One-pot three-component synthesis of novel spirooxindoles with potential cytotoxic activity against triple-negative breast cancer MDA-MB-231 cells

Article abstract of DOI:10.1080/14756366.2017.1417276

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited treatment options due to its heterogeneity and the lack of well-defined molecular targets. In our endeavour towards the development of novel anti-TNBC agents, herein we report a one-pot three-component synthesis of novel spirooxindoles 6a–p, and evaluation of their potential anti-proliferative activity towards TNBC MDA-MB-231 cells. Spirooxindoles 6a, 6e and 6i emerged as the most potent analogues with IC₅₀ = 6.70, 6.40 and 6.70 μM, respectively. Compounds 6a and 6e induced apoptosis in MDA-MB-231 cells, as evidenced by the up-regulation of the Bax and down-regulation of the Bcl-2, besides boosting caspase-3 levels. Additionally, 6e displayed significant increase in the percent of annexin V-FITC positive apoptotic cells from 1.34 to 44%. Furthermore, spirooxindoles 6e and 6i displayed good inhibitory activity against EGFR (IC₅₀ = 120 and 150 nM, respectively). Collectively, these data demonstrated that 6e might be a potential lead compound for the development of effective anti-TNBC agents.

Full text of DOI:10.1080/14756366.2017.1417276

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
One-pot three-component synthesis of novel
spirooxindoles with potential cytotoxic activity
against triple-negative breast cancer MDA-MB-231
cells
Wagdy M. Eldehna, Dina H. EL-Naggar, Ahmed R. Hamed, Hany S. Ibrahim,
Hazem A. Ghabbour & Hatem A. Abdel-Aziz
To cite this article: Wagdy M. Eldehna, Dina H. EL-Naggar, Ahmed R. Hamed, Hany S. Ibrahim,
Hazem A. Ghabbour & Hatem A. Abdel-Aziz (2018) One-pot three-component synthesis of
novel spirooxindoles with potential cytotoxic activity against triple-negative breast cancer MDA-
MB-231 cells, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 309-318, DOI:
10.1080/14756366.2017.1417276
To link to this article: https://doi.org/10.1080/14756366.2017.1417276
© 2017 The Author(s). Published by Informa
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Group.
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Date: 28 December 2017, At: 03:59

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 309–318
https://doi.org/10.1080/14756366.2017.1417276
RESEARCH PAPER
One-pot three-component synthesis of novel spirooxindoles with potential
cytotoxic activity against triple-negative breast cancer MDA-MB-231 cells
Wagdy M. Eldehnaa, Dina H. EL-Naggarb, Ahmed R. Hamedc,d, Hany S. Ibrahime, Hazem A. Ghabbourf,g and
Hatem A. Abdel-Azizb
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh, Egypt; Department of Applied Organic
c
d
Chemistry, National Research Center, Giza, Egypt; Department of Phytochemistry, National Research Center, Giza, Egypt; Biology Unit, Central
e
Laboratory of the Pharmaceutical & Drug Industries Research Division, National Research Center, Giza, Egypt; Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo, Egypt; Department of Medicinal Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura, Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi
f
g
Arabia
ABSTRACT
ARTICLE HISTORY
Received 4 November 2017
Accepted 11 December 2017
Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited treatment options due
to its heterogeneity and the lack of well-defined molecular targets. In our endeavour towards the develop-
ment of novel anti-TNBC agents, herein we report a one-pot three-component synthesis of novel spiroox-
indoles 6a–p, and evaluation of their potential anti-proliferative activity towards TNBC MDA-MB-231 cells.
Spirooxindoles 6a, 6e and 6i emerged as the most potent analogues with IC50 ¼ 6.70, 6.40 and 6.70 mM,
respectively. Compounds 6a and 6e induced apoptosis in MDA-MB-231 cells, as evidenced by the up-regu-
lation of the Bax and down-regulation of the Bcl-2, besides boosting caspase-3 levels. Additionally, 6e dis-
played significant increase in the percent of annexin V-FITC positive apoptotic cells from 1.34 to 44%.
Furthermore, spirooxindoles 6e and 6i displayed good inhibitory activity against EGFR (IC50 ¼ 120 and
150 nM, respectively). Collectively, these data demonstrated that 6e might be a potential lead compound
for the development of effective anti-TNBC agents.
KEYWORDS
Triple-negative breast
cancer; spirooxindoles; anti-
proliferative activity;
apoptosis; EGFR
Introduction
lines, compound (i) (Figure 1) showed cytotoxic activity with
Breast cancer is the fifth most common cause of death in women IC50 ¼ 11 mM against MDA-MB-231 cancer cell line9, while spirooxin-
world-wide. In fact, it represents about 12% of all new cancer dole–pyranopyrimidine derivative (ii) (Figure 1) possessed cytotox-
icity against MDA cell lines with IC50 ¼ 6.9 mM10. Moreover,
cases and 25% of all cancers in women with nearly 1.7 million
new cases diagnosed in 20121. Routine breast cancer case showed compound (iii) exhibited cytotoxic activity against ordinary breast
cancer (MCF-7; IC50 ¼ 8.6 mM) and TNBC (MDA-MB-231;
an expression of three distinctive receptors which are oestrogen
receptor (ER), progesterone receptor (PR) and human epidermal
IC50 ¼ 6.4 mM)11 while compound (iv), Figure 1, displayed cytotox-
icity against MDA-MB-231 with IC50 ¼ 4.2 mM). Unfortunately, there
growth factor 2 receptor (Her2). About 15–20% of the women
is no trial till now to investigate the mechanism of action of spi-
rooxindole derivative with cytotoxic activity against TNBC cell lines.
Based on the aforementioned findings and as a continuation of
our research program on the development of novel effective anti-
TNBC candidates12–14, it was thought worthwhile to extend our
investigations to probe for novel spirooxindoles possessing prom-
ising anti-proliferative activity towards TNBC. In the present work
we report the synthesis of a novel series of spirooxindoles 6a–p,
Figure 1, and their in vitro efficacy against the proliferation of the
aggressive TNBC MDA-MB-231 cell line. In addition, spirooxindoles
were further investigated regarding their potential apoptotic
induction and their effects on cell cycle progression in the MDA-
MB-231 cancer cells to acquire a perception for the mechanism of
the antitumor activity of target spirooxindoles. Since the epidermal
growth factor receptor (EGFR) is frequently overexpressed in
TNBC15,16, the most potent spirooxindoles in this study will be
assayed for their potential inhibitory activity towards EGFR.
diagnosed with breast cancer lacked in overexpression of these
three receptors (ER, PR and Her2)2. This case is known as triple-
negative breast cancer (TNBC) and it does not respond to normal
protocol for the treatment of normal breast cancer. As a result,
TNBC is responsible for high incident among the total percent of
deaths regarded to breast cancer3,4. Unfortunately, TNBC patients
were subjected to traditional cytotoxic therapies during their treat-
ment protocol as till now there is no approved targeted cytotoxic
drug for TNBC5.
On the other hand, spirooxindole is considered as a scaffold of
interest to produce many derivatives with anticancer activity6. For
example, Spirotryprostatins B (Figure 1), naturally isolated alkaloids
from Aspergillus fumigatus, inhibit the cell cycle progression of
tsFT210 cells at G2/M phase with IC50 ¼ 14.0 mM7. Other synthetic
spirooxindole derivative (MI-888) (Figure 1) in preclinical trials on
xenograft models as mdm2-p53 inhibitor (Ki ¼ 0.44 nM)8. Regarding
cytotoxicity of some spirooxindole derivatives against TNBC cell
CONTACT Wagdy M. Eldehna
wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh,
Egypt
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

310
W. M. ELDEHNA ET AL.
F
Cl
H
N
O
F
H
O
H2N
O
O
H
N
OH
O
N
NH
O
O
O
N
(H3C)2HCOOC
Cl
O
O
O
N
NH
O
O
N
H
N
H
N O
H
Cl
O
F
(i)
(ii)
MI-888
Spirotryprostatin B
H
N
S
S
R
O
N
N
N
N
O
NC
Ar
N
N
Cl
O
O
N
H
O
N
H
Ph
N
H
(vi)
Cl
6a-p
(iii)
Figure 1. Structures of some reported anticancer spirooxindoles and target spirooxindoles 6a–p.
5.86 (s, 1H, H4-pyrazole), 7.19 (t, 1H, H4-thiophene, J ¼ 4.8 Hz),
7.34–7.65 (m, 7H, Ar-H); 13 C NMR (DMSO-d6) d ppm: 87.54, 123.48
(2 C), 124.33, 125.23, 126.88, 127.99, 129.46 (2 C), 137.54, 139.46,
146.38, 148.69; Anal. calcd. For C13H11N3S: C, 64.71; H, 4.59; N,
17.41; Found C, 64.92; H, 4.54; N, 17.33.
Materials and methods
Chemistry
Melting points were measured with a Stuart melting point apparatus
and were uncorrected. Infrared (IR) Spectra were recorded as KBr
disks using Schimadzu FT-IR 8400 S spectrophotometer. Mass spec-
tral data are given by GCMS-QP1000 EX spectrometer at 70 e.V. NMR
1
General procedure for the synthesis of target compounds (6a–p)
A mixture of 1H-indole-2,3-diones 1a–d (1 mmol) with an equi-
molar amount of 3-oxo-3-phenylpropanenitriles 3a–d and 1-
phenyl-3-(thiophen-2-yl)-1H-pyrazol-5-amine 5 in 15 ml of HOAc/
H2O (1:1 v/v) at 120 ꢀC was stirred for 8–11 h (the reaction
progress was monitored by TLC). Upon completion, the reac-
tion mixture was cooled to room temperature. The solid
formed was filtered, dried and crystallized from acetonitrile to
produce compounds 6a–p.
Spectra were recorded on a Varian Mercury NMR spectrometer. H
spectrum was run at 400 MHz and 13 C spectrum was run at 100 MHz
in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are
expressed in values (ppm) using the solvent peak as internal stand-
ard. All coupling constant (J) values are given in hertz. The abbrevia-
tions used are as follows: s, singlet; d, doublet; m, multiplet.
Elemental analyses were carried out at the Regional Center for
Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt.
Analytical thin layer chromatography (TLC) on silica gel plates con-
taining UV indicator was employed routinely to follow the course of
reactions and to check the purity of products. Some representative
NMR charts are provided in the Supplementary material.
2-Oxo-1',6'-diphenyl-3'-(thiophen-2-yl)-1',7'-dihydrospiro[indoline-
3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6a)
Yield 68%, m.p. 295–297 ꢀC; IR (KBr, ꢀ cmꢁ1): 3227 (NH), 2205
1
(CꢂN), 1709 (C¼O); H NMR (DMSO-d6) d (ppm): 6.21 (d, 1H, Ar-H,
Synthesis of 3-oxo-3-phenylpropanenitriles (3)
J ¼ 2.8 Hz), 6.82 (dd, 1H, Ar-H, J ¼ 4.8, 3.6 Hz), 6.92 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 7.05 (t, 1H, Ar-H, J ¼ 7.2 Hz), 7.29 (d, 1H, Ar-H,
J ¼ 7.6 Hz), 7.32 (d, 1H, Ar-H, J ¼ 7.6 Hz), 7.40 (d, 1H, Ar-H,
J ¼ 5.2 Hz), 7.44–7.62 (m, 8H, Ar-H), 7.68 (d, 2H, Ar-H, J ¼ 7.6 Hz),
10.45 (s, 1H, NH, D2O exchangeable), 10.74 (s, 1H, NH, D2O
exchangeable); 13 C NMR (DMSO-d6) d ppm: 51.83, 83.74, 97.41,
110.47, 118.66, 123.28, 124.69 (2 C), 125.44, 125.80, 126.98, 127.62,
128.44, 128.89, 129.35 (2 C), 129.60, 129.90, 130.01 (2 C), 130.99,
133.78, 134.48, 135.64, 138.00, 139.66, 141.70, 142.97, 151.53,
178.02 (C¼O); MS m/z [%]: 497 [Mþ, 26.91], 77 [100]; Anal. calcd.
For C30H19N5OS: C, 72.42; H, 3.85; N, 14.08; Found C, 72.59; H, 3.79;
N, 13.95.
To ethyl benzoates 2a–d (100 mmol) and acetonitrile (4.1 ml,
100 mol) in dry benzene (250 ml) and dimethylformamide (10 ml),
sodium hydride (4.8 g, 60%) was added. The reaction mixture was
heated under reflux for 4 h then allowed to cool to room tempera-
ture. The solid formed was collected by filtration, washed with
ether and dried. This solid was dissolved in water and then neu-
tralized with conc. HCl to pH 7. The precipitated product was col-
lected by filtration, washed with water and dried. Recrystallization
from ethanol gave compounds 317.
Synthesis of 1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-5-amine (5)
To a stirred solution of 3-oxo-3-(thiophen-2-yl)propanenitrile 4
(1.5 g, 10 mmol) in ethyl alcohol (15 ml), phenylhydrazine (1.96 ml,
2.16 g, 20 mmol) was added. The reaction mixture was heated
under reflux for 1 h. The solid product obtained upon cooling was
2-Oxo-1'-phenyl-3'-(thiophen-2-yl)-6'-(p-tolyl)-1',7'-dihydrospiro[in-
doline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6 b)
filtered off, washed with cold water and recrystallized from metha- Yield 75%, m.p. 285–287 ꢀC; IR (KBr, ꢀ cmꢁ1): 3317 (NH), 2206
nol to furnish compound 5 (yield 65%), m.p. 113–115 ꢀC; IR (KBr, ꢀ (CꢂN), 1712 (C¼O); 1H NMR (DMSO-d6) d (ppm): 2.33 (s, 3H,
cmꢁ1): 3351 (NH2); 1H NMR (DMSO-d6) d (ppm): 5.51 (s, 2H, NH2), CH3), 6.15 (d, 1H, Ar-H, J ¼ 3.2 Hz), 6.78 (dd, 1H, Ar-H, J ¼ 4.8,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
311
3.6 Hz), 6.87 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.00 (t, 1H, Ar-H, J ¼ 7.6 Hz), 5-Chloro-6'-(4-methoxyphenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-
7.27 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.37–7.47 (m, 6H, Ar-H), 7.53 (t, 2H,
1',7'-dihydrospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carboni-
Ar-H, J ¼ 8.0 Hz), 7.64 (d, 2H, Ar-H, J ¼ 8.0 Hz), 10.33 (s, 1H, NH,
trile (6 g)
D2O exchangeable), 10.68 (s, 1H, NH, D2O exchangeable); 13 C
Yield 68%, m.p. > 300 ꢀC; IR (KBr, ꢀ cmꢁ1): 3213 (NH), 2206 (CꢂN),
1708 (C¼O); 1H NMR (DMSO-d6) d (ppm): 3.78 (s, 3H, OCH3), 6.31
NMR (DMSO-d6) d ppm: 21.37 (CH3), 51.76, 82.85, 97.40, 110.40,
132.22, 124.61 (2 C), 125.35, 125.75, 126.93, 127.59, 128.36,
(d, 1H, Ar-H, J ¼ 3.6 Hz), 6.83 (dd, 1H, Ar-H, J ¼ 5.2, 3.6 Hz), 6.87 (d,
129.24 (2 C), 129.33 (2 C), 129.54, 129.85 (2 C), 129.94, 130.88,
1H, Ar-H, J ¼ 8.4 Hz), 7.02 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.29 (dd, 1H, Ar-
134.48, 135.63, 138.00, 139.64, 140.84, 141.68, 142.90, 151.46,
H, J ¼ 8.0, 2.0 Hz), 7.34–7.63 (m, 7H, Ar-H), 7.66 (d, 2H, Ar-H,
177.99 (C¼O); MS m/z [%]: 511 [Mþ, 16.08], 482 [100]; Anal.
J ¼ 8.0 Hz), 10.31 (s, 1H, NH, D2O exchangeable), 10.79 (s, 1H, NH,
calcd. For C31H21N5OS: C, 72.78; H, 4.14; N, 13.69; Found C,
D2O exchangeable); 13 C NMR (DMSO-d6) d ppm: 55.84, 55.91
72.54; H, 4.11; N, 13.81.
(OCH3), 82.13, 97.08, 111.89, 114.15, 114.56, 121.93, 124.71, 125.40,
125.64, 125.87, 127.12, 127.15, 127.63, 128.42, 129.82, 129.98,
130.14, 130.94, 131.47, 134.31, 137.35, 138.01, 138.49, 139.77,
140.64, 142.73, 161.45, 161.55, 177.90 (C¼O); Anal. calcd. For
C31H20ClN5O2S: C, 66.25; H, 3.59; N, 12.46; Found C, 66.41; H, 3.63;
N, 12.35.
6'-(4-Methoxyphenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-1',7'-dihy-
drospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6c)
Yield 75%, m.p. 296–298 ꢀC; IR (KBr, ꢀ cmꢁ1): 3361 (NH), 2206
(CꢂN), 1715 (C¼O); 1H NMR (DMSO-d6) d (ppm): 3.81 (s, 3H,
OCH3), 6.19 (d, 1H, Ar-H, J ¼ 3.6 Hz), 6.81 (dd, 1H, Ar-H, J ¼ 4.8,
3.6 Hz), 6.91 (d, 1H, Ar-H, J ¼ 7.6 Hz), 7.03–7.06 (m, 3H, Ar-H),
7.28–7.32 (m, 2H, Ar-H), 7.40 (d, 1H, Ar-H, J ¼ 4.8 Hz), 7.46 (t, 1H,
Ar-H, J ¼ 7.6 Hz), 7.55–7.60 (m, 4H, Ar-H), 7.68 (d, 2H, Ar-H,
J ¼ 7.6 Hz), 10.29 (s, 1H, NH, D2O exchangeable), 10.70 (s, 1H, NH,
D2O exchangeable); MS m/z [%]: 527 [Mþ, 18.5], 498 [100]; Anal.
calcd. For C31H21N5O2S: C, 70.57; H, 4.01; N, 13.27; Found C,
70.78; H, 3.96; N, 13.38.
5-Chloro-6'-(4-chlorophenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-1',7'-
dihydrospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile
(6 h)
Yield 70%, m.p. 281–283 ꢀC; IR (KBr, ꢀ cmꢁ1): 3324 (NH), 2202
1
(CꢂN), 1715 (C¼O); H NMR (DMSO-d6) d (ppm): 6.31 (d, 1H, Ar-H,
J ¼ 3.2 Hz), 6.85 (t, 1H, Ar-H, J ¼ 8.4 Hz), 6.90 (d, 1H, Ar-H,
J ¼ 8.4 Hz), 7.32 (dd, 1H, Ar-H, J ¼ 8.4, 2.0 Hz), 7.38–7.57 (m, 7H, Ar-
H), 7.62 (d, 2H, Ar-H, J ¼ 8.4 Hz), 7.72–7.83 (m, 2H, Ar-H), 9.09 (s,
1H, NH, D2O exchangeable), 10.83 (s, 1H, NH, D2O exchangeable);
13 C NMR (DMSO-d6) d ppm: 55.32, 77.19, 96.50, 111.86, 112.87,
117.51, 122.10, 125.32, 125.99, 127.04, 127.14, 128.63, 128.80,
129.14, 129.77, 130.34, 131.30, 131.66, 132.09, 135.94, 136.52,
136.72, 140.51, 141.34, 142.70, 148.51, 150.43, 154.33, 159.62,
178.10 (C¼O); Anal. calcd. For C30H17Cl2N5OS: C, 63.61; H, 3.03; N,
12.36; Found C, 63.38; H, 3.05; N, 12.49.
6'-(4-Chlorophenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-1',7'-dihydro-
spiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6d)
Yield 70%, m.p. 279–281 ꢀC; IR (KBr, ꢀ cmꢁ1): 3283 (NH), 2202
(CꢂN), 1716 (C¼O); 1H NMR (DMSO-d6) d (ppm): 6.20 (dd, 1H, Ar-
H, J ¼ 3.6, 0.8 Hz), 6.81 (dd, 1H, Ar-H, J ¼ 4.8, 3.6 Hz), 6.91 (d, 1H,
Ar-H, J ¼ 7.6 Hz), 7.04 (t, 1H, Ar-H, J ¼ 7.6 Hz), 7.29–7.34 (m, 2H, Ar-
H), 7.41 (dd, 1H, Ar-H, J ¼ 4.8, 0.8 Hz), 7.45 (t, 1H, Ar-H, J ¼ 7.6 Hz),
7.56–7.64 (m, 6H, Ar-H), 7.68 (d, 2H, Ar-H, J ¼ 7.6 Hz), 10.44 (s, 1H,
NH, D2O exchangeable), 10.75 (s, 1H, NH, D2O exchangeable);
Anal. calcd. For C30H18ClN5OS: C, 67.73; H, 3.41; N, 13.16; Found C,
67.61; H, 3.46; N, 13.25.
5-Bromo-2-oxo-1',6'-diphenyl-3'-(thiophen-2-yl)-1',7'-dihydrospir-
o[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6i)
Yield 75%, m.p. 275–277 ꢀC; IR (KBr, ꢀ cmꢁ1): 3293 (NH), 2206
1
(CꢂN), 1713 (C¼O); H NMR (DMSO-d6) d (ppm): 6.33 (d, 1H, Ar-H,
5-Chloro-2-oxo-1',6'-diphenyl-3'-(thiophen-2-yl)-1',7'-dihydrospir-
o[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6e)
Yield 78%, m.p. 289–291 ꢀC; IR (KBr, ꢀ cmꢁ1): 3205 (NH), 2206
(CꢂN), 1708 (C¼O); 1H NMR (DMSO-d6) d (ppm): 6.33 (dd, 1H, Ar-
H, J ¼ 3.6, 0.8 Hz), 6.86 (dd, 1H, Ar-H, J ¼ 5.2, 3.6 Hz), 6.91 (d, 1H,
Ar-H, J ¼ 8.4 Hz), 7.33 (dd, 1H, Ar-H, J ¼ 8.4, 2.4 Hz), 7.44–7.62 (m,
10H, Ar-H), 7.68 (d, 2H, Ar-H, J ¼ 7.6 Hz), 10.52 (s, 1H, NH, D2O
exchangeable), 10.87 (s, 1H, NH, D2O exchangeable); MS m/z [%]:
532 [Mþ, 5.28], 534 [Mþþ2, 1.81], 502 [100]; Anal. calcd. For
C30H18ClN5OS: C, 67.73; H, 3.41; N, 13.16; Found C, 67.49; H, 3.44;
N, 13.22.
J ¼ 3.2 Hz), 6.87–6.91 (m, 2H, Ar-H), 7.44–7.62 (m, 11H, Ar-H), 7.69
(d, 2H, Ar-H, J ¼ 7.6 Hz), 10.52 (s, 1H, NH, D2O exchangeable), 10.89
(s, 1H, NH, D2O exchangeable); 13 C NMR (DMSO-d6) d ppm: 52.02,
82.85, 96.88, 112.46, 114.88, 118.57, 124.69, 124.90, 125.47, 126.98,
127.22, 127.68, 128.54, 128.65, 128.86, 129.39, 129.85, 131.01,
132.83, 133.63, 134.29, 137.70, 137.95, 139.72, 140.53, 141.04,
141.89, 142.79, 151.92, 177.73 (C¼O); MS m/z [%]: 576 [Mþ, 3.93],
77 [100]; Anal. calcd. For C30H18BrN5OS: C, 62.51; H, 3.15; N, 12.15;
Found C, 62.63; H, 3.20; N, 12.03.
5-Bromo-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-6'-(p-tolyl)-1',7'-dihydro-
spiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6j)
Yield 80%, m.p. 282–284 ꢀC; IR (KBr, ꢀ cmꢁ1): 3207 (NH), 2204
(CꢂN), 1708 (C¼O); 1H NMR (DMSO-d6) d (ppm): 2.33 (s, 3H, CH3),
6.30 (d, 1H, Ar-H, J ¼ 2.8 Hz), 6.83–6.87 (m, 2H, Ar-H), 7.29 (d, 2H,
Ar-H, J ¼ 8.0 Hz), 7.41–7.50 (m, 6H, Ar-H), 7.55 (t, 2H, Ar-H,
J ¼ 8.0 Hz), 7.65 (d, 2H, Ar-H, J ¼ 7.6 Hz), 10.39 (s, 1H, NH, D2O
exchangeable), 10.82 (s, 1H, NH, D2O exchangeable); 13 C NMR
(DMSO-d6) d ppm: 21.38 (CH3), 51.95, 82.44, 96.85, 112.41, 114.82,
124.82 (2 C), 125.38, 127.18, 127.66, 128.47, 128.59, 129.27, 129.31,
129.57, 129.59, 129.77, 129.82 (2 C), 130.73, 132.77, 134.30, 137.70,
137.94, 139.72, 140.87, 141.01, 142.73, 151.86, 177.72 (C¼O); Anal.
5-Chloro-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-6'-(p-tolyl)-1',7'-dihydro-
spiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6f)
Yield 75%, m.p. 286–288 ꢀC; IR (KBr, ꢀ cmꢁ1): 3259 (NH), 2205
(CꢂN), 1712 (C¼O); 1H NMR (DMSO-d6) d (ppm): 2.36 (s, 3H, CH3),
6.32 (dd, 1H, Ar-H, J ¼ 4.0, 0.8 Hz), 6.86 (dd, 1H, Ar-H, J ¼ 5.2,
4.0 Hz), 6.90 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.30–7.35 (m, 3H, Ar-H),
7.42–7.47 (m, 3H, Ar-H), 7.48 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.56 (t, 2H,
Ar-H, J ¼ 8.0 Hz), 7.67 (d, 2H, Ar-H, J ¼ 8.0 Hz), 10.43 (s, 1H, NH, D2O
exchangeable), 10.86 (s, 1H, NH, D2O exchangeable); Anal. calcd.
For C31H20ClN5OS: C, 68.19; H, 3.69; N, 12.83; Found C, 67.92; H,
3.70; N, 12.89.

312
W. M. ELDEHNA ET AL.
calcd. For C31H20BrN5OS: C, 63.06; H, 3.41; N, 11.86; Found C, 5-Methoxy-6'-(4-methoxyphenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-
62.83; H, 3.48; N, 11.79.
1',7'-dihydrospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carboni-
trile (6o)
Yield 75%, m.p. 279–281 ꢀC; IR (KBr, ꢀ cmꢁ1): 3361 (NH), 2204
(CꢂN), 1710 (C¼O); 1H NMR (DMSO-d6) d (ppm): 3.70 (s, 3H,
OCH3), 3.81 (s, 3H, OCH3), 6.27 (d, 1H, Ar-H, J ¼ 2.8 Hz), 6.78–6.91
(m, 4H, Ar-H), 7.05 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.41–7.48 (m, 2H, Ar-
H), 7.53–7.60 (m, 4H, Ar-H), 7.70 (d, 2H, Ar-H, J ¼ 7.6 Hz), 10.28 (s,
1H, NH, D2O exchangeable), 10.57 (s, 1H, NH, D2O exchangeable);
13 C NMR (DMSO-d6) d ppm: 52.32, 55.86 (OCH3), 56.02 (OCH3),
83.12, 97.47, 110.99, 112.44, 114.19, 114.72, 118.99, 122.03,
124.57, 125.45, 125.86, 126.90, 127.66, 128.34, 129.75, 129.88,
129.96, 130.97, 131.50, 134.60, 135.05, 136.84, 138.09, 139.78,
142.98, 151.21, 156.19, 161.44, 177.95 (C¼O); Anal. calcd. For
C32H23N5O3S: C, 68.93; H, 4.16; N, 12.56; Found C, 69.06; H, 4.19;
N, 12.65.
5-Bromo-6'-(4-methoxyphenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-
1',7'-dihydrospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carboni-
trile (6k)
Yield 81%, m.p. > 300 ꢀC; IR (KBr, ꢀ cmꢁ1): 3387 (NH), 2202
(CꢂN), 1716 (C¼O); 1H NMR (DMSO-d6) d (ppm): 3.78 (s, 3H,
OCH3), 6.31 (d, 1H, Ar-H, J ¼ 3.2 Hz), 6.83 (d, 2H, Ar-H, J ¼ 8.4 Hz),
7.02 (d, 2H, Ar-H, J ¼ 8.8 Hz), 7.40–7.55 (m, 8H, Ar-H), 7.66 (d, 2H,
Ar-H, J ¼ 8.0 Hz), 10.30 (s, 1H, NH, D2O exchangeable), 10.80 (s,
1H, NH, D2O exchangeable); 13 C NMR (DMSO-d6) d ppm: 51.96,
55.84 (OCH3), 82.08, 96.96, 112.41, 114.15 (2 C), 114.80, 118.83,
124.74 (2 C), 125.39, 125.62, 127.17, 127.65, 128.43, 128.56,
129.83 (2 C), 130.95 (2 C), 132.74, 134.31, 137.72, 137.99, 139.77,
141.04, 142.73, 151.57, 161.45, 177.76 (C¼O); MS m/z [%]: 606
[Mþ, 2.24], 608 [Mþþ2, 1.20], 468 [100]; Anal. calcd. For
C31H20BrN5O2S: C, 61.39; H, 3.32; N, 11.55; Found C, 61.57; H,
3.36; N, 11.39.
6'-(4-Chlorophenyl)-5-methoxy-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-
1',7'-dihydrospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carboni-
trile (6p)
Yield 80%, m.p. 255–257 ꢀC; IR (KBr, ꢀ cmꢁ1): 3273 (NH), 2206
(CꢂN), 1712 (C¼O); 1H NMR (DMSO-d6) d (ppm): 3.68 (s, 3H,
OCH3), 6.31 (d, 1H, Ar-H, J ¼ 2.8 Hz), 6.74–6.89 (m, 3H, Ar-H), 7.05
(d, 1H, Ar-H, J ¼ 8.8 Hz), 7.05 (d, 1H, Ar-H, J ¼ 8.8 Hz), 7.42 (d, 1H,
Ar-H, J ¼ 7.6 Hz), 7.50–7.67 (m, 2H, Ar-H), 7.69 (d, 2H, Ar-H,
J ¼ 8.0 Hz), 7.95 (d, 2H, Ar-H, J ¼ 8.4 Hz), 8.23 (d, 2H, Ar-H,
J ¼ 8.4 Hz), 10.33 (s, 1H, NH, D2O exchangeable), 10.59 (s, 1H, NH,
D2O exchangeable); Anal. calcd. For C31H20ClN5O2S: C, 66.25; H,
3.59; N, 12.46; Found C, 65.98; H, 3.61; N, 12.58.
5-Bromo-6'-(4-chlorophenyl)-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-1',7'-
dihydrospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile
(6 l)
Yield 76%, m.p. 293–294 ꢀC; IR (KBr, ꢀ cmꢁ1): 3225 (NH), 2206
1
(CꢂN), 1715 (C¼O); H NMR (DMSO-d6) d (ppm): 6.30 (d, 1H, Ar-H,
J ¼ 3.3 Hz), 6.84 (d, 2H, Ar-H, J ¼ 7.2 Hz), 7.43–7.54 (m, 3H, Ar-H),
7.57–7.63 (m, 7H, Ar-H), 7.66 (d, 2H, Ar-H, J ¼ 8.1 Hz), 10.49 (s, 1H,
NH, D2O exchangeable), 10.86 (s, 1H, NH, D2O exchangeable);
Anal. calcd. For C30H17BrClN5OS: C, 58.98; H, 2.80; N, 11.46; Found
C, 59.15; H, 2.77; N, 11.61.
Biological evaluation
In vitro anti-proliferative activity assay
5-Methoxy-2-oxo-1',6'-diphenyl-3'-(thiophen-2-yl)-1',7'-dihydrospir-
o[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6 m)
Yield 75%, m.p. 276–278 ꢀC; IR (KBr, ꢀ cmꢁ1): 3319 (NH), 2202
(CꢂN), 1716 (C¼O); 1H NMR (DMSO-d6) d (ppm): 3.68 (s, 3H,
OCH3), 6.24 (d, 1H, Ar-H, J ¼ 2.8 Hz), 6.79–6.90 (m, 3H, Ar-H),
7.38–7.62 (m, 10H, Ar-H), 7.65 (d, 2H, Ar-H, J ¼ 7.6 Hz), 10.35 (s, 1H,
NH, D2O exchangeable), 10.51 (s, 1H, NH, D2O exchangeable); 13 C
NMR (DMSO-d6) d ppm: 56.00, 56.48 (OCH3), 83.92, 97.31, 110.96,
112.44, 114.72, 118.52, 122.10, 122.31, 124.68, 125.40, 126.90,
127.63, 128.39, 128.82, 129.06, 129.35, 129.84, 129.90, 129.97,
130.92, 133.86, 134.52, 134.95, 136.74, 137.97, 139.69, 142.94,
156.16, 177.83 (C¼O); MS m/z [%]: 527 [Mþ, 16.84], 498 [93.20];
Anal. calcd. For C31H21N5O2S: C, 70.57; H, 4.01; N, 13.27; Found C,
70.41; H, 3.98; N, 13.35.
Synthetic spirooxindoles 6a–p were tested for their anti-prolifera-
tive potency on TNBC cells (MDA-MB-231). Cells lines were main-
tained as monolayers in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml
penicillin and 100 mg/ml streptomycin sulfate. Cells were sub-cul-
tured with trypsine/EDTA solution, counted with haemocytometer
and plated onto 96-well plates (5000 cells/well) and left overnight
to form a semi-confluent monolayer. We employed a modified
method utilizing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-
trazolium bromide dye (Carbosynth, UK) that is based on the
reduction of the dye by mitochondrial dehydrogenases of meta-
bolically active cells to insoluble formazan crystals18,19. Briefly, cell
monolayers were treated in quadrates with vehicle (DMSO, 0.1%
v/v), test samples or Adriamycin as positive control for an expos-
ure time of 48 h. At the end of exposure, MTT solution in PBS
(5 mg/ml) was then added to all wells including no cell blank and
left to incubate for 90 min20. The formation of formazan crystals
were visually confirmed using phase contract microscopy. DMSO
(100 ml/well) was added to dissolve the formazan crystals with
shaking for 10 min after which the absorbance was read at 590 nm
against no cell blanks on a FLuo Star Optima microplate reader
(BMG Technologies, Germany). Cell proliferation was calculated
comparing the OD values of the DMSO control wells and those of
the samples represented as % proliferation to the control. Dose-
response experiment was performed on samples producing ꢃ50%
loss of cell proliferation using five serial 2-fold dilutions (50, 25,
5-Methoxy-2-oxo-1'-phenyl-3'-(thiophen-2-yl)-6'-(p-tolyl)-1',7'-dihy-
drospiro[indoline-3,4'-pyrazolo[3,4-b]pyridine]-5'-carbonitrile (6n)
Yield 78%, m.p. 268–269 ꢀC; IR (KBr, ꢀ cmꢁ1): 3314 (NH), 2206
1
(CꢂN), 1712 (C¼O); H NMR (DMSO-d6) d (ppm): 2.34 (s, 3H, CH3),
3.68 (s, 3H, OCH3), 6.26 (d, 1H, Ar-H, J ¼ 2.8 Hz), 6.79–6.87 (m, 4H,
Ar-H), 7.27 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.37–7.48 (m, 4H, Ar-H), 7.52 (t,
2H, Ar-H, J ¼ 8.0 Hz), 7.65 (d, 2H, Ar-H, J ¼ 7.2 Hz), 10.21 (s, 1H, NH,
D2O exchangeable), 10.45 (s, 1H, NH, D2O exchangeable); 13 C NMR
(DMSO-d6) d ppm: 21.73 (CH3), 52.07, 56.00 (OCH3), 83.47, 97.31,
110.93, 112.43, 114.69, 118.77, 124.64 (2 C), 125.39, 126.89, 127.63,
128.34, 129.27, 129.31, 129.57, 129.77, 129.83 (2 C), 130.92, 134.61,
134.99, 136.78, 138.01, 139.68, 140.79, 142.94, 151.43, 156.15, 12.5, 6.25 and 3.125 mM) of the sample. IC50 values (concentration
177.83 (C¼O); Anal. calcd. For C32H23N5O2S: C, 70.96; H, 4.28; N, of sample causing 50% loss of cell proliferation of the vehicle con-
12.93; Found C, 71.27; H, 4.22; N, 12.79.
trol) were calculated using non-linear regression curve fitting of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
313
the dose response plots on GraphPad Prism V.6.0 software Annexin V–FITC apoptosis assay
(Graphpad Inc, San Diego, CA). Assessment of morphological
changes of MDA-MB-231 cells following treatment with the most
active hits were performed using phase contrast inverted micro-
scope (Carl Zeiss Microscopy GmbH, Gottingen, Germany) and
photomicrographs were taken using digital camera.
Phosphatidylserine externalization was measured using Annexin
V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA)
according to the manufacturer's instructions, as reported earlier22.
MDA-MB-231cells were treated with 6e at defined concentrations
for 24 h.
In vitro cytotoxic activity WI-38 cells (human lung fibroblast nor-
mal cell line)
EGFR kinase ELISA assay
EGFR enzyme inhibition was measured using a BPS Biosciences
Colorimetric 96-well EGFR assay kit (catalog no. 40321), according
to the manufacturer's instructions. Percent inhibition was calcu-
lated by the comparison of compounds treated to control incuba-
tions. The concentration of the test compound causing 50%
inhibition (IC50) was calculated from the concentration–inhibition
response curve (triplicate determinations) and the data were com-
pared with Erlotinib as standard EGFR inhibitor.
WI-38 cells (normal breast cells), were obtained from American
Type Culture Collection. The cells were propagated in DMEM sup-
plemented with 10% heat-inactivated FBS (Hyclone), 10 lg/ml of
insulin (Sigma), and 1% penicillin-streptomycin. All of the other
chemicals and reagents were from Sigma, or Invitrogen.
Cytotoxicity was determined using MTT assay following a reported
procedure18. The 50% inhibitory concentration (IC50) was esti-
mated from graphic plots of the dose response curve for each
conc. using Graphpad Prism software (San Diego, CA).
Results and discussion
Chemistry
Assay of the apoptosis markers (Bax, caspase-3 and Bcl-2) levels
The levels of the apoptotic markers (Bax, caspase-3) as well as the
anti-apoptotic marker (Bcl-2) were assessed using ELISA colorimet-
ric kits per the manufacturer’s protocol and referring to reported
The proposed synthetic routes to prepare the target compounds
are shown in Scheme 1. Synthesis was initiated in Scheme 1 by
reacting ethyl benzoates 2a–d and acetonitrile in dry benzene and
DMF, in the presence of sodium hydride under reflux temperature
to afford 3-oxo-3-phenylpropanenitriles 3a–d. Also, synthesis of 1-
phenyl-3-(thiophen-2-yl)-1H-pyrazol-5-amine 5 was accomplished
via heterocyclocondensation of 3-oxo-3-(thiophen-2-yl)propaneni-
instructions21,22
.
Cell cycle analysis
The MDA-MB-231 cells were treated with 6.40 lM of compound trile 4 with phenylhydrazine in refluxing absolute ethyl alcohol.
6e for 24 h. Then cells were washed twice with ice-cold phos- Preparation of the target spirooxindoles 6a–p was achieved via a
phate buffered saline (PBS). Subsequently, the treated cells one-pot three-component reaction of 1H-indole-2,3-diones 1a–d
were collected by centrifugation, fixed in ice-cold 70% (v/v) with an equimolar amount of 3-oxo-3-phenylpropanenitriles 3a–d
ethanol, washed with PBS, re-suspended with 0.1 mg/mL RNase, and 1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-5-amine 5 in hot HOAc/
stained with 40 mg/mL PI, and analyzed by flow cytometry H2O (1:1 v/v), with 68–81% yield (Scheme 1).
using FACS Calibur (Becton Dickinson, BD, USA). The cell cycle
Postulated structures of the newly synthesized spirooxindoles
distributions were calculated using CellQuest software (Becton 6a–p were in full agreement with their spectral and elemental
Dickinson).
analyses data. IR spectra of the latter products displayed
O
O
CN
Ar
O
2a-d
4
S
H
N
(i)
(ii)
H2N
Ph
H
N
O
Ph
O
S
R
R
(iii)
O
N
N N
+
CN
+
NC
Ar
O
Ar
N
H2N
N
H
N
S
3a-d
Ar;
: C6H5
b: 4-CH3-C6H4
: 4-CH3O-C6H4
1a-d
5
H
Ph
R;
6a-p
a
a
: H
R
Ar
C6H5-
R
Ar
b: Cl
c: Br
d
6
a
b
c
d
e
f
6
i
j
k
l
m
n
o
p
c
H
H
H
H
Cl
Cl
Cl
Cl
Br
Br
Br
Br
C6H5-
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
d: 4-ClC6H4
: OMe
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
OCH3 C6H5-
OCH3 4-CH3-C6H4-
OCH3 4-OCH3-C6H4-
OCH3 4-Cl-C6H4-
g
h
Scheme 1. Synthesis of target compounds 6a–p; Reagents and conditions: (i) CH3CN, DMF, NaH, benzene, reflux 4 h; (ii) Ethanol, phenylhydrazine, reflux 1 h; (iii) HOAc/
H2O (1:1 v/v), heating at 120 ꢀC, 8–11 h.

314
W. M. ELDEHNA ET AL.
absorption bands of the (NH) groups in the region compounds 6c, 6 g, 6k and 6 m–p were displayed as singlet sig-
3205–3387 cmꢁ1, in addition to the carbonyl bands in the region
nals in the range d 3.68–3.81 ppm, whereas, the methyl (-CH3) pro-
1709–1716 cmꢁ1 and CꢂN bands in the region 2202–2206 cmꢁ1
.
tons of compounds 6 b, 6f, 6j and 6n appeared as singlet signals
around d 2.35 ppm. Moreover, 13 C NMR spectra of spirooxindoles
6a–p showed signals resonating in the range d 177.72–178.10 ppm
attributable for the carbon of the carbonyl (C¼O) groups, whereas
the carbons of the methoxy (-OCH3) groups of compounds 6 g, 6k
and 6 m–o and carbons of the methyl (-CH3) groups of com-
pounds 6 b, 6j and 6n appeared as two signals around d 56.0 and
21.4 ppm, respectively.
1H NMR spectra of 6a–p showed two singlet D2O-exchangeable
signals attributable to two (NH) protons at range d 10.09–10.52
and 10.45–10.89 ppm. In addition, the methoxy (-OCH3) protons of
Table 1. In vitro anti-proliferative activity of the newly synthesized spirooxin-
doles 6a–p against MDA-MB-231 cell line.
H
N
S
R
O
N
Biological evaluation
NC
Ar
N
In vitro anti-proliferative activity
N
H
The in vitro anti-proliferative activity of the newly synthesized spi-
rooxindoles 6a–p was examined against TNBC MDA-MB-231 cells.
This assay was performed utilizing the 3–(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as
described by Mosmann18. Adriamycin was included in this assay as
a reference drug. The results were expressed as median growth
inhibitory concentration (IC50) values that represent the compounds
concentrations required to afford a 50% inhibition of cell growth
after 48 h of incubation, compared to untreated controls (Table 1).
As shown in Figure 2, morphological assessment using bright
field phase contrast microscopy revealed adverse effects on cell
morphology such as monolayer disruption in addition to cell
shrinkage and rounding, as indicated by arrows.
From the obtained results, it was obvious that various prepared
spirooxindoles displayed excellent to modest growth inhibitory
activity against MDA-MB-231 cells. Spirooxindoles 6a, 6e and 6i
emerged as the most potent analogues towards TNBC MDA-MB-
231 cells through this study with IC50 ¼ 6.70, 6.40 and 6.70 mM,
respectively. Besides, compounds 6d, 6f, 6 h, 6j, 6k and 6 m were
moderately active against MDA-MB-231 cells with IC50 range
(12.00 – 18.20 mM). While, spirooxindoles 6 b, 6c, 6 g and 6 l
Ph
6a-p
IC50 (mM)
MDA-MB-231
Compound
R
Ar
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
C6H5-
6.70
29.70
37.80
12.00
6.40
18.20
24.10
16.60
6.70
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-OCH3-C6H4-
4-Cl-C6H4-
Cl
Cl
Cl
Cl
Br
Br
Br
Br
OCH3
OCH3
OCH3
OCH3
13.50
12.70
30.60
17.50
NAa
NAa
NAa
Adriamycin
0.12
aNA: Compounds having IC50 value >50 mM.
Figure 2. Photomicrographs showing morphological assessment of the effects of compounds 6a and 6e on MDA-MB-231 cell monolayers. Cells were treated with
vehicle (DMSO, 0.1%) or increasing concentrations of the compounds (50, 25, 12.5, 6.25 and 3.125mM). Arrows indicate morphological signs of cytotoxicity including
R
cell rounding and/or disintegrated monolayer compared to the DMSO control. Photomicrographs were taken using ZeissVPrimovert (Carl Zeiss Microscopy GmbH,
Gottingen, Germany) equipped with a digital camera. Total magnification is 150ꢄ.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
315
possessed weak anti-proliferative activity against MDA-MB-231 their own termination. Accordingly, cellular integrity is conserved
cells (IC50 ¼ 29.70, 37.80, 24.10, and 30.60 mM, respectively), com- by this finely tuned, self-automated death23. Consequently, the
pounds 6n–p did not display growth inhibitory activity against success of cancer cells to proliferate unconditionally is allied to its
MDA-MB-231 cells.
ability to halt apoptosis. Thus, targeting apoptosis induction is a
successful strategy for combating tumour progression.
To further elucidate the mechanism of cell death induced by
the target spirooxindoles and as a part of our ongoing efforts to
develop novel pro-apoptotic agents21,22,24–26, we evaluated ability
of compounds 6a and 6e to provoke apoptosis in MDA-MB-231
cells through determination of the hallmark parameters of
apoptosis.
Structure activity relationship (SAR)
Based on the aforementioned results of the biological anti-prolif-
erative activity assay, important structure activity relationships
could be deduced. Firstly, we investigated the impact of the C-5
substitution of the indoline moiety. The abolished activity of 5-
methoxyindoline derivatives 6n–p along with the decreased IC50
value of 6m, with incorporated unsubstituted phenyl group,
(17.50 mM) than that of its corresponding members 6a, 6e and 6i,
with unsubstituted, 5-chloro substituted and 5-bromo substituted
indoline moiety, (6.70, 6.40 and 6.70 mM, respectively) indicated
that unsubstitution or C-5 substitution of the indoline moiety with
electron withdrawing groups (EWGs), as 5-Cl or 5-Br, is more bene-
ficial than incorporation of electron-donating substituents, as 5-
OCH3, to the growth inhibitory activity against MDA-MB-231 cells.
We then examined the effect of the substitution of the pendant
phenyl group at C-6 of pyrazolo[3,4-b]pyridine moiety on the pre-
pared spirooxindoles activities. Incorporation of unsubstituted phe-
nyl group led to compounds 6a (IC50 ¼ 6.70 mM), 6e (IC50 ¼ 6.40 mM),
6i (IC50 ¼ 6.70 mM) and 6m (IC50 ¼ 17.50 mM) with superior activity to
their corresponding 4-substituted-phenyl analogous 6c–d (IC50
range: 12.00–37.80mM), 6f–h (IC50 range: 16.60–24.10mM), 6j–l
(IC50 range: 12.70–30.60 mM) and 6n–p (IC50 > 50mM) against MDA-
MB-231 cells, implying that incorporation of unsubstituted phenyl
group is indispensable for the anti-proliferative activity.
Effects on the levels of active caspase-3
Caspases, a family of cysteineaspartic proteases, are the crucial
apoptosis mediators that provide essential links in cell regulatory
networks controlling cell death. Caspase-3 is the key executioner
caspase which modifies proteins ultimately responsible for apop-
tosis27. Accordingly, the effect of spirooxindoles 6a and 6e on the
level of caspase 3 was evaluated, to give insight to the pro-apop-
totic effect of the prepared spirooxindoles (Table 3).
Results in Table 3 showed that treatment of MDA-MB-231 cells
with compounds 6a and 6e resulted in a significant elevation in
the level of active caspase-3 by about 31.5 and 36.5 folds, respect-
ively, compared to control.
Effects on mitochondrial apoptosis pathway (Bcl-2 family) proteins
Bcl-2 family comprises a group of crucial regulatory factors in
apoptosis that finely tune the apoptotic switch on/off mechanism.
Based on their functional and structural criteria, the members are
divided into two major classes; group I proteins that are anti-
apoptotic and group II proteins that are apoptotic. Group I anti-
apoptotic proteins exert their function by inhibiting group II apop-
totic proteins through simply binding to them. Group I proteins
bind selectively to the active conformations of group II proteins to
prevent them from being inserted into the mitochondria and thus
cease the release of pro-apoptotic factors such as cytochrome c,
ultimately aborting apoptosis [25]. Thus, the inhibition of group I
proteins and/or the activation of group II proteins can successfully
induce apoptosis. Herein, we evaluated the impact of compounds
6a and 6e on the level of Bcl2, as a representative group I mem-
ber, and the level of Bax, as a representative group II member
(Table 3).
Finally, we can deduce that the substitution pattern over the
synthesized spirooxindoles is a crucial element for the anti-prolif-
erative activity towards TNBC MDA-MB-231 cell line. Incorporation
of unsubstituted or 5-EWG-substituted indoline moiety along with
unsubstituted phenyl group at C-6 of pyrazolo[3,4-b]pyridine moi-
ety greatly enhances the growth inhibitory activity of the target
spirooxindoles 6a–p.
In vitro cytotoxicity towards human normal WI-38 cells
Three potent compounds 6a, 6e and 6i were evaluated for their
ability to induce cytotoxic effect against human normal lung fibro-
blast cell line (WI-38 cells), to investigate their safety adopting the
MTT assay procedures18. The results, expressed as (IC50) values,
and the calculated selectivity index were displayed in Table 2.
The tested spirooxindoles 6a, 6e and 6i showed non-significant
cytotoxic effect with IC50 values of 78.1, 43.2 and 39.3 mM, respect-
ively, thereby providing a high-safety profile as anti-proliferative
agents with good selectivity index rang (11.7, 6.8 and 5.9
respectively).
As presented in Table 3 compound 6a induced the protein
expression of Bax with 506.8 folds of the control while 442.1 folds
were recorded with compound 6e. Parallel to this, the protein
expression of the antiapoptotic marker Bcl-2 was down-regulated
to 14.7% compared to that of the basal level in the control by
compound 6a while compound 6e produced down-
Apoptosis induction in TNBC MDA-MB-231 cells
Cell-cycle analysis
Antiproliferative agents abort cell growth by arresting its prolifer-
ation at certain well-known checkpoints. Distinguish cells in
Apoptosis is a programed routine that proves to be an essential
physiological process for tissue development, immune response,
redundant cells clearance and homeostasis by which cells signal
Table 3. Effect of compounds 6a and 6e on the active caspases-3 level, and the
expression levels of Bcl-2 and Bax in MDA-MB-231 cancer cells treated with each
compound at its IC50 concentration.
Table 2. In vitro cytotoxic activity of compounds 6a, 6e and 6i against WI-38
cells, and Selectivity index for the tested compounds.
IC50 (mM)
Comp.
Caspase-3 (ng/ml)
Bax (Pg/ml)
Bcl-2 (ng/ml)
Compound
WI-38
MDA-MB-231
Selectivity Index
ꢅ
ꢅ
ꢅ
6a
6e
Control
ꢅ
0.3501 (31.5)
0.4058 (36.5)
0.0111
405.5 (506.8)
353.7 (442.1)
0.80
0.3958 (0.147)
0.7449 (0.276)
2.692
ꢅ
ꢅ
ꢅ
6a
6e
6i
78.1
43.2
39.3
6.7
6.4
6.7
11.7
6.8
5.9
Numbers given between parentheses are the number of folds of control.

316
W. M. ELDEHNA ET AL.
Figure 3. DNA-flow cytometry analysis for MDA-MB-231 cells treated with compound 6e for 24 h at their IC50 concentrations. The experiments were done in triplicates.
various phases of cell cycle can be detected upon treatment of
In conclusion, the enhanced expression of the pro-apoptotic
cancer cells with anticancer agents28. Compound 6e was investi- protein Bax and the reduced expression of the anti-apoptotic pro-
gated for its activity to disrupt the cell cycle of MDA-MB-231 tein Bcl-2 as well as the up-regulated active caspase-3 level
cancer cell lines. This effect was illustrated by DNA flow cytomet- together with a harmonized increase in the Bax/Bcl-2 ratio, high-
ric analysis which MDA-MB-231 cells was treated with compound lighted that the anti-proliferative activity of the target spirooxin-
6e at concentration equals to the IC50 for 24 h. Figure 3 showed doles 6 might be attributed, at least in part, to the induction of
that compound 6e expressed significant decrease in the G0-G1 the intrinsic apoptotic mitochondrial pathway.
phase by approximately 0.5 folds related to the control.
Compound 6e displayed no significant change in the S phase
In vitro EGFR kinase ELISA assay
while G2-M phase was arrested by 2.65 folds with 19.1% with
respect to control (7.2%). Alteration of the Pre-G phase and
arrest of G2-M phase were significant remarks for compound 6e
to induce apoptosis.
On account of its overexpression in a significant number of
TNBC, epidermal growth factor receptor (EGFR) emerged as an
attractive target for developing effective therapeutic strategies
for treatment of TNBC patients28–30. In this study the most
potent anti-proliferative spirooxindoles 6a, 6d, 6e and 6i–k were
selected to evaluate their potential inhibitory activity against
Annexin V-FITC apoptosis assay
Externalization of the phospholipid phosphatidylserine at the cell EGFR by use of a colorimetric Enzyme-Linked Immunosorbent
membrane is a one of the well-recognized hallmarks of cells going Assay (ELISA). Erlotinib, a clinically used EGFR inhibitor, was
into apoptosis17,29. In our study, the apoptotic effect of compound taken as the reference drug. The results were reported as a 50%
6e was further assessed by Annexin V-FITC/PI (AV/PI) dual staining inhibition concentration values (IC50) which determined as tripli-
assay to examine the occurrence of phosphatidylserine externaliza- cate determinations from the standard curve and summarized in
tion and also to comprehend whether cell death is due to physio- Table 4.
logical apoptosis or nonspecific necrosis (Figure 4).
Results revealed that the tested compounds exhibited EGFR
Flow cytometric analysis revealed that MDA-MB-231 cells inhibitory activity with IC50 values ranging from 0.12 to 0.51 lM.
treated with compound 6e displayed a significant increase in the Compound 6i emerged as the most potent EGFR inhibitor in this
percent of annexin V-FITC positive apoptotic cells (UR þ LR) from study that showed comparable potency (IC50 ¼ 0.12 0.01 lM) to
1.34% to 44% which comprises about 32.8 folds compared to the reference drug Erlotinib (IC50 ¼ 0.11 0.01 lM). Besides, com-
control.
pound 6e displayed good activity (IC50 ¼ 0.15 0.02 lM).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
317
Figure 4. Effect of compound 6e on the percentage of annexin V–FITC-positive staining in MDA-MB-231 cells. The four quadrants identified as: LL: viable; LR: early
apoptotic; UR: late apoptotic; UL: necrotic. The experiments were done in triplicates.
(WI-38 lung fibroblast) where none of them displayed significant
cytotoxic effect, thereby providing good safety profile.
Table 4. IC50 values for the inhibitory activity of spiroox-
indoles 6a, 6d, 6e and 6i–k against – EGFR.
a
IC50 (lM)a
Subsequently, 6a and 6e were further estimated for their apop-
tosis induction potential. Both proved to induce apoptosis, which
evidenced via the reduced expression of the anti-apoptotic protein
Bcl-2 in addition to the enhanced expression of the pro-apoptotic
protein Bax as well as the up-regulated active caspase-3 level.
Moreover, 6e displayed a significant increase in the percent of
annexin V-FITC positive apoptotic cells from 1.34% to 44% which
comprises about 32.8 folds compared to control. As the EGFR is
frequently overexpressed in TNBC, six potent spirooxindoles was
assayed for their potential inhibitory activity towards EGFR.
Compounds 6e and 6i displayed potent inhibitory activity against
EGFR with IC50 values of 120 and 150 nM, respectively.
Compound
EGFR
6a
6d
6e
6i
6j
6k
Erlotinib
0.43 0.04
0.36 0.02
0.15 0.02
0.12 0.01
0.51 0.04
0.31 0.02
0.11 0.01
aIC50 values are the mean SD of three separate
experiments.
Conclusions
In summary, we have synthesized a novel series of sixteen spiroox-
indoles 6a–p through a one-pot three-component reaction, with
the prime aim of developing potent anti-TNBC agents. All the
newly synthesized spirooxindoles 6a–p was evaluated for their in
vitro anti-proliferative activity towards TNBC MDA-MB-231 cells.
Spirooxindoles 6a, 6e and 6i were the most potent members
against MDA-MB-231 cells with IC50 ¼ 6.70, 6.40 and 6.70 mM,
respectively. Besides, compounds 6d, 6f, 6h, 6j, 6k and 6m were
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors would like to extend their sincere appreciation to the
moderately active against MDA-MB-231 cells with IC50 rang (12.00 Deanship of Scientific Research at King Saud University for its
– 18.20 mM). Moreover, the cytotoxicity of the active counterparts funding of this research through the Research Group Project no.
6a, 6e and 6i was examined against normal human cell line RG-1436-038.

318
W. M. ELDEHNA ET AL.
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