Synthesis and evaluation of cytotoxicity of 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles

Article abstract of DOI:10.1134/S1068162016020047

Several derivatives of 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles were synthesized via Biginelli type reaction and tested for their anti-proliferative activity on human breast cancer (MCF-7) and human colon carcinoma (HT29) cell

Full text of DOI:10.1134/S1068162016020047

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 3, pp. 316–322. © Pleiades Publishing, Ltd., 2016.
Synthesis and Evaluation of Cytotoxicity of 6ꢀAminoꢀ4ꢀArylꢀ2ꢀ
Thioxoꢀ1,2,3,4ꢀTetrahydropyrimidineꢀ5ꢀCarbonitriles1
Hoda AtapourꢀMashhada, Mohammad Soukhtanloob, Abdolhossien Massoudia,
2
Ali Shiric, and Mehdi Bakavolic,
aChemistry Department, Payame Noor University, 19395ꢀ4697 Tehran, I. R. Iran
bDepartment of Clinical Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
cDepartment of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, 91775ꢀ1436 Iran
Received June 5, 2015; in final form, August 10, 2015
Abstract—Several derivatives of 6ꢀaminoꢀ4ꢀarylꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahydropyrimidineꢀ5ꢀcarbonitriles
were synthesized via Biginelli type reaction and tested for their antiꢀproliferative activity on human breast
cancer (MCFꢀ7) and human colon carcinoma (HT29) cell lines. Malignant and nonꢀmalignant cells were
cultivated in RPMI medium and incubated with different concentrations of these pyrimidines. Cell viability
was evaluated by MTT assay. Apoptotic cells were determined using DAPI (4'ꢀ6ꢀdiamidinoꢀ2ꢀphenylindole)
and propidium iodide staining of DNA fragmentation by flow cytometry (subꢀG1 peak). 6ꢀAminoꢀ4ꢀ(4ꢀ
chlorophenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahydropyrimidineꢀ5ꢀcarbonitrile
and
6ꢀaminoꢀ4ꢀ[4ꢀdimethyꢀ
lamino)phenyl]ꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahydropyrimidineꢀ5ꢀcarbonitrile decreased the viability of MCFꢀ7
and HT29 cells, in contrast to L929 cells. These compounds induced a subꢀG1 peak inflow cytometry histoꢀ
grams of treated cells indicating that apoptosis is involved in their toxicity.
Keywords: apoptosis, cytotoxic evaluation, pyrimidines, toxicity
DOI: 10.1134/S1068162016020047
INTRODUCTION
oplastic agents due to its inhibition of DNA synthesis
[9, 10].
Cancer is a group of diseases characterized by
uncontrolled growth and spread of abnormal cells. If
the spread is not controlled, it can result in a death.
Breast cancer is the most common cause of cancer
death among women (522000 deaths in 2012) and the
most frequently diagnosed cancer among women in
140 of 184 countries worldwide [1]. Similarly, the colꢀ
orectal cancer continues to be a major health problem
worldwide [2, 3]. At present, with the development of
molecular biology, chemotherapy is becoming a more
important therapeutic method. Hence, the search for
new drugs has always been in demand in order to
Thioꢀsubstituted pyrimidine derivatives occupy a
special position among these compounds. They are
also identified as potential antimicrobial and anticanꢀ
cer [11–18], as well as antiꢀinflammatory agents [19].
Prompted by these reports and in continuation of our
endeavor on potential anticancer agents [20, 21], we
have now focused our attention on preparing a series
of 6ꢀaminoꢀ4ꢀphenylꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahydroꢀ
pyrimidineꢀ5ꢀcarbonitriles (I)–(X) and screened
them for antiproliferative activity against human
breast cancer (MCFꢀ7), human colon carcinoma
(HT29), and nonꢀmalignant mouse fibroblast (L929)
improve the efficiency of response to anticancer cheꢀ cell lines. The MCFꢀ7 and HT29 cell lines are known
as widely used model systems for the study of breast
[22] and colorectal [23] cancers.
motherapy.
Being a building unit of DNA and RNA, pyrimiꢀ
dine derivatives are found to be associated with a variꢀ
ety of chemotherapeutic effects, including antineoꢀ
plastic [4, 5], angiogenic [6], and enzyme inhibitory
effects [7, 8]. Literature survey revealed that 5ꢀfluoꢀ
rouracil (5ꢀFU), one of the earlyꢀprepared metaboꢀ
lites, has become one of the most widely used antineꢀ
RESULTS AND DISCUSSION
Chemistry
Several methods are well known for the syntheꢀ
sis of pyrimidine derivatives. In the current study,
Biginelli type reaction was utilized for the syntheꢀ
sis of substituted pyrimidine derivatives [24, 25].
According to the recent report by Ghodasara et al.
[17], several derivatives of 6ꢀaminoꢀ4ꢀphenylꢀ2ꢀ
1
The article is published in the original.
2
Corresponding author: phone: eꢀmail: mbakavoli@um.ac.ir,
mbakavoli@yahoo.com.
316

SYNTHESIS AND EVALUATION OF CYTOTOXICITY
317
thioxoꢀ1,2,3,4ꢀtetrahydropyrimidineꢀ5ꢀcarboniꢀ
trile, and thiourea in EtOH in the presence of
triles were prepared by oneꢀpot and threeꢀcompoꢀ sodium ethoxide at room temperature as shown in
nent reaction between aryladehydes, malononiꢀ Scheme.
H
H2N
NC
N
S
NH
NC2
NC
NC
NH
EtONa/EtOH
rt
+
+
S
CHO
NH
NC
R
2
(
(
(
(
I
): R = H; (II): R = 3ꢀNO2; (III): R = 4ꢀOMe;
IV): R = 4ꢀOH; ( ): R = 2ꢀCl; (VI): R = 4ꢀCl;
VII): R = 4ꢀMe; (VIII): R = 3ꢀBr; (IX): R = 4ꢀBr;
): R = 4ꢀN(Me)2
R
V
(I)–(X)
X
Scheme.
The results show that the electron withdrawing subꢀ decreased cell viability in a timeꢀ and concentrationꢀdeꢀ
stituents on phenyl ring can facilitate the cyclization pendent manner. Dose inducing 50% cell growth inhiꢀ
process, while the electron donating substituents have bition (IC50) for the tested compounds was calculated
adverse effects.
and presented in Table 1.
Structure–activity relationship studies revealed
that biological activity is largely dependent on the R
The structural assignments of the newly syntheꢀ
sized compounds were based upon spectral and eleꢀ
mental analysis data. The previously reported comꢀ
pounds were identified by comparing their physical and
spectral data with those of the authentic samples. For exꢀ
substituents. Compound (I) showed a doseꢀ and timeꢀ
dependent growth inhibition against MCFꢀ7 and HT29
cell lines. Compounds (II) and (VII) showed similar efꢀ
fects. Our results reveal that compounds containing
halogen groups showed good toxicity effects on two cell
ample, the IR spectrum of compound (
X
) showed the
stretching vibration bands of C=S at 1157 cm–1, C
≡N at
2179 cm–1, NH2 at 3406 and 3328 cm–1, and NH at lines in comparison with the phenyl analog. Among
3201 cm–1, respectively. The 1H NMR spectrum of comꢀ
pound ( ) in DMSOꢀ 6 showed the D2O exchangeable
peaks of NH2 and two NH at 6.08, 9.63, and 9.91 ppm,
respectively; the signals of two methyl groups were asꢀ
signed at 2.88 ppm and the singlet signal of C–H, at
4.84 ppm. Two characteristic doublet signals around
6.70–7.05 ppm were determined for aromatic ring
protons. In 13C NMR spectrum, the appearance of the
carbon signals of the aryl moiety at 142.1, 130.4,
125.2, and 123.0 ppm, as well as the thiocarbonyl at
174.3, nitrile at 119.8, and methyl groups at
35.6 ppm, confirmed the heterocyclization. The
mass spectrum of compound ( ) showed the molecuꢀ
lar ion peak at
273 (M+) corresponding to the moꢀ
lecular formula C13H15N5S.
halogenated derivatives, compounds (
onstrated more potent antiproliferative activities than
the other halogenated compounds. Similarly, comꢀ
pound ( ), containing dimethylamino group, showed
various effects on two cell lines and had better antiproꢀ
liferative activity on HT29 cell line after both 48 and
72 h treatment. The other analogs showed no significant
toxicity. Generally speaking, compounds (VI) and (X)
were found to be more effective than other derivatives.
In order to compare the cytotoxicity of compounds
V) and (VI) demꢀ
X
d
δ
X
δ
δ
δ
(VI) and (X) against malignant and nonꢀmalignant
δ
δ
cells, another MTT assay was carried out using mouse
fibroblast (L929) cells. The results showed that both
compounds decreased cell viability in malignant cells
more than in nonꢀmalignant ones (Fig. 1).
δ
X
m/z
The role of apoptosis in HT29 cells treated with 6ꢀ
aminoꢀ4ꢀarylꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahydropyrimidineꢀ
5ꢀcarbonitrile derivatives (I)–(X). Recently pyrimidine
Pharmacology
Cytotoxicity of 6ꢀaminoꢀ4ꢀarylꢀ2ꢀthioxoꢀ1,2,3,4ꢀ derivatives were discovered as potent apoptosis inducers
tetrahydropyrimidineꢀ5ꢀcarbonitrile derivatives (I)–(X). [29, 30]. Apoptotic cells exhibit several biochemical
Many nucleoside analogs have been reported to be potent modifications such as protein cleavage, protein crossꢀ
growth inhibitors in cell cultures [26–28]. Correspondꢀ linking, DNA fragmentation, and phagocytic recogniꢀ
ingly, in this study, the target products (
screened in vitro against MCFꢀ7 and HT29 using MTT pathology [31]. Herein, to determine whether apoptoꢀ
assay. First, malignant cells were incubated with various sis is involved in the toxicity of compounds (VI) and
concentrations of compounds ( )–( ) (15–500 M) for ) against MCFꢀ7 cells, DNA fragmentation was inꢀ
48 and 72 h. The results showed that these compounds vestigated in these cells. The cells were incubated with
I)–(X) were tion that altogether result in the distinctive structural
I
X
µ
(X
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 3
2016

318
HODA ATAPOURꢀMASHHAD et al.
Table 1. Doses inducing 50% cell growth inhibition (IC50) of MCFꢀ7 and HT29 cells for compounds (I)–(X)
IC50, μM
Entry
R
MCFꢀ7
HT29
48 h
72 h
48 h
72 h
(
I
)
)
)
)
)
)
)
)
)
)
H
251
791
353
521
95
211
143
267
289
92
157
154
364
453
42
47
70
87
(
II
III
IV
3ꢀNO2
4ꢀOMe
4ꢀHO
2ꢀCl
(
(
309
29
25
78
35
59
3
(
V
(
VI
VII
VIII
IX
4ꢀCl
78
70
30
(
4ꢀMe
3ꢀBr
454
120
129
453
170
72
181
61
(
(
4ꢀBr
111
80
74
(
X
4ꢀN(Me)2
21
Results are Mean SEM (n = 3).
30
μ
M of compound (VI) or (
X
) for 48 h, stained with
CONCLUSION
A series of 6ꢀaminoꢀ4ꢀarylꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetꢀ
rahydropyrimidineꢀ5ꢀcarbonitriles ( )–( ) were preꢀ
pared and evaluated for the antiprolifrative activity
against MCFꢀ7, HT29, and L929 cell lines by a cellꢀ
DAPI, and evaluated by fluorescent microscopy. As
shown in Fig. 2, cells stained with equal intensity of
DAPI were condensed and presented strong blue
staining under the fluorescent inverted microscope afꢀ
ter treatment with compounds (VI) and (
confirmed the occurrence of apoptosis.
I
X
X), which
based screening method. Compounds (VI) and (X)
were found to be more potent than their other analogs,
with apoptosis playing an important role in cell death
induced by these compounds.
It has been reported that DNA fragmentation creꢀ
ates small fragments of DNA that can be eluted folꢀ
lowing incubation in a hypotonic phosphate–citrate
buffer. When cells are stained with a quantitative
DNAꢀbinding dye, such as PI, to detect the subꢀG1
peak resulting from DNA fragmentation, the cells that
have lost DNA would take up less stain and appear to
the left of the G1 peak [32]. So apoptosis that followed
EXPERIMENTAL
Chemistry
Melting points were recorded on a Krüss Melting
Point Meter in an open capillary tube and are uncorꢀ
rected. The IR spectra (νmax, cm–1) were recorded in
for KBr pellets on a Buck Scientific spectrophotomeꢀ
the treatment with compounds (VI) and (
and 60 M) was measured with PI staining and flow
cytometry to detect the subꢀG1 peak resulting from
DNA fragmentation. Flow cytometry histograms of
cells treated with compounds (VI) and ( ) exhibited a
subꢀG1 peak in HT29 cells. It is the indication of the
involvement of an apoptotic process in compound
VI)ꢀ and ( )ꢀinduced cell death (Fig. 3 and Table 2).
X) (15, 30,
μ
ter. The 1H NMR and 13C NMR spectra (
δ
, ppm) were
recorded on a Bruker (400 MHz) spectrometer using
DMSOꢀ 6 or CDCl3 as solvents and TMS as the interꢀ
X
d
nal reference standard. Mass spectra were taken on an
LKB 9000 mass spectrometer. Chemical ionization
mass spectra were recorded on an Eager 300 elemental
analyzer. The purification of compounds was routinely
checked by TLC using silica gel and the spots were exꢀ
posed in iodine vapor for visualization.
(
X
Table 2. Apoptotic cells determined using PI staining of
DNA fragmentation and flow cytometry (subꢀG1 peak)
Apoptosis, %
General Procedure for Synthesis of 6ꢀAminoꢀ4ꢀArylꢀ
2ꢀThioxoꢀ1,2,3,4ꢀTetrahydropyrimidineꢀ5ꢀ
Concentration,
HT29
µ
M
Carbonitriles (I)–(X)
(VI)
(X)
To a magnetically stirred solution of appropriate
benzaldehyde (1 mmol), malononitrile (1 mmol,
0.066 g), and thiourea (1 mmol, 0.076 g) in ethanol
(5 mL), sodium ethoxide (2 mmol) was added and the
mixture was stirred at room temperature. When the reꢀ
0
6.1
38.8
36.6
46.0
6.1
29.7
35.1
38.2
15
30
60
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 3
2016

SYNTHESIS AND EVALUATION OF CYTOTOXICITY
319
Compound
(
VI
)
(
VI
)
MCFꢀ7_48 h
HT29_48 h
L929_48 h
MCFꢀ7_72 h
HT29_72 h
L929_72 h
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
**
***
**
***
***
***
***
C
15
31
Concentration,
62 125 250 500
µ
C
15
31
Concentration, M
62 125 250 500
µ
M
(
X
)
(
X
)
MCFꢀ7_48 h
HT29_48 h
L929_48 h
MCFꢀ7_72 h
HT29_72 h
L929_72 h
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
**
31
**
***
**
15
**
***
***
**
**
*
**
**
31
**
***
***
***
*** ***
C
15
62 125 250 500
µ
C
62 125 250 500
Concentration, µM
Concentration,
M
Fig. 1. Doseꢀdependent growth inhibition of MCFꢀ7, HT29, and L929 cells by compounds (VI) and (
and 72 h. Viability was quantified by MTT assay. Results are Mean SEM
compared with control.
X
) (15–500 ) after 48
µ
M
(
n
= 3). *
P
< 0.05, **
P
< 0.01, and *** P < 0.001,
Fig. 2. The evaluation of morphological changes after administrating of compounds (VI) and (
exposed to 30 of compounds (VI) and ( ), incubated for 48 h. Cells stained with an equal intensity of DAPI were condensed
and presented strong blue staining under the fluorescent inverted microscope (magnification ×100). Left to right: control of
MCFꢀ7 cells; compound (VI) (30 ); and compound ( ) (30 ).
X) in MCFꢀ7 cells. Cells were
µ
M
X
µ
M
X
µ
M
action was completed, which was monitored by TLC
using CHCl3 : MeOH (9 : 1), the solvent was removed
under reduced pressure. Then, water (5 mL) was addꢀ
ed to the resulting precipitate and the mixture was
neutralized by 0.1 N HCl solution. The solid was filꢀ
6ꢀAminoꢀ4ꢀphenylꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahydroꢀ
pyrimidineꢀ5ꢀcarbonitrile (I). Yield 85%, mp 154
°C
(lit. 150–152°C [17, 25]).
6ꢀAminoꢀ4ꢀ(3ꢀnitrophenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetꢀ
rahydropyrimidineꢀ5ꢀcarbonitrile (II). Yield 56%, mp
137°C (lit. 131–133°C [17]).
tered and washed with CH2Cl2 (2 × 10 mL).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 3
2016

320
HODA ATAPOURꢀMASHHAD et al.
(a)
(b)
128
64
Gm: 46.35
CV: 50.71
[33ꢀ639] 401 (6.1%)
Gm: 71.57
CV: 30.16
[33ꢀ639] 1528 (38.0%)
M1
M1
0
100
0
100
101
102
FL2ꢀH
103
104
101
102
FL2ꢀH
(d)
103
104
(c)
32
64
Gm: 62.46
CV: 32.86
[33ꢀ639] 1304 (36.6%)
Gm: 66.72
CV: 32.82
[33ꢀ639] 2293 (46.0%)
M1
M1
0
100
0
100
101
102
FL2ꢀH
103
104
101
102
FL2ꢀH
103
104
(e)
(f)
64
128
Gm: 94.21
CV: 28.88
[33ꢀ639] 1375 (29.7%)
Gm: 41.77
CV: 40.90
[33ꢀ639] 3509 (35.1%)
M1
M1
0
100
0
100
101
102
FL2ꢀH
103
104
101
102
FL2ꢀH
103
104
(VI)
(X)
(g)
HT29
32
50
45
40
35
30
25
20
15
10
5
Gm: 52.70
CV: 41.67
[33ꢀ639] 1136 (38.2%)
M1
0
100
0
101
102
FL2ꢀH
103
104
Control 15 30
Treatments, µM
60
Fig. 3. Flowꢀcytometry histograms of apoptosis assays by PI method in HT29 cells. Cells were treated with compounds (VI) and
) (15, 30, and 60 ) for 48 h. SubꢀG1 peak, as indication of apoptotic cells, was induced in cells treated with compounds (VI
and ( ), but not in control cells. (a) control; (b) compound (VI) (15 M); (c) compound (VI) (30 M); (d) compound (VI
(60 M); (e) compound ( ) (15 M); (f) compound ( ) (30 M); (g) compound ( ) (60 M).
(X
µ
M
)
)
V
µ
µ
µ
X
µ
X
µ
X
µ
6ꢀAminoꢀ4ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀ
tetrahydropyrimidineꢀ5ꢀcarbonitrile (III). Yield 41%, tetrahydropyrimidineꢀ5ꢀcarbonitrile (IV). Yield 37%,
mp 124 C (lit.118–120 C [17, 25]). mp 146 C (lit. 141–143 C [17]).
6ꢀAminoꢀ4ꢀ(4ꢀhydroxyphenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀ
°
°
°
°
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 3
2016

SYNTHESIS AND EVALUATION OF CYTOTOXICITY
321
6ꢀAminoꢀ4ꢀ(2ꢀchlorophenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀ
centration used as solvent control did not exceed 1%
tetrahydropyrimidineꢀ5ꢀcarbonitrile (V). Yield 75%, throughout the study. The fluorescent probes propidiꢀ
mp 154
6ꢀAminoꢀ4ꢀ(4ꢀchlorophenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀ
tetrahydropyrimidineꢀ5ꢀcarbonitrile (VI). Yield 78%,
mp 127 C (lit. 123–125 C [17]).
6ꢀAminoꢀ4ꢀ(4ꢀmethylphenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀ
tetrahydropyrimidineꢀ5ꢀcarbonitrile (VII). Yield 81%,
mp 129 C (lit. 126–128 C [17]).
6ꢀAminoꢀ4ꢀ(3ꢀbromophenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀ
tetrahydropyrimidineꢀ5ꢀcarbonitrile (VIII). Yield
71%, mp 130 C; IR spectrum: 3417–3317 (NH2),
3193 (NH), 2195 (C
(DMSOꢀ 6, 400 MHz): 5.42 (s, 1H, CHꢀAr), 6.17 (s,
°C (lit. 148–150°C [17]).
um iodide (PI) and 4'ꢀ6ꢀdiamidinoꢀ2ꢀphenylindole
(DAPI), 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl
tetrazolium (MTT), sodium citrate, and Triton Xꢀ100
were purchased from Sigma (St Louis, MO, USA).
RPMI and fetal calf serum were purchased from Gibꢀ
co (Grand Island, New York, USA).
°
°
Cell culture. Human breast cancer cell line (MCFꢀ7)
and human colon carcinoma cell line (HT29) cancerꢀ
ous and mouse fibroblast (L929) nonꢀmalignant cells
were obtained from the Pasteur Institute (Tehran,
°
°
°
Iran) and maintained at 37°C in a humidified atmoꢀ
1
≡
N), 1168 (C=S); H NMR
sphere (90%) containing 5% CO2. Cell line was culꢀ
tured in RPMIꢀ1640 medium with 5% (v/v) fetal boꢀ
vine serum, 100 units/mL of penicillin, and
d
2H, NH2, D2O exchangeable), 7.27–7.49 (m, 4H,
AromꢀH), 9.66 and 10.08 (s, 1H, NH, D2O exchangeꢀ
100
night and then incubated with various concentrations
149.5 (C–NH2), 143.3 (CH of Ph), 129.0 (2CH–CH–Br of compounds (
)–( ) for 48 and 72 h. Cells were
µ
g/mL of streptomycin. Cells were seeded overꢀ
able); 13C NMR (DMSOꢀ
d6, 100 MHz): 174.4 (C=S),
I
X
seeded at 5000 cells/well onto 96ꢀwell culture plates
for MTT assay. For each concentration course study,
there was a control sample that remained untreated.
of Ph), 128.2 (CH of Ph), 122.1 (Br–CH of Ph), 120.0
(CN), 54.8 (C–CN), 39.9 (CH–Ph); EIꢀMS m/z 308
[M]+ (100); anal. calcd. for C11H9N4SBr (%): C, 42.73;
H, 2.91; N, 18.12; found: C, 42.59; H, 3.06; N, 18.03.
Cell viability. Cell viability was determined using a
modified MTT assay [33, 34]. Briefly, cells were seedꢀ
ed (5000 cells/well) onto flatꢀbottomed, 96ꢀwell culꢀ
ture plates, allowed to grow for 24 h, and treated with
6ꢀAminoꢀ4ꢀ(4ꢀbromophenyl)ꢀ2ꢀthioxoꢀ1,2,3,4ꢀtetrahyꢀ
dropyrimidineꢀ5ꢀcarbonitrile (IX). Yield 71%, mp
127
2183 (C
°C; IR spectrum: 3406–3325 (NH2), 3193 (NH),
compounds (
I
)–(
X
) (15–500 M) for 48 and 72 h.
μ
≡
N), 1164 (C=S); 1H NMR (DMSOꢀ
d6, 400
MTT solution (5 mg/mL in phosphateꢀbuffered saꢀ
line; PBS) was added to the wells, cells were incubated
for 4 h, plates with adhesive cells were sharply turned
MHz,): 5.003 (s, 1H, CH–Ar), 6.19 (s, 2H, NH2,
D2O exchangeable), 7.16–7.60 (dd, 4H, AromꢀH),
13
9.77 and 10.05 (s, 1H, NH, D2O exchangeable); C
upside down removing the medium. DMSO (100
was added to dry wells. The absorption was measured
at 570 nm (620 nm as a reference) in an enzymeꢀ
Ph), 129.3 (2CH of Ph), 123.0 (Br–CH of Ph), 119.9 linked immunosorbent assay reader.
µ
L)
NMR (DMSOꢀd6, 100 MHz): 174.4 (C=S), 149.4
(C–NH2), 143.3 (CH of Ph), 130.2 (2CH–CHꢀBr of
(CN), 54.7 (C–CN), 39.8 (CH–Ph); EIꢀMS m/z 308
[M]+ (100); anal. calcd. for C11H9N4SBr (%): C,
42.73; H, 2.91; N, 18.12; found: C, 42.61; H, 2.89; N,
18.00.
Apoptosis
DAPI staining. Nuclear morphology of MCFꢀ7
cells was analyzed with DAPI (4'ꢀ6ꢀdiamidinoꢀ2ꢀpheꢀ
nylindole) staining [35]. MCFꢀ7 cells were seeded onꢀ
to glass cover slips in a six well plate. Twentyꢀfour
6ꢀAminoꢀ4ꢀ[4ꢀ(dimethylamino)phenyl]ꢀ2ꢀthioxoꢀ
1,2,3,4ꢀtetrahydropyrimidineꢀ5ꢀcarbonitrile (X).
Yield 40%, mp 154
(NH2), 2179 (C
≡
°C; IR spectrum: 3159–2816
N), 1157 (C=S); 1H NMR (DMSOꢀd6
,
hours later, cells were treated with compounds (VI
)
400 MHz,): 2.88 (s, 6H, CH3), 4.84 (s, 1H, CH–Ar),
6.08 (s, 2H, NH2, D2O exchangeable), 6.70–7.05 (dd,
4H, AromꢀH), 9.63 and 9.91 (s, 1H, NH, D2O exꢀ
changeable); 13C NMR (DMSOꢀ
d6, 100 MHz): 174.3
and ( ) for 48 h.The cells were fixed in 4% formaldeꢀ
X
hyde for 30 min and permeabilized with 0.1% Triton
Xꢀ100 for 30 min. Nuclei were then stained with DAPI,
and examined under the fluorescent inverted microꢀ
scope. Cells with condensed or fragmented nuclei
were considered to be apoptotic.
(C=S), 149.5 (C–NH2), 142.1 (CH of Ph), 130.4
(2CH of Ph), 125.2 (2CH–CH–N of Ph), 123.0 (N–
CH of Ph), 119.8 (CN), 54.9 (C–CN), 39.6 (CH–Ph),
PI staining. Apoptotic cells were detected using PI
staining of treated cells followed by flow cytometry to
detect the soꢀcalled subꢀG1 peak [36, 37]. Briefly,
HT29 cells were cultured overnight in a 24ꢀwell plate
35.6 (2CH3–N); EIꢀMS
m/z
273 [M]+ (100); anal.
calcd. for C13H15N5S (%): C, 57.14; H, 5.49; N, 25.64;
found: C, 57.01; H, 5.63; N, 25.58.
and treated with compounds (VI) and (
Floating and adherent cells were then harvested and
incubated at 4 C overnight in the dark with 750 L of
a hypotonic buffer (50 g/mL of PI in 0.1% sodium
citrate plus 0.1% Triton Xꢀ100) before the flow cytomꢀ
dium before each experiment. The final DMSO conꢀ etry analysis using a FACS can flow cytometer (BD
X) for 48 h.
Cell Assays
°
μ
Each of compounds (
centration of 50 mM in dimethyl sulfoxide (DMSO) as
stock solution, stored at –20 C and diluted with meꢀ
I)–(X) were dissolved at a conꢀ
μ
°
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 3
2016

322
HODA ATAPOURꢀMASHHAD et al.
17. Ghodasara, H.B., Trivedi, A.R., Kataria, V.B.,
Patel, B.G., and Shah, V.H., Med. Chem. Res., 2013,
vol. 22, pp. 6121–6128.
18. Prachayasittikul, S., Sornsongkhram, N., Pingarew, R.,
Techatanachai, S., Ruchirawat, S., and Prachayasitꢀ
tikul, V., Eur. J. Sci. Res., 2009, vol. 36, pp. 236–245.
19. Tozkoparan, B., Yarim, M., Saraç, S., Ertan, M., Keliꢀ
cen, P., Altinok, G., and Demirdamar, R., Arch. der
Pharmazie, 2000, vol. 333, pp. 415–420.
20. Bakavoli, M., Rahimizadeh, M., Shiri, A., Akbarzaꢀ
deh, M., Mousavi, S. H., AtapourꢀMashhad, H., and
TayaraniꢀNajaran, Z., J. Chem. Res., 2010, pp. 403–
406.
Biosciences, Franklin Lakes, New Jersey, USA) was
conducted. A total of 10000 events were acquired with
fluorescenceꢀactivated cell sorting (FACS).
ACKNOWLEDGMENTS
The authors would like to thank Dr. Seyed Hadi
Mousavi and Dr. Zahra TayaraniꢀNajaran for preparaꢀ
tion of PI solution and for their assistance in flow
cytometry.
21. Bakavoli, M., Rahimizadeh, M., Shiri, A., Akbarzaꢀ
deh, M., Mousavi, S. H., TayaraniꢀNajaran, Z.,
AtapourꢀMashhad, H., and Nikpour, M., J. Het.
Chem., 2011, vol. 48, pp. 183–187.
REFERENCES
1. The International Agency for Research on Cancer
(IARC) Latest World Cancer Statistics, 2013, Decemꢀ
ber 12. http://www.iarc.fr/en/mediaꢀcentre/pr/2013/
pdfs/pr223_E.pdf
22. Pisani, P., Parkin, D.M., and Ferlay, J., Int. J. Cancer,
1993, vol. 55, pp. 891–903.
23. Fogh, J., Fogh, J.M., and Orfeo, T., J. Natl. Cancer.
Inst., 1977, vol. 59, pp. 221–226.
24. Navio, J.L.G., Lorente, A., and Soto, J.L., Heterocyꢀ
cles, 1982, vol. 19, pp. 305–311.
2. Goldstein,C.M., Mehren, M.V., Gradel, T., Kilꢀ
patrick, D., Vaders, L., Brady, D., and Vogel, L.,
Breast Cancer Research, Fox Chase Cancer Center
Scientific Report, Philadelphia, 2000.
3. Parkin, D.M., Bray, F., Ferlay, J., and Pisani, P., CA 25. Fadda, A.A., ElꢀLatif, E.A., Bondock, S., and
Cancer. J. Clin., 2005, vol. 55, pp. 74–108.
Samir, A., Synth. Commun., 2008, vol. 38, pp. 4352–
4368.
4. Eidinoff, M.L., Knoll, J.E., Marano, B.J., and
26. Jung, M.H., Kim, H., Choi, W.K., ElꢀGamal, M.I.,
Park, J.H., Yoo, K.H., Sim, T.B., Lee, S.H., Baek, D.,
Hah, J.M., Cho, J.H., and Oh, C.H., Bioorg. Med.
Chem. Lett., 2009, vol. 19, pp. 6538–6543.
27. Zhao, L., Chen, Z., Wang, J., Yang, L., Zhao, Q.,
Wang, J., Qi, Q., Mu, R., You, Q.D., and Guo, QL.,
Cancer. Chemother. Pharmacol., 2009, vol. 10,
pp. 1053–1052.
Klein, D., Cancer. Res., 1961, vol. 21, pp. 1377–1385.
5. Callery, P., and Gannett, P., in Cancer and Cancer
Therapy, William, D.A. and Lemke, T.L., Eds., Philaꢀ
delphia, PA, USA: Lippincot Williams and Wilkins,
2002, pp. 934–935.
6. Donnini, S., Monti, M., Castagnini, C., Solito, R.,
Botta, M., Schenone, S., Giachetti, A., and Ziche, M.,
Int. J. Cancer, 2006, vol. 120, pp. 995–1004.
28. Yu, B., Shi, X.J., Zheng, Y.F., Fang, Y., Zhang, E.,
Yu, D.Q., and Liu, H.M., Eur. J. Med. Chem., 2013,
vol. 69, pp. 323–330.
7. Bazgir, A., Khanaposhtani, M. M., and Soorki, A.A.,
Bioorg. Med. Chem., 2008, vol. 18, pp. 5800–5803.
8. Shaban, N.Z., Masoud, M.S., Mawlawi, M.A.,
Awad, D., and Sadek, O.M., J. Physiol. Biochem.,
2012, vol. 68, pp. 475–484.
29. Kemnitzer, W., Sirisoma, N., May, C., Tseng, B.,
Drewe, J., and Cai, S.X., Bioorg. Med. Chem. Lett.
,
2009, vol. 1, pp. 3536–3540.
30. AtapourꢀMashhad, H., TayaraniꢀNajaran, Z., Davoodꢀ
nia, A., Moloudi, R., and Mousavi, S. H., Drug. Chem.
Toxicol., 2011, vol. 34, pp. 271–276.
9. Sawyer, R.C., Stolfi, R.L., Martin, D.S., and
Balis, M.E., Biochem. Pharmacol., 1989, vol. 38,
pp. 2305–2311.
31. Mousavi, S.H., TayaraniꢀNajaran, Z., and Hersey, P.,
10. Ghoshal, K., and Jacob, S.T., Biochem. Pharmacol.
1997, vol. 53, pp. 1569–1575.
,
Iran. J. Basic. Med. Sci., 2008, vol. 11, pp. 121–142.
32. Brohem, C.A., Sawada, T.C., Massaro, R.R.,
Almeida, R.L., Rivelli, D.P., Ropke, C.D., da Silva, V.V.,
de Lima, T.M., Curi, R., Barros, S.B., and Mariaꢀ
Engler, S.S., Toxicol. In Vitro, 2009, vol. 23, pp. 111–
119.
11. Yang, S., Li, Z., Jin, L., Song, B., Liu, G., Chen, J.,
Chen, Z., Hu, D., Xue, W., and Xu, R., Bioorg. Med.
Chem. Lett., 2007, vol. 17, pp. 2193–2196.
12. Prachayasittikul, S., Worachartcheewan, A., Nantaseꢀ
namat, C., Chinworrungsee, M., Sornsongkhram, N.,
Ruchirawat, S., and Prachayasittikul, V., Eur. J. Med.
Chem., 2011, vol. 46, pp. 738–742.
13. Sharma, S.K., Kumar, P., Narasimhan, B.,
Ramasamy, K., Mani, V., Mishra, R.K., and
Majeed, A.B., Eur. J. Med. Chem., 2012, vol. 48,
pp. 16–25.
33. Mosmann, T., J. Immunol. Meth., 1983, vol. 65,
pp. 55–63.
34. Mousavi,S. H., TavakkolꢀAfshari, J., Brook, A., and
JafariꢀAnarkooli, I., Food. Chem. Toxicol., 2009,
vol. 47, pp. 1909–1913.
35. Mousavi, S.H., AtapourꢀMashhad, H., Bakavoli, M.,
Shiri, A., Akbarzadeh, M., and TayaraniꢀNajaran, Z.,
Russ. J. Bioorg. Chem., 2015, vol. 41, pp. 201–208.
36. Mousavi, S.H., TavakkolꢀAfshari, J., Brook, A., and
JafariꢀAnarkooli, I., Food. Chem. Toxicol., 2009,
vol. 47, pp. 855–859.
14. Taher, A.T. and Helwa, A.A., Chem. Pharm. Bull.,
2012, vol. 60, pp. 521–530.
15. Taher, A.T. and AbouꢀSeri, S.M., Molecules, 2012,
vol. 17, pp. 9868–9886.
16. Fargualy, A.M., Habib, N.S., Ismail, K.A., 37. TayaraniꢀNajaran, Z., Mousavi, S.H., Asili, J., and
Hassan, A.M., and Sarg, M.T., Eur. J. Med. Chem. Emami, S.A., Food. Chem. Toxicol., 2010, vol. 48,
2013, vol. 66, pp. 276–295. pp. 599–604.
,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 3
2016