Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors and mitochondrial apoptotic inducers

Article abstract of DOI:10.1039/c4ob02606c

A series of phenstatin/isocombretastatin-chalcones were synthesized and screened for their cytotoxic activity against various human cancer cell lines. Some representative compounds exhibited significant antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI₅₀ values in the range of 0.11 to 19.0 μM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds (3a-l and 4a-l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7 and MDA-MB-231 with IC₅₀ values in the range of 0.5 to 19.9 μM. Moreover, the tubulin polymerization assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e exhibited significant inhibitory effect on the tubulin assembly with an IC₅₀ value of 0.8 μM and 0.6 μM respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding site of tubulin. A cell cycle assay revealed that these compounds arrest at the G₂/M phase and lead to apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9, DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking studies indicated that compounds like 3e occupy the colchicine binding site of tubulin. This journal is

Full text of DOI:10.1039/c4ob02606c

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of phenstatin/isocombretastatin–
chalcone conjugates as potent tubulin
polymerization inhibitors and mitochondrial
apoptotic inducers†
Cite this: Org. Biomol. Chem., 2015,
3, 3963
1
a,b
a
a
a
Ahmed Kamal,* G. Bharath Kumar, M. V. P. S. Vishnuvardhan, Anver Basha Shaik,
a
a
a
Vangala Santhosh Reddy, Rasala Mahesh, Ibrahim Bin Sayeeda and
Jeevak Sopanrao Kapure
b
A series of phenstatin/isocombretastatin–chalcones were synthesized and screened for their cytotoxic
activity against various human cancer cell lines. Some representative compounds exhibited significant
antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI50 values in the
range of 0.11 to 19.0 µM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative
efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds
(
3a–l and 4a–l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7
and MDA-MB-231 with IC50 values in the range of 0.5 to 19.9 µM. Moreover, the tubulin polymerization
assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e
exhibited significant inhibitory effect on the tubulin assembly with an IC50 value of 0.8 μM and 0.6 μM
respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding
Received 15th December 2014,
Accepted 10th February 2015
site of tubulin. A cell cycle assay revealed that these compounds arrest at the G /M phase and lead to
2
apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9,
DOI: 10.1039/c4ob02606c
DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking
www.rsc.org/obc
studies indicated that compounds like 3e occupy the colchicine binding site of tubulin.
5
ment of a variety of human cancers. More recently, taxanes
such as paclitaxel and docetaxel have shown strong activity in
Introduction
6
The discovery of natural as well as synthetic compounds that
are capable of interfering with the microtubule assembly or
disassembly has gained much attention because microtubules
constitute an attractive chemotherapeutic target for anticancer
treating tumors in breast, ovaries, lung, and other tissues.
Vinca alkaloids like vinblastine and vincristine are major
drugs for the treatment of leukemia, lymphoma and a variety
7
of other diseases.
1
,2
drugs. Microtubules are present in all eukaryotic cells and
play an essential role in the formation of mitotic spindles as
Combretastatin A-4 (CA-4, 1a, Fig. 1) is another micro-
8
tubule-destabilizing agent, isolated from Combretum caffrum,
3
well as cell division. The classical microtubule-targeting
agents are antimitotic drugs that affect cellular migration,
intracellular trafficking and cell secretion. For example,
taxanes, vinca alkaloids and the new spindle-specific drugs are
inhibitors of Kinesin-5 (aka KSP, Eg5, KIF11), Polo kinase-1
and Aurora kinases and they play a central role in the treat-
which exhibits promising cytotoxicity towards a wide range of
human cancer cell lines, including multidrug-resistant cancer
4
9
cells which binds to the β-tubulin at the colchicine binding
1
0
site and significantly inhibits tubulin polymerization.
However, the main drawbacks associated with CA-4 are bio-
availability and isomerization of the biologically active Z-con-
figured double bond into the inactive E-configuration during
1
1
a
long storage and administration. To overcome this, the
ethenyl bridge of the stilbene moiety was replaced by a biologi-
cally stable keto group (phenstatin) and bioisosteric replace-
ment of the (Z)-1,2-ethylene group with a 1,1-ethylene bridge
of the stilbene moiety (isocombretastatin) that resulted in the
retention of biological activity with improved physical
Medicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical
Technology, Hyderabad 500007, India. E-mail: ahmedkamal@iict.res.in;
Fax: (+)91-40-27193189; Tel: (+)91-40-27193157
b
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research, Hyderabad 500 037, India
1
13
†
Electronic supplementary information (ESI) available: H NMR and C NMR
spectra of all phenstatin/isocombretastatin–chalcones (3a–l and 4a–l) are shown
1
2
in ESI. See DOI: 10.1039/c4ob02606c
properties.
This journal is © The Royal Society of Chemistry 2015
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Organic & Biomolecular Chemistry
Results and discussion
Chemistry
The 5-iodo salicylic acid (5) was converted to aryl iodo ester 6.
The obtained aryl iodo ester 6 is reduced with DIBAL to
produce the corresponding alcohol (7). This alcohol 7 pro-
tected with TBDMS to give tert-butyl (5-iodo-2-methoxy-
phenoxy)dimethylsilane (8). The resulting aryl iodide 8 was
reacted with trimethoxy benzaldehyde (9) in the presence of
n-BuLi in dry THF to generate benzhydrol derivative 10. The
obtained benzhydrol derivative 10 on oxidation to give ketone
1
1
3a and subsequent witting methylation afforded the olefin
3b. These compounds 13a and 13b were oxidized with the
swern method to produce phenstatin 3-aldehyde/isocombretas-
tatin 3-aldehyde (14a,b) (Scheme 1).
The Claisen–Schmidt condensation of phenstatin 3-alde-
hyde/isocombretastatin 3-aldehyde (14a,b) with substituted
acetophenones (15a–l) in the presence of KOH (10%) produced
phenstatin/isocombretastatin–chalcone conjugates (3a–l and
Fig. 1 Chemical structures of microtubule targeting agents. CA-4 (1a),
phenstatin (1b), phenstatin amine (1c), isocombretastatin A-4 (1d), iso-
combretastatin A-4 amine (1e), chalcone (2), and phenstatin/isocom-
bretastatin–chalcone conjugates (3a–l and 4a–l).
Phenstatin (1b, Fig. 1) and its derivatives reported by the 4a–l) as shown in Scheme 1. All the new compounds syn-
Pettit group exhibited substantial anticancer and antimitotic thesized were characterized by spectral analysis.
1
3
activity, comparable to CA-4. In addition, phenstatin and its
derivatives were easier to synthesize than the combretastatins
Biological studies
(which require control of geometric selectivity) and have
Antiproliferative activity. The phenstatin–chalcones (3a–l)
greater pharmacological potential due to improved metabolic and isocombretastatin–chalcones (4a–l) were evaluated for
1
4
stability. Moreover, the carbonyl oxygen of phenstatin is their cytotoxic activity against a panel of 60 human cancer cell
capable of forming crucial interactions at the colchicine lines at the National Cancer Institute (NCI, Bethesda, MD,
1
5
binding site of the tubulin.
Recently, disclosed CA-4 USA). This panel is organized into nine sub-panels represent-
analogues isocombretastatin A-4 (isoCA-4, 1d, Fig. 1) and its ing leukemia, melanoma, lung, colon, kidney, ovary, breast,
derivatives displayed potent antiproliferative activity against prostate, and central nervous system. Among the twenty-four
various human cancer cell lines and inhibited tubulin compounds, nine compounds (3a–e, 3g, 3j, 4a and 4c) were
polymerization at the micromolar level, and arrested cancer selected for the preliminary test at a single concentration
1
6,12b
cells in the G
2
/M phase of the cell cycle.
(10 μM) screen. These nine compounds exhibited significant
Chalcones (2, Fig. 1) are naturally occurring compounds mean growth inhibitory effect and the results are illustrated in
that belong to the flavonoid family and have displayed a wide Table 1. Apart from 3a, other compounds (3b–e, 3g, 3j, 4a and
variety of biological activities. Notable anticancer potential of 4c) were further evaluated in the secondary screening at five
the chalcones involves their capacity to induce apoptosis in a concentrations with 10-fold dilutions. These eight compounds
1
7,18
variety of cell types, including breast cancers.
Recently, manifested significant cytotoxic activity with GI50 values
Ducki and coworkers synthesized and developed CA-4 type ranging from 0.11 to 19.0 µM and the results are listed in
chalcones wherein the cis-double bond of CA-4 was replaced by Table 2.
an olefin conjugated to a carbonyl group. These chalcone
Particularly, the presence of a carbonyl group between A
analogs showed potent inhibition of tubulin polymerization and B rings in 3e and 3g shows noticeable cytotoxicity. Com-
and arrested the cell at the G /M phase of the cell cycle and pound 3e having strong electron donating substituents like
2
1
9
displayed promising antiproliferative activity. The prodrugs meta-amine and para-methoxy groups on the C-ring exhibited
2
0
of these chalcones are currently under preclinical evaluation.
a broad spectrum of inhibition with GI50 values in the nano-
More recently, a series of resveratrol–chalcone conjugates were molar range against most of the cell lines. More particularly,
shown to possess significant cytotoxic activity against the this conjugate displayed excellent cytotoxic activity with GI50
2
1
various human cancer cell lines.
values of 350 nM, 350 nM, 370 nM, 110 nM, 380 nM and
Therefore as described above in view of their potential as 330 nM against CCRF-CEM (leukemia), NCI-H522 (non-small
anticancer agents, we envisaged to combine the structural cell lung), HCT-15 (colon), UACC-62 (melanoma), NCI/ADR-RES
features of these three pharmacophores for developing newer (ovarian), and MCF-7 (breast) cancer cells respectively. In com-
more effective cytotoxic compounds. Interestingly, these new parison, compound 3g with electron withdrawing substituents
molecules exhibited promising cytotoxic activity and prompted like fluoro at the para position on the C-ring exhibited com-
us to investigate their role in the proliferation and apoptosis of paratively less cytotoxicity with GI50 values of 2.24 μM,
human breast cancer cells (MCF-7). We also investigated their 2.02 μM, 2.14 μM, 2.01 μM, 3.69 μM and 2.30 μM respectively
effect on the proteins that regulate the cell-cycle progression.
against the above cancer cells. This reveals that replacing the
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Scheme 1 Reagents & conditions: (a) DMS, K
2
CO
3
, acetone, 60 °C, 8 h; (b) DIBAL-H (1 N), THF, 5 °C, 3 h; (c) TBDMSCl, imidazole, CH
2 2
Cl , 3 h; (d)
n-BuLi (1.6 N), THF −78 °C; (e) IBX, DMSO, 10 °C, 2 h; (f) CH
78 °C to rt, Et N; (i) EtOH, 10% KOH (aq.), 6–12 h, rt.
2
PPh I, t-ButOK, THF, 5 °C, 8 h; (g) TBAF (1 N), THF, 5 °C, 6 h; (h) oxalyl chloride, DMSO,
3
−
3
electron donating by withdrawing groups on the C-ring signifi-
cantly influences their antiproliferative activities.
However, the presence of a bicyclic ring system on 3c and
c like the 3,4-methlenedioxy group on the C-ring resulted in
Table 1 Single dose screening of the nine selected compounds of
phenstatin–chalcones (3a–l) and isocombretastatin–chalcones (4a–l) in
NCI-60 cell line
4
varied cytotoxic activity. Compound 3c having a carbonyl
group between the A and B rings manifested significant
antiproliferative activity in leukemia, colon and breast cancer
subpanels of NCI, whereas compound 4c having an ethylene
bridge in between rings A and B displayed slightly better anti-
proliferative efficacy than 3c against prostate and lung cancer
cells.
However, the remaining conjugates like 3b, 3d, 3j and 4a
having electron donating substituents like 3,4-dimethoxy,
4-methoxy, 4-hydroxy and 3,4,5-trimethoxy respectively on the
C-ring show moderate to good cytotoxic activity against most
of the cell lines. Specifically, compound 3e exhibited promis-
ing cytotoxicity against leukemia, colon, ovarian and breast
cancer cells within the submicromolar range (Fig. 2, Table 2).
Mean growth
percentage
a
S.no
Compound
01
02
03
04
05
06
07
08
09
3a
3b
3c
3d
3e
3g
3j
85.71
24.41
8.85
55.88
−21.93
b
55.77
22.23
−8.50
b
4a
4c
13.72
a
Determined at 10 μM: data were obtained from the NCI in vitro
b
disease-oriented human tumor cell screen. Negative value indicates
cell death.
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Table 2 In vitro cytotoxic effect of conjugates 3b, 3c, 3d, 3e, 3j, 4a and 4c against a panel of 60 human cancer cell lines
a
GI50 values (μM)
b
c
d
j
e
f
g
h
i
Cancer panel/cell line
3b
3c
3d
3e
3g
3j
4a
4c
Leukemia
CCRF-CEM
HL60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.46
0.40
0.66
0.58
0k.29
0.58
2.16
0.37
1.61
1.04
0.40
—
—
0.35
1.48
0.38
0.61
0.41
0.46
2.24
4.80
2.34
3.24
2.32
2.04
0.87
0.69
0.35
0.84
1.56
0.43
0.35
2.03
0.92
1.61
0.45
0.46
0.91
2.67
0.78
3.08
1.56
0.90
k
2k.86
—
2.54
1.08
—
Non-small cell lung
A549/ATCC
HOP-62
k
j
—
4.39
4.40
12.0
2.23
2.67
4.04
3.09
1.28
—
3.20
2.03
2.94
2.14
1.42
1.80
2.13
0.35
16.5
1.87
21.1
1.81
2.27
1.14
1.41
1.95
2.93
1.44
1.10
3.54
4.16
1.78
1.73
3.20
4.73
2.34
1.55
3.41
1k.69
11.8
5.8
3.92
2.70
3.57
6.20
15.4
16.0
2.02
HOP-92
1.21
2.50
2.93
6.03
2.56
2.69
NCI-H226
NCI-H23
NCIH322M
NCI-H460
NCI-H522
Colon
COLO-205
HCC-2998
HCT-116
HCT-15
—
4.j34
4.13
8.89
2.43
0.68
—j
—
j
—
2.34
j
3.00
4.14
0.41
0.42
0.49
1.14
0.32
3.51
2.09
1.65
1.38
1.68
1.67
0.39
—j
1.47
1.27
0.66
0.37
0.42
0.55
0.37
5.18
7.29
1.75
2.14
2.44
2.79
2.53
15.1
2.03
1.84
0.60
0.55
0.62
1.16
0.60
4.15
2.19
2.01
1.68
2.98
2.51
1.92
—
1.66
1.58
2.15
5.35
2.44
2.51
2.00
2.j54
—j
HT29
KM12
SW-620
—
3.07
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
3.00
5.26
3.08
2.70
3.86
1.14
2.95
3.43
2.02
1.65
3.11
1.51
3.51
6.27
1.37
2.30
1.53
1.06
1.16
1.02
3.86
7.21
1.98
2.26
3.18
1.85
3.98
6.50
2.72
4.42
1.62
4.58
1.46
2.90
1.41
1.39
0.89
0.77
3.18
4.14
2.36
2.79
1.64
2.54
—j
4.54
4.75
3.11
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian
0k.55
1.43
2.38
2.09
2.10
2.81
2.13
1.71
3.55
1.63
2.j29
0.74
1.47
0.75
1.19
1.63
1.36
1.02
1.32
0.11
1.78
2.76
3.39
2.88
9.89
2.48
4.31
1.75
9.69
1.98
1.18
5.01
4.75
1.46
7.77
1.69
0.58
1.88
1.59
0.69
2.58
1.52
1.18
1.63
0.49
1.58
—
j
—
—
2.79
2.29
2.86
4.59
1.63
3.19
1.14
4.92
—j
6.j04
2.05
0.60
4.25
2.55
1.97
6.69
1.32
—j
—
—j
12.3
2.01
2.05
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal
3.08
1.12
1.95
3.67
1.85
0.46
5.76
3.52
1.96
5.05
2.64
2.32
1.46
5.70
8.82
2.07
0.44
0.98
1.33
0.84
0.38
3.16
2.59
4.83
4.55
2.67
3.70
3.69
9.94
3.00
2.73
2.07
1.53
3.70
2.07
0.71
0.93
3.60
4.31
2.98
3.93
4.40
2.89
2.77
5.07
—j
j
—j
—
11.2
4.61
2k.93
4.48
2.68
1.63
—
17.9
786-0
4.71
2.42
1.32
0.j82
2.28
2.43
1.61
2.56
1.94
3.07
3.92
2.04
7.70
7.33
3k.19
1.54
2.14
1.08
0.66
1.50
1.62
3.61
0.76
2.15
14.0
1.66
3.14
5.22
3.07
3.57
2.66
1.18
3.20
1.68
1.48
1.26
1.28
1.11
1.87
2.56
1.07
2.16
2.72
1.70
3.02
1.98
3.84
4.79
3.10
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
1.68
2.78
7.29
2.62
4.43
1.77
—
—
1.47
—j
1.j40
1.65
4.16
5.76
—
Prostate
PC-3
DU-145
5.90
2.21
4.10
4.25
—j
—
1.80
1.03
3.30
7.72
1.69
1.88
1.30
1.32
2.91
5.54
j
Breast
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
0.41
0.65
11.1
0.90
0.38
2.35
4.56
1.82
3.05
0.32
0.71
1.39
0.82
2.30
4.72
4.48
3.27
3.16
4.02
18.3
2.01
0.44
0.99
1.46
0.89
0.91
5.97
4.65
2.15
3.70
4.30
2.85
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Table 2 (Contd.)
a
GI50 values (μM)
b
c
d
e
f
g
h
i
Cancer panel/cell line
3b
3c
3d
3e
3g
3j
4a
4c
k
k
k
T-47D
MDA-MB-435
2.76
1.25
—
—
—
3.54
4.63
19.0
2.07
1.38
0.70
2.66
1.65
1.44
3.03
0.33
a
Values are reported as GI50, the micromolar concentration of the compound required to cause 50% inhibition of cell growth after an incubation
b
c
d
e
f
g
h
i
j
time of 48 h. NSC 777185. NSC 777187. NSC 777182. NSC 777186. NSC 780196. NSC 780175. NSC 780194. NSC 780198. Not active.
k
Not tested.
(
1
IC50 value – 19.9 µM) as well as MCF-7 cells (IC50 value –
5.8 µM). Notably, compounds containing methylene (4a–l)
instead of carbonyl inhibited cell growth with IC50 values in
the range 0.7 to 19.7 µM. Compound 4e that possesses an
amino group at the meta position and methoxy at the para
position on the C-ring inhibited the growth of breast cancer
cells with IC50 values of 0.7 µM (MCF-7) and 2.2 µM
(
MDA-MB-231). Taken together, the compounds that contain
para-methoxy and meta-amino groups on the C-ring exhibited
substantial cytotoxic effect against most of the cancer cells
apart from breast cancer cells. Thus, based on these results,
we considered it worthwhile to investigate the mechanism of
action of these conjugates (Fig. 2).
Fig. 2 Structure–activity relationship of phenstatin/isocombretastatin–
chalcone conjugates (3a–l and 4a–l).
In addition, we have evaluated the antiproliferative activi-
ties of the other compounds of this series, apart from the com-
pound screened by the NCI. Interestingly, similar to NCI
results, compounds (3a–l) harboring a carbonyl group in
Cell cycle analysis
between A and B rings showed profound cytotoxic activity with In order to understand the role of some of the potential conju-
an IC50 value in the range 0.5 to 19.9 µM against two human gates like 3b, 3c and 3e in the cell cycle, an FACS analysis was
breast cancer cells such as MCF-7 and MDA-MB-231 and the performed in breast cancer cells (MCF-7). The cells were
results are summarized in Table 3. Particularly, compound 3e treated for 24 h with these compounds (3b, 3c and 3e) at
which possesses an amino group at the meta position and 0.5 µM and 1 µM concentrations. It was observed that MCF-7
methoxy at the para position on the C-ring inhibited the cells showed 31.30%, 31.64%, 33.78% at 0.5 µM and 39.36%,
growth of breast cancer cells with an IC50 value of 0.5 µM 38.39%, 36.18% at 1 µM G /M cell cycle arrest respectively,
2
(MCF-7) and 1.2 µM (MDA-MB-231). However, substitution of whereas the control (untreated cells) exhibited 21% and the
bromo at the para position of the C-ring as in 3i drastically positive control phenstatin has shown 35.22% cell cycle arrest
2
2
reduced its antiproliferative activity against MDA-MB-231 cells at 0.5 µM as illustrated in Fig. 3 (Table 4).
Table 3 In vitro cytotoxicity of phenstatin–chalcone and isocombretastatin–chalcones (3a–l) (4a–l)
a
a
IC50 (μM)
IC50 (μM)
b
c
b
c
Compound
MCF-7
MDA-MB-231
Compound
MCF-7
MDA-MB-231
3
3
3
3
3
3
3
3
3
3
3
3
1
a
b
c
d
e
f
g
h
i
9.5 ± 0.52
1.0 ± 0.03
0.9 ± 0.08
5.4 ± 0.21
0.5 ± 0.06
3.9 ± 0.12
12.7 ± 1.09
12.5 ± 1.15
15.8 ± 1.14
3.9 ± 0.29
5.4 ± 0.35
3.1 ± 0.9
7.8 ± 0.25
1.8 ± 0.39
1.6 ± 0.24
7.6 ± 0.91
1.2 ± 0.09
4.7 ± 0.22
10.9 ± 0.65
19.9 ± 1.13
19.9 ± 1.21
2.6 ± 0.12
6.5 ± 0.15
3.9 ± 0.12
1.5 ± 0.05
4a
4b
4c
4d
4e
4f
4g
4h
4i
4.2 ± 0.36
7.3 ± 0.25
7.9 ± 0.54
7.7 ± 0.99
0.9 ± 0.01
10.1 ± 0.57
16.7 ± 0.32
2.6 ± 0.59
16.2 ± 1.2
4.9 ± 0.12
10.1 ± 1.12
9.1 ± 1.05
0.8 ± 0.05
8.7 ± 0.25
9.3 ± 1.2
5.0 ± 0.99
10.7 ± 0.54
2.2 ± 0.36
13.4 ± 0.75
19.7 ± 1.1
5.2 ± 0.05
19.5 ± 1.19
10.5 ± 1.25
12.5 ± 1.36
12.3 ± 1.25
1.5 ± 0.05
j
k
l
4j
4k
4l
b
0.8 ± 0.05
1b
a
Concentration required to inhibition 50% cell growth following 48 h treatment with the tested drug from three different experiments performed
b
c
in triplicates. MCF-7. MDA-MB-231-breast cancer cell lines.
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Fig. 3 Effects of compounds A–Control, 1b, 3b, 3c and 3e on DNA content/cell following treatment of MCF-7 cells at 0.5 µM and 1 µM for 24 h.
Cell cycle distribution was analyzed by the standard propidium iodide procedure as described in the Experimental section.
Immunohistochemistry of tubulin
Effect of compounds on the tubulin polymerization assay
In order to substantiate the observed effects of these conju- One of the ways in which these compounds exhibit cytotoxic
gates on the inhibition of tubulin polymerization to functional activity as well as G /M cell cycle arrest is by the inhibition of
2
2
4
microtubules, immunohistochemistry studies were carried out tubulin polymerization as has been observed in many anti-
to examine the effect of these conjugates like 3b, 3c and 3e on mitotic agents such as combretastatins and phenstatin.
2
3
cellular microtubules in MCF-7 cancer cells. Thus, MCF-7 Hence, it was considered of interest to investigate the tubulin
cells were treated with 3b, 3c and 3e at 1 µM concentration for polymerization inhibition capacity of these compounds. As
4
8 h. In this study, untreated human breast cancer cells dis- tubulin subunits heterodimerize and self-assemble to form
played normal distribution of microtubules (Fig. 4). However, microtubules in a time dependent manner, we investigated the
cells treated with compounds 3b, 3c and 3e showed disrupted progression of tubulin polymerization by monitoring the
microtubule organization as seen in Fig. 4, thus demonstrating increase in fluorescence emission at 420 nm (excitation wave-
the inhibition of tubulin polymerization.
length is 360 nm) in a 384 well plate for 1 h at 37 °C with and
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Table 4 Cell-cycle distribution of MCF-7 cells in the presence of 3b,
Table 5 Inhibition of tubulin polymerization (IC50) of compounds 3b,
3c, 3e, 1b and 1a
a
3
c, 3e and 1e at a conc dependent manner
Compound
0
G /G
1
S-PHASE
2
G /M
Tubulin polymerization
inhibition IC50 ± SD (µM)
Compound
Control (DMSO)
75.85
47.40
38.26
46.95
40.90
46.25
41.91
43.79
8.45
18.71
20.91
18.78
18.43
17.36
19.29
18.43
13.48
31.30 3b
39.06 3c
31.64 3e
38.29 1b
33.78 1a (CA-4)
36.19
35.22
3
3
3
3
3
3
1
b-0.5 µM
b-1.0 µM
c-0.5 µM
c-1.0 µM
e-0.5 µM
e-1.0 µM
b-0.5 µM
1.3 ± 0.10
0.8 ± 0.02
0.6 ± 0.03
0.7 ± 0.01
1.0 ± 0.05
a
Effect of conjugates on tubulin polymerization. IC50 values for 3b, 3c,
e, 1b and 1a were determined from tubulin polymerization assays.
3
1
.3 µM, 0.8 µM, and 0.6 µM, respectively, compared to phen-
2
5
statin (0.7 μM) and combretastatin A-4 (1.0 μM) (Table 5).
Competitive tubulin-binding assay
To confirm the above observations, we investigated the effect
of 3b, 3c and 3e on tubulin polymerization using an in vitro
tubulin polymerization assay (Fig. 6) and it was observed that
they inhibited polymerization of tubulin similar to that of
phenstatin. Therefore, we further assessed the ability of these
compounds to compete with the colchicine for binding to
tubulin via competitive binding assays. Phenstatin was used as
Fig. 4 IHC analyses of compounds on the microtubule network: a positive control and paclitaxel as a negative control. As the
MCF-7 cells were treated with compounds 3b, 3c and 3e at 1 µM con-
intrinsic fluorescence of colchicine increases upon binding to
the tubulin it was used as an index for competition with col-
centration for 48 h followed by staining with α-tubulin antibody. Micro-
tubule organization was clearly observed by green color tubulin
network like structures in control cells and was found to be disrupted in
cells treated with compounds 3b, 3c and 3e as the positive control.
26
chicine in tubulin binding, where paclitaxel did not affect the
binding to tubulin as shown in Fig. 6. However, the fluore-
scence of a colchicine–tubulin complex was reduced in the
presence of these compounds in a dose-dependent manner.
These observations indicate that they inhibit the binding of
colchicine to the tubulin, thereby suggesting that compounds
(
3b, 3c and 3e) bind at the colchicine binding site. In this, F/F0
represents inhibition rate (IR = F/F ) whereas F refers to
0
0
fluorescence of the 5 µM colchicine–tubulin complex, and
F describes the fluorescence of a given concentration (5 µM,
10 µM, and 15 µM) of compounds 3b, 3c, 3e and taxol compe-
tition with the 5 µM colchicine–tubulin complex.
Fig. 5 Effect on tubulin polymerization: the tubulin polymerization
assay was carried out in a reaction mixture that contained PEM buffer
and GTP (1 µM) in the presence or absence of test compounds 3b, 3c,
3
e, 1b and 1a (CA-4) at 5 µM concentration. The reaction was initiated
by the addition of GTP to all the wells. Tubulin polymerization was
monitored by the increase in fluorescence at 420 nm (excitation wave-
length of 360 nm) that was measured for 1 h at 1 min intervals in a multi-
mode plate reader (Tecan) at 37 °C.
without the compounds at 5 µM concentration. These conju-
gates inhibited tubulin polymerization by 58.4%, 55.4 and
5
6.8%, respectively, compared to phenstatin and combretasta-
tin A-4 (Fig. 5).
Fig. 6 Fluorescence based colchicine competitive binding assay of
conjugates 3b, 3c and 3e was carried out at various concentrations con-
taining 5 μM of tubulin and colchicine for 60 min at 37 °C. Phenstatin
was used as a positive control whereas taxol was used as a negative
control which binds at the taxane site. Fluorescence values are normal-
Furthermore, these three potential compounds (3b, 3c and
3
e) were evaluated in an in vitro tubulin polymerization assay
at different concentrations. These molecules showed potent
inhibition of tubulin polymerization with IC50 values of ized to DMSO (control).
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Fig. 7 Western blot analysis of tubulin in the soluble and polymerized
fractions of MCF-7 cells treated with 1 µM concentration of control (C),
Fig. 8 Effect of compounds 3b, 3c and 3e on caspase-9 activity:
MCF-7 cells were treated for 48 h with 2 µM concentrations of com-
pounds 3b, 3c and 3e. Values indicate the mean ± SD of two different
experiments performed in triplicates.
3
e and 1b. Ratio of soluble versus polymerized tubulin.
Western blot analysis
1
µM concentration for 24 h. Manual field quantification of
The microtubules continuously undergo polymerization and
depolymerization which are mediated by α- and β-tubulin; as
inhibition of tubulin polymerization disturbs the assembly of
microtubules, we analyzed the levels of soluble versus polymer-
ized forms of tubulin in MCF-7 cells. In order to extend the in
vitro effects of compounds like 3e and 1b on tubulin polymeri-
zation to the cellular effects, Western blot analyses of tubulin
apoptotic cells based on cytoplasmic condensation, the pres-
ence of apoptotic bodies, nuclear fragmentation and relative
fluorescence of the test compounds (3b, 3c and 3e) indicates
cell death by apoptosis (Fig. 9).
Effect on mitochondrial depolarization
in MCF-7 were performed by treating compounds 3e and 1b at Mitochondrial membrane potential serves as a marker to esti-
µM for 48 h. Following this incubation, the medium was mate the overall function of mitochondria during apoptosis. It
1
removed, cells were washed with PBS, and soluble (containing is well established that at an early stage, apoptotic stimuli alter
free tubulin, supernatant) and insoluble (containing tubulin the mitochondrial transmembrane potential (Δψmt) and this
from microtubules, pellet) fractions were collected as was monitored by the fluorescence of the dye 5,5′,6,6′-tetra-
2
7
described in the Experimental section. Our results indicate chloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1). It
that tubulin in the supernatant fraction showed an increased was observed that with normal cells (high Δψmt), JC-1 displays
level of free tubulin at 3 µM of 3e and 1b, whereas the pellet a red fluorescence (590 nm), which is caused by the spon-
has shown a steady decrease in the polymerized tubulin. taneous and local formation of aggregates that are associated
Therefore, our results suggest that compounds 3e and 1b func- with a large shift in the emission. In contrast, when the mito-
tion as microtubule-destabilizing agents under both in vitro chondrial membrane is depolarized (low Δψmt), JC-1 forms
and intracellular conditions (Fig. 7).
monomers that emit at 530 nm. As shown in Fig. 10, com-
pounds 3b, 3c and 3e induced an increase in the proportion of
cells to 11.1%, 19.1% and 20.3% respectively with depolarized
Activation of caspase-9
3
0
mitochondria (Fig. 10).
2
It is well known that the cell cycle arrest at the G /M phase is
often associated with induction of cellular apoptosis; hence it
was considered of interest to examine whether the cytotoxicity
of 3b, 3c and 3e is by virtue of apoptotic cell death. The MCF-7
cell line lacks endogenous caspase-3, whereas caspase-9 plays
an important role in mediating drug-induced apoptosis. Thus
the role of caspases was examined in MCF-7 cells treated with
conjugates 3b, 3c and 3e at 2 µM for 48 h. Cell lysates were
analyzed for active caspase-9 expression levels by a fluore-
scence-based caspase-9 assay. Up-regulation of caspase-9 was
observed for cells treated with these conjugates relative to the
Annexin V-FITC/propidium iodide analysis of apoptosis
The apoptotic effect of these conjugates was also evaluated by
an Annexin V FITC/PI (AV/PI) dual staining assay to examine
the occurrence of phosphatidylserine externalization and to
understand whether it is due to physiological apoptosis or
2
8
control, as shown in Fig. 8.
Hoechst staining
Apoptosis is one of the major pathways that lead to the
process of cell death. Chromatin condensation and fragmen-
ted nuclei are known to be the classic characteristics of apop-
29
tosis.
It was considered of interest to investigate the
apoptotic inducing effect of these compounds (3b, 3c and 3e)
by the Hoechst staining (H33258) method in the MCF-7 cancer
Fig. 9 Hoechst staining of the conjugates (3b, 3c and 3e) at 1 µM in
cell line. Therefore cells were treated with 3b, 3c and 3e at breast cancer cells (MCF-7).
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Fig. 10 Assessment of Δψmt after treatment of MCF-7 cells with com-
pounds 3b, 3c and 3e Representative histograms of both control cells
and cells incubated for 48 h in the presence of 3b, 3c and 3e as indi-
cated and stained with the fluorescent probe JC-1 after treatment. The
horizontal axis shows fluorescence intensity of the JC-1 monomer, and
the vertical axis shows fluorescence of JC-1 aggregates.
Fig. 12 DNA fragmentation of compounds 3b, 3c and 3e in the MCF-7
cell line. C is the control (untreated).
that these conjugates have the ability to significantly induce
apoptosis in MCF-7 cells.
DNA fragmentation assay
Apoptosis was also assessed by electrophoresis of extracted
3
2
genomic DNA from cells. Endonuclease mediated cleavage of
nuclear DNA results in the formation of an oligonucleosomal
DNA fragment (180–200 base pairs long), a biochemical hall-
mark of apoptosis in many cell types. A DNA laddering assay
was performed with MCF-7 cells by treatment of 3b, 3c and 3e
at 3 µM concentration for 48 h, then the genomic DNA was iso-
lated and electrophoreses was carried out in 1.8% agarose gel.
All the tested compounds induce DNA fragmentation and
a characteristic ladder pattern was observed in MCF-7 cells,
while no laddering was observed in the control cells as shown
Fig. 11 Annexin V-FITC staining. MCF-7 cells were treated with com- in Fig. 12.
pounds 3b, 3c and 3e.
Molecular modelling studies
To investigate the possible binding mode on the colchicine
3
1
33
non-specific necrosis. In this study MCF-7 cells were treated binding site on tubulin, a molecular modeling study was per-
with 3b, 3c and 3e for 48 h at 3 µM concentration to examine formed for the potent molecule 3e of the series and the coordi-
the apoptotic effect. It was observed that they showed signifi- nates of the protein structure of tubulin–colchicine were
cant apoptosis against MCF-7 cells (Fig. 11). Results indicate obtained from the Protein Data Bank (PDB ID 3E22). Docking
that compounds 3b, 3c and 3e showed 28.5%, 21.0% and was accomplished into the colchicine binding sites of tubulin
3
4
2
3.8% of apoptosis respectively, whereas 10.9% was observed using AutoDock 4.2 software Fig. 13 shows that trimethoxy-
in the control (untreated cells). This experiment also suggests benzoyl (A ring) of 3e gets buried in the hydrophobic pocket by
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ted significant antiproliferative activity against a panel of sixty
human cancer cell lines of the NCI, with GI50 values in the
range of 0.11 to 18.3 µM. Three compounds (3b, 3c and 3e)
showed a broad spectrum of antiproliferative efficacy on most
of the cell lines within the sub-micromolar range. In addition,
all the synthesized compounds (3a–l and 4a–l) displayed mod-
erate to excellent cytotoxicity against human breast cancer cell
lines such as MCF-7 and MDA-MB-231 with IC50 values in the
range of 0.5 to 19.9 µM. The tubulin polymerization assay and
immunofluorescence analysis results suggest that these com-
pounds (3b, 3c and 3e) exhibit a strong inhibitory effect on the
tubulin assembly with an IC50 value of 1.3 μM, 0.8 μM and
Fig. 13 Interaction of compound 3e with the colchicine binding site of
tubulin. The probable hydrogen bonds found are shown in red color.
This figure has been generated using the software PYMOL from the
tubulin–colchicine crystal structure.
0.6 μM respectively, similar to that of phenstatin (1b). A cell
cycle assay revealed that these compounds arrest at the G /M
2
phase of the cell cycle, thereby leading to apoptotic cell death.
Occurrence of apoptotic cell death was confirmed by Hoechst
33258 staining, activation of caspase 9, DNA fragmentation,
Annexin V-FITC and mitochondrial membrane depolarization.
The competitive binding assay and docking studies indicate
that these conjugates effectively bind at the colchicine binding
site of tubulin. Therefore, it may be concluded that phen-
statin/isocombretastatin–chalcones were potent inhibitors of
tubulin and apoptotic inducers that are also amenable to
further structural modifications in the discovery and develop-
ment of effective chemotherapeutic agents.
Fig. 14 Compound 3e (green) and colchicine (orange) share the same
binding site on tubulin, β-chain shown in pink colour and α-chain shown
in blue color.
Experimental section
Chemistry
βLeu242, βLeu255, βAla316, βAla250 βVal238, βLeu248 and
βLys352 residues located in β-tubulin similar to that of the
trimethoxyphenyl group of the colchicine. Whereas B and C All chemicals and reagents were obtained from Sigma-Aldrich,
rings interact with the α and β tubulin interface; however ring Lancaster (Alfa Aesar, Johnson Matthey Company) or Spectro-
C extends towards the α-tubulin. Methoxyphenyl (B ring) of 3e chem Pvt. Ltd and were used without further purification.
shows hydrophobic contacts with αVal181, αThr179 and Reactions were monitored by TLC performed on silica gel
αSer178 residues where the 7-membered ring of the colchicine coated glass plates containing 60 GF254 with visualization
binds. The amine group of the C ring (4-methoxy 3-amine achieved by a UV light or iodine indicator. Column chromato-
1
benzoyl) establishes hydrogen bonding interaction with graphy was performed with Merck 60–120 mesh silica gel. H
1
3
αGly11, βLys254, and βAsn248 and its methoxy group shows and C spectra were recorded on Bruker UXNMR/XWIN-NMR
hydrogen bonding interaction with αGly11, whereas the C ring (300 MHz) or Inova Varian-VXR-unity (400, 500 MHz) instru-
makes hydrophobic contacts with βLeu248. Thus a docking ments. Chemical shifts (δ) are reported in ppm downfield from
investigation suggests that conjugate 3e interacted with both an internal TMS standard. ESI spectra were recorded on a
α- and β-tubulin in the colchicine pocket. Fig. 14 shows that Micromass Quattro LC using ESI+ software with a capillary
the proposed binding of conjugate 3e (green) is very similar to voltage of 3.98 kV and an ESI mode positive ion trap detector.
the pose of the colchicine (orange) with trimethoxybenzoyl High-resolution mass spectra (HRMS) were recorded on a
placed in the β-tubulin (pink) chain and the 4-methoxy QSTAR XL hybrid MS–MS mass spectrometer. Melting points
3
-amine benzoyl ring placed towards the α-tubulin (blue) were determined with an Electro thermal melting point appar-
chain.
atus, and are uncorrected.
Preparation of methyl 5-iodo-2-methoxybenzoate (6). To a
solution of 5-iodosalicylic acid (5, 5.0 g, 0.019 mol) in dry
acetone (30 mL) was added anhydrous K CO (7.85 g,
2
3
Conclusion
0.056 mol), dimethyl sulphate (7.16 g, 0.056 mol) at 0 °C. The
In summary, a new class of phenstatin/isocombretastatin– reaction mixture was stirred at reflux temperature for 6 h. The
chalcones (3a–l and 4a–l) were designed and synthesized via reaction was monitored by TLC using ethyl acetate–hexane
Claisen–Schmidt condensation and were investigated for their (1 : 19). After completion of the reaction as indicated by TLC,
cytotoxic activity against various human cancer cell lines.
2 3
K CO was removed by filtration and the solvent was concen-
Some investigated compounds (3b–e), 3g, 3j, 4a and 4c exhibi- trated under vacuum, diluted with water and extracted with
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ethyl acetate. The combined organic phases were dried 5.75 (s, 1H), 4.72 (s, 2H), 3.82 (s, 6H), 3.81 (s, 3H), 3.80 (s, 3H),
+
(
Na SO ) and evaporated under vacuum and the residue was 0.91 (s, 9H), 0.05 (d, J = 1.67 Hz, 6H); ESI-MS: m/z 449 [M + 1] .
2 4
purified by column chromatography (40% EtOAc–hexane) to
Preparation of (3-((tert-butyldimethylsilyloxy)methyl)-4-
afford compound 6 as a white solid (5.1 g, 92%); mp: methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (11). To a
1
4
2
8
7–50 °C; H NMR (CDCl , 300 MHz) δ (ppm): 8.06 (d, J = solution of (3-((tert-butyldimethylsilyloxy)methyl)-4-methoxy-
3
.28 Hz, 1H), 7.72 (dd, J = 2.28 Hz, 8.85 Hz, 1H), 6.73 (d, J = phenyl)(3,4,5-trimethoxyphenyl)methanol
.85 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H); ESI-MS: m/z 292 2.2 mmol) in dry DMSO (5 mL) was added, a solution of
(10,
1.0
g,
+
[M + 1] .
2-iodoxy-benzoic acid (IBX) (0.686 g, 2.45 mmol) in dimethyl
Preparation of (5-iodo-2-methoxyphenyl)methanol (7). To a sulfoxide (DMSO) (10 mL) at 10–15 °C. The reaction mixture
solution of methyl 5-iodo-2-methoxybenzoate (6, 5.0 g, was stirred at room temperature for 2 h. The reaction was
.017 mol) in dry CH Cl (50 mL) was added dropwise DIBAL monitored by TLC using ethyl acetate–hexane (1 : 4). After com-
0
2
2
(34 mL, 1.0 M in hexane, 0.034 mol) at 0 °C. The reaction pletion of the reaction as indicated by TLC, an appropriate
mixture was stirred at room temperature for 2 h. The reaction amount of water was added to the reaction mixture and fil-
was monitored by TLC using ethyl acetate–hexane (4 : 6). After tered through the celite bead. The aqueous layer was extracted
completion of the reaction as indicated by TLC, saturated with ethyl acetate. The organic layer was washed with water
ammonium chloride solution was added to the reaction and brine, dried over anhydrous Na
mixture. Salts were removed by filtration and the solvent was a vacuum to obtain crude compounds. The residue was puri-
dried (Na SO ) and evaporated under vacuum and it was taken fied by column chromatography (10% concentrated under
as such for the next step without further purification. vacuum, the combined organic phases EtOAc–hexane) to
Preparation of tert-butyl (5-iodo-2-methoxybenzyloxy)- afford compound 11 as a light yellow solid (820 mg, 82%); mp:
2 4
SO , and evaporated using
2
4
1
dimethylsilane (8). To a solution of (5-iodo-2-methoxyphenyl)- 48–49 °C; H NMR (CDCl
methanol (7, 4.0 g, 0.015 mol) in dry CH Cl (30 mL) was 2.3 Hz, 1H), 7.80 (dd, J = 2.3 Hz, 8.3 Hz, 1H), 7.02 (s, 2H), 6.90
added imidazole (1.16 g, 0.017 mol) and TBDMSCl (2.56 g, (d, J = 8.3 Hz, 1H), 4.76 (s, 2H), 3.92 (s, 3H), 3.91 (s, 3H),
.017 mol) at 0 °C. The reaction mixture was stirred at room 3.87 (s, 6H), 0.90 (s, 9H), 0.09 (s, 6H); ESI-MS: m/z 447
3
, 300 MHz) δ (ppm): 7.97 (d, J =
2
2
0
+
temperature for 3 h. The reaction was monitored by TLC using [M + 1] .
ethyl acetate–hexane (1 : 10). After completion of the reaction
Preparation of tert-butyl(2-methoxy-5-(1-(3,4,5-trimethoxy-
as indicated by TLC, water was added to the reaction mixture phenyl)vinyl)benzyloxy) dimethylsilane (12). To a solution of
and the organic layer was separated. The solvent was dried PPh
3 3
CH Br (725.8 mg, 2.02 mmol) in dry THF (15 mL) was
(
Na SO ) and evaporated under vacuum and the residue was added potassium tert-butoxide (188.4 mg, 1.68 mmol) at
2
4
purified by column chromatography (5% concentrated under 10–15 °C under an argon atmosphere. The yellow colour
vacuum, the combined organic phases EtOAc–hexane) to mixture was stirred for 4 h. Then, (3-((tert-butyldimethylsilyl-
afford compound 8 as a colourless oil (5.2 g, 91%); bp: oxy)methyl)-4-methoxyphenyl)(3,4,5-trimethoxyphenyl)metha-
1
2
2
8
6
87–289 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 7.72 (d, J = none (11, 500 mg, 1.12 mmol) in THF was added dropwise at
.26 Hz, 1H), 7.48 (dd, J = 2.26 Hz, 8.31 Hz, 1H), 6.56 (d, J = 10–15 °C and the reaction mixture was allowed to be stirred for
.31 Hz, 1H), 4.68 (s, 2H), 3.78 (s, 3H), 0.95 (s, 9H), 0.11 (s, another 3 h. The reaction was monitored by TLC using ethyl
H); ESI-MS: m/z 379 [M + 1] .
+
acetate–hexane (1 : 5). After completion of the reaction as indi-
Preparation of (3-((tert-butyldimethylsilyloxy)methyl)-4- cated by TLC, an appropriate amount of saturated ammonium
methoxyphenyl)(3,4,5-trimethoxyphenyl)methanol (10). To a chloride solution was added to the reaction mixture and it was
solution of tert-butyl(5-iodo-2-methoxybenzyloxy)dimethyl- concentrated. The aqueous layer was extracted with ethyl
silane (8, 4.0 g, 0.011 mol) in dry THF (20 mL) was added drop- acetate. The organic layer was washed with water, dried over
wise n-BuLi (7.5 mL, 1.6 M in hexane, 0.012 mol) at −78 °C. anhydrous Na SO , and evaporated using a vacuum to obtain
2
4
After 1 h, a solution of 3,4,5-trimethoxybenzaldehyde (9, crude compounds. The residue was purified by column chrom-
.96 g, 0.01 mol) in dry THF (4 mL) was added dropwise at atography (10% concentrated under vacuum, the combined
78 °C. After 1 h stirring at rt, the reaction was monitored by organic phases EtOAc–hexane) to afford compound 12 as a
1
−
1
TLC using ethyl acetate–hexane (4 : 6). After completion of the light yellow solid (400 mg, 80%); mp: 60–63 °C; H NMR
reaction as indicated by TLC, saturated ammonium chloride (CDCl , 300 MHz) δ (ppm): 7.48 (d, J = 2.26 Hz, 1H), 7.21 (dd, J
3
solution (10 mL) was added to the reaction mixture. The reac- = 2.26 Hz, 8.49 Hz, 1H), 6.78 (d, J = 8.49 Hz, 1H), 6.64 (s, 2H),
tion mass solvent was evaporated under vacuum, diluted with 5.39 (d, J = 1.13 Hz, 1H), 5.28 (d, J = 1.32 Hz, 1H), 4.74 (s, 2H),
water and extracted with ethyl acetate. The combined organic 3.85 (s, 3H), 3.84 (s, 3H), 3.81 (s, 6H), 0.88 (s, 9H), 0.07 (s, 6H);
+
layers were washed with 1 N HCl and then brine water (20 mL), ESI-MS: m/z 445 [M + 1] .
dried over Na
2
SO
4
, filtered, and concentrated under vacuum.
Preparation of (3-(hydroxymethyl)-4-methoxyphenyl)(3,4,5-
Purification of the residue by column chromatography on trimethoxyphenyl)methanone (13a). To a solution of (3-((tert-
silica gel (hexane–EtOAc, 7 : 3) afforded the desired product 10 butyldimethylsilyloxy)methyl)-4-methoxyphenyl)(3,4,5-trimethoxy-
1
as a white colour solid (5.8 g, 63%); mp: 41–43 °C; H NMR phenyl)methanone (11, 800 mg, 1.79 mmol) in dry THF
(CDCl
3
, 300 MHz) δ (ppm): 7.41 (d, J = 1.98 Hz, 1H), 7.25 (dd, (10 mL) was added a solution of tetra butyl ammonium fluor-
J = 1.98 Hz, 8.39 Hz, 1H), 6.79 (d, J = 8.39 Hz, 1H), 6.62 (s, 2H), ide (2.15 mL, 1.0 M in THF, 2.15 mmol) at 10–15 °C. The reac-
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tion mixture was stirred at room temperature for 3 h. The reac- (CDCl
3
, 300 MHz) δ (ppm): 10.48 (s, 1H), 7.88 (d, J = 2.26 Hz,
tion was monitored by TLC using ethyl acetate–hexane (4 : 6). 1H), 7.52 (dd, J = 2.26 Hz, 8.49 Hz, 1H), 6.98 (d, J = 8.49 Hz,
After completion of the reaction as indicated by TLC, an appro- 1H), 6.50 (s, 2H), 5.41 (dd, J = 0.92 Hz, 14.3 Hz, 2H), 3.96 (s,
+
priate amount of saturated ammonium chloride solution was 3H), 3.88 (s, 3H), 3.81 (s, 6H); ESI-MS: m/z 329 [M + 1] .
added to the reaction mixture and it was concentrated. The
aqueous layer was extracted with ethyl acetate. The organic trimethoxyphenyl)prop-2-en-1-one (3a). A 10% aqueous solu-
layer was washed with water, dried over anhydrous Na SO tion of KOH (5 mL) was added to a stirred solution of
and evaporated using a vacuum to obtain crude compounds. 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzaldehyde (14a,
(E)-3-(2-Methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl)-1-(3,4,5-
2
4
,
The residue was purified by column chromatography (30% 100 mg, 0.303 mmol) and 3,4,5-trimethoxyphenyl acetophe-
concentrated under vacuum, the combined organic phases none (15a, 63.6 mg, 0.303 mmol) in ethanol (20 mL). The reac-
EtOAc–hexane) to afford compound 13a as a light yellow solid tion mixture was stirred at room temperature (27 °C) for 6 h
1
(
450 mg, 76%); mp: 45–47 °C; H NMR (CDCl
3
, 300 MHz) δ and monitored by TLC. After completion of the reaction the
(
ppm): 7.91 (d, J = 2.26 Hz, 1H), 7.77 (dd, J = 2.26 Hz, 8.31 Hz, solvent was evaporated under vacuum, and then the residue
1
H), 6.97 (s, 2H), 6.85 (d, J = 8.31 Hz, 1H), 4.71 (s, 2H), 3.89 (s, was dissolved in EtOAc–H O. The organic layer was washed
2
+
3
H), 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)- column chromatography using EtOAc–Hex (1 : 1) as a solvent
vinyl)phenyl)methanol (13b). This compound was prepared system to obtain the pure product as a light yellow colour solid
with brine and evaporated. This was further purified by
−1
according to the method described for compound 13a, (120 mg, 76% yield); mp: 181–183 °C; IR (KBr): ¯ν max/cm
–
employing tert-butyl(2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)- 2945, 2837, 1653, 1587, 1504, 1465, 1441, 1415, 1336, 1287,
vinyl)benzyloxy)dimethyl silane (12, 500 mg, 1.124 mmol) to 1262, 1237, 1192, 1129, 1035, 1020, 989, 938, 862, 841, 811,
1
obtain the pure product 13b as a light yellow solid (300 mg, 797, 764, 747, 551; H NMR (CDCl
3
, 300 MHz) δ (ppm): 8.19
1
8
1%); mp: 59–61 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 7.32 (d, J = 1.88 Hz, 1H), 8.13 (d, J = 15.8 Hz, 1H), 7.87 (dd, J = 1.88
(d, J = 2.26 Hz, 1H), 7.23 (d, J = 2.26 Hz, 1H), 6.86 (d, J = Hz, 8.67 Hz, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.28 (s, 2H), 7.04 (s,
8
4
3
.49 Hz, 1H), 6.54 (s, 2H), 5.35 (dd, J = 1.13 Hz, 12.8 Hz, 2H), 2H), 7.03 (d, J = 8.69 Hz, 1H), 4.02 (s, 3H), 3.98 (s, 9H), 3.95 (s,
1
3
.69 (s, 2H), 3.89 (s, 3H), 3.88 (s, 3H), 3.81 (s, 6H); ESI-MS: m/z 3H), 3.89 (s, 6H); C NMR (CDCl
31 [M + 1] .
3
, 75 MHz) δ (ppm): 194.2,
188.6, 161.5, 152.7, 153.1, 149.0, 137.9, 133.7, 132.7, 130.7,
Preparation of 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benz- 130.1, 124.1, 123.3, 123.0, 110.6, 110.4, 109.8, 107.3, 60.8, 56.2,
+
+
aldehyde (14a). A solution of DMSO (0.258 g, 3.3 mmol) in dry 56.0, 55.9; ESI-MS: m/z 545 [M + Na] ; HRMS (ESI m/z) for
CH Cl (3 mL) was added dropwise to a magnetically stirred C H O Na calcd: 545.17820, found: 545.17746 [M + Na] .
+
2
2
29 30 9
solution of oxalyl chloride (209 mg, 1.65 mmol) in dry CH
5.0 mL) kept at −78 °C under an argon atmosphere. The benzoyl)phenyl)prop-2-en-1-one (3b). This compound was
mixture was stirred for another 15 min. The CH Cl of prepared according to the method described for compound
2
Cl
2
(E)-1-(3,4-Dimethoxyphenyl)-3-(2-methoxy-5-(3,4,5-trimethoxy-
(
2
2
(3-(hydroxymethyl)-4-methoxyphenyl)(3,4,5-trimethoxyphenyl)- 3a, employing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benz-
methanone (13a, 500 mg, 1.5 mmol) was added dropwise and aldehyde (14a, 100 mg, 0.303 mmol) and 3,4-dimethoxy aceto-
the reaction mixture was allowed to stir for 3 h. Triethylamine phenone (15b, 54.5 mg, 0.303 mmol) to obtain the pure
(
607 mg, 6.0 mmol) was added dropwise over 5 min, and the product 3b as a yellow solid (122 mg, 82% yield); mp:
−
1
stirred solution was allowed to warm to room temperature. An 178–180 °C; IR (KBr): ¯ν max/cm – 3080, 2998, 2938, 2835,
appropriate amount of water was added to reaction mixture 2608, 2029, 1655, 1578, 1515, 1459, 1412, 1330, 1261, 1129,
1
and the organic layer was separated and the aqueous phase 1020, 857, 827, 767, 746, 700, 671, 634, 601, 545; H NMR
was extracted with CH
layer was washed with brine (10 mL), dried over anhydrous = 15.8 Hz, 1H), 7.87 (dd, J = 1.88 Hz, 8.67 Hz, 1H), 7.70–7.63
Na SO and evaporated using a vacuum to obtain a crude com- (m, 3H), 7.05 (s, 2H), 7.01 (d, J = 8.87 Hz, 1H), 6.93 (d, J = 8.31
pound. The residue was purified by column chromatography Hz, 1H), 4.02 (s, 3H), 3.98 (s, 6H), 3.95 (s, 3H), 3.89 (s, 6H);
30% concentrated under vacuum, the combined organic NMR (CDCl , 75 MHz) δ (ppm): 194.2, 188.6, 161.5, 152.7,
2 2 3
Cl (3 × 20 mL). The combined organic (CDCl , 300 MHz) δ (ppm): 8.19 (d, J = 1.88 Hz, 1H), 8.13 (d, J
2
4
1
3
C
(
3
phases EtOAc–hexane) to afford compound 14a as a light 153.1, 149.0, 137.9, 133.7, 132.7, 130.7, 130.1, 124.1, 123.3,
1
yellow solid (420 mg, 84%); mp: 74–76 °C; H NMR (CDCl
3
(
3
,
123.0, 110.6, 110.4, 109.8, 107.3, 60.8, 56.2, 56.0, 55.9; ESI-MS:
+
00 MHz) δ (ppm): 10.49 (s, 1H), 8.29 (d, J = 2.26 Hz, 1H), 8.13 m/z 515 [M + Na] ; HRMS (ESI m/z) for C H O Na calcd:
2
8
28 8
+
dd, J = 2.26 Hz, 8.87 Hz, 1H), 7.15 (d, J = 8.87 Hz, 1H), 7.01 515.16764, found: 515.16695 [M + Na] .
s, 2H), 4.05 (s, 3H), 3.95 (s, 3H), 3.87 (s, 6H); ESI-MS: m/z 331 (E)-1-(Benzo[d][1,3]dioxol-5-yl)-3-(2-methoxy-5-(3,4,5-trimethoxy-
benzoyl)phenyl)prop-2-en-1-one (3c). This compound was pre-
Preparation of 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)- pared according to the method described for compound
(
+
[M + 1] .
benzaldehyde (14b). This compound was prepared according 3a, employing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benz-
to the method described for compound 14a, employing aldehyde (14a, 100 mg, 0.303 mmol) and 1-(benzo[d][1,3]-
(
(
2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)methanol dioxol-5-yl)ethanone (15c, 49.7 mg, 0.303 mmol) to obtain the
13b, 500 mg, 1.51 mmol) to obtain the pure product 14b as a pure product 3c as a yellow solid (115 mg, 80% yield); mp:
1
−1
light yellow solid (419 mg, 84%); mp: 89–92 °C; H NMR 181–179 °C; IR (KBr): ¯ν max/cm – 2945, 2837, 1653, 1587,
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Paper
1
1
504, 1465, 1441, 1415, 1336, 1287, 1262, 1237, 1192, 1129, aldehyde (14a, 100 mg, 0.303 mmol) and 4-amino acetophe-
1
035, 1020, 989, 938, 862, 841, 811, 797, 764, 747, 551;
H
none (15f, 41.3 mg, 0.303 mmol) to obtain the pure product 3f
NMR (CDCl
3
, 500 MHz) δ (ppm): 8.16 (d, J = 2.13 Hz, 1H), 8.10 as a yellow solid (105 mg, 78% yield); mp: 140–143 °C; IR
−
1
(
(
d, J = 15.8 Hz, 1H), 7.86 (dd, J = 2.13 Hz, 8.54 Hz, 1H), 7.66 (KBr): ¯ν max/cm – 3466, 3366, 3234, 2938, 2839, 1649, 1604,
dd, J = 1.67 Hz, 8.24 Hz, 1H), 7.62 (d, J = 15.8 Hz, 1H), 7.53 (d, 1582, 1561, 1503, 1464, 1414, 1334, 1286, 1260, 1234, 1216,
1
J = 1.67 Hz, 1H), 7.04 (s, 2H), 7.01 (d, J = 8.54 Hz, 1H), 6.90 (d, 1172, 1126, 1016, 1004, 985, 864, 834, 802, 768, 751, 559; H
J = 8.24 Hz, 1H), 6.07 (s, 2H), 4.02 (s, 3H), 3.95 (s, 3H), 3.89 (s, NMR (CDCl
3
, 500 MHz) δ (ppm): 8.17 (s, 1H), 8.11 (d, J = 15.8
1
3
6
1
1
1
H);
C NMR (CDCl , 75 MHz) δ (ppm): 193.4, 187.5, Hz, 1H), 7.91 (d, J = 8.24 Hz, 2H), 7.83 (d, J = 8.54 Hz, 1H), 7.67
3
60.9, 151.1, 147.6, 141.0, 137.6, 133.7, 132.1, 132.1, 130.2, (d, J = 15.7 Hz, 1H), 7.05 (s, 2H), 7.02 (d, J = 8.54 Hz, 1H), 6.69
29.5, 124.1, 122.8, 123.2, 110.2, 107.5, 107.2, 106.6, (d, J = 8.39 Hz, 2H), 4.27 (bp, 2H), 4.00 (s, 3H), 3.95 (s, 3H),
01.2, 60.1, 55.6, 55.4; ESI-MS: m/z 499 [M + Na] ; HRMS (ESI 3.89 (s, 6H); C NMR (CDCl , 75 MHz) δ (ppm): 194.3, 188.1,
+
13
3
m/z) for C27
H
24
O
8
Na calcd: 499.13634, found: 499.13549 161.5, 152.7, 151.2, 137.1, 133.5, 132.8, 131.0, 130.6, 130.1,
+
[M + Na] .
128.2, 124.3, 123.7, 113.5, 110.4, 107.3, 60.8, 55.8, 56.2;
+
(
E)-3-(2-Methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl)-1-(4- ESI-MS: m/z 448 [M + 1] ; HRMS (ESI m/z) for C H NO calcd:
2
6
26
6
+
methoxyphenyl)prop-2-en-1-one (3d). This compound was pre- 448.17546, found: 448.17355 [M + 1] .
pared according to the method described for compound 3a, (E)-1-(4-Fluorophenyl)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)-
employing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benz- phenyl)prop-2-en-1-one (3g). This compound was prepared
aldehyde (14a, 100 mg, 0.303 mmol) and 4-methoxy acetophe- according to the method described for compound 3a, employ-
none (15d, 45.5 mg, 0.303 mmol) to obtain the pure product ing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzaldehyde (14a,
3
d as a yellow solid (110 mg, 79% yield); mp: 166–164 °C; IR 100 mg, 0.303 mmol) and 4-fluoro acetophenone (15g,
−
1
(KBr): ¯ν max/cm – 3003, 2941, 2837, 1660, 1646, 1606, 1589, 41.8 mg, 0.303 mmol) to obtain the pure product 3g as a white
−
1
1
1
5
505, 1457, 1411, 1338, 1308, 1288, 1268, 1255, 1223, 1168, solid (99 mg, 73% yield); mp: 114–116 °C; IR (KBr): ¯ν max/cm
130, 1020, 989, 977, 867, 839, 805, 764, 748, 666, 642, 615, – 3607, 2942, 2836, 2624, 1665, 1600, 1582, 1504, 1410, 1333,
1
3
49, 508; H NMR (CDCl , 500 MHz) δ (ppm): 8.17 (d, J = 1.88 1287, 1258, 1230, 1214, 1171, 1157, 1130, 1007, 978, 868, 834,
1
Hz, 1H), 8.12 (d, J = 15.8 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.84 763, 747, 703, 665; H NMR (CDCl
3
, 500 MHz) δ (ppm): 8.18
(dd, J = 2.07 Hz, 6.6 Hz, 1H), 7.66 (d, J = 15.8 Hz, 1H), (d, J = 2.13 Hz, 1H), 8.13 (d, J = 15.8 Hz, 1H), 7.96 (d, J =
7
.04–7.03 (m, 2H), 7.00 (d, J = 9.06 Hz, 3H), 4.01 (s, 3H), 3.95 8.54 Hz, 2H), 7.86 (dd, J = 2.14 Hz, 8.69 Hz, 1H), 7.61 (d, J =
1
3
(s, 3H), 3.89 (s, 9H); C NMR (CDCl3, 75 MHz) δ (ppm): 192.2, 15.8 Hz, 1H), 7.47 (d, J = 8.54 Hz, 2H), 7.04 (s, 2H), 7.01 (s,
1
3
1
1
5
86.7, 161.9, 160.0, 158.1, 151.3, 140.1, 136.3, 132.3, 131.2, 1H), 4.02 (s, 3H), 3.95 (s, 3H), 3.89 (s, 6H); C NMR (CDCl3,
29.3, 129.2, 128.6, 122.3, 122.0, 112.4, 109.4, 105.9, 59.1, 54.7, 75 MHz) δ (ppm): 194.3, 189.1, 161.7, 152.9, 139.2, 134.4,
+
4.1; ESI-MS: m/z 463 [M + 1] ; HRMS (ESI m/z) for C27
H
26
O
7
134.1, 132.8, 131.1, 130.3, 123.9, 123.3, 115.8, 115.6, 110.5,
107.4, 107.3, 61.0, 56.3, 56.0; ESI-MS: m/z 451 [M + 1] ; HRMS
+
+
calcd: 463.1756, found: 463.1752 [M + 1] .
(
6
E)-1-(3-Amino-4-methoxyphenyl)-3-(2-methoxy-5-(3,4,5-tri- (ESI m/z) for C26H24FO calcd: 451.15514, found: 451.15293
+
methoxybenzoyl)phenyl) prop-2-en-1-one (3e). This compound [M + 1] .
was prepared according to the method described for com- (E)-1-(4-Chlorophenyl)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)-
pound 3a, employing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)- phenyl)prop-2-en-1-one (3h). This compound was prepared
benzaldehyde (14a, 100 mg, 0.303 mmol) and 4-methoxy according to the method described for compound 3a, employ-
3
-amino acetophenone (15e, 50.4 mg, 0.303 mmol) to obtain ing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzaldehyde (14a,
the pure product 3e as a yellow solid (105 mg, 73% yield): mp: 100 mg, 0.303 mmol) and 4-chloro acetophenone (15h,
−
1
1
1
52–154 °C; IR (KBr): ¯ν max/cm – 3416, 2932, 2839, 1654, 46.8 mg, 0.303 mmol) to obtain the pure product 3h as a white
−
1
597, 1578, 1506, 1462, 1442, 1414, 1335, 1293, 1277, 1259, solid (125 mg, 88% yield); mp: 170–172 °C; IR (KBr): ¯ν max/cm
229, 1192, 1131, 1020, 990, 863, 840, 817, 796, 763, 751, 556; – 2942, 2836, 1667, 1642, 1596, 1504, 1454, 1413, 1334, 1287,
H NMR (CDCl , 500 MHz) δ (ppm): 8.18 (s, 1H), 8.12 (d, J = 1262, 1234, 1214, 1129, 1090, 1010, 865, 830, 803, 769, 745,
3
1
1
1
1
7
8
3
5.7 Hz, 1H), 7.86 (d, J = 8.55 Hz, 1H), 7.64 (d, J = 15.5 Hz, 1H), 665, 641, 562, 527; H NMR (CDCl , 500 MHz) δ (ppm): 8.17
3
.49 (d, J = 8.39 Hz, 1H), 7.44 (s, 1H), 7.05 (s, 2H), 7.01 (d, J = (d, J = 2.14 Hz, 1H), 8.13 (d, J = 15.8 Hz, 1H), 8.08–8.03 (m,
.69 Hz, 1H), 6.85 (d, J = 8.39 Hz, 1H), 4.01 (s, 3H), 3.95 (s, 2H), 7.87 (dd, J = 2.14 Hz, 8.69 Hz, 1H), 7.63 (d, J = 15.8 Hz,
1
3
H), 3.94 (s, 3H), 3.89 (s, 6H); C NMR (CDCl , 75 MHz) δ 1H), 7.18 (t, J = 8.69 Hz, 14.8 Hz, 2H), 7.04 (s, 2H), 7.02 (d, J =
3
1
3
(
ppm): 194.3, 189.0, 161.5, 152.8, 151.2, 137.4, 136.3, 133.5, 8.39 Hz, 1H), 4.02 (s, 3H), 3.95 (s, 3H), 3.89 (s, 6H); C NMR
1
1
32.8, 131.2, 130.1, 130.6, 124.2, 123.6, 120.4, 114.2, 110.4, (CDCl
3
, 75 MHz) δ (ppm): 193.2, 188.3, 161.1, 152.7, 141.1,
09.1, 107.4, 60.8, 56.2, 55.8, 55.5; ESI-MS: m/z 478 [M + 1] ; 138.7, 138.3, 135.8, 133.6, 132.1, 130.3, 129.6, 129.5, 128.3,
calcd: 478.18603, found: 123.1, 122.6, 110.3, 106.8, 60.1, 55.6, 55.1; ESI-MS: m/z 467
+
HRMS (ESI m/z) for C27H28NO
7
+
+
4
78.18372 [M + 1] .
E)-1-(4-Aminophenyl)-3-(2-methoxy-5-(3,4,5-trimethoxy- found: 467.1245 [M + 1] .
benzoyl)phenyl)prop-2-en-1-one (3f). This compound was pre- (E)-1-(4-Bromophenyl)-3-(2-methoxy-5-(3,4,5-trimethoxybenzoyl)-
pared according to the method described for compound 3a, phenyl)prop-2-en-1-one (3i). This compound was prepared
employing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benz- according to the method described for compound 3a, employ-
6
[M + 1] ; HRMS (ESI m/z) for C26H24ClO calcd: 467.1261,
+
(
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ing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzaldehyde (14a, ing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benzaldehyde (14a,
1
00 mg, 0.303 mmol) and 4-bromo acetophenone (15i, 100 mg, 0.303 mmol) and 1-(naphthalen-2-yl)ethanone (15l,
6
0.3 mg, 0.0303 mmol) to obtain the pure product 3i as a 51.6 mg, 0.303 mmol) to obtain the pure product 3l as a white
−
1
white solid (129 mg, 84% yield): mp: 128–130 °C; IR (KBr): solid (130 mg, 89% yield): mp: 145–148 °C; IR (KBr): ¯ν max/cm
¯
νmax/cm – 3002, 2924, 2853, 1660, 1646, 1606, 1589, 1505, – 3058, 2973, 2941, 2839, 1664, 1638, 1582, 1503, 1465, 1412,
−
1
1
1
457, 1411, 1338, 1308, 1288, 1268, 1255,1223, 1168, 1130, 1335, 1287, 1262, 1232, 1184, 1125, 1010, 984, 893, 854, 828,
020, 989, 977, 867, 839, 805, 772, 763, 666, 642, 615, 549, 507; 753, 672, 644, 562, 478; H NMR (CDCl , 300 MHz) δ (ppm):
3
1
1
H NMR (CDCl , 500 MHz) δ (ppm): 8.17 (d, J = 2.14 Hz, 1H), 8.56 (s, 1H), 8.25 (d, J = 2.26 Hz, 1H), 8.18 (d, J = 15.8 Hz, 1H),
3
8
7
7
.10 (d, J = 15.8 Hz, 1H), 8.04 (dd, J = 1.98 Hz, 8.85 Hz, 2H), 8.10 (d, J = 1.51 Hz, 8.31 Hz, 1H), 8.00 (d, J = 8.31 Hz, 1H),
.85 (dd, J = 2.14 Hz, 8.69 Hz, 1H), 7.66 (d, J = 15.8 Hz, 1H), 7.96–7.85 (m, 3H), 7.79 (d, J = 15.8 Hz, 1H), 7.63–7.53 (m, 2H),
.04 (s, 2H), 7.01 (d, J = 8.69 Hz, 1H), 6.98 (dd, J = 1.98 Hz, 8.85 7.06 (s, 2H), 7.02 (d, J = 9.1 Hz, 1H), 4.03 (s, 3H), 3.96 (s, 3H),
13
13
Hz, 2H), 4.02 (s, 3H), 3.95 (s, 3H), 3.89 (s, 6H); C NMR 3.90 (s, 6H); C NMR (CDCl
3
, 75 MHz) δ (ppm): 194.3, 190.3,
3
CDCl , 75 MHz) δ (ppm): 194.1, 188.6, 163.2, 161.5, 152.7, 161.6, 152.8, 141.7, 138.7, 135.4, 133.9, 132.8, 130.8, 130.3,
(
1
1
41.6, 137.9, 133.6, 132.7, 130.7, 130.1, 124.0, 123.4, 113.6, 130.0, 129.4, 128.4, 127.7, 126.7, 124.4, 124.1, 123.7, 110.5,
+
10.4, 107.8, 107.2, 107.1, 63.8, 55.8, 55.1; ESI-MS: m/z 533 107.4, 60.9, 56.2, 55.9; ESI-MS: m/z 505 [M + Na] ; HRMS (ESI
+
+
[M + Na] ; HRMS (ESI m/z) for C26
H
23BrO
81
6
calcd: 533.0575, m/z) for C30
H
26
O
6
Na calcd: 505.1627, found: 505.1643 [M + Na] .
+
+
found: 533.0552 [M + Na] and 535.0466 [M + Na] .
(E)-3-(2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)-
(
E)-1-(4-Hydroxyphenyl)-3-(2-methoxy-5-(3,4,5-trimethoxy- 1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4a). This com-
benzoyl)phenyl)prop-2-en-1-one (3j). This compound was pound was prepared according to the method described for
prepared according to the method described for compound compound 3a, employing 2-methoxy-5-(1-(3,4,5-trimethoxyphe-
3
a, employing 2-methoxy-5-(3,4,5-trimethoxybenzoyl)benz- nyl)vinyl)benzaldehyde (14b, 100 mg, 0.305 mmol) and 3,4,5-
aldehyde (14a, 100 mg, 0.303 mmol) and 1-(4-((tert-butyldi- trimethoxy acetophenone (15a, 64.05 mg, 0.305 mmol) to
methylsilyl)oxy)phenyl)ethanone (15j, 81.6 mg, 0.333 mmol) to obtain the pure product 4a as a white solid (130 mg, 82%
−
1
obtain the pure product 3j as a white solid (88 mg, 65% yield); yield); mp: 160–162 °C; IR (KBr): ¯ν max/cm – 3064, 2968, 2938,
−
1
mp: 152–154 °C; IR (KBr): ¯ν max/cm – 3419, 2927, 1651, 1606, 2836, 1726, 1659, 1581, 1502, 1460, 1411, 1339, 1281, 1260,
1
1
8
579, 1504, 1457, 1411, 1335, 1284, 1260, 1223, 1166, 1129, 1229, 1188, 1159, 1123, 1068, 1023, 998, 980, 963, 922, 881,
1
1
018, 984, 839, 763, 749; H NMR (CDCl
3 3
, 300 MHz) δ (ppm): 851, 819, 779, 752, 725, 713; H NMR (CDCl , 500 MHz)
.29 (m, 2H), 7.69 (d, J = 8.31 Hz, 2H), 7.83–7.69 (m, 2H), 7.19 δ (ppm): 8.05 (d, J = 15.8 Hz, 1H), 7.63 (d, J = 1.52 Hz, 1H),
(
(
d, J = 8.68 Hz, 1H), 7.03 (s, 2H), 6.87 (d, J = 8.49 Hz, 2H), 3.85 7.53 (d, J = 15.8 Hz, 1H), 7.38 (dd, J = 1.83 Hz, 8.54 Hz, 1H),
s, 6H), 3.83 (s, 3H), 3.35 (s, 3H); C NMR (CDCl , 75 MHz) 7.26 (d, J = 3.05 Hz, 2H), 6.94 (d, J = 8.69 Hz, 1H), 6.56 (s, 2H),
3
1
3
δ (ppm): 191.8, 186.1, 160.7, 159.7, 151.1, 144.9, 135.5, 132.1, 5.40 (d, J = 9.01 Hz, 2H), 3.95 (s, 3H), 3.94 (s, 9H), 3.89 (s, 3H),
1
3
1
1
31.06, 129.3, 128.7, 128.4, 122.2, 121.9, 113.8, 113.6, 109.7, 3.82 (s, 6H); C NMR (CDCl , 75 MHz) δ (ppm): 189.7, 152.7,
3
+
05.7, 58.8, 54.5; ESI-MS: m/z 471 [M + Na] ; HRMS (ESI m/z) 152.5, 142.9, 140.8, 136.6, 137.0, 134.6, 133.6, 133.2, 132.6,
130.1, 129.2, 107.6, 106.2, 105.7, 104.4, 103.3, 60.7, 60.6, 56.0,
E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(2-methoxy-5-(3,4,5-tri- 55.7, 55.5; ESI-MS: m/z 521 [M + 1] ; HRMS (ESI m/z) for
+
for C26
H
24
O
7
Na calcd: 471.14142, found: 471.14145 [M + Na] .
+
(
+
methoxybenzoyl)phenyl) prop-2-en-1-one (3k). This com-
C
30
H
33
O
8
calcd: 521.21699, found: 521.21657 [M + 1] .
pound was prepared according to the method described for
(E)-1-(3,4-Dimethoxyphenyl)-3-(2-methoxy-5-(1-(3,4,5-tri-
compound 3a, employing 2-methoxy-5-(3,4,5-trimethoxyben- methoxyphenyl)vinyl)phenyl)prop-2-en-1-one (4b). This com-
zoyl)benzaldehyde (14a, 100 mg, 0.303 mmol) and 1-(3-((tert- pound was prepared according to the method described for
butyldimethylsilyl)oxy)-4-methoxyphenyl)ethanone
(15k, compound 3a, employing 2-methoxy-5-(1-(3,4,5-trimethoxyphe-
9
1.6 mg, 0.333 mmol) to obtain the pure product 3k as a white nyl)vinyl)benzaldehyde (14b, 100 mg, 0.305 mmol) and 3,4-
−
1
solid (89 mg, 62% yield); mp: 184–186 °C; IR (KBr): ¯ν max/cm
dimethoxyacetophenone (15b, 54.9 mg, 0.305 mmol) to obtain
–
1
5
3429, 2934, 2839, 1651, 1580, 1504, 1455, 1412, 1334, 1261, the pure product 4b as a white solid (126 mg, 84% yield); mp:
1
−1
230, 1170, 1125, 1018, 860, 836, 764, 749; H NMR (CDCl , 145–147 °C; IR (KBr): ¯ν max/cm – 2958, 2837, 1725, 1654,
3
00 MHz) δ (ppm): 8.16 (d, J = 1.98 Hz, 1H), 8.09 (d, J = 15.7 1597, 1580, 1513, 1462, 1415, 1343, 1268, 1162, 1127, 1023,
1
Hz, 1H), 7.85 (dd, J = 1.98 Hz, 8.56 Hz, 1H), 7.65–7.62 (m, 3H), 893, 845, 820, 767, 708, 551; H NMR (CDCl
3
, 500 MHz) δ
7
.05 (s, 2H), 7.01 (d, J = 8.54 Hz, 1H), 6.93 (d, J = 8.85 Hz, 1H), (ppm): 8.08 (d, J = 15.8 Hz, 1H), 7.67–7.61 (m, 4H), 7.36 (dd, J
.82 (s, 1H), 4.01 (s, 3H), 3.97 (s, 3H), 3.95 (s, 3H), 3.89 (s, 6H); = 8.39 Hz, 1.98 Hz, 1H), 6.93 (dd, J = 2.59 Hz, 8.69 Hz, 2H),
5
1
3
3
C NMR (CDCl , 75 MHz) δ (ppm): 194.2, 188.9, 161.6, 152.8, 6.57 (s, 2H), 5.42 (d, J = 7.63 Hz, 2H), 3.96 (s, 6H), 3.95 (s, 3H),
1
3
1
1
5
50.6, 145.5, 141.8, 138.2, 133.7, 132.8, 131.7, 130.9, 130.2, 3.89 (s, 3H), 3.82 (s. 6H); C NMR (CDCl , 75 MHz) δ (ppm):
3
24.1, 123.5, 122.1, 114.6, 110.5, 109.9, 107.4, 60.9, 56.2, 56.0, 188.1, 157.8, 152.5, 148.4, 138.5, 137.1, 136.3, 133.1, 130.9,
+
5.9; ESI-MS: m/z 501 [M + Na] ; HRMS (ESI m/z) for 128.1, 122.8, 122.5, 122.1, 112.6, 110.4, 110.2, 109.7, 104.9,
+
+
C H O Na calcd: 501.15199, found: 501.15244 [M + Na] .
60.3, 55.4, 55.3, 55.2; ESI-MS: m/z 491 [M + 1] ; HRMS (ESI m/z)
2
7
26 8
+
(
E)-3-(2-Methoxy-5-(3,4,5-trimethoxybenzoyl)phenyl)-1-(naph- for C29
thalen-2-yl)prop-2-en-1-one (3l). This compound was prepared
according to the method described for compound 3a, employ- methoxyphenyl)vinyl) phenyl)prop-2-en-1-one (4c). This com-
H
31
O
7
calcd: 491.2069, found: 491.2032 [M + 1] .
(E)-1-(Benzo[d][1,3]dioxol-5-yl)-3-(2-methoxy-5-(1-(3,4,5-tri-
3976 | Org. Biomol. Chem., 2015, 13, 3963–3981
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Paper
+
pound was prepared according to the method described for 60.8, 56.1, 55.6; ESI-MS: m/z 476 [M + 1] ; HRMS (ESI m/z) for
+
compound 3a, employing 2-methoxy-5-(1-(3,4,5-trimethoxyphe- C H NO calcd: 476.20676, found: 476.20639 [M + 1] .
2
8
30
6
nyl)vinyl)benzaldehyde (14b, 100 mg, 0.305 mmol) and
-(benzo[d][1,3]dioxol-5-yl)ethanone (15c, 50.1 mg, 0.305 mmol) phenyl)vinyl)phenyl)prop-2-en-1-one (4f). This compound was
to obtain the pure product 4c as a white solid (125 mg, 87% prepared according to the method described for compound 3a,
(E)-1-(4-Aminophenyl)-3-(2-methoxy-5-(1-(3,4,5-trimethoxy-
1
−
1
yield); mp: 153–155 °C; IR (KBr): ¯ν max/cm – 3087, 2934, 2842, employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benz-
2
1
8
1
355, 1652, 1605, 1582, 1502, 1443, 1410, 1343, 1323,1288, aldehyde (14b, 100 mg, 0.305 mmol) and 4-amino acetophe-
246, 1191, 1128, 1038, 1024, 1001, 937, 910, 890, 851, 833, none (15f, 41.5 mg, 0.305 mmol) to obtain the pure product 4f
1
07, 784, 722; H NMR (CDCl
3
, 300 MHz) δ (ppm): 8.06 (d, J = as a yellow colour solid (100 mg, 74% yield); mp: 115–117 °C;
−
1
5.8 Hz, 1H), 7.65–7.63 (m, 2H), 7.56 (d, J = 15.8 Hz, 1H), 7.52 IR (KBr): ¯ν max/cm – 3355, 2924, 1603, 1341, 1248, 1172, 1126,
1
(
(
(
d, J = 1.32 Hz, 1H), 7.35 (dd, J = 2.07 Hz, 8.49 Hz, 1H), m 6.90 1021; H NMR (CDCl , 300 MHz) δ (ppm): 8.03 (d, J = 15.8 Hz,
t, J = 8.49 Hz, 16.6 Hz, 2H), 6.56 (s, 2H), 6.05 (s, 2H), 5.41 1H), 7.89 (d, J = 7.55 Hz, 2H), 7.63–7.58 (m, 2H), 7.31 (d, J =
d, J = 3.02 Hz, 2H), 3.95 (s, 3H), 3.89 (s, 3H), 3.82 (s, 6H);
3
1
3
C
8.31 Hz, 1H), 6.91 (d, J = 8.31 Hz, 1H), 6.66 (d, J = 7.55 Hz, 2H),
NMR (CDCl , 75 MHz) δ (ppm): 188.7, 158.7, 152.9, 149.0, 6.56 (d, 2H), 5.39 (d, J = 3.02 Hz, 2H), 4.02 (bp, 2H), 3.93 (s,
3
13
1
1
4
3
39.6, 137.0, 133.1, 131.4, 129.1, 124.6, 123.6, 122.8, 113.1, 3H), 3.89 (s, 3H), 3.82 (s, 6H); C NMR (CDCl , 125 MHz) δ
10.8, 108.4, 107.8, 105.5, 101.7, 60.8, 56.1, 55.6; ESI-MS: m/z (ppm): 188.5, 158.3, 152.8, 151.1, 149.1, 138.4, 137.7, 137.0,
+
75 [M + 1] ; HRMS (ESI m/z) for C H O calcd: 475.17513, 133.7, 131.1, 130.9, 128.8, 128.5, 129.9, 123.1, 113.7, 113.0,
2
8
27 7
+
+
found: 475.17477 [M + 1] .
E)-3-(2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)- (ESI m/z) for C27H28NO
5
1
110.7, 105.4, 60.8, 56.6, 56.1; ESI-MS: m/z 446 [M + 1] ; HRMS
(
calcd: 446.19620, found: 446.19521
+
-(4-methoxyphenyl)prop-2-en-1-one (4d). This compound was [M + 1] .
prepared according to the method described for compound 3a,
(E)-1-(4-Fluorophenyl)-3-(2-methoxy-5-(1-(3,4,5-trimethoxy-
employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benz- phenyl)vinyl)phenyl)prop-2-en-1-one (4g). This compound was
aldehyde (14b, 100 mg, 0.305 mmol) and 4-methoxyacetophe- prepared according to the method described for compound 3a,
none (15d, 45.8 mg, 0.305 mmol) to obtain the pure product employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benz-
4
d as a white solid (130 mg, 93% yield); mp: 142–146 °C; IR aldehyde (14b, 100 mg, 0.305 mmol) and 4-fluoro acetophe-
−
1
(KBr): ¯ν max/cm – 3002, 2964, 2937, 2835, 1721, 1654, 1603, none (15g, 42.1 mg, 0.305 mmol) to obtain the pure product
1
1
593, 1577, 1505, 1455, 1412, 1347, 1321, 1287, 1252, 1233, 4g as a white colour solid (98 mg, 72% yield); mp: 120–122 °C;
1
−1
217, 1169, 1126, 1032, 1008, 984, 844, 827, 610; H NMR IR (KBr): ¯ν max/cm – 3001, 2912, 1687, 1601, 1555, 1526, 1496,
1
(CDCl , 500 MHz) δ (ppm): 8.06 (d, J = 15.8 Hz, 1H), 8.02 (d, 1428, 1345, 1303, 1229, 1187, 1162, 1089, 998, 979; H NMR
3
J = 8.85 Hz, 2H), 7.64–7.60 (m, 2H), 7.34 (dd, J = 2.29 Hz, 8.54 (CDCl
3
, 300 MHz) δ (ppm): 8.09 (d, J = 15.8 Hz, 1H), 8.06–8.02
Hz, 1H), 6.97 (d, J = 8.85 Hz, 2H), 6.91 (d, J = 8.85 Hz, 1H), 6.56 (m, 2H), 7.63 (d, J = 2.26 Hz, 1H), 7.59 (d, J = 15.8 Hz. 1H), 7.36
(
(
1
1
1
4
s, 2H), 5.40 (d, J = 7.78 Hz, 2H), 3.95 (s, 3H), 3.89 (s, 3H), 3.88 (dd, J = 2.26 Hz, 8.31 Hz, 1H), 7.20–7.13 (m, 2H), 6.91 (d, J =
1
3
3
s, 3H), 3.82 (s, 6H); C NMR (CDCl , 125 MHz) δ (ppm): 9.06 Hz, 1H), 6.55 (s, 2H), 5.40 (d, J = 1.51 Hz, 2H), 3.96 (s,
1
3
3
89.2, 163.2, 158.4, 156.6, 152.8, 149.0, 139.3, 137.0, 133.8, 3H), 3.89 (s, 3H), 3.82 (s, 6H); C NMR (CDCl , 75 MHz) δ
31.3, 130.7, 130.3, 129.0, 127.6, 123.7, 122.9, 113.7, 113.0, (ppm): 189.9, 161.7, 152.8, 152.7, 141.8, 139.2, 134.2, 133.1,
12.3, 110.8, 109.1, 105.4, 60.8, 56.1, 56.0, 55.6; ESI-MS: m/z 132.7, 131.1, 131.0, 123.8, 123.2, 115.7, 110.5, 107.4, 60.9, 56.2,
+
+
61 [M + 1] ; HRMS (ESI m/z) for C28
H
29
O
6
calcd: 461.19587, 55.9; ESI-MS: m/z 449 [M + 1] ; HRMS (ESI m/z) for C27
H
26FO
5
+
+
found: 461.19495 [M + 1] .
calcd: 449.17588, found: 449.17591 [M + 1] .
(E)-1-(3-Amino-4-methoxyphenyl)-3-(2-methoxy-5-(1-(3,4,5-tri-
(E)-1-(4-Chlorophenyl)-3-(2-methoxy-5-(1-(3,4,5-trimethoxy-
methoxyphenyl)vinyl) phenyl)prop-2-en-1-one (4e). This com- phenyl)vinyl)phenyl)prop-2-en-1-one (4h). This compound
pound was prepared according to the method described for was prepared according to the method described for com-
compound 3a, employing 2-methoxy-5-(1-(3,4,5-trimethoxyphe- pound 3a, employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)-
nyl)vinyl)benzaldehyde (14b, 100 mg, 0.305 mmol) and vinyl)benzaldehyde (14b, 100 mg, 0.305 mmol) and 4-chloro
4
-methoxy 3-aminoacetophenone (15e, 50.6 mg, 0.305 mmol) acetophenone (15h, 47.1 mg, 0.305 mmol) to obtain the pure
to obtain the pure product 4e as a yellow colour solid (106 mg, product 4h as a white colour solid (125 mg, 88% yield); mp:
−
1
−1
7
2
1
3% yield); mp: 120–122 °C; IR (KBr): ¯ν max/cm – 3358, 2934, 150–152 °C; IR (KBr): ¯ν max/cm – 2994, 2958, 2935, 2838,
837, 1654, 1579, 1504, 1440, 1410, 1346, 1318, 1290, 1248, 1676, 1659, 1596, 1582, 1493, 1458, 1412, 1398, 1347, 1329,
171, 1125, 1020, 895, 822, 709; H NMR (CDCl
1
3
, 500 MHz) δ 1290, 1266, 1248, 1234, 1208, 1189, 1172, 1129, 1029, 1088,
(
(
ppm): 8.06 (d, J = 15.8 Hz, 1H), 7.63 (d, J = 2.26 Hz, 1H), 7.59 1011, 987, 966, 914, 857, 966, 914, 857, 842, 825, 782, 769, 720;
1
d, J = 15.8 Hz, 1H), 7.46 (dd, J = 1.51 Hz, 8.31 Hz, 1H), 7.42 (d,
H NMR (CDCl , 300 MHz) δ (ppm): 8.08 (d, J = 15.8 Hz, 1H),
3
J = 1.51 Hz, 1H), 7.33 (dd, J = 2.26 Hz, 8.31 Hz, 1H), 6.91 (d, J = 7.93 (d, J = 8.31 Hz, 2H), 7.63 (d, J = 2.26 Hz, 1H), 7.57 (d, J =
8
4
6
1
1
.31 Hz, 1H), 6.82 (d, J = 8.31 Hz, 1H), 6.56 (s, 2H), 5.40 (d, J = 15.8 Hz, 1H), 7.45 (d, J = 8.31 Hz, 2H), 7.35 (dd, J = 2.26 Hz,
.53 Hz, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H), 3.82 (s, 8.31 Hz, 1H), 6.94 (d, J = 9.06 Hz, 1H), 6.58 (s, 2H), 5.41 (d, J =
13
13
H); C NMR (CDCl
51.1, 149.1, 138.8, 137.8, 137.1, 136.3, 133.7, 131.6, 131.2, (CDCl
28.8, 123.8, 123.1, 120.4, 114.4, 113.1, 110.8, 109.2, 105.5, 138.9, 137.8, 136.9, 136.6, 133.9, 131.8, 129.8, 129.1, 128.7,
3
, 75 MHz) δ (ppm): 189.5, 158.4, 152.9, 1.51 Hz, 2H), 3.95 (s, 3H), 3.89 (s, 3H), 3.82 (s, 6H); C NMR
3
, 75 MHz) δ (ppm): 189.7, 158.5, 152.7, 148.9, 140.7,
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1
4
C
C
27.5, 122.5, 110.8, 113.2, 105.4, 60.8, 56.0, 55.6; ESI-MS: m/z NMR (CDCl
3
, 75 MHz) δ (ppm): 189.2, 152.8, 150.3, 148.9,
+
+
65 [M + 1] and m/z 467 [M + 3] ; HRMS (ESI m/z) for 145.3, 139.4, 137.0, 133.7, 131.9, 131.4, 129.0, 122.8, 121.9,
+
27
H
H
26ClO
26 ClO
5
calcd: 465.14633, found: 465.14624 [M + 1] and 114.6, 113.1, 110.8, 109.8, 105.4, 60.8, 56.0, 55.6; ESI-MS: m/z
3
7
+
+
27
5
calcd: 467.14338, found: 467.14339 [M + 3] .
477 [M + 1] ; HRMS (ESI m/z) for C28
H
29
O
7
calcd: 477.1913,
+
(
E)-1-(4-Bromophenyl)-3-(2-methoxy-5-(1-(3,4,5-trimethoxy- found: 477.1874 [M + 1] .
phenyl)vinyl)phenyl)prop-2-en-1-one (4i). This compound was
(E)-3-(2-Methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)phenyl)-
prepared according to the method described for compound 3a, 1-(naphthalen-2-yl)prop-2-en-1-one (4l). This compound was
employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benz- prepared according to the method described for compound 3a,
aldehyde (14b, 100 mg, 0.305 mmol) and 4-bromoacetophe- employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benz-
none (15i, 60.7 mg, 0.305 mmol) to obtain the pure product 4i aldehyde (14b, 100 mg, 0.305 mmol) and 1-(naphthalen-2-yl)-
as a white colour solid (130 mg, 84% yield); mp: 132–134 °C; ethanone (15l, 51.9 mg, 0.305 mmol) to obtain the pure
−
1
IR (KBr): ¯ν max/cm – 2998, 2842, 1689, 1586, 1468, 1416, 1377, product 4l as a white colour solid (128 mg, 87% yield); mp:
1
−1
1
266, 1234, 1162, 1155, 1029, 1088, 976, 966, 914; H NMR 126–128 °C; IR (KBr): ¯ν max/cm – 2998, 2960, 2935, 2829,
(
CDCl , 300 MHz) δ (ppm): 8.08 (d, J = 15.8 Hz, 1H), 7.86 (d, J 1653, 1628, 1594, 1578, 1506, 1494, 1459, 1413, 1346, 1327,
3
=
7
6
3
1
1
1
5
8.54 Hz, 2H), 7.64–7.62 (m, 3H), 7.55 (d, J = 15.8 Hz, 1H), 1291, 1266, 1251, 1236, 1211, 1184, 1124, 1025, 1009, 985, 966,
.35 (dd, J = 2.28 Hz, 8.54 Hz, 1H), 6.91 (d, J = 8.69 Hz, 1H), 910, 895, 851, 825, 813, 784, 769, 762, 709, 627, 599, 556; H
1
.55 (s, 2H), 5.40 (dd, J = 1.07 Hz, 3.66 Hz, 2H), 3.95 (s, 3H), NMR (CDCl , 300 MHz) δ (ppm): 8.52 (s, 1H), 8.16 (d, J = 15.8
3
1
3
3
.89 (s, 3H), 3.82 (s, 6H); C NMR (CDCl , 125 MHz) δ (ppm): Hz, 1H), 8.08 (dd, J = 1.51 Hz, 8.31 Hz, 1H), 8.01–7.88 (m, 3H),
89.9, 158.6, 152.9, 152.7, 148.9, 140.8, 137.9, 137.1, 136.9, 7.76 (d, J = 15.8 Hz, 1H), 7.70 (d, J = 1.51 Hz, 1H), 7.63–7.53
33.9, 131.2, 130.2, 130.0, 129.5, 129.2, 127.6, 122.6, 113.2, (m, 2H), 7.36 (dd, J = 2.26 Hz, 9.06 Hz, 1H), 6.92 (d, J = 8.31
+
10.9, 105.5, 60.8, 56.1, 55.1; ESI-MS: m/z 509 [M + 1] and m/z Hz, 1H), 6.58 (s, 2H), 5.42 (d, J = 5.28 Hz, 2H), 3.96 (s, 3H),
+
13
11 [M + 3] ; HRMS (ESI m/z) for C27
H
26BrO
5
calcd: 509.09581, 3.89 (s, 3H), 3.83 (s, 6H); C NMR (CDCl
3
, 75 MHz) δ (ppm):
+
81
found: 509.09567 [M + 1] and C H
BrO calcd: 511.09377, 190.9, 152.9, 149.0, 140.2, 137.8, 136.9, 135.9, 135.7, 135.3,
2
7
26
5
found: 511.09375.
E)-1-(4-Hydroxyphenyl)-3-(2-methoxy-5-(1-(3,4,5-trimethoxy-
phenyl)vinyl)phenyl)prop-2-en-1-one (4j). This compound was + 1] ; HRMS (ESI m/z) for C H O calcd: 481.20095, found:
133.8, 132.5, 131.6, 129.8, 129.4, 128.4, 127.7, 126.6, 124.5,
(
123.6, 113.2, 110.8, 105.4, 60.9, 56.1, 55.7; ESI-MS: m/z 481 [M
+
3
1
29 5
+
prepared according to the method described for compound 3a, 481.20049 [M + 1] .
employing 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)vinyl)benz-
aldehyde (14b, 100 mg, 0.305 mmol) and 1-(4-((tert-butyldi-
Biology
methylsilyl)oxy)phenyl)ethanone (15j, 82.3 mg, 0.336 mmol) to
Cytotoxic activity. The cytotoxicity activity of the compounds
4
obtain the pure product 4j as a white colour solid (95 mg, 70% was determined using the MTT assay. 1 × 10 cells per well
−
1
yield); mp: 142–144 °C; IR (KBr): ¯ν max/cm – 2999, 2989, 2888, were seeded in 200 µL DMEM, supplemented with 10% FBS in
1
1
659, 1601, 1549, 1524, 1434, 1389, 1222, 1089, 875; H NMR each well of 96-well microculture plates and incubated for 24 h
(
CDCl , 500 MHz) δ (ppm): 8.11 (d, J = 15.7 Hz, 1H), 7.97 (d, J at 37 °C in a CO incubator. Compounds, diluted to the
3
2
=
1
3
7
1
1
+
4
7.78 Hz, 2H), 7.63 (d, J = 10.9 Hz, 2H), 7.35 (d, J = 7.78 Hz, desired concentrations in culture medium, were added to the
H), 6.96–6.90 (m, 3H), 6.56 (s, 2H), 5.40 (d, J = 4.42 Hz, 2H), wells with the respective vehicle control. After 48 h of incu-
1
3
.92 (s, 3H), 3.89 (s, 3H), 3.81 (s, 6H); C NMR (CDCl
3
,
bation, 10 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
5 MHz) δ (ppm): 190.0, 161.3, 158.5, 152.8, 149.0, 139.8, tetrazolium bromide) (5 mg mL− ) was added to each well and
37.6, 137.1, 133.7, 131.6, 131.1, 130.4, 128.9, 123.5, 122.8, the plates were further incubated for 4 h. Then the super-
15.5, 113.2, 110.8, 105.4, 60.8, 56.0, 55.9; ESI-MS: m/z 447 [M natant from each well was carefully removed; formazon crystals
1] ; HRMS (ESI m/z) for C H O calcd: 447.1807, found: were dissolved in 100 µL of DMSO and the absorbance at
47.1817 [M + 1] .
E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(2-methoxy-5-(1-(3,4,5-
1
+
2
7
27 6
+
540 nm wavelength was recorded.
Cell cycle analysis. A flow cytometric analysis (FACS) was
(
trimethoxyphenyl)vinyl) phenyl)prop-2-en-1-one (4k). This performed to evaluate the distribution of the cells through the
compound was prepared according to the method described cell cycle phases. Human breast cancer cells (MCF-7) were
for compound 3a, employing 2-methoxy-5-(1-(3,4,5-trimethoxy- incubated with compounds (3b, 3c and 3e) at 0.5 µM and 1 μM
phenyl)vinyl)benzaldehyde (14b, 100 mg, 0.305 mmol)and concentrations for 48 h. Untreated and treated cells were har-
1
-(3-((tert-butyldimethylsilyl)oxy)-4-methoxyphenyl)ethanone vested, washed with PBS, fixed in ice-cold 70% ethanol and
15k, 92.4 mg, 0.335 mmol) to obtain the pure product 4k as a stained with propidium iodide (Sigma–Aldrich). Cell cycle was
white colour solid (96 mg, 66% yield); mp: 148–146 °C; IR performed by flow cytometry (Becton Dickinson FACS Caliber)
(
−
1
(
1
KBr): ¯ν max/cm – 3388, 2929, 1669, 1579, 1508, 1459, 1412, as earlier described.
1
330, 1273, 1170, 1125, 1019, 821, 764; H NMR (CDCl
3
,
In vitro tubulin polymerization assay. A fluorescence based
5
7
6
00 MHz) δ (ppm): 8.07 (d, J = 15.8 Hz, 1H), 7.63–7.61 (m, 3H), in vitro tubulin polymerization assay was performed according
.59 (d, J = 15.8 Hz, 1H), 7.35 (dd, J = 2.07 Hz, 8.49 Hz, 1H), to the manufacturer’s protocol (BK011, Cytoskeleton, Inc.).
.95–6.90 (m, 2H), 6.56 (s, 2H), 5.70 (s, 1H), 5.40 (d, J = 4.15 Briefly, the reaction mixture in a total volume of 10 µL con-
1
3
Hz, 2H), 3.97 (s, 3H), 3.94 (s, 3H), 3.89 (s, 3H), 3.82 (s, 6H);
C
tained PEM buffer, GTP (1 μM) in the presence or absence of
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test compounds (final concentration 5 μM). Tubulin polymeri- soluble (S) tubulin. Pellets of polymerized “P” tubulin were
zation was followed by a time dependent increase in fluo- resuspended in a volume of lysis buffer equal to the soluble
rescence due to the incorporation of a fluorescence reporter “S” fraction, and resolved in 7% SDS/PAGE as described
into microtubules as the polymerization proceeds. Fluo- earlier. After transfer to an NC membrane, immunoblotting
rescence emission at 420 nm (excitation wavelength of was performed with an anti-mouse α-tubulin antibody [DMIA,
3
60 nm) was measured using a Varioscan multimode plate Sigma, St. Louis, MO], followed by an FITC-conjugated second-
reader (Thermo Scientific Inc.). Phenstatin was used as a posi- ary antibody (Sigma). The blot was imaged using a phosphor
tive control in each assay. The IC50 value was defined as the imager (Fugifilm, Japan). Quantitative analysis of the soluble
drug concentration required for inhibiting 50% of tubulin and polymer fractions was done by densitometry using Gene-
assembly compared to the control. The reaction mixture for box (Syngene).
−
1
these experiments include: tubulin (3 mg mL ) in PEM
Hoechst staining. Cells were seeded at a density of 10 000
buffer, GTP (1 μM), in the presence or absence of test com- cells over 18 mm cover slips and incubated for 24 h. After incu-
pounds at various concentrations. Polymerization was moni- bation, cells were treated with compounds 3b, 3c and 3e at 1
tored by the increase in fluorescence as mentioned above at µM concentration for 24 h. Hoechst 33258 (Sigma Aldrich) was
added to the cells at a concentration of 0.5 mg mL− and incu-
1
3
7 °C.
Immunohistochemistry. MCF-7 cells were seeded on glass bated for 30 min at 37 °C. Later, cells were washed with phos-
coverslips, incubated for 48 h in the presence or absence of phate buffered saline (PBS). Cells from each cover slip were
test compounds 3b, 3c and 3e at 1 µM concentration. Follow- captured from randomly selected fields under a fluorescent
ing the termination of incubation, cells were fixed with 3% microscope (Leica, Germany) to qualitatively determine the
paraformaldehyde, 0.02% glutaraldehyde in PBS and permea- proportion of viable and apoptotic cells based on their relative
bilized by dipping the cells in 100% methanol followed by fluorescence and nuclear fragmentation.
overnight incubation at 4 °C. Later, cover slips were blocked
Caspase 9 activity. To determine the caspase-9 activity of 3b,
with 1% BSA in phosphate buffered saline for 1 h followed by 3c and 3e for the detection of apoptosis in the breast cancer
incubation with a primary antitubulin (mouse monoclonal) cell line (MCF-7), a commercially available apoptosis detection
antibody and a FITC conjugated anti-mouse-IgG secondary kit (Sigma-Caspase 9 Assay kit, Fluorometric) was used. MCF-7
antibody. Photographs were taken using a fluorescence micro- cells were treated with compounds 3b, 3c and 3e at 2 µM con-
scope, equipped with FITC settings and the pictures were ana- centration for 48 h. Here the substrate used is Ac-LEHD-AFC to
lyzed for the integrity of the microtubule network.
the cell lysate and incubation was carried out at 37 °C for 1 h.
Competitive tubulin-binding assay. For the colchicine com- Readings were taken at an excitation wavelength of 400 nm
petitive binding assay, tubulin was co-incubated with indicated and an emission wavelength of 505 nm.
6
concentrations of MPSP-001 and taxol at 37 °C for 1 h. Then
DNA fragmentation analysis. Cells were seeded (1 × 10 ) in
colchicine was added to a final concentration of 5 μmol L−
1
.
six well plates and incubated for 24 h. After incubation, cells
Fluorescence was determined using a Hitachi F-2500 spectro- were treated with compounds 3b, 3c and 3e at 3 µM concen-
fluorometer (Tokyo, Japan) at the excitation wavelength of tration for 48 h. After 48 h of drug treatment cells were col-
3
(
65 nm and the emission wavelength of 435 nm. Blank values lected and centrifuged at 2500 rpm for 5 min at 4 °C. Pellets
buffer alone) as the background were subtracted from all were collected and washed with phosphate buffered saline
samples. Then the inhibition rate (IR) was calculated as (PBS), added 100 µL of lysis buffer, centrifuged at 3000 rpm
−
1
follows: IR = F/F where F is the fluorescence of 5 μmol L
for 5 min at 4 °C and collected supernatant. 10 µL of 10% SDS
0
0
colchicine–tubulin complex, and F is the fluorescence of a and 10 µL of (50 mg mL− ) RNase-A were added and incubated
1
−
1
−1
given concentration of phenstatin or taxol (12.5 μmol L
2
with 5 μmol L
,
for 2 h at 56 °C. After that, 10 µL of Proteinase K (25 mg mL
)
5 μmol L− , 50 μmol L and 100 μmol L ) in competition was added and incubated at 37 °C for 2 h. After incubation, 65
1
−1
−1
−1
colchicine–tubulin complex. Taxol, not µL of 10 M ammonium acetate and 500 µL of ice cold ethanol
binding in the colchicine site of tubulin, was added as a nega- were added and mixed well. This sample was incubated at
tive control. 80 °C for 1 h. After that, samples were centrifuged at 12 000
Analysis of soluble versus polymerized tubulin in cells. Cells rpm for 20 min at 4 °C and washed with 80% ethanol followed
plated in 24-well dishes, grown to 60–80% confluency, and by air drying for 10 min at room temperature. The pellet was
treated with compounds 3e and 1a at 1 μM concentration were dissolved in 50 µL of TE buffer. After that, DNA laddering was
used as positive controls. Cells were incubated with the drug determined by 2% agarose gel electrophoresis.
for 48 h, and later the medium was removed, cells were rinsed
in 1× PBS at 22 °C, harvested at the same temperature in lysis The mitochondrial membrane potential was measured using
buffer containing 0.1 M Pipes, 1 mM EGTA, 1 mM MgSO the lipophilic cationic dye JC-1 (Molecular Probes), as
0% glycerol, 5% DMSO, 5 mM GTP, 0.125% NP-40, and pro- described. Cultures were treated with the test drugs for 48 h.
Measurement of the mitochondrial membrane potential.
4
,
3
−
1
tease inhibitors, including aprotinin [200 units mL ], pH 6.9, After drug treatment the cells were incubated with JC-1 dye for
and then centrifuged at 15 000g at 22 °C for 30 min in a 20 min at 37 °C. After incubation, cultures were used for
Sorvall Legend Micro 21R model temperature controlled cen- measuring the mitochondrial membrane potential (ΔΨm),
trifuge (Thermo Scientific), to separate polymerized (P) from according to the manufacturer’s instructions. The shift of
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membrane potential was measured in flow cytometry using
JC-1 (molecular probe) as previously described.
Flow cytometric evaluation of apoptosis. MCF-7 (1 × 10 )
were seeded in six-well plates and allowed to grow overnight.
The medium was then replaced with a complete medium con-
taining 3 µM concentration of compounds 3b, 3c and 3e for
7 (a) R. Crazzolara, A. Cisterne, M. Thien, J. Hewson,
R. Baraz, K. F. Bradstock and L. J. Bendall, Blood, 2009,
113(14), 3297–3306; (b) S. O. Brien, G. Schiller, J. Lister,
L. Damon, S. Goldberg, W. Aulitzky, D. Ben-Yehuda,
W. Stock, S. Coutre, D. Douer, L. T. Heffner, M. Larson,
K. Seiter, S. Smith, S. Assouline, P. Kuriakose, L. Maness,
A. Nagler, J. Rowe, M. Schaich, O. Shpilberg, K. Yee,
G. Schmieder, J. A. Silverman, D. Thomas, S. R. Deitcher
and H. Kantarjian, J. Clin. Oncol., 2013, 31(6), 676–
683.
8 (a) G. R. Pettit, G. M. Cragg, D. L. Herald, J. M. Schmidt
and P. Lohavanijaya, Can. J. Chem., 1982, 60, 1374–1376;
(b) G. R. Pettit, S. B. Singh, M. R. Boyd, E. Hamel,
R. K. Pettit, J. M. Schmidt and F. Hogan, J. Med. Chem.,
1995, 38, 1666–1672.
6
4
8 h along with the vehicle alone (0.001% DMSO) as the
control. After 48 h of drug treatment, cells from the super-
natant and adherent monolayer cells were harvested by trypsi-
nization, and washed with PBS at 3000 rpm. Then the cells
6
(
1 × 10 ) were stained with Annexin V-FITC and propidium
iodide using an Annexin-V-PI apoptosis detection kit (Invitro-
gen). Flow cytometry was performed using a FACScan (Becton
Dickinson) equipped with a single 488 nm argon laser as
described earlier. Annexin V-FITC was analyzed using exci-
tation and emission settings of 488 nm and 535 nm (FL-1
channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and
clumps were gated out using forward and orthogonal light
scatter.
9 (a) A. T. McGown and B. W. Fox, Cancer Chemother. Pharma-
col., 1990, 26, 79; (b) C. Shustik, W. Dalton and P. Gros,
Mol. Aspects Med., 1995, 16, 1; (c) O. Fardel, V. Lecureur and
A. Guillouzo, Gen. Pharmacol., 1996, 27, 1283.
Molecular modelling experimental procedure. All the com- 10 J. A. Woods, J. A. Hadfield, G. R. Pettit, B. W. Fox and
pounds under study and reference compounds were 3D struc- A. T. McGown, Br. J. Cancer, 1995, 71, 705–711.
tures built and optimized using Gaussian 03W. These 11 (a) K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka,
optimized 3D structures were utilized for docking. All the com-
pounds were docked using the AutoDockTools software
package. The co-crystallized structure of the colchicine site of
tubulin downloaded from the PDB data bank (http://www.rcsb.
org/pdb/index.html; PDB code: 3E22).
Y. Morinaga, Y. Nihei, K. Ohishi, Y. Suga, Y. Akiyama and
T. Tsuji, J. Med. Chem., 1998, 41, 3022; (b) G. R. Pettit,
B. E. Toki, D. L. Herald, M. R. Boyd, E. Hamel and
R. K. Pettit, J. Med. Chem., 1999, 42, 1459–
1465; (c) G. R. Pettit, M. R. Rhodes, D. L. Herald,
D. J. Chaplin, M. R. L. Stratford, E. Hamel, R. K. Pettit,
J. C. Chapuis and D. Oliva, Anti-Cancer Drug Des., 1998, 13,
9
81–993.
Acknowledgements
1
2 (a) M. Alami, J. D. Brion, O. Provot, J. F. Peyrat,
S. Messaoudi, A. Hamze, A. Giraud, J. Bignon, J. Bakala
and J. M. Liu, PCT WO2008/122620A1, 2008;
G.B.K., V.S.R. and A.B.S. acknowledge CSIR, New Delhi for the
award of a senior research fellowship. We also acknowledge
CSIR for financial support under the 12th Five Year plan
project “Affordable Cancer Therapeutics (ACT)” (CSC0301).
We thank Dr Rajeev S. Menon for reviewing this manuscript.
(b) S. Messaoudi, B. Treguier, A. Hamze, O. Provot,
J. F. Peyrat, J. R. D. Losada, J. M. Liu, J. Bignon,
J. Wdzieczak-Bakala, S. Thoret, J. Dubois, J.-D. Brion and
M. Alami, J. Med. Chem., 2009, 52, 4538–4542;
(c) C. Mousset, A. Giraud, O. Provot, A. Hamze, J. Bignon,
J. M. Liu, S. Thoret, J. Dubois, J. D. Brion and M. Alami,
Bioorg. Med. Chem. Lett., 2008, 18, 3266–3271;
Notes and references
(d) A. Hamze, A. Giraud, S. Messaoudi, O. Provot,
1
R. Romagnoli, P. G. Baraldi, M. D. Carrion, O. Cruz-Lopez,
C. L. Cara, G. Basso, G. Viola, M. Khedr, J. Balzarini,
S. Mahboobi, A. Sellmer, A. Brancale and E. Hamel, J. Med.
Chem., 2009, 52(7), 5551–5555.
P. Singh, K. Rathinasamy, R. Mohan and D. Panda, IUBMB
Life, 2008, 60(6), 368–375.
A. Jordan, J. A. Hadfield, N. J. Lawrence and A. T. McGown, 14 G. R. Pettit, P. M. Grealish, D. L. Herald, M. R. Boyd,
Med. Res. Rev., 1998, 18, 259–296.
J. J. Field, A. Kanakkanthar and J. H. Miller, Bioorg. Med.
Chem., 2014, 22, 5050–5059.
J. Shi, Y. Zhou, H. C. Huang and T. J. Mitchison, Cancer
Res., 2011, 71(13), 4518–4526.
J. F. Peyrat, J. Bignon, J. M. Liu, J. Wdzieczak-Bakala,
S. Thoret, J. Dubois, J.-D. Brion and M. Alami, ChemMed-
Chem, 2009, 4, 1912–1924; (e) B. Trguier, A. Hamze,
O. Provot, J. D. Brion and M. Alami, Tetrahedron Lett., 2009,
50, 6549–6552.
2
3
4
5
6
13 G. R. Pettit and B. Toki, PCT/US 1999/000475, 2005.
E. Hamel and R. K. Pettit, J. Med. Chem., 2000, 43, 2731–
2737.
15 (a) G. R. Pettit, J. W. Lippert and D. L. Herald, J. Org.
Chem., 2000, 65, 7438–7444; (b) G. R. Pettit, B. Toki,
D. L. Herald, P. Verdier-Pinard, M. R. Boyd, E. Hamel and
R. K. Pettit, J. Med. Chem., 1998, 41, 1688–1695.
R. N. Hansen, S. D. Ramsey, D. Lalla, A. Masaquel,
T. Kamath, M. Brammer, S. A. Hurvitz and S. D. Sullivan, 16 A. Giraud, O. Provot, A. Hamze, J. D. Brion and M. Alami,
Springerplus, 2014, 3, 259. Tetrahedron Lett., 2008, 49, 1107–1110.
3980 | Org. Biomol. Chem., 2015, 13, 3963–3981
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Paper
1
7 M. L. Go, X. Wu and X. L. Liu, Curr. Med. Chem., 2005, 12,
83–499 (chalcons).
8 (a) A. C. Claude, C. L. Jean, T. Patrick, P. Christelle,
A. B. Shaik, G. B. Kumar, C. Kishor, M. K. Reddy,
A. Nagabhushana, K. V. S. Ramakrishna, A. Addlagatta and
S. Kotamraju, ChemMedChem, 2013, 8, 2015–2025.
4
1
H. Gerard, J. C. Albert and L. D. Jean, Anticancer Res., 2001, 26 (a) C. Zhang, N. Yang, C. H. Yang, H. S. Ding, C. Luo,
2
1, 3949–3956; (b) T. Takahashi, N. Takasuka, M. Iigo,
Y. Zhang, et al., PLoS One, 2009, 4, e4881; (b) Z. L. Liu,
W. Tian, Y. Wang, S. Kuang, X. M. Luo and Q. Yu, Acta
Pharmacol. Sin., 2012, 33, 261–270.
M. Baba, H. Nishino, H. Tsuda and T. Okuyama, Cancer
Sci., 2004, 95, 448–453.
1
2
9 (a) S. Ducki, G. Mackenzie, B. Greedy, S. Armitage, 27 (a) A. M. Minotti, S. B. Barlow and F. Cabral, J. Biol. Chem.,
J. F. Chabert, E. Bennett, J. Nettles, J. P. Snyder and
N. J. Lawrence, Bioorg. Med. Chem., 2009, 17, 7711–7722;
1991, 266, 3987–3994; (b) N. Jain, D. Yada, T. B. Shaik,
G. Vasantha, P. S. Reddy, S. V. Kalivendi and B. Sreedhar,
ChemMedChem, 2011, 6, 859–868.
(b) A. Kamal, M. K. Reddy and A. Viswanath, Expert Opin.
Drug Discovery, 2013, 8, 289–304.
28 P. Costantini, J. M. Bruey, M. Castedo, D. Metivier,
M. Loeffler, S. A. Susin, L. vagnan, N. Zamzami, C. Garrido
and G. Kroemer, Cell Death Differ., 2002, 9, 82.
0 S. Sale, R. G. Tunstall, K. C. Ruparelia, P. C. Butler,
G. A. Potter, W. P. Steward and A. J. Gescher, Invest New
Drugs., 2006, 24, 459–464.
29 E. Pasquier and M. Kavallaris, IUBMB Life, 2008, 60, 165.
2
1 D. Kumar, K. K. Raj, S. V. Malhotrab and D. S. Rawat, 30 R. Romagnoli, P. G. Baraldi, O. Cruz-Lopez, C. Lopez-Cara,
MedChemComm, 2014, 5, 528–535.
2 (a) P. S. Frisa and J. W. Jacobberger, PLoS One, 2009, 4,
e7064; (b) A. Kamal, Y. V. V. Srikanth, T. B. Shaik,
M. D. Carrion, A. Brancale, E. Hamel, L. Chen,
R. Bortolozzi, G. Basso and G. Viola, J. Med. Chem., 2010,
53, 4248–4258.
2
M. N. A. Khan, Md. Ashraf, M. K. Reddy and 31 (a) L. J. Browne, C. Gude, H. Rodriguez, R. E. Steele and
A. S. Kalivendi, MedChemComm, 2011, 2, 819–823.
3 A. Kamal, Y. V. V. Srikanth, T. B. Shaik, M. N. A. Khan,
M. Ashraf, M. K. Reddy, K. A. Kumar and S. V. Kalivendi,
MedChemComm, 2011, 2, 819.
4 M. A. Reddy, N. Jain, D. Yada, C. Kishore, J. R. Vangala,
R. P. Surendra, A. Anthony, S. V. Kalivendi and B. Sreedhar,
J. Med. Chem., 2011, 54, 6751–6760.
A. Bhatnager, J. Med. Chem., 1991, 34, 725–736;
(b) N. M. Weir, K. Selvendiran, V. K. Kutala, L. Tong,
S. Vishwanath, M. Rajaram, S. Tridandapani, S. Anant and
P. Kuppusamy, Cancer Biol. Ther., 2007, 6, 178–184;
(c) A. Kamal, G. B. Kumar, V. L. Nayak, V. S. Reddy,
B. S. Anver, Rajender and M. K. Reddy, MedChemComm,
2015, DOI: 10.1039/C4MD00400K.
2
2
2
5 (a) A. Kamal, G. B. Kumar, S. Polepalli, A. B. Shaik, 32 S. M. Konstantinov and M. R. Berger, Cancer Lett., 1999,
V. S. Reddy, M. K. Reddy, Ch. R. Reddy, R. Mahesh, 144, 153–160.
J. S. Kapure and N. Jain, ChemMedChem, 2014, 9, 2565– 33 R. B. Ravelli, B. Gigant, P. A. Curmi, I. Jourdain, S. Lachkar,
579; (b) A. Kamal, M. K. Reddy, T. B. Shaik, Rajender, A. Sobel and M. Knossow, Nature, 2004, 428, 198–202.
Y. V. V. Srikanth, V. S. Reddy, G. B. Kumar and 34 G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
2
S. V. Kalivendi, Eur. J. Med. Chem., 2012, 50, 9–17;
c) A. Kamal, V. S. Reddy, S. Karnewar, S. S. Chourasiya,
R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput.
Chem., 2009, 30, 2785.
(
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