Synthesis and anticancer activity of some new s-glycosyl and s-Alkyl 1,2,4-triazinone derivatives

Article abstract of DOI:10.3390/molecules16075682

A series of S-glycosyl and S-alkyl derivatives of 4-amino-3-mercapto-6-(2- (2- thienyl)vinyl)-1,2,4-triazin-5(4H)-one (1) were synthesized using different halo compounds such as preacetylated sugar bromide, 4-bromobutylacetate, 2-acetoxyethoxymethyl bromide, 3-chloropropanol, 1,3-dichloro-2-propanol, epichlorohydrin, allyl bromide, propargyl bromide, phthalic and succinic acids in POCl₃. The structures of the synthesized compounds have been deduced from their elemental analysis and spectral (IR, ¹H-NMR, and ¹³C-NMR) data. Some of the synthesized compounds were screened as anticancer agents. Significant anticancer activities were observed in vitro for some members of the series, and compounds 4-Amino-3-(3-hydroxypropylthio)-6-(2- (2- thienyl)vinyl)-1,2,4-triazin-5(4H)-one (12) and 3-(4-Oxo-3-(2-(2-thienyl) vinyl)-4H- [1,3,4]thiadiazolo-[2,3-c][1,2,4]tr-iazin-7-yl)propanoic acid (18) are active cytotoxic agents against different cancer cell lines.

Full text of DOI:10.3390/molecules16075682

Molecules 2011, 16, 5682-5700; doi:10.3390/molecules16075682
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Anticancer Activity of Some New S-Glycosyl and
S-Alkyl 1,2,4-Triazinone Derivatives
Hosam A. Saad 1,2,* and Ahmed H. Moustafa 2
1
Department of Chemistry, Faculty of Science, Taif University, Taif, 21974,
Kingdom of Saudi Arabia
2
Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, 44511, Egypt
* Author to whom correspondence should be addressed; E-Mail: h_saadzag@yahoo.com;
Tel.: +96-65-5342-0899; +20-10-537-3242; Fax: +20-55-228-5374.
Received: 31 May 2011; in revised form: 27 June 2011 / Accepted: 29 June 2011 /
Published: 4 July 2011
Abstract: A series of S-glycosyl and S-alkyl derivatives of 4-amino-3-mercapto-6-(2-(2-
thienyl)vinyl)-1,2,4-triazin-5(4H)-one (1) were synthesized using different halo
compounds such as preacetylated sugar bromide, 4-bromobutylacetate, 2-acetoxyethoxy-
methyl bromide, 3-chloropropanol, 1,3-dichloro-2-propanol, epichlorohydrin, allyl
bromide, propargyl bromide, phthalic and succinic acids in POCl3. The structures of the
synthesized compounds have been deduced from their elemental analysis and spectral (IR,
13
1H-NMR, and C-NMR) data. Some of the synthesized compounds were screened as
anticancer agents. Significant anticancer activities were observed in vitro for some
members of the series, and compounds 4-Amino-3-(3-hydroxypropylthio)-6-(2-(2-
thienyl)vinyl)-1,2,4-triazin-5(4H)-one (12) and 3-(4-Oxo-3-(2-(2-thienyl)vinyl)-4H-
[1,3,4]thiadiazolo-[2,3-c][1,2,4]tr-iazin-7-yl)propanoic acid (18) are active cytotoxic
agents against different cancer cell lines.
Keywords: S-glycosyl; S-alkyl triazine; 1,2,4-triazinone; thiadiazolotriazines; anticancer
activities

Molecules 2011, 16
5683
1. Introduction
The chemistry and diverse applications of heterocyclic glycosyl derivatives have received much
attention due to their pronounced biological activity. 6-Aza analogues of the naturally occurring
nucleic acids components represent isosteres with the C-6 carbon atom of the pyrimidine nucleoside
base being replaced by nitrogen [1]. They became attractive to scientists after it was discovered that
6-azauracil and 6-azauridine are potent inhibitors of tumor growth, and it was determined that
6-azauridine is converted in the organism into 6-azauridylphosphate which blocks the enzyme orotic
acid decarboxylase, and thus the conversion of orotidylic acid into uridylic acid is impaired [2,3].
2-Glycosyl derivatives of 1,2,4-triazine-3,5(2H,4H)-diones and their thiones (6-azauracil derivatives)
possess [4-6] biological activity as cytotoxic, antiviral, enzyme inhibiting, immunosuppressive,
antiphlogestic and bacteriostatic agents [7,8]. As will as, thiophene-containing compounds are well
known to exhibit various biological activities such as anti-HIV PR inhibitors [9], anti breast cancer
(MCF-7) [10], anti-inflammatory [11-13]. anti-protozoal [14], antitumor [15], antitubercular with
antimycobacterial activity [16]. On the other hand, the 1,2,4-triazine moiety has also attracted the
attention of chemists because many 1,2,4-triazines are biologically active [17-22] and are used in
medicine, especially as anti AIDS, anticancer [23,24], and antitubercular agents [25], for their
anti-anxiety and anti-inflammatory activities [26,27], as well as in agriculture [28-31].
2. Results and Discussion
For the above reasons we sought to synthesize a series of S-glycosyl and S-alkyl of 4-amino-3-
mercapto-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (1) [32] which should combine all the above
benefits in one target and to then test the anticancer activity of these synthesized compounds against a
panel of human cell lines including hepatocellular carcinoma (Hep-G2), colon carcinoma (HCT-116),
and histiocytic lymphoma and breast adenocarcinoma (MCF-7) (ATCC, VA, USA). In our previous
work we prepared compound 1 by two different methods [33]. The synthetic routes to S-glycosides
3–7 via microwave irradiation (MWI, 2,450 MHz, 800 W) and conventional methods are illustrated in
Scheme 1.
Scheme 1. Synthesis of some S-glycosyl 1,2,4-triazinone.
OAc
O
R
AcO
R
R
S
R
S
AcO
O
O
N
N
OAc
O
O
MeOH / TEA
2
Br
N
N
N
N
N
N
MeOH / TEA
N
N
H2N
KOH/ Acetone
N
N
H2N
H2N
H2N
OH
S
HO
S
AcO
O
OAc
O
O
O
AcO
AcO
HO
HO
OH OH
OAc OAc
OAc
OH
AcO
7
6
OAc
3
4
O
H2N
O
peracetylated
glucose
c
b
N
e
OAc OAc
HS
MWI 5min
R
a
5
N N
R =
S
MWI
5min
d
1

Molecules 2011, 16
5684
The appearance of signals at δ 1.87, 1.92, 1.95, 2.02 ppm due to the four CH3 of four OAc groups
and at δ 6.21 ppm due to the anomeric proton of sugar with J = 9.60 Hz in the 1H-NMR confirms the
formation of the β-configuration of S-glucoside 3. This was also confirmed by the IR spectrum which
showed a bands at 3,310, 1,739 and 1,660 cm−1 for NH2 and two ester and amide C=O groups.
The 13C-NMR spectrum of 3 showed signals at δ 61.63, 66.24, 68.47, 72.88, 73.13 ppm
characteristic for sp3 carbon in a sugar moiety, in addition to δ 86.73 ppm for the anomeric carbon
(C1). In compound 6 the IR spectrum showed a band at 1,737 cm−1 for the acetoxy groups of the sugar
1
and the H-NMR displayed a signal at δ 6.46 ppm with J = 2.80 Hz characteristic for the anomeric
protons, in addition, in the 13C-NMR spectrum the anomeric carbon appear at δ 87.6 ppm.
The deacetylated compounds 4 and 7 showed in the IR spectra the disappearance of the acetoxy
groups and the appearance of bands at 3,434 and 3,421 cm−1 for the resulting free hydroxyl groups.
1
The H-NMR spectra and elemental analysis confirmed the structure as shown in the Experimental
section. When compound 1 was treated with alkylating agents such as 4-bromobutylacetate, 2-acetoxy-
ethoxymethyl bromide [34], 3-chloropropanol, 1,3-dichloro-2-propanol and epichlorohydrin using
DMF as a solvent in the presence of K2CO3, acetyloxy S-alkyl 1,2,4-triazinones 8, 10 and S-alkyl
1,2,4-triazinones 12-14 were obtained (Scheme 2). Deacetylation of compounds 8 and 10 in the
presence of TEA/MeOH and few drops of water [35-37] yielded the deacetylated S-alkyl 1,2,4-
triazinone 9 and 11, respectively (Scheme 2).
Scheme 2. Synthesis of some S alkyl 1,2,4-triazinones.
R
O
N
R
O
N
OAc
MeOH / TEA
Br
N
N
OAc
OH
DMF / K2CO3
N
S
N
S
NH2
NH2
8
9
OAc
O
R
N
N
R
O
N
N
MeOH / TEA
OAc
O
Br
OH
DMF / K2CO3
O
N
S
R
N
S
O
NH2
NH2
O
N
N
10
11
N
H2N
R
O
N
Cl
OH
SH
N
h
f
DMF / K2CO3
1
N
S
OH
g
NH2
12
Cl
Cl
R
N
OH
Hf
Hg
N
Hi
Hh
DMF / K2CO3
Hj
Cl
O
N
S
NH2
OH
13
R
Cl
O
N
O
O
N
S
DMF / K2CO3
N
H2N
c
b
14
e
R =
a
S
d

Molecules 2011, 16
5685
The structures of compounds 8 and 10 were confirmed by the presence of the (two) C=O bands in
the IR spectrum at 1,735 and 1,969 cm−1 for the acetoxy and amide groups. Compounds 9, 10, 12 and
1
13 showed characteristic OH group bands at 3,450–3,486 cm−1. In addition, the H-NMR spectrum of
8 showed signals at δ 1.91 and 4.06 ppm characteristic for CH3CO and SCH2 groups, respectively,
while compound 10 showed the deshielded signal at δ 4.45 ppm for the SCH2O group.
The 1H-NMR spectrum of compound 12 gave signals at δ 1.98, 3.53 and 4.48 ppm characteristic for
CH2(g), CH2(f) and CH2(h) groups, in addition to a triplet signal at δ 4.85 ppm for the OH group which
13
exchanged with D2O. Its C-NMR showed signals at δ 30.30, 50.08, 58.02 ppm characteristic for
1
CH2(g), CH2(f) and CH2(h), respectively., while the H-NMR spectrum of 14 showed signals at δ
4.05–4.18, 4.25 ppm for the CH2-O and CH-O of an epoxypropyl moiety, in addition to signals at δ
1
4.67 and 4.87 ppm for the diastereotropic protons of the S-CH2 grouping. IR, H-NMR and elemental
analysis data for compounds 9, 11 and 13 were in agreement with the assigned structures as shown in
the Experimental section. Alkylation of compound 1 with allyl and propargyl bromide in the presence
of K2CO3/acetone afforded S-alkyl triazine derivatives 15 and 16 (Scheme 3).
Scheme 3. Synthesis of S alkyl 1,2,4-triazinone and fused systems.
Br
R
N
N
Acetone / K2CO
3 O
N
S
NH2
15
Br
R
N
N
O
R
S
N
N
Acetone / K2CO3
N
N
H2N
O
NH2
SH
16
COOH
COOH
1
HOOC
N
O
S
N
N
POCl3
N
R
O
17
HO
OH
N
N
N
O
R
S
POCl3
N
O
COOH
c
b
e
18
R =
a
S
d
The 1H-NMR spectrum of 15 showed doublet signals at δ 5.06, 5.24 and 5.31 ppm characteristic for
SCH2 and the terminal olefin =CH2 protons, while its 13C-NMR spectrum gave a signal at δ 60.1 ppm
13
for SCH2 carbon. Menawhile, the C-NMR for compound 16 showed signals at δ 48.3 and 76.6,
77.1 ppm for SCH2 and C≡C carbons.
In the reaction of compound 1 with phthalic and succinic acids in the presence of POCl3, an
intramolecular cyclization took place giving the thiadiazolo[2,3-c][1,2,4]triazine derivatives 17 and 18,
respectively (Scheme 3). The structures of compound 17 and 18 were supported by elemental analysis
and spectral data. The IR spectra of 17 and 18 showed the absence of the (NH2) bands and the presence
of C=O and OH bands of acids in the 1,710–1,715 and 3,445–3,463 cm−1 range. In addition, the

Molecules 2011, 16
5686
1H-NMR spectra of these compounds revealed the presence of signals at δ 12.18 and 12.28 ppm for the
OH group of an acid, while in compound 18 there are two triplet signals at δ 1.82 and 2.68 ppm for the
13
CH2-CH2 protons, respectively. In addition the C-NMR spectrum showed signals at δ 26.80 and
32.50 ppm for CH2-CH2 carbon and eleven sp2 signals for the aromatic carbons, three C=N and two
C=O groups.
2.1. Pharmacological Studies
2.1.1. Cytotoxicity of the compounds against Hep-G2 Cells
Using the MTT assay we studied the effect of the compounds on the viability of cells after 48 h
incubation. Incubation of Hep-G2 cell line with gradual doses of all the compounds led to insignificant
change in the growth of Hep-G2 cells, as indicated from their IC50 values (>20 µg/mL), except for the
compounds 12 and 18, which produced an inhibition in the viability of Hep-G2 cells compared with
the growth of untreated control cells, as concluded from their low IC50 values, indicated by black bars
in Figure 1. The positive control, paclitaxol, which is a known anti-cancer drug, show high
cytotoxicity against Hep-G2 cells with an IC50 value of 643 ng/mL (Table 1, Figure 1).
Table 1. Cytotoxicity test using MTT assay against three different human cancer cell lines.
Mean IC50 (µg/mL)
MCF-7
cells
SE
MCF-7
cells
1.62
2.08
0.65
2.07
1.48
2.01
2.18
Compd.
No.
Hep-G2
cells
HCT-116
Hep-G2
cells
2.25
1.88
0.45
2.49
2.32
1.54
2.77
1.65
0.58
HCT-116
cells
1.45
cells
32.6
27.23
6.51
1
3
23.51
30.12
9.44
21.04
39.2
2.70
0.69
1.85
1.77
1.50
1.99
1.46
0.80
12
13
14
15
16
17
18
10.06
26.78
25.69
21.67
28.88
21.09
11.65
30.02
21.51
29.14
31.64
30.47
12.13
36.12
33.67
18.38
40.08
23.86
8.34
2.10
0.84
Figure 1. Cytotoxicity (IC50, µg/mL) of different tested compounds against human
Hep-G2 cells 48 hours of incubation. The grey bars represent non-cytotoxic compounds,
and the black bars represent the promising cytotoxic compounds. Data represent mean
value of IC50 SE.
Cytotoxicity against human hepatocellular carcinoma Hep-G2 cells
40
35
30
25
20
15
10
5
0
1
3
12
13
14
15
16
17
18

Molecules 2011, 16
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2.1.2. Cytotoxicity of the compounds against MCF-7 Cells
Using the MTT assay we studied the effect of the compounds on the viability of cells after 48 h
incubation. Incubation of MCF-7 cell line with most of the tested compounds led to insignificant
changes in the growth of MCF-7 cells, as indicated from their IC50 values (>20 µg/mL), except for
compounds 12 and 18, which possessed an inhibitory effect on MCF-7 cells viability, compared with
the growth of untreated control cells, as concluded from their low IC50 values, indicated by black bars
in Figure 2. The positive control, paclitaxol, which is a known anti-cancer drug, resulted in high
cytotoxicity against MCF-7 cells with IC50 value of 452 ng/mL (Table 1, Figure 2).
Figure 2. Cytotoxicity (IC50, µg/mL) of different tested compounds against human
MCF-7 cells 48 hours of incubation. The grey bars represent non-cytotoxic compounds,
and the black bars represent the promising cytotoxic compounds. Data represent mean
value of IC50 SE.
Cytotoxicity against human breast cancer MCF-7 cells
45
40
35
30
25
20
15
10
5
0
1
3
12
13
14
15
16
17
18
2.1.3. Cytotoxicity of the compounds against HCT-116 Cells
The effect of the compounds on the viability of cells after 48 h incubation was studied by MTT
assay. Incubation of HCT-116 cell line with gradual doses of some tested compounds led to
insignificant change in the growth of HCT-116 cells as indicated from their IC50 values (>20 µg/mL).
On the other hand, the compounds 12, 15 and 18 resulted in a significant inhibition in the viability of
HCT-116 cells, compared with the growth of untreated control cells, as concluded from their low IC50
values, as indicated by black bars in Figure 3. The positive control, paclitaxol, which is a known
anti-cancer drug, resulted in high cytotoxicity against HCT-116 cells with IC50 value of 709 ng/mL
(Table 1, Figure 3).
2.1.4. Percentage of induced apoptotic and necrotic cells in Hep-G2 Cells
According to the cytotoxicity experiments, compounds 12 and 18 possessed potent cytotoxic effects
against Hep-G2 cells. To detect the type of cell death induced in the cells by those compounds,
Hep-G2 cells were treated with the IC50 values of each compound for 6 h and the apoptosis and
necrosis cell population percentages was recorded using acridine orange/ethidium bromide staining. As

Molecules 2011, 16
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shown in Figure 4, both of the tested compounds led to an apoptosis-dependant cell death (84–86% of
the total dead cell number), while the percentage of necrotic cells were only 14–16% of the total dead
cell number (Table 2, Figure 4).
Figure 3. Cytotoxicity (IC50, µg/mL) of different tested compounds against human
HCT-116 cells after 48 h of incubation. The grey bars represent non-cytotoxic compounds,
and the black bars represent the promising cytotoxic compounds. Data represent mean
value of IC50 SE.
Cytotoxicity against human colon cancer HCT-116 cells
45
40
35
30
25
20
15
10
5
0
1
3
12
13
14
15
16
17
18
Table 2. Apoptosis and necrosis assay for cytotoxic compounds only.
Hep-G2
Compd. No.
Apoptotic cells
Necrotic cells
12
18
84
86
16
14
Figure 4. The type of cell death was investigated in Hep-G2 cells after the treatment with
the promising cytotoxic compounds, using acridine orange/ethidium bromide staining to
compare between the percentage of necrotic cells (grey segment) and the apoptotic cells
(black segment). Data represent mean value SE.
Percentage of apoptosis and necrosis in Hep-G2 cell population
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
12
18
Apoptotic
cells
Necrotic
cells

Molecules 2011, 16
5689
2.1.5. Percentage of induced apoptotic and necrotic cells in MCF-7 Cells
According to the findings of the cytotoxicity experiment, compounds 12 and 18 possessed a potent
cytotoxic effect against MCF-7 cells. To detect the type of cell death induced in the cells by those
compounds, MCF-7 cells were treated with the IC50 values of each compound for 6 h and the apoptosis
and necrosis cell population percentages was recorded using acridine orange/ethidium bromide
staining. As shown in Figure 5, both of the tested compounds led to an apoptosis-dependant cell death
(85–92% of the total dead cell number), while the percentage of necrotic cells were only 8–15% of the
total dead cell number (Table 3, Figure 5).
Table 3. Apoptosis and necrosis assay for cytotoxic compounds only.
MCF-7
Compd. No.
Apoptotic cells
Necrotic cells
12
18
92
85
8
15
Figure 5. The type of cell death was investigated in MCF-7 cells after the treatment with
the promising cytotoxic compounds, using acridine orange/ethidium bromide staining to
compare between the percentage of necrotic cells (grey segment) and the apoptotic cells
(black segment). Data are representing mean value SE.
Percentage of apoptosis and necrosis in MCF-7
cell population
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
12
18
Apoptotic
cells
Necrotic
cells
2.1.6. Percentage of induced apoptotic and necrotic cells in HCT-116 Cells
According to the cytotoxicity experiments, compounds 12, 15, and 18 possessed potent cytotoxic
effects against HCT-116 cells. To detect the type of cell death induced in the cells by those
compounds, HCT-116 cells were treated with the IC50 values of each compound for 6 h and the
apoptosis and necrosis cell population percentages was recorded using acridine orange/ethidium
bromide staining. As shown in Figure 6, the tested compounds 12, 15, and 18 resulted in an

Molecules 2011, 16
5690
apoptosis-dependant cell death (61–94% of the total dead cell number), while the percentage of
necrotic cells were only 6–39% of the total dead cell number (Table 4, Figure 6).
Table 4. Apoptosis and necrosis assay for cytotoxic compounds only.
HCT-116
Compd. No.
Apoptotic cells
Necrotic cells
12
15
18
94
61
82
6
39
18
Figure 6. The type of cell death was investigated in HCT-116 cells after the treatment with
the promising cytotoxic compounds, using acridine orange/ethidium bromide staining to
compare between the percentage of necrotic cells (grey segment) and the apoptotic cells
(black segment). Data are representing mean value SE.
Percentage of apoptosis and necrosis in HCT-116 cell population
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
12
15
18
Apoptotic
cells
Necrotic
cells
3. Experimental
3.1. General
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus.
1
The IR spectra (KBr) discs were recorded on a Perkin-Elmer 1650 spectrometer. H-, 13C-NMR
spectra were determined on Bruker AC-300 MHz instrument. Chemical shifts are expressed as δ (ppm)
relative to TMS as internal standard and DMSO-d6 as solvent. The elemental analysis and mass spectra
were carried by the Micro-analytical Center, Cairo University. Mass spectra were recorded on a
Shimadzu GC-MS-QP 1000 EX spectrometer. A domestic microwave oven was used (2,450 MHz, 800 W).
The pharmacological studies were carried out in National Research Center (Center of Excellence for
Advanced Sciences, Cancer Biology Research Laboratory). All chemicals were from Sigma.

Molecules 2011, 16
5691
3.2. Material and Methods for Pharmacological Activity
3.2.1. Cell culture
Several human cell lines were used in testing the anti-cancer activity including: hepatocellular
carcinoma (Hep-G2), colon carcinoma (HCT-116), and histiocytic lymphoma and breast
adenocarcinoma (MCF-7) (ATCC, VA, USA). HCT-116 cells were grown in Mc Coy's medium, while
all cells were routinely cultured in DMEM (Dulbeco’s Modified Eagle’s Medium) at 37 °C in
humidified air containing 5% CO2. Media were supplemented with 10% fetal bovine serum (FBS),
2 mM L-glutamine, containing 100 units/mL penicillin G sodium, 100 units/mL streptomycin sulphate,
and 250 ng/mL amphotericin B. Monolayer cells were harvested by trypsin/EDTA treatment, while
and leukemia cells were harvested by centrifugation. Compound dilutions were tested before assays for
endotoxin using Pyrogent® Ultra gel clot assay, and they were found endotoxin free. All experiments
were repeated four times, unless mentioned, and the data was represented as (mean S.D.). Cell
culture material was obtained from Cambrex BioScience (Copenhagen, Denmark), and all chemicals
were from Sigma (USA).
3.2.2. Cytotoxicity assay
Cytotoxicity of tested samples against different types of cells was measured using the MTT Cell
Viability Assay. MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay is based
on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium
rings of the yellow MTT and form a dark blue insoluble formazan crystals which is largely
impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilization of
the cells results in the liberation of crystals, which are then solubilized. The number of viable cells is
directly proportional to the level of soluble formazan dark blue color. The extent of the reduction of
MTT was quantified by measuring the absorbance at 570 nm [38].
3.2.2.1. Reagents preparation
MTT solution: 5 mg/mL of MTT in 0.9% NaCl; Acidified isopropanol: 0.04 N HCl in absolute
isopropanol.
3.2.2.2. Procedure
Cells (0.5 × 105 cells/ well) in serum-free media were plated in a flat bottom 96-well microplate,
and treated with 20 µL of different concentrations of each tested compound for 48 h at 37 °C, in a
humidified 5% CO2 atmosphere. After incubation, media were removed and 40 µL MTT solution/well
were added and Incubated for an additional 4 h. MTT crystals were solubilized by adding 180 µL of
acidified isopropanol/well and plate was shacked at room temperature. Followed by photometric
determination of the absorbance at 570 nm using microplate ELISA reader. Triplicate repeats were
performed for each concentration and the average was calculated. Data were expressed as the

Molecules 2011, 16
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percentage of relative viability compared with the untreated cells compared with the vehicle control,
with cytotoxicity indicated by <100% relative viability.
3.2.2.3. Calculations
Percentage of relative viability was calculated using the following equation:
[Absorbance of treated cells / Absorbance of control cells)] × 100
Then the half maximal inhibitory concentration IC50 was calculated from the equation of the dose
response curve.
3.2.3. Apoptosis and necrosis staining
The type of cell death was investigated in compound-treated and untreated cells using acridine
orange/ethidium bromide staining [39,40]. In brief, cells were treated with The IC50 value of each
promising compound for 6 h and collected to be treated with acridine orange/ethedium bromide
mixture. The vital, necrotic, and apoptotic cells were counted. A mixture of 100 µg/mL acridine
orange and 100 µg/mL ethidium bromide was prepared in PBS. The cell uptake of the stain was
monitored under a fluorescence microscope, and the apoptotic, necrotic, and viable cells were counted.
The early apoptotic cells had yellow chromatin in nuclei that were highly condensed or fragmented.
Apoptotic cells also exhibited membrane blebbing. The late apoptotic cells had orange chromatin with
nuclei that were highly condensed and fragmented. The necrotic cells had bright orange chromatin in
round nuclei. Only cells with yellow, condensed, or fragmented nuclei were counted as apoptotic cells
in a blinded, nonbiased manner.
4-Amino-3-mercapto-6-[2-(2-thienyl)vinyl]-1,2,4-triazin-5(4H)-one (1). Method A: A mixture of
2-oxo-4-(2-thienyl)but-3-enoic acid (0.01 mol), thiocarbohydrazide (0.01 mol) in glacial acetic acid
(25 mL) was stirred under reflux for 2 h, cooled to room temperature, the precipitate separated
collected by filtration to give a yellowish crystals. Yield 62%; m.p. 252–255 °C. Method B: A mixture
of 2-oxo-4-(2-thienyl)but-3-enoic acid (0.01 mol) and thiocarbohydrazide (0.01 mol), was dissolved in
a mixture of methylene chloride/methanol (80:20, 15 mL) then silica gel (1.0 g, 200–400 mesh) was
added, the solvent was removed by evaporation, the dried residue was transferred into a glass beaker
and irradiated for (1.5–2.0 min) in a domestic microwave oven (2,450 MHz, 800 W). The product was
chromatographed on a silica gel column, using methylene chloride as eluent. Yield 98%; m.p.
1
254–255 °C; IR (KBr): 3,295–3,201 cm−1 (NH2), 1,666 cm−1 (C=O amide); H-NMR (DMSO-d6):
δ = 6.51 (s, 2H, NH2), 6.74 (d, 1H, J = 15.9 Hz, CH=CHe), 7.07 (dd, 1H, J = 3.60, 4.80 Hz, thiophene-
Hb) 7.36 (d, 1H, J = 3.6 Hz, thiophene–Hc), 7.57 (d, 1H, J = 5.1 Hz, thiophene–Ha), 7.92 (d, 1H,
13
J = 16.0 Hz, CHd=CH), 14.00 (s, 1H, SH); C-NMR (DMSO-d6): δ = 118.2, 127.7, 128.3, 128.6,
129.5, 141.0, 141.1, 148.0 and 167.2 (Ar-C, C=C, C=N and C=O); Anal. Calcd for C9H8N4OS2
(252.31): C, 42.84; H, 3.20; N, 22.20; found C, 42.90; H, 3.16; N, 22.03; MS m/e (int. %): 250 (82.8),
251 (90.8), 250 (100), 135 (87.4), 134 (65.5), 69 (80.5), 59 (82.8), 58 (93.1), 51 (75.9), 50 (62.1).

Molecules 2011, 16
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3.2.4. General method for preparation of compounds 3 and 6
A mixture of compound 1 (10 mmol) and peracetylated sugar (10 mmol) was dissolved in
methylene chloride (20 mL), then silica gel (1 g, 200–400 mesh) was added, the solvent was removed
by evaporation and the dried residue was irradiated for 5 min in a domestic microwave oven
(2,450 MHz, 800 W). The product was extracted with methylene chloride, evaporated to dryness and
purified by recrystallization from ethanol.
Method A: 4-Amino-3-(2`,3`,4`,6`-tetra-O-acetyl-β-D-glucopyranosylthio)-6-[2-(2-thienyl)vinyl]-1,2,4-
triazin-5(4H)-one (3). Yield 21%; m.p. 86–87 °C; IR (KBr): 3,310 cm−1 (NH2), 1,739 cm−1 (C=O,
1
acetoxy groups), 1,660 cm−1 (C=O, amide); H-NMR (DMSO-d6): δ = 1.87, 1.92, 1.95 and 2.02 (4s,
12H, 4 CH3CO), 4.03 (m, 2H, H-5` and H-6''), 4.28 (m,1H, H-6'), 5.06 (t, 1H, J = 9.80 Hz, H-4'), 5.61
(t, 1H, J = 9.80 Hz, H-2'), 5.69 (t, 1H, J = 8.65 Hz, H-3'), 6.21 (d, 1H, J1`,2` = 9.60 Hz, H-1'), 6.64 (s,
2H, NH2, exchanged with D2O), 6.76 (d, 1H, J = 15.9 Hz, CH=CHe), 7.13 (dd, 1H, J = 3.90, 4.80 Hz,
thiophene-Hb), 7.45 (d, 1H, J = 3.30 Hz, thiophene-Hc), 7.66 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.01
(d, 1H, J = 15.9 Hz, CHd=CH); 13C-NMR (DMSO-d6): δ = 19.70, 20.30, 20.46 and 21.14 (4 CH3CO),
61.36 (C-6'), 66.24 (C-4'), 68.47 (C-3'), 72.88 (C-2'), 73.13 (C-5') and 86.73 (C-1'), 116.9, 128.4,
130.0, 130.1, 140.3, 140.7, 146.7, 146.8, 168.7, 169.0, 169.4, 169.5 and 170.0 (Ar-C, C=C, 2C=N and
5 C=O); Anal. Calcd for C23H26N4O10S2 (582.60): C, 47.42; H, 4.50; N, 9.62. Found: C, 47.40; H,
4.50; N, 9.61. Method B: A solution of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl bromide 2
(10 mmol) in acetone (30 mL) was added to a solution of compound 1 (10 mmol) in aqueous (distilled
water, 10 mL) potassium hydroxide (10 mmol). The reaction mixture was stirred overnight at room
temperature. The solvent was removed under reduced pressure, the residue was washed with distilled
water to remove the inorganic residue and then the formed solid product dried and crystallized from
ethanol. Yield 25%; m.p. 85–87 °C.
3.2.5. General procedure for preparation of compounds 4,7,9 and 10
A mixture of any of the compounds 3, 6, 8 or 10 (10 mmol) in methanol (20 mL), triethylamine
(1 mL) and few drops of water were stirred overnight at room temperature, and then the solvent was
evaporated under reduced pressure. The residue was purified by crystallization from ethanol.
4-Amino-3-(β-D-glucopyranosylthio)-6-[2-(2-thienyl)vinyl]-1,2,4-triazin-5(4H)-one (4). Yield 85%;
m.p. 140–142 °C; IR (KBr): 3,434 cm−1 (OH), 3,225 cm−1 (NH2), 1,665 cm−1 (C=O, amide); 1H NMR
(DMSO-d6): δ = 3.14–3.64 (m, 6H, H-6', H-6'', H-5', H-4', H-3' and H-2'), 4.48 (d, 1H, OH-2'), 5.08 (d,
1H, OH-3'), 5.23 (d, 1H, OH-4'), 5.48 (t, 1H, OH-6'), 5.59 (d, 1H, J1`,2` = 7.82 Hz, H-1'), 6.65 (s, 2H,
NH2), 6.77 (d, 1H, J = 15.9 Hz, CH=CHe), 7.10 (dd, 1H, J = 3.90, 4.80 Hz, thiophene-Hb), 7.35 (d, 1H,
J = 3.30 Hz, thiophene-Hc), 7.65 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.01 (d, 1H, J = 15.9 Hz,
CHd=CH); Anal. Calcd for C15H18N4O6S2 (414.46): C, 43.47; H, 4.38; N, 13.52. Found: C, 43.45; H,
4.39; N, 13.50.
4-Amino-3-(2`,3`,5`-tri-O-acetyl-β-
D-ribofuranosylthio)-6-[2-(2-thienyl)vinyl]-1,2,4-triazin-5(4H)-one
(6). Yield 20%; m.p. 92–94 °C; IR (KBr): 3,328 cm−1 (NH2), 1,737 cm−1 (C=O, acetoxy groups),

Molecules 2011, 16
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1,675 cm−1 (C=O, amide); 1H-NMR (DMSO-d6): δ = 1.70, 2.11 and 2.14 (3s, 9H, 3 CH3CO), 4.09 (m,
2H, H-5' and H-5''), 4.37 (m, 1H, H-4'), 5.44 (t, 1H, J2`,3` = 5.90, J3`,4` = 6.00 Hz, H-3'), 5.79 (t, 1H,
J = 2.60 Hz, H-2'), 6.46 (d, 1H, J1`,2` = 2.80 Hz, H-1'), 6.52 (s, 2H, NH2, exchange with D2O), 6.82 (d,
1H, J = 15.9 Hz, CH=CHe), 7.10 (dd, 1H, J = 3.80, 4.82 Hz, thiophene-Hb), 7.41 (d, 1H, J = 3.30 Hz,
thiophene-Hc), 7.60 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.01 (d, 1H, J = 15.9 Hz, CHd=CH); 13C-NMR
(DMSO-d6): δ = 20.20, 20.41 and 20.74 (3 CH3CO), 63.10 (C-5'), 70.02 (C-3'), 73.54 (C-2'), 79.01
(C-4'), 87.60 (C-1'), 118.3, 127.8, 128.4, 128.6, 129.6, 141.1, 141.2, 148.1, 167.3, 168.7, 169.4 and
169.9 (Ar-C, C=C, 2 C=N and 4 C=O); Anal. Calcd for C20H22N4O8S2 (510.54): C, 47.05; H, 4.34; N,
10.97. Found: C, 47.04; H, 4.35; N, 10.98.
4-Amino-3-(β-D-ribofuranosylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (7). Yield 85%; m.p.
1
137–139 °C; IR (KBr): 3,421 cm−1 (OH), 3,280 cm−1 (NH2), 1,668 cm−1 (C=O, amide); H-NMR
(DMSO-d6): δ = 3.98–4.07 (m. 5H, H-5', H-5'', H-4', H-3' and H-2'), 4.20 (t, 1H, OH-5'), 4.26 (d, 1H,
OH-3'), 4.30 (d, 1H, OH-2'), 6.03 (d, 1H, J1`,2` = 3.80 Hz, H-1'), 6.64 (s, 2H, NH2), 6.75 (d, 1H,
J = 15.9 Hz, CH=CHe), 7.09 (dd, 1H, J = 3.80, 4.82 Hz, thiophene-Hb), 7.34 (d, 1H, J = 3.30 Hz,
thiophene-Hc), 7.63 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.10 (d, 1H, J = 15.9 Hz, CHd=CH); Anal.
Calcd for C14H16N4O5S2 (384.43): C, 43.74; H, 4.20; N, 14.57. Found: C, 43.76; H, 4.22; N, 14.56.
3.2.6. General procedure for synthesis of S-alkyl compounds 8, 10, 12, 13 and 14
Method A: An appropriate alkyl halide such as 4-bromobutylacetate, 2-acetoxyethoxymethyl bromide,
3-chloropropanol, 1,3-dichloro-2-propanol and/or epichlorohydrin (10 mmol) was added to a well
stirred mixture of 1 (10 mmol) and potassium carbonate (10 mmol) in dry DMF (15 mL). The reaction
mixture was stirred overnight at room temperature, the mixture was filtered off, and the solvent
evaporated under reduced pressure. The residue was crystallized from ethanol. Method B: The
appropriate alkyl halide (10 mmol) was added to a well stirred mixture of 1 (10 mmol) and potassium
carbonate (10 mmol) in dry DMF (15 mL). The reaction mixture was stirred under reflux for 2–8 h, the
mixture was filtered off, and the solvent evaporated under reduced pressure. The residue was
crystallized from ethanol.
4-(4-Amino-5-oxo-6-(2-(2-thienyl)vinyl)-4,5-dihydro-1,2,4-triazin-3-ylthio)butyl acetate (8). Method
B: Yield 60%; m.p. 90–92 °C; IR (KBr): 3280 cm−1 (NH2), 1735 cm−1 (C=O, acetoxy), 1668 cm−1
(C=O, amide); 1H-NMR (DMSO-d6): δ = 1.63 (m, 2H, CH2), 1.83 (m, 2H, CH2), 1.91 (s, 3H, CH3CO),
4.06 (t, 2H, J = 6.60 Hz, SCH2), 4.49 (t, 2H, J = 6.90 Hz, CH2OCO), 6.67 (s, 2H, NH2, exchanged with
D2O), 6.76 (d, 1H, J = 15.9 Hz, CH=CHe), 7.10 (dd, 1H, J = 3.80, 4.80 Hz, thiophene-Hb), 7.35 (d, 1H,
J = 3.30 Hz, thiophene-Hc), 7.61 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.06 (d, 1H, J = 15.9 Hz,
CHd=CH); Anal. Calcd for C15H18N4O3S2 (366.46): C, 49.16; H, 4.95; N, 15.29. Found: C, 49.18; H,
4.96; N, 15.28.
4-Amino-3-(4-hydroxybutylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (9). Yield 85%; m.p.
125–126 °C; IR (KBr): 3,450 cm−1 (OH), 3,228 cm−1 (NH2), 1,665 cm−1 (C=O, amide); 1H- NMR
(DMSO-d6): δ = 1.58 (m, 2H, CH2), 1.75 (m, 2H, CH2), 3.96 (t, 2H, J = 6.62 Hz, SCH2), 4.38 (t, 2H,

Molecules 2011, 16
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J = 6.82 Hz, CH2O), 4.82 (br, 1H, OH), 6.68 (s, 2H, NH2), 6.75 (d, 1H, J = 15.9 Hz, CH=CHe), 7.12
(dd, 1H, J = 3.80, 4.80 Hz, thiophene-Hb), 7.41 (d, 1H, J = 3.30 Hz, thiophene-Hc), 7.63 (d, 1H,
J = 4.80 Hz, thiophene-Ha), 8.08 (d, 1H, J = 15.9 Hz, CHd=CH); Anal. Calcd for C13H16N4O2S2
(324.42): C, 48.13; H, 4.97; N, 17.27. Found: C, 48.15; H, 4.96; N, 17.29.
2-((4-Amino-5-oxo-6-(2-(2-thienyl)vinyl)-4,5-dihydro-1,2,4-triazin-3-ylthio)methoxy)ethyl acetate (10).
Method A: Yield 60%; m.p. 85–86 °C; IR (KBr): 3,295 cm−1 (NH2), 1,735 cm−1 (C=O, acetoxy),
1
1,669 cm−1 (C=O, amide); H-NMR (DMSO-d6): δ = 1.94 (s, 3H, CH3CO), 3.72 (t, 2H, J = 5.80 Hz,
OCH2), 4.28 (t, 2H, J = 6.00 Hz, CH2OCO), 4.45 (s, 2H, SCH2O), 6.65 (s, 2H, NH2, exchanged with
D2O), 6.72 (d, 1H, J = 15.9 Hz, CH=CHe), 7.09 (dd, 1H, J = 3.80, 4.80 Hz, thiophene-Hb), 7.39 (d, 1H,
J = 3.30 Hz, thiophene-Hc), 7.58 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.03 (d, 1H, J = 15.9 Hz,
CHd=CH); 13C-NMR (DMSO-d6): δ = 20.5 (CH3CO), 61.7 (OCH2), 62.9 (CH2OCO), 63.04 (SCH2O),
117.6, 128.1, 128.4, 129.9, 139.5, 140.8, 146.8, 147.1, 168.9 and 170.2 (Ar-C, C=C, 2 C=N and 2
C=O); Anal. Calcd for C14H16N4O4S2 (368.43): C, 45.64; H, 4.38; N, 15.21. Found: C, 45.65; H, 4.36;
N, 15.20.
4-Amino-3-((2-hydroxyethoxy)methylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (11). Yield
90%; m.p. 128–130 °C; IR (KBr): 3,486 cm−1 (OH), 3,275 cm−1 (NH2), 1,672 cm−1 (C=O, amide); 1H-
NMR (DMSO-d6): δ = 3.68 (t, 2H, J = 5.82 Hz, OCH2), 4.08 (t, 2H, J = 6.00 Hz, CH2OH), 4.50 (s, 2H,
SCH2O), 4.82 (br, 1H, OH), 6.62 (s, 2H, NH2), 6.68 (d, 1H, J = 15.9 Hz, CH=CHe), 7.10 (dd, 1H,
J = 3.80, 4.80 Hz, thiophene-Hb), 7.36 (d, 1H, J = 3.30 Hz, thiophene-Hc), 7.53 (d, 1H, J = 4.80 Hz,
thiophene-Ha), 8.10 (d, 1H, J = 15.9 Hz, CHd=CH); Anal. Calcd for C12H14N4O3S2 (326.39): C, 44.16;
H, 4.32; N, 17.17. Found: C, 44.18; H, 4.34; N, 17.18.
4-Amino-3-(3-hydroxypropylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (12). Method B: Yield
50%; m.p. 125–127 °C; IR (KBr): 3,450 cm−1 (OH), 3,281 cm−1 (NH2), 1,683 cm−1 (C=O, amide);
1H- NMR (DMSO-d6): δ = 1.98 (m, 2H, CH2(g)), 3.53 (t. 2H, J = 6.30 Hz, CH2(f)), 4.48 (t, 2H,
J = 6.90 Hz, CH2(h)), 4.85 (t, 1H, J = 5.80 Hz, OH), 6.68 (s, 2H, NH2), 6.77 (d, 1H, J = 15.9 Hz,
CH=CHe), 7.11 (dd, 1H, J = 3.80, 4.80 Hz, thiophene-Hb), 7.43 (d, 1H, J = 3.30 Hz, thiophene-Hc),
7.62 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.02 (d, 1H, J = 15.9 Hz, CHd=CH); 13C-NMR (DMSO-d6):
δ = 30.30 (CH2(g)), 50.08 (CH2(f)), 58.02 (CH2(h)), 118.1, 128.0, 128.5, 129.4, 129.8, 140.0, 140.9,
147.0 and 166.4 (Ar-C, C=C, 2 C=N and C=O); Anal. Calcd for C12H14N4O2S2 (310.40): C, 46.43; H,
4.55; N, 18.05. Found: C, 46.44; H, 4.53; N, 18.03.
4-Amino-3-(3-chloro-2-hydroxypropylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one
(13).
Method B: yield 55%; m.p. 160–162 °C; IR (KBr): 3,452 cm−1 (OH), 3,280 cm−1 (NH2), 1,672 cm−1
(C=O, amide); 1H-NMR (DMSO-d6): δ = 2.90 (m, 1H, H(g)), 3.18 (m. 1H, H(f)), 3.42 (m, 1H, H(j)), 3.68
(m, 1H, H(i)), 3.82 (m, 1H, H(h)), 4.81 (d, 1H, J = 5.30 Hz, OH), 6.62 (s, 2H, NH2), 6.71 (d, 1H,
J = 15.9 Hz, CH=CHe), 7.10 (dd, 1H, J = 3.80, 4.80 Hz, thiophene-Hb), 7.35 (d, 1H, J = 3.30 Hz,
thiophene- Hc), 7.60 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.06 (d, 1H, J = 15.9 Hz, CHd=CH); Anal.
Calcd for C12H13ClN4O2S2 (344.84): C, 41.80; H, 3.80; N, 16.25. Found: C, 41.81; H, 3.82; N, 16.23.
4-Amino-3-(oxiran-2-ylmethylthio)-6-(2-(2-thienyl)-1,2,4-triazin-5(4H)-one (14). Method A: Yield
65%; m.p. 130–132 °C; IR (KBr): 3,285 cm−1 (NH2), 1,672 cm−1 (C=O, amide); 1H-NMR (DMSO-d6):

Molecules 2011, 16
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δ = 4.05–4.18 (m, 2H, CH2O, oxirane ring), 4.25 (m, 1H, CH-O, oxirane ring), 4.67 (t, 1H, J = 5.52,
1.80 Hz, SCHH, diastereotropic proton), 4.87 (d, 1H, J = 5.33 Hz, SCHH, diastereotropic proton), 6.54
(s, 2H, NH2, exchange with D2O), 6.67 (d, 1H, J = 15.9 Hz, CH=CHe), 7.11 (dd, 1H, J = 3.80, 4.80 Hz,
thiophene-Hb), 7.41 (d, 1H, J = 3.30 Hz, thiophene-Hc), 7.62 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.08
(d, 1H, J = 15.9 Hz, CHd=CH); Anal. Calcd for C12H12N4O2S2 (308.38): C, 46.74; H, 3.92; N, 18.17.
Found: C, 46.73; H, 3.90; N, 18.18.
3.2.7. General procedure for synthesis of compounds 15 and 16
The appropriate alkyl halide (10 mmol) allyl bromide and/or propargyl bromide was added portion-
wise to a well stirred mixture of 1 (10 mmol) and potassium carbonate (10 mmol) in dry acetone
(15 mL). The reaction mixture was stirred overnight at room temperature, the mixture was filtered off,
and the solvent evaporated under reduced pressure. The residue was crystallized from ethanol.
3-(Allylthio)-4-amino-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (15). Yield 62%; m.p. 123–125 °C; IR
1
(KBr): 3,280 cm−1 (NH2), 1,668 cm−1 (C=O, amide); H-NMR (DMSO-d6): δ = 5.06 (d, 2H,
J = 5.10 Hz, SCH2), 5.24 (d, 1H, J = 3.00 Hz, =CHH), 5.31 (d, 1H, J = 10.8 Hz, =CHH), 5.96 (m, 1H,
CH=CH2), 6.67 (s, 2H, NH2, exchanged with D2O), 6.76 (d, 1H, J = 15.9 Hz, CH=CHe), 7.10 (dd, 1H,
J = 3.90, 4.80 Hz, thiophene-Hb), 7.43 (d, 1H, J = 3.30 Hz, thiophene-Hc), 7.62 (d, 1H, J = 4.80 Hz,
13
thiophene-Ha), 8.01 (d, 1H, J = 15.9 Hz, CHd=CH); C-NMR (DMSO-d6): δ = 60.1 (SCH2), 118.0,
118.5, 128.1, 128.5, 129.5, 129.8, 131.0, 140.3, 140.9, 147.0 and 166.6 (Ar-C, C=C, 2C=N and C=O);
Anal. Calcd for C12H12N4OS2 (292.38): C, 49.29; H, 4.14; N, 19.16. Found: C, 49.28; H, 4.15;
N, 19.15.
4-Amino-3-(prop-2-ynylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (16). Yield 70%; m.p.
160–162 °C; IR (KBr): 3,295 cm−1 (NH2), 1,662 cm−1 (C=O, amide); 1H-NMR (DMSO-d6): δ = 3.45 (t,
1H, J = 1.50 Hz, ≡CH), 5.24 (d, 2H, J = 2.40 Hz, SCH2), 6.65 (s, 2H, NH2, exchanged with D2O), 6.78
(d, 1H, J = 15.9 Hz, CH=CHe), 7.01 (dd, 1H, J = 3.90, 4.80 Hz, thiophene-Hb), 7.41 (d, 1H,
J = 3.30 Hz, thiophene-Hc), 7.65 (d, 1H, J = 4.80 Hz, thiophene-Ha), 8.08 (d, 1H, J = 15.9 Hz,
13
CHd=CH); C-NMR (DMSO-d6): δ = 48.3 (SCH2), 76.6, 77.1 (C≡C), 117.6, 120.5, 127.5, 128.3,
129.7, 130.1, 141.2, 147.0 and 166.5 (Ar-C, C=C, 2C=N and C=O); Anal. Calcd for C12H10N4OS2
(290.36): C, 49.64; H, 3.47; N, 19.30. Found: C, 49.65; H, 3.49; N, 19.30.
3.2.8. General procedure for compounds 17 and 18
Compound 1 (10 mmol) and the appropriate dicarboxylic acid (10 mmol) in POCl3 (15 mL) were
gently heated for 6 h, the reaction mixture was concentrated to 1/3 volume under reduced pressure and
leave to cool, cold water (20 mL) was added to the reaction mixture at 0 °C, and the solid product that
formed was collected by filtration and crystallized from ethanol.
2-(4-Oxo-3-(2-(2-thienyl)vinyl)-4H-[1,3,4]thiadiazolo-[2,3-c][1,2,4]tri-azin-7-yl)benzoic acid (17).
Yield 60%; m.p. 300–302 °C; IR (KBr): 3,463 cm−1 (br, OH, acid), 1,715 cm−1 (C=O, acid), 1,680
1
cm−1 (C=O, amide); H-NMR (DMSO-d6): δ = 6.72 (d, 1H, J = 15.9 Hz, CH=CHe), 6.98 (dd, 1H,
J = 3.80, 4.80 Hz, thiophene-Hb), 7.38 (d, 1H, J = 3.30 Hz, thiophene-Hc), 7.56 (d, 1H, J = 4.80 Hz,

Molecules 2011, 16
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thiophene-Ha), 7.72–8.37 (m, 4H, Ar-H and CHd=CH), 12.18 (s, 1H, OH, exchange with D2O); Anal.
Calcd for C17H10N4O3S2 (382.42): C, 53.39; H, 2.64; N, 14.65. Found: C, 53.38; H, 2.63; N, 14.64.
3-(4-Oxo-3-(2-(2-thienyl)vinyl)-4H-[1,3,4]thiadiazolo-[2,3-c][1,2,4]tr-iazin-7-yl)propanoic acid (18).
Yield 60%, m.p. 340–342 °C; IR (KBr): 3,445 cm−1 (br, OH, acid), 1,715 cm−1 (C=O, acid),
1,667 cm−1 (C=O, amide); 1H-NMR (DMSO-d6): δ = 1.82 (t, 2H, CH2), 2.68 (t, 2H, CH2CO), 6.75 (d,
1H, J = 15.9 Hz, CH=CHe), 7.09 (dd, 1H, J = 3.80, 4.80 Hz, thiophene-Hb), 7.32 (d, 1H, J = 3.30 Hz,
thiophene-Hc), 7.59 (d, 1H, J = 4.80 Hz, thiophene-Ha), 7.99 (d, 1H, J = 15.9 Hz, CHd=CH), 12.28 (s,
1H, OH, exchanged with D2O); 13C-NMR (DMSO-d6): δ = 26.80 (CH2), 32.50 (CH2CO), 118.2, 127.7,
128.4, 128.6, 129.6, 141.0, 141.2, 148.0, 148.5, 165.8 and 167.2 (Ar-C, C=C, 3C=N and 2C=O); Anal.
Calcd for C13H10N4O3S2 (334.37): C, 46.70; H, 3.01; N, 16.76. Found: C, 46.72; H, 3.00; N, 16.77.
4. Conclusions
In this work we prepared the S-glycosyl and S-alkyl derivatives of 1,2,4-triazinone, in addition to
thiadiazolotriazines. From the anticancer tested compounds its revealed that compounds 12 and 18 are
active cytotoxic agents against different cancer cell lines, to a variable extent. This cytotoxic effect
was found to be mainly due to apoptosis, which indicated that those compounds may act as promising
candidate anticancer agents. From the chemistry point of view, the cytotoxic effects may be due to the
presence of free CH2CH2CH2OH or CH2CH2COOH groups in these compounds.
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