Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization inhibitors and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmc.2015.05.060

A new series of 2-aryl 1,2,4-oxadiazolo-benzimidazole conjugates have been synthesized and evaluated for their antiproliferative activity in the sixty cancer cell line panel of the National Cancer Institute (NCI). Compounds 5l (NSC: 761109/1) and 5x (NSC:

Full text of DOI:10.1016/j.bmc.2015.05.060

Accepted Manuscript
Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization
inhibitors and apoptosis inducing agents
Ahmed Kamal, T. Srinivasa Reddy, M.V.P.S. Vishnuvardhan, K. Nimbarte,
A.V. Subba Rao, Vunnam Srinivasulu, Nagula Shankaraiah
PII:
S0968-0896(15)00495-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.060
BMC 12357
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2015
29 May 2015
30 May 2015
Please cite this article as: Kamal, A., Srinivasa Reddy, T., Vishnuvardhan, M.V.P.S., Nimbarte, K., Subba Rao,
A.V., Srinivasulu, V., Shankaraiah, N., Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin
polymerization inhibitors and apoptosis inducing agents, Bioorganic & Medicinal Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bmc.2015.05.060
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Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization
inhibitors and apoptosis inducing agents
Ahmed Kamal,*a,b T. Srinivasa Reddy,a,b M. V. P. S. Vishnuvardhan,a Vijay K. Nimbarte, a,b A.
V. Subba Rao,a Vunnam Srinivasulu,a Nagula Shankaraiahb
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
Abstract: A new series of 2-aryl 1,2,4-oxadiazolo-benzimidazole conjugates have been
synthesized and evaluated for their antiproliferative activity in the sixty cancer cell line panel of
the National Cancer Institute (NCI). Compounds 5l (NSC: 761109 / 1) and 5x (NSC: 761814 /
1) exhibited remarkable cytotoxic activity against most of the cancer cell lines in the one dose
assay and were further screened at five dose concentrations (0.01, 0.1, 1, 10 and 100 µM) which
showed GI50 values in the range of 0.79 – 28.2 µM. Flow cytometric data of these compounds
showed increased cells in G2/M phase, which is suggestive of G2/M cell cycle arrest. Further,
compounds 5l and 5x showed inhibition of tubulin polymerization and disruption of the
formation of microtubules. These compounds induce apoptosis by DNA fragmentation and
chromatin condensation as well as by mitochondrial membrane depolarization. In addition,
structure activity relationship studies within the series are also discussed. Molecular docking
studies of compounds 5l and 5x into the colchicine-binding site of the tubulin, revealed the
possible mode of interaction by these compounds.
Keywords: Benzimidazole, 1,2,4-oxadiazole, cytotoxicity, tubulin polymerization, apoptosis,
molecular docking.
∗,a,bCorresponding author. Tel.: +91-40-27193157; fax: +91-40-27193189 (A.K.); e-mail:
ahmedkamal@iict.res.in (A. Kamal).
1

1. Introduction
Benzimidazoles have attracted significant interest in medicinal chemistry as they exhibit a broad
range of biological activities such as antiulcer,1 antimicrobial,2 antioxidant,3 antiviral,4 anti-
inflammatory5 and anticancer activity.6 For instance, Hoechst 33258 (1, Fig. 1),7 nocodazole (2)8
and bendamustin (3)9 are some of the benzimidazole based anticancer drugs which are in clinical
or preclinical studies. Benzimidazole substituted derivatives are also known inhibitors of tubulin
polymerization that inhibit cell proliferation in the treatment of cancer.8,9 Recently, we have
reported the synthesis and anticancer activity of terphenyl benzimidazoles as tubulin
polymerization inhibitors.10 Among the benzimidazoles with anticancer potential, 2-
aryl/heteroaryl benzimidazole based molecules exhibited pronounced activity, therefore design
and synthesis of newer 2-aryl benzimidazoles is of much significance.11,12
Recently, there has been wide interest in compounds containing the 1,2,4-oxadiazole scaffold
because of their wide range of biological activities such as antimicrobial,13 antiviral,14 anti-
inflammatory15 and antineoplastic.16 Moreover, 1,2,4-oxadiazole heterocycles are widely used
for the bioisosteric replacement of an ester or amide functionality with substantial improvement
in biological activity by participating in hydrogen bonding interactions with different
receptors.17Among the oxadiazoles, 3,5-disubstituted-1,2,4-oxadiazoles (4) are reported in the
literature for their anticancer potential.18
< Insert Figure.1>
On the basis of these observations and in continuation to our previous efforts aiming at finding
promising new leads with potential anticancer activity, it was considered worthwhile to design
and synthesize new hybrids that contain the 2-arylbenzimidazole as well as 1,2,4-oxadiazole ring
systems. Furthermore, benzimidazole attached with other heterocyclic moieties resulted in
compounds (hybrid molecules) with improved pharmacological profile.19 Therefore, we have
synthesized a series of new 2-arylbenzimidazole conjugates linked to the 1,2,4-oxadiazole
moiety and evaluated for their cytotoxic activity at the National Cancer Institute (NCI), USA.
Sixteen compounds were evaluated for their antiproliferative activity as per the NCI protocol and
the results are shown in Table 2. In addition, cell cycle analysis and tubulin polymerization
2

assay were carried out for the most potent compounds of the series (5l and 5x) apart from the
molecular docking study on the tubulin protein.
2. Chemistry
2-Arylbenzimidazole linked 1,2,4-oxadiazole conjugates 5a–x were synthesized as shown in
Scheme 1. The benzimidazole precursors 4-(1H-benzo[d]imidazol-2-yl)benzonitriles (8a–d)
were synthesized by reacting 4-cyano benzaldehyde (7) with different substituted o-
phenylenediamines (6a–d) in the presence of sodium metabisulphate, which were then reacted
with hydroxylamine hydrochloride in ethanol under refluxing condition that resulted in the
formation of corresponding amidoximes 9a–d. The reaction of amidoximes 9a–d with different
aromatic carboxylic acids in the presence of carbonyl diimidazole (CDI) produced in situ o-
acylamidoximes, which upon thermal cyclization afforded the desired target conjugates 5a–x in
good to moderate yields (39–68%).
< Insert Scheme.1>
3. Pharmacology
3.1 Anticancer activity
Primary screening of one-dose assay (10-5 M) was performed using a panel of about 60 human
tumor cell lines which includes nine tumor subpanels namely; leukemia, non-small cell lung,
colon, CNS, melanoma, ovarian, renal, prostate, and breast cancer cells, in accordance with the
protocol of National Cancer Institute (NCI), USA. The compounds were added at a 10 µM
concentration and the culture was incubated for 48 h. End point determinations were made with a
protein binding dye, sulforhodamine B. Results for each compound were reported as a mean
graph of the growth percent (GP) of the treated cells and the results obtained are illustrated in
Table. 1.
< Insert Table.1>
The results indicated a selective influence of some compounds on proliferation of a number of
cancer cells with broad-spectrum of activity. Compound 5l was highly active on leukemia cancer
cell line (RPMI-8226) with GP = –15.28%, whereas compounds 5h, 5x and 5u showed selective
potency against ovarian cancer (OVACAR-4) cell line with GP = –8.43%, GP = –53.44, and GP
3

= –21.59% respectively. The majority of the tested compounds displayed growth inhibition on
ovarian cancer cell line OVACAR-4 (5h, 5x, 5t, and 5u) and different cell lines of leukemia (5f,
5j, 5l, 5s). Close examination of the data presented in Table. 1 indicated that marked growth
inhibition was observed with the compounds 5l and 5x whereas 5f, 5h, and 5u displayed
moderate activity and all the remaining compounds were least active.
Among the tested compounds, 5l and 5x were further explored for the five dose assay at 10-fold
dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100 µM). Data of the five dose
assay for compounds 5l and 5x is shown in Tables 2 and 3 respectively. The antiproliferative
activity of tested compounds is given by three response parameters (GI50, TGI and LC50) for each
cell line. The GI50 value (growth inhibitory activity) corresponds to the concentration of the
compound that inhibits 50% net cell growth; the TGI value (cytostatic activity) is the
concentration of the compound leading to total growth inhibition and the LC50 value (cytotoxic
activity) is the concentration of the compound causing 50% net cell death at the end of the
incubation period of 48 h. Furthermore, a mean graph midpoint (MG-MID) value is calculated
giving an averaged activity parameter for all the cell lines.
< Insert Table.2>
< Insert Table.3>
Compound 5l under investigation exhibited potent anticancer activity against all the tested cancer
cell lines representing nine different subpanels with GI50 values between 1.30–8.27 µM (Table.
2). With regard to the sensitivity of the compound 5l against individual cell line, the highest
growth inhibitory activity was observed against the NCI-H322M cell line of non-small cell lung
cancer with GI50 value 1.30 µM and minimum growth inhibitory activity against TK-10 cell line
of renal cancer with GI50 value 8.27 µM. The compound 5x also showed potent and highest
growth inhibitory activity against BT-549 (breast cancer) cell line with GI50 value of 0.52 µM,
SR (leukemia) cell line with GI50 value of 0.71 µM and least activity on A-498 (renal) cell line
with GI50 value of 28.2 µM (Table. 3). Moreover, compound 5x is selectively sensitive towards
all the leukemia cancer cell lines with GI50 values not more than 6.02 µM concentrations. The
remaining subpanel cell lines showed maximum sensitivity towards these compounds with not
4

more than 28 µM concentration. Overall, the screening process resulted in GI50 values of less
than 28.2 µM indicating an exceptional activity and the values of LC50 are >100 µM in most of
the cell lines, which indicates that these compounds are selectively toxic to cancer cells.
The criterion for selectivity of the compound towards a cancer cell line is based on the ratio
obtained by dividing the full panel MG-MID (the average sensitivity of all cell lines towards the
test agent) of the compounds by their individual subpanel MG-MID (the average sensitivity of all
cell lines of a particular subpanel towards the test agent). Ratios between 3 and 6 refer to
moderate selectivity, ratios greater than 6 indicate high selectivity toward the corresponding cell
line, while compounds not meeting either of these criteria are rated non-selective.20 As per this
criterion, 5x appears to be moderately selective towards leukemia cancer subpanel with selective
index of 3.46 and 5l was found to be non-selective with broad spectrum of antitumor activity
against the nine tumor subpanels with selectivity ratios ranging between 0.74–1.35 at the GI50
levels.
Structure activity relationship was established based on the number of cell lines that showed
sensitivity towards each of the newly synthesized compounds. Among all conjugates, the
compounds 5f, 5l, and 5x having methoxy groups at R1, R2, and R3 positions have shown better
antiproliferative activity. The activity was also influenced by the presence of weak ring de-
activating groups like fluorine and electron releasing groups like methoxy at R substitution of
benzimidazole ring. For instance, compounds 5h and 5l having fluoro group showed increased
sensitivity on OVCAR-4 (ovarian cancer) cell line (–8.43% at 10 µM) and RPMI-8226
(leukemia) cell line (–15.28% at 10 µM) when compared to the unsubstituted derivatives.
Furthermore, the presence of electron withdrawing groups like chloro (5r) on benzimidazole ring
resulted in a dramatic loss of growth inhibitory effect. In view of the promising antiproliferative
activity and higher therapeutic ratio of the compounds 5l and 5x, it was considered of interest to
understand the detailed biological implicating by these compounds.
3.2. Effect on cell cycle arrest
To investigate the mechanism underlying the antiproliferative effect of these conjugates 5l and
5x, we examined the effect of these compounds on cell cycle progression by flow-cytometry in
5

MCF-7 cells and nocodazole was used as a positive control. In this study, MCF-7 cells were
treated with the compounds 5l and 5x at concentrations of 1 µm and 3 µm for a period of 48 h.
Cells were harvested and the percentage of cells in each phase were analyzed by flowcytomety.
The results are shown in Figure 2A and 2B. The data obtained clearly indicated that, these
compounds arrest the cells in G2/M phase of the cell cycle in comparison to control. It was found
that a large proportion of cells treated with the conjugates 5l and 5x accumulated in the G2/M
phase i.e., 35.83% at 1 µM, 44.01 at 3 µM and 38.94% at 1 µM, 52.15 at 3 µM respectively.
< Insert Figure.2A>
< Insert Figure.2B>
3.3 Effect on tubulin polymerization
Tubulin polymerization dynamics within the cells are critical for completion of mitosis and
frequently targeted by agents that induce mitotic arrest by interfering with the mitotic spindle
formation.20 If the function of microtubules is blocked, cell differentiation will be arrested which
leads to the accumulation of cells at G2/M phase in cell cycle analysis and it eventually leads to
cell death.21 Therefore, the two potent compounds in the series 5l and 5x, which induced cell
cycle arrest at G2/M phase were evaluated for their effects on tubulin polymerization along with
the positive control, nocodazole. The 3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-phenyl-1,2,4-
oxadiazole conjugates (5l, 5x) and nocodazole were employed at a concentration of 3 µm in
tubulin assembly assays. The compounds 5l and 5x have exhibited potent inhibition of
microtubule assembly with 55.6% and 52.1% inhibition respectively, these values are
comparable to that of standard nocodazole. Furthermore, the IC50 values of these conjugates
were determined for their ability to inhibit tubulin assembly and the two lead molecules 5l and
5x showed pronounced inhibition of tubulin polymerization with IC50 values of 1.5 µM and 1.8
µM respectively as compared with 1.7 µM for nocodazole (Table. 4). Overall, these results
suggest that the compounds 5l and 5x significantly inhibit the tubulin polymerization.
< Insert Table.4>
3.4 Immunohistochemistry (IHC) of tubulin
6

In order to substantiate the observed cytotoxic effects of these compounds on the inhibition of
tubulin polymerization to functional microtubules, immunohistochemistry studies have been
carried out to examine the in situ effects of compounds 5l and 5x on cellular microtubules.
Therefore, MCF-7 cells were treated with 5l and 5x at 1 µM concentration for 48 h. In this study,
untreated human breast cancer cells displayed the normal distribution of microtubules. However,
cells treated with 5l and 5x showed disrupted microtubule organization as seen in Figure. 3,
thereby demonstrating the inhibition of tubulin polymerization. This immunofluorescence study
showed that the level of tubulin polymerization inhibition was comparable to that of nocodazole
for these conjugates (5l and 5x).
< Insert Figure.3>
3.5 Competitive colchicine binding assay
As the compounds 5l and 5x showed significant inhibitory effects on the tubulin polymerization,
we investigated their mode of binding to the tubulin by fluorescence based assay.22 The results
from the figure. 4 indicated that that the compound 5x as well as nocodazole showed significant
affinity towards the colchicine site on the tubulin, whereas 5l showed moderate or low binding
affinity in comparison to nocodazole. Moreover, taxol was used as a negative control, which is
known to bind at a different site and shows no effect on tubulin–colchicine complex.
< Insert Figure.4>
3.6 Effect on chromatin condensation
It is well-known that the cell cycle arrest at G2/M phase is shown to induce cellular apoptosis.23
Hence, it was considered of interest to investigate whether the cytotoxicity of these conjugates
(5l and 5x) is by virtue of apoptotic cell death. During apoptosis, nuclear fragmentation and
chromatin condensation takes place; therefore Hoechst-33258 staining was used to visualize
nuclear condensation. It was observed that the 3-(4-(1H-benzo[d]imidazol-2-yl) phenyl)-5-
phenyl-1,2,4-oxadiazole conjugates 5l and 5x caused significant nuclear condensation in MCF-7
cells upon treatment at 1 µM concentration for 24 h which is comparable to that of reference
standard nocodazole (Figure. 5).
< Insert Figure.5>
7

3.7 Effect on mitochondrial membrane potential
Mitochondria plays an essential role in the propagation of apoptosis and its dysfunction within
the apoptotic process is often associated with loss or depolarization of mitochondrial membrane
potential (DΨm), which leads to collapse of mitochondrial functions ensuing cell death.24
Therefore, in this study we examined the effect of 3-(4-(1H-benzo[d]imidazol-2-yl) phenyl)-5-
phenyl-1,2,4-oxadiazole conjugates on DΨm. The MCF-7 cells were treated with 5l and 5x at 1
µm concentration for 24 h and stained with JC-1 dye. In control cells the dye concentrates in the
mitochondrial matrix where it forms red fluorescent aggregates (J-aggregates) because of the
electrochemical potential gradient. In cells treated with compounds which induce apoptosis, the
mitochondrial membrane is depolarized thus preventing the accumulation of the JC-1 dye in the
mitochondria. Therefore, the dye in the monomeric form is dispersed throughout the entire cell
leading to a shift from red (J-aggregates) to green fluorescence (JC-1 monomers). Thus,
apoptotic cells showing primarily green fluorescence are easily differentiated from healthy cells
which show red and green fluorescence. It was observed that these conjugates significantly
depolarize the mitochondrial membrane potential (Figure 6A) as indicated by the green
fluorescence in the cells treated by these conjugates, whereas control (untreated cells) showed
red colored florescence because of intact mitochondrial membrane potential. Further, the loss of
mitochondrial membrane potential was quantified by flow cytometry. As shown in Figure 6B,
there is a remarkable shift in the green fluorescence that has increased to 24.0%, and 25.7%
compared with control cells which is showing 2.2%, indicating that 5l and 5x induces apoptosis
through depolarization of the mitochondrial membrane potential in MCF-7 cells.
< Insert Figure.6A>
< Insert Figure.6B>
3.8 DNA Fragmentation analysis
Endonuclease mediated cleavage of nuclear DNA results in the formation of oligonucleosomal
DNA fragment (180–200 base pairs long), a biochemical hallmark of apoptosis.25 DNA
laddering assay was performed on MCF-7 cells treated with 2 µM concentration of the
compounds 5l and 5x. The chromosomal DNA extracted from MCF-7 cells and used for agarose
8

gel electrophoresis. The results from the Figure. 7 indicated that 5l and 5x induces DNA
fragmentation in MCF-7 treated cells which lead to a smear formation in gel lanes.
< Insert Figure.7 >
3.9 Annexin V(FITC)-Propidium Iodide Dual Staining Assay
The apoptotic inducing ability of the conjugates 5l and 5x was also investigated by Annexin V
FITC/PI (AV/PI) dual staining assay to examine the occurrence of phosphatidylserine
externalization and also to understand whether it is due to physiological apoptosis or nonspecific
necrosis.26 In this study, the MCF-7 cells were treated with 5l and 5x for 24 h at 1 µM
concentration to examine the apoptotic effect. It was observed that these compounds showed
significant apoptotic effect against MCF-7 cells. As shown in Figure 8, the compounds 5l and 5x
showed 16.9% and 17.7% of apoptotic cells, respectively, whereas 2.2% of apoptotic cells were
observed in the control (untreated cells). This experiment further suggests that these compounds
significantly induced apoptosis in MCF-7 cells.
< Insert Figure.8 >
4.0 Molecular Docking Studies
To investigate the possible mode of binding of these new conjugates, we carried out docking
studies of conjugates 5l and 5x in the colchicines binding site of α,β-tubulin (PDB:3E22).
27-30
Conjugates 5l and 5x were docked using Auto dock 4.2 docking tool,
which implements a
genetic algorithm based search method. As depicted in Figure 9, the conjugates 5l and 5x shared
the binding pocket of colchicine displaying interactions with βCys241, βMet259, βIle378,
βLys352, βAsn249, αThr179, αSer178 and αGlu71 in the α,β-tubulin.
< Insert Figure.9 >
Docking studies show that conjugates 5l and 5x occupied the colchicine binding site of
α,β-tubulin mostly buried in the β-subunits (Figure 10). The trimethoxy phenyl group of these
conjugates occupies the hydrophobic binding site in the tubulin with βLys352, βMet259 and
βCys241. Some hydrogen-bonding interactions were observed between their benzimidazole ring
with that of βAsn249, βAsn258 and αAsn101 in the range of 2.3-2.9Å. Furthermore, some
9

electrostatic interactions were also observed in between benzimidazole ring and αAsp98.
Whereas oxadiazole ring of these conjugates is involved in the hydrophobic interactions with
αThr179 apart from π-π* stacking interaction with βAsn258. In addition, the 4-methoxy group
on benzimidazole ring is involved in hydrogen-binding with that of αGlu71.
< Insert Figure.10 >
5.0 Conclusion
In conclusion, a series of new 3-(4-(1H-benzo[d]imidazol-2-yl) phenyl)-5-phenyl-1,2,4-
oxadiazole conjugates 5 a-x were synthesized and tested for their antiproliferative activity. Most
of the compounds showed moderate to remarkable cytotoxic activity against all the tested tumor
cell lines. Two of the conjugates 5l and 5x have emerged as potential candidates with broad
spectrum of cytotoxic activity against most of the tumor cell lines. The flow cytometric analysis
indicated that they induced G2/M cell cycle arrest in MCF-7 cells and inhibited tubulin
polymerization comparable to a reference standard nocodazole apart from disruption of
microtubule network formation. Apoptosis induced by these compounds was also determined by
characteristic morphological changes such as DNA fragmentation and chromatin condensation.
Furthermore, depolarization of mitochondrial membrane potential (MMP) was also observed,
which indicated that the mitochondrial pathway was also involved in the apoptosis signaling
pathway. The molecular modeling study on tubulin demonstrated that these molecules bind well
with the tubulin and are involved in a series of interactions with the proteins. Thus these
conjugates could be considered as potential leads in the development of a new class of
antimitotic anti-cancer agents.
6. Experimental Section
All the reagents, solvents used were of commercial grade and were used without any further
purification. The progress of the reactions was monitored by thin layer chromatography (TLC),
performed on silica gel glass plates containing 60 F-254, and visualization on TLC was achieved
by UV light or iodine indicator. Melting points were measured with an Electrothermal melting
1
13
point apparatus, and are uncorrected. 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)
10

instruments. Chemical shifts (d) are reported in ppm downfield from internal TMS standard.
Signal multiplicities are represented by s (singlet), d (doublet), t (triplet), ds (double singlet), dd
(double doublet), m (multiplet) and br s (broad singlet). ESI spectra were recorded on Micro
mass, Quattro LC using ESI+ software with capillary voltage 3.98 kV and ESI mode positive ion
trap detector. IR spectra were recorded on KBr disc using a FTIR bruker Vector 22
Spectrophotometer
6.1. General procedure for the synthesis of 4-(1H-benzo[d]imidazol-2-yl)benzonitrile 8 (a-d)
The starting nitrile compounds 8 (a-d) were prepared according to the literature method. To a
mixture of appropriate o-phenylenediamines 7 a-d (0.5 mmol) and 4-cyano benzaldehyde 6 (0.5
mmol) in ethanol was added a solution of Na2S2O5 (4 mmol) in H2O (1.6 mL). The resulting
mixture was stirred at reflux for 4 h, after completion of reaction; the solution was poured onto
crushed ice. The resulting solid was filtered, washed with cold water, dried and recrystallized
from ethanol to afford compounds 8(a-d). Among the synthesized 4-(1H-benzo[d]imidazol-2-yl)
benzonitriles 8(a-d), compounds 8b and 8d were reported for the first time and were
characterized by NMR (1H, 13C) and the physical and spectral data of the compounds 8a and 8c
were in complete agreement with the reported data.31
4-(5-fluoro-1H-benzo[d]imidazol-2-yl)benzonitrile (8b)
This compound was prepared by method described in section 6.1 employing 4-cyano
benzaldehyde (1.31 g, 1 mmol) and 4-Flouro-o-phenylenediamine (1.26 mg, 1 mmol) to afford
1
8b, as light yellow solid 1.65 g, in 69 % yield. Mp 218-221 ºC. H NMR (300 MHz, DMSO) δ
7.12 (d, 1H, J = 7.65 Hz), 7.52 (s, 1H), 7.67 (d, 1H, J = 7.71 Hz), 8.02 (d, 2H, J = 8.3 Hz), 8.13
(d, 2H, J = 8.3 Hz). MS (ESI): m/z 238 [M+H]+.
4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzonitrile (8d)
This compound was prepared by method described in section 6.1 employing 4-cyano
benzaldehyde (1.31 g, 1 mmol) and 4-methoxy-o-phenylenediamine (1.38 g, 1 mmol) to afford
8d, as light yellow solid, 1.66 g, in 66 % yield. Mp. 205-207 ºC. 1H NMR (300 MHz, DMSO) δ
11

3.81 (s, 3H), 6.92 (d, 1H, J = 7.29 Hz), 7.21 (s, 1H), 7.58 (d, 1H, J = 7.27 Hz), 8.08 (d, 2H, J =
8.24 Hz), 8.21 (d, 2H, J = 8.26 Hz). MS (ESI): m/z 250 [M+H]+.
6.2. General Procedure for the Synthesis of (Z)-4-(1H-benzo[d]imidazol-2-yl)-N'-
hydroxybenzimidamide 9 (a-d)
To a solution of appropriate benzonitrile (1.0 mmol) in ethanol (5 ml) was added Hydroxylamine
hydrochloride (1.1 mmol, 764 mg) and sodium hydroxide (1.1 mmol, 444 mg), each dissolved in
water (10 mL) sequentially, for a period of 20 min at 0 °C. Then the resulting mixture was
allowed to reflux with stirring for 18 h. The pH of the solution was adjusted to 2 with 1N HCl
and the aqueous phase was washed with ethyl acetate (2X35 mL). Upon cooling (0 ºC) and
neutralization with sodium bicarbonate gave precipitate which was filtered, washed and dried to
afford pure amidoximes 9 (a-d).
(Z)-4-(1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide (9a)
This compound was prepared by method described in section 6.2 employing 4-(1H-
benzo[d]imidazol-2-yl)benzonitrile (8a, 2.18 g, 1 mmol) to afford 9a as light brown solid, 1.87 g,
in 74 % yield. Mp; 227-229 ºC. 1H NMR (300 MHz, DMSO) δ 5.57 (s, 2H), 7.11-7.29 (m, 2H),
7.65-7.74 (m, 2H), 7.89 (d, 2H, J = 8.12 Hz), 8.24 (dd, 4H, J = 8.32, 5.65 Hz), 9.31 (s, 1H). 13C
NMR (75 MHz, DMSO) δ 162.3, 154.1, 151.8, 139.7, 139.5, 135.6, 128.4, 127.2, 123.6, 123.4,
116.3, 114.9. MS (ESI): m/z 253 [M+H]+.
9(Z)-4-(5-fluoro-1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide (9b)
This compound was prepared by method described in section 6.2 employing 4-(5-fluoro-1H-
benzo[d]imidazol-2-yl)benzonitrile (8b, 2.37 g, 1 mmol) to afford 9b as off light brown solid
1
2.06 g, in 76% yield. Mp;. 223-225 ºC. H NMR (300 MHz, DMSO) δ 5.61 (bs, 2H), 7.11 (d,
1H, J = 7.63 Hz), 7.47 (s, 1H), 7.66 (d, 1H, J = 7.67 Hz), 7.97 (d, 2H, J = 8.27 Hz), 8.14 (d, 2H,
13
J = 8.26 Hz), 9.35 (s, 1H). C NMR (75 MHz, DMSO) δ. 162.5, 155.7, 154.0, 151.9, 138.7,
135.7, 134.2, 128.6, 127.3, 116.1, 11.4, 103.3.; MS (ESI): m/z 271 [M+H]+.
(Z)-4-(5-chloro-1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide (9c)
12

This compound was prepared by method described in section 6.2 employing 4-(5-chloro-1H-
benzo[d]imidazol-2-yl)benzonitrile (8c, 2.49 g, 1 mmol) to afford 9c as brown solid 2.21 g, in
77% yield. Mp; 236-238 ºC. 1H NMR (300 MHz, DMSO) δ 5.58 (bs, 1H), 7.14 (d, 1H, J = 8.49
13
Hz), 7.41-7.60 (m, 2H), 7.92 (d, 2H, J = 8.30 Hz), 8.16 (d, 2H, J = 8.49 Hz) 9.30 (s, 1H). C
NMR (75 MHz, DMSO) δ 162.3, 154.2, 151.7, 137.7, 135.9, 133.5, 129.8, 128.6, 127.4, 123.3,
116.7, 116.3. MS (ESI): m/z 287 [M+H]+.
(Z)-N'-hydroxy-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide (9d)
This compound was prepared by method described in section 6.2 employing (4-(5-methoxy-1H-
benzo[d]imidazol-2-yl)benzonitrile) (8d, 2.49 g, 1 mmol) to afford 9d as light yellow solid 1.97
g, in 69% yield. Mp; 218-221 ºC. 1H NMR (300 MHz, DMSO) δ 3.78 (s, 3H), 5.51 (s, 2H), 6.92-
7.18 (m, 2H), 7.61 (d, 1H, J = 7.26 Hz), 8.02 (d, 2H, J = 8.26 Hz), 8.17 (d, 2H, J = 8.26 Hz),
9.35 (s, 1H).13C NMR (75 MHz, DMSO) δ 162.3, 155.8, 154.3, 151.8, 138.6, 136.1, 135.5,
128.6, 127.7,114.6, 113.2, 101.7. MS (ESI): m/z 283 [M+H]+.
6.3. General procedure for the Synthesis of compounds 5 (a-x)
To a solution of appropriate carboxylic acid (0.5 mmol) in dry DMF (3 mL) was added Carbonyl
diimidazole (0.6 mmol) under nitrogen atmosphere and the reaction mixture was stirred at room
temperature for 1 h. Then appropriate amidoxime 9 a-d (0.6 mmol) was added and reaction
mixture was heated at 110 ºC for about 18 h (monitored by TLC). The contents of the reaction
were cooled to 25 ºC and poured into ice-cold water (25 mL), extracted by ethyl acetate (3X15.0
mL) and the combined organic phase was washed with brine, dried over anhydrous sodium
sulfate., filtered and concentrated in vacuo. The obtained residue was purified by column
chromatography using ethyl acetate–hexane (0–30%) as eluent to furnish pure 1,2,4-oxadiazoles
5 (a-x) in moderate yields.
(3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-phenyl-1,2,4-oxadiazole) 5a
This compound was prepared by method described in section 6.3 employing ((Z)-4-(1H-
benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9a (152 mg, 0.6 mmol) and benzoic acid (61
1
mg, 0.5 mmol) to afford 5a as off white solid 121 mg, in 59% yield. Mp; 187-189 ºC. H NMR
13

(300 MHz, CDCl3) δ 5.38 (bs, 1H), 7.01 (dd, 2H, J = 6.0, 3.1 Hz), 7.29-7.49 (m, 4H), 7.63-7.67
(m, 1H), 8.00 (d, 2H, J = 7.9 Hz), 8.06 (d, 2H, J = 8.2 Hz), 8.14 (d, 2H, J = 8.5 Hz); 13C NMR
(125 MHz, CDCl3) δ 174.8, 167.9, 151.3, 138.7, 138.5, 134.6, 130.2, 128.9, 128.4, 128.2, 127.2,
126.8, 126.6, 123.5, 123.3, 116.4, 116.2. MS (ESI): m/z 339 [M+H]+. HRMS (ESI) calcd for
C21H14N4O [M+H]+ 339.12471; found: 339.12132.
(3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-fluorophenyl)-1,2,4-oxadiazole) 5b
This compound was prepared by method described in section 6.3 employing ((Z)-4-(1H-
benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9a (152 mg, 0.6 mmol), 4-flouro benzoic
acid (70 mg, 0.5 mmol) to afford 5b as off white solid 115 mg, in 53% yield. Mp; 181-183 ºC.
1H NMR (300 MHz, CDCl3) δ 5.29 (bs, 1H), 7.16 - 7.24 (m, 2H), 7.50 - 7.55 (m, 2H), 7.56 (d,
2H, J = 8.23 Hz), 7.92 (d, 2H, J = 8.23 Hz), 8.19-8.30 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
174.9, 167.9, 164.9, 151.3, 138.8, 138.5, 134.7, 130.2, 128.5,128.3, 127.1, 124.0, 123.6, 123.3,
116.4, 116.1, 115.8.; MS (ESI): m/z 357 [M+H]+. HRMS (ESI) calcd for C21H14FN4O [M+H]+
357.11508; found: 357.11463.
(3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-chlorophenyl)-1,2,4-oxadiazole) 5c
This compound was prepared by method described in section 6.3 employing ((Z)-4-(1H-
benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9a (152 mg, 0.6 mmol), p-chloro benzoic
acid (78 mg, 0.5 mmol) to afford 5c as off white solid 127 mg, in 56% yield. Mp; 195-196 ºC. 1H
NMR (500 MHz, CDCl3) δ 5.32 (bs, 1H), 7.18-7.24 (m, 2H), 7.52-7. 63 (m, 4H), 7.87 (d, 2H, J =
8.12 Hz), 8.18-8.30 (dd, 4H, J = 8.30, 5.61 Hz); 13C NMR (125 MHz, CDCl3) δ 174.8, 168.0,
151.2, 138.8, 138.4, 138.1, 134.6, 129.7, 129.4, 128.5, 128.4, 127.3, 127.1, 123.5, 123.3 116.3,
116.1.; MS (ESI): m/z 373 [M+H]+. HRMS (ESI) calcd for C21H14ClN4O [M+H]+ 373.08481;
found: 373.08495.
(3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-methoxyphenyl)-1,2,4-oxadiazole) 5d
This compound was prepared by method described in section 6.3 employing ((Z)-4-(1H-
benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9a (152 mg, 0.6 mmol), p-methoxy benzoic
acid (76 mg, 0.5 mmol) to afford 5d as light yellow solid 108 mg, in 48.6% yield. Mp; 197-201
1
ºC. H NMR (300 MHz, CDCl3) δ 3.84 (s, 3H), 5.29 (bs, 1H), 6.89 (d, 2H, J = 8.68 Hz), 7.21-
14

13
7.42 (m, 4H), 7.81 (d, 2H, J = 8.12 Hz), 8.19-8.28 (m, 4H); C NMR (125 MHz, CDCl3) δ
174.8, 167.9, 161.3, 151.1, 138.6, 138.2, 134.6, 128.6, 128.4, 127.0, 125.6, 123.4, 123.2, 118.9,
116.3, 116.1, 114.2, 56.0.; MS (ESI): m/z 369 [M+H]+. HRMS (ESI) calcd for C22H17N4O2
[M+H]+ 369.13495; found: 369.13482.
(3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-p-tolyl-1,2,4-oxadiazole) 5e
This compound was prepared by method described in section 6.3 employing ((Z)-4-(1H-
benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9a (152 mg, 0.6 mmol), p-tolylbenzoic acid
(68 mg, 0.5 mmol) to afford 5e as off white solid 103 mg, in 48.3% yield. Mp; 182-184 ºC. 1H
NMR (500 MHz, CDCl3) δ 2.39 (s, 3H), 5.21 (bs, 1H), 7.21-7.36 (m, 4H), 7.51 (d, 2H, J = 8.87
Hz), 7.58-7.64 (m, 2H), 8.20-8.29 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 174.7, 167.8, 151.1,
141.1, 138.6, 138.4, 134.4, 129.1, 128.4, 128.2, 127.7, 127.1, 125.7, 123.5, 123.3, 116.3, 116.2,
21.2. MS (ESI): m/z 353 [M+H]+; HRMS (ESI) calcd for C22H17N4O [M+H]+ 353.14002; found:
353.14034.
(3-(4-(1H-benzo[d]imidazol-2-yl)phenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,4-oxadiazole) 5f
This compound was prepared by method described in section 6.3 employing ((Z)-4-(1H-
benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9a (152 mg, 0.6 mmol), 3,4,5-methoxy
benzoic acid (106 mg, 0.5 mmol) to afford 5f as light yellow solid 146 mg, in 56.5% yield. Mp;
187-189 ºC. 1H NMR (300 MHz, CDCl3) δ 3.87 (s, 3H), 3.91 (s, 6H), 5.27 (bs, 1H), 7.04 (s, 2H),
13
7.20-7.31 (m, 2H), 7.41-7.49 (m, 2H), 8.19-8.28 (dd, 4H, J = 8.30, 7.93 Hz); C NMR (125
MHz, CDCl3) δ 174.8, 167.9, 151.4, 151.2, 143.2, 138.6, 138.5, 134.4, 129.1, 128.4, 127.0,
123.4, 123.3, 121.9, 116.3, 116.2, 106.8, 60.7, 56.3.; MS (ESI): m/z 429 [M+H]+. HRMS (ESI)
calcd for C24H21N4O4 [M+H]+ 429.15627; found: 429.15616.
(3-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-5-phenyl-1,2,4-oxadiazole) 5g
This compound was prepared by method described in section 6.3 employing 9(Z)-4-(5-fluoro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9b (162 mg, 0.6 mmol), benzoic acid (
61 mg, 0.5 mmol) to afford 5g as light brown solid 127 mg, in 59.6% yield. Mp; 177-179 ºC. 1H
NMR (300 MHz, CDCl3) δ 5.31 (bs, 1H), 7.02 (d, 1H, J = 7.29 Hz), 7.32-7.41 (m, 4H) 7.59 (d,
1H, J = 7.29 Hz), 7.79 (d, 2H, J = 8.12 Hz), 8.19-8.30 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
15

174.8, 167.9, 155.9, 151.2, 139.6, 134.6, 133.2, 130.2, 128.8, 128.4, 128.1, 127.2, 126.7, 126.5,
116.2, 112.8, 103.9.; MS (ESI): m/z 357 [M+H]+. HRMS (ESI) calcd for C21H14FN4O [M+H]+
357.11509; found: 357.11535.
(3-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-fluorophenyl)-1,2,4-oxadiazole) 5h
This compound was prepared by method described in section 6.3 employing 9(Z)-4-(5-fluoro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9b (162 mg, 0.6 mmol), p-flouro
benzoic acid (70 mg, 0.5 mmol) to afford 5h as light brown solid 131 mg, in 58.6% yield. Mp;
1
165-168 ºC. H NMR (300 MHz, CDCl3) δ 5.31 (bs, 1H), 7.11 (d, 1H, J = 7.26 Hz), 7.36-7.45
(m, 3H) 7.61 (s, 1H), 7.83 (d, 2H, J = 8.28 Hz), 8.21-8.33 (m, 4H); 13C NMR (125 MHz, CDCl3)
δ 174.9, 167.9, 164.9, 155.9, 151.2, 139.6, 134.6, 133.1, 130.1, 128.4, 128.2, 127.1, 123.9, 116.2,
115.8, 112.9, 103.8.; MS (ESI): m/z 375 [M+H]+. HRMS (ESI) calcd for C21H13F2N4O [M+H]+
375.10556; found: 375.10545.
(5-(4-chlorophenyl)-3-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,4-oxadiazole) 5i
This compound was prepared by method described in section 6.3 employing 9(Z)-4-(5-fluoro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9b (162 mg, 0.6 mmol), p-chloro
benzoic acid (78 mg, 0.5 mmol) to afford 5i as light brown solid 142 mg, in 60.8% yield. Mp;
186-188 ºC. 1H NMR (300 MHz, CDCl3) δ 5.31 (bs, 1H), 7.07 (d, 1H, J = 7.3 Hz), 7.41-7.57 (m,
13
5H) 7.59 (d, 1H, J = 7.3 Hz), 8.21-8.33 (m, 4H); C NMR (125 MHz, CDCl3) δ 174.7, 167.9,
155.8, 151.2, 139.7, 138.2, 134.7, 129.7, 129.4, 128.8, 128.4, 127.3, 127.0, 116.1, 133.2, 112.7,
103.9.; MS (ESI): m/z 391 [M+H]+. HRMS (ESI) calcd for C21H13ClFN4O [M+H]+ 391.07613;
found: 391.07594.
(3-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-methoxyphenyl)-1,2,4-oxadiazole) 5j
This compound was prepared by method described in section 6.3 employing 9(Z)-4-(5-fluoro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9b (162 mg, 0.6 mmol), p- methoxy
benzoic acid (76 mg, 0.5 mmol) to afford 5j as light yellow solid 147 mg, in 63.7% yield. Mp;
175-177 ºC. 1H NMR (500 MHz, CDCl3) δ 3.89 (s, 3H), 7.08-7.21 (m, 3H), 7.58 (d, 1H, J = 7.24
Hz), 7.68 (s, 1H), 7.91 (d, 2H, J = 8.28 Hz), 8.27-8.32 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
174.9, 167.8, 161.2, 155.7, 151.2, 139.7, 134.6, 133.2, 128.6, 128.4, 126.9, 125.5, 118.8, 116.2,
16

114.3, 112.8, 103.7, 56.1.; MS (ESI): m/z 387 [M+H]+. HRMS (ESI) calcd for C22H16FN4O2
[M+H]+ 387.12567; found: 387.12551.
(3-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-5-p-tolyl-1,2,4-oxadiazole) 5k
This compound was prepared by method described in section 6.3 employing 9(Z)-4-(5-fluoro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9b (162 mg, 0.6 mmol), p-tolyl benzoic
acid (68 mg, 0.5 mmol) to afford 5k as off white solid 123 mg, in 55.6% yield. Mp; 172-175 ºC.
1H NMR (300 MHz, CDCl3) δ 2.37 (s, 3H), 7.02-7.25 (m, 3H), 7.57 (d, 1H, J = 7.18 Hz), 7.64
(s, 1H), 7.87 (d, 2H, J = 8.23 Hz), 8.16-8.29 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 175.0,
167.8, 155.6, 151.2, 141.4, 139.7, 134.6, 133.3, 129.3, 128.6, 128.2, 127.7, 127.2, 125.8, 116.4,
112.9, 103.6, 21.3.; MS (ESI): m/z 371 [M+H]+. HRMS (ESI) calcd for C22H16N4F [M+H]+
371.13067; found: 371.13025.
(3-(4-(5-fluoro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,4-
oxadiazole) 5l
This compound was prepared by method described in section 6.3 employing 9(Z)-4-(5-fluoro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9b (162 mg, 0.6 mmol), 3,4,5-methoxy
benzoic acid ( 106 mg, 0.5 mmol) to afford 5l as light yellow solid 160 mg, in 60% yield. Mp;
166-168 ºC. 1H NMR (500 MHz, CDCl3) δ 3.81 (s, 3H), 3.87 (s, 6H), 6.98-7.21 (m, 3H), 7.46 (s,
13
1H), 7.55 (d, 1H, J = 7.28 Hz), 8.19-8.30 (dd, 4H, J = 8.30, 7.93 Hz); C NMR (125 MHz,
CDCl3) δ 174.9, 167.8, 155.7, 151.4, 151.1, 143.3, 139.6, 134.3, 133.1, 129.2, 128.3, 127.1,
122.0, 116.4, 112.8, 106.9, 103.8, 60.8, 56.3. MS (ESI): m/z 447 [M+H]+. HRMS (ESI) calcd
for C24H20FN4O4 [M+H]+ 447.14677; found: 447.14642.
3-(4-(5-Chloro-1H-benzo[d]imidazol-2-yl)phenyl)-5-phenyl-1,2,4-oxadiazole 5m
This compound was prepared by method described in section 6.3 employing ((Z)-4-(5-chloro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9c (172 mg, 0.6 mmol), benzoic acid (61
mg, 0.5 mmol) to afford 5m as off white solid 134 mg, in 60.1% yield. Mp; 163-165 ºC. 1H
NMR (500 MHz, CDCl3) δ 5.28 (bs, 1H), 7.10 (t, 1H, J = 7.27 Hz), 7.13-7.24 (m, 3H), 7.76 (d,
13
2H, J = 8.27 Hz), 7.96 (d, 2H, J = 8.28 Hz), 8.24 (dd, 4H, J = 8.32, 5.65 Hz). C NMR (125
MHz, CDCl3) δ 174.9, 167.8, 151.1, 138.1, 134.7, 133.2, 130.2, 129.7, 128.9, 128.3, 128.2,
17

127.1, 126.6, 122.9, 116.7, 116.2.; MS (ESI): m/z 373 [M+H]+. HRMS (ESI) calcd for
C21H14ClN4O [M+H]+ 373.08556; found: 373.08561.
(3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-fluorophenyl)-1,2,4-oxadiazole) 5n
This compound was prepared by method described in section 6.3 employing ((Z)-4-(5-chloro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9c (172 mg, 0.6 mmol), p-flouro benzoic
acid (70 mg, 0.5 mmol) to afford 5n as light brown solid 127 mg, in 54.4% yield. Mp; 156-158
1
ºC. H NMR (300 MHz, CDCl3) δ 7.21-7.35 (m, 3H), 7.57 (d, 1H, J = 7.23 Hz), 7.75 (s, 1H),
7.87 (d, 2H, J = 8.30 Hz), 8.26-8.30 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 174.8, 167.9,
164.8, 151.3, 138.2, 134.7, 133.1, 130.1, 129.8, 128.4, 128.2, 127.2, 124.0, 123.0, 116.7, 116.2,
115.8.; MS (ESI): m/z 355 [M+H]+. HRMS (ESI) calcd for C21H13ClFN4O [M+H]+ 391.07578;
found: 391.07556.
(3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-chlorophenyl)-1,2,4-oxadiazole) 5o
This compound was prepared by method described in section 6.3 employing ((Z)-4-(5-chloro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9c (172 mg, 0.6 mmol), p-chloro
benzoic acid (78 mg, 0.5 mmol) to afford 5o as light brown solid 134 mg, in 55.1% yield. Mp;
159-162 ºC. 1H NMR (300 MHz, CDCl3) δ 7.22 (d, 1H, J = 7.24 Hz), 7.41-7.54 (m, 3H), 7.83 (s,
1H), 7.91 (d, 2H, J = 8.12 Hz), 8.18-8.29 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 174.9, 168.0,
151.4, 138.8. 138.2, 134.6, 133.2, 129.8, 129.7, 129.6, 128.6, 128.3, 127.2, 126.9, 122.9, 116.8,
116.3.; MS (ESI): m/z 407 [M+H]+. HRMS (ESI) calcd for C21H13Cl2N4O [M+H]+ 407.04676;
found: 407.04689.
(3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-methoxyphenyl)-1,2,4-oxadiazole)
5p
This compound was prepared by method described in section 6.3 employing ((Z)-4-(5-chloro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9c (172 mg, 0.6 mmol), p-methoxy
benzoic acid (76 mg, 0.5 mmol) to afford 5p as light yellow solid 126 mg, in 52.3% yield. Mp;
166-169 ºC. 1H NMR (300 MHz, CDCl3) δ 3.87 (s, 3H), 7.12-7.24 (m, 3H), 7.54 (d, 1H, J = 7.32
Hz), 7.77 (s, 1H), 7.93 (d, 2H, J = 8.21 Hz), 8.27-8.32 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
175.0, 167.9, 161.3, 151.2, 138.2, 134.4, 133.3, 129.6, 128.7, 128.6, 126.9, 125.6, 122.9, 118.9,
18

116.6, 116.2, 114.2, 56.2.; MS (ESI): m/z 403 [M+H]+. HRMS (ESI) calcd for C22H16ClN4O2
[M+H]+ 403.09602; found: 403.09578.
(3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-5-p-tolyl-1,2,4-oxadiazole) 5q
This compound was prepared by method described in section 6.3 employing ((Z)-4-(5-chloro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9c (172 mg, 0.6 mmol), p-tolyl benzoic
acid (68 mg, 0.5 mmol) to afford 5q as off white solid 138 mg, in 59.7% yield. Mp; 179-182 ºC.
1H NMR (300 MHz, CDCl3) δ 2.39 (s, 3H), 7.29-7.37 (m, 3H), 7.54-7.65 (m, 3H), 7.78 (s, 1H),
8.06-8.21 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 175.1, 167.9, 151.3, 141.4, 138.3, 134.6,
133.2, 129.6, 129.3, 128.5, 128.1, 127.7, 127.2, 125.9, 122.8, 116.5, 116.4, 21.2.; MS (ESI): m/z
387 [M+H]+. HRMS (ESI) calcd for C22H16ClN4O [M+H]+ 387.10118; found: 387.10109.
(3-(4-(5-chloro-1H-benzo[d]imidazol-2-yl)phenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,4-
oxadiazole) 5r
This compound was prepared by method described in section 6.3 employing ((Z)-4-(5-chloro-
1H-benzo[d]imidazol-2-yl)-N'-hydroxybenzimidamide) 9c (172 mg, 0.6 mmol), 3,4,5-trimethoxy
benzoic acid (106 mg, 0.5 mmol) to afford 5r as yellow solid 176 mg, in 65% yield. Mp; 169-
172 ºC. 1H NMR (300 MHz, CDCl3) δ 3.89 (s, 3H), 3.93 (s, 6H), 7.16-7.25 (m, 3H), 7.57 (d, 1H,
J = 7.2 Hz), 7.81 (s, 1H), 8.20-8.29 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 174.9, 167.8, 151.2,
151.4, 143.4, 138.2, 134.3, 133.2, 129.6, 129.3, 128.3, 127.2, 122.8, 122.1, 116.6, 116.2, 106.8,
60.7, 56.3.; MS (ESI): m/z 463 [M+H]+. HRMS (ESI) calcd for C24H20ClN4O4 [M+H]+
463.11722; found: 463.11694.
(3-(4-(5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-5-phenyl-1,2,4-oxadiazole) 5s
This compound was prepared by method described in section 6.3 employing ((Z)-N'-hydroxy-4-
(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide) 9d (169 mg, 0.6 mmol), benzoic acid
(61 mg, 0.5 mmol) to afford 5s as yellow solid 139 mg, in 63% yield. Mp; 172-175 ºC. 1H NMR
(300 MHz, CDCl3) δ 3.88 (s, 3H), 7.12-7.23 (m, 2H), 7.32-7.51 (m, 4H), 7.79 (d, 1H, J = 7.23
Hz), 8.23-8.30 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 175.0, 167.9, 155.7, 151.2, 138.4, 134.7,
130.1, 128.9, 128.4, 128.3, 127.1, 126.6, 114.5, 113.7, 101.9, 55.9.; MS (ESI): m/z 369 [M+H]+.
HRMS (ESI) calcd for C22H17N4O2 [M+H]+ 369.13507; found: 369.13534.
19

(5-(4-fluorophenyl)-3-(4-(5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,4-oxadiazole) 5t
This compound was prepared by method described in section 6.3 employing ((Z)-N'-hydroxy-4-
(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide) 9d (169 mg, 0.6 mmol), p-flouro
benzoic acid (70 mg, 0.5 mmol) to afford 5t as yellow solid 129 mg, in 55.7% yield. Mp; 162-
1
166 ºC. H NMR (300 MHz, CDCl3) δ 3.85 (s, 3H), 7.04-7.21 (m, 2H), 7.57-7.62 (m, 3H), 7.71
13
(d, 1H, J = 7.24 Hz), 8.19-8.28 (m, 4H); C NMR (125 MHz, CDCl3) δ 174.8, 167.9, 164.8,
155.7, 151.3, 138.4, 135.5, 134.7, 130.2, 128.4, 128.2, 127.3, 124.0, 115.9, 114.5, 113.6, 101.8,
56.0. MS (ESI): m/z 387[M+H]+. HRMS (ESI) calcd for C22H16FN4O2 [M+H]+ 387.12557;
found: 387.12516.
(5-(4-chlorophenyl)-3-(4-(5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-1,2,4-oxadiazole)
5u
This compound was prepared by method described in section 6.3 employing ((Z)-N'-hydroxy-4-
(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide) 9d (169 mg, 0.6 mmol), p-chloro
benzoic acid (78 mg, 0.5 mmol) to afford 5u as yellow solid 119 mg, in 49 % yield. Mp; 192-196
1
ºC. H NMR (500 MHz, CDCl3) δ 3.83 (s, 3H), 7.02 (d, 1H, J = 7.21 Hz), 7.21 (s, 1H), 7.54 (d,
2H, J = 8.11 Hz), 7.59 (d, 1H, J = 7.24 Hz), 7.83 (d, 2H, J = 8.12 Hz), 8.21-8.32 (dd, 4H, J = 8.3,
5.62 Hz); 13C NMR (125 MHz, CDCl3) δ 174.9, 168.0, 155.6, 151.4, 139.8, 138.6, 135.6, 134.5,
129.9, 129.7, 128.3, 128.6, 127.3, 126.8, 114.4, 113.6, 101.8, 55.9. MS (ESI): m/z 403 [M+H]+.
HRMS (ESI) calcd for C22H16ClN4O2 [M+H]+ 403.09603; found: 403.09576.
(3-(4-(5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-5-(4-methoxyphenyl)-1,2,4-oxadiazole)
5v
This compound was prepared by method described in section 6.3 employing ((Z)-N'-hydroxy-4-
(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide) 9d (169 mg, 0.6 mmol), p-methoxy
benzoic acid (76 mg, 0.5 mmol) to afford 5v as pale yellow solid 125 mg, in 52.4% yield. Mp;
179-182 ºC. 1H NMR (300 MHz, CDCl3) δ 3.87 (s, 3H), 3.89 (s, 3H), 6.91 (d, 1H, J = 7.24 Hz),
7.15 (s, 1H), 7.46-7.52 (m, 2H), 7.57 (d, 1H, J = 7.26 Hz), 7.89-8.12 (m, 2H), 8.27-8.32 (m, 4H);
13C NMR (125 MHz, CDCl3) δ 174.8, 167.9, 161.4, 155.6, 151.2, 138.6, 135.4, 134.6, 128.7,
128.5, 126.9, 125.5, 118.9, 118.8, 114.4, 114.3, 113.7, 101.9, 56.3. MS (ESI): m/z 399 [M+H]+.
HRMS (ESI) calcd for C23H19N4O3 [M+H]+ 399.14565; found: 399.14559
(3-(4-(5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-5-p-tolyl-1,2,4-oxadiazole)5w
20

This compound was prepared by method described in section 6.3 employing ((Z)-N'-hydroxy-4-
(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide) 9d (169 mg, 0.6 mmol), p-tolyl benzoic
acid (68 mg, 0.5 mmol) to afford 5w as yellow solid 137 mg, in 57.6% yield. Mp; 184-187 ºC.
1H NMR (300 MHz, CDCl3) δ 2.36 (s, 3H), 3.78 (s, 3H), 6.99 (d, 1H, J = 8.26 Hz), 7.18 (s, 1H),
7.32- 7.46 (m, 4H), 7.61 (d, 1H, J = 8.23 Hz), 8.21-8.31 (m, 4H); 13C NMR (125 MHz, CDCl3) δ
175.1, 167.9, 155.5, 151.3, 141.3, 138.5, 135.4, 134.6, 129.4, 128.5, 128.4, 127.7, 127.2, 125.8,
114.6, 113.7, 101.7, 56.2, 21.3. MS (ESI): m/z 383 [M+H]+. HRMS (ESI) calcd for C23H19N4O2
[M+H]+ 383.15046; found: 383.15032.
(3-(4-(5-methoxy-1H-benzo[d]imidazol-2-yl)phenyl)-5-(3,4,5-trimethoxyphenyl)-1,2,4-
oxadiazole) 5x
This compound was prepared by method described in section 6.3 employing ((Z)-N'-hydroxy-4-
(5-methoxy-1H-benzo[d]imidazol-2-yl)benzimidamide) 9d (169 mg, 0.6 mmol), 3,4,5-
trimethoxy benzoic acid (106 mg, 0.5 mmol) to afford 5x as yellow solid 157 mg, in 57.2%
1
yield. Mp; 187-189 ºC. H NMR (500 MHz, CDCl3) δ 3.86 (s, 3H), 3.90 (s, 3H), 3.99 (s, 6H),
6.84 (d, 1H, J = 8.68 Hz), 7.12 (s, 1H), 7.42 (s, 2H), 7.50 (d, 1H, J = 8.68 Hz), 8.30 (dd, 4H, J =
13
8.30, 7.93 Hz); C NMR (125 MHz, CDCl3) δ 174.9, 167.7, 155.5, 151.9, 151.3, 143.3, 138.6,
135.5, 134.4, 129.3, 128.4, 127.1, 122.2, 114.5, 113.7, 106.9, 101.9, 56.4, 60.8. MS (ESI): m/z
459 [M+H]+. HRMS (ESI) calcd for C25H23N4O5 [M+H]+ 459.16665; found: 459.16643.
7.0 Biology
7.1 In Vitro Anti proliferative activity (SRB assay)
The compounds were evaluated for their in vitro anti-proliferative activity at National Cancer
Institute (NCI), USA against full NCI 60 cell lines panel representing on full nine human
systems as leukemia, melanoma and cancers of lung, colon, brain, breast, ovary, kidney and
prostate in accordance with their applied protocol (used SRB assay).The screening, begins with
the evaluation of the compounds against the 60 cell lines at one dose of 10 mM. The single dose
screen results were reported as a mean graph. Compounds that exhibited significant growth
inhibition in single dose screening were further evaluated against the 60 cell panel at five
concentration levels. The human tumor cell lines of the screening panel were grown in RPMI
21

1640 medium containing 5% fetal bovine serum and 2 m ML-glutamine. For a typical screening
experiment, cells seeded into 96 well microtiter plates in 100 µL at plating densities ranging
from 5000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell
seeding, the plates incubated at 37 ºC, 5% CO2, 95% air and 100% relative humidity for 24 h
prior to addition of experimental drugs. After 24 h, two plates of each cell line fixed in situ with
TCA, to represent a measurement of the cell population for each cell line at the time of drug
addition (Tz). Test compounds solubilized in dimethyl sulfoxide at 400-fold the desired final
maximum test concentration and stored frozen prior to use. At the time of drug addition, an
aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 mg/mL gentamicin. Additional four, 10-fold
or log serial dilutions are made to provide a total of five drug concentrations plus control.
Aliquots of 100 µL of these different drug dilutions added to the appropriate microtiter wells
already containing 100 µL of medium, resulting in the required final drug concentrations.
Following drug addition, the plates were incubated for an additional 48 h at 37 ºC, 5% CO2, 95%
air, and 100% relative humidity. For adherent cells, the assay terminated by the addition of cold
TCA. Cells fixed in situ by the addition of 50 µL of cold 50% (w/v) TCA (final concentration,
10% TCA) and incubated for 60 min at 4 ºC. The supernatant was discarded, and the plates
washed five times with tap water and then air dried. Then, 0.4% (w/v) solution of
Sulforhodamine B (SRB) in 1% acetic acid, (100 µL) was added to each well and plates
incubated for 10 min at room temperature. Unbound dye removed by washing five times with
1% acetic acid and then the plates air dried. Bound stain solubilized with 10 mM trizma base and
the absorbance was recorded on an automated plate reader at a wavelength of 515 nm. For Non
adherent cells, the methodology was same except that the assay terminated by fixing settled cells
at the bottom of the wells by adding 50 µL of 80% TCA (final concentration, 16% TCA). Using
the seven absorbance measurements [time zero, (Tz), control growth, (C) and test growth in the
presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at
each of the test concentrations levels using the following formula:
(Ti - Tz)/(C - Tz) x 100 for concentrations for which Ti > / = Tz
(Ti - Tz) /Tz x 100 for concentrations for which Ti < Tz:
22

Three dose response parameters (GI50, TGI and LC50) were calculated for each experimental
agent. Growth inhibition of 50% (GI50) calculated from [(T i- Tz)/(C-Tz)] x100 = 50, which was
the drug concentration resulting in a 50% reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation. The drug concentration resulting in
total growth inhibition (TGI) calculated from Ti = Tz. The LC50 (concentration of drug resulting
in a 50% reduction in the measured protein at the end of the drug treatment as compared to that
at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti-
Tz)/Tz] x 100 = -50. Values were calculated for each of these three parameters, if the level of
activity was reached; however, if the effect is not reached or was exceeded, the value for that
parameter expressed as greater or less than the maximum or minimum concentration tested.
7.2 Cell Cycle Analysis
MCF-7 cells (1x106 cells/well) were seeded in six well plates and treated with compounds 5l, 5x
and Nocodazole at concentrations of 1 µM and 3 µM for 48h. After the treatment, both floating
and trypsinised adherent cells were collected, washed with PBS and fixed with 70% ethanol.
After fixation cells were washed with PBS and stained with 50 mg/mL propidium iodide in
hypotonic lysis buffer (0.1% sodium citrate, 0.1% Triton X-100) containing DNase-free RNase-
A for 20 min. Stained cells were analyzed using fluorescence-activated cell sorter caliber
(Becton Dickinson).
7.3 In Vitro Tubilin Polymerization
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, GTP (1 µM) in the presence or absence of test
compounds 5l, 5x and nocodazole (final concentration of 3µM). 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 Varioscan multimode plate reader
(Thermo scientific Inc.). The IC50 value was defined as the drug concentration required to inhibit
50% of tubulin assembly compared to control. The reaction mixture for these experiments
include: tubulin (3 mg/mL) in PEM buffer, GTP (1 mM), in the presence or absence of test
23

compounds at various concentrations. Polymerization was monitored by increase in the
fluorescence as mentioned above at 37 °C.
7.4 Immunohistochemistry of Tubulin
MCF-7 cells were seeded on glass cover slips, incubated for 48 h in the presence or absence of
test compounds 5l, 5x and nocodazole at a concentration of 1 µM. Following the termination of
incubation, cells were fixed with 3% formaldehyde, 0.02% glutaraldehyde in PBS and
o
permeabilized by dipping the cells in 100% methanol (-20 C). Later, cover slips were blocked
with 1% BSA in phosphate buffered saline for 1h followed by incubation with a primary
antitubulin (mouse monoclonal) antibody and FITC conjugated secondary mouse anti IgG
antibody. Photographs were taken using the confocal microscope, equipped with FITC settings
and the pictures were analyzed for the integrity of microtubule network.
7.5 Colchicine competitive binding assay
The test compounds 5l, 5x, nocodazole and taxol of various concentrations 0, 5, 10 and 15 µM
were co-incubated with 4 µM colchicine in 30 mM Tris buffer containing 3 µM tubulin for 60
min at 37 ºC. Nocodazole was used as a positive control whereas taxol was used as negative
control which does not bind at colchicine site. After incubation the fluorescence of tubulin–
colchicine complex was determined by using Tecan multimode reader at excitation wavelength
at 350 nm and emission wavelength at 435 nm. 30 mM Tris buffer was used as blank which was
subtracted from all the samples. The experiment was carried out both in the presence and
absence of colchicine to obtain fluorescence values of the desired tubulin–colchicine complex.
The fluorescence values of tubulin–conjugates complex was subtracted from the tubulin–
conjugates–colchicine complex. Fluorescence values are normalized to DMSO control
7.6 Hoechst Staining
MCF-7 Cells were seeded at a density of 10,000 cells over 18-mmcoverslips and incubated for
24h. Then, the medium was replaced, and cells were treated with 1 µM concentration of
compounds 5l, 5x and nocodazole for 24h. Cells treated with vehicle (0.001% DMSO) were
included as controls for all experiments. Cells were fixed with 4 % paraformaldehyde and
stained with solution of Hoechst 33342 (Sigma Aldrich) 5 µg/mL in PBS. After incubation for
24

30 min at 37 °C, excess dye was washed with PBS and cells from each dish were captured from
randomly selected fields under fluorescent microscope (Leica, Germany) to qualitatively
determine the proportion of viable and apoptotic cells based on their relative fluorescence and
nuclear fragmentation.
7.7 Mitochondrial membrane potential
MCF-7 (1x106 cells/well) cells were cultured in six-well plates and treated with compounds 5l
and 5x at 1 µM concentration for 24 h. After treatment, cells were collected by trypsinization and
washed with PBS followed by suspending in 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-
imidacarbocyanine iodide (JC-1 dye) solution (10 mM/L) and incubated at 37 ºC for 15 min.
Cells were rinsed three times with medium and suspended in pre warmed medium. The cells
were then subjected fluorescence-activated cell sorter caliber (Becton Dickinson) in the FL1,
FL2 channel to detect mitochondrial potential
7.8 DNA Fragmentation
MCF-7 Cells were seeded (1x106) in six well plates and allowed to adhere for 24 h. Cells were
treated with compounds 5l and 5x at 2 µM concentration fore 24 h. Cells were collected and
centrifuged at 2500 rpm for 5 min at 4 ºC. Pellet was collected and washed with PBS, added 250
µl of lysis buffer (100mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0, 0.25 % SDS)
º
containing 400 µg/mL DNase free RNase A and incubated at 37 C for 90 min followed by
incubation with proteinase K (200 ug/mL) at 50 oC for 1h. Centrifuged at 3000 rpm for 5 min at
4 ºC and collected supernatant. Then added 65 µl of 10 M Ammonium acetate and 500 µl of ice
o
cold ethanol and mixed well. And these samples were incubated at -80 C for 1 h. After
o
incubation samples were centrifuged at 12000 rpm for 20 min at 4 C. After centrifuge, pellet
was washed with 80% ethanol and air dried for 10 min at room temperature. Dissolved the pellet
in 50 µl of TE buffer and DNA laddering was determined by using 2% agarose gel
electrophoresis in TE Buffer.
7.9 Annexin V–FITC assay
6)
MCF-7 cells (1x10
were seeded in six-well plates and allowed to adhere overnight. The
medium was then replaced with medium containing 1 µM concentrations of compounds 5l and
25

5x and incubated for 24 h. Cells treated with DMSO (0.001%) was used as control. After 24 h of
drug treatment, cells were harvested, washed with PBS. Then the cells (1x 106) were stained with
Annexin V-FITC and propidium iodide using the Annexin-V-PI apoptosis detection kit (Sigma
Aldrich-India). Flow cytometry was performed using a FACScan (Becton Dickinson) equipped
with a single 488 nm argon laser. Annexin V-FITC was analyzed using excitation and emission
settings of 488 nm and 535 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2 channel). Debris
and clumps were gated out using forward and orthogonal light scatter.
7.10. Molecular modeling procedure
All the geometries were optimized in Gaussian 09 using PM3 semi-empirical method.27 Protein
structure was downloaded from Protein Data Bank (PDB ID: 3E22).28 Docking studies were
performed using AutoDock 4.2 software.29 The analysis of intermolecular interactions has been
performed using Pymol, v. 0.99.30
Acknowledgements
T.S.R, V.K.N thanks NIPER, Hyderabad, India for the award of research fellowship and
A.V.S.R, V.S thanks CSIR,New DELhi for the award of the senior research fellowships. We also
thank CSIR for financial support under the 12th Five Year plan project “Affordable Cancer
Therapeutics” (CSC0301) and “Small Molecules in Lead Exploration (SMiLE)” (CSC0111).
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