Design, synthesis of phenstatin/isocombretastatin-oxindole conjugates as antimitotic agents

Article abstract of DOI:10.1016/j.bmc.2016.02.047

A series of phenstatin/isocombretastatin-oxindole conjugates was synthesized and tested for their cytotoxic activity against five human cancer cells such as prostate (DU-145), lung (A549), colon (HT-29), breast (MCF-7), liver (HepG2) cancer cells with IC₅₀ values ranging from 0.049 to 38.90 μM. Amongst them, two conjugates (5c and 5d) showed broad spectrum of antiproliferative efficacy on lung cancer cells with an IC₅₀ value of 79 nM and 93 nM, respectively, whereas on colon cancer cells with an IC₅₀ values 45 nM and 49 nM, respectively. In addition, cell cycle assay revealed that these conjugates (5c and 5d) arrest at the G₂/M phase and leads to apoptotic cell death which was confirmed by Annexin V-FITC and mitochondrial membrane depolarization. Further, the tubulin polymerization assay analysis results suggest that these conjugates particularly 5c and 5d exhibit significant inhibitory effect on the tubulin assembly with an IC₅₀ value of 1.23 μM and 1.01 μM, respectively. Molecular docking studies indicated that these compounds (5c and 5d) occupy the colchicine binding site of the tubulin.

Full text of DOI:10.1016/j.bmc.2016.02.047

Accepted Manuscript
Design, Synthesis of Phenstatin/isocombretastatin-Oxindole Conjugates as An-
timitotic agents
G. Bharath Kumar, V. Lakshma Nayak, Ibrahim Bin Sayeed, Vangala Santhosh
Reddy, Anver Basha Shaik, Mirza Feroz Baig, Mohd Adil Shareef, A.
Ravikumar, Rasala Mahesh, Ahmed Kamal
PII:
S0968-0896(16)30137-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.02.047
BMC 12849
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 December 2015
29 February 2016
29 February 2016
Please cite this article as: Bharath Kumar, G., Lakshma Nayak, V., Sayeed, I.B., Reddy, V.S., Shaik, A.B., Baig,
M.F., Shareef, M.A., Ravikumar, A., Mahesh, R., Kamal, A., Design, Synthesis of Phenstatin/isocombretastatin-
Oxindole Conjugates as Antimitotic agents, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.02.047
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Design, Synthesis of Phenstatin/isocombretastatin-Oxindole
Conjugates as Antimitotic agents
G. Bharath Kumar,[a] V Lakshma Nayak,[a] Ibrahim Bin Sayeed,[a] Vangala Santhosh Reddy,[a]
Anver Basha Shaik,[a] Mirza Feroz Baig,[a] Mohd Adil Shareef,[a] A Ravikumar[b], Rasala
Mahesh,[a] Ahmed Kamal,*[a,b,c]
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
cCatalytic Chemistry Chair, Chemistry Department College of Science, King Saud University,
Riyadh (Saudi Arabia)
Abstract :
A series of phenstatin/isocombretasatin-oxindole conjugates was synthesized and tested for their
cytotoxic activity against five human cancer cells such as prostate (DU-145), lung (A549), colon
(HT-29), breast (MCF-7), liver (HepG2) cancer cells with IC50 values ranging from 0.049-38.90
M. Amongst them, two conjugates (5c and 5d) showed broad spectrum of antiproliferative
efficacy on lung cancer cells with an IC50 value of 79 nM and 93 nM respectively, whereas on
colon cancer cells with an IC50 values 45 nM and 49 nM respectively. In addition, cell cycle
assay revealed that these conjugates (5c and 5d) arrest at the G2/M phase and leads to apoptotic
cell death which was confirmed by Annexin V-FITC and mitochondrial membrane
depolarization. Further, the tubulin polymerization assay analysis results suggest that these
conjugates particularly 5c and 5d exhibit significant inhibitory effect on the tubulin assembly
with an IC50 value of 1.23 M and 1.01 M respectively. Molecular docking studies indicated
that these compounds (5c and 5d) occupy the colchicine binding site of the tubulin
Keywords: phenstatin/isocombretastatin-oxindole, tubulin depolymerization, Annexin V-FITC
and mitochondrial membrane depolarization
Corresponding authors Tel: +91-40-27193157; Fax: +91-40-27193189, E-mail:
ahmedkamal@iict.res.in (A. Kamal)
1

Introduction
Microtubules have become an important target in chemotherapy that are present in all
eukaryotic cells and play an essential role in the formation of mitotic spindles, cell division etc.1
The mitotic spindles are generated by noncovalent polymerization of -tubulin and -tubulin
heterodimers that have become an attractive target to treat many types of malignancies.2 The
majority of drugs that binds at -tubulin of the microtubule, are known to inhibit tubulin
polymerization or depolymerization.3 In particular, three major binding domains on -tubulin
subunit of microtubule, namely the vinca binding site, the taxane binding site and the colchicine
binding site for mostly lipophilic ligands and some high affinity agents have been developed as
antiproliferative drugs.3,4 More particularly, compounds such as the paclitaxel, docetaxal and
epothilones, that bind to the taxane binding site prevent microtubule disassembly by stabilizing
microtubules and are used in the treatment of carcinomas, such as lung, breast, ovarian, and
bladder.5 In contrast, compounds like colchicinium, vinca alkaloids and combretastatin A-4 (1,
Figure 1) and nocodazole, that bind to the vinca or colchicine binding sites inhibit cancer cell
proliferation and tubulin assembly by destabilizing microtubules and are used in the treatment of
leukemia, lymphoma and a variety of other diseases.6 However, many of such agents manifest
different limitations in their clinical utility, therefore development of new microtubule targeting
agents is of significance.
Phenstatin (2a, Figure 1) is a benzophenone motif reported by Pettit and coworkers and it
is also well known microtubule-destabilizing agent. Phenstatin (2a, Figure 1) and its derivatives
exhibits the most potent activity at the colchicine binding site and significantly inhibits tubulin
polymerization and exerts profound antiproliferative activity against various human cancer cell
lines, including multidrug-resistant cancer cells.7 Recently, a new class of benzophenone series
with insertion of small heterocyclic groups in the B-ring like indole, quinoline, carbazole,
thiophenes displayed excellent cytotoxic activity as well as significant inhibition of tubulin
polymerization.8 Recently, isocombretastatin A-4 (iso CA-4, 2c) (Figure 1) is a non isomerized C
A-4, reported as antimitotic agent. In addition, isocombretastatin and its derivatives shows
substantial cytotoxic activity towards selected human cancer cell lines and cells are accumulated
in the G2/M phase of the cell cycle.9
2

Oxindoles are versatile moieties that display diverse biological activities, including
anticancer activity. Bis-indole alkaloid indirubin (3, Figure 1) is interesting natural
pharmacophore and traditional Chinese medicine recipe used in the treatment of leukemias. This
has broad spectrum of activity and it is mainly recognized as kinase inhibitor. In addition,
indirubins are potent inhibitors of cyclin-dependent kinases (CDKs) like glycogen synthase
kinase-3b (GSK-3b) and CDK1/cyclin B.10 A-432411 (4, (Z)-3-((1H-pyrrol-2-yl)methylene)-6-
(4-hydroxy-3-methoxyphenyl)indolin-2-one) (Figure 1) is an indolinone that is structurally
different from other known synthetic microtubule inhibitors. This compound is efficacious
against a variety of human cancer cell lines including drug-resistant HCT-15 that over expresses
Pgp170. These compete with the colchicine-binding site on the tubulin thereby inhibiting
microtubule polymerization and causing G2-M arrest and induces apoptosis.11
Figure 1. Chemical structures of microtubule targeting agents. Combretastatin A-4 (1),
phenstatin (2a) phenstatin amine (2b) isocombretastatin A-4 (2c) isocombretastatin A-4 amine
(2d), Indirubin (3), A-432411 (4) Phenstatin\isoCombretastatin-Oxindole conjugates (5a-h and
6a-h)
3

Thus Based on these observations,, it was considered of interest to explore newer
antitubulin agents by linking different pharmacophores. In this study, we linked the substituted
oxindoles with phenstatin and isocombretastatin A-4 to generate phenstatin/isocombretastatin A-
4 oxindoles. The promising activity observed prompted us to investigate the role of these new
compounds in the proliferation and apoptosis of human colon cancer cell line (HT-29). We also
investigated the effect of these compounds on proteins that regulate cell-cycle progression.
Results and Discussion
Chemistry
These phenstatin/iso-combretastatin-oxindole conjugates (5a-h and 6a-h) were prepared
employing Knoevenagel reaction between equimolar mixtures of substituted oxindoles (17a-h)
and phenstatin 3-aldehyde/isocombretastatin 3-aldehyde (16a-b) in the presence of piperidine as
shown in Scheme 1. The compound structures were confirmed by means of 1H NMR, 13C NMR,
HRMS and IR spectra.
The phenstatin 3-aldehyde/iso-combretastatin 3-aldehyde (16a-b) were obtained via
Swern’s oxidation of the corresponding (3-(hydroxymethyl)-4-methoxyphenyl)(3,4,5-
trimethoxyphenyl)methanone
(15a)
and
(2-methoxy-5-(1-(3,4,5-
trimethoxyphenyl)vinyl)phenyl)methanol (15b).
The ketone 13 is generated from the
benzhydrol derivative 12 via oxidation and subsequent Witting methylation afforded the olefin
14. The benzhydrol derivative 12 was in turn, obtained via the addition of the aryl lithium
reagent generated from the iodide 10 to aldehyde 11. The aryl iodide 10 was obtained via a three
step synthetic manipulation of 5-iodo salicylic acid 7 (Scheme 1).
4

Scheme 1. Reagents and Conditions: (a) DMS, K2CO3, Acetone, 60 ºC, 8 h; (b) DIBAL-H (1N), THF
05 ºC, 3 h; (c) TBDMSCl, imidazole, CH2Cl2 3 h; (d) n-But-Li (1.6N), THF -78 ºC; (e) IBX, DMSO, 10
ºC, 2 h; (g) TBAF (1N), THF 05 ºC, 6 h; (g) CH2PPh3I, t-ButOK, dry THF 05 ºC, 8 h; (h) Oxalyl
chloride, DMSO, -78 ºC to rt, Et3N; (i) piperidine, EtOH, reflux 3–8 h.
Biological studies
Antiproliferative activity
In an attempt to examine the structure activity relationship of phenstatin/iso-
combretastatin-oxindole conjugates (5a-h and 6a-h) consisting A, B, C, D-rings and X (Fig 2) as
5

shown in scheme 1. Sixteen compounds were prepared with respect to different modifications
made on the D-ring and X which were evaluated for cytotoxic activity against a panel of five
human cancer cell lines such as A549 (lung), DU-145 (prostate cancer), HT-29 (colon cancer),
MCF-7 (breast cancer) and HepG2 (liver cancer) by employing MTT assay.12 Combretastatin A-
4 was used as reference drug and the results are summarized in Table 1 and expressed as IC50
values.
Table 1: aIC50 values (in µM) for compounds in selected human cancer cell lines
Compound
DU-145b
26.78
35.55
7.943
9.705
29.21
32.64
19.14
12.02
25.60
19.49
5.035
3.133
38.90
11.40
19.14
20.70
0.007
A549c
6.698
8.912
0.079
0.093
9.660
11.93
9.772
4.570
11.38
5.754
1.479
1.174
10.93
7.413
7.430
12.58
0.051
HT-29d
2.728
5.128
0.045
0.049
3.235
13.42
8.609
3.715
6.456
1.339
0.107
0.081
4.265
2.269
5.623
8.317
0.049
MCF-7e
10.70
12.27
0.269
0.386
14.25
15.88
30.82
14.69
20.32
13.18
3.162
3.981
15.16
11.74
18.20
21.00
0.046
HepG2f
3.801
6.918
0.095
0.186
2.511
15.84
8.128
4.017
7.673
3.715
0.897
0.084
5.623
3.235
8.317
10.47
0.023
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
6g
6h
CA-4
a 50% Inhibitory concentration after 48 h of drug treatment; b human prostate cancer; c human lung
cancer; human colon cancer; e human breast cancer; f human liver cancer.
d
The in vitro screening results revealed that these conjugates possess excellent to
moderate cytotoxic activity with IC50 values ranging from 0.049-38.90 M. Particularly, the
6

phenstatin-oxindole conjugates harboring carbonyl group in between A and B rings showed
profound cytotoxic activity with an IC50 value in the range 0.045-35.55 µM. More particularly,
the conjugate 5c having electron donating substituent like para-methoxy group on D-ring possess
excellent cytotoxicity against lung and colon cancer cells with IC50 value 79 nM and 45 nM
respectively. In contrast, substitution with nitro at para position of D-ring as in 5b proved
deleterious for its antiproliferative activity against human prostate cancer like Du145 cells (IC50
value is 35.55 µM). However, isocombretastatin-oxindole conjugates (6a-h) harboring ethylene
group in between A and B ring displayed potent antiproliferative activity with an IC50 value in
the range 0.081-38.9 µM. In addition, the conjugate 6c having para-methoxy group on D-ring as
electron donating substituent exhibited significant cytotoxic activity towards colon cancer
(HT29) with an IC50 value 81 nM.
These results suggest that phenstatin-oxindole conjugates exhibit better cytotoxicity
than iso-combretastatin-oxindoles (6a-h) against tested cell lines.
Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the cell cycle
at a particular checkpoint of the cell cycle or by induction of apoptosis or a combined effect of
both cycle block and apoptosis.13-14 Furthermore, regulation of the cell cycle and apoptosis are
considered to be effective cancer therapeutic methods.15 Therefore, it was considered of interest
to understand whether this inhibition of cell growth was on account of cell cycle arrest. In this
study DU-145 cells were treated with compounds 5c and 5d at 10 and 50 nM concentrations for
48 h, and CA-4 was used a reference compound in this study. The data obtained clearly indicated
that this compound arrested cell cycle at G2/M phase as compared to the untreated control
(Figure 1). These compounds (5c and 5d) showed 21.69 and 47.12 % of cell accumulation in
G2/M phase, respectively, at 10 nM concentration, whereas it exhibited 49.80 and 56.82 % of the
cell accumulation in G2/M phase at 50 nM concentration (Figure 2).
7

Cell cycle distribution of HT29 cells in presence of Compound 5c, 5d and CA-4 at concentrate
dependant manner
Sample
Sub G1 %
G0/G1 %
S %
G2/M %
A: Control
1.00
0.16
0.43
0.62
0.35
0.18
83.01
51.46
73.61
48.01
50.06
41.27
3.02
2.62
4.87
0.91
2.47
1.36
12.47
45.76
21.69
49.80
47.12
56.82
B: CA-4 (10 nM)
B: 5c (10 nM)
C: 5c (50 nM)
D: 5d (10 nM)
E: 5d (50 nM)
Figure 2. Cell cycle analysis of 5c and 5d on HT-29 cells. A: Control cells (HT-29), B: Ca-4 (10
nM), C: 5c (10 nM), D: 5c (50 nM), E: 5d (10 nM) and F: 5d (50 nM).
Effect of compounds on tubulin polymerization
In general G2/M cell cycle arrest is strongly associated with inhibition of tubulin
polymerization.16 Since compounds like 5c and 5d cause cell cycle arrest at G2/M phase cell
8

cycle arrest, it was considered of interest to investigate their microtubule inhibitory function.
Tubulin subunits are known to heterodimerize and self-assemble to form microtubules in a time
dependent manner. The progression of tubulin polymerization17-18 was thus examined by
monitoring the increase in fluorescence emission at 420 nm (excitation wavelength is 360 nm) in
o
384 well plate for 1 h at 37 C with and without the conjugate in comparison with reference
compound CA-4. The test compounds (5c and 5d) efficiently inhibited tubulin polymerization by
68.78 and 72.06%, respectively, comparable to 71.52 % inhibition exhibited by CA-4 (Figure. 3).
This was followed by the evaluation of IC50 values for these compounds and results indicate that
compounds 5c and 5d showed better tubulin-assembly inhibition with an IC50 value of 1.23 µM
and 1.01 µM respectively.
80
60
40
20
0
Control
5c
5d
Ca-4
Figure 3. Effect of compounds on tubulin polymerization: tubulin polymerization was
monitored by the increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h
at 37 °C. CA-4 was used as the reference compound in this study. Values indicated are the mean
± SD of two different experiments performed in triplicate.
Table 2. Inhibition of tubulin polymerization (IC50) for compounds 5c and 5d.
Compound
a IC50 ± SD (in μM)
1.23±0.12
5c
5d
1.01±0.08
Ca-4
1.06±0.12
a Concentration of drug to inhibit 50% of tubulin assembly.
9

Measurement of mitochondrial membrane potential (ΔΨm)
Figure 4. Compounds 5c and 5d triggers mitochondrial injury. Drops in membrane potential
(ΔΨm) was assessed by JC-1 staining of HT-29 cells treated with test compound and samples
were then subjected to flow cytometry analysis on a FACScan (Becton Dickinson) in the FL1,
FL2 channel to detect mitochondrial potential. A: Control cells (HT-29), B: Ca-4 (10 nM), C: 5c
(10 nM), D: 5c (50 nM), E: 5d (10 nM) and F: 5d (50 nM).
The maintenance of mitochondrial membrane potential (∆Ψm) is significant for mitochondrial
integrity and bioenergetic function.19 Mitochondrial changes, including loss of mitochondrial
membrane potential (∆Ψm), are key events that take place during drug-induced apoptosis.
10

Mitochondrial injury by compounds 5c and 5d was evaluated by detecting drops in
mitochondrial membrane potential (ΔΨm). In this study, we have investigated the involvement
of mitochondria in the induction of apoptosis by these compounds. After 48 h of drug treatment
with these compounds, it was observed that reduced mitochondrial membrane potential (ΔΨm)
of HT-29 cells, assessed by JC-1 staining (Figure 4).
Annexin V-FITC for apoptosis
Sample
UL
UR
LL
LR
A: Control
B: CA-4
C: 5c (10 nM)
D: 5c (50 nM)
E: 5d (10 nM)
F: 5d (50 nM)
0.82
2.93
0.83
3.52
0.98
2.92
4.06
20.21
8.34
18.70
9.67
24.46
93.82
73.40
88.68
72.80
86.47
67.20
1.30
3.46
2.16
4.97
2.88
5.42
11

Figure 5: Annexin V-FITC staining. A: Control cells (HT-29), B: CA-4 (10 nM), C: 5c (10 nM),
D: 5c (50 nM), E: 5d (10 nM) and F: 5d (50 nM).
The apoptotic effect of 5c and 5d was further evaluated by Annexin V FITC/PI (AV/PI) dual
staining assay20 to examine the occurrence of phosphatidylserine externalization and to
understand whether it is due to physiological apoptosis or nonspecific necrosis. In this study,
HT-29 cells were treated with these compounds for 48 h at 10 and 50 nM concentrations to
examine the apoptotic effect. It was observed that these compounds showed significant apoptosis
against HT-29 cells as shown in Figure 5. Results indicated that compounds 5c and 5d showed
10.50 and 12.55 % at 10 nM concentration, whereas they exhibited 23.67 and 29.88 % apoptosis
at 50 nM concentration respectively for 48 h (Figure 5).
Molecular Docking
Here, the main objective of molecular docking was to predict the accurate binding poses of these
new Phenstatin-Oxindole conjugates within the constraints of receptor binding site. Cytotoxic
results of these conjugates (especially 5c and 5d on human colon cancer cell lines) encouraged
us to perform their molecular docking studies. Docking studies revealed that these conjugates fit
well in the LOC binding site present at β subunit at α subunit interface of the tubulin (Figure
6A).
The cyclic amide proton in these conjugates exhibited hydrogen bonding (red dashed line in
figure 6C and 6D) with phenolic oxygen of Tyr224A ( A and B represents α subunit and β
subunit of tubulin) amino acid residue. The hydrogen bond length for 5c and 5d conjugates
was1.8Å and 2.2Å respectively. Besides this, these conjugates also showed hydrophobic
interactions (figure 6C and 6D) with the target protein. As similar to colchicine, the trimethoxy
substituted aromatic ring in these conjugate precisely fitted into the hydrophobic pocket (figure
6B) which consists of Cys241B, Ala250B, Lys254B, Leu255B, Ala317B, Val318B, and
Ala354B residues of β subunit. In addition, Thr353B is part of hydrophobic pocket for conjugate
5d. The substituted oxyindolin-2-one rings in these conjugate showed interactions with
Gln247B, Leu248B, Ser178A, Lys352B and GTP600B. Along with that,5-methoxy oxyindolin-
2-one ring present in conjugate5c also interacted with Thr353B. 1,5-disubstituted 2-
12

methoxybenzylidene ring present in these conjugates interacted with Ala180A, Thr179A and
Asn101A and also conjugate 5d also interact with Lys 254B.
Figure 6: A) Surface binding pose of conjugate 5C in LOC binding pocket of β subunit of
tubulin; B) Surface binding pose of co-crystal (colchicine) ligand and conjugate 5c; C) Binding
pose of conjugate 5c; D)Binding pose of conjugate 5d. Conjugate 5c and co-crystal ligand are
shown in stick and coloured by the atom type (Conjugates 5c, 5d and co-crystal ligand are shown
in stick and coloured by the atom type [carbon: cyan (5c and 5d) and yellow (co-crystal ligand);
oxygen: red;hydrogen: white; nitrogen: blue; fluorine: ice blue]
Conclusion
In the present study, phenstatin/isocombretasatin-oxindole conjugates were designed and
synthesized via Knoevenagel reaction and they were investigated for their cytotoxic activity
against five human cancer cells such as prostate (DU-145), lung (A549), colon (HT-29), breast
(MCF-7), liver (HepG2) cancer cells with IC50 values ranging from 0.045-38.90 M. Among
them, two conjugates like 5c and 5d displayed remarkable antiproliferative activity towards lung
(79 nM and 93 nM respectively) and colon (45 nM and 49 nM respectively) cancer cells in
nanomolar range. The flow cytometric analysis revealed that these conjugates cause cell cycle
arrest at G2/M phase. Interestingly, the mitochondrial membrane potential and Annexin V FITC
13

assay suggested that these compounds (5c and 5d) induced cell death by apoptosis. The tubulin
polymerization assay analysis results suggest that these conjugates like 5c and 5d exhibit
significant inhibitory effect on the tubulin assembly with an IC50 value of 1.23 M and 1.01 M
respectively. The binding of these compounds (5c and 5d) was at the colchicine binding site of
tubulin, as indicated by molecular docking studies. Thus, these conjugates could be considered as
potential scaffolds that are useful newer leads for the treatment of prostate cancer.
Experimental Section
I. Chemistry
All chemicals and reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA), Lancaster
(Alfa Aesar, 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 coated glass plates containing 60 GF254 with visualisation achieved by
UV light or iodine indicator. Column chromatography was performed with Merck 60–120 mesh
1
silica gel. H and 13C spectra were recorded on Bruker UXNMR/ XWIN-NMR (300 MHz) or
Inova Varian-VXR-unity (400, 500 MHz) instruments. Chemical shifts () are reported in ppm
downfield from an internal TMS standard. ESI spectra were recorded on a 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 a QSTAR XL Hybrid MS–MS mass
spectrometer. Melting points were determined with an Electro thermal melting point apparatus,
and are uncorrected.
Preparation of methyl 5-iodo-2-methoxybenzoate (8):
To a solution of iodo salicilic acid (7,5.0 g, 0.019 mol) in dry acetone (30 ml), was added
anhydrous K2CO3 (7.85 g, 0.056 mol), dimethyl sulphate (7.16 g, 0.056 mol) at 0 °C. The
reaction mixture was stirred at reflux temperature for 6 h. The reaction was monitored by TLC
using Ethylacetate:hexane (1:19). After completion of the reaction as indicated by the TLC, after
completion of the reaction as indicated by the TLC, K2CO3 was removed by filtration and the
solvent was concentrated under the vacuum, diluted with water and extracted with ethyl acetate.
The combined organic phases were dried (Na2SO4) and evaporated under vacuum and the
residue was purified by column chromatography (40% EtOAc-hexane) to afford compound 8 as
white solid (5.1 g, 92 %); mp: 47-50 °C; 1H NMR (CDCl3, 300 MHz) (ppm): 8.06 (d, J = 2.28
14

Hz, 1H), 7.72 (dd, J = 2.28 Hz, 8.85 Hz, 1H), 6.73 (d, J = 8.85 Hz, 1H), 3.88 (s, 3H), 3.87 (s,
3H); ESI-MS: m/z 292 [M+1]+.
Preparation of (5-iodo-2-methoxyphenyl)methanol (9):
To a solution of methyl 5-iodo-2-methoxybenzoate (8, 5.0 g, 0.017 mol) in dry CH2Cl2 (50 ml)
was added drop wise DIBAL (34 mL, 1.0M in hexane, 0.034 mol) at 0 °C. The reaction mixture
was stirred at room temperature for 2 h. The reaction was monitored by TLC using ethyl acetate-
hexane (4:6). After completion of the reaction as indicated by the TLC, Saturated ammonium
chloride solution was added to the reaction mixture. Salts were removed by filtration, the
solvent was dried (using Na2SO4) and evaporated under vacuum, and it was taken as such for the
next step without further purification.
Preparation of tert-butyl (5-iodo-2-methoxybenzyloxy)dimethylsilane (10):
To a solution of (5-iodo-2-methoxyphenyl)methanol (9, 4.0 g, 0.015 mol) in dry CH2Cl2(30 ml)
was added imidazole (1.16 g, 0.017 mol) and TBDMSCl (2.56 g, 0.017 mol) at 0 °C. The
reaction mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC
using ethyl acetate-hexane (1:10). After completion of the reaction as indicated by the TLC, The
water was added to the reaction mixture and separates the organic layer. The solvent was dried
(Na2SO4) and evaporated under vacuum and the residue was purified by column chromatography
(5% concentrated under the vacuum, the combined organic phases EtOAc-Hexane) to afford
1
compound 10 as colourless oil (5.2 g, 91%); bp: 287-289 °C; H NMR (CDCl3, 300 MHz)
(ppm): 7.72 (d, J = 2.26 Hz, 1H), 7.48 (dd, J = 2.26 Hz, 8.31 Hz, 1H), 6.56 (d, J = 8.31 Hz,
1H), 4.68 (s, 2H), 3.78 (s, 3H), 0.95 (s, 9H), 0.11 (s, 6H); ESI-MS: m/z 379 [M+1]+.
Preparation
of
(3-((tert-butyldimethylsilyloxy)methyl)-4-methoxyphenyl)(3,4,5-
trimethoxyphenyl)methanol (12)
To a solution of tert-butyl(5-iodo-2-methoxybenzyloxy)dimethylsilane (10, 4.0 g, 0.011 mol) in
dry THF (20 ml) was added dropwise n-BuLi (7.5 mL, 1.6 Min hexane, 0.012 mol) at -78 °C.
After 1 h, a solution of 3,4,5 trimethoxybenzaldehyde (11, 1.96 g, 0.01 mol) in dry THF (4 mL)
was added drop wise at -78 °C. After 1 h stirring at rt, The reaction was monitored by TLC using
ethyl acetate-hexane (4:6). After completion of the reaction as indicated by the TLC, Saturated
ammonium chloride solution (10 mL) was added to the reaction mixture. The reaction mass
solvent was evaporated under the vacuum, diluted with water and extracted with ethyl acetate.
The combined organic layers were washed with 1 N HCl and then brine water (20 mL), dried
15

over Na2SO4, filtered, and concentrated in vacuum. Purification of the residue by column
chromatography on silica gel (hexane/EtOAc, 7:3) afforded the desired product 12 as a white
colour solid (5.8 g, 63%); mp: 41-43 °C; 1H NMR (CDCl3, 300 MHz) (ppm): 7.41 (d, J = 1.98
Hz, 1H), 7.25 (dd, J = 1.98 Hz, 8.39 Hz, 1H), 6.79 (d, J = 8.39 Hz, 1H), 6.62 (s, 2H), 5.75 (s,
1H), 4.72 (s, 2H), 3.82 (s, 6H), 3.81 (s, 3H), 3.80 (s, 3H), 0.91 (s, 9H), 0.05 (d, J = 1.67 Hz, 6H);
ESI-MS: m/z 449 [M+1]+.
Preparation
trimethoxyphenyl)methanone (13)
To solution of (3-((tert-butyldimethylsilyloxy)methyl)-4-methoxyphenyl)(3,4,5-
of
(3-((tert-butyldimethylsilyloxy)methyl)-4-methoxyphenyl)(3,4,5-
a
trimethoxyphenyl)methanol (12, 1.0 g, 2.2 mmol) in dry DMSO (5 ml) was added, a solution of
2-iodoxy-benzoic acid (IBX) (0.686 g, 2.45 mmol) in dimethyl sulfoxide (DMSO) (10 mL) at
10-15 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction was
monitored by TLC using ethyl acetate-hexane (1:4). After completion of the reaction as indicated
by the TLC, Appropriate amount water was added to the reaction mixture and filtered through
the celite bead. The aqueous layer was extracted with ethyl acetate. The organic layer was
washed with water, brine, dried over anhydrous Na2SO4, and evaporated by using vacuum to get
crude compounds. The residue was purified by column chromatography (10% concentrated
under the vacuum, the combined organic phases EtOAc-hexane) to afford compound 13 as light
1
yellow solid (820 mg, 82%); mp: 48-49 °C; H NMR (CDCl3, 300 MHz) (ppm): 7.97 (d, J =
2.3 Hz, 1H), 7.80 (dd, J = 2.3 Hz, 8.3 Hz, 1H), 7.02 (s, 2H), 6.90 (d, J = 8.3 Hz, 1H), 4.76 (s,
2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.87 (s, 6H), 0.90 (s, 9H), 0.09 (s, 6H); ESI-MS: m/z 447
[M+1]+.
Preparation
of
tert-butyl(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzyloxy)
dimethylsilane (14)
A solution of PPh3CH3Br (725.8 mg, 2.02 mmol) in dry THF (15 mL) was added, potassium tert-
butoxide (188.4 mg, 1.68 mmol) at 10-15 °C under an argon atmosphere. The yellow colour
mixture was stirred for 4 h. The THF of (3-((tert-butyldimethylsilyloxy)methyl)-4-
methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (13, 500 mg, 1.12 mmol) was added drop
wise at 10-15 °C and reaction mixture was allowed to stirred for another 3 h. The reaction was
monitored by TLC using ethyl acetate-hexane (1:5). After completion of the reaction as indicated
by the TLC, Appropriate amount of saturated ammonium chloride solution was added to the
16

reaction mixture and concentrate it. The aqueous layer was extracted with ethyl acetate. The
organic layer was washed with water, dried over anhydrous Na2SO4, and evaporated by using
vacuum to get crude compounds. The residue was purified by column chromatography (10%
concentrated under the vacuum, the combined organic phases EtOAc-hexane) to afford
1
compound 14 as light yellow solid (400 mg, 80%); mp: 60-63 °C; H NMR (CDCl3, 300 MHz)
(ppm): 7.48 (d, J = 2.26 Hz, 1H), 7.21 (dd, J = 2.26 Hz, 8.49 Hz, 1H), 6.78 (d, J = 8.49 Hz,
1H), 6.64 (s, 2H), 5.39 (d, J = 1.13 Hz, 1H), 5.28 (d, J = 1.32 Hz, 1H), 4.74 (s, 2H), 3.85 (s, 3H),
3.84 (s, 3H), 3.81 (s, 6H), 0.88 (s, 9H), 0.07 (s, 6H); ESI-MS: m/z 445 [M+1]+.
Preparation of (3-(hydroxymethyl)-4-methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone
(15a)
To
a
solution
of
(3-((tert-butyldimethylsilyloxy)methyl)-4-methoxyphenyl)(3,4,5-
trimethoxyphenyl)methanone (13, 800 mg, 1.79 mmol) in dry THF (10 ml) was added, a
solution of tetra butyl ammonium fluoride (2.15 mL, 1.0 M in THF, 2.15 mmol) at 10-15 °C. The
reaction mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC
using ethyl acetate-hexane (4:6). After completion of the reaction as indicated by the TLC,
Appropriate amount of saturated ammonium chloride solution was added to the reaction mixture
and concentrate it. The aqueous layer was extracted with ethyl acetate. The organic layer was
washed with water, dried over anhydrous Na2SO4, and evaporated by using vacuum to get crude
compounds. The residue was purified by column chromatography (30% concentrated under the
vacuum, the combined organic phases EtOAc-hexane) to afford compound 15a as light yellow
solid (450 mg, 76%); mp: 45-47 °C; 1H NMR (CDCl3, 300 MHz) (ppm): 7.91 (d, J = 2.26 Hz,
1H), 7.77 (dd, J = 2.26 Hz, 8.31 Hz, 1H), 6.97 (s, 2H), 6.85 (d, J = 8.31 Hz, 1H), 4.71 (s, 2H),
3.89 (s, 3H), 3.86 (s, 3H), 3.82 (s, 6H); ESI-MS: m/z 333[M+1]+.
Preparation of (2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)methanol (15b)
This compound was prepared according to the method described for compound 15a, employing
tert-butyl(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzyloxy)dimethyl silane (14, 500
mg, 1.124 mmol) to obtain the pure product 15b as light yellow solid (300 mg, 81%); mp: 59-61
1
°C; H NMR (CDCl3, 300 MHz) (ppm): 7.32 (d, J = 2.26 Hz, 1H), 7.23 (d, J = 2.26 Hz, 1H),
6.86 (d, J = 8.49 Hz, 1H), 6.54 (s, 2H), 5.35 (dd, J = 1.13 Hz, 12.8 Hz, 2H), 4.69 (s, 2H), 3.89 (s,
3H), 3.88 (s, 3H), 3.81 (s, 6H); ESI-MS: m/z 331 [M+1]+.
Preparation of 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzaldehyde (16a)
17

A solution of DMSO (0.258 g, 3.3 mmol) in dry CH2Cl2(3 mL) was added drop wise to a
magnetically stirred solution of oxalyl chloride (209 mg, 1.65 mmol) in dry CH2Cl2(5.0 mL) kept
at -78 °C under an argon atmosphere. The mixture was stirred for another 15 min. The CH2Cl2 of
(3-(hydroxymethyl)-4-methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (15a, 500 mg, 1.5
mmol) was added drop wise and reaction mixture was allowed to stirred for 3 h. Triethylamine
(607 mg, 6.0 mmol) was added drop wise over 5 min, and the stirred solution was allowed to
warm to room temperature. The appropriate amount of water was added to reaction mixture and
organic layer was separated and the aqueous phase extracted with CH2Cl2 (3 ×20 mL). The
combined organic layer were washed with brine (10 mL), dried over anhydrous Na2SO4 and
evaporated by using vacuum to get crude compound. The residue was purified by column
chromatography (30% concentrated under the vacuum, the combined organic phases EtOAc-
1
hexane) to afford compound 16a as light yellow solid (420 mg, 84%); mp: 74-76 °C; H NMR
(CDCl3, 300 MHz) (ppm): 10.49 (s, 1H), 8.29 (d, J = 2.26 Hz, 1H), 8.13 (dd, J = 2.26 Hz, 8.87
Hz, 1H), 7.15 (d, J = 8.87 Hz, 1H), 7.01 (s, 2H), 4.05 (s, 3H), 3.95 (s, 3H), 3.87 (s, 6H); ESI-MS:
m/z 331 [M+1]+.
Preparation of 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzaldehyde (16b)
This compound was prepared according to the method described for compound 15a, employing
(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)methanol (15b, 500 mg, 1.51 mmol)to
1
obtain the pure product 16b as light yellow solid (419 mg, 84%); mp: 89-92 °C; H NMR
(CDCl3, 300 MHz) (ppm): 10.48 (s, 1H), 7.88 (d, J = 2.26 Hz, 1H), 7.52 (dd, J = 2.26 Hz, 8.49
Hz, 1H), 6.98 (d, J = 8.49 Hz, 1H), 6.50 (s, 2H), 5.41 (dd, J = 0.92 Hz, 14.3 Hz, 2H), 3.96 (s,
3H), 3.88 (s, 3H), 3.81 (s, 6H); ESI-MS: m/z 329 [M+1]+.
General Preparation of phenstatin/isocombretatstatin-oxindoles (5a-h and 6a-h)
To the phenstatin-3-aldehyde/isocombretastatin-3-aldehyde (16a-b) (0.303mmol/0.304 mmol)
prepared in the above step was added corresponding substituted oxindoles (17a-h) (0.303mmol)
and catalytic amount of piperidine (1.0 ml) in ethanol. Heated the reaction mixture to reflux for 4
h at 85 °C. The solid compounds obtained in the reaction vessel were filtered and washed with
ethanol for 4-5 times. After complete air drying the final compounds
phenstatin/isocombretastatin-oxindole analogues (5a-h and 6a-h) were obtained as pure solids
(yield 71-90%).
Synthesis of (E)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5a)
18

(E)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5a) was obtained
using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303 mmol) and
catalytic amount of piperidine to the oxindole (17a, 40.3 mg, 0.303 mmol) in ethanol as yellow
1
solid (120 mg, yield: (89%); mp: 190-192 °C; H NMR (CDCl3 + 1 drop [D]TFA, 300 MHz)
(ppm): 8.81 (s, 1H), 8.06-8.02 (m, 1H), 7.96 (d, J = 8.12 Hz, 1H), 7.61 (d, J = 7.36 Hz, 1H),
7.30 (d, J = 7.55 Hz, 1H), 7.13 (d, J = 7.55 Hz, 1H), 7.10-7.06 (m, 3H), 6.98 (d, J = 7.74 Hz,
1H), 4.03 (s, 3H), 3.98 (s, 3H), 3.88 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz)
(ppm): 194.3, 161.7, 152,6, 149.3, 144.3, 142.6, 139.6, 137.6, 131,2, 129.7, 127.4, 124.5,
123.3, 121.6, 121.4, 119.9, 119.4, 111.1, 109.7, 107.8,60.9, 56.4, 56.1; ESI-MS: m/z 446
[M+H]+; HRMS (ESI m/z) for C26H24O6N calcd: 446.15981, found: 446.15833 [M+ H]+.
(E)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)-5-nitroindolin-2-one (5b)
(E)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)-5-nitroindolin-2-one
(5d)
was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 5-nitro oxindole (17b) (49.4 mg, 0.303 mmol) in
1
ethanol as light yellow solid (105 mg, yield 72%); mp: 204-205 °C; H NMR (CDCl3 + 1 drop
[D]TFA, 300 MHz) (ppm): 9.04 (d, J = 0.75 Hz, 1H), 8.52 (d, J = 1.5 Hz, 1H), 8.31 (s, 1H),
8.27 (dd, J = 1.5 Hz, 8.3 Hz, 1H), 8.02 (d, J = 9.06 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.09-7.07
(m, 3H), 4.11 (s, 3H), 4.01 (s, 3H), 3.89 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75
MHzppm): 196.6, 162.8, 152.5, 145.2, 143.5, 141.8, 138.3, 136.5, 136.6, 132.8, 128.8,
126.3, 125.6, 122.7, 121.4, 118.8, 115.7, 110.6, 108.1, 61.4, 56.5, 56.3; ESI-MS: m/z 491
[M+H]+; HRMS (ESI m/z) for C26H23O8N2 calcd: 491.14489, found: 491.14338 [M+ H]+.
(E)-5-methoxy-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5c):
(E)-5-methoxy-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5c) was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 5-methoxy oxindole (17c) (53.9 mg, 0.303
1
mmol) in ethanol as light red solid (112 mg, yield 78%); mp: 189-191 °C; H NMR (CDCl3 + 1
drop [D]TFA, 500 MHz) (ppm): 8.32 (d, J = 1.2 Hz, 1H), 8.03 (s, 1H), 7.95 (dd, J = 1.8 Hz,
8.69 Hz, 1H), 7.13-7.15 (m, 2H), 7.02 (s, 2H), 6.95-6.93 (m, 1H), 6.85 (dd, J = 2.1 Hz, 8.5 Hz,
1H), 4.01 (s, 3H), 3.98 (s, 3H), 3.87 (s, 6H), 3.70 (s, 3H); 13C NMR (CDCl3 + 1 drop [D]TFA,
75 MHzppm):195.8, 161.8, 152.8, 146.9, 141.8, 135.4, 135.1, 132.3, 132.1, 129.4, 128.1,
19

123.4, 123.1, 122.0, 115.8, 113.4, 111.7, 110.5, 109.5, 107.6, 61.1, 56.2, 55.9; ESI-MS: m/z 476
[M+H]+; HRMS (ESI m/z) for C27H26O7N calcd: 476.17038, found: 476.16928 [M+ H]+.
Synthesis of (E)-5-fluoro-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-
one (5d)
(E)-5-fluoro-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5d) was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 5-fluoro oxindole (17d, 45.7 mg, 0.303 mmol)
1
in ethanol as yellow solid (118 mg, yield: (84%); mp: 201-203 °C; H NMR (CDCl3 + 1 drop
[D]TFA, 300 MHz) (ppm): 8.81-8.79 (m, 1H), 8.16 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.72 (d, J
= 8.3 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 7.09-7.07 (m, 3H), 4.03 (s, 3H),
3.98 (s, 3H), 3.88 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHzppm): 194.9, 164.1,
160.9, 159.7, 159.1, 152.8, 142.2, 140.1, 134.4, 133.7, 131.6, 130.7, 122.5, 120.4, 116.6, 116.4,
112.8, 112.5, 112.1, 107.7, 61.1, 57.0, 56.9; ESI-MS: m/z 464 [M+H]+; HRMS (ESI m/z) for
C26H23O6NF calcd: 464.15039, found: 464.14934 [M+ H]+.
(E)-5-chloro-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5e)
(E)-5-chloro-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5e) was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 5-chloro oxindole (17e) (50.6 mg, 0.303 mmol)
1
in ethanol as light red solid (109 mg, yield 75%); mp: 188-190 °C; H NMR (CDCl3 + 1 drop
[D]TFA, 500 MHz) (ppm): 8.89 (d, J = 0.9 Hz, 1H), 8.07 (s, 1H), 7.98 (dd, J = 1.4 Hz, 8.2 Hz,
1H), 7.57 (d, J = 1.86 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 7.09-7.07 (m, 3H), 6.92 (d, J = 8.2 Hz,
1H), 4.05 (s, 3H), 3.99 (s, 3H), 3.88 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75
MHzppm): 198.0, 162.6, 152.5, 135.9, 135.1, 133.1, 132.7, 132.3, 130.3, 129.3, 128.7,
123.3, 122.5, 121.8, 121.7, 120.2, 111.7, 110.5, 107.8, 61.2, 56.3, 56.1; ESI-MS: m/z 480
[M+H]+; HRMS (ESI m/z) for C26H23O6NCl calcd: 480.12084, found: 480.11969 [M+ H]+.
(E)-6-chloro-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5f)
(E)-6-chloro-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5f) was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 6-chloro oxindole (17f) (50.6 mg, 0.303 mmol)
1
in ethanol as light red solid (113 mg, yield 78%); mp: 179-182 °C; H NMR (CDCl3 + 1 drop
[D]TFA, 300 MHz) (ppm): 8.86-8.84 (m, 1H), 8.04 (s, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.52 (d, J
20

= 8.1 Hz, 1H), 7.11 (d, J = 7.9 Hz, 1H), 7.09-7.07 (m, 3H), 7.00 (s, 1H), 4.04 (s, 3H), 3.99 (s,
13
3H), 3.88 (s, 6H); C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz) (ppm): 198.1, 161.4, 152.8,
138.8, 136.3, 134.9, 132.3, 129.8, 128.4, 124.6, 123.3, 122.6, 120.1, 113.5, 111.8, 110.5, 109.9,
107.9, 107.4, 61.0, 56.2, 56.0; ESI-MS: m/z 480 [M+H]+; HRMS (ESI m/z) for C26H23O6NCl
calcd: 480.12084, found: 480.11969 [M+ H]+.
(E)-5-bromo-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5g)
(E)- 5-bromo-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5g) was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 5-bromo oxindole (17g) (63.6 mg, 0.303 mmol)
in ethanol as light red solid (123 mg, yield 78%); mp: 184-186 °C; 1H NMR (CDCl3 + 1 drop
[D]TFA, 500 MHz) (ppm): 8.89-8.87 (m, 1H), 8.06 (s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.65 (d, J
= 7.8 Hz, 1H), 7.30 (d, J = 7.55 Hz, 1H), 7.23 (d, J = 7.55 Hz, 1H), 7.10-7.07 (m, 3H), 4.05 (s,
3H), 3.99 (s, 3H), 3.88 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHzppm):197.2,
162.7, 152.5, 136.4, 136.0, 132.3, 132.1, 128.9, 128.6, 126.1, 125.4, 123.1, 121.8, 116.1, 114.5,
112.7, 112.2, 110.9, 110.5, 108.1, 107.7, 61.2, 56.8; ESI-MS: m/z 480 [M+H]+; HRMS (ESI
m/z) for C26H23O6NBr calcd: 524.07033, found: 524.06896 [M+ H]+ and C26H23O6N81Br calcd:
526.06828, found: 526.06694 [M+ H]+.
(E)-6-bromo-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5h)
E)- 6-bromo-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzylidene)indolin-2-one (5h) was
obtained using above described method by adding phenstatin-3-aldehyde (16a) (100 mg, 0.303
mmol) and catalytic amount of piperidine to the 6-bromo oxindole (17h) (63.6 mg, 0.303 mmol)
1
in ethanol as light yellow solid (123 mg, yield 78%); Yield: 119 mg (75%); mp: 181-185 °C; H
NMR (CDCl3 + 1 drop [D]TFA, 300 MHz) (ppm): 8.90-8.89 (m, 1H), 8.06 (s, 1H), 7.98 (d, J =
8.68 Hz, 1H), 7.70 (s, 1H), 7.39 (d, J = 8.68 Hz, 1H), 7.10-7.07 (m, 3H), 6.84 (d, J = 8.31 Hz,
1H), 4.05 (s, 3H), 3.99 (s, 3H), 3.88 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz)
(ppm):197.3, 162.3, 161.7, 161.1, 152.4, 141.3, 140.9, 136.2, 135.5, 134.8, 132.8, 126.6, 124.9,
123.2, 122.1, 121.2, 119.9, 116.1, 112.4, 108.6, 108.4, 61.4, 56.4; HRMS (ESI m/z) for
C26H23O6NBr calcd: 524.07033, found: 524.06896 [M+ H]+ and C26H23O6N81Br calcd:
526.06828, found: 526.06694 [M+ H]+.
(E)-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one (6a)
21

(E)-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6a)
was
obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100 mg,
0.304 mmol) and catalytic amount of piperidine to the oxindole (17a) (40.4 mg, 0.304 mmol) in
ethanol as light red solid (119 mg, yield 88%); mp: 179-180 °C;1H NMR (CDCl3 + 1 drop
[D]TFA, 500 MHz) (ppm): 8.10 (s, 1H), 7.73 (d, J = 1.7 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H),
7.47 (dd, J = 1.9 Hz, 8.7 Hz, 1H), 7.25-7.20 (m, 1H), 6.99 (t, J = 7.63 Hz, 16.1 Hz, 2H), 6.89 (t,
J = 7.6 Hz, 15.2 Hz, 1H), 6.58 (s, 2H), 5.40 (d, J = 7.5 Hz, 1H), 5.36 (d, J = 7.3 Hz, 1H), 3.93
(s, 3H), 3.91 (s, 3H), 3.83 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz) (ppm): 170.0,
157.9, 152.9, 149.1, 141.2, 137.7, 137.6, 133.4, 131.3, 129.6, 124.5, 123.3, 122.8, 121.8, 121.5,
113.4, 110.6, 110.2, 110.0, 105.4, 60.8, 56.0, 55.6; ESI-MS: m/z 444 [M+H]+; HRMS (ESI m/z)
for C27H26O5N calcd: 444.18055, found: 444.17838 [M+ H]+.
(E)-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)-5-nitroindolin-2-one
(6b)
(E)-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)-5-nitroindolin-2-one
(6b)
was obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100
mg, 0.304 mmol) and catalytic amount of piperidine to the 5-nitro oxindole (17b) (54.2 mg,
0.304 mmol) in ethanol as yellow solid (109 mg, yield 73%); mp: 181-183 °C; 1H NMR
(CDCl3 + 1 drop [D]TFA, 500 MHz) (ppm): 8.57 (d, J = 1.9 Hz, 1H), 8.28 (s, 1H), 8.22 (dd, J
= 2.1 Hz, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.45 (dd, J = 1.8 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 7.02
(d, J = 8.7 Hz, 1H), 6.58 (s, 2H), 5.49 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 3.95 (s, 3H),
3.90 (s, 3H), 3.83 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHzppm): 158.3, 152.4,
145.1, 143.5, 140.5, 136.4, 133.9, 133.5, 129.5, 122.1, 121.8, 118.6, 114.4, 111.3, 111.1, 110.9,
110.8,, 107.7, 105.7, 61.1, 56.1, 55.8; ESI-MS: m/z 489 [M+H]+; HRMS (ESI m/z) for
C27H25O7N2 calcd: 489.16563, found: 489.16464 [M+ H]+.
(E)-5-methoxy-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6c)
(E)-5-methoxy-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6c) was obtained using above described method by adding isocombretastatin-3-aldehyde (16b)
(100 mg, 0.304 mmol) and catalytic amount of piperidine to the 5-methoxy oxindole (17c) (49.6
1
mg, 0.304 mmol) in ethanol as yellow solid (118 mg, yield 81%); mp: 178-180 °C; H NMR
(CDCl3 + 1 drop [D]TFA, 500 MHz)  (ppm): 7.97 (s, 1H), 7.80 (d, J = 2.26 Hz, 1H), 7.34 (dd, J
22

= 2.26 Hz, 9.1 Hz, 1H), 7.16 (d, J = 2.26 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.79 (d, J = 8.3 Hz,
1H), 6.75 (dd, J = 2.26 Hz, 8.3 Hz, 1H), 6.54 (s, 2H), 5.36 (d, J = 13.5 Hz, 2H), 3.90 (s, 3H),
13
3.87 (s, 3H), 3.81 (s, 6H), 3.62 (s, 3H); C NMR (CDCl3 + 1 drop [D]TFA, 75 MHzppm):
170.2, 157.8, 154.4, 152.9, 149.0, 137.0, 135.3, 133.9, 133.2, 131.4, 129.4, 123.3, 122.7, 114.9,
113.2, 110.6, 110.3, 109.3, 105.3, 60.9, 56.0, 55.6, 55.7; ESI-MS: m/z 474 [M+H]+; HRMS (ESI
m/z) for C28H28O6N calcd: 474.19111, found: 474.19001 [M+ H]+.
(E)-5-fluoro-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6d)
(E)-5-fluoro-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one (6d)
was obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100
mg, 0.304 mmol) and catalytic amount of piperidine to the 5-fluoro oxindole (17d) (45.9 mg,
0.304 mmol) in ethanol as yellow solid (120 mg, yield 86%); mp: 178-179 °C; 1H NMR
(CDCl3 + 1 drop [D]TFA, 500 MHz) (ppm): 8.13 (s, 1H), 7.70 (d, J = 1.13 Hz, 1H), 7.44 (dd, J
= 2.1 Hz, 8.7 Hz, 1H), 7.25 (dd, J = 2.4 Hz, 9.0 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.94 (dd, J =
2.4 Hz, 8.7 Hz, 1H), 6.90-6.87 (m, 1H), 6.54 (s, 2H), 5.41 (d, J = 7.5 Hz, 1H), 5.38 (d, J = 7.5
13
Hz, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.82 (s, 6H); C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz)
(ppm): 170.3, 160.7, 159.6, 152.5, 138.8, 138.1, 136.4, 135.9, 133.6, 132.7, 129.3, 126.0,
122.2, 116.5, 116.1, 112.7, 111.9, 111.1, 110.1, 109.1, 105.5, 61.1, 56.1, 55.8; ESI-MS: m/z 462
[M+H]+; HRMS (ESI m/z) for C27H25O5NF calcd: 462.17113, found: 462.16957 [M+ H]+.
(E)-5-chloro-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6e)
(E)-5-chloro-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one (6e)
was obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100
mg, 0.304 mmol) and catalytic amount of piperidine to the 5-chloro oxindole (17e) (50.9 mg,
0.304 mmol) in ethanol as yellow solid (110 mg, yield 76%); mp: 178-180 °C; 1H NMR
(CDCl3 + 1 drop [D]TFA, 500 MHz) (ppm): 8.11 (s, 1H), 7.72 (d, J = 1.7 Hz, 1H), 7.48 (dd, J
= 3.6 Hz, 8.7 Hz, 1H), 7.29 (dd, J = 2.1 Hz, 8.9 Hz, 1H), 6.99 (d, J = 8.7 Hz, 1H), 6.94 (dd, J =
3.5 Hz, 8.9 Hz, 1H), 6.91-6.88 (m, 1H), 6.54 (s, 2H), 5.41 (d, J = 7.5 Hz, 1H), 5.38 (d, J = 7.5
13
Hz, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.82 (s, 6H); C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz)
(ppm): 172.9, 156.5, 154.5, 150.2, 138.6, 138.4,133.9, 133.1, 130.1, 129.1, 128.2, 128.0, 122.8,
23

123.1, 122.1, 121.5, 111.9, 111.6, 110.5, 110.0, 107.4, 106.5, 60.9, 56.0, 55.6; ESI-MS: m/z 478
[M+H]+; HRMS (ESI m/z) for C27H25O5NCl calcd: 478.14158, found: 478.14158 [M+ H]+.
(E)-6-chloro-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6f)
(E)-6-chloro-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one (6f)
was obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100
mg, 0.304 mmol) and catalytic amount of piperidine to the 6-chloro oxindole (17f) (50.9 mg,
0.304 mmol) in ethanol as yellow solid (116 mg, yield 79%); mp: 179-182 °C; 1H NMR
(CDCl3 + 1 drop [D]TFA, 500 MHz) (ppm): 8.13 (s, 1H), 7.81 (d, J = 1.5 Hz, 1H), 7.51 (d, J =
8.9 Hz, 1H), 7.31 (d, J = 8.9 Hz, 1H), 6.89 (d, J = 6.58 Hz, 1H), 6.68 (d, J = 2.4 Hz, 1H), 6.90-
6.87 (m, 1H), 6.54 (s, 2H), 5.41 (d, J = 7.5 Hz, 1H), 5.38 (d, J = 7.5 Hz, 1H), 3.92 (s, 3H), 3.88
(s, 3H), 3.82 (s, 6H); 13C NMR (CDCl3 + 1 drop [D]TFA, 75 MHz) (ppm): 173.6, 161.1, 160.5,
159.9, 151.8, 149.4, 147.5, 140.0, 134.5, 130.2, 129.5, 127.2, 123.2, 116.4, 115.4, 112.6, 111.2,
111.1, 109.0, 106.9, 61.3, 56.1, 55.7; ESI-MS: m/z 478 [M+H]+; HRMS (ESI m/z) for
C27H25O5NCl calcd: 478.14158, found: 478.14158 [M+ H]+.
(E)-5-bromo-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6g)
(E)-5-bromo-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one (6g)
was obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100
mg, 0.304 mmol) and catalytic amount of piperidine to the 5-bromo oxindole (17g) (64.5 mg,
0.304 mmol) in ethanol as yellow solid (119 mg, yield 75%); mp: 184-186 °C; 1H NMR
(CDCl3 + 1 drop [D]TFA, 500 MHz) (ppm):8.09 (s, 1H), 7.77 (dd, J = 1.8 Hz, 8.5 Hz, 2H),
7.40-7.35 (m, 2H), 6.96 (d, J = 8.5 Hz, 1H), 6.55 (s, 2H), 6.48 (dd, J = 2.3 Hz, 18.1 Hz, 1H),
13
5.42 (d, J = 18.1 Hz, 2H), 3.92 (s, 3H), 3.88 (s, 3H), 3.82 (s, 6H); C NMR (CDCl3 + 1 drop
[D]TFA, 75 MHz) (ppm): 176.7, 160.6, 160.0, 157.1, 151.8, 149.4, 140.3, 137.7, 136.2, 134.5,
133.5, 132.3, 131.1, 126.7, 122.8, 120.7, 116.3, 115.9, 112.5, 109.1, 106.6, 61.2, 56.0, 55.6; ESI-
MS: m/z 522 [M+H]+; HRMS (ESI m/z) for C27H25O5NBr calcd: 522.09106, found: 522.09033
[M+ H]+ and C26H23O6N81Br calcd: 524.08902, found: 524.08823 [M+ H]+.
(E)-6-bromo-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one
(6h)
24

(E)-6-bromo-3-(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benzylidene)indolin-2-one (6h)
was obtained using above described method by adding isocombretastatin-3-aldehyde (16b) (100
mg, 0.304 mmol) and catalytic amount of piperidine to the 6-bromo oxindole (17h) (64.5 mg,
0.304 mmol) in ethanol as yellow solid (121 mg, yield 76%); Yield: 120 mg (76%); mp: 181-
1
185 °C; H NMR (CDCl3 + 1 drop [D]TFA, 500 MHz) (ppm): 8.10 (s, 1H), 7.65 (s, 1H), 7.47
(dd, J = 1.5 Hz, 8.9 Hz, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.11 (s, 1H), 7.02-6.97 (m, 2H), 6.55 (s,
2H), 5.38-5.35 (m, 2H), 3.93 (s, 3H), 3.91 (s, 3H), 3.83 (s, 6H); 13C NMR (CDCl3 + 1 drop
[D]TFA, 75 MHz) (ppm): 178.1, 159.9, 157.9, 157.7, 150.7, 148.5, 139.7, 135.4, 133.1,
132.9, 132.5, 133.1, 124.1, 122.4, 119.9, 115.3, 111.9, 111.7, 109.2, 105.9, 61.1, 59.9, 55.7;
ESI-MS: m/z 522 [M+H]+; HRMS (ESI m/z) for C27H25O5NBr calcd: 522.09106, found:
522.09023 [M+ H]+ and C26H23O6N81Br calcd: 524.08902, found: 524.08814 [M+ H]+.
II. Biology
Anticancer activity
The cytotoxic activity of the compounds was determined using MTT assay.21 Cells were seeded
in 100 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. After incubation cells were treated with test
compounds for 48 h. 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.
Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells through
the cell-cycle phases. HT-29 cells were incubated for 48 h with compounds 5c and 5d at
concentrations of 10 and 50 nM. 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).22
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
25

compounds. Tubulin polymerization was followed by a time dependent increase in fluorescence
due to the incorporation of a fluorescence reporter into microtubules as polymerization proceeds.
Fluorescence emission at 420 nm (excitation wavelength is 360 nm) was measured by using a
Varioscan multimode plate reader (Thermo scientific Inc.). CA-4 was used as reference
compound in this study. To determine the IC50 values of the compounds against tubulin
polymerization, the compounds were pre-incubated with tubulin at varying concentrations.
Assays were performed under similar conditions as employed for polymerization assays as
described above.23
Mitochondrial membrane potential
HT-29 cells were cultured in six-well plates after treatment with compounds 5c and 5d at 10 and
50 nM concentrations for 48 h. After 48 h of treatment, cells were collected by trypsinization and
washed with PBS followed by resuspending in JC-1 (5 µg/ml) and incubated at 37 0C for 15 min.
Cells were rinsed three times with medium and suspended in pre warmed medium. The cells
were then subjected to flow cytometric analysis on a flow cytometer (Becton Dickinson) in the
FL1, FL2 channel to detect mitochondrial potential.24
Annexin staining assay for apoptosis
HT-29 cells were seeded in six-well plates and allowed to grow overnight. The medium was then
replaced with complete medium containing compounds 5c and 5d at 10 and 50 nM
concentrations. After 48 h of drug treatment, cells from the supernatant and adherent monolayer
cells were harvested by trypsinization, washed with PBS at 5000 rpm. Then the cells were
stained with Annexin VFITC and propidium iodide using the Annexin-V-FITC apoptosis
detection kit (Sigma aldrich). Flow cytometry was performed for this study as described earlier.25
Molecular docking
The molecular docking studies were performed at the colchicine (LOC) binding site of the
tubulin (PDB ID: 3E22).26 The co-ordinates of the crystal structure were obtained from RCSB-
Protein Data Bank and suitable corrections to it were made by using Protein Preparation Wizard
from Schrödinger package. In regard to the ligands, molecules were constructed using
ChemBio3D Ultra 12.0 and their geometries were optimized using molecular mechanics. Finally,
docking studies were performed on the most active molecules (5c and 5d) by using AutoDock
4.2 docking software27and the results were visualized through PyMOL.28
26

Acknowledgement
G.B.K, V.S.R and A.B.S, acknowledge CSIR, New Delhi 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) and King Saud University, Riyadh
(Saudi Arabia).
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