Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases

Article abstract of DOI:10.1016/j.bmcl.2012.05.098

Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described, along with X-ray crystallographic, anti-proliferati

Full text of DOI:10.1016/j.bmcl.2012.05.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4599–4604
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent
and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases
Les A. Dakin a, Michael H. Block a, Huawei Chen a, Erin Code a, James E. Dowling a, Xiaomei Feng a,
Andrew D. Ferguson b, Isabelle Green c, Alexander W. Hird a, Tina Howard d, Erika K. Keeton a,
a
c
d
a
a
Michelle L. Lamb a, , Paul D. Lyne , Hannah Pollard , Jon Read , Allan J. Wu , Tao Zhang ,
⇑
Xiaolan Zheng a
a Oncology iMed Sciences Group, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
b Discovery Sciences – Structure and Biophysics, AstraZeneca, 35 Gatehouse Drive, Waltham, MA 02451, USA
c Discovery Sciences – Reagents and Assay Development, AstraZeneca, Alderley Park, Cheshire SK10 4TG, UK
d Discovery Sciences – Structure and Biophysics, AstraZeneca, Alderley Park, Cheshire SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel substituted benzylidene-1,3-thiazolidine-2,4-diones (TZDs) have been identified as potent and
highly selective inhibitors of the PIM kinases. The synthesis and SAR of these compounds are described,
along with X-ray crystallographic, anti-proliferative, and selectivity data.
Ó 2012 Published by Elsevier Ltd.
Received 26 March 2012
Revised 18 May 2012
Accepted 29 May 2012
Available online 6 June 2012
Keywords:
PIM kinases
Hit-to-lead
Small molecule inhibitor
Thiazolidine-2,4-dione
X-ray crystallography
Cancer
The PIM kinases (PIM-1, 2, and 3) (proviral integration site for
moloney murine leukemia virus) are a family of three serine/thre-
onine kinases which constitute a small family of enzymes with a
high degree of sequence and structural homology. They were iden-
tified as oncogenes1 and found to synergize with c-Myc for lym-
phomagenesis in transgenic mouse models.2 The PIM kinases are
primarily regulated by growth factor and cytokine signaling down-
stream of PTK/STAT activation.3 In contrast to the majority of other
kinases, PIM kinases are constitutively active enzymes whose
activity is largely regulated by their post-transcriptional and
post-translational (protein stability) levels. These kinases are also
unusual in the structure of their kinase domains, where the hinge
region of the ATP-binding pocket contains a proline residue not
found in other kinases. This results in a molecular recognition mo-
tif that is unique among the kinase family.4 Additionally, the PIM
kinases have pleiotropic roles, but the primary functions of these
kinases are in promoting proliferation, through direct phosphory-
lation of cell cycle regulators (Cdc25A, Cdc25C, p21 and p27),
and cell survival, at least in part through phosphorylation and inac-
tivation of the pro-apoptotic BAD protein.5 Gene knockout studies
have demonstrated that mice deficient in all three PIM kinases are
small but viable and fertile.6 PIM kinases are also overexpressed in
a number of human tumors. PIM-1 and PIM-2 proteins are com-
monly found in haematological malignancies and prostate tumors,
while elevated PIM-3 expression is observed with several different
solid tumors. Deregulated PIM kinase expression is linked to genet-
ic aberrations such as FLT3-ITD in AML7 and BCR-ABL in CML.8 In
addition, aberrant somatic hypermutation of PIM-1 is characteris-
tic of diffuse large B-cell lymphoma, as well as follicular lym-
phoma, Hodgkin’s lymphoma and MALT (mucosa-associated
lymphoid tissue).9 PIM proteins have been found to interact with
the transcription factor Myc in promoting oncogenesis in prostate
cancer,10 as well as lymphoma,2 and high PIM-1 expression levels
have been correlated to poor radiation therapy response in squa-
mocellular head and neck cancer.11 These and other accumulating
evidence support the importance of the PIM kinases in tumor ini-
tiation, progression, and maintenance.
On embarking on the design of potent PIM inhibitors, a key con-
sideration was the proline in the hinge region of the ATP binding
site which is not found in other kinases. This cyclic residue alone
sequesters the backbone nitrogen required to donate a hydrogen
⇑
Corresponding author.
E-mail address: Michelle.Lamb@astrazeneca.com (M.L. Lamb).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.05.098

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L. A. Dakin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4599–4604
bond to the adenine ring of ATP, and thus, conventional ATP-com-
petitive kinase inhibitors are not optimized for this site. Although
this feature presented a challenge, we felt this also afforded an
opportunity to develop inhibitors selective against other kinases.
Our goal was to identify inhibitors capable of pan-PIM inhibition,
since the biological roles of the isozymes could be redundant in
some circumstances, and the active site homology between family
members appeared to support this strategy (PIM-1 and PIM-2 are
85% identical; PIM-1 and PIM-3, 93%). A broad high-throughput
screen against PIM-2 to identify ATP competitive compounds was
undertaken and (5Z)-5-(3-ethoxy-4-hydroxybenzylidene)-1,3-thi-
azolidine-2,4-dione (1), which is commercially available from the
O
HN
O
O
H
H
O
R1
R2
S
H
N
i
O
R1
R2
O
Eq.1
Eq.2
S
R3
R3
4
2
3
O
H
ii
amines
R2
F
R2
R3
R3
5
ChemBridge Corporation, was identified as a 0.16 lM inhibitor of
the PIM-2 kinase (Fig. 1). Interestingly, other thiazolidine-2,4-
3
diones (TZDs) have independently been reported in the literature
Scheme 1. Reagents and conditions: (i) piperidine (5 mol %), ethanol, reflux, (50–
90%), Z/E (>98:2); (ii) amine, K2CO3, MeCN (reflux) or DMSO (80 °C) (40–88%).
as PI3Kc
, PIM, and CDK2 kinase inhibitors.12–14
We chose to pursue 1 for further studies since it was highly
ligand efficient15 (by pIC50/number of heavy atoms method,
LE = 0.38) and there were opportunities to expand the SAR around
the aryl ring. We were interested to determine whether or not we
could remove the phenol from the series, as phenols often give
poor pharmacokinetic and drug-like properties. To do this we first
synthesized a variety of substituted benzylidene-1,3-thiazolodine-
2,4-diones (TZDs) via a Knoevenagel condensation16 with 2,4-thia-
zolidinedione (2) and a variety of substituted benzaldehydes (3)
(Scheme 1, Eq. 1).17
Given the observed importance of both the acidic TZD and basic
amine to potency, the binding of these ligands was expected to in-
volve an interaction of the TZD moiety with the conserved lysine
residue (Lys 67) and placement of the amine into the carboxyl-
ate-rich ribose binding pocket. Indeed, several small molecule
PIM inhibitors have been disclosed to date that indicate the impor-
tance of a ligand–protein interaction with the conserved lysi-
ne.13b,19 This binding mode would place the chlorine of 12
adjacent to hydrophobic residues within the site, near the region
known as the solvent channel. We chose next to examine further
the effect of hydrophobic substitution at this position of the scaf-
fold. To develop the SAR, we maintained the optimal (3R)-piperi-
din-3-amine base and synthesized derivatives varying the size
and nature of the group at the R2 position of the scaffold. The hal-
ogenated derivatives (22, 23) were synthesized according to
Scheme 1 starting from commercially available benzaldehydes.
The alkoxy derivatives (24–27), could readily be synthesized from
commercially available 2-fluoro-3-hydroxybenzonitrile (16).
Alkylation of the phenol with the appropriate alkyl bromide and
K2CO3 in acetonitrile at 60 °C afforded alkylated derivatives 17 in
good yield. Functional group interconversion of the nitrile to the
aldehyde with Dibal-H provided the desired 2-fluoro-3-alkoxy-
benzaldehyde substrates (18) (Scheme 2, Eq. 1). The benzalde-
hydes 18 were then subjected to the SNAr reaction with
tert-butyl (3R)-piperidin-3-ylcarbamate to yield the desired Knoe-
venagel substrates. The aromatic (28) and heteroaromatic (29)
derivatives were synthesized starting from 3-bromo-2-fluorobenz-
aldehyde (19). First, a SNAr reaction with tert-butyl (3R)-piperidin-
3-ylcarbamate was performed using conditions described previ-
ously. This furnished the amine substituted compound 20, which
could then be converted to the desired derivatives, 21, via a Suzuki
coupling under standard conditions (Scheme 2, Eq. 2). The result-
ing substituted benzaldehydes (21) were then converted into their
corresponding final compounds via the Knoevenagel protocol illus-
trated in Scheme 1 followed by Boc-deprotection of the (3R)-piper-
idin-3-amine.
The compounds initially synthesized, examples 6 and 7 (Ta-
ble 1), were still modestly potent which suggested to us that the
TZD was the critical binding element for this scaffold. This moiety
was expected to be quite acidic, and the pKa of the unsubstituted
analog 7 was found to be 6.14. Structure-based design, utilizing
SAR from additional HTS hits, led us to next investigate effects of
amine substitution ortho to the TZD moiety on the aromatic core
(position R1). The required benzaldehyde substrates were readily
prepared via a nucleophilic aromatic substitution with various
amine bases in the presence of K2CO3 in heated DMSO or refluxing
acetonitrile (Scheme 1, Eq. 2). Incorporation of the basic amine,
N,N-dimethylpyrrolidin-3-amine, at the R1 position of the scaffold
afforded compound 8, which had potency against all three iso-
forms of the enzyme comparable to compound 1 and was devoid
of the phenol. The Boc protected analog 9 was significantly less po-
tent than its deprotected counterpart (11), and notably, all other
synthesized analogs that did not incorporate a basic amine on
the molecule showed significantly diminished activity (not
shown). Of all the amines explored, cyclic primary amines, such
as the (3S)-pyrrolidin-3-amine (11) and (3R)-piperidin-3-amine
(15) examples, were most effective. Interestingly, these chiral
examples showed only a slight potency advantage over their corre-
sponding enantiomers. It was also discovered that incorporation of
chlorine adjacent the (3S)-pyrrolidine-3-amine at position R2 (12)
conferred a 10-fold increase in enzyme potency over its matched
pair (11), with inhibitory potencies of this example approaching
the limit of detection of our Km [ATP] enzyme assays.18 Substitu-
tion instead at the R3 position of the ring (13) had a less pro-
nounced effect on enzyme potency.
Continued exploration of the R2 position of the scaffold proved
an effective strategy since incorporation of hydrophobes at this po-
sition, (chlorine (22), bromine (23), alkoxy (24–27), and phenyl
(28)), afforded inhibitors at or beyond the level of detection of
our Km [ATP] enzyme assays, < 3 nM against all PIM isoforms
(Table 2). Notably, more polar heteroaromatic groups such as pyr-
idine (29) were less tolerated, especially against PIM-2.
An X-ray crystal structure of 27 in PIM-1 (residues 29–313) was
obtained to confirm its binding mode (Fig. 2). There is, as expected,
a close interaction of the TZD with the conserved lysine (TZD N–Lys
NZ 2.9 Å) and also the backbone NH of Asp 186 (TZD O–Asp N 2.9 Å,
O
HN
O
S
OEt
OH
1, PIM-2 IC50: 0.16 µM
Figure 1. The TZD PIM-2 HTS hit.

L. A. Dakin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4599–4604
4601
N
N
Table 1
O
H
18
SAR of the aromatic core (IC50, lM, [ATP] at Km)
F
i
F
F
ii
Eq.1
O
HN
OR
OH
OR
O
16
17
18
S
O
H
O
H
O
H
Ar
F
N
iv
iii
N
Eq.2
NHBoc
NHBoc
Br
Br
Ar
19
20
21
Compd
Ar
PIM-1
0.16
PIM-2
0.16
PIM-3
0.39
Scheme 2. Reagents and conditions: (i) alkyl bromide, K2CO3, MeCN, 60 °C (65–
88%); (ii) Dibal-H, PhMe, 0 °C?rt (65–90%); (iii) tert-butyl (3R)-piperidin-3-
ylcarbamate, K2CO3, MeCN, reflux (70%); (iv) ArB(OH)2, Pd(dppf)Cl2, Na2CO3,
dioxane/water (5:1), reflux, (50–75%).
1
OEt
OH
Table 2
18
Effect of substitution at the R2 position (IC50
,
l
M, [ATP] at Km)
6
0.95
2.5
0.28
OMe
OMe
O
HN
O
7
8
9
0.89
0.061
1.5
4.7
0.50
15
0.73
0.11
3.2
S
N
NH2
NMe2
N
N
R2
Compd
R2
PIM-1
PIM-2
PIM-3
NHBoc
15
22
23
24
25
26
27
28
29
H
CI
Br
OMe
OEt
i-BuO
i-PrO
Ph
0.009
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
0.014
0.005
0.009
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
0.004
<0.003
0.005
<0.004
<0.004
<0.003
0.005
NH
N
N
10
11
12
0.028
0.036
0.18
0.25
0.025
0.036
0.042
NH2
4-Pyridyl
0.019
NH2
NH2
N
shown), and Leu 174. For PIM-2, the residue analogous to Val
126 is an alanine, and the increased potency against this isozyme
with substitutions at R2 (compared to 15, R2 = H, Tables 2 and 3)
can be rationalized by an increase in contact at this position. The
observed binding mode for 27 is consistent with the experimental
pKa values determined for compound 28, (5.95 for the N–H of the
TZD and 9.83 for the (3R)-piperidin-3-amine), and differs from ear-
lier TZD-PIM-1 binding modes that have been described in which
the thiazolidine-2,4-dione is found to interact with the lysine with
the carbonyl at the 4-position of the ring directed into the selectiv-
ity pocket.13b While this manuscript was being prepared, two addi-
tional TZD crystal structures were reported that further support
the influence of the substitution pattern of the phenyl ring on
the observed binding modes in PIM-1.20
<0.006
<0.005
Cl
N
13
0.012
0.079
0.005
Cl
NH
N
14
15
0.018
0.009
0.082
0.014
0.026
0.009
Cl
N
NH2
In order to differentiate between the most potent examples (15,
22–28) we evaluated them in enzyme assays with high substrate
concentration ([ATP] = 5 mM) to better approximate intracellular
concentrations. Additionally, we further profiled these inhibitors
in cell proliferation, metabolic stability, aqueous solubility, and
hERG activity assays (Table 3). Our inhibitors showed good anti-
proliferative activity in a megakaryoblastic leukemia cell line,
MOLM-16, with GI50 values less than 100 nM.22 All of the leading
not shown). The more buried carbonyl oxygen at the 2-position of
the TZD displaces a water molecule often found in other PIM-1-li-
gand structures, for example that interacting with a triazolopyrid-
azine in PDB: 3BGQ.19b The primary amine is positioned between
the receptor carboxylate of Asp 128 and the backbone oxygen of
Glu 171 in the ribose pocket. Two crystallographic waters are near-
by, one of which interacts with the sidechain of Glu 171. An addi-
tional water molecule is located near the 4-carbonyl of the TZD and
is centered beneath the sidechain of the P-loop Phe 49 (water
O–Phe ring center, 3.4 Å). The hydrophobic isopropyl group is posi-
tioned between the hydrophobic residues Val 126, Leu 44 (not
examples exhibited low in vitro rat clearances (<12.1
lL/min/
mg), excellent aqueous solubilities, and low activity in the hERG
ion channel assay, which predicts for cardiac QT prolongation.23
An early representative of the series with potency against all
three isozymes (Table 2), compound 23 was tested against a panel
of 441 kinases at a single concentration of 1.0 lM to assess its

4602
L. A. Dakin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4599–4604
Figure 2. Human PIM-1 kinase in complex with 27 (PDB code: 4DTK). The structure is oriented with the N-terminal lobe towards the top left, the C-terminal lobe towards the
bottom right and the hinge region on the bottom left. Main chain is shown as a cartoon and selected residues of the protein are depicted with yellow carbon atoms. Carbon
atoms of 27 are shown in green. The electron density (2Fo À Fc), contoured at 1r, is shown as a wire mesh. Polar interactions 63.0 Å are shown as dotted lines. The P-loop has
been removed for the purposes of clarity.21
Table 3
Leading examples tested in PIM-1,-2, and -3 enzyme assays at high [ATP] = 5 mM (IC50
M), and hERG channel inhibition (IC50 M)
,
l
M), MOLM-16 (GI50, lM), rat microsomal stability (Clint, lL/min/mg), aqueous solubility
(l
, l
O
HN
O
S
N
NH2
R2
Compd
R2
PIM-1
PIM-2
PIM-3
MOLM-16
Microsomal stability
Solubility
hERG
15
22
23
24
25
26
27
28
29
H
CI
Br
OMe
OEt
i-BuO
i-PrO
Ph
0.10
0.02
0.01
0.02
0.01
0.008
<0.004
<0.003
0.004
1.20
0.31
0.15
0.32
0.16
0.14
0.05
0.15
0.69
0.24
0.02
0.01
0.49
0.19
0.10
0.21
0.08
0.08
0.02
0.02
2.28
<4.8
8.8
<4.8
3.9
12.1
6.9
<4.8
<4.8
—
782
527
>720
>1000
>1000
337
378
263
142
>30
>30
>30
>30
>30
23
>30
>30
>30
0.01
0.006
<0.003
<0.003
0.009
0.038
4-Pyr
selectivity profile (Fig. 3). The Ambit KINOMEscan™ technology
employs a competition binding assay that measures the ability of
a compound to compete with an immobilized, active-site directed
ligand.24 The three PIM kinases were the top hits with the highest
percentage of competition, and only 13 additional kinases (13/441,
In summary, we have described a novel class of TZDs which
are potent and selective inhibitors of the PIM family of kinases.
Starting from the micromolar PIM-2 HTS hit 1, we were able
to advance the series to examples with low nanomolar activity
against all three PIM isoforms. We have elucidated the binding
mode, and in so doing, demonstrated the significance of the
interaction between the TZD and the conserved lysine. Addition-
2.9%) were inhibited by greater than 50%. Neither PI3K
were inhibited at this concentration.
c nor CDK2

L. A. Dakin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4599–4604
4603
Figure 3. Example 23 tested in the Ambit kinase selectivity panel. Red circles correspond to kinases that are inhibited >65%, with size proportional to activity.25
11. Peltola, K.; Hollmen, M.; Maula, S.-M.; Rainio, E.; Ristamäki, R.; Luukkaa, M.;
Sandholm, J.; Sundvall, M.; Elenius, K.; Koskinen, P. J.; Grenman, R.; Jalkanen, S.
Neoplasia 2009, 11, 629.
ally, we have shown the beneficial effects on the potency of this
series through the incorporation of basic amines into the ribose
pocket and the strategic placement of hydrophobic groups. Sig-
nificantly, these inhibitors have been demonstrated to have good
anti-proliferative activity in the MOLM-16 cell line, high in vitro
stability, limited hERG activity, and high aqueous solubilities. As
such, this series of PIM inhibitors is currently under further
investigation, and those findings will be reported in due course.
12. Knight, S. D.; Adams, N. D.; Burgess, J. L.; Chaudhari, A. M.; Darcy, M. G.;
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R.; Kershner, K. L.; Luo, L.; Raha, K.; Sherk, C. S.; Sung, C.-M.; Sutton, D.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
05.098.
14. Richardson, C. M.; Nunns, C. L.; Williamson, D. S.; Parratt, M. J.; Dokurno, P.;
Howes, R.; Borgognoni, J.; Drysdale, M. J.; Finch, H.; Hubbard, R. E.; Jackson, P.
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[ATP] = 5 M; PIM-3 Km [ATP] = 50 M.
lM; PIM-2 Km
l
l
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a division of DiscoveRx Corporation,
ÓDISCOVERX CORPORATION 2010.