In vitro antitumor activity evaluation of some 1,2,4-triazine derivatives bearing piperazine amide moiety against breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2014.10.002

A series of 1,2,4-triazine derivatives bearing piperazine amide moiety has been synthesized and investigated for their potential anticancer activities. 1-[4-(5,6-Bis(4-subtituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-(3-substituted phenyl)pipera

Full text of DOI:10.1016/j.bmc.2014.10.002

Bioorganic & Medicinal Chemistry 22 (2014) 6313–6323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vitro antitumor activity evaluation of some 1,2,4-triazine
derivatives bearing piperazine amide moiety against breast cancer
cells
a,
⇑
b
c
c
Leyla Yurtta ßs , Sß eref Demirayak , Sinem Ilgın , Özlem Atlı
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey
Department of Pharmaceutical Chemistry, School of Pharmacy, Medipol University, 34083 Istanbul, Turkey
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,2,4-triazine derivatives bearing piperazine amide moiety has been synthesized and investi-
gated for their potential anticancer activities. 1-[4-(5,6-Bis(4-subtituted phenyl)-1,2,4-triazin-3-yl)pip-
erazin-1-yl]-2-[4-(3-substituted phenyl)piperazin-1-yl]ethanone derivative (1–32) compounds were
synthesized by a four step synthetic procedure. The activity studies were evaluated using XTT method,
BrdU method and flow cytometric analysis on MCF-7 breast cancer cells and NIH/3T3 (mouse embryonic
fibroblast cells) healthy cells. Compounds 5 with 3-chlorophenyl and compound 7 with 4-chlorophenyl
substitutions were found to be promising antiproliferative agents comparing with an effective anticancer
drug, cisplatin.
Received 5 August 2014
Revised 26 September 2014
Accepted 2 October 2014
Available online 13 October 2014
Keywords:
1
,2,4-Triazine
Piperazine amide
Breast cancer
Ó 2014 Elsevier Ltd. All rights reserved.
Antiproliferative activity
Apoptosis
XTT method
1
. Introduction
Breast cancer is one of the most common cancer in females all
components represent isosteres with the C-6 carbon atom of the
pyrimidine nucleoside base being replaced by nitrogen.7 Certain
azanucleosides, 6-azauracil and 6-azacytosine), structurally based
on 1,2,4-triazine nucleus have displayed an impressive array of
,8
over the world. According to the latest data observed in United
Kingdom, it was shown that breast cancer is the most common
cancer in women (31%).1 Additionally, according to the data
obtained from United States in 2012, it was suggested that breast
9
–19
23
20
biological activities, among which antitumor,
antiviral, anti-
21
22
24
microbial,
antiinflammatory,
antiplatelet,
antimalarial,
2
5
and antifungal properties. Additionally, tertiary amines in the
linkers reported to be positively charged at physiological pH in
order to favor electrostatic interactions in the minor groove with
2
cancer occurred in 29% of newly diagnosed cancers in women.
Treatments against breast cancer are surgery, drugs (hormonal
therapy and chemotherapy), radiation and/or immunotherapy.
However, an effective drug for therapy and prognosis after surgery
still do not exist. Therefore an effective new drug development
process is seemed to be necessary.
2
6
the anionic phosphate DNA backbone.
On the other hand, a comprehensive literature study shows that
two carbon linker piperazines have potential antiproliferative
3
2
7,28
effect.
Additionally, structure–activity relationships (SARs) of a
One important approach to antineoplastic agents is the design
of an antimetabolite drug whose structure is related those of
pyrimidines and purines involved in the biosynthesis of DNA. Anti-
metabolites often similar in structure to the metabolite that they
interfere with DNA synthesis and consequently block crucial met-
abolic pathways essential for cell growth.4 Various nucleosides
containing interchanged nitrogen and carbon atoms in their base
moieties have shown considerable activity as antimetabolic agents.
farnesyltransferase inhibitor anticancer drug lonafarnib (SCH
66336) have been explored and several studies have established that
piperazine is a suitable replacement for the piperidine core, produc-
ing compounds with comparable potency and pharmacokinetic (PK)
profiles. In fact, compounds including
structure improves therapeutic level of these compounds.
a-alanine piperazine amide
–6
29
Based on the literature above, we designed a novel series of poly-
amines existing a ‘Y shape’ pharmacophore model that contains
three basic center, three hydrophobic domains, and an amide linker
and synthesized thirty two 1-[4-(5,6-bis(4-subtituted phenyl)-1,2,4-
triazin-3-yl)piperazin-1-yl]-2-[4-(3-substituted phenyl)piperazin-
6
-Aza analogues of the naturally occurring nucleic acids
⇑
Corresponding author. Tel.: +90 222 3350580/3783; fax: +90 222 3350750.
E-mail address: lyurttas@anadolu.edu.tr (L. Yurtta ßs ).
1-yl]ethanone derivatives by adopting the bioisosteric-replacement
http://dx.doi.org/10.1016/j.bmc.2014.10.002
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

6
314
L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
principle. Cytotoxic activities of the titled compounds were evalu-
ated against human breast adenocarcinoma cell line, MCF-7 and
mouse embryonic fibroblast cell line, NIH3T3. To determine poten-
tial anticancer activity, DNA synthesis inhibition abilities and apop-
totic properties of the indicated compounds were also specified and
reported within the study.
chlorine atom were observed at about 3.49–3.50 ppm. In the spec-
tra of the final compounds (1–32), same protons were seen much
further to low-field and at about 3.28–3.42 ppm. Piperazine CH
2
protons were assigned in between 2.59–4.01 ppm as broad singlets
and two or four overlapping protons. Aromatic protons were
observed at the range of 6.34–8.08 ppm, changing to substituents
1
3
on phenyl rings. In the C NMR spectra of the final compounds,
signals belonging to aliphatic carbon atoms were assigned at about
2
2
. Results and discussion
2
0.70–60.76 ppm; and signals for aromatic carbon atoms were
observed at 101.36–162.27 ppm. The carbonyl carbon of amide
function was seen at about 167 ppm. In mass spectra of the com-
.1. Chemistry
+
pounds, [M+1] peaks were observed for all final compounds and
Compounds Ia–Id were synthesized by the condensation
also elemental analyses results for C, H, and N elements were sat-
isfactory within calculated values of the compounds.
reaction of benzil derivatives and S-methylthiosemicarbazide
hydrogen iodide with sodium carbonate in methanol to give 5,6-
bis(4-substituted phenyl)-3-methylthio-1,2,4-triazine (Scheme 1).
The obtained 3-methylthio derivatives of 1,2,4-triazines were
reacted with twofold excess piperazine in pyridine to give
2
2
2
.2. Biological evaluation
.2.1. Cytotoxicity test
According to results, the compounds 2, 3, 5, 7, 10, 13, 14, 15, 23,
6, 27 and 28 showed antiproliferative activities against MCF-7 cell
5,6-bis(4-substituted phenyl)-3-(piperazin-1-yl)-l,2,4-triazine (IIa–
IId) derivatives. The amino group of the piperazine ring was acet-
ylated with chloroacetyl chloride in dimethylformamide and basic
medium was provided by triethylamine to gain intermediate prod-
ucts IIIa–IIId. Lastly, final compounds 1–32 were obtained by
the reaction of 2-chloro-1-[4-(5,6-bis(4-substituted phenyl)-
line. Compounds 2, 5, 7, 10, 14 and 28 were found to have the high-
est cytotoxic activity against MCF-7 cell line. The IC50 values of the
compounds are represented in Table 1.
Furthermore, the cytotoxicity of the compounds 2, 5, 7, 10, 14
and 28, which were determined to have the highest selective
cytotoxic activity against MCF-7 cell line, were measured against
NIH/3T3 cell line. According to the results of the XTT test against
NIH/3T3 cell line, the compounds 2, 5, 7, 10, 14 and 28 showed
cytotoxicity against NIH/3T3 cell line in higher doses than their
antiproliferative doses against MCF-7 cell line. The IC50 values
of the compounds against NIH/3T3 cell line are represented in
Table 2.
1
,2,4-triazin-3-yl)piperazin-1-yl]ethanone derivatives (IIIa–IIId)
and 1-substituted piperazine derivatives in triethylamine and
dimethylformamide by refluxing and yielding 66–83%. All final
compounds were characterized by IR, H NMR, C NMR elemental
analyses and mass spectra. In IR spectrum of the compounds,
1
13
ꢀ1
bands which were seen in between 1645–1669 cm and 1296–
ꢀ1
1
612 cm were confirmed the presence of amide C@O and also
1
C@C, C@N bonds, respectively. In H NMR of the intermediate com-
pounds IIIa–IIId, signals belonging to COCH
2
protons vicinal to
R
R
O
O
H N
2
N
NH
i
N
+
HI
HN
SCH3
N
SCH3
Ia-Id
R
R
3 3
R=H, CH , OCH , Cl
R
ii
N
N
Ia-Id
+
HN
NH
N
N
NH
IIa-IId
R
R
iii
N
N
IIa-IId
2
+ ClCOCH Cl
Cl
N
N
N
O
R
IIIa-IIId
iv
IIIa-IIId
+
HN
N
1-32
R'
R
N
R'=H, 2-CH , 3-CH , 4-CH ,
3
3
3
N
N
N
2
-OCH , 3-OCH , 4-OCH ,
3
3
3
R'
N
N
2-Cl, 3-Cl, 4-Cl
N
O
R
1
-32
Scheme 1. The synthesis of the compounds. Reactants and conditions: (i) NaHCO
h.
3
, CH
3
OH, reflux 3 h; (ii) pyridine, reflux 6 h; (iii) Et N, DMF, rt, 45 min; (iv) Et N, DMF, rt,
3 3
5

L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
6315
Table 1
Cytotoxic activity of the compounds against MCF-7 cell line (
l
g/mL and
lM)
Compound
IC50
(l
g/mL)
IC50
(l
M)
Compound
IC50
(lg/mL)
IC50 (lM)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
>500
>963.4
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
>500
>500
>500
>500
>500
>500
372.7 ± 6.8
>500
>500
203.7 ± 3.9
246.4 ± 3.7
91.9 ± 4.2
>500
>500
>500
>863.6
>821.0
>814.3
>821.0
>814.3
>821.0
607.0 ± 11.0
>801.2
>850.3
329.6 ± 6.3
395.8 ± 10.1
148.7 ± 6.8
>803.2
>809.0
>803.2
91.7 ± 3.4
145.9 ± 2.7
>500
31.2 ± 4.5
>500
31.2 ± 3.8
>500
500 ± 6.5
34.3 ± 2.2
>500
167.0 ± 6.2
263.3 ± 4.9
>910.7
56.3 ± 8.1
>910.7
56.3 ± 6.9
>886.5
914 ± 11.9
59.4 ± 4.4
859.1
0
1
2
3
4
5
6
>500
866.6
463.8 ± 3.5
30.6 ± 4.0
384.2 ± 4.1
>500
796.9 ± 6.0
53.0 ± 6.9
659.8 ± 7.0
844.6
>500
>789.9
Cisplatin
29.3 ± 2.5
97.7 ± 8.3
The effects of compounds 2, 5, 7, 10, 14 and 28 were evaluated
on DNA synthesis and apoptosis because they were determined to
be the most active derivatives against MCF cell line.
2
.2.2. DNA synthesis inhibition assay
Figure 1 shows the DNA synthesis inhibitory activity % of the
compounds 2, 5, 7, 10, 14, 28 and cisplatin against MCF-7 cells
for 24 h. According to the assay, compounds 2 and 14 were deter-
mined as the most active compounds. Compound 14 was found to
have 56.94, 77.82 and 88.52% DNA synthesis inhibition, respec-
tively compound 2 was found to have 22.44, 76.35 and 81.61%
DNA synthesis inhibition at IC50/2, IC50 and 2 ꢁ IC50 values after
2
4 h of incubation, respectively whereas cisplatin was found to
have 59.69, 65.96 and 76.53% inhibition at IC50/2, IC50 and
ꢁ IC50 values, respectively. According to these results, it was
2
observed that compounds 2 and 14 showed more antiproliferative
activity against MCF-7 cells than cisplatin at IC50 and 2 ꢁ IC50 val-
ues. Compound 5 was found to have 26.06, 31.36 and 41.48% DNA
synthesis inhibition at IC50/2, IC50 and 2 ꢁ IC50 values, respectively.
Additionally, it was determined that compounds 7 and 28 showed
less antiproliferative activity than other compounds against the
MCF-7 cell line.
Figure 1. DNA synthesis inhibitory activity % of the compounds 2, 5, 7, 10, 14, 28
and cisplatin against MCF-7 cells for 24 h incubation period. Mean percent
absorbance of untreated control cells were assumed to be 0% and three different
concentrations (IC50/2, IC50, 2 ꢁ IC50) of test compounds and cisplatin were given.
Data points represent means for two independent experiments ± SD of four
independent wells. p <0.05.
The DNA synthesis inhibitory activity % of the compounds 2, 5,
7
, 10, 14, 28 and cisplatin against MCF-7 cells is shown in Figure 2
for 48 h. DNA inhibition % was increased with 48 h incubation per-
iod for all compounds. These increases are obviously significant for
the compounds 5, 7 and cisplatin. It was observed that compounds
2 and 14 showed the highest DNA synthesis inhibitory activities on
Table 2
Cytotoxic activity of the compounds against NIH/3T3 cell line (
l
g/mL and
M)
>963.4
443.5 ± 10.2
349.6 ± 5.1
361.2 ± 8.7
177.8 ± 15.7
>910.7
lM)
Compound
IC50
(l
g/mL)
IC50
(l
Compound
IC50
(
lg/mL)
IC50 (lM)
1
2
3
4
5
6
7
8
9
>500
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
>500
>500
>500
>500
>500
>500
>500
>500
>500
250 ± 4.2
>500
125.0 ± 4.8
>500
>863.5
>821.0
>814.3
>821.0
>814.3
>821.0
>814.3
>801.3
>850.3
404.5 ± 8.4
>803.2
202.3 ± 7.8
>803.2
243.5 ± 5.6
193.7 ± 2.8
198.3 ± 4.8
98.5 ± 4.7
>500
62.5 ± 4.9
>500
>500
112.8 ± 8.8
>886.5
>914.1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
76.9 ± 6.5
>500
>500
500 ± 6.4
66.8 ± 3.3
330.1 ± 5.5
>500
133.3 ± 11.3
>859.1
>866.5
859.1 ± 11.0
115.8 ± 5.7
567.2 ± 9.5
>844.6
>500
>500
>500
>809.1
>803.2
>789.9
Cisplatin
388.2 ± 4.7
1294.0 ± 15.7

6
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L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
MCF-7 cells. Otherwise, cisplatin showed the most inhibitory activ-
ity with 93.31% at a concentration of 62.4 g/mL. Compound 10
showed 78.50% and 78.71% inhibitory activity after 48 h incubation
period, at concentrations of 22.09 and 44.18 g/mL, respectively
whereas compound 14 showed 76.73%, 85.99% and 88.91% inhibi-
tions at concentrations of 15.32, 30.63 and 61.26 g/mL, respec-
tively. Compound 5 was found to have 56.59%, 61.16% and
0.18% DNA synthesis inhibition at IC50/2, IC50 and 2 ꢁ IC50 values,
respectively whereas compound 7 was found to have 24.98%,
2.05% and 62.34% DNA synthesis inhibition after 48 h incubation
l
l
l
7
5
period at IC50/2, IC50 and 2 ꢁ IC50 values, respectively. Further-
more, it was determined that compound 28 showed less antiprolif-
erative activity than other compounds against the MCF-7 cell line.
2
.2.3. Induction of apoptosis
FITC Annexin V staining is a technique to show the loss of mem-
brane integrity which accompanies the latest stages of cell death
resulting from either apoptotic or necrotic processes. Hence,
Annexin V-FITC staining is typically accompanied by a vital dye
staining such as PI to identify the formation of apoptotic cells in
the early stages (Annexin V-FITC positive, PI negative).Viable cells
with intact membranes exclude PI, whereas the membranes of dead
Figure 2. DNA synthesis inhibitory activity % of the compounds 2, 5, 7, 10, 14, 28
and cisplatin against MCF-7 cells for 48 h incubation period. Mean percent
absorbance of untreated control cells were assumed to be 0% and three different
concentrations (IC50/2, IC50, 2 ꢁ IC50) of test compounds and cisplatin were given.
Data points represent means for two independent experiments ± SD of four
independent wells. p <0.05.
Figure 3. The flow cytometric analysis diagram of compound 2, 5, 7, 10, 14, 28 and cisplatin for MCF-7 cell line. Annexin V-PI analysis in MCF-7 cells, following 24 h
incubation period of compounds 2, 5, 7, 10, 14, 28 and cisplatin by Annexin-V-FITC method at IC50 concentrations, which are 91.71, 31.20, 31.20, 34.27, 30.63, 91.85 and
2
9.31
and 2.2% for compound 5; 11.2, 10.3, 76.4 and 2.1% for compound 7; 28.2, 5.8, 63.4 and 2.6% for compound 10; 26.7, 6.2, 66.5 and 0.6% for compound 14; 24.5, 3.3, 71.3 and
.9% for compound 28; 7.0, 9.1, 80.9 and 3.0% for compound cisplatin, respectively.
lg/mL, respectively. Percentages of Q1, Q2, Q3, and Q4 are measured as 3.66, 1.52, 94.36, and 0.46% for control; 17.1, 0.4, 82.5 and 0% for compound 2; 11.5, 9.4, 76.9
0

L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
6317
and damaged cells are permeable to PI. After this procedure, cells
can be classified from FITC Annexin V and PI negative (viable, or
no measurable apoptosis), to FITC Annexin V positive and PI nega-
tive (early apoptosis, membrane integrity is present) and finally
(Merck KGaA, Darmstadt, Germany). All melting points (mp) were
determined by Electrothermal 9100 digital melting point appara-
tus (Electrothermal, Essex, UK) and are uncorrected. All the reac-
tions were monitored by thin-layer chromatography (TLC) using
Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany).
Spectroscopic data were recorded with the following instruments:
IR, Shimadzu 8400S spectrophotometer (Shimadzu, Tokyo, Japan);
3
0
to FITC Annexin V and PI positive (end stage apoptosis and death).
In Figure 3, the flow cytometric analysis diagram is showed for
MCF-7 cell line. Compound 7 showed the highest population of
apoptotic cells (12.3%) whereas cisplatin showed 12.1% apoptotic
cells. Compound 5 produced a comparable population of apoptotic
cells with a percentage of 11.6% according to cisplatin’s percentage.
However compounds 2, 10, 14 and 28 provoked necrotic induction
in MCF-7 cells after 24 h treatment.
1
H NMR, Bruker DPX 500 NMR spectrometer (Bruker Bioscience,
1
3
Billerica, MA, USA); C NMR, VARIAN Mercury 100 FT spectrome-
ter (Varian Inc, Palo Alto, CA, USA) in DMSO-d , using TMS as inter-
6
nal standard; M+1 peaks were determined by AB Sciex-3200
Q-TRAP LC/MS/MS system (AB Applied Biosystems Co., MA, USA).
Elemental analyses were performed on a Leco TruSpec Micro
CHN/CHNS elemental analyzer (Leco, Michigan, USA).
3
. Conclusion/discussion
4
.2. General procedure for the synthesis of 5,6-bis(4-substituted
One of the important criteria for an anticancer agent, is to show
phenyl)-3-methylthio-l,2,4-triazine (Ia–Id)
minimum or no side-effects on healthy cells of the patients receiv-
ing chemotherapy. We evaluated the antiproliferative potential of
newly synthesized compounds on human estrogen-dependent
MCF-7 human breast adenocarcinoma cell line. In addition, the
cytotoxic activities of these compounds were also evaluated
against normal mouse embryonic fibroblast cell line, NIH/3T3, for
determining the selectivity of potential anticancer agents. Signifi-
cant differences in IC50 values of the compounds 2, 5, 7, 10, 14
and 28 were observed between breast carcinoma and normal cell
lines.
0
Equimolar quantities of 4,4 -substituted benzil (0.3), S-methyl-
thiosemicarbazide hydrogen iodide (0.3 mol) and sodium bicar-
bonate (0.3 mol) were refluxed in methanol (600 mL) for 3 h.
After cooling, the mixture was filtrated to obtain raw product.
The obtained product was washed with water and ethanol then
3
1
crystallized from ethanol.
4
.3. General procedure for the synthesis of 5,6-bis(4-substituted
phenyl)-3-(piperazin-1-yl)-l,2,4-triazine (IIa–IId)
Structures of the final compounds differ from each other due to
substituents on phenyl rings and belong to four main structures
including 5,6-diphenyl-1,2,4-triazine (1–8); 5,6-bis(4-methyl-
phenyl)-1,2,4-triazine (9–15); 5,6-bis(4-methoxyphenyl)-1,2,4-tri-
azine (16–23) and 5,6-bis(4-chlorophenyl)-1,2,4-triazine (24–32).
Considering the substituent effect on cytotoxic activity, com-
pounds 5 and 7 bearing 3- and 4-chloro phenyl piperazines has
attracted attention with higher activities. Also compounds 10
and 14 containing 2- and 4-methoxy phenyl piperazine moiety
had remarkable influence on the cytotoxic activities. Compounds
A mixture of 5,6-bis(4-substituted phenyl)-3-methylthio-l,2,4-
triazine (Ia–Id) derivative (0.2 mol) and piperazine (0.4 mol) in
pyridine (400 mL) was heated at reflux for 6 h until there was no
odor of mercaptan. After cooling, the mixture was treated with
water and the separated precipitate was collected by filtration,
washed with water, and dried to provide product. Recrystallization
3
2
from ethanol was afforded for purification.
0
0
4.4. General procedure for the synthesis of 2-chloro-1-[4-(5,6-
bis(4-substituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]
ethanone (IIIa–IIId)
synthesized from 4,4 -dimethoxybenzil (16–23) and 4,4 -dichloro-
benzil (24–32) did not show prominent activity. Additionally, elec-
tron withdrawing substituent nitro on phenyl ring produced
inactive compounds (8, 16, 24 and 32).
Chloroacetyl chloride (0.035 mol) in 50 mL dimethylformamide
was added drop wise over 45 minutes to a magnetically stirred
solution of compound IIa–IId (0.03 mol) and triethylamine
According to the DNA synthesis inhibition studies, compounds 2,
, 7, 10 and 14 inhibited DNA synthesis against MCF-7 cell line and
5
there was a time dependent increase of inhibition ratios, especially
in compounds 5 and 7. Otherwise, the compounds 5 and 7 were
determined that they affected breast cancer cell by the apoptotic
pathway and compounds 2, 10 and 14 by the necrotic pathway.
Apoptosis, or programmed cell death, plays an essential role in con-
trolling cell death. Our results illustrated that compounds 5 and 7
exhibits significant antiproliferative effect on MCF-7 human breast
cells concomitant with the induction of apoptosis.
Our present in vitro studies demonstrate the antiproliferative
effects of newly synthesized compounds 5 and 7 against MCF-7
and NIH/3T3 cell lines. Comparing compounds on the basis of bio-
logical activity, it was revealed that the molecules with -meta and
(
0.035 mol) in dimethylformamide (300 mL). After completion of
reaction, the solvent was evaporated to dryness under reduced
pressure. Water was added to wash the resulting solid and the
mixture was filtered, dried and recrystallized from ethanol to give
compounds IIIa–IIId.
4
1
.4.1. 2-Chloro-1-[4-(5,6-diphenyl-1,2,4-triazin-3-yl)piperazin-
-yl]ethanone (IIIa)
Yield 76–79%, mp 205–207 °C. IR (KBr, cmꢀ1):
m
maks 3031 (aro-
matic C–H), 2887 (aliphatic C–H), 1668 (C@O), 1556–1323 (C@C
1
and C@N), 1235–1012 (C–N).
ppm): d 3.67 (t, 4H, J: 5.0 Hz, piperazine-H), 3.95 (br s, 2H, pipera-
zine-H), 4.0 (br s, 2H, piperazine-H), 4.49 (s, 2H, –CO–CH ), 7.36–
.7.39 (m, 7H, Ar-H), 7.44–7.48 (m, 3H, Ar-H). MS [M+1] : m/z 394.
6
H NMR (500 MHz, DMSO-d ,
-
para chloro substituted phenyl ring displayed the highest activity.
Further studies are required to determine the anticancer activity of
the compounds 5 and 7 in animal models. Besides, it is thought to
design new compounds with -meta and -para chloro aryl substitu-
tion, improving synthetic and activity screening methods.
2
+
7
4
.4.2. 2-Chloro-1-[4-(5,6-bis(4-methylphenyl)-1,2,4-triazin-3-yl)
piperazin-1-yl]ethanone (IIIb)
4
4
. Experimental section
Yield 75–77%, mp 198–201 °C. IR (KBr, cmꢀ1):
mmaks 3025 (aro-
matic C–H), 2879 (aliphatic C–H), 1669 (C@O), 1569–1333 (C@C
1
.1. General
and C@N), 1237–1014 (C–N).
ppm): d 2.33 and 2.33 (2s, 6H, CH
H
NMR (500 MHz, DMSO-d
6
,
3
), 3.66 (t, 4H, J: 5.0 Hz, pipera-
All chemicals were purchased from Sigma–Aldrich Chemical Co
Sigma–Aldrich Corp., St. Louis, MO, USA) and Merck Chemicals
zine-H), 3.93 (br s, 2H, piperazine-H), 3.98 (br s, 2H, piperazine-
H), 4.49 (s, 2H, –CO–CH ), 7.18 (d, 4H, J: 8.25 Hz, Ar-H), 7.27 (d,
(
2

6
318
L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
+
2
4
H, J: 8.0 Hz, Ar-H), 7.38 (d, 2H, 2H, J: 8.0 Hz, Ar-H). MS [M+1] : m/z
22.
35 7 2
167.70. For C32H N O calculated: 69.92% C, 6.42% H, 17.84% N;
found: 69.91% C, 6.48% H, 17.80% N. MS [M+1] : m/z 550.
+
4
.4.3. 2-Chloro-1-[4-(5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-
4.5.3. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
yl)piperazin-1-yl]ethanone (IIIc)
(2-chlorophenyl)piperazin-1-yl)]ethanone (3)
Yield 81–83%, mp 166–169 °C. IR (KBr, cmꢀ1):
m
maks 3042 (aro-
Yield 69–73%, mp 225–226 °C. IR (KBr, cm ):
ꢀ1
m
maks 3016 (aro-
matic C–H), 2913 (aliphatic C–H), 1671 (C@O), 1571–1315 (C@C
matic C–H), 2842 (aliphatic C–H), 1662 (C@O), 1587–1319 (C@C
1
1
and C@N), 1243–1015 (C–N). H NMR (500 MHz, DMSO-d
ppm): d 3.66 (br s, 4H, piperazine-H), 3.78 (s, 6H, OCH ), 3.92 (br
s, 2H, piperazine-H), 3.97 (br s, 2H, piperazine-H), 4.50 (s, 2H,
6
,
and C@N), 1235–976 (C–N). H NMR (500 MHz, DMSO-d
d 2.65 (br s, 4H, piperazine-H), 3.02 (br s, 4H, piperazine-H), 3.39
(s, 2H, –CO–CH ), 3.67 (br s, 2H, piperazine-H), 3.78 (br s, 2H,
6
, ppm):
3
2
–
7
CO–CH
2
), 6.93–6.96 (m, 4H, Ar-H), 7.33 (d, 2H, J: 8.0 Hz, Ar-H),
piperazine-H), 3.94 (br s, 2H, piperazine-H), 4.01 (br s, 2H, pipera-
zine-H), 7.16 (t, 1H, J: 7.5 Hz, Ar-H), 7.18 (d, 1H, J: 8.0 Hz, Ar-H),
+
.47 (d, 2H, J: 8.5 Hz, Ar-H). MS [M+1] : m/z 454.
7
.29 (t, 1H, J: 7.5 Hz, Ar-H), 7.37–7.39 (m, 7H, Ar-H), 7.42 (d, 1H,
1
3
4
.4.4. 2-Chloro-1-[4-(5,6-bis(4-chlorophenyl)-1,2,4-triazin-3-yl)
J: 8.0 Hz, Ar-H), 7.44–7.49 (m, 3H, Ar-H). C NMR (100 MHz,
DMSO-d , ppm) d 40.87, 43.08, 43.70, 44.75, 50.82, 52.57, 60.56,
piperazin-1-yl]ethanone (IIId)
6
Yield 82–84%, mp 176–181 °C. IR (KBr, cmꢀ1):
m
maks 3024 (aro-
120.81, 123.76, 127.54, 127.94, 128.16, 128.19, 128.83, 129.33,
130.14, 130.21, 136.05, 136.14, 148.45, 148.90, 155.31, 159.22,
matic C–H), 2876 (aliphatic C–H), 1667 (C@O), 1572–1328 (C@C
1
and C@N), 1237–1014 (C–N). H NMR (500 MHz, DMSO-d
ppm): d 3.67 (t, 4H, J: 5.0 Hz, piperazine-H), 3.95 (br s, 2H, pipera-
zine-H), 4.0 (br s, 2H, piperazine-H), 4.49 (s, 2H, –CO–CH ), 7.40–
.42 (m, 2H, Ar-H), 7.46–7.48 (m, 2H, Ar-H), 7.50 (s, 4H, Ar-H).
6
,
7
167.67. For C31H32ClN O calculated: 67.20% C, 5.82% H, 17.70% N;
+
found: 67.25% C, 5.96% H, 17.81% N. MS [M+1] : m/z 554.
2
7
4.5.4. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
(3-methoxyphenyl)piperazin-1-yl)]ethanone (4)
+
MS [M+1] : m/z 462.
ꢀ
1
Yield 66–69%, mp 126–128 °C. IR (KBr, cm ):
mmaks 3024 (aro-
4
.5. General procedure for the synthesis of 1-[4-(5,6-bis(4-
matic C–H), 2836 (aliphatic C–H), 1661 (C@O), 1581–1321 (C@C
1
subtituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-(3-
substituted phenyl)piperazin-1-yl]ethanone derivatives (1–32)
and C@N), 1235–976 (C–N). H NMR (500 MHz, DMSO-d
d 2.61 (br s, 4H, piperazine-H), 3.16 (br s, 4H, piperazine-H), 3.38
s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.71 (s, 3H,
OCH ), 3.77 (br s, 2H, piperazine-H), 3.94 (br s, 2H, piperazine-
6
, ppm):
(
2
2
-Chloro-1-[4-(5,6-bis(4-substituted phenyl)-1,2,4-triazin-3-
yl)piperazin-1-yl]ethanone derivative (IIIa–IIId) (1.5 mmol) and
-substituted piperazine derivative (1.6 mmol) were stirred in
3
H), 3.99 (br s, 2H, piperazine-H), 6.38 (d, 1H, J: 8.0 Hz, Ar-H),
6.45 (br s, 1H, Ar-H), 6.53 (d, 1H, J: 8.0 Hz, Ar-H), 7.10 (t, 1H, J:
8.0 Hz, Ar-H), 7.36–7.37 (m, 7H, Ar-H), 7.43–7.47 (m, 3H, Ar-H).
1
dimethylformamide with the presence of triethylamine (1.6 mmol)
for 5 h. After duration of reaction, the solvent was evaporated and
the residue was reacted with water. Filtrated product was crystal-
lized, after drying.
1
3
6
C NMR (100 MHz, DMSO-d , ppm) d 40.86, 43.08, 43.69, 44.73,
48.18, 52.38, 54.78, 60.56, 101.44, 104.14, 108.0, 128.14, 128.18,
128.82, 129.32, 129.47, 130.11, 136.04, 136.14, 148.44, 152.29,
1
55.29, 159.22, 160.13, 167.62. For
C
32
H
35
N
7
O
2
calculated:
4
.5.1. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-(4-
69.92% C, 6.42% H, 17.84% N; found: 69.46% C, 6.44% H, 17.85% N.
MS [M+1] : m/z 550.
+
phenylpiperazin-1-yl)ethanone (1)
Yield 70–76%, mp 153–154 °C. IR (KBr, cmꢀ1):
mmaks 3024 (aro-
matic C–H), 2836 (aliphatic C–H), 1664 (C@O), 1587–1316 (C@C
and C@N), 1237–976 (C–N). 1H NMR (500 MHz, DMSO-d
, ppm):
d 2.64 (br s, 4H, piperazine-H), 3.17 (br s, 4H, piperazine-H), 3.42
s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.77 (br s, 2H,
piperazine-H), 3.94 (br s, 2H, piperazine-H), 3.99 (br s, 2H, pipera-
zine-H), 6.78 (t, 1H, J: 8.0 Hz, Ar-H), 6.94 (d, 2H, J: 8.0 Hz, Ar-H),
4.5.5. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
6
(3-chlorophenyl)piperazin-1-yl)]ethanone (5)
Yield 66–69%, mp 149–150 °C. IR (KBr, cm 1):
ꢀ
mmaks 3026 (aro-
(
2
matic C–H), 2825 (aliphatic C–H), 1659 (C@O), 1593–1325 (C@C
1
and C@N), 1240–977 (C–N). H NMR (500 MHz, DMSO-d
d 2.60 (br s, 4H, piperazine-H), 3.20 (br s, 4H, piperazine-H), 3.31
(s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.77 (br s, 2H,
6
, ppm):
7
.21 (t, 2H, J: 7.5 Hz, Ar-H), 7.36–7.38 (m, 7H, Ar-H), 7.43–7.47
2
1
3
(
m, 3H, Ar-H).
C NMR (100 MHz, DMSO-d
6
, ppm) d 40.84,
piperazine-H), 3.93 (br s, 2H, piperazine-H), 3.99 (br s, 2H, pipera-
zine-H), 6.78 (d, 1H, J: 8.0 Hz, Ar-H), 6.90 (d, 1H, J: 8.5 Hz, Ar-H),
6.95 (br s, 1H, Ar-H), 7.21 (t, 1H, J: 8.0 Hz, Ar-H), 7.34–7.37 (m,
4
1
1
3.05, 43.66, 44.73, 48.17, 52.40, 55.92, 60.55, 115.32, 118.74,
28.16, 128.19, 128.80, 128.81, 129.33, 130.14, 148.42, 150.89,
1
3
55.31, 159.18, 162.20, 167.59. For C31
H
33
N
7
O calculated: 71.65%
7H, Ar-H), 7.43–7.47 (m, 3H, Ar-H). C NMR (100 MHz, DMSO-
, ppm) d 40.85, 43.08, 43.67, 44.72, 47.66, 52.18, 60.43, 113.57,
114.49, 117.94, 128.15, 128.18, 128.82, 129.31, 130.12, 130.27,
33.72, 136.04, 136.14, 148.44, 152.14, 155.29, 159.22, 167.59.
For C31 32ClN O calculated: 67.20% C, 5.82% H, 17.70% N; found:
C, 6.40% H, 18.87% N; found: 71.74% C, 6.42% H, 18.83% N. MS
d
6
+
[
M+1] : m/z 520.
1
4
.5.2. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
H
7
+
(
2-methoxyphenyl)piperazin-1-yl)]ethanone (2)
67.23% C, 5.86% H, 17.81% N. MS [M+1] : m/z 554.
ꢀ1
Yield 68%, mp 187 °C. IR (KBr, cm ):
H), 2838 (aliphatic C–H), 1659 (C@O), 1571–1315 (C@C and
C@N), 1243–976 (C–N). 1H NMR (500 MHz, DMSO-d
, ppm): d
.60 (br s, 4H, piperazine-H), 3.15 (br s, 4H, piperazine-H), 3.39
s, 2H, –CO–CH2), 3.66 (br s, 2H, piperazine-H), 3.72 (s, 3H,
mmaks 3031 (aromatic C–
4.5.6. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
(4-methoxyphenyl)piperazin-1-yl)]ethanone (6)
6
ꢀ1
2
(
Yield 69–75%, mp 100–103 °C. IR (KBr, cm ):
mmaks 3041 (aro-
matic C–H), 2883 (aliphatic C–H), 1665 (C@O), 1603–1315 (C@C
1
OCH3), 3.78 (br s, 2H, piperazine-H), 3.94 (br s, 2H, piperazine-
H), 3.99 (br s, 2H, piperazine-H), 6.41–6.43 (m, 2H, Ar-H), 6.56
and C@N), 1253–1008 (C–N).
ppm): d 2.65 (br s, 4H, piperazine-H), 3.06 (br s, 4H, piperazine-
H), 3.33 (s, 2H, –CO–CH ), 3.67 (br s, 2H, piperazine-H), 3.69 (s,
3H, OCH ), 3.77 (br s, 2H, piperazine-H), 3.93 (br s, 2H, pipera-
6
H NMR (500 MHz, DMSO-d ,
(
7
d, 1H, J: 8.0 Hz, Ar-H), 7.13 (t, 1H, J: 8.0 Hz, Ar-H), 7.38–7.39 (m,
2
H, Ar-H), 7.45–7.49 (m, 3H, Ar-H). 13C NMR (100 MHz, DMSO-
3
d
1
1
6
, ppm) d 40.88, 43.08, 43.70, 44.75, 50.04, 52.72, 55.28, 60.76,
11.99, 117.93, 120.77, 122.30, 128.15, 128.18, 128.83, 129.32,
30.12, 136.05, 136.15, 141.17, 148.45, 151.95, 155.30, 159.22,
zine-H), 3.99 (br s, 2H, piperazine-H), 6.82 (d, 2H, J: 8.0 Hz,
Ar-H), 6.91 (d, 2H, J: 8.0 Hz, Ar-H), 7.36–7.39 (m, 6H, Ar-H),
7.43–7.47 (m, 4H, Ar-H). C NMR (100 MHz, DMSO-d , ppm)
6
1
3

L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
6319
d 40.84, 43.06, 43.65, 44.73, 49.57, 52.50, 55.06, 60.61, 114.12,
2H, piperazine-H), 3.98 (br s, 2H, piperazine-H), 6.84–6.97 (m,
1
1
17.28, 128.16, 128.19, 128.81, 129.32, 130.14, 135.99, 136.10,
45.27, 148.41, 152.79, 155.31, 159.18, 167.62. For C32 cal-
4H, Ar-H), 7.18 (d, 4H, J: 8.0 Hz, Ar-H), 7.27 (d, 2H, J: 8.0 Hz, Ar-
H), 7.38 (d, 2H, J: 8.0 Hz, Ar-H). C NMR (100 MHz, DMSO-d ,
6
1
3
H
35
N
7
O
2
culated: 69.92% C, 6.42% H, 17.84% N; found: 69.93% C, 6.46% H,
ppm) d 20.70, 20.80, 40.85, 43.06, 43.66, 44.75, 50.03, 52.70,
55.25, 60.75, 111.91, 117.91, 120.74, 122.30, 128.64, 128.76,
+
1
7.79% N. MS [M+1] : m/z 550.
1
28.83, 129.29, 133.22, 133.43, 137.53, 140.08, 141.13, 148.35,
4
.5.7. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
151.92, 155.0, 159.12, 167.68. For C34 calculated: 70.69%
39 7 2
H N O
(
4-chlorophenyl)piperazin-1-yl)]ethanone (7)
C, 6.80% H, 16.84% N; found: 70.67% C, 6.85% H, 16.87% N. MS
[M+1] : m/z 578.
Yield 75–78%, mp 169–171 °C. IR (KBr, cm 1):
ꢀ
m
maks 3033 (aro-
+
matic C–H), 2868 (aliphatic C–H), 1663 (C@O), 1588–1303 (C@C
1
and C@N), 1252–1012 (C–N).
ppm): d 2.61 (br s, 4H, piperazine-H), 3.16 (br s, 4H, piperazine-
H), 3.30 (s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.76 (br
H
NMR (500 MHz, DMSO-d
6
,
4.5.11. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(2-chlorophenyl)piperazin-1-yl]ethanone (11)
ꢀ1
2
Yield 75–76%, mp 152–154 °C. IR (KBr, cm ):
mmaks 3022 (aro-
s, 2H, piperazine-H), 3.93 (br s, 2H, piperazine-H), 3.99 (br s, 2H,
matic C–H), 2873 (aliphatic C–H), 1659 (C@O), 1591–1309 (C@C
1
piperazine-H), 6.95 (d, 2H, J: 8.0 Hz, Ar-H), 7.22 (d, 2H, J: 7.5 Hz,
and C@N), 1244–1010 (C–N).
ppm): d 2.31 and 2.32 (2s, 6H, CH
H
NMR (500 MHz, DMSO-d
), 2.64 (br s, 4H, piperazine-H),
2
3.0 (br s, 4H, piperazine-H), 3.30 (s, 2H, –CO–CH ), 3.65 (br s, 2H,
6
,
1
3
Ar-H), 7.34–7.37 (m, 7H, Ar-H), 7.43–7.47 (m, 3H, Ar-H).
NMR (100 MHz, DMSO-d , ppm) d 40.88, 43.10, 43.68, 43.67,
4.75, 48.04, 52.23, 60.49, 109.46, 116.78, 122.25, 128.17, 128.21,
28.50, 128.84, 129.34, 130.15, 136.05, 136.14, 148.45, 149.73,
55.32, 159.22, 167.61. For C31 32ClN O calculated: 67.20% C,
.82% H, 17.70% N; found: 67.26% C, 5.88% H, 17.73% N. MS
C
3
6
4
1
1
5
piperazine-H), 3.77 (br s, 2H, piperazine-H), 3.91 (br s, 2H, pipera-
zine-H), 3.99 (br s, 2H, piperazine-H), 7.04 (t, 1H, J: 8.0 Hz, Ar-H),
7.16–7.18 (m, 5H, Ar-H), 7.26–7.30 (m, 3H, Ar-H), 7.38 (d, 2H, J:
H
7
1
3
8.0 Hz, Ar-H), 7.41 (d, 1H, J: 8.0 Hz, Ar-H). C NMR (100 MHz,
DMSO-d , ppm) d 20.72, 20.82, 40.84, 43.05, 43.66, 44.75, 50.80,
2.56, 60.56, 120.80, 123.77, 127.51, 127.94, 128.64, 128.77,
128.84, 129.31, 130.21, 133.21, 133.41, 137.54, 140.10, 148.33,
148.87, 155.0, 159.10, 167.62. For 36ClN calculated:
+
[
M+1] : m/z 554.
6
5
4
.5.8. 1-[4-(5,6-Diphenyl-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[4-
(
4-nitrophenyl)piperazin-1-yl)]ethanone (8)
C
33
H
7
O
Yield 68–70%, mp 188–190 °C. IR (KBr, cmꢀ1):
mmaks 3018 (aro-
68.09% C, 6.23% H, 16.84% N; found: 68.12% C, 6.27% H, 16.80% N.
+
matic C–H), 2875 (aliphatic C–H), 1662 (C@O), 1613–1302 (C@C
MS [M+1] : m/z 582.
1
and C@N), 1241–1012 (C–N).
ppm): d 2.61 (br s, 4H, piperazine-H), 3.32 (s, 2H, –CO–CH
.49 (br s, 4H, piperazine-H), 3.65 (br s, 2H, piperazine-H), 3.77
br s, 2H, piperazine-H), 3.94 (br s, 2H, piperazine-H), 4.0 (br s,
6
H NMR (500 MHz, DMSO-d ,
2
),
4.5.12. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(3-methoxyphenyl)piperazin-1-yl]ethanone (12)
3
(
ꢀ1
Yield 68–71%, mp 111–112 °C. IR (KBr, cm ): mmaks 3025
2
7
H, piperazine-H), 7.04 (d, 2H, J: 9.5 Hz, Ar-H), 7.35–7.47 (m,
H, Ar-H), 7.38 (d, 3H, J: 8.0 Hz, Ar-H), 8.06 (d, 2H, J: 9.5 Hz,
(aromatic C–H), 2871 (aliphatic C–H), 1664 (C@O), 1582–1305
(C@C and C@N), 1256–1009 (C–N). H NMR (500 MHz, DMSO-
1
1
3
Ar-H). C NMR (100 MHz, DMSO-d
6
, ppm) d 40.84, 43.08, 43.66,
4.71, 46.33, 51.89, 60.09, 112.58, 125.59, 128.16, 128.19,
28.82, 129.32, 130.15, 136.04, 136.12, 136.84, 148.45, 154.64,
55.31, 159.22, 167.52. For C31 calculated: 65.94% C,
.71% H, 19.85% N; found: 65.87% C, 5.83% H, 19.80% N. MS
d
6
, ppm): d 2.31 and 2.32 (2s, 6H, CH
zine-H), 3.15 (br s, 4H, piperazine-H), 3.28 (s, 2H, –CO–CH
3.65 (br s, 2H, piperazine-H), 3.71 (s, 3H, OCH ), 3.77 (br s,
3
), 2.59 (br s, 4H, pipera-
4
1
1
5
2
),
3
32
H N
8
O
3
2H, piperazine-H), 3.91 (br s, 2H, piperazine-H), 3.97 (br s, 2H,
piperazine-H), 6.36 (d, 1H, J: 8.0 Hz, Ar-H), 6.45 (s, 1H, Ar-H),
6.52 (d, 1H, J: 7.5 Hz, Ar-H), 7.09 (t, 1H, J: 8.0 Hz, Ar-H), 7.16
+
[
M+1] : m/z 565.
(
d, 4H, J: 7.5 Hz, Ar-H), 7.29 (d, 2H, J: 8 Hz, Ar-H), 7.38 (d, 2H,
1
3
4
1
.5.9. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)piperazin-
-yl]-2-(4-phenylpiperazin-1-yl)ethanone (9)
6
J: 8.5 Hz, Ar-H). C NMR (100 MHz, DMSO-d , ppm) d 20.71,
20.72, 40.13, 40.90, 43.11, 43.70, 44.80, 48.21, 52.43, 54.79,
60.64, 101.46, 104.13, 108.04, 128.69, 128.81, 128.88, 129.36,
129.52, 133.26, 133.47, 137.57, 140.14, 148.37, 152.32, 155.02,
Yield 73–75%, mp 203–205 °C. IR (KBr, cmꢀ1):
mmaks 3021 (aro-
matic C–H), 2890 (aliphatic C–H), 1664 (C@O), 1589–1302 (C@C
1
and C@N), 1231–1003 (C–N).
H
NMR (500 MHz, DMSO-d
), 2.61 (br s, 4H, piperazine-H),
), 3.65 (br s,
H, piperazine-H), 3.76 (br s, 2H, piperazine-H), 3.91 (br s, 2H,
6
,
39 7 2
159.14, 160.14, 167.63. For C34H N O calculated: 70.69% C,
ppm): d 2.31 and 2.32 (2s, 6H, CH
3
6.80% H, 16.97% N; found: 70.72% C, 6.86% H, 16.91% N. MS
+
3
2
.20 (br s, 4H, piperazine-H), 3.30 (s, 2H, –CO–CH
2
[M+1] : m/z 578.
piperazine-H), 3.97 (br s, 2H, piperazine-H), 6.77 (t, 1H, J: 7.5 Hz,
Ar-H), 6.94 (d, 1H, J: 8.0 Hz, Ar-H), 7.16–7.22 (m, 6H, Ar-H), 7.27
4.5.13. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(3-chlorophenyl)piperazin-1-yl]ethanone (13)
1
3
ꢀ1
(
(
4
1
1
d, 2H, J: 8.0 Hz, Ar-H), 7.38 (d, 2H, J: 8.0 Hz, Ar-H). C NMR
100 MHz, DMSO-d , ppm) d 20.70, 20.80, 40.85, 43.05, 43.66,
4.72, 48.20, 52.40, 60.55, 115.32, 118.72, 128.63, 128.77, 128.79,
28.83, 129.28, 133.22, 133.42, 137.52, 140.07, 148.35, 150.91,
55.0, 159.12, 167.61. For C33 O calculated: 72.37% C, 6.81%
Yield 71–76%, mp 173–175 °C. IR (KBr, cm ):
m
maks 3013 (aro-
6
matic C–H), 2873 (aliphatic C–H), 1663 (C@O), 1612–1319 (C@C
1
and C@N), 1252–1005 (C–N).
ppm): d 2.31 and 2.32 (2s, 6H, CH
H), 3.20 (br s, 4H, piperazine-H), 3.30 (s, 2H, –CO–CH
2
H
NMR (500 MHz, DMSO-d
), 2.60 (br s, 4H, piperazine-
), 3.65 (br
6
,
3
H
37
N
7
+
H, 17.90% N; found: 72.47% C, 6.88% H, 17.82% N. MS [M+1] : m/z
s, 2H, piperazine-H), 3.75 (br s, 2H, piperazine-H), 3.91 (br s,
5
48.
2H, piperazine-H), 3.97 (br s, 2H, piperazine-H), 6.78 (d, 1H, J:
7
.5 Hz, Ar-H), 6.90 (d, 1H, J: 8.5 Hz, Ar-H), 6.94 (s, 1H, Ar-H),
4
.5.10. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
7.16–7.22 (m, 5H, Ar-H), 7.26 (d, 2H, J: 8.0 Hz, Ar-H), 7.37 (d,
1
3
zin-1-yl]-2-[4-(2-methoxyphenyl)piperazin-1-yl]ethanone (10)
2H, J: 8.0 Hz, Ar-H).
6
C NMR (100 MHz, DMSO-d , ppm) d
ꢀ1
Yield 71–77%, mp 134–137 °C. IR (KBr, cm ):
matic C–H), 2882 (aliphatic C–H), 1661 (C@O), 1581–1313 (C@C
and C@N), 1252–927 (C–N). 1H NMR (500 MHz, DMSO-d
, ppm):
d 2.31 and 2.32 (2s, 6H, CH ), 2.61 (br s, 4H, piperazine-H), 2.98
br s, 4H, piperazine-H), 3.30 (s, 2H, –CO–CH ), 3.65 (br s, 2H,
piperazine-H), 3.77 (br s, 5H, piperazine-H and OCH ), 3.91 (br s,
m
maks 3028 (aro-
20.71, 20.81, 40.84, 43.05, 43.63, 44.73, 47.62, 52.18, 60.42,
113.56, 114.46, 117.93, 128.63, 128.76, 128.83, 129.30, 130.27,
133.19, 133.40, 133.69, 137.53, 140.09, 148.33, 152.12, 155.0,
6
3
7
159.09, 167.55. For C33H36ClN O calculated: 68.09% C, 6.23% H,
+
(
2
16.84% N; found: 68.15% C, 6.29% H, 16.78% N. MS [M+1] : m/z
3
582.

6
320
L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
4
.5.14. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
131.05, 148.04, 150.92, 154.34, 159.05, 159.20, 160.91, 167.61.
For C33 calculated: 68.37% C, 6.43% H, 16.91% N; found:
68.38% C, 6.48% H, 16.88% N. MS [M+1] : m/z 580.
zin-1-yl]-2-[4-(4-methoxyphenyl)piperazin-1-yl]ethanone (14)
37 7 3
H N O
ꢀ1
+
Yield 69–74%, mp 126–129 °C. IR (KBr, cm ):
m
maks 3017 (aro-
matic C–H), 2839 (aliphatic C–H), 1663 (C@O), 1587–1305 (C@C
1
and C@N), 1261–1002 (C–N).
ppm): d 2.31 and 2.32 (2s, 6H, CH
3
H
NMR (500 MHz, DMSO-d
), 2.59 (br s, 4H, piperazine-H),
), 3.65 (br s,
), 3.78 (br s, 2H, piperazine-
6
,
4.5.18. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)piper-
azin-1-yl]-2-[4-(2-methoxyphenyl)piperazin-1-yl]ethanone (18)
3
ꢀ
1
.15 (br s, 4H, piperazine-H), 3.28 (s, 2H, –CO–CH
H, piperazine-H), 3.72 (s, 3H, OCH
H), 3.90 (br s, 2H, piperazine-H), 3.98 (br s, 2H, piperazine-H),
.36 (d, 1H, J: 8.0 Hz, Ar-H), 6.85 (d, 2H, J: 8.5 Hz, Ar-H), 6.94 (d,
H, J: 8.0 Hz, Ar-H), 7.35–7.38 (m, 3H, Ar-H), 7.40–7.43 (m, 4H,
2
Yield 65–67%, mp 114–118 °C. IR (KBr, cm ):
mmaks 3034 (aro-
2
3
matic C–H), 2839 (aliphatic C–H), 1661 (C@O), 1603–1302 (C@C
1
and C@N), 1258–1006 (C–N).
ppm): d 2.61 (br s, 4H, piperazine-H), 2.99 (br s, 4H, piperazine-
H), 3.29 (s, 2H, –CO–CH ), 3.65 (br s, 2H, piperazine-H), 3.77–
3.79 (m, 11H, piperazine-H and OCH ), 3.92 (br s, 2H, piperazine-H),
6
H NMR (500 MHz, DMSO-d ,
6
2
2
1
3
Ar-H). C NMR (100 MHz, DMSO-d
6
, ppm) d 20.72, 20.82, 40.84,
3.05, 43.65, 44.75, 49.59, 52.52, 55.06, 60.63, 114.12, 117.29,
28.64, 128.77, 128.83, 129.30, 133.20, 133.41, 137.53, 140.10,
45.28, 148.33, 152.81, 154.99, 159.10, 167.63. For C34 cal-
3
4
1
1
3.98 (br s, 2H, piperazine-H), 6.85–6.97 (m, 8H, Ar-H), 7.33
(d, 2H, J: 7.0 Hz, Ar-H), 7.47 (d, 2H, J: 8.5 Hz, Ar-H). 13C NMR
H
39
N
7
O
2
6
(100 MHz, DMSO-d , ppm) d 40.86, 43.05, 43.67, 44.77, 50.01,
culated: 70.69% C, 6.80% H, 16.97% N; found: 70.75% C, 6.89% H,
52.69, 55.03, 55.22, 55.92, 60.76, 111.81, 113.66, 113.77, 117.88,
120.71, 122.31, 128.08, 128.70, 129.99, 131.07, 141.07, 148.02,
+
1
6.90% N. MS [M+1] : m/z 578.
1
39 7 4
51.88, 154.35, 159.08, 159.16, 160.88, 167.64. For C34H N O cal-
4
.5.15. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
culated: 66.98% C, 6.45% H, 16.08% N; found: 66.88% C, 6.50% H,
16.82% N. MS [M+1] : m/z 610.
+
zin-1-yl]-2-[4-(4-chlorophenyl)piperazin-1-yl]ethanone (15)
ꢀ1
Yield 72–75%, mp 117–118 °C. IR (KBr, cm ):
matic C–H), 2872 (aliphatic C–H), 1669 (C@O), 1602–1303 (C@C
and C@N), 1251–925 (C–N). 1H NMR (500 MHz, DMSO-d
, ppm):
d 2.31 and 2.32 (2s, 6H, CH ), 2.61 (br s, 4H, piperazine-H), 3.16
br s, 4H, piperazine-H), 3.31 (s, 2H, –CO–CH ), 3.65 (br s, 2H,
mmaks 3015 (aro-
4.5.19. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(2-chlorophenyl)piperazin-1-yl]ethanone (19)
6
ꢀ
1
3
Yield 69–72%, mp 109–110 °C. IR (KBr, cm ):
mmaks 3018 (aro-
(
2
matic C–H), 2837 (aliphatic C–H), 1656 (C@O), 1601–1305 (C@C
1
piperazine-H), 3.75 (br s, 2H, piperazine-H), 3.91 (br s, 2H, pipera-
zine-H), 3.97 (br s, 2H, piperazine-H), 6.95 (d, 2H, J: 9.0 Hz, Ar-H),
and C@N), 1251–974 (C–N). H NMR (500 MHz, DMSO-d
d 2.65 (br s, 4H, piperazine-H), 3.01 (br s, 4H, piperazine-H), 3.31
(s, 2H, –CO–CH ), 3.65 (br s, 2H, piperazine-H), 3.75–3.79 (m, 8H,
piperazine-H and OCH ), 3.91 (br s, 2H, piperazine-H), 3.98 (br s,
6
, ppm):
7
.17 (d, 4H, J: 7.5 Hz, Ar-H), 7.22 (d, 2H, J: 9.0 Hz, Ar-H), 7.26 (d, 2H,
2
13
J: 8.0 Hz, Ar-H), 7.37 (d, 2H, J: 8.0 Hz, Ar-H). C NMR (100 MHz,
DMSO-d , ppm) d 20.79, 20.88, 30.74, 35.75, 40.91, 43.11, 43.71,
4.79, 48.05, 52.28, 60.52, 116.81, 122.29, 128.55, 128.70, 128.84,
28.91, 129.36, 133.27, 133.47, 137.60, 140.16, 148.40, 149.76,
55.06, 159.17, 162.27, 167.62. For 36ClN calculated:
8.09% C, 6.23% H, 16.84% N; found: 68.06% C, 6.28% H, 16.83% N.
3
6
2H, piperazine-H), 6.92–6.96 (m, 4H, Ar-H), 7.04 (t, 1H, J: 8.0 Hz,
Ar-H), 7.17 (d, 1H, J: 7.5 Hz, Ar-H), 7.29 (t, 1H, J: 8.0 Hz, Ar-H),
7.33 (d, 2H, J: 7.5 Hz, Ar-H), 7.41 (d, 1H, J: 8.0 Hz, Ar-H), 7.47 (d,
4
1
1
6
1
3
C
33
H
7
O
6
2H, J: 8.5 Hz, Ar-H). C NMR (100 MHz, DMSO-d , ppm) d 40.85,
43.05, 43.66, 44.77, 50.80, 52.55, 55.03, 55.21, 60.55, 113.67,
113.77, 120.80, 123.77, 127.49, 127.94, 128.08, 128.69, 129.98,
+
MS [M+1] : m/z 582.
1
30.21, 131.07, 148.02, 148.86, 154.35, 159.01, 159.16, 160.88,
4
.5.16. 1-[4-(5,6-Bis(4-methylphenyl)-1,2,4-triazin-3-yl)pipera-
167.61. For C33 36ClN calculated: 64.54% C, 5.91% H, 15.96%
N; found: 64.58% C, 6.0% H, 16.02% N. MS [M+1] : m/z 614.
H
7 3
O
+
zin-1-yl]-2-[4-(4-nitrophenyl)piperazin-1-yl]ethanone (16)
ꢀ1
Yield 70–72%, mp 254–255 °C. IR (KBr, cm ):
mmaks 3034 (aro-
matic C–H), 2884 (aliphatic C–H), 1667 (C@O), 1584–1303 (C@C
4.5.20. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(3-methoxyphenyl)piperazin-1-yl]ethanone (20)
1
and C@N), 1256–1005 (C–N). H NMR (500 MHz, DMSO-d
d 2.31 and 2.32 (2s, 6H, CH ), 2.61 (br s, 4H, piperazine-H), 3.31 (s,
H, –CO–CH ), 3.49 (br s, 4H, piperazine-H), 3.65 (br s, 2H, pipera-
zine-H), 3.76 (br s, 2H, piperazine-H), 3.92 (br s, 2H, piperazine-H),
6
, ppm):
ꢀ1
3
Yield 68–71%, mp 91–92 °C. IR (KBr, cm ):
mmaks 3028 (aromatic
2
2
C–H), 2833 (aliphatic C–H), 1645 (C@O), 1604–1300 (C@C and
1
C@N), 1244–974 (C–N). H NMR (500 MHz, DMSO-d
2.60 (br s, 4H, piperazine-H), 3.15 (br s, 4H, piperazine-H), 3.29 (s,
2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.71 (s, 3H, OCH ),
3.75–3.78 (m, 8H, piperazine-H and OCH ), 3.91 (br s, 2H, pipera-
6
, ppm): d
3.98 (br s, 2H, piperazine-H), 7.04 (d, 2H, J: 9.0 Hz, Ar-H), 7.18 (d,
4H, J: 8.0 Hz, Ar-H), 7.27 (d, 2H, J: 8.0 Hz, Ar-H), 7.38 (d, 2H, J:
8
.0 Hz, Ar-H), 8.06 (d, 2H, J: 9.0 Hz, Ar-H). 13C NMR (100 MHz,
2
3
3
DMSO-d
6
, ppm) d 20.71, 20.73, 40.85, 43.06, 43.66, 44.70, 45.09,
6.35, 51.90, 60.08, 112.59, 125.58, 128.64, 128.77, 128.83, 129.29,
33.24, 133.44, 136.87, 137.54, 140.09, 148.36, 154.64, 155.0,
59.14, 167.51. For C33 calculated: 66.87% C, 6.12% H,
zine-H), 3.96 (br s, 2H, piperazine-H), 6.34 (d, 1H, J: 8.0 Hz, Ar-H),
6.45 (s, 1H, Ar-H), 6.53 (d, 1H, J: 8.0 Hz, Ar-H), 6.92–6.97 (m, 4H,
Ar-H), 7.10 (t, 1H, J: 8.0 Hz, Ar-H), 7.33 (d, 2H, J: 8.5 Hz, Ar-H),
4
1
1
1
1
3
H
36
N
8
O
3
7.46 (d, 2H, J: 7.5 Hz, Ar-H). C NMR (100 MHz, DMSO-d
40.85, 43.05, 43.65, 44.75, 48.13, 52.36, 54.74, 55.03, 55.20, 60.55,
01.36, 104.10, 107.96, 113.66, 113.76, 128.07, 128.70, 129.47,
129.98, 131.06, 148.02, 152.27, 154.35, 159.01, 159.16, 160.07,
160.87, 167.58. For C34 calculated: 66.98% C, 6.45% H,
6
, ppm) d
+
8.91% N; found: 66.78% C, 6.18% H, 18.82% N. MS [M+1] : m/z 593.
1
4
.5.17. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-(4-phenylpiperazin-1-yl]ethanone (17)
39 7 4
H N O
ꢀ1
+
Yield 70–73%, mp 174–176 °C. IR (KBr, cm ):
matic C–H), 2846 (aliphatic C–H), 1663 (C@O), 1581–1307 (C@C
and C@N), 1254–976 (C–N). 1H NMR (500 MHz, DMSO-d
, ppm):
d 2.62 (br s, 4H, piperazine-H), 3.16 (br s, 4H, piperazine-H), 3.32
s, 2H, –CO–CH ), 3.65 (br s, 2H, piperazine-H), 3.73–3.78 (m, 8H,
piperazine-H and OCH ), 3.90 (br s, 2H, piperazine-H), 3.96 (br s,
H, piperazine-H), 6.78 (t, 1H, J: 7.5 Hz, Ar-H), 6.91–6.96 (m, 6H,
Ar-H), 7.21 (t, 2H, J: 8.0 Hz, Ar-H), 7.32 (d, 2H, J: 8.5 Hz, Ar-H),
.46 (d, 2H, J: 9.0 Hz, Ar-H). 13C NMR (100 MHz, DMSO-d
, ppm)
d 40.87, 43.08, 43.69, 44.75, 48.22, 52.50, 55.05, 55.22, 60.57,
13.68, 113.78, 115.32, 118.72, 128.13, 128.75, 128.79, 129.99,
m
maks 3016 (aro-
16.08% N; found: 66.88% C, 6.48% H, 16.04% N. MS [M+1] : m/z 610.
6
4.5.21. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(3-chlorophenyl)piperazin-1-yl]ethanone (21)
ꢀ1
(
2
Yield 66–70%, mp 186–188 °C. IR (KBr, cm ):
mmaks 3020 (aro-
3
matic C–H), 2892 (aliphatic C–H), 1664 (C@O), 1581–1304 (C@C
1
2
and C@N), 1264–1010 (C–N).
ppm): d 2.60 (br s, 4H, piperazine-H), 3.20 (br s, 4H, piperazine-
H), 3.31 (s, 2H, –CO–CH ), 3.65 (br s, 2H, piperazine-H),
3.74–3.78 (m, 8H, piperazine-H and OCH ), 3.91 (br s, 2H, pipera-
zine-H), 3.96 (br s, 2H, piperazine-H), 6.83 (d, 1H, J: 8.0 Hz,
6
H NMR (500 MHz, DMSO-d ,
7
6
2
3
1

L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
6321
Ar-H), 6.89–6.97 (m, 6H, Ar-H), 7.21 (t, 1H, J: 8.5 Hz, Ar-H), 7.33 (d,
2
(s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.77 (br s, 2H,
1
3
2
H, J: 8.5 Hz, Ar-H), 7.47 (d, 2H, J: 7.5 Hz, Ar-H).
C NMR
piperazine-H), 3.96 (br s, 2H, piperazine-H), 3.99 (br s, 2H, pipera-
zine-H), 6.78 (t, 1H, J: 7.5 Hz, Ar-H), 6.94 (d, 2H, J: 8.0 Hz, Ar-H),
7.21 (t, 2H, J: 8.5 Hz, Ar-H), 7.41 (d, 2H, J: 8.5 Hz, Ar-H), 7.45–
(
100 MHz, DMSO-d , ppm) d 40.85, 43.07, 43.65, 44.74, 47.62,
6
5
1
1
2.17, 55.03, 55.21, 60.43, 113.56, 113.66, 113.76, 114.46, 117.93,
28.07, 128.70, 129.99, 130.28, 131.06, 133.69, 148.02, 152.12,
1
3
6
7.48 (m, 5H, Ar-H). C NMR (100 MHz, DMSO-d , ppm) d 40.81,
54.34, 159.02, 159.16, 160.88, 167.54. For C33
H
36ClN
7
O
3
calcu-
43.05, 43.66, 44.71, 48.17, 52.39, 60.56, 115.29, 118.73, 128.41,
128.79, 130.59, 131.24, 133.15, 134.65, 134.75, 135.22, 143.75,
135.22, 147.15, 150.88, 154.25, 159.10, 167.60. For C31H31Cl N O
2 7
lated: 64.54% C, 5.91% H, 15.96% N; found: 64.59% C, 6.01% H,
+
1
6.03% N. MS [M+1] : m/z 614.
calculated: 63.26% C, 5.31% H, 16.66% N; found: 63.29% C, 5.37%
+
4
.5.22. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
H, 16.64% N. MS [M+1] : m/z 588.
zin-1-yl]-2-[4-(4-methoxyphenyl)piperazin-1-yl]ethanone (22)
ꢀ1
Yield 66–69%, mp 113–115 °C. IR (KBr, cm ):
m
maks 3034 (aro-
4.5.26. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)pipera-
matic C–H), 2886 (aliphatic C–H), 1668 (C@O), 1574–1307 (C@C
zin-1-yl]-2-[4-(2-methoxyphenyl)piperazin-1-yl]ethanone (26)
1
ꢀ1
and C@N), 1243–1011 (C–N).
ppm): d 2.65 (br s, 4H, piperazine-H), 3.06 (br s, 4H, piperazine-
H), 3.31 (s, 2H, –CO–CH ), 3.65 (br s, 5H, piperazine-H and
OCH ), 3.75–3.79 (m, 8H, piperazine-H and OCH ), 3.91 (br s, 2H,
H
NMR (500 MHz, DMSO-d
6
,
Yield 65–70%, mp 197–200 °C. IR (KBr, cm ):
mmaks 3034 (aro-
matic C–H), 2896 (aliphatic C–H), 1669 (C@O), 1582–1301 (C@C
1
2
and C@N), 1268–1002 (C–N).
ppm): d 2.61 (br s, 4H, piperazine-H), 2.99 (br s, 4H, piperazine-
H), 3.30 (s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.78 (br
s, 2H, piperazine-H and OCH ), 3.94 (br s, 2H, piperazine-H), 4.0
(br s, 2H, piperazine-H), 6.85–6.97 (m, 4H, Ar-H), 7.41 (d, 2H, J:
6
H NMR (500 MHz, DMSO-d ,
3
3
piperazine-H), 3.98 (br s, 2H, piperazine-H), 6.83 (d, 2H, J: 8.0 Hz,
Ar-H), 6.91–6.96 (m, 6H, Ar-H), 7.33 (d, 2H, J: 8.0 Hz, Ar-H), 7.47
2
3
(
d, 2H, J: 8.5 Hz, Ar-H). 13C NMR (100 MHz, DMSO-d
6
, ppm) d
0.93, 43.13, 43.72, 44.82, 48.07, 52.29, 55.09, 55.27, 56.0, 60.55,
1
3
4
1
1
C
6
7.5 Hz, Ar-H), 7.46–7.50 (m, 6H, Ar-H).
C NMR (100 MHz,
13.74, 113.84, 116.82, 122.30, 128.15, 128.56, 128.78, 130.06,
31.14, 149.77, 154.42, 159.09, 159.24, 160.96, 167.63. For
DMSO-d , ppm) d 40.82, 43.05, 43.67, 44.73, 50.01, 52.69, 55.22,
6
60.76, 111.81, 117.85, 120.70, 122.31, 128.41, 130.59, 131.24,
133.16, 134.65, 134.75, 135.23, 141.06, 147.15, 151.87, 154.25,
34
39 7 4
H N O calculated: 66.98% C, 6.45% H, 16.08% N; found:
+
6.87% C, 6.34% H, 16.03% N. MS [M+1] : m/z 610.
159.09, 167.68. For C32
2 7 2
H33Cl N O calculated: 62.14% C, 5.38% H,
+
1
5.85% N; found: 62.19% C, 5.33% H, 15.74% N. MS [M+1] : m/z 618.
4
.5.23. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(4-chlorophenyl)piperazin-1-yl]ethanone (23)
4.5.27. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(2-chlorophenyl)piperazin-1-yl]ethanone (27)
ꢀ1
Yield 69–73%, mp 110–113 °C. IR (KBr, cm ):
mmaks 3033 (aro-
ꢀ1
matic C–H), 2877 (aliphatic C–H), 1663 (C@O), 1588–1305 (C@C
Yield 68–71%, mp 199–200 °C. IR (KBr, cm ):
mmaks 3036 (aro-
1
and C@N), 1252–1018 (C–N).
ppm): d 2.60 (br s, 4H, piperazine-H), 3.16 (br s, 4H, piperazine-
H), 3.29 (s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.73–3.78
m, 8H, piperazine-H and OCH ), 3.90 (br s, 2H, piperazine-H),
.96 (br s, 2H, piperazine-H), 6.91–6.96 (m, 6H, Ar-H), 7.23
6
H NMR (500 MHz, DMSO-d ,
matic C–H), 2868 (aliphatic C–H), 1658 (C@O), 1563–1325 (C@C
1
and C@N), 1252–976 (C–N). H NMR (500 MHz, DMSO-d
d 2.65 (br s, 4H, piperazine-H), 3.12 (br s, 4H, piperazine-H), 3.30
(s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.77 (br s, 2H,
6
, ppm):
2
(
3
3
2
piperazine-H), 3.94 (br s, 2H, piperazine-H), 4.01 (br s, 2H, pipera-
zine-H), 7.04 (t, 1H, J: 7.5 Hz, Ar-H), 7.17 (d, 1H, J: 8.0 Hz, Ar-H),
7.29 (t, 1H, J: 8.0 Hz, Ar-H), 7.41 (d, 2H, J: 8.5 Hz, Ar-H), 7.47–
(
d, 2H, J: 9.0 Hz, Ar-H), 7.33 (d, 2H, J: 8.5 Hz, Ar-H), 7.47 (d, 2H, J:
.5 Hz, Ar-H). C NMR (100 MHz, DMSO-d , ppm) d 40.89, 42.97,
6
3.48, 44.51, 52.39, 55.03, 55.08, 55.22, 113.68, 113.78, 114.17,
17.41, 128.06, 128.68, 129.99, 131.07, 131.54, 132.09, 149.12,
7 3
51.32, 154.65, 159.22, 159.69, 160.49, 167.31. For C33H36ClN O
1
3
8
4
1
1
1
3
6
7.50 (m, 7H, Ar-H). C NMR (100 MHz, DMSO-d , ppm) d 40.81,
43.05, 43.67, 44.71, 50.79, 52.55, 60.54, 120.78, 123.77, 127.50,
127.94, 128.43, 130.21, 130.59, 131.25, 133.16, 134.66, 134.75,
calculated: 64.54% C, 5.91% H, 15.96% N; found: 64.59% C, 6.05%
H, 16.04% N. MS [M+1] : m/z 614.
3 7
135.24, 147.17, 148.85, 154.27, 159.11, 167.64. For C31H30Cl N O
+
calculated: 59.77% C, 4.85% H, 15.74% N; found: 59.79% C, 4.87%
+
H, 15.64% N. MS [M+1] : m/z 622.
4
.5.24. 1-[4-(5,6-Bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(4-nitrophenyl)piperazin-1-yl]ethanone (24)
4.5.28. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)piperazin-
1-yl]-2-[4-(3-methoxyphenyl)piperazin-1-yl]ethanone (28)
ꢀ1
Yield 66–72%, mp 210–213 °C. IR (KBr, cm ):
mmaks 3048 (aro-
ꢀ1
matic C–H), 2895 (aliphatic C–H), 1669 (C@O), 1572–1296 (C@C
Yield 71–72%, mp 182–186 °C. IR (KBr, cm ):
mmaks 3019 (aro-
1
and C@N), 1241–1013 (C–N).
H
NMR (500 MHz, DMSO-d
), 3.49
br s, 4H, piperazine-H), 3.65 (br s, 2H, piperazine-H), 3.74–3.78
m, 8H, piperazine-H and OCH ), 3.91 (br s, 2H, piperazine-H),
.97 (br s, 2H, piperazine-H), 6.92–6.96 (m, 4H, Ar-H), 7.04 (d,
6
,
matic C–H), 2873 (aliphatic C–H), 1668 (C@O), 1575–1298 (C@C
1
ppm): d 2.60 (br s, 4H, piperazine-H), 3.31 (s, 2H, –CO–CH
2
and C@N), 1232–1021 (C–N). H NMR (500 MHz, DMSO-d
d 2.59 (br s, 4H, piperazine-H), 3.15 (br s, 4H, piperazine-H), 3.31
(s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.71 (s, 3H, OCH ),
6
, ppm):
(
(
3
2
8
3
2
3
3.77 (br s, 2H, piperazine-H), 3.94 (br s, 2H, piperazine-H), 3.98
(br s, 2H, piperazine-H), 6.36 (d, 1H, J: 8.0 Hz, Ar-H), 6.45 (br s,
1H, J: 8.0 Hz, Ar-H), 6.54 (d, 1H, J: 8.5 Hz, Ar-H), 7.11 (t, 1H, J:
H, J: 9.0 Hz, Ar-H), 7.32 (d, 2H, J: 8.5 Hz, Ar-H), 7.47 (d, 2H, J:
.5 Hz, Ar-H), 8.07 (d, 2H, J: 7.5 Hz, Ar-H). 13C NMR (100 MHz,
1
3
DMSO-d
6
, ppm) d 40.83, 43.06, 43.64, 44.71, 46.29, 51.89, 55.03,
5.20, 60.10, 112.54, 113.66, 113.76, 125.59, 128.07, 128.70,
29.99, 131.06, 136.78, 148.02, 154.33, 154.62, 159.01, 159.16,
60.89, 167.47. For C33 calculated: 63.45% C, 5.81% H,
8.0 Hz, Ar-H), 7.40–7.47 (m, 8H, Ar-H).
C NMR (100 MHz,
5
1
1
1
DMSO-d , ppm) d 40.82, 43.05, 43.66, 44.70, 48.13, 52.36, 54.74,
6
60.55, 101.38, 104.08, 107.96, 128.42, 129.47, 130.59, 131.23,
133.15, 134.65, 134.75, 135.22, 147.15, 152.26, 154.26, 159.11,
36 8 5
H N O
+
7.94% N; found: 63.49% C, 5.87% H, 17.84% N. MS [M+1] : m/z 625.
160.07, 167.60. For C32
2 7 2
H33Cl N O calculated: 62.14% C, 5.38% H,
+
1
5.85% N; found: 61.19% C, 5.34% H, 15.84% N. MS [M+1] : m/z 619.
4
1
.5.25. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)piperazin-
-yl]-2-(4-phenylpiperazin-1-yl]ethanone (25)
4.5.29. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)pipera-
zin-1-yl]-2-[4-(3-chlorophenyl)piperazin-1-yl]ethanone (29)
Yield 69–74%, mp 193–194 °C. IR (KBr, cmꢀ1):
m
maks 3018 (aro-
matic C–H), 2869 (aliphatic C–H), 1664 (C@O), 1582–1319 (C@C
and C@N), 1212–975 (C–N). 1H NMR (500 MHz, DMSO-d
, ppm):
d 2.61 (br s, 4H, piperazine-H), 3.17 (br s, 4H, piperazine-H), 3.31
ꢀ1
Yield 70–74%, mp 160–262 °C. IR (KBr, cm ):
mmaks 3032 (aro-
6
matic C–H), 2876 (aliphatic C–H), 1666 (C@O), 1574–1300 (C@C
1
and C@N), 1254–1005 (C–N).
6
H NMR (500 MHz, DMSO-d ,

6
322
L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
ppm): d 2.59 (br s, 4H, piperazine-H), 3.20 (br s, 4H, piperazine-H),
.31 (s, 2H, –CO–CH ), 3.65 (br s, 2H, piperazine-H), 3.76 (br s, 2H,
piperazine-H), 3.94 (br s, 2H, piperazine-H), 3.99 (br s, 2H, pipera-
the metabolic activity of viable cells. Tetrazolium salts are reduced
to formazan by mitochondrial succinate dehydrogenase, an
enzyme which is only active in cells with an intact metabolism
and respiratory chain. The formazan is quantified photometrically
and correlates with the number of viable cells. NIH/3T3 and MCF-
7 cells line were used for cytotoxicity tests. NIH/3T3 and MCF-7
cells were incubated in RPMI medium (Hyclone, Thermo Scientific,
USA) supplemented with fetal calf serum (Hyclone, Thermo Scien-
tific, USA), 100 IU/mL penicillin and 100 mg/mL streptomycin
(Hyclone, Thermo Scientific, USA) at 37 °C in a humidified atmo-
3
2
zine-H), 6.78 (d, 1H, J: 7.5 Hz, Ar-H), 6.91 (d, 1H, J: 8.5 Hz, Ar-H),
3
3
6
8
.95 (br s, 1H, Ar-H), 7.21 (t, 1H, J: 8.0 Hz, Ar-H), 7.41 (d, 2H, J:
13
.5 Hz, Ar-H), 7.45–7.49 (m, 6H, Ar-H).
, ppm) d 40.82, 43.05, 43.66, 44.70, 47.61, 52.17, 60.41,
13.56, 114.46, 117.94, 128.42, 130.27, 130.59, 131.23, 133.16,
33.69, 134.65, 134.75, 135.23, 147.15, 152.11, 154.26, 159.10,
67.58. For C31 O calculated: 59.77% C, 4.85% H, 15.74%
C NMR (100 MHz,
DMSO-d
6
1
1
1
3 7
H30Cl N
+
N; found: 59.75% C, 4.87% H, 15.64% N. MS [M+1] : m/z 623.
2
sphere of 95% air and 5% CO . NIH/3T3 and MCF-7 cells were
4
seeded at 2 ꢁ 10 cells into each well of 96-well plates. After
4
.5.30. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)pipera-
24 h of incubating period, the culture mediums were removed
zin-1-yl]-2-[4-(4-methoxyphenyl)piperazin-1-yl]ethanone (30)
and compound was added to culture medium at 3.9–500 lg/ml
ꢀ1
Yield 71–76%, mp 187–190 °C. IR (KBr, cm ):
m
maks 3027 (aro-
concentrations. After 24 h of incubation, cytotoxicity test was per-
formed using the In Cytotox-XTT 1 Parameter Cytotoxicity Kit
(Xenometrix AG, Gewerbertrasse, Switzerland), which measures
mitochondrial activity in NIH/3T3 and MCF-7 cells. Firstly, the cells
were washed phosphate buffer saline (PBS) and were added
matic C–H), 2900 and 2825 (aliphatic C–H), 1646 (C@O), 1525–
1
1
379 (C@C and C@N), 1252–974 (C–N). H NMR (500 MHz,
DMSO-d , ppm): d 2.61 (br s, 4H, piperazine-H), 3.04 (br s, 4H,
piperazine-H), 3.31 (s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-
H), 3.68 (s, 3H, OCH ), 3.77 (br s, 2H, piperazine-H), 3.94 (br s,
H, piperazine-H), 3.99 (br s, 2H, piperazine-H), 6.81 (d, 2H, J:
.0 Hz, Ar-H), 6.89 (d, 2H, J: 9.0 Hz, Ar-H), 7.40 (d, 2H, J: 8.5 Hz,
6
2
3
200
mixed at 1:100 ratio. Then, 50
wells. The plate was incubated for 3 h at 37 °C, 5% CO
l
L/well of fresh culture medium. XTTI and XTTII solution were
L of this mixture was added to all
. After 3 h,
2
9
l
2
1
3
Ar-H), 7.45–7.49 (m, 6H, Ar-H). C NMR (100 MHz, DMSO-d
ppm) d 40.83, 43.05, 43.66, 44.71, 49.57, 52.50, 55.06, 60.61,
6
,
the content of the well was mixed by pipetting up and down. Then,
OD of the plate was read at 480 nm with a reference wave length at
680 nm. Inhibition % was calculated each concentration of com-
pound. IC50 value was estimated by non-linear regression analysis.
Cisplatin was used as positive control. Stock solutions of com-
pounds were prepared in dimethyl sulfoxide (DMSO) and further
dilutions were made with fresh culture medium. The final DMSO
concentration was under 0.1%. All experiments were performed
in duplicate.
1
1
14.11, 117.27, 128.41, 130.59, 131.24, 133.15, 134.65, 134.75,
35.22, 145.26, 147.15, 152.79, 154.26, 159.10, 167.63. For C32
calculated: 62.14% C, 5.38% H, 15.85% N; found: 62.20% C,
H
33
2 7 2
Cl N O
+
5
.37% H, 15.74% N. MS [M+1] : m/z 619.
4
1
.5.31. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)piperazin-
-yl]-2-[4-(4-chlorophenyl)piperazin-1-yl]ethanone (31)
ꢀ1
Yield 72–75%, mp 199–203 °C. IR (KBr, cm ):
mmaks 3025 (aro-
matic C–H), 2877 (aliphatic C–H), 1662 (C@O), 1581–1310 (C@C
4.6.2. DNA synthesis inhibition assay
1
0
and C@N), 1259–1003 (C–N). H NMR (500 MHz, DMSO-d
d 2.60 (br s, 4H, piperazine-H), 3.16 (br s, 4H, piperazine-H), 3.31
s, 2H, –CO–CH ), 3.66 (br s, 2H, piperazine-H), 3.76 (br s, 2H, piper-
azine-H), 3.94 (br s, 2H, piperazine-H), 3.99 (br s, 2H, piperazine-H),
.95 (d, 2H, J: 9.0 Hz, Ar-H), 7.23 (d, 2H, J: 9.0 Hz, Ar-H), 7.41 (d, 2H,
6
, ppm):
The 5-bromo-2 -deoxy-uridine (BrdU) cell proliferation assay
was used to detect the proliferation of MCF-7 cells. Sensitive and
reproducible colorimetric alternative to quantitate cell prolifera-
tion based on the measurement of BrdU incorporation during
DNA synthesis in proliferating cells. This technique is based on
the incorporation of the pyrimidine analogue BrdU instead of thy-
(
2
6
13
J: 8.5 Hz, Ar-H), 7.46–7.50 (m, 6H, Ar-H). C NMR (100 MHz,
DMSO-d , ppm) d 40.81, 43.05, 43.66, 44.70, 47.98, 52.21, 60.47,
16.74, 122.22, 128.42, 128.43, 128.48, 130.59, 131.24, 133.16,
34.65, 134.75, 135.24, 147.16, 149.68, 154.26, 159.11, 167.58.
O calculated: 59.77% C, 4.85% H, 15.74% N; found:
3
4
6
midine into the DNA of proliferating cells. This study was per-
formed on six compounds 2, 5, 7, 10, 14, and 28, which were
determined to have the highest selective cytotoxic activity by the
XTT test. Cisplatin was used as positive control. MCF-7 cells were
incubated with the related compounds in three different concen-
trations (IC50/2, IC50, and IC50 ꢁ 2) for 24 h and 48 h. MCF-7 cells
were seeded into the each well of the 96-well plates at a density
1
1
3 7
For C31H30Cl N
+
5
9.79% C, 4.87% H, 15.64% N. MS [M+1] : m/z 623.
4
.5.32. 1-[4-(5,6-Bis(4-chlorophenyl)-1,2,4-triazin-3-yl)pipera-
4
zin-1-yl]-2-[4-(4-nitrophenyl)piperazin-1-yl]ethanone (32)
of 2 ꢁ 10 cells. After 24 h of incubation, cytotoxicity test was per-
ꢀ1
Yield 74–78%, mp 210–214 °C. IR (KBr, cm ):
m
maks 3024 (aro-
formed using the Cell Proliferation ELISA, BrdU (Colorimetric) Kit
(Roche Diagnostics GmbH, Mannheim, Germany). Firstly, BrdU
solution was added and cells were reincubated 2 h at 37 °C. After
reincubation, anti-BrdU-POD was added and incubated for
90 min. Then, plates were washed with PBS for three times and
the cells were incubated with substrate solution until the color
development was adequate. Optical density of the samples were
measured at 492 nm. Finally, the percentage of the cell growth
was measured versus medium controls. All experiments were per-
formed in duplicate.
matic C–H), 2856 (aliphatic C–H), 1659 (C@O), 1591–1309 (C@C
1
and C@N), 1244–1010 (C–N).
ppm): d 2.61 (br s, 4H, piperazine-H), 3.32 (s, 2H, –CO–CH
br s, 4H, piperazine-H), 3.66 (br s, 2H, piperazine-H), 3.77 (br s,
H, piperazine-H), 3.94 (br s, 2H, piperazine-H), 4.01 (br s, 2H,
piperazine-H), 7.04 (d, 2H, J: 9.0 Hz, Ar-H), 7.41 (d, 2H, J: 8.5 Hz,
H
NMR (500 MHz, DMSO-d
6
,
2
), 3.49
(
2
1
3
Ar-H), 7.45–7.49 (m, 6H, Ar-H), 8.08 (d, 2H, J: 9.5 Hz, Ar-H).
NMR (100 MHz, DMSO-d , ppm) d 40.81, 43.04, 43.64, 44.66,
5.04, 46.26, 51.88, 60.05, 112.56, 125.60, 128.43, 130.59, 131.23,
33.17, 134.65, 134.74, 135.24, 136.80, 147.16, 154.26, 154.61,
59.11, 167.48. For C31 calculated: 58.77% C, 4.77% H,
C
6
4
1
1
1
H30Cl
2
N
8
O
3
4.6.3. Flow cytometric analysis of apoptosis and necrosis using
Annexin V/PI dual staining
+
7.69% N; found: 58.81% C, 4.67% H, 17.64% N. MS [M+1] : m/z 634.
Apoptosis is a normal physiologic process occurs during embry-
onic development and in maintenance of tissue homeostasis. The
apoptotic process is characterized by loss of plasma membrane
asymmetry and attachment, condensation of the cytoplasm and
4
4
.6. Experimental procedure for antiproliferative activity
.6.1. Cytotoxicity test
The tetrazolium salt XTT (2,3-bis-(2-methoxy-4-nitro-5-sulf-
3
5,36
nucleus, and internucleosomal cleavage of DNA.
Apoptosis
ophenyl)-2H-tetrazolium-5-carboxanilide) can be used to measure
was detected by using the FITC-Annexin V Apoptosis Detection

L. Yurtta ßs et al. / Bioorg. Med. Chem. 22 (2014) 6313–6323
6323
Kit (BD, Pharmingen™, United States) according to the manufac-
turer’s instruction. Apoptosis was induced by adding cisplatin
and the related compounds 2, 5, 7, 10, 14 and 28, which were
determined to have the highest selective cytotoxic activity by the
XTT test, in their IC50. After 24 h incubation, cells were harvested
by centrifugation at 1200g for 5 min at room temperature. Then,
cells were rinsed with cold water twice and resuspended at a con-
6. Thurston, D. A. The Chemistry and Pharmacology of Anticancer Drugs; CRC Press:
Florida, 2007.
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8
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centration of 1 ꢁ 10 cells/mL in Annexin V-FITC binding buffer.
1
1
3. Abdel-Rahman, R. M. Pharmazie 2001, 56, 18.
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at various stages. The areas in the diagrams represent; necrotic
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tive); the late apoptotic cells in the upper right quadrant (Q2;
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lower left quadrant (Q3; Annexin V-negative/PI-negative) and the
early apoptotic cells in the lower right quadrant (Q4; Annexin V-
positive/PI-negative), respectively. The experiment was performed
in duplicate.
1
1
1
1
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7
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