C-Glycosylated cinnamoylfuran derivatives as novel anti-cancer agents

Article abstract of DOI:10.1039/c7ra04207h

A series of C-glycosylated cinnamoylfuran derivatives were synthesized in excellent yield from free sugars. The C-glycosylated furan derivatives were prepared under aqueous reaction conditions. The anticancer potentials of the synthesized compounds were evaluated on the basis of their comparative cytotoxic effects on cancer cells (MCF-7 and HeLa) and normal cells (NKE). Among 28 compounds evaluated for their cytotoxic effects on cancer cells, three compounds (compounds 8, 24 and 28) were shown to have significant cytotoxic effects on MCF-7 and HeLa cell lines and comparatively less toxicity against normal NKE cell line. Based on its selectivity index, compound 24 was considered the most promising anticancer agent amongst the above three compounds. Further biochemical studies with compound 24 showed that both intrinsic and extrinsic pathways of apoptosis contribute to compound 24 mediated cytotoxicity.

Full text of DOI:10.1039/c7ra04207h

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C-Glycosylated cinnamoylfuran derivatives as
novel anti-cancer agents†
Cite this: RSC Adv., 2017, 7, 28853
Ananya Dutta,a Debashis Dhara,‡a Pravat Kumar Parida,a Anshupriya Si,a
b
a
a
*
*
Ravichandran Yesuvadian, Kuladip Jana and Anup Kumar Misra
A series of C-glycosylated cinnamoylfuran derivatives were synthesized in excellent yield from free sugars.
The C-glycosylated furan derivatives were prepared under aqueous reaction conditions. The anticancer
potentials of the synthesized compounds were evaluated on the basis of their comparative cytotoxic
effects on cancer cells (MCF-7 and HeLa) and normal cells (NKE). Among 28 compounds evaluated for
their cytotoxic effects on cancer cells, three compounds (compounds 8, 24 and 28) were shown to have
significant cytotoxic effects on MCF-7 and HeLa cell lines and comparatively less toxicity against normal
NKE cell line. Based on its selectivity index, compound 24 was considered the most promising anticancer
agent amongst the above three compounds. Further biochemical studies with compound 24 showed
that both intrinsic and extrinsic pathways of apoptosis contribute to compound 24 mediated cytotoxicity.
Received 13th April 2017
Accepted 20th May 2017
DOI: 10.1039/c7ra04207h
rsc.li/rsc-advances
larial agents.22 Inspired by these earlier reports, it was
decided to synthesize a series of C-glycosylated cinnamoylfuran
Introduction
derivatives to evaluate their potential against cancer cell lines to
develop novel anticancer agents. It was envisioned that the
presence of a C-glycosylated furan moiety as well as an aryl
cinnamoyl functionality in the molecules could improve their
efficacy to act as effective cytotoxic agents against cancer cells.
The C-glycosylated furan derivatives containing an a-methyl-
carbonyl functionality were prepared from commercially avail-
able reducing sugars under aqueous reaction conditions,
following an earlier report.23,24 The furan derivatives were
treated with a number of aryl aldehydes in the presence of
a base to furnish the desired compounds. Some selected C-
cinnamoylated products were acetylated to check whether the
O-acetyl group has any inuence on the biological activities. The
synthesized compounds (7–34) were evaluated for their cyto-
toxicity potential against two cancer cell lines (MCF-7 and HeLa)
as well as a normal cell line (NKE). The synthesis of a series of
novel C-glycosyl cinnamoylfuran derivatives and their potential
as anticancer agents are reported herein.
Cancer is a leading cause of death worldwide, representing
a number of diseases due to uncontrolled cell division.1 Cervical
cancer and breast cancer are the most frequent among several
organ-specic cancers found in women throughout the world
and their management strategies are quite expensive.2,3
Although several anticancer therapeutics are available in
clinics, they suffer from poor prognosis due to their nonspecic
action and cytotoxic effects towards normal cells.4–6 The use of
many commonly used chemotherapeutic agents is limited
because of drug resistance.7 Therefore, the development of
novel therapeutics with better efficacy and lower toxicity is
a thrusting area in medicinal chemistry. Over the years, several
chemotherapeutic agents have been developed against
a number of organ-specic cancers, taking their lead from
natural products or synthetic intermediates.8,9 In the recent
past, C-glycosylated heterocycles, such as C-glycosylfuran
derivatives and related compounds, have been reported for their
therapeutic potential.10–15 In addition, a variety of cinnamoy-
lated C-glycoside derivatives have been synthesized with
a similar sugar moiety but different aryl groups and evaluated
for their potential as anti-mycobacterial agents,16,17 anti-cancer
agents,18 lectin inhibitors,19,20 enzyme inhibitors,21 and anti-
Results and discussion
Chemistry
Synthesis of C-glycosylated cinnamoylfuran derivatives. A
series of C-glycosylated cinnamoyl furan derivatives (7–34) were
synthesized from D-glucose, D-galactose and D-xylose following
a two-step reaction sequence. Following earlier reported reaction
conditions,23 treatment of D-glucose (1), D-galactose (2) and D-
xylose (3) with pentane-2,4-dione in the presence of CeCl3$7H2O
in water furnished the corresponding C-glycosylated furan
derivatives, such as 3-acetyl-5-C-glycosyl-2-methylfurans 4 (90%)
aBose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M,
Kolkata-700054, India. E-mail: akmisra69@gmail.com; Fax: +91 33 2355 3886; Tel:
+91 33 2569 3240
bDepartment of Biotechnology, School of Bioengineering, SRM University,
Kattankulathur – 603203, Tamil Nadu, India
† Electronic supplementary information (ESI) available: Copies of 1D and 2D
NMR spectra of compounds 7–34. See DOI: 10.1039/c7ra04207h
‡ Contributed equally.
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Table 1 Optimization of the preparation of C-glycosylated cinnamoyl
furan derivatives
Sl. no.
Base
Equiv.
Solvent
Time (h)
Yield (%)
1
2
3
4
5
NaOH
1.0
1.0
1.0
1.0
1.0
CH3OH
CH3OH
C2H5OH
CH3OH
CH3OH
12
12
16
24
24
50
75
60
50
40
NaOCH3
NaOC2H5
Piperidine
DBU
Scheme 2 Reagents and conditions: (a) pentane-2,4-dione, CeCl3-
$7H2O, H2O, 90 ꢀC, 6 h, 90%; (b) ArCHO, CH3ONa, CH3OH, rt, 12 h; (c)
acetic anhydride, pyridine, rt, 3 h.
and 5 (90%) and 3-acetyl-5-(1,2,3-trihydroxypropyl)-2-methylfuran
6 (85%), respectively, with an a-methylcarbonyl functionality
attached to them. In order to synthesize a series of cinnamoyl
derivatives under a base mediated aldol condensation of aryl
aldehydes with compounds 4–6, compound 4 was initially
allowed to react with 4-methylbenzaldehyde in the presence of
a series of bases (NaOH, NaOCH3, NaOEt, piperidine, DBU) (1.0
equiv.) in alcohol (Table 1). Aer a set of optimizations, it was
found that the use of 1.0 equiv. of sodium methoxide furnished
an excellent yield (75%) of the desired product (7) in 12 h.
Therefore, compound 4 was allowed to condense with a series of
aromatic aldehydes in the presence of sodium methoxide in
methanol to give C-glycosylated cinnamoyl furan derivatives (7–
24) in excellent yield. The formation of the trans-cinnamoyl
double bonds (E-isomers) in the molecules was conrmed from
a
1H NMR spectral analysis [doublets with large coupling
constant (J ¼ 15.5–16.0 Hz)]. It is noteworthy that the formation
of other isomers was not observed under the aldol reaction
conditions. Acetylation of compounds 23 and 24 using acetic
Scheme 3 Reagents and conditions: (a) pentane-2,4-dione, CeCl3-
$7H2O, H2O, 90 ꢀC, 6 h, 85%; (b) ArCHO, CH3ONa, CH3OH, rt, 12 h; (c)
acetic anhydride, pyridine, rt, 3 h.
anhydride and pyridine furnished acetylated derivatives 25 and
26 in quantitative yield (Scheme 1). Following similar reaction
conditions, compounds 27 and 28 were prepared from
compound 5 in very good yield, which were then acetylated to
give compounds 29 and 30 (Scheme 2). Compounds 31 and 32
were synthesized from D-xylose derived compound 6 in very good
yield, which were acetylated to give compounds 33 and 34
(Scheme 3). Earlier it was observed that O-acetylated cinnamoyl
C-glycoside derivatives showed better antilarial activities via an
apoptotic pathway than non-O-acetylated compounds.22 This
could happen due to the increase in lipophilicity in the
compounds because of the presence of acetyl groups. Based on
this observation, we have prepared some acetylated derivatives
(25, 26, 29, 30, 33 and 34) to compare the cytotoxic activities of O-
acetylated and non-O-acetylated compounds against cancer and
normal cell lines. All synthesized compounds were characterized
using spectral analysis.
Scheme 1 Reagents and conditions: (a) pentane-2,4-dione, CeCl3-
$7H2O, H2O, 90 ꢀC, 6 h, 90%; (b) ArCHO, CH3ONa, CH3OH, rt, 12 h; (c)
acetic anhydride, pyridine, rt, 3 h.
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Table 2 The IC50 (mM) values of the compounds for the inhibition of
Out of 28 tested compounds, compound 8 (IC50: 17.77 ꢁ 1.46
mM), compound 24 (IC50: 9.588 ꢁ 5.25 mM) and compound 28
(IC50: 22.98 ꢁ 4.19 mM) showed higher efficacy than etoposide
(IC50: 24.77 ꢁ 3.4 mM) in MCF-7 cells. In another set of HeLa
cells, compound 8 (IC50: 15.98 ꢁ 1.85 mM), compound 24 (IC50:
14.22 ꢁ 3.94 mM), compound 28 (IC50: 22.29 ꢁ 4.03 mM) and also
compound 9 (IC50: 10.93 ꢁ 5.36 mM) exerted lower IC50 as
compared to etoposide (IC50: 29.12 ꢁ 8.56 mM). From these
results, it was found that compound 24 was more potent in both
MCF-7 and HeLa cells as compared to compound 8 and
compound 28, whereas compound 9 was only effective in HeLa
cells. Consequently, an evaluation of the cytotoxic effect of
compounds 8, 24 and 28 against NKE cells showed signicantly
higher IC50 values than their IC50 values against MCF-7 and
HeLa cells [compound 24 (IC50: 54.14 ꢁ 1.56 mM), compound 8
(IC50: 46.03 ꢁ 10.56 mM) and compound 28 (IC50: 40.29 ꢁ 11.87
mM)]. Subsequently, when the selectivity indexes (SI) were
calculated based on the IC50 value ratio of compounds in NKE
vs. MCF-7, it was found that compound 24 (SI ¼ 7.93 ꢁ 4.18)
possessed higher SI in comparison to compound 8 (SI ¼ 2.56 ꢁ
0.38) and compound 28 (SI ¼ 4.05 ꢁ 0.06). In the case of HeLa
cells, compound 24 also showed a better SI value than the other
two compounds [compound 24 (SI ¼ 3.42 ꢁ 0.30), 8 (SI ¼ 3.33 ꢁ
0.34) and 28 (SI ¼ 1.71 ꢁ 0.20)].
proliferationa
Compd
MCF-7
HeLa
NKE
7
$60
$60
—
8
9
17.77 ꢁ 1.46
15.98 ꢁ 1.85
10.93 ꢁ 5.36
$60
$60
$60
46.03 ꢁ 10.56
$60
109 ꢁ 7.85
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Etoposide
64.54 ꢁ 13.16
$60
$60
75.07 ꢁ 27.83
—
—
$60
$60
—
$60
68.75 ꢁ 12.84
90.77 ꢁ 105.53
54.07 ꢁ 6.49
$60
81.48 ꢁ 76.22
35.93 ꢁ 10.13
$60
$60
$60
59.05 ꢁ 59.05
$60
—
—
$60
—
$60
43.49 ꢁ 7.39
47.58 ꢁ 10.37
$60
$60
45.37 ꢁ 14.31
33.0 ꢁ 1.5
$60
$60
$60
$60
99.92 ꢁ 27.89
24.38 ꢁ 11.38
—
9.588 ꢁ 5.25
14.22 ꢁ 3.94
54.14 ꢁ 1.56
$60
$60
—
—
$60
$60
$60
32.35 ꢁ 3.6
22.98 ꢁ 4.19
34.79 ꢁ 6.86
$60
$60
22.29 ꢁ 4.03
40.29 ꢁ 11.87
$60
$60
$60
—
The activity of compound 24 was further tested against MDA-
MB-231 cells. Interestingly, it was also observed that compound
24 was very effective in inhibiting the proliferation of MDA-MB-
231 cells and the calculated IC50 value was 19.16 ꢁ 5.97 mM
(Fig. 1). Based on the preliminary MTT experimental data and
selectivity index, compound 24 has been selected as a promising
anti-cancer agent and considered for further evaluation.
43.84 ꢁ 7.66
30.25 ꢁ 6.76
30.23 ꢁ 23
$60
$60
—
91.27 ꢁ 53.63
$60
24.77 ꢁ 3.4
$60
$60
29.12 ꢁ 8.56
23.84 ꢁ 17.45
—
—
a
IC50 ¼ concentration at which 50% inhibition in motility was
achieved.
Treatment with compound 24 exhibited
a signicant
decrease in the cell reproductive death (colony formation) in
MCF-7 cells. Clonogenic assay or colony formation assay,29 an in
vitro cell survival assay, was performed in MCF-7 cells using
compound 24 to check the ability of a single cell to grow into
a colony in the presence of different doses of the compound
(0 nM, 100 nM, 500 nM, 1.25 mM, 2.5 mM and 5 mM) for 15 days.
Interestingly, counting of colonies clearly demonstrated that
compound 24 signicantly decreased the colony numbers in
MCF-7 cells even at a dose of 1.25 mM (Fig. 2). At a dose of 5 mM
of compound 24, not a single colony was observed, as shown in
Fig. 2.
Biology
Cytotoxic effect of C-glycosyl cinnamoylfuran derivatives by
MTT cell proliferation assay. The newly synthesized C-glycosyl-
3-cinnamoylfuran derivatives (7–34) were screened in vitro for
their anti-proliferative effect on MCF-7 (Human breast carci-
noma), HeLa (Human cervical carcinoma) and NKE (Normal
Kidney Epithelial) cells. MCF-7 is the most studied human
breast cancer cell line and results from this cell line have had
a fundamental impact upon breast cancer research and patient
outcomes. The MCF-7 cell line is one of a very few breast cancer
cell lines that expresses substantial levels of ER, mimicking the
majority of invasive human breast cancers that express ER.25
Similarly, HeLa has been widely used as a model for cervical
cancer over the past 50 years of cervical cancer research.26
Owing to these facts we chose these two cells lines for our
experiments. Etoposide was used as a standard control,
considering its tremendous anti-proliferative activity.27 In this
experiment, MCF-7, HeLa and NKE cells were treated with
various concentrations (0 mM, 5 mM, 10 mM, 20 mM, 30 mM and
50 mM) of the C-glycoside derivatives and the cell viability was
measured by MTT assay.28 The results are summarized in Table
2 and expressed in terms of IC50 values (half-maximal inhibitory
concentration).
Fig. 1 Determination of dose-dependent cytotoxicity of compound
24 on various human cell lines.
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Fig. 3 Compound 24 induced G2/M phase cell cycle arrest in MCF-7
cells.
PI+ when treated with concentrations of 5 mM, 10 mM, 20 mM, 30
mM, respectively, in comparison with a vehicle control (4.22 ꢁ
0.45%). Thus, the apoptotic potential of compound 24 has been
established by this experiment. MCF-7 cells were shown to be
prone to apoptosis in a dose-dependent manner except for
doses of 10 mM and 20 mM, where a dose-dependent increase in
Fig. 2 Effects of compound 24 on colony formation on MCF-7 cells.
The results show a minimum of three independent experiments and
the bar graph shows mean ꢁ SEM (*p < 0.05, **p < 0.01, ***p < 0.001,
ns ¼ not significant).
Compound 24 mediated G2/M arrest contributes to growth
inhibition in MCF-7 cells. The cell cycle process has a critical
role in the cellular growth process. In order to check whether
compound 24 mediated inhibition of cancer cell proliferation
has any relationship with cell cycle arrest, MCF-7 cells were
treated with varying concentrations of compound 24 (0 mM, 5
mM, 10 mM, 20 mM and 30 mM) for 24 h and stained with pro-
pidium iodide and the cell cycle prole checked in FACs.
Interestingly, it was found that there was an increase in the
percentage of cells in the G2/M phase of the cell cycle (19.4%,
29.6%, 34.2%, 37.1%, 45.2%), as is evident from Fig. 3. 2 mM of
nocodazole was used as a positive control30 for G2/M arrest,
where 81.7% cells were found in the G2/M phase. From this
experiment it was conrmed that compound 24 mediated
apoptosis G2/M arrest contributes to the growth inhibition in
MCF-7 cells.
Treatments with compound 24 induced signicant
apoptosis in MCF-7 cells. Apoptosis is a crucial phenomenon
which blocks the proliferation of cells.31 In order to check
whether compound 24 mediated inhibition of cancer cell
proliferation has any relationship with apoptosis, we treated
MCF-7 cells with varying concentrations of compound 24 (0 mM,
5 mM, 10 mM, 20 mM and 30 mM) and stained with annexin V-
FITC/PI for analysis of apoptotic cells. Interestingly, 6.11 ꢁ
0.11%, 14.71 ꢁ 1.29%, 15.82 ꢁ 1.78%, 28.46 ꢁ 10.36% cells
were found to have annexin V-FITC+/PIꢂ and annexin V-FITC+/
Fig. 4 Demonstrates the apoptosis-inducing ability of compound 24
in the MCF-7 cell line. After treatment with compound 24, the MCF-7
cells were incubated with 5 ml of annexin V-FITC and 5 ml of PI for
15 min at room temperature and flow cytometric analysis was
performed.
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Fig. 6 Caspase-3 cleavage activity in MCF-7 cell extracts after treat-
ment with compound 24. Caspase-3 activity was measured by
determining the ability of cell extracts to cleave the colorimetric
substrate, Ac-DEVD-pNA, and plotted as a concentration in mM of the
cleaved pNA in the extract. Data represent the means ꢁ SEM of 2
independent experiments. *P < 0.01 and ns ¼ non significant represent
significant differences compared to the control condition.
proteins (TNFR1, CD4/TRAIL-1, CD5/TRAIL-2, CD95/Fas) that
help in the receptor-mediated programmed cell death process.
More importantly, a signicant increase in the expression of
cleaved caspase 3 was observed under treated conditions as
compared to the control condition (Fig. 4). In addition,
a human apoptosis proteome prole array using MCF-7 cells
showed that treatment with compound 24 resulted in an
increase in the expression of several pro-apoptotic proteins
(Bax, p21 p53 etc.) and a decrease in the expression of anti-
apoptotic proteins (survivin, claspin, bcl-2, etc.), as shown in
Fig. 4. This imbalance between pro-apoptotic and anti-apoptotic
proteins within the cells leads to apoptosis. It is well established
that p53 phosphorylation at ser15 (DNA damage response)32,33
ser 46 (induction of apoptosis)34,35 and ser 392 (growth
suppression)36 plays an important role in cell proliferation.
Interestingly, an increased level of total p53 along with p-p53
ser15, ser46, and ser392 was observed from proteome
proling and Western-immunoblotting analysis (Fig. 5). These
ndings suggested that both intrinsic as well as extrinsic
apoptosis pathways contribute to compound 24 mediated
apoptosis.
Caspase 3 activity assay. Since, caspase-3 activity is the key
factor in apoptosis induction, it was decided to carry out a cas-
pase-3/7 activity assay (colorimetric) using compound 24 to
establish its potential to induce apoptosis.37 In MCF-7 cells, a 2-
fold increase in caspase activity was measured at a concentra-
tion of 30 mM when compared with the control. Interestingly,
such a level of caspase activation can be considered sufficient to
cause cell death and the results clearly indicated that the cell
death was induced by increased caspase-3 activities (Fig. 6).
Quantitative structure activity relationship analysis. In order
to gain further information about the structure–function rela-
tionship of the active compounds, 3D-QSAR analyses of the
Fig. 5 The Human Apoptosis Array detects the expression of multiple
apoptosis-related proteins in treated and untreated MCF-7 cells. The
bar graphs indicate the band intensity. Data are representative of two
independent experiments and bar graph shows mean ꢁ SEM (*p <
0.05, **p < 0.01, ***p < 0.001, ns ¼ not significant).
apoptosis was not prominent. This signicantly elevated level of
apoptosis, conrmed through annexin V binding with trans-
located phosphatidylserine, provided a clue that the process of
apoptosis denitely contributes to the compound 24 mediated
cytotoxicity (Fig. 4).
Human apoptosis proteome prole array demonstrated
compound 24 induced increase in pro-apoptotic and decrease
in anti-apoptotic proteins in MCF-7 cell line. Annexin V-FITC
staining conrmed that apoptosis is one of the key processes
contributing to the compound 24 mediated cytotoxicity. In
order to gain a comprehensive idea regarding the molecular
changes in the expression level of several proteins associated
with apoptosis machinery, a Human Apoptosis Array kit (R&D
Biosystem) was used. MCF-7 cells were treated with 30 mM of
compound 24 for 48 hours and lysates were used to prole the
expression level of 35 proteins associated with the apoptosis
machinery. Analysis of membranes indicated the differential
expression of several apoptosis-related proteins, as shown in
Fig. 5. Interestingly, the dot images of the Human Apoptosis
Array kit showed signicant down-regulation of several anti-
apoptotic proteins, including Bcl-2, survivin, cIAP1, cIAP2 as
well as claspin. In contrast, pro-apoptotic protein Bax and p21
were signicantly up-regulated due to treatment with
compound 24. Since the Bax/Bcl-2 ratio plays a critical role in
the apoptosis process, compound 24 probably mediates
apoptosis by changing the Bax/Bcl-2 ratio. In addition, we also
observed a signicant increase in the expression of some
¨
active compounds were carried out using the Schrodinger
program.38 Model building was performed using partial least-
squares regression (PLS).39 The 3D-QSAR study described the
pharmacophoric features of C-glycosylated cinnamoylfuran
derivatives that contribute most towards the elicitation of an
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Table 3 Statistical results for 3D-QSAR modela
exhibit the interactions of their functional groups with all
pharmacophoric features (Figure not shown). Substitution with
an electron-donating alkyl group in the meta- and para- posi-
tions of the aromatic group (phenyl ring) increased the anti-
cancer activity (compounds 8 and 24). The methyl group of
the furan ring imparts hydrophobic interaction, which is also
essential for improving affinity towards the target as an anti-
cancer compound.
Training set
Test set
N1 ¼ 18
N2 ¼ 9
Corr. coefficient ¼ 0.9407
RMSD ¼ 0.1995
Corr. coefficient ¼ 0.8694
RMSD ¼ 0.2631
Max. t value ¼ 7.9529
Max. t value ¼ 3.9561
Weight ¼ 1.1276
Weight ¼ 1.1257
a
N1 and N2: number of molecules in the training and test set,
respectively; RMSD: root-mean square deviation.
Conclusions
In summary, a series of C-glycosyl cinnamoylfuran derivatives
were synthesized and evaluated for their in vitro cytotoxic
activity against cancer cells (MCF-7 and HeLa) and normal cells
(NKE). Three compounds (8, 24 and 28) showed signicant
cytotoxic effects on MCF-7 and HeLa cell lines. Based on the
selectivity index, compound 24 was considered the most
promising anticancer agent. Further biochemical studies
showed that compound 24 mediated cytotoxicity appeared to be
due to both intrinsic and extrinsic pathways of apoptosis. QSAR
studies have also been carried out to establish the structure–
activity relationships of the active compounds.
Experimental
General methods
All reactions were monitored by thin layer chromatography over
silica gel coated TLC plates. The spots on TLC were visualized by
warming ceric sulphate (2% Ce(SO4)2 in 2 N H2SO4) sprayed
plates on a hot plate. Silica gel 230–400 mesh was used for
column chromatography. 1H and 13C NMR, 2D COSY, HSQC
spectra were recorded on a Bruker Avance 500 MHz spectrom-
eter using CDCl3 as solvent and TMS as the internal reference
unless stated otherwise. Chemical shi values are expressed in
d ppm. ESI-MS were recorded on a Micromass mass spectrom-
eter. Elementary analysis was carried out on a Carlo Erba
analyzer. Optical rotations were measured at 25 ꢀC on a Jasco P-
2000 polarimeter. Biological experiments were carried out in
Fig. 7 3D-QSAR analysis of the compounds evaluated for anti-cancer
activities. (A): Regression plot representing the statistical analysis with
reference to the predicted IC50 value and theoretical IC50 values. (B):
Alignment of the molecules showing conserved C-glycosylfuran
moiety and variable aryl groups.
a
Shimadzu UV-2401PC spectrophotometer. Commercially
available grades of organic solvents of adequate purity were
used in many reactions. The cell culture media along with
ingredients were purchased from HiMedia (India). FBS was
procured from Gibco (USA). DMSO was purchased from SRL
(India). An annexin V-FITC apoptosis assay kit was bought from
BD Bioscience (India). A Caspase 3 Assay Kit (ab39401) and
Human Apoptosis Array Kit were purchased from AbCam (UK)
and R&D Systems (USA), respectively. MCF-7, MDA-MB-231
(human breast cancer) cells, HeLa (human cervical cancer)
cells were obtained from the central cell repository of National
Center for Cell Science (NCCS), Pune, India. Normal Kidney
Epithelial cell (NKE) was a kind gi from Dr K. Biswas, Bose
Institute, Kolkata. The cell lines were cultured in RPMI-1640 or
DMEM supplemented with 10% FBS, 1 mM sodium pyruvate,
2 mM L-glutamine, non-essential amino acids, 100 units per ml
penicillin, 50 mg mlꢂ1 streptomycin and 50 mg mlꢂ1 gentamicin
anti-cancer property. To analyze the anti-cancer property of the
selected data set compounds (28 molecules) by 3D-QSAR anal-
yses, they were categorized into a training set (19 molecules)
and a test set (9 molecules). The statistical signicance of this
model is given in Table 3. The training set molecules exhibited
a higher t value and correlation coefficient and a lower RMSD
value, which denote the excellence of accommodating the active
groups of the molecules in the pharmacophoric features. The
correlation between the experimental IC50 and the estimated
IC50 values of the training set and the test set molecules are
shown in Fig. 7A. The alignment of the molecular conforma-
tions conrmed that the C-glycosylated furan moiety present in
all molecules is the essential feature eliciting an anti-cancer
response (Fig. 7B). Mapping of the most active compounds 24
and 8 with the pharmacophore model has shown excellent
interaction with all pharmacophoric features. Whereas
mapping of the least active compounds 15 and 19 did not
ꢀ
sulfate at 37 C in a humidied incubator containing 5% CO2
atmosphere.
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15.5 Hz, COCH ¼ CH), 7.00 (d, 2H, J ¼ 9.0 Hz, Ar-H), 6.72 (s, 1H,
H-4), 5.12 (brs, 2H, PhCH2), 4.69 (d, 1H, J ¼ 6.5 Hz, H-10), 4.43–
4.38 (m, 2H, H-40), 4.30–4.27 (m, 1H, H-30), 3.93 (dd, 1H, J ¼ 3.0,
10.5 Hz, H-20), 2.65 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3):
d 185.7 (C]O), 160.7 (C-4), 150.2 (C-1), 143.2 (COCH ¼ CH),
136.3, 130.1, 128.6 (Ar-C), 128.1 (COCH ¼ CH), 127.7, 127.4 (Ar-
C), 121.5 (C-2), 115.2 (Ar-C), 109.0 (C-3), 77.2 (C-10), 74.8 (C-30),
73.2 (C-40), 71.0 (C-20), 70.0 (PhCH2), 14.6 (CH3); ESI-MS: 443.11
[M + Na]+; anal. calcd for C25H24O6 (420.45): C, 71.41; H, 5.75%;
found: C, 71.30; H, 5.87%.
General experimental condition for the preparation of
compound 7–34
To a solution of compound 4 or 5 or 6 (1 mmol) in CH3OH (10
ml) were added aromatic aldehyde (1.05 mmol) and NaOCH3
(100 mg) and the reaction mixture was allowed to stir at room
temperature for 12 h. The reaction mixture was diluted with
water and acidied with 1 N HCl. The white solid precipitated
out from the solution, which was ltered and crystallized from
EtOH to give pure products 7–34 in appropriate yield, as
mentioned in the schemes.
Compound 11. Yellow solid; Rf: 0.24 (hexane–EtOAc; 1 : 2);
25
1
ꢀ
mp 124–125 C [EtOH]; [a]D ꢂ15 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 7.56 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.43 (d,
Analytical data of the compounds 7–34
Compound 7. Yellow solid; Rf: 0.20 (hexane–EtOAc; 1 : 2); mp 2H, J ¼ 8.0 Hz, Ar-H), 7.28 (d, 2H, J ¼ 8.0 Hz, Ar-H), 7.04 (d, 1H, J
120–122 ꢀC [EtOH]; [a]D25 ꢂ51 (c 1.0, CHCl3); 1H NMR (500 MHz, ¼ 15.5 Hz, COCH ¼ CH), 6.62 (s, 1H, H-3), 4.58 (d, 1H, J ¼
CDCl3): d 7.71 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.50 (d, 2H, J ¼ 6.5 Hz, H-10), 4.31–4.27 (m, 2H, H-40), 4.18–4.15 (m, 1H, H-30),
7.5 Hz, Ar-H), 7.21 (d, 2H, J ¼ 7.5 Hz, Ar-H), 7.14 (d, 1H, J ¼ 3.81 (dd, 1H, J ¼ 2.5, 10.0 Hz, H-20), 2.54 (s, 3H, CH3); 13C NMR
15.5 Hz, COCH ¼ CH), 6.72 (s, 1H, H-4), 4.68 (d, 1H, J ¼ 6.5 Hz, (125 MHz, CDCl3): d 186.3 (C]O), 161.1 (C-4), 150.0 (C-1), 141.9
H-10), 4.42–4.38 (m, 2H, H-40), 4.29–4.26 (m, 1H, H-30), 3.92 (dd, (COCH ¼ CH), 136.2, 133.2, 129.5, 128.4, 129.2 (Ar-C), 124.0
1H, J ¼ 3.0, 10.5 Hz, H-20), 2.65 (s, 3H, CH3), 2.41 (s, 3H, CH3); (COCH ¼ CH), 122.2 (C-2), 108.9 (C-3), 77.1 (C-10), 74.8 (C-30),
13C NMR (125 MHz, CDCl3): d 185.7 (C]O), 159.7 (C-4), 150.9 73.1 (C-40), 71.0 (C-20), 14.6 (CH3); ESI-MS: 371.0 [M + Na]+; anal.
(C-1), 143.5 (COCH ¼ CH), 140.8, 132.2, 129.6, 128.4 (Ar-C), calcd for C18H17ClO5 (348.78): C, 61.99; H, 4.91%; found: C,
122.6 (COCH ¼ CH), 122.3 (C-2), 109.0 (C-3), 77.2 (C-10), 74.8 61.84; H, 5.02%.
(C-30), 73.1 (C-40), 71.0 (C-20), 21.5, 14.6 (2C, CH3); ESI-MS: 351.1
Compound 12. Yellow solid; Rf: 0.35 (hexane–EtOAc; 1 : 3);
25
1
[M + Na]+; anal. calcd for C19H20O5 (328.36): C, 69.50; H, 6.14%; mp 122–123 C [EtOH]; [a]D ꢂ10 (c 1.0, CHCl3); H NMR (500
ꢀ
found: C, 69.45; H, 6.34%.
MHz, CDCl3): d 8.54 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 8.21 (d,
Compound 8. Yellow solid; Rf: 0.22 (hexane–EtOAc; 1 : 2); mp 1H, J ¼ 8.0 Hz, Ar-H), 7.86–7.76 (m, 2H, Ar-H), 7.55–7.42 (m, 4H,
128–130 ꢀC [EtOH]; [a]D25 ꢂ34 (c 1.0, CHCl3); 1H NMR (500 MHz, Ar-H), 7.21 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.71 (s, 1H, H-4),
CDCl3): d 7.58 (d, 1H, J ¼ 16.0 Hz, COCH ¼ CH), 7.10 (d, 1H, J ¼ 4.67 (d, 1H, J ¼ 6.0 Hz, H-10), 4.35–4.34 (m, 2H, H-40), 4.22–4.19
7.5 Hz, Ar-H), 7.03 (s, 1H, Ar-H), 6.96 (d, 1H, J ¼ 15.5 Hz, COCH (m, 1H, H-30), 3.87–3.85 (m, 1H, H-20), 2.63 (s, 3H, CH3); 13C
¼ CH), 6.79 (d, 1H, J ¼ 8.0 Hz, Ar-H), 6.65 (s, 1H, H-4), 4.61 (d, NMR (125 MHz, CDCl3): d 185.2 (C]O), 160.0 (C-4), 150.3 (C-1),
1H, J ¼ 6.0 Hz, H-10), 4.32–4.29 (m, 2H, H-40), 4.19–4.17 (m, 1H, 140.2 (COCH ¼ CH), 133.7, 132.1, 131.8, 130.7, 128.7, 127.5,
H-30), 3.86–3.82 (m, 7H, H-20, 2 OCH3), 2.55 (s, 3H, CH3); 13C 126.4, 125.3, 125.1, (Ar-C), 123.5 (COCH ¼ CH), 122.3 (C-2),
NMR (125 MHz, CDCl3): d 185.7 (C]O), 159.7 (C-4), 151.5 (C-1), 108.9 (C-3), 77.1 (C-10), 74.8 (C-30), 73.2 (C-40), 71.0 (C-20), 14.7
150.0, 149.2 (Ar-C), 143.8 (COCH ¼ CH), 127.6 (Ar-C), 122.2 (CH3); ESI-MS: 387.1 [M + Na]+; anal. calcd for C22H20O5
(COCH ¼ CH), 122.3 (C-2), 121.5 (Ar-C), 110.1, 110.2 (Ar-C), (364.39): C, 72.51; H, 5.53%; found: C, 72.42; H, 5.65%.
109.1 (C-3), 77.1 (C-10), 74.7 (C-30), 73.1 (C-40), 71.0 (C-20), 55.7
Compound 13. Yellow solid; Rf: 0.15 (hexane–EtOAc; 1 : 1);
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1
(2C, 2 OCH3), 14.6 (CH3); ESI-MS: 397.1 [M + Na]+; anal. calcd for mp 103–104 C [EtOH]; [a]D ꢂ26 (c 1.0, CHCl3); H NMR (500
ꢀ
C
20H22O7 (374.38): C, 64.16; H, 5.92%; found: C, 64.42; H, MHz, CDCl3): d 7.60 (d, 1H, J ¼ 9.0 Hz, Ar-H), 7.58 (d, 1H, J ¼
6.01%.
15.5 Hz, COCH ¼ CH), 7.18 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH),
Compound 9. Yellow solid; Rf: 0.31 (hexane–EtOAc; 1 : 2); mp 6.81–6.79 (m, 2H, Ar-H), 6.60 (s, 1H, H-4), 4.77 (d, 1H, J ¼ 7.0 Hz,
141–142 ꢀC [EtOH]; [a]D25 ꢂ41 (c 1.0, CHCl3); 1H NMR (500 MHz, H-10), 4.52–4.46 (m, 2H, H-40), 4.38–4.35 (m, 1H, H-30), 4.01–3.98
CDCl3): d 7.63 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.05 (d, 1H, J ¼ (m, 1H, H-20), 2.75 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3):
15.5 Hz, COCH ¼ CH), 6.81 (s, 2H, Ar-H), 6.73 (s, 1H, H-4), 4.69 d 185.2 (C]O), 160.0 (C-4), 150.3 (C-1), 144.7 (COCH ¼ CH),
(d, 1H, J ¼ 7.0 Hz, H-10), 4.43–4.38 (m, 2H, H-40), 4.30 (dd, 1H, J 129.3 (C-2), 121.2 (COCH ¼ CH), 116.0, 112.6 (Ar-C), 109.0 (C-3),
¼ 5.0, 10.0 Hz, H-30), 3.92–3.84 (m, 10H, H-20, 3 OCH3), 2.66 (s, 77.2 (C-10), 74.7 (C-30), 73.1 (C-40), 71.0 (C-20), 14.6 (CH3); ESI-MS:
3H, CH3); 13C NMR (125 MHz, CDCl3): d 185.4 (C]O), 159.8 (C- 327.0 [M + Na]+; anal. calcd for C16H16O6 (304.29): C, 63.15; H,
4), 153.4 (Ar-C), 149.9 (C-1), 143.6 (COCH ¼ CH), 137.7 (Ar-C), 5.30%; found: C, 63.10; H, 5.42%.
130.2 (C-2), 126.2 (Ar-C), 123.0 (COCH ¼ CH), 109.0 (C-3),
Compound 14. Yellow solid; Rf: 0.33 (hexane–EtOAc; 1 : 3);
105.7 (2C, Ar-C), 77.2 (C-10), 74.7 (C-30), 73.2 (C-40), 71.0 (C-20), mp 93–95 ꢀC [EtOH]; [a]D25 ꢂ37 (c 1.0, CHCl3); 1H NMR (500
60.9, 56.1 (3C, 3OCH3), 14.6 (CH3); ESI-MS: 427.1 [M + Na]+; MHz, CDCl3): d 7.76 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.58 (d,
anal. calcd for C21H24O8 (404.41): C, 62.37; H, 5.98%; found: C, 2H, J ¼ 9.0 Hz, Ar-H), 7.05 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH),
62.47; H, 6.11%.
6.78–6.75 (m, 3H, Ar-H), 6.66 (s, 1H, H-4), 4.73 (d, 1H, J ¼ 7.0 Hz,
Compound 10. Yellow solid; Rf: 0.28 (hexane–EtOAc; 1 : 2); H-10), 4.50–4.44 (m, 2H, H-40), 4.37–4.32 (m, 1H, H-30), 3.99 (dd,
1
mp 99–101 ꢀC [EtOH]; [a]D25 ꢂ21 (c 1.0, CHCl3); H NMR (500 1H, J ¼ 3.0, 10.0 Hz, H-20), 3.15 (s, 6H, N (CH3)2), 2.46 (s, 3H,
MHz, CDCl3): d 7.72 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.56 (d, CH3); 13C NMR (125 MHz, CDCl3): d 185.7 (C]O), 159.7 (C-4),
2H, J ¼ 9.0 Hz, Ar-H), 7.44–7.34 (m, 5H, Ar-H), 7.07 (d, 1H, J ¼ 149.7 (C-1), 148.6 (Ar-C), 144.7 (COCH ¼ CH), 130.4 (2C, Ar-C),
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122.6 (COCH ¼ CH), 121.9 (Ar-C), 118.5 (C-2), 111.9 (2C, Ar-C),
109.0 (C-3), 77.1 (C-10), 74.7 (C-30), 73.2 (C-40), 71.1 (C-20), 40.1
(2C, N (CH3)2), 14.6 (2C, CH3); ESI-MS: 380.1 [M + Na]+; anal.
calcd for C20H23NO5 (357.40): C, 67.21; H, 6.49%; found: C,
67.14; H, 6.60%.
Compound 19. Yellow solid; Rf: 0.32 (hexane–EtOAc; 1 : 3);
25
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ꢀ
mp 138–140 C [EtOH]; [a]D ꢂ31 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 7.57 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.43 (d,
2H, J ¼ 8.5 Hz, Ar-H), 6.93 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.70
(d, 2H, J ¼ 8.0 Hz, Ar-H), 6.60 (s, 1H, H-3), 4.58 (d, 1H, J ¼ 6.0 Hz,
H-10), 4.32–4.27 (m, 2H, H-40), 4.17–4.15 (dd, 1H, J ¼ 4.5,
10.0 Hz, H-30), 3.99 (q, 2H, J ¼ 7.0 Hz each, OCH2CH3), 3.81 (dd,
1H, J ¼ 3.0, 10.0 Hz, H-20), 2.52 (s, 3H, CH3), 1.36 (t, 3H, J ¼
7.0 Hz, OCH2CH3); 13C NMR (125 MHz, CDCl3): d 185.7 (C]O),
161.0 (Ar-C), 159.6 (C-4), 149.8 (C-1), 143.5 (COCH ¼ CH), 130.2,
127.2 (Ar-C), 122.4 (C-2), 121.2 (COCH ¼ CH), 114.8 (Ar-C), 109.1
(C-3), 77.1 (C-10), 74.7 (C-30), 73.1 (C-40), 71.0 (C-20), 63.5
(OCH2CH3), 14.7 (CH3), 14.6 (OCH2CH3); ESI-MS: 381.1 [M +
Na]+; anal. calcd for C20H22O6 (358.39): C, 67.03; H, 6.19%;
found: C, 66.90; H, 6.27%.
Compound 15. Yellow solid; Rf: 0.25 (hexane–EtOAc; 1 : 2);
25
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ꢀ
mp 102–104 C [EtOH]; [a]D ꢂ20 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 7.80 (d, 1H, J ¼ 16.0 Hz, COCH ¼ CH), 7.69–7.68
(m, 2H, Ar-H), 7.50–7.48 (m, 3H, Ar-H), 7.27 (d, 1H, J ¼ 16.0 Hz,
COCH ¼ CH), 6.81 (s, 1H, H-4), 4.78 (d, 1H, J ¼ 6.5 Hz, H-10),
4.51–4.47 (m, 2H, H-40), 4.38–4.35 (m, 1H, H-30), 4.01 (dd, 1H, J
¼ 3.0, 10.0 Hz, H-20), 2.73 (s, 3H, CH3); 13C NMR (125 MHz,
CDCl3): d 185.5 (C]O), 159.9 (C-4), 150.0 (C-1), 143.5 (COCH ¼
CH), 134.7, 130.4, 128.9, 128.4 (Ar-C), 123.6 (COCH ¼ CH), 122.3
(C-2), 109.0 (C-3), 77.2 (C-10), 74.8 (C-30), 73.2 (C-40), 71.0 (C-20),
14.6 (CH3); ESI-MS: 337.1 [M + Na]+; anal. calcd for C18H18O5
(314.33): C, 68.78; H, 5.77%; found: C, 68.65; H, 5.90%.
Compound 20. Yellow solid; Rf: 0.24 (hexane–EtOAc; 1 : 2);
25
1
ꢀ
mp 125–127 C [EtOH]; [a]D ꢂ40 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 7.56 (d, 1H, J ¼ 16.0 Hz, COCH ¼ CH), 7.21–7.15
(m, 1H, Ar-H), 7.07–6.98 (m, 3H, COCH ¼ CH, Ar-H), 6.83 (d, 1H,
J ¼ 8.0 Hz, Ar-H), 6.62 (s, 1H, H-3), 4.58 (d, 1H, J ¼ 6.5 Hz, H-10),
4.28–4.26 (m, 2H, H-40), 4.16–4.12 (m, 1H, H-30), 3.80–3.73 (m,
4H, OCH3, H-20), 2.50 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3):
d 185.7 (C]O), 159.9 (Ar-C), 159.9 (C-4), 150.0 (C-1), 143.5
(COCH ¼ CH), 136.0, 129.9, 123.9 (Ar-C), 122.2 (C-2), 121.0
(COCH ¼ CH), 116.2, 113.4 (Ar-C), 109.1 (C-3), 77.1 (C-10), 74.8
(C-30), 73.1 (C-40), 71.0 (C-20), 55.2 (OCH3), 14.6 (CH3); ESI-MS:
367.1 [M + Na]+; anal. calcd for C19H20O6 (344.36): C, 66.27; H,
5.85%; found: C, 66.10; H, 5.99%.
Compound 16. Yellow solid; Rf: 0.30 (hexane–EtOAc; 1 : 3);
25
1
ꢀ
mp 108–110 C [EtOH]; [a]D ꢂ21 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 13.2 (s, 1H, NH), 6.76 (d, 1H, J ¼ 14.5 Hz, COCH
¼ CH), 6.75 (d, 1H, J ¼ 8.0 Hz, Ar-H), 6.67 (s, 1H, H-4), 6.53–6.49
(m, 3H, COCH ¼ CH, Ar-H), 4.77 (d, 1H, J ¼ 6.5 Hz, H-10), 4.48–
4.46 (m, 2H, H-40), 4.37–4.34 (m, 1H, H-30), 3.98 (dd, 1H, J ¼ 2.5,
10.5 Hz, H-20), 2.65 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3):
d 185.2 (C]O), 157.1 (C-4), 149.0 (C-1), 138.9 (C-2), 128.5 (COCH
¼ CH), 123.9 (Ar-C), 118.9 (COCH ¼ CH), 111.4 (Ar-C), 109.9 (C-
3), 107.7, 103.2 (Ar-C), 77.3 (C-10), 74.6 (C-30), 73.0 (C-40), 71.0 (C-
20), 14.0 (CH3); ESI-MS: 326.1 [M + Na]+; anal. calcd for
C
16H17NO5 (303.31): C, 63.36; H, 5.65%; found: C, 63.20; H,
5.48%.
Compound 17. Pale yellow solid; Rf: 0.30 (hexane–EtOAc;
Compound 21. Yellow solid; Rf: 0.27 (hexane–EtOAc; 1 : 2);
25
1
ꢀ
mp 155–157 C [EtOH]; [a]D ꢂ16 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 8.12 (d, 1H, J ¼ 16.0 Hz, COCH ¼ CH), 7.69 (dd,
1H, J ¼ 2.0, 7.0 Hz, Ar-H), 7.44 (dd, 1H, J ¼ 1.0, 7.5 Hz, Ar-H),
7.34–7.24 (m, 2H, Ar-H), 7.16 (d, 1H, J ¼ 16.0 Hz, COCH ¼
CH), 6.74 (s, 1H, H-3), 4.71 (d, 1H, J ¼ 6.5 Hz, H-10), 4.45–4.39
(m, 2H, H-40), 4.30–4.27 (m, 1H, H-30), 3.92 (dd, 1H, J ¼ 3.0,
10.0 Hz, H-20), 2.65 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3):
d 186.2 (C]O), 160.3 (C-4), 149.9 (C-1), 139.3 (COCH ¼ CH),
135.5, 133.1, 131.1, 130.3, 127.7, 127.0 (Ar-C), 126.4 (COCH ¼
CH), 122.1 (C-2), 109.1 (C-3), 77.2 (C-10), 74.8 (C-30), 73.3 (C-40),
71.0 (C-20), 14.7 (CH3); ESI-MS: 371.0 [M + Na]+; anal. calcd for
2 : 5); mp 145–147 ꢀC [EtOH]; [a]D25 ꢂ32 (c 1.0, CHCl3); 1H NMR
(500 MHz, CDCl3): d 8.26 (d, 2H, J ¼ 6.5 Hz, Ar-H), 7.75 (d, 1H, J
¼ 16.0 Hz, COCH ¼ CH), 7.73 (d, 2H, J ¼ 7.5 Hz, Ar-H), 7.29 (d,
1H, J ¼ 16.0 Hz, COCH ¼ CH), 6.76 (s, 1H, H-3), 4.71 (d, 1H, J ¼
7.0 Hz, H-10), 4.46–4.41 (m, 2H, H-40), 4.32–4.22 (m, 1H, H-30),
3.94 (dd, 1H, J ¼ 3.0, 10.0 Hz, H-20), 2.65 (s, 3H, CH3); 13C NMR
(125 MHz, CDCl3): d 185.7 (C]O), 159.7 (C-4), 142.1 (C-1), 140.1
(COCH ¼ CH), 132.2, 129.6, 128.4 (Ar-C), 127.4 (COCH ¼ CH),
124.2 (Ar-C), 123.1 (C-2), 108.8 (C-3), 77.2 (C-10), 74.8 (C-30), 73.3
(C-40), 71.0 (C-20), 14.7 (CH3); ESI-MS: 382.1 [M + Na]+; anal.
calcd for C18H17NO7 (359.33): C, 60.17; H, 4.77%; found: C,
60.00; H, 4.95%.
C
18H17ClO5 (348.78): C, 61.99; H, 4.91%; found: C, 61.82; H,
5.05%.
Compound 22. Yellow solid; Rf: 0.18 (hexane–EtOAc; 1 : 2);
25
1
ꢀ
Compound 18. Yellow solid; Rf: 0.28 (hexane–EtOAc; 1 : 2);
mp 121–123 C [EtOH]; [a]D ꢂ18 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 7.61–7.39 (m, 5H, J ¼ 16.0 Hz, COCH ¼ CH, Ar-
H), 7.13 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.64 (s, 1H, H-3), 4.59
(d, 1H, J ¼ 6.5 Hz, H-10), 4.31–4.27 (m, 2H, H-40), 4.18–4.15 (m,
1H, H-30), 3.81 (dd, 1H, J ¼ 3.0, 9.0 Hz, H-20), 2.54 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3): d 185.0 (C]O), 160.3 (C-4), 150.2
(C-1), 141.4 (COCH ¼ CH), 138.1, 128.4, 126.9, 125.8 (Ar-C),
125.8 (COCH ¼ CH), 122.2 (C-2), 108.9 (C-3), 77.1 (C-10), 74.8
(C-30), 73.2 (C-40), 71.0 (C-20), 14.7 (CH3); ESI-MS: 405.1 [M +
Na]+; anal. calcd for C19H17F3O5 (382.33): C, 59.69; H, 4.48%;
found: C, 59.58; H, 4.60%.
25
1
ꢀ
mp 133–135 C [EtOH]; [a]D ꢂ35 (c 1.0, CHCl3); H NMR (500
MHz, CDCl3): d 7.59 (d, 1H, J ¼ 16.5 Hz, COCH ¼ CH), 7.45 (d,
2H, J ¼ 8.5 Hz, Ar-H), 6.95 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.82
(d, 2H, J ¼ 8.0 Hz, Ar-H), 6.61 (s, 1H, H-3), 4.59 (d, 1H, J ¼ 6.5 Hz,
H-10), 4.32–4.28 (m, 2H, H-40), 4.19 (dd, 1H, J ¼ 4.5, 10.0 Hz, H-
30), 3.82 (dd, 1H, J ¼ 3.0, 10.0 Hz, H-20), 3.75 (s, 3H, CH3), 2.53 (s,
3H, CH3); 13C NMR (125 MHz, CDCl3): d 185.2 (C]O), 161.2 (Ar-
C), 159.0(C-4), 151.1 (C-1), 143.3 (COCH ¼ CH), 130.1, 127.4 (Ar-
C), 123.5 (C-2), 121.4 (COCH ¼ CH), 114.4 (Ar-C), 109.1 (C-3),
77.2 (C-10), 74.7 (C-30), 73.2 (C-40), 71.0 (C-20), 55.3 (OCH3),
14.7 (CH3); ESI-MS: 367.1 [M + Na]+; anal. calcd for C19H20O6
(344.36): C, 66.27; H, 5.85%; found: C, 66.07; H, 6.00%.
Compound 23. Yellow solid; Rf: 0.27 (hexane–EtOAc; 1 : 2);
25
1
ꢀ
mp 167–169 C [EtOH]; [a]D ꢂ29 (c 1.0, CHCl3); H NMR (500
28860 | RSC Adv., 2017, 7, 28853–28864
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MHz, CDCl3): d 8.35 (s, 1H, Ar-H), 8.12 (d, 1H, J ¼ 8.5 Hz, Ar-H), CH3CO), 14.6 (CH3); ESI-MS: 557.1 [M + Na]+; anal. calcd for
7.76 (d, 1H, J ¼ 7.5 Hz, Ar-H), 7.61 (d, 1H, J ¼ 15.5 Hz, COCH ¼
C30H30O9 (534.55): C, 67.41; H, 5.66%; found: C, 67.30; H,
CH), 7.50 (t, 1H, J ¼ 8.0 Hz each, Ar-H), 7.19 (d, 1H, J ¼ 15.5 Hz, 5.80%.
COCH ¼ CH), 6.67 (s, 1H, H-3), 4.60 (d, 1H, J ¼ 6.5 Hz, H-10),
Compound 27. Yellow solid; Rf: 0.26 (hexane–EtOAc; 1 : 3);
25
1
4.31–4.28 (m, 2H, H-40), 4.18–4.15 (m, 1H, H-30), 3.82 (dd, 1H, J mp 123–125 C [EtOH]; [a]D +17 (c 1.0, CHCl3); H NMR (500
¼ 2.5, 10.5 Hz, H-20), 2.54 (s, 3H, CH3); 13C NMR (125 MHz, MHz, CDCl3): d 8.46 (s, 1H, Ar-H), 8.25 (d, 1H, J ¼ 7.5 Hz, Ar-H),
CDCl3): d 184.6 (C]O), 160.6 (C-4), 150.3 (C-1), 148.7 (Ar-C), 7.89 (d, 1H, J ¼ 8.0 Hz, Ar-H), 7.76 (d, 1H, J ¼ 15.5 Hz, COCH ¼
140.2 (COCH ¼ CH), 136.5, 134.3, 129.9, 126.2, 124.5 (Ar-C), CH), 7.62 (t, 1H, J ¼ 8.0 Hz each, Ar-H), 7.32 (d, 1H, J ¼ 15.5 Hz,
122.2 (COCH ¼ CH), 122.0 (C-2), 108.8 (C-3), 77.0 (C-10), 74.8 COCH ¼ CH), 6.87 (s, 1H, H-3), 5.15 (d, 1H, J ¼ 3.0 Hz, H-10),
(C-30), 73.2 (C-40), 71.0 (C-20), 14.7 (CH3); ESI-MS: 382.1 [M + 4.50 (brs, 1H, H-20), 4.42–4.38 (m, 1H, H-40a), 4.26 (brs, 1H, H-
Na]+; anal. calcd for C18H17NO7 (359.33): C, 60.17; H, 4.77%; 40b), 3.87 (d, 1H, J ¼ 10.0 Hz, H-20), 2.67 (s, 3H, CH3); 13C NMR
ꢀ
found: C, 60.05; H, 4.95%.
(125 MHz, CDCl3): d 184.7 (C]O), 160.5 (C-4), 148.7 (C-1), 148.4
Compound 24. Yellow solid; Rf: 0.33 (hexane–EtOAc; 1 : 2); (Ar-C), 140.3 (COCH ¼ CH), 136.5, 134.2, 123.0, 126.3, 124.6 (Ar-
1
mp 98–100 ꢀC [EtOH]; [a]D25 ꢂ21 (c 1.0, CHCl3); H NMR (500 C), 122.3 (COCH ¼ CH), 122.1 (C-2), 109.2 (C-3), 78.0 (C-10), 77.2
MHz, CDCl3): d 7.59 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.46–7.12 (C-20), 76.8 (C-30), 73.9 (C-40), 14.6 (CH3); ESI-MS: 382.1 [M +
(m, 5H, Ar-H), 7.15–7.12 (m, 2H, Ar-H), 6.91 (d, 1H, J ¼ 15.5 Hz, Na]+; anal. calcd for C18H17NO7 (359.33): C, 60.17; H, 4.77%;
COCH ¼ CH), 6.86 (d, 2H, J ¼ 8.5 Hz, Ar-H), 6.66 (s, 1H, H-4), found: C, 60.05; H, 4.95%.
5.16 (br s, 2H, PhCH2), 4.67 (d, 1H, J ¼ 6.0 Hz, H-10), 4.37–
Compound 28. Yellow solid; Rf: 0.18 (hexane–EtOAc; 1 : 1);
25
1
4.35 (m, 2H, H-40), 4.25–4.23 (m, 1H, H-30), 3.90–3.88 (m, 4H, H- mp 131–133 C [EtOH]; [a]D +12 (c 1.0, CHCl3); H NMR (500
20, OCH3), 2.58 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3): d 185.7 MHz, CDCl3): d 7.70 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.59–7.41
(C]O), 159.6 (C-4), 152.1 (C-1), 150.0, 148.3 (Ar-C), 143.2 (COCH (m, 5H, Ar-H), 7.27 (d, 1H, J ¼ 8.0 Hz, Ar-H), 7.23 (d, 1H, J ¼
¼ CH), 136.7, 128.6, 128.0, 127.5, 127.3, (Ar-C), 123.3 (COCH ¼ 1.5 Hz, Ar-H), 7.05 (d, 1H, J ¼ 15.0 Hz, COCH ¼ CH), 6.98 (d, 1H,
CH), 122.4 (Ar-C), 121.5 (C-2), 113.1, 111.5 (Ar-C), 109.1 (C-3), J ¼ 8.5 Hz, Ar-H), 6.78 (s, 1H, H-3), 5.28 (brs, 2H, PhCH2), 4.78
77.1 (C-10), 74.7 (C-30), 73.2 (C-40), 71.2 (PhCH2), 71.0 (C-20), (d, 1H, J ¼ 4.5 Hz, H-10), 4.50–4.46 (m, 2H, H-30, H-20), 4.23 (dd,
55.9 (OCH3), 14.6 (CH3); ESI-MS: 473.1 [M + Na]+; anal. calcd for 1H, J ¼ 4.5, 10.0 Hz, H-40b), 4.10 (dd, 1H, J ¼ 2.5, 10.0 Hz, H-40a),
ꢀ
C
26H26O7 (450.48): C, 69.32; H, 5.82%; found: C, 69.18; H, 4.02 (s, 3H, OCH3), 2.71 (s, 3H, CH3); 13C NMR (125 MHz,
6.00%.
CDCl3): d 185.6 (C]O), 159.4 (C-4), 152.1 (C-1), 148.3 (Ar-C),
Compound 25. White solid; Rf: 0.40 (hexane–EtOAc; 1 : 1); 143.6 (COCH ¼ CH), 136.7, 128.6, 128.0, 127.3 (Ar-C), 123.6
25
1
ꢀ
mp 113–115 C [EtOH]; [a]D ꢂ68 (c 1.0, CHCl3); H NMR (500 (COCH ¼ CH), 122.5 (C-2), 121.5, 113.1, 113.0, 111.5, 109.5 (Ar-
MHz, CDCl3): d 8.57 (s, 1H, Ar-H), 8.36 (dd, 1H, J ¼ 1.5, 8.5 Hz, C), 108.5 (C-3), 81.2 (C-10), 80.1 (C-20), 78.2 (C-30), 73.5 (C-40), 71.2
Ar-H), 7.99 (d, 1H, J ¼ 7.5 Hz, Ar-H), 7.86 (d, 1H, J ¼ 15.5 Hz, (PhCH2), 55.9 (OCH3), 14.6 (CH3); ESI-MS: 473.1 [M + Na]+; anal.
COCH ¼ CH), 7.73 (t, 1H, J ¼ 8.0 Hz each, Ar-H), 7.39 (d, 1H, J ¼ calcd for C26H26O7 (450.48): C, 69.32; H, 5.82%; found: C, 69.20;
15.5 Hz, COCH ¼ CH), 6.88 (s, 1H, H-3), 5.64–5.58 (m, 2H, H-30, H, 5.95%.
H-20), 5.03 (d, 1H, J ¼ 6.5 Hz, H-10), 4.51 (dd, 1H, J ¼ 5.0, 10.5 Hz,
Compound 29. Yellow oil; Rf: 0.40 (hexane–EtOAc; 1 : 1);
H-40a), 4.09 (dd, 1H, J ¼ 3.5, 10.5 Hz, H-40b), 2.79 (s, 3H, CH3), [a]2D5 +26 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.58 (s, 1H,
2.24, 2.22 (2 s, 6H, CH3CO); 13C NMR (125 MHz, CDCl3): d 184.3 Ar-H), 8.57 (dd, 1H, J ¼ 1.0, 8.0 Hz, Ar-H), 7.99 (dd, 1H, J ¼ 3.0,
(C]O), 169.7, 169.5 (CH3CO), 160.9 (C-4), 148.8 (C-1), 148.7 (Ar- 7.5 Hz, Ar-H), 7.87 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.73 (t, 1H,
C), 140.2 (COCH ¼ CH), 136.6, 134.1, 129.9, 126.2, 124.5 (Ar-C), J ¼ 7.5, 8.0 Hz, Ar-H), 7.40 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.90
122.2 (COCH ¼ CH), 122.0 (C-2), 109.2 (C-3), 74.9 (C-10), 73.7 (C- (s, 1H, H-3), 5.57 (d, 1H, J ¼ 3.0 Hz, H-20), 5.36–5.34 (m, 1H, H-
30), 71.2 (C-20), 70.7 (C-40), 20.6, 20.5 (CH3CO), 14.7 (CH3); ESI- 30), 4.91 (d, 1H, J ¼ 4.0 Hz, H-10), 4.33 (dd, 1H, J ¼ 5.0, 10.5 Hz,
MS: 466.1 [M + Na]+; anal. calcd for C22H21NO9 (443.40): C, H-40a), 4.20 (dd, 1H, J ¼ 2.0, 11.5 Hz, H-40b), 2.77 (s, 3H, CH3),
59.59; H, 4.77%; found: C, 59.47; H, 4.88%.
2.24, 2.23 (2 s, 6H, CH3CO); 13C NMR (125 MHz, CDCl3): d 184.7
Compound 26. Yellow oil; Rf: 0.25 (hexane–EtOAc; 2 : 1); (C]O), 158.7 (C-4), 149.8 (C-1), 140.2 (COCH ¼ CH), 136.8,
1
[a]2D5 ꢂ20 (c 1.0, CHCl3); H NMR (500 MHz, CDCl3): d 7.73 (d, 134.2, 129.9, 126.3, 124.5 (Ar-C), 122.2 (COCH ¼ CH), 122.0 (C-
1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.58 (d, 2H, J ¼ 7.0 Hz, Ar-H), 7.51 2), 109.2 (C-3), 108.7 (Ar-C), 79.7 (C-10), 78.4 (C-20), 77.9 (C-30),
(t, 2H, J ¼ 7.5 Hz each, Ar-H), 7.44 (d, 1H, J ¼ 7.0 Hz, Ar-H), 7.30 72.0 (C-40), 20.8, 20.5 (2C, CH3CO), 14.7 (CH3); ESI-MS: 466.1 [M
(dd, 1H, J ¼ 1.5, 8.0 Hz, Ar-H), 7.24 (d, 1H, J ¼ 1.5 Hz, Ar-H), 7.04 + Na]+; anal. calcd for C22H21NO9 (443.40): C, 59.59; H, 4.77%;
(d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.01 (d, 1H, J ¼ 8.5 Hz, Ar-H), found: C, 59.45; H, 4.90%.
6.81 (s, 1H, H-4), 5.63–5.58 (m, 2H, H-20, H-30), 5.30 (brs, 2H,
Compound 30. Yellow oil; Rf: 0.23 (hexane–EtOAc; 1 : 3);
PhCH2), 5.02 (d, 1H, J ¼ 6.5 Hz, H-10), 4.50 (dd, 1H, J ¼ 5.0, [a]D25 +29 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.73 (d, 1H,
10.0 Hz, H-40), 4.08 (dd, 1H, J ¼ 3.5, 10.0 Hz, H-40), 4.04 (s, 3H, J ¼ 15.5 Hz, COCH ¼ CH), 7.58–7.23 (m, 7H, Ar-H), 7.05 (d, 1H, J
OCH3), 2.74 (s, 3H, CH3), 2.24, 2.19 (2 s, 6H, CH3CO); 13C NMR ¼ 15.5 Hz, COCH ¼ CH), 7.01 (d, 1H, J ¼ 2.0 Hz, Ar-H), 6.99 (s,
(125 MHz, CDCl3): d 185.4 (C]O), 169.7, 169.5 (2C, CH3CO), 1H, H-3), 5.58 (d, 1H, J ¼ 3.0 Hz, H-20), 5.34 (dd, 1H, J ¼ 2.5,
159.8 (C-4), 152.2 (C-1), 148.3 (Ar-C), 143.2 (COCH ¼ CH), 136.7, 4.5 Hz, H-30), 5.29 (brs, 2H, PhCH2), 4.91 (d, 1H, J ¼ 4.0 Hz, H-
128.6, 128.0, 127.6, 127.3, (Ar-C), 123.4 (COCH ¼ CH), 122.5 (C- 10), 4.32 (dd, 1H, J ¼ 5.0, 10.5 Hz, H-40a), 4.18 (dd, 1H, J ¼ 2.0,
2), 121.6, 113.1, 111.5 (Ar-C), 109.5 (C-3), 74.9 (C-10), 73.7 (C-20), 12.5 Hz, H-40b), 4.05 (s, 3H, OCH3), 2.72 (s, 3H, CH3), 2.23, 2.20
71.3 (C-30), 71.1 (C-40), 70.7 (PhCH2), 55.9 (OCH3), 20.6, 20.5 (2C, (2 s, 6H, CH3CO); 13C NMR (125 MHz, CDCl3): d 185.2 (C]O),
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170.0, 169.5 (CH3CO), 159.5 (C-4), 152.0 (C-1), 148.3, 147.1 (Ar- H), 7.75 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.62 (t, 1H, J ¼ 8.0 Hz
C), 143.3 (COCH ¼ CH), 136.8, 128.6, 128.0, 127.6, 127.3 (Ar- each, Ar-H), 7.28 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.85 (s, 1H,
C), 123.4 (COCH ¼ CH), 122.5 (C-2), 121.6, 113.2, 111.5 (Ar-C), H-3), 6.02 (d, 1H, J ¼ 3.5 Hz, H-10), 5.53–5.50 (m, 1H, H-20), 4.43
109.0 (C-3), 79.7 (C-10), 78.3 (C-20), 77.9 (C-30), 72.0 (C-40), 71.2 (dd, 1H, J ¼ 3.0, 12.5 Hz, H-30a), 3.94 (dd, 1H, J ¼ 5.5, 12.0 Hz, H-
(PhCH2), 55.9 (OCH3), 14.6 (CH3); ESI-MS: 557.1 [M + Na]+; anal. 30b), 2.68 (s, 3H, CH3), 2.12, 2.11, 2.09 (3 s, 9H, CH3CO); 13C NMR
calcd for C30H30O9 (534.55): C, 67.41; H, 5.66%; found: C, 67.31; (125 MHz, CDCl3): d 183.8 (C]O), 170.0, 169.8, 169.1 (3C,
H, 5.70%.
CH3CO), 160.6 (C-4), 148.7 (C-1), 140.3 (COCH ¼ CH), 136.4,
Compound 31. Yellow solid; Rf: 0.18 (hexane–EtOAc; 1 : 3); 133.9, 129.7, 127.9, 126.0, 125.5 (Ar-C), 122.2 (COCH ¼ CH),
mp 99–101 ꢀC [EtOH]; [a]D25 +13 (c 1.0, CHCl3); 1H NMR (500 122.8 (C-2), 110.5 (C-3), 70.1 (C-10), 66.1 (C-20), 61.6 (C-30), 20.7,
MHz, CDCl3): d 7.70 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.56 (d, 20.6, 20.5 (3C, CH3CO), 14.6 (CH3); ESI-MS: 496.1 [M + Na]+;
1H, J ¼ 7.5 Hz, Ar-H), 7.49 (t, 2H, J ¼ 8.0, 7.5 Hz, Ar-H), 7.41 (d, anal. calcd for C23H23NO10 (473.43): C, 58.35; H, 4.90%; found:
1H, J ¼ 7.5 Hz, Ar-H), 7.27 (d, 1H, J ¼ 8.0 Hz, Ar-H), 7.22 (d, 1H, J C, 58.47; H, 5.10%.
¼ 2.0 Hz, Ar-H), 7.03 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 6.97 (d,
1H, J ¼ 8.5 Hz, Ar-H), 6.80 (s, 1H, H-3), 5.27 (brs, 2H, PhCH2),
MTT cell proliferation assay
4.83 (d, 1H, J ¼ 5.5 Hz, H-10), 4.12 (m, 1H, H-20), 4.02 (s, 3H,
The anti-proliferative effects of the C-glycosyl-3-cinnamoylfuran
OCH3), 3.88 (dd, 1H, J ¼ 3.0, 12.5 Hz, H-30a), 3.76 (dd, 1H, J ¼
derivatives (7–34) were assessed by a MTT proliferation assay.26
5.5, 12.5 Hz, H-30b), 2.69 (s, 3H, CH3); 13C NMR (125 MHz,
For this experiment, cells were seeded in triplicate in 96 well
CDCl3): d 185.7 (C]O), 158.9 (C-4), 152.1 (C-1), 151.3, 148.3 (Ar-
plates and incubated overnight. Post incubation cells were
C), 143.5 (COCH ¼ CH), 136.7, 128.6, 128.0, 127.5, 127.3 (Ar-C),
treated with compounds at various concentrations (5 mM, 10
123.5 (COCH ¼ CH), 122.0 (C-2), 121.5, 113.1, 111.5 (Ar-C), 108.1
mM, 20 mM and 50 mM) for 48 h. Aer the treatment, 200 ml of
(C-3), 72.9 (C-10), 71.2 (PhCH2), 68.3 (C-20), 63.5 (C-30), 55.9
MTT solution (0.5 mg mlꢂ1) were added to each well and
(OCH3), 14.5 (CH3); ESI-MS: 461.1 [M + Na]+; anal. calcd for
ꢀ
incubated at 37 C for 3 h. Subsequently, the MTT media was
C
25H26O7 (438.47): C, 68.48; H, 5.98%; found: C, 68.30; H,
discarded and purple colored formazan crystals were dissolved
using DMSO. The absorbance was measured at 570 nm in a 96
well micro-plate reader (Thermo, Multiskan Go).
6.13%.
Compound 32. Yellow solid; Rf: 0.15 (hexane–EtOAc; 1 : 3);
mp 94–96 ꢀC [EtOH]; [a]D25 +7 (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3): d 8.44 (s, 1H, Ar-H), 8.22 (d, 1H, J ¼ 8.0 Hz, Ar-H), 7.85
(d, 1H, J ¼ 8.5 Hz, Ar-H), 7.73 (d, 1H, J ¼ 15.5 Hz, COCH ¼ CH),
Clonogenic survival assay
7.58 (t, 1H, J ¼ 8.0, 8.5 Hz, Ar-H), 7.26 (d, 1H, J ¼ 15.5 Hz, COCH In 6 well plates around 200 MCF-cells were seeded in each of the
¼ CH), 6.74 (s, 1H, H-3), 4.72 (d, 1H, J ¼ 5.0 Hz, H-10), 3.99–3.79 wells and cells were allowed to attach to the plates for 2 h. Then
(m, 1H, H-20), 3.88 (dd, 1H, J ¼ 3.0, 12.5 Hz, H-30a), 3.76 (dd, 1H, the cells were treated with different concentrations of
J ¼ 5.5, 12.5 Hz, H-30b), 2.68 (s, 3H, CH3); 13C NMR (125 MHz, compound 24 (0 nM, 100 nM, 500 nM, 1.25 mM, 2.5 mM and 5
CDCl3): d 185.6 (C]O), 158.7 (C-4), 149.8 (C-1), 139.9 (COCH ¼ mM) for the next 15 days with continuous change of media on
CH), 135.5, 133.6, 129.6, 126.3, 124.2 (Ar-C), 123.5 (COCH ¼ every 3rd day with or without compounds. Aer 15 days the cells
CH), 122.1 (C-2), 107.5 (C-3), 72.8 (C-10), 68.4 (C-20), 63.3 (C-30), were washed with 1X PBS and incubated with 3 ml of a mixture
14.4 (CH3); ESI-MS: 370.26 [M + Na]+; anal. calcd for C17H17NO7 of 3.7% formaldehyde in 1X PBS and 0.5% crystal violet for 30
(347.32): C, 58.79; H, 4.93%; found: C, 58.62; H, 5.10%.
minutes. The formaldehyde crystal violet mixture was carefully
Compound 33. Yellow oil; Rf: 0.38 (hexane–EtOAc; 1 : 1); removed by rinsing with tap water and plates were allowed to
[a]2D5 +12 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.73 (d, 1H, dry at room temperature. Finally, the image was captured using
J ¼ 15.5 Hz, COCH ¼ CH), 7.59 (d, 2H, J ¼ 7.0 Hz, Ar-H), 7.52 (t, Gel Doc XR + (Bio-Rad) and colony numbers were counted.
2H, J ¼ 7.5 Hz each, Ar-H), 7.45 (d, 1H, J ¼ 7.0 Hz, Ar-H), 7.30
(dd, 1H, J ¼ 1.5, 8.5 Hz, Ar-H), 7.25 (d, 1H, J ¼ 2.0 Hz, Ar-H), 7.02
Cell cycle arrest assay by propidium iodide staining
(d, 1H, J ¼ 15.5 Hz, COCH ¼ CH), 7.01 (d, 1H, J ¼ 8.5 Hz, Ar-H),
MCF-7 cells were treated with increasing concentrations (0, 5
6.87 (s, 1H, H-3), 6.15 (d, 1H, J ¼ 8.0 Hz, H-10), 5.70–5.68 (m, 1H,
mM, 10 mM, 20 mM, 30 mM) of compound 24 for 24 h and 2 mM of
H-20), 5.31 (brs, 2H, PhCH2), 4.48 (dd, 1H, J ¼ 8.5, 12.5 Hz, H-
nocodazole was used as a positive control for G2 arrest. Post
30a), 4.05–4.02 (m, 4H, OCH3, H-30b), 2.74 (s, 3H, CH3), 2.2, 2.18
treatment cells were converted into single cell suspension and
(2 s, 6H, CH3CO); 13C NMR (125 MHz, CDCl3): d 184.9 (C]O),
170.1, 169.7, 169.4 (3C, CH3CO), 159.9 (C-4), 152.1 (C-1), 148.4,
146.3 (Ar-C), 143.5 (COCH ¼ CH), 136.8, 128.6, 128.0, 127.3 (Ar-
ꢀ
xed with 75% ethanol overnight at ꢂ20 C. Cells were centri-
fuged and resuspended in 1X PBS for 2 h followed by RNase A
ꢀ
(20 mm) treatment for 2 h at 37 C. Finally, propidium iodide
C), 123.5 (COCH ¼ CH), 122.5 (C-2), 121.4, 113.0, 111.5 (Ar-C),
was added and incubated for 20 minutes at room temperature.
110.6 (C-3), 71.2 (PhCH2), 70.2 (C-10), 66.4 (C-20), 62.0 (C-30),
Flow cytometric analysis was immediately performed using
55.9 (OCH3), 20.8, 20.7, 20.6 (3C, CH3CO), 14.5 (CH3); ESI-MS:
a FACS Verse instrument (BD).
587.2 [M + Na]+; anal. calcd for C31H32O10 (564.58): C, 65.95;
H, 5.71%; found: C, 65.86; H, 5.89%.
Analysis of apoptosis by annexin V-FITC/PI
Compound 34. Yellow oil; Rf: 0.44 (hexane–EtOAc; 1 : 1);
[a]2D5 +11 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.48 (s, 1H, To determine the apoptosis-inducing ability of compound 24 on
Ar-H), 8.26 (d, 1H, J ¼ 8.0 Hz, Ar-H), 7.89 (d, 1H, J ¼ 7.5 Hz, Ar- MCF-7 cells, a double staining method with annexin V-FITC and
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PI was performed using an assay kit (BD bioscience kit). MCF-7 Bhunia, Bose Institute is thankfully acknowledged for valuable
cells were seeded in a 6 well plate and incubated overnight discussions regarding QSAR analysis.
followed by treatment with various concentrations of
compound 24 (5 mM, 10 mM, 20 mM and 30 mM) for 48 h. Aer
treatment, the cells were washed with 1X PBS and resuspended
Notes and references
in 100 ml of binding buffer. Finally, cells were incubated with 5
ml of annexin V-FITC and 5 ml of PI for 15 min at room
temperature in the dark and ow cytometric analysis was per-
formed using a BD Verse FACS machine.
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˜
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Proteome Proler array
A Human Apoptosis Array Kit from R&D Systems (USA) was used
for determining the apoptosis-related proteins following the
manufacturer's protocol. Briey, the cells were treated with
compound 24 at 30 mM concentration for 48 h and a DMSO
control was kept. 250 mg protein from each sample was incu-
bated overnight with the array. Finally, the data was measured
by developing the membrane and then the pixel density was
calculated using Image J soware. Data are representative of
two independent experiments and the bar graph shows mean ꢁ
SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ns ¼ not signicant).
Caspase 3 activity assay
A colorimetric assay following the manufacturer's protocol
(Caspase 3 Assay Kit (Colorimetric) (ab39401)) was used to
measure the caspase activity. MCF-7 cells were treated with
compound 24 at a concentration of 30 mM for 24 h and 48 h. The
RIPA buffer method was used to prepare the cell lysates. Each
cell lysate was incubated with 50 ml of 2ꢃ reaction buffer (con-
taining 10 mM DTT) and 5 ml of the 4 mM DEVD-p-NA substrate
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ꢀ
(200 mM) and kept at 37 C for 2 h. The OD was measured at
400 nm in a micro-plate reader and then the graph was plotted.
Quantitative structure activity relationship (QSAR) analysis
Based on the experimental results, the molecular structures
were compared with the IC50 value to elucidate a quantitative
structure-activity relationship. The 3D-QSAR module of the
¨
Schrodinger program was used to derive the atom-based anal-
ysis. All molecular structures were prepared and minimized
using Maestro GUI. The models were processed by categorizing
all 28 molecules into a training set and a test set. The model
divides the space of grids into uniform-sized cubes that reect
the biological relationship using structural features, corre-
sponding through positive (blue) and negative (red) contours.
Characterization of molecules is performed using independent
variables for structural components to reect statistically
signicant results. Model building was performed using partial
least-squares regression (PLS). Detailed methodology and
principles are similar to the reports published earlier.39
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Acknowledgements
D. D., P. P. and A. S. thank Council of Scientic and Industrial 23 A. K. Misra and G. Agnihotri, Carbohydr. Res., 2004, 339,
Research (CSIR), India for providing Senior Research Fellow- 1381–1387.
ships. This work is supported by CSIR, India [Grant No. 24 J. S. Yadav, B. V. S. reddy, M. Sreenivas and G. Sathees,
02(0237)/15/EMR-II (AKM)] and Bose Institute. Dr Anirban
Synthesis, 2007, 1712–1716.
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