Design, synthesis, and biological evaluation of novel 3-pyrrolo[b] cyclohexylene-2-dihydroindolinone derivatives as potent receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.08.037

A novel series of 3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives targeting VEGFR-2, PDGFR-β and c-Kit kinases were designed and synthesized. The molecular design was based on the SAR features of indolin-2-ones as kinase inhibitors. SAR study of the series allowed us to identify compounds possessing more potent inhibitory activities against the three kinases than sunitinb with IC₅₀ values in the low nanomolar range in vitro. Additionally, some compounds also showed favorable antiproliferative activities against a panel of cancer cell lines (BXPC-3, T24, BGC, HEPG2 and HT29).

Full text of DOI:10.1016/j.bmcl.2013.08.037

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5630–5633
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of novel
3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives
as potent receptor tyrosine kinase inhibitors
Lei Ding a,b, , Feng Tang b, , Wei Huang b, Qiu Jin b, Han Shen b, Ping Wei a,
⇑
a The College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 30 Puzhu Road(S), Nanjing 211800, China
b Jiangsu Simcere Pharmaceutical Co., Ltd, Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18 Xuan Wu Avenue, Nanjing 210042, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives targeting VEGFR-2, PDGFR-
b and c-Kit kinases were designed and synthesized. The molecular design was based on the SAR features
of indolin-2-ones as kinase inhibitors. SAR study of the series allowed us to identify compounds possess-
ing more potent inhibitory activities against the three kinases than sunitinb with IC50 values in the low
nanomolar range in vitro. Additionally, some compounds also showed favorable antiproliferative activi-
ties against a panel of cancer cell lines (BXPC-3, T24, BGC, HEPG2 and HT29).
Received 28 April 2013
Revised 21 July 2013
Accepted 7 August 2013
Available online 15 August 2013
Keywords:
Receptor tyrosine kinase
Inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
2-Dihydroindolinone derivatives
Antiproliferative
Molecular docking
Over the past 15 years, protein kinases have become the most
important drug targets in the field of anti-tumor drugs. So far,
more than 20 kinase-targeted drugs have been used in clinical
treatment, and hundreds of kinase-targeted candidates are in clin-
ical trials.1 Receptor tyrosine kinases (RTKs) play important roles in
intracellular signal transduction. RTKs modulate cellular responses
to external stimuli and influence a series of biological processes of
cells, such as cell cycle, migration, metabolism, survival, prolifera-
tion and differentiation, etc.2 Normally, signal transduction of RTKs
is strictly regulated.3 However, abnormal expression or activation
of RTKs, especially excessive activations of the VEGF/VEGFR (vas-
cular endothelial growth factor receptor) signaling pathway,4
PDGF/PDGFR (platelet derived growth factor receptor) signaling
pathway5,6 and SCF/Kit signaling pathway,7 will lead to series of
tumor-related events, such as carcinogenesis, tumor angiogenesis,
cancer metastasis, as well as disease progression and poor progno-
sis.3,8 As cancer is caused by an accumulation of mutations in dif-
ferent genes, its growth and survival depend on more than one
receptor or one signaling pathway. So theoretically multi-targeting
drugs might show better curative effect than highly selective sin-
gle-targeting agents. Furthermore, using a multi-targeting drug
can avoid the issue of drug-drug interactions which is often a con-
cern when combinations of single-targeting drugs are used. In fact,
developing multi-targeting drugs has been considered to be a
promising strategy in cancer therapy,9,10 of which some VEGFR
inhibitors have been developed in recent years, such as Sorafenib11
Sunitinib12, Axitinib13 and Regorafenib14 (Fig. 1).
,
Among numerous ATP-competitive VEGFR inhibitors15,16, the
scaffold of Sunitinib, 3-[(substituted pyrrol-2-yl)methylide-
nyl]indolin-2-one has been extensively studied and the struc-
ture–activity relationships (SAR) are described clearly based on
lots of indolin-2-one derivatives (Fig. 2).17–24 All these compounds
5–13 keep the similar structural features: Firstly, the amide frag-
ments of indolin-2-ones, as the key pharmacophore, provide criti-
cal hydrogen bond acceptor and donor to interact with the amino
acid residues of the kinase hinge region and remain unsubstituted
in all structures. Secondly, the double bond outside indolin-2-one
ring retains cis-configuration. Moreover, the intramolecular hydro-
gen bond is formed between oxygen atom of the indolin-2-one and
NH of the pyrrole ring to stabilize the planar conformation of
whole molecule.16,25 Furthermore, three-dimensional model of
SU5402 at the binding site of FGFR shows a highly conservative
phenylalanine residue (Phe489) in the loop extends toward the
indolin-2-one moiety, capping the hydrophobic pocket in which
the indolin-2-one anchors.26 There is enough space available to
accommodate a larger group between exocyclic methylene and
Phe489. Recently, Kim et al.,24 reported a series of indolin-2-one
structures with methylene substituted by phenyl, among which
compound 13 showed very strong inhibitory activities against
⇑
Corresponding author. Tel.: +86 25 58139908.
E-mail addresses: dinglei@simcere.com (L. Ding), weiping@njut.edu.cn (P. Wei).
VEGFR-2 (IC50 = 10 nM), FGFR-1 (IC50 = 11 nM) and PDGFR-
a
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.037

L. Ding et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5630–5633
5631
conformation similar to sunitinib and it’s analogs 5–13. In addition,
free rotation of the single bond between the exocyclic double bond
and 50 position of the pyrrole ring was further restricted by
introducing a rigid six-membered ring which would help to make
pharmacophoric conformation more stable. Our primary objective
was to maintain the planar conformation of the molecule by intro-
ducing a rigid ring structure while retaining the key intramolecular
hydrogen bond, and improve the biological activities of these novel
scaffold compounds as kinase inhibitors. Additionally, this study
helped to expand the structural diversity of indolin-2-ones as
kinase inhibitors and establish more specific structure–activity
relationships.
O
N
HN
N
H
3'
Cl
O
4'
5'
O
O
2'
N
N
H
F
F3C
N
H
N
H
O
N
H
1, Sorafenib
2, Sunitinib
NH
O
H
S
HN
N
N
Cl
F3C
O
O
N
O
N
N
H
H
F
Compound 18 is
a
key intermediate in building
N
3-pyrrolo[b]cyclohexylene-2-dihydroindolinones scaffold. Here-
in, 6-amino-5-oxohexanoic acid hydrochloride27, as the starting
material, was cyclized with ethyl acetoacetate to form pyrrole
ring 16, which underwent intramolecular dehydration to give
compound 17 in the presence of P2O5. Hydrolysis of compound
17 under alkaline condition produced 2-methyl-7-oxo-4,5,6,7-
tetrahydro-1H-indole-3-carboxylic acid 18. The substituted indo-
lin-2-ones 19 was either commercially available or easy to be
prepared according to the procedures described in literature.28,29
The final products were synthesized through the following steps:
firstly, the condensation of substituted indolin-2-ones with com-
pound 18 was catalyzed by TiCl4 in pyridine to generate interme-
diate 20; which reacted with primary or secondary amines in the
presence of coupling reagent EDCI/HOBt (Scheme 1) to afford the
final products 21–44. In addition, to verify the configuration of
the exocyclic double bond of the oxyindole ring, The two dimen-
sional (2D) NMR spectroscopy of compound 22 was conducted
which showed a ROESY correlation between proton 60 and proton
4. Accordingly, the double bond was assigned as configuration Z.
This configuration was further confirmed by single-crystal X-ray
diffraction of compound 22 methanol solvate as shown in Figure
3.30
The in vitro inhibitory activities against VEGFR-2, PDFR-b and
c-Kit of the synthesized compounds were shown in Table 1. Most
of 3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives
exhibited potent inhibitory activities. Herein, compound 22 exhib-
ited better inhibitory activities against VEGFR-2, PDGFR-b and
c-Kit than the reference compound, sunitinib, which indicated that
our design was reasonable. To further investigate the influence of
different substituents at the scaffold and gain insight into SAR for
this series of molecules, we firstly kept the R2 as N,N-diethyl ethy-
lenediamine and modified the substituents of indolin-2-ones moi-
ety. A total of 11 compounds 21–31 were prepared, whose
inhibitory activities against the VEGFR-2, PDGFR-b and c-Kit were
tested with IC50 values in the range of 2.3–330.3 nM, 7.6–395.7 nM
and 2.3–397.6 nM, respectively. The results indicated that chang-
ing the substituents on indolin-2-ones would significantly affect
the inhibitory activity of the compounds.
4, Regorafenib
3, Axitinib
Figure 1. Approved multi-targeted VEGFR inhibitors.
OH
O
O
OH
N
H
N
H
N
H
O
O
O
N
H
N
H
N
H
7, SU6668
5, SU5416
6
, SU5402
O
N
O
N
H
OH
N
H
N
H
O
F
O
O
N
H
N
H
N
H
N
H
8, SU14813
9
O
O
N
OH
N
O
H
N
n
N
H
F
N
H
N
H
O
n=1, 2, 3
O
N
N
H
H
10, NP506
11
OH
O
O
N
H
N
N
H
N
H
O
Br
N
N
H
O
N
H
12
13
Figure 2. Representative of 3-[(substituted pyrrol-2-yl)methylidenyl]indolin-2-
ones.
(IC50 = 6 nM). This further confirms that hydrophobic groups are
tolerated at the region between the 40 position of pyrrole ring
and exocyclic methylene of indolin-2-one.
Since the ATP-binding domain of FGFR, VEGFR-2 and PDGFR is
highly homological and conservative, the binding mode and SAR
based on FGFR can also be applied to other kinases.18 We designed
Introducing fluorine atom to the 4 or 7 position of the indolin-
2-ones moiety led to significant decrease of the inhibitory activities
to VEGFR-2, PDGFR-b and c-Kit. For example, the inhibitory activity
of compound 31 to all three kinases decreased significantly, with
IC50 all greater than 100 nM. Moreover, since the indolin-2-one
moiety stretched deeply into the ATP-binding pocket, the introduc-
tion of large substituents at 5 position, such as methyl carboxylic
ester 28 and sulfonyl diethylamine 29, would hinder the binding
of the ligand to receptor sterically and significantly reduce the
inhibitory activity. On the other hand, the compounds with smaller
substituents at the 5 position of indolin-2-ones, such as fluorine 22,
chlorine 23, bromine 24 and methyl 25, exhibited better inhibitory
activities against VEGFR-2 and c-Kit than the compound without
any substitutions 21, and different halogen substitutions had little
effect on the potency.
and synthesized
a novel class of 3-pyrrolo[b]cyclohexylene-
2-dihydroindolinone derivatives 21–44 (Table 1) according to the
known SARs and binding information. The inhibitory activities
against VEGFR-2, PDGFR-b and c-Kit were then tested. In the new
scaffold, indolin-2-one as the key pharmacophore was retained
and a six member ring was formed by connecting the methyl group
at 40 position of the pyrrole ring and the double bond outside of the
indolinone via two methylenes. Meanwhile, the intramolecular
hydrogen bond between oxygen atom of the indolin-2-one and
NH of the pyrrole ring was kept to stabilize the planar

5632
L. Ding et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5630–5633
Table 1
IC50 values (nM) of kinase inhibition for compounds 21–44 and Sunitinib
Compd
R1
R2
Biochemical activity (IC50, nM)a
VEGFR-2
PDGFR-b
c-Kit
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
H
5-F
5-Cl
5-Br
5-CH3
5-OCH3
5-NO2
5-COOCH3
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2N(CH2CH3)2
NH(CH2)2-morpholin-4-yl
NH(CH2)2-piperidin-1-yl
NH(CH2)2-pyrrolidin-1-yl
NH(CH2)2N(CH3)2
9.4
2.3
2.6
3.9
89.9
10.1
12.1
7.6
2.4
2.7
2.3
4.1
35.5
6.9
255.7
397.6
43.1
180.8
2.0
5.8
7.4
6.6
1.4
2.3
15.6
101.4
13.3
70.3
395.7
62.1
260.3
13.2
19.4
41.6
22.7
12.7
160.6
21.3
18.0
64.8
19.3
40.1
24.6
25.7
167.2
330.3
15.0
112.4
1.6
4.3
9.7
3.0
1.1
11.4
2.7
1.0
3.8
2.5
5-SO2N(CH2CH3)2
4-F
7-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
NH(CH2)2OH
NH(CH2)2-pyridin-2-yl
NH(CH2)3N(CH2CH3)2
NH(CH2)3-morpholin-4-yl
NH(CH2)3-pyrrolidin-1-yl
NHCH2CH(OH)CH2N(CH2CH3)2
Morpholin-4-yl
37.7
9.9
5.7
10.7
4.1
2.1
1.1
N
N
43
5-F
8.0
40.7
7.8
N
44
5-F
—
11.8
4.0
118.4
10.6
8.5
8.9
N
b
Sunitinib
—
a
IC50 values were determined by at least two separate tests with deviation less than 20% and are reported as mean values.
IC50 value obtained under our experimental conditions.
b
O
In addition, we prepared 13 compounds (22, 32–44) with 5 po-
sition of indolin-2-ones fixed as fluorine to explore the influence of
different side-chain substituents at 30 position. Their inhibitory
activities against the VEGFR-2, PDGFR-b, and c-Kit were tested
and their ranges of IC50 were 1–11.8 nM, 10.1–160.6 nM and 1.4–
37.7 nM, respectively, which suggested that changes of substitu-
ents at the side chain had slight effect on the activity against
VEGFR-2 but had significant effect on the activities against
PDGFR-b and c-Kit. Especially, when R2 was NH(CH2)2-pyridin-
2-yl 37, the inhibition against PDGFR-b and c-Kit decreased nota-
bly, with IC50 at 160.6 nM and 37.7 nM, respectively.
HO
O
O
O
O
a
O
NH2
+
HCl
O
HO
O
71%
N
H
16
15
14
R1
O
O
O
N
H
OH
O
c
19
b
61%
d
N
74%
N
H
21% ~ 35%
O
H
O
17
18
O
O
OH
R2
4'
5'
To understand how these multi-targeted kinase inhibitors inter-
act with the receptor, the compound 22 and VEGFR-2 (PDB:
3C7Q31) was submitted for docking studies using Surflex-dock in
SYBYL7.3. The results revealed that compound 22 bound to the
ATP-binding site in the cleft between the NH2 and COOH terminal
lobes of the kinase domain and kept the same molecular orienta-
tion as original ligand BIBF 1120 (Fig. 4a). The indolinone moiety
stretched into the receptor-binding pocket deeply, while the side
chain oriented towards outside. The indolinone scaffold formed
3'
2'
1o or 2o amine
6'
R1
N
R1
5
4
N
H
H
e
O
63% ~ 84%
O
N
H
6
N
H
7
20
21~44
Scheme 1. Reagents and conditions: (a) NaH2PO4, H2O, reflux, 0.5 h; (b) P2O5, PPA,
70 °C, 48 h; (c) LiOH, 80 °C, 24 h; (d) TiCl4, Py, 90 °C, 12 h; (e) EDCI, HOBt, DMF, rt,
24 h.
(a)
(b)
Figure 4. Compound 22 and BIBF 1120 in the ATP binding pocket of VEGFR-2 (PDB
entry 3C7Q). (a) Compound 22 (green) was overlapped with BIBF 1120 (cyan) in
binding pocket. (b) The interaction between compound 22 and VEGFR-2. The
picture is prepared in Benchware 3D Explorer 2.3 (Tripos, St. Louis, MO).
Figure 3. Molecular structure of compound 22 methanol solvate.

L. Ding et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5630–5633
5633
Table 2
Supplementary data
Antiproliferative activity of the target compounds and Sunitinib against selected
cancer cell lines
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
08.037.
Compd
Cell lines IC50
BGC
(lM)
BXPC-3
T24
HEPG2
HT29
21
22
23
25
0.52
0.52
1.95
2.43
3.63
3.56
3.31
1.83
NTb
6.73
6.11
2.03
3.15
4.78
7.73
7.27
3.14
NT
0.54
1.2
6.48
1.73
1.47
References and notes
a
Sunitinib
2.44
5.61
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IC50 value obtained under our experimental conditions.
Not tested.
b
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two hydrogen bonds with the backbone nitrogen of Cys919 and the
backbone carbonyl oxygen of Glu917 in the hinge region. A similar
intramolecular hydrogen bond was also formed between the NH-10
and carbonyl at the C-2 position of the indolin-2-one just like com-
pounds 2 and 5–13 (Fig. 4b).
Moreover, some of the newly synthesized indolin-2-ones (21,
22, 23, 25) were evaluated for in vitro antiproliferative activity
against five human cancer cell lines, including BXPC-3 (primary
pancreatic adenocarcinoma), T24 (bladder carcinoma), BGC (Gas-
tric carcinoma), HEPG2 (liver hepatocellular carcinoma) and
HT29 (Colon Carcinoma) using a standard MTT assay (Table 2).32
They exhibited potent anti-proliferative activities against all five
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Thierauch, K. H.; Zopf, D. Int. J. Cancer 2011, 129, 245.
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cell lines with IC50 values in the range of 0.52–7.73 lM, and espe-
cially more effective than the reference drug in inhibiting the
growth of the BXPC-3 cell lines. In particular, compound 21
17. 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.
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fold more potent than Sunitinib in BXPC-3.
Furthermore, compound 22 had similar kinase selectivity as
sunitinib. In addition to VEGFR-2, PDGFR-b and c-Kit, it also had
strong inhibitory activities against VEGFR-1, VEGFR-3, Ret and
Flt-3, with IC50 ranging from 1.3 to 9.2 nM. PK studies in rats, dogs
and cynomolgus monkeys revealed that compound 22 had higher
oral bioavailability (ꢀ90%) in cynomolgus monkeys than in rats
or dogs. Compound 22 also showed strong efficacy in multiple
human xenograft mouse tumor models, including HT-29, A549,
MDA-MB-231, SK-OV-3, SW-620, Bel-7402, BGC-823, HePG2 and
NCI-H526.
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In summary, we reported herein the design and synthesis of novel
3-pyrrolo[b]cyclohexylene-2-dihydroindolinone derivatives based
on the SAR features of indolin-2-ones as kinase inhibitors. Most of
the newly synthesized compounds exhibited potent inhibitory activ-
ities against VEGFR-2, PDGFR-b and c-Kit in biochemical assays. Ini-
tial SAR study suggested that structural modifications at phenyl
ring of indolin-2-ones had a dramatic effect on the potency of these
agents. Furthermore, molecular docking study indicated that indo-
lin-2-one moiety of compound 22 formed two key hydrogen bonds
with the backbone nitrogen of Cys919 and the backbone carbonyl
oxygen of Glu917 in the ATP binding site of VEGFR-2. Additionally,
some compounds also showed favorable antiproliferative activity
against a panel of cancer cell lines (BXPC-3, T24, BGC, HEPG2 and
HT29). Further PK and in vivo efficacy studies showed that compound
22 was potentially an ideal drug candidate for cancer treatment.
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C.; Hseu, T. H.; Chang, C. N.; Hsu, H. C.; Lin, H. C.; Shih, Y. C.; Chou, S. H.; Tseng,
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Acknowledgments
This work was supported by grants from Scientific
&
Technical Innovation and Commercialization of Jiangsu Province
(BE2011602).
We thank Dr. Huan-ming Chen, Dr. Bai-ming, Xiao and Dr. Yong,
Zhao, JiangSu Simcere Pharmaceutical Co., Ltd, for their manuscript
reading.