Design and Synthesis of Potent and Selective PIM Kinase Inhibitors by Targeting Unique Structure of ATP-Binding Pocket

Article abstract of DOI:10.1021/acsmedchemlett.6b00518

In the development of kinase inhibitors, one of the major concerns is selectivity. An effective strategy to achieve high selectivity is to utilize structural differences among kinases to inform inhibitor design. Here, we set out to improve the PIM (proviral integration site for Moloney murine leukemia virus) kinase-inhibitory selectivity of our previously reported 7-azaindole derivative 2, which has promising ADMET properties, by targeting a unique bulge in the ATP-binding pocket. 6-Substituted 7-azaindoles, especially the 6-chlorinated derivatives, proved to be potent and selective PIM kinase inhibitors and appear to be promising lead compounds for future drug discovery.

Full text of DOI:10.1021/acsmedchemlett.6b00518

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Letter
Design and Synthesis of Potent and Selective PIM Kinase
Inhibitors by Targeting Unique Structure of ATP–binding Pocket
Hirofumi Nakano, Tsukasa Hasegawa, Hirotatsu Kojima, Takayoshi Okabe, and Tetsuo Nagano
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00518 • Publication Date (Web): 03 Apr 2017
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Design and Synthesis of Potent and Selective PIM Kinase Inhibitors
by Targeting Unique Structure of ATP–binding Pocket
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Hirofumi Nakano, Tsukasa Hasegawa, Hirotatsu Kojima, Takayoshi Okabe, and Tetsuo Nagano*
Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
KEYWORDS: PIM kinase, kinase inhibitor, kinase selectivity, structure–based drug design, ADMET
ABSTRACT: In the development of kinase inhibitors, one of the major concerns is selectivity. An effective strategy to
achieve high selectivity is to utilize structural differences among kinases to inform inhibitor design. Here, we set out to
improve the PIM (proviral integration site for Moloney murine leukemia virus) kinase-inhibitory selectivity of our previ-
ously reported 7-azaindole derivative 2, which has promising ADMET properties, by targeting a unique bulge in the ATP-
binding pocket. 6-Substituted 7-azaindoles, especially the 6-chlorinated derivatives, proved to be potent and selective
PIM kinase inhibitors, and appear to be promising lead compounds for future drug discovery.
5
–7
Selective kinase inhibitors are useful both as tools to
probe signal cascades and as candidate drugs to treat dis-
eases. Kinases form a large family in the human proteome
gen donor. The lack of this hydrogen donor, a signifi-
cant structural difference from other kinases, offers an
opportunity to design PIM-selective inhibitors.
1
(
>500 members), and regulate cellular events by phos-
The PIM kinase family consists of three constitutively
8
–10
phorylating a wide range of substrates. Over-activation or
over-expression of kinases may lead to excessive phos-
phorylation of their substrates, which in turn may lead to
hyper-activation of signal cascades and physiological
active proto-oncogenic serine/threonine kinases.
PIM
kinases share substrates such as BAD, p21, p27 and MYC,
and regulate apoptosis, the cell cycle and cell survival.
Typically, PIM1 is mainly overexpressed in acute myeloid
leukemia and PIM2 plays a major role in multiple myelo-
2
changes such as tumorigenesis and inflammation. Be-
1
1
cause kinases share ATP as a common substrate, devel-
opment of ATP-competitive inhibitors (“type I” inhibi-
tors) is a general strategy to inhibit over-activated kinases.
However, the ATP-binding pockets of kinases are general-
ly similar, making the design of selective ATP-competitive
kinase inhibitors challenging. To achieve high selectivity,
it has been proposed to target the non-ATP-competitive
pocket of the inactive form of kinases (“type II” inhibi-
tors) or the allosteric pocket (“type III and IV” inhibitors),
ma. PIM3 is reported to be overexpressed in endoderm-
1
2
derived organ cancers, including pancreatic cancer. PIM
triple knockout mice are reported to be healthy, although
1
3
their size is smaller than usual. Various PIM inhibitors
1
4–16
have been reported to date,
however, none of them
has been marketed so far. SGI-1776 is a representative
first-generation PIM inhibitor, which had been under
1
7–19
clinical trials for leukemia and prostate cancer.
While
most of the first-generation PIM inhibitors are PIM1-
selective, there is currently great interest in the potential
of pan-PIM inhibitors to treat cancer, because the three
3
because these pockets are not shared among kinases.
However, targeting the active conformation is the only
available strategy to develop inhibitors of kinases that do
not form either inactive conformations or allosteric pock-
ets.
20
PIM kinases are reported to function redundantly. Rep-
resentative pan-PIM inhibitors are AZD1208 from Astra-
21,22
23
Zeneca,
and PIM447 from Novartis. Clinical trials of
Typically, ATP-competitive type-I kinase inhibitors
form a pair of hydrogen bonds in the ATP-binding pocket
of kinases, in a similar way to ATP. The hydrogen-
PIM447 are underway.
4
bonding partners are a carbonyl O (acceptor) and an am-
ide NH (donor) pair in the hinge region, which connects
the N-lobe and the C-lobe of the protein.
PIM (proviral integration site for Moloney murine leu-
kemia virus) kinases are the only kinases that cannot form
the canonical bidentate hydrogen bonds with ATP and
ATP-competitive inhibitors, because they have a proline
residue in the hinge region at the position of the hydro-
Figure 1. Structures of compounds 1 and 2.
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In previous study, we reported a 2-azaindole (indazole)
derivative 1 and a 7-azaindole derivative 2 as potent PIM1
1
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24
inhibitors (Figure 1). A schematic representation of 1-
PIM1 binding is depicted in Figure 2. Compound 1 was
confirmed to be highly PIM1-selective, whereas 2 was
poorly selective. The difference between 1 and 2 is the
position of the nitrogen atom in the indole ring. In 1, the
highly selective PIM1 inhibitor, there is no hydrogen
bond-accepting nitrogen at the 7-position of the ring,
which is proximal to the gatekeeper + 3 residue, the ca-
Figure 3. Basis of our plan for improving the kinase selectivi-
24
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nonical hydrogen donor of general kinases (Figure 2).
ty of 2. Substituents at the 6-position of the 7-azaindole ring
would interfere with hydrogen bonding between the inhibi-
tor and off-target kinases.
On the other hand, the 7-position nitrogen in 2 can ac-
cept hydrogen from off-target kinases, which lowers the
selectivity. However, 2 showed more favorable in vitro
ADMET properties, such as higher aqueous solubility and
membrane permeability, and lower hERG inhibition (Ta-
ble 3, vide infra). Therefore, we set out to improve the
kinase selectivity of 2, which is promising in terms of
ADMET properties.
Table 1. Effect of substituents at the 6-position of the
7-azaindole ring on PIM1-inhibitory potency
R2
HN
N
N
NH
R1
O
O
1
2
a
b
c
Comp.
R
R
IC50 (nM)
cLogP
2.8
3.3
LLE
6.3
5.9
5.1
d
2
3
OMe
H
0.84
Me
Et
0.64
1.1
4
5
3.9
4.4
4.9
4.9
3.1
n-Pr
n-Bu
Ph
F
4.5
2.1
3.9
3.8
2.9
6.4
5.8
6.2
5.4
4.3
6.0
5.6
6
7
16
Figure 2. Schematic representation of the complex of com-
pound 1 and PIM1 kinase (PDB ID: 3UMW). Dashed arrows
indicate hydrogen bonds between compound 1 and PIM1.
Pro123, the non-hydrogen donor in the gatekeeper + 3 posi-
tion, is colored red. Glu124 and Pro125, two inserted residues,
are colored blue.
8
9
10
0.28
0.41
2.0
3.8
16
Cl
3.6
2.5
OH
H
11
Me
Et
3.0
3.5
12
To improve the kinase selectivity of the 7-azaindole de-
rivative, we targeted structurally distinct features in the
13
F
1.8
2.7
3.3
5
–7
14
Cl
1.4
ATP-binding pocket of PIM kinases. First, PIM kinases
are the only kinases that have a proline at the gatekeeper
+ 3 position and cannot donate hydrogen, as mentioned
above. Second, in contrast to most kinases, PIM kinases
have one or two extra amino acids inserted downstream
of the ATP-binding pocket to create a bulge at the end of
the hinge region, which should accommodate substitu-
ents at the 6-position of the heterocyclic ring (Figure 2).
We hypothesized that introduction of a suitable 6-
substituent in the 7-azaindole ring would be sterically
tolerated by PIM, but not by off-target kinases, and hy-
drogen bonding between the 7-position nitrogen in the 7-
azaindole ring and off-target kinases would be blocked,
thereby increasing the selectivity for PIM kinase. There-
fore, we planned to introduce substituents at the 6-
position of the 7-azaindole ring in order to improve the
poor kinase selectivity of 2 (Figure 3).
a
All compounds were evaluated in the same experiment (n
= 4). [ATP] = 30 ꢀM.
b
Calculated logarithm of the octanol/water distribution
coefficient using ChemBioDraw Ultra Version 13.0.
c
d
LLE = –log(IC50) – cLogP. In a previous study (ref. 24),
the IC50 was reported as 2 nM.
To test our hypothesis, we introduced various substitu-
ents at the 6-position of the 7-azaindole ring, and exam-
ined whether or not the resulting compounds retained
PIM1-inhibitory potency. Initially, we introduced neutral
alkyl and aromatic groups at the 6-position of the 7-
azaindole ring in 2 as steric shields, because such neutral
groups would not form unwanted extra hydrogen bonds
and/or polar interactions with off-targets (Table 1, com-
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ing and electronegative chlorine substituent, showed a
pounds 3–7). As we expected, the introduction of a small
1
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8
9
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6
methyl group did not affect the potency, while the intro-
duction of a bulky phenyl ring reduced the potency. Un-
expectedly, longer alkyl groups such as ethyl, n-propyl
and n-butyl groups also did not reduce the potency, com-
pared with the unsubstituted analogue 2, although LLE
very clean selectivity profile, as expected. Indeed, com-
pound 9 inhibited only three off-target kinases by more
than 50% (PDGFRα: 61%; DAPK1: 80%; PKD2: 62%) while
it completely inhibited PIM1 (99%; IC50 = 0.49 nM) and
PIM3 (>99%; IC50 = 0.40 nM). Moreover, 14, a less lipo-
philic analog of 9, showed an even cleaner profile; no off-
targets were inhibited potently (>75%), and only two off-
target kinases were inhibited by more than 50% by 14
(PDGFRα: 63%; DAPK1: 57%). PKD2 was not inhibited by
14 (30% inhibition). Importantly, 14 completely inhibited
PIM1 (99%; IC50 = 1.3 nM) and PIM3 (>99%; IC50 = 0.88
nM). The hinge of PDGFRα and DAPK1 are one amino
acid residue shorter than that of PIM, therefore we could
not find a structural rationale why 14 still inhibits the two
off-targets. To our knowledge, PDGFRα and DAPK1 are
not common off-targets of PIM inhibitors. Further cell–
based analysis may clarify whether the two off-targets are
inhibited by 14 in cell, and whether inhibition of the two
off-targets has any influence on the development of PIM
inhibitor. In addition to their clean profiles, 9 and 14 in-
hibited PIM2 to the extents of 41% and 58%, respectively,
whereas 3 and 4, 6-alkylated derivatives, inhibited PIM2
by only 25% and 23%.
25
scores were significantly decreased with longer alkyl
groups. Because there is only a small hydrophobic space
in the pocket, there could be unfavorable steric clash be-
tween the longer alkyl chains and the protein. We specu-
lated that the sub-optimal binding modes of the deriva-
tives due to unfavorable steric clash of alkyl chains were
compensated by hydrophobic interaction of the flexible P-
loop and the methoxy group on the benzofuranone core
0
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2
6
(
Figure 2). Indeed, when the methoxy group was re-
placed with a hydroxyl group to eliminate the hydropho-
bic interaction, a 6-methyl substituent was tolerated, but
a 6-ethyl substituent, only one carbon atom larger, was
not tolerated (compounds 10–12). Further, we introduced
small halogen atoms in the same position. Fluorine or
chlorine substitution did not affect the PIM1-inhibitory
potency, irrespective of the substituents on the benzo-
furanone core (compounds 8, 9, 13 and 14).
We then confirmed that the poor kinase selectivity of 2
was significantly improved by the introduction of substit-
uents at the 6-position of the azaindole ring, as we had
hypothesized (Figure 4 and Table S1, Supporting Infor-
mation). The selectivity of each inhibitor was evaluated at
200 nM concentration against a panel of 52 kinases at
Carna Biosciences. In the case of 2, nine off-target kinases
were inhibited by more than 50%, in addition to PIM1 and
3
. Among the nine off-targets, seven were potently inhib-
ited (>75%). In particular, PKCα and ROCK1 were com-
pletely inhibited (right-side of the graph) by 2. Compared
with 2, compounds 3 and 4, having sterically demanding
short-alkyl substituents, showed improved selectivity pro-
files. Six off-targets were inhibited by more than 50% by
the methylated derivative 3, and only four off-targets were
inhibited by more than 50% by the ethylated derivative 4.
One kinase (DAPK1: 91%) was potently inhibited by 3, and
two kinases (DAPK1: 77% and PKD2: 90%) were potently
inhibited by 4. It is noteworthy that PKCα and ROCK1,
which are two major off-targets of 2, were inhibited by 3
and 4 with significantly reduced potency (PKCα: ≈ 10%
inhibition and ROCK1: ≈ 50% inhibition). Because halo-
gens are electron-withdrawing atoms, it is possible that
they reduce the hydrogen-accepting ability of the 7-
nitrogen atom sterically as well as electronically, thereby
significantly improving the kinase selectivity. Indeed, 8,
with small but electronegative fluorine substitution, also
showed an improved selectivity profile compared with 2.
Six off-targets were inhibited by more than 50%, and
three of them were potently inhibited (PDGFRα: 79%;
DAPK1: 97%; ROCK1: 94%) by 8. PKD2, the major off-
target of 4, was only modestly (59%) inhibited by 8. These
results show that the poor kinase selectivity profile of 2
could be greatly improved by introducing sterically de-
manding substituents (3 and 4) or an electronegative sub-
stituent (8). Gratifyingly, 9, having the sterically demand-
Figure 4. Kinase selectivity profiles of compounds 2, 3, 4, 8, 9
and 14. Experiments were carried out at Carna Biosciences.
All compounds were tested against a panel of 52 kinases at
200 nM concentration. In each graph, the x-axis shows %in-
hibition of each kinase. Red bars represent %inhibition of
PIM1, and blue bars represent %inhibition of PIM2. Black
bars next to the blue bars represent %inhibition of PIM3.
Because it seemed probable that substituents at the 6-
position of the 7-azaindole ring would influence not only
the kinase selectivity but also the inhibitory potency to-
ward PIM2, we further determined the IC50 values of our
compounds for PIM2 (Table 2). For comparison, the IC50
value of the indazole derivative 1, a selective PIM1 inhibi-
24
tor, is also shown in Table 2. Irrespective of the substit-
uents on the benzofuranone core, 9 and 14 (the chlorinat-
ed derivatives) achieved a significant increase of PIM2
inhibition, as expected from the results of the kinase pan-
el study. Under the assay conditions used, the IC50 values
of 9 and 14 for PIM2 were 140 nM. Therefore, 9 and 14 are
about three times as potent as the non-substituted ana-
logues 2 and 10, and about eight times as potent as the
indazole derivative 1. Glu124 and Val126 in PIM1 are re-
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Page 4 of 7
o
placed by Leu and Ala in PIM2, which may change lipo-
philicity and steric accessibility of the available space in
out in the presence of piperidine in MeOH at 60 C, and
the Boc protective group was removed under acidic con-
ditions to obtain the final products 3–6, and 8–14. The
Knoevenagel product obtained from 6-chloro-7-
azaindole-3-carbaldehyde 18g was reacted with phenyl-
boronic acid under Suzuki coupling conditions and the
Boc protective group was removed to yield 6-phenyl-7-
azaindole derivative 7.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
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5
6
26–28
the pocket.
However, improvement of PIM2 inhibition
by the chlorine substituent cannot be explained simply in
terms of lipophilic and steric effects, because the alkylat-
ed derivatives 3, 4 and 11 did not achieve any noticeable
gain in PIM2-inhibitory potency compared with their
non-substituted analogues 2 and 10. These results indi-
cate that the space utilized by the 6-chloro moiety of the
7
-azaindole ring could be a key to the design of other po-
a
Scheme 1. Synthesis of compounds 3–14
0
1
2
3
4
5
6
7
8
9
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9
0
tent and selective pan-PIM kinase inhibitors in the future.
The in vitro ADMET properties are summarized in Ta-
ble 3. Compared with indazole analogue 1, the 7-azaindole
analogue 2 shows a better in vitro ADMET profile, with
higher aqueous solubility, higher membrane permeability
and lower hERG inhibition, although it is a non-selective
kinase inhibitor. Compound 3, the 6-methylated deriva-
tive, retained acceptable solubility and a low hERG inhibi-
tion profile, but showed poor membrane permeability. At
present, we have no idea why the membrane permeability
is decreased so severely as a result of simple methylation.
Compound 9, the 6-chloro derivative of 2, was also
scarcely membrane-permeable, and showed very poor
aqueous solubility compared with 2. The lower solubility
might be due to higher lipophilicity of 9. Compound 14,
the less lipophilic phenolic derivative of 9, showed better
aqueous solubility, as well as greater membrane permea-
bility. A plausible explanation of the recovery of permea-
bility is the formation of an intra-molecular hydrogen
bond between phenol and piperazine nitrogen. Overall, 14
showed superior ADMET properties to 1, including lower
hERG inhibition and higher membrane permeability in
Caco-2 cells, and had better kinase selectivity than 2.
To further evaluate the potential of 14, cell–based assay
was carried out with a leukemia cell line MOLM–16,
which was used to evaluate the representative pan-PIM
a
21
Reagents and conditions: (a) trimethylboroxine or alkyl-
boronic acid, PdCl (dppf), K CO , 1,4-dioxane, 140 C or
160 C, microwave, 35–66%; (b) hexamethylenetetramine,
AcOH/H O, 120 C, 35–99%; (c) piperidine, MeOH, 60 C,
inhibitor AZD1208 in the literature (Table 4). In MOLM–
6 growth inhibition assay, IC50 of 14 was 200 nM, which
o
2
2
3
1
o
was weaker than that of AZD1208, but stronger than that
of SGI-1776, the representative first-generation PIM inhib-
itor. Reflecting the clean kinase selectivity profile, 14
scarcely inhibited the growth of WI–38, a human diploid
lung fibroblast cell line, which was used as a surrogate for
general toxicity.
o
o
2
3
0%–quant.;(d) TFA, CH Cl , rt, 32–89%; (e) PdCl (dppf), 2M
2
2
2
o
aq. Na CO , 1,2-dimethoxyethane, 140 C, microwave, 98%; (f)
TFA, CH Cl , rt, 28%; (g) piperidine, MeOH, 60 C, 37–75%;
2
3
o
2
2
(
h) TFA, CH Cl , rt, 7–59%.
2 2
Compounds were synthesized as shown in Scheme 1.
24
In this study, we successfully transformed our previous
Synthesis of 1 and 2 was reported previously by us. Syn-
thesis of two active methylene compounds for
Knoevenagel condensation, 15 and 16, was also reported
previously. 7-Azaindole-3-carbaldehydes 18a–g, coupling
partners of 15 and 16, were prepared as follows, except for
commercially available 18a. Methyl 6-chloro-1H-
pyrrolo[2,3-b]pyridine-1-carboxylate was reacted with
trimethylboroxine or alkylboronic acids under Suzuki
coupling conditions to yield 6-alkyl-7-azaindoles 17b–e.
Then, 17b–e were formylated with hexamethylenetetra-
mine to obtain 6-alkyl-7-azaindole-3-carbaldehydes 18b–e.
non-selective 7-azaindole derivative 2 into potent and
selective PIM inhibitors by means of substitution target-
ing the unique structural features of PIM. It is noteworthy
that chlorine substitution not only reduced unfavorable
interactions with off-targets but also improved the inhibi-
tory potency toward PIM2. In addition, the chloro-
substituted derivative 14 showed acceptable in vitro
ADMET properties, indicating that our PIM inhibitors
have potential for further development. These results
suggest that the space occupied by the chlorine atom is a
promising target for the design of more sophisticated
pan-PIM inhibitors, although the effect of altering the
chlorine substituent on the improved PIM2 inhibition
24
29
1
8f and g (R = halogen) were also synthesized by formyla-
tion of commercially available 17f and g. Then,
Knoevenagel condensation of 15/16 and 18a–g was carried
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remains to be clarified. More detailed analyses, such as
crystallographic, calorimetric and kinetic studies, should
also be helpful for further development of rationally de-
signed PIM inhibitors.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Table 2. Results of PIM2 inhibition assay
Comp.
1
2
3
4
9
10
11
14
b
PIM2 IC50 (nM)
1200
390
300
290
140
420
510
140
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
Carried out at Carna Biosciences. [ATP] = 5 ꢀM. Ref. 24.
a
Table 3. ADMET profiling data
Comp.
1
2
≧200
19
3
9
14
130
30
1.7
82
63
b
f
Aqueous solubility (ꢀM)
190
60
130
14
21
c
hERG inhibition @ 10 ꢀM (%)
22
0.31
82
-
6
d
Caco-2 permeability (x 10 cm/s)
0.89
68
2.1
0.13
74
e
Stability in human liver microsomes (%)
Stability in mouse liver microsomes (%)
90
e
83
72
69
58
a
b
c
Carried out at Cerep. Solubility in PBS, pH 7.4. Final DMSO concentration was 2%. A patch-clamp method was used to
d
e
evaluate hERG inhibition.
cubated with 0.3 mg/mL protein at 37 C for 1 h. Final DMSO concentration was 10%.
Compound concentration was 10 ꢀM. %Remaining of the compound. 1 ꢀM compound was in-
o
f
Table 4. Cell growth inhibitory potency of 14, AZD1208 and SGI-1776
leukemia cell line
MOLM-16 IC50 (nM)
AZD1208
diploid lung fibroblast cell line
a
b
WI-38 IC50 (nM)
AZD1208
Compound 14
00
SGI-1776
600
Compound 14
>10,000
SGI-1776
1,700
2
12
>10,000
a
b
Incubated for 72 h. Incubated for 48 h.
ASSOCIATED CONTENT
Supporting Information
ABBREVIATIONS
ADMET, absorption, distribution, metabolism, excretion,
toxicity; BAD, Bcl–2–associated death promoter; DAPK1,
death-associated
The Supporting Information is available free of charge on the
ACS Publications website.
protein
kinase
1;
dppf,
[1,1’-
bis(diphenylphosphino)ferrocene]; hERG, human ether–a–
go–go related gene; LLE, ligand lipophilicity efficiency; MYC,
myelocytomatosis oncogene; PDGFRα, platelet–derived
growth factor receptor alpha; PIM, proviral integration site
for Moloney murine leukemia virus; PKCα, protein kinase C
alpha; PKD2, protein kinase D2; ROCK1, Rho–associated
coiled–coil containing protein kinase 1; TFA, trifluoroacetic
acid.
Raw data of the kinase selectivity studies (compounds 3, 4, 8,
9
and 14), protocols of biological assays and details of chem-
istry. (PDF)
AUTHOR INFORMATION
Corresponding Author
*
Phone: +81-3-5841-1960; E-mail: tlong@mol.f.u-tokyo.ac.jp
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This work was supported by the Platform Project for Sup-
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for Drug Discovery, Informatics, Structural Life Science)
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