Convenient synthesis of fused heterocyclic 1,3,5-triazines from some N-Acyl imidates and heterocyclic amines as anticancer and antioxidant agents

Article abstract of DOI:10.1002/ardp.200400964

N-Acyl imidates (2), reacting with 5-amino pyrazole (3), 2-aminobenzimidazole (4), 3-amino-1,2,4-triazole (5), 3,5-diamino-1,2,4-triazole (6), and 5-aminotetrazole (7) give pyrazolo[1,5-a][1,3,5]triazine (8), benzo[4,5]imidazo[1,2-a][1,3,5]triazine (9), [1,2,4]triazolo [2,3-a][1,3,5]triazine (10), [1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamine (12), and tetrazolo-[1,5-a][1,3,5]triazine (14) derivatives, respectively. The synthesized compounds were characterized on the basis of IR, ¹H-NMR, ¹³C-NMR, and mass spectral data and elemental analyses results. Five of the newly synthesized compounds, 8a, 9a, 10a, 12a, and 14a, were selected by National Cancer Institute and screened for their anticancer activity against three cancer cell lines MCF7, NCI-H460, and SF-268, where 12a exhibited moderate antiproliferation potential. 12a was, thus, further tested for anticancer activity against 60 human cancer cell lines and showed moderate growth inhibition potency. 12a showed a high growth inhibitory activity against A498 renal cancer cell line. All of the newly synthesized compounds 8-10, 12 and 14 were tested for their antioxidant capacity where they exhibited very high activity, even higher than the widely used reference antioxidants butylated hydroxytoluene and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) Compound 12a also showed the highest antioxidant activity.

Full text of DOI:10.1002/ardp.200400964

Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
DOI 10.1002/ardp.200400964
365
Convenient Synthesis of Fused Heterocyclic
1,3,5-Triazines from Some N-Acyl Imidates and
Heterocyclic Amines as Anticancer and Antioxidant
Agents
Olcay Bekircana, Murat Küxükb, Bahittin Kahvecic, Sevgi Kolaylıb
a Department of Chemistry, Giresun Faculty of Arts & Sciences, Karadeniz Technical University, Giresun,
Turkey
b Department of Chemistry, Faculty of Arts & Sciences, Karadeniz Technical University, Trabzon, Turkey
c Department of Chemistry, Rize Faculty of Arts & Sciences, Karadeniz Technical University, Rize, Turkey
N-Acyl imidates (2), reacting with 5-amino pyrazole (3), 2-aminobenzimidazole (4), 3-amino-1,2,4-
triazole (5), 3,5-diamino-1,2,4-triazole (6), and 5-aminotetrazole (7) give pyrazolo[1,5-a][1,3,5]triazine
(8), benzo[4,5]imidazo[1,2-a][1,3,5]triazine (9), [1,2,4]triazolo [2,3-a][1,3,5]triazine (10), [1,2,4]tri-
azolo[2,3-a][1,3,5]triazin-5-ylamine (12), and tetrazolo-[1,5-a][1,3,5]triazine (14) derivatives, respec-
1
tively. The synthesized compounds were characterized on the basis of IR, H-NMR, 13C-NMR, and
mass spectral data and elemental analyses results. Five of the newly synthesized compounds, 8a, 9a,
10a, 12a, and 14a, were selected by National Cancer Institute and screened for their anticancer activity
against three cancer cell lines MCF7, NCI-H460, and SF-268, where 12a exhibited moderate anti-
proliferation potential. 12a was, thus, further tested for anticancer activity against 60 human cancer
cell lines and showed moderate growth inhibition potency. 12a showed a high growth inhibitory activity
against A498 renal cancer cell line. All of the newly synthesized compounds 8-10, 12 and 14 were
tested for their antioxidant capacity where they exhibited very high activity, even higher than the
widely used reference antioxidants butylated hydroxytoluene and 6-hydroxy-2,5,7,8-tetramethylchro-
man-2-carboxylic acid (Trolox®) Compound 12a also showed the highest antioxidant activity.
Keywords: Fused heterocycle; N-Acyl imidate; 1,3,5-Triazine; Anticancer; Antioxidant
Received: November 30, 2004; Accepted: May 15, 2005
Introduction
syncytial virus [8]. s-Triazine derivatives of polyamines have
been reported as potential drugs for Human African Try-
panosomasis and the activity highly dependent on the tri-
azine moity [9]. Triazines have long been known with herbi-
cidal activity, since the resistance of weeds was recognized
in 1960s. Vicentini and co-workers recently presented such
an activity for fused ring triazine structures as photosyn-
thetic electron transport inhibitors, which reflects the poten-
tial for use against photosynthetic organisms [10]. Fused
triazine derivatives have also been reported to be good
corticotropin-releasing factor (CRF) antagonists, which
makes them drug candidates for depression and anxiety [11,
12]. Many such fused heterocyclic compounds, as pyrazolo-
triazines, triazolo-triazines, and benzimidazo-triazines, have
been synthesized by several methods [13Ϫ23].
Triazine derivatives are one group of the compound types
possessing a wide array of biological activities. Many in-
vestigations revealed the anticancer potential of various
types of triazine derivatives [1Ϫ4]. Nucleoside analogues of
triazolo-triazines [1], also structurally related to the anti-
biotic formycin A, amino substituted triazine derivatives
[2, 3], and platinum(II)-complexes of imidazo-triazine de-
rivatives [4] have been shown to exhibit antitumor activity.
Triazine derivatives have also been shown to have inhibitory
activity against the enzymes phosphodiesterase (PDE) [5,
6], which is expected to be the target for the treatment of
various diseases including asthma, diabetes mellitus, and
thrombosis, and against dihydrofolate reductase (DHFR),
inhibition of which leads to cell death [7]. Golankiewicz and
co-workers have reported antiviral imidazo-triazine deriva-
tives as potential agents against influenza A and respiratory
The current study was aimed at the synthesis of new fused
heterocyclic 1,3,5-triazine derivatives by starting from N-
acyl imidates, which have been known to be synthesized
from the reaction of imidoesters with acylchlorides [24Ϫ26].
N-Acyl imidates have been found to yield s-triazines, 3,5-
disubstituted-4H-1,2,4-triazoles, 1,3,5-trisubstituted-1,2,4-
triazoles, 3,5-disubstituted-1,3,4-oxadiazoles, and pyrazolo
Correspondence: Olcay Bekircan, Department of Chemistry, Gire-
sun Faculty of Arts & Sciences, Karadeniz Technical University,
28049-Giresun, Turkey. Phone: ϩ90 533 737-1938, Fax: ϩ90 454
216-4518, e-mail: obekircan@ktu.edu.tr
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

366
Bekircan et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
Scheme 1. Synthesis of N-acyl imidates.
Scheme 2. Synthesis routes for fused 1,3,5-triazine derivatives.
and triazolo-triazines from their reactions with amidines,
hydrazine, some hydrazine derivatives, hydroxylamine, and
some heterocyclic amines, respectively [27-30]. In the cur-
rent study, N-Acyl imidates (2) were synthesized, from the
reaction of imidoethyl esters (1) with benzoyl chloride
(Scheme 1) and then further derivatized by reacting with 5-
amino pyrazole (3), 2-amino benzimidazole (4), 3-amin-
1,2,4-triazole (5), 3,5-diamino-1,2,4-triazole (6), and 5-
aminotetrazole (7) (Scheme 2).
The synthesized compounds were evaluated for their phar-
macological activity by three cell line anticancer prescreen-
ing, a 60 human cancer cell line full anticancer screening,
and two antioxidant activity methods. Compounds 8a, 9a,
10a, 12a, and 14a, selected by the National Cancer Institute
(NCI), USA, were screened for their in vitro anticancer ac-
tivities. TLC-fluorescence persistence time antioxidant
method was used together with lipid peroxidation inhibition
method for better monitoring the antioxidant capacity of
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
Biologically Active, Fused Heterocyclic 1,3,5-Triazines
367
all the compounds. Lipid peroxidation inhibitory activity
measurement is widely used in many disciplines for the as-
sessment of antioxidant capacity of a sample either ob-
tained by synthesis or isolated from biological sources.
(9a), 2-phenyl-4-p-tolyl-benzo[4,5]imidazo[1,2-a][1,3,5]tri-
azine (9b), 5,7-diphenyl-tetrazolo[1,5-a][1,3,5]triazine (14a),
and 5-phenyl-7-p-tolyl-tetrazolo[1,5-a][1,3,5]triazine (14b)
with good yields. In addition, the condensation of com-
pounds 2 with heterocyclic amine 5 in an oil bath at
160Ϫ165°C are expected to give 5,7-diphenyl and 5-phenyl-
7-p-tolyl substituted [1,2,4]triazolo[2,3-a][1,3,5]triazine (10a
and 10b, respectively) and/or 5,7-diphenyl and 7-phenyl-5-p-
tolyl substituted [1,2,4]triazolo[4,3-a][1,3,5]triazine (11a and
11b). Similarly, the condensation of compounds 2 with het-
erocyclic amine 6 in an oil bath at 160-165°C are expected
to give 5,7-diphenyl and 5-phenyl-7-p-tolyl substituted
[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamine (12a and 12b)
and/or 5,7-diphenyl and 7-phenyl-5-p-tolyl substituted
[1,2,4]triazolo[4,3-a][1,3,5]triazin-5-ylamine (13a and 13b,
respectively). However, because the N(2) atom is more nu-
Results and discussion
Compound 2a, used as one of the starting agent in the
syntheses, is found in the literature, and the novel com-
pound 2b was synthesized according to the literature meth-
ods similar to 2a [24-26]. In the succeeding step, the cyclo-
condensation reaction of compounds 2 with heterocyclic
amines (3, 4, and 7) in an oil bath at 160Ϫ165°C gave the
novel compounds 2,4-diphenyl-pyrazolo[1,5-a][1,3,5]tri-
azine (8a), 2-Phenyl-4-p-tolyl-pyrazolo[1,5-a][1,3,5]triazine
(8b),
2,4-diphenyl-benzo[4,5]imidazo[1,2-a][1,3,5]triazine
Table 1. Sixty human cancer cell line anticancer screening results for compound 12a (NCI code: NSC 731071)†.
§
§
Cancer
Cell Line
GI50
TGI‡
[μM]
Cancer
Cell Line
GI50
TGI‡
[μM]
[μM]
[μM]
Leukemia
HL-60 (TB)
K-562
MOLT-4
>100
>100
>100
>100
>100
>100
Melanoma
LOX IMVI
7.78
>100
81.5
>100
>100
>100
>100
M14
SK-MEL-2
RPMI-8226
SR
>100
>100
>100
>100
SK-MEL-28
SK-MEL-5
64.5
19.7
>100
>100
Non-small
cell lung
cancer
A549/ATCC
EKVX
47.2
23.2
>100
>100
UACC-257
UACC-62
69.8
28.0
95.4
HOP-92
Ovarian
cancer
>100
17.6
26.2
54.1
15.3
35.8
73.3
>100
40.7
42.9
48.0
40.9
84.5
25.6
66.2
81.0
>100
41.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
IGROV1
85.6
25.9
25.9
24.9
37.3
44.4
0.493
18.2
41.2
44.7
18.9
12.2
>100
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCT-116
HCT-15
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
66.2
>100
57.6
>100
>100
30.6
Renal cancer
786-0
A498
Colon cancer
CNS cancer
ACHN
CAKI-1
RXF 393
SN12C
TK-10
97.8
>100
>100
50.3
39.3
>100
>100
HT29
KM12
SW-620
SF-268
UO-31
Prostate
cancer
SF-295
SF-539
SNB-19
SNB-75
U251
30.5
17.6
23.7
13.6
17.2
>100
52.1
>100
72.3
82.7
PC-3
DU-145
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
82.4
21.3
31.6
25.0
27.0
30.8
>100
23.4
>100
>100
>100
74.9
98.2
>100
72.7
Breast Cancer
†
50% lethal concentration (LC50, μM) values for all cell lines were larger than 100 μM. Mean-graph midpoint (MG-MID) values for GI50
and TGI for full panel were 35.5 and 87.1 μM, respectively. Insensitive cell lines were included with the highest concentration tested in the
calculation of MG-MID value.
GI50: The concentration of the test compound providing 50% inhibition of the growth.
TGI: The concentration of the test compound providing total growth inhibition.
§
‡
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

368
Bekircan et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
cleophilic than the N(4) atom in 1,2,4-triazole ring [31, 32],
the cyclocondensation reaction proceeds through 3-amino-
2H-1,2,4-triazole (5) and 3,5-diamino-2H-1,2,4-triazole (6)
structures and, thus, compounds 10 and 12 are more likely
to form. Therefore, the products of the above-mentioned
reactions are taken as compound 10 and 12 throughout this
article, but not compounds 11 and 13.
Results for each test agent are reported as the percent
growth of the tested cells when compared with the untreated
control cells. Only the compounds exhibiting 32% growth
inhibition or a higher activity are considered for further an-
ticancer testing by NCI. Compound 12a, which reduced the
growth of NCI-H460 cell line to 26% (νegative members
would indicate cell killing) was passed on for evaluation in
the full panel of 60 cancer cell lines.
The synthesized compounds were investigated from the
point of two biological activities: their anticancer and anti-
oxidant potential. The effectiveness of compounds of high
antioxidant activity in the prevention of many cancer types
is well known and proved in many studies though the use
of antioxidant agents as adjunctive in cancer therapy is still
controversial because of conflicting reports [33]. However,
the antioxidant and anticancer activities of synthesized
compounds or compounds isolated from natural sources are
still focus of many investigations because the effectiveness
of cancer prevention and inhibition of proliferation by the
test agents are both reflected by these two activities.
Compound 12a (NSC 731071) was tested by NCI with 60
human tumor cell lines over a 5-log dose range (five 10-fold
dilutions) [35Ϫ37]. Following incubation for 48 h, the cell
viability and growth was estimated with sulforhodamine B
(SRB) protein assay. The growth inhibitory activity is ex-
pressed as the μM concentration of the agent providing 50%
inhibition of the growth of the tumor cells, denoted as GI50
in Table 1. The GI50 values of 12a ranged from 0.4 to above
100 μM. Its growth inhibition activity was high towards A-
498 (renal cancer) cell line, as evident from its low GI50
(required concentration for 50 per cent growth inhibition)
and TGI (required concentration for total growth inhi-
bition) values of 0.493 and 30.6 μM, respectively (Table 1).
Though compound 12a was not referred to the Biological
Evaluation Committee for further testing to be a drug can-
didate, it adds to the understanding of the anticancer poten-
tial of compounds with specific groups within a series,
showing the higher efficacy of an amino-substituted triazole
fused ring when compared to pyrazole, benzimidazole, un-
substituted triazole, and tetrazole structures fused with sub-
stituted triazines.
Anticancer activity
Five compounds 8a, 9a, 10a, 12a, and 14a selected by the
National Cancer Institute (NCI), USA, given the NCI codes
NSC 731070, NSC 731073, NSC 731072, NSC 731071,
NSC 731069, respectively, were screened for their growth
inhibitory properties on three human tumor cell lines, breast
cancer (MCF7), non small cell lung cancer (NCI-H460),
and CNS cancer (SF-268) at NCI, Bethesda, Maryland,
USA under the Developmental Therapeutics Program of
NCI in a primary one dose anticancer assay (Figure 1) [34].
Antioxidant activity
TLC-plate fluorescence persistence time
The synthesized compounds 8Ϫ10, 12, and 14 were tested
for their antioxidant potential as a prescreening test with
TLC-plate fluorescence persistence time measurement. All
of the 10 compounds showed antioxidant activities higher
than the reference antioxidants butylated hydroxytoluene
(BHT) and 6ϪhydroxyϪ2,5,7,8ϪtetramethylchromanϪ
2Ϫcarboxylic acid (Trolox®) in this method (Figure 2). The
phenyl substituted forms were generally more active with
the only exception of compounds 14. The activity of
5-phenyl-7-p-tolyl-tetrazolo[1,5-a][1,3,5]triazine (14b) was
more than twice as much as that of 5,7-diphenyl-tetra-
zolo[1,5-a][1,3,5]triazine (14a). The 5,7-diphenyl-[1,2,4]tri-
azolo[2,3-a][1,3,5]triazin-5-ylamine (12a), which was found
to posses higher anticancer activity among the five phenyl
substituted compounds tested, also exhibited the highest
antioxidant activity with the longest period of time required
for fluorescence to disappear pointing to the delayed induc-
tion of lipid peroxidation.
Figure 1. Three cell line anticancer prescreening results for
the five compounds selected by NCI, 8a, 9a, 10a, 12a, and
14a given the NCI codes NSC 731070, NSC 731073, NSC
731072, NSC 731071, and NSC 731069, respectively. Per-
cent growth values are calculated by comparing the cell
growth in the presence and absence of test compounds. The
growth percentage of 26% of 12a (NSC 731071), for exam-
ple, means 74% cancer growth inhibition.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
Biologically Active, Fused Heterocyclic 1,3,5-Triazines
369
among all test compounds, including the standards BHT
and Trolox®. Compounds 8a, 8b, 9a, 12a, 14a, and 14b were
more active than the standards as in TLC-plate method
(Figures 2 and 3). The antioxidant activity of compounds
10a and 10b were relatively low in both antioxidant assays.
Conclusion
By starting from N-acyl imidates, 10 new fused heterocyclic
1,3,5-triazine derivatives with expected biological activity
were synthesized. All of the compounds showed high anti-
oxidant activity in two different assays. The anticancer ca-
pacity of five of the compounds were tested in the Develop-
mental Therapeutics Program by NCI, USA and compound
12a (NSC 731071) was found to be highly active against the
renal cancer cell line A-498, though not to the extent to be
referred to the Biological Evaluation Committee to be a
drug candidate. The higher antioxidant activity of 12a in
this series reflects structure-activity relationship among
analogous compounds. Interestingly, compound 12a, di-
phenyl substituted [1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-
amine, was found to have higher antioxidant activity in both
antioxidant assays. The findings suggest amino substituted
triazole fused triazine derivative (12a) as a good antioxidant
and cancer antiproliferative agent, to be developed further
with various ring substitutions.
Figure 2. TLC-plate fluorescence persistence time anti-
oxidant assay results. The samples were at 1.0 mg/mL con-
centration. Percent relative activity values are calculated by
taking the time period of fluorescence disappearance with
BHT as 100% (BHT: butylated hydroxytoluene;% Relative
Activity = [tsample/tBHT]x100).
Acknowledgments
We are grateful to Karadeniz Technical University Research
Fund (Project # 2002.122.001.2) for the financial support.
We also thank to National Cancer Institute (NCI) at Be-
thesda, USA for the anticancer screening tests of our com-
pounds.
Figure 3. Lipid peroxidation inhibition assay results. The
samples were at 10 mg/mL concentration. Percent inhibition
values were calculated by using the absorbance values of
both sample and control experiments (BHT: butylated hydro-
xytoluene; %Inhibition = [(Acontrol-Asample)/Acontrol] ϫ 100).
Experimental
Lipid peroxidation inhibitory activity
Chemistry
This assay is the second antioxidant method used to better
evaluate the antioxidant capacity of the compounds. This
method finds more and wider application in the literature
for the assessment of the antioxidant activity of synthetic
or natural compounds as well as for determining the total
antioxidant capacity of biological fluids. The results of the
tests confirmed the results obtained with the previous
method. All 10 compounds showed antioxidant activity at
various extents (Figure 3). The antioxidant capacities of the
test compounds were comparable with BHT and Trolox®.
Among compounds 9 and 12, phenyl substituted forms
showed considerably higher activity. Compound 12a, after
14b, exhibited the second highest antioxidant activity
All chemicals used in this study were of high purity and purchased
from Merck (Darmstadt, Germany) and Fluka (Buchs, Switzerland)
companies. Melting points were determined on a Büchi oil heated
melting point apparatus (Büchi Labortechnik, Flawil, Switzerland)
and are uncorrected. 1H NMR and 13C NMR spectra (δ, ppm)
were recorded on a Varian-Mercury 200 MHz spectrophotometer
(Varian Inc., Palo Alto, CA, USA) using tetramethylsilane as an
internal reference. The IR spectra (λ, cmϪ1) were run on a Perkin-
Elmer 1600 FTIR spectrophotometer (Perkin-Elmer, Beaconfields,
England) by using KBr pellets. The microanalyses were performed
on a Carlo Erba 1106 elemental analyzer (Carlo Erba, Milan, Italy.
Mass spectra were recorded on a Micromass Quattro LC-MS/MS
spectrophotometer at 70 eV (Micromass, Manchester, U.K.). Ab-
sorbance measurements were made with ATI Unicam UV2 UV-vis
spectrophotometer (ATI Unicam, Cambridge, U.K.).
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Bekircan et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
General method for the synthesis of compounds 2
2,4-Diphenyl-benzo[4,5]imidazo[1,2-a][1,3,5]triazine (9a)
The yellow solid product of the general procedure above with 2-
aminobenzimidazole (4) was recrystallized from benzene-ethyl ether
(1:2) to give the desired compound (yield 85%, mp 170Ϫ171°C).
IR (KBr) cmϪ1: 1658, 1572 (νCϭN), 763, 699 (νarm. ring). 1H NMR
(DMSO-d6) δ: 7.34 (m, 7H, Ar-H), 7.84 (m, 7H, Ar-H). 13C NMR
(DMSO-d6) δ: 127.33 (2C), 128.09 (2C), 128.22 (2C), 128.61,
128.83, 129.00 (2C), 131.11 (2C), 131.84 (2C), 134.11, 135.32,
144.57, 158.87, 161.57, 167.76 (2C). LC-MS/MS, m/z: 322.96 (%12)
[M]ϩ, 344.97 (%38) [MϩNa]ϩ. Anal. calcd. for C21H14N4: C, 78.24;
H, 4.38; N, 17.38. Found: C, 78.14; H, 4.11, N, 17.75.
Imidoethyl ester (1) (0.5 mol) was dissolved in CH2Cl2 (100 mL)
containing freshly distilled triethylamine (8.42 mL, 0.060 mol). To
this mixture, cooled in an ice/salt bath, was added (dropwise over
30 min) a solution of benzoyl chloride (5.98 mL, 0.051 mol) in
CH2Cl2 (50 mL). The mixture was stirred and kept at or below 0°C
for an additional 2 h and then at room temperature for 16 h. The
solution was filtered to remove triethyl ammonium chloride and
evaporated. The residue was then extracted with anhydrous diethyl
ether to get rid of triethyl ammonium chloride. The resulting solu-
tion was evaporated and dried in vacuo. The solid residue was
recrystallized from the appropriate solvent to give the desired com-
pounds (2a and 2b).
2-Phenyl-4-p-tolyl-benzo[4,5]imidazo[1,2-a][1,3,5]triazine (9b)
The yellow solid product of the general procedure above with 2-
aminobenzimidazole (4) was recrystallized from benzene-ethyl ether
(1:2) to give the desired compound (yield 88%, mp 267Ϫ268°C).
IR (KBr) cmϪ1: 1653, 1588 (νCϭN), 810, 753, 710 (νarm. ring). 1H
NMR (DMSO-d6) δ: 2.40 (s, 3H, CH3), 7.20 (m, 3H, Ar-H), 7.45
(m, 2H, Ar-H), 7.64(m, 4H, Ar-H), 7.90 (m, 4H, Ar-H). 13C NMR
(DMSO-d6) δ: 20.85, 125.50 (2C), 127.25, 127.94 (2C), 128.21,
128.52 (2C), 128.87 (2C), 129.20 (2C), 129.56, 129.68 (2C), 141.56,
141.81, 142.63, 147.62, 152.68, 162.15. LC-MS/MS, m/z: 360.97
(%48) [MϩNa]ϩ. Anal. calcd. for C22H16N4: C, 78.55; H, 4.79; N,
16.65. Found: C, 78.21; H, 4.85; N, 16.94.
N-Benzoyl ethyl imidobenzoate (2a)
Recrystallized from ethanol-water (3:1); yield 35%, mp. 65Ϫ66°C,
ref. [3] mp 65°C. IR (KBr) cmϪ1: 1677 (νCϭO), 1647 (νCϭN), 735,
697, 782, 716 (νarm. ring) 1H NMR (CDCl3) δ 1.56 (t, 3H, CH3), 4.44
(q, 2H, CH2), 7.42 (m, 8H, Ar-H), 8.10 (m, 2H, Ar-H).
N-Benzoyl ethyl imido-p-methylbenzoate (2b)
Recrystallized from ethanol water (3:1); yield 40%, mp 87Ϫ88°C.
IR (KBr) cmϪ1: 1670 (νCϭO), 1648 (νCϭN), 730, 690, 831 (νarm. ring
)
1H NMR (CDCl3) δ 1.44 (t, 3H, CH3), 2.24 (s, 3H, CH3), 4.24 (q,
2H, CH2), 6.90 (d, 2H, Ar-H), 7.36 (m, 5H, Ar-H), 7.84 (d, 2H,
Ar-H).
5,7-Diphenyl-[1,2,4]triazolo[2,3-a][1,3,5]triazine (10a)
The white solid product of the general procedure above with 3-
amino-1,2,4-triazole (5) was recrystallized from benzene-ethyl ether
(1:2) to give the desired compound (yield 76%, mp 147Ϫ148°C).
IR (KBr) cmϪ1: 1604, 1594 (CϭN), 789, 710, 766, 689 (νarm. ring).
1H NMR (DMSO-d6) δ: 7.72 (m, 6H, Ar-H), 8.64 (m, 2H, Ar-H),
8.84 (s, 1H, CH), 8.87 (d, 2H, Ar-H). 13C NMR (DMSO-d6) δ:
128.25 (4C), 128.49 (2C), 128.94, 130.68 (2C), 132.01, 133.40,
134.59, 154.14, 156.71, 157.15, 161.86. LC-MS/MS, m/z: 273.91
(%36) [M]ϩ, 295.94 (%100) [MϩNa]ϩ. Anal. calcd. for C16H11N5:
C, 70.32; H: 4.06; N, 25.63. Found: C, 69.32; H: 4.05; N: 25.11.
General procedure for the synthesis of compounds 8Ϫ10, 12, 14
Compounds 2 (0.01 mol) were heated in an oil bath with the appro-
priate heterocyclic amine (3Ϫ7) (0.01 mol) at 165Ϫ170°C for 2 h
and then allowed to cool. The formed solid was recrystallized sev-
eral times from the appropriate solvent to give the desired com-
pounds (8Ϫ10, 12, and 14).
2,4-Diphenyl-pyrazolo[1,5-a][1,3,5]triazine (8a)
5-Phenyl-7-p-tolyl-[1,2,4]-triazolo[2,3-a][1,3,5]triazine (10b)
The yellow solid product of the general procedure above with 5-
amino pyrazole (3) was recrystallized from ethanol-water (2:1) to
give the desired compound (yield 83%, mp 124Ϫ125°C). IR (KBr)
The white solid product of the general procedure above with 3-
amino-1,2,4-triazole (5) was recrystallized from benzene-ethyl ether
(1:2) to give the desired compound (yield 80%, mp 210Ϫ211°C).
IR (KBr) cmϪ1: 1622, 1598 (CϭN), 774, 690, 836 (νarm. ring). 1H
NMR (DMSO-d6) δ: 2.41 (s, 3H, CH3), 7.38 (d, 2H, Ar-H), 7.75
(m, 3H, Ar-H), 8.48 (d, 2H, Ar-H), 8.85 (s, 1H, CH), 8.88 (d, 2H,
Ar-H). 13C NMR (DMSO-d6) δ: 20.73, 128.62, 129.34 (2C), 129.55
(2C), 130.96 (2C), 131.16 (2C), 132.26, 133.64, 142.33, 154.14,
156.72, 157.86, 161.79. LC-MS/MS, m/z: 287.99 (%100) [M]ϩ.
Anal. calcd. for C17H13N5: C, 71.07; H: 4.56; N: 24.37. Found: C,
71.51; H: 4.84; N: 23.57.
cmϪ1: 1595 (νCϭN), 680, 742 (νarm. ring 1H NMR (DMSO-d6) δ:
)
6.87 (s, 1H, CH), 7.24 (m, 5H, Ar-H), 7.60 (s, 1H, NϭCH), 8.53
(m, 3H, Ar-H), 8.83 (m, 2H, Ar-H). 13C NMR (DMSO-d6) δ: 96.47,
127.98 (2C), 128.26 (2C), 128.62 (2C), 130.05, 130.92 (2C), 131.35,
132.99, 135.45, 147.66, 150.18, 153.68, 156.10. LC-MS/MS, m/z:
272.93 (%100) [M]ϩ, 273.93 (%22) [Mϩ1]ϩ.Anal. calcd. for
C17H12N4: C, 74.98; H, 4.44; N, 20.57. Found: C, 75.57; H, 4.11;
N, 20.32.
2-Phenyl-4-p-tolyl-pyrazolo[1,5-a][1,3,5]triazine (8b)
5,7-Diphenyl-[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamine (12a)
The yellow solid product of the general procedure above with 5-
amino pyrazole (3) was recrystallized from ethanol-water (2:1) to
give the desired compound (yield 85%, mp 118Ϫ119°C). IR (KBr)
cmϪ1: 1596 (νCϭN), 688, 742, 766, 801 (νarm. ring). 1H NMR
(DMSO-d6) δ: 2.45 (s, 3H, CH3), 6.84 (s, 1H, CH), 7.54 (m, 4H,
Ar-H), 7.56 (s, 1H, NϭCH), 8.46 (m, 3H, Ar-H), 8.80 (m, 2H, Ar-
H). 13C NMR (DMSO-d6) d: 24.91, 100.08, 131.71 (2C), 132.34
(2C), 132.61 (2C), 132.95, 133.82, 134.75 (2C), 139.27, 145.10,
151.30, 154.11, 156.83, 160.01. LC-MS/MS, m/z: 286.98 (%21)
[M]ϩ. Anal. calcd. for C18H14N4: C, 75.51; H, 4.93; N, 19.57.
Found: C, 75.78; H, 4.15; N, 20.07.
The white solid product of the general procedure above with 3,5-
diamino-1,2,4-triazole (6) was recrystallized from ethanol-water
(2:1) to give the desired compound (yield 70%, mp 255Ϫ256°C).
IR (KBr) cm1: 3375, 3338 (νNH2), 1653, 1610 (νCϭN), 767, 694
(νarm. ring). 1H NMR (DMSO-d6) δ: 7.03 (s, 2H, NH2), 7.52 (m, 6H,
Ar-H), 7.56 (m, 2H, Ar-H), 8.91 (m, 2H, Ar-H). 13C NMR (DMSO-
d6) δ: 128.78 (2C), 129.00 (2C), 129.30 (2C), 130.51, 131.23 (2C),
132.25, 133.60, 136.18, 150.75, 159.12, 161.15, 168.74. LC-MS/MS,
m/z: 288.93 (%100) [M]ϩ. Anal. calcd. for C16H12N6: C, 66.66; H,
4.20; N, 29.15. Found: C, 67.02; H, 4.01; N, 28.97.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 365−372
Biologically Active, Fused Heterocyclic 1,3,5-Triazines
371
5-Phenyl-7-p-tolyl-[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamin
(12b)
mg/mL) was spotted on the plate twice with a semiautomatic pi-
pette, drying in between. The plate was then plunged into α-linoleic
acid solution (3%) in hexane twice, drying in between and at the
end. The dried plate was then placed 2.5 cm below a UV (254 nm)
light source and the background of the spots appeared within the
first 15 min under continuous irradiation. The TLC plate was ob-
served every 15 min under continuous irradiation, and the time each
fluorescent spot disappeared was considered to be the induction
period for lipid peroxidation. The antioxidant activities of the
samples and Trolox® were evaluated by comparing their fluor-
escence disappearance times with that of the reference standard
BHT and given as percent of the activity of BHT (Figure 2).
The white solid product of the general procedure above with 3,5-
diamino-1,2,4-triazole (6) was recrystallized from ethanol to give
the desired compound (yield 77%, mp 260Ϫ261°C). IR (KBr)
cmϪ1
: 3380, 3337 (νNH2), 1657, 1605 (νCϭN), 805, 771, 696
1
(νarm. ring). H NMR (DMSO-d6) δ: 2.47 (s, 3H, CH3), 7.00 (s, 2H,
NH2), 7.62 (m, 5H, Ar-H), 8.46 (m, 2H, Ar-H), 8.87 (m, 2H, Ar-
H). 13C NMR (DMSO-d6) δ: 21.04, 128.19 (2C), 128.40 (2C),
129.30, 129.92, 130.62 (2C), 130.70 (2C), 133.01, 141.79, 150.02,
158.78,160.83, 168.08. LC-MS/MS, m/z: 325.01 (%83) [MϩNa]ϩ.
Anal. calcd. for C17H14N6: C, 67.54; H, 4.67; N, 27.80. Found: C,
68.45; H; 4.25, N, 27.30.
Antioxidant activity by lipid peroxidation inhibition
5,7-Diphenyl-tetrazolo[1,5-a][1,3,5]triazine (14a)
In the determination of lipid peroxide formation inhibitory activity
of the compounds, the reaction mixture contained rat liver homo-
genate (0.1 mL, 25%, w/v) in Tris-HCl buffer (40 mM, pH 7.0), KCl
(30 mM), ferrous iron (0.16 mM), the test compound and positive
controls (10 mg/mL), and ascorbic acid (0.06 mM) in a final volume
of 0.5 mL. All the samples and positive controls had their own
control reactions, containing all related reagents except the test
compounds. The mixtures were then incubated at 37°C for 1 h.
The lipid peroxide formation was then monitored [39]. For this, the
reaction mixture (0.4 mL) was treated with sodium dodecyl sulfate
(SDS) (0.2 mL, 8.1%), thiobarbituric acid (1.5 mL, 0.8%), and
acetic acid solution (1.5 mL, 20%) adjusted to pH 3.5 with NaOH.
The total volume was then made up to 4 mL by adding distilled
water and kept in a water-bath at 95°C for 1h. After cooling, dis-
tilled water (1 mL) and n-butanol:pyridine mixture (5 mL, 15:1,
v/v) were added and the mixture was shaken vigorously. Following
centrifugation at 4000 rpm for 10 min, the organic layer was taken
and its absorbance at 532 nm was measured. The percent inhibition
of lipid peroxidation was determined by comparing the results of
the test compounds with those of controls not containing the test
compounds (Figure 3). BHT and Trolox® were used as standards
for comparison.
The white solid product of the general procedure above with 5-ami-
notetrazole (7) was recrystallized from ethyl acetate-n-hexane (1:3)
to give the desired compound (yield 65%, mp 272Ϫ273°C). IR
(KBr) cmϪ1: 1595 (CϭN), 701, 743 (νarm. ring). 1H NMR (DMSO-
d6) δ: 8.13 (m, 4H, Ar-H), 7.63 (m, 6H, Ar-H). 13C NMR (DMSO-
d6) δ: 128.23 (4C), 128.58 (4C), 131.58 (2C), 132.92 (2C), 150.26,
165.40 (2C). LC-MS/MS, m/z: 274.85 (%15) [M]ϩ, 297.87 (%28)
[MϩNa]ϩ. Anal. calcd. for C15H10N6: C, 65.69; H, 3.67; N, 30.64.
Found: C, 65.05; H, 3.97; N: 30.98.
5-Phenyl-7-p-tolyl-tetrazolo[1,5-a][1,3,5]triazine (14b)
The white solid product of the general procedure above with 5-ami-
notetrazole (7) was recrystallized from ethyl acetate-n-hexane (1:3)
to give the desired compound (yield 71%, mp 287Ϫ288°C). IR
(KBr) cmϪ1: 1594 (CϭN), 802, 744, 701 (νarm. ring). 1H NMR
(DMSO-d6) δ: 2.53 (s, 3H, CH3), 7.37 (m, 1H, Ar-H), 7.59 (m, 4H,
Ar-H), 8.15 (m, 4H, Ar-H). 13C NMR (DMSO-d6) δ: 21.15, 128.29
(4C), 128.66 (4C), 131.65 (2C), 133.02, 143.55, 150.35, 165.50 (2C).
LC-MS/MS, m/z: 288.93 (%17) [M]ϩ, 311.85 (%35) [MϩNa]ϩ.
Anal. calcd. for C16H12N6: C, 66.66; H, 4.20; N, 29.15. Found: C,
66.41; H, 3.95; N, 29.64.
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