Synthesis and biological evaluation of novel indolin-2-one derivatives as protein tyrosine phosphatase 1b inhibitors

Article abstract of DOI:10.2174/157017811796504909

3-Substituted indolin-2-one derivatives had been designed and synthesized as a novel class ofprotein tyrosine phosphatase 1B (PTP1B) inhibitors. These compounds had been evaluated for their inhibitory activities against PTP1B in vitro with IC₅₀ values in a low micromolar range.Compound 36, the lowest, bore an IC₅₀ value of 3.48 μM. Preliminary structure-activity relationship was summarized.

Full text of DOI:10.2174/157017811796504909

526
Letters in Organic Chemistry, 2011, 8, 526-530
Synthesis and Biological Evaluation of Novel Indolin-2-One Derivatives as
Protein Tyrosine Phosphatase 1B Inhibitors
Hou-Ling Daia, Qiang Shenb, Jian-Bin Zheng*,a, Jing-Ya Lib, Ren Wen*,a and Jia Li*,b
aSchool of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P.R.
China
bNational Center of Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Science,
Chinese Academy of Science, Shanghai 201203, P.R. China
Received April 07, 2010: Revised September 16, 2010: Accepted October 06, 2010
Abstract: 3-Substituted indolin-2-one derivatives had been designed and synthesized as a novel class of protein tyrosine
phosphatase 1B (PTP1B) inhibitors. These compounds had been evaluated for their inhibitory activities against PTP1B in
vitro with IC50 values in a low micromolar range. Compound 36, the lowest, bore an IC50 value of 3.48 μM. Preliminary
structure-activity relationship was summarized.
Keywords: 3-substituted indolin-2-ones, diabetes, inhibitor, protein tyrosine phosphatase 1B, structure-activity relationship.
INTRODUCTION
effectively inhibited PTP1B in a low micromolar range.
Therefore, we intended to design a novel series of indolin-2-
one derivatives to simplify this lead compound.
Protein tyrosine phosphatase 1B (PTP1B) is a member of
the family of protein tyrosine phosphatase and has been
identified as an attractive drug target for type II diabetes and
obesity for about a decade [1-2]. PTP1B is involved in the
insulin receptor (IR) dephosphorylation process, regulates IR
and leptin signaling, and eventually acts as an important me-
diator for control of blood glucose levels and body weight
[3-5]. The development of PTP1B inhibitors has become a
potential way to treat type II diabetes. Although many
PTP1B inhibitors had been developed, the further develop-
ment to clinical trials was restricted due to their low cell
permeability and bioavailability. Therefore, there is a need to
develop new potential drug candidates targeting PTP1B.
In the work described herein, we synthesized novel
classes of 3-substituted indolin-2-one derivatives and evalu-
ated their inhibitory activity against PTP1B. Preliminary
structure and activity relationship of this series compounds
were discussed.
RESULTS AND DISCUSSION
The synthesis of 3-substituted indolin-2-one derivatives
is shown in Scheme 1. Firstly, aniline 3a or 4-methylaniline
3b was treated with chloral hydrate and hydroxylamine hy-
drochloride to give the corresponding hydroxyimino pheny-
lacetamide 4. Compounds 5a and 5b were achieved from 4
through cyclization with concentrated H2SO4 followed by
hydrolysis [9]. Wolff-Kishner reduction of 5a and 5b with
hydrazine hydrate yielded 6a and 6b respectively [10].
Compound 6c was prepared by bromination of 6a with NBS
in CH3CN at 0°C [11-12]. Compound 6d was prepared by
nitration of 6a at -5 °C to 0°C followed by recrystallization
in aqueous acetic acid [13]. Mixture of 5-chloro methyl-
furan-2-carbaldehyde 7 and corresponding acids was treated
with K2CO3 in DMF to give compound 8a-k. Compounds 9-
52 were synthesized by condensation of indolin-2-one 6a-d
with the equivalent amount of esters 8a-k in EtOH in the
presence of piperidine. The target products were purified by
silica gel column chromatography. The structures of final
compounds including the known compound 17 [14] were
unambiguously confirmed by spectroscopic methods.
The active site of PTP1B is P-loop, which binds the
phosphate group and contains the catalytic Cys215. Another
loop is WPD loop, which recognizes the peptide substrate
and contains the general acid/base Asp181. Other important
residues offer electrostatic, hydrogen-bonding and hydro-
phobic interactions [6-7].
According to the literature, PTP1B inhibitors can be di-
vided into three parts in structure: (1) a polar and hydrophilic
binding group interacting with PTP1B catalytic site; (2) an
aromaticity group to act on WPD loop; and (3) a hydropho-
bic binding group acting with other amino acid residues.
Additionally, It had been reported that indolin-2-one com-
pound 1 (Fig. 1) inhibited the catalytic activity of PTP1B
with IC50 at 3.55 μM [8]. Our laboratory had discovered that
compound 2 containing furan and indolin-2-one fragments
*Address correspondence to these authors at the School of Pharmacy, East
China University of Science and Technology, 130 Meilong Road, Shanghai
200237, P.R. China; Tel: +86 21 64251984;
The signal from H4 of 3-substituted indolin-2-one deriva-
tives was markedly shifted downfield relative to the H4 sig-
nal of the parent 1,3-dihydroindolin-2-one 6 due to an inter-
action with the ring current of the furanyl substituent de-
shielding this proton [15]. Therefore, the geometrical con-
figurations of compounds 9-52 had been assigned as E-
E-mails: jianbinz@ecust.edu.cn, wenren.sh@gmail.com
National Center of Drug Screening, Shanghai Institute of Materia Medica,
Shanghai Institute for Biological Science, Chinese Academy of Science,
Shanghai 201203, P.R. China; Tel: +86 21 50801313x341;
E-mail: jli@mail.shcnc.ac.cn
1570-1786/11 $58.00+.00
© 2011 Bentham Science Publishers

Synthesis of Novel Indolin-2-Ones Derivatives
Letters in Organic Chemistry, 2011, Vol. 8, No. 7
527
HOOC
NH
COOR
N
N
O
HOOC
O
O
HN
N
R= Me, Et, etc
N
H
O
O
1
2
Fig. (1). Structures of compound 1 and 2.
O
R1
H
NOH
O
R1
R1
R1
a
c
b
O
O
N
N
N
H
NH2
H
H
3a: R1=H
4a
5a
5b
6a
6b
3b: R1=CH3
4b
d
e
6c: R1=Br
6d: R1=NO2
R2
Cl
O
H
f
H
O
O
O
O
O
O
R2
7
8a-k
R1
O
5
6
R1
R2
4
7
O
O
g
H
O
+
3
O
O
N
HN
1
H
O
2
H
O
6a-d
8a-k
9-52
Scheme 1. Preparations of 3-substituted indolin-2-one derivatives.
Reagents and conditions: (a) chloral hydrate, Na2SO4, NH2OH•HCl, HCl, reflux, 2 min, 82-93%; (b) H2SO4, 80ꢀ, 10min, 58-78%; (c)
NH2NH2•H2O, 140-160ꢀ, 4 h, 54-77%; (d) NBS, CH3CN, -10ꢀ, 1 h and then 0ꢀ, 2 h, 81%; (e) KNO3, H2SO4, -5ꢀ to 0ꢀ, 30 min, 58%;
(f) R2COOH (for R2 see Table 1), K2CO3, DMF, rt, overnight, 71-94%; (g) piperidine, EtOH, rt, 26-96%.
isomer. Compound 5-methyl-3-(5-acetoxymethylfurfuryl-
idenemethyl)indolin-2-one 28 was selected to perform
ROESY experiments in order to verify our conclusions. No
NOE effect was observed between the proton at the C-4 po-
sition (ꢀ= 8.28 ppm) of the indoline ring and the vinyl proton
(ꢀ= 7.39 ppm), which was in agreement with the assigned
configuration.
inhibition against PTP1B than 5-unsubstituted indolin-2-
ones 9-19, 5-methylindolin-2-ones 20-30 and 5-bromo-
indolin-2-ones 31-41. Of the nine compounds with IC50 < 10
μM, seven compounds contained nitro at C-5 of the indolin-
2-one and two compounds contained bromo at C-5 of the
indolin-2-one. Thus, it was assumed that introduction of
electron-withdrawing groups at C-5 greatly increased PTP1B
inhibitory activity. In addition, as seen from Table 1, most of
the aromatic esters had higher inhibitory potency on PTP1B
than aliphatic esters with the exception of caproates.
All target compounds were evaluated as PTP1B inhibi-
tors and biological results are shown in Table 1. Among the
forty-four compounds we synthesized, nine compounds ex-
hibited strong inhibitory effects (IC50 <10 μM). Moreover,
another sixteen compounds showed medium inhibitory ef-
fects (IC50 = 10-20 μM). The most potent 3-substituted indo-
lin-2-one compounds are compounds 36 and 42, which had
IC50 in low micromolar range (36 IC50 = 3.48 μM; 42 IC50 =
3.67 μM).
As for 5-unsubstituted indolin-2-one derivatives, the
most potent compound, 3-(5-p-nitrobenzoyloxymethyl fur-
furylidene methyl)indolin-2-one (12) gave an IC50 value of
14.01 μM. It means that electron-withdrawing substitutions
on para position of the ester phenyl ring could produce a
beneficial effect. As for 5-methylindolin-2-one derivatives,
all of aromatic esters showed similar inhibitory activity and
incorporation of different substituents at aromatic ring did
not affect activity, as analogues 21 and 23 were essentially
The basic SAR study of 3-substituted indolin-2-one de-
rivatives was summarized from compounds 9-52. In general,
5-nitroindolin-2-one derivatives (42-52) expressed a better

528 Letters in Organic Chemistry, 2011, Vol. 8, No. 7
Dai et al.
Table 1. Inhibitory Activity of 3-Substituted Indolin-2-One Derivatives Against PTP1B (IC50 [μM])
O
R2
R1
O
O
HN
H
O
Compound
R1
R2
IC50 (μM)
9
H
H
Ph
p-CH3O-C6H4
p-CH3-C6H4
p-NO2-C6H4
o-CH3-C6H4
o-NO2- m-CH3-C6H4
Phenylmethyl
Phenylethyl
Methyl
25.47 ± 5.56
41.72 ± 1.97
45.00 ± 4.71
14.01 ± 2.94
22.14 ± 0.72
25.35 ± 0.38
18.99 ± 1.27
31.86 ± 2.56
>60
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
H
H
H
H
H
H
H
Ethyl
>60
H
n-Pentyl
15.81 ± 2.32
15.03 ± 1.44
16.17 ± 0.28
12.84 ± 0.72
11.78 ± 2.09
13.16 ± 1.99
13.07 ± 0.66
21.48 ± 1.56
13.43 ± 1.45
>60
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Br
Ph
p-CH3O-C6H4
p-CH3-C6H4
p-NO2-C6H4
o-CH3-C6H4
o-NO2- m-CH3-C6H4
Phenylmethyl
Phenylethyl
Methyl
Ethyl
>60
n-Pentyl
23.92 ± 1.55
20.41 ± 2.55
11.59 ± 1.53
13.45 ± 1.72
5.27 ± 0.17
14.76 ± 0.78
3.48 ± 0.55
14.49 ± 0.64
15.25 ± 1.05
41.99 ± 3.58
22.83 ± 0.51
18.36 ± 1.16
3.67 ± 0.34
7.43 ± 1.10
Ph
Br
p-CH3O-C6H4
p-CH3-C6H4
p-NO2-C6H4
o-CH3-C6H4
o-NO2-m-CH3-C6H4
Phenylmethyl
Phenylethyl
Methyl
Br
Br
Br
Br
Br
Br
Br
Br
Ethyl
Br
n-Pentyl
NO2
NO2
Ph
p-CH3O-C6H4

Synthesis of Novel Indolin-2-Ones Derivatives
Letters in Organic Chemistry, 2011, Vol. 8, No. 7
529
(Table 1). Contd…..
Compound
R1
R2
IC50 (μM)
44
45
46
47
48
49
50
51
52
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
p-CH3-C6H4
p-NO2-C6H4
o-CH3-C6H4
o-NO2- m-CH3-C6H4
Phenylmethyl
Phenylethyl
Methyl
7.35 ± 1.15
5.45 ± 0.22
8.18 ± 1.97
4.68 ± 0.44
8.45 ± 1.29
21.01 ± 2.15
34.60 ± 0.70
20.85 ± 2.36
21.25 ± 1.52
4.58 ± 0.44
Ethyl
n-Pentyl
a
Na3VO4
aUsed as a positive control.
equally active. All of 5-bromoindolin-2-one derivatives ex-
hibited potential inhibitory activities while substitution of the
ester phenyl ring of 31 with ortho-nitro or para-nitro sub-
stituents gave a 4-fold and 6-fold increase in PTP1B inhibi-
tory, respectively (34 IC50 = 5.27 μM; 36 IC50 = 3.48 μM).
Among aliphatic esters, prolonging the carbon chain may
provide the possibility to improve inhibitory activity. Such
an effect has similarly been described for 5-nitroindolin-2-
one compounds. Furthermore, all of benzoates and pheny-
lacetate showed high potency with IC50 value < 10 μM.
General Procedure for the Synthesis of 3-(5-
acyloxymethylfurfurylidene-methyl) indolin-2-ones 9-52
A stirred solution of acid (1.08 equiv) in DMF was
treated with K2CO3 (1.08 equiv), and the mixture was stirred
for 30 min, and was then added to 5-chloromethyl-furan-2-
carbaldehyde 7 (1 equiv). The reaction mixture was stirred
overnight. The mixture was then poured into water and ex-
tracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4 and concentrated in vacuo. The
residue was purified by flash column chromatography (PE/
EtOAc = 8:1) to give 8 in over 70% yield. A reaction mix-
ture of the proper oxindole 6 (1 equiv), 8 (1.07 equiv) and
piperidine (0.1 equiv) was stirred at room temperature for
3h. The precipitate was filtered off and dried. The crude
solid was purified by column chromatography to afford the
target compound, in 26-96% yields. The analytical data re-
ported here are for the major isomer.
CONCLUSIONS
In conclusion, a novel series of 3-substituted indolin-2-
one derivatives were synthesized by a facile and convenient
method. These compounds have assessed in vitro inhibitory
activities on PTP1B. Twenty-five of forty-four compounds
exhibited potent activities with IC50 < 20 μM. Thereinto, 5-
bromo-3-(5-o-nitro-m-methylbenzoyloxymethylfurfuryliden-
emethyl) indolin-2-one (36) and 5-nitro-3-(5-benzoyloxyme-
thylfurfurylidenemethyl)indolin-2-one (42) showed the most
potent inhibitory activity (IC50 = 3.48 μM and 3.67 μM re-
spectively). Taken together, these analogues showed poten-
tial direction of 3-substituted indolin-2-one analogues as
PTP1B inhibitors for the treatment of diabetes.
The spectroscopic data of representative compound are
reported here. (E)-3-(5-benzoyloxymethylfurfurylidene-
methyl)indolin-2-one (9). Yellow solid. EI-MS m/z 345.1
1
[M]+. H NMR (DMSO-d6) ꢀ: 5.59 (s, 2H, CH2O), 6.79 (t,
1H, J = 8 Hz, Ph-H), 6.84 (d, 1H, J = 8 Hz, Ph-H), 6.92 (d,
1H, J = 3.2 Hz, furan-H), 7.20 (t, 1H, J = 8 Hz, Ph-H), 7.26
(d, 1H, J = 3.2 Hz, furan-H), 7.32 (s, 1H, CH=), 7.55 (t, 2H,
J = 7.6 Hz, Ph-H), 7.69 (t, 1H, J = 7.6 Hz, Ph-H), 8.06 (d,
2H, J = 7.6 Hz, Ph-H), 8.35 (d, 1H, J = 8 Hz, Ph-H), 10.56
(s, 1H, CONH). Anal. Calcd for C21H15NO4: C, 73.03; H,
4.38; N, 4.06; O, 18.53. Found: C, 72.62; H, 4.39; N, 3.87.
EXPERIMENTAL
General
Reagents and solvents were used as obtained from the
supplier without further purification. Column chromatogra-
phy was carried out on a silica gel column. Thin-layer chro-
matography (TLC) was performed on Huanghai TLC Silica
GF254 with detection by UV quenching at 254nm. Synthetic
PTP1B Biological Assay
PTP1B inhibitor screening assay was performed as de-
scribed [16,17]. GST-fusion human PTP1B was cloned into
pGEX-KG plasmid. PTPs were overexpressed as GST-
fusion proteins in Escherichia coli BL21-Conden plus (DE3)
and purified through affinity chromatography. The enzy-
matic activities of PTP1B catalytic domain were determined
at 30 ºC by monitoring the hydrolysis of pNPP. Dephos-
phorylation of pNPP generates product pNP, which can be
monitored at an absorbance for 405 nm. We used Na3VO4 as
positive control and DMSO as negative control to evaluate
our HTS (high-throughput screening) system. For the 50%
1
yields of compounds were not optimized. H NMR spectra
were recorded on a Bruker INOVA 400 NMR spectrometer.
Chemical shifts (ꢀ) were given in ppm from the internal
standard, tetramethylsilane, and coupling constants were
given in hertz (Hz). Mass spectra were recorded by Micro-
mass GCT spectrometer. Elemental analyses were performed
on an elementar vario EL III elemental analyzer.

530 Letters in Organic Chemistry, 2011, Vol. 8, No. 7
Dai et al.
[8]
Wu, J.; Lawrence, N.J.; Sebti, S.M.; Lawrence, H.R. Inhibition of
SHP2/PTPN11 protein tyrosine phosphatase by NSC-87877, NSC-
117199 and their analogs. U.S. Patent 20,080,176,309, July 24,
2008.
Marvel, C.S.; Hiers, G.S. Isatin. Org. Synth., 1925, 5, 71-74.
Sureyya, O.; Eiichi, A.; Dogu, N. Synthesis and anti-tyrosine
kinase activity of 3-(substituted-benzylidene)-1,3-dihydro-indolin
derivatives: investigation of their role against p60c-Src receptor tyro-
sine kinase with the application of receptor docking studies. Far-
maco, 2005, 60(6-7), 497-506.
inhibition concentration (IC50) calculation, inhibition assays
were performed with 30 nmol/L recombinant enzyme,
2mmol/L pNPP in 50 mmol/L MOPs (3-[N-morpholino]
propane-sulfonic acid) at pH 6.5, and the inhibitors diluted
around the estimated IC50 values. The IC50 was calculated
with Prism 4 software according to the formula: % Inhibition
= 100/(1+[IC50/[I]]k), where k is the Hill coefficient.
[9]
[10]
[11]
Sun, L.; Tran, N.; Tang, F.; App, H.; Hirth, P.; McMahon, G.;
Tang, C. Synthesis and biological evaluations of 3-substituted indo-
lin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit
selectivity toward particular receptor tyrosine kinases. J. Med.
Chem., 1998, 41(14), 2588-2603.
REFERENCES
[1]
[2]
[3]
Johnson, T.O.; Ermolieff, J.; Jirousek, M.R. Protein tyrosine phos-
phatase 1B inhibitors for diabetes. Nat. Rev. Drug Discov., 2002,
1(9), 696-709.
Barford, D.; Flint, A.J.; Tonks, N.K. Crystal structure of human
protein tyrosine phosphatase 1B. Science, 1994, 263(5152), 1397-
1404.
Zabolotny, J.M.; Haj, F.G.; Kim, Y.B.; Kim, H.J.; Shulman, G.I.;
Kim, J.K.; Neel, B.G.; Kahn, B.B. Transgenic overexpression of
protein-tyrosine phosphatase 1B in muscle causes insulin resis-
tance, but overexpression with leukocyte antigen-related phospha-
tase does not additively impair insulin action. J. Biol. Chem., 2004,
279(23), 24844-24851.
Zabolotny, J.M.; Bence-Hanulec, K.K.; Stricker-Krongrad, A.; Haj,
F.; Wang, Y.; Minokoshi, Y.; Kim, Y.B.; Elmquist, J.K.; Tartaglia,
L.A.; Kahn, B.B.; Neel, B.G. PTP1B regulates leptin signal trans-
duction in vivo. Dev. Cell, 2002, 2(4), 489-495.
Seely, B.L.; Staubs, P.A.; Reichart, D.R.; Berhanu, P.; Milarski,
K.L.; Saltiel, A.R.; Kusari, J.; Olefsky, J.M. Protein tyrosine phos-
phatase 1B interacts with the activated insulin receptor. Diabetes,
1996, 45(10), 1379-1385.
Burke, T.R.; Zhang, Z.Y. Protein-tyrosine phosphatases: Structure,
mechanism, and inhibitor discovery. Biopolymers, 1998, 47(3),
225-241.
[12]
[13]
[14]
Wolfgang, F.; Barbara, M.; Tobias, M.B.; Laura, C.; Heidi, S.;
Pierfausto, S. Regiospecific synthesis of mono-N-substituted in-
dolopyrrolocarbazoles. Org. Lett., 2005, 7(21), 4573-4576.
Torsten, K.; Manuela, K.; Frank, W. Facile synthesis of various
nitrosubstituted derivatives of semaxinib (SU5416). Synth. Com-
mun., 2008, 38(17), 3017-3022.
Arnaiz, D.; Bryant, J.; Chou, Y.L.; Feldman, R.; Hrvatin, P.; Islam,
I.; Kochanny, M.; Lee, W.; Polokoff, M.; Yu, H.; Yuan, S. Indoli-
none derivatives and their use in treating disease-states such as
cancer. U.S. Patent, 2,005,090,541, April 28, 2005.
Stetinova, J.; Kovac, J.; Sura, J.; Dandarova, M. Stereochemistry
and photoisomerization of furfurylideneoxoindoles. Chem. Com-
mun., 1977, 42, 2201-2206.
Zhang, W.; Hong, D.; Zhou, Y.Y.; Zhang, Y.N.; Shen, Q.; Li, J.Y.;
Hu, L.H.; Li, J. Ursolic acid and its derivative inhibit protein tyro-
sine phosphatase 1B, enhancing insulin receptor phosphorylation
and stimulating glucose uptake. Biochim. Biophys. Acta, 2006,
1760(10), 1505-1512.
[4]
[5]
[15]
[16]
[6]
[7]
[17]
Shi, L.; Yu, H.P.; Zhou, Y.Y.; Du, J.Q.; Shen, Q.; Li, J.Y.; Li, J.
Discovery of a novel competitive inhibitor of PTP1B by high-
throughput screening. Acta Pharmacol. Sin., 2008, 29(2), 278-284.
Taylor, S.D. Inhibitors of protein tyrosine phosphatase 1B
(PTP1B). Curr. Top. Med. Chem., 2003, 3(7), 759-782.