Inhibitors of Src homology-2 domain containing protein tyrosine phosphatase-2 (Shp2) based on oxindole scaffolds

Article abstract of DOI:10.1021/jm8002526

Screening of the NCI diversity set of compounds has led to the identification of 5 (NSC-117199), which inhibits the protein tyrosine phosphatase (PTP) Shp2 with an IC₅₀ of 47 μM. A focused library incorporating an isatin scaffold was designed a

Full text of DOI:10.1021/jm8002526

4948
J. Med. Chem. 2008, 51, 4948–4956
Inhibitors of Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (Shp2) Based
on Oxindole Scaffolds
Harshani R. Lawrence,*,§,†,| Roberta Pireddu,§,† Liwei Chen,†, Yunting Luo,†,| Shen-Shu Sung,| Ann Marie Szymanski,
,#,O
M. L. Richard Yip,|,‡ Wayne C. Guida,|,#, Saïd M. Sebti,§,# Jie Wu,§,
and Nicholas J. Lawrence*,§,#
Drug DiscoVery Program, Molecular Oncology Program, and High Throughput Screening and Chemistry Core Facility, Moffitt Cancer Center,
12901 Magnolia DriVe, Tampa, Florida 33612, and Departments of Oncologic Sciences, Chemistry, and Molecular Medicine, UniVersity of
South Florida, Tampa, Florida, 33612
ReceiVed March 7, 2008
Screening of the NCI diversity set of compounds has led to the identification of 5 (NSC-117199), which
inhibits the protein tyrosine phosphatase (PTP) Shp2 with an IC50 of 47 µM. A focused library incorporating
an isatin scaffold was designed and evaluated for inhibition of Shp2 and Shp1 PTP activities. Several
compounds were identified that selectively inhibit Shp2 over Shp1 and PTP1B with low to submicromolar
activity. A model for the binding of the active compounds is proposed.
Introduction
The function of many proteins, particularly those involved
in signal transduction pathways, is dependent upon their tyrosine
phosphorylation status, which is finely regulated by the action
of protein tyrosine kinases and protein tyrosine phosphatases.
Many protein tyrosine kinases such as Bcr-Abl,a c-kit, ErbB,
and VEGFR are validated drug targets for cancer therapy.1 Less
attention has been paid to the development of protein tyrosine
phosphatase inhibitors as an alternative strategy to modulate
key target protein phosphorylation states.2,3 The protein tyrosine
phosphatase (PTP) Shp2 is a nonreceptor PTP that plays a
positive role in cell signaling by growth factors and cytokines
and is involved in regulation of cell proliferation, differentiation,
and migration.4,5 In particular, Shp2 mediates activation of Erk1
and Erk2 MAP kinases by receptor tyrosine kinases such as
ErbB1, ErbB2, and c-Met that are involved in the pathogenesis
Figure 1. Known nonselective inhibitors of Shp2.
of human carcinoma.6–11 Furthermore, gain-of-function Shp2
develop Shp2 inhibitors. Furthermore, the development of small
mutants are found in childhood hematological malignancies such
molecule Shp2 inhibitors would allow us to investigate the role
as juvenile myelomonocytic leukemia, some cases of solid
of Shp2 in normal and malignant processes.
tumors, and are associated with ∼50% cases of Noonan
syndrome.12 It has also been reported that Shp2 is a key mediator
The structure of the Shp2 phosphatase has been determined
by X-ray crystallography at 2.0 Å resolution.15 Shp2 contains
two Src homology-2 (SH2) domains (N-SH2, C-SH2) at its
of the oncogenic CagA protein of Helicobactor pylori, which
causes gastric cancer.13,14 These studies suggest that Shp2 PTP
N-terminal region.4 In the absence of a tyrosine-phosphorylated
is a molecular target for cancer therapy and prompted us to
binding partner, the N-SH2 domain blocks the catalytic domain
until the upstream signaling effectors bind to Shp2.15 Conse-
* To whom correspondence should be addressed. For H.R.L.: phone,
quently, Shp2 is basally inactive because of autoinhibition, and
inhibition of Shp2 by a small molecule is not predicted to affect
resting cells, making it an attractive target for drug design.
Several compounds have been reported to nonselectively
inhibit Shp2 (Figure 1). These include the potent PTP1B
inhibitor 1,16 the bis(trifluorosulfonamide) 2, which also inhibits
PTP1B17 and the tetrazole 3 (NAT6-297775)18 discovered from
a screen of a natural product-like library. Recently, we found
that 4 (NSC-87877),26 identified as a hit from the NCI Diversity
set, is a potent Shp2 and Shp1 inhibitor.19 The development of
a Shp2-specific inhibitor that does not cross-inhibit Shp1 is an
important goal of the present study. Shp1 is mostly expressed
in hematopoietic and epithelial cells and functions as a negative
regulator of signaling pathways in lymphocytes.20,21 The crystal
structure of ligand-free Shp1 shows a similar arrangement of
tandem SH2 domains that adopt a conformation blocking the
PTP catalytic site.22 Shp1 and Shp2 share 60% overall sequence
813-745-6076;
fax,
813-745-6748;
e-mail,
Harshani.
Lawrence@moffitt.org. For N.J.L.: phone, 813-745-6037; fax, 813-745-
6748; e-mail, Nicholas.Lawrence@moffitt.org.
§
Drug Discovery Program, Moffitt Cancer Center.
These authors contributed equally.
High Throughput Screening and Chemistry Core Facility, Moffitt Cancer
†
|
Center.
Molecular Oncology Program, Moffitt Cancer Center.
Present address: HTS Core and Division of Molecular Medicine,
‡
Beckman Research Institute, City of Hope National Medical Center, 1500E
Duarte Road, Duarte, CA 91010.
#
Department of Oncologic Sciences, University of South Florida.
Department of Chemistry, University of South Florida.
O
Department of Molecular Medicine, University of South Florida.
a Abbreviations: Bcr-Abl, breakpoint cluster region Abelson; CagA,
cytotoxin-associated gene A; c-MET, hepatocyte growth factor receptor;
DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DIPEA, diisopro-
pylethylamine; ErbB, avian erythroblastosis oncogene B; ERK, externally
regulated kinase; GLIDE, grid-based ligand docking with energetics; NBS,
N-bromosuccinimide; PTP, protein tyrosine phosphatase; SH2, Src homol-
ogy-2; VEGFR, vascular endothelial growth factor receptor.
10.1021/jm8002526 CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

Inhibitors of Src Homology-2 Domain
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4949
Figure 2. (A) Model of 5 representing possible important structural features for activity. (B) Structure 6 representing the oxindole phamacophore
for new inhibitor design.
Scheme 1a
a Reagents and conditions: (a) POCl3, tetramethylenesulfone, Ar, 60 °C,
3 h; (b) THF, DIPEA, RNH2, Ar, room temp, overnight; (c) EtOH,
ArNHNH2, HCl (aqueous 1 M, 2 drops) 80 °C, 4 h; (d) EtOH, ArNHNH2,
HCl (aqueous 1M, 2 drops), microwave, 120 °C, 15 min.
Figure 3. Overlay of 5 (blue) and 14a (green) docked in the Shp2
PTP active site.
site, suggesting that the hydrazone unit should not be replaced
by longer spacer groups (Figures 2 and 3). The model also
suggested the sulfonic acid could be replaced with small polar
groups, e.g., sulfonamide and carboxylamide. Thus, our library
was therefore biased to include small polar replacements of the
nitro and sulfonic acid groups to interact with the two polar
binding sites (Figure 2B). Other larger replacements (e.g., benzyl
sulfonamides and benzylamides at the 5-position) were included
to test to the model and to see whether only a single polar group
was required. These modifications were mostly at the 2 and 3
positions of the A-ring and the C5 of the indole ring,
respectively. Inclusion of sulfonamide or carboxylamide groups
at the 5-position, accessible from readily available precursors,
adds a useful point of diversity into the oxindole pharmacophore.
The hydrogen bonding interactions of the oxindole N-H group
with Asp425 was thought to be optimal, and variation of N-H
was limited to N-Me. The hydrazone linker was varied by
inclusion of enamine analogues (X ) CH).
Several libraries of hydrazones (10, 14, and 16) were prepared
by combining a 5-substituted isatin with commercially available
hydrazines; the sulfonyl and carboxyl groups were elaborated
with a further set of amines to provide sulfonamides and amides.
The novel oxindole hydrazone sulfonamide library 10 was
developed using commercially available building blocks as
shown in Scheme 1. The oxindolesulfonyl chloride 8 was
obtained from commercially available isatin-5-sulfonic acid
according to a literature reported procedure.31 Isatin-5-sulfonyl
chloride was coupled to a series of requisite amines to obtain
the sulfonamide library 9. We were able to isolate and analyze
(NMR, mass) some members of the isatin library 9. Attempts
to purify and isolate other members of the library 9 were not
successful. However, use of the crude isatinsulfonamides 9 was
successful. The hydrazone library 10 was obtained by micro-
wave assisted coupling of the crude library 9 with an appropriate
set of hydrazines in moderate yields.
identity and approximately 75% similarity in their PTP
domains.15,22 The Shp1 and Shp2 PTP domains are known to
have different substrate specificity,23,24 indicating that the
domains are not identical. Furthermore, the surface electrostatic
potential of the catalytic cleft is much more positive in human
Shp2 than in human Shp1.25 Although amino acid residues
presented at the base of Shp1 and Shp2 PTP catalytic clefts are
identical, all four sides of the catalytic cleft contain one or more
residues that are different between Shp1 and Shp2. These
differences suggest that the development of a Shp2-specific PTP
inhibitor, while challenging, is a realistic endeavor.
Inhibitor Design and Synthesis
We now report the development of another class of Shp2
inhibitors based on a hit from our initial screen of the NCI
Diversity set. The oxindole 5 (NSC-117199)26 was found to be
a hit with only moderate potency (IC50 ) 47 µM). Nevertheless,
5 was considered a reasonable hit, since it can be easily modified
for library synthesis. Compounds with an oxindole core have
been studied by other groups as potential therapeutic agents.27,28
On the basis of 5, we have developed PTP inhibitors that display
selectivity for Shp2 over Shp1 inhibition. Our primary aim, in
preparing the first generation library based on the oxindole 5,
was to improve the Shp2 activity and Shp2/Shp1 selectivity.
Inspection of 5 docked to Shp2 furnished a number of guidelines
for analogue design. The model was obtained by docking ligands
to the Shp2 PTP domain (from PDB 2SHP15) using GLIDE29
and methods we have described previously.19 The model reveals
the hydrazone aromatic ring system is pointing into the active
site PTP signature motif30 (VHCSAGIGRTG) with the polar
nitro group mimicking the substrate phosphate group (Figures
2 and 3). The sulfonic acid group of the 5 is hydrogen-bonded
with the basic residues Arg362 and Lys366 (Figure 2A).
The o-nitro group exhibited hydrogen bonding interactions
with backbone atoms of the PTP loop residues Ser460, Cys459,
and Arg465. The oxindole 5 appeared to fit well in the catalytic
The carboxylic acid library 14 was prepared from methyl
indole-5-carboxylate 11a (Scheme 2). Treatment of 11a with
NBS provided the dibromooxindole intermediate32 12 in moder-
ate yield. This was then converted into the desired library 14

4950 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lawrence et al.
Scheme 2a
a Reagents and conditions: (a) NBS, isopropanol/H2O (95:5), 0 °C, 45 min; (b) (i) HCl (aqueous 4 M, 1-3 mL), microwave, 150 °C, 5 min; (ii) HCl
(aqueous 4 M, 1-3 mL), ArNHNH2, microwave, 150 °C, 5 min; (c) NaOH, EtOH/H2O (1:1), 96%; (d) pentafluorophenyl trifluoroacetate, pyridine, DMF,
inert conditions, room temp, 70%; (e) RNH2, pyridine, acetonitrile, room temp; (f) NBS, isopropanol/H2O (95:5), 0 °C, 30 min; (g) dry MeOH, ArNHNH2,
microwave, 150 °C, 5 min (yield from 20-60%); (h) HCl (aqueous 1 M, 2 drops) EtOH, microwave, ArNHNH2, 120 °C, 15 min (for Z-18b irradiation time
was only 2 min).
in good yields by microwave assisted coupling with the requisite
hydrazines. This one-pot process occurs by hydrolysis of 12 to
give the intermediate isatin 13. Carboxylamides 15 were
prepared from the pentafluorophenyl ester33 of indole-5-car-
boxylic acid 11b (Scheme 2). These amides were then reacted
with NBS to form the dibromooxindole32 intermediates, which
were subsequently reacted with the requisite hydrazines to obtain
the final library 16 as shown in Scheme 2. The syntheses of the
most potent compounds are highlighted in Schemes 1 and 2.
The 5-unsubstituted oxindoles 18 were synthesized in high yield
by microwave assisted coupling of isatin and the required
hydrazines as shown in Scheme 2. The libraries 10, 14, and 16
were analyzed by NMR (1H and 13C) and low and high
we have not been able to produce the major isomer of the 18b
as a single compound. The enamine analogues 6 (X ) CH)
were prepared using the methods described by Kuyper and co-
workers.33 None of the enamine compounds were active
(synthetic details will be presented elsewhere).
Results and Discussion
The libraries were evaluated for Shp2 PTP inhibition by in
vitro PTP assay using DiFMUP as the substrate.34 The
compounds that displayed low micromolar inhibitory activity
against Shp2 are shown in Table 1. These compounds were
further screened against Shp1 and PTP1B to determine their
selectivity. It was found that the carboxylic acid, sulfonamides,
and carboxylamides at the 5-position of the oxindole moiety
and nitro or carboxylic acid functional groups at the ortho, meta,
or para position of the phenylhydrazone moiety gave rise to
the best Shp2 PTP inhibitory activity. Compounds 10a-r from
the sulfonamide library with either carboxylic acid or nitro
(ortho, meta, or para) groups on the aromatic hydrazine moiety
showed good Shp2 inhibitory activities; IC50 ) 1-10 µM with
>5 fold Shp2 over Shp1 selectivity. Compounds from the
sulfonamide library 10 displayed better solubility under the assay
conditions. The bis-carboxylic acid derivatives 14a and 14b
displayed IC50 of 0.8 and 15 µM inhibitory activity, respectively,
with 20- and 5-fold Shp2 selectivity. The compounds that lack
the 5-position carboxylic acid, carboxyamides or sulfonamide
groups (18a-e, Scheme 2) showed poor activity (IC50 > 60
µM, not shown in Table 1), indicating that the 5-substitution
with polar groups (carboxylic acid, sulfonamide, or carboxya-
mide) is important for activity and suggesting that the interac-
tions in this region with Lys366 and Arg362 (Figure 3) are
pivotal. Members of both libraries 10 and 14 that possessed
hydrogen, halogens (chloro or fluoro), primary amide or alkyl
groups on the aromatic hydrazine moiety showed poor Shp2
inhibitory activity (not reported here), further demonstrating the
importance of a carboxylate moiety (phosphotyrosine mimic)
at this position and its critical role in interacting with Cys459
and Arg465. The carboxyl group when positioned at the 3 and
4 positions for the sulfonamide library generally leads to better
activity. It is likely that a number of binding modes are possible,
1
resolution mass spectroscopy. The H NMR spectra of these
compounds indicated formation a single stereoisomer with
>95% purity. We did not observe hydrolysis of the hydrazones
in routine handling and analysis of the samples. The compounds
are chemically and configurationally stable in DMSO for
extended periods of time at room temperature as determined
by NMR (e.g., 10c and 10h). Isatin hydrazones have been
reported to exist in the Z configuration in solution, presumably
because of the intramolecular hydrogen bonding between NH
of the hydrazone linkage and the carbonyl group of the
indolinone.33 The analysis of the 13C NMR spectra of the final
compounds of the library 10 (10a-e, 10h, 10i, 10m, 10o, 10q)
revealed oxindole carbonyl chemical shifts around 163 ppm,
indicative of the Z-hydrazone stereochemical configuration.
Similarly, 13C NMR analysis of the library 14 also revealed
carbonyl shifts around 163 ppm indicating that the members
are also configured as Z. However, the reaction of isatin 17 with
2-hydrazinobenzoic acid afforded a mixture of isomers. The 1H
NMR of the compound 18b indicated formation of a mixture
of stereoisomers at approximately 1:3 ratio. Upon modifying
the reaction conditions (microwave heating, 120 °C, 2 min),
1
we see the exclusive formation (by H and 13C NMR) of the
E-configured hydrazone that was previously the minor isomer
(18c) in 48% yield. The assignment of stereochemistry was
based on observation of an NOE between the hydrazone NH
and H-4 of the isatin (see Supporting Information). For
comparison, no such NOE was observed for isatin 5. So far,

Inhibitors of Src Homology-2 Domain
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4951
Table 1. Shp2 Active Compounds from the Isatin Library
IC50 (µM)a
compd
R1
R2
Shp2
Shp1
PTP1B
5
SO3H
CO2H
CO2H
2-NO2
46.8 ( 10.2
0.8 ( 0.2
15.8 ( 6.6
22.3 ( 5.2
4.5 ( 1.7
4.5 ( 1.0
11.9 ( 1.5
4.4 ( 2.4
5.0 ( 1.9
1.3 ( 0.9
3.8 ( 0.2
1.4 ( 0.6
5.9 ( 1.4
7.4 ( 2.4
5.5 ( 0.4
22.9 ( 7.0
1.0 ( 0.2
6.3 ( 2.3
4.8 ( 0.6
5.2 ( 1.7
8.3 ( 1.7
10.6 ( 1.5
68 ( 17.1
15.4 ( 2.1
72.5 ( 19.3
19.4 ( 1.8
15.7 ( 1.7
27.7 ( 9.0
103.7 ( 17.4
40.9 ( 15.3
32.6 ( 8.5
7.1 ( 1.1
42.5 ( 7.2
18.0 ( 7.2
18.1 ( 3.7
60.8 ( 26.1
15.9 ( 1.7
32.4 ( 17.9
18.3 ( 5.2
10.8 ( 4.1
122.8 ( 74.5
73.4 ( 36.9
43.1 ( 7.2
74.2 ( 8.0
96.7 ( 29.9
1.5 ( 0.6
38.2 ( 13.9
>300
37.1 ( 4.9
9.3 ( 1.6
156.6 ( 14.1
9.8 ( 2.6
11.3 ( 4.1
2.5 ( 1.5
15.4 ( 6.2
18.8 ( 5.1
7.5 ( 1.1
11.7 ( 2.4
19.4 ( 10.2
25.7 ( 6.6
14.5 ( 1.5
11.4 ( 0.4
54.9 ( 13.3
19.3 ( 13.8
32.8 ( 8.0
63.1 ( 24.7
14a
14b
16
2-CO2H
3-CO2H
3-CO2H
3-CO2H
4-CO2H
2-NO2
CONHCH2(4-ClC6H4)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
SO2NHiPr
SO2NHiPr
SO2NH2
SO2NHCH2(4-ClC6H4)
SO2NHCH2(4-MeC6H4)
SO2NHCH2C6H5
2-NO2
3-CO2H
4-CO2H
3-CO2H
3-CO2H
4-CO2H
4-CO2H
3-CO2H
2-CO2H
3-CO2H
4-CO2H
3-CO2H
3-CO2H
4-CO2H
3-CO2H
SO2NHCH2(3-ClC6H4)
SO2NHCH2(4-ClC6H4)
SO2NHCH2(4-ClC6H4)
SO2NHCH2(2-ClC6H4)
SO2NHCH2(2-ClC6H4)
SO2NHCH2(3-CF3-4-Cl-C6H4)
SO2NHCH2(4-FC6H4)
SO2NHCH2(4-FC6H4)
SO2NHCH2(3-Cl-4-F-C6H4)
SO2NH(CH2)2(2-Cl-4-ClC6H3)
SO2NHCH2(3-CF3-4-Cl-C6H4)
SO2NHCH2(3-CF3-4-Cl-C6H4)
a Values are mean values and standard deviations of at least four experiments, each performed in duplicate.
since the shallow binding pocket contains multiple positively
charged residues available for interaction with a carboxyl group
(e.g., Lys364, Lys366, Arg 465, Arg362, Arg 465, His426,
Lys280). Varying degrees of selectivity for Shp2 versus PTP1B
phosphatase inhibition were observed. For example, the potent
compounds 14a and 10f show a 2-fold Shp2/PTP1B selectivity,
whereas halosulfonamides 10g, 10h, and 10m show a better 4-
to15-fold difference.
A comparison of the docking modes of 5 and 14a is shown
in Figure 3. The 5-substituents of the oxindole ring are
superimposed and in the model display favorable interactions
with both Lys366 and Arg362 residues and most likely
contribute toward the Shp2 affinity of the ligands. The lack of
activity found for all five derivatives of 18 (IC50 > 60 µM,
Scheme 2), which lack any 5-substituents, is consistent with
this observation.
The orientation of the hydrazine aromatic ring positions the
carboxylic acid moiety of 14a (Figure 3) so that it is capable
of undergoing additional hydrogen bond interactions with
Cys459, Gly464, and Ilu463 in the catalytic site. This site binds
substrate phosphotyrosine residues; therefore, it is not surprising
that the carboxylate ion binds better than the nitro group. These
interactions may explain the 40-fold increased potency of 14a
compared to 5 (IC50 values of 47 µM vs 0.8 µM). The indole
nitrogen atoms do not overlay well, but both show a hydrogen
bonding interaction with Shp2 amino acid residues (Asp425 in
the case of 5 and Glu361 with 14a). These observations are in
agreement with our design strategy to include both a polar group
and phosphate mimic as a model for Shp2 inhibition.
herein provides the basis for further optimization of the isatin
library, which will be reported in due course.
Experimental Section
General. All reagents were purchased from commercial suppliers
and used without further purification. Melting points were deter-
mined using a Barnstead international melting point apparatus and
1
remain uncorrected. H NMR spectra were recorded on a Varian
Mercury 400 MHz spectrometer with CDCl3 or DMSO-d6 as the
solvent. 13C NMR spectra are recorded at 100 MHz. All coupling
constants are measured in hertz (Hz), and the chemical shifts (δH
and δC) are quoted in parts per million (ppm) relative to TMS
(δ 0), which was used as the internal standard. High resolution
mass spectroscopy was carried out on an Agilent 6210 LC/MS (ESI-
TOF). Microwave reactions were performed in CEM 908005 model
and Biotage initiator 8 machines. HPLC analysis was performed
using a JASCO HPLC system equipped with a PU-2089 Plus
quaternary gradient pump and a UV-2075 Plus UV-vis detector,
using an Alltech Kromasil C-18 column (150 mm × 4.6 mm, 5
µm). Thin layer chromatography was performed using silica gel
60 F254 plates (Fisher), with observation under UV when necessary.
Anhydrous solvents (acetonitrile, dimethyl formamide, ethanol,
isopropanol, methanol, and tetrahydrofuran) were used as purchased
from Aldrich. HPLC grade solvents (methanol, acetontrile, and
water) were purchased from Burdick and Jackson for HPLC and
mass analysis.
2,3-Dioxoindoline-5-sulfonyl Chloride 8. This compound was
prepared according to a procedure reported by Lee et al.31 Mp )
200-202 °C (lit. mp ) 188-190 °C);35 1H NMR (400 MHz,
CD3CN/CDCl3 1:1) δ 9.49 (br s, NH, 1H), 8.24 (dd, J ) 8.4, 2.0
Hz, 1H), 8.16 (d, J ) 2.0 Hz, 1H), 7.22 (d, J ) 8.4 Hz, 1H).
Synthesis of Isatin Library 9. The DIPEA (2.0 mmol) and
appropriate amine (1.3 mmol) were added to a solution of 8 (1.0
mmol) in anhydrous THF (8 mL) at 0 °C under inert atmosphere.
The reaction mixture was warmed to room temperature and stirred
overnight (some reactions required 3 days for completion). The
crude reaction mixture was poured into water (10 mL) and extracted
In summary, we have identified a series of isatins capable of
selectively and potently inhibiting (one compound 14a with
submicromolar activity) Shp2 over Shp1 in vitro. Furthermore
the p-halosulfonamides 10h and 10m also display significant
Shp2/Shp1 and Shp2/PTP1B selectivity. The SAR developed

4952 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lawrence et al.
with EtOAc (3 × 15 mL). The organic phase was dried (MgSO4)
and evaporated to obtain a beige/yellow solid. This crude product
was taken to next stage without purification in the majority of the
cases (see general procedure for library 10) except compounds 9a,
9b, 9c, which were purified by trituration with cold ethyl acetate.
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonamide (9a). Yellow
solid,36 47%. Mp ) 198-200 °C, decomposed; 1H NMR (400
MHz, DMSO-d6) δ 7.02 (d, J ) 8.2 Hz, 1H), 7.38 (s, 2H), 7.82 (d,
J ) 1.8 Hz, 1H), 7.95 (dd, J ) 8.2, 1.8 Hz, 1H), 11.35 (s, 1H);
13C NMR (DMSO-d6) δ 112.90, 118.50, 122.44, 135.74, 139.13,
153.34, 160.26, 184.00; HRMS (ESI +ve) m/z calculated for
C8H7N2O4S (M + H)+ 227.0127, found 227.0133.
HRMS (ESI -ve) m/z calculated for C18H17N4O5S (M - H)-
401.0925, found 401.0929.
4-[N′-(5-Isopropylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidene)-
hydrazino]benzoic Acid (10b). 10b was obtained by method B.
1
Yellow solid, 75%. Mp ) 290 °C, decomposed; H NMR (400
MHz, DMSO-d6) δ 12.77 (s, 1H, disappeared on D2O shake), 12.74
(bs, 1H, disappeared on D2O shake), 11.47 (s, 1H, disappeared on
D2O shake), 7.95 (m, 1H), 7.94 (d, J ) 8.0 Hz, 2H), 7.70 (d, J )
8.0 Hz, 1H), 7.56 (1H, d, J ) 8.0 Hz, 2H), 7.48 (d, J ) 7.2 Hz,
1H), 7.07 (d, J ) 8.0 Hz, 1H), 0.93 (d, J ) 6.0 Hz, 6H); 13C NMR
(DMSO-d6) δ 23.91, 45.90, 111.35, 114.77, 117.83, 121.95, 125.72,
128.35, 129.08, 131.78, 136.02, 143.59, 146.57, 163.70, 167.56;
HPLC 99.9% (tR ) 1.20, 90% methanol in acetonitrile); HRMS
(ESI -ve) m/z calculated for C18H17N4O5S 401.0925 (M - H)-,
found 401.0932.
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic Acid Isopropy-
1
lamide (9b). Yellow solid, 70%. Mp ) 184-186 °C; H NMR
(400 MHz, DMSO-d6) δ 0.94 (d, J ) 6.8 Hz, 6H), 3.11 (sept, J )
6.8 Hz, 1H), 7.04 (d, J ) 8.2 Hz, 1H), 7.58 (d, J ) 6.8 Hz, 1H),
7.77 (d, J ) 1.8 Hz, 1H), 7.94 (dd, J ) 8.2, 1.8 Hz, 1H), 11.38 (s,
1H); 13C NMR (DMSO-d6) δ 23.90, 45.99, 113.15, 118.73, 123.00,
136.53, 136.65, 153.65, 160.20, 183.90; HRMS (ESI +ve) m/z
calculated for C11H13N2O4S (M + H)+ 269.0596, found 269.0594.
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonic Acid 4-Chloroben-
zylamide (9c). Yellow solid, 57%. Mp ) 250 °C, decomposed;
1H NMR (400 MHz, DMSO-d6) δ 3.97 (d, J ) 6.2 Hz, 2H), 7.00
(d, J ) 8.1 Hz, 1H), 7.20 (d, J ) 8.4 Hz, 2H), 7.29 (d, J ) 8.4 Hz,
2H), 7.68 (d, J ) 1.9 Hz, 1H), 7.89 (dd, J ) 8.1, 1.9 Hz, 1H), 8.22
(t, J ) 6.2 Hz, 1H), 11.41 (s, 1H); 13C NMR (DMSO-d6) δ 46.08,
113.10, 118.57, 123.24, 128.85, 130.20, 132.40, 135.52, 136.76,
137.12, 153.75, 160.15, 183.72; HRMS (ESI +ve) m/z calculated
for C15H12ClN2O4S (M + H)+ 351.0206, found 351.0203.
General Procedure for Synthesis of Isatin Library 10. Method
A. A mixture of the crude intermediate from library 9 (100 mg)
and hydrazinobenzoic acid (1 equiv) in ethanol (1 mL) with
hydrochloric acid (2 drops 1 M aqueous) was heated in the Biotage
microwave reactor at 120 °C for 15 min. A yellow solid precipitated
on cooling the reaction vial in an ice bath. The solid obtained was
filtered and washed with methanol to give the pure product 10.
The yields for these two steps were in the range of 15-80%.
Method B. A mixture of the pure isatin 9a-c (0.419 mmol)
and the appropriate hydrazine (0.461 mmol) in ethanol (3 mL) with
hydrochloric acid (2 drops 1 M aqueous) was heated in the Biotage
microwave reactor at 120 °C for 15 min. A yellow solid was
precipitated on cooling in an ice bath. The solid obtained was
filtered and washed with methanol to provide the pure final product.
(Z)-3-(2-(2-Nitrophenyl)hydrazono)-2-oxoindoline-5-sulfon-
ic Acid 5. Isatin-5-sulfonic acid (7) (400 mg, 1.72 mmol) and
2-nitrophenylhydrazine (296 mg, 1.65 mmol) were suspended in
ethanol (10 mL), to which was added hydrochloric acid (aqueous
4 M, 4 drops). The mixture was heated under reflux for 12 h. The
bright-orange precipitate obtained was filtered and washed with
ethanol to provide the required product 5 (0.597 g, 93%). Mp )
272 °C, decomposed; 1H NMR (400 MHz, DMSO-d6) δ 14.21 (s,
1H, disappeared on D2O shake), 11.23 (s, 1H, disappeared on D2O
shake), 8.24 (dd, J ) 8.4, 0.8 Hz, 2H), 8.21 (dd, J ) 8.4, 1.2 Hz,
1H), 7.84 (appd, J ) 1.6 Hz, 1H), 7.79 (t, J ) 7.6 Hz, 1H), 7.57
(dd, J ) 8.0, 1.6 Hz, 1H), 7.19-7.14 (m, 1H), 6.87 (d, J ) 8.0
Hz, 1H); 13C NMR (DMSO-d6) δ 110.61, 116.59, 118.11, 120.25,
122.31, 126.46, 128.58, 133.59, 133.86, 137.33, 140.02, 142.14,
143.31, 163.38; HPLC 100% (tR ) 1.38, 80% methanol in water);
HRMS (ESI -ve) m/z calculated for C14H9N4O6S (M - H)-
361.0248, found 361.0250.
3-[(2-Nitrophenyl)hydrazono]-2-oxo-2,3-dihydro-1H-indole-
5-sulfonamide (10c). 10c was obtained by method B. Yellow solid,
58%. Mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 14.24 (s,
1
1H, disappeared on D2O shake), 11.53 (s, 1H, disappeared on D2O
shake), 8.21-8.24 (m, 1H), 8.07 (d, J ) 1.6 Hz, 1H), 7.81-785
(m, 1H), 7.77 (dd, J ) 8.0, 2.0 Hz, 1H), 7.30 (s, 2H), 7.19-7.24
(m, 1H), 7.03 (d, J ) 8.4 Hz, 1H); 13C NMR (DMSO-d6) δ 111.27,
116.44, 118.04, 121.18, 122.78, 126.56, 128.56, 132.74, 133.87,
137.31, 138.72, 139.63, 144.25, 163.26; HPLC 99% (tR ) 2.28,
90% acetonitrile in water); HRMS (ESI -ve) m/z calculated for
C14H10N5O5S (M - H)- 360.0408, found 360.0412.
3-[(2-Nitrophenyl)hydrazono]-2-oxo-2,3-dihydro-1H-indole-
5-sulfonic Acid 4-Chlorobenzylamide (10d). 10d was obtained
1
by method B. Yellow solid, 43%. Mp > 300 °C; H NMR (400
MHz, DMSO-d6) δ 14.23 (s, 1H), 11.56 (s, 1H), 8.23-8.28 (m,
2H), 8.13 (t, J ) 6.4 Hz, 1H), 7.90 (d, J 1.6 Hz, 1H), 7.82 (t, J )
8.4 Hz, 1H), 7.70 (dd, J ) 8.2, 1.8 Hz, 1H), 7.23-7.27 (m, 5H),
7.06 (d, J ) 8.0 Hz, 1H), 3.98 (d, J ) 6.4 Hz, 2H); 13C NMR
(DMSO-d6) δ 46.14, 111.62, 116.69, 118.94, 121.39, 122.89,
126.58, 128.78, 129.59, 130.18, 132.38, 132.58, 133.95, 135.11,
137.32, 137.34, 139.69, 144.73, 163.26; HPLC 99% (tR ) 2.55,
90% acetonitrile in water); HRMS (ESI +ve) m/z calculated for
C21H20ClN6O5S (M + NH4)+ 503.0904, found 503.0909; calculated
for C21H17ClN5O5S (M + H)+ 486.0639, found 486.0643.
(Z)-3-(2-(5-(N-(4-Methylbenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10e). 10e was obtained by
method A. Yellow solid, 27%. Mp ) 297 °C, decomposed; 1H
NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O
shake), 11.43 (s, 1H disappeared on D2O shake), 8.07 (s, 1H), 8.02
(t, J ) 6.4 Hz, NH, 1H disappeared on D2O shake), 7.84 (s, 1H),
7.72-7.62 (m, 3H), 7.49 (t, J ) 8.0 Hz, 1H), 7.09 (d, J ) 8.4 Hz,
2H), 7.05-7.02 (m, 3H), 5.75 (s, 1H disappeared on D2O shake),
3.90 (d, J ) 6.0 Hz, 2H, CH2, singlet on D2O shake), 2.19 (s, CH3,
3H); HPLC 92% (tR ) 1.57, 90% methanol in acetonitrile); HRMS
(ESI -ve) m/z calculated for C23H19N4O5S (M - H)- 463.1082,
found 463.1087.
(Z)-4-(2-(5-(N-Benzylsulfamoyl)-2-oxoindolin-3-ylidene)hy-
drazinyl)benzoic Acid (10f). 10f was obtained by method A.
1
Yellow solid, 79%. Mp > 300 °C; H NMR (400 MHz, DMSO-
d6) δ 12.78 (s, 1H disappeared on D2O shake), 11.48 (s, 1H
disappeared on D2O shake), 8.06 (t, J ) 8.0 Hz, NH, 1H
disappeared on D2O shake), 7.95 (d, J ) 8.0 Hz, 2H), 7.93 (s, 1H),
7.70 (dd, J ) 8.0, 2.0 Hz, 1H), 7.57 (d, J ) 8.0 Hz, 2H), 7.29-7.19
(m, 5 H), 7.06 (d, J ) 8.0 Hz, 1H), 5.70 (s, 1H disappeared on
D2O shake), 3.96 (d, J ) 8.0 Hz, 2H, CH2, singlet on D2O shake);
13C NMR (DMSO-d6) δ 46.84, 111.33, 114.78, 118.01, 121.99,
125.73, 127.77, 128.28, 128.33, 128.54, 128.88, 131.77, 134.81,
138.28, 143.71, 146.57, 163.70, 167.56; HPLC 99% (tR ) 1.15,
90% methanol in acetonitrile); HRMS (ESI -ve) m/z calculated
for C22H17N4O5S (M - H)- 449.0925, found 449.0940.
3-[N′-(5-Isopropylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidene)-
hydrazino]benzoic Acid (10a). 10a was obtained by method B.
1
Yellow solid, 57%. Mp ) 275 °C, decomposed; H NMR (400
MHz, DMSO-d6) δ 12.76 (s, 1H, disappeared on D2O shake), 11.43
(s, 1H, disappeared on D2O shake), 8.05-8.06 (m, 1H), 7.92 (d, J
) 2.0 Hz, 1H), 7.67-7.70 (m, 3H), 7.47-7.51 (d, J ) 7.6 Hz,
2H), 7.45 (d, J ) 8.0 Hz, 1H), 7.05 (d, J ) 8.4 Hz, 1H), 3.21
(sept, J ) 6.8 Hz, 1H), 0.93 (d, J ) 6.8 Hz, 6H)); 13C NMR
(DMSO-d6) δ 23.88, 45.88, 111.18, 115.36, 117.44, 119.75, 122.20,
124.74, 127.90, 127.97, 130.43, 132.79, 135.89, 143.24, 143.30,
163.76, 167.72; HPLC 99% (tR ) 5.20, 75% methanol in water);
(Z)-3-(2-(5-(N-(3-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10g). 10g was obtained by
method A. Yellow solid, 40%. Mp ) 295 °C, decomposed; 1H
NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O
shake), 11.44 (s, 1 H disappeared on D2O shake), 8.17 (t, J ) 6.4
Hz, NH, 1H disappeared on D2O shake), 8.06 (s, 1H), 7.85 (d, J )

Inhibitors of Src Homology-2 Domain
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4953
1.6 Hz, 1H), 7.70 (d, J ) 6.4 Hz, 1H), 7.63 (d, J ) 8.0 Hz, 2H),
7.49 (t, J ) 8.0 Hz, 1H), 7.28-7.17 (m, 4H), 7.01 (d, J ) 8.4 Hz,
1H), 5.75 (s, 1H), 4.00 (d, J ) 6.4 Hz, 2H, CH2, singlet on D2O
shake); HPLC 99% (tR ) 1.63, 90% methanol in acetonitrile);
HRMS (ESI -ve) m/z calculated for C22H16ClN4O5S (M - H)-
483.0535, found 483.0550.
(Z)-3-(2-(5-(N-(4-Fluorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10m). 10m was obtained by
method A. Yellow solid, 40%. Mp > 300 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.76 (s, 1H disappeared on D2O shake), 11.43 (s,
1H disappeared on D2O shake), 8.10 (t, J ) 6.4 Hz, NH, 1H
disappeared on D2O shake), 8.06 (s, 1H), 7.86 (s, 1H), 7.70 (d, J
) 7.6 Hz, 1H), 7.64 (t, J ) 8.8 Hz, 2H), 7.49 (t, J ) 7.6 Hz, 1H),
7.25 (dd, J ) 8.0, 5.6 Hz, 2H), 7.08-7.02 (m, 3H), 3.94 (d, J )
6.4 Hz, 2H, CH2, singlet on D2O shake); 13C NMR (DMSO-d6) δ
46.07, 111.12, 115.42, 115.55 (d, J ) 21 Hz), 117.56, 119.78,
122.20, 124.76, 127.75, 128.16, 130.29 (d, J ) 8 Hz), 130.45,
132.79, 134.49 (d, J ) 3 Hz), 134.64, 143.31, 143.35, 161.97 (d,
J ) 241 Hz), 163.72, 167.72; HPLC 99% (tR ) 1.45, 90% methanol
in acetonitrile); HRMS (ESI -ve) m/z calculated for C22H16FN4O5S
(M - H)- 467.0831, found 467.0844.
(Z)-4-(2-(5-(N-(4-Fluorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10n). 10n was obtained by
method A. Yellow solid, 41%. Mp > 300 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.46 (s, 1
H disappeared on D2O shake), 8.07 (t, J ) 6.4 Hz, NH, 1H
disappeared on D2O shake), 7.93 (d, J ) 8.8 Hz, 2H), 7.88 (s, 1H),
7.67 (dd, J ) 8.4, 1.2 Hz, 1H), 7.56 (d, J ) 8.4 Hz, 2H), 7.27-7.23
(m, 2H), 7.08-7.03 (m, 3H), 3.95 (d, J ) 6.4 Hz, 2H, CH2, singlet
on D2O shake); HPLC 96% (tR ) 1.43, 90% methanol in
acetonitrile); HRMS (ESI -ve) m/z calculated for C22H16FN4O5S
(M - H)- 467.0831, found 467.0846.
(Z)-3-(2-(5-(N-(4-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10h). 10h was obtained by
method A. Yellow solid, 57%. Mp > 300 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake), 11.43 (s, 1
H disappeared on D2O shake), 8.14 (t, J ) 6.4 Hz, NH, 1H
disappeared on D2O shake), 8.06 (s, 1H), 7.82 (s, 1H), 7.71 (d, J
) 8.4 Hz, 1H), 7.64 (t, J ) 7.2 Hz, 2H), 7.49 (t, J ) 8.0 Hz, 1H),
7.25 (dd, J ) 19.2, 8.4 Hz, 4 H), 7.03 (d, J ) 8.4 Hz, 1H), 5.75 (s,
1H disappeared on D2O shake), 3.96 (d, J ) 6.0 Hz, 2H CH2, singlet
on D2O shake); 13C NMR (DMSO-d6) δ 46.12, 111.12, 115.46,
117.59, 119.77, 122.19, 124.76, 127.77, 128.14, 128.75, 130.45,
130.46, 132.65, 132.82, 134.68, 137.36, 143.36, 143.77, 163.74,
167.72; HPLC 98.9% (tR ) 1.18, 90% methanol in acetonitrile);
HRMS (ESI -ve) m/z calculated for C22H16N4O5SCl (M - H)-
483.0535, found 483.0536.
(Z)-4-(2-(5-(N-(4-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10i). 10i was obtained by
method A. Yellow solid, 55%. Mp > 300 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.78 (s, 1H disappeared on D2O shake), 11.47 (s,
1H disappeared on D2O shake), 8.12 (t, J ) 6.4 Hz, NH, 1H
disappeared on D2O shake), 7.94 (d, J ) 8.0 Hz, 2H,), 7.84 (s,
1H), 7.66 (d, J ) 8.0 Hz, 1H), 7.57 (d, J ) 8.0 Hz, 2H,), 7.25 (dd,
J ) 17.6, 8.4 Hz, 4H), 7.04 (d, J ) 7.6 Hz, 1H), 5.75 (s, 1H
disappeared on D2O shake), 3.97 (d, J ) 6.4 Hz, 2H, CH2, singlet
on D2O shake); 13C NMR (DMSO-d6) δ 46.12, 11.29, 114.79,
117.96, 121.94, 125.73, 128.76, 128.92, 130.15, 131.79, 131.82,
132.34, 134.82, 137.34, 143.71, 146.61, 163.68, 167.57; HRMS
(ESI -ve) m/z calculated for C22H16ClN4O5S (M - H)- 483.0535,
found 483.0540.
(Z)-3-(2-(5-(N-(3-Chloro-4-fluorobenzyl)sulfamoyl)-2-oxoin-
dolin-3-ylidene)hydrazinyl)benzoic Acid (10o). 10o was obtained
1
by method B. Yellow solid, 87%. Mp ) 293-295 °C; H NMR
(400 MHz, DMSO-d6) δ 12.76 (s, 1H disappeared on D2O shake),
11.43 (s, 1 H, disappeared on D2O shake), 8.17 (t, J ) 5.6 Hz,
NH, 1H disappeared on D2O shake), 8.01 (s, 1H), 7.79 (s, 1H),
7.70 (d, J ) 6.8 Hz, 1H), 7.64 -7.58 (m, 2H), 7.49 (t, J ) 7.6 Hz,
1H), 7.30 (d, J ) 8.0 Hz, 1H), 7.24-7.22 (m, 2H), 6.99 (d, J )
8.8 Hz, 1H), 3.99 (d, J ) 5.6 Hz, 2H, CH
2, singlet on D2O shake);
13C NMR (DMSO-d6) δ 45.59, 111.06, 115.44, 117.03, 117.24,
117.51, 119.61, 119.79, 122.17, 124.77, 127.68, 128.11, 128.98 (d,
J ) 7 Hz), 130.35 (d, J ) 15 Hz), 132.79, 134.72, 136.15 (d, J )
3 Hz), 143.34 (d, J ) 6 Hz), 156.88 (d, J ) 244 Hz), 163.70,
167.72; HRMS (ESI -ve) m/z calculated for C22H15ClFN4O5S (M
- H)- 501.0441, found 501.0437.
(Z)-4-(2-(5-(N-(2-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10j). 10j was obtained by
method A. Yellow solid, 28%. Mp > 300 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.77 (s, 1H, disappeared on D2O shake), 11.47 (s,
1H, disappeared on D2O shake), 8.14 (t, J ) 5.6 Hz, NH, 1H
disappeared on D2O shake), 7.94 (d, J ) 8.8 Hz, 2H), 7.92 (s, 1H,),
7.69 (dd, J ) 8.4, 1.6 Hz, 1H), 7.56 (d, J ) 8.8 Hz, 2H), 7.42 (d,
J ) 7.6 Hz, 1H), 7.34 (dd, J ) 7.6, 1.6 Hz, 1H), 7.29-7.21 (m,
2H), 7.05 (d, J ) 8.0 Hz, 1H), 4.04 (d, J ) 5.6 Hz, 2H, CH2,
singlet on D2O shake); HRMS (ESI -ve) m/z calculated for
C22H16ClN4O5S (M - H)- 483.0535, found 483.0538.
(Z)-3-(2-(5-(N-(2-Chlorobenzyl)sulfamoyl)-2-oxoindolin-3-
ylidene)hydrazinyl)benzoic Acid (10k). 10k was obtained by
method A. Yellow solid, 35%. Mp > 300 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.77 (s, 1H, disappeared on D2O shake), 11.44 (s,
1H, disappeared on D2O shake), 8.16 (t, J ) 5.6 Hz, NH, 1H
disappeared on D2O shake), 8.07 (s, 1H,), 7.90 (d, J ) 1.2 Hz,
1H,), 7.69 (dt, J ) 8.4, 2.0 Hz, 2H), 7.63 (d, J ) 8.0 Hz, 1H), 7.49
(t, J ) 8.0 Hz, 1H), 7.38 (dd, J ) 7.6, 2.0 Hz, 1H), 7.33 (dd, J )
7.2, 1.2 Hz, 1H), 7.29-7.21 (m, 2H), 7.05 (d, J ) 8.0 Hz, 1H),
5.75 (s, 1H, disappeared on D2O shake), 3.96 (d, J ) 5.6 Hz, 2H,
CH2, singlet on D2O shake); HRMS (ESI -ve) m/z calculated for
C22H16ClN4O5S (M - H)- 483.0535, found 483.0541.
(Z)-2-(2-(5-(N-(4-Chloro-3-(trifluoromethyl)benzyl)sulfamoyl)-
2-oxoindolin-3-ylidene)hydrazinyl)benzoic Acid (10l). 10l was
obtained by method A. Yellow solid, 35%. Mp > 300 °C; 1H NMR
(400 MHz, DMSO-d6) δ 14.25 (s, 1H disappeared on D2O shake),
11.30 (s, 1H, disappeared on D2O shake), 8.24 (t, J ) 6.4 Hz, NH,
1H disappeared on D2O shake), 8.04 (d, J ) 8.4 Hz, 1H), 7.95
(dd, J ) 8.0, 1.6 Hz, 1H), 7.74 (d, J ) 1.6 Hz, 1H), 7.66, (t, J )
7.6 Hz, 1H,), 7.60-7.52 (m, 4H), 7.12 (t, J ) 7.6 Hz, 1H), 6.95
(d, J ) 8.0 H, 1H), 4.14 (d, J ) 6.4 Hz, 2H, CH2, singlet on D2O
shake); HRMS (ESI -ve) m/z calculated for C23H15ClF3N4O5S (M
- H)- 551.0409, found 551.0414.
(Z)-3-(2-(5-(N-(2,4-Dichlorophenethyl)sulfamoyl)-2-oxoindo-
lin-3-ylidene)hydrazinyl)benzoic Acid (10p). 10p was obtained
1
by method A. Yellow solid, 14%. Mp ) 290-292 °C; H NMR
(400 MHz, DMSO-d6) δ 12.77 (s, 1H disappeared on D2O shake),
11.43 (s, 1H disappeared on D2O shake), 8.07 (s, 1H), 7.82 (s,
1H), 7.71 (d, J ) 6.4 Hz, 2H), 7.64-7.61 (m, 2H, integrated to1H
on D2O shake), 7.50 (t, J ) 8.4 Hz, 1H), 7.44 (d, J ) 1.6 Hz 1H),
7.31-7.25 (m, 2H), 7.03 (d, J ) 8.4 Hz, 1H), 5.75 (s, 1H
disappeared on D2O shake), 2.99 (q, J ) 6.4 Hz, 2H, CH2, triplet
on D2O shake), 2.76 (t, J ) 6.4 Hz, 2H, CH2); HRMS (ESI -ve)
m/z calculated for C23H17Cl2N4O5S (M - H)- 531.0302, found
531.0304.
(Z)-4-(2-(5-(N-(4-Chloro-3-(trifluoromethyl)benzyl)sulfamoyl)-
2-oxoindolin-3-ylidene)hydrazinyl)benzoic Acid (10q). 10q was
obtained by method A. Yellow solid, 34%. Mp > 300 °C; 1H NMR
(400 MHz, DMSO-d6) δ 12.76 (s, 1H disappeared on D2O shake),
11.44 (s, 1 H disappeared on D2O shake), 8.25 (t, J ) 6.4 Hz, NH,
1H disappeared on D2O shake), 7.94 (d, J ) 8.8 Hz, 2H), 7.70 (s,
1H), 7.58-7.52 (m, 6H), 6.95 (d, J ) 8.4 Hz, 1H), 5.75 (s, 1H
disappeared on D2O shake), 4.13 (d, J ) 6.4 Hz, 2H, CH2, singlet
on D2O shake); 13C NMR (DMSO-d6) δ 45.72, 111.15, 114.76,
117.90, 121.79, 125.73, 126.04 (q, J ) 272 Hz), 126.64, 126.94,
127.46 (d, J ) 5 Hz), 128.37, 128.72, 131.76, 131.93, 134.10,
134.93, 138.27, 143.68, 146.59, 163.64, 167.58; HRMS (ESI -ve)
m/z calculated for C23H15ClF3N4O5S (M - H)- 551.0409, found
551.0404.
(Z)-3-(2-(5-(N-(4-Chloro-3-(trifluoromethyl)benzyl)sulfamoyl)-
2-oxoindolin-3-ylidene)hydrazinyl)benzoic Acid (10r). 10r was
obtained by method A. Yellow solid, 40%. Mp ) 292-294 °C;
1H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H disappeared on

4954 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lawrence et al.
1
D2O shake), 11.41 (s, 1 H disappeared on D2O shake), 8.28 (t, J )
6.4 Hz, NH, 1H disappeared on D2O shake), 8.05 (s, 1H), 7.70-7.68
(m, 2H), 7.63 (d, J ) 7.6 Hz, 1H), 7.57-7.47 (m, 5H), 6.95 (d, J
) 8.0 Hz, 1H), 4.12 (d, J ) 6.4 Hz, 2H, CH2, singlet on D2O
shake); HRMS (ESI -ve) m/z calculated for C23H15ClF3N4O5S (M
- H)- 551.0409, found 551.0420.
was used in the next step without further purification. H NMR
(400 MHz, DMSO-d
6) δ 11.40 (s, 1H, disappeared on D2O
shake), 11.32 (s, 1H), 8.91 (t, J ) 6.4 Hz, 1H, changed on D2O
shake), 8.15 (s, 1H), 7.64 (dd, J ) 8.4, 1.2 Hz, 1H), 7.40-7.31
(m, 5H), 6.51 (m, 1H), 4.45 (d, J ) 6.0 Hz, 2H, singlet on D2O
shake).
3,3-Dibromo-2-oxo-2,3-dihydro-1H-indole-5-carboxylic Acid
Methyl Ester (12). N-Bromosuccinimide (13.41 g, 74.91 mmol)
was added portionwise to a solution of 5-methyl indole-2-
carboxylate (11a) (4.50 g, 25.71 mmol) in isopropanol/H2O (95:5,
350 mL) over 45 min under argon at room temperature. After the
addition, the solvent was removed under reduced pressure and the
solid residue was triturated with cold acetone (150 mL) to give the
3,3-Dibromo-N-(4-chlorobenzyl)-2-oxoindoline-5-carboxam-
ide, Intermediate for 16. The indole 15 (252 mg, 0.89 mmol) was
dissolved in aqueous isopropanol/H2O (95:5, 5 mL), and NBS
(0.471 g, 2.65 mmol) was added portionwise over 30 min with
stirring under argon atmosphere. The reaction was monitored by
TLC (EtOAc/hexane, 1:1). TLC indicated the disappearance of the
starting material. The reaction mixture was concentrated at room
temperature and diluted with ether (approximately 10 mL). The
succinimide precipitate was filtered and washed with ether. The
ether phase was concentrated to obtain a pale-yellow solid (225
mg, 55%). Attempts to purify this intermediate were not successful.
The crude compound was used in the next step.
1
pure product as a yellow solid (4.90 g, 55%). Mp > 300 °C; H
NMR (400 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.05 (d, J ) 1.5 Hz,
1H), 7.98 (dd, J ) 8.2, 1.5 Hz, 1H), 7.06 (d, J ) 8.2 Hz, 1H), 3.86
(s, 3H); 13C NMR (DMSO-d6) δ 52.92, 111.96, 125.37, 126.83,
132.08, 134.15, 143.28 (2C), 165.88, 171.27.
(Z)-3-(2-(2-Carboxyphenyl)hydrazono)-2-oxoindoline-5-car-
boxylic Acid (14a). Methyl dibromooxindole carboxylate 9 (40 mg,
0.114 mmol) was suspended in HCl (aqueous 4 M, 2.00 mL) in a
microwave vial and heated at 150 °C for 5 min. The intermediate
13 was not isolated (Scheme 2). 2-Hydrazinylbenzoic acid (23 mg,
0.126 mmol) was added to the reaction mixture and heated using
the Biotage microwave reactor at 150 °C for 15 min. The solid
obtained upon cooling the reaction vial was filtered and washed
with DCM to obtain the pure 14a (15 mg, 40%) as an off-white
(Z)-3-(2-(5-(4-Chlorobenzylcarbamoyl)-2-oxoindolin-3-ylidene)-
hydrazinyl)benzoic Acid (16). The crude dibromoisatinamide
intermediate from the above experiment (70 mg, 0.152 mmol) was
suspended in dry MeOH (2.0 mL) in a microwave vial (CEM, 10.0
mL), and 3-hydrazinylbenzoic acid (31 mg, 0.166 mmol) was added
and irradiated for 5 min at 150 °C in the CEM microwave reactor.
The reaction vial was left in an ice bath until a precipitate formed.
The compound 16 (15 mg, 22%) was isolated as a pale-yellow solid.
1
1
Mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 13.10 (broad s,
solid. Mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 14.22 (s,
1H, disappeared on D2O shake), 12.79 (s, 1H, disappeared on D2O
shake), 11.31 (s, 1H, disappeared on D2O shake), 9.13 (broad t,
1H), 8.12 (s, 1H), 8.04 (s, 1H), 7.84 (d, J ) 8.4 Hz, 1H), 7.67 (d,
J ) 6.8 Hz, 1H), 7.61 (d, J ) 7.6 Hz, 1H), 7.48 (t, J ) 6.8 Hz,
1H), 7.37-7.33 (m, 4H), 6.99 (d, J ) 8.0 Hz, 1H), 4.45 (d, J )
5.2 Hz, 2H,); HPLC 95% (tR ) 1.70, 80% methanol in water);
LRMS (ESI +ve) m/z 449 [100, (M + H+)]; HRMS (ESI -ve)
m/z calculated for C23H17ClN4O4 (M - H)- 447.0860, found
447.0861.
1H, disappeared on D2O shake), 11.27 (s, 1H, disappeared on D2O
shake), 8.11 (appd, J ) 1.2 Hz, 1H), 8.07 (d, J ) 8.4 Hz, 1H),
7.93 (dd, J ) 8.0, 1.2 Hz), 7.87 (dd, J ) 8.0, 1.2 Hz, 1H), 7.63 (t,
J ) 8.4 Hz, 1H), 7.09 (t, J ) 7.2 Hz, 1H), 6.99 (d, J ) 8.4 Hz,
1H); 13C NMR (DMSO-d6) δ 110.94, 114.55, 114.88, 120.71,
121.93, 122.48, 124.98, 129.82, 131.53, 131.97, 135.21, 144.74,
145.26, 163.04, 167.82, 168.91; HPLC 100% (tR ) 1.55, 80%
methanol in water); HRMS (ESI -ve) m/z calculated for
C16H10N3O5 (M - H)- 324.0625, found 324.0622.
(Z)-3-(2-(3-Carboxyphenyl)hydrazono)-2-oxoindoline-5-car-
boxylic Acid (14b). The procedure was same as for the compound
14a. 3-Hydrazinylbenzoic acid (23 mg, 0.126 mmol) was used to
obtain the final compound 14b (20 mg, 54%) as an off-white solid.
(Z)-3-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)benzoic Acid (18a).
A mixture of isatin (0.111 g, 0.775 mmol) and 3-carboxylphenyl-
hydrazine (0.085 g, 0.160 mmol) and HCl (aqueous 1 M, 2 drops)
in ethanol (3 mL) was heated in the Biotage microwave at 120 °C
for 15 min. After the mixture was cooled to room temperature,
pure product 18a was collected as a yellow precipitate by filtration
and dried in vacuo (0.184 g, 0.65 mmol, 84%). Yellow solid, 84%.
1
Mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 12.97 (broad s,
1H, disappeared on D2O shake), 12.70 (s, 1H, disappeared on D2O
shake), 11.37 (s, 1H, disappeared on D2O shake), 8.06 (s, 1H), 8.00
(s, 1H), 7.86 (dd, J ) 8.0, 1.2 Hz, 1H), 7.72 (d, J ) 8.0 Hz, 1H),
7.60 (d, J ) 7.6 Hz, 1H), 7.49-7.45 (m, 1H), 7.00 (d, J ) 8.4 Hz,
1H); HPLC 100% (tR ) 1.60, 80% methanol in water); HRMS
(ESI -ve) m/z calculated for C16H10N3O5 (M - H)- 325.0625,
found 324.0635.
1
Mp > 300 °C; H NMR (400 MHz, DMSO-d6) δ 13.05 (broad s,
1H), 12.77 (s, 1H), 11.04 (s, 1H), 9.91 (d, J ) 8.0 Hz, 1H), 7.96
(s, 1H), 7.54-7.64 (m, 3H), 7.44 (t, J ) 8.0 Hz, 1H), 7.24 (t, J )
7.2 Hz, 1H), 7.04 (t, J ) 8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) δ 111.23, 115.06, 119.16, 119.54, 121.66, 122.62, 124.12,
129.24, 129.52, 130.39, 132.74, 140.77, 143.57, 163.75, 167.75;
HRMS (ESI -ve) m/z calculated for C15H10N3O3 (M - H)-
280.0727, found 280.0736.
Pentafluorophenyl 1H-Indole-5-carboxylate, Intermediate for
15. To a solution of 5-indolecarboxylic acid (0.5 g, 3.10 mmol) in
DMF (3.00 mL) was added pentafluorophenyl trifluoroacetate (1.068
mL, 6.20 mmol) followed by pyridine (0.281 mL). The reaction
mixture (a suspension was obtained at this stage) was stirred at
room temperature under inert atmosphere for approximately 30 min.
The reaction mixture was poured into ether (40 mL) and diluted
with ethyl acetate (2 × 50 mL). The organic phase was washed
with water, dried (Na2SO4), and concentrated to obtain an off-white
solid (720 mg, 70%), TLC Rf ) 0.71 (EtOAc/hexane, 1:1). No
purification was necessary: 1H NMR (400 MHz, DMSO-d6) δ 11.71
(s, 1H, NH), 8.49 (s, 1H), 7.85 (dd, J ) 8.4, 2.0 Hz, 1H), 7.59-7.55
(m, 2H), 6.67 (broad t, 1H).
N-(4-Chlorobenzyl)-1H-indole-5-carboxamide (15). The pen-
tafluorophenyl ester (200 mg, 0.61 mmol) from the above experi-
ment was suspended in dry acetonitrile under argon. Pyridine (0.075
mL, 0.85 mmol) was added followed by 4-chlorobenzylamine (121
mg, 0.85 mmol) and stirred overnight (approximately 12 h). The
resulting cloudy solution was diluted with EtOAc and washed
with HCl (4 M aqueous, 6 mL). The organic phase was separated,
washed with water, dried (Na2SO4), and concentrated to give
the intermediate 15 (252 mg) as an orange solid. This compound
(E) and (Z)-2-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)benzoic
Acid (18b). A mixture of isatin (17) (0.074 g, 0.517 mmol),
2-carboxylphenylhydrazine (0.107 g, 0.568 mmol), and HCl (aque-
ous 1 M, 2 drops) in ethanol (3 mL) was heated in the Biotage
microwave reactor at 120 °C for 15 min. After the mixture was
cooled to room temperature, hydrazone 18b (0.057 g, 0.20 mmol,
72%) was collected as a mixture of isomers (Z/E 3:1) as a yellow
1
precipitate by filtration and dried in vacuo. H NMR (400 MHz,
DMSO-d6) δ 14.19 (s, 1H), 10.92 (s, 1H), 8.01 (d, J ) 8.0 Hz,
1H), 7.92 (d, J ) 7.6 Hz, 1H), 7.58-7.69 (m, 2H, overlap with
the minor isomer), 7.25 (t, J ) 7.6 Hz, 1H), 7.21-7.11 (m, 2H,
overlap with the minor isomer), 6.90 (d, J ) 6.8 Hz, 1H); minor
isomer (E) (25%) δ 12.39 (s, 1H), 10.72 (s, 1H), 8.04 (d, J ) 8.0
Hz, 1H), 7.97 (dd, J ) 8.0 Hz, 1H), 7.85 (d, J ) 8.4 Hz, 1H),
7.58-7.69 (m, 1H, overlap with the major isomer), 7.37 (t, J )
7.2 Hz, 1H), 7.21-7.11 (m, 2H, overlap with the major isomer),
6.94 (d, J ) 7.2 Hz, 1H); HRMS (ESI -ve) m/z calculated for
C15H10N3O3 (M - H)- 280.0727, found 280.0732.

Inhibitors of Src Homology-2 Domain
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4955
(E)-2-(2-(2-Oxoindolin-3-ylidene)hydrazinyl)benzoic Acid (18b).
A mixture of isatin (17) (0.060 g, 0.419 mmol), 2-carboxylphe-
nylhydrazine (0.085 g, 0.461 mmol), and HCl (aqueous 1 M, 2
drops) in ethanol (3 mL) was heated in the Biotage microwave
reactor at 120 °C for 2 min. After the mixture was cooled to room
temperature, pure product 18b was collected as a yellow precipitate
by filtration and dried in vacuo (0.057 g, 0.20 mmol, 48%). Mp >
300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 10.71 (s,
1H), 8.04 (d, J ) 6.8 Hz, 1H), 7.97 (dd, J ) 1.2, 8.0 Hz, 1H), 7.85
(d, J ) 8.4 Hz, 1H), 7.64-7.68 (m, 1H), 7.37 (t, J ) 7.6 Hz, 1H),
7.06 (m, 2H), 6.94 (d, J ) 7.6 Hz, 1H); 13C NMR (DMSO-d6) δ
111.33, 133.33, 114.38, 116.43, 121.97, 122.21, 122.82, 131.72,
131.97, 132.72, 135.56, 142.87, 146.20, 165.60, 170.63; HRMS
(ESI -ve) m/z calculated for C15H10N3O3 (M - H)- 280.0727,
found 280.0736.
description of the molecular modeling studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
Note Added in Proof. Birchmeier and co-workers have recently
described the virtual screening based discovery of several phenyl-
hydrazonpyrazolone derivatives as Shp2 inhibitors. See Hellmuth,
K.; Grosskopf, S.; Lum, C. T.; Wuertele, M.; Roeder, N.; von Kries,
J. P.; Rosario, M.; Rademann, J.; Birchmeier, W. Specific inhibitors
of the protein tyrosine phosphatase Shp2 identified by high-
throughput docking. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7275-
7280.
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1
solid (145 mg, 38%). Mp ) 279 °C, decomposed; H NMR (400
MHz, DMSO-d6) δ 11.56 (s, 1H, disappeared on D2O shake), 10.84
(s, 1H, disappeared on D2O shake), 8.22 (d, J ) 7.2 Hz, 1H), 8.01
(d, J ) 8.4 Hz, 1H), 7.87-7.80 (m, 2H), 7.44-7.40 (m, 1H), 7.18
(q, J ) 14.4, 7.2 Hz, 2H), 6.96 (d, J ) 7.6 Hz, 1H); 13C NMR
(DMSO-d6) δ 111.76, 116.27, 116.66, 122.44, 122.68, 123.69,
126.60, 132.92, 134.18, 135.96, 137.43, 140.57, 143.85, 165.17;
HRMS (ESI +ve) m/z calculated for C14H11N4O3 (M + H)+
283.0826, found 283.0834.
(Z)-3-(2-(3-Nitrophenyl)hydrazine)indolin-2-one (18d). A mix-
ture of isatin (17) (200 mg, 1.35 mmol), 2-nitrophenylhydrazine
(226 mg, 1.48 mmol), and HCl (aqueous 4 M, 4 drops) in ethanol
(26.0 mL) was heated under reflux for 2 h. The reaction mixture
was cooled to room temperature and the yellow solid was filtered
and washed with ethanol to obtain the pure product 18d (279 mg,
73%) as a yellow solid. Mp ) 267-269 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.75 (s, 1H), 11.07 (s, 1H), 8.25 (t, J ) 4.0 Hz,
1H), 7.85 (ddd, J ) 8.0, 2.8, 0.8 Hz, 1H), 7.81 (ddd, J ) 8.0, 2.0,
0.8 Hz, 1H), 7.62-7.57 (m, 2H), 7.26 (td, J ) 7.6, 1.2 Hz, 1H),
7.04 (td, J ) 7.2, 1.2 Hz, 1H), 6.90 (d, J ) 7.6 Hz, 1H); 13C NMR
(DMSO-d6) δ 108.91, 111.30, 117.34, 119.95, 121.14, 122.71,
130.07, 130.53, 131.53, 141.24, 144.24, 144.80, 149.46, 163.49;
HRMS (ESI +ve) m/z calculated for C14H11N4O3 (M + H)+
283.0826, found 283.0835.
Phosphatase Activity. PTP activity was measured using the
fluorogenic 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP,
from Molecular Probes) as the substrate. Each reaction contained
25 mM MOPS (pH 7.0), 50 mM NaCl, 0.05% Tween-20, 1 mM
DTT, 20 µM DiFMUP, 10 nM Microcystin LR, 20 nM PTP (Shp2,
Shp1 or PTP1B),19 and 5 µL of test compound or dimethyl sulfoxide
(DMSO, solvent) in a total reaction volume of 100 µL in a black
96-well plate. Reaction was initiated by addition of DiFMUP, and
the incubation time was 30 min at room temperature. DiFMUP
fluorescence signal was measured at an excitation of 355 nm and
an emission of 460 nm with a Wallac Victor2 1420 plate reader.
IC50 was defined as the concentration of an inhibitor that caused a
50% decrease in the PTP activity. For IC50 determination, eight
concentrations of compounds at 1/3 dilution (∼0.5 log) were tested.
Each experiment was performed in duplicate, and IC50 data were
derived from at least four independent experiments. The curve-
fitting program Prism 4 (GraphPad Software) was used to calculate
the IC50 value.
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