Synthesis and Structure-Activity Relationship of Tetra-Substituted Cyclohexyl Diol Inhibitors of Proviral Insertion of Moloney Virus (PIM) Kinases

Article abstract of DOI:10.1021/acs.jmedchem.0c01279

Overexpression of PIM 1, 2, and 3 kinases is frequently observed in many malignancies. Previously, we discovered a potent and selective pan-PIM kinase inhibitor, compound 2, currently in phase I clinical trials. In this work, we were interested in replacing the amino group on the cyclohexane ring in compound 2 with a hydroxyl group. Structure-based drug design led to cellularly potent but metabolically unstable tetra-substituted cyclohexyl diols. Efforts on the reduction of Log D by introducing polar heterocycles improved metabolic stability. Incorporating fluorine to the tetra-substituted cyclohexyl diol moiety further reduced Log D, resulting in compound 14, a cellularly potent tetra-substituted cyclohexyl diol inhibitor with moderate metabolic stability and good permeability. We also describe the development of efficient and scalable synthetic routes toward synthetically challenging tetra-substituted cyclohexyl diol compounds. In particular, intermediate 36 was identified as a versatile intermediate, enabling a large-scale synthesis of highly substituted cyclohexane derivatives.

Full text of DOI:10.1021/acs.jmedchem.0c01279

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Article
Synthesis and Structure−Activity Relationship of Tetra-Substituted
Cyclohexyl Diol Inhibitors of Proviral Insertion of Moloney Virus
(PIM) Kinases
Wooseok Han,* Yu Ding, Zheng Chen, John L. Langowski, Cornelia Bellamacina, Alice Rico,
Gisele A. Nishiguchi, Jiong Lan, Gordana Atallah, Mika Lindvall, Song Lin, Richard Zang, Paul Feucht,
Tatiana Zavorotinskaya, Yumin Dai, Pablo Garcia, and Matthew T. Burger
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01279
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ABSTRACT: Overexpression of PIM 1, 2, and 3 kinases is frequently observed in many malignancies. Previously, we discovered a
potent and selective pan-PIM kinase inhibitor, compound 2, currently in phase I clinical trials. In this work, we were interested in
replacing the amino group on the cyclohexane ring in compound 2 with a hydroxyl group. Structure-based drug design led to
cellularly potent but metabolically unstable tetra-substituted cyclohexyl diols. Efforts on the reduction of Log D by introducing polar
heterocycles improved metabolic stability. Incorporating fluorine to the tetra-substituted cyclohexyl diol moiety further reduced Log
D, resulting in compound 14, a cellularly potent tetra-substituted cyclohexyl diol inhibitor with moderate metabolic stability and
good permeability. We also describe the development of efficient and scalable synthetic routes toward synthetically challenging tetra-
substituted cyclohexyl diol compounds. In particular, intermediate 36 was identified as a versatile intermediate, enabling a large-scale
synthesis of highly substituted cyclohexane derivatives.
cancers, a pan-PIM kinase inhibitor would be predicted to offer
the greatest potential clinical utility.11 In addition, elevated
expression of PIM2 specifically has been described in multiple
myeloma relative to other hematologic cancers, and this
expression has been shown to be required for proliferation,
presenting an effective pharmacological target for inhibition.12
However, identification of pan-PIM inhibitors retaining potent
PIM2 inhibitory capacity in myeloma cells is challenging given
the 10−100 times lower Km for ATP possessed by PIM2
relative to PIM1 and PIM3.13−21
INTRODUCTION
■
Proviral insertion of Moloney virus (PIM) kinases is a family of
three serine/threonine kinases (PIM1, PIM2, and PIM3)
initially identified as frequent sites of integration in viral-
induced murine lymphomas.1 In a normal setting, PIM
expression is induced in response to growth factors and
cytokines to promote the survival and proliferation of
hematopoietic cells, and their activity is primarily regulated
by the balance between synthesis and degradation.2 However,
the overexpression of PIMs in vitro or in transgenic mouse
models has confirmed a role for this family in tumorigenesis of
hematological tissues, either alone or in synergy with other
oncogenes.3−5 In humans, increased expression of PIM1 is
reported in acute lymphoblastic leukemia, acute myeloid
leukemia, and diffuse B-cell lymphoma, while high PIM2
expression is noted in multiple myeloma.6 In addition,
increased expression or dysfunction of all three PIMs has
been reported in solid tumors including those of prostate,
hepatocellular, colon, and gastric origin.2,7−10
In the course of our lead optimization efforts on the
pyridylcarboxamide scaffold, we reported the discovery of
compound 1,13 a potent pan-PIM inhibitor and in vivo tool
compound, through the structure-guided optimization of a
Received: August 11, 2020
Given the apparent functional redundancy of PIMs in
oncogenesis and a differential expression across a wide array of
https://dx.doi.org/10.1021/acs.jmedchem.0c01279
© XXXX American Chemical Society
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singleton HTS hit (Figure 1). Compound 1 demonstrated in
vivo efficacy in multiple myeloma (KMS-11.luc) and acute
Figure 1. Novartis pan-PIM Inhibitors.
myeloid leukemia (EOL-1) xenograft mouse models. However,
it showed high clearance in human liver microsomes, and the
piperidine A-ring was identified as the major metabolic moiety
by metabolic identification studies. Replacements of the
piperidine A-ring with other rings such as heteroaryl22 and
cyclohexyl were explored, and cyclohexane rings improved
metabolic stability in liver microsomes, resulting in the
identification of an orally available, potent, and selective pan-
PIM kinase inhibitor (PIM447), compound 2 (Figure 1).23 We
recently disclosed the phase I clinical trial data for PIM447 that
displayed good tolerability and single agent efficacy in several
patients with relapsed/refractory multiple myeloma.24
A key structural motif of compound 2 is the cyclohexyl
amine moiety. The X-ray co-crystal structure in PIM1 showed
that the amino group participated in polar H-bond interactions
with the Asp128 side chain carboxylate and the Glu171
backbone carbonyl in the ribose patch, which are crucial for
PIM potency.23 In the early stage of the discovery of
compound 2, we were interested in replacing the amino
group with non-basic isosteres such as a hydroxyl group as an
alternative H-bond acceptor and donor. Without an ionizable
group, such molecules could differentiate and display distinct
overall drug properties.25 Herein, we present the medicinal
chemistry efforts on optimizing cyclohexyl diol inhibitors of
pan-PIM kinases focusing on improving metabolic stability and
cellular potency and describe the development of a diverse set
of efficient synthetic routes to access synthetically challenging
highly substituted cyclohexyl diol intermediates.
Figure 2. X-ray structure of compound 2 in PIM1 (PDB code:
5DWR).
piperidinyl amine series, a hydroxyl group adjacent to the
amino group was successfully implemented to improve drug-
like properties by lowering Log D.13 Based on this knowledge,
we synthesized the trans-diol analogue, compound 4, with a
reduced Log D value (Log D = 3.0). The introduction of the
additional hydroxyl group favorably influenced microsomal
intrinsic clearance while having no negative impact on Caco-2
permeability and efflux, and a slight gain in cellular potency
(EC50 = 1.88 μM) was achieved relative to compound 3
(Table 1). Next, we employed a structure-based drug design
approach to further improve on the potency of compound 4.
As previously reported for compounds 1 and 2,13,23 the methyl
group at the 5-position fills a hydrophobic dimple in the
glycine rich loop, resulting in the potency increase with
minimal increase in lipophilicity. Therefore, we pursued the
same strategy by examining the glycine rich loop region in the
docking pose of compound 4. It was anticipated that the axial
methyl group at the 4-position would make a hydrophobic
contact with the Phe49 residue of the glycine rich loop (Figure
3a). We synthesized compound 5 and observed an increase in
the cellular potency by 6-fold (EC50 = 0.32 μM, Table 1) with
minimal increase in lipophilicity (Log D = 3.2). Since
compounds 4 and 5 have similar Caco-2 values, it was thought
that the increased cellular potency mainly resulted from the
increase in the binding affinity of compound 5 to the PIM
kinases by incorporating the 4-methyl group. We obtained the
X-ray co-crystal structure of compound 5 in PIM1 (Figure 3b),
which demonstrated that the methyl group at the 4-carbinol
position of the cyclohexane ring clearly made a hydrophobic
interaction with Phe49 residue of the P-loop, as predicted by
the docking model (Figure 3a). The X-ray structure also
revealed that the 4-hydroxyl group participates in a hydrogen
bond network with the Asp128 residue, an interaction that was
not present in the X-ray structure of compound 1 illustrating
the flexibility of the side chain of Asp128.
RESULTS AND DISCUSSION
■
Medicinal Chemistry. Our medicinal chemistry efforts of
the cyclohexyl diol series began with replacement of the amino
group of compound 2 with a hydroxyl group. We initially
performed molecular docking studies of compound 3 with
PIM1 kinase, which displayed a single H-bond interaction
between the hydroxyl group and the Glu171 residue, unlike the
amino group of compound 2 that makes two H-bond
interactions with Glu171 and Asp128 residues, as shown in
the X-ray co-crystal structure (Figure 2).
As predicted from the docking result, the potency of
compound 3 decreased in both biochemical and cellular assays
(EC50 = 3.41 μM, >20-fold decrease in potency in the KMS-
11.luc assay, Table 1). Because the biochemical potency
reached the lowest limit of detection in the series, the cellular
potency was used as to guide SAR. The permeability profile of
compound 3 was encouraging [Caco-2 assay: Papp (A−B) =
26.5 × 10−6 cm/s, ER = 0.9]; however, the increased
lipophilicity (Log D = 4.6) resulted in high intrinsic clearance
in liver microsomes across species (Table 1).26 In the previous
However, while compound 5 exhibited increased potency
relative to compound 4, it showed high in vitro and in vivo
clearance28 in rats (CLint = 187 μL/min/mg and CL = 56 mL/
min/kg, respectively), which correlated with the increased Log
D (Log D for compound 5 = 3.2, Table 1). Having identified
the 4-axial methyl group in the cyclohexyl diol A ring as a
driver of potency, we next focused on improving metabolic
stability of compound 5 by focusing on physicochemical
properties, especially lowering Log D by introducing polar C
and/or D rings. C ring heterocycle analogues of compound 5
B
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Table 1. Potency, Clearance, Permeability, and Physicochemical Property for PIM Compounds
CLint
b
Ki (mM)
PIM2
(mL/min/kg)
ER_H
Caco-2
Cpd
no.
cell prolif KMS-11 luc
EC50 (mM)
log
tPSA
(Å)
P
appA−B
ER
a
c
PIM1
PIM3
D7.4
rat
human
rat
human
(10−6 cm/s)
(PappB−A/PappA−B)
1
2
3
4
5
6
7
8
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.005
<0.001
<0.001
<0.001
0.001
<0.003
<0.003
0.004
<0.003
<0.003
0.003
<0.003
<0.003
0.003
<0.003
<0.003
<0.003
0.004
<0.003
<0.003
<0.003
0.004
0.017
0.17
3.41
1.88
0.32
>10
>10
1.06
0.93
0.45
3.24
1.71
1.74
0.25
2.2
1.9
4.2
3
104
81
75
95
95
198
28
375
107
183
60
13
352
61
0.88
0.51
0.93
0.8
0.77
0.42
0.95
0.77
0.73
8.2
8.5
26.5
28.8
27.6
16
13.4
28.1
10.1
23.5
6.6
30.3
14.6
28
2.6
2.6
0.9
1.1
1.1
1.8
6.6
1.4
5.6
0.9
6.9
1.1
2.9
1.7
3.2
2.5
1.3
2.6
2.8
3.7
2.3
3.2
1.8
1.4
48
0.87
95
N/D
N/D
0.74
0.013
108
121
134
121
134
134
134
134
<0.003
<0.003
<0.003
0.003
0.011
<0.003
<0.003
77
149
75
35
133
41
44
11
67
30
62
8
0.71
0.39
0.79
0.62
0.78
0.3
9
0.85
0.73
0.56
0.83
0.6
10
11
12
13
14
0.001
<0.001
<0.001
0.005
<0.003
<0.003
96
29
0.78
0.62
a
b
c
Measured Log D at pH 7.4. ER_H (hepatic extraction ratio) = CLh/Qh (see the Experimental Section). ER: Efflux ratio (PappB−A/PappA−B).
were first explored. Compounds 6 and 7, thiazole, and
pyrimidine C ring analogues were not potent in KMS-11.luc
cellular assay, but the corresponding analogues having a hinge
amino group on the C-ring, compounds 8 and 9, showed
around 1 μM in the cellular proliferation assay (Table 1).
Interestingly, while the hinge amino group in thiazole and
pyrimidine C rings provided a positive effect on cellular
potency, pyridine C ring analogues showed the opposite trend;
des-hinge amino compound 5 was slightly more potent than
compound 10. Our previous observation in other series was
that the hinge amino group negatively affected permeability
and/or efflux, but the cyclohexyl diol series did not show much
difference on the Caco-2 values irrespective of the presence of
hinge amino group. In addition, the introduction of a hinge
amino group to the C ring did not affect liver microsomal
clearance profiles, except for clearance in human liver
microsomes for compound 9 (Table 1). We then investigated
heterocyclic D rings such as thiazole, pyrazole, and pyridine in
the presence of hinge amino C rings. Among them, compound
11, a fluoropyridine D ring analogue, displayed moderate
extraction ratio in rat and human liver microsomes in
alignment with a lower Log D value (Log D for compound
11 = 2.3, Table 1). It was the first compound that showed a
modest clearance in rat liver microsomes among cell active
compounds in the series. A subsequent rat PK study of
compound 11 showed good oral bioavailability (% F = 49) and
moderate clearance (CL = 18 mL/min/kg). However, the
cellular proliferation EC50 of compound 11 was not satisfactory
(3.24 μM, Table 1), probably due to the reduced permeability
and increased efflux [Caco-2 assay: Papp (A−B) = 6.61 × 10−6
cm/s, ER = 6.9] relative to compound 5 [Caco-2 assay: Papp
(A−B) = 27.6 × 10−6 cm/s, ER = 1.1]. Since compound 11
has a hinge amino group that might deteriorate selectivity
against other kinases, we assessed its kinase selectivity using an
internal panel of 36 kinases (see Supporting Information).
Gratifyingly, compound 11 was highly selective against 35
kinases (IC50s > 10 μM), while maintaining >100-fold
selectivity against GSK3β (IC50 = 0.54 μM).29
Having demonstrated that oral bioavailablity in the cyclo-
hexyl diol series could be achieved, we turned our attention
back to the diol functionality on the A-ring to further enhance
the cellular potency. We hypothesized that incorporation of
additional lipophilicity to the quaternary methyl group would
increase membrane permeability by shielding the polar
group30,31 or via an intramolecular hydrogen bond.32 Docking
studies demonstrated that a methyl or fluorine would
accommodate into the ligand pocket surface of the glycine
rich residues.33
Therefore, we synthesized ethyl and fluoromethyl analogues
of 11, compounds 12 and 13. Compared to compound 11
[EC50 = 3.24 μM, Papp(A−B) = 6.61, ER = 6.9, Table 1], both
compounds improved the permeability and reduced the efflux
C
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Figure 4. X-ray co-crystal structure of compound 13 in PIM1 (PDB
code: 1ozrb).34
We continued to evaluate combinations of other C and/or D
ring analogues of compound 13. Among them, compound 14
with the aminopyrimidine C ring displayed a low Log D value
(1.4, Table 1) and maintained good permeability and low
efflux potential in Caco-2 assay that translated to improved cell
proliferation activity (Table 1). Gratifyingly, compound 14
showed an EC50 of 0.25 μM in KMS-11.luc assay that was
comparable to 0.18 μM for the clinical candidate, compound 2.
In comparison with its des-fluoro analogue, compound 9, the
fluorine effect35 on compound 14 was more pronounced with
respect to permeability and Log D. The permeability and efflux
profiles were improved even with a lower Log D value by 1.4
unit (Log D for compound 14: 1.4), which apparently resulted
in the 3.7 fold increase in cellular potency, while still
maintaining moderate intrinsic clearances. However, this fluoro
addition reduced solubility (HT-Eq Sol. at pH 7.4: 6 μM for
compound 14; 19 μM for compound 9) but improved the
CYP inhibition profile of compound 14 (IC50s of CYP450
3A4, 2C9, 2D6, and 1A2 isozymes: >30 μM for compound 14;
26, 9.8, 9.9, and >30 μM, respectively, for compound 9). Also,
compound 14 demonstrated a superior permeability and efflux
profile to the basic amine analogues, compounds 1 and 2
(Table 1). Due to the advancement of compound 2 (PIM447)
to clinical stage, we made no further effort to pursue the
cyclohexyl diol series. Overall, we demonstrated the improve-
ment of permeability and microsomal stability by reducing Log
D values through the structural modification of diol
functionality and the introduction of polar C and/or D ring,
resulting in the achievement of a cellularly potent pan-PIM
inhibitor in the cyclohexyl diol series. In general, these
cyclohexyl diol analogues did not show strong inhibitory
activities against CYP450 enzymes and hERG potassium
channel.36
Figure 3. Docking model of compound 4 and the X-ray structure of
compound 5 in PIM1. (a) Docking model of 4-methyl analogue of
compound 4 in PIM1. (b) X-ray co-crystal structure of compound 5
in PIM1. The hydroxyl groups at the 3- and 4-positions on the
cyclohexyl A-ring form H-bond interactions with the backbone
residues Glu171 and Asp128, respectively. While the methyl group at
the carbinol carbon made a hydrophobic interaction with Phe49, the
methyl group at the 5-position occupies the hydrophobic dimple
under the P-loop. The pyridine nitrogen at the B-ring interacts with
the catalytic Lys67 residue (PDB code: 1oblv).27
ratio in Caco-2 permeability assay as hypothesized, but
surprisingly, it did not significantly translate into the cellular
potency (EC50 = 1.71 and 1.74 μM for 12 and 13, Table 1).
While the increased Log D value of compound 12 (Log D =
3.2) had a positive effect on the permeability in Caco-2 assay, it
resulted in high microsomal clearances in rat and human liver
microsomes. Compound 13 maintained the moderate intrinsic
clearances that were explicable because it has a lower Log D
value (1.8 vs 2.3) than compound 11.
We obtained the X-ray co-crystal structure of compound 13
in PIM1 protein (Figure 4). The binding pose was almost
identical to that of compound 5 with a newly formed hydrogen
bond between Glu121 residue with the hinge amino group of
compound 13. As shown in compound 5, the 4-hydroxyl group
makes a hydrogen bond interaction with Asp128. The fluoro
group in the fluoromethyl faces toward Phe49 residue, being in
the gouche and anti-conformations with 3- and 4-hydroxyl
groups, respectively. In the fluoropyridine D-ring, the pyridine
nitrogen is located in the opposite side of the C ring pyridine
nitrogen.
CHEMISTRY
■
Our strategy for the synthesis of hydroxy- or di-hydroxy-
substituted cyclohexyl analogues was to utilize ( )-5-methyl-3-
(3-nitropyridin-4-yl)cyclohex-2-en-1-one 15, a key intermedi-
ate for cyclohexyl amine A−B ring of compound 2 (Scheme
1),23 to develop efficient and scalable syntheses to various
tetra-substituted cyclohexyl diol intermediates. The final
D
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a
Scheme 1. Synthesis of Compounds 3 and 4
a
Reagents and conditions: (a) chiral separation; (b) TBDMSCl, imidazole, >99%; (c) H2, Pd on carbon, EtOAc, MeOH, 2 h, 85%; (d) EDC,
HOAt, DMF; (e) HCl, THF, MeOH, 37% (over 2 steps); (f) tBuOK, THF, 90%; (g) AcOH (cat.), MeCN, water, 33%; (h) TBDMSCl, imidazole,
50%; (i) H2, Pd on carbon, EtOAc, EtOH, 50%; (j) chiral separation, 43%; (k) 18, EDC, HOAt, DMF, >99%; (l) HCl, THF, MeOH, 57% (over 2
steps).
a
Scheme 2. Synthesis of Compound 4
a
Reagents and conditions: (a) LiHMDS, TMSCl, THF, 0 °C, 96%; (b) Oxone, NaHCO3, acetone, water, EtOAc, 2 h, 61% (24/24-cis = 1.3:1);
(c) acetic anhydride, pyridine, >99%; (d) NaBH4, CeCl3·7H2O, THF, MeOH, 97%; (e) TBDMSCl, imidazole, DCM, 84%; (f) H2, Pd on carbon,
EtOAc, EtOH, 88%; (g) chiral separation, 45%; (h) EDC, HOAt, DMF; (i) HCl, THF, MeOH; (j) LiOH, THF, water, 17% (over 3 steps).
compounds were synthesized through amide coupling reaction
between A−B rings and the corresponding C−D ring acids.
When C−D ring acid intermediates were not accessible, C ring
acids were used for amide coupling reaction with A−B rings
followed by Suzuki coupling reaction to introduce the D ring.
A racemic mixture of final compounds or intermediates was
resolved by chiral HPLC. On the basis of X-ray co-crystal
structural information and/or biochemical results, we assigned
absolute stereochemistry for the final compounds.
The synthesis of compound 3 started from intermediate 16.
The desired enantiomer of 16, benzyl (4-((1R,3S,5S)-3-
hydroxy-5-methylcyclohexyl)pyridin-3-yl)carbamate, was pro-
tected with a TBDMS group followed by Cbz group
deprotection. The resulting amino intermediate 17 underwent
amide coupling with 18 and subsequent deprotection of the
TBDMS group under acidic conditions to yield compound 3.37
To synthesize the A-B ring diol intermediate 22, the
bromohydrin intermediate 1923 was subjected to basic
conditions to produce an epoxide intermediate 20. An acid-
catalyzed epoxide ring opening reaction afforded a mixture of
trans/cis cyclohexene diols in ∼3:2 ratio. After protecting the
alcohols with TBDMS, a mixture of trans and cis isomers was
separated by a series of purification methods: silica column
chromatography and preparative reverse phase HPLC. The
desired trans diastereomer 21 underwent palladium-catalyzed
hydrogenation followed by chiral resolution to yield
enantiomerically pure intermediate 22. After amide coupling
between 22 and 18 followed by desilylation compound 4 was
obtained. However, the current method to intermediate 22
E
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a
Scheme 3. Synthesis of Compound 5
a
Reagents and conditions: (a) Ac2O, pyridine, 83%; (b) NaBH4, CeCl3·7H2O, THF, MeOH, 82%; (c) TBDMSCl, imidazole, DCM, 75%; (d) H2,
Pd on carbon, EtOAc, EtOH, >99%; (e) Boc2O, DMAP, DCM, 65%; (f) LiOH, THF, 60 °C, 99%; (g) TBDMSCl, imidazole, DCM, 55%; (h)
Dess-Martin reagent, DCM, 90%; (i) TMSCH2MgCl, THF, 35 °C, 92%; (j) HCl (cat.), acetone, 97%; (k) OsO4 (cat.), NMO, 77%; (l) MsCl,
pyridine, DCM; (m) LiAlH4, THF, 98% (over 2 steps); (n) TBDMSCl, imidazole, DCM, 70%; (o) TFA, DCM, >99%; (p) EDC, HOAt, DMF;
(q) HCl, THF, MeOH, 45% (over 2 steps); (r) chiral separation, 39%.
gave a very poor yield (3% yield over 8 steps from 15) that was
not amenable for analogue and large scale. To improve the
overall yield, we considered an orthogonal protection on the
diol functionality strategy that would facilitate selective
functionalization of A-ring diol. To execute this, we examined
several different α-hydroxylation reactions of intermediate 15.
Among them, Rubottom oxidation of 23 successfully provided
the desired α-hydroxy enone 24. Whereas the reaction with
mCPBA gave poor yield, in situ-generated DMDO38 afforded
good yield of a mixture of trans and cis hydroxy cyclo-
hexenones (1.3:1 ratio) The facial selectivity was slightly
favored toward the desired trans isomer 24, which was readily
separable by silica gel chromatography.
The trans isomer 24 was protected with an acetyl group, and
the resulting acetate intermediate underwent NaBH4 reduction
in the presence of CeCl3 which selectively yielded trans
acetoxy cyclohexenol 25. Subsequent TBDMS protection and
palladium-catalyzed hydrogenation gave a racemic mixture of
aniline intermediates that underwent chiral separation to give
the desired aniline (26). Compound 26 was then coupled with
intermediate 18 to yield compound 4 after the deprotection of
TBDMS and acetyl groups. When compared to the previous
route to 22, this new synthetic route largely improved the yield
of intermediate 26 with one less step (11% yield over 7 steps
from 15), enabling the scale-up of compound 4 for in vivo
pharmacokinetic studies.
Dess−Martin oxidation reaction to afford a mixture of 3- and
4-cyclohexanone intermediates. The desired 4-cyclohexanone
28 was separated by silica column chromatography (20% yield
over 4 steps from 27, Scheme 3). To establish a tertiary alcohol
at the 4-position, we tested the addition of MeMgBr to
cyclohexanone 28 to examine axial/equatorial selectivity,
which led to the predominant formation of undesired cis-
cyclohexane diol resulting from the equatorial attack of the
methyl group. On the basis of this outcome, we thought that
an exo-methylene group at the 4-position would provide a
handle to install hydroxyl group. The exo-methylene
intermediate 29 was successfully synthesized through Peterson
olefination reaction of intermediate 28. While the epoxidation
of 29 with mCPBA or DMDO mostly formed the N-oxide of
29 as a byproduct, osmium-catalyzed dihydroxylation provided
the triol intermediate 30 in a highly stereoseletive manner.40
The mesylation of the triol 30 selectively occurred at the
primary hydroxy group, followed by LiAlH4 reduction to yield
the diol intermediate 31. Sequential selective TBDMS
protection and Boc de-protection provided intermediate 32,
which underwent amide coupling with 18. After TBDMS
deprotection, the coupling product was resolved by chiral
column chromatography to yield compound 5. The exo-
methylene strategy successfully led to the desired trans-diol
intermediate 32 having a quaternary carbon center, but the
overall yield to 32 was only 4% over 18 steps from 15.
For the synthesis of compound 5 with a quaternary
stereogenic center at the 4-position on the cyclohexane ring,
we used intermediate 27 (Scheme 3) to modify the 4-position
hydroxyl group selectively. Intermediate 27 was synthesized
from the cis isomer of the Rubottom oxidation of 23 (24-cis),
(51% yield over 4 steps, Scheme 2). The amino group of 27
was then protected with a Boc group to yield a mixture of
mono- and bis-Boc protected products. Under basic con-
ditions, the acetyl group underwent hydrolysis to give a 4-
hydroxyl intermediate, which concomitantly removed one Boc
group from the bis-Boc protected intermediate. The resulting
4-hydroxyl intermediate underwent 1,4 O → O silyl
migration39 leading to a mixture of 3- and 4-hydroxyl
intermediates. The mixture was subsequently subjected to
To further improve the current synthetic route, we sought
the possibility of early establishment of the exo-methylene
moiety on intermediate 15. We explored several different
carbon-carbon bond formation reactions of 15; however,
reactions requiring a strong base, such as LiHMDS turned
rapidly to a dark purple solution, which was supposedly due to
the electron-withdrawing nitropyridine ring, leading to
decomposition of the starting material. We found a suitable
example for the introduction of an exo-methylene group to the
α-position of cyclohexenone using Eschenmoser’s salt.41 In
order to implement this strategy in our route, we prepared the
silyl enol ether 23 from the common cyclohexenone
intermediate 15, which reacted with Eschenmoser’s salt to
give dimethylaminomethylation intermediate 33. Subsequent
F
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a
Scheme 4. Synthesis of Exo-methylene Intermediate 36 and Compound 5
a
Reagents and conditions: (a) Eschenmoser’s salt, DCM, 18 h, 99%; (b) Iodomethane, THF, 18 h; (c) NaHCO3, 5 h, 63%; (d) NaBH4, CeCl3·
7H2O, MeOH, 72%; (e) TBDMSCl, imidazole, DCM, 86%; (f) mCPBA, NaHCO3, DCM, 14 h, 90%; (g) NBS, THF, water, 3 h, 56%; (h)
TBDMSCl, imidazole, DMF, 75%; (i) K2CO3, MeOH, water, 1 h, 97%; (j) H2 (200 psi), Pd(OH)2 on carbon, MeOH, 4 d; 88%; (k) H2, Pd on
carbon, pyridine, MeOH, 24 h; 70%; (l) 18, EDC, HOAt, DMF, >99%; (m) HCl, THF, MeOH, 86% (over 2 steps); (n) chiral resolution, 31%;
(o) TBAF, THF.
methylation gave a quaternary ammonium salt 34, which
underwent elimination under basic conditions to yield α-exo-
methylene cyclohexenone 35. For further functionalization of
35, we performed selective reduction of the carbonyl group
under Luche conditions, which successfully yielded cis-
cyclohexenol intermediate 36 as the major isomer.
Our initial idea for creating a quarternary carbinol
stereogenic center from the exo-methylene group of 36 was
to employ an epoxidation/ring-opening strategy. To do this,
after TBDMS group protection of 36, the epoxidation of 37
was performed with mCPBA to afford epoxide intermediate 37
in good yield without forming a pyridin N-oxide byproduct
that was observed in the epoxidation of NHBoc-pyridine
compound 29 (Scheme 3). However, it turned out that the
exo-methylene intermediate 37 underwent axial epoxidation,
providing the epoxide 38 with unwanted regioselectivity that
was confirmed by a 1H NMR NOE study of 38. This result was
well supported by literature precedents42 for similar exo-
methylene cyclohexanes, which also described the equatorial
epoxide formation through a halohydrin/ring closing reaction
from exo-methylene cyclohexanes. Then, we carried out the
bromohydrin formation by reacting intermediate 36 with N-
bromosuccinimide in the presence of water, which resulted in
the selective formation of a bromohydrin product 39 at the
exo-methylene group with a decent facial selectivity toward the
desired trans diol (∼2.5:1). The stereochemistry of 39 was
confirmed with the structure of the epoxide 41 that was
synthesized from a ring closing reaction of TBDMS-protected
bromohydrin 40 under basic conditions. The 1H NMR spectra
of 41 was clearly different from that of the epoxide 38 from
mCPBA reaction.43 Having established a trans relationship
between the vicinal diol group of 39, we next tried to find a
global reduction condition for de-halogenation and hydro-
genation of nitro and alkene groups. Under high pressure
palladium-catalyzed hydrogenation conditions, we were able to
obtain intermediate 32 in a decent yield with a good facial
selectivity (∼4:1). The amide coupling of 32 with 18 followed
by deprotection of TBDMS group on under acidic conditions
G
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a
Scheme 5. Synthesis of Compound 12
a
Reagents and conditions: (a) OsO4, NMO, acetone, water, 1 h, 95%; (b) Dess−Martin periodinane, DCM, 72 h, 77%; (c) Bestmann−Ohira’s
reagent, MeOH, K2CO3, 1.5 h, 96%; (d) H2, Pd on carbon, MeOH, 12 h, 33%; (e) chiral separation, 33%; (f) EDC, HOAt, DMF, 16 h; (g)
4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), Pd2(dba)3, PCy3, KOAc, dioxane, K2CO3, μW, 120 °C, 10 min; (h) 2-bromo-3-
fluoropyridine, PdCl2(dppf), DME, Na2CO3, μW, 120 °C, 10 min; (i) HCl, THF, MeOH, 1 h, 30% (over 3 steps).
a
Scheme 6. Synthesis of Compounds 13 and 14
a
Reagents and conditions: (a) Et3N·3HF, 100 °C, 8 h, 46%; (b) H2, Pd on carbon, MeOH, 12 h, 49%; (c) 3-amino-6-bromopicolinic acid 47,
EDC, HOAt, DMF, 16 h, 85%; (d) 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), Pd2(dba)3, PCy3, KOAc, dioxane, K2CO3, μW, 120
°C, 10 min; (e) 2-bromo-3-fluoropyridine, PdCl2(dppf), DME, Na2CO3, μW, 120 °C, 10 min, 10% (over 3 steps); (f) chiral resolution, 24%; (g)
mucobromic acid, NaOEt, EtOH, 40 °C, 2 h; (h) NH4OH, CuSO4, 110 °C, 1 h, 25% (over 2 steps); (i) 51, EDC, HOAt, DMF, 12 h, 25%; (j)
chiral resolution, 34%.
gave compound 5 after chiral resolution. When compared to
the previous synthesis in Scheme 3 (4% yield over 17 steps
from 15), this new synthetic method to intermediate 32 made
significant improvements in the overall yield as well as the
number of reaction steps (15% yield over 7 steps from 15).
However, the hydrogenation reaction of 40 was very slow even
under high pressure of hydrogen gas (200 psi). We reasoned
that hydrogen bromide generated in situ may slow down the
reactions. We continued to examine milder conditions using
intermediate 39 and discovered that in the presence of
pyridine, palladium-catalyzed hydrogenation went to comple-
tion even at room temperature and under atmospheric
hydrogen pressure. Pearlman’s catalyst was also replaced by
Pd on carbon. The diol intermediate 42 was obtained in good
yield with similar levels of selectivity (trans/cis = ∼4:1). We
also found that chiral SFC could resolve the diol 42 in the
absence of the TBDMS group, saving one additional step. The
desired enantiomer was used for the synthesis of compound 5.
This newly developed synthetic route facilitated the scale-up of
compound 5 for in vivo animal studies. Compounds 6 to 11
were synthesized using the desired diols of 32 or 42 and the
corresponding carboxylic acids.
For the synthesis of compound 12, intermediate 37
underwent Upjohn dihydroxylation to give a triol intermediate
43 with high levels of selectivity. Subsequent oxidation
reaction yielded the aldehyde 44, which was then reacted
with Bestmann−Ohira’s reagent for one carbon homologa-
tion.44,45 The resulting alkyne 45 was subjected to a palladium-
catalyzed hydrogenation followed by chiral resolution to afford
the desired aniline intermediate 46. The amide coupling of 46
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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 × 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.
with 3-amino-6-bromopicolinic acid 47 followed by boronic
ester formation yielded intermediate 48, which underwent
Suzuki coupling reaction with 2-bromo-3-fluoropyridine to
afford compound 12.46
To synthesize fluoromethyl aniline 49, the ring-opening
reaction of epoxide intermediate 41 was performed in the
presence of triethylamine trihydrofluoride (Scheme 6).47−50
Subsequent Pd-catalyzed hydrogenation yielded intermediate
49. Compound 14 was synthesized through amide coupling of
49 with pyrimidine acid intermediate 51 followed by chiral
resolution. Compound 13 was prepared from 49 and 3-amino-
6-bromopicolinic acid 47 (Scheme 6) using the same synthetic
procedures used for the synthesis of compound 12 (Scheme
5). For the synthesis of intermediate 51, intermediate 50
underwent a cyclization reaction with mucobromic acid to give
5-bromo-2-(2,6-difluorophenyl)pyrimidine-4-carboxylic acid,
which turned to intermediate 51 through copper-catalyzed
amination.
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 × 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) analysis was performed on
some of the compounds with a Varian 300 or 400 MHz NMR and a
Bruker 500 MHz NMR. The spectral reference was either TMS or the
known chemical shift of the solvent.
CONCLUSIONS
■
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, C18 reversed phase column, 30 × 50 mm,
flow 75 mL/min. Typical solvents employed for the Teledyne ISCO
chromatography system and flash column chromatography were
dichloromethane, methanol, ethyl acetate, hexane, 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% TFA. The purity
of all compounds screened in the biological assays was examined by
LC−MS analysis and was found to be ≥ 95%.
Our goal to identify a pan-PIM inhibitor bearing a cyclohexyl
diol A-ring moiety was successfully achieved, leading to a
cellularly potent compound 14 with moderate metabolic
clearance profile. Structure-based design guided us to find
compound 5 with the axial methyl group at the 4-position of
the cyclohexyl A-ring that improved cellular potency but
suffered from poor oral bioavailability and high in vivo
clearance in rats. We focused on the modulation of
physicochemical properities, especially, the reduction of Log
D value by introducing more polar C and/or D ring to improve
metabolic stability. In parallel, the introduction of a fluorine
atom near the diol functionality to improve permeability
resulted in a cellularly potent cyclohexyl diol compound 14
with moderate metabolic stability. For our structure−activity
relationship (SAR) exploration, we established efficient and
scalable synthetic routes to complex cyclohexyl diol ring
systems that contain contiguous stereogenic centers including
a tertiary alcohol. In particular, we identified ( )-cis-5-methyl-
6-methylene-3-(3-nitropyridin-4-yl)cyclohex-2-en-1-ol 36 as a
versatile intermediate for the synthesis of all the cyclohexyl
analogues, which could be employed to synthesize a diverse set
of cyclohexane ring systems.
Synthesis of Compound 3. 4-((1R,3S,5S)-3-(tert-Butyldimethyl-
silyloxy)-5-methylcyclohexyl)pyridin-3-amine (17). ( )-benzyl 4-
((1R,3S,5S)-3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate 16
was resolved by chiral HPLC (4.5 mg/1 mL ethanol, heptane/
isopropanol = 85:15, 1 mL/min, IA column). Benzyl 4-((1R,3S,5S)-3-
hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate was eluted at
8.526 min. To a solution of benzyl 4-((1R,3S,5S)-3-hydroxy-5-
methylcyclohexyl)pyridin-3-ylcarbamate (423 mg, 1.243 mmol) in
DMF (1.5 mL) was added TBDMSCl (243 mg, 1.615 mmol) and
imidazole (186 mg, 2.73 mmol). The reaction mixture was stirred at
room temperature overnight. After diluted with water, the reaction
mixture was extracted with EtOAc (15 mL) three times. The
combined organic layers were washed with water and brine, dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo to yield crude
benzyl 4-((1R,3S,5S)-3-(tert-butyldimethylsilyloxy)-5-
methylcyclohexyl)pyridin-3-ylcarbamate (571 mg, >99% yield).
LCMS (m/z): 455.4 [M + H]+. To a solution of crude benzyl 4-
((1R,3S,5S)-3-(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)-
pyridin-3-ylcarbamate (571 mg, 1.256 mmol) in EtOAc (3 mL) was
added Pd on carbon (134 mg, 0.126 mmol) and MeOH (6 mL). The
mixture was degassed with nitrogen stream for 10 min, purged with
hydrogen gas, and equipped with hydrogen balloon. The reaction
mixture was stirred for 3 h. The mixture was filtered through 1μm
PTFE Acrodisc filter, eluting with EtOAc (30 mL). The filtrate was
concentrated in vacuo to yield crude 4-((1R,3S,5S)-3-(tert-butyldi-
methylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine 17 (403.4 mg,
98% yield). 6-(2,6-difluorophenyl)-5-fluoro-N-(4-((1R,3S,5S)-3-hy-
droxy-5-methylcyclohexyl)pyridin-3-yl)picolinamide (3). To a sol-
ution of 4-((1R,3S,5S)-3-(tert-butyldimethylsilyloxy)-5-
methylcyclohexyl)pyridin-3-amine 17 (50 mg, 0.156 mmol) in
DMF (1.5 mL) was added 6-(2,6-difluorophenyl)-5-fluoropicolinic
acid 18 (47.4 mg, 0.187 mmol), HOAt (25.5 mg, 0.187 mmol), and
EDC (35.9 mg, 0.187 mmol). The reaction mixture was stirred at
room temperature overnight. LCMS showed the coupling reaction
was completed. LCMS (m/z): 556.4 [M + H]+. After partitioned
between EtOAc (10 mL) and water (10 mL), the crude product was
EXPERIMENTAL SECTION
■
General. The compounds and/or intermediates were character-
ized by high performance liquid chromatography (HPLC) using a
Waters Millenium chromatography system with a 2695 Separation
Module (Milford, MA). The analytical columns were Alltima C18
reverse phase, 4.6 × 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% 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, for example, Baker-Flex Silica Gel
1B2-F flexible sheets. TLC results were readily detected visually under
ultraviolet light or by employing well known iodine vapor and other
various staining techniques.
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 × 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;
I
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extracted with EtOAc. The combined organic layers were washed with
water and brine, dried over anhydrous Na2SO4, filtered off, and
concentrated in vacuo to yield crude N-(4-((1R,3S,5S)-3-(tert-
butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-di-
fluorophenyl)-5-fluoropicolinamide. The crude product (87 mg, 0.157
mmol) was dissolved in THF (1 mL) and methanol (0.5 mL)
followed by addition of 6 N HCl aqueous solution (0.048 mL, 1.57
mmol). The reaction mixture was stirred at room temperature for 2 h.
After neutralized with saturated Na2CO3 aqueous solution, the
mixture was extracted with EtOAc. The combined organic layers were
washed with water and brine, dried over anhydrous Na2SO4, filtered
off, and concentrated in vacuo. The crude product was purified by
reverse phase HPLC. After pure fractions were combined and free-
based with saturated Na2CO3 aqueous solution, the crude product
was extracted with EtOAc. The combined organic layers were dried
over anhydrous Na2SO4, filtered off, and concentrated in vacuo. After
dissolving in water and acetonitrile (1:1, 2 mL) and adding 1
equivalent of 1 N HCl soltuion, the solution was lyophilized to yield
6-(2,6-difluorophenyl)-5-fluoro-N-(4-((1R,3S,5S)-3-hydroxy-5-
methylcyclohexyl)pyridin-3-yl)picolinamide 3 as its HCl salt (60.9
mg, 0.137 mmol, 87% yield over 2 steps). LCMS (m/z): 442.2 [M +
H]+. HRMS (m/z): calcd for C24H23N3O2F3 [M + H]+, 442.1742;
oxy)-5-methylcyclohexyl)pyridin-3-amine (22). To a solution of
trans-( )-4-((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methyl-
cyclohex-1-enyl)-3-nitropyridine 21 (550 mg, 1.149 mmol) in ethanol
(5.7 mL) and EtOAc (5.7 mL) was added Pd on carbon (122 mg).
After degassed for 15 min, the reaction mixture was charged with
hydrogen at room temperature and stirred at room temperature
overnight. The reaction mixture was filtered through Celite pad and
washed with EtOAc. The filtrate was concentrated in vacuo. The
crude product was purified by ISCO (heptane/EtOAc) to yield trans-
(
)-4-((1R,3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-
methylcyclohexyl)pyridin-3-amine (260 mg, 58% yield). LCMS (m/
z): 451.2 [M + H]+. 260 mg of the pure racemate was resolved by
chiral HPLC (IC column, 1 mL/min, 5% IPA in heptane). Peak 1: 4-
((1S,3S,4S,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-
methylcyclohexyl)pyridin-3-amine 22-ent (114 mg, 99% ee, Rt = 6.1
min). LCMS (m/z): 451.2 [M + H]+. Peak 2: 4-((1R,3R,4R,5S)-3,4-
bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine
22 (112 mg, >99% ee, Rt = 7.83 min). LCMS (m/z): 451.2 [M + H]+.
6-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-5-
methylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide (4). To a reac-
tion vial with a stir bar was added 4-((1R,3R,4R,5S)-3,4-bis(tert-
butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-amine 22 (17
mg, 0.038 mmol), 6-(2,6-difluorophenyl)-5-fluoropicolinic acid 18
(10.50 mg, 0.041 mmol), and HOAt (6.15 mg, 0.045 mmol) followed
by DMF (0.4 mL). After the reaction mixture was homogeneously
mixed, EDC (8.67 mg, 0.045 mmol) was added to the reaction
mixture in a single portion. The reaction mixture was stirred at room
temperature overnight. After quenched with water, the reaction
mixture was extracted with EtOAc. The organic layers were washed
with water, 1 M NaOH aqueous solution, and brine, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. Crude
N-(4-((1R,3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-
methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicoli-
namide was obtained in >99% yield (26 mg, 0.038 mmol). LCMS
(m/z): 686.4 [M + H]+. To a solution of crude N-(4-((1R,3R,4R,5S)-
3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohexyl)pyridin-3-yl)-
6-(2,6-difluorophenyl)-5-fluoropicolinamide (26 mg, 0.038 mmol) in
MeOH (126 μL) and THF (253 μL) was added HCl (120 μL, 0.360
mmol) at room temperature. The reaction mixture was stirred at
room temperature for 1 h. After quenched with saturated NaHCO3
solution, the reaction mixture was extracted with EtOAc. The organic
layers were washed with water and brine, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. After preparative
HPLC purification and lyophilzation, 6-(2,6-difluorophenyl)-N-(4-
((1R,3R,4R,5S)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-5-flu-
oropicolinamide 4 was obtained as its TFA salt (12.4 mg, 0.021
mmol, 57% over 2 steps). LCMS (m/z): 458.3 [M + H]+. HRMS (m/
z): calcd for C24H23N3O3F3 [M + H]+, 458.1692; found, 458.1691.
1H NMR (400 MHz, DMSO-d6): δ ppm 10.5 (s, 1H), 8.88 (s, 1H),
8.51 (d, J = 5.83 Hz, 1H), 8.31 (dd, J = 8.61, 3.91 Hz, 1H), 8.16 (t, J
= 9.0 Hz, 1H), 7.65 (m, 2H), 7.29 (t, J = 8.22 Hz, 2H), 3.15 (ddd, J =
10.96, 8.61, 4.3 Hz, 1H), 2.94 (d, J = 12.3 Hz, 1H), 2.75 (t, J = 9.0
Hz, 1H), 1.81 (m, 1H), 1.58 (d, J = 12.3, 2.54 Hz, 1H), 1.46 (m, 1H),
1.23 (m, 2H), 0.84 (d, J = 6.26 Hz, 3H).
1
found, 442.1745. H NMR (400 MHz, DMSO-d6): δ ppm 10.6 (s,
1H), 9.09 (s, 1H), 8.71 (d, J = 5.85 Hz, 1H), 8.38 (dd, J = 8.6, 3.92
Hz, 1H), 8.24 (t, J = 8.81 Hz, 1H), 7.78 (d, J = 6.24 Hz, 1H), 7.65−
7.76 (m, 2H), 7.36 (t, J = 8.21 Hz, 2H), 3.41 (m, 1H), 2.95 (m, 1H),
1.75 (m, 2H), 1.61 (m, 1H), 1.4 (m, 1H), 1.31 (m, 1H), 0.99 (m,
1H), 0.89 (m, 1H), 0.86 (d, J = 6.63 Hz, 3H).
Synthesis of Compound 4 (Scheme 1). ( )-4-((1S,5S)-5-
Methyl-7-oxabicyclo[4.1.0]hept-2-en-3-yl)-3-nitropyridine (20). To a
solution of ( )-(1S,5S,6S)-6-bromo-5-methyl-3-(3-nitropyridin-4-yl)-
cyclohex-2-enol 19 (1.05 g, 3.35 mmol) in THF (33.5 mL) was added
potassium tert-butoxide (0.564 g, 5.03 mmol) at room temperature.
The reaction mixture was stirred for 10 min. The reaction mixture was
quenched with NH4Cl solution and extracted with EtOAc by washing
with water and brine. The organic layers were dried over anhydrous
sodium sulfate, filtered off, and dried in vacuo. The crude product 20
(700 mg, 3.01 mmol, 90%) was used in the next step without further
purification. Rf = 0.5 (50% EtOAc/hexanes). LCMS (m/z): 251.2 [M
+ H]+ (detected as a diol). To a solution of crude ( )-4-((1S,5S)-5-
methyl-7-oxabicyclo[4.1.0]hept-2-en-3-yl)-3-nitropyridine 20 (700
mg, 3.01 mmol) in CH3CN (20.1 mL) and H2O (10.1 mL) was
added a catalytic amount of acetic acid (0.052 mL, 0.904 mmol) at
room temperature. The reaction mixture was stirred for 16 h at room
temperature. After quenched with NaHCO3 solution, the reaction
mixture was concentrated to remove the majority of CH3CN and the
residue was partitioned between EtOAc and water. The combined
organic layers were washed with water and brine, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. A
mixture of trans- and cis-( )-6-methyl-4-(3-nitropyridin-4-yl)-
cyclohex-3-ene-1,2-diols was obtained as a white solid in 33.1%
yield (250 mg, 0.99 mmol) by flash column chromatography. Rf = 0.3
(100% EtOAc; diols were not separable on TLC). LCMS (m/z):
251.1 [M + H]+. To a solution of a mixture of trans- and cis-( )-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-diols (250 mg, 0.99
mmol) in DMF (3.33 mL, 0.3 M) was added TBDMSCl (1.05 g, 7.0
mmol), imidazole (612 mg, 9 mmol) at room temperature. The
reaction mixture was stirred at room temperature overnight. After
quenched with saturated NaHCO3, the reaction mixture was extracted
with EtOAc. The combined organic layers were washed with water
and brine, dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The trans and cis isomers were separated by
ISCO (gradient eluting with EtOAc and hexanes) and subsequent
preparative reverse phase HPLC (55−95% acetonitrile in water, then
run 5−95% acetonitrile in water) to yield 130 mg of cis-( )-4-
((3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-
enyl)-3-nitropyridine (27.2% yield) and 240 mg of trans-( )-4-
((3R,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylcyclohex-1-
enyl)-3-nitropyridine 21 (50.2% yield). LCMS (m/z): 479.0 [M +
H]+, respectively. 4-((1R,3R,4R,5S)-3,4-Bis((tert-butyldimethylsilyl)-
Synthesis of Compound 4 (Scheme 2). Trans-( )-(5S,6R)-6-
hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone (23). A
solution of ( )-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-enone 15
(3 g, 12.9 mmol) and TMSCl (1.65 mL, 12.9 mmol) in THF was
added LiHMDS (1.0 M solution in THF, 13.56 mL, 13.56 mmoL) at
0 °C slowly over 1 h. The reaction mixture was warmed up to room
temperature and stirred for 2 h. The reaction mixture was quenched
with NaHCO3 aqueous solution and removed THF in vacuo. The
residue was extracted with EtOAc 3 times. The organic layers were
washed with water and brine, dried over anhydrous K2CO3, filtered,
and concentrated in vacuo to yield crude ( )-4-(5-methyl-3-
(trimethylsilyloxy) cyclohexa-1,3-dienyl)-3-nitropyridine 23 (3.6 g,
11.83 mmol, 96% yield). 1H NMR (400 MHz, CDCl3): δ ppm 9.14−
9.00 (m, 1H), 8.80−8.64 (m, 1H), 7.42−7.25 (m, 1H), 6.00−5.88
(m, 1H), 4.98 (br s, 1H), 2.86−2.53 (m, 1H), 2.51−2.29 (m, 1H),
2.27−2.03 (m, 1H), 1.21−1.03 (m, 3H), 0.36−0.15 (m, 9H). A
J
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solution of ( )-4-(5-methyl-3-(trimethylsilyloxy) cyclohexa-1,3-dien-
yl)-3-nitropyridine (3.6 g, 10.39 mmol), sodium bicarbonate (4.36 g,
51.9 mmol), acetone (7.63 mL, 104 mmol), water (51.9 mL), and
ethyl acetate (51.9 mL) was vigorously stirred at 0 °C. To this, a
solution of Oxone (6.39 g, 10.39 mmol) in water (45 mL was slowly
added via dropping funnel for 1 h 30 min. After addition, the reaction
mixture was allowed to stir at room temperature for 2 h. The pH of
the mixture was adjusted to around 4 by adding 1 N HCl aqueous
solution. After stirring for 30 min, the reaction mixture was extracted
with EtOAc. The combined organic layers were washed with water
and brine, dried over anhydrous Na2SO4 and filtered, and
concentrated in vacuo. A mixture of crude trans and cis products
was purified by ISCO (50% EtOAc in heptane). For trans-
( )-(5S,6R)-6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-2-
vacuo. The crude product was purified by ISCO (gradient EtOAc in
heptane) to yield ( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-
((tert-butyldimethylsilyl)oxy)-6-methylcyclohexyl acetate 26-rac
(335 mg, 0.885 mmol, 88% yield). LCMS (m/z): 379.4 [M + H]+.
1H NMR (400 MHz, CDCl3): δ ppm 8.06 (s, 1H), 8.04 (d, J = 4.0
Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 4.69 (t, J = 8.0 Hz, 1H), 3.7 (m,
1H), 3.61 (s, 1H), 2.65 (m, 1H), 2.11 (s, 3H), 2.05 (m, 3H), 1.82 (m,
1H), 0.95 (d, J = 8.0 Hz, 3H), 0.86 (s, 9H), 0.06 (s, 6H). 335 mg of
26-rac was resolved by chiral HPLC (AS-H column, 1 mL/min,
heptane/EtOH = 98:2). Peak 1: (1S,2S,4S,6R)-4-(3-aminopyridin-4-
yl)-2-(tert-butyldimethylsilyloxy)-6-methylcyclohexyl acetate 26-ent
(115.7 mg, >99% ee, Rt = 6.72 min). LCMS (m/z): 379.0 [M + H]+.
Peak 2: (1R,2R,6S)-2-(tert-butyldimethylsilyloxy)-6-methyl-4-(3-ni-
tropyridin-4-yl)cyclohex-3-enyl acetate 26 (154.7 mg, 99% ee, Rt =
11.31 min). LCMS (m/z): 379.0 [M + H]+. 6-(2,6-Difluorophenyl)-
N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-
yl)-5-fluoropicolinamide (4). To a reaction vial with a stir bar was
added (1R,2R,6S)-2-(tert-butyldimethylsilyloxy)-6-methyl-4-(3-nitro-
pyridin-4-yl)cyclohex-3-enyl acetate 26 (40 mg, 0.106 mmol), 6-(2,6-
difluorophenyl)-5-fluoropicolinic acid 18 (29.4 mg, 0.116 mmol), and
HOAt (18.7 mg, 0.137 mmol) followed by DMF (0.2 mL). After the
reaction mixture was homogeneously mixed, EDC (30.4 mg, 0.158
mmol) was added to the reaction mixture in a single portion. The
reaction mixture was stirred at room temperature for 18 h. After
quenched with water, the reaction mixture was extracted with EtOAc.
The organic layers were washed with water and brine, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo to
afford crude (1R,2R,4R,6S)-2-(tert-butyldimethylsilyloxy)-4-(3-(6-
(2,6-difluorophenyl)-5-fluoropicolinamido)pyridin-4-yl)-6-methylcy-
clohexyl acetate. LCMS (m/z): 614.3 [M + H]+. The crude product
was dissolved in THP (1 mL) and MeOH (0.5 mL) followed by 1 N
HCl aqueous solution (0.5 mL). After stirring for 3 h, the mixture was
neutralized with saturated NaHCO3 solution and extracted with
EtOAc. The combined organic layers were dried over sodium sulfate,
filtered, and concentrated in vacuo. The crude product was purified by
reverse phase HPLC. The pure fractions were combined and
lyophilized to yield (1R,2R,4R,6S)-4-(3-(6-(2,6-difluorophenyl)-5-
fluoropicolinamido)pyridin-4-yl)-2-hydroxy-6-methylcyclohexyl ace-
tate (19.4 mg, 0.039 mmol). LCMS (m/z): 500.2 [M + H]+. The
acetate product was dissolved in MeOH (0.5 mL), and THF (0.5 mL)
was added 1 N LiOH solution (0.5 mL) at room temperature. The
reaction mixture was stirred at room temperature overnight. After
neutralized with 1 N HCl aqueous solution, the reaction mixture was
extracted with EtOAc. The organic layers were washed with water and
brine, dried over anhydrous sodium sulfate, filtered, and concentrated
in vacuo. After preparative HPLC purification and lyophilzation, 6-
(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-5-
methylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 4 was obtained
as its TFA salt (10 mg, 0.018 mmol, 17% over 3 steps). LCMS (m/z):
458.1 [M + H]+.
1
enone 24 (890 mg, 34.5% yield), LCMS (m/z): 249 [M + H]+. H
NMR (400 MHz, CDCl3): δ ppm 9.34 (s, 1H), 8.9 (d, J = 5.2 Hz,
1H), 7.28 (s, 1H), 6.09 (d, J = 2.4 Hz, 1H), 3.99 (d, J = 12.4 Hz, 1H),
3.68 (d, J = 2.0 Hz, 1H), 2.61−2.52 (m, 2H), 2.39 (m, 1H), 1.27 (d, J
= 8.0 Hz, 3H). For cis-( )-(5S,6R)-6-hydroxy-5-methyl-3-(3-nitro-
pyridin-4-yl)cyclohex-2-enone 24-cis (680 mg, 26.4% yield), LCMS
(m/z): [M + H]+. 1H NMR (400 MHz, CDCl3): δ ppm 9.32 (s, 1H),
8.9 (d, J = 4.0 Hz, 1H), 7.28 (s, 1H), 6.06 (d, J = 2.8 Hz, 1H), 4.53
(d, J = 12.0 Hz, 1H), 3.60 (m, 1H), 3.11 (m, 1H), 2.8 (m, 1H), 2.47
(d, J = 2.0 Hz, 1H), 1.09 (d, J = 8 Hz, 3H). (1R,2R,6S)-2-Hydroxy-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enyl acetate (25). To a
solution of ( )-(5S,6R)-6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)-
cyclohex-2-enone 24 (4.2 g, 16.9 mmol) in pyridine (56.4 mL) was
slowly added acetic anhydride (3.19 mL, 33.8 mmol). The reaction
mixture was stirred at room temperature overnight. After all volatile
materials were removed in vacuo, the residue was diluted with EtOAc.
The organic layers were washed with 1 N aqueous HCl solution and
water, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo. The crude product was purified by ISCO
(gradient EtOAc in heptane) to yield ( )-(1R,6S)-6-methyl-4-(3-
nitropyridin-4-yl)-2-oxocyclohex-3-enyl acetate (4.9 g, 16.9 mmol,
1
>99% yield). LCMS (m/z): 291.1 [M + H]+. H NMR (400 MHz,
CDCl3): δ ppm 9.35 (s, 1H), 8.9 (d, J = 4.0 Hz, 1H), 7.27 (s, 1H),
6.04 (s, 1H), 5.56 (d, J = 12.0 Hz, 1H), 2.65−2.55 (m, 3H), 2.25 (s,
3H), 1.16 (d, J = 4.0 Hz, 3H). To a suspension of ( )-(1R,6S)-6-
methyl-4-(3-nitropyridin-4-yl)-2-oxocyclohex-3-enyl acetate (580 mg,
2 mmol) in THF (4 mL) and MeOH (6 mL) was added cerium(III)
chloride heptahydrate (744 mg, 2 mmol). The reaction mixture was
stirred for 2 h at room temperature. At 0 °C, NaBH4 (76 mg, 2 mmol)
was slowly added to the reaction mixture, which was then stirred at
room temperature overnight. The reaction mixture was extracted with
EtOAc (20 mL) twice. The combined organic layers were washed
with water and NaHCO3 solution and brine. The crude product was
purified by ISCO (0−40% EtOAc in heptane) to yield
( )-(1R,2R,6S)-2-hydroxy-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-
3-enyl acetate 25 (567 mg, 1.94 mol, 97% yield). LCMS (m/z): 293.0
1
[M + H]+. H NMR (400 MHz, CDCl3): δ ppm 9.16 (s, 1H), 8.76
Synthesis of Compound 5 (Scheme 3). cis-( )-(1S,2R,4R,6S)-
4-(3-Aminopyridin-4-yl)-2-(tert-butyldimethylsilyloxy)-6-methylcy-
clohexyl acetate (27). For step a, cis-( )-(1S,6S)-6-methyl-4-(3-
nitropyridin-4-yl)-2-oxocyclohex-3-enyl acetate was synthesized from
cis-( )-(5S,6R)-6-hydroxy-5-methyl-3-(3-nitropyridin-4-yl)cyclohex-
2-enone (24-cis) according to step c in Scheme 2 (83% yield), LCMS
(m/z): 291.1 [M + H]+. For step b, cis-( )-(1S,2R,6S)-2-hydroxy-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enyl acetate was synthesized
according to step d in Scheme 2 (82% yield), LCMS (m/z): 293.0 [M
+ H]+. For step c, cis-( )-(1S,2R,6S)-2-(tert-butyldimethylsilyloxy)-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enyl acetate was synthesized
according to step e in Scheme 2 (75% yield), LCMS (m/z): 407.2 [M
+ H]+. For step d, cis-( )-(1S,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-
(tert-butyldimethylsilyloxy)-6-methylcyclohexyl acetate 27 was syn-
thesized according to step f in Scheme 2 (>99% yield), LCMS (m/z):
379.4 [M + H]+. ( )-tert-Butyl (4-((1R,3R,5S)-3-((tert-
butyldimethylsilyl)oxy)-5-methyl-4-oxocyclohexyl)pyridin-3-yl)-
carbamate (28). To a solution of ( )-(1S,2R,4R,6S)-4-(3-amino-
pyridin-4-yl)-2-(tert-butyldimethylsilyloxy)-6-methylcyclohexyl ace-
tate (2.8 g, 7.40 mmol) in DCM (37.0 mL) was added DMAP
(d, J = 4.8 Hz, 1H), 7.27 (s, 1H), 6.04 (s, 1H), 5.3 (m, 1H on alkene),
4.75 (dd, J = 10.8, 7.2 Hz, 1H on acetoxy attached carbon), 4.38 (m,
1H on hydroxyl attached carbon), 2.35 (s, 1H), 2.21 (m, 2H), 2.19 (s,
3H), 1.16 (d, J = 6.0 Hz, 3H). To a solution of (1.34 g, 4.58 mmol) in
DCM (9.17 mL) was added imidazole (0.468 g, 6.88 mmol) and
TBDMSCl (0.829 g, 5.50 mmol). The reaction mixture was stirred at
room temperature overnight. After diluted with water, the mixture was
extracted with EtOAc (50 mL). The combined organic layers were
washed with water and NaHCO3 solution and brine. The crude
product was purified by ISCO (0−40% EtOAc in heptane) to yield
( )-(1R,2R,6S)-2-(tert-butyldimethylsilyloxy)-6-methyl-4-(3-nitro-
pyridin-4-yl)cyclohex-3-enyl acetate (1.56 g, 3.84 mmol, 84% yield).
LCMS (m/z): 407.1 [M + H]+. To a solution of ( )-(1R,2R,6S)-2-
(tert-butyldimethylsilyloxy)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-
3-enyl acetate (411 mg, 1.011 mmol) in ethanol (1.7 mL) and EtOAc
(1.7 mL) was added Pd on carbon (108 mg). The reaction mixture
was equipped with hydrogen gas balloon. The reaction mixture was
stirred at room temperature overnight, filtered through Celite pad,
and washed with EtOAc, and then, the filtrate was concentrated in
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(0.181 g, 1.48 mmol) and Boc2O (4.29 mL, 18.49 mmol). The
reaction mixture was stirred at room temperature over-the-weekend.
After diluted with water, the mixture was extracted with DCM (10
mL). The combined organic layers were washed with water and
NaHCO3 solution and brine. The crude product was purified by
ISCO (0−50% EtOAc in heptane) to yield ( )-(1S,2R,4R,6S)-4-(3-
(bis(tert-butoxycarbonyl)amino)pyridin-4-yl)-2-(tert-butyldimethylsi-
lyloxy)-6-methylcyclohexyl acetate (2.8 g, 4.84 mmol, 65% yield).
LCMS (m/z): 579.3 [M + H]+. To a solution of ( )-(1S,2R,4R,6S)-
4-(3-(bis(tert-butoxycarbonyl)amino)pyridin-4-yl)-2-(tert-butyldime-
thylsilyloxy)-6-methylcyclohexyl acetate (2.8 g, 4.84 mmol) in THF
(16.12 mL) was added LiOH (9.67 mL, 9.67 mmol). The reaction
mixture was heated at 60 °C overnight. LCMS still showed the
starting material remained. Next day, after adding 4 mL of LiOH
more, the reaction was heated at 60 °C again overnight. LCMS
showed the reaction went to completion, but two product peaks
indicated that a half of TBDMS was migrated to de-acetylated alcohol
(∼1:1). After neutralized with 1 N HCl aqueous solution, the mixture
was extracted with EtOAc. The combined organic layers were dried
over sodium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by ISCO (gradient EtOAc in heptane) to afford
a mixture of ( )-tert-butyl 4-((1R,3R,4S,5S)-3-(tert-butyldimethylsi-
lyloxy)-4-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate and
( )-tert-butyl 4-((1R,3R,4S,5S)-4-(tert-butyldimethylsilyloxy)-3-hy-
droxy-5-methylcyclohexyl)pyridin-3-ylcarbamate (2.1 g, 4.81 mmol,
99%). LCMS (m/z): 437.2 [M + H]+, 0.98 min and 1.0 min. To a
solution of a mixture of ( )-tert-butyl 4-((1R,3R,4S,5S)-3-(tert-
butyldimethylsilyloxy)-4-hydroxy-5-methylcyclohexyl)pyridin-3-ylcar-
bamate and ( )-tert-butyl 4-((1R,3R,4S,5S)-4-(tert-butyldimethylsily-
loxy)-3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate (2.1 g,
4.81 mmol) in DCM (16 mL) was added Dess−Martin periodinane
(2.55 g, 6.01 mmol) at room temperature. The reaction mixture was
stirred at room temperature overnight. LCMS showed the reaction
went to completion. After quenched with NaHCO3 solution/Na2S2O3
solution (8:1), the mixture was vigorously stirred for 2 h and extracted
with EtOAc. The combined organic layers were dried over sodium
sulfate, filtered, and concentrated in vacuo. The desired cyclo-
hexanone product was separated by ISCO (0−80% EtOAc in DCM)
to yield ( )-tert-butyl 4-((1R,3R,5S)-3-(tert-butyldimethylsilyloxy)-5-
methyl-4-oxocyclohexyl)pyridin-3-ylcarbamate (577 mg, 1.328 mmol,
55% yield). LCMS (m/z): 435.2 [M + H]+, 0.96 min. The undesired
cyclohexanone, ( )-tert-butyl 4-((1R,3R,4S,5S)-4-(tert-butyldimethyl-
silyloxy)-3-hydroxy-5-methylcyclohexyl)pyridin-3-ylcarbamate, was
obtained in 34% yield (405 mg, 0.932 mmol). LCMS (m/z): 435.2
[M + H]+, 1.01 min. ( )-tert-Butyl 4-((1R,3R,5S)-3-hydroxy-5-
methyl-4-methylenecyclohexyl)pyridin-3-ylcarbamate (29). To a
solution of ( )-tert-butyl 4-((1R,3R,5S)-3-(tert-butyldimethylsily-
loxy)-5-methyl-4-oxocyclohexyl)pyridin-3-ylcarbamate (1.09 g, 2.508
mmol) in THF (12.5 mL) was added trimethylsilylmethylmagnesium
chloride (25.1 mL, 25.1 mmol) at room temperature. The reaction
mixture was heated to 35 °C overnight. After quenched with saturated
NH4Cl aqueous solution, the mixture was extracted with EtOAc. The
combined organic layers were dried over sodium sulfate, filtered, and
concentrated in vacuo. The crude product was purified by ISCO
(gradient EtOAc in heptane) to yield ( )-tert-butyl 4-((1R,3R,4S,5S)-
3-(tert-butyldimethylsilyloxy)-4-hydroxy-5-methyl-4-((trimethylsilyl)-
methyl)cyclohexyl)pyridin-3-ylcarbamate (1.2 g, 2.3 mmol, 92%).
LCMS (m/z): 523.2 [M + H]+. 1H NMR (400 MHz, CDCl3): δ ppm
8.6 (s, 1H), 8.38 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.0 Hz, 1H), 6.11 (s,
1H), 4.69 (t, J = 8.0 Hz, 1H), 2.41 (m, 1H), 2.31 (s, 1H), 1.79 (m,
2H), 1.61 (m, 1H), 1.52 (s, 9H), 1.37 (m, 1H), 1.28 (m, 1H), 1.06
(d, J = 8.0 Hz, 3H), 0.9 (s, 9H), 0.88 (m, 2H), 0.13 (s, 4H), 0.09 (s,
9H). To a solution of ( )-tert-butyl 4-((1R,3R,4S,5S)-3-(tert-
butyldimethylsilyloxy)-4-hydroxy-5-methyl-4-((trimethylsilyl)-
methyl)cyclohexyl)pyridin-3-ylcarbamate (1.2 g, 2.295 mmol) in
acetone (7.65 mL) was added 0.191 mL of 1 N HCl aqueous solution.
The mixture was stirred at room temperature for 20 min. After
neutralized with saturated NaHCO3 aqueous solution, the mixture
was extracted with EtOAc. The combined organic layers were dried
over sodium sulfate, filtered, and concentrated in vacuo to yield crude
( )-tert-butyl 4-((1R,3R,5S)-3-hydroxy-5-methyl-4-
methylenecyclohexyl)pyridin-3-ylcarbamate (710 mg, 2.23 mmol,
97%). LCMS (m/z): 319.0 [M + H]+. 1H NMR (400 MHz,
CDCl3): δ ppm 8.69 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 4.0
Hz, 1H), 6.2 (s, 1H), 5.15 (s, 1H), 4.88 (s, 1H), 4.23 (m, 1H), 3.08
(m, 1H), 2.27 (m, 2H), 1.91 (m, 2H), 1.79 (m, 2H), 1.65 (m, 1H),
1.52 (s, 9H), 1.18 (d, J = 8.0 Hz, 3H). ( )-tert-Butyl 4-
((1R, 3R,4S,5S)-3, 4-dihydroxy-4-(hydroxymethyl)-5-
methylcyclohexyl)pyridin-3-ylcarbamate (30). To a solution of
(
)-tert-butyl 4-((1R,3R,5S)-3-hydroxy-5-methyl-4-
methylenecyclohexyl)pyridin-3-ylcarbamate (490 mg, 1.539 mmol)
in acetone (24.6 mL) and water (6.15 mL) was added 4-
methylmorpholine 4-oxide (NMO) (541 mg, 4.62 mmol) followed
by osmium tetroxide (4% in water) (0.097 mL, 0.308 mmol) at room
temperature. The reaction was stirred overnight but did not attain
completion. After adding the same amounts of the reagents, the
reaction was stirred overnight once more. The reaction was quenched
with saturated Na2SO3 aqueous solution (30 mL) and stirred for
additional 1 h. After evaporation of acetone in vacuo, the mixture was
extracted with EtOAc. The combined organic layers were dried over
sodium sulfate, filtered, and concentrated in vacuo. The crude product
was purified by ISCO (gradient EtOAc in heptane) to yield ( )-4-
((1R, 3R,4S,5S)-3, 4-dihydroxy-4-(hydroxymethyl)-5-
methylcyclohexyl)pyridin-3-ylcarbamate 30 (416 mg, 1.18 mmol, 77%
yield). LCMS (m/z): 353.1 [M + H]+. 1H NMR (400 MHz, CDCl3)
δ ppm 8.62 (s, 1H), 8.34 (d, J = 4.8 Hz, 1H), 7.15 (d, J = 5.2 Hz, 1H),
6.86 (s, 1H), 4.20 (d, J = 11.6 Hz, 1H), 3.82−3. 72 (m, 2H), 2.97 (m,
1H), 2.08 (m, 1H), 2.04 (m, 1H), 1.75 (m, 2H), 1.68 (m, 1H), 1.65
(m, 1H), 1.51 (s, 9H), 1.0 (d, J = 7.2 Hz, 3H). ( )-tert-Butyl 4-
((1R,3R,4R,5S)-3,4-dihydroxy-4,5-dimethylcyclohexyl)pyridin-3-yl-
carbamate (31). To a solution of ( )-tert-butyl 4-((1R,3R,4S,5S)-3,4-
dihydroxy-4-(hydroxymethyl)-5-methylcyclohexyl)pyridin-3-ylcarba-
mate (84 mg, 0.238 mmol) in DCM (794 μL) was added pyridine (96
μL, 1.192 mmol). After cooling down to 0 °C, methanesulfornyl
chloride (18.57 μL, 0.238 mmol) was added to the reaction mixture,
which was stirred overnight. However, LCMS showed that a small
amount of the product formed. One equivalent of methanesulfornyl
chloride was added to the mixture, which was stirred for 2 h. LCMS
showed no starting material remained. After quenched with water, the
mixture was extracted with EtOAc. The combined organic layers were
dried over sodium sulfate, filtered, and concentrated in vacuo to yield
( )-((1S,2R,4R,6S)-4-(3-(tert-butoxycarbonylamino)pyridin-4-yl)-
1,2-dihydroxy-6-methylcyclohexyl)methyl methanesulfonate. The
crude product was used in the next step without further purification.
LCMS (m/z): 337.1 [M + H]+. To a solution of ( )-((1S,2R,4R,6S)-
4-(3-(tert-butoxycarbonylamino)pyridin-4-yl)-1,2-dihydroxy-6-
methylcyclohexyl)methyl methanesulfonate (85 mg, 0.197 mmol) in
THF (1.974 mL) was slowly added LiAlH4 (14.99 mg, 0.395 mmol)
at room temperature. The reaction mixture was stirred at room
temperature overnight. After quenched with water slowly, the reaction
was extracted with EtOAc. The combined organic layers were dried
over sodium sulfate, filtered, and concentrated in vacuo to yield
(
)-tert-butyl 4-((1R,3R,4R, 5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-ylcarbamate 31 (65 mg, 0.193 mmol,
98%). The crude product was used in the next step without further
purification. ( )-(1S,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butyl-
dimethylsilyloxy)-1,6-dimethylcyclohexanol (32). To solution of
(
)-tert-butyl 4-((1R,3R,4R, 5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-ylcarbamate (77 mg, 0.229 mmol) in
DCM (0.458 mL) was added imidazole (23.37 mg, 0.34 mmol) and
TBDMSCl (37.9 mg, 0.252 mmol). The reaction mixture was stirred
at room temperature overnight. After quenched with water, the
mixture was extracted with EtOAc. The combined organic layers were
dried over sodium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by ISCO (gradient EtOAc in heptane) to
yield ( )-tert-butyl 4-((1R,3R,4R,5S)-3-(tert-butyldimethylsilyloxy)-
4-hydroxy-4,5-dimethylcyclohexyl)pyridin-3-ylcarbamate (72 mg,
1
70% yield). LCMS (m/z): 451.4 [M + H]+. H NMR (400 MHz,
CDCl3): δ ppm 8.67 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 7.15 (d, J = 5.2
Hz, 1H), 6.12 (s, 1H), 3.61 (dd, J = 11.2 Hz, 4 Hz, 1H), 2.88 (m,
L
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1H), 2.1 (s, 1H), 1.89 (m, 1H), 1.76 (m, 1H), 1.68 (m, 1H), 1.52 (s,
9H), 1.23 (m, 2H), 1.14 (s, 3H), 1.01 (d, J = 7.2 Hz, 3H), 0.91 (s,
9H), 0.11 (s, 3H), 0.79 (s, 3H). To a solution of ( )-tert-butyl 4-
((1R,3R,4R,5S)-3-(tert-butyldimethylsilyloxy)-4-hydroxy-4,5-
dimethylcyclohexyl)pyridin-3-ylcarbamate (72 mg, 0.16 mmol) in
DCM (1.2 mL) was added TFA (0.4 mL). The reaction mixture was
stirred for 1 h 30 min. After diluted with toluene, the volatile materials
were removed in vacuo, neutralized with saturated NaHCO3 aqueous
solution, and extracted with EtOAc. The combined organic layers
were dried over sodium sulfate, filtered, and concentrated in vacuo to
yield crude ( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butyl-
dimethylsilyloxy)-1,6-dimethylcyclohexanol 32 (56 mg, 0.16 mmol,
>99%). The crude product was used in the next step without further
purification. LCMS (m/z): 351.3 [M + H]+. 6-(2,6-Difluorophenyl)-
N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-4,5-dimethylcyclohexyl)pyridin-
3-yl)-5-fluoropicolinamide (5). To solution of ( )-(1R,2R,4R,6S)-4-
(3-aminopyridin-4-yl)-2-(tert-butyldimethylsilyloxy)-1,6-dimethylcy-
clohexanol 32 (32 mg, 0.091 mmol) in DMF (114 μL) was added 6-
(2,6-difluorophenyl)-5-fluoropicolinic acid 18 (25.4 mg, 0.1 mmol),
HOAt (16.2 mg, 0.119 mmol) and EDC (26.2 mg, 0.137 mmol). The
reaction mixture was stirred at room temperature for 18 h. After
quenched with water, the mixture was extracted with EtOAc. The
combined organic layers were dried over sodium sulfate, filtered, and
concentrated in vacuo. LCMS (m/z): 586.3 [M + H]+. The crude
product was dissolved in THF (1 mL) and MeOH (0.5 mL) followed
by 3 N HCl aqueous solution (0.5 mL). The reaction mixture was
stirred for 3 h. After neutralized with saturated NaHCO3 aqueous
solution, the mixture was extracted with EtOAc. The combined
organic layers were dried over sodium sulfate, filtered, and
concentrated in vacuo. The crude product was purified by ISCO
(0−10% MeOH in EtOAc) to yield 24 mg of ( )-6-(2,6-
difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 5-rac (45%
18 h to yield the trimethyl ammonium salt intermediate 34. After
saturated NaHCO3 aqueous solution (35.9 g in water) was added, the
reaction mixture was stirred at room temperature for 5 h, diluted with
EtOAc, and stirred at room temperature for another 6 h. After the
organic layers were separated, aqueous phase was extracted with
EtOAc 3 times, the combined organic layers were washed with water
and brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The crude product was purified by ISCO (gradient EtOAc in
heptane) to afford ( )-5-methyl-6-methylene-3-(3-nitropyridin-4-
yl)cyclohex-2-enone 35 (14.7 g, 60.2 mmol, 63%). LCMS (m/z):
1
245 [M + H]+. H NMR (400 M Hz, CDCl3): δ ppm 9.33 (s, 1H),
8.88 (d, J = 5.1 Hz, 1H), 6.19 (m, 1H), 6.16 (s, 1H), 5.42 (s, 1H),
3.15 (m, 1H), 2.59 (dd, J = 17.4, 5.3 Hz, 1H), 2.43 (ddd, J = 7.3, 9.5,
2.2 Hz, 1H), 1.31 (d, J = 6.7 Hz, 3H). ( )-cis-5-Methyl-6-methylene-
3-(3-nitropyridin-4-yl)cyclohex-2-enol (36). To a solution of ( )-5-
methyl-6-methylene-3-(3-nitropyridin-4-yl)cyclohex-2-enone 35
(13.7 g, 56.1 mmol) in methanol (260 mL) was added cerium (III)
chloride heptahydrate (23 g, 61.7 mmol). The reaction mixture was
stirred at room temperature for 1 h. After cooled down to at 0 °C,
NaBH4 (2.1 g, 56.1 mmol) was added slowly and stirred for 30 min.
After quenched with water, the volatile materials were removed in
vacuo, and saturated NaHCO3 aqueous solution was added into
mixture with vigorous stirring. The reaction mixture was extracted
with EtOAc, and the organic layers were washed with brine and dried
over anhydrous sodium sulfate, filtered off, and concentrated in vacuo.
The crude product was purified by ISCO (heptane/EtOAc, 80:20 to
20:80) to give ( )-cis-5-methyl-6-methylene-3-(3-nitropyridin-4-yl)-
cyclohex-2-enol 36 as a yellow solid (10 g, 40.76 mmol, 72% yield).
1
LCMS (m/z): 247 [M + H]+. H NMR (400 MHz, CDCl3): δ ppm
9.13 (s, 1H), 8.75 (d, J = 4.7 Hz, 1H), 7.26 (s, 1H), 5.72 (m, 1H),
5.25 (s, 1H), 5.03 (m, 1H), 4.86 (br s, 1H), 2.67 (m, 1H), 2.39 (dd, J
= 16.6, 4.9 Hz, 1H), 2.11 (br s, 1H), 1.91 (m, 1H), 1.23 (d, J = 6.7
Hz, 3H). 1D NOE: selective irradiation of δ 4.86 ppm (H at 3-
position) revealed the strongest NOE to δ 2.67 ppm (H at 5-position)
(Scheme 4).
1
yield over 2 steps). LCMS (m/z): 472.2 (MH+). H NMR (CDCl3,
400 MHz): δ 9.96 (br s, 1H), 9.34 (s, 1H), 8.44 (d, J = 4.0 Hz, 1H),
8.42 (m, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.52 (m, 1H), 7.19 (d, J = 4
Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 3.57 (dd, J = 12 Hz, 4 Hz, 1H),
2.99 (m, 1H), 2.02 (m, 1H), 1.78−1.52 (m, 4H), 1.36−1.26 (m, 1H),
1.12 (s, 3H), 0.9 (d, J = 4 Hz, 3H). 24 mg of 5-rac was resolved by
chiral HPLC (AD-H column, heptane/EtOH = 75:25, 1 mL/min).
Peak 1: 6-(2,6-difluorophenyl)-N-(4-((1S,3S,4S,5R)-3,4-dihydroxy-
4,5-dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 5-ent (9.7
mg, >99% ee, Rt = 4.45 min). LCMS (m/z): 472.2 [M + H]+. Peak 2
6-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 5 (9.3 mg,
>99% ee, Rt = 6.056 min). LCMS (m/z): 472.2 [M + H]+. HRMS
(m/z): calcd for C25H25N3O3F3 [M + H]+, 472.1848; found,
472.1850.
( )-4-((3R,5S)-3-(tert-Butyldimethylsilyloxy)-5-methyl-4-methyle-
necyclohex-1-enyl)-3-nitropyridine (37). To solution of ( )-(1R,5S)-
5-methyl-6-methylene-3-(3-nitropyridin-4-yl) cyclohex-2-enol 36 (5.0
g, 20.3 mol) in DCM (77 mL) was added imidazole (2.07 g, 30.5
mmol) and TBDMSCl (3.37 g, 22.33 mol). The reaction mixture was
stirred for 18 h at room temperature. The volatile materials were
removed in vacuo, and the residue was partitioned between EtOAc
and water. The combined organic layers were washed with water and
brine and dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The crude material was purified by flash
column chromatography (gradient EtOAc in heptane) to yield ( )-4-
((3R,5S)-3-(tert-butyldimethylsilyloxy)-5-methyl-4-methylenecyclo-
hex-1-enyl)-3-nitropyridine 37 (6.3 g, 17.47 mmol, 86% yield). LCMS
1
Synthesis of Compound 5 (Scheme 4). ( )-5-Methyl-6-
methylene-3-(3-nitropyridin-4-yl)cyclohex-2-enone (35). To a sol-
ution of Eschenmoser’s salt (19.33 g, 105 mmol) in DCM (140 mL)
was added ( )-4-(5-methyl-3-(trimethylsilyloxy)cyclohexa-1, 3-dien-
yl)-3-nitropyridine 23 in DCM (100 mL) at 0 °C slowly over 60 min.
The reaction mixture was allowed to warm up to room temperature
and stirred for 18 h. After the reaction mixture was transferred to a
larger vessel and diluted with DCM (100 mL), 1 M HCl aqueous
solution (160 mL) was added to the reaction mixture, which was
stirred for 20 min at 0 °C. After 2 N NaOH aqueous solution (80
mL) was slowly added at 0 °C, the reaction mixture was stirred for 1 h
and pH of the mixture was adjusted to 12 by adding 3 N NaOH
aqueous solution. After organic layers were separated, aqueous phase
was extracted with DCM 3 times. The combined organic layers were
dried over anhydrous Na2SO4, filtered off, and concentrated in vacuo
to yield crude ( )-6-((dimethylamino)methyl)-5-methyl-3-(3-nitro-
pyridin-4-yl)cyclohex-2-enone 33 (27.5 g, 95 mmol, 99% yield).
LCMS (m/z): 290.0 [M + H]+. To a solution of ( )-6-
((dimethylamino) methyl)-5-methyl-3-(3-nitropyridin-4-yl) cyclo-
hex-2-enone 33 (27.5 g, 95 mmol) in THF (190 mL) was added
iodomethane (7.13 mL, 114 mmol) slowly at 0 °C. The reaction
mixture was allowed to warm up to room temperature and stirred for
(m/z): 361.0 [M + H]+. H NMR (400 MHz, CDCl3): δ ppm 9.12
(s, 1H), 8.73 (d, J = 5.1 Hz, 1H), 7.27 (d, J = 5.1 Hz, 1H), 5.57 (t, J =
2.5 Hz, 1H), 5.24−5.20 (m, 1H), 4.98−4.94 (m, 1H), 4.84−4.92 (m,
1H), 2.57−2.72 (m, 1H), 2.37 (dd, J = 16.6, 5.3 Hz, 1H), 2.11−2.01
(m, 1H), 1.20 (d, J = 6.7 Hz, 3H), 0.94 (m, 9H), 0.15 (s, 3H), 0.12
(s, 3H). ( )-4-((3R,4R,8S)-4-(tert-Butyldimethylsilyloxy)-8-methyl-
1-oxaspiro[2.5]oct-5-en-6-yl)-3-nitropyridine (38). To a solution of
( )-4-((3R,5S)-3-(tert-butyldimethylsilyloxy)-5-methyl-4-methylene-
cyclohex-1-enyl)-3-nitropyridine 37 (90 mg, 0.250 mmol) in DCM
(832 μL) was added sodium bicarbonate (31.5 mg, 0.374 mmol).
After cooling down to 0 °C, mCPBA (61.5 mg, 0.275 mmol) was
added to the reaction mixture. The reaction was allowed to warm up
to room temperature for 14 h. After quenched with NaHCO3
solution, the reaction mixture was extracted with EtOAc. The organic
layers were washed with saturated NaHCO3 aquesous solution and
brine, dried over anhydrous sodium sulfate, filtered off, and dried in
vacuo. The crude product was purified by ISCO (gradient EtOAc in
heptane) to yield ( )-4-((3R,4R,8S)-4-(tert-butyldimethylsilyloxy)-8-
methyl-1-oxaspiro[2.5]oct-5-en-6-yl)-3-nitropyridine 38 (85 mg,
1
0.225 mmol, 90% yield). LCMS (m/z): 377.1, [M + H]+. H NMR
(400 MHz, CDCl3): δ ppm 9.15 (s, 1H), 8.77 (d, J = 4.0 Hz, 1H),
7.36 (d, J = 4.0 Hz, 1H), 5.65 (m, 1H), 4.53 (m, 1H), 2.95 (d, J = 8.0
M
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Hz, 1H), 2.84 (d, J = 4.0 Hz, 1H), 2.39−2.25 (m, 3H), 0.96 (d, J =
8.0 Hz, 3H), 0.9 (s, 9H), 0.1 (s, 3H), 0.08 (s, 3H). 1D NOE: selective
irradiation of δ 4.83 ppm (H at 3-position) revealed the strong NOEs
to δ 2.95 ppm (H at epoxide) and δ 2.34 ppm (H at position 5)
(Scheme 4).
yield). LCMS (m/z): 263.1 [M + H]+. 1H NMR (400 MHz, CDCl3):
δ ppm 9.17 (s, 1H), 8.78 (d, J = 4.0 Hz, 1H), 7.31 (d, J = 4.0 Hz, 1H),
5.75 (m, 1H), 4.62 (m, 1H), 3.10 (d, J = 4.0 Hz, 1H), 2.83 (d, J = 4.0
Hz, 1H), 2.51 (m, 1H), 2.28 (m, 2H), 0.96 (d, J = 4.0 Hz, 3H).
( 1 R , 2 R , 4 R , 6 S ) - 4 - ( 3 - A m i n o p y r i d i n - 4 - y l ) - 2 - ( (t e r t -
butyldimethylsilyl)oxy)-1,6-dimethylcyclohexan-1-ol (32). To a steel
bomb reactor was added a solution of ( )-(1R,2R,6S)-1-(bromo-
methyl)-2-(tert-butyldimethylsilyloxy)-6-methyl-4-(3-nitropyridin-4-
yl)cyclohex-3-enol 40 (3.7 g, 8.09 mmol) in methanol (27 mL). After
degassed by nitrogen for 10 min followed by addition of 10%
Pd(OH)2 on carbon (2.8 g), the reaction mixture was charged with
hydrogen to 200 psi and stirred at room temperature for 4 days. The
reaction mixture was filtered through Celite pad, and the filtrate was
neutralized with saturated NaHCO3 aqueous solution. After volatile
materials were removed in vacuo, the mixture was extracted with
EtOAc. The organic layers were washed with water and brine, dried
over anhydrous sodium sulfate, filtered off, and concentrated in vacuo.
The crude product was purified by ISCO (gradient EtOAc containing
2% TEA in DCM) to yield ( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-
yl)-2-((tert-butyldimethylsilyl)oxy)-1,6-dimethylcyclohexanol 32 (2.5
( )-(3R,4R,8S)-8-Methyl-6-(3-nitropyridin-4-yl)-1-oxaspiro[2.5]-
oct-5-en-4-ol 36-o was synthesized from ( )-(1R,5S)-5-methyl-6-
methylene-3-(3-nitropyridin-4-yl)cyclohex-2-enol 36 according to
step f in Scheme 4 (96% yield). LCMS (m/z): 263.1 [M + H]+.
1H NMR (400 MHz, CDCl3): δ ppm 9.16 (s, 1H), 8.77 (d, J = 4.0
Hz, 1H), 7.29 (d, J = 4.0 Hz, 1H), 5.72 (m, 1H), 4.50 (m, 1H), 3.11
(d, J = 8.0 Hz, 1H), 2.97 (d, J = 4.0 Hz, 1H), 2.53 (m, 1H), 2.27 (m,
2H), 0.91 (d, J = 8.0 Hz, 3H). TBDMS protecting group of 38 was
1
deprotected to give 36-o, H NMR spectra of which was matched
with that of 36-o from 36 (Scheme 4). (1R,2R,6S)-1-(bromomethyl)-
2-((tert-butyldimethylsilyl)oxy)-6-methyl-4-(3-nitropyridin-4-yl)-
cyclohex-3-en-1-ol (40). To a solution of ( )-(1R,5S)-5-methyl-6-
methylene-3-(3-nitropyridin-4-yl)cyclohex-2-enol 36 (9 g, 36.5
mmol) in THF (91 mL) and water (91 mL) was slowly added N-
bromosuccinimide (7.16 g, 40.2 mmol) at 0 °C. The reaction mixture
was stirred at 0 °C for 3 h. After quenched with saturated sodium
thiosulfite aqueous solution (300 mL), the reaction mixture was
extracted with EtOAc. The organic layers were washed with NaHCO3
solution, water and brine, dried over anhydrous sodium sulfate,
filtered off, and concentrated in vacuo to give a mixture of
diastereomers (∼2.5:1). The crude product was purified by ISCO
(0−5% MeOH/DCM, 330 g column) to yield (1R,2R,6S)-1-
(bromomethyl)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-
diol 39 (7 g, 20.4 mmol, 56% yield). LCMS (m/z): 342.8, 344.9 [M +
H]+. 1H NMR (400 MHz, CDCl3): δ ppm 9.13 (s, 1H), 8.77 (d, J =
4.0 Hz, 1H), 7.29 (d, J = 4.0 Hz, 1H), 5.73 (m, 1H), 4.28 (m, 1H),
4.06 (d, J = 8.0 Hz, 1H), 3.79 (d, J = 12.0 Hz, 1H), 2.75 (m, 2H),
2.42 (m, 1H), 2.27 (m, 1H), 2.12 (m, 1H), 1.19 (d, J = 8.0 Hz, 3H).
To a solution of ( )-(1R,2R,6S)-1-(bromomethyl)-6-methyl-4-(3-
nitropyridin-4-yl)cyclohex-3-ene-1,2-diol 39 (12 g, 35 mmol) in DMF
(70 mL) was added TBDMSCl (7.91 g, 52.5 mmol), imidazole (4.76
g, 69.9 mmol) at room temperature. The reaction mixture was stirred
for 24 h. After quenched with NaHCO3, the reaction mixture was
extracted with EtOAc 3 times. The organic layers were washed with
water and brine, dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The crude product was purified by ISCO
(gradient EtOAc in heptane) to afford ( )-(1R,2R,6S)-1-(bromo-
methyl)-2-(tert-butyldimethylsilyloxy)-6-methyl-4-(3-nitropyridin-4-
yl)cyclohex-3-enol 40 (12 g, 26.2 mmol, 75%). LCMS (m/z): 457,
459.0 [M + H]+. 1H NMR (400 MHz, CDCl3): δ ppm 9.11 (s, 1H),
8.75 (d, J = 5.1 Hz, 1H), 7.31−7.25 (m, 1H), 5.61 (br s, 1H), 4.15−
4.08 (m, J = 3.5 Hz, 1H), 3.95 (d, J = 10.6 Hz, 1H), 3.76 (d, J = 10.2
Hz, 1H), 2.81 (dd, J = 17.6, 5.9 Hz, 1H), 2.35 (s, 1H), 2.32−2.23 (m,
1H), 2.06 (dd, J = 17.6, 3.5 Hz, 1H), 1.20 (d, J = 7.4 Hz, 3H), 0.83−
0.97 (m, 9H), 0.13 (s, 3H), 0.08 (s, 3H). ( )-4-((3S,4R,8S)-4-(tert-
butyldimethylsilyloxy)-8-methyl-1-oxaspiro[2.5]oct-5-en-6-yl)-3-ni-
tropyridine (41). To a solution of ( )-(1R,2R,6S)-1-(bromomethyl)-
2-(tert-butyldimethylsilyloxy)-6-methyl-4-(3-nitropyridin-4-yl)-
cyclohex-3-enol 40 (3.0 g, 6.56 mmol) in MeOH (20 mL) and water
(2 mL) was added potassium carbonate (1.36 g, 9.84 mmol). The
reaction mixture was vigorously stirred for 1 h at room temperature.
After the volatile material was evaporated, the reaction mixture was
extracted with EtOAc. The combined organic layers were washed with
water and brine, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo to yield crude ( )-4-((3S,4R,8S)-4-(tert-
butyldimethylsilyloxy)-8-methyl-1-oxaspiro[2.5]oct-5-en-6-yl)-3-ni-
tropyridine 41 (2.4 g, 6.4 mmol, 97% yield). LCMS (m/z): 377.1 [M
+ H]+. 1H NMR (400 MHz, CDCl3): δ ppm 9.14 (s, 1H), 8.76 (d, J =
4.0 Hz, 1H), 7.31 (d, J = 4.0 Hz, 1H), 5.59 (s, 1H), 4.49 (br s, 1H),
2.99 (d, J = 4.0 Hz, 1H), 2.73 (d, J = 4.0 Hz, 1H), 2.5 (m, 1H), 2.41
(m, 1H), 2.25 (m, 1H), 0.94 (d, J = 8.0 Hz, 1H), 0.89 (s, 9H), 0.08
(m, 6H). ( )-(3S,4R,8S)-8-methyl-6-(3-nitropyridin-4-yl)-1-
oxaspiro[2.5]oct-5-en-4-ol 39-o was synthesized from
( )-(1R,2R,6S)-1-(bromomethyl)-6-methyl-4-(3-nitropyridin-4-yl)-
cyclohex-3-ene-1,2-diol 39 according to step i in Scheme 4 (53%
1
g, 7.13 mmol, 88% yield). LCMS (m/z): 351.1 [M + H]+. H NMR
(400 MHz, CDCl3): δ ppm 8.06 (s, 1H), 8.03 (m, 1H), 6.99 (m, 1H),
3.62 (m, 1H), 2.69 (m, 1H), 1.88 (m, 1H), 1.81−1.65 (m, 4H), 1.13
(s, 3H), 1.01 (d, J = 8.0 Hz, 3H), 0.89 (s, 9H), 0.11 (s, 3H), 0.03 (s,
3H).
( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-((tert-
butyldimethylsilyl)oxy)-1,6-dimethylcyclohexanol 32 (850 mg, 2.42
mmol) was resolved by chiral HPLC (AD column, heptane/IPA =
95:5, 1 mL/min). Peak 1: (1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-
((tert-butyldimethylsilyl)oxy)-1,6-dimethylcyclohexanol 32a (340 mg,
>99% ee, Rt = 2.74 min). LCMS (m/z): 351.1 [M + H]+. Peak 2:
(1S,2S,4S,6R)-4-(3-aminopyridin-4-yl)-2-((tert-butyldimethylsilyl)-
oxy)-1,6-dimethylcyclohexanol 32b (360 mg, 99% ee, Rt = 4.28 min).
LCMS (m/z): 351.1 [M + H]+. 6-(2,6-Difluorophenyl)-N-(4-
((1R,3R,4S,5S)-3,4-dihydroxy-4,5-dimethylcyclohexyl)pyridin-3-yl)-
5-fluoropicolinamide (5). To solution of ( )-(1R,2R,4R,6S)-4-(3-
aminopyridin-4-yl)-2-((tert-butyldimethylsilyl)oxy)-1,6-dimethylcy-
clohexanol 32 (56 mg, 0.160 mmol) in DMF (200 μL) was added 6-
(2,6-difluorophenyl)-5-fluoropicolinic acid (44.5 mg, 0.176 mmol),
HOAt (28.3 mg, 0.208 mmol) and EDC (45.9 mg, 0.240 mmol). The
reaction mixture was stirred at room temperature for 16 h. After the
mixture was quenched with water and extracted with EtOAc, the
combined organic layers were washed with water and brine, dried over
anhydrous sodium sulfate, filtered off, and concentrated in vacuo to
yield crude N-(4-((1R,3R,4S,5S)-3-(tert-butyldimethylsilyloxy)-4-hy-
droxy-4,5-dimethylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-
fluoropicolinamide (93.5 mg, >99% yield). LCMS (m/z): 586.5 [M +
H]+. The crude product was dissolved in THF (2 mL) and MeOH (1
mL) followed by addition of 3 N HCl aqueous solution (0.5 mL).
The reaction mixture was stirred for 3 h. After neutralized with
saturated NaHCO3 aqueous solution, the reaction mixture was
extracted with EtOAc. The combined organic layers were washed with
water and brine, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo. The crude produce was purified by ISCO
(gradient EtOAc in DCM followed by 10% MeOH in EtOAc) to yield
( )-6-(2,6-difluorophenyl)-N-(4-((1R,3R,4S,5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 5-rac (65 mg,
1
0.137 mmol, 86% yield). LCMS (m/z):472.3 [M + H]+. H NMR
(CDCl3, 400 MHz): δ 9.90 (br s, 1H), 9.34 (s, 1H), 8.43 (d, J = 4 Hz,
1H), 8.41 (m, 1H), 7.78 (t, J = 8.4 Hz, 1H), 7.51 (m, 1H), 7.19 (d, J
= 4 Hz, 1H), 7.1 (dd, J = 8.4, 7.6 Hz, 1H), 3.57 (m, 1H), 3.0 (m, 1H),
2.01 (m, 1H), 1.76−1.51 (m, 4H), 1.35−1.26 (m, 1H), 1.12 (s, 3H),
0.9 (d, J = 6.8 Hz, 3H). 65 mg of 5-rac was resolved by chiral HPLC
(AD-H column, heptane/EtOH = 75:25, 1 mL/min). Peak 1: 6-(2,6-
difluorophenyl)-N-(4-((1S,3S,4R,5R)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 5-ent (26.8
mg, 98% ee, Rt = 3.8 min). LCMS (m/z): 472.0 [M + H]+. Peak
2: 6-(2,6-difluorophenyl)-N-(4-((1R,3R,4S,5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)-5-fluoropicolinamide 5 (27 mg,
99% ee, Rt = 5.5 min). LCMS (m/z): 472.0 [M + H]+. All analytical
and biological data of compounds 5 and 5-ent that were synthesized
N
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by procedures as described in Schemes 3 and 4 were matched.
Compounds 5 and 5-ent were synthesized respectively from two
enantiomers 32a and 32b of 32 using the same procedures described
above (Scheme 4). All the data of compound 5 from enantiomer 32a
were matched with those of compound 5 synthesized in Scheme 3.
(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-1,6-dimethylcyclohexane-1,2-
diol (42). To a solution of ( )-(1R,2R,6S)-1-(bromomethyl)-6-
methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-diol 39 (2.3 g, 67
mmol) in ethanol (100 mL) was added Pd on carbon (content 5%,
4.28 g) and pyridine (5.42 mL, 59.4 mmol). The reaction vessel was
degassed with nitrogen stream for 15 min, purged, and flushed with
hydrogen gas three times. After equipped with hydrogen balloon, the
reaction was stirred for 24 h. The reaction mixture was diluted with
methanol and filtered. The residue was washed with 100 mL of
methanol. The volatile materials were removed in vacuo. The crude
product was purified by ISCO (95:5:0.5 DCM/MeOH/NH4OH to
80:19:1 DCM/MeOH/NH4OH over 15 min, 80 g silica cartridge) to
give ( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-1,6-dimethylcyclo-
hexane-1,2-diol 42 (1.1 g, 4.65 mmol, 70% yield). LCMS (m/z):
237.2 [M + H]+. 1H NMR (CD3OD, 400 MHz): δ 7.92 (s, 1H), 7.77
(d, J = 4.8 Hz, 1H), 7.09 (d, J = 5.2 Hz, 1H), 4.8 (s, 4H), 3.64 (m,
1H), 2.91 (m, 1H), 1.95 (m, 1H), 1.8−1.62 (m, 4H), 1.31 (m, 1H),
1.12 (s, 3H), 0.98 (d, J = 6.8 Hz, 3H). 950 mg of ( )-(1R,2R,4R,6S)-
4-(3-aminopyridin-4-yl)-1,6-dimethylcyclohexane-1,2-diol 42 was re-
solved by chiral HPLC (OJ-H column, heptane/IPA = 85:15, 1 mL/
min). Peak 1: (1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-1,6-dimethylcy-
clohexane-1,2-diol 42a (370 mg, >99% ee, Rt = 7.6 min). LCMS (m/
z): 237.3 [M + H]+. Peak 2: (1S,2S,4S,6R)-4-(3-aminopyridin-4-yl)-
1,6-dimethylcyclohexane-1,2-diol 42b (340 mg, >99% ee, Rt = 10.2
min). LCMS (m/z): 237.3 [M + H]+. The amide coupling between
42a and 18 yielded compound 5. All analytical and biological data
were matched with those for compound 5 obtained from intermediate
32.
( )-(1R,2R,6S)-2-(tert-butyldimethylsilyloxy)-1-hydroxy-6-methyl-4-
(3-nitropyridin-4-yl)cyclohex-3-enecarbaldehyde 44 (1.58 g, 4.03
mmol) in MeOH (130 mL) was added Bestmann−Ohira’s reagent
(1.55, 8.05 mmol) in MeOH (70 mL) followed by addition of
potassium carbonate (2.78 g, 20.13 mmol) at room temperature. The
reaction mixture was stirred for 1.5 h. After removing 90% of MeOH
in vacuo, the mixture was partitioned with water and EtOAc, the
organic layers were washed with saturated NH4Cl solution and brine,
dried with anhydrous sodium sulfate, filtered off, and concentrated.
The crude compound was purified via ISCO (0−30% EtOAc in
heptane) to yield ( )-(1S,2R,6S)-2-(tert-butyldimethylsilyloxy)-1-
ethynyl-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enol 45 (1.5 g,
1
3.86 mmol, 96% yield). LCMS (m/z): 389.2 [M + H]+. H NMR
(400 MHz, CDCl3): δ ppm, 9.12 (s, 1 H) 8.74 (d, J = 5.09 Hz, 1 H)
7.29 (d, J = 5.09 Hz, 1 H) 5.44 (s, 1 H) 4.33 (dt, J = 3.33, 1.86 Hz, 1
H) 2.66 (s, 1 H) 2.45 (s, 1 H) 2.38−2.30 (m, 2 H) 2.28−2.19 (m, 1
H) 1.17 (d, J = 6.26 Hz, 3 H) 0.93 (s, 9 H) 0.17−0.09 (m, 6 H).
(1R,2R,4R,6S)-4-(3-Aminopyridin-4-yl)-2-(tert-butyldimethylsily-
loxy)-1-ethyl-6-methylcyclohexanol (46). To a solution of
( )-(1S,2R,6S)-2-(tert-butyldimethylsilyloxy)-1-ethynyl-6-methyl-4-
(3-nitropyridin-4-yl)cyclohex-3-enol 45 (1.1 g, 2.83 mmol) in MeOH
(28 mL) was added 10% Pd on carbon (0.9 g). After degassed by
nitrogen for 10 min and equipped with hydrogen balloon, the reaction
mixture was stirred at room temperature for 12 h. The reaction
mixture was filtered through Celite and washed by MeOH and
EtOAc. After the filtrate was concentrated in vacuo, the crude product
was purified by ISCO (gradient EtOAc in DCM) to yield
( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butyldimethylsily-
loxy)-1-ethyl-6-methylcyclohexanol 46-rac (336 mg, 0.922 mmol,
1
33% yield). LCMS (m/z): 365.2 [M + H]+. H NMR (400 MHz,
CDCl3): δ ppm 8.05 (s, 1H), 8.03 (m, 1H), 7.0 (m, 1H), 3.65 (m,
1H), 2.72 (m, 1H), 2.11−1.62 (m, 5H), 1.35 (m, 2H), 1.05 (d, J = 8.0
Hz, 3H), 0.91 (s, 9H), 0.89 (m, 3H), 0.12 (s, 3H), 0.05 (s, 3H). 336
mg of ( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butyldime-
thylsilyloxy)-1-ethyl-6-methylcyclohexanol 46-rac was resolved by
chiral SFC (Chiralpak, 10 × 250, 15 mL/min, CO2/EtOH + 0.1%
DEA, 85/15. 40 °C). Peak 1: (1S,2S,4S,6R)-4-(3-aminopyridin-4-yl)-
2-(tert-butyldimethylsilyloxy)-1-ethyl-6-methylcyclohexanol 46-ent
(97 mg, 99% ee, Rt = 1.49 min). LCMS (m/z): 365.1 [M + H]+.
Peak 2: (1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-(tert-butyldimethyl-
silyloxy)-1-ethyl-6-methylcyclohexanol 46 (112 mg, 99% ee, Rt = 1.91
min). LCMS (m/z): 365.1 [M + H]+. 3-Amino-N-(4-((1R,3R,4R,5S)-
3-(tert-butyldimethylsilyloxy)-4-ethyl-4-hydroxy-5-methylcyclohexyl)-
pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
picolinamide (48). To solution of (1R,2R,4R,6S)-4-(3-aminopyridin-
4-yl)-2-(tert-butyldimethylsilyloxy)-1-ethyl-6-methylcyclohexanol 46
(37 mg, 0.101 mmol) in DMF (127 μL) was added 3-amino-6-
bromopicolinic acid 47 (102 mg, 0.471 mmol), HOAt (17.96 mg,
0.132 mmol), and EDC (29.2 mg, 0.152 mmol). The reaction mixture
was stirred at room temperature for 16 h. After quenched with water,
the reaction mixture was extracted with EtOAc. The combined
organic layers were washed with water and brine, dried over
anhydrous sodium sulfate, filtered off, and concentrated in vacuo to
yield crude 3-amino-6-bromo-N-(4-((1R,3R,4R,5S)-3-(tert-butyldime-
thylsilyloxy)-4-ethyl-4-hydroxy-5-methylcyclohexyl)pyridin-3-yl)-
picolinamide (56 mg, 99%). LCMS (m/z): 563.1, 565.1 [M + H]+.
To a microwave vessel was added 3-amino-6-bromo-N-(4-
((1R,3R,4R,5S)-3-(tert-butyldimethylsilyloxy)-4-ethyl-4-hydroxy-5-
methylcyclohexyl)pyridin-3-yl)picolinamide (56 mg, 0.099 mmol),
4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (50.5 mg,
0.199 mmol), tricyclohexylphosphine (6.13 mg, 0.022 mmol),
Pd2(dba)3 (10.0 mg, 10.9 μmol), and dioxane (497 μL). The
reaction was degassed by nitrogen stream for 5 min followed by
addition of potassium acetate (29.3 mg, 0.298 mmol). The reaction
mixture was microwaved at 120 °C for 10 min. After diluted with
EtOAc, the mixture was filtered through Celite pad. The volatile
materials were removed in vacuo to yield crude 3-amino-N-(4-
((1R,3R,4R,5S)-3-(tert-butyldimethylsilyloxy)-4-ethyl-4-hydroxy-5-
methylcyclohexyl)pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)picolinamide 48 (60.7 mg, 0.099 mmol, >99%). LCMS (m/
Synthesis of Compound 12. ( )-(1S,2R,6S)-2-(tert-Butyldime-
thylsilyloxy)-1-(hydroxymethyl)-6-methyl-4-(3-nitropyridin-4-yl)-
cyclohex-3-enol (43). To a solution of ( )-4-((3R,5S)-3-(tert-
butyldimethylsilyloxy)-5-methyl-4-methylenecyclohex-1-enyl)-3-nitro-
pyridine 37 (6.3 g, 17.47 mmol) in acetone (132 mL) and water (43
mL) was added osmium tetroxide (10 mL, 2.5% in water) and NMO
(13.06 g, 112 mmol). The reaction mixture was stirred at room
temperature for 1 h. After quenched with Na2S2O3 aqueous solution
(21 g in 80 mL of water), the mixture was stirred for 1 h. After
acetone was removed in vacuo, the reaction mixture was extracted
with EtOAc. The combined organic layers were washed with water
and brine, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo. The crude material was purified by ISCO
(gradient EtOAc in heptane) to yield ( )-(1S,2R,6S)-2-(tert-
butyldimethylsilyloxy)-1-(hydroxymethyl)-6-methyl-4-(3-nitropyri-
din-4-yl)cyclohex-3-enol 43 (7.0 g, 17.7 mmol, 95% yield). LCMS
(m/z): 395.0 [M + H]+. ( )-(1R,2R,6S)-2-(tert-butyldimethylsily-
loxy)-1-hydroxy-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-enecar-
baldehyde (44). To a solution of ( )-(1S,2R,6S)-2-(tert-butyldime-
thylsilyloxy)-1-(hydroxymethyl)-6-methyl-4-(3-nitropyridin-4-yl)-
cyclohex-3-enol 43 (3.4 g, 8.62 mmol) in DCM (28.7 mL, 0.3 M) was
added Dess−Martin periodinane (4.02 g, 9.48 mmol). The reaction
mixture was stirred at room temperature for 72 h. After quenched
with Na2S2O3 and NaHCO3 solution (1:8) and vigorously stirred for
1 h, the reaction mixture was extracted with EtOAc, the organic layer
was washed with water and brine, and dried by anhydrous sodium
sulfate, filtered and concentrated in vacuo, and the crude product was
purified by ISCO (0−40% EtOAc in heptane) to afford
( )-(1R,2R,6S)-2-(tert-butyldimethylsilyloxy)-1-hydroxy-6-methyl-4-
(3-nitropyridin-4-yl)cyclohex-3-enecarbaldehyde 44 (2.6 g, 6.62
1
mmol, 77% yield). LCMS (m/z): 393.1 [M + H]+. H NMR (400
MHz, CDCl3): δ ppm 9.94−9.89 (m, 1H), 9.18 (s, 1H), 8.81 (d, J =
4.7 Hz, 1H), 7.32 (d, J = 5.1 Hz, 1H), 5.67 (s, 1H), 4.46−4.55 (m,
1H), 3.86−3.80 (s, 1H), 2.54 (d, J = 3.1 Hz, 1H), 2.49−2.32 (m,
2H), 0.97 (d, J = 6.7 Hz, 3H), 0.83 (s, 9H), 0.12−0.05 (m, 6H).
( )-(1S,2R,6S)-2-(tert-butyldimethylsilyloxy)-1-ethynyl-6-methyl-4-
(3-nitropyridin-4-yl)cyclohex-3-enol (45). To a solution of
O
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z): 529.2 [M + H]+ for boronic acid. 5-Amino-N-(4-((1R,3R,4R,5S)-
4-ethyl-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-3′-fluoro-2,2′-
bipyridine-6-carboxamide (12). To a microwave vessel, 3-amino-N-
(4-((1R,3R,4R,5S)-3-(tert-butyldimethylsilyloxy)-4-ethyl-4-hydroxy-5-
methylcyclohexyl)pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)picolinamide 48 (60.7 mg, 0.099 mmol), 2-bromo-3-
fluoropyridine (26.2 mg, 0.149 mmol), PdCl2(dppf) (10.91 mg,
0.015 mmol), DME (745 μL), and 2 M Na2CO3 solution (248 μL)
were added. The reaction mixture was degassed by N2 stream for 5
min. The reaction was microwaved at 120 °C for 10 min. The
reaction mixture was extracted with EtOAc. The combined organic
layers were washed with water and brine, dried over anhydrous
sodium sulfate, filtered off, and concentrated in vacuo to crude
product. LCMS (m/z): 580.2 [M + H]+. The crude product was
dissolved in THF and MeOH (1:1, 1 mL) followed by addition of 3
N HCl (0.5 mL). The reaction mixture was stirred for 1 h. After
added with saturated Na2CO3 aqueous solution until the mixture
turned to pH 8, the reaction mixture was extracted with EtOAc. The
combined organic layers were washed with water and brine, dried over
anhydrous sodium sulfate, filtered off, and concentrated in vacuo. The
crude product was purified by prep HPLC. The combined pure
fractions were lyophilized to yield 5-amino-N-(4-((1R,3R,4R,5S)-4-
ethyl-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-3′-fluoro-2,2′-
bipyridine-6-carboxamide 12 as its TFA salt (17.4 mg, 30% yield over
3 steps). LCMS (m/z): 466.1 [M + H]+. HRMS (m/z): calcd for
hydroxy-5-methylcyclohexyl)pyridin-3-yl)picolinamide (120 mg,
0.265 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaboro-
lane) (134 mg, 0.529 mmol), tricyclohexylphoshpine (13.36 mg,
0.048 mmol), Pd2(dba)3 (19.39 mg, 0.021 mmol), and dioxane (0.88
mL). After the reaction was degassed by nitrogen stream for 5 min,
potassium acetate (78 mg, 0.794 mmol) was added. The reaction
mixture was microwaved at 120 °C for 10 min. LCMS (m/z): 419.0
[M + H]+ for boronic acid. The reaction mixture was diluted with
EtOAc, which was filtered through Celite pad. The volatile materials
were removed to yield ( )-3-amino-N-(4-((1R,3R,4R,5S)-4-(fluoro-
methyl)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-6-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)picolinamide (132 mg, 0.264
mmol, >99%), which was used in the next step without further
purification. 5-Amino-3′-fluoro-N-(4-((1R,3R,4R,5S)-4-(fluorometh-
yl)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-[2,2′-bipyridine]-
6-carboxamide (13). To solution of
( )-3-amino-N-(4-
((1R,3R,4R,5S)-4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)-
pyridin-3-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
picolinamide (132 mg, 0.264 mmol) in DME (2 mL) and 2 M
Na2CO3 aqueous solution (0.66 mL) was added 2-bromo-3-
fluoropyridine (55.7 mg, 0.317 mmol) and PdCl2(dppf) (19.3 mg,
0.026 mmol). The reaction mixture was heated at microwave for 10
min at 120 °C. After diluted with EtOAc and water, the mixture was
extracted with EtOAc. The combined organic layers were washed with
water and brine, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo. The crude product was purified by prep
HPLC, and pure fractions were free based followed by lyophilization
to yield ( )-5-amino-3′-fluoro-N-(4-((1R,3R,4R,5S)-4-(fluorometh-
yl)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-2,2′-bipyridine-6-
carboxamide (12.8 mg, 10% yield) 13-rac. LCMS (m/z): 470.1 [M +
H]+. 1H NMR (400MHz, CDCl3): δ ppm 10.32 (s, 1H), 9.31 (s, 1H),
8.52 (d, J = 4.3 Hz, 1H), 8.40 (d, J = 5.1 Hz, 1H), 8.18 (d, J = 8.6 Hz,
1H), 7.54 (m, 1H), 7.32 (m, 1H), 7.21 (m, 2H), 6.26 (br s, 2H), 5.30
(s, 3H), 4.89−4.61 (m, 2H), 3.85 (d, J = 11.3 Hz, 1H), 3.21 (br s,
1H), 2.72 (br s, 1H), 2.29 (br s, 1H), 2.14 (m, 1H), 1.97−1.68 (m,
3H), 1.59 (m, 1H), 1.06 (d, J = 8.0 Hz, 3H). 12.8 mg of 13-rac was
resolved by chiral SFC (OJ column, 5 mL/min, EtOH + 0.1% DEA =
20%). Peak 1: 5-amino-3′-fluoro-N-(4-((1R,3R,4R,5S)-4-(fluorometh-
yl)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-2,2′-bipyridine-6-
carboxamide 13 (3.1 mg, >99% ee, Rt = 1.52). LCMS (m/z): 470.1
[M + H]+. HRMS (m/z): calcd for C24H26N5O3F2 [M + H]+,
470.2004; found, 470.2003. Peak 2: 5-amino-3′-fluoro-N-(4-
((1S,3S,4S,5R)-4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)-
pyridin-3-yl)-2,2′-bipyridine-6-carboxamide 13-ent (3.6 mg, >99% ee,
Rt = 1.92). LCMS (m/z): 470.1 [M + H]+.
Synthesis of Compound 14. 5-Amino-2-(2,6-difluorophenyl)-
pyrimidine-4-carboxylic acid (46). A solution of 2,6-difluorobenzimi-
damide hydrochloride 50 (3.8 g, 19.73 mmol) in anhydrous ethanol
(63 mL) was cooled to 0 °C in an ice bath. Sodium ethoxide solution
(16.14 mL, 41.2 mmol, 20%) was added slowly via syringe, the ice
bath was removed, and the reaction mixture was warmed to room
temperature and stirred for 1 h. A solution of mucobromic acid, (E)-
2,3-dibromo-4-oxobut-2-enoic acid (3.54 g, 13.73 mmol), in ethanol
(69 mL) was added dropwise via syringe, and the reaction was heated
in a 40 °C oil bath for 2 h. The volatile materials were removed in
vacuo and water (100 mL) and 6 N NaOH aqueous solution (50 mL)
was added to dissolve the crude product. The basic aqueous solution
was extracted with EtOAc. Then, the aqueous layer was acidified to
pH 4.0 by adding 3 N HCl aqueous solution slowly and extracted with
EtOAc. The combined organic layers were washed with water and
brine, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo to afford crude 5-bromo-2-(2,6-
difluorophenyl)pyrimidine-4-carboxylic acid (4.3 g, 13.7 mmol,
>99% yield) as a reddish brown solid, which was used in the next
step without purification. LCMS (m/z): 314.9, 316.9 [M + H]+. To a
steel bomb reactor was added 5-bromo-2-(2,6-difluorophenyl)-
pyrimidine-4-carboxylic acid (4.3 g, 13.7 mmol) dissolved in 28%
NH4OH solution (10 mL) and copper (II) sulfate (0.219 g, 1.37
mmol). The reactor was placed in a preheated bath (110 °C) and
stirred for 1 h. After cooling down, the reaction vessel was unsealed.
1
C25H29N5O3F [M + H]+, 466.2254; found, 466.2251. H NMR (400
MHz, DMSO-d6): δ ppm 10.52 (s, 1H), 9.35 (s, 1H), 8.51 (m, 2H),
8.11 (d, J = 8 Hz, 1H), 7.85 (d, J = 8 Hz, 1H), 7.78 (m, 1H), 7.51 (m,
1H), 7.43 (d, J = 8 Hz, 1H), 7.26 (br s, 2H), 3.12 (m, 1H), 1.76−1.55
(m, 5H), 1.47−1.34 (m, 2H), 0.92 (t, J = 8 Hz, 3H), 0.85 (d, J = 4
Hz, 3H).
Synthesis of Compound 13. ( )-(1R,2R,6S)-1-(Fluoromethyl)-
6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-diol (49). A sol-
ution of ( )-4-((3S,4R,8S)-4-(tert-butyldimethylsilyloxy)-8-methyl-1-
oxaspiro[2.5]oct-5-en-6-yl)-3-nitropyridine 41 (520 mg, 1.381 mmol)
in triethylamine trihydrofluoride (2.25 mL, 13.81 mmol) in a stainless
steel reactor was heated at 100 °C for 8 h. After cooled down and
quenched by saturated NaHCO3 aqueous solution carefully, the
reaction mixture was partitioned between EtOAc and water. The
mixture was extracted with EtOAc. The combined organic layers were
washed with water and brine, dried over anhydrous sodium sulfate,
filtered off, and concentrated in vacuo to yield ( )-(1R,2R,6S)-1-
(fluoromethyl)-6-methyl-4-(3-nitropyridin-4-yl)cyclohex-3-ene-1,2-
diol (180 mg, 0.638 mmol, 46% yield). LCMS (m/z): 283.0 [M +
H]+. A solution of ( )-(1R,2R,6S)-1-(fluoromethyl)-6-methyl-4-(3-
nitropyridin-4-yl)cyclohex-3-ene-1,2-diol (180 mg, 0.638 mmol) in
MeOH (5.3 mL) was degassed by nitrogen stream for 10 min
followed by addition of 10% Pd on carbon (68 mg). The reaction
mixture was stirred at room temperature for 12 h under hydrogen
balloon. The reaction mixture was filtered through Celite pad and
washed by MeOH and EtOAc, and the filtrate was concentrated in
vacuo to give ( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-1-(fluoro-
methyl)-6-methylcyclohexane-1,2-diol 49 (79 mg, 0.311 mmol, 49%
yield). LCMS (m/z): 255.0 [M + H]+. 5-Amino-3′-fluoro-N-(4-
((1R,3R,4R,5S)-4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)-
pyridin-3-yl)-[2,2′-bipyridine]-6-carboxamide (13). A solution of
( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-1-(fluoromethyl)-6-
methylcyclohexane-1,2-diol 49 (79 mg, 0.311 mmol) and 3-amino-6-
bromopicolinic acid 47 (101 mg, 0.466 mmol), HOAt (76 mg, 0.559
mmol) and EDC (107 mg, 0.559 mmol) in DMF (0.62 mL) was
stirred for 12 h at room temperature. The reaction mixture was
quenched with water and extracted with EtOAc. The organic layers
were washed by saturated NaHCO3 aqueous solution, water and
brine, dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuo to yield crude ( )-3-amino-6-bromo-N-(4-
((1R,3R,4R,5S)-4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)-
pyridin-3-yl)picolinamide (120 mg, 0.265 mmol, 85% yield). The
crude product was used in the next step without further purification.
LCMS (m/z): 453, 455 [M + H]+. To a microwave vessel was added
( )-3-amino-6-bromo-N-(4-((1R,3R,4R,5S)-4-(fluoromethyl)-3,4-di-
P
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The reaction mixture was transferred to Erlenmeyer flask by washing
with water, which was acidified by pH 4.0 with 3 N HCl aqueous
solution, and extracted with EtOAc. The combined organic layers
were washed with water and brine, dried over anhydrous sodium
sulfate, filtered off, and concentrated in vacuo to yield a crude reddish
solid (863 mg, 3.4 mmol, 25% yield over 2 steps). The crude product
was used in the next step without further purification. LCMS (m/z):
(9). LCMS (m/z): 470.2 [M + H]+. HRMS (m/z): calcd for
C25H27N4O3F2 [M + H]+, 470.2004; found, 470.2005. 1H NMR
(DMSO-d6, 400 MHz): δ 10.4 (s, 1H), 8.95 (s, 1H), 8.38 (d, J = 4.0
Hz, 1H), 8.14 (m, 2H), 7.82 (m, 1H), 7.52 (m, 1H), 6.88 (d, J = 12.0
Hz, 1H), 3.8 (s, 1H), 2.93 (m, 1H), 1.67 (m, 1H), 1.53−1.38 (m,
3H), 1.17 (m, 1H), 0.89 (s, 3H), 0.75 (d, J = 8.0 Hz, 3H). 3-Amino-
6-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)picolinamide (10). LCMS (m/z):
469.1 [M + H]+. HRMS (m/z): calcd for C25H27N4O3F2 [M + H]+,
252.0 [M
+
H]+. 5-Amino-2-(2,6-difluorophenyl)-N-(4-
((1R,3R,4R,5S)-4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)-
pyridin-3-yl)pyrimidine-4-carboxamide (14). To a solution of
( )-(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-1-(fluoromethyl)-6-
methylcyclohexane-1,2-diol 49 (50 mg, 0.197 mmol) in DMF (0.4
mL) was added 5-amino-2-(2,6-difluorophenyl)pyrimidine-4-carbox-
ylic acid 51 (64.2 mg, 0.256 mmol), HOAt (40.1 mg, 0.295 mmol)
and EDC (56.5 mg, 0.295 mmol). The reaction mixture was stirred
for 12 h at room temperature. After partitioned between EtOAc and
water, the mixture was extracted with EtOAc. The combined organic
layers were washed by water and brine, dried over anhydrous sodium
sulfate, filtered off, and concentrated in vacuo. The crude material was
purified by reverse phase HPLC. The combined pure fractions were
free-based and extracted with EtOAc. The organic layers were dried
over anhydrous sodium sulfate, filtered off, and concentrated in vacuo
to afford ( )-5-amino-2-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-
4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)pyridin-3-yl)-
pyrimidine-4-carboxamide 14-rac (24 mg, 0.049 mmol, 25%). LCMS
(m/z): 488.1 [M + H]+. 1H NMR (400 MHz, CDCl3): δ ppm: 10.08
(br s, 1H), 9.29 (s, 1H), 8.56 (s, 1H), 8.41 (d, J = 5.1 Hz, 1H), 7.35−
7.45 (m, 1H), 7.20 (d, J = 5.1 Hz, 1H), 7.05 (t, J = 8.0 Hz, 2H), 6.12
(br s, 2H), 4.87−4.58 (m, 2H), 3.77 (d, J = 11.0 Hz, 1H), 3.07 (br s,
1H), 2.70 (br s, 1H), 2.44 (br s, 1H), 2.06 (m, 1H), 1.89−1.72 (m,
4H), 1.65−1.51 (m, 1H), 1.02 (d, J = 6.3 Hz, 3H). 20 mg of 14-rac
was resolved by chiral HPLC (AD-H column, EtOH/heptane =
15:85, 1 mL/min). Peak 1: 5-Amino-2-(2,6-difluorophenyl)-N-(4-
((1S,3S,4S,5R)-4-(fluoromethyl)-3,4-dihydroxy-5-methylcyclohexyl)-
pyridin-3-yl)pyrimidine-4-carboxamide 14-ent (5.5 mg, >99% ee, Rt =
7.83 min), LCMS (m/z): 488.1 [M + H]+. Peak 2: 5-amino-2-(2,6-
difluorophenyl)-N-(4-((1R,3R,4R,5S)-4-(fluoromethyl)-3,4-dihy-
droxy-5-methylcyclohexyl)pyridin-3-yl)pyrimidine-4-carboxamide 14
(6.7 mg, >99% ee, Rt = 9.28 min). LCMS (m/z): 488.1 [M + H]+.
HRMS (m/z): calcd for C24H25N3O3F3 [M + H]+, 488.1909; found,
488.1913.
Synthesis of Compounds 6, 7, 8, 9, and 10. According to steps
l and m in Scheme 4, compounds 6−10 were synthesized using
(1R,2R,4R,6S)-4-(3-aminopyridin-4-yl)-2-((tert-butyldimethylsilyl)-
oxy)-1,6-dimethylcyclohexanol 32a and the corresponding acids, 2-
(2,6-difluorophenyl)thiazole-4-carboxylic acid, 2-(2,6-
difluorophenyl)pyrimidine-4-carboxylic acid, 5-amino-2-(2,6-
difluorophenyl)thiazole-4-carboxylic acid, 5-amino-2-(2,6-
difluorophenyl)pyrimidine-4-carboxylic acid 51, and 3-amino-6-(2,6-
difluorophenyl)picolinic acid, respectively. The desired products were
obtained as their TFA salts through reverse phase preparative HPLC.
2-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihydroxy-4,5-
dimethylcyclohexyl)pyridin-3-yl)thiazole-4-carboxamide (6). LCMS
(m/z): 460.0 [M + H]+. HRMS (m/z): calcd for C23H24N3O3F2S [M
+ H]+, 460.1506; found, 460.1505. 1H NMR (DMSO-d6, 400 MHz):
δ 10.2 (s, 1H), 8.67 (m, 2H), 8.48 (m, 1H), 7.68 (m, 2H), 7.36 (m,
2H), 3.31 (m, 1H), 3.04 (m, 1H), 1.75 (m, 1H), 1.58−1.17 (m, 4H),
0.96 (s, 3H), 0.85 (d, J = 6.8 Hz, 3H). 2-(2,6-Difluorophenyl)-N-(4-
((1R,3R,4R,5S)-3,4-dihydroxy-4,5-dimethylcyclohexyl)pyridin-3-yl)-
pyrimidine-4-carboxamide (7). LCMS (m/z): 455.1 [M + H]+.
HRMS (m/z): calcd for C24H25N4O3F2 [M + H]+, 455.1895; found,
455.1894. 5-Amino-2-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-
dihydroxy-4,5-dimethylcyclohexyl)pyridin-3-yl)thiazole-4-carboxa-
mide (8). LCMS (m/z): 475.1 [M + H]+. HRMS (m/z): calcd for
C23H25N4O3F2S [M + H]+, 475.1615; found, 475.1620. 1H NMR
(DMSO-d6, 400 MHz): δ 8.93 (s, 1H), 8.44 (d, J = 4.0 Hz, 1H), 7.63
(br s, 2H), 7.55 (m, 2H), 7.28 (m, 2H), 3.05 (m, 1H), 1.78 (m, 1H),
1.59−1.41 (m, 3H), 1.27 (m, 1H), 0.97 (s, 3H), 0.88 (d, J = 8.0 Hz,
3H). 5-Amino-2-(2,6-difluorophenyl)-N-(4-((1R,3R,4R,5S)-3,4-dihy-
droxy-4,5-dimethylcyclohexyl)pyridin-3-yl)pyrimidine-4-carboxamide
1
469.2051; found, 469.2056. H NMR (DMSO-d6, 400 MHz): δ 10.3
(s, 1H), 9.14 (s, 1H), 8.45 (d, J = 4.0 Hz, 1H), 7.68 (d, J = 4.0 Hz,
1H), 7.58 (m, 1H), 7.45 (m, 1H), 7.20 (m, 1H), 3.0 (m, 1H), 1.72
(m, 1H), 1.54−1.35 (m, 3H), 1.24 (m, 1H), 0.94 (s, 3H), 0.77 (d, J =
4.0 Hz, 3H).
Synthesis of Compound 11. 5-Amino-N-(4-((1R,3R,4R,5S)-3,4-
dihydroxy-4,5-dimethylcyclohexyl)pyridin-3-yl)-3′-fluoro-2,2′-bipyri-
dine-6-carboxamide (11). According to steps f, g, h, and i in Scheme
5, compound 11 was synthesized using (1R,2R,4R,6S)-4-(3-amino-
pyridin-4-yl)-2-((tert-butyldimethylsilyl)oxy)-1,6-dimethylcyclohexa-
nol 32a. The desired product was obtained as its TFA salt through
reverse phase preparative HPLC. LCMS (m/z): 452.2 [M + H]+.
HRMS (m/z): calcd for C24H27N5O3F [M + H]+, 452.2098; found,
1
452.2096. H NMR (DMSO-d6, 400 MHz): δ 10.2 (s, 1H), 9.09 (s,
1H), 8.52 (m, 1H), 8.31 (d, J = 4.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H),
7.72 (m, 1H), 7.48 (m, 1H), 7.41 (m, 1H), 7.26 (br s, 1H), 4.48 (s,
2H), 4.07 (s, 1H), 2.97 (m, 1H), 1.75 (m, 1H), 1.68−1.41 (m, 3H),
1.28 (m, 1H), 0.94 (s, 3H), 0.84 (d, J = 8.0 Hz, 3H).
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 alamethicin/mg microsomal 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 30 min incubation.
Phenolphthalein was used as a reference control. Data were obtained
utilizing semi-quantitative 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 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
mL/min/kg and 20.7 mL/min/kg were used for rat and human.
Calculation of hepatic extraction ratio: ER_H = CLh/Qh.
PIM Enzymatic Assays. Kinase-Glo Pim1, Pim2, Pim3 ATP
depletion assays: Pim1, Pim2 & Pim3 Kinase-Glo assays using ATP (at
or below ATP Km) were used as previously described to determine
the biochemical activity of compounds 1−7 and compounds 9−12, as
shown in Table 1.13 High ATP Pim1, Pim2, Pim3 AlphaScreen Assays:
Pim1, Pim2 & Pim3 AlphaScreen assays using high ATP (11−125 X
ATP Km) were used as previously described to determine the
biochemical activity of compounds 8, 13, and 14, as shown in Table
1.13
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,
Ontario, Canada. KMS-11.luc was cultured in RPMI-1640 (ATCC,
Manassas VA) supplemented with 10% FBS.
Cellular Proliferation Assays. Proliferation assays in KMS-11.luc
were conducted as previously described with the concentration at
which 50% maximal inhibition is reached reported as EC50.13
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01279.
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c01279
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Pharmaceuticals, South San Francisco, California 94080,
United States
Kinase selectivity profiles for compounds 5 and 11 and
LCMS and HPLC spectra for representative compounds
5, 11, and 14 (PDF)
Molecular formula strings and statistical analysis of the
associated biochemical data (CSV)
Yumin Dai − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Bristol Myers
Squibb, Redwood City, California 94158, United States
Pablo Garcia − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Circle Pharma,
Inc., South San Francisco, California 94080, United States
Matthew T. Burger − Novartis Institutes for BioMedical
Research, Emeryville, California 94608, United States
(XLSX)
Accession Codes
PDB codes of compounds 5 and 13 in PIM1 are 1oblv and
1ozrb, respectively. The atomic coordinates will be released
upon article publication.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01279
AUTHOR INFORMATION
■
Author Contributions
All authors have given approval to the final version of the
manuscript.
Corresponding Author
Wooseok Han − Novartis Institutes for BioMedical Research,
Global Discovery Chemistry, Emeryville, California 94608,
United States; orcid.org/0000-0002-7567-1883;
Email: wooseok.han@gmail.com
Notes
The authors declare no competing financial interest.
Authors
ACKNOWLEDGMENTS
■
Yu Ding − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; BeiGene, Ltd.,
San Mateo, California 94403, United States
Zheng Chen − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Boston
Analytical, Salem, New Hampshire 03079, United States
John L. Langowski − Novartis Institutes for BioMedical
Research, Emeryville, California 94608, United States; Kite, a
Gilead Company, Emeryville, California 94608, United
States
Cornelia Bellamacina − Novartis Institutes for BioMedical
Research, Emeryville, California 94608, United States;
Crystallographic Consulting, Berkeley, California 94704,
United States
Alice Rico − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Exelixis,
Alameda, California 94502, United States
Gisele A. Nishiguchi − Novartis Institutes for BioMedical
Research, Emeryville, California 94608, United States; St.
Jude Children’s Research Hospital, Memphis, Tennessee
38105, United States
Jiong Lan − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Genfleet
Therapeutics, Inc., Shanghai 201203, China
Gordana Atallah − Novartis Institutes for BioMedical
Research, Emeryville, California 94608, United States;
Pharmacyclics, an AbbVie Company, Sunnyvale, California
94085, United States
Mika Lindvall − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Recursion
Pharmaceuticals, Salt Lake City, Utah 84101, United States
Song Lin − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Astex
Pharmaceuticals Inc., Pleasanton, California 94588, United
States
The authors thank Weiping Jia and Dahzi Tang for analytical
chemistry support, Alice Wang for chiral resolution, Kent
Wong for in vitro DMPK support, Jee-Yeon Wong for data
analysis, and Fiorella Ruggiu for fruitful discussion about
membrane permeability.
ABBREVIATIONS
■
BOC, tert-butoxycarbonyl; Cbz, benzyloxycarbonyl; CL,
clearance; Clint, intrinsic clearance; DCM, dichloromethane;
DIEA, diisopropylethylamine; DMAP, dimethylaminopyridine;
DMDO, dimethyldioxirane; DME, 1,2-dimethoxyethane;
DMF, dimethylformamide; ee, enantiomeric excess; EDC, N-
ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochlor-
ide; ER, efflux ratio; ER_H, extraction ratio; EtOAc, ethyl
acetate; Log D7.4, log of the octanol−water distribution
coefficient at pH 7.4 of ionized and unionized compound;
HOAt, 1-hydroxy-7-azabenzotriazole; HPLC, high perform-
ance liquid chromatography; HT-Eq Sol., high throughput
equilibrium solubility; ISCO, Teledyne ISCO chromatography
system; LCMS, liquid chromatography mass spectrometry;
mCPBA, meta-chloroperoxybenzoic acid; NOE, nuclear over-
hauser effect; PSA, polar surface area; SFC, supercritical fluid
chromatography; TBDMS, tert-butyldimethylsilyl; TEA, trie-
thylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TLC, thin layer chromatography; TMS, trimethylsilyl
REFERENCES
■
(1) Cuypers, H. T.; Selten, G.; Quint, W.; Zijlstra, M.; Maandag, E.
R.; Boelens, W.; Van Wezenbeek, P.; Melief, C.; Berns, A. Murine
leukemia virus-induced T-cell lymphomagenesis: integration of
proviruses in a distinct chromosomal region. Cell 1984, 37, 141−150.
(2) Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.;
Schwaller, J. PIM serine/threonine kinases in the pathogenesis and
therapy of hematologic malignancies and solid cancers. Haematologica
2010, 95, 1004−1015.
Richard Zang − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States; Global Blood
Therapeutics, South San Francisco, California 94080, United
States
Paul Feucht − Novartis Institutes for BioMedical Research,
Emeryville, California 94608, United States
(3) Berns, A.; Breuer, M.; Verbeek, S.; Van Lohuizen, M. Transgenic
mice as a means to study synergism between oncogenes. Int. J. Cancer
Suppl. 1989, 44, 22−25.
(4) Nawijn, M. C.; Alendar, A.; Berns, A. For better or for worse: the
role of Pim oncogenes in tumorigenesis. Nat. Rev. Cancer 2011, 11,
23−34.
Tatiana Zavorotinskaya − Novartis Institutes for BioMedical
Research, Emeryville, California 94608, United States; ORIC
(5) van Lohuizen, M.; Verbeek, S.; Krimpenfort, P.; Domen, J.; Saris,
C.; Radaszkiewicz, T.; Berns, A. Predisposition to lymphomagenesis
R
https://dx.doi.org/10.1021/acs.jmedchem.0c01279
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
in pim-1 transgenic mice: cooperation with c-myc and N-myc in
murine leukemia virus-induced tumors. Cell 1989, 56, 673−682.
(6) Garcia, P. D.; Langowski, J. L.; Wang, Y.; Chen, M.; Castillo, J.;
Fanton, C.; Ison, M.; Zavorotinskaya, T.; Dai, Y.; Lu, J.; Niu, X.-H.;
Basham, S.; Chan, J.; Yu, J.; Doyle, M.; Feucht, P.; Warne, R.;
Narberes, J.; Tsang, T.; Fritsch, C.; Kauffmann, A.; Pfister, E.;
Drueckes, P.; Trappe, J.; Wilson, C.; Han, W.; Lan, J.; Nishiguchi, G.;
Lindvall, M.; Bellamacina, C.; Aycinena, J. A.; Zang, R.; Holash, J.;
Burger, M. T. Pan-PIM Kinase Inhibition Provides a Novel Therapy
for Treating Hematologic Cancers. Clin. Cancer Res. 2014, 20, 1834−
1845.
(7) Chen, W. W.; Chan, D. C.; Donald, C.; Lilly, M. B.; Kraft, A. S.
Pim family kinases enhance tumor growth of prostate cancer cells.
Mol. Cancer Res. 2005, 3, 443−451.
(8) Fujii, C.; Nakamoto, Y.; Lu, P.; Tsuneyama, K.; Popivanova, B.
K.; Kaneko, S.; Mukaida, N. Aberrant expression of serine/threonine
kinase Pim-3 in hepatocellular carcinoma development and its role in
the proliferation of human hepatoma cell lines. Int. J. Cancer 2005,
114, 209−218.
(9) Popivanova, B. K.; Li, Y.-Y.; Zheng, H.; Omura, K.; Fujii, C.;
Tsuneyama, K.; Mukaida, N. Proto-oncogene, Pim-3 with serine/
threonine kinase activity, is aberrantly expressed in human colon
cancer cells and can prevent Bad-mediated apoptosis. Cancer Sci.
2007, 98, 321−328.
(10) Zheng, H.-C.; Tsuneyama, K.; Takahashi, H.; Miwa, S.;
Sugiyama, T.; Popivanova, B. K.; Fujii, C.; Nomoto, K.; Mukaida, N.;
Takano, Y. Aberrant Pim-3 expression is involved in gastric adenoma-
adenocarcinoma sequence and cancer progression. J. Canc. Res. Clin.
Oncol. 2008, 134, 481−488.
(11) Blanco-Aparicio, C.; Carnero, A. Pim kinases in cancer:
diagnostic, prognostic and treatment opportunities. Biochem.
Pharmacol. 2013, 85, 629−643.
(12) 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. Pim2 is required for maintaining multiple myeloma cell growth
through modulating TSC2 phosphorylation. Blood 2013, 122, 1610−
1620.
(13) 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. Structure guided optimization, in
vitro activity, and in vivo activity of pan-PIM kinase inhibitors. ACS
Med. Chem. Lett. 2013, 4, 1193−1197.
(14) Nishiguchi, G. A.; Atallah, G.; Bellamacina, C.; Burger, M. T.;
Ding, Y.; Feucht, P. H.; Garcia, P. D.; Han, W.; Klivansky, L.;
Lindvall, M. Discovery of novel 3,5-disubstituted indole derivatives as
potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases. Bioorg.
Med. Chem. Lett. 2011, 21, 6366−6369.
(15) Nakano, H.; Hasegawa, T.; Kojima, H.; Okabe, T.; Nagano, T.
Design and synthesis of potent and selective PIM kinase inhibitors by
targeting unique structure of ATP-binding pocket. ACS Med. Chem.
Lett. 2017, 8, 504−509.
Hickman, D.; Laszlo, J., III; 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. Discovery and Optimization of Macrocyclic Quinoxaline-
pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors.
ACS Med. Chem. Lett. 2016, 7, 408−412.
(19) Wang, X.; Kolesnikov, A.; Tay, S.; Chan, G.; Chao, Q.; Do, S.;
Drummond, J.; Ebens, A. J.; Liu, N.; Ly, J.; Harstad, E.; Hu, H.;
Moffat, J.; Munugalavadla, V.; Murray, J.; Slaga, D.; Tsui, V.; Volgraf,
M.; Wallweber, H.; Chang, J. H. Discovery of 5-azaindazole (GNE-
955) as a potent pan-Pim inhibitor with optimized bioavailability. J.
Med. Chem. 2017, 60, 4458−4473.
(20) Wang, H.-L.; Andrews, K. L.; Booker, S. K.; Canon, J.; Cee, V.
J.; Chavez, F., Jr; Chen, Y.; Eastwood, H.; Guerrero, N.; Herberich,
B.; Hickman, D.; Lanman, B. A.; Laszlo, J., III; Lee, M. R.; Lipford, J.
R.; Mattson, B.; Mohr, C.; Nguyen, Y.; Norman, M. H.; Pettus, L. H.;
Powers, D.; Reed, A. B.; Rex, K.; Sastri, C.; Tamayo, N.; Wang, P.;
Winston, J. T.; Wu, B.; Wu, Q.; Wu, T.; Wurz, R. P.; Xu, Y.; Zhou, Y.;
Tasker, A. S. Discovery of (R)-8-(6-Methyl-4-oxo-1,4,5,6-
tetrahydropyrrolo[3,4-b]pyrrol-2-yl)-3-(1-methylcyclopropyl)-2-((1-
methylcyclopropyl)amino)quinazolin-4(3H)-one, a potent and selec-
tive Pim-1/2 kinase inhibitor for hematological malignancies. J. Med.
Chem. 2019, 62, 1523−1540.
(21) Wang, X.; Blackaby, W.; Allen, V.; Chan, G. K. Y.; Chang, J. H.;
̀
Chiang, P.-C.; Diene, C.; Drummond, J.; Do, S.; Fan, E.; Harstad, E.
B.; Hodges, A.; Hu, H.; Jia, W.; Kofie, W.; Kolesnikov, A.; Lyssikatos,
J. P.; Ly, J.; Matteucci, M.; Moffat, J. G.; Munugalavadla, V.; Murray,
J.; Nash, D.; Noland, C. L.; Del Rosario, G.; Ross, L.; Rouse, C.;
Sharpe, A.; Slaga, D.; Sun, M.; Tsui, V.; Wallweber, H.; Yu, S.-F.;
Ebens, A. J. Optimization of pan-Pim kinase activity and oral
bioavailability leading to diaminopyrazole (GDC-0339) for the
treatment of multiple myeloma. J. Med. Chem. 2019, 62, 2140−2153.
(22) 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. Design, synthesis and structure
activity relationship of potent pan-PIM kinase inhibitors derived from
the pyridyl carboxamide scaffold. Bioorg. Med. Chem. Lett. 2016, 26,
2328−2332.
(23) 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.; Lindvall, 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. Identification ofN-(4-((1R,3S,5S)-3-Amino-5-
methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicoli-
namide (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. J. Med. Chem. 2015,
58, 8373−8386.
(24) Raab, M. S.; Thomas, S. K.; Ocio, E. M.; Guenther, A.; Goh, Y.-
T.; Talpaz, M.; Hohmann, N.; Zhao, S.; Xiang, F.; Simon, C.;
Vanasse, K. G.; Kumar, S. K. The first-in-human study of the pan-PIM
kinase inhibitor PIM447 in patients with relapsed and/or refractory
multiple myeloma. Leukemia 2019, 33, 2924−2933.
(25) Meanwell, N. A. Synopsis of some recent tactical application of
bioisosteres in drug design. J. Med. Chem. 2011, 54, 2529−2591.
(26) Smith, D. A.; Beaumont, K.; Maurer, T. S.; Di, L. Clearance in
drug design. J. Med. Chem. 2019, 62, 2245−2255.
(27) Structure of compound 5 in PIM1 submitted under PDB
accession code 1oblv.
(28) All animal experiments performed in the manuscript were
conducted in compliance with institutional guidelines.
(29) Compound 5 having no hinge amino group showed a similar
selectivity profile towards these kinases: IC50 for GSK3β was 0.15 μM
(see the Supporting Information).
(16) 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. Synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine
compounds as potent and selective Pim-1 inhibitors. ACS Med. Chem.
Lett. 2015, 6, 63−67.
(17) Pettus, L. H.; Andrews, K. L.; Booker, S. K.; Chen, J.; Cee, V. J.;
Chavez, F.; Chen, Y.; Eastwood, H.; Guerrero, N.; Herberich, B.;
Hickman, D.; Lanman, B. A.; Laszlo, 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. Discovery and optimization of quinazolinone-pyrrolopyrrolones
as potent and orally bioavailable pan-Pim kinase inhibitors. J. Med.
Chem. 2016, 59, 6407−6430.
(30) Matsson, P.; Doak, B. C.; Over, B.; Kihlberg, J. Cell
permeability beyond the rule of 5. Adv. Drug Deliv. Rev. 2016, 101,
42−61.
(18) Cee, V. J.; Chavez, F., Jr; Herberich, B.; Lanman, B. A.; Pettus,
L. H.; Reed, A. B.; Wu, B.; Wurz, R. P.; Andrews, K. L.; Chen, J.;
S
https://dx.doi.org/10.1021/acs.jmedchem.0c01279
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(31) Dalvit, C.; Vulpetti, A. Intermolecular and intramolecular
hydrogen bonds involving fluorine atoms: implications for recog-
nition, selectivity, and chemical properties. ChemMedChem 2012, 7,
262−272.
(32) Desai, P. V.; Raub, T. J.; Blanco, M.-J. How hydrogen bonds
impact P-glycoprotein transport and permeability. Bioorg. Med. Chem.
Lett. 2012, 22, 6540−6548.
(33) Han, W.; Menezes, D. L.; Xu, Y.; Knapp, M. S.; Elling, R.;
Burger, M. T.; Ni, Z.-J.; Smith, A.; Lan, J.; Williams, T. E.; Verhagen,
J.; Huh, K.; Merritt, H.; Chan, J.; Kaufman, S.; Voliva, C. F.; Pecchi, S.
Discovery of imidazo[1,2- a ]-pyridine inhibitors of pan-PI3 kinases
that are efficacious in a mouse xenograft model. Bioorg. Med. Chem.
Lett. 2016, 26, 742−746.
(34) Structure of compound 13 in PIM1 submitted under PDB
accession code 1ozrb.
(35) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.;
Meanwell, N. A. Applications of fluorine in medicinal chemistry. J.
Med. Chem. 2015, 58, 8315−8359.
(36) For compounds 4, 5, 11, and 12, IC50 values for hERG
potassium channel were >30 μM.
(37) The synthesis of intermediates 16 and 18 was previously
reported (see ref 23).
(38) Hashimoto, N.; Kanda, A. Practical and environmentally
friendly epoxidation of olefins using oxone. Org. Process Res. Dev.
2002, 6, 405−406.
(39) Perali, R. S.; Mandava, S.; Chunduri, V. R. An unexpected
migration of O-silyl group under Mitsunobu reaction conditions.
Tetrahedron Lett. 2011, 52, 3045−3047.
(40) VanRheenen, V.; Kelly, R. C.; Cha, D. Y. An improved catalytic
OsO4 oxidation of olefins to -1,2-glycols using tertiary amine oxides
as the oxidant. Tetrahedron Lett. 1976, 17, 1973−1976.
(41) Kraus, G.; Kim, J. A direct preparation of 6-methylene-2-
cyclohexenones. Synthesis 2004, 1737−1738.
(42) Berti, G. Stereochemical aspects of the synthesis of 1,2-
epoxides. In Topics in Stereochemistry; Allinger, N. L., Eliel, E. L., Eds.;
Wiley: New York, London, Sydney, Toronto, 1973; Vol. 7, p 93.
(43) To further confirm the stereochemistry, we synthesized two
regioisomeric epoxides through either direct mCPBA reaction or
bromohydrin/ring closing reactions of 36 (see the Experimental
Section). Also, see ref 38 for similar examples.
(44) Muller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J. An
̈
improved one-pot procedure for the synthesis of alkynes from
aldehydes. Synlett 1996, 521−522.
(45) Ohira, S. Methanolysis of dimethyl (1-diazo-2-oxopropyl)
phosphonate: generation of dimethyl (diazomethyl) phosphonate and
reaction with carbonyl compounds. Synth. Commun. 1989, 19, 561−
564.
(46) Han, W.; Ding, Y.; Xu, Y.; Pfister, K.; Zhu, S.; Warne, B.; Doyle,
M.; Aikawa, M.; Amiri, P.; Appleton, B.; Stuart, D. D.; Fanidi, A.;
Shafer, C. M. Discovery of a Selective and Potent Inhibitor of
Mitogen-Activated Protein Kinase-Interacting Kinases 1 and 2
(MNK1/2) Utilizing Structure-Based Drug Design. J. Med. Chem.
2016, 59, 3034−3045.
̈
(47) Wolker, D.; Haufe, G. Synthesis of optically active vicinal
fluorohydrins by lipase-catalyzed deracemization. J. Org. Chem. 2002,
67, 3015−3021.
(48) Junnemann, J.; Lundt, I.; Thiem, J.; Ivanov, C. B.; Atanasova,
̈
R.; Napoli, A.; Sindona, G.; Aksnes, D. W.; Francis, G. W. Reaction of
Epoxyaldonolactones with HF–Amine Complexes. Acta Chem. Scand.
1994, 48, 265−268.
(49) Richardson, P. Fluorination methods for drug discovery and
development. Expet Opin. Drug Discov. 2016, 11, 983−999.
(50) Yerien, D. E.; Bonesi, S.; Postigo, A. Fluorination methods in
drug discovery. Org. Biomol. Chem. 2016, 14, 8398−8427.
T
https://dx.doi.org/10.1021/acs.jmedchem.0c01279
J. Med. Chem. XXXX, XXX, XXX−XXX