Discovery of di-indolinone as a novel scaffold for protein tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.054

A series of di-indolinone derivatives was designed and synthesized to optimize our lead compounds basing on molecular docking study as PTP1B inhibitors. Successive enzymatic assay identified the synthetic di-indolinone as novel PTP1B inhibitors with low micromole-ranged inhibitory activity and at least several-fold selectivity over other tested homologous PTPs.

Full text of DOI:10.1016/j.bmcl.2012.10.054

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7440–7443
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of di-indolinone as a novel scaffold for protein tyrosine
phosphatase 1B inhibitors
Hou-ling Dai a, Li-xin Gao b, Ying Yang a, Jing-ya Li b, Jia-gao Cheng a, , Jia Li b, , Ren Wen a,
⇑
⇑
Yan-qing Peng a, Jian-bin Zheng a,
⇑
a Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
b National Center of Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Science, Chinese Academy of Science, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of di-indolinone derivatives was designed and synthesized to optimize our lead compounds bas-
ing on molecular docking study as PTP1B inhibitors. Successive enzymatic assay identified the synthetic
di-indolinone as novel PTP1B inhibitors with low micromole-ranged inhibitory activity and at least sev-
eral-fold selectivity over other tested homologous PTPs.
Received 25 April 2012
Revised 9 October 2012
Accepted 11 October 2012
Available online 17 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Protein tyrosine phosphatase 1B
Molecular docking
Inhibitors
Di-indolinone
Protein tyrosine phosphatases (PTPs), a large family of signaling
enzymes, act in opposition with protein tyrosine kinases to control
phosphorylation level of protein tyrosine, which have been associ-
ated with the pathogenesis of a wide variety of acquired or inher-
ited human diseases, including cancer, diabetes and obesity.1,2
Protein tyrosine phosphatase 1B (PTP1B), as a member of protein
tyrosine phosphatase family, interacts directly with activated insu-
lin receptor and is a negative regulator of the insulin and leptin-
signaling pathway.3,4 PTP1B knockout mice showed enhanced
insulin sensitivity, lower plasma glucose and insulin levels, and
resistance to high-fat diet induced obesity.5–8 In addition, PTP1B
was reported to regulate the phosphorylation state of the
Tyr1162/Tyr1163 site in the activation loop of the IR PTK catalytic
domain in vivo.9,10 Based on these studies, PTP1B was proposed as
an attractive drug target for therapy of non-insulin dependent dia-
betes and obesity.11
Over the past two decades, numerous PTP1B inhibitors have
been developed while two compounds, ertiprotafib and trodusque-
mine, were progressed to clinical trials. However, ertiprotafib had
been discontinued in phase II clinical trials due to lack of efficacy
and side effect.4,12 The further development of most PTP1B inhibi-
tors was restricted due to their low cell permeability and poor bio-
availability. Therefore, there is a need to develop novel potential
drug scaffolds targeting PTP1B with desirable physicochemical
properties and in vivo efficacies.
High throughput screening of our own laboratory compounds
as PTP1B inhibitor identified that 3-substituted indolin-2-one
1(Fig. 1) shown to inhibit PTP1B weakly (14% inhibition at 20 lg/
mL). However, when a furyl-containing group was incorporated
to 1-position of 1, inhibition ratio (compound 2) was increased
to 55% at the same concentration. Inspiringly, when another indo-
lin-2-one was incorporated to 2, inhibition ratio (compound 3) was
increased to 75% at 20 lg/mL (synthesis scheme and spectrum data
of compounds 1-3 see Supplementary data).
Basing on these interesting results in this series of structure
modifications, in this letter, we used molecular docking studies
to illustrate the binding modes and structure–activity relation of
these compounds and designed novel molecules as PTP1B inhibi-
tors. And then, the target compounds were synthesized and evalu-
ated on PTPs inhibitory assays.
The X-ray structure of PTP1B was download from the Protein
Data Bank (PDB code: 3EB1).13 The co-crystallized inhibitor and
water molecules were removed from the structure of 3EB1. Then
the protein structure was prepared using the ‘protein preparation
wizard’ panel (Schrödinger, LLC, New York, NY, 2009). The com-
pounds were prepared using LigPrep (VERSION 2.3, 2009; Schrö-
dinger, LLC, New York, NY) with OPLS_2005 force field. The
binding pocket was set as residues within 20 Å around the virtual
atom center defined by the crucial residues Asp48, Phe182, Cys215
and Gly259.14 The docking studies were carried out using Glide5.5
program (Schrödinger, LLC, New York, NY).
⇑
Corresponding authors. Tel./fax: +86 21 64251984. (J.C. and J.Z.); tel./fax: +86 21
50801552 (J.L.).
E-mail addresses: jgcheng@ecust.edu.cn (J. Cheng), jli@mail.shcnc.ac.cn (J. Li),
jianbinz@ecust.edu.cn (J. Zheng).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.054

H. Dai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7440–7443
7441
Figure 1. Structures of lead compounds.
The docking results were depicted in Figure 2. It could be found
that all of the three compounds tended to interact with PTP1B in
the substrate binding pocket (Fig. 2A). The docking studies of com-
pound 1 showed that it was slightly deviated from the pocket and
only one N–Hꢀ ꢀ ꢀO hydrogen bonding interaction could be observed
between the carbonyl group and the positive side chain of Lys120
(Fig. 2B, Supplementary Fig. S1-A). Whereas, it can be seen from
Fig. 2C that compound 3 with the substitution on the indoline N
atom bound to the catalytic site strongly. Three N–Hꢀ ꢀ ꢀO hydrogen
bonds were formed with the positive charged side chains from
Arg221 and Lys120. Another N–Hꢀ ꢀ ꢀO hydrogen bonding interac-
tion could also be observed with the main chain carbonyl group
of Gly183. These hydrogen bonds played an important role to the
binding between the inhibitor and the active site. Hydrophobic
Figure 2. Docking results of the compounds 1, 3, 4 to PTP1B (PDB code: 3EB1). (A) alignment of compounds 1, 3, 4 with the PTP1B enzyme; (B) The interaction of compound 1
with the key residues of 3EB1; (C) The interaction of compound 3 with the key residues of 3EB1; (D) The interaction of compound 4 with the key residues of 3EB1; Carbon
atoms of the compounds are show in warm pink (1), yellow (3) and green (4) sticks; and those for PTP1B are shown in palegreen sticks, Nitrogen atoms are shown in blue and
oxygen atoms in red. Hydrogen bonds are shown in warm pink dashed lines. The figures were prepared using PyMOL.

7442
H. Dai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7440–7443
Scheme 1. Reagents and conditions: (a) chloral hydrate, Na2SO4, NH2OHꢁHCl, HCl, reflux, 2 min; (b) H2SO4, 80 °C, 10 min; (c) NH2NH2ꢁH2O, 140–160 °C, 4 h; (d) NBS, CH3CN,
ꢂ10 °C, 1 h, and then 0 °C, 2 h; (e) KNO3, H2SO4, 0 °C, 1 h; (f) 5-chloromethylfuran-2-carbaldehyde, K2CO3, DMF, rt, 8 h; (g) indolin-2-one 14–18, piperidine, EtOH, rt, 3 h.
interactions with residues Lys116, Ser118, Tyr46 further stabilized
the binding to the active site.
Table 1
Inhibitory activity of compounds 19-21, 4, 22-32 on PTP1B
a
However, the docking result showed that 3-substituded group
on the indolin-2-one moiety of compound 3 stretched outside of
the binding pocket (Fig. 2C) and no evident interaction was ob-
served with residues around the active site, except a weak hydro-
phobic interaction with Lys116 (Supplementary Fig. S1-B). So, the
structure of 3 could be simplified to di-indolindione fragment. Suc-
cessive docking study showed that the di-indolindione compound
4 possessed a higher inhibiting activity than 3. The binding mode
of compound 4 (Fig. 2D) further validated our assumption. As de-
picted in Figure 2D, a network of N–Hꢀ ꢀ ꢀO hydrogen bonds were
found with the guanidium group of Arg221, the backbone carbonyl
group of Gly183 and the ammonium group of Lys120, which were
apparently the strong interactions for enzyme-inhibitor complexa-
tion. The other residues Thr263, Gly220, Ile219, Ala217, Tyr46,
were also found to be involved in hydrophobic contacts with com-
pound 4 (Supplementary Fig. S1-C).
Compound
R1
R2
IC50 (lM)
19
20
21
4
H
CH3
Cl
H
H
H
H
H
CH3
CH3
CH3
CH3
Cl
—
—
—
H
>60
>60
>60
7.63 0.58
2.80 0.04
9.82 1.09
5.35 0.60
4.41 1.04
6.71 0.68
5.23 0.94
5.70 0.83
3.46 0.53
4.59 0.47
3.37 0.38
2.30 0.48
5.73 0.32
4.58 0.44
22
23
24
25
26
27
28
29
30
31
32
33b
Br
CH3
NO2
Cl
H
Br
CH3
Cl
H
CH3
Cl
Cl
Cl
b
Na3VO4
a
Values are means of three experiments.
Used as a positive control.
To validate the docking study assumption, some of the di-ind-
olinones were synthesized as depicted in Scheme 1. 5-Substituted
and not substituted indoline-2,3-diones 11-13, which were ob-
tained from substituted anilines by isatin reaction, were reacted
with hydrazine hydrate yielded indoline-2-ones 14-16.15,16 Com-
pound 17 and 18 were prepared by bromination or nitration of
compound 14, respectively.17,18 Reaction of indoline-2,3-diones
11-13 and 5-chloromethylfuran-2-carbaldehyde in DMF afforded
the corresponding indoline-2,3-diones 19–21. Di-indolinones 4
and 22–32 were synthesized by condensation of indolin-2-ones
14–18 with 19–21 in EtOH in the presence of piperidine. As re-
ported in previous papers,17,19–21 the predominant condensation
product of indolin-2-one with furan-2-aldehyde was E-isomer,
which may be due to the electrostatic repulsion between the C-2
carbonyl oxygen atom of the indolin-2-one and the O-1 of furan
in their Z-isomer form.17 The configuration of 3-[(2-furanyl)methy-
lidenyl]indolin-2-one could be determined by markedly 1H NMR
b
only product,22 which could be confirmed by 1H NMR and NOE
experiments. The proton signals from H4’ of compounds 4 and
22–32 were markedly shifted downfield relative to the H4 signal
of indolin-2,3-diones 11–13 due to interaction with the ring cur-
rent of the furyl substituent deshielding this proton. Compound
25, 26, 30 were further subjected to NOE experiments to verified
our conclusions. Obvious NOE effects were observed between the
proton at the C-40 position of the indoline ring (about 8.2 ppm)
and the CH2 proton (about 5.2 ppm) (See Supplementary Fig. S2
1H NMR and NOE spectrums in Supplementary data). These results
were in agreement with the designed configurations. Therefore,
the geometrical configurations of compounds 4 and 22–32 had
been assigned as E-isomer.
difference of H4’ in two isomers (E: 8.45 ppm; Z: 7.44 ppm)19 and
And then, the target compounds were evaluated for their inhib-
itory activity against PTP1B by the method of pNPP23,24 using com-
pound 3325 (structure see Fig. S3 in Supplementary data) and
Na3VO4 as positive control. The assay results were summarized
21
0
NOE experiments on the H4 .
We had reported that the condensation reaction of substituted
indoline-2-one and furan-2-carbaldehyde gave the E-isomer as

H. Dai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7440–7443
7443
Table 2
Supplementary data
Inhibitory activity of compounds 22 and 32 on PTPs
a
Compound
IC50
(l
M)
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.054.
PTP1B
TCPTP
SHP-1
LAR
22
32
2.80 0.04
2.30 0.48
5.73 0.32
4.58 0.44
11.47 1.43
18.81 1.94
5.35 0.21
2.58 0.31
>100
>100
>100
18.65 1.49
>100
>100
>100
24.63 1.65
33b
Na3VO4
References and notes
b
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a
Values are means of three experiments.
Used as a positive control.
b
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in Table 1. As predicted, the initial set of compounds 19–21 with-
out further structural modifications were first determined not to
inhibit PTP1B-catalyzed dephosphorylation. However, while an-
other indolinone moiety was connected to these compounds, the
resulting di-indolinones exhibited potent inhibitory activities on
5. Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.; Loy, A. L.;
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PTP1B with IC50 values below 10 lM. In general, it seemed that
the substitutes on the C5 and C5’ positions didn’t affect the inhib-
itory activity obviously. Compound 32, with a Cl-substituent at C5
and C50 position, respectively, resulted in the lowest IC50 value
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(IC50 = 2.30 lM) in these series.
Next, compounds 22 and 32 owning best PTP1B inhibitory
activities among this series were then subjected to the inhibitory
assays on a panel of homologous PTPs including the TCPTP, SHP-
1 and LAR for evaluating their specificity24 (Table 2). As shown in
Table 2, both of the compounds 22 and 32 were more selective
over TCPTP (fourfold and eightfold, respectively). Finally, both
compounds were determined not to be inhibitors of SHP-1and
12. Dipam, P.; Mukul, J.; Shailesh, R. S.; Rajesh, B.; Pradip, J.; Brijesh, D.; Yemaidu,
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LAR considering their >100 lM IC50 values.
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In summary, a novel series of di-indolinone derivatives were
discovered and identified as potent PTP1B inhibitors in a low
micromolar IC50 range based on molecular docking studies. In
addition, successive specificity assessment indicated that the se-
lected inhibitors 22 and 32 shown at least several-fold selectivity
over other tested homologous PTPs. This preliminary result could
provide a novel scaffold for further development of more promis-
ing inhibitors for PTP1B.
21. Mologni, L.; Rostagno, R.; Brussolo, S.; Knowles, P. P.; Kjaer, S.; Murray-Rust, J.;
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23. Zhang, W.; Hong, D.; Zhou, Y. Y.; Zhang, Y. N.; Shen, Q.; Li, J. Y.; Hu, L. H.; Li, J.
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Acknowledgments
24. Shi, L.; Yu, H. P.; Zhou, Y. Y.; Du, J. Q.; Shen, Q.; Li, J. Y.; Li, J. Acta Pharmacol. Sin.
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25. Zhang, Y. N.; Zhang, W.; Hong, D.; Shi, L.; Shen, Q.; Li, J. Y.; Li, J.; Hu, L. H. Bioorg.
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Project supports by National Natural Science Foundation of Chi-
na (Grant No. 21102044 and Grant No. 21172070) and Shanghai
Committee of Science and Technology (Grant No. 11DZ2260600).