Irreversible Nek2 kinase inhibitors with cellular activity

Article abstract of DOI:10.1021/jm200222m

A structure-based approach was used to design irreversible, cysteine-targeted inhibitors of the human centrosomal kinase, Nek2. Potent inhibition of Nek2 kinase activity in biochemical and cell-based assays required a noncatalytic cysteine residue (Cys22), located near the glycine-rich loop in a subset of human kinases. Elaboration of an oxindole scaffold led to our most selective compound, oxindole propynamide 16 (JH295). Propynamide 16 irreversibly inhibited cellular Nek2 without affecting the mitotic kinases, Cdk1, Aurora B, or Plk1. Moreover, 16 did not perturb bipolar spindle assembly or the spindle assembly checkpoint. To our knowledge, 16 is the first small molecule shown to inactivate Nek2 kinase activity in cells.

Full text of DOI:10.1021/jm200222m

ARTICLE
pubs.acs.org/jmc
Irreversible Nek2 Kinase Inhibitors with Cellular Activity
†
,†,‡
Jeffrey C. Henise and Jack Taunton*
†
‡
Program in Chemistry and Chemical Biology, Department of Cellular and Molecular Pharmacology, and Howard Hughes Medical
Institute, University of California, San Francisco, California 94158, United States
S Supporting Information
b
ABSTRACT: A structure-based approach was used to design
irreversible, cysteine-targeted inhibitors of the human centro-
somal kinase, Nek2. Potent inhibition of Nek2 kinase acti-
vity in biochemical and cell-based assays required a noncatalytic
cysteine residue (Cys22), located near the glycine-rich loop in a
subset of human kinases. Elaboration of an oxindole scaffold led
to our most selective compound, oxindole propynamide 16
(
JH295). Propynamide 16 irreversibly inhibited cellular Nek2 without affecting the mitotic kinases, Cdk1, Aurora B, or Plk1.
Moreover, 16 did not perturb bipolar spindle assembly or the spindle assembly checkpoint. To our knowledge, 16 is the first small
molecule shown to inactivate Nek2 kinase activity in cells.
1
8
’
INTRODUCTION
lymphoma. These studies have motivated the development of
Nek2 inhibitors as potential therapeutic leads.
Nek2 is a homodimeric serine/threonine kinase that localizes
1
,2
Previously reported Nek2 inhibitors include a series of amino-
to centrosomes, the microtubule organizing centers of the cell.
At the onset of mitosis, Nek2 phosphorylates the intercentro-
some linker proteins C-Nap1 and rootletin. Nek2 is thus
thought to play a role in bipolar spindle assembly driven by the
microtubule motor protein Eg5 (also known as “kinesin-5” or
1
9
20
pyrazines, a thiophene-based Plk1 inhibitor, a wortmannin-
2
1
3
,4
like series, and the sunitinib-like oxindole inhibitor 1
22
(
SU11652, Figure 1A). The aminopyrazines were extensively
characterized in biochemical assays and were found to bind to an
inactive conformation of the isolated Nek2 kinase domain by
X-ray crystallography. However, none of the aminopyrazines
were active in cells, possibly because of insufficient membrane
permeability conferred by a critical carboxylic acid moiety. The
wortmannin-like compounds were reported to antagonize the
effects of Nek2 overexpression on centrosome separation in
cells; however, it is not clear whether these effects were caused
by inhibition of Nek2 or of other cellular targets.
The selective alkylation of poorly conserved, noncatalytic
cysteines has emerged as a powerful strategy for enhancing the
potency and especially the selectivity of kinase inhibitors.
least six cysteine-targeted kinase inhibitors have entered clinical
trials for various cancer indications.
useful tool compounds have resulted from this strategy.
kinome-wide structural bioinformatics analysis carried out by our
group revealed a previously untargeted cysteine located near the
glycine-rich loop in 11 out of the ∼500 human kinases, including
Rsk1ꢀ4, Msk1/2, Plk1ꢀ3, Mekk1, and Nek2. On the basis of the
presence of this cysteine, along with a threonine in the gatekeeper
position, we designed an irreversible fluoromethylketone inhi-
bitor that is highly selective for Rsk1/2/4.
report the structure-based design of propynamide oxindole 16
JH295), which to our knowledge is the first reported inhibitor
5
“
kinesin spindle protein”). Moreover, Nek2 knockdown by
RNA interference (RNAi) was found to partially compromise
the spindle assembly checkpoint (SAC). The SAC pathway
functions early in mitosis (metaphase) to monitor the strength
and orientation of microtubule/chromosome connections and
mediates mitotic arrest in response to inhibitors of Eg5 and
microtubule dynamics. It is subject to regulation by multiple
protein kinases (e.g., Plk1, AurB, and Mps1)
interest as a potential point of intervention for anticancer drugs.
The cellular roles of Nek2, including its putative role in the SAC
pathway, have been defined primarily by RNAi-mediated knock-
down approaches. The lack of cell-active Nek2 inhibitors has
hindered attempts to elucidate its kinase activity-dependent
functions.
Like many protein kinases with roles in mitosis, Nek2 has been
implicated in cancer. Knockdown of Nek2 inhibited the prolif-
eration of cholangiocarcinoma and breast cancer cell lines in
tissue culture and in mouse tumor xenografts while having no
effect on normal fibroblasts.
the ability of oncogenic H-Ras(G12V) to induce centrosome
amplification. Forced overexpression of Nek2 in nontrans-
formed breast epithelial cells induced the formation of multi-
nucleated cells with increased numbers of centrosomes, a phenotype
6
1
9
7
21
8
8
ꢀ12
and is of great
2
3ꢀ26
At
24,27,28
Moreover, several
2
9ꢀ31
A
13,14
Nek2 knockdown also abrogated
29,30,32
1
5
Herein, we
(
1
6
that irreversibly inactivates Nek2 kinase activity in cells.
associated with mitotic errors, aneuploidy, and oncogenesis.
Finally, Nek2 overexpression at the mRNA and/or protein level
has been detected in primary breast tumors, cholangio-
1
6
Received: February 26, 2011
Published: May 31, 2011
1
3
17
carcinoma, testicular seminoma, and diffuse large B-cell
r 2011 American Chemical Society
4133
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|J. Med. Chem. 2011, 54, 4133–4146

Journal of Medicinal Chemistry
ARTICLE
Figure 1. (A) Oxindole pyrrole 1 guides the design of irreversible Nek2 inhibitors: E = electrophile. (B) Crystal structure of 1 bound to Nek2 (PDB
22
code 2JAV), showing the key cysteine (Cys22), the gatekeeper (Met86), and hydrogen bonds to the hinge region.
’
RESULTS AND DISCUSSION
acrylamide electrophile (compound 7), used extensively in the
context of irreversible EGFR and BTK inhibitors, was ineffective
against Nek2. By contrast, the more reactive propynamide
Structure-Based Design of Electrophilic Oxindoles. A
crystal structure of the Nek2 kinase domain bound to oxindole
provided a starting point for the design of irreversible inhibi-
(
compound 8) provided nearly complete inhibition.
1
To test the requirement of Cys22 for potent inhibition by 2
2
2
tors (Figure 1). Because this structure represents an unusual
inactive conformation of the isolated monomeric kinase domain,
its relevance to full-length Nek2 is unclear. We therefore used
this structure as a rough guide to predict the orientation of key
residues relative to the oxindole scaffold. Our basic design started
with the oxindole-pyrrole core found in 1, which forms three
hydrogen bonds to the Nek2 hinge region (Figure 1). This
structural feature, found in many kinase inhibitors, is predicted to
be critical for binding. Alkylation of the oxindole NH group
should thus prevent binding to Nek2 and most other kinases, a
property we exploited to control for nonspecific effects of the
reactive electrophiles (see below).
Oxindole positions 6 and 7 form close contacts with Met86,
the “gatekeeper” residue, and were therefore left unsubstituted.
By contrast, the 5-chloro substituent of oxindole 1 is ∼6 Å from
the thiol of Cys22 (Figure 1B). We hypothesized that Cys22
would be within striking distance of electrophilic substituents
and 8, we prepared a C22V mutant of Nek2 in which the target
cysteine was replaced by valine (found at the equivalent position
in most kinases). Comparison of dose-response curves for 2 and
8
against WT and C22V Nek2 revealed a strong dependence on
Cys22 for potent inhibition. Whereas chloromethylketone 2 and
propynamide 8 inhibited WT Nek2 with submicromolar po-
tency, they were significantly less potent against the C22V
mutant (Figure 2C). Under our standard in vitro kinase assay
conditions, chloromethylketone 2 was much more potent than
propynamide 8, but this advantage disappeared in cell-based
assays (see below). The increased potency of both compounds
toward WT Nek2 suggests that they function through alkylation
of Cys22. However, the data do not rule out other possible
explanations, such as steric occlusion by the bulkier valine side
chain or enhanced affinity of the C22V mutant for ATP.
To test for Cys22-dependent alkylation of Nek2, we employed
an electrospray mass spectrometry assay that reports the molec-
ular mass of the Nek2 kinase domain (amino acids 1ꢀ271), plus
that of any covalent adducts, with high accuracy. This assay does
not detect reversible Nek2/inhibitor complexes, as the protein is
denatured during the reversed-phase liquid chromatography step
prior to mass spectrometric detection. At physiological pH, a 10-
fold molar excess of 2 or 8 alkylated WT Nek2 at a single site,
giving the predicted increase in mass (Figure 3). No labeling of
the C22V mutant was detected under identical conditions,
consistent with the unique site of modification being Cys22.
These results are remarkable given that the Nek2 kinase domain
has five cysteines, at least three of which are exposed to solvent.
Thus, alkylation of Nek2 by 2 and 8 likely occurs via formation of
an initial complex in which the electrophilic carbon is proximal to
Cys22, as predicted by the orientation of oxindole 1 in the Nek2
crystal structure (Figure 1).
0
0
placed at the oxindole 5-position. Finally, the pyrrole 3 -, 4 -, and
0
5
-positions (Figure 1) represent opportunities for increasing the
selectivity for Nek2. The pyrrole 5 -methyl group of 1 forms close
0
ꢀ
contacts with Nek2 (∼3.8 Å from Tyr88, Figure 1D) and points
toward a small cleft near the hinge region. Alkyl groups at this
position may be tolerated by Nek2 to a greater extent than off-
target kinases.
On the basis of the design in Figure 1A, we first synthesized
and tested a series of seven oxindole pyrroles, each sub-
stituted with a different electrophile at the oxindole 5-position
(
(
Figure 2A). The electrophiles included a chloromethylketone
2), a chloroacetamide (3), and five Michael acceptors (4ꢀ8). In
vitro kinase assays using a single compound concentration of
5
μM and a fixed 30 min preincubation period revealed that
chloromethylketone 2 and propynamide 8 were the most potent
0
inhibitors in this initial series, inhibiting Nek2 by more than 80%
2 -Imidazole Substituents Confer Improved Selectivity for
(
Figure 2B). Intrinsic chemical reactivity, coupled with the
Nek2. Several oxindole kinase inhibitors have been shown to
33
location of the electrophilic carbon relative to the oxindole, likely
accounts for the observed potency differences. Notably, the
potently inhibit Cdk1. Because Cdk1 inhibitors block mitotic
34 35
entry and rapidly trigger mitotic exit, oxindoles that inhibit
4
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ARTICLE
Figure 2. (A) First-generation electrophilic oxindoles. (B) Percent inhibition of Nek2 kinase activity by compounds 2ꢀ8 (5 μM, 30 min). (C) Dose-
response curves from in vitro kinase assays with 2 and 8 vs WT and C22V Nek2. Specific kinase activities of WT and C22V Nek2 were indistinguishable.
Figure 3. Electrospray mass spectrometry reveals stoichiometric alkylation of WT, but not C22V, Nek2 kinase domain (aa 1ꢀ271) by compounds 2
and 8 (10 equiv). Note that the observed molecular weight of C22V Nek2 kinase domain differs from that predicted by þ79 Da, presumably because of
efficient autophosphorylation.
Cdk1 would not be useful for studying the mitotic roles of
Nek2. On the basis of the Nek2/compound 1 crystal structure
imidazole ring, respectively, could destabilize interactions with a
subset of off-target kinases, including the cyclin-dependent
kinases. Consistent with this idea, sunitinib, which contains a
(
Figure 1), as well as the structure of an imidazole oxindole
33
0
bound to Cdk2 (PDB code 1PF8), it seemed plausible that
alkyl substituents on the 5 - or 2 -position of the pyrrole or
5 -methyl group (similar to 1) did not bind detectably to the
0
0
36
Cdk1-related kinase, Cdk2 (K > 10 μM).
d
4
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Journal of Medicinal Chemistry
ARTICLE
a
Table 1. In Vitro Kinase Assays with Nek2 and Cdk1/Cyclin B
inactive against Cdk1 (IC > 20 μM). By contrast, propynamide
50
0
15 lacks the 2 -ethyl group and demonstrated inverse selectivity,
being 400-fold more potent toward Cdk1 compared to Nek2.
0
Comparison of propynamides 15 and 16 reveals that the 2 -ethyl
group confers a >20000-fold decrease in potency toward Cdk1
0
(
Table 1). All of the oxindoles lacking a 2 -ethyl group proved to
be low or subnanomolar Cdk1 inhibitors.
In vitro kinase assays with compounds 12 and 17 (Table 1,
R3 = Me) confirmed the importance of the oxindole NH (Table 1),
which forms a hydrogen bond with the Nek2 hinge region in the
crystal structure with compound 1 (Figure 1). An electrophilic
moiety was also essential for potent Nek2 inhibition, as
revealed by the sharply reduced activities of methyl ketone 13
(
IC50 > 20 μM) and 2-butynamide 18 (IC50 ≈ 20 μM). We
envisioned that these “negative control” compounds would be useful
for ruling out off-target effects in cell-based assays, including assays
for cellular Nek2 kinase activity, as described below.
Finally, we tested propynamide 16, which had the highest
selectivity for Nek2 vs Cdk1, against the kinases Rsk2 and Plk1.
Both kinases have a cysteine that is structurally equivalent to
Cys22 of Nek2. It was especially important to test Plk1, as this
mitotic kinase is an essential regulator of bipolar spindle assem-
bly, among other mitotic functions also proposed to be regulated
9
by Nek2. In vitro kinase assays (under identical conditions to
the Nek2 assays) revealed that 16 was ∼5-fold selective for Nek2
vs Rsk2 and was essentially inactive toward Plk1 (IC > 20 μM;
50
see Supporting Information). With the exception of Nek2 and
the polo-like kinases (Plk1ꢀ3), kinases containing a cysteine
equivalent to Cys22 of Nek2 (i.e., Rsk1ꢀ4, Msk1/2, and Mekk1)
are not thought to be critical mitotic regulators in human cells.
Irreversible Inhibition of Nek2 Kinase Activity in Cells. We
next tested our inhibitors against endogenous Nek2 in human
A549 lung cancer cells. This was accomplished by treating live
cells with inhibitors for 45 min at 5 μM, followed by cell lysis and
immunoprecipitation (IP) of endogenous Nek2. Immunopreci-
pitated Nek2 was then subjected to in vitro kinase assays
a
Each IC50 is the mean of three determinations (SD < 20%, unless
otherwise indicated).
(
Table 2). It is important to note that the IP process, which
includes several wash and dilution steps, allows reversibly bound
inhibitors to dissociate and is thus well suited for detecting
irreversible kinase inhibition in cells that had previously been
treated with inhibitors. Electrophilic oxindoles with potent
activity against Nek2 in vitro (IC50 < 1 μM) demonstrated up
to 95% inhibition of cellular Nek2 at 5 μM. By contrast, the
negative control compounds (12, 13, 17, and 18), including the
N-methyloxindoles bearing reactive electrophiles (12 and 17),
were significantly less active (10ꢀ25% inhibition at 5 μM).
Despite being much more potent against purified Nek2 in vitro,
the chloromethylketones were somewhat less efficacious than the
propynamides in intact cells (Table 2), most likely because of
their reduced stability toward cellular thiols (e.g., glutathione).
Compared to the propynamides, the chloromethylketones are
∼20 times more reactive toward 10 mM β-mercaptoethanol (pH
7.5; chloromethylketone 11, t1 ≈ 3 min; propynamide 16, t
Seeking alternatives to the pyrrole found in compounds 2 and
, we prepared two series of imidazole-substituted oxindoles
Table 1). Each series retained either the chloromethylketone
8
(
(
9ꢀ11) or propynamide (14ꢀ16) at the oxindole 5-position.
We noted that the IC50 values of these inhibitors were time
dependent, as expected for irreversible inhibitors. To make
meaningful comparisons among inhibitors, we therefore strictly
standardized the duration of the assays. In vitro kinase assays with
the imidazole-based inhibitors revealed different potency trends
depending on the electrophile (Table 1). In the chloromethylk-
etone series, replacement of the pyrrole of 2 with the imidazoles
of 9ꢀ11 led to a 10- to 20-fold decrease in potency. By contrast,
the same imidazole replacements had little effect on potency in
the propynamide series. Thus, propynamide 16, which contains a
0
0
2
-ethyl, 4 -methylimidazole appended to the oxindole core,
/2
1/2
inhibited Nek2 with an IC50 of ∼800 nM, similar to the
unsubstituted pyrrole 8. Mass spectrometry analysis revealed
that both chloromethylketone 11 and propynamide 16 reacted
stoichiometrically with WT but not C22V Nek2 (Supporting
Information).
≈ 60 min).
To confirm that propynamide 16 inhibits cellular Nek2 in a
Cys22-dependent manner, we generated cell lines that stably
express hemagglutinin (HA) epitope-tagged WT or C22V Nek2
under the control of a tetracycline-inducible promoter. Using an
IP kinase assay similar to that used for endogenous Nek2, we
found that 16 inhibited WT Nek2 in cells with an IC of ∼1.3 μM,
A counterscreen against Cdk1 (bound to its activator, cyclin B)
0
revealed that the 2 -ethyl group in compounds 11 and 16 is essential
5
0
for selective Nek2 inhibition (Table 1). Propynamide 16, which
exhibited the highest selectivity toward Nek2, was completely
whereas it had little effect on the C22V mutant (Figure 4). These
results suggest that inhibition of cellular Nek2 by 16 occurs via
4
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ARTICLE
Table 2. Cellular Effects of Electrophilic Oxindoles
Figure 4. Hek293 cells containing tetracycline (tet.) inducible, HA-
tagged WT (A) and C22V (B) Nek2 were pretreated with propynamide
1
6 (45 min) prior to cell lysis and IP kinase assays. After separation by
32
SDSꢀPAGE, P-labeled β-casein was visualized by phosphor imaging.
Anti-HA Western blot and Coomassie blue (CB) staining confirmed
equivalent amounts of Nek2-HA and β-casein, respectively.
a
IP kinase assays of Nek2 from cells that had been pretreated with 5 μM
b
of the indicated compounds for 45 min (single measurement). Per-
centage of cells that exited mitosis after treatment with 5 μM of the
indicated compounds for 45 min, assessed by immunofluorescence
microscopy (see text and Figure 5 for details).
alkylation of Cys22 and is not an indirect effect of inhibiting
kinases that regulate Nek2.
Effect of Electrophilic Oxindoles on the Spindle Assembly
Checkpoint. The spindle assembly checkpoint (SAC) is a
complex signaling network that prevents progression into ana-
phase (the stage of mitosis in which chromosome separation
occurs, just prior to cell division) until proper attachment of
Figure 5. A549 cells were arrested in metaphase (monastrol, 18 h) and
treated with the indicated compounds (5 μM) for 45 min. Cells were
then fixed and processed for immunofluorescence microscopy for
detection of phospho-Ser10 histone H3 (red), chromatin (blue), and
tubulin (green). Scale bar, 10 μm.
8
sister chromatids to spindle microtubules occurs in metaphase.
Small molecules that prevent bipolar spindle assembly, such as
microtubule or Eg5 kinesin inhibitors (e.g., monastrol ), arrest
stained for immunofluorescence analysis, and counted to assess
the percentage of cells that had exited mitosis (Table 2; >75 cells
were counted for each condition). We included the Cdk1
inhibitor 19 (RO3306) as a positive control for inducing mitotic
7
cells in metaphase in a SAC-dependent manner. Inhibitors of
kinases required for SAC maintenance, such as AurB, promote
10
34
rapid exit from mitosis without cell division. As Nek2 had been
previously implicated in SAC regulation by RNAi-mediated knock-
exit. Representative images of cells treated with 19 and
propynamides 15 and 16 are shown in Figure 5. Whereas cells
treated with DMSO (control) and the more selective Nek2
inhibitor 16 stained brightly with antibodies against phospho-
Ser10 histone H3, cells treated with 19 and propynamide 15 were
devoid of this mitotic marker (Figure 5). Consistent with their
potent activity toward Cdk1 in vitro (Table 1), compounds
6
down, we were motivated to test the effect of our inhibitors on
this process.
To test for a role of Nek2 in SAC maintenance, we treated
monastrol-arrested A549 cells with the electrophilic oxindoles
shown in Table 2 for 45 min, at which point the cells were fixed,
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Figure 6. Propynamide 16 does not perturb bipolar spindle assembly or chromosome congression. (A) Nek2 IP kinase assay from A549 cells arrested
with CDK1 inhibitor 19 and treated with the indicated inhibitors for 30 min. (B) Quantification of cells with bipolar spindles 1 h after 19 washout into
MG132-containing media: N > 160 cells/condition; mean ( SD from three experiments. (C) Immunofluorescence images of bipolar spindles and
chromosomes aligned at the metaphase plate. Cells were treated as in (a) fixed and stained 1 h after release from 19 into MG132-containing media. Scale
bar, 10 μm.
0
lacking a 2 -ethyl group, including propynamide 15, triggered
mitotic exit in 78ꢀ99% of the monastrol-arrested cells (Table 2).
The strong correlation between Cdk1 inhibition in vitro and
rapid induction of mitotic exit in monastrol-arrested cells sug-
gests that Cdk1 is a relevant target of these compounds in cells.
with 16 (5 μM) for 30 min, conditions that irreversibly inhibit
Nek2, as revealed by parallel IP kinase assays (Figure 6A).
Finally, the cells were released from G2-arrest by washing into
fresh media containing a proteasome inhibitor (MG132), which
allows cells to transit mitosis until they stably arrest in late
metaphase with bipolar spindles and chromosomes aligned along
the equator (termed “congression”). This well-established pro-
tocol provides a sensitive test for bipolar spindle assembly and
0
Conversely, the 2 -ethyl group of 16 is sufficient to prevent
inhibition of Cdk1 in cells, consistent with the results from Cdk1
kinase assays (Table 1).
Taken together, our results demonstrate that inhibition of
Nek2 kinase activity (Figure 4) is not sufficient to override
the SAC or induce mitotic exit (Figure 5). We further con-
clude that 16 does not significantly inhibit cellular kinases
required for SAC maintenance, such as AurB or Mps1, since
inhibitors of these kinases rapidly trigger mitotic exit under
35
chromosome congression.
In the presence of sufficient concentrations of 16 to inhibit
cellular Nek2 kinase activity by greater than 95%, we observed
no defects in bipolar spindle assembly or chromosome con-
gression nor any reduction in the total number of mitotic cells
(Figure 6). Thus, Nek2 kinase activity may not be required for
these processes, although it is possible that the trace amount
of active Nek2 remaining after inhibitor treatment is sufficient
to carry out important mitotic functions. Previously reported
mitotic defects may have resulted from depletion of the entire
Nek2 protein or from off-target effects caused by RNAi or
Nek2 overexpression. The lack of mitotic defects also sup-
ports the conclusion that 16 does not significantly inhibit
1
0ꢀ12
similar conditions.
Effect of Propynamide 16 on Bipolar Spindle Assembly
and Chromosome Congression. RNAi knockdown and domi-
nant negative overexpression experiments have implicated
Nek2 in two critical mitotic functions: bipolar spindle assembly
and chromosome congression at the “metaphase plate”.
These conclusions were challenged in a recent study (also based
on RNAi), which argued that Nek2 plays a supporting rather than
37,38
Plk1 in cells, as Plk1 inhibition causes cells to arrest in early
5
39ꢀ41
an obligatory role in mitosis. To test whether propynamide
metaphase with monopolar spindles.
Selectivity vs Plk1
1
6 perturbs these processes, we first arrested cells in late G2
is not readily explained by available crystal structures and is
surprising given the presence of a cysteine equivalent to
Cys22 in Nek2.
phase (after DNA replication and just prior to mitosis) with the
Cdk1 inhibitor 19 (Figure 5). We then treated the arrested cells
4
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ARTICLE
a
Scheme 1. Synthesis of Pyrrole-Substituted Electrophilic Oxindoles 3ꢀ5 and 6ꢀ8
a
2 3 2 3
Reagents and conditions: (a) RCHdCH (4 equiv), Ph P (0.6 equiv), Pd(OAc) (0.3 equiv), Et N (5 equiv), DMF, 85 °C, 2 h (yield, 4, 48%; 5, 34%);
(
(
b) ClCH COCl (1.3 equiv), pyridine (1.5 equiv), THF, 0 °C to room temp, 30 min; (c) CH dCHCOCl (1.2 equiv), THF, 0 °C to room temp, 10 min;
2
2
d) R
1
CO
2
H (1.2 equiv), EDC (2 equiv), acetonitrile, room temp, 0.5ꢀ2 h; (e) pyrrole-2-carboxaldehyde (5ꢀ10 equiv), piperidine (0.1ꢀ0.2 equiv),
THF, 75 °C, 4ꢀ20 h (yield, two steps, 3, 28%; 6, 28%; 7, 23%; 8, 12%).
’
CONCLUSIONS
Scheme 2. Synthesis of Imidazole-Substituted Electrophilic
Oxindoles 9ꢀ11 and 14ꢀ16
a
Starting with an oxindole scaffold that has been frequently
used to generate reversible kinase inhibitors, we have developed
cell-active, irreversible inhibitors of the centrosomal kinase,
Nek2. A crystal structure of Nek2 bound to oxindole 1 guided
the design of compounds with electrophilic substituents at the
5
-position, which we hypothesized would be within striking
distance of Cys22, located near the C-terminal end of the
glycine-rich loop in only 11 human kinases. From a set of seven
electrophilic functional groups tested at this position, propyna-
mides proved superior in terms of balancing potency, selectivity,
and chemical stability. Both propynamide- and chloromethylk-
etone-based inhibitors irreversibly alkylated Nek2 with 1:1
stoichiometry in a Cys22-dependent manner, consistent with
our structural hypothesis.
Our primary goal was to generate a chemical tool that could be
used to inhibit Nek2 acutely in living cells without inhibiting
other kinases that have critical mitotic functions. Our initial
compounds were problematic in that they inhibited Cdk1 with
subnanomolar potency and caused cells to rapidly exit mitosis,
precluding their use as Nek2 inhibitors. This problem was solved
a
Reagents and conditions: (a) RCHO (1.2 equiv), piperidine (0.3
equiv), THF, 75 °C, 7ꢀ20 h; (b) aq HCl, THF, room temp, 5ꢀ24 h
(
1
yield, two steps, 9, 41%; 10, 49%; 11, 49%); (c) R CHO (1.1 equiv),
0
by adding a 2 -ethyl group to the imidazole moiety. The resulting
piperidine (0.2 equiv), THF, 75 °C, 20 h; (d) anhydrous HCl (excess),
MeOH, room temp, 23ꢀ48 h; (e) propiolic acid (1.5ꢀ6 equiv), DIPEA
(2 equiv), EDC (2 equiv), DMF, 0 °C, 2ꢀ2.5 h (yield, three steps, 14,
propynamide 16 was inactive against Cdk1 in vitro. Moreover, at
concentrations that resulted in complete and irreversible inhibi-
tion of Nek2, propynamide 16 did not significantly inhibit
mitotic entry, the spindle assembly checkpoint, bipolar spindle
assembly, or chromosome congression. These data strongly
suggest that 16 does not inhibit cellular Cdk1, Plk1, AurB, or
Mps1. Unraveling the cellular roles of Nek2 will likely benefit
from acute small-molecule inhibition and high-resolution live
imaging experiments, which may reveal effects on centrosome,
microtubule, and chromosome dynamics. Propynamide 16 and
Nek2-inactive compounds 17 and 18 provide an initial toolkit for
undertaking these studies.
7
%; 15, 27%; 16, 32%).
pyrrole-2-carboxaldehyde (Scheme 1). To prepare imidazole-
substituted oxindoles 9ꢀ11, the 5-chloroacetyl group was first
protected to give dioxolane 21 prior to Knoevenagel condensa-
0
tion with the requisite imidazole 5 -carboxaldehydes (Scheme 2).
Finally, imidazole-substituted propynamides 14ꢀ16 were synthe-
sized from Boc-protected 5-aminooxindole 22 in three steps: (1)
Knoevenagel condensation, (2) Boc deprotection, and (3)
coupling of the resultant amine with propiolic acid (Scheme 2).
Methyl ketone 13, 2-butynamide 18, and N-methyloxindoles 12
and 17 were synthesized by analogous routes (see Experimental
Section for details).
’
CHEMISTRY
Vinyl sulfone 4 and vinyl ketone 5 were synthesized by
coupling the requisite olefins to iodide 20 under Heck conditions
’
EXPERIMENTAL SECTION
(
Scheme 1). Amide-linked electrophilic oxindoles 3 and 6ꢀ8
were prepared from 5-aminooxindole in two steps: (1) acylation
of the 5-amino group and (2) Knoevenagel condensation with
In Vitro Kinase Assays with Nek2, Cdk1, Plk1, and Rsk2.
Inhibitors were incubated for 30 min at room temperature with WT or
4
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Journal of Medicinal Chemistry
ARTICLE
C22V Nek2 (15 nM) in 20 μL of kinase reaction buffer (100 μM ATP,
additional 1.5 h at 4 °C. The beads were washed (200 μL per wash for
5 min with rocking at 4 °C) once with lysis buffer, once with lysis
buffer plus 0.5 M NaCl, and twice with kinase buffer (20 mM HEPES,
2
0 mM HEPES, pH 7.5, 5 mM MgCl , 0.1 mM EDTA, 0.08 mg/mL
2
BSA) with 3% DMSO. Next, 5 μL of kinase reaction buffer containing
32
0
.5 mg/mL β-casein (Sigma) and 2 μCi [γ- P]ATP (PerkinElmer) was
pH 7.5, 5 mM MgCl
2
, 0.1 mM EDTA, 1 mM DTT, 1ꢁ PhosSTOP).
added. Kinase reactions were allowed to proceed for 30 min at room
temperature. Next, 5 μL of the mixture was blotted onto nitrocellulose
membrane (Bio-Rad) that had been prewashed with wash buffer (1 M
The beads were suspended in the last wash buffer and divided in half.
One half was used for Nek2 Western blot analysis (Supporting
Information), and the other half was used for a kinase assay. For
the kinase assay, beads were incubated in 30 μL of kinase buffer
containing 0.5 mg/mL β-casein (Sigma), 100 μM ATP, and 4 μCi
3 4
NaCl, 0.1% H PO ) and dried. The membrane was washed three times
with wash buffer and dried under a heat lamp. The dry blots were
exposed to a storage-phosphor screen (GE Healthcare), imaged using a
Typhoon scanner (GE Healthcare), and quantified using Image Quant
software (GE Healthcare), and the data were processed and plotted
using Excel (Microsoft) and GraphPad (Prism) software.
3
2
[γ- P]ATP for 1 h at room temperature. Reactions were quenched
with 8 μL of 5ꢁ sample buffer (300 mM Tris, pH 6.8, 10% SDS, 50%
glycerol, 0.01% bromophenol blue, 0.5 M DTT). Substrate proteins
were resolved by SDSꢀPAGE and detected by Coomassie staining
and phosphor imaging.
IP kinase assays of HA-tagged Nek2 from tetracycline-inducible
Hek293 cell lines were carried out exactly as for endogenous Nek2 with
the following changes. Tet-inducible Hek293 cells expressing either WT
or C22V Nek2-3HA were plated at 630 000 cells per well in six-well plates
(Nunc), grown for 20 h, and then treated with 1 μg/mL tetracycline for
7 h to induce Nek2 expression. Cells (one well per condition) were treated
with propynamide 16 in 2 mL of media for 45 min, washed twice with
DPBS (Gibco), and stored at ꢀ80 °C. Normalized cell lysates were
immunoprecipitated with 1.6 μg of 12CA5 anti-HA antibody (Roche).
Western blot conditions and methods for generating these cell lines are
presented in the Supporting Information.
Mitosis Assays in A549 Cells. For assessing mitotic exit induced
by acute treatment with kinase inhibitors, A549 cells (180 000 in 1 mL of
media) were plated on 18 mm round polylysine-coated coverslips in 12-
well plates (Nunc), grown for 24 h, and treated with 100 μM monastrol
for 18 h. The monastrol-arrested cells were then treated with 5 μM
kinase inhibitors in the continued presence of 100 μM monastrol for an
additional 45 min. Cells were then processed for immunofluorescence
microscopy as described in the Supporting Information.
For assessing mitotic defects induced by propynamide 16, A549 cells
(180 000 in 1 mL media) were plated on 18 mm round polylysine-coated
coverslips in 12-well plates (Nunc), grown for 24 h, and treated with
5 μM 19 for 20 h. Cells arrested with 19 were treated with 5 μM 16 for
30 min in the presence of 5 μM 19, washed twice compound-free media,
then treated with 10 μM MG132 for 1 h. Cells were then processed
for immunofluorescence microscopy as described in the Supporting
Information.
Cdk1 assays were conducted using the method for Nek2 with the
following substitutions: Cdk1/cyclin-B complex (2 nM, Millipore),
3
4
8 μM ATP, 0.2 mg/mL histone-H1 (Millipore) as the substrate, and
μCi [γ- P]ATP.
32
Plk1 assays were conducted using the method for Nek2 with the follow-
ing substitutions: Plk1 (8.5 nM, Millipore), 3.8% DMSO, 2.5 mg/mL
dephosphorylated R-casein as the substrate (Sigma), and 0.75 μCi
3
2
[
γ- P]ATP.
For Rsk2 assays, inhibitors were incubated for 30 min at room tempera-
29
ture with the Rsk2 C-terminal kinase domain (15 nM) in 15 μL of
kinase reaction buffer (100 μM ATP, 20 mM HEPES, pH 7.5, 5 mM
MgCl
2
, 0.02 mg/mL BSA) with 5% DMSO. Next, 10 μL of
kinase reaction buffer containing 0.2 mg/mL BSA, 417 μM substrate
32
peptide (RRQLFRGFSFVAK), and 3 μCi [γ- P]ATP was added.
Kinase reactions were allowed to proceed for 30 min at room temperature,
and 5 μL of the mixture was blotted onto P81 filtermat (Whatman). The
filtermat was washed once with 10% acetic acid and twice with 0.1%
phosphoric acid, dried under a heat lamp, and further processed exactly
as for Nek2 kinase assays.
Mass Spectrometry Based Covalent Binding Assays. In-
hibitors (500 nM) were incubated with 50 nM WT or C22V Nek2 kinase
domain in pH 7.4 PBS with 5% DMSO for 5 h at room temperature, then
acidified to pH 3 with 0.5 M HCl. The samples were analyzed by
LCꢀMS using linear gradient elution (5ꢀ95% acetonitrile with 0.2%
formic acid over 3 min) on a Microtrap C18 protein column (Michrom
Bioresources) with a Waters 1525μ solvent delivery system and a Waters
LCT Premier ESI mass spectrometer for detection. Proteins or protein-
adducts were detected as multiply charged ions and computationally
deconvoluted to provide a single mass for each species using MassLynx
software (Waters).
General Chemistry. All reactions were conducted in oven-dried
glassware under an atmosphere of air unless otherwise noted. Reagent
grade solvents DCM, DMF, and THF were dried over molecular sieves
Immunoprecipitation Kinase Assays. All cells were grown in
media (high glucose DMEM, Gibco 11965) containing 10% fetal bovine
serum (Culture Pure, Axenia Biologix) and penicillin/streptomycin
prior to use. Reagent grade solvents carbon disulfide, EtOAc, Et
hexanes, methanol, and acetonitrile were used as purchased. The organic
bases Et N and DIPEA were dried over solid NaOH prior to use. All
2
O,
(Gibco 15140) at 37 °C in an atmosphere of 10% CO
2
. For IP kinase
3
assays with endogenous Nek2, A549 cells were plated at 400000/well in
six-well plates (Nunc) and grown for 20 h. Cells (three wells per
condition) were treated with 5 μM Nek2 inhibitors for 45 min, washed
twice with DPBS (Gibco), and stored at ꢀ80 °C. Alternatively, cells
other reagents and starting materials were purchased and used as
obtained. Reactions were monitored by TLC using K65 250 μm glass-
backed silica gel plates with a fluorescent indicator (Whatman). Flash
column chromatography was carried out on 140ꢀ400 mesh silica gel as
the solid phase (Fisher Scientific). Preparative reverse phase HPLC was
preformed on a Peeke Scientific Combi-A 5 μm preparative C18 column
(50 mm ꢁ 22 mm, flow rate 10 mL/min) using a Varian Prostar 210
solvent delivery system equipped with a Varian Prostar 335 full spectrum
UV/vis detector. Analytical HPLC was performed using the same
system equipped with a Varian Pursuit XRs 5 C18 column (150 mm ꢁ
4.6 mm, flow rate 1 mL/min), using acetonitrile/water linear gradient
elution, 5ꢀ80% acetonitrile over 20 min. By use of this method, the
purity of all compounds used in bioassays was determined to be >95%.
(
three wells per condition) were arrested in late G2 phase by incubation
with 5 μM 19 for 20 h. Cells were then treated with 5 μM Nek2
inhibitors plus 5 μM 19 for 30 min, washed twice with DPBS (Gibco),
and stored at ꢀ80 °C. The cells were thawed at room temperature for
5
min, treated with 200 μL/well of ice-cold lysis buffer (50 mM HEPES,
pH 7.6, 150 mM NaCl, 0.1% Triton X100, 1 mM DTT, 1ꢁ Roche
PhosSTOP phosphatase inhibitors, 1ꢁ Complete Roche protease
inhibitors) and bath-sonicated for 30 s. Lysates from a single condition
were combined and clarified for 10 min (20000g) at 4 °C. The clarified
lysates were normalized for total protein concentration. Next, 600 μL of
normalized lysate was incubated with 0.4 μg of anti-Nek2 antibody (H-235,
Santa Cruz) with rocking for 1.5 h at 4 °C. Next, 30 μL of protein A
Dynabeads (Invitrogen) was added, and rocking was continued for an
1
H NMR spectra were obtained using a Varian Inova 400 MHz
spectrometer. Spectral data are reported as follows: chemical shift (as
ppm referenced to the residual DMSO solvent signal at 2.50 ppm),
multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet,
4
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Journal of Medicinal Chemistry
ARTICLE
q = quartet, m = multiplet), coupling constant, and integration value.
Fully assigned H NMR spectra are reported for every compound synthe-
(Z)-3-((1H-Pyrrol-2-yl)methylene)-5-((E)-3-oxobut-1-enyl)-
indolin-2-one (4). Under an atmosphere of argon, indolinone 20
(50.0 mg, 0.149 mmol, 1 equiv), triphenylphosphine (23.0 mg, 0.0876
mmol, 0.6 equiv), triethylamine (75.0 mg, 0.741 mmol, 5 equiv), methyl
vinyl ketone (42.0 mg, 0.599 mmol, 4 equiv), and palladium(II) acetate
(10.0 mg, 0.0445 mmol, 0.3 equiv) were added to anhydrous DMF
(2 mL). The mixture was sparged with dry argon, then heated to 85 °C
for 2 h. After cooling to room temperature, the mixture was diluted with
water (50 mL) and extracted four times with EtOAc (40 mL). The
combined EtOAc extract was washed with water, then saturated NaCl
and concentrated under vacuum to give a crude orange solid. This
material was further purified by flash chromatography on silica gel
(hexanes/EtOAc 1:1) to give 4 (20 mg, 48%) as a yellow solid. A small
amount of this material was further purified by preparative HPLC
1
sized. High resolution mass spectra (HRMS) were recorded on a
Thermo Electron Corporation LTQFT using electrospray ionization
with FT resolution set to 30 000. HRMS spectra are reported for every
compound synthesized. The following compounds were synthesized
4
2
42
according to published procedures: 5-aminooxindole, 5-acetyloxindole,
43
44
45
tert-butyl hypochlorite, monastrol, and 19.
Z)-3-((1H-Pyrrol-2-yl)methylene)-5-(2-chloroacetyl)indolin-
-one (2). Chloroacetyl chloride (21.7 g, 192 mmol, 1.7 equiv) was
added dropwise to a stirred suspension of anhydrous AlCl (90.0 g, 675
(
2
3
mmol, 6 equiv) in carbon disulfide (420 mL). Stirring was allowed to
continue at room temperature for 15 min. Indolin-2-one (15.0 g, 113 mmol,
1
equiv) was then added, and the mixture was brought to reflux for 2.5 h,
then cooled on ice. After removal of the solvent by decantation the
remaining thick brown residue was treated with ice cold water (500 mL).
The resulting precipitate was recovered by filtration, washed with water,
(acetonitrile/water, linear gradient elution, 5ꢀ80% acetonitrile over
1
2
6
0 min) for use in bioassays. H NMR (DMSO-d
6
): δ 2.33 (s, 3H),
.38ꢀ6.40 (m, 1H), 6.80 (d, J = 16 Hz, 1H), 6.84ꢀ6.86 (m, 1H), 6.93
and dried under vacuum to provide pure 5-(2-chloroacetyl)indolin-2-
(
d, J = 8.1 Hz, 1H), 7.39ꢀ7.41 (m, 1H), 7.47 (dd, J = 8.2, 1.6 Hz, 1H),
1
one (21.6 g, 92%) as a white solid. H NMR (DMSO-d
6
): δ 3.57 (s, 2H),
7
.61 (d, J = 16 Hz, 1H), 7.91 (s, 1H), 8.15 (d, J = 1.5 Hz, 1H), 11.1 (s, 1H),
þ
5
1
.08 (s, 2H), 6.93 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.88 (d, J = 8.1 Hz, 1H),
0.8 (bs, 1H). HRMS calcd for C10H ClNO 210.0316; found (ESI )
9 2
1
3.2 (bs, 1H). HRMS calcd for C H N O 279.1134; found (ESI )
1
7 15 2 2
þ
(
M þ H) 279.1130.
Z)-3-((1H-Pyrrol-2-yl)methylene)-5-((E)-2-(methylsulfonyl)-
vinyl)indolin-2-one (5). Under an atmosphere of argon, indolinone 20
50.0 mg, 0.149 mmol, 1 equiv), triphenylphosphine (23.0 mg, 0.0876
(
M þ H) 210.0319.
(
5
-(2-Chloroacetyl)indolin-2-one (750 mg, 3.58 mmol, 1 equiv) was
added to a solution of pyrrole-2-carboxaldehyde (3.42 g, 36.3 mmol, 10
equiv) and piperidine (61.0 mg, 0.716 mmol, 0.2 equiv) in THF (125 mL).
The resulting mixture was heated to 60 °C in a sealed jar for 45 min.
After the mixture was cooled to room temperature, the solvent was
removed under vacuum to give a thick red oil that was dissolved in
EtOAc. Treating this solution with hexanes (30 mL) produced a tarry
red precipitate that was removed by filtration and discarded. The filtrate
was then cooled on ice to produce an orange precipitate that was
collected by filtration. This crude product was further purified by flash
chromatography on silica gel (hexanes/EtOAc 3:1) followed by recrys-
(
mmol, 0.6 equiv), triethylamine (75.0 mg, 0.741 mmol, 5 equiv), methyl
vinyl sulfone (63.0 mg, 0.594 mmol, 4 equiv), and palladium(II) acetate
(10.0 mg, 0.0445 mmol, 0.3 equiv) were added to anhydrous DMF (2 mL).
The mixture was sparged with dry argon, then heated to 85 °C for 2 h. After
the mixture was cooled to room temperature, the DMF was removed under
vacuum and the remaining black residue was purified by flash chromatog-
raphy on silica gel (hexanes/EtOAc 2:1, then hexanes/EtOAc 1:1) to give
1
pure 5 (16 mg, 34%) as a yellow solid. H NMR (DMSO-d ): δ 3.09 (s,
6
3H), 6.39ꢀ6.41 (m, 1H), 6.84ꢀ6.86 (m, 1H), 6.94 (d, J = 8.1 Hz, 1H),
7.34 (d, J = 15 Hz, 1H), 7.40ꢀ7.42 (m, 1H), 7.47 (d, J = 15 Hz, 1H), 7.49
(dd, J= 8.1, 1.5 Hz, 1H), 7.84 (s, 1H), 8.14 (d, J = 1.1 Hz, 1H), 11.2 (s, 1H),
tallization from EtOAc to give 2 (200 mg, 20%) as a crystalline orange
1
solid. H NMR (DMSO-d
6
8
(
(
6
): δ 5.16 (s, 2H), 6.40ꢀ6.42 (m, 1H), 6.92ꢀ
þ
.93 (m, 1H), 7.01 (d, J = 8.1 Hz, 1H), 7.42ꢀ7.44 (m, 1H), 7.84 (dd, J =
13.2 (s, 1H). HRMS calcd for C H N O S 315.0803; found (ESI )
1
6 15 2 3
.3, 1.7 Hz, 1H), 7.99 (s, 1H), 8.34 (d, J = 1.5 Hz, 1H), 11.3 (s, 1H), 13.2
(M þ H) 315.0798.
þ
bs, 1H). HRMS calcd for C H ClN O 287.0587; found (ESI )
(E)-Ethyl 4-((Z)-3-((1H-Pyrrol-2-yl)methylene)-2-oxoindo-
lin-5-ylamino)-4-oxobut-2-enoate (6). A solution of monoethyl
fumarate (175 mg, 1.21 mmol, 1.2 equiv) in acetonitrile (3 mL) was
added dropwise over 10 min to a stirred solution of 5-aminoindolin-2-
one (150 mg, 1.01 mmol, 1 equiv) and EDC (387 mg, 2.02 mmol, 2
equiv) in acetonitrile (50 mL) at room temperature. After the mixture
was stirred for 105 min, water (5 mL) was added and most of the aceto-
nitrile removed under vacuum. The resulting precipitate was recovered
by filtration, washed with water, and dried under vacuum to give pure
15
12
2 2
M þ H) 287.0584.
Z)-N-(3-((1H-Pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)-
-chloroacetamide (3). Pyridine (120 mg, 1.52 mmol, 1.5 equiv)
(
2
was added to a solution of 5-aminoindolin-2-one (150 mg, 1.01 mmol,
equiv) in anhydrous THF (50 mL). The mixture was cooled to 0 °C, and
1
chloroacetyl chloride (148 mg, 1.31 mmol, 1.3 equiv) was added drop-
wise over the course of 5 min. The mixture was allowed to come to room
temperature for 30 min. Next water (10 mL) was added and most of the
THF was removed under vacuum. The resulting precipitate was recovered
by filtration and washed with water to give pure 2-chloro-N-(2-oxoin-
(E)-ethyl 4-oxo-4-(2-oxoindolin-5-ylamino)but-2-enoate (153 mg, 55%) as
1
a white solid. H NMR (DMSO-d
6
): δ 1.26 (t, J = 7.1 Hz, 3H), 3.48 (s,
1
dolin-5-yl)acetamide (102 mg, 45%) as a white solid. H NMR (DMSO-
2H), 4.21 (q, J = 7.2 Hz, 2H), 6.67 (d, J = 15 Hz, 1H), 6.78 (d, J = 8.4 Hz,
1H), 7.19 (d, J = 15 Hz, 1H), 7.45 (dd, J = 8.2, 2.0 Hz, 1H), 7.61 (s, 1H),
d6): δ 3.47 (s, 2H), 4.20 (s, 2H), 6.76 (d, J = 8.4 Hz, 1H), 7.33 (dd, J =
8
.4, 1.8 Hz, 1H), 7.49 (bs, 1H), 10.1 (s, 1H), 10.3 (s, 1H). HRMS calcd
10.3 (s, 1H), 10.4 (s, 1H). HRMS calcd for C14
found (ESI ) (M þ H) 275.1029.
H
15
N
2
O
4
275.1032;
þ
þ
for C10
H
10ClN
2
O
2
225.0425; found (ESI ) (M þ H) 225.0429.
2
-Chloro-N-(2-oxoindolin-5-yl)acetamide (40.0 mg, 0.178 mmol,
(E)-Ethyl 4-oxo-4-(2-oxoindolin-5-ylamino)but-2-enoate (40.0 mg,
0.198 mmol, 1 equiv) was added to a solution of pyrrole-2-carboxalde-
hyde (188 mg, 1.98 mmol, 10 equiv) and piperidine (3.5 mg, 0.041
mmol, 0.2 equiv) in THF (6 mL). The resulting mixture was heated to
75 °C in a sealed jar for 20 h. After the mixture was cooled to room
temperature, the solvent was removed under vacuum. The residue was
then triturated in 1:1 hexanes/EtOAc, recovered by filtration, and dried
under vacuum. This crude product was further purified by dissolving in
THF (2 mL) and treating with hexanes (2.5 mL). The resulting
precipitate was recovered by filtration, washed with hexanes, and dried
1
0
equiv) was added to a solution of pyrrole-2-carboxaldehyde (84.7 mg,
.891 mmol, 5 equiv) and piperidine (1.5 mg, 0.018 mmol, 0.1 equiv) in
THF (6 mL). The resulting mixture was heated to 75 °C in a sealed jar
for 5 h. After the mixture was cooled to room temperature, the solvent
was removed under vacuum. The residue was then triturated in EtOAc,
recovered by filtration, and dried under vacuum to give pure 3 (34 mg,
1
63%) as a yellow/orange solid. H NMR (DMSO-d
6
): δ 4.24 (s, 2H),
6
.34ꢀ6.37 (m, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.91ꢀ6.94 (m, 1H), 7.20
(
(
d, J = 8.4 Hz, 1H), 7.35ꢀ7.38 (m, 1H), 7.65 (s, 1H), 7.92 (s, 1H), 10.2
1
s, 1H), 10.9 (s, 1H), 13.3 (bs, 1H). HRMS calcd for C15
H
13ClN
3
O
2
under vacuum to give pure 6 (35 mg, 50%) as an orange solid. H NMR
þ
3
02.0696; found (ESI ) (M þ H) 302.0694.
(DMSO-d ): δ 1.27 (t, J = 7.1 Hz, 3H), 4.22 (q, J = 7.1 Hz, 2H),
6
4
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Journal of Medicinal Chemistry
ARTICLE
6
6
1
1
3
.35ꢀ6.38 (m, 1H), 6.7 (d, J = 15 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H),
.94ꢀ6.97 (m, 1H), 7.24 (d, J = 15 Hz, 1H), 7.27 (dd, J = 8.2, 1.6 Hz,
H), 7.35ꢀ7.38 (m, 1H), 7.65 (s, 1H), 8.08 (d, J = 1.8, 1H), 10.5 (s,
for 7 h. After the mixture was cooled to room temperature, concentrated
HCl (300 μL) was added and stirring continued for 4.5 h. The mixture
was treated with EtOAc (5 mL) and the resulting precipitate collected by
filtration, triturated in 2 N NaOH, washed with water, and dried under
vacuum to give pure 9 (23 mg, 41%) as a yellow solid. H NMR (DMSO-
d ): δ 5.16 (s, 2H), 7.03 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 7.89 (d, J = 8.4
6
H), 10.9 (s, 1H), 13.3 (bs, 1H). HRMS calcd for C19
H
18
N
3
O
4
þ
1
52.1297; found (ESI ) (M þ H) 352.1296.
(
Z)-N-(3-((1H-Pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)-
acrylamide (7). Acryloyl chloride (191 mg, 2.11 mmol, 1.2 equiv) was
added dropwise over the course of 5 min to a solution of 5-aminoindolin-
Hz, 1H), 8.06 (s, 1H), 8.09 (s, 1H), 8.37 (s, 1H), 11.4 (s, 1H), 13.5 (bs,
þ
1H). HRMS calcd for C14
H) 288.0532.
H
11ClN
3
O
2
288.0540; found (ESI ) (M þ
2
0
-one (261 mg, 1.76 mmol, 1 equiv) in anhydrous THF (100 mL) at
°C. The mixture was allowed to warm to room temperature for 5 min,
(Z)-5-(2-Chloroacetyl)-3-((4-methyl-1H-imidazol-5-yl)methyl-
ene)indolin-2-one (10). Indolinone 21 (50.0 mg, 0.197 mmol, 1
equiv) was added to a suspension of 4-methyl-1H-imidazole-5-carbalde-
hyde (23.9 mg, 0.216 mmol, 1.1 equiv) and piperidine (5.0 mg,
0.059 mmol, 0.3 equiv) in THF (3 mL). The resulting mixture was
heated to 75 °C in a sealed jar for 9 h. After the mixture was cooled to
room temperature, concentrated HCl (300 μL) was added and stirring
continued for 24 h. The resulting precipitate was collected by filtration,
washed with EtOAc, triturated in 2 N NaOH, washed with water, and
then filtered to remove some insoluble material. The filtrate was then
concentrated under vacuum, and the remaining residue was dissolved
in EtOAc. This solution was washed with 5% NaHCO
3
, then water
and concentrated under vacuum to give pure N-(2-oxoindolin-5-yl)-
1
acrylamide (147 mg, 41%) as a white solid. H NMR (DMSO-d
6
): δ
3
6
2
.47 (s, 2H), 5.71 (dd, J = 9.9, 2.2 Hz, 1H), 6.21 (dd, J = 17, 2.2 Hz, 1H),
.40 (dd, J = 17, 10 Hz, 1H), 6.75 (d, J = 8.4 Hz, 1H), 7.41 (dd, J = 8.4,
.2 Hz, 1H), 7.59 (d, J = 1.8 Hz, 1H), 9.98 (s, 1H), 10.3 (bs, 1H). HRMS
1
þ
dried under vacuum to give pure 10 (29 mg, 49%) as a yellow solid. H
calcd for C H N O 203.0821; found (ESI ) (M þ H) 203.0818.
11 11 2 2
6
NMR (DMSO-d ): δ 2.51 (s, 3H), 5.18 (s, 2H), 7.02 (d, J = 8.4 Hz, 1H),
N-(2-Oxoindolin-5-yl)acrylamide (40.0 mg, 0.198 mmol, 1 equiv) was
7
1
3
.86 (dd, J = 8.3, 1.7 Hz, 1H), 7.97 (s, 1H), 7.99 (s, 1H), 8.52 (d, J =
added to a solution of pyrrole-2-carboxaldehyde (94.2 mg, 0.990 mmol,
.8 Hz, 1H), 11.4 (s, 1H), 13.7 (bs, 1H). HRMScalcd forC H ClN O
02.0696; found (ESI ) (M þ H) 302.0693.
5
equiv) and piperidine (1.7 mg, 0.020 mmol, 0.1 equiv) in THF (6 mL).
15 13
3
2
þ
The resulting mixture was heated to 75 °C in a sealed jar for 4 h. After the
mixture was cooled to room temperature, the solvent was removed
under vacuum. The residue was then triturated in EtOAc, recovered by
(Z)-5-(2-Chloroacetyl)-3-((2-ethyl-4-methyl-1H-imidazol-
5-yl)methylene)indolin-2-one (11). Indolinone 21 (50.0 mg,
0.197 mmol, 1 equiv) was added to a suspension of 2-ethyl-4-methyl-
filtration, and dried under vacuum to give pure 7 (31 mg, 56%) as a
1
yellow/orange solid. H NMR (DMSO-d ): δ 5.74 (d, J = 10 Hz, 1H),
1H-imidazole-5-carbaldehyde (33.0 mg, 0.239 mmol, 1.2 equiv) and
piperidine (5.0 mg, 0.059 mmol, 0.3 equiv) in THF (5 mL). The resulting
mixture was heated to 75 °C in a sealed jar for 20 h. After the mixture was
cooled to room temperature, concentrated HCl (300 μL) was added and
stirring continued for 20 h. The resulting precipitate was collected by
filtration, triturated in 1 N NaOH, washed with water, and dried under
6
6
6
7
1
.25 (d, J = 17 Hz, 1H), 6.34ꢀ6.37 (m, 1H), 6.44 (dd, J = 17, 10 Hz, 1H),
.84 (d, J = 8.4 Hz, 1H), 6.91ꢀ6.94 (m, 1H), 7.26 (d, J = 8.1 Hz, 1H),
.34ꢀ7.38 (m, 1H), 7.62 (s, 1H), 8.04 (s, 1H), 10.1 (s, 1H), 10.9 (s,
þ
H), 13.3 (bs, 1H). HRMS calcd for C H N O 280.1086; found (ESI )
1
6 14 3 2
(
M þ H) 280.1083.
Z)-N-(3-((1H-Pyrrol-2-yl)methylene)-2-oxoindolin-5-yl)-
propiolamide (8). A solution of propiolic acid (114 mg, 1.63 mmol,
.2 equiv) in acetonitrile (10 mL) was added dropwise over 10 min to a
1
(
vacuum to give pure 11 (32 mg, 49%) as a yellow solid. H NMR
6
(DMSO-d ): δ 1.28 (t, J = 7.5 Hz, 3H), 2.47 (s, 3H), 2.78 (q, J = 7.6 Hz,
1
2H), 5.18 (s, 2H), 7.02 (d, J = 8.4 Hz, 1H), 7.84 (dd, J = 8.2, 1.6 Hz, 1H),
7.94 (s, 1H), 8.49 (d, J = 1.6 Hz, 1H), 11.4 (s, 1H), 13.7 (bs, 1H). HRMS
stirred solution of 5-aminoindolin-2-one (200 mg, 1.35 mmol, 1 equiv)
and EDC (518 mg, 2.70 mmol, 2 equiv) in acetonitrile (70 mL) at room
temperature. Stirring was continued for 30 min. Water (20 mL) was added
and most of the acetonitrile removed under vacuum. The resulting
precipitate was recovered by filtration, washed with water, and dried
þ
calcd for C H ClN O 330.1009; found (ESI ) (M þ H) 330.1000.
1
7
17
3 2
(Z)-5-(2-Chloroacetyl)-3-((2-ethyl-4-methyl-1H-imidazol-
5-yl)methylene)-1-methylindolin-2-one Hydrochloride (12).
Chloroacetyl chloride (1.95 g, 17.3 mmol, 1.7 equiv) was added
under vacuum to give pure N-(2-oxoindolin-5-yl)propiolamide (108 mg,
dropwise to a stirred suspension of anhydrous AlCl (8.10 g, 60.9 mmol,
6 equiv) in carbon disulfide (40 mL). Stirring was allowed to continue at
3
1
4
6
1
0%) as a white solid. H NMR (DMSO-d
.75 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 8.4, 1.8 Hz, 1H), 7.49 (d, J = 1.8 Hz,
6
): δ 3.46 (s, 2H), 4.35 (s, 1H),
room temperature for 15 min. 1-Methylindolin-2-one (1.50 g, 10.2
mmol, 1 equiv) was then added, and the mixture was brought to reflux
for 2 h, then cooled on ice. After the solvent was decanted, the remaining
thick brown residue was treated with ice cold water. The resulting
precipitate was recovered by filtration, washed with water, and dried
under vacuum to provide pure 5-(2-chloroacetyl)-1-methylindolin-2-
H), 10.3 (s,1H), 10.7 (s, 1H). HRMS calcd for C H N O 201.0659;
11 9 2 2
þ
found (ESI ) (M þ H) 201.0661.
N-(2-Oxoindolin-5-yl)propiolamide (40.0 mg, 0.200 mmol, 1 equiv)
was added to a solution of pyrrole-2-carboxaldehyde (190 mg, 2.00
mmol, 10 equiv), and piperidine (3.3 mg, 0.039 mmol, 0.2 equiv) in
THF (6 mL). The resulting mixture was heated to 75 °C in a sealed jar
for 18 h. After the mixture was cooled to room temperature, the solvent
was removed under vacuum. The residue was purified by flash chroma-
tography on silica gel (hexanes/EtOAc 2:1) to give 8 (16 mg, 29%) as an
orange solid. A small amount of this material was further purified by
1
one (2.0 g, 88%) as a white solid. H NMR (DMSO-d ): δ 3.17 (s, 3H),
6
3.64 (s, 2H), 5.11 (s, 2H), 7.12 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 1.1 Hz,
1H), 7.99 (dd, J = 8.4, 1.8 Hz, 1H). HRMS calcd for C H ClNO
1
1
11
2
þ
224.0478; found (ESI ) (M þ H) 224.0475.
Ethylene glycol (555 mg, 8.94 mmol, 2 equiv) and pTSA H O (170
3
2
preparative HPLC (acetonitrile/water, linear gradient elution, 5ꢀ80%
mg, 0.894 mmol, 0.2 equiv) were added to a suspension of indolinone
5-(2-chloroacetyl)-1-methylindolin-2-one (1.00 g, 4.47 mmol, 1 equiv)
in benzene (200 mL). The mixture was brought to reflux using a
DeanꢀStark trap and stirred vigorously for 5 h. After cooling to room
1
acetonitrile over 20 min) for use in bioassays. H NMR (DMSO-d ): δ
6
4
.37 (s, 1H), 6.35ꢀ6.37 (m, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.93ꢀ6.95
(
7
m, 1H), 7.22 (dd, J = 8.2, 2.0 Hz, 1H), 7.35ꢀ7.38 (m, 1H), 7.61 (s, 1H),
.91 (d, J = 1.8 Hz, 1H), 10.7 (s, 1H), 10.9 (s, 1H), 13.3 (bs, 1H). HRMS
temperature, the mixture was washed with 5% NaHCO , then saturated
3
þ
calcd for C16
H
11
N
3
O
Z)-3-((1H-Imidazol-5-yl)methylene)-5-(2-chloroacetyl)-
2
277.0851; found (ESI ) (M þ) 277.0848.
NaCl and concentrated under vacuum to give 5-(2-(chloromethyl)-1,3-
1
(
dioxolan-2-yl)-1-methylindolin-2-one (1.2 g, 100%) as a tan solid. H
indolin-2-one (9). Indolinone 21 (50.0 mg, 0.197 mmol, 1 equiv) was
added to a suspension of 1H-imidazole-5-carbaldehyde (20.8 mg, 0.216
mmol, 1.1 equiv) and piperidine (5.0 mg, 0.059 mmol, 0.3 equiv) in
THF (4 mL). The resulting mixture was heated to 75 °C in a sealed jar
NMR (DMSO-d ): δ 3.06 (s, 3H), 3.50 (s, 2H), 3.76ꢀ3.80 (m, 2H),
6
3.82 (s, 2H), 4.02ꢀ4.06 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 7.31 (s, 1H),
7.33 (d, J = 8.4 Hz, 1H). HRMS calcd for C13
H
15ClNO
3
268.0740;
þ
found (ESI ) (M þ H) 268.0737.
4
142
dx.doi.org/10.1021/jm200222m |J. Med. Chem. 2011, 54, 4133–4146

Journal of Medicinal Chemistry
-(2-(Chloromethyl)-1,3-dioxolan-2-yl)-1-methylindolin-2-one (50.0 mg,
ARTICLE
5
0.592 mmol, 0.2 equiv) in THF (45 mL). The resulting mixture was
heated to 75 °C in a sealed jar for 20 h, then concentrated under vacuum.
The residue was triturated in EtOAc, collected by filtration, washed with
1:1 hexanes/EtOAc, and then treated with 80 mL of methanolic HCl
(prepared fresh by dissolving 4% v/v acetyl chloride in MeOH) for 48 h.
The mixture was concentrated to dryness under vacuum to give pure
0
1
.187 mmol, 1 equiv) was added to a suspension of 2-ethyl-4-methyl-
H-imidazole-5-carbaldehyde (25.0 mg, 0.181 mmol, 0.97 equiv) and
piperidine (4.8 mg, 0.056 mmol, 0.3 equiv) in THF (6 mL). The
resulting mixture was heated to 75 °C in a sealed jar for 18 h. After the
mixture was cooled to room temperature concentrated HCl (300 μL)
was added, and stirring was allowed to continue for an additional 18 h.
The resulting precipitate was collected by filtration, washed with THF,
(Z)-5-amino-3-((4-methyl-1H-imidazol-5-yl)methylene)indolin-2-one
1
dihydrochloride (781 mg, 83%) as a yellow solid. H NMR (DMSO-d
6
):
and dried under vacuum to give pure 12 (40 mg, 60%) as an orange solid.
δ 2.63 (s, 3H), 7.02 (d, J = 8.1 Hz, 1H), 7.30 (dd, J = 8.1, 1.6 Hz, 1H),
1
H NMR (DMSO-d
6
): δ 1.30 (t, J = 7.7 Hz, 3H), 2.41 (s, 3H), 2.76 (q,
7.86 (d, J = 1.8 Hz, 1H), 8.00 (s, 1H), 9.01 (s, 1H), 11.5 (bs, 1H). HRMS
þ
J = 7.7 Hz, 2H), 3.18 (s, 3H), 4.29 (s, 1H), 6.89 (d, J = 8.4 Hz, 1H), 7.24
dd, J = 8.4, 2.2 Hz, 1H), 7.43 (s, 1H), 9.70 (d, J = 1.8 Hz, 1H), 10.5 (s,
(free base) calcd for C13
241.1081.
H
13
N
4
O 241.1089; found (ESI ) (M þ H)
(
1
H), 12.3 (bs, 1H). HRMS calcd for C H ClN O 344.1166; found
DIPEA (41.4 mg, 0.320 mmol, 2 equiv) was added to a suspension of
(Z)-5-amino-3-((4-methyl-1H-imidazol-5-yl)methylene)indolin-2-one
dihydrochloride (50.0 mg, 0.160 mmol, 1 equiv) in DMF (5 mL) at 0 °C
with stirring. The resulting red solution was treated with propiolic acid
(16.8 mg, 0.240 mmol, 1.5 equiv), then EDC (61.3 mg, 0.320 mmol,
2 equiv). After being stirred for 2.5 h, the mixture was diluted with 5%
18
19
3 2
þ
(ESI ) (M þ H) 344.1153.
(
Z)-5-Acetyl-3-((2-ethyl-4-methyl-1H-imidazol-5-yl)methyl-
ene)indolin-2-one (13). 5-Acetylindolin-2-one (100 mg, 0.571 mmol,
equiv) was added to a suspension of 2-ethyl-4-methyl-1H-imidazole-5-
carbaldehyde (71.0 mg, 0.514 mmol, 0.9 equiv) and piperidine (14.0 mg,
.164 mmol, 0.3 equiv) in THF (8 mL). The resulting mixture was
1
0
NaHCO (50 mL) and extracted three times with EtOAc (50 mL). The
3
heated to 75 °C in a sealed jar for 16 h. After the mixture was cooled to
room temperature, the product was collected by filtration and washed
with THF and EtOAc to give 13 (57 mg, 34%) as an orange solid. A small
amount of this material was further purified by preparative HPLC
combined EtOAc extract was washed with water, then saturated NaCl
and concentrated under vacuum. The residue was triturated in 1:1
hexanes/EtOAc, recovered by filtration, and washed with EtOAc to give
15 (15 mg, 32%) as an orange solid. A small amount of this material was
further purified by preparative HPLC (acetonitrile/water, linear gradi-
(
acetonitrile/water, linear gradient elution, 5ꢀ80% acetonitrile over
1
1
2
3
1
1
0 min) for use in bioassays. H NMR (DMSO-d ): δ 1.28 (t, J = 7.7 Hz,
ent elution, 5ꢀ80% acetonitrile over 20 min) for use in bioassays. H
6
H), 2.46 (s, 3H), 2.59 (s, 3H), 2.78 (q, J = 7.6 Hz, 2H), 6.98 (d, J = 8.4 Hz,
H), 7.82 (dd, J = 8.2, 1.6 Hz, 1H), 7.94 (s, 1H), 8.46 (d, J = 1.8 Hz, 1H),
NMR (DMSO-d ): δ 2.44 (s, 3H), 4.36 (s, 1H), 6.85 (d, J = 8.4 Hz, 1H),
6
7.27 (dd, J = 8.4, 2.2 Hz, 1H), 7.62 (s, 1H), 7.93 (s, 1H), 7.94 (d, J = 1.8
1.3 (bs, 1H), 13.7 (bs, 1H). HRMS calcd for C17
H
18
N
3
O
2
296.1394;
Hz, 1H), 10.7 (s, 1H), 11.0 (s, 1H), 13.8 (bs, 1H). HRMS calcd for
þ
þ
found (ESI ) (M þ H) 296.1396.
C H N O 292.0960; found (ESI ) (M þ) 292.0965.
1
6 12 4 2
(
Z)-N-(3-((1H-Imidazol-5-yl)methylene)-2-oxoindolin-5-yl)-
(Z)-N-(3-((2-Ethyl-4-methyl-1H-imidazol-5-yl)methylene)-
propiolamide (14). Indolinone 22 (750 mg, 3.02 mmol, 1 equiv) was
added to a suspension of 1H-imidazole-5-carbaldehyde (320 mg, 3.33
mmol, 1.1 equiv) and piperidine (50.4 mg, 0.592 mmol, 0.2 equiv) in
THF (45 mL). The resulting mixture was heated to 75 °C in a sealed jar
for 20 h, then concentrated under vacuum. The residue was triturated in
EtOAc, collected by filtration, washed with 1:1 hexanes/EtOAc, and
then treated with 80 mL of methanolic HCl (prepared fresh by
dissolving 4% v/v acetyl chloride in MeOH) for 23 h. The mixture
was concentrated under vacuum to give pure (Z)-3-((1H-imidazol-5-yl)-
2-oxoindolin-5-yl)propiolamide (16). Indolinone 22 (750 mg,
3.02 mmol, 1 equiv) was added to a suspension of 2-ethyl-4-methyl-1H-
imidazole-5-carbaldehyde (459 mg, 3.32 mmol, 1.1 equiv) and piper-
idine (50.4 mg, 0.592 mmol, 0.2 equiv) in THF (45 mL). The resulting
mixture was heated to 75 °C in a sealed jar for 20 h, then concentrated
under vacuum. The residue was triturated in EtOAc, collected by filtra-
tion, washed with 1:1 hexanes/EtOAc, and treated with 80 mL of
methanolic HCl (prepared fresh by dissolving 4% v/v acetyl chloride in
MeOH) for 48 h. The mixture was concentrated to dryness under vacuum
to give pure (Z)-5-amino-3-((2-ethyl-4-methyl-1H-imidazol-5-yl)methyl-
methylene)-5-aminoindolin-2-one dihydrochloride (750 mg, 83%) as a
1
1
yellow solid. H NMR (DMSO-d
6
): δ 7.02 (d, J = 8.1 Hz, 1H), 7.26 (d,
ene)indolin-2-one dihydrochloride (740 mg, 72%) as a yellow solid. H
J = 8.4 Hz, 1H), 7.59 (s, 1H), 7.94 (s, 1H), 8.36 (s, 1H), 8.99 (s, 1H),
NMR (DMSO-d ): δ 1.36 (t, J = 7.5 Hz, 3H), 2.60 (s, 3H), 3.04 (q, J =
6
1
1.4 (s, 1H). HRMS (free base) calcd for C12
H
11
N
4
O 227.0933; found
7.6 Hz, 2H), 7.01 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.78 (bs,
þ
(ESI ) (M þ H) 227.0926.
1H), 7.99 (s, 1H), 11.5 (s, 1H). HRMS (free base) calcd for C H N O
1
5 17 4
þ
Under an atmosphere of argon, DIPEA (45.5 mg, 0.352 mmol, 2.1
269.1402; found (ESI ) (M þ H) 269.1394.
equiv) was added to a stirred suspension of (Z)-3-((1H-imidazol-5-yl)-
methylene)-5-aminoindolin-2-one dihydrochloride (50.0 mg, 0.167 mmol,
1
and then treated with propiolic acid (70.2 mg, 1.00 mmol, 6 equiv)
followed by EDC (67.5 mg, 0.352 mmol, 2.1 equiv). After being stirred
for 2 h, the mixture was diluted with EtOAc (150 mL), washed three
times with water (50 mL), then with saturated NaCl, and concen-
trated under vacuum to give a crude orange solid. This material was
further purified by preparative HPLC (acetonitrile/water, linear
DIPEA (45.5 mg, 0.352 mmol, 2 equiv) was added to a stirred
suspension of (Z)-5-amino-3-((2-ethyl-4-methyl-1H-imidazol-5-yl)methyl-
ene)indolin-2-one dihydrochloride (60.0 mg, 0.176 mmol, 1 equiv) in
DMF (5 mL) at 0 °C. The resulting red solution was treated with
propiolic acid (18.5 mg, 0.264 mmol, 1.5 equiv), followed by EDC (67.5
mg, 0.352 mmol, 2 equiv). After stirring was continued for 2.5 h, the
mixture was allowed to warm to room temperature over 1.5 h. The
equiv) in DMF (5 mL). The resulting red solution was cooled to 0 °C
mixture was then diluted with 5% NaHCO (50 mL) and extracted three
3
times with EtOAc (50 mL). The combined EtOAc extract was washed
gradient elution, 5ꢀ to 80% acetonitrile over 20 min) to give pure
with water, then saturated NaCl, and concentrated under vacuum. The
resulting residue was triturated in EtOAc, recovered by filtration, washed
with EtOAc, and dried under vacuum to give pure 16 (25 mg, 44%) as a
1
1
4
1
1
4 (3.8 mg, 8.2%) as a yellow/orange solid. H NMR (DMSO-d ): δ
6
.38 (s, 1H), 6.86 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.74 (s,
H), 7.74 (s, 1H), 7.96 (s, 1H), 8.02 (s, 1H), 10.7 (s, 1H), 11.0 (bs,
1
yellow/orange solid. H NMR (DMSO-d ): δ 1.27 (t, J = 7.5 Hz, 3H),
6
H), 13.7 (bs, 1H). HRMS calcd for C15
H
11
N
4
O
2
279.0882; found
2.40 (s, 3H), 2.76 (q, J = 7.6 Hz, 2H), 4.36 (s, 1H), 6.85 (d, J = 8.4 Hz,
1H), 7.24 (dd, J = 8.4, 1.5 Hz, 1H), 7.56 (s, 1H), 7.92 (d, J = 1.8 Hz, 1H),
þ
(
ESI ) (M þ H) 279.0876.
(
Z)-N-(3-((4-Methyl-1H-imidazol-5-yl)methylene)-2-ox-
10.6 (s, 1H), 10.9 (bs, 1H), 13.8 (bs, 1H). HRMS calcd for C18
H
17
N
4
O
2
þ
oindolin-5-yl)propiolamide (15). Indolinone 22 (750 mg, 3.02
mmol, 1 equiv) was added to a suspension of 4-methyl-1H-imidazole-5-
carbaldehyde (366 mg, 3.32 mmol, 1.1 equiv) and piperidine (50.4 mg,
321.1352; found (ESI ) (M þ H) 321.1368.
(Z)-N-(3-((2-Ethyl-4-methyl-1H-imidazol-5-yl)methylene)-
1-methyl-2-oxoindolin-5-yl)propiolamide (17). HNO (70%
3
4
143
dx.doi.org/10.1021/jm200222m |J. Med. Chem. 2011, 54, 4133–4146

Journal of Medicinal Chemistry
ARTICLE
aqueous, 1.41 g, 22.4 mmol, 1.7 equiv) was slowly added to a solution of
’ ASSOCIATED CONTENT
Supporting Information. Experimental protocols for
purification of Nek2 constructs, generation of stable cell lines,
immunofluorescence microscopy, and Western blotting; mass
spectrometry data of Nek2 adducts with compounds 12 and 16;
and raw data from IP kinase assays. This material is available free
of charge via the Internet at http://pubs.acs.org.
1-methylindolin-2-one (2.00 g, 13.6 mmol, 1 equiv) in concentrated
S
H
2
SO
4
(8 mL) at 0 °C with stirring. After being stirred for 30 min, the
b
mixture was poured onto a water/ice mixture (200 mL). The resulting
precipitate was collected by filtration and washed with water (250 mL)
to give pure 1-methyl-5-nitroindolin-2-one (2.3 g, 88%) as a tan solid. H
1
6
NMR (DMSO-d ): δ 3.19 (s, 3H), 3.70 (s, 2H), 7.19 (d, J = 8.4 Hz, 1H),
8
1
.14 (s, 1H), 8.26 (d, J = 8.8 Hz, 1H). HRMS calcd for C
9
H
7
N
2
O
3
ꢀ
91.0462; found (ESI ) (M ꢀ H) 191.0464.
1-Methyl-5-nitroindolin-2-one (100 mg, 0.520 mmol, 1 equiv) was
’
AUTHOR INFORMATION
added to a suspension of 2-ethyl-4-methyl-1H-imidazole-5-carbalde-
hyde (68.3 mg, 0.494 mmol, 0.95 equiv) and piperidine (13.3 mg, 0.156
mmol, 0.3 equiv) in THF (7 mL). The resulting mixture was heated to
Corresponding Author
*Phone: 415-514-2004. Fax: 415-514-0822. E-mail: taunton@
cmp.ucsf.edu.
7
5 °C in a sealed jar for 18 h. After the mixture was cooled to room
temperature, the resulting precipitate was collected by filtration and
washed with EtOAc to give pure (Z)-3-((2-ethyl-4-methyl-1H-imidazol-
’
ACKNOWLEDGMENT
5
-yl)methylene)-1-methyl-5-nitroindolin-2-one (113 mg, 73%) as an
1
orange solid. H NMR (DMSO-d
6
): δ 1.43 (t, J = 7.5 Hz, 3H), 2.45 (s,
We gratefully acknowledge the UCSF Mass Spectrometry
3H), 2.75 (q, J = 7.6 Hz, 2H), 3.29 (s, 3H), 7.16 (d, J = 8.4 Hz, 1H), 7.55
Facility (A. L. Burlingame, Director, NIH Grant NCRR
P41RR001614), Stefan Knapp (University of Oxford, U.K.) for
Nek2 expression plasmids, Nick Hertz (UCSF) for RO3306, and
Rand Miller (UCSF) for help with kinase assays. J.C.H. acknowl-
edges the ARCS Foundation for funding and members of the
Taunton lab for support. This work was funded by the NIH
(
1
3
s, 1H), 8.15 (dd, J = 8.8, 2.6 Hz, 1H), 10.6 (d, J = 2.6 Hz, 1H), 12.5 (bs,
þ
H). HRMS calcd for C16
13.1293.
H
17
N
4
O
3
313.1301; found (ESI ) (M þ H)
Zinc dust (1.02 g, 15.6 mmol, 47 equiv) was added to a suspension
of (Z)-3-((2-ethyl-4-methyl-1H-imidazol-5-yl)methylene)-1-methyl-5-
nitroindolin-2-one (105 mg, 0.336 mmol, 1 equiv) in THF (40 mL) and
(
Grant GM071434).
saturated aqueous NH
mixture was vigorously stirred for 10 min, then diluted with EtOAc
150 mL), washed with water, then saturated NaCl, and concentrated
4
Cl (20 mL) at room temperature. The resulting
’
ABBREVIATIONS USED
(
BME, β-mercaptoethanol; Cdk1, cyclin dependent kinase 1;
C-Nap1, centrosomal Nek2 associated protein 1; DCM, dichlor-
omethane; DIPEA, N,N-diisopropylethylamine; DMF, dimethyl-
formamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride; Eg5, kinesin family member 11; HA, hemagglutinin;
IP, immunoprecipitation;Mps1, monopolarspindlekinase1; Nek2,
NIMA related kinase 2; Plk1, polo-like kinase 1; Rsk2, p90
ribosomal S6 protein kinase 2; SAC, spindle assembly check-
point; SDSꢀPAGE, sodium dodecyl sulfateꢀpolyacrylamide gel
electrophoresis; TLC, thin layer chromatography; WT, wild type
under vacuum to give pure (Z)-5-amino-3-((2-ethyl-4-methyl-1H-imi-
dazol-5-yl)methylene)-1-methylindolin-2-one (67 mg, 71%) as a red/
1
orange solid. H NMR (DMSO-d ): δ 1.32 (t, J = 7.7 Hz, 3H), 2.38
6
(s, 3H), 2.75 (q, J = 7.6 Hz, 2H), 3.11 (s, 3H), 4.50 (bs, 2H), 6.50 (dd,
J = 8.1, 2.6 Hz, 1H), 6.62 (d, J = 8.1 Hz, 1H), 7.32 (s, 1H), 8.81 (d,
J = 2.2 Hz, 1H), 12.2 (bs, 1H). HRMS calcd for C16
H
19
N
4
O 283.1553;
þ
found (ESI ) (M þ H) 283.1552.
EDC (80.3 mg, 0.419 mmol, 2 equiv) was added to a solution of
indolinone (Z)-5-amino-3-((2-ethyl-4-methyl-1H-imidazol-5-yl)methyl-
ene)-1-methylindolin-2-one (59.1 mg, 0.209 mmol, 1 equiv) and
propiolic acid (22.2 mg, 0.317 mmol, 1.5 equiv) in DMF (4 mL) at
0
°C. After stirring for 2 h, the mixture was diluted with EtOAc
’
REFERENCES
(100 mL), washed with water, then saturated NaCl, and concentrated
(
1) Fry, A. M.; Meraldi, P.; Nigg, E. A. A centrosomal function for
under vacuum to give a crude orange solid. This material was further
purified by preparative HPLC (acetonitrile/water, linear gradient elu-
the human Nek2 protein kinase, a member of the NIMA family of cell
cycle regulators. EMBO J. 1998, 17, 470–481.
tion, 5ꢀ80% acetonitrile over 20 min) to give pure 17 (16 mg, 23%) as
(
2) O’Regan, L.; Blot, J.; Fry, A. M. Mitotic regulation by NIMA-
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1
an orange solid. H NMR (DMSO-d
6
): δ 1.30 (t, J = 7.7 Hz, 3H), 2.41
(s, 3H), 2.76 (q, J = 7.7 Hz, 2H), 3.18 (s, 3H), 4.29 (s, 1H), 6.89 (d, J =
(
8.4 Hz, 1H), 7.24 (dd, J = 8.4, 2.2 Hz, 1H), 7.43 (s, 1H), 9.70 (d, J = 1.8 Hz,
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1H), 10.5 (s, 1H), 12.3 (bs, 1H). HRMS calcd for C19
H
19
N
4
O
2
335.1508;
þ
found (ESI ) (M þ H) 335.1493.
(
Z)-N-(3-((2-Ethyl-4-methyl-1H-imidazol-5-yl)methylene)-
2
-oxoindolin-5-yl)but-2-ynamide (18). To a solution of (Z)-5-
amino-3-((2-ethyl-4-methyl-1H-imidazol-5-yl)methylene)indolin-2-
one dihydrochloride (50.0 mg, 0.147 mmol, 1 equiv), EDC (112 mg,
(
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0
2
.584 mmol, 4 equiv), HOBt (20.0 mg, 0.148 mmol, 1 equiv), and
-butynoic acid (74.0 mg, 0.880 mmol, 6 equiv) in DMF (6 mL) was
2
010, 12, 1166–1176.
6) Draviam, V. M.; Stegmeier, F.; Nalepa, G.; Sowa, M. E.; Chen, J.;
added DIPEA (57.0 mg, 0.441 mmol, 3 equiv). After being stirred for 2 h,
the mixture was diluted with 5% NaHCO (20 mL) and extracted with
EtOAc. The EtOAc extract was washed with water, then saturated NaCl
and concentrated to dryness to give pure 18 (25 mg, 51%) as an orange
(
3
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solid. H NMR (DMSO-d ): δ 1.27 (t, J = 7.7 Hz, 3H), 2.04 (s, 3H), 2.40
6
(s, 3H), 2.75 (q, J = 7.6 Hz, 2H), 6.83 (d, J = 8.4. 1H), 7.22 (dd, J = 8.2,
2
1
.0 Hz, 1H), 7.53 (s, 1H), 7.92 (d, J = 1.8 Hz, 1H), 10.4 (s, 1H), 10.9 (bs,
H), 13.8 (s, 1H). HRMS calcd for C19
H
19
N
4
O
2
335.1503; found
þ
(ESI ) (M þ H) 335.1496.
4
144
dx.doi.org/10.1021/jm200222m |J. Med. Chem. 2011, 54, 4133–4146

Journal of Medicinal Chemistry
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