The design, synthesis, and biological evaluation of PIM kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.04.025

A series of substituted benzofuropyrimidinones with pan-PIM activities and excellent selectivity against a panel of diverse kinases is described. Initial exploration identified aryl benzofuropyrimidinones that were potent, but had cell permeability limitation. Using X-ray crystal structures of the bound PIM-1 complexes with 3, 5m, and 6d, we were able to guide the SAR and identify the alkyl benzofuropyrimidinone (6l) with good PIM potencies, permeability, and oral exposure.

Full text of DOI:10.1016/j.bmcl.2012.04.025

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3732–3738
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design, synthesis, and biological evaluation of PIM kinase inhibitors
⇑
Amy Lew Tsuhako , David S. Brown, Elena S. Koltun, Naing Aay, Arlyn Arcalas, Vicky Chan,
Hongwang Du, Stefan Engst, Maurizio Franzini, Adam Galan, Ping Huang, Stuart Johnston, Brian Kane,
Moon H. Kim, A. Douglas Laird, Rui Lin, Lillian Mock, Iris Ngan, Michael Pack, Gordon Stott,
Thomas J. Stout, Peiwen Yu, Cristiana Zaharia, Wentao Zhang, Peiwen Zhou, John M. Nuss,
Patrick C. Kearney, Wei Xu
Exelixis, Department of Drug Discovery, 210 East Grand, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 11 April 2012
A series of substituted benzofuropyrimidinones with pan-PIM activities and excellent selectivity against a
panel of diverse kinases is described. Initial exploration identified aryl benzofuropyrimidinones that were
potent, but had cell permeability limitation. Using X-ray crystal structures of the bound PIM-1 complexes
with 3, 5m, and 6d, we were able to guide the SAR and identify the alkyl benzofuropyrimidinone (6l) with
good PIM potencies, permeability, and oral exposure.
Keywords:
PIM-1
PIM-2
PIM-3
Ó 2012 Elsevier Ltd. All rights reserved.
CK2 inhibitor
Cdc7 inhibitor
Benzofuropyrimidinone
Proviral integration site of moloney murine leukemia virus
(PIM) is a constitutively active serine/threonine protein kinase
whose family consists of three highly related proteins, PIM-1, 2,
and 3.1,2 Since PIM kinases do not need prior post-translational
modifications for the induction of kinase activity, they are tightly
regulated at the transcriptional and translational level by cyto-
kines, mitogens, and growth factors in the JAK/STAT pathway.3
PIM’s role in the cell is to suppress apoptosis through the direct
phosphorylation of BCL-2 associated agonist of cell death (BAD).4
Aside from its role in regulating cell survival, overexpression of
PIM’s has been reported in select solid tumors and many hemato-
logic cancers such as chronic lymphocytic leukemia, Flt3-mediated
acute myelogenous leukemia and diffuse B cell lymphoma.5–8
These evidence strongly suggest PIM as an interesting target for
cancer therapy.
identified from our internal CK2 inhibitor program, but showed
cross reactivity with PIM (PIM-1: IC50 = 75 nM, CK2: IC50 = 32 nM).
Synthesis of an analog that hybridized 1 and 2, by incorporating a 2-
chloro phenyl group to HTS hit 1, gave compound 3, an analog with
significantly improved PIM-1 and PIM-3 activities and modest
selectivity over CK2 (Fig. 1).
ATP competition studies found 3 to be an ATP competitive
inhibitor. Furthermore, co-crystallization of 3 with PIM-1 revealed
that while 3 binds in the ATP binding pocket, unlike typical kinase
inhibitors that bind in this region, there are no hydrogen bond
interactions with the hinge. This is a unique feature of PIM inhib-
itors as the hinge sequence of PIM has a proline insertion such that
the hinge region bulges away from the ATP-binding site by up to
4 Å.24–26 Consequently, the primary interactions for 3 that derive
Several groups have reported PIM inhibitors in the literature.9–23
PIM-2 has been intrinsically more difficult to target primarily due to
its high Km for ATP.17 Our efforts towards designing a pan-PIM
inhibitor began with a high throughput screen (HTS) of our internal
small molecule library against PIM-1 and PIM-2. These screens led
to the discovery of benzofuropyrimidinone 1 (Fig. 1). While 1 was
only moderately potent against PIM (PIM-1: IC50 = 526 nM, PIM-
2: IC50 = 2963 nM, PIM-3: IC50 = 346 nM), this compound showed
structural similarities to uncyclized 2, a pyrimidinone that was
O
O
N
O
N
O
O
N
NH Cl
NH
NH
1
HO
2
Cl
3
Br
Br
PIM-1: 17 nM
PIM-2: 781 nM
PIM-3: 18 nM
CK2: 231 nM
PIM-1: 526 nM
PIM-2: 2963 nM
PIM-3: 346 nM
PIM-1: 75 nM
CK-2: 32 nM
⇑
Corresponding author. Tel.: +1 6508378042.
E-mail addresses: alew@exelixis.com, amy.lew@gmail.com (A.L. Tsuhako).
Figure 1. Early PIM leads.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.025

A. L. Tsuhako et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3732–3738
3733
Figure 2. (A) CPK view of the crystal structure of 3 in PIM-1 solved to 2.6 Å showing the close packing of the bromide against the hinge (PDB code 4ALU). (B) Compound 3
crystallized in PIM-1 (Pink). The 2-Cl phenyl is disordered (Orange atoms indicate non-resolved disorder).
Table 1
Biochemical inhibitory activity for left hand modifications
O
O
G loop
NH
6
7
R1
N
R2
9
8
a
ID
R1
R2
Kinase inhibitory activity IC50 (nM)
PIM-1
PIM-2
PIM-3
CK2
231
5000
371
3
8-Br
H
8-Cl
8-Me
8-OMe
8-OH
8-CN
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
17
5000
28
781
5000
858
18
5000
30
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
99
61
3934
1451
3755
3429
5000
5000
2330
5000
5000
5000
2377
5000
5000
104
31
106
204
88
286
102
4393
1244
1168
71
2625
312
232
281
3963
293
23
5000
2982
5000
205
5000
1871
1288
5000
5000
5000
5000
5000
5000
5000
2485
5000
3471
8-Ph
8-OCF3
8-CF3
8-NH2
8-NHMe
8-NHAc
7-OH
7-OMe
9-OMe
DFG pocket
5000
850
4p
2-Cl, 6-F
497
3330
166
3457
a
Values reported are the average of at least two independent dose–response
curves; variation was generally 15%.29,30
PIM potency are from van der Waals interactions of the 8-bromo
Substrate
benzofuropyrimidinone core with the linker region, a potential
p
Binding Pocket
stacking between the 2-chloro phenyl and Phe49, and a salt bridge
that forms between the pyrimidinone C@O and Lys67 (Fig. 2).
To optimize the potency of 3 for PIM over CK2, we first looked
towards modifying the left-hand side (Table 1). We found that
the 8-Br was necessary for activity, as the des-Br analog 4a was
inactive. Replacement of the 8-Br with 8-Cl was tolerated (4b),
but replacement with larger functionalities such as a cyano or phe-
nyl were not (4f, 4g). This was expected, as the crystal structure of
3 in PIM-1 showed that the 8-Br was packed tightly against the
hinge region (Fig. 2A). Interestingly, the 8-CF3 analog was more po-
tent against PIM-1 than the non-halogenated 8-Me analog 4c, indi-
cating that a dipole interaction between the ligand and protein was
important in this region. However, this dipole is very directed as
8-OCF3 (4h) was less potent than 8-OMe (4d).
Figure 3. Comparison of aryl versus alkyl benzofuropyrimidinone binding modes in
PIM-1. Compound 5m co-crystallized with PIM-1 (PDB code 4ALV) is shown in pink
(2.59 Å) and overlaid with compound 6d co-crystallized in PIM-1 (PDB code 4ALW)
shown in green (1.92 Å). (A) Side view showing the opening of the G-loop. (B) Front
view showing the aryl piperidine group on 5m pointing in a different direction from
that of the alkyl piperazine on 6d.
8-NH2 was more potent than lead compound 3. However, the crys-
tal structure of 3 in PIM-1 showed that the carbonyl backbone of
Glu121 in the linker region was accessible for hydrogen bonding
if a hydroxyl group was placed in the 7-position instead of 8-posi-
tion of the benzofuropyrimidinone (Fig. 2B). Unfortunately, the 7-
OH analog (4m) was less potent than lead compound 3. Since the
SAR on the left-hand side of the benzofuropyrimidinone 3 was
Aside from space constraints, there were also no hydrogen
bonding opportunities in the 8-position as neither 8-OH, nor

3734
A. L. Tsuhako et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3732–3738
steep, we decided to switch our focus to optimizing the right-hand
side while holding the left-hand side constant as the 8-Br.
To further improve pan-PIM potency and selectivity, we looked
towards molecular modeling studies. These modeling studies
hinted that we could improve pan-PIM potency by designing ana-
logs that could hydrogen bond with the three non-conserved acidic
residues (Asp128, Asp 131, Glu171) in the PIM substrate binding
pocket (Fig. 2B). A number of analogs with different 1°, 2° and 3°
amines tethered via an aniline, amide or methylene off of the
para-position of the 2-Cl phenyl were synthesized and representa-
tive examples are shown in Table 2 (5j–5m). Indeed, we found that
these basic amines improved PIM-1, 2 and 3 activities, especially
PIM-2 activities; but although these aryl benzofuropyrimidinones
had biochemical potencies below 10 nM for PIM-1/-3, and below
100 nM for PIM-2, they were not potent in cells. Evaluation of this
series of amine tethered benzofuropyrimidinone analogs in MDCK
cells indicates permeability as an issue, presumably due to the
higher molecular weight (487–530 amu), and high lipophilicity
(clogP 4.9–5.2).
To improve the cell permeability, we wanted to replace the
large right-hand di-substituted aryl groups with simpler alkyl ami-
no groups that would still allow access to the three acidic residues
in the substrate binding pocket, but might offer improved physico-
chemical properties. Initially, simple alkyl amines such as a piper-
idine, pyrrolidine, azetidine and methyl-piperazine tethered via a
methylene group to the benzofuropyrimidinone core were synthe-
sized (Table 3: 6a–6d). We were encouraged to find that the alkyl
amino benzofuropyrimidinones were as potent in PIM as the aryl
Based on the X-ray crystal structure of 3, the right-hand portion
of the molecule appeared to leave more room for modification than
the left-hand side (Fig. 2A). The 2-Cl phenyl group can favorably
interact with the nearby Phe49; although the 2-Cl phenyl was dis-
ordered in both the PIM-1 (Fig. 2B) and CK2 crystal structures (data
not shown). MS/MS and LC/MS analysis of 3, and the protein com-
plex with 3, demonstrated that the 2-Cl phenyl group was intact
(data not shown). Once the 2-Cl phenyl was substituted with larger
functional groups (i.e., 5m, Fig. 3), no disorder of this moiety was
observed in PIM complex crystal structure.
Modification of the right-hand side began with surveying
replacements for the 2-Cl functionality. Unfortunately, none of
these replacements were fruitful. For example, 2-OMe and 2-iPr
phenyl were both inactive against PIM (Table 2, 5a and b). On
the other hand, di-substituted phenyl analogs with a 2-Cl group,
maintained potency and selectivity for PIM-1 and PIM-3 (5c–5h).
Especially noteworthy was 5f, where the PIM-1 and PIM-3 poten-
cies were below 10 nM, and CK2 activity was >10-fold of PIM activ-
ities. Despite having potencies in PIM-1 and PIM-3, 5f did not have
PIM-2 potency. Understandably, PIM-2 inhibition will be more dif-
ficult to attain due to the significant differences in affinity for ATP
between these proteins. The Km of ATP for PIM-1 is 343
1 mM of substrate, while the Km of ATP for PIM-2 is 2.3
the same substrate concentration.27
l
l
M at
M at
Table 2
Biochemical inhibitory activity for right hand modifications of the aryl ring
O
N
O
NH
R2
Br
a
ID
R2
Kinase inhibitory activity IC50 (nM)
MDCK (nm/s)
PIM-1
PIM-2
PIM-3
CK2
231
3
2-Cl
17
>5000
376
24
14
35
8
11
15
3
781
18
>5000
810
23
13
23
8
17
21
8
—
5a
5b
5c
5d
5e
5f
5g
5h
5i
2-OMe
2-iPr
>5000
5000
850
653
1068
598
293
1741
87
5000
1419
243
173
743
269
95
—
—
2-Cl, 4-F
2-Cl, 5-F
2-Cl, 6-F
2-Cl, 3-Cl
2-Cl, 4-Cl
2-Cl, 3-OMe
2-Cl, 4-CH2NH2
289
42
336
77
11
197
93
575
184
2-Cl, 4-
O
5j
4
4
134
29
20
8
94
8
3
N
N
H
2-Cl, 4-
NH
5k
339
N
H
2-Cl, 4-
H
5l
3
7
68
29
5
8
194
68
4
N
N
O
NH
H
N
5m
2122
a
Values reported are the average of at least two independent dose–response curves; variation was generally 15%.29,30

A. L. Tsuhako et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3732–3738
3735
Table 3
Biochemical inhibitory activity for right hand modifications with alkyl groups
O
N
O
NH
R3
Br
a
ID
R3
Kinase inhibitory activity IC50 (nM)
MDCK (nm/s)
PIM-1
32
PIM-2
PIM-3
41
CK2
6a
730
147
481
N
6b
6c
13
9
194
68
12
10
92
848
499
N
N
115
N
N
6d
6e
27
16
197
503
11
35
2950
3151
475
461
N
N
N
6f
10
3
110
162
9
582
452
68
N
N
N
6g
18
129
NH2
6h
4
49
11
68
10
N
N
NH2
6i
6
18
11
72
534
121
6
31
10
535
484
28
9
83
6j
N
OH
6k
172
N
OH
OH
6l
5
68
4
95
87
152
81
N
N
6m
10
142
11
a
Values reported are the average of at least two independent dose–response curves; variation was generally 15%.29,30
Table 4
benzofuropyrimidinones; and more importantly, showed im-
proved cellular permeability. For example, azetidine analog 6c
showed pan-PIM activities with excellent MDCK value.
Biochemical inhibitory activity for left hand modifications in the alkyl benzofuro-
pyrimidinone series
O
Interestingly, the crystal structure of PIM-1 with 6d revealed
that the alkyl benzofuropyrimidinones did not bind in the ATP
pocket in the same manner as the aryl benzofuropyrimidinones
(Fig. 3). Rather than the piperazine occupying the substrate bind-
ing pocket as predicted, the molecule is shifted closer to Lys67,
and the piperazine forces the G-loop and Phe49 out, allowing the
distal amine on the piperazine to make a hydrogen-bond contact
with the Asp186 of the DFG loop. In fact, the presence of the distal
amine and the hydrogen bonding capability with Asp186 is what
drives this G-loop movement. We were encouraged by this finding
as it showed that there was more room in the DFG pocket than
originally thought.
O
NH
N
N
N
R4
a
ID
R4
Kinase inhibitory activity IC50 (nM)
PIM-1
PIM-2
PIM-3
CK2
6d
7a
7b
Br
Cl
I
27
77
24
197
480
140
11
34
11
2950
5000
2800
7c
580
840
160
5000
Increasing the steric bulk around the piperazine to maximize
van der Waals contact with the expanded DFG pocket resulted in
analogs 6e–6g that showed improved PIM-1 activities compared
to the parent piperazine analog 6d, but did not improve PIM-2 or
3 activities. Consequently, rather than maximizing van der Waals
O
7d
640
620
250
5000
a
Values reported are the average of at least two independent dose–response
curves; variation was generally 15%.29,30

3736
A. L. Tsuhako et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3732–3738
Table 5
Cell potency, mouse and rat pk properties of compounds with pan-PIM activities
ID
Inhibition of phosphorylation in
HEL 92.1.7
BAD S112 IC50 (nM)
Mouse HTS PK dosed at
100 mg/kg (po)
Rat PK
a
Plasma exposure (lM) 1h/4h
Dose (mg/ CL (mL/h/ Vd (L/
T1/2
F
Cmax
M)iv/po
AUC/dose (
mg)iv/po
lM h kg/
kg)
kg)
kg)
(h)iv/po
(%)
(l
6c
6f
6i
6l
1051
2412
2582
1218
13/5
11/7
6/5
93/35
78/22
2.5
5
5
5
2.5
1837
2044
3371
793
3.8
7.1
6.2
2.3
2.4
1.0/1.0
2.6/2.6
1.4/1.6
2.7/4.7
8.4/3.7
77
86
13
79
56
1.8/0.8
1.5/0.4
1.8/0.1
7.6/3.1
3.5/1.2
1.7/1.3
1.2/0.9
0.8/0.1
3.8/2.9
2.6/1.5
6m 1963
810
a
IC50 values reported are the average of at least two independent dose–response curves.31
O
O
R5
O
O
OH
CN
O
O
NH
a
NH2
b
NH2
c or d
N
NH2
CN
R5
Cl
R5
R5
8
9
10
3, 4a-4o
Scheme 1. Reagents and conditions: (a) Bromoacetamide, Cs2CO3, DMA, 75 °C; (b) KOH, EtOH, 75 °C; (c) 2-chloro-benzaldehyde, cat. concd HCl, 150 °C; (d) 2-
chlorobenzaldehyde, CuCl2, EtOH, 120 °C microwave, 20 min
1° amine with a hydroxyl (6k, 6l) substantially improved the
MDCK and cell potency while maintaining PIM activities (Tables
3 and 5).
OBoc
OH
OH
N
N
N
a, b
c
Since the alkyl benzofuropyrimidinones bind in the ATP pocket
differently from the aryl benzofuropyrimidinones, we wanted to go
back and re-optimize the left-hand side 8-position to see if meth-
ylpiperazine derivatives leave more room for modifications. Unfor-
tunately, even replacement of the 8-bromide with an 8-chloride
which was previously tolerated in the aryl series (4b), was now sig-
nificantly less potent in the alkyl series (7a). A larger halide such as
the 8-iodo analog (7b) was equipotent to the 8-bromo analog, but a
non-halide such as the 8-cyclopropyl (7c) was inactive, similar to
what we had observed for the aryl series (4p vs 5e).
Br
8a
11
12
Scheme 2. Reagents and conditions: (a) Boc2O, DMAP, DCM, ACN, 99% yield; (b)
cyclopropyl boronic acid, Pd(OAc)2, PCy3, K3PO4, toluene, H2O, 67% yield; (c) TFA,
Et3SiH, DCM.
contacts, we decided to optimize the hydrogen-bond contact with
the Asp of the DFG pocket by installing an amino-pyrrolidine or
installing a 3-piperidine (6h–6j). While this strategy gave pan-
PIM active analogs 6h and 6i, the cell permeabilities were compro-
mised due to the presence of primary amines. Replacement of the
Compounds with pan-PIM activities, and P10-fold selectivity
for CK2 with respect to both PIM-1 and PIM-3, were then evaluated
in PK studies (Table 5). Compound 6l showed high plasma expo-
sures (1h/4h) in mouse high-throughput screening PK dosed at
O
O
O
O
O
O
NH2
b or c
a
Br
NH
Br
NH
NH2
N
N
14
13
10a
Br
Cl
Cl
f
NH2
NO2
d or e
O
N
O
NH Cl
O
O
R7
NH
Br
N
Br
15: R7 = OMe
5j: R7 =
O
g,h
Cl
N
NH
N
5l, 5m
R6
H
Scheme 3. Reagents and conditions: (a) 2-Chloro-4-nitrobenzaldehyde (2.3 equiv), NaHSO3 (7 equiv), DMSO, 150 °C, 5 h; (b) sodium dithionite, THF/H2O, rt 15 min; (c)
SnCl2ÁH2O, MeOH, EtOAc, H2O, 80 °C, 1 h; (d) alkyl aldehyde, NaBH(OAc)3, HOAc, DCE; (e) acid chloride, pyridine, DCM, rt; (f) methyl 3-chloro-4-formylbenzoate, CuCl2,
MeOH, 120 °C microwave, 20 min; (g) LiOH, 50 °C; (h) HATU, DIEA, DMF, various amines.

A. L. Tsuhako et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3732–3738
3737
O
O
N
In summary, we describe a novel class of benzofuropyrimidi-
nones for PIM kinase inhibition. Using structure based design, we
were able to successfully optimize lead 3 from a PIM-1/-3 inhibitor
to a pan-PIM inhibitor, 6l, with cellular potency and good oral
exposure. In addition, through the SAR, we discovered that the
G-loop of PIM-1 was flexible, opening up the pocket for hydrogen
bonding opportunities with the DFG motif.
O
O
NH2
NH
NH
b
a
Cl
10a
Cl
O
Br
Br
17
16
c
Acknowledgments
We greatly appreciate the contributions to the biochemical and
pharmacokinetic data from members of Exelixis Genome Biochem-
istry, New Lead Discovery, Pharmacology and Compound Reposi-
tory. In addition, we greatly appreciate Jia Li for additional PIM-1
co-crystals not reported in this manuscript and Lai Man Adams
for technical expertise in crystallization.
O
N
O
NH R9
N
R8
Br
6a - 6m
Scheme 4. Reagents and conditions: (a) Chloroacetyl chloride, 40 °C, 30 min, 90%
yield; (b) 2 N NaOH, 40 °C, 20 min, 63% yield; (c) alkyl amine, EtOH, 80 °C.
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(MDZ), 2C8, 2C9, 2C19, 2D6 IC50’s >20 lM; CYP1A2 IC50 = 5.1 lM.
In mechanistic cellular assays with HEL92.1.7 cells, 6l caused a
dose dependent inhibition of the phosphorylation of BAD at the
PIM kinase specific site S112. This inhibitory effect in cells is be-
lieved to be due to PIM activity since 6l is not biochemically active
against JAK2 or Flt-3 (IC50 >3600 nM).
The compounds in Table 1 were synthesized according to
Scheme 1 where an appropriately substituted hydroxybenzonitrile
(8) was alkylated with bromoacetamide under mildly basic condi-
tions at 75 °C to give the corresponding cyanophenoxy-acetamide
9.28 Heating of 9 under basic conditions induced cyclization to give
aminobenzofuran-carboxamide 10. Formation of the benzofuro-
pyrimidinone required cyclization under high temperatures with
2-chlorobenzaldehyde and a catalytic amount of acid, either concen-
trated HCl or copper(II) chloride. Withoutthe high temperatures, the
reaction stopped at the 2,3-dihydrobenzofuropyrimidinone stage.
Synthesis of the cyclopropyl analog 4p (Scheme 2) required Boc pro-
tection of the phenol, followed by Suzuki coupling of the bromide
with the corresponding cyclopropyl boronic acid to give 11. Boc
deprotection gave the corresponding phenol that was then used to
synthesize 4p and 7c.
15. Grey, R.; Pierce, A. C.; Bemis, G. W.; Jacobs, M. D.; Moody, C. S.; Jajoo, R.; Mohal,
N.; Green, J. Bioorg. Med. Chem. Lett. 2009, 19, 3019.
The compounds in Table 2 were synthesized according to
Scheme 3 where instead of cyclization with 2-chlorobenzaldehyde,
10a was cyclized with 2-chloro-4-nitrobenzaldehyde to give inter-
mediate 13. The nitro group was then reduced with sodium dithio-
nite or tin(II) chloride to give aniline 14, which was either acylated
with a variety of acid chlorides, or reductively alkylated with a
variety of aldehydes. The reverse amides in this table were made
by reacting methyl-3-chloro-4-formylbenzoate with 10a to give
the corresponding benzofuropyrimidinone 15. The methyl ester
was then saponified and the corresponding acid was activated with
HATU for couplings with amines.
The compounds in Table 3 were synthesized according Scheme 4
where intermediate 10a was reacted with chloroacetyl chloride to
give intermediate 16. Cyclization under basic conditions occurs rap-
idly to give chloromethyl benzofuropyrimidinone 17, which can
then be displaced with a variety of alkyl amines. The compounds
in Table 4 were also synthesized in an analogous manner where
the starting hydroxy-bromobenzonitrile was substituted with the
corresponding chloro, iodo or cyclopropyl hydroxybenzonitrile.
16. Sliman, F.; Blairvacq, M.; Durieu, E.; Meijer, L.; Rodrigo, J.; Desmaele, D. Bioorg.
Med. Chem. Lett. 2010, 20, 2801.
17. Qian, K.; Wang, L.; Cywin, C. L.; Farmer, B. T.; Hickey, E.; Homon, C.; Jakes, S.;
Kashem, M. A.; Lee, G.; Leonard, S.; Li, J.; Magboo, R.; Wang, M.; Pack, E.; Peng,
C.; Prokopowicz, A.; Welzel, M.; Wolak, J.; Morwick, T. J. Med. Chem. 1814, 2009,
52.
18. Tong, Y.; Syewart, K. D.; Thomas, S.; Przytulinska, M.; Johnson, E. F.; Klinghofer,
V.; Leverson, J.; McCall, O.; Soni, N. B.; Luo, Y.; Lin, N.; Sowin, T. J.; Giranda, V. L.;
Penning, T. D. Bioorg. Med. Chem. Lett. 2008, 18, 5206.
19. Pierce, A. C.; Jacobs, M.; Stuver-Moody, C. J. Med. Chem. 1972, 2008, 51.
20. Cheney, I. W.; Yan, S.; Appleby, T.; Walker, H.; Vo, T.; Yao, N.; Hamatake, R.;
Hong, Z.; Wu, J. Z. Bioorg. Med. Chem. Lett. 2007, 17, 1679.
21. Holder, S.; Zemskova, M.; Zhang, C.; Tabrizizad, M.; Bremer, R.; Neidigh, J. W.;
Lilly, M. B. Mol. Cancer Ther. 2007, 6, 163.
22. Pogacic, V.; Bullock, A. N.; Fedorov, O.; Filippakopoulos, P.; Gasser, C.; Biondi,
A.; Meyer-Monard, S.; Knapp, S.; Schwaller, J. Cancer Res. 2007, 67, 6916.
23. Pastor, J.; Oyarzabal, J.; Saluste, G.; Alvarez, R. M.; Rivero, V.; Ramos, F.; Cendón,
E.; Blanco-Aparicio, C.; Ajenjo, N.; Cebria, A.; Albarrán, M. I.; Cebrián, D.;
Corrionero, A.; Fominaya, J.; Montoya, G. Mazzorana; M Bioorg. Med. Chem. Lett.
2012, 22, 1591.
24. Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.; Marsden, B. D.;
Knapp, S. J. Med. Chem. 2005, 48, 7604.
25. Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.; Peng, C.; Kronkaitis,
A.; Li, J.; White, A.; Mische, S.; Farmer, B. J. Biol. Chem. 2005, 280, 6130.

3738
A. L. Tsuhako et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3732–3738
26. Jacobs, M. D.; Black, J.; Futer, O.; Swenson, L.; Hare, B.; Fleming, M.; Saxena, K. J.
Biol. Chem. 2005, 280, 13728.
30. Munagala, N.; Nguyen, S.; Lam, W.; Lee, J.; Joly, A.; McMillan, K.; Zhang, W.
Assay Drug Dev. Technol. 2007, 5, 65.
27. Data generated in house.
31. BAD Phosphorylation ELISA Analysis: HEL92.1.7 cells (AML) stably transfected
with wildtype BAD (clone 2B8) were seeded at 1.5 Â 105 cells/well onto 96-
well plates (Costar, 3628), in serum-free RPMI (ATCC) containing 1% penicillin–
streptomycin (Cellgro). Dimethyl sulfoxide (DMSO, ATCC) or serial dilutions of
compounds in fresh serum-free medium were added to the cells and incubated
for 1 h at 37 °C, 5% CO2. Medium was removed and the cells were lysed with
28. Detailed experimentals for this manuscript are found in the following patent:
Brown, S.D.; Du, H.; Franzini, M.; Galan, A.A.; Huang, P.; Kearney, P.; Kim, M.H.;
Koltun, E.S.; Richards, S.J.; Tsuhako, A.L.; Zaharia, C.A. WO 2009086264 A1
2009.
29. Human PIM1 (E32-D292), PIM2 (E26-A306) and PIM3 (K30-L326) were
expressed as N-terminally His-tagged proteins in E. coli and purified using
100
and 20
were incubated overnight at 4 °C in 96-well high-binding plates (Pierce,
15042), coated with mouse anti-FLAG M2 (1 g/mL, Sigma Aldrich, F3165).
l
L cold lysis buffer (0.1% Triton X-100 lysis buffer). Protein lysates (20
lL
nickel affinity chromatography. Human CK2
a
isoform A (amino acids: R8–
lL) for detection of phosphorylated BAD and total BAD, respectively,
R333) and full-length b subunits were expressed separately as N-terminally
MBP-tagged proteins in E. coli and purified using amylose Sepharose
chromatography. The purified subunits were reconstituted to form the
l
Immuno-complexes were washed three times with TBST (25 mM Tris–HCl, pH
7.2, 150 mM NaCl, 0.1% Tween-20) and incubated for 3 h in TBST buffer
containing either rabbit anti-pBAD (Ser116) (1:1,500, Cell Signaling) or total
BAD (1:1,000, Epitomics,) antibodies. Immuno-complexes were washed and
incubated for 1 h in TBST containing HRP-conjugated goat anti-rabbit IgG
(1:10,000, Pierce). Immuno-complexes were then washed, followed by the
tetrameric
a2b2 CK2 holoenzyme. Protein concentration was determined by
the Bradford assay and identification was confirmed by trypsin digestion and
mass spectrometry. Kinase activity and compound inhibition were determined
using the luciferase-luciferin-coupled chemiluminescence assay and measured
as the percentage of ATP utilized following the kinase reaction in a 384-well
format as described previously (Ref. 29). The final PIM kinase assay condition
addition of
measurements were taken in
a
luminol-based substrate solution (Pierce, 37069) before
Victor Wallac multiwavelength
was 0.5
lM ATP, 10
lM peptide substrate (AKRRRLSA), 20 mM Hepes pH 7.4,
a
V
10 mM MgCl2, 0.03% Triton, and 1 mM DTT. The kinase concentrations were
2.4 nM for PIM1, 4 nM for both PIM2 and PIM3. The final CK2 kinase assay
condition was 4 nM CK2 holoenzyme, 2 lM ATP, 2 lM casein, 20 mM Hepes
measurement reader (Perkin Elmer). The percentage inhibition of BAD
phosphorylation was normalized to total BAD and the IC50 values were
calculated from six different compound concentrations.
pH 7.5 10 mM MgCl2, 0.03% Triton, 1 mM DTT and 0.1 mM NaVO3. All kinase
reactions were incubated at room temperature for 1–2 h.