Pyrimidooxadiazine and triazolopyrimidooxadiazine derivatives: Synthesis and cytotoxic evaluation in human cancer cell lines

Article abstract of DOI:10.1134/S1068162015020077

In vitro antiproliferative activities of some pyrimido[4,5-e][1,3,4]oxadiazine and [1,2,4]triaz-olo[4',3':1,2]pyrimido[4,5-e][1,3,4]oxadiazine derivatives were examined in human malignant cancer cell lines. All synthesized compounds inhibited the growth o

Full text of DOI:10.1134/S1068162015020077

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 2, pp. 201–208. © Pleiades Publishing, Ltd., 2015.
Pyrimidooxadiazine and Triazolopyrimidooxadiazine Derivatives:
Synthesis and Cytotoxic Evaluation in Human Cancer Cell Lines1
2
SeyedꢀHadi Mousavia, Hoda AtapourꢀMashhadb, Mehdi Bakavolic, Ali Shiric, ,
2
Marzieh Akbarzadehc, and Zahra TayaraniꢀNajarana,
a Pharmacological Research Center of Medicinal Plants, School of Medicine,
Mashhad University of Medical Sciences, Mashhad, Iran
b Department of Chemistry, Payame Noor University, Mashhad, Iran
c Department of Chemistry, School of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
Received April 23, 2014; in final form, October 20, 2014
Abstract—In vitro antiproliferative activities of some pyrimido[4,5ꢀ
olo[4',3':1,2]pyrimido[4,5ꢀ ][1,3,4]oxadiazine derivatives were examined in human malignant cancer cell
lines. All synthesized compounds inhibited the growth of malignant cells in a dose dependent manner, but
among them 1,5,7ꢀtrimethylꢀ3ꢀphenylꢀ1 ꢀ[1,2,4]triazolo[4',3':1,2]pyrimido[4,5ꢀ ][1,3,4]oxadiazine
and [(1,5ꢀdimethylꢀ3ꢀphenylꢀ1 ꢀ[1,2,4]triazolo[4',3':1,2]pyrimido[4,5ꢀ ][1,3,4]oxadiazinꢀ7ꢀyl)sulfaꢀ
e][1,3,4]oxadiazine and [1,2,4]triazꢀ
e
H
e
H
e
nyl]acetonitrile, both with triazole moiety, were found to be more effective than other compounds; they
also induced a subꢀG1 peak in the flow cytometry histogram of treated cells compared to controls, indicatꢀ
ing that apoptotic cell death is involved in toxicity they induce. The results showed that compounds with
triazole moiety fused to pyrimido[4,5ꢀe][1,3,4]oxadiazine derivatives are more active than those bearing
chlorine or pyrrolidine groups at Cꢀ7 position.
Keywords: pyrimidooxadiazine, triazolopyrimidooxadiazine, cytotoxicity, tumor cell line, apoptosis
DOI: 10.1134/S1068162015020077
2 1
INTRODUCTION
lar basal epithelial (A549), human cervix carcinoma
(HeLa), human liver carcinoma (HepG2), and
human breast cancer (MCFꢀ7) cell lines. The most
active derivatives were further studied to determine the
role of apoptosis in the cytotoxic activity.
Oxadiazines bearing heteroatoms at positions 1, 2,
and 4 or 1, 3, and 4 are interesting and promising hetꢀ
erocyclic compounds, as they possess diverse biologiꢀ
cal activities [1]. There are some reports in the literaꢀ
ture on their cardiovascular, antibacterial, insecticidal,
and anticonvulsive activities [2, 3]. In addition, oxadiꢀ
azines are useful intermediates in the synthesis of
RESULTS AND DISCUSSION
tenidap prodrugs or ꢀlactam antibiotics, particularly,
β
Chemical Synthesis
the synthesis of carbapenems and penems [4, 5].
7ꢀChloroꢀ1,5ꢀdimethylꢀ3ꢀphenylꢀ1
Hꢀpyrimido
On the other hand, pyrimidineꢀcontaining comꢀ
pounds have been reported to offer a variety of antiꢀ
cancer potentials including antitumor [6], antineoꢀ
plastic [7], antiproliferative [8, 9], and cyclinꢀdepenꢀ
dent kinase [10], angiogenesis [11] and dihydrofolate
reductase [12] inhibitory activities.
[4,5ꢀ ][1,3,4]oxadiazine (III) was prepared by the
e
reaction of 5ꢀbromoꢀ2ꢀchloroꢀ6ꢀmethylꢀ4ꢀ(1ꢀmethꢀ
ylhydrazino)pyrimidine (II) with benzoyl chloride in
the presence of K2CO3 according to previous published
method [13]. Subsequent treatment of compound (III
with pyrrolidine in boiling ethanol led to the replaceꢀ
ment of chorine atom to give 1,5ꢀdimethylꢀ3ꢀphenylꢀ7ꢀ
(pyrrolidinꢀ1ꢀyl)ꢀ1 ꢀpyrimido[4,5ꢀ ][1,3,4]oxadiazꢀ
ine (IV). On the other hand, compound (III) was treatꢀ
ed with hydrazine hydrate and then with triethyꢀ
lorthoacetate in boiling acetic acid to give compound ( ).
)
The promising therapeutic potential of this class of
heterocycles prompted us to synthesize various pyrimꢀ
idooxadiazine and triazolopyrimidooxadiazine derivꢀ
atives and evaluate the antiproliferative profiles of the
obtained derivatives against a panel of four human
solid tumor cell lines: adenocarcinomic human alveoꢀ
H
e
V
Moreover, heating the hydrazino derivative of comꢀ
pound (III) and CS2 in dry pyridine and conversion of
the resulted product into the corresponding alkyl deꢀ
rivatives by reaction with ethylbromide and chloroacꢀ
1
The article is published in the original.
Corresponding authors: eꢀmail: alishiri@um.ac.ir (A. Shiri);
2
tayaraninz@mums.ac.ir.
201

202
SEYEDꢀHADI MOUSAVI et al.
etonitrile provided the desired compounds (VIa) and compounds (III)–(VI) are based upon the spectroꢀ
(VIb), respectively. The structural assignments of scopic and microanalytical data (Scheme 1).
CH3
N
N
Cl
O
H2N
N
Cl
+
Br
CH3
(
II
)
(I)
K2CO
3
CH3CN
CH3
N
N
Cl
N
N
O
CH3
1) N H , EtOH
2
2) CS , Pyridine
4
(
III)
Pyrrolidine
EtOH
2
3) RX, Et N
3
1) N H , EtOH
2
4
2) CH C(OEt)
DMF/CH CN
3
3
AcOH
3
CH3
CH3
N
N
N
N
N
N
N
N
CH3
N
N
N
N
N
O
N
O
N
N
CH3
SR
CH3
N
O
(IV)
(
(
VIa): R = –CH2CH3
VIb): R = –CH2CN
CH3
CH3
(V)
Scheme 1.
Cytotoxicity of the Synthesized
Compounds (III)–(VI
against a panel of four human tumor cell lines includꢀ
ing A549, HeLa, HepꢀG2, and MCFꢀ7. The IC50
)
against A549 and HeLa for compound (
lated to be 82.64 and 43.75
V) were calcuꢀ
µ
mol L–1, respectively
The cytotoxicities of the synthesized comꢀ
pounds (III)–(VI) were examined in malignant cells.
At first, HeLa cells were incubated with various conꢀ
centrations of compounds (III)–(VI) for 24 h. The
results showed that these compounds decreased the viaꢀ
bility of cells in a concentrationꢀdependent manner.
(Fig. 2), while the IC50 for compound (VIb) against
A549, HeLa, HepG2, and MCFꢀ7 cells were 79.85,
<25, 135.1, and 156.2 μ
mol L–1, respectively, after 48 h
(Fig. 3).
Among them, compounds (V) and (VIb) were found to
The Role of Apoptosis in HeLa Cells Treated
with Compounds (III)–(VI
be more effective than the other derivatives (
P
V
< 0.001)
), (VIa),
)
(Fig. 1). The doses of compounds (III)–(
To determine whether apoptosis is involved in cell
toxicity of compounds ( ) and (VIb) in A549 and
HeLa cell lines, cellular morphological changes
and DNA fragmentation were investigated in these
cells. HeLa cells were exposed to 60
mol L–1 of comꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium broꢀ pound (
), incubated for 48 h, stained with DAPI, and
mide) method for their inꢀvitro cytotoxic effect examined with fluorescent microscopy. The same proꢀ
and (VIb) that induced 50% cell growth inhibition
IC50) against HeLa cells were 1092.0, 244.4, 91.06,
122.0, and 49.95
mol L–1, respectively (Fig. 1). Among
all newly synthesized compounds, compounds ( ) and
VIb) were selected to be evaluated via MTT (3ꢀ(4,5ꢀ
V
(
µ
V
(
µ
V
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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PYRIMIDOOXADIAZINE AND TRIAZOLOPYRIMIDOOXADIAZINE DERIVATIVES
203
120
100
80
60
40
20
0
120
HeLa
HeLa
VIa
VIb
III
IV
V
100
80
60
40
20
0
*
**
*
**
*
*
***
*
*
*
*
*
*
*
**
***
C
62.5
Concentration,
125
mol L–1
250
C
15
Concentration,
31.2 62.5 125
mol L–1
250
µ
µ
–1
Fig. 1. Doseꢀdependent growth inhibition of HeLa cells by compounds (III)–(VI) at different concentrations (μmol L ) after
24 h. Viability was quantitated by MTT assay. IC against HeLa cells for compounds (III)–(VI) were 1092.0, 244.4, 91.06, 122.0, and
50
–1
49.95
µ
mol L , respectively. Results are mean SEM (
n
= 3). *
P
< 0.05, **
P
< 0.01, and ***
P
< 0.001 compared to control (C).
120
120
100
80
60
40
20
0
HeLa
100
80
60
40
20
0
A549
*
*
*
*
***
*
**
**
***
C
25
50
Concentration,
75
100 125 150
mol L–1
C
25
50
Concentration,
100 150 200
mol L–1
µ
µ
–1
Fig. 2. Doseꢀdependent growth inhibition of A549 and HeLa cells by compound (
V
) at different concentration (
) were 82.64 and
43.75 mol L , respectively. Results are mean SEM (n = 3). * P < 0.05, ** P < 0.01, and ***P < 0.001 compared to control (C).
µ
mol L ) after
48 h. Viability was quantitated by MTT assay. IC values against A549 and HeLa cells for compound (
V
50
–1
µ
mol L–1) on
apoptotic process in compounds (
duced cell death (Figs. 5, 6).
V) and (VIb)ꢀinꢀ
tocol was used for compound (VIb) (60
A549 cells. As follows from Figure 4, after treatment
with compounds ( ) and (VIb), the cells stained with
equal intensity of DAPI were condensed and presentꢀ
ed strong blue staining under the fluorescent inverted
microscope confirming that apoptosis had occurred.
After the treatment with compound (VIb) (50 μ
mol L–1),
µ
V
In this study, we investigated the potential antituꢀ
mor activity of fused heterocyclic compounds of pyꢀ
rimidooxadiazine and triazolopyrimidooxadiazine deꢀ
rivatives. Different concentrations of compounds (III)–
(
VI) were tested for their antiproliferative activities.
Structural changes on Cꢀ7 and a triazole moiety fused
to pyrimido[4,5ꢀ ][1,3,4]oxadiazine heterocyclic ring
apoptosis of A549 cells was measured with PI staining
and flow cytometry. The same protocol was used for
e
appear to have considerable effect on the biological
activity of the synthesized compounds. The results
showed that among compounds (III)–(VI), comꢀ
pounds bearing fused triazole ring are more reactive
than those bearing chlorine or pyrrolidine substituents
compounds (
subꢀG1 peak resulting from DNA fragmentation.
Flow cytometry histograms of compounds ( ) and
VIb)ꢀtreated cells exhibited a subꢀG1 peak in A549
V) and (VIb) in HeLa cells to detect the
V
(
and HeLa cells. This indicates the involvement of an on the Cꢀ7 position of the compound (III).
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204
SEYEDꢀHADI MOUSAVI et al.
120
120
100
80
60
40
20
0
HeLa
A549
100
80
60
**
25
**
***
***
**
**
**
40
20
0
**
**
***
C
25 50 75 100 125 150 175
Concentration,
mol L–1
C
50 100 150 200
µ
µ
Concentration,
mol L–1
120
100
80
60
40
20
0
120
100
80
60
40
20
0
HepG2
MCFꢀ7
*
*
**
***
*
***
***
***
C
15.6 31.2 62.5 125 250 500
Concentration,
mol L–1
C
15.6 31.2 62.5 125 250 500
Concentration,
mol L–1
µ
µ
Fig. 3. Doseꢀdependent growth inhibition of A549, HeLa, HepG2, and MCFꢀ7 cells by compound (VIb) at different concentraꢀ
–1
tions (
for compound (VIb) were 79.85, <25, 135.1, and 156.2
** < 0.01, and *** < 0.001 compared to control (C).
µ
mol L ) after 48 h. Viability was quantitated by MTT assay. IC values against A549, HeLa, HepG2, and MCFꢀ7
50
µ
–1
mol L , respectively. Results are mean SEM (n = 3). * P < 0.05,
P
P
Based on these observations, our efforts were foꢀ DNA fragmentation, cells that have lost DNA take up
cused on compounds ( ) and (VIb). They were evaluꢀ less stain and appear to the left of the G1 peak.
ated for their inhibitory activity on A549, HepG2, and
MCFꢀ7 cell lines. We then explored the role of apopꢀ
tosis in compounds ( ) and (VIb)ꢀinduced toxicity. In
the present study, compounds ( ) and (VIb)ꢀinduced
V
CONCLUSION
V
V
In summary, we have identified 1,5,7ꢀtrimethylꢀ
apoptosis was involved in the induction of cell death.
Apoptotic cells exhibit several biochemical modificaꢀ
tions such as protein cleavage, protein crossꢀlinking,
DNA fragmentation, and phagocytic recognition,
which together result in the distinctive structural paꢀ
thology [18]. PIꢀstained cell nuclei permit simple,
quantitative, and reproducible measurement of apopꢀ
tosis. The reduced DNA content of apoptotic nuclei
resulted in an unequivocal hypodiploid DNA peak in
the red fluorescence channels. According to the previꢀ
ous results [18], DNA fragmentation creates small
fragments of DNA that can be eluted and incubated in
a hypotonic phosphate–citrate buffer. When cells are
stained with a quantitative DNAꢀbinding dye, such as
3ꢀphenylꢀ1
][1,3,4]oxadiazine (
ꢀ[1,2,4]triazolo[4',3':1,2]pyrimido[4,5ꢀe
adiazinꢀ7ꢀyl)sulfanyl]acetonitrile (VIb) analogs as a
novel class of antiproliferative agents by a cellꢀbased
screening method.
H
ꢀ[1,2,4]triazolo[4',3':1,2]pyrimido[4,5ꢀ
) and [(1,5ꢀdimethylꢀ3ꢀphenylꢀ
][1,3,4]oxꢀ
e
1
V
H
EXPERIMENTAL
Chemistry
1H and 13C NMR spectra were recorded on a Brukꢀ
er Avance DRXꢀ400 Fourier transformer spectrometer
in CDCl3 at a resonant frequency of 100 MHz using
PI, in order to detect the subꢀG1 peak resulted from tetramethylsilane (TMS) as internal standard; chemiꢀ
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PYRIMIDOOXADIAZINE AND TRIAZOLOPYRIMIDOOXADIAZINE DERIVATIVES
205
(a)
(b)
(c)
(d)
Fig. 4. Evaluation of morphological changes after administrating of compound (VIb) in HeLa and A549 cells. HeLa cells were
–1
exposed to 60
was used for compound (VIb) (60
presented strong blue staining under the fluorescent inverted microscope (magnification 100
µ
mol L of compound (VIb), incubated for 48 h, and examined for apoptosis by DAPI staining. The same protocol
–1
µ
mol L ) in A549 cells. Cells stained with an equal intensity of DAPI were condensed and
). (a) Control of A549 cells,
×
–1
–1
(b) compound (VIb), 60
µ
mol L , (c) control of HeLa cells, (d) compound (VIb), 60 mol L .
µ
cal shifts are reported in
δ
, ppm. The infrared (IR)
Synthesis of 1,5,7ꢀtrimethylꢀ3ꢀphenylꢀ1
Hꢀ[1,2,4]triꢀ
spectra (νmax, cm–1) were recorded on an Avatar 370
FTꢀIR Thermo Nicolet equipment using KBr discs.
The mass spectra were recorded on a Varian Mat
CHꢀ7 instrument with 70 eV ionizing energy. Elemenꢀ
azolo[4',3':1,2]pyrimido[4,5ꢀ ][1,3,4]oxadiazine (V).
e
To a solution of 7ꢀchloroꢀderivative (III) (3.7 mmol,
1.1 g) in ethanol (20 mL), hydrazine hydrate (2 mL)
was added, and the solution was refluxed for 5 h. The
tal analysis was performed on a Thermo Finnigan resulting precipitate was filtered off and recrystallized
Flash EA microanalyzer.
from ethanol. Then, to a solution of the prepared hyꢀ
drazine derivative in HOAc (4 mL), triethylorthoaceꢀ
tate (4 mmol) was added. The reaction solution was
heated under reflux for 4 h. After the completion of the
reaction which was monitored by TLC using chloroꢀ
form–methanol (9 : 1), the mixture was cooled to
room temperature. Water (10 mL) was added and the
mixture was neutralized by saturated NaHCO3 soluꢀ
tion. The collected solid was recrystallized from ethaꢀ
Synthesis of 1,5ꢀdimethylꢀ3ꢀphenylꢀ7ꢀpyrrolidinoꢀ
1
H
ꢀpyrimido[4,5ꢀ
of 7ꢀchloroꢀ1,5ꢀdimethylꢀ3ꢀphenylꢀ1
][1,3,4]oxadiazine (III) (1 mmol, 0.27 g) [13] and pyrꢀ
e][1,3,4]oxadiazine (IV). A mixture
H
ꢀpyrimido[4,5ꢀ
e
rolidine (2.2 mmol) in ethanol (5 mL) was heated unꢀ
der reflux for about 2 h. After the mixture was cooled,
water was added to the solution. The resulting solid
was collected by filtration, washed with water, and reꢀ
nol. Yield 72%; mp 324–326°
C; 1H NMR: 2.60 (s,
crystallized in ethanol. Yield 79%; mp 168
J
°C;
= 4 Hz, 4H, CH2), 2.21 (s, 3H,
1H NMR : 1.93 (t,
CH3ꢀpyrimidine), 3.24 (s, 3H, CH3ꢀoxadiazine), 3.51
3H, CH3ꢀpyrimidine), 2.75 (s, 3H,CH3ꢀtriazol), 3.42
(s, 3H, CH3–N), 7.32–7.81 (m, 5H, ph), 13C NMR:
15.5 (CH3ꢀtriazole), 16.7 (CH3ꢀpyrimidine), 42.4
(N–CH3), 128.2, 128.4, 128.8, 128.9, 129.8, 131.3
(C of phenyl ring), 136.7 (O–C), 149.5 (N–C=N),
149.9 (O–C=N), 152.4 (N–C=N), 155.6 (C=N),
157.2 (N=C–N); IR: 3000, 2900, 1610, 1550, 1490,
13
(t, 4H, CH2), 7.4–7.8 (m, 5H, phenyl); C NMR:
18.5 (CH3), 25.5 (CH2), 25.6 (CH2), 42.7 (N–CH3),
54.3 (NCH2), 54.5 (NCH2), 126.7 (O–C), 128.1,
128.2, 128.8, 128.9, 129.6, 131.1 (C of phenyl ring),
147.4 (O–C=N), 152.9 (N–C=N), 155.8 (C=N),
1005; MS (
C15H14N6O (%): C, 61.22; H, 4.79; N, 28.55. Found:
m/z): 294 (M
+). Anal. calcd. for
158.2 (N–C–N); IR: 3065, 2940, 1155; MS (
309 (
+). Anal. calcd. for C17H19N5O (%): C, 66.00;
H, 6.19; N, 22.64. Found: C, 65.79; H, 6.12; N, 22.50. C, 61.10; H, 4.72; N, 28.48.
m/z):
M
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SEYEDꢀHADI MOUSAVI et al.
200
160
120
80
100
80
60
40
20
0
22.5%
Sub G1 Peak 5.3%
40
0
0.1
1
10
100
1000
0.1
1
10
100
1000
Fluorescent intensity
Fig. 5. Flow cytometry histograms of apoptosis assay by PI method in A549 cells. Cells were treated with compound (VIb
)
–1
(50
µ
mol L ) for 48 h. SubꢀG1 peak as an indicative of apoptotic cells was induced in compound (VIb)ꢀtreated, but not in conꢀ
trol cells. (Left to right: control; compound (VIb).)
400
320
240
160
80
200
160
120
80
200
160
120
80
14.51%
40.54%
38.25%
40
40
0
0.1
0
0.1
0
100 1000 0.1
1
10
100 1000
1
10
1
10
100 1000
Fluorescent intensity
100
80
200
160
120
80
60
40
20
46.81%
42.14%
40
0
0.1
0
100 1000 0.1
1
10
1
10
100 1000
Fig. 6. Flow cytometry histograms of apoptosis assays by PI method in HeLa cells. Cells were treated with compounds (V) and
–1
(
VIb) (30 and 60
μ
mol L ) for 48 h. SubꢀG1 peak as an indicative of apoptotic cells was induced in compounds (
V
) and (VIb)ꢀ
–1
–1
treated, but not in control cells. (Left to right (up): control; compound (
to right (down): compound (VIb), 30
V), 30
µ
mol L ; and compound (
V), 60
µ
mol L . Left
–1
–1
µ
mol L , and compound (VIb), 60 µmol L ).)
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PYRIMIDOOXADIAZINE AND TRIAZOLOPYRIMIDOOXADIAZINE DERIVATIVES
207
General Procedure for the Preparation
of 7ꢀAlkylsulfinylꢀ1,5ꢀDimethylꢀ3ꢀPhenylꢀ1Hꢀ
[1,2,4]triazolo [4',3':1,2]pyrimido[4,5ꢀ
cin. The cells were seeded overnight and then incubated
with various concentrations of compounds (III)–(VI
)
for 24 h and 48 h. The cells were seeded at
5000 cells/well onto 96ꢀwell culture plates for MTT
assay and at 100000 cells/well onto a 24ꢀwell plate for
apoptosis assay. There was a control sample for time
course study in each concentration and they also reꢀ
ceived the equal volume of medium.
e][1,3,4]oxadiazines (VIa) and (VIb
3ꢀPhenylꢀ1,5ꢀdimethylꢀ7ꢀhydrazinoꢀ1
do[4,5ꢀ ][1,3,4]oxadiazine (1 mmol, 0.27 g) prepared
)
Hꢀpyrimiꢀ
e
from the previous part and CS2 (1 mL) was refluxed in
dry pyridine (7 mL) for 6 h. Then, the mixture was
cooled to room temperature and the resulting solid
was filtered off and recrystallized from ethanol. Conꢀ
sequently, the precipitant and an appropriate alkyl haꢀ
lide (0.9 mmol) and triethylamine (0.9 mmol) were
heated in a mixture of DMF–MeCN (1 : 5) (12 mL)
as solvent for 4 h. Then, the solvent was removed under
reduced pressure. The crude solid was recrystallized
from ethanol.
Cell viability. Cell viability was determined using a
modified MTT assay [14, 15]. Briefly, the cells were
seeded (5000 cells/well) onto flatꢀbottomed 96ꢀwell
culture plates and allowed to grow for 24 h followed by
treatment with compounds (III)–(VI). After removꢀ
ing the medium, the cells were labeled with MTT soꢀ
lution [5 mg mL–1 in phosphate buffered saline (PBS)]
for 4 h and the resulting formazan was dissolved in
DMSO (100 L). The absorption was measured at 570
nm (620 nm as a reference) in an enzymeꢀlinked imꢀ
munosorbent assay (ELISA) reader.
μ
7ꢀEthylsulfinyl derivative (VIa). Yield 67%, mp
221–223°
C, 1H NMR: 1.43 (t, 3H, CH3), 2.73 (s, 3H,
CH3ꢀpyrimidine), 3.28 (q, 2H, CH2), 3.45 (s, 3H,
CH3–N), 7.36–7.53 (m, 3H, ph), 7.74–7.88 (m, 2H,
DAPI staining. The cancer cells were incubated on
glass cover slips in a six well plate. Twentyꢀfour hours
13
ph); C NMR: 13.6 (CH3), 15.3 (CH3ꢀpyrimidine),
later, the cells were treated with compounds (
V) and
30.4 (S–CH2), 43.1 (N–CH3), 128.2, 128.3, 128.7,
128.8, 129.7, 131.2 (C of phenyl ring), 136.9 (O–C),
146.5 (S–C=N), 149.8 (O–C=N), 152.3 (N–C=N),
155.7 (C=N), 157.3 (N=C–N); IR: 3010, 2980, 1590;
(VIb) for 48 h. After washing the cells with PBS for
three times, they were fixed in 4% formaldehyde for
30 min and permeabilized with 3% Triton Xꢀ100 for
30 min. The cell nuclei were then stained with 4',6ꢀdiꢀ
amidinoꢀ2ꢀphenylindole (DAPI, Sigma) and examꢀ
ined under the fluorescent inverted microscope. Cells
with condensed or fragmented nuclei were considered
to be apoptotic.
MS (m/z): 340 (M
+), Anal. calcd. for C16H16N6OS (%):
C, 56.45; H, 4.74; N, 24.69; S, 9.42. Found: C, 56.40;
H, 4.62; N, 24.51; S, 9.33.
Cyanomethyl derivative (VIb). Yield 63%, mp 238–
240°
C, 1H NMR: 2.77 (s, 3H, CH3ꢀpyrimidine), 3.47
Apoptosis. Apoptotic cells were detected using PI
staining of treated cells followed by flow cytometry to
detect the soꢀcalled subꢀG1 peak [16, 17]. It has been
reported that DNA fragmentation creates small fragꢀ
ments of DNA that can be eluted and incubated in a
hypotonic phosphateꢀcitrate buffer. When stained
with a quantitative DNAꢀbinding dye such as PI, cells
that have lost DNA will take up less stain and appear
to the left of the G1 peak. Briefly, A549 and HeLa cells
were cultured overnight in a 24ꢀwell plate and treated
(s, 3H, CH3–N), 4.03 (s, 2H, CH2CN), 7.40–7.85
(m, 5H, ph); 13C NMR: 15.3 (CH3ꢀpyrimidine), 29.1
(S–CH2), 42.8 (N–CH3), 117.5 (CN), 128.1, 128.3,
128.6, 128.7, 129.8, 131.3 (C of phenyl ring), 137.3
(O–C), 146.7 (S–C=N), 149.7 (O–C=N), 152.4
(N–C=N), 155.6 (C=N), 157.5 (N=CꢀN); IR: 3020,
2980, 2220, 1570; MS (m/z): 351 (M
+); Anal. calcd.
for C16H13N7OS (%): C, 54.69; H, 3.73; N, 27.90; S,
9.13. Found: C, 54.60; H, 3.70; N, 27.87; S, 9.02.
with compounds (
adherent cells were then harvested and incubated at
C overnight in the dark with 750 L of a hypotonic
buffer (50
g mL–1 PI in 0.1% sodium citrate plus
0.1% Triton Xꢀ100) before flow cytometric analysis.
Cells were evaluated in a FACScalibur flow cytometer
V) and (VIb) for 48 h. Floating and
The newly synthesized compounds (III)–(VI
)
were dissolved at a concentration of 25 mmol L–1 in
dimethylsulfoxide (DMSO) as a stock solution that
4°
μ
μ
was stored at –20°C and diluted with cell medium beꢀ
fore each experiment.
Cell culture. The fluorescent probe propidium ioꢀ
dide (PI), sodium citrate, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
2,5ꢀdiphenyl tetrazolium (MTT), and Triton Xꢀ100
were purchased from Sigma (St Louis, MO, USA).
RPMI and FCS were purchased from Gibco (Grand
Island, USA). A549, HeLa, HepG2, and MCFꢀ7 cell
lines were obtained from Pasteur Institute (Tehran,
(Partec, Münster, Germany) using the Flomax softꢀ
ware.
ACKNOWLEDGMENTS
The authors would like to thank Research Affairs of
Mashhad University of Medical Sciences and Ferꢀ
dowsi University of Mashhad for financially supportꢀ
ing this work. We also thank Dr. H. Nasirli for her kind
assistance in flow cytometry. We are also grateful for
Iran) and maintained at 37°C in a humidified atmoꢀ
sphere (90%) containing 5% CO2. The cell lines were
cultured in Dulbecco’s modified Eagle’s medium
(RPMI) with 5% (v/v) fetal bovine serum (FBS),
100 units mL–1 penicillin, and 100 g mL–1 streptomyꢀ the editorial assistance of Dr. N. TayaraniꢀNajaran.
μ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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2015

208
SEYEDꢀHADI MOUSAVI et al.
10. Verma, S., Nagarathnam, D., Shao, J., Zhang, L.,
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