Design, synthesis and evaluation of novel benzimidazoles, benzothiazoles and benzofurans incorporating pyrazole moiety as antiangiogenic agents

Article abstract of DOI:10.1055/s-0031-1295483

Novel benzimidazoles, benzothiazoles and benzofurans incorporating pyrazole moiety have been synthesized and screened for their antiangogenic activities, by testing their ability to inhibit human umbilical vein endothelial cell (HUVEC) proliferation, cord formation and migration in response to chemoattractant. 3 compounds 19, 23 and 26 showed antiangiogenic activities at non-cytotoxic concentrations. Compound 19 was the most active with chemotaxis activity data nearly comparable to that of the positive control, TNP-470. Compound 42 showed a significant cytotoxic effect on the tested cancer cell lines and less antiangiogenesis activity compared to compounds 19, 23 and 26. All the tested compounds, in contrary to TNP-470, interfered with the migratory function of HUVECs in response to vascular endothelial growth factor rather than the endothelial cells proliferation or cord formation. Moreover, a docked pose of compounds 19 and 26 was obtained bound to kinase insert domain receptor using Molecular Operating Environment module. Georg Thieme Verlag KG · Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1295483

Original Article 63
Design, Synthesis and Evaluation of Novel
Benzimidazoles, Benzothiazoles and Benzofurans
Incorporating Pyrazole Moiety as Antiangiogenic
Agents
Authors
S. M. Rida1, A. M. Youssef1, M. H. Badr1, A. Malki2, Z. A. Sherif3, A. S. Sultan2,3
Affiliations
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
2 Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
3 Department of Oncology, Georgetown University Medical Center, Washington DC, USA
Key words
Abstract
▼
positive control, TNP-470. Compound 42 showed
▶
● Benzimidazole
a significant cytotoxic effect on the tested can-
cer cell lines and less antiangiogenesis activity
compared to compounds 19, 23 and 26. All the
tested compounds, in contrary to TNP-470, inter-
fered with the migratory function of HUVECs in
response to vascular endothelial growth factor
rather than the endothelial cells proliferation or
cord formation. Moreover, a docked pose of com-
pounds 19 and 26 was obtained bound to kinase
insert domain receptor using Molecular Operat-
ing Environment module.
▶
● benzothiazole
Novel benzimidazoles, benzothiazoles and
benzofurans incorporating pyrazole moiety
have been synthesized and screened for their
antiangogenic activities, by testing their abil-
ity to inhibit human umbilical vein endothelial
cell (HUVEC) proliferation, cord formation and
migration in response to chemoattractant. 3
compounds 19, 23 and 26 showed antiangiogenic
activities at non-cytotoxic concentrations. Com-
pound 19 was the most active with chemotaxis
activity data nearly comparable to that of the
▶
● benzofuran and pyrazole
derivatives
▶
● human umbilical vein
endothelial cell (HUVEC)
▶
● vascular endothelial growth
factor receptor (VEGF-2)
▶
● Antiangiogenic activity
▶
● Antiproliferative effect
received 12.08.2011
accepted 07.11.2011
Bibliography
Introduction
▼
factors and cytokines [12,13]. Until recently,
investigations in this area have focused on VEGF
and its receptor tyrosin kinase VEGFR-2 (or KDR,
kinase insert domain receptor) [14,15]. Thus inhi-
bition of the VEGF signaling pathway has emerged
as one of the most valuable new approaches in the
treatment of cancers as approximately 60% of
malignant tumors express high concentrations of
VEGF [16]. VEGF is perhaps the most specific for
endothelial cells. When VEGF binds to its recep-
tor it triggers signaling pathways that result in
endothelial cell migration, differentiation and
proliferation, increased vascular permeability
and release of endothelial cell precursors from
the bone marrow, VEGF also prevents endothelial
cell apoptosis [17].
Strategies to inhibit the VEGF pathway include
antibodies directed against VEGF or VEGFR, solu-
ble VEGFR/VEGFR hybrids, soluble analogues of
the VEGFR (VEGF-Trap) and tyrosine kinase
inhibitors. One of the earliest strategies to inhibit
VEGF activity involved the use of antibodies
directed against VEGFRs. For example, preclinical
data with anti-VEGFR-2 antibodies demonstrated
decreased VEGF-induced signaling, decreased
angiogenesis and decreased primary and meta-
static growth in a variety of tumor systems [18].
DOI http://dx.doi.org/
10.1055/s-0031-1295483
Arzneimittelforschung 2012;
62: 63–74
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Angiogenesis, the formation of new capillaries
from existing vasculature, is a normal physiologi-
cal event that occurs during embryonic growth,
wound healing and the menstrual cycle. Abnor-
mal regulation of angiogenesis has been found to
be involved in the pathogenesis of several disor-
ders including diabetic retinopathy [1], inflam-
mation [2], rheumatoid arthritis [3], psoriasis [4],
age-related macular degeneration [5], and cancer
[6,7].
It has been well recognized that angiogenesis is
essential for solid tumor growth and metastasis
[8,9]. Tumors that lack an adequate vasculature
become necrotic or apoptotic and do not grow
beyond a limited size. Thus, new means to retard
angiogenesis have shown promise as potential
cancer therapies. Antiangiogenic therapy, which
targets activated endothelial cells, presents sev-
eral advantages over therapy directed against
tumor cells; it targets genetically stable, normal
endothelial cells that should not develop resist-
ance [10,11].
Correspondence
Amal M. Youssef
Associate Prof
Department of Pharmaceutical
Chemistry
Faculty of Pharmacy
Alexandria University
1 Elkhartoum square-Azarita
21521 Alexandria
Egypt
Tel.: +2/(03)/4871 317
Fax: +2/(03)/4873 273
ayoussef65@yahoo.com
Ahmed S. Sultan
Associate Prof
Department of Biochemistry
Faculty of Science
Alexandria University
Alexandria
Angiogenesis is regulated by the expression of a
variety of growth factors including vascular
endothelial growth factor (VEGF), other growth
Egypt
dr_asultan@WHO.net
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

64 Original Article
On the other hand, the importance of COX-2 in carcinogenesis
and brain tumor progression is highlighted by the detection of
COX-2 in brain tumor and COX-2 overexpression in gliomas
associated with poor prognosis. Targeting COX-2 with selective
COX inhibitors as celecoxib has proven effective to reduce human
glioblastoma cell viability in vivo [19]. Celecoxib is the only drug
approved by FDA for adjuvant treatment of patients with familial
adenomatous polyposis [20]. Several mechanisms have been
proposed in various tumor models, celecoxib activates apoptotic
protein P53 leading to antiproliferative effect and tumor angio-
genesis [21].
The target compounds incorporate in their molecules the phar-
macophores that are believed to be responsible for the biological
significance of some relevant chemotherapeutic agents such as
the trisubstituted pyrazole functionality as in angiogenic COX-2
inhibitor; celecoxib. The substitution pattern of the pyrazole
ring was carefully selected so as to confer different electronic
environments to the molecules. So we report herein the synthe-
sis and the in vitro antiangiogenic activity of some novel struc-
ture hybrids incorporating both the benzimidazole moiety or its
bioiostere with the pyrazole ring through different linkages. In
the first scheme, the benzimidazole or benzothiazole scaffold is
linked with substituted pyrazole through a vinyl 2 carbon bridge
or through azavinyl linker, while in the second scheme the ben-
zofuran ring is directly attached to the pyrazole moiety through
a single bond. This combination was suggested in an attempt to
investigate the influence of such hybridization and structure
variation on the anticipated biological activity hoping to obtain
synergestic chemotherapeutic activity with higher selectivity
and less toxicity.
According to a scientific report, a novel 1,3,4- trisubstituted
▶
pyrazole derivative a ( ● Fig. 1) were found to exhibit significant
antiangiogenic profile at non-cytotoxic doses and was superior
to celecoxib [22].
Careful literature survey revealed that a 2-aminobenzimidazole
▶
derivative b ( ● Fig. 1) was identified as potent inhibitor of
VEGFR-2. The modeling study proved that the benzimidazole
core is oriented in the hydrophobic pocket neighboring the ATP
binding site [23].
In a recent approach, some novel benzimidazole urea derivatives
▶
c ( ● Fig. 1) were discovered as potent inhibitors of TIE-2 (tyro-
Materials and Methods
sine kinase containing Ig and EGF homology domains) and
▼
VEGFR-2 which are implicated in angiogenesis [24].
Chemistry
▶
Moreover, novel benzimidazole derivatives d ( ● Fig. 1) with
Melting points were determined in open glass capillaries on a
Gallenkamp melting point apparatus and were uncorrected. The
infrared (IR) spectra were recorded on Perkin-Elmer 1430 infra-
unique antiangiogenic characteristics have been reported where
they inhibited VEGF [25].
Furthermore, a 2-arylimidazo[2,1-b][1,3]benzothiazole deriva-
1
red spectrophotometer using the KBr plate technique. H-NMR
▶
tive e ( ● Fig. 1) was reported as a selective growth inhibitor of
spectra were determined on Jeol spectrometer (500MHz) at the
Microanalytical unit, Faculty of Science, Alexandria University
and on a Varian spectrometer (300MHz), Faculty of Science,
Cairo University using tetramethylsilane (TMS) as the internal
standard and DMSO-d6 as the solvent (Chemical shifts in δ,
endothelial cells [26].
In view of the above mentioned findings, the aim of the present
study is to synthesize and investigate the in vitro antiangiogenic
activity of some novel benzimidazoles and their bioisosteres.
Fig. 1 Structure of lead compounds a–e.
a
b
c
d
e
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

Original Article 65
ppm). Splitting patterns were designated as follows: s: singlet;
d: doublet; m: multiplet. MS were run on a Finnigan mass spec-
trometer model SSQ/7000 (70eV, Thermo Electron Corporation).
Microanalyses were performed at the Microanalytical Unit, Fac-
ulty of Science, Cairo University, Egypt and the found values
were within±0.4% of the theoretical values. Follow-up of the
reactions and checking the homogeneity of the compounds
were made by TLC on silica gel-protected glass plates and the
spots were detected by exposure to UV-lamp at λ 254.
ane/EtOH). IR (cm−1): 3229 (NH); 2226 (C≡N); 1630 (C=N).
1H-NMR (δ ppm): 7.19–7.22 (m, 2H, benzimidazole-C5,6-H);
7.41–7.63 (m, 7H, benzimidazole-C4,7-H and phenyl-H); 7.72 (d,
J=8.4Hz, 2H, p-chlorophenyl-C2,6-H); 7.91 (d, J=8.4Hz, 2H,
p-chlorophenyl-C3,5-H); 8.09 (s, 1H, =CH); 9.19 (s, 1H, pyrazole-
C5-H); 13.10 (s, 1H, NH, D2O exchangeable). Anal. Calcd for
C25H16ClN5 (421.88): C, 71.17; H, 3.82; N, 16.60. Found: C, 70.88;
H, 3.62; N, 16.56.
2-(1H-Benzimidazol-2-yl)-3-(3-(4-bromophenyl)-1-phenyl-1H-
3-(4-Methylphenyl)-1-(4-nitrophenyl)-1H-pyrazole-4-
carbaldehyde (14)
pyrazol-4-yl)acrylonitrile (20). Yield: 69%, mp:>300°C (diox-
1
ane). IR (cm−1): 3264 (NH); 2243 (C≡N); 1618 (C=N). H-NMR
To an ice-cold dimethylformamide (25g, 30ml, 0.34mole), POCl3
(10g, 6ml, 0.066° mole) was added dropwise with stirring over
a period of 30min. Stirring was continued for further 1h keeping
the reaction temperature at 0°C. Compound 6 [27] (8.1g,
0.03mole) was then added and the reaction mixture was heated
at 60–70°C with stirring for 4h, allowed to cool and poured onto
ice-water mixture. The obtained mixture was boiled and the
obtained precipitate was filtered, washed with water, dried and
crystallized from dioxane. Yield: 76%, mp: 181–183°C. IR
(cm−1): 1700 (C=O); 1632 (C=N). 1H-NMR (δ ppm): 1.54 (s, 1H,
CH3); 7.57 (d, J=8.4Hz, 2H, p-tolyl-C3,5-H); 7.83 (d, J=8.4Hz, 2H,
p-tolyl-C2,6-H); 8.24 (d, J=9.2Hz, 2H, p-nitrophenyl-C2,6-H); 8.39
(d, J=9.2Hz, 2H, p-nitrophenyl-C3,5-H); 9.23 (s, 1H, pyrazole-
C5-H); 9.62 (s, 1H, CHO). Anal. Calcd for C17H13N3O3 (307.31): C,
66.44; H, 4.26; N, 13.67. Found: C, 66.76; H, 4.31; N, 13.45.
(δ ppm): 7.21–7.24 (m, 2H, benzimidazole-C5,6-H); 7.50–7.65
(m, 7H, benzimidazole-C4,7-H and phenyl- H); 7.74 (d, J=8.4Hz,
2H, p-bromophenyl-C2,6-H); 7.95 (d, J=8.4Hz, 2H, p-bromophe-
nyl-C3,5-H); 8.13 (s, 1H, =CH); 9.21 (s, 1H, pyrazole-C5-H); 12.88
(s, 1H, NH, D2O exchangeable). Anal. Calcd for C25H16BrN5
(466.33): C, 64.39; H, 3.46; N, 15.02. Found: C, 64.45; H, 3.67; N,
14.77.
2-(1H-Benzimidazol-2-yl)-3-(1-(4-nitrophenyl)-3-phenyl-1H-
pyrazol-4-yl)acrylonitrile (21). Yield: 75%, mp:>300°C (diox-
ane/EtOH). IR (cm−1): 3240 (NH); 2229 (C≡N); 1626 (C=N).
1H-NMR (δ ppm): 7.22–7.25 (m, 2H, benzimidazole-C5,6-H);
7.49–7.62 (m, 5H, benzimidazole-C4,7-H and phenyl-C3,4,5-H);
7.71 (d, J=7.65Hz, 2H, phenyl-C2,6-H); 8.14 (s, 1H, =CH); 8.21
(d, J=9.2Hz, 2H, p-nitrophenyl-C2,6-H); 8.43 (d, J=9.2Hz, 2H,
p-nitrophenyl-C3,5-H); 9.30 (s, 1H, pyrazole-C5-H); 13.02 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C25H16N6O2 (432.43): C,
69.44; H, 3.73; N, 19.43. Found: C, 69.72; H, 3.54; N, 19.67.
2-(1H-Benzimidazol-2-yl)-3-(1,3-diaryl-1H-pyrazol-4-yl)
acrylonitrile (17–28)
To a stirred solution of benzazol-2-ylacetonitrile (0.002mole) in
absolute ethanol (10ml), triethylamine (0.2ml) and the appro-
priate pyrazole aldehyde 9–16 (0.002mole) were added. The
reaction mixture was heated under reflux for 1–3h during which
yellow crystals separated out. The crystalline product was fil-
tered, washed with water, dried and crystallized from the proper
solvent.
2-(1H-Benzimidazol-2-yl)-3-(1-(4-nitrophenyl)-3-p-tolyl-1H-
pyrazol-4-yl)acrylonitrile (22). Yield: 65%, mp:>300°C (diox-
ane/EtOH). IR (cm−1): 3250 (NH); 2251 (C≡N); 1626 (C=N).
1H-NMR (δ ppm): 2.43 (s, 1H, CH3); 7.23–7.26 (m, 2H, benzimi-
dazole-C5,6-H); 7.41 (d, J=7.8Hz, 2H, p-tolyl-C3,5-H); 7.62–7.65
(m, 4H, benzimidazole-C5,6-H and p-tolyl-C2,6-H); 8.14 (s,
1H, =CH); 8.24 (d, J=9.2Hz, 2H, p-nitrophenyl-C2,6-H); 8.45 (d,
J=9.2Hz, 2H, p-nitrophenyl-C3,5-H); 9.40 (s, 1H, pyrazole-C5-H).
Anal. Calcd for C26H18N6O2 (446.46): C, 69.95; H, 4.06; N, 18.82.
Found: C, 70.14; H, 4.09; N, 18.53.
2-(1H-Benzimidazol-2-yl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acry-
lonitrile (17). Yield: 72%, mp:>300°C (EtOH). IR (cm−1): 3300
(NH); 2247 (C≡N); 1609 (C=N). 1H-NMR (δ ppm): 7.18–7.21 (m,
2H, benzimidazole-C5,6-H); 7.45–7.71 (m, 10H, benzimidazole-
C4,7-H and phenyl-H); 7.77 (d, J=7.2Hz, 2H, phenyl-C2,6-H); 7.99
(s, 1H, =CH); 9.29 (s, 1H, pyrazole-C5-H); 12.80 (s, 1H, NH, D2O
exchangeable). MS, m/z (rel. Abund. %): 388 (18.5) M∙++1; 387
(77.8) M∙+; 386 (47.7); 311 (25.9); 310 (100); 283 (11.1); 282
(7.6); 281 (8.8); 180 (7.1); 77 (23.9); 51 (16.0). Anal. Calcd for
C25H17N5 (387.44): C, 77.50; H, 4.42; N, 18.08. Found: C, 77.27;
H, 4.15; N, 17.89.
2-(1H-Benzimidazol-2-yl)-3-(3-(4-chlorophenyl)-1-(4-nitrophenyl)-
1H-pyrazol-4-yl)acrylonitrile (23). Yield: 73%, mp:>300°C (diox-
1
ane). IR (cm−1): 3259 (NH); 2233 (C≡N); 1619 (C=N). H-NMR
(δ ppm): 7.22–7.25 (m, 2H, benzimidazole-C5,6-H); 7.49–7.52
(m, 2H, benzimidazole-C4,7-H); 7.62 (d, J=8.4Hz, 2H, p-chlo-
rophenyl-C2,6-H); 7.89 (d, J=8.4Hz, 2H, p-chlorophenyl-C3,5-H);
8.11 (s, 1H, =CH); 8.22 (d, J=9.2Hz, 2H, p-nitrophenyl-C2,6-H);
8.44 (d, J=9.2Hz, 2H, p-nitrophenyl-C3,5-H); 9.36 (s, 1H, pyra-
zole-C5-H); 12.99 (s, 1H, NH, D2O exchangeable). Anal. Calcd for
C25H15ClN6O2 (466.88): C, 64.31; H, 3.24; N, 18.00. Found: C,
64.63; H, 3.45; N, 18.21.
2-(1H-Benzimidazol-2-yl)-3-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)
acrylonitrile (18). Yield: 86%, mp: >300°C (EtOH). IR (cm−1):
3254(NH); 2231 (C≡N); 1627 (C=N). 1H-NMR (δ ppm): 2.36 (s,
1H, CH3); 7.20–7.23 (m, 2H, benzimidazole-C5,6-H); 7.39 (d,
J=7.4Hz, 2H, p-tolyl-C3,5-H); 7.55–7.62 (m, 9H, benzimidazole-
C4,7-H, phenyl-H and p-tolyl-C2,6-H); 8.12 (s, 1H, =CH); 9.31 (s,
1H, pyrazole-C5-H); 12.91 (s, 1H, NH, D2O exchangeable). Anal.
Calcd for C26H19N5 (401.46): C, 77.79; H, 4.77; N, 17.44. Found: C,
77.52; H, 4.98; N, 17.08.
2-(1H-Benzimidazol-2-yl)-3-(3-(4-bromophenyl)-1-
(4-nitrophenyl)-1H-pyrazol-4-yl)acrylonitrile (24). Yield: 74%,
mp:>300°C (DMF/H2O). IR (cm−1): 3247 (NH); 2227 (C≡N);
1609 (C=N). 1H-NMR (δ ppm): 7.24–7.27 (m, 2H, benzimida-
zole-C5,6-H); 7.50–7.53 (m, 2H, benzimidazole-C4,7-H); 7.66 (d,
J=8.4Hz, 2H, p-bromophenyl-C2,6-H); 7.91 (d, J=8.4Hz, 2H,
p-bromophenyl-C3,5-H); 8.13 (s, 1H, =CH); 8.24 (d, J=9.2Hz, 2H,
p-nitrophenyl-C2,6-H); 8.46 (d, J=9.2Hz, 2H, p-nitrophenyl-C3,5-H);
2-(1H-Benzoimidazol-2-yl)-3-(3-(4-chlorophenyl)-1-phenyl-1H-
pyrazol-4-yl)acrylonitrile (19). Yield: 63%, mp:>300°C (Diox-
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

66 Original Article
9.29 (s, 1H, pyrazole-C5-H); 12.75 (s, 1H, NH, D2O exchangeable).
Anal. Calcd for C25H15BrN6O2 (511.33): C, 58.72; H, 2.96; N,
16.44. Found: C, 59.01; H, 2.79; N, 16.57.
C4,7-H and p-tolyl-C2,6-H); 8.31 (d, J=8.4Hz, 2H, p-nitrophenyl-
C2,6-H); 8.47 (d, J=8.4Hz, 2H, p-nitrophenyl-C3,5-H); 9.43 (s, 1H,
pyrazole-C5-H); 9.82 (s, 1H, N=CH); 12.10 (s, 1H, D2O exchange-
able). Anal. Calcd for C24H18N6O2 (422.44): C, 68.24; H, 4.29; N,
19.89. Found: C, 68.59; H, 4.40; N, 20.07.
2-(Benzothiazol-2-yl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)acryloni-
trile (25). yield: 87%, mp: 170–172°C (dioxane/EtOH). IR
(cm−1): 2237 (C≡N); 1608 (C=N); 1298, 1026 (C-S-C). 1H-NMR
(δ ppm): 7.21–7.24 (m, 3H, phenyl-C3,4,5-H); 7.43–7.61 (m, 9H,
benzothiazole-H and phenyl-H); 7.78 (d, J=7.2Hz, 2H, phenyl-
C2,6-H); 8.45 (s, 1H, =CH); 9.31 (s, 1H, pyrazole-C5-H). Anal.
Calcd for C25H16N4S (404.49): C, 74.23; H, 3.99; N, 13.85; S 7.93.
Found: C, 74.50; H, 4.04; N, 13.56; S 8.22.
N-{[3-(4-Chlorophenyl)-1-(4-nitrophenyl)-1H-pyrazol-4-yl]
methylene}-1H-benzimidazole-2-amine (30). Yield: 59%,
mp:>300°C (dioxane/EtOH). IR (cm−1): 3330 (NH); 1628 (C=N).
1H-NMR (δ ppm): 7.07–7.10 (m, 2H, benzimidazole-C5,6-H);
7.63 (d, J=8.2Hz, 2H, p-chlorophenyl-C2,6-H); 7.79–8.12 (m, 4H,
benzimidazole-C4,7-H and p-chlorophenyl-C3,5-H); 8.26 (d,
J=8.4Hz, 2H, p-nitrophenyl-C2,6-H); 8.38 (d, J=8.4Hz, 2H,
p-nitrophenyl-C3,5-H); 9.55 (s, 1H, pyrazole-C5-H); 9.99 (s, 1H,
N=CH); 11.22 (s, 1H, D2O exchangeable). Anal. Calcd for
C23H15ClN6O2 (442.86): C, 62.38; H, 3.41; N, 18.98. Found: C,
62.49; H, 3.31; N, 18.67.
2-(Benzothiazol-2-yl)-3-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)
acrylonitrile (26). Yield: 78%, mp: 196–198°C (dioxane/
EtOH). IR (cm−1): 2242 (C≡N); 1630 (C=N); 1255, 1085 (C-S-C).
1H-NMR (δ ppm): 2.33 (s, 1H, CH3); 7.35 (d, J=7.4Hz, 2H, p-tolyl-
C3,5-H); 7.53–7.72 (m, 11H, benzothiazole-H, phenyl-H and
p-tolyl-C2,6-H); 8.39 (s, 1H, =CH); 9.28 (s, 1H, pyrazole-C5-H).
MS, m/z (rel. Abund. %): 371 (3.9); 370 (20.4); 369 (35.5); 368
(100); 367 (44.1); 366 (60.5); 352 (8.6); 148 (12.5); 136 (16.4);
118 (15.1); 116 (13.8); 109 (11.8); 90 (16.4); 78 (28.9); 77 (96.7);
76 (14.5); 69 (13.8); 65 (48.7); 51 (64.5). Anal. Calcd for
C26H18N4S (418.51): C, 74.62; H, 4.34; N, 13.39; S 7.66. Found: C,
74.99; H, 4.25; N, 13.71; S 7.81.
N-(1-Benzofuran-2yl-ethylidene)-N’-phenylhydrazine (32)
and N-(1-benzofuran-2yl-ethylidene)-N’-(4-nitrophenyl)
hydrazine (33)
A mixture of 2-acetylbezofuran 31 (0.96g, 0.006mole) and sub-
stituted phenyl hydrazine (0.006mole) in absolute ethanol
(10ml) containing few drops of glacial acetic acid was heated
under reflux for 3h. After cooling, the precipitated product was
filtered, washed with ethanol, dried and crystallized from etha-
nol.
2-(Benzothiazol-2-yl)-3-(3-(4-chlorophenyl)-1-phenyl-1H-pyra-
zol-4-yl)acrylonitrile (27). Yield: 73%, mp: 184–186°C (diox-
ane). IR (cm−1): 2219 (C≡N); 1642 (C=N); 1283, 1061 (C-S-C).
1H-NMR (δ ppm): 7.31–7.34 (m, 3H, phenyl-C3,4,5-H); 7.52–7.63
(m, 8H, benzothiazole-H, phenyl-C2,6-H and p-chloro-C2,6-H);
7.95 (d, J=8.4Hz, 2H, p-chlorophenyl-C3,5-H); 8.22 (s, 1H, =CH);
9.26 (s, 1H, pyrazole-C5-H). Anal. Calcd for C25H15ClN4S (438.93):
C, 68.41; H, 3.44.; N, 12.76; S 7.31. Found: C, 68.73; H, 3.47; N,
12.95; S 7.59.
N-(1-Benzofuran-2 yl-ethylidene)-N’-phenylhydrazine
(32). yield: 85%, mp: 86–88°C [reported 85–87°C] [28]. IR
1
(cm−1): 3269 (NH); 1642 (C=N); 1213, 1042 (C-O-C). H-NMR
(δ ppm): 2.31 (s, 3H, CH3); 7.15–7.24 (m, 3H, phenyl-C3,4,5-H);
7.27–7.36 (m, 3H, benzofuran-C3,5,6-H); 7.39 (d, J=7.2Hz, 2H,
phenyl-C2,6-H); 7.66 (d, J=8.4Hz, 1H, benzofuran-C4-H); 7.69 (d,
J=7.65Hz, 1H, benzofuran-C7-H); 10.28 (s, 1H, NH, D2O
exchangeable).
2-(Benzothiazol-2-yl)-3-(1-(4-nitrophenyl)-3-phenyl-1H-pyra-
zol-4-yl)acrylonitrile (28). Yield: 81%, mp: 176–178°C (diox-
ane). IR (cm−1): 2246 (C≡N); 1615 (C=N); 1290, 1088 (C-S-C).
1H-NMR (δ ppm): 7.19–7.21 (m, 3H, phenyl-C3,4,5-H); 7.43–7.51
(m, 4H, benzothiazole-H); 7.88 (d, J=7.1Hz, 2H, phenyl-C2,6-H);
8.27 (d, J=9.2Hz, 2H, p-nitrophenyl-C2,6-H); 8.37 (d, J=9.2Hz,
2H, p-nitrophenyl-C3,5-H); 8.55 (s, 1H, =CH); 9.32 (s, 1H, pyra-
zole-C5-H). Anal. Calcd for C25H15N5O2S (449.48): C, 66.80; H,
3.36; N, 15.58; S 7.13. Found: C, 66.67; H, 3.49; N, 15.71; S 7.45.
N-(1-Benzofuran-2yl-ethylidene)-N’-(4-nitrophenyl)hydrazine
(33). yield: 89%, mp: 98–100°C [reported 97–99°C] [28]. IR
1
(cm−1): 3300 (NH); 1630 (C=N); 1245, 1073 (C-O-C). H-NMR
(δ ppm): 2.33 (s, 3H, CH3); 7.22–7.33 (m, 3H, benzofuran-C3,5,6-H);
7.36 (d, J=9.2Hz, 2H, p-nitrophenyl-C2,6-H); 7.60 (d, J=8.4Hz,
1H, benzofuran-C4-H); 7.63 (d, J=7.65Hz, 1H, benzofuran-
C7-H); 8.15 (d, J=9.2Hz, 2H, p-nitrophenyl-C3,5-H); 10.39 (s, 1H,
NH, D2O exchangeable).
N-{[1-(4-nitrophenyl)-3-p-tolyl-1H-pyrazol-4-yl]
methylene}-1H-benzimidazole-2-amine (29) and N-{[3-
(4-Chlorophenyl)-1-(4-nitrophenyl)-1H-pyrazol-4-yl]
methylene}-1H-benzimidazole-2-amine (30)
A mixture of 2-aminobenzimidazole (0.26g, 0.002mole) and the
appropriate pyrazole aldehyde 14, 15 (0.002mole) in dry diox-
ane (10ml) containing few drops of conc. H2SO4 was heated
under reflux for 10h. After cooling, the precipitated product was
filtered, washed with ethanol, dried and crystallized from the
proper solvent.
3-(Benzofuran-2yl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(34) and 3-(benzofuran-2yl)-1-(4-nitrophenyl)-1H-
pyrazole-4-carbaldehyde (35)
To an ice-cold dimethylformamide (2.5g, 3ml, 0.034mole),
POCl3 (1g, 6ml, 0.0066mole) was added dropwise with stirring
over a period of 30min. Stirring was continued for further 1h
keeping the reaction temperature at 0°C. Compound 32, 33
(0.003mole) was then added and the reaction mixture was
heated at 60–70°C with stirring for 4h, allowed to cool and
poured onto ice-water mixture. The obtained mixture was
boiled and the obtained precipitate was filtered, washed with
water, dried and crystallized from the proper solvent.
N-{[1-(4-Nitrophenyl)-3-p-tolyl-1H-pyrazol-4-yl]methylene}-1H-
benzimidazole-2-amine (29). Yield: 62%, mp:>300°C (diox-
ane). IR (cm−1): 3327 (NH); 1631 (C=N). 1H-NMR (δ ppm): 2.31
(s, 1H, CH3); 7.16–7.19 (m, 2H, benzimidazole-C5,6-H); 7.42 (d,
J=7.8Hz, 2H, p-tolyl-C3,5-H); 7.63–7.67 (m, 4H, benzimidazole-
3-(Benzofuran-2 yl)-1-phenyl-1H-pyrazole-4-carbaldehyde
(34). Yield: 79%, mp: 118–120°C (EtOH) [reported 116–118°C]
[28]. IR (cm−1): 1710 (C=O); 1622 (C=N); 1240, 1061 (C-O-C).
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

Original Article 67
1H-NMR (δ ppm): 7.32 (t, J=7.2Hz, phenyl-C4-H); 7.34–7.43 ((m,
3H, benzofuran-C3,5,6-H); 7.60 (t, J=7.8Hz, 2H, phenyl-C3,5-H);
7.71 (d, J=8.1Hz, 1H, benzofuran-C4-H); 7.77 (d, J=7.65Hz, 1H,
benzofuran-C7-H); 8.00 (d, J=8.4Hz, 2H, phenyl-C2,6-H); 9.40 (s,
1H, pyrazole-C5-H); 10.22 (s, 1H, CHO). Anal. Calcd for
C18H12N2O2 (288.30): C, 74.99; H, 4.20; N, 9.72. Found: C, 75.06;
H, 4.28; N, 9.83.
12.16 (s, 1H, NH, D2O exchangeable). Anal. Calcd for C24H17N5O2
(407.42): C, 70.75; H, 4.21; N, 17.19. Found: C, 70.57; H, 4.15; N,
17.03.
3-(3-Benzofuran-2-yl)-1-aryl -1H-pyrazol-4-yl)-1-arylprop-
2-en-1-one (40–42)
An equimolar mixture of the substituted pyrazolecarboxalde-
hyde 34, 35 (0.01mole) and the appropriate acetophenone in
ethanolic KOH (3%) (30mL) was stirred at room temperature for
4h. The precipitate formed was filtered, washed with EtOH,
dried and crystallized from the appropriate solvent.
3-(Benzofuran-2yl)-1-(4-nitrophenyl)-1H-pyrazole-4-carbalde-
hyde (35). Yield: 81%, mp: 126–128°C (dioxane) [reported
125–127°C] [28]. IR (cm−1): 1712 (C=O); 1610 (C=N); 1225,
1041 (C-O-C). 1H-NMR (δ ppm): 7.29–7.40 ((m, 3H, benzofuran-
C3,5,6-H); 7.67 (d, J=8.1Hz, 1H, benzofuran-C4-H); 7.75 (d,
J=7.65Hz, 1H, benzofuran-C7-H); 8.12 (d, J=9.2Hz, 2H, p-nitro-
phenyl-C2,6-H); 8.35 (d, J=9.2Hz, 2H, p-nitrophenyl-C3,5-H);
9.38 (s, 1H, pyrazole-C5-H); 10.31 (s, 1H, CHO). Anal. Calcd for
C18H11N3O4 (333.30): C, 64.86; H, 3.33; N, 12.61. Found: C, 64.90;
H, 3.56; N, 12.80.
3-(3-Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-phenylprop-
o
2-en-1-one (40). Yield: 67%, mp: 215–217 C (dioxane/EtOH).
IR (cm-1): 1665 (C=O), 1620 (C=N); 1244, 1050 (C-O-C). 1H-
NMR (δ ppm): 7.21–7.81 (m, 17H, aromatic-H and CH=CH);
9.39 (s, 1H, pyrazole-C5-H). Anal. Calcd for C26H18N2O2 (390.43):
C, 79.98.; H, 4.65; N, 7.17. Found: C, 80.17; H, 4.83; N, 7.44.
General procedure for preparation of compounds
(36–39)
3-(3-Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)-1-(4-chloroph-
enyl)prop-2-en-1-one (41). Yield: 77%, mp: 210–212 oC
(EtOH). IR (cm-1): 1660 (C=O), 1625 (C=N); 1267, 1072 (C-O-C).
1H-NMR (δ ppm): 7.22–7.89 (m, 16H, aromatic-H and CH=CH);
9.41 (s, 1H, pyrazole-C5-H). MS, m/z (rel. Abund. %): 426 (6.0)
M∙++2; 425 (6.0) M∙++1; 424 (13.4) M∙+; 286 (19.8); 285 (100);
182 (7.4); 155 (7.4); 139 (29.7); 127 (8.1); 113 (10.2); 111 (27.2);
89 (9.5); 77 (56.2); 75 (18.0); 69 (9.9); 63 (10.2); 51 (26.1); 50
(10.6). Anal. Calcd for C26H17ClN2O2 (424.88): C, 73.50; H, 4.03;
N, 6.59. Found: C, 73.19; H, 4.19; N, 6.63.
A mixture of the appropriate aldehyde 34, 35 (0.002mole) and
the appropriate thiosemicarbazide or acid hydrazide in ethanol
(10ml) containing few drops of glacial acetic acid was refluxed
for 4h. After cooling, the precipitated product was filtered,
washed with ethanol, dried and crystallized from the proper sol-
vent.
1-((3-Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-
cyclohexylthiosemicarbazide (36). Yield: 83%, mp: 264–
266°C (dioxane). IR (cm−1): 3296 (NH); 1632 (C=N); 1266,
3-(3-Benzofuran-2-yl)-1-(4-nitrophenyl)-1H-pyrazol-4-yl)-1-(4-
chlorophenyl)prop-2-en- 1-one (42). Yield: 81%, mp: 234–236
oC (dioxane). IR (cm−1): 1662 (C=O), 1621 (C=N); 1247, 1061
(C-O-C). 1H-NMR (δ ppm): 1H-NMR (δ ppm): 7.25–8.07 (m, 11H,
aromatic-H and CH=CH); 8.12 (d, J=9.2Hz, 2H, p-nitrophenyl-
C2,6-H); 8.39 (d, J=9.2Hz, 2H, p-nitrophenyl-C3,5-H); 9.54 (s, 1H,
pyrazole-C5-H). Anal. Calcd for C26H16ClN3O4 (469.88): C, 66.46;
H, 3.43; N, 8.94. Found: C, 66.75; H, 3.44; N, 8.68.
1
1088 (C-O-C). H-NMR (δ ppm): 1.10–1.22 (m, 4H, cyclohexyl-
C3,5-H); 1.51–1,72 (m, 6H, cyclohexyl-C2,4,6-H); 2.53 (m, 1H,
cyclohexyl-C1-H); 7.18–7.88 (m, 10H, aromatic-H); 8.71 (s, 1H,
CH=N); 9.27 (s, 1H, pyrazole-C5-H); 9.75 and 11.48 (2 s, each 1H,
2NH, D2O exchangeable). Anal. Calcd for C25H25N5OS (443.56): C,
67.69; H, 5.68; N, 15.79; S 7.23. Found: C, 67.55; H, 5.79; N,
15.55; S 7.51.
1-((3-Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-
phenylthiosemicarbazide (37). Yield: 79%, mp: 240–242°C
(dioxane). IR (cm−1): 3300 (NH); 1611 (C=N); 1258, 1047 (C-O-
C). 1H-NMR (δ ppm): 7.20–7.91 (m, 15H, aromatic-H); 8.74
(s, 1H, CH=N); 9.31 (s, 1H, pyrazole-C5-H); 9.98 and 11.93 (2 s,
each 1H, 2NH, D2O exchangeable). Anal. Calcd for C25H19N5OS
(437.52): C, 68.63; H, 4.38; N, 16.01; S 7.33. Found: C, 68.83; H,
4.37; N, 15.83; S 7.46.
Antiangiogenesis activity[29]
In Vitro cytotoxicity assay
MCF-7 human breast cancer cell line was grown in RPMI 1640
medium containing 10% fetal bovine serum and 2mM
L-glutamine. Colo-205 human colon cancer and Hep-2G cells
were grown in DMEM medium containing 10% fetal bovine
serum and 2mM L-glutamine. Human umbilical venous
endothelial cells (HUVECs) were grown in M-199 supplemented
with heparin (5U·mL−1) endothelial cell growth supplement
(ECGS, 200mg·mL−1) and 20% FBS. Cells were inoculated into 96
well microtiter plates in 100μl at plating densities ranging from
5000 to 40000cells/well. After cell inoculation, the microtiter
plates were incubated at 37°C, 5% CO2, 95% air and 100% relative
humidity for 24h. After 24h, a plate of MCF-7 and HUVEC lines
were fixed in situ with trichloroacetic acid (TCA), to represent a
measurement of the cell population at the time of compounds
addition. Tested compounds were solubilized in dimethyl sul-
foxide at 400-fold the desired final tested concentrations. Serial
dilutions were made to provide a total of different concentra-
tions of tested compounds plus control. Aliquots of 100μl of
tested compound dilutions were added to the appropriate
microtiter wells containing 100μl of medium, resulting in the
required final drug concentrations. The plates were incubated
N’-((3-Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)
benzohydrazide (38). Yield: 76%, mp: 246–248°C (dioxane/
EtOH). IR (cm−1): 3250 (NH); 1660 (C=O); 1609 (C=N); 1250,
1
1061 (C-O-C). H-NMR (δ ppm): 7.25–8.70 (m, 15H, aromatic-
H); 8.80 (s, 1H, CH=N); 9.23 (s, 1H, pyrazole-C5-H); 12.08 (s, 1H,
NH, D2O exchangeable). Anal. Calcd for C25H18N4O2 (406.44): C,
73.88; H, 4.46; N, 13.78. Found: C, 73.69; H, 4.31; N, 13.91.
N’-((3-Benzofuran-2-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)
isonicotinohydrazide (39). Yield: 71%, mp: 236–238°C
(EtOH). IR (cm−1): 3207 (NH); 1667 (C=O); 1621 (C=N); 1236,
1
1069 (C-O-C). H-NMR (δ ppm): 7.28–7.85 (m, 10H, aromatic-
H); 7.89 (d, J=7.4Hz, 2H, pyridyl-C2,6-H); 8.78 (d, J=7.4Hz, 2H,
pyridyl-C3,5-H); 8.82 (s, 1H, CH=N); 9.11 (s, 1H, pyrazole-C5-H);
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

68 Original Article
for an additional 48h at 37°C, 5% CO2, 95% air, and 100% relative
humidity. Cells were fixed in situ by the gentle addition of 50μl
of 50% (w/v) 10% TCA and incubated for 60min at 4°C. The
supernatant was discarded, and the plates were washed 5 times
with PBS and dried. Sulforhodamine B (SRB) solution (100μl) at
0.4% (w/v) in 1% acetic acid was added to each well, and plates
were incubated for 10min at room temperature. After staining,
unbound dye was removed by washing several times with 1%
acetic acid and the plates were air dried. Bound stain is subse-
quently solubilized with 10mM trizma base, and the absorbance
was read on an automated plate reader at a wavelength of
515nm. Dose–response cure was calculated.
concentration using a Bioquant Image Analysis system. Drug
effect (IC50) is assessed compared to untreated controls by meas-
uring the length of cords formed and number of junctions.
Cell migration assay [31]
Migration is assessed using the 48-well Boyden chamber and
8μm pore size collagen-coated according to Sultan et al. RPMI
1640 medium alone (control) or medium containing chemoat-
tractant VEGF 20ng/ml were added to the bottom chambers.
HUVEC suspension (2×105 cells/ml) were prepared in (RPMI
1640+1% BSA) with or without test compounds and added to
the top chambers. After 24h incubation at 37°C, the membrane
is rinsed in PBS, fixed and stained in Diff-Quick solutions. The
filter is placed on a glass slide with the migrated cells facing
down and cells on top are removed using a Kimwipe. The testing
is performed in triplicates and 5 fields are counted from each
well. Unstimulated control values are subtracted from stimu-
lated control and drug treated values and data is plotted as mean
migrated cell ±S.D. IC50 is calculated from the plotted data.
HUVECs growth inhibition assay [30]
HUVECs at a concentration of (2×103) were plated in a 96-well
plate in 200ml of M-199 medium. After 24h, the test compound
(100μl) was added to each well at different concentrations in
M-199 medium. On day 0, one plate is stained with 0.5% crystal
violet in 20% methanol for 10min, rinsed with PBS. The remain-
ing plates are incubated for 72h at 37°C. After 72h, plates were
stained with 0.5% crystal violet in 20% methanol, rinsed with
PBS and dried. The stain is eluted with (1:1) solution of ethanol:
0.1M sodium citrate (including day 0 plate), and absorbance is
measured at 540nm with an ELISA reader. Day 0 absorbance is
subtracted from the 72h plates and data is plotted as percentage
of control proliferation (vehicle treated cells). IC50 (drug concen-
tration causing 50% inhibition) was calculated from the plotted
data.
Modeling Studies
▼
Computer-assisted simulated docking experiments were carried
out under an MMFF94X force field in KDR structure (PDB ID:
2QU5) using Chemical Computing Group’s Molecular Operating
Environment (MOE-dock 2009) software, Montréal, Canada.
Cord formation assay [31]
Results and Discussion
Matrigel (100μl of 5mg/ml) was placed in each well of an ice-
cold 96-well plate. The plate is allowed to sit at room tempera-
ture for 15min then incubated at 37°C for 30min to permit the
matrigel to polymerize. HUVEC were prepared in M-199 medium
at a concentration of (2×105) cells/ml. The tested compounds
were prepared at different concentrations (5 concentration lev-
els) in the same medium. Equal amounts of cells suspension and
tested compounds (500μl/each) are mixed and 200μl of this sus-
pension are placed in triplicate on the polymerized matrigel.
After 24h incubation, pictures were taken in triplicate for each
▼
Chemistry
The synthetic strategies to obtain the target compounds are
depicted in ● Fig. 5, 6. The key starting materials 1,3-diphenyl-
▶
1H-pyrazole-4-carbaldehyde 9 [32], 1-phenyl-3-p-tolyl-1H-pyra-
zole-4-carbaldehyde 10 [32], 3-(4-chlorophenyl)-1-phenyl-1H-
pyrazole-4-carbaldehyde 11 [32], 3-(4-bromophenyl)-1-phenyl-
1H-pyrazole-4-carbaldehyde 12 [32], 1-(4-nitrophenyl)-3-phenyl-
1H-pyrazole-4-carbaldehyde 13 [33], 1-(4-nitrophenyl)-3-p-
tolyl-1H-pyrazole-4-carbaldehyde 14, 3-(4-chlorophenyl)-1-(4-
Fig. 2 Binding mode for 2-aminobenzimidazole
derivative B docked and minimized in KDR binding
pocket using MOE software.
Glu
917
Phe
918
Ile
892
Gly
922
Thr
Glu
Leu
916
885
1035
Ile
Cys
919
888
H
N
N
H
N
Lys
H
868
N
N
F
F
O
F
O
Asp
1046
Val
848
Cys
1045
Val
CI
898
Leu
Leu
Ala
889
840
866
Phe
1047
Leu
1019
Val
899
Ile
1044
His
1026
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

Original Article 69
nitrophenyl)-1H-pyrazole-4-carbaldehyde 15 [34], and 3-(4-
bromophenyl)-1-(4-nitrophenyl)-1H-pyrazole-4-carbaldehyde 16
[22] were prepared from the reaction of acetophenone or 4-substi-
tuted acetophenones with phenyl hydrazine or 4-nitrophenyl
hydrazine to produce the corresponding hydrazones 1–8, followed
by Vilsmeier-Haack reaction [35].
Heating the appropriate pyrazole aldehyde with the correspond-
ing 2-(1H-benzimidazol-2-yl)acetonitrile or 2-(benzothiazol-
2-yl)acetonitrile in absolute ethanol in the presence of
triethylamine afforded the corresponding acrylonitrile 17–28.
Analogously, reaction of the pyrazole aldehyde with 2-amino-
1H-benzoimidazole in dioxane in presence of few drops of conc.
Fig. 3 Binding mode for compound 19 docked
and minimized in KDR binding pocket using MOE
software.
Ala
881
Ser
Cl
884
Ile
888
Ala
1050
N
N
Glu
885
Leu
1019
H
N
Ile
Ile
N
1025
892
Cys
1024
N
Leu
Leu
1049
889
His
1026
Asp
Lys
1046
868
Cys
1045
Val
Thr
914
916
Fig. 4 Binding mode for compound 26 docked
and minimized in KDR binding pocket using MOE
software.
Val
898
Val
899
Ile
1044
Glu
885
N
N
N
Leu
1035
S
Phe
N
1047
Asp
1046
Cys
1045
His
1026
Thr
916
Lys
868
Leu
889
Ala
1050
Ala
866
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

70 Original Article
H2SO4 yielded the corresponding pyrazolemethylenebenzimida-
Table 1 Growth% after incubation with 10−4 M of tested compounds.
▶
zol-2-amine 29, 30 (● Fig. 5) .
Compounds
MCF-7 (Breast)
Hep2G (Liver)
Colo 205
Condensation of 2-acetylbenzofuran [36] with the appropriate
phenyl hydrazine afforded the parent hydrazones 32, 33 [28].
The latter hydrazones were then treated with a mixture of phos-
phorousoxychloride and DMF to obtain the key intermediates;
3-(benzofuran-2-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 34
[28] and 3-(benzofuran-2-yl)-1-(4-nitrophenyl)-1H-pyrazole-4-
carbaldehyde 35 [28]. These key intermediates were then
reacted with the appropriate thiosemicarbazide, acid hydrazide
or acetophenone to afford the corresponding thiosemicarbazone
36, 37, hydrazone 38, 39 and chalcone derivatives 40–42;
18
19
22
23
26
27
30
35
37
39
41
42
29
89
22
83
94
19
25
20
26
22
28
15
33
75
29
78
82
27
37
31
29
28
33
11
19
71
36
90
98
36
39
44
32
38
41
18
▶
respectively (● Fig. 6) .
Antiangiogenesis activity
Angiogenesis is a complex process that includes endothelial cells
proliferation, migration, alignment and formation of capillary
like structures. Endothelial cells rather than tumor cells are con-
sidered as good drug candidates for diseases associated with
antiangiogenesis. 12 compounds 18, 19, 22, 23, 26, 27, 30, 35,
37, 39, 41, and 42 were initially evaluated for their cytotoxicity
in 3 cancer cell lines tested at 10−4 M drug concentration
Table 2 Cell growth inhibition in 3 different cancer cell lines.
Compounds
Cytotoxicity (IC50 μg mL−1
)
MCF-7
Hep2G
Colo 205
AVR-C
HUVEC
19
4.9
3.6
3.25
2.56
2.44
1.69
1.0
3.9
0.29
0.35
0.41
1.82
0.21
23
3.79
2.39
1.35
0.4
2.8
3.05
2.5
▶
(● Table 1). Cancer cell lines adopted in this prescreen were
26
2.68
1.43
1.2
42
1.49
0.86
MCF-7 (Breast), Hep2G (Liver), and Colo205 (Colon). The pre-
screen assay aims to preliminary testing a large proportion of
tested compounds and eliminating the inactive compounds.
Data for each compound are reported as the percent of growth of
the treated cells compared to the untreated control cells. Only
compounds that reduce the growth of any one of the tested can-
cer cell lines to approximately 32% or less are considered
Pos. con
IC50: A compound’s concentration produces 50% reduction in cell growth. The
values were the averages from a triplicate experiment with the individual value less
than 10%. AVR-C: average values of IC50 of 3 tested cancer cell lines
Table 3 In vitro antiangiogenesis activities of selected compounds on tested
HUVECS.
▶
active. ● Table 1 revealed that all the tested compounds were
cytotoxic against one or more of the tested cancer cell lines
except compounds 21, 25 and 28 which showed no cytotoxic
effects.
Furthermore, compounds 19, 23, 26, and 42 were selected for
the antiangiogenesis testing, using HUVEC. Compounds 19, 23,
and 26 showed no cytotoxic effects compared to compound 42
that showed a significant cytotoxic effect on the tested cancer
IC50 (μM)
Compounds
Proliferation
inhibition
Cord formation
inhibition
Chemotaxis
19
5.1
8.1
0.73
1.1
23
9.3
11.0
11.6
43.0
1.12
26
10.8
34.6
0.005
1.6
42
21.3
0.42
▶
cell lines (● Table 1) .
TNP-470 (ST)
The IC50 values for the tested compounds 19, 23, 26, and 42 were
determined as 0.29, 0.35, 0.41, and 1.82μg·mL−1, respectively as
described under the experimental section. Compounds 19, 23,
26, and 42 were assayed at 3 different concentrations, one using
▶
than that of TNP-470, positive control (● Table 3). It is interest-
ing to point out that compounds 19, 23, and 26 showed no anti-
tumor activity in the 3 tested cancer cell line, indicating that
they can exert antiangiogenic activities at non-cytotoxic con-
▶
IC50 for each tested compound as mentioned in ● Table 2 and
other 2 lower concentrations of 0.1, and 0.05μg·mL−1 respec-
tively. TNP-470, an established angiogenesis inhibitor, was used
as a positive inhibitory control at the same above mentioned
▶
centrations (● Table 3) .
To mimic the final events during angiogenesis, in which HUVEC
can form network of capillaries in a 3-dimensional Matrigel cul-
ture (3D), all tested compounds were examined for their ability
to interfere with HUVEC function crucial to angiogenesis, the
alignment of endothelial cells in a capillary-like structure.
HUVEC were plated on (3D) of Matrigel culture where they
aligned, forming cords, which were evident a few hours after
plating. Compounds 19, 23, and 26 exhibited cord formation
inhibitory properties but still lower than that of the positive
▶
concentration as that of the tested compounds (● Table 2).
Moreover, the above concentrations of the tested compounds
showed complete inhibitory effects on HUVEC tube formation at
their IC50 concentrations and at 0.1μg·mL−1, indicating that
0.1μg·mL−1 concentration showed no cytotoxic effect while the
complete inhibition was still observed. This concentration was
ranged from 2.9~18.2-fold lower than the IC50 value of the tested
compounds against HUVECs measured under the same condi-
tions. Besides, the comparison between IC50 values of the tested
compounds and the 0.1μg·mL−1 concentration in cancer cell
lines revealed that this concentration was more than 13.5 fold
lower than the average cytotoxicity (AVR-C) against the 3 tested
▶
control, TNP-470 (● Table 3) .
The chemotaxis inhibition test mainly measures the ability to
inhibit HUVEC cells migration in response to the specific chem-
oatrractant VEGF. Compounds 19, 23, and 26 showed significant
activity with IC50 values within the range of 0.73~1.6μM, and
were less than TNP-470 activity under the same experimental
▶
cancer cell lines (● Table 2) .
In addition, using HUVEC cells proliferation inhibition test, com-
▶
conditions as shown in ● Table 3. Compound 19 was the most
pounds 19, 23, and 26 showed an antiproliferative effect but less
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

Original Article 71
CH3
O
H
N
H
N
i
N
NH2
+
CH3
R
R1
R
R 1
R1 = H, CH3, Cl, Br
R = H, NO2
1: R = H, R1= H; 2: R = H, R1 = CH3
3: R = H, R1= Cl; 4: R = H, R1 = Br
5: R = NO2 , R1 = H; 6: R = NO2, R1 = CH3
7: R = NO 2, R 1 = Cl; 8: R = NO2, R
1 = Br
R1
ii
O
N
N
R
iv
iii
9: R = H, R1= H; 10: R = H, R1 = CH3
11: R = H, R1 = Cl; 12: R = H, R1 = Br
13: R = NO2, R1 = H; 14: R = NO2, R1 = CH3
15: R = NO2, R1 = Cl; 16: R = NO2, R1 = Br
R1
R1
H
N
CN
X
N
N
N
N
N
N
N
29: R 1 = CH3
30: R 1 = Cl
NO2
R
17: X = NH, R = H, R1 = H; 18: X = NH, R = H, R1 = CH
19: X = NH, R = H, R1 = Cl; 20: X = NH, R = H, R1 = Br 3
21: X = NH, R = NO2, R1 = H; 22: X = NH, R = NO2, R1 = CH3
23: X = NH, R = NO2, R1 = Cl; 24: X = NH, R = NO2, R1 = Br
25: X = S, R = H, R1 = H; 26: X = S, R = H, R1 = CH3
27: X = S, R = H, R1 = Cl; 28: X = S, R = NO2, R 1 = H
Fig. 5 Reagents and reaction conditions: i gl HAc, EtOH, reflux; ii POCL3/DMF, 60–70°C; iii benzazole-2-ylacetonitrile, Et3N, EtOH, reflux, 1–3h.; iv 2-ami-
nobenzimidazole, H2SO4, dioxane, reflux, 10h.
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

72 Original Article
R
H
N
O
N
N
H
NH2
i
+
O
CH3
R
O
CH3
32: R = H
33: R = NO2
R = H, NO2
31
O
ii
N
O
N
34: R = H
35: R = NO2
R
iii
v
34: R = H
R1
S
H
N
34: R = H
iv
N
N
N
R1
H
O
N
N
O
O
N
O
36: R1 = C6H11
37: R1 = C6H5
R
40: R = H, R1 = H
41: R = H, R1 = Cl
42: R = NO2 , R1 = Cl
R1
N
N
H
N
N
O
38: R1= C 6H5
39: R1= 4-pyridyl
Fig. 6 Reagents and reaction conditions: i gl HAC, EtOH, reflux, 3h. ii POCL3/DMF, 60–70°C; iii R1NHCSNHNH2, gl HAC, EtOH, reflux, 4h.; iv R1CONHNH2,
gl HAC, EtOH, reflux, 4h.; v R1C6H4COCH3, KOH, EtOH, r.t.
active compared to compounds 23, 26, and 42 and data of chem-
otaxis activity of compound 19 was nearly comparable to TNP-
Introduction of nitro group at the 4-positon of the phenyl ring
(compound 23) or replacement of the benzimidazole moiety
with its bioisostere, benzthiazole nucleus and introduction of
methyl group at the 4-position of the phenyl ring at position-3 of
the pyrazole moiety (compound 26) resulted in reduction of
antiangiogenic activity.
▶
470 (● Table 3). Structurally, the most active compound 19 has
benzimidazole nucleus, unsubstituted phenyl ring at position-1
of the pyrazole moiety and phenyl ring at position-3 substituted
with chloro group (electron withdrawing group of small size).
Rida SM et al. Design, Synthesis and Evaluation… Arzneimittelforschung 2012; 62: 63–74

Original Article 73
It is well known that antiangiogenic activity pattern of TNP-470
is due to its effect on endothelial cells proliferation rather than
motility and cord formation. In contrast, the antiangiogenic
activity pattern of compounds 19, 23 and 26 was apparently
attributed to cell motility inhibition rather than proliferation,
indicating that the antiangiogenic action of the above tested
compounds may be mediated through different mechanisms of
action from that reported to previously known compounds.
Conclusion
▼
In the present study, new series of benzimidazoles, benzothia-
zoles and benzofurans incorporating pyrazole moiety were syn-
thesized and tested for their antiangiogenic effect. Our data
indicated that benzimidazole and benzothiazole derivatives
linked with substituted pyrazole through a vinyl 2 carbon
bridge; 19, 23, and 26, represent a new class of compounds
which are non-cytotoxic but have an antiangiogenic activity
profile, mainly through inhibiting the motility of endothelial
cells rather than its proliferation. Compound 42, a benzofuran
ring directly attached to the pyrazole moiety through a single
bond, showed a cytotoxic activity against the tested 3 cancer cell
lines, but its antiangiogenesis activity was less than that of com-
pounds 19, 23, and 26.
Docking Study
▼
The docking study was carried out using the enzyme parameters
obtained from the crystallographic structure of the complex
between KDR with the co-crystallized 2-aminobenzimidazole
derivative B (PDB ID: 2QU5) [23]. The docking simulation for the
ligands was carried out using molecular operating environment
(MOE) software supplied by the Chemical Computing Group,
Inc., Montréal Canada [37].
Conflict of Interest
▼
The X-ray co-crystal structure of the complex between KDR and
2-aminobenzimidazole derivative reveals that the benzimida-
zole core is oriented in the hydrophobic pocket surrounded by
Asp 1046 and Phe 1047 in addition, it displayed arene-arene
interaction with Lys 868. The endocyclic nitrogen of the benz-
imidazole hydrogen bonds with the NH of Asp 1046 and either
the exocyclic or the endocyclic NH may interact with the side
chain of Glu 885. Hydrogen bonds are also observed between the
hinge-region Cys 919 and both the nitrogen of the pyridine and
The authors report no conflict of interest.
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