Synthesis of ring-C modified oleanolic acid derivatives and their cytotoxic evaluation

Article abstract of DOI:10.1016/j.bioorg.2016.08.001

Ring-C of oleanolic acid was chemically modified by treating with NBS under a variety of experimental conditions. The structures of the synthesized compounds were established by spectral analysis (¹H &¹³C NMR and Mass). All the compounds were evaluated against a panel of five human cancer cell lines by using MTT assay. Among the tested compounds, 2 and 7 showed significant activity against breast cancer cell line, MCF-7. Most significantly, compound 7 showed several folds enhanced activity against MCF-7 cancer cell lines (IC₅₀: 2.96?μM) than that of the parent (1) and the intermediate compound (6). Flow cytometric analysis revealed that these compounds arrested the cell cycle in G0/G1 phase and induced mitochondrial mediated apoptosis.

Full text of DOI:10.1016/j.bioorg.2016.08.001

Accepted Manuscript
Synthesis of ring-C modified oleanolic acid derivatives and their cytotoxic eval-
uation
Banita Pattnaik, Vadithe Lakshma Nayak, Sistla Ramakrishna, Uppuluri
Venkata Mallavadhani
PII:
S0045-2068(16)30191-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.08.001
YBIOO 1935
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
24 April 2016
31 July 2016
3 August 2016
Please cite this article as: B. Pattnaik, V.L. Nayak, S. Ramakrishna, U.V. Mallavadhani, Synthesis of ring-C modified
oleanolic acid derivatives and their cytotoxic evaluation, Bioorganic Chemistry (2016), doi: http://dx.doi.org/
1
0.1016/j.bioorg.2016.08.001
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Revised
Synthesis of ring-C modified oleanolic acid derivatives and their
cytotoxic evaluation
a
b
b
Banita Pattnaik , Vadithe Lakshma Nayak , Sistla Ramakrishna , Uppuluri Venkata
a*
Mallavadhani
a
Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology,
Hyderabad-500007, India.
b
Medicinal Chemistry & Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad-500007, India
_
____________________________________________________________________________
ABSTRACT
Ring-C of oleanolic acid was chemically modified by treating with NBS under a variety of
experimental conditions. The structures of the synthesized compounds were
1
13
established by spectral analysis ( H & C NMR and Mass). All the compounds were
evaluated against a panel of five human cancer cell lines by using MTT assay. Among
the tested compounds, 2 and 7 showed significant activity against breast cancer cell
line, MCF-7. Most significantly, compound 7 showed several folds enhanced activity
against MCF-7 cancer cell lines (IC50:2.96 µM) than that of the parent (1) and the
intermediate compound (6). Flow cytometric analysis revealed that these compounds
arrested the cell cycle in G0/G1 phase and induced mitochondrial mediated apoptosis.
Keywords: Oleanolic acid, N-bromosuccinimide, Anticancer activity, Apoptosis,
Caspase 9 activity
_
____________________________________________________________________
*Corresponding author Tel: +91-4027193167; Fax: +91-4027191882; E. Mail address:
mallavadhani@iict.res.in (U. V. Mallavadhani)
1

1
. Introduction
Oleanolic acid (3-Hydroxy-olea-12-en-28-oic acid), a natural pentacyclic
triterpenoid (PCT) widely exists in plants in free or glycosidic forms [1]. It attracted the
attention of researchers, due to its broad spectrum bioactivity and it plays a key role in
human health care with high efficacy and low toxicity [2]. Literature search reveals that,
it has been isolated from more than 1620 plant species including many food and
medicinal plants [3]. The hepatoprotective effects of oleanolic acid allow its use in
China as an over-the-counter oral drug to treat liver disorders [4]. In addition to this,
oleanolic acid has also been found to exhibit highly potent anti-inflammatory, antitumor,
anti-HIV, cardiovascular and glycogen phosphorylase inhibitory activities [5,6]. Recently,
oleanolic acid was patented in China as an injectable for hepatitis B and liver cancer [7].
In view of the above pharmacological properties, oleanolic acid proves to be a good
starter molecule for further synthetic modifications [8,9]. Oleanolic acid has three
‘‘active’’ sites, i.e. the C-3 hydroxyl,C12-C13 double bond in ring-C and C-28 carboxylic
acid, which are amenable for chemical modifications to make a series of new chemical
entities [10,11]. The ring-C has an advantage with a double bond present between
C12-C13, leaving the allylic C-11 position for substitution reactions such as
halogenation. The resultant C-11 halo substituted intermediates can be transformed
further into a range of products such as dienes, ,-unsaturated ketones, saturated and
unsaturated lactones. It has been reported in literature that modification of ring-C
enhances the biological activities, such as anti-inflammatory, anti-diabetic, nephropathy
and cytotoxicity [12,13]. Further, Siewert et al. reported some 12-hydroxy or halo
2
8→13 lactones by modifying ring-C of oleanane type triterpenes with significantly
enhanced in vitro cytotoxicity [14]. Recently, we have reported the synthesis of some
2

ring-C modified analogues of ursolic acid, the regioisomer of oleanolic acid with
enhanced cytotoxicity [15]. With this background and in continuation of our discovery
programme to identify highly potent and selective antitumor agents based on
pentacyclic triterpenic acids, a series of ring-C modified frameworks of oleanolic acid
were synthesized and evaluated for their cytotoxic potential against A-549 (Colon),
DU-145 (Prostate), MCF-7 (Breast), HeLa (Cervical) and ACHN (Renal) human cancer
cell lines. The potent molecules were subjected to apoptosis and other cytometric
studies.
2
. Results and discussions
2
.1. Chemistry
The protocols employed for the synthesis of ring-C analogues of oleanolic acid
are presented in Scheme 1. Oleanolic acid (1) when treated with N-bromosuccinimide in
-
1
CCl
4
at room temperature afforded 3-Hydroxy-olean-28→13-olide [2, IR (1760 cm )
13
and C NMR ( 178.78 ppm)] in 45 % yield. The same reaction was then extended to
oleanolic acid protected at C-3 hydroxyl or C-17 carboxylic acid or both in CCl
dioxane-CaCO solvent systems. The C-3 hydroxyl protected oleanolic acid acetate (3)
on treatment with NBS yielded an unsaturated lactone 4 [IR: 1758 (-lactone), 1720 &
4
or
3
1
3
1
240 (C-3-acetate); C NMR: 178.15, 156.96, 121.76 ppm] in CCl
4
and a saturated
lactone 5 [IR: 1760 cm ; C NMR: 178.87 (-lactone)] in aq. dioxane-CaCO each in
7% yield. Interestingly, the C-17 carboxylic acid protected oleanolic acid methyl ester
6) under identical experimental conditions yielded the same methyl-3hydroxy-olean-
-
1 13
3
4
(
1
3
9
1
(11),12-dien-28-oate [7, C NMR at 170.96 (C=O), 115.78 (C12), 120.44 (C9),
45.14 (C11), 154.16 (C13)] in both the cases with 32% and 30% yields respectively. In
3

another set of reaction, both the C-17 carboxylic acid and C-3 hydroxyl protected 3-
acetoxy-17-carbomethoxy-oleanolic acid (8) was subjected to similar NBS reactions. In
aq. dioxane, it afforded methyl-3acetoxy-olean-11-oxo-12-ene-28-oate 9 [IR: 1720
-
1 13
cm ; C NMR: 200.15 (C11), 177.49 (C28) ppm] in 50% yield. Surprisingly, it did not
yield any product in CCl , but only underwent decomposition.
4
2
.2 Biology
.2.1 In vitro cytotoxicity
The synthesized compounds (2-9) along with the parent (1) were evaluated for
2
their in vitro anticancer activity against a panel of five human cancer cell lines by
employing MTT assay using doxorubicin as the standard [16]. The results were
summarized in Table 1. The cytotoxicity values were expressed as IC50 in µM
concentration. The in vitro screening results revealed that, most of the tested
compounds showed potent anticancer activity with IC50 values ranging from 2.10±0.65
to 9.42±1.47µM. From the IC50 values, we concluded that:
a) Among the tested compounds, 2, 4, 5 and 7 showed highly potent anticancer
activities against all the cancer cell lines used.
b) Compound 7 was found to be the most active one exhibiting highest activity against
four cell lines namely MCF-7 (IC50:2.96 µM), HeLa (IC50:6.30 µM), DU-145 (IC50:6.77
µM) and ACHN (IC50:5.58 µM). The anticancer activity of this compound against MCF-7
cell line is 10.5 fold higher than intermediate compound 6 and several folds higher than
the parent compound (1). Close evaluation of the IC50 values of the compound (7)
suggests that, introduction of a conjugated double bond system in ring-C of the parent
acid increases the in vitro cytotoxicity potency to several folds.
4

c) Compound 2 showed highest activity against A-549 cell line (IC50:2.10M) followed
by MCF-7 (IC50:4.6 M). In fact, this compound exhibited maximum activity against A-
5
49 among the tested compounds with several fold enhancement over the parent
compound and more than the standard doxorubicin (IC50:2.17M).
d) Among the lactones 2 and 5, the lactone (2) with a C-3 hydroxyl group showed higher
activity against MCF-7 & A-549, whereas lactone 5 with C-3 acetoxy functionality found
to exhibit higher activity against Hela, DU-145 and ACHN.
e) Similarly, between the lactones 4 and 5, the unsaturated lactone (4) found to be more
active against MCF-7, A-549, DU-145, ACHN, whereas the saturated one (5) is more
active against Hela cell line.
As we have been working on identifying the potent leads for breast cancer cell
lines [17], compound 7 with highest activity has been taken up for further biological
evaluation.
2
.2.2 Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by
arresting the cell cycle at a particular checkpoint or by induction of apoptosis or a
combined effect of both cell cycle arrest and apoptosis [18,19]. Furthermore, regulation
of the cell cycle and apoptosis are considered to be effective cancer therapeutic
methods [20]. In vitro screening results revealed that compound 7 showed significant
anticancer activity against human breast cancer cell line, MCF-7. Therefore, it was
considered of interest whether the inhibition of cell growth was on account of cell cycle
arrest. In this study MCF-7 cells were treated with compound (7) at 2.5 and 5 µM
concentrations along with compound 6 at 5 µM concentration for 48h. The data obtained
5

from the cytometric profile clearly indicated that, compound 7 arrested cell cycle at
G0/G1 phase (86.99%, 92.15%) compared to the untreated control (84.46%) and the
intermediate 6 (86.21%) (Figure 1).The population of G2 phase decreased (from 6.92%
to 3.37%) with increase in concentration from 2.5 µM to 5.0 µM for compound 7,
whereas for compound 6 at 5 M concentration it was (8.44%) compared to control
(
8.59%).
2
.2.3 Measurement of mitochondrial membrane potential (∆Ψm)
The maintenance of mitochondrial membrane potential ((∆Ψm) is significant for
mitochondrial integrity and bio energetic function [21]. Mitochondrial changes, including
loss of mitochondrial membrane potential (∆Ψm), are key events that take place during
drug-induced apoptosis. Mitochondrial injury by 6 and 7 was evaluated by detecting fall
in mitochondrial membrane potential (∆Ψm). In this study we have investigated the
involvement of mitochondria in the induction of apoptosis by 6 and 7. After 48h of drug
treatment with these compounds, it reduced the mitochondrial membrane potential
(
∆Ψm) of MCF-7 cells, as observed by JC-1 staining (Figure2).
2
.2.4 Effect on intracellular ROS generation
Many anticancer agents have demonstrated to exert their cytotoxic effects by the
generation of reactive oxygen species (ROS) [22,23], which is considered as one of the
key mediators of apoptotic signaling. Therefore, we decided to investigate the role of
these compounds 6 and 7 in inducing the production of ROS that could potentially lead
to the cytotoxic effect in the MCF-7 cells. In order to demonstrate the role of test
compounds on ROS generation during apoptosis process, production of ROS was
6

examined by using an oxidant-sensitive fluorescent probe, DCFDA (2',
’dichlorofluorescin diacetate). After treatment with these compounds, the level of ROS
was significantly increased (Figure 3). The ratio of DCF-positive cells for compound 6 is
.6 % at 5 µM and for compound 7 are 7.3 and 12.0 % at 2.5 and 5 µM respectively.
7
7
The test results evidenced that these compounds have enhanced the generation of
ROS in MCF-7 cells.
2
.2.5 Effect on caspase-9
The activation of caspases plays an important role in the process of programmed
cell death or apoptosis. Caspases, or cysteine aspartic protease, are a family of
cysteine proteases, which are crucial mediators of apoptosis. The MCF-7 cells lack
endogenous caspase-3, whereas caspase-9 plays an important role in mediating drug-
induced apoptosis [24]. MCF-7 cells were treated with these compounds (6 and 7) for
4
8h. The results demonstrated that there was 2-3 fold induction in caspase-9 activity
when compared to untreated control (Figure 4), which suggests that they have the
ability to induce cell death by apoptosis in MCF-7 cells.
3
. Conclusion
The chemical transformation of ring C of oleanolic acid was resulted in the
synthesis of some modified frameworks. The cytotoxicity evaluation of these
compounds revealed that the diene functionality in ring C (7) drastically enhances the
activity against four of the tested cell lines and most effective towards the MCF-7, breast
cancer cell line. Flow cytometric analysis revealed that this compound arrested the cell
cycle at G0/G1 phase and induced mitochondrial mediated apoptosis. Therefore,
7

compound 7 can be considered as lead molecule for further development against breast
cancer.
4
. Experimental
4
.1. Chemistry
Melting points were determined in open capillaries on a Buchi melting point
apparatus and are uncorrected. IR spectra were recorded in KBr disks on JASCO FTIR
-
1
1
13
5
300 spectrophotometer and wave numbers are expressed in cm . Hand C NMR
spectra were recorded on Bruker500 MHz spectrometers and chemical shifts were
reported as part per million (ppm) with tetramethylsilane as an internal standard. Mass
spectra were obtained on Waters Synapt mass instrument. Column chromatography
was performed on ACME grade 60-120 mesh silica gel and Merck pre-coated silica gel
6
4
0 F254 plates were used for thin layer chromatography.
.1.1. Oleanolic acid 1
Oleanolic acid was isolated from the leaves of Diospyros melanoxylon in 0.1%
yield, by our reported procedure [25].
.2.2 General procedure for NBS reaction in CCl
To a stirred solution of 1, 3, 6 and 8(0.4 mmol) in CCl
4
4
4
(5ml), freshly
recrystallized NBS (0.6 mmol) were added at room temperature. After continuous
stirring for 8h, the reaction mixture was filtered and concentrated under reduced
pressure to yield the crude product and chromatographed over silica gel column to
afford compound 4.
8

4
1
.2.3 3-Hydroxy-olean-28→13-olide(2)
Colorless solid (45 %); mp: 235-238 C; IR (KBr)  max: 3400, 2950, 1760, 1710,
0
-
1 1
460, 1380 cm ; H NMR (500 MHz, CDCl , ppm):0.78 (3H, s), 0.89(3H, s), 0.91(3H,
3
13
s), 1.00 (3H, s), 1.22 (3H, s), 1.44 (3H, s), 1.99 (3H, s), 2.34(1H, m) 3.26(1H, m) ; C
NMR (125 MHz, CDCl ):  15.38 (C25), 16.91(C27), 17.70 (C ), 19.07 (C11), 21.09 (C26),
1.34 (C16), 23.55 (C24), 27.18 (C23), 27.55 (C22), 27.97 (C15), 29.18 (C ), 30.46 (C20),
0.87 (C30), 31.86 (C29), 33.25 (C12), 33.89 (C21), 34.64 (C ), 36.57 (C10), 38.35 (C19),
3
6
2
3
3
5
2
7
8.90 (C
1
), 39.95 (C
), 78.72 (C
455.3525, found 455.3543.
.2.4 3Acetoxy-olean-11-ene-28→13-olide (4) [26]
Colorless solid (0.046 g, 46.9%); mp: 221-225 C; IR(KBr)  max: 2950, 1900,
8
), 42.45 (C
4
), 43.42 (C14), 45.58 (C18), 52.35 (C17), 55.23 (C
9
),
+
6.48 (C
5
3
), 91.61 (C13, CH
2
), 178.78 (C28, CO);ESI-HRMS (m/z) [M - H]
calcd for C30
47
H O
3
4
0
-
1
1
1
0
6
758, 1720, 1460, 1380, 1240 cm ; H NMR (500 MHz, CDCl
3
): 0.88, 0.91, 0.94,
.97, 1.01, 1.18, 2.06 , 2.95 (1H m), 4.52 (1H, m), 5.55 (d, J = 6.04 Hz, 1H), 5.58 (d, J =
13
.04 Hz, 1H); C NMR(125 MHz, CDCl
26), 25.64 (C ), 26.03 (C16 &CH CO), 27.11 (C15), 29.16 (C23), 29.70(C24), 30.83 (C11),
1.80 (C30), 32.14 (C29), 32.20 (C ), 33.83 (C22), 33.89 (C20), 37.03 (C21), 37.46 (C19),
8.34 (C10), 39.47 (C ), 41.43 (C ), 43.58 (C ), 47.49 (C14), 50.73 (C17), 51.09 (C18),
1.62 (C ), 55.51 (C ), 65.38 (C & C13), 121.76 (C11 & C12), 156.96 (CH CO), 178.15
3
):  14.95 (C27), 19.86 (C
6
), 20.61 (C25), 21.29
(
C
2
3
3
3
5
7
4
1
8
9
5
3
3
+
(
C
28, CO); ESI-HRMS (m/z) [M + H] calcd for C32
H
49
O 497.3631, found 497.3633.
4
4
.2.5 General procedure for NBS reaction in aq dioxane-CaCO
3
To a stirred solution of 1, 3, 6 and 8(0.4 mmol) in dioxane (5ml), freshly
recrystallized NBS (0.6mmol)were added at room temperature with water (0.5 ml)
9

followed by CaCO (0.4 mmol). After continuous magnetic stirring for 18h, the reaction
3
mixture was filtered. The resultant filtrate was diluted with ice cold water (5 ml) and
extracted with ethyl acetate (3 x5 ml). The combined organic layer was dried over
anhydrous sodium sulphate and concentrated under reduced pressure to yield the
crude product. Then the crude product was chromatographed over silica gel column to
afford compound 5, 7 and 9.
4
.2.6 3Acetoxy-olean-28→13-olide(5) [27]
Colorless amorphous solid (0.546 g, 92%); mp: 178-180 C; IR (KBr)  max:
0
-
1
1
2
0
950, 1900, 1760, 1450, 1380, 1210 cm ; H NMR (500 MHz, CDCl ): 0.86, 0.88,
3
1
3
.90, 0.92, 0.97, 1.00, 1.22, 1.44, 1.99, 2.05, 2.35 (1H, m), 4.55 (1H, m) C NMR (125
):  16.49 (C26), 17.03 (C24), 17.59 (C25), 19.10 (C ), 21.13 (C27), 21.32
12), 23.45 (CH CO), 23.57 (C16), 27.54 (C30), 27.88 (C11), 29.18 (C15), 29.70 (C23),
0.45 (C ), 30.92 (C20), 31.87 (C22), 33.26 (C ), 33.87 (C29), 34.58 (C21), 36.47 (C10),
7.83 (C ), 38.04 (C ), 39.93 (C ), 42.45 (C19), 43.41 (C14), 45.53 (C18), 52.33 (C17),
5.32 (C ), 56.22 (C ), 80.59 (C ), 91.65 (C13), 170.97 (CH CO), 178.87 (C28, CO); ESI-
499.3787, found 499.3767.
.2.7 Methyl-3hydroxy-olean-9(11), 12-dien-28-oate(7) [28]
Colourless amorphous solid (0.546 g, 92%); mp: 138-140 C; IR (KBr)  max:
MHz, CDCl
3
6
(
C
3
3
3
5
2
7
4
1
8
9
5
3
3
+
HRMS (m/z) [M + H] calcd for C32
50
H O
4
4
0
-
1
1
3
460-3380, 1720, 1450, 1360, 1260, 1220 cm ; H NMR (500 MHz, CDCl
3
): 0.83,
0
.87, 0.92, 0.98, 1.00, 1.02, 1.18, 3.04 (br. dd, J = 17.37, 3.77 Hz, 1H) , 3.64 (s, 3H,
1
3
OCH
3
), 5.63 (d, J = 6 Hz, 1H), 5.65 (d, J = 6 Hz, 1H); C NMR (125 MHz, CDCl
), 18.10 (C25), 20.09 (C26), 21.29 (C15), 23.67 (C24), 24.16 (C23), 25.04 (C16),
5.10 (C27), 26.86 (C30), 28.09 (C29), 30.64 (C ), 32.05 (C20), 32.13 (C22), 32.92 (C ),
3
): 
1
2
6.72 (C
6
2
7
10

3
4
3.67 (C21), 36.70 (C
5.75 (C19), 46.05 (C17), 51.18 (OCH
12), 145.14 (C
469.3682, found 469.3673.
1
), 37.82 (C
4
), 38.63 (C10), 39.61 (C
8
), 40.63 (C18), 42.26 (C14),
3
), 51.64 (C ), 80.51 (C
5
3
), 115.78 (C11), 120.44
+
(
C
9
), 154.16 (C13), 178.13 (C28, CO); ESI-HRMS (m/z) [M + H] calcd for
C
31
H
49
O
3
4
.2.8 Methyl-3acetoxy-olean-11-oxo-12-ene-28-oate (9) [29]
Colorless amorphous solid (0.546 g, 50%); mp: 217-220 C; IR (KBr)  max:
0
-
1
1
2
0
2
960, 2925, 2850, 1720, 1460, 1360, 1240 cm ; H NMR (500 MHz, CDCl
3
): 0.87,
.91, 0.93, 1.13, 1.35, 1.55-1.75 (m 9H), 2.05 (m, 1H), 2.34 (s, 3H), 2.81-2.84 (m, 1H),
13
.98 (d, J = 3.77 Hz, 1H), 3.03 (d, J = 3.77 Hz, 1H), 3.64 (s, 1H), 5.56 (s, 1H); C NMR
): 16.24 (C24), 16.68 (C25), 17.33 (C ), 18.96 (C26), 21.29 (C16), 22.98
CO), 23.53 (C27), 23.59 (C ), 27.77 (C15), 28.09 (C23), 29.69 (C20),
0.68 (C22), 31.63 (C29), 32.86 (C ), 33.74 (C21), 37.17 (C10), 38.07 (C ), 38.81 (C ),
1.63 (C18), 43.50 (C14), 44.27 (C19), 45.07 (C ), 46.25 (C17), 51.87 (C31), 55.13 (C ),
(
(
125 MHz, CDCl
3
6
C
30), 23.45 (CH
3
2
3
4
6
7
4
1
8
5
1.72 (C
9
), 80.65 (C
3
), 127.89 (C12), 168.63 (C13), 177.49 (C28, & C32, CO), 200.15 (C11,
+
CO) ; ESI-HRMS (m/z) [M + H] calcd for C33
H
51
O 527.3736, found 527.3734.
5
4
.3 Anticancer activity
The anticancer activity of the compounds was determined using MTT assay.
4
Cells(1×10 ) per well were seeded in 100µl DMEM, supplemented with 10% FBS in
each well of 96-well micro culture plates and incubated for 24h at 37°C in a CO
2
incubator. Compounds, diluted to the desired concentrations in culture medium, were
added to the wells with respective vehicle control. After 48h of incubation, a volume of
1
0 µl MTT [3-(4, 5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] (5 mg/mL)
was added to each well and the plates were further incubated for 4h. The supernatant
11

from each well was carefully removed, formazon crystals were dissolved in 100µl of
DMSO and absorbance at 540 nm wavelength was recorded.
4
.3.1 Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of
the cells through the cell cycle phases. MCF-7, Breast cancer cells were incubated with
compounds 6 and 7 for 48h. Untreated and treated cells were harvested, washed with
PBS, fixed in ice-cold 70% ethanol and stained with propidium iodide (Sigma Aldrich).
Cell cycle was performed by flow cytometry (Becton Dickinson FACS Caliber) as earlier
described [30].
4
.3.2 Mitochondrial membrane potential
6
MCF-7 (1×10 cells/well) cells were cultured in six-well plates after treatment with
compounds 6 and 7 for 48h. Cells were collected by trypsinization after 48h of treatment
and washed with PBS followed by resuspending in JC-1 (5 µg/ml) and incubated at
0
3
7 C for 15 min. Cells were rinsed three times with medium and suspended in pre
warmed medium. The cells were then subjected to flow cytometric analysis on a flow
cytometer (Becton Dickinson) in the FL1, FL2 channel to detect mitochondrial potential
[
31].
.3.3. ROS generation
The production of ROS (reactive oxygen species) was measured by flow
4
cytometry using 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously
described [32]. In this study MCF-7 cells were treated with compound 6 and 7 for 48h.
0
After treatment, cells were incubated with DCF-DA at 37 C for 30 min and then
measured with the flow cytometer (FACS).
12

4
.3.4 Activation of Caspase 9
To determine the caspase-9 activity of compounds 6 and 7 for detection of
apoptosis in breast cancer cell line (MCF-7), the commercially available apoptosis
detection kit (Caspase 9 Assay kit, Sigma Aldrich, St Louis, USA) was used. MCF-7
cells were treated with compounds 6 and 7 for 48h. After 48h of drug treatment assay
was performed according to manufactures introduction and readings were taken at
excitation wavelength 400 nm and emission wavelength 505 nm.
Acknowledgements
We are thankful to CSIR, New Delhi for financial support under Network project
(
NWP 009). We are also thankful to Director, CSIR-IICT for support and
encouragement.
13

References
[
[
1]
2]
J. Liu, J. Ethnopharmacol. 49 (1995) 57.
Z. Ovesná, A. Vachalkova, K. Horvathova, D. Tothova, Neoplasma 51 (2004)
327-333.
[
[
[
[
3]
4]
5]
6]
J. Pollier, A. Goossens, Phytochem. 77 (2012) 10.
J. Liu, J.Ethnopharmacol.100 (2005) 92.
Biostar Pharmaceuticals, Patent -201410182757.7,18 (2014).
P. Dzubak, M.Hajduch, D. Vydra, A. Hustova, M. Kvasnica, D. Biedermann, L.
Markova, M. Urban, J. Sarek, Nat. Prod. Rep. 23 (2006) 394.
J. Chen, J. Liu, L. Zhang, G. Wu, W. Hua, X. Wu, H. Sun, Bioorg. Med.Chem.
Lett. 16 (2006) 2915.
[
[
7]
8]
A.E. Place, N. Suh, R. Risingsong, T. Honda, Y. Honda, G.W. Gribble, L.M.
Leesnitzer, J.B. Stimmel, T.M. Willson, E. Rosen, M.B. Sporn, Clin. Cancer Res. 9
(
2003) 2798.
[
[
[
9]
M.B. Sporn, K.T. Liby, M.M. Yore, L. Fu, J.M. Lopchuk, G.W. Gribble, J. Nat.
Prod. 74 (2011) 537.
10] B. Bednarczyk–Cwynar, L. Zaprutko, P. Ruszkowski, B. Hładoń, Org. Biomol.
Chem.10 (2012) 2201.
11] A. García-Granados, P.E. López, E. Melguizo, A. Parra, Y. Simeó, Tetrahedron
6
0 (2004) 1491.
[
[
12] M.B. Sporn, K.T. Liby, Pharmacol. Rev. 64 (2012) 972.
13] N. Suh, Y. Wang, T. Honda, G.W. Gribble, E. Dmitrovsky, W.F. Hickey, R.A.
Maue, A.E. Place, D.M. Porter, M.J. Spinella, C.R. Williams, G. Wu, A.J.
14

Dannenberg, K.C. Flanders, J.J. Letterio, D.J. Mangelsdrof, C.F. Nathan,
L. Nguyen, W.W. Porter, R.F. Ren, A. Roberts, N.S. Roche, K. Subbaramaiah,
M.B. Sporn, Cancer Res. 56 (1999) 336.
[
[
14] B. Siewert, J. Wiemann, A. Kowitsch, R. Csuk, Eur. J. Med. Chem. 72 (2014) 84.
15] U.V. Mallavadhani, B. Pattnaik, N. Suri, A.K. Saxena, Nat. Prod. Commun. 9
(
2014) 1687.
[
[
[
16] M. Botta, S. Armaroli, D. Castagnolo, G. Fontana, P. Pera, E. Bombardelli,
Bioorg. Med. Chem. Lett. 17 (2007) 1579.
17] U.V. Mallavadhani, N.R. Vanga, M.K., Jeengar, V.G.M. Naidu, Eur. J. Med.
Chem. 74 (2014) 398.
18] K.T. Chan, F.Y. Meng, Q.Li, C.Y. Ho, T.S. Lam, Y. To, W.H. Lee, M. Li, K.H. Chu,
M. Toh, Cancer Lett. 294 (2010) 118.
[
[
[
19] J.-K.Shen, H.-P. Du, M. Yang, Y.-G.Wang, J. Jin, Ann. Hematol. 88 (2009) 743.
20] M. Blank, Y. Shiloh, Cell Cycle 6 (2007) 686.
21] K. Gonda, H. Tsuchiya, T. Sakabe, Y. Akechi, R. Ikeda, R. Nishio, K.
Terabayashi, K. Ishii, Y. Matsumi, A.A. Ashla, H. Okamoto, K. Takubo, S.
Matsuoka, Y. Watanabe, Y. Hoshikawa, A. Kurimasa, G. Shiota, Biochem.
Biophys. Res. Commun. 370 (2008) 629.
[
[
22] Y. Sanchez, G.P. Simon, E. Calvino, E. de Blas, P. Aller,J. Pharmacol. Exp. 335
(
2010) 114.
23] K. Liu, D. Zhang, J. Chojnacki, Y. Du, H. Fu, S. Grant, S. Zhang, Org. Biomol.
Chem. 11 (2013) 4757.
15

[
24] P. Costantini, J.-M. Bruey, M. Castedo, D. Metivier, M. Loeffler, S.A. Susin, L.
Ravagnan, N. Zamzami, C. Garrido, G. Kroemer, Cell Death Differ. 9 (2002) 82.
25] U.V. Mallavadhani, A.K. Panda, Y.R. Rao, Pharm. Biol. 39 (2001) 20.
[
[
[
[
26] J.M.Narvaez-Mastache, C. Soto, G. Delgado, Biol. Pharm. Bull. 30 (2007) 1503.
27] H.M. G.Al-Hazmi, G.A. Miana, M.S.H.Deep, Phytochem. 26 (1987) 1091.
28] M.E.P. DeLampasona, C.A.N. Catalan, T.E. Gedris, W. Herz, Phytochem. 49
(
1998) 2061.
[
[
[
[
29] M. Szumilak, A. Szulawska-Mroczek, K. Koprowska, M. Stasiak, W. Lewgowd,
A. Stanczak, M. Czyz, Eur. J. Med. Chem. 45 (2010) 5744.
30] B. Chakravarti, R. Maurya, J.A. Siddiqui, H.K. Bid, S.M. Rajendran, P.P. Yadav,
R. Konwar, J. Ethnopharmacol. 142 (2012) 72.
31] G. Ding,F. Liu, T. Yang, Y. Jiang, H. Fu, Y. Zhao, Bioorg. Med. Chem.14 (2006)
3
766.
32] Y. Tampo, S. Kotamraju, C.R. Chitambar, S.V., Kalivendi, A. Keszler,
J. Joseph, B. Kalyanaraman, Circu. Res. 92 (2003) 56.
16

Captions
Scheme 1: Synthesis of oleanolic acid ring C modified frameworks.
Table 1: In vitro anticancer activity of compounds 1-9.
Figure 1: Cell cycle analysis of compounds 6 and 7 on MCF-7 cells.
Figure 2: Mitochondrial membrane potential of compound 6 and 7 in MCF-7 cells.
Figure 3: The effect of 6 and 7 on the ROS production in MCF-7 cells.
Figure 4: Effect of compounds 6 and 7 on caspase-9 activity.
17

Scheme 1.Reagents and conditions a. NBS-CCl
4 3 3 2
, rt, 8h b. NBS-aq.Dioxane-CaCO , rt 18h c. (CH CO) O-
Py, rt, 12h, 98% d. CH , Diethyl ether, 12h, 96%.
2 2
N
18

Table 1. IC50 values (in µM) of compounds in selected human cancer cell lines.
Compounds
MCF-7
> 100
A-549
> 100
Hela
DU-145
ACHN
> 100
1
> 100
53.49±2.04
2
4.6±0.20
> 100
2.10±0.65 30.96±0.75 33.05±0.36 30.10±0.98
> 100 > 100 > 100 > 100
15.30±2.83 13.41±0.29 12.38±2.19 10.63±0.35
3
4
9.42±1.47
5
15.05±1.33 17.00±2.69 7.04 ±0.49 28.31±1.73 27.71±1.26
31.29±0.04 48.88±1.62 36.49±0.052 51.45±1.02 55.80±2.83
6
7
2.96±0.15
> 100
19.60±1.80 6.30±0.13
> 100 > 100
6.77±2.57
> 100
5.85±0.32
> 100
8
9
16.68±0.45 44.44±0.22 32.86±0.61 24.06±1.61
0.90±0.37 2.17±0.54 5.05±0.48 1.83±0.75
> 100
Doxorubicin
2.74±0.36
Values represent mean + SD, n=3
19

Sample
SubG1%
0.76
G0/G1%
84.46
S%
G2/M%
8.59
Control (MCF-7)
4.56
2.61
4.19
2.10
6
7
7
(5 µM)
(2.5 µM)
(5 µM)
0.79
86.21
8.44
0.69
86.99
6.92
0.62
92.15
3.37
Figure 1: Cell cycle analysis of compounds 6 and 7 on MCF-7 cells; A: Untreated control cells
MCF-7), B: 6 (5 µM), C: 7 (2.5 µM) and D: 7 (5 µM).
(
20

Figure 2: Drops in mitochondrial membrane potential (∆Ψm) was assessed by JC-1 staining of
MCF-7 cells treated with compound 6 and 7 and samples were then subjected to flow cytometry
analysis on a FACScan (Becton Dickinson); A: Untreated control cells (MCF-7), B:6 (5 µM), C: 7
(2.5 µM) and D: 7 (5 µM).
21

Figure 3: The effect of 6 and 7 on the ROS production in human breast cancer cells (MCF-7);
A:Untreated control cells (MCF-7), B: 6 (5 µM), C: 7 (2.5 µM) and D: 7 (5 µM).
22

Figure 4. Effect of compounds 6 and 7 on caspase-9 activity.
23

Graphical Abstract
GRAPHICAL ABSTRACT
Synthesis of ring-C modified oleanolic acid derivatives and their cytotoxic evaluation
a
b
b
a*
Banita Pattnaik , V. Lakshma Nayak , Sistla Ramakrishna , Uppuluri Venkata Mallavadhani
IC50 : 2.10±0.65 (A-549); 4.6±0.20 (MCF-7)
IC50 : >100 (A-549; MCF-7; ACHN ; Hela)
IC50 : 2.96±0.15 (MCF-7); 5.85±0.32 (ACHN); 6.30±0.13 (Hela)

Synthesis of ring-C modified oleanolic acid derivatives and their
cytotoxic evaluation
Highlights


Oleanolic acid ring-C was chemically modified.
Dienes, , -unsaturated ketone, saturated and unsaturated lactones were
synthesized.



The synthesized compounds showed significant anti-cancer activity.
Compounds 2 and7 found to active in A-549 and MCF-7cells respectively.
Compound 7 arrested the cell cycles at G0/G1 phase by inducing apoptosis.
24