Discovery of CX-6258. A potent, selective, and orally efficacious pan-pim kinases inhibitor

Article abstract of DOI:10.1021/ml200259q

Structure-activity relationship analysis in a series of 3-(5-((2-oxoindolin-3-ylidene)methyl)furan-2-yl)amides identified compound 13, a pan-Pim kinases inhibitor with excellent biochemical potency and kinase selectivity. Compound 13 exhibited in vitro synergy with chemotherapeutics and robust in vivo efficacy in two Pim kinases driven tumor models.

Full text of DOI:10.1021/ml200259q

Letter
pubs.acs.org/acsmedchemlett
Discovery of CX-6258. A Potent, Selective, and Orally Efficacious pan-
Pim Kinases Inhibitor
Mustapha Haddach,* Jerome Michaux, Michael K. Schwaebe, Fabrice Pierre, Sean E. O’Brien,
Cosmin Borsan, Joe Tran, Nicholas Raffaele, Suchitra Ravula, Denis Drygin, Adam Siddiqui-Jain,
Levan Darjania, Ryan Stansfield, Chris Proffitt, Diwata Macalino, Nicole Streiner, Joshua Bliesath,
May Omori, Jeffrey P. Whitten, Kenna Anderes, William G. Rice, and David M. Ryckman
Cylene Pharmaceuticals Inc., 5820 Nancy Ridge Drive, Suite 200, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Structure−activity relationship analysis in a series of 3-
(5-((2-oxoindolin-3-ylidene)methyl)furan-2-yl)amides identified com-
pound 13, a pan-Pim kinases inhibitor with excellent biochemical
potency and kinase selectivity. Compound 13 exhibited in vitro
synergy with chemotherapeutics and robust in vivo efficacy in two Pim
kinases driven tumor models.
KEYWORDS: CX-6258, Pim-1, Pim-2, Pim-3, MV-4-11, PC3
im kinases (Provirus Integration site for Moloney murine
leukemia virus) are a family of serine/threonine kinases
development of Pim-2 selective inhibitors still lags behind
despite the high sequence similarity of the ATP-binding sites of
Pim-1 and Pim-2. Only a small number of Pim-2 kinase
inhibitors have been reported, and they inhibit the Pim-2 kinase
at double-digit nanomolar IC50 levels. No selective Pim-3
kinase inhibitors have been described in the patent or open
literature. To date, only two reports32,33 have described
inhibitors of all three Pim kinases; however, their in vivo
anticancer activities remain to be disclosed. Herein, we report
our findings of a novel class of oxindole-based derivatives that
selectively inhibit all three Pim kinases and show robust in vivo
anticancer activity in Pim kinase related xenograft models.
High-throughput screening (HTS) of an internal compound
collection identified 5 (Table 1), a molecule containing an
oxindole group, as an inhibitor of Pim-1 kinase (IC50 = 386
nM). From the same collection of compounds, we discovered
that replacing the hydrogen bond donor in the lactam NH of
compound 5 with a methyl group led to loss of Pim-1
inhibitory activity (compound 6, 21% inhibition at 500 nM
ATP concentration, Table 1), indicating the importance of this
hydrogen bond interaction with the Pim-1 enzyme. To
determine the orientation of compound 5 within the Pim-1
ATP active site, we hypothesized that the lactam NH and
carbonyl of compound 5 might be interacting with Lys67 and
Asp186 in Pim-1 (Figure1). In this proposed binding model of
compound 5, the carboxylate moiety was deemed an
appropriate position to incorporate polar groups, such as
aliphatic basic amines. This change was predicted to improve
the kinase potency due to the possible additional interactions of
the polar groups with Asp128 and/or Glu171.
P
that regulate cell survival.1,2 This family of kinases is composed
of three different isoforms (Pim-1, Pim-2, and Pim-3) that
share 60−70% sequence identity in their kinase domains.3,4
The Pim kinases are tightly regulated at the level of
transcription and translation,5 and their expression is mediated
by the JAK/STAT signaling pathway, which is activated by
various cytokines and hormones.6 In addition, several
oncogenes, including Flt3-ITD and Bcr/Abl, were shown to
upregulate the expression of Pims.7 The Pim kinase family
members are considered oncogenes and have been implicated
in tumorigenesis either alone or operating synergistically with c-
Myc.8−10 Pim kinases are known to suppress apoptosis by the
direct phosphorylation and inhibition of pro-apoptotic Bcl-2
antagonist of cell death (BAD).11−13 Overexpression of Pim
kinases has been reported in leukemia and lymphoma
tumors.3,14 They were also found to be overexpressed in solid
tumors, including pancreatic cancer and prostate can-
cer.8−10,13,15−20
Recent evidence reveals the overlapping and compensatory
nature of Pim-1, Pim-2, and Pim-3 functions, highlighting the
importance of inhibiting all three Pim kinase isoforms.21 Mice
mutants for all three Pim kinases are viable and have minimal
phenotype differences,21,22 suggesting that pan-Pim kinase
inhibitors should possess favorable toxicity profiles. Moreover,
the distinct ATP binding pocket in the active site of Pim
kinases should allow for the design of specific and selective
inhibitors. In light of these findings, the simultaneous inhibition
of Pim-1, Pim-2, and Pim-3 kinases is emerging as a promising
strategy for anticancer drug development.
Several structurally distinct chemotypes of inhibitors have
been described.23−31 However, the majority of these molecules
were reported to be only potent inhibitors of Pim-1 kinase. The
Received: October 31, 2011
Accepted: December 27, 2011
Published: December 27, 2011
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Table 1. SAR around Regions R1, R2, and R3
compd
R1
R2
R3
Pim-1, IC50 (μM)
Pim-2, IC50 (μM)
5
H
Me
H
H
H
H
H
H
H
H
OH
OH
0.386
*
ND
6
H
ND
7
H
2-(pyrrolidin-1-yl)ethanamine
2-(pyrrolidin-1-yl)ethanamine
2-(pyrrolidin-1-yl)ethanamine
2-(pyrrolidin-1-yl)ethanamine
cyclopropylamine
0.043
0.021
0.0744
0.045
0.506
0.025
0.005
0.005
ND
8
1-Cl
1-F
2-Cl
1-F
1-Cl
1-Cl
1-Cl
0.062
0.127
0.445
0.109
0.038
0.025
0.022
9
10
11
12
13
14
3-aminopropan-1-ol
1-methyl-1,4-diazepane
1,4-diazepane
H
a
*
ND = not determined. 21% inhibition at 500 nM conc ATP.
a
Scheme 1. Synthesis of Analogues
a
Conditions: Analogues 7−17; (a) Piperidine, oxindole, EtOH; (b)
CDI, DMF, NHR6R7. Intermediate 3; Y1 = B(OH)2; (c) Oxalyl
chloride, CH2Cl2 and then Et3N, N-methylhomopiperazine; (d) 5-
formylfuran-2-ylboronic acid, Na2CO3, PdCl2(dppf)2, DMF, 80 °C.
Analogues 18 and 19; intermediate 2; Y1 = Br; N,O-dimethylhydroxyl-
amine hydrochloride, amide coupling reagents, CH2Cl2 and then Me-
or Et-MgBr, ether; (c) 3-Methoxycarbonylphenylboronic acid,
Na2CO3, PdCl2(dppf)2, DME, 80 °C; (d) 6 M NaOH, EtOH, rt.
(a) Amide coupling with methyl homopiperazine; (b) Titanium
tetrachloride, THF, 5-chloroxindole, diisopropylethylamine, −78 °C to
rt.
Figure 1. Proposed molecular model of analogue 5. GLU171 and
ASP128 are represented in magenta and pink, respectively.
To probe this hypothesis, we synthesized several compounds
via amide coupling between the carboxylate in compound 5 and
amine reagents. Analogues in Table 1 and analogue 17 were
synthesized by condensing a commercially available intermedi-
ate 3 (3-(5-formylfuran-2-yl)benzoic acid or 4-chloro-3-(5-
formylfuran-2-yl)benzoic acid) with indolin-2-one derivatives,
followed by attachment of various amines to the carboxylate
under a standard amide coupling reaction to give the desired
analogues (Scheme 1).
Addition of various aliphatic basic amines to compound 5 via
amide coupling with the carboxylate in compound 5 led to an
increase in potency at inhibiting Pim-1 kinase. As shown by
representative examples in Table 1, analogue 7 resulting from
the addition of 2-(pyrrolidin-1-yl)ethanamine led to a 9-fold
increase in potency at inhibiting Pim-1 versus analogue 5.
Addition of a chloro group to the C-1 position (analogue 8)
improved further the Pim-1 potency and showed an IC50 of
0.062 μM at inhibiting Pim-2. Replacement of the chloro group
with a fluoro group (analogue 9) at the C-1 position led to a
decrease of potency against both Pim-1 and Pim-2. However, a
chloro group at the C-2 position (analogue 10) resulted in a
significant loss of Pim-2 potency, whereas the Pim-1 potency
loss was only 2-fold, indicating that the chloro group at the C-2
position caused a steric clash within the Pim-2 binding site.
This representative SAR clearly showed the importance of the
basicity of the side chain for Pim-1 potency. Indeed,
replacement of the basic side chain in analogue 9 with
The intermediate 3 used in the synthesis of the analogues in
Table 2 was prepared by applying the palladium-catalyzed cross
coupling reaction to intermediates 1 and 2 (Scheme 1). The
exocyclic double bond is exclusively in the E configuration for
the majority of analogues, as exemplified by the crystal structure
of analogue 13 (Figure 2) and as described previously.34
Analogues 18 and 19 are mixtures of E and Z configurations
1
with 3 to 1 ratio by H NMR, respectively (data not shown).
The analogues described in Scheme 1 were tested in a
radiometric enzyme assay using recombinant human Pim-1 and
Pim-2, and the results are shown in Tables 1 and 2.
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ACS Medicinal Chemistry Letters
Letter
Table 2. SAR of the Core Structure
compd
R4
R5
X1
X2
Pim-1 IC50 (μM)
Pim-2 IC50 (μM)
15
16
17
18
19
H
H
Cl
F
N
N
0.067
0.021
0.003
0.057
0.131
0.225
0.217
0.005
0.038
0.082
H
CH
CH
CH
CH
CH
CH
CH
CH
H
Me
Et
H
H
a
Table 3. Mouse PK Profile of 13 as an Anticancer Agent
IV dosing
oral dosing
AUC
1016
Cls
2.1
Vd
T1/2
%F
23
85
27
a
Units: Cls, L/h/kg; Vd, L/kg; T1/2, h; and AUC(0−24 h), (ng h)/
mL; vehicle, D5W; data reported as an average of four animals.
cellular activity performed with Pim kinases inhibitors. The Flt-
3 IC50 values of analogues 13 and 14 were therefore
determined, and it was found that 13 and 14 inhibited Flt-3
with IC50 values of 0.134 μM and 0.150 μM, respectively. In
addition, the Pim-3 IC50 values of both analogues were
measured. Analogues 13 and 14 inhibited Pim-3 with IC50
values of 0.016 μM and greater than 1 μM, respectively. Since
the Pim-3 isoform is overexpressed in pancreatic, gastric, and
colon cancer,15 analogue 13 could be beneficial as an anticancer
agent against these malignancies.
Taken together, these studies led to the identification of
analogue 13 as an E-isomer (Figure 2) for further investigation.
To assess its selectivity profile, analogue 13 was screened
against 107 kinases (Supporting Information). Using a
concentration of 0.5 μM, only Pim-1, Pim-2, Pim-3, and Flt-3
of the 107 kinases were inhibited by more than 80%,
demonstrating excellent selectivity of 13. This analogue was
also shown to be a reversible inhibitor of Pim-1 with a Ki of
0.005 μM.
The antiproliferative activity of 13 was examined against a
panel of cell lines derived from human solid tumors and
hematological malignancies. Analogue 13 demonstrated robust
antiproliferative potencies against all cell lines tested. Cell lines
derived from acute leukemias were the most sensitive to 13
(Supporting Information).
In mechanistic cellular assays with MV-4-11 human AML
cells, 13 caused dose dependent inhibition of the phosphor-
ylation of two pro-survival proteins, Bad and 4E-BP1, at the
Pim kinase specific sites S112 and S65 and T37/46, respectively
(Figure 3). Using ELISA against phospho-Flt3 (Y591), we
demonstrated that 13 does not inhibit Flt3 activity in MV-4-11
cells at relevant concentrations (IC50 > 10 μM). These data
indicate that the effect of 13 on phosphorylation of Bad and
4E-BP1 results from direct inhibition of Pim kinases rather than
being a secondary consequence of Flt3 inhibition.
Figure 2. Crystal structure of 13 as the HCl salt coordinated to a water
molecule, demonstrating the exocyclic double bond is exclusively in
the E configuration.
cyclopropylmethanamine (analogue 11) resulted in a 6.8-fold
decrease of Pim-1 potency, whereas Pim-2 potency was not
affected. Introduction of a hydroxy group to the side chain
(analogue 12) restored the Pim-1 potency, providing further
support for the proposed binding hypothesis that polar or basic
groups might be interacting favorably with Asp128 and/or
Glu171 in Pim-1 enzyme. Addition of rigidity to the side chain
resulted in potent inhibition of both Pim-1 and Pim-2
(analogues 13 and 14).
We next examined the SAR of the core structure (Table 2)
and found that pyrazine analogue 15 exhibited lesser potency
than 13 against both enzymes. Introduction of a chloro group
at the R5 position (analogue 16) caused a loss of potency
against both enzymes, signifying the importance of coplanarity
between the phenyl and furan rings of the core structure for
potency in both enzymes. Unexpectedly, introduction of a
fluoro group to the R5 position led to the most potent inhibitor
of Pim-1 and Pim-2. Finally, the SAR around the alkene region
revealed that addition of methyl (analogue 18) led to loss of
potency in Pim-1 while maintaining most of the potency in
Pim-2. However, the addition of an ethyl moiety (analogue 19)
led to a more pronounced loss of potency against both Pim-1
and Pim-2, indicting a steric interaction with both enzymes.
Although analogues 14 and 17 exhibited promising in vitro
activity profiles (data not shown), both analogues displayed
poor oral absorptions. Surprisingly, analogue 13 demonstrated
not only a good in vitro activity profile but also a moderate PK
profile (Table 3). In addition, analogue 14 was identified as one
of the metabolites of 13 (data not shown) and it was expected
to contribute positively to the in vivo activity of 13.
The Pim kinase family offers a great opportunity in
combination therapy, since Pim kinases can modulate chemo-
therapy resistance through suppression of apoptosis and
enhancing the expression of Pgp pumps.39−41 To explore the
Since Flt-3 regulates the Pim kinases in leukemia,35, its level
of inhibition is important information for the interpretation of
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ACS Medicinal Chemistry Letters
Letter
antiproliferative activity. In addition, 13 displays significant in
vivo efficacy in two xenograft models representing the diseases
where Pim kinases have been shown to play an important role.
Inhibition of Pim kinases is expected to have a beneficial effect
as cancer therapy, and compound 13 is undergoing further
preclinical studies to determine its viability for human clinical
trials.
ASSOCIATED CONTENT
■
Figure 3. Effect of 13 on phosphorylation of Pim kinase substrates in
MV-4-11 cells. MV-4-11 cells were treated for 2 h with various doses
of 13. Cells were lysed, and the relative levels of phospho-proteins
were measured using Western hybridization.
S
* Supporting Information
Descriptions of the biological assays, table of the antiprolifer-
ative activity of 13, graph of the in vivo efficacy of 13, and
details of the preparation and characterization of CX-6258. This
material is available free of charge via the Internet at http://
pubs.acs.org.
potential of using 13 in combination with other drugs, analogue
13 was evaluated for its ability to enhance the activity of
chemotherapeutics against prostate adenocarcinoma PC3 cells.
As single agents, 13, doxorubicin, and paclitaxel inhibited the
viability of PC3 cells with the IC50 values equal to 452 nM, 114
nM, and 2.5 nM, respectively. Combinations of 13 with
doxorubicin (10:1 molar ratio) and 13 with paclitaxel (100:1
molar ratio) produced synergistic cell killing with combination
index (CI50) values equal to 0.4 and 0.56, respectively,
supporting the role of Pim kinases in modulating the
chemosensitivity of cancer cells. To our knowledge, this the
first report that described the use of a pan-Pim kinase inhibitor
in combination with chemotherapeutics.
In an in vivo setting, analogue 13 was evaluated in two
human tumor xenograft growth efficacy models, acute myeloid
leukemia MV-4-11, and prostate adenocarcinoma PC3. These
particular models were selected because of the important roles
that Pim kinases were shown to play in both diseases. Mice
carrying MV-4-11 xenografts were administered a single oral
dose daily. The drug exhibited dose dependent efficacy, with a
50 mg/kg dose producing 45% tumor growth inhibition (TGI)
and a 100 mg/kg dose producing 75% TGI (Figure 4).
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mhaddach@san.rr.com or mhaddach@cylenepharma.
com.
Notes
The authors declare no competing financial interest.
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