Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretastatin conjugates as mitochondrial mediated apoptosis inducers

Article abstract of DOI:10.1016/j.bmc.2014.08.008

A series of 1,2,3-triazole linked aminocombretastatin conjugates were synthesized and evaluated for cytotoxicity, inhibition of tubulin polymerization and apoptosis inducing ability. Most of the conjugates exhibited significant anticancer activity against

Full text of DOI:10.1016/j.bmc.2014.08.008

Accepted Manuscript
Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretasta-
tin conjugates as mitochondrial mediated apoptosis inducers
Ahmed Kamal, Bajee Shaik, V. Lakshma Nayak, Burri Nagaraju, Jeevak
Sopanrao Kapure, M. Shaheer Malik, Thokhir Basha Shaik, B. Prasad
PII:
S0968-0896(14)00585-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.08.008
BMC 11756
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 April 2014
23 July 2014
11 August 2014
Please cite this article as: Kamal, A., Shaik, B., Lakshma Nayak, V., Nagaraju, B., Kapure, J.S., Shaheer Malik, M.,
Shaik, T.B., Prasad, B., Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretastatin conjugates
as mitochondrial mediated apoptosis inducers, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bmc.2014.08.008
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of 1,2,3-triazole linked
aminocombretastatin conjugates as mitochondrial mediated apoptosis
inducers
Ahmed Kamal,*a,b Bajee Shaik,a V. Lakshma Nayak,a Burri Nagaraju,a Jeevak Sopanrao
Kapure,a,b M. Shaheer Malik,a Thokhir Basha Shaik, B. Prasada
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
Abstract: A series of 1,2,3-triazole linked aminocombretastatin conjugates were synthesized and
evaluated for cytotoxicity, inhibition of tubulin polymerization and apoptosis inducing ability.
Most of the conjugates exhibited significant anticancer activity against some representative
human cancer cell lines and two of the conjugates 6d and 7c displayed potent cytotoxicity with
IC50 values of 53 nM and 44 nM against A549 human lung cancer respectively, and were
comparable to combretastatin A-4 (CA-4). SAR studies revealed that 1-benzyl substituted
triazole moiety with an amide linkage at 3-position of B-ring of the combretastatin subunit are
more active compared to 2-position. G2/M cell cycle arrest was induced by these conjugates 6d
and 7c and the tubulin polymerization assay (IC50 of 1.16 µM and 0.95 µM for 6d and 7c,
respectively) as well as immunofluorescence analysis showed that these conjugates effectively
inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Colchicine
competitive binding assay suggested that these conjugates bind at the colchicine binding site of
tubulin as also observed from the docking studies. Further, mitochondrial membrane potential,
ROS generation, caspase-3 activation assay, Hoechst staining and DNA fragmentation analysis
revealed that these conjugates induce cell death by apoptosis.
Keywords: CA-4, cell cycle, cytotoxicity, triazoles, tubulin polymerization.
∗,aCorresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189 (A.K.); e-mail:
ahmedkamal@iict.res.in (A. Kamal).
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1. Introduction
Tubulin, a globular protein, has emerged as one of the valuable molecular targets in anticancer
drug discovery. It exists as dimeric (α and β) subunits in the microtubules, which plays a pivotal
role in many cellular process such as maintenance of skeletal integrity of cell, cell signaling and
segregation of chromosomes during mitosis.1,2 Several chemical agents are known to target the
microtubule dynamics by binding to different domains of the tubulin protein and prevent the
polymerization or depolymerization of the microtubules resulting in mitotic spindle arrest3. In
nature, the bark of the South African tree Combretum caffrum contains cis stilbene derivatives
named as combrestatins, which are well known to effectively bind at the colchicine site of the
tubulin and inhibit the polymerization resulting in the arrest of cell proliferation.4 Combretastatin
A-4 (CA-4, 1) is the lead compound of this class with high cytotoxic potency against murine
lymphocytic leukemia, human ovarian and colon cancer cell lines. However it suffers from
limitation of poor aqueous solubility and to overcome this, prodrug CA-4P (2) and other
synthetic analogues have been developed, which are undergoing clinical trials.5-9 The structure
activity relationship studies of CA-4 suggested the importance of trimethoxy substitution on the
ring A and the crucial role played by cis-configuration of the olefic bond to impart activity to this
pharmacophoric structure has been well established. The ring B has been reported to be tolerant
to structural modification to enhance the pharmacological profile of the molecule. Particularly,
the aminocombretastatins such as AVE-8063(3), AVE-8062 (4) with an amino and amino-serine
groups replacing the hydroxyl group at the C-3 position of ring B showed potent antitubulin
activity and cytotoxicity.
In recent years 1,2,3-triazoles have emerged as attractive and desirable scaffolds in the
development of potential drug molecules in medicinal chemistry. Molecules with triazole
nucleus are known to display broad spectrum of biological properties such as antifungal,
anticancer,10-13 anti-allergic, antibacterial, anti-HIV, anticonvulsant, anti-inflammatory and
antitubercular activities. Interestingly, triazole hybrids of some biologically important natural
products14-18 have been developed and recently N-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)arylamide (5) have been evaluated as antimicrotubule agents against selective cancer
cell lines.19
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<Insert Figure 1>
We have been involved in the design and synthesis of new heterocyclic based compounds as
potential anticancer agents that could target the tubulin polymerization process.20 In the present
work, we have explored the structural modification of the ring B of CA-4 by incorporating the
triazole moiety on different positions via an amide linkage. These conjugates are evaluated for
their cytotoxic activity and studied for the mechanistic aspects which include the inhibition of
tubulin polymerization and apoptosis induction.
2. Results and Discussions
2.1. Chemistry.
The synthesis of these triazole linked combretastatin conjugates (6a-e, 7a-e, and 8a-e) was
accomplished as illustrated in Schemes 1 and 2. The synthesis of conjugates with amino group at
C-3 of ring B (6a-e and 7a-e) started with a double bond forming Wittig reaction between the
nitro benzaldehydes (10, 11) and 3,4,5-trimethoxybenzyl triphenylphosphonium bromide (9) in
presence of sodium hydride in anhydrous dichloromethane to provide a mixture of nitro Z-
stilbenes (12,13) and E-stilbenes (14,15) as depicted in Scheme 1. The starting materials (9-11)
were prepared by a standard protocol as reported in the literature.21 The nitro Z-stilbenes (12, 13)
were subjected to reduction with zinc-ammonium formate in methanol to give the corresponding
amino Z-stilbenes (16,17). These amino stilbenes were coupled with different substituted benzyl-
1H-1,2,3-triazole-4-carboxylic acids (18a-e)22 using 3-ethyl-1(N,N-dimethyl)aminopropyl
carbodiimide (EDCI) /1-hydroxybenzotriazole (HOBT) in dimethyl formamide to afford the
desired conjugates (6a-e and 7a-e).
<Insert scheme 1>
The desired conjugates with amino group at C-2 of ring B (8a-e) were synthesized following a
similar synthetic route as shown in Scheme 2. In this route, a silyl protected nitrobenzaldehyde
(19) prepared from a standard literature protocol23 was employed as the starting material. A
double bond was formed employing phosphonium bromide 9 and aldehyde 19 to afford a
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mixture of nitro Z-stilbenes (20) and E-stilbenes (21), this was followed by the reduction of nitro
functionality of Z isomer 20 to provide the amino stilbenes 22. This was coupled with different
substituted carboxylic acids (18a-e) and followed by deprotection of silyl group with tetra-n-
butylammonium fluoride to afford the desired conjugates 8a-e.
<Insert scheme 2>
The key benzyl-1H-1,2,3-triazole-4-carboxylic acids (18a-e) intermediates with different
substitution were synthesized with a standard copper (I) catalyzed azide-alkyne cycloaddition
protocol yielding 1,4-adduct exclusively (Scheme 3).
<Insert scheme 3>
3. Biology
3.1 Cytotoxicity
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay24 was
performed to evaluate the cytotoxic potential of these compounds against selected human cancer
cell lines which include HeLa (cervical) HepG2 (liver) A549 (lung), ACHN (renal) and MCF-7
(breast). The results are summarized as IC50 values in Table 1 and are compared with CA-4. The
assay revealed that these compounds showed significant cytotoxic activity with IC50 values
ranging from 0.044-9.5 µM against different cancer cell lines. The most active compounds 6d
and 7c with triazole moiety at the 3-position of B-ring exhibited promising antiproliferative
activity with IC50 values ranging from 0.053 to 1.659 µM and 0.044 to 1.698 µM, respectively.
Particularly the compound 6d and 7c exhibited remarkable cytotoxicity comparable to CA-4 in
A549 cell line with IC50 values of 0.053 and 0.044 µM, respectively.
The electron donating alkoxy groups are present in many naturally occurring and
synthetic tubulin inhibitors and apoptosis inducers which are reported as anticancer agent. In this
study some selected substitutions, which includes 3 and 4-methoxy, 3-phenoxy, 3,4,5-trimethoxy
and an electron withdrawing 4-fluoro, were employed on the basis of literature precedence.19
SAR studies have revealed that the conjugates 6a-e, 7a-e with triazole moiety attached as amide
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at C-3 position of B-ring of CA-4 exhibited significantly enhanced activity than conjugates 8a-e
with triazole moiety attached at C-2 position. In addition the para substitution on the benzyl ring
of the 1,2,3-triazole moiety was crucial in imparting more potency to these conjugates.
<Insert Table 1>
3.2 Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combined
effect of both cycle block and apoptosis.25,26 Furthermore regulation of the cell cycle and
apoptosis are considered to be effective cancer therapeutic methods.27 The in vitro screening
results revealed that compounds, 6d and 7c showed significant antiproliferative activity against
lung cancer cell line A549. Therefore, it was considered of interest to understand whether this
inhibition of cell growth was on account of cell cycle arrest. In this study A549 cells were treated
with compounds 6d and 7c at concentrations of 25 and 50 nM for 48 h. The data obtained clearly
indicated that these compounds arrested cell cycle at G2/M phase as compared to the untreated
control. CA-4 was used as reference compound in this study. These compounds (6d and 7c)
showed 17.57 and 21.37 % of cell accumulation in G2/M phase respectively at 25 nM
concentration, whereas it exhibited 25.73 and 26.63 % of cell accumulation in G2/M phase at 50
nM concentration (Figure 2). Interestingly, at higher concentration both the compounds exhibited
G2/M cell cycle arrest comparable to the standard CA-4 (26.48 %)
<insert Figure 2>
<insert Table 2>
3.3 Effect of compounds on tubulin polymerization
In general G2/M cell cycle arrest is strongly associated with inhibition of tubulin
polymerization28 and since compounds 6d and 7c cause cell cycle arrest at G2/M cell cycle
arrest, their effect on microtubule inhibitory function was investigated. Tubulin subunits are
known to heterodimerize and self-assemble to form microtubules in a time dependent manner.
The progression of tubulin polymerization29,30 was thus examined by monitoring the increase in
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fluorescence emission at 420 nm (excitation wavelength is 360 nm) in 384 well plate for 1 h at
o
37 C with and without the conjugates at 3 µM concentration in comparison with reference
compound CA-4. The test compounds (6d and 7c) efficiently inhibited tubulin polymerization by
66.82 and 70.86 %, respectively, comparable to 70.51 % inhibition exhibited by CA-4 (Figure 3).
This was followed by evaluation of IC50 values for these compounds and results are shown in
Table 3. The compound 7c, which displayed higher tubulin-assembly inhibition was more potent
with an IC50 value of 0.95 µM and was superior to standard CA-4 (IC50=1.08 µM). The effect of
these compounds on the inhibition of tubulin assembly correlated well with their strong
antiproliferative activity.
<Insert Figure 3>
<Insert Table 3>
3.4 Competitive colchicine binding assay
Compounds 6d and 7c showed similar inhibitory effects on tubulin polymerization comparable
to CA-4 and this prompted us to investigate whether these compounds bind to the colchicine site
of tubulin as CA-4 by fluorescence based assay. Tubulin (3 µM) was incubated with the various
concentrations of compounds 6d and 7c (0-20 µM) in the presence of colchicine (3 µM) with
o
CA-4 as positive control at 37 C for 60 min and the fluorescence of tubulin-colchicine complex
was monitored at 435 nm when excited at 350 nm. We observed an increase in fluorescence of
tubulin-colchicine complex in the presence of tested CA-4 analogues (6d and 7c) and CA-4. This
is in the agreement with a previous investigation in which CA-4 is reported to give fluorescence
upon binding at the colchicine site.31a, b Therefore, the experiment was carried out both in the
presence and absence of colchicine to obtain fluorescence values of the desired tubulin-
colchicine complex, the fluorescence values of tubulin-test compounds complex was subtracted
from the tubulin-test compounds-colchicine complex. It was observed that at low concentrations
the test compounds (6d and 7c) and CA-4 showed significant affinity towards colchicine site,
whereas at higher concentration 7c showed a steady increase while 6d and CA-4 showed lower
in the binding affinity. Compared to the positive control CA-4, compound 7c exhibited more
affinity towards colchicine site whereas 6d has less affinity. Taxol was used as a negative
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control, which is known to bind at different site and shows no effect on tubulin-colchicine
complex. Therefore this study indicates that 6d and 7c bind at the colchicine site on tubulin.
<insert Figure 4>
3.5 Immunohistochemistry (IHC) studies on tubulin
In addition to in vitro tubulin polymerisation studies, we investigated alterations in the
microtubule network induced by conjugates 6d and 7c in A549 cell culture by
immunofluoresence microscopy, as most antimitotic agents affect microtubules.32 Therefore,
A549 cells were treated with these compounds at 25 nM concentration for 48 h. The test results,
demonstrated a well organized microtubular network in control cells, however cells treated with
these compounds (6d, 7c and CA-4) showed disrupted microtubule organization as seen in
Figure 4, thus demonstrating the inhibition of tubulin polymerization.
. <insert Figure 5>
3.6 Hoechst staining for the effect on apoptosis
Disruption of microtubule formation leads to cell-cycle arrest in the G2/M phase, followed by
apoptotic cell death.33 Apoptosis is one of the major pathways that lead to the process of cell
death. Chromatin condensation, nuclear shrinking and fragmented nuclei are known as classic
characteristics of apoptosis. In the present investigation the apoptotic inducing effect of
compounds 6d and 7c was studied by Hoechst staining (H 33258) in A549 cancer cell line.
Therefore cells were treated with these compounds at 25 nM concentration for 48 h and the
results revealed typical apoptotic characteristics, including condensation of chromatin and
appearance of nuclear fragmentation and apoptotic bodies. These results clearly demonstrated
that compounds 6d and 7c were also effective in inducing cellular apoptosis (Figure 5).
<insert Figure 6>
3.7 Measurement of Mitochondrial Membrane Potential (∆Ψm)
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The maintenance of mitochondrial membrane potential (∆Ψm) is significant for mitochondrial
integrity and bio energetic function.34 Mitochondrial changes, including loss of mitochondrial
membrane potential (∆Ψm), are key events that take place during drug-induced apoptosis. To
determine the changes in mitochondrial membrane potential (∆Ψm), we examined the lipophilic
dye JC-1, specific for mitochondria. Mitochondria that maintain normal mitochondrial
membrane potential (∆Ψm) concentrate JC-1 into aggregates indicates red fluorescence.
However, JC-1 forms monomers (green fluorescence) in depolarized mitochondria. A549 cells
were treated with compounds 6d and 7c at 25 nM concentration and incubated for 48 h. After 48
h of treatment with these conjugates, it was observed that decrease in the mitochondrial
membrane potential (∆Ψm) when compared to untreated control (Figure 6)
<insert Figure 7>
3.8 Effect of 6d and 7c on intracellular ROS generation
Generation of intracellular reactive oxygen species (ROS) is considered one of the key mediators
of apoptotic signaling for the most anti tumor agents.35 In order to demonstrate the role of test
compounds on ROS generation (6d and 7c) during apoptosis process, production of ROS was
examined by using an oxidant-sensitive fluorescent probe, DCFDA (2',7'dichlorofluorescin
diacetate). The results showed that treatment with test compounds at 25 nM concentration
significantly increased the intensity of the DCF signal when compared to control cells (Figure
7).
<insert Figure 8>
3.9 Effect on activation of caspase 3
From literature precedence, it is well established that molecules affecting microtubule
polymerization cause mitotic arrest and ultimately lead to apoptosis.36 Caspases, are a family of
cysteine-aspartic proteases that are crucial mediators of apoptosis. Among them, caspase-3 is the
most studied in the mammalian caspases in terms of its specificity role in apoptosis.37 Some
reports38,39 indicated that the cell cycle arrest at G2/M phase takes place by the induction of
cellular apoptosis and in the present study an investigation to understand the correlation of
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cytotoxicity and apoptosis by compounds 6d and 7c was undertaken. A549 cells were treated
with 6d and 7c at 25 and 50 nM concentrations for 48 h. CA-4 (50 nM) was used as reference
compound in this study and examined for the activation of caspase-3 activity. Results indicate
that there was nearly 3 to 7-fold induction in caspase-3 levels compared to the control and
compound 6d exhibited superior caspase activation compared to CA-4 (Figure 8).
<insert Figure 9>
3.10 DNA Fragmentation analysis
Apoptosis is a programmed cell death, characterized with chromatin condensation and
internucleosomal DNA fragmentation.40 DNA fragmentation is well known and a typical
biochemical hallmark of apoptotic cell death. During apoptosis DNA is cleaved into small
fragments by endonucleases and these fragments can be observed by gel electrophoresis as
ladders. In this context, the endonucleolytic DNA cleavage was checked by agarose gel
electrophoresis to further confirm 6d and 7c induced cell apoptosis. A549 cells were treated with
these compounds at 25 nM concentration for 48 h. After treatment it was observed that
compounds 6d and 7c produced significant DNA fragmentation, which is indicative of apoptosis
(Figure 9).
. <insert Figure 10>
4. Molecular modeling studies:
A molecular modeling study was performed to explain the binding mode of conjugates 6d and 7c
within tubulin protein domains to correlate the polymerization inhibitory activities of these
compounds (Figure 10). Coordinates of protein structure of tubulin-colchicine was obtained
from the Protein Data Bank (PDB ID 3E22). Docking was accomplished into the colchicine
binding sites of tubulin using AutoDock 4.2 software and the important interactions are
discussed. Results showed that the triazole ring of compound 6d exhibited important
hydrophobic interaction with αSER178, αTYR224. Carbonyl oxygen of amide group present
between ring B and triazole ring establishes hydrophobic interaction with αThr179. The ring A
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(trimethoxyphenyl group) buried in a hydrophobic pocket interacted with the βAla250, βLys254,
βLeu255, βLys352 residues similar to that of trimethoxyphenyl group of colchicine and ring B
binded with αVal181, βALA250, αAsn258, αAsn101 residues. The fluorobenzyl group on
triazole ring shows hydrophobic interaction with αSer178, αVal177, and αTyr224. Similar to 6d
carbonyl oxygen of amide of conjugate 7c establishes hydrophobic interaction through a
different residue βThr353. Methoxybenzyl group on triazole ring is involved in important
hydrophobic interaction with αAsn101 and βLys254, whereas the ring A of conjugate 7c
occupies the same position as the corresponding moiety of the colchicine analogue. Ring B
shows hydrophobic interaction with βLys352 and βLeu255, where as the triazole ring of 7c
establishes hydrophobic interaction with αSer178 and βLeu255. The molecular docking
simulations suggest that the conjugates 6d and 7c interact with αβ tubulin interface in the
colchicine binding as pocket seen from the colchicine competitive binding assay. <Insert Figure
11>
5. Conclusion
In conclusion, a new series of 1,2,3-triazole linked aminocombretastatin conjugates (6a-e to 8a-
e) have been synthesized and evaluated for their anticancer potential against selected human
cancer cell lines. All the synthesized compounds showed potent anticancer activity against the
tested cancer cell lines and two of them (6d and 7c) showed IC50 values 0.053 and 0.044 µM,
respectively, against A549 cancer cell line. Flow cytometric analysis of these compounds
showed the arrest of the cell cycle in the G2/M phase leading to caspase-3 dependent apoptotic
cell death. These active compounds (6d and 7c) showed potent inhibition of tubulin
polymerization with IC50 values 1.16 and 0.95 µM, respectively. The immunohistochemistry
study of tubulin showed that the level of tubulin polymerization inhibition was comparable to
that of CA-4 for the compounds 6d and 7c. The colchicine competitive binding assay and
docking studies clearly indicate that these conjugates bind at colchicine binding site of the
tubulin. Further, Hoechst staining, loss of mitochondrial membrane potential, significant
activation and cleavage of caspase-3 activity, DNA fragmentation analysis and the production of
reactive oxygen species (ROS) suggested that cytotoxic efficacy of these compounds is mainly
due to inhibition of tubulin polymerization and apoptosis. The results demonstrate that
compounds 6d and 7c are potential leads and could be taken up for further evaluation as in the
development of tubulin polymerization inhibitors.
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6. Experimental protocol
All chemicals and reagents were obtained from Aldrich (Sigma– Aldrich, S t. Louis, MO, USA),
Lancaster (AlfaAesar, Johnson Matthey Company, Ward Hill, MA, USA) or Spectrochem Pvt.
Ltd (Mumbai, India) and were used without further purification. Reactions were monitored by
TLC, performed on silica gel glass plates containing 60 G F -254, and visualization was achieved
by UV light or iodine indicator. Column chromatography was performed with Merck 60–120
1
mesh silica gel. H spectra were recorded on AVANCE-300 (300 MHz) or JCAMP (300/500
MHz) instruments. Chemical shifts (δ) were reported in ppm with TMS as internal standard. EI-
MS spectra were recorded on Micro mass, Quattro LC using ESI+ software with capillary
voltage 3.98 kV and ESI mode positive ion trap detector. High -resolution mass spectra (HRMS)
were recorded on QSTAR XL Hybrid MS /MS mass spectrometer. The purity of tested
compounds was ≥95% as determined by HPLC performed on a Shimadzu LCMS-2020
apparatus equipped with a SPD-M20A diode array detector and a Shimadzu SIL-20AC auto
injector using C18 column (Phenomenex luna 5µm C18, 4.6 mm × 250 mm column). Elution
conditions: mobile phase A(75%)-acetonitrile; mobile phase B(25%)-water containing 0.1%
formic acid + 10 mmol NH4OAc. The flow rate was 1.0 ml/min and the injection volume was 5
µl at 25 °C and detection at 254 nm.
6.1 Procedure for the synthesis of nitro Z-stilbenes (12, 13, 20)
To a stirred solution of nitrobenzaldehydes (10, 11, 19, 1 mmol) and 3,4,5-trimethoxybenzyl
triphenylphosphonium bromide (9, 1.1 mmol) in anhydrous dichloromethane, sodium hydride (4
o
mmol) was added at 0 C. The reaction mixture was stirred at room temperature for 18 h and
monitored by TLC. After completion of the reaction appropriate amount of water, until foaming
stopped, was added at 0 oC. The organic layer was separated and the aqueous layer was extracted
with chloroform. The combined organic layers were washed with brine solution, dried over
anhydrous Na2SO4, filtered and the solvent was removed under reduced pressure to get crude
compounds, nitro Z-and E-stilbenes isomeric mixture. The isomers were separated by using
column chromatography to give pure Z- stilbenes.
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1
6.1.1 (Z)-1,2,3-Trimethoxy-5-(4-methoxy-3-nitrostyryl)benzene (12): bright yellow solid. H
NMR (300 MHz, CDCl3) δ (ppm); 7.78 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 2.1, 8.4 Hz, 1H), 6.95
(d, J = 8.4 Hz, 1H), 6.58 (d, J = 12.8 Hz, 1H), 6.48 (s, 2H), 6.45 (d, J = 12.8 Hz, 1H), 3.95 (s,
3H), 3.84 (s, 3H), 3.72 ( s, 6H); MS (ESI, m/z): 346 [M+1]+.
6.1.2 (Z)-5-(3,4-Dimethoxy-5-nitrostyryl)-1,2,3-trimethoxybenzene(13): bright yellow solid,
1H NMR (300 MHz, CDCl3) δ (ppm) 7.21 (s, 1H), 6.93 (s, 1H), 6.56 (d, J = 11.8 Hz, 1H), 6.43
(s, 2H), 6.40 (d, J = 11.8 Hz, 1H), 3.91 (s, 3H), 3.79 (s, 3H), 3.71 (s, 6H), 3.66 (s, 3H); MS (ESI,
m/z): 376 [M+1]+.
6.1.3(Z)-tert-Butyl(6-methoxy-2-nitro-3-(3,4,5-trimethoxystyryl)phenoxy)dimethylsilane
(20): yellow liquid; 1H NMR (300 MHz, CDCl3) δ (ppm); 6.77 (s, 2H), 6.61 (d, J = 12.0 Hz, 1H),
6.41 (s, 2H), 6.38 (d, J = 12.0 Hz, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.67 (s, 6H), 0.94 (s, 9H), 0.21
(s, 6H). MS (ESI, m/z): 476 [M+1]+.
6.2 Procedure for the synthesis of amino Z-stilbenes (16, 17, 22)
Zinc powder (3 mmol) and ammoniumformate (3mmol) were added portion wise to a solution of
nitro Z-stilbenes (12, 13, 20, 1 mmol) in methanol at 0 °C and the reaction mixture was stirred at
room temperature for 12 hrs. After completion of reaction, the reaction mixture was filtered to
remove the residual zinc and the solvent was evaporated under reduced pressure. The residue
was redissolved in ethyl acetate and washed with water, brine solution and dried over anhydrous
Na2SO4. The resulting solution was filtered and the solvent was removed under reduced pressure
followed by the purification of residue by column chromatography using ethyl acetate and
hexane as eluents to give pure products.
1
6.2.1 (Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)aniline (16): H NMR (300 MHz, CDCl3.) δ
(ppm); 6.70 (s, 1H), 6.67 (s, 2H), 6.55 (s, 2H), 6.47 (d, J = 12.0 Hz, 1H,), 6.36 (d, J = 12.0 Hz,
1H,), 3.84 (s, 3H), 3.82 (s, 3H), 3.70 (s, 6H,); MS (ESI, m/z): 316 [M+1]+.
6.2.2 (Z)-2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)aniline (17): 1H NMR (300 MHz, CDCl3) δ
(ppm); 6.47 (s, 2H), 6.39 (d, J = 12.0 Hz, 1H), 6.36 (d, J = 12.0 Hz, 1H), 6.27 (d, J = 1.5 Hz,
12

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1H), 6.19 (d, J = 1.5 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 3.63 (s, 6H), 3.59 (s,3H); MS (ESI,
m/z): 346 [M+1]+.
6.2.3 (Z)-2-(tert-Butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)aniline (22): 1H
NMR (300 MHz, CDCl3) δ (ppm); 6.70 (d, J = 8.3 Hz, 1H), 6.51 (s, 2H), 6.47 (d, J = 12.0 Hz,
1H), 6.43 (d, J = 12.0 Hz ,1H), 6.27 (d, J = 8.3 Hz, 1H), 3.81 (brs, 2H), 3.81 (s, 3H), 3.76 (s,
3H), 3.63 (s, 6H), 1.00 (s, 9H), 0.17 (s, 6H), MS (ESI, m/z): 446 [M+1]+.
6.3 Procedure for the synthesis of benzyl-1H-1,2,3-triazole-4-carboxylic acids (18a-e)
Appropriate azide (1 mmol), sodium ascorbate (0.1mmol) and copper sulfate pentahydrate (0.01
mmol) were added sequentially to a solution of propiolic acid (1 mmol), in tert-butanol and
water (1:1). The resulting reaction mixture was stirred at room temperature for 12h. The
precipitate formed was filtered and washed with water to yield pure products.
1
6.3.1 1-(3-Methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (18a): H NMR (300 MHz,
DMSO-d6) δ (ppm); 8.26 (s, 1H), 7.29 (t, J = 7.8 Hz, 1H), 6.90-6.85 (m, 3H), 5.57 (s, 2H), 3.78
(s, 3H); MS (ESI, m/z): 234 [M+1]+.
1
6.3.2 1-(3-Phenoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (18b): H NMR (300 MHz,
DMSO-d6) δ (ppm); 8.27 (s, 1H), 7.37-7.30 (m, 3H), 7.12 (t, J = 7.3 Hz, 1H), 7.03-6.93 (m, 5H),
5.58 (s, 2H); MS (ESI, m/z): 296 [M+1]+.
1
6.3.3 1-(4-Methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (18c): H NMR (300 MHz,
DMSO-d6) δ (ppm); 8.29 (s, H), 7.32 (d, J = 7.8 Hz, 2H), 6.93 (d, J = 7.8 Hz, 2H), 5.56 (s, 2H),
3.75 (s, 3H); MS (ESI, m/z): 234 [M+1]+.
1
6.3.4 1-(4-Fluorobenzyl)-1H-1,2,3-triazole-4-carboxylic acid (18d): H NMR (300 MHz,
DMSO-d6) δ (ppm); 8.27 (s, 1H), 7.38-7.33 (m, 2H), 7.07 (t, J = 8.5 Hz, 2H), 5.60 (s, 2H); MS
(ESI, m/z): 222 [M+1]+.
13

ACCEPTED MANUSCRIPT
1
6.3.5 1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (18e): H NMR (300
MHz, DMSO-d6) δ (ppm); 8.25 (s, 1H), 6.59 (s, 2H), 5.52 (s, 2H), 3.83 (s, 6H), 3.79 (s, 3H); MS
(ESI, m/z): 294 [M+1]+.
6.4 General procedure for the synthesis of triazole linked aminocombretastatins (6a-e, 7a-e)
To a solution of substituted benzyl-1H-1,2,3-triazole-4-carboxylic acids (18a-e, 1 mmol) in dry
dimethylformamide, EDCI (1.2 mmol) and HOBT (1.2mmol) were added and the reaction
mixture was stirred for 20 min. To the reaction mixture, amino Z-stilbenes (16, 17, 1 mmol) was
added and stirred at room temperature for 12 h. The solvent was removed and the residue was
dissolved in dichloromethane (50 mL), washed with 5% HCl (2 x 20 mL), 5% NaHCO3 (2 x 20
mL), and brine solution (20 mL). The organic layer was separated, dried over anhydrous Na2SO4,
filtered, and the solvent was removed to give a crude product which was purified by
chromatography (ethyl acetate/hexanes) to give the desired product (6a-e, 7a-e).
6.4.1 (Z)-N-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(3-methoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (6a)
The compound 6a was prepared according to the general procedure by employing 1-(3-
methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (74 mg, 0.32 mmol) and (Z)-2-methoxy-5-
(3,4,5-trimethoxystyryl)aniline (100 mg, 0.32 mmol) as white solid. Yield 117 mg (69%); m.p:
83-85 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.49 (s, 1H), 8.43 (s, 1H), 8.02 (s, 1H), 7.31 (t, J
= 7.9 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.93-6.86 (m, 2H), 6.82-6.75 (m, 2H), 6.56-6.52 (m,
3H), 6.42 (d, J = 12.0 Hz, 1H), 5.54 (s, 2H), 3.91 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.68 (s, 6H);
13C NMR (75 MHz, CDCl3) δ (ppm): 160.1, 157.4, 152.7, 147.4, 144.0, 135.0, 132.7, 130.3,
129.9, 129.9, 129.0, 126.9, 125.5, 125.5, 124.5, 120.6, 120.2, 120.2, 114.4, 113.8, 109.5, 105.9,
60.8, 55.7, 55.2, 54.4; IR (KBr): ῡmax/cm-1 = 3350, 3007, 2950, 1652, 1566, 1490, 1448, 1325,
1250, 1125, 1078, 1042, 1001, 855; MS (ESI, m/z): 531 [M+1]+; HRMS (ESI, m/z) Calculated
for, C29H31O6N4: 531.22337; found: 531.22373 [M+1]+. HPLC: tR 6.21 min, purity 98.6%.
6.4.2 (Z)-N-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(3-phenoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (6b)
14

ACCEPTED MANUSCRIPT
The compound 6b was prepared according to the general procedure by employing 1-(3-
phenoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (94 mg, 0.32 mmol) and (Z)-2-methoxy-5-
(3,4,5-trimethoxystyryl)aniline (100 mg, 0.32 mmol) as reddish solid. Yield: 123 mg (65%); m.p;
159-161 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.48 (s, 1H), 8.43 (s, 1H), 8.03 (s, 1H), 7.38-
7.29 (m, 3H), 7.14 (d, J = 7.5 Hz, 1H), 7.04-6.93 (m, 6H), 6.74 (s, 1H), 6.56 (d, J = 12.2 Hz,
13
1H), 6.52 (s, 2H), 6.46 (d, J = 12.2 Hz, 1H), 5.54 (s, 2H), 3.91 (s, 6H), 3.68 (s, 6H); C NMR
(125 MHz, CDCl3) δ (ppm): 158.2, 156.2, 153.3, 152.8, 147.5, 144.1, 135.5, 132.7, 130.6, 129.9,
129.6, 129.1, 125.5, 124.6, 123.9, 122.5, 120.7, 119.3, 118.8, 118.1, 117.2, 110.1, 109.6, 105.9,
103.2, 60.8, 56.0, 55.8, 54.2, IR (KBr): ῡmax/cm-1 = 3373, 3136, 2931, 1683, 1585, 1560, 1528,
1485, 1455, 1326, 1252, 1212, 1125, 1041, 1007, 964, 899, 874, 821; MS (ESI , m/z): 593
[M+1]+. HPLC: tR 9.52 min, purity 95.2%.
6.4.3 (Z)-N-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(4-methoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (6c)
The compound 6c was prepared according to the general procedure by employing 1-(4-
methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (94 mg, 0.32 mmol) and (Z)-2-methoxy-5-
(3,4,5-trimethoxystyryl)aniline (100 mg, 0.32 mmol) as dark brown solid. Yield: 115 mg (68%);
1
m.p: 85-87 °C; H NMR (500 MHz, CDCl3) δ (ppm): 9.48 (s, 1H), 8.43 (s, 1H), 7.97 (s, 1H),
7.26 (d, J = 7.7 Hz, 2H), 7.02 (d, J = 8.4 Hz, 1H), 6.92-6.80 (m, 2H), 6.77 (d, J = 8.2 Hz, 1H),
6.55-6.52 (m, 3H), 6.43 (d, J = 12.0 Hz, 1H), 5.50 (s, 2H), 3.91 (s, 3H), 3.82 (s, 6H), 3.67 (s,
6H); 13C NMR (75 MHz, CDCl3) δ (ppm): 160.1, 157.5, 152.7, 147.4, 143.8, 136.9, 132.7,
129.9, 129.7, 129.6, 129.0, 126.9, 125.3, 124.5, 120.6, 114.5, 109.5, 105.9, 103.2, 60.8, 56.0,
55.7, 55.2, 54.0; IR (KBr): ῡmax/cm-1 = 3369, 3137, 2995, 2940, 2836, 1666, 1608, 1565, 1527,
1484, 1430, 1415, 1327, 1252, 1177, 1119, 1045, 1033, 894, 877, 845; MS (ESI, m/z): 531
[M+1]+; HRMS (ESI, m/z) Calculated for, C29H31O6N4: 531.22364; found: 531.22381 [M+1]+.
HPLC: tR 7.64 min, purity 96.8%.
6.4.4 (Z)-1-(4-Fluorobenzyl)-N-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1H-1,2,3-
triazole-4-carboxamide (6d)
The compound 6d was prepared according to the general procedure by employing 1-(4-
fluorobenzyl)-1H-1,2,3-triazole-4-carboxylic acid (70 mg, 0.32 mmol) and (Z)-2-methoxy-5-
15

ACCEPTED MANUSCRIPT
(3,4,5-trimethoxystyryl)aniline (100 mg, 0.32 mmol) as brown red solid. Yield: 117 mg (71%);
1
m.p: 85-87 °C; H NMR (500 MHz, CDCl3) δ (ppm): 9.48 (s, 1H), 8.42 (s, 1H), 8.03 (s, 1H),
7.31 (t, J = 8.5 Hz, 2H), 7.02 (t, J = 8.5 Hz, 2H), 7.03 (d, J = 8.5 Hz, 1H), 6.76 (d, J = 8.5 Hz,
1H), 6.52 (d, J = 12.0 Hz, 1H), 6.52 (s, 2H), 6.43 (d, J = 12.0 Hz, 1H), 5.55 (s, 2H), 3.91 (s, 3H),
3.82 (s, 3H), 3.68 (s, 6H); 13C NMR (75 MHz, CDCl3) δ (ppm): 157.4, 152.8, 147.5, 144.1,
137.0, 132.7, 130.2, 130.0, 129.6, 129.1, 127.7, 126.9, 125.4, 124.7, 120.7, 116.4, 116.1, 109.6,
106.0, 103.3, 60.8, 56.0, 55.8, 53.8; IR (KBr): ῡmax/cm-1 = 3378, 3133, 2937, 2835, 1681, 1583,
1560, 1508, 1459, 1431,1326, 1231, 1183, 1157, 1125, 1041, 1007, 900, 874, 850; MS (ESI,
m/z): 541 [M+Na]+; HRMS (ESI, m/z) Calculated for, C28H27O5N4FNa: 541.18598; found:
541.18585 [M+Na]+. HPLC: tR 7.90 min, purity 95.3%.
6.4.5 (Z)-N-(2-Methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(3,4,5-trimethoxybenzyl)-1H-
1,2,3-triazole-4-carboxamide (6e)
The compound 6e was prepared according to the general procedure by employing 1-(3,4,5-
trimethoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (93 mg, 0.32 mmol) and (Z)-2-methoxy-
5-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.32 mmol) as red solid. Yield: 126 mg (67%); m.p:
90-92 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.49 (s, 1H), 8.49 (s,1H), 8.06 (s, 1H),7.02 (d, J
= 8.4 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.62-6.52 (m, 5H), 6.44 (d, J = 12.0 Hz, 1H), 5.49 (s,
2H), 3.91 (s, 3H), 3.84 (s, 9H), 3.82 (s, 3H), 3.67 (s, 6H); 13C NMR (75 MHz, CDCl3 + DMSO-
d6) δ (ppm): 156.3, 152.3, 151.5, 146.3, 142.9, 131.3, 128.8, 128.6, 128.3, 127.9, 126.9, 125.7,
125.1, 123.4, 118.9, 117.7, 108.7, 104.9, 104.5, 59.3, 54.9, 54.8, 54.6, 53.2; IR (KBr): ῡmax/cm-1
= 3379, 2937, 1682, 1586, 1559, 1528, 1505, 1459, 1425, 1385, 1327, 1239, 1186, 1156, 1126,
1004, 901; MS (ESI, m/z): 591 [M+1]+; HRMS (ESI, m/z) Calculated for; C31H35O8N4:
591.24565; found : 591.24560 [ M+1]+. HPLC: tR 5.35 min, purity 95.6%.
6.4.6 (Z)-N-(2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(3-methoxybenzyl)-1H-
1,2,3-triazole-4-carboxamide (7a)
The compound 7a was prepared according to the general procedure by employing 1-(3-
methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (67 mg, 0.29 mmol) and (Z)-2,3-dimethoxy-
5-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.29 mmol) as light orange solid. Yield: 112 mg
(69%); m.p :120-122 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.56 (s, 1H), 8.06-8.04 (m, 2H),
16

ACCEPTED MANUSCRIPT
7.41-7.33 (m, 3H), 6.90 (t, J = 7.5 Hz, 1H), 6.63 (s, 1H), 6.55 (d, J = 12.2 Hz, 1H), 6.52 (s, 2H),
6.50 (d, J = 12.2 Hz, 1H), 5.54 (s, 2H), 3.92 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.69 (s, 6H), 3.61
13
(s, 3H); C NMR (125 MHz, CDCl3) δ (ppm): 161.6, 160.1, 152.8, 151.3, 143.8, 137.0, 135.0,
133.1, 132.6, 131.4, 130.4, 129.8, 126.4, 125.6, 120.4, 114.5, 113.9, 113.5, 111.4, 108.4, 106.0,
60.9, 60.8, 55.9, 55.6, 55.3, 54.5; IR (KBr): ῡmax/cm-1 = 3290, 3098, 1670, 1607, 1553, 1452,
1384, 1271, 1231, 1187, 1157, 1129, 1104, 985, 863; MS (ESI, m/z) : 561 [M+1]+; HRMS (ESI,
m/z) Calculated for C30H33O7N4: 561.23507; found: 561.23530 [M+1]+. HPLC: tR 7.76 min,
purity 95.8%.
6.4.7 (Z)-N-(2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(3-phenoxybenzyl)-1H-
1,2,3-triazole-4-carboxamide (7b)
The compound 7b was prepared according to the general procedure by employing 1-(3-
phenoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (85 mg, 0.29 mmol) and (Z)-2,3-dimethoxy-
5-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.29 mmol) to obtained cream white solid. Yield :117
1
mg (65%); m.p: 135-137 °C; H NMR (500 MHz, CDCl3) δ (ppm): 9.56 (s, 1H), 8.07 (s, 1H),
8.06 (s, 1H), 7.37-7.32 (m, 3H), 7.14 (t, J = 7.10 Hz, 1H), 7.10-6.95 (m, 5H), 6.64 (s, 1H), 6.54
(d, J = 12.1 Hz, 1H), 6.53 (s, 2H), 6.52 (d, J = 12.1 Hz, 1H), 5.45 (s, 2H), 3.92 (s, 3H), 3.81 (s,
3H), 3.70 (s, 6H), 3.62 (s, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm): 158.2, 157.7, 156.2, 152.8,
151.3, 143.9, 137.2, 137.1, 135.5, 133.1, 132.6, 131.4, 130.6, 129.8, 125.6, 123.9, 122.5, 119.3,
118.8, 118.1, 113.5, 108.4, 106.1, 60.9, 60.8, 55.9, 55.6, 54.2; IR (KBr): ῡmax/cm-1 = 3305, 3095,
2938, 1651, 1555, 1449, 1381, 1270, 1233, 1186, 1160, 1132, 1108, 988; MS (ESI, m/z): 623
[M+1]+; HRMS (ESI, m/z) Calculated for, C35H35O7N4: 623.25042; found : 623.25036 [M+1]+.
HPLC: tR 7.36 min, purity 97.0%.
6.4.8 (Z)-N-(2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(4-methoxybenzyl)-1H-
1,2,3-triazole-4-carboxamide (7c)
The compound 7c was prepared according to the general procedure by employing 1-(4-
methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (67 mg, 0.29 mmol) and (Z)-2,3-dimethoxy-
5-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.29 mmol) as white solid. Yield: 103 mg (64%);
1
m.p: 115-117 °C; H NMR (300 MHz, CDCl3) δ (ppm): 9.54 (s, 1H), 8.04 (s, 1H), 7.99 (s, 1H),
7.27 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 6.63 (s, 1H), 6.58-6.45 (m, 4H), 5.50 (s, 2H),
17

ACCEPTED MANUSCRIPT
3.91 (s, 3H), 3.81 (s, 3H), 3.81 (s, 3H), 3.69 (s, 6H), 3.61 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
(ppm): 160.1, 157.3, 152.7,151.3, 143.7, 136.9, 133.0, 132.6, 131.4, 129.8, 129.8, 129.7, 125.5,
125.3, 120.8, 114.5, 113.4, 108.2, 105.8, 60.9, 60.8, 55.8, 55.5, 55.3, 54.1; IR (KBr): ῡmax/cm-1 =
3284, 3095, 1673, 1615, 1556, 1450, 1392, 1359, 1270, 1233, 1185, 1160, 1132, 1108, 989, 845,
859, 845; MS (ESI, m/z): 561 [M+1]+; HRMS (ESI, m/z) Calculated for, C30H33O7N4:
561.23503; found: 561.23516 [M+1]+. HPLC: tR 7.33 min, purity 98.9%.
6.4.9 (Z)-N-(2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(4-fluorobenzyl)-1H-1,2,3-
triazole-4-carboxamide (7d)
The compound 7d was prepared according to the general procedure by employing 1-(4-
fluorobenzyl)-1H-1,2,3-triazole-4-carboxylic acid (64 mg, 0.29 mmol) and (Z)-2,3-dimethoxy-5-
(3,4,5-trimethoxystyryl)aniline (100 mg, 0.29 mmol) as white solid. Yield: 111 mg (70%); m.p:
1
85-87 °C; H NMR (300 MHz, CDCl3) δ (ppm): 9.54 (s, 1H), 8.05 (s, 1H),8.03 (s, 1H), 7.35-
7.28 (m, 2H), 7.13-7.05 (m, 2H), 6.64 (s, 1H), 6.56 (d, J = 12.2 Hz, 1H), 6.52 (s, 2H), 6.48 (d, J
= 12.2 Hz, 1H), 5.55 (s, 2H), 3.91 (s, 3H), 3.81 (s, 3H), 3.69 (s, 6H), 3.62 (s, 3H); 13C NMR (75
MHz, CDCl3) δ (ppm): 164.6, 161.3, 157.6, 152.8, 151.3, 144.0, 137.1, 133.1, 132.6, 131.3,
130.1, 129.8, 129.6, 125.5, 116.4, 116.1, 113.5, 108.5, 106.1, 60.9, 60.8, 55.9, 55.6, 53.8; IR
(KBr): ῡmax/cm-1 = 3359, 3122, 2935, 2836, 1670, 1586, 1565, 1511, 1457, 1424, 1330, 1235,
1183, 1129, 1080, 1044, 998, 883, 862; MS (ESI, m/z): 549 [M+1]+; HRMS (ESI, m/z)
Calculated for C29H30O6N4F: 549.21491; found 549.21490 [M+1]+. HPLC: tR 7.52 min, purity
96.7%.
6.4.10 (Z)-N-(2,3-Dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-1-(3,4,5-trimethoxybenzyl)-
1H-1,2,3-triazole-4-carboxamide (7e)
The compound 7e was prepared according to the general procedure by employing 1-(3,4,5-
trimethoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (84 mg, 0.29 mmol) and (Z)-2,3-
dimethoxy-5-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.29 mmol) as yellow solid. Yield: 129
1
mg (72%); m.p: 96-98 °C; H NMR (300 MHz, CDCl3) δ (ppm): 9.56 (s, 1H), 8.06 - 8.05 (m,
2H), 6.63 (s, 1H), 6.59-6.46 (m, 6H), 5.49 (s, 2H), 3.92 (s, 3H), 3.84 (s, 9H), 3.81 (s, 3H), 3.69
(s, 6H) 3.62 (s, 3H); 13C NMR (75 MHz, CDCl3) δ (ppm): 157.7, 153.7, 152.8, 151.3, 143.8,
138.6, 137.1, 137.0, 133.1, 132.6, 131.3, 129.8, 129.0, 125.5, 113.4, 108.4, 106.0, 105.5, 60.9,
18

ACCEPTED MANUSCRIPT
60.7, 56.1, 55.8, 55.5, 54.8; IR (KBr): ῡmax/cm-1 = 3373, 2937, 2835, 1651, 1590, 1561, 1530,
1505, 1462, 1423, 1327, 1236, 1183, 1126, 1079, 1044, 1000, 878; MS (ESI, m/z): 621 [M+1]+;
HRMS (ESI, m/z) Calculated for C32H37O9N4: 621.25659; found: 621.25657 [M+1]+. HPLC: tR
6.31 min, purity 96.9%.
6.5 Procedure for the synthesis of triazole linked aminocombretastatins (8a-e)
a) To a solution of substituted benzyl-1H-1,2,3-triazole-4-carboxylic acids (18a-e,1 mmol) in dry
dimethylformamide, EDCI (1.2 mmol) and HOBT (1.2mmol) were added and the reaction
mixture was stirred for 20 min. To the reaction mixture amino Z-stilbene (22, 1 mmol) was
added and stirred at room temperature for 12 h. The solvent was removed and the residue was
dissolved in dichloromethane (50 mL), washed with 5% HCl (2 x 20 mL), 5% NaHCO3 (2 x 20
mL), and brine solution (20 mL). The organic layer was separated, dried over anhydrous Na2SO4,
filtered, and the solvent was removed to give crude products (23a-e), which was used without
purification.
b) To a solution of compounds (23a-e, 1 mmol) in dry tetrahydrofuran, TBAF (1.0 M in THF,
o
2.5 mmol) was added at 0 C and stirred at room temperature for 1h. After completion of
reaction, the reaction mixture was diluted with ethyl acetate, washed with water and brine
solution, dried over anhydrous Na2SO4. The solution was filtered and the solvent was removed
under reduced pressure followed by the purification of reside by column chromatography using
ethyl acetate and hexane as eluents to afford respective desired products (8a-e).
6.5.1 (Z)-N-(2-Hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-1-(3-methoxybenzyl)-
1H-1,2,3-triazole-4-carboxamide (8a)
The compound 8a was prepared according to the general procedure by employing 1-(3-
methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (50 mg, 0.22 mmol) and (Z)-2-(tert-
butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.22 mmol) as
1
brown solid. Yield: 84 mg (70%); m.p: 153-155 °C; H NMR (500 MHz, CDCl3) δ (ppm): 9.05
(s, 1H), 9.02 (s, 1H), 7.85 (s, 1H), 7.35-7.30 (m, 1H), 6.95-6.82 (m, 5H), 6.67 (d, J = 12.0 Hz,
1H), 6.56 (d, J = 12.0 Hz, 1H), 6.29 (s, 2H), 5.48 (s, 2H), 3.91 (s, 3H), 3.80 (s, 3H), 3.70 (s, 3H),
3.53 (s, 6H); 13C NMR (75 MHz, CDCl3) δ (ppm): 160.2, 159.3, 152.3, 141.7, 139.9, 137.1,
133.0, 131.0, 130.4. 128.9, 127.8, 126.0, 125.8, 124.2, 121.0, 120.9, 120.5, 114.5, 114.2, 109.6,
19

ACCEPTED MANUSCRIPT
105.5, 60.7, 56.2, 55.3, 54.4; IR (KBr): ῡmax/cm-1 = 3355, 3138, 2933, 2835, 1650, 1577, 1494,
1459, 1327, 1240, 1125, 1076, 1043, 1002, 856; MS (ESI, m/z): 547 [M+1]+; HRMS (ESI, m/z)
Calculated for, C29H31O7N4: 547.21928; found: 547.21919 [M+1]+. HPLC: tR 4.86 min, purity
95.7%.
6.5.2 (Z)-N-(2-Hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-1-(3-phenoxybenzyl)-
1H-1,2,3-triazole-4-carboxamide (8b)
The compound 8b was prepared according to the general procedure by employing 1-(3-
phenoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (64 mg, 0.22 mmol) and (Z)-2-(tert-
butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.22 mmol) as
yellow solid. Yield: 90 mg (68%); m.p: 151-152 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 9.03
(s, 1H), 9.01 (s, 1H), 7.87 (s, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.17-7.13
(m, 1H), 7.03-6.95 (m, 5H), 6.89-6.83 (m, 3H), 6.66 (d, J = 12.6 Hz, 1H), 6.55 (d, J = 12.6 Hz,
13
1H), 6.29 (s, 2H), 5.48 (s, 2H), 3.92 (s, 3H), 3.70 (s, 3H), 3.53 (s, 6H); C NMR (125 MHz,
CDCl3) δ (ppm): 159.4, 153.8, 152.3, 150.1, 141.7, 139.9, 138.6, 137.5, 133.0, 132.9, 131.6,
130.7, 129.9, 128.4, 125.8, 125.7, 124.3, 124.0, 123.6, 123.1, 122.6, 121.0, 119.4, 119.4, 118.8,
109.6, 105.7, 60.8, 56.1, 55.5, 54.8; IR (KBr): ῡmax/cm-1 = 3350, 2936, 2835, 1649, 1580, 1504,
1460, 1422, 1328, 1241, 1186, 1126, 1042, 1003, 856; MS (ESI, m/z): 609 [M+1]+; HRMS (ESI,
m/z) Calculated for, C34H33O7N4: 609.23496; found: 609.23569 [M+1]+. HPLC: tR 4.34 min,
purity 95.2%.
6.5.3 (Z)-N-(2-Hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-1-(4-methoxybenzyl)-
1H-1,2,3-triazole-4-carboxamide (8c)
The compound 8c was prepared according to the general procedure by employing 1-(4-
methoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (51 mg, 0.22 mmol) and (Z)-2-(tert-
butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.22 mmol) as
1
brick red solid. Yield: 85 mg (71%); m.p: 142-144 °C; H NMR (400 MHz, CDCl3) δ (ppm):
9.07 (s, 1H), 9.02 (s, 1H), 7.79 (s, 1H), 7.27 (d, J = 9.4 Hz, 2H), 6.93-6.82 (m, 4H), 6.66 (d, J =
12.6 Hz, 1H), 6.56 (d, J = 12.6Hz, 1H), 6.29 (s, 2H), 5.45 (s, 2H), 3.91 (s, 3H), 3.81 (s, 3H), 3.70
13
(s, 3H), 3.53 (s, 6H); C NMR (125 MHz, CDCl3) δ (ppm): 160.2, 159.4, 152.3, 150.1, 141.6,
139.9, 137.5, 133.0, 131.6, 130.1, 125.6, 125.0, 124.2, 123.5, 123.2, 120.9, 114.7, 109.6, 105.5,
20

ACCEPTED MANUSCRIPT
60.7, 56.2, 55.5, 55.3, 54.1; IR (KBr): ῡmax/cm-1 = 3246, 3136, 3007, 2835, 1646, 1606, 1575,
1513, 1505, 1460, 1428, 1331, 1243, 1178, 1121, 1121, 1080, 1043, 1023, 997, 854. MS (ESI,
m/z): 547[M+1]+; HRMS (ESI, m/z) Calculated for, C29H31O7N4: 547.21874; found: 547.21919
[M+1]+. HPLC: tR 4.77 min, purity 95.2%.
6.5.4 (Z)-1-(4-Fluorobenzyl)-N-(2-hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-
1H-1,2,3-triazole-4-carboxamide (8d)
The compound 8d was prepared according to the general procedure by employing 1-(4-
fluorobenzyl)-1H-1,2,3-triazole-4-carboxylic acid (48 mg, 0.22 mmol) and (Z)-2-(tert-
butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.22 mmol) as
1
white solid. Yield: 88 mg (75%); m.p: 141-143 °C; H NMR (300 MHz, CDCl3 + DMSO-d6) δ
(ppm): 9.37 (s, 1H), 8.45 (s, 1H), 8.41 (s, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 8.3Hz, 1H),
7.21-7.06 (m, 4H), 6.94-6.87 (m, 2H), 6.68 (s, 2H), 5.62 (s, 2H), 3.91 (s, 3H), 3.81 (s, 6H), 3.80
(s, 3H); 13C NMR (75 MHz, CDCl3 + DMSO-d6) δ (ppm):158.9, 152.7, 151.3, 150.5, 143.6,
141.3, 140.8, 136.7, 128.7, 126.0, 125.1, 122.8, 121.3, 121.1, 117.9, 114.1, 113.8, 108.9, 101.8,
58.5, 54.3, 54.0, 52.7; IR (KBr): ῡmax/cm-1 = 3462, 3355, 3152, 2934, 2835, 1650, 1574, 1509,
1461, 1325, 1234, 1130, 1073, 854; MS (ESI, m/z): 535 [M+1]+; HRMS (ESI, m/z) Calculated
for, C28H28O6N4F: 535.19897; found: 535.19902 [M+1]+. HPLC: tR 4.87 min, purity 95.6%.
6.5.5 (Z)-N-(2-Hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-1-(3,4,5-
trimethoxybenzyl)-1H-1,2,3-triazole-4-carboxamide (8e)
The compound 8e was prepared according to the general procedure by employing 1-(3,4,5-
trimethoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid (64 mg, 0.22 mmol) and (Z)-2-(tert-
butyldimethylsilyloxy)-3-methoxy-6-(3,4,5-trimethoxystyryl)aniline (100 mg, 0.22 mmol) as
brick red solid. Yield: 93mg (70%); m.p:95-97 °C; 1H NMR (500 MHz, CDCl3) δ (ppm): 9.03 (s,
1H), 9.01 (s, 1H), 7.87 (s, 1H) ,7.36 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H) 7.27 (s, 2H),
6.69 (d, J = 12.0 Hz, 1H), 6.55 (d, J = 12.0 Hz, 1H ), 6.29 (s, 2H), 5.84 (s, 2H), 3.92 (s, 6H), 3.70
13
(s, 6H), 3.53 (s, 9H); C NMR (75 MHz, CDCl3) δ (ppm): 158.3, 152.3, 151.9, 135.0, 133.0,
131.6, 130.7, 129.9, 129.5, 125.8, 124.2, 124.0, 123.1, 122.6, 121.0, 119.4, 118.8, 118.3, 109.7,
105.6, 60.7, 56.2, 55.5, 54.2; IR (KBr): ῡmax/cm-1 = 3343, 2932, 2834, 1648, 1579, 1489, 1455,
1327, 1244, 1125, 1077, 1043, 1003, 858; MS (ESI, m/z): 607 [M+1]+; HRMS (ESI, m/z)
21

ACCEPTED MANUSCRIPT
Calculated for, C31H35O9N4: 607.24140; found: 607.24149 [M+1]+. HPLC: tR 7.49 min, purity
96.0%.
7. Biology
7.1 Evaluation of in vitro anti-cancer activity
The cytotoxic 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 of 96-well
microculture plates and incubated for 24 h at 370 C in a CO2 incubator. Compounds, diluted to
the desired concentrations in culture medium, were added to the wells with respective vehicle
control. After 48 h of incubation, 10 µl MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl
tetrazolium 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 570 nm wavelength was recorded.
7.2 Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells
through the cell-cycle phases. A549 cells, Lung cancer cells were incubated for 48 h with
compounds 6d and 7c at concentrations of 25 and 50 nM, and also with CA-4 at 50 nM
concentration. Untreated and treated cells were harvested, washed with phosphate-buffered
saline (PBS), fixed in ice-cold 70% ethanol, and stained with propidium iodide (Sigma–Aldrich).
Cell-cycle analysis was performed by flow cytometry (Becton Dickinson FACS Caliber
instrument).41
7.3 Tubulin polymerization assay
A fluorescence based in vitro tubulin polymerization assay was performed according to the
manufacturer’s protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture in a total
volume of 10 µL contained PEM buffer, GTP (1 µM) in the presence or absence of test
compounds (final concentration of 3 µM). Tubulin polymerization was followed by a time
22

ACCEPTED MANUSCRIPT
dependent increase in fluorescence due to the incorporation of a fluorescence reporter into
microtubules as polymerization proceeds. Fluorescence emission at 420 nm (excitation
wavelength is 360 nm) was measured by using a Varioscan multimode plate reader (Thermo
scientific Inc.). CA-4 was used as positive control in each assay. The IC50 value was defined as
the drug concentration required inhibiting 50% of tubulin assembly compared to control. The
reaction mixture for these experiments include: tubulin (3 mg/ml) in PEM buffer, GTP (1 µM),
in the presence or absence of test compounds at 2.5, 5, 10, and 15 µM concentrations.
Polymerization was monitored by increase in the Fluorescence as mentioned above at 37 0C.
7.4 Colchicine competition assay
The test compounds (6d, 7f) of various concentrations 5 µM, 10 µM, 15 µM and 20 µM were
incubated with 3 µM tubulin in the presence and absence of 3 µM colchicine in 30 mM Tris
o
buffer for 60 min at 37 C. CA-4 was used as a positive control whereas taxol was used as
negative control which binds at the taxane site. After incubation the fluorescence of tubulin-
colchicine complex was determined by using Tecan multimode reader with excitation
wavelength of 350 nm and emission wavelength of 435 nm. 30 mM Tris buffer was used as
blank which was subtracted from all the samples and the fluorescence values are normalized to
DMSO (control)42.
7.5 Immunohistochemistry
A549 cells were seeded on glass cover slips, incubated for 48 h in the presence or absence of test
compounds 6d and 7c 25 nM concentrations. Following the termination of incubation, cells were
fixed with 3% paraformaldehyde, 0.02% glutaraldehyde in PBS and permeabilized by dipping
the cells in 100% methanol followed by overnight incubation in _40C. Later, cover slips were
blocked with 1% BSA in phosphate buffered saline for 1 h followed by incubation with a
primary anti tubulin (mouse monoclonal) antibody and FITC conjugated secondary mouse anti
IgG antibody. Photographs were taken using the fluorescence microscope, equipped with FITC
settings and the pictures were analyzed for the integrity of microtubule network. In parallel
experiments, CA-4 (25 nM) was used as positive control for analyzing microtubule integrity
7.6 Morphological analysis for apoptosis with Hoechst staining
23

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A549 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 6d and 7c at 25 nM concentration for
48 h. Hoechst 33258 (Sigma Aldrich) was added to the cells at a concentration of 0.5 mg/mL and
incubated for 30 min at 370C. Later, cells were washed with phosphate buffered saline (PBS).
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.43
7.7 Mesurement of Mitochondrial Membrane Potential
Mitochondrial membrane potential (∆Ψm) was determined by using JC-1 dye. Cell-permeable
cationic carbocyanine dye JC-1 (Sigma aldrich India, Catlog- CS0390), also known as 5,5′,6,6′-
tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodine, emits green fluorescence (525
nm) in its monomeric form. However, upon transfer to the membrane environment of a
functionally active mitochondrion, it exhibits an aggregation dependent orange-red fluorescence
(emission at 590 nm). Briefly, cultures were treated with the test drugs for 48 h. After drug
treatment the cells were incubated with JC-1 dye for 20 min at 37 °C. After incubation cultures
were used for the measuring mitochondrial membrane potiential (∆Ψm), according to the
manufacturer’s instructions.
7.8 ROS generation
To evaluate intracellular reactive oxygen species (ROS) levels, 2’,7’-dichlorofluorescein
diacetate (DCFH-DA, Molecular Probes) fluorescent dye was used to clarify this issue. The
nonpolar DCFH-DA is converted to the polar derivative DCFH by esterases when it is taken up
by the cell. DCFH is nonfluorescent but is rapidly oxidized to the highly fluorescent DCF by
intracellular H2O2 or nitric oxide. A549 cells were treated with compound 6d, 7c and CA-4 at 25
nM concentration for 48 h. To determine the production of ROS, cells were treated with 5 µM of
DCFH2-DA at 370C for 30 min, and the fluorescence of DCF was measured at 530 nm after
excitation at 485 nm (DCFH2-DA, after deacetylation to DCFH2, is oxidized intracellularly to
its fluorescent derivative, DCF).44
7.9 Caspase 3 activity
24

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Caspase-3 assay was conducted for detection of apoptosis in Lung cancer cell line (A549). The
commercially available apoptosis detection kit (Sigma-Caspase 3 Assay kit, Colorimetric) was
used. A549 cells were treated with compounds 6d and 7c at 25 and 50 nM concentrations for 48
h. After 48 h of treatment, cells were collected by centrifugation, washed once with PBS, and
cell pellets were collected. Suspended the cell pellet in lysis buffer and incubated for 15 min.
After incubation, cells were centrifuge at 20,000 rpm for 15 min and collected the supernatant.
Supernatants were used for measuring caspase 3 activities using an ELISA-based assay,
according to the manufacturer’s instructions.
7.10 Determination of the Apoptotic DNA Ladder
Cells were seeded (1×106) in six well plates and incubated for 24 h. After incubation, cells were
treated with compounds 6d and 7c along with CA-4 at 25 nM concentration for 48 h. After 48 h
of drug treatment cells were collected and centrifuged at 2500 rpm for 5 min at 40C. Pellet was
collected and washed with Phosphate buffered saline (PBS). And added 100 µl of Lysis buffer,
o
centrifuged at 3000 rpm for 5 min at 4 C and collected supernant. And add 10 µl of 10% SDS
and 10 µl of (50 mg/mL) RNase-A and incubated for 2 h at 560c. After that 10 µl of Proteinase K
(25 mg/mL) was added and incubated at 370c for 2 h. After incubation, 65 µl of 10 M
Ammonium acetate and 500 µl of ice cold ethanol was added and mixed well. And this sample
o
was incubated in _80 C for 1 h. After that samples were centrifuged at 12000 rpm for 20 min at
40c and washed with 80% ethanol followed by air dried for 10 min at room temperature.
Dissolved pellet in 50µl of TE buffer. After that, DNA laddering was determined by 2% agarose
gel electrophoresis.45
8.0 Molecular modeling studies
All the compounds under study and reference compounds 3D structures were built and
optimised using Gaussian 03W. These optimised 3D structures were utilised for Docking. All the
compounds were Docked using AutoDockTools46 software package. The co crystallised structure
25

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of colchicine site tubulin downloaded from the PDB data bank (http://
www.rcsb.org/pdb/index.html; PDB code: 3E22).47
Acknowledgement
B.S acknowledge CSIR-UGC, New Delhi and for the award of Senior Research Fellowship. We
also acknowledge CSIR for financial support under the 12th Five Year plan project “Affordable
Cancer Therapeutics (ACT)” (CSC0301).
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