (NH ). LC/MS : purity: 100%, t =2.42, (ESI)

N-H 2 2 R

+

m/z [M+H] 336.27.

4

.2. Biological studies

.2.1. Radioligand binding assays

Human histamine H receptor: The affinity of new compounds to human histamine H R

4

was examined in the in vitro radioligand binding assays. In these competition binding tests,

3

[

H]histamine in 10 nM concentration (566.1 GBq/mmol) was used as a radioligand. Cell

ACCEPTED MANUSCRIPT

membranes were prepared from Sf9 insect cells, transiently expressing human H R with co-

4

expressed G-protein subunits (G_α₁₂and G ), as described before [39]. Sedimentation of cell

βy2

membranes was carried out before the start of experiment by centrifugation (10 min, +4° C,

1

6,000 g) and obtained pellet was subsequently resuspended in binding buffer (1 mM EDTA,

1

2.5 mM MgCl , 75 mM Tris-HCl pH=7.4). Cell membranes (35 µg protein/well) were

2

incubated for 60 min at +25° C with 0.1-100,000 nM concentrations of test compound in the

3

presence of 10 nM [ H]histamine in a total assay volume of 200 µl. Each assay was

performed in triplicates and for at least 2 times. To determine non-specific binding, 100 nM

non-labeled JNJ7777120 was used. The bound radioactivity was separated by filtration

through GF/C filtermats. Filters were pretreated with 0.3% PEI and washed three times ice-

cold binding buffer (0.3 ml, +4°C). The radioactivity retained on the filters was measured by

liquid scintillation counting. K values were determined from Cheng-Prusoff equation [40],

i

after fitting the experimental data using non-linear least squares fit and presented as average ±

standard deviation (SD). GraphPad Prism™ (2000, version 3.02, San Diego, CA, USA)

software were used for processing competition binding data.

Mouse histamine H receptor: Radioligand binding studies were performed at Griffin

4

Discoveries BV (Amsterdam, The Netherlands), according to the following procedure: human

embryonic kidney (HEK) 293 cells were transiently transfected using 25 kDA linear

polyethyleneimine (PEI) and mouse histamine H receptor cDNA cloned in pcDEF3 plasmid.

4

Two days after the transfection cells were harvested for membrane preparation in ice cold

Phosphate Buffered Saline (PBS). The scrapped cellular material was isolated by centrifuging

samples at 3000 rpm (Heraeus Multifuge 3S-R) for 10 minutes at 4°C, and resuspended in

ice-cold PBS, making aliquots. These aliquots were centrifuged again at the same conditions

and cell pellets were stored at -20°C until use. For the assays the crude membrane extract was

3

resuspended in 50 mM Tris-HCl, pH 7.4 and sonicated for 10 seconds. [ H]Histamine binding

assays were performed in 50 mM Tris-HCl buffer (pH 7.4 at room temperature), in the

presence or absence of competing ligand. The reaction mixtures, in a total assay volume of

1

00 ꢀl in 96-well plates, were incubated for one hour at room temperature (22°C) and then

harvested on 96-well glass fiber C plates that were pretreated with 0.5% PEI, followed by

washing three times with ice-cold 50 mM Tris-HCl buffer (pH 7.4 at 4°C). The radioactivity

retained on the filters was measured by liquid scintillation counting in a Microbeta counter

(

PerkinElmer). The K value of the tested compound was calculated using nonlinear

i

regression GraphPad Prism™ (2012, version 6.0, San Diego, CA, USA). The K value of

d

3

[

H]histamine for the mouse H R used for the K calculations is 90.5 nM [40].

4 i

Human histamine H receptor: In binding studies to histamine H R, membranes

3

preparation from HEK293 cells stably expressing hH R were employed as a source for

3

receptor proteins. HEK293-hH R cells were grown in standard conditions until reaching 80-

3

9

0% confluence. After medium removal, cells were washed with sterile PBS buffer and kept

for at least 2 h at -80°C. In the next step, cells were thawed and collected in 5 mM Tris-HCl

and 2 mM EDTA, pH 7.4 and homogenized using Ultra-Turrax homogenizer. The resulting

suspension was centrifuged at 1000×g for 10 min to remove unbroken cells and nuclei. The

ACCEPTED MANUSCRIPT

supernatant was homogenized again with Ultra-Turrax and subsequently centrifuged at

4

7

8,000×g for 60 min. The resulting pellets were resuspended in 50 mM Tris-HCl buffer, pH

.4.

Binding experiments were carried out as follows: membranes (30 ꢀg protein per well)

3

were incubated with 1 nM [ H]-N-α-methylhistamine and different concentrations of the

respective test ligand, ranging from 0.1 to 100 ꢀM., in total volume of 400 ꢀl of assay buffer

(

50 mM Tris-HCl, 5 mM MgCl , pH 7.4). Incubations were performed for 60 min at 25°C

2

with shaking (400 rpm). Experiments were run two times in duplicates. Non-specific binding

was determined in the presence of 20 µM (R)(-)-α-methylhistamine. Bound radioligand was

separated from free radioligand by filtration through GF/B filtermats, pretreated with 0.5%

PEI and washed eight times with 0.4 ml of ice-cold washing buffer (50 mM Tris-HCl, pH

7

.4). The collection of results and data analysis were performed as in case of human histamine

H R binding experiments described before.

4

.2.2. cAMP accumulation assay in cells expressing hH R

4

Efficacy of selected compounds was determined in cAMP accumulation assay, as

already described elsewhere [21]. Briefly, CHO cells stably expressing human H R were

4

incubated (30 min, room temperature) with histamine (140 nM), forskolin (10 ꢀM) and

evaluated compounds (10 appropriate concentrations in a range spanning over 5 log units) in

presence of phosphodiesterase inhibitor (RO-201724, 100ꢀM). Subsequently, intracellular

cAMP accumulation was measured with homogenous TR-FRET immunoassay, using

LANCE Ultra cAMP kit (PerkinElmer) and following manufacturer’s instructions. TR-FRET

signal, inversely proportional to cAMP concentration in sample, was measured using EnSpire

microplate reader (PerkinElmer) and translated into actual quantities of produced cAMP, on

the basis of cAMP standard curve. Sigmoidal dose-response curve fitting was performed,

using GraphPad Prism™ software (version 5.01, San Diego, CA, USA), for data expressed as

%

of maximal response in function of used ligand concentration. Showed results represent the

mean of three separate experiments, each performed in triplicates.

4

.2.3. Cytotoxicity

Cell lines: Neuroblastoma IMR-32 cell line was provided by Department of

Oncogenomics, Academisch Medisch Centrum, Amsterdam, Holland [41, 42]. 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).

Cell culture: 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

-1

serum (FBS), 100 mg×ml streptomycin and 100 U×ml penicillin. Cells were cultured at

3

7°C in an atmosphere containing 5% CO₂.

In vitro antiproliferative assay: The cells were seeded in 96-well plates at a

4

concentration of 3×10 cells/well (IMR-32) or 2×10 cells/well (HEK-293) in 200 ꢀl culture

medium and incubated in standard growth conditions. Next, compound 6 dissolved in DMSO

was added into microplates at the final concentrations 0.01-250 ꢀM (the concentration of

ACCEPTED MANUSCRIPT

DMSO did not exceed 1%) and the cells were incubated with 6 for 48 h. Then, 20 ꢀl of EZ4U

labeling mixture (Biomedica) was added and the cells were incubated for another 5 h at 37°C

and 5% CO . After incubation period, the absorbance of samples was measured using EnSpire

2

microplate reader (PerkinElmer) at 492 nm. The activity of the standard drug doxorubicin was

estimated as described previously [43].

4

.2.4. Ames test

Ames microplate fluctuation protocol (MPF) assay was performed with Salmonella

typhimurium strain TA98, enabling the detection of frame shift mutations. Bacterial strain as

well as exposure and indicator medium were purchased from Xenometrix AG (Allschwil,

Switzerland). The mutagenic potential of tested structures was evaluated by incubation of

bacteria, incapable of producing histidine, with particular concentration of compound for 90

min in exposure medium, containing limited amount of histidine. The occurrence of reversion

events to histidine prototrophy was observed as a growth of bacteria in the indicator medium

without histidine after 48 h of incubation in room temperature. Bacterial growth in 384-well

plates was visualized by color change of medium from violet to yellow due to addition of pH

indicator dye. Compound was classified as mutagenic, if the fold increase in number of

positive wells over the solvent control baseline (FIB) was greater than 2.0. The FIB was

determined by dividing the mean number of revertants for tested compound by the solvent

control baseline. The solvent control baseline was defined as the mean number of positive

wells in the negative control sample, increased by one standard deviation (SD). DMSO was

used as a negative and 10 ꢀM 2-nitrofluorene as positive control in performed experiments.

4

.2.5. Metabolic stability and interaction with CYP3A4

Reaction with recombinant human liver microsomes (HLMs): The biotransformation

was carried out using 1 mg/ml human (adult male and female) liver microsomes (Sigma-

Aldrich) in 200 ꢀl of reaction buffer, containing 0.1 M Tris-HCl (pH 7.4), NADPH

regeneration system (Promega) and 6 in final concentration of 50 µM. The reaction was

initiated after 5 min of sample preincubation at 37°C by adding 50 µl of NADPH regeneration

system. Next, the samples were incubated for 120 min at 37°C and 200 µl of cold methanol

was added to terminate the reaction. The mixture was centrifuged at 13,000 rpm for 15 min.

Finally, the LC/MS analysis of the supernatant was performed, using LC/MS system,

consisted of a Waters Acquity UPLC coupled to a Waters TQD mass spectrometer

(electrospray ionization mode ESI-tandem quadrupole).

CYP3A4 P450-Glo Assay: The protocol of luminescent CYP3A4 P450-Glo Assay

(Promega) was provided by manufacturer. The enzymatic reactions were performed in white

polystyrene, flat-bottom 96-well microplates (Thermo-Scientific). The luminescence signal

was measured with a microplate reader in luminescence mode (PerkinElmer). The IC value

5

0

of the reference compound - ketoconazole (Sigma-Aldrich) was determined as we described

previously [21]. The final concentrations of 6 were 0.025-25 ꢀM and for CYP3A4 inhibitor -

ketoconazole 0.025-10 µM.

ACCEPTED MANUSCRIPT

In silico study: The metabolic biotransformation of compound 6 was studied in silico by

using computational procedure MetaSite 4.1.1 in liver model (Molecular Discovery Ltd) [27,

2

8].

4

.2.6. In vivo pharmacology: carrageenan-induced edema mice model

Animals: The experiments were carried out on male albino Swiss mice (18-25 g). 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.

Determination of anti-inflammatory activities of investigated compounds using

carrageenan-induced hind paw edema test: Male Swiss albino mice (18–26 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, according to the modified method of C. A. Winter [34]

and P. Lence [35]. 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 compounds were administered at doses of 10, 25,

0 mg/kg, intraperitoneally (as a suspension in methylcellulose), prior to carrageenan

injection. Methylcellulose was administered by the same route to the control group

methylcelullose had no effect on edema, data not shown). After these administrations, paw

5

(

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 volumes measured 1, 2 and 3 hours after injection of carrageenan to the control group

–

paw volumes at the beginning.

N’: paw volumes measured 1, 2, and 3 hours after injection of carrageenan to the test groups –

paw volumes at the beginning.

Data Analysis: Data are expressed as mean ± SEM (Table 2: Supplemental materials).

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 Prism GraphPad 6.0 (GraphPad

Software Inc.) was used to process data.

4

.3. X-ray structure crystal determination

The relevant crystals were obtained from methanol solutions. Pertinent crystallographic

parameters are summarized in Table 3, whereas selected metric parameters are presented in

Table 4 and Table 5 (supplemental material).The structures were solved by direct methods

ACCEPTED MANUSCRIPT

and refined with SHELXTL [44]. E-map provided positions for all non-H-atoms. The full-

2

matrix least-squares 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

iso

eq

attached to nitrogens were located in difference Fourier map and refined with the restraints N-

H = 0.86 Å and U = 1.2U (N).

iso

eq

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:

1

047578 and 1047579 (Fax: Int code + (1223)336-033; E-mail: deposit@ccdc.cam.ac.uk).

Crystal data for 5: C H N Cl, M = 330.82, monoclinic, space group P2 /c, a = 20.018(4)

1

6

19

6

1

3

Å, b = 4.7670(9) Å, c = 19.114(4) Å, β= 115.436(3)°, V = 1647.2(6) Å , Z = 4, D = 1.334 g

x

-3

-1

cm , T = 296 K, µ = 0.241 mm , λ = 0.71073 Å, data/parameters = 1751/ 210; final R =

1

0

.0289.

Crystal data for 17: C₁₈H₂₁N , M = 335.42, orthorhombic, space group P2 2 2 , a =

7

1 1 1

3

7

.4562(3) Å, b = 13.7860(6) Å, c = 16.8044(7) Å, V = 1727.3(1) Å , Z = 4, D = 1.290

x

g cm , T = 196 K, µ = 0.658 mm , λ = 0.71073 Å, data/parameters = 3023/227; final R =

-3

-1

1

0

.0282, Flack x = 0.3(3).

4

.4. Docking studies

Homology model: Histamine H R homology model used in these studies was built on

4

the basis of previously described and published crystal structure of histamine H R (PDB

1

entry: 3RZE), paying attention to mutagenesis data described by Jongejan et al. [36]. After the

alignment of sequences, a total of 100 models was obtained using Modeller [45]. Models were

then fitted to H R crystal structure using Schrödinger software [46]. Missing side chains were

1

added using Prime module. H-bonds optimization and energy minimization (using OPLS2005

forcefield) were then performed using Protein Preparation Wizard, implemented in

Schrödinger suite. Structures evaluation and stereochemical analyses were performed using

PROCHECK [47] and Ramachandran plots. Further docking studies allowed to choose one

model used for subsequent docking procedures. Obtained model lacks the large intracellular

loop, due to its length difference in desired (H R) and template (H R) model.

4

1

Ligands: In current 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

by Feng [37]. Docking studies were performed via GlideDock module. Results were

interpreted by the means of Docking Score functions. Receptor-ligand interactions were

visualized using PyMOL [48].

ACCEPTED MANUSCRIPT

Acknowledgments

Partial supports by COST Actions BM0806, CM1207 financed by EU-FP7, Grant No.

5

94/N-COST/2009/0, DFG INST 2018/664-1 FUGG and Polish National Science Center

funds, granted on the basis of decisions No: DEC-2011/02/A/NZ4/00031 and

DEC-2011/01/N/NZ4/01126 are greatly acknowledged.

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Table 1. 2-(Arylethenyl)-1,3,5-triazine-2-amine derivatives (1-18) produced via Scheme 1d.

b

hH

4

R

a

c

No.

Conditions

Substituents

(±) SD

K

i

[nM]

phenyl-group

2

3

4

R

1

2

3

4

5

6

7

8

9

(I): 22h reflux

(I): 15h reflux

(I): 24h reflux

(I): 24,5h reflux

(II): T=80°C (4h)

(I): 23h reflux

H

CH

H

Cl

H

Cl

H

CH

H

Cl

H

CH

H

Cl

H

5 210

2 230

703

37 400

4 860

253

4 210

9 040

1 780

1 140

3 520

2 450

2 640

2 570

376

1 360

365

5 040

1 320

75

1 500

2 080

33

191

18

967

78

3

(I): 15h reflux

(I): 26,5h reflux

(I): 15h reflux

(II): T=80°C (6,5h); T=90°C (1h); p=10bar

(I); 27h reflux

H

Cl

Br

F

CF

CN

NO

1

0

1

2

3

4

(II): T=80°C (4h)

(II): T=80°C (2h)

(I): 36h reflux

H

3

H

2

1278

naphthyl-group

1

5

6

(I): 18,5h reflux

(I): 17h reflux

1-naphthyl

2-naphthyl

29 985

3 794

12 664

1 427

indolyl-group

1

7

(I): 18h reflux

1H-indolyl

2 525

174

a

(

[

I) - method I; (II) - method II;

b

3

4 1 2

H]Histamine displacement assay with membrane preparation of Sf9 cells expressing hH R, co-expressed with G-protein Gαi2 and Gβ γ

subunits;

c

SD - standard deviation value at least after two experiments.

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Fig. 1. Structural formula of the first selective, non-imidazole H R antagonist – JNJ 7777120.

4

ACCEPTED MANUSCRIPT

Fig. 2. On the left: Structural formulas of known and potent H R ligands in the group of

4

aminopyrimidines [19] and 1,3,5-triazines [20]. On the right: General structure of designed

compounds

ACCEPTED MANUSCRIPT

Fig. 3. Structural formulas of the new 1,3,5-triazin-2-amine derivatives.

ACCEPTED MANUSCRIPT

Fig. 4. cAMP accumulation studies in CHO cells expressing the human histamine H₄

receptor, co-treated with forskolin, histamine and tested compounds: 3 (A) and 6 (B).

ACCEPTED MANUSCRIPT

Fig. 5. Activity of doxorubicin (standard) and 6 against HEK-293 cell line.

ACCEPTED MANUSCRIPT

Fig. 6. Activity of doxorubicin (standard) and 6 against IMR-32 cell line.

Article Doi

(2-Arylethenyl)-1,3,5-triazin-2-amines as a novel histamine H4 receptor ligands

DOI: 10.1016/j.ejmech.2015.08.014

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Article abstract of DOI:10.1016/j.ejmech.2015.08.014

Full text of DOI:10.1016/j.ejmech.2015.08.014

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