Identification and structure-activity relationships of substituted pyridones as inhibitors of Pim-1 kinase

Article abstract of DOI:10.1016/j.bmcl.2006.12.086

A novel series of highly potent substituted pyridone Pim-1 kinase inhibitors is described. Structural requirements for in vitro activity are outlined as well as a complex crystal structure with the most potent Pim-1 inhibitor reported (IC₅₀ = 5

Full text of DOI:10.1016/j.bmcl.2006.12.086

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1679–1683
Identification and structure–activity relationships of substituted
pyridones as inhibitors of Pim-1 kinase
I. Wayne Cheney,* Shunqi Yan, Todd Appleby, Heli Walker, Todd Vo, Nanhua Yao,
Robert Hamatake, Zhi Hong and Jim Z. Wu
Valeant Pharmaceuticals Research and Development, 3300 Hyland Avenue, Costa Mesa, CA 92626, USA
Received 24 October 2006; revised 20 December 2006; accepted 22 December 2006
Available online 4 January 2007
Abstract—A novel series of highly potent substituted pyridone Pim-1 kinase inhibitors is described. Structural requirements for
in vitro activity are outlined as well as a complex crystal structure with the most potent Pim-1 inhibitor reported (IC50 = 50 nM).
A hydrogen bond matrix involving the Pim-1 inhibitor, two water molecules, and the catalytic core, together with a potential weak
hydrogen bond between an aromatic hydrogen on the R1 phenyl ring and a main-chain carbonyl of Pim-1, accounts for the overall
potency of this inhibitor.
Ó 2007 Elsevier Ltd. All rights reserved.
The pim-1 proto-oncogene was initially identified as a
frequent site of integration for the slowly transforming
Maloney murine leukemia virus in murine T cell lym-
phomas.1 Pim-1 is an oncogenic serine/threonine kinase
that is a member of a family of proteins containing
homologues Pim-2 and Pim-3, and is transcriptionally
regulated by cytokines, mitogens, and numerous growth
factors.2–4 Elevated expression of Pim kinases has been
described predominantly in murine and human leukemi-
as and lymphomas.5 The enzymatic substrates reported
for Pim kinases are diverse (p100,6 Bad,7 NFATc1,8–10
p21/Cip1,11 Cdc25a,12 SOCS,13,14 and others), suggest-
ing that Pim plays a central regulatory role in numerous
processes. Indeed, Pim kinases have defined biological
roles in cell survival, proliferation, and differentiation,4
particularly for cells of hematopoietic lineage. Studies
using either pim transgenic or pim knockout mice sug-
gest that overlapping functions exist for Pim-1 and
Pim-2, and that the full transforming potential of Pim
kinases cannot be realized in vivo without cooperation
from other oncogenes.15–19 Perhaps the most compelling
biological roles described for Pim-1 and Pim-2 include
the regulation of rapamycin-resistant T cell growth
and survival18 as well as the recently reported impairment
of mutant Fms-like tyrosine kinase 3 (FLT3)10,20 or
drug-resistant BCR/ABL-transformed hematopoietic
cell survival,21 upon functional suppression of Pim
kinases. From these perspectives, inhibitors of Pim
kinases may have immunomodulatory potential alone
or in combination with immunosuppressive drugs such
as rapamycin, or as an alternative method to treating
patients who have developed resistance to small-
molecule protein tyrosine kinase inhibitors for cancer.
X-ray crystallographic studies of Pim-1 support a high
degree of main chain similarity to other structurally
defined serine/threonine kinases, with characteristic
fold-hinge-fold domains.22–24 However, Pim kinases
possess a unique proline residue, Pro123, in the hinge
region where other hydrophobic amino acids are more
typically found, as well as insertion of one or two resi-
dues in the hinge region following Pro123. As Pim-1
adopts a unique structure in the hinge region, which acts
in part as a boundary for positioning ATP binding, it
may be possible to identify highly selective ATP-com-
petitive Pim kinase inhibitors for potential use as anti-
cancer or immunomodulatory agents. Small molecule
Pim-1 kinase inhibitors derived from various chemical
scaffolds have been previously described.23–25 None of
these inhibitors have progressed to the clinic. Here we
report the identification of a novel series of Pim-1 kinase
inhibitors based on a substituted pyridone scaffold and
outline the potential mechanism by which this type of
inhibitor could compete and interfere with Pim-1 ATP
utilization.
Keywords: Pim-1 kinase; Pim-1 kinase inhibitors; Pyridone; Fused-ring
pyridone; X-ray complex structure; Pim-1; Pim.
*
Corresponding author. Current address: Ardea Biosciences, 3300
Hyland Avenue, Costa Mesa, CA 92626, USA. Tel.: +1 714 729
5555; fax: +1 714 729 5578; e-mail: wcheney@ardeabiosciences.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.086

1680
I. W. Cheney et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1679–1683
˚
In order to identify alternative scaffolds we screened a
compound library of approximately 160,000 molecules
using an enzymatic luminescent assay for Pim-1 inhibi-
tion.26 A substantial number of Pim-1-inhibitory hits
were identified, many of which had IC50 values less than
1 lM. Several potent chemical library hits along with a
number of newly synthesized derivatives of these com-
pounds are listed in Table 1. Corresponding IC50 values
were determined by radioactive filter-plate assay using
histone proteins as the substrate for Pim-1 kinase.27
Compound 1, a substituted pyridone scaffold, had the
lowest IC50 at 0.05 lM. This molecule served both as a
starting point for SAR chemical syntheses and was used
for co-crystallization with Pim-1 protein. A limited
number of compounds with highly similar chemical
composition were also identified from our library
including compounds 4, 6, 7, 8, and 10. Although none
of these compounds produced IC50 values superior to
compound 1 they were informative with respect to the
SAR. Specifically, modification of R1 contributed more
substantially to the loss of in vitro activity compared to
alteration of R2.
resolved to 2.5 A. Analysis of the electron density plot
for 1 supported the conformation of the complex struc-
ture without ambiguity (Fig. 1). Compound 1 appeared
to adopt a partially flat conformation with co-planar R1
phenyl and central pyridone rings, while the R2 phenyl
ring was twisted perpendicularly with respect to the
other rings. We contend that the possibility for H-bond-
ing between the 2-OH group on the R1 phenyl ring and
the pyridone N is high and within acceptable H-bonding
˚
distance (1.9 A). This internal H-bonding may serve to
fix the conformation of the two rings into a nearly pla-
nar relationship.
Compound 1 bound convincingly within the ATP-bind-
ing site of Pim-1 suggesting an ATP-competitive inhibi-
tory mechanism (Fig. 2). Perhaps the most significant
finding from this complex structure was the prominent
interaction of the carbonyl group on the pyridone ring
with both the Lys67 side chain (interacts with phos-
phates from ATP) of Pim-1 together with a hydrogen
bond to a water molecule (Wat1) that appeared to play
an integral part of a larger H-bonding network in this
region. The apparent H-bonding network further con-
sisted of a second conserved water molecule (Wat2) that
interacted with both the Pim-1 kinase catalytic residue
Phe187 and Wat1 (Fig. 2). Interestingly, the only poten-
tial interaction of 1 with the hinge region occurred on
the left side of the R1 phenyl ring where the complex
To help comprehend the inhibitory mechanism of action
for compound 1, we took advantage of a highly purified
and enzymatically active Pim-1 protein28 to initiate com-
plex structure studies by X-ray crystallography.29,30 The
structure of Pim-1 co-crystallized with compound 1 was
Table 1. Pyridone-based inhibitors of Pim-1 kinase
O
N
HN
R1
R2
R1
R2
IC50 (lM)
a
Compound
1
2
3
4
5
6
7
8
9
3-Br-6-OH-Ph
3-Me-6-OH-Ph
Ph
Ph
Ph
0.05
0.34
0.37
0.43
1.03
1.14
0.99
3.54
16.00
3-Me-6-OH-Ph
4-OH-Ph
3-Cl-Ph
CF3
6-OH-Ph
3-Et-6-OH-Ph
4-OMe-5-Br-Ph
Thiophene
CF3
Thiophene
Thiophene
CF3
NH
Furan
N
N
10
4.41
HO
N
HN
NH
OH
N
11
1.77
Br
a Values are means of three experiments.

I. W. Cheney et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1679–1683
1681
O
N
O
O
N
O
O
a
HN
N
OH
HN
R1
R2
NH2
R1
R2
2
I
II
1 - 3, 5, 9
5
Cl
NHNH2
N
N
d
Br
b
c
N
N
R1
R2
R1
R2
Figure 1. X-ray electron density plot of compound 1 in complex with
˚
Pim-1 obtained at a resolution of 2.5 A. Structure of 1 is shown to the
right.
III
IV
N
HN
NH2
N
R1
R2
11
Scheme 1. The synthetic route to 1–3, 5, 9 and 11. Reagents and
conditions: (a) DBU, toluene, 90 °C, 16 h, 50-70%; (b) POCl3,
pyridine, dioxane, 80 °C, 14 h, 16%; (c) NH2NH2 in H2O, DMF,
90 °C, 14 h, 90%; (d) CH3COOH, EtOH, 90 °C, 14 h. Refer to Table 1
for R1 and R2 designations.
˚
Figure 2. X-ray complex structure of compound 1 with Pim-1 at 2.5 A
resolution. Hydrogen and side-chain atoms are omitted for clarity.
The synthesized pyridone derivatives 1–3, 5, and 9 were
obtained from a cyclo-condensation reaction in one step
(Scheme 1). Reaction of commercially available dike-
tone I with cyanoacetamide II, in the presence of
DBU in toluene at 90 °C for 16 h, readily afforded the
title compounds which were purified by HPLC and
structure defined a weak H-bond between the main-
chain carbonyl of Glu121 and an aromatic hydrogen
1
˚
(C-3 position) on R (2.6 A C@Oꢀ ꢀ ꢀH–C). Although sig-
1
˚
nificantly less favorable at 3.0 A, potential H-bonding
characterized by H NMR and MS.
between the same carbonyl of Glu 121 and the aromatic
H at the C-4-position may also contribute to stabiliza-
tion of 1 with the hinge. Hydrogen bonding of this nat-
ure was also reported for interactions between Pim-1
Glu121 with the known phosphatidylinositol 3-kinase
inhibitor LY294002.23 The role of the Br atom on R1
is less well defined from a SAR perspective although
its importance is exemplified by the fact that replace-
ment of the Br as in 2 by a methyl group leads to a
7-fold loss of inhibition. It is our interpretation that sta-
bilization of 1 by a robust H-bonding network involving
two water molecules accounts for the majority of the
inhibitor’s potency. Hence compound 1 binds and com-
petitively inhibits ATP utilization thereby suppressing
Pim-1 kinase function.
The synthesis of ring-fused product 11 started with 1
combined with three additional steps including sequen-
tial chlorination, displacement, and cyclization reac-
tions. Thus, reaction of 1 with POCl3 with pyridine
in dioxane at 80 °C for 14 h formed chloro intermedi-
ate III, which was displaced by hydrazine to give IV.
Treatment of IV with acetic acid in ethanol under
reflux for 14 h formed the desired product 11, which
1
was HPLC purified and confirmed by H, 2D NMR
and MS.
Rigidification of the R1 phenyl ring in relation to the
central pyridone through cyclization (similar to phen-
anthrene, although not fully aromatic) (Table 2) was
envisioned to mimic the proposed intra-molecularly
H-bonded co-planar rings of 1 when complexed with
Pim-1, without the R1 2-OH moiety. Although com-
pounds 12–14 produced IC50 values less than 1 lM,
they failed to reach low nanomolar potencies. Com-
pounds containing an O rather than S at position X
were seemingly more potent (12 vs 19) when the R
group was thiophene. Lack of halogenation on the
left-side phenyl ring may account in part for these
lower efficacies. Although the R group was never an
unsubstituted phenyl ring (as in R2 of 1), the proba-
bility of significant interaction with the protein in this
section of the compounds was likely minimal due to
the predominant solvent exposure observed near R2
of Pim-1/compound 1.
Newly synthesized compounds based on the planar com-
plex structure of 1, including compounds 2, 3, 5, 9, and
11, are listed in Table 1. However, highly potent com-
pounds were not identified within this sub-series of
SAR molecules. Disruption of the pyridone carbonyl
by ring closure (11) led to a 35-fold reduction in potency
compared to 1, substantiating the fragile nature of the
proposed H-bonding network in this area. Contribu-
tions to inhibitor activity are less obvious for the cyano
group although its strong electron-withdrawing poten-
tial may result in modification to the electronics, and
hence H-bonding capability, of the pyridone ring. The
key reactions necessary to synthesize the SAR deriva-
tives are outlined in Scheme 1.

1682
I. W. Cheney et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1679–1683
4. Wang, Z.; Bhattacharya, N.; Weaver, M.; Petersen, K.;
Table 2. Inhibitors of Pim-1 kinase
Meyer, M.; Gapter, L.; Magnuson, N. S. J. Vet. Sci. 2001,
2, 167.
O
N
5. Amson, R.; Sigaux, F.; Przedborski, S.; Flandrin, G.;
Givol, D.; Telerman, A. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 8857.
HN
6. Leverson, J.; Koskinen, P. J.; Orrico, F.; Rainio, E.-M.;
Jalkanen, K.; Dash, A.; Eisenman, R.; Ness, S. Mol. Cell.
1998, 2, 417.
R
7. Lilly, M.; Sandholm, J.; Cooper, J. J.; Koskinen, P. J.;
Kraft, A. Oncogene 1999, 18, 4022.
X
a
8. Rainio, E.-M.; Sandholm, J.; Koskinen, P. J. J. Immunol.
2002, 168, 1524.
9. Aho, T. L.; Sandholm, J.; Peltola, K. J.; Mankonen, H. P.;
Lilly, M.; Koskinen, P. J. FEBS Lett. 2004, 571, 43.
10. Kim, K.-T.; Levis, M.; Small, D. Br. J. Hematol. 2006,
134, 500.
11. Wang, Z.; Bhattacharya, N.; Mixter, P. F.; Wei, W.;
Sedivy, J.; Magnuson, N. S. Biochem. Biophys. Acta 2002,
1593, 45.
12. Mochizuki, T.; Kitanaka, C.; Noguchi, K.; Muramatsu,
T.; Asai, A.; Kuchino, Y. J. Biol. Chem. 1999, 274, 18659.
13. Chen, X. P.; Losman, J. A.; Cowan, S.; Donahue, E.; Fay,
S.; Vuong, B. Q.; Nawijn, M. C.; Capece, D.; Cohan, V.
L.; Rothman, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
2175.
Compound
R
X
IC50 (lM)
12
13
14
15
16
17
18
19
20
21
22
23
Thiophene
Furan
4-Cl-Ph
O
O
O
O
O
O
O
S
0.46
0.87
0.92
1.23
>20
20.0
>20
1.49
4.79
20.0
>20
>20
6-Cl-Ph
4-OMe-Ph
4-Pyridine
2-Pyridine
Thiophene
3-Pyridine
2-Cl-Ph
S
S
4-F-Ph
4-Pyridine
S
CH2
O
14. Peltola, K. J.; Paukku, K.; Aho, T. L.; Ruuska, M.;
Silvennoinen, O.; Koskinen, P. J. Blood 2004, 103, 3744.
15. van Lohuizen, M.; Verbeek, S.; Krimpenfort, P.; Domen,
J.; Saris, C.; Radaszkiewicz, T.; Berns, A. Cell 1989, 56,
673.
N
O
O
24
>20
O
O
16. Domen, J.; van der Lugt, N. M.; Laird, P. W.; Saris, C. J.;
Berns, A. Leukemia 1993, 7(Suppl. 2), S108.
17. Mikkers, H.; Nawijn, M.; Allen, J.; Brouwers, C.; Verho-
even, E.; Jonkers, J.; Berns, A. Mol. Cell. Biol. 2004, 24,
6104.
18. Fox, C. J.; Hammerman, P. S.; Thompson, C. B. J. Exp.
Med. 2005, 201, 259.
19. Hammerman, P. S.; Fox, C. J.; Birnbaum, M. J.;
Thompson, C. B. Blood 2005, 105, 4477.
20. Kim, K.-T.; Baird, K.; Ahn, J.-Y.; Melzer, P.; Lilly, M.;
Levis, M.; Small, D. Blood 2005, 105, 1759.
21. Adam, M.; Pogacic, V.; Bendit, M.; Chappuis, R.; Nawijn,
M. C.; Duyster, J. C. F.; Thompson, C. B.; Cools, J.;
Schwaller, J. Cancer Res. 2006, 66, 3828.
22. Qian, K. C.; Studts, J.; Wang, L.; Barringer, K.; Kronka-
itis, A.; Peng, C.; Baptiste, A.; LaFrance, R.; Mische, S.;
Farmer, B. Acta Crystallogr., Sect. F 2005, 61, 96.
23. Jacobs, M. D.; Black, J.; Futer, O.; Swenson, L.; Hare, B.;
Fleming, M.; Saxena, K. J. Biol. Chem. 2005, 80, 13728.
24. Kumar, A.; Mandiyan, V.; Suzuki, Y.; Zhang, C.; Rice, J.;
Tsai, J.; Artis, D. R.; Ibrahim, P.; Bremer, R. J. Mol. Biol.
2005, 348, 183.
a Values are means of three experiments.
In summary, a series of substituted pyridone molecules
was identified from a high throughput screen as potent
inhibitors of Pim-1 kinase. Because of its compelling
in vitro efficacy, a complex structure and mechanism
of action were defined for compound 1. The pyridone
carbonyl group was critically positioned within the
Pim-1 ATP binding site effecting kinase inhibition. Pre-
liminary data further suggested that 1 lacked in vitro
inhibitory activity toward related serine/threonine ki-
nases Pim-2 and MEK1/2 (IC50 > 20 lM; data not
shown). Hence, small molecules similar to compound 1
may serve as useful starting scaffolds for the develop-
ment of other improved yet selective Pim-1 inhibitors.
Acknowledgment
25. Bullock, A. N.; Debreczeni, J. E.; Federov, O. Y.; Nelson,
A.; Marsden, B. D.; Knapp, S. J. Med. Chem. 2005, 48,
7604.
The authors thank Dr. J. M. Vernier for insightful com-
ments pertaining to this manuscript.
26. High throughput screening assay: A purified recombinant
GST–Pim-1 fusion protein together with histone proteins
(Sigma) as substrate was used for the Kinase-Glo assay
(Promega). Briefly, reaction conditions were 25 mM Hepes
(pH 7.5), 10 mM MgCl2, 5 mM DTT, 4% DMSO, 15 lM
ATP, 1 lg GST-Pim-1, 5 lg calf thymus histones plus the
test compound in a 20 ll total volume. Reactions were
performed in a 384-well plate for 1 h at room temperature
and were terminated by the addition of the Kinase-Glo
reagent then counted on the LJL Analyst plate reader. The
Z0 for the assay was 0.8.
References and notes
1. Cuypers, H. T.; Selten, G.; Quint, W.; Zijlstra, M.;
Robanus-Manndag, E.; Boelens, W.; van Wezenbeek, P.;
Berns, A. Cell 1984, 37, 141.
2. van der Lugt, N. M.; Domen, J.; Verhoeven, E.; Linders,
K.; van der Gulden, H.; Allen, J.; Berns, A. EMBO
J. 1995, 14, 2536.
3. Feldman, J. D.; Vician, L.; Crispino, M.; Tocco, G.;
Marcheselli, V. L.; Bazan, N. G.; Baudry, M.; Herschman,
H. R. J. Biol. Chem. 1998, 273, 16535.
27. Radioactive filter plate assay: Pim-1 kinase reaction con-
ditions included 25 mM Hepes (pH 7.5), 10 mM MgCl2,

I. W. Cheney et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1679–1683
1683
5 mM DTT, 4% DMSO, 25 lM ATP, 0.5 lCi [c-33P] ATP,
100 nM enzyme, and 2.5 lg calf thymus histone substrate in
a 25 ll reaction volume. Reaction mixtures proceeded for
1 h at 23 °C and were terminated by the addition of 20%
trichloroacetic acid/1% pyrophosphate. Reactions were
transferred to a 96-well filter plate, washed, and dried, then
scintillation cocktail was added and the radioactivity
counted on a TopCount plate reader (Perkin-Elmer).
Compounds were titrated using these conditions and the
IC50 values were determined.
filtration column. Using this process we recovered
milligram quantities of Pim-1 with purity sufficient for
attempting crystallization.
29. The Pim-1/inhibitor complex was obtained by combining
25 ll protein (12 mg/ml) with 0.35 ll of 20 mM compound
1 in DMSO. The samples were incubated at 4 °C for 1–2 h.
Co-crystals were grown using a sitting-drop vapor diffu-
sion tray with a crystallization solution containing 1.0 M
ammonium phosphate (dibasic), 300 mM sodium chloride,
and 100 mM citrate buffer (pH 5.0). Crystallization plates
were incubated at 23 °C and optimal crystal size was
reached in approximately 10 days. Data acquisition was
performed on a Rigaku R-Axis IV+ detector mounted on
a Rigaku RUH3R generator operating at 50 kV and
100 mA. Measurement intensities were integrated, scaled,
and merged using HKL software (Z. Otwinoski and W.
Minor).
28. Protein purification: Full-length Pim-1 (residues 1–313)
was PCR cloned into the bacterial expression vector
pET28a in-frame with the N-terminal hexahistidine tag.
Expression was carried out in BL21 (DE3) pLysS
Escherichia coli in LB medium after induction with
IPTG for 4 h at 28 °C. Following cell lysis, the His-
tagged Pim-1 was subjected to a sequential purification
scheme initially incorporating FPLC using a nickel-
chelating Sepharose column followed by a Resource Q
ion exchange column and lastly a Superdex 75 gel
30. Coordinates for the complex structure of Pim-1/com-
pound 1 have been deposited with the Protein Data Bank
(www.rcsb.org) under PDB code 2OBJ.