Synthesis of novel ring-A fused hybrids of oleanolic acid with capabilities to arrest cell cycle and induce apoptosis in breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2013.12.040

Six novel oleanolic acid ring-A fused hybrids (5-10) have been synthesized by employing a four step protocol with the introduction of benzylidene functionality at C-2 as the key step. Their structures were established by high resolution NMR and Mass spectral data. The synthesized compounds have been screened against seven human cancer cell lines including ME-180 & HeLa (cervix), MCF-7, MDA-MB-453 & MDA-MB-231 (breast), PC-3 (prostate) and HT-29 (colon) using MTT assay. Most significantly, compound 10 showed potent activity against the three breast cancer cell lines. The IC₅₀ value (10.60 μM) of compound 10 against MCF-7 found to be much lower than that of the standards and parent compound. Flow cytometric analysis reveals that compound 10 arrests cell cycle in S phase and induces apoptosis in MCF cells.2014 Elsevier Masson SAS. All rights reserved.

Full text of DOI:10.1016/j.ejmech.2013.12.040

European Journal of Medicinal Chemistry 74 (2014) 398e404
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel ring-A fused hybrids of oleanolic acid with
capabilities to arrest cell cycle and induce apoptosis in breast cancer
cells
,
a
b
Uppuluri Venkata Mallavadhani a , Nagi Reddy Vanga , Manish Kumar Jeengar ,
*
V.G.M. Naidu b
a Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Andhra Pradesh, India
b Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education & Research, Hyderabad 5000 037, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Six novel oleanolic acid ring-A fused hybrids (5e10) have been synthesized by employing a four step
protocol with the introduction of benzylidene functionality at C-2 as the key step. Their structures were
established by high resolution NMR and Mass spectral data. The synthesized compounds have been
screened against seven human cancer cell lines including ME-180 & HeLa (cervix), MCF-7, MDA-MB-453
& MDA-MB-231 (breast), PC-3 (prostate) and HT-29 (colon) using MTT assay. Most significantly, com-
Received 5 November 2013
Received in revised form
29 December 2013
Accepted 31 December 2013
Available online 8 January 2014
pound 10 showed potent activity against the three breast cancer cell lines. The IC50 value (10.60 mM) of
compound 10 against MCF-7 found to be much lower than that of the standards and parent compound.
Flow cytometric analysis reveals that compound 10 arrests cell cycle in S phase and induces apoptosis in
MCF cells.
Keywords:
Oleanolic acid
Oleanolic acid hybrids
Anti-cancer activity
Apoptosis
Ó 2014 Elsevier Masson SAS. All rights reserved.
Cell cycle arrest
1. Introduction
groups like enones, oximes and amides have been introduced [7].
When it comes to ring-A, simple oxidation or alkylation of the C-3
Oleanolic acid [3
b
-hydroxy-olean-12-ene-28-oic acid] (1), a
hydroxyl group has been reported [8,9]. Most of the synthesized
analogues have been reported to exhibit newer or enhanced ac-
tivities than the parent oleanolic acid. Of the various synthesized
analogues of oleanolic acid, ring-A modified analogues have been
reported to be much more potent than the other analogues. The
most important example of this class of analogues is Bardoxolone
methyl (CDDO-Methyl ester), which is in advanced stage of clinical
development for the treatment of cancer and chronic kidney dis-
ease [10]. Ring-A of oleanolic acid is still the attractive site to
perform chemical modifications to generate diverse hybrid struc-
natural pentacyclic triterpenic acid, has been found widespread in
plant kingdom [1]. It has been in clinical use as an anti-hepatitis
drug in China for more than 20 years [2]. In addition to its excel-
lent hepatoprotective effects, oleanolic acid has also been found to
exhibit highly potent anti-inflammatory, antitumour, anti-HIV,
cardiovascular and glycogen phosphorylase inhibitory activities
[3,4]. In view of high natural abundance, significant bio-activities
and relatively non-toxic nature, oleanolic acid has become a high-
ly useful lead compound. It has three active sites such as C-3 hy-
droxyl, ring-C double bond and C-28 carboxylic acid, which are
amenable for a wide range of chemical transformations. A number
of oleanolic acid analogues have been reported so far by trans-
forming these functionalities. The C-28 carboxylic acid has been
utilised to prepare different types of esters, alcohols, amides, ni-
triles, and triazoles [5,6]. In case of ring-C, variety of functional
tures. The 3b-hydroxyl functionality, through its oxidation product,
can be utilised to introduce a benzylidene functionality at C-2. The
resultant exocyclic enone system can then be exploited to synthe-
size a range of hybrid structures by condensing with appropriate
amino functionalities. This concept is novel and the resultant
hybrid structures are expected to exhibit potent biological activ-
ities. With this background and in continuation to our research
programs on synthesizing triterpenic acid based new chemical
entities [6,8,11], we have synthesized some novel 2,3 ring-A fused
heterocyclic hybrids of oleanolic acid and screened them against a
panel of seven human cancer cell lines. The synthesis, anti-cancer
* Corresponding author. Tel./fax: þ91 4027191882.
E-mail
addresses:
uvmavadani@yahoo.com,
mallavadhani@iict.res.in
(U.V. Mallavadhani).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.040

U.V. Mallavadhani et al. / European Journal of Medicinal Chemistry 74 (2014) 398e404
399
activity and mode of action of the synthesized compounds are
presented in this paper.
periplanar orientation. The stereo chemistry of compound 5 was
further confirmed by its NOESY spectrum, which reveals that the
proton H-2 correlates with 24-CH3 and 25-CH3 groups. The above
observations suggest that H-2 is
(Fig. 1).
b a proton
proton and H-20 is
2. Results and discussion
The benzylidene compound (4), when reacted with hydroxyl-
amine hydrochloride yielded an isoxazole (7) in 56% yield. Its
structure was confirmed by its 13C NMR, which showed the char-
2.1. Chemistry
The synthetic protocols employed for the synthesis of the novel
hybrid structures are presented in Scheme 1. Oleanolic acid (1) was
treated with methyl iodide and potassium carbonate in acetone to
form compound 2 in 90% yield, which was then oxidized with PCC
in dichloromethane to afford compound 3 in 88% yield. The ben-
zylidene compound 4, which is the key precursor for the synthesis
of the title compounds, was prepared by Claisen Schmidt conden-
sation of compound 3 with benzaldehyde in presence of ethanolic
potassium hydroxide in 92% yield. Compound 4 showed a sharp
acteristic carbon signals of isoxazole ring at
d 109.14, 166.24 and
169.46. Compound 4 when treated separately with urea, thiourea
and guanidine yielded the corresponding 2-oxo-pyrimidine (8),
pyrimidine-2-thione (9) and 2-amino-pyrimidine (10) in 73%, 76%
and 82% yields respectively. The structures of compounds 8e10
were confirmed by their respective 13C NMR spectra, which showed
the characteristic C-2 carbon signals at
respectively.
d 100.32, 105.69 and 128.32
peak at 1677 cmꢀ1 in IR spectrum indicating the presence of
unsaturated ketone functionality, which was further confirmed by
the presence of olefinic proton signal at
7.53 in 1H NMR and
carbonyl & olefinic carbon signals at
207.80, 137.38 & 133.66 in 13
a,b
2.2. Biology
d
d
C
2.2.1. Anti-cancer activity
NMR spectra. This benzylidene compound (4) with an exocyclic
enone system is a versatile precursor. It can form fused heterocyclic
ring systems such as pyrazolines, pyrimidines, isoxazoles on
treatment with dinucleophiles. With this objective, compound 4
was treated with hydrazines, ureas and hydroxylamine hydro-
chloride to prepare a range of ring-A fused hybrids of oleanolic acid.
Interestingly, compound 4 reacted differently with hydrazine and
phenylhydrazine and yielded pyrazoline (5, 72%) and pyrazole (6,
58%) hybrids respectively. Compound 6 showed the characteristic
The anti-cancer activity of the synthesized compounds (3e10)
was studied using ME-180 & HeLa (cervix), MCF-7, MDA-MB-453 &
MDA-MB-231 (breast), PC-3 (prostate) and HT-29 (colon) cancer
cell lines by employing MTT assay [15]. Doxorubicin and etoposide
along with compound 1 were taken as reference standards in this
study. From the close analysis of the IC50 values (Table 1), it is
evident that the synthesized compounds showed selectivity in
exhibiting significant anti-cancer activity against the 3 breast
cancer cell lines. Especially, the synthesized compounds found
active against MCF-7 cell line. Among the synthesized compounds,
the pyrimidine compounds (8e10) showed much higher activity
than pyrazoline (5), pyrazole (6) and isoxazole (7) analogues. Most
significantly, 2-amino-pyrimidine (10) showed potent activity
against all the three breast cancer cell lines with IC50 values ranging
carbon signals of pyrazole ring at
d 174.31, 161.98 and 113.65 in
addition to the expected oleanane carbon signals in its 13C NMR
spectrum. Whereas, compound 5 showed a clean doublet at
d 4.28
(J ¼ 11.042 Hz) in its 1H NMR spectrum and diagnostic signals at
d
164.86, 70.03 & 38.33 in 13C NMR spectrum confirming a pyr-
azoline structure to the compound with H-2 and H-20 in anti-
from 10.60 to 16.27
mM. Compound 10 showed highest anti-cancer
COOH
COOCH3
COOCH3
a
b
HO
O
HO
1
3
2
c
e
COOCH3
COOCH3
H
N
H
COOCH3
d
N
N
HN
O
4
5
6
h
f
g
COOCH3
COOCH3
COOCH3
N
N
O
H2N
N
X
N
H
N
10
8.
X=O
7
9. X=S
Scheme 1. Reagents and conditions a. CH3I, K2CO3, acetone, rt, 90%, b. PCC, DCM, rt, 88% c. PheCHO, KOH, ethanol, rt, 92% d. N2H4$H2O, Ethanol, reflux, 72% e. PhNHNH2$HCl, KOH,
ethanol, reflux, 58% f. NH2OH$HCl, KOH, ethanol, reflux, 56% g. Urea or thiourea, KOH, ethanol, reflux, 73e76% h. NH2C(NH)NH2$HCl, KOH, ethanol, reflux, 82%.

400
U.V. Mallavadhani et al. / European Journal of Medicinal Chemistry 74 (2014) 398e404
30
showed increase in percentage of early apoptotic cells (Fig. 2b).
Cells treated with 10 M for 48 h showed a significant increase in
percentage of late apoptotic/necrotic cells. Fluorescence micro-
scopic images (Fig. 2c) of cells treated with 10 M of compound 10
clearly showed the morphological changes such as cell shrinkage,
membrane blebbing, chromatin condensation with destructive
fragmentation of the nucleus, suggesting that the compound 10
induced cell death in breast cancer cells by apoptosis.
29
m
20
m
19
21
22
H
13
12
26
18
11
17
16
H
25
H
COOCH3
28
14
15
The effects of compound 10 on cell cycle progression were
examined by propidium iodide staining method [17]. When the
1
4
9
2'
2
10
8
7
HN
H
27
MCF-7 cells were treated with 5 and 10 mM of compound 10 for 48 h
3
5
6
resulted in a marked accumulation of cells in S-phase (1.94 fold)
and a slight reduction in G1 Phase as shown in Fig. 3. These results
indicated that the compound 10 inhibited the growth of the cancer
cells by inhibiting the cell cycle.
N
H
24
23
Fig. 1. Selected NOESY correlations of compound 5.
3. Conclusion
activity against MCF-7 breast cell line (IC50:10.60
almost equal to that of the standard anti-cancer drug doxorubicin
(10.90 M). More interestingly this activity is 2 times higher than
that of etoposide (23.9 M) and several times than that of the
parent compound 1 (>100 M). Compound 10 also showed potent
activity against ME-180 (16.72 M), MDA-MB-453 (12.60 M) and
MDA-MB-231 (16.27 M) cell lines. In case of MDA-MB-231 cell
line, it showed much higher activity than the standard etoposide
(24.22 M) and the parent compound 1 (>100), but less than
doxorubicin (1.69 M). However, in case of ME-180 and MDA-MB-
453, compound 10 showed much higher activity than the parent
compound 1, but less than doxorubicin and etoposide standards.
Among the remaining synthesized analogues, compound 9 only
showed significant activity against MCF-7, MDA-MB-453 & MDA-
MB-231 (breast), HeLa (cervix) and HT-29 (colon) cancer cell lines.
mM), which is
In summary, six novel oleanolic acid ring-A fused hybrids
have been synthesized by employing a four step protocol with
introduction of benzylidene functionality at C-2 as the key step
and evaluated for their anti-cancer activity against seven human
cancer cell lines. The new compounds were found to be prom-
ising lead compounds against the majority of the studied cancer
cell lines, exhibiting IC50 values much lower than that of the
parent compound. Notably, compound 10 found to be as potent
as the reference drugs doxorubicin (DOX) and etoposide against
breast cancer cells MCF-7, MDA-MB 231 and MDA-MB 453 by
arresting the cell cycle at S phase. Fused pyrimidine moiety
seems to be important for enhanced antiproliferative activity of
oleanolic acid.
m
m
m
m
m
m
m
m
4. Experimental
2.2.2. Morphological observation by Acridine Orange and Ethidium
Bromide (AO/EB) staining & cell cycle analysis
4.1. Chemistry
The morphological abnormalities induced by compound 10 in
MCF-7 cells were studied under fluorescence microscopy using AO/
EB staining technique [16]. AO permeates the intact cell membrane
and stains the nuclei green, whereas EB is taken up only when the
membrane integrity is deteriorated, and stains the nucleus red.
Intact cells therefore exhibit homogeneous green nuclei, whereas
apoptotic cells show condensed or fragmented chromatin. Late
apoptotic or necrotic cells have orange to red nuclei. Compared
with spontaneous apoptosis observed in control cells (early
apoptotic 3.19%, 0% late apoptotic and 0% necrotic cells) (Fig. 2a),
Melting points were determined in open capillaries on a Buchi
melting point apparatus and are uncorrected. IR spectra were
recorded in KBr disks on Nicolet 740 FTIR spectrophotometer. 1H
and 13C NMR spectra were recorded on Bruker 300 MHz and Varian
500 MHz spectrometers and chemical shifts were reported as parts
per million (ppm) with tetramethylsilane as an internal standard.
Mass spectra were obtained on Agilent ESI-QTOF instrument. Col-
umn chromatography was performed on ACME grade 60e
120 mesh silica gel and Merck pre-coated silica gel 60 F254 plates
were used for thin layer chromatography.
MCF-7 cells treated with 5 mM for 48 h with the compound 10
Table 1
Anti-cancer activity (IC50 values) of compounds 1, 3 & 4e10.
Compound
IC50 (mM)
ME-180
MCF-7
HeLa
MDA-MB 453
MDA-MB 231
PC-3
HT-29
1
>100
>100
>100
>100
>100
>100
>100
3
4
5
6
>100
>100
>100
>100
78.7 ꢁ 5.26
71.7 ꢁ 7.35
96.6 ꢁ 3.19
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7
8
9
10
>100
>100
>100
16.7 ꢁ 0.32
0.39 ꢁ 0.01
8.9 ꢁ 0.3
88.2 ꢁ 2.31
78.9 ꢁ 4.46
30.9 ꢁ 1.09
10.6 ꢁ 0.13
10.9 ꢁ 1.76
23.9 ꢁ 0.3
>100
>100
75.4 ꢁ 3.31
45.1 ꢁ 2.94
0.36 ꢁ 0.02
4.71 ꢁ 1.4
>100
>100
>100
75.54 ꢁ 1.32
16.27 ꢁ 0.12
1.69 ꢁ 0.56
24.22 ꢁ 2.94
>100
>100
>100
69 ꢁ 3.25
30.7 ꢁ 1.34
1.76 ꢁ 0.23
21.45 ꢁ 3.87
96.7 ꢁ 1.01
40.55 ꢁ 0.93
12.6 ꢁ 0.35
1.99 ꢁ 1.75
NA
87.4 ꢁ 5.83
>100
75.3 ꢁ 3.54
0.19 ꢁ 0.01
14.4 ꢁ 3.23
Doxorubicin
Etoposide
NA: Not analysed
Initially all the compounds were tested for cytotoxicity activity in all the human cancer cell lines at concentration of 100
were further tested for dose response study and their IC50 values were calculated.
m
M. The compounds which showed ꢂ50% cytotoxicity

U.V. Mallavadhani et al. / European Journal of Medicinal Chemistry 74 (2014) 398e404
401
Fig. 2. Morphological changes in MCF-7 cells treated with and without compound 10 for 48 h. (a) Green live cells with intact membrane and nuclei; (b) cells treated with 5 mM
showed early signs of apoptosis characterized by condensed chromatin, cell membrane blebbing, and destructive fragmentation of the nuclei; (c) cells treated with 10 mM showed
late apoptotic cells/necrotic cells characterized with orange-red staining of the nucleus after treatment and (d) apoptotic rate of MCF-7 cells treated with and without compound 10
for 48 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4.1.1. Oleanolic acid (1)
Oleanolic acid was isolated from the leaves of Diospyros mela-
noxylon in 0.1% yield, by our reported procedure [12].
Solvent was evaporated under reduced pressure and reaction
mixture was purified by silica gel column chromatography to afford
compound 3 as colourless crystals (0.876 g, 88%); mp: 186e188 ꢃC.
Its identity was confirmed by comparing its physical and spectral
data with reported values [14].
4.1.2. Methyl 3b-hydroxy-olean-12-en-28-oate (2)
To a stirred solution of 1 (2 g, 4.38 mmol) in acetone (18 ml),
anhydrous K2CO3 (0.6 g, 4.38 mmol) and CH3I (0.4 ml, 6.57 mmol)
were added at room temperature. After continuous stirring for 12 h,
the reaction mixture was diluted with water and extracted with
chloroform. The organic layer was dried over anhydrous sodium
sulphate, concentrated under reduced pressure and chromato-
graphed over silica gel column to afford compound 2 as colourless
crystals (1.85 g, 90%); mp: 202e204 ꢃC. Its identity was confirmed by
comparing its physical and spectral data with reported values [13].
4.1.4. Methyl 3-oxo-2-benzylidene-olean-12-en-28-oate (4)
Compound 3 (0.5 g, 1.06 mmol) in absolute ethanol (8 ml) was
stirred with benzaldehyde (0.2 ml, 2.13 mmol) in presence of KOH
(0.12 g, 2.13 mmol) at room temperature for 2 h. The reaction
mixture was neutralized with aq. HCl (1:1) and organic layer was
extracted with chloroform (3 ꢄ 50 ml). The combined organic layer
was dried over anhydrous sodium sulphate and concentrated under
reduced pressure to give crude reaction mixture, which was chro-
matographed over silica gel column to afford compound 4 as col-
ourless amorphous solid (0.546 g, 92%); mp: 178e180 ꢃC; IR (KBr)
4.1.3. Methyl 3-oxo-olean-12-en-28-oate (3)
PCC (0.687 g, 3.18 mmol) was added to the solution of 2 (1 g,
2.12 mmol) in DCM (12 ml) and stirred for 2 h at room temperature.
nmax: 2947, 2864, 1727, 1677 cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d
0.79, 0.86 0.91, 0.93,1.14,1.17,1.2 (21H, 7ꢄ s, 7ꢄ CH3),1.2e1.8 (15H,

402
U.V. Mallavadhani et al. / European Journal of Medicinal Chemistry 74 (2014) 398e404
Fig. 3. Effect of MCF-7 on cell cycle progression of breast cancer cells, (a) MCF-7 control cells; (b, c) MCF-7 cells treated with 5 and 10
mM for 48 h followed by analysis of cell cycle
distribution using propidium iodide cell staining method. Cell population in each cell cycle phase was numerically depicted. Data represent one of two independent experiments.
m, CH2, CH), 1.96e2.03 (3H, m), 2.32 (1H, d, J ¼ 17.9 Hz, H-1), 2.9
(1H, dd, J ¼ 3.9, 13.9 Hz, H-18), 3.02 (1H, d, J ¼ 15.9 Hz, H-1), 3.63
(3H, s, COOCH3), 5.35 (1H, t, J ¼ 3.9 Hz, H-12), 7.33 (1H, t, J ¼ 6.9 Hz,
AreH), 7.38e7.43 (4H, m, AreH), 7.53 (1H, s, exocyclic ene-H); 13C
ESI-HRMS (m/z) [M þ H]þ calcd for C38H55O2N2 571.4258, found
571.4242.
4.1.5.2. Methyl 2,3-(1,5-diphenyl)-1H-pyrazolo-olean-12-en-28-oate
(6). Pale yellow amorphous solid, yield: 58%; mp: 221e223 ꢃC; IR
(KBr) nmax: 2925, 2865, 1726, 1598, 1498 cmꢀ1; 1H NMR (300 MHz,
NMR (125 MHz, CDCl3):
d 15.19, 16.41, 20.33, 22.62, 23.07, 23.64,
25.7, 26.87, 27.64, 29.7, 30.68, 31.81, 32.27, 33.07, 33.81, 36.22, 39.14,
41.48, 41.92, 44.07, 45.11, 45.35, 45.87, 46.77, 51.55, 52.96, 122.07,
128.36, 128.42, 130.34, 133.66, 135.88, 137.38, 143.93, 178.16, 207.8;
ESI-HRMS (m/z) [M þ H]þ calcd for C38H53O3 557.3989, found
557.3986.
CDCl3):
d
0.67, 0.82, 0.85, 0.92, 1.02, 1.03, 1.31 (21H, 7ꢄ s, 7ꢄ CH3),
1.2e1.7 (17H, m, CH2, CH), 2.06 (2H, m), 2.41 (1H, d, J ¼ 16.2 Hz, H-
1), 2.79 (1H, dd, J ¼ 4.5, 13.5 Hz, H-18), 3.55 (3H, s, COOCH3), 5.22
(1H, t, J ¼ 3.77 Hz, H-12), 6.67 (1H, t, J ¼ 7.5, 6.7 Hz, AreH), 6.88 (2H,
d, J ¼ 7.5 Hz, AreH), 7.05 (2H, t, J ¼ 6.7, 9.06 Hz, AreH), 7.21e7.32
(5H, m, AreH); 13C NMR (125 MHz, CDCl3):
d 15.31, 16.96, 18.61,
4.1.5. General procedure for the synthesis of ring A fused
22.34, 23.01, 23.62, 25.62, 25.85, 26.97, 27.64, 29.67, 30.67, 32.32,
33.09, 36.04, 37.75, 38.41, 39.44, 41.74, 45.8, 46.34, 46.69, 47.23,
51.53, 52.99, 57.35, 113.65, 121.94, 122.26, 125.63, 125.83, 126.07,
127.34, 128.67, 129.07, 143.22, 144.09, 161.98, 174.31, 178.32; ESI-
HRMS (m/z) [M þ H]þ calcd for C44H57O2N2 645.4414, found
645.4426.
heterocyclic analogues of oleanolic acid (5, 6, 7, 8, 9 and 10)
Compound 4 (1 eq.) in absolute ethanol was refluxed with
appropriate amino functionalities (2 eq.) in presence of KOH (2 eq.)
for 2e12 h. After completion, reaction mixture was neutralized
with aq. HCl (1:1) and organic layer was extracted three times with
chloroform. The combined organic layer was dried over anhydrous
sodium sulphate and concentrated under reduced pressure to give
crude reaction mixture, which was chromatographed over silica gel
column to afford pure compounds 5, 6, 7, 8, 9 and 10.
4.1.5.3. Methyl 2,3-(5-phenyl)-isoxazolo-olean-12-en-28-oate (7).
Colourless amorphous solid, yield: 56%; mp: 174e176 ꢃC; IR (KBr)
nmax: 2927, 2856,1726,1636,1459 cmꢀ1; 1H NMR (500 MHz, CDCl3):
d
0.73, 0.84, 0.92, 0.97, 0.99, 1.07, 1.25 (21H, 7ꢄ s, 7ꢄ CH3), 1.2e1.8
(18H, m, CH2, CH), 2.03 (1H, d, J ¼ 16.6 Hz, H-1), 2.45 (1H, d,
J ¼ 16.3 Hz, H-1), 2.87 (1H, d, J ¼ 13.2 Hz, H-18), 3.61 (3H, s,
4.1.5.1. Methyl 2,3-(5-phenyl)-4,5-dihydro-1H-pyrazolo-olean-12-
en-28-oate (5). Colourless amorphous solid, yield: 72%; mp: 182e
COOCH3), 5.3 (1H, t, J ¼ 3.8 Hz, H-12), 7.33e7.38 (5H, m, AreH); 13
C
184 ꢃC; IR (KBr) nmax: 3426 (br), 2946, 2870, 1727, 1592, 1455 cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d 0.76, 0.9, 0.92, 0.97, 1.06, 1.14, 1.3 (21H,
NMR (125 MHz, CDCl3):
d 14.07, 16.33, 19.68, 21.95, 23.14, 23.58,
25.72, 27.61, 28.94, 29.69, 30.71, 31.77, 32.4, 33.1, 33.85, 38.27, 38.83,
39.27, 41.41, 41.79, 44.95, 45.83, 46.77, 46.9, 51.5, 54.02, 109.14,
122.13, 128.3, 128.81, 129.31, 132.8, 143.8, 166.24, 169.46, 178.21;
ESI-HRMS (m/z) [M þ H]þ calcd for C38H52O3N 570.3941, found
570.3951.
7ꢄ s, 7ꢄ CH3), 1.2e1.7 (16H, m, CH2, CH), 1.9 (2H, dd, J ¼ 3.3, 3.8 Hz),
1.99 (2H, m), 2.87 (1H, dd, J ¼ 4.4, 14.3 Hz, H-18), 2.96 (1H, m, H-2),
3.62 (3H, s, COOCH3), 4.28 (1H, d, J ¼ 11.04 Hz, H-20), 5.28 (1H, t,
J ¼ 3.3 Hz, H-12), 5.6 (1H, s, NH), 7.29e7.41 (5H, m, AreH); 13C NMR
(75 MHz, CDCl3):
d 15.32, 16.94, 18.57, 22.71, 23.03, 23.5, 23.64,
25.88, 27.01, 27.67, 30.71, 32.34, 32.42, 33.12, 33.83, 37.45, 38.33,
39.45, 41.3, 41.78, 44.33, 45.84, 46.71, 47.25, 48.62, 51.57, 56.16,
72.03, 122.09, 126.98, 127.6, 128.6, 141.74, 143.95, 164.86, 178.27;
4.1.5.4. Methyl 2,3-(4-phenyl)-1H-2-pyrimidonyl-olean-12-en-28-
oate (8). Colourless amorphous solid, yield: 73%; mp: 185e

U.V. Mallavadhani et al. / European Journal of Medicinal Chemistry 74 (2014) 398e404
403
186 ꢃC; IR (KBr) nmax: 3450 (br), 2947, 1727, 1680, 1463 cmꢀ1
;
1H
medium. Then the above media was replaced with 90
serum free media and 10
were incubated at 37 ꢃC for 4 h, there after the above media was
replaced with 200
l of DMSO and incubated at 37 ꢃC for 10 min.
The absorbance at 570 nm was measured on a spectrophotometer
(spectra max, Molecular devices). IC50 values were determined
from plot: % cell viability (from control) versus concentration.
m
l of fresh
NMR (300 MHz, DMSO-d6): 0.69, 0.83, 0.84, 0.92, 1.02, 1.03, 1.25
d
ml of MTT reagent (5 mg/ml) and plates
(21H, 7ꢄ s, 7ꢄ CH3), 1.2e1.6 (17H, m, CH2, CH),1.98 (2H, m), 2.45
(1H, d, J ¼ 17.2 Hz, H-1), 2.75 (1H, d, J ¼ 13.2 Hz, H-18), 3.53 (3H, s,
COOCH3), 5.16 (1H, t, J ¼ 3.5 Hz, H-12), 7.24e7.35 (5H, m, AreH),
m
7.81 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6):
d 15.36, 16.24, 19.03,
21.05, 22.62, 22.76, 24.31, 25.43, 27.07, 27.83, 30.03, 31.62, 31.94,
32.71, 33.15, 34.97, 35.61, 41.02, 41.32, 42.18, 45.32, 45.62, 46.11,
51.48, 52.19, 60.24, 100.32, 121.69, 126.61, 127.25, 128.46, 134.31,
143.28, 153.49, 156.66, 172.08, 177.16; ESI-HRMS (m/z) [M þ H]þ
calcd for C39H53O3N2 597.4052, found 597.4049.
4.2.2. Morphological observation by Acridine Orange and Ethidium
Bromide (AO/EB) staining
AO/EB dual staining was carried by using the method of Ribble
et al. [16]. Cellular morphological changes were investigated by AO/
EB staining using fluorescence microscopy. Briefly, MCF-7 cells
were plated at a concentration of 1 ꢄ 106 cell/ml and treated with
different concentration of compound 10. Experimental control
(untreated cells) was employed. Plates were incubated in an at-
4.1.5.5. Methyl 2,3-(4-phenyl)-1H-2-thiopyrimidonyl-olean-12-en-
28-oate (9). Colourless amorphous solid, yield: 76%; mp: 233e
235 ꢃC; IR (KBr) nmax: 3446 (br), 2947, 1726, 1192, 1559, 1461 cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d 0.6, 0.71, 0.92, 0.94,1.07,1.13,1.16 (21H,
mosphere of 5% CO2 at 37 ꢃC for 48 h. 10
containing Acridine Orange (AO) and Ethidium Bromide (EB) added
into each well in equal volumes (10 g/ml) respectively and within
ml of fluorescent dyes
7ꢄ s, 7ꢄ CH3), 1.3e1.7 (18H, m, CH2, CH), 1.95 (1H, m), 2.42 (1H, d,
J ¼ 16.2 Hz, H-1), 2.82 (1H, dd, J ¼ 3.13 Hz, H-18), 3.58 (3H, s,
COOCH3), 5.2 (1H, t, J ¼ 3.4 Hz, H-12), 7.15 (1H, s, NH), 7.3e7.37 (5H,
m
m, AreH); 13C NMR (125 MHz, CDCl3):
d 14.85, 16.44, 19.45, 20.11,
30 min the cells were visualized under a fluorescent microscope
(Nikon, Japan), using a blue filter. At least 200 cells were randomly
counted in various fields. The tests were repeated three times. The
percentage of apoptotic cells was calculated by the following
formula:
23.01, 23.56, 25.67, 27.56, 28.2, 30.62, 31.93, 32.26, 33.04, 33.79,
34.67, 35.76, 39.11, 40.02, 41.35, 41.71, 45.8, 45.97, 46.7, 51.46, 52.37,
60.23, 105.69, 121.92, 127.33, 128.54, 128.91, 132.6, 143.64, 151.44,
154.2, 174.06, 178.15; ESI-HRMS (m/z) [M
39H53O2N2S 613.3824, found 613.3831.
þ
H]þ calcd for
C
The apoptotic rate ð%Þ ¼ ðnumbers of early apoptotic cells
þ numbers of late apoptotic cellsÞ
4.1.5.6. Methyl 2,3-(2-amino,4-phenyl)-pyrimidinyl-olean-12-en-28-
oate (10). Colourless amorphous solid, yield: 82%; mp: 282e
284 ꢃC; IR (KBr) nmax: 3500e3250 (br), 2948, 1686, 1564, 1449,
= ðnumbers of all the cells countedÞ:
1411 cmꢀ1 1H NMR (500 MHz, DMSO-d6):
; d 0.73, 0.82, 0.91, 1.08,
1.13, 1.27, 1.36 (21H, 7ꢄ s, 7ꢄ CH3), 1.2e1.65 (17H, m, CH2, CH), 1.8
(1H, m), 2.2 (1H, d, J ¼ 17.4 Hz, H-1), 2.6 (1H, d, J ¼ 16.8 Hz, H-1),
2.75 (1H, dd, J ¼ 4.8, 14.2 Hz, H-18), 3.56 (3H, s, COOCH3), 5.23 (1H,
t, 3.5 Hz, H-12), 7.20e7.40 (5H, m, AreH), 7.72 (2H, s, NH2); 13C NMR
4.2.3. Cell cycle analysis
Cell cycle analysis was carried out using method given by Lee
et al. [17] with slight modifications. To determine the effect of
compound 10 on the cell cycle, cells were seeded in 6-well plates at
a density of 1 ꢄ 105 cells/ml for 24 h. After incubation, cells were
treated with different concentration of compound 10 for 48 h. Then
the cells were collected, washed and fixed in 70% ethanol in PBS
at ꢀ20 ꢃC. After overnight, fixed cells were pelleted and stained
with cell cycle analysis reagent as per the manufacturer in-
structions for 30 min at 37 ꢃC in dark, and about 5000 events were
analysed on a MuseÔ Cell Analyzer (Merck-Millipore, Germany).
(125 MHz, DMSO-d6):
d 15.35, 16.25, 19.09, 22.95, 23.34, 25.46,
27.12, 27.42, 27.68, 30.54, 31.61, 31.98, 32.75, 33.19, 35.01, 35.19,
35.62, 41.05, 41.37, 42.09, 45.37, 46.15, 51.48, 52.34, 55.86, 57.28,
121.64, 126.72, 127.12, 128.32, 128.84, 132.79, 143.32, 151.68, 162.71,
174.11, 177.19; ESI-HRMS (m/z) [M þ H]þ calcd for C39H54O2N3
596.4212, found 596.4221.
4.2. Biology
MCF-7 (Breast cancer) was obtained from National Center for
Cell Science (NCCS), Pune, India. DMEM, MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Trypsin,
EDTA, Acridine orange, Ethidium bromide were purchased from
Sigma Chemicals Co (St. Louis, MO), Fetal bovine serum was pur-
chased from Gibco, USA, MuseÔ Cell Cycle reagent from Milliopore,
6 well flat bottom tissue culture plates were purchased from
Tarson.
Acknowledgements
We are thankful to Director, CSIR-IICT for support and encour-
agement. We are also thankful to CSIR and UGC, New Delhi for
financial support under Network project, ORIGIN (CSC 0108) and
fellowship to VNR respectively.
Appendix A. Supplementary data
4.2.1. Cytotoxicity screening using MTT assay
MTT assay was performed according to the method of Naidu
et al. [15]. MTT assay is a standard colorimetric assay for measuring
cellular proliferation. MTT is a tetrazolium salt, which is yellow in
colour and is photosensitive. MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide] is taken by the living cells and
reduced by a mitochondrial dehydrogenase enzyme to a purple
formazan product that is impermeable to the cell membrane. Sol-
ubilization with solvents like DMSO leads to liberation of product
and amount of purple formazan product is directly related to the
cell viability. 1 ꢄ 104 cells (counted by Trypan blue exclusion dye
method) in 96-well plates were incubated with series of concen-
trations of compounds for 48 h at 37 ꢃC in DMEM with 10% FBS
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.040.
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