Rational evolution of a novel type of potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor from a screening-hit compound

Article abstract of DOI:10.1021/jm3001289

Serine/threonine kinase PIM1 is an emerging therapeutic target for hematopoietic and prostate cancer therapy. To develop a novel PIM1 inhibitor, we focused on 1, a metabolically labile, nonselective kinase inhibitor discovered in our previous screening study. We adopted a rational optimization strategy based mainly on structural information for the PIM1-1 complex to improve the potency and selectivity. This approach afforded the potent and metabolically stable PIM1-selective inhibitor 14, which shows only a marginal increase in molecular weight compared with 1 but has a significantly decreased cLogP. The validity of our design concept was confirmed by X-ray structure analysis. In a cellular study, 14 potently inhibited the growth of human leukemia cell line MV4-11 but had a negligible effect on the growth of WI-38 (surrogate for general toxicity). These results demonstrate the effectiveness of our design strategy for evolving the screening-hit compound 1 into a novel type of PIM1 inhibitor, 14.

Full text of DOI:10.1021/jm3001289

Article
pubs.acs.org/jmc
Rational Evolution of a Novel Type of Potent and Selective Proviral
Integration Site in Moloney Murine Leukemia Virus Kinase 1 (PIM1)
Inhibitor from a Screening-Hit Compound
Hirofumi Nakano,† Nae Saito,† Lorien Parker,‡ Yukio Tada,† Masanao Abe,† Keiko Tsuganezawa,‡
Shigeyuki Yokoyama,‡,§,∥ Akiko Tanaka,†,‡ Hirotatsu Kojima,† Takayoshi Okabe,†
and Tetsuo Nagano*,†,⊥
†Open Innovation Center for Drug Discovery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan
§Laboratory of Structural Biology, and ∥Department of Biophysics and Biochemistry, Graduate School of Science, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
⊥Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
S
* Supporting Information
ABSTRACT: Serine/threonine kinase PIM1 is an emerging
therapeutic target for hematopoietic and prostate cancer
therapy. To develop a novel PIM1 inhibitor, we focused on 1,
a metabolically labile, nonselective kinase inhibitor discovered in
our previous screening study. We adopted a rational
optimization strategy based mainly on structural information
for the PIM1-1 complex to improve the potency and selectivity.
This approach afforded the potent and metabolically stable
PIM1-selective inhibitor 14, which shows only a marginal increase in molecular weight compared with 1 but has a significantly
decreased cLogP. The validity of our design concept was confirmed by X-ray structure analysis. In a cellular study, 14 potently
inhibited the growth of human leukemia cell line MV4−11 but had a negligible effect on the growth of WI-38 (surrogate for
general toxicity). These results demonstrate the effectiveness of our design strategy for evolving the screening-hit compound 1
into a novel type of PIM1 inhibitor, 14.
toxicity, i.e., cardiac QT prolongation, was identified (http://
clinicaltrials.gov/ct2/show/study/NCT00848601). Although
other chemical classes of PIM1 inhibitor have been
reported,25−42 none is currently under clinical investigation to
our knowledge. Therefore, there is still great interest in novel
types of small-molecular PIM1 inhibitors.
We identified compound 1 (Figure 1) as a PIM1 inhibitor in
a previous screening study (Table 1).43 But, although its
potency was acceptable as a starting point for optimization
(IC50 = 410 nM), compound 1 showed a poor kinase selectivity
profile toward a panel of 50 kinases (see Figure 2 and Table S1,
Supporting Information). Because over 500 protein kinases are
encoded in the human genome, selectivity is a major concern in
the design of kinase inhibitors.44 In addition, the metabolic
stability of 1 was mediocre in both human and mouse liver
microsome systems, with only about 40% recovery after
incubation for 1 h. The calculated lipophilicity (cLogP) of 1
is high, 5.3,45 and this is considered to be related to the
metabolic instability.46 Therefore, it is important to minimize
any increase in cLogP, or to reduce it if possible, during
INTRODUCTION
■
PIM1, a member of serine/threonine kinase, is an emerging
target for cancer therapy.1,2 Its expression is increased in
patients with hematopoietic and prostate cancer.3,4 Enforced
expression of PIM1 leads to inhibition of apoptosis and
enhancement of cell proliferation,5 and PIM1-transgenic mice
show increased susceptibility to tumorigenesis.6,7 PIM1 has
been reported to phosphorylate multiple proteins, such as
BAD,8 c-MYC,9 histone H3,10 p21waf1
,
p27kip1
,
CDC25A,13
11
12
CDC25C,14 and CXCR4,15 resulting in enhanced cell survival,
proliferation, and migration. PIM1 also phosphorylates and
stabilizes drug efflux transporters, such as P-glycoprotein16 and
BCRP,17 resulting in drug resistance. Because PIM1-deficient
mice do not display any overt abnormalities,18 these
experimental results indicate the potential importance of
PIM1 as a therapeutic target for cancer therapy.
There has been increasing interest in PIM1 inhibitors,19 and
typical PIM1 inhibitors reported so far have fused rings as a
core such as the imidazo[1,2-b]pyridazine ring.20,21 Among
these inhibitors, SGI-1776 was the most advanced drug
candidate22−24 and it is the only PIM1 inhibitor to have been
the subject of clinical trials. However, clinical investigation of
SGI-1776 was discontinued in 2010 because dose-limiting
Received: January 30, 2012
Published: April 27, 2012
© 2012 American Chemical Society
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inhibited the growth of human leukemia cell line MV4−11 and
induced apoptosis and G1 phase arrest. These results indicate
that 14 is a promising lead compound for further study.
RESULTS AND DISCUSSION
■
Structural Basis of the Inhibitor Design. We solved the
crystal structure of PIM1 complexed with 1, which will be
reported separately.49 The structure revealed that 1 binds in the
ATP-binding cleft. Figure 1 shows a schematic representation
of the complex. Briefly, the major interactions with the protein
involve the benzofuranone core and the indole ring. Both rings
form hydrophobic interactions with the protein: the benzofur-
anone core with the P-loop and the indole ring through a
hydrophobic pocket lined by residues Leu-44, Val-52, Ala-65,
Val-126, Leu-174, Ile-185, and Cα, Cβ, Cδ, and Cγ of Arg-122
and Cδ of Pro-123. Both rings also form hydrogen bonds with
the protein: the 3-keto group on the benzofuranone core with
the side chain of the catalytically essential Lys-6750 and the NH
in the indole ring with the backbone carbonyl of the hinge
residue Glu-121, which usually forms a critical hydrogen bond
with the adenine amino group of ATP.51 These two amino acid
residues, Lys-67 and Glu-121 are typically involved in
interactions with PIM1 inhibitors.52 The phenolic hydroxyl
on the left-hand side of the core is located near the edge of the
ATP-binding pocket and seems to have no significant
interaction with the protein.
The homopiperidine ring on the top side of the core extends
into regions following the hinge. These regions have been
termed the specificity surface and the ribose-binding region. It
has been proposed that targeting the specificity surface can
improve the kinase selectivity of inhibitors.53−55
A unique structural feature of PIM kinases is the presence of
proline, which cannot donate a hydrogen atom, as the
“gatekeeper (gk) +3” residue (Pro-123 in PIM1, Figure 1) at
the hinge region.51 In most cases, kinase inhibitors accept a
hydrogen bond from the backbone amide of the gk+3 residue.56
It has therefore been proposed that the unusual lack of a
hydrogen bond donor at the gk+3 position in PIM can be
targeted to design more selective inhibitors.36,52
Figure 1. A schematic representation of compound 1 complexed with
PIM1 kinase. Dashed lines indicate hydrogen bonds between
compound 1 and PIM1.
Table 1. Properties of Compound 1
a
kinase inhibition
metabolic stability
b
c
d
e
compd PIM1 IC50 (nM) kinase selectivity HLM (%) MLM (%)
410 low 36 44
1
a
Carried out at Cerep. Compound was incubated at 37 °C for 1 h with
0.3 mg/mL protein. % remaining was determined by HPLC-MS/MS.
b
IC50 value is shown as the mean of duplicate experiments. [ATP] =
c
30 μM. For details, see Figure 2 and Table S1, Supporting
Information. Human liver microsomes. Mouse liver microsomes.
d
e
optimization of 1. In general, lower cLogP as well as lower
molecular weight (MW) are preferable to achieve good
ADMET (absorption, distribution, metabolism, excretion, and
toxicity) properties.47 However, cLogP and MW generally tend
to increase during optimization.48
So, as an optimization strategy, we decided to focus first on
introducing specific hydrogen-bonding interactions between
the compound and the protein in order to improve potency
and selectivity and second on reducing the lipophilicity in order
to improve the ADMET properties. Further, we aimed to
modify the compound in as simple a way as possible in order to
minimize the increase of molecular weight. To help us achieve
these goals, we planned to utilize structural information, mainly
from X-ray analysis of the PIM1-1 complex, to guide the
modifications.
Here, we report the rational evolution of 1 into a potent,
selective, and metabolically stable PIM1 inhibitor, 14, using the
strategy described above. Compared with the initial screening-
hit compound 1, the new inhibitor 14 showed greatly increased
potency and selectivity for PIM1 over other kinases, as well as a
significant reduction in cLogP value, with only a marginal
increase in molecular weight. In cellular assay, 14 potently
On the basis of the above structural information, we sought
to modify compound 1 to improve its potency and selectivity
while retaining key interactions.
Modification of the Top Side of 1. On the basis of the
above structural analysis, we initially modified the top side (7-
position) of the benzofuranone core (Table 2). As shown in
Figure 1, the homopiperidine ring of 1 lies in close proximity to
the acidic amino acid residues Asp-128 and Glu-171. Targeting
Figure 2. Kinase selectivity profiles of compounds 1, 3, 6, 10, and 14. The color-coding scheme is as follows: <1% inhibition (green), 50% inhibition
(black), and >99% inhibition (magenta). Selective inhibitor profiles contain fewer magenta elements and more green elements. A complete table
with percent inhibition data is available in Supporting Information (Table S1).
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expected to be useful as a synthetic handle. From a medicinal
chemistry point of view, capping a possibly metabolically labile
phenolic hydroxyl group is a favorable modification.
Table 2. Structure−Activity Relationship of 7-Substitutes on
Benzofuran-3-one core
The P-loop is the only region of the protein within potential
binding distance from the phenolic hydroxyl group of 1. In the
P-loop, there are only two polar amino acids, Ser-46 and Ser-51.
In the crystal structure, the hydroxyl side chains of these
residues point away from 1, toward the aqueous region.49
Backbone carbonyls and amides in the P-loop also point toward
the aqueous region. Therefore, it would be difficult to introduce
additional hydrogen-bonding interactions between the protein
and the compound at this site. As an alternative approach, we
targeted the P-loop through hydrophobic interactions. The
phenolic hydroxyl is located near the edge of the ATP-binding
pocket, and so modification with smaller lipophilic substituents
seemed preferable. Also, the smaller the lipophilic substituent,
the smaller the increase in lipophilicity, which is a key factor for
metabolic lability.
As we expected, the size of the lipophilic group was critical
for inhibitory potency (Table 3). Removal of the hydroxyl
a
IC50 values are shown as the mean of duplicate experiments. [ATP] =
Table 3. Structure−Activity Relationship of 6-Substitutes on
Benzofuran-3-one core
b
30 μM. Calculated logarithm of the octanol/water distribution
coefficient using ChemBioDraw Ultra version 11.0.1. LE = −1.4·log-
(IC50)/HAC; HAC = heavy atom count. LLE = −log(IC50) − cLogP.
c
d
Asp-128 is especially attractive in terms of kinase selectivity
because Asp-128 is positioned in the specificity surface. We
therefore planned to introduce additional hydrogen bonds with
Asp-128 and Glu-171 by replacing the homopiperidine ring
with a structure having an additional basic amino group.
Introduction of an amino group is also expected to reduce the
cLogP value of 1. We first synthesized 2, which has a 7-
membered homopiperazine ring with an amino group at the
lateral of the molecule. As expected, 2 showed a 4-fold
improvement in potency compared to 1. Next, we synthesized
3, which has a 6-membered piperazine ring, and this resulted in
a further 6-fold increase in potency (IC50 = 17 nM). We have
solved the structure of representative compounds at each step
of the ligand-design process and confirmed the lateral NH
group on the piperazine ring forms direct hydrogen bonds with
the side-chain carboxyl of Asp-128 and the backbone carbonyl
of Glu-171 throughout the optimization. In sharp contrast,
methylation of the lateral amino group on the piperazine ring,
as in compound 4, dramatically decreased the potency. The
piperazine ring in 3 was also found to contribute to the
improvement of kinase selectivity (Figure 2). Further, the
transition from 1 to 3 reduced the cLogP value from 5.3 to 3.4.
Throughout the optimization process, we monitored the
effectiveness of our synthetic effort by calculating ligand
efficiency (LE) and ligand lipophilicity efficiency (LLE) values:
LE is a measure of the potency with respect to the molecular
size, while LLE indicates the balance between potency and
lipophilicity.57−59 Because we aimed to improve potency while
minimizing the increase in MW and reducing cLogP, the easily
calculable values of LE and LLE were expected to be useful
guides. In the evolution of 1 to 3, the LE and LLE values were
improved from 0.31 to 0.39 and from 1.1 to 4.4, respectively.
Modification of the Left-Hand Side of the Molecule.
We considered that the left-hand side of 1 might be modified in
a variety of ways, as there were no obvious interactions between
the compound and the protein. Therefore, we selected the
phenolic hydroxyl at the 6-position of the core because it was
a
b
c
d
compound
R
PIM1 IC50 (nM)
cLogP
LE
LLE
3
5
6
7
8
9
OH
H
17
20
2
3.4
3.8
3.8
4.3
4.9
5.6
0.39
0.40
0.42
0.38
0.36
0.29
4.4
3.9
4.9
3.9
3.0
1.8
OMe
OEt
OPr
OBn
6
12
43
a
IC50 values are shown as the mean of duplicate experiments. [ATP] =
30 μM. Calculated logarithm of the octanol/water distribution
coefficient using ChemBioDraw Ultra Version 11.0.1. LE =
−1.4·log(IC50)/HAC; HAC = heavy atom count. LLE = −log(IC50)
b
c
d
− cLogP.
group did not significantly affect the potency, as observed for
compound 5, while methylation increased the potency by 8.5-
fold, as in compound 6. The quality indicators, LE and LLE,
were increased to 0.42 and 4.9, respectively. The inhibitory
potency decreased as the size of the lipophilic group was
increased (compounds 7−9). Benzyl substitution at this
position significantly decreased the potency. A possible
explanation is a steric clash between the larger lipophilic
groups and the P-loop, which is located 3 Å away from the
methyl group at the closest point. Therefore, to be
accommodated inside the pocket, bigger groups might have
to adopt less energetically favored, possibly twisted con-
formations.
Compound 6, which has the 6-methoxy group on the left-
hand side of the core, was selected for further modifications
based on the improvement of potency, LE, and LLE, compared
with 1.
Modification of the Right-Hand Side of the Molecule.
The right-hand side of 1 offers fewer options for modification
because the parent indole ring forms the critical hydrogen bond
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with Glu121 and has various hydrophobic interactions with the
protein. Therefore, we sought to modify the right-hand side
without disrupting these critical interactions. Researchers at
Plexxikon have reported the crystal structure of the PIM1
complex with a 7-azaindole compound. Their compound binds
to the ATP-binding site of PIM1 in the same orientation as 1,
forming a hydrogen bond between the NH group and Glu-121
(PDB ID: 3C4E).60 Thus, we replaced the indole ring of 6 with
a 7-azaindole ring. The resulting 7-azaindole derivative, 10,
inhibited PIM1 as potently as 6 (Table 4). In general,
the serine/threonine kinase GSK3β more potently than PIM1.
In addition, 1 inhibited other tyrosine kinases, EGFR, KDR,
and serine/threonine kinases Aurora A, MLK1, and MST1, as
potently as PIM1.
Introduction of a piperazine ring on the top side of the core
resulted in a marked improvement in selectivity. The selectivity
profile of 3 showed that it inhibited PIM1 most potently among
the kinases examined. However, FLT3, PDGFRα, and SYK
were still potently inhibited by 3. Modification of the left-hand
side of the core, specifically methylation of the hydroxyl at the
6-position, did not significantly change the selectivity (3 vs 6).
The two azaindole derivatives, 10 and 14, showed a
significant difference in the kinase selectivity profile. As
expected, the 7-azaindole derivative 10 showed a nonspecific
selectivity profile compared to 3 and 6, presumably owing to
hydrogen bond formation with the backbone amide of the gk+3
residue in off-target kinases. In addition to FLT3 and PDGFRα,
10 potently inhibited serine/threonine kinases DAPK1,
GSK3β, PKCα, PKD2, and ROCK1. In contrast, 14, the
indazole (2-azaindole) derivative showed a much cleaner
selectivity profile. Other than PIM1, only FLT3 was potently
inhibited by 14. Under the assay conditions used, 14 inhibited
PIM1 and FLT3 with IC50 values of 6 nM and 47 nM,
respectively. The clinically tested PIM1 inhibitor SGI-1776
shows similar potency and selectivity characteristics with
respect to FLT3.22−24
Table 4. Kinase Inhibitory Activity of Azaindole Derivatives
All our compounds tested showed selectivity for PIM1 and
PIM3 over PIM2 (Table 5). In the case of 14, the IC50 values
a
Table 5. Pim Family Selectivity Study
c
rate of inhibition (%)
b
kinase
ATP concn (μM)
1
3
6
10
14
a
IC50 values are shown as the mean of duplicate experiments. [ATP] =
PIM1
PIM2
PIM3
500
5
77
46
88
98
30
97
99
30
98
>99
39
99
19
98
b
30 μM. Calculated logarithm of the octanol/water distribution
coefficient using ChemBioDraw Ultra version 11.0.1. LE = −1.4·log-
(IC50)/HAC; HAC = heavy atom count. LLE = −log(IC50) − cLogP.
c
d
150
99
a
b
Carried out at CarnaBiosciences. ATP concentration was approx-
c
imate Km value of each kinase. The rate of enzyme inhibition was
examined by an electrophoretic mobility shift assay. Each compound
was tested at the concentration which is 50 times higher than its PIM1
IC50 value. The concentration of each compound was; 1, 22500 nM; 3,
600 nM; 6, 250 nM; 10, 200 nM; 14, 300 nM. PIM1 IC50 values for
this study were determined at CarnaBiosciences.
replacement of a carbon atom with a nitrogen atom, while
maintaining the molecular size, reduces the lipophilicity.
However, as can be deduced from Figure 1, introduction of a
nitrogen atom that faces the hinge region, as in 10, may cause a
kinase selectivity problem because the nitrogen atom can act as
a hydrogen bond acceptor for kinases with a hydrogen bond
donor at this site.
With this selectivity concern in mind, we next explored other
azaindole derivatives. The 6-, 5-, and 4-azaindole derivatives,
11, 12, and 13, showed decreased potency (Table 4), while the
indazole (2-azaindole) derivative, 14, inhibited PIM1 as
potently as 6 or 10.
The cLogP values for 10 and 14 are 2.8 and 3.3, respectively,
both of which are lower than that of 6. The reduction in cLogP
values of 10 and 14 was associated with an improvement of the
LLE values to 5.9 and 5.2, respectively, from the value of 4.9 in
6, and the LE values were maintained.
Kinase Selectivity Study. Kinase selectivity data are
summarized in Figure 2 and Table S1, Supporting Information.
Each compound was tested against a panel of 20 tyrosine
kinases and 30 serine/threonine kinases at a concentration 50-
fold higher than the IC50 value in the PIM1 inhibitory assay
(carried out at CarnaBiosciences, Japan). The ATP concen-
tration used was approximately equal to the Km value for each
kinase. As mentioned earlier, 1 showed a poor kinase selectivity
profile, inhibiting tyrosine kinases FLT3, PDGFRα, SYK, and
for PIM2 and PIM3 were 1160 and 13 nM, respectively. Again,
the same tendency has been reported for SGI-1776.22−24 At the
amino acid level, PIM2 is 53% identical to PIM1 and PIM3 is
71% identical to PIM1.1,19 PIM2 is mainly expressed in
hematological and prostate cancer cells as PIM1, and PIM3 is
highly expressed in hepatic and pancreatic cancer cells.1 The
concept of the inhibition of all three PIM family members has
been proposed in consideration of the functional redundancy
and potential compensatory mechanism of PIM family
members.36 However, the functional redundancy of PIM family
members in cancer biology remains unclear.1 For example,
PIM1 deficient mice show a lower level of CXCR4 expression
on the surface of the bone marrow cells, which is not
compensated by the presence of PIM2.15
Finally, compound 14, which showed potent PIM1-
inhibitory activity and a clean kinase selectivity profile, was
selected as the most promising lead candidate.
Cellular Study. To evaluate the cellular activity of 14, we
carried out growth inhibition assay using the human leukemia
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Figure 3. Cellular study of compound 14. (A) Growth inhibition assay with MV4−11 cells. Cells were counted with a Cell Counting Kit-8. WI-38
cells were used as a control. (B) Inhibition of phosphorylation of BAD at Ser-112. MV4−11 cells were treated with various concentrations of 14 for 1
h, and protein extracts were subjected to immunoblotting. (C) MV4−11 cells were treated with 14 for 48 h. Induction of apoptosis was evaluated
with Annexin V and PI staining. (D) Cell cycle distribution of MV4−11 cells after 48 h treatment with 14.
a
Table 6. ADMET Profiling Data for Compound 14
b
metabolic stability (%)
e
f
c
d
liver microsomes
hepatocytes
mouse
89
CYP inhibition (%)
hERG inhibition (%)
human
>99
mouse
92
1A2
33
2C9
<1
2C19
54
2D6
11
3A4
10
10 μM
1 μM
60
13
a
b
ADMET study was carried out at Cerep. Compound concentration was 1 μM. %Remainings of the compound was determined by HPLC-MS/MS.
c
d
e
Compound concentration was 10 μM. A patch-clamp method was carried out to evaluate hERG inhibition. Compound was incubated with 0.3
f
mg/mL protein at 37 °C for 1 h. Cryopreserved hepatocytes. Compound was incubated with 1 million cells/mL at 37 °C for 2 h.
cell line MV4−11, which is frequently used to evaluate the
efficiency of PIM1 inhibitors (Figure. 3A). Compound 14
potently inhibited the growth of MV4−11 (GI50 = 43 nM) and
had only a negligible effect on the growth of the normal human
diploid lung fibroblast cell line WI-38, which was used as a
surrogate for general toxicity. In comparison, 6, which inhibited
PIM1 as potently as 14, showed a reduced ability to inhibit the
growth of MV4−11 (GI50 = 95 nM). Compound 3 showed
weak inhibition of MV4−11 cell growth (GI50 = 270 nM),
which was not unexpected as it is a weaker PIM1 inhibitor. On
the basis of these results, 14 was prioritized for further study.
Next, we examined the phosphorylation status of BAD to
confirm the inhibition of PIM1 in MV4−11 cells. PIM1 is
reported to phosphorylate BAD at Ser-112, thereby deactivat-
ing the pro-apoptotic function of BAD.61 Compound 14 was
incubated with MV4−11 for 1 h, and cells were lysed for
analysis of their phosphorylation status by Western blotting. As
shown in Figure 3B, 14 dose-dependently inhibited the
phosphorylation of BAD at Ser-112 but not at Ser-136. It
was also confirmed to induce apoptosis (Figure 3C) and cell-
cycle arrest at the G1 phase (Figure 3D) in a dose-dependent
manner, as has been found for other PIM1 inhibitors.22−24,29
ADMET Study. Compound 14 showed an acceptable
ADMET profile (Table 6). First, its metabolic stability in
liver microsomes was greatly improved compared with that of
1, as expected from the reduction in cLogP by 2 log units. It
was very stable and was almost quantitatively recovered after
incubation with both human (>99%) and mouse liver
microsomes (92%), whereas recovery of 1 was only about
40% in both cases (Table 1). Other potent and selective PIM1
inhibitors, compound 3 and 6, were less stable compared to 14.
Compound 3 was recovered in 90% in human liver microsomes
and 71% in mouse liver microsomes. Compound 6 was
recovered in 82% in human liver microsomes and 86% in
mouse liver microsomes. In addition, 14 was stable in the
presence of cryopreserved hepatocytes, which mimic in vivo
metabolic conditions due to their retention of a complete
ensemble of metabolizing enzymes and other cellular
components.62 Second, 14 did not potently inhibit five major
cytochrome P450 (CYP) enzymes at 10 μM, indicating that it
has low potential for drug−drug interactions. Third, 14 showed
only moderate inhibition of potassium channel hERG (human
ether-a-go-go related gene). hERG is a cardiac potassium ion
channel, and its inhibition may trigger life-threatening torsades
de pointes (TdP) arrhythmia through QT prolongation.62
Compound 14 inhibited hERG to the extent of 60% at 10 μM
and only 13% at 1 μM, whereas SGI-1776, whose clinical
investigations were discontinued due to QT prolongation, is
reported to inhibit hERG more potently than this (IC50 = 0.98
μM).63
Structural Study. Compound 14 (PDB ID: 3UMW) binds
in the ATP-binding cleft at the same site as compound 1
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(Figure 4), and it retains the representative hydrophobic
interactions and hydrogen bonds seen in the complex of 1 and
showed less well-defined electron density, presumably due to
increased flexibility of the 7-membered ring compared to that of
the 6-membered ring.49 It seems likely that the homopiperazine
ring would behave similarly and may also bind with both Asp-
128 and Glu-171, but there would be a higher entropy penalty
as compared to the piperazine ring in compound 14. Although
the contribution to selectivity was not mentioned, Qian and co-
workers reported that the side-chain carboxyl of Asp-128 and
backbone carbonyl of Glu-171 exhibit a water-mediated
interaction with their PIM1 inhibitors.31 In addition, during
the preparation of this manuscript, Xiang and co-workers
reported that hydrogen bonds with those two amino acid
residues are key interactions of their potent PIM1 inhibitors.35
On the left-hand side of the molecule, the methyl group at
the 6-position of the core is accommodated inside the
hydrophobic pocket and participates in interactions between
the core and the P-loop, which improves the potency, as seen in
the comparison of 3 and 6 (Table 3).
Judging from currently available information, no other
reported compound makes as many simultaneous interactions
with PIM1 as 14, namely direct H-bonds with Lys-67, Glu-121,
Asp-128, and Glu-171.
Chemistry. Compounds 2 and 3 were synthesized as shown
in Scheme 1. Mannich reaction of 6-hydroxy-2H-benzofuran-3-
one with paraformaldehyde and N-Boc-homopiperazine or N-
Boc-piperazine provided intermediates 15 and 16. Knoevenagel
condensation with 1H-indole-3-carbaldehyde afforded 17 and
18. Subsequent acidic removal of a Boc group furnished 2 and
3.
An analogue unsubstituted at the 6-position of the
benzofuranone core, 5, was synthesized according to Scheme
2. Basic cyclization of 2-chloro-1-(2-hydroxy-3-methylphenyl)-
ethanone64 afforded 19. Bromination at the benzyl position of
19 was carried out with N-bromosuccinimide (NBS) and
benzoyl peroxide (BPO) in refluxing CCl4 to afford 20.
Replacement of bromide with N-Boc-piperazine furnished 21,
which was condensed with 1H-indole-3-carbaldehyde to afford
22. Finally, acidic removal of the Boc group of 22 furnished 5.
6-Alkoxylated benzofuranone derivatives were synthesized
according to Scheme 3. The 6-hydroxyl group of 16 was
alkylated by Mitsunobu reaction with various alcohols.
Figure 4. Electron density and hydrogen-bonding interactions
between compound 14 and PIM1 kinase. Compound 14 is
represented by green sticks, while the protein residues are colored
gray. Nitrogen and oxygen are colored blue and red, respectively.
Water molecules are displayed as green spheres. Interactions are drawn
as dashed lines, and distances are given in Å. Electron density is
represented as a green mesh and was calculated using an omit map
contoured to 1-σ.
PIM1. Importantly, the distance between the ring and Glu-121
is shorter in 14 than in 1.49
On the top side, the piperazine ring, which is a critical
substituent for potency and selectivity, forms two hydrogen
bonds with the protein, as anticipated. The nitrogen atom at
the top of the ring forms hydrogen bonds with the side-chain
carboxyl of Asp-128 and the backbone carbonyl of Glu-171.
The decreased potency of 4, in which the lateral nitrogen atom
in the piperazine ring is methylated, supports the importance of
both hydrogen bonds. Replacement of the 6-membered
piperazine with 7-membered homopiperazine resulted in a 6-
fold reduction of potency, as seen with 2 (Table 2). In the
structure of the compound 1 complex, the 7-membered ring
a
Scheme 1. Synthesis of 7-Modified Derivatives 2 and 3
a
Reagents and conditions: (a) paraformaldehyde, EtOH, 120 °C under microwave irradiation, 15% (15) or reflux, 52% (16); (b) 1H-indole-3-
carbaldehyde, piperidine, MeOH, 60 °C, 28% (17) and 68% (18); (c) 4N HCl in dioxane, CH2Cl2, rt, 50% (2) and 75% (3).
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a
Scheme 2. Synthesis of 6-Unsubstituted Derivative 5
a
Reagents and conditions: (a) K2CO3, CH3CN, rt, 43%; (b) NBS, BPO, CCl4, reflux, 36%; (c) N-Boc-piperazine, CH2Cl2, rt, quant; (d) 1H-indole-
3-carbaldehyde, piperidine, MeOH, 60 °C, 76%; (e) 4N HCl in dioxane, CH2Cl2, rt, 67%.
a
Scheme 3. Synthesis of 6-Alkoxy Derivatives 6−9
a
Reagents and conditions: (a) ROH, PPh3, DEAD, toluene, 110 °C, 28−43%; (b) 1H-indole-3-carbaldehyde, piperidine, MeOH, 60 °C, 53−77%;
(c) 4N HCl in dioxane, CH2Cl2, rt, 53−80%.
Subsequent Knoevenagel condensation and acidic deprotection
afforded compounds 6−9.
Azaindole analogues 10−14 were synthesized as shown in
Scheme 4. 23 was condensed with various azaindole-3-
carbaldehydes. Subsequent removal of the Boc group afforded
10−14. Among azaindole-3-carbaldehydes, 7-azaindole-3-car-
baldehyde and indazole-3-carbaldehyde were purchased. 4-
Azaindole-3-carbaldehyde and 5-azaindole-3-aldehyde were
synthesized according to the literature.65 6-Azaindole-3-
carbaldehyde was synthesized by formylation of a parent 6-
azaindole similarly to 4-azaindole-3-carbaldehyde.
CONCLUSIONS
■
a
Scheme 4. Synthesis of Azaindole Derivatives 10−14
We evolved 1 into 14, which is about 100 times more potent
and much more selective for PIM1, by means of simple
chemical modifications guided by structural information. We
employed a rational optimization strategy, aiming to introduce
specific hydrogen-bonding interactions between the compound
and the protein to improve potency and selectivity and to
modify the compound in as simple a way as possible in order to
minimize the increase of molecular weight as well as to reduce
lipophilicity. The validity of our design concept was confirmed
by X-ray crystallography, which showed that the incorporated
groups participate in interactions with the protein as expected.
The molecular weight was increased by only 2 Da, while cLogP
was greatly reduced by 2 log units. The reduction of cLogP is
mirrored by a significant improvement in metabolic stability.
The effectiveness of our modifications was also reflected in the
quality indicators LE and LLE: the LE value was increased from
0.31 to 0.41 and LLE from 1.1 to 5.2. In addition, 14 was active
in cellular assay, potently inhibiting the growth of the leukemia
cell line MV4−11. Further cell-based analysis using MV4−11
confirmed that 14 inhibited the phosphorylation of the typical
PIM1 substrate BAD and induced apoptosis and cell cycle
arrest at the G1 phase.
a
Reagents and conditions: (a) RCHO, piperidine, MeOH, 60 °C, 33−
82%; (b) 4N HCl in dioxane, CH2Cl2, rt, 12−78%.
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HRMS (ESI+) calcd for (C23H24N3O3) [M + H]+ 390.1818; found
390.1771.
Compounds 2, 3, 5−14 were synthesized as follows.
Our results provide an example of successful rational
compound evolution through simple, structure-guided chemical
modifications. By introducing hydrogen-bonding and hydro-
phobic modifications at appropriate sites, while minimizing the
increase of molecular size and decreasing the lipophilicity, we
achieved significant improvements in potency, selectivity, and
metabolic stability.
tert-Butyl 4-[(6-Hydroxy-3-oxo-2,3-dihydrobenzofuran-7-
yl)methyl]-1,4-diazepane-1-carboxylate (15). A mixture of 6-
hydroxyl-2H-benzofuran-3-one (1.50 g, 10.0 mmol), paraformalde-
hyde (0.300 g, 10.0 mmol), and N-Boc-homopiperazine (2.00 g, 10.0
mmol) in EtOH (10 mL) was heated at 120 °C under microwave
irradiation for 20 min. The mixture was concentrated in vacuo, and the
resulting residue was purified by column chromatography (0−100%
EtOAc/hexane) to afford the title compound (549 mg, 15%).
1H NMR (300 MHz, CD3OD) δ 1.47 (s, 9H), 1.78−1.85 (m, 2H),
2.91−2.94 (m, 2H), 2.96−2.99 (m, 2H), 3.47−3.52 (m, 2H), 3.58 (t, J
= 5.1 Hz, 2H), 4.65 (s, 2H), 4.92 (s, 2H), 6.49 (d, J = 8.8 Hz, 1H),
7.42 (d, J = 8.8 Hz, 1H).
EXPERIMENTAL SECTION
■
General Information. Reagents and solvents were purchased from
commercial sources and used without further purification. All reactions
were performed under an argon atmosphere unless otherwise noted.
Microwave irradiation was carried out with an Initiator (Biotage).
Reactions were monitored by thin layer chromatography (TLC) on
precoated TLC glass plates (silica gel 60 F254, 250 μm thickness).
Column chromatography was carried out with Yamazen YFLC wprep
2XY on silica gel unless otherwise noted. 1H NMR spectra were
recorded on either a JEOL JNM-LA300 or a JEOL JNM-LA400;
chemical shifts (δ) are reported relative to a signal of Me4Si. Data for
NMR are reported as follows: chemical shift (δ ppm), multiplicity (s =
singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, dd =
doublet of doublets, m = multiplet or overlapping), coupling constant
(Hz), integration. Mass spectra (ESI+) were measured with a JEOL
JMS-T100LC mass spectrometer. The purity of compounds was
determined by HPLC analysis. The HPLC instrument was composed
of a Leap HTS PAL 96-well plate injector (CTC Analysis), HPLC
pump model P580 (Dionex), HPLC column thermostat STH 585
(Dionex), HPLC photodiode array detector (Dionex), and Chrome-
leon HPLC data system (Dionex), and was equipped with a Keystone
Scientific Prism RPN 4 mm × 20 mm guard column. The column
temperature was 21 °C. The purity measurements were done by
measuring peak area at 230 nm, and purity was calculated based on the
observed target peak area as a percentage of total area. Mobile phase A
consisted of 12.0 mM ammonium formate/5.9 mM formic acid in
water. Mobile phase B consisted of 6.7 mM ammonium formate/3.3
mM formic acid in water/CH3CN (5/40, v/v). Gradient conditions
were as follows: 0% mobile phase B at time of injection; linear increase
to 82.5% B at 1.20 min; linear increase to 90% B at 1.60 min; maintain
90% B until 1.80 min. The flow rate was 2.5 mL/min. For compound
12, ACQUITY UPLC (Waters) equipped with an ACQUITY UPLC
BEH C18 column was used to determine the purity. The column
temperature was 40 °C. Mobile phase A consisted of 5.0 mM
ammonium acetate in water. Mobile phase B consisted of CH3CN.
Gradient conditions were as follows: 5% mobile phase B at time of
injection; linear increase to 95% B at 1.00 min; maintain 95% B until
1.50 min. The flow rate was 0.7 mL/min. The purity measurements
were done by measuring peak area at 230 nm, and purity was
calculated based on the observed target peak area as a percentage of
total area. All compounds assayed were ≥95% pure.
tert-Butyl 4-[(6-Hydroxy-3-oxo-2,3-dihydrobenzofuran-7-
yl)methyl]piperazine-1-carboxylate (16). A mixture of 6-
hydroxy-2H-benzofuran-3-one (10.0 g, 66.6 mmol), paraformaldehyde
(2.00 g, 66.6 mmol), and N-Boc-piperazine (12.4 g, 66.6 mmol) in
EtOH (70 mL) was refluxed for 4 h. The mixture was concentrated in
vacuo, and the resulting residue was purified by column chromatog-
raphy (0−50% EtOAc/hexane) to afford 15.6 g of a crude product.
The crude product was recrystallized from EtOAc to furnish the title
compound (12.1 g, 52%).
1H NMR (300 MHz, DMSO-d6) δ 1.38 (s, 9H), 2.44 (m, 4H), 3.32
(m, 4H), 3.66 (s, 2H), 4.73 (s, 2H), 6.59 (d, J = 8.8 Hz, 1H), 7.40 (d, J
= 8.8 Hz, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-6-hydroxy-3-
oxo-2,3-dihydrobenzofuran-7-yl}methyl)-1,4-diazepane-1-car-
boxylate (17). A mixture of 15 (155 mg, 0.428 mmol), 1H-indole-3-
carbaldehyde (62.1 mg, 0.428 mmol), and piperidine (3.64 mg, 0.0428
mmol) in MeOH (2 mL) was heated at 60 °C for 2 h. The mixture
was cooled to room temperature and concentrated in vacuo, and the
resulting residue was purified by column chromatography (0%-2%
MeOH/CHCl3) to afford the title compound (59.1 mg, 28%).
1H NMR (400 MHz, CDCl3) δ 1.48 (s, 9H), 1.95 (m, 2H), 2.82−
2.89 (m, 4H), 3.51−3.63 (m, 4H), 4.08 (d, J = 17.6 Hz, 2H), 6.66 (d, J
= 8.8 Hz, 1H), 7.23−7.30 (m, 3H), 7.45 (d, J = 7.8 Hz, 1H), 7.63 (d, J
= 8.3 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 10.8 Hz, 1H), 9.11
(br s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-6-hydroxy-3-
oxo-2,3-dihydrobenzofuran-7-yl}methyl)piperazine-1-carbox-
ylate (18). A mixture of 16 (348 mg, 1.00 mmol), 1H-indole-3-
carbaldehyde (145 mg, 1.00 mmol), and piperidine (68.1 mg, 0.0800
mmol) in MeOH (4 mL) was heated at 60 °C for 2 h. The mixture
was cooled to room temperature, and the precipitate was collected by
filtration to afford the title compound (324 mg, 68%).
1H NMR (300 MHz, DMSO-d6) δ 1.38 (s, 9H), 2.53 (m, 4H), 3.35
(m, 4H), 3.87 (s, 2H), 6.72 (d, J = 8.8 Hz, 1H), 7.14−7.25 (m, 3H),
7.50 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 8.09 (d, J = 8.1 Hz,
1H), 8.16 (d, J = 2.9 Hz, 1H), 11.98 (br s, 1H).
(Z)-7-[(1,4-Diazepan-1-yl)methyl]-2-[(1H-indol-3-yl)-
methylene]-6-hydroxybenzofuran-3(2H)-one (2). To 17 (56.0
mg, 0.114 mmol) in CH2Cl2 (6 mL) was added 4 N HCl in dioxane (6
mL) at room temperature. The mixture was stirred at the same
temperature for 2 h and concentrated in vacuo. Then, 4 mL of
triethylamine and 6 mL of toluene were added to the flask, and the
resulting mixture was concentrated in vacuo. This process was
repeated twice. The residue was purified by column chromatography
(0−10% MeOH/CHCl3) on NH2-modified silica gel to afford the title
compound (22.2 mg, 50%).
Compound 1 was purchased from InterBioScreen Ltd. (catalogue
ID: STOCK6S-34005). Compound 4 was purchased from Princeton
Biomolecular Research, Inc. (catalogue ID: OSSK_865988). 7-
azaindole-3-carbaldehyde and indazole-3-carbaldehyde were purchased
from Aldrich and Apollo Scientific, respectively.
(Z)-2-[(1H-Indol-3-yl)methylene]-7-(azepan-1-ylmethyl)-6-
1
hydroxybenzofuran-3(2H)-one (1). H NMR (300 MHz, DMSO-
d6) δ 1.62 (m, 4H), 1.72 (m, 4H), 2.93 (t, J = 5.1 Hz, 4H), 4.21 (s,
2H), 6.52 (d, J = 8.7 Hz, 1H), 7.11 (s, 1H), 7.15−7.25 (m, 2H), 7.45−
7.51 (m, 2H), 8.02 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 2.4 Hz, 1H), 11.89
(br s, 1H).
1H NMR (400 MHz, DMSO-d6) δ 1.78 (m, 2H), 2.88−2.94 (m,
8H), 4.11 (s, 2H), 6.46 (d, J = 8.3 Hz, 1H), 7.04 (s, 1H), 7.15−7.24
(m, 2H), 7.41 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 8.03 (d, J
= 7.3 Hz, 1H), 8.10 (s, 1H).
HRMS (ESI+) calcd for (C23H24N3O3) [M + H]+ 389.1865; found
389.1817.
HRMS (ESI+) calcd for (C23H24N3O3) [M + H]+ 390.1818; found
390.1791.
(Z)-2-[(1H-Indol-3-yl)methylene]-6-hydroxy-7-[(4-methylpi-
perazin-1-yl)methyl]benzofuran-3(2H)-one (4). 1H NMR (300
MHz, DMSO-d6) δ 2.17 (s, 3H), 2.31−2.43 (m, 4H), 2.59−2.69 (m,
4H), 3.94 (s, 2H), 6.67 (d, J = 8.7 Hz, 1H), 7.15−7.26 (m, 3H), 7.49−
7.53 (m, 2H), 8.08 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 2.1 Hz, 1H), 11.96
(br s, 1H).
(Z)-2-[(1H-Indol-3-yl)methylene]-6-hydroxy-7-(piperazin-1-
ylmethyl)benzofuran-3(2H)-one (3). To a suspension of 18 (143
mg, 0.300 mmol) in CH2Cl2 (14 mL) was added 4 N HCl in dioxane
(14 mL) at room temperature. The mixture was stirred at the same
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temperature for 2 h and concentrated in vacuo. Then 14 mL of
saturated aqueous NaHCO3 was added. The precipitated yellow
powder was collected by filtration, washed with water, and dried in
vacuo to afford the title compound as a yellow powder (85.2 mg,
75%).
(1.97 g, 7.50 mmol), and 40% diethylazodicarboxylate (DEAD) in
toluene (3.27 g, 7.50 mmol) in toluene (20 mL) was stirred at 110 °C
for 5 h, then cooled to room temperature and concentrated in vacuo.
The resulting residue was purified by column chromatography (0−
75% EtOAc/CHCl3). The crude product obtained was further purified
by column chromatography (0−50% EtOAc/hexane) to afford the title
compound (790 mg, 43%).
1H NMR (300 MHz, DMSO-d6) δ 2.59 (m, 4H), 2.80 (m, 4H),
3.92 (s, 2H), 6.59 (d, J = 8.1 Hz, 1H), 7.12 (s, 1H), 7.14−7.26 (m,
2H), 7.46−7.51 (m, 2H), 8.07 (d, J = 8.1 Hz, 1H), 8.14 (s, 1H), 11.95
(br s, 1H).
1H NMR (300 MHz, CDCl3) δ 1.44 (s, 9H), 2.48 (m, 4H), 3.42 (t,
J = 5.1 Hz, 4H), 3.70 (s, 2H), 3.93 (s, 3H), 4.64 (s, 2H), 6.70 (d, J =
8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H).
HRMS (ESI+) calcd for (C22H22N3O3) [M + H]+ 376.1661; found
376.1645.
tert-Butyl 4-[(6-Ethoxy-3-oxo-2,3-dihydrobenzofuran-7-yl)-
methyl]piperazine-1-carboxylate (24). From 16 (522 mg, 1.50
mmol) and EtOH (0.105 mL, 1.80 mmol), the title compound was
obtained (158 mg, 28%) in the same manner as described for 23.
1H NMR (300 MHz, CDCl3) δ 1.44−1.48 (m, 12H), 2.49 (m, 4H),
3.41 (m, 4H), 3.72 (s, 2H), 4.15 (q, J = 7.3 Hz, 2H), 4.64 (s, 2H), 6.67
(d, J = 8.8 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H).
7-Methylbenzofuran-3(2H)-one (19). A mixture of 2-chloro-1-
(2-hydroxy-3-methylphenyl)ethanone (6.40 g, 34.7 mmol) and K2CO3
(14.4 g, 104 mmol) in CH3CN (170 mL) was stirred at room
temperature for 1 h, then filtered. The filtrate was concentrated in
vacuo, and the resulting residue was purified by column chromatog-
raphy (0−10% EtOAc/hexane) to afford the title compound (2.23 g,
43%).
tert-Butyl 4-[(3-Oxo-6-propoxy-2,3-dihydrobenzofuran-7-
yl)methyl]piperazine-1-carboxylate (25). From 16 (522 mg,
1.50 mmol) and n-propanol (0.135 mL, 1.80 mmol), the title
compound was obtained (205 mg, 35%) in the same manner as
described for 23.
1H NMR (300 MHz, CDCl3) δ 2.33 (s, 3H), 4.64 (s, 2H), 7.00 (m,
1H), 7.42 (d, J = 7.3 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H).
7-(Bromomethyl)benzofuran-3(2H)-one (20). A mixture of
benzoyl peroxide (BPO) (8.90 mg, 0.0500 mmol) and N-
bromosuccinimide (NBS) (291 mg, 1.20 mmol) in CCl4 (10 mL)
was refluxed. A solution of 19 (227 mg, 1.00 mmol) in CCl4 (5 mL)
was added to the refluxing mixture, and the mixture was further
refluxed for 8 h, then cooled to room temperature and concentrated.
The resulting residue was purified by column chromatography (0−
10% EtOAc/hexane) to afford the title compound (82.0 mg, 36%).
1H NMR (300 MHz, CDCl3) δ 4.57 (s, 2H), 4.72 (s, 2H), 7.09 (m,
1H), 7.63−7.65 (m, 2H).
1H NMR (300 MHz, CDCl3) δ 1.07 (t, J = 7.3 Hz, 3H), 1.44 (s,
9H), 1.80−1.91 (m, 2H), 2.48 (m, 4H), 3.41 (t, J = 5.1 Hz, 4H), 3.71
(s, 2H), 4.04 (t, J = 6.6 Hz, 2H), 4.64 (s, 2H), 6.67 (d, J = 8.8 Hz,
1H), 7.59 (d, J = 8.8 Hz, 1H).
tert-Butyl 4-{[6-(Benzyloxy)-3-oxo-2,3-dihydrobenzofuran-7-
yl]methyl}piperazine-1-carboxylate (26). From 16 (522 mg, 1.50
mmol) and benzyl alcohol (0.186 mL, 1.80 mmol), the title compound
was obtained (259 mg, 39%) in the same manner as described for 23.
1H NMR (300 MHz, CDCl3) δ 1.44 (s, 9H), 2.48 (m, 4H), 3.40
(m, 4H), 3.73 (s, 2H), 4.65 (s, 2H), 5.20 (s, 2H), 6.76 (d, J = 8.8 Hz,
1H), 7.35−7.45 (m, 5H), 7.60 (d, J = 8.8 Hz, 1H).
tert-Butyl 4-[(3-Oxo-2,3-dihydrobenzofuran-7-yl)methyl]-
piperazine-1-carboxylate (21). To a solution of N-Boc-piperazine
(130 mg, 0.697 mmol) in CH2Cl2 (2 mL) was added dropwise a
solution of 20 (72.0 mg, 0.317 mmol) in CH2Cl2 (1 mL) at room
temperature. The mixture was stirred at room temperature for 12 h
and concentrated in vacuo. The resulting residue was purified by
column chromatography (0−2% MeOH/CHCl3) to afford the title
compound (105 mg, quant.).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-6-methoxy-3-
oxo-2,3-dihydrobenzofuran-7-yl}methyl)piperazine-1-carbox-
ylate (27). A mixture of 23 (181 mg, 0.500 mmol), 1H-indole-3-
carbaldehyde (72.6 mg, 0.500 mmol), and piperidine (34.1 mg, 0.400
mmol) in MeOH (2 mL) was stirred at 60 °C for 2 h, then cooled to
room temperature. The precipitate was collected by filtration to afford
the title compound (165 mg, 67%).
1H NMR (300 MHz, CDCl3) δ 1.45 (s, 9H), 2.46 (t, J = 5.1 Hz,
4H), 3.45 (t, J = 5.1 Hz, 4H), 3.64 (s, 2H), 4.65 (s, 2H), 7.09 (t, J =
7.3 Hz, 1H), 7.59−7.65 (m, 2H).
1H NMR (300 MHz, DMSO-d6) δ 1.37 (s, 9H), 2.48 (m, 4H), 3.31
(m, 4H), 3.78 (s, 2H), 3.95 (s, 3H), 7.01 (d, J = 8.8 Hz, 1H), 7.16−
7.27 (m, 3H), 7.51 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 8.15
(d, J = 7.3 Hz, 1H), 8.20 (s, 1H), 12.06 (br s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-3-oxo-2,3-di-
hydrobenzofuran-7-yl}methyl)piperazine-1-carboxylate (22).
A mixture of 21 (79.6 mg, 0.239 mmol), 1H-indole-3-carbaldehyde
(34.7 mg, 0.239 mmol), and piperidine (16.3 mg, 0.191 mmol) in
MeOH (1 mL) was heated at 60 °C for 2 h, then cooled to room
temperature and concentrated in vacuo. The resulting residue was
purified by column chromatography (0−2% MeOH/CHCl3) to afford
the title compound (83.8 mg, 76%).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-6-ethoxy-3-
oxo-2,3-dihydrobenzofuran-7-yl}methyl)piperazine-1-carbox-
ylate (28). From 24 (138 mg, 0.367 mmol), the title compound was
obtained (137 mg, 74%) in the same manner as described for 27.
1H NMR (300 MHz, DMSO-d6) δ 1.36−1.42 (m, 12H), 2.47 (m,
4H), 3.31 (m, 4H), 3.80 (s, 2H), 4.22 (q, J = 6.6 Hz, 2H), 6.98 (d, J =
8.8 Hz, 1H), 7.15−7.27 (m, 3H), 7.51 (d, J = 8.1 Hz, 1H), 7.70 (d, J =
8.8 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 2.9 Hz, 1H), 12.04
(br s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-3-oxo-6-pro-
poxy-2,3-dihydrobenzofuran-7-yl}methyl)piperazine-1-car-
boxylate (29). From 25 (163 mg, 0.417 mmol), the title compound
was obtained (166 mg, 77%) in the same manner as described for 27.
1H NMR (300 MHz, DMSO-d6) δ 1.05 (t, J = 7.3 Hz, 3H), 1.37 (s,
9H), 1.74−1.86 (m, 2H), 2.51 (m, 4H), 3.30 (m, 4H), 3.79 (s, 2H),
4.12 (t, J = 5.9 Hz, 2H), 6.98 (d, J = 8.8 Hz, 1H), 7.18 (t, J = 7.3 Hz,
1H), 7.22−7.27 (m, 2H), 7.51 (d, J = 7.3 Hz, 1H), 7.70 (d, J = 8.8 Hz,
1H), 8.14 (d, J = 8.1 Hz, 1H), 8.19 (d, J = 2.9 Hz, 1H), 12.03 (br s,
1H).
1H NMR (300 MHz, DMSO-d6) δ 1.38 (s, 9H), 2.47 (m, 4H), 3.37
(m, 4H), 3.84 (s, 2H), 7.17−7.30 (m, 3H), 7.38 (s, 1H), 7.52 (d, J =
8.1 Hz, 1H), 7.69−7.72 (m, 2H), 8.17 (d, J = 8.0 Hz, 1H), 8.26 (d, J =
2.2 Hz, 1H), 12.12 (br s, 1H).
(Z)-2-[(1H-Indol-3-yl)methylene]-7-(piperazin-1-ylmethyl)-
benzofuran-3(2H)-one (5). To a suspension of 22 (56.0 mg, 0.122
mmol) in CH2Cl2 (7 mL) was added 4 N HCl in dioxane (7 mL) at
room temperature. The mixture was stirred at the same temperature
for 2 h and concentrated in vacuo, and then 7 mL of saturated aqueous
NaHCO3 was added. The precipitated yellow powder was collected by
filtration, washed with water, and dried in vacuo to afford the title
compound as an orange powder (29.6 mg, 67%).
1H NMR (300 MHz, DMSO-d6) δ 2.46 (m, 4H), 2.73 (m, 4H),
3.78 (s, 2H), 7.18−7.29 (m, 3H), 7.38 (s, 1H), 7.52 (d, J = 8.1 Hz,
1H), 7.69 (d, J = 7.3 Hz, 2H), 8.19 (d, J = 8.1 Hz, 1H), 8.26 (s, 1H),
12.16 (br s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Indol-3-yl)methylene]-6-(benzyl-
oxy)-3-oxo-2,3-dihydrobenzofuran-7-yl}methyl)piperazine-1-
carboxylate (30). From 26 (120 mg, 0.319 mmol), the title
compound was obtained (86.4 mg, 53%) in the same manner as
described for 27.
HRMS (ESI+) calcd for (C22H22N3O2) [M + H]+ 360.1712; found
360.1736.
1H NMR (300 MHz, DMSO-d6) δ 1.37 (s, 9H), 2.48 (m, 4H), 3.30
(m, 4H), 3.81 (s, 2H), 5.34 (s, 2H), 7.10 (d, J = 8.8 Hz, 1H), 7.16−
7.27 (m, 3H), 7.34−7.46 (m, 3H), 7.50−7.55 (m, 3H), 7.72 (d, J = 8.8
tert-Butyl 4-[(6-Methoxy-3-oxo-2,3-dihydrobenzofuran-7-
yl)methyl]piperazine-1-carboxylate (23). A mixture of 16 (1.74
g, 5.00 mmol), MeOH (0.243 mL, 6.00 mmol), triphenylphosphine
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Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.20 (d, J = 2.9 Hz, 1H), 12.05 (br
s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Pyrrolo[3,2-c]pyridin-3-yl)-
methylene]-6-methoxy-3-oxo-2,3-dihydrobenzofuran-7-yl}-
methyl)piperazine-1-carboxylate (33). From 23 (46.8 mg, 0.129
mmol) and 5-azaindole-3-carbaldehyde (18.8 mg, 0.129 mmol), the
title compound was obtained (51.5 mg, 81%) in the same manner as
described for 31.
(Z)-2-[(1H-Indol-3-yl)methylene]-6-methoxy-7-(piperazin-1-
ylmethyl)benzofuran-3(2H)-one (6). To a suspension of 27 (78.3
mg, 0.160 mmol) in CH2Cl2 (3 mL) was added 4 N HCl in dioxane (3
mL) at room temperature. The mixture was stirred at the same
temperature for 2 h and concentrated in vacuo, then 3 mL of saturated
aqueous NaHCO3 was added. The precipitated yellow powder was
collected by filtration, washed with water, and dried in vacuo to afford
the title compound as a yellow powder (50.0 mg, 80%).
1H NMR (300 MHz, DMSO-d6) δ 2.44 (m, 4H), 2.66 (m, 4H),
3.72 (s, 2H), 3.94 (s, 3H), 7.00 (d, J = 8.8 Hz, 1H), 7.17 (m, 1H),
7.20−7.26 (m, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H),
8.14 (d, J = 8.0 Hz, 1H), 8.20 (s, 1H), 12.09 (br s, 1H).
HRMS (ESI+) calcd for (C23H24N3O3) [M + H]+ 390.1818; found
390.1845.
1H NMR (300 MHz, DMSO-d6) δ 1.36 (s, 9H), 2.47 (m, 4H), 3.30
(m, 4H), 3.76 (s, 2H), 3.95 (s, 3H), 7.03 (d, J = 8.8 Hz, 1H), 7.29 (s,
1H), 7.48 (d, J = 5.9 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 8.21 (s, 1H),
8.32 (d, J = 5.9 Hz, 1H), 9.54 (s, 1H), 12.29 (br s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Pyrrolo[3,2-b]pyridin-3-yl)-
methylene]-6-methoxy-3-oxo-2,3-dihydrobenzofuran-7-yl}-
methyl)piperazine-1-carboxylate (34). From 23 (49.7 mg, 0.137
mmol) and 4-azaindole-3-carbaldehyde (20.0 mg, 0.137 mmol), the
title compound was obtained (49.6 mg, 73%) in the same manner as
described for 31.
1H NMR (300 MHz, DMSO-d6) δ 1.36 (s, 9H), 2.47 (m, 4H), 3.30
(m, 4H), 3.80 (s, 2H), 3.95 (s, 3H), 7.02 (d, J = 8.8 Hz, 1H), 7.26 (dd,
J = 4.4 Hz, 8.0 Hz, 1H), 7.31 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.92
(dd, J = 1.5 Hz, 8.0 Hz, 1H), 8.39 (s, 1H), 8.48 (dd, J = 1.5 Hz, 4.4 Hz,
1H), 12.00 (br s, 1H).
(Z)-2-[(1H-Indol-3-yl)methylene]-6-ethoxy-7-(piperazin-1-
ylmethyl)benzofuran-3(2H)-one (7). From 28 (106 mg, 0.210
mmol), the title compound was obtained (65.3 mg, 77%) in the same
manner as described for 6.
1H NMR (300 MHz, DMSO-d6) δ 1.39 (t, J = 7.3 Hz, 3H), 2.46
(m, 4H), 2.67 (m, 4H), 3.74 (s, 2H), 4.20 (q, J = 7.3 Hz, 2H), 6.97 (d,
J = 8.8 Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H), 7.21−7.26 (m, 2H), 7.50 (d,
J = 7.3 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 8.15 (d, J = 7.3 Hz, 1H),
8.19 (s, 1H), 12.08 (br s, 1H).
(Z)-tert-Butyl 4-({2-[(1H-Indazol-3-yl)methylene]-6-methoxy-
3-oxo-2,3-dihydrobenzofuran-7-yl}methyl)piperazine-1-car-
boxylate (35). From 23 (181 mg, 0.500 mmol) and 1H-indazole-3-
carbaldehyde (73.1 mg, 0.500 mmol), the title compound was
obtained (171 mg, 69%) in the same manner as described for 31.
1H NMR (300 MHz, DMSO-d6) δ 1.35 (s, 9H), 2.46 (m, 4H), 3.29
(m, 4H), 3.74 (s, 2H), 3.97 (s, 3H), 7.05−7.08 (m, 2H), 7.26 (t, J =
7.3 Hz, 1H), 7.47 (t, J = 7.3 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.80 (d,
J = 8.8 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H), 13.86 (br s, 1H).
(Z)-2-[(1H-Pyrrolo[2,3-b]pyridin-3-yl)methylene]-6-methoxy-
7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one (10). To a sus-
pension of 31 (147 mg, 0.300 mmol) in CH2Cl2 (14 mL) was added 4
N HCl in dioxane (14 mL) at room temperature. The mixture was
stirred at the same temperature for 2 h and concentrated in vacuo, and
then 14 mL of saturated aqueous NaHCO3 was added to the residue.
The mixture was extracted with EtOAc (×5), and the combined
organic layers were dried over Na2SO4. Evaporation of the solvent
afforded the title compound (14.6 mg, 12%).
HRMS (ESI+) calcd for (C24H26N3O3) [M + H]+ 404.1974; found
404.1968.
(Z)-2-[(1H-Indol-3-yl)methylene]-7-(piperazin-1-ylmethyl)-6-
propoxybenzofuran-3(2H)-one (8). From 29 (70.5 mg, 0.136
mmol), the title compound was obtained (30.6 mg, 53%) in the same
manner as described for 6.
1H NMR (300 MHz, DMSO-d6) δ 1.05 (t, J = 7.3 Hz, 3H), 1.73−
1.85 (m, 2H), 2.46 (m, 4H), 2.66 (m, 4H), 3.72 (s, 2H), 4.11 (t, J =
5.9 Hz, 2H), 6.97 (d, J = 8.8 Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H), 7.21−
7.26 (m, 2H), 7.50 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 8.15
(d, J = 8.1 Hz, 1H), 8.19 (s, 1H), 12.08 (br s, 1H).
HRMS (ESI+) calcd for (C25H28N3O3) [M + H]+ 418.2131; found
418.2175.
(Z)-2-[(1H-Indol-3-yl)methylene]-6-(benzyloxy)-7-(piperazin-
1-ylmethyl)benzofuran-3(2H)-one (9). From 30 (123 mg, 0.217
mmol), the title compound was obtained (72.0 mg, 71%) in the same
manner as described for 6.
1H NMR (300 MHz, DMSO-d6) δ 2.46 (m, 4H), 2.72 (m, 4H),
3.72 (s, 2H), 3.95 (s, 3H), 7.02 (d, J = 8.8 Hz, 1H), 7.20−7.24 (m,
2H), 7.74 (d, J = 8.8 Hz, 1H), 8.27 (s, 1H), 8.35 (d, J = 4.4 Hz, 1H),
8.68 (d, J = 8.0 Hz, 1H).
1H NMR (300 MHz, DMSO-d6) δ 2.46 (m, 4H), 2.68 (m, 4H),
3.76 (s, 2H), 5.33 (s, 2H), 7.09 (d, J = 8.8 Hz, 1H), 7.16−7.27 (m,
3H), 7.33−7.45 (m, 3H), 7.49−7.56 (m, 3H), 7.71 (d, J = 8.8 Hz,
1H), 8.16 (d, J = 8.1 Hz, 1H), 8.20 (s, 1H), 12.08 (br s, 1H).
HRMS (ESI+) calcd for (C29H28N3O3) [M + H]+ 466.2131; found
466.2120.
HRMS (ESI+) calcd for (C22H23N4O3) [M + H]+ 391.1770; found
391.1783.
(Z)-2-[(1H-Pyrrolo[2,3-c]pyridin-3-yl)methylene]-6-methoxy-
7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one (11). To a sus-
pension of 32 (52.1 mg, 0.106 mmol) in CH2Cl2 (5 mL) was added 4
N HCl in dioxane (5 mL) at room temperature. The mixture was
stirred at the same temperature for 2 h and concentrated in vacuo, and
then 5 mL of saturated aqueous NaHCO3 was added to the residue.
The mixture was extracted with CHCl3 (×5), and the combined
organic layers were dried over Na2SO4. Evaporation of the solvent
afforded the title compound (13.1 mg, 31%).
(Z)-tert-Butyl 4-({2-[(1H-Pyrrolo[2,3-b]pyridin-3-yl)-
methylene]-6-methoxy-3-oxo-2,3-dihydrobenzofuran-7-yl}-
methyl)piperazine-1-carboxylate (31). A mixture of 23 (478 mg,
1.32 mmol), 7-azaindole-3-carbaldehyde (193 mg, 1.32 mmol), and
piperidine (90.3 mg, 1.06 mmol) in MeOH (5 mL) was stirred at 60
°C for 2 h, then cooled to room temperature. The precipitate was
collected by filtration to afford the title compound (220 mg, 33%).
1H NMR (300 MHz, DMSO-d6) δ 1.36 (s, 9H), 2.46 (m, 4H), 3.30
(m, 4H), 3.76 (s, 2H), 3.95 (s, 3H), 7.02 (d, J = 8.8 Hz, 1H), 7.23 (dd,
J = 4.4 Hz, 8.0 Hz, 1H), 7.24 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H), 8.27 (s,
1H), 8.35 (dd, J = 1.5 Hz, 4.4 Hz, 1H), 8.66 (dd, J = 1.5 Hz, 8.0 Hz,
1H), 12.45 (br s, 1H).
1H NMR (300 MHz, DMSO-d6) δ 2.54 (m, 4H), 2.81 (m, 4H),
3.78 (s, 2H), 3.96 (s, 3H), 7.03 (d, J = 8.8 Hz, 1H), 7.27 (s, 1H), 7.75
(d, J = 8.8 Hz, 1H), 8.20 (d, J = 5.1 Hz, 1H), 8.27 (d, J = 5.1 Hz, 1H),
8.36 (s, 1H), 8.86 (s, 1H).
HRMS (ESI+) calcd for (C22H23N4O3) [M + H]+ 391.1770; found
391.1723.
(Z)-2-[(1H-Pyrrolo[3,2-c]pyridin-3-yl)methylene]-6-methoxy-
7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one (12). From 33
(36.9 mg, 0.0752 mmol), the title compound was obtained (18.2 mg,
61%) in the same manner as described for 11.
(Z)-tert-Butyl 4-({2-[(1H-Pyrrolo[2,3-c]pyridin-3-yl)-
methylene]-6-methoxy-3-oxo-2,3-dihydrobenzofuran-7-yl}-
methyl)piperazine-1-carboxylate (32). From 23 (61.6 mg, 0.170
mmol) and 6-azaindole-3-carbaldehyde (24.8 mg, 0.170 mmol), the
title compound was obtained (68.6 mg, 82%) in the same manner as
described for 31.
1H NMR (300 MHz, DMSO-d6) δ 2.55 (m, 4H), 2.71 (m, 4H),
3.73 (s, 2H), 3.96 (s, 3H), 7.03 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.48
(d, J = 5.9 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 8.23 (s, 1H), 8.32 (d, J =
5.9 Hz, 1H). 9.52 (s, 1H).
1H NMR (300 MHz, DMSO-d6) δ 1.36 (s, 9H), 2.47 (m, 4H), 3.30
(m, 4H), 3.78 (s, 2H), 3.95 (s, 3H), 7.02 (d, J = 8.8 Hz, 1H), 7.26 (s,
1H), 7.74 (d, J = 8.8 Hz, 1H), 8.17 (d, J = 5.9 Hz, 1H), 8.26 (d, J = 5.9
Hz, 1H), 8.35 (s, 1H), 8.85 (s, 1H), 12.44 (br s, 1H).
HRMS (ESI+) calcd for (C22H23N4O3) [M + H]+ 391.1770; found
391.1785.
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(Z)-2-[(1H-Pyrrolo[3,2-b]pyridin-3-yl)methylene]-6-methoxy-
7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one (13). From 34
(33.0 mg, 0.0673 mmol), the title compound was obtained (19.9 mg,
75%) in the same manner as described for 10.
boiled for 5 min, and the lysates were applied to 12.5% polyacrylamide
gels (Cosmobio) and separated by SDS polyacrylamide gel electro-
phoresis. The gels were blotted onto polyvinylidene difluoride
membrane (BIO-RAD, Hercules, CA), and the membrane was
incubated with the primary antibody, Total BAD, Phospho-BAD
(Ser-112), or Phospho-BAD (Ser-136) (Cell Signaling Technology,
Danvers, MA) for 16 h at 4 °C. The membrane was washed with TBS-
T and incubated with Anti-Rabbit IgG HRP conjugates (GE
Healthcare, Buckinghamshire, UK) for 1 h at room temperature.
Chemiluminescence signals of protein obtained on addition of ECL
Plus (GE Healthcare) reagent were analyzed with an ImageQuant LAS
4000 (GE Healthcare).
Apoptosis and Cell-Cycle Analysis. MV4−11 was maintained in
Iscove’s modified Dulbecco’s medium with 10% FBS and penicillin
(5000 U/mL)−streptomycin (5 mg/mL) (Sigma) at 37 °C in 5%
CO2. MV4−11 cells (1 × 105 cells/mL) were cultured with the
compounds for 48 h and then harvested. Cells were stained with
ApoAlert AnnexinV (Clontech 630109) for apoptosis analysis and
Cycle TEST PLUS DNA Reagent Kit (Becton, Dickinson) for cell
cycle analysis according to the manufacturers’ instructions. Cells were
analyzed with a flow cytometer (CaliburTM, Becton, Dickinson and
Company, Franklin Lakes, NJ). Data analysis was performed using
FlowJo software (Tree Star, Ashland, Oregon).
1H NMR (300 MHz, DMSO-d6) δ 2.45 (m, 4H), 2.66 (m, 4H),
3.74 (s, 2H), 3.94 (s, 3H), 7.01 (d, J = 8.8 Hz, 1H), 7.26 (dd, J = 4.4
Hz, 8.1 Hz, 1H), 7.31 (s, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.91 (dd, J =
1.5 Hz, 8.1 Hz, 1H), 8.39 (s, 1H), 8.48 (dd, J = 1.5 Hz, 4.4 Hz, 1H).
HRMS (ESI+) calcd for (C22H23N4O3) [M + H]+ 391.1770; found
391.1770.
(Z)-2-[(1H-Indazol-3-yl)methylene]-6-methoxy-7-(piperazin-
1-ylmethyl)benzofuran-3(2H)-one (14). From 35 (125 mg, 0.255
mmol), the title compound was obtained (77.7 mg, 78%) in the same
manner as described for 10.
1H NMR (400 MHz, DMSO-d6) δ 2.44 (m, 4H), 2.67 (m, 4H),
3.68 (s, 2H), 3.96 (s, 3H), 7.04 (d, J = 8.8 Hz, 1H), 7.07 (s, 1H), 7.25
(t, J = 7.3 Hz, 1H), 7.47 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.78 (d, J =
8.8 Hz, 1H), 8.62 (d, J = 8.8 Hz, 1H).
HRMS (ESI+) calcd for (C22H23N4O3) [M + H]+ 391.1770; found
391.1740.
6-Azaindole-3-carbaldehyde. A mixture of 6-azaindole (118 mg,
1.00 mmol), hexamethylenetetramine (210 mg, 1.50 mmol), and acetic
acid (0.6 mL) in H2O (1.2 mL) was heated at 120 °C for 6 h, then
cooled to room temperature and basified by the addition of saturated
aqueous NaHCO3. After extraction with AcOEt (×3), the combined
organic layers were washed with brine, dried over MgSO4, and
concentrated. The resulting residue was washed with CH2Cl2 to afford
the title compound (47.9 mg, 32%).
ADMET Study. Metabolic stability assay with liver microsomes and
hepatocytes, CYP inhibition assay, and hERG inhibition assay were
carried out at Cerep (http://www.cerep.fr/Cerep/Users/).
PIM1 Crystallization. The kinase domain (residues 29−313) of
human PIM-1 was synthesized in an Escherichia coli cell-free system,
using the dialysis method.66−68 The protein was purified as described
previously43 and dialyzed against buffer containing 20 mM Tris-HCl
(pH 8.0), 200 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 5 mM
DTT, then concentrated to 12 mg/mL. Crystallization was carried out
under conditions previously reported by Jacobs et al.69 Briefly, the
protein was crystallized by the hanging drop vapor diffusion method at
20 °C, and the compound was allowed to soak into the crystal for 24 h.
Data for the PIM1-14 complex were collected on beamline BL26B2,
SPring-8, Harima, Japan. The crystals belonged to the spacegroup P65,
with cell dimensions of a, b, c = 98.28, 98.28, 80.64, respectively, and α
= β = 90° and γ = 120°. Statistics are presented in Table S2,
Supporting Information.
1H NMR (300 MHz, DMSO-d6) δ 7.99 (d, J = 5.9 Hz, 1H), 8.32 (d,
J = 5.9 Hz, 1H), 8.51 (s, 1H), 8.87 (s, 1H), 10.00 (s, 1H), 12.57 (br s,
1H).
PIM1 Inhibition Assay. Enzyme inhibition was examined by
means of electrophoretic mobility shift assay. An aqueous suspension
containing 1.5 μM FL-Peptide 20, 5-FAM-RSRHSSYPAGT-CONH2
(Caliper Life Science, Hopkinton, MA), 30 μM ATP, 33 ng/mL
PIM1,43 50 mM Hepes (pH 7.4), 10 mM MgCl2, 1 mM DTT, 1%
Protease Inhibitor Cocktail Set V (Merck, Darmstadt, Germany), 1%
Phosphatase Inhibitor Cocktail Set III (Merck), 0.01% Brij-35, and a
test compound (in 5% DMSO) was incubated for 2 h at room
temperature, and the reaction was stopped by addition of 140 mM
EDTA. Phosphorylated and unphosphorylated peptides obtained were
separated and detected with a Lab-Chip EZ Reader II (Caliper Life
Science).
PIM1 Structure Solution. The resolution was 2.08 Å. The data
were processed and integrated with the Mosflm70 program and scaled
with the Scala71 program. Molecular replacement was performed with
Phaser,72 using PDB code 3A99 as the search model, with the peptide
removed. There is one molecule in the asymmetric unit. Refinement
was performed using Refmac (CCP4),73 and model building was
accomplished with COOT.74 The electron density of the compound
was immediately evident in the Fo − Fc and 2Fo − Fc maps, and the
compound was built using Monomer Library Sketcher (CCP4). The
ligand was fitted with 100% occupancy and refined with an average B-
factor of 24.4. The average temperature factor for the protein is 28.26
for the main chain and 30.16 for the side chains. The final R-factor and
Rfree values were 17.35 and 20.03, respectively. Statistics for the
complex are presented in Table S2, Supporting Information.
Kinase Selectivity Assay. Kinase selectivity was evaluated against
a panel of 20 protein tyrosine kinases and 30 serine/threonine kinases
at CarnaBiosciences (http://www.carnabio.com). The ATP concen-
tration used was approximately equal to the Km value for each kinase.
Each compound was tested at a concentration 50-fold higher than its
IC50 value in the PIM1-inhibitory assay (1, 22500 nM; 3, 600 nM; 6,
250 nM; 10, 200 nM; 14, 300 nM). For these experiments, the IC50
values for PIM1 of each compound were determined at CarnaBio-
sciences. Kinase inhibition was examined by electrophoretic mobility
shift assay except for JNK2, MAP2K1, MLK1, and RAF1, which were
examined by ELISA.
Cell Growth Inhibition Assay. MV4−11 (ATCC, Manassas, VA)
and WI-38 (RIKEN BioResource Center, Japan) cells were cultured in
Iscove’s modified Dulbecco’s medium (Invitrogen, Carlsbad, CA)
containing 10% fetal bovine serum (FBS, Invitrogen) and in minimum
essential medium (Invitrogen) containing 10% FBS, respectively, at 37
°C in 5% CO2. The aqueous suspension containing MV4−11 (2 × 105
cells/mL) or WI-38 (3 × 104 cells/mL) was precultured for 24 h,
followed by incubation for 48 h in the presence of the test compound
(in 0.5% DMSO). Cell growth inhibition was measured with a Cell
Counting Kit-8 (Dojindo, Japan) according to the manufacturer’s
instruction. Absorbance at 450 nm was monitored with PHERAstar
(BMG Labtech, Germany).
ASSOCIATED CONTENT
■
S
* Supporting Information
Raw data of the kinase selectivity study and crystallographic
data collection and refinement statistics. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
Western Blotting. MV4−11 cells precultured for 24 h were
incubated in the presence of the test compound (in 0.5% DMSO) for
1 h. The cells were washed with PBS and dispersed in β-ME Sample
Treatment for Tris SDS (Cosmobio, Japan). The suspension was
The atomic coordinates and structure factors have been
deposited in the Protein Data Bank, www.rcsb.org (PDB ID
codes 3UMW and 3UMX).
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(9) Zhang, Y.; Wang, Z.; Li, X.; Magnuson, N. S. Pim kinase-
dependent inhibition of c-Myc degradation. Oncogene 2008, 27, 4809−
4819.
(10) Zippo, A.; De Robertis, A.; Serafini, R.; Oliviero, S. PIM1-
dependent phosphorylation of histone H3 at serine 10 is required for
MYC-dependent transcriptional activation and oncogenic trans-
formation. Nature Cell Biol. 2007, 9, 932−944.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-3-5841-4850. Fax: +81-3-5841-4855. E-mail:
tlong@mol.f.u-tokyo.ac.jp. Address: Graduate School of
Pharmaceutical Sciences, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
■
(11) Zhang, Y.; Wang, Z.; Magnuson, N. S. Pim-1 kinase-dependent
phosphorylation of p21Cip1/WAF1 regulates its stability and cellular
localization in H1299 cells. Mol. Cancer Res. 2007, 5, 909−922.
(12) Morishita, D.; Katayama, R.; Sekimizu, K.; Tsuruo, T.; Fujita, N.
Pim kinases promote cell cycle progression by phosphorylating and
down-regulating p27Kip1 at the transcriptional and posttranscriptional
levels. Cancer Res. 2008, 68, 5076−5085.
(13) Mochizuki, T.; Kitanaka, C.; Noguchi, K.; Muramatsu, T.; Asai,
A.; Kuchino, Y. Physical and functional interactions between Pim-1
kinase and Cdc25A phosphatase. J. Biol. Chem. 1999, 274, 18659−
18666.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Targeted Proteins Research
Program from the Ministry of Education, Culture, Sports,
Science and Technology of Japan. L.P. is supported by a
National Health and Medical Research Council of Australia
(NHMRC). C.J. Martin Overseas Postdoctoral Fellowship. We
thank Mio Inoue, Noboru Ohsawa, and Tahako Terada for
preparation of the PIM1 expression plasmid. We also thank the
staff at beamline BL26, Spring-8 Harima, Japan, for their
assistance with crystallographic data collection.
(14) Bachmann, M.; Kosan, C.; Xing, P. X.; Montenarh, M.;
Hoffmann, I.; Moroy, T. The oncogenic serine/threonine kinase Pim-1
̈
̈
directly phosphorylates and activates the G2/M specific phosphatase
Cdc25C. Int. J. Biochem. Cell Biol. 2006, 38, 430−443.
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ABBREVIATIONS USED
■
PIM, proviral integration site in Moloney murine leukemia
virus; BAD, Bcl-2 antagonist of call death; CDC25, cell division
cycle 25; CXCR4, CXC chemokine receptor 4; BCRP, breast
cancer resistance protein; FLT3, Fms-like tyrosine kinase 3;
PDGFα, platelet-derived growth factor receptor α; SYK, spleen
tyrosine kinase; GSK3β, glycogen synthase kinase 3 β; EGFR,
epidermal growth factor receptor; KDR, kinase insert domain
receptor; MLK1, mixed-lineage kinase 1; MST1, mammalian
homologue Ste20-like kinase 1; DAPK1, death-associated
protein kinase 1; PKCα, protein kinase C α; PKD2, protein
kinase D2; ROCK1, Rho-associated, coiled-coil containing
protein kinase 1
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Linders, K.; McWhir, J.; Berns, A.; Hooper, M. In vivo analysis of Pim-
1 deficiency. Nucleic Acids Res. 1993, 21, 4750−4755.
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