UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor

Article abstract of DOI:10.1021/jm500749d

We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.

Full text of DOI:10.1021/jm500749d

Article
pubs.acs.org/jmc
Open Access on 07/28/2015
UNC2025, a Potent and Orally Bioavailable MER/FLT3 Dual Inhibitor
Weihe Zhang,† Deborah DeRyckere,∥ Debra Hunter,§ Jing Liu,† Michael A. Stashko,†
Katherine A. Minson,∥ Christopher T. Cummings,∥ Minjung Lee,∥ Trevor G. Glaros,⊥
Dianne L. Newton,⊥ Susan Sather,∥ Dehui Zhang,† Dmitri Kireev,† William P. Janzen,† H. Shelton Earp,‡,§
Douglas K. Graham,∥ Stephen V. Frye,*,†,§ and Xiaodong Wang*,†
†Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman
‡
§
School of Pharmacy, Department of Pharmacology, School of Medicine, and Lineberger Comprehensive Cancer Center,
Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599,
United States
∥Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80045, United States
⊥Biological Testing Branch, Developmental Therapeutics Program, Leidos Biomedical Research, Inc, Frederick National Laboratory
for Cancer Research, Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: We previously reported a potent small molecule Mer tyrosine kinase inhibitor
UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the
sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly
orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral
dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic
blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular
selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an
additional important target in acute myelogenous leukemia (AML), with pharmacologically useful
selectivity versus other kinases examined.
challenging to optimize the DMPK profile for a given
compound while retaining the required pharmacological profile.
This manuscript presents our approach to improve the DMPK
of an in vitro tool compound to generate an orally bioavailable
lead targeting two receptor tyrosine kinases, Mer and the Fms-
like tyrosine kinase 3 (Flt3).
INTRODUCTION
■
Drug metabolism and pharmacokinetics (DMPK) are key
elements to be optimized in drug development. Poor PK
properties have historically been identified as one of the main
contributors to failure in advancing new compounds toward
approval as medicines, along with drug safety issues and lack of
phase II efficacy. On the basis of a survey conducted by the U.S.
Food and Drug Administration (FDA) in 1991, 39% of clinical
failure resulted from unfavorable PK properties of clinical
candidates, including poor bioavailability, high clearance, low
solubility, and difficult formulation.1 Since that time, medicinal
chemists have focused on improvement of DMPK in the early
drug discovery phase, allowing unsuitable compounds to be
filtered out as these properties are optimized. This change was
enabled by major improvements utilizing mass spectrometry of
unlabeled compounds and has been further facilitated by the
introduction of higher throughput in vitro and in vivo DMPK
methodologies as well as in silico modeling techniques to help
predict the effects that structural changes have on individual PK
parameters.2 Consequently, by the year 2000, the attrition rate
of compounds due to poor DMPK dropped to less than 10%.1
Although multiple reports of medicinal chemistry efforts to
improve DMPK properties of selected compounds exist,3 the
process relies heavily on trial and error, and it remains
Mer receptor tyrosine kinase (RTK) belongs to the Tyro3,
Axl, and Mer (TAM) family of RTKs.4 Abnormal expression
and activation of Mer has been implicated in the oncogenesis of
many human cancers,5 including acute lymphoblastic leukemia
(ALL),6 acute myeloid leukemia (AML),7 nonsmall cell lung
cancer (NSCLC),8 melanoma,9 and glioblastoma,10 where Mer
functions to increase cancer cell survival, thereby promoting
tumorigenesis and chemoresistance.7−9,10a,11 Mer has recently
been identified as a potential therapeutic target in leukemia and
several types of solid tumors by demonstration that shRNA-
mediated Mer inhibition abrogated oncogenic phenotypes,
including decreased clonogenic growth, enhanced chemo-
sensitivity, and delayed tumor progression in animal models.
Similarly, activating mutations in Flt3, especially internal
tandem duplications (ITD) in the juxtamembrane domain,
Received: May 14, 2014
Published: July 28, 2014
© 2014 American Chemical Society
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are detected in approximately 30% of adult and 15% of
childhood AMLs.12 In AML, Flt3 ITD is considered to be a
classic oncogenic driver.12 Clinical responses to early Flt3
inhibitors were largely limited to transient reductions in
peripheral blood and bone marrow blasts.13 This has been
attributed to insufficient Flt3 inhibitory activity and high
toxicity of early compounds due to broad spectrum kinase
inhibition.14 Subsequently, enhanced potency Flt3 inhibitors
with more selective kinase inhibitory profiles have been
advanced and have demonstrated significant clinical activity,
though none have been approved to date for the treatment of
AML.14 Since the Mer RTK is aberrantly expressed in ALL, and
widely expressed in non-Flt3 mutant AML, an inhibitor
demonstrating potent activity against both Mer and Flt3 with
selectivity versus other kinases could be widely applicable in
leukemias. A compound with this profile would additionally
provide a chemical tool to assess the degree to which combined
antisurvival and antichemoresistance activity, due to Mer
inhibition, can augment inhibition of an oncogenic driver
such as the Flt3-ITD mutation.
Scheme 1. Pyrrolopyrimidine Analogue 2
imidines to enable late-stage variation at each substituent
position,15a,18 the trans-4-hydroxycyclohexyl group was most
efficiently attached to the nitrogen at the N1 position of the
pyrrole ring early in the synthesis of pyrrolopyrimidines. In
addition, the N-alkylation reaction to introduce a trans-4-
hydroxycyclohexyl group at the N1 position of 1 did not work
for 2. Instead, a Mitsunobu reaction was used to introduce this
substituent. As shown in Scheme 2, commercially available 5-
bromo-2-chloro-7H-pyrrolo[2,3-d]pyrimidine (3) was chosen
as the starting material and was converted to intermediate 5 by
treatment with mono-TBS protected cis-cyclohexane-1,4-diol 4
in the presence of freshly prepared cyanomethylenetrimethyl
phosphorane (CMMP) in 72% yield.18 The SNAr replacement
of the chloride in 5 with butylamine under microwave
irradiation at 150 °C yielded compound 6. Suzuki-Miyaura
coupling reaction of 6 with boronic acid 7 led to compound 8.
Finally, compound 2 was obtained in good yield (36% over 3
steps) following removal of the TBS group from 8 by treatment
with 1% HCl.
PK Property Improvement of Pyrrolo[2,3-d]-
pyrimidines. It was determined that compound 2 had similar
activity and selectivity profiles (IC50’s: Mer, 0.93 nM; Axl, 29
nM; Tyro3, 37 nM; Flt3, 0.69 nM) as 1 within the TAM family.
In addition, 2 had a lower melting point (215.4−216.2 °C)
than 1 (234.2−234.6 °C), which suggested it would have better
solubility.19 Indeed, 2 proved more soluble in DMPK
formulations, and following intravenous (iv) or oral (po)
administration in mice, 2 had better oral exposure as compared
to 1 (Table 1). Mice were chosen for PK studies because they
are an appropriate species for determination of therapeutic
effects in preclinical leukemia models. Taken together, these
data indicated that this scaffold modification approach to
improving the PK properties of 1 was promising. However, the
structure of 2 needed to be fine-tuned to further address PK
limitations such as high clearance.
In order to decrease the metabolic clearance of 2, we
considered modifications at each substituent position to either
decrease log P, increase solubility, or decrease cytochrome
P450 oxidation while retaining Mer/Flt3 potency. Our
hypothesis that P450 oxidation was the dominant route of
clearance was based on prior experience with compounds of
this log P and molecular weight.20 Re-examination of the SAR
of the pyrazolopyrimidine scaffold revealed that a trans-4-
hydroxycyclohexyl group at the N1 position was optimal, while
alternative substitutions at this position either compromised the
Mer potency or introduced undesired hERG activity.15a,b
Therefore, this substituent was fixed. However, SAR in the
pyrazolopyrimidines demonstrated that the butylamine group
at the C6 position could be replaced with other aliphatic groups
RESULTS AND DISCUSSION
■
Pyrrolo[2,3-d]pyrimidine Scaffold Improves DMPK. To
date, there are only a few kinase-targeted compounds that have
been designed intentionally as Mer inhibitors,15 such as
UNC1062 (1),15b while others were developed for different
purposes but have Mer inhibitory activity as part of their kinase
profiles.16 Consequently, none of the latter reported inhibitors
are believed to demonstrate pharmacology primarily related to
Mer inhibition. We previously showed that compound 1 is a
potent Mer inhibitor (IC50 1.1 nM) that blocked Mer
phosphorylation in cell-based assays, including 697 B-ALL,
BT-12 pediatric rhabdoid tumor, NSCLC, and melanoma cell
lines.13b This compound also decreased colony formation in
solid tumor cell lines.9a,15b Surprisingly, kinome profiling
revealed that 1 was also very potent against Flt3 (IC50 3.0
nM) despite the relatively low overall homology between Mer
and Flt3 kinase domains (42% identity) and significant
differences within their ATP binding sites. While Flt3 activity
lessens the utility of this lead as a specific chemical probe for
Mer kinase,17 the potential therapeutic utility of a dual inhibitor
is compelling and warranted further development. Separate
optimization efforts are being focused on development of even
more selective Mer specific compounds. In addition, because of
low solubility and absence of oral exposure, compound 1 was
subjected to further chemical improvement to render it suitable
for in vivo study. Therefore, the solubility and PK properties of
1 were addressed, while its activity and kinome profile were
maintained in order to advance a Mer/Flt3 inhibitor as an agent
to treat AML and ALL.
On the basis of the reported X-ray structure of the UNC569/
Mer complex,15a the N2 nitrogen on the pyrazole ring appears
to make no specific interactions with the Mer protein and
therefore replacement with a carbon to introduce a new core
structure, a pyrrolopyrimidine, was investigated. As shown in
Scheme 1, the corresponding analogue of 1 in the
pyrrolopyrimdine scaffold is 2 and we subsequently discovered
that this modification resulted in improved solubility for
members of this series during formulation for DMPK studies.
Synthesis of Pyrrolo[2,3-d]pyrimidines. Although there
is only one difference (N vs CH) between compounds 1 and 2,
the synthesis of 2 is distinct (Scheme 2). While the reaction
sequence could be varied during the synthesis of pyrazolopyr-
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Scheme 2. Synthesis of 2
Table 1. In Vivo PK Parameters of 1 and 2 (n = 3 Mice Per Time Point)
a
b
iv
po
compound
T1/2 (h)
Cmax (μM)
AUClast (h μM)
Vss (L/kg)
CL (mL/min/kg)
Tmax (h)
Cmax (μM)
AUClast (h μM)
%F
c
1
2.3
16
3.3
0.43
0.78
30
70
0.5
0.013
0.16
0.01
0.12
0.3
8.4
d
2
0.23
7.9
1.4
0.25
a
b
c
d
iv Dose at 3 mg/kg. po Dose at 3 mg/kg. iv Formulation: 7.5% v/v N-methyl pyrrolidone; 20% cremophor EL in water. iv Formulation: 5%
NMP, 5% solutol HS in normal saline.
Figure 1. Structures and enzymatic activity of 9−12.
Table 2. In Vivo Pharmacokinetic Parameters of 9−12 (n = 3 Mice Per Time Point)
iv
po
dose
(mg/kg)
T1/2
(h)
Cmax
(μM)
AUClast
(h μM)
Vss
(L/kg)
CL
dose
(mg/kg)
Tmax
(h)
Cmax
(μM)
AUClast
(h μM)
compound
(mL/min/kg)
% F
a
9
1
3
3
3
0.80
1.2
0.40
1.2
0.27
1.0
5.5
5.3
2.3
2.0
103
76
10
3
0.25
0.25
0.50
1.0
0.39
0.42
1.6
0.66
0.69
9.2
25
67
b
10
c
11
3.8
4.1
9.2
9.2
3
100
c
12
4.4
6.5
12
7.2
3
1.3
9.0
78
a
b
c
iv Formulation: 7.5% v/v N-methyl pyrrolidone; 40% v/v PEG-400 in normal saline. iv Formulation: 5% DMSO, 5% solutol in normal saline. iv
Formulation: normal saline (0.9% NaCl).
and Mer potency retained. Additionally, substituents at the C3
position were positioned toward the solvent front in X-ray
cocrystal structures, and we reasoned that different solubilizing
groups might be well-tolerated at this position.15b We therefore
proceeded to make simultaneous changes in the C3 and C6
positions in order to rapidly explore their combined effect on
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Scheme 3. Scale-up Route for 11
DMPK properties in an economically feasible fashion. This
modification strategy led to analogues 9−12 (Figure 1), which
were synthesized using the synthetic route presented in Scheme
2.
cyclopropyl ethyl side-chain resulted in 12, which demonstrated
a further modest decrease in clearance, consistent with some
contribution of P450 metabolism of the C3 side-chain to
metabolic stability but with very similar overall PK properties to
11. With excellent solubility and PK properties, as well as a
much less expensive C3 substituent versus 12, analogue 11 was
chosen for further studies, including kinome selectivity
profiling, cell-based assays, and pharmacodynamic assays
using a mouse model to determine the activity of the
compound in leukemic blasts in vivo.
Scale-up Route for 11. In vivo studies require gram
quantities of compound, and although the synthetic route
presented in Scheme 2 was successfully applied to prepare
analogs for SAR purposes, it was costly and difficult to perform
on a multigram scale, especially the Mitsunobu reaction. Large-
scale preparation of the CMMP required for this reaction was
also challenging. Therefore, an alternative synthetic route for
the large-scale synthesis of 11 was developed, as shown in
Scheme 3. Starting with readily available 5-bromo-2,4-
dichloropyrimidine (13), compound 14 was obtained in
quantitative yield after an SNAr replacement of the 4-chloro
group with trans-4-aminocyclohexanol. A Sonogashira coupling
reaction between 14 and ethynyltrimethylsilane yielded
intermediate 15, which was converted to intermediate 16 by
in situ deprotection of the TMS group and formation of the
pyrrole ring in 77% yield. To ease purification in the next few
steps, the hydroxyl group of 16 was protected with a TBS
protecting group to yield 17. Bromination of 17 with NBS
provided compound 5 which was converted to 6 via a second
SNAr replacement reaction with butylamine in high yield.
Finally, analogue 11 was obtained by a Suzuki-Miyaura cross
coupling reaction of 6 with 4-(4-methylpiperazino)-
methylphenylboronic acid pinacol ester 18 in the presence of
Similar to 2, these analogues were potent against both Mer
and Flt3 and had some selectivity over Axl and Tyro3.
Compound 9 incorporated a basic, solubilizing N-methyl
piperazine group on the phenyl ring in addition to a 3-fluoro
substituent. As shown in Table 2, this led to increased oral
bioavailability (25%, 3 fold better than 2), although the
clearance of 9 was still high (103 mL/min/kg) (Table 2). To
further improve the metabolic stability of 9, we tried to identify
and block potential metabolic hot spots in the molecule. Two
positions were explored simultaneously: fluorination of the
meta position of the sulfonamide group on the phenyl ring and
replacement of the butyl side chain of 9 by cyclopropyl ethyl to
create analogue 10.21 On the basis of published studies with
simple alkanes, the C−H bond strength of the cyclopropyl ring
exceeds that of the terminal −CH3 of the butyl group in 9 by
approximately 7 kcal/mol, suggesting that this substituent
might result in diminished P450-mediated oxidation liability for
this aliphatic chain.22 Indeed, analogue 10 demonstrated
reduced clearance (76 mL/min/kg) and better oral bioavail-
ability (67% vs 25% for 9). We hypothesized that removing the
sulfonamide group of 9 might be another way to further
increase its solubility and improve PK properties, as
sulfonamides are known to have high melting points relative
to the corresponding amines.23 As a result, analogue 11
(UNC2025) was prepared and demonstrated excellent PK
properties: low clearance (9.2 mL/min kg), longer half-life (3.8
h), and high oral exposure (100%) (Table 2). Furthermore, the
HCl salt of 11 was highly soluble in normal saline (kinetic
solubility: 38 μg/mL, pH = 7.4). Substitution of 11 with a
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Table 3. Carna IC50 of the Top 10 kinases and Met kinase Inhibited by 11
Kinase
FLT3
MER
AXL
TRKA
TRKC
QIK
TYRO3
SLK
NuaK1
KIT
Met
IC50 (nM)
0.35
0.42
0.46
1.0
1.65
0.70
1.67
0.37
4.38
0.34
5.75
0.24
5.83
0.65
6.14
0.36
7.97
0.25
8.18
0.41
364
sequence identity
0.48
Pd(PPh3)4 and K2CO3 followed by deprotection of the TBS
group. Although this reaction sequence is longer compared to
the one shown in Scheme 2, each reaction in this sequence can
be easily scaled up and the overall yield is comparable (25% vs
26%). More than 170 g of 11 have been prepared via this route.
Selectivity Profiling. As most kinase inhibitors are ATP
competitive and bind at a functionally conserved site, an
understanding of selectivity within the context of the kinome is
important for clinical development for two different but
important reasons: (1) it is critical to understand which kinases
are inhibited by in vivo concentrations of a drug candidate and
contribute to the observed pharmacology in order to target the
appropriate patients based on kinase mutational status and
preclinical target validation based on shRNA and other gene-
based approaches. (2) Multikinase inhibitors have demon-
strated significant toxicity, and even though definition of
individual kinase “anti-targets” is not well-developed, broad
spectrum inhibitors are generally undesirable. Of these two
issues, we focused on addressing the kinase pharmacology
attributable to 11 most thoroughly at this stage. As there are
numerous methods available for kinome profiling,24 in vitro and
in cells, we sought to obtain a data set for correlation across in
vitro and cellular assays in order to best predict the
pharmacology that would emerge from the kinome profile of
11.
whether the potency of 11 versus the 8 other kinases in Table 3
(or kinases even less potently inhibited) could contribute
significantly to its pharmacology. To begin to address this
question, we decided to examine how well in vitro potency
translated to inhibition of phospho-protein signaling in cells
where the presence of serum protein, high intercellular
concentrations of ATP, and cellular membrane permeability
can significantly modulate compound activity.
Cellular Kinase Inhibition. In 697 B-ALL cells, 11
mediated potent inhibition of Mer phosphorylation with an
IC50 of 2.7 nM (Figure 2). Similarly, in Flt3-ITD positive
Therefore, the overall kinome profile of 11 was assessed in
duplicate versus 305 kinases at Carna Biosciences using a
microcapillary electrophoresis assay similar to our in house
assay. A concentration of 100 nM was used as it is more than
100-fold above the IC50 determined in our in vitro assay for
Mer kinase and would therefore capture other kinases that
could be partially inhibited when Mer is inhibited by >90%.
Sixty-six kinases were inhibited by more than 50% at this
concentration.25 IC50 values against these kinases were
determined at their ATP Km, and the top 10 kinases inhibited
by 11 are shown in Table 3. Gratifyingly, of the 305 kinases
tested, 11 inhibited Mer and Flt3 with the greatest potency.
Interestingly, the IC50 values against other kinases did not
correlate with a sequence similarity to the Mer protein.26 On
the basis of protein sequence within the kinase domain, Met is
the closest kinase to the TAM family, and many Met inhibitors
also inhibit the TAMs.26 However, 11 was more than 700-fold
less active against Met compared to Mer, while it was equally
potent against Flt3. In addition, the modest degree of selectivity
of 11 for Mer over Axl and Tyro3 in this external profiling was
roughly consistent with the selectivity estimated from our
determination of their Morrison Ki’s [Mer, 0.16 0.06 nM (n
= 9); Axl, 13.3 8.3 nM (n = 4); Tyro3, 4.67 2.82 nM (n =
5); Flt3, 0.59 0.32 nM (n = 4)].27 The in vitro observation
that Mer and Flt3 were the kinases most potently inhibited by
11 was confirmed for Mer kinase in B-ALL 697 cell lysates
using the ATP ActivX probe assay,28 where it demonstrated an
IC50 of 0.05 nM for Mer (although the redundancy of Mer and
Tyro3 peptides does not distinguish these kinases). Despite the
basic differences between these two assays, both indicated that
Mer was a primary target for 11. Reflecting on the selectivity
considerations discussed above, we were curious to assess
Figure 2. 11 Inhibits activation of Mer in acute leukemia cells. 697
Cells were treated with the indicated concentrations of 11 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 (p-Mer) were
detected by immunoblot. (A) Representative Western blots. (B)
Relative levels of p-Mer and Mer proteins were determined by
densitometry. Mean values
standard error derived from 3
independent experiments are shown. IC50 = 2.7 nM with a 95%
confidence interval from 1.8 to 4.2 nM.
Molm-14 acute myeloid leukemia cells, treatment with 11
resulted in decreased phosphorylation of Flt3 with an IC50 of
14 nM (Figure 3). This phospho-protein readout is predictive
of biological consequences attributable to these kinases, as
incubation with 11 resulted in significant inhibition of colony
formation in soft agar cultures of the A549 NSCLC and Molm-
14 AML cell lines, which are known to be dependent on Mer8
and Flt3,29 respectively, for optimal expression of oncogenic
phenotypes (Figure 4). In contrast, a negative control
compound 20, a structurally similar but much weaker Mer
and Flt3 inhibitor (Figure 4C), had no significant effect on
colony-forming potential in either cell line. It is noteworthy that
the correlation of biological effect as compared to phospho-
protein inhibition IC50 correlates most closely for the ITD
driver mutation in Flt3 (EC50 for colony formation inhibition =
IC50 for p-Flt3 inhibition, Figure 4B), while the biological effect
attributed to Mer inhibition is right-shifted as compared to
phospho-protein inhibition (EC50 for colony formation
inhibition > IC50 for p-Mer inhibition, Figure 4A). Since it is
difficult to reject the hypothesis that this right-shift is due to the
need to inhibit a kinase other than Mer in the NSCLC colony
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difference in activity for Mer relative to Axl and Tyro3 in Carna
IC50 profiling (Table 3) translated to 40- to 100-fold selectivity
for Mer over Axl and Tyro3, respectively, in phospho-protein
readouts in cell-based assays. The simplest explanation for this
difference in fold selectivity is that the Mer IC50 (0.46 nM)
slightly underestimates the true potency of 11 as seen when the
method of Morrison is used to determine its Ki, which is equal
to 160 picomolar, while the Carna IC50’s are more reflective of
the potency of 11 versus Flt3, Axl, and Tyro3. In fact, a plot of
in vitro potency versus cellular phospho-protein potency that
utilizes the Mer Ki and the Carna IC50’s for Flt3, Axl, and Tyro3
(Figure 6) results in a correlation coefficient (R2) of 0.98 with a
predicted 50-fold shift in potency in the cellular assay
environment relative to Carna IC50’s. With this correlation in
mind, we proceeded to evaluate how effectively 11 could inhibit
Mer phosphorylation in vivo in order to establish pharmacody-
namic evidence of target engagement and assess the drug
concentrations required and how they relate to potential
engagement of other kinase targets.
Pharmacodynamic Evaluation. To determine whether 11
can mediate inhibition of target proteins in vivo, we generated
mice with human leukemia xenografts. In these mice, a single 3
mg/kg dose of 11 administered orally was sufficient to decrease
Mer phospho-protein levels in bone marrow leukemia cells by
greater than 90% (Figure 7). The plasma concentration of 11 at
the time of bone marrow collection can be estimated to be
approximately 1.6 μM based on the PK data shown in Table 2.
In order to relate this concentration to the IC50 versus p-Mer,
we determined the plasma protein binding of 11 in mice to be
98.6% 0.4% (n = 3), resulting in a free fraction concentration
of approximately 22 nM, 30 min after a 3 mg/kg oral dose. As
this is roughly 10-fold above the cellular IC50 versus p-Mer
(Figure 2), inhibition of p-Mer in vivo by >90% at a 3 mg/kg
oral dose is consistent with expectations at a 30 min time point
after dosing (see the Supporting Information for calculation of
% I versus free fraction based on Ki and cellular potency for
11).
Figure 3. 11 Inhibits activation of Flt3 in acute leukemia cells. Flt3-
ITD positive Molm-14 cells were treated with the indicated
concentrations of 11 for 1 h. Pervanadate was added to cultures for
3 min to stabilize the phosphorylated form of Flt3. Flt3 was
immunoprecipitated from cell lysates and total Flt3 protein and Flt3
phosphoprotein (p-Flt3) were detected by immunoblot. (A)
Representative Western blots. (B) Relative levels of p-Flt3 and Flt3
proteins were determined by densitometry. Mean values standard
error derived from 3 independent experiments are shown. IC50 = 14
nM with a 95% confidence interval from 8.4 to 24 nM.
formation assay, we decided to further explore the potency of
11 versus other kinases appearing in the selectivity data of
Table 3.
Because of their high degree of similarity to Mer, our overall
interest in the TAM family, and the significant inhibition by 11
in enzymatic assays, Axl and Tyro3 were chosen from Table 3
as sentinel kinases to further evaluate the selectivity of 11 in
cell-based phospho-protein assays. In order to facilitate this
comparison in a systematic fashion, chimeric proteins
consisting of the extracellular and transmembrane domains
from the epidermal growth factor receptor (EGFR) and the
intracellular domain from Mer, Axl, or Tyro3 were expressed in
32D cells such that all three proteins could be identically
stimulated with the EGF ligand for direct comparison. In this
system, 11 mediated potent inhibition of the chimeric Mer
protein with an IC50 of 2.7 nM (Figure 5), identical to its
activity against endogenous Mer in 697 cells and consistent
with the validity of this system for evaluation of selectivity. In
contrast, much higher concentrations of 11 were required to
effectively inhibit phosphorylation of Axl (IC50 = 122 nM) and
Tyro3 (IC50 = 301 nM). Thus, the approximately 4- to 13-fold
These data for in vivo inhibition of p-Mer enable an estimate
of the kinome pharmacology profile of 11 in vivo using the
following assumptions: (1) in vitro potency translates to
cellular phospho-protein potency for all kinases in the same
way as for the TAMs and Flt3 (Figure 6); (2) pharmacological
effects in vivo resulting from inhibition of a particular kinase
require >90% inhibition of phospho-protein signaling from that
kinase; (3) selectivity estimated at Cmax is indicative of overall
pharmacological selectivity. Figure 8 is a ranked plot of the
Figure 4. 11 Inhibits colony formation in Mer-dependent and Flt3-dependent tumor cell lines. (A) A549 NSCLC cells or (B) Molm-14 AML cells
were cultured in 0.35% soft agar overlaid with medium containing 11, a negative control (20) (300 nM for A549 NSCLC cells and 50 nM for Molm-
14 AML cells), or vehicle. Medium and compounds were refreshed 3 times per week. Colonies were stained and counted. Mean values standard
error derived from 3 to 4 independent experiments are shown. Statistically significant differences were determined using the student’s paired t test (*
p < 0.05, ** p ≤ 0.005, ***P < 0.0005 relative to vehicle only). (C) Structure and enzymatic IC50’s of compound 20.
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Figure 5. 11 Selectively inhibits Mer in cell-based assays. 32D Cells stably expressing chimeric receptors consisting of the extracellular ligand-binding
and transmembrane domains of the EGF receptor and the intracellular kinase domain of Mer, Axl, or Tyro3 were treated with 11 or vehicle for 1 h
prior to stimulation for 15 min with 100 ng/mL EGF. Chimeric proteins were immunoprecipitated from whole cell lysates and phospho-tyrosine-
containing and total proteins were detected by Western blot. (A) Representative Western blots are shown. (B and C) Phosphorylated and total
protein levels were determined by densitometry. Mean values standard error derived from 3 independent experiments are shown. IC50 values and
95% confidence intervals were determined by nonlinear regression and are 2.7 nM (1.7−4.2 nM) for Mer, 122 nM (64−230 nM) for Axl, and 301
nM (110−820 nM) for Tyro3.
Figure 7. 11 Inhibits Mer phosphorylation in bone marrow leukemia
cells in vivo. NOD/SCID/gamma mice were transplanted with 697
acute leukemia cells and allowed to engraft for 14 days. Leukemic mice
were then treated with a single 3 mg/kg dose of 11 or saline vehicle
Figure 6. Correlation of in vitro and phospho-protein potency for 11
versus Mer, Flt3, Axl, and Tyro3 utilizing the Morrison Ki for Mer and
the IC50’s from Table 3 for all others.
administered by oral gavage. Femurs were collected 30 min later. Bone
marrow cells were flushed and incubated for 10 min in the presence of
kinases predicted to be most potently inhibited by 11 versus
the free concentration required for 90% inhibition. This rank
order differs from that of Table 3 due to the effect of varying
ATP Km’s on the predicted inhibition of each kinase in vivo.
For example, KIT has a particularly high Km for ATP (370 μM)
and is therefore predicted to be relatively easy to inhibit. At an
oral dose of 3 mg/kg, 11 results in a Cmax at 30 min of 22 nM
(vertical line in Figure 8). Only Flt3 and Mer are inhibited by
90%, while TRKA, KIT, and Axl kinases are predicted to be
partially inhibited at this dose. Tyro3 and the other kinases are
minimally inhibited and would require free concentrations at
least 10-fold higher for >90% inhibition to occur. Therefore, the
pharmacology of 11 is most likely to be dominated by effects
on Mer and Flt3, while Axl pharmacology can serve as an
indicator of potential activity emerging from partial inhibition
of other kinases such as KIT and TRKA. With the use of the
data from a recent study of the selectivity of nine clinically
approved RTK inhibitors for comparison,25 11 falls in the
20% FBS and pervanadate phosphatase inhibitor to stabilize Mer
phosphoprotein. Cell lysates were prepared and Mer was immuno-
precipitated. (A) Phosphorylated and total Mer proteins were detected
by Western blot. (B) Phosphorylated and total Mer protein levels were
determined by densitometry. Mean values standard error are shown.
Mer phosphoprotein was significantly decreased in leukemia cells
collected from mice treated with 11 relative to mice treated with
vehicle (0.07
unpaired t test; n = 6).
0.04 versus 1.00
0.29; *(* p = 0.01, student’s
midground of selectivity profiles, being more selective than
dasatinib, less selective than imatinib, and similar to pazopanib
in terms of the number of other kinases inhibited when the
most potently inhibited kinases are >90% inhibited (see
Supporting Information for comparisons). Additionally, the
unique rank order of kinases inhibited by 11 provides the basis
for differentiation compared to these other RTK inhibitors,
both for effectiveness when targeting Mer and Flt3 and perhaps
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spectrometric analysis, peptides were resuspended in 25 μL of 0.1%
TFA. Details on mass spectrometry analysis and data analysis are
provided in the Supporting Information.
Cell-Based Assays for Kinase Inhibition. 697 B-ALL cells and
Molm-14 AML cells were cultured in the presence of 11 or vehicle-
only for 1.0 h. 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 for 3 min prior to
collection, and 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 Biochem-
icals, no. 11836153001). Mer and Flt3 proteins were immunopreci-
pitated with anti-Mer (R&D Systems, no. MAB8912) or anti-Flt3
(Santa Cruz Biotechnology no. sc-480) antibody and Protein G
agarose beads (InVitrogen). Phospho-proteins were detected by
Western blot using an antiphospho-Mer antibody raised against a
peptide derived from the triphosphorylated activation loop of Mer8
(Phopshosolutions, Inc.) or an antibody specific for phosphorylated
Flt3 (Cell Signaling Technology, no. 3461). Nitrocellulose membranes
were stripped and total proteins were detected using a second anti-Mer
antibody (Epitomics Inc., no. 1633-1) or anti-Flt3 antibody (Santa
Cruz Biotechnology no. sc-480). Relative phosphorylated and total
protein levels were determined by densitometry using ImageJ, and
IC50 values were calculated by nonlinear regression.
32D Cells expressing a chimeric EGFR-Mer, EGFR-Axl, or EGFR-
Tyro3 receptor were cultured in the presence of 11 or vehicle-only for
1.0 h 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). Mer protein was immunoprecipitated using a
custom polyclonal rabbit antisera raised against a peptide derived from
the C-terminal catalytic domain of Mer and Protein A agarose beads
(Santa Cruz Biotechnology). Axl and Tyro3 proteins were
immunoprecipitated using an antibody directed against a FLAG
epitope engineered into the chimeric proteins (Sigma-Aldrich, no.
F1804). Phosphotyrosine-containing proteins were detected by
Western blot with a monoclonal HRP-conjugated antiphosphotyrosine
antibody (Santa Cruz Biotechnology, no. sc-508). Antibodies were
stripped from membranes, and total proteins were detected with the
same antibodies used for immunoprecipitation.
Figure 8. Predicted free concentration of 11 required in vivo for 90%
inhibition of the top 10 kinases from Table 3. The vertical line
corresponds to the measured free concentration of 11 at 30 min
following a 3 mg/kg oral dose (Cmax, Table 2). (See the Supporting
Information for methods and comparison to clinically approved RTK
profiles assessed in the same manner.)
the side effect profile. While further efficacy studies and DMPK
assessments in treated mice will be needed to fully validate the
kinome basis for the preclinical pharmacology of 11, this
analysis provides a transparent and quantitative basis for study
design and hypothesis testing.
In conclusion, we have successfully generated a potent,
pharmacologically selective, and orally bioavailable Mer/Flt3
dual inhibitor 11 with improved solubility and DMPK
properties relative to a previous in vitro tool compound 2.
Importantly, oral treatment with 11 resulted in effective target
inhibition in bone marrow leukemia cells in an animal model. A
cost-effective synthetic route for preparation of 11 was also
developed to support in vivo preclinical studies.
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 and NMR to be >95%.
Kinome Profiling Using ActivX ATP/ADP Probes. Cellular
lysate, inhibitor treatment, labeling reactions, digestion, and peptide
capture were performed according to manufacturer’s published
protocols with modifications detailed below. Briefly, 697 B-ALL cells
were gently pelleted, washed twice with PBS, lysed using MPER
supplemented with HALT protease/phosphatase inhibitor cocktail
(Pierce), and subjected to Zeba (Pierce) gel filtration spin columns to
remove residual ATP and ADP. Following filtration, the final protein
concentration was adjusted to 5.0 mg/mL using reaction buffer and
supplemented with additional 1X HALT protease and phosphatase
inhibitor cocktail. Lysate was aliquoted, snap frozen in liquid nitrogen,
and stored at −80 °C until labeling. Prior to labeling, 2.5 mg of total
lysate (final volume, 500 μL) was thawed to room temperature and
treated with 10 μL of 1 M MnCl2 for 1 min. Then the lysate was
treated with or without 11 [0, 0.01, 0.1, 1.0, 10, 100, and 1000 nM] for
10 min. Following treatment, the ATP probe was added for 10 min at
a final concentration of 5 μM. The labeling reaction was quenched
with 500 μL of 10 M urea in MPER, 10 μL of 500 mM DTT, and
heated to 65 °C for 30 min with shaking. Samples were cooled to
room temperature and alkylated with 40 μL of a 1 M iodoacetamide
solution for 30 min protected from light. The solution was then
subjected to Zeba (Pierce) gel filtration and digested with 20 μg of
trypsin at 37 °C for 2 h with shaking. 50 μL of a 50% high capacity
streptavidin agarose slurry was added and allowed to incubate for 1 h
at room temperature with constant mixing on a rotator. Agarose beads
were then captured, washed, and eluted. Purified peptides were frozen,
lyophilized, and stored at −80 °C. Immediately before mass
Soft Agar Colony Formation Assays. A549 or Molm-14 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 11 or DMSO
vehicle only. Medium and 11 or vehicle were refreshed 3 times per
week. Colonies were stained with nitrotetrazolium blue chloride
(Sigma-Aldrich, no. N6876) and counted after 2 weeks.
Pharmacodynamic Studies. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
(NSG) mice were transplanted with 2 × 106 697 B-ALL cells by
intravenous injection into the tail vein, and leukemia was established
for 14 days prior to treatment with a single dose of 3 mg/kg 11 or an
equivalent volume (10 mL/kg) of saline vehicle. Pervanadate solution
was prepared fresh, as described above. Femurs were collected from
mice 30 min after treatment, and bone marrow cells were flushed with
1 mL of room temperature RPMI medium + 20% FBS + 1 μM MgCl2
+ 100 untis/ml DNase + 240 μM pervanadate and incubated at room
temperature in the dark for 10 min. Bone marrow cells were collected
by centrifugation at 4 °C, lysates were prepared, Mer protein was
immunoprecipitated, and total and phospho-Mer proteins were
detected and quantitated by Western blot, as described above.
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and characterization of all compounds and
biological methods. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Authors
*Tel: 919-843-5486. E-mail: svfrye@email.unc.edu.
*Tel: 919-843-8456. E-mail: xiaodonw@unc.edu.
Notes
The authors declare the following competing financial
interest(s): D.K., W.P.J., H.S.E., D.K.G., S.V.F., and X.W.
have stock in Meryx, Inc.
■
ACKNOWLEDGMENTS
■
We thank Dr. Nancy Cheng, Ms. Wendy M. Stewart, and Ms.
Yingqiu Zhou for their help with MCE assays. This work was
supported by the University Cancer Research Fund and Federal
Funds from the National Cancer Institute, National Institute of
Health, under Contract HHSN261200800001E. Additional
support was provided by NIH 1R01CA137078 (D.G.) and the
BCRF (H.S.E.). 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 endorsement by
the U.S. Government.
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