Design, synthesis and biological evaluation of novel indolin-2-ones as potent anticancer compounds

Article abstract of DOI:10.1016/j.bmcl.2017.06.019

The indolin-2-one core is a privileged structure for antitumor agents, especially kinase inhibitors. Twenty-three novel indolin-2-ones were designed by molecular dissection of the anticancer drug indirubin. Seventeen of them exhibited significant inhibition against the tested cell lines, and two of them (1c and 1h) showed IC₅₀ values at the submicromolar level against HCT-116 cells. Compounds 1c and 2c were also potent inhibitors of the triple-negative breast cancer (TNBC) cell line MDA-MB-231. Flow cytometry was utilized to explore the antitumor mechanism of 1c and 2c with MDA-MB-231 cells, and distinct effects were observed on 2c. Furthermore, immunocytochemical examination of 1c suggested a destabilization of microtubules, which was significantly different from the effect of IM, an indirubin derivative.

Full text of DOI:10.1016/j.bmcl.2017.06.019

Accepted Manuscript
Design, synthesis and biological evaluation of novel indolin-2-ones as potent
anticancer compounds
Andong Zhou, Lei Yan, Fangfang Lai, Xiaoguang Chen, Masuo Goto, Kuo-
Hsiung Lee, Zhiyan Xiao
PII:
S0960-894X(17)30621-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.019
BMCL 25053
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 April 2017
16 May 2017
4 June 2017
Please cite this article as: Zhou, A., Yan, L., Lai, F., Chen, X., Goto, M., Lee, K-H., Xiao, Z., Design, synthesis and
biological evaluation of novel indolin-2-ones as potent anticancer compounds, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.019
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, synthesis and biological evaluation of
novel indolin-2-ones as potent anticancer
compounds
Leave this area blank for abstract info.
Andong Zhou, Lei Yan, Fangfang Lai, Xiaoguang Chen, Masuo Goto, Kuo-Hsiung Lee, Zhiyan Xiao

Bioorganic & Medicinal Chemistry Letters
Design, synthesis and biological evaluation of novel indolin-2-ones as potent
anticancer compounds
a,1
a,1
b
b
c
Andong Zhou , Lei Yan , Fangfang Lai , Xiaoguang Chen , Masuo Goto , Kuo-
c,d
a,
Hsiung Lee and Zhiyan Xiao 
a
Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China
b
Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Sciences
&
Peking Union Medical College, Beijing 100050, PR China
c
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568, USA
Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
d
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The indolin-2-one core is a privileged structure for antitumor agents, especially kinase
inhibitors. Twenty-three novel indolin-2-ones were designed by molecular dissection of the
anticancer drug indirubin. Seventeen of them exhibited significant inhibition against the tested
cell lines, and two of them (1c and 1h) showed IC50 values at the submicromolar level against
HCT-116 cells. Compounds 1c and 2c were also potent inhibitors of the triple-negative breast
cancer (TNBC) cell line MDA-MB-231. Flow cytometry was utilized to explore the antitumor
mechanism of 1c and 2c with MDA-MB-231 cells, and distinct effects were observed.
Keywords:
Indolin-2-one
Privileged structure
Cytotoxicity
Furthermore, immunocytochemical examination of 1c suggested
a destabilization of
microtubules, which was significantly different from the effect of IM, an indirubin derivative.
2
017 Elsevier Ltd. All rights reserved.
Molecular mechanism
—
——
*
Corresponding author. Tel.: +86-10-63189228.
E-mail address: xiaoz@imm.ac.cn (Z. Xiao).
These authors contribute to this work equally.
1

2
3
The indolin-2-one core is regarded as a privileged structure
for antitumor agents, in particular, kinase inhibitors, and
numerous indolin-2-one derivatives have been reported as potent
Sunitinib and Toceranib phosphate have been approved for
4 5
cancer therapy by the US FDA, and SU5416 , SU6668 , SU5614,
SU14813, SU9516, and SU4984 have been investigated in either
clinical trials or preclinical development.
1
1
kinase inhibitors . Among the examples shown in Figure 1,
Figure 1. Representative indolin-2-ones as kinase inhibitors
Indirubin (Figure 2) is a natural product with the bis-indole
scaffold. As the active ingredient of the traditional Chinese
prescription Danggui Longhui Wan, indirubin is used to treat
6,7
chronic myeloid leukemia in China . It is also reported to
8
possess various biological activities, including anti-HIV , anti-
9
10,11
angiogenesis and anti-inflammatory
effects. However, the
therapeutic application of indirubin and its derivatives is often
hampered by their poor solubility, which is at least in part
attributed to the bis-indole scaffold.
We have previously explored the SAR of a series of indirubin-3'-
12
Figure 2. Structure of indirubin and IM
monoxime (IM, Figure 2) derivatives . As a follow-up study and
also to expand the SAR of this compound class, two series of
indolin-2-one derivatives were designed by molecular dissection
of indirubin (Figure 3). Series 1 was designed by dividing
between N1' and C2' to provide a 2-oxo-2-phenylethylidene side
chain. From previous SAR studies, fluoro-substitution on ring B
1
2
might be favored for antitumor activity . Thus, fluorine was
1
maintained at R for most series 1 compounds and substituted
2
phenyl and other aromatic rings were introduced in R . In
compound 1a, an isosteric OH group replaced the residual N1'
amino. In contrast, series 2 was obtained by breaking the
covalent bond between C2' and C3' to leave a (phenylamino)-
methylene side chain. To enhance water-solubility, 3-(p-
4
methylpiperazinyl) phenyl was incorporated as R in series 2
Figure 3. Molecular design of the target compounds based on indirubin
compounds, whereas various aromatic and aliphatic substituents
3
were introduced as R .
The series 1 and 2 compounds were prepared following
one (6, Scheme 2). After treating 6 with ethylorthoformate and
acetic anhydride, the enol ether intermediate 7 was obtained, and
7 was subsequently converted into the enamine 8. Hydrolysis of
the acetyl and reduction of the nitro group in 8 afforded
intermediate 10, which was reacted with different acids to give
series 2 compounds.
13,14
previously published protocols
as presented in Schemes 1 and
2
1
. Briefly, nucleophilic addition of indoline-2,3-dione 3 (Scheme
) and various ethanones 4 provided the key intermediate 5.
2
Elimination of H O from 5 produced series 1 compounds. The
preparation of series 2 compounds began from 5-nitroindolin-2-
2 2 5 2 5
Scheme 1. Synthesis of compounds 1a-1p. Reagents and conditions: (a) Et NH, C H OH, r.t.; (b) hydrochloric acid, C H OH, 60 C, 2-24h.

Scheme 2. Synthesis of compounds 2a-2g. Reagents and conditions: (a) CH(OEt)
3
2 2
, acetic anhydride, 150 C, 1h; (b) R NH , dry DMF, 110 C, 10h; (c) 1 M
5
NaOH (aq), CH
3
OH, r.t., 3-4 h; (d) Pd/C, H
2
, CH
3
OH/CH
2
Cl
2
, r.t.; (e) R COOH, HATU, triethylamine, DMSO, r.t.
All compounds in both series 1 and 2 were obtained as a single
lines, and compounds 1c and 1h exhibited submicromolar IC50
stereoisomer. The downfield chemical shifts of H-4 and H-2'
values against HCT-116 cells. According to the data given in
Table 1, it appears that R can accommodate a range of different
substituents and might have little effect on the tumor inhibitory
1
(
Figure 3) suggested an (E)-configuration in series 1 compounds
15,16
,
which was further confirmed by the lack of correlation
between H-4 and H-2' in the NOESY spectra of compound 1c
Supporting Data). Similarly, the stereochemistry of compounds
activity of this compound class (1e vs 1f, 1g vs 1h). In contrast,
2
(
the R substituents have a significant impact on the tumor
in series 2 was assigned as (Z)-configuration based on the
downfield chemical shifts of H-4 and the apparent correlation
between H-4 and H-2' in compound 2c (Supporting Data).
inhibitory activity, although no obvious trends could be deduced
from the currently available data. Five compounds in series 2
also displayed significant inhibitory activities against the tested
tumor cell lines (Table 2). As observed in series 1, many various
5
To explore their potential as antitumor agents, compounds in
series 1 and 2 were tested in an MTT assay for their inhibitory
activity against HCT-116, HepG2, BGC-823, NCI-H1650 and
A2780 tumor cell lines. The results are shown in Tables 1 and 2.
With regard to series 1 in Table 1, ten compounds showed
significant inhibitory activity against all the tested tumor cell
substituents were well-tolerated at R in series 2. However,
aliphatic substituents and heteroaromatic rings might be
unfavorable for the cytotoxicity of this compound class (2a-2d vs
2e-2g). Surprisingly, the indirubin derivative IM was tested in
parallel and was inactive against all the tested cell lines.
Table 1
Inhibitory activity of compounds 1a-1p against selected tumor cell lines by the MTT assay
1a-1p
IC50 (μM)
BGC-823
0.001
1
2
Compd.
R
R
#
HCT-116
0.03
HepG-2
0.02
NCI-H1650
0.07
A2780
0.03
Taxol
IM
-
-
-
-
*
NA
NA
NA
NA
NA
1
a
F
F
F
NA
1.07
0.76
NA
3.44
2.28
NA
6.12
4.09
NA
3.46
1.22
NA
1.58
1.12
1
b
1
c
1
d
F
F
NA
2.52
2.32
NA
2.84
3.99
NA
4.45
5.90
NA
2.73
3.86
NA
2.31
2.09
1
e
f
1
NO
2

1
1
g
F
3.01
0.86
1.86
2.11
8.99
9.44
6.58
1.27
2.44
2.08
h
H
1
1
i
F
F
NA
NA
NA
NA
NA
j
1.38
3.12
6.42
2.84
2.17
1
k
F
F
NA
NA
NA
NA
NA
1
l
1.84
8.78
7.75
3.22
2.75
1
m
F
2.45
3.10
2.66
3.56
2.14
1
n
o
F
F
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1
1
p
F
1.41
3.09
8.02
2.73
2.37
*
NA: not active. No significant inhibition against tumor cell growth was observed at the concentration of 10 μM.
#
HCT-116 (human colon cancer), HepG2 (liver hepatocellular cells), BGC-823 (human gastric cancer cell line), NCI-H1650 (non-small-cell carcinoma), A2780
(human ovarian cancer).
Table 2
Inhibitory activity of compounds 2a-2g against selected tumor cell lines by the MTT assay
2
a-2g
IC50 (μM)
BGC-823
5
Compd.
R
#
HCT-116
0.03
HepG-2
0.02
NCI-H1650
0.07
A2780
0.03
Taxol
IM
-
-
0.001
NA
*
NA
NA
NA
NA
2
a
2.01
1.78
3.17
1.87
2.05
2
b
2.74
6.72
8.50
4.28
2.91
2
c
2.15
3.03
2.67
1.63
2.03
2
d
2.28
NA
2.37
5.37
2.24
NA
2.17
7.56
2.14
4.46
2
e
2
f
9.41
4.67
5.07
NA
6.08
NA
5.87
NA
3.37
NA
2
g

*
NA: not active. No significant inhibition against tumor cell growth was observed at the concentration of 10 μM.
#
HCT-116 (human colon cancer), HepG2 (liver hepatocellular cells), BGC-823 (human gastric cancer cell line), NCI-H1650 (non-small-cell carcinoma), A2780
(human ovarian cancer).
1
2
Compounds 1c and 2c, the most active compound from each
series, were selected as representative compounds for further
evaluation against MDA-MB-231, MCF-7, A594, KB and KB-
vin cells in a sulforhodamine B (SRB) assay, and IM was tested
in parallel (Table 3). Notably, all three compounds were potent
against MDA-MB-231, which are triple-negative breast cancer
Since IM is an effective inhibitor of CDK2 and CDK9 ,
compounds in series 1 and 2 were initially evaluated against
CDK2/Cyclin E1 and CDK9/Cyclin T1 at a concentration of 10
μM. However, neither compound inhibited either CDK system.
This observation implied that the molecular dissection of the bis-
indole scaffold in IM to the indolin-2-one core in 1c and 2c
resulted in significantly altered pharmacological profiles and an
implicit shift in molecular mechanism.
(TNBC) cells, a clinically aggressive form of breast cancer and
generally unresponsive to chemotherapies.
Table 3
Inhibitory activity of compounds 1c, 2c and IM against selected tumor cell lines by the SRB assay
IC50 (μM)
Compd.
#
MDA-MB-231
7.59 ± 0.71
MCF-7
A549
KB
KB-VIN
IM
6.81 ± 0.21
5.82 ± 0.05
5.34 ± 0.17
5.17 ± 0.07
1
2
c
c
5
.31 ± 0.22
>10
>10
>10
>10
4.43 ± 0.19
4.44 ± 0.10
4.75 ± 0.04
4.40 ± 0.14
7.86 ± 0.08
#
MDA-MB-231(human breast carcinoma cell line), MCF (Michigan Cancer Foundation-7), A549 (lung cancer), KB (nasopharyngeal carcinoma) and KBvin
vincristine-resistant KB subline).
(
To shed light on the underlying antitumor mechanism of
compounds 1c and 2c, their effects on cell cycle progression were
evaluated in the TNBC cell line MDA-MB-231 by employing
1
7
flow cytometry . As depicted in Figure 4, the ratio of G2/M was
slightly increased in cells treated with compounds 1c and IM at a
concentration five-fold of the corresponding IC50 value. The sub-
G1 phase was dramatically increased in cells treated with
compound 2c, suggesting that 2c might induce apoptosis quickly
in a target-independent manner. Furthermore, cells treated with
Figure 4. Effects of compounds 1c, 2c and IM on cell cycle progression.
TNBC MDA-MB-231 cells were treated for 24 h with compounds at a
concentration of five-fold the IC50 value. Combretastatin A-4 (CA-4) was
used at 200 nM as a reference for induction of cell cycle arrest in G2/M. Cells
were fixed and stained with propidium iodide (PI) followed by analysis of
cell cycle using a flow cytometer.
1c and IM were observed with confocal fluorescence microscopy
to examine their effects on microtubules (Figure 5). The
microtubule network in interphase cells (unstained by antibody to
phosphorylated histone H3) was clearly observed in control cells
(DMSO), while unclear in cells treated with 1c, suggesting that
1c destabilized microtubules. In contrast, pseudopodia elongation
was detected when the cells were treated with IM, suggesting no
significant effect on microtubule destabilization. These
observations implied different mechanisms of action for the two
compounds.
In summary, twenty-three novel indolin-2-one compounds were
designed, synthesized and evaluated for their cytotoxic activity.
Most compounds showed significant antiproliferative activity
against the tested cell lines, and notably, two compounds showed
potent inhibition against the TNBC cell line MDA-MB-231. To
explore the antitumor mechanism of this compound class, flow
cytometry and confocal microscopy were utilized and molecular
mechanisms different from that of IM were implied. The results
suggest that the privileged structure indolin2-one could be a good
chemical phenotype for both structural diversity and mechanistic
novelty.
Figure 5. Immunocytochemical examination of cells treated with compound
1
c and IM. TNBC MDA-MB-231 cells were treated for 24 h with compound
at a concentration five-fold the IC50 value. Cells were fixed and stained with
antibodies to α-tubulin (green) as a marker for the chromosome condensation,
and DAPI for DNA (blue). Phosphorylated histone H3 was undetectable in
interphase cells. Images were captured by Zeiss LSM700 confocal
fluorescence microscope using ×63 (A) or ×40 (B) objective lens. Bar, 25
µm.
Acknowledgments
We wish to thank the Microscopy Service Laboratory
(UNC-CH) for their expertise in confocal microscopy. This
investigation was supported by the National Natural Science
Foundation of China (81172985 and 81261120391) and CAMS

Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-009)
awarded to Z.X. This study was also supported in part by NIH
Grant CA177584 from the National Cancer Institute awarded to
K.H.L, and by the Eshelman Institute for Innovation, Chapel Hill,
North Carolina awarded to M.G.
5.
6.
Laird, A. D.; Vajkoczy, P.; Shawver, L. K.; Thurnher, A.; Liang,
C.; Mohammadi, M.; Schlessinger, J.; Ullrich, A.; Hubbard, S. R.;
Blake, R. A.; Fong, T. A.; Strawn, L. M.; Sun, L.; Tang, C.;
Hawtin, R.; Tang, F.; Shenoy, N.; Hirth, K. P.; McMahon, G.;
Cherrington, J. M. Cancer Res. 2000, 60, 4152.
Eisenbrand, G.; Hippe, F.; Jakobs, S.; Muehlbeyer, S. J Cancer
Res. Clin. Oncol. 2004, 130, 627.
7
8
.
.
Xiao, Z.; Hao, Y.; Liu, B.; Qian, L. Leuk. Lymphoma 2002, 43,
1
763.
Supplementary data
Lee, M. J.; Kim, M. Y.; Mo, J. S.; Ann, E. J.; Seo, M. S.; Hong, J.
A.; Kim, Y. C.; Park, H. S. Cancer Lett. 2008, 265, 215.
Supplementary data (All target compounds were characterized by
9.
Yoon, J. H.; Kim, S. A.; Kwon, S. M.; Park, J. H.; Park, H. S.;
Kim, Y. C.; Yoon, J. H.; Ahn, S.G. Biochim. Biophys. Acta. 2010,
1
melting point,
H
NMR, and mass spectra analyses.
1
800, 352.
Supplementary information on general preparation procedures
and spectroscopic data of the target compounds are available
online. Biological assays were performed by following
experimental protocols in the cited references and brief
descriptions) associated with this article can be found, in the
online version, at
1
1
0. Babcock, A. S.; Anderson, A. L.; Rice, C. D. Toxicol. Appl.
Pharmacol. 2013, 266, 157.
1. Pergola, C.; ꢀaboriaꢁd-ꢂoꢃarꢄ ꢅ.ꢆ ꢇestꢈdtꢄ ꢅ.ꢆ ꢂꢉniꢊ, S.;
Kritsanida, M.; Schaible, A. M.; Li, H.; Garscha, U.; Weinigel, C.;
Barz, D.; Albring, K. F.; Huber, O.; Skaltsounis, A. L.; Werz, O.;
Albring, K. F. J. Med. Chem. 2014, 57, 3715.
1
1
2. Yan, L.; Lai, F.; Chen, X.; Xiao, Z. Bioorg. Med. Chem. Lett.
2
015, 25, 2447.
3. Klöck, C.; Jin, X.; Choi, K.; Khosla, C.; Madrid, P. B.; Spencer,
A.; Raimundo, B. C.; Boardman, P.; Lanza, G.; Griffin, J. H.
Bioorg. Med. Chem. Lett. 2011, 21, 2692.
References and notes
1
.
Prakash, C. R.; Theivendren, P.; Raja, S. Pharm. Pharmacol.
012, 3, 62.
Carter S. K. Oncologist. 2000, 5 Suppl 1, 51.
London, C. A.; Malpas, P. B.; Wood-Follis, S. L.; Boucher, J. F.;
Rusk, A. W.; Rosenberg, M. P.; Henry, C. J.; Mitchener, K. L.;
Klein, M. K.; Hintermeister, J. G.; Bergman, P. J.; Couto, G. C.;
Mauldin, G. N. and Michels, G. M. Clin. Cancer Res. 2009, 15,
14. Bramson, H. N.; Corona, J.; Davis, S. T.; Dickerson, S. H.;
Edelstein, M.; Frye, S. V.; Gampe, R. T. Jr; Harris, P. A.; Hassell,
A.; Holmes, W. D.; Hunter, R. N.; Lackey, K. E.; Lovejoy, B.;
Luzzio, M. J.; Montana, V.; Rocque, W. J.; Rusnak, D.;
Shewchuk, L.; Veal, J. M.; Walker, D. H.; Kuyper, L. F. J. Med.
Chem. 2001, 44, 4339.
2
2
3
.
.
15. Faita, G.; Mella, M.; Righetti, P.; Tacconi, G. Tetrahedron 1994,
50, 10955.
3
856.
4
.
Mendel, D. B.; Laird, A. D.; Smolich, B. D.; Blake, R. A.; Liang,
C.; Hannah, A. L.; Shaheen, R. M.; Ellis, L. M.; Weitman, S.;
Shawver, L. K.; Cherrington, J. M. Anticancer Drug Des. 2000, 15,
16. Klöck, C.; Jin, X.; Choi, K.; Khosla, C.; Madrid, P. B.; Spencer,
A.; Raimundo, B. C.; Boardman, P.; Lanza, G.; Griffin, J. H.
Bioorg. Med. Chem. Lett. 2011, 21, 2692.
2
9.
17. Nakagawa-Goto, K.; Oda, A.; Hamel, E.; Ohkoshi, E.; Lee, K. H;
Goto, M. J. Med. Chem. 2015, 58, 2378.