Design and synthesis of new bioisosteres of spirooxindoles (MI-63/219) as anti-breast cancer agents

Article abstract of DOI:10.1016/j.bmc.2014.12.037

We report herein the design and synthesis of bioisosteres of spirooxindole (MI-63/219), a small-molecule inhibitors of the MDM2-p53 interaction as anti-breast cancer agents. Compound 5b has been exhibiting significant anti-proliferative activity in nude mice bearing MCF-7 xenograft tumor. The compound 5b was found to act via modulation of MDM2 and p53 expression in breast cancer cells expressing wild type p53. Compound 5b stimulated p53 activation, caused modulation of downstream effectors p21, pRb, and cyclin D1 which regulate cell cycle. Thus, compound triggered G1-S phase cell cycle arrest, which was evident by flow cytometric analysis of treated breast cancer cells. Thus, compound 5b restores the p53 function, which triggers molecular events consistent with cell cycle arrest at G1/S phase.

Full text of DOI:10.1016/j.bmc.2014.12.037

Accepted Manuscript
Design and synthesis of new bioisosteres of Spirooxindoles (MI-63/219) as anti-
breast cancer agents
Atul Kumar, Garima Gupta, Ajay Kumar Bishnoi, Ruchi Saxena, Karan Singh
Saini, Rituraj Konwar, Sandeep Kumar, Anila Dwivedi
PII:
S0968-0896(14)00885-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.037
BMC 11969
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 September 2014
15 December 2014
16 December 2014
Please cite this article as: Kumar, A., Gupta, G., Bishnoi, A.K., Saxena, R., Saini, K.S., Konwar, R., Kumar, S.,
Dwivedi, A., Design and synthesis of new bioisosteres of Spirooxindoles (MI-63/219) as anti-breast cancer agents,
Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.037
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical abstract:
Design and synthesis of new bioisosteres of Spirooxindoles (MI-63/219) as anti-
breast cancer agents

Design and synthesis of new bioisosteres of
Spirooxindoles (MI-63/219) as anti-breast
cancer agents
Atul Kumar*,#,‡, Garima Gupta‡, Ajay Kumar Bishnoi‡#, Ruchi Saxena§,
Karan Singh Saini§,Rituraj Konwar§,Sandeep Kumar||,Anila Dwivedi *#§
‡
§
Medicinal and Process Chemistry Division, Endocrinology Division CSIR-Central Drug Research
Institute, (CSIR-CDRI), Sector 10, Jankipuram , Sitapur Road, Lucknow, 226 031, || Department of
Surgery, CSM Medical University, Lucknow, #Academy of Scientific & Innovative Research (AcSIR), New
Delhi, India
*Corresponding author. Tel.: +91 522-2612411; Fax: +91-522-26234051.
Email: dratulsax@gmail.com, atul_kumar@cdri.res.in
Abstract
We report herein the design and synthesis of bioisosteres of spirooxindole (MI-63/219) ,
a small-molecule inhibitors of the MDM2-p53 interaction as anti-breast cancer agents.
Compound 5b has been exhibiting significant anti-proliferative activity in nude mice
bearing MCF-7 xenograft tumor. The compound 5b was found to act via modulation of
MDM2 and p53 expression in breast cancer cells expressing wild type p53. Compound
5b stimulated p53 activation, caused modulation of downstream effectors p21, pRb, and
2

cyclinD1 which regulate cell cycle. Thus, compound triggered G1-S phase cell cycle
arrest, which was evident by flow cytometric analysis of treated breast cancer cells. Thus,
compound 5b restores the p53 function, which triggers molecular events consistent with
cell cycle arrest at G1/S phase.
Keywords: Spirooxindoles, MDM2- p53, Bioisosteres, Breast Cancer, Anticancer
Agents.
Introduction
Breast cancer is the most commonly diagnosed malignancy among women with more
than one million new cases diagnosed per year throughout the world.1,2 Despite advances
in the early detection of breast cancer and the advent of novel targeted therapies, breast
cancer still remains a significant public health problem due to the involvement of
multiple aberrant and redundant signaling pathways in the tumorigenesis and the
development of resistance to the existing therapeutic agents. Hence, there is still an
increasing need to develop safe and efficacious agents for breast cancer therapy. The
overexpression of MDM2 has been observed in a wider range of tumor types, including
brain (11%), breast (5-40%), soft tissue tumors (17%).3-8 One attractive pharmacological
approach to p53 reactivation is to use a small molecule to block the MDM2–p53
interaction.9 Several selective small-molecule inhibitors have been previously reported
that block the MDM2-p53 protein-protein interaction and activation of p53 as a potential
cancer therapeutic strategy.10 Recently, the sprirooxindole-containing compounds enticed
the academics and pharmaceutical industry because of their ubiquity in natural products
3

and medicinally important molecules (Figure 1).11 Sprirooxindole-containing compounds
are potent and specific inhibitors that block the MDM2-p53 protein-protein interaction.
Some sprirooxindole such as, MI-219 binds to MDM2 with low nanomolar affinity,
efficaciously blocks the MDM2-p53 protein-protein interaction in cells, reactivates p53
in cancer cells.12 However, these compounds are still in their pre-clinical stage and also
do not exhibit significant activity in breast cancer cell lines with mutated p53.
The key structural characteristic of MI-43 and MI-63 are oxindole core with spiro
pyrrolidine ring having Nitrogen at N3 position spiro[indoline-3,3'-pyrrolidin]-2-one
motifs.13 With a view to identify small molecule inhibitors of MDM2-p53, as possible
anti-breast cancer agents, we herein report the bioisosteres of MI-43 and MI-63 in which
oxindole core with spiro pyrrolidine ring having Nitrogen at N2 position “spiro[indoline-
3,2'-pyrrolidin]-2-one” as main pharmacophore. It is also interesting to point out here,
that all the high-profile selective estrogen modulators have 1, 2 diaryl system as well as
some MDM2 inhibitors such as nutlin-3, and AM-8553 also contains diaryl system with
N- heterocycleic ring as main pharmacophore.14-15 Based on these molecules, we have
designed a new scaffold of diarylsprio-oxyindoles. The diarylsprio-oxyindole has
spriopyrrolidine-oxyindole as a core nucleus with 1, 2- diaryl substitutions as present in
Nutlins, Am8553 and SERMs and spriopyrrolidine nucleus with Nitrogen at N2 position,
which makes the design scaffold of the bioisostere of MI -43/63. (Figure-2)
The present study focuses on study of anti-proliferative potential of novel series of Spiro-
oxindoles compounds in wild type p53 expressing breast cancer MCF-7 cells. The most
active compound was evaluated for in vitro efficacy in primary culture cells expressing
wild type p53, obtained from breast cancer patients and for in vivo efficacy in nude mice
4

model. Since spiro-oxindoles are known inhibitors of MDM2, so we have characterized
the effect of lead compound 5b on p53 activation and further downstream events i.e. cell
cycle regulation leading to growth inhibitory activity in breast cancer cells.
Results and discussion
Chemistry
The designed compounds were synthesized via multi component reaction of isatin,
benzyl amine and various , unsaturated ketones to generate diastereoselective
spiropyrrolidine-oxindole scaffold at room temperature in high diastereoselectivity.
All the , unsaturated ketones derivatives (chalcone) were synthesized by using
known methodology.16 Synthesis of alkyl amino substituted , unsaturated ketones
were achieved by reaction of , unsaturated ketones with chloro alkyl amine chain in
base. In order to develop one pot process for amino alkyl substituted spiropyrrolidine-
oxindole, we have incorporated alkyl amino side chain in , unsaturated ketones and
then utilized in multi component reaction with isatin, benzyl amine. (Scheme 1).
Scheme 1
Antiproliferative activity in vitro
The synthesized compounds were first screened for cytotoxic effect in breast
cancer cell lines MCF-7 and MDA-MB 231cells using MTT assay (summary of IC50
values (µM) in Table 1). Out of these, four compounds (5a, 5b, 5i, 5k) caused loss of
viability of breast cancer cells at concentrations that were comparable to Nutlin III.
Because specific cytotoxicity towards the cancer cells without affecting normal cell
growth is a key safety feature of cancer chemotherapy, assessments of cytotoxicity of all
active compounds were tested in human kidney epithelial cell line (HEK-293) and
5

monkey kidney epithelial cells (Vero). All the four compounds did not affect the growth
of normal cells significantly. The 5b exhibited 3.7 (MDA-MB231) and 6.5 (MCF-7) µM
IC50 values, which indicated that the 5b compound is more potent than Nutlin-III, OH-
Tamoxifen in MDA-MB231, MCF-7 cell lines respectively (Table 1). Since compound
5b displayed better efficacy compared to the other three compounds, it was further
evaluated for anti-proliferative activity in primary culture cells obtained from patient
tumor samples (Figure 3). Therefore, 5b was selected as the lead compound for detailed
studies on the mechanism in breast cancer cells and for in vivo efficacy in nude mice.
Figure 3.
Compound 5b modulates the expression of mdm2 and p53
Because the interaction between MDM2 and P53 is a primary mechanism for
inhibition of the p53 function in cancers retaining wild-type p53, targeting the MDM2-
P53 interaction by small molecules to reactivate p53 has emerged as a promising new
cancer therapeutic strategy. So, we analysed the expression of p53 mRNA (Figure 4).
Increased p53 expression led to increase in the expression of MDM2 both at RNA and
protein level (Figure 4) which is a result of an autoregulatory feedback loop that
maintains the levels of p53 andMDM2.
Figure 4.
Stimulation of p53 activity by compound 5b induced cell cycle arrest via p21
activation
Activation of p53 can halt cell cycle progression in the G1- S boundaries of cell
cycle through the up-regulation of the p21. Transition into the S-phase requires cyclin-
6

dependent kinases (CDK), such as CDK2, which phosphorylates and inactivates
Retinoblastoma protein (Rb), rendering E2F free and transcriptionally active, leading to
cell cycle progression. Here we report that stimulation of p53 leads to increased
expression of p53-dependent genes that cause cell cycle arrest. So further, we analyzed
the activity and expression of p53 dependent gene i.e. p21 which is involved in G1/S cell
phase arrest.
An increase in p21 promoter activity assessed by transient transfection of p21-reporter
construct in MCF-7 cells was observed in a concentration dependent manner on treatment
with the compound 5b (Figure 5(A)). Real time-quantitative PCR analysis indicated that
the compound significantly increased mRNA levels of p21 (p<0.001 at 5 and 7.5µ M
concentrations) in MCF-7 cells (Figure 5(B)).
Figure 5:
Similarly, the protein expression analyzed by immunoblotting was also found to increase
by approximately 2.5 folds at 7.5µM concentration (p<0.001) in both MCF-7 cells and
primary breast cancer cells (Figure 6). These results suggested that the compound might
cause growth arrest by p53 mediated p21 induction.
The cyclin dependent kinase inhibitor p21 is known to inhibit formation of cyclin D1, a
cell cycle regulatory protein required for transition from G1 to S phase. Thus, the
expression levels of cyclin D1 and activation status of its downstream effector namely,
Rb were studied using immunoblotting (Figure 6). p21 induction is likely to cause the
decrease in levels of cyclin D1 and can prevent the Rb phosphorylation.
Figure 6:
7

Since hypophosphorylated Rb sequesters and inactivates E2F preventing
transcription of E2F dependent genes required for G1/S cell cycle progression. Thus,
further, we analysed the G1/S phase arrest of breast cancer cells by flow cytometry of PI
stained cells. AT 7.5µM concentration of 5b approx 90% of MCF-7 and primary breast
cancer cells were in G1 phase as opposed to 60% of cells in the control group (p<0.01)
(Figure 7).
Figure 7.
G1/S phase arrest was further confirmed by studying the expression of various
cyclins required for cell cycle progression and are specific phase specific. Real time
analysis of mRNA levels indicated that the compound led to a decrease in the expression
of cyclin D1 and E1 which are required throughout the cycle and it also led to decrease in
the expression of cyclin A1 which is required for G1/S transition. However, the
expression of cyclin B1 required specifically for G2/M was not significantly altered,
suggesting G1/S phase arrest (Figure 8).
Figure 8.
In vivo studies
Spiro-oxindole derivative 5b suppressed the growth of tumor xenograft in nude
mice (In vivo studies).
To examine the effect of compound in vivo, xenograft tumors were induced in
athymic nude mice. Figure 9(A) shows that the compound significantly reduced tumor
8

volume in xenograft mice model. It was found that compound at dose 20 mg/kg body
weight/day significantly decreased tumor volume and average tumor size in this group
was 66% lower after 14 days of treatment as compared with that of vehicle treated group
(Figure 9(B)). No significant change was observed in body weight in the vehicle and
treated group (Figure 9(C).
Figure 9.
Histomorphology did not show any marked change in liver, lungs, spleen, uterus
and kidney of the compound treated mice as compared to vehicle treated group
suggesting that the compound did not have any toxic effect at the effective dose (Figure
10).
Figure 10.
5b modulated the expression of p53- mediated cell cycle proteins.
The expression of p53 mediated cell cycle proteins was observed in xenograft
tissue and the compound increased the expression of p21 (Figure 11). This led to
significant decrease in the expression of cyclin D1 and phosphorylated Rb proteins
resulting in G1/S phase arrest. These findings correlated well with the findings of in vitro
experiments.
Figure 11.
The diarylspiropyrrolidine-oxindole derivative exhibited promising anticancer
activity against human breast cancer cells. The study investigates the anti-proliferative
activity of 5b, a new, potent and specific spiro-oxindole in breast cancer cell lines and
9

selectivity in normal cells and also the in vivo efficacy as antitumor agent. The study on
mechanism of action revealed that 5b mediated anti-proliferative and anti-tumor activity
via activation of p53 function. Both in vitro and in vivo studies provide evidence that the
activation of P53 function caused by compound leads to increased expression and activity
of p21 that induces G1/S phase arrest and thus inhibiting the growth of breast cancer
cells.
The results demonstrate that 5b represents a potent and specific small-molecule inhibitor
of the MDM2 and a promising lead compound for further optimization for the
development of a new class of anti-cancer therapy for the treatment of breast and other
types of human cancer.
Summary and conclusions
The Diaryl spiroxindole derivative exhibited promising anticancer activity against
human breast cancer cells. The study investigates the anti-proliferative activity of 5b, a
new, potent and specific spiro-oxindole in breast cancer cell lines and selectivity in
normal cells and also the invivo efficacy as antitumor agent. The study on mechanism of
action revealed that 5b mediated anti-proliferative and anti-tumor activity via activation
of p53 function. Both in vitro and in vivo studies provide evidence that the activation of
p53 caused by compound leads to increased expression and activity of p21 that induces
G1/S phase arrest and thus inhibiting the growth of breast cancer cells.
The results demonstrate that 5b represents a potent and specific small-molecule inhibitor
of the MDM2 and a promising lead compound for further optimization for the
10

development of a new class of anti cancer therapy for the treatment of breast and other
types of human cancer.
Experimental Section
1. General procedure for the synthesis of amino alkyl substituted chalcone.
A mixture of 1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one and chloro alkyl amine
hydrochloride in presence of anhydrous K2CO3 in dry acetone (20.0 mL) were refluxed
for 8-10 h. After completion of reaction as evidenced by TLC, solvent was removed in
vacuo. The residue obtained was suspended in ethyl acetate and extracted with water.
Aqueous layer was further extracted with ethyl acetate. Combined organic layers were
dried with Na2SO4, filtered and solvent was removed in vacuo to give different amino
alkyl substituted chalcones.
2. General procedure for the synthesis of substituted 3'-benzoyl-4',5'-
diphenylspiro[indoline- 3,2'-pyrrolidin]-2-one.
A mixture of isatin (1.0 mmol), benzyl amine (1.1 mmol) and chalcone (1.0 mmol)
were stirred at room temperature for 18-22 h in ethanol (10.0 mL). After completion of
reaction as evidenced by TLC solvent was removed in vacuo to yield a crude residue,
which upon purification via silica gel column chromatography using MeOH/CHCl3
gave pure products (4a-4c).
General procedure for the synthesis of amino alkyl chain derivatives of 3'-
benzoyl-4',5'- diphenylspiro[indoline-3,2'-pyrrolidin]-2-one.
A mixture of isatin (1.0 mmol), benzyl amine (1.1 mmol) and amino alkyl substituted
chalcone (1.0 mmol) were stirred at room temperature for 20-26 h in ethanol (10.0
11

mL). After completion of reaction as evidenced by TLC solvent was removed in vacuo
to yield a crude residue, which upon purification via silica gel column chromatography
using MeOH/CHCl3 gave pure products (5a-5l).
2.1. 3'-benzoyl-4'-(4-methoxyphenyl)-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one.
(4a)
1
Physical state: white solid. ESI MS (m/z)= 475 (M+H); HNMR (300 MHz, CDCl3):
H 7.72-7.69 (m, 1H, ArH), 7.43-7.23 (m, 12H, ArH), 6.97 6.94 (m, 2H, ArH), 6.79-
6.75 (m, 2H, ArH), 6.48 (d, J= 7.4 Hz, 1H, ArH), 5.05-4.98 (m, 1H, CH), 4.70 (d, J =
13
10.7Hz, 1H, CH), 4.17-4.06 (m, 1H, CH), 3.72 (s, 3H, OCH3); C NMR (75 MHz;
CDCl + DMSO-d6 ) C 197.5, 182.4, 158.5, 141.6, 141.2, 137.3, 133.0, 131.3, 130.2,
3
129.5, 129.0, 128.6, 128.4, 128.2, 127.7, 127.4, 126.0, 122.3, 114.0, 113.5, 109.9,
68.4, 67.9, 62.6, 55.2, 54.9. IR 3033, 2362, 1704, 1682, 757 Analysis calculated for
C31H26N2O3 : C 78.46, H 5.52, N 5.90; Found: C 78.50, H 5.55, N 5.85.
2.2. 3'-benzoyl-4'-(4-hydroxyphenyl)-5'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one.
(4b)
1
Physical state: white solid. ESI MS (m/z) = 461 (M+H); HNMR (300 MHz, DMSO-d 6
): 7.33-7.21 (m, 12H, ArH), 6.99-6.86 (m, 3H, ArH), 6.62-6.59 (m, 2H, ArH), 6.53-
H
6.50 (m, 1H, ArH), 5.00 (d, J = 10.1 Hz, 1H, CH), 4.54 (d, J = 10.5 Hz, 1H, CH), 4.08-
4.04 (m, 1H, CH); 13C NMR (75 MHz; DMSO-d 6 ) C 195.0, 182.4, 162.4, 142.0, 141.9,
140.1, 130.8, 130.2, 128.9, 128.8, 128.6, 128.4, 127.7, 127.6, 127.1, 126.5, 121.9, 115.4,
109.4, 68.2, 67.4, 61.8, 55.9. IR 3431, 3021, 2362, 1634, 763. Analysis calculated for
C30H24N2O3 : C 78.24, H 5.25, N 6.08; Found: C 78.28, H 5.30, N 6.10.
2.3. 3'-(4-nitrobenzoyl)-4',5'-diphenylspiro[indoline-3,2'-pyrrolidin]-2-one. (4c)
12

Physical state: yellow solid. ESI MS (m/z) = 490 (M+H); 1HNMR (300 MHz, CDCl3): δH
8.11-8.08 (m, 2H, ArH), 7.61 (d, J = 8.6 Hz, 2H, ArH), 7.42-7.18 (m, 11H, ArH), 6.99-
6.84 (m, 2H, ArH), 6.45 (d, J = 7.5 Hz, 1H, ArH), 5.04-4.99 (m, 1H, CH), 4.66 (d, J =
10.5 Hz, 1H, CH), 4.20-4.13 (m, 1H, CH); 13C NMR (75 MHz; DMSO-d6) δC 197.3,
182.0, 147.8, 146.9, 141.8, 140.9, 136.7, 133.7, 130.2, 130.0, 129.3, 128.8, 128.7, 128.1,
127.6, 127.5, 126.2, 124.0, 122.1, 109.6, 68.1, 67.4, 62.3, 55.5. IR 3021, 2359, 1630,
768. Analysis calculated for C H23N3O4 : C 73.61, H 4.74, N 8.58; Found: C 73.65, H
30
4.80, N 8.52.
2.4. 4',5'-diphenyl-3'-(4-(2-(piperidin-1-yl)ethoxy)benzoyl)spiro[indoline-3,2'-
pyrrolidin]-2-one. (5a)
1
Physical state: oily. ESI MS (m/z) = 572 (M+H); HNMR (300 MHz, CDCl3): δH 7.46-
738 (m, 7H, ArH), 7.28-7.22 (m, 4H, ArH), 7.18-7.10 (m, 2H, ArH), 7.06-6.98 (m, 2H,
ArH), 6.64 (d, J = 8.6 Hz, 2H, ArH), 6.52-6.49 (m, 1H, ArH), 5.12 (d, J = 10.7 Hz, 1H,
CH), 4.79 (d, J = 8.4 Hz, 1H, CH), 4.31-4.24 (m, 1H, CH), 4.03 (t, J = 5.4 Hz, 2H, CH2),
2.73-2.69 (m, 2H, CH2), 2.49- 2.48 (m, 4H, 2CH2), 1.61-1.58 (m, 4H, 2CH2), 1.46-1.44
13
(m, 2H, CH2); C NMR (75 MHz; CDCl3) δC 194.8, 181.8, 162.1, 141.3, 141.2, 139.4,
130.0, 129.7, 129.4, 128.3, 128.1, 128.0, 127.8, 127.1, 126.9, 126.5, 125.8, 121.3, 113.7,
108.9, 67.7, 67.1, 65.8, 61.4, 57.0, 54.3, 25.4, 23.7. IR 2933, 2362, 1706, 1598, 755.
Analysis calculated for C37H37N3O3 : C 77.73, H 6.52, N 7.35; Found: C 77.70, H 6.55, N
.7.37.
2.5. 5-chloro-4',5'-diphenyl-3'-(4-(2-(piperidin-1-yl)ethoxy)benzoyl)spiro[indoline-
3,2'- pyrrolidin]-2-one. (5b)
13

Physical state: oily. ESI MS (m/z) = 606 (M+H); 1HNMR (300 MHz, CD3 OD): δH 7.55-
7.49 (m, 3H, ArH), 7.37-7.34 (m, 5H, ArH), 7.28-7.21 (m, 6H, ArH), 7.04- 7.01 (m, 1H,
ArH), 6.89 (d, J = 8.6 Hz, 1H, ArH), 6.52 (d, J = 8.2 Hz, 1H, ArH), 5.10 (d, J = 10.5 Hz,
1H, CH), 4.75 (d, J = 10.7 Hz, 1H, CH), 4.35-4.34 (m, 1H, CH), 4.12-4.10 (m, 2H, CH2),
13
2.16-2.15 (m, 2H, CH2), 2.02-2.00 (m, 4H, 2CH2), 1.27-1.22 (m, 6H, 3CH2); C NMR
(75 MHz; CD3OD) δC 197.2, 183.9, 166.4, 163.0, 141.6, 141.2, 140.2, 133.1, 132.2,
131.2, 130.0, 129.6, 129.5, 129.3, 129.1, 128.7, 128.6, 128.4, 128.1, 127.6, 115.3, 111.8,
69.8, 69.2, 63.4, 61.5, 56.9, 54.9, 24.0, 22.5. IR 3021, 2359, 1756, 1629, 766. Analysis
calculated for C37H36ClN3O3 : C 73.31, H 5.99, N 6.93; Found: C 73.40, H 5.92, N 6.95.
2.6. 5-nitro-4',5'-diphenyl-3'-(4-(2-(piperidin-1-yl)ethoxy)benzoyl)spiro[indoline-3,2'-
pyrrolidin]-2-one. (5c)
1
Physical state: oily. ESI MS (m/z) = 617 (M+H); HNMR (300 MHz, CDCl3): 7.96-
H
7.92 (m, 1H, ArH), 7.44-7.36 (m, 7H, ArH), 7.27 7.20 (m, 6H, ArH), 6.59-6.56 (m, 3H,
ArH), 5.12 (d, J = 10.3 Hz, 1H, CH), 4.78 (d, J = 10.7 Hz, 1H, CH), 4.30-4.23 (m, 1H,
CH), 3.97-3.96 (m, 2H, CH2), 2.68-2.67 (m, 2H, CH2), 2.47-2.46 (m, 4H, 2CH2), 1.56-
1.55 (m, 4H, 2CH2), 1.44-1.43 (m, 2H, CH2); 13C NMR (75 MHz; CDCl3) C 194.6, 182.6,
162.7, 146.1, 143.6, 139.9, 138.2, 131.5, 130.1, 130.0, 128.6, 128.4, 127.8, 127.1, 125.9,
122.4, 114.1, 109.2, 68.2, 68.1, 65.8, 62.0, 57.4, 55.2, 54.8, 25.5, 23.9. IR 3021, 2359,
1630, 768. Analysis calculated for C37H36N4O5 : C 72.06, H 5.88, N 9.08; Found: C 72.11,
H 5.90, N 9.12.
2.7. 3'-(4-(2-morpholinoethoxy)benzoyl)-4',5'-diphenylspiro[indoline-3,2'-
pyrrolidin]-2-one. (5d)
14

1
Physical state: oily. ESI MS (m/z) = 574 (M+H); HNMR (300 MHz, CDCl3): δH 7.66-
7.63 (m, 1H, ArH), 7.38-7.29 (m, 5H, ArH), 7.19-7.13 (m, 5H, ArH), 6.96-6.92 (m, 3H,
ArH), 6.74 6.71 (m, 1H, ArH), 6.55 (d, J = 8.6 Hz, 2H, ArH), 6.46-6.43 (m, 1H, ArH),
5.03 (d, J = 10.6 Hz, 1H, CH), 4.70 (d, J = 10.6 Hz, 1H, CH), 4.23-4.16 (m, 1H, CH),
3.93-3.90 (m, 2H, CH2), 3.63 3.60 (m, 4H, 2CH2), 2.67-2.63 (m, 2H, CH2), 2.49-2.48 (m,
4H, 2CH2); 13C NMR (75 MHz; CDCl3) δC 195.2, 182.4, 162.2, 140.4, 140.0, 138.9,
130.6, 130.5, 129.9, 129.0, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.6, 127.1, 126.9,
126.2, 123.0, 113.9, 109.5, 68.7, 66.7, 65.6, 62.1, 57.2, 55.5, 54.0. IR 3444, 2362, 1713,
1669, 769. Analysis calculated for C36H35N3O4 : C 75.37, H 6.15, N 7.32; Found: C
75.35, H 6.25, N 7.28.
2.8. 5-chloro-3'-(4-(2-morpholinoethoxy)benzoyl)-4',5'-diphenylspiro[indoline-3,2'-
pyrrolidin]- 2-one. (5e)
Physical state: oily. ESI MS (m/z) = 608 (M+H); 1HNMR (200 MHz, CDCl3): 7.50-
H
7.47 (m, 3H, ArH), 7.41-7.37 (m, 4H, ArH), 7.28-7.25 (m, 5H, ArH), 7.10-7.09 (m, 1H,
ArH), 7.04-7.00 (m, 1H, ArH), 6.71 (d, J = 8.6 Hz, 2H, ArH), 6.46 (d, J = 8.4 Hz, 1H,
ArH ), 5.13 (d, J = 10.6 Hz, 1H, CH), 4.76-4.73 (m, 1H, CH), 4.26-4.23 (m, 1H, CH),
4.08-4.04 (m, 2H, CH2), 3.74-3.71 (m, 4H, 2CH2), 2.78-2.75 (m, 2H, CH2), 2.57-2.54 (m,
13
4H, 2CH2); C NMR (75 MHz; DMSO-d6) 194.9, 181.5, 162.3, 141.1, 140.3, 139.2,
C
132.5, 129.5, 129.3, 128.4, 128.2, 128.0, 127.7, 127.5, 127.1, 126.7, 126.0, 125.4, 114.2,
110.4, 67.6, 66.8, 66.1, 65.3, 61.4, 56.6, 53.5. IR 3021, 2362, 1604, 766. Analysis
calculated for C36H34ClN3O4 : C 71.10, H 5.64, N 6.91; Found: C 71.15, H 5.70, N 6.94.
2.9. 3'-(4-(2-morpholinoethoxy)benzoyl)-5-nitro-4',5'-diphenylspiro[indoline-3,2'-
pyrrolidin] 2- one. (5f)
15

Physical state: oily. ESI MS (m/z) = 619 (M+H; 1HNMR (300 MHz, CDCl3): δH 7.47 (d,
J = 8.4 Hz, 2H, ArH), 7.40-7.37 (m, 5H, ArH), 7.32-7.14 (m, 7H, ArH), 6.68-6.64 (m,
3H, ArH), 5.14 (d, J = 10.3 Hz, 1H, CH), 4.79 (d, J = 10.7 Hz, 1H, CH), 4.32-4.25 (m,
1H, CH), 4.05-4.02 (m, 2H, CH2), 3.72-3.67 (m, 4H, 2CH2), 2.77-2.73 (m, 2H, CH2),
2.56-2.53 (m, 4H, 2CH2); 13C NMR (75 MHz; CDCl3) δC 194.6, 182.5, 162.6, 145.9,
143.7, 139.8, 138.2, 131.2, 130.1, 130.0, 128.6, 128.4, 128.3, 127.9, 127.1, 125.9, 122.5,
117.3, 114.1, 109.2, 68.0, 66.9, 66.6, 65.7, 61.9, 57.2, 55.3, 53.9. IR 3021, 2361, 1643,
764. Analysis calculated for C36H34N4O6: C 69.89, H 5.54, N 9.06; Found: C 69.90, H
5.52, N 9.10.
2.10.
1-ethyl-3'-(4-(2-morpholinoethoxy)benzoyl)-4',5'-diphenylspiro[indoline-
3,2'-pyrrolidin] 2- one. (5g)
1
Physical state: oily. ESI MS (m/z) = 602 (M+H); HNMR (300 MHz, CDCl3): 7.43-
H
7.37 (m, 7H, ArH), 7.28-7.22 (m, 6H, ArH), 7.18-7.11 (m, 1H, ArH), 7.08-7.03 (m, 1H,
ArH), 6.64 (d, J = 8.6 Hz, 2H, ArH), 6.51 (d, J = 8.4 Hz, 1H, ArH), 5.14 (d, J = 10.5 Hz,
1H, CH), 4.78 (d, J = 10.8 Hz, 1H, CH), 4.31-4.24 (m, 1H, CH), 4.06-4.03 (m, 2H, CH2),
3.74-3.71 (m, 4H, 2CH2), 3.67-3.60 (m, 2H, CH2), 2.78-2.74 (m, 2H, CH2), 2.56-2.53 (m,
4H, 2CH2), 1.18 (t, J = 7.2 Hz, 3H, CH3);13C NMR (75 MHz; CDCl3) C 195.2, 179.8,
162.2, 141.8, 140.5, 139.0, 130.5, 129.9, 129.7, 129.0, 128.5, 128.4, 128.2, 127.5, 127.1,
126.8, 126.0, 122.9, 113.8, 107.7, 68.3, 68.2, 66.8, 65.9, 62.4, 57.3, 55.4, 54.0, 34.8, 12.5.
IR 2934, 2362, 1703, 1673, 754. Analysis calculated for C38H39N3O4: C 75.85, H 6.53, N
6.98; Found: C 75.90, H 6.58, N 6.95.
2.11.
4',5'-diphenyl-3'-(4-(2-(pyrrolidin-1-yl)ethoxy)benzoyl)spiro[indoline-3,2'
pyrrolidin]-2- one. (5h)
16

1
Physical state: oily. ESI MS (m/z) = 558 (M+H); HNMR (300 MHz, CDCl3): δH 7.45-
7.28 (m, 8H, ArH), 7.26-7.21 (m, 6H, ArH), 7.00-6.97 (m, 1H, ArH), 6.64 (d, J = 8.6 Hz,
2H, ArH), 6.49-6.47 (m, 1H, ArH), 5.10 (d, J = 10.6 Hz, 1H, CH), 4.78-4.75 (m, 1H,
CH), 4.30-4.26 (m, 1H, CH), 4.04-4.03 (m, 2H, CH2), 2.89-2.88 (m, 2H, CH2), 2.65-2.64
13
(m, 4H, 2CH2), 1.81-1.80 (m, 4H, 2CH2); C NMR (75 MHz; CDCl3) δC 195.3, 182.4,
162.3, 141.5, 140.5, 140.3, 139.0, 130.4, 130.1, 129.9, 129.0, 128.5, 128.4, 128.2, 128.0,
127.8, 127.6, 127.1, 126.8, 126.1, 122.8, 113.8, 109.5, 68.7, 68.2, 66.7, 62.2, 55.5, 54.5,
23.4. IR 3021, 2358, 1714, 1620, 766. Analysis calculated for C36H35N3O3 : C 77.53, H
6.33, N 7.53; Found: C 77.60, H 6.35, N 7.60.
2.12.
3'-(4-(3-(dimethylamino)propoxy)benzoyl)-4',5'-diphenylspiro[indoline-
3,2'-pyrrolidin]-2- one. (5i)
1
Physical state: oily. ESI MS (m/z) = 546 (M+H); HNMR (300 MHz, CDCl3): 7.33-
H
7.19 (m, 7H, ArH), 7.15-7.10 (m, 6H, ArH), 6.98-6.94 (m, 2H, ArH), 6.62 (d, J = 8.6 Hz,
2H, ArH), 6.49-6.47 (m, 1H, ArH), 5.11 (d, J = 10.5 Hz, 1H, CH), 4.78-4.75 (m, 1H,
CH), 4.29-4.27 (m, 1H, CH), 3.89-3.84 (m, 2H, CH2), 2.41-2.36 (m, 2H, CH2), 2.22 (s,
13
6H, 2CH3), 1.87-1.84 (m, 2H, CH2); C NMR (75 MHz; CDCl3) C 195.4, 182.6, 169.9,
162.6, 140.5, 140.4, 139.0, 133.3, 131.9, 129.8, 129.0, 128.6, 128.5, 128.3, 127.9, 127.4,
127.1, 126.8, 126.0, 122.8, 113.8, 109.6, 68.8, 68.2, 66.1, 62.2, 55.9, 55.5, 45.3, 26.9. IR
3021, 2362, 1629, 769. Analysis calculated for C35H35N3O3: C 77.04, H 6.47, N 7.70;
Found: C 77.10, H 6.45, N 7.72.
2.13.
3'-(4-(2-(dimethylamino)ethoxy)benzoyl)-5-nitro-4',5'
diphenylspiro[indoline-3,2'-pyrrolidin]-2-one. (5j)
1
Physical state: oily. ESI MS (m/z) = 577 (M+H); HNMR (300 MHz, CDCl3): 7.38-
H
7.35 (m, 7H, ArH), 7.27-7.23 (m, 7H, ArH), 6.63-6.60 (m, 3H, ArH), 5.12 (d, J = 10.4
17

Hz, 1H, CH), 4.78 (d, J = 10.7 Hz, 1H, CH), 4.30-4.23 (m, 1H, CH), 3.98-3.97 (m, 2H,
CH 2 ), 2.70-2.69 (m, 2H, CH2), 2.30 (s, 6H, 2CH3); 13C NMR (75 MHz; CDCl3) C 194.6,
182.5, 162.6, 146.1, 143.6, 139.9, 138.2, 132.2, 131.3, 130.4, 130.1, 130.0, 128.9, 128.6,
128.4, 127.9, 127.1, 125.9, 122.3, 117.0, 114.1, 109.3, 68.1, 65.8, 62.1, 57.7, 55.2, 45.5,
43.6. IR 3021, 2360, 1601, 1522, 762. Analysis calculated for C34H32N4O5 : C 70.82, H
5.59, N 9.72; Found: C 70.86, H 5.58, N 9.75.
2.14.
3'-(4-(2-(diethylamino)ethoxy)benzoyl)-4',5'-diphenylspiro[indoline-3,2'-
pyrrolidin]-2 one.(5k)
1
Physical state: oily. ESI MS (m/z) = 560 (M+H); HNMR (300 MHz, CDCl3): 7.46-
H
7.40 (m, 7H, ArH), 7.28-7.22 (m, 6H, ArH), 7.03-7.00 (m, 2H, ArH), 6.69 (d, J = 8.6 Hz,
2H, ArH), 6.52- 6.49 (m, 1H, ArH), 5.13 (d, J = 10.6 Hz, 1H, CH), 4.78 (d, J = 10.5 Hz,
1H, CH), 4.32-4.24 (m, 1H, CH), 4.02-4.00 (m, 2H, CH2), 2.88-2.86 (m, 2H, CH2), 2.69-
13
2.62 (m, 4H, 2CH2), 1.13-1.05 (m, 6H, 2CH3); C NMR (75 MHz; CDCl3)
195.7,
C
182.9, 162.9, 141.0, 140.7, 139.5, 131.4, 130.9, 130.6, 130.3, 129.4, 128.8, 128.4, 128.0,
127.5, 127.2, 126.5, 124.4, 123.2, 114.6, 109.4, 68.6, 66.8, 62.6, 56.0, 54.3, 51.8, 48.1,
11.8. IR 3021, 2359, 1736, 1607, 770. Analysis calculated for C36H37N3O3 : C 77.25, H
6.66, N 7.51; Found: C 77.28, H 6.70, N 7.55.
2.15.
3'-(4-(1-(dimethylamino)propan-2-yloxy)benzoyl)-4',5'-
diphenylspiro[indoline-3,2'- pyrrolidin]-2-one. (5l)
1
Physical state: oily. ESI MS (m/z) = 546 (M+H); HNMR (300 MHz, CDCl3): δH 8.06-
8.00 (m, 3H, ArH), 7.84-7.83 (m, 1H, ArH), 7.77-7.76 (m, 1H, ArH), 7.66-7.58 (m, 3H,
ArH), 7.42-7.39 (m, 5H, ArH), 7.28-7.26 (m, 1H, ArH), 7.02-6.97 (m, 4H, ArH), 4.69-
4.61 (m, 1H, CH), 4.11-4.08 (m, 1H, CH), 3.96-3.93 (m, 1H, CH), 3.05-2.96 (m, 1H,
18

13
CH), 2.70-2.64 (m, 2H, CH2), 2.37 (s, 6H, 2CH3), 1.36 (d, J = 8.4 Hz, 3H, CH3); C
NMR (75 MHz; CDCl3) δC 188.5, 162.7, 161.8, 143.8, 135.0, 132.9, 131.2, 131.1, 130.9,
130.8, , 130.6, 130.2, 129.6, 129.5, 128.9, 128.3, 128.2, 127.8, 127.7, 121.8, 115.3, 114.2,
72.3, 69.9, 64.4, 58.2, 46.0, 45.9, 41.3, 18.2. Analysis calculated for C35H35N3O3 : C
77.04, H 6.47, N 7.70; Found: C 77.10, H 6.50, N 7.65.
Acknowledgments
Authors .G.G., A.K.B. and R.S. are thankful to CSIR-UGC New Delhi for the award of
senior research fellowship. Authors also acknowledge to SAIF-CDRI and BHU for
providing the spectral and analytical data. Authors thank Mr. A. L. Vishwakarma, SAIF
facility, CDRI for help in flowcytometric analysis. The authors are grateful to Director,
INMAS and Dr. B. G. Roy, INMAS (DRDO) for providing facility to perform nude mice
work. Financial support was provided by Ministry of Health and Family Welfare, Govt of
India and CSIR Net Work Project BSC 0108/0104.
Supporting information
1H NMR and 13C NMR spectral data of compounds is available free of charge via the
Internet at http:// pubs.acs.org.
19

References and notes:
1) DeSantis, C. E.; Lin, C. C.; Mariotto, A. B. Siegel, R. L.; Stein, K. D.; Kramer, J.
L.; Alteri, R.; Robbins, A. S.; Jemal, A. Cancer treatment and
survivorship statistics, 2014, 64, 252-71.
2) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer, 2002, 2, 48–58. (b)
Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M.
Nat. Rev. Drug Discovery 2006, 5, 219–234.
3) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. J. Clin.,
2011, 61, 69−90. (b) Siegel, R.; Ma, J. M.; Zou, Z. H.; Jemal, J. Clin., 2014, 64,
9−29.
4) (a) Gottesman, M. M.; Fojo, T.; Bates, S. E. Nat. Rev. Cancer, 2002, 2, 48–58. (b)
Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M.
Nat. Rev. Drug Discovery 2006, 5, 219–234.
5) (a) Wade, M.; Wang, Y. V.; Wahl, G. M. Trends Cell Biol. 2010, 20, 299-309. (b)
Bond, G. L.; Hu, W.; Levine, A. J. Curr. Cancer Drug Targets, 2005, 5, 3-8. (c)
Vousden, K. H.; Lane, D. P. Nature Rev. Mol. Cell Biol. 2007, 8, 275-283. (d)
Hainaut, P.; Hollstein, M. Adv. Cancer Res. 2000, 77, 81-137.
6) (a) Fridman, J. S.; Lowe, S. W. Oncogene, 2003, 22, 9030–9040. (b) Vousden, K.
H.; Lu, X. Nat Rev Cancer, 2002, 2, 594-604. (c) Vogelstein, B.; Lane, D.;
Levine, A. J. Nature, 2000, 408, 307–310.
20

7) (a) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A.
J.; et al. Science, 1996, 274, 948–953. (b) Momand, J.; Zambetti, G. P.; Olson, D.
C.; George, D.; Levine, A. J. Cell, 1992, 69, 1237–1245.
8) Harris, S. L.; Levine, A. J. Oncogene, 2005, 24, 2899–2908.
9) Shangary, S.; Ding, K.; Qiu, S.; Nikolovska-Coleska, Z.; Bauer, J. A.; Liu, M.;
Wang, G.; Lu, Y.; McEachern, D.; Bernard, D.; Bradford, C. R.; Carey, T. E.;
Wang, S. Mol Cancer Ther., 2008, 7, 1533–1542.
10) (a) Ramos, Y. F.; Stad, R.; Attema, J.; Peltenburg, L. T.; Jochemsen, A. G.
Cancer Res, 2001, 61,1839–42. (b) Momand, J.; Jung, D.; Wilczynski, S.; Niland,
J. Nucleic Acids Res, 1998, 26, 3453–9. (c) Bartel, F.; Meye, A.; Wurl, P.;
Kappler, M.; Bache, M.; Lautenschlager, C. et al. Intl J Cancer, 2001, 95, 168–
175.
11) (a) Wu, X.; Bayle, J. H.; Olson, D.; Levine, A. J. Genes Dev. 1993, 7, 1126–
1132. (b) Chene, P. Nat. Rev Cancer. 2003, 3, 102–109.
12) (a) Ding, Q.; Zhang, Z.; Liu, J.; Jiang, N.; Zhang, J.; Ross, T. M.; Chu, X. J.;
Bartkovitz, D.; Podlaski, F.; Janson, C.; Graves, B. J. Med. Chem. 2013, 56,
5979-5983. (b) Yu, S.; Qin, D.; Shangary, S.; Chen, J.; Wang, G.; Kang, S.; Yang,
D.; Wang, S. J. Med. Chem. 2009, 52, 7970–7973. (c) Gonzalez, A. Z. et al. J.
Med. Chem. 2014, 57, 2472-2488. (d) Rew, Y.; Sun, D. J. Med. Chem. 2014, 57,
6332-6341. (e) Zhuang, C.; Miao, Z.; Zhu, L.; Dong, G.; Guo, Z.; Wang, S.;
Zhang, Y.; Wu, Y.; Yao, J.; Sheng, C.; Zhang, W. J. Med. Chem. 2012, 55, 9630-
9642. (f) Zhao, Y.; Yu, S.; Sun, W.; Liu, L.; Lu, J.; McEachern, D.; Shargary, S.;
21

Bernard, D.; Li, X.; Zhao, T.; Zou, P.; Sun, D.; Wang, S. J. Med. Chem. 2013, 56,
5553-5561. (g) Sun, D. et al. J. Med. Chem. 2014, 57, 1454-1472.
13) (a) Lo, M. M. C.; Neumann, C. S.; Nagayama, S.; Perlstein, E. O.; Schreiber, S.
L. J. Am. Chem. Soc. 2004, 126, 16077-16086.(b) Antonchick, A. P.; Gerding-
Reimers, C.; Catarinella, M.; Schu¨ rmann, M.; Preut, H.; Ziegler, S.; Rauh, D.;
Waldmann, H. Nat. Chem. 2010, 2, 735-740. (c) a) Ashimori, A.; Bachand, B.;
Overmann, L.; Poon, D. J. J. Am. Chem. Soc. 1998, 120, 6477. (d) Edmondson,
S.; Danishefsky, S. J.; Sepp-Lorenzino, L.; Rosen, N. J. Am. Chem. Soc. 1999,
121, 2147-2155.
14) Shangary, S.; Qin, D.; Wang, D. PNAS, 2008, 105, 3933–3938. (b) Zheng, M.;
Yang, J.; Xu, X.; Sebolt, J.; Wang, S.; Sun, Y. ANTICANCER RESEARCH, 2010,
30, 3321-3332.
15) (a) Shangary, S.; Ding, K.; Qiu, S.; Nikolovska-Coleska, Z.; Bauer, J. A.; Liu,
M.; Wang, G.; Lu, Y.; McEachern, D.; Bernard, D.; Bradford, C. R.; Carey, T. E.;
Wang, S. Mol Cancer Ther., 2008, 7, 1533–1542.
16) Siew, K. T.; Richard, P.; Rohana, Y.; Halijah, I,; Norzulaani, K.; Abd, R. N.
Bioorg. Med. Chem. 2006, 16, 3337-3340.
22

Captions to illustrations.
Figure 1. Important biologically active compounds possessing Spiro-oxindoles motif.
Figure 2. Designing Strategy of Novel Spiro-oxindoles
Figure 3. 5b suppresses the growth of breast cancer cells MCF-7 and primary breast
adenocarcinoma cells
Figure 4. Analysis of p53 and MDM2 by real time quantitative PCR and protein
expression.
Figure 5. Transcriptional activation of the p21, Real Time Quantitative PCR analysis and
Western Blot Analysis
Figure 6. Western blot analysis to demonstrate the effect of compound on the expression
of cell cycle regulatory proteins in MCF-7 and primary breast cancer cells
Figure 7. Cell cycle analysis by flow cytometry
Figure 8. Real time analysis of cell cycle specific genes
Figure 9. Effect of compound 5b on tumor regression in a MCF-7 xenograft mouse
model.
Figure 10. Photomicrographs of histological sections
Figure 11. Western blot analysis
Scheme 1. Synthesis of Spiro-oxindoles
Table 1. Synthesis of Spiro-oxindoles derivatives and their antiproliferative activity.
23

Ph
Ph
OC2H4OH
O
O
N
O
CHO
O
O
OMe
NH
H
N
N
N
H
N
N
O
N
H
O
O
O
N
CO2C2H4OMe
(-)- Spirotryprostatin
Salacin
Schreiber's Lead Compound
Cl
OMe
MeO
OH
O
OMe
NH
O
MeO
OH
NH
O
F
NH
N
H
Br
O
N
H
Cl
Coerulescine
O
N
H
Waldmann's
mitosis arresting agent
MI-219
Figure 1. Important biologically active compounds possessing Spiro-oxindoles motif
Cl
OH
Cl
O
F
OH
O
NH
NH
O
Cl
N
O
N
F
N
O
NH
N
Cl
O
N
H
N
Cl
HN
O
N
H
O
MI-219
Cl
O
Nutlin-3
MI-63
HO
O
O
O
N
R4
OH
N
Br
O
Cl
AM-8553
N
n
NH
Cl
HN
R1
O
R3
N
OH
N
O
N
R2
Cl
O
N
H
Synthesized Compound
Tamoxifen
MI-43
Figure 2. Designing Strategy of Novel Spiro-oxindoles
24

Figure 3. 5b suppresses the growth of breast cancer cells MCF-7 and primary breast
adenocarcinoma cells. Cells were treated with various concentrations of compound 5b
and Nutilin-3a for 48h. Cell viability was measured using MTT cell viability assay.
Results are expressed as mean SEM, n = 5. P values are a-p<0.001, b<0.01, c-p<0.05
and d-p>0.05 vs. control.
25

Figure 4. Analysis of p53 and MDM2 mRNA expression in vehicle treated and 5b
treated MCF-7 cells by real time quantitative PCR. MCF-cells (left panel) and primary
breast cancer cells (right panel). Data are expressed as mean of three different
experiments; SEM, p values are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs.
control.
Fig. 5: (A) Transcriptional activation of the p21 promoter in MCF-7 cells transiently
transfected with p21-luc reporter plasmid in response to compound 5B. pRL-luc plasmid
was used as internal control. Results are described as % of normalized relative luciferase
unit (RLU).(B) Real Time Quantitative PCR analysis for the expression of p21 mRNA in
MCF-7 cells treated as indicated. Results are expressed as mean SEM, n = 3. p values
are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs. control.
26

Fig. 6: Western blot analysis to demonstrate the effect of compound on the expression of
cell cycle regulatory proteins in (A) MCF-7 and (B) primary breast cancer cells.
Representative blots are shown in the upper panel and densitometric quantitation of
protein expression levels are shown as fold changes in the lower panel. Results are
expressed as mean SEM, n = 3 .p values are a-p<0.001, b<0.01, c-p< 0.05and d-p> 0.05
vs. control.
27

Figure 7. Cell cycle analysis by flow cytometry. After incubation with 7.5µM of 5b, cells
were stained with propidium iodide (PI) and subjected to flow cytometric analysis to
determine the percentage of cell at each phase of the cell cycle. Representative images of
flow cytometric analysis are shown in the left panel and the percentage of cells with SEM
calculated based on three independent experiments are shown in the right panel. p values
are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs. control.
28

Figure 8. Real time analysis of cell cycle specific genes. MCF-7 cells were treated with
compound 5b as shown in the figure and mRNA expression was studied by Realtime -
PCR to compare changes in the levels of cyclin D1, cyclin A1, cyclin B1 and cyclin E1
mRNA in the compound treated MCF-7 cells. Results are expressed as mean SEM, n =
3. p values are a-p < 0.001, b-p < 0.01, c-p < 0.05 and d-p > 0.05 vs. control.
29