The Synthesis of 1,3,5-triazine Derivatives and JNJ7777120 Analogues with Histamine H4 Receptor Affinity and Their Interaction with PTEN Promoter

Article abstract of DOI:10.1111/cbdd.12752

The involvement of histamine and H₄ receptor (H₄R) in cancer has been investigated recently using the H₄R agonists and antagonists. The scope of the research project was synthesis and exploration of the consequences of a g

Full text of DOI:10.1111/cbdd.12752

Chem Biol Drug Des 2016
Research Article
The Synthesis of 1,3,5-triazine Derivatives and
JNJ7777120 Analogues with Histamine H4 Receptor
Affinity and Their Interaction with PTEN Promoter
Gniewomir Latacz1, Petros Kechagioglou2, Rigini
Key words: 1,3,5-triazines, histamine H4 receptor ligands,
JNJ7777120 analogues, PTEN promoter
2
1
1
_
ꢀ
Papi , Dorota Łazewska , Małgorzata Wiecek ,
z
1
1
Katarzyna Kaminska , Przemysław Wencel ,
Received 24 November 2015, accepted for publication 8
February 2016
Tadeusz Karcz1, Johannes S. Schwed3,4
,
Holger Stark3,4, Dimitrios A. Kyriakidis2 and
1,
ꢀ
Katarzyna Kiec-Kononowicz *
Phosphatase and tensin homolog deleted on chromosome
ten (PTEN) is one of the most frequently mutated tumor
suppressor genes in human cancer. In 1997 PTEN, also
known as MMAC1 (mutated in multiple advanced can-
cers-1) or TEP1 (tensin-like phosphatase-1), was identified
by three independent research groups as a tumor sup-
pressor gene located at the 10q23 region (1–3). The pro-
tein encoded by PTEN is a phosphatase (PTEN, EC
3.1.3.67), member of the large protein tyrosine phos-
phatase family. It is a non-redundant, plasma membrane
phosphatase with a relative molecular mass of 47 kDa and
characterized as a dual specificity phosphatase. Although
it can dephosphorylate proteins, its primary targets are
highly specialized membrane lipids, mainly facilitating the
removal of the phosphate group from the inositol rings.
More specifically, PTEN acts on the phosphatidylinositol-
3,4,5-triphosphate (PIP3) that is formed through the action
of phosphoinositide-3 lipid kinase (PI3K). PIP3 is an impor-
tant lipid second messenger in tumorigenesis. PTEN
reduces the pool of PIP3, inhibiting growth and survival
signals and suppressing tumor formation. This way, an
‘on-off’ switch is evolved that appears to regulate a critical
signaling pathway for oncogenesis (4).
1Department of Technology and Biotechnology of Drugs,
Jagiellonian University Medical College, Medyczna 9,
ꢀ
Krakow 30-688, Poland
2Laboratory of Biochemistry, Institute of Pharmaceutical
Chemistry, Aristotle University of Thessaloniki, Thessaloniki
GR-54124, Greece
3Biozentrum, Goethe University, Max-von-Laue-Str. 9,
Frankfurt/Main 60438, Germany
4Institute of Pharmaceutical and Medicinal Chemistry,
Heinrich-Heine-University, Universitaetsstr. 1, Duesseldorf
40225, Germany
ꢀ
*Corresponding author: Katarzyna Kiec-Kononowicz,
mfkonono@cyf-kr.edu.pl
The involvement of histamine and H4 receptor (H4R) in
cancer has been investigated recently using the H4R
agonists and antagonists. The scope of the research
project was synthesis and exploration of the conse-
quences of a group of compounds with histamine H4
receptor (H4R) affinity on the promoter of PTEN gene
encoding the antitumor PTEN protein. The series of
novel compounds based either on H4R antagonists
JNJ7777120 structure or 1,3,5-triazine scaffold were
synthesized, evaluated for histamine H4R affinity and
used in this study. Compounds 5 and 7 belonging to
the group of JNJ7777120 analogues showed the high-
est interaction with the promoter of PTEN gene and
weak affinity against H4R with Ki value >100 lM. These
compounds showed no significant effect on neurob-
lastoma IMR-32 cells viability indicating no correlation
between PTEN gene promoter affinity and antitumor
activity. Compound 6, another JNJ7777120 analogue,
showed the highest effect on IMR-32 viability with cal-
culated IC50 = 23.27 lM. The 1,3,5-triazine derivatives
exhibited generally low or medium interaction with
PTEN gene promoter. However, the 1,3,5-triazine
derivative 11 with the para-bromo substituent showed
the highest affinity against H4R with Ki value of
520 nM and may be considered as a new lead struc-
ture.
Histamine [2-(4-imidazolyl)-ethylamine] (HIS) is one of the
most important biogenic amines and it was first identified
as a mediator in biological functions in the early 1900s.
HIS liberation is a recognized feature of various allergic
conditions and for many years its importance was limited
to that field. However, HIS is involved in the regulation of
numerous physiological and pathophysiological processes
including gastric acid secretion, hypersensitivity reactions,
bronchial asthma, multiple elements of immune regulation
and tumor genesis (5–7). HIS performs its activation via
different types of histamine receptors (HR). Four metabo-
tropic HR types have been cloned so far (H1R, H2R, H3R,
and H4R). These receptors belong to the rhodopsin-like
family of G protein coupled receptors. Histamine H4R is
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12752
1

Latacz et al.
the latest identified member of the HIS receptor subfamily.
The H4R is widely distributed, especially in organs associ-
ated with the immune system. It is preferentially expressed
in intestinal tissue, spleen, thymus, medullary cells, bone
marrow, and peripheral hematopoietic cells, including eosi-
nophils, basophils, mast cells, T lymphocytes, leukocytes,
and dendritic cells. These cell types are primarily involved
with the development and continuation of allergic
responses. The human H4R, reported as a sequence of
390 amino acid residues, possesses all of the highly con-
served sequence motifs of the class A of the G protein-
coupled receptor (GPCR) superfamily. The H4R is coupled
to Gi/o proteins and shows higher endogenous ligand affin-
ity than the H1 and H2 receptors (8). The H4R gene is
located on chromosome 18q11.2. The amino acid
sequence of the H4R shares approximately 26%, 27%,
and 58% homology in the transmembrane regions with the
H1, H2, and H3 receptors respectively (9).
cially with pyrimidine moiety are important group (25).
Recently, some derivatives of 1,3,5-triazine showed H4R
nanomolar affinities (25–27).
The scope of the research was the synthesis and explo-
ration of the consequences of a group of compounds
obtained as ligands of histamine H4R modifying
JNJ7777120 structure or based on 1,3,5-triazine scaffold,
on the promoter of PTEN gene encoding the antitumor
PTEN protein. Next, the antiproliferative assay was applied
to determine the influence of the selected compounds on
a human embryonic kidney HEK-293 and neuroblastoma
IMR-32 cell lines viability.
Methods and Materials
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 (JASCO, Easton, MD,
USA). Mass spectra (LC/MS) were performed on Waters
TQ Detector mass spectrometer (Waters, Milford, MA,
USA). 1H-NMR and 13C NMR spectra were recorded on
Varian-Mercury 300 MHz spectrometer in DMSO-d6.
Chemical shifts in 1H-NMR spectra are expressed in ppm
downfield from deuterated solvent signal treated as refer-
ence. Data are reported in the following order: multiplicity
(br, broad; def, deformed; s, singlet; d, doublet; t, triplet;
m, multiplet; Im, imidazole, Bzt, benzothiophene Naph,
naphthalene, Ph, phenyl; Pp, piperazine; Py, pyridine, Tr,
triazine), 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
determined (%). Elemental analyses (C, H, N) were mea-
sured on Elemental Vario-EL III instrument and are within
Æ0.5% of the theoretical values. TLC was carried out using
silica gel F254 plates (Merck, Darmstadt, Germany). The
spots were visualized with Dragendorff’s reagent or by UV
absorption at 254 nm. Abbreviations: AcOEt (ethyl acetate),
CDI (carbonyldiimidazole), CH3CN (acetonitrile), Et2O (di-
ethyl ether).
Although little is known about the physiology of the H4R,
its role in colorectal cancer, melanoma, human breast can-
cer has been recently explored using the H4R agonists
and antagonists. In several studies, different carcinoma
cells showed varying expression of H4R and this might
indicate that H4R may play a special role in the develop-
ment of human breast neoplasms. The HIS may regulate
through H4R proliferation of mammary epithelial cells, and
a loss of that control may contribute to neoplasia (10,11).
Moreover, an interestingly number of studies have shown
that up-regulation of the enzyme L-histidine decarboxylase
(HDC; E.C. 4.1.1.22), responsible for HIS synthesis from
its precursor histidine, is correlated with growth of several
types of human tumors such as: melanoma (12), small cell
lung carcinoma (13), breast carcinoma (14), endometrial
cancer (15), and colorectal carcinoma (16). The structural
analysis revealed that natural compounds might be able to
inhibit HIS synthesis by binding to specific regions of HDC
(17). All of recent results indicate that HDC can be a
potential target for therapeutic intervention of many inflam-
matory diseases, some neurological and neuroendocrine
diseases, osteoporosis and even different carcinoma types
and neuroendocrine tumors (18).
Much of the drug research in H4R field is focused on the
search for H4R antagonists because of possibilities for the
treatment of different inflammatory conditions, e.g. skin
diseases, airway diseases or bowel diseases (19–21).
However, preclinical results show also utility of H4R ligands
in cancer, neuropathic pain or vestibular disorder (20). For
years JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-
methylpiperazine; Table 1), the first potent and selective
H4R ligand, has been used as a reference antagonist in
in vitro and in vivo studies (22). Recently, JNJ7777120 has
been identified as partial agonist in a b-arrestin recruitment
assay (23). On the basis of JNJ7777120 structure varieties
of structurally diverse classes of compounds have been
developed (20,24). Among them azine derivatives, espe-
Amide and urea derivatives
(2-Chlorophenyl)(4-methylpiperazin-1-yl)methanone
(1). To the solution of 2-chlorobenzoyl chloride (1.75 g,
10 mmol) in 30 mL of benzene 4-methylpiperazine
(1.00 g, 10 mmol), natrium carbonate (6.36 g, 60 mmol)
and 20 mL of water was added and stirred in the room
temperature for 12 h. The solid was filtered off, separated
and the organic phase was washed with 30 mL of 0.5%
NaOH and water. After drying with Na2SO4, the filter was
evaporated to dryness. The product was crystallized from
Et2O. White solid. Mp. 60–63 °C. Yield: 29%.
C
12H15N2OCl (MW = 238.71) (lit. (28)) C12H15N2OCl x HCl
2
Chem Biol Drug Des 2016

H4 Receptor Ligands – Synthesis and Interaction with PTEN
Table 1: Structures and histamine H4R affinities of synthesized compounds
Histamine H4R
Histamine H4R
Compound
Ki Æ SE [lM]
Compound
Ki Æ SE [lM]
Cl
O
NH2
1
>100
3.88a
7.55a
9
14.61 Æ 1.78
8.26 Æ 1.50
0.52 Æ 0.16b
H
N
O
N
N
N
N
N
N
N
O
N
O
NH2
2
3
10
11
Cl
N
N
N
N
N
N
N
N
N
O
NH2
N
N
N
N
Cl
N
Br
3.52a
12
5.60 Æ 1.32
Cl
NH2
4
O
N
N
N
N
H
N
N
N
N
N
O
NH2
5
6
>100
>100
13
14
7.50 Æ 1.37
N
N
N
H
NH
O
N
Cl
N
N
N
NH2
Cl
10.61 Æ 1.33
N
N
N
N
H
O
S
N
N
N
N
Cl
Cl
Cl
NH2
O
7
8
>100
15
3.19 Æ 0.02
N
N
N
H
N
N
H
N
N
N
N
N
2.36 Æ 0.90b
JNJ7777120
0.0045c
NH2
O
N
N
N
NH
N
Cl
N
N
N
NC
aKi values were determined in duplicates in one experiment;
bData from (27);
cData from (22).
Mp. 292–294 °C). Anal. Calcd for C12H15N2OCl: C60.37;
H6.33; N11.73; Found: C60.14; H6.64; N11.62.
J = 7.2 Hz; J = 1.29 (1.79) Hz, Ph-5-H), 3.50–3.70 (br s,
2H, Pp-2,6-He), 3.18–3.40 (br s, 2H, Pp-2,6-Ha), 2.20–
2.40 (br s, 4H, Pp-3,5-H), 2.17 (s, 3H, Pp-CH3). Anal.
Calcd for C12H15N2OCl: C60.37; H6.33; N11.73; Found:
C60.15; H6.38; N11,65.
(3-Chlorophenyl)(4-methylpiperazin-1-yl)methanone
(2). Synthesized as
(1.75 g, 10 mmol). White solid. Mp. 75–77 °C. Yield: 49%.
C12H15N2OCl (MW = 238.71) 1H-NMR [DMSO-d6]
1 from 3-chlorobenzoyl chloride
d
(4-Chlorophenyl)(4-methylpiperazin-1-yl)methanone
(3). Synthesized as
(ppm): 7.39–7.53 (m, 3H, Ph-2,4,6-H), 7.32 (def dt, 1H,
1
from 4-chlorobenzoyl chloride
Chem Biol Drug Des 2016
3

Latacz et al.
(1.75 g, 10 mmol). White solid. Mp. 70–72 °C. Yield: 10%.
white solid (1.52 g, 41%; Mp. 133–136 °C) was filtered,
washed with n-hexane and used for further reaction with
any more purification.
C
12H15N2OCl (MW = 238.71) (lit. (28) C12H15N2OCl x HCl
1
Mp. 279–281 °C). H-NMR [DMSO-d6] d (ppm): 7.46–7.51
(m, 2H, Ph-2,6-H), 7.36–7.42 (m, 2H, Ph-3.5-H), 3.50–
3.70 (br s, 2H, Pp-2,6-He), 3.20–3.40 (br s, 2H, Pp-2,6-
Ha), 2.20–2.40 (br s, 4H, Pp-3,5-H), 2.17 (s, 3H, Pp-CH3).
Anal. Calcd for C12H15N2OCl: C60.37; H6.33; N11.73;
Found: C60.47; H6.44; N11,75.
3,6-Dichloro-N-(3-(4-methylpiperazin-1-yl)propyl)benzo
[b]thiophene-2-carboxamide (6). 3,6-dichlorobenzo[b]
thiophene-2-carbonyl chloride (0.40 g, 1.5 mmol) and 1-(3-
aminopropyl)-4-methylpiperazine (0.24 g, 1.5 mmol) in
10 mL of absolute CH3CN was stirring in r.t. for 4 h and
heated to reflux for 20 h. The solvent was evaporated,
and to the residue 35 mL AcOEt was added and
washed with 35 mL of 5% HCl. The acidic layer was alka-
lized (10% NaHCO3) and extracted with CH2Cl2. The
organic layer was then dried over anhydrous Na2SO4, fil-
tered and after concentration converted into hydrochloride
(3 mL of acetone and concentrated HCl). Cream solid. Mp.
254 °C (dec). Yield: 41%. C17H21N3SOCl2 9 2HCl x 2H2O
N-(4-Chlorophenyl)-4-methylpiperazine-1-carboxamide
(4). To 4-methylpiperazine (0.25 g, 2.5 mmole) in 15 mL
of CH3CN was added 1-chloro-4-isocyanatobenzene
(0.38 g, 2.5 mmole) and refluxed for 8 h. The white precipi-
tate was filtered, dissolved in acetone and two drops of
conc. hydrochloric acid was added. The solid was filtered.
White crystals. Mp. 252 °C (dec). Yield: 40%. C12H16N3OCl
1
x HCl 9 0.25H2O (MW = 294.69). H-NMR [DMSO- d6]: d
1
(ppm) 8.98 (s, 1H, CO-NH), 7.52 (d, J = 8.7 Hz, 2H, Ph-
3,5-H), 7.30 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.32 (br s, 4H,
Pp-2,6-H), 2.76 (s, 3H, Pp-CH3), 2.50 (m, 4H, DMSO-
(MW = 493.30). H-NMR [DMSO-d6] d (ppm): 11.73 (s, 1H,
Pp-H+), 8.60 (t, 1H, J = 5.4 Hz, -CONH), 8,31 (s, 1H, Bzt-
7-H), 7.79 (d, 1H, J = 8.7 Hz, Bzt-5-H), 7.61 (dd, 1H,
J = 8.7 Hz J = 1.8 Hz, Bzt-4-H), 4.10-3.10 (m, 12 H, Pp-
2,3,5,6-H + Pp-CH2 + -CONH-CH2), 2.80 (s, 3H, CH3),
1.99 (br s, 2H, Pp-CH2-CH2). 13C NMR (DMSO-d6): d
(ppm) 160.5, 138.3, 135.3, 133.6, 132.8, 127.1, 124.5,
d6 + Pp-3,5-H). 13C-NMR [DMSO-d6]:
d (ppm) 154.8,
139.7, 128.6, 126.0, 121.5, 52.5, 42.4, 41.5. LC/MSÆ: pur-
ity (100%); tR = 3.04, (ESI) m/z [M+H]+ = 254.24.
5-Chloro-N-(3-(4-methylpiperazin-1-yl)propyl)-1H-indole-
2-carboxamide (5). 5-Chloroindole-2-carboxylic acid
(0.49 g, 2.5 mmol) and CDI (0.41 g, 2.5 mmol) in 3 mL of
absolute CH3CN was stirred at r.t. for 3 h. Then the mix-
ture was cooled to 00 C and 1-(3-Aminopropyl)-4-methyl-
piperazine (0.39 g, 2.5 mmol) in 2 mL of absolute CH3CN
was added dropwise and stirred at this temperature for
0.5 h and at r.t. for 72 h. The solid was filtered and crys-
tallized from CH3CN (with carbon). Light yellowish crystals.
123.5, 119.1, 50.1, 48.6, 37.3, 23.8. IR (KBr) [cmÀ1
]
v = 3502, 3455 (N-H), 1624 (C=O, amide), 1541 (N-H,
amide). LC/MSÆ: purity (100%); tR = 4.03, (ESI) m/z
[M]+ = 386.33.
1-(3-Chlorophenyl)-3-(3-(4-methylpiperazin-1-yl)propyl)
urea
(7). 1-chloro-3-isocyanatobenzene
(0.38 g,
2.5 mmol)
and 1-(3-aminopropyl)-4-methylpiperazine
(0.40 g, 2.5 mmol) in 5 mL of absolute CH3CN was
heated to reflux for 6 h. After evaporation to dryness the
residue was dissolved in CH2Cl2, purified by CC (eluent
CH2Cl2:MeOH:NH3 – 90:9:1) and after concentration crys-
tallized as salt of oxalic acid from EtOH/Et2O. White solid.
Mp. 165–168 °C. Yield: 46%. C15H23N4OCl 9 0.8C2H2O4
(MW = 382.86). 1H-NMR [DMSO-d6] d (ppm): 9.04 (s, 1H,
Ph-CO-NH), 7.66 (s, 1H, Ph-2-H), 7.20 (d, 2H,
J = 3.6 Hz, Ph-5,6-H), 6.85–6.92 (m, 1H, Ph-4-H), 6.65 (t,
1H, J = 5.4 Hz, NH-CO-NH), 3.08 (q, 2H, J = 6.4 Hz,
CO-NH-CH2), 2.70–2.80 (br s, 4H, Pp-2,6-H), 2.60–2.70
(br s, 4H, Pp-3,5-H), 2.49 (def t, 2H, Pp-CH2-CH2), 2.44
(s, 3H, Pp-CH3), 1,60 (qt, 2H, J = 6.9 Hz, Pp-CH2-CH2-
CH2). 13C NMR (DMSO-d6): d (ppm) 165.6, 155.6, 142.8,
133.5, 130.6, 117.3, 116.4, 55.0, 53.5, 51.2, 44.2, 37.6,
26.9. IR (KBr) [cmÀ1] v = 3288 (N-H), 1695 (C=O, amide),
1592 (N-H, amide). LC/MS: m/z (%) = 310.99 ([M.]+,100).
Anal. Calcd for C15H23N4OCl 9 0.8C2H2O4: C52.08;
H6.48; N14.63; Found: C52.02; H6.53, N14.76.
Mp.
182–185 °C.
Yield:
44%.
C17H23N4OCl
(MW = 334.85).1H-NMR [DMSO-d6] d (ppm): 11.75 (br s,
1H, indole-1-H), 8.54 (t, 1H, J = 5.6 Hz, CO-NH), 7.67 (d,
1H, J = 2.1 Hz, indole-4-H), 7.40 (d, 1H, J = 8.7 Hz,
indole-7-H), 7.15 (dd, 1H, J = 8.7 Hz, J = 2.1 Hz indole-
6-H), 7.06 (s, 1H, indole-3-H), 3.31 (m, 2H, CONH-CH2),
2.50–2.20 (m, 10H, Pp-CH2 + Pp-2,3,5,6-H), 2.12 (s, 3H,
Pp-CH3), 1.66 (qu, 2H, J = 6.9 Hz, Pp-CH2-CH2). 13C
NMR (DMSO-d6): d (ppm) 161.1, 135.2, 133.9, 128.6,
124.6, 123.7, 120.9, 114.3, 102.2, 56.1, 55.3, 37.9, 26.9.
IR (KBr) [cmÀ1] v = 3222 (N-H), 1645 (C=O, amide), 1550
(N-H, amide). LC/MSÆ: purity (100%); tR = 3.42, (ESI) m/z
[M+H]+ = 335.30. Anal. Calcd for C17H23N4OCl: C60.98;
H6.92; N16.73; Found: C61.23; H7.02; N16.90.
3,6-Dichloro-N-(3-(4-methylpiperazin-1-yl)propyl)benzo
[b]thiophene-2-carboxamide (6). 3,6-Dichlorobenzo[b]
thiophene-2-carbonyl chloride (6a)—Compound was
obtained according to the method described by Kaizerman
et al. (29).
Triazine derivatives
To 4-chlorocinnamic acid (2.56 g, 14 mmol) in 15 mL of
chlorobenzene, thionyl chloride (8.63 g, 720 mmol) and
slowly pyridine (0.14 g, 2 mmol) were added. The solution
was refluxed for 95 h, and n-hexane was added. The
All compounds were obtained as described previously by
_
Łazewska et al. (27). Compounds 8 and 11 are described
in lit. (27). Physical properties and spectrum data of all tri-
azine derivatives are included in Appendix S1.
4
Chem Biol Drug Des 2016

H4 Receptor Ligands – Synthesis and Interaction with PTEN
In vitro [3H]histamine binding assay on human H4R
Prior to the experiments, cell membranes were sedimented
by a 10 min centrifugation at 4 °C and 16 0009 g and re-
suspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA
and 75 mM Tris/HCl, pH 7.4). Competition binding experi-
ments were carried out by incubating membranes, 35 lg/
well (prepared from Sf9 cells transiently expressing human
H4R, co-expressed with G proteins Gai2 and Gb1ᵞ2 subunits)
in a final volume of 0.2 mL containing binding buffer and
[3H]histamine 29 HCl (10 nM, 15.3 Ci/mmol, PerkinElmer,
Waltham, MA, USA) in a 96 well microtitre plate. Assays
were run in duplicates or triplicates with four or seven appro-
priate concentrations between 100 nM or 0.1 nM and
100 lM of the test compounds. Amount of experiments was
at least 2. Only exceptions were for substance 2, 3, and 4
where only one experiment in duplicates was performed.
Incubations were performed for 60 min at 25 °C and shak-
ing at 250 rpm. Non-specific binding was determined in the
presence of 100 lM unlabeled JNJ7777120. Bound radioli-
gand was separated from free radioligand by filtration
through GF/B filters pretreated with 0.3% (mass/vol) poly-
ethyleneimine and washed three times with 5 mL of ice-cold
binding buffer (4 °C). The filters were soaked in a sample
bag with 9 mL scintillator liquid. The amount of radioactivity
collected on the filter was determined by liquid scintillation
counting. Competition binding data were analysed by the
software GRAPHPAD PRISM 3.02 (San Diego, CA, USA) using
non-linear least squares fit. Ki values were calculated from
the IC50 values according to the Cheng-Prusoff equa-
tion (30). Mean values of three independent experiments in
triplicate are stated in the Table 1, with the exception for
compounds 2, 3 and 4.
Electrophoretic mobility shift assay
DNA extraction
Isolation of genomic DNA from whole human blood was per-
formed using the Kit NucleoSpin Blood XL (MACHEREY-
NAGEL, Du¨ ren, Germany).
Promoter’s amplification
Specific primers were designed to amplify PTEN promoter.
The primers that were used for the amplification of each
promoter were as follows:
5΄-CAGAAAGACTTGAAGCGTAT-3΄ forward,
5΄-AACGGCTGAGGGAACCTC-3΄ reverse
primer for PTEN promoter. PCR reactions using genomic
DNA were performed on a MJ Research P200 thermal
cycler (MJ Research Inc., Waltham, MA, USA).
The PCR reactions for PTEN promoter were performed in
a mix containing 1.5 mM MgCl2, 1 unit per reaction of
DyNAzyme EXT DNA polymerase, 0.5 mM of each primer,
0.2 mM of each dNTP and approximately 30 pg of tem-
plate DNA in a total reaction volume of 20 lL. After an ini-
tial denaturing step for 3 min at 98 °C, amplification
comprised of 35 cycles of 30 seconds at 98 °C, 1, 30 min
at 50 °C and 5 min at 72 °C. Final elongation was for
7 min at 72 °C. PTEN promoter was amplified using
human genomic DNA as template that was isolated from
whole blood.
Scheme 1: Synthetic route of amides (1-3,
5, 6). Reagents and conditions: (i) benzene/
water/Na2CO3, r. t. 12 h; (ii) CH3CN, r.t 4 h,
reflux 20 h; (iii) carboxylic acid, CH3CN, CDI,
r.t. 3 h; amine added at 0 °C, 0.5 h at 0 °C,
r.t. 72 h.
Scheme 2: Synthetic route of ureas (4, 7).
Reagents and conditions: (i) CH3CN, reflux
6–8 h.
Chem Biol Drug Des 2016
5

Latacz et al.
Scheme 3: Synthetic route of 2,4,6-
trisubstituted 1,3,5-triazines (8-15). Reagents
and conditions: (i) BuOH, temperature
gradually increased from 50 to 90 °C during
1 h, 5 h reflux; (ii) MeONa, reflux from 15 to
30 h.
A
Band shift assay
Each compound, at a final concentration of 2 lM, was
incubated with an equal amount of PCR product at 24 °C
for 30 min. Subsequently, the mixtures were elec-
trophoresed on 2% w/v agarose gel.
Antiproliferative assay
Human embryonic kidney HEK-293 cell line (ATCC
€
CRLÀ1573) was kindly donated by Prof. Dr. Christa Muller
B
(Pharmaceutical Institute, Pharmaceutical Chemistry I,
University of Bonn). Neuroblastoma IMR-32 cell line was
provided by Department of Oncogenomics, Academisch
Medisch Centrum, Amsterdam, Holland (31,32). The cell
lines were seeded in 96-well flat-bottomed microtitre
plates at a concentration of 1.5 9 104 cells/well (HEK-
293) and 2 9 104 cells/well (IMR-32) and cultured in
200 lL of Dulbecco’s Modified Eagle’s Medium – DMEM
(Gibco, Gaithersburg, MD, USA) supplemented with 10%
fetal bovine serum (FBS), 100 mg/mL streptomycin and
100 U/mL penicillin at 37 °C in a humidified atmosphere
(5% CO2, 95% air) at 37 °C for 24 h to reach 60% conflu-
ence. The stock solution of H4R ligand in DMSO (25 mM)
was diluted into the fresh growth medium and added to
the wells at final concentrations 0.01–250 lM. Final DMSO
concentration did not exceed 1%. After 48 h of incubation,
the EZ4U (EZ4U Non-radioactive cell proliferation and
cytotoxicity assay, Biomedica, Vienna, Austria) labeling
mixture (20 lL) was added to each well and the cells were
incubated under the same conditions for 5 h. The absor-
bance of the samples was measured using a microplate
reader (EnSpire, PerkinElmer, Waltham, MA, USA) at
492 nm. The IC50 values of the reference compound dox-
orubicin (DX), used as a standard during this study,
against HEK-293 and IMR-32 cell lines were obtained and
calculated as we described previously (33).
C
D
Figure 1: Agarose gel electrophoresis (2% w/v) of PTEN
promoter, JNJ7777120, and compounds 1, 2, (A), PTEN
3
promoter and compounds 4, 5, 6, 7 (B), PTEN promoter and
compounds 8, 9,10 (C), PTEN promoter and compounds 11, 12,
13, 14, 15 (D).
Results and Discussion
Chemistry
amines (Scheme 1) giving amides 1-3, 6, whereas in the
second one amide (5) was produced from the carboxylic
acid and amine in the presence of CDI as an activator
Two different strategies for the synthesis of the novel
amides (1, 2, 3, 5, 6) were employed. In the first aminoly-
sis approach: acid chlorides reacted with the appropriate
6
Chem Biol Drug Des 2016

H4 Receptor Ligands – Synthesis and Interaction with PTEN
Table 2: The antiproliferative effect and the interaction of com-
pounds with PTEN promoter: not examined (NE), no (À), low (+),
medium (++), high (+++) effect
Commercially available isocyanates were refluxed in
CH3CN with the appropriate amines to give ureas 4 and 7
(Scheme 2).
Effect
2,4,6-Trisubstituted 1,3,5-triazines (8-15) were obtained as
the result of cyclization of appropriate esters with 4-
Anti-proliferative effect
PTEN promoter
interaction
methylpiperazin-1-yl
biguanide
dihydrochloride
as
Comp.
IMR-32
HEK-293
described previously (Scheme 3) (27). Biguanide dihy-
drochloride intermediate was obtained by the heating of
cyanoguanidine and 4-methylpiperazine dihydrochloride in
1-butanol (34). All acid methyl or ethyl esters were
commercially available (except 9E and 14E that were
synthesized according to described procedures (35,36).
The structures of all synthesized final compounds are
shown in Table 1 with their affinities at human H4R.
1
À
NE
NE
NE
À
NE
NE
NE
À
2
À
3
À
4
++
+++
5
+
+
a
6
À
++
À
++
À
7
+++
+
8
NE
NE
NE
À
NE
NE
NE
+
9
10
+
+
++
+
+
++
+
À
Histamine H4R screening
11
12
NE
NE
+
NE
NE
+
NE
+
As depicted in Table 1, all compounds showed moderate
to low human H4R affinities, except compound 1, 5, 6 and
7. In JNJ7777120 analogues series (1-7) the most potent
were compounds 2 (Ki = 3.88 lM) and 4 (Ki = 3.52 lM).
The introduction of a carbon chain (propyl linker) between
amide/urea moiety (5-7) caused the strongest decrease of
H4R affinities (Ki > 100 lM). In the triazine series (8-15) the
most potent was phenyl derivative with the para-bromo
substituent 11 with Ki of 0.52 lM. The change of the phe-
nyl moiety for a pyridine ring 15 or an aliphatic chain 10 as
13
14
15
JNJ7777120
NE
À
aDNA and 6 probably precipitated during the incubation process.
(Scheme 1). Non-commercially available acid chloride 6a
was synthesized according to the procedure described by
Kaizerman et al. (29).
Figure 2: The effect of compounds 6 and 11 on IMR-32 (left) and HEK-293 (right) cell lines viability. All data are included in Appendix S1.
Chem Biol Drug Des 2016
7

Latacz et al.
well as the introduction of the methylene linker between
triazine moiety and the substitutent (hydantoin 9, naphthyl
12, phenyl 13, or phenoxy 14) decreased the affinity
(Ki > 3 lM).
effect on normal HEK-293 cell line is an additional benefit
in terms of preliminary ADME-Tox parameters determina-
tion (Figure 2).
Conclusions
Electrophoretic mobility shift assay
The electrophoretic mobility shift assay (EMSA), also
known as band shift assay was applied to study potential
promoter’s PCR products – substances interactions. This
procedure determines if proteins or compounds are cap-
able of binding to a given DNA or RNA sequence (37).
Regarding PTEN promoter, among the group of
JNJ7777120 analogues, no effect for 1-3 was observed,
as well as JNJ7777120 also did not reduce PTEN pro-
moter mobility (Figure 1A). On the other hand, the highest
interaction among all tested compounds was observed for
other JNJ7777120 analogues 5 and 7 followed by 4, while
6 had no effect. However, the effect of 6 on PTEN pro-
moter was ambiguous due to its potential ability to induce
DNA precipitation during the incubation process (Fig-
ure 1B). Among 1,3,5-triazine derivatives the agarose gel
electrophoretic separation showed, that all compounds
exhibited the effect on PTEN promoter mobility, where the
11 and 14 derivatives reduced the mobility of the PTEN
PCR product to a greater extent than 8, 9, 10, 12, 13 and
15 (Figure 1C,D). All data were summarized in Table 2.
The high and medium interactions with PTEN gene pro-
moter were determined for five synthesized compounds –
three JNJ7777120 analogues (4, 5 and 7) and two 1,3,5-
triazine derivatives (11 and 14). The JNJ7777120 ana-
logues (5 and 7), with the weakest potency against H4R
among all synthesized compounds, showed the highest
interaction with the PTEN gene promoter. However, no
significant effect of these compounds on neuroblastoma
IMR-32 cell line proliferation and no correlation between
PTEN promoter interaction and antitumor activity were
shown. Compound 6 showed the highest antiproliferative
effect against tumor IMR-32 as well as normal HEK-293
cell lines. On the other hand, the similar IC50 values of 6
against tumor and normal cell lines excluded its potential
specific antitumor activity. Though, we conclude that fur-
ther study should be conducted to determine potential
inhibition or activation effect of the most interacting with
PTEN promoter JNJ7777120 analogues on PTEN protein
expression in several tumor cell lines. Almost all synthe-
sized and examined compounds showed moderate to low
human H4R affinities. Generally, the group of triazines
showed higher potency as H4R ligands in compare to the
urea/amide series designed as JNJ7777120 analogues.
Moreover, compound 11, the 1,3,5-triazine phenyl deriva-
tive with the para bromine substituent, showed the highest
affinity against H4R among all compounds and no cytotox-
icity, therefore may be considered as a promising lead
structure for further investigations.
The antiproliferative assay
The antiproliferative colorimetric assay was applied to
determine potentially correlation between interaction of
compounds with PTEN gene promoter and tumor activity
in reference to the cytostatic effect of doxorubicine (DX).
The influence of synthesized molecules on the viability of
normal HEK-293 cell line was also examined. To this study
were chosen compounds which reduced the mobility of
PTEN promoter with either medium (4, 11, 14) or strong
(5, 7) manner (Table 2). Additionally, the antiproliferative
effect of the compound 6 and JNJ7777120 was deter-
mined. The obtained data showed, that 6 reduced most
significantly cells viability among all compounds with calcu-
Acknowledgments
The authors acknowledge the partial support of ESF COST
Action BM0806 ‘Recent advances in histamine receptor
H4R research’, financed by EU-FP7, of Grant No. 594/N-
COST/2009/0, National Science Center, granted on the
basis of decision No DEC-2011/02/A/NZ4/00031, of the
European COST Actions CM1103, CM1207, Fraunhofer
IME, Projektgruppe Translationale Medizin und Phar-
makologie TMP (Fh-TMP), and DFG INST 206/664-1.
lated
IC50 = 23.27 lM
against
IMR-32
and
IC50 = 36.61 lM against HEK-293. However, 6 did not
possess potent antitumor activity compare with the refer-
ence drug DX (calculated DX IC50 = 0.000325 lM against
IMR-32, DX IC50 = 9.5 lM against HEK-293) (Figure 2).
Moreover, comparable IC50 values of 6 against normal as
well as tumor cells indicated no specific antitumor mecha-
nism of action. Interestingly, despite of their high interac-
tion with PTEN promoter the other examined JNJ7777120
analogues showed very low (5) or no significant (4, 7) anti-
tumor and antiproliferative activity. The examined com-
pounds from the triazine series, 11 and 14, showed
comparable, very low cytotoxic effect (IC50 ≥ 100 lM).
However, regarding 11, the derivative with the highest
affinity against H4R among all compounds presented in
that study, as the new lead structure, its low cytotoxic
Conflict of Interest
Declared none.
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Figure S2. The effect of compounds on cell lines viability.
10
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