Synthesis of new heterocyclic compounds containing benzimidazole moiety as inhibitors of breast cancer cell growth

Article abstract of DOI:10.1515/hc-2013-0013

A series of new benzimidazole derivatives, namely 2-acylbenzimidazoles 2- 9, a dihydroquinoxaline 10, a benzoxazine 11, quinolines 13 - 15 and fused 1,2,4-triazines 17 - 24 were synthesized. Structure elucidation of the compounds was conducted using IR, ¹H NMR, ¹³C NMR, mass spectral data and elemental analysis. These products were evaluated for in vitro antitumor activity against MCF7 cell line (human breast cancer). Compounds 13 - 15 and 24 manifested significant antitumor activity.

Full text of DOI:10.1515/hc-2013-0013

ꢁꢁꢁꢂ
DOI 10.1515/hc-2013-0013ꢀ ꢀHeterocycl. Commun. 2013; 19(2): 125–131
Atif Tantawy, Alaa-eldin Barghash, Sahar Badr* and Rania Gomaa
Synthesis of new heterocyclic compounds
containing benzimidazole moiety as inhibitors
of breast cancer cell growth
Abstract: A series of new benzimidazole derivatives, Certain quinolines [17–20] and 1,2,4-triazines [21–23] are
namely 2-acylbenzimidazoles 2–9, a dihydroquinoxaline also active. Hence, the aim of the current study was the
10, a benzoxazine 11, quinolines 13–15 and fused 1,2,4-tri- synthesis of novel benzimidazole derivatives that incorpo-
azines 17–24 were synthesized. Structure elucidation of rate benzoxazine, quinoxaline, quinoline and 1,2,4-tria-
1
the compounds was conducted using IR, H NMR, 13C zine moieties. It was reasoned that this type of molecu-
NMR, mass spectral data and elemental analysis. These lar combination might lead to finding compounds with
products were evaluated for in vitro antitumor activ- improved antitumor activity.
ity against MCF7 cell line (human breast cancer). Com-
pounds 13–15 and 24 manifested significant antitumor
activity.
Results and discussion
Keywords: antitumor; benzimidazoles; benzoxazines;
quinolines; quinoxalines; 1,2,4-triazines; synthesis.
Chemistry
The target compounds were synthesized as depicted
in Scheme 1 and Equations 1–2. The key intermediate,
1-(1H-benzimidazol-2-yl)-2-bromoethanone (1), was syn-
thesized as previously reported [24]. Treatment of 1 with
the appropriate substituted anilines gave the correspond-
ing products 2–5 (Scheme 1). The reaction of 1 with sodium
benzoate or its 2-hydroxy derivative yielded the respec-
tive benzoate 6 or 7. In addition, the desired hydrazides
8 and 9 were synthesized by treatment of the substrate
1 with the appropriate hydrazides. By contrast, the reac-
*Corresponding author: Sahar Badr, Faculty of Pharmacy,
Department of Pharmaceutical Organic Chemistry, University of
Mansoura, Mansoura 35516, Egypt, e-mail: saharmbadr@yahoo.com
Atif Tantawy, Alaa-eldin Barghash and Rania Gomaa: F a c u l t y
of Pharmacy, Department of Pharmaceutical Organic Chemistry,
University of Mansoura, Mansoura 35516, Egypt
Introduction
Cancer represents the largest cause of death worldwide tion of 1 with 1,2-phenylenediamine or 2-aminophenol
[1]. Despite progress in the efficacy of surgical opera- gave 3-(1H-benzimidazol-2-yl)-1,2-dihydroquinoxaline (10)
tions to remove tumors, treatment with chemotherapy is and 3-(1H-benzimidazol-2-yl)-2H-benzo[b][1,4]oxazine (11),
considered to be one of the most important approaches. respectively. All compounds 2–11 were fully characterized
A literature survey has revealed the importance of by spectroscopic methods and elemental analysis.
benzimidazoles as antitumor agents [2–12] including ben-
Known 2-acetylimidazoles 12a,b [25] were allowed to
damustine, dovitinib, Hoechst 33342 and Hoechst 33258 react with isatins to furnish the desired 2-(1H-benzimi-
(Figure 1). Recently, benzoxazines [13, 14] and quinoxa- dazole-2-yl)-6-substituted quinoline-4-carboxylic acids
lines [15, 16] have been identified as anticancer agents. 13–15 (Equation 1).
(1)
R1
R
N
N
R1
KOH
R
O
N
HN
Δ
N
H
N
H
Me
O
COOH
O
13-15
12a,b
13: R= H, R1= Cl
14: R= Me, R1= H
15: R= Me, R1= Me
12a: R=H
12b: R=Me
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM

ꢁꢁꢁꢂ
126ꢀ
ꢀA. Tantawy etꢃal.: Benzimidazole compounds as inhibitors of breast cancer cell growth
R
Cl
NH2
Me
Cl
N
N
COOH
O
R
NH
2-5
N
N
N
N
NH
F
N
Δ
anh. EtOH,
N
H
O
N
H
Me
Bendamustine
Me
H2N
2: R= 4-Br; 3: R= 4-Cl; 4: R=4-OMe; 5: R= 2-SMe
Dovitinib
COONa
O
O
R
N
N
N
N
R
DMF, rt
N
H
O
N
6,7
N
H
N
6
8
7
: R= H; : R= OH
H
CONHNH2
R
OR
O
R
CH2Br
O
N
Hoechst 33342 R = Et
Hoechst 33258 R = H
N
H
1
NHNH
N
anh. EtOH, Δ
Figure 1ꢀStructures of some antitumor drugs containing benzimida-
N
H
O
8,9
zole moiety.
9
: R= H; : R= OH
NH2
N
N
XH
Finally, the substrates 16a [26–28] and 16b were used
in the preparation of benzimidazo[1,2-d][1,2,4]triazine-
4-carbonitriles 17–24 (Equation 2). Again, the given struc-
tures of the synthesized compounds 12–24 were fully
consistent with the spectroscopic and elemental analysis
data.
EtOH, Δ
N
H
X
10,11
10: X= NH; 11: X= O
Scheme 1
R1
O
O
OEt
N N
N
Cl
R1
R
N
N
R
N
(2)
pyridine
CN
N
CN
N
16a,b
17-24
1
1
16a
: R= H
17
18
19
20
: R= H, R = 2-Cl
21
22
: R= Me, R = 2-Cl
1
16b: R= CH3
1
: R= H, R = 3-Cl
: R= Me, R = 3-Cl
1
23: R= Me, R1= 4-OMe
24: R= Me, R1= 2-SMe
: R= H, R = 4-OMe
1
: R= H, R = 2-SMe
The IC50 values for the active compounds 13–15 and 24
are also given in Table 1.
Biology
All compounds 2–15 and 17–24 were evaluated for their
in vitro antitumor activity against human MCF7 cell line
(breast cancer) using a one dose primary anticancer in
vitro assay [29, 30]. The results are presented in Table Conclusion
1. The requirement for antitumor activity set by the
National Cancer Institute is that the fraction of surviving A series of benzimidazoles 2–11, 13–15 and 17–24 were
tumor cells is 30% or less, which corresponds to inhibi- synthesized and evaluated for their in vitro antitumor
tion of 70% or more. According to this definition it may activity against MFC7 cell line. Compounds 13–15 and 24
be concluded that compounds 13–15 and 24 are active. exhibit significant activity.
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM

ꢂꢁꢁꢁ
A. Tantawy etꢃal.: Benzimidazole compounds as inhibitors of breast cancer cell growthꢀ
ꢀ127
Table 1ꢁAntitumor activity of compounds 2–11, 13–15 and 17–24
against MCF7 cell line.
dried and crystallized from absolute ethanol to afford the title com-
pound 2–5.
1-(1H-Benzimidazol-2-yl)-2-[(4-bromophenyl)amino]ethanone
(2)ꢀYellow compound; yield 68%; mp 158–160°C; 1H NMR: δ 4.40 (s,
2H, CH2), 6.28 (s, 1H, NH, D2O exchangeable), 6.48 (d, J = 8, 2H), 7.12
(d, J = 8, 2H), 7.24 (d, J = 8.7, 2H), 7.66 (d, J = 8.7, 2H), 12.81 (s, 1H,
NH, D2O exchangeable); MS: m/z 330 [M+], 332 [M++2]. Anal. Calcd
for C15H12BrN3O: C, 54.56; H, 3.66; N, 12.73. Found: C, 54.49; H, 3.61;
N, 12.82.
Compound
% Surviving
% Inhibition
IC50b, μg/mL
2
3
40.5
33.2
32.9
48.4
39.5
34.3
31.2
30.8
47.4
42.7
28.1
26.0
27.0
41.4
32.9
40.6
34.4
37.1
32.8
33.2
29.9
59.5
66.8
67.1
51.6
60.5
65.7
68.8
69.2
52.6
57.3
71.9a
74.0a
73.0a
58.6
67.1
59.4
65.6
62.9
67.2
66.8
70.1a
4
5
6
7
8
1-(1H-Benzimidazol-2-yl)-2-[(4-chlorophenyl)amino]ethanone
(3)ꢀYellowish brown compound; yield 72%; mp 182–184°C; 1H NMR:
δ 5.12 (s, 2H, CH2), 6.24 (s, 1H, NH, D2O exchangeable), 6.54 (d, J = 7. 5,
2H), 7.17 (d, J = 7.5, 2H), 7.27 (d, J = 8.7, 2H), 7.59 (d, J = 8.7, 2H), 12.81
(s, 1H, NH, D2O exchangeable); MS: m/z 285 [M+], 287 [M++2]. Anal.
Calcd for C15H12ClN3O: C, 63.05; H, 4.23; N, 14.71. Found: C, 63.11; H,
4.17; N, 14.68.
9
10
11
13
14
15
17
18
19
20
21
22
23
24
16.3
15.5
12.7
1-(1H-Benzimidazol-2-yl)-2-[(4-methoxyphenyl)amino]ethanone
(4)ꢀBrown compound; yield 70%; mp 199–201°C; 1H NMR: δ 3.80 (s,
3H, OCH3), 4.19 (s, 2H, CH2), 6.50 (s, 1H, NH, D2O exchangeable), 6.70
(d, J = 7.8, 2H), 6.90 (d, J = 7.8, 2H), 7.22 (d, J = 8.4, 2H), 7.59 (d, J =
8.4, 2H), 12.81 (s, 1H, NH, D2O exchangeable); MS: m/z 281 [M+]. Anal.
Calcd for C16H15N3O2: C, 68.31; H, 5.37; N, 14.94. Found: C, 68.24; H,
5.45; N, 15.03.
16.7
aCompounds showing significant antitumor activity. bIC50 for active
antitumor compounds.
1-(1H-Benzimidazol-2-yl)-2-[(2-methylthiophenyl)amino]eth-
anone (5) Brown compound; yield 79%; mp 151–153°C; 1H NMR: δ
2.33 (s, 3H, SCH3), 5.18 (s, 2H, CH2), 6.22 (s, 1H, NH, D2O exchange-
able), 6.28 (t, J = 7.8, 1H), 6.57 (t, J = 7.8, 1H), 6.82 (d, J = 7.8, 1H),
7.10 (d, J = 7.8, 1H), 7.22 (d, J 8.7, 2H), 7.59 (d, J = 8.7, 2H), 12.81
(s, 1H, NH, D2O exchangeable); MS: m/z 297 [M+]. Anal. Calcd for
C16H15N3OS: C, 64.62; H, 5.08; N, 14.13. Found: C, 64.66; H, 5.13; N,
14.09.
Experimental
General
Unless otherwise noted, all materials were obtained from commercial
suppliers (Aldrich and Merck companies) and used without further
purification. Melting points were recorded using an Electrothermal
C14500 apparatus and were uncorrected. Microanalyses were per-
formed at the microanalytical unit, Cairo University. IR spectra were
1-(1H-Benzimidazol-2-yl)-2-oxoethyl
benzoates 6, 7
1
recorded on a Mattson 5000 FT-IR spectrometer using KBr disks. H
Sodium benzoate or sodium 2-hydroxybenzoate (0.002 mol) was
added at room temperature to a solution of 1-(1H-benzimidazol-2-yl)-
2-bromoethanone 1 [24] (0.5 g, 0.002 mol) in DMF. Stirring at room
temperature was continued for another 8 h. Then the mixture was
poured into water and the precipitated product was filtered, washed
thoroughly with water, dried and crystallized from DMF to afford the
title compound 6, 7.
NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a
Bruker Avance-400 spectrometer in DMSO-d6 at Georgia State Univer-
sity, Atlanta, GA, USA and the Korea Institute of Science and Technol-
ogy, Republic of Korea. Mass spectral analyses were performed on a
JOEL JMS-600H spectrometer at Cairo University. Reaction progress
was monitored using TLC on silica gel plates 60 F245 E. Merck, and
the spots were visualized by UV light at 366 nm or 245 nm. Substrates
1 [24], 12a,b [25] and 16a [26–28] were synthesized according to re-
ported methods.
1-(1H-Benzimidazol-2-yl)-2-oxoethyl benzoate (6)ꢀOrange com-
pound; yield 72%; mp 136–138°C; IR: 1660 (-CO-CH2), 1740 (-COO-
1
CH2), 2985, 2960 (CH2), 3150 cm-1 (NH); H NMR: δ 5.86 (s, 2H, CH2),
7.22 (d, J = 8.7, 2H), 7.46 (t, J = 8Hz, 1H), 7.59 (d, J = 8.7, 2H), 7.66 (t, J =
8Hz, 2H), 8.03 (d, J = 8Hz, 2H), 12.01 (s, 1H, NH, D2O exchangeable);
MS: m/z 280 [M+]. Anal. Calcd for C16H12N2O3: C, 68.56; H, 4.32; N, 9.99.
Found: C, 68.51; H, 4.28; N, 10.11.
1-(1H-Benzimidazol-2-yl)-2-[(substituted
phenyl)amino]ethanones 2–5
A mixture of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.5 g,
0.002 mol), a substituted aniline (0.002 mol) and sodium bicarbonate
(1.55 g) in anhydrous ethanol (50 mL) was heated under reflux for 4 h.
Then the mixture was allowed to cool to room temperature and the
resultant precipitate was filtered, washed thoroughly with water,
1-(1H-Benzimidazol-2-yl)-2-oxoethylꢀ2-hydroxybenzo-
ateꢀ(7)ꢀYellow compound; yield 75%; mp 158–160°C; IR: 1650 (-CO-
CH2), 1740 (-COO-CH2), 2940, 2875 (CH2), 3100 (NH), 3500 cm-1 (OH);
1H NMR: δ 5.89 (s, 2H, CH2), 6.88 (d, J = 7.7 Hz, 1H), 7.01 (t, J = 7.7 Hz,
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM

ꢁꢁꢁꢂ
128ꢀ ꢀA. Tantawy etꢃal.: Benzimidazole compounds as inhibitors of breast cancer cell growth
3-(1H-Benzimidazol-2-yl)-2H-benzo[b][1,4]oxazineꢀ(11)ꢀBrown
compound; yield 76%; mp 211–213°C; 1H NMR: δ 4.0 (s, 2H, CH2), 6.68
(d, J = 8.2 Hz, 1H), 7.02 (t, J = 8.2 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.43
(t, J = 8.2 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 8.2 Hz, 1H), 12.01
(s, 1H, NH, D2O exchangeable); MS: m/z 249 [M+]. Anal. Calcd for
C15H11N3O: C, 72.28; H, 4.45; N, 16.86. Found: C, 72.21; H, 4.34; N, 16.82.
1H), 7.12 (t, J = 7.7 Hz, 1H), 7.24 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.7 Hz,
2H), 7.88 (d, J = 7.7 Hz, 1H), 10.29 (s, 1H, OH), 12.01 (s, 1H, NH, D2O
exchangeable); 13C NMR: δ 60.2, 111.6, 114.3, 116.2, 120.2, 122.1, 129.2,
130.1, 132.4, 150.2, 155.1, 160.9, 163.2; MS: m/z 296 [M+]. Anal. Calcd
for C16H12N2O4: C, 64.86; H, 4.08; N, 9.46. Found: C, 64.91; H, 4.11; N,
9.39.
2-(1H-Benzimidazol-2-yl)quinoline-4-carbox-
N′-[2-(1H-Benzimidazol-2-yl)-2-oxoethyl]
benzohydrazides 8, 9
ylic acids 13–15
A solution of potassium hydroxide (1.02 g, 0.018 mol) in water (5 mL)
was added dropwise to the appropriate isatin derivatives (0.003 mol)
in ethanol (10 mL) over 15 min. The appropriate benzimidazole de-
rivatives 12a,b [25] (0.003 mol) were added and the reaction mixture
was heated under reflux for 18 h, then cooled to room temperature
and the solvent was removed under vacuum. The resulting solid was
dissolved in water, washed with diethyl ether, cooled in ice-cold wa-
ter and acidified with acetic acid. The separated solid was filtered and
recrystallized from acetic acid to afford the title compounds 13–15.
A mixture of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.5 g,
0.002 mol), an acid hydrazide (0.002 mol) and sodium bicarbonate
(1.55 g) in absolute ethanol (50 mL) was heated under reflux for 6 h.
Then the mixture was allowed to cool to room temperature and the
resulting precipitate was filtered, washed thoroughly with water,
dried and crystallized from absolute ethanol to afford the title com-
pound 8, 9.
N′-[2-(1H-Benzimidazol-2-yl)-2-oxoethyl]benzohydrazideꢀ(8)ꢀ
Yellow compound; yield 74%; mp 150–152°C; IR: 1662 (-CO-CH2),
1685 (C=O hydrazino), 2960, 2890 (CH2), 3150, 3350, 3410 cm-1 (NH);
1H NMR: δ 3.34 (s, 2H, CH2), 5.0 (s, 1H, NH, D2O exchangeable), 7.22
(d, J = 8.7 Hz, 2H), 7.42 (t, J = 8 Hz, 2H), 7.59 (d, J = 8.7 Hz, 2H), 7.63 (t,
J = 8 Hz, 1H), 7.84 (d, J = 8 Hz, 2H), 8.0 (s, 1H, NH, D2O exchangeable),
12.10 (s, 1H, NH, D2O exchangeable); MS: m/z 294 [M+]. Anal. Calcd for
C16H14N4O2: C, 65.30; H, 4.79; N, 19.04. Found: C, 65.37; H, 4.71; N, 19.13.
2-(1H-Benzimidazol-2-yl)-6-chloroquinoline-4-carboxylicꢀacid
(13)ꢀYellow compound; yield 69%; mp 257–259°C; IR: 1725 (CO),
3200 (NH), 3400 cm-1 (OH); 1H NMR: δ 7.22 (d, J = 8.4 Hz, 2H), 7.65 (d,
J = 8.4 Hz, 2H), 7.86 (d, J = 8.6 Hz, 1H), 8.26 (d, J = 8.6 Hz, 1H), 8.47
(s, 1H), 9.07 (s, 1H), 12.81 (s, 1H, NH, D2O exchangeable), 13.21 (s, 1H,
COOH); 13C NMR: δ 114.8, 122.3, 123.1, 124.0, 124.5, 126.8, 129.0, 130.1,
133.2, 140.2, 145.1, 152.7, 154.2, 162.1; MS: m/z 323 [M+], 325 [M++2].
Anal. Calcd for C17H10ClN3O2: C, 63.07; H, 3.11; N, 12.98. Found: C,
62.96; H, 3.18; N, 13.09.
N ′ - [ 2 - ( 1 H - B e n z i m i d a z o l - 2 - y l ) - 2 - o x o e t h y l ] - 2 -
hydroxybenzohydrazide (9)ꢀYellow compound; yield 71%; mp 177–
179°C; IR: 1662 (-CO-CH2), 1685 (C=O hydrazino), 2972, 2860 (CH2), 3150,
3350, 3410 (NH), 3500 cm-1 (OH); 1H NMR: δ 3.34 (s, 2H, CH2), 5.0 (s, 1H,
NH, D2O exchangeable), 6.95 (d, J = 7.7 Hz, 1H), 7.03 (t, J = 7.7 Hz, 1H),
7.22 (d, J = 8.7 Hz, 2H), 7.32 (t, J = 7.7 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.86
(d, J = 7.7 Hz, 1H), 8.0 (s, 1H, NH, D2O exchangeable), 11.50 (s, 1H, OH),
12.10 (s, 1H, NH, D2O exchangeable); MS: m/z 310 [M+]. Anal. Calcd for
C16H14N4O3: C, 61.93; H,4.55; N, 18.06. Found: C, 61.99; H, 4.50; N, 17.97.
2-[(5-Methyl)-1H-benzimidazol-2-yl]quinoline-4-carboxylic acid
(14)ꢀBuff compound; yield 70%; mp 133–135°C; IR: 1725 (CO), 3200
(NH), 3500 cm-1 (OH); 1H NMR: δ 2.44 (s, 3H, CH3), 7.15 (d, J = 8.4 Hz,
1H), 7.33 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 8.6 Hz, 1H), 7.98 (d,
J = 8.6 Hz, 1H), 8.10 (t, J = 8.6 Hz, 1H), 8.20 (d, J = 8.6 Hz, 1H), 8.47
(s, 1H), 12.77 (s, 1H, NH, D2O exchangeable), 13.19 (s, 1H, COOH); 13C
NMR: δ 20.8, 114.5, 119.2, 122.8, 124.5, 124.9, 126.5, 128.0, 128.8, 130.0,
135.4, 135.7, 138.2, 145.1, 152.7, 153.2, 162.8; MS: m/z 303 [M+]. Anal.
Calcd for C18H13N3O2: C, 71.28; H, 4.32; N, 13.85. Found: C, 71.36; H,
4.25; N, 13.71.
3-(1H-Benzimidazol-2-yl)-1,2-dihydroqui-
noxaline (10) and 3-(1H-benzimidazol-2-yl)-
2H-benzo[b][1,4]oxazine (11)
A mixture of 1-(1H-benzimidazol-2-yl)-2-bromoethanone 1 [24] (0.01
mol), 1,2-phenylenediamine or 2-aminophenol (0.011 mol), and etha-
nol (20 mL) was heated under reflux for 3 h. Afer cooling, the sepa-
rated solid was filtered and washed with ethanol. The precipitate was
then suspended in water, stirred with saturated sodium bicarbonate
solution (30 mL, 5%), filtered, washed thoroughly with water, dried
and crystallized from ethyl acetate/ethanol (1:1) to afford the title
compound 10 or 11.
2-(5-Methyl-1H-benzimidazol-2-yl)-6-methylquinoline-4-car-
boxylic acid (15)ꢀYellow compound; yield 65%; mp 204–206°C; IR:
1
1725 (CO), 3200 (NH), 3500 cm-1 (OH); H NMR: δ 2.51 (s, 6H, 2CH3),
7.12 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.76 (d, J =
8.6 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 8.36 (s, 1H), 8.48 (s, 1H), 12.81
(s, 1H, NH, D2O exchangeable), 13.21 (s, 1H, COOH); 13C NMR: δ 20.7,
20.9, 114.9, 122.1, 123.0, 124.2, 125.1, 128.2, 129.4, 132.1, 132.4, 134.2,
134.8, 137.8, 144.2, 152.1, 152.4, 163.7; MS: m/z 317 [M+]. Anal. Calcd for
C19H15N3O2: C, 71.91; H, 4.76; N, 13.24. Found: C, 72.12; H, 4.72; N, 13.28.
3-(1H-Benzimidazol-2-yl)-1,2-dihydroquinoxalineꢀ(10)ꢀYellow
1
compound; yield 79%; mp 227–229°C; H NMR: δ 3.2 (s, 2H, CH2), 5.0
Ethyl 2-cyanomethyl-5-methyl-1H-benzimi-
(s, 1H, NH, D2O exchangeable), 6.89 (d, J = 8.8 Hz, 1H), 7.01 (t, J = 8.8
Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.32 (t, J = 8.8 Hz, 1H), 7.45 (d, J = 8.8
Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 12.0 (s, 1H, NH, D2O exchangeable);
MS: m/z 248 [M+]. Anal. Calcd for C15H12N4: C, 72.56; H, 4.87; N, 22.57.
Found: C, 72.62; H, 4.75; N, 22.69.
dazole-1-carboxylate (16b)
A solution of 5-methyl-2-cyanomethylbenzimidazole [27, 28] (0.01
mol) in pyridine (20 mL) was stirred in an ice bath and treated drop-
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM

ꢂꢁꢁꢁ
A. Tantawy etꢃal.: Benzimidazole compounds as inhibitors of breast cancer cell growthꢀ
ꢀ129
wise with ethyl chloroformate (2.7 g, 0.025 mol). Stirring was con- ethanol; yellow compound; yield 72%; mp 150–152°C; IR: 1600, 1630
1
tinued for an additional 10 min and then the mixture was poured on (C=N), 1700 (C=O), 2225 cm-1 (C≡N); H NMR: δ 2.47 (s, 3H, CH3), 7.39
cold water (400 mL). The resultant precipitate of 16b was collected (d, J = 8.4 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.90
by filtration, dried and crystallized from ethanol: yellow com- (d, J = 7.5 Hz, 1H), 8.0 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 8.21
pound; yield 74%; mp 76–78°C; IR: 2220 (CN), 1715 (CO), 1620 cm-1 (s, 1H); MS: m/z 335 [M+], 337 [M++2]. Anal. Calcd for C17H10ClN5O: C,
1
(C=N); H NMR: δ 1.25 (t, J = 7 Hz, 3H, CH2CH3), 2.34 (s, 3H, CH3), 60.81; H, 3.00; N, 20.86. Found: C, 60.89; H, 3.12; N, 20.71.
3.67 (s, 2H, CH2-CN), 4.17 (q, J = 7 Hz, 2H, CH2CH3), 7.12 (d, J = 8.7
Hz, 1H), 7.33 (s, 1H), 7.54 (d, J = 8.7 Hz, 1H); MS: m/z 243 [M+]. Anal.
Calcd for C13H13N3O2: C, 64.19; H, 5.39; N, 17.27. Found: C, 64.24; H,
5.45; N, 17.33.
8-Methyl-1-oxo-2-(3-chlorophenyl)-1,2-dihydrobenzimidazo[1,2-
d][1,2,4]triazine-4-carbonitrile (22)ꢀCrystallized from methanol,
yellowish brown compound; yield 88%; mp 147–149°C; IR: 1605, 1640
1
(C=N), 1700 (C=O), 2220 cm-1 (C≡N); H NMR: δ 2.43 (s, 3H, CH3), 7.39
(d, J = 8.4 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.91
(d, J = 7.5 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 8.12 (s, 1H), 8.21 (s, 1H); MS:
m/z 335 [M+], 337 [M++2]. Anal. Calcd for C17H10ClN5O: C, 60.81; H, 3.00;
N, 20.86. Found: C, 60.87; H, 3.09; N, 20.89.
1-Oxo-2-(substituted phenyl)-
1,2-dihydrobenzimidazo[1,2-d][1,2,4]tria-
zine-4-carbonitriles 17–24
To a solution of 16a [26] or 16b (0.01 mol) in pyridine (20 mL) in an
ice bath, a solution of a diazonium hydrochloride [amine (0.01 mol),
36% HCl (3 mL), ice water (10 mL) and NaNO2 (0.7 g, 0.01 mol)] was
added with stirring. Afer 24 h, the mixture was diluted with ice-cold
water (200 mL), and the resultant precipitate of 17–24 was collected
by filtration, dried and crystallized.
8-Methyl-1-oxo-2-(4-methoxyphenyl)-1,2-dihydrobenzimidazo
[1,2-d][1,2,4]triazine-4-carbonitrile (23)ꢀCrystallized from abso-
lute ethanol, buff compound; yield 85%; mp 166–168°C; IR: 1600,
1640 (C=N), 1680 (C=O), 2220 cm-1 (C≡N); 1H NMR: δ 2.56 (s, 3H, CH3),
3.85 (s, 3H, O-CH3), 7.12 (d, J = 7.8 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.69
(d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.15 (s, 1H); 13C NMR: 20.9,
55.0, 111.7, 113.7, 113.9, 119.9, 127.2, 127.6, 128.4, 131.9, 136.3, 140.7, 159.0;
MS: m/z 331 [M+]. Anal. Calcd for C18H13N5O2: C, 65.25; H, 3.95; N, 21.14.
Found: C, 65.20; H, 3.87; N, 21.18.
1-Oxo-2-(2-chlorophenyl)-1,2-dihydrobenzimidazo[1,2-d][1,2,4]
triazine-4-carbonitrile (17)ꢀCrystallized from absolute ethanol;
yellow compound; yield 71%; mp 151–153°C; IR: 1600, 1640 (C=N),
1700 (CO), 2220 cm-1 (C≡N); 1H NMR: δ 7.63 (d, J = 8.4 Hz, 2H), 7.80 (d,
J = 8.4 Hz, 2H), 7.92 (t, J = 7.5 Hz, 1H), 8.01 (t, J = 7.5 Hz, 1H), 8.15 (d,
J = 7.5 Hz, 1H), 8.35 (d, J = 7.5 Hz, 1H); MS: m/z 321 [M+], 323 [M++2].
Anal. Calcd for C16H8ClN5O: C, 59.73; H, 2.51; N, 21.77. Found: C, 59.60;
H, 2.55; N, 21.73.
8-Methyl-1-oxo-2-(2-thiomethylphenyl)-1,2-dihydrobenzimidazo
[1,2-d][1,2,4]triazine-4-carbonitrile (24)ꢀCrystallized from aque-
ous ethanol; brown compound; yield 77%; mp 162–164°C; IR: 1600,
1630 (C=N), 1700 (C=O), 2220 cm-1 (C≡N); 1H NMR: δ 2.34 (s, 3H, CH3),
2.53 (s, 3H, S-CH3), 7.42 (d, J = 7.8 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.78
(t, J = 7.8 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.91(d, J = 7.8 Hz, 1H), 8.11 (d,
J = 8.4 Hz, 1H), 8.20 (s, 1H); MS: m/z 347 [M+]. Anal. Calcd for
C18H13N5OS: C, 62.23; H, 3.77; N, 20.16. Found: C, 62.34; H, 3.73; N, 20.19.
1-Oxo-2-(3-chlorophenyl)-1,2-dihydrobenzimidazo[1,2-d][1,2,4]
triazine-4-carbonitrile (18)ꢀCrystallized from absolute ethanol;
orange compound; yield 69%; mp 183–185°C; IR: 1600, 1640 (C=N),
1680 (CO), 2225 cm-1 (C≡N); 1H NMR: δ 7.63 (d, J = 8.4 Hz, 2H), 7.72 (d,
J = 8.4 Hz, 2H),7.88 (t, J = 7.5 Hz, 1H), 8.11 (d, J = 7.5 Hz, 1H), 8.28 (d, J =
7.5 Hz, 1H), 8.37 (s, 1H); MS: m/z 321 [M+], 323 [M++2]. Anal. Calcd for
C16H8ClN5O: C, 59.73; H, 2.51; N, 21.77. Found: C, 59.79; H, 2.48; N, 21.69.
Biology
All materials were obtained from Sigma Chemical Co.
(USA). Human tumor cell lines were obtained frozen in
liquid nitrogen (-180°C) from the American Type Culture
Collection. The tumor cell lines were maintained in the
National Cancer Institute, Cairo, Egypt, by serial sub-
culturing. In vitro antitumor activity against human
MCF7 (breast cancer cell line) was determined using the
Sulforhodamine B assay [29, 30]. Cells were plated in a
96-multiwell plate (104 cells/well) for 24 h before treatment
with the compounds to allow attachment of the cell to the
1-Oxo-2-(4-methoxylphenyl)-1,2-dihydrobenzimidazo[1,2-d]
[1,2,4]triazine-4-carbonitrile (19)ꢀCrystallized from methanol;
orange compound; yield 75%; mp 200–202°C; IR: 1605, 1640 (C=N),
1680 (CO), 2225 cm-1 (C≡N); 1H NMR: δ 3.86 (s, 3H, OCH3), 7.02 (d, J = 7. 8
Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 8.7 Hz, 2H), 8.35 (d, J = 7. 8
Hz, 2H); MS: m/z 317 [M+]. Anal. Calcd for C17H11N5O2: C, 64.35; H, 3.49;
N, 22.07. Found: C, 64.44; H, 3.45; N, 22.17.
1-Oxo-2-(2-thiomethylphenyl)-1,2-dihydrobenzimidazo[1,2-d]
[1,2,4]triazine-4-carbonitrile (20)ꢀCrystallized from absolute
ethanol; yellow compound; yield 66%; mp 194–196°C; IR: 1560, 1614 wall of the plate. Monolayer cells were incubated with
(C=N), 1743 (C=O), 2220 cm-1 (C≡N); 1H NMR: δ 2.51 (s, 3H, S-CH3), 7.44
the compounds for 48 h at 37°C in a humidified incubator
with 5% CO2. Cells were fixed with trichloroacetic acid and
stained for 30 min with 0.4% (wt/vol) Sulforhodamine B
(SRB) stain dissolved in 1% acetic acid. Unbound dye was
washed with 1% acetic acid and protein bound dye was
extracted with Tris EDTA. The optical density (OD) of each
well was measured spectrophotometrically at 564 nm with
(d, J = 8.7 Hz, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.91 (t,
J = 7.8 Hz, 1H), 8.04 (d, J = 8.7 Hz, 2H), 8.36 (d, J = 7.8 Hz, 1H); MS: m/z
333 [M+]. Anal. Calcd for C17H11N5OS: C, 61.25; H, 3.33; N, 21.01. Found:
C, 61.33; H, 3.28; N, 21.07.
8-Methyl-1-oxo-2-(2-chlorophenyl)-1,2-dihydrobenzimidazo[1,2-
d][1,2,4]triazine-4-carbonitrile (21)ꢀCrystallized from absolute
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM

ꢁꢁꢁꢂ
130ꢀ ꢀA. Tantawy etꢃal.: Benzimidazole compounds as inhibitors of breast cancer cell growth
an ELISA microplate reader (Meter tech. Σ 960, USA). Cell the survival curve of the tumor cell line after the specified
survival was calculated as follows: survival fraction = OD compound (Table 1).
(treated cells)/OD (control cells) (Table 1). For determi-
Acknowledgments: The authors would like to express
nation of IC50, different concentrations of the compound
their gratitude and thanks to the National Cancer Institute
under test (0, 1, 2.5, 5 and 10 μg/mL) were added to the
(NCI), Cancer Biology Department, Pharmacology Unit,
Cairo University, Egypt for performing antitumor activity.
vidual dose. The absorbance of each well was determined
cell monolayer wells which were prepared for each indi-
by an ELISA reader. The relation between surviving frac-
tion and compound concentration was plotted to obtain
Received January 20, 2013; accepted January 28, 2013; previously
published online March 21, 2013
References
[1] EL-Naggar, S. A.; El-Barbary, A. A.; Mansour, M. A.;
Abdel-Shafy, F.; Talat, S. Anti-tumor activity of some
1,3,4-thiadiazoles and 1,2,4-triazine derivatives against
Ehrlichs ascites carcinoma. Int. J. Cancer Res. 2011, 7,
278–288.
[2] Patel, O. B.; Patel, L. J. Microwave assisted synthesis and
biological evaluation of benzimidazole derivatives as
anticancer agents. Int. J. Pharm. Appl. Sci. 2011, 2, 15–19.
[3] Xiang, P.; Zhou, T.; Wang, L.; Sun, C. Y.; Hu, J.; Zhao, Y. L.;
Yang, L. Novel benzothiazole, benzimidazole and benzoxazole
derivatives as potential antitumor agents: synthesis and
preliminary in vitro biological evaluation. Molecules 2012, 17,
873–883.
[4] Algul, O.; Ozc¸ B.; Abbasoglu, U.; Gumus, F. Synthesis, charac-
terization and genotoxicity of platinum(II)complexes with
substituted benzimidazole ligands. Turk. J. Chem. 2005, 29,
607–615.
[5] Siddiqui, N.; Bhrigu, B.; Pathak, D. R.; Alam, M. S. R.; Ali, R.
Cytotoxicity and enzymes estimation of some newer benzimi-
dazoles. Ann. Biol. Res. 2011, 2, 194–199.
[6] Abdel-Hafez, A. A. Benzimidazole condensed ring systems:
new synthesis and antineoplastic activity of substituted
3,4-dihydro and 1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]
pyrimidine derivatives. Arch. Pharm. Res. 2007, 30,
678–684.
[7] Alpan, A. S.; Gunes, H. S.; Topcu, Z. 1H-Benzimidazole
derivatives as mammalian DNA topoisomerase I inhibitors.
Anticancer Res. 2007, 54, 561–565.
[8] Nofal, Z. M.; Soliman, E. A.; Abdelkarim, S. S.; Elzahar, M. I.;
Srour, A. M.; Sethumadhavan, S.; Maher, T. J. Novel
benzimidazole derivatives as expected anticancer agents. Acta
Pol. Pharm. Drug Res. 2011, 68, 519–534.
[9] Ramla, M. M.; Omar, M. A.; Elkhamry, A. M.; Eldiwani, H. I.
Synthesis and antitumor activity of 1-substituted-2-methyl-
5-nitrobenzimidazoles. Bioorg. Med. Chem. 2006, 14,
7224–7332.
[12] Alper, S.; Arpaci, O. T.; Aki, E. S.; Yalc, I. Some new bi- and
ter-benzimidazole derivatives as topoisomerase I inhibitors. IL
Farmaco 2003, 58, 497–507.
[13] Varga, A.; Akisener, E.; Yalcin, I.; Temizarpaci, O.;
Tekinergulbas, B.; Cherepnev, G.; Molnar, J. Induction of
apoptosis and necrosis by resistance modifiers benzazoles
and benzoxazines on tumour cell line mouse lymphoma L5718
cells. In Vivo 2005, 19, 1087–1092.
[14] Korolyov, A.; Dorbes, S.; Azema, J.; Guidetti, B.; Danel, M.;
Theys, D. L.; Gras, T.; Dubois, J.; Kiss, R.; Martino, R.; et al.
Novel lipophilic 7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-
carboxylic acid derivatives as potential antitumor agents:
improved synthesis and in vitro evaluation. Bioorg. Med.
Chem. 2010, 18, 8537–8548.
[15] Ghorab, M. M.; Ragab, F. A.; Heiba, H. I.; Elgazzar, M. G.
Synthesis, in vitro anticancer screening and radiosensitizing
evaluation of some new 4-[3-(substituted)thioureido]-
N-(quinoxalin-2-yl)benzenesulfonamide derivatives. Acta
Pharm. 2011, 61, 415–425.
[16] Noolvi, M. N.; Patel, H. M.; Bhardwaj, V.; Chauhan, A. Synthesis
and in vitro antitumor activity of substituted quinazoline and
quinoxaline derivatives: search for anticancer agent. Eur. J.
Med. Chem. 2011, 46, 2327–2346.
[17] Abuhashem, A. A.; Aly, A. S. Synthesis of new pyrazole,
triazole, and thiazolidine-pyrimido [4,5-b] quinoline
derivatives with potential antitumor activity. Arch. Pharm. Res.
2012, 35, 437–445.
[18] Srivastava, S. K.; Jha, A.; Agarwal, S. K.; Mukherjee, R.;
Burman, A. C. Synthesis and structure activity relationships
of potent antitumor active quinoline and naphthyridine
derivatives. Anticancer Agents Med. Chem. 2007, 7, 685–709.
[19] Rashad, A. E.; Elsayed, W. A.; Mohamed, A. M.; Ali, M. M.
Synthesis of new quinoline derivatives as inhibitors of human
tumor cells growth. Arch. Pharm. 2010, 343, 440–448.
[20] Utsugi, T.; Aoyagi, K.; Asao, T.; Okazaki, S.; Aoyagi, Y.;
Sano, M.; Wierzba, K.; Yamada, Y. Antitumor activity of a
novel quinoline derivative, TAS-103, with inhibitory effects
on topoisomerases I and II. Jpn. J. Cancer Res. 1997, 88,
992–1002.
[10] Nawrocka, W.; Sztuba, B.; Kowalska, M. W.; Liszkiewicz, H.;
Wietrzyk, J.; Nasulewicz, A.; Pelczynska, M.; Opolski, A.
Synthesis and antiproliferative activity in vitro of 2-aminoben-
zimidazole derivatives. IL Farmaco 2004, 59, 83–91.
[11] Leoni, L. M. Bendamustine: rescue of an effective antineo-
plastic agent from the mid-twentieth century. Semin. Hematol.
2011, 48, 4–11.
[21] Sztanke, K.; Pasternak, K.; Rzymowska, J.; Sztanke, M.;
Szerszen, M. K. Synthesis, structure elucidation and identi-
fication of antitumor properties of novel fused 1,2,4-triazine
aryl derivatives. Eur. J. Med. Chem. 2008, 43, 1085–1094.
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM

ꢂꢁꢁꢁ
A. Tantawy etꢃal.: Benzimidazole compounds as inhibitors of breast cancer cell growthꢀ
ꢀ131
[22] Ruel, R.; Thibeault, C.; Heureux, A. L.; Martel, A.;
Cai, Z. W.; Wei, D.; Qian, L.; Barrish, J. C.; Mathur, A.;
Arienzo, C. D.; etꢃal. Discovery and preclinical studies of
5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-
1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-
4-amine (BMS-645737), an in vivo active potent
VEGFR-2 inhibitor. Bioorg. Med. Chem. Lett. 2008, 18,
2985–2989.
[23] Sharma, N. K.; Kumar, Y.; Sahi, S. 3D QSAR studies of
pyrrolo[2,1-f][1,2,4] triazines as tyrosine kinase inhibitors. Int.
J. Pharm. Pharm. Sci. 2010, 2, 118–121.
[24] Serafin, B.; Szymanowska, E. Benzimidazole derivatives. Part
I. Bromination of 2-acetyl benzimidazoles and its derivatives.
Roczn. Chem. 1975, 49, 791–798.
[25] Cheeseman, G. W. H. 2-Acetylbenzimidazole. J. Chem. Soc.
1964, 4645–4646.
[26] Slouka, J. Cyclisierungsreaktionen von hydrazonen I. Synthese
von 1-oxo-2-aryl-1,2-dihydro-(as-triazino)[5,4-a]benzimidazol-
4-carbonsäurenitrilen. Tetrahedron Lett. 1968, 9, 4007–4008.
[27] Ralph, A. B.; Allan, R. D. The preparation and reactions of
2-benzimidazolecarboxylic acid and 2-benzimidazoleacetic
acid. J. Am. Chem. Soc. 1943, 65, 1072–1075.
[28] Sawlewicz, J.; Milczarska, B. Reactions of cyanometh-
ylbenzimidazoles. Part I. Synthesis of 1- and
2-cyanomethylbenzimidazoles and some of their derivatives.
Pol. J. Pharmacol. Pharm. 1974, 26, 639–646.
[29] Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon,
J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd,
M. R. New colorimetric cytotoxicity assay for anticancer drug
screening. Natl. Cancer Inst. 1990, 82, 1107–1112.
[30] Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for
cytotoxicity screening. Nat. Protoc. 2006, 1, 1112–1116.
Brought to you by | Lulea University of Technology
Authenticated | 130.240.43.43
Download Date | 7/26/13 9:51 PM