Discovery of imidazopyridazines as potent Pim-1/2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.09.067

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers, suggesting that inhibition of Pim signaling could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal using a screening hit (rac-1) as a starting point. Modification of the indazole ring resulted in the discovery of a series of imidazopyridazine-based Pim inhibitors exemplified by compound 22m, which was found to be a subnanomolar inhibitor of the Pim-1 and Pim-2 isoforms (IC₅₀values of 0.024?nM and 0.095?nM, respectively) and to potently inhibit the phosphorylation of BAD in a cell line that expresses high levels of all Pim isoforms, KMS-12-BM (IC₅₀?=?28?nM). Profiling of Pim-1 and Pim-2 expression levels in a panel of multiple myeloma cell lines and correlation of these data with the potency of compound 22m in a proliferation assay suggests that Pim-2 inhibition would be advantageous for this indication.

Full text of DOI:10.1016/j.bmcl.2016.09.067

Accepted Manuscript
Discovery of imidazopyridazines as potent Pim-1/2 kinase inhibitors
Ryan P. Wurz, Christine Sastri, Derin C. D’Amico, Brad Herberich, Claire L.
M. Jackson, Liping H. Pettus, Andrew S. Tasker, Bin Wu, Nadia Guerrero, J.
Russell Lipford, Jeffrey T. Winston, Yajing Yang, Paul Wang, Yen Nguyen,
Kristin L. Andrews, Xin Huang, Matthew R. Lee, Christopher Mohr, J. D.
Zhang, Darren L. Reid, Yang Xu, Yihong Zhou, Hui-Ling Wang
PII:
S0960-894X(16)31016-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.067
BMCL 24291
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 July 2016
23 September 2016
27 September 2016
Please cite this article as: Wurz, R.P., Sastri, C., D’Amico, D.C., Herberich, B., M. Jackson, C.L., Pettus, L.H.,
Tasker, A.S., Wu, B., Guerrero, N., Russell Lipford, J., Winston, J.T., Yang, Y., Wang, P., Nguyen, Y., Andrews,
K.L., Huang, X., Lee, M.R., Mohr, C., D. Zhang, J., Reid, D.L., Xu, Y., Zhou, Y., Wang, H-L., Discovery of
imidazopyridazines as potent Pim-1/2 kinase inhibitors, Bioorganic & Medicinal Chemistry Letters (2016), doi:
http://dx.doi.org/10.1016/j.bmcl.2016.09.067
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Discovery of imidazopyridazines as potent Pim-1/2 kinase inhibitors
Ryan P. Wurz,*,a Christine Sastri,*,b Derin C. D’Amico,a Brad Herberich,a Claire L. M. Jackson,a Liping
H. Pettus,a Andrew S. Tasker,a Bin Wu,a Nadia Guerrero,b J. Russell Lipford,b Jeffrey T. Winston,b
Yajing Yang,b Paul Wang,c Yen Nguyen,d Kristin L. Andrews,e Xin Huang,e Matthew R. Lee,e
Christopher Mohr,e J. D. Zhang,e Darren L. Reid,f Yang Xu,g Yihong Zhou,h and Hui-Ling Wang.a
Amgen Inc. One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
a) Department of Therapeutic Discovery; b) Department of Oncology Research; c) Department of Discovery Technologies;
d) Department of Discovery Attribute Sciences; e) Department of Molecular Engineering; f) Department of Pre-pivotal Drug
Product; g) Department of Clinical Pharmacology; h) Department of Pharmacokinetics and Drug Metabolism.
* To whom correspondence should be addressed:
Ryan P. Wurz: Tel +1-805-313-5400; Fax 805-480-3016; email: rwurz@amgen.com
Christine Sastri: Tel +1-805-313-5629; email: csastri@amgen.com
This is where the receipt/accepted dates will go; Received Month XX, 2016; Accepted Month XX, 2016 [BMCL RECEIPT]
Abstract—High levels of Pim expression have been implicated in several hematopoietic
and solid tumor cancers, suggesting that inhibition of Pim signaling could provide patients
with therapeutic benefit. Herein, we describe our progress towards this goal using a
screening hit (rac-1) as a starting point. Modification of the indazole ring resulted in the
discovery of a series of imidazopyridazine-based Pim inhibitors exemplified by compound
22m, which was found to be a subnanomolar inhibitor of the Pim-1 and Pim-2 isoforms
(IC50 values of 0.024 nM and 0.095 nM, respectively) and to potently inhibit the
phosphorylation of BAD in a cell line that expresses high levels of all Pim isoforms, KMS-12-BM (IC50 = 28 nM). Profiling of Pim-1 and
Pim-2 expression levels in a panel of multiple myeloma cell lines and correlation of these data with the potency of compound 22m in a
proliferation assay suggests that Pim-2 inhibition would be advantageous for this indication.
Proviral Integration site of Moloney (Pim) murine leukemia virus kinases are constitutively active
serine/threonine kinases that are involved in cell survival and proliferation as well as a number of other
signal transduction pathways.1,2,3 The Pim-1, -2 and -3 isoforms share a high level of sequence
homology and appear largely redundant in function. High levels of Pim expression have been
implicated as oncogenic drivers in hematologic and solid malignancies4 including multiple myeloma,5
acute myeloid leukemia, prostate cancer, and gastric and liver carcinomas.6 These findings, in
1

conjunction with the observation that the triple knockout of Pim proteins has a mild phenotype, suggest
that inhibition of Pim signaling by a pan-Pim inhibitor could provide patients with therapeutic benefit.3
All three Pim isoforms share the unique feature among kinases of having a proline residue in the
hinge, which results in only one hydrogen bond interaction with ATP.7 This property can be exploited
to derive selectivity over other kinases. However, among the Pim isoforms, the KM,ATP for Pim-2 is 136-
fold higher affinity than that for Pim-18 which makes the design of Pim inhibitors with potent cellular
activities in pan-Pim expressing cell lines very challenging. Pim directly phosphorylates Bcl-2-
associated death promoter (BAD) which becomes sequestered in pBAD-(14-3-3) protein heterodimers.
Inhibition of Pim-mediated BAD phosphorylation is expected to lead to high levels of free BAD which
can bind to the antiapoptotic Bcl-2 protein, causing release of the proapoptotic Bax and BAK.9
Phosphorylation of BAD at the serine 112 site is a non-redundant function of the Pim kinases.10 To
evaluate the cellular potency of novel Pim inhibitors, a multiple myeloma cell line, KMS-12-BM11, was
used to develop a cellular assay to advance compounds in this study using the inhibition of
phosphorylation of BAD at the serine 112 site (pBAD(s112)) as a gauge for activity.12
The development of Pim inhibitors13 has been very active in recent years resulting in several clinical
compounds including: SGI-1776,14 AZD-120815 and PIM447.16 Herein, we describe our progress
towards the development of a novel class of Pim-1/2 inhibitors based on an imidazopyridazine scaffold.
In previous communications, we described our efforts in the development of Pim inhibitors based on
aminothiadiazole 2,17 aminooxadiazole 3,18 quinazolinone-pyrrolopyrrolones 4,19 and macrocyclic
quinoxaline-pyrrolo-dihydropiperidinone scaffold 5.20 Although several of these compounds possessed
attractive cellular activities, limited solubility and/or undesirable pharmacokinetic properties precluded
their further development. Rac-1 represented a previously identified lead from a high-throughput
screening campaign and resulted in the discovery of indazole 6.21 The basic amine present in indazole 6
was beneficial for the simulated intestinal fluids (SIF) solubility of these Pim inhibitors and this
favorable property could enable improved exposures in high dose toxicology studies. However, its
2

presence likely contributed to a large shift in enzyme to cellular potency resulting in a compound that
was >1 µM in the KMS-12 pBAD cellular assay.
H
H
N
N
N
N
N
NH
N
NH2
N
O
S
N
N
N
NH
2
3
Pim-1 IC50 2.0 nM
Pim-2 IC50 10 nM
Pim-1 IC50 1.4 nM
Pim-2 IC50 2.4 nM
KMS-12 BM IC50 ND
KMS-12 BM IC50 0.607
Sol. (SIF) 135
M
Sol. (SIF) 418
M
M
O
O
N
O
N
HN
N
N
HN
NH
NH
HN
HN
5
4
Pim-1 IC50 0.3 nM
Pim-2 IC50 0.5 nM
Pim-1 IC50 0.3 nM
Pim-2 IC50 0.2 nM
KMS-12 BM IC50 0.025
M
KMS-12 BM IC50 0.031
M
Sol. (SIF) 295
M
Sol. (SIF) 104
M
HN
N
N
N
F
O
F
N
6
H
Pim-1 IC50 0.4 nM
Pim-2 IC50 1.1 nM
KMS-12 BM IC50 1.41
Sol. (SIF) 320
M
M
Figure 1. Recent literature reports of Pim inhibitors from Amgen.
A previously reported x-ray co-crystal structure of (rac)-1 in the ATP-binding pocket of Pim-1
protein revealed that the (S)-isomer of the 3-aminopiperidine was the preferred enantiomer for binding
(Fig. 2). In addition, this compound showed three strong electrostatic interactions that likely
contributed to its potency including: 1) engagement of the indazole N-H in a hydrogen bonding
interaction with the carbonyl of Glu121 residue (3.0 Å) of the hinge region; 2) formation of a hydrogen
bond with the nitrogen of the pyrazine and the catalytic Lys67 (3.2 Å); 3) strong hydrogen bonding
interactions of the amino group in the 3-position of the piperidine ring with the Asp186 and Asn172
residues, at 3.2 Å and 3.3 Å, respectively. Inclusion of the protein surface of the binding pocket
suggested that additional van der Waals interactions could be obtained by further exploiting
hydrophobic dimples on the roof of the binding pocket.
3

Figure 2. X-ray of compound (S)-1 bound in the ATP binding site of unphosphorylated Pim-1 protein determined to 3.0 Å r
esolution. PDB code: 4RPV.21
Dashed lines indicate hydrogen bonds. For the inhibitor, C: green; N: blue; F: light blue.
Molecular modeling guided modification of rac-121 led us to prepare compound 7 bearing an amide
tethered to the 3-position of an aza-indole. Compound 7 possessed similar Pim-1 potency (Pim-1, IC50 =
65 nM), however, this modification led to a 100-fold decrease in its Pim-2 potency (Pim-2, IC50 = 314
nM). This scaffold is similar to recently disclosed Pim inhibitors from Novartis 916,22 Biogen 1023 and
Genentech 11.24 Removal of the amide carbonyl and modification of the nitrogen substitution pattern of
the bicyclic ring yielded a novel 7-amino-imidazopyridazine core represented by 8 (Fig. 3).25 This ring
system was designed to favor co-planarity of the core while maintaining key interactions for Pim
binding.
N
N
N
HN
Ar
N
N
N
O
HN
SAR
N
H
N
H
N
N
N
N
NH2
Table 1
N
F
F
NH2
F
F
NH2
F
1
7
8
rac-
New Scaffold
O
N
N
O
HN
O
F
N
O
N
N
N
H
N
N
H
N
H
S
N
N
F
F
F
HN
F
NH
NH
NH2
F
9
10
Biogen Idec
11
(PIM447)
Novartis
Genentech
F
Figure 3. Preliminary SAR leading to Amgen’s starting point from (rac-1) and structures of publicly disclosed Pim inhibitors.
As a starting point for investigation of the new scaffold 8, we chose to introduce a benzene ring (Ar =
benzene) to append the imidazopyridazine scaffold and to present the basic aminopiperidine motif to the
acidic patch of residues in the ATP binding pocket in the Pim-1 protein (compound 8a). This
4

compound had micromolar activity in the cell (KMS-12 pBAD IC50 = 3.4 µM). Introduction of a 3-
pyridyl ring (compound 8b) resulted in subnanomolar potency in the Pim-1 and Pim-2 enzymatic assays
and a modest submicromolar activity in the KMS-12-BM cellular assay (IC50 = 403 nM). The
introduction of other heterocyclic motifs indicated that the positioning and presence of the 3-pyridyl
nitrogen was critical for activity. For example, placement of the nitrogen in this heterocycle in the 4-
position (compound 8c) was >1 µM in the Pim-1 and Pim-2 enzymatic assays. Introduction of an
additional nitrogen to the 3-pyridyl ring in the 5-position (compound 8d) as well as replacement of the
3-pyridyl ring with a bioisostere, such as a thiazole ring (compound 8e), were also not well tolerated.
Table 1. SAR of central ring.a
KMS-12
Pim-1
Pim-2
R/HLM CLint
Cmpd
8a
Ar
pBAD
(µL/min*mg)
IC50 (nM) IC50 (nM)
IC50 (nM)
2.2b
6.5b
3440b
403
63/82
N
0.10
>1000b
19b
0.50
130/66
61/67
8b
8c
8d
>1000b
110b
ND
>15,800b
110/28
5

56
148
ND
167/28
8e
a Data represents an average of at least two separate determinations and the enzyme assay details are available in
the Supporting information. b Single determination.
A representative synthesis of this class of Pim inhibitors is depicted in Scheme 1 wherein compound
8b was prepared in a six linear step sequence.26 Commercially available 3-chloro-6-methylpyridazine
(12) was converted to 3-iodo-6-methylpyridazine (13) upon heating with hydroiodic acid. Negishi
cross-coupling of 13 and 2,6-difluorobenzenezinc bromide catalyzed by AmPhosPdCl2 afforded 3-
substituted pyridazine 14 in 94% yield. Benzylic chlorination mediated by trichloroisocyanuric acid
was used to prepare 6-chloromethyl-pyridazine 15 which upon treatment with sodium azide afforded the
desired benzylic azide 16 in high yield. The synthesis of the right-hand-side of the molecule
commenced with a SNAr displacement of 4-chloro-3-nitropyridine with commercially available (S)-3-
(N-Boc-amino)-piperidine (17) followed by a nitro reduction to furnish the corresponding 3-
aminopyridine 18. Treatment of this compound with 1,1’-thiocarbonylimidazole afforded the
corresponding isothiocyanate 19 which could be isolated in modest yields following chromatography on
silica gel. An aza-Staudinger reaction between isothiocyanate 19 and the benzylic azide 16 facilitated
by trimethylphosphine formed the desired imidazopyridazine 20.27 Finally, HCl-mediated Boc-
deprotection afforded Pim inhibitor 8b.
6

Scheme 1. Synthesis of compound 8b.
Reagents and conditions: a) 57 wt% aq. HI (11 equiv.), 100 °C, 1.5 h, 92%; b) 2,6-difluoro-C6H3ZnBr (1.4 equiv.), AmPhosPdCl2 (0.04 equiv.), THF, 60 °C,
1 h, 94%; c) trichloroisocyanuric acid (0.35 equiv.), 1,2-DCE, 90 °C, 1.5 h; d) NaN3 (1.3 equiv.), DMF, r.t., 3 h, 94% (2 steps); e) 4-chloro-3-nitropyridine
(1.5 equiv.), 2-propanol, DIEA (2.5 equiv.), 60 °C, 3 h, 99%; f) Pd/C, EtOH, 30 psi H2, 6 h, r.t., 89%; g) 1,1’-thiocarbonylimidazole (2 equiv.), THF, 60 °C,
1 h, 67%; h) PMe3 (1.05 equiv.), THF, 0 °C – r.t., 3 h, 88%; i) HCl, EtOAc, r.t., 92%.28
The x-ray crystal structure of compound 8b in the ATP-binding pocket of Pim-1 protein revealed
a number of key interactions with the Pim-1 protein that likely contributed to its potency (Fig. 4). The
C−H at C4 of the imidazopyridazine engages in a favorable interaction (3.4 Å) with the carbonyl of the
Glu121 residue of the hinge. A water mediated hydrogen bond is also formed between N6 of the
imidazopyridizine and the backbone N−H of Asp186 and the rest of the imidazopyridazine ring sits
against the gatekeeper residue Leu120 and potentially gains favorable van der Waals interactions. The
nitrogen of the 3-pyridyl group forms a hydrogen bond with the catalytic Lys67 (3.1 Å) and helps
understand the steep decline in activity observed with compound 8c wherein the position of the nitrogen
is moved to the 4-position of this ring. The amino group in the 3-position of the piperidine ring forms
strong hydrogen bonding interactions with the Asp128 and Glu171 residues, at 2.7 Å and 2.7 Å,
respectively. Additional favorable hydrophobic contacts are observed between the fluorine atoms of the
2,6-difluorobenzene motif appended to the C2 of the imidazopyridazine ring and Val126 and Leu174
residues.
7

Figure 4. X-ray of compound 8b bound in the ATP b
the inhibitor, C: magenta; N: blue; F: light blue.
Next, the substitution patterns at C
compound 8b exhibited excellent P
modest cellular potency. In additio
The synthetic precursor to these ana
activity in the Pim-1 and Pim-2 enzy
the bottom of the ATP binding pocke
systematic analysis of the 2,6-difluo
fluorine atoms (compound 21b) w
compared to compound 8b. Replac
21c) was also found to be detrime
difluorobenzene substitution (compo
a 2,4-dichlorobenzene substitution
introduction of heterocycles at C2
Consequently, introduction of a 2-py
the parent compound but this modif
fluoro-2-pyridyl group 21g showed
compound 8b. This compound al

Conversely, its regioisomer, a 2-fluoro-3-pyridyl ring (compound 21h), was less potent but maintained
its microsomal stability. A 6-chloro-2-pyridyl group (compound 21i) was similar in potency to the
parent compound and displayed an improvement in microsomal stability in a similar fashion to its fluoro
analog. A thiazole ring was also introduced (compound 21j) and this substitution resulted in a
compound that was <2-fold shifted from the parent compound but had improved microsomal stability.
Table 2. SAR for substitution at C2 of the imidazopyridazinea
Lys67
KMS-12
Pim-2
pBAD
R/HLM CLint
Pim-1
R
Cmpd
IC50
µL/(min*mg)b
IC50 (nM)
IC50
(nM)
(nM)
0.07
25c
0.47
99c
403
130/66
27/18
8b
Cl
ND
21a
21b
0.23c
1.6c
1536c
120/48
0.89c
0.38c
3.4c
3.1c
2668c
1761c
122/90
132/31
21c
21d
9

1.8c
11c
5007c
260/100
21e
1.5c
6.4c
1.1c
12c
1760c
286
<10/<10
40/36
21f
21g
21h
0.23c
0.83c
6599c
39/34
Cl
0.94c
1.0c
2.6c
2.9c
620
602
58/52
54/27
21i
21j
N
a Data represents an average of at least two separate determinations and the enzyme assay details
are available in the Supporting information. b Single experimental value; estimated clearance from
percent parent compound (1 µM) remaining following a 30 min incubation in liver microsomes
(0.25 mg/mL) and NADPH (1 mM). c Single determination.
In an attempt to further improve cellular potency, we decided to explore modifications to the
piperidine ring (Table 3). The corresponding (R)-isomer of the 3-aminopiperidine (compound 22a) was
>100-fold weaker in the enzymatic assay than the (S)-isomer and less stable in H/RLM. Removal of the
3-amino group from the piperidine (compound 22b) resulted in significant loss in potency, as did
replacement of this amine with an alcohol (compound 22c). Replacement of the piperidine nitrogen
with a carbon atom resulted in a compound (rac-22d) that was 10-fold weaker in the cellular assay than
the parent compound 8b and additional analogues of this nature were not further pursued. Mono-
methylation of the amine (compound 22e) also resulted in loss of enzymatic activity. Ring contraction
to a 3-aminopyrrolidine ring (compound 22f) resulted in significant erosion in potency although this
modification was beneficial for the compound’s microsomal stability. We then focused SAR on
10

introducing substituents to reinforce the desired piperidine conformation for optimal binding as well as
trying to make additional van der Waals contacts with hydrophobic dimples on the roof of the ATP
binding pocket (Fig. 4). With this goal in mind, introduction of a methyl group at all positions of the
piperidine ring was explored. Accordingly, the cis- and trans-2-methyl-(3S)-aminopiperidine analogs
(compounds 22g and 22h) were prepared. The trans-diastereomer (compound 22h) provided a 2-fold
improvement in cellular potency (KMS-12 pBAD IC50 = 221 nM) while the cis-isomer (compound 22g)
was 10-fold weaker in the cellular assay. A racemic mixture of the geminally methylated analog (rac-
22i) was not well tolerated. Introduction of a trans-methyl-group in the 4-position of the piperidine ring
(compound 22j) was very potent in the enzymatic and cellular assays but this modification was
somewhat detrimental to the microsomal stability (R/HLM = 219/71 µL/(min*mg)). The corresponding
cis-4-methyl analog (compound 22k) was 10-fold weaker in the cellular assay. The trans-4-fluoro
substituted analog (compound 22l) was prepared in an attempt to improve the enzyme to cellular shift
and hERG binding (Table 5) of the 3-aminopiperidine by attenuation of its pKa but this modification,
unfortunately, resulted in a significant loss in enzymatic, cellular potency and microsomal stability.
Introduction of a methyl-group into the 5-position of the piperidine ring with a cis-disposition with
respect to the amino group (compound 22m) resulted in a significant improvement in enzymatic potency
and low double digit cellular potency (KMS-12 pBAD IC50 = 28 nM). Introduction of a cis-fluoro-
substitution in the 5-position of the piperidine ring29 (compound 22n) was also inferior. The
corresponding trans-5-methyl-piperidine analog (compound 22o) was also reasonably potent but still
20-fold less potent than the corresponding cis-analog in the cellular assay. Finally, we explored analogs
bearing a 6-methyl-3-aminopiperidine substitution pattern. Again, the cis-6-methyl-substitution
(compound 22p) appeared to be preferred over its trans-diastereomer (compound 22q), however, both
compounds had poor microsomal stability. Introduction of a 4-hydroxy-5-methyl-substitution
(compound 22r) on the piperidine ring resulted in another compound with low double digit nanomolar
potency in the cellular assay (KMS-12 pBAD = 31 nM).30
11

Table 3. SAR of the 3-aminopiperidine ring.a
KMS-12
pBAD
R/HLM CLint
Pim-1
Pim-2
Cmpd
R
IC50 (nM)b IC50 (nM)
µL/(min*mg)b
IC50 (nM)a
0.07
0.47
403
130/66
8b
7.6c
30c
35c
undef.
ND
280/90
22a
22b
22c
undef.
30c
>399/399
>399/472
2.9c
4807c
rac-
22d
3.7c
2.0c
27c
31c
5120c
106/<10
185/66
undef.
22e
22f
569c
>1000c
ND
<14/15
12

0.51c
0.062c
10c
5.4c
0.29
2.2c
2530c
221
312/102
344/79
311/91
22g
22h
rac-22i
undef.
0.035c
0.26c
7.4c
0.18c
1.6c
49c
212
219/71
22j
22k
22l
2982c
6100c
>399/130
>399/443
0.024c
0.78c
0.094c
3.2c
28
>399/69
143/51
22m
22n
22o
22p
983
522
49
0.39c
0.79c
0.31c
347/111
>399/203
0.021c
13

0.037c
0.04
1.0c
142
31
477/80
125/65
22q
22r
0.09
a Data represents an average of at least two separate determinations and the enzyme assay details are available in
the Supporting information. b Single experimental value; estimated clearance from percent parent compound (1
µM) remaining following a 30 min incubation in liver microsomes (0.25 mg/mL) and NADPH (1 mM). c Single
determination.
A representative synthesis for the substituted piperidine analogs is depicted in Scheme 2 using 22m as
an example.26 3-Amino-5-methylpyridine (23) was mono-Boc-protected using Boc-anhydride and
LiHMDS as a base. Hydrogenation of the 5-methyl-3-N-Boc-aminopyridine (24), with a combination
of PtO2 and Rh/C in acetic acid, afforded a racemic mixture of the corresponding cis- and trans-5-
methyl-3-N-Boc-aminopiperidines (25 and 26) in a ratio of 85:15 favoring the cis-isomer. Treatment of
this crude mixture with 4-chloro-3-nitropyridine enabled easier purification and furnished the desired
cis-5-methyl-N-Boc-piperidinyl(3-nitropyridine) (rac-27) in modest yields. Nitro reduction to the
corresponding 3-aminopyridine (rac-28) followed by treatment with 1,1’-thiocarbonylimidazole
produced the requisite isothiocyanate rac-29 as a coupling partner for the aza-Staudinger reaction.
Following cyclization with the desired benzylic azide (16), the enantiomers were separated and the Boc-
protecting group removed with TFA to furnish Pim inhibitor 22m.
14

Scheme 2. Synthesis of compound 22m. Reagents and conditions: a) LiHMDS (2.2 equiv.), Boc2O (1.25 equiv.), THF, r.t., 16 h, 39%; b) PtO2 (0.05
equiv.), 5% Rh/C (0.10 equiv.), H2 (200 psi), AcOH, 70 °C, 1 h; c) 4-Cl-3-NO2-pyridine, IPA, 60 °C, 3 h, 34% (2 steps); d) Pd/C, H2, EtOH, 2 h, quant.; e)
1,1’-thiocarbonylimidazole (2.0 equiv.), THF, 60 °C, 2 h, 34%. f) 13, PMe3 (1.1 equiv.), THF, r.t., 45 min, then chiral separation by SFC, 38%; g) TFA,
DCM, 0 °C, 30 min, 99%.31
Because it brought a good balance of potency and stability, we then prepared compounds containing
the 6-fluoro-2-pyridyl substitution pattern at the C2-position of the imidazopyridazine along with some
of the most promising substituted piperidines identified in Table 3. Accordingly, compound 30a
exhibited excellent cellular potency as well as an improvement in microsomal stability over its 2,6-
difluorobenzene analog at C2 (Table 4, compound 30a versus 22m). Compound 30b was also found to
have modest cellular potency (KMS-12 pBAD IC50 = 258 nM) as did compound 30c and both exhibited
an improvement in microsomal stability relative to compound 22m.
Table 4. Selected analogs containing a 6-fluoro-2-pyridyl-substitution at C2.a
KMS-12
Pim-1
Pim-2
R/HLM CLint
pBAD
Cmpd
R
IC50
(nM)
IC50
(nM)
µL/(min*mg)b
IC50
(nM)
0.06c
0.15c
0.43c
1.3c
45
91/33
30a
30b
258
194/71
0.29c
1.3c
115
50/21
30c
15

a Data represents an average of at least two separate determinations and the enzyme assay details are available in
the Supporting information. b Single experimental value; estimated clearance from percent parent compound (1
µM) remaining following a 30 min incubation in liver microsomes (0.25 mg/mL) and NADPH (1 mM). c Single
determination.
Rat pharmacokinetic (PK) studies were conducted with selected compounds in an attempt to further
differentiate some of the potent compounds (Table 5). Rat PK of the initial lead compound 8b showed
moderate iv clearance (CL = 1.4 L/kg/h, 40% of liver blood flow) with relatively low bioavailability
(%F = 16). Due to the presence of the basic amine, the hERG binding affinity was also monitored and
compound 8b was found to be a modest binder (hERG = 3.6 µM). Replacement of the 2,6-
difluorobenzene motif with a 6-fluoro-2-pyridyl group (compound 21g) resulted in an improvement in
iv clearance (CL = 0.7 L/kg/h, 21% of liver blood flow) and in bioavailability (%F = 28) with little
affect on hERG binding (hERG = 2.8 µM). Compound 22m, which showed the best overall cellular
potency, had a rat iv clearance (CL = 1.4 L/kg/h, 40% of liver blood flow) similar to the parent
compound 8b and similar bioavailability (%F = 17). Unfortunately, compound 22p bearing a 6-cis-
methyl-substitution on the piperidine ring had rapid iv clearance (CL = 5.5 L/kg/h) while the 4-hydroxy-
5-methyl analog 22r exhibited higher clearance than the parent compound (CL = 1.8 L/kg/h) with only
modest bioavailability (%F = 25) although the introduction of the 4-hydroxy group appeared beneficial
for hERG binding (hERG = 10.4 µM).
Introduction of the 6-fluoro-2-pyridyl group at C2 of the imidazopyridazine had little influence
on the overall clearance of the compound as compared to its 2,6-difluorobenzene analogs (30a versus
22m and 22r versus 30c), however, its introduction generally led to an improvement in oral
bioavailability and in the case of compound 30c greatly improved its hERG binding profile (hERG =
11.5 µM). Compound 22m was selected as a representative compound within this series for further
profiling. This compound showed low permeability and high efflux (4.5 x 10-6 cm/s and ER = 7.4,
respectively in a rat LLC-PK1 cell line transfected with a MDR1 gene). The aqueous solubility of
compound 22m was: 200 µg/mL, 180 µg/mL and 150 µg/mL in 0.01N HCl, PBS (pH 7.4) and FaSIF
(pH 6.8), respectively.32
16

The selectivity of compound 22m against a panel of protein and lipid kinases and their common
mutants was examined in the DiscoveRX scanMAX platform.33 In this panel, the competitive binding
of 22m was measured as a percentage of control (POC) indicated high kinase selectivity [S(35) = 0.078,
S(10) = 0.047 at 10 µM)]. For all of the kinases assayed, competitive binding (POC <10%) by 22m was
observed for 16 kinases including CSNK2A1 (4.2%), CSNK2A2 (2%), DYRK (3.4%), ERK8 (0.65%),
FLT3(D835Y) (6.1%), GSK3A (1.4%), GSK3B (2.3%), HIPK1 (7%), HIPK2 (1.8%), HIPK3 (1.2%),
p38δ (8.4%), PIP5K1C (7%), TAOK1 (0.6%), TAOK2 (7.8%), TAOK3 (0.4%) and VRK2 (9.2).34
Table 5. Pharmacokinetic properties and hERG IC50 of selected compoundsa
Cmpd
ivb
Vdss
poc
AUC(0-inf.)
(µM*h)
3.7
CL
MRT
(h)
F
hERG
(µM)d
3.6
(L/h/kg)
(L/kg)
(%)
8b
21g
22m
22p
22r
30a
30c
1.4
0.7
1.4
5.5
1.8
1.6
1.3
7.9
3.3
7.3
21.7
6.1
6.7
5.5
5.6
4.1
4.3
4.0
3.5
4.3
4.3
16
28
2.8
7.3
1.0
3.5
17
19.7
10.4
1.2
3.3
141
25
3.2
3.2
60
11.5
1.2
30
a Pharmacokinetic parameters following administration in male Sprague Dawley rats; mean values from 3 animals per dosing
b
c
route. Dosed at 2 mg/kg as a solution in DMSO. po doses were 5 mg/kg in 1% Tween 80, 2% hydroxypropyl
d
methylcellulose (HPMC), 97% water/methanesulfonic acid pH 2.2. hERG binding was assessed in HeK293 cells using
radiolabeled dofetilide (8 nM).
Achieving Pim-2 inhibition in the cellular context was the biggest challenge in development of
compound 22m. We then chose to profile its single-agent activity in a panel of human multiple
myeloma (MM) cell lines. Multiple myeloma has higher Pim-2 expression than any other cancer type,
and Pim-2 has a well-established role in proliferation and survival of these cells.35 In addition to
profiling the panel for sensitivity to our agent, basal Pim-1 and Pim-2 protein levels were determined
using sandwich ELISA assays that we developed with Meso Scale Diagnostics (MSD) (Table 6).
17

Consistent with the literature, high expression of Pim-2 protein was found to be present in all the
multiple myeloma cell lines. Interestingly, the cell lines that did not respond to compound 22m as a
single-agent have mutations that allow them to signal around the Pim pathway. We found that even
these lines could be sensitized to Pim inhibition when co-treated with another targeted molecule or
Standard of Care agent, which will be discussed in detail in a future publication.36
Table 6. Proliferation Activity of Compound 22m Correlation with Pim-1/2 Expression Levels.
Cell Linea
Basal Pim-1 levelsc
Basal Pim-2 levelsc
Proliferation IC50 (µΜ)b
MOLP-8
0.058
462 138.6
5,008 311.8
KMS-28-BM
KMS-11
0.086
0.100
0.130
0.162
0.216
0.228
0.332
0.332
0.337
0.361
0.375
0.393
0.457
0.569
2.12
470 53.7
1,338 82.0
2,126 105.4
341 40.3
13,069 442.6
24,763 1013.3
19,982 444.1
6,586 337.3
19,067 1074.1
8,064 195.2
12,309 455.4
9,624 529.6
56,645 2712.5
31,778 994.9
14,899 57.3
36,867 705.0
31,935 2507.4
23,228 413.7
57,425 2341.2
10,289 310.4
27,416 471.6
KMS-12-PE
Karpas620
KMS-12-BM
SK-MM-2
L-363
3,547 68.6
870 30.4
454 6.4
MOLP-2
2,061 88.4
612 28.3
NCI-H929
MM1.S
3,419 212.1
866 59.4
JJN-3
KMS-26
1,916 46.0
2,401 40.3
1,188 4.2
3,311 34.6
264 16.3
MM1.R
RPMI8226
KMM-1
OPM-2 (PTEN null)
U266-B1 (BRAF
mutant)
4.38
10.00
1479 147.1
a
b
Multiple myeloma cell line. IC50 value corresponds to 72 h incubation of compound 22m in the CellTiter-Glo®
proliferation assay. c Basal Pim-1 and Pim-2 protein levels as measured by MSD assays (n = 2 separate determinations) using
electrochemiluminescence (ECL) per 15 µg protein (see: Supporting information for further details).
Western blot analysis of KMS-12-BM cell lysates treated with compound 22m revealed an increase
in Pim-1/2/3 protein levels37 and a decrease in p-BAD(s112) while maintaining total-BAD levels
18

constant, consistent with specific inhibition of the Pim pathway. We also observed an increase in
cleaved-PARP and active caspase levels over time, indicating that Pim pathway inhibition results in
apoptosis.
Figure 5. Western blot analysis of KMS-12-BM cell lysates treated with 3.3 µM compound 22m.
In conclusion, a structurally novel class of Pim-1/2 inhibitors containing an imidazopyridazine motif
has been described by Amgen in 2012 which was followed by a similar structural class of Pim inhibitors
reported by Novartis in 2014.38 Compounds from Amgen’s series demonstrated improved solubility
attributed to the presence of the basic amine motif as compared to the previously reported scaffolds.
Optimization of the substitution patterns on the piperidine ring helped improve enzymatic potency to the
low double digit picomolar range resulting in the discovery of compound 22m which had superior
cellular potency over previously identified compounds (KMS-12 pBAD IC50 = 28 nM). The moderate
to high clearance of this class of compounds precluded their further advancement into preliminary
toxicology studies.
19

Supplementary data X-Ray data for the co-crystal structure of compound 8b in Pim-1 protein is
provided.
References and Notes
1. Nawijn, M. C.; Alendar, A.; Berns, A. Nat. Rev. Cancer 2011, 11, 23-33.
2. Santio, N. M.; Vahakoski, R. L.; Rainio, E.-M.; Sandholm, J. A.; Virtanen, S. S.; Prudhomme, M.; Anizon, F.; Moreau, P.;
Koskinen, P. J. Mol. Cancer 2010, 9, 279-292.
3. Magnuson, N. S.; Wang, Z.; Ding, G.; Reeves, R. Future Onc. 2010, 6, 1461-1478,
4. Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.; Schwaller, J. Haematologica 2010, 95, 1004-1015.
5. Lu, J.; Zavorotinskaya, T.; Dai, Y.; Niu, X.-H.; Castillo, J.; Sim, J.; Yu, J.; Wang, Y.; Langowski, J. L.; Holash, J.;
Shannon, K.; Garcia, P. D. Blood, 2013, 122, 1610-1620.
6. Tursynbay, Y.; Zhang, Y.; Tokay, T.; Zhumadilov, Z.; Wu, D.; Xie, Y. Biomed. Rep. 2015, DOI: 10.3892/br.2015.561.
7. Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.; Marsden, B. D.; Knapp, S. J. Med. Chem. 2005, 48, 7604-
7614.
8. Pim-1 KM,ATP = 407 µM; Pim-2 KM,ATP = 3 µM see: Qian, K.; Wang, L.; Cywin, C. L.; Farmer II, B. T.; Hickey, E.; Homon,
C.; Jakes, S.; Kashem, M. A.; Lee, G.; Leonard, S.; Li, J.; Magboo, R.; Mao, W.; Pack, E.; Peng, C.; Prokopowicz III, A.;
Welzel, M.; Wolak, J.; Morwick, T. J. Med. Chem. 2009, 52, 1814-1827.
9. Yan, B.; Zemskova, M.; Holder, S. J. Biol. Chem. 2003, 278, 45358-45367.
10. Macdonald, A.; Campbell, D. G.; Toth, R.; McLauchlan, H.; Hastie, C. J.; Arthur, J. S. C. BMC Cell Biol. 2006, 7, 1-14.
11. Ohtsuki, T.; Yawata, Y.; Wada, H.; Suqihara, T.; Mori, M.; Namba, M. Br. J. Haematol. 1989, 73, 199-204.
12. A cell-free kinase assay with recombinant Pim-1 and Pim-2 was used to assess the ability of test compounds to inhibit
Pim phosphorylation of a short sequence of BAD, and a cellular assay constructed with KMS-12-BM, a multiple myeloma
cell line with relatively high endogenous levels of Pim-1/2, was used to asses the ability of test compounds to inhibit Pim
phosphorylation of Ser-112 of the full-length BAD protein.
13. For reviews on Pim kinase inhibitors see: (a) Merkel, A. L.; Meggers, E.; Ocker, M. Expert Opin. Investig. Drugs 2012,
21, 425-436. (b) Arunesh, G. M.; Shanthi, E.; Krishna, M. H.; Kumar, J. S.; Viswanadhan, V. N. Expert Opin. Ther. Patents
2014, 24, 5-17. (c) Drygin, D.; Haddach, M.; Pierre, F.; Ryckman, D. M. J. Med. Chem. 2012, 55, 8199-8208. (d) Morwick,
T. Expert Opin. Ther. Patents 2010, 20, 193-212.
14. (a) Mumenthaler, S. M.; Ng, P. Y.; Hodge, A.; Bearss, D.; Berk, G.; Kanekal, S.; Redkar, S.; Taverna, P.; Agus, D. B.;
Jain, A.; Mol. Cancer Ther. 2009, 8, 2882-2893. (b) Xu, Y.; Brenning, B. G.; Kultgen, S. G.; Foulks, J. M.; Clifford, A.; Lai,
S.; Chan, A.; Merx, S.; McCullar, M. V.; Kanner, S. B.; Ho, K.-K. ACS Med. Chem. Lett. 2015, 6, 63-67. (c) Chen, L. S.;
Redkar, S.; Taverna, P.; Cortes, J. E.; Gandhi, V. Blood 2011, 118, 693-702.
15. Dakin, L. A.; Block, M. H.; Chen, H.; Code, E.; Dowling, J. E.; Feng, X.; Ferguson, A. D.; Green, I.; Hird, A. W.;
Howard, T. Bioorg. Med. Chem. Lett. 2012, 22, 4599-4604.
16. For a report on the discovery of PIM447 (also known as LGH447) see: (a) Burger, M. T.; Nishiguchi, G.; Han, W.; Lan,
J.; Simmons, R.; Atallah, G.; Ding, Y.; Tamez, V.; Zhang, Y.; Mathur, M.; Muller, K.; Bellamacina, C.; Lindval, M. K.;
Zang, R.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Basham, S.; Chan, J.; Ginn, E.; Aycinena, A.; Holash, J.; Castillo,
J.; Langowski, J. L.; Wang, Y.; Chen, M. Y.; Lambert, A.; Fritsch, C.; Kauffmann, A.; Pfister, E.; Vanasse, K. G.; Garcia, P.
D. J. Med. Chem. 2015, 21, 8373-8386. See also: (b) Garcia, P. D.; Langowski, J. L.; Wang, Y.; Chen, M.; Castillo, J.;
Fanton, C.; Ison, M.; Zavorotinskaya, T.; Dai, Y.; Lu, J.; Niu, X.; Basham, S.; Chan, J.; Yu, J.; Doyle, M.; Feycht, P.; Warne,
R.; Narberes, J.; Tsang, T.; Fritsch, C.; Kauffmann, A.; Pfister, E.; Drueckes, P.; Trappe, J.; Wilson, C.; Han, W.; Lan, J.;
20

Nishiguchi, G.; Lindvall, M.; Bellamacina, C.; Aycinena, A.; Zang, R.; Holash, J.; Burger, M. T. Clin. Can. Res. 2014, 20,
1834-1845. (c) Burger, M. T.; Han, W.; Lan, J.; Nishiguchi, G.; Bellamacina, C.; Lindval, M.; Atallah, G.; Ding, Y.; Mathur,
M.; McBride, C.; Beans, E. L.; Muller, K.; Tamez, V.; Zhang, Y.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Holash,
J.; Castillo, J.; Langowski, J.; Wang, Y.; Chen, M. Y.; Garcia, P. D. ACS Med. Chem. Lett. 2013, 4, 1193-1197. (d)
Nishiguchi, G. A.; Burger, M. T.; Han, W.; Lan, J.; Atallah, G.; Tamez, V.; Lindvall, M.; Bellamacina, C.; Garcia, P.;
Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Wong, K. Bioorg. Med. Chem. Lett. 2016, 26, 2328-2332.
17. Wu, B.; Wang, H.-L.; Cee, V. J.; Lanman, B. A.; Nixey, T.; Pettus, L.; Reed, A. B.; Wurz, R. P.; Guerrero, N.; Sastri, C.;
Winston, J.; Lipford, J. R.; Lee, M. R.; Mohr, C.; Andrews, K. L.; Tasker, A. S. Bioorg. Med. Chem. Lett. 2015, 25, 775-780.
18. Wurz, R. P.; Pettus, L. H.; Jackson, C.; Wu, B.; Wang, H.-L.; Herberich, B.; Cee, V.; Lanman, B. A.; Reed, A. B.;
Chavez, Jr.; F.; Nixey, T.; Laszlo III, J.; Wang, P.; Nguyen, Y.; Sastri, C.; Guerrero, N.; Winston, J.; Lipford, J. R.; Lee, M.
R.; Andrews, K. L.; Mohr, C.; Xu, Y.; Zhou, Y.; Reid, D. L.; Tasker, A. S. Bioorg. Med. Chem. Lett. 2015, 25, 847-855.
19. Pettus, L. H.; Andrews, K. L.; Booker, S. K.; Chen, J.; Cee, V. J.; Chavez, Jr., F.; Chen, Y.; Eastwood, H.; Guerrero, N.;
Herberich, B.; Hickman, D.; Lanman, B. A.; Laszlo III, J.; Lee, M. R.; Lipford, J. R.; Mattson, B.; Mohr, C.; Nguyen, Y.;
Norman, M. H.; Powers, D.; Reed, A. B.; Rex, K.; Sastri, C.; Tamayo, N.; Wang, P.; Winston, J. T.; Wu, B.; Wu, T.; Wurz,
R. P.; Xu, Y.; Zhou, Y.; Tasker, A. S.; Wang, H.-L. J. Med. Chem. 2016, 59, 6407-6430.
20. Cee, V. J.; Chavez, Jr., F.; Herberich, B.; Lanman, B. A.; Pettus, L. H.; Reed, A. B.; Wu, B.; Wurz, R. P.; Andrews, K.
L.; Chen, J.; Hickman, D.; Laszlo, III, J.; Lee, M. R.; Guerrero, N.; Mattson, B. K.; Nguyen, Y.; Mohr, C.; Rex, K.; Sastri, C.
E.; Wang, P.; Wu, Q.; Wu, T.; Xu, Y.; Zhou, Y.; Winston, J. T.; Lipford, J. R.; Tasker, A. S.; Wang, H.-L. ACS Med. Chem.
Lett. 2016, 7, 408-412.
21. Wang, H.-L.; Cee, V. J.; Chavez, Jr. F; Lanman, B. A.; Reed, A. B.; Wu, B.; Guerrero, N.; Lipford, J. R.; Sastri, C.;
Winston, J.; Andrews, K. L.; Huang, X.; Lee, M. R.; Mohr, C.; Xu, Y.; Zhou, Y.; Tasker, A. S. Bioorg. Med. Chem. Lett.
2015, 25, 834-840.
22. For additional structurally related examples see: (a) Burger, M.; Lindvall, M.; Han, W.; Lan, J.; Nishiguchi, G.; Shafer,
C.; Bellamacina, C.; Huh, K.; Atallah, G.; McBride, C.; Antonios-McCrea, W. Jr.; Zavorotinskaya, T.; Walter, A.; Garcia, P.
WO 2008/106692, 2008. (b) Burger, M.; Lan, J.; Lindvall, M.; Nishiguchi, G.; Tetalman, M.; Von Sprecher, G. WO
2009/109576, 2009. (c) Buger, M.; Lan, J. WO 2010/026122, 2010.
23. Ishchenko, A.; Zhang, L.; LeBrazidec, J.-Y.; Fan, J.; Chong, J.-H.; Hingway, A.; Raditsis, A.; Singh, L.; Elenbaas, B.;
Hong, V.; Marcotte, D.; Silvian, L.; Enyedy, I.; Chao, J. Bioorg. Med. Chem. Lett. 2015, 25, 474-480.
24. Wang, X.; Magnuson, S.; Pastor, R.; Fan, E.; Hu, H.; Tsui, V.; Deng, W.; Murray, J.; Steffek, M.; Wallweber, H.;
Moffat, J.; Drummond, J.; Chan, G.; Harstad, E.; Ebens, A. J. Bioorg. Med. Chem. Lett. 2013, 23, 3149-3153.
25. The (S)-enantiomer of the 3-aminopiperidine was found to be more potent than the corresponding (R)-isomer in all cases
(data not shown).
26. For additional experimental details, see: (a) D’Amico, D. C.; Herberich, B. J.; Jackson, C. L. M.; Pettus, L. H.; Tasker,
A.; Wang, H.-L.; Wu, B.; Wurz, R. US 9187486, 2015. (b) D’Amico, D. C.; Herberich, B. J.; Jackson, C. L. M.; Pettus, L.
H.; Tasker, A.; Wang, H.-L.; Wu, B.; Wurz, R. WO 2012/148775, 2012.
27. This synthesis was inspired by the report for the synthesis of 2,3-dihydroimidazo[1,5-b]pyridazin-3-one prepared via
thermolysis of an acyl azide, see: Iwao, M.; Kuraishi, T. J. Het. Chem. 1979, 16, 689-698.
28. Compound 8b was isolated as a white crystalline solid as its tris(TFA) salt. MS (ESI, pos. ion) m/z: 422.0 [M+H]. 1H
NMR (400 MHz, D2O) ppm 1.28 (qq, J=10.80, 3.70 Hz, 1 H) 1.58 (dtd, J=13.30, 10.30, 10.30, 4.10 Hz, 1 H) 1.74 (dt,
J=14.18, 3.86 Hz, 1 H) 2.03 (dq, J=12.60, 4.20 Hz, 1 H) 3.08 (ddd, J=13.30, 10.95, 2.74 Hz, 1 H) 3.15 (dd, J=12.30, 9.98
Hz, 1 H) 3.23 (tt, J=9.80, 3.70 Hz, 1 H) 3.80 (br. dt, J=13.30, 3.60, 3.60 Hz, 1 H) 4.09 (br. d, J=10.40 Hz, 1 H) 6.95 (d,
J=9.59 Hz, 1 H) 7.13 (t, J=8.41 Hz, 2 H) 7.35 (d, J=7.04 Hz, 1 H) 7.43 (s, 1 H) 7.54 (tt, J=8.40, 6.50 Hz, 1 H) 8.01 (d,
J=9.78 Hz, 1 H) 8.23 (dd, J=7.04, 0.98 Hz, 1 H) 8.39 (d, J=0.98 Hz, 1 H). 19F NMR (377 MHz, D2O) ppm -117.35 (t,
J=7.44 Hz, 2 F) -78.50 (s, 9 F).
29. (a) Déchamps, I.; Pardo, D. G.; Cossy, J. Synlett 2007, 263-267. (b) Déchamps, I.; Pardo, D. G.; Cossy, J. Eur. J. Org.
Chem. 2007, 4224-4234. (c) Roudeau, R.; Pardo, D. G.; Cossy, J. Tetrahedron 2006, 62, 2388-2394.
30. For the synthesis of this 3,4,5-trisubstituted piperidine see: Burger, M. T.; Han, W.; Lan, J.; Nishiguchi, G. US
2010/0056576, 2010.
21

1
31. Compound 22m was isolated as an orange amorphous solid. MS (ESI, pos. ion) m/z: 436.2 (M+1). H NMR (400 MHz,
MeOH) δ ppm 9.27 (1 H, s), 8.12 (1 H, d, J=5.3 Hz), 8.01 (1 H, d, J=9.6 Hz), 7.54 - 7.66 (1 H, m), 7.37 (1 H, s), 7.17 - 7.25
(2 H, m), 7.14 (1 H, d, J=5.3 Hz), 6.69 (1 H, d, J=9.4 Hz), 3.38 - 3.44 (1 H, m), 3.28 (1 H, d, J=9.8 Hz), 2.89 - 3.01 (1 H, m),
2.45 (1 H, t, J=10.7 Hz), 2.21 (1 H, t, J=11.3 Hz), 1.99 (1 H, d, J=12.9 Hz), 1.72 - 1.88 (1 H, m), 0.89 - 0.99 (1 H, m), 0.81 -
0.88 (3 H, m). 19F NMR (376 MHz, MeOH) δ ppm -115.13 (2 F, s).
32. Aqueous equilibrium solubility assay was conducted on Symyx Solubility System. 1 mg solid compound was added to
each of the three media, equilibrated at room temperature for 48-72 hours, centrifuged and the supernatants were analyzed by
HPLC and compared against a standard curve.
33. The scanMAX panel includes: 453 human kinases, 3 pathogen kinases, 56 disease relevant mutants, 132 tyrosine kinase
assays, and 20 lipid kinases. For more information see: http://www.discoverx.com/technology/technology-kinomescan.php.
34. The POC values for Pim-1, Pim-2 and Pim-3 were 0.9%, 4.6% and 1.1%, respectively.
35. Lu, J.; Zavorotinskaya, T.; Dai, Y.; Niu, X.-H.; Castillo, J.; Sim, J.; Yu, J.; Wang, Y.; Langowski, J. L.; Holash, J.;
Shannon, K.; Garcia, P. D. Blood 2013, 122, 1610–1620.
36. Sastri, C. E.; Guerrero, N.; Yu, D.; Mattson, B.; Dellamaggiore, K.; Yang, Y.; Hughes, P.; Wang, H.-L.; Cee, V.;
Lanman, B. A.; Pettus, L.; Reed, A. B.; Wu, B.; Wurz, R. P.; Tasker, A.; Huang, L.-Y.; Branstetter, D.; Rex, K.; Winston, J.;
Burgess, T. L.; Kendal, R.; Lipford III, J. R. AACR Annual Meeting: Philadelphia, PA, 2015; Abstract 5396.
37. Pim-3 IC50 = 0.027 nM for compound 22m. An increase in Pim protein levels by Western blot analysis has been
previously reported upon inhibition of Pim signaling, see: Cen, B.; Xiong, Y.; Song, J. H.; Mahajan, S.; DuPont, R.;
McEachern, K.; DeAngelo, D. J.; Cortes, J. E.; Minden, M. D.; Ebens, A.; Mims, A.; LaRue, A. C.; Kraft, A. S. Mol. Cell.
Biol. 2014, 34, 2517-2532.
38. Burger, M.; Nishiguchi, G.; Rico, A.; Taft, B. WO 2014/099880, 2014.
22