Synthesis of different heterocycles-linked chalcone conjugates as cytotoxic agents and tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.07.031

A series of new heterocycles-linked chalcone conjugates has been designed and synthesized by varying different alkane spacers. These conjugates were tested for their in vitro cytotoxic potential against a panel of selected human cancer cell lines namely,

Full text of DOI:10.1016/j.bmc.2017.07.031

Accepted Manuscript
Synthesis of different heterocycles-linked chalcone conjugates as cytotoxic
agents and tubulin polymerization inhibitors
Nagula Shankaraiah, Shalini Nekkanti, Uma Rani Brahma, Niggula Praveen
Kumar, Namrata Deshpande, Daasi Prasanna, Kishna Ram Senwar, Uppu Jaya
Lakshmi
PII:
S0968-0896(17)31242-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.031
BMC 13868
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 June 2017
13 July 2017
15 July 2017
Please cite this article as: Shankaraiah, N., Nekkanti, S., Brahma, U.R., Praveen Kumar, N., Deshpande, N.,
Prasanna, D., Senwar, K.R., Lakshmi, U.J., Synthesis of different heterocycles-linked chalcone conjugates as
cytotoxic agents and tubulin polymerization inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.07.031
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Synthesis of different heterocycles-linked chalcone conjugates as cytotoxic
agents and tubulin polymerization inhibitors
Nagula Shankaraiah,a* Shalini Nekkanti,a Uma Rani Brahma,b Niggula Praveen Kumar,a
Namrata Deshpande,a Daasi Prasanna,a Kishna Ram Senwar,a Uppu Jaya Lakshmib
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad-500 037, India
bDepartment of Pharmacology & Toxicology, National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad-500 037, India
∗
Corresponding author. Tel.: (+91) 040 23073740; Fax: (+91) 040 23073751; E-mail:
shankar@niperhyd.ac.in; shankarnbs@gmail.com
Abstract: A series of new hetercycles-linked chalcone conjugates has been designed and
synthesized by varying different alkane spacers. These conjugates were tested for their in vitro
cytotoxic potential against a panel of selected human cancer cell lines namely, lung (A549 and
NCI-H460), prostate (DU-145 and PC-3), colon (HCT-15 and HCT-116), and brain (U-87
glioblastoma) by MTT assay. Notably, among all the tested compounds, 4a exhibited potent
cytotoxicity on NCI-H460 (lung cancer) cells with IC50 of 1.48 0.19 µM. The compound 4a
showed significant inhibition of tubulin polymerization and disruption of the formation of
microtubules (IC50 of 9.66 0.06 µM). Moreover, phase contrast microscopy and DAPI staining
studies indicated that compound 4a can induce apoptosis in NCI-H460 cells. Further, the flow-
cytometry analysis revealed that compound 4a arrests NCI-H460 cells in the G2/M phase of the
cell cycle. In addition, molecular docking studies of the most active compounds 4a and 4b
selectively bind into colchicine site of the tubulin, revealed the possible mode of interaction by
these new conjugates.
Keywords: Heterocycles, anticancer activity, chalcones, aldol condensation, tubulin
polymerization inhibitor, molecular modeling.
1

1. Introduction
Tubulin is an α,β-heterodimeric protein that forms the cytoskeletal framework of microtubules.1
It is one of the most active molecular targets of numerous anticancer ligands that cause cell cycle
arrest at the G2/M phase by interfering with the microtubule polymerization/depolymerization
process.2,3 Many clinically used natural product anticancer agents such as colchicines (A, Figure
1), MDL-27048, taxanes, combretastatin A-4 (B), podophyllotoxin and vinca alkaloids are
tubulin-binding agents.4 Antimitotic agents generally interact with tubulin at three known
binding sites namely, the colchicine, the vinca alkaloid and the paclitaxel binding sites. Agents
that target the colchicine site (e.g., colchicines, combretastatin A-4 and podophyllotoxin)5 or the
vinca alkaloid site (e.g., vinblastine)6 are known as microtubule destabilizing agents. On the
other hand, agents that bind to the paclitaxel site (e.g., paclitaxel)7 are referred to as microtubule-
stabilizing agents. As microtubules play a vital role in several cellular functions such as the
formation of the mitotic spindle, cytoplasmic organelle movement, maintaining cell shape, intra-
cellular transport and cell replication, interfering with microtubule dynamics lead to severe side
effects.8–10 Therefore, it is essential to develop new tubulin-binding agents and antimitotic agents
with novel modes of action to overcome side effects as well as drug resistance.
Chalcones (1,3-diaryl-2-propen-1-ones) are precursors of flavonoids and isoflavonoids
that possess a wide range of biological activities such as anticancer, anti-diabetic, anti-
hypertensive, anti-viral, anti-inflammatory, anti-tuberculosis, anti-oxidant, anti-leishmanial, anti-
filarial, anti-malarial, anti-bacterial and anti-fungal activities.11–14 The biological activities of
chalcones are largely attributed to the presence of α,β-unsaturated ketone functionality, as
removal of this group makes them inactive. The extensive exploration of chalcones can be
attributed to their ease of synthesis, relatively simpler chemical architecture, being precursors for
important synthetic manipulations and promising biological activities. Naturally occurring
chalcones and their synthetic analogs displayed significant cytotoxic activity against various
cancer cells.15 One of the most widely proposed anticancer mechanisms of chalcones is the
prevention of tubulin polymerization by binding to the colchicine-binding site.16–18 Due to their
promising anti-cancer activities, considerable efforts have been dedicated to discovering new
potential chalcone-based drug candidates during the last decade. A majority of these naturally
occurring anti-cancer compounds are substituted with electron donating hydroxy and/or methoxy
2

groups at various positions around the chalcone scaffold.19,20 In order to ascertain more advanced
structure–activity relationships (SARs) of chalcones, it is of great importance to synthesize new
compounds with more diverse substitutions patterns.
In continuation of our earlier efforts in the field of anti-cancer drug discovery,21–25 herein,
we report the synthesis of a series of novel hetercycles-linked chalcone conjugates (C, Figure 1)
with diverse substitutions and evaluated for their in vitro cytotoxic activity on selected human
cancer cell lines.
OMe
MeO
MeO
O
MeO
MeO
OMe
OMe
OMe
Me
NH
OMe
A
B
O
O
OMe
R1
A
B
O
N
n
C
R
Fig. 1. The structures of colchicine (A), combretastatin A-4 (B), and heterocycles-linked new
chalcone conjugates 4a–y.
2. Results and discussion
2.1. Chemistry
Two core structural components (i) an aldehyde, and (ii) a ketone are required for achieving the
target chalcone conjugates 4a–y. The synthetic route for the preparation of the hetercycles-linked
chalcone conjugates 4a–y was outlined in Scheme 1. Initially, the etherification of vanillin (1)
was carried out with dibromo alkane spacers of varying lengths (n = 2,3,4), in the presence of
K2CO3 as the base to give the intermediates 2a–c. Next, various nitrogen containing heterocycles
were tethered to the alkane spacer of 2 by refluxing in acetonitrile in the presence of K2CO3, to
afford the aldehyde intermediates 3a–h in quantitative yields. Finally, aldol condensation
reaction was performed between intermediates 3a–h and a variety of substituted acetophenones
by using Ba(OH)2 as the base to furnish the chalcone derivatives 4a–y in good yields. All the
newly synthesized compounds were characterized by HRMS, 1H and 13C NMR spectroscopy.
3

Almost similar pattern was observed in 1H and 13C NMR spectra of all the final synthesized
compounds of this series. The double bond of the chalcone moiety was in the trans-configuration
as denoted by the coupling constant (J) values of 15–16 Hz. In the 13C NMR spectrum, the
characteristic peaks corresponding to the chalcone moiety were observed: the carbonyl carbon
appeared in the range δ 183–192 whereas the aromatic carbons along with the olefinic carbons
appeared in the range of δ 163–104. The HRMS (ESI) of all the compounds showed an [M + H]+
peak equivalent to their molecular formulae.
R
R
NH
Br
n
N
OH
K2CO3
CH3CN
−
O
O
n
Br
n = 3−4
+
n Br
K2CO3/CH3CN
OHC
OMe
OHC
OMe
OMe
OHC
O
reflux, 4 5 h
reflux, 4−5 h
1
2a−c
3a−h
Ba(OH)2/EtOH
rt, 12 h
Me
R1
Me
HN
Me
Me
O
O
R
=
NH
R
N
O
N
H
N
H
N
H
N
H
N
H
Me
R1
n
N
OMe
R1 = H, Cl, Br, CH3, OCH3, CF3, NH2, OH
O
4a−y
1
4a
4b
4c
; R = piperidinyl, n = 3, R = 4-methoxy
1
; R = piperidinyl, n = 3, R = 2-hydroxy
4n; R = 2,6-dimethylmorpholin yl, n = 3, R1 = 2-methoxy
4o; R = 2,6-dimethylmorpholin yl, n = 3, R1 = 2,4-dichloro
4p; R = 2,6-dimethylmorpholin yl, n = 3, R1 = 4-methyl
1
; R = piperidinyl, n = 3, R = 4-bromo
4d; R = piperidinyl, n = 4, R1 = H
4e; R = piperidinyl, n = 4, R1 = 4-bromo
4f; R = piperidinyl, n = 4, R1 = 4-chloro
1
4q
; R = indolinyl, n = 3, R = 4-methoxy
1
; R = indolinyl, n = 3, R = 2,4-dichloro
4r
1
; R = 1,2,3,4-tetrahydroquinolin yl, n = 3, R = H
4s
4g; R = piperidinyl, n = 4, R1 = 2,4-dichloro
1
4t
; R = 1,2,3,4-tetrahydroquinolin yl, n = 3, R = 4-methoxy
1
4h
; R = morpholinyl, n = 2, R = 4-methoxy
1
4u; R = 1,2,3,4-tetrahydroquinolin yl, n = 3, R1 = 4-bromo
4v; R = 1,2,3,4-tetrahydroquinolin yl, n = 3, R1 = 4-chloro
4w; R = 1,2,3,4-tetrahydroquinolin yl, n = 3, R1 = 2,4-dichloro
4i
4j
; R = morpholinyl, n = 2, R = 4-bromo
1
; R = morpholinyl, n = 2, R = 2,4-dichloro
1
; R = morpholinyl, n = 2, R = 4-trifluoromethyl
4l; R = morpholinyl, n = 3, R1 = 4-methoxy
4m; R = 2,6-dimethylmorpholin yl, n = 3, R1 = 4-methoxy
4k
1
4x
4y
; R = 1,2,3,4-tetrahydroquinolin yl, n = 3, R = 4-amino
1
; R = 3,5-dimethyl-1 H-pyrazolyl, n = 3, R = 4-methoxy
Scheme 1. Synthesis of different heterocycles-linked new chalcone conjugates 4a–y.
2.2. Biological evaluation
2.2.1. In vitro cell growth inhibitory activity
The new chalcone conjugates 4a–y were evaluated for their in vitro antiproliferative activity
against a panel of seven human cancer cell lines namely, lung (A549 and NCI-H460), prostate
(DU-145 and PC-3), colon (HCT-15 and HCT-116) and brain (U-87 glioblastoma) by using 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Concentration-
4

response course analysis was carried out to establish the drug concentration required to inhibit
the growth of cancer cells by 50% (IC50) after incubation for 48 h. Combretastatin A-4 (CA-4)
was used as the reference standard and the screening results were summarized in Table 1.
It is noticeable from the initial screening that the hybrids 4a, 4b, 4l and 4y displayed a
broad range of activities against all the tested human cancer cell lines with IC50 values ranging
from 1.48 0.19 µM to 10.98 0.81 µM. Among them, the most potent compound was 4a with
IC50 values of 1.48 0.19 and 1.73 0.16 µM against NCI-H460 and HCT-116 cell lines,
respectively. Moreover, the compound 4a was also found to be active against all the tested
cancer cell lines with IC50 values less than 10 µM. Interestingly, the compound 4b was found to
be active in all the seven cancer cell lines with IC50 values in the range of 1.63 0.15 to 3.24
1.2 µM. The compounds 4b and 4y showed potent cytotoxic activity on the HCT-15 cell line,
with IC50 values less than 5 µM, which was significant compared to the activity of CA-4 (IC50
8.87 0.88 µM). Except for 4l, 4m, 4n, 4p and 4y, all other chalcone conjugates were found to
be inactive on the HCT-116 cell line. These five compounds 4l, 4m, 4n, 4p and 4y were also
effective against the U-87 and A549 cell lines. It was also observed that, in DU-145 cell line, the
compounds 4l, 4m and 4n displayed potent cytotoxicities with IC50 values of 2.02 0.14, 3.27
1.16 and 3.87 1.0 µM, respectively. In the PC-3 cell line, except 4b (IC50 1.95 0.02 µM), none
of the compounds showed better activity than CA-4 (IC50 3.53 1.1 µM). The compounds 4r–w
were found to be inactive against all the cell lines, with IC50 values greater than 15 µM.
Based on the in vitro cytotoxic activities, the structural activity relationships (SARs) of
the chalcones 4a–y have been proposed. In order to comprehend the SARs, we varied the
substitutions on both the A and B rings, the length of the linkers as well as the nitrogen
heterocycles in these conjugates. It could be easily observed from the in vitro cytotoxicity data
that the conjugates with piperidine (4a and 4b) and morpholine (4l) linked to the B ring elevated
the biological response, compared to those with other nitrogen heterocycles. The presence of
larger nitrogen heterocycles such as indoline and tetrahydroquinoline (4q–x) completely
abolished the cytotoxic activity. The effect of substitution on ring A is interesting in the light of
the results of the SAR study, which demonstrates that the presence of electron-donating groups
such as 2-hydroxy and 4-methoxy (4a, 4b, 4l, 4m and 4y) is favorable for activity, in contrast,
electron withdrawing groups such as 4-chloro, 4-bromo (4c, 4e–g, etc) resulted in significant loss
5

of activity. The importance of linker length on cytotoxic activity is demonstrated by the fact that
analogues with the linker length of three showed enhanced cytotoxicity than those with linker
lengths of two or four. Based on the promising cytotoxic activity, the most active compounds 4a
from this series was taken-up for detailed further studies.
Table 1. Cytotoxic activity (IC50 in µM)a of heterocycles-linked new chalcone conjugates 4a–y.
HCT-15b
5.34 0.06
3.24 1.2
>20
NCI-H460c HCT-116d
U-87e
DU145f
PC-3g
A549h
Compound
1.48 0.19
1.63 0.15
8.16 0.98
>20
1.73 0.16
2.01 1.1
>20
6.30 0.33
2.87 0.16
>20
5.08 0.05
2.73 0.14
9.53 0.01 3.29 0.74
1.95 0.02 3.13 0.03
4a
4b
4c
17.09 0.65 8.93 0.26 16.52 0.96
>20
>20
>20
>20
9.18 0.82 >20
4d
4e
8.97 1.0
8.11 1.0
>20
>20
>20
4.01 0.09
8.01 0.39
6.09 1.10
7.09 0.65
>20
>20
7.54 1.1
9.06 0.04
10.19 0.62 >20
16.32 0.35 >20
7.74 1.01
5.73 0.51
8.74 0.01
9.74 0.09
6.74 0.61
>20
6.84 0.91 8.99 0.65
7.29 0.22 9.48 0.07
9.19 0.39 8.21 0.67
8.89 0.98 12.08 0.29
5.12 0.96 >20
4f
4g
4h
4i
7.54 0.45
>20
9.96 0.18
>20
>20
>20
18.23 0.68 >20
19.93. 0.62 >20
4j
>20
>20
>20
> 20
6.17 0.26 19.09 0.63
7.89 0.71 5.36 0.02
4k
4l
10.98 0.8
8.64 0.6
6.36 0.1
>20
9.89 0.9
4.18 0.55
8.1 0.2
>20
6.78 0.4
7.87 0.29
6.48 0.16
>20
7.12 0.2
5.67 0.08
5.03 0.09
>20
2.02 0.14
3.27 1.16
3.87 1.0
>20
>20
7.42 0.44
5.32 0.01
4m
4n
4o
4p
4q
4r
> 20
8.87 0.19 >20
>20 9.12 0.30
8.69 0.4
>20
5.45 0.3
>20
4.21 0.45
> 20
19.92 0.89 >20
15.21 0.82 16.08 0.05 >20
18.68 0.13
> 20
>20
>20
19.23 0.86
> 20
19.80 0.11 >20
19.75 0.67 >20
18.21 0.92 >20
>20
>20
>20
>20
>20
>20
>20
4s
>20
>20
>20
> 20
4t
>20
>20
>20
> 20
>20
19.08 0.85
>20
4u
4v
4w
4y
CA-4i
>20
>20
>20
16.21 0.82 18.09 0.65 >20
>20
>20
>20
>20
>20
8.17 0.78 >20
4.78 0.06 3.70 0.11
3.53 1.1 9.29 0.36
4.03 0.17
8.87 0.88
8.96 0.18
3.97 1.04
6.93 0.74
9.75 0.63
5.74 1.0
0.28 0.15
0.048 0.02 9.12 0.52
a 50% inhibitory concentration after 48 h of compound treatment. b,d Colon cancer cells.
c,h Lung cancer cells. e Glioblastoma cancer cells. f,g Prostate cancer cells. CA-4: Combretastatin A-4, reference
compound.
6

2.2.2. Morphological observations using phase contrast microscope
The induction of the formation of apoptotic bodies has always been an ideal criterion in
developing anti-cancer therapeutics. To observe whether the treatment with the compounds could
lead to loss of cell viability and induction of apoptosis, NCI-H460 cells were treated with the
most active compound 4a. Cells were observed and photographs were taken with phase contrast
microscope. Compound 4a treated NCI-H460 cells showed cell wall deformation, cell shrinkage
and resulted in reduced number of viable cells, whereas these distinctive morphological features
were absent in control cells as shown in Fig. 2. This indicates that the compound 4a induces
marked morphological changes in NCI-H460 cells.
Fig. 2. Effect of the compound 4a [(a) 0.0 µM; (b) 1.25 µM; (c) 2.5 µM; (d) 5.0 µM] on the
morphology and cell viability of NCI-H460 cells.
2.2.3. DAPI nucleic acid staining
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye capable of strong binding to
A–T rich sequences of DNA and aids in the visualization of chromatin condensation or nuclear
damage. It distinguishes live cells from apoptotic cells by staining the characteristic condensed
7

nuclei of the latter bright blue.26 Therefore, this staining technique was performed to detect the
induction of apoptosis by the compound 4a in NCI-H460 cells. From Fig. 3, it was established
that the nuclear structure of untreated control cells was intact whereas NCI-H460 cells treated
with 4a displayed condensed, horse-shoe shaped or fragmented nuclei.
Fig. 3. Nuclear morphology in NCI-H460 cells stained with DAPI. NCI-H460 cells treated with
compound 4a [(a) 0.0 µM; (b) 2.5 µM; (c) 5.0 µM; (d) 10.0 µM] for 24 h were stained with
DAPI. The images were captured with a fluorescence microscope.
2.2.4. Generation of Reactive Oxygen Species (ROS)
The generation of ROS is a characteristic feature of many chemotherapeutic agents, thereby
initiating oxidative damage to the mitochondrial permeability and membrane potential. Hence,
DCFDA staining method27 has been used to assess the generation of intracellular ROS of
compound 4a in NCI-H460 cells. The treatment of compound 4a for 6 h resulted in enhanced
DCFDA fluorescence in a dose-dependent manner, indicating the capability of compounds in
accumulating ROS (Fig. 4). On the other hand, a decreased fluorescence intensity was observed
when NCI-H460 cells were treated with N-acetyl cysteine (NAC) prior to treatment with 4a,
indicating the compound-induced cytotoxicity by ROS generation. Moreover, H2O2 treatment of
8

NCI-H460 cells led to the increased fluorescence when compared to the control due to the
generation of free radicals.
Fig. 4. Effect of compound 4a on Reactive Oxygen Species’ (ROS) levels. Dose-dependent
increment of fluorescence was observed compared to control. Each bar
represents mean SD from three independent experiments.
2.2.5. Flow-cytometry analysis
Usually, anticancer therapeutics prevent the proliferation of cancer cells by blockade of the cell
cycle at a specific checkpoint. From the in vitro screening results, it was evident that the
compound 4a showed significant activity against NCI-H460 cells. Hence, it was our interest to
figure out whether this cytotoxicity was due to cell cycle arrest, through cell cycle analysis.28
NCI-H460 cells were treated with compounds 4a, at concentrations of 1.25, 2.5 and 5 µM for 24
h, stained with propidium iodide and further analyzed by using BD flow cytometry. The results
from Fig. 5 indicated that the NCI-H460 untreated control cells exposed to DMSO showed
34.71% cells in G2/M phase, whereas cells treated with 4a showed 39.30, 42.88 and 48.85%
increase in G2/M population at 1.25, 2.5 and 5 µM concentrations respectively after 24 h. A
simultaneous decreased number of cells in G0/G1 phase were also observed. These results
clearly indicated that treatment of NCI-H460 cells with compound 4a resulted in G2/M phase
arrest.
9

A
B
Fig. 5. Cell cycle analysis of NCI-H460 cancer cells treated with compound 4a for 24 h. (A) The
cell cycle distribution was analysed by using propidium iodide staining method and
analysed by flow cytometry (BDc6 accuri). (B) Data of 10,000 cells was collected for each data
file. The percentage of the cell population in G0/G1, S and G2/M phase were calculated by
using the BDc6accuri software.
10

2.2.6. Effect on tubulin polymerization
To investigate whether the synthesized compounds can interact with microtubule system in order
to elicit antiproliferative activity, the compound 4a was evaluated for its tubulin polymerization
inhibitory activity in a cell-free in vitro assay.29 The compound 4a displayed G2/M cell cycle
arrest which is a hallmark of tubulin polymerization. Hence, we investigated the ability of
compound 4a to inhibit tubulin polymerization by monitoring the increase in fluorescence
emission at 440 nm (excitation wavelength is 360 nm) for 1 h at 37 oC (Fig. 6). In this assay,
Combretastatin A-4 (CA-4) and paclitaxel, were used as reference standards at 3 µM
concentration. The compound 4a were included at final concentrations of 10, 5, 2.5, 1.25, 0.625
µM respectively. The experiment performed in duplicates. The IC50 value was calculated from
the drug concentration required to inhibit 50% of tubulin assembly compared to control. The
compound 4a was found to be a potent inhibitor of tubulin polymerization with an IC50 value of
9.66 0.06 µM (Fig. 7).
Fig. 6. Effect of compound 4a on the tubulin polymerization: tubulin polymerization was
monitored by the increase in fluorescence at 360 nm (excitation) and 440 nm (emission)
for 1 h at 37 oC.
11

Fig. 7. % Dose-response inhibition of tubulin polymerization by compound 4a at final
concentrations of 10, 5, 2.5, 1.25, 0.625 µM. Combretastatin A-4 (CA-4) and paclitaxel, were
used as reference standards at 3 µM concentration.
2.3. Molecular docking
To elucidate the mode of binding with tubulin, the synthesized compounds were docked into the
colchicine-binding site of tubulin (PDB ID: 1SA0) located at the interface of the α,β subunits.30
Co-crystallized ligand colchicine was re-docked into tubulin as to validate the docking
protocol, with a root mean squared deviation (RMSD) of 0.67 Å. Colchicine interacted strongly
with the critical amino acid residues Leu248, Leu255, Asn258 and Lys352 in the colchicine-
binding site of tubulin. Therefore, these amino acid residues were very important for the binding
of tubulin inhibitors. To confirm the correct binding mode and ensure a geometric fit,
compounds 4a and 4b were docked into the colchicine-binding site of tubulin (Fig. 8).
Compounds 4a and 4b exhibited strong hydrophobic interactions with Thr179, Ala316, Lys254
and Met259, as well as critical amino acid residues including Leu248, Leu255, Asn258 and
Lys352. We can clearly see that the hydrophobic pockets of colchicine binding site are occupied
by A ring and the carbonyl group of the α,β-unsaturated ketone moiety. The ring B and the linker
region of molecules extend into the interface between the α- and β-chains which are surrounded
by Gln176, Ser178, Tyr210 and Pro255 of the α-subunit and Asp229, Met325 and Val355 of the
β-subunit. More importantly, the protonated nitrogen of piperidine formed hydrogen-bonding
with the side chain of Asp 329 in both compounds. Moreover, the oxygen atom of the α,β-
unsaturated carbonyl system formed a hydrogen-bonding with the side chain of Asn101 (N–
12

H…..O = 1.971 Å). In compound 4b, it is the phenolic OH that forms hydrogen-bonding with the
side chain of Asn101 as well as the backbone of Thr179. Moreover, the ring A formed cation-π
stacking with the nitrogen in the sidechain of Lys254. Asn101 played a very important role
during the protein-ligand binding process, possibly explaining the stronger inhibitory activity of
compounds against all the cancer cell lines.
Fig. 8. (A), (B) Docking poses of ligands 4a, 4b respectively and the interacting amino acids at
the colchicine binding domain of tubulin. (C), (D) Poses represent the superimposition of 4a and
4b with colchicine at the colchicine binding domain.
Based on the modeling, it is clear that these molecules fit well in the tubulin α,β interface
with favorable interactions. An understanding of this interaction between tubulin and the hit
compounds will aid in the development of new inhibitors with potent antitumor activities. The
results of the molecular docking study showed linear linker played a bridging role between the
phenyl and heterocyclic rings leading to a successful docking at the target binding site. The
13

optimal linker length was three; linkers longer or shorter were not efficient in binding to the
target site. To gain further insight of our findings, we demonstrated the superimposition of
colchicine with the ligands and the corresponding superimposition poses suggest that methoxy
phenyl ring of ligands under investigation occupied a similar position with respect to 2-
methoxycyclohepta-2,4,6-trien-1-one ring of colchicine and the carbonyl group in 4a and 4b
superimposes with the amide carbonyl of the colchicines. All these interactions together
contribute to the hypothesis that the synthesized compounds exhibit efficient binding into the
colchicines binding site on tubulin and thus, the ensuing antimitotic activity.
3. Conclusion
In summary, a series of heterocycles-linked chalcone conjugates were synthesized, and evaluated
for their in vitro anticancer potential. Interestingly, the compound 4a showed the most potent
antiproliferative activity on the NCI-H460 cell line. Based on the in vitro cytotoxic activities, the
structure activity relationships (SARs) of the chalcones 4a–y have been proposed. The presence
of piperidine (4a and 4b) and morpholine (4l) linked to the ring B together with the presence of
electron-donating groups on ring A elevated the biological response. The importance of linker
length on cytotoxic activity is demonstrated by the fact that analogues with the linker length of
three showed enhanced cytotoxicity than those with linker lengths of two or four. Additionally,
4a effectively inhibited the polymerization of tubulin through binding at the colchicine binding
site. It also induced apoptosis in NCI-H460 cells through the generation of ROS and arresting the
G2/M phase of the cell cycle. Overall, the current studies demonstrate that the heterocycles-
linked new chalcone conjugate 4a has the potential to be developed as lead and their further
structural modifications may produce promising new cancer therapeutics.
4. Experimental protocols
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. Analytical thin layer chromatography (TLC) was performed on MERCK
precoated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization of the spots on TLC plates
was achieved by UV light. 1H and 13C NMR spectra were recorded on bruker 300 and 500 MHz
spectrometers using tetramethyl silane (TMS) as the internal standard. Chemical shifts for 1H and
14

13C are reported in parts per million (ppm) downfield from tetra methyl silane. Spin multiplicities
are described as s (singlet), bs (broad singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet), and m (multiplet). Coupling constant (J) values are reported in hertz (Hz). HRMS were
determined with Agilent QTOF mass spectrometer 6540 series instrument.
Wherever required, column chromatography was performed using silica gel (60–120 or 100–
200) or neutral alumina.
4.2. General experimental procedure for the synthesis of
heterocycles-linked chalcone conjugates 4a–y
Aldehyde intermediate 3, acetophenone (1 mmol), and barium hydroxide (1 mmol) were
dissolved in EtOH (5 mL). The reaction mixture was stirred for 12 h at room temperature and
then evaporated in vacuo, water (5 mL) was added and the mixture was neutralized with HCl (1
M, 1.75 mL) and extracted with EtOAc. The organic layer was separated, washed with water,
dried, and evaporated in vacuo. The residue yielded crude chalcones (4a-y) which were purified
by column chromatography (Silica gel, 60–120 mesh, 9:1 hexane/ethyl acetate).
4.2.1. (E)-3-(3-Methoxy-4-(3-(piperidin-1-yl)propoxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-
one (4a). Yellow liquid, yield 81%; 1H NMR (500 MHz, CDCl3): δ 8.03 (d, 2H, J = 9.0 Hz),
7.75 (d, 1H, J = 16.0 Hz), 7.39 (d, 1H, J = 16.0 Hz), 7.20 (dd, 1H, J = 2.0, 8.5 Hz), 7.15 (d, 1H,
J = 2.0 Hz), 6.98 (d, 2H, J = 9.0 Hz), 6.92 (d, 1H, J = 8.0 Hz), 4.12 (t, 2H, J = 7.0 Hz), 3.92 (s,
3H), 3.88 (s, 3H), 2.51 (t, 2H, J = 7.0 Hz), 2.46–2.39 (m, 4H), 2.10–2.04 (m, 2H), 1.94–1.87 (m,
4H), 1.64–1.56 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 189.3, 151.3, 149.6, 145.7, 138.9,
136.8, 129.9 (2C), 128.9 (2C), 127.5, 123.3, 119.3, 112.4, 110.6, 68.8, 59.0, 56.1, 54.6 (2C),
27.2, 25.9 (2C), 24.4, 23.4; HRMS (ESI); m/z calcd. for C25H31NO4, 410.2331, found 410.2326
[M + H]+.
4.2.2. (E)-1-(2-Hydroxyphenyl)-3-(3-methoxy-4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-
one (4b). Yellowish brown liquid, yield 88%; 1H NMR (500 MHz, CDCl3): δ 12.94 (s, 1H), 7.94
(dd, 1H, J = 1.5, 8.0 Hz), 7.88 (d, 1H, J = 15.0 Hz), 7.53–7.48 (m, 2H), 7.25 (dd, 1H, J = 1.9, 8.3
Hz), 7.17 (d, 1H, J = 2.0 Hz), 7.03 (dd, 1H, J = 1.0, 8.5 Hz), 6.96–6.93 (m, 2H), 4.14 (t, 2H, J =
7.0 Hz), 3.94 (s, 3H), 2.49 (t, 2H, J = 7.0 Hz), 2.45–2.37 (m, 4H), 2.08–2.03 (m, 2H), 1.61–1.57
(m, 4H), 1.50–1.41 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 189.5, 151.3, 149.6, 145.7, 137.3,
15

131.9 (2C), 130.0 (2C), 129.7, 127.5, 123.3, 119.3, 112.6, 110.7, 67.6, 56.1, 55.7, 54.5 (2C),
26.5, 25.9 (2C), 24.4; HRMS (ESI): m/z calcd. for C24H30NO4, 396.2175, found 396.2165 [M +
H]+.
4.2.3. (E)-1-(4-Bromophenyl)-3-(3-methoxy-4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-
one (4c). Yellow liquid, yield 80%; 1H NMR (500 MHz, CDCl3): δ 7.88 (d, 1H, J = 8.5 Hz), 7.80
(d, 1H, J = 8.5 Hz), 7.76 (d, 1H, J = 15.5 Hz), 7.64 (d, 1H, J = 8.5 Hz), 7.58 (d, 1H, J = 8.5 Hz),
7.32 (d, 1H, J = 15.5 Hz), 7.21 (dd, 1H, J = 2.0, 8.5 Hz ), 7.14 (d, 1H, J = 2 Hz), 6.93 (d, 1H, J
= 8.5 Hz), 4.13 (t, 2H, J = 6.5 Hz), 3.92 (s, 3H), 2.50 (t, 2H, J = 7.5 Hz), 2.43– 2.37 (m, 4H),
2.09–2.01 (m, 4H), 1.62–1.57 (m, 4H); 13C NMR (125 MHz, CDCl3): δ 189.5, 151.3, 149.6,
145.7, 137.3, 131.9 (2C), 130.0 (2C), 129.7, 127.6, 123.3, 119.3, 112.6, 110.7, 67.6, 56.1, 55.7,
54.6 (2C), 26.5, 25.9 (2C), 24.4; HRMS (ESI): m/z calcd. for C24H28BrNO3, 458.1331, found
458.1327 [M + H]+.
4.2.4. E)-3-(3-Methoxy-4-(4-(piperidin-1-yl)butoxy)phenyl)-1-phenylprop-2-en-1-one (4d).
1
Brown semi-solid; H NMR (500 MHz, CDCl3): δ 8.02 (dd, 2H, J = 5.0, 3.2 Hz), 7.78 (d, 1H, J
= 15.6 Hz), 7.61–7.58 (m, 1H), 7.52 (t, 2H, J = 7.8 Hz), 7.40 (d, 1H, J = 15.1 Hz), 7.25–7.22 (dd,
1H, J = 1.9, 8.3 Hz), 7.18 (d, 1H, J = 1.9 Hz), 6.91 (d, 1H, J = 2.0 Hz), 4.11 (t, 2H, J = 6.7 Hz),
3.94 (s, 3H), 2.48–2.36 (m, 6H), 1.94–1.82 (m, 2H), 1.75–1.68 (m, 2H), 1.64–1.59 (m, 4H),
1.48–1.42 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 190.7, 151.1, 149.6, 145.2, 138.5, 132.5,
128.6 (2C), 128.4 (2C), 127.7, 123.2, 120.0, 112.4, 110.6, 68.8, 58.9, 56.1, 54.6 (2C), 27.2, 25.9
(2C), 24.4, 23.3; HRMS (ESI) calcd. for C25H32NO3, 394.2382, found 394.2388 [M + H]+.
4.2.5. (E)-1-(4-Bromophenyl)-3-(3-methoxy-4-(4-(piperidin-1-yl)butoxy)phenyl)prop-2-en-1-one
(4e). Brown semi-solid; 1H NMR (500 MHz, CDCl3): δ 8.12 (t, J = 1.7 Hz, 1H), 8.12 (t, J = 1.6
Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 15.5 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.34 – 7.31
(m, 1H), 7.15 (d, J = 1.9 Hz, 1H), 6.90 (d, J = 8.3 Hz 1H), 6.77 (d, J = 2.0 Hz, 1H), 4.09 (t, J =
6.7 Hz, 2H), 3.94 (s, 3H), 2.43 – 2.36 (m, 6H), 1.90 – 1.83 (m, 2H), 1.63 – 1.57 (m, 4H), 1.48 –
1.41 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 189.1, 151.3, 149.6, 146.1, 140.4, 136.0, 135.4,
131.2, 130.2, 126.9, 126.6, 123.5, 119.2, 112.4, 110.6, 68.8, 58.9, 56.1, 54.5 (2C), 27.2, 25.8
(2C), 24.4, 23.3; HRMS (ESI) calcd. for C25H31BrNO3, 472.1487, found 472.1486 [M + H]+.
16

4.2.6. (E)-1-(4-Chlorophenyl)-3-(3-methoxy-4-(4-(piperidin-1-yl)butoxy)phenyl)prop-2-en-1-one
(4f). Yellow solid; Mp: 79–80 oC; 1H NMR (500 MHz, CDCl3): δ 7.95 (d, 2H, J = 6.8 Hz), 7.76
(d, 1H , J = 15.6 Hz), 7.47 (d, 2H, J = 5.6 Hz), 7.33 (d, 1H, J = 15.6 Hz), 7.21 (dd, 1H, J = 8.3,
1.8 Hz), 7.15 (s, 1H), 6.90 (d, 1H, J = 7.4 Hz), 4.09 (t, 2H, J = 6.7 Hz), 3.93 (s, 3H), 2.51–2.27
(m, 7H), 1.95–1.85 (m, 2H), 1.74–1.64 (m, 2H), 1.64–1.51 (m, 4H), 1.47–1.40 (m, 2H); 13C
NMR (125 MHz, CDCl3): δ 189.3, 151.2, 149.6, 145.7, 138.9, 136.8, 129.8 (2C), 128.9 (2C),
127.5, 123.3, 119.3, 112.4, 110.6, 68.8, 58.9, 56.0, 54.6 (2C), 27.2, 25.9 (2C), 24.4, 23.4; HRMS
(ESI) calcd. for C25H31ClNO3, 428.1992, found 428.1990 [M + H]+.
4.2.7. (E)-1-(2,4-Dichlorophenyl)-3-(3-methoxy-4-(4-(piperidin-1-yl)butoxy)phenyl)prop-2-en-1-
one (4g). Yellow solid; Mp: 79–80 oC; 1H NMR (500 MHz, CDCl3): δ 7.48 (d, 1H, J = 1.9 Hz),
7.42–7.33 (m, 3H), 7.12 (dd, 1H, J = 8.8, 1.9 Hz), 7.07 (d, 1H, J = 1.9 Hz), 6.96 (d, 1H, J = 15.9
Hz), 6.88 (d, 1H, J = 8.4 Hz), 4.08 (t, 2H, J = 6.7 Hz), 3.89 (s, 3H), 2.48–2.37 (m, 6H), 1.92–
1.83 (m, 2H), 1.72–1.66 (m, 2H), 1.64–1.57 (m, 4H), 1.47–1.42 (m, 2H); 13C NMR (125 MHz,
CDCl3): δ 192.8, 151.5, 149.6, 147.1, 137.7, 136.6, 132.3, 130.2, 130.1, 127.2, 127.0, 123.9,
123.6, 112.4, 110.5, 68.8, 58.8, 56.0, 54.5 (2C), 27.1, 25.7 (2C), 24.3, 23.2; HRMS (ESI) calcd
for C25H30Cl2NO3, 462.1603, found 462.1618 [M + H] +.
4.2.8. (E)-3-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
(4h). Yellow liquid, yield 78%; 1H NMR (500 MHz, CDCl3): δ 8.06–8.00 (m, 2H), 7.75 (d, 1H, J
= 15.5 Hz), 7.41 (d, 1H, J = 15.5 Hz), 7.21 (dd, 1H, J = 2.0 Hz, 9.0 Hz), 7.16 (d 1H, J = 2.0
Hz), 7.01–6.96 (m, 2H), 6.91 (d, 1H, J = 8.3 Hz), 4.20 (t, 2H, J = 6.0 Hz), 3.92 (s, 3H), 3.89 (s,
3H) 3.75–3.73 (m, 4H), 2.86 (t, 2H, J = 6.0 Hz), 2.61–2.59 (m, 4H); 13C NMR (125 MHz,
CDCl3): δ 188.7, 163.2, 150.4, 149.6, 144.0, 131.2, 130.7 (2C), 128.4, 122.7, 119.9, 113.7 (2C),
112.8, 110.7, 66.8 (2C), 66.6, 57.3, 56.0, 55.4, 54.0 (2C); HRMS (ESI): m/z calcd. for
C23H28NO5, 398.1967, found 398.1969 [M + H]+.
4.2.9. (E)-1-(4-Bromophenyl)-3-(3-methoxy-4-(2-morpholinoethoxy)phenyl)prop-2-en-1-one (4i).
Yellow liquid, yield 83%; 1H NMR (500 MHz, CDCl3): δ 7.89–7.85 (m, 1H), 7.80 (d, 1H, J =
8.5 Hz), 7.76 (d, 1H, J = 15.5 Hz), 7.66–7.63 (m, 1H), 7.59 (d, 1H, J = 9.0 Hz), 7.33 (d, 1H, J =
15.5 Hz), 7.21 (dd, 1H, J = 2.0 Hz, 8.0 Hz ), 7.15 (d, 1H, J = 2.0 Hz), 6.91 (d, 1H, J = 8.5 Hz),
4.21 (t, 2H, J = 6.0 Hz), 3.92 (s, 3H), 3.75–3.72 (m, 4H), 2.87 (t, 2H, J = 6.0 Hz), 2.61–2.59 (m,
17

4H); 13C NMR (125 MHz, CDCl3): δ 188.8, 163.3, 150.4, 149.6, 144.0, 131.3, 130.7 (2C), 128.5,
122.8, 120.0, 113.8 (2C), 113.0, 110.8, 66.8, 66.6, 57.3, 56.0, 55.5, 54.0 (2C); HRMS (ESI): m/z
calcd. for C22H25BrNO4, 446.0967, found 446.0960 [M + H]+.
4.2.10. (E)-1-(2,4-Dichlorophenyl)-3-(3-methoxy-4-(2-morpholinoethoxy)phenyl)prop-2-en-1-
one (4j). Yellow liquid, yield 86%; 1H NMR (CDCl3, 500 MHz): δ 7.48 (d, 1H, J = 2.0 Hz), 7.41
(d, 1H, J = 8.0 Hz), 7.38–7.34 (m, 2H), 7.13 (dd, 1H, J = 2.0, 8.5 Hz), 7.07 (d, 1H, J = 2.0 Hz),
6.96 (d, 1H, J = 16.0 Hz), 6.89 (d, 1H, J = 8.0 Hz), 4.19 (t, 2H, J = 6.0 Hz), 3.89 (s, 3H), 3.74–
3.72 (m, 4H), 2.86 (t, 2H, J = 6.0 Hz), 2.60–2.58 (m, 2H); 13C NMR (125 MHz, CDCl3): δ
192.7, 151.1, 149.7, 146.8, 137.7, 136.7, 132.2, 130.2, 130.1, 127.5, 127.2, 124.1, 123.4, 112.8,
110.6, 66.9 (2C), 66.8, 57.3, 56.0, 54.1 (2C); HRMS (ESI): m/z calcd. for C22H24Cl2NO4,
436.1082, found 436.1078 [M + H]+.
4.2.11. (E)-3-(3-Methoxy-4-(2-morpholinoethoxy)phenyl)-1-(4-(trifluoromethyl)phenyl) prop-2-
en-1-one (4k). Yellow liquid, yield 88%; 1H NMR (500 MHz, CDCl3): δ 8.08 (d, 2H, J = 8.0
Hz), 7.79–7.74 (m, 3H), 7.33 (d, 1H J = 16.0 Hz), 7.22 (d, 1H, J = 6.5 Hz), 7.15 (s, 1H), 6.92 (d,
1H, J = 8.0 Hz), 4.21 (t, 2H, J = 6.0 Hz), 3.93 (s, 3H), 3.74–3.71 (m, 4H), 2.89–2.84 (m, 2H),
2.62–2.59 (m, 4H); 13C NMR (125 MHz, CDCl3): δ 189.8, 151.0, 149.7, 146.2, 141.4, 128.7
(2C), 128.6, 128.5, 127.9, 125.8, 125.7, 125.6, 125.6, 123.3, 119.7, 112.9, 110.7, 66.8 (2C), 66.6,
57.3, 56.1, 54.0 (2C); HRMS (ESI): m/z calcd. for C23H25F3NO4, 436.1736, found 436.1727 [M
+ H]+.
4.2.12. (E)-3-(3-Methoxy-4-(3-morpholinopropoxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
(4l). Yellow liquid, yield 75%; 1H NMR (500 MHz, CDCl3): δ 8.04 (d, 2H, J = 7.5 Hz), 7.74 (d,
1H, J =15.0 Hz), 7.40 (d, 1H, J =15.0 Hz), 7.20 (d, 1H, J = 8.0 Hz), 7.16 (s, 1H), 6.98 (d, 2H, J
=11.0 Hz), 6.91 (d, 1H, J = 5.5 Hz), 4.14 (t, 2H, J = 5.5 Hz), 3.93 (s, 3H), 3.89 (s, 3H), 3.77–
3.72 (m, 4H), 2.58 (t, 2H, J = 6.5 Hz), 2.55–2.50 (m, 4H), 2.08–2.01 (m, 2H); 13C NMR (125
MHz, CDCl3): δ 188.8, 163.3, 150.7, 149.5, 144.3, 131.3, 130.7 (2C), 128.1, 122.9, 119.9, 113.8
(2C), 112.7, 110.8, 67.1, 66.6, 56.1, 55.5 (2C), 55.4, 53.5 (2C), 25.9; HRMS (ESI): m/z calcd for
C24H30NO5, 412.2124, found 412.2113 M + H]+.
18

4.2.13. (E)-3-(4-(3-(2,6-Dimethylmorpholino)propoxy)-3-methoxyphenyl)-1-(4-methoxyphenyl)
prop-2-en-1-one (4m). Yellow semi-solid, yield 85%; 1H NMR (500 MHz, CDCl3): δ 8.03 (d,
2H, J = 6.5 Hz ), 7.75 (d, 1H, J = 15.5 Hz), 7.40 (d, 1H, J = 15.5 Hz), 7.20 (d, 1H, J = 8.0 Hz),
7.16 (s, 1H), 6.97 (d, 2H, J = 8.5 Hz), 6.91 (d, 1H, J = 8.0 Hz), 4.13 (t, 2H, J = 6.5 Hz), 3.92 (s,
3H), 3.88 (s, 3H), 3.70–3.65 (m, 2H), 2.76 (d, 2H, J = 11.0 Hz), 2.51 (t, 2H, J = 7.0 Hz), 2.08–
2.02 (m, 2H), 1.74 (t, 2H, J = 11.0 Hz), 1.16 (s, 3H), 1.15 (s, 3H); 13C NMR (125 MHz, CDCl3):
δ 188.7, 163.2, 150.7, 149.5, 144.1, 131.2, 130.6 (2C), 128.0, 122.8, 119.7, 113.7 (2C), 112.5,
110.6, 71.5 (2C), 67.2, 59.4 (2C), 56.0, 55.4, 54.8, 26.1, 19.1 (2C); HRMS (ESI): m/z calcd. for
C26H34NO5, 440.2437, found 440.2457 [M + H]+.
4.2.14. (E)-3-(4-(3-(2,6-Dimethylmorpholino)propoxy)-3-methoxyphenyl)-1-(2-
methoxyphenyl)prop-2-en-1-one (4n). Yellow semi-solid, yield 80%; 1H NMR (500 MHz,
CDCl3): δ 7.93 (d, 2H, J = 8.0 Hz), 7.75 (d, 1H, J = 15.5 Hz), 7.38 (d, 1H, J = 15.5 Hz), 7.30 (d,
2H, J = 8.5 Hz), 7.20 (d, 1H, J = 8.5 Hz), 7.16 (s, 1H), 6.91 (d, 1H, J = 8.5 Hz), 4.13 (t, 2H, J =
6.5 Hz), 3.93 (s, 3H), 3.70–3.65 (m, 2H), 2.76 (d, 2H, J = 12.0 Hz), 2.51 (t, 2H, J = 7.0 Hz), 2.44
(s, 3H), 2.07–2.00 (m, 2H), 1.74 (t, 2H, J = 11.0 Hz), 1.17 (s, 3H) 1.15(s, 3H); 13C NMR (125
MHz, CDCl3): δ 190.1, 150.8, 149.5, 144.6, 143.4, 135.9, 129.2 (2C), 128.6 (2C), 127.9, 123.0,
120.0, 112.6, 110.6, 71.6 (2C), 67.2, 59.4 (2C), 56.0, 54.9, 26.1, 21.6, 19.1 (2C); HRMS (ESI):
m/z calcd. for C26H34NO5, 440.2437, found 440.2432 [M + H]+.
4.2.15. (E)-1-(2,4-Dichlorophenyl)-3-(4-(3-(2,6-dimethylmorpholino)propoxy)-3-methoxyphenyl)
prop-2-en-1-one (4o). Yellow solid, yield 78%; Mp: 98–100 oC; 1H NMR (500 MHz, CDCl3): δ
7.48 (d, 1H, J = 2.0 Hz ), 7.41 (d, 1H, J = 8.0 Hz), 7.38–7.33 (m, 2H), 7.11 (dd, 1H, J = 2.0, 8.5
Hz), 7.07 (d, 1H, J = 2.0 Hz), 6.96 (d, 1H, J = 16.0 Hz), 6.89 (d, 1H, J = 8.5 Hz), 4.12 (t, 2H, J =
6.5 Hz), 3.89 (s, 3H), 3.68–3.65 (m, 2H), 2.75 (d, 2H, J = 10.5 Hz), 2.49 (t, 2H, J = 7.0 Hz),
2.06–2.01 (m, 2H), 1.73 (t, 2H, J = 10.5 Hz), 1.16 (s, 3H), 1.15 (s, 3H); 13C NMR (125 MHz,
CDCl3): δ 192.7, 151.4, 149.6, 147.0, 137.7, 136.6, 132.2, 130.2, 130.1, 127.2, 127.1, 123.9,
123.5, 112.5, 110.5, 71.6 (2C), 67.2, 59.5 (2C), 56.0, 54.8, 26.1, 19.1 (2C); HRMS (ESI): m/z
calcd. for C25H30Cl2NO4, 478.1552, found 478.1547 [M + H]+.
4.2.16. (E)-3-(4-(3-(2,6-Dimethylmorpholino)propoxy)-3-methoxyphenyl)-1-p-tolylprop-2-en-1-
one (4p). Yellow semi-solid, yield 80%; 1H NMR (500 MHz, CDCl3): δ 7.93 (d, 2H, J = 7.5 Hz),
19

7.75 (d, 1H, J = 15.5 Hz), 7.39 (d, 1H, J = 15.5 Hz), 7.30 (d, 2H, J = 7.5 Hz), 7.21 (d, 1H, J =
8.0 Hz), 7.16 (s, 1H), 6.92 (d, 1H, J = 8.0 Hz), 4.13 (t, 2H, J = 6.5 Hz), 3.93 (s, 3H), 3.73–3.67
(m, 2H), 2.75 (d, 2H, J = 11.0 Hz), 2.50 (t, 2H, J = 12.0 Hz), 2.43 (s, 3H), 2.07–2.02 (m, 2H),
1.74 (t, 2H, J = 11.5 Hz), 1.17 (s, 3H), 1.15 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 190.1,
150.8, 149.5, 144.6, 143.4, 135.9, 129.2 (2C), 128.6 (2C), 127.9, 123.0, 120.0, 112.6, 110.6, 71.6
(2C), 67.2, 59.5 (2C), 56.0, 54.9, 26.2, 21.6, 19.1 (2C); HRMS (ESI): m/z calcd. for C26H34NO4,
424.2488, found 424.2477 [M + H]+.
4.2.17. (E)-3-(4-(3-(Indolin-1-yl)propoxy)-3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-
one (4q). Yellow solid, yield 90%; Mp: 227–229 oC; 1H NMR (500 MHz, CDCl3): δ 8.04 (d, 2H,
J = 9.0 Hz), 7.76 (d, 1H, J =15.5 Hz), 7.41 (d, 1H, J = 15.5 Hz), 7.21 (d, 1H, J = 8.5 Hz), 7.17 (s,
1H), 7.09–7.03 (m, 2H), 6.99 (d, 2H, J = 8.5 Hz), 6.91 (d, 1H, J = 8.5 Hz), 6.65 (t, 1H, J = 7.5
Hz), 6.51 (d, 1H, J = 8.0 Hz), 4.19 (t, 2H, J = 6.0 Hz), 3.96 (s, 3H), 3.90 (s, 3H), 3.37 (t, 2H, J =
8.5 Hz), 3.30 (t, 2H, J = 7.0 Hz), 2.97 (t, 2H, J = 8.5 Hz), 2.17 (m, 2H); 13C NMR (125 MHz,
CDCl3): δ 188.8, 163.3, 152.6, 150.8, 149.6, 144.2, 131.3, 130.7 (2C), 129.9, 128.1, 127.3,
124.4, 122.9, 119.8, 117.6, 113.8 (2C), 112.7, 110.7, 106.9, 66.7, 56.1, 55.5, 53.3, 46.0, 28.6,
27.3; HRMS (ESI): m/z calcd. for C28H30NO4, 444.2175, found 444.2193 [M + H]+.
4.2.18. (E)-1-(2,4-Dichlorophenyl)-3-(4-(3-(indolin-1-yl)propoxy)-3-methoxyphenyl)prop-2-en-
1-one (4r). Off-white solid, yield 89%; Mp: 92–94 oC; 1H NMR (500 MHz, CDCl3): δ 7.48 (d,
1H, J = 1.5 Hz), 7.41 (d, 1H, J = 8.5 Hz), 7.38–7.34 (m, 2H), 7.11–7.01 (m, 4H), 6.96 (d, 1H, J =
16.0 Hz), 6.88 (d, 1H, J = 8.0 Hz), 6.64 (t, 1H, J = 7.0 Hz), 6.49 (d, 1H, J = 8.0 Hz), 4.18 (t, 2H,
J = 6.5 Hz), 3.92 (s, 3H), 3.35 (t, 2H, J = 8.5 Hz), 3.28 (t, 2H, J = 6.5 Hz), 2.96 (t , 2H, J = 8.5
Hz), 2.16 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 192.7, 152.6, 151.4, 149.7, 147.1, 137.7,
136.6, 132.3, 130.3, 130.1, 130.0, 127.3, 127.2 (2C), 124.4, 124.0, 123.6, 117.6, 112.6, 110.5,
106.9, 66.7, 56.0, 53.4, 46.0, 28.6, 27.3; HRMS (ESI): m/z calcd. for C27H26Cl2NO3, 482.1290,
found 482.1302 [M + H]+.
4.2.19. (E)-3-(4-(3-(3,4-Dihydroquinolin-1(2H)-yl)propoxy)-3-methoxyphenyl)-1-phenylprop-2-
en-1-one (4s). Yellow solid; Mp: 111–113 oC; 1H NMR (500 MHz, CDCl3): δ 8.01 (d, 2H, J =
7.3 Hz), 7.76 (d, 1H, J = 15.6 Hz), 7.58 (t, 1H, J = 7.3 Hz), 7.51 (t, 2H, J = 7.5 Hz), 7.39 (d, 1H,
J = 15.6 Hz), 7.19 (d, 1H, J = 7.0 Hz), 7.18 (s, 1H), 7.00 (t, 1H, J = 7.5 Hz), 6.94 (d, 1H, J = 7.1
20

Hz), 6.88 (d, 1H, J = 8.3 Hz), 6.64 (d, 1H, J = 8.2 Hz), 6.55 (t, 1H, J = 7.2 Hz), 4.14 (t, 2H, J =
5.9 Hz), 3.95 (s, 3H), 3.50 (t, 2H, J = 6.9 Hz), 3.29 (t, 2H, J = 4.9 Hz), 2.75 (t, 2H, J = 6.3 Hz),
2.20–2.10 (m, 2H), 1.97–1.89 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 190.7, 150.9, 149.7,
145.2, 145.1, 138.5, 132.6, 129.2, 128.6 (2C), 128.5 (2C), 128.0, 127.1, 123.2, 122.4, 120.1,
115.6, 112.8, 110.7, 110.6, 66.6, 56.1, 49.7, 48.0, 28.2, 26.3, 22.3; HRMS (ESI) calcd. for
C28H30NO3, 428.2226, found 428.2284 [M + H]+.
4.2.20. (E)-3-(4-(3-(3,4-Dihydroquinolin-1(2H)-yl)propoxy)-3-methoxyphenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one (4t). Yellow solid; Mp: 91–92 oC; 1H NMR (500 MHz, CDCl3):
δ 8.04 (d, 2H, J = 8.7 Hz), 7.75 (d, 1H, J = 15.5 Hz), 7.41 (d, 1H, J = 15.5 Hz), 7.20 (d, 1H, J =
6.0 Hz), 7.18 (s, 1H), 7.04–6.95 (m, 3H), 6.93 (d, 1H, J = 7.2 Hz), 6.88 (d, 1H, J = 8.2 Hz), 6.64
(d, 1H, J = 8.2 Hz), 6.55 (t, 1H, J = 7.2 Hz), 4.13 (t, 2H, J = 5.7 Hz), 3.95 (s, 3H), 3.89 (s, 3H),
3.50 (t, 2H, J = 6.9 Hz), 3.29 (t, 2H, J = 4.7 Hz), 2.75 (t, 2H, J = 6.3 Hz), 2.22–2.08 (m, 2H),
2.00–1.87 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 188.8, 163.3, 150.7, 149.7, 145.2, 144.2,
131.4, 130.7 (2C), 129.2, 128.2, 127.1, 122.9, 122.4, 119.8, 115.6, 113.8 (2C), 112.3, 110.8,
110.7, 66.6, 56.1, 55.5, 49.7, 48.0, 28.2, 26.3, 22.3; HRMS (ESI) calcd. for C29H32NO4,
458.2331, found 458.2328 [M + H]+.
4.2.21. (E)-1-(4-Bromophenyl)-3-(4-(3-(3,4-dihydroquinolin-1(2H)-yl)propoxy)-3-
1
methoxyphenyl)prop-2-en-1-one (4u). Yellow solid; Mp: 95–97 oC; H NMR (500 MHz,
CDCl3): δ 7.88 (d, 2H, J = 8.1 Hz), 7.76 (d, 1H, J = 15.6 Hz), 7.64 (d, 2H, J = 8.1 Hz), 7.32 (d,
1H, J = 8.5 Hz), 7.20 (d, 1H, J = 7.3 Hz), 7.17 (s, 1H), 7.00 (t, 1H, J = 7.7 Hz), 6.94 (d, 1H, J =
7.3 Hz), 6.88 (d, 1H, J = 8.3 Hz), 6.64 (d, 1H, J = 8.2 Hz), 6.56 (t, 1H, J = 7.3 Hz), 4.14 (t, 2H, J
= 6.0 Hz), 3.95 (s, 3H), 3.50 (t, 2H, J = 6.9 Hz), 3.29 (t, 2H, J = 4.9 Hz), 2.75 (t, 2H, J = 6.3 Hz),
2.20–2.09 (m, 2H), 1.97–1.88 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 189.5, 151.1, 149.7,
145.7, 145.2, 137.2, 131.9 (2C), 130.0 (2C), 129.2, 127.8, 127.6, 127.1, 123.3, 122.4, 119.4,
115.7, 112.7, 110.8, 110.7, 66.6, 56.1, 49.7, 48.0, 28.2, 26.3, 22.2; HRMS (ESI) calcd. for
C28H29BrNO3, 506.1331, found 506.1328 [M + H]+.
4.2.22. (E)-1-(4-Chlorophenyl)-3-(4-(3-(3,4-dihydroquinolin-1(2H)-yl)propoxy)-3-
methoxyphenyl)prop-2-en-1-one (4v). Yellow solid; Mp: 103–105 oC; 1H NMR (500 MHz,
CDCl3): δ 7.96 (d, 2H, J = 8.3 Hz), 7.76 (d, 1H, J = 15.6 Hz), 7.47 (d, 2H, J = 8.3 Hz), 7.34 (d,
21

1H, J = 15.6 Hz), 7.20 (d, 1H, J = 8.3 Hz), 7.17 (s, 1H), 7.00 (t, 1H, J = 7.7 Hz), 6.94 (d, 1H, J =
7.3 Hz), 6.88 (d, 1H, J = 8.3 Hz), 6.63 (d, 1H, J = 8.2 Hz), 6.55 (t, 1H, J = 7.3 Hz), 4.14 (t, 2H, J
= 6.0 Hz), 3.95 (s, 3H), 3.50 (t, 2H, J = 6.9 Hz), 3.28 (t, 2H, J = 5.5 Hz), 2.75 (t, 2H, J = 6.2
Hz,), 2.21–2.08 (m, 2H), 2.05–1.83 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 189.3, 151.1,
149.7, 145.6, 145.2, 139.0, 136.8, 129.9 (2C), 129.2, 128.9 (2C), 127.8, 127.1, 123.3, 122.4,
119.4, 115.7, 112.7, 110.8, 110.7, 66.6, 56.1, 49.7, 48.0, 28.1, 26.3, 22.3; HRMS (ESI) calcd. for
C28H29ClNO3, 462.1836, found 462.1836 [M + H]+.
4.2.23. (E)-1-(2,4-Dichlorophenyl)-3-(4-(3-(3,4-dihydroquinolin-1(2H)-yl)propoxy)-3-
1
methoxyphenyl)prop-2-en-1-one (4w). Yellow solid; Mp: 92–94 oC; H NMR (500 MHz,
CDCl3): δ 7.49 (s, 1H), 7.43–7.33 (m, 3H), 7.13–7.08 (m, 2H), 7.01–6.91 (m, 3H), 6.86 (d, 1H, J
= 8.2 Hz), 6.63 (d, 1H, J = 8.2 Hz), 6.55 (t, 1H, J = 7.3 Hz), 4.13 (t, 2H, J = 6.0 Hz), 3.92 (s,
3H), 3.49 (t, 2H, J = 6.9 Hz), 3.28 (t, 2H, J = 5.5 Hz), 2.74 (t, 2H, J = 6.3 Hz), 2.19–2.09 (m,
2H), 1.97–1.88 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 188.0, 146.6, 145.0, 142.3, 140.7,
140.2, 133.0, 131.9, 127.5, 125.5, 125.4, 124.5, 122.5, 122.4, 119.3, 118.8, 117.7, 110.9, 107.9,
105.9, 61.9, 51.3, 45.0, 43.2, 23.4, 21.6, 17.5; HRMS (ESI) calcd. for C28H28Cl2NO3, 496.1446,
found 496.1446 [M + H] +.
4.2.24. (E)-1-(4-Aminophenyl)-3-(4-(3-(3,4-dihydroquinolin-1(2H)-yl)propoxy)-3-
methoxyphenyl)prop-2-en-1-one (4x). Brown semi-solid; 1H NMR (500 MHz, CDCl3): δ 7.93 (d,
2H, J = 8.6 Hz), 7.73 (d, 1H, J = 15.5 Hz), 7.41 (d, 1H, J = 15.5 Hz), 7.21–7.14 (m, 2H), 7.00 (t,
1H, J = 7.7 Hz), 6.94 (d, 1H, J = 7.1 Hz), 6.87 (d, 1H, J = 8.2 Hz,) 6.70 (d, 2H, J = 8.5 Hz), 6.64
(d, 1H, J = 8.2 Hz), 6.55 (t, 1H, J = 7.3 Hz), 4.13 (t, 2H, J = 6.1 Hz), 3.94 (s, 3H), 3.50 (t, 2H, J
= 6.9 Hz), 3.29 (t, 2H, J = 5.6 Hz), 2.75 (t, 2H, J = 6.3 Hz), 2.22–2.04 (m, 2H), 1.97–1.85 (m,
2H), 1.67 (brs, 2H); 13C NMR (125 MHz, CDCl3): δ 183.5, 146.3, 145.8, 144.9, 140.5, 138.6,
126.3 (2C), 124.4, 124.0, 123.7, 122.4, 118.0, 117.6, 115.2, 110.9, 109.2 (2C), 108.1, 106.0,
105.9, 61.9, 51.4, 45.0, 43.3, 23.4, 21.6, 17.5; HRMS (ESI) calcd. for C28H31N2O3, 443.2335,
found 443.2333 [M + H]+.
4.2.25. (E)-3-(4-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)propoxy)-3-methoxyphenyl)-1-(4-methoxy
phenyl)prop-2-en-1-one (4y). Yellow semi-solid, yield 80%; 1H NMR (500 MHz, CDCl3): δ 8.03
(d, 2H, J = 9.0 Hz), 7.73 (d, 1H, J =15.5 Hz), 7.39 (d, 1H, J =15.5 Hz), 7.19 (dd, 1H, J = 1.7,
22

10.0 Hz), 7.15 (d, 1H, J = 1.6 Hz), 6.98 (d, 2H, J =8.5 Hz), 6.83 (d, 1H, J = 8.0 Hz), 5.75 (s, 1H),
4.19 (t, 2H, J = 6.5 Hz), 3.98 (t, 2H, J = 6.0 Hz), 3.93 (s, 3H), 3.89 (s, 3H), 2.37–2.32 (m, 2H),
2.21 (s,3H), 2.18 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 188.8, 163.3, 150.4, 149.5, 147.6,
144.1, 139.2, 131.3, 130.7 (2C), 128.2, 122.8, 119.9, 113.8 (2C), 112.7, 110.6, 104.7, 65.4, 55.9,
55.5, 44.6, 29.6, 13.5, 10.7; HRMS (ESI): m/z calcd. for C25H29N2O4, 421.2127, found 421.2120
[M + H]+.
4.3. Biology
4.3.1. Cell cultures
Cells were procured from National Centre for Cell Science (NCCS) Pune, India and stocks were
maintained in the sterile laboratory conditions. Lung (A549 and NCI-H460), prostate (DU-145
and PC-3), colon (HCT-15 and HCT-116) and brain (U-87 glioblastoma) cancer cells were
grown in tissue culture flasks in DMEM (Dulbecco modified Eagle medium, Sigma) or MEM
(Minimum Essential Medium, Sigma) supplemented with 10% fetal bovine serum with 1X
stabilized antibiotic-antimycotic solution (Sigma) in a CO2 incubator at 37 oC with 5% CO2 and
90% relative humidity.
4.3.2. MTT assay
The cytotoxic activity of 4a–y was determined using MTT assay. 1 x 104 cells per well were
seeded in 100 µL DMEM, supplemented with 10% FBS in each well of 96-well microculture
plates and incubated for 48 h, at 37 oC in a CO2 incubator. Compounds, diluted to the required
concentrations in culture medium, were added to the wells with respective vehicle control. After
48 h of incubation, 10 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) (5 mg/mL) was added to all the plates and incubated for 4 h. Then, the
supernatant from each well was carefully decanted, formazon crystals were dissolved in 100 µL
of DMSO and absorbance was recorded at 570 nm wavelength.
4.3.3. Morphological observations using phase contrast microscope
NCI-H460 cells were plated in 6 well culture plates with a density of 1x105 cells/mL and allowed
to adhere for overnight. Cells were incubated with various concentrations of 4a. After 48 h, cells
23

were observed for morphological changes and images were taken with phase contrast microscope
(Nikon).
4.3.4. DAPI nucleic acid staining
To clearly examine nuclear morphological changes induced by compounds 4a on NCI-H460
cells, DAPI nuclear staining was performed. Blue-fluorescent DAPI is a nuclear
stain, which can cross intact membrane of live cells and stains the nucleus of the live cells as
light blue, wherein the apoptotic cell nuclei appears as bright blue due to chromatin
condensation. Thus, it was of our interest to examine nuclear fragmentation or chromatin
condensation induced by compound 4a. For this, NCI-H460 cells were stained with DAPI
following the treatment with the compound 4a and cells were observed under fluorescence
microscope for nuclear morphological changes. The results indicate that the nuclear structure of
untreated cells was intact while 4a treated cells showed nuclear shrinkage, fragmented, pyknotic
and horse shoe shaped nuclei (white arrow) and chromatin condensation, and these are the
remarkable features of apoptosis. These findings demonstrate that these compounds could induce
apoptosis in NCI-H460 cells.
4.3.5. Generation of Reactive Oxygen Species (ROS)
NCI-H460 cells were plated in 24 well plates at a density of (1 x 106 cells/mL) and allowed to
adhere for overnight. Then the cells were treated with 1.25 µM, 2.5 µM and 5.0 µM of active
compound 4a for 24 h. The media was replaced with culture medium containing DCFDA dye
(10 µM) and incubated for 30 min dark. The fluorescence intensity from samples was analysed
by spectrofluorometer at an excitation and emission wavelength of 488 and 525 nm, respectively.
4.3.6. Flow-cytometry analysis
NCI-H460 cells (1 x 106 cells/well) in 6 well plate were treated with different 1.25, 2.5 and 5 µM
mM concentrations of the compound 4a for 24 h and cells were collected by trypsinisation,
washed with 150 mM PBS and were fixed with 70% ethanol for 30 min at 4 oC. After fixing,
cells were washed with PBS and stained with 400 µL of propidium
24

iodide staining buffer [PI (200 µg), Triton X (100 µL), DNAse-free RNAse A (2 mg) in 10 mL
PBS] for 15 min at room temp in dark. The samples were then analyzed for propidium iodide
fluorescence from 15,000 events by flow cytometry using BD Accuri C6 flowcytometer.
4.3.7. Effect on tubulin polymerization
Tubulin polymerization kit was purchased from Cytoskeleton, Inc. (BK011). To evaluate the
effect of compound 4a, fluorescence based in vitro tubulin polymerization assay was conducted
following the manufacturer's protocol. The reaction mixture having porcine brain tissue (2
mg/mL) in 80 mM PIPES at pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA,1.0 mM GTP
and glycerol in the presence and absence of test compound 4a (final concentration of 10 µM)
was prepared and added to each well of 96-well plate. 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
440 nm (excitation wavelength is 360 nm) was measured by using a Spectramax M4 Multi mode
Micro plate Detection System. CA-4 was used as positive control in the assay at 3µM final
concentrartion. The IC50 value was calculated from the drug concentration required for inhibiting
50% of tubulin assembly compared to control.
4.3.8. Molecular docking
The crystal co-ordinates of α,β-tubulin subunits were retrieved from the protein data bank (PDB
ID: 3E22). As the active site i.e., colchicine binding site of tubulin is located at the interface of
the subunits, both the chains were considered for molecular modelling studies. The protein
preparation tool was used for the preparation of receptor model (Schrödinger 2015-4). The tool
adds missing side chains and loops and also removes water molecules with a distance of more
than 5 Å. Then, restrained minimization was performed utilizing the OPLS 2005 force field to
RMSD of 0.3 Å and an active site pocket of colchicine with 20 Å equally in each
direction of X, Y, and Z was used for receptor grid generation. The potential ligands 4a and 4b
were sketched by using 2D sketcher and, prepared for docking using Ligprep and a total number
of 10 conformers were generated for each of the ligand. The ligands were docked into the active
site of tubulin using GLIDE-XP (Extra Precision) flexible program.
25

Acknowledgements
SN, NPK, ND, DP and KRS are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of
India, New Delhi, for the award of NIPER fellowship. Dr. N. Shankaraiah thankful to SERB,
DST, Govt. of India for the start-up grant (YSS-2015-001709).
References
1. Mandelkow E, Mandelkow EM. Microtubular structure and tubulin polymerization. Curr.
Opin. Cell. Biol. 1989;1:5–9.
2. Jordan MA, Wilson L. Microtubules and actin filaments: dynamic targets for cancer
chemotherapy. Curr. Opin. Cell Biol. 1998;10:123-130.
3. Giannakakou P, Sackett D, Fojo T. Tubulin/microtubules: still a promising target for new
chemotherapeutic agents. J. Natl. Cancer Inst. 2000;92:182-183.
4. (a) Biswas BB, Sen K, Choudhury GG, Bhattacharyya B. Molecular biology of tubulin:
Its interaction with drugs and genomic organization. J. Biosci. 1984;6:431-457; (b)
Shankaraiah N, Kumar NP, Amula SB, Nekkanti S, Jeengar MK, Naidu VG, Reddy TS,
Kamal A. One-pot synthesis of podophyllotoxin–thiourea congeners by employing NH2
SO3H/NaI: Anticancer activity, DNA topoisomerase-II inhibition, and apoptosis inducing
agents. Bioorg. Med. Chem. Lett. 2015;25:4239–4244; (c) Kamal A, Reddy TS, Polepalli
S, Shalini N, Reddy VG, Rao AS, Jain N, Shankaraiah N. Synthesis and biological
evaluation of podophyllotoxin congeners as tubulin polymerization inhibitors. Bioorg.
Med. Chem. 2014;22:5466–5475; (d) Kamal A, Suresh P, Ramaiah MJ, Reddy TS,
Kapavarapu RK, Rao BN, Imthiajali S, Reddy TL, Pushpavalli SN, Shankaraiah N, Pal-
Bhadra M. 4β-[4′-(1-(Aryl) ureido) benzamide] podophyllotoxins as DNA topoisomerase
I and IIα inhibitors and apoptosis inducing agents. Bioorg. Med. Chem. 2013;21:5198–
5208; (e) Kamal A, Reddy TS, Polepalli S, Paidakula S, Srinivasulu V, Reddy VG, Jain
N, Shankaraiah N. Synthesis and biological evaluation of 4-aza-2,3-
dihydropyridophenanthrolines as tubulin polymerization inhibitors. Bioorg. Med. Chem.
Lett. 2014;24:3356–3360; (f) Kamal A, Shankaraiah N, Prabhakar S, Reddy ChR,
Markandeya N, Reddy KL, Devaiah V. Solid-phase synthesis of new
pyrrolobenzodiazepine-chalcone conjugates: DNA-binding affinity and anticancer
activity. Bioorg. Med. Chem. Lett. 2008;18:2434–2439.
5. terHaar E, Rosenkranz HS, Hamel E, Day BW. Computational and molecular modeling
evaluation of the structural basis for tubulin polymerization inhibition by colchicine site
agents. Bioorg. Med. Chem. 1996;4:1659-1671; (b) Kamal A, Reddy TS, Vishnuvardhan
MVPS, Nimbarte VK, Rao AVS, Srinivasulu V, Shankaraiah N. Synthesis of 2-aryl-
26

1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization inhibitors and apoptosis
inducing agents. Bioorg. Med. Chem. 2015;23:4608–4623.
6. Rai SS, Wolf JJ. Localization of the vinblastine-binding site on beta-tubulin. J. Biol.
Chem. 1996;271:14707-14711.
7. Andreu JM, Barasaoin I. The interaction of baccatin III with the taxol binding site of
microtubules determined by a homogeneous assay with fluorescent taxoid. Biochemistry
2001;40:11975-11984.
8. Downing KH, Nogales E. Tubulin structure: insights into microtubule properties and
functions. Curr. Opin. Struct. Biol. 1998;8:785-791.
9. Downing KH. Structural basis for the interaction of tubulin with proteins and drugs that
affect microtubule dynamics. Annu. Rev. Cell. Dev. Biol. 2000;16:89-111.
10. Sorger PK, Dobles M, Tournebize R, Hyman AA. Coupling cell division and cell death to
microtubule dynamics. Curr. Opin. Cell Biol. 1997;9:807-814.
11. Sahu NK, Balbhadra SS, Choudhary J, Kohli DV. Exploring pharmacological
significance of chalcone scaffold: a review. Curr. Med. Chem. 2012;19:209-225.
12. Ni L, Meng CQ, Sikorski JA. Recent advances in therapeutic chalcones. Expert Opin.
Ther. Pat. 2004;14:1669-1691.
13. Batovska DI, Todorova IT. Trends in utilization of the pharmacological potential of
chalcones. Curr. Clin. Pharmacol. 2010;5:1-29.
14. Dimmock JR, Elias DW, Beazely MA, Kandepu NM. Bioactivities of chalcones. Curr.
Med. Chem. 1999;6:1125-1149.
15. (a) Mirzaei H, Emami S. Recent advances of cytotoxic chalconoids targeting tubulin
polymerization: Synthesis and biological activity. Eur. J. Med. Chem. 2016;121:610-639;
(b) Kamal A, Prabhakar S, Ramaiah MJ, Reddy PV, Reddy ChR, Mallareddy A,
Shankaraiah N, Reddy TLN, Pushpavalli SNCVL, Bhadra M-P. Synthesis and anticancer
activity of new chalcone-pyrrolobenzodiazepine conjugates linked via 1,2,3-triazole ring
side-armed alkane spacers. Eur. J. Med. Chem. 2011;46:3820–3831.
16. Go ML, Wu X, Liu XL. Chalcones: An Update on Cytotoxic and Chemoprotective
Properties. Curr. Med. Chem. 2005;12:483–499.
17. Ducki S. Antimitotic Chalcones and Related Compounds as Inhibitors of Tubulin
Assembly. Med. Chem. 2009;9:336–347.
27

18. Dyrager C, Wickstrom M, Friden-Saxin M, Friberg A, Dahlen K, Wallen EA, Gullbo J,
Grotli M, Luthman K. Inhibitors and promoters of tubulin polymerization: Synthesis and
biological evaluation of chalcones and related dienones as potential anticancer agents.
Bioorg. Med. Chem. 2011;19:2659–2665.
19. Ducki S, Rennison D, Woo M. Kendall A, Chabert JFD, McGown AT,
Lawrence NJ. Combretastatin-like chalcones as inhibitors of microtubule polymerization.
Part 1: Synthesis and biological evaluation of antivascular activity. Bioorg. Med. Chem.
2009;17:7698–7710.
20. Boumendjel A, Boccard J, Carrupt PA, Nicolle E, Blanc M, Geze A,
Choisnard L, Wouessidjewe D, Matera EL, Dumontet C. Antimitotic and antiproliferative
activities of chalcones: forward structure-activity relationship. J. Med. Chem.
2008;51:2307–2310.
21. (a) Shankaraiah N, Siraj KP, Nekkanti S, Srinivasulu V, Satish M, Sharma P, Senwar
KR, Vishnuvardhan MVPS, Ramakrishna S, Kamal A. Synthesis, DNA-Binding Affinity
and Cytotoxic Activity of New C3-Linked Chalcone-β-Carboline Hybrids. Bioorg. Chem.
2015;59:130–139; (b) Kumar NP, Nekkanti S, Kumari SS, Sharma P, Shankaraiah N,
Design and synthesis of 1,2,3-triazolo-phenanthrene hybrids as cytotoxic agents. Bioorg.
Med. Chem. Lett. 2017;27:2369–2376.
22. Kamal A, Srinivasulu V, Nayak VL, Sathish M, Shankaraiah N, Bagul C, Reddy NVS,
Rangaraj N, Nagesh N. Design and synthesis of C3-pyrazole/chalcone linked β-carboline
hybrids: Antitopoisomerase I, DNA interactive and apoptosis inducing anticancer agents.
ChemMedChem. 2014;9:2084–2098.
23. (a) Shankaraiah N, Jadala C, Nekkanti S, Senwar KR, Nagesh N, Shrivastava S, Naidu V
GM, Sathish M, Kamal A. Design and synthesis of C3-tethered 1,2,3-triazolo-β-carboline
derivatives: Anticancer activity, DNA-binding ability, viscosity and molecular modeling
studies. Bioorg. Chem. 2016;64:42–50; (b) Kumar NP, Sharma P, Reddy TS. Nekkanti S,
Shankaraiah N, Lalitha G, Sujanakumari S, Bhargava SK, Naidu VGM, Kamal A.
Synthesis of 2,3,6,7-tetramethoxyphenanthren-9-amine: An efficient precursor to access
new 4-aza-2,3-dihydropyridophenanthrenes as apoptosis inducing agents. Eur. J. Med.
Chem. 2017;127:305–317.
24. Shankaraiah N, Nekkanti S, Chudasama KJ, Senwar KR, Sharma P, Jeengar MK, Naidu
VGM, Srinivasulu V, Kamal A. Design, Synthesis and Anticancer Evaluation of
Tetrahydro-β-Carboline-hydantoin Hybrids. Bioorg. Med. Chem. Lett. 2014;24:5413–
5417.
28

25. Nekkanti S, Veeramani K, Kumari SS, Tokala R, Shankaraiah N. A Recyclable and
Water Soluble Copper(I)-Catalyst: One-pot Synthesis of 1,4-Disubstituted 1,2,3-Triazoles
and their Biological Evaluation. RSC Adv. 2016;6:103556–103566.
26. Kennedy SG, Kandel ES, Cross TK, Hay N. Akt/Protein kinase B inhibits cell death by
preventing the release of cytochrome c from mitochondria. Mol. Cell Biol.1999;19:5800–
5810.
27. Eruslanov E, Kusmartsev S. Identification of ROS using oxidized DCFDA and flow-
cytometry. Methods Mol Biol. 2010;594:57–72.
28. Lawrence NJ, McGown AT, Ducki S, Hadfield JA. The interaction of chalcones with
tubulin. Anticancer Drug Des. 2000;15:135–141.
29. Kanthou C, Greco O, Stratford A, Cook I, Knight R, Benzakour O, Tozer G.
The tubulin-binding agent combretastatin A-4-phosphate arrests endothelial cells in
mitosis and induces mitotic cell death. Am. J. Pathol. 2004;165:1401–1411.
30. Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, Knossow M. Insight
into tubulin regulation from a complex with colchicine and a stathmin-like domain.
Nature 2004;428:198–202.
29