Salicylic acid based small molecule inhibitor for the oncogenic src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2)

Article abstract of DOI:10.1021/jm901645u

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

Full text of DOI:10.1021/jm901645u

2482 J. Med. Chem. 2010, 53, 2482–2493
DOI: 10.1021/jm901645u
Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing
Protein Tyrosine Phosphatase-2 (SHP2)†
Xian Zhang,‡,^ Yantao He,‡ Sijiu Liu,‡ Zhihong Yu,‡ Zhong-Xing Jiang,‡ Zhenyun Yang,§ Yuanshu Dong,‡
Sarah C. Nabinger,§ Li Wu,‡, Andrea M. Gunawan, Lina Wang,‡ Rebecca J. Chan,§ and Zhong-Yin Zhang*,‡,
‡Department of Biochemistry and Molecular Biology, §Herman B. Wells Center for Pediatric Research, and Chemical Genomics Core Facility,
Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202, and ^Center for Chemical Genetics and Drug Discovery
and College of Chemistry, Nankai University, Tianjin, China
Received November 6, 2009
The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in
growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan
syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as
a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combina-
torial library approach aimed at binding both active site and unique nearby subpockets for enhanced
affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08
(compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated
ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that
chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment.
X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular
determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.
Introduction
factor, cytokine, integrin, and hormone signaling pathways
regulating processes such as cell proliferation, differentiation,
adhesion, migration, and apoptosis.6,7 In nearly all cases, the
catalytic activity of SHP2 is required for full activation of the
Ras-ERK1/2 cascade that is mediated through dephosphory-
lation of substrates that are negatively regulated by tyrosine
phosphorylation.6-8 The critical role of SHP2 in cell physiol-
ogy is further underscored by the identification of mutations
within SHP2, which are linked to several human diseases.
Thus, germline mutations in SHP2 that cause hyperactivation
of its phosphatase activity are associated with 50% Noonan
syndrome, an autosomal dominant disorder that is estimated
to occur in 1:1000 to 1:2500 live births in the U.S. and world-
wide.9,10 Many Noonan syndrome patients display hematolo-
gic abnormalities, including myeloid disorders and juvenile
myelomonocytic leukemia (JMML).10 Somatic gain-of-func-
tion mutations in SHP2 occur in 35% of individuals with
JMML, as well as in acute myeloid leukemia (AML, 4%),
myelodysplastic syndrome (10%), and acute lymphoid leuke-
mia (7%).11-15 In addition to childhood leukemia, SHP2
mutations also occur in adult AML (6%) as well as in solid
tumors including lung adenocarcinoma, colon cancer, neuro-
blastoma, melanoma, and hepatocellular carcinoma.16-18
Finally, SHP2 is also implicated in gastric ulcer and, ulti-
mately, gastric carcinoma caused by Helicobacter pylori, which
harbors a key virulence factor CagA that promotes SHP2
activation when it is tyrosine phosphorylated by the host Src
family kinases.19 Collectively, these genetic and biochemical
observations identify SHP2 as the first bona fide oncogene in
the PTP superfamily.
Reversible tyrosine phosphorylation, catalyzed by the co-
ordinated actions of protein tyrosine kinases (PTKsa) and
protein tyrosine phosphatases (PTPs), is of critical importance
to the regulation of signaling events that underlie virtually all
essential cellular processes.1,2 Not surprisingly, disturbance of
the normal balance between PTK and PTP activity results in
aberrant tyrosine phosphorylation, which has been linked to
the etiology of several human diseases, including cancer.3,4 For
example, abnormal expression and activation of many PTKs,
such as epidermal growth factor receptor and proto-oncogene
products Src and Abl, are causative factors for a number of
malignancies.5 By catalyzing the removal of the phosphoryl
group from tyrosine residues, the PTPs are generally viewed as
negative regulators due to their ability to reverse the actions
of PTKs, which usually drive signaling cascades. However,
mounting genetic and biochemical evidence suggests that
some PTPs can potentiate, rather than antagonize, signaling
mediated by the PTKs. This mode of synergy enhances
mitogenic processes, leading to cell transformation.
The Src homology-2 domain containing protein tyrosine
phosphatase-2, SHP2, is a positive transducer of growth
†The coordinates for the structure of the SHP2 II-B08 (compound 9)
complex (accession number 3JRL) have been deposited in the Protein
Data Bank.
*To whom correspondence should be addressed. Phone: 317-274-
8025. Fax: 317-274-4686. E-mail: zyzhang@iupui.edu.
aAbbreviations: SHP2, Src homology-2 domain containing protein
tyrosine phosphatase-2; PTP, protein tyrosine phosphatase; PTK, pro-
tein tyrosine kinase; ERK, extracellular signal-regulated protein kinase;
EGF, epidermal growth factor; GM-CSF, granulocyte macrophage
colony stimulating factor.
3
In view of the importance of SHP2 in various kinds of
leukemias and solid tumors, SHP2 represents an exciting
r
pubs.acs.org/jmc
Published on Web 02/19/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2483
target for anticancer and antileukemia therapy. Moreover,
giventhe obligatoryrequirement of SHP2 function in multiple
oncogenic receptor tyrosine kinase pathways, inhibition of
SHP2 may also prove effective for cancers with coactivation
of receptor PTKs, which respond poorly to kinase inhibitor
monotherapy.20 Understandably, there is increasing interest
in targeting SHP2 for therapeutic development.21-25 How-
ever, obtaining SHP2 inhibitors with optimal potency and
pharmacological properties has been difficult primarily be-
cause of the highly conserved and positively charged nature of
the active site pocket shared by all PTP family members. We
describe here a salicylic acid based combinatorial library
approach that led to the identification of a SHP2 inhibitor
II-B08 (compound 9) with highly efficacious cellular activity.
This proof-of-principle SHP2 inhibitor blocks growth factor
stimulated ERK1/2 activation and hematopoietic progenitor
proliferation, validating the concept that chemical inhibition
of SHP2 may be therapeutically useful for anticancer and
antileukemia treatment. X-ray crystallographic analysis of
compound 9-bound SHP2 structure together with structure
and activity studies reveal molecular determinants that can be
exploited for the acquisition of more potent and selective
SHP2 inhibitors.
Figure 1. Strategy for the construction of an indole salicylic acid
based library using click chemistry.
lopment because they are evolutionarily selected and vali-
dated for interfering and interacting with biological targets.
The indole nucleus is a prominent structural scaffold found
in numerous natural products and synthetic compounds with
vital medicinal value.31,32 To this end, we have focused our
attention on indole-based salicylic acids as PTP active-site-
directed pTyr mimetics. We hypothesized that an indole-
based salicylic acid may bind the active site and interact in the
desired inhibitory fashion with the PTPs, and additional
diversity elements to which the salicylic core is attached to
should render the inhibitors PTP isozyme-selective.
To target both the active site and an adjacent, peripheral
secondary binding site in SHP2, we have developed a salicylic
acid based combinatorial library approach that entails the
following criteria: (1) each member will be of a modular,
bidentate structure containing both an active-site-directed
core (the salicylic acid derivative) and a diversity element for
interaction with adjacent peripheral sites in SHP2; (2) both
the core group and the diversity elements will possess favor-
able pharmacological properties; (3) each member will be
efficiently assembled in situ for facile screening against
SHP2. We chose click chemistry for library construction
because it offers an expedient way to connect two compo-
nents together with high yield and purity under extremely
mild conditions.33 More importantly, the click reaction can
be conducted in aqueous solution in the absence of deleter-
ious reagents, thus allowing direct screening and identifica-
tion of hits from the library. In fact, click chemistry has
found increasing applications in lead discovery and optimi-
zation for a number of enzymes including the PTPs.34-39
Figure 1 depicts a focused library-based strategy for the
acquisition of potent and selective SHP2 inhibitory agents
that are capable of bridging both the active site and an
adjacent peripheral site. The library contains (a) a salicylic
acid core to engage the PTP active site and (b) four alkyl
linkers of one to four methylene units to tether the pTyr
mimetic to (c) a structurally diverse set of amines and
hydrazines aimed at capturing additional interactions with
adjacent pockets surrounding the active site. In the interest
of keeping the library to a reasonable size, we selected
53 amines and hydrazines that vary by molecular weight,
charge, polarity, hydrophobicity, sterics, etc. and therefore
provide a reasonable (albeit limited) structural diversity to
increase the number and strength of noncovalent interac-
tions between SHP2 and the inhibitor.
Results and Discussion
Identification of Compound 9 as a SHP2 Inhibitor from a
Salicylic Acid Based Combinatorial Library. As discussed
above, SHP2 is an outstanding target for oncology. How-
ever, the common architecture of the active site (i.e., pTyr-
binding pocket) shared by all PTPs poses a significant
problem for the acquisition of selective SHP2 inhibitors.
Fortunately, it has been recognized that pTyr alone is not
sufficient for high-affinity binding and residues flanking
pTyr are important for PTP substrate recognition.26 These
findings point to a conceptual framework for PTP inhibitor
design, namely, bidentate ligands capable of engaging both
the active site and an adjacent less-conserved subpocket for
enhanced affinity and selectivity. In principle, active site-
directed, potent, and selective PTP inhibitors can be devised
by tethering a nonhydrolyzable pTyr mimetic to appropri-
ately functionalized moieties to bind both the active site and
a unique nearby subpocket.26,27 Indeed, a number of potent
and selective PTP1B inhibitors have been developed using
this strategy.28
Unfortunately, the highly positively charged pTyr-bind-
ing pocket impedes the development of inhibitors with
favorable bioavailability. Most of the pTyr mimetics de-
scribed in the literature are not druglike and thus are
deficient in cell membrane permeability. Indeed, the lack of
cellular efficacy of existing PTP inhibitors represents a major
obstacle in developing phosphatase-based therapeutics.
Consequently, there is continued interest in developing pTyr
mimetics with more acceptable physicochemical properties.
We recently discovered from an in silico DOCK screening
campaign that the natural product salicylic acid can serve as
a pTyr mimetic.29 We further demonstrated that naphthyl
and polyaromatic salicylic acid derivatives exhibit enhanced
affinity for PTPs relative to the corresponding single ring
compounds.29,30 Therefore, we sought to develop bicyclic
salicylic acid based PTP inhibitors that carry sufficient polar
and nonpolar interactions with the active site and yet possess
improved pharmacological properties. Bioactive natural
products are very promising starting points for drug deve-
As shown in Scheme 1, the salicylic acid core 1 was
synthesized in eight steps with Sonogashira coupling and
an electrophilic cyclization as key steps. After esterification
of 4-aminosalicylic acid with dimethyl sulfate, compound 2
was isolated in 86% yield which was then treated with
paraformaldehyde and NaCNBH3 to give the product N,
N-dimethylaniline 3 with good yield. Selective iodination of

2484 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Zhang et al.
Scheme 1. Synthesis of the Alkyne-Containing Salicylic Acid
Core 1a
a Conditions: (a) Me2SO4, Na2CO3, acetone, room temp, 86%;
(b) (CH2O)n, NaCNBH3, AcOH, room temp, 78%; (c) I2, K2CO3,
water, room temp, 42%; (d) phenylacetylene, Pd(PPh3)2Cl2, CuI,
Et3N, DMF, 89%; (e) I2, CH2Cl2, room temp, 87%; (f) trimethylsilyla-
cetylene, Pd(PPh3)2Cl2, CuI, Et3N, DMF, room temp, 73%; (g) TBAF,
THF, room temp, 69%; (h) KOH, THF/H2O, reflux, 38%.
compound 3 with iodine gave methyl 4-(dimethylamino)-
2-hydroxy-5-iodobenzoate 4, which was then coupled with
the commercially available phenylacetene with Sonogashira
coupling to afford compound 5 with excellent yield. Com-
pound 6 was constructed by electrophilic cyclization of 5
with iodine in 89% yield. Key compound 7 was generated
through another Pd(0)-catalyzed Sonogashira coupling of
compound 6 with trimethylsilyacetylene. Finally, sequential
removal of the protective groups in compound 7 with TBAF
and KOH gave the salicylic acid core 1.
To increase potency and selectivity, the strategically posi-
tioned alkyne in salicylic acid core 1 was tethered to 212
different azide-containing diversity elements (53 discrete
amines and hydrazines (Figure 2) with four alkyl linkers
of one to four methylenes length), using click chemistry
or the Cu(I)-catalyzed [3 þ 2] azide-alkyne cycloaddition
reaction (Figure 1). The azide-containing building blocks
were synthesized in a one-pot procedure (Scheme 2) in which
amines or hydrazines were reacted with the acyl chloride
linkers in N,N-dimethylformamide (DMF) followed by SN2
reaction with sodium azide to generate the corresponding
azides.
Figure 2. Chemical structures of 53 amines and hydrazines.
Scheme 2. Synthesis of Azide-Tagged Diversity Elements
To construct the 212-member library, each azide (32 mM)
was coupled with the alkyne containing core 1 (26.6 mM) in
the presence of 1.33 mM CuSO4 and 6.64 mM sodium
ascorbate and 0.798 mM TBTA (tris(benzyltriazolylmethyl)-
amine) in 0.5 mL solvent (THF/t-BuOH/H2O = 1:1:1) at
room temperature for 12 h. The products were assessed by
LC-MS and used directly for screening without further
purification (the purity was in the range 40-70%).
The ability of the library compounds to inhibit the SHP2-
catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) was
assessed at pH 7 and 25 ꢀC. Out of the 212-member library,
5 compounds displayed measurable inhibitory activity at
∼10 μM. Resynthesis of the hits confirmed that they were
genuine inhibitors for SHP2 with IC50 values in the low
micromolar range (Table 1). These values compare very
favorable to the alkyne-containing salicylic acid core, which
displays an IC50 of 212 ( 23 μM for SHP2. The results
indicate that the linker and amine diversity element contri-
bute significantly to SHP2 binding. In addition, the structure
and activity data revealed that compounds with substituted
biphenyls as the diversity elements were preferentially se-
lected by SHP2. Compound 9 (Table 1) appeared to be the
most potent for SHP2 with an IC50 of 5.5 ( 0.4 μM and was
selected for further characterization. Kinetic analysis indi-
cated that 9 is a reversible and noncompetitive inhibitor for
SHP2 with a Ki of 5.2 ( 0.3 μM (Figure 3). To determine
the specificity for 9, its inhibitory activity toward a panel of
mammalian PTPs including cytosolic PTPs, SHP1, PTP1B,
Lyp, HePTP, and FAP1, the receptor-like PTPs, CD45,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2485
Table 1. IC50 Values (μM) of Top Hits for SHP2 Identified from the
Librarya
Figure 3. Lineweaver-Burk plot for compound 9-mediated SHP2
inhibition. Compound 9 is a noncompetitive inhibitor of SHP2.
Compound 9 concentrations were 0 (b), 1.0 (O), and 5.0 μM (1),
respectively.
Table 2. Selectivity of 9 against a Panel of PTPsa
PTP
IC50 (μM)
SHP2
SHP1
5.5 ( 0.4
15.7 ( 2.1
14.3 ( 1.5
>50
PTP1B
HePTP
Lyp
a All measurements were made using pNPP as a substrate at pH 7.0,
25 ꢀC, and I = 0.15 M.
25.0 ( 3.6
20.3 ( 1.3
30.0 ( 6
>50
FAP1
CD45
LAR
LAR, and PTPR, the dual specificity phosphatases VHR,
and Cdc14, and the low molecular weight PTP was mea-
sured. As shown in Table 2, compound 9 exhibits at least
several-fold selectivity for SHP2 over all PTPs examined.
Most importantly, compound 9 displays excellent cellular
activity as shown below.
PTPR
VHR
>50
>50
Cdc14A
LMWPTP
>50
31.1 ( 1.9
a All measurements were made using pNPP as a substrate at pH 7.0,
Cellular Activity of Compound 9. Our ultimate goal is to
develop potent and specific SHP2 inhibitors as anticancer
and antileukemia agents. Given the excellent potency and
selectivity of 9 toward SHP2, we proceeded to evaluate its
ability to inhibit SHP2-dependent signaling inside the cell.
Previous studies have demonstrated that SHP2 promotes
growth-factor-stimulated ERK1/2 activation and cell pro-
liferation.6,7 We have initially used human embryonic kidney
293 (HEK293) cells, as these cells are relatively easy to
culture and express high levels of the EGF receptor, a path-
way in which SHP2 is known to positively participate in.40,41
Briefly, HEK293 cells were serum-deprived for 16 h followed
by incubation with vehicle or 10 μM compound 9 for 3 h.
Cells were then stimulated with EGF at 2 ng/mL for 5-60
min, total cellular lysates were resolved by SDS-PAGE, and
levels of phospho-ERK1/2 were examined. As shown in
Figure 4A, 9 strongly inhibits the sustained phase of
ERK1/2 activation (60 min after EGF stimulation), which
has been shown to depend on SHP2,42 but not the initial
transient phase (5-15 min), which is SHP2-independent and
mediated by recruitment of growth factor receptor bound
protein 2 (Grb2) and the Ras guanine nucleotide exchange
factor Son of Sevenless (Sos).43 Similar results were also
obtained in NIH3T3 cells (data not shown). To investigate
whether this inhibitory effect of 9 on SHP2-mediated signal-
ing is dose-dependent, we pretreated HEK293 cells with
0-20 μM II-B08, followed by EGF stimulation for 60 min.
Indeed, a concentration-dependent inhibition of the EGF-
25 ꢀC, and I = 0.15 M.
Figure 4. Compound 9 inhibits SHP2-dependent ERK1/2 activa-
tion: (A) immunoblot showing phospho-ERK1/2 levels from 0 to
60 min time points of EGF stimulation of HEK293 cells in the
presence or absence of 10 μM compound 9; (B) concentration
dependence of inhibition of the EGF-induced sustained ERK1/2
activation by 9 in HEK293 cells.
induced ERK1/2 activation by 9 was observed (Figure 4B).
To ensure that the cellular activity displayed by 9 is not due
to nonspecific effects, we also evaluated a structurally related
but inactive II-B05 (compound 14), which has an IC50 value

2486 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Zhang et al.
Table 3. Crystallographic Data and Refinement Statistics for SHP2 9
3
Table 4. Structure and Activity for Compound 9a
Complex
parameter
space group
cell dimensions
P21
˚
a (A)
39.5
75.8
48.2
98.5
˚
b (A)
˚
c (A)
β (deg)
data collection
wavelength for data collection
˚
resolution (A)
˚
highest resolution shell (A)
0.97915
50.0-2.0
2.06-2.0
18 718
99.4 (99.1)a
3.4
7.5 (30.8)a
13.7 (2.2)a
no. of unique reflections
completeness (%)
redundancy
b
Rmerge
ÆI/δ(I)æ
refinement
˚
resolution range (A)
50-2.0
18 673
2075
no. of reflections used (F g 2.0σ (F))
no. of protein atoms
no. of inhibitor
1
142
no. of water
d
Rworkc/Rfree
rms deviations from ideal geometry
19.1/25.1
˚
bond length (A)
bond angle (deg)
0.006
1.22
31.7
a All measurements were made using pNPP as a substrate at pH 7.0,
25 ꢀC, and I = 0.15 M.
2
average B (A )
˚
a The value in parentheses corresponds to the highest resolution shell.
P P
P P
P
b Rmerge
F(h)obsd|/ hF(h)obsd, where F(h)calcd and F(h)obsd were the refined
=
i|I(h)i - ÆI(h)æ|/
=
h|F(h)calcd -
c
iI(h)i. Rwork
(Figure 5). In the presence of 10 μM compound 9, the
hyperproliferation of mutant SHP2-expressing progenitors
in response to GM-CSF was substantially reduced to levels
similar to that observed with wild-type SHP2-expressing
progenitors (Figure 5). These studies demonstrate that 9
is effective at significantly reducing GM-CSF-stimulated
hyperproliferation induced by activating SHP2 mutations.
Collectively, the results described above indicate that 9 is
highly efficacious in cell-based assays and capable of block-
ing SHP2 activity inside the cell.
h
h
P
d
calculated and observed structure factors, respectively. Rfree was
calculated for a randomly selected 3.8% of the reflections that were
omitted from refinement.
of 86 μM for SHP2 (Table 4). As expected, compound 14 at
20 μM elicits no appreciable change in pERK1/2 level when
normalized by total ERK1/2 protein level (Figure 4B). The
observation that 9 inhibits SHP2 in intact cells with similar
potency as that toward isolated enzyme is remarkable, since
previous PTP inhibitors have shown 100- to 10000-fold loss
of potency from biochemical to cellular assays. Together, the
data demonstrate that 9 is cell permeable and can effectively
inhibit a major pathway positively regulated by SHP2.
As we observed inhibition of EGF-stimulated ERK1/2
activation in HEK293 cells, we next investigated whether 9
could inhibit gain-of-function SHP2 mutant-induced hema-
topoietic progenitor hyperproliferation in response to gran-
ulocyte macrophage colony stimulating factor (GM-CSF).
Peripheral blood hematopoietic progenitors from JMML
patients are hypersensitive to the cytokine GM-CSF.44 Stu-
dies from our lab have demonstrated that somatic gain-of-
function SHP2 mutations E76K, D61V, and D61Y induce
hematopoietic progenitor hypersensitivity to GM-CSF.45
On the basis of these findings, we predicted that hemato-
poietic progenitors expressing gain-of-function SHP2 mu-
tants would display reduced proliferation in response to
GM-CSF when treated with 9. We compared the effect of
10 μM compound 9 on GM-CSF-stimulated hypersensitivity
of wild-type SHP2, SHP2/E76K-, and SHP2/D61Y-expres-
sing hematopoietic progenitors using [3H]thymidine incor-
poration assays in response to increasing concentrations of
GM-CSF. As previously found, expression of SHP2/E76K
and SHP2/D61Y induced GM-CSF-stimulated hyperproli-
feration compared to cells expressing wild-type SHP2
Molecular Basis of SHP2 Inhibition by Compound 9. To
provide insight into the molecular basis for SHP2 inhibition
by 9 and to aid the design of SHP2 inhibitors with improved
potency and selectivity, we determined the crystal structure
of the catalytic domain of SHP2 (residues 262-528) in
complex with 9. The SHP2 9 complex crystallized in space
3
group P21 with one molecule in the asymmetric unit. The
˚
crystals of the SHP2 9 complex diffracted to 2.0 A resolu-
3
tion, and the structure was solved by molecular replacement,
using the apo-form of SHP2 catalytic domain (PDB acces-
sion 3B7O)46 as the search model. The three-dimensional
structure of SHP2 9 was refined to a crystallographic
3
R-factor of 19.1% (Rfree = 25.1%), and the statistics for
the data collection and refinement are shown in Table 3. The
atomic model includes SHP2 residues 262-312 and 324-527
and all atoms of the inhibitor (Figure 6). Unambiguous
electron densities are observed for all surface loops, includ-
ing the PTP signature motif or the P-loop (residues 458-465)
which harbors the active site nucleophile C459 and R465 for
recognition of the phosphoryl moiety in the substrate), the
pTyr recognition loop (residues 277-284) which confers
specificity to pTyr, the WPD loop (residues 421-431) which
contains the general acid-base catalyst D425, and the
Q-loop (residues 501-507) which contains the conserved
Q506 required to position and activate a water molecule for
hydrolysis of the phosphoenzyme intermediate.47

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2487
Figure 6. Crystal structure of SHP2 in complex with 9. (A) Ribbon
diagram of SHP2 catalytic domain in complex with inhibitor 9. The
molecule is oriented with the C-terminal end of the β8 strand toward
the viewer. R-Helices and β-strands are colored in red and yellow,
respectively. The P-loop is shown in green and the WPD loop in
blue. Compound 9 is shown in stick-and-bond mode. (B) Fo - Fc
difference map of SHP2 contoured at the 3.0σ level.
participates in a W2-mediated H-bond network with the
backbone carbonyls of P424, D425, and G427 in the WPD
loop whereas O1 is hydrogen-bonded to W1, which makes a
H-bond with the main chain amide of S460 in the P-loop and
a polar interaction with the side chain of K366. In addition to
these polar interactions, there are also van der Waals con-
tacts between SHP2 and the salicylic acid core portion of 9.
For example, the indole ring of 9 interacts with the side
chains of S460 and R465; the methyl group immediately
attached to the indole ring has hydrophobic interactions with
the side chains of Y279 and A461.
Although the salicylic acid core occupies the active site, the
manner by which it interacts with SHP2 differs significantly
from those observed between pTyr and PTP1B (Figure 8).
Binding of pTyr or nonhydrolyzable pTyr mimetics to the
PTPs usually accompanies the closure of the flexible WPD
loop,27,48,49 which harbors the catalytically essential general
acid/base D425.52 A strong stacking interaction between
R465 in the P-loop and W423 of the WPD loop is found in
the pTyr-bound PTP structures, which plays an important
role in stabilizing the closed conformation. Interestingly, the
WPD loop in SHP2 adopts an open conformation when 9 is
bound. Compared to pTyr in PTP1B, the salicylic acid core
Figure 5. Compound 9 inhibits GM-CSF-stimulated bone marrow
low density mononuclear cell proliferation. (A, B) Bone marrow low
density mononuclear cells retrovirally transduced with wild-type
SHP2, SHP2/D61Y, or SHP2/E76K were serum- and growth
factor-deprived, stimulated with increasing concentrations of GM-
CSF (0-1 ng/mL) in the presence or absence of 10 μM compound 9
and subjected to [3H]thymidine incorporation to measure cellular
proliferation: n = 3, (/) p < 0.005 for DMSO vs 9 for both SHP2/
D61Y- and SHP2/E76K-expressing cells at all concentrations of
GM-CSF.
The overall structure of SHP2 9 is similar to the ligand-
3
free SHP2 structure used for molecular replacement, with the
root-mean-square-derivation (rmsd) for all R-carbon posi-
tions between the two being 0.5 A. The major difference
˚
moves ∼3.5 A toward the WPD loop (Figure 8). As a result,
˚
the carboxylate of the salicylic acid core makes direct polar
contacts with the guanidinium group of R465 and the side
chain of W423, precluding a favorable interaction between
R465 and W423 to enable WPD loop closure. In addition,
the W2-mediated interactions between O3 and the backbone
carbonyls of P424, D425, and G427 further prevent WPD
loop closure. Molecular modeling suggests that substrate
pNPP can still be accommodated by SHP2’s active site in the
presence of 9. Since the WPD loop open conformation is
catalytically incompetent, our structural observation that 9
binds SHP2 in the WPD loop open conformation is consis-
tent with 9 being a noncompetitive inhibitor.
between the two structures is electron density in the SHP2
active site corresponding to 9, which was confirmed by
analyzing the |Fo| - |Fc| difference map contoured at 3.0σ
(Figure 6B). This represents the first three-dimensional
structure of SHP2 in complex with a small molecule ligand.
As expected, the salicylic acid moiety of 9 is found in the
SHP2 active-site pocket and forms extensive interactions
with residues in the P-loop, the pTyr recognition loop, and
the WPD loop (Figure 7). Specifically, the phenolic oxygen
atom O1 within the salicylic acid core makes a hydrogen
bond with the main chain amide of R465 in the P-loop; the
carboxylate O2 forms two polar interactions with the side
chains of R465 and Q510, and O3 contributes two H-bonds
with the side chains of R465 and W423, respectively. Ad-
ditionally, the salicylic acid moiety also engages in five more
polar interactions with SHP2, mediated by two water mole-
cules (W1 and W2) in the active site (Figure 7). Thus, O3 also
Further inspection of the structure of the SHP2 9 complex
3
revealed that, in addition to the salicylic acid core-dependent
interactions, the distal phenyl ring P4 in the biphenyl diver-
sity element is sandwiched between the side chains of R362
and K364 in the β5-β6 loop (residues 362-365) (Figure 7).
Interestingly, the β5-β6 loop is highly divergent among the

2488 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Zhang et al.
Figure 7. Detailed interactions between SHP2 and 9. The P-loop and WPD loop are shown in green and blue, respectively. Atomic colors are as
follows: oxygen, red; carbon, gray; sulfur, orange; nitrogen, blue. Ligand’s carbon atoms are colored yellow. Polar interactions are highlighted
with red dash lines.
Figure 8. Superposition of SHP2 9 and PTP1B pY (PDB entry code 1EEN). The superposition was calculated with active site residues
without the ligands. Active site of PTP1B is in the WPD loop closed conformation. pY is shown in yellow, the WPD loop is in blue, and the
3
3
P-loop and other residues are in green. For the SHP2 9 structure, the WPD loop is in the open conformation, with 9 shown in red, WPD loop in
orange, the P-loop in green, Y279 and R465 in pink, and W423, P424, D425, H426, and G427 in gray.
3
PTPs (Figure 9) and has been implicated in substrate recog-
nition by SHP1.50 These additional nonpolar interactions
between P4 and residues R362 and K364 are likely respon-
sible for the enhanced potency and selectivity of 9. To
directly show the involvement for these peripheral site inter-
actions in SHP2 binding, we prepared and analyzed several
compounds structurally related to 9 (Table 4). Consistent
with the observed binding mode, 9 binds SHP2 with a 38.5-
fold higher affinity than that of the salicylic acid core 1 alone
(Table 4). Moreover, when the distal P4 ring was removed
from 9, the IC50 value for the resulting compound 14
increased 15.6-fold. The importance of the interactions
between P4 and R362/K364 was further manifest by the
observation that either a decrease or an increase in the linker
size by one methylene unit led to a ∼2-fold reduction in the
IC50 values for II-A08 (9a) and II-C08 (9b), respectively
(Table 4). These results are in excellent agreement with the
structural observation, supporting the conclusion that inter-
actions between P4 and R362/K364 contribute to the en-
hanced potency of 9 for SHP2.
tions at residue K364, which corresponds to an Arg in SHP1
and a Ser in PTP1B (Figure 9). Substitutions at K364 are
expected to perturb the interactions with the distal end of 9.
Kinetic analysis yielded an IC50 of 9.0 ( 0.3 μM for SHP2/
K364R and of 10.8 ( 0.2 μM for SHP2/K364S. These IC50
values are 1.6- and 2.0-fold higher than that of wild-type
SHP2, in accord with K364 playing a contributing role to
compound 9 selectivity. They are also slightly lower than
those determined for SHP1 and PTP1B, indicating that
additional variable residues in SHP2 may also be involved
in controlling compound 9 selectivity. Collectively, the
structure and activity data plus results from mutational
experiments are consistent with the structural observation
that 9 achieves its potency and specificity by directly target-
ing regions that are structurally divergent in PTPs.
In summary, we describe here a salicylic acid based
combinatorial library approach designed to target both the
PTP active site and a unique nearby subpocket for enhanced
affinity and selectivity. High throughput screening of the
library led to the identification of a SHP2 inhibitor 9 with
promising potency and selectivity. More importantly, 9
possesses highly efficacious cellular activity and is capable
of blocking growth factor stimulated ERK1/2 activation as
To determine whether the interactions between P4 and
R362/K364 also play a role in controlling compound 9
selectivity, we studied two SHP2 mutants bearing substitu-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2489
by Fisher (Fair Lawn, NJ). For organic synthesis, reagents were
used as purchased (Aldrich, Acros, Alfa Aesar, TCI) except
where noted. 1H and 13C NMR spectra were obtained on a
Bruker Avance II 500 MHz NMR spectrometer with TMS or
residual solvent as standard. All column chromatography was
performed using Dynamic Adsorbents 230-400 mesh silica gel
(SiO2) with the indicated solvent system unless otherwise noted.
TLC analysis was performed using 254 nm glass-backed plates
and visualized using UV light (254 nm). HPLC purification was
carried out on a Waters Delta 600 equipped with a Sunfire Prep
C18 OBD column (30 mm ꢀ 150 mm, 5 μm) with methanol-
water (both containing 0.1% TFA) as mobile phase (gradient:
50-100% methanol, flow 10 mL/min). The purity of all final
tested compounds was established to be >95% by reverse-phase
HPLC on a Waters Breeze HPLC system with a SunFire C18
analytical column (4.6 mm ꢀ 150 mm, 5 μm) using acetonitrile-
water (both containing 0.1% TFA) as the mobile phase
(gradient: 30-100% acetonitrile, flow 1.5 mL/min), with UV
monitoring at the fixed wavelength of 254 nm. Low-resolution
mass spectra were obtained using an Agilent Technologies 6130
quadrupole LC/MS. HRMS data were obtained at the Mass
Spectrometry Facility at Indiana University Chemistry Depart-
ment (http://msf.chem.indiana.edu) on a Waters/Macromass
LCT (electrospray ionization ESI).
Azide Synthesis. Commercially available amine/hydrazine
(20 mmol) was dissolved in dry DMF (40 mL) under nitrogen.
To this solution was added Et3N (20 mmol) and acid chloride
(20 mmol) via syringe at 0 ꢀC under nitrogen. The mixture was
stirred at 0 ꢀC for 30 min and then at room temperature for
another 2 h. Next, solid NaN3 (30 mmol) was added and the
reaction mixture was stirred overnight. The mixture was poured
into water (100 mL) and extracted with EtOAc (3 ꢀ 100 mL).
The combined extracts were washed with water (3 ꢀ 50 mL) and
brine (50 mL). The organic layer was dried over Na2SO4, fil-
tered, and evaporated under reduced pressure. Silica gel flash
chromatography (10% ethyl acetate in hexanes) gave the desired
azide product in 50-90% isolated yield.
Salicylic Acid Library Synthesis. To 2 mL vials were added
133 μL of core 1 (100 mM in THF), 167 μL of appropriate azide
(100 mM in t-BuOH), 33 μL of TBTA (12 mM in DMF), 67 μL
of CuSO4 (10 mM in water), and 100 μL of sodium ascorbate
(33.3 mM in water). Thus, the total volume of the reaction was
0.5 mL and the final concentration was 26.6 mM for core 1,
32 mM for each azide, 0.798 mM for TBTA, 1.33 mM for
CuSO4, and 6.64 mM for sodium ascorbate. The vials were then
capped and shaken at room temperature for 12 h. Subsequently,
1.0 mL DMSO was added to each vial, and the solutions were
then transferred to 386-well plates for screening.
Methyl 4-Amino-2-hydroxybenzoate (2). Dimethyl sulfate
(11.4 mL, 0.12 mol) was added to a mixture of 4-aminosalicylic
acid (15.3 g, 0.1 mol), anhydrous sodium carbonate (12.96 g,
0.12 mol), and acetone (300 mL) at room temperature. After
being stirred for 24 h at room temperature, the reaction mixture
was filtered. Filtrate was concentrated under vacuum. The
residue was purified by silica gel column chromatography to
provide the product 2 (16.79 g, 86%).
Figure 9. Amino acid sequence alignment of the β5-β6 loop among
the PTPs.
well as blocking SHP2 gain-of-function mutant-induced
hematopoietic progenitor hyperproliferation in response to
GM-CSF. The results suggest that the bicyclic salicylic acid
pharmacophore chemistry platform may provide a potential
solution to overcome the bioavailability issue that has
plagued the PTP drug discovery field for many years.
X-ray crystallographic analysis of compound 9-bound
SHP2 structure reveals that the salicylic acid core occupies
the PTP active site, while the distal biphenyl ring makes
hydrophobic contacts with a region highly divergent among
the PTPs. The atomic-level information on SHP2 9 and
3
studies of compound 9 derivatives and SHP2 mutants fur-
nish a solid foundation for the design of more potent and
selective SHP2-based small molecule therapeutics. Given the
ease and versatility of the click chemistry based library
construction methodology described in this report, it is
expected that cell permeable, salicylic acid based small
molecule inhibitors will become available for other PTPs as
well.
Methyl 4-(Dimethylamino)-2-hydroxybenzoate (3). Parafor-
maldehyde (15.3 g, 0.51 mol) and NaCNBH3 (16 g, 0.254 mol)
were added portionwise to a solution of 2 (8.5 g, 50.8 mmol) in
acetic acid (150 mL) at 0 ꢀC, and the mixture was stirred at room
temperature for 4 h. After evaporation, the residue was dis-
solved in ethyl acetate, washed with saturated NaHCO3, brine,
and dried over Na2SO4. After evaporation of solvent, the
residue was purified by silica gel column chromatography to
1
afford 3 (7.74 g, 78%). H NMR (500 MHz, CDCl3) δ 10.93
Experimental Section
(s, 1H), 7.63 (d, J = 9.05 Hz, 1H), 6.19 (dd, J = 9.05, 2.5 Hz,
1H), 6.11 (d, J = 2.5 Hz, 1H), 3.86 (s, 3H), 2.99 (s, 6H); 13C
NMR (125 MHz, CDCl3) δ 170.67, 163.27, 155.61, 131.01,
104.13, 100.82, 97.87, 51.53, 39.93. Mass calculated for
C10H13NO3 [M], 195, found [M þ H]þ, 196.
Materials and General Procedures. Polyethylene glycol
(PEG3350) and buffers for crystallization were purchased from
Hampton Research Co. p-Nitrophenyl phosphate (pNPP) was
purchased from Fluke Co. Dithiothreitol (DTT) was provided

2490 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Zhang et al.
Methyl 4-(Dimethylamino)-2-hydroxy-5-iodobenzoate (4).
A solution of iodine (2.9 g, 11.4 mmol) in ether (15 mL) was
added dropwise to a suspension of compound 3 (2.5 g, 12.8
mmol) and K2CO3 (2.6 g, 18.8 mmol) in water (15 mL) at room
temperature over 2 h. The reaction mixture was then stirred at
room temperature for an additional 30 min. Then, the reaction
mixture was acidified to pH 3 with 2 N HCl and extracted with
diethyl ether. The combined organic extractions were washed
with brine, dried over Na2SO4, and concentrated in vacuo.
Purification by flash chromatography (SiO2) afforded com-
silica gel column chromatography to provide compound 8
(4.58 g, 69%). H NMR (500 MHz, CDCl3) δ 10.90 (s, 1H),
1
8.24 (s, 1H), 7.60 (m, 2H), 7.51 (m, 2H), 6.82 (m, 1H), 3.98 (s,
1H), 3.19 (s, 1H), 3.17 (s, 1H); 13C NMR (125 MHz, CDCl3) δ
171.30, 158.24, 145.71, 141.73, 130.10, 129.96, 128.84, 128.52,
122.47, 122.34, 107.63, 96.59, 96.31, 80.05, 52.11, 31.64. Mass
calculated for C19H15NO3 [M], 305, found [M þ H]þ, 306.
3-Ethynyl-6-hydroxy-1-methyl-2-phenyl-1H-indole-5-carboxy-
lic Acid (1). Compound 8 (6.20 g, 20.31 mmol) was dissolved in
80 mL of THF, followed by the addition of a KOH solution
(4.56 g, 81.24 mmol KOH in 80 mL of water). The mixture was
refluxed for 2 h, cooled to room temperature, acidified to pH 3,
and extracted with EtOAc three times. The organic layers were
combined, washed with brine, dried over sodium sulfate, and
concentrated in vacuum. This crude product was quickly taken
to click reaction without purification. 1H NMR (500 MHz,
CDCl3) δ 11.42 (s, 1H), 8.83 (s, 1H), 7.61 (m, 3H), 7.56 (m, 2H),
7.07 (s, 1H), 3.36 (s, 1H), 2.18 (s, 3H). Mass calculated for
C18H13NO3 [M], 291, found [M þ H]þ, 292.
1
pound 4 (1.73 g, 42%). H NMR (500 MHz, CDCl3) δ 10.73
(s, 1H), 8.22 (s, 1H), 6.55 (s, 1H), 3.91 (s, 3H), 2.84 (s, 6H); 13
C
NMR (125 MHz, CDCl3) δ 169.28, 162.82, 161.29, 141.68,
108.55, 108.11, 80.07, 52.24, 44.26. Mass calculated for C10H12-
INO3 [M], 321, found [M þ H]þ, 322.
Methyl 4-(Dimethylamino)-2-hydroxy-5-(phenylethynyl)ben-
zoate (5). A mixture of methyl 4-(dimethylamino)-2-hydroxy-
5-iodobenzoate 4 (6.5 g, 20.2 mmol), phenylacetylene (3.10 g,
30.4 mmol), bis(triphenylphosphine)palladium(II) chloride
(0.288 g, 0.41 mmol), and CuI (0.155 g, 0.81 mmol) was loaded
in a flask, which was degassed and backfilled with nitrogen.
Then Et3N (11.3 mL, 80.8 mmol) and solvent DMF were added.
The resulting mixture was stirred under a nitrogen atmosphere
at room temperature for 4 h. The reaction was monitored by
TLC to ensure completion. After removal of solvent under
reduced pressure, the residue was purified by column chroma-
tography on silica gel to afford 5 (5.32 g, 89%). 1H NMR (500
MHz, CDCl3) δ 10.93 (s, 1H), 7.94 (s, 1H), 7.48 (m, 2H), 7.31 (m,
3H), 6.31 (s, 1H), 3.90 (s, 3H), 3.12 (s, 6H); 13C NMR (125 MHz,
CDCl3) δ 169.95, 162.79, 159.36, 137.37, 131.03, 128.46, 127.96,
124.03, 104.45, 104.06, 103.02, 92.63, 88.88, 51.99, 42.66. Mass
calculated for C18H17NO3 [M], 295, found [M þ H]þ, 296.
Methyl 6-Hydroxy-3-iodo-1-methyl-2-phenyl-1H-indole-5-car-
boxylate (6). To a solution of 5 (5.2 g, 17.6 mmol) in CH2Cl2
(100 mL) was added iodine (8.9 g, 35 mmol). After the resulting
mixture was stirred at room temperature for an additional 4 h,
100 mL of CH2Cl2 was added, and the resulting mixture was
washed with 10% aqueous NaS2O3 solution (3 ꢀ 50 mL), brine
(50 mL), dried over Na2SO4, and concentrated in vacuo. Pur-
ification by flash chromatography afforded 6 (6.23 g, 87%). 1H
NMR (500 MHz, CDCl3) δ 10.88 (s, 1H), 7.96 (s, 1H), 7.45 (m,
5H), 6.78 (s, 1H), 3.98 (s, 3H), 3.54 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 171.29, 158.34, 142.88, 142.59, 131.22, 130.82, 129.06,
128.59, 124.30, 124.20, 107.64, 96.26, 59.53, 52.26, 32.23. Mass
calculated for C17H14INO3 [M], 407, found [M þ H]þ, 408.
Methyl 6-Hydroxy-1-methyl-2-phenyl-3-((trimethylsilyl)ethy-
nyl)-1H-indole-5-carboxylate (7). To a solution of 6 (6.52 g, 16.0
mmol), bis(triphenylphosphine)palladium(II) chloride (225 mg,
0.320 mmol), and CuI (122 mg, 0.640 mmol) in DMF were
added (trimethylsilyl)acetylene (2.35 g, 24.01 mmol) and Et3N
(6.48 g, 64.04 mmol), and the reaction was stirred at room
temperature under nitrogen for 4 h. The reaction was quenched
with water, and the aqueous phase was extracted with ethylene
chloride three times. The combined organic extract was dried
over sodium sulfate, and the solvent was removed under va-
cuum. The residue was purified by silica gel column chroma-
tography using 15:1 hexanes/ethyl acetate to provide compound
7 (4.4 g, 73%). 1H NMR (500 MHz, CDCl3) δ 10.91 (s, 1H), 8.23
(s, 1H), 7.60 (m, 2H), 7.48 (m, 3H), 6.83 (s, 1H), 4.00 (s, 3H), 3.64
(s, 3H), 0.21 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 171.36,
158.23, 145.76, 141.86, 130.13, 129.99, 128.65, 122.68, 122.05,
107.57, 98.98, 97.91, 96.28, 52.13, 31.81, 0.14. Mass calculated
for C22H23NO3Si [M], 377, found [M þ H]þ, 378.
6-Hydroxy-1-methyl-3-(1-(3-oxo-3-(phenylamino)propyl)-1H-
1,2,3-triazol-4yl)-2-phenyl-1H-indole-5-carboxylic Acid (14). A
mixture of core 1 (50 mg, 0.172 mmol), 3-azido-N-phenylpro-
panamide (49 mg, 0.26 mmol), TBTA (2.7 mg, 0.005 mmol),
CuSO4 5H2O (2.2 mg, 0.0088 mmol), sodium ascorbate (8.5 mg,
3
0.043 mmol) in THF (1 mL), t-BuOH (1 mL), and H2O (1 mL)
was stirred at room temperature under nitrogen for 24 h. The
solvent was removed in vacuo. The resultant solid was then
washed three times with water and purified by reversed-phase
HPLC to yield the title compound as a white solid (33 mg, 40%).
1H NMR (500 MHz, DMSO-d6) δ 13.64 (s, 1H), 11.38 (s, 1H),
9.99 (s, 1H), 8.77 (s, 1H), 7.47 (m, 8H), 7.31 (t, J = 7.5 Hz, 2H),
7.27 (s, 1H), 7.05 (t, J = 7.5 Hz, 1H), 4.58 (t, J = 2.0 Hz, 2H),
3.48 (s, 3H), 2.89 (t, J = 2.0 Hz, 2H); 13C NMR (125 MHz,
DMSO-d6) δ 173.05, 167.95, 157.35, 141.39, 138.87, 138.36,
130.60, 130.36, 129.06, 128.84, 128.66, 124.35, 123.25, 120.45,
119.24, 119.11, 107.10, 104.92, 99.49, 95.84, 45.39, 36.49, 30.79.
HRMS (ESI) mass calculated for C27H24N5O4 [M þ H]þ,
482.1828, found [M þ H]þ, 482.1813.
3-(1-(2-(Biphenyl-4-ylamino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)-
6-hydroxy-1-methyl-2-phenyl-1H-indole-5-carboxylic Acid (9a).
A mixture of core 1 (50 mg, 0.172 mmol), 2-azido-N-(biphenyl-
4-yl)acetamide (65 mg, 0.26 mmol), TBTA (2.7 mg, 0.005
mmol), CuSO4 5H2O (2.2 mg, 0.0088 mmol), sodium ascorbate
3
(8.5 mg, 0.043 mmol) in THF (1 mL), t-BuOH (1 mL), and H2O
(1 mL) was stirred at room temperature under nitrogen for 24 h.
The solvent was removed in vacuo. The resultant solid was then
washed three times with water and purified by reversed-phase
HPLC to yield the title compound as a white solid (31 mg, 33%).
1H NMR (500 MHz, DMSO-d6) δ 11.39 (s, 1H), 10.52 (s, 1H),
8.79 (s, 1H), 7.65 (m, 6H), 7.56 (m, 3H), 7.52 (m, 2H), 7.44 (t, J =
7.5 Hz, 2H), 7.41 (s, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.03 (s, 1H),
5.29 (s, 2H), 3.51 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ
141.94, 141.82, 140.02, 138.90, 138.33, 135.90, 131.25, 131.03,
129.67, 129.45, 129.40, 127.56, 126.73, 122.45, 120.09, 119.77,
105.40, 100.02, 52.65, 31.33. HRMS (ESI) mass calculated for
C32H26N5O4 [M þ H]þ, 544.1985, found [M þ H]þ, 544.2001.
3-(1-(3-(Biphenyl-4-ylamino)-3-oxopropyl)-1H-1,2,3-triazol-
4-yl)-6-hydroxy-1-methyl-2-phenyl-1H-indole-5-carboxylic Acid
(9). A mixture of core 1 (50 mg, 0.172 mmol), 3-azido-
N-(biphenyl-4-yl)propanamide (69 mg, 0.26 mmol), TBTA
(2.7 mg, 0.005 mmol), CuSO4 5H2O (2.2 mg, 0.0088 mmol),
3
sodium ascorbate (8.5 mg, 0.043 mmol) in THF (1 mL), t-BuOH
(1 mL), and H2O (1 mL) was stirred at room temperature under
nitrogen for 24 h. The solvent was removed in vacuo. The
resultant solid was then washed three times with water and
purified by reversed-phase HPLC to yield the title compound as
a white solid (42 mg, 44%). 1H NMR (500 MHz, DMSO-d6) δ
11.35 (s, 1H), 10.09 (s, 1H), 8.76 (s, 1H), 7.64 (m, 7H), 7.50-7.41
(m, 7H), 7.33 (m, 1H), 7.28 (s, 1H), 7.00 (s, 1H), 4.59 (t, J = 6.5
Hz, 2H), 3.47 (s, 3H), 2.90 (t, J = 6.5 Hz, 2H); 13C NMR (125
Methyl 3-Ethynyl-6-hydroxy-1-methyl-2-phenyl-1H-indole-5-
carboxylate (8). To a solution of 7 (8.22 g, 21.77 mmol) in THF
(100 mL) was added TBAF (6.86 g, 21.77 mmol). The solution
was allowed to stir for 4 h before addition of 200 mL of
methylene chloride to the reaction. The resulting solution was
washed with water and brine and dried over sodium sulfate. The
solvent was removed under vacuum. The residue was purified by

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2491
MHz, DMSO-d6) δ 173.08, 168.07, 157.38, 141.44, 141.42,
139.70, 138.42, 138.36, 134.97, 130.64, 130.41, 129.13, 128.89,
127.03, 126.90, 126.26, 124.36, 120.52, 119.52, 119.28, 107.13,
104.95, 99.53, 95.88, 45.44, 36.57, 30.83. HRMS (ESI): mass
calculated for C33H28N5O4 [M þ H]þ, 558.2141, found [M þ
H]þ, 558.2122.
3-(1-(4-(Biphenyl-4-ylamino)-4-oxobutyl)-1H-1,2,3-triazol-4-yl)-
6-hydroxy-1-methyl-2-phenyl-1H-indole-5-carboxylic Acid (9b).
A mixture of core 1 (50 mg, 0.172 mmol), 4-azido-N-(biphenyl-
4-yl)butanamide (73 mg, 0.26 mmol), TBTA (2.7 mg, 0.005
EDTA with an ionic strength of 0.15 M adjusted by NaCl. The
salicylic acid based library was screened in a 96-well format at
10 μM compound concentration. The reaction was initiated by
the addition of 5 μL of the enzyme to 195 μL of reaction mixture
containing 10 μM test compound and various concentrations of
pNPP and quenched after 5 min by the addition of 50 μL of 5 N
NaOH. The nonenzymatic hydrolysis of pNPP was corrected
by measuring the control without the addition of enzyme. The
amount of product p-nitrophenol was determined from the
absorbance at 405 nm detected by a Spectra MAX340 micro-
plate spectrophotometer (Molecular Devices) using a molar
mmol), CuSO4 5H2O (2.2 mg, 0.0088 mmol), sodium ascorbate
3
extinction coefficient of 18 000 M-1 cm-1
.
(8.5 mg, 0.043 mmol) in THF (1 mL), t-BuOH (1 mL), and H2O
(1 mL) was stirred at room temperature under nitrogen for 24 h.
The solvent was removed in vacuo. The resultant solid was then
washed three times with water and purified on reversed phase
HPLC to yield the desired title compound as a white solid
(46 mg, 47%). 1H NMR (500 MHz, DMSO-d6) δ 11.38 (s,
1H), 10.01 (s, 1H), 8.77 (s, 1H), 7.63 (m, 6H), 7.53 (m, 3H), 7.50
(m, 2H), 7.44 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 1H), 7.31
(s, 1H), 7.03 (s, 1H), 4.36 (t, J = 7.0 Hz, 2H), 3.52 (s, 3H), 2.31 (t,
J = 7.0 Hz, 2H), 2.06 (t, J = 7.0 Hz, 2H); 13C NMR (125 MHz,
DMSO-d6) δ 173.53, 170.61, 157.84, 141.94, 140.17, 139.09,
138.94, 135.18, 131.13, 130.99, 129.59, 129.36, 127.45, 127.31,
126.67, 124.80, 120.86, 119.91, 119.80, 107.60, 105.53, 99.99,
96.36, 49.20, 33.23, 31.33, 29.60. HRMS (ESI): mass calcu-
lated for C34H30N5O4 [M þ H]þ, 572.2298, found [M þ H]þ,
572.2274.
Compounds exhibiting more than 50% of inhibitory activity
against SHP2 were selected for IC50 measurement. The reaction
was started by the addition of 5 μL of the enzyme to 195 μL of
reaction mixture containing 2.9 mM (the Km value) of pNPP and
various concentrations of the inhibitor. The reaction was
quenched after 5 min by the addition of 50 μL of 5 N NaOH,
and then 200 μL of reaction mixture was transferred to a 96-well
plate. The absorbance at 405 nm was detected by a Spectra
MAX340 microplate spectrophotometer (Molecular Devices).
IC50 values were calculated by fitting the absorbance at 405 nm
versus inhibitor concentration to the following equation:
AI=A0 ¼ IC50=ðIC50 þ ½IꢁÞ
where AI is the absorbance at 405 nm of the sample in the
presence of inhibitor, A0 is the absorbance at 405 nm in the
absence of inhibitor, and [I] is the concentration of the inhibitor.
For selectivity studies, the PTPs, including PTP1B, SHP1,
FAP1, Lyp, HePTP, VHR, LMWPTP, Cdc14, LAR, PTPR,
and CD45, were expressed and purified from E. coli. The
inhibition assay for these PTPs were performed under the same
conditions as SHP2 except using a different pNPP concentration
corresponding to the Km of the PTP studied. Inhibitor concen-
trations used for IC50 measurements cover the range from 0.2ꢀ
to 5ꢀ of the IC50 value.
SHP2 Cloning, Expression, and Purification. A PCR-based
strategy was used to amplify the sequence coding for the
catalytic domain of SHP2 (residues 262-528) from the full
length construct. The PCR products were gel purified, digested
with Nde I and Xho I restriction enzymes (Invitrogen), and
ligated to the linearized pET-21a(þ) vector (Novagen). The
ligation products were used to transform E. coli BL21(DE3)
plys competent cells (Novagen). Gene sequence was confirmed
by DNA sequencing carried out by the DNA Sequencing
Facility at Indiana University School of Medicine. For protein
expression, transformed E. coli cells were grown at 37 ꢀC in
Luria broth (LB) containing 100 μg/mL ampicillin for 4 h to an
absorbance of 0.6 at 600 nm and then induced for protein
production with 0.4 mM IPTG overnight at 20 ꢀC. Cells were
harvested by centrifugation (6500 rpm for 15 min at 4 ꢀC), and
the cell pellets from 1 L LB medium were suspended in 30 mL of
ice-cold lysis buffer consisting of 5 mM imidazole, 500 mM
NaCl, 20 mM Tris-HCl (pH 7.9), 0.05 mg/mL trypsin inhibitor,
and 0.1 mM PMSF. The suspensions were passed twice through
a French press at 1200 psi, and the cell lysates were centrifuged at
4 ꢀC for 45 min at 16 000 rpm. The supernatants were mixed with
2 mL of Ni-NTA Agarose (His*Bind resin) (Qiagen) at 4 ꢀC for
50 min, washed with 200 mL of binding buffer (5 mM imidazole,
500 mM NaCl, 20 mM Tris-HCl (pH 7.9)), followed by 20 mL of
washing buffer (20 mM imidazole, 500 mM NaCl, 20 mM Tris-
HCl (pH 7.9)), and then eluted with 20 mL of elution buffer (200
mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)). The
elute was dialyzed for 6 h at 4 ꢀC against 1 L of buffer A (100 mM
NaCl, 20 mM MES (pH 6.0), 1 mM EDTA), and then loaded
onto a Mono S column equilibrated at 4 ꢀC with buffer A. The
column was washed with 10 mL of buffer A and then eluted with
a 40 mL linear gradient of 0 to 1 M NaCl in buffer A. SHP2 was
eluted at 0.35 M NaCl. The column fractions were analyzed by
measuring the absorbance at 280 nm and by carrying out
SDS-PAGE analysis. The fractions were combined, dialyzed
against 1 L of buffer A, concentrated at 4 ꢀC to 7 mg/mL using
an Amicon concentrator, and then stored at -80 ꢀC. The
SHP2 protein was shown to be homogeneous by SDS-PAGE
analysis.
To evaluate potential nonspecific promiscuous inhibition, we
first determine whether the compound displays time-depen-
dence inhibition by measuring the IC50 values with and without
enzyme preincubation with the inhibitor. We also determine the
effect of bovine serum albumin (0.1 mg/mL) on IC50 values.
Finally, we also assess whether the IC50 values can be influenced
by the presence of detergents such as 0.1% of Triton X-100.
A well-behaved classical and reversible active site directed
inhibitor should not exhibit any dependence on time, BSA,
and detergents.
Ki Measurement. The SHP2-catalyzed hydrolysis of pNPP in
the presence of 9 was assayed at 25 ꢀC and in the assay buffer
described above. The mode of inhibition and Ki value were
determined in the following manner. At various fixed concen-
trations of inhibitor (0-3 Ki), the initial rate at a series of pNPP
concentrations was measured by following the production of
p-nitrophenol as describe above, ranging from 0.2- to 5-fold the
apparent Km values. The data were fitted to appropriate equa-
tions using SigmaPlot-Enzyme Kinetics to obtain the inhibition
constant and to assess the mode of inhibition.
Retroviral Vectors and Retroviral Transduction. The wild-type
SHP2 cDNA was mutated at nucleotides 226 (G > A) or 181
(G > T) using QuikChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA) to yield amino acid changes E76K
or D61Y, respectively.45 Each cDNA was subcloned into the
murine stem cell virus (MSCV)-based bicistronic retroviral
vector, pMIEG3, in tandem with enhanced green fluorescent
protein (EGFP). Ecotropic retroviral supernatants (pMIEG3-
wild-type SHP2, pMIEG3-SHP2/E76K, and pMIEG3-SHP2/
D61Y) were prepared using Eco-Phoenix packaging cells. Bone
marrow low density mononuclear cells from C57Bl/6 mice
(Jackson Laboratories, Bar Harbor, ME) were purified using
a Ficoll gradient. Cells were prestimulated at a concentration of
2 ꢀ 106 cells/mL in IMDM, 20% fetal calf serum (Hyclone,
Kinetic Analysis of SHP2 Inhibition by the Salicylic Acid
Based Library. The phosphatase activity of SHP2 was assayed
using p-nitrophenyl phosphate (pNPP) as a substrate at 25 ꢀC in
50 mM 3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM

2492 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Zhang et al.
Logan, UT), 2 mM glutamine, 1% penicillin/streptomycin,
stem cell factor (SCF), granulocyte-colony stimulating factor
(G-CSF), and thrombopoietin (Tpo) all at 100 ng/mL (growth
factors from Peprotech, Rocky Hill, NJ) for 24 h. Cells were
then transferred to retroviral supernatant on plates coated with
fibronectin fragments (Retronectin, Takara, Madison, WI) with
SCF, G-CSF, and Tpo 100 ng/mL for an additional 48 h.
Transduced cells were collected using fluorescence activated cell
sorting (FACS) for EGFP positive cells.
progressed. They were assigned in the |Fo| - |Fc| difference
Fourier maps with a 3σ cutoff level for inclusion in the model.
Acknowledgment. This work was supported by National
Institutes of Health Grants CA69202, CA126937, and
HL82981 and the Clarian Values Fund for Research (Grant
VFR-245). X.Z. was also supported in part by the State
Scholarship Fund from China Scholarship Council, and
S.C.N. was supported by the National Institutes of Health
(Grant F31AG031648).
[3H]Thymidine Incorporation. Proliferation was assessed by
conducting thymidine incorporation assays on EGFP-sorted
wild-type SHP2-, SHP2/D61Y-, or SHP2/E76K-expressing he-
matopoietic progenitors. Briefly, cells were washed and starved
in 0.2% BSA with or without any growth factors for 4 h. Then
5 ꢀ 104 cells were cultured in 96-well plates in various concen-
trations of granulocyte-macrophage colony-stimulating factor
(GM-CSF) in the absence or presence of 9. Cells were cultured
for 16 h and subsequently pulsed with 1.0 μCi of [3H]thymidine
for 6-8 h. Cells were harvested using an automated 96-well cell
harvester (Brandel, Gaithersburg, MD), and thymidine incor-
poration was determined as counts per minute (CPM).
Cell Culture and Immunoblot Analysis. HEK293 cells were
cultured in Dulbecco modified Eagle medium (DMEM) with
10% fetal calf serum. Then 4 ꢀ 105 cells were plated into each
well of 12-well plates and cultured for 24 h. For biochemical
studies, cells were serum-deprived for 16 h followed by treat-
ment with vehicle, 9, or 14 for 3 h. The cells were then stimulated
with epidermal growth factor (EGF, 2 ng/mL) for the indicated
time followed by preparation of total cell protein lysates.
Lysates (30 μg) were electrophoresed on a 10% polyacrylamide
gel, transferred to a nitrocellulose membrane, and probed with
antiphospho-ERK1/2 and anti-ERK1/2 (Cell Signaling Tech-
nology, Beverly, MA).
Supporting Information Available: Figure showing the inhibi-
tion curves used to determine the IC50 values for compounds
listed in Table 4. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Hunter, T. The Croonian lecture 1997. The phosphorylation of
proteins on tyrosine: its role in cell growth and disease. Philos.
Trans. R. Soc. London, Ser. B 1998, 353, 583–605.
(2) Tonks, N. K.; Neel, B. G. Combinatorial control of the specificity
of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 2001, 13,
182–195.
(3) Zhang, Z.-Y. Protein tyrosine phosphatases: prospects for thera-
peutics. Curr. Opin. Chem. Biol. 2001, 5, 416–423.
(4) Ventura, J. J.; Nebreda, A. R. Protein kinases and phosphatases as
therapeutic targets in cancer. Clin. Transl. Oncol. 2006, 8, 153–160.
(5) Krause, D. S.; Van Etten, R. A. Tyrosine kinases as targets for
cancer therapy. N. Engl. J. Med. 2005, 353, 172–187.
(6) Neel, B. G.; Gu, H.; Pao, L. The “Shp” ing news: SH2 domain-
containing tyrosine phosphatases in cell signaling. Trends Biochem.
Sci. 2003, 28, 284–293.
(7) Chan, R. J.; Feng, G. S. PTPN11 is the first identified proto-
oncogene that encodes a tyrosine phosphatase. Blood 2007, 109,
862–867.
(8) Tiganis, T.; Bennett, A. M. Protein tyrosine phosphatase function:
the substrate perspective. Biochem. J. 2007, 402, 1–15.
(9) Tartaglia, M.; Mehler, E. L.; Goldberg, R.; Zampino, G.; Brunner,
H. G.; Kremer, H.; van der Burgt, I.; Crosby, A. H.; Ion, A.;
Jeffery, S.; Kalidas, K.; Patton, M. A.; Kucherlapati, R. S.; Gelb,
B. D. Mutations in PTPN11, encoding the protein tyrosine phos-
phatase SHP-2, cause Noonan syndrome. Nat. Genet. 2001, 29,
465–468.
(10) Tartaglia, M.; Gelb, B. D. Noonan syndrome and related disor-
ders: genetics and pathogenesis. Annu. Rev. Genomics Hum. Genet.
2005, 6, 45–68.
(11) Tartaglia, M.; Niemeyer, C. M.; Fragale, A.; Song, X.; Buechner,
J.; Jung, A.; Hahlen, K.; Hasle, H.; Licht, J. D.; Gelb, B. D.
Somatic mutations in PTPN11 in juvenile myelomonocytic leuke-
mia, myelodysplastic syndromes and acute myeloid leukemia. Nat.
Genet. 2003, 34, 148–150.
(12) Tartaglia, M.; Martinelli, S.; Cazzaniga, G.; Cordeddu, V.;
Iavarone, I.; Spinelli, M.; Palmi, C.; Carta, C.; Pession, A.; Arico,
M.; Masera, G.; Basso, G.; Sorcini, M.; Gelb, B. D.; Biondi, A.
Genetic evidence for lineage-related and differentiation stage-
related contribution of somatic PTPN11 mutations to leukemo-
genesis in childhood acute leukemia. Blood 2004, 104, 307–313.
(13) Loh, M. L.; Vattikuti, S.; Schubbert, S.; Reynolds, M. G.; Carlson,
E.; Lieuw, K. H.; Cheng, J. W.; Lee, C. M.; Stokoe, D.; Bonifas,
J. M.; Curtiss, N. P.; Gotlib, J.; Meshinchi, S.; Le Beau, M. M.;
Emanuel, P. D.; Shannon, K. M. Mutations in PTPN11 implicate
the SHP-2 phosphatase in leukemogenesis. Blood 2004, 103, 2325–
2331.
(14) Loh, M. L.; Reynolds, M. G.; Vattikuti, S.; Gerbing, R. B.; Alonzo,
T. A.; Carlson, E.; Cheng, J. W.; Lee, C. M.; Lange, B. J.;
Meshinchi, S. Children’s Cancer Group. PTPN11 mutations in
pediatric patients with acute myeloid leukemia: results from the
Children’s Cancer Group. Leukemia 2004, 18, 1831–1834.
(15) Kratz, C. P.; Niemeyer, C. M.; Castleberry, R. P.; Cetin, M.;
Crystallization of SHP2 with 9 and X-ray Data Collection. All
crystallization experiments were carried out at room tempera-
ture using the hanging drop vapor diffusion method. For
cocrystallization, 100 μL of SHP2 stock (7.0 mg/mL) in 20
mM Tris-HCL (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, and
3.0 mM DTT was mixed with 1 μL of compound 9 stock solution
(50 mM in DMSO). Crystals of SHP2 9 complex were obtained
3
at room temperature by vapor diffusion in hanging drops.
Protein drops were equilibrated against a reservoir solution
containing 20% w/v polyethylene glycol 3350, 200 mM magne-
sium acetate tetrahydrate, and 100 mM HEPES buffer (pH 7.7).
The space group of the crystals is P21 (Table 3). For X-ray data
collection, the crystals were transferred into 5 μL of cryopro-
tectant buffer containing 30% w/v polyethylene glycol 3350,
100 mM NaCl, and 100 mM HEPES (pH 7.7) and were allowed
to soak for 30 min. The crystals were then flash-cooled by liquid
nitrogen. X-ray data were collected at 23 ID beamline at APS
(Argonne, IL). Data were processed using the program
HKL2000,51 and the statistics are provided in Table 3.
Structural Determination and Refinement. The structure of
SHP2 9 was solved by molecular replacement using the pro-
3
gram AMoRe.52 The structure of SHP2 (PDB entry code
3B7O),46 without the solvent molecules and the first 16 residues,
was used as a search model. The resulting difference Fourier
map indicated some alternative tracing, which was incorporated
into the model. The map revealed the density for the bound 9 in
˚
the active site of SHP2. The structure was refined to 2.0 A
resolution with the program CNS,53 first using simulated an-
nealing at 2500 K and then alternating positional and individual
temperature factor refinement cycles. The progress of the re-
finement was evaluated by the improvement in the quality of the
electron density maps, and the reduced values of the conven-
€
Bergstrasser, E.; Emanuel, P. D.; Hasle, H.; Kardos, G.; Klein,
C.; Kojima, S.; Stary, J.; Trebo, M.; Zecca, M.; Gelb, B. D.;
Tartaglia., M.; Loh., M. L. The mutational spectrum of PTPN11
in juvenile myelomonocytic leukemia and Noonan syndrome/
myeloproliferative disease. Blood 2005, 106, 2183–2185.
P
P
tional R factor (R=
Fo| - |Fc
/
h|Fo|) and the free R factor
h
(3.8% of the reflections omitted from the refinement).54 Elec-
tron density maps were inspected, and the model was modified
on an interactive graphics workstation with the program O.55
Finally, water molecules were added gradually as the refinement
(16) Bentires-Alj, M.; Paez, J. G.; David, F. S.; Keilhack, H.; Halmos,
B.; Naoki, K.; Maris, J. M.; Richardson, A.; Bardelli, A.;
Sugarbaker, D. J.; Richards, W. G.; Du, J.; Girard, L.; Minna, J.

Article
D.; Loh, M. L.; Fisher, D. E.; Velculescu, V. E.; Vogelstein, B.;
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2493
human alpha-1,3-fucosyltransferase via click chemistry. J. Am.
Chem. Soc. 2003, 125, 9588–9589.
Meyerson, M.; Sellers, W. R.; Neel, B. G. Activating mutations of
the Noonan syndrome-associated SHP2/PTPN11 gene in human
solid tumors and adult acute myelogenous leukemia. Cancer Res.
2004, 64, 8816–8820.
ꢀ
ꢀ
(36) Manetsch, R.; Krasinski, A.; Radic, Z.; Raushel, J.; Taylor, P.;
Sharpless, K. B.; Kolb, H. C. In situ click chemistry: enzyme
inhibitors made to their own specifications. J. Am. Chem. Soc.
2004, 126, 12809–12818.
(17) Chan, G.; Kalaitzidis, D.; Neel, B. G. The tyrosine phospha-
tase Shp2 (PTPN11) in cancer. Cancer Metastasis Rev. 2008, 27,
179–192.
(18) Miyamoto, D.; Miyamoto, M.; Takahashi, A.; Yomogita, Y.;
Higashi, H.; Kondo, S.; Hatakeyama, M. Isolation of a distinct
class of gain-of-function SHP-2 mutants with oncogenic RAS-like
transforming activity from solid tumors. Oncogene 2008, 27, 3508–
3515.
(37) Srinivasan, R.; Uttamchandani, M.; Yao, S. Q. Rapid assembly
and in situ screening of bidentate inhibitors of protein tyrosine
phosphatases. Org. Lett. 2006, 8, 713–716.
(38) Xie, J.; Seto, C. T. A two stage click-based library of protein
tyrosine phosphatase inhibitors. Bioorg. Med. Chem. 2007, 15, 458–
473.
(39) Yu, X.; Sun, J.-P.; He, Y.; Guo, X.-L.; Liu, S.; Zhou, B.; Hudmon,
A.; Zhang, Z.-Y. Structure, inhibitor, and regulatory mechanism
of Lyp, a lymphoid-specific tyrosine phosphatase implicated in
autoimmune diseases. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
19767–19772.
(40) Deb, T. B.; Wong, L.; Salomon, D. S.; Zhou, G.; Dixon, J. E.;
Gutkind, J. S.; Thompson, S. A.; Johnson, G. R. A common
requirement for the catalytic activity and both SH2 domains of
SHP-2 in mitogen-activated protein (MAP) kinase activation by
the ErbB family of receptors. A specific role for SHP-2 in map, but
not c-Jun amino-terminal kinase activation. J. Biol. Chem. 1998,
273, 16643–16646.
(19) Hatakeyama, M. Oncogenic mechanisms of the Helicobacter pylori
CagA protein. Nat. Rev. Cancer 2004, 4, 688–694.
(20) Stommel, J. M.; Kimmelman, A. C.; Ying, H.; Nabioullin, R.;
Ponugoti, A. H.; Wiedemeyer, R.; Stegh, A. H.; Bradner, J. E.;
Ligon, K. L.; Brennan, C.; Chin, L.; DePinho, R. A. Coactivation
of receptor tyrosine kinases affects the response of tumor cells to
targeted therapies. Science 2007, 318, 287–290.
(21) Chen, L.; Sung, S. S.; Yip, M. L.; Lawrence, H. R.; Ren, Y.; Guida,
W. C.; Sebti, S. M.; Lawrence, N. J.; Wu, J. Discovery of a novel
shp2 protein tyrosine phosphatase inhibitor. Mol. Pharmacol.
2006, 70, 562–570.
(22) Lawrence, H. R.; Pireddu, R.; Chen, L.; Luo, Y.; Sung, S. S.;
Szymanski, A. M.; Yip, M. L.; Guida, W. C.; Sebti, S. M.; Wu, J.;
Lawrence, N. J. Inhibitors of Src homology-2 domain containing
protein tyrosine phosphatase-2 (Shp2) based on oxindole scaffolds.
J. Med. Chem. 2008, 51, 4948–4956.
(41) Qu, C. K.; Yu, W. M.; Azzarelli, B.; Feng, G. S. Genetic evidence
that Shp-2 tyrosine phosphatase is a signal enhancer of the
epidermal growth factor receptor in mammals. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 8528–8533.
(42) Maroun, C. R.; Naujokas, M. A.; Holgado-Madruga, M.; Wong,
A. J.; Park, M. The tyrosine phosphatase SHP-2 is required for
sustained activation of extracellular signal-regulated kinase and
epithelial morphogenesis downstream from the met receptor tyro-
sine kinase. Mol. Cell. Biol. 2000, 20, 8513–8525.
(43) Egan, S. E.; Giddings, B. W.; Brooks, M. W.; Buday, L.; Sizeland,
A. M.; Weinberg, R. A. Association of Sos Ras exchange protein
with Grb2 is implicated in tyrosine kinase signal transduction and
transformation. Nature 1993, 363, 45–51.
(44) Emanuel, P. D.; Bates, L. J.; Castleberry, R. P.; Gualtieri, R. J.;
Zuckerman, K. S. Selective hypersensitivity to granulocyte-macro-
phage colony-stimulating factor by juvenile chronic myeloid leu-
kemia hematopoietic progenitors. Blood 1991, 77, 925–929.
(45) Chan, R. J.; Leedy, M. B.; Munugalavadla, V.; Voorhorst, C. S.;
Li, Y.; Yu, M.; Kapur, R. Human somatic PTPN11 mutations
induce hematopoietic-cell hypersensitivity to granulocyte-macro-
phage colony-stimulating factor. Blood 2005, 105, 3737–3742.
(46) Barr, A. J.; Ugochukwu, E.; Lee, W. H.; King, O. N.;
Filippakopoulos, P.; Alfano, I.; Savitsky, P.; Burgess-Brown,
N. A.; Muller, S.; Knapp, S. Large-scale structural analysis of
the classical human protein tyrosine phosphatome. Cell 2009, 136,
352–363.
(47) Zhang, Z.-Y. Mechanistic studies on protein tyrosine phospha-
tases. Prog. Nucleic Acid Res. Mol. Biol. 2003, 73, 171–220.
(48) Jia, Z.; Barford, D.; Flint, A. J.; Tonks, N. K. Structural basis for
phosphotyrosine peptide recognition by protein tyrosine phospha-
tase 1B. Science 1995, 268, 1754–1758.
(49) Sun, J.-P.; Fedorov, A. A.; Lee, S.-Y.; Guo, X.-L.; Shen, K.;
Lawrence, D. S.; Almo, S. C.; Zhang, Z.-Y. Crystal structure of
PTP1B in complex with a potent and selective bidentate inhibitor.
J. Biol. Chem. 2003, 278, 12406–12414.
€
€
(23) Hellmuth, K.; Grosskopf, S.; Lum, C. T.; Wurtele, M.; Roder, N.;
von Kries, J. P.; Rosario, M.; Rademann, J.; Birchmeier, W.
Specific inhibitors of the protein tyrosine phosphatase Shp2 iden-
tified by high-throughput docking. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 7275–7280.
(24) Yu, W. M.; Guvench, O.; Mackerell, A. D.; Qu, C. K. Identifica-
tion of small molecular weight inhibitors of Src homology 2
domain-containing tyrosine phosphatase 2 (SHP-2) via in silico
database screening combined with experimental assay. J. Med.
Chem. 2008, 51, 7396–7404.
(25) Wu, D.; Pang, Y.; Ke, Y.; Yu, J.; He, Z.; Tautz, L.; Mustelin, T.;
Ding, S.; Huang, Z.; Feng, G.-S. A conserved mechanism for
control of human and mouse embryonic stem cell pluripotency
and differentiation by shp2 tyrosine phosphatase. PLoS One 2009,
4, e4914.
(26) Zhang, Z.-Y. Protein tyrosine phosphatases: structure and func-
tion, substrate specificity, and inhibitor development. Annu. Rev.
Pharmacol. Toxicol. 2002, 42, 209–234.
(27) Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.; Almo, S. C.;
Zhang, Z.-Y. Identification of a second aryl phosphate-binding site
in protein-tyrosine phosphatase 1B: a paradigm for inhibitor
design. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13420–13425.
(28) Zhang, S.; Zhang, Z.-Y. PTP1B as a drug target: recent develop-
ment in PTP1B inhibitor discovery. Drug Discovery Today 2007,
12, 373–381.
(29) Sarmiento, M.; Wu, L.; Keng, Y.-F.; Song, L.; Luo, Z.; Huang, Z.;
Wu, G.-Z.; Yuan, A. K.; Zhang, Z.-Y. Structure-based discovery
of small molecule inhibitors targeted to protein tyrosine phospha-
tase 1B. J. Med. Chem. 2000, 43, 146–155.
(30) Liang, F.; Huang, Z.; Lee, S.-Y.; Liang, J.; Ivanov, M. I.; Alonso,
A.; Bliska, J. B.; Lawrence, D. S.; Mustelin, T.; Zhang, Z.-Y.
Aurintricarboxylic acid blocks in vitro and in vivo activity of
YopH, an essential virulent factor of Yersinia pestis, the agent of
plague. J. Biol. Chem. 2003, 278, 41734–41741.
(50) Yang, J.; Cheng, Z.; Niu, T.; Liang, X.; Zhao, Z. J.; Zhou, G. W.
Structural basis for substrate specificity of protein-tyrosine phos-
phatase SHP-1. J. Biol. Chem. 2000, 275, 4066–4071.
(51) Otwinowski, Z.; Minow, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307–
326.
(31) Horton, D. A.; Bourne, G. T.; Smythe, M. L. The combinatorial
synthesis of bicyclic privileged structures or privileged substruc-
tures. Chem. Rev. 2003, 103, 893–930.
(52) Navaza, J. AMoRe: an automated package for molecular replace-
(32) Gul, W.; Hamann, M. T. Indole alkaloid marine natural products:
an established source of cancer drug leads with considerable
promise for the control of parasitic, neurological and other dis-
eases. Life Sci. 2005, 78, 442–453.
ment. Acta Crystallogr. A 1994, 50, 157–163.
€
(53) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: a new software suite for macro-
molecular structure determination. Acta Crystallogr. D 1998, 54,
905–921.
(33) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click chemistry: diverse
chemical function from a few good reactions. Angew. Chem., Int.
Ed. 2001, 40, 2004–2021.
ꢀ
€
(34) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier,
(54) Brunger, A. T. The free R value: a novel statistical quantity for
P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click chemistry in
situ: acetylcholinesterase as a reaction vessel for the selective
assembly of a femtomolar inhibitor from an array of building
blocks. Angew. Chem., Int. Ed. 2002, 41, 1053–1057.
assessing the accuracy of crystal structures. Nature 1992, 355, 472–
475.
(55) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, G. J. Improved
methods for building protein models in electron density maps and
the location of errors in these models. Acta Crystallogr. A. 1991, 47,
110–119.
(35) Lee, L. V.; Mitchell, M. L.; Huang, S. J.; Fokin, V. V.; Sharpless,
K. B.; Wong, C. H. A potent and highly selective inhibitor of