An Easy Access to Construct Some New Fused 1,2,4-Triazines with Ring Junction Nitrogen Systems and Their Biological Evaluation

Article abstract of DOI:10.1002/jhet.2718

The chemical reactivity of 4-amino-6-benzyl-3-mercapto-1,2,4-triazine-5(4H)-one (1) towards various aliphatic or/and mono and bis aromatic carboxylic acid derivatives to give the corresponding fused heterocyclic systems, 1,3,4-thiadiazoles 4, 5, 6, which incorporating 1,2,4-triazine moiety was achieved. Moreover, compound 1 was subjected to reaction either with halo acetic acids or bromo ester to afford the respective fused nitrogen ring junction systems, thiadiazole 2 and 3 or thiadiazine 7. However, while the tetracyclic ring system 9 was furnished through condensation reaction of isatine with triazine 1. In addition, some of the new synthesized compounds were evaluated as an antioxidant and antitumor agents.

Full text of DOI:10.1002/jhet.2718

Month 2016 An Easy Access to Construct Some New Fused 1,2,4-Triazines with Ring
Junction Nitrogen Systems and Their Biological Evaluation
*
Wafaa Hamama, Ghada El-Bana, Saad Shaaban, O. M. O. Habib, and Hanafi Zoorob
Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhoria Street, Mansoura ET-35516, Egypt
*
E-mail: wshamama53@gmail.com
Received June 7, 2015
DOI 10.1002/jhet.2718
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
The chemical reactivity of 4-amino-6-benzyl-3-mercapto-1,2,4-triazine-5(4H)-one (1) towards various
aliphatic or/and mono and bis aromatic carboxylic acid derivatives to give the corresponding fused
heterocyclic systems, 1,3,4-thiadiazoles 4-6, which incorporating 1,2,4-triazine moiety was achieved.
Moreover, compound 1 was subjected to reaction either with halo acetic acids or bromo ester to afford the
respective fused nitrogen ring junction systems, thiadiazole 2 and 3 or thiadiazine 7. However, while the
tetracyclic ring system 9 was furnished through condensation reaction of isatine with triazine 1. In addition,
some of the new synthesized compounds were evaluated as an antioxidant and antitumor agents.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
Triazine 1 has two competing reactive sites at both the
thiol–thioxo and amino groups, which can be used for the
synthesis of nitrogen ring junction triazines, concordant
with similar reactions [17]. Refluxing 1 with various ali-
phatic acid, namely, chloroacetic acid, dichloroacetic acid,
and N-acetylglycine, respectively, in phosphorus
oxy-chloride (POCl3) afforded the corresponding [1,3,4]
thiadiazolo[2,3-c] [1,2,4]triazine-4-ones 2–4. Furthermore,
triazine 1 was refluxed with the corresponding mono and
bis aromatic acids, namely, 3-hydroxy benzoic acid and
isophthalic acid under the same experimental condition
used for 2–4 to afford 3-benzyl-7-(3-hydroxyphenyl)-4H-
[1,3,4]thiadiazolo[2,3-c][1,2,4] triazine-4-one (5) and
7,7′-(1,3-phenylene) bis (3-benzyl-4H-[1,3,4]thiadiazolo
[2,3-c][1,2,4]triazine-4-one (6), respectively.
A great attention has been directed to the synthesis of
fused 1,2,4-triazines with ring junction nitrogen systems
because they constitute an important class of natural
products and exhibit many useful biological activity [1,2].
1,3,4-Thiadiazoles have been used in the pharmaceutical
area as an antibacterial with known sulfonamide drugs,
antimicrobial, antidepressant, antitumor, antioxidant, anti-
HIV, antileishmanial, antidiabetic, and anti-inflammatory
[3–10]. Interesting biological activity of 1,3,4-thiadiazole
probably attributed to N–C–S linkage [5].
Heterocyclic fused ring systems consisting of
1,2,4-triazole or 1,3,4-thiadiazine represent a class of
compounds that possess a broad spectrum of biological
activities such as anti-inflammatory, antiviral, antimicrobial,
and antitumor activity [11–15].
1
The H NMR showed new peaks at 1.223, 4.823, and
6.915 ppm attributed to CH2N, CH2Cl, and CHCl2
indicating that the carboxylic group of the acids used
reacted with SH and NH2 groups to afford [1,3,4]
thiadiazolo[2,3-c] [1,2,4] triazine-4-ones 2–4, respectively.
13C NMR spectra of compounds 2 and 3 showed signals at
58.437 and 64.033ppm attributed to CH2Cl and CHCl2, re-
spectively. Furthermore, the mass spectrum of compound 2
showed a molecular ion peak at m/z [292/294, (100/38)].
The IR spectrum of compound 5 showed a broad band
for the OH group at 3420cmꢀ1, which observed at
Consequently, in view of this fact and as a part of an
ongoing investigation into biologically more active and less
toxic substances, we focused our attention on the synthesis
of a series of new 1,3,4-thiadiazolo[2,3-c]1,2,4-triazin-4-
ones and 1,2,4-triazolo[3,4-b]1,3,4-thiadiazines in order to
investigate their biological activity.
RESULT AND DISCUSSION
1
5.859 ppm in its H NMR. Whereas, 13C NMR spectrum
The target compounds were synthesized as outlined in
Schemes 1 and 2. The starting material 4-amino-6-ben-
zyl-3-mercapto-1,2,4-triazine-5(4H)-one (1) was consistent
as obtained previously [16], but we can separate the precip-
itate formed in the reaction mixture while it was still hot.
of 5 showed the presence of carbon signals at appropriate
chemical shift values.
Furthermore, when triazine 1 refluxed with ethyl
bromoacetate in acetic acid in the presence of anhydrous
sodium acetate afforded 3-benzyl-[1,2,4]triazino[3,4-b]
© 2016 Wiley Periodicals, Inc.

W. S. Hamama, G. G. El-Bana, S. Shaaban, O. M. O. Habib, and H. H. Zoorob
Vol 000
Scheme 1. Transformation of trazine 1 to the corresponding thiadiazolotriazine systems.
[1,3,4]thiadiazine-4,7(6H,8H)-dione (7). The 1H NMR
spectrum of compound 7 showed singlet signals at
4.140 ppm for CH2–S protons. While, fusion of 1 with
diethyl oxalate at 210–220°C furnished 8. The IR spectra
for compounds 7 and 8 showed bands at 3380cmꢀ1 (NH)
and 1690 (C = O), 1677 (O = C–NH amide), 1640 (C = O
amide), and 1535 (NH, bending).
The rate and/or the extent of the process measured in
terms of the decrease in X. concentration would be related
to the ability of the added compounds to trap free radicals.
The decrease in color intensity of the free radical solution
according to the scavenging of the free radical by the
antioxidant material is measured calorimetrically at a
specific wavelength. The actual assay employs the radical
cation derived from 2,2′-azino-bis(3-ethyl benzthiazoline-
6-sulfonic acid) (ABTS) as stable free radical to measure
the antioxidant potential of the isolated compounds and
extracts. The main advantage of ABTS-derived free radical
method over other methods is that the product color
remains stable for more than 1 hr and the reaction is
stoichiometric.
The antioxidant activity of the new synthesized
compounds was evaluated using the procedure
developed by Lissi et al. [19]. Some of the 1,2,4-triazine
derivatives exhibited antioxidant activity as shown in
Table 1. Compared with the control compound
(L-ascorbic acid), the antioxidant potency of compound
In
addition,
3-benzyl-[1,2,4]triazino[3,4-b][1,3,4]
thiadiazine-4,7,8(6H)-trione (9) was furnished with heating
compound 1 with isatine in glacial acetic acid. The IR
spectrum of 9 showed band at 1691cmꢀ1 for C = O, and
its 1H NMR spectrum showed signals at 7.273–
8.042 ppm for the aromatic protons.
Because of the very low solubility of compounds 4, 6, 7, 8,
and 9, we were unable to perform their 13C NMR spectra [18].
Biological activity
Antioxidant activity for ABTS. The antioxidant activity
assay employed here is one of the several assays that
depend on measuring the consumption of stable free
radicals, that is, assesses the free radical scavenging
activity of the investigated component. The methodology
assumes that the consumption of the stable free radical
(X) will be determined by reactions as follows:
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Fused 1,2,4-Triazines with Ring Junction Nitrogen Systems
Scheme 2. Conversions of triazine 1 to the corresponding triazinothiadiazine systems.
8 was found to be high, whereas compounds 4, 6, and 7
showed moderate antioxidant activity, and the rest of
the tested compound showed weak activities (Figs. 1
and 2).
moieties incorporated with the 1,2,4-triazine skeleton
enhanced the antioxidant activity of 1,2,4-triazine
derivatives.
Antitumor activity
From the structure activity relationships, we found that
the presence of 1,3,4-thiadiazolo and 1,3,4-thiadiazine
Effect of drugs on the viability of Ehrlich ascites carcinoma
cells in vitro. Compounds (2–9) were tested for potential
antitumor activity against Ehrlich ascites carcinoma
(EAC) in vitro [20,21]. The results for the IC100, IC50,
and IC25 values of the tested compounds are summarized
in Table 2.
Table 1
2,2′-Azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) (ABTS) antioxi-
dant activity assay for 1,2,4-triazine analogues.
The data clearly showed that all the tested compounds
showed more toxicity than the 5-FU in vitro studies.
Interestingly, compound 6 was increased around 10
times more toxic than the 5-FU, and compounds 3, 4,
5, 7, and 9 were increased around four times more toxic
than the 5-FU. The experimental potential antitumor
activity of the tested compounds reported in this study
to their structures and the following structure activity
relationships were postulated: (a) compound 6 contains
two 1,2,4-triazinothiadiazole moiety. (b) Also, in case
for compounds 3, 4, and 5, the high cytotoxic activity
may be attributed to the presence of 1,2,4-triazinothiadiazole
moiety, which is in agreement with that reported [6].
(c) Moreover, the high cytotoxic activity of compounds 7
and 9 may be attributed to the presence of 1,2,4-triazino-
thiadiazine moiety (Figs. 3 and 4).
ABTS
Compounds
Absorbance of samples
% inhibition
Control of ABTS
Ascorbic acid
0.520
0.065
0.390
0.418
0.212
0.284
0.129
0.186
0.080
0.337
0
87.50
25.00
19.61
59.23
45.38
75.19
64.23
84.61
35.19
2
3
4
5
6
7
8
9
Of the tested compounds, 50 mL were prepared (1 mg/mL) in spectro-
scopic grade MeOH/phosphate buffer reaction mixture (1 mL, 1:1 v/v),
and 50 mL of (2 mm) of ascorbic acid was used in spectroscopic grade
Me–OH/phosphate buffer reaction mixture (1 mL, 1:1 v/v).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

W. S. Hamama, G. G. El-Bana, S. Shaaban, O. M. O. Habib, and H. H. Zoorob
Vol 000
Figure 1. Structure of the most potent compounds 4, 6, 7, and 8.
EXPERIMENTAL
thiocarbahydrazide (1.06 g, 10mmol) in warm water
(30 mL). The reaction mixture was refluxed on a water bath
for 1 h. The solid that precipitated hot was filtered off, dried,
and recrystallized to afford 1.
All melting points are in degree centigrade (uncorrected)
and were determined on Gallenkamp electric melting point
apparatus. Elemental analyses were carried out at Micro
analytical Center, Faculty of Science, Cairo University.
IR spectra were recorded (KBr), (ύ cmꢀ1) on a Mattson
5000 FTIR Spectrophotometer at Micro analytical Center
White sheets (0.9 g, 85%): (lit. Yield 78%); mp
205–206°C: (lit. mp 195–198°C) [16] [ethanol]; Rf = 0.37
[pet. ether (60:80)/ethyl acetate (4:1)]; IR (KBr):
ν cmꢀ1 = 3298, 3209 (NH2), 1686 (C = O); 1H NMR
(300 MHz, DMSO-d6) δ (ppm): 2.528 (s, 1H, SH), 3.925
(s, 2H, CH2–Ph), 6.585 (s, 2H, NH2), 7.220–7.335 (m,
5H, H–Ar); 13C NMR (75 MHz, DMSO-d6) δ: 36.357
(1C, CH2Ph ), 127.126 (1C, CAr), 128.880 (2CH, CAr),
129.672 (2CH, CAr), 136.832 (1C, CAr), 147.459 (1C,
C-6), 148.941 (1C, C-3), 169.002 (1C, CO). 13C NMR
(dept135, DMSO-d6) δ: 35,543 (1C, CH2).
1
Faculty of Science, Mansoura University. The H NMR
and 13C NMR spectra were measured on a Varian Spectro-
photometer at 300 and 75 MHz, respectively. Using TMS
as an internal reference and DMSO-d6 as solvent at
Chemistry Department, Faculty of Science, Cairo
University. The chemical shifts (δ) are reported in parts
per million and were referenced to the residual solvent
peak. Mass spectra were recorded on (Kratos, 70ev) MS
equipment and/or a Varian MAT 311A Spectrometer at
Micro analytical Center, Faculty of Science, Cairo
University. Reaction mixtures were monitored by thin
layer chromatography using EM science silica gel-coated
plates with visualization by irradiation with ultraviolet
lamp. Biological testing was carried out at Drug Depart-
ment, Faculty of Pharmacy, Mansoura University,
Mansoura, Egypt. Ehrlich cells (EAC) were derived from
ascetic fluid from diseased mice (the cells were purchased
from the National Cancer Institute, Cairo, Egypt).
Synthesis of thiadiazolotriazines 2-5
General procedure.
A mixture of triazine 1 (0.5 g,
2.14 mmol) and the proper monocarboxylic acid
derivatives (2.14mmol) in phosphorus oxychloride
(7.5 mL) was refluxed for 12h over a water bath. The
reaction mixture was cooled and poured onto crushed ice.
The resulting solid was filtered off and washed with
aqueous sodium bicarbonate solution (2%) till to
neutralize at pH 7 and then water (30 mL). It was dried
and recrystallized from the proper solvent to yield 2–5.
4-Amino-6-benzyl-3-mercapto-1,2,4-triazine-5(4H)-one (1).
A solution of phenyl pyruvic acid (1.64g, 10mmol) in ethanol
Table 2
In vitro potential antitumor activity of 1,2,4-triazine analogues using EAC
(20 mL) was added dropwise to
a
solution of
assay.
Death
Compound
no
IC100 (μM)
IC50 (μM)
IC25 (μM)
5-Fu
0.762
0.324
0.245
0.273
0.276
0.089
0.286
0.326
0.240
0.461
0.164
0.119
0.142
0.141
0.04
0.149
0.17
0.118
0.283
0.085
0.064
0.078
0.077
0.026
0.076
0.097
0.068
2
3
4
5
6
7
8
9
IC100, IC50, and IC25 are the inhibitor concentration at 25, 50, and
100 μM, respectively, of the compounds used. 5-FU is 5-fluorouracil, a
well-known cytotoxic agent.
Figure 2. Antioxidant activity screening assay. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
EAC, Ehrlich ascites carcinoma.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Fused 1,2,4-Triazines with Ring Junction Nitrogen Systems
Figure 3. Structure of the most potent compounds 3, 4, 5, 6, 7, and 9.
3-Benzyl-7-(chloromethyl)-4H-[1,3,4]thiadiazolo[2,3-c]
[1,2,4]triazin-4-one (2).
monochloroacetic acid (0.2 mL, 2.14mmol).
(ppm): 4.307 (s, 2H, CH2–Ph), 6.915 (s, 1H, CHCl2),
7.207–7.459 (m ,5H , H–Ar); 13C NMR (75 MHz,
DMSO-d6) δ: 35.323 (1C, CH2Ph), 64.033 (1C, CHCl2),
125.841 (1CH, CAr), 128.223 (2CH, CAr), 129.095 (2CH,
CAr), 136.643 (1C, CAr), 148.052 (1C, C-3), 155.754
(1C, C-6), 159.805 (1C, C-3), 161.370 (1C, CO); MS
(EI, 70ev) m/z (%)= 326/328/330 (M+, 63/43/10), 144
(35.1), 117 (46.8), 142 (14.4), 94 (18.9), 85 (16.2), 83
(40.5), 76 (31.5), 52 (18.9), 50 (100.0); Anal. Calcd for
C12H8Cl2N4OS (325.98): C, 44.05; H, 2.46; N, 17.12 %.
Found: C, 44.03; H, 2.43; N, 17.10%.
An acid derivative used was
Yellow powder (0.5 g, 80%); mp 225–227°C; [ethanol];
Rf =0.45 [pet. ether (60:80)/ethyl acetate (4:1)]; IR (KBr):
1
ν cmꢀ1 =1697 (C =O); H NMR (300MHz, DMSO-d6) δ
(ppm): 4.327 (s, 2H, CH2–Ph), 4.823 (s, 2H, CH2–Cl),
7.259–7.486 (m, 5H, H–Ar); 13C NMR (75MHz,
DMSO-d6) δ: 36.264 (1C, CH2Ph), 58.447 (1C, CH2Cl),
126.110 (1CH, CAr), 128.781 (2CH, CAr), 129.026 (2CH,
CAr), 142.850 (1C, CAr), 152.810 (1C, C-3), 153.715 (1C,
C-6), 160.842 (1C, C-3), 165.477 (1C, CO); MS (EI, 70 ev)
m/z (%) =292/294 (M+, 100.0/83.7), 217 (23.8), 117 (61.5),
91 (54.3), 78 (8.5), 77 (57.1), 76 (18.5), 75 (13.4), 52 (10.9),
50 (22.4); Anal. Calcd for C12H9ClN4OS (292.02): C, 49.24;
H, 3.10; N, 19.14 %. Found: C, 49.22; H, 3.08; N, 19.13%.
3-Benzyl-7-(dichloromethyl)-4H-[1,3,4]thiadiazolo[2,3-c][1,2,4]
triazin-4-one (3). An acid derivative used was dichloroacetic
acid (0.27 mL, 2.14 mmol).
N-((3-benzyl-4-oxo-4H-[1,3,4]thiadiazolo[2,3-c][1,2,4]
triazin-7-yl)methyl) acetamide (4).
An acid derivative
used was mono N-acetyl glycine (0.25 g, 2.14 mmol).
Brown powder (0.46g, 68%); mp 237–239°C; [EtOH/
DMF (1:2)]; Rf = 0.34 [ethyl acetate/pet. ether (60:80)
(4:2)]; IR (KBr): ν/cmꢀ1 = 3453 (enolic OH) and 1709
1
(C = O); H NMR (300MHz, DMSO-d6) δ (ppm): 1.221
(s, 2H, CH2–N), 1.999 (S, 3H, CH3), 3.682 (s, 1H, OH),
4.175 (s, 2H, CH2–Ph), 7.249–7.317 (m, 5H, H–Ar),
10.40 (br, 1H, NH); MS (EI, 70ev) m/z (%) = 315 (M+,
1.7), 131 (16.8), 129 (6.0), 117 (69.6), 94 (0.9), 91
(100.0), 76 (21.0), 60 (30.7), 58 (15.6), 52 (13.9), 50
(32.1); Anal. Calcd for C14H13N5O2S (315.08): C, 53.32;
H, 4.16; N, 22.21 %. Found: C, 53.35; H, 4.18; N, 22.23%.
3-Benzyl-7-(3-hydroxyphenyl)-4H-[1,3,4]thiadiazolo[2,3-
c][1,2,4]triazin-4-one (5). An acid derivative used was
mono 3-hydroxy benzoic acid (0.3 g, 2.14 mmol).
Brown crystals (0.57 g, 82%); mp 160–161°C; [ethanol];
Rf = 0.5 [pet. ether (60:80)/ethyl acetate (4:1)]; IR (KBr):
1
ν cmꢀ1 =1697 (C =O); H NMR (300 MHz, DMSO-d6) δ
Orange powder (0.56 g, 78%); mp 243–244°C [EtOH/
DMF (1:2)]; Rf = 0.36 [ethyl acetate/pet. ether (60:80)
(4:2); IR (KBr): ν/cmꢀ1 = 3420 (OH) and 1702 (C = O);
1H NMR (300MHz, DMSO-d6) δ (ppm): 4.903 (s, 2H,
CH2–Ph), 5.852 (s, 1H, OH, D2O exchangeable), 7.215–
7.977 (m, 9H, H–Ar); 13C NMR (75 MHz, DMSO-d6)
δ:36.308 (1C, CH2Ph), 121.314 (2CH, CAr), 125.803
(2CH, CAr), 128.280 (2CH, CAr), 129.125 (2CH, CHAr),
129.888 (1CH, CAr), 136.844 (2CH, CAr), 148.035 (1C,
C-3,), 155.119 (1C, C-6), 159.816 (1C, C-3), 160.030
Figure 4. Antitumor activity for the novel prepared compounds. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

W. S. Hamama, G. G. El-Bana, S. Shaaban, O. M. O. Habib, and H. H. Zoorob
Vol 000
(1C, CAr–OH), 165.493 (1C, CO); MS (EI, 70 ev) m/z (%)
= 336 (M+, 20.4), 131 (16.8), 129 (6.0), 117 (69.6), 94
(0.9), 91 (100.0), 76 (21.0), 60 (30.7), 58 (15.6), 52
(13.9), 50 (32.1); Anal. Calcd for C17H12N4O2S (336.37):
C, 60.70; H, 3.60; N, 16.66%. Found: C, 60.72; H, 3.61;
N, 16.67%.
(s, 2H, CH2–Ph), 7.252–7.664 (m, 5H, H–Ar) 7.953 (s,
1H, NH); MS (EI, 70ev) m/z (%) =289 (M++1, 0.1), 117
(0.71), 101 (0.9), 94 (1.4), 88 (1.0), 89 (3.9), 50 (100.0);
Anal. Calcd for C12H8N4O3S (288.03): C, 50.00; H, 2.80;
N, 19.44%. Found: C, 50.02; H, 2.81; N, 19.45%.
3-Benzyl-[1,2,4]triazino[3,4-b][1,3,4]thiadiazine-4,7,8(6H)-
trione (9). A mixture of triazine 1 (0.5 g, 2.14 mmol) and
isatine (0.31 g, 2.14mmol) in glacial acetic acid (20 mL) in
the presence of sodium acetate (0.2 g, 2.4 mmol) was
refluxed for 24h, cooled to room temperature, and poured
onto ice. The formed precipitate was separated by
filtration, washed with hot ethanol filtered (10 mL,
threefold), and recrystallized to furnish 9.
Black powder (0.5 g, 68%); mp >300°C [EtOH/DMF
(1:2)]; Rf = 0.5 [ethyl acetate/EtOH (4:5)]; IR (KBr):
ν/cmꢀ1 = 1691 (C = O amide); 1H NMR (300MHz,
DMSO-d6) δ (ppm): 4.163 (s, 2H, CH2–Ph), 7.273–8.042
(m, 9H, H–Ar); MS (EI, 70ev) m/z (%) =346 (M++1,
0.1), 117 (16.4), 116 (18.0), 114 (2.7),94 (0.9), 91 (20.0),
52 (49.3), 50 (100.0); Anal. Calcd For C18H11N5OS
(345.28): C, 62.60; H, 3.21; N, 20.28%. Found: C, 62.58;
H, 3.19; N, 20.27%.
7,7′-(1,3-Phenylene)bis(3-benzyl-4H-[1,3,4]thiadiazolo[2,3-c]
[1,2,4]triazin-4-one) (6).
A mixture of triazine 1 (0.5g,
2.14mmol) and isophthalic acid (0.18g, 1.07mmol) in
phosphorus oxychloride (7.5ml) was refluxed for 12h
over a water bath. The reaction mixture was cooled and
poured onto crushed ice. The resulting solid was filtered
off and washed with aqueous (2%) sodium bicarbonate
solution till to neutralize at pH 7 and then water (30mL).
It was dried and recrystallized to afford 6.
Yellow powder (1.06 g, 88%); mp 220–222°C [EtOH/
DMF (1:2)]; Rf = 0.3 [pet. ether (60:80)/EtOH) (1:2)]; IR
(KBr): ν/cmꢀ1 = 1698 (2 C =O) ; 1H NMR (300MHz,
DMSO-d6) δ (ppm): 4.065 (s, 2H, CH2), 7.201–7.789 (m,
9H, H–Ar); MS (EI, 70 ev) m/z (%)= 563 (M++1, 9.5),
377 (10.1), 320 (19.9), 186 (15.8), 117 (12.0), 94 (21.4),
52 (56.4), 50 (100.0); Anal. Calcd for C28H18N8O2S2
(562.10): C, 59.77; H, 3.22; N, 19.92%. Found: C, 59.75;
H, 3.20; N, 19.90%.
Pharmacological screening for antitumor activity using
Ehrlich ascites in vitro assay
3-Benzyl-[1,2,4]triazino[3,4-b][1,3,4]thiadiazine-4,7(6H,8H)-
dione (7). A mixture of triazine 1 (0.5g, 2.14mmol) and
ethyl bromoacetate (0.23 mL, 2.14mmol) in glacial acetic
acid (20 mL) in the presence of sodium acetate (0.2g,
2.4mmol) was refluxed 8 h, cooled to room temperature,
and poured onto ice. The formed precipitate was
separated by filtration, washed with hot ethanol (10mL,
threefold) and recrystallized to yield 7.
Dark red powder (0.37 g, 63%); mp >300°C [EtOH/
DMF (1:2)]; Rf = 0.32 [ethyl acetate/EtOH (4:6)]; IR
(KBr): ν/cmꢀ1 = 3380 (NH) and 1691 (O = C–NH amide),
1647 (O = C amide); 1535 (NH, bending); 1H NMR
(300 MHz, DMSO-d6) δ (ppm): 3.903 (s, 2H, CH2–Ph),
4.140 (s, 2H, –CH2–S), 7.280–7.796 (m, 6H, H–Ar, NH);
MS (EI, 70ev) m/z (%)=274 (M+, 20.4), 183 (3.9),117
(11.8), 94 (6.6), 91 (14.7), 52 (43.8), 50 (8.1), 46 (18.5), 45
(100.0); Anal. Calcd for C12H10N4O2S (274.05): C, 52.54;
H, 3.67; N, 20.34%. Found: C, 52.56; H, 3.70; N, 20.36%.
3-Benzyl-[1,2,4]triazino[3,4-b][1,3,4]thiadiazine-4,7,8(6H)-
trione (8). A mixture of triazine 1 (0.5 g, 2.14 mmol)
and diethyl oxalate (0.3 mL, 2.14 mmol) was fused in an
oil bath at (210–220°C) for 7 h. The solid product that
obtained was left to cool at room temperature, filtered
off, then washed with hot ethanol (10 mL, threefold),
dried, and recrystallized to give 8.
ABTS antioxidant screening assay
Reagents.
Vitamin C was obtained from Sigma, ABTS
was purchased from Wak, and all other chemicals were of
the highest quality available.
For each of the investigated compounds, 2 mL of ABTS
solution (60 μM) was added to 3M MnO2 solution (25 mg/
mL) all prepared in phosphate buffer (pH 7, 0.1 M). The
mixture was shaken, centrifuged, filtered, and the absor-
bance [A (control)] of the resulting green-blue solution
(ABTS radical solution) was adjusted at ca. 0.5 at λ
734 nm. Then, 50 μL of (2 mm) solution of the test com-
pound in spectroscopic grade methanol/phosphate buffer
(1:1) was added. The absorbance [A (test)] was measured,
and the reduction in color intensity was expressed as %
inhibition. The inhibition for each compound was
calculated from the following equation.
% Inhibition ¼ ½A ðcontrolÞ - A ðtestÞ = A ðcontrolÞꢁ X 100
Vitamin C was used as a standard antioxidant (positive
control). Blank sample was run without ABTS and using
methanol/phosphate buffer (1:1) instead of samples. The
negative control sample was run with methanol/phosphate
buffer (1:1) instead of tested compound.
Antitumor activity.
Ehrlich cells were derived from
ascetic fluid from diseased mouse (purchased from the
National Cancer Institute, Cairo, Egypt). The cells were
grown in suspension culture, partly floating and partly
attached, in RPMI 1640 medium, supplemented with
10% fetal bovine serum. They were maintained at 37°C
in a humidified atmosphere with 5% CO2. The viability
Black powder (0.43 g, 70%); mp 260–261°C [EtOH/
DMF (1:2)]; Rf = 0.37 [pet. ether (60:80)/EtOH (1:2)]; IR
(KBr): ν/cmꢀ1 = 3380 (NH, stretching), 1701(C = O),
1691 (O = C–NH amide), 1640 (C = O amide), 1535 (NH,
bending) ;1H NMR (300 MHz, DMSO-d6) δ (ppm): 3.826
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Fused 1,2,4-Triazines with Ring Junction Nitrogen Systems
[10] Joshi, S.; Manikpuri, A. D.; Khare, D. J Indian Chem Soc
2008, 85, 508.
[11] Khan, I.; Zaib, S.; Ibrar, A.; Rama, N. H.; Simpson, J.; Iqbal, J.
Eur J Med Chem 2014, 78, 167.
[12] Agarwal, A.; Srivastava, K.; Puri, S. K.; Chauhan, P. M. S.
Bioorg Med Chem Lett 2005, 15, 531.
of the cells used in control experiments (DMSO only
without drug) exceeded 95% as determined by trypan
blue. Test compounds were prepared initially at
concentration of 1 mg/mL DMSO.
[13] Kuo, G.-H.; DeAngelis, A.; Emanuel, S.; Wang, A.; Zhang,
Y.; Connolly, P. J.; Chen, X.; Gruninger, R. H.; Rugg, C.;
Fuentes-Pesquera, A.; Middleton, S. A.; Jolliffe, L.; Murray, W. V. J Med
Chem 2005, 48, 4535.
[14] Srinivas, K.; Srinivas, U.; Bhanuprakash, K.; Harakishore, K.;
Murthy, U. S. N.; Jayathirtha Rao, V. Eur J Med Chem 2006, 41, 1240.
[15] McKay, G. A.; Reddy, R.; Arhin, F.; Belley, A.; Lehoux, D.;
Moeck, G.; Sarmiento, I.; Parr, T. R.; Gros, P.; Pelletier, J.; Far, A. R.
Bioorg Med Chem Lett 2006, 16, 1286.
REFERENCES AND NOTES
[1] Kumar, R.; Sirohi, T. S.; Singh, H.; Yadav, R.; Roy, R. K.;
Chaudhary, A.; Pandeya, S. N. Mini Rev Med Chem 2014, 14, 168.
[2] Khan, I.; Ibrar, A.; Abbas, N. Eur J Med Chem 2013, 63, 854.
[3] Düğdü, E.; Ünver, Y.; Ünlüer, D.; Sancak, K. Molecules 2014,
19, 2199.
[4] Khan, M. R. A.; Ahmed, B. Bioorg Med Chem 2008, 16, 8029.
[5] Hamama, W. S.; Gouda, M. A.; Badr, M. H.; Zoorob, H. H.
J Heterocyclic Chem 2013, 50, 787.
[6] Hamama, W. S.; Gouda, M. A.; Badr, M. H.; Zoorob, H. H.
J Heterocyclic Chem 2014, 51, 1558.
[7] Cressier, D.; Prouillac, C.; Hernandez, P.; Amourette, C.;
Diserbo, M. Bioorg Med Chem 2009, 17, 5275.
[8] Poorrajab, F.; Ardestani, S. K.; Emami, S.; Behrouzi-
Fardmoghadam, M.; Shafiee, A.; Foroumadi, A. Eur J Med Chem 2009,
44, 1758.
[16] Holla, B. S.; Gonsalves, R.; Sarojini, B. K. Ind J Chem, Sect B
1997, 36, 943.
[17] Ashok, M.; Holla, B. S. Phosphorus, Sulfur, Silicon Relat
Elem 2007, 182, 981.
[18] Bazgir, A.; Tisseh, Z. N.; Mirzaei, P. Tetrahedron Lett 2008,
49, 5165.
[19] Lissi, E. A.; Modak, B.; Torres, R.; Escobar, J.; Urza, A. Free
Rad Res 1999, 30, 471.
[20] Saluja, M. S.; Sangameswaran, B.; Sharma, A. Int J Pharm
Tech Res 2010, 2, 1369.
[9] Joseph, A.; Joseph, B.; Narayanab, B. J Indian Chem Soc
2008, 85, 479.
[21] Patel, K. C.; Rami, C. S.; Panigrahi, B.; Patel, C. N. J Chem
Pharm Res 2010, 2, 73.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet