Identification of N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (PIM447), a potent and selective proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitor in clinical trials for hematological malignancies

Article abstract of DOI:10.1021/acs.jmedchem.5b01275

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

Full text of DOI:10.1021/acs.jmedchem.5b01275

Drug Annotation
pubs.acs.org/jmc
Identification of N‑(4-((1R,3S,5S)‑3-Amino-5-
methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-
fluoropicolinamide (PIM447), a Potent and Selective Proviral
Insertion Site of Moloney Murine Leukemia (PIM) 1, 2, and 3 Kinase
Inhibitor in Clinical Trials for Hematological Malignancies
Matthew T. Burger,*,† Gisele Nishiguchi,† Wooseok Han,† Jiong Lan,† Robert Simmons,†
Gordana Atallah,† Yu Ding,† Victoriano Tamez,† Yanchen Zhang,† Michelle Mathur,† Kristine Muller,†
Cornelia Bellamacina,† Mika K. Lindvall,† Richard Zang,† Kay Huh,† Paul Feucht,‡
Tatiana Zavorotinskaya,‡ Yumin Dai,‡ Steve Basham,‡ Julie Chan,‡ Elaine Ginn,† Alex Aycinena,†
Jocelyn Holash,‡ Joseph Castillo,‡ John L. Langowski,‡ Yingyun Wang,‡ Min Y. Chen,‡ Amy Lambert,§
Christine Fritsch,∥ Audry Kauffmann,∥ Estelle Pfister,∥ K. Gary Vanasse,⊥ and Pablo D. Garcia‡
‡
§
†Global Discovery Chemistry, Oncology, Oncology Research, and Preclinical Safety, Novartis Institutes for Biomedical Research,
4560 Horton Street, Emeryville, California 94608, United States
∥Oncology Research, Novartis Institutes for Biomedical Research, CH-4056, Basel, Switzerland
⊥Translational Clinical Oncology, Novartis Institutes for Biomedical Research, 220 Massachusetts Avenue, Cambridge Massachusetts
02139, United States
S
* Supporting Information
ABSTRACT: Pan proviral insertion site of Moloney murine
leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently
begun to be tested in humans to assess whether pan PIM
kinase inhibition may provide benefit to cancer patients.
Herein, the synthesis, in vitro activity, in vivo activity in an
acute myeloid leukemia xenograft model, and preclinical
profile of the potent and selective pan PIM kinase inhibitor
compound 8 (PIM447) are described. Starting from the
reported aminopiperidyl pan PIM kinase inhibitor compound
3, a strategy to improve the microsomal stability was pursued
resulting in the identification of potent aminocyclohexyl pan
PIM inhibitors with high metabolic stability. From this
aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several
phase 1 trials of cancer patients with hematological malignancies.
disease, high expression and/or dysfunction of the three PIMs
has been implicated in the progression of hematopoietic and
solid tumor cancers.1,6 High expression of PIM1 has been
reported in acute lymphoblastic leukemia, acute myeloid
leukemia (AML), and diffuse large B-cell lymphoma, while
high expression of PIM2 has been reported in multiple
myeloma.7 The high expression of PIM in cancer, particularly
hematopoietic cancers, is thought to play a role in promoting
survival and proliferation while suppressing apoptosis. As PIMs
display functional redundancy in oncogenesis and are differ-
entially overexpressed in different cancer cell types, a pan-PIM
kinase inhibitor rather than an isoform-selective inhibitor would
INTRODUCTION
■
Proviral insertion site of Moloney murine leukemia virus
kinases, or PIMs 1, 2, and 3 kinases are serine/threonine
kinases that normally function in the survival, proliferation, and
differentiation of hematopoietic cells in response to growth
factors and cytokines.1 Cytokine signaling through the JAK/
STAT pathway leads to increase transcription of the PIM genes
with subsequent translation of constitutively active proteins.1
PIM1 was originally discovered as an oncogene by frequent
proximal proviral insertions of murine leukemia virus in
lymphomas of infected mice.2,3 Similar unbiased insertional
mutagenesis screens were used to demonstrate the oncogenic
potential of PIM24 and PIM35 in PIM1 and PIM1/2 knockout
mice, respectively, and in doing so demonstrated the potential
for functional redundancy of the PIM isoforms. In human
Received: August 14, 2015
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Drug Annotation
be expected to provide superior antitumor efficacy.8 In addition
to cancer, PIM kinases have been reported to play a role in
several autoimmune diseases.9 Not surprisingly, PIM kinases
have emerged as attractive therapeutic targets and have elicited
several groups to investigate and report novel inhibitors of
PIM10−18 including the clinical compounds SGI-177611 and
AZD1208,12 compound 1 and compound 2, respectively,
Figure 1. PIM kinases share a high level of sequence homology
While compound 3 has served as a valuable in vitro and in
vivo tool for studying PIM biology, the moderate to high in
vitro clearance, vide infra, could pose a developability risk,
especially since the PK−PD efficacy relationship observed
supports maximal efficacy being driven by the maintenance of
continuous free plasma levels above cellular antiproliferative
EC50’s.7 In vitro met ID studies in human liver microsomes
indicate metabolism of compound 3 involves oxidations on
carbons of the piperidine ring adjacent to both the internal and
external nitrogens. This eventually leads to the removal the
piperidine ring altogether, leaving the piperidine nitrogen
behind, funneling into a 4-amino 3-aminoacyl pyridyl
metabolite, Figure 2. In addition to the high in vitro CL of
compound 3, the formation of an electron rich amino,
aminoacyl pyridyl metabolite was not deemed as positive
with respect to developability/toxicity. Thus, the challenge for
medicinal chemistry was to improve the metabolic stability and
reduce or eliminate the formation of a diaminopyridyl
metabolite while maintaining the potency and selectivity
achieved with compound 3.
The medicinal chemistry strategy to achieve this goal focused
on removing the substituted piperidine portion of the molecule
and replacing it with a similarly substituted cyclohexyl ring.
With respect to potency, from the SAR developed leading up to
compound 3, it was known that the pyridyl nitrogen in the
diaminopyridine ring and the primary amino group on the
piperidine were important for potency. The X-ray crystal
structure of compound 3 in PIM 1 (PDB code 4N70),20 Figure
3, indicates these moieties participate in three hydrogen bonds
Figure 1. PIM clinical compounds and starting point 3.
within the family (>61%), and all share the unique feature of
being the only kinases with a proline in the hinge,19 which
results in only one hydrogen bond interaction with ATP. As the
ATP Km for PIM2 is 10−100× lower than that for PIM1 and
PIM3, cell active pan PIM inhibitors have been more
challenging to identify than PIM1/3 inhibitors.
Recently, we described the optimization of a highly kinase
selective aminopiperidine, pyridylcarboxamide scaffold,20 which
binds in the PIMs ATP pocket, lacks any hydrogen bonds to
the hinge, and exhibits in vitro pan PIM biochemical and
cellular potencies greater than compound 1 and compound 2.
From this scaffold, compound 3, Figure 1, has been
characterized in multiple myeloma and AML in vivo
hematological mouse xenograft models7,21 where a PK−PD
efficacy relationship has been established. Described herein are
our further efforts on the pyridylamide scaffold that have
focused on the improvement of metabolic stability. These
efforts have led to the identification of N-(4-((1R,3S,5S)-3-
amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophen-
yl)-5-fluoropicolinamide compound 8 (PIM447) which exhibits
pan PIM potency, pharmacokinetic properties, and druglike
properties suitable for clinical development. Compound 8
entered clinical trials in 2012 and is currently in several phase I
trials. Compound 8 was previously known as and is
synonymous with LGH447.
Figure 3. Structure of compound 3 in PIM1.
to the protein (NH2 to Asp128 side chain and Glu171
backbone carbonyl, and pyridyl to Lys67). Substitution of the
cyclohexyl for piperidyl could possibly allow for a similar
display of the primary amino group for hydrogen bonding to
Asp128 and Glu171 if the piperidine to cyclohexyl change does
not change the relative conformation of the saturated six-
membered ring relative to the attached N-acylpyridine.
Figure 2. Metabolism of compound 3 in human liver microsomes.
B
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Journal of Medicinal Chemistry
Drug Annotation
Additionally, it was unclear a priori what effect the piperidine to
cyclohexyl change might have on potency with respect to the
hydrogen bond interaction of the pyridyl nitrogen and Lys67,
as the electron density and basicity of the pyridine would be
altered. With respect to metabolism, while the piperidine to
cyclohexyl change would remove the α or α′ N dealkylation
metabolic spots adjacent to the piperidine ring nitrogen, it was
unclear a priori whether this would improve overall metabolic
stability or whether metabolism would just be shifted toward N-
dealkylative oxidation α to the primary amino group. This α N-
dealkylative oxidation (followed by loss of NH2 leading to the
corresponding ketone) had also been observed in the
microsomal studies of compound 3 as one of several piperidine
metabolites. Attempts to suppress the metabolism α to the
primary amine via installation of an α methyl group led to a loss
in PIM potency, unpublished results. Thus, it was unclear
beforehand whether the proposed strategy of replacing
piperidine with cyclohexyl would yield compounds with
improved microsomal stability and with sufficient pan PIM
potency. This uncertainty withstanding, cyclohexyl compounds
were pursued and synthetic routes were developed.
Aminocyclohexyl targets containing the 5 position methyl
group on the cyclohexyl ring were prepared as described in
Scheme 2. (5)-Methylcyclohexanedione was converted via the
a
Scheme 2
a
Reagents and conditions: (a) Na2CO3, Tf2O, CH2Cl2, 0 °C, 2 h, 78%;
CHEMISTRY
(b) KOAc, Pd(dppf)Cl2, pinacolatodiboron, dioxane, 80 °C, 10 h; (c)
4-chloro-3-nitropyridine, Pd(pddf)Cl2, dioxane/2 M Na2CO3, 110 °C,
1 h, 48% (2 steps); (d) NaBH4, CeCl3·7H2O, EtOH, 0 °C, 1 h, 72%;
(e) TBDMSCl, imidazole, DMF, 18 h, 77%; (f) H2, Pd/C, MeOH, 15
h, 93%; (g) Cbz-OSu, DMAP, CH2Cl2, 90 h, 81%; (h) HCl, EtOH, 1
h, 94%; (i) Dess−Martin periodinane, CH2Cl2, 1 h, 91%; (j)BnNH2,
LiBH4, MeOH, 3.5 h; (k) H2, Pd/C, MeOH, Boc2O, 46 h, 47% (2
steps); (l) chiral separation; (m) heteroaryl acid, EDC, HOAT, DMF;
12 h, 91%; (n) 4 M HCl/dioxane, 17 h, 80% or 25% TFA/CH2Cl2, 0.5
h, 90%.
■
Initially, aminocyclohexyl targets without additional ring
substitution were prepared to establish the viability of the
synthetic route, Scheme 1. Cyclohexanedione was converted via
a
Scheme 1
monotriflate to the corresponding cyclohexenoneboronate ester
which underwent palladium mediated carbon bond formation
with 4-chloro-3-nitropyridine to yield after 1,2 reduction of the
enone a cis cyclohexenol IV. tert-Butyldimethylsilyl protection
of the alcohol, hydrogenation of the nitro and alkene groups
followed by carbobenzyloxyamine protection yielded the
racemic all cis, equatorial, trisubstituted cyclohexane V.
Deprotection of the silyl ether, Dess−Martin oxidation of the
alcohol to the cis disubstituted cyclohexanone, followed by
lithium borohydride22 mediated reductive amination with
benzylamine reestablished the all cis equatorial cyclohexyl
relative stereochemistry. Removal of the carbobenzyloxy and
benzyl groups under hydrogenation conditions followed by in
situ primary aliphatic amine Boc protection and chiral
resolution yielded the desired all cis trisubstituted Boc
protected aminocyclohexylpyridylaniline VI. Amide coupling
followed by acetate and Boc deprotection yielded target
compounds 6, 8, and 14.
a
Reagents and conditions: (a) Na2CO3, Tf2O, CH2Cl2, 0 °C, 3 h; (b)
KOAc, Pd(dppf)Cl2, pinacolatodiboron, dioxane, 80 °C, 2 h; (c) 4-
chloro-3-nitropyridine, Pd(pddf)Cl2, dioxane/2 M Na2CO3, 110 °C, 2
h, 55% (3 steps); (d) NaBH4, CeCl3·7H2O, MeOH, 0 °C, 2 h, 94%;
(e) PhthNH, Ph3P, (C4H9O2CN)2, THF, 0 °C, 2 h, 50%; (f) 400 PSI
H2, Pd/C, MeOH, 50 °C, 48 h, 53%; (g) chiral separation; (h)
heteroaryl acid, EDC, HOAT, DMF; 12 h, 91%; (i) H2NNH2, MeOH,
50 °C, 2 h, 90%.
Aminocyclohexyl targets with the additional hydroxy
substitution present in compound 3 were prepared as described
in Scheme 3. Cyclohexenol IV was dehydrated to yield a diene
which reacted with N-bromosuccinimide at the less hindered
alkene to yield bromohydrin VII with the bromine cis to the
methyl group. Upon base treatment intramolecular cyclization
yielded the epoxide, trans to the methyl, which was opened
with sodium azide yielding azido alcohol VIII, therein
establishing the relative 1,2 trans, 1,3 cis azido, hydroxy, methyl
stereochemical relationship. Subsequent azide reduction, Boc
amine protection, hydroxyl acetylation, hydrogenation of the
nitro and alkene groups, and chiral resolution yielded the chiral
tetrasubstituted, all equatorial, aminohydroxypyridylaniline IX.
the monotriflate to the corresponding cyclohexenoneboronate
ester which underwent palladium mediated carbon bond
formation with 4-chloro-3-nitropyridine to yield nitropyridyl-
cylohexenone I. Subsequent 1,2 reduction of the enone yielded
the allylic alcohol II. The protected amine group was
introduced under Mitsunobu conditions with phthalimide.
Reduction of the alkene and nitro groups under hydrogenation
conditions followed by chiral separation yielded the enantio-
pure cis substituted cyclohexylpyridylaniline III. Amide
coupling followed by phthalimide deprotection yielded target
compounds 10, 12, and 16.
C
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Journal of Medicinal Chemistry
Drug Annotation
a
tested in PIM biochemical and PIM cellular assays to assess the
effect of cyclohexyl replacement on target potency. PIM1−3
kinase activity was assessed in a kinase-Glo assay, with [ATP] at
or below ATP Km for each isoform. Potent PIM inhibition,
picomolar and nanomolar, for all PIM isoforms was retained for
the aminocyclohexyl compounds, Table 1. While many
compound matched pairs were at the limits of detection in
the low ATP enzymatic assay, for the few (9−14) that were not
there was a ∼3-fold potency difference between piperidine and
cyclohexyl, with the piperidine analog being the more potent
member of each pair. Cellular activity was assessed in the
multiple myeloma derived, PIM2 driven, KMS11-luc cell line
where antiproliferative activity was obtained for all compounds
except 9 and 10 and target modulation as assessed by
phosphorylation of S6 ribosomal protein (pS6RP) was
obtained for 3, 4, 8, 14, and 16. S6RP, which is not a direct
PIM substrate but is downstream from PIM phosphorylation of
TSC2, was chosen as a biomarker as it is modulation correlated
best with in vivo antiproliferative activity in preclinical
models.7,21 Consistent with prior reports,7,20,23 there was a
large shift upward relative to the low [ATP] enzymatic assays.
Additionally, as in the enzymatic assay, the piperidine
component of any matched pair was more potent than the
corresponding cylcohexyl. The greater potency difference of
matched pair 3 and 4 which contain the hydroxyl and amino
groups relative to other matched pairs which lack the hydroxyl
group is noteworthy, highlighting subtleties of hydrogen
bonding, desolvation, and amine basicity for interactions in
this region of the protein. Despite the potency decrease in
replacing piperidine with cyclohexyl, aminocyclohexyl com-
pounds still exhibit picomolar enzymatic and significant cell
potency.
Scheme 3
a
Reagents and conditions: (a) p-TsOH, dioxane, 100 °C, 3 h, 68%;
(b) NBS, H2O, THF, 0.5 h, 80%; (c) KO-t-Bu, THF, 0.5 h; (d) NaN3,
NH4Cl, EtOH, H2O, 14 h, 55%; (e) PMe3, pyridine, NH4OH, 2 h; (f)
Boc2O, dioxane, NaHCO3(sat.), 2 h, 59% (2 steps); (g) Ac2O, pyridine,
14 h; (h) H2, Pd/C, MeOH, 72 h, 75% (2 steps); (i) chiral separation;
(j) heteroaryl acid, EDC, HOAT, DMF, 14 h; (k) K2CO3, EtOH, 14 h;
(l) 25% TFA/CH2Cl2, 0.5 h, 80% (3 steps).
Amide coupling followed by acetate and Boc deprotection
yielded the target compound 4. The piperidyl compound 3 was
previously described,20 and compounds 5, 7, 9, 11, 13, and 15
were prepared following a similar route; see experimental part
for details.
RESULTS AND DISCUSSION
The aminocyclohexanes (4, 6, 8, 10, 12, 14, 16) along with
matched pair aminopiperidines (3, 5, 7, 9, 11, 13, 15) were
The microsomal stability for the piperidine and cyclohexyl
matched pairs 3−16 (Table 2) was assessed in human and rat
liver microsomes where in addition to NADPH, UDPGA and
■
Table 1. PIMs 1, 2, and 3 Enzymatic and Cellular Activity of Compounds 3−16
Ki (μM)
EC50 (μM)
KMS11luc pS6RP
compd
X
R1
R2
R3
PIM1
PIM2
PIM3
KMSIlluc
3
N
CH3
CH3
CH3
CH3
CH3
CH3
H
OH
OH
H
F
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.005
<0.003
<0.003
<0.003
0.004
<0.003
<0.003
<0.003
<0.003
<0.003
<0.003
0.003
0.02
0.41
1.1
0.03
0.91
4
(R)-CH
N
F
5
H
H
F
6
(R)-CH
N
H
1.4
7
H
<0.003
<0.003
0.004
0.05
0.17
8
(R)-CH
N
H
F
0.18
9
H
10
11
12
13
14
15
16
(R)-CH
N
H
H
0.023
0.014
H
H
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.005
<0.003
0.004
1.2
(R)-CH
N
H
H
0.029
5.0
CH3
CH3
H
H
<0.003
0.006
<0.003
0.004
0.39
0.91
1.4
(R)-CH
N
H
0.32
0.71
H
F
F
<0.003
0.005
<0.003
<0.003
(R)-CH
H
H
0.54
D
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Journal of Medicinal Chemistry
Drug Annotation
Table 2. Human and Rat Microsomal Stability of
Compounds 3−16
Table 3. Selected SAR of Compound 8 and Comparison to
Compounds 1 and 2
CL_H
Pim1 Ki
Pim2 Ki
Pim3 Ki
KMS11luc EC50
T1/2 (min)
(mL min−1 kg−1
)
ER_H
human
compd
(nM)
(nM)
(nM)
(μM)
1
2
8
16
610
24
5
compd
rat
human
rat
human
rat
0.017
0.006
0.160
0.018
0.230
0.009
0.67
0.17
3
7
92
11
92
20
50
3
23
90
49
20
45
20
40
28
52
47
49
21
47
24
44
30
16
10
18
<6
14
9
0.88
0.36
0.82
0.36
0.72
0.51
0.94
0.86
0.89
0.38
0.85
0.44
0.79
0.55
0.77
0.46
0.86
4
5
13
6
>180
40
<0.3
7
0.66
0.42
0.85
0.48
0.52
0.39
0.61
8
106
14
9
18
10
11
8
10
11
12
13
14
15
16
8
85
6
70
85
9
121
49
13
<6
16
9
65
13
42
>180
25
<0.3
0.75
0.42
107
alemethicin were added to capture PhII glucoronidation. The
compounds were tested at 1 μM for 30 min, and t1/2’s were
measured from which scaled CL’int, hepatic CL (CL_H), and
hepatic extraction ratio (ER_H) were determined using a well-
stirred liver model. The measured t1/2’s, hepatic CL, and
hepatic extraction ratio are listed in Table 2. In human
microsomes there is a consistent increase in stability when
piperidine is replaced with cyclohexyl with t1/2’s increasing and
extraction ratio decreasing. For example, the t1/2 of the
cyclohexyl analog of compound 3, compound 4, is 3 times
more stable in human microsomes than compound 3, leading
to a ∼2-fold decrease in ER value (∼0.77 versus 0.46). The
stability improvement was evident in rat microsomes as well.
Improved stability in preclinical species was viewed as a positive
with respect to ensuring necessary exposures could be reached
in safety studies. The high metabolic stability was also observed
in hepatocytes, as compound 8 exhibits clearance values of 12
and 3 mL min−1 kg−1 in rat and human hepatocytes. From the
enzymatic potency, cellular potency, and metabolic stability
data depicted in Tables 1 and 2, it is evident replacing the
aminopiperidine of aminopiperidine carboxamide inhibitors
with an aminocyclohexyl can yield pan PIM potent, cell active
compounds with high metabolic stability in human liver
microsomes. Of the aminocyclohexanes described in Tables 1
and 2, compound 8 stood out with respect to cellular potency
and metabolic stability and was selected for further profiling.
As the biochemical potency for compound 8 reached the
limits of detection in the low [ATP] kinase Glo assay format,
the potency was further assessed in a high ATP AlphaScreen
assay using [ATP]’s of 2800, 500, and 2800 μM for PIMs 1, 2,
and 3, respectively. Under these conditions, the Ki’s of
compound 8 were determined to be 6, 18, and 9 pM for
PIMs 1, 2, and 3, respectively. When compared alongside the
clinical compounds 1 and 2, Table 3, compound 8 is more
potent in enzymatic and cellular assays.
Figure 4. Kinase activity of compound 8 against 68 kinases.
μM (>105-fold differential relative to the Ki on PIMs). The
biochemical IC50 for all other kinases tested in this panel was
>9 μM. In follow-up cellular assays of GSK3β inhibition,7
compound 8 was tested up to 20 μM and was not active.
Additional kinase profiling of compound 8 at 1 μM against
442 kinases using the KINOMEscan binding displacement
assay, Figure 5, indicated high kinase selectivity [S(35) = 0.021
Figure 5. KINOMEscan binding of 8 at 1 μM against 442 kinases.
at 1 μM, 386 nonmutant kinases including PIMs 1, 2, and 3;
S(35) = 0.013 at 1 μM, 383 nonmutant kinases not including
PIMs 1, 2, and 3], Figure 5. The unlabeled smaller red dots in
the kinome tree, Figure 5, correspond to ERK8, GSK3β, and
PRKD. The high selectivity of compound 8 is consistent with
the scaffold class as reported earlier.7
The kinase selectivity of compound 8 was first determined in
biochemical assays for a panel of 68 diverse protein kinases that
included PIM2 (Figure 4) as well as 9 lipid kinases. In this
panel, only PIM2 was significantly inhibited by compound 8
with an IC50 of <0.003 μM, the lowest sensitivity range for the
assay. Compound 8 also inhibited GSK3β, PKN1, and PKCτ,
but at a significantly lower potency with IC50 between 1 and 5
A crystal structure of compound 8 in PIM1 was obtained,24
Figure 6. The overall binding mode is the same as for 1 with the
central amide making no direct hydrogen bonds to the protein
E
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Journal of Medicinal Chemistry
Drug Annotation
compound 8, a dose dependent modulation of pS6RP was
evident (Figure 7) .
Figure 6. Structure of 8 in PIM1.
Figure 7. In vitro pS6RP target modulation by compound 8 in KG-1
acute myeloid leukemia cell line.
and displaying the two sides of the molecule toward the hinge/
lower hinge and catalytic Lys region/ribose patch. The amino
group on the cyclohexyl ring makes two hydrogen bonds to the
Asp128 side chain carboxylate and the Glu171 backbone
carbonyl in the ribose patch. Additionally, the methyl group fills
a hydrophobic dimple in the glycine rich loop above. The
acylaminopyridine ring nitrogen makes a hydrogen bond with
the catalytic Lys67. The C ring fluoropyridine fills the Pro123
hinge region with no hydrogen bond interactions, and the
difluorophenyl ring extends into the lower hinge region.
With an expanded understanding of the kinase selectivity of
compound 8 and the structural features contributing to PIM
potency in hand, the cellular activity of compound 8 across an
expanded panel of 26 AML cell lines was assessed, Table 4. The
The activity of compound 8 in an in vivo KG-1 AML
xenograft mouse model was assessed next. Following a single
oral dose of compound 8 (30 or 100 mg/kg) plasma and tumor
samples were collected for pharmacokinetic and pharmacody-
namic analysis, respectively. PIM kinase inhibition was
determined by assessing the modulation of pS6RP in tumor
lysate using the quantitative Meso Scale assay, and the results
were expressed as a ratio of their phosphorylated to
unphosphorylated forms (Figure 8). The plasma exposure
Table 4
cell line
GI50 (μM)
cell line
OCI-M1
GI50 (μM)
Molm16
KG-1
0.01
0.01
0.01
0.05
0.09
0.13
0.28
0.48
1.03
1.39
1.66
1.96
1.99
2.57
2.69
2.92
5.06
5.19
5.31
5.53
7
SKM-1
EOL-1
M-07e
OCI-AML3
P31/FUJ
Mono-MAC-1
THP-1
UKE-1
MV-4-11
CMK
OCI-AML2
NB-4
Set-2
CMK-11-5
Molm-13
Hel92.1.7
TF-1
PL-21
8.56
8.66
10
Figure 8. Single dose pS6RP target modulation and plasma exposure
of compound 8 in KG-1 acute myeloid leukemia subcutaneous mouse
tumor model.
SIG-M5
NOMO-1
OCI-AML5
F-36P
10
Mutz8
10
(Figure 8) was roughly dose proportional with a significant
difference in exposures at the 24 h time point in particular.
With mouse plasma protein binding of 95%, the 30 and 100
mg/kg doses maintain free plasma concentrations of ≥15 and
300 nM, respectively, throughout 24 h. At both doses there was
a ∼50% reduction in pS6RP at 1, 8, and 24 h. The lack of a
dose response in the target modulation may be a reflection of
dynamic range of the detection method, as the ∼50% reduction
of pS6RP was the maximal signal seen across multiple xenograft
PK−PD studies utilizing compounds in this scaffold class even
when dosed up to 300 mg/kg, data not shown. Additionally, in
the higher bar KMS11.luc multiple myeloma mouse xenograft
model, a dose response in pS6RP modulation at 24 h was
observed when compound 8 was dosed at 25 and 100 mg/kg.25
The in vivo target modulation translated to an in vivo
antiproliferative effect in the KG-1 AML xenograft model when
compound 8 was assessed in an 11-day study at 30 and 100
mg/kg QD doses. At the 30 mg/kg daily regimen of compound
antiproliferative activity spanned a range from low nM to
insensitivity at 10 μM, with approximately 1/3 sensitive at
≤500 nM, 1/3 sensitive between 500 nM and 3 μM, and 1/3
sensitive at >3 μM. The activity of compound 8 across the
expanded AML panel was consistent with what has been
reported for selective PIM inhibitors such as compound 223 and
compound 37 wherein a range of sensitivities is evident, with
highly sensitive cell lines correlating with the highest
expressions of PIM1.
Within the most sensitive AML cell lines, the activity of
compound 8 in KG-1 cells was further evaluated both in vitro
and in vivo, as a KG-1 in vivo xenograft model was available and
synergy with the AML standard of care cytarabine could be
assessed given the minimal activity observed with cytarabine at
the maximum tolerated dose in the model. Upon treatment of
KG-1 cells in vitro at concentrations of 0.05, 0.5, and 5 μM
F
DOI: 10.1021/acs.jmedchem.5b01275
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Journal of Medicinal Chemistry
Drug Annotation
8, near stasis was observed with only minor increase in tumor
volume relative to the vehicle control (Figure 9). In contrast,
were observed in mouse, rat, and dog, respectively. The volume
of distribution was consistently large across species, with Vss of
5.3, 6.4, and 3.6 L/kg observed in mouse, rat, and dog,
respectively. Additionally, compound 8 exhibited high oral
bioavailability across species, as 84%, 70%, and 71% was
observed in mouse, rat, and dog, respectively.
The melting point of compound 8 as a free base is 139 °C.
Its log D (pH 6.8) is 1.1, and its pKa values are 9.8 and 4.2. The
solubility of crystalline free base at pH 1, 3, and 6.8 is >45, 4,
and 0.5 mM. The stability of compound 8 in human plasma is
high, >90% after a 3 h incubation, and the human plasma
protein binding of compound 8 is 95%. In the manual patch
clamp hERG assay compound 8 exhibited a 12 μM IC50 and in
dog telemetry studies demonstrated no cardiosafety signals
wherein a 2× mouse efficacious free Cmax plasma concentration,
1 μM, was achieved. With a mouse efficacious unbound Cmax of
∼0.5 μM, the window between the free Cmax in mouse models
and hERG IC50 is ∼20-fold.
On the basis of favorable cellular potency, kinase selectivity,
manageable preclinical pharmacology and pharmacokinetic,
physical properties, and preclinical safety profiles, compound 8
was advanced into clinical trials in 2012, where a wide
therapeutic window demonstrating single agent activity and a
tolerable safety profile has been observed in multiple myeloma
patients.26 As opposed to that seen with halted clinical
compounds 1 and 2, the observation of early clinical activity
and favorable safety profile with compound 8 may be due in
part to its increased potency and/or selectivity.
Figure 9. Antitumor activity and tolerability of single agent compound
8 and compound 8 in combination with Ara-C in KG-1 acute myeloid
leukemia subcutaneous mouse tumor model.
tumor regression of 31% was observed with the 100 mg/kg QD
regimen. Both doses of compound 8 were well tolerated, and
the activity observed was similar to that of compound 3 in the
same model.7 In addition to single agent activity, a combination
of compound 8 with the nucleoside analog cytarabine (Ara-C),
a standard-of-care in the clinical treatment of AML, was
assessed. KG-1 tumors exhibited a slight but statistically
significant response to Ara-C alone when delivered at 100
mg/kg daily. While this dose results in clinically relevant
exposures, it is near the maximum tolerated dose in mice and
required a 2-day dosing holiday. When the 30 mg/kg QD dose
of compound 8 which led to tumor stasis was combined with
the 100 mg/kg QD dose of Ara-C, a synergistic effect resulting
in significant regression was achieved (Figure 9). When the
higher 100 mg/kg QD dose was similarly combined with Ara-C,
the effect did not reach statistical significance compared to the
100 mg/kg dose of compound 8 alone, as this dose achieved
regression as a single agent. Similar to the single-agent Ara-C
cohort, the combination of Ara-C with compound 8 at both
doses induced significant body weight loss following the fifth
day of administration which required dosing to be halted only
in these arms for 2 days before resuming treatment. Taken
together, these results demonstrate the efficacy of compound 8
both as a single agent and in combination with Ara-C, even in a
model that is refractory to this standard-of-care.
CONCLUSION
■
In summary, the metabolic stability of a selective pan PIM
kinase active aminopiperidine pyridyl carboxamide series
exemplified by compound 3 was improved by replacing the
chiral trisubstituted aminopiperidine with di-, tri-, and
tetrasubstituted aminocyclohexanes. Synthetic routes were
developed for the preparation of the substituted cylcohexanes
with control of relative stereochemistry. The improved in vitro
metabolic stability translated in vivo as cyclohexyl compound 8
exhibits low to moderate CL in mouse, rat, and dog. The
metabolically stable aminocyclohexyl pyridyl carboxamides
described exhibit picomolar pan PIM enzymatic activity, high
kinase selectivity, and nanomolar cellular activity. With the
combination of potent in vitro activity and low to moderate CL,
compound 8 demonstrates in vivo target modulation (pS6RP),
single agent antitumor activity in a KG-1 AML mouse xenograft
model, and druglike properties suitable for development.
Compound 8 advanced into humans in 2012 and is currently
being assessed in several phase I trials.27
EXPERIMENTAL SECTION
■
With these encouraging mouse pharmacology activities,
compound 8 was profiled further with respect to developability
and druglike properties. The pharmacokinetic properties of
compound 8 were evaluated across multiple species (Table 5).
Low to moderate in vivo CL was observed for compound 8
across species, as CL values of 20, 28, and 8 mL min−1 kg−1
The compounds and/or intermediates were characterized by high
performance liquid chromatography (HPLC) using a Waters
Millenium chromatography system with a 2695 separation module
(Milford, MA). The analytical columns were Alltima C-18 reversed
phase, 4.6 mm × 50 mm, flow 2.5 mL/min, from Alltech (Deerfield,
IL). A gradient elution was used, typically starting with 5%
acetonitrile/95% water and progressing to 100% acetonitrile over a
period of 10 min. All solvents contained 0.1% trifluoroacetic acid
(TFA). Compounds were detected by ultraviolet light (UV)
absorption at either 220 or 254 nm. HPLC solvents were from
Burdick and Jackson (Muskegan, MI) or Fisher Scientific (Pittsburgh,
PA). In some instances, purity was assessed by thin layer
chromatography (TLC) using glass or plastic backed silica gel plates,
such as Baker-Flex silica gel 1B2-F flexible sheets. TLC results were
Table 5. PK Properties of Compound 8 across Species
species
iv t1/2 (h)
CL (mL min−1 kg−1
)
Vss (L/kg)
po F (%)
mouse
rat
3.3
2.8
6.7
20
28
8
5.3
6.4
3.6
84
70
71
dog
G
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Journal of Medicinal Chemistry
Drug Annotation
readily detected visually under ultraviolet light or by employing well-
known iodine vapor and other various staining techniques.
MgSO4, and the volatiles were removed in vacuo. The product was
purified on silica utilizing the ISCO 0−65% gradient of hexane/EtOAc
to yield methyl 3-amino-6-cyclohexylpicolinate. LCMS (m/z): 235.2
(MH+). To a solution of methyl 3-amino-6-cyclohexylpicolinate (1.2 g,
5.1 mmol) in THF (10 mL) was added 1 M LiOH (20.4 mL, 20.4
mmol). After stirring for 4 h at room temperature, 1 N HCl (20.4 mL,
20.4 mmol) was added and the THF was removed in vacuo. The
resulting solid was filtered, rinsed with cold H2O (3 × 20 mL),
yielding 3-amino-6-cyclohexylpicolinic acid (203 mg, 18%). LCMS
(m/z): 221.0 (MH+).
2-(2,6-Difluorophenyl)thiazole-4-carboxylic Acid. A solution
of 2,6-difluorobenzamide (3.14 g, 0.2 mol) and Lawesson’s reagent
(4.04 g, 0.1 mol) in of toluene (100 mL) was heated at 90 °C for 14 h.
Upon cooling, the volatiles were removed in vacuo and purified by
SiO2 chromatography (25% EtOAc/hexanes) yielding 2,6-difluor-
obenzothioamide (3.97 g) as a light yellow solid LCMS (m/z): 174.1
(MH+). The material was used as is assuming 3.46 g. A solution of 2,6-
difluorobenzothioamide (3.46 g, 0.2 mol) and ethyl bromopyruvate
(2.5 mL, 0.2 mol) in ethanol (20 mL) was heated in the microwave at
130 °C for 30 min. Upon removal of volatiles in vacuo, ethyl acetate
was added and the solution was washed with Na2CO3(sat.), with
NaCl(sat.), was dried over MgSO4, filtered, and concentrated yielding
ethyl 2-(2,6-difluorophenyl)thiazole-4-carboxylate (4.5 g, 84%). LCMS
(m/z): 270.1 (MH+). To a solution of ethyl 2-(2,6-difluorophenyl)-
thiazole-4-carboxylate (4.5 g, 0.167 mol) in 2:1 THF/MeOH (100
mL) was added 1.0 M LiOH (33.5 mL, 0.335 mol). After standing for
16 h, 1.0 M HCl (33.5 mL, 0.335 mol) was added and the THF/
MeOH was removed in vacuo. The resulting solid was filtered, rinsed
with H2O, and dried, yielding 2-(2,6-difluorophenyl)thiazole-4-
carboxylic acid (3.54 g, 88%). LCMS (m/z): 251.1 (MH+). 1H
NMR (400 MHz, DMSO-d6) δ ppm 8.72 (s, 1 H), 7.65 (tt, J = 8.51,
6.36 Hz, 1 H), 7.28−7.39 (m, 2 H).
3-(3-Nitropyridin-4-yl)cyclohex-2-enone (I). To a solution of
cyclohexane-1,3-dione (40 g, 357 mmol) in DCM (1 L) was added
Na2CO3 (41.6 g, 392 mmol), and the heterogeneous solution was
cooled to 0 °C. A solution of Tf2O (66.3 mL, 392 mmol) in DCM
(100 mL) was added dropwise over 1 h at room temperature under a
nitrogen atmosphere with an internal temperature of <2 °C. Upon
addition, the reaction was stirred for 2 h (dark red solution). The
solution was filtered, and to the filtrate was added saturated NaHCO3
(carefully), then extracted the organics, dried with brine, then Na2SO4,
and concentrated. The crude material was used directly assuming 55.5
g of 3-oxocyclohex-1-enyl trifluoromethanesulfonate LC/MS = 244.9/
286.0 (M + H and M + CH3CN). To a solution of 3-oxocyclohex-1-
enyl trifluoromethanesulfonate (55.5 g, 227 mmol) in degassed
dioxane (1 L) was added bis(pinacolato)diboron (87 g, 341 mmol),
KOAc (66.9 g, 682 mmol), and Pd(dppf)Cl2−DCM (9.28 g, 11.4
mmol). The sealed reaction was heated to 80 °C for 2 h, cooled to rt,
and then filtered. The dioxane solution was used for the next step
without further purification. LC/MS = 140.9 (M + H of boronic acid).
To a solution of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
cyclohex-2-enone (50.5 g, 227 mmol) in degassed dioxane (1.4 L)
and 2 M Na2CO3 (335 mL) were added 4-chloro-3-nitropyridine (57.7
g, 364 mmol) and Pd(dppf)Cl2−DCM (9.28 g, 11.4 mmol) The
reaction was placed under a reflux condenser and heated in an oil bath
to 110 °C for 2 h. The mixture was cooled to room temperature,
filtered through a pad of Celite. The pad was washed with ethyl
acetate, and the filtrate was concentrated under vacuo. The residue was
partitioned between brine and ethyl acetate, the layers were separated,
the aqueous phase was further extracted with ethyl acetate (4×), the
organics were combined, dried over sodium sulfate, filtered, and
concentrated. The crude was purified via silica gel chromatography to
yield 3-(3-nitropyridin-4-yl)cyclohex-2-enone I (27.42 g, 55%). LC/
MS = 219.0 (M + H). 1H NMR (400 MHz, CDCl3) δ ppm 9.32 (s, 1
H), 9.21−9.42 (m, 1 H), 8.88 (d, J = 4.69 Hz, 1 H), 7.28 (s, 1 H), 6.01
(t, J = 1.37 Hz, 1 H), 2.49−2.61 (m, 5 H), 2.17−2.31 (m, 2 H).
3-(3-Nitropyridin-4-yl)cyclohex-2-enol (II). A solution of 3-(3-
nitropyridin-4-yl)cyclohex-2-enone (8.0 g, 36.7 mmol) and CeCl3·
7H2O (17.4 g, 46.8 mmol) in MeOH (367 mL) was cooled at 0 °C,
and NaBH4 (1.39 g, 36.7 mmol) was added in portions. The reaction
Mass spectrometric analysis was performed on one of two LCMS
instruments: a Waters system (Alliance HT HPLC and a Micromass
ZQ mass spectrometer; column, Eclipse XDB-C18, 2.1 mm × 50 mm;
solvent system, 5−95% (or 35−95% or 65−95% or 95−95%)
acetonitrile in water with 0.05% TFA; flow rate 0.8 mL/min;
molecular weight range 200−1500; cone voltage 20 V; column
temperature 40 °C) or a Hewlett-Packard system (series 1100 HPLC;
column, Eclipse XDB-C18, 2.1 mm × 50 mm; solvent system, 1−95%
acetonitrile in water with 0.05% TFA; flow rate 0.8 mL/min; molecular
weight range 150−850; cone voltage 50 V; column temperature 30
°C). All masses were reported as those of the protonated parent ions.
HRMS ESI-MS data were recorded using a Synapt G2 HDMS
(TOF mass spectrometer, Waters) with electrospray ionization source.
The resolution of the MS system was approximately 15 000. Leucine
enkephalin was used as lock mass (internal standards) infused from
lockspray probe. The compound was infused into the mass
spectrometer by UPLC (Acquity, Waters) from sample probe. The
separation was performed on Acquity UPLC BEH C18 1 mm × 50
mm column at 0.2 mL/min flow rate with the gradient from 5% to
95% in 3 min. Solvent A was water with 0.1% formic acid, and solvent
B was acetonitrile with 0.1% formic acid. The mass accuracy of the
system has been found to be <5 ppm with lock mass.
1H nuclear magnetic resonance (NMR) analyses described herein
were performed on some of the compounds with a Varian 400 MHz
NMR (Palo Alto, CA). The spectral reference was either TMS or the
known chemical shift of the solvent. 13C NMR spectra were recorded
using a Bruker AVANCE-500 NMR spectrometer operating at a
frequency of 125.77 MHz for 13C equipped with a 5 mm BBO probe.
Preparative separations were carried out using a Teledyne ISCO
chromatography system, by flash column chromatography using silica
gel (230−400 mesh) packing material, or by HPLC using a Waters
2767 sample manager, C-18 reversed phase column, 30 mm × 50 mm,
flow 75 mL/min. Typical solvents employed for the Teledyne ISCO
chromatography system and flash column chromatography were
dichloromethane, methanol, ethyl acetate, heptane, acetone, aqueous
ammonia (or ammonium hydroxide), and triethylamine. Typical
solvents employed for the reverse phase HPLC were varying
concentrations of acetonitrile and water with 0.1% trifluoroacetic acid.
The purity of all compounds screened in the biological assays was
examined by LC−MS analysis and was found to be ≥95%.
(S)-tert-Butyl 1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate, tert-
butyl (3S,5R)-1-(3-aminopyridin-4-yl)-5-methylpiperidin-3-ylcarba-
mate, tert-butyl (3R,4R,5S)-1-(3-aminopyridin-4-yl)-4-(tert-butyldime-
thylsilyloxy)-5-methylpiperidin-3-ylcarbamate, N-(4-((3R,4R,5S)-3-
amino-4-hydroxy-5-methylpiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluoro-
phenyl)-5-fluoropicolinamide (1), and 6-(2,6-difluorophenyl)-5-fluo-
ropicolinic acid were prepared as previously described.19
5-Fluoro-6-(2-fluorophenyl)picolinic Acid. To a solution of 6-
bromo-5-fluoropicolinic acid (699 mg, 3.2 mmol) in DME and 2 M
Na2CO3 (3:1, 16 mL) were added 2-fluorophenylboronic acid (672
mg, 4.8 mmol) and Pd(dppf)Cl2-DCM (130 mg, 0.16 mmol) in a
microwave vial. The vial was heated in the microwave at 120 °C for 30
min. The mixture was diluted with ethyl acetate, and 1 N NaOH was
added. The organic phase was separated and extracted three more
times with 1 N NaOH and once with 6 N NaOH. The combined
aqueous phases were filtered and acidified to pH 1 by the addition of
concentrated HCl and extracted with ethyl acetate. The organic layer
was dried over magnesium sulfate, filtered, and concentrated to yield
5-fluoro-6-(2-fluorophenyl)picolinic acid (301 mg, 40%). LC/MS =
236.1 (M + H). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.19 (dd, J =
8.61, 3.91 Hz, 1 H), 8.02 (dd, J = 9.39, 8.61 Hz, 1 H), 7.54−7.69 (m, 2
H), 7.35−7.44 (m, 2 H).
3-Amino-6-cyclohexylpicolinic Acid. A solution of methyl 3-
amino-6-bromopicolinate (1.2 g, 5.2 mmol), cyclohexylzinc bromide
0.5 M solution in THF (13 mL, 6.5 mmol), and tetrakis(triphenyl-
phosphine)palladium (0.25 g, 0.2 mmol) was stirred at 50 °C for 15
min. The reaction was filtered and washed with EtOAc. The organic
was washed with H2O (100 mL), NaCl(sat.) (50 mL), dried over
H
DOI: 10.1021/acs.jmedchem.5b01275
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Journal of Medicinal Chemistry
Drug Annotation
was stirred for 2 h at 0 °C and then quenched by adding water. The
volatiles were removed in vacuo, and the residue was dissolved in
EtOAc, washed with brine, dried over Na2SO4, and concentrated to
yield 3-(3-nitropyridin-4-yl)cyclohex-2-enol II (7.59 g, 94%). LC/MS
= 221.1 (M + H). 1H NMR (400 MHz, CDCl3) δ ppm 9.11 (s, 1 H),
8.75 (d, J = 4.99 Hz, 1 H), 7.27 (d, J = 4.74 Hz, 1 H), 5.72−5.85 (m, 1
H), 4.25−4.45 (m, 1 H), 2.13−2.34 (m, 2 H), 1.86−2.06 (m, 2 H),
1.68−1.86 (m, 2 H), 1.64 (d, J = 6.90 Hz, 1 H).
next step without further purification. LC/MS = 155.1 (M + H of
boronic acid). To a solution of 5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)cyclohex-2-enone (82 g, 347 mmol) in degassed
dioxane (694 mL, 0.5 M) and 2 M Na2CO3 (335 mL, 694 mmol) were
added 4-chloro-3-nitropyridine (71.5 g, 451 mmol) and Pd(dppf)Cl2−
DCM (14.2 g, 17.4 mmol). The reaction was placed under a reflux
condenser and heated in an oil bath to 110 °C for 1 h. The mixture
was cooled to room temperature, filtered through a pad of Celite. The
pad was washed with ethyl acetate, and the filtrate was concentrated
under vacuo. The residue was further pumped at 80 °C on a rotary
evaporator for 1 h to remove boronate byproducts (M + H = 101) via
sublimation. The residue was partitioned between brine and ethyl
acetate, the layers were separated, the aqueous phase was further
extracted with ethyl acetate (4×), the organics were combined, dried
over sodium sulfate, filtered, and concentrated. The crude was purified
via silica gel chromatography loading in DCM and eluting with 2−50%
ethyl acetate and hexanes. The pure fractions were concentrated in
vacuo to yield an orange oil. The oil was placed under high vacuum
(∼500 mTorr) with seed crystals overnight to yield an orange solid.
The solid was further purified via trituration in hexanes to yield 5-
methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (39 g, 48% over 2
2-(3-(3-Aminopyrin-4-yl)cyclohexyl)isoindoline-1,3-dione
(III). To a homogeneous solution of 3-(3-nitropyridin-4-yl)cyclohex-2-
enol (7.4 g, 33.6 mmol), triphenylphosphine (13.22 g, 50.4 mmol),
and phthalimide (7.42 g, 50.4 mmol) in THF (168 mL) cooled to 0
°C was added di-tert-butyl azodicarboxylate (11.61 g, 50.4 mmol) in
THF (32 mL). The mixture was stirred at 0 °C for 2 h. The reaction
was concentrated in vacuo. The residue was purified by SiO2 column
chromatography (5% methanol in 1:1 ethyl acetate and hexanes) and
trituration with DCM and hexanes to yield a solid, which was further
triturated with DCM and hexanes to yield 2-(3-(3-nitropyridin-4-
yl)cyclohex-2-enyl)isoindoline-1,3-dione (5.83 g, 50%). LC/MS (m/
z): MH+ = 350.2. A solution of 2-(3-(3-nitropyridin-4-yl)cyclohex-2-
enyl)isoindoline-1,3-dione (5.63 g, 16.1 mmol) in MeOH (160 mL)
was purged with argon for 10 min while in a steel bomb. At this time
10% Pd/C (2.57 g, 2.4 mmol) was added and the steel bomb was
sealed and placed under 400 psi of H2. The reaction mixture was
stirred at 50 °C for 2 days. Upon reaction completion, the solids were
removed by filtration over Celite, then rinsed with EtOAc and MeOH.
The filtrate was concentrated, diluted with EtOAc, and washed 2×
with sat. aq 2 M Na2CO3, dried with MgSO4, filtered, concentrated,
purified by SiO2 chromatography (10% MeOH in EtOAc), and
triturated from EtOAc/hexanes to yield 2-(3-(3-aminopyrin-4-yl)-
cyclohexyl)isoindoline-1,3-dione (III) (2.75 g, 53%). LC/MS (m/z):
MH+ = 322.2. 1H NMR (400 MHz, CDCl3) δ ppm 8.04 (s, 1 H), 8.01
(d, J = 5.09 Hz, 1 H), 7.80−7.87 (m, 2 H), 7.67−7.75 (m, 2 H), 7.05
(d, J = 5.09 Hz, 1 H), 4.34 (tt, J = 12.28, 3.77 Hz, 1 H), 3.66 (br s, 2
H), 2.60−2.72 (m, 1 H), 2.40−2.50 (m, 1 H), 2.34 (qd, J = 12.52, 3.91
Hz, 1 H), 2.03−2.14 (m, 1 H), 1.82−1.98 (m, 3 H), 1.45−1.65 (m, 2
H). Separation of the enantiomers under the following conditions.
Column: Chiralpak OJ (25 × 2 cm). Solvent: hexane/ethanol (70:30).
Flow rate: 15 mL/min. Injection volume: 5 mL (10 mg/mL solution
in 1:1 MeOH/EtOH). UV: 210 nm. Peak 1: 2-((1S,3R)-3-(3-
aminopyridin-4-yl)cyclohexyl)isoindoline-1,3-dione. Peak 2: 2-
((1R,3S)-3-(3-aminopyridin-4-yl)cyclohexyl)isoindoline-1,3-dione.
cis-( )-5-Methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (IV).
To a solution of 5-methylcyclohexane-1,3-dione (56.1 g, 445 mmol)
in DCM (880 mL, 0.5 M) was added Na2CO3 (51.8 g, 863 mmol) and
cooled to 0 °C. Tf2O (132 g, 468 mmol) in DCM (150 mL, 5.0 M)
was added dropwise over 1 h at 0 °C under a nitrogen atmosphere.
Upon addition, the reaction was stirred for 1 h at room temperature
(dark red solution). The solution was filtered and the filtrate was
quenched by careful addition of saturated NaHCO3 (∼400 mL) with
vigorous stirring until pH = 7. The solution was transferred to a
separatory funnel, and the layers were separated. The organic layer was
washed with brine, dried with Na2SO4, filtered, concentrated under
vacuo, and dried under high vacuum to yield 5-methyl-3-oxocyclohex-
1-enyl trifluoromethanesulfonate as light yellow oil in 78% yield (89.6
g). The triflate decomposes upon storage and should be used
immediately for the next reaction. LC/MS = 259.1/300.1 (M + H and
M + CH3CN). 1H NMR (400 MHz, CdCl3) δ ppm: 6.05 (s, 1H), 2.70
(dd, J = 17.2, 4.3, 1H), 2.53 (dd, J = 16.6, 3.7, 1H), 2.48−2.31 (m,
2H), 2.16 (dd, J = 16.4, 11.7, 1H), 1.16 (d, J = 5.9, 3H). To a solution
of 5-methyl-3-oxocyclohex-1-enyl trifluoromethanesulfonate (89.6 g,
347 mmol) in degassed dioxane (478 mL, 0.7 M) was added
bis(pinacolato)diboron (176 g, 693 mmol), KOAc (102 g, 1.04 mol),
and Pd(dppf)Cl2−DCM (8.5 g, 10 mmol). The reaction was heated to
80 °C for 10 h (initial heating at large scale results in exothermic
formation of an orange foam on top of the solution, and the heating
bath should be removed until the foam retracts; reheating to 80 °C at
this point appears to be fine), then cooled to room temperature and
filtered through a coarse frit glass funnel. The cake was rinsed with
more dioxane (∼250 mL), and the filtrate solution was used for the
1
steps). LC/MS = 233.2 (M + H). H NMR (400 MHz, CDCl3) δ
ppm: 9.31 (s, 1H), 8.88 (d, J = 5.1, 1H), 7.30 (d, J = 5.1, 1H), 6.00 (d,
J = 2.4, 1H), 2.62 (dd, J = 16.4, 3.5, 1H), 2.53−2.34 (m, 3H), 2.23 (dd,
J = 16.1, 11.7, 1H), 1.16 (d, J = 6.3, 3H). To a solution of 5-methyl-3-
(3-nitropyridin-4-yl)cyclohex-2-enone (46 g, 198 mmol) in EtOH
(990 mL) was added CeCl3·7H2O (89 g, 238 mmol). The reaction was
cooled to 0 °C, then NaBH4 (8.99 g, 238 mmol) was added in
portions. The mixture was stirred for 1 h at 0 °C, then quenched by
adding water, concentrated to remove the EtOH, added EtOAc,
extracted the organics, washed with brine, then dried with Na2SO4,
filtered, and concentrated and purified by SiO2 chromatography (0−
10% MeOH/CH2Cl2) to yield cis-( )-5-methyl-3-(3-nitropyridin-4-
1
yl)cyclohex-2-enol IV (33.4 g, 72%). LC/MS = 235.2 (M + H). H
NMR (400 MHz, DMSO-d6) δ ppm 9.07 (s, 1H), 8.79 (d, J = 4.70 Hz,
1H), 7.50 (d, J = 5.09 Hz, 1H), 5.65 (s, 1H), 4.97 (d, J = 5.48 Hz, 1H),
4.21 (d, J = 4.30 Hz, 1H), 2.04−2.15 (m, 1H), 1.75−1.98 (m, 3H),
1.07−1.19(m, 1H), 0.98 (d, J = 6.26 Hz, 3H).
Synthesis of cis-( )-Benzyl 4-3-(tert-Butyldimethylsilyloxy)-
5-methylcyclohexyl)pyridin-3-ylcarbamate (V). To a solution of
5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol (IV) (21.9 g, 93
mmol) in DMF (93 mL, 1.0 M) were added imidazole (14 g, 206
mmol) and TBDMSCl (18.3 g, 122 mmol). After stirring for 18 h the
solution was portioned between EtOAc and H2O and the layers were
separated. After washing further with H2O (3×) and NaCl(sat.), drying
over MgSO4, filtering, and removal of solvents, the crude was purified
via silica gel column chromatography, eluting with 0−70% ethyl
acetate in hexanes to afford cis-( )-4-(3-(tert-butyldimethylsilyloxy)-5-
methylcyclohex-1-enyl)-3-nitropyridine (27.3 g, 77%). LC/MS =
349.2 (M + H). To a solution of cis-( )-4-(3-(tert-butyldimethylsi-
lyloxy)-5-methylcyclohex-1-enyl)-3-nitropyridine (23.1 g, 66.4 mmol)
in methanol (221 mL), at a concentration of 0.3 M, was added 10%
palladium on carbon (Degussa type, 101NE/W, 1.4 g). The resultant
heterogeneous solution was put under an atmosphere of hydrogen and
was stirred for 15 h. At this time the mixture was filtered through a pad
of Celite, eluting with methanol. The volatiles were removed in vacuo
yielding all cis-( )-4-(3-(tert-butyldimethylsilyloxy)-5-
methylcyclohexyl)pyridin-3-amine (19.7 g, 93%). LCMS (m/z):
1
321.3 (MH+). H NMR (400 MHz, CDCl3) δ ppm: 8.03 (s, 1H),
8.00 (d, J = 5.1, 1H), 7.0 (d, J = 5.1, 1H), 3.80−3.69 (m, 1H), 3.63 (bs,
1H), 3.48 (d, J = 5.1, 1H), 2.55 (tt, J = 12.2, 3.1, 1H), 1.89−2.05 (m,
2H), 1.78 (d, J = 12.9, 1H), 1.52−1.73 (m, 1H), 1.36−1.50 (m, 1H),
1.00−1.16 (m, 1H), 0.98 (d, J = 6.7, 3H), 0.89 (s, 9H), 0.08 (s, 3H),
0.06 (s, 3H). To a solution of cis-( )-4-(3-(tert-butyldimethylsilyloxy)-
5-methylcyclohexyl)pyridin-3-amine (20.5 g, 64 mmol) in dichloro-
methane (213 mL) were added benzyl 2,5-dioxopyrrolidin-1-yl
carbonate (28.7 g, 115 mmol) and DMAP (0.78 g, 6.4 mmol). After
stirring for 90 h the solution was filtered through a glass frit funnel,
diluted with 300 mL of DCM, washed with water (2×), brine, dried
over sodium sulfate, filtered, and concentrated. The crude material was
I
DOI: 10.1021/acs.jmedchem.5b01275
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
purified via silica gel column chromatography, eluting with 10−100%
ethyl acetate in hexanes to yield cis-( )-benzyl-4-3-(tert-butyldime-
thylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarbamate V (23.6 g, 81%
mm × 500 mm). Solvent: heptane/ethanol/methanol (70:25:5). Flow
rate: 60 mL/min. Injection volume: 1 g/10 mL 1:1 MeOH/mobile
phase. UV: 210 nm. Analytical chiral HPLC on Chiracel OD 10 μM;
solvent, heptane/EtOH (80:20); flow rate = 1.2 mL/min where peak 1
comes at 12.12 min and peak 2 comes at 19.07 min. Peak 1 = tert-butyl
(1R,3S,5R)-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate.
Peak 2 = tert-butyl (1S,3R,5S)-3-(3-aminopyridin-4-yl)-5-
methylcyclohexylcarbamate
1
yield). LC/MS = 455.3 (M + H). H NMR (400 MHz, CDCl3) δ
ppm: 8.77 (bs, 1H), 8.37 (d, J = 5.1, 1H), 7.29−7.49 (m, 5H), 7.17 (d,
J = 5.1, 1H), 5.22 (s, 2H), 3.59−3.75 (m, 1H), 2.74−2.67 (m, 1H),
1.94−1.91 (m, 2H), 1.67−1.50 (m, 2H), 1.36−1.45 (m, 1H), 1.09−
1.00 (m, 2H), 0.95 (d, J = 6.7, 3H), 0.87 (s, 9H), 0.06 (s, 3H), 0.04 (s,
3H).
(
)-6-Bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-
enol (VII). To a solution of 5-methyl-3-(3-nitropyridin-4-yl)cyclohex-
2-enol (IV) (28.9 g, 123 mmol) in dioxane (1.2 L, 0.1 M) was added
p-TSA (25.8 g, 135 mmol), and the reaction was stirred at 100 °C for
3 h. The solution was cooled to room temperature, then passed
through a column of neutral alumina eluting with EtOAc to yield
( )-4-(5-methylcyclohexa-1,3-dienyl)-3-nitropyridine as a yellow oil in
cis-( )-tert-Butyl (3-(3-Aminopyridin-4-yl)-5-
methylcyclohexylcarbamate (VI). A solution of cis-( )-benzyl 4-
3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-ylcarba-
mate (V) (18.8 g, 41.3 mmol) in 1:2:1 6 N HCl/THF/MeOH (138
mL) was stirred at rt for 1 h. The pH was then adjusted to pH = 7 by
addition of 6 N NaOH, and the volatiles were removed in vacuo. The
residue was partitioned between ethyl acetate and water, the aqueous
layer was extracted with EtOAc, and the organic phase was washed
with NaCl(sat.), dried over MgSO4, filtered, and upon removal of the
volatiles in vacuo, silica gel column chromatography eluting with 0−
20% (1:1 DCM/MeOH) in DCM afforded 13.2 g of cis-( )benzyl 4-
(3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate in 94% yield.
1
68% yield (14.7 g). H NMR indicated about 12−15% contamination
1
of another diene isomer. LC/MS = 217.1 (M + H). H NMR (400
MHz, CDCl3) δ ppm 9.01 (s, 1H), 8.70 (d, J = 5.1, 1H), 7.29 (d, J =
5.3, 1H), 6.14 (dd, J = 5.4, 0.85, 1H), 6.05 (dd, J = 5.4, 2.3, 1H), 5.89
(dd, J = 9.6, 3.7, 1H), 2.55−2.68 (m, 1H), 2.33−2.43 (m, 1H), 2.17−
2.27 (m, 1H), 1.1 (d, J = 7.1, 3H). To a solution of 4-(5-
methylcyclohexa-1,3-dienyl)-3-nitropyridine (14.7 g, 67 mmol) in
THF and water (1:1, 515 mL) was added NBS (18.2 g, 102 mmol),
and the reaction was stirred at room temperature for 30 min. Upon
completion, ethyl acetate and water were added to the reaction, the
organic phase was washed with brine, then dried with sodium sulfate,
filtered, and concentrated. The crude material was purified via silica gel
column chromatography, eluting with ethyl acetate and hexanes (1:1)
to give ( )-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enol
VII as a yellow oil in 80% yield (17.9 g). LC/MS = 315.0/313.0 (M +
1
LC/MS = 341.2 (M + H), LC = 2.38 min. H NMR (300 MHz,
CDCl3) δ J = 5.3, 1H), 6.70 (bs, 1H), 5.20 (s, 2H), 3.72−3.65 (m,
1H), 2.79−2.74 (m, 1H), 2.04−1.97 (m, 2H),1.69−1.50 (m, 2H),
1.37−1.26 (m, 1H), 1.01−0.94 (m, 2H), 0.97 (d, J = 6.5, 3H). To a 0
°C solution of cis-( )-benzyl 4-(3-hydroxy-5-methylcyclohexyl)-
pyridin-3-ylcarbamate (13.2 g, 38.8 mmol) in wet CH2Cl2 (194 mL)
was added Dess-Martin Periodinane (16.5 g, 38.9 mmol) and the
solution was stirred for 1 h as it warmed to rt. The solution was
partitioned between EtOAc and 1:1 10% Na2S2O3/NaHCO3(sat.) and
separated. Upon further washing with 1:1 10% Na2S2O3/NaHCO3(sat.)
(2x) and NaCl(sat.), drying over MgSO4, filtering, removal of solvents,
cis-( )-benzyl-4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate
was obtained (11.9 g, 91%). LC/MS = 339.2 (M + H), LC: 2.47 min.
1H NMR (300 MHz, CDCl3) δ ppm: 8.70 (bs, 1H), 8.43 (d, J = 5.3,
1H), 7.30−7.45 (m, 5H), 7.20 (d, J = 5.3, 1H), 6.90 (s, 1H), 5.19 (s,
2H), 3.27−3.04 (m, 1H), 2.52−2.22 (m, 3H), 2.13−1.79 (m, 3H),
1.52 (q, J = 12.4, 1H), 1.06 (d, J = 6.2, 3H). A solution of cis-
( )-benzyl-4-(3-methyl-5-oxocyclohexyl)pyridin-3-ylcarbamate (11.9
g, 35.2 mmol) and benzylamine (11.3 g, 105 mmol) in MeOH (117
mL) was stirred at rt for 2 h. Upon cooling in a −78 °C bath, LiBH4
(19.3 mL, 38.6 mmol, 2.0 M in THF) was added dropwise and the
solution was allowed to warm to rt with stirring over 1.5 h. The
solution was partitioned between EtOAc and NaHCO3(sat.), separated,
washed further with NaHCO3(sat.) and NaCl(sat.), dried over MgSO4,
filtered, and after removal of volatiles in vacuo, cis-( )- benzyl 4-(-3-
(benzylamino)-5-methylcyclohexyl)pyridin-3-ylcarbamate was ob-
tained as a 4:1 mixture of isomers, with the all cis as predominant
(15.3 g, >99% yield). LC/MS = 430.3 (M + H), LC = 2.38 min. To a
solution of cis-( )- benzyl 4-(-3-(benzylamino)-5-methylcyclohexyl)-
pyridin-3-ylcarbamate (15.1 g, 35.2 mmol) in methanol (176 mL) was
added 20% palladium hydroxide on carbon (4.9 g, 0.2 equiv). The
resultant heterogeneous solution was put under an atmosphere of
hydrogen and was stirred for 14 h. At this time the reaction was
purged with Ar, Boc2O (15.4 g, 70.6 mmol) was added, and the
solution was stirred for 8 h. Additional Boc2O (7.7 g, 35.3 mmol) was
added and the solution was stirred for 16 more hours. At this time the
mixture was filtered through a pad of Celite eluting with methanol.
Upon removal of volatiles in vacuo, purification by silica gel
chromatography eluting with 0−20% (1:1 MeOH/DCM) in DCM
yielded 7 g of a white foam. Further recrystallization from 10%
EtOAc/hexanes yielded cis-( )-tert-butyl (3-(3-aminopyridin-4-yl)-5-
methylcyclohexylcarbamate (5 g, 47%). LCMS (m/z): 306.3 (MH+),
1
H). H NMR (400 MHz, CDCl3) δ ppm 9.17 (s, 1 H), 8.78 (d, J =
4.70 Hz, 1 H), 7.30 (d, J = 4.70 Hz, 1 H), 5.74−5.79 (m, 1 H), 4.55 (t,
J = 3.72 Hz, 1 H), 4.27 (t, J = 2.54 Hz, 1 H), 2.59 (br s, 1 H), 2.28−
2.36 (m, 1 H), 2.22−2.28 (m, 2 H), 1.18 (d, J = 6.26 Hz, 3 H).
( )-2-Azido-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol
(VIII). To a solution of ( )-6-bromo-5-methyl-3-(3-nitropyridin-4-
yl)cyclohex-2-enol (VII) (16 g, 51.5 mmol) in THF (510 mL) was
added potassium tert-butoxide (8.6 g, 77 mmol) in portions. The
reaction turned from orange to black almost immediately. By TLC, the
formation of product is clean in 30 min. The reaction was quenched by
adding saturated ammonium chloride and ethyl acetate. The organic
phase was washed with brine, then dried with sodium sulfate, filtered,
and concentrated. The crude product was dissolved in ethanol and
water (3:1, 400 mL, 0.1 M), and ammonium chloride (5.5 g, 103
mmol) and sodium azide (6.6 g, 103 mmol) were added. The dark
orange reaction was stirred at room temperature overnight. The
conversion to product is clean as indicated by LC/MS. The reaction
was concentrated to remove the ethanol, ethyl acetate and water were
added, the organic phase was dried with sodium sulfate, filtered, and
concentrated. The crude material was purified via silica gel column
chromatography, eluting with ethyl acetate and hexanes (1:1) to give
1
VIII in 55% yield (12 g). LC/MS = 276.0 (M + H). H NMR (400
MHz, CDCl3) δ ppm 9.18 (s, 1 H), 8.78 (d, J = 5.09 Hz, 1 H), 7.25 (d,
J = 5.09 Hz, 1 H), 5.57 (t, J = 2.15 Hz, 1 H), 4.01−4.09 (m, 1 H),
3.45−3.58 (m, 1 H), 2.26−2.41 (m, 2 H), 2.06−2.22 (m, 2 H),1.12−
1.17 (m, 3 H).
( )-4-(3-Aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-
6-methylcyclohexyl Acetate (IX). To a solution of ( )-2-azido-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol (VIII) (12 g, 44 mmol)
in pyridine and ammonium hydroxide (8:1, 225 mL) was added
trimethylphosphine (9.95 g, 131 mmol), and the brown solution was
stirred at room temperature for 2 h (bubbling). Upon completion,
EtOH (50 mL) was added and the solution was concentrated in vacuo.
More ethanol (50 mL) was added, and the reaction was concentrated
again. Dioxane and sat. NaHCO3 (1:1, 200 mL, 0.08 M) were added
to the crude, followed by Boc2O (9.5 g, 44 mmol). The reaction
mixture was stirred at room temperature for 2 h, then water and ethyl
acetate were added. The organic phase was dried with MgSO4, and
concentrated. The crude product was purified via silica gel column
chromatography, eluting with ethyl acetate and hexanes (1:1) to afford
1
LC tR = 2.59 min. H NMR (400 MHz, CDCl3) δ ppm 8.04 (s, 1H),
7.98 (d, J = 5.1, 1H), 6.94 (d, J = 5.1, 1H), 4.3 (m, 1H), 3.77−3.54 (m,
2H), 2.68−2.54 (m, 1H), 2.19 (d, J = 12.5, 1H), 2.06 (d, J = 12.2, 1H),
1.83 (d, J = 12.9, 1H), 1.76−1.62 (m, 1H), 1.44 (s, 9H), 1.20−1.09
(m, 2H), 0.99 (d, J = 6.7, 3H), 0.86 (q, J = 12.0, 1H). Pure
enantiomers were by chiral HPLC. Separation of the enantiomers was
performed under the following conditions. Column: Chiralpak AD (50
J
DOI: 10.1021/acs.jmedchem.5b01275
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
( )-tert-butyl 6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-
enylcarbamate (7.9 g, 59%). LC/MS = 350.1 (M + H).1H NMR
(400 MHz, CDCl3) δ ppm 9.12 (s, 1 H), 8.75 (d, J = 5.09 Hz, 1 H),
7.24 (d, J = 4.70 Hz, 1 H), 5.46 (t, J = 2.35 Hz, 1 H), 4.77 (m, 1 H),
4.27 (m, 1 H), 3.48 (m, 1 H), 3.36−3.41 (m, 1 H), 1.95−2.34 (m, 4
H), 1.43−1.50 (m, 9 H), 1.14 (d, J = 6.26 Hz, 3 H). To a solution of
( )-tert-butyl 6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-
enylcarbamate (7.7 g, 22 mmol) in pyridine (220 mL) was added
Ac2O (6.2 mL, 66 mmol), and the reaction was stirred at room
temperature overnight. Upon completion, the reaction was concen-
trated to dryness, then worked up with ethyl acetate and water. The
organic phase was washed with brine, then dried with sodium sulfate,
filtered, and concentrated to give ( )-2-(tert-butoxycarbonylamino)-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enyl acetate in 99% yield
ether (1:21). The mixture was sonicated, and the precipitate was
filtered. The precipitate was dried under high vacuum for several days
to yield N-(4-((1R,3S,5S)-3-amino-5-methylcyclohexyl)pyridin-3-yl)-
6-(2,6-difluorophenyl)-5-fluoropicolinamide as the HCl salt in 80%
yield. The free base was obtained by partitioning the salt between
EtOAc and Na2CO3 (sat.), washing with brine, drying over MgSO4,
and removal of volatiles in vacuo. LCMS (m/z): 441.3 (MH+). HRMS
1
(m/z) calcd for C24H24F3N4O (MH+) 441.1902, found 441.1905. H
NMR (400 MHz, DMSO-d6, bis-HCl salt) δ 10.59 (s, 1H), 8.92 (s,
1H), 8.62 (d, J = 6.0, 1H), 8.37 (dd, J = 8.8, 4.0, 1H), 8.23 (t, J = 8.8,
1H), 8.19 (bs, 2H), 7.68−7.71 (m, 2H), 7.36−7.40 (m, 2H),3.01−
3.10 (m, 2H), 2.01−2.05 (m, 1H), 1.94−1.97 (m, 1H), 1.72−1.76 (m,
1H), 1.46−1.53 (m, 2H), 1.01−1.13 (m, 2H), 0.89 (d, J = 6.8, 3H).
13C NMR (125.77 MHz, CDCl3, free base) 160.8, 160.5, 159.8, 18,
131.8, 131.4, 125.4, 124.8, 120.7, 111.9, 111.8, 50.3, 44.3, 41.6, 39.8,
36.5, 31.6, 22.1. In variations of this general method, the Boc group
could be deprotected by treatment with 25% TFA/CH2Cl2 for 30 min,
and upon concentration of the volatiles in vacuo, purification via RP-
HPLC yielded final compounds as TFA salts after lyophilization.
Compounds in free base form were obtained by partitioning HPLC
purified fractions between EtOAc and Na2CO3(s), washing with brine,
drying over MgSO4, and removal of volatiles in vacuo. For compounds
10, 12, and 16 the phthalimide group was removed by treating with
hydrazine (10 equiv) in MeOH (0.1M) at 60 °C for 2 h or overnight
at rt.
1
(8.5 g). LC/MS = 392.2 (M + H). H NMR (400 MHz, CDCl3) δ
ppm 9.14 (s, 1 H), 8.75 (d, J = 5.09 Hz, 1 H) 7.23 (d, J = 5.09 Hz, 1
H), 5.50−5.59 (m, 1 H), 4.81 (t, J = 9.78 Hz, 1 H), 4.72 (d, J = 9.00
Hz, 1 H), 4.40−4.53 (m, 1 H), 2.18−2.46 (m, 4 H), 2.13 (s, 3 H), 1.42
(s, 9 H), 1.01 (d, J = 5.87 Hz, 3 H). To a degassed solution of ( )-2-
(tert-butoxycarbonylamino)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-
3-enyl acetate (8.5 g, 22 mmol) in MeOH and EtOAc (1:1, 200 mL)
was added 10% Pd/C (2.3 g, 2.2 mmol), and the reaction was stirred
at room temperature under a hydrogen balloon for 3 days. Upon
completion, the solution was filtered through a pad of Celite, the pad
was washed with ethyl acetate, and the filtrate was concentrated. The
crude material contained about 10% of the undesired isomer. The
crude was dissolved in ethyl acetate (∼20%) and hexanes and heated
until all dissolved. The solution was allowed to sit at room temperature
for 2 days. The precipitate was then collected to give ( )-4-(3-
aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-6-methylcyclohexyl
acetate as the pure product in 75% yield (5.9 g). LC/MS = 364.3 (M +
H), tR = 0.63 min. 1H NMR (400 MHz, CDCl3) δ ppm 8.05 (s, 1 H),
8.00 (d, J = 5.09 Hz, 1 H), 6.93 (d, J = 5.09 Hz, 1 H), 4.64 (d, J = 8.61
Hz, 1 H), 4.52 (t, J = 10.17 Hz, 1 H), 3.77−3.83 (m, 1 H), 3.66 (br s,
2 H), 2.68−2.74 (m, 1 H), 2.26 (ddd, J = 12.91, 6.85, 2.93 Hz, 1 H),
2.12 (s, 3 H), 1.83−1.99 (m, 2 H), 1.42 (s, 9 H), 1.39−1.27 (m, 2 H),
0.99 (d, J = 6.26 Hz, 3 H). Separation of the enantiomers was
performed under the following conditions. Column: Chiralpak AD (50
mm × 500 mm). Solvent: heptane/2-propanol (80:20). Flow rate: 80
mL/min. Injection volume: 10 mL. UV: 210 nm. Peak 1,
(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-((tert-butoxycarbonyl)-
amino)-6-methylcyclohexyl acetate (IX), at 8.043 min. Peak 2,
(1S,2S,4S,6R)-4-(3-aminopyridin-4-yl)-2-((tert-butoxycarbonyl)-
amino)-6-methylcyclohexyl acetate, at 14.581 min.
N - ( 4 - ( ( 1 R , 3 R , 4 R , 5 S ) - 3 - A m i n o - 4 - h y d r o x y - 5 -
methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-
fluoropicolinamide (4). Following the general procedure,
(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butoxycarbonylamino)-
6-methylcyclohexyl acetate and 6-(2,6-difluorophenyl)-5-fluoropico-
linic acid were coupled and deprotected to yield 2. The acetate group
was removed prior to Boc deprotection by treating with K2CO3 (1.2
equiv) in EtOH (0.1 M) overnight. LC/MS = 457.3 (M + H). HRMS
1
(m/z) calcd for C24H24F3N4O2 (MH+) 457.1851, found 457.1855. H
NMR (DMSO-d6, HCl salt) δ 10.42 (s, 1H), 8.65 (s, 1H), 8.45 (d, J =
5.1, 1H), 8.33 (dd, J = 8.6, 3.9, 1H), 8.19 (t, J = 8.8, 1H), 8.0 (bs, 2H),
7.69 (m, 1H), 7.39−7.32 (m, 3H), 3.07−2.94 (m, 2H), 2.82 (m, 1H),
2.03−2.00 (m, 1H), 1.74−1.59 (m, 2H), 1.40 (m, 1H), 1.23 (m, 1H),
0.93 (d, J = 6.3, 3H).
N-(4-((3S,5R)-3-Amino-5-methylpiperidin-1-yl)pyridin-3-yl)-
5-fluoro-6-(2-fluorophenyl)picolinamide (5). Following the gen-
eral procedure tert-butyl (3S,5R)-1-(3-aminopyridin-4-yl)-5-methylpi-
peridin-3-ylcarbamate and 5-fluoro-6-(2-fluorophenyl)picolinic acid
were coupled and deprotected to yield 5. LC/MS = 424.1 (M + H).
HRMS (m/z) calcd for C23H24F2N5O (MH+) 424.1949, found
General Procedure for Amide Coupling and Amine
Deprotection for Preparing 4−16. tert-Butyl (1S,3R,5S)-3-(3-
(6-(2,6-Difluorophenyl)-5-fluoropicolinamido)pyridin-4-yl)-5-
methylcyclohexylcarbamate (8). A solution of tert-butyl
(1S,3R,5S)-3-(3-aminopyridin-4-yl)-5-methylcyclohexylcarbamate
(1.46 g, 4.78 mmol), 6-(2,6-difluorophenyl)-5-fluoropicolinic acid
(1.57 g, 6.21 mmol), EDC−HCl (1.2 g, 6.21 mmol), and HOAt (0.85
g, 6.21 mmol) in DMF (24 mL, 0.2 M) was stirred at room
temperature overnight. The reaction was poured into water and
extracted with ethyl acetate (4×). The combined organics were
washed with 0.5 N NaOH (3×), brine, dried over sodium sulfate,
filtered, and concentrated to afford tert-butyl (1S,3R,5S)-3-(3-(6-(2,6-
difluorophenyl)-5-fluoropicolinamido)pyridin-4-yl)-5-
methylcyclohexylcarbamate in 91% yield (2.34 g). LCMS (m/z): 541.0
1
424.1953. H NMR (400 MHz, DMSO-d6, TFA salt) δ ppm 10.39
(s, 1 H), 9.13 (s, 1 H), 8.39 (d, J = 6.21 Hz, 1 H), 8.34 (dd, J = 8.58,
3.84 Hz, 1 H), 8.18 (t, J = 8.97 Hz, 1 H), 7.99 (br s, 2 H), 7.76−7.84
(m, 1 H), 7.60−7.72 (m, 1 H), 7.41−7.50 (m, 2 H), 7.32 (d, J = 6.26
Hz, 1 H), 3.66−3.79 (m, 1 H), 3.57 (d, J = 10.27 Hz, 1 H), 3.18 (br s,
1 H), 2.85 (t, J = 11.30 Hz, 1 H), 2.28−2.41 (m, 1 H), 1.82 (d, J =
12.32 Hz, 1 H), 1.70 (d, J = 6.46 Hz, 1 H), 1.05 (q, J = 11.88 Hz, 1 H),
0.59 (d, J = 6.55 Hz, 3 H).
N-(4-((1R,3S,5S)-3-Amino-5-methylcyclohexyl)pyridin-3-yl)-
5-fluoro-6-(2-fluorophenyl)picolinamide (6). Following the gen-
eral procedure tert-butyl (1S,3R,5S)-3-(3-aminopyridin-4-yl)-5-
methylcyclohexylcarbamate and 5-fluoro-6-(2-fluorophenyl)picolinic
acid were coupled and deprotected to yield 6. LC/MS = 423.1 (M
+ H). HRMS (m/z) calcd for C24H25F2N4O (MH+) 423.1996, found
423.1996. 1H NMR (400 MHz, DMSO-d6, TFA salt) δ ppm 10.42 (s,
1 H), 8.72 (s, 1 H), 8.47 (d, J = 5.18 Hz, 1 H), 8.27 (dd, J = 8.61, 3.91
Hz, 1 H), 8.13 (t, J = 9.05 Hz, 1 H), 7.89 (td, J = 7.52, 1.79 Hz, 1 H),
7.81 (br s, 2 H), 7.59−7.70 (m, 1 H), 7.35−7.49 (m, 3 H), 3.09 (d, J =
3.62 Hz, 1 H), 2.96 (t, J = 12.13 Hz, 1 H), 2.00 (d, J = 11.88 Hz, 1 H),
1.89 (d, J = 12.91 Hz, 1 H), 1.75 (d, J = 12.52 Hz, 1 H), 1.52 (br s, 1
H), 1.41 (q, J = 11.95 Hz, 1 H), 0.95−1.12 (m, 2 H), 0.89 (d, J = 6.50
Hz, 3 H).
1
(MH+). H NMR (400 MHz, CDCl3) δ ppm 9.86 (s, 1H), 9.34 (s,
1H), 8.43 (dd, J = 8.6, 3.9, 1H), 8.39 (d, J = 5.1, 1H), 7.77 (t, J = 8.4,
1H), 7.58−7.43 (m, 1H), 7.17−7.05 (m, 3H), 4.43 (bs, 1H), 3.55 (bs,
1H), 2.94−2.80 (m, 1H), 2.13 (d, J = 12.2, 1H), 2.07−1.94 (m, 1H),
1.83 (d, J = 12.9, 1H), 1.60−1.47 (m, 1H), 1.43 (m, 9H), 1.40−1.30
(m, 1H), 1.05−0.89 (m, 1H), 0.86 (d, J = 6.3, 3H), 0.82−0.71 (m,
1H). A solution of 4 M HCl in dioxane (13.4 mL, 53.6 mmol) was
added to tert-butyl (1S,3R,5S)-3-(3-(6-(2,6-difluorophenyl)-5-
fluoropicolinamido)pyridin-4-yl)-5-methylcyclohexylcarbamate (2.9 g,
5.36 mmol), and the reaction mixture was stirred at room temperature
for 17 h. The heterogeneous solution was concentrated in vacuo to
yield a white solid which was further dissolved in MeOH and diethyl
N-(4-((3S,5R)-3-Amino-5-methylpiperidin-1-yl)pyridin-3-yl)-
6-(2,6-difluorophenyl)-5-fluoropicolinamide (7). Following the
general procedure tert-butyl (3S,5R)-1-(3-aminopyridin-4-yl)-5-meth-
K
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Drug Annotation
ylpiperidin-3-ylcarbamate and 6-(2,6-difluorophenyl)-5-fluoropicolinic
acid were coupled and deprotected to yield 7. LC/MS = 442.3 (M +
H). HRMS (m/z) calcd for C23H23F3N5O (MH+) 442.1855, found
442.1856. 1H NMR (400 MHz, methanol-d4, TFA salt) δ ppm 9.29 (s,
1 H), 8.47 (dd, J = 8.68, 3.94 Hz, 1 H), 8.39 (d, J = 6.46 Hz, 1 H), 8.07
(t, J = 8.68 Hz, 1 H), 7.59−7.76 (m, 1 H), 7.48 (d, J = 6.55 Hz, 1 H),
7.25 (t, J = 8.56 Hz, 2 H), 3.81−3.97 (m, 1 H), 3.72 (d, J = 12.67 Hz,
1 H), 3.32−3.41 (m, 1 H), 3.01 (t, J = 11.32 Hz, 1 H), 2.47 (t, J =
12.25 Hz, 1 H), 2.00 (d, J = 12.28 Hz, 1 H), 1.79−1.93 (m, 1 H), 1.20
(q, J = 12.13 Hz, 1 H), 0.73 (d, J = 6.60 Hz, 3 H).
the general procedure tert-butyl (1S,3R,5S)-3-(3-aminopyridin-4-yl)-
5-methylcyclohexylcarbamate and 2-(2,6-difluorophenyl)thiazole-4-
carboxylic acid were coupled and deprotected to yield 14. LC/MS =
429.2 (M + H). HRMS (m/z) calcd for C22H23F2N4OS (MH+)
1
429.1561, found 429.1561. H NMR (400, DMSO-d6, HCl salt): δ
10.21 (s, 1H), 8.74 (s, 1H), 8.64 (bs, 1H), 8.47 (d, J = 4.8, 1H), 7.99
(bs, 2H), 7.64−7.12 (m, 1H), 7.35−7.42 (m, 3H), 3.11 (bs, 1H),
2.93−3.00 (m, 1H), 2.01 (d, J = 12.4, 1H), 1.92 (d, J = 11.6, 1H), 1.74
(d, J = 12.4, 1H), 1.52−1.60 (m, 1H), 1.42 (q, J = 12.0, 1H), 1.00−
1.10 (m, 2H), 0.92 (d, J = 6.4, 3H).
(S)-N-(4-(3-Aminopiperidin-1-yl)pyridin-3-yl)-6-(2,6-difluoro-
phenyl)-5-fluoropicolinamide (15). Following the general proce-
dure (S)-tert-butyl 1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate
and 6-(2,6-difluorophenyl)-5-fluoropicolinic acid were coupled and
deprotected to yield 3. LC/MS = 428.1 (M + H). HRMS (m/z) calcd
(S)-3-Amino-N-(4-(3-aminopiperidin-1-yl)pyridin-3-yl)-6-
cyclohexylpicolinamide (9). Following the general procedure (S)-
tert-butyl 1-(3-aminopyridin-4-yl)piperidin-3-ylcarbamate and 3-
amino-6-cyclohexylpicolinic acid were coupled and deprotected to
yield 9. LC/MS = 395.2 (M + H). HRMS (m/z) calcd for C22H31N6O
1
for C22H21F3N5O (MH+) 428.1698, found 428.1699. H NMR (400
1
(MH+) 395.2559, found 395.2560. H NMR (400 MHz, methanol-d4,
MHz, methanol-d4) δ ppm 9.15 (s, 1 H), 8.46 (dd, J = 8.66, 3.91 Hz, 1
H), 8.38 (d, J = 6.70 Hz, 1 H), 8.06 (t, J = 8.68 Hz, 1 H), 7.59−7.71
(m, 1 H), 7.50 (d, J = 6.75 Hz, 1 H), 7.23 (t, J = 8.49 Hz, 2 H), 3.88
(dd, J = 12.10, 3.40 Hz, 1 H), 3.66 (d, J = 13.25 Hz, 1 H), 3.38−3.48
(m, 1 H), 3.11−3.29 (m, 2 H), 1.98−2.09 (m, 1 H), 1.75−1.86 (m, 1
H), 1.58−1.75 (m, 2 H).
N-(4-((1R,3S)-3-Aminocyclohexyl)pyridin-3-yl)-6-(2,6-difluor-
ophenyl)-5-fluoropicolinamide (16). Following the general
procedure 2-(3-(3-aminopyrin-4-yl)cyclohexyl)isoindoline-1,3-dione
and 6-(2,6-difluorophenyl)-5-fluoropicolinic acid were coupled and
deprotected to yield 4. LC/MS = 427.2 (M + H). HRMS (m/z) calcd
for C23H22F3N4O (MH+) 427.1746, found 427.1749. 1H NMR
(CDCl3, free-base): δ 9.93(s, 1H), 9.38(s, 1H), 8.40−8.45(m,1H),
8.40(d, 1H), 7.74−7.80(m, 1H), 7.47−7.55(m, 1H), 7.19(d, 1H),
7.06−7.13(m, 2H), 2.68−2.83(m, 2H), 1.97−2.05(m, 1H), 1.65−
1.95(m, 5H), 1.22−1.40(m, 3H), 1.04−1.15(m, 1H).
Microsomal Stability Testing. The test compounds were
incubated at a concentration of 1 μM in the presence of a 0.5 mg/
mL microsomal protein suspension, 1 mM UDPGA, 3 mM MgCl2, 1
mM NADPH, and 25 μg of alamethicin/mg microsomal of protein.
Over the time course of the incubation, duplicate samples were
collected at intervals of 0, 5, 15, and 30 min for determination of
metabolic rate and parent compound remaining at the end of a 30 min
incubation. Phenolphthalein was used as a reference control. Data
were obtained utilizing semiquantitative LC−MS/MS methods.
Calculation of scaled intrinsic clearance: CLint = 0.693 × [1/t1/2
(min)] × (g of liver weight/kg of body weight) × (mL incubation/mg
of microsomal protein) × (45 mg of microsomal protein/g of liver
weight) with rat = 45 g of liver per kg of body weight and with human
= 25.7 g of liver per kg of body weight. Hepatic clearance was
extrapolated from microsomal data using well-stirred liver model CLh
= [Qh × (fub/fui) × CLint]/Qh + (fub/fui) × CLint, where assumption is
fub(fraction unbound in blood) = fui(fraction unbound in incubation)
and Qh values of 55 and 20.7 mL min−1 kg−1 were used for rat and
human. Calculation of hepatic extraction ratio was with E = CLh/Qh.
PIM Enzymatic Assays. Kinase-Glo Pim1, Pim2, Pim3 ATP
Depletion Assays. Pim1, Pim2, and Pim3 kinase-Glo assays using
ATP (at or below ATP Km) were used as previously described20 to
determine the biochemical activity of the compounds 1−14 as shown
in Table 1.
TFA salt) δ ppm 9.33 (s, 1 H), 8.37 (d, J = 6.50 Hz, 1 H), 7.50 (d, J =
6.50 Hz, 1 H), 7.16−7.34 (m, 2 H), 3.83 (d, J = 12.08 Hz, 1 H), 3.65
(d, J = 12.76 Hz, 1 H), 3.51−3.60 (m, 1 H), 3.04−3.18 (m, 2 H),
2.61−2.73 (m, 1 H), 2.26 (dd, J = 13.30, 3.42 Hz, 1 H), 1.86−2.07 (m,
5 H), 1.81 (d, J = 12.37 Hz, 1 H), 1.64−1.77 (m, 1 H), 1.26−1.62 (m,
6 H).
3-Amino-N-(4-((1R,3S)-3-aminocyclohexyl)pyridin-3-yl)-6-
cyclohexylpicolinamide (10). Following the general procedure 2-
(3-(3-aminopyrin-4-yl)cyclohexyl)isoindoline-1,3-dione and 3-amino-
6-cyclohexylpicolinic acid were coupled and deprotected to yield 10.
LC/MS = 394.2 (M + H). HRMS (m/z) calcd for C23H32N5O (MH+)
1
394.2607, found 394.2610. H NMR (400 MHz, cd3od, TFA salt) δ
ppm 9.47 (s, 1 H), 8.49 (d, J = 5.67 Hz, 1 H), 7.77 (d, J = 5.72 Hz, 1
H), 7.16−7.33 (m, 2 H), 3.11−3.24 (m, 1 H), 2.63−2.74 (m, 1 H),
2.26 (d, J = 11.98 Hz, 1 H), 2.11−2.22 (m, 2 H), 1.96−2.11 (m, 3 H),
1.91 (d, J = 12.81 Hz, 2 H), 1.77−1.86 (m, 1 H), 1.66−1.77 (m, 2 H),
1.40−1.66 (m, 6 H), 1.23−1.40 (m, 2 H).
(S)-N-(4-(3-Aminopiperidin-1-yl)pyridin-3-yl)-2-(2,6-
difluorophenyl)thiazole-4-carboxamide (11). Following the
general procedure (S)-tert-butyl 1-(3-aminopyridin-4-yl)piperidin-3-
ylcarbamate and 2-(2,6-difluorophenyl)thiazole-4-carboxylic acid were
coupled and deprotected to yield 11. LC/MS = 416.1 (M + H).
HRMS (m/z) calcd for C20H20F2N5OS (MH+) 416.1357, found
1
416.1354. H NMR (400 MHz, DMSO-d6, HCl salt) δ ppm 10.25 (s,
1 H), 8.88 (s, 1 H), 8.80 (s, 1 H), 8.41 (d, J = 6.80 Hz, 1 H), 8.33 (br
s, 3 H), 7.64−7.76 (m, 1 H), 7.34−7.46 (m, 3 H), 3.86 (d, J = 9.34 Hz,
1 H), 3.56 (d, J = 13.84 Hz, 1 H), 3.37 (br s, 2 H), 3.27 (br s, 1 H),
1.93−2.05 (m, 1 H), 1.79−1.93 (m, 1 H), 1.51−1.77 (m, 2 H).
N-(4-((1R,3S)-3-Aminocyclohexyl)pyridin-3-yl)-2-(2,6-
difluorophenyl)thiazole-4-carboxamide (12). Following the
general procedure 2-(3-(3-aminopyrin-4-yl)cyclohexyl)isoindoline-
1,3-dione and 2-(2,6-difluorophenyl)thiazole-4-carboxylic acid were
coupled and deprotected to yield 12. LC/MS = 415.0 (M + H).
HRMS (m/z) calcd for C21H21F2N4OS (MH+) 415.1404, found
415.1403. 1H NMR (400 MHz, DMSO-d6, TFA salt) δ ppm 10.21 (s,
1 H), 8.74 (s, 1 H), 8.64 (s, 1 H), 8.49 (d, J = 5.23 Hz, 1 H), 7.82 (br
s, 3 H), 7.63−7.77 (m, 1 H), 7.32−7.47 (m, 3 H), 3.10 (br s, 1 H),
2.94 (t, J = 11.79 Hz, 1 H), 1.91−2.05 (m, 2 H), 1.71−1.90 (m, 2 H),
1.46 (q, J = 12.23 Hz, 1 H), 1.26−1.38 (m, 3 H).
N-(4-((3S,5R)-3-Amino-5-methylpiperidin-1-yl)pyridin-3-yl)-
2-(2,6-difluorophenyl)thiazole-4-carboxamide (13). Following
the general procedure tert-butyl (3S,5R)-1-(3-aminopyridin-4-yl)-5-
methylpiperidin-3-ylcarbamate and 2-(2,6-difluorophenyl)thiazole-4-
carboxylic acid were coupled and deprotected to yield 13. LC/MS =
430.0 (M + H). HRMS (m/z) calcd for C21H22F2N5OS (MH+)
430.1513, found 430.1514. 1H NMR (400 MHz, DMSO-d6, HCl salt)
δ ppm 9.85 (s, 1 H), 9.24 (s, 1 H), 8.81 (s, 1 H), 8.33 (d, J = 5.53 Hz,
1 H), 8.12 (br s, 3 H), 7.65−7.76 (m, 1 H), 7.41 (t, J = 8.83 Hz, 2 H),
7.22 (d, J = 5.53 Hz, 1 H), 3.51 (d, J = 9.19 Hz, 1 H), 3.31−3.32 (m, 2
H), 2.64−2.75 (m, 1 H), 2.34 (t, J = 11.57 Hz, 1 H), 2.06 (d, J = 12.18
Hz, 1 H), 1.95 (d, J = 6.31 Hz, 1 H), 1.13 (q, J = 12.05 Hz, 1 H), 0.80
(d, J = 6.55 Hz, 3 H).
High ATP Pim1, Pim2, Pim3 AlphaScreen Assays. Pim1, Pim2, and
Pim3 AlphaScreen assays using high ATP (11−125 × APP Km) were
used as previously described20 to determine the biochemical activity of
compounds 1, 2, and 8 as shown in Table 3.
Biochemical Kinase Specificity Profile of 8. The kinase
specificity profile for compound 8 reported in Table 5 was determined
as previously described.28 Compound 8 was assessed in the Ambit
KINOMEscan binding assay at 1 μM with activity presented as % of
control = [(test compound signal − positive control signal)/(negative
control signal − positive control signal]] × 100. Data for the 442
kinase KINOMEscan are in Supporting Information.
Cell Lines and Reagents. The KMS-11.luc human multiple
myeloma tumor cell line, a KMS-11 clone expressing firefly luciferase,
was obtained from the University Health Network (UHN), Toronto,
N-(4-((1R,3S,5S)-3-Amino-5-methylcyclohexyl)pyridin-3-yl)-
2-(2,6-difluorophenyl)thiazole-4-carboxamide (14). Following
L
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Drug Annotation
Ontario, Canada. KMS-11.luc was cultured in RPMI-1640 (ATCC,
Manassas, VA) supplemented with 10% FBS. The AML cell line KG-1
and all others included in Table 5 were obtained from commercial
sources (ATCC or DSMZ) and were cultured in RPMI or IMDM plus
10−20% FBS (Invitrogen) as supplier recommended.
Cellular Proliferation Assays. Proliferation assays in KMS-11.luc
were conducted as previously described20 with the concentration at
which 50% maximal inhibition is reached reported as EC50.
Proliferation assays in KG-1 cells lines as well as for cell lines in the
expanded AML panel, Table 5, were conducted as previously
described7 with the concentration at which growth is inhibited by
50% reported as GI50.
Western Blot Analysis. Following treatment of KG-1 cells with 8
for 2 h at the indicated concentrations, cells were lysed in RIPA buffer.
Protein concentration was determined using a BCA assay (Thermo
Scientific 23227), and 50 μg of lysate was separated by SDS−PAGE
using 10% bis-Tris gels. Proteins were transferred onto 0.2 μm
nitrocellulose membrane (Invitrogen LC2002), and pS6RP was
detected using an antibody from Cell Signaling Technology (CST
no. 4857) while total S6RP was detected with CST no. 2217.
Following incubation with secondary antibodies, antibody binding was
detected using ECL Advance (GE Healthcare RPN2135).
Phosphoprotein Assays. Commercial electrochemiluminescence
(ECL) assay kits from Meso Scale Discovery (MSD; Rockville, MD)
were used to quantify the effects of compounds on the levels of
phosphorylated S6RP in KG-1 cells and KMS11-luc cells. For in vitro
assays, KMS11-luc cells were seeded in 96-well tissue culture plates,
and then compounds were serially diluted and added to cell plates to
achieve a final concentration range of 10 μM to 2 nM in 0.1% DMSO
and incubated with cells for 1 h at 37 °C. Cells were pelleted by
centrifugation for 7 min at 1500 rpm. Medium was gently aspirated,
and MSD lysis buffer was added. Cells were lysed by placing plates on
a DELFIA (PerkinElmer, Waltham MA) plate shaker at 4 °C and
shaking the plates for 30 min at 600 rpm. For in vivo assays, MDS lysis
buffer (MSD, Rockville, MD) was added to frozen pulverized tumor
samples on ice and homogenates were prepared using the MagNA
Lyser bead instrument (Roche Applied Science, Indianapolis, IN) by
disrupting the samples with four cycles of 6000 rpm for 30 s at 4 °C.
Supernatants were created following centrifugation at 11 000 rpm for
15 min at 4 °C, and protein concentration was determined using the
BCA protein assay kit according to the manufacturer’s instructions
(Pierce Chemical Company, Rockford, IL). For both assays, samples
were transferred to ECL assay plates previously blocked with 3% BSA,
sealed, and incubated at 4 °C overnight while undergoing gentle
shaking on a DELFIA (PerkinElmer, Waltham MA) plate shaker. After
overnight incubation, assay plates were processed according to
manufacturer instructions.
AUTHOR INFORMATION
Corresponding Author
*E-mail: matthew.burger@novartis.com. Phone: 510-923-3537.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Weiping Jia and Dahzi Tang for analytical
chemistry support and Kent Wong for in vitro DMPK support.
■
ABBREVIATIONS USED
■
AML, acute myeloid leukemia; ATP, adenosine triphospate;
[ATP], concentration of adenosine triphospate; Ara-C,
cytarabine; BAD, proapoptotic protein of the BCl2 family;
BOC, tert-butoxycarbonyl; CL, clearance; ER, extraction ratio;
hERG, human-ether-a-go-go-related gene; JAK/STAT, Janus
kinase and signal transducer and activator of transcription; Km,
Michaelis constant; log D7.4, log of the octanol−water
distribution coefficient at pH 7.4 of ionized and un-ionized
compound; PD, pharmacodynamics; PIM, proviral insertion of
Moloney virus; PK, pharmacokinetics; PSA, polar surface area;
S6RP, ribosomal protein S6; S(35), number of nonmutant
kinases tested with ≥65% inhibition/number nonmutant
kinases tested
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■
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ASSOCIATED CONTENT
■
S
* Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01275.
Data for the 442 kinase KINOMEscan (PDF)
Accession Codes
Coordinates and structure factors have been deposited in the
PDB with accession code 5DWR for the PIM1:8 complex.
M
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Journal of Medicinal Chemistry
Drug Annotation
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N
DOI: 10.1021/acs.jmedchem.5b01275
J. Med. Chem. XXXX, XXX, XXX−XXX