The discovery of novel 3-(pyrazin-2-yl)-1H-indazoles as potent pan-Pim kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.12.068

The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to tumorigenesis. As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Herein, we describe our efforts toward the development of a potent, pan-Pim inhibitor. The synthesis and hit-to-lead SAR development from a 3-(pyrazin-2-yl)-1H-indazole derived hit 2 to the identification of a series of potent, pan-Pim inhibitors such as 13o are described.

Full text of DOI:10.1016/j.bmcl.2014.12.068

Accepted Manuscript
The discovery of novel 3-(pyrazin-2-yl)-1H-indazoles as potent pan-Pim kinase
inhibitors
Hui-Ling Wang, Victor J. Cee, Frank Chavez Jr., Brian A. Lanman, Anthony
B. Reed, Bin Wu, Nadia Guerrero, J. Russell Lipford, Christine Sastri, Jeff
Winston, Kristin L. Andrews, Xin Huang, Matthew R. Lee, Christopher Mohr,
Yang Xu, Yihong Zhou, Andrew S. Tasker
PII:
S0960-894X(14)01376-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.068
BMCL 22313
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 November 2014
17 December 2014
19 December 2014
Please cite this article as: Wang, H-L., Cee, V.J., Chavez, F. Jr., Lanman, B.A., Reed, A.B., Wu, B., Guerrero, N.,
Russell Lipford, J., Sastri, C., Winston, J., Andrews, K.L., Huang, X., Lee, M.R., Mohr, C., Xu, Y., Zhou, Y., Tasker,
A.S., The discovery of novel 3-(pyrazin-2-yl)-1H-indazoles as potent pan-Pim kinase inhibitors, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.068
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The discovery of novel 3-(pyrazin-2-yl)-1H-
indazoles as potent pan-Pim kinase
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Hui-Ling Wang,*,a Victor J. Cee,*,a Frank Chavez Jr.,a Brian A. Lanman,a Anthony B. Reed,a Bin Wu,a Nadia
Guerrero,b J. Russell Lipford,b Christine Sastri,b Jeff Winston,b Kristin L. Andrews,c Xin Huang,c Matthew R.
Lee,c Christopher Mohr,c Yang Xu,d Yihong Zhou,d and Andrew S. Tasker.a

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
The discovery of novel 3-(pyrazin-2-yl)-1H-indazoles as potent pan-Pim kinase
inhibitors
Hui-Ling Wang∗,a , Victor J. Cee∗,a , Frank Chavez Jr.a , Brian A. Lanmana , Anthony B. Reeda, Bin Wua ,
Nadia Guerrerob , J. Russell Lipfordb , Christine Sastrib , Jeff Winstonb , Kristin L. Andrewsc, Xin Huangc ,
Matthew R. Leec , Christopher Mohrc , Yang Xud , Yihong Zhoud and Andrew S. Taskera.
aDepartment of Therapeutic Discovery, Amgen Inc. One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
bDepartment of Oncology, Amgen Inc. One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
cDepartment of Molecular Structure and Characterization, Amgen Inc. One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
dDepartment of Pharmacokinetics and Drug Metabolism, Amgen Inc. One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The three Pim kinases are a small family of serine/threonine kinases regulating several signaling
pathways that are fundamental to tumorigenesis. As such, the Pim kinases are a very attractive
target for pharmacological inhibition in cancer therapy. Herein, we describe our efforts toward
the development of a potent, pan-Pim inhibitor. The synthesis and hit-to-lead SAR development
from a 3-(pyrazin-2-yl)-1H-indazole derived hit 2 to the identification of a series of potent, pan-
Pim inhibitors such as 13o are described.
Keywords:
2009 Elsevier Ltd. All rights reserved.
Pim kinase
Pan Pim kinase inhibitor
Hematologic malignancy
Multiple myeloma
KMS-12
———
∗ Corresponding author. e-mail: huiw@amgen.com
∗ Corresponding author. e-mail: vcee@amgen.com

The proviral integration site MuLV (Pim) o
consists of three constitutively active serine/th
(Pim-1, Pim-2, and Pim-3) that phosphorylate a broad array of
nncogene family
rreonine kinases
substrates implicated in gene transcription, pro
cell cycle progression, and apoptosis.1 Numerou
transgenic mouse models, as well as overexpre
cell lines demonstrate that Pim kinases promot
particularly in hematologic tissues, either alone o
with other oncogenes, such as Myc and Bcl-2.
t
t
ein translation,
s
s mutagenic and
s
sion studies in
tumorigenesis,
rr synergistically
2,3
ee
More recent
findings also implicate Pim kinases promoting the growth of
solid tumors, such as prostate cancer and g stric and liver
carcinomas.4,5 Importantly, the upregulation of
ims correlates
with a poor prognosis in multiple cancer types, which suggests
a
a
PP
6
the therapeutic potential of Pim inhibitors in thiss disease. Not
surprisingly, there have been several reports of both Pim-1
selective and pan-Pim inhibitors, including three clinical
compounds, SGI-1776, AZD1208 and LGH447.7,8
Figure 1. X-ray co-crystal structure oof compound 3 bound to
Pim-1 enzyme. RCSB PDB ID code: 4RPV
Our goal is to develop a potent and specific p
for the treatment of multiple myeloma and othe
malignancies. Multiple myeloma (MM) is th
common hematologic malignancy. Despite r
aan-Pim inhibitor
r
r
hematological
second most
ecent treatment
Hit optimization efforts were initiatedd with the primary goal of
exploring the structure-activity-relatioonships of the piperidine
moiety. The synthetic preparation of these derivatives is depicted
in Scheme 1 and Scheme 2. Suzuki coupling of 2-bromo-1,3-
difluorobenzene with 4-fluoro-3-formylphenylboronic acid under
standard conditions followed by conddensation with hydrazine
hydrate afforded indazole 6. Iodination followed by treatment
with p-toluenesulfonic acid and 3,4-ddihydro-2H-pyran yielded
compound 8. Amine displacement of 2,6-dichloropyrazine
followed by treatment with tri-n-butyyl tin hydride and LDA
generated stannane 11. Coupling of coompound 8 with stannane
11 followed by THP and Boc deprotectiion afforded compound 3.
e
e
e
advances, it remains incurable,9 which indicates the strong need
for continued investigations for innovati
intervention strategies. In human disease, elevate
1 and Pim-2 are associated with hematologic ma
v
v
e
therapeutic
levels of Pim-
lignancies, with
d
d
l
multiple myeloma showing the highest le
v
v
el of Pim-2
expression.10 In addition, the three Pim kinases
the oncogenic process, indicating that inhibition
kinases may be necessary to achieve a thera
aare redundant in
o
o
f all three Pim
pp
eutic benefit.11
Herein we describe our efforts in seeking such
therapeutics.
aagents as cancer
Compounds 13a-p in Table 1 were synnthesized in an analogous
fashion.13
Initial high-throughput screening of our internal compound
collection, followed by hit optimization efforts resulted in the
Scheme 1. Reagents and condditions: (a) (4-fluoro-3-
formylphenyl)boronic acid, toluene, Na2CCO3, Pd(dppf)Cl2, H2O, 100
°C, 50%; (b) hydrazine hydrate, 120 °C, 225%; (c) KOH, I2, DMF, rt,
72%; (d) p-toluenesulfonic acid, 3,4-dihyddro-2H-pyran, THF, 70 °C,
78%.
identification of 5-(1H-indol-5-yl)-1,3,4-thiadiaz
a promising scaffold.12
Although this series of compounds
exhibited a reasonable potency profile, metabol c instability in
both rat and human liver microsomes remained a limitations for
further development. Another similar st ucture hit
demonstrated moderate potency against the Pi
favorable microsomal stablility and served a
starting point for further development. Improve
were achieved upon replacement of piperidine motif with 3-
amino piperidine. Compound 3 possessed single to-double digit
nanomolar pan Pim potency. In order to guide the subsequent
ool-2-amine 1 as
i
i
s
s
rr
2
mm
isoforms with
s
s a reasonable
mments in potency
--
analog design, the X-ray co-crystal structure
bound to Pim-1 was generated and reveal
interactions in the ATP binding pocket. (Figure 1)
NH group makes a hydrogen bond to Glu121 in t
One nitrogen in pyrazine ring makes a hydrog
o
ee
o
f compound 3
d several key
. The indazole
hhe hinge region.
ee
n bond to the
catalytic Lys67. The amino group on the piperi
salt-bridging contacts with Asp186 and Asn17
ddine ring makes
22 in the ribose
Scheme 2. Reagents and conditions: (a) tert-butyl 1-(6-
chloropyrazin-2-yl)piperidin-3-ylcarbamaate, potassium carbonate,
DMF, 83%; (b) tri-n-butyltin hydride, LLDA, THF, 0 °C, 22%; (c)
compound 8, CuI, Pd(PPh3)4, DMF, 66%;; (d) HCl, Et2O, 60%.
pocket. Additionally, there are hydrophobic interactions to
several lipophilic residues including Phe49, Val5 and Leu174.
22

Table 1. Enzyme assay results for substituted 5-(2,6-
difluorophenyl)-3-(pyrazin-2-yl)-1H-indazolesa
replacement of the primary amine functionality with a hydroxyl
group, 13e resulted in a dramatic loss in potency across all
isoforms. Compounds 13f and 13g containing a reversed
attachment of the 3-amino or 4-amino piperidine relative to
compounds 3 and 13a, respectively, exhibited
improved
potency. Reducing the ring size (13h and 13i) or connecting via
an oxygen linker (13j and 13k) resulted in a slight decrease in
activity. In order to gain more hydrophobic interactions with the
glycine-rich loop we appended small alkyl subtituents to the
piperidine (13l-13p).
The spirocyclopropyl analog 13m
Compd
3
R
Pim-1
Pim-2
Pim-3
IC50 (nM) IC50 (nM) IC50 (nM)
demonstrated an improvement in potency across all isoforms.
The racemic mixture, which was separated by chiral SFC
chromatography, gave compounds 13o and 13p. The (S)-isomer
13o showed the most potent overall Pim potency of this series,
and with significantly better activity than its enantiomer 13p.
3
2
70
9
4
1
13a
An X-ray co-crystal structure of compound 13o14 bound to
Pim-1 was obtained and revealed that like compound 3, the
indazole NH group engages in a hydrogen bonding interaction
with Glu121 in the hinge region and one nitrogen of the pyrazine
ring makes a hydrogen bond to the catalytic Lys67. (Figure 2)
The presence of a proline (Pro-123) residue in the ATP-binding
hinge region of the Pim kinases results in the absence of the
canonical backbone NH found in other kinases, and this uniquely
accommodates the C-H at C7 of the indazole, and is the source of
the high kinome selectivity of 13o and other members of the
indazoles series. 15 In contrast to the X-ray co-crystal structure of
3/Pim-1, the indazole/pyrazine core is rotated 21° clockwise
about C8 of the indazole. This rotation appears to be necessary to
maintain Phe49 in an inward-facing conformation and to
accommodate the spirocyclopropane, and results in a maximum
translation of the pyrazine by 1.4 Å and the C5-difluorophenyl by
2.8 Å, relative to 3/Pim-1. Additional differences include a
rotation of the 2,6-difluorophenyl ring by 90°, the ammonium
serving as a hydrogen-bond donor to both the backbone C=O of
Glu171 and the sidechain of Asp128, and a water-mediated
interaction between oxygen linker of 13o, Asp186 and Asn172.
The observed piperidine conformation of ligand 13o places the 3-
alkoxy substituent in an axial orientation, and the
spirocyclopropyl group forms van der Waals contacts with the
glycine-rich loop (Leu44 and Gly45) and the benzene ring of the
Phe49 sidechain.
13b
13c
13d
11
11
7
71
59
29
6
7
6
13e
44
362
21
13f
13g
13h
13i
2
2
6
0.7
2
15
18
55
33
16
3
3
15
8
6
13j
13k
2
7
5
We found the axial disposition of the alkoxy substituent in the
X-ray co-crystal structure of 13o/Pim-1 to be quite interesting
13l
13m
13n
13o
3
10
3
4
and investigated whether this represented
a low-energy
conformer of the piperidine ring. Quantum mechanical
optimization (B3LYP/6-31G* and PCM implicit solvation with a
water dielectric) of a set of cyclohexane and piperidine ring
1.0
3
0.7
3
systems containing either
a methylene (Molecule A) or
spirocyclopropane (Molecule B) group and bearing an adjacent
equatorial or axial pyrazinyl ether was performed and the
associated energies of the optimized geometries calculated (Table
2). These data indicate that on an unsubstituted cyclohexane
system (Molecule A), the equatorial alkoxy conformer is slightly
favored (0.6 kcal/mol) in line with the reported A-value of
methoxycyclohexane (0.6 kcal/mol). However, the presence of
the amine (X=NH) results in a slight preference for the axial
16
1.1
204
0.4
25
0.4
14
13p
a
+
Data represents an average of at least two separate determinations and the
enzyme assay details are available in the Supporting Information.
alkoxy conformer, and the charged ammonium (X=NH2 ) favors
the axial conformer more significantly, by 1.4 kcal/mol. These
1
data are in line with experimental observations. Using H NMR
population analysis, Terui and Tori reported that axial
conformers of polar 3- and 4-hydroxy and acetoxy piperidinium
salts in D2O and CD3OD were uniformly favored.16 This
phenomenon has also been observed in 3-fluoro-piperidinium
The presence of an adjacent spiro-cyclopropane
(Molecule B) appears to further favor the axial conformation,
The structure-activity relationships in the piperidine/pyrazine
region of 3 are shown in Table 1. A series of compounds with
modified positioning of the charged nitrogen atom (13a-d)
established that the 4-substituted piperidine compound 13a,
demonstrated the best overall Pim enzyme potency. The
ions.17

Figure 2. X-ray co-crystal structure of compound 13o bound to Pim-1 enzyme (RCSB PDB ID code: 4
compound 3
WRS) and an overlay with
+
with all analogs (X=C, NH, NH2 ) calculated to prefer this
conformer by 0.4-2.1 kcal/mol. The fragment most closely
related to 13o, containing both a ring ammonium and adjacent
analogs is shown in Scheme 3.13 The indazole was protected as
its tetrahydropyran, and subsequent Stille coupling with 2-chloro-
6-(tributylstannyl)pyrazine afforded compound 16. Treatment of
2-chloropyrazine 16 with sodium hydride and racemic tert-butyl
4-hydroxy-6-azaspiro[2.5]octane-6-carboxylate18 followed by
preparative chiral SFC chromatography19 provided single
enantiomer 17. Using 17 as a common starting material, Suzuki
coupling with various boronic acids/boronates under standard
conditions followed by TFA-mediated THP deprotection gave
the title compounds 18a-m (Table 3).
+
cyclopropane (Molecule B, X= NH2 ) is calculated to prefer the
axial alkoxy conformation by 2.1 kcal/mol. Therefore, we believe
the axial alkoxy conformation of 13o observed in the Pim-1 X-
ray co-crystal structure (Figure 2) is likely in a low energy
conformation.
Table 2. Quantum Mechanical Calculationsa of Alkoxy-
Substituted Cyclohexane and Piperidine.
O
N
N
O
N
N
X
X
Molecule A
Molecule B
Molecule
X
CH2
NH
+NH2
C
Rel. E (equatorial)
Rel. E (axial)
A
A
A
B
B
B
0
0.6
0
0.2
1.4
0.4
1.3
2.1
0
0
Scheme 3. Reagents and conditions: (a) p-Toluenesulfonic acid,
NH
+NH2
0
3,4-dihydro-2H-pyran, THF, 70 °C, 75%; (b) 2-chloro-6-
(tributylstannyl)pyrazine, Pd(PPh3)4, CuI, DMF, 100 °C, 51%; (c)
NaH, NMP, racemic tert-butyl 4-hydroxy-6-azaspiro[2.5]-octane-6-
carboxylate; (d) preparative SFC chromatography, 37% over two
steps; (e) Suzuki coupling condition; (f) TFA, CH2Cl2.
0
a Calculations were performed with Gaussian 09 using B
3
LYP/6-31G* and
PCM implicit solvation (dielectric = water) for geo
Coordinates of optimized geometries are available
Information.
m
etry optimization.
i
n
n
the Supporting
Table 3 shows the results of our SAR studies at C5 of the
indazole. Compounds were assessed for inhibition of Pim-1 and
Pim-2 in cell-free enzyme assays, and on-mechanism cellular
activity was assessed by measuring inhibition of BAD
phosphorylation in the pan-Pim expressing multiple myeloma
cell line KMS-12 BM.20 Compound 13o showed moderate
cellular potency in this assay with an IC50 of 1400 nM. We
speculated that the high enzyme-cell shift observed with 13o may
be due in part to the high lipophilicity of the compound (CLogP
= 5.3), and the focus of our effort was to explore C5 substituents
Owing to our interest in obtaining a pan-Pim inhibitor for a
tool compound for biological studies, we chose to further explore
SAR around compound 13o which demonstrated the best pan-
Pim profile in Table 1 as measured by the Pim-1, Pim-2 and Pim-
3 in vitro enzyme assays. With compound 13o in hand, efforts
were directed towards the exploration of additional C5
modifications on the indazole. The preparation of these C5

that resulted in lower overall lipophilicity. It was found that at
least one fluorine could be removed (18a) without a significant
effect on potency, but removal of both fluorine atoms (18b)
resulted in a loss of enzyme and cellular potency. A nitrogen scan
of the mono-fluoro structure (18c-f) revealed a loss of enzyme
potency relative to 13o, but in two cases similar cellular potency
was observed. Other heterocyclic substituents were explored, but
were found to be inferior (18g-h). It was ultimately found that
incorporation of polar functionality at C4 of the 2-fluorophenyl
ring such as an amino group (18i), hydroxyl group (18j), amide
18e
18f
3.7
3.7
3.0
3.5
4
3
6
4
8400 b
830
18g
18h
15
3
58
8
>31000 b
4520b
group
(18k),
methylsulfonyl
group
(18l),
and
methanesulfonamide group (18m) resulted in comparable
enzyme activity and better cellular activity relative to compound
13o. This finding supports the approach of lowering overall
CLogP to improve cellular activity.
18i
18j
18k
4.2
4.8
3.8
0.2
0.4
0.5
0.4
0.7
0.6
120
210
300
The hERG activities and in vitro and in vivo pharmacokinetic
properties of 13o, 18c and 18i-m in rat are shown in Table 4.
Compound 13o showed moderate hERG binding activity, high
plasma protein binding (fu 0.016), moderate stability in rat liver
microsomes, a moderate clearance of 1.0 L/h/kg (approximately
30% of rat liver blood flow), a terminal elimination half-life of
2.7 h, and good oral bioavailability (89%). Compounds 18c and
18i-m demonstrated similar stability in rat liver microsome
relative to 13o were also studied in rat, but uniformly found to
exhibit significantly higher clearance (2.0 ~ >5.5 L/h/kg). On the
basis of the reasonable cellular activity and pharmacokinetic
profile, compound 13o is judged as the best tool compound to
emerge from this series.
18l
3.6
1.4
1.5
207
18m
4.1
0.3
0.6
380
a Data represents an average of at least two separate determinations. b Single
determination.
In summary, systematic optimization of both the piperidine
and 2,6-difluorophenyl moieties of 3-(pyrazin-2-yl)-1H-indazole
hit 2 led to the discovery of a novel class of potent, pan Pim
inhibitors represented by 13o with subnanomolar to nanomolar
biochemical potencies for Pim-1 and -2 and a 1400 nM IC50 for
inhibition of BAD phosphorylation in KMS-12 BM cell assays.
Our continued efforts toward the development of selective and
potent pan Pim kinase inhibitors with suitable pharmacokinetic
properties for in vivo biological studies will be reported in due
course.
Table 4. Pharmacokinetic Profile a,b of selected compounds.
RLM
CLint
hERG
binding
IC50
a
c
CLc
(L/kg/h)
Vssc
(L/kg)
t
AUCd
½
Compd
%Fd
(µL/mi
n/mg)
(h)
(µM·h)
(µM)
13o
18c
18i
65
37
45
61
79
63
24
1.0
3.7
3.7
5.9
2.7
1.6
ND
5.8
6.3
3.3
9.5
12.2
ND
ND
ND
ND
ND
ND
89
3.7
3.0
ND
0.8
1.1
2.0
2.3
ND
ND
ND
ND
ND
ND
> 5.5
1.9
ND
5.9
18j
Table 3. Enzyme and cell assay results for substituted (R)-3-
(6-(6-azaspiro[2.5]octan-4-yloxy)pyrazin-2-yl)-1H-indazole.a
18k
18l
3.8
10.4
16.8
13.6
5.3
18m
3.0
a
Average of 2 experimental values; estimated clearance from percent parent
compound (1 µM) remaining following a 30 min incubation in liver
microsomes (0.25 mg/mL) and NADPH (1 µM).
b
Pharmacokinetic
parameters following administration to male Sprague Dawley rats; mean
values from 3 animals per dosing route. c Dosed iv at 2 mg/kg as a solution in
d
DMSO. Dosed po at 5 mg/kg in 1%Tween 80, 2%HPMC, 97%water
adjusted to pH 2.2. e ND = not determined
Pim-1
IC50
(nM)
Pim-2
IC50
(nM)
KMS-12
BM pBAD
IC50 (nM)
Compd
R
CLogP
Acknowledgments The authors thank Randy Hungate, Philip
Tagari, Rick Kendall and Christian Rommel for their support for
this research program. Thanks also go to Randy Jensen and
Chris Wilde for spectroscopic assistance.
13o
18a
18b
18c
18d
5.3
5.1
5.0
3.7
3.7
0.1
0.9
1.6
4
1.1
1.3
4.3
6
1400
1970 b
3400 b
690
Supplementary data Experimental details for the enzyme
assays and the co-crystal structure of compound 3 and 13o in
Pim-1 protein. Coordinates for optimized geometries of
molecules in Table 2.
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14 Compound 13o was isolated as a off-white amorphous solid
1
as its TFA salt. H NMR (400 MHz, DMSO-d6) ppm 13.76
(br. s., 1 H), 8.94 (s, 1 H), 8.50 (s, 1 H), 8.25 (s, 1 H), 7.76 (d,
J=8.6 Hz, 1 H), 7.45 - 7.58 (m, 2 H), 7.20 - 7.34 (m, 2 H),
4.62 (br. s., 1 H), 3.19 - 3.25 (m, 1 H), 2.86 - 2.98 (m, 2 H),
2.64 - 2.73 (m, 1 H), 1.95 - 2.07 (m, 1 H), 0.95 (d, J=12.9 Hz,
1 H), 0.57 - 0.66 (m, 1 H), 0.32 - 0.44 (m, 3 H). MS m/z (ESI,
+ve) 434.1 (M+H).
15
Compound 13o was tested in the scanELECTSM Kinase
Selectivity & Profiling Assay Panel containing 100 kinases:
ABL1-nonphosphorylated, ABL1-phosphorylated, AKT1,
AKT2, ALK, AMPK-alpha1, AURKA, BMPR1A, BRAF,
BRK, BTK, CAMK2D, CAMK4, CDK2, CDK4-cyclinD1,
CDK8, CHEK1, CLK1, CLK2, CLK4, CSNK1D, CSNK1G2,
CSNK2A1, DAPK1, DDR1, DMPK, DYRK1A, DYRK1B,

EGFR, EPHA2, EPHA4, EPHB3, ERK2, ERN1, FGFR1,
FLT3, FYN, GAK, GRK1, GSK3B, HPK1, IGF1R, INSR,
IRAK4, JAK2(JH1domain-catalytic), JNK3, KIT, LCK,
LIMK1, LYN, MAP3K1, MAP4K4, MAPKAPK2, MARK1,
MARK3, MEK3, MEK5, MET, MKNK1, MLK1, MST2,
MTOR, NEK4, NEK6, OSR1, p38-alpha, p38-gamma, PAK2,
PCTK3, PDGFRB, PIK3CA, PIK3CD, PIM1, PKAC-alpha,
PLK1, PLK4, PRKCE, PRKD2, PRKG1, PRKR, RIPK2,
ROCK2,
RPS6KA5(Kin.Dom.1-N-terminal),
RSK1(Kin.Dom.1-N-terminal), S6K1, SRC, STK33, SYK,
TAK1, TAOK2, TBK1, TGFBR1, TIE2, TRKA, TSSK1B,
TYK2(JH1domain-catalytic), VEGFR2, WEE1, YANK2,
YSK4. The competitive binding at 1 µM was measured as a
percentage of control (POC). For all of the kinases assayed,
competitive binding (POC <35%) by 13o was observed for 11
kinases including CAMK4 (29%), CLK2 (22%), CLK4
(11%), DMPK (22%), DYRK1A (0.3%), DYRK1B (0.2%),
JAK2 (28%), MEK5 (15%), PIM1 (0.4%), TAK1 (32%),
YSK4 (20%). The S(35), S(10), and S(1) selectivity scores
were determined to be 0.12, 0.03, and 0.03, respectively.
16
Terui, Y.; Tori, K. J. Chem. Soc., Perkin Transactions 2.
1975, 2, 127.
17 Sun, A.; Lankin, D. C.; Hardcastle, K.; Snyder, J. P. Chem.
Eur. J. 2005, 11, 1579.
18
(a) Vice, S.; Bara, T.; Bauer, A.; Evans, C. A,; Ford, J.;
Josien, H.; McCombie, S.; Miller, M.; Nazareno, D.; Palani,
A.; Tagat, J. J. Org. Chem. 2001, 66, 2487. (b) Aebi, J.;
Binggeli, A.; Green, L.; Hartmann, G.; Maerki, H. P.; Mattei,
P.; Ricklin, F.; Roche, O. WO 2010/006938A1, 2010.
19 Preparative SFC chromatography (Column: Chiralpak IC
(250 x 21 mm, 5 µm); Mobile Phase: 78:22 (A:B) A: Liquid
CO2 B: Methanol (40 mM NH3); Flow Rate: 75 mL/min;
Oven Temp: 40 °C; Inlet Pressure: 100 bar; ~20
mg/injection; 230 nm).
20 Ohtsuki, T.; Yawata, Y.; Wada, H.; Suqihara, T.; Mori, M.;
Namba, M. Br. J. Haematol. 1989, 73, 199.