A versatile synthesis of novel pan-PIM kinase inhibitors with initial SAR study

Article abstract of DOI:10.1016/j.tetlet.2015.01.119

Herein, we describe the versatile synthesis of (Z)-5-((2-aminopyrimidin-4-yl)methylene)thiazolidine-2,4-dione inhibitors (1) of the PIM family of kinases. This chemistry strategy was a key element in the multi-variable optimization program with the goal of identifying high quality leads for the development of a treatment for cancer.

Full text of DOI:10.1016/j.tetlet.2015.01.119

Accepted Manuscript
A versatile synthesis of novel pan-PIM kinase inhibitors with initial SAR study
Yvonne Flanders, Stephane Dumas, Justin Caserta, Robb Nicewonger, Michael
Baldino, Chee-Seng Lee, Carmen M. Baldino
PII:
S0040-4039(15)00157-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.01.119
TETL 45789
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 November 2014
14 January 2015
19 January 2015
Please cite this article as: Flanders, Y., Dumas, S., Caserta, J., Nicewonger, R., Baldino, M., Lee, C-S., Baldino,
C.M., A versatile synthesis of novel pan-PIM kinase inhibitors with initial SAR study, Tetrahedron Letters (2015),
doi: http://dx.doi.org/10.1016/j.tetlet.2015.01.119
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Graphical Abstract
A versatile synthesis of novel pan-PIM
kinase inhibitors and initial SAR study
Leave this area blank for abstract info.
Yvonne Flanders, Stephane Dumas, Justin Caserta, Robb Nicewonger, Michael Baldino, Chee-Seng Lee, and
Carmen M. Baldino*
R2
N
O
N
R1
NH
N
S
O
1

1
Tetrahedron Letters
journal homepage: www.els evier.com
A versatile synthesis of novel pan-PIM kinase inhibitors with initial SAR study
Yvonne Flandersa, Stephane Dumasa, Justin Casertaa, Robb Nicewongerb, Michael Baldinoa, Chee-Seng
Leea, and Carmen M. Baldinoa*
aJasco Pharmaceuticals, LLC, 10-N Roessler Road, Woburn MA 01801
bEisai, Inc., 4 Corporate Drive, Andover, MA 01810
ARTICLE INFO
ABSTRACT
Article history:
Received
Herein,
we
describe
the
versatile
synthesis
of
(Z)-5-((2-aminopyrimidin-4-
yl)methylene)thiazolidine-2,4-dione inhibitors (1) of the PIM family of kinases. This chemistry
strategy was a key element in the multi-variable optimization program with the goal of
identifying high quality leads for the development of a treatment for cancer.
Received in revised form
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
PIM
Kinase
Multiple myeloma
The three Pim kinases (PIM1, PIM2 and PIM3) are a small
family of serine/threonine kinases regulating several signaling
pathways that are fundamental to cancer development and
progression1. Over-expression of the PIM kinases has been
reported in several hematological and solid tumors2. Novel small
molecule pan-PIM inhibitors developed by AstraZeneca (AZD-
1208)3 and Novartis (LGH-447)4 have entered Phase I/2 clinical
trials, validating these very attractive targets for pharmaceutical
development in the treatment of cancer. In fact, Novartis has
described PIM2 as critical for multiple myeloma cell survival.
However, the PIMs do exhibit redundant activity, which in part
has led to the fact that all known PIM inhibitors show activity
against the entire family.
Our design efforts centered around utilizing the thiazolidine-2,4-
dione component as a key element in a novel chemical scaffold.
One thought was to investigate heteroaromatic moieties attached
to the thiazolidine and to achieve this in a modular approach that
would facilitate hit-to-lead efforts on active molecules.
A
pyrimidine replacement of the substituted benzene ring was of
keen interest since aminopyrimidines have been identified by a
number of groups to provide potent, selective, and orally
available small molecule inhibitors of various kinases6.
A
literature search identified a process that described the synthesis
of a suitably elaborated pyrimidine system, compound 67. The
synthesis, detailed in Figure 2, started with the condensation of
the readily available 1,1-dimethoxy-4-dimethylaminobut-3-en-2-
one 4 and thiourea. The resulting substituted pyrimidine could
then be methylated to provide intermediate 5. After acid
catalyzed removal of the dimethyl acetal, the aldehyde 6 was
condensed with thiazolidine-2,4-dione affording the novel
intermediate 7, exclusively as the Z-isomer. Finally, mild
oxidation conditions using oxone were developed to cleanly
provide the library template 8, which served as a versatile starting
point for the development of a general experimental procedure
for the displacement of the methylsulfoxide leaving group8. The
thermal conditions allowed for the use of both primary and
secondary amines. In addition, amines containing free hydroxyl
groups and heterocycles also performed well in the displacement.
However, anilines and heteroaromatic amino groups were not
effective in the displacement reaction under the general
procedure affording this new class of molecules.
O
O
F3C
NH
NH
S
S
O
O
O
2
3
Figure 1. Known benzylidenethiazolidine-2,4-dione PIM
inhibitors.
A number of unique chemical structures have demonstrated
the ability to inhibit PIM kinase enzymatic activity. The known
benzylidenethiazolidine-2,4-diones,5 2 and 3 (Figure 1) were
among the early classes of potent PIM inhibitors to be described,
which exhibited micromolar IC50’s against the family of PIM
isoforms.
∗
Corresponding author. Tel.: +1-617-794-2733; e-mail:
cbaldino@jascopharma.com

2
Figure 2. Synthesis of pyrimidine library template.
To this end, an initial set of analogs were designed to
incorporate a diamine component into the molecule. A
representative set of these analogs are described in Table 1
along with PIM inhibition (IC50s) and permeability data
(PAMPA assay). Incorporation of an end terminal NH
moiety dramatically increased the enzymatic inhibition by
comparison of the IC50 data against PIM1, PIM2 and PIM3
between compound 10 and 11 as well as IC50 data of compound
13 versus 14 in Table 1. However, introduction of the third NH
group in the molecule (16 vs 13) or methylation of the NH group
at the alpha-position of the pyrimidine (12 vs 11) has no
significant affect on the inhibitory activity. We believe that the
terminal NH group at this length and position in compound 13 is
likely necessary to form a hydrogen bond with an enzyme residue
of the target resulting in the observed activity increase. In
addition, when the terminal basic NH group was replaced with an
amide group (comparing 17 with 13), the IC50 data was
substantially decreased by about 10-fold indicating that basicity
of the terminal NH group plays an important role in the
enzymatic activity.
Each new compound, described in Figure 3, was purified
using reverse phase HPLC (yields not optimized) and
characterized by LCMS and/or NMR. This initial set of analogs
was screened against the PIM kinases (isoforms 1, 2, & 3) using
a standard radiometric assay, which identified a number of
micromolar inhibitors. The general procedure was then utilized
to synthesize more than 120 analogs using commercially
available amines to further explore the chemical series. This data
led to a more detailed knowledge of the SAR (Structure Activity
Relationship). The pattern of activity generated an interest in
better understanding the impact of incorporating a wider range of
potential binding elements to increase potency and selectivity.
One approach involved using mono-protected diamines, which
could provide terminal basic NH groups.
Unfortunately, the most active compound 13 in Table 1
has very poor permeability as measured in PAMPA assay9, which
was not desired for achieving good cell-based potency or oral
bioavailability in vivo. Encouraged by the much improved
permeability of compound 15, which included
a fused
hydrophobic benzene ring into the diamine linker, as compared
with 13 in Table 1 (74.1 vs 0.4), we started to explore the
incorporation of aromatic groups attached to the end of the
molecule for increasing hydrophobicity while retaining the basic
NH moiety at the same position as that in compound 13.
Table 1. PIM kinase inhibition via diamine analogs.
Figure 3. Library synthesis of 2-aminopyrimidine analogs.
H
N
R1
R2
O
MeS
O
R1
N
O
O
cat. piperidine
DMSO/110oC
N
N
R2
NH
NH
N
S
N
S
O
O
8
9a-n

3
Table 2. PIM kinase inhibition via modified diamine analogs.
Table 3. Cell proliferation assay data for compound 22.
Cell Line
DU145
IC50 (uM)
Cancer
Prostate
3.7
5.9
6.4
6.9
5.6
LNCaP
Prostate
K562
Leukemia
Myeloma
Myeloma
RPMI-8226
NCI-H929
In conclusion, we have identified a novel class of (Z)-5-((2-
aminopyrimidin-4-yl)methylene)thiazolidine-2,4-diones as potent
and selective inhibitors of the PIM family of kinases. The PIM
kinases have been implicated as critical to cancer cell survival
and proliferation confirming them as attractive biological targets
for pharmaceutical drug development. This report demonstrates
the ability to optimize initially identified active molecules within
this series to provide lead structures with enhanced PIM kinase
inhibition and other pharmaceutical properties.
Selected
compounds such as 22 have exhibited a high degree of selectivity
for inhibition of the PIM kinases with the ability to inhibit cancer
cell proliferation in both hematologic malignancies and solid
tumors. A comprehensive description of the lead optimization
effort to afford a targeted drug candidate for the treatment of
cancer will be disclosed in due course.
A focused set of diamine analogs were designed and
synthesized to address the need to improve both permeability and
PIM kinase inhibition (Table 2). A newly developed and general
reductive amination procedure10 was utilized to provide all of the
analogs 18-22, which introduce a heteroaromatic group but
maintain the basic nature of the NH. These analogs all have both
enhanced permeability and increased PIM inhibition. The best
compound 22 was determined to have a Ki of 7.7 nM against
PIM1. Compound 22 also demonstrated a clear selectivity
profile across a panel of 150 oncology relevant kinases11 (Figure
4) when screened at 0.5 uM.
References and notes
1. Asano, J,; Nakano, A.; Oda, A.; Amou, H.; Hiasa, M.; Takeuchi,
K.; Miki, H.; Nakamura, S.; Harada, T.; Fujii, S.; Kagawa, K.;
Endo, I.; Yata, K.; Sakai, A.; Ozaki, S.; Matsumoto, T.; and
Abe, M. Leukemia, 2011, 25(7), 1182. Ebens, A. Jr.; and Wang,
X. 4-Substituted Pyridin-3-yl-carboxamide compounds and
methods of use, International Patent Application
WO2011/0059961A1. Brault, L.; Gasser, C.; Bracher, F.; Huber,
K.; Knapp, S.; and Schwaller, J. Heamatologica, 2010, 95(6),
1004.
2. Nawijn, M. C.; Alendar, A.; and Berns, A. Nature Reviews
Cancer, 2010 11, 23. Chen, W. W.; Chan, D. C.; Donald, C.;
Lilly, M. B.; and Kraft, A. S. Mol. Cancer Res., 2005, 3(8), 443.
Adam, M.; Pogacic, V.; Bendit, M.; Chappuis, R.; Nawijn, M. C.;
Duyster, J.; Fox, C. J.; Thompson, C. B.; Cools, J.; and Schwaller,
J. Cancer Res., 2006, 66(7), 3828.
Figure 4. Kinase selectivity panel for compound 22.
n 93-100% inhibition
n 50-70% inhibition
n <50% inhibition
3. Keeton, E. K.; McEachem, K.; Dillman, K. S.; Palakurthi, S.; Cao,
Y.; Grondine, M. R.; Kaur, S.; Wang, S.; Chen, Y.; Wu, A.; Shen.
M.; Gibbons, F. D.; Lamb, L. M.; Zheng, X.; Stone, R. M.;
DeAngelo, D. J.; Platanias, L. C.; Dakin, L. A.; Chen, H.; Lyme,
P. D.; and Huszar, D. Blood, 2014, 123(6), 905. Lee, J.; Park, J.;
and Hong, V. S. Chem. Pharm. Bull. 2014, 62(9), 906. Mondello,
P.; Cuzzocea, S.; and Miam, M. J. of Hematology & Oncology
2014, 7, 95.
4. Lu, J.; Zavorotinskaya, T.; Dai, Y.; Niu, X. H.; Castillo, J.; Sim,
J.; Yu, J.; Wang, Y.; Langowski, J. L.; Holash, J.; Shannon, K.;
and Garcia, P. D. Blood, 2013, 122(9), 1610.
5.
Beharry, Z.; Zemskova, M.; Mahajan, S.; Zhang, F.; Ma, J.; Xia,
Z.; Lilly, M.; Smith, C. D.; and Kraft, A. S. Molecular Cancer
Therapeutics, 2009, 8(6), 1473.
Pim-1, Pim-2, &
Pim-3
6. Saylea, K.L.; Bentley, J.; Boyle, F. T.; Calvert, A. H.; Cheng, Y.;
Curtin, N. J.; Endicott, J. A.; Golding, B. T.; Hardcastle, I. R.;
Jewsbury, P.; Mesguiche, V.; Newell, D. R.; Noble, M. E. M.;
Parsons, R. J.; Pratt, D. J.; Wang, L. Z.; and Griffin, R. J. BioOrg.
Med. Chem. Lett. 2003, 13(18), 3079. Binch, H.; Mortimore, M.;
Fraysse, D.; Davis, C.; O'Donnell, M.; Everitt, S,; Robinson, D.;
Pinder, J.; Miller, A. Aminopyrimidines useful as kinase
In addition, 22 also exhibited potent IC50’s against a number of
cancer cell lines in prostate, leukemia, and multiple myeloma
(Table 3). An in vivo rat PK study, dosing the animals both i. v.
and orally with 5 mg/kg of compound 22, demonstrated a plasma
half-life (T1/2) of 3.1 h with very good oral bioavailability (F =
49.9%).
inhibitors, United States Patent US 11/592,119.
7. Garrick, L. M.; Hauze, D. B.; Kees, K. I.; Lundquist, J. T.; Mann,
C. W.; Mehlmann, J. F.; Pelletier, J. C.; Rogers, J. F.; and Wrobel,
J. F. Gonadotropin releasing hormone receptor antagonists,
International Patent Application WO2006/009734A1.

4
8. General Displacement Procedure: 2 dram round bottomed vials
were charged with (Z)-5-((2-(methylsulfonyl)pyrimidin-4-
yl)methylene)thiazolidine-2,4-dione (25 mg, 0.0877 mmol)
prepared according to the general procedure, DMSO (1 mL, 0.08
M), diisopropylethylamine (50 µL, 0.288 mmol, 3.2 equiv.), and
the appropriate amine (0.0877 mmol, 1.0 equiv.). The reaction
mixture was heated to 110oC and shaken for 24 h. The solvent was
removed under reduced pressure (genvac HT-4) and the crude
residues were purified using reverse phase HPLC (MS-triggered
fraction collection) with an acetonitrile/water gradient and
trifluoroacetic acid as a modifier. The pure fractions were then
concentrated under reduced pressure (Genevac HT-4). General
De-Protection Procedure: The crude protected products were
prepared using the General Displacement Procedure and were then
treated with 2 mL DCE and 500 uL of TFA and shaken for 24 h.
The solvent was removed under reduced pressure (Genevac HT-4)
and the crude residues were purified using reverse phase HPLC
(MS-triggered fraction collection) with an acetonitrile/water or
methanol/water gradient and trifluoroacetic acid as a modifier.
The pure fractions were then concentrated under reduced pressure
(Genevac HT-4).
onto a P30 filtermat and washed three times for 5 minutes in 75
mM phosphoric acid and once in methanol prior to drying and
scintillation counting.
9. PAMPA assay kit: BD Biosciences GentestTM pre-coated PAMPA
plate system, catalog number 353015. Parallel Artificial
Membrane Permeability Assays (PAMPA) conditions: The target
concentration in the assay was 200 µM, prepared by diluting (50-
fold) the 10 mM stock solution of each compound in DMSO into
PBS, pH 7.4. The final DMSO concentration was 2%. The 200
µM solutions were added, 300 µL, to wells in the donor plate. The
receiver plate, which contained 200 µL of PBS, pH 7.4 per well,
was placed in the donor plate and the assembly was incubated for
5 hours at ambient temperature. At the end of the incubation
period the plates were separated and the compound concentrations
in each solution were determined by LC/MS/MS. The assay was
performed in triplicate. Dexamethasone and verapamil were used
as reference compounds.
10. General Reductive Amination Procedure 1: A 2-dram round
bottomed vial was charged with the crude amine/TFA salt
intermediate prepared using the general displacement procedure
followed by the general TFA de-protection procedure (0.115
mmol), DCE (2 mL), DIPEA (6 eq. 0.690 mmol), DMF (1 mL),
the aldehyde (1 equiv., 0.115 mmol), and the reaction mixture was
shaken for 1 h at RT. The reaction mixture was then treated with
NaBH(OAc)
3 (2.5 equiv., 0.230 mmol)
and the reaction was
shaken 16 h at RT. The reaction mixture was then diluted with
DCE (2 mL) and NaHCO3 (2 mL). The aqueous layer was back
extracted with DCE (2 x 2 mL) and the combined organic layer
was concentrated under reduced pressure (Genevac HT-4) and the
crude residue was purified using reverse phase HPLC (MS-
triggered fraction collection) with an acetonitrile/water or
methanol/water gradient and triflouroacetic acid as the modifier.
The pure fractions were then concentrated under reduced pressure
(Genevac HT-4) to afford the pure products as the TFA salt.
11. EMD Millipore Kinase ProfilerTM Services,
www.emdmillipore.com . Assay conditions: Pim-1 (h): Pim-1
(h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 100
µM KKRNRTLTV, 10 mM MgAcetate and [γ-33P-ATP] (specific
activity approx. 500 cpm/pmol, concentration as required). The
reaction is initiated by the addition of the MgATP mix. After
incubation for 40 minutes at room temperature, the reaction is
stopped by the addition of 3% phosphoric acid solution. 10 µL of
the reaction is then spotted onto a P30 filtermat and washed three
times for 5 minutes in 75 mM phosphoric acid and once in
methanol prior to drying and scintillation counting. Pim-2 (h):
Pim-2 (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA,
300 µM RSRHSSYPAGT, 10 mM MgAcetate and [γ-33P-ATP]
(specific activity approx. 500 cpm/pmol, concentration as
required). The reaction is initiated by the addition of the MgATP
mix. After incubation for 40 minutes at room temperature, the
reaction is stopped by the addition of 3% phosphoric acid solution.
10 µL of the reaction is then spotted onto a P30 filtermat and
washed three times for 5 minutes in 75 mM phosphoric acid and
once in methanol prior to drying and scintillation counting. Pim-3
(h): Pim-3 (h) is incubated with 8 mM MOPS pH 7.0, 0.2 mM
EDTA, 0.1% Triton X-100, 300 µM RSRHSSYPAGT, 10 mM
MgAcetate and [γ-33P-ATP] (specific activity approx. 500
cpm/pmol, concentration as required). The reaction is initiated by
the addition of the MgATP mix. After incubation for 40 minutes at
room temperature, the reaction is stopped by the addition of 3%
phosphoric acid solution. 10 µL of the reaction is then spotted