Synthesis, characterization and evaluation of benzimidazole derivative and its precursors as inhibitors of MDA-MB-231 human breast cancer cell proliferation

Article abstract of DOI:10.1016/j.bmcl.2007.08.078

A novel series of trisubstituted benzimidazole and its precursors (3-7) were synthesised and characterized by using ¹H NMR, LC/MS, FTIR and elemental analysis techniques. The title compounds were evaluated for inhibition against MDA-MB-231 brea

Full text of DOI:10.1016/j.bmcl.2007.08.078

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 432–435
Synthesis, characterization and evaluation of benzimidazole
derivative and its precursors as inhibitors of MDA-MB-231
human breast cancer cell proliferation
N. R. Thimmegowda,a S. Nanjunda Swamy,a,c C. S. Ananda Kumar,a Y. C. Sunil Kumar,a
S. Chandrappa,a George W. Yipb,* and K. S. Rangappaa,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
bDepartment of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore
cDepartment of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore 570006, India
Received 16 March 2007; revised 14 August 2007; accepted 15 August 2007
Available online 24 October 2007
1
Abstract—A novel series of trisubstituted benzimidazole and its precursors (3–7) were synthesised and characterized by using H
NMR, LC/MS, FTIR and elemental analysis techniques. The title compounds were evaluated for inhibition against MDA-MB-
231 breast cancer cell proliferation. The results revealed that the compound N-(4-cyano-3-(trifluoromethyl) phenyl)-4-fluoro-3-
nitrobenzamide (3) was the potent inhibitor.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Breast carcinoma is the most common cancer in women
worldwide and remains the most frequent cause of
malignancy-associated deaths among women. The benz-
imidazole nucleus is an important pharmacophore in
drug discovery.1 Benzimidazoles are very useful interme-
diates/subunits for the development of molecules of
pharmaceutical or biological interest. Substituted benz-
imidazole derivatives have diverse therapeutic applica-
tions as they exhibit antihistamine,2,3 antiulcerative,4
anti-inflammatory, analgesic,5–9 antioxidant,10 anti-
HIV-1,11–13 antibacterial,14–20 antiproliferative,21,22
antiallergic,23,24 antitumour,25–27 antikinase28,29 and po-
tential anticancer activities.30–33 The biological relevance
of these heteroaromatic groups is due to their being
good bioisosteres of biomolecules. Benzimidazoles are
also used as biomimetics of guanine residues34 and benz-
imidazole derivatives selectively inhibit endothelial cell
growth and suppress angiogenesis in vitro and in vivo.35
active heterocycles, we describe here the synthesis of tri-
substituted benzimidazole and its precursors and their
inhibition of proliferation of MDA-MB-231 human
breast cancer cells.
Synthesis of trisubstituted benzimidazole is outlined in
Scheme 1. 4-Amino-2-trifluoromethyl-benzonitrile (1)
was reacted with 4-fluoro-3-nitro-benzoyl chloride (2)
to produce an amide compound 3. This reaction re-
quired the use of N,N-dimethyl acetamide both as base
and as solvent. Nucleophilic aromatic substitution of
fluorine group with 4-fluoro benzyl amine was carried
out using N,N-diisopropyl ethylamine as base and
N,N-dimethyl formamide as solvent at room tempera-
ture producing 4. The aryl nitro group was then reduced
to obtain compound 5 by using 5 equiv of aqueous
NH4Cl solution and 20% iron (II) powder using isopro-
pyl alcohol as solvent. The selective acylation of primary
aromatic amino moiety in compound 5 was carried out
with 2,4-dichloro benzoyl chloride using triethylamine
as base and methylene dichloride as solvent producing
the compound 6. Finally,1-(4-fluorobenzyl)-2-(2,4-
dichlorophenyl)-N-(4-cyano-3-trifluoromethyl)phenyl)-
1H-benzo[d]imidazole-5-carboxamide (7) was obtained
by cyclisation of compound 6 with glacial acetic acid.
All the precursors and substituted benzimidazole com-
pound were obtained in good yield ranging from 81%
to 91% after recrystallization with diethyl ether and
Due to broad spectrum of activities reported so far and
in continuation of our research on the synthesis of bio-
Keywords: Trisubstituted benzimidazole; MDA-MB-231; Cell
proliferation.
*
Corresponding authors. Tel.: +91 821 2412191; fax: +91 821 2412191
(K.S.R.); tel.: +65 65163206; fax: +65 67787643 (G.W.Y.); e-mail
addresses: georgeyip@nus.edu.sg; rangappaks@gmail.com; rangap-
paks@chemistry.uni-mysore.ac.in
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.078

N. R. Thimmegowda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 432–435
433
CF3
CF3
CF3
O
NC
NC
NC
NO2
F
i
ii
O
O
Cl
+
NH2
NO2
NH
NO2
F
N
H
N
H
1
2
3
4
F
iii
CF3
CF3
CF3
NC
NC
iv
O
Cl
O
Cl
O
NC
O
F
N
N
NH2
NH
N
H
v
N
H
Cl
N
H
N
H
Cl
NH
F
F
7
6
5
Scheme 1. Reagents and conditions: (i) DMA, rt, 4 h; (ii) 4-fluorobenzyl amine, DIPEA, DMF, rt, 3 h; (iii) Fe powder, NH4Cl, IPA, 70 ꢁC, 6 h; (iv)
2,4-dichlorobenzoyl chloride, TEA, MDC, rt, 3 h; (v) CH3COOH, 90 ꢁC, 8 h.
the final compound 7 was purified by silica gel column
chromatography using ethyl acetate/hexane (4:6) as elu-
ent. All the compounds obtained were characterized by
1H NMR, FTIR, LC/MS and elemental analysis.36
bition by compound 3 may be due to the presence of
strong electronegative groups fluorine (F) at para posi-
tion and nitro (–NO2 ) at meta position to amide.
Replacement of fluorine by electron donating amine
decreased the inhibition in compound 4, reduction of
nitro group to amine again increased the inhibition in
compound 5, whereas the selective acylation at meta
position reduced the inhibition in compound 6. The
presence of benzimidazole nucleus may be responsible
for the moderate inhibition shown by compound 7.
We have synthesised the target molecule, substituted
benzimidazole 7, using 4-amino-2-trifluoromethyl-
benzonitrile (1), a starting material, which is used for
the synthesis of the well-known antineoplastic drug
bicalutamide, and 4-fluoro-3-nitro-benzoyl chloride (2)
as starting materials to get compound 3. The latter was
confirmed by the presence of –NH peak at 11.19 d in pro-
ton NMR. Similarly, the presence of –N–H stretching fre-
quency at 3352 cmÀ1 and C–O stretching frequency at
1685 cmÀ1 in FTIR spectrum and mass of the compound
were confirmed by LC/MS analysis. The intermediate
compound 4 was obtained by nucleophilic substitution
reaction of compound 3 with 4-fluoro benzylamine. Com-
pound 4 was confirmed by the presence of benzylic proton
From the above summary, it can be concluded that the
presence of electron withdrawing groups at para posi-
tion and electron donating groups at meta position to
amide may be responsible for the inhibition. But exact
interaction of electron withdrawing and donating
groups with human breast cancer cells is not known.
Further studies are needed to develop still best anti-
breast cancer drugs.
1
at 4.70 d and additional aromatic protons in H NMR
spectrum and further verified by LC/MS analysis. The
compound 5 was obtained by reducing the nitro group
in compound 4 and confirmed by the presence of –NH2
peak at 4.88 d and the absorption peak at 3439 cmÀ1 in
FTIR spectra and further confirmed by LC/MS analysis.
The compound 6 was obtained by acylation of precursor 5
and this was confirmed by the absence of primary amine
peak in FTIR spectrum and presence of additional aro-
matic proton peak in 1H NMR spectrum. The final target
molecule7obtainedbycyclisationofcompound6wascon-
firmedby 1H NMR, FTIR, LC/MSandelementalanalysis.
In summary, synthesis of trisubstituted benzimidazole
and its precursors was carried out with good yield and
purity. Compound 3 significantly inhibited MDA-MB-
231 human breast cancer cell proliferation, which may
be due to presence of electron withdrawing groups at
para position and electron donating groups at meta po-
sition in amide. Further, synthesis of novel benzimid-
azole derivatives by changing the substituents at 1st,
2nd and 5th position of the benzimidazole nucleus to
enhance the anticancer activity is under progress at
our laboratory (Table 1).
Our results revealed that the title compounds showed
inhibition of proliferation of MDA-MB-231 human
breast cancer cells.37 Among the compounds tested,
the inhibitory activity was found in the order
3 > 7 > 5. Compound 3 showed maximum inhibition
of 60%. Compounds 7 and 5 showed moderate inhibi-
tion of 26.1% and 9.8% respectively, and compounds 4
and 6 did not show any inhibition. The significant inhi-
Table 1. Percentage inhibition of MDA-MB-231 cell proliferation
Compounds
Inhibition (%)
P Value (Student’s ‘t’-test)
3
4
5
6
7
60
0
0.00
0.00
0.08
0.46
0.024
9.8
0
26.1

434
N. R. Thimmegowda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 432–435
24. Nakano, H.; Inoue, T.; Kawasaki, N.; Miyataka, H.;
Acknowledgments
Matsumoto, H.; Taguchi, T.; Inagaki, N.; Nagai, H.;
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The authors are grateful to UGC, Government of India,
for financial support under the project UGC-SAP
(Phase-I) Programme No. F. 540/10/DRS/2004(SAP-I)
and to the National Medical Research Council, Singa-
pore, for financial support from Grants NMRC/0772/
2003.DST Project No. DV6/15/DST/2005-06 New Delhi
and NMRC/1023/2005 (G.W.Y.) and DST-FIST (order
No. SR/FST/CSI-051/2002) grants for the instrument
elemental analyser.
28. Sondhi, S. M.; Singh, N.; Lahoti, A. M.; Bajaj, K.;
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36. Experimental: Melting points were determined using
SELACO-650 hot stage melting point apparatus and the
values are not corrected. Infrared (IR) spectra were
recorded using Jasco FTIR-4100 series. Nuclear magnetic
resonance (1H NMR) spectra were recorded on Shimadzu
AMX 400-Bruker, 400 MHz spectrometer using DMSO-
d6 and CDCl3 as solvent and TMS as internal standard
(chemical shift in d ppm). Spin multiplets are given as s
(singlet), d (doublet), t (triplet) and m (multiplet). Mass
and purity were recorded on a LC-MSD-Trap-XCT.
Elemental (CHNS) analyses were done on Vario EL III
Elementar. Silica gel column chromatography was per-
formed using Merck 7734 silica gel (60–120 mesh) and
Merck made TLC plates.
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Synthesis of N-(4-cyano-3-(trifluoromethyl)phenyl)-4-flu-
oro-3-nitrobenzamide (3):
A solution of 4-amino-2-
(trifluoromethyl)benzonitrile (1) (10.0 g, 53.7 mmol) in
N,N-dimethyl acetamide (100 mL) was taken and cooled
from À5 to 0 ꢁC in an ice bath. 4-Fluoro-3-nitro-benzoyl
chloride (2) (10.9 g, 53.7 mmol) was added to the cold
reaction mixture and stirred for 30 min, and then the
reaction mixture was allowed to attain room temperature
under stirring for 4 h. The reaction was monitored by
TLC. Upon completion, the reaction mixture was
quenched with water and filtered under reduced pressure,
and the brownish red solid compound obtained (3)
was recrystallized with diethyl ether. Yield: 90%
(17.08 g). Mp 144–146 ꢁC. 1H NMR. (DMSO-d6, 400
MHz) d: 11.19 (s, 1H, –CO–NH–), 8.78 (d, 1H, Ar-H),
8.43–8.39 (m, 2H, Ar-H), 8.29–8.26 (d, 1H, Ar-H), 8.18–
8.16 (d, 1H, Ar-H), 7.84–7.79 (d, 1H, Ar-H). MS: 354.0.
LC purity: 98.43% IR (KBr, cmÀ1): 3392 (CON–H str),
3059 (–C–H str), 2229 (–CN str), 1685 (–CO str), 1529
and 1326 (–N–O str), 1051 (–C–F str). Anal. Calcd for
C15H7F4N3O3 (in %): C, 51.00; H, 2.00; N, 11.90. Found:
C, 51.04; H, 2.01; N, 11.86.
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N. R. Thimmegowda et al. / Bioorg. Med. Chem. Lett. 18 (2008) 432–435
435
Synthesis of 4-(4-fluorobenzylamino)-N-(4-cyano-3-(tri-
fluoromethyl)phenyl)-3-nitrobenzamide (4): A solution of
N-(4-cyano-3-(trifluoromethyl)phenyl)-4-fluoro-3-nitrob-
enzamide (3) (2.0 g, 5.66 mmol) in N,N-dimethyl form-
amide was taken. N,N-diisopropyl ethylamine (2.19 g,
16.98 mmol) was added to the reaction mixture and
stirred for 10 min, and then 4-fluorobenzyl amine (0.71 g,
5.66 mmol) was added, and the reaction mixture was
stirred for 3 h at room temperature. The reaction was
monitored by TLC. Upon completion, the reaction
mixture was quenched with water and filtered under
reduced pressure and the obtained yellow solid compound
(4) was recrystallized with diethyl ether. Yield: 91% (2.35
g). Mp 212–214 ꢁC. 1H NMR (DMSO-d6, 400 MHz) d:
10.9 (s, 1H, –CO–NH–), 9.1 (t, 1H, –NH–), 8.84 (d, 1H,
Ar-H), 8.40 (s, 1H, Ar-H), 8.25 (d, 1H, Ar-H), 8.11 (d, 1H,
Ar-H), 8.01 (d, 1H, Ar-H), 7.46–7.42 (m, 2H, Ar-H), 7.2–
7.15 (m, 2H, Ar-H), 7.05 (d, 1H, Ar-H), 4.70 (d, 2H,
–CH2–) MS: 459.1. LC purity: 99.47%. IR (KBr, cmÀ1):
3353 (CON–H str), 3088 (–C–H str), 2224 (–CN str), 1681
(–CO str), 1521 and 1474 (–N–O str), 1049 (–C–F str).
Anal. Calcd for C22H14F4N4O3 (in %): C, 57.65; H, 3.08;
N, 12.22. Found: C, 57.60; H, 3.05; N, 12.25.
The solvent was removed under reduced pressure, and
the obtained white amorphous powder compound (6) was
recrystallized with diethyl ether. Yield: 86% (1.81 g). Mp
187–189 ꢁC. 1H NMR (DMSO-d6, 400 MHz) d: 10.14
(s, 1H, –CO–NH–), 9.01 (s, 1H, –CO–NH–), 8.75 (s, 1H,
Ar-H), 8.27 (s, 1H, Ar-H), 8.17–8.14 (d, 1H, Ar-H), 8.03–
8.01 (d, 1H, Ar-H), 7.75–7.73 (d, 1H, Ar-H), 7.44–7.39
(t, 1H, Ar-H), 7.27–7.23 (d, 1H, Ar-H), 6.98–6.95 (d, 2H,
Ar-H). 6.93–6.91 (t, 4H, Ar-H), 5.22 (s, 2H, –CH2), 5.11 (t,
1H, –NH) MS: 601. LC purity: 97.12%. IR (KBr, cmÀ1):
3430 (N–H str), 3379 (CON–H str), 3128 (–C–H str), 2226
(–CN str), 1681 (–CO str), 1048 (–C–F str), 747 (–C–Cl).
Anal. Calcd for C29H18Cl2F4N4O2 (in %): C, 57.92, H,
3.02, N, 9.32. Found: C,57.85; H, 3.07; N, 9.29.
Synthesis of 1-(4-fluorobenzyl)-2-(2,4-dichlorophenyl)-N-
(4-cyano-3-(trifluoromethyl)phenyl)-1H-benzo[d]imidaz-
ole-5-carboxamide (7): Obtained compound 6 (1.5 g,
2.5 mmol) was taken in glacial acetic acid and allowed
the reaction to proceed at 90 ꢁC for 8 h. The reaction was
monitored by TLC. Upon completion, the reaction
mixture was quenched with water and filtered under
reduced pressure, and the obtained white amorphous
powder compound 7 was purified by silica gel column
chromatography using ethyl acetate and hexane as eluent
(4:6). Yield: 81% (1.18 g). Mp 222–224 ꢁC. 1H NMR
(DMSO-d6, 400 MHz) d: 10.14 (s, 1H, –CO–NH–), 8.75
(s, 1H, Ar-H), 8.27 (s, 1H, Ar-H), 8.17–8.14 (d, 1H, Ar-
H), 8.03–8.01 (d, 1H, Ar-H), 7.75–7.73 (d, 1H, Ar-H),
7.44–7.39 (d, 1H, Ar-H), 7.27–7.23 (d, 1H, Ar-H), 6.98–
6.95 (d, 2H, Ar-H), 6.93–6.91 (m, 4H, Ar-H), 5.22 (s, 2H,
–CH2) MS: 583.0. LC purity: 99.12%. IR (KBr, cmÀ1):
3379 (CON–H str), 3128 (–C–H str), 2226 (–CN str), 1681
(–CO str), 1048 (–C–F str), 747 (–C–Cl). Anal. Calcd for
C29H16Cl2F4N4O (in %): C, 59.71; H, 2.76; N, 9.60.
Found: C, 59.65; H, 2.70; N, 9.55.
Synthesis of 4-(4-fluorobenzylamino)-3-amino-N-(4-cyano-
3-(trifluoromethyl) phenyl) benzamide (5): A solution of
4-(4-fluorobenzylamino)-N-(4-cyano-3-(trifluoromethyl)-
phenyl)-3-nitrobenzamide (4) (2.0 g) in isopropyl alcohol
was taken, 20% (W/W) of iron powder and saturated
ammonium chloride solution (5.0 equiv) were added to the
reaction mixture and heated to 90 ꢁC for 6 h. The reaction
was monitored by TLC. Upon completion, the reaction
mixture was filtered through Celite bed, isopropyl alcohol
was removed under reduced pressure and residue was
extracted with ethyl acetate. Solvent was again removed
under reduced pressure to get crude product and recrystal-
lized with diethyl ether to get pure compound 5. The product
obtained was a brown solid. Yield: 87% (1.63 g). Mp 176–
37. Biology: Culturing human breast cancer cell line
MDA-MB-231 human breast cancer cells were grown in
RPMI medium (Sigma), supplemented with 10% foetal
bovine serum (FBS). The cells were maintained at 37 ꢁC in
a 5% CO2 incubator. They were subsequently dislodged
from the substratum by 1· trypsin treatment for 5 min
followed by inactivation using FBS.
1
178 ꢁC. H NMR (DMSO-d6, 400 MHz) d: 10.45 (s, 1H,
–CO–NH–), 8.23 (d, 1H, Ar-H), 8.06 (d, 1H, Ar-H), 7.41–
7.38 (m, 2H, Ar-H), 7.19–7.13 (m, 4H, Ar-H), 6.40–6.38
(d, 1H, Ar-H), 5.99–5.97 (t, 1H, –N–H), 4.88 (s, 2H, NH2),
4.41–4.39 (d, 2H, –CH2). MS: 429.1. LC purity: 95.0%. IR
(KBr, cmÀ1): 3439 (–N–H str), 3370 (CON–H str), 3198
(–C–H str), 2228 (–CN str), 1689 (–CO str), 1050 (–C–F str),
750 (–C–Cl). Anal. Calcd for C22H16F4N4O (in %): C, 61.68;
H, 3.76; N,13.08. Found: C, 61.65; H, 3.72; N, 13.05.
Cell proliferation assay: The MTS assay was performed
using the Promega CellTiter 96ꢂ aqueous non-radioac-
tive cell proliferation assay kit as previously described.38
Briefly, MDA-MB-231 cells were seeded onto the wells
of 96-well plates at a density of 2000 cells/well. The test
compounds at 2 mM final concentration in 1% DMSO
were added and the cultures continued for 72 h.
Replenishment with fresh compounds was done after
the first 48 h. At the end of 72 h of treatment, the
cultured medium was removed and 20 ll/well of com-
bined MTS/PMS solution was added and incubated for
4 h. The absorbance at 490 nm was recorded using an
ELISA plate reader.
Synthesis of 3-(2,4-dichloro-benzoylamino)-N-(4-cyano-3-
trifluoromethyl-phenyl)-4-(4-fluoro-benzylamino)- benzam-
ide (6): The compound 5 (1.5 g, 3.5 mmol) in methylene
dichloride (15 mL) and triethylamine (1.06 g, 10.50 mmol)
were taken and cooled from À5 to 0 ꢁC in an ice bath. 2,4-
Dichloro benzoyl chloride (0.73 g, 3.5 mmol) was added to
the cold reaction mixture and stirred for 30 min, then the
reaction mixture was allowed to attain room temperature
with stirring for 3 h. The reaction was monitored by TLC.
Upon completion, the reaction mixture was quenched
with water and extracted with methylene dichloride.
38. Zou, X. H.; Foong, W. C.; Cao, T.; Bay, B. H.; Ouyang,
H. W.; Yip, G. W. J. Dent. Res. 2004, 83, 880.