Hit to lead account of the discovery of a new class of inhibitors of pim kinases and crystallographic studies revealing an unusual kinase binding mode

Article abstract of DOI:10.1021/jm801242y

A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 KM(ATP) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.

Full text of DOI:10.1021/jm801242y

1814
J. Med. Chem. 2009, 52, 1814–1827
Hit to Lead Account of the Discovery of a New Class of Inhibitors of Pim Kinases and
Crystallographic Studies Revealing an Unusual Kinase Binding Mode†
Kevin Qian,‡ Lian Wang,‡ Charles L. Cywin, Bennett T. Farmer, II, Eugene Hickey, Carol Homon, Scott Jakes,
Mohammed A. Kashem, George Lee, Scott Leonard, Jun Li, Ronald Magboo, Wang Mao, Edward Pack, Charlene Peng,
Anthony Prokopowicz, III, Morgan Welzel,§ John Wolak, and Tina Morwick*
Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06801-0368
ReceiVed September 30, 2008
A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the
hit validation and lead generation process and structure-activity relationship (SAR) studies are presented.
Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly
homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the
Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both
Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the
literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 KM(ATP)
values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural
features which may contribute to the association are discussed.
Introduction
signaling pathway, implicating this pathway to some extent in
the functional activities and expression of Pim kinases.3a,6,7
The Pima kinases include three gene-encoded isoforms: Pim-
1, -2, and -3 with translational variants reported for Pim-1 and
-2 resulting from utilization of alternative starting codons.1 These
enzymes form a distinct family of serine/threonine kinases with
several unique features and limited homology to other members
of the kinase gene family (<30%).2 A high level of homology
exists within the family, however, and studies have suggested
some degree of functional redundancy.1b,3 The Pim kinases
retain a unique consensus hinge region sequence LERPXPX
(L is the gatekeeper residue) which affords some interesting
consequences, and several cocrystal structures of Pim-1 with
ATP analogues or inhibitors have now been reported, demon-
strating some remarkable binding features.4 These proteins have
exhibited high levels of expression in hematopoietic tissue1b,5
and have been implicated as having a functional role in cell
survival.6 This has provided a general mechanism for the
association of Pim kinases with both cancer and immune
regulation. Also noteworthy is the lack of post-translational
regulatory modifications for these kinases which are reportedly
constitutively active with regulation enabled at the level of
expression and proteosomal degradation.1a,4e,6 Transcription of
Pim kinases is induced by a number of cytokines, mitogens,
and growth factors, many of which intersect with the JAK-STAT
As a part of our continuing efforts to identify potential protein
targets involved in the regulation of inflammation and immunol-
ogy, we have previously reported that the serine/threonine kinase
Pim-2 is a novel NF-κB-responsive gene.8 DNA microarray
technology comparing gene expression in 70Z/3 murine pre-B
cells during NF-κB-driven pre-B to immature B cell transition
versus an IKK signaling-defective variant (1.3E2) revealed a
number of novel genes with altered expression, including Pim-
2. The NF-κB signaling pathway has been well documented as
having a critical role in inflammation and immunity as well as
other important physiological processes.9 NF-κB transcriptional
activity is regulated by its association with a cytoplasmic
chaperone IκB, the details of which have been comprehensively
delineated.10 Gold has also recently reported that the related
Pim-1 was identified as a target of CD40 signaling in B cells,
and the increase in the level of Pim-1 expression observed as a
consequence of CD40 signaling was regulated via the activation
of NF-κB.11 We have also found that Pim-2 kinase exhibited
enhanced levels of expression in a variety of inflammatory states.
For example, a comparison of inflamed versus noninflamed
tissue from patients with IBD demonstrated an elevated level
of expression associated with inflammation.12 A role for Pim-1
and -2 in cytokine-induced T-cell growth and survival has also
been reported.13 In a comparison of the sensitivity of stimulated
T-cells from Pim-1-/-Pim-2-/- mice to the sensitivity of those
from wild-type mice following treatment with the immunosup-
pressant rapamycin, it was found that the Pim-1-/-Pim-2-/-
T-cells but not those from wild-type mice were unable to mount
an immune response, suggesting that Pim kinases provide a
PI3K/Akt/TOR-independent pathway promoting lymphocyte
growth and survival. Therefore, Pim kinases may represent an
attractive target in immunology.
†
Coordinates for the Pim-1-DAPPA structure have been deposited in
the Protein Data Bank (PDB) as entry 3F2A.
* To whom correspondence should be addressed. Phone: (203) 798-5751.
Fax: (203) 798-5297. E-mail: tmorwick@rdg.boehringer-ingelheim.com.
‡
These authors contributed equally to this work.
Boehringer Ingelheim summer intern.
§
a Abbreviations: BAD, Bcl-2 antagonist of cell death; CK2, casein kinase-
2; Cot, cancer osaka thyroid; DAPPA, diazepanylpyrazinylphenylacrylic
acid; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein;
HtL, hit to lead; HTS, high-throughput screening; IBD, inflammatory bowel
disease; IκB, inhibitor of κB; IKK, IκB kinase; JAK-STAT, Janus kinase-
signal transducers and regulators of transcription; NF-κB, nuclear factor
κB; PI3K, phosphatidylinositol 3-kinase; Pim, proviral insertion site in
Moloney murine leukemia virus; PKB (Akt), protein kinase B; SAR,
structure-activity relationship; SEM, standard error of the mean (standard
deviation of the result values divided by the square root of the number of
independent results); TOR, target of rapamycin.
The prosurvival function of the Pim proteins in immunology
is parallel to their more established role in oncology whereby
abnormal expression of these kinases has been linked to various
human cancers, including prostate, pancreatic, colon, chronic
lymphocytic leukemia, non-Hodgkin’s lymphoma, and multiple
10.1021/jm801242y CCC: $40.75
2009 American Chemical Society
Published on Web 03/03/2009

New Class of Inhibitors of Pim Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1815
Figure 1. Inhibitors of Pim-1 kinase with reported IC50 values (28, 29, 31, and 33-35) or Ki values (30 and 32).
myeloma.14 The name Pim derives from the original identifica-
tion of Pim-1 as a common proviral insertion site of the Moloney
murine leukemia virus,15 and Pim-2 has also demonstrated a
similar propensity.1b Constitutive expression of Pim-2 in B-cells
has resulted in resistance to various apoptotic stimuli, suggesting
that Pim-2 participates in cell transformation primarily by
positive regulation of survival, unlike other growth factor-
regulated oncogenes such as Myc which induces proliferation,
but is proapoptotic.1a,6 Suppression of the apoptotic component
of Myc function can lead to malignancy, and studies with
transgenic mice have shown that Pim kinases potently cooperate
withMyc,blockapoptosis,andinduceoncogenictransformation.3b,16
Pim kinases may regulate resistance to apoptosis by phospho-
rylation of apoptotic regulatory proteins such as BAD and
4E-BP1.14d,17 It is interesting to note the evolving role of NF-
κB has implicated these transcription factors in oncogenic
transformation in addition to their more established role in
immunology.18 Studies suggest that NF-κB promotes cell
proliferation and transformation, functions as an antiapoptotic
regulator by transcription of target genes known to block
induction of apoptosis, and stimulates angiogenesis and me-
tastasis. Both Pim-2 and Akt, which engage distinct hemato-
poietic cell survival pathways, reportedly can positively regulate
NF-κB-dependent transcription by inducing phosphorylation of
the serine/threonine kinase Cot, thereby enhancing IKK activity,
a key enzyme leading to release of NF-κB transcription factors.
It has been further suggested that the activation of NF-κB is
critical for mediation of the antiapoptotic role of Pim-2 as well
as its ability to cooperate with the oncogenic protein Myc.6,19
Thus, Pim kinases are critical mediators of hematopoeitic cell
survival in both immunology and oncology, and the development
of inhibitors of these kinases has potential utility for treatment
of associated pathologies as well as enhancing an understand-
ing of the function of this unique family. In this paper, we
describe our HtL efforts to identify inhibitors of Pim-2 kinase
and our discovery of a series of pyrazinylcinnamic acids as
potent Pim-2 inhibitors.
is observed (29, 31, and 34), which is surprising considering
the degree of homology between these enzymes.
Results and Discussion
HTS Assay and Hit Validation. The corporate compound
collection was screened in a homogeneous luciferase assay using
GST-Pim-2,AKRRRLSAasasubstrate,21 andaluciferin-luciferase
detection reagent to quantify residual ATP. Dose-response data
on hits showing reproducible activity from the screen were
generated for both Pim-2 and Pim-1 using the same assay
format. Pim-2 shares 62% sequence identity with Pim-1 in the
kinase domain, and we were interested in selective inhibitors
(Pim-2 vs Pim-1) as well as compounds which potently inhibited
both kinases. Generic criteria for hit validation have been
detailed in a recent publication.22 Initial filtering of the hits is
often accomplished by utilization of selectivity assays. However,
since members of the the Pim family have limited homology
to other protein kinases, the choices were not obvious. Therefore,
proteins for selectivity filtering were chosen by identifying
kinases with the most significant inhibitor cross reactivity with
Pim-2 from a large in-house kinase data set and included
GSK3B, MAP2K6, MAP3K11, PDPK1, STK12, and SYK.
With the exception of typical promiscuous kinase structural
classes, many of the Pim-2 hits were selective in these assays.
After removal of nonselective inhibitors, additional filtering
included confirmation of structural identity, purity, and stability,
removal of structures with reactive chemotypes, and inspection
of historical data for expanded selectivity filtering and identi-
fication of frequent hitters. Activity was then confirmed on
samples of available solids. Actives were also characterized in
terms of drug-likeness and novelty, and representatives were
examined for ATP competition using a radiometric assay
(Supporting Information). The Pim hit set was unusual compared
to screening results from other kinases in that a significant
proportion of the hits were found to have carboxylic acid
functionality or were acid isosteres (data not shown). Since
selectivity is a common issue for kinase inhibitors as the
predominant binding occurs in the ATP site, some of these
carboxylate-bearing inhibitor scaffolds were selected for further
profiling in anticipation of identifying a uniquely selective class
of inhibitors. One such series identified for follow-up was
distinguished by the cinnamic acid functionality and is shown
in Figure 2. Cinnamic acid is itself a natural product arising
from the deamination of phenylalanine and, interestingly, has
Inhibitors of Pim-1 kinase have only recently been described
in the literature (Figure 1). Examples include the promiscuous
kinase ligand staurosporine (28) and related bisindolylmaleim-
ides, disubstituted bicyclics 29, 30, and 32, trisubstituted
pyridone 31, and various flavonoids such as quercetin (33),
quercetagetin (34), and the related 35 (LY294002).4a-d,f,h,20
Interestingly, for publications which provide selectivity data
comparing Pim-1 and Pim-2, selectivity for Pim-1 over Pim-2

1816 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
Structural Studies) having a potency within 4-fold of that of
hit compound 1.
Results reported in Table 4 summarize our efforts to
determine the importance of the homopiperazine fragment and
execute its optimization. Complete removal of the fragment (26)
suggested the methylhomopiperazine contributed a >50-fold
improvement to binding. That the improvement was most likely
due to the presence of the distal amine was suggested by the
homopiperidine analogue 8. Attempts to replace the distal
nitrogen of related analogues with oxygen also resulted in a
significant loss of potency (11 vs 12, 14 vs 19, and 23 vs 25).
Additionally, reduction of the basicity of the amine by acylation
was also detrimental (1 vs 9 and 13 vs 18). Removal of the
methyl from the distal amine of 1 modestly improved the
potency (7), and a similar trend was seen for acyclic amines 14
and 16, but not for piperidine analogues 10 and 11 which had
similar potency. Several compounds demonstrated inhibition
within 3-fold of our standard staurosporine, a potent and
promiscuous kinase inhibitor (7, 15, and 22-24).24 Additionally,
compound 27 shows that the pyrazine core can be replaced with
pyridine with no significant effect on binding.
Figure 2. Original dose-response data: IC50 ) 270 nM (Pim-2), and
IC50 ) 340 nM (Pim-1). Dose-response data for the resynthesized
solid: IC50 ) 40 nM (Pim-2), and IC50 ) 57 nM (Pim-1).
Table 1. Selectivity Data for Inhibitor 1
kinase
ITK
ANKRD3
LYN
VEGFR1
EGFR
PRKCD
DAPK1
KI (µM)a
kinase
LK
PTK2
CLK1
MST4
KD (µM)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
MAP3K11
STK12
a Calculated from IC50.
Compounds from this series demonstrated similar IC50 values
for both Pim-2 and Pim-1 using the same assay conditions. Some
examples are listed in Table 5. We could not find Pim-2 data
for all the Pim-1 inhibitor classes described in the literature
(Figure 1), but for those reporting this information (29, 31, and
34), selectivity for Pim-1 over Pim-2 is observed.25 On the basis
of the reported selectivity, it was suggested that Pim-2 may be
more difficult to target. The observed selectivity reported as
IC50 values likely results in part from significant differences in
the affinity of ATP for these proteins (Pim-1 KM,ATP ) 407 µM;
Pim-2 KM,ATP ) 3 µM).26 The divergent KM values are quite
surprising considering the high degree of homology and can
affect the interpretation of in vitro selectivity as determined by
IC50 comparisons for these reversible ATP competitive inhibi-
tors. Our Pim-1 and Pim-2 assays used an ATP concentration
of 1 µM (below KM for both enzymes), while IC50 values for
29, 31, and 34 were determined using ATP concentrations of
100, 25, and 10 µM, respectively, for both Pim-1 and Pim-2.
The IC50 values will depend on the intrinsic affinity of the
inhibitor (Ki) as well as the level of competition from ATP.
The relationship between these variables has been derived to
describe classical inhibition (Cheng-Prusoff equation):27
been shown to induce cytostasis and reverse malignant properties
of human tumor cells in vitro.23
Lead Generation and SAR Studies. Hits from this series
were part of a general library stored in solution; therefore, hit
1 was resynthesized, and activity was confirmed on the solid
(Figure 2). ATP competition studies on the original library hit
had found 1 to be an ATP competitive inhibitor. The presence
of the cinnamic acid component prompted us to evaluate
reversibility. Repeating the ATP competition assay with an
initial 30 min preincubation of compound and protein, we again
found that ATP could attenuate inhibition in a concentration-
dependent manner, suggesting reversibility. Additionally, in-
cubation of 1 with Pim-2 protein at 4 °C for 24 h followed by
direct analysis by mass spectrometry revealed no adduct with
1 (Supporting Information). The absence of covalent modifica-
tion of the protein was subsequently corroborated by X-ray
crystallography (see Structural Studies). This series showed no
activity against kinases in the hit validation panel (all >10 µM).
Additional selectivity was evaluated for 1 using an in-house
panel of 13 kinases (Table 1), and the results demonstrated good
selectivity for the series. Early in vitro profiling to assess
DMPK-related properties for inhibitor 1 is shown in Table 2.
The compound was found to have good solubility (>2 mg/mL),
acceptable permeability, a good half-life in human liver mi-
crosomes, and no CYP inhibition below 30 µM.
Early modeling efforts could not provide a conclusive
binding mode for this series of inhibitors, and therefore, initial
SAR studies were designed to explore key features of the
pharmacophore. As mentioned previously, the unusual preva-
lence of carboxylic acids or isosteres in the Pim-2 hit set
suggested the importance of this functional group, and
modifications were therefore incorporated to assess this
observation. The results in Table 3 are conclusive. Substitu-
tion of the carboxyl group with the ester or amide (2 and 3)
was generally detrimental. Saturation of the linker was also
adverse (4), and complete removal of the cinnamic acid
portion expectedly generated an inactive compound (6).
Comparing this analogue to 26 (Table 4) suggests the
cinnamic acid fragment contributes more significantly to the
potency than does the homopiperazine. Interestingly, the is-
osteric tetrazole (5) can apparently engage the network of
interactions observed for the acid to some extent (see
IC50 ) Ki(1 + [ATP]/KM,ATP
)
It can be seen from this equation that only a small variation
in IC50 occurs as the ATP concentration falls below the KM.
However, as the concentration increases above KM, the impact
becomes nearly proportional.28 Using an alternative assay
format, a comparison of Pim-1 and Pim-2 IC50 values for 1 was
made at ATP concentrations near the KM for each protein (Pim-1
at 400 µM and Pim-2 at 5 µM), and alternatively at 100 µM
for both proteins, which for Pim-2 was well above the KM, but
below the KM for Pim-1. The data are listed in Table 6.29 These
results demonstrate the variability in observed selectivity for 1
when comparing IC50 values generated at different ATP
concentrations. Considering these results, it can be seen that
when the ATP concentration (100 µM) is significantly above
the KM for Pim-2 but below the KM for Pim-1, it appears that 1
is moderately selective for Pim-1 over Pim-2. However, when
experiments were conducted near the KM,ATP for both proteins,
the IC50 results show a slight preference for Pim-2. The trend
is also observed for our standard staurosporine (28), which in
our hands exhibited similar IC50 values for the two enzymes at
1 µM ATP (7.5 nM for Pim-1 and 7.0 nM for Pim-2), but when

New Class of Inhibitors of Pim Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1817
Table 2. Profiling Data for Inhibitor 1
solubility
pH 7.4 (mg/mL)
solubility
pH 2.0 (mg/mL)
caco-2 A-B
caco-2 B-A
HLM t1/2
(min)
CYPa
IC50 (µM)
(×10-6 cm/s)
(×10-6 cm/s)
>2
>2
2.73
3.01
159
>30
a CYP isoforms tested: 1A2, 2C9, 2C19, 3A4 (BQ, BFC), and 2D6.
Table 3. SAR of the Pyrazine Series with Variation of the Acid
Kinase Selectivity Assessment. An expanded selectivity
assessment for the pyrazine series was carried out during the
course of the lead generation sequence. In addition to the
selectivity testing already completed (>10 µM for 17 kinases),
1 was further evaluated against another 34 kinases, and as
initially anticipated, the series demonstrated remarkable selectiv-
ity [>50% of control at 10 µM for 33 of the 34 proteins
(Supporting Information)]. The one notable exception was CK2,
whereby 1 demonstrated potent inhibition (0% of control at 10
µM). Dose-response data were subsequently generated (IC50
) 663 nM for CK2R1, and IC50 ) 701 nM for CK2R2).33 Like
Pim-1, CK2 has been reported to be constitutively active, and
regulation of this pleiotropic protein has been the subject of
intensive research. CK2 promotes cell survival, is antiapoptotic,
and has exhibited an elevated level of expression in a variety
of human tumors, also analogous to Pim kinases.34 Additionally,
CK2 and Pim kinases have been associated with activation of
NF-κB transcriptional activity in cancer.18a Potent inhibitors of
CK2 are sparse, but for those, reported carboxylic acids are well
represented,35 similar to what was observed for the Pim-2 hit
set. It would be interesting to speculate that endogenous acids
may exist with some functional responsibility for regulating
these constitutively active kinases through competitive inhibi-
tion. Polyphenols represent another typical class of CK2
inhibitors,36 and this was likewise a well-represented motif for
Pim-2 actives in our screen and is seen in reported Pim-1
inhibitors.4b,h The flavenoids and related phenolic derivatives
have, however, demonstrated a more comprehensive propensity
for inhibition of protein kinases and other enzyme systems.37
The homology between Pim-2 and CK2 is not significant (20%
identity in the kinase domain), so the cross reactivity was
surprising, but not unprecedented.4f,38
Fragmenta
a Assay values reported as the mean ( SEM of n g 2 replicates.
tested at 100 µM ATP using an alternative assay format, 28
demonstrated a 10-fold selectivity for Pim-1 (24.6 nM for Pim-1
and 345 nM for Pim-2).29 It should be noted, therefore, when
considering relative potency in cells where ATP concentrations
may be in the low millimolar range,30 Pim-2, having a greater
affinity for ATP, should be intrinsically more difficult to target
with an ATP competitive inhibitor.31
Structural Studies. As mentioned previously, Pim kinases
retain a consensus hinge region sequence LERPXPX which is
unique within the kinase gene family. The vast majority of
kinase inhibitors bind in the ATP-binding pocket and require
hydrogen bond interactions with the hinge. Crystal structures
of these inhibitors typically demonstrate a minimum consensus
donor interaction from a specific hinge residue (equivalent to
residue 123 of Pim-1).39 For the Pim kinases, this residue is a
proline, thereby precluding the interaction. The unusual land-
scape for this critical binding region hampered our initial
modeling efforts and obligated the use of X-ray crystallography
to identify the binding mode of our inhibitors. While our
crystallization efforts with Pim-2 were not successful,40 we were
able to obtain a cocrystal structure of 1 bound to the homologous
Pim-1 protein (Pim-1-DAPPA) determined at 1.9 Å resolution
by soaking experiments using a previously described protocol
for crystal formation and structure determination.2,4e While the
ATP KM values for Pim-1 and -2 are significantly different, our
inhibitors consistently demonstrated a more comparable affinity
for the two enzymes, as revealed by our SAR studies. Therefore,
we anticipated the Pim-1 structure would provide a common
binding mode, and in fact, the observed binding interactions
are consistent with our Pim-2 SAR.
Chemistry. A general method for preparation of the pyrazine
inhibitors is shown in Scheme 1 exemplifying the synthesis of
1. Monosubstitution of dichloropyrazine was achieved with
excess N-methylhomopiperazine. Initial nucleophilic substitution
with the amine deactivates the second electrophilic site,
discouraging formation of a disubstituted product. Suzuki
coupling of the intermediate with a boronic acid-substituted
cinnamic acid provided the target inhibitor.
The ester (2) was prepared analogously using a boronic acid-
substituted methylcinnamate. Other carboxyl variations were
synthesized directly from the acid (Scheme 2). For example,
the amide (3) and the propionic acid (4) were prepared from
the acid (1) either by aminolysis of an activated intermediate
or by hydrogenation. Scheme 3 illustrates synthesis of the
tetrazole (5) which was prepared by initial Suzuki coupling of
a monosubstituted pyrazine to generate a benzaldehyde inter-
mediate. This was converted to an acrylonitrile via Horner-
Emmons chemistry. Conversion of the nitrile to the tetrazole
by traditional procedures using sodium azide and HCl or acetic
acid yielded complex mixtures from which the desired inhibitor
could not be isolated. A more serviceable option was found
using a Lewis acid, zinc bromide, with sodium azide in a mixture
of water and DMF.32
The Pim-1-DAPPA complex structure reported here is very
similar to our apo Pim-1 kinase structure as well as that bound
with ANP-PNP reported previously.2,4e It adopts the typical

1818 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Table 4. SAR of the Pyrazine Series with Variation of the Homopiperazinea
Qian et al.
Table 5. IC50 Values for Pim-2 versus Pim-1 at 1 µM ATP
a
a
Pim-2 IC50 (nM)
Pim-1 IC50 (nM)
1
40 ( 5.8
22 ( 2.8
480 ( 15
12 ( 0.5
46 ( 3.5
57 ( 1.8
33 ( 3.0
370 ( 10
13 ( 0.5
50 ( 0.5
15
20
23
27
a Assay values reported as the mean ( SEM of n g 2 replicates.
Table 6. IC50 Values for 1 (Pim-2 or Pim-1) at Different ATP
Concentrationsa
[ATP]
Pim-2 IC50 (nM)
Pim-1 IC50 (nM)
near KM
100 µM
153 (5 µM ATP)
760
305 (400 µM ATP)
161
a Data generated at Invitrogen.29
kinase canonical fold with the N-lobe mainly comprised of
ꢀ-sheets and the C-lobe predominantly R-helices. The ligand is
bound in the cleft formed between the two lobes as shown in
Figure 3a. The structural motifs that interact with 1 include the
hinge region, the RC helix, and several ꢀ sheets as labeled. A
surface representation (Figure 3b) containing the bound inhibitor
inside the cleft illustrates that all inhibitor atoms are located
within the cleft, resulting in strong van der Waals interactions
between the inhibitor and Pim-1 kinase. The diazepanylpyrazine
group of the inhibitor binds near the edges of the cleft, while
the acid and a Mg2+ bind toward the bottom, an area rendered
acidic by the presence of two carboxyl residues (Glu89 and
Asp186). The edges of the pocket are comprised of many
secondary structural elements common to protein kinases,
including the hinge, ꢀ1, ꢀ6, the catalytic loop, and the DFG
motif.
The ATP binding site in protein kinases has been subdivided
into specific regions characterized by ATP or inhibitor moieties
with which they interact. These have been generally defined
by Williams as the adenine, ribose, and hydrophobic pockets,
the phosphate groove, and the specificity surface.41 Liao has
further subdivided and characterized this binding region an-
notating key domains.42 In the front cleft, A (adenine), R
(ribose), and P (phosphate) are used by ATP. Not used by ATP
is a small region in the deep front pocket (K). In the back cleft
are various regions (BP-I-IV) not utilized by ATP but often
exploited to gain ligand selectivity such as the hydrophobic
region (BP-I) used for kinase targets with a small gatekeeper.
In the Pim-1-DAPPA complex, the acrylic acid binds in the K
pocket, the basic diazepanylpyrazine spans both the specificity
surface and the ribose pocket, and the phenyl moiety of the
inhibitor binds in the adenine pocket. Regions in the back cleft
are not occupied (Figure 4). These interactions, as elaborated
below, are consistent with our key SAR findings, including the
importance of both the acid and basic amine as well as the
absence of evidence for a hydrogen bonding interaction with
the hinge.
The acrylic acid forms a salt bridge with a bound Mg2+, which
in turn forms another salt bridge with Glu89 from the RC helix,
creating a dual salt bridge system (Figure 4a). In this system,
Mg2+ plays a critical role as a counterion connecting these
negatively charged species. Structurally, the dual salt bridge is
at the bottom of the inhibitor-binding cleft (Figure 3) and
therefore concealed from solvent. As a result, this dual salt
bridge is rendered quite stable and may contribute significantly
to the potency of 1. Additionally, the carboxyl group of the
inhibitor hydrogen bonds directly with Lys67 (not shown).
Another important structural feature at this location is the critical
salt bridge formed between Lys67 (from ꢀ3, N-lobe) and
a Assay values reported as the mean ( SEM of n g 2 replicates.

New Class of Inhibitors of Pim Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1819
Scheme 1a
a (i) N-Methylhomopiperazine, methylene chloride; (ii) 3-[(E)-2-carboxyvinyl]benzeneboronic acid, bistriphenylphosphinepalladium(II) chloride, 2 M
Na2CO3, DMF, µW.
Scheme 2a
a (i) N-[3-(Dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride, benzotriazole-1-ol, N-methylmorpholine, NH3, dioxane; (ii) ammonium formate,
palladium/C, ethanol.
Scheme 3a
direct interaction is in agreement with the variety of basic groups
which are tolerated at this position (Table 4).
The phenyl group of 1 occupies the adenine pocket (Figure
4c). This pocket is bounded by the hinge. The vast majority of
kinase inhibitors form one or more hydrogen bonding interac-
tions with the hinge from functional groups residing in this
pocket. While the existence of aromatic CH · · ·O hydrogen
bonds has been asserted,43 there is no evidence to support
hydrogen bonding interactions with the hinge for the Pim-
1-DAPPA complex. All aromatic carbon atoms of 1 are more
than 3 Å from hinge carbonyl oxygens. The predominant binding
interactions between the phenyl group and the hinge are van
der Waals contacts. This is in contrast to Pim-1 structures of
30-32 and 35 in which aromatic CH· · ·O interactions with the
main chain carbonyl of Glu121 are reported.4a,c,f On the other
hand, 29, an inhibitor structurally related to 30, affords no
hydrogen bonding interactions from its aromatic ring, while
quercetagetin (34) and staurosporine (28) demonstrate hydrogen
bond donation to the Glu121 main chain carbonyl by a hydroxyl
and pyrrolidinone proton, respectively.4b,d,f
There had been some concern that the acrylic acid moiety
may potentially act as a Michael acceptor enhancing the affinity
of our compounds by covalent modification of the protein. We
had evaluated reversibility of 1 by a time-dependent assay with
varying concentrations of ATP, and the results suggested
reversible binding. Our structure also confirms this observation,
as an examination of the area near the carbon ꢀ to the acid
reveals no residues within bonding distance of the protein.
One final observation explained by our cocrystal structure is the
inconsequential replacement of the central pyrazine with a pyridine
(1 and 27). In concert with this SAR finding, our structure shows
no residues within 4 Å for this solvent-exposed nitrogen.
The unique features for Pim-1 ligand binding described herein
provide opportunities for identification of selective inhibitors
such as 1. While 1 demonstrated selectivity against 49 protein
kinases, it exhibited interesting cross reactivity with CK2 (20%
identity to Pim-2 in the kinase domain). A structural basis for
this observation is suggested by comparing the Pim-1-DAPPA
complex with a carboxyl-bearing liganded CK2 structure
(CK2-IQA, PDB entry 1OM1). An overlay of the structures
demonstrates good overlap with key residues associated with
a (i) 3-Formylphenylboronic acid, bistriphenylphosphinepalladium(II)
chloride, 2 M Na2CO3, DMF, µW; (ii) cyanomethylphosphonic acid diethyl
ester, sodium hexamethyldisilazane, THF; (iii) sodium azide, zinc bromide,
water, DMF.
Asp186 (from the DFG motif, C-lobe). These two residues are
highly conserved in the human kinome and believed to be
essential for kinase activity. This salt bridge functions to
maintain an active conformation by bringing together the two
kinase lobes. It may also further shield the network of
interactions described above from solvent. The strong interaction
of the acid group with Pim-1 explains why analogues from the
series lacking this group are essentially inactive (Table 3), and
why the carboxyl group was a predominant feature of the hit
set. Additionally, the involvement of multiple components
associated with this binding network necessitates a precise
location and orientation for the carboxyl group and is consistent
with the fact that saturated analogue 4 also loses activity.
The basic nitrogen of the homopiperazine group of 1 is also
important, and elimination of this feature results in a significant
loss of potency (Table 4). In the crystal structure, this nitrogen
binds near the kinase specificity surface and the ribose pocket
but is involved in no direct interaction with the protein. It is
located in a region aligned with three acidic residues [Asp131
(RD), Asp128 (RD), and Glu171 (catalytic loop)] and forms
water-mediated hydrogen bonds with two of the residues (Figure
4b). It is reasonable to assume that the basic group also
contributes to Pim-1 binding by neutralizing some of the
negative charges associated with these residues. The lack of a

1820 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
Figure 3. Overview of the Pim-1-DAPPA complex structure. (a) A ribbon diagram shows that 1 binds in the cleft between two lobes of Pim-1.
Structural elements directly involved in binding of 1 are annotated. A Mg2+ (shown as a blue sphere) forms a salt bridge with the carboxylic acid
of 1. (b) A surface representation rendered with electrostatic potential (red for negative and blue for positive) shows that 1 binds in a cleft that is
highly negatively charged. The acrylic acid binds near the bottom of the cleft, the phenyl group near the hinge region, and the diazepanylpyrazine
at the surface of the pocket.
Figure 4. Binding interactions of Pim-1 with 1. (a) The carboxyl group of the inhibitor forms a salt bridge with a bound Mg2+ that in turn forms
a salt bridge with Glu89 from the RC helix. The Lys67-Asp186 salt bridge shields Mg2+ bridges from solvent. (b) The basic group does not
interact with Pim-1 directly but forms water-mediated hydrogen bonds and binds in a region that is lined up with three acidic residues. (c) The
phenyl group binds in the adenine pocket. Inhibitor atoms and Pim-1 residues at the hinge region do not form any hydrogen bond interactions.
the carboxylic acid: Lys67, Glu89, and Asp186 (Lys68, Glu81,
and Asp175 in CK2). Additionally, a water molecule in CK2
occupies a space similar to that of the Mg2+ in Pim-1 (Figure
5). Therefore, the carboxylate ion of 1 can likely propagate a
comparable network of hydrogen bonds in CK2. Furthermore,
both ligands orient a hydrophobic face toward the hinge where
van der Waals interactions support binding. Like the Pim-
1-DAPPA complex, the CK2-IQA complex does not engage
a hydrogen bond with the hinge.
The notable similarities in the binding hypothesis for the
carboxylic acid of 1 with CK2 in comparison with Pim-1 suggest
a comparable distinction. An empirical confirmation was subse-
quently completed using ester (2) and amide (3). The data listed
in Table 7 show a significant reduction in potency for these
structurally related non-acids, in agreement with the hypothesis.
of inhibitor cross reactivity may offer a more aggressive approach
to overcoming antiapoptotic mechanisms (and may be one aspect
of the association of flavenoids with induction of apoptosis).37a,c
Additionally, we have described some interesting binding interac-
tions identified by X-ray crystallography, including an unusual
network of chelation and hydrogen bonding associated with the
carboxylic acid of the inhibitor series. The lack of hinge binding
is also unusual, but not unprecedented for Pim-1 inhibitors. The
Pim kinases, having exhibited potentially important roles in cell
survival, are becoming a more recognized target, especially in
oncology, but more information is needed to fully define the
mechanistic contributions for this family in signaling pathways.
The identification of potent and selective inhibitor classes and an
understanding of important structural features associated with these
inhibitors offer opportunities to design and develop compounds
which can more fully delineate these mechanisms.
Conclusion
Experimental Section
In summary, we have identified a potent series of inhibitors of
Pim kinases. The findings provide a new class of inhibitors for
this unique family of proteins, for which limited examples are
currently available. While highly selective within the kinase gene
family (49 kinases), the series demonstrates unexpected cross
reactivity with CK2, a protein of limited homology. As these
kinases collectively participate in cell survival pathways, this pattern
1
General Methods. H and 13C NMR spectra were recorded on
a Bruker UltraShield-400 MHz spectrometer operating at 400 and
100 MHz, respectively, in solvents as noted. Mass spectra were
recorded on a Waters ZQ single quadrupole mass spectrometer using
electrospray positive/negative ionization. The HPLC system used
for analytical purposes (methods A and B) consisted of an Agilent
1100 system (binary pump, diode array detector, and autosampler)

New Class of Inhibitors of Pim Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1821
product was characterized by mass and used directly. 3-[(E)-2-
carboxyvinyl]Benzeneboronic acid (1-2 equiv) and bistriph-
enylphosphinepalladium(II) chloride (0.01-0.028 equiv) were
combined in a microwave reaction tube. The intermediate prepared
previously was added as a solution in DMF, and 2 M Na2CO3 (5-15
equiv) was then added. The tube was sealed, and the reaction
mixture was heated in a microwave reactor for 10-20 min at
100-150 °C. The reaction mixture was then filtered through a plug
of Celite and purified directly using HPLC with an acetonitrile/
water/formic acid, acetonitrile/water/TFA, or acetonitrile/water
gradient, preparative TLC with a methanol/ammonia/dichlo-
romethane gradient, or flash chromatography using a methanol/
dichloromethane gradient. Products having a basic nitrogen purified
by preparative HPLC using a gradient incorporating formic acid
were typically isolated as formates. These were further characterized
as HCl salts formed by dissolution of a sample of the formate in 1
N HCl followed by concentration.
The general method was used for the preparation of compounds
1, 7-20, 22, 23, and 25. Modifications to the general method or
alternative methods are described for compounds 2-6, 21, 24, 26,
and 27.
Figure 5. Structural overlay of Pim-1-DAPPA (blue) and CK2R1-IQA
(orange) complexes. The comparison shows good alignment with key
residues. No hydrogen bond interactions with the hinge are observed
for either ligand.
(E)-3-{3-[6-(4-Methyl[1,4]diazepan-1-yl)pyrazin-2-yl]phenyl}acr-
ylic Acid (1). Purified by preparative HPLC with an acetonitrile/
Table 7. CK2 Percent Inhibition for 2 and 3 at 10 µMa
1
water/formic acid gradient: yield 67%; H NMR (DMSO-d6, 400
% inhibition for CK2R1
% inhibition for CK2R2
MHz) δ 8.49 (formate), 8.32 (s, 1H), 8.20 (s, 1H), 8.15 (s, 1H),
8.11 (d, J ) 7.7 Hz, 1H), 7.78 (d, J ) 7.6 Hz, 1H), 7.68 (d, J )
16 Hz, 1H), 7.54 (dd, J ) 7.6, 7.8 Hz, 1H), 6.66 (d, J ) 16 Hz,
1H), 3.86-3.89 (m, 2H), 3.72-3.75 (m, 2H), 2.74-2.77 (m, 2H),
2.57-2.60 (m, 2H), 2.32 (s, 3H), 1.94-2.00 (m, 2H). HCl salt: 1H
NMR (DMSO-d6, 400 MHz) δ 11.0 (s, br, 1H), 8.61 (s, 1H), 8.38
(s, 1H), 8.26 (s, 1H), 8.16 (d, J ) 7.8 Hz, 1H), 7.81 (d, J ) 7.8
Hz, 1H), 7.71 (d, J ) 16 Hz, 1H), 7.56 (dd, J ) 7.8, 7.8 Hz, 1H),
6.70 (d, J ) 16 Hz, 1H), 4.40-4.45 (m, 1H), 3.80-3.90 (m, 2H),
3.67-3.76 (m, 1H), 3.55-3.64 (m, 1H), 3.45-3.52 (m, 1H),
3.15-3.29 (m, 2H), 2.78 (d, J ) 4.8 Hz, 3H), 2.39-2.50 (m, 1H),
2.15-2.26 (m, 1H); 13C NMR APT (DMSO-d6, 100 MHz) δ
167.5(+), 152.8(+), 147.8(+), 143.6(-), 137.0(+), 134.9(+),
129.4(-), 128.9(-), 128.2(-), 128.1(-), 127.4(-), 126.5(-),
120.0(-), 55.7(+), 54.8(+), 45.1(+), 43.2(-), 40.8(+), 23.3(+);
ESMS m/z 339 (M + H); HPLC method A, tR ) 5.40 min (100%).
1-(6-Chloropyrazin-2-yl)-4-methylperhydro-1,4-diazepine (inter-
2
3
14 ( 1
42 ( 1
7 ( 1
36 ( 1
a The reported percent inhibition is the mean ( SEM of n ) 2 replicates
(data for 1, IC50 ) 663 nM for CK2R1 and IC50 ) 701 nM for CK2R2).33
with a Zorbax Eclipse XDB-C18 column (4.6 mm × 150 mm, 5
µm, part no. 993967-902). Detection was conducted at 254 nm,
and integration used Agilent Chemstation software, version A 10.02.
The mobile phase was a gradient using either a 10:90 A/B to 95:5
A/B mixture over 11 min and hold (method A) or a 4:96 A/B to
95:5 A/B mixture over 8 min and hold (method B), and a common
flow rate of 1 mL/min. Solvent A was 0.1% formic acid in
acetonitrile, and solvent B was 0.1% formic acid in water.
Purification by flash chromatography was accomplished using Redi
Sep prepacked disposable columns run on the Isco Combiflash.
Preparative TLC was done using Uniplate silica gel plates purchased
from Analtech. Microwave reactions were conducted in a PS3 Smith
Synthesizer microwave reactor. Purification by reverse-phase HPLC
utilized a mass-directed preparative scale LC/MS Fractionlynx
system controlled by Masslynx version 3.5. The system consisted
of a 2525 high-performance liquid chromatography (HPLC) pump,
a ZQ single quadrupole mass spectrometer, two 515 high-
performance liquid chromatography (HPLC) pumps for at column
dilution and a makeup pump, a 2767 Sample Manager, and a 2996
photo diode array detector (Waters Corp., Milford, MA). All
samples were purified on an Atlantis prep dC18 (5 µm particle
diameter) 19 mm × 50 mm column. The mobile phases consisted
of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1%
formic acid. The at column dilution solvent used was methanol
with 0.1% formic acid at a constant flow rate of 1 mL/min and an
overall system flow rate of 21 mL/min. All solvents were purchased
from EM Science. All reagents were purchased from Aldrich
Chemical Co. with the following exceptions: N-methylhomopip-
erazine, 1,4-diaminobutane, and 3-aminopropionamide from TCI,
N-acetylhomopiperazine from Fluka, 1-BOC-4-(aminomethyl)pip-
eridine from Lancaster, 1-N-BOC-cis-1,4-cyclohexyldiamine from
Albany Molecular Research, and 3-[(E)-2-carboxyvinyl]benzenebo-
ronic acid and {3-[(E)-3-methoxy-3-oxo-1-propen-1-yl]phenyl}boro-
nic acid from Combiblocks.
1
mediate): yield 55%; H NMR (DMSO-d6, 400 MHz) δ 8.10 (s,
1H), 7.77 (s, 1H), 3.71 (dd, J ) 5, 5 Hz, 2H), 3.61 (dd, J ) 6.1,
6.1 Hz, 2H), 2.60 (dd, J ) 5, 5 Hz, 2H), 2.47 (dd, J ) 5.4, 5.4 Hz,
2H), 2.25 (s, 3H), 1.90-1.84 (m, 2H); 13C NMR APT (DMSO-d6,
100 MHz) 153.3, 145.4, 128.2, 129.0, 56.6, 56.5, 46.1, 45.9, 45.8,
26.4; ESMS m/z 227 (M + H).
(E)-3-{3-[6-(4-Methyl[1,4]diazepan-1-yl)pyrazin-2-yl]phenyl}acr-
ylic Acid Methyl Ester (2). {3-[(E)-3-Methoxy-3-oxo-1-propen-1-
yl]phenyl}boronic acid (125 mg, 0.61 mmol) and bistriphenylphos-
phinepalladium(II) chloride (30 mg, 0.057 mmol) were combined
in a microwave reaction tube. Subsequently, 1-(6-chloropyrazin-
2-yl)-4-methyl[1,4]diazepane (130 mg, 0.57 mmol) was added as
a solution in DMF (8 mL) followed by an aqueous solution of
Na2CO3 (2 M, 3 mL, 6 mmol). The tube was sealed, and the reaction
mixture was heated in the microwave at 120 °C for 20 min. The
reaction mixture was then filtered through a plug of Celite and
purified by flash chromatography using a dichloromethane/methanol/
ammonia gradient to provide the title compound (124 mg, 61%):
1H NMR (DMSO-d6, 400 MHz) δ 8.48 (s, 1H), 8.36 (s, 1H), 8.14
(s, 1H), 8.12 (d, J ) 7.7 Hz, 1H), 7.82 (d, J ) 7.7 Hz, 1H), 7.77
(d, J ) 16 Hz, 1H), 7.56 (dd, J ) 7.7, 7.7 Hz, 1H), 6.79 (d, J )
16 Hz, 1H), 3.83-3.88 (m, 2H), 3.72-3.78 (m, 2H), 3.75 (s, 3H),
2.64-2.69 (m, 2H), 2.48-2.53 (m, 2H), 2.27 (s, 3H), 1.90-1.97
(m, 2H); 13C NMR APT (methanol-d4, 100 MHz) δ 169.0(+),
155.0(+), 150.3(+), 146.0(-), 139.2(+), 136.3(+), 130.5(-),
129.8(-), 129.7(-), 128.9(-), 128.5(-), 127.7(-), 119.3(-),
58.8(+), 58.3(+), 52.2(-), 47.2(+), 46.7(-), 46.3(+), 28.0(+);
ESMS m/z 353 (M + H); HPLC method A, tR ) 6.21 min (96.9%).
General Procedure for the Synthesis of Aminopyrazin-2-
ylphenylacrylic Acids. 2,6-Dichloropyrazine was dissolved in
dichloromethane (0.2-0.5 mmol/mL). An amine (1.2-4 equiv) was
added, and the resulting mixture was agitated for 18-72 h at
ambient temperature. The reaction mixture was directly loaded onto
a flash column and eluted with a methanol/ammonia/dichlo-
romethane gradient. The solvent was concentrated, and the resultant

1822 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
(E)-3-{3-[6-(4-Methyl[1,4]diazepan-1-yl)pyrazin-2-yl]phenyl}acryl-
amide (3). Compound 1(36 mg, 0.106 mmol) was dissolved in DMF
(1 mL) in a reaction tube. N-[3-(Dimethylamino)propyl]-N′-
ethylcarbodiimide hydrochloride (38.3 mg, 0.2 mmol), benzotriazol-
1-ol (27 mg, 0.2 mmol), and N-methylmorpholine (0.02 mL, 0.18
mmol) were added, and the tube was sealed and agitated for 15
min. The amine (1.0 mL, 0.5 M in dioxane, 0.5 mmol) was added,
and the resulting mixture was agitated at 35 °C for 72 h. The
reaction mixture was concentrated, and the product was purified
by preparative HPLC with an acetonitrile/water/formic acid gradient
to provide the title compound (17 mg, 47%): 1H NMR (DMSO-d6,
400 MHz) δ 8.43 (s, 1H), 8.21 (s, 1H), 8.15 (s, 1H, formate), 8.14
(s, 1H), 8.05 (d, J ) 7.9 Hz, 1H), 7.64 (d, J ) 7.6 Hz, 1H), 7.56
(s, br, 1H), 7.48-7.56 (m, 2H), 7.14 (s, br, 1H), 6.70 (d, J ) 16
Hz, 1H), 3.88-3.92 (m, 2H), 3.72-3.77 (m, 2H), 2.84-2.90 (m,
2H), 2.69-2.74 (m, 2H), 2.41 (s, 3H), 1.98-2.05 (m, 2H); 13C
NMR APT (methanol-d4, 100 MHz) δ 170.7(+), 169.7 (formate),
154.6(+), 150.3(+), 142.3(-), 138.8(+), 136.8(+), 130.5(-),
129.7(-), 129.6(-), 129.2(-), 127.5(-), 125.4(-), 122.2(-),
58.3(+), 57.3(+), 46.7(+), 45.1(-), 42.8(+), 25.9(+). HCl salt:
1H NMR (DMSO-d6, 400 MHz) δ 10.74 (s, br, 1H), 8.55 (s, 1H),
8.27 (s, 1H), 8.25 (s, 1H), 8.10 (d, J ) 7.7 Hz, 1H), 7.69 (d, J )
7.7 Hz, 1H), 7.64 (s, br, 1H), 7.55 (dd, J ) 7.7, 7.7 Hz, 1H), 7.53
(d, J ) 16 Hz, 1H), 7.18 (s, br, 1H), 6.74 (d, J ) 16 Hz, 1H),
4.40-4.43 (m, 1H), 3.7-4.0 (3H buried with water), 3.55-3.66
(m, 1H), 3.42-3.52 (m, 1H), 3.15-3.3 (m, 2H), 2.80 (d, J ) 4.7
Hz, 3H), 2.35-2.47 (m, 1H), 2.20-2.30 (m, 1H); ESMS m/z 338
(M + H); HPLC, method A, tR ) 5.62 min (100%).
THF was added. After 15 min, the solvents were evaporated and
the residue was diluted with water (5 mL) and extracted with ethyl
acetate (2 × 5 mL). The organic extracts were combined, washed
with brine, dried with sodium sulfate, and concentrated. The residue
was purified by flash chromatography to provide (E)-3-{3-[6-(4-
methyl[1,4]diazepan-1-yl)pyrazin-2-yl]phenyl}acrylonitrile (40 mg,
62%): ESMS m/z 320 (M + H). This intermediate (25 mg, 0.08
mmol) was dissolved in DMF (0.5 mL) and added to a solution of
NaN3 (13.2 mg, 0.2 mmol) and ZnBr (44 mg, 0.2 mmol) in water
(1.5 mL). The reaction mixture was then stirred for 72 h at 110
°C, treated with 1 N HCl (0.2 mL), and concentrated. The crude
product was purified by preparative HPLC with an acetonitrile/
water/formic acid gradient to provide the title compound (7 mg,
24% yield): 1H NMR (methanol-d4, 400 MHz) δ 8.65 (s, 1H), 8.44
(s, 1H), 8.36 (s, 1H), 8.20 (d, J ) 7.7 Hz, 1H), 7.89 (d, J ) 7.7
Hz, 1H), 7.82 (d, J ) 16 Hz, 1H), 7.66 (d, d, J ) 7.7, 7.7 Hz, 1H),
7.42 (d, J ) 16 Hz, 1H), 4.57-4.65 (m, 1H), 3.84-4.13 (m, 4H),
3.63-3.70 (m, 1H), 3.40-3.51 (m, 2H), 2.99 (s, 3H), 2.38-2.52
(m, 2H); 13C NMR APT (methanol-d4, 100 MHz) δ 160.9(+),
154.5(+), 150.6(+), 138.6(+), 138.5(+), 134.2(-), 130.4(-),
129.9(-), 129.0(-), 128.9(-), 127.6(-), 126.2(-), 116.6(-),
58.3(+), 57.3(+), 46.7(+), 45.0(-), 42.5(+), 25.9(+); ESMS m/z
363 (M + H); HPLC, method A, tR ) 5.34 min (95.2%).
1-(Pyrazin-2-yl)-4-methylperhydro-1,4-diazepine (6). A solution
of 2-chloropyrazine (0.075 mL, 1 mmol) in DMF (2 mL) was
treated with N-methylhomopiperazine (228 mg, 2 mmol), and the
resulting mixture was heated at 70 °C for 16 h. The reaction mixture
was then concentrated and purified directly by flash chromatography
using a methanol/ammonia/dichloromethane gradient. The isolated
product was contaminated with the N-methylhomopiperazine
reagent which was removed by transfer of an ethyl acetate solution
of the mixture to an SPE cartridge with 800 mg of Si-isocyanate.
A dichloromethane/methanol/HCl eluant (1 × 2 mL) provided the
title compound as an HCl salt following concentration of the
solvents (30 mg, 13% yield): 1H NMR (methanol-d4, 400 MHz) δ
8.59 (d, J ) 3 Hz, 1H), 8.39 (s, 1H), 7.97 (d, J ) 3 Hz, 1H),
4.44-4.49 (m, 1H), 3.96-4.02 (m, 1H), 3.75-3.87 (m, 3H),
3.62-3.65 (m, 1H), 3.32-3.46 (m, 2H), 2.97 (s, 3H), 2.39-2.50
(m, 2H); ESMS m/z 193 (M + H); HPLC, method A, tR ) 2.50
min (99.1%).
3-{3-[6-(4-Methyl[1,4]diazepan-1-yl)pyrazin-2-yl]phenyl}propio-
nic Acid (4). Compound 1 (30 mg, 0.09 mmol) was combined with
Pd/C (2 mg) and ammonium formate (31.5 mg, 0.5 mmol) in 1
mL of ethanol in a reaction tube. The tube was sealed and heated
at 70 °C for 72 h. The reaction mixture was cooled, filtered, and
concentrated. The crude product was purified using preparative
HPLC with an acetonitrile/water/formic acid gradient to provide
the title compound (17 mg, 55% yield): 1H NMR (DMSO-d6, 400
MHz) δ 8.35 (s, 1H), 8.19 (s, formate), 8.10 (s, 1H), 7.90 (s, 1H),
7.87 (d, J ) 7.6 Hz, 1H), 7.40 (dd, J ) 7.6, 7.6 Hz, 1H), 7.31 (d,
J ) 7.7 Hz, 1H), 3.83-3.86 (m, 2H), 3.73 (t, J ) 6.1 Hz, 2H),
2.91 (t, J ) 7.6, 2H), 2.68-2.72 (m, 2H), 2.60 (t, J ) 7.6 Hz, 2H),
2.56-2.51 (m, 2H), 2.30 (s, 3H), 1.92-1.99 (m, 2H); 13C NMR
APT (methanol-d4, 100 MHz) δ 179.4(+), 169.5 (formate),
154.3(+), 150.8(+), 143.6(+), 137.8(+), 131.0(-), 130.0(-),
129.5(-), 128.4(-), 127.9(-), 125.5(-), 58.5(+), 57.2(+), 46.8(+),
(E)-3-[3-(6-[1,4]Diazepan-1-ylpyrazin-2-yl)phenyl]acrylic Acid
(7). Purified by preparative HPLC with an acetonitrile/water/TFA
1
gradient: yield 50%. TFA salt: H NMR (DMSO-d6, 400 MHz) δ
12.5 (s, br, 1H), 8.70 (s, br, 2H), 8.58 (s, 1H), 8.36 (s, 1H), 8.26
(s, 1H), 8.14 (d, J ) 7.8 Hz, 1H), 7.81 (d, J ) 7.7 Hz, 1H), 7.71
(d, J ) 16 Hz, 1H), 7.56 (dd, J ) 7.8, 7.7 Hz, 1H), 6.68 (d, J )
16 Hz, 1H), 3.99-4.06 (m, 2H), 3.80-3.84 (m, 2H), 3.32-3.40
(m, 2H), 3.20-3.28 (m, 2H), 2.07-2.16 (m, 2H); 13C NMR APT
(DMSO-d4, 100 MHz) δ 167.6(+), 158.0(+) (q, J ) 34 Hz, TFA
carbonyl), 152.4(+), 147.3(+), 143.7(-), 137.3(+), 134.8(+),
129.4(-), 129.2(-), 128.8(-) (2C), 128.2(-), 126.4 (-), 119.9(-),
45.2(+), 45.0(+), 44.8(+), 42.6(+), 24.8(+) (CF3 carbon [TFA
salt] not observed); ESMS m/z 325 (M + H); HPLC, method A, tR
) 5.38 min (100%). 1-(6-Chloropyrazin-2-yl)perhydro-1,4-diaz-
epine (intermediate): yield 66%; ESMS m/z 213 (M + H).
(E)-3-[3-(6-Azepan-1-ylpyrazin-2-yl)phenyl]acrylic Acid (8). Pu-
rified by flash chromatography using a methanol/dichloromethane
gradient followed by trituration with methanol: yield 18%; 1H NMR
(DMSO-d6, 400 MHz) δ 8.44 (s, 1H), 8.32 (s, 1H), 8.12 (s, 1H),
8.10 (d, J ) 7.7 Hz, 1H), 7.77 (d, J ) 7.7 Hz, 1H), 7.67 (d, J )
16 Hz, 1H), 7.54 (dd, J ) 7.7, 7.7 Hz, 1H), 6.65 (d, J ) 16 Hz,
1H), 3.74 (t, J ) 6 Hz, 4H), 1.76-1.84 (m, 4H), 1.49-1.51 (m,
4H); ESMS m/z 324 (M + H); HPLC, method B, tR ) 12.42 min
(98.9%). 1-(6-Chloropyrazin-2-yl)perhydroazepine (intermediate):
yield 53%; ESMS m/z 212 (M + H).
1
44.8(-), 42.1(+), 38.3(+), 32.7(+), 25.4(+). HCl salt: H NMR
(DMSO-d6, 400 MHz) δ 10.82 (s, br, 1H), 8.48 (s, 1H), 8.22 (s,
1H), 7.95 (s, 1H), 7.91 (d, J ) 7.7 Hz, 1H), 7.42 (dd, J ) 7.7, 7.7
Hz, 1H), 7.34 (d, J ) 7.7 Hz, 1H), 4.38-4.43 (m, 1H), 3.7-4.0
(3H buried with water), 3.63-3.56 (m, 1H), 3.43-3.52 (m, 1H),
3.18-3.26 (m, 2H), 2.92 (m, 2H), 2.79 (d, J ) 4.8 Hz, 3H), 2.62
(t, J ) 7.6 Hz, 2H), 2.38-2.48 (m, 1H), 2.15-2.26 (m, 1H); ESMS
m/z 341 (M + H); HPLC, method A, tR ) 5.28 min (98.6%).
1-Methyl-4-(6-{3-[(E)-2-(1H-tetrazol-5-yl)vinyl]phenyl}pyrazin-
2-yl)[1,4]diazepane (5). 1-(6-Chloropyrazin-2-yl)-4-methyl[1,4]-
diazepane (360 mg, 1.59 mmol), 3-formylphenylboronic acid (235
mg, 1.58 mmol), and bistriphenylphosphinepalladium(II) chloride
(21 mg, 0.03 mmol) were dispersed in 10 mL of DMF in a reaction
tube. Subsequently, 1.5 mL of 2 M Na2CO3 was added. The tube
was sealed, and the reaction mixture was heated at 90 °C for 3 h.
The reaction mixture was then filtered through a plug of Celite
and diluted with ethyl acetate and water (25 mL), and the organic
phase was separated. The aqueous phase was extracted with 2 ×
10 mL of ethyl acetate. The organic extracts were combined, washed
with brine, dried, and concentrated. The residue was purified by
flash chromatography to provide the intermediate aldehyde [337
mg, 72%; ESMS m/z 297 (M + H)]. Sodium hexamethyldisilazane
(0.22 mL, 1 M in THF, 0.22 mmol) was added to a solution of
cyanomethylphosphonic acid diethyl ester (0.034 mL, 0.21 mmol)
in 1 mL of THF under an atmosphere of N2. The mixture was stirred
for 2 h, after which time a solution of the intermediate, 3-[6-(4-
methyl[1,4]diazepan-1-yl)pyrazin-2-yl]benzaldehyde, in 2 mL of
(E)-3-{3-[6-(4-Acetyl[1,4]diazepan-1-yl)pyrazin-2-yl]phenyl}-
acrylic Acid (9). Purified by preparative HPLC using an acetoni-
trile/water/formic acid gradient followed by trituration with metha-
nol: yield 34%; 1H NMR peak doubling observed at room
temperature for certain protons indicative of slow isomeric inter-
conversion (DMSO-d6, 400 MHz, 100 °C) δ 11.5-12.5 (s, br, 1H),

New Class of Inhibitors of Pim Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1823
8.43 (s, 1H), 8.26 (s, 1H), 8.17 (s, 1H), 8.08 (d, J ) 7.8 Hz, 1H),
7.71 (d, J ) 7.8 Hz, 1H), 7.68 (d, J ) 16 Hz, 1H), 7.54 (dd, J )
17.7, 7.7 Hz, 1H), 6.58 (d, J ) 16 Hz, 1H), 3.95 (s, br, 1H),
3.75-3.87 (m, br, 3H), 3.73 (t, J ) 6 Hz, 2H), 3.48 (t, J ) 6 Hz,
2H), 1.80-2.00 (m, br, 5H); ESMS m/z 367 (M + H); HPLC,
method A, tR ) 7.23 min (100%). 1-[4-(6-Chloropyrazin-2-
yl)perhydro-1,4-diazepin-1-yl]ethanone (intermediate): yield 44%;
ESMS m/z 255 (M + H).
(E)-3-[3-(4-Methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl-6′-
yl)phenyl]acrylic Acid (10). Purified by preparative TLC using a
methanol/ammonia/dichloromethane gradient: yield 74%; 1H NMR
(DMSO-d6, 400 MHz) δ 8.54 (s, 1H), 8.30 (s, 1H), 8.29 (s, 1H),
8.08 (d, J ) 7.8 Hz, 1H), 7.74 (d, J ) 7.8 Hz, 1H), 7.56 (d, J )
16 Hz, 1H), 7.52 (dd, J ) 7.8, 7.7 Hz, 1H), 6.67 (d, J ) 16 Hz,
1H), 3.64-3.72 (m, 4H), 2.43-2.46 (m, 4H), 2.23 (s, 3H); ESMS
m/z 325 (M + H); HPLC, method A, tR ) 5.24 min (98.7%). 6′-
Chloro-4-methyl-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl (intermedi-
ate): yield 66%; ESMS m/z 213 (M + H).
(E)-3-{3-[6-(4-Aminobutylamino)pyrazin-2-yl]phenyl}acrylic Acid
(15). Purified by preparative HPLC with an acetonitrile/water/formic
acid gradient: yield 30%; 1H NMR (DMSO-d6 and D2O, 400 MHz)
δ 8.44 (s, formate), 8.33 (s, 2H), 8.00 (d, J ) 7.8 Hz, 1H), 7.91 (s,
1H), 7.60 (d, J ) 7.7 Hz, 1H), 7.54 (dd, J ) 7.7, 7.7 Hz, 1H), 7.31
(d, J ) 16 Hz, 1H), 6.52 (d, J ) 16 Hz, 1H), 3.43-3.51 (m, 2H),
2.90-2.97 (m, 2H), 1.68-1.76 (m, 4H). HCl salt: 1H NMR
(DMSO-d6, 400 MHz) δ 8.39 (s, 1H), 8.32 (s, 1H), 8.10 (d, J )
7.7 Hz, 1H), 7.94 (s, 1H), 7.83 (s, br, 3H), 7.78 (d, J ) 7.7 Hz,
1H), 7.68 (d, J ) 16 Hz, 1H), 7.55 (dd, J ) 7.7, 7.7 Hz, 1H), 7.38
(s, br, 1H), 6.67 (d, J ) 16 Hz, 1H), 3.37-3.46 (m, 2H), 2.79-2.88
(m, 2H), 1.61-1.70 (m, 4H); ESMS m/z 313 (M + H); HPLC,
method A, tR ) 5.33 min (96.1%). N1-(6-Chloropyrazin-2-yl)butane-
1,4-diamine (intermediate): yield 47%; ESMS m/z 201 (M + H).
(E)-3-(3-{6-[3-(Dimethylamino)propylamino]pyrazin-2-yl}phenyl)-
acrylic Acid (16). Purified by preparative HPLC with an acetonitrile/
1
water/formic acid gradient: yield 80%; H NMR (DMSO-d6 and
D2O, 400 MHz) δ 8.42 (s, formate), 8.31 (s, 1H), 8.18 (s, 1H),
8.00 (d, J ) 7.8 Hz, 1H), 7.91 (s, 1H), 7.65 (d, J ) 7.8 Hz, 1H),
7.54 (dd, J ) 7.8, 7.8 Hz, 1H), 7.32 (d, J ) 16 Hz, 1H), 6.53 (d,
J ) 16 Hz, 1H), 3.44-3.48 (m, 2H), 2.72-2.77 (m, 2H), 2.46 (s,
6H), 1.87-1.97 (m, 2H). HCl salt: 1H NMR (DMSO-d6, 400 MHz)
δ 10.07 (s, br, 1H), 8.41 (s, 1H), 8.32 (s, 1H), 8.12 (d, J ) 7.8 Hz,
1H), 7.95 (s, 1H), 7.79 (d, J ) 7.8 Hz, 1H), 7.69 (d, J ) 16 Hz,
1H), 7.55 (dd, J ) 7.8, 7.8 Hz, 1H), 7.43 (s, br, 1H), 6.68 (d, J )
16 Hz, 1H), 3.43-3.50 (m, 2H), 3.12-3.19 (m, 2H), 2.46 (d, J )
4.9 Hz, 6H), 1.97-2.05 (m, 2H); ESMS m/z 327 (M + H); HPLC,
method B, tR ) 4.93 min (98.6%). N1-(6-Chloropyrazin-2-yl)-N,N-
dimethylpropane-1,3-diamine (intermediate): yield 35%; ESMS m/z
215 (M + H).
(E)-3-[3-(3,4,5,6-Tetrahydro-2H-[1,2′]bipyrazinyl-6′-yl)pheny-
l]acrylic Acid (11). Purified by preparative HPLC with an aceto-
1
nitrile/water/formic acid gradient: yield 56%; H NMR (DMSO-
d6, 400 MHz) δ 8.43 (s, 1H), 8.40 (formate), 8.20 (s, 1H), 8.15 (s,
1H), 7.96 (d, J ) 7.9 Hz, 1H), 7.60 (d, J ) 7.7 Hz, 1H), 7.42 (dd,
J ) 7.8, 7.7 Hz, 1H), 7.35 (d, J ) 16 Hz, 1H), 6.52 (d, J ) 16 Hz,
1
1H), 3.50-3.56 (m, 4H), 2.74-2.80 (m, 4H). HCl salt: H NMR
(DMSO-d6, 400 MHz): δ 9.37 (s, br, 2H), 8.66 (s, 1H), 8.40 (s,
1H), 8.38 (s, 1H), 8.15 (d, J ) 7.8 Hz, 1H), 7.81 (d, J ) 7.8 Hz,
1H), 7.70 (d, J ) 16 Hz, 1H), 7.56 (dd, J ) 7.8, 7.8 Hz, 1H), 6.70
(d, J ) 16 Hz, 1H), 3.93-3.96 (m, 4H), 3.20-3.29 (m, 4H); ESMS
m/z 311 (M + H); HPLC, method A, tR ) 5.17 min (98.8%). 6′-
Chloro-3,4,5,6-tetrahydro-2H-[1,2′]bipyrazinyl (intermediate): yield
18%; ESMS m/z 199 (M + H).
(E)-3-[3-(6-{[3-(Dimethylamino)propyl]methylamino}pyrazin-
2-yl)phenyl]acrylic Acid (17). Purified by preparative HPLC with
1
an acetonitrile/water/formic acid gradient: yield 81%; H NMR
(E)-3-[3-(6-Morpholin-4-ylpyrazin-2-yl)phenyl]acrylic Acid (12).
Purified by flash chromatography using a methanol/dichloromethane
gradient: yield 31%; 1H NMR (DMSO-d6, 400 MHz) δ 12.5 (s, br,
1H), 8.60 (s, 1H), 8.35 (s, 1H), 8.32 (s, 1H), 8.13 (d, J ) 7.9 Hz,
1H), 7.79 (d, J ) 7.9 Hz, 1H), 7.69 (d, J ) 16 Hz, 1H), 7.54 (dd,
J ) 7.7, 7.8 Hz, 1H), 6.67 (d, J ) 16 Hz, 1H), 3.74-3.77 (m, 4H),
3.62-3.65 (m, 4H); ESMS m/z 312 (M + H); HPLC, method A,
tR ) 7.81 min (100%). 4-(6-Chloropyrazin-2-yl)morpholine (in-
termediate): yield 20%; ESMS m/z 200 (M + H).
(DMSO-d6, 400 MHz) δ 8.47 (s, 1H), 8.35 (s, 1H), 8.22 (s, 1H),
8.13 (s, formate), 8.12 (d, J ) 7.8 Hz, 1H), 7.77 (d, J ) 7.8 Hz,
1H), 7.66 (d, J ) 16 Hz, 1H), 7.54 (dd, J ) 7.8, 7.8 Hz, 1H), 6.65
(d, J ) 16 Hz, 1H), 3.66 (t, J ) 7.4, 2H), 3.14 (s, 3H), 2.40 (t, J
1
) 7.0, 2H), 2.23 (s, 6H), 1.74-1.81 (m, 2H). HCl salt: H NMR
(DMSO-d6, 400 MHz) δ 10.51 (s, br, 1H), 8.55 (s, 1H), 8.38 (s,
1H), 8.22 (s, 1H), 8.16 (d, J ) 7.8 Hz, 1H), 7.81 (d, J ) 7.8 Hz,
1H), 7.70 (d, J ) 16 Hz, 1H), 7.57 (dd, J ) 7.8, 7.8 Hz, 1H), 6.70
(d, J ) 16 Hz, 1H), 3.74 (t, J ) 7.0 Hz, 2H), 3.17 (s, 3H),
3.07-3.16 (m, 2H), 2.72 (d, J ) 4.9 Hz, 6H), 2.00-2.08 (m, 2H);
ESMS m/z 341 (M + H); HPLC, method A, tR ) 5.61 min (96.4%).
N1-(6-Chloropyrazin-2-yl)-N,N,N-trimethylpropane-1,3-diamine (in-
termediate): yield 65%; ESMS m/z 229 (M + H).
(E)-3-{3-[6-(2-Aminoethylamino)pyrazin-2-yl]phenyl}acrylic Acid
(13). Purified by preparative HPLC with an acetonitrile/water/formic
acid gradient: yield 33%; 1H NMR (DMSO-d6 and D2O, 400 MHz)
δ 8.43 (s, formate), 8.39 (s, 1H), 8.21 (s, 1H), 8.02 (d, J ) 7.9 Hz,
1H), 7.97 (s, 1H), 7.67 (d, J ) 7.6 Hz, 1H), 7.55 (dd, J ) 7.7, 7.7
Hz, 1H), 7.33 (d, J ) 16 Hz, 1H), 6.54 (d, J ) 16 Hz, 1H), 3.68
(E)-3-{3-[6-(2-Acetylaminoethylamino)pyrazin-2-yl]phenyl}-
acrylic Acid (18). Purified by preparative HPLC with an acetonitrile/
1
1
(t, J ) 6.2 Hz, 2H), 3.15 (t, J ) 6.2 Hz, 2H). HCl salt: H NMR
water/formic acid gradient: yield 35%; H NMR (DMSO-d6, 400
(DMSO-d6, 400 MHz) δ 8.47 (s, 1H), 8.34 (s, 1H), 8.14 (d, J )
7.8 Hz, 1H), 8.00 (s, br, 4H), 7.80 (d, J ) 7.7 Hz, 1H), 7.70 (d, J
) 16 Hz, 1H), 7.55 (dd, J ) 7.8, 7.7 Hz, 1H), 7.47 (s, br, 1H),
6.69 (d, J ) 16 Hz, 1H), 3.58-3.67 (m, 2H), 3.04-3.13 (m, 2H);
ESMS m/z 285 (M + H); HPLC, method B, tR ) 4.70 min (100%).
N1-(6-Chloropyrazin-2-yl)ethane-1,2-diamine (intermediate): yield
43%; ESMS m/z 173 (M + H).
MHz) δ 8.17 (s, 1H), 8.14 (s, 1H), 7.99 (d, J ) 8.0 Hz, 1H), 7.73
(s, 1H), 7.64 (d, J ) 16 Hz, 1H), 7.58 (d, J ) 7.8 Hz, 1H), 7.42
(dd, J ) 7.8, 7.8 Hz, 1H), 6.51 (d, J ) 16 Hz, 1H), 3.53 (t, J ) 6.2
Hz, 2H), 3.37 (t, J ) 6.2, 2H), 1.81 (s, 3H); ESMS m/z 327 (M +
H); HPLC, method A, tR ) 6.42 m (100%). N-[2-(6-Chloropyrazin-
2-ylamino)ethyl]acetamide (intermediate): yield 31.2%; ESMS m/z
215 (M + H).
(E)-3-{3-[6-(3-Hydroxypropylamino)pyrazin-2-yl]phenyl}acr-
ylic Acid (19). Purified by preparative HPLC with an acetonitrile/
water/formic acid gradient: yield 63%; H NMR (DMSO-d6, 400
(E)-3-{3-[6-(3-Aminopropylamino)pyrazin-2-yl]phenyl}acrylic Acid
(14). Purified by preparative HPLC with an acetonitrile/water/TFA
gradient: yield 64%. TFA salt: H NMR (DMSO-d6, 400 MHz) δ
1
1
MHz) δ 12.50 (s, br, 1H), 8.34 (s, 1H), 8.29 (s, 1H), 8.08 (d, J )
7.8 Hz, 1H), 7.89 (s, 1H), 7.74 (d, J ) 7.8 Hz, 1H), 7.65 (d, J )
16 Hz, 1H), 7.51 (dd, J ) 7.8, 7.8 Hz, 1H), 7.13 (t, J ) 5.3 Hz,
1H), 6.64 (d, J ) 16 Hz, 1H), 4.50 (s, br, 1H), 3.53 (t, J ) 6.3,
2H), 3.45-3.38 (m, 2H), 1.76 (t, t, J ) 6.6, 6.6 Hz, 2H); ESMS
m/z 300 (M + H); HPLC, method B, tR ) 7.43 min (95.9%). 3-(6-
Chloropyrazin-2-ylamino)propan-1-ol (intermediate): yield 60%;
ESMS m/z 188 (M + H).
8.40 (s, 1H), 8.32 (s, 1H), 8.11 (d, J ) 7.9 Hz, 1H), 7.93 (s, 1H),
7.79 (d, J ) 7.6 Hz, 1H), 7.68 (d, J ) 16 Hz, 1H), 7.54 (dd, J )
7.7, 7.7 Hz, 1H), 7.30 (t, J ) 5.7 Hz, 1H), 6.67 (d, J ) 16 Hz,
1H), 7-8 (br, NH3+), 3.43-3.50 (m, 2H), 2.87-2.90 (m, 2H),
1.85-1.95 (m, 2H); 13C NMR APT (methanol-d4, 100 MHz) δ
170.3(+), 163.1.0(+) (q, J ) 34 Hz, TFA carbonyl), 156.2(+),
150.4(+), 145.8(-), 139.2(+), 136.5(+), 132.9(-), 130.6(-),
130.0(-), 129.5(-), 128.8(-), 127.6(-), 120.3(-), 38.8(+),
38.6(+), 28.7(+), (CF3 carbon [TFA salt] not observed); ESMS
m/z 299 (M + H); HPLC, method A, tR ) 5.12 min (98.7%). N1-
(6-Chloropyrazin-2-yl)propane-1,3-diamine (intermediate): yield
84%; ESMS m/z 187 (M + H).
(E)-3-{3-[6-(3-Methoxypropylamino)pyrazin-2-yl]phenyl}acry-
lic Acid (20). Purified by preparative HPLC with an acetonitrile/
1
water/formic acid gradient: yield 31%; H NMR (DMSO-d6, 400
MHz) δ 8.29 (s, 1H), 8.23 (s, 1H), 8.09 (d, J ) 7.8 Hz, 1H), 7.83

1824 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Qian et al.
(s, 1H), 7.78 (d, J ) 16 Hz, 1H), 7.69 (d, J ) 7.8 Hz, 1H), 7.54
(dd, J ) 7.8, 7.8 Hz, 1H), 6.60 (d, J ) 16 Hz, 1H), 3.55-3.60 (m,
4H), 3.38 (s, 3H), 1.93-2.00 (m, 2H); ESMS m/z 314 (M + H);
HPLC, method A, tR ) 7.74 min (100%). (6-Chloropyrazin-2-yl)(3-
methoxypropyl)amine (intermediate): yield 55%; ESMS m/z 202
(M + H).
yl]carbamic acid tert-butyl ester according to the standard method.
In this example, the reaction mixture was heated at 45 °C for
24 h, concentrated, and purified by flash chromatography (yield
41%). This intermediate was then converted to (E)-3-{3-[6-(cis-4-
tert-butoxycarbonylaminocyclohexylamino)pyrazin-2-yl]phenyl}-
acrylic acid according to the standard protocol. After purification
by preparative TLC with a methanol/ammonium hydroxide/dichlo-
romethane eluant, the isolated intermediate was taken up in 5 mL
of a 20% TFA/dichloromethane solution. The reaction mixture was
stirred at room temperature for 16 h and concentrated, and the
isolate was purified by preparative HPLC using an acetonitrile/
water gradient to provide the title compound: yield 91%; 1H NMR
(DMSO-d6, 400 MHz) δ 8.37 (s, 1H), 8.28 (s, 1H), 8.06 (d, J )
7.8 Hz, 1H), 8.00 (s, 1H), 7.74 (d, J ) 7.7 Hz, 1H), 7.63 (d, J )
16 Hz, 1H), 7.52 (dd, J ) 7.8, 7.7 Hz, 1H), 7.16 (d, J ) 5.1 Hz,
1H), 6.62 (d, J ) 16 Hz, 1H), 3.96-4.04 (m, 1H), 3.10-3.18 (m,
1H), 1.92-2.03 (m, 2H), 1.66-1.80 (m, 6H); ESMS m/z 339 (M
+ H); HPLC, method A, tR ) 5.58 min (95.0%).
(E)-3-{3-[6-(4-Hydroxycyclohexylamino)pyrazin-2-yl]phenyl}-
acrylic Acid (25). Purified by preparative HPLC using an acetoni-
trile/water gradient: yield 40%; 1H NMR (DMSO-d6, 400 MHz) δ
8.33 (s, 1H), 8.27 (s, 1H), 8.06 (d, J ) 7.8 Hz, 1H), 7.87 (s, 1H),
7.74 (d, J ) 7.8 Hz, 1H), 7.61 (d, J ) 16 Hz, 1H), 7.53 (dd, J )
7.8, 7.6 Hz, 1H), 7.04 (d, J ) 7.3 Hz, 1H), 6.63 (d, J ) 16 Hz,
1H), 4.58 (s, br, 1H), 3.63-3.70 (m, 1H), 3.39-3.45 (m, 1H),
1.98-2.05 (m, 2H), 1.84-1.92 (m, 2H), 1.21-1.39 (m, 4H); ESMS
m/z 340 (M + H); HPLC, method A, tR ) 7.03 min (98.0%). 4-(6-
Chloropyrazin-2-ylamino)cyclohexanol (intermediate): yield 13%;
ESMS m/z 228 (M + H).
(E)-3-(3-{6-[(Piperidin-4-ylmethyl)amino]pyrazin-2-yl}phenyl)acr-
ylic Acid (21). 4-(Aminomethyl)piperidine-1-carboxylic acid tert-
butyl ester (200 mg, 0.93 mmol) and 2,6-dichloropyrazine (160
mg, 1.07 mmol) were dissolved in DMF (5 mL), and diisopropy-
lethylamine (500 µL, 2.9 mmol) was added. The reaction mixture
was stirred at ambient temperature for 2 days and diluted with water
(15 mL), and the resulting mixture was extracted with ethyl acetate
(3 × 10 mL). The combined extracts were washed with brine (1 ×
10 mL), dried with sodium sulfate, and concentrated to afford 4-[(6-
chloropyrazin-2-ylamino)methyl]piperidine-1-carboxylic acid tert-
butyl ester (150 mg, 43% yield): ESMS m/z 327 (M + H). This
intermediate was subsequently dissolved in dichloromethane (10
mL) and treated with TFA (2 mL). The resulting mixture was stirred
at ambient temperature for 2 h and concentrated, and the residue
was purified by flash chromatography with a methanol/ammonia/
dichloromethane gradient to provide 6-(chloropyrazin-2-yl)piperi-
din-4-ylmethylamine (85 mg, 82% yield). This intermediate was
converted to the title compound by the general method. Purified
by preparative HPLC with an acetonitrile/water/formic acid gradi-
ent: yield 35%: 1H NMR (DMSO-d6, 400 MHz) δ 8.42 (s, formate),
8.34 (s, 1H), 8.26, (s, 1H), 8.02 (d, J ) 7.8 Hz, 1H), 7.92 (s, 1H),
7.67 (d, J ) 7.8 Hz, 1H), 7.47-7.52 (m, 2H), 7.35 (t, J ) 5.6,
1H), 6.60 (d, J ) 16 Hz, 1H), 3.30-3.33 (m, 2H), 3.22 (d, J )
12.1 Hz, 2H), 2.76 (t, J ) 11.7 Hz, 2H), 1.82-1.97 (m, 3H),
1.34-1.43 (m, 2H); 13C NMR APT (methanol-d4, 100 MHz) δ
172.8(+), 169.4(-) (formate), 156.3(+), 150.5(+), 143.3(-),
139.1(+), 137.1(+), 132.5(-), 130.4(-), 129.7(-), 128.7(-),
128.4(-), 127.1(-), 123.6(-), 46.5(+), 45.1(+), 35.5(-), 28.1(+).
HCl salt: 1H NMR (DMSO-d6, 400 MHz) δ 8.78-8.87 (br, s, 1H),
8.45-8.55 (s, br, 1H), 8.39 (s, 1H), 8.32, (s, 1H), 8.10 (d, J ) 7.8
Hz, 1H), 7.95 (s, 1H), 7.78 (d, J ) 7.8 Hz, 1H), 7.69 (d, J ) 16
Hz, 1H), 7.54 (dd, J ) 7.8, 7.8 Hz, 1H), 7.40-7.52 (br, s, 1H),
6.67 (d, J ) 16 Hz, 1H), 3.30-3.33 (m, 2H), 3.20-3.34 (m, 2H),
2.73-2.80 (m, 2H), 1.82-1.92 (m, 3H), 1.32-1.45 (m, 2H); ESMS
m/z 339 (M + H); HPLC, method A, tR ) 5.49 min (96.0%).
(E)-3-(3-{6-[4-(Aminomethyl)piperidin-1-yl]pyrazin-2-yl}phenyl)-
acrylic Acid (22). Purified by preparative HPLC with an acetonitrile/
water/formic acid gradient: yield 71%. Converted to an HCl salt:
1H NMR (DMSO-d6, 400 MHz) δ 8.52 (s, 1H), 8.35 (s, 1H), 8.34
(s, 1H), 8.12 (d, J ) 7.8 Hz, 1H), 8.10-8.00 (s, br, 3H), 7.79 (d,
J ) 7.8 Hz, 1H), 7.68 (d, J ) 16 Hz, 1H), 7.54 (dd, J ) 7.8, 7.7
Hz, 1H), 6.67 (d, J ) 16 Hz, 1H), 4.51 (d, J ) 13.3 Hz, 2H), 2.95
(dd, J ) 11.6, 12.0 Hz, 2H), 2.70-2.75 (m, 2H), 1.83-1.92 (m,
3H), 1.18-1.30 (m, 2H); 13C NMR APT (methanol-d4, 100 MHz)
δ 170.2(+), 155.7(+), 150.5(+), 145.9(-), 139.0(+), 136.5(+),
130.6(-), 130.1(-), 130.0(-), 129.6(-), 129.1(-), 127.6(-),
120.1(-), 45.6(+), 45.3(+), 36.0(-), 29.9(+); ESMS m/z 339 (M
+ H); HPLC, method A, tR ) 4.98 min (95.4%). C-[1-(6-
Chloropyrazin-2-yl)piperidin-4-yl]methylamine (intermediate): yield
62%; ESMS m/z 227 (M + H).
(E)-3-(3-Pyrazin-2-ylphenyl)acrylic Acid (26). 26 was prepared
as described in the second step of the general procedure from
2-chloropyrazine and 3-[(E)-2-carboxyvinyl]benzeneboronic acid.
The product was purified by preparative TLC using a methanol/
1
ammonia/dichloromethane eluant: yield 25%; H NMR (DMSO-
d6, 400 MHz) δ 9.36 (d, J ) 1.0 Hz, 1H), 8.75 (dd, J ) 2.0, 1.0
Hz, 1H), 8.64 (d, J ) 2 Hz, 1H), 8.41 (s, 1H), 8.17 (d, J ) 7.8 Hz,
1H), 7.80 (d, J ) 7.8 Hz, 1H), 7.62 (d, J ) 16 Hz, 1H), 7.58 (dd,
J ) 7.8, 7.8 Hz, 1H), 6.70 (d, J ) 16 Hz, 1H); ESMS m/z 227 (M
+ H); HPLC, method B, tR ) 7.68 min (95.6%).
(E)-3-{3-[6-(4-Methyl[1,4]diazepan-1-yl)pyridin-2-yl]phenyl}acr-
ylic Acid (27). 2,6-Dichloropyridine (200 mg, 1.35 mmol) was
dissolved in DMF (5 mL). N-Methylhomopiperazine (500 µL, 4.02
mmol) was added, and the resulting mixture was heated at 105 °C
for 16 h. The reaction mixture was concentrated, loaded onto a
flash column, and eluted with a methanol/dichloromethane gradient.
The intermediate, 1-(6-chloropyridin-2-yl)-4-methylperhydro-1,4-
diazepine, was isolated by evaporation of the solvent and used
directly (160 mg, 53%): ESMS m/z 226 (M + H). 3-[(E)-2-
Carboxyvinyl]benzeneboronic acid (50 mg, 0.26 mmol) and bis-
triphenylphosphinepalladium(II) chloride (10 mg, 0.01 mmol) were
combined in a microwave reaction tube. The intermediate prepared
above (55 mg, 0.24 mmol) was added as a solution in DMF (3
mL), and 1 mL of 2 M Na2CO3 was then added. The tube was
sealed, and the reaction mixture was heated in a microwave reactor
for 10 min at 100 °C. The reaction mixture was then filtered through
a plug of Celite and purified directly using HPLC with an
acetonitrile/water/formic acid gradient. The product was further
purified by preparative TLC using a methanol/dichloromethane
eluant. The title compound was collected as a white solid (10 mg,
(E)-3-{3-[6-(trans-4-Aminocyclohexylamino)pyrazin-2-yl]phenyl}-
acrylic Acid (23). Purified by preparative HPLC with an acetonitrile/
water gradient: yield 24%; 1H NMR (DMSO-d6, 400 MHz) δ 8.24
(s, 1H), 8.14 (s, 1H), 7.91 (d, J ) 7.7 Hz, 1H), 7.81 (s, 1H), 7.59
(d, J ) 7.7 Hz, 1H), 7.41 (dd, J ) 7.8, 7.7 Hz, 1H), 7.34 (d, J )
16 Hz, 1H), 7.05 (d, J ) 7.1 Hz, 1H), 6.50 (d, J ) 16 Hz, 1H),
3.68 (s, br, 1H), 2.89 (s, br, 1H), 2.00-2.10 (m, 2H), 1.90-2.00
(m, 2H), 1.20-1.47 (m, 4H); ESMS m/z 339 (M + H); HPLC,
method B, tR ) 5.35 min (95.3%). trans-[4-(6-Chloropyrazin-2-
ylamino)cyclohexyl]carbamic acid tert-butyl ester (intermediate):
yield 11%; ESMS m/z 227 (M + H).
1
12%) following evaporation of the solvent: H NMR (DMSO-d6,
400 MHz) δ 8.16 (s, 1H), 7.99 (d, J ) 7.8 Hz, 1H), 7.61 (d, J )
7.6 Hz, 1H), 7.49-7.54 (m, 2H), 7.41 (dd, J ) 7.7, 7.7 Hz, 1H),
7.15 (d, J ) 7.4 Hz, 1H), 6.55 (d, J ) 8.4 Hz, 1H), 6.53 (d, J )
16 Hz, 1H), 3.74-3.77 (m, 2H), 3.61 (t, J ) 6.2 Hz, 2H), 2.39-2.43
(m, 2H), 2.40-2.43 (m, 2H), 2.20 (s, 3H), 1.84-1.89 (m, 2H);
ESMS m/z 338 (M + H); HPLC, method A, tR ) 6.05 min (99.1%).
Pim-2/Pim-1 Dose-Response Assay. The activity of Pim-2 and
Pim-1 kinases was measured utilizing Cambrex PKLight ATP
Detection Reagent (Cambrex catalog no. LT27-200), a homoge-
neous assay technology using luciferin-luciferase to quantify
residual ATP. The assay was performed in 96-well half-area, white,
(E)-3-{3-[6-(cis-4-Aminocyclohexylamino)pyrazin-2-yl]phenyl}-
acrylic Acid (24). The mono-BOC-protected diamine, cis-(4-
aminocyclohexyl)carbamic acid tert-butyl ester, was used to gener-
ate the intermediate cis-[4-(6-chloropyrazin-2-ylamino)cyclohex-

New Class of Inhibitors of Pim Kinases
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1825
(3) (a) Mikkers, H.; Nawijn, M.; Allen, J.; Brouwers, C.; Verhoeven, E.;
Jonkers, J.; Berns, A. Mice deficient for all PIM kinases display
reduced body size and impaired responses to hematopoietic growth
factors. Mol. Cell. Biol. 2004, 24, 6104–6115. (b) Allen, J. D.;
Verhoeven, E.; Domen, J.; van der Valk, M.; Berns, A. Pim-2
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c-myc. Oncogene 1997, 15, 1133–1141.
nonbinding surface microtiter plates (Corning, catalog no. 3642)
in assay buffer consisting of 25 mM HEPES (pH 7.5), 10 mM
MgCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 µM Na3VO4,
and 200 µM TCEP. Test compounds received as 5 mg/mL DMSO
stocks were serially diluted 1:3 in DMSO for the 10-point
concentration response. The DMSO dilutions were further diluted
in the assay buffer, and 15 µL of this dilution was added to the
assay plate, for a final starting assay concentration of 5 µg/mL in
1% DMSO. A 30 µL volume of a mixture of 2 µM ATP and 100
µM peptide substrate (Biotin-AKRRRLSA, ANASPEC) diluted in
assay buffer was added to the assay plate for final concentrations
of 1 µM ATP and 50 µM peptide, respectively. A kinase stock
solution (GST-Pim2, 48 µM stock; and GST-Pim-1, 19.4 µM
stock) was diluted to 40 nM in assay buffer, and 15 µL of this
dilution was added to the assay, for a final concentration of 10 nM
in a total kinase reaction volume of 60 µL. The kinase reaction
mixture was incubated for 15 min at room temperature. Following
incubation, 60 µL of ATP detection reagent diluted in reconstitution
buffer was added to the assay plates and incubated at room
temperature for 15 min, and the relative light unit (RLU) signal
was measured on the Analyst (Molecular Devices) in luminescence
mode. The RLU signals were converted to percent of control (POC)
values using the formula POC ) 100 (BCTRL - signal)/(BCTRL
- PCTRL), where signal is the test well signal, BCTRL is the
average of background (negative control) well signals on the plate,
and PCTRL is the average of positive control well signals on the
plate. For the concentration-responsive compounds, POC values
as a function of test compound concentration were fitted to a four-
parameter logistic equation of the form Y ) A + (B - A)/[1 +
(x/C)D], where A, B, C, and D are fitted parameters (parameter B is
fixed at zero POC) and x and y are the independent and dependent
variables, respectively. The IC50 (50% inhibitory concentration) was
determined as the inflection point parameter, C. Results for 5 and
7 were generated using Kinase Glo Plus ATP detection reagent
(Promega) and ARRRHLSY substrate (ANASPEC) as substitutes
for the Cambrex reagent and the AKRRRLSA substrate. These
substitutes were also used for some replicates of 21 and 22.
Crystallography Studies. Details of cloning, expression, purifica-
tion, and crystallization of Pim-1 have been published.2 Cocomplex
crystals at 1.9 Å resolution were obtained by soaking Pim-1 apo
crystals with 1. The soaking procedure, data collection, and analysis
methods are identical to those previously described for the Pim-
1-ANP-PNP cocomplex structure.4e
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imidazo[1,2-b]pyridazines as PIM kinase inhibitors with in Vitro
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K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.; Peng, C.;
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Zhang, C.; Rice, J.; Tsai, J.; Artis, D. R.; Ibrahim, P.; Bremer, R.
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somatic hypermutations in diffuse large cell lymphoma. J. Mol. Biol.
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O. Y.; Nelson, A.; Marsden, B. D.; Knapp, S. Structural basis of
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Acknowledgment. We thank Chris Pargellis for helpful
discussions on enzyme kinetics and John Miglietta for kinase
sequence alignments.
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response. Oncogene 2006, 25, 6758–6780. (b) Karin, M.; Yamamoto,
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Note Added after ASAP Publication. This manuscript was
released March 3, 2009 with typographical errors. The revised
version was posted on March 5, 2009.
Supporting Information Available: ATP competition and
reversible binding studies, additional kinase selectivity panel, target-
independent profiling methods, table of HPLC data, HPLC traces,
and NMR spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
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