Synthesis and evaluation of novel inhibitors of pim-1 and pim-2 protein kinases

Article abstract of DOI:10.1021/jm800937p

The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. 5-(3-Trifluoromethylbenzylidene) thiazolidine-2,4-dione (4a) was identified by screening to be a Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although 4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases demonstrated that it has high selectivity for Pim kinases. Computational docking of 4a to Pim-1 provided a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized. The most potent new compounds exhibited IC ₅₀s of 13 nM for Pim-1 and 2.3 μM for Pim-2. Additional compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim- 2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall, these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.

Full text of DOI:10.1021/jm800937p

74
J. Med. Chem. 2009, 52, 74–86
Synthesis and Evaluation of Novel Inhibitors of Pim-1 and Pim-2 Protein Kinases
Zuping Xia,† Christian Knaak,† Jian Ma,‡ Zanna M. Beharry,† Campbell McInnes,§ Wenxue Wang,† Andrew S. Kraft,*,‡ and
Charles D. Smith*,†
Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, Medical UniVersity of South Carolina,
280 Calhoun Street, MSC140, Charleston, South Carolina, Department of Medicine, Medical UniVersity of South Carolina,
Charleston, South Carolina, Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, UniVersity of South
Carolina, Columbia, South Carolina
ReceiVed July 25, 2008
The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and
lymphoma. 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (4a) was identified by screening to be a
Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although
4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases
demonstrated that it has high selectivity for Pim kinases. Computational docking of 4a to Pim-1 provided
a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized.
The most potent new compounds exhibited IC50s of 13 nM for Pim-1 and 2.3 µM for Pim-2. Additional
compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim-
2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall,
these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.
Introduction
death from factor withdrawal-induced apoptosis.8 This result
may be explained by the observation that Pim can phosphorylate
the proapoptotic protein BAD, leading to sequestration by
14-3-3 proteins and inhibition of apoptosis.9-11 This phos-
phorylation event abrogates the ability of BAD to regulate the
activity of the Bcl-xL protein. It has now been shown that Pim-1
plays an important role in the ability of the FLT-3 protein kinase
to stimulate leukemic growth by phosphorylating and inactivat-
ing BAD and preventing cell death.12
Pim-1a and Pim-2 are serine/threonine protein kinases that
were originally cloned as proviral insertions in murine (Pim) T
cell lymphomas.1 The Pim-2 gene is 53% identical to Pim-1,
with the greatest divergence occurring at the amino and carboxy
termini of the encoded proteins. These kinases share the ability
to transform lymphoma cells, although it is not clear that they
have completely overlapping biochemical mechanisms of action.
The Pim protein kinases were first implicated in the development
of prostate cancer by DNA microarray analyses that showed
that Pim-1 is overexpressed in human prostate cancer and that
its expression correlates with clinical outcome.2 In humans,
enhanced levels of nuclear Pim-2 in the tumor cells has been
associated with a higher risk of prostate-specific antigen
recurrence and with perineural invasion of the prostate gland.3
Consistently, overexpression of Pim-1 has been reported to be
related to the grade of prostate cancer,4 with moderate-to-strong
cytoplasmic staining of Pim-1 being seen in tumors of 68% of
patients with a Gleason score of 7 or higher.5 Pim-1 also is
overexpressed in prostate intraepithelial neoplasia, and Pim
staining may be helpful in differentiating benign glands from
intraepithelial neoplasia.6
In spite of the interest in Pim-mediated signaling, there are
few known inhibitors of these enzymes. The potential for the
development of Pim-selective inhibitors is enhanced by the
crystal structure of Pim-1, which has been recently solved by
multiple groups.13-15 Importantly, the hinge region that contains
the ATP binding site has a novel architecture, containing an
additional amino acid residue not found in other protein kinases.
This residue, proline-123, is incapable of making hydrogen
bonds with ATP because of lack of a key amide H-bond donor.
This structural feature is a key determinant in the binding mode
of Pim inhibitors,13 and several groups have reported structurally
novel Pim kinase inhibitors. For example, the Meggers group
identified ruthenium half-sandwich complexes, which mimic
staurosporine and inhibit Pim-1 and other kinases.16,17 Also,
imidazo[1,2-b]pyridazines that inhibit Pim and block the growth
Additionally, overexpression of Pim-1 and Pim-2 in human
prostate cancer cells markedly enhances their growth as tumors
in nude mice7 and enforced expression of Pim-1 prevents cell
of leukemia cells in vitro have recently been described.18,19
A
similar chemotype is being developed for Pim inhibition by the
SuperGen company.20,21 In 2007, substituted pyridones22 and
specific flavinoids23,24 were shown to be Pim inhibitors.
However, it is well-known that staurosporine analogues and
flavinoids inhibit a variety of protein kinases, leaving the
therapeutic usefulness of such compounds unclear. Therefore,
improved inhibitors of Pim-1 are required not only for basic
research, but also as lead compounds for developing novel
anticancer agents. Pierce, et al. recently reported the use of
virtual screening for the identification of several Pim-1 inhibitors
with a variety of structures.25 The pharmacologic properties of
these agents remain to be described. The present report describes
* To whom correspondence should be addressed. For C.D.S.: phone,
(843) 792-3420; fax, (843) 792-9588; E-mail, smithchd@musc.edu. For
A.S.K.: phone, (843) 792-8284; fax, (843) 792-9456; E-mail, kraft@musc.edu.
Address: Hollings Cancer Center, 86 Jonathan Lucas Street, Charleston,
SC 29425.
†
Department of Pharmaceutical and Biomedical Sciences, South Carolina
College of Pharmacy, Medical University of South Carolina.
‡
Department of Medicine, Medical University of South Carolina.
Department of Pharmaceutical and Biomedical Sciences, South Carolina
§
College of Pharmacy, University of South Carolina.
a Abbreviations: 4E-BP1, eukaryotic translation initiation factor 4E
binding protein 1; mTOR, mammalian target of rapamycin; MTS, (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt; MTS); Pim, proviral insertions in murine; S6K, S6
protein kinase.
10.1021/jm800937p CCC: $40.75
2009 American Chemical Society
Published on Web 12/15/2008

Inhibitors of Pim-1 and Pim-2 Protein Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 75
rimidin-2-ylsulfanyl)-1-(4-fluoro-3-methylphenyl)ethanone (F7)
and 4-[3-(4-phenylaminophenyl)thioureidomethyl]benzenesulfo-
namide (H4) (Table 1), were tested for their activities against
Pim-1 in an independent in vitro assay and in a cellular assay.
Pim-1 protein kinase when overexpressed in prostate cancer cells
has been shown to increase the phosphorylation of 4E-BP1.7
On the basis of this in vivo activity, 4E-BP1 protein was shown
to be a good substrate for Pim protein kinases in vitro as well
(unpublished data). In the first assay, recombinant human Pim-1
protein was incubated with His-tagged 4E-BP1 protein and the
test compounds to determine their effects toward a full-length
substrate protein. As indicated in Figure 1, all three of the
compounds inhibited Pim-1 activity toward 4E-BP1, with 4a
and H4 being somewhat more potent than F7. As in the
screening assays, (S)-6, a staurosporine-based Pim inhibitor,16,17
was used as a positive control, and this compound inhibited
Pim-1 activity by approximately 50% at a concentration of 0.5
µM. Therefore, the inhibitory activities of the new compounds
were confirmed by an independent assay.
Figure 1. Inhibition of Pim-1 in a cell-free kinase assay. His-tagged
4E-BP1 protein was incubated with 0.1 µg Pim-1 protein kinase for
1 h at 30 °C along with [γ-32P]ATP and DMSO (solvent control, lane
1), 1.5 µM 4a (lane 2), 3 µM 4a (lane 3), 2.5 µM F7 (lane 4), 5 µM
F7 (lane 5), 2.5 µM H4 (lane 6), 5 µM H4 (lane 7), or 0.5 µM (S)-6
(lane 8), respectively, as described in the Experimental Section. As a
negative control, the sample in lane 9 lacked 4E-BP1. Radiolabeled
4E-BP1 was visualized by gel electrophoresis and autoradiography.
the identification of a new chemotype of isozyme-selective Pim
inhibitors with high potency.
Results and Discussion
Identification and Characterization of the Novel Pim-1
Inhibitor, 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-
dione (4a). Because of the relative lack of high-affinity inhibitors
of Pim kinases, we conducted a high-throughput screen for novel
inhibitors using recombinant human Pim-1 and S6 kinase/Rsk-2
peptide 2 as the substrate. Compounds from the DIVERSet
collection of the ChemBridge Corporation were tested at a final
concentration of 100 µM, and hits were defined as compounds
that inhibited Pim-1 activity by at least 50%. The known Pim-1
inhibitor (S)-616,17 was used as a positive control (Figure 6).
Nearly 50000 compounds were screened, and 10 compounds
with IC50s of 20 µM or less were identified (0.034%). This low
hit rate reflects the assay design that utilized a relatively high
concentration of ATP to select for allosteric or high-affinity
ATP-binding site-directed compounds. The four most-potent
screening hits are shown in Table 1. Of the Pim-1 inhibitors
with IC50s < 20 µM, four compounds contain a benzylidene-
linked thiazolidine or imidazolidine nucleus substituted at the
2- and 4- positions by oxo- or thioxo-moieties.
The potency of 4a for inhibition of Pim-1 kinase activity was
determined using 4E-BP1 as the substrate. 4a caused a dose-
dependent reduction in Pim-1-induced 4E-BP1 phosphorylation,
with an IC50 of approximately 125 nM (data not shown). It is
interesting to note that this is considerably more potent than
was determined in the conditions of the screening assay, i.e., 3
µM. This difference in potency may arise from the higher
catalytic efficiency of Pim-1 toward the full-length protein
substrate in comparison with the S6K peptide used in the
screening assay. Similarly, Pim-1 was found to be much more
active using a substrate peptide that corresponds to Bad
compared with the S6K peptide used in the initial studies. With
the Bad peptide, the IC50s for 4a and most of the analogues
discussed below were much lower than that for the S6K peptide,
reflecting excellent inhibition of the enzyme operating under
optimal conditions.
The most potent compound of this chemotype (4a), as well
as two structurally dissimilar compounds (2-(4,6-dimethylpy-
Table 1. Structures and Potencies of Pim-1 Inhibitors from the ChemBridge Library

76 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Xia et al.
Figure 2. Inhibition of Pim-1 autophosphorylation in intact cells.
cDNA-encoding Flag-Pim-1 was transfected into HEK-293T cells and
cells were labeled with 32PO4 for 3 h. The following compounds (0.5
µM) were added and the incubations were continued for an additional
1 h: (S)-6 (lane 1), H4 (lane 2), 4a (lane 3), F7 (lane 4), or DMSO
(solvent control, lane 5). Pim-1 was immunoprecipitated with Flag beads
and 1/10 of the beads were analyzed by Western blotting (input), while
the remaining material was analyzed by SDS-PAGE and autoradiog-
raphy to visualize the autophosphorylation level of Pim-1 as described
in the Experimental Section.
Figure 4. Computational docking of 4a to Pim-1. Left panel: docking
of 5-[3-(trifluoromethyl)benzylidene]-1,3-thiazolidine-2,4-dione (4a)
with the crystal structure of Pim-1 kinase (1XWS) showing interacting
residues and pharmacophore centers near the ATP binding site. Right
panel: 2-D pharmacophore for 4a bound to Pim-1.
Table 2. Selectivity of 4a and 16aa
Figure 3. Kinetics of inhibition of Pim-1 by 4a. Pim-1 kinase activity
was measured using the coupled assay in the presence of the indicated
concentrations of ATP and 0 (b), 5 (9), or 10 (2) µM 4a as described
in the Experimental Section.
Importantly, the abilities of the compounds to inhibit Pim-1
activity in intact cells were also determined. In this assay, Pim-1
activity was measured as the extent of its autophosphorylation.
293T cells were transfected with Flag-Pim-1 and labeled with
32PO4 and then treated with the test compound for 1 h. As
demonstrated in Figure 2, the amount of Flag-Pim-1 recovered
from the cells did not change upon exposure to the test
compounds. Pronounced autophosphorylation of Pim-1 was
evident in all of the samples except those from cells treated
with 4a, indicating that this compound is a very effective
inhibitor of intracellular Pim-1 autophosphorylation. The lack
of inhibition by the known Pim-1 inhibitor (S)-6 confirms that
this compound is poorly delivered to the intracellular kinase,
which is consistent with its known high-binding to serum
proteins. As it is clearly advantageous that the Pim-1 inhibitor
be cell permeable, subsequent studies were focused on 4a and
its congeners.
To test for competition with ATP, the effects of 4a at different
ATP concentrations were determined. As indicated in Figure
3, 4a acts as a competitive inhibitor with respect to ATP, with
a calculated Ki of 0.6 µM. To test the selectivity of 4a, it was
sent to Caliper Life Sciences for profiling against a diverse panel
of protein kinases. As shown in Table 2, 5 µM 4a inhibited
Pim-1 and Pim-2 but did not significantly inhibit the other 47
serine/threonine- or tyrosine-kinases tested. Similar testing of
a 4a analogue 16a indicated that this compound also is highly
selective for Pim kinases, although the kinase DYRK1R was
inhibited to a similar extent as Pim-1 and Pim-2. This finding
is similar to the recent findings of Pagano et al.,26 who found
Pim kinases and DYRK1R to be similarly sensitive to a series
a The effects of 4a or 16a on the indicated protein kinases were
determined through the Rapid KinaseAdvisor profiling service from Caliper
Life Sciences. Values represent the percent inhibition of each kinase at 5
µM of the test compound.
of 4,5,6,7-tetrabromon-1H-benzimidazoles. Together, the data
indicate that this chemotype has excellent selectivity for Pim
kinases.
Molecular Docking of 4a to Pim-1. The molecular structure
for compound 4a, 5-[3-(trifluoromethyl)benzylidene]-1,3-thia-
zolidine-2,4-dione, was built and docked with the crystal
structure of Pim-1 (PDB code: 1XWS) using the suite of
programs within the DiscoveryStudio interface (Accelrys)
(Figure 4). The ligand and protein structures were prepared for
docking by the addition of hydrogens, followed by the assign-
ment of atom types and charges within the CHARMm forcefield.
This was followed by creation of the docking grids within a 6
Å radius of the native ligand and molecular mechanics/energy
minimization docking of flexible 4a into the rigid binding site
of Pim-1. After generation of 100 poses, plausible binding
modes were generated through energetic ranking by CDocker
nonbonded energies and similarity to known interactions for
Pim-1 ligands within the RCSB protein databank. Evaluation
with these criteria suggested a binding mode for 4a in which
the thiazolidine NH group donates a H-bond to the carbonyl
oxygen of Glu121 and the trifluorophenyl group makes numer-

Inhibitors of Pim-1 and Pim-2 Protein Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 77
romethylbenzen (6a1) or 2-methoxyethoxy-1-trifluoromethyl-
benzene (7a1). Because methoxymethoxy and methoxyethoxy
groups are strongly ortho-directing,29 further intermediates were
ortho-metalated with nBuLi. The corresponding arylaldehydes
(6a2 and 7a2) were obtained by addition of DMF to the
metalated intermediates, followed by hydrolysis with water. The
arylaldehydes were then condensed with 2,4-thiazolidinedione
to afford the compounds 6a and 7a with quite low yields,
probably due to the steric hindrance of the arylaldehydes by
the 2-position substitution.
Also suggested by computational modeling, compounds 5a
and 19a were synthesized via a three-component condensation
in a one-pot reaction using the ionic solvent 1-n-butyl-3-
methylimidazolium hexafluorophosphate as depicted in Scheme
3.30 However, an attempt to synthesize 3-(2-fluorobenzyl)-5-
(4-methoxybenzylidene)thiazolidine-2,4-dione by this reaction
was unsuccessful, producing only compound 25b, 3-(2-fluo-
robenzyl)thiazolidine-2,4-dione.
5-Arylidene-3-benzylhydantoins (26c-31c) were also sug-
gested by computational modeling and were synthesized using
the strategy as described in Scheme 4. Although this is similar
to that used for the preparation of 5-arylidenethiazolidine-2,4-
diones, the reactions did not always proceed smoothly and often
resulted in a mixture of Z/E isomers, which were difficult to
separate by routine silica gel chromatography. As expected, the
E-isomer was the major compound in the Z/E-mixtures.31
Figure 5. Antitumor activity of 16a. Female Balb/C mice were injected
subcutaneously with JC cells (1 × 106) suspended in PBS. After
palpable tumor growth, animals were treated five days per week by
intraperitoneal injection of vehicle alone (1) or 50 mg/kg of 16a (9).
Values represent the mean ( standard error tumor volumes. n ) 5
mice per group.
ous van der Waals contacts with the hydrophobic cleft. These
results suggested that the thiazolidine ring in particular is a good
candidate for modification, as according to the pharmacophore
model shown in Figure 4 right, this ring system only contributes
a hydrogen bond to the hinge region while making few other
nonbonded contacts. While the thiazolidine ring lacks scope for
diversity, the alternative imidazoline ring has potential for
substitution on the N1 with hydrophobic groups as observed
with the XWS ligand. The carbonyl groups could be replaced
with hydrophobic functionality in order to exploit contacts with
Pro123 and Leu44 among other residues. Appropriate modifica-
tion of the aromatic ring also was indicated in initial modeling
results to have potential for increasing potency of this series
and an initial set of derivatives was proposed. Further refinement
of the binding mode was completed subsequent to obtaining
SAR information from the synthesized compounds.
Synthesis of 4a Analogues. As indicated above, the com-
pound 4a and a few congeners were identified as Pim-1
inhibitors through screening a commercial library of compounds.
Therefore, we synthesized a series of congeners to begin SAR
studies of this chemotype, and the approach to the synthesis of
5-arylidene-2,4-thiazolidinediones (1a-4a, 8a-18a, and
20a-24a, as indicated in the Table 3) is shown in Scheme 1.
As described by Momose et al.27 and Bruno et al.,28 2,4-
thiazolidinedione can undergo a Knoevenagel condensation with
a variety of meta- and para-substituted benzaldehydes to
produce 5-arylidene-2,4-thiazolidinones (Scheme 1). In this
piperidine-catalyzed reaction, the exocyclic double bond is
exclusively in the Z configuration because of the high degree
of thermodynamic stability of this isomer. Thirty-three com-
pounds within the benzylidene-thiazolidine-2,4-dione chemotype
were synthesized and evaluated, demonstrating that these
compounds can be made through a Knoevenagel condensation
with a variety of meta- and para-substituted benzaldehydes with
good-to-excellent yields.
Evaluation of 4a Analogues. Each of these compounds was
tested for its ability to inhibit recombinant Pim-1 using the ATP-
depletion assay described above, except that the ATP concentra-
tion was 0.1 µM and the Bad peptide was used as the phosphate
acceptor. The results are summarized in Table 3. The data
demonstrate that several new compounds are significantly more
potent than the original screening hit, with IC50s for inhibition
of Pim-1 ranging from 0.013 to 45 µM and for inhibition of
Pim-2 ranging from 0.02 to >100 µM. Substitution of the
thiazolidine nitrogen decreased the potency of the compounds
by more than 1000-fold, e.g., 4a vs 5a. Benzylidines with Cl-
or F-substitutions at the 4-position were approximately 10-fold
more potent than the corresponding 3-substituted compounds,
while 3-Br- and 4-Br-congeners had poor Pim-1 inhibitory
activity but substantial Pim-2 inhibitory activity. Trifluo-
romethoxy-substituted compounds were 10- to 15-fold more
potent than the corresponding methoxy compounds, e.g., 11a
vs 10a. It is also interesting to note that certain compounds are
significantly more potent toward either Pim-1 or -2 than the
other isozyme. For example, 9a is more than 2500-fold selective
for Pim-1, and 24a is more than 400-fold selective for Pim-2.
This provides the opportunity to pharmacologically distinguish
the roles of the kinase in prostate cancer biology. Compounds
that inhibit both isozymes, e.g., 16a, may be more effective
as therapeutic agents if there is redundancy in the functions of
Pim-1 and Pim-2.
After the synthesis and evaluation of Pim inhibitors based
on the initial computational design, retrospective analysis of
the structure-activity relationship and further refinement of the
3-D pharmacophore was carried out. This analysis showed that
a refined binding mode as shown in Figure 4 best explained
the SAR and provided insights for the design of next generation
compounds. The potency increase of the triflouromethoxy and
the smaller halogen substitutions on the benzylidine can be
attributed to more optimal interactions of these groups with
Phe49 of the Pim-1 active site. The decreased binding affinity
of analogues derivatized at R3 is explained by steric clashes of
the ortho substitutions with Val52 or Ile185. This was not
Compounds 6a and 7a were suggested by the initial round
of computational modeling based on the docking of the
screening hit 4a. They were prepared with a three-step reaction
applying ortho-lithiation strategies illustrated in Scheme 2.
2-Trifluoromethylphenol was treated with sodium hydride,
followed by reaction with chloromethylmethyl ether or chlo-
roethylmethyl ether to produce 2-methoxymethoxy-1-trifluo-

78 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Table 3. Biological effects of the new compoundsa
Xia et al.
geometric
IC50 for Pim-1
IC50 for Pim-2
IC50 for PC3
compound
isomer
R1
CH3
H
CF3
H
H
H
H
C2H5
(CH3)2CH
CH3O
CF3O
H
H
C2H5O
H
C3H7O
(CH3)2N
F
F
H
Cl
H
Br
H
R2
R3
R4
(µΜ)
(µΜ)
cells (µΜ)
1a
2a
3a
4a
5a
6a
7a
8a
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
H
CH3
H
CF3
CF3
CF3
CF3
H
H
H
H
H
H
H
H
H
H
0.06 ( 0.02
0.16 ( 0.04
0.33 ( 0.13
0.024 ( 0.006
28 ( 22
0.46 ( 0.32
0.28 ( 0.13
0.6 ( 0.1
0.04 ( 0.03
5.1 ( 5.0
0.3 ( 0.2
0.65 ( 0.05
0.067 ( 0.061
0.17 ( 0.04
0.073 ( 0.053
0.15 ( 0.11
6.0 ( 1.7
4.4 ( 0.2
0.1 ( 0.1
0.08 ( 0.02
0.1 ( 0.3
>100
1.5 ( 0.1
42 ( 12
0.3 ( 0.1
>100
0.3 ( 0.1
0.5 ( 0.2
0.04 ( 0
0.9 ( 0.4
43 ( 18
2.2 ( 1.1
0.02 ( 0.01
0.1 ( 0.1
2.3 ( 0.1
>100
>100
97 ( 0
38 ( 31
17 ( 6
>100
68 ( 18
80 ( 12
65 ( 18
28 ( 13
11 ( 1
48 ( 27
73 ( 13
6.4 ( 2.4
65 ( 17
3.2 ( 0.5
48 ( 8
CH2C6H5-4-OCF3
CH3OCH2O
CH3O(CH2)2O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9a
H
H
H
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25b
26c
27c-1
27c-2
28c
29c-1
30c
31c
CH3O
CF3O
H
CF2HCF2O
H
H
H
H
F
H
Cl
H
Br
66 ( 13
93 ( 5
H
0.013 ( 0.01
45 ( 15
CH2C6H4-2-F
H
>100
0.13 ( 0.06
0.04 ( 0.03
0.60 ( 0.51
28 ( 23
0.4 ( 0.2
0.2 ( 0.1
0.04 ( 0.15
0.09 ( 0.04
0.1 ( 0.02
>100
83 ( 9
H
H
H
H
49 ( 8
63 ( 6
85 ( 12
58 ( 11
88 ( 10
67 ( 28
74 ( 20
>100
45 ( 11
CH2C6H4-2-F
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
63 ( 3.0
E/Z (9/1)
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
69 ( 6.5
>100
E
CH3O
CH3O
C2H5O
Br
(CH3)2N
H
38 ( 23
>100
E/Z (83/17)
E/Z (4/1)
E
75 ( 5.0
>100
73 ( 7.5
>100
18 ( 9.7
68 ( 22
57 ( 23
80 ( 15
58 ( 7.5
>100
E
78 ( 7.5
>100
E/Z (5/1)
CH3O
78 ( 2.5
>100
a Inhibition of Pim-1 and Pim-2 enzymatic activity and PC3 cell proliferation by arylidene-2,4-thiazolidinediones (1a-24a), 3-(2-fluorobenzyl)thiazolidine-
2,4-dione (25b), and arylidenehydantoins (26c-31c) are shown. Data represents the mean ( SEM for at least 3 experiments.
Scheme 1. General Synthesis of 5-Arylidene-2,4-thiazolidinediones
predicted in the initial binding hypothesis, which described a
different conformation of the benzylidine group. Further opti-
mization of activity and selectivity is possible through insights
provided by the refined binding mode.
The cytotoxicity of each of the compounds toward PC3
prostate carcinoma cells was determined. The compounds
exhibited IC50s that ranged from 3 to >100 µM, with the least
effective Pim inhibitors generally having no toxicity at the
highest dose tested. Because the IC50 for proliferation reflects
both the affinity of the compound for the target and its ability
to enter the cells, this series of compounds appears to have
reasonably poor penetration. Alternately, inhibition of Pim alone
may not be sufficient to drive these cells into apoptosis, and
combinatorial studies with other signaling inhibitors and cyto-
toxic drugs will be conducted.
Finally, we have begun to characterize the in vivo toxicity
and efficacy of a representative Pim inhibitor. Compound 16a
was chosen because it has excellent potency for inhibition of
both Pim-1 and Pim-2. Dose-escalation studies indicated that
mice tolerate intraperitoneal doses of the compound of 50 mg/

Inhibitors of Pim-1 and Pim-2 Protein Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 79
Scheme 2. Synthesis of Compounds 6a and 7aa
a (a) NaH/DMF/Cl(CH2)nOCH3; (b) (1) nBuLi, -78 °C, Ar, (2) DMF, RT; (c) piperidine, reflux.
Scheme 3. Synthesis of 3-Aryl-5-arylidene-2,4-thiazolidinediones
Scheme 4. Synthesis of 5-Arylidene-3-benzylhydantoins
Table 4. Toxicologic Evaluation of 16aa
parameter
units
control
3 mg/kg 16a
10 mg/kg 16a
50 mg/kg 16a
white blood cells
lymphocytes
monocytes
granulocytes
red blood cells
hemoglobin
albumin
109/L
3.55–9.83
3.44–8.88
0.04–0.36
0.06–0.36
10.37–11.76
13.2–14
3.2–3.4
3.73–7.99
3.61–6.78
0.11–0.34
0.01–0.91
10.37–11.33
12.8–14
3.3–3.4
4.95–7.03
3.94–5.84
0.08–0.28
0.61–0.93
9.22–10.93
11.7–14.1
2.9–3.4
3.65–7.1
3.5–5.62
0.04–0.35
0.08–1.13
8.99–10.98
12.2–13.5
2.6–3.1
109/L
109/L
109/L
1012/L
g/dL
g/dL
alkaline phosphatase
alanine aminotransferase
amylase
blood urea nitrogen
phosphate
U/L
U/L
U/L
mg/dL
mg/dL
mMg/dL
mmol/L
mmol/L
mg/dL
87–134
47–87
926–981
15–19
5.7–9.5
0.2–0.3
150–155
7.7–7.9
85–127
40–134
878–1206
17–19
5.1–5.6
0.2–0.4
150–155
7.3–7.5
85–113
44–46
816–1054
11–16
5.7–6.2
0.2–0.3
144–151
7.2–8.2
80–86
37–52
760–967
14–22
5.5–8.1
0.2–0.3
145–153
6.3–6.7
creatinine
Na+
K+
glucose
70–94
80–90
87–129
136–163
a The indicated doses of compound 16a were administered by intraperitoneal injection daily for 7 days and blood was collected after an additional 7 days
of observation. The ranges of values for cell counts and blood chemistry are given.
kg daily for 5 days, while 100 mg/kg is overtly toxic. Therefore,
toxicity and antitumor studies were limited to doses of or below
50 mg/kg. As indicated in Table 4, subchronic dosing with 16a
did not affect the levels of red or white blood cells, including
lymphocytes, monocytes, and granulocytes, indicating that the
compound does not have myelosuppressive effects. The blood
chemistry profile also indicated that 16a does not have toxicity
toward the liver as the albumin, alkaline phosphatase, and
alanine aminotransferase levels were unchanged. Similarly,
pancreatic function, monitored by amylase activity, and kidney
function, monitored by electrolyte, blood urea nitrogen, and
creatinine levels, were also unaltered in the 16a-treated mice.
However, elevations in blood glucose levels were detected in
mice treated with the highest dose of 16a, suggesting an
alteration in metabolism, which is consistent with inhibition of
signaling through mTOR, which is known to be regulated by
Pim activity.32 To evaluate the antitumor activity of 16a, the
compound was administered to Balb/c mice bearing tumors of
JC murine mammary adenocarcinoma cells. As indicated in
Figure 5, treatment of the animals with 16a for 5 days per week
did not cause a loss of body weight, consistent with the
toxicology studies described above. However, the compound

80 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Xia et al.
Proton magnetic resonance and carbon magnetic resonance were
recorded on Varian INOVA 400 MHz spectrometer. High resolution
mass spectra were determined by the Department of Chemistry at
the University of South Carolina. Radial chromatography was
performed on a Harrison Research Chromatotron using EMD 60
PF254 containing gypsum. Thin layer chromatography was per-
formed with Sigma-Aldrich silica gel aluminum-backed plate with
UV visualization. The following synthetic conditions have not been
optimized.
(Z)-5-(4-methylbenzylidene)thiazolidine-2,4-dione (1a). 2,4-
Thiazolidinedione (118 mg, 1 mmol) and 4-tolualdehyde (119 µL,
121 mg, 1 mmol) were dissolved in ethanol (7 mL), followed by
addition of piperidine (79 µL, 0.80 mmol). After refluxing for 24 h,
the mixture was poured into water (∼60 mL) and acidified with
acetic acid to a pH of 3-4 and left at 4 °C overnight. After filtration
and washing with methanol, a yellow solid was collected (150 mg,
0.68 mmol, 68% yield). 1H (DMSO) δ 7.76 (s, 1 H), 7.49 (d, J )
8.0 Hz, 2 H), 7.35 (d, J ) 8.0 Hz, 2 H), 2.36 (s, 3 H). 13C NMR
(DMSO) δ 168.1, 167.6, 140.9, 132.0, 130.5, 130.2 (2 C), 130.1
(2 C), 122.5, 21.2.
(Z)-5-(3-methylbenzylidene)thiazolidine-2,4-dione (2a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and m-tolualdehyde (118 µL,
120 mg, 1 mmol) were dissolved in ethanol (7 mL), followed by
addition of piperidine (79 µL, 0.8 mmol). After refluxing for 20 h,
the yellow solution was poured into water (∼60 mL) and acidified
with acetic acid to a pH of 3-4, and left at 4 °C overnight. After
filtration, the collected solid was carefully washed with methanol
1
to afford an orange product (135 mg, 0.62 mmol, 62% yield). H
(DMSO) δ 12.59 (bs, 1 H), 7.75 (s, 1 H), 7.37-7.45 (m, 3 H),
7.30 (d, J ) 7.2 Hz, 1 H), 2.37 (s, 3 H). 13C NMR (DMSO) δ
168.1, 167.5, 138.8, 133.2, 132.0, 131.3, 130.7, 129.4, 127.2, 123.5,
21.1.
(Z)-5-(4-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (3a).
2,4-Thiazolidinedione (119 mg, 1 mmol) and 4-trifluoromethyl-
benzaldehyde (136 µL, 177 mg, 1 mmol) were dissolved in ethanol
(7 mL), followed by addition of piperidine (79 µL, 0.8 mmol). The
mixture was refluxed for 24 h. The yellow solution was poured
into water (∼60 mL), and a yellow precipitate appeared im-
mediately. The mixture was acidified with acetic acid to a pH of
3-4 and then left at 4 °C overnight. After filtration, the yellow
solid was washed with methanol to afford the product (147 mg,
Figure 6. Structures and potencies of reported Pim-1 inhibitors.
reduced the growth of tumors by approximately 50%. Therefore,
the Pim inhibitors of this chemotype have good potential for
further optimization as anticancer agents.
1
0.54 mmol, 54% yield). H (DMSO) δ 7.89 (d, J ) 8.4 Hz, 2 H),
7.87 (s, 1 H), 7.81 (d, J ) 8.4 H, 2 H). 13C NMR (DMSO) δ 167.7,
167.3, 137.2, 130.6 (2 C), 130.0, 129.7, 126.9, 126.2 (t, J ) 3.8
Hz, 2 C), 120.4 (t, J ) 271 Hz, 1 C).
Conclusions
Pim protein kinases are receiving increasing attention because
of their roles in prostate cancer and leukemias. Consequently,
several types of Pim-1 inhibitors have recently been described
(Figure 6), with several examples that take advantage of the
unique structure of the ATP binding domain of these kinases.
Substituted thiazolidine-2,4-diones provide a new chemotype
that inhibits the Pim family of protein kinases with excellent
selectivity in spite of their acting as ATP competitors. A binding
mode that rationalizes this selectivity has been proposed by
computational docking and provides a model for the further
refinement of this chemotype. Interestingly, the current com-
pounds include examples that selectively target Pim-1 or Pim-
2, thereby providing new pharmacologic probes that may be
useful for differentiating the biological roles of these Pim
isozymes. Initial in vivo testing of a potent Pim-1 and Pim-2
inhibitor demonstrates the ability of these compounds to
attenuate tumor growth in the absence of undue toxicity to the
host. Therefore, the new Pim inhibitors have considerable
promise for development as new antitumor and antileukemia
agents.
(Z)-5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (4a).
2,4-Thiazolidinedione (119 mg, 1 mmol) and 3-trifluoromethyl-
benzaldehyde (136 µL, 177 mg, 1 mmol) were dissolved in ethanol
(7 mL), followed by addition of piperidine (79 µL, 0.8 mmol). After
reflux for 20 h, the yellow solution was poured into water (∼60
mL) and acidified with acetic acid to a pH of 3-4. After filtration,
the solid was purified by radial chromatography (silica) eluted with
5% methanol in chloroform to afford an off-white white solid (79
mg, 0.29 mmol, 29% yield). Rf ) 0.25, silica, 3% methanol in
chloroform. 1H (CDCl3) δ 7.88 (s, 1 H), 7.75 (s, 1 H), 7.70 (d, J )
7.6 Hz, 1 H), 7.68 (d, J ) 7.6 H, 1 H), 7.63 (J1 ) J2 ) 7.6 Hz, 1
H). 13C NMR (CDCl3) δ 166.5, 166.3, 133.8, 132.7, 132.5, 132.0
(t, J ) 32.6 Hz, 1 C), 129.9, 127.1 (t, J ) 3.8 Hz, 1 C), 127.0 (t,
J ) 3.8 Hz, 1 C), 124.6, 123.5 (t, J ) 271 Hz, 1 C).
(Z)-3-(4-Trifluoromethoxybenzyl)-5-(3-trifluoromethylben-
zylidene)thiazolidine-2,4-dione (5a). 2,4-Thiazolidinedione (59
mg, 0.5 mmol) and m-trifluorotolualdehyde (67 µL, 87 mg, 0.5
mmol) were dissolved in 1-butyl-3-methylimidazolium hexafluo-
rophosphate, [bmim]PF6, (2 mL), followed by addition of Et3N (84
µL, 0.6 mmol) and 4-trifluoromethoxybenzyl bromide (0.6 mmol,
96 µL). The reaction was carried out at 60 °C for 17 h. After cooling
down to room temperature, the mixture was extracted with ether
(4 × 15 mL). After filtration, the concentrated residue was purified
by radial chromatography (silica) eluted with 15-25% ethyl acetate
in hexane to afford a semisolid (5 mg, 0.011 mmol, 2% yield). Rf
Experimental Section
Chemistry. All chemical reagents were purchased from Sigma-
Aldrich except ethanol (200 proof) that was from EMD Chemicals.
1
) 0.28, 25% ethyl acetate in hexane. H (CDCl3) δ 7.92 (s, 1 H),

Inhibitors of Pim-1 and Pim-2 Protein Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 81
7.73 (s, 1 H), 7.59-7.70 (m, 3 H), 7.48 (d, J ) 8.0 Hz, 2 H), 7.19
(d, J ) 8.0 Hz, 2 H), 4.90 (s, 2 H). 13C NMR (CDCl3) δ 167.3,
165.9, 149.5, 134.2, 133.8, 132.8, 132.5, 132.1 (q, J ) 33 Hz, 1
C), 130.8 (2 C), 130.1, 127.0-127.2 (m, 2 C), 123.8, 123.7 (q, J
) 271 Hz, 1 C), 121.5 (2 C), 120.6 (q, J ) 256 Hz, 1 C), 44.8.
HR-MS (EI): calcd 447.0364 for C19H11F6NO3S, found 447.0360.
1-Methoxymethoxy-2-trifluoromethoxybenzene (6a1). A sus-
pension of 60% NaH (1.07 g, 27 mmol) in anhydrous DMF (15
mL) was chilled to 0 °C under argon, and 2-trifluoromethylphenol
(3.72 g, 23 mmol) in DMF (5 mL) was added to it dropwise. The
the mixture was allowed to warm to RT and stirred for 1 h, followed
by the slow addition of chloromethyl methyl ether (2.97 g, 37 mmol)
and the suspension was stirred overnight. Note: smoke was produced
during this addition. Ice-water (30 mL) was then slowly added,
and the suspension became clear solution. The mixture was
extracted with ether (3 × 30 mL), and the collected ether was
washed with 1 N NaOH (30 mL), 1 N HCl (30 mL), and 10%
NaCl (30 mL) sequentially and then dried over sodium sulfate. After
passing through a pad of silica eluted with ether, the solvent was
removed with nitrogen flushing to afford a colorless oil (3.42 g,
72%). Rf ) 0.44, 25% ethyl acetate in hexane. 1H (CDCl3) δ 7.57
(d, J ) 7.6 Hz, 1 H), 7.46 (dd, J1 ) 8.4 Hz, J2 ) 7.6 Hz, 1 H),
7.22 (d, J ) 8.4 Hz, 1 H), 7.04 (dd, J1 ) J2 ) 7.6 Hz, 1 H), 5.25
(s, 2 H), 3.49 (s, 3 H). 13C NMR (CDCl3) δ 155.0 (m, 1 C), 133.3,
127.1 (q, J ) 5.3 Hz, 1 C), 123.8 (q, J ) 270 Hz, 1 C), 121.2,
119.7 (q, J ) 30.3 Hz, 1 C), 115.3, 94.3, 56.4.
132.3, 129.5, 129.4 (q, J ) 5.3 Hz, 1 C), 128.4, 125.8 (q, J ) 30
Hz, 1 C), 125.0, 124.9, 123.2 (q, J ) 271 Hz, 1 C), 102.5, 58.3.
HR-MS (EI): clcd 333.0283 for C13H10F3NO4S, found 333.0283.
1-Methoxyethoxy-2-trifluoromethylbenzene (7a1). A suspen-
sion of 60% NaH (1.05 g, 26 mmol) in DMF (15 mL) was chilled
to 0 °C under argon, and 2-trifluoromethylphenol (3.38 g, 21 mmol)
in DMF (5 mL) was added to it dropwise. The mixture was allowed
to warm to RT and was stirred for 1 h, followed by the slow addition
of bromoethyl methyl ether (3.59 g, 26 mmol). The reaction was
continued for 18 h, and then ice-water (30 mL) was slowly added
to it. The mixture was extracted with ether (3 × 30 mL), and the
collected ether was washed with 1 N NaOH (30 mL), 1 N HCl (30
mL), and 10% NaCl (30 mL) sequentially and then dried over
sodium sulfate. After filtration through a funnel with cotton, the
solvent was removed with nitrogen flushing to afford a colorless
1
oil (3.55 g, 77% yield). Rf ) 0.62, ether. H (CDCl3) δ 7.55 (d, J
) 7.6 Hz, 1 H), 7.46 (dd, J1 ) J2 ) 7.6 Hz, 1 H), 6.97-7.01 (m,
2 H), 4.18 (t, d ) 4.8 Hz, 2 H), 3.78 (d, J ) 4.8 Hz, 2 H), 3.45 (s,
3 H). 13C NMR (CDCl3) δ 156.9 (m, 1 C), 133.3, 127.1 (q, J )
5.3 Hz, 1 C), 123.8 (q, J ) 270 Hz, 1 C), 120.4, 119.2 (q, J )
30.3 Hz, 1 C), 113.3, 71.0, 68.9, 59.6.
2-Methoxyethoxy-3-trifluoromethylbenzaldehyde (7a2). A
dried (100 mL) three-necked flask was charged with freshly dried
THF (20 mL) and chilled to <-70 °C under argon. A solution of
N,N,N′,N′-tetramethylethylenediamine (960 µL, 6.5 mmol) and 2.5
M nBuLi in hexane (2.6 mL, 6.5 mmol) was then added and the
mixture was recooled to reached <-70 °C. 1-Methoxyethoxy-2-
trifluoromethylbenzene (972 mg, 4.4 mmol) in THF (5 mL) was
then added dropwise, keeping the temperature <-70 °C, and the
mixture was stirred for 1 h. DMF (501 µL, 6.5 mmol) was then
added and the cooling bath was removed after 5 min, allowing the
temperature to gradually rise to RT for 20 h. After the dropwise
addition of saturated NH4Cl (25 mL), the mixture was extracted
with ethyl acetate (3 × 25 mL), and the combined ethyl acetate
was washed with 1 N HCl (25 mL), water (25 mL), and 10% NaCl
(25 mL) and dried over sodium sulfate. After passing through a
pad of silica gel eluted with 25% ethyl acetate in hexane, the
concentrated residue was purified by radial chromatography (silica)
eluted with 5-15-25-50-100% ethyl acetate in hexane to afford
an oil (Rf ) 0.09, chloroform). A second cycle of radial chroma-
tography (silica) eluted with hexane-chloroform was necessary to
afford the product (32 mg, 0.13 mmol, 3% yield). Rf ) 0.27, 1%
methanol in chloroform. 1H (CDCl3) δ 10.5 (bd, J ) 0.8 Hz, 1 H),
8.06 (dd, J1 ) 7.6 Hz, J2 ) 1.2 Hz, 1 H), 7.86 (dd, J1 ) 7.6 Hz,
J2 ) 1.2 Hz, 1 H), 7.36 (dd, J1 ) J2 ) 7.6 Hz, 1 H), 4.24 (t, J )
4.4 Hz, 2 H), 3.79 (t, J ) 4.4 Hz, 2 H), 3.45 (s, 3 H). 13C NMR
(CDCl3) δ 189.3, 160.4, 133.0 (q, J ) 5.2 Hz, 1 C), 132.9, 131.1,
125.5 (q, J ) 31 Hz, 1 C), 124.7, 123.3 (q, J ) 272 Hz, 1 C),
78.4, 71.6, 59.3.
Methoxymethoxy-3-trifluoromethylbenzaldehyde (6a2). A
dried (100 mL) three-necked flask was charged with freshly dried
THF (20 mL) and chilled to <-70 °C under argon. N,N,N′,N′-
tetramethylethylenediamine (480 µL, 3.25 mmol) and 2.5 M nBuLi
in hexane (1.3 mL, 3.25 mmol) were then added and the system
was recooled to <-70 °C. 1-Methoxymethoxy-2-trifluoromethyl-
benzene (432 mg, 2.1 mmol) in anhydrous THF (5 mL) was then
added dropwise so that the temperature was kept below -70 °C
and the mixture was stirred for 1 h. DMF (250 µL, 3.25 mmol)
was then added to it, and the cooling bath was removed after 5
min. The temperature was gradually raised to room temperature
and the color of the mixture changed to red from yellow and the
reaction was continued for 20 h at room temperature. After dropwise
addition of saturated NH4Cl (25 mL), the mixture was extracted
with ethyl acetate (3 × 25 mL). The combined ethyl acetate was
washed with 1 N HCl (25 mL), water (25 mL), and 10% NaCl (25
mL) sequentially and then dried over sodium sulfate. After passing
through a pad of silica gel eluted with 25% ethyl acetate in hexane,
the concentrated residue was purified by radial chromatography
(silica) eluted with 5-15-25-50-100% ethyl acetate in hexane
to afford a yellowish oil (Rf ) 0.43, 25% ethyl acetate in hexane).
NMR spectra indicated that the compound was not pure, so it was
further purified by radial chromatography (silica) eluted with
hexane-chloroform to afford the pure compound (100 mg, 20%
1
(Z)-5-(2-Methoxyethoxy-3-trifluoromethyl-benzylidene)thia-
zolidine-2,4-dione (7a). Piperidine (5.6 µL, 0.056 mmol) was added
to a mixture of 2,4-thiazolidinedione (8 mg, 0.07 mmol) and
2-methoxyethoxy-3-trifluoromethyl-benzaldehyde (17 mg, 0.07
mmol) in ethanol (1.2 mL) and refluxed overnight, during which it
became yellow. The mixture was poured into water (∼12 mL) and
acidified with acetic acid to a pH of 3-4 and then precipitated at
4 °C. After centrifugation and filtration, the collected yellow solid
was dissolved in chloroform. After passing through a pad of silica
eluted with 5% methanol in chloroform, the concentrated residue
was purified by radial chromatography (silica) eluted with
0-1-3-5% methanol in chloroform to afford a white solid (11
mg, 45% yield). Rf ) 0.24, 5% methanol in chloroform; Rf ) 0.13,
3% methanol in chloroform. 1H (CDCl3) δ 8.88 (bs, 1 H), 8.30 (s,
1 H), 7.69 (d, J ) 7.6 Hz, 1 H), 7.67 (d, J ) 7.6 Hz, 1 H), 7.34
(dd, J1 ) J2 ) 7.6 Hz, 1 H), 4.08 (t, J ) 4.8 Hz, 2 H), 3.80 (t, J
) 4.8 Hz, 2 H), 3.51 (s, 3 H). 13C NMR (CDCl3) δ 166.8, 165.9,
157.3, 132.6, 129.3 (q, J ) 4.6 Hz, 1 C), 129.2, 128.6, 125.6 (q, J
) 30 Hz, 1 C), 125.5, 124.7, 123.3 (q, J ) 272 Hz, 1 C), 76.6,
71.4, 59.4. HR-MS (EI): calcd 347.0439 for C14H12F3NO4S, found
347.0441.
yield). Rf ) 0.19, chloroform. H (CDCl3) δ 10.32 (bd, J ) 0.4
Hz, 1 H), 8.07 (dd, J1 ) 7.6 Hz, J2 ) 1.2 Hz, 1 H), 7.88 (dd, J1 )
7.6 Hz, J2 ) 1.2 Hz, 1 H), 7.38 (dd, J1 ) J2 ) 7.6 Hz, 1 H), 5.13
(s, 2 H), 3.64 (s, 3 H). 13C NMR (CDCl3) δ 189.6, 158.7, 132.9 (q,
J ) 5.3 Hz, 1 C), 132.7, 131.7, 124.9, 123.2 (q, J ) 171 Hz, 1 C),
102.7 (2 C), 58.2.
(Z)-5-(2-Methoxymethoxy-3-trifluoromethylbenzylidene)thi-
azolidine-2,4-dione (6a). Piperidine (15 µL, 0.15 mmol) was added
to a mixture of 2,4-thiazolidinedione (18 mg, 0.15 mmol) and
2-methoxymethoxy-3-trifluoromethyl-benzaldehyde (35 mg, 0.15
mmol) in ethanol (3 mL) and refluxed overnight. After cooling to
room temperature, the mixture was poured into water (∼30 mL)
and acidified with acetic acid to a pH of 3-4. After filtration, the
collected solid was dissolved in chloroform and passed through a
pad of silica eluted with 5% methanol in chloroform. The
concentrated residue was purified by radial chromatography (silica)
eluted with 0-1-3% methanol in chloroform to afford the
compound 6a (7 mg, 14% yield). Rf ) 0.24, 3% methanol in
chloroform. 1H (CDCl3) δ 8.20 (s, 1 H), 7.71 (d, J ) 8.0 Hz, 1 H),
7.69 (d, J ) 8.0 Hz, 1 H), 7.36 (dd, J1 ) J2 ) 8.0 Hz, 1 H), 5.05
(s, 2 H), 3.70 (s, 3 H). 13C NMR (DMSO) δ 167.0, 166.3, 156.2,

82 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Xia et al.
1
(Z)-5-(4-ethylbenzylidene)thiazolidine-2,4-dione (8a). 2,4-Thia-
zolidinedione (119 mg, 1 mmol) and 4-ethoxybenzaldehyde (139
µL, 136 mg, 1 mmol) were dissolved in ethanol (7 mL), followed
by the addition of piperidine (79 µL, 0.8 mmol). After refluxing
for 20 h, the yellow solution was poured into water (∼60 mL),
acidified with acetic acid to a pH of 3-4, and incubated at 4 °C
overnight. After filtration, the solid was washed with methanol to
afford a yellow product (116 mg, 0.50 mmol, 50% yield). 1H
(DMSO) δ 7.76 (s, 1 H), 7.51 (d, J ) 8.4 Hz, 2 H), 7.38 (d, J )
8.4 Hz, 2 H), 2.66 (q, d ) 7.6 Hz, 2 H), 1.20 (t, J ) 7.6 Hz, 3 H).
13C NMR (DMSO) δ 168.1, 167.6, 147.0, 132.0, 130.7, 130.4 (2
C), 128.9 (2 C), 122.6, 28.3, 15.4.
(Z)-5-(4-Isopropylbenzylidene)thiazolidine-2,4-dione (9a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and 4-methoxybenzaldehyde
(148 mg, 151 µL, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (79 µL, 0.8 mmol). After
refluxing for 24 h, the yellow solution was poured into water (∼60
mL), and acidified with acetic acid to a pH of 3-4. After filtration,
it was subjected to radial chromatography (silica) eluted with 3-5%
methanol in chloroform to afford the product (125 mg, 0.51 mmol,
51% yield). Rf ) 0.31, 3% methanol in chloroform. 1H (CDCl3) δ
7.87 (s, 1 H), 7.44 (d, J ) 8.0 Hz, 2 H), 7.34 (d, J ) 8.0 Hz, 2 H),
2.96 (tt, J1 ) J2 ) 6.8 Hz, 1 H), 1.27 (d, J ) 6.8 Hz, 6 H). 13C
NMR (CDCl3) δ 167.8, 167.4, 152.6, 135.0, 130.8 (2 C), 130.7,
127.7 (2 C), 121.3, 34.4, 23.9 (2 C).
(Z)-5-(4-methoxybenzylidene)thiazolidine-2,4-dione (10a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and 4-methoxybenzaldehyde
(136 mg, 123 µL, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (79 µL, 0.8 mmol). After
refluxing for 20 h, the yellow solution was poured into water (∼60
mL), acidified with acetic acid to a pH of 3-4, and incubated at 4
°C overnight. After filtration, the yellow solid was washed with
methanol to afford the product (90 mg, 0.38 mmol, 38% yield). 1H
(DMSO) δ 7.74 (s, 1 H), 7.56 (d, J ) 8.8 Hz, 2 H), 7.10 (d, J )
8.8 Hz, 2 H), 3.83 (s, 3 H). 13C NMR (DMSO) δ 168.4, 168.1,
160.9, 132.0 (2 C), 131.6, 125.6, 114.9 (2 C), 55.5.
in chloroform. H (CDCl3) δ 7.88 (bs, 1 H), 7.83 (s, 1 H), 7.52
(dd, J1 ) J2 ) 8.0 Hz, 1 H), 7.43 (d, J ) 8.0 Hz, 1 H), 7.34 (s, 1
H), 7.29 (d, J ) 8.0 Hz, 1 H). 13C NMR (CDCl3) δ 167.5, 167.3,
150.0 (m, 1 C), 135.1, 132.8, 130.9, 128.4, 124.8, 123.0, 122.5,
120.6 (q, J ) 257.3 Hz, 1 C).
(Z)-5-(4-Ethoxybenzylidene)thiazolidine-2,4-dione (14a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and 4-ethoxybenzaldehyde
(150 mg, 139 µL, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (79 µL, 0.8 mmol). After
refluxing for 20 h, the yellow solution was poured into water (∼60
mL) and acidified with acetic acid to a pH of 3-4. After filtration,
the solid was purified by precipitation from methanol-hexane to
1
afford the yellow product (60 mg, 0.24 mmol, 24% yield). H
(DMSO) δ 12.50 (bs, 1 H), 7.73 (s, 1 H), 7.54 (d, J ) 8.6 Hz, 2
H), 7.08 (d, J ) 8.6 Hz, 2 H), 4.10 (q, J ) 6.8 Hz, 2 H), 1.35 (t,
J ) 6.8 Hz, 3 H). 13C NMR (DMSO) δ 168.5, 168.3, 160.4, 132.2
(2 C), 131.6, 125.6, 121.0, 115.4 (2 C), 63.7, 14.7.
(Z)-5-[3-(1,1,2,2-Tetrafluoroethoxy)benzylidene]thiazolidine-
2,4-dione (15a). 2,4-Thiazolidinedione (117 mg, 1 mmol) and
3-(1,1,2,2-tetrafluoroethoxy)benzaldehyde (160 µL, 222 mg, 1
mmol) were dissolved in ethanol (7 mL), followed by the addition
of piperidine (79 µL, 0.80 mmol). After refluxing for 20 h, the
mixture was poured into water (∼60 mL) and acidified with acetic
acid to a pH of 3-4. After filtration, the solid dissolved in
chloroform was purified by radial chromatography (silica) eluted
with 3-5% methanol in chloroform to afford a solid product (101
mg, 0.31 mmol, 31% yield). Rf ) 0.23, 3% methanol in chloroform.
1H (DMSO) δ 12.70 (bs, 1 H), 7.85 (s, 1 H), 7.65 (dd, J1 ) J2 )
8.0 Hz, 1 H), 7.59 (d, J ) 8.0 Hz, 1 H), 7.52 (s, 1 H), 7.40 (dd, J1
) 8.0 Hz, J2 ) 1.2 Hz, 1 H), 6.84 (tt, J1 ) 52 Hz, J2 ) 3.2 Hz, 1
H). 13C NMR (DMSO) δ 167.6, 167.3, 148.7, 135.4, 131.3, 130.3,
128.1, 125.7, 123.3, 122.9, 116.6 (tt, J1 ) 270 Hz, J2 ) 28.1 Hz,
1 C), 107.9 (tt, J1 ) 247 Hz, J2 ) 40.2 Hz, 1 C). HR-MS (EI):
calcd 321.0083 for C12H7F4NO3S, found 321.0080.
(Z)-5-(4-Propoxybenzylidene)thiazolidine-2,4-dione (16a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and 4-propoxybenzaldehyde
(164 mg, 158 µL, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (79 µL, 0.8 mmol). After
refluxing for 20 h, the yellow solution was poured into water (∼60
mL), acidified with acetic acid to a pH of 3-4, and incubated at 4
°C overnight. After filtration, the solid was washed with methanol
to afford the product (114 mg, 0.43 mmol, 43% yield). 1H (DMSO)
δ 12.50 (bs, 1 H), 7.75 (s, 1 H), 7.54 (d, J ) 8.8 Hz, 2 H), 7.09 (d,
J ) 8.8 Hz, 2 H), 4.01 (t, J ) 6.8 Hz, 2 H), 1.75 (m, 2 H), 0.98 (t,
J ) 7.6 Hz, 3 H). 13C NMR (DMSO) δ 168.1, 167.6, 160.7, 132.3
(2 C), 132.1, 125.5, 120.3, 115.5 (2 C), 69.5, 22.1, 10.5. HR-MS
(EI): calcd 263.0616 for C13H13NO3S, found 263.0618.
(Z)-5-(4-Dimethylaminobenzylidene)thiazolidine-2,4-dione (17a).
2,4-Thiazolidinedione (121 mg, 1 mmol) and 4-trifluoromethyl-
benzaldehyde (154 mg, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (80 µL, 0.81 mmol). The
mixture was refluxed for 3 h, and the resulting orange suspension
was poured into water (∼60 mL), acidified with acetic acid to a
pH of 3-4, and incubated overnight at 4 °C. After filtration, the
yellow solid was washed with methanol to afford the product (175
mg, 0.71 mmol, 71% yield). 1H (DMSO) δ 7.66 (s, 1 H), 7.44 (d,
J ) 8.8 Hz, 2 H), 6.82 (d, J ) 8.8 Hz, 2 H), 3.32 (s, 6 H). 13C
NMR (DMSO) δ 168.4, 167.8, 151.6, 133.1, 132.3 (2 C), 120.0,
115.9, 112.2 (2 C), 39.5 (2 C).
(Z)-5-(4-Fluorobenzylidene)thiazolidine-2,4-dione (18a). 2,4-
Thiazolidinedione (119 mg, 1 mmol) and 4-trifluoromethylbenzal-
dehyde (107 µL, 126 mg, 1 mmol) were dissolved in ethanol (7
mL), followed by the addition of piperidine (79 µL, 0.80 mmol).
After refluxing for 24 h, the mixture was poured into water (∼60
mL) and acidified with acetic acid to a pH of 3-4. After filtration,
the solid was purified by radial chromatography (silica) eluted with
3% methanol in chloroform to afford a yellow solid (91 mg, 0.41
(Z)-5-(4-Trifluoromethoxybenzylidene)thiazolidine-2,4-di-
one (11a). 2,4-Thiazolidinedione (119 mg, 1 mmol) and 4-trifluo-
romethoxybenzaldehyde (144 µL, 192 mg, 1 mmol) were dissolved
in ethanol (7 mL), followed by the addition of piperidine (79 µL,
0.8 mmol). After refluxing for 20 h, the solution was poured into
water (∼60 mL) and acidified with acetic acid to a pH of 3-4.
After filtration, the solid dissolved in chloroform and was purified
by radial chromatography (silica) eluted with 3-5% methanol in
chloroform to afford the product (80 mg, 0.28 mmol, 28% yield).
1
Rf ) 0.28 (3% methanol in chloroform). H (CDCl3) δ 7.85 (s, 1
H), 7.55 (d, J ) 8.4 Hz, 2 H), 7.32 (d, J ) 8.4 H, 2 H). 13C NMR
(CDCl3) δ 167.0, 166.8, 150.6 (t, J ) 2.2 Hz, 1 H), 132.8, 131.9
(2 C), 131.4, 123.3, 121.4 (2 C), 120.4 (t, J ) 257 Hz, 1 C). HR-
MS (EI): calcd 289.0020 for C11H6F3NO3S, found 289.0014.
(Z)-5-(3-Methoxybenzylidene)thiazolidine-2,4-dione (12a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) was dissolved in ethanol (8
mL), followed by addition of m-anisaldehyde (122 µL, 1 mmol)
and piperidine (79 µL, 0.8 mmol). After refluxing for 20 h, the
yellow solution was poured into water (∼60 mL), acidified with
acetic acid to a pH of 3-4, and incubated at 4 °C overnight. After
filtration, the yellow solid was washed with methanol to afford the
1
product (115 mg, 0.49 mmol, 49% yield). H (DMSO) δ 7.77 (s,
1 H), 7.45 (dd, J1 ) J2 ) 8.0 Hz, 1 H), 7.16 (d, J ) 8.0 Hz, 1 H),
7.15 (s,1 H), 7.07 (d, J ) 8.0 Hz, 1 H), 3.81 (s, 3 H). 13C NMR
(DMSO) δ 168.2, 167.7, 160.1, 134.9, 132.2, 130.9, 124.4, 122.3,
116.8, 115.8, 55.7.
(Z)-5-(3-Trifluoromethoxybenzylidene)thiazolidine-2,4-di-
one (13a). 2,4-Thiazolidinedione (118 mg, 1 mmol) and 4-tolual-
dehyde (143 µL, 190 mg, 1 mmol) were dissolved in ethanol (7
mL), followed by the addition of piperidine (79 µL, 0.80 mmol).
After refluxing for 24 h, the mixture was poured into water (∼60
mL) and acidified with acetic acid to a pH of 3-4. After filtration,
the yellow solid was purified by radial chromatography (silica)
eluted with 3% methanol in chloroform to afford a white solid
product (78 mg, 0.27 mmol, 27% yield). Rf ) 0.27, 3% methanol
1
mmol, 41% yield). Rf ) 0.27, 3% methanol in chloroform. H
(DMSO) δ 7.81 (s, 1 H), 7.67 (dd, J1 ) 8.8 Hz, J2 ) 5.6 Hz, 2 H),
7.39 (dd, J1 ) J2 ) 8.8 Hz, 2 H). 13C NMR (DMSO) δ 168.0,
167.6, 163.0 (d, J ) 249 Hz, 1 C), 132.6 (d, J ) 8.4 Hz, 2 C),

Inhibitors of Pim-1 and Pim-2 Protein Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 83
130.8, 129.9 (d, J ) 3 Hz, 1 C), 123.6 (d, J ) 2.2 Hz, 1 C), 116.7
(d, J ) 22 Hz, 2 C).
(s, 1 H), 7.73 (d, J ) 8.4 Hz, 2 H), 7.54 (d, J ) 8.4 Hz, 2 H).C
NMR (DMSO) δ 168.0, 167.8, 132.6, 132.5 (2 C), 131.9 (2 C),
131.4, 130.5, 124.0.
(Z)-3-(2-Fluorobenzyl)-5-(4-fluorobenzylidene)thiazolidine-
2,4-dione (19a). 2,4-Thiazolidinedione (117 mg, 1 mmol) and
2-fluorobenzbenzaldehyde (127 µL, 149 mg, 1.2 mmol) were
dissolved in 1-butyl-3-methylimidazolium hexafluorophosphate,
[bmim]PF6, (2 mL), followed by the addition of Et3N (170 µL, 1.2
mmol) and 2-fluorobenzyl chloride (127 µL, 1.2 mmol). The
mixture was stirred at 60 °C for 20 h. After cooling to room
temperature, the mixture was extracted with ether (4 × 15 mL),
passed through a pad of silica eluted with 25% ethyl acetate in
hexane, and the concentrated residue was purified by radial
chromatography (silica) eluted with 15-25% ethyl acetate in hexane
to afford two products based on differences in their Rfs. One
component is a semisolid identified as 3-(2-fluorobenzyl)thiazoli-
dine-2,4-dione (25b). The other component was not pure and was
purified again by radial chromatography (silica) eluted with hexane
and chloroform to afford a white solid (5 mg, 0.02 mmol, 2% yield)
(Z)-5-(3-Bromobenzylidene)thiazolidine-2,4-dione (24a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and m-bromobenzaldehyde
(185 mg, 117 µL, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (79 µL, 0.8 mmol). After
refluxing for 20 h, the yellow solution was poured into water (∼60
mL), acidified with acetic acid to a pH of 3-4 and incubated at 4
°C overnight. After filtration, the solid was suspended in chloroform,
followed by centrifugation and discarding the supernatant to afford
an off-white product (155 mg, 0.55 mmol, 55% yield). 1H (DMSO)
δ 12.69 (bs, 1 H), 7.82 (dd, J1 ) J2 ) 2.0 Hz, 1 H), 7.78 (s, 1 H),
7.68 (ddd, J1 ) 8.0 Hz, J2 ) 2.0 Hz, J3 ) 0.8 Hz, 1 H), 7.58 (d,
J ) 8.0 Hz, 1 H), 7.50 (dd, J1 ) J2 ) 8.0 Hz, 1 H). 13C NMR
(DMSO) δ 167.7, 167.3, 135.7, 133.0, 132.9, 131.5, 130.2, 128.3,
125.6, 122.6.
3-(2-Fluorobenzyl)thiazolidine-2,4-dione (25b). 2,4-Thiazo-
lidinedione (59 mg, 0.5 mmol) and p-methoxybenzaldehyde (61
µL, 68 mg, 0.5 mmol) were dissolved in 1-butyl-3-methylimida-
zolium hexafluorophosphate, [bmim]PF6, (2 mL), followed by the
addition of Et3N (84 µL, 0.6 mmol) and 2-fluorobenzyl chloride
(0.6 mmol, 72 µL). The reaction was carried out at 60 °C for 17 h,
cooled to room temperature, and then extracted with ether (4 × 15
mL). TLC (25% ethyl acetate in hexane, silica) demonstrated the
presence of two major UV spots: Rf1 ) 0.26 (same Rf of
p-methoxybeznaldehyde) and Rf2 ) 0.19. After passing through a
pad of silica eluted with 25% ethyl acetate in hexane, the
concentrated residue was purified by radial chromatography (silica)
eluted with 15-25% ethyl acetate in hexane to afford a yellowish
semisolid (Rf2) (55 mg, 0.24 mmol, 24% yield). NMR spectra
confirmed it was 3-(2-fluorobenzyl)thiazolidine-2,4-dione rather
than 3-(2-fluorobenzyl)-5-(4-methoxybenzylidene)thiazolidine-2,4-
1
(Rf ) 0.33, silica, chloroform). H (CDCl3) δ 7.88 (s, 1 H), 7.50
(dd, J1 ) 8.4 Hz, 2 H), 7.27-7.37 (m, 2 H), 7.17 (dd, J1 ) J2 )
8.4 Hz, 2 H), 7.04-7.14 (m, 2 H), 5.00 (s, 2 H). 13C NMR (CDCl3)
δ 167.4, 166.1, 163.9 (d, J ) 253 Hz, 1 C), 160.9 (d, J ) 247 Hz,
1 C), 133.2, 132.5 (d, J ) 8.4 Hz, 2 C), 130.4 (d, J ) 3.8 Hz, 1
C), 130.2 (d, J ) 7.6 Hz, 1 C), 129.7 (d, J ) 3.8 Hz, 1 C), 124.5
(d, J ) 3.8 Hz, 1 C), 122.0 (d, J ) 14.4 Hz, 1 C), 121.1 (d, J )
2.3 Hz, 1 C), 116.8 (d, J ) 22.0 Hz, 2 C), 115.9 (d, J ) 21.2 Hz,
1 C), 39.3 (d, J ) 4.5 Hz, 1 C). HR-MS (EI): calcd 331.0479 for
C17H11F2NO2S, found 331.0477.
(Z)-5-(3-Fluorobenzylidene)thiazolidine-2,4-dione (20a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and 3-fluorobenzaldehyde (124
mg, 105 µL, 1 mmol) were dissolved in ethanol (7 mL), followed
by the addition of piperidine (79 µL, 0.8 mmol). After refluxing
for 20 h, the mixture was poured into water (∼60 mL) and acidified
with acetic acid to a pH of 3-4. After filtration, the solid dissolved
in chloroform was purified by radial chromatography eluted with
5% methanol in chloroform to afford a yellowish product (100 mg,
0.45 mmol, 45% yield). Rf ) 0.36, 5% methanol in chloroform.
1H (DMSO) δ 12.68 (bs, 1 H), 7.80 (s, 1 H), 7.59 (m, 1 H),
7.42-7.48 (m, 2 H), 7.34 (ddd, J1 ) J2 ) 8.0 Hz, J3 ) 2 Hz, 1 H).
13C NMR (DMSO) δ 167.9, 167.6, 162.4 (d, J ) 243.6 Hz, 1 C),
135.7 (d, J ) 8.3 Hz, 1 C), 131.5 (d, J ) 8.3 Hz, 1 C), 130.3,
125.7 (2 C), 117.3 (d, J ) 21.2 Hz, 1 C), 116.8 (d, J ) 22.8 Hz,
1 C).
1
dione. H (CDCl3) δ 7.24-7.32 (m, 2 H), 7.00-7.11 (m, 2 H),
4.85 (s, 2 H), 3.97 (s, 2 H). 13C NMR (CDCl3) δ 171.4, 171.1,
160.8 (d, J ) 247 Hz, 1 C), 130.5 (d, J ) 3.0 Hz, 1 C), 130.2 (d,
J ) 8.3 Hz, 1 C), 124.4 (d, J ) 3.8 Hz, 1 C), 122.0 (d, J ) 14 Hz,
1 C), 115.8 (d, J ) 21 Hz, 1 C), 39.3 (d, J ) 4.6 Hz, 1 C), 33.9.
(E/Z)-1-Benzyl-5-(4-chlorobenzylidene)imidazolidine-2,4-di-
one (26c). 1-Benzylhydantoin (190 mg, 1 mmol) and p-chloroben-
zaldehyde (142 mg, 1 mmol) were dissolved in ethanol (7 mL),
followed by the addition of piperidine (79 µL, 0.8 mmol). The
mixture was refluxed for 24 h, and the yellowish solution was
poured into water (∼60 mL) and acidified with acetic acid to a pH
of 5 and then was extracted with chloroform (3 × 30 mL). The
collected organic solution was dried over sodium sulfate. After
passing through a pad of silica column eluted with 25% ethyl acetate
in hexane, the concentrated residue was purified by radial chro-
matography (silica) eluted with 15-50% ethyl acetate in hexane
to afford three components: (A) Rf ) 0.42 (25% ethyl acetate in
hexane); (B) Rf ) 0.14 (25% ethyl acetate in hexane); (C) 0.20
(50% ethyl acetate in hexane). Component (B) was the desired
product (20 mg, 6% yield, E/Z ≈ 9/1). According to the NMR
(Z)-5-(4-Chlorobenzylidene)thiazolidine-2,4-dione (21a). 2,4-
Thiazolidinedione (119 mg, 1 mmol) and 4-chlorobenzaldehyde
(142 mg, 1 mmol) were dissolved in ethanol (8 mL), followed by
the addition of piperidine (79 µL, 0.8 mmol). After refluxing for
20 h, the yellow solution was poured into water (∼60 mL), acidified
with acetic acid to a pH of 3-4 and incubated at 4 °C overnight.
After filtration, the yellow solid was washed with methanol to afford
1
the product (152 mg, 0.63 mmol, 63% yield). H (DMSO) δ 7.79
(s, 1 H), 7.61 (m, 4 H). 13C NMR (DMSO) δ 167.8, 167.4, 135.1,
132.1, 131.8 (2 C), 130.5, 129.5 (2 C), 124.6.
1
spectra, the (E)-isomer was the major component: H (DMSO) δ
(Z)-5-(3-Chlorobenzylidene)thiazolidine-2,4-dione (22a). 2,4-
Thiazolidinedione (119 mg, 1 mmol) and 3-chlorobenzaldehyde
(118 µL, 1 mmol) were dissolved in ethanol (8 mL), followed by
the addition of piperidine (79 µL, 0.8 mmol). After refluxing for
20 h, the yellow solution was poured into water (∼60 mL), acidified
with acetic acid to a pH of 3-4 and incubated at 4 °C overnight.
After filtration, the yellow solid was washed with methanol to afford
8.87 (bs, 1 H), 7,27-7.40 (m, 7 H), 6.14 (s, 1 H), 4.91 (s, 2 H).
13C NMR (DMSO) δ 162.0, 153.4, 135.2, 135.0, 131.8 (2 C), 130.7,
129.3 (2 C), 128.8, 128.6 (2 C), 128.3, 127.2 (2 C), 117.9, 43.8.
Z-isomer is the minor component: 1H (DMSO) δ 7,25-7.40 (m, 1
H), 7.24 (d, J ) 8.0 Hz, 2 H), 7.14 (d, J ) 8.0 Hz, 2 H), 6.97 (d,
J ) 8.0 Hz, 2 H), 6.80 (s, 1 H), 6.61 (dd, J1 ) 8.0 Hz, J2 ) 1.6
Hz, 2 H), 4.73 (s, 2 H). HR-MS (EI): calcd 312.0666 for
C17H13ClN2O2, found 312.0663.
1
the product (113 mg, 0.47 mmol, 47% yield). H (DMSO) δ 7.79
(s, 1 H), 7.68 (s, 1 H), 7.52-7.60 (m, 3 H). 13C NMR (DMSO) δ
167.8, 167.4, 135.5, 134.1, 131.3, 130.3, 130.2, 130.1, 128.0, 125.7.
(Z)-5-(4-Bromobenzylidene)thiazolidine-2,4-dione (23a). 2,4-
Thiazolidinedione (117 mg, 1 mmol) and 4-bromobenzaldehyde
(185 mg, 1 mmol) were dissolved in ethanol (7 mL), followed by
the addition of piperidine (79 µL, 0.8 mmol). After refluxing for
20 h, the yellow solution was poured into water (∼60 mL), acidified
with acetic acid to a pH of 3-4 and incubated at 4 °C overnight.
After filtration, the yellow solid was washed with methanol to afford
(E)-1-Benzyl-5-(4-methoxybenzylidene)imidazolidine-2,4-di-
one (27c-1) and (E/Z)-1-Benzyl-5-(4-methoxybenzylidene)imi-
dazolidine-2,4-dione (27c-2). 1-Benzylhydantoin (190 mg, 1 mmol)
and p-methoxybenzaldehyde (136 mg, 1 mmol) were dissolved in
ethanol (7 mL), followed by the addition of piperidine (100 µL, 1
mmol). The mixture was refluxed for 22 h, and the yellowish
solution was poured into water (∼60 mL) and acidified with acetic
acid to a pH of 3-4. After incubation at 4 °C for a few hours, the
sample wash filtered and washed with cold water, producing an
1
the product (220 mg, 0.77 mmol, 77% yield). H (DMSO) δ 7.76

84 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Xia et al.
off-white solid, which quite dissolved in methanol or chloroform.
TLC (25% ethyl acetate in hexane, silica) suggested two products,
one was UV absorbent with an Rf ) 0.06 and the other was
fluorescent with an Rf ) 0.02. When 50% ethyl acetate in hexane
was used as the developing solution, TLC was showed only one
UV tailing spot, Rf ) 0.28 (no fluorescent spot). Similarly, when
1% or 3% methanol in chloroform was used as developing solution,
TLC analyses indicated only one UV spot. Products were purified
by radial chromatography (silica) eluted with 25% ethyl acetate in
hexane (collection-1) and 3% methanol in chloroform (collection-
2). NMR was shown that collection-1 was a mixture composed of
83% E-isomer and 17% Z-isomer, whereas collection-2 contained
only the E-isomer (19 mg (collection-2) + 107 mg (collection-1)
(100 mg, 31% yield) was obtained. 1H (DMSO) δ 11.35 (bs, 1 H),
7.87 (d, J ) 9.2 Hz, 2 H), 7.24-7.40 (m, 5 H), 7.65 (d, J ) 9.2
Hz, 2 H), 6.28 (s, 1 H), 4.87 (s, 2 H), 2.94 (s, 6 H). 13C NMR
(DMSO) δ 162.4, 152.9, 149.9, 136.1, 131.5 (2 C), 128.2 (2 C),
126.8, 126.5 (2 C), 124.2, 119.8, 118.3, 110.7 (2 C), 41.4, 39.2 (2
C). HR-MS (EI): calcd 321.1477 for C19H19N3O2, found 321.1472.
(E/Z)-1-Benzyl-5-(2-methoxybenzylidene)imidazolidine-2,4-di-
one (31c). 1-Benzylhydantoin (190 mg, 1 mmol) and o-methoxy-
benzaldehyde (122 µL, 136 mg, 1 mmol) were dissolved in ethanol
(7 mL), followed by the addition of piperidine (100 µL), and the
mixture was refluxed for 48 h. The solution was poured into water
(∼60 mL), acidified with acetic acid to a pH of 3, and kept at 4 °C
overnight. After filtration and washing with water, a white solid
was collected. The solid was dissolved in 50% ethyl acetate in
hexane and passed a pad of silica column eluted with 50% ethyl
acetate in hexane to afford a solid compound (145 mg, 0.47 mmol,
47% yield, E/Z ≈ 5/1). Rf ) 0.37, 50% ethyl acetate in hexane; Rf
) 0.24, 3% methanol in chloroform. The E-isomer: 1H (CDCl3) δ
9.15 (bs, 1 H), 7.62 (m, 1 H), 7.11-7.39 (m, 7 H), 6.85-6.88 (m,
1 H), 6.19 (s, 1 H), 4.91 (s, 2 H), 3.82 (s, 3 H). 13C NMR (CDCl3)
δ 162.2, 159.5, 153.7, 135.1, 133.5, 129.3, 129.2 (2 C), 128.2, 127.2
(2 C), 126.9, 123.6, 119.6, 116.0, 114.9, 55.5, 43.7. HR-MS (EI):
calcd 308.1161 for C18H16N2O3, found 308.1155.
Pim Kinase Assays. Pim protein kinase assays were conducted
using multiple methods to ensure that the effects of the compounds
were not due to any experimental artifacts. The primary screen and
evaluation of the compounds shown in Table 3 was conducted using
an ATP-depletion assay. Briefly, recombinant human Pim-1 (Up-
state) was incubated with S6 kinase/Rsk-2 peptide 2 (KKRN-
RTLTK) (Upstate) as the substrate in the presence 100 µM of
compounds from the screening library (DIVERSet collection of the
ChemBridge Corporation), 1 µM ATP and 10 mM MgCl2 for 1 h.
The Kinase-Glo luciferase kit (Promega) was used to measure
residual ATP levels after the kinase reaction. For experiments that
required higher ATP concentrations, Pim-1 kinase activity was
monitored spectrophotometrically using a coupled assay in which
ADP production is coupled to NADH oxidation catalyzed by
pyruvate kinase and lactate dehydrogenase. Assays were carried
out in 20 mM MOPS pH 7 containing 100 mM NaCl, 10 mM
MgCl2, 2.5 mM phosphoenolpyruvate, 0.2 mM NADH, 30 µg/mL
pyruvate kinase, 10 µg/mL lactate dehydrogenase, 2 mM dithio-
threitol, 25 nM Pim-1, 100 µM S61 peptide (RRLSSLRA, American
Peptide Company), and varying concentrations of ATP. Activity
was measured by monitoring NADH oxidation as the decrease at
340 nm in a VersaMax microplate reader (Molecular Devices) at
25 °C. Reactions were initiated by the addition of ATP (typically
100 µM). Inhibitors (final 1% DMSO) were added just prior to the
addition of ATP. In either case, IC50 values were determined using
nonlinear regression with the program GraphPad Prism. In some
experiments, Pim-1 kinase activity was determined using His-tagged
4E-BP1 as the substrate. The active Pim-1 protein (Upstate) was
resuspended in kinase reaction buffer (10 mM MOPS, pH 7.4, 100
µM ATP, 15 mM MgCl2, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT,
and protease inhibitor cocktail). In each reaction (30 µL), 3 µg of
His-4E-BP1 protein was used as substrate, and 10 µCi of [γ-32P]
ATP were then added. Incubation was carried out at 30 °C for 30
min with agitation. The samples were then subjected to SDS-PAGE
and 32P labeled 4E-BP1 was visualized by autoradiography. Finally,
Pim-1 activity in intact cells was measured in some experiments.
HEK-293T cells were transfected with Flag-Pim-1 for 24 h, and
then were trypsined and divided into smaller dishes for overnight.
Cells were washed once and incubated with phosphate-free media
containing 10% phosphate-free FBS (Invitrogen, Carlsbad, CA) for
1 h. Cells were then incubated in medium containing 50 µCi/ml
1
) 126 mg, 0.41 mmol, 41% yield). E-isomer: H (CDCl3) δ 7.79
(d, J ) 8.8 Hz, 2 H), 7.27-7.39 (m, 5 H), 6.85 (d, J ) 8.8 Hz, 2
H), 6.17 (s, 1 H), 4.91 (s, 2 H), 3.81 (s, 3 H). 13C NMR (CDCl3)
δ 162.0, 160.7, 153.1, 135.4, 132.5 (2 C), 129.2 (2 C), 128.2, 127.2
(2 C), 126.8, 125.1, 119.9, 113.9 (2 C), 55.5, 43.8. HR-MS (EI):
calcd 308.1161 for C18H16N2O3, found 308.1159.
(Z/E)-1-Benzyl-5-(4-ethoxybenzylidene)imidazolidine-2,4-di-
one (28c). 1-Benzylhydantoin (190 mg, 1 mmol) and p-ethoxy-
bezyaldehyde (139 µL, 150 mg, 1 mmol) were dissolved in ethanol
(7 mL), followed by the addition of piperidine (100 µL, 1 mmol).
The mixture was refluxed for 24 h, and the yellowish solution was
poured into water (∼60 mL) and acidified with acetic acid. After
filtration, the solid dissolved in chloroform was purified by radial
chromatography (silica) eluted with 0-1-3% methanol in chlo-
roform to afford a yellowish solid (136 mg, 0.50 mmol, 50% yield).
Rf ) 0.21, 3% methanol in chloroform. NMR analyses demonstrated
a mixture of Z- and E-compounds (Z/E ≈ 1/4). The E-compound:
1H (CDCl3) δ 8.94 (bs, 1 H), 7.80 (d, J ) 8.8 Hz, 2 H), 7.28-7.38
(m, 5 H), 6.84 (d, J ) 8.8 Hz, 2 H), 6.17 (s, 1 H), 4.91 (s, 2 H),
4.04 (q, d ) 6.8 Hz, 2 H), 1.40 (t, J ) 6.8 Hz, 3 H). 13C NMR
(CDCl3) δ 162.4, 160.1, 153.5, 135.4, 132.6 (2 C), 129.2 (2 C),
128.1, 127.2 (2 C), 126.6, 124.8, 120.1, 114.4 (2 C), 63.7, 43.7,
14.9. The Z-compound: 1H (CDCl3) δ 9.02 (bs, 1 H), 7.13 (d, J )
8.4 Hz, 2 H), 7.05 (d, J ) 8.4 Hz, 2 H), 6.81-6.86 (m, 4 H), 6.67
(dd, J1 ) 8.0 Hz, J2 ) 2.0 Hz, 2 H), 4.79 (s, 2 H), 4.06 (q, J ) 7.2
Hz, 2 H), 1.45 (t, J ) 7.2 Hz, 3 H). 13C NMR (CDCl3) δ 162.3,
159.5, 155.7, 135.4, 131.2 (2 C), 128.6 (2 C), 127.9, 127.7 (2 C),
124.8, 114.7, 114.4 (2 C), 63.8, 45.2, 14.9. HR-MS (EI): calcd
322.1317 for C19H18N2O3, found 322.1309.
(E)-1-Benzyl-5-(4-bromobenzylidene)imidazolidine-2,4-di-
one (29c-1) and (E/Z)-1-Benzyl-5-(4-bromobenzylidene)imida-
zolidine-2,4-dione (29c-2). 1-Benzylhydantoin (190 mg, 1 mmol)
and p-bromobenzaldehyde (185 mg, 1 mmol) were dissolved in
ethanol (7 mL), followed by the addition of piperidine (100 µL, 1
mmol). The mixture was refluxed for 36 h, and the yellowish
solution was poured into water (∼60 mL) and acidified with acetic
acid to a pH of 3 and kept at 4 °C overnight. After filtration and
washing with methanol, a yellow solid was collected (9 mg, 0.025
mmol, 3% yield) and shown to be the E-isomer (29c-1). The
remaining solution was passed through a pad of silica column and
eluted with chloroform. The concentrated residue was purified by
radial chromatography (silica) eluted with 0-1-3% methanol in
chloroform to provide a solid which was a mixture of E/Z-isomer
(37 mg, 0.1 mmol, 13% yield, E/Z ≈ 10/1). Rf ) 0.45, 50% ethyl
acetate in hexane; Rf ) 0.31, 3% methanol in chloroform. The
1
E-isomer: H (CDCl3) δ 7.60 (d, J ) 8.4 Hz, 2 H), 7.44 (d, J )
8.4 Hz, 2 H), 7.28-7.40 (m, 5 H), 6.12 (s, 1 H), 4.91 (s, 2 H). 13
C
NMR (CDCl3) δ 161.5, 152.8, 135.0, 132.0 (2 C), 131.6 (2 C),
131.1, 129.3 (2 C), 128.9, 128.3, 127.2 (2 C), 123.6, 117.9, 43.8.
HR-MS (EI): calcd 356.0160 for C17H13BrN2O2, found 356.0154.
(E)-1-Benzyl-5-(4-dimethylaminobenzylidene)imidazolidine-
2,4-dione (30c). 1-Benzylhydantoin (190 mg, 1 mmol) and p-
dimethylaminobenzaldehyde (149 mg, 1 mmol) were dissolved in
ethanol (7 mL), followed by the addition of piperidine (100 µL).
The mixture was refluxed for 6 h, resulting in a precipitate in a
yellow solution. The reaction mixture was poured into water (∼60
mL), acidified with acetic acid to a pH of 3, and kept at 4 °C
overnight. After filtration and washing with methanol, a yellow solid

Inhibitors of Pim-1 and Pim-2 Protein Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 85
[32P]orthophosphate for 4 h, in which the test compounds were
added for the final 1 h. To immunoprecipitate Pim-1, anti-Flag M2
agarose was added to the cell lysate and incubated for 3 h. A portion
(10%) of the immunoprecipitates was used for Western blotting
with anti-Flag antibodies (input). The other 90% of each sample
was subjected to SDS-PAGE, and 32P-labeled Pim-1 was visualized
by autoradiography.
Cytotoxicity Assays. Human prostate cancer PC3 cells were
seeded in 96-well tissue culture dishes at approximately 10%
confluency and allowed to attach and recover for 24 h. Varying
concentrations of the test compounds are then added to each well,
and the plates were incubated for an additional 48 h. The number
of surviving cells was determined by the MTS assay (Promega).
The percentage of cells killed was calculated as the percentage
decrease in MTS metabolism compared with control cultures.
Toxicity Evaluations. Six week-old female Swiss Webster mice
(approximately 25 g) were purchased from Charles River Labora-
tories. Groups of mice were injected intraperitoneally daily with
vehicle (30% PEG-400, 5% Tween-80, 65% DMSO), or 3, 10, or
50 mg/kg 16a for a total of 7 days. Animals were monitored for an
additional 7 days, and on the final day, blood was collected and
analyzed for complete blood count and blood chemistry (Drug
Metabolism and Clinical Pharmacology Core, MUSC).
Antitumor Assay. A syngeneic mouse tumor model that uses a
transformed murine mammary adenocarcinoma cell line (JC, ATCC
number CRL-2116) and Balb/C mice (Charles River) was performed
as previously described.33 Animal care and procedures were in
accordance with guidelines and regulations of the IACUC of the
Medical University of South Carolina. Tumor cells (1 × 106) were
implanted subcutaneously, and tumor volume was calculated using
the equation: (L × W2)/2. Upon detection of tumors, mice were
randomized into treatment groups. Treatment was then administered
once per day, five days per week, thereafter consisting of intrap-
eritoneal doses of 0 or 50 mg 16a/kg or vehicle (50% DMSO: 50%
phosphate-buffered saline). Whole body weights and tumor volume
measurements were performed three times per week.
Computational Modeling. The molecular structure for com-
pound 4a was built and docked to the crystal structure of Pim-1
(PDB code: 1XWS) using the suite of programs within the
DiscoveryStudio interface (Accelrys). The ligand and protein
structures were prepared for docking by the addition of hydrogens,
followed by the assignment of atom types and charges within the
CHARMm forcefield. This was followed by creation of the docking
grids within a 6 Å radius of the native ligand and molecular
mechanics/energy minimization docking of flexible 4a into the rigid
binding site of Pim-1.
(8) Lilly, M.; Kraft, A. Enforced expression of the Mr 33,000 Pim-1 kinase
enhances factor-independent survival and inhibits apoptosis in murine
myeloid cells. Cancer Res. 1997, 57 (23), 5348–5355.
(9) Aho, T. L.; Sandholm, J.; Peltola, K. J.; Mankonen, H. P.; Lilly, M.;
Koskinen, P. J. Pim-1 kinase promotes inactivation of the pro-apoptotic
Bad protein by phosphorylating it on the Ser112 gatekeeper site. FEBS
Lett. 2004, 571 (1-3), 43–49.
(10) Macdonald, A.; Campbell, D. G.; Toth, R.; McLauchlan, H.; Hastie,
C. J.; Arthur, J. S. Pim kinases phosphorylate multiple sites on Bad
and promote 14-3-3 binding and dissociation from Bcl-XL. BMC Cell
Biol. 2006, 7, 1.
(11) Yan, B.; Zemskova, M.; Holder, S.; Chin, V.; Kraft, A.; Koskinen,
P. J.; Lilly, M. The PIM-2 kinase phosphorylates BAD on serine 112
and reverses BAD-induced cell death. J. Biol. Chem. 2003, 278 (46),
45358–45367.
(12) Kim, K. T.; Levis, M.; Small, D. Constitutively activated FLT3
phosphorylates BAD partially through pim-1. Br. J. Hamaetol. 2006,
134 (5), 500–509.
(13) Jacobs, M. D.; Black, J.; Futer, O.; Swenson, L.; Hare, B.; Fleming,
M.; Saxena, K. Pim-1 ligand-bound structures reveal the mechanism
of serine/threonine kinase inhibition by LY294002. J. Biol. Chem.
2005, 280 (14), 13728–13734.
(14) Kumar, A.; Mandiyan, V.; Suzuki, Y.; Zhang, C.; Rice, J.; Tsai, J.;
Artis, D. R.; Ibrahim, P.; Bremer, R. Crystal structures of proto-
oncogene kinase Pim1: a target of aberrant somatic hypermutations
in diffuse large cell lymphoma. J. Mol. Biol. 2005, 348 (1), 183–193.
(15) Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.;
Peng, C.; Kronkaitis, A.; Li, J.; White, A.; Mische, S.; Farmer, B.
Structural basis of constitutive activity and a unique nucleotide
binding mode of human Pim-1 kinase. J. Biol. Chem. 2005, 280
(7), 6130–6137.
(16) Bregman, H.; Meggers, E. Ruthenium half-sandwich complexes as
protein kinase inhibitors: an N-succinimidyl ester for rapid derivati-
zations of the cyclopentadienyl moiety. Org. Lett. 2006, 8 (24), 5465–
5468.
(17) Debreczeni, J. E.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.;
Bregman, H.; Knapp, S.; Meggers, E. Ruthenium half-sandwich
complexes bound to protein kinase Pim-1. Angew. Chem., Int. Ed.
2006, 45 (10), 1580–1585.
(18) Pogacic, V.; Bullock, A. N.; Fedorov, O.; Filippakopoulos, P.; Gasser,
C.; Biondi, A.; Meyer-Monard, S.; Knapp, S.; Schwaller, J. Structural
analysis identifies imidazo[1,2-b]pyridazines as PIM kinase inhibitors
with in vitro antileukemic activity. Cancer Res. 2007, 67 (14), 6916–
6924.
(19) Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.; Marsden,
B. D.; Knapp, S. Structural basis of inhibitor specificity of the human
protooncogene proviral insertion site in moloney murine leukemia virus
(PIM-1) kinase. J. Med. Chem. 2005, 48 (24), 7604–7614.
(20) Bearss, D.; Liu, X.-H.; Grand, C.; Gourley, E.; Lamb, J.; Lloyd, M.;
Vankayalapati, H. Discovery and characterization of a small molecule
inhibitor for Pim-1 kinase. In American Society of Hematology
National Meeting, 2006; poster presentation.
(21) Lamb, J.; Gourley, E.; Liu, X.-H.; Grand, C.; Vankayalapati, H.;
Warner, S.; Bearss, D. A small molecule inhibitor of Pim-1 kinase
with activity in both hematological and solid tumor malignancies. In
AACR-NCI-EOTRC International Conference on Molecular Targets
and Cancer Therapeutics, San Francisco, CA, 2007.
(22) Cheney, I. W.; Yan, S.; Appleby, T.; Walker, H.; Vo, T.; Yao, N.;
Hamatake, R.; Hong, Z.; Wu, J. Z. Identification and structure-activity
relationships of substituted pyridones as inhibitors of Pim-1 kinase.
Bioorg. Med. Chem. Lett. 2007, 17 (6), 1679–1683.
(23) Holder, S.; Lilly, M.; Brown, M. L. Comparative molecular field
analysis of flavonoid inhibitors of the PIM-1 kinase. Bioorg. Med.
Chem. 2007, 15 (19), 6463–6473.
(24) Holder, S.; Zemskova, M.; Zhang, C.; Tabrizizad, M.; Bremer, R.;
Neidigh, J. W.; Lilly, M. B. Characterization of a potent and selective
small-molecule inhibitor of the PIM1 kinase. Mol. Cancer Ther. 2007,
6 (1), 163–172.
(25) Pierce, A. C.; Jacobs, M.; Stuver-Moody, C. Docking study yields
four novel inhibitors of the protooncogene Pim-1 kinase. J. Med. Chem.
2008, 51 (6), 1972–1975.
References
(1) Selten, G.; Cuypers, H. T.; Berns, A. Proviral activation of the putative
oncogene Pim-1 in MuLV induced T-cell lymphomas. EMBO J. 1985,
4 (7), 1793–1798.
(2) Dhanasekaran, S. M.; Barrette, T. R.; Ghosh, D.; Shah, R.; Varambally,
S.; Kurachi, K.; Pienta, K. J.; Rubin, M. A.; Chinnaiyan, A. M.
Delineation of prognostic biomarkers in prostate cancer. Nature 2001,
412 (6849), 822–826.
(3) Dai, H.; Li, R.; Wheeler, T.; Diaz de Vivar, A.; Frolov, A.; Tahir, S.;
Agoulnik, I.; Thompson, T.; Rowley, D.; Ayala, G. Pim-2 upregulation:
biological implications associated with disease progression and perinu-
eral invasion in prostate cancer. Prostate 2005, 65 (3), 276–286.
(4) Xu, Y.; Zhang, T.; Tang, H.; Zhang, S.; Liu, M.; Ren, D.; Niu, Y.
Overexpression of PIM-1 is a potential biomarker in prostate
carcinoma. J. Surg. Oncol. 2005, 92 (4), 326–330.
(5) Valdman, A.; Fang, X.; Pang, S. T.; Ekman, P.; Egevad, L. Pim-1
expression in prostatic intraepithelial neoplasia and human prostate
cancer. Prostate 2004, 60 (4), 367–371.
(6) Cibull, T. L.; Jones, T. D.; Li, L.; Eble, J. N.; Ann Baldridge, L.;
Malott, S. R.; Luo, Y.; Cheng, L. Overexpression of Pim-1 during
progression of prostatic adenocarcinoma. J. Clin. Pathol. 2006, 59
(3), 285–288.
(7) Chen, W. W.; Chan, D. C.; Donald, C.; Lilly, M. B.; Kraft, A. S. Pim
family kinases enhance tumor growth of prostate cancer cells. Mol.
Cancer Res. 2005, 3 (8), 443–451.
(26) Pagano, M.; Bain, J.; Kazimierczuk, Z.; Sarno, S.; Ruzzene, M.; di
Maira, G.; Elliott, M.; Orzeszko, A.; Cozza, G.; Meggio, F.; Pinna,
L., The selectivity of inhibitors of protein kinase CK2: an update.
Biochem. J. 2008, 415, 353-365.
(27) Momose, Y.; Meguro, K.; Ikeda, H.; Hatanaka, C.; Oi, S.; Sohda, T.
Studies on antidiabetic agents. X. Synthesis and biological activities
of pioglitazone and related compounds. Chem. Pharm. Bull. 1991, 39
(6), 1440–1445.
(28) Bruno, G.; Costantino, L.; Curinga, C.; Maccari, R.; Monforte, F.;
Nicolo, F.; Ottana, R.; Vigorita, M. G. Synthesis and aldose reductase
inhibitory activity of 5-arylidene-2,4-thiazolidinediones. Bioorg. Med.
Chem. 2002, 10 (4), 1077–1084.

86 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Xia et al.
(29) Wada, A.; Kanatomo, S.; Nagai, S. ortho-Directed lithiation of (2-
methoxy)ethoxy- and (2-dimethylamino)ethoxy-arenes. Chem. Pharm.
Bull. 1985, 33 (3), 1016–1022.
(32) Hammerman, P. S.; Fox, C. J.; Birnbaum, M. J.; Thompson, C. B.
Pim and Akt oncogenes are independent regulators of hematopoietic
cell growth and survival. Blood 2005, 105 (11), 4477–4483.
(30) Yang, D.; Chen, Z.; Chen, S.; Zheng, Q. Organic reactions in ionic
liquids: ionic liquid-accelerated three-component reaction: a rapid, one-
pot synthesis of 3-alkyl-5-[(Z)-arylmethylidene]-1,3-thiazolidine-2,4-
diones. Synthesis 2003, 12, 1891–1894.
(33) Lee, B. D.; French, K. J.; Zhuang, Y.; Smith, C. D. Development of
a syngeneic in vivo tumor model and its use in evaluating a novel
P-glycoprotein modulator, PGP-4008. Oncol. Res. 2003, 14 (1), 49–60.
(31) Tan, S.-F.; Ang, K.-P.; Fong, Y.-F. (Z)- and (E)-5-Arylmethylenehy-
dantoins: spectroscopic properties and configuration assignment.
J. Chem. Soc., Perkin Trans. 2 1986, 1941–1944.
JM800937P