Synthesis, antitumor activity and SAR study of novel [1,2,4]triazino[4,5-a] benzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.03.028

A series of novel 1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]benzimidazoles carrying variety of aryl and heteroaryl groups at position 1 were synthesized. The newly synthesized compounds were tested in vitro on human breast adenocarcinoma cell line (MCF7). Some of the test compounds showed potent antitumor activity, especially compound 3c [1-(2-chlorophenyl) derivative] which displayed the highest activity among the test compounds.

Full text of DOI:10.1016/j.ejmech.2012.03.028

European Journal of Medicinal Chemistry 53 (2012) 22e27
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitumor activity and SAR study of novel [1,2,4]triazino[4,5-a]
benzimidazole derivatives
*
Hala Bakr El-Nassan
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, 33 Kasr El-Aini Street, Cairo 11562, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]benzimidazoles carrying variety of aryl and
heteroaryl groups at position 1 were synthesized. The newly synthesized compounds were tested in vitro
on human breast adenocarcinoma cell line (MCF7). Some of the test compounds showed potent anti-
tumor activity, especially compound 3c [1-(2-chlorophenyl) derivative] which displayed the highest
activity among the test compounds.
Received 9 January 2012
Received in revised form
12 March 2012
Accepted 14 March 2012
Available online 23 March 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzimidazole
[1,2,4]Triazino[4,5-a]benzimidazole
Antitumor activity
MCF7
1. Introduction
[12e16]. However, little work had been published on the biological
activity of [1,2,4]triazino[4,5-a]benzimidazoles [13,17e19].
Breast cancer (BC) is the most frequent cause of cancer among
women in both developed and undeveloped societies. It is also the
primary cause of cancer death among women worldwide. Its inci-
dence has been rising in several developing countries over the past
few years [1e5].
Currently, many anticancer drugs are used clinically and indeed
many of these compounds are used successfully for the treatment
of several neoplastic diseases such as leukemia or testicular cancer.
However, their effect on solid tumors such as BC has been poor.
Since the response of solid tumors to available anticancer chemo-
therapy has been reduced, new drugs with improved efficacy are
desired.
Benzimidazole derivatives represent one of the chemical classes
that showed potent anticancer activity especially against breast
cancer cell line (MCF7) [6e10]. Benzimidazole nucleus was used as
biomimetic of guanine residues and many benzimidazole deriva-
tives were reported to selectively inhibit endothelial cell growth
and suppress angiogenesis both in vitro and in vivo [11].
Recently, many fused benzimidazoles were reported to exhibit
promising anticancer activity [12e19]. Several publications repor-
ted the synthesis and biological activities of [1,2,4]triazino[2,3-a]
benzimidazoles as well as [1,2,4]triazino[4,3-a]benzimidazoles
In 2008, Styskala et al. [17] reported the anticancer activity of 2-
aryl-1,2-dihydro[1,2,4]triazino[4,5-a]benzimidazol-1-one deriva-
tives I (Fig. 1) against several cell lines including breast cancer cell
line (MCF7). The authors stated that the synthesized compounds
showed preferential activity against solid tumors compared to
leukemia cells. They also studied the effect of the 2-aryl substitu-
ents and reported that compounds with the lipophilic substituents
phenyl, 4-methylphenyl and 4-methoxyphenyl were highly active,
while, substitutions with 4-chlorophenyl or 4-nitrophenyl groups
substantially decreased the cytotoxic potency [17]. Since then, no
further study of the anticancer activity or the effect of substituents
on other positions of this tricyclic nucleus had been published.
Encouraged by these findings, we aim in this study to synthesize
the novel tricyclic 1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]benz-
imidazole derivatives as a new cytotoxic lead structure. The aim
also involves studying the effect of the different aryl and heteroaryl
groups at position 1 of the nucleus on the anticancer activity
against breast cancer cell line (MCF7).
2. Results and discussion
2.1. Chemistry
The target compounds depicted in Scheme 1 were obtained by
reacting the starting material 2-(chloromethyl)-1H-benzimidazole
* Tel.: þ20 2 23632245; fax: þ20 2 23635140.
E-mail address: hala_bakr@hotmail.com.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.028

H.B. El-Nassan / European Journal of Medicinal Chemistry 53 (2012) 22e27
23
singlet signals at
d 10e12 ppm corresponding to two NH protons.
O
Besides, a singlet signal appeared at
CH proton at position 1.
d 4e5 ppm corresponding to
Ar
N
N
N
The mass spectra of compounds 3aes showed the corre-
sponding molecular ion peaks and most of them were character-
ized by the presence of peaks due to [ArCHNH]þ.
A possible mechanism for the formation of compounds 3aes
was outlined in Fig. 2.
N
R
N
N
I
2.2. In vitro anticancer screening
All the synthesized tricyclic derivatives were evaluated for their
in vitro cytotoxic activity against human breast cancer cell line,
MCF7, using Sulforhodamine-B stain (SRB) assay [20].
Ar =C H , 4-CH C H , 4-CH OC H , 4-ClC H , 4-NO C H
4
6
5
3
6
4
3
6
4
6
4
2
6
R= H, COOC H
2
5
The relationship between surviving fraction of MCF7 and drug
concentration was plotted and the response parameter IC50 was
calculated. IC50 value corresponds to the concentration required for
50% inhibition of cell viability. The IC50 of the test compounds are
shown in Table 1 and the results are represented graphically in
Fig. 3.
Structurally, the test compounds differ only in the nature of the
aryl group at position 1 of the tricyclic nucleus, being (substituted
phenyl) in compounds 3aeo and heteroaryl in compounds 3pes.
The obtained data revealed that most of the synthesized
compounds showed potent antitumor activity. The best result was
obtained by compound 3c [R ¼ 2-Cl]. Whilst, compound 3h [R ¼ 3-
OH] exhibited the least cytotoxic activity among the test
compounds.
Regarding the 1-(substituted phenyl) derivatives 3aeo, better
result was obtained by the 2-substituted derivatives (3c, 3g, 3l and
3n). While, the 3-substituted derivatives (3h and 3m) demon-
strated the least antitumor activity against MCF7 cell line. Besides,
better results were obtained by electron withdrawing groups
especially at the 2 position of the benzene nucleus (3l and 3n) and
also by halides [Br and Cl] (3be3d).
Fig. 1. Structure of previously synthesized potent antitumor [1,2,4]triazino[4,5-a]
benzimidazoles.
(1) with 4 molar equivalent of hydrazine hydrate (99%) for 45 min
to give 2-(hydrazinylmethyl)-1H-benzimidazole (2) in 75% yield.
The formation of compound 2 was confirmed by 1H NMR that
showed two exchangeable singlet signals at
d 4.38 and 10.38 ppm
corresponding to NH2 and NH protons, respectively. Moreover, the
mass spectrum of compound 2 displayed molecular ion peak at m/z
162 (Mþ).
Searching the literature revealed that no previous work had
been published on the cyclization reactions of 2-(hydrazi-
nylmethyl)-1H-benzimidazole to afford the tricyclic [1,2,4]triazino
[4,5-a]benzimidazole.
In this work, 2-(hydrazinylmethyl) derivative 2 was subjected
to cycloaddition condensation reaction using different aryl and
heteroaryl aldehydes in ethanol and triethylamine to give 1-aryl-
1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]benzimidazoles 3aes.
1H NMR spectra of compounds 3aes revealed the disappearance
of the NH2 signal at d 4.38 and the appearance of two exchangeable
Scheme 1.

24
H.B. El-Nassan / European Journal of Medicinal Chemistry 53 (2012) 22e27
O
Ar
N
NHNH
2
Ar
H
N
H
N
H
H
N
N
NH
NH
N
H
N
Ar
2
N
3a-s
Fig. 2. A possible mechanism for the formation of compound 3aes.
On the other hand, the 1-(heteroaryl) derivatives 3pes showed
activity. 3) The heteroaryl groups exhibited similar anticancer
activity compared to the substituted phenyl groups. Up to our
knowledge, this is the first published work on the synthesis and
the antitumor activity of 1-aryl-1,2,3,4-tetrahydro[1,2,4]triazino
[4,5-a]benzimidazoles.
better antitumor activities compared to the unsubstituted phenyl
derivative 3a. The six membered pyridine derivatives (3q and 3r)
showed better antitumor activity than the five membered thienyl
or the indole derivative. Within the pyridyl derivatives, the 4-
pyridyl derivative 3r exhibited better cytotoxic activity than the
2-pyridyl one 3q.
The results of the anticancer screening suggested the following
general structural requirements for the anti-proliferative action of
1-aryl-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]benzimidazoles on
MCF7 cell line:
4. Experimental part
4.1. General
Melting points were determined using a Griffin apparatus and
were uncorrected. IR spectra were recorded on Shimadzu IR 435
spectrophotometer and values were represented in cmꢀ1. 1H NMR
and 13C NMR were carried out on Varian Gemini 300 MHz spec-
trophotometer, Cairo University, Cairo, Egypt, using TMS as an
internal standard and chemical shifts were recorded in ppm on
1. An ortho-substitution on the aromatic nucleus at position 1
with electron withdrawing groups like CF3 and NO2 or halogens
especially Cl enhanced the anticancer activity.
2. Substitutions at the meta-position reduced the anti-prolifera-
tive activity.
d
scale. The electron impact (EI) mass spectra were recorded on
3. The heteroaryl groups exhibited similar anticancer activity
compared to the substituted phenyl groups.
Hewlett Packard 5988 spectrometer, Microanalytical center, Cairo
University, Cairo, Egypt. Analytical thin layer chromatography (TLC)
on silica gel plates containing UV indicator was employed routinely
to follow the course of reactions and to check the purity of prod-
ucts. All reagents and solvents were purified and dried by standard
techniques.
3. Conclusion
The aim of the present study was to synthesize novel 1,2,3,4-
tetrahydro[1,2,4]triazino[4,5-a]benzimidazoles carrying different
aryl and heteroaryl groups at position 1 in order to examine
the effect of the substitution at position 1 on the antitumor
activity. The newly synthesized compounds were tested in vitro
on human breast adenocarcinoma cell line (MCF7). Some of the
test compounds showed potent antitumor activity, especially
compound 3c [1-(2-chlorophenyl) derivative] which displayed
the highest activity among the test compounds. The results of the
anticancer screening suggested the following general conclu-
sions: 1) An ortho-substitution on the aromatic nucleus at posi-
tion 1 with electron withdrawing groups like CF3 and NO2 or
halogens especially Cl enhanced the anticancer activity. 2)
Substitutions at the meta-position reduced the anti-proliferative
4.1.1. 2-(Hydrazinylmethyl)-1H-benzimidazole (2)
2-(Chloromethyl)-1H-benzimidazole (1) (3.33 g, 20 mmol) and
hydrazine hydrate (99%, 4 mL, 80 mmol) were heated under reflux
for 45 min. The solid formed on hot was cooled, triturated with
ethanol (95%, 20 mL), filtered, dried and crystallized from ethanol.
Yield: 75%; mp: 197e198 ꢁC; IR (cmꢀ1): 3302, 3190 (NH/NH2), 2920,
2893 (CH aliphatic); 1H NMR (DMSO-d6)
d ppm 3.91 (s, 2H, CH2),
4.38 (br s, 2H, NH2, D2O exchangeable), 6.48e6.84 (m, 4H, AreH),
9.27 (s, 1H, NH, D2O exchangeable), 10.38 (s, 1H, NH, D2O
exchangeable); MS m/z: 162 [Mþ, 6.58%], 161 [(Mþ ꢀ 1), 64.55%],
133 [100%].
4.1.2. General procedure for the synthesis of 1-aryl-1,2,3,4-tetrahy-
dro[1,2,4]triazino[4,5-a]benzimidazoles 3aes
A
mixture of 2-(hydrazinylmethyl)-1H-benzimidazole (2)
Table 1
(0.25 g, 1.5 mmol) and the appropriate aldehyde (1.5 mmol) in
absolute ethanol (10 mL) and triethylamine (1 mL) were heated
under reflux for 12 h. The separated solid was filtered, dried and
crystallized from acetic acid.
Results of in vitro cytotoxic activity of the synthesized compounds on human breast
adenocarcinoma cell line (MCF7).
Compound no.
IC50 in
m
Ma
Compound no.
IC50 in m
Ma
3a
3b
3c
3d
3e
3f
3g
3h
3i
0.0752
0.0547
0.0390
0.0545
0.0713
0.0746
0.0583
0.0835
0.0766
0.0703
3k
3l
0.0639
0.0505
0.0711
0.0518
0.0529
0.0702
0.0501
0.0450
0.0675
3m
3n
3o
3p
3q
3r
4.1.2.1. 1-Phenyl-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]benzimidaz-
ole (3a). Yield: 86%; mp: 253e254 ꢁC; IR (cmꢀ1): 3371, 3186 (NH),
2918, 2870 (CH aliphatic); 1H NMR (DMSO-d6)
d ppm 3.98 (s, 2H,
CH2), 4.48 (s, 1H, CH), 6.48e8.23 (m, 9 H, AreH), 10.41 (s, 1H, NH,
D2O exchangeable), 11.58 (s, 1H, NH, D2O exchangeable); MS m/z:
250 [Mþ, 9.94%], 249 [(M ꢀ 1)þ, 11.60%], 173 [(MeC6H5)þ, 10.50%],
160 [(MeC6H5CH)þ, 62.43%], 133 [100%], 105 [C6H5CHNHþ, 7.37%],
90 [C6H5CHþ, 11.79%], 77 [C6Hþ5 , 37.94%].
3s
3j
a
The values given are means of three experiments.

H.B. El-Nassan / European Journal of Medicinal Chemistry 53 (2012) 22e27
25
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 3s
Compound number
Fig. 3. IC50 of the synthesized compounds in mM on the human breast adenocarcinoma cell line (MCF7).
4.1.2.2. 1-(4-Bromophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3b). Yield: 79%; mp: 268e269 ꢁC; IR (cmꢀ1):
3429, 3190 (NH), 2924, 2854 (CH aliphatic); 1H NMR (DMSO-
115.6, 115.9, 117.9, 122.8, 127.0, 129.0, 130.6, 146.0 (aromatic
carbons); MS m/z: 268 [Mþ, 0.93%], 123 [FC6H4CHNHþ, 17.98%],
108 [FC6H4CHþ, 10.19%], 95 [FC6H4þ, 61.72%], 76 [C6Hþ4 , 5.11%], 57
[100%].
d6)
d ppm 3.96 (s, 2H, CH2), 4.48 (s, 1H, CH), 6.45e8.20 (m,
8 H, AreH), 10.40 (s, 1H, NH, D2O exchangeable), 11.63 (s, 1H,
NH, D2O exchangeable); MS m/z: 330 [(M þ 2)þ, 6.86%], 328
4.1.2.7. 1-(2-Hydroxyphenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3g). Yield: 70%; mp: 280e281 ꢁC; IR (cmꢀ1): 3352,
3221, 3182 (NH/OH), 2924, 2854 (CH aliphatic); 1H NMR (DMSO-d6)
[Mþ, 4.62%], 221 [100%], 185
[
81BrC6H4CHNHþ, 4.41%], 183
[
79BrC6H4CHNHþ, 3.50%], 157 [81BrC6Hþ4 , 8.52%], 155 [79BrC6Hþ4 ,
8.08%], 76 [C6Hþ4 , 20.04%].
d
ppm 3.97 (s, 2H, CH2), 4.45 (s, 1H, CH), 6.56e8.44 (m, 8 H, AreH),
10.06 (s, 1H, OH, D2O exchangeable), 10.40 (s, 1H, NH, D2O
exchangeable), 11.50 (s, 1H, NH, D2O exchangeable); MS m/z: 266
[Mþ, 1.97%], 121 [HOC6H4CHNHþ, 5.05%], 106 [HOC6H4CHþ, 5.00%],
64 [100%].
4.1.2.3. 1-(2-Chlorophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3c). Yield: 72%; mp: 266e267 ꢁC; IR (cmꢀ1): 3448,
3190 (NH), 2920, 2854 (CH aliphatic); 1H NMR (DMSO-d6)
d ppm
4.92 (s, 1H, CH), 5.35 (s, 2H, CH2), 7.14e8.59 (m, 8 H, AreH), 12.00
(s, 1H, NH, D2O exchangeable), 12.21 (s, 1H, NH, D2O exchange-
able); MS m/z: 286 [(Mþ2)þ, 0.54%], 284 [Mþ, 2.29%], 173
[(MeClC6H4)þ, 1.20%], 160 [(MeClC6H4CH)þ, 1.64%], 145
[(MeClC6H4CHNH)þ, 1.74%], 69 [100%].
4.1.2.8. 1-(3-Hydroxyphenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3h). Yield: 51%; mp: 273e274 ꢁC; IR (cmꢀ1): 3421,
3309, 3194 (NH/OH), 2924, 2850 (CH aliphatic); 1H NMR (DMSO-d6)
d
ppm 3.98 (s, 2H, CH2), 4.47 (s, 1H, CH), 6.57e8.44 (m, 8 H, AreH),
10.06 (s, 1H, OH, D2O exchangeable), 10.40 (s, 1H, NH, D2O
exchangeable), 11.78 (s, 1H, NH, D2O exchangeable); MS m/z: 266
[Mþ, 0.18%], 121 [HOC6H4CHNHþ, 12.79%], 106 [HOC6H4CHþ, 2.29%],
93 [HOC6Hþ4 , 14.37%], 76 [C6Hþ4 , 3.68%], 58 [100%].
4.1.2.4. 1-(4-Chlorophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3d). Yield: 83%; mp: 271e272 ꢁC; IR (cmꢀ1):
3271, 3190 (NH), 2912, 2858 (CH aliphatic); 1H NMR (DMSO-d6)
d
ppm 3.97 (s, 2H, CH2), 4.54 (s, 1H, CH), 6.47e8.53 (m, 8 H, AreH),
10.41 (s, 1H, NH, D2O exchangeable), 11.81 (s, 1H, NH, D2O
exchangeable); MS m/z: 286 [(M þ 2)þ, 2.28%], 284 [Mþ, 7.52%],
249 [(MeCl)þ, 12.26%], 165 [100%], 160 [(MeClC6H4CH)þ, 6.55%],
141 [37ClC6H4CHNHþ, 6.97%], 139 [35ClC6H4CHNHþ, 18.57%], 113
4.1.2.9. 1-(4-Hydroxy-3-methoxyphenyl)-1,2,3,4-tetrahydro[1,2,4]tri-
azino[4,5-a]benzimidazole (3i). Yield: 20%; mp: 247e248 ꢁC; IR
(cmꢀ1): 3417, 3275, 3217 (NH/OH), 2927, 2839 (CH aliphatic); 1H
NMR (DMSO-d6)
d ppm 3.83 (s, 3H, CH3), 4.83 (s, 1H, CH), 5.31 (s,
[
37ClC6Hþ4 , 26.58%], 111 [35ClC6H4þ, 79.25%], 76 [C6Hþ4 , 17.84%].
2H, CH2), 6.80e8.08 (m, 7 H, AreH), 9.55 (s, 1H, OH, D2O
exchangeable), 11.65 (s, 1H, NH, D2O exchangeable), 12.21 (s, 1H,
NH, D2O exchangeable); MS m/z: 296 [Mþ, 54.07%], 295 [(M ꢀ 1)þ,
38.52%], 294 [(M ꢀ 2)þ, 46.67%], 151 [4eOHe3-CH3OC6H3CHNHþ,
42.22%], 109 [100%].
4.1.2.5. 1-(4-Dimethylaminophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino
[4,5-a]benzimidazole (3e). Yield: 81%; mp: 237e238 ꢁC; IR
(cmꢀ1): 3200, 3178 (NH), 2897, 2854 (CH aliphatic); 1H NMR
(DMSO-d6)
CH), 6.44e8.07 (m,
exchangeable), 11.26 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) ppm 40.2 (CH3), 53.0 (C-4), 72.0 (C-1), 114.7, 117.8,
d
ppm 3.33 (s, 6H, CH3), 3.98 (s, 2H, CH2), 4.42 (s, 1H,
8
H, AreH), 10.39 (s, 1H, NH, D2O
4.1.2.10. 1-(2-Methoxyphenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-
a]benzimidazole (3j). Yield: 83%; mp: 233e234 ꢁC; IR (cmꢀ1):
3394, 3209 (NH), 2924, 2854 (CH aliphatic); 1H NMR (DMSO-d6)
d
121.2, 122.8, 126.5, 128.2, 129.3, 134.8, 144.2, 151.2 (aromatic
carbons); MS m/z: 293 [Mþ, 1.60%], 148 [(CH3)2NC6H4CHNHþ,
21.02%], 133 [(CH3)2NC6H4CHþ, 100%].
d ppm 3.86 (s, 3H, OCH3), 4.87 (s, 1H, CH), 5.32 (s, 2H, CH2),
6.81e8.53 (m, 8 H, AreH), 10.36 (s, 1H, NH, D2O exchangeable),
11.78 (br s, 1H, NH, D2O exchangeable); MS m/z: 280 [Mþ, 8.29%],
135 [CH3OC6H4CHNHþ, 100%].
4.1.2.6. 1-(4-Flourophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3f). Yield: 84%; mp: 235e236 ꢁC; IR (cmꢀ1): 3383,
4.1.2.11. 1-(4-Methoxyphenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-
3190 (NH), 2985, 2920 (CH aliphatic); 1H NMR (DMSO-d6)
d
ppm
a]benzimidazole (3k). Yield: 87%; mp: 250e251 ꢁC; IR (cmꢀ1): 3437,
3.97 (s, 2H, CH2), 4.47 (s, 1H, CH), 6.45e8.23 (m, 8 H, AreH), 10.40
3190 (NH), 2924, 2839 (CH aliphatic); 1H NMR (DMSO-d6)
d ppm
(s, 1H, NH, D2O exchangeable), 11.58 (s, 1H, NH, D2O exchange-
3.80 (s, 3H, OCH3), 4.88 (s, 1H, CH), 5.31 (s, 2H, CH2), 7.00e8.01 (m,
8 H, AreH), 11.69 (s, 1H, NH, D2O exchangeable), 12.20 (s, 1H, NH,
able); 13C NMR (DMSO-d6)
d ppm 52.6 (C-4), 72.0 (C-1), 111.1,

26
H.B. El-Nassan / European Journal of Medicinal Chemistry 53 (2012) 22e27
D2O exchangeable); MS m/z: 280 [Mþ, 2.50%], 279 [(M ꢀ 1)þ,
12.09%], 173 [(MeCH3OC6H4)þ, 15.16%], 160 [(MeCH3OC6H4CH)þ,
11.52%], 135 [CH3OC6H4CHNHþ, 6.91%], 133 [CH3OC6H4CNþ, 100%],
120 [CH3OC6H4CHþ, 7.29%], 107 [CH3OC6Hþ4 , 9.60%], 76 [C6Hþ4 ,
5.18%].
9.90%], 106 [C5H4NCHNHþ, 9.27%], 91 [C5H4NCHþ, 11.06%], 78
[C5H4Nþ, 17.00%].
4.1.2.19. 1-(Thiophen-2-yl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]ben-
zimidazole (3s). Yield: 73%; mp: 252e253 ꢁC; IR (cmꢀ1): 3200, 3194
(NH), 2947, 2850 (CH aliphatic); 1H NMR (DMSO-d6)
d ppm 3.97 (s,
4.1.2.12. 1-(2-Nitrophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3l). Yield: 73%; mp: 246e247 ꢁC; IR (cmꢀ1): 3275,
3190 (NH), 2924, 2854 (CH aliphatic), 1523, 1342 (NO2); 1H NMR
2H, CH2), 4.37 (s, 1H, CH), 6.43e8.44 (m, 7 H, AreH), 10.39 (s, 1H, NH,
D2O exchangeable), 11.51 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6)
d ppm 53.0 (C-4), 70.5 (C-1), 117.9, 118.4, 122.8, 126.8,
(DMSO-d6)
d
ppm 3.96 (s, 2H, CH2), 4.48 (s, 1H, CH), 6.47e8.38 (m,
127.8,128.8,130.9,134.7,138.7,142.0 (aromatic carbons); MS m/z: 256
[Mþ, 1.60%], 133 [100%], 111 [C4H3SCHNHþ, 3.52%], 96 [C4H3SCHþ,
16.90%].
8 H, AreH), 10.41 (s, 1H, NH, D2O exchangeable), 11.84 (s, 1H, NH,
D2O exchangeable); MS m/z: 295 [Mþ, 0.11%], 150 [NO2C6H4
CHNHþ, 1.02%], 135 [NO2C6H4CHþ, 35.13%], 76 [C6Hþ4 , 46.60%], 57
[100%].
4.2. Biological testing
4.1.2.13. 1-(3-Nitrophenyl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
4.2.1. Materials and methods
The human breast adenocarcinoma cell line (MCF7) was
obtained as a gift from NCI, MD, USA.
benzimidazole (3m). Yield: 78%; mp: 283e284 ꢁC; IR (cmꢀ1):
3336, 3190 (NH), 2920, 2854 (CH aliphatic), 1523, 1350 (NO2); 1
H
NMR (DMSO-d6)
d ppm 3.96 (s, 2H, CH2), 4.48 (s, 1H, CH),
All chemicals and solvents were purchased from SigmaeAldrich.
6.45e8.00 (m, 8 H, AreH), 10.41 (s, 1H, NH, D2O exchangeable),
11.63 (s, 1H, NH, D2O exchangeable); MS m/z: 295 [Mþ, 52.94%],
173 [(MeNO2C6H4)þ, 60.73%], 150 [NO2C6H4CHNHþ, 55.88%], 76
[C6Hþ4 , 55.88%], 57 [100%].
4.2.2. Measurement of potential cytotoxicity
The cytotoxic activity of the newly synthesized compounds was
examined in vitro on human breast adenocarcinoma cell line
(MCF7) using Sulforhodamine-B stain (SRB) assay applying the
method of Skehan et al. [20].
4.1.2.14. 1-[(2-Triflouromethyl)phenyl]-1,2,3,4-tetrahydro[1,2,4]triazi-
no[4,5-a]benzimidazole (3n). Yield: 74%; mp: 248e249 ꢁC; IR
(cmꢀ1): 3213, 3155 (NH), 2920, 2858 (CH aliphatic); 1H NMR
Cells were plated in 96-multiwell plate (104 cells/well) for 24 h
before treatment with the test compounds to allow attachment of
the cells to the wall of the plate. Test compounds were dissolved
in DMSO and diluted with saline to the appropriate volume.
Different concentrations of the test compound (5, 12.5, 25 and
(DMSO-d6)
d ppm 3.96 (s, 2H, CH2), 4.52 (s, 1H, CH), 6.49e8.58 (m,
8 H, AreH), 10.41 (s, 1H, NH, D2O exchangeable), 11.81 (s, 1H, NH,
D2O exchangeable); MS m/z: 318 [Mþ, 0.18%], 158 [F3CC6H4CHþ,
4.20%], 145 [F3CC6Hþ4 , 12.20%], 133 [100%].
50
mg/mL) were added to the cell monolayer. Triplicate wells
prepared for each individual dose. Monolayer cells were incu-
bated with the test compound for 48 h at 37 ꢁC in atmosphere of
5% CO2. After 48 h, cells were fixed with trichloroacetic acid,
washed with water and stained for 30 min with 0.4% (wt/vol)
Sulforhodamine-B stain dissolved with 1% acetic acid. Excess stain
was removed by four washes with 1% acetic acid and attached
stain was recovered with Tris EDTA buffer. Color intensity was
measured in ELISA reader. The relation between surviving fraction
and compound concentration was plotted and IC50 [the concen-
tration required for 50% inhibition of cell viability] was calculated
for each compound and results are given in Table 1 and repre-
sented graphically in Fig. 3.
4.1.2.15. 1-(3,4,5-Trimethoxyphenyl)-1,2,3,4-tetrahydro[1,2,4]triazino
[4,5-a]benzimidazole (3o). Yield: 95%; mp: 223e224 ꢁC; IR
(cmꢀ1): 3228, 3159 (NH), 2939, 2839 (CH aliphatic); 1H NMR
(DMSO-d6)
d ppm 3.69 (s, 3H, OCH3), 3.82 (s, 6H, OCH3 two
groups), 3.98 (s, 2H, CH2), 4.49 (s, 1H, CH), 6.47e8.15 (m, 6 H,
AreH), 10.40 (s, 1H, NH, D2O exchangeable), 11.54 (s, 1H, NH, D2O
exchangeable); MS m/z: 340 [Mþ, 3.04%], 221 [100%], 195
[(CH3O)3C6H2CHNH þ, 10.12%], 180 [(CH3O)3C6H2CH þ, 13.61%],
160 [(Me(CH3O)3C6H2CH)þ, 5.30%].
4.1.2.16. 1-(1H-Indol-3-yl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]
benzimidazole (3p). Yield: 90%; mp: 262e263 ꢁC; IR (cmꢀ1):
3290, 3197, 3155 (NH), 2931, 2897 (CH aliphatic); 1H NMR
Acknowledgments
(DMSO-d6) d ppm 3.96 (s, 2H, CH2), 4.44 (s, 1H, CH), 6.39e8.30 (m,
9 H, AreH), 10.33 (s, 1H, NH, D2O exchangeable), 11.15 (s, 1H, NH,
D2O exchangeable), 11.48 (s, 1H, NH, D2O exchangeable); MS m/z:
289 [Mþ, 65.82%], 288 [(M ꢀ 1)þ, 73.42%], 213 [100%].
The author is deeply thankful to the staff members of the
department of Cancer Biology, National Cancer Institute, Cairo,
Egypt, for carrying out the anticancer screening of the newly
synthesized compounds.
4.1.2.17. 1-(Pyridin-2-yl)-1,2,3,4-tetrahydro[1,2,4]triazino[4,5-a]ben-
zimidazole (3q). Yield: 67%; mp: 208e209 ꢁC; IR (cmꢀ1): 3336,
3201 (NH), 2939, 2870 (CH aliphatic); 1H NMR (DMSO-d6)
d ppm
References
3.89 (s, 2H, CH2), 4.43 (s,1H, CH), 6.39e8.52 (m, 8 H, AreH),10.32 (s,
1H, NH, D2O exchangeable), 11.68 (s, 1H, NH, D2O exchangeable);
MS m/z: 251 [Mþ, 21.53%], 160 [(MeC5H4NCH)þ, 22.34%], 145
[(MeC5H4NCHNH)þ, 16.89%], 133 [100%], 106 [C5H4NCHNHþ,
25.07%], 91 [C5H4NCHþ, 31.06%], 78 [C5H4Nþ, 41.96%].
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