Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent Tubulin Polymerization inhibitors

Article abstract of DOI:10.1016/j.bioorg.2016.02.007

A new class of combretastatin linked 1,3,4-oxadiazoles were designed, synthesized and screened for their cytotoxic activity against five human cancer cell lines such as HeLa, DU-145, A549, MDA-MB-231 and B16. These compounds showed significant cytotoxicity with IC₅₀ values in the range 0.118-54.32 μM. Conjugate 5m displayed potent antiproliferative activity against DU-145 cell line. Flow cytometric analysis revealed that these compounds arrested the cell cycle in G2/M phase. Moreover, the tubulin polymerization assay and immunofluorescence analysis indicate that 5m exhibits potent inhibitory effect on the tubulin assembly. Further, DNA fragmentation and Hoecst staining assays confirm that 5m induces apoptosis. Molecular docking studies and competitive binding assay indicated that 5m effectively bind at the colchicine binding site of the tubulin.

Full text of DOI:10.1016/j.bioorg.2016.02.007

Accepted Manuscript
Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent Tubulin Poly-
merization inhibitors
Ahmed Kamal, P.S. Srikanth, M.V.P.S. Vishnuvardhan, G. Bharath Kumar, M.
Korrapati Suresh Babu, S.M. Ali Hussaini, Jeevak Sopanrao Kapure, Abdullah
Alarifi
PII:
S0045-2068(16)30015-3
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.02.007
YBIOO 1883
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 January 2016
23 February 2016
24 February 2016
Please cite this article as: A. Kamal, P.S. Srikanth, M.V.P.S. Vishnuvardhan, G. Bharath Kumar, M. Korrapati
Suresh Babu, S.M. Ali Hussaini, J.S. Kapure, A. Alarifi, Combretastatin linked 1,3,4-oxadiazole conjugates as a
Potent Tubulin Polymerization inhibitors, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.
2016.02.007
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Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent
Tubulin Polymerization inhibitors
Ahmed Kamal,*a,b,c,d P. S. Srikanth,a,b M. V. P. S. Vishnuvardhan,a G. Bharath Kumar,a M
Korrapati Suresh Babu,a S. M. Ali Hussaini,a Jeevak Sopanrao Kapure,a,c Abdullah Alarifi d
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India
bAcademy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology,
Hyderabad, India
cDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
dCatalytic Chemistry Chair, Chemistry Department, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia
Abstract: A new class of combretastatin linked 1,3,4-oxadiazoles were designed, synthesized
and screened for their cytotoxic activity against five human cancer cell lines such as HeLa, DU-
145, A549, MDA-MB-231 and B16. These compounds showed significant cytotoxicity with
IC50 values in the range 0.118–54.32 M. Conjugate 5m displayed potent antiproliferative
activity against DU-145 cell line. Flow cytometric analysis revealed that these compounds
arrested the cell cycle in G2/M phase. Moreover, the tubulin polymerization assay and
immunofluoresence analysis indicate that 5m exhibits potent inhibitory effect on the tubulin
assembly. Further, DNA fragmentation and Hoecst staining assays confirm that 5m induces
apoptosis. Molecular docking studies and competitive binding assay indicated that 5m
effectively bind at the colchicine binding site of the tubulin.
Keywords: 1,3,4-oxadiazole, tubulin polymerization, apoptotic cell death, colchicine binding and
molecular modeling.
Corresponding authors Tel: +91-40-27193157; fax: +91-40-27193189, e-mail:
ahmedkamal@iict.res.in (A. Kamal)

1. Introduction
Microtubules are essential for various cellular processes including motility, cell
signalling and division, shape maintenance and intracellular transport.[1, 2] They are formed by
the polymerization of tubulin heterodimers and dissociate back during normal functioning of the
cell. This process is highly aberrant in case of cancer thereby rendering it to be an attractive
target for the development of newer anticancer agents.[3, 4] Drugs such as paclitaxel and
vinblastine that are used for the treatment of several types of cancers act by interfering with
microtubule dynamics. Their clinical success indicates the immense potential of this therapeutic
target and attracted interest of numerous research groups across the globe to find out microtubule
targeting agents.[5, 6]
Combretastatin A-4 (CA-4,1, Figure 1) is a cis-stilbene natural product isolated by Pettit
and coworkers from the bark of Combretum caffrum.[7] It showed cytotoxicity against a wide
range of human cancer cell lines including multidrug-resistant cancer cells.[8] It is a
microtubule-destabilizing agent and interacts strongly with the -tubulin by binding into the
colchicine binding site.[9] Structure–activity relationship (SAR) studies revealed that 3,4,5-
trimethoxyphenyl ring (ring-A) and cis configuration of the stilbene connecting the ring A to
ring B are essential for antiproliferative activity.[10-12] Whereas the 3ˈ-hydroxy group of ring B
is considered to be nonessential for the interaction of CA-4 with the tubulin and therefore it has
been replaced by other suitable moieties however, CA-4 suffers from bioavailability and stability
issues.[13] The cis double bond readily isomerizes to the stable trans form that leads to loss of
both cytotoxicity as well as antitubulin activity.[14] Therefore, several attempts made to
overcome the stability issues led to the development of phenstatin and iso CA-4 derivatives (1b-
e).[15, 16] Similarly, strategy of replacing the olefinic bridge with several carbocycles and
heterocycles has also been adapted to overcome the isomerization. Some of the heterocycles
include pyrazole, isoxazole, oxadiazole, imidazole, aminothiazole, benzofuran etc. This
replacement was found to be fruitful as both cytotoxicity and antitubulin activity have not only
been retained but also improved in most of the cases.[17-19]
On the other hand, oxadiazoles are known to possess various biological activities such as
antibacterial, anti-inflammatory and anticancer etc.[20-27] 1,3,4-Oxadiazole moiety shows lower
lipophilicity and high metabolic stability, hERG inhibition and aqueous solubility compared to
1,2,4-oxadiazole.[28] One of the compounds containing an oxadiazole ring, zibotentan (2) is a
pyridine based sulfonamide that exhibits remarkable antiproliferative activity.[29] Considering
their biological potential we have previously developed and reported newer anticancer agents by

conjugating it to other pharmacophores such as 2-anilinonicotine and pyrazole.[30, 31] These
conjugates effectively inhibited tubulin polymerization which indicates the immense potential of
oxadiazole structure to be useful in the development of microtubule destabilizing agents.
Therefore, in order to develop newer anticancer agents based on CA-4 scaffold an attempt has
been made to rigidify its unstable ethenyl bridge by introducing substituted oxadiazole moiety
adjacent to the ring A. We herein report the synthesis and cytotoxic activities of these oxadiazole
linked combretastatin congeners along with a brief study to determine their mode of action.
Fig. 1 Chemical structures of CombretastatinA-4 (1a), phenstatin (1b), phenstatin amine (1c),
isocombretastatin A-4 (1d), isocombretastatin A-4 amine (1e), Zibotentan (2), anilinonicotinyl–
oxadiazole conjugate (3), pyrazole–oxadiazole conjugate (4), combretastatin linked oxadiazole
conjugates (5).
2. Results and discussion
2.1 Chemistry
The desired target molecules (5a-p) were obtained from the starting material
trimethoxyphenylacetic acid (6) prepared by Perkins condensation of 3,4,5-

trimethoxyphenylacetic acid (6) and 4-methoxybenzaldehyde (7) in the presence of acetic
anhydride and triethylamine under refluxing conditions to afford (E)-3-(4-methoxyphenyl)-2-
(3,4,5-trimethoxyphenyl)acrylic acid (8).[32] The hydrazide intermediate (11) was prepared
from substituted aromatic and hetero aromatic carboxylic acids by esterification followed by
their reaction with hydrazine hydrate and H2SO4 in EtOH.[33] The formation of oxadiazole
scaffold from carboxylic acids and hydrazides has been well investigated by various
researchers.[34-38] Therefore, the desired combretastatin-oxadiazole sconjugates (5a-p) were
obtained by following one of the known methods i.e., condensation of aroylhydrazides (11a-p)
and acid (12) in POCl3.
Scheme 1: Reagents and conditions: (a) Ac2O, TEA, 120 °C 12 h, 40%; (b) H2SO4, EtOH, 70 °C, 3 h, 86-
94%; (c) NH2NH2.5H2O, EtOH, 70 °C, 6 h, 80-92%; (d) RCONHNH2, POCl3, 70 °C, 5-6 h, 80–93%.
2.2 Antiproliferative activity
The synthesized conjugates 5a-p were evaluated for their antiproliferative activity
against five human cancer cell lines namely cervix (HeLa), prostate (DU-145), lung
adenocarcinoma (A549), breast adenocarcinoma (MDA-MB-231) and mouse macrophages (B-
16) cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay.[39] The results expressed as IC50 values are summarized in Table 1 and CA-4 has been

employed as a positive control for comparison. Results indicate that some of the compounds
exhibit significant cytotoxicity in almost all the tested cell lines with IC50 values ranging
between 0.118 and 54.3 µM. Particularly, compounds 5l and 5m showed significant cytotoxity
against all the tested cell lines displaying IC50 below 3 µM. In fact, 5m containing 3,4,5-
rimethoxy substituent was found to be the most effective compound from the series against the
prostate cancer cell line (IC50 = 0.118 µM). Similarly, 5k exhibited significant cytotoxicity
particularly against HeLa, DU-145 and A549 cells.
Table 1. Cytotoxicity of combretastatin linked 1,3,4-oxadiazole conjugates (5a–p) against a
panel of human cancer and normal cell lines.
aIC50 (µM)
dA549
Comp.
eMDA-MB-
231
bHeLa
cDU145
fB-16
12.51 ± 0.41
13.68 ± 0.14
15.90 ± 0.52
28.24 ± 0.44
17.35 ± 0.19
14.84 ± 0.67
32.26 ± 0.26
35.23 ± 0.83
3.521 ± 0.74
2.687 ± 1.04
2.184 ± 0.48
1.117 ± 0.37
0.365 ± 0.23
24.45 ± 1.17
16.42 ± 0.63
13.34 ± 1.58
0.004 ± 0.03
19.27 ± 0.22
13.23 ± 0.46
16.7 ± 1.63
25.37 ± 2.22
14.65 ± 2.55
16.60 ± 0.4
27.39 ± 0.71
28.47 ± 3.26
5.103 ± 0.23
2.138 ± 1.71
0.538 ± 0.02
0.274 ± 0.52
0.118 ± 0.37
32.13 ± 1.6
18.48 ± 1.7
9.433 ± 0.5
0.270 ± 0.08
18.65 ± 0.32
8.48 ± 0.92
33.44 ± 0.14
11.76 ± 2.4
28.93 ± 0.25
55.28 ± 0.84
15.83 ± 2.84
39.28 ± 0.58
50.41 ± 0.84
39.82 ± 1.32
5.257 ± 1.24
5.548 ± 1.74
2.869 ± 0.82
0.415 ± 0.54
0.321 ± 0.87
27.17 ± 0.26
23.68 ± 0.77
18.75 ± 0.46
0.045 ± 0.04
25.47 ± 0.32
15.26 ± 0.15
25.22 ± 0.53
43.24 ± 0.72
13.75 ± 0.68
29.73 ± 0.24
40.75 ± 0.37
36.33 ± 0.16
16.247 ± 0.18
10.386 ± 0.92
5.536 ± 0.47
2.632 ± 0.82
0.537 ± 0.61
28.62 ± 0.45
15.59 ± 0.28
20.33 ± 0.21
5a
5b
5c
5d
5e
15.89 ± 0.54
20.83 ± 0.39
24.75 ± 0.62
24.51 ± 0.15
54.32 ± 0.32
51.78 ± 0.18
4.372 ± 0.82
3.382 ± 0.86
1.373 ± 0.78
2.023 ± 0.27
0.261 ± 0.91
15.87 ± 0.95
12.18 ± 0.46
3.541 ± 0.21
0.180 ± 0.06
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
CA-4
-
a
b
Each data represents mean + S.D. from three different experiments performed in triplicates. HeLa: human cervix
c
d
cancer cell line. DU145: human prostate cancer cell line. A549: human lung adenocarcinoma epithelial cell line.
eMDA-MB-231: human breast adenocarcinoma cell line. fB-16: Mouse macrophages cell line.
Based on the cytotoxicity data, the structure–activity relationship (SAR) for these
combretastatin linked 1,3,4-oxadiazole conjugates has been elucidated. Keeping the stilbene
pharmacophore constant the effect of modifications on the phenyl ring at the second position of
1,3,4-oxadiazole moiety has been examined. The phenyl ring containing various substituents
with electron donating and electron withdrawing nature have been employed in this study. In
addition, the effect of hetero aromatic rings such as thiophenyl, 2-furfuryl and 3-benzofuran
have also been investigated. Compounds containing electron donating groups exhibited very
good activity. Compound 5m having trimethoxy substituent on C-ring displayed excellent
anticancer efficacy towards DU-145 cell line with IC50 value 0.118 µM. The cytotoxicity was
found to decrease with the removal of methoxy group which is evident from the activities of 5l

and 5k containing 3,4-dimethoxy and 4-methoxy substituents respectively (IC50= 0.274 and
0.538 µM respectively against DU-145). Further decrease in the activity was observed when
methoxy group is replaced by a N,N-dimethylamino and methyl group as in 5i and 5j (IC50=
2.138 and 5.108 µM respectively against DU-145). Compounds with weak electron withdrawing
groups such as halogen substituents exhibited moderate efficacy. It was observed that
cytotoxicity decreases with the increase in size of the halogen atom and thus cytotoxicity for
fluoro, chloro and bromo is in the order F>Cl>Br. On the other hand, compounds with electron
withdrawing groups like nitro at meta and para position as in 5g and 5h showed reduced
cytotoxicity against the all five tested human cancer cell lines.
Fig. 2 Structure–activity relationship of combretastatin linked 1,3,4-oxadiazole conjugates (5a–p).
These observations indicate that presence of electron donating groups on the phenyl ring
of oxadiazole moiety is useful for cytotoxicity which diminishes with the increase in electron
withdrawing nature of the substituents. However, the presence of heteroaromatic groups do not
show profound effect on the cytotoxicity with 3-benzofuran as in 5p being more suitable
compared to 2-thiophenyl (5n) and 2-furfuryl (5o) groups. As compound 5m is the most
effective conjugate from the series exhibiting a mean IC50 of 0.398µM and IC50 of 0.11µM
against DU-145 it is taken up for detailed studies to investigate the mechanism of action.

2.3 Effect of compounds on cell cycle analysis
In order to understand the role of these conjugates 5m in cell cycle, FACS analysis [40]
was conducted in prostrate cancer cells (DU-145). It is well known that the polymerization
inhibitors arrest the cell cell cycle at the G2/M phase. Thus preventing further cell division or
subsequently resulting in mitotic slippage, which eventually leads to mitotic catastrophe or
apoptosis.
Fig 3. Flow cytometric analysis in DU-145 prostate cancer cell lines after treatment with compound 5m
at 0.5 μM concentrations for 24 h. CA-4 were is used as the positive controls.
To determine whether the cytotoxic effects induced by the treatment of these conjugates
were due to cell cycle arrest, we performed flow cytometry analysis. Prostrate cancer cells (DU-
145) were treated with compound 5m, at concentrations of 0.5 μM for duration of 24 h. Later,
cells were harvested and analyzed by flow cytometry and interestingly, major populations of the
cells treated with 5m arrested at the G2/M phase. These results confirm the growth inhibitory
effects of 5m which is comparable to CA-4 on prostate cancer (DU-145) cells showed 26.46%
(27.11%) whereas control (untreated cells) exhibited 12.19% as shown in Fig 3 and Table 2.

Table: 2 Cell cycle distribution of DU-145 cells (%) for 5m and CA-4
Compound
Control
5m
Sub-G1
1.5
G1
S
G2/M
1.2
92.7
50.9
49.1
2.9
3.0
3.9
2.0
41.2
43.8
CA-4
1.1
2.4 Effect of compounds on Tubulin polymerization
Fig. 4 Effect of compound on tubulin polymerization: Tubulin polymerization assay was carried out in a
reaction mixture that contained PEM buffer and GTP (1 mM) in the presence or absence of test conjugate
5m and CA-4 at 5 µM concentration. The reaction was initiated by the addition of GTP to all the wells.
Tubulin polymerization was monitored by the increase in fluorescence at 420 nm (excitation wavelength
is 360 nm) was measured for 30 min at 1 min interval in a multimode plate reader (Tecan) at 37 oC.

One of the possibilities that these compounds exhibit anticancer activity as well as G2/M cell
cycle arrest is by the inhibition of tubulin polymerization,[41, 42] this as has been observed in
many antimitotic agents such as combretastatins. Hence it was considered of interest to
investigate the tubulin polymerization aspect. As tubulin subunits heterodimerize and self-
assemble to form microtubules in a time dependent manner, we have investigated the
progression of tubulin polymerization by monitoring the increase in fluorescence emission at
420 nm (excitation wavelength is 360 nm) in 384 well plate for 30 min at 37 °C with and
without the compounds at 5 µM concentration. Compound 5m inhibited tubulin polymerization
by 53.26% and in comparison to CA-4 by 54.76%, compared to control, (Fig 4).
2.5 Immunohistochemistry of tubulin
Fig. 5 IHC analyses of compounds on the microtubule network: DU-145cells were treated with
compound 5m at 0.5 μM concentration for 48 h followed by staining with α-tubulin antibody.
Microtubule organization was clearly observed by green color tubulin network like structures in control
cells and was found to be disrupted in cells treated with compound 5m and CA-4 as the positive control.
In order to determine whether these conjugates interact directly with tubulin through binding to
the colchicine-binding site, we further examined the effect of 5m on cellular microtubule
network using immunofluorescence techniques.[43] The effect of 5m was further examined on

the cellular microtubules in DU-145 cancer cells by immunohistochemistry. It is clear from the
cytotoxicity studies that these compounds inhibited the growth of DU-145 cells. Therefore, DU-
145 cells were treated with 5m at 0.5 µM concentration for 48 h. As shown in Fig 5,
microtubules displayed normal arrangement and organization in untreated prostate cancer cells.
However, cells treated with 5m and CA-4 showed disrupted microtubule organization with
spherical morphology thus demonstrating the inhibition of tubulin polymerization as shown in
Fig 5.
2.6 Competitive tubulin binding assay
Compounds like E7010 and CA-4 bind to the colchicine site of the tubulin. It was
considered of interest, whether 5m also posses the similar ability, hence competitive binding
assay was carried out. Since the intrinsic fluorescence of colchicine increases upon binding to
tubulin,[44, 45] it was used as an index for competition with colchicine in tubulin binding. As
shown in Fig 6 taxol did not affect the binding to tubulin.
Fig. 6 Fluorescence based colchicine competitive binding assays ofcompound 5m were carried out at
various concentrationscontaining 5 μM of tubulin and colchicine for 60 min at 37 °C. CA-4 was used as a
positive control whereas taxol was used as a negativecontrol which binds at the taxane site. Fluorescence
values are normalized to the control.
2.7 Hoechst staining for morphological analysis of apoptosis
Disruption of microtubule formation leads to cell-cycle arrest in the G2/M phase, followed by
apoptotic cell death. Apoptosis is one of the major pathways that leads to the process of cell
death. Chromatin condensation, nuclear shrinking and fragmented nuclei are known as classic
characteristics of apoptosis.[46] In the present data the apoptotic inducing effect of compounds

5m and CA-4 was studied in comparison to CA-4 by Hoechst staining (H 33258) in the DU-145
cancer cell. The results revealed typical apoptotic characteristics, including condensation of
chromatin and appearance of nuclear fragmentation and apoptotic bodies. These results clearly
demonstrate that 5m and CA-4 are also effective in inducing apoptotic cell death (Fig 7).
Fig. 7 Hoechst staining of DU145 cells treated with compound 5m and CA-4.
2.8 DNA Fragmentation assay
Apoptosis was also assessed by electrophoresis of extracted genomic DNA from cells.
Endonuclease mediated cleavage of nuclear DNA results in the formation of oligonucleosomal
DNA fragments a biochemical hallmark of apoptosis in many cell types.[47] DNA laddering
assay was performed with DU-145 cells by treatment of 5m and CA-4 at 1µM concentration
for 48 h, then the genomic DNA was isolated and electrophoresis in a 1.8% agarose gel. The
tested compound 5m induce DNA fragmentation which is observed as characteristic ladder
pattern in DU-145 cells at 1 µM, while no laddering was observed in control cells indicating that
5m induces apoptotic cell death (Fig 8).

Fig 8 DNA fragmentations of conjugate 5m and CA-4 in DU-145 cell line, C is the control (untreated).
2.9 Molecular docking studies
To further investigate the possible binding mode of colchicine binding site on
tubulin,[48] we performed docking studies on most active conjugates 5m and 5l of the series.
Coordinates of protein structure of tubulin-colchicine were obtained from the Protein Data Bank
(PDB ID 3E22). Docking was accomplished into the colchicine binding site of tubulin using
Auto Dock 4.2 software.[49] Fig 9a shows that ring A of conjugate 5m ring was buried in
hydrophobic pocket by βAla354, βVal318, βCys241, βThr353 and βLeu255 residues whereas
methoxy group of ring A established hydrogen bonding interaction with the βAla254 residue.
Ring B of conjugate 5m surrounded by residues at α, β interface of the tubulin by βLys352,
βAsn258, βLeu255, αVal181 and αAla180 residues. Ring C of conjugate 5m and
trimethoxyphenyl ring at 2nd position of ring C buried at α and β interface of tubulin by βAla250,
βLeu248, βAsn249, βLys254, Ser178, αAsn101 and αThr179 residues. However
trimethoxyphenyl ring at 2nd position of ring C extended more towards α-tubulin and shows
interactions with αGln11, αTyr224, and αVal177 residues. In Fig 9b, ring A of conjugate 5l
showed hydrogen bonding interactions with βAla254 and βLeu248. Conjugate 5l established
similar binding position like conjugate 5m and surrounded by similar amino acid residues. Fig
9c shows overlapping of conjugate 5m and 5l, Fig 9d shows that the proposed binding of

conjugate 5m (green) is very similar to the pose of the colchicine (yellow) and CA-4 (red) with
different orientation at interface of α-tubulin (blue) and β-tubulin (pink). Thus docking
investigation suggests that conjugate 5m and 5l interacted with both α-and β-tubulin in
colchicine binding pocket.
Fig 9: (a) and (b) Interaction of the conjugate 5m and 5l with colchicines binding site of tubulin. This
figure has been generated using the software PYMOL from the tubulin-colchicine crystal structure. (c)
Superimposition of conjugate 5m (green) and 5l (magneta) (d) Overlapping of conjugate 5m (green) with
of colchicine (yellow) and CA-4 (red) share the similar binding site on tubulin. β-tubulin shows in pink
and α-tubulin shows in blue color.
3. Conclusion
A new series of combretastatin linked 1,3,4-oxadiazoles conjugates comprised of a three
ring scaffold were synthesized by dehydroxylation and evaluated for their antiproliferative
against five human cancer cell lines and inhibition of tubulin polymerization activities. The
majority of the conjugates exhibited significant antiproliferative activity against various cancer
cell lines with IC50 values in the range 0.118–54.32 M. The lead compound 5m exhibited
potent antiproliferative activity with IC50 values ranging from 0.118 to 0.537 µM inhibited
tubulin polymerization in both in vitro assay as well as in cells as observed by the
immunofluoroscence assay. Further studies indicated that cell cycle arrest at G2/M phase leading
to apoptotic cell death. Furthermore, DNA fragmentation and hoechst staining revealed that
compound induced the apoptotic cell death. Docking studies indicated that the compound
extensively binds to the tubulin at colchicine binding site which was confirmed by competitive
colchicine binding assay. Based on these interesting results the work reported herein is expected
to promote the process of developing newer chemical agents to combat cancer.
4. Experimental Section
4.1 General information
All the chemicals and reagents used in the reactions are of best grade commercially
available and were used without further purification. TLC was performed on 0.25 mm silica gel

60-F254 plates. Spots were visualized by UV light. All the melting points were taken by
Electrothermal apparatus and the values are uncorrected. 1H and 13C NMR spectra were
recorded on 300 MHz, 400 MHz and 500 MHz NMR instruments using tetramethylsilane as the
internal standard. Chemical shifts (δ) are reported in parts per million (ppm) downfield from
tetramethylsilane. HRMS analyses were acquired on single quadruple and carried out using the
ESI techniques at 70 eV. Wherever required column chromatography was performed using silica
gel of 60–120 mm with hexane, ethyl acetate and methanol as eluents. Melting points are
determined with an Electro thermal melting point apparatus, and values are uncorrected.
4.2. Chemistry
experimental procedure for the synthesis of Combretastatin linked 1,3,4-oxadiazole
conjugates (5a-p):
A mixture of (E)-3-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acrylic acid (8, 688.8 mg, 2
mmol) and substituted hydrazide (11) (2 mmol) in POCl3 (10 ml) was refluxed for 5-6 hr. The
reaction mixture was cooled to room temperature and then the contents were poured on to
crushed ice and neutralized with bicarbonate solution. The resulting solid collected and washed
with water extracted with EtOAc dried over Na2SO4. The residue obtained was purified by
column chromatography by using ethyl acetate and hexane to afford the desired compounds
combretastatin linked oxadiazoles (5a-p)
4.2.1 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-phenyl-1,3,4-oxadiazole
(5a)
White solid (775 mg, 87%); Mp: 182186 °C; 1H (500 MHz, CDCl3): δ 8.09 (dd, J = 5.47, 1.88
Hz, 2H), 7.61 (s, 1H), 7.51 (d, J = 2.07 Hz, 2H), 7.49 (s, 1H), 7.06 (d, J = 8.87 Hz, 2H), 6.73 (d,
J = 8.87 Hz, 2H), 6.56 (s, 2H), 3.91 (s,3H), 3.79 (s,9H); 13C (75 M Hz, CDCl3): δ 166.5, 160.1,
153.8, 135.0, 131.9, 131.6, 130.3, 130.1, 128.9, 127.1, 126.8, 123.9, 113.8, 106.7, 104.7, 60.9,
56.1, 55.2; IR (KBr): 3415, 2947, 2834, 1634, 1553, 1471, 1433, 1242, 1136, 1120 cm-1; ESI-
MS: m/z 445 (M + H)+: HRMS (ESI) m/z for C26H25N2O5 calculated m/z: 445.17635, found m/z:
445.17348.
4.2.2 (E)-2-(4-fluorophenyl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-
oxadiazole (5b)
White solid (760 mg, 82%); Mp: 152156 °C; 1H (500 MHz, CDCl3): δ 8.078.12 (m, 2H), 7.64
(s, 1H), 7.24 (t, J = 8.68 Hz, 2H), 7.11 (d, J = 8.68 Hz, 2H), 6.76 (d, J = 8.87 Hz, 2H), 6.60 (s,

2H), 3.93 (s, 3H), 3.79 (s, 3H), 3.78 (s, 6H); 13C (75 M Hz, CDCl3): δ 167.0, 163.8 (d, J = 257.03
Hz), 161.8, 160.3, 153.8, 153.2, 135.5, 132.7 (d, J = 9.08 Hz), 132.0, 129.9, 127.0, 122.9, 119.7
(d, J = 3.63 Hz), 118.7, 118.5, 119.7, 114.8 (d, J = 21.80 Hz), 106.7, 60.9, 56.1, 55.2; IR (KBr):
 3438, 2968, 2822, 1642, 1562, 1440, 1405, 1258, 1168, 1121, 999 cm-1; ESI-MS: m/z 505 (M
+ H)+: HRMS (ESI) m/z for C26H24O5N2F calculated m/z: 463.16638, found m/z: 463.16322.
4.2.3 (E)-2-(4-chlorophenyl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-
oxadiazole (5c)
1
Pale yellow solid (794 mg, 83%); Mp: 164169 °C; H (300 MHz, CDCl3): δ 8.20 (d, J =7.91
Hz, 2H), 7.77 (d, J = 7.91 Hz, 2H), 7.64 (s,1H), 7.08 (d, J = 7.91 Hz, 2H), 6.73 (d, J = 7.91 Hz,
2H), 6.57 (s, 2H), 3.92 (s,3H), 3.79 (s,9H); 13C (75 M Hz, CDCl3): δ 165.1, 161.6, 158.8, 152.3,
136.5,136.1, 139.9, 130.6, 128.8, 128.1, 126.8, 125.5, 121.2, 120.9, 112.4, 105.5, 59.2, 54.7,
53.8; IR (KBr):  3447, 3192, 2992, 2939, 1836, 1599, 1520, 1465, 1414, 1259, 1178, 1127,
1093, 1004 cm-1; ESI-MS: m/z 479 (M + H)+: HRMS (ESI) m/z for C26H24O5N2Cl calculated
m/z: 479.13683, found m/z: 479.13473.
4.2.4 (E)-2-(4-bromophenyl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-
oxadiazole (5d)
1
Pale yellow solid (930 mg, 89%); Mp: 178182 °C; H (500 MHz, CDCl3): δ 7.95 (d, J = 8.27
Hz, 2H), 7.64 (d, J = 8.27 Hz, 2H), 7.6 (s, 1H), 7.07 (d, J = 8.27 Hz, 2H), 6.73 (d, J = 8.27 Hz,
2H), 6.56 (s, 1H), 3.91 (s,3H), 3.79 (s, 9H); 13C (75 M Hz, CDCl3): δ 165.6, 162.3, 159.3, 152.8,
137.2, 134.4, 131.4, 131.0, 129.2, 127.3, 126.1, 125.2, 121.9, 112.9, 106.0, 59.8, 55.2, 54.3; IR
(KBr):  3347, 2928, 2840, 1598, 1518, 1456, 1413, 1297, 1177, 1128, 1090, 1004 cm-1; ESI-
MS: m/z 523 (M + H)+: HRMS (ESI) m/z for C26 H24 O5 N2 Br calculated m/z: 523.08686, found
m/z: 523.08564.
4.2.5 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-(4-
(trifluoromethyl)phenyl)-1,3,4-oxadiazole (5e)
White solid (830 mg, 81%); Mp: 146151 °C; 1H (300 MHz, CDCl3+DMSO-d6): δ 7.98 (d, J =
8.0 Hz, 2H), 7.57 (s, 1H), 7.4 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 7.0 Hz, 2H), 6.88 (d, J =8.0 Hz,
13
2H), 6.53(s, 2H), 3.88(s, 3H), 3.75 (s, 9H); C (75 M Hz, DMSO+CDCl3): δ 165.3, 161.1,
158.7, 152.2, 132.3 (q, J = 272.9 Hz), 128.6, 127.0, 125.7, 125.4, 124.6 (q, J = 2.7 Hz), 123.9,
123.7 (q, J = 12.1 Hz), 121.1, 120.3, 112.3, 105.6, 59.0, 54.6, 53.7; IR (KBr):  3444, 3074,

2940, 1602, 1521, 1410, 1324, 1257, 1180, 1125, 1063, 1013 cm-1; ESI-MS: m/z 513 (M + H)+:
HRMS (ESI) m/z for C27 H24 O5 N2 F3 calculated m/z: 513.16318, found m/z: 513.15937.
4.2.6 (E)-2-(2-chloro-4-fluorophenyl)-5-(2-(4-methoxyphenyl)-1-(3,4,5trimethoxyphenyl)vinyl)-
1,3,4-oxadiazole (5f)
Pale yellow solid (836 mg, 84%); Mp: 170175 °C; 1H (500 MHz, CDCl3): δ 8.07 (m, 1H), 7.67
(s,1H), 7.30 (dd, J = 8.03, 2.26 Hz, 1H), 7.15 (dd, J = 7.55, 2.26Hz, 1H), 7.10 (d, J = 9.06 Hz,
2H), 6.74 (d, J = 9.06 Hz, 2H), 6.61(s, 2H), 3.92 (s, 3H), 3.79 (s, 9H); 13C (75 MHz, CDCl3): δ
167.1, 163.8 (d, J = 257.0 Hz), 161.8, 160.3, 153.8, 139.3, 138.1, 135.9, 134.3, 132.8 (d, J = 9.9
Hz), 132.1, 130.4, 130.0, 127.0, 123.4, 122.7, 118.5 (d, J = 25.4 Hz), 114.74 (d, J = 21.8 Hz),
113.8, 106.7, 60.9, 56.1, 55.2; IR (KBr):  3460, 2947, 2834, 1611, 1534, 1467, 1414, 1254,
1184, 1128, 1019 cm-1; ESI-MS: m/z 497 (M+H)+: HRMS (ESI) m/z for C26H23ClFN2O5
calculated m/z: 497.12740, found m/z: 497.12484.
4.2.7 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-(3-nitrophenyl)-1,3,4-
oxadiazole (5g)
Pale Yellow solid (791 mg, 81%); Mp: 175180 °C;1H (300 MHz, CDCl3): δ 8.9 (m,1H), 8.48
(dt, J = 7.9,1.1 Hz,1H), 8.40 (ddd, J = 8.3,1.1,0.9 Hz,1H), 7.75 (t, J = 8.12 Hz, 1H), 7.71 (s, 1H),
13
7.14 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 6.61 (s, 1H) , 3.94 (s, 3H) , 3.80 (s, 9H); C
(75 M Hz, CDCl3): δ 167.2, 162.2, 160.4, 153.9, 148.6, 135.2, 136.0, 132.4, 132.2, 130.3, 129.8,
126.9, 125.9, 125.6, 122.6, 121.6, 113.9, 106.7, 60.9, 56.1, 55.2; IR (KBr):  3475, 2926, 2853,
1647, 1538, 1482, 1443, 1252, 1194, 1187, 1043 cm-1; ESI-MS: m/z 490 (M + H)+: HRMS (ESI)
m/z for C26 H24 O7 N3 calculated m/z: 490.10688, found m/z: 490.15838.
4.2.8 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-(4-nitrophenyl)-1,3,4-
oxadiazole (5h)
1
Yellow solid (785 mg, 80%); Mp: 183186 °C; H (300 MHz, CDCl3): δ 8.39 (d, J = 8.87 Hz,
2H), 8.25 (d, J = 8.87 Hz, 2H), 7.66 (s,1H), 7.07 (d, J = 8.68 Hz, 2H), 6.72 (d, J = 8.68 Hz, 2H),
13
6.55 (s, 2H), 3.92 (s,3H) , 3.79 (s,9H); C (75 M Hz, CDCl3): δ 167.4, 162.3, 160.5, 153.9,
149.4, 138.2, 136.2, 132.2, 129.8, 129.4, 127.7, 124.3, 122.5, 113.9, 106.6, 104.9, 60.9, 56.1,
55.2; IR (KBr):  3442, 2974, 2856, 1611, 1582, 1473, 1445, 1384, 1192, 1138, 1048 cm-1; ESI-
MS: m/z 490 (M + H)+: HRMS (ESI) m/z for C26H24O7N3 calculated m/z: 490.10688, found
m/z: 490.15832.

4.2.9 (E)-4-(5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-oxadiazol-2-yl)-
N,N-dimethylaniline (5i)
1
White solid (898 mg, 92%); Mp: 194198 °C; H (300 MHz, CDCl3): δ 7.93 (d, J = 9.00 Hz,
2H), 7.59 (s, 1H), 7.08 (d, J = 8.69 Hz, 2H), 6.75 (d, J = 8.54 Hz, 4H), 6.61 (s, 2H) , 3.92 (s,
3H), 3.79 (s, 3H), 3.78 (s, 6H), 3.06 (s, 6H); 13C (75 M Hz, CDCl3): δ 165.4, 164.8, 159.9, 153.7,
152.4, 137.1, 133.0, 131.8, 130.4, 128.3, 127.4, 123.6, 113.7, 111.5, 110.9, 106.8, 60.9, 56.1,
55.2, 40.0; IR (KBr):3440, 2923, 2852, 1610, 1581, 1459, 1412, 1369, 1301, 1244, 1175,
1125, 1029, 959 cm-1; ESI-MS: m/z 488 (M + H)+: HRMS (ESI) m/z for C28H30O5N3 calculated
m/z: 488.21855, found m/z: 488.21508.
4.2.10 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-(p-tolyl)-1,3,4-oxadiazole
(5j):
1
White solid (735 mg, 80%); Mp: 202206 °C; H (500 MHz, CDCl3): δ 7.97 (d, J = 8.30 Hz,
2H), 7.64 (s, 1H), 7.30 (d, J = 8.30 Hz, 2H), 7.09 (d, J = 9.06 Hz, 2H), 6.74 (d, J = 8.30 Hz, 2H),
13
6.60 (s, 1H), 3.93, (s, 3H), 3.79 (s, 3H), 3.78 (s, 6H), 2.44 (s, 3H); C (75 M Hz, CDCl3): δ
166.2, 164.2, 160.1, 153.7, 142.11, 138.0, 134.7, 131.9, 130.2, 129.7, 127.2, 126.8, 123.2, 121.1,
113.7, 106.7, 60.9, 56.1, 55.2, 21.6; IR (KBr): 3436, 2923, 2796, 1611, 1503, 1474, 1420,
1258, 1167, 1132, 1036 cm-1; ESI-MS: m/z 459 (M + H)+: HRMS (ESI) m/z for C27H27N2O5
calculated m/z: 459.19200, found m/z: 459.19145.
4.2.11 (E)-2-(4-methoxyphenyl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-
oxadiazole (5k):
White solid (834 mg, 88%); Mp: 182186 °C; 1H (500 MHz, CDCl3): δ 7.988.03 (m, 2H), 7.57
(s, 1H), 7.047.09 (m, 2H), 6.967.0 (m, 2H), 6.696.74 (m, 2H), 6.56 (s, 1H), 3.90 (s, 1H),
13
3.88 (s, 1H), 3.78 (s, 1H); C (75 M Hz, CDCl3): δ 166.07, 164.10, 162.27, 160.11, 153.81,
134.59, 131.91, 130.29, 128.68, 127.31, 123.38, 116.53, 114.44, 113.81,106.90, 60.95, 56.13,
55.45, 55.26; IR (KBr): 3428, 2939, 2838, 1603, 1500, 1304, 1258, 1237, 1175, 1130, 1026,
999cm-1; ESI-MS: m/z 452 (M + H)+: HRMS (ESI) m/z for C27H27O6 N2 calculated m/z:
475.18636, found m/z: 475.18358.
4.2.12 (E)-2-(3,4-dimethoxyphenyl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-
1,3,4-oxadiazole (5l)

Pale yellow solid (938 mg, 93%); Mp: 125130 °C; 1H (500 MHz, CDCl3): δ 7.567.61 (m, 3H),
7.07 (d, J = 8.03 Hz, 2H), 6.2 (d, J = 8.03 Hz, 1H), 6.72 (d, J = 9.06 Hz, 2H), 6.56 (s, 2H), 3.98
(s, 3H) 3.94 (s, 3H), 3.9 (s, 3H), 3.78 (s, 9H); 13C (75 M Hz, CDCl3): δ 166.13, 164.09, 160.08,
153.75, 151.93, 149.27, 138.02, 134.67, 131.89, 130.20, 127.20, 123.24, 120.36, 113.77, 110.97,
109.44, 106.80, 60.92, 56.38, 56.08, 55.22; IR (KBr):3427, 2935, 2834, 1603, 1501, 1460,
1410, 1261, 1176, 1125, 1024 cm-1; ESI-MS: m/z 505 (M + H)+: HRMS (ESI) m/z for
C28H29O7N2 calculated m/z: 505.19693, found m/z: 505.19364.
. 4.2.13 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-(3,4,5-
trimethoxyphenyl)-1,3,4-oxadiazole (5m)
1
Pale yellow solid (960 mg, 90%); Mp: 165170 °C; H (500 MHz, CDCl3): δ 7.57 (s, 1H),
7.257.30 (m, 2H), 7.07 (d, J = 9.60 Hz, 2H), 6.67 (d, J = 9.60 Hz, 2H), 6.57 (s, 2H), 3.95 (s,
6H) 3.90 (s, 6H), 3.78 (s, 9H); 13C (75 M Hz, CDCl3): δ 166.37, 164.0, 160.1, 153.7, 153.6,
138.1, 137.0, 134.9, 131.9, 130.3, 127.1, 123.1, 119.0, 113.8, 106.8, 104.2, 60.9, 56.4, 56.1,
55.2; IR (KBr):  3424, 2936, 2837, 1598, 1499, 1460, 1414, 1309, 1240, 1123, 1033, 1002 cm-
1; ESI-MS: m/z 535 (M + H)+: HRMS (ESI) m/z for C29H31O8N2 calculated m/z: 535.20749,
found m/z: 535.20418.
4.2.14 (E)-2-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-5-(thiophen-2-yl)-1,3,4-
oxadiazole (5n)
Yellow solid (740 mg, 82%) yield; Mp: 205210 °C; 1H (500 MHz, CDCl3): δ 7.79 (d, J = 3.58
Hz, 1H) 7.60 (s, 1H), 7.55 (d, J = 5.09 Hz, 1H), 7.167.19 (m, 1H), 7.1(d, J = 8.68 Hz, 2H), 6.75
13
(d, J = 8.68 Hz, 2H), 6.59 (s, 2H), 3.98 (s, 3H), 3.78 (s, 9H); C (75 MHz, CDCl3): δ 165.96,
160.4, 160.2, 153.8, 138.8, 135.1, 132.0, 130.0, 129.7, 128.1, 127.2, 125.3, 122.9, 113.8, 106.8,
60.9, 56.1, 55.3; IR (KBr): 3444, 3084, 2932, 2845, 1581, 1520, 1459, 1410, 1300, 1245,
1175, 1129, 1034, 999 cm-1; ESI-MS: m/z 451 (M + H)+: HRMS (ESI) m/z for C24H23O5N2S
calculated m/z: 451.13222, found m/z: 451.12939.
4.2.15 (E)-2-(furan-2-yl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-
oxadiazole (5o)
Yellow solid (705 mg, 81%); Mp: 180185 °C; 1H (500 MHz, CDCl3): δ 7.66 (d, J = 15.29 Hz,
1H), 7.20 (d, J = 3.20 Hz, 1H), 7.10 (d, J = 8.54 Hz, 2H), 6.74 (d, J = 8.54 Hz, 2H), 6.61 (m,
13
1H), 6.59 (s, 2H), 3.92 (s, 3H), 3.78 (s, 9H); C (75 MHz, CDCl3): δ 55.2, 56.0, 60.9, 106.7,
112.1, 113.8, 114.0, 122.6, 127.0, 130.0, 132.0, 135.3, 138.0, 139.4, 145.6, 153.8, 156.9, 160.2,

165.8; IR (KBr):3434, 2941, 2847, 1613, 1527, 1453, 1421, 1253, 1169, 1142, 1075 cm-1;
ESI-MS: m/z 435 (M + H)+: HRMS (ESI) m/z for C24H23O6N2 calculated m/z: 435.15506, found
m/z: 435.15172.
4.2.16 (E)-2-(benzofuran-3-yl)-5-(2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)vinyl)-1,3,4-
oxadiazole (5p)
White solid (892 mg, 92%); Mp: 145150 °C; 1H (500 MHz, CDCl3): δ 7.727.70 (m, 2H), 7.64
(dd, J = 8.39, 0.61Hz, 1H), 7.56 (d, J = 0.76 Hz, 1H), 7.477.44 (m, 1H), 7.377.31 (m, 1H),
7.13 (d, J = 8.85Hz, 2H), 6.76 (d, J = 8.85 Hz, 2H),6.61 (s, 2H) 3.94 (s, 3H), 3.80 (s, 9H); 13C
(75 M Hz, CDCl3): δ 166.1, 164.1, 160.1, 153.7, 151.93, 149.3, 138.0, 134.7, 131.9, 130.2,
127.2, 123.2, 120.4, 113.8, 111.0, 109.4, 106.8, 60.9, 56.4, 56.1, 55.2; IR (KBr):  3431, 2942,
2832, 1612, 1532, 1443, 1483, 1258, 1143, 1176, 1039 cm-1; ESI-MS: m/z 485 (M + H)+:
HRMS (ESI) m/z for C28H24O6N2 calculated m/z: 485.17071, found m/z: 485.16802.
4.3 Biology
4.3.1 Anticancer activity MTT Assay
The cytotoxicity activity of the compounds was determined using MTT assay. 1×104
cells/well were seeded in 200 µl DMEM, supplemented with 10% FBS in each well 96 well
micro culture plates and incubated for 24 h at 37 °C in a CO2 incubator. Compounds are diluted
to the desired concentrations in culture medium than added to the wells with respective vehicle
control. After 48 h of incubation, 10 µL of MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-
Diphenyltetrazolium Bromide) (5 mg/mL) was added to each well and the plates were further
incubated for 4 h. Then the supernatant from each well was carefully removed; formazon
crystals were dissolved in 100 µL of DMSO and absorbance at 540 nm wavelengths was
recorded.
4.3.2 Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells
through the cell cycle phases. Human prostrate cancer cells (DU-145) were incubated with
compounds 5m and CA-4 at 0.5 M concentration for 24h. Control and treated cells were
harvested, washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide

(Sigma Aldrich). Cell cycle anlaysis was performed by flow cytometry (Becton Dickinson
FACS Caliber) as described earlier.
4.3.3 Hoechst staining
Cancer cells were seeded at a density of 10,000 cells over 18-mm cover slips and
incubated for 24 h. After incubation, cells were treated with the compounds 5m and CA-4 at 0.5
µM concentration for 24 h. Hoechst 33258 (Sigma Aldrich) was added to the cells at a
concentration of 0.5 mg/mL and incubated for 30 min at 37 °C. Later, cells were washed with the
phosphate buffered saline (PBS). Then cells from each cover slip 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.
4.3.4 DNA fragmentation analysis
Prostate Cancer cells were seeded in six well plates and incubated for 24 h. After
incubation, cells were treated with compounds 5m and CA-4 at a 0.5µM concentration for 24 h.
Subsequently, cells were collected and centrifuged at 664 g for 5 min at 4 °C. Pellet was
collected and washed with Phosphate buffered saline (PBS), added 100 µl of Lysis buffer,
centrifuged at 3000 rpm for 5 min at 4 °C, collected supernatant and added 10 µl of 10% SDS
and 10 µl of (50 mg/mL) RNase-A and incubated for 2 h at 56 °C. Later 10 µl of Proteinase K
(25 mg/mL) was added and incubated at 37 °C for 2 h. After incubation, 65 µl of 10 M
Ammonium acetate and 500 µl of ice cold ethanol were added than mixed well and this sample
was incubated at 80 °C for 1 h. Further, samples were centrifuged at 12000 rpm for 20 min at 4
°C and washed with 80% ethanol followed by air dried for 10 min at room temperature.
Dissolved pellet in 50 µL of TE buffer, after that DNA laddering was determined by 2% agarose
gel electrophoresis.
4.3.5 In vitro tubulin polymerization
A fluorescence based tubulin polymerization assay was performed according to the
manufacturers protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture was taken
within a total volume of 10 µL contained PEM buffer, GTP (1µM) in the presence or absence of
test compounds 5m and CA-4 at a final concentration of 5 µM. In vitro 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 Biotek multimode plate
reader. Polymerization was finally monitored by increase in the fluorescence mentioned above at
37 0C.
4.3.6 Immunohistochemistry of tubulin
Prostate cancer cells were seeded on glass cover slips, incubated for 48 hours in the
presence or absence of test compounds 5m and CA-4 at a concentration of 0.5µM. Following the
termination of incubation, cells were fixed with 3% formaldehyde, 0.02%glutaraldehyde in PBS
and permeabilized by dipping the cells in100% methanol and kept overnight at 40C. Later the
cover slips were blocked with 1%BSA in phosphate buffered saline for 1 h followed by
incubation with a primary antibody α-tubulin (mouse monoclonal) and later with FITC
conjugated secondary mouse anti IgG antibody. Photographs were taken using the Fluoroscence
microscope, equipped with FITC settings and the pictures were analyzed for the integrity of
microtubule network
4.3.7 Colchicine competitive binding assay
The promising compound 5m and standard CA-4 and taxol of different concentrations 0,
5, 10 and 15 µM were co-incubated with 5 µM colchicine in 30 mM Tris buffer containing 5 µM
tubulin for 60 min at 37 °C. Whereas a non binding site taxol was used as negative control at
colchicines. After incubation, the fluorescence of tubulin–colchicine complex was determined by
using Biotek multimode reader at excitation wavelength at 350 nm and emission wavelength at
435nm. 30mM Tris buffer solution was used as blank which was subtracted from all the
samples. The experiment was carried out in the presence and absence of colchicine to obtain
fluorescence values of the desired tubulin–colchicine complex. The fluorescence values of the
tubulin-conjugates complex was subtracted from the tubulin conjugates-colchicine complex and
finally the Fluorescence values are normalized to control.
4.3.8 DNA fragmentation analysis.
The cells were seeded in six well plates than incubated for 24 h. After incubation, cells are
treated with compound 5m at 3 μM concentration for 48 h. After 48 h of drug treatment cells
were collected and centrifuged at 664 g for 5 min at 4 °C. Pellets were collected and washed
with phosphate buffered saline (PBS), added 100 μL of lysis buffer, centrifuged at 956 g for 5
min at 4 °C and collected supernatant. 10 μL of 10% SDS and 10 μL of (50 mg mL−1) RNase-A
were added and incubated for 2 h at 56 °C. After that, 10 μL of Proteinase K (25 mg mL−1) was

added and incubated at 37 °C for 2 h. After incubation, 65 μL of 10 M ammonium acetate and
500 μL of ice cold ethanol were added and mixed well. This sample was incubated at 80 °C for 1
h. After that, samples were centrifuged at 15294 g for 20 min at 4 °C and washed with 80%
ethanol followed by air drying for 10 min at room temperature. The pellet was dissolved in 50
μL of TE buffer. After that, DNA laddering was determined by 2% agarose gel electrophoresis.
4.3.9. Molecular modelling experimental procedure
The 3D structures for the compounds under study were built and optimized by Gaussian 03W.
These optimized 3D structures are utilized for docking. The docking of compounds was done by
using the AutoDock 4.2 package. The co-crystallized structure of the colchicine site of tubulin
downloaded from the PDB data bank (http://www.rcsb.org/pdb/index.html; PDB code: 3E22).
Acknowledgements
P.S.Srikanth is thankful to CSIR-New Delhi for the award of senior research fellowship. We
acknowledge funding received from the project entitled ‘Affordable Cancer Therapeutics
(ACT)’ (CSC0301) under XIIth five year plan. This project was also supported by King Saud
University, Saudi Arabia, Deanship of Scientific Research, Research Chair.
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Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent
Tubulin Polymerization inhibitors
Ahmed Kamal,*a,b,c,d P. S. Srikanth,a,b G. V. P. S. Vishnuvardhan,a G. Bharath Kumar,a Korrapati
Suresh Babu,a S. M. Ali Hussaini,a Jeevak Sopanrao Kapure,a,c Abdullah Alarifi d
A new class of combretastatin linked to oxadiazole have synthesized and evaluated for their anticancer
activity. The tubulin polymerization assay and immunofluoresence analysis indicate that exhibits potent
inhibitory effect on the tubulin assembly also these are effectively bind at the colchicine binding site of
the tubulin.

Highlights



A new series of combretastatin linked 1,3,4-oxadiazoles were designed and synthesized.
These conjugates are screened for their cytotoxic activity.
Conjugate 5m displayed potent antiproliferative activity against DU-145 cell line with IC50 value
0.118 µM.


These conjugates induced cell cycle arrest at G2/M phase in cell cycle analysis.
These conjugates effectively inhibit tubulin polymerization.