Synthesis of potential anticancer derivatives of pyrido[1,2-a] benzimidazoles

Article abstract of DOI:10.1007/s00044-011-9636-y

In this study, the starting compounds, 2-cyanomethyl benzimidazoles (1 or 2) were reacted with ethyl acetoacetate, ethyl benzoylacetate, or 2-acetylbutyrolactone to give the novel series of 4-cyano-3-substituted-1-oxo- 1H, 5H-pyrido[1,2-a]benzimidazole (3-6, 15, 16). The latter was chlorinated to give compounds 7-10, 17, 18 then aminated with 4-(2-fluorophenyl) piperazine to afford compounds 11-14, 19, 20. The structures of the new compounds were confirmed by elemental analysis as well as ¹H-NMR, IR, and mass data. All the synthesized products were subjected to in vitro anticancer screening that revealed that all the tested compounds exhibited antitumor activity against human breast adenocarcinoma (MCF7) cell line, with IC₅₀'s 3.43-14.70 μg/ml. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9636-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1253–1260
DOI 10.1007/s00044-011-9636-y
ORIGINAL RESEARCH
Synthesis of potential anticancer derivatives
of pyrido[1,2-a]benzimidazoles
Hanan M. Refaat
Received: 6 January 2011 / Accepted: 25 March 2011 / Published online: 10 April 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, the starting compounds, 2-cy-
anomethyl benzimidazoles (1 or 2) were reacted with ethyl
acetoacetate, ethyl benzoylacetate, or 2-acetylbutyrolac-
tone to give the novel series of 4-cyano-3-substituted-1-
oxo-1H, 5H-pyrido[1,2-a]benzimidazole (3–6, 15, 16). The
latter was chlorinated to give compounds 7–10, 17, 18 then
aminated with 4-(2-fluorophenyl) piperazine to afford
compounds 11–14, 19, 20. The structures of the new
compounds were confirmed by elemental analysis as well
2010). The results obtained from their in vitro screening
have shown interesting percent tumor-growth inhibition on
various cell lines. All of these compounds exhibited sig-
nificant values of percent growth inhibition at \10 lg/ml.
These results were in accordance with the previously
reported papers signifying the 5-chloro or 5-carboxylic
acid benzimidazole as potent anticancer agents (And-
rzejewska et al., 2002; Pinar et al., 2004; Ram et al., 1992;
Galal et al., 2009; Graham et al., 2007).
1
as H-NMR, IR, and mass data. All the synthesized prod-
A literature survey described a variety of substituted
pyrido[1,2-a]benzimidazole ring system that received a
great deal of attention for their anticancer activity (Soliman
et al., 1984; Rida et al., 1988a, b; Badawey and Kappe,
1995, 1999; Badawey et al. 1989; EL-Hawash et al., 1999;
Demirayak and Mohsen, 1998; Dupuy et al., 1998, 2001). It
was concluded from these investigations that the pyr-
ido[1,2-a]benzimidazole nucleus is not the only factor
essential for the anticancer activity, and that by proper
substitution, good cytotoxic candidates can be obtained. Of
particular interest, 1-chloro-2-(2-chloroethyl)-3-methyl-
pyrido[1,2-a]benzimidazole (NSC 649900) (Badawey and
Kappe, 1995), 1-(4-fluorophenylamino)-3-phenylpyrido[1,2-a]
benzimidazole (NSC 699944), 4-(4-fluorophenylamino)-
2,3-dihydro-1H-cyclopenta[40,50:2,3]pyrido[1,2-a]benzimidazole
(NSC 682011) (EL-Hawash et al., 1999) (Fig. 1) displayed good
sensitivity and selectivity against leukemia cell lines in vitro.
The above-mentioned results prompted us to continue
our investigation on benzimidazole in order to achieve new
lead compounds for future development as antitumor. In
this context, new series of pyrido[1,2-a]benzimidazole
having 5-chloro or 5-underivatized carboxylic acid group
together with 3-methyl or 3-phenyl were synthesized and
their antitumor activity was evaluated.
ucts were subjected to in vitro anticancer screening that
revealed that all the tested compounds exhibited antitumor
activity against human breast adenocarcinoma (MCF7) cell
line, with IC50’s 3.43–14.70 lg/ml.
Keywords 4-Cyano-1-oxo-pyrido[1,2-a]benzimidazoles ꢀ
1-Chloro-4-cyanopyrido[1,2-a]benzimidazoles ꢀ
4-Cyano-1-(4-(2-fluorophenyl)piperazin-yl)-3-
methylpyrido[1,2-a]benzimidazoles ꢀ Anticancer
Introduction
As a part of our ongoing research program on benzimid-
azole derivatives, which may serve as leads for designing
novel chemotherapeutic agents, we have recently reported
the synthesis, anticancer evaluation of various series of
2-substituted benzimidazole bearing a chloro or carboxylic
acid at the 5 position of the benzimidazole ring (Refaat,
H. M. Refaat (&)
Department of Organic Chemistry, Faculty of Pharmacy,
Cairo University, Cairo 11562, Egypt
e-mail: hanan-refaat@hotmail.com
It is worth mentioning that pyrido[1,2-a]benzimidazole
ring system was recently reported as centrally active
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N
8-Chloro-4-cyano-3-methyl-1-oxo-1H, 5H-pyrido
CN
[1,2-a]benzimidazole (3)
CH3
N
Yield: 82%; mp: 230–232°C; IR (cm-1): 3200–2660, 2212
1
(C:N), 1660 (C=O); H-NMR (DMSO-d6, d ppm): 2.35
Cl
Cl
(s, 3H, CH3), 5.90 (s, 1H, C2-H), 7.31 (d, 1H, J = 8.1 Hz,
C6-H), 7.63 (d, 1H, J = 8.1 Hz, C7-H), 8.49 (s, 1H, C9-H),
13.62 (brs, 1H, NH, D2O exchangeable); Anal. Calcd. for
C13H8ClN3O (257.67): C, 60.59, H, 3.12, N, 16.30. Found:
C, 60.61, H, 3.15, N, 16.15.
NSC 649900
N
CN
C6H5
N
F
NH
4-Cyano-3-methyl-1-oxo-1H, 5H-pyrido
[1,2-a]benzimidazole-8-carboxylic acid (4)
NSC 699944
Yield: 70%; mp: 278–280°C; IR (cm-1): 3340–2660, 2210
(C:N), 1662, 1654 (2C=O); 1H-NMR (DMSO-d6, d ppm):
2.33 (s, 3H, CH3), 5.90 (s, 1H, C2-H), 7.84 (d, 1H,
J = 7.5 Hz, C6-H), 8.09 (d, 1H, J = 7.5 Hz, C7-H), 8.35
(s, 1H, C9-H), 12.20–13.42 (brs, 2H, NH and COOH, D2O
exchangeable); Anal. Calcd. for C14H9N3O3 (267.23): C,
62.92, H, 3.39, N, 15.72. Found: C, 62.86, H, 3.42, N,
15.62.
N
CN
N
F
NH
NSC 682011
Fig. 1 Structures of potent anticancer pyrido[1,2-a]benzimidazoles
General procedure for the preparation of compounds 5
and 6
antagonists (Wu et al., 2006; Lazareno et al., 2002; Jordan
et al., 2002; Reitz et al., 2004).
Substituted 2-cyanomethyl benzimidazoles (1 or 2)
(10 mmol), ammonium acetate (1.54 g, 20 mmol) and
ethyl benzoylacetate (1.9 g, 10 mmol) were heated at
170–180°C for 15 h. The residue was broken up and
extracted with ether. The residue was crystallized from
dimethylformamide-water.
Materials and methods
Chemistry
Melting points were obtained on a Griffin apparatus and are
uncorrected. Microanalyses for C, H, and N were carried
out at the microanalytical center, Cairo University. IR
spectra were recorded on a Shimadzu 435 spectrometer,
8-Chloro-4-cyano-3-phenyl-1-oxo-1H, 5H-pyrido
[1,2-a]benzimidazole (5)
Yield: 68%; mp: 238–240°C; IR (cm-1): 3190–3066, 2210
(C:N), 1666 (C=O); 1H-NMR (DMSO-d6, d ppm): 6.1 (s,
1H, C2-H), 7.33 (d, 1H, J = 8.4 Hz, C6-H), 7.40–7.53 (m,
5H, Ar-H), 7.69 (d, 1H, J = 8.4 Hz, C7-H), 7.90 (s, 1H,
C9-H), 13.62 (brs, 1H, NH, D2O exchangeable); Anal.
Calcd. for C18H10ClN3O (319.74): C, 67.61, H, 3.15, N,
13.14. Found: C, 67.75, H, 3.18, N, 13.22.
1
using KBr discs. H-NMR spectra were performed on a
Varian Mercury-300 (300 MHz) spectrometer, using TMS
as the internal standard. Mass spectra were recorded on a
GCMP-QP1000 EX Mass spectrometer. Progress of the
reactions was monitored by TLC using precoated alumi-
num sheet silica gel MERCK 60F 254 and was visualized
by UV lamp.
4-Cyano-3-phenyl-1-oxo-1H, 5H-pyrido
General procedure for the preparation of compounds 3
and 4
[1,2-a]benzimidazole-8-carboxylic acid (6)
Yield: 91%; mp: [300°C; IR (cm-1): 3400–3066, 2208
(C:N), 1666, 1654 (2C=O); 1H-NMR (DMSO-d6, d ppm):
5.95 (s, 1H, C2-H), 7.40–7.67 (m, 5H, Ar-H), 7.81 (d, 1H,
J = 8.2 Hz, C6-H), 7.96 (d, 1H, J = 8.2 Hz, C7-H), 8.12
(s, 1H, C9-H), 12.61–13.02 (brs, 2H, NH, and COOH, D2O
exchangeable); Anal. Calcd. for C19H11N3O3 (329.30): C,
Substituted 2-cyanomethyl benzimidazoles (1 or 2) (10 mmol),
ammonium acetate (1.54 g, 20 mmol) and ethyl acetoace-
tate (1.3 g, 10 mmol) were heated at 170–180°C for 7 h.
The residue was broken up and extracted with ether. The
residue was crystallized from dimethylformamide-water.
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1255
69.29, H, 3.36, N, 12.76. Found: C, 69.41, H, 3.31, N,
12.84.
(50 ml). The residue was filtered, and crystallized from
dimethylformamide-water.
General procedure for preparation of compounds 7–10
8-Chloro-4-cyano-1-(4-(2-fluorophenyl)piperazin-yl)-
3-methylpyrido[1,2-a]benzimidazole (11)
The desired compounds 3–6 (10 mmol) were refluxed with
phosphorus oxychloride (15 ml) for 3 h. The excess phos-
phorus oxychloride was removed under vacuum, and the
residue was treated with ice-water (50 ml). The residue was
filtered, and crystallized from dimethylformamide-water.
Yield: 76%; mp: 267–269°C; IR (cm-1): 2222 (C:N);
1H-NMR (DMSO-d6, d ppm): 2.49 (s, 3H, CH3), 2.95 (t, 2H,
J = 5.4 Hz, piperazine-H), 2.99 (t, 2H, J = 5.4 Hz, piper-
azine-H), 3.52 (t, 4H, J = 5.5 Hz, piperazine-H), 6.81 (s,
1H, C2-H), 7.01–7.17 (m, 3H, Ar-H), 7.31 (d, 1H, C6-H),
7.67 (d, 1H, J = 7.8 Hz, C7-H), 7.94 (s, 1H, Ar-H), 8.06 (s,
1H, C9-H); MS: m/z 419.9 (M?, 100%), 422.0 (M ? 2,
32.8%); Anal. Calcd. for C23H19ClFN5 (419.89): C, 65.79,
H, 4.56, N, 16.68. Found: C, 65.63, H, 4.62, N, 16.79.
1,8-Dichloro-4-cyano-3-methylpyrido
[1,2-a]benzimidazole (7)
Yield: 88%; mp: 279–281°C; IR (cm-1): 2229 (C:N);
1H-NMR (DMSO-d6, d ppm): 2.35 (s, 3H, CH3), 7.51–7.63
(m, 3H, Ar-H), 8.50 (s, 1H, C9-H); Anal. Calcd. for
C13H7Cl2N3 (276.12): C, 56.54, H, 2.55, N, 15.21. Found:
C, 56.43, H, 2.61, N, 15.32.
4-Cyano-1-(4-(2-fluorophenyl)piperazin-yl)-3-
methylpyrido[1,2-a]benzimidazole-8-carboxylic
acid (12)
1-Chloro-4-cyano-3-methylpyrido
Yield: 79%; mp: 236–238°C; IR (cm-1): 2210 (C:N),
1690 (C=O); H-NMR (DMSO-d6, d ppm): 2.43 (s, 3H,
1
[1,2-a]benzimidazole-8-carboxylic acid (8)
CH3), 2.91 (t, 4H, J = 6.1 Hz, piperazine-H), 3.49 (t, 4H,
J = 6.1 Hz, piperazine-H), 6.89 (s, 1H, C2-H), 6.94–7.12
(m, 3H, Ar-H), 7.36 (d, 1H, C6-H), 7.71 (d, 1H, C7-H), 7.94
(s, 1H, Ar-H), 8.01 (s, 1H, C9-H), 13.30 (brs, 1H, COOH,
D2O exchangeable); MS: m/z 429.0 (M?, 0.053%); Anal.
Calcd. for C24H20FN5O2 (429.43): C, 67.12, H, 4.69, N,
16.30. Found: C, 67.23, H, 4.62, N, 16.47.
Yield: 75%; mp: 285–287°C; IR (cm-1): 2223 (C:N),
1690 (C=O); H-NMR (DMSO-d6, d ppm): 2.35 (s, 3H,
CH3), 7.51–7.63 (m, 3H, Ar-H), 8.61 (s, 1H, C9-H); Anal.
Calcd. for C14H8ClN3O2 (285.68): C, 58.85, H, 2.82, N,
14.70. Found: C, 58.97, H, 2.89, N, 14.64.
1
1,8-Dichloro-4-cyano-3-phenylpyrido
[1,2-a]benzimidazole (9)
8-Chloro-4-cyano-1-(4-(2-fluorophenyl)piperazin-yl)-
3-phenylpyrido[1,2-a]benzimidazole (13)
Yield: 75%; mp: 262–264°C; IR (cm-1): 2225(C:N);
1H-NMR (DMSO-d6, d ppm): 7.46–7.58 (m, 5H, Ar-H),
7.60–7.85 (m, 3H, Ar-H), 8.02 (s, 1H, C9-H); Anal. Calcd.
for C18H9Cl2N3 (338.19): C, 63.92, H, 2.68, N, 12.42.
Found: C, 64.02, H, 2.74, N, 12.49.
Yield: 73%; mp: 288–290°C; IR (cm-1): 2212 (C:N);
1H-NMR (DMSO-d6, d ppm): 2.98 (m, 4H, piperazine-H),
3.59 (m, 4H, piperazine-H), 6.81 (s, 1H, C2-H), 7.00–7.17
(m, 3H, Ar-H), 7.31 (d, 1H, C6-H), 7.49–7.52 (m, 5H, Ar-
H), 7.61 (d, 1H, C7-H), 7.91 (s, 1H, Ar-H), 7.94 (s, 1H, C9-
H); MS: m/z 482.0 (M?, 44.8%), 483.9 (M ? 2, 13.6%);
Anal. Calcd. for C28H21Cl FN5 (481.94): C, 69.78, H, 4.39,
N, 14.53. Found: C, 69.65, H, 4.48, N, 14.65.
1-Chloro 4-cyano-3-phenylpyrido
[1,2-a]benzimidazole-8-carboxylic acid (10)
Yield: 80%; mp: [300°C; IR (cm-1): 2225(C:N), 1698
1
(C=O); H-NMR (DMSO-d6, d ppm): 7.48–7.60 (m, 5H,
4-Cyano-1-(4-(2-fluorophenyl)piperazin-yl)-3-
phenylpyrido[1,2-a]benzimidazole-8-carboxylic
acid (14)
Ar-H), 7.61–7.92 (m, 3H, Ar-H), 8.60 (s, 1H, C9-H); Anal.
Calcd. for C19H10ClN3O2 (347.75): C, 65.62, H, 2.89, N,
12.08. Found: C, 65.75, H, 2.95, N, 12.14.
Yield: 82%; mp: 296–298°C; IR (cm-1): 2224 (C:N),
1
General procedure for preparation of compounds 11–14
1678 (C=O); H-NMR (DMSO-d6, d ppm): 2.88 (t, 4H,
J = 6.5 Hz, piperazine-H), 3.53 (m, 4H, J = 6.5 Hz,
piperazine-H), 7.89–7.10 (m, 4H, C2-H and Ar-H),
7.48–7.59 (m, 5H, Ar-H), 7.79–7.89 (m, 2H, C6-H and C7-
H), 8.10 (s, 1H, Ar-H), 8.41 (s, 1H, C9-H), 13.15 (brs, 1H,
COOH, D2O exchangeable); MS: m/z 491.3 (M?, 36.6%);
A mixture of the appropriate compound 7–10 (10 mmol),
4-(2-fluorophenyl) piperazine (1.8 g, 10 mmol) in dimeth-
ylformamide (10 ml) was stirred at room temperature for
72 h. The reaction mixture was poured onto ice-water
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Med Chem Res (2012) 21:1253–1260
Anal. Calcd. for C29H22FN5O2 (491.50): C, 70.86, H, 4.51,
N, 14.24. Found: C, 70.01, H, 4.49, N, 14.15.
(s, 1H, C9-H); Anal. Calcd. for C15H10Cl3N3 (338.61): C,
53.20, H, 2.97, N, 12.40. Found: C, 53.33, H, 3.05, N,
12.35.
General procedure for the preparation of compounds 15
and 16
1-Chloro-4-cyano-2-(2-chloroethyl)-3- methyl
pyrido[1,2-a]benzimidazole-8-carboxylic acid (18)
Substituted 2-cyanomethyl benzimidazoles (1 or 2) (10 mmol),
ammonium acetate (1.54 g, 20 mmol) and 2-acetyl butyro-
lactone (1.3 g, 10 mmol) were heated at 170–180°C for
24 h. The residue was broken up and extracted with ether.
The residue was crystallized from dimethylformamide-
water.
Yield: 93%; mp: 284–286°C; IR (cm-1): 2220 (C:N),
1
1685 (C=O); H-NMR (DMSO-d6, d ppm): 2.49 (s, 3H,
CH3), 2.78 (m, 2H, CH2), 3.06 (m, 2H, CH2), 7.69 (d, 1H,
J = 8.1 Hz, C6-H), 8.01 (d, 1H, J = 8.1 Hz, C7-H), 8.32
(s, 1H, C9-H), 13.34 (brs, 1H, COOH, D2O exchangeable);
Anal. Calcd. for C16H11Cl2N3O2 (348.18): C, 55.19, H,
3.18, N, 12.06. Found: C, 55.28, H, 3.25, N, 12.15.
8-Chloro-4-cyano-2-(2-hydroxyethyl)-3- methyl-1-oxo-
1H, 5H-pyrido[1,2-a]benzimidazole (15)
Yield: 68%; mp: 238–240°C; IR (cm-1): 2210 (C:N), 1662
(C=O); H-NMR (CF3COOD, d ppm): 2.14 (s, 3H, CH3),
General procedure for preparation of compounds 19–20
1
The same procedure as described for 11–14 using 4-(2-
fluorophenyl) piperazine (3.6 g, 20 mmol).
2.38 (m, 2H,CH2), 3.08 (m, 2H,CH2), 4.56 (s, 1H,OH), 7.30
(d, 1H, J = 8.8 Hz, C6-H), 7.64 (d, 1H, J = 8.9 Hz, C7-H),
8.11 (s, 1H, C9-H), 12.96 (brs, 1H, NH, D2O exchangeable);
Anal. Calcd. for C15H12ClN3O2 (301.72): C, 59.71, H, 4.00,
N, 13.92. Found: C, 59.89, H, 4.05, N, 14.05.
8-Chloro-4-cyano-1-(4-(2-fluorophenyl)piperazin-yl)-
2-[2-(4-(2-fluorophenyl)piperazin-yl)ethyl]-3-
methylpyrido[1,2-a]benzimidazole (19)
4-Cyano-2-(2-hydroxyethyl)-3-methyl-1-oxo-1H, 5H-
pyrido[1,2-a]benzimidazole-8-carboxylic acid (16)
Yield: 72%; mp: 287–289°C; IR (cm-1): 2216 (C:N); 1H-
NMR (CF3COOD, d ppm): 2.44 (s, 3H, CH3), 2.73 (m, 2H,
CH2), 2.93 (t, 4H, J = 5.1 Hz, piperazine-H), 2.98 (t, 4H,
J = 5.1 Hz, piperazine-H), 3.16 (m, 2H, CH2), 3.44 (t, 4H,
J = 5.1 Hz, piperazine-H), 3.52 (t, 4H, J = 5.1 Hz,
piperazine-H), 6.90–7.05 (m, 6H, Ar-H), 7.53–7.68 (m, 2H,
C6-H and C7-H), 7.81–7.83 (m, 2H, Ar-H), 7.94 (s, 1H, C9-
H); MS: m/z 626.2 (M?, 18.65%), 628.3 (M ? 2, 6.87%);
Anal. Calcd. for C35H34ClF2N7 (626.12): C, 67.13, H, 5.47,
N, 15.65. Found: C, 67.25, H, 5.55, N, 15.79.
Yield: 93%; mp: [300°C; IR (cm-1): 3400–3066, 2208
(C:N), 1666, 1654 (2C=O); 1H-NMR (DMSO-d6, d ppm):
1.90 (s, 3H, CH3), 2.30 (t, 2H, J = 5.2 Hz, CH2), 2.74 (t,
2H, J = 5.2 Hz, CH2), 4.55 (brs, 1H, OH), 7.67 (d, 1H,
J = 8.6 Hz, C6-H), 8.07 (d, 1H, J = 8.6 Hz, C7-H), 8.28
(s, 1H, C9-H), 12.67–13.54 (brs, 2H, NH and COOH, D2O
exchangeable); Anal. Calcd. for C16H13N3O4 (311.29): C,
61.73, H, 4.20, N, 13.49. Found: C, 61.61, H, 4.05, N,
13.35.
General procedure for preparation of compounds 17
and 18
4-Cyano-1-(4-(2-fluorophenyl)piperazin-yl)-2-[2-(4-(2-
fluorophenyl)piperazin-yl)ethyl]-3-methyl pyrido[1,2-
a]benzimidazole-8-carboxylic acid (20)
The desired compound 15 or 16 (10 mmol)was refluxed with
phosphorus oxychloride (15 ml) for 3 h. The excess phos-
phorus oxychloride was removed under vacuum, and the
residue was treated with ice-water (50 ml). The residue was
filtered, and crystallized from dimethylformamide-water.
Yield: 72%; mp: [300°C; IR (cm-1): 2216 (C:N), 1698
(C=O); H-NMR (CF3COOD, d ppm): 2.46 (s, 3H, CH3),
1
2.72 (m, 2H, CH2), 2.92(t, 4H, J = 5.4 Hz, piperazine-H),
2.98 (t, 4H, J = 5.4 Hz, piperazine-H), 3.15 (m, 2H, CH2),
3.44 (t, 4H, J = 5.4 Hz, piperazine-H), 3.52 (t, 4H,
J = 5.4 Hz, piperazine-H), 7.00–7.16 (m, 6H, Ar-H),
7.58–7.81 (m, 2H, C6-H and C7-H), 7.93–8.05 (m, 2H, Ar-
H), 8.10 (s, 1H, C9-H), 13.25 (brs, 1H, COOH, D2O
exchangeable); MS: m/z 635.8 (M?, 41.89%); Anal. Calcd.
for C36H35F2N7O2 (635.68): C, 68.01, H, 5.54, N, 15.42.
Found: C, 68.27, H, 5.39, N, 15.59.
1,8-Dichloro-4-cyano-2-(2-chloroethyl)-3-
methylpyrido[1,2-a]benzimidazole (17)
Yield: 87%; mp: 225–227°C; IR (cm-1): 2216 (C:N); 1H-
NMR (DMSO-d6, d ppm): 2.48 (s, 3H, CH3), 2.89 (m, 2H,
CH2), 3.20 (m, 2H, CH2), 7.59–7.81 (m, 2H, Ar-H), 8.10
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Med Chem Res (2012) 21:1253–1260
Cytotoxic activity studies
1257
1H-NMR and IR spectra. The H-NMR spectra of com-
1
pounds 3–6 showed a sharp singlet at d 5.90–6.10 ppm due to
C2 proton, whereas the H-NMR spectra of compounds 15
1
Anticancer activity studies were done at Cairo University,
National Cancer Institute, Cancer Biology Department,
Pharmacology Unit.
and 16 displayed two multiplet at d 2.30–2.38 and
2.74–4.08 ppm. On the other hand, the IR spectra of com-
pounds 3–6, 15 and 16 showed the characteristic C=O
stretching absorption.
Compounds 3–20 were tested at concentrations between
1 and 10 lg/ml using SulfoRhodamine-B (SRB) assay for
cytotoxic activity against breast carcinoma cell line
(MCF7).
On refluxing 4-cyano-3-substituted-1-oxo-1H, 5H-pyr-
ido[1,2-a]benzimidazole 3–6, 15, 16, with phosphorus oxy-
chloride, the corresponding 1-chloro derivatives 7–10, 17, 18
were produced. The IR spectra of compounds 7–10, 17, and
18 proved as useful in tracing the disappearance of the C=O
Measurement of potential cytotoxicity by SRB assay
Potential cytotoxicity of the compounds was tested using
the method of Skehan et al. (1990) as follows:
H
N
Cells were plated in 96 multiwell plate (104 cells/well)
for 24 h before treatment with the compound(s) to allow
attachment to the wall of the plate. Different concentrations
of the compounds (0, 1, 2.5, 5, and 10 lg/ml) were added
to the cell monolayer triplicate wells were prepared for
each individual dose. Monolayer cells were incubated with
the compound(s) for 48 h at 37°C in atmosphere of 5%
CO2. After 48 h, cells were fixed, washed and stained with
SulfoRhodamine-B stain. Excess stain was washed with
acetic acid and attached stain was recovered with Tris
EDTA buffer. Color intensity was measured in an ELISA
reader.
CN
N
R
1,2
R'COCH2COOC2H5
H
N
CN
R'
N
R
O
3 - 6
The relation between surviving fraction and drug con-
centration is plotted to get the survival curve of each tumor
cell line after the specified compound.
POCl3
N
CN
Statistical analysis
R'
N
R
Differences between different treatment groups were ana-
lyzed using ANOVA followed by Dunnett t-test. P values
of less than 0.05 were considered to represent a significant
difference.
Cl
7 - 10
F
HN
N
Results and discussion
N
CN
Chemistry
R'
N
R
The reaction sequence employed for synthesis of the target
compounds is shown in Schemes 1 and 2. The starting
materials, substituted 2-cyanomethyl benzimidazoles
(1 and 2) were conveniently prepared according to reported
methods (Refaat, 2010). Compounds 1 and 2 were cyclized
to 4-cyano-3-substituted-1-oxo-1H, 5H-pyrido[1,2-a]benz-
imidazoles (3–6, 15, 16), by heating it with ethyl aceto-
acetate, ethyl benzoylacetate or 2-acetyl butyrolactone in
presence of ammonium acetate at 170–180°C. The cycli-
zation of 4-cyano-3-substituted-1-oxo-1H,5H-pyrido[1,2-a]
benzimidazoles (3–6, 15, 16) was evidenced by its
N
N
F
11 - 14
1 R = Cl
3, 7, 11 R= Cl
4, 8, 12 R= COOH , R' = CH3
5, 9, 13 R= Cl , R' = C6H5
6, 10, 14 R= COOH , R' = C6H5
, R' = CH3
2 R = COOH
Scheme 1 Synthesis of compounds 3–14
123

1258
Med Chem Res (2012) 21:1253–1260
H
N
Cytotoxic activity
CN
O
The cytotoxicity of compounds 3–20 was evaluated against
human breast adenocarcinoma cell line (MCF7). For com-
parison purposes, the cytotoxicity of doxorubicin, a standard
antitumor drug, was evaluated under the same conditions.
The IC50 values (dose of the compound which caused a 50%
reduction of survival values) are shown in Table 1. The
results are represented graphically in Figs. 2, 3, 4, 5, 6, and 7.
All the tested compounds were found to possess potential
antitumor activities against all of the tested tumor cell lines,
with IC50’s 3.43–14.70 lg/ml (Table 1). Among the
4-cyano-1-oxo-pyrido[1,2-a]benzimidazole series 3–6, 15,
and 16, compounds 3, 5, and 6 showed significant activity,
compound 16 was moderately potent, whereas compounds 4
and 15 were the least active. This was of particular interest
since all the previously prepared 4-cyano-1-oxo-pyrido
[1,2-a]benzimidazoles and their 6,7-dimethyl analogs were
inactive as anticancer agents (Rida et al., 1988a, b; Badawey
and kappe, 1995, 1999; EL-Hawash et al., 1999). This
observation confirms the importance of the structure
parameters for the benzimidazole ring substituent in deter-
mining the potential antitumor activity.
N
R
1,2
CH3
O
H
N
CN
CH3
N
R
O
OH
15 , 16
POCl3
N
CN
CH3
N
R
Cl
Cl
17 , 18
Table 1 IC50 values of compounds 3–20 against MCF7
F
Compd. no
IC50 (lg/ml)
HN
N
3
3.43 0.03
14.70 0.07
3.58 0.10
3.99 0.04
14.62 0.07
10.55 0.07
8.61 0.03
12.82 0.04
3.89 0.07
3.58 0.07
7.09 0.04
9.07 0.05
5.72 0.01
12.60 0.04
10.40 0.03
17.65 0.07
14.00 0.08
9.90 0.04
2.97 0.03
0.000*
N
CN
CH3
4
5
N
R
6
15
16
7
N
N
F
N
N
8
19 , 20
F
9
10
17
18
11
12
13
14
19
20
DOX
P value
1, 15, 17, 19 R= Cl
2, 16, 18, 20 R= COOH
Scheme 2 Synthesis of compounds 15–20
stretching absorption of the parent compounds 3–6, 15, and
16.
On the other hand, the synthesis of 4-cyano-1-(4-(2-fluoro-
phenyl)piperazin-yl)-3-methylpyrido[1,2-a]benzimidazoles
11–14 and 4-cyano-1-(4-(2-fluorophenyl) piperazin-yl)-2-
[2-(4-(2-fluorophenyl)piperazin-yl)ethyl]-3-methylpyrido[1,2-
a]benzimidazoles 19 and 20 was achieved via the reaction of
the chloro derivatives 7–10, 17, 18 with 4-(2-flourophenyl)
IC50 (lg/ml): dose of the compound which caused a 50% reduction of
survival. Values were calculated from dose-response curves done in
triplicate for each compound. Values were given standard devia-
tion. MCF7 human breast adenocarcinoma, Dox Doxorubicin
1
piperazine. The H-NMR and mass spectra were consistent
* There is a significant difference by using one way ANOVA at
P \ 0.05
with the proposed structures.
123

Med Chem Res (2012) 21:1253–1260
1259
Fig. 2 Cytotoxicity of 3–6, 15, 16, and doxorubicin against human
breast adenocarcinoma cell line (MCF7)
Fig. 5 IC50 values of compounds 3–6, 15, and 16 against MCF7
Fig. 3 Cytotoxicity of 7–10, 17, 18, and doxorubicin against human
breast adenocarcinoma cell line (MCF7)
Fig. 6 IC50 values of compounds 7–10, 17, and 18 against MCF7
Fig. 4 Cytotoxicity of 11–14, 19, 20, and doxorubicin against human
breast adenocarcinoma cell line (MCF7)
Fig. 7 IC50 values of compounds 11–14, 19, and 20 against MCF7
On the other hand, in the 1-(4-(2-fluorophenyl)pipera-
zin-yl)pyrido[1,2-a]benzimidazole 11–14 and 1-(4-(2-fluo-
rophenyl)piperazin-yl)-2-[2-(4-(2-fluorophenyl)piperazin-yl)
ethyl]pyrido[1,2-a]benzimidazole 19, 20 series, the most
Within the 1-chloro-4-cyano-pyrido[1,2-a]benzimid-
azole 7–10 and the 1,8-dichloro 17, 18 series, compounds 9
and10 displayed a notable potency while compounds 17, 7,
18, and 8 were moderately potent.
123

1260
Med Chem Res (2012) 21:1253–1260
The pyrido[10,20:1,2]imidazo[4,5-h]quinazolines. Chem Pharm
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EL-Hawash SAM, Badawey EAM, Kappe T (1999) Benzimidazole
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pronounced activity was recorded for compound 11, while
compounds 20, 13, and 12 were moderately active and
compounds 19 and 14 were the least active. Apparently, the
activity exhibited by compounds 11–14, 19, and 20 was not
improved by the incorporation of 2-fluorophenylpiperazinyl
moiety, as estimated, however, they showed lower activity
than their precursor 1-oxo and 1-chloro derivatives.
Conclusion
The present research study reports the successful synthesis,
antitumor study of two new series of pyrido[1,2-a]benz-
imidazoles derivatives. Their screening results revealed
that all the compounds showed moderate to very good
activities against human breast adenocarcinoma (MCF7)
cell line. On this basis, it can be concluded that a combi-
nation of 5-chloro or 5-underivatized carboxylic acid group
to pyrido[1,2-a]benzimidazoles core showed an increased
antitumor activity and hence they are ideally suited for
further modifications to obtain more effective antitumor
compounds.
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