Pseudo-cyclization through intramolecular hydrogen bond enables discovery of pyridine substituted pyrimidines as new mer kinase inhibitors

Article abstract of DOI:10.1021/jm401387j

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design appr

Full text of DOI:10.1021/jm401387j

Article
pubs.acs.org/jmc
Pseudo-Cyclization through Intramolecular Hydrogen Bond Enables
Discovery of Pyridine Substituted Pyrimidines as New Mer Kinase
Inhibitors
Weihe Zhang,†,‡,# Dehui Zhang,†,‡,# Michael A. Stashko,†,‡ Deborah DeRyckere,⊥ Debra Hunter,∥
Dmitri Kireev,†,‡ Michael J. Miley,§ Christopher Cummings,⊥ Minjung Lee,⊥ Jacqueline Norris-Drouin,†,‡
Wendy M. Stewart,†,‡ Susan Sather,⊥ Yingqiu Zhou,‡ Gregory Kirkpatrick,⊥ Mischa Machius,§
William P. Janzen,†,‡ H. Shelton Earp,∥,§ Douglas K. Graham,⊥ Stephen V. Frye,†,‡,∥
and Xiaodong Wang*,†,‡
†Center for Integrative Chemical Biology and Drug Discovery and ‡Division of Chemical Biology and Medicinal Chemistry, Eshelman
School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
§Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,
United States
∥Department of Medicine, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, United States
⊥Department of Pediatrics, School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045,
United States
S
* Supporting Information
ABSTRACT: Abnormal activation or overexpression of Mer receptor tyrosine kinase has
been implicated in survival signaling and chemoresistance in many human cancers.
Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug
design approach using a pseudo-ring replacement strategy was developed and validated to
discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through
SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation
based on high selectivity against other kinases and good pharmacokinetic properties. When
applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an
IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein.
Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and
NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation
of this compound for therapeutic application in patients with cancer.
expression is abrogated with shRNA in non-small-cell lung
cancer (NSCLC) cells.6 In addition, treatment of melanoma
cells with a small molecule Mer inhibitor UNC10627 results in
effects comparable to shRNA-mediated Mer inhibition,
including reduced colony formation in soft agar and decreased
invasion into collagen matrix.8 Taken together, these data
indicate that Mer is a novel therapeutic target for the treatment
of ALL and other cancers that ectopically or overexpress Mer.
Therefore, Mer inhibition with small molecule inhibitors may
have clinical benefit either alone or in combination with
chemotherapeutic agents.
We have previously developed several potent small molecule
Mer inhibitors within the pyrazolopyrimidine scaffold.7,9
However, we were motivated to explore other scaffolds to
potentially increase selectivity within the TAM family of
kinases9a and address solubility and pharmacokinetic properties
required for further in vivo studies.7 In order to design new
INTRODUCTION
■
Mer belongs to the TAM (Tyro3, Axl, Mer) family of receptor
tyrosine kinases (RTK) which share at least one common
biological ligand: growth arrest-specific-6 (Gas6). Other
ligands, including Protein S,1 Tubby,2 TULP-1,2 and
Galectin-3,3 can also stimulate Mer. Under normal physiologic
conditions, Mer mediates the second phase of platelet
aggregation, macrophage and epithelial cell clearance of
apoptotic cells, modulation of macrophage cytokine synthesis,
cell motility, and cell survival.4 Abnormal activation or
overexpression of Mer RTK has been implicated in neoplastic
progression of many human cancers and has been correlated
with poorer prognosis. For example, Mer is ectopically
expressed in both B-cell and T-cell acute lymphoblastic
leukemia (ALL), the most common pediatric malignancy, but
is not expressed in normal mouse and human T- and B-
lymphocytes at any stage of development.5 Inhibition of Mer by
si/sh-RNA knockdown sensitizes cells to chemotherapy-
induced apoptosis and doubles survival in a xenograft model
of acute leukemia.5c Similar effects are observed when Mer
Received: September 9, 2013
Published: November 6, 2013
© 2013 American Chemical Society
9683
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
Figure 1. Structure-based design of a scaffold that relies on pseudo-ring formation through an intramolecular hydrogen bond: (A) X-ray structure of
1 in complex with Mer protein (kinase domain) (PDB code 3TCP); (B) docking model (based on X-ray structure PDB code 3TCP) of the designed
molecule 2.
Scheme 1. General Synthetic Routes for Pyridinepyrimidine Analogues II
Mer inhibitors, we revisited the cocrystal structure of Mer in
complex with 1 (UNC569) (Figure 1A).9a Wherein the
inhibitor 1 is fully confined to the relatively small adenine
pocket, including its flexible lipophilic butyl side chain, the
pyrazole ring of 1 does not interact with any amino acid residue
directly, suggesting that its main role might be to maintain the
molecule’s overall conformation. Replacing the pyrazole ring
with a pseudo-ring10 constrained by an intramolecular hydro-
gen bond as shown in compound 2 (Figure 1B) could
potentially generate potent Mer inhibitors displaying different
physicochemical properties when compared to pyrazolopyr-
imidines. Additionally, because the pseudo-ring in 2 is formed
through a hydrogen bond between the pyridine nitrogen and
the 4-amino group, it is less rigid than pyrazolopyrimidine and
therefore is more likely to undergo “induced fit” to optimize its
interaction with the binding pocket. This scaffold would also
simplify analogue synthesis and facilitate exploration of
structure−activity relationships (SARs).
CHEMISTRY
■
The proposed pyridinepyrimdines were synthesized using the
route shown in Scheme 1. Starting with the commercially
available compound 5-bromo-2,4-dichloropyrimidine, a SNAr
displacement of the chloride at the C4 position of the
pyrimidine ring with an amine (R2NH2) introduces substituents
at the R2 position in I. The second SNAr reaction at a higher
temperature installs the R3 group at the C2 of the pyrimidine
ring to yield the intermediate II. Finally, cross-coupling
between II and a lithium trihydroxy borate salt of 2-pyridine
or its derivatives provides the desired analogues III. We used 2-
9684
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
Scheme 2. General Synthetic Route To Explore SAR at the R3 Position
Figure 2. Structures and enzymatic activities of compound 2 and its close analogues.
pyridyl trihydroxyborate salts for the coupling, as they are easily
prepared.11 In contrast, the corresponding boronic acid/ester of
2-pyridine and its derivatives are either very expensive or not
readily available. Alternatively, I can also be converted to the
corresponding boronic ester IV and subsequently be coupled
with 2-chloropyridine or its derivatives to introduce the R1
group at the C5 of the pyrimidine ring.12 Overall, this route is
optimal to explore the SAR at the R1 site; it can also be used for
SAR exploration at the R2 and R3 sites.
In addition, we developed an alternative route to synthesize
analogues with different R3 groups as shown in Scheme 2
because the most efficient way to prepare analogues was to
place the exploration step at the last step of the synthesis. The
SNAr replacement of the chloride of commercially available 5-
bromo-4-chloro-2-(methylthio)pyrimidine provides the inter-
mediate V which can be transformed to VI via a Suzuki−
Miyaura cross-coupling reaction with a lithium trihydroxyborate
salt of 2-pyridinyl compounds. m-CPBA oxidation of VI
followed by the second SNAr replacement by various amines
(R3NH2) yields the final products III.
also rendered the corresponding 6 much less active (890-fold).
Collectively, these results validate that the pseudo-ring design
through an intramolecular hydrogen bond maintains a
conformation similar to the pyrazolopyrimidine. Compared to
1, compounds 2 and 3 are also more selective for Mer relative
to Axl (38-fold and 67-fold, respectively) and Tyro3 (10-fold
and 37-fold, respectively). This enhanced selectivity was
fortuitous, but subsequent cocrystal structures (see below)
may provide an explanation.
To develop potent Mer inhibitors with improved properties,
we investigated the SAR with this newly discovered
pyridinepyrimidine scaffold at the R1, R2, and R3 positions.
First, different substituents at the R1 position were explored
while fixing the R2 site as a trans-4-hydroxycyclohexyl group
and the R3 site as a butyl group. As shown in Table 1, a fluorine
at the 5-position of the pyridine ring did not affect the Mer
activity of analogue 7 significantly while a larger group such as
N-methylpiperazine at the same position led to a 6-fold more
active analogue 8. An extra methylene group between pyridine
and N-methylpiperazine (9) was also tolerated. The N-
methylpiperazine ring could be replaced by a morpholine ring
to yield an equipotent analogue 10 (UNC2250). Interestingly,
the substitution pattern on the pyridine ring played an
important role in the activity of these compounds. The most
active analogue 10 was generated from substitution at the 5-
position of the pyridine ring, while a substituent at the 3-
position (11) totally abolished the Mer activity, possibly
because of steric clash of the substituents with the Mer protein
backbone. The substitutions at the 4-position (12) and the 6-
position of the pyridine ring (13) were tolerated but were not
as active as when substituted at the 5-position (10). In addition,
N-methylpiperazine at the 5-position of the pyridine in 9 could
also be replaced with other groups such as 4,4-difluoropiper-
idine (14) and dimethylamine (15) without disrupting activity.
Furthermore, other functionalities such as the sulfonamide
(16), amide (17), or reversed amide (18) could be used to link
the pyridine with another group such as morpholine (16) or N-
methylpiperazine (17 and 18), resulting in similarly active
analogues. However, the amide bond in 17 was not as stable to
RESULTS AND DISCUSSION
■
To validate the pseudo-ring strategy, we first synthesized
compound 2 and its structurally close analogues (Figure 2)
using the route shown in Scheme 1. Their activities against Mer
kinase were then determined in a microfluidic capillary
electrophoresis (MCE) assay.7,9a,13 The IC50 value of 2 was
18 nM, which was in the same range as that of the parent
pyrazolopyrimidine compound 1. Replacing the trans-4-amino-
cyclohexylmethylamino substitution in 2 with a trans-4-
hydroxycyclohexylamino group also resulted in a potent Mer
inhibitor 3. This will likely diminish the undesired hERG
activity present in 1 as previously demonstrated with
UNC1062.7 In contrast, the Mer activity decreased dramatically
when the intramolecular hydrogen bond was disrupted either
by moving the nitrogen of 2-pyridinyl to the C6 position of the
pyridine ring (4, 34-fold) or by replacing it with a CH group (5,
56-fold). The complete removal of the 2-pyridinyl substitution
9685
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
Table 1. Preliminary SAR at the R1 Position
a
Values are the mean of two or more independent assays.
hydrolysis as the reversed amide bond in 18. 4-Amino-N-
methylpiperidine was partially replaced by the solvent
isopropanol during the workup of the amide bond coupling
reaction (the reaction was quenched with a 1.0 N aqueous
solution of NaOH or a saturated aqueous solution of NaHCO3
and extracted with a mixture of isopropyl alcohol and CH2Cl2
(1:10)). In addition, the secondary amines cyclohexylamino
(19), cyclopropylamino (20), and even 2-hydroxyethylamino
group (21) could serve as substituents on the methylene group
at the 5-position of the pyridine ring. This is consistent with
our docking model in which the R1 group is exposed mostly to
solvent, and there is space to accommodate a variety of groups.
This feature makes this position ideal for substitutions that
modify the physical and pharmacokinetic (PK) properties of
these analogues. In general, this series is more potent for Mer
than the other TAM kinases, and some are quite Mer-specific.
For example, 10 is 160-fold more active for Mer versus Axl and
60-fold versus Tyro3.
cyclohexyl substituent in compound 3 to produce analogue 22
dramatically decreased Mer activity (74-fold), while introducing
an amino group at the same position of the cyclohexyl ring
increased the activity of analogue 23. Even an aminomethyl
group at this position yielded a similarly active analogue 24.
However, introducing an aniline group at the R2 position
resulted in a 10-fold less inhibitory analogue (25 versus 23). A
secondary amino group was less tolerated in analogues 26 and
27. In addition, the cyclohexyl ring itself may have favorable
hydrophobic interactions with the protein. When the ring size
was reduced to a five- or four-membered ring, the activities of
the corresponding analogues decreased (28 and 29) and the
open-chained analogues 30 and 31 were further reduced in
activity. In addition, the proton on the amino group which
connects the substituents at the R2 site to the pyrimidine ring
played a key role in our design, as it forms an intramolecular
hydrogen bond with the 2-pyridinyl maintaining the overall
conformation of the molecule. Without this proton (32), the
Mer activity was completely lost. Again, several of the analogues
in Table 2 are quite Mer-selective (e.g., 23 has 300-fold
selectivity for Mer over Axl and 50-fold over Tyro3).
Next, the SAR at the R2 site was explored (Table 2). As
predicted from the docking model, the polar group on the R2
site is critical and can form a hydrogen bond with the carbonyl
of Glu595. For example, removing the hydroxyl group from the
9686
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
Table 2. SAR Study of R2 Site
Table 3. Further SAR Study of R3
a
Values are the mean of two or more independent assays.
Analogues were also synthesized to investigate the SAR at
the R3 position using the synthetic route shown in Scheme 2
(Table 3). On the basis of the X-ray crystal structure of 1 in
complex with the Mer protein (Figure 1A), the NH from the
butylamino side chain forms a hydrogen bond with the hinge
region of the Mer protein. When a sulfur atom was placed at
this position, the corresponding analogue 33 was much less
active than analogue 34. This was due to absence of this key
hydrogen bond. Analogue 34 was a less potent Mer inhibitor
compared to 3 because of the diminished hydrophobic
interaction at the R3 site. When the alkyl side chain at the R3
position was made longer, the resulting analogues 35 and 36
gained activity until the side chain reached five carbons in
length (37). Analogue 37 was equally potent to 3. A branched
methyl group at the α-position of the NH (38) decreased the
activity slightly. However, the activity dropped dramatically
(22-fold) when the hydrogen on the NH was replaced with a
a
b
Values are the mean of two or more independent assays. Racemate.
methyl group (39), which further confirmed the importance of
the hydrogen bond formed between the NH and the hinge
region of the Mer kinase domain. This effect was more evident
when a larger piperidine group was placed at the R3 position;
the corresponding analogue 40 lost almost all activity against
Mer. A cycloalkyl group at this position had a similar effect as a
branched alkyl side chain; analogue 41 had similar potency to
38. A phenyl ring was also tolerated at the side chain,
comparing analogues 42 to 35 and 43 to 36. In addition, a
polar hydroxyl group at the end of the alkyl side chain
decreased the activity of the resulting analogues because of a
weaker binding caused by a less favorable hydrophobic
9687
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
contribution (44 vs 36, 45 vs 3). When the length of the side
alkyl chain reached four carbons, the corresponding analogue
46 regained its activity comparable to 37, probably because of
the vicinity of the polar hydroxyl group to the solvent front.
Again, these analogues were quite Mer-selective compared to
the pyrazolopyrimdines.
To provide a deeper insight into the binding interactions of
the pyridine−pyrimidine inhibitors and afford structural
guidance for further chemical optimization, a cocrystal structure
of Mer in complex with compound 3 was obtained (Figure 3).
(residues Pro672 and Met674). Similar to the previously
published structure of the Mer/1 complex, the aminobutyl R3
substituent is bent within the adenine subpocket. Unlike the
former structure, the hydroxyl group of the R2 substituent
forms a hydrogen bond with the backbone carbonyl of Glu595
(the analogous R2 amino group of 1 interacts with Arg727).
This difference is most likely because the cyclohexyl group was
reoriented because of the intramolecular H-bond between the
pyridine and 4-amino groups. It is noteworthy that one of the
gate-forming residues, Ile650, is not conserved in the other two
TAM family members. Axl and Tyro3 feature methionine and
alanine, respectively, at this position. This variability is one
likely reason for significant intrafamily selectivity. Consistent
with the SAR outlined above, the R1 substituent interacts
mostly with the solvent and does not significantly impact the
activity. Consequently, it can be utilized for tuning solubility
and PK properties. Nevertheless, the freedom to modify R1 has
its limits, as demonstrated by compound 11, whose bulky
pyridine−morpholine R1 substituent precludes an efficient
interaction with the hinge.
Four potent analogues (10, 14, 16, and 20) with distinctively
different R1 groups were chosen for PK studies (Table 4). The
in vivo PK properties of these compounds were assessed in
mice following intravenous (iv) or oral (po) administration
(Table 4). All four compounds had high systemic clearance
(69−149% of normal liver blood flow in mice). Among them
20 had the longest half-life (4.71 h) but also had an extremely
high volume of distribution (56-fold higher than the normal
volume of total body water in mice) and low plasma
concentrations. Compound 14 had the best oral bioavailability
(55%) but a high volume of distribution (20-fold), while 16 had
a short half-life and low oral exposure. Compound 10 had a
moderate half-life, clearance, and volume of distribution as well
Figure 3. X-ray structure of 3 in complex with Mer kinase domain
(PDB code 4M3Q).
In this structure, the inhibitor 3 demonstrates a binding mode
in which the pyrimidine-2-amine group mimics the interactions
of the cofactor’s adenine with the backbone atoms of the hinge
Table 4. PK Profiles of 10, 14, 16, and 20
a
A dose of 3 mg/kg for both routes.
9688
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
Figure 4. Kinase tree.
as reasonable oral bioavailability and good solubility and was
thus chosen for characterization of kinase selectivity and further
evaluation in cell-based studies of Mer activity.
data suggest that the functional antiproliferative activity
mediated by 10 resulted from Mer inhibition rather than a
consequence of off-target inhibition of other kinases.
The inhibitory activity of 10 against 30 kinases (detailed in
Supporting Information) was determined at a concentration
100-fold above its Mer IC50 (Figure 4). The kinase panel
selected emphasized tyrosine kinases along with a selection of
serine/threonine kinases, providing a broad survey of kinase
families. Five out of 30 kinases were inhibited greater than 50%
in the presence of 180 nM 10, and none of the serine/
threonine kinases were appreciably inhibited.
In cell-based assays, 10 mediated inhibition of Mer
phosphorylation in 697 B-ALL cells with an IC50 value of 9.8
nM (Figure 5). Mer phosphorylation was also inhibited in
Colo699 NSCLC cells (Figure 6A). In addition to these effects
on steady-state levels of Mer tyrosine phosphorylation, 10
efficiently inhibited ligand-dependent phosphorylation of a
chimeric protein consisting of the extracellular and trans-
membrane domains of the epidermal growth factor (EGF)
receptor and the intracellular tyrosine kinase domain of Mer
(Figure 6B). Moreover, 10 incubation inhibited colony
formation in soft agar cultures of the BT-12 rhabdoid tumor
and the Colo699 NSCLC cell lines (Figure 7). In the Colo699
NSCLC cell line, the concentrations of 10 required to inhibit
colony formation and Mer phosphorylation were similar. These
CONCLUSIONS
■
A new pyridinepyrimidine scaffold has been discovered and
developed to create potent Mer kinase inhibitors by applying a
structure-based drug design approach using a pseudo-ring
replacement strategy. A cocrystal structure of Mer with one
inhibitor (3) has been obtained and supports the predicted
binding mode where an intramolecular hydrogen bond is used
to induce conformational rigidity. The selectivity and PK
properties of the lead compound 10 are promising, and further
optimization of this series is ongoing. When applied to live
cells, 10 efficiently inhibited both steady state and ligand-
stimulated phosphorylation of Mer. Furthermore, treatment
with 10 was sufficient to reduce colony-forming potential in
both rhabdoid tumor cells and NSCLC cells, confirming
functional antitumor activity mediated by 10 and suggesting the
tractability of this new series to deliver clinically useful agents.
EXPERIMENTAL SECTION
Details on the synthesis of all compounds are given in the Supporting
Information. The purity of all tested compounds was determined by
LC/MS to be >95%.
■
9689
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
Figure 5. 10 inhibits Mer tyrosine kinase activation in acute leukemia
cells. 697 B-ALL cells were treated with the indicated concentrations
of 10 for 1 h. Pervanadate was added to cultures for 3 min to stabilize
the phosphorylated form of Mer. Mer was immunoprecipitated from
cell lysates, and total Mer protein and Mer phosphoprotein were
detected by immunoblot. (A) Representative Western blots. (B)
Relative levels of phospho-Mer and Mer proteins were determined.
Mean values
standard error derived from three independent
Figure 7. 10 inhibits colony formation in solid tumor cell lines. (A)
BT-12 rhabdoid tumor cells were cultured in soft agar containing 10
nM to 3 μM 10 or vehicle only and overlaid with medium containing
10 or vehicle only. Medium and 10 were refreshed twice weekly.
Colonies were stained and counted. Representative plates are shown.
(B) Colo699 NSCLC cells were cultured in 0.35% soft agar overlaid
with medium containing 10 or vehicle. Medium and 10 were refreshed
3 times per week. Colonies were stained and counted. Mean values
SEM derived from four independent experiments are shown.
Statistically significant differences were determined using the Student’s
paired t test ((∗) p < 0.02, (∗∗) p < 0.01, (∗∗∗) P < 0.001 relative to
vehicle only).
experiments are shown. IC50 = 9.8 nM with a 95% confidence interval
of 7.9−12.3 nM.
Table 5. Assay Conditions for MCE Assays
kinase
peptide substrate
kinase (nM) ATP (μM)
Mer
Axl
5-FAM-EFPIYDFLPAKKK-CONH2
5-FAM-KKKKEEIYFFF-CONH2
5-FAM-EFPIYDFLPAKKK-CONH2
2.0
120
10
5.0
65
21
Tyro
Figure 6. 10 inhibits Mer activation in adherent tumor cell lines. (A)
Colo699 NSCLC cells were treated with 10 or vehicle for 72 h
followed by treatment with pervanadate for 3 min. Whole cell lysates
were prepared. Mer protein was immunoprecipitated, and phosphory-
lated and total Mer proteins were detected by Western blot. (B) 32D
cells stably expressing a chimeric receptor consisting of the
extracellular ligand-binding and transmembrane domains of the EGF
receptor and the intracellular kinase domain of Mer were treated with
10 or vehicle for 1 h prior to stimulation for 15 min with 100 ng/mL
EGF. Mer was immunoprecipitated from whole cell lysates and
phosphotyrosine containing and total Mer proteins were detected by
Western blot. Results shown are representative of four (A) or three
(B) independent experiments.
LabChip EZ Reader equipped with a 12-sipper chip. Data were
analyzed using EZ Reader software.
Cell Based Assays for Mer Kinase Inhibition. 697 B-ALL, 32D
cells expressing a chimeric EGFR-MerTK receptor, BT-12 rhabdoid
tumor, or Colo699 NSCLC cells were cultured in the presence of 10
or vehicle only for 1.0 h (697 B-ALL, 32D, BT-12 rhabdoid tumor) or
72 h (Colo699 NSCLC). Pervanadate solution was prepared fresh by
combining 20 mM sodium orthovanadate in 0.9× PBS in a 1:1 ratio
with 0.3% (w/w) hydrogen peroxide in PBS for 15−20 min at room
temperature. Cultures were treated with 120 μM pervanadate prior to
collection for preparation of whole cell lysates, immunoprecipitation of
Mer, and analysis by Western blot.
697 B-ALL and Colo699 NSCLC cells were treated with
pervanadate for 3.0 and 1.0 min, respectively. Cell lysates were
prepared in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM EDTA,
10% glycerol, and 1% Triton X-100, supplemented with protease
inhibitors (Roche Molecular Biochemicals, no. 11836153001). Mer
protein was immunoprecipitated with a monoclonal anti-Mer antibody
(R&D Systems, no. MAB8912) and protein G agarose beads
(InVitrogen). Phospho-Mer was detected by Western blot using a
polyclonal anti-phospho-Mer antibody raised against a peptide derived
from the triphosphorylated activation loop of Mer. Nitrocellulose
membranes were stripped, and total Mer protein was detected using a
Microfluidic Capillary Electrophoresis (MCE) Assay.9a Activity
assays were performed in a 384-well, polypropylene microplate in a
final volume of 50 μL of 50 mM Hepes, pH 7.4, containing 10 mM
MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% bovine serum
albumin (BSA), containing 1.0 μM fluorescent substrate (Table 5) and
ATP at the Km for each enzyme (Table 5). All reactions were
terminated by addition of 20 μL of 70 mM EDTA. After an 180 min
incubation, phosphorylated and unphosphorylated substrate peptides
(Table 5) were separated in buffer supplemented with 1× CR-8 on a
9690
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
second anti-Mer antibody (Epitomics Inc., no. 1633-1). For 697 B-
ALL cells, relatively phosphorylated and total Mer protein levels were
determined by densitometry using Image J software, and IC50 values
were calculated by nonlinear regression.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 919-843-8456. E-mail: xiaodonw@unc.edu.
Confluent BT-12 rhabdoid tumor cultures were treated with
pervanadate for 15 min. Confluent 32D cells expressing EGFR-
MerTK chimeric protein were treated with 100 ng/mL EGF ligand for
15 min and were collected without pervanadate treatment. Cell lysates
were prepared in 20 mM HEPES, pH 7.5, 50 mM sodium flouride, 500
mM NaCl, 5.0 mM EDTA, 10% glycerol, and 1% Triton X-100,
supplemented with protease inhibitors (10 μg/mL leupeptin, 10 μg/
mL phenylmethylsulfonyl fluoride, and 20 μg/mL aprotinin) and
phosphatase inhibitors (50 mM sodium fluoride and 1.0 mM sodium
orthovanadate), and Mer protein was immunoprecipitated using a
polyclonal rabbit anti-Mer antisera raised against a peptide derived
from the C-terminal 100 amino acids of human Mer expressed as AST
protein and protein A agarose beads (Santa Cruz Biotechnology).
Phosphotyrosine-containing proteins were detected by Western blot
with a monoclonal HRP-conjugated anti-phosphotyrosine antibody
(Santa Cruz Biotechnology, no. sc-508). Antibodies were stripped
from membranes, and total Mer levels were determined using a
custom polyclonal rabbit anti-Mer antibody raised against a peptide
derived from the catalytic domain of Mer.
Author Contributions
#W.Z. and D.Z. contributed equally.
Notes
The authors declare the following competing financial
interest(s): D.D., D.K., W.P.J., H.S.E., D.K.G., S.V.F, and
X.W. hold equity in Meryx, Inc.
ACKNOWLEDGMENTS
■
We thank Dr. Andrew McIver for his help on manuscript
writing. This work was supported by the University Cancer
Research Fund and Federal Funds from the National Cancer
Institute, National Institutes of Health, under Contract No.
HHSN261200800001E. The content of this publication does
not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does mention of trade
names, commercial products, or organizations imply endorse-
ment by the U.S. Government.
32D-EMC suspension cultures were treated with the indicated of 10
or vehicle before stimulation with 100 ng/mL EGF (BD Biosciences
no. 354010) for 15 min. Cells were centrifuged at 1000g for 5 min and
washed with 1× PBS. Cell lysates were prepared in 20 mM HEPES
(pH 7.5), 50 mM NaF, 500 mM NaCl, 5.0 mM EDTA, 10% glycerol,
and 1% Triton X-100, supplemented with protease inhibitors (10 μg/
mL leupeptin, 10 μg/mL phenylmethylsulfonyl fluoride, and 20 μg/
mL aprotinin) and phosphatase inhibitors (50 mM NaF and 1.0 mM
sodium orthovanadate), and Mer protein was immunoprecipitated
using the polyclonal rabbit anti-Mer C-terminus antisera and protein A
agarose beads (Santa Cruz Biotechnology). Phosphotyrosine-contain-
ing proteins were detected by Western blot with a monoclonal HRP-
conjugated anti-phosphotyrosine antibody (Santa Cruz Biotechnology,
no. sc-508). Antibodies were stripped from membranes, and total Mer
levels were determined using the custom polyclonal rabbit anti-Mer
antibody raised against a peptide derived from the catalytic domain of
Mer.
Soft Agar Colony Formation Assays. BT-12 rhabdoid tumor
cells (10 000 cells) were cultured in 2.0 mL of 0.35% soft agar
containing 0.5× RPMI medium, 7.5% FBS, and the indicated
concentrations of 10 or DMSO vehicle only and overlaid with 0.5
mL of 1× RPMI medium containing 15% FBS and 10 or DMSO
vehicle only. Medium and 10 or vehicle were refreshed 2 times per
week. Colonies were stained with thiazolyl blue tetrazolium bromide
(Sigma Aldrich, no. M5655) and counted after 3 weeks.
REFERENCES
■
(1) Chen, J.; Carey, K.; Godowski, P. J. Identification of Gas6 as a
ligand for Mer, a neural cell adhesion molecule related receptor
tyrosine kinase implicated in cellular transformation. Oncogene 1997,
14 (17), 2033−2039.
(2) Caberoy, N. B.; Zhou, Y.; Li, W. Tubby and Tubby-like protein 1
are new MerTK ligands for phagocytosis. EMBO J. 2010, 29 (23),
3898−3910.
(3) Caberoy, N. B.; Alvarado, G.; Bigcas, J.-L.; Li, W. Galectin-3 is a
new MerTK-specific eat-me signal. J. Cell. Physiol. 2012, 227 (2), 401−
407.
(4) (a) Linger, R. M. A.; Keating, A. K.; Earp, H. S.; Graham, D. K.
TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and
Potential Therapeutic Targeting in Human Cancer. In Advances in
Cancer Research; Academic Press: New York, 2008; Vol. 100, pp 35−
83. (b) Linger, R. M. A.; Keating, A. K.; Earp, H. S.; Graham, D. K.
Taking aim at Mer and Axl receptor tyrosine kinases as novel
therapeutic targets in solid tumors. Expert Opin. Ther. Targets 2010, 14
(10), 1073−1090. (c) Brandao, L.; Migdall-Wilson, J.; Eisenman, K.;
Graham, D. K. TAM receptors in leukemia: expression, signaling, and
therapeutic implications. Crit. Rev. Oncogen. 2011, 16 (1−2), 47−63.
(5) (a) Graham, D. K.; Salzberg, D. B.; Kurtzberg, J.; Sather, S.;
Matsushima, G. K.; Keating, A. K.; Liang, X.; Lovell, M. A.; Williams,
S. A.; Dawson, T. L.; Schell, M. J.; Anwar, A. A.; Snodgrass, H. R.;
Earp, H. S. Ectopic expression of the proto-oncogene Mer in pediatric
T-cell acute lymphoblastic leukemia. Clin. Cancer Res. 2006, 12 (9),
2662−2269. (b) Yeoh, E. J.; Ross, M. E.; Shurtleff, S. A.; Williams, W.
K.; Patel, D.; Mahfouz, R.; Behm, F. G.; Raimondi, S. C.; Relling, M.
V.; Patel, A.; Cheng, C.; Campana, D.; Wilkins, D.; Zhou, X.; Li, J.;
Liu, H.; Pui, C. H.; Evans, W. E.; Naeve, C.; Wong, L.; Downing, J. R.
Classification, subtype discovery, and prediction of outcome in
pediatric acute lymphoblastic leukemia by gene expression profiling.
Cancer Cell 2002, 1 (2), 133−143. (c) Linger, R. M.; Lee-Sherick, A.
B.; Deryckere, D.; Cohen, R. A.; Jacobsen, K. M.; McGranahan, A.;
Colo699 NSCLC cells (15 000 cells) were cultured in 1.5 mL of
0.35% soft agar containing 1× RPMI medium and 10% FBS and
overlaid with 2.0 mL of 1× RPMI medium containing 10% FBS and
the indicated concentrations of 10 or DMSO vehicle only. Medium
and 10 or vehicle were refreshed 3 times per week. Colonies were
stained with nitrotetrazolium blue chloride (Sigma Aldrich, no.
N6876) and counted after 2 weeks.
ASSOCIATED CONTENT
■
Brandao, L.; Winges, A.; Sawczyn, K. K.; Liang, X.; Keating, A. K.;
̃
S
* Supporting Information
Tan, A. C.; Earp, H. S.; Graham, D. K. Mer receptor tyrosine kinase is
a therapeutic target in pre-B cell acute lymphoblastic leukemia. Blood
2013, 122, 1599−1609.
Experimental details, characterization of all compounds, and
selectivity profiling. This material is available free of charge via
the Internet at http://pubs.acs.org.
(6) Linger, R. M.; Cohen, R. A.; Cummings, C. T.; Sather, S.;
Migdall-Wilson, J.; Middleton, D. H.; Lu, X.; Baron, A. E.; Franklin, W.
A.; Merrick, D. T.; Jedlicka, P.; Deryckere, D.; Heasley, L. E.; Graham,
D. K. Mer or Axl receptor tyrosine kinase inhibition promotes
apoptosis, blocks growth and enhances chemosensitivity of human
non-small cell lung cancer. Oncogene 2013, 32, 3420−3431.
Accession Codes
The atomic coordinates for the X-ray crystal structure of 3 have
been deposited with the RCSB Protein Data Bank under the
accession code 4M3Q.
9691
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692

Journal of Medicinal Chemistry
Article
(7) Liu, J.; Zhang, W.; Stashko, M. A.; DeRyckere, D.; Cummings, C.
T.; Hunter, D.; Yang, C.; Jayakody, C. N.; Cheng, N.; Simpson, C.;
Norris-Drouin, J.; Sather, S.; Kireev, D.; Janzen, W. P.; Earp, H. S.;
Graham, D. K.; Frye, S. V.; Wang, X. UNC1062, a new and potent
Mer inhibitor. Eur. J. Med. Chem. 2013, 65, 83−93.
(8) Schlegel, J.; Sambade, M. J.; Sather, S.; Moschos, S. J.; Tan, A. C.;
Winges, A.; Deryckere, D.; Carson, C. C.; Trembath, D. G.; Tentler, J.
J.; Eckhardt, S. G.; Kuan, P. F.; Hamilton, R. L.; Duncan, L. M.; Miller,
C. R.; Nikolaishvili-Feinberg, N.; Midkiff, B. R.; Liu, J.; Zhang, W.;
Yang, C.; Wang, X.; Frye, S. V.; Earp, H. S.; Shields, J. M.; Graham, D.
K. MERTK receptor tyrosine kinase is a therapeutic target in
melanoma. J. Clin. Invest. 2013, 123 (5), 2257−2267.
(9) (a) Liu, J.; Yang, C.; Simpson, C.; DeRyckere, D.; Van, D. A.;
Miley, M. J.; Kireev, D.; Norris-Drouin, J.; Sather, S.; Hunter, D.;
Korboukh, V. K.; Patel, H. S.; Janzen, W. P.; Machius, M.; Johnson, G.
L.; Earp, H. S.; Graham, D. K.; Frye, S. V.; Wang, X. Discovery of small
molecule mer kinase inhibitors for the treatment of pediatric acute
lymphoblastic leukemia. ACS Med. Chem. Lett. 2012, 3, 129−134.
(b) Wang, X.; Liu, J.; Yang, C.; Zhang, W.; Frye, S.; Kireev, D.
Preparation of Pyrazolopyrimidine Compounds for the Treatment of
Cancer. WO2011146313A1, 2011.
(10) (a) Mathieu, S.; Gradl, S. N.; Ren, L.; Wen, Z.; Aliagas, I.;
Gunzner-Toste, J.; Lee, W.; Pulk, R.; Zhao, G.; Alicke, B.; Boggs, J. W.;
Buckmelter, A. J.; Choo, E. F.; Dinkel, V.; Gloor, S. L.; Gould, S. E.;
Hansen, J. D.; Hastings, G.; Hatzivassiliou, G.; Laird, E. R.; Moreno,
D.; Ran, Y.; Voegtli, W. C.; Wenglowsky, S.; Grina, J.; Rudolph, J.
Potent and selective aminopyrimidine-based B-Raf inhibitors with
favorable physicochemical and pharmacokinetic properties. J. Med.
Chem. 2012, 55 (6), 2869−2881. (b) Zhang, G.; Ren, P.; Gray, N. S.;
Sim, T.; Liu, Y.; Wang, X.; Che, J.; Tian, S.-S.; Sandberg, M. L.;
Spalding, T. A.; Romeo, R.; Iskandar, M.; Chow, D.; Martin Seidel, H.;
Karanewsky, D. S.; He, Y. Discovery of pyrimidine benzimidazoles as
Lck inhibitors: part I. Bioorg. Med. Chem. Lett. 2008, 18 (20), 5618−
5621. (c) Furet, P.; Caravatti, G.; Guagnano, V.; Lang, M.; Meyer, T.;
Schoepfer, J. Entry into a new class of protein kinase inhibitors by
pseudo ring design. Bioorg. Med. Chem. Lett. 2008, 18 (3), 897−900.
(11) Chen, K.; Peterson, R.; Math, S. K.; LaMunyon, J. B.; Testa, C.
A.; Cefalo, D. R. Lithium trihydroxy/triisopropoxy-2-pyridylborate
salts (LTBS): synthesis, isolation, and use in modified Suzuki−
Miyaura cross-coupling reactions. Tetrahedron Lett. 2012, 53 (36),
4873−4876.
(12) Wang, L.; Cui, X.; Li, J.; Wu, Y.; Zhu, Z.; Wu, Y. Synthesis of
biaryls through a one-pot tandem borylation/Suzuki−Miyaura cross-
coupling reaction catalyzed by a palladacycle. Eur. J. Org. Chem. 2012,
595−603.
(13) (a) Pommereau, A.; Pap, E.; Kannt, A. Two simple and generic
antibody-independent kinase assays: comparison of a bioluminescent
and a microfluidic assay format. J. Biomol. Sceening 2004, 9 (5), 409−
416. (b) Dunne, J.; Reardon, H.; Trinh, V.; Li, E.; Farinas, J.
Comparison of on-chip and off-chip microfluidic kinase assay formats.
Assay Drug Dev. Technol. 2004, 2 (2), 121−129. (c) Bernasconi, P.;
Chen, M.; Galasinski, S.; Popa-Burke, I.; Bobasheva, A.; Coudurier, L.;
Birkos, S.; Hallam, R.; Janzen, W. P. A chemogenomic analysis of the
human proteome: application to enzyme families. J. Biomol. Screening
2007, 12 (7), 972−982.
9692
dx.doi.org/10.1021/jm401387j | J. Med. Chem. 2013, 56, 9683−9692