Synthesis and biological evaluation of benzimidazole-oxindole conjugates as microtubule-targeting agents

Article abstract of DOI:10.1016/j.bioorg.2015.09.003

A series of benzimidazole-oxindole conjugates were synthesized and evaluated for their cytotoxic activity. The cytotoxicity assay results suggest that conjugates 5c and 5p exhibit promising cytotoxicity against human breast cancer cell line (MCF-7). The Cell cycle analysis revealed that these conjugates induced cell cycle arrest at G2/M phase in MCF-7 cells. The tubulin polymerization assay results suggested that these conjugates inhibit tubulin polymerization with IC₅₀ values 1.12 and 1.59 μM respectively. Immunofluorescence analysis also suggested that these conjugates effectively inhibited the microtubule assembly in MCF-7 cells. Further, molecular docking studies indicated that these conjugates 5c and 5p interact and binds efficiently with the tubulin protein. By and large, the results demonstrated that these benzimidazole-oxindole conjugates possess cytotoxic property by inhibiting the tubulin polymerization.

Full text of DOI:10.1016/j.bioorg.2015.09.003

Accepted Manuscript
Synthesis and Biological Evaluation of Benzimidazole-Oxindole Conjugates as
Microtubule-Targeting Agents
Ahmed Kamal, B. Nagaseshadri, V. Lakshma Nayak, Vunnam Srinivasulu,
Manda Sathish, Jeevak Sopanrao Kapure, C. Suresh Reddy
PII:
S0045-2068(15)30018-3
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.09.003
YBIOO 1841
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 August 2015
21 September 2015
22 September 2015
Please cite this article as: A. Kamal, B. Nagaseshadri, V. Lakshma Nayak, V. Srinivasulu, M. Sathish, J.S. Kapure,
C. Suresh Reddy, Synthesis and Biological Evaluation of Benzimidazole-Oxindole Conjugates as Microtubule-
Targeting Agents, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.09.003
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Synthesis and Biological Evaluation of Benzimidazole-Oxindole
Conjugates as Microtubule-Targeting Agents
Ahmed Kamal,*a,b B. Nagaseshadri,a V. Lakshma Nayak,a Vunnam Srinivasulu,a Manda
Sathish,a Jeevak Sopanrao Kapure,a,b and C. Suresh Reddy.c
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad 500 037, India
cDepartment of Chemistry Sri Venkateswara University, Tirupati 517 502 India
Abstract: A series of benzimidazole-oxindole conjugates were synthesized and evaluated for
their cytotoxic activity. The cytotoxicity assay results suggest that conjugates 5c and 5p
exhibit promising cytotoxicity against human breast cancer cell line (MCF-7). The Cell cycle
analysis revealed that these conjugates induced cell cycle arrest at G2/M phase in MCF-7
cells. The tubulin polymerization assay results suggested that these conjugates inhibit tubulin
polymerization with IC50 values 1.12 and 1.59 µM respectively. Immuofluorescence analysis
also suggested that these conjugates effectively inhibited the microtubule assembly in MCF-7
cells. Further, molecular docking studies indicated that these conjugates 5c and 5p interact
and binds efficiently with the tubulin protein. By and large, the results demonstrated that
these benzimidazole-oxindole conjugates possess cytotoxic property by inhibiting the tubulin
polymerization.
Keywords: Benzimidazoles-Oxindoles; Cytotoxic activity; Cell cycle; Tubulin
polymerization; Docking.
,aCorresponding author. Tel.: +91-40-27193157; fax: +91-40-27193189; E-mail:
ahmedkamal@iict.res.in.
1

1. Introduction
Microtubule is an important component of the cytoskeleton formed by α and β tubulin
heterodimers, and are critical in many cellular processes such as cell shape maintenance,
intracellular transport, cell division, cell growth and mitosis [1–3]. As the key structural basis
of microtubule, tubulin is considered as a highly attractive target for anticancer therapy [4–
12]. Several tubulin inhibitors have been approved as the first line chemotherapeutic agents
for different types of human cancer [13,14]. Tubulin inhibitors are categorized as three types
based on their binding site in the tubulin protein, that includes taxol-site inhibitors, (paclitaxel
1, and epothilones) [15,16], vinblastine-site inhibitors, including (vinblastine, 2, and
vincristine) [17] and colchicine-site inhibitors (colchicines [18] and combretastatin [19,20]
A-4) as shown in Fig. 1. Most of the tubulin-binding agents are derived from natural products
with complex chemical structures that restrict chemical modifications, however compounds
with relatively simple chemical structures could be valuable candidates for the development
of newer molecules.
<Insert Figure 1>
Benzimidazole is a bicyclic heteroaromatic molecule which is structural isostere of
naturally occurring nucleotides hence, it has been extensively utilized as a useful scaffold in
the medicinal chemistry. Benzimidazole derivatives have shown different therapeutic
properties including antitumor activity [21], and exert their antitumor activity by acting on
various targets such as topoisomerases inhibitors [22], DNA-alkylating agents [23], tubulin
polymerization inhibitors [24] and antiangiogenic agents [25]. Nocodazole (3, Fig. 1) having
benzimidazole moiety as its basic scaffold is a well known agent that inhibits tubulin
polymerization. On the other hand oxindoles are versatile molecules that display diverse
biological activities, including anticancer activity [26,27]. For example, Indirubin (4, Fig. 1)
which has an oxindole moiety as its basic structure, is reported as a potent anticancer agent
that inhibits cyclin dependent kinase (CDK) [28]. Recently, it has been reported that
oxindoles not only inhibited CDK but also tubulin polymerization by binding at the
colchicine binding site [29]. Some of the potent hybrid molecules that have been recently
developed as new anticancer agents are obtained by the combination of different
pharmacophores [26,30]. The promising biological activity exhibited by these conjugates
prompted us to explore newer conjugates by combining benzimidazole and oxindole
2

scaffolds with a view to enhance the cytotoxic activity. In this context, we have synthesized
such conjugates (5a–t and 6a–d) and evaluated them for their cytotoxic potential.
Interestingly, some of the conjugates such as 5c, 5d, 5f, 5i, 5k, 5m, 5o, 5p and 5s exhibited
considerable antiproliferative activity with GI50 0.26–28 µM against different human cancer
cell lines (Table 2). Further, the results of cell cycle and mitotic arrest showed that these
conjugates affect the G2/M phase along with the inhibition of tubulin assembly. The flow
cytometric analysis was performed to determine the effect of active conjugates in altering the
cell cycle phase distribution in MCF-7 cells.
2. Results and Discussion
2.1. Chemistry
The synthetic strategy for the preparation of benzimidazole-oxindole conjugates is
depicted in Scheme 1. Initially, oxidative cyclization of methyl ester of 3,4-diaminobenzoic
acid (8) with various substituted benzaldehydes (9a‒e) and thiophene-2-carbaldehyde in
the presence of Na2S2O5 afforded the corresponding methyl benzimidazole carboxylate
derivatives (10a‒e and 13). The reduction of these esters 10a‒e and 13 with lithium
aluminium hydride in dry THF produced the corresponding alcohols, which were taken to
further oxidation by Dess–Martin periodinane in CH2Cl2 to the respective benzimidazole
carbaldehydes (11a‒e and 14) in good yields. Finally, the required benzimidazole-oxindole
conjugates (5a‒t and 6a‒d) were synthesized by employing the Knoevenagel condensation
between equimolar mixture of these carbaldehydes (11a‒e and 14) and oxindoles (12a‒d)
1
as shown in Scheme 1. The compound compounds were characterized by means of H
13
NMR, C NMR, HRMS, and IR spectral data. All the compounds were obtained as Z
isomers and confirmed by comparing the data of the previous reports [31,32].
<Insert Scheme 1>
2.2. Cytotoxicity
To evaluate the cytotoxic potential of these conjugates (MTT) assay [33] was
performed against four human cancer cell lines, A549 (lung), MCF-7 (breast), DU-145
(prostate) and HT-29 (colon) in comparison to nocodazole and the results are summarized as
IC50 values in Table 1. The results revealed that these conjugates showed promising cytotoxic
activity with IC50 values ranging from 1.84‒19.9 µM against different cancer cell lines. In
3

this assay conjugates 5c and 5p showed significant anticancer activity against human breast
cancer cell line (MCF-7) with IC50 values 1.84 µM and 1.97 µM respectively.
<Insert Table 1 >
To further validate their potency in other cell lines, benzimidazole-oxindole
conjugates were evaluated by NCI-USA with regard to anticancer activity against 60 cancer
cell lines some of the conjugates (5c, 5d, 5f, 5i, 5k, 5m, 5o, 5p and 5s) in this series showed
noticeable cytotoxicity and were taken up for the five dose screening for further evaluation.
These selected conjugates 5c, 5d, 5f, 5i, 5k, 5m, 5o, 5p and 5s exhibited considerable
cytotoxicity with GI50 values ranging from 0.28-28.3 µM. Interestingly, conjugates 5c and 5p
exhibited GI50 values ranging from 0.28‒13.8 and 0.57‒6.34 µM respectively and these
results are showed in Table 2. Conjugate 5c, which contains a methoxy group at C4 position
of the C-ring and chloro group at C5 position of the B-ring showed significant cytotoxic
activity. However change the position of chloro group from C5 to C6 in the B-ring decreased
the activity as in case of in 5d. Conjugate 5p bearing fluoro groups at C3 and C5 positions of
the C-ring and chlorine at C6 position of the B-ring showed prominent activity. On the other
hand, change in the position of chloro group from C6 to C5 in the B-ring resulted in moderate
activity. On the basis of the structure of nocodazole (4, Fig. 1), we decided to replace the C-
ring with 2-thienyl scaffold, while the corresponding compounds (6a‒d) were found to be
moderate by active. The SAR revealed that among these compounds, conjugates with chloro
group on the B-ring showed better activity than other compounds, while substituents on the
C-ring have little effect on the activity. By and large, the conjugates 5c and 5p were
considered as promising among these conjugates assayed and were taken up for detailed
biological studies like cell cycle analysis, tubulin polymerization studies and
immunohistochemistry studies. Finally, the molecular docking studies also performed to find
out the mode binding on colchicines binding site on tubulin.
<Insert Table 2 >
2.3. Cell cycle analysis
Anticancer drugs are known to interact with cells leading to cell growth arrest or cell
death. To shed more light on the mechanism responsible for anticancer activity the potential
4

conjugates 5c and 5p were examined for their effect on the cell cycle analysis [34]. In this
study, MCF-7 cells were treated with these conjugates for 48 h and nocodazole was used as
the reference compound in this study. Interestingly, the results obtained from this assay
clearly indicated that these conjugates showed G2/M cell cycle arrest as compared to the
untreated control cells. These conjugates showed 52.41% and 37.62% of cell accumulation in
G2/M phase at 1 µM concentration, whereas they exhibited 63.20 and 53.00 % of cell
accumulation in G2/M phase at 2 µM concentration, respectively (Fig. 2 and Table 2). The
reference compound nocodazole showed 54.65 % cell accumulation in G2/M phase at 2 µM
concentration, whereas in control (untreated cells) 11.41 % of G2/M phase was observed.
<Insert Figure 2 >
<Insert Table 3 >
2.4. Tubulin polymerization study
Conjugates that alter cell-cycle parameters with preference to G2/M blockade are
known to exhibit effects on tubulin assembly. Moreover, inhibition of tubulin polymerization
is strongly associated with G2/M cell cycle arrest [35]. These conjugates arrest the cell cycle
at G2/M phase, hence it was considered important to investigate the tubulin polymerization
aspect. As tubulin subunits heterodimerize and self-assemble to form microtubules in a time
dependent manner and have investigated the progression of tubulin polymerization [36] by
monitoring the increase in fluorescence emission at 420 nm (excitation wavelength is 360
nm) in 384 well plate for 1 h at 37 °C with and without the conjugates at 1 μM concentration.
In this assay, conjugates 5c and 5p inhibited tubulin polymerization by 70.5 and 65.9 %,
respectively, and in comparison, 68.2 % of inhibition was observed with nocodazole. (Fig. 3).
This was followed by the evaluation of IC50 values for these compounds and the results
showed that both 5c and 5p displayed inhibition of tubulin assembly in comparison to
nocodazole as shown in Table 3. Moreover, the effect of these conjugates on the inhibition of
tubulin assembly correlated well with their significant antiproliferative activity.
<Insert Figure 3 >
<Insert Table 4 >
5

2.5. Immunohistochemistry studies on tubulin
We also investigated alterations in the microtubule network in the MCF-7 cells
induced by these conjugates (5c and 5p) by carrying out immunohistochemistry studies using
fluorescence microscope, as most antimitotic agents affect microtubules [37]. Therefore,
MCF-7 cells were treated with 5c and 5p at 1 µM concentration for 48 h and the results
demonstrated a well-organized microtubular network in control cells. However, cells treated
with these conjugates and using nocodazole as standard showed disrupted microtubule
organization as shown in Fig. 4, thus confirming the inhibition of tubulin polymerization.
<Insert Figure 4 >
2.6. Molecular modeling studies
The conjugates that have been synthesised in the present study are designed based on
nocodazole an aminobenzimidazole scaffold a well known tubulin polymerization inhibitor.
It has been proved that the binding of nocodazole to the tubulin affects the colchicine
binding, indicating that its binding site is closer to colchicine, that is between the α and β
tubulin subunits [38]. We performed docking studies to investigate the possible binding mode
of these conjugates. Coordinates of protein structure of tubulin-colchicine were obtained from
the Protein Data Bank (PDB ID 3E22) [39] and the docking was accomplished into the
colchicine binding site of the tubulin using AutoDock 4.2 software and analyzed by PYMOL
software [40]. Fig. 5 shows that carbonyl group on the oxindole ring of 5c establishes
hydrogen binding with αGly11, βAsn249 and βLys254 and this oxindole ring is surrounded
by αTyr224, αSer178 and αThr179 residues. Whereas the benzimidazole ring of 5c shows
hydrophobic contacts with βLeu254, βLeu255 and αAsn101 and the methoxyphenyl group of
the benzimidazole ring is buried in the hydrophobic pocket by βLys352 βAsn258, βAsn350,
βVal351, βVal181 and βMet259 residues located in β-tubulin, this is similar to that of
trimethoxyphenyl group in case of colchicines. Similarly carbonyl group on oxindole moiety
in conjugates 5p showed hydrogen binding with βAsn249 and βLys254. Figure 6 shows the
molecular docking pose of conjugate 5p (red) and 5c (yellow) overlaying with that of
nocodazole (pink). Whereas Fig. 7 shows that the binding of conjugate 5c (yellow) and 5p
(red) is very similar to the pose of the colchicine (green) with oxindole ring of both 5c and 5p
placed towards the α-tubulin (blue) chain. Thus these docking studies suggest that these
conjugates interact with both α-and β-tubulin in the colchicine-binding pocket.
6

<Insert Table 5 >
<Insert Figure 5 >
<Insert Figure 6 >
<Insert Figure 7 >
3. Conclusion
In conclusion, we have designed, synthesized some benzimidazole-oxindole
conjugates that were evaluated for their cytotoxic activity and tubulin polymerization
inhibitory potential. Conjugates 5c and 5p showed significant cytotoxic activity against
human breast cancer cell line (MCF-7) with IC50 values of 1.84 and 1.97 µM respectively.
Further, the flow cytometric analysis revealed that these conjugates caused cell cycle arrest
and accumulated cells in the G2/M phase. Furthermore, these conjugates effectively inhibited
microtubule assembly in MCF-7 cells. Moreover, docking experiments showed that they
interact and bind efficiently with the tubulin protein. The results suggest that these new
benzimidazole-oxindole conjugates are potent inhibitors of tubulin polymerization, and are
amenable for further structural modifications in the discovery and development of effective
chemotherapeutics for the treatment of breast cancer.
4. Experimental
4.1. General
The majority of the solvents were purified by distillation under nitrogen from the indicated
drying agent and used fresh: dichloromethane (calcium hydride), tetrahydrofuran (sodium
benzophenone ketyl), acetone (potassium permanganate), and acetonitrile (phosphorous
pentoxide). Reaction progress was monitored by thin-layer chromatography (TLC) using
GF254 silica gel with fluorescent indicator on glass plates. Visualization was achieved with
UV light and iodine vapor unless otherwise stated. Chromatography was performed using
1
13
Acme silica gel (100–200 mesh). H and C NMR spectra were recorded on INOVA (400
MHz) or Gemini Varian-VXR-unity (200 MHz) or Bruker UXNMR/XWIN-NMR (300
MHz) spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shifts
are reported in parts per million (ppm) downfield from tetramethylsilane. Spin multiplicities
7

are described as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), and m
(multiplet). Coupling constants are reported in Hertz (Hz). Mass spectra were recorded on a
VG-7070H Micromass mass spectrometer at 200 _C, 70 eV with trap current of 200 lA, and 4
kV acceleration voltage. FABMS spectra were recorded on LSIMS-VG-AUTOSPEC-
Micromass. Melting points were recorded on Electrothermal 9100 and are uncorrected. All
computational studies were done with a Red Hat Enterprise Linux version 5.0 using Maestro
software version 9.5 (Schrödinger, LLC, New York, NY, 2013).
4.2. Chemistry
4.2.1. Methyl 2-(4-methoxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (10a).
A mixture of methyl 3,4-diaminobenzoate (8, 830 mg, 5 mmol) and 4-methoxy benzaldehyde
(9a, 680 mg, 5 mmol) in ethanol (25 mL) was heated with an aqueous solution of sodium
pyrosulfite (1.42 gm, 1.5 mmol, 5 mL) and then refluxed for 4 to 6 h, until TLC indicated that
the reaction was complete. The ethanol was evaporated under vacuum and the residue was
dissolved in CHCl3 (2x15 mL) and washed with water. The combined organic phases were
dried over anhydrous Na2SO4,and the solvent was removed under vacuum and purified by
column chromatography MeOH-CH2Cl2 (0.5: 9.5) to obtain the pure product 10a as a white
o
solid (952 mg, 68%); mp: 231‒234 C; 1H NMR (500 MHz, CDCl3): δ 8.28 (s, 1H), 8.12 (d,
J = 9.1 Hz, 2H), 7.92 (d, J = 9.0 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 2H),
3.94 (s, 3H), 3.85 (s, 3H); MS (ESI): m/z 283 [M+H]+.
4.2.2. Methyl 2-(3-fluorophenyl)-1H-benzo[d]imidazole-5-carboxylate (10b).
Compound 10b was prepared according to the method described for compound 10a,
employing 3-fluorobenzaldehyde (9b, 620 mg, 5 mmol) and methyl 3,4-diaminobenzoate (8,
830 mg, 5mmol) to obtain the pure product 10b as a white solid (938 mg, 70%); mp:
238‒240 oC; 1H NMR (500 MHz, CDCl3): δ 8.36 (s, 1H), 8.28 (dd, J = 8.3, 5.4 Hz, 2H), 7.97
(d, J = 8.4 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.25 (t, J = 8.3 Hz, 2H), 3.98 (s, 3H); MS (ESI):
m/z 271 [M+H]+.
4.2.3. Methyl 2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-carboxylate (10c).
Compound 10c was prepared according to the method described for compound 10a,
employing 4-(trifluoromethyl)benzaldehyde (9c, 870 mg, 5 mmol) and methyl 3,4-
diaminobenzoate (8, 830 mg 5 mmol mmol) to obtain the pure product 10c as a white solid
(1.14 gm, 72%); mp: 275‒277 oC; 1H NMR (300 MHz, CDCl3+DMSO‒d6): δ 8.39 (d, J = 7.8
8

Hz, 2H), 7.95 (t, J = 7.9 Hz, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.72 (s, 1H), 7.56 (d, J = 8.3 Hz,
1H), 3.94 (s, 3H); MS (ESI): m/z 331 [M+H]+.
4.2.4. Methyl 2-(3,5-difluorophenyl)-1H-benzo[d]imidazole-5-carboxylate (10d).
Compound 10d was prepared according to the method described for compound 10a,
employing 3,5-difluorobenzaldehyde (9d, 710 mg, 5 mmol) and methyl 3,4-diaminobenzoate
(8, 830 mg 5 mmol) to obtain the pure product 10d as a white solid (1.0 gm, 70%); mp:
295‒297 oC; 1H NMR (300 MHz, CDCl3): δ 8.44 (d, J = 8.3 Hz, 2H), 7.98 (t, J = 8.6 Hz, 1H),
7.90–7.75 (m, 3H), 3.98 (s, 3H); MS (ESI): m/z 289 [M+H]+.
4.2.5. Methyl 2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole-5-carboxylate.(10e).
Compound 10e was prepared according to the method described for compound 10a,
employing 3,4,5-trimethoxybenzaldehyde (9e, 980 mg, 5 mmol) and methyl 3,4-diamino
benzoate (8, 830 mg, 5 mmol) to obtain the pure product 10e as a white solid (1.1 gm, 68%);
o
mp: 243‒245 C; 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 8.1 Hz, 1H), 7.63 (s, 1H), 7.53
(s, 2H), 7.46 (s, 1H), 3.98 (s, 6H), 3.95 (s, 3H), 3.91 (s, 3H); MS (ESI): m/z 343 [M+H]+.
4.2.6. Methyl 2-(thiophen-2-yl)-1H-benzo[d]imidazole-5-carboxylate (13).
Compound 13 was prepared according to the method described for compound 10a,
employing thiophene-2-carbaldehyde (560 mg, 5 mmol) and methyl 3,4-diaminobenzoate (8,
830 mg, 5 mmol) to obtain the pure product 13 as a white solid (782 mg, 62%); mp: 212‒214
oC; 1H NMR (300 MHz, DMSO‒d6): 8.23 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.07 (s, 1H), 7.84
(s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 3.86 (s, 3H); MS (ESI): m/z 259
[M+H]+.
4.3. General procedure for the preparation of compounds (11a-e & 14).
LiAlH4 (4 mmol) was added to a fine suspension of compounds 10a–e & 13 (3 mmol) in dry
THF (60 mL) at 0 oC and was stirred for 4 h at RT. The reaction mixture was then quenched
with saturated aq NH4Cl and diluted with EtOAc (150 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated under vacuum. The obtained crude was used directly for
the next step without any further purification. The crude obtained was taken in dry CH2Cl2
(100 mL). To that Dess–Martine periodinane (DMP) (4 mmol) was added, stirred at room
temperature for about 3 h. The reaction mixture was then quenched with a 1:1 mixtureof aq
sodium thiosulfate and NaHCO3 (40 mL) and dilutedwith CH2Cl2 (50 mL). The organic layer
9

was dried over anhydrous Na2SO4 and concentrated under vacuum to obtain the crude
product. The resulting crude product was purified by column chromatography to afford
products 11a–e & 14 in high purity.
4.3.1. 2-(4-Methoxyphenyl)-1H-benzo[d]imidazole-5-carbaldehyde (11a).
Compound 11a was prepared according to the general procedure. Compound 11a obtained as
a white solid: (604 mg, 80% yield); mp: 179‒181 oC; 1H NMR (500 MHz, CDCl3): δ 9.98 (s,
1H), 8.28 (s, 1H), 8.02 (d, J = 9.0 Hz, 2H), 7.94 (dd, J = 9.0, 1.1 Hz, 1H), 7.06 (d, J = 8.3
Hz, 1H), 6.95 (d, J = 8.3 Hz, 2H), 3.83 (s, 1H); MS (ESI): m/z 253 [M+H]+.
4.3.2. 2-(3-Fluorophenyl)-1H-benzo[d]imidazole-5-carbaldehyde (11b).
Compound 11b was prepared according to the general procedure. Compound 11b obtained as
1
a white solid: (547 mg, 76% yield); mp: 188‒190 oC; H NMR (300 MHz,
CDCl3+DMSO‒d6): δ 10.04 (s, 1H), 8.34–8.15 (m, 2H), 7.90 (s, 1H), 7.78 (s, 1H), 7.64 (s,
1H), 7.26 (t, J = 8.6 Hz, 2H); MS (ESI): m/z 241 [M+H]+.
4.3.3. 2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-carbaldehyde (11c).
Compound 11c was prepared according to the general procedure. Compound 11c obtained as
o
a white solid: (617 mg, 71% yield) ; mp: 208‒210 C; 1H NMR (500 MHz, CDCl3): δ 10.05
(s, 1H), 8.38 (d, J = 7.5 Hz, 2H), 8.23 (s, 1H), 7.84–7.80 (m, 2H), 7.78 (d, J = 7.8 Hz, 2H),
7.63 (d, J = 8.1 Hz, 1H); MS (ESI): m/z 291 [M+H]+.
4.3.4. 2-(3,5-Difluorophenyl)-1H-benzo[d]imidazole-5-carbaldehyde (11d).
Compound 11d was prepared according to the general procedure. Compound 11d obtained as
o
1
a white solid (526 mg, 68% yield) yield; mp: 217‒219 C; H NMR (300 MHz,
CDCl3+DMSO‒d6): δ 10.05 (s, 1H), 8.38 (d, J = 8.2 Hz, 2H), 7.78 (t, J = 8.4 Hz, 1H), 7.84–
7.71 (m, 3H); MS (ESI): m/z 259 [M+H]+.
4.3.5. 2-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazole-5-carbaldehyde (11e).
Compound 11e was prepared according to the general procedure. Compound 11e obtained as
a white solid: (651 mg, 70% yield); mp: 216‒218 oC; 1H NMR (300 MHz,
CDCl3+DMSO‒d6): δ 10.07 (s, 1H), 8.16 (s, 1H), 7.84 (d, J = 7.4 Hz, 1H), 7.42 (s, 2H), 7.28
(s, 1H), 3.91 (s, 3H), 3.79 (s, 6H); MS (ESI): m/z 313 [M+H]+.
10

4.3.6. 2-(Thiophen-2-yl)-1H-benzo[d]imidazole-5-carbaldehyde (14).
Compound 14 was prepared according to the general procedure. Compound 14 obtained as a
white solid: (471 mg, 69% yield); mp: 196‒198 oC; 1H NMR (300 MHz, DMSO‒d6): δ 10.38
(s, 1H), 8.32 (s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.13 (s, 1H), 7.86 (s, 1H), 7.78 (dd, J = 6.4, 3.6
Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H); MS (ESI): m/z 229 [M+H]+.
4.4. General procedure for the preparation of compounds (5a-t and 6a‒d).
4.4.1. (Z)-3-((2-(4-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-one (5a).
A mixture of aldehyde 11a (126 mg, 0.5 mmol) and indolin-2-one (12a, 66.5 mg, 0.5 mmol)
were dissolved in EtOH (10 mL) and piperidine (2–3 drops) was added. The reaction mixture
was refluxed for 3–5 h. After the completion of the reaction, the reaction mixture was
allowed to cool to room temperature and the precipitated product was collected by vacuum
filtration and washed with EtOH (5 mL), then recrystallized from ethanol to afford pure
o
compound 5a as a yellow solid in 113 mg, 62% yield. mp: 172‒174 C; IR (KBr): 3420,
3157, 2931, 2357, 1683, 1674, 1570, 1470, 1432, 1257, 1174, 1027, 964, 847 cm-1; 1H NMR
(300 MHz, DMSO−d6): δ 10.45 (bs, 1H), 8.20–8.07 (m, 3H), 7.96 (d, J = 7.5 Hz, 1H), 7.78
(s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.59 (t, J = 8.3 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.14 (d, J
13
= 7.5 Hz, 2H), 6.89–6.74 (m, 2H), 3.86 (s, 3H); C NMR (75 MHz, DMSO−d6): δ 168.9,
160.7, 153.1, 142.5, 137.4, 129.3, 128.1, 127.9, 127.7, 125.5, 124.0, 123.8, 122.1, 121.8,
121.2, 120.7, 114.1, 109.8, 55.1; MS (ESI): m/z 368 [M+H]+; HRMS (ESI): calcd for
C23H18O2N3 m/z 368.13869 [M+H]+; found 368.13935.
4.4.2. (Z)-5-Methoxy-3-((2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-
2-one (5b).
Compound 5b was prepared according to the method described for compound 5a, employing
aldehyde 11a (126 mg, 0.5 mmol) and 5-methoxyindolin-2-one (12b, 81 mg, 0.5 mmol) to
o
obtain the pure product 5c as a yellow solid (114 mg, 58%); mp: 177‒179 C; IR (KBr):
3422, 3153, 2931, 2352, 1681, 1642, 1576, 1474, 1435, 1254, 1179, 1029, 961, 864 cm-1; 1H
NMR (300 MHz, DMSO−d6): δ 10.41 (bs, 1H), 8.25–8.16 (m, 3H), 7.82 (s, 1H), 7.72 (d, J =
8.1 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.40 (s, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.82–6.70 (m,
2H), 3.90 (s, 3H), 3.68 (s, 3H); 13C NMR (75 MHz, DMSO−d6): δ 168.9, 160.6, 154.3, 153.7,
153.1, 137.4, 136.2, 128.0, 127.9, 127.6, 126.1, 124.5, 123.7, 122.0, 121.9, 113.9, 109.9,
108.5, 55.0; MS (ESI): m/z 398 [M+H]+; HRMS (ESI): calcd for C24H20O3N3 m/z 398.14923
[M+H]+; found 398.14992.
11

4.4.3. (Z)-5-Chloro-3-((2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (5c).
Compound 5c was prepared according to the method described for compound 5a, employing
aldehyde 11a (126 mg, 0.5 mmol) and 5-chloroindolin-2-one (12c, 83 mg, 0.5 mmol) to
o
obtain the pure product 5c as a yellow solid (128 mg, 64%); mp: 194‒196 C; IR (KBr):
3422, 3170, 2359, 1702, 1665, 1594, 1477, 1250, 1175, 1114, 1026, 967, 740 cm-1; 1H NMR
(300 MHz, DMSO−d6): δ 10.76 (bs, 1H), 8.17 (d, J = 8.3 Hz, 2H), 8.09 (s, 1H), 7.88 (s, 1H),
7.77 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H),7.58 (s, 1H), 7.25 (dd, J = 8.6, 1.8 Hz, 1H),
7.14 (d, J = 8.6 Hz, 2H), 6.94–6.75 (m, 1H), 3.85 (s, 3H); 13C NMR (75 MHz, DMSO‒d6): δ
168.5, 167.1, 160.9, 153.1, 141.3, 140.5, 138.6, 129.1, 128.2, 127.4, 125.2, 124.7, 122.8,
122.0, 121.2, 119.2, 114.3, 111.3, 110.4, 55.3; MS (ESI): m/z 402 [M+H]+; HRMS (ESI):
calcd for C23H17O2N3Cl m/z 402.09674 [M+H]+; found 402.09587.
4.4.4. (Z)-6-Chloro-3-((2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (5d).
Compound 5d was prepared according to the method described for compound 5a, aldehyde
11a (126 mg, 0.5 mmol) and 6-chloroindolin-2-one (12d, 83 mg, 0.5 mmol) to obtain the
o
pure product 5d as a yellow solid (122 mg, 61%); mp: 199‒201 C; IR (KBr): 3426, 3172,
2360, 1669, 1647, 1587, 1469, 1257, 1170, 1118, 1028, 961, 742 cm-1; 1H NMR (300 MHz,
DMSO−d6): δ 10.46 (bs, 1H), 8.16 (d, J = 8.6 Hz, 2H), 7.86 (s, 1H), 7.77 (d, J = 8.3 Hz, 2H),
7.68 (d, J = 7.1 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H), 6.97–6.75 (m,
2H), 3.90 (s, 3H); 13C NMR (75 MHz, DMSO−d6): δ 168.9, 160.7, 153.1, 142.5, 137.4,
129.3, 128.1, 127.9, 127.7, 125.5, 124.0, 123.8, 122.1, 121.8, 121.2, 120.7, 114.1, 109.8,
55.1; MS (ESI): m/z 402 [M+H]+; HRMS (ESI): calcd for C23H17O2N3Cl m/z 402.09674
[M+H]+; found 402.09587.
4.4.5. (Z)-3-((2-(3-Fluorophenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-one (5e).
Compound 5e was prepared according to the method described for compound 5a, employing
aldehyde 11b (120 mg,0.5 mmol) and indolin-2-one (12a, 66.5 mg, 0.5 mmol) to obtain the
o
pure product 5e as a yellow solid (106 mg, 60%); mp: 194‒196 C; IR (KBr): 3420, 3223,
1
3160, 2356, 1703, 1610, 1466, 1347, 1222, 1164, 1113, 964, 864, 742 cm-1; H NMR (300
MHz, DMSO−d6): δ 10.67 (bs, 1H), 8.37–8.19 (m, 2H), 7.97 (s, 1H), 7.82 (s, 1H), 7.73 (d, J
= 7.5 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 8.8 Hz, 2H),7.17
(t, J = 7.3 Hz, 1H), 6.95–6.80 (m, 2H); 13C NMR (75 MHz, DMSO−d6): δ 168.9, 164.3 and
12

161.2 (d, J = 248.1 Hz ) 152.2, 142.7, 140.1, 137.4, 129.7, 129.0, 128.9 (d, J = 8.2 Hz ),
128.2, 126.3 (d, J = 2.7 Hz ), 125.8, 124.3, 121.9, 121.2 ,120.9, 116.0 (d, J = 22.0 Hz ),
110.0, 109.1; MS (ESI): m/z 356 [M+H]+; HRMS (ESI): calcd for C22H15ON3F m/z
356.11868 [M+H]+; found 356.11937.
4.4.6. (Z)-3-((2-(3-Fluorophenyl)-1H-benzo[d]imidazol-6-yl)methylene)-5-methoxyindolin-2-
one (5f).
Compound 5f was prepared according to the method described for compound 5a, employing
aldehyde 11b (120 mg,0.5 mmol) and 5-methoxyindolin-2-one (12b, 111 mg, 0.83 mmol) to
o
obtain the pure product 5f as a yellow solid (109 mg, 57%); mp: 196‒198 C; IR (KBr):
3424, 3167, 2359, 2340, 1697, 1608, 1452, 1414, 1231, 1207, 1020, 840, 746 cm-1; 1H NMR
(300 MHz, DMSO–d6): δ 10.43 (bs, 1H), 8.29–8.22 (m, 2H), 7.99 (s, 1H), 7.79 (s, 1H), 7.76
(d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.44 (t, J = 8.8 Hz, 2H), 7.32 (d, J = 1.5 Hz,
13
1H), 6.89–6.72 (m, 2H), 3.62 (s, 3H); C NMR (75 MHz, DMSO–d6): δ 168.8, 164.9 and
161.6 (d, J = 248.4 Hz ), 153.8, 152.0, 137.4, 136.4, 129.0 (d, J = 8.2 Hz ), 128.3, 126.5,
125.9, 124.3, 121.9, 116.0 (d, J = 22.0 Hz ), 14.6, 110.2, 108.6, 55.2; MS (ESI): m/z 386
[M+H]+; HRMS (ESI): calcd for C22H17O2N3F m/z 386.12946 [M+H]+; found 386.12993.
4.4.7. (Z)-5-Chloro-3-((2-(3-fluorophenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (5g).
Compound 5g was prepared according to the method described for compound 5a, employing
aldehyde 11b (120 mg,0.5 mmol) and 5-chloroindolin-2-one (12c, 83 mg, 0.5 mmol) to
o
obtain the pure product 5g as a yellow solid (126 mg, 65%); mp: 195‒197 C; IR (KBr):
3420, 3166, 2924, 2351, 1708, 1672, 1607, 1556, 1415, 1235, 1159, 1072, 1019, 847 cm-1;
1H NMR (300 MHz, DMSO−d6): δ 10.77 (bs, 1H), 8.33–8.23 (m, 2H), 7.99 (s, 1H), 7.83 (s,
1H), 7.75 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.42 (t, J =
8.8Hz, 2H), 7.05–6.81 (m, 2H); 13C NMR (75 MHz, DMSO–d6): δ 168.8, 164.9 and 161.6 (d,
J = 247.5 Hz ), 152.3, 144.0, 141.2, 138.3, 133.6, 132.2, 129.0 (d, J = 8.2 Hz ), 128.1 (d, J =
12.1 Hz ), 124.4, 123.1, 120.7, 120.1, 116.0 (d, J = 21.4 Hz ), 109.9, 109.1; MS (ESI): m/z
390 [M+H]+; HRMS (ESI): calcd for C22H14ON3ClF m/z 390.07317 [M+H]+; found
390.07482.
4.4.8. (Z)-6-Chloro-3-((2-(3-fluorophenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (5h).
13

Compound 5h was prepared according to the method described for compound 5a, employing
aldehyde 11b (120mg, 0.5 mmol) and 6-chloroindolin-2-one (12d, 83 mg, 0.5 mmol) to
o
obtain the pure product 5h as a yellow solid (122 mg, 63%); mp: 206‒208 C; IR (KBr):
3427, 3160, 2918, 1710, 1678, 1614, 1550, 1420, 1230, 1162, 1020, 840, 749 cm-1; 1H NMR
(300 MHz, DMSO–d6): δ 10.78 (bs, 1H), 8.33–8.21 (m, 2H), 7.99 (s, 1H), 7.80 (s, 1H), 7.74
(d, J = 7.5 Hz, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.42 (t, J = 8.6 Hz,
13
2H), 7.00–6.78 (m, 2H); C NMR (75 MHz, DMSO–d6): δ 168.8, 165.0 and 161.5 (d, J =
248.6 Hz ), 152.1, 144.0, 138.1, 133.6, 129.0 (d, J = 8.2 Hz ), 128.1, 126.1 (d, J = 2.4 Hz ),
124.8, 123.1, 120.7, 120.1, 116.1 (d, J = 22.0 Hz ), 109.9, 108.9; MS (ESI): m/z 390 [M+H]+;
HRMS (ESI): calcd for C22H14ON3ClF m/z 390.07317 [M+H]+; found 390.07482.
4.4.9. (Z)-3-((2-(4-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (5i).
Compound 5i was prepared according to the method described for compound 5a, employing
aldehyde 11c (145mg, 0.5 mmol) and indolin-2-one (12a, 66.5 mg, 0.5 mmol) to obtain the
o
pure product 5i as a yellow solid (129 mg, 64%); mp: 313‒315 C; ; IR (KBr): 3420, 3170,
2352, 1668, 1628, 1468, 1320, 1115, 1010, 847, 809, 740 cm-1; 1H NMR (300 MHz, DMSO–
d6): δ 10.69 (bs, 1H), 9.21 (s, 1H), 8.43 (d, J = 7.9 Hz, 2H), 8.17 (d, J = 8.1 Hz, 1H), 7.94 (d,
J = 9.1 Hz, 3H), 7.75 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.19 (t, J = 7.3 Hz, 1H), 6.99 (t, J =
7.3 Hz, 1H), 6.83 (d, J = 7.5 Hz, 1H); 13C NMR (75 MHz, DMSO–d6): δ 167.4, 151.6, 140.2,
138.2, 133.4, 130.1, 129.7, 129.2, 128.9, 128.3, 127.2, 125.9 (q, J = 2.2 Hz ), 125.3, 124.7,
119.3, 109.1; MS (ESI): m/z 406 [M+H]+; HRMS (ESI): calcd for C23H15ON3F3 m/z
406.10782 [M+H]+; found 406.10587.
4.4.10.
(Z)-5-Methoxy-3-((2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-
yl)methylene) indolin-2-one (5j).
Compound 5j was prepared according to the method described for compound 5a, employing
aldehyde 11c (145 mg,0.5 mmol) and 5-methoxyindolin-2-one (12b, 81, 0.5 mmol) to obtain
the pure product 5j as a yellow solid (132 mg, 61%); mp: 293‒295 oC; IR (KBr): 3420, 3160,
2355, 1672, 1618, 1470, 1328, 1113, 1015, 915, 845, 741 cm-1; 1H NMR (300 MHz, DMSO–
d6): δ 10.48 (bs, 1H), 9.22 (s, 1H), 8.44 (d, J = 8.1 Hz, 2H), 8.17 (s, 1H), 7.99 (s, 1H), 7.96
(d, J = 8.1 Hz, 2H), 7.72 (d, J = 8.3 Hz, 1H), 7.45 (s, 1H), 6.87–6.68 (m, 2H), 3.79 (s, 3H);
13C NMR (75 MHz, CDCl3+DMSO–d6): δ 168.8, 154.6, 153.8, 138.4, 137.3, 136.4, 134.0,
133.4, 127.2, 126.6, 125.9 , (q, J = 2.7 Hz ), 125.2, 125.0, 122.9, 121.9, 114.9, 110.2, 109.6,
14

108.6, 105.5, 55.2; MS (ESI): m/z 436 [M+H]+; HRMS (ESI): calcd for C24H17ON3F3 m/z
436.12073 [M+H]+; found 436.12437.
4.4.11. (Z)-5-Chloro-3-((2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-yl) methylene)
indoli-2-one (5k).
Compound 5k was prepared according to the method described for compound 5a, employing
aldehyde 11c (145 mg,0.5 mmol) and 5-chloroindolin-2-one (12c, 83 mg, 0.5 mmol) to obtain
the pure product 5k as a yellow solid (151 mg, 69%); mp: 317‒319 oC; IR (KBr): 3417, 3166,
2351, 1668, 1628, 1471, 1325, 1115, 1015, 947, 847, 741 cm-1; 1H NMR (300 MHz, DMSO–
d6): δ 10.63 (bs, 1H), 8.39 (d, J = 7.9 Hz, 2H), 7.95 (s, 1H), 7.87–7.80 (m, 3H).), 7.76 (s, 1H),
13
7.64 (s, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.15 (dd J = 8.3, 1.7 Hz, 1H), 6.89–675(m, 1H); C
NMR (75 MHz, CDCl3+DMSO–d6): δ 168.7, 151.2, 141.2, 135.0, 133.4, 132.3, 130.1, 128.9,
128.2, 127.2, 125.9 (q, J = 2.2 Hz ), 123.6, 123.1, 122.2, 120.7, 120.6, 118.6, 115.3, 109.9,
109.0; MS (ESI): m/z 440 [M+H]+; HRMS (ESI): calcd for C23H14ON3ClF3 m/z 440.07648
[M+H]+; found 440.07691.
4.4.12. (Z)-6-Chloro-3-((2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-6-yl)methylene)
indolin-2-one (5l).
Compound 5l was prepared according to the method described for compound 5a, employing
aldehyde 11c (145 mg,0.5 mmol) and 6-chloroindolin-2-one (12d, 83 mg, 0.5 mmol) to
obtain the pure product 5l as a yellow solid (146 mg, 67%); mp: 318‒320 oC; IR (KBr): 3420,
1
3160, 2358, 1670, 1637, 1471, 1320, 1114, 1020, 845, 742 cm-1; H NMR (300 MHz,
DMSO–d6): δ 10.83 (bs, 1H), 8.41 (d, J = 7.7 Hz, 2H), 8.06 (d, J = 7.9 Hz, 1H), 7.99 (s, 1H),
7.94 (d, J = 7.3 Hz, 2H), 7.84 (s, 1H), 7.75 (d, J = 7.9 Hz, 2H), 7.62 (s, 1H), 7.08–6.80 (m,
13
2H); C NMR (75 MHz, CDCl3+DMSO–d6): δ 168.5, 151.4, 141.2, 139.7, 138.6, 133.2,
128.8, 128.3, 127.5, 126.9, 125.5, 125.2 (q, J = 2.8 Hz), 124.8, 123.5, 122.6, 121.9, 121.3,
119.0, 110.9, 110.1; MS (ESI): m/z 440 [M+H]+; HRMS (ESI) calcd for C23H14ON3ClF3 m/z
440.07648 [M+H]+; found 440.07720.
4.4.13.
(Z)-3-((2-(3,5-Difluorophenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-one
(5m).
Compound 5m was prepared according to the method described for compound 5a, employing
aldehyde 11d (129 mg,0.5 mmol) and indolin-2-one (12a, 66.5 mg, 0.5 mmol) to obtain the
o
pure product 5m as a yellow solid (111 mg, 60%); mp: 306‒308 C; IR (KBr): 3427, 3165,
15

1
2924, 2352, 1664, 1620, 1556, 1454, 1414, 1121, 945, 866, 740 cm-1; H NMR (300 MHz,
DMSO–d6): δ 10.63 (bs, 1H), 8.02 (s, 1H), 7.90 (d, J = 6.1 Hz, 2H), 7.80 (s, 1H), 7.75 (d, J =
2.8 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.43 (t, J = 9.0 Hz, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.00
13
(t, J = 7.3 Hz, 1H), 6.93–6.80 (m, 2H); C NMR (75 MHz, CDCl3+DMSO–d6): δ 168.8,
164.4 and 161.1(d, J = 245.9 Hz), 164.2 and 160.9 (d, J = 246.4 Hz) , 150.7, 142.7, 138.1,
137.1, 132.9, 129.8, 128.9 (d, J = 7.7 Hz),, 128.3, 126.1, 124.6, 122.0, 121.1 (t, J = 7.8 Hz),
110.0, 109.7 (d, J = 8.8 Hz), 109.5 (d, J = 8.8 Hz), 109.1, 105.4; MS (ESI): m/z 374 [M+H]+;
HRMS (ESI): calcd for C22H14ON3F2 m/z 374.10965 [M+H]+; found 374.10995.
4.4.14.
(Z)-3-((2-(3,5-Difluorophenyl)-1H-benzo[d]imidazol-6-yl)methylene)-5-
methoxyindolin-2-one (5n).
Compound 5n was prepared according to the method described for compound 5a, employing
aldehyde 11d (129 mg,0.5 mmol) and 5-methoxyindolin-2-one (12b, 81 mg, 0.5 mmol) to
o
obtain the pure product 5n as a yellow solid (110 mg, 55%); mp: 296‒298 C; IR (KBr):
1
3425, 3165, 2925, 2357, 1673, 1640, 1560, 1410, 1282, 1190, 1120, 942, 866, 745 cm-1; H
NMR (300 MHz, DMSO–d6): δ 10.69 (bs 1H), 8.04 (s, 1H), 7.91 (s, 3H), 7.75 (s, 1H), 7.62
(s, 2H), 7.47 (t, J = 7.9 Hz, 1H), 7.05–6.87 (m, 2H), 3.79 (s, 3H); 13C NMR (75 MHz,
CDCl3+DMSO–d6): δ 168.8, 164.4 and 161.3 (d, J = 246.1 Hz), 163.7 and 161.7 (d, J = 246.1
Hz) 160.7,154.6, 149.7, 141.2, 137.5, 134.6, 128.7 (d, J = 4.9 Hz), 127.6, 125.4, 124.8, 122.7,
120.6, 118.6, 109.8, 109.4, 105.7, 55.2; MS (ESI): m/z 404 [M+H]+; HRMS (ESI): calcd for
C23H16O2N3F2 m/z 404.12308 [M+H]+; found 404.12758.
4.4.15. (Z)-5-Chloro-3-((2-(3,5-difluorophenyl)-1H-benzo[d]imidazol-6-
yl)methylene)indolin-2-one (5o).
Compound 5o was prepared according to the method described for compound 5a, employing
aldehyde 11d (129 mg,0.5 mmol) and 5-chloroindolin-2-one (12c, 83 mg, 0.5 mmol) to
o
obtain the pure product 5o as a yellow solid (127 mg, 63%); mp: 316‒318 C; IR (KBr):
3427, 3164, 2924, 2357, 2340, 1673, 1642, 1570, 1410, 1280, 1190, 1121, 956, 866 cm-1; 1H
NMR (300 MHz, DMSO–d6): δ 10.79 (bs 1H), 8.09 (s, 1H), 7.88 (s, 3H), 7.75 (bs, 1H), 7.62
(s, 2H), 7.44 (t, J = 8.1 Hz, 1H), 7.34–7.17 (m, 1H), 6.95–6.78 (m, 1H); 13C NMR (75 MHz,
CDCl3+DMSO–d6): δ 168.6, 164.3 and 161.0 (d, J = 246.4Hz), 164.1 and 160.9 (d, J =
245.9Hz), 150.7, 140.1, 138.7, 134.5, 129.1, 128.6 (d, J = 3.3 Hz), 128.4 (d, J = 4.9 Hz),
127.5, 127.2, 125.2, 124.7, 123.6, 121.1 (d, J = 4.9 Hz), 122.7, 119.3, 110.4, 109.7 (d, J = 9.3
16

Hz), 109.5 (d, J = 9.3 Hz), 105.7; MS (ESI): m/z 408 [M+H]+; HRMS (ESI): calcd for
C22H13ON3ClF2 m/z 408.07004 [M+H]+; found: 408.07097.
4.4.16. (Z)-6-Chloro-3-((2-(3,5-difluorophenyl)-1H-benzo[d]imidazol-6-
yl)methylene)indolin-2-one (5p).
Compound 5p was prepared according to the method described for compound 5a, employing
aldehyde 11d (129 mg,0.5 mmol) and 6-chloroindolin-2-one (12d, 83 mg, 0.5 mmol) to
o
obtain the pure product 5p as a yellow solid (123 mg, 61%); mp: 315‒317 C; IR (KBr):
1
3428, 3170, 2930, 2354, 1667, 1645, 1556, 1476, 1320, 1116, 1011, 953, 847, 752 cm-1; H
NMR (300 MHz, DMSO–d6): δ 10.09 (s, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.24 (s, 1H), 7.12 (s,
2H), 7.06 (d, J = 7.4 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.67 (dt, J =
13
9.1, 1.9 Hz, 1H), 6.27 (d, J = 8.0 Hz, 1H), 6.06 (d, J = 1.6 Hz, 1H); C NMR (75 MHz,
CDCl3+DMSO–d6): δ 168.7, 163.7 and 161.7 (d, J = 246.1 Hz), 163.6 and 161.5 (d, J = 246.9
Hz), 150.5, 141.3, 139.1, 132.9, 129.1, 128.3 (t, J = 5.4 Hz), 124.3, 123.7, 120.8, 120.7 (d, J
= 5.4 Hz), 120.6, 118.7, 109.9, 109.7 (d, J = 4.5 Hz), 109.5 (d, J = 4.5 Hz), 105.5; MS (ESI):
m/z 408 [M+H]+; HRMS (ESI) calcd for C22H13ON3ClF2 m/z 408.07004 [M+H]+; found
408.07097.
4.4.17. (Z)-3-((2-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (5q).
Compound 5q was prepared according to the method described for compound 5a, employing
aldehyde 11e (156 mg, 0.5 mmol) and indolin-2-one (12a, 66.5 mg, 0.5 mmol) to obtain the
o
pure product 5q as a yellow solid (132 mg, 62%); mp: 171‒173 C; IR (KBr): 3419, 3169,
1
2935, 2357, 1694, 1642, 1590, 1477, 1421, 1284, 1127, 1035, 999, 840, 749 cm-1; H NMR
(300 MHz, DMSO–d6): δ 10.50 (bs, 1H), 7.90 (s, 1H), 7.78 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H),
7.63 (d, J = 8.2 Hz, 1H), 7.58 (s, 1H), 7.54 (s, 2H), 7.18 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 7.7
13
Hz, 1H), 3.92 (s, 6H), 3.76 (s, 3H); C NMR (75 MHz, CDCl3+DMSO–d6): δ 168.8, 152.9,
139.7, 134.2, 133.7, 127.6, 126.5, 124.8, 121.9, 119.4, 114.7, 110.9, 108.8, 103.6, 59.5, 55.6;
MS (ESI): m/z 428 [M+H]+; HRMS (ESI): calcd for C25H22O4N3 m/z 428.15878 [M+H]+;
found 428.16048.
17

4.4.18. (Z)-5-Methoxy-3-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-6-yl) methylene)
indolin-2-one (5r).
Compound 5r was prepared according to the method described for compound 5a, employing
aldehyde 11e (156 mg,0.5 mmol) and 5-methoxyindolin-2-one (12b, 81 mg, 0.5 mmol) to
o
obtain the pure product 5r as a yellow solid (134 mg, 59%); mp: 168‒166 C; IR (KBr):
3425, 3174, 2354, 1690, 1640, 1575, 1488, 1435, 1280, 1120, 1032, 992, 846, 742 cm-1; 1H
NMR (300 MHz, DMSO−d6): δ 10.34 (bs, 1H), 7.89 (s, 1H), 7.77 (s, 1H), 7.66 (d, J = 8.1 Hz,
1H), 7.58 (s, 1H), 7.54 (s, 2H), 7.36 (s, 1H), 6.89–6.66 (m, 2H), 3.92 (s, 6H), 3.76 (s, 3H),
13
3.62 (s, 3H); C NMR (75 MHz, CDCl3+DMSO−d6): δ 168.9, 153.7, 153.0, 139.0, 137.3,
136.3, 128.2, 127.7, 126.2, 124.8, 121.9, 114.2, 113.8, 111.2, 111.1, 110.0, 108.9, 108.5,
103.8, 59.9, 55.8, 55.0; MS (ESI): m/z 458 [M+H]+; HRMS (ESI) calcd for C26H24O5N3 m/z
458.16992 [M+H]+; found 458.17105.
4.4.19. (Z)-5-Chloro-3-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)
indolin-2-one (5s).
Compound 5s was prepared according to the method described for compound 5a, employing
aldehyde 11e (156 mg, 0.5 mmol) and 5-chloroindolin-2-one (12c, 83 mg, 0.5 mmol) to
o
obtain the pure product 5s as a yellow solid (147 mg, 64%); mp: 185‒187 C; IR (KBr):
1
3420, 3172, 2930, 2357, 1693, 1642, 1594, 1478, 1292, 1132, 1039, 987, 845, 753 cm-1; H
NMR (300 MHz, DMSO−d6): δ 10.70 (bs, 1H), 8.10 (s, 1H), 7.93 (s, 1H), 7.78 (d, J = 7.7 Hz,
1H), 7.68 (d, J = 1.7 Hz, 1H), 7.63 - 7.56 (m, 3H), 7.31–7.21 (m, 1H), 6.88 (dd, J = 30.6, 8.6
13
Hz, 1H), 3.94 (s, 6H), 3.77 (s, 3H); C NMR (75 MHz, CDCl3+DMSO−d6): δ 168.8, 153.0,
142.7, 139.2, 134.6, 133.2, 127.6, 124.8, 124.2, 122.7, 120.5, 119.6, 112.4, 109.7, 103.7,
59.7, 55.6; MS (ESI): m/z 462 [M+H]+; HRMS (ESI): calcd for C25H21O4N3Cl m/z 462.12079
[M+H]+; found 462.12151.
4.4.20. (Z)-6-Chloro-3-((2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazol-6-yl)methylene)
indolin-2-one (5t).
Compound 5t was prepared according to the method described for compound 5a, employing
aldehyde 11e (156 mg, 0.5 mmol) and 6-chloroindolin-2-one (12d, 83 mg, 0.5 mmol) to
o
obtain the pure product 5t as a yellow solid (144 mg, 63%); mp: 189–191 C; IR (KBr):
1
3427, 3170, 2357, 1692, 1642, 1590, 1470, 1425, 1284, 1127, 1035, 999, 840, 748 cm-1; H
NMR (300 MHz, DMSO−d6): δ 10.73 (bs, 1H), 8.06–7.88 (m, 2H), 7.82 (s, 1H), 7.74 (s, 1H),
7.55 (s, 3H), 7.08–6.79 (m, 2H), 3.92 (s, 6H), 3.75 (s, 3H); 13C NMR (75 MHz,
18

CDCl3+DMSO−d6): δ 168.5, 152.7, 143.5, 138.8, 134.1, 133.2, 127.8, 126.2, 124.5, 124.3,
122.6, 120.1, 119.7, 112.2, 110.7, 109.5, 103.6, 59.6, 55.5; MS (ESI): m/z 462 [M+H]+;
HRMS (ESI): calcd for C25H21O4N3Cl m/z 462.12079 [M+H]+; found 462.12273.
4.4.21. (Z)-3-((2-(Thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-one (6a).
Compound 6a was prepared according to the method described for compound 5a, employing
aldehyde 14 (114 mg, 0.5 mmol) and indolin-2-one (12a, 66.5 mg, 0.5 mmol) to obtain the
o
pure product 6a as a yellow solid (102 mg, 60%); mp: 185‒187 C; IR (KBr): 3417, 2927,
1
2355, 1689, 1603, 1476, 1435, 1308, 1203, 1089, 1035, 860 cm-1; H NMR (300 MHz,
DMSO−d6): δ 10.70 (bs, 1H), 7.96 (s, 1H), 7.91 (d, J = 3.2 Hz, 1H), 7.83–7.74 (m, 3H), 7.70
13
(d, J = 8.3 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.29–7.17 (m, 2H), 6.93–6.81 (m, 2H); C
NMR (75 MHz, CDCl3+DMSO−d6): δ 168.8, 148.9, 142.6, 140.1, 138.4, 137.4, 133.2, 129.7,
129.3, 128.4, 128.2, 127.3, 125.7, 124.3, 121.9, 121.1, 120.9, 110.0, 109.1; MS (ESI): m/z
344 [M+H]+; HRMS (ESI): calcd for C20H14ON3S m/z 344.08466 [M+H]+; found 344.08521.
4.4.22. (Z)-5-Methoxy-3-((2-(thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (6b).
Compound 6b was prepared according to the method described for compound 5a, employing
aldehyde 14 (114 mg, 0.5 mmol) and 5-methoxyindolin-2-one (12b, 81 mg, 0.5 mmol) to
o
obtain the pure product 6b as a yellow solid (104 mg, 56%); mp: 189‒191 C; IR (KBr):
3420, 3152, 2920, 2330, 1689, 1640, 1603, 1476, 1430, 1308, 1209, 1092, 1037, 862 cm-1;
1H NMR (300 MHz, DMSO−d6): δ 10.33 (bs, 1H), 7.94–7.83 (m, 2H), 7.75 (s, 1H), 7.68 (s,
1H), 7.67 (s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.20 (t, J = 4.9 Hz, 1H),
13
6.84–6.64 (m, 2H), 3.62 (s, 3H); C NMR (75 MHz, CDCl3+DMSO−d6): δ 168.8, 153.7,
148.6, 137.2, 136.3, 133.0, 128.8, 127.0, 126.3, 124.0, 121.9, 114.1, 113.6, 109.9, 109.4,
108.6, 55.0; MS (ESI): m/z 374 [M+H]+; HRMS (ESI): calcd for C21H16O2N3S m/z
374.09561 [M+H]+; found 374.09577.
4.4.23. (Z)-5-Chloro-3-((2-(thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (6c).
Compound 6c was prepared according to the method described for compound 5a, employing
aldehyde 14 (114 mg,0.5 mmol) and 5-chloroindolin-2-one (12c, 83 mg, 0.5 mmol) to obtain
the pure product 6c as a yellow solid (120 mg, 64%); mp: 204‒206 oC; IR (KBr): 3425, 3417,
2927, 2350, 1697, 1642, 1605, 1470, 1438, 1312, 1210, 1097, 1042, 872 cm-1; 1H NMR (300
19

MHz, DMSO−d6): δ 10.76 (bs, 1H), 8.08 (s, 1H), 7.98–7.84 (m, 3H), 7.78 (d, J = 4.9 Hz,
1H), 7.75–7.55 (m, 2H), 7.33–7.17 (m, 2H), 6.94–6.80 (m, 1H); 13C NMR (75 MHz,
CDCl3+DMSO−d6): δ 168.5, 141.4, 139.3, 138.7, 133.1, 129.4, 129.1, 128.3, 127.4, 125.2,
124.9, 124.7, 123.2, 122.8, 121.2, 124.9, 124.7, 123.2, 122.8, 121.2, 119.2, 111.3, 110.4; MS
(ESI): m/z 378[M+H]+; HRMS (ESI): calcd for C20H13ON3ClS m/z 378.04627 [M+H]+; found
378.04514.
4.4.24. (Z)-6-Chloro-3-((2-(thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methylene)indolin-2-
one (6d).
Compound 6d was prepared according to the method described for compound 5a, employing
aldehyde 14 (114 mg,0.5 mmol) and 6-chloroindolin-2-one (12d, 83 mg, 0.5 mmol) to obtain
the pure product 6d as a yellow solid (116 mg, 62%); mp: 210‒212 oC; IR (KBr): 3425, 3162,
1
2936, 2924, 2360, 1692, 1648, 1607, 1478, 1435, 1310, 1210, 1095, 1038, 845 cm-1; H
NMR (300 MHz, DMSO−d6): δ 10.74 (bs, 1H), 8.04 (s, 1H), 7.87–7.82 (m, 2H), 7.85 (d, J =
3.9 Hz, 1H), 7.76 (d, J = 4.9 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.28–7.17 (m, 2H), 6.91–6.78
(m, 1H); 13C NMR (75 MHz, CDCl3+DMSO−d6): δ 168.8, 148.7, 143.9, 141.1, 139.3, 138.0,
133.6, 133.0, 132.1, 129.0, 128.1, 127.2, 124.6, 124.2, 122.9, 120.5, 120.0, 109.8, 109.0; MS
(ESI): m/z 378[M+H]+; HRMS (ESI): calcd for C20H13ON3ClS m/z 378.04627 [M+H]+; found
378.04514.
4.5. Biology
4.5.1. Evaluation of in vitro anti-cancer activity
The anticancer activity of the compounds was determined using MTT (3-(4, 5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reduction assay [33]. 1×106
cells/well were seeded in 100 µl DMEM, supplemented with 10% FBS in each well of 96-
o
well micro culture plates and incubated for 24 h at 37 C in a CO2 incubator. Compounds,
diluted to the desired concentrations in culture medium, were added to the wells with
respective vehicle control. After 48 h of incubation, MTT (10 µl, 5 mg/mL) was added to
each well and the plates were further incubated for 4 h. The supernatant from each well was
carefully removed, formazon crystals were dissolved in DMSO (100 µl) and absorbance at
570 nm wavelength was recorded.
20

4.5.2 In vitro growth inhibition
The screening of anticancer activity was evaluated by the NCI, USA, according to standard
procedures (http://dtp.nci.nih.gov/ branches/btb/ivclsp.html) [41].
4.5.3. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells
through the cell-cycle phases. MCF-7 cells were incubated for 48 h with compounds 5c and
5p at concentrations of 1 and 2 µM. The reference compound nocodazole was treated at 2
µM concentration in this experiment. Untreated and treated cells were harvested, washed
with phosphate-buffered saline (PBS), fixed in ice-cold 70% ethanol, and stained with
propidium iodide (Sigma–Aldrich). Cell-cycle analysis was performed by flow cytometry
(Becton Dickinson FACS Caliber instrument) [34].
4.5.4 Tubulin polymerization assay
A fluorescence based in vitro tubulin polymerization assay was performed according to the
manufacturer’s protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture in a total
volume of 10 µl contained PEM buffer (General tubulin buffer, contains 80 mM PIPES pH
6.9, 2 mM MgCl2 and 0.5 mM EGTA and used as a tubulin polymerization buffer), GTP (1
µM) in the presence or absence of test compounds 5c and 5p (final concentration of 1 µM).
Tubulin polymerization was followed by a time dependent increase in fluorescence due to the
incorporation of a fluorescence reporter into microtubules as polymerization proceeds.
Fluorescence emission at 420 nm (excitation wavelength is 360 nm) was measured by using a
Vario scan multimode plate reader (Thermo scientific Inc.). Nocodazole was used as positive
control in each assay. The IC50 value is defined as the drug concentration required inhibiting
50% of tubulin assembly compared to control. The reaction mixture for these experiments
include: tubulin (3 mg/mL) in PEM buffer, GTP (1 µM), in the presence or absence of tested
compounds at varying concentrations. Polymerization was monitored by increase in the
fluorescence as mentioned above at 370C [36,37].
4.5.5. Immunohistochemistry
A549 cells were seeded on glass cover slips and incubated for 48 h in the presence or absence
of the test compounds 5c, 5p, and nocodazole (1 µM). After treatment, the cover slips were
fixed with a paraformaldehyde solution (4% in 1×PBS) for 20 min at room temperature. Cell
permeabilization was achieved by administration of a Triton X-100 solution (0.2% in 1×PBS)
for 5 min. The cover slips were left in 100% MeOH overnight at -40C. Subsequently, the
21

cover slips were blocked with a 1% bovine serum albumin (BSA) solution for 60 min and
then incubated with anti-α-tubulin antibody (1:1000) at room temperature for 2 h. The slides
were washed three times for 5 min each with PBST. Next, the cover slips were incubated
with FITC-conjugated anti-mouse secondary antibody (Sigma–Aldrich) for 1 h and then
washed three times with PBST solution. Finally, the cells were observed under a fluorescence
microscope (Leica, Germany), and the pictures were analyzed for the integrity of the
microtubule network [37].
4.5.6. Molecular modelling experimental procedure
Optimizations of all compounds were performed in Gaussian 09 by using PM3 semiempirical
methods. The protein cocrystal structure was downloaded from RSCB-Protein Data Bank
with ligand colchicine (PDB ID 3E22) [39]. Autodock 4.2 software [42] was used to perform
the docking studies. The visualization and analysis of the interactions were performed by
using PyMOL (ver. 0.99) [40].
Acknowledgements
The authors B.N.S, M.S, V.S and J.S.K are thankful to the National Cancer Institute (NCI),
USA for providing the data on the sixty cell panel of human cancer cell lines. The authors are
also grateful to CSIR, New Delhi, for the award of Research Fellowships.
22

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25

Figures
Fig. 1. Structures of standard anticancer molecules Paclitaxel (1), vinblastine (2), indirubin (3),
nocodazole (4) and benzimidazole–oxindole analogues (5a‒t and 6a‒d).
26

Fig. 2. Flow cytometric analysis in the MCF-7 breast cancer cell line after treatment with
conjugates5c and 5p at 1 µM and 2 µM concentrations for 48 h; A. control; B. Nocodazole (2
µM); C. 5c (1 µM); D. 5c (2 µM); E. 5p (1 µM) and F. 5p (2 µM).
27

Fig. 3. Effect of conjugates on tubulin polymerization: tubulin polymerization was monitored by
the increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h at 37°C. All the
conjugateswere included at a final concentration of 1 µM. Nocodazole was used as a positive
control. Values indicated are the mean ± SD of two different experiments performed in triplicates.
28

Fig. 4. IHC analysis of conjugates on the microtubule network: MCF-7 cells were treated
with conjugates 5c, 5p and nocadazole at 1 µM concentration for 48 h.
29