3-(2,6-Dichloro-benzyloxy)-11-oxo-olean-12-ene-29-oic acid, a semisynthetic derivative of glycyrrhetic acid: Synthesis, antiproliferative, apoptotic and anti-angiogenesis activity

Article abstract of DOI:10.1039/c4md00344f

Glycyrrhetic acid (2, 3β-hydroxyl-11-oxo-olean-12-ene-29-oic acid), a pentacyclic triterpenoid isolated from Glycyrrhiza glabra, is known to possess a wide range of biological activities. Herein, we report the synthesis and antiproliferative activity of 3-O-ether derivatives of glycyrrhetic acid. The cytotoxicity of the prepared derivatives was investigated in three cancer cell lines, including human pancreatic (MIAPaCa-2), prostate (PC-3) and human hepatocellular liver carcinoma (HepG-2). Amongst the tested compounds, the 2,6-dichlorobenzyl 5b and 2,4-dichlorobenzyl derivative 5r displayed significant cytotoxicity in PC-3 cells with IC₅₀ values of 6 and 18 μM, respectively. The dichlorobenzyl derivative 5b also displayed cytotoxicity in MIAPaCa-2 (IC₅₀: 7 μM) and HepG-2 cells (IC₅₀: 19 μM). Further, compound 5b was investigated for apoptosis-induction by cell cycle analysis, nuclear morphological changes and mitochondrial membrane potential loss in PC-3 cells. Compound 5b led to an increase in the sub-G1 population in PC-3 cells, which is indicative of its apoptotic properties. Interestingly, compound 5b also arrested the S-phase of the cell cycle. The nuclear morphology of PC-3 cells after treatment with compound 5b was also investigated which confirmed the formation of apoptotic bodies. Compound 5b induced apoptosis through both intrinsic and extrinsic apoptotic pathways in PC-3 cells, which was confirmed by mitochondrial membrane potential loss, inhibition of pro-caspase-3, 8 and 9 and cleavage of PARP-1. Furthermore, there was a significant decrease in the Bcl-2/Bax ratio induced by compound 5b in PC-3 cells. Interestingly, compound 5b also inhibited the VEGF-induced PC-3 cell migration and decreased the wound closure percentage from 100 to 12% at 30 μM. Similarly, compound 5b inhibited the angiogenesis-dependent cell migration in HUVEC cells and decreased wound closure from 100 to 20% at 30 μM, indicating its anti-angiogenic activity. This journal is

Full text of DOI:10.1039/c4md00344f

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GRAPHICAL ABSTRACT
O
H
O
HO
HO
O
O
H
H
Cl
H
H
O
HO
H
Cl
Glycyrrhetic acid (2)
5b
IC50: 6 µM (PC-3)
120
100
80
IC50: >20 µM (PC-3)
*
60
40
20
*
*
30
0
0
10 20
5b, μM
Synthesis and biological evaluation of semisynthetic analogs of glycyrrhetic acid is described.

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Concise Article
3-(2,6-Dichloro-benzyloxy)-11-oxo-olean-12-ene-29-oic acid, a
semisynthetic derivative of glycyrrhetic acid: Synthesis,
antiproliferative, apoptotic and anti-angiogenesis activity
Rajni Sharma,ab Santosh K. Guru,c Shreyans K. Jain,ab Anup Singh Pathania,c Ram A. Vishwakarma,abd*
Shashi Bhushanbc* and Sandip B. Bharatebd*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Glycyrrhetic acid (2, 3β-hydroxyl-11-oxo-olean-12-ene-29-oic acid), a pentacyclic triterpenoid isolated from Glycyrrhiza glabra is
known to possess wide range of biological activities. Herein, we report synthesis and antiproliferative activity of 3-O-ether derivatives of
10 glycyrrhetic acid. The cytotoxicity of prepared derivatives was investigated in three cancer cell lines, including human pancreatic
(MIAPaCa-2), prostate (PC-3) and human hepatocellular liver carcinoma (HepG-2). Amongst tested compounds, the 2,6-dichlorobenzyl
5b and 2,4-dichlorobenzyl derivative 5r displayed significant cytotoxicity in PC-3 cells with IC50 values of 6 and 18 µM, respectively.
The dichlorobenzyl derivative 5b also displayed cytotoxicity in MIAPaCa-2 (IC50: 7 µM) and HepG-2 cells (IC50:19 µM). Further, the
compound 5b was investigated for apoptosis-induction by cell cycle analysis, nuclear morphological changes and mitochondrial
15 membrane potential loss in PC-3 cells. Compoound 5b led to increase in sub-G1 population in PC-3 cells, which is indicative of its
apoptotic property. Interestingly, compound 5b also arrested S-phase of the cell cycle. The nuclear morphology of PC-3 cells after
treatment with compound 5b was also investigated which confirmed the formation of apoptotic bodies. The compound 5b induced
apoptosis through both intrinsic and extrinsic apoptotic pathways in PC-3 cells, which was confirmed by mitochondrial membrane
potential loss, inhibition of pro-caspase-3, 8 and 9 and cleavage of PARP-1. Furthermore, there was a significant decrease in Bcl-2/Bax
20 ratio by compound 5b in PC-3 cells. Interestingly, compound 5b also inhibited the VEGF-induced PC-3 cell migration and decreased
wound closure percentage from 100 to 12% at 30 µM. Similarly, compound 5b inhibited angiogenesis-dependent cell migration in
HUVEC cells and decreased wound closure from 100 to 20% at 30 µM, indicating its anti-angiogenic activity.
Introduction1
cell under the action of the TPA (12-O-tetradecanoylphorbol-13-
acetate; a tumor promotor),4 and also suppressed estrogen-related
endometrial cancer by inhibiting COX-2, IL-1α and TNF-α.5
25 Glycyrrhiza glabra (Licorice) is a tall shrub of the Leguminosae
family, widely cultivated throughout Europe, the Middle East and
Asia.1 The ethnomedical use of G. glabra has been documented
Glycyrrhetic acid (2, 3β-hydroxyl-11-oxo-olean-12-ene-29-oic
in several traditional systems of medicine. The rhizomes of 40 acid; also called as glycyrrhetinic acid), is a aglycone of
licorice have been used worldwide as an herbal medicine and
30 natural sweetener (30-50 times sweeter than sucrose).2 G. glabra
and its active components are reported to possess wide range of
biological activities,3 however the most active component which
glycyrrhizin, and is also present in G. glabra.6 Glycyrrhetic acid
belongs to the class of ursane-type pentacyclic triterpenoid and
has wide range of biological activities including anti-
inflammatory,7 anti-ulcer, analgesic, anti-type IV allergic,3d,
8
is responsible for its medicinal properties is a triterpene saponin 45 prevention of metabolic and vascular diseases9 and anticancer
9
glycyrrhizin (1, also called as glycyrrhizinic acid and glycyrrhizic
35 acid).3d Glycyrrhizin also inhibits specific changes that occur in a
activity.3c, The glycyrrhetic acid (2) is reported to possess
cytotoxicity, and apoptosis-inducing activity in different hepatic,
stomach, melanoma, breast and leukemia cancer cell lines.10 It
showed cytotoxic activity in HL-60 cells with IC50 of 63.2 µM.11
a
Natural Products Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu-180001, India
b
Academy of Scientific & Innovative Research (AcSIR), CSIR-Indian
50 Glycyrrhetic acid also showed protection against UV-induced
skin cancer,12 and also increased natural killer cell activity in
metastatic tumor.13 Another interesting property of glycyrrhetic
acid is chemosensitizing effect with various clinical oncology
drugs. It partly reversed multidrug resistance (MDR) in Pgp-
55 expressing cells (KB-C2) by increasing the intracellular
accumulation of antitumor drugs.14 Based on these findings, it is
likely that glycyrrhetic acid derivatives elicit antitumor activity
through multiple signaling pathways. In general, the cytotoxic or
apoptotic activity of glycyrrhetic acid in most of the studies was
Institute of Integrative Medicine, Canal Road, Jammu-180001, India
c
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative
Medicine, Canal Road, Jammu-180001, India
d
Medicinal Chemistry Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu-180001, India
*E-mail: sbharate@iiim.ac.in; sbhushan@iiim.ac.in; ram@iiim.ac.in
¤
IIIM Publication number IIIM/1670/2014
Fax: +91-191-2569333; Tel: +91-191-2569111
#
Electronic supplementary information (ESI) available: Experimental
details and NMR spectra of all new compounds. See DOI: 10.1039/xxxx
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O
O
moderate; thus research efforts were mainly focused on the
identification of its derivatives with improved activity.11b, 15 The
structure-activity relationship of glycyrrhetic acid has been
reviewed recently.11b The modifications on ring A were found to
be more effective. A 2-trifluoromethyl derivative displayed
potent cytotoxicity in 253JB-V (IC50= 0.67 µM), KU7 (IC50=
0.38 µM), PANC-1 (IC50= 0.82 µM) and PANC-28 (IC50= 1.14
µM) cell lines.16 Glycyrrhetic acid derivative with C-3
alkoxyimino group and C-30 carboxylic acid methyl ester showed
HO
HO
O
O
a
H
H
HOOC
O
H
HO
HO
H
5
O
O
HO
H
HOOC
HO
HO
O
H
1
2
OH
b
O
HO
10 improvement in cytotoxicity in HL-60 cells from IC50 of 63 to 19
µM.11a Fused heterocyclic rings at C-2 and C-3 positions of
glycyrrhetic acid led to improvement in cytotoxicity by 20-fold.17
In general, the C-3 modifications were more successful, and led
to identification of two antiulcer drugs carbenoxolone (3) and
15 acetoxolone (4).18 Carbenoxolone has also been reported to
possess chemopreventive activity.19 The literature precedence
indicated that substitution of lipophilic groups at C-3 position is
beneficial. Thus, herein we aimed to prepare new C-3 ether
derivatives of glycyrrhetic acid and investigate their cytotoxicity
20 in various cancer cell lines. Furthermore, the most promising
cytotoxic compound was then mechanistically investigated in
detail, in a panel of assays for apoptosis-inducing activity and
anti-angiogenesis activity.
O
H
H
H
R
O
5a-r
35 Scheme 1. Synthesis of derivatives 5a-r from glycyrrhizic acid (1).
Reagents and conditions: (a) 5% Aq. HCl, MeOH, 90 oC, 20 h. (b) K2CO3
(1.2 equiv), R-Cl (for R, see Table 1, 5a to 5r) (1 equiv), dry acetone, 0
°C, 2 h, yield: see Table 1.
40 A series of ethers were synthesized and characterized by NMR
and MS analysis. The list of synthesized derivatives is shown in
Table 1.
O
30
29
HO
Table 1. Synthesized glycyrrhetic acid derivatives
O
H
20
19
21
22
E
HO
11
O
25
Entry R
% yield
Entry R
% yield
92
18
12
17
D 16
15
13
14
28
C
O
H
HOOC
1
9
Cl
Cl
O
3 A10
B
6
2
8
7
27
HO
HO
5
26
4
5a
5j
O
O
92
H
HOOC
HO
HO
O
23
24
F
HO
OH
2
1
O
Br
O
HO
5b
5k
HO
F
95
97
O
H
Cl
O
H
H
O
O
H
H
5c
5l
HO
Cl
F
O
90
88
95
95
O
H
O
4
3
I
5d
5m
25 Figure 1. Chemical structures of glycyrrhizin (1), its aglycone
glycyrrhetic acid (2) and known derivatives 3-4 of glycyrrhetic acid
Br
Cl
O2N
5e
5f
5n
5o
95
94
97
95
Results and discussion
O
O
NO2
Glycyrrhizin (1) was isolated from G. glabra using reported
30 procedure.20 Aglycone 2 was then obtained by acid hydrolysis of
glycyrrhizin (1). The C-3 etherification was achieved by adding
different benzyl or alkyl chlorides in a solution of glycyrrhetic acid
Br
5g
5p
MeO
90%
90
(2) in dry acetone, under alkaline conditions (Scheme 1).
MeO
MeO
CF3
5q
5r
90
85
90
92
5h
5i
F3C
Cl
H3C
Cl
2
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All compounds were evaluated for antiproliferative activity in
three human cancer cell lines including pancreatic (MIAPaCa-2),
propidium iodide (PI) staining. As depicted in Figure 3, the
regions marked with different colors represent % population at
prostate (PC-3) and hepatocellular carcinoma (HepG-2). 40 different phases of the cell cycle. PC-3 cells exposed to
Cytotoxicity results of most promising compounds are shown in
Table 2. Results for all compounds are shown in ESI (Table S1).
compound 5b for 24 h exhibited a dose-dependent increase in
sub-G1 fraction (<2n DNA), which may comprise both apoptosis
and debris fraction implying together the extent of cell death
(Figure 3). The sub-G1 apoptotic population was found to be 8,
45 12 and 37% following 5, 10 and 20 µM of 5b treatment compared
to control (untreated cells - 3%). Interestingly, compound 5b
significantly arrested the S-phase of the cell cycle in a dose-
dependent manner in PC-3 cells, which ultimately results in
blockage of cell (DNA) division.
5
Table 2. Cytotoxicity of most active compounds in different
cancer cell lines a,b,c,d
Entry
IC50 (µM)
PC-3
MIAPaCa-2
11 0.97
7.0 0.56
12 1.0
>100
0.1 0.003
0.19 0.008
HepG2
>100
19 1.18
>100
>100
5a
5b
5c
5r
10 1.0
6.0 0.46
9.0 0.49
18 1.11
0.012 0.012
1.2 0.067
5b, 5 µM
600
500
400
300
200
(b).
Control
3% Apo
50% G1
30% S
20% G2
(a).
800
600
400
200
8% Apo
59% G1
40% S
Apoptosis
Apoptosis
Dip G1
Dip G2
Dip S
Dip G1
Dip G2
Dip S
Paclitaxel
Camptothecin
>100
0.2 0. 01
1% G2
aCells were grown in 96-well culture plates and treated with various
100
10 concentrations of each test compounds for 48 h. Thereafter, cells were
incubated with MTT solution for 2 h and the optical density of formazan
b
crystals was measured as described in the experimental section. HepG2,
80
Channels (FL2-A)
20
40
60
100
30
60
90
120
Channels (PE-A)
hepatocellular carcinoma cells; MIAPaCa-2, pancreatic cancer cells; PC-
c
3, prostate cancer cells. Compounds 2, and 5d-5q were inactive against
15 all cell lines used (IC50 > 20 µM). d Data are Mean SD (n= 3).
5b, 10 µM
5b, 20 µM
(c).
(d).
500
800
12% Apo
51% G1
42% S
37% Apo
44% G1
53% S
Apoptosis
Dip G1
Dip G2
Dip S
Apoptosis
400
300
200
Dip G1
Dip G2
Dip S
600
400
7% G2
4% G2
All the tested compounds were found to be more sensitive against
prostate cancer PC-3 cells in comparison to MIAPaCa-2 and
HepG-2 cells. Compound 5b (2,6-dichloro-benzyloxy derivative)
20 showed significant cytotoxicity in all three tested cell lines and it
caused concentration and time-dependent inhibition of PC-3 cell
proliferation. It showed varying cytotoxicity potential (IC50) at
different time points as depicted in Figure 2. The main purpose of
this experiment was to check the IC50 value of compound 5b at
25 24 h time point, for the purpose of cell cycle, MMP loss,
microscopy, wound scratch and western blot experiments.
200
100
100
150
50
120
30
60
90
Channels (FL2-A)
Channels (FL2-A)
50
Control
(e).
5b, 5 µM
60
50
40
30
20
10
0
*
5b, 10 µM
5b, 20 µM
*
*
*
500
*
400
370
*
*
400
*
*
300
200
G0
G1
G2
S
DifferentPhasesof the Cell Cycle
100
Figure 3. DNA cell cycle analyses in PC-3 cells exposed to compound
5b. PC-3 cells were treated with different concentrations (5, 10 and 20
µM) of compound 5b for 24 h and stained with Propidium iodide, PI (10
100
15
24
6
0
6
12
18
48
55 µg/ml) to determine DNA fluorescence and cell cycle phase distribution
as described in Materials and Methods. Data were analyzed by Modfit
software (Verity Software House Inc., Topsham, ME) for the proportions
of different cell cycle phases. The fraction of cells from apoptosis (sub-
G1/G0), G1, S and G2 phases analyzed from FL2- A vs. cell counts are
60 shown in %. Data are representative of one of three similar experiments.
Time, h
Figure 2. Cytotoxicity of compound 5b in PC-3 cells at different time
points. The cells were grown in 96-well culture plate and treated with
30 different concentrations of compound 5b for indicated time intervals.
Cells were incubated with MTT solution and optical density of formazan
crystals was measured as described in Materials and Methods. Data are
Mean SD (n= 8 wells), and representative of three similar experiments.
The apoptosis induction results obtained from cell cycle analysis
were further corroborated by studying nuclear morphological
changes of cells by fluorescence microscopy. After the treatment
65 at 5, 10, and 20 µM of compound 5b, characteristic changes of
apoptosis such as nuclear condensation, membrane blebbing and
35 In order to address the cell death caused by compound 5b, the
extent of apoptotic death in PC-3 cells was assessed using flow
cytometry through determination of sub-G1 cell population by
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formation of apoptotic bodies were observed in the morphology
of treated cells in a concentration-dependent manner, whereas
cytometer in FL-1 channel. In the untreated control cells, almost
all cells were functionally active with high Rh-123 fluorescence.
untreated cells nuclei were found to be of normal intact 25 Mitochondrial damage results in decrease in Rh-123
morphology. The results suggest that compound 5b was able to
Fluorescence. Compound 5b at 10 µM caused mitochondrial
damage and hence led to increase in the mitochondrial membrane
potential loss by about 15%, which was further increased to 38%
at 20 µM (Figure 5). The loss of mitochondrial membrane
30 potential (∆ψm) is largely due to the opening of mitochondrial
permeability transition pores (PTP), which conduit the leakage of
proapoptoic proteins from mitochondria to cytosol.21
5
induce apoptotic cell morphology in PC-3 cells (Figure 4).
5b, 5 µM
Control
300
5b, 5 μM
Control
300
250
200
(b).
(a).
250
200
150
12%
150
100
5%
100
50
0
50
0
105
102
104
103
FL1-H
104
102
103
FL1-H
105
250
200
300
5b, 10 μM
5b, 20 μM
(d).
250
(c).
200
150
100
50
150
100
50
15%
38%
0
0
5b, 10 µM
5b, 20 µM
102
103
104
105
102
103
FL1-H
104
105
FL1-H
50
40
30
20
10
0
(e).
*
*
0
5
10
20
5b, µM
35 Figure 5 Compound 5b induced mitochondrial membrane potential loss
in prostate cancer PC-3 cells. Cells were treated with compound 5b at 5,
10, and 20 µM concentration for 24 h time period. Cells were stained with
Rhodamine-123 (200 nM) dye for 30 min and analyzed in FL-1 vs. counts
channels of flow cytometer. Data are representative of one of three similar
40 experiments at different time period.
Figure 4. Effect of compound 5b on cellular and nuclear morphology of
PC-3 cells. Cells were treated with indicated concentrations of compound
Next, the effect of compound 5b on key mitochondrial apoptotic
proteins (pro-caspases and PARP-1) and Bcl-2/Bax ratio was
investigated. The expression of anti-apoptotic protein Bcl-2 was
45 significantly decreased by compound 5b at 10 and 20 µM
concentrations in PC-3 cells (Figure 6a). The downregulation of
anti-apoptotic Bcl-2 protein caused the structural deformation of
mitochondria, which opens mitochondrial permeability transition
pores and release pro-apoptotic proteins to the cytosol and
50 translocation of Bax from cytosol to mitochondria. Activation of
Bax leads to cleavage of pro-caspase-9, activation of pro-caspase-
3 and finally cleavage of downstream target poly(ADP-ribose)
polymerase-1 (PARP-1). Compound 5b was also found to inhibit
the pro-caspase-8 expression in PC-3 cells. Hence, both intrinsic
55 and extrinsic apoptotic pathways seemed to play a role in the
10 5b for 24 h time period and subsequently stained with Hoechst 33258 as
described in experimental section and visualized for nuclear morphology
and apoptotic bodies’ formation. Data are representative of one of three
similar experiments and magnification of the pictures was 30X on
Olympus 1X 70 inverted microscopes.
15
Compound 5b induced apoptosis through both intrinsic and
extrinsic apoptotic pathways, which was confirmed by
mitochondrial membrane potential (MMP) loss. Mitochondrial
damage to cells results in perturbation of MMP. The loss in MMP
20 (∆ψm) of PC-3 cells by compound 5b was studied using
rhodamine123 dye, which was reduced by healthy mitochondria
into fluorescent probe whose fluorescence was measured by flow
4
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activation of executioner pro-caspase-3. The Bcl-2/Bax ratio in
PC-3 cells was also determined. The compound 5b drastically
reduced the ratio from 5 to 0.5 (Figure 6b), indicating significant
apoptotic behavior of the compound 5b.
In conclusion, the semisynthetic derivative 5b displayed
significant antiproliferative activity in human prostate PC-3 cells.
Mechanistic study revealed that, derivative 5b also possesses
potent apoptotic and anti-angiogenesis potential in PC-3 cells. It
50 induces apoptosis which is confirmed by apoptotic bodies’
formation, increase of sub-G1 population and induction of
various pro-apoptotic proteins. Compound 5b caused disruption
of mitochondrial membrane potential, rendered Bcl-2 inhibition,
Bax translocation, decrease Bcl-2/Bax ratio, and released pro-
55 apoptotic factors from the mitochondria. Mitochondria and Bcl-2
family of proteins play a pivotal role in the induction of
apoptosis. The cell death regulated by Bcl-2 associated proteins
have both pro-apoptotic and anti-apoptotic effects in cancer cells.
These proteins regulate mitochondrial outer membrane potential
60 and control the release of many apoptotic factors originating in
the mitochondria. These events are accompanied by activation of
pro-caspases-3, 8, -9, which cleave PARP-1 and finally induce
apoptosis. Compound 5b also inhibit pro-caspase-8 level,
therefore it induces apoptosis through both intrinsic and extrinsic
65 apoptotic pathways. These results provide the basis for further in-
depth drug targeted studies, while the pro-apoptotic feature of
0
5
10
20
5b, µM
4
3
2
1
0
Pro-caspase-3
Pro-caspase-8
*
5
Pro-caspase-9
Parp-1
*
*
Bax
Bcl-2
0
10
15
20
5b, µM
β-Actin
(a).
(b).
5
Figure 6. Influence of compound 5b on the expression of important
proteins involved in the initiation of apoptosis. PC-3 cells were treated
with 5-20 µM concentrations of compound 5b for 24h. Protein lysates
were prepared and electrophoresis as described in Materials and Methods.
10 β-actin was used as an internal control to represent the same amount of
proteins applied for SDS-PAGE. Specific antibodies were used for
detection of the indicated proteins in designated cell lysates. (a)
Compound 5b induced differential activation of different caspases,
mitochondrial apoptotic proteins PARP-1 in PC-3 cells. Western blot
15 analyses of the indicated proteins were performed in the whole cell lysate.
Data are representative of one of three similar experiments. (b) Influence
compound 5b raises its potential as
a future anticancer
therapeutics.
Experimental Section
of compound 5b on the Bcl-2/Bax ratio in PC-3 cells. Mitochondria and 70 General. All chemicals were obtained from Sigma-Aldrich
1
Bcl-2 family of proteins play a pivotal role in the induction of apoptosis.21
Bcl-2 associated proteins have both pro-apoptotic and anti-apoptotic
20 effects in cancer cells. These proteins regulate mitochondrial outer
membrane potential and control the release of many apoptotic factors
originating in the mitochondria. Compound 5-b decreases the expression
of anti-apoptotic protein Bcl-2 and increase the expression of pro-
apoptotic protein Bax in a dose dependent manner. The relative density
25 of each band was measured as arbitrary units by Quantity One software of
Bio-RAD gel documentation system. Data expressed as mean SD of
three independent experiments.
Company and used as received. H, 13C and DEPT NMR spectra
were recorded on Bruker-Avance DPX FT-NMR 500 and 400
MHz instruments. Chemical data for protons are reported in parts
per million (ppm) downfield from tetramethylsilane and are
75 referenced to the residual proton in the NMR solvent (CDCl3,
7.26 ppm). Carbon nuclear magnetic resonance spectra (13C
NMR) were recorded at 125 MHz or 100 MHz: chemical data for
carbons are reported in parts per million (ppm, δ scale) downfield
from tetramethylsilane and are referenced to the carbon resonance
80 of the solvents (CDCl3: 77.36 ppm). ESI-MS and HRMS spectra
were recorded on Agilent 1100 LC-Q-TOF and HRMS-6540-
UHD machines. IR spectra were recorded on Perkin-Elmer IR
spectrophotometer. Melting points were recorded on digital
melting point apparatus. HPLC analysis was done on Waters and
85 Shimadzu HPLC systems (detailed method provided in ESI).
Angiogenesis is one of the common hallmark manifestations of
30 all cancers and it is an elementary event in the development of
tumor growth and malignancy. To appraise the anti-angiogenic
property of compound 5b in vitro, the chemotactic motility of
PC-3 cells was examined by wound-healing migration assay. Cell
migration is necessary for tumor growth and metastasis. It was
35 observed that compound 5b significantly inhibited VEGF-
induced HUVEC migration and decrease wound closure
percentage from 100% to 12% at 20 µM concentrations (Figure
7a-b). The effect of compound 5b was also investigated in
angiogenesis dependent cell migration in HUVEC cells. Results
40 are shown in Figure 7c-d, which indicated that compound 5b
inhibited angiogenesis-dependent cell migration in HUVEC cells
Plant material. The dried Glycyrrhiza glabra roots were
purchased from the local market of Jammu and it was
authenticated by B. K. Kapahi, Biodiversity and Applied Botany
Division of Indian Institute of Integrative Medicine (CSIR),
90 Jammu. A specimen sample (accession number: 13485) was
preserved in Janaki Ammal Herbarium at the CSIR-IIIM, Jammu,
India.
Isolation of glycyrrhizin. G. glabra roots were extracted with
water: ethanol (8: 2) under warm condition (55-60 °C). The crude
in a dose-dependent manner. The significant % wound closure 95 extract was filtered and concentrated, which was washed with
was observed at 10 and 20 µM of 5b treatment.
cold dichloromethane and cold acetone to remove other low
molecular weight phenolics. By repeating this procedure
glyccyrrhizin-enriched fraction (80% glyccyrrhizin) was
obtained. This material was directly used for further hydrolysis.22
45 Conclusion
100 Hydrolysis of glycyrrhizin. The glycyrrhizin was hydrolyzed by
5% aq. HCl to get glycyrrhetic acid, which was purified over
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DOI: 10.1039/C4MD00344F
(a)
(b)
Control,
5b, 5 µM
5b, 10 µM
5b, 20 µM
(c)
(d)
Figure 7. (a-b). Effect of compound 5b on VEGF arbitrated in vitro angiogenesis in PC-3 cells. Compound 5b inhibits PC-3 migration in the wound
healing assay. PC-3 cells were scratched by a sterile micro tip and the areas were quantified in three random fields in the terms of wound closure
percentage. (c-d). Effect of compound 5b on angiogenesis-dependent cell migration in HUVEC cells. Data were mean S.D. of three independent
experiments.
silica column in EtOAc: hexane – 25: 75. Pure glycyrrhetic acid
was characterized by comparison of m.p. and spectral data with
literature values.6 The glycyrrhetic acid was then used for
modifications at C-3 position for the synthesis of O-
alkylated/benzylated glycyrrhetic acid derivatives.
layer was decanted off and the remaining solid residue was
washed 5-6 times with chloroform. Combined chloroform layer
was evaporated under reduced pressure and the residue obtained
was purified by silica gel (#100-200) column chromatography
20 using hexane-ethyl acetate as a eluent to yield the different
alkylated products 5a-r.
5
General method for preparation of O-alkylated/benzylated
glycyrrhetic acid ( 3β-hydroxy-11-oxoolean-12-en-30-olic
acid). To the solution of 3β-hydroxy-11-oxoolean-12-en-30-olic
acid (2, 100 mg, 1 mmol) in dry acetone (5 mL) was added
3-(2-Chloro-6-fluoro-benzyloxy)-11-oxo-olean-12-ene-29-oic
acid (5a): white solid; HPLC: tR = 11.9 min (100% purity); yield:
92%; m.p. 236-238 οC; IR (CHCl3): νmax 3844, 3900, 3400, 2852,
10 K2CO3 (1.2 mmol) followed by addition of different alkyl and 25 2923, 1731, 1657, 1609, 1582, 1385, 1259, 1311, 1259, 1209,
1
benzyl halides (1 mmol). The mixture was stirred at room
temperature for 8 h under inert atmosphere and concentrated
under reduced pressure. The reaction mixture was diluted with
chloroform. Water was added to the resultant mixture leading to
1153, 1083, 1039 cm-1; H NMR (400 MHz, CDCl3):
δ (ppm)
7.39-7.29 (m, 3H, Ar-4´,5´,6´), 5.47 (s, 1H, CH-12), 5.32-5.47
(m, 2H, CH2-1´), 3.20 (brs,1H, CH-3), 2.74 (tt, 1H, CH-18), 2.28
(s, 1H, CH-8), 2.05 (m, 2H, CH-2), 2.01 (m, 2H, CH-1), 1.32 (s,
15 formation of a white precipitate in the aqueous layer. The organic 30 3H, Me-27), 1.17(s, 3H, Me-25), 1.13 (s, 3H, Me-29), 1.11 (s,
6
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3H, Me-26), 0.99 (s, 3H, Me-23), 0.80 ( s, 3H, Me-24), 0.80 (s,
3H, Me-28 ), 0.69 (d, 1H, CH-5); 13C NMR (100 MHz, CDCl3):
1´), 3.16 (d, J = 4.0, 12.0 Hz, 1H, CH-3), 2.73 (tt, 1H, CH-18),
δ
2.25 (s, 1H, CH-9), 1.18 (s, 3H, Me-27), 1.13 (s, 3H, Me-25),
(ppm) 200.9 (C-11), 176.0 (C-30), 169.0 (C-13), 136.8 (C-3´, 7´), 60 1.06 (s, 3H, Me-29), 1.04 (s, 3H, Me-26), 0.93 (s, 3H, Me-23),
131.4 (C-2´), 130.6 (C-5´), 128.5 (C-4´, 6´, 12), 78.7 (C-3), 61.7
(C-9 ), 61.7 (C-9), 54.8 (C-5), 48.2 (C-18), 45.3 (C-14), 44.3 (C-
20), 43.1 (C-8), 40.9 (C-19), 39.1 (C-1, C-4), 37.6 (C-22), 37.0
(C-10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 28.4 (C-29), 28.4
0.73 (s, 3H, Me-24), 0.71 (s, 3H, Me-28), 0.63 (d, 1H, CH-5); 13
C
5
NMR (100 MHz, CDCl3): δ 200.1 (C-11), 175.8 (C-30), 168.4
(C-13), 139.6 (C-2´), 138.5 (C-4´), 130.3 (C-5´), 129.7 (C-7´),
128.6 (C-12), 120.5 (C-6´), 78.8 (C-3), 70.1 (C-1´), 61.8 (C-9),
(C-28), 28.3 (C-23), 27.2( C-2), 26.4 (C-15), 26.3 (C-16), 23.2 65 54.9 (C-5), 48.2 (C-18), 45.3 (C-14), 44.1 (C-20), 43.1 (C-8),
(C-27), 18.6 (C-26), 17.4 (C-6), 16.3 (C-25), 15.5 (C-24); HR-
+
41.1 (C-19), 39.1 (C-1, C-4), 37.7 (C-22), 37.1 (C-10), 32.8 (C-
7), 31.8 (C-17), 31.2 (C-21), 28.4 (C-29), 28.1 (C-23, C-28), 27.5
(C-2), 26.4 (C-15), 26.2 (C-16) , 23.3 (C-23), 18.7 (C-26), 17.5
(C-6), 16.3 (C-25), 15.5 (C-24); HR-ESIMS 687.2917 [M+H]+
70 calcd for C37H51IO4 + H+ (687.2904).
10 ESIMS: m/z 613.3459 [M+H]+ calcd for C37H50ClFO4 + H
(613.3454).
3-(2,6-Dichloro-benzyloxy)- 11-oxo-olean-12-ene-29-oic acid
(5b): white solid; HPLC: tR = 26.7 min (97% purity); yield: 95%;
m.p. 226-228 οC; IR (CHCl3): νmax 3441, 2926, 2865, 1730, 1655,
15 1565, 1582, 1461, 1438, 1280, 1255, 1209, 1151, 1039, 1082 cm-
3-(3-Nitro-benzyloxy)- 11-oxo-olean-12-ene-29-oic acid (5e):
Cream solid; HPLC: tR = 49.6 min (90% purity); yield: 95%; m.p.
186-188 °C; IR (CHCl3): νmax 3448, 3053, 2928, 2868, 2304,
1; 1H NMR (400 MHz, CDCl3):
δ (ppm) 7.39 (d, 1H, Ar-4´), 7.37
(d, 1H, Ar-6´), 7.27 (m, 1H, Ar-5´), 5.42 (s, 1H, CH-12), 5.32-
5.47 (m, 2H, CH2-1´), 3.22 (dd, J = 4.0, 12.0 Hz, 1H, CH-3), 2.77 75 1210, 1149, 1039 cm-1; H NMR (400 MHz, CDCl3):
1730, 1656, 1619, 1585, 1462, 1386, 1327, 1313, 12679, 1262,
1
δ 8.20 (d,
(tt, 1H, CH-18), 2.01 (m, 2H, CH-2),1.98 (m, 2H, CH-1), 1.32 (s,
20 3H, Me-27), 1.17 (s, 3H, Me-25), 1.13 (s, 3H, Me-29), 1.11 (s,
3H, Me-26), 0.99 (s, 3H, Me-23 ), 0.80 (s, 3H, Me-24), 0.80 (s,
3H, Me-28), 0.69 (d, 1H, CH-5); 13C NMR (100 MHz, CDCl3): δ
1H, Ar-3´, 5´), 7.71 (m, 2H, Ar-7´), 7.59 (m, 1H, Ar-6´), 5.55 (s,
1H, CH-12), 5.24 (m, 2H, CH2-1´), 3.23 (dd, J = 4.0, 8.0 Hz,1H,
CH-3), 2.80 (tt, 1H, CH-18), 2.33 (s, 1H, CH-9), 1.36 (s, 3H,
Me-27), 1.19 (s, 3H, Me-25), 1.13 (s, 3H, Me-29), 1.11 (s, 3H,
(ppm) 200.1 (C-11), 176.1 (C-30), 168.9 (C-13), 136.8(C-3´,7´), 80 Me-26), 1.00 (s, 3H, Me-23), 0.80 (s, 3H, Me-24 ), 0.76 (s, 3H,
131.5 (C-2´), 130.7 (C-5´), 128.5 (C-4´, 6´, C-12), 78.8 (C-3),
25 61.8 C-9), 61.3 (C-1´), 54.2 (C-5), 48.2 (C-18), 45.3 (C-14), 44.3
(C-20), 43.1 (C-8), 40.9 (C-19), 39.1 (C-1, C-4), 37.6 (C-22),
37.0 (C-10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 28.4 (C-29),
Me-28), 0.71 (dd, 1H, CH-5); 13C NMR (100 MHz, CDCl3): δ
(ppm) 200.4 (C-11), 176.1 (C-30), 168.9 (C-13), 148.7 (C-4),
138.6 (C-2´), 134.2 (C-7´), 130.1 (C-6´), 128.9 (C-12), 123.6 (C-
3´), 123.1 (C-5´), 79.1 (C-3), 65.1 (C-1´), 62.1 (C-9), 55.2 (C-5),
28.3 (C-28), 28.0 (C-23), 27.3 (C-2), 26.5 (C-15), 26.4 (C-16), 85 48.6 (C-18), 45.7 (C-14), 44.4 (C-20), 43.5 (C-8), 41.4 (C-19),
23.3 (C-27), 18.6 (C-26), 17.4 (C-6), 16.3 (C-25), 15.5(C-24).
30 HR-ESIMS: m/z 629.3161 [M+H]+ calcd for C37H50Cl2O4 + H+
(629.3158).
39.9 (C-1,C-4), 38.0 (C-22), 37.4 (C-10), 33.0 (C-7), 31.4 (C-
17), 31.4 (C-21), 28.8 (C-29), 28.6 (C-28), 28.4 (C-23), 27.6 (C-
2), 26.9 (C-15), 26.7 (C-16), 23.7 (C-27), 18.9 (C-26), 17.5 (C-
6), 16.7 (C-25), 15.9 (C-24); HR-ESIMS: m/z 606.3786 [M+H]+
90 calcd for C37H51N O6 + H+ (606.3789).
3-(4-Fluoro-benzyloxy)- 11-oxo-olean-12-ene-29-oic acid (5c):
white solid; HPLC: tR = 8.4 min (96% purity); yield: 90%; m.p.
238-240 °C; IR (CHCl3): νmax 3790, 3663, 3436, 2922, 2853,
3-(2-Nitro-benzyloxy)-11-oxo-olean-12-ene-29-oic acid (5f):
white solid; HPLC: tR = 7.8 min (100% purity); yield: 94%;
m.p.186-188 °C; IR (CHCl3): νmax 3448, 3053, 2928, 2868, 2304,
35 1730, 1650, 1579, 1617, 1513, 1345, 1386, 1084, 1215, 1260 cm-
1
;
1H NMR (400 MHz, CDCl3):
δ (ppm) 7.36 (m, 2H, Ar-3´,7´),
7.08 (m, 2H, Ar-4´,6´), 5.56 (s, 1H, CH-12), 5.10-5.12 (dd, J = 12
Hz, 2H, CH2-1´), 3.23 (dd, J = 4.0, 8.0 Hz,1H, CH-3), 2.81 (tt, 95 1210, 1141, 1039 cm-1; H NMR (400 MHz, CDCl3):
1730, 1656, 1619, 1585, 1462, 1386, 1327, 1313, 12679, 1262,
1
δ (ppm)
1H, CH-18), 2.32 (s, 1H, CH-9), 1.35 (s, 3H, Me-27), 1.14 (s, 3H,
40 Me-26), 1.14 (s, 3H, Me-29), 1.11 (s, 3H, Me-26), 1.00 (s, 3H,
Me-23), 0.81 (s, 3H, Me-24), 0.73 (s, 3H, Me-28), 0.71 (s, 1H,
CH-5); 13C NMR (100 MHz, CDCl3): δ (ppm) 200.1 (C-11),
8.05 (d, 1H, Ar-3´), 7.63 (m, 1H, Ar-5´), 7.61 (m, 1H, Ar-6´),
7.45 (m, 1H, Ar-4´), 5.51 (s, 1H, CH-12), 5.43 (m, 2H, CH2-1´),
3.16 (dd, J = 8.0, 12.0 Hz, 1H, CH-3), 2.70 (tt, 1H, CH-18), 2.26
(s, 1H, CH-9), 1.91 (m, 2H, CH-2), 1.20 (s, 3H, Me-27), 1.18 (s,
176.1 (C-30), 169.0 (C-13), 163.9 (d, 1JCF = 246 Hz, C-5´), 132.0 100 3H, Me-25), 1.14 (s, 3H, Me-29), 1.14 (s, 3H, Me-26), 1.04 (s,
(C-2´), 130.3 (d, 3JCF = 9 Hz, C-3´, C-7´), 128.5 (C-12), 115.6 (d,
2
45 JCF = 21 Hz, C-4´, C-6´), 78.5 (C-3), 65.4 (C-1´), 61.8 (C-9),
3H, Me-23), 0.93 (s, 3H, Me-24), 0.93 (s, 3H, Me-28), 0.73 (d,
1H, CH-5); 13C NMR (100 MHz, CDCl3): δ (ppm) 200.7 (C-11),
175.7 (C-30), 168.9 (C-13), 148.7 (C-3´), 133.8 (C-6´), 132.0
(C-2´), 129.3 (C-7´), 129.0 (C-5´), 128.6 (C-4´), 125.1 (C-12),
54.9 (C-5), 48.2 (C-18), 45.3 (C-14), 43.9 (C-20) 43.1 (C-8), 41.0
(C-19), 39.1 (C-1, C-4), 37.6 (C-22), 37.0 (C-10), 32.7 (C-7),
31.7 (C-17), 31.1 (C-21), 28.4 (C-29), 28.2 (C-28), 28.1 (C-23), 105 78.7 (C-3), 63.1 (C-1´), 61.8 (C-9 ), 54.9 (C-5), 48.3 (C-18), 45.3
27.3 (C-2), 26.4 (C-15), 26.3 (C-16), 23.3 (C-23), 18.6 (C-26),
50 17.4 (C-6), 16.3 (C-25), 15.5 (C-24); HR-ESIMS: m/z 579.3846
[M+H]+ calcd for C37H51FO4 + H+ (579.3844).
(C-14), 44.2 (C-20), 43.2 (C-8), 41.0 (C-19), 39.1 (C-1,C-4), 37.6
(C-22), 37.0 (C-10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 28.4
(C-29), 28.4 (C-28), 28.1 (C-23), 27.3 (C-2), 26.4 (C-15), 26.3
(C-16), 23.4 (C-27), 18.6 (C-26), 17.4 (C-6), 16.4 (C-25), 15.5
110 (C-24); HR-ESIMS: m/z 606.3802 [M+H]+ calcd for C37H51NO6
+ H+ (606.3789).
3-(2-Iodo-benzyloxy)- 11-oxo-olean-12-ene-29-oic acid (5d):
white solid; HPLC: tR = 48.4 min (98% purity) yield: 88%; m.p.
228-230 °C; IR (CHCl3): νmax 3435, 2923, 2852, 1729, 1656,
55 1463, 1384, 1311, 1259, 1210, 1150, 1045, 1084 cm-1; 1H NMR
3-(3-Methyl-but-2-enyloxy)-11-oxo-olean-12-ene-29-oic acid
(5g): cream colored oil; HPLC: tR = 48.4 min (98% purity);
yield: 90%; IR (CHCl3): νmax 3441, 2927, 2867, 1724, 1658,
(400 MHz, CDCl3):
δ 7.81 (d, 1H, Ar-4´), 7.33 (m, 2H, Ar-5´,
7´), 6.96 (m, 1H, Ar-6´), 5.51 (s, 1H, CH-12), 5.10 (s, 2H, CH2-
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1658, 1619, 1454, 1385, 1327, 1310, 1257, 1210, 1152, 1084,
1046 cm-1; 1H NMR (400 MHz, CDCl3):
(ppm) 5.63 (s, 1H,
CH-12), 5.34 (t, 1H, CH-2´), 4.62 (m, 2H, CH2-1´), 3.23 ( dd,1H, 60 1H, CH-12), 5.27 (t, 1H, CH-2,), 5.01 (t, 1H, CH-6,), 4.54 (m,
yield: 92%; IR (CHCl3): νmax 3442, 2925, 2854, 1725, 1660,
δ
1620, 1454, 1385 cm-1; 1H NMR (400 MHz, CDCl3):
δ 5.57 (s,
CH-2), 2.81 (tt, 1H, CH-18), 2.34 (s, 1H, CH-9), 1.77 (s, 3H, CH-
3´), 1.72 (s, 3H, CH-4´), 1.36 (s, 3H, Me-27), 1.26 (s, 3H, Me-
25), 1.14 (s, 3H, Me-29), 1.13 (s, 3H, Me-26), 1.01(s, 3H, Me-
23), 0.81 (s, 3H, Me-24), 0.80 (s, 3H, Me-28) , 0.71 (d, 1H, CH-
2H, CH-1,), 3.16 (dd, J = 8.0, 12.0 Hz, 1H, CH-3), 2.74 (tt, 1H,
CH-18), 2.26 (s, 1H, CH-9), 1.65 (s, 3H, Me-8′), 1.60 (s, 3H, Me-
9′), 1.52 (s, 3H, Me-10′), 1.27 (s, 3H, Me-27), 1.18 (s, 3H, Me-
25), 1.07 (s, 3H, Me-29), 1.06 (s, 3H, Me-26), 0.93 (s, 3H, Me-
5
5), 13C NMR (100 MHz, CDCl3): δ (ppm) 200.2 (C-11), 176.4 65 23), 0.73 (s, 1H, Me-24), 0.73 (s, 3H, Me-28), 0.71 (d, 1H, CH-
(C-30), 169.3 (C-13), 128.4 (C-3′), 118.6 (C-2′), 78.7 (C-3′),
5);13C NMR (100 MHz, CDCl3): δ (ppm) 200.2 (C-11), 179.4 (C-
30), 169.3 (C-13),142.4 (C-7′), 131.7 (C-3′), 128.5 (C-12), 123.7
(C-6′), 118.4 (C-2′), 78.7 (C-3), 61.8 (C-9), 61.2 (C-1′), 54.9 (C-
5), 48.3 (C-18), 45.3 (C-14), 43.9 (C-20), 43.1 ( C-8), 41.1 (C-
10 61.8 (C-1′), 61.2 (C-9), 54.9 (C-5), 48.3 (C-18 ), 45.3 (C-14),
44.2 (C-20),43.9 (C-2) , 43.2 (C-8), 41.1 (C-19), 39.1 (C-1, C-4),
37.7 (C-22), 37.0 (C-10), 32.7 (C-7), 31.8 (C-17), 29.7 (C-21),
28.5 (C-29), 28.3 (C-28), 28.1 (C-23), 27.3 (C-2), 26.5 (C-15), 70 19), 39.5 (C-4′), 39.1 (C-1, C-4), 37.7 (C-22), 37.0 (C-10), 32.7
26.4 (C-16), 25.7 (C-5´), 23.3 (C-27), 18.1 (C-26), 17.5 (C-6),
15 16.6 (C-4´), 16.4 (C-25), 15.5 (C-24); HR-ESIMS: m/z 539.4106
[M+H]+ calcd for C35H54O4 + H+ (539.4094).
(C-7), 31.8 (C-17), 31.2 (C-21), 29.7 (C-5′)28.5 (C-29), 28.3 (C-
28), 28.1 (C-27), 27.3 (C-2), 26.4 (C-9′), 26.4 (C-15), 26.3 (C-
16), 25.7 (C- 10′), 23.3 (C-23), 18.6 (C-26), 17.4 (C-6), 16.5 (C-
8′), 16.3 (C-25), 15.6 (C-24); HR-ESIMS: m/z 607.4734 [M+H]+
75 calcd for C40H62O4H+ (607.4720).
3-(2,4-Bis-trifluoromethyl-benzyloxy)- 11-oxo-olean-12-ene-
29-oic acid (5h): white solid; HPLC: tR = 42.4 min (98% purity);
yield: 90%; m.p. 254-256 °C; IR (CHCl3): νmax 3448, 3053,
20 2928, 2868, 2304, 1730, 1656, 1619, 1585, 1462, 1386, 1327,
3-(3-Bromo-4-fluoro-benzyloxy)- 11-oxo-olean-12-ene-29-oic
acid (5k): white solid; HPLC: tR = 10.1 min (100% purity); yield:
97%; m.p. 298-299 °C; IR (CHCl3): νmax 3400, 2923, 2852, 1729,
1
1313, 12679, 1262, 1210, 1149, 1039 cm-1; H NMR (400 MHz,
1
CDCl3): δ (ppm) 7.95 (d, 1H, Ar-4´), 7.87 (d, 1H, J = 8 Hz, Ar-
1655, 1498, 1463, 1385, 1248, 1209, 1151, 1083, 1047 cm-1; H
6´), 7.72 (s, 1H, J = 8 Hz, Ar-7´), 5.60 (s, 1H, CH-12), 5.36 (m, 80 NMR (400 MHz, CDCl3): δ (ppm) 7.56 (dd, 1H, Ar-3´), 7.29
2H, CH2-1′), 3.23 (dd, J = 8.0, 12.0 Hz 1H, CH-3), 2.80 (tt, 1H,
25 CH-18), 2.33 (s, 1H, CH-9) 1.35 (s, 3H, Me-27), 1.21 (s, 3H, Me-
25), 1.13 (s, 3H, Me-29), 1.12 (s, 3H, Me-26), 1.00 (s, 3H, Me-
23), 0.81 (s, 1H, Me-24), 0.80 (s, 3H, Me-28), 0.71 (d, 1H, CH-
(m, 1H, Ar-7´), 7.13 (t, 1H, Ar-6´), 5.58 (s, 1H, CH-12), 5.08 (m,
2H, CH2-1´), 3.23 (dd, J = 8.0, 12.0 Hz, 1H, CH-3), 2.79 (tt, 1H,
CH-18), 2.33 (s, 1H, CH-9), 1.92 (dd, 2H, CH-2), 1.82 (dd, 2H,
CH-1),1.35 (s, 3H, Me-27), 1.15 (s, 3H, Me-25), 1.14 (s, 3H, Me-
5); 13C NMR (100 MHz, CDCl3): δ (ppm) 200.1 (C-11), 175.7 85 29), 1.12 (s, 3H, Me-26), 1.00 (s, 3H, Me-23), 0.81 (s, 1H, Me-
(C-30), 168.7 (C-13), 138.5 (C-2´), 132.3 (C-5´), 130.1 (C-3´),
30 129.3 (C-7´, 129.0 (C-4´), 128.6 (C-12), 123.8 (CF3), 123.4 (CF3)
78.5 (C-3), 61.9 (C-1´), 61.8 (C-9), 54.9 (C-5), 48.2 (C-18), 45.3
(C-14), 43.9 (C-20), 41.0 (C-19 ), 39.1 (C-1, C-4), 37.7 (C-22),
24), 0.75 (s, 3H, Me-28), 0.68 (d, 1H, CH-5); 13C NMR (100
MHz, CDCl3): δ (ppm) 200.1 (C-11), 176.0 (C-30), 168.9 (C-13),
159.8 (d, 1JCF = 198 Hz, C-5´), 133.7 (d, 3JCF = 3 Hz, C-3´), 133.5
2
(C-11), 129.0 (d, JCF = 6 Hz, C-6´),128.5 (C-7´), 116.7 (C-12),
37.0 (C-10), 32.7 (C-7 ), 31.8 (C-17), 31.0 (C-21), 28.4 (C-29), 90 109.2 (d, 2JCF = 17 Hz, C-4´), 78.7 (C-3), 64.6 (C-1´), 61.8 (C-9),
28.3 (C-28 ), 28.1 (C-23), 27.3 (C-2 ), 26.4 (C-15 ), 26.3 (C-16 ),
35 23.4 (C-27), 18.6 (C-26), 17.4 (C-6), 16.3 (C-25), 15.5 (C-24);
HR-ESIMS: m/z 697.3702 [M+H]+ calcd for C39H50F6,O4 + H+
(697.3686).
54.9 (C-5), 48.3 (C-18), 45.3 (C-14), 44.0 (C-20), 43.1 (C-8),
41.1 (C-19), 39.1 (C-4), 39.1 (C-1), 37.6 (C-22), 37.0 (C-4 ), 32.7
(C-7), 31.7 (C-10), 31.1 (C-21), 29.7 (C-5), 28.4 (C-29), 28.2 (C-
28), 28.1 (C-23), 27.2 (C-2), 26.4 (C-15), 26.3 (C-16), 23.3 (C-
95 27), 18.6 (C-26), 17.4 (C-6), 16.4 (C-25), 15.6 (C-24); HR-
3-Methoxy-11-oxo-olean-12-ene-29-oic acid (5i): white solid; ;
HPLC: tR = 49.6 min (95%); yield: 85%; m.p. 262-264 °C; IR
40 (CHCl3): νmax 3340, 2931, 2869, 1722, 1657, 1618, 1465, 1386,
ESIMS: m/z 657.2944 (M+H+) calcd for C37H 50BrF,O4
+
H+(657.2949).
1
1358, 1323, 1246, 1189, 1086, 1040 cm-1; H NMR (400 MHz,
3-(4-Chloro-benzyloxy)- 11-oxo-olean-12-ene-29-oic acid (5l):
CDCl3): δ (ppm) 5.59 (s, 1H, CH-12), 3.62 (s, 3H, Me-1′), 3.16
cream colored sticky solid; HPLC: tR = 12.1 min (100% purity)
(dd, J = 8.0, 12.0 Hz, 1H, CH-3), 2.74 (tt, 1H, CH-18), 2.27 (s, 100 yield: 95%; m.p. 260-261 °C, IR (CHCl3): νmax 3400, 2923, 2852,
1H, CH-9), 1.55 (m, 2H, CH-2), 1.18 (s, 3H, Me-27), 1.08 (s, 3H,
45 Me-25), 1.08 (s, 3H, Me-29), 1.07 (s, 3H, Me-26), 1.06 (s, 3H,
Me-23), 0.94 (s, 1H, Me-24), 0.93 (s, 3H, Me-28), 0.74 (d, 1H,
CH-5); 13C NMR (100 MHz, CDCl3): δ (ppm) 200.3 (C-11),
1729, 1656, 1463, 1463, 1385, 1256, 1210, 1151, 1084, 1018 cm-
1; H NMR (400 MHz, CDCl3): δ (ppm) 7.36-7.27 (m, 4H, Ar-
3´,4´,6´,7´), 5.59 (s, 1H, CH-12), 5.10 (m, 2H, CH2-1´), 3.23 (dd,
1
J = 4.0, 8.0 Hz, 1H, CH-3), 2.78 (tt, 1H, CH-18), 2.33 (s, 1H,
177.0 (C-30), 169.3 (C-13), 128.5 (C-12), 78.8 (C-3), 61.8 (C- 105 CH-9), 1.98 (dd, 2H, CH-2), 1.35 (s, 3H, Me-27), 1.15 (s, 3H,
9), 54.9 (C-5), 51.8 (C-1´), 48.4 (C-18), 45.4 (C-14), 44.0 (C-20),
50 43.2 (C-8), 41.0 (C-19), 39.1 (C-1, C-4), 37.7 (C-22), 37.0 (C-
10), 32.7 (C-7), 31.8 (C-17), 31.1 (C-21), 28.5 (C-29), 28.3 (C-
28), 28.1 (C-23), 27.3 (C-2), 26.4 (C-15), 26.4 (C-16), 23.4 (C-
Me-25), 1.14 (s, 3H, Me-29), 1.12 (s, 3H, Me-26), 1.00 (s, 3H,
Me-23), 0.98 (s, 1H, Me-24), 0.81 (s, 3H, Me-28), 0.71 (d, 1H,
CH-5); 13C NMR (100 MHz, CDCl3): δ (ppm) 200.2 (C-11),
176.1 (C-30), 169.0 (C-13), 134.6 (C-5´), 134.5 (C-2´), 129.6 (C-
27), 18.7 (C-26), 17.4 (C-6), 16.4 (C-25), 15.5 (C-24); HR- 110 3´,7´), 128.8 (C-4´, C-6´), 128.4 (C-12), 78.7 (C-3), 65.4 (C-1´),
ESIMS: m/z 485.3616 [M+H]+ calcd for C31H48O4
+
H+
61.8 (C-9), 54.9 (C-5), 48.2 (C-18), 45.3 (C-14), 44.0 (C-20),
43.1 (C-8), 41.0 (C-19), 39.1 (C-1, C-17), 37.6 (C-4), 37.7 (C-
22), 32.7 (C-7), 31.7 (C-10), 31.1 (C-21), 28.4 (C-29), 28.2 (C-
28), 28.1 (C-23), 27.3 (C-2), 26.4 (C-15), 26.3 (C-16), 23.3 (C-
55 (485.3625).
3-(3,7-Dimethyl-octa-2,6-dienyloxy)-
11-oxo-olean-12-ene-
29-oic acid (5j): yellow oil; HPLC: tR = 8.0 min (100% purity);
8
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DOI: 10.1039/C4MD00344F
27), 18.6 (C-26), 17.4 (C-6), 16.3 (C-25), 15.5 (C-24); HR-
ESIMS: m/z 595.3547 [M+H]+ calcd for C37H51ClO4 + H+
(595.3548).
43.1 (C-8), 41.0 (C-19), 39.1 (C-1, C-4), 37.6 (C-22), 37.0 (C-
10), 32.7 (C-7), 31.7 (C-17), 31.1 (C-21), 28.4 (C-29), 28.2 (C-
60 28), 28.1 (C-23), 27.2 (C-2), 26.4 (C-15), 26.4 (C-16), 23.3 (C-
27), 18.6 (C-26), 17.4 (C-6), 16.4 (C-25), 15.6 (C-24); HR-
ESIMS: m/z 605.3835 [M+H]+ for calcd for C38H51O6 + H+
(605.3836).
3-(4-Bromo-benzyloxy)- 11-oxo-olean-12-ene-29-oic
acid
5
(5m): colorless oil; HPLC: tR = 12.7 min (95% purity); yield:
95%; IR (CHCl3): νmax 3411, 2925, 2855, 1729, 1657, 1488,
1
1455, 1385, 1279, 1256, 1210, 1150, 1083, 1071, 1014 cm-1; H
3-(3-Bromo-4-methoxy-benzyloxy)-11-oxo-olean-12-ene-29-
NMR (400 MHz, CDCl3): δ (ppm) 7.51-7.49 (m, 2H, Ar-3´,4´), 65 oic acid (5p): white solid; HPLC: tR = 10.6 min (100% purity);
7.22-7.24 (m, 2H, Ar-6´,7´), 5.59 (s, 1H, CH-12), 5.09 (m, 2H,
yield: 90%; m.p. 300-301 °C; IR (CHCl3): νmax 3400, 2924,
2853, 1726, 1656, 1500, 1462, 1385, 1280, 1209, 1151, 1053,
1021 cm-1; 1H NMR (400 MHz, CDCl3): δ 7.55 (m, 1H, Ar-3´),
7.30 (m, 1H, CH-7´), 6.90 (m, 1H, CH-5´), 5.54 (s, 1H, CH-12),
10 CH2-1´), 3.23 (dd, J = 8.0, 12.0 Hz, 1H, CH-3), 2.78 (tt, 1H, CH-
18), 2.33 (s, 1H, CH-9), 2.01 (dd, 2H, CH-2),1.65 (dd, 2H, CH-
1), 1.35 (s, 3H, Me-27), 1.15 (s, 3H, Me-25), 1.14 (s, 3H, Me-29),
1.12 (s, 3H, Me-26), 1.00 (s, 3H, Me-23), 0.98 (s, 1H, Me-24), 70 5.09 (dd, 1H,CH2-1´), 3.91 (s, 3H, OMe), 3.23 (dd, J = 4.0 Hz,
0.81 (s, 3H, Me-28), 0.75 (d, 1H, CH-5); 13C NMR (100 MHz,
15 CDCl3): δ (ppm) 200.2 (C-11), 176.1 (C-30), 168.9 (C-13), 135.1
(C-2´), 131.7 (C-4´, C-6´), 129.9(C-3´, C-7´), 128.5 (C-12), 78.7
(C-3), 65.4 (C-1´), 61.8 (C-9), 54.9 (C-5), 48.2 (C-18), 45.3 (C-
8.0 Hz 1H, CH-3), 2.78 (tt, 1H, CH-18), 2.32 (s, 1H, CH-9), 1.34
(s, 3H, Me-27), 1.14 (s, 3H, Me-25), 1.13 (s, 3H, Me-29), 1.11 (s,
3H, Me-26), 1.00 (s, 3H, Me-23), 0.80 (s, 1H, Me-24), 0.74 (s,
3H, Me-28), 0.68 (d, 1H, CH-5); 13C NMR (100 MHz, CDCl3): δ
14), 44.0 (C-20), 43.2 (C-8), 41.0 (C-19), 39.1 (C-1, C-4), 37.8 75 (ppm) 200.2 (C-11), 176.3 (C-30), 169.1 (C-13), 156.0 (C-5´),
(C-22), 37.1 (C-10), 32.7 (C-7), 31.7 (C-17), 31.1 (C-21), 28.4
20 (C-29, C-28), 28.1 (C-23), 27.3 (C-2), 26.4 (C-15), 26.3 (C-16),
23.4 (C-27), 18.6 (C-26), 17.4 (C-6), 16.4 (C-25), 15.5 (C-24);
HR-ESIMS: m/z 639.3048 and 641.3035 [M+H]+ calcd for
C37H51BrO4 + H+ (639.3045 and 641.3029).
133.6 (C-3´,2´), 128.9 (C-7´), 128.5 (C-12), 112.0 (C-6´),111.7
(C-4´), 78.9 (C-3), 65.2 (C-1´), 61.9 (C-9), 56.4 (C-5), 55.7 (-
OCH3), 48.3 (C-18), 45.4 (C-14), 44.0 (C-20), 43.3 (C-8), 41.2
(C-19), 39.2 (C-1, C-4), 37.7 (C-22), 37.2 (C-10), 32.8 (C-7),
80 31.9 (C-17), 31.2 (C-21), 28.6 (C-29), 28.3 (C-28), 28.2 (C-23),
27.4( C-2), 26.5 (C-15), 26.5 (C-16), 23.5 (C-27), 18.8 (C-26),
17.6 (C-6), 16.5 (C-25), 15.7 (C-24); HR-ESIMS: m/z 669.3114
and 671.3098 [M+H]+ calcd for C38H53BrO5 + H+ (669.3149 and
671.3128).
3-(3-Chloro-benzyloxy)-11-oxo-olean-12-ene-29-oic acid (5n):
25 cream colored sticky solid; HPLC: tR = 13.7 min (96% purity);
yield: 97%; m.p. 260-262 °C; IR (CHCl3): νmax 3416, 2923, 2852,
1729, 1656, 1463, 1463, 1385, 1256, 1210, 1151, 1084, 1018 cm-
1; 1H NMR (400 MHz, CDCl3): δ (ppm) 7.36-7.28 (m, 4H, Ar- 85 3-(3,4-Dimethoxy-benzyloxy)-11-oxo-olean-12-ene-29-oic
4´,5´,6´,7´), 5.59 (s, 1H, CH-12), 5.10 (m, 2H, CH2-1´), 3.23 (dd,
30 J = 4.0, 8.0 Hz, 1H, CH-3), 2.78 (tt, 1H, CH-18), 2.33 (s, 1H,
CH-9), 2.01 (dd, 2H, CH-2),1.65 (dd, 2H, CH-1), 1.35 (s, 3H,
Me-27), 1.15 (s, 3H, Me-25), 1.14 (s, 3H, Me-29), 1.12 (s, 3H,
acid (5q): colorless oil; HPLC: tR = 7.8 min (100 % purity);
yield: 90%; IR (CHCl3): νmax 3434, 2930, 2867, 1724, 1656,
1
1517, 1463, 1385, 1263, 1210, 1159, 1084, 1024 cm-1; H NMR
(400 MHz, CDCl3): δ 6.95-6.85 (m, 3H, Ar-3´,6´,7´), 5.57 (s,
Me-26), 0.98 (s, 3H, Me-23), 0.81 (s, 1H, Me-24), 0.75 (s, 3H, 90 1H, CH-12), 5.11 (dd, 1H, CH2-1´ ), 3.89 (s, 6H, OMe), 3.22 (dd,
Me-28), 0.71 (d, 1H, CH-5); 13C NMR (100 MHz, CDCl3): δ
35 (ppm) 200.2 (C-11), 176.1 (C-30), 169.0 (C-13), 134.6 (C- 2´),
134.1 (C- 3´), 129.6 (C-7´), 128.8 (4´,5´,6´) ,128.5 (C-12), 78.7
(C-3), 65.4 (C-1´), 61.8 (C-9), 54.9 (C-5), 48.2 (C-18), 45.3 (C-
J = 8.0, 12.0 Hz, 1H, CH-3), 2.77 (tt, 1H, CH-18), 2.32 (s, 1H,
CH-9), 1.35 (s, 3H, Me-27), 1.14 (s, 3H, Me-25), 1.13 (s, 3H,
Me-29), 1.10 (s, 3H, Me-26), 1.00 (s, 3H, Me-23), 0.80 (s, 1H,
Me-24), 0.72 (s, 3H, Me-28), 0.67 (d, 1H, CH-5); 13C NMR: (100
14), 44.0 (C-20), 43.1 (C-8), 41.0 (C-19) , 39.1 (C-1, C-4), 37.6 95 MHz, CDCl3): δ (ppm) 200.1 (C-11), 176.3 (C-30), 169.2 (C-13),
(C-22), 37.0 (C-10), 32.7 (C-7), 31.7 (C-17), 31.1 (C-21), 28.4
40 (C-29), 28.2 (C-28), 28.1 (C-23), 27.3 (C-2), 26.4 (C-15), 26.3
(C-16), 23.3 (C-27), 18.6 (C-26), 17.4 (C-6), 16.3 (C-25), 15.5
149.0 (C-5´), 148.8 (C-4´), 128.7 (C-2´), 128.4 (C-12), 121.3 (C-
7´), 111.6 (C-3´), 111.0 (C-6´), 78.9 (C-3), 66.2 (C-1´), 61.9 (C-
9), 55.9 (C-5), 55.5 (OMe), 54.9 (OMe), 48.2 (CH-18), 45.3 (C-
14), 43.9 (C-20), 43.1 (C-8), 41.0 (C-19), 39.1 (C-1, C-4), 37.6
100 (C-22), 37.0 (C-10), 32.7 (C-7), 31.7 (C-17), 31.1 (C-21), 28.4
(C-29), 28.2 (C-28), 28.1 (C-23), 27.2( C-2), 26.4(C-15), 26.3 (C-
16), 23.3 (C-27), 18.6 (C-26), 17.4 (C-6), 16.3 (C-25), 15.6 (C-
(C-24); HR-ESIMS: m/z 595.3549 [M+H]+
C37H51ClO4 + H+ (595.3548).
calcd for
3-(Benzo[1,3]dioxol-5-ylmethoxy)-11-oxo-olean-12-ene-29-oic
45 acid (5o): colorless oil; HPLC: tR = 12.6 min (96% purity);
yield: 95%; IR (CHCl3): νmax 3391, 2924, 2876, 1723, 1649,
1443, 1490, 1386, 1249, 1211, 1154, 1095, 1018 cm-1; 1H NMR
24); HR-ESIMS: m/z 621.4145 [M+H]+ for calcd for C39H56O6
+
H+ (621.4149).
(400 MHz, CDCl3): δ (ppm) 6.87-6.77 (m, 3H, Ar-3´,4´,7´), 5.97 105 3-(2,4-Dichloro-benzyloxy)-11-oxo-olean-12-ene-29-oic acid
(s, 2H, CH-8´), 5.52 (s, H, CH-12), 5.10 (m, 2H, CH2-1´), 3.23
50 (dd, J = 4.0, 12.0 Hz, 1H, CH-3), 2.80 (tt,1H, CH-18), 2.32 (s,
1H, CH-9),1.35 (s, 3H, Me-27), 1.14 (s, 3H, Me-25), 1.13 (s, 3H,
Me-29), 1.11 (s, 3H, Me-26), 1.00 (s, 3H, Me-23), 0.81 (s, 1H,
(5r): yellow oil; HPLC: tR = 7.3 min (100% purity); yield: 92%;
IR (CHCl3): νmax 3454, 2927, 2866, 1730, 1654, 1590, 1464,
1
1386, 1365, 1326, 1312, 1279, 1102, 1084, 1048 cm-1; H NMR
(400 MHz, CDCl3): δ (ppm) 7.43-7.27 (m, 3H, Ar-4´,6´,7´),
Me-24), 0.80 (s, 3H, Me-28), 0.75 (d, 1H, CH-5); 13C NMR (100 110 5.60 (s, 1H, CH-12), 5.30-5.19 (dd, 2H, CH2-1´), 3.23 (dd, J = 4.0
MHz, CDCl3): δ (ppm) 200.1 (C-11), 176.2 (C-30), 169.1 (C-13),
55 147.8 (C-6′), 147.8 (C-5´), 129.9 (C-2´), 128.5 (C-11), 122.2 (C-
3´), 109.0 (C-7´), 108.3 (C-4´), 101.2 (C-8´), 78.7 (C-3), 66.1 (C-
1´), 61.7 (C-9), 54.9 (C-5), 48.2 (C-18), 45.3 (C-14), 44.9 (C-20),
Hz, 8.0 Hz 1H, CH-3), 2.80 (tt, 1H, CH-18), 2.33 (s, 1H, CH-9),
1.36 (s, 3H, Me-27), 1.15 (s, 3H, Me-25), 1.13 (s, 3H, Me-29),
1.12 (s, 3H, Me-26), 1.00 (s, 3H, Me-23), 0.81 (s, 1H, Me-24),
0.80 (s, 3H, Me-28), 0.77 (d, 1H, CH-5);13C NMR (100 MHz,
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DOI: 10.1039/C4MD00344F
CDCl3): δ (ppm) 200.1 (C-11), 176.0 (C-30), 168.9 (C-13), 134.8 55 temperature. After 30 min slides were washed with distilled water
(C-5´), 134.5 (CH-3´), 132.3 (C-2´) , 130.9 (C-7´), 129.5 (C-4´),
128.6 (C-12), 127.2 (C-6´), 78.7 (C-3), 63.8 (C-1´) ,61.8 (C-9),
54.9 (C-5), 48.2 (C-18), 45.3 (C-14), 44.1 (C-20), 43.2 (C-8),
41.0 (C-19), 39.1 (C-1, C-4), 37.7 (C-22), 37.0 (C-10), 32.7 (C-
followed by in PBS. While wet, 40 µl of mounting fluid (PBS:
glycerol, 1/1) was poured over the slide and covered with a glass
cover slip and sealed with nail polished. Cells were observed
under microscope for any nuclear morphological changes occur
5
7), 31.8 (C-17), 31.1 (C-21), 28.4 (C-29), 28.3 (C-28), 28.1 (C- 60 in apoptosis.21
23), 27.3 (C-2), 26.4 (C-15), 26.4 (C-16), 23.3 (C-27), 18.6 (C-
26), 17.4 (C-6), 16.3 (C-25), 15.5 (C-24); HR-ESIMS: m/z
629.3164 [M+H]+ calcd for C37H50Cl 2O4 + H+ (629.3158).
Flow cytometric determination of mitochondrial membrane
potential: Changes in mitochondrial transmembrane potential
(∆ψm) as a result of mitochondrial perturbation were measured
after staining with Rhodamine-123.21 PC-3 cells were incubated
10 Cell culture and growth conditions: PC-3 prostate cancer,
MIAPaCa-2 pancreatic cancer, and HepG2 hepatocellular 65 with the indicated doses of compound 5b for 24 h. Rhodamine-
carcinoma cells were originated from EACC, UK (European
Collection of Cell Culture) and were purchased through Sigma-
Aldrich India. The cells were grown in RPMI- 1640 or MEM
15 medium supplemented with 10% heat-inactivated fetal bovine
123 (5 µM) was added 1 h before the termination of the
experiment and cells were collected, washed in PBS. The
fluorescence intensity of 10,000 events was analyzed in FL-1
channel on a BD FACS Calibur (Becton Dickinson, USA) flow
70 cytometer. The decrease in fluorescence intensity caused by loss
of mitochondrial membrane potential was analyzed in FL-1
channel.
serum, penicillin (100 units/mL), streptomycin (100 µg/mL),
glutamine (0.3 mg/mL), pyruvic acid (0.11 mg/mL), and 0.37%
NaHCO . Cells were grown in a CO incubator (Thermocon
Electron Corporation, MA, USA) at 37 °C in an atmosphere of
20 95% air and 5% CO with 98% humidity. Camptothecin and
L-
3
2
2
Western blot analysis: Cells were treated with different
concentration of compound 5b for 24 h. Cells were collected at
75 400×g at 4 °C, washed in PBS twice and cell pellets were
incubated with cold RIPA buffer (Sigma Aldrich, India))
containing 50 mM NaF, 0.5 mM NaVO4, 2 mM PMSF and 1%
protease inhibitor cocktail for 40 min. Cells were centrifuged at
12000 x g for10 min at 4 oC and the supernatant was collected as
paclitaxel were used as a positive controls in this study.
Cell proliferation assay: MTT assay was performed to
determine the cell viability. Cells were seeded in 96 well plates
and exposed to different concentrations of synthesize molecules
25 for 48 h. MTT dye (2.5 mg/ml in PBS) was added 4 hours priors
to experiment termination. The plates were then centrifuged at 80 whole cell lysates for western blot analysis of various proteins.
1500 RPM for 15 min and the supernatant was discarded, while
the MTT formazan crystals were dissolved in 150 µL of DMSO.
The OD measured at 570 nm with reference wavelength of 620
Protein was measured employing Bio-Rad protein assay kit using
bovine serum albumin as standard. Proteins aliquots (30-70 µg)
were resolved on SDS-PAGE and then electro transferred to
PVDF membrane overnight at 4 °C at 30V. Nonspecific binding
85 was blocked by incubation with 5% non-fat milk in Tris-buffered
saline containing 0.1% Tween-20 (TBST) for 1 h at room
temperature. The blots were probed with respective primary
antibodies (purchased from Santacruz Biotech) for 2 h and
washed three times with TBST. The blots were then incubated
90 with horseradish peroxidase conjugated mouse or rabbit
secondary antibodies (purchased from Santacruz Biotech.) for 1
h, washed again three times with TBST and signals detected
using ECL plus chemiluminescence’s kit on X-ray film.21
30 nm.21
DNA content and cell cycle phase distribution: PC-3 cells were
treated with 5, 10 and 20 µM concentrations of compound 5b for
24 h. Cells were collected, washed in PBS, fixed at 70% cold
ethanol and placed at −20 °C overnight. Cells were again washed
o
35 with PBS, subjected to RNase digestion (400 µg/ml) at 37 C for
45 min. Finally, cells were incubated with propidium iodide (10
µg/ml) for 30 min and analyzed immediately on a flow cytometer
FACSCalibur (Becton Dickinson, USA). The data were collected
in list mode on 10,000 events and illustrated in a histogram,
40 where the number of cells (counts) is plotted against the relative
Wound healing migration assay: The wound-healing migration
fluorescence intensity of PI (FL-2; λem: 585 nm; red 95 assay was performed as described previously.23 Briefly, PC-3
fluorescence). Resulting DNA distributions were analyzed by
Modfit (Verity Software House Inc., Topsham, ME) for the
proportions of cells in apoptosis, G1-phase, S- phase, and G2-M
45 phases of the cell cycle.21
cells were treated with mitomycin-C to inactivate cell
proliferation, wounded by micro tip, washed with PBS,
supplemented with fresh medium treated with SAD. Images of
the cells were taken after ∼0 to 24 h of incubation and the
100 percentage of wound closure was expressed with respect to
untreated cells consider 100%. The wound-healing migration
assay in HUVEC cells was also done using similar protocol.
Fluorescence microscopy: PC-3 cells were treated with 5, 10
and 20 µM concentrations of compound 5b for 24 h. After
treatment cells were collected, washed with PBS twice and fixed
in 400 µl cold acetic acid: methanol (1+3, v/v) overnight at 4 °C.
50 Next day cells washed with fixing solution and dispensed in 50 µl
Statistical analysis. Data expressed as mean
SD or
representative of one of three similar experiments unless
of fixing solution. Spread cells on a clean slide and dried 105 otherwise indicated. Comparisons were made between control
overnight at room temperature. Cells were stained with Hoechst
33258 (5 µg/ml in 0.01 M citric acid and 0.45 M disodium
phosphate containing 0.05% Tween 20) for 30 min at room
and treated groups or the entire intra group using one way
ANOVA with post Bonferroni test through GraphPad Prism
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Acknowledgements
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the award of Senior Research Fellowships.
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