Fragment-Based Discovery of Pyrazolopyridones as JAK1 Inhibitors with Excellent Subtype Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.0c00359

Herein, we report the discovery of a series of JAK1-selective kinase inhibitors with high potency and excellent JAK family subtype selectivity. A fragment screening hit 1 with a pyrazolopyridone core and a JAK1 bias was selected as the starting point for our fragment-based lead generation efforts. A two-stage strategy was chosen with the dual aims of improving potency and JAK1 selectivity: Optimization of the lipophilic ribose pocket-targeting substituent was followed by the introduction of a variety of P-loop-targeting functional groups. Combining the best moieties from both stages of the optimization afforded compound 40, which showed excellent potency and selectivity. Metabolism studies in vitro and in vivo together with an in vitro safety evaluation suggest that 40 may be a viable lead compound for the development of highly subtype-selective JAK1 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.0c00359

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Article
Fragment-based discovery of pyrazolopyridones as
JAK1 inhibitors with excellent subtype selectivity
Bettina Borreschmidt Hansen, Tue Heesgaard Jepsen, Mogens Larsen, Rikke Sindet, Thomas Vifian,
Mia Nørreskov Burhardt, Jens Larsen, Jimmi Gerner Seitzberg, Martin A Carnerup, Anders Jerre,
Christina Mølck, Paola Lovato, Sanjay Rai, Venkatarathnam Reddy Nasipireddy, and Andreas Ritzén
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00359 • Publication Date (Web): 28 May 2020
Downloaded from pubs.acs.org on May 28, 2020
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Journal of Medicinal Chemistry
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Fragment-based discovery of pyrazolopyridones as JAK1 inhibitors
with excellent subtype selectivity
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Bettina Borreschmidt Hansen,† Tue Heesgaard Jepsen,† Mogens Larsen,† Rikke Sindet,† Thomas
Vifian,† Mia Nørreskov Burhardt,† Jens Larsen,† Jimmi Gerner Seitzberg,† Martin A. Carnerup,‡
Anders Jerre,§,# Christina Mølck,§ Paola Lovato,§ Sanjay Rai,‖ Venkatarathnam Reddy Nasipireddy,‖
and Andreas Ritzén*,†
†MedChem II, ‡DMPK, and §Skin Research, LEO Pharma A/S, Industriparken 55, 2750 Ballerup, Denmark; ‖GVK
Biosciences Private Limited, Medicinal Chemistry, 28 A, IDA Nacharam, Hyderabad, India
ABSTRACT: Herein we report the discovery of a series of JAK1-selective kinase inhibitors with high potency and excellent
JAK family subtype selectivity. A fragment screening hit 1 with a pyrazolopyridone core and a JAK1 bias was selected as the
starting point for our fragment-based lead generation efforts. A two-stage strategy was chosen with the dual aims of
improving potency and JAK1 selectivity: Optimization of the lipophilic ribose pocket-targeting substituent was followed by
introduction of a variety of P-loop-targeting functional groups. Combining the best moieties from both stages of the
optimization afforded compound 40, which showed excellent potency and selectivity. Metabolism studies in vitro and in vivo
together with an in vitro safety evaluation suggest that 40 may be a viable lead compound for the development of highly
subtype-selective JAK1 inhibitors.
inhibitors, abrocitinib, has been evaluated in Phase III
clinical trials in atopic dermatitis with a favorable efficacy
and safety profile.12,13
INTRODUCTION
The Janus kinase (JAK) family of tyrosine kinases plays a
central role in the cytokine-dependent regulation of
proliferation, survival, differentiation and function of
diverse cells. It consists of four closely related proteins
(JAK1, JAK2, JAK3 and TYK2) that transduce signaling from
cytokine receptors by phosphorylation and subsequent
activation of signal transducers and activators of
transcription (STATs).1,2 JAKs are critically involved in
immune response in health and disease.1,3 The
understanding of the role of cytokines in the
pathophysiology of immune-mediated diseases and the
identification of the critical role of JAKs in cytokine
signaling prompted the idea that inhibiting the activity of
JAKs might provide a new immunomodulatory therapeutic
approach. Tofacitinib, a first-generation JAK inhibitor
targeting the activity of JAK1, JAK2 and JAK3, was the first
drug with this mechanism of action that was approved by
the FDA for the treatment of the autoimmune disease
rheumatoid arthritis.4 Tofacitinib has been investigated in
a wide range of autoimmune diseases including juvenile
arthritis, inflammatory bowel disease, and dermatological
disorders e.g. psoriasis, atopic dermatitis, alopecia and
vitiligo.5-7 Today, five JAK inhibitors (ruxolitinib,
tofacitinib, baricitinib, peficitinib, and upadacitinib) are
approved as drugs and several more are in late-stage
clinical development.8-11
We recently reported the discovery of a series of pan-
JAK inhibitors that were designed with topical application
in mind.14 The starting point for that work was a fragment
screen of a kinase-targeted library of ca. 500 fragments
screened against JAK2 at a single concentration (100 μM)
using SPR. Hits were validated by determination of KD
using SPR and determination of IC50 in a biochemical
enzyme inhibition assay for JAK1. In general, a good
correlation was observed between these two
measurements suggesting that most fragments had little or
no selectivity for JAK1 over JAK2. However, one compound
(1) deviated from this trend: its potency towards JAK1
(IC50 = 16 µM) was higher than expected from its affinity to
JAK2 (KD = 45 µM). Because of the JAK1 bias and good
potency of this unique fragment we selected it as the
starting point for the discovery of subtype-selective JAK1
inhibitors reported herein.
RESULTS AND DISCUSSION
Hit expansion of compound 1 by commercial and
synthesized analogs yielded a preliminary map of the SAR
of the scaffold (Table 1). Deletion of the fluorine atoms was
tolerated with a limited drop in potency (compound 2),
while removal of the pyrazole methyl group was not
(compound 3). Methylation of either of the nitrogen atoms
of the pyrazole ring also abolished all activity (compounds
4 and 5), suggesting that hydrogen bonding interactions
with this moiety are important for binding.
Subtype-selective inhibitors are currently in clinical
trials with the rationale of maintaining the clinical efficacy
of pan-JAK inhibitors while improving safety. In particular,
selectivity over JAK2 should have the advantage of
avoiding hematological side effect like neutropenia and
anemia.8,9 One of the most advanced selective JAK1
In the absence of crystallographic evidence, we assumed
that compounds 1 and 2 interact with the kinase hinge
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region. In principle, docking should give a good indication
subtype-selective JAK inhibitors,14 at the time compound 6
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8
of the binding mode of these compounds, but this scaffold
can plausibly exist in multiple tautomeric forms, and each
of these tautomers would have distinct binding modes.
Prediction of the dominant tautomer in solution by
quantum mechanical computations was attempted
following the method of Ribeiro et al,15 see Table 2. Gas
phase (vacuum) molecular geometries were optimized at
the M06-2X/6-311G**+ level and were used to calculate
solvation energies in water using the SM8 method.16
Relative energies in water were calculated using the M06-
2X functional17 and two different basis sets, but the results
were ambiguous. In vacuum, the 1H-pyrazolopyridinol
tautomer was strongly favored, but in water, either this
tautomer or the 2H-pyrazolopyridone tautomer was
favored depending on the basis set. Furthermore, the
bioactive tautomer may be different from the dominant
tautomer in solution, and because of these complications
we were unable to rely on docking to determine the
binding mode of the scaffold. Instead, a pragmatic
approach was taken: given the SAR outlined above, and
with the assumption that the scaffold binds to the kinase
hinge region, we set out to grow the difluoromethyl
substituent of 1 by substituting larger lipophilic moieties
that might be able to interact with the ribose pocket of the
kinase.
was prepared we had only access to crystallography for
JAK2. Because 6 showed a very modest level of selectivity
for JAK1 over JAK2, we reasoned that a crystal structure of
this compound in complex with JAK2 would be sufficient to
establish its binding mode across the JAK kinase family.
Thus, an X-ray crystal structure of compound 6 bound to
the kinase domain of JAK2 was obtained, see Figure 1. This
unequivocally established the bioactive tautomer of the
scaffold as the 2H-pyrazolopyridone structure (Table 2).
Compound 6 engages the kinase hinge region with three
hydrogen bonds to the backbone amides of Glu 930 and
Leu 932, and a fourth water-mediated hydrogen bond to
Pro 933 and the side chain hydroxyl of Tyr 931. Except for
the last one, these interactions would be expected to be
identical in JAK1 because the only difference in the hinge is
Tyr 931 in JAK2 that corresponds to Phe 958 in JAK1. The
ligand carbonyl oxygen also nucleates a water network
that connects it loosely to the side chain carboxylate of Asp
939 in JAK2. This residue is a glutamic acid (Glu 966) in
JAK1 but remains an aspartic acid in JAK3 and TYK2.18,19
The X-ray structure of compound 6 is consistent with the
SAR derived in Table 1. Methylation of either nitrogen of
the pyrazole would disrupt the hinge interaction, and
deletion of the pyrazole methyl group would remove a
productive lipophilic interaction. Methyl group deletion
may even create a high energy void that cannot be filled by
protein side chain rearrangement or a low energy water
molecule. Such effects could explain the dramatic loss of
potency of 3 compared to 1.
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Table 1. Fragment hit and first set of analogs.a
Compound
Structure
JAK1 IC50 (µM)
Table 2. Tautomers of compound 2 with relative
energies in kcal/mol calculated using DFT.a
1
16
2
3
30
>100
aGeometries were optimized in vacuum with the density
functional M06-2X17 and the 6-311G**+ basis set, and these
geometries were used for the solvation energy calculations.
Methods: A, M06-2X/6-311G**+ in vacuum; B, M06-2X/6-
31G* with the SM8 water solvation model; C, M06-2X/6-
311G**+ with the SM8 water solvation model.15,16
4
5
>500
>900
aIC50 values were determined in a TR-FRET biochemical
assay for kinase inhibition with [ATP] = Km. All values
represent the geometric mean of at least two independent
experiments, see Supporting information for details.
Gratifyingly, replacing the difluoromethyl group with a
phenyl group (compound 6) yielded
a seven-fold
improvement in potency relative to 1, see Table 3. Because
this series originated from our earlier work on non-
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Figure 2. Strategy for lead generation.
To optimize the lipophilic ribose pocket substituent of 6,
the phenyl group was replaced by a selection of mono- and
bicyclic aliphatic ring systems, see Table 3. The cyclohexyl
analog 7 showed dramatically improved potency and also
increased LLE, but very low solubility. This compound was
the first with activity in the cellular STAT6 assay.
Importantly, compound 7 exhibited a 10-fold selectivity
for JAK1 over JAK2 in the biochemical assays. Although the
reason for this jump in selectivity relative to compound 6
is unclear, this result prompted a thorough investigation
into this chemical series. Smaller, less lipophilic
substituents e.g. cyclopentyl (8) and cyclobutyl methyl (9)
afforded similar levels of potency with improved LLE and
solubility. The racemic cis-methyl cyclohexyl analog 10
possessed improved potency in both enzymatic and
cellular assays, albeit with a significant log D penalty. The
design of this compound was inspired by the similar
positioning of the methyl group of tofacitinib (20),4,20 see
Figure 3 and the discussion below. Racemic norbornane
analogs 11 and 12 showed similar or even higher potency
with slightly reduced log D and thus improved LLE with
the same number of sp3 carbons as 10. Importantly,
compared with 7, compounds 11 and 12 achieved a four-
fold improvement in cellular potency with no increase in
lipophilicity and much improved solubility. The racemic
bicyclo[2.2.2]octane analog 13 displayed no further
improvement in potency, and its increased lipophilicity
caused a drop in LLE and solubility. Thus, the norbornanes
11 and 12 were the best performing analogs as they
combined excellent potency with LLE on par with the
smaller, less potent analogs 8 and 9. The log D advantage
of bicyclic over monocyclic scaffolds has been noted for
bicyclic morpholine and piperazine analogs, although the
effects for those compounds are partly due to increased
basicity.21 Synthetic access to norbornane analogs of
specific stereochemistry is restricted, so for synthetic
expedience, we initially chose to optimize the cyclohexane
derivative 7. This would allow us to explore substitution
towards the solvent front and the P-loop in detail, and if
necessary, we envisaged that optimized substituents could
be transferred back to the norbornane analogs.
Figure 1. X-ray crystal structure of compound 6 in complex
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with JAK2 (PDB entry 6TPD, 2.0 Å resolution). The ligand 6 is
colored green, the hinge loop is colored purple, the P-loop is
colored orange, the DFG loop is colored blue and the αC helix
is colored red. Hydrogen bonds are shown as green dotted
lines.
With the binding mode and key interactions established,
structure-based optimization could be initiated, see Figure
2. The phenyl group of 6 does not appear to make optimal
interactions with the lipophilic ribose pocket. Aliphatic
moieties with more three-dimensional shape would likely
offer a better fit. Thus, the first step should be optimizing
this moiety by adding sp3 carbons and simultaneously
keeping lipophilicity at a minimum. The methyl group of 6
seems to fit well, nevertheless, a limited set of analogs with
alternative small alkyl groups were targeted to map SAR at
this position. The last accessible vector, i.e. the carbon
atom next to the carbonyl, points towards the solvent
front. It could potentially be exploited to modulate
solubility and lipophilicity, and perhaps to further engage
Glu 966 (the JAK1 residue corresponding to Asp 939 in
JAK2) to improve selectivity for JAK1. Eventually,
extending the ligand to contact the P-loop of the kinase
was planned to further improve potency.
The biochemical kinase assay used in this work was
performed with an ATP concentration close to the Km of
the respective enzyme (JAK1: 7 µM, JAK2: 4 µM, JAK3: 2
µM, TYK2: 13 µM). However, the intracellular
concentration of ATP can reach millimolar, and for an ATP-
competitive enzyme inhibitor this leads to a dramatic
increase in the IC50 in a cellular assay compared to the
biochemical assay used here. In addition, low permeability
across the cell membrane can further reduce or abolish the
activity of the tested compound. Thus, cellular STAT
reporter gene assays were established for JAK1-STAT6
signaling (to assess JAK1 inhibition) and JAK2-STAT5
signaling (to asses JAK2 inhibition), and compounds were
tested in these assays in parallel with the biochemical
assays during the optimization campaign reported here.
As noted above, a methyl substituent on the pyrazole
appeared optimal based on the X-ray structure of 6, but a
limited exploration seemed justified to confirm this
hypothesis. Deletion of the methyl group in 7 yielded a
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compound (14) with measurable, but ~110-fold reduced position of the cyclohexyl ring were considered. With the
1
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4
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6
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8
JAK1 potency, thus confirming the importance of this
substituent (compare compounds 1 and 3 discussed
above). Opposing trends in the enzymatic and cellular
assays were observed for the series methyl (7), ethyl (15)
and cyclopropyl (16), perhaps reflecting improved
permeability of the more lipophilic analogs. The increased
cellular potency of 15 and 16 was negated by increased
lipophilicity as the LLE was unchanged compared with 7.
Thus, the methyl group was retained at this position
throughout the rest of the optimization.
bioactive tautomer and binding mode known, docking
could now be leveraged to inform the design of analogs.
One example is shown in Figure 3. Here, the published X-
ray crystal structure of the JAK inhibitor tofacitinib (20)20
overlaid on the X-ray structure of 6 (Figure 1) suggested
that an acetonitrile appended to compound 7 might
overlay with the acetonitrile moiety of tofacitinib. Indeed,
docking this hypothetical compound 21 into the tofacitinib
X-ray structure indicated a fairly close overlay of the
nitriles in the two compounds, see Figure 3. The reported
SAR of tofacitinib (20) indicates that its nitrile moiety
contributes significantly to the binding affinity.4 Thus, we
synthesized and tested compound 21, and it afforded a
three-fold improvement in cellular potency relative to 7
with a concomitant increase in LLE from 2.6 to 4.4, see
Table 4. Because the nitrile of 21 appears to be slightly
further from the P-loop than the corresponding nitrile in
20 (see Figure 3), a one-carbon homolog, 22, was prepared
to try to reach closer towards the P-loop, but potency was
essentially unchanged. On the other hand, contracting the
nitrile back to the cyclohexane caused a large drop in
activity because this compound (23) cannot reach the P-
loop and the nitrile does not add other interactions with
the kinase. The desolvation penalty of the nitrile may
explain the six-fold drop in potency relative to the
unsubstituted cyclohexane 7.
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The only unsubstituted carbon atom of the core scaffold
offered a vector towards the solvent front and the
selectivity-modulating residue Glu 966, and this position
was targeted next. A hydroxymethyl substituent (17) was
tolerated: no major difference in cellular and enzymatic
potency compared to 7 was observed, and LLE and
solubility were improved. Interestingly, 17 exhibited a
two-fold improvement in selectivity over JAK2 compared
to 7, suggesting a productive interaction with Glu 966 of
JAK1 or a disruption of the water network connecting the
ligand to Asp 939 in JAK2. Thus, we noted that a
hydroxymethyl substituent might be useful later in the
optimization campaign, should we need to solve issues
resulting from high log D, e.g. low solubility or poor
metabolic stability. Hydroxyethyl (18) and cyano (19)
analogs were less potent and were not considered further.
To better understand the postulated interaction
between the nitrile of 21 and the P-loop of the kinase, an
X-ray crystal structure of 21 in complex with the JAK1
kinase domain was solved, see Figure 4. The core scaffold
and cyclohexane moieties in the crystal structure overlaid
perfectly with the predicted binding mode from docking
(Figure 3). However, the nitrile functional group was not
defined by the electron density suggesting that it adopts
several conformations. Thus, it is unlikely that it engages
the P-loop like the nitrile in tofacitinib (20), which has a
clearly defined electron density (PDB entry 3EYG). With
this result in hand we set out to investigate other options
for reaching the P-loop and forming productive
interactions with it. To this end, compounds with amide,
ester and sulfonamide linkers connecting the cyclohexane
moiety with small cycloalkyl and fluorocycloalkyl moieties
were synthesized (Table 4). The cyclohexyl carboxamide
24 possessed modest JAK1 potency, slightly lower than for
7, while the potency of reversed amide 25 was much
lower. Increasing the steric bulk of 24 to cyclopentane 26
only afforded a two-fold improvement. Thus, 26 was no
better than 7 with respect to JAK1 potency. Gratifyingly,
introduction of a gem-difluoro motif to 24 greatly
improved JAK1 potency, particularly in the S enantiomer
27, but the less active R enantiomer 28 also showed a
slight improvement relative to 24.
Figure 3. Docking pose of 21 (cyan) overlaid with the X-ray
structures of 6 (green) bound to JAK2 (PDB entry 6TPD) and
tofacitinib 20 (yellow) bound to JAK1 (PDB entry 3EYG). The
protein from PDB entry 3EYG is shown, and this was used to
dock 21. The hinge loop is colored purple, the P-loop is
colored orange, the DFG loop is colored blue and the αC helix
is colored red. Hydrogen bonds are shown as green dotted
lines.
Having optimized the lipophilic substituents of the core,
we proceeded to grow the prototypical analog 7 towards
the P-loop. In the spirit of fragment-based drug design –
start small and make every added atom count – small
substituents with balanced polarity were added to the
cyclohexyl analog 7, see Table 4. To direct the substituent
towards the P-loop while avoiding the complication of
fluctuation
between
two
axial/equatorial
chair
conformers, only trans-configured substituents at the 4-
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Figure 4. X-ray crystal structure of compound 21 (cyan) in
complex with JAK1 (PDB entry 6TPE, 2.9 Å resolution). The
ligand electron density is shown in blue mesh. Hydrogen
bonds are shown as green dotted lines. The nitrile functional
group is modelled in an arbitrary rotameric state, because no
electron density was observed for it.
Unfortunately, the high potency of 27 in the enzymatic
assay did not translate to the cellular STAT6 assay. For the
compounds discussed up to this point, on average a ca. 30-
fold drop in potency was observed going from enzymatic
to cellular assays, largely consistent with the different ATP
concentrations in these assays as discussed above.
However, for compound 27, the drop was close to 3,000-
fold, suggesting a permeability limitation for this analog.
We suspected that the polar nature of the scaffold core
together with the additional hydrogen bond donor of 27
might be responsible for the putative permeability
limitation. To remove one hydrogen bond donor, the ester
analog 29 was prepared and tested. This compound
retained most of the potency in the JAK1 assay and showed
a potency in the STAT6 assay consistent with the expected
30-fold drop discussed above. However, this compound
had poor solubility and was extremely rapidly degraded in
human liver microsomes, presumably due to ester
hydrolysis. Replacing the amide linker with a sulfonamide
was briefly investigated (compounds 30 and 31) but did
not result in an improvement in potency or lower drop in
activity from biochemical to cellular assays. For
completeness, we tested the building blocks 32 and 33
used to prepare compounds 24 and 26-31, but they were
much less potent than 7. Very large desolvation penalties
of the hydroxy and amino groups of these compounds in
the absence of productive interactions with the protein
may explain this result.
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Table 3. Optimization of the lipophilic substituents.a
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STAT6 STAT5 JAK1 JAK2 JAK3 TYK2
IC50 IC50 IC50 IC50 IC50 IC50
(µM) (µM) (µM) (µM) (µM) (µM)
Aq. sol.
(µM)b
STAT6
LLE
b
Cpd
R1
R2
R3
H
Log D7.4
6
Me
Ph
>50
n.d.
2.2
4.2b
2.6b
3.9b
2.2
>90
4
<2.1
3.5c
10
11
12
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7
Me
cyclohexyl
cyclopentyl
H
3.7
>40
0.13
1.3
0.55
0.67
2.8
2.6
4.1c
8
9
Me
Me
H
H
4.3
3.4
>40
>40
0.072
0.12
1.3
0.67
0.48
0.83
1.1
2.2
2.2
39
16
3.2
0.57
3.3
10
Me
H
1.3
8.9
0.040 0.34
0.11
0.70
3.0
8
2.9
rac-
11
12
Me
Me
H
H
1.0
>40
22
0.044 0.38
0.046 0.43
0.18
0.24
0.15
0.43
2.7
2.8
33
64
3.3
3.3
rac-
rac-
0.85
13
Me
H
0.89
9.1
0.054 0.33 0.065 0.22
3.3
8
2.7
rac-
14
15
16
17
18
19
H
Et
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
H
>50
2.5
1.8
4.8
8.5
20b
n.d.
>40
>40
>40
>40
>40
15
35b
2.7
1.3
2.8
3.9
17
12b
1.6
24b
n.d.
1.6b
0.65
3.4
n.d.
3.0
3.1
2.1
2.6
n.d.
n.d.
n.d.
n.d.
100
11
n.d.
2.6
2.6
3.2
2.5
n.d.
H
H
0.46
0.50
0.14
0.49
2.1
c-Pr
Me
Me
Me
0.98
1.4
CH2OH
CH2CH2OH
CN
0.99
5.4
11
n.d.
aSTAT IC50 values were determined in a cellular reporter gene assay and JAK IC50 values were determined in a TR-FRET
biochemical assay for kinase inhibition with [ATP] = Km. All values represent the geometric mean of at least two independent
experiments unless otherwise noted, see Supporting information for details. Log D7.4 was determined using a shake flask assay and
aqueous solubility was determined using an HPLC assay from DMSO stock solutions of test compounds diluted in phosphate buffer
at pH 7.4. bOnly one determination was made. cLLE based on JAK1 IC50.
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Journal of Medicinal Chemistry
Table 4. Addition of polar substituents projecting towards the P-loop.a
1
2
3
4
5
6
7
8
9
STAT6
IC50 (µM) IC50 (µM)
STAT5
JAK1 IC50 JAK2 IC50 JAK3 IC50 TYK2 IC50
Aq. sol.
(µM)b
STAT6
LLE
b
Cpd
R
Log D7.4
(µM)
0.040
0.032
0.73
(µM)
0.36
0.16
5.1
(µM)
0.27
0.28
11
(µM)
0.13
n.d.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21
22
23
CH2CN
CH2CH2CN
CN
1.2
1.2
n.d.
>40
13
1.5
1.8
n.d.
59
18
4.4
4.1
n.d.
n.d.
n.d.
n.d.
24
25
>14
>40
>14
>40
0.22
2.0
2.1
9.4
7.0
1.1
2.8b
14b
1.2
22
<3.7
n.d.
n.d.
n.d.
26
27
9.1
1.7
>30
>7
0.11
1.6
4.8
6.8b
n.d.
1.3
n.d.
43
n.d.
4.5
0.0064
0.12
0.48
0.22b
28
29
>40
>40
>25
0.078
0.014
1.7
2.7
2.7b
n.d.
1.3
29
3
<3.1
n.d.
0.42
0.12
0.091
n.d.
30
31
n.d.
3.2
n.d.
>10
0.40
2.5
6.6
1.7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.040
0.57
32
33
NH2
OH
>40
20
>40
>40
0.82
0.49
18
11
16b
4.2
n.d.
0.8
n.d.
>85
n.d.
3.8
4.6
1.7
aSTAT IC50 values were determined in a cellular reporter gene assay and JAK IC50 values were determined in a TR-FRET
biochemical assay for kinase inhibition with [ATP] = Km. All values represent the geometric mean of at least two independent
experiments unless otherwise noted, see Supporting information for details. Log D7.4 was determined using a shake flask assay and
aqueous solubility was determined using an HPLC assay from DMSO stock solutions of test compounds diluted in phosphate buffer
at pH 7.4. bOnly one determination was made.
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Journal of Medicinal Chemistry
Page 8 of 33
colored orange, and the DFG loop is colored blue. Hydrogen
To confirm the binding mode of 27 and understand the
details of its interaction with the JAK1 kinase domain, an X-
ray crystal structure of this compound in complex with
JAK1 was solved, see Figure 5. The pyrazolopyridone core
makes the same interactions with the hinge loop residues
as do compounds 6 and 21. The higher resolution of this X-
ray structure allows the water network to be observed. A
water-mediated contact with Arg 879 and Pro 960 is
observed, but no interaction with Glu 966 is seen.
Moreover, the two protein molecules in the
crystallographic unit cell show different side chain
conformations for this residue. In contrast, the Arg 879
side chain has a very similar conformation in both protein
molecules. This suggests that the JAK1 selectivity of the
scaffold derives from the interaction with Arg 879. This
residue is Gln in JAK2, Ser in JAK3, and Arg in TYK2.
However, the combination of Phe 958 (Tyr in JAK2, JAK3
and TYK2) and Arg 879 is unique to JAK1. Comparing the
binding modes of 6 in JAK2 and 27 in JAK1 may give a clue
to the reason for the JAK1 selectivity of the scaffold: The
carbonyl oxygen of 6 (Figure 1) makes a water-mediated
contact with the JAK2 Tyr 931 hydroxyl while 27 makes a
very similar contact with the JAK1 Arg 879 side chain. If
the latter interaction is more favorable, that would explain
the preference of the scaffold for JAK1 binding.
1
2
3
4
5
6
7
8
bonds are shown as green dotted lines. Side chain
conformations from the second protein molecule in the
crystallographic unit cell are shown in cyan for Arg 879 and
Glu 966.
The gem-difluoro moiety of 27 does contact the P-loop,
however the reason for the 34-fold increase in potency
observed when adding these two fluorine atoms to 24 is
not obvious from visual inspection of the X-ray structure. A
hydrogen bonding interaction between the amide NH and
the backbone carbonyl of the Asp 1021 residue of the DFG
loop is present, but it probably contributes little to the
binding affinity, considering that the ester analog 29 is
almost as potent. Thus, to improve permeability by
removing one hydrogen bond donor, the amide NH of 27
was replaced by CH2 and a tertiary amide nitrogen was
introduced on the other side of the carbonyl, see Table 5.
With this change, the direct analog of 27 would be an
aziridine amide, which would be expected to have poor
chemical stability. Thus, we targeted azetidine, pyrrolidine
and piperidine amides instead.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To maintain lipophilicity under control, the nitrile
functional group was re-introduced as a P-loop interaction
moiety. Both azetidine 34 and pyrrolidine 35 showed
respectable levels of JAK1 potency, but unfortunately
cellular potency was not achieved. It is worth noting that
35 is racemic, and the pure enantiomers were not
prepared because of the lack of sufficient cellular activity.
Both compounds possessed good aqueous solubility and
were stable towards human liver microsomes. To improve
cellular potency at the expense of increasing log D, the
nitrile was replaced by more lipophilic substituents, e.g.
benzyl (36), phenyl (37) and gem-dimethyl (38). Although
these compounds showed excellent JAK1 potency and
cellular potency in the STAT6 assay, consistent with the
expected 30-fold drop, solubility and in particular
metabolic stability were very poor. Clearly, the polar
nature of the core scaffold limits the amount of additional
polarity that can be tolerated in the compounds without
losing cellular potency. On the other hand, compounds
with sufficient lipophilicity to achieve cellular activity
showed poor solubility and metabolic stability.
Other strategies for achieving high selectivity for JAK1
have been pursued in the literature, and a large body of
work by many groups has been reviewed.18,19 In particular,
targeting Glu 966 in JAK1 has been shown to be a viable
approach, although other amino acid differences between
the JAK family subtype enzymes have also been exploited.
For a comprehensive discussion of these approaches, see
Reference 19. Alternatively, it has been suggested that the
P-loop is more flexible in JAK1 than in JAK2. Extending the
ligand towards the P-loop thus causes clash with this
moiety in JAK2, thus rendering compounds JAK1-
selective.12 Although, as noted above, interaction with Glu
966 seems unlikely to contribute to the selectivity of the
series reported herein, a contribution from the differential
P-loop flexibility may well augment the inherent JAK1 bias
of compound 27.
Figure 5. X-ray crystal structure of compound 27 in complex
with JAK1. (PDB entry 6TPF, 2.3 Å resolution). The ligand 27
is colored green, the hinge loop is colored purple, the P-loop is
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1
Journal of Medicinal Chemistry
Table 5. Tertiary amides projecting towards the P-loop.a
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
STAT6
IC50
(µM)
STAT5
IC50
(µM)
JAK1
IC50
(µM)
JAK2
IC50
(µM)
JAK3
IC50
(µM)
Aq. sol.
(µM)b
HLM Clapp
STAT6
LLE
b
Cpd
X
Log D7.4
(ml/min/kg)b
34
>30
14
>30
>45
0.013
0.011
0.064
0.089
0.097
0.15
0.5
540
85
<8
<8
<4.0
4.2
35
36
0.7
3.0
rac-
0.25
5.1
0.0030
0.015
0.088
2.4
87
3.6
37
38
0.11
0.11
2.4
3.4
0.0021
0.0034
0.019
0.032
0.063
0.057
3.0
2.9
n.d.
n.d.
75
70
4.0
4.0
rac-
aSTAT IC50 values were determined in a cellular reporter gene assay and JAK IC50 values were determined in a TR-FRET
biochemical assay for kinase inhibition with [ATP] = Km. All values represent the geometric mean of at least two independent
experiments, see Supporting information for details. Log D7.4 was determined using a shake flask assay and aqueous solubility was
determined using an HPLC assay from DMSO stock solutions of test compounds diluted in phosphate buffer at pH 7.4. HLM Clapp is
the intrinsic clearance in human liver microsomes. bOnly one determination was made.
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Table 6. Norbornane nitriles compared to benchmark pan-JAK and selective JAK1 inhibitors.a
1
2
3
STAT6
STAT5 JAK1 IC50 JAK2 IC50 JAK3 IC50 TYK2 IC50
Aq. sol.
HLM Clapp STAT6
b
Compound
Log D7.4
IC50 (µM) IC50 (µM)
(µM)
(µM)
(µM)
(µM)
(µM)b (ml/min/kg)b LLE
4
5
6
7
8
39
40
41
42
0.77
0.077
1.2
15
2.8
24
0.011
0.093
0.42
0.18
n.d.
n.d.
89
n.d.
<8
<8
<8
n.d.
5.6
4.4
4.0
0.0010
0.030
0.047
0.012
0.15
0.36
0.088
0.22
0.97
0.020
0.22
0.33
1.5
1.5
1.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>92
290
2.7
>43
0.087
0.12
0.0031
0.0027
0.0015
0.072
1.9
n.d.
<8
5.2
Tofacitinib (20)
0.18
1.4
0.0051
0.036
0.48
0.16
2.0
n.d.
<8
4.7
Abrocitinib
aCompounds 39 and 40 are enantiomers of unknown absolute configurations, as are compounds 41 and 42. STAT IC50 values
were determined in a cellular reporter gene assay and JAK IC50 values were determined in a TR-FRET biochemical assay for kinase
inhibition with [ATP] = Km. All values represent the geometric mean of at least two independent experiments, see Supporting
information for details. Log D7.4 was determined using a shake flask assay and aqueous solubility was determined using an HPLC
assay from DMSO stock solutions of test compounds diluted in phosphate buffer at pH 7.4. HLM Clapp is the intrinsic clearance in
human liver microsomes. bOnly one determination was made.
To overcome these issues, we set out to transfer the best
P-loop-targeting substituents discovered above back to the
norbornane scaffold (compounds 11 and 12). Thus, we
hoped to significantly improve the potency of compound
21 without compromising solubility and metabolic
stability. Although no difference in potency between the
unsubstituted exo (11) and endo (12) norbornanes was
observed, once a second substituent is appended to the
norbornane this must be directed towards the P-loop to
achieve the desired potency boost. Although docking could
not conclusively direct our efforts towards a single regio-
and stereoisomer, it appeared unlikely that the core hinge
binder could occupy an endo vector. To closely mimic the
arrangement of the core and nitrile of 21, the four
stereoisomers 39-42 were targeted for synthesis, see
Table 6. The compounds were prepared as racemates and
afforded high enzymatic and cellular potency combined
with high metabolic stability in human liver microsomes
and good solubility. Importantly, 40 showed excellent
selectivity for JAK1 over JAK2 in both assay formats: In the
enzymatic assays with ATP at Km, a 12-fold selectivity was
found, and in the STAT cellular assays, a 36-fold selectivity
was observed. The enantiomer of 40, i.e. compound 39,
and the enantiomeric pair of endo,exo diastereomers 41
and 42 all showed much lower potency.
the
enantiomers
were
separated
by
chiral
chromatography. The absolute configurations of these
compounds were not determined. Gratifyingly, one
enantiomer of the exo,exo diastereomer, compound 40,
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Journal of Medicinal Chemistry
approach. Throughout the lead generation campaign, a
1
2
3
4
5
6
7
8
strict focus was kept on adding the minimum amount of
molecular weight and lipophilicity at every step. During
the progression from 1 to 40, the molecular weight
increased from 199 to 282, the number of non-hydrogen
atoms increased from 14 to 21, and JAK1 potency
increased 16,000-fold (4.2 log units). The resulting lead
compound 40 displayed potency in the cellular STAT6
assay below 100 nM, 36-fold selectivity over JAK2
signalling as measured in the cellular STAT assays, and
physicochemical properties that satisfied our lead
compound criteria.
9
10
11
12
13
14
CHEMISTRY
To support the fragment-based lead generation
campaign reported above, a flexible and convenient
synthetic route to the 2,7-dihydropyrazolo[3,4-b]pyridin-
6-one scaffold was needed. Synthetic routes to this scaffold
have been reviewed22 and our initial attempt at re-
synthesis of compound 1 was based on the condensation
between 3-amino-5-methylpyrazole (43c) and ethyl
difluoroacetoacetate (49) in acetic acid, see Scheme 1.
Based on the reported regiochemical outcome of reactions
of 3-aminopyrazoles (43a and 43b) and ethyl
trifluoroacetoacetate (44) that yielded 45 and 46
respectively,23-25 we expected to obtain 1. However, the
product of this reaction was not identical to the
commercial sample of 1 and was inactive in the JAK1
assay. Based on 1H-13C HMBC NMR and predicted 13C
chemical shifts we assigned structure 50 to the product,
see supporting information for details. This result is
contrary to the reported result that only when the 4-
position was blocked was this regiochemistry observed,
e.g. pyrazole 47 was reported to give 48 when reacted
with 44.23 To avoid the formation of 50, the pyrazole NH
position was protected with a t-Bu group.26 Reaction of the
protected pyrazole 51 with 49 afforded 55, which upon
Figure 6. Rat PK study with compound 40. T = 0.3 h, Cl = 53
½
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mL/min/kg, Vβ
concentration on a logarithmic scale vs. time.
=
1.2 L/kg,
F
=
14%. Inset: Blood
To benchmark the selectivity profile of compound 40,
we assayed the pan-JAK inhibitor tofacitinib (20)4 and the
selective JAK1 inhibitor abrocitinib12 that recently
completed Phase III clinical trials in atopic dermatitis.13 As
expected, tofacitinib (20) showed no selectivity for JAK1
over JAK2, nor did it show any selectivity in the cellular
STAT reporter gene assays. Abrocitinib on the other hand
showed a 7-fold selectivity for JAK1 over JAK2, and 8-fold
selectivity in the cellular STAT reporter gene assays.
Compound 40 was selected for further profiling to
assess its potential as a lead compound. In a panel of 50
kinases broadly covering the kinome it only inhibited one
kinase more than 30% (FGFR1: 32%) when tested at 1 µM.
All assays in the panel were conducted with 1 mM ATP, see
Supporting Information for details. Compound 40 did not
inhibit the cytochrome P450 enzymes Cyp1A2, 2C9, 2C19,
2D6, or 3A4 up to 25 µM and showed no effect in a hERG
binding assay at 30 µM. Good in vitro metabolic stability
was found in rat liver microsomes (Clapp < 15 mL/min/kg)
and rat hepatocytes (Clapp = 25 mL/min/kg), and rat
plasma protein binding was moderate (fu = 22%). A low
permeability (Papp = 0.7 × 10-6 cm/s) and a high efflux ratio
= 40 was observed in the Caco-2 assay suggesting a
potential permeability limitation for oral absorption from
the gut. Thus, compound 40 was tested in an early
discovery rat PK study (only one animal for each route of
administration), see Figure 6, and was found to exhibit
high clearance, short half-life and fairly low oral
bioavailability (F = 14%). Assuming no elimination in the
gut, the fraction of dose absorbed (Fabs) was calculated to
approx. 41%. This confirms that 40 has a permeability-
limited absorption as suggested by the Caco-2 data. The in
vitro to in vivo extrapolation using the well stirred model
adjusted for protein binding was poor: The predicted in
vivo unbound clearance based on rat microsomal or
hepatocyte intrinsic clearance was <2.0 and 10
mL/min/kg, respectively, indicating extrahepatic clearance
mechanisms.
deprotection with TFA yielded
1 identical to the
commercial sample and with the same JAK1 potency.
Compounds 3, 6 and 14 were similarly prepared starting
from the appropriate pyrazoles (51 and 52) and β-
ketoesters (49, 53 and 54).
Although a regiochemically unambiguous route was now
available, the harsh conditions needed for both the cyclo-
condensation and the deprotection steps, and the necessity
of constructing β-keto ester starting materials lead us to
explore alternatives. A three-component reaction between
an aminopyrazole (43), an aldehyde (59) and Meldrum’s
acid (60) to afford intermediate 6127,28 appeared
attractive, see Scheme 2. When aromatic α-keto aldehydes
were employed, air oxidation of 61 in either acidic or basic
media furnished the desired 2,7-dihydropyrazolo[3,4-
b]pyridin-6-ones.28 In our hands, air oxidation of 61 was
inefficient with aliphatic R2 groups, but oxidation with
DDQ readily afforded the desired products under mild
conditions. This method allowed access to the target
compounds in two steps from readily available starting
materials and was used to prepare all final compounds
reported herein except 1-6, 14 and 19. In many cases we
observed the formation of a by-product (61p) in the three-
component reaction. Compound 61p is presumably
CONCLUSIONS
A fragment screening hit 1 was developed into lead
compound 40 using a structure-guided fragment growing
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Journal of Medicinal Chemistry
Page 12 of 33
formed from in situ generated acetone deriving from diastereomeric ratio. Hydrogenation of the olefin thus
1
2
3
4
5
6
7
8
Meldrum’s acid (60). The formation of 61p from 43a, 60
and acetone in refluxing methanol has been reported in the
literature.29 In some cases, 61p was the only product
formed when the reaction was carried out in ethanol.
Switching solvent to pyridine with 10 mol% piperidine for
the condensation of aldehyde 59 with Meldrum’s acid 60
followed by addition of pyrazole 43c and ethanol allowed
formation of the desired product in those problematic
cases (61c and 61n).
afforded a ca. 1:1 mixture of racemic exo,exo-norbornane
and endo,exo-norbornane diastereomers. This mixture was
subjected to chiral SFC separation to afford the four
stereoisomers
39-42
individually.
Relative
stereochemistry was assigned from 1H NMR NOE (see
Supporting information for details) but no attempt to
assign absolute configuration was made. Thus, although 39
and 40 are enantiomers, the assignment of absolute
configuration shown in Scheme 9 is arbitrary. This also
holds for 41 and 42.
9
To access compounds 17-19 with the carbonyl α-carbon
functionalized, two methods were used. Compounds 17
and 18 were prepared from intermediate 61a by
simultaneous chlorination of the amide functionality and
Vilsmeier-Haack formylation of the α-carbon as shown in
Scheme 3. The resulting chloride 62 was converted to the
corresponding methoxide 63, which was reduced to
alcohol 64 without isolation and demethylated to yield 17.
Alternatively, methoxide 63 was subjected to a Wittig
reaction yielding alkene 65. Hydroboration-oxidation then
afforded alcohol 66 which was demethylated to give 18. To
prepare compound 19, a cyclocondensation between
aminopyrazole 43c and malononitrile 67 was employed to
yield intermediate 68, see Scheme 4. Oxidation by
bromination followed by elimination of HBr afforded
compound 19.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Although most of the aldehydes 59 (see Scheme 2)
required to prepare the final compounds were
commercially available, 59d-f, 59j and 59o needed to be
synthesized. The routes used to access these intermediates
are shown in schemes 10-13. Starting from the known
ester 79,30 the racemic aldehyde 59d was prepared by
LiAlH4 reduction to furnish alcohol 80, which was oxidized
with Dess-Martin periodinane (Scheme 10). The
norbornanecarbaldehydes 59e and 59f were prepared
from commercially available norbornene carboxylic acid
81 (exo/endo mixture, predominantly endo) as shown in
Scheme 11.31 After esterification with isopropanol, the exo
and endo esters 82 and 83 could be separated by silica gel
column chromatography. The two isomers were separately
treated with LiAlH4 to afford alcohols 84 and 86, which
were hydrogenated to afford norbornane methanols 85
and 87. Swern oxidation furnished the required aldehyde
building blocks 59e and 59f. To synthesize building block
59j the route in Scheme 12 was employed. Mesylation of
alcohol 88 afforded intermediate 89, which was treated
with KCN to yield nitrile 90. Chemoselective reduction of
the ester moiety with LiBH4 gave alcohol 91, which upon
Swern oxidation furnished aldehyde 59j. The route to
aldehyde 59o is outlined in Scheme 13. Analogous to the
preparation of esters 82 and 83 (Scheme 11), the
corresponding benzyl esters 92 and 93 were prepared
from norbornene carboxylic acid 81 and separated using
silica gel column chromatography. The exo isomer 93 was
subjected to hydroboration/oxidation to afford a separable
mixture of regioisomers 94 and 95.32 Dess-Martin
periodinane oxidation of the desired regioisomer 94
afforded ketone 96, which was converted to alkene 97 by
means of a HWE olefination. Chemoselective reduction of
the ester moiety with LiBH4 gave alcohol 98, which upon
Swern oxidation furnished aldehyde 59o.
Nitrile 22 was prepared by homologation of the nitrile
intermediate 61j, see Scheme 5. Protection of the NH
protons with 4-methoxybenzyl chloride gave 69, which
1
appeared to be a single regioisomer according to the H
NMR spectrum. The regiochemistry of the PMB groups was
not investigated. Structure 69 and the following structures
show one plausible regioisomer, but no attempt to
determine if this is the correct one was made. Oxidation of
69 with DDQ gave 70, which upon reduction with DIBAL-H
yielded aldehyde 71. This was further reduced to the
alcohol 72, mesylated to yield 73, and treated with NaCN
to afford the homologated nitrile 74. Deprotection in
refluxing TFA finally yielded compound 22.
The syntheses of the amides 24, 26-28, sulfonamides 30
and 31, and ester 29 are outlined in Scheme 6. The amides
were prepared by HATU coupling of amine 32 with the
respective acids, and the sulfonamides were prepared by
reacting 32 with the respective sulfonyl chlorides. The
ester 29 was prepared by HATU/DMAP coupling of alcohol
33 with (1S)-2,2-difluorocyclopropanecarboxylic acid. To
prepare the inverted amide 25, the route in Scheme 7 was
used. Protection of the ester intermediate 61n afforded 75,
which was oxidized with DDQ to afford 76, which
1
appeared to be a single regioisomer according to the H
NMR spectrum (see the discussion of 69 above).
Hydrolysis of the methyl ester yielded acid 77 and amide
coupling with HATU followed by deprotection gave
compound 25. Amides 34-38 were prepared in a library
format from acid intermediate 78 and the respective
amines, see Scheme 8.
The acetonitrile norbornanes 39-42 were prepared from
the common intermediate 39a, see Scheme 9. Compound
39a is a mixture of four stereoisomers: it consists of the
enantiomeric pairs of the (Z)-olefin/exo-norbornane and
(E)-olefin/exo-norbornane diastereomers in a ca. 1:1
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Journal of Medicinal Chemistry
Scheme 1. First route to the pyrazolopyridone scaffolda
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (a) AcOH, reflux, 76−82%; (b)
AcOH, reflux, 88%; (c) AcOH, 135 °C (microwave heating),
23%; (d) AcOH, 110 °C, 12−98%; (e) TFA, 70 °C, 16−68%.
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Scheme 2. Three-component reaction route to the pyrazolopyridone scaffolda
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (a) EtOH, 60−70 °C or reflux, 23−86%; (b) DDQ, 1,4-dioxane, 100 °C, 12−87%; (c) 4 M HCl in 1,4-
dioxane, rt, 86%; (d) 1 M TBAF in THF, DCM, 40 °C, 40%; (e) H2, Pt/C, MeOH, rt.
Scheme 3. Synthesis of compounds 17 and 18a
aReagents and conditions: (a) POCl3, DMF, 100 °C, then MnO2, MeCN, rt, 15%; (b) 4.4 M NaOMe in MeOH, 120 °C, microwave
heating, then NaBH4, MeOH, rt, 81%; (c) 5 M HBr in AcOH, 40 °C, 23%; (d) 4.4 M NaOMe in MeOH, 120 °C, microwave heating, 99%;
(e) PPh3MeBr, 2.5 M n-BuLi, THF, 0 °C−rt, 91%; (f) 0.5 M 9-BBN, THF, then H2O2, NaOH, rt, 47%; (g) Pyridine hydrochloride, neat,
90 °C, 4.4%.
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Scheme 4. Synthesis of compound 19a
1
2
3
4
5
6
7
8
9
aReagents and conditions: (a) MeOH, 60 °C, 47%; (b) NBS, DBU, MeCN, rt, 9.1%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5. Synthesis of compound 22a
aReagents and conditions: (a) Cs2CO3, PMBCl, DMF, rt, 53%; (b) DDQ, 1,4-dioxane, 100 °C, 50%; (c) 1.0 M DIBAL-H, toluene, 0 °C,
82%; (d) NaBH4, THF, rt, 71%; (e) MsCl, Et3N, DCM, rt, 69%; (f) NaCN, DMF, 90 °C, 66%; (g) TFA, reflux, 25%.
Scheme 6. Synthesis of compounds 24 and 26-31a
aReagents and conditions: (a) RCO2H, HATU, DIPEA, DMF, rt, library format; (b) RSO2Cl, DIPEA, DMSO, rt, library format; (c) (1S)-
2,2-difluorocyclopropanecarboxylic acid, HATU, DMAP, DMF, rt, 13%.
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1
2
Scheme 7. Synthesis of compound 25a
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (a) Cs2CO3, PMBCl, DMF, rt, 92%; (b) DDQ, 1,4-dioxane, 100 °C, 87%; (c) LiOH, H2O, THF, 40 °C, 50%;
(d) cyclopropylamine, HATU, DIPEA, DMF, rt, then TFA, 90 °C, 9%.
Scheme 8. Synthesis of compounds 34-38a
Scheme 10. Synthesis of aldehyde building block 59da
aReagents and conditions: (a) LiAlH4, THF, −30 °C, 83%; (b)
Dess-Martin periodinane, NaHCO3, DCM, rt, 30%.
aReagents and conditions: (a) Conc. HCl, 1,4-dioxane, 100
°C, 83%; (b) Amine, HATU, DIPEA, DMF, rt, library format.
Scheme 9. Synthesis of compounds 39-42a
aReagents and conditions: (a) H2, Pt/C, MeOH, rt, then chiral
SFC separation of stereoisomers. Compound 39a is a mixture
of E and Z isomers and the E and Z isomers are racemic.
Compounds 39 and 40 are enantiomers of unknown absolute
configurations, as are compounds 41 and 42.
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1
Journal of Medicinal Chemistry
2
3
Scheme 11. Synthesis of aldehyde building blocks 59e and 59fa
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (a) 2-Propanol, EDCI, DMAP, 1,2-dichloroethane, rt, 14% (82), 55% (83); (b) LiAlH4, THF, 40 °C; (c)
H2, Pd/C, EtOAc, rt, 96%; (d) (COCl)2, DMSO, Et3N, DCM, −78 °C, 68%.
Scheme 12. Synthesis of aldehyde building block 59ja
aReagents and conditions: (a) MsCl, Et3N, DCM, 0 °C, 99%; (b) KCN, DMSO, rt, 81%; (c) LiBH4, THF, 40 °C, 84%; (d) (COCl)2,
DMSO, Et3N, DCM, −78 °C, 75%.
Scheme 13. Synthesis of aldehyde building block 59oa
aReagents and conditions: (a) Benzyl alcohol, DCC, DMAP, DCM, rt, 26% (92), 10% (93); (b) BH3 . THF, THF, −5 °C, then KH2PO4,
H2O2, −5 °C, 25% (94), 25% (95); (c) Dess-Martin periodinane, DCM, rt, 54%; (d) NCCH2PO(OEt)2, t-BuOK, THF, rt, 68%; (e) LiBH4,
THF, rt, 87%; (f) Dess-Martin periodinane, DCM, rt, 94%.
The housing and husbandry of animals was undertaken
in compliance with the European Committee Directive
2010/63/EU and performed under permit 2015-15-0201-
00595 from the Danish authorities. Our animal welfare
body inspects all experiments in our animal facility on a
regular basis in order to secure implementation of the 3Rs
in all experimental procedures.
EXPERIMENTAL
General biology experimental procedures are detailed in
the supporting information. Most of the IC50 values
reported in Tables 1 and 3-6 are geometric means of at
least three independent experiments. The corresponding
pIC50 values, their standard errors of the mean (SEM), and
the number of experiments (N) are provided in the
Molecular formula strings of the Supporting information.
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Reagents and solvents were commercially available and
with brine, dried over Na2SO4, filtered and concentrated.
The yellow solid residue was dried under high vacuum to
afford 1-tert-butyl-4-(difluoromethyl)-3-methyl-7H-
pyrazolo[3,4-b]pyridin-6-one 55 (263 mg, 98%) that was
used without purification. 1H NMR (300 MHz, CDCl3) δ 9.81
(br s, 1H), 6.74 (t, J = 55.0 Hz, 1H), 6.51 (s, 1H), 2.48 (s, 3H),
1.73 (s, 9H).
1
2
3
4
5
6
7
8
were used without purification unless otherwise noted.
Chromatographic purification was performed using a
Grace REVELERIS system or a Teledyne ISCO CombiFlash
Rf system with pre-packed silica gel cartridges, or
manually using silica gel 60. Mass spectra of intermediates
were recorded using a Shimadzu LCMS2020 system
equipped with a LCMS2020 mass spectrometer operating
in positive electrospray ionization mode. A Chromolith
SpeedROD RP-18e column (50 × 4.6 mm, 2 µm particle
A solution of 1-tert-butyl-4-(difluoromethyl)-3-methyl-
7H-pyrazolo[3,4-b]pyridin-6-one 55 (200 mg, 0.744 mmol)
in TFA (2 mL) was heated at 70 °C for 19 h. After cooling,
volatiles were evaporated and sat. NaHCO3 solution was
added to the residue. The mixture was extracted with
EtOAc (2 × 30 mL) and the combined organic layers were
washed with brine and dried over Na2SO4. Upon
concentration to a volume of ~3 mL a white solid
precipitated. This was collected by filtration, washed with
a small amount of EtOAc, and dried to afford 4-
(difluoromethyl)-3-methyl-2,7-dihydropyrazolo[3,4-
9
size) at 40 °C was used (mobile phase:
A
=
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH3CN/H2O/HCO2H = 10/90/0.05 and B = CH3CN/H2O/
HCO2H = 90/10/0.05; flow rate: 3.0 mL/min; gradient: 0.8
min @ 10% B, 2.7 min gradient (10-95% B), then 0.8min @
95% B). Alternatively, UPLC-MS analyses of intermediates
were performed using a Waters Acquity UPLC system with
a 2.1 × 50 mm Acquity UPLC HSS T3 1.8 µm column and an
Acquity SQ Detector operated in positive ionization
electrospray mode. The mobile phase consisted of 0.1%
formic acid in an aqueous 10 mM ammonium acetate
solution for buffer A and 0.1% formic acid in acetonitrile
for buffer B. A binary gradient (A:B 95:5→5:95) over 1.4
min was used with a flow rate of 1.2 mL/min and the
column temperature was 60 °C. Purities and high
resolution mass spectra of all compounds tested in
biological assays were determined using a Waters Acquity
UPLC system with a 2.1 × 50 mm Acquity UPLC HSS T3 1.8
µm column, a Waters PDA detector, and a Waters XEVO
b]pyridin-6-one 1 (55.3 mg, 35%) as a white solid. 1H NMR
(300 MHz, DMSO-d6) δ 13.05 (br s, 1H), 11.88 (br s, 1H),
7.11 (t, J = 54.2 Hz, 1H), 6.31 (s, 1H), 2.43 (s, 3H). UPLC tR =
1.65 min, Purity UV254 >99%. HRMS (ESI) m/z: [M + H]+
Calcd for C8H8F2N3O 200.0630; Found 200.0638.
4-(Difluoromethyl)-2,7-dihydropyrazolo[3,4-b]pyridin-6-
one (3). Obtained as a white solid (86% in step 1, 16% in
step 2) from 2-tert-butylpyrazol-3-amine 52 (400 mg, 2.9
mmol) and ethyl 4,4-difluoro-3-oxo-butanoate 49 (760 mg,
4.6 mmol) following the experimental procedure described
G2-XS QTof or
a Waters LCT Premier XE mass
1
for 1. H NMR (600 MHz, DMSO-d6) δ 13.34 (br s, 1H),
spectrometer. The same buffers A and B as above were
used, but with a slower gradient (A:B 99:1→1:99 over 4.8
min; 0.7 mL/min; column temp. 30 °C). All compounds
tested in biological assays were ≥95% pure by integration
of peaks in the UV total absorbance chromatogram trace
(240-400 nm) except compounds 17, 37, and 41 that were
12.01 (br s, 1H), 8.11 (s, 1H), 7.07 (t, J = 54.6 Hz, 1H), 6.34
(s, 1H). UPLC tR = 1.53 min, Purity UV254 98%. HRMS (ESI)
m/z: [M + H]+ Calcd for C7H6F2N3O 186.0479; Found
186.0461.
3-Methyl-4-phenyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-
one (6). Obtained as a white solid (47% in step 1, 68% in
step 2) from 2-tert-butyl-5-methyl-pyrazol-3-amine 51
(400 mg, 2.61 mmol) and methyl 3-oxo-3-phenyl-
propanoate 53 (1.01 g, 5.25 mmol) following the
1
94% pure, and compound 34 that was 81% pure. H NMR
spectra were recorded on Bruker instruments at 300, 400,
or 600 MHz with tetramethylsilane as reference.
Attempted synthesis of 4-(difluoromethyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (1).23-25 A solution of
ethyl 4,4-difluoro-3-oxo-butanoate (171 mg, 1.03 mmol)
and 3-methyl-1H-pyrazol-5-amine 43c (100 mg, 1.03
mmol) in acetic acid (1 mL) was heated in a microwave
synthesizer at 135 °C for 30 min. The reaction mixture
partially solidified. Volatiles were evaporated and the
residue was diluted with water (5 mL). The solid was
collected by filtration and washed with water. It was then
dried in vacuo overnight and recrystallized from methanol
to afford 7-(difluoromethyl)-2-methyl-4H-pyrazolo[1,5-
1
experimental procedure described for 1. H NMR (600
MHz, DMSO-d6) δ 12.87 (br s, 1H), 11.59 (br s, 1H), 7.52 –
7.45 (m, 5H), 5.86 (s, 1H), 2.00 (s, 3H). UPLC tR = 1.83 min,
Purity UV254 >99%. HRMS (ESI) m/z: [M + H]+ Calcd for
C13H12N3O 226.0975; Found 226.0961.
4-Cyclohexyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-one
(14). Obtained as a white solid (12% in step 1, 18% in step
2) from 2-tert-butylpyrazol-3-amine 52 (305 mg, 2.19
mmol) and 3-cyclohexyl-3-oxo-propanoate 54 (645 mg,
3.50 mmol) following the experimental procedure
1
1
described for 1. The product was purified by HPLC. H
a]pyrimidin-5-one 50 (48 mg, 23%) as a white solid. H
NMR (600 MHz, DMSO-d6) δ 13.03 (br s, 1H), 11.42 (br s,
1H), 8.19 (s, 1H), 5.86 (s, 1H), 2.68 – 2.58 (m, 1H), 1.88 –
1.76 (m, 4H), 1.74 – 1.68 (m, 1H), 1.49 – 1.36 (m, 4H), 1.33
– 1.22 (m, 1H). UPLC tR = 1.89 min, Purity UV254 > 99%.
HRMS (ESI) m/z: [M + H]+ Calcd for C12H16N3O 218.1288;
Found 218.1287.
NMR (300 MHz, DMSO-d6) δ 12.94 (br s, 1H), 7.03 (t, J =
53.5 Hz, 1H), 6.10 (s, 1H), 6.00 (s, 1H), 2.32 (s, 3H).
4-(Difluoromethyl)-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one (1).26 To a solution of 2-tert-butyl-5-
methyl-pyrazol-3-amine 51 (153 mg, 1.00 mmol) in acetic
acid (1 mL) was added ethyl 4,4-difluoro-3-oxo-butanoate
49 (332 mg, 2.00 mmol) under argon. The resulting pale
yellow solution was stirred at 110 °C for 17 h, cooled to rt,
and volatiles were evaporated. To the residue was added
sat. NaHCO3 solution and the mixture was extracted twice
with EtOAc. The combined organic layers were washed
4-Cyclohexyl-3-methyl-2,7-dihydropyrazolo[3,4-b]pyridin-
6-one (7). A 25 mL round-bottom flask was charged with
Meldrum’s acid 60 (144 mg, 1.00 mmol) and ethanol (3
mL) under argon. To the white suspension was added
cyclohexanecarbaldehyde 59a (0.121 mL, 1.00 mmol). The
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Journal of Medicinal Chemistry
resulting suspension was stirred for 5 min at rt, 3-methyl-
4-(Cyclobutylmethyl)-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one (9). To solution of
1
2
3
4
5
6
7
8
1H-pyrazol-5-amine 43c (97.1 mg, 1.00 mmol) was added,
and the mixture was heated to reflux. Initially, a pale
yellow solution was formed. After 2.5 h at reflux, the
resulting pale yellow suspension was cooled to rt. The
solid was collected by filtration and was washed with
ethanol (2 × 0.4 mL). The filter cake was dried under high
vacuum (freeze dryer) overnight to afford 4-cyclohexyl-3-
methyl-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-6-one
61a (147 mg, 63%) as a white solid.
a
cyclobutaneacetaldehyde 59c (49 mg, 0.50 mmol) in
pyridine (1 mL) was added piperidine (4.9 µL, 0.050
mmol) and Meldrum’s acid 60 (72 mg, 0.50 mmol). The
mixture was stirred at rt for 30 min. To the reaction
mixture was added ethanol (1 mL) and 3-methyl-1H-
pyrazol-5-amine 43c (48 mg, 0.50 mmol) and the mixture
was heated at 70 °C for 1 hour. Volatiles were evaporated
and the residue was purified by chromatography (ISCO
CombiFlash, 12 g silica gel column, heptane:EtOAc 100:0 →
0:100) to afford 4-(cyclobutylmethyl)-3-methyl-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61c (25 mg, 23%)
as a solid.
9
To
a suspension of 4-cyclohexyl-3-methyl-2,4,5,7-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tetrahydropyrazolo[3,4-b]pyridin-6-one 61a (35.0 mg,
0.150 mmol) and 1,4-dioxane (0.35 mL) was added DDQ
(51.1 mg, 0.225 mmol). The mixture was heated at 100 °C
for 2 h and was then cooled to rt over 2 h. Volatiles were
removed by evaporation. To the residue was added sat. aq.
NaHCO3 solution (2 mL) and the mixture was extracted
with DCM/MeOH 9:1 (5 × 3 mL). Because some solid
remained in the water phase, the mixture was filtered. The
organic phases from the extraction and the filter cake were
combined, diluted with DCM/MeOH 7:3 (50 mL) and dried
over Na2SO4. Filtration and evaporation afforded a crude
product which was suspended in ethanol (0.5 mL). The
solid was collected by filtration and the filter cake was
washed with ethanol (4 × 0.15 mL). The filter cake was
then dried under high vacuum (freeze dryer) overnight to
To a suspension of 4-(cyclobutylmethyl)-3-methyl-
2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-6-one 61c (25
mg, 0.11 mmol) in 1,4-dioxane (1 mL) was added DDQ (44
mg, 0.19 mmol) and the mixture was heated at 100 °C for
20 min. After cooling to rt, the precipitated solid was
collected by filtration, dissolved in DMSO and purified by
HPLC to afford 4-(cyclobutylmethyl)-3-methyl-2,7-
1
dihydropyrazolo[3,4-b]pyridin-6-one 9 (3.0 mg, 12%). H
NMR (600 MHz, DMSO-d6) δ 12.70 (br s, 1H), 11.30 (br s,
1H), 5.72 (s, 1H), 2.77 (d, J = 7.3 Hz, 2H), 2.64 – 2.55 (m,
1H), 2.45 (s, 3H), 2.09 – 2.01 (m, 2H), 1.89 – 1.79 (m, 2H),
1.76 – 1.68 (m, 2H). UPLC tR = 1.89 min, Purity UV254 >99%.
HRMS (ESI) m/z: [M + H]+ Calcd for C12H16N3O 218.1288;
Found 218.1308.
afford
4-cyclohexyl-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one 7 (20.9 mg, 57%) as a pale yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ 12.72 (br s, 1H), 11.36 (br s,
1H), 5.79 (s, 1H), 2.82 – 2.67 (m, 1H), 2.46 (s, 3H), 1.93 –
1.77 (m, 4H), 1.73 (d, J = 13.0 Hz, 1H), 1.48 – 1.16 (m, 5H).
UPLC tR = 1.95 min, Purity UV254 96%. HRMS (ESI) m/z: [M
+ H]+ Calcd for C13H18N3O 232.1444; Found 232.1469.
3-Methyl-4-[(1S*,2R*)-2-methylcyclohexyl]-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (10). To a solution of
(1S*,2R*)-2-methylcyclohexanecarbaldehyde 59d (0.600 g,
4.75 mmol) in ethanol (10 mL) was added Meldrum’s acid
60 (0.685 g, 4.75 mmol). The reaction mixture was stirred
at rt for 15 min followed by the addition of 3-methyl-1H-
pyrazol-5-amine 43c (0.369 g, 3.80 mmol) and the
resulting mixture was stirred at 80 °C for 3 h. After cooling
to rt the mixture was concentrated under reduced
pressure. The residue was washed with diethyl ether. The
solid thus obtained was dried under vacuum to afford 3-
methyl-4-[(1S*,2R*)-2-methylcyclohexyl]-2,4,5,7-
4-Cyclopentyl-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one
(8).
To
a
solution
of
cyclopentanecarbaldehyde 59b (150 mg, 1.53 mmol) in
ethanol (2 mL) was added Meldrum’s acid 60 (220 mg,
1.53 mmol) and the mixture was stirred for 15 min at rt.
Another portion of ethanol (3 mL) was added and the
mixture was stirred for a further 10 min at rt. To the
reaction mixture was added 3-methyl-1H-pyrazol-5-amine
43c (148 mg, 1.53 mmol) and the mixture was heated at
70 °C for 1.5 hours during which time a precipitate formed.
The mixture was cooled to rt and the solid was collected by
filtration and dried under high vacuum (freeze dryer) to
tetrahydropyrazolo[3,4-b]pyridin-6-one 61d (0.250 g,
1
27%) as an off-white solid (mixture of diastereomers). H
NMR (400 MHz, CDCl3) δ 11.69 (s, 1H), 10.06 (s, 1H), 2.54
(m, 1H), 2.25 (m, 2H), 2.05 (s, 3H) 1.75 – 1.45 (m, 6H), 1.45
– 1.20 (m, 2H), 0.9 (m, 3H).
afford
4-cyclopentyl-3-methyl-2,4,5,7-
To
a
solution
of
3-methyl-4-[(1S*,2R*)-2-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61b (239 mg,
71%) as a white solid.
methylcyclohexyl]-2,4,5,7-tetrahydropyrazolo[3,4-
b]pyridin-6-one 61d (0.250 g, 1.01 mmol) in 1,4-dioxane
(10 mL) was added DDQ (0.456 g, 1.52 mmol). The
reaction mixture was stirred at 100 °C for 3h. After cooling,
the mixture was concentrated under reduced pressure.
The residue was purified by HPLC to afford 3-methyl-4-
[(1S*,2R*)-2-methylcyclohexyl]-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one 10 (0.040 g, 16%) as an off-white solid. 1H
NMR (500 MHz, DMSO-d6) δ 12.72 (br s, 1H), 11.30 (br s,
1H), 5.65 (s, 1H), 3.01 (br d, J = 11.5 Hz, 1H), 2.15 – 2.04
(m, 1H), 1.87 – 1.56 (m, 4H), 1.54 – 1.31 (m, 4H), 0.72 (d, J
= 7.1 Hz, 3H). UPLC tR = 2.02 min, Purity UV254 >99%.
HRMS (ESI) m/z: [M + H]+ Calcd for C14H19N3O 246.1601;
Found 246.1622.
To
a suspension of 4-cyclopentyl-3-methyl-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61b (25 mg, 0.11
mmol) in 1,4-dioxane (2 mL) was added DDQ (44 mg, 0.19
mmol) and the mixture was heated at 100 °C for 25 min.
After cooling to rt, the formed precipitate was collected by
filtration, dissolved in DMSO and purified by HPLC (acidic
method)
to
afford
4-cyclopentyl-3-methyl-2,7-
1
dihydropyrazolo[3,4-b]pyridin-6-one 8 (7.8 mg, 32%). H
NMR (600 MHz, DMSO-d6) δ 12.69 (br s, 1H), 11.35 (br s,
1H), 5.84 (s, 1H), 3.28 (p, J = 7.7 Hz, 1H), 2.49 (s, 3H), 2.03
– 1.92 (m, 2H), 1.78 – 1.53 (m, 6H). UPLC tR = 1.87 min,
Purity UV254 >99%. HRMS (ESI) m/z: [M + H]+ Calcd for
C12H16N3O 218.1288; Found 218.1347.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
3-Methyl-4-[(1R*,2R*,4S*)-norbornan-2-yl]-2,7-
Page 20 of 33
[(1R*,2S*,4S*)-norbornan-2-yl]-2,7-dihydropyrazolo[3,4-
1
1
2
3
4
5
6
7
8
dihydropyrazolo[3,4-b]pyridin-6-one (11). To a solution of
norbornane-2-exo-carbaldehyde 59e (137 mg, 1.10 mmol)
in ethanol (2 mL) was added Meldrum’s acid 60 (159 mg,
1.10 mmol). The reaction mixture was stirred at rt for 5
min followed by the addition of 3-methyl-1H-pyrazol-5-
amine 43c (107 mg, 1.10 mmol) and the resulting mixture
was stirred at reflux for 4 h. After cooling to rt the mixture
was concentrated under reduced pressure. The residue
was stirred with EtOAc (3 mL) for 30 min. The solid thus
obtained was collected by filtration, washed with EtOAc (2
× 0.5 mL) and dried under vacuum to afford 3-methyl-4-
[(1R*,2R*,4S*)-norbornan-2-yl]-2,4,5,7-
b]pyridin-6-one 12 (29.8 mg, 34%) as a yellow solid. H
NMR (300 MHz, DMSO-d6) δ 12.69 (br s, 1H), 11.34 (br s,
1H), 5.89 (s, 1H), 3.53 – 3.40 (m, 1H), 2.50 (s, 3H), 2.40 –
2.24 (m, 2H), 1.82 – 1.69 (m, 1H), 1.69 – 1.60 (m, 1H), 1.58
– 1.36 (m, 3H), 1.33 – 1.15 (m, 3H). UPLC tR = 1.95 min,
Purity UV254 99%. HRMS (ESI) m/z: [M + H]+ Calcd for
C14H18N3O 244.1444; Found 244.1438.
4-(2-Bicyclo[2.2.2]octanyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (13). To a solution of
bicyclo[2.2.2]octane-2-carbaldehyde 59g (74.3 mg, 0.473
mmol) in ethanol (1.4 mL) was added Meldrum’s acid 60
(68.2 mg, 0.473 mmol). The reaction mixture was stirred at
rt for 5 min followed by the addition of 3-methyl-1H-
pyrazol-5-amine 43c (45.9 mg, 0.473 mmol) and the
resulting mixture was stirred at reflux for 4 h. The
resulting pale yellow suspension was cooled in an ice bath.
The solid was collected by filtration and was washed with
ice cold ethanol (2 × 0.3 mL). Drying under high vacuum
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tetrahydropyrazolo[3,4-b]pyridin-6-one 61e (180 mg,
60%) as a white solid (mixture of diastereomers).
To
a
suspension of 3-methyl-4-[(1R*,2R*,4S*)-
norbornan-2-yl]-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-
6-one 61e (180 mg, 0.66 mmol) in 1,4-dioxane (3 mL) was
added DDQ (255 mg, 1.12 mmol) and the mixture was
heated at 100 °C for 2 h. After cooling to rt the mixture was
concentrated under reduced pressure. The residue was
suspended in sat. NaHCO3 solution (20 mL) and the
mixture was extracted with DCM:MeOH 9:1 (3 × 40 mL).
The combined organic phases were washed with brine (20
mL), dried over Na2SO4, and volatiles were evaporated. The
residue was purified by column chromatography (silica gel
column, DCM:MeOH 96:4 → 94:6) to afford 3-Methyl-4-
[(1R*,2R*,4S*)-norbornan-2-yl]-2,7-dihydropyrazolo[3,4-
afforded
4-(2-bicyclo[2.2.2]octanyl)-3-methyl-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61g (63.3 mg,
49%) as a white solid (mixture of diastereomers).
To a suspension of 4-(2-bicyclo[2.2.2]octanyl)-3-methyl-
2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-6-one 61g (59.0
mg, 0.227 mmol) in 1,4-dioxane (1.0 mL) was added DDQ
(77.5 mg, 0.341 mmol) and the mixture was heated at 100
°C for 1.5 h. After cooling to rt the mixture was
concentrated under reduced pressure. The residue was
dissolved in DCM:MeOH 95:5 (8 mL), washed with sat.
NaHCO3 solution (10 mL) and the aqueous layer was
extracted with DCM:MeOH 95:5 (4 mL). The combined
organic phases were dried over Na2SO4 and volatiles were
evaporated. The residue was purified by column
chromatography (silica gel column, DCM:MeOH 96:4 →
94:6) to afford 4-(2-bicyclo[2.2.2]octanyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one 13 (25.8 mg, 42%)
1
b]pyridin-6-one 11 (39.2 mg, 22%) as a yellow solid. H
NMR (600 MHz, DMSO-d6) δ 12.71 (br s, 1H), 11.30 (br s,
1H), 5.76 (br s, 1H), 2.92 (br s, 1H), 2.49 (s, 3H), 2.32 (br s,
1H), 2.29 (br d, J = 4.1 Hz, 1H), 1.67 – 1.50 (m, 4H), 1.45 –
1.37 (m, 2H), 1.32 – 1.26 (m, 1H), 1.20 – 1.14 (m, 1H).
UPLC tR = 1.97 min, Purity UV254 >99%. HRMS (ESI) m/z:
[M + H]+ Calcd for C14H18N3O 244.1444; Found 244.1427.
3-Methyl-4-[(1R*,2S*,4S*)-norbornan-2-yl]-2,7-
1
dihydropyrazolo[3,4-b]pyridin-6-one (12). To a solution of
norbornane-2-endo-carbaldehyde 59f (107 mg, 0862
mmol) in ethanol (1.5 mL) was added Meldrum’s acid 60
(124 mg, 0.862 mmol). The reaction mixture was stirred at
rt for 5 min followed by the addition of 3-methyl-1H-
pyrazol-5-amine 43c (83.7 mg, 0.862 mmol) and the
resulting mixture was stirred at reflux for 4 h. The
resulting pale yellow suspension was cooled in an ice bath.
The solid was collected by filtration and was washed with
ice cold ethanol (3 × 0.2 mL). Drying under high vacuum
as a white solid. H NMR (300 MHz, DMSO-d6) δ 12.63 (br
s, 1H), 11.37 (br s, 1H), 5.97 (s, 1H), 3.30 – 3.19 (m, 1H),
2.45 (s, 3H), 1.88 – 1.39 (m, 11H), 1.37 – 1.22 (m, 1H).
UPLC tR = 2.03 min, Purity UV254 >99%. HRMS (ESI) m/z:
[M + H]+ Calcd for C15H20N3O 258.1601; Found 258.1567.
4-Cyclohexyl-3-ethyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-
one (15). To a solution of cyclohexanecarbaldehyde 59a
(63 mg, 0.56 mmol) in ethanol (2.7 mL) was added
Meldrum’s acid 60 (81 mg, 0.56 mmol). The reaction
mixture was stirred at rt for 5 min followed by the addition
of 3-ethyl-1H-pyrazol-5-amine 43d (62 mg, 0.56 mmol)
and the resulting mixture was stirred at 60 °C overnight.
The reaction mixture was cooled, concentrated to half its
volume, and ether (2 mL) was added to afford a white
precipitate. The white solid was collected by filtration and
was washed with ether. Drying under high vacuum
afforded
[(1R*,2S*,4S*)-norbornan-2-yl]-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61f (88.5 mg,
35%) as a white solid (mixture of diastereomers).
To
a
suspension of 3-methyl-4-[(1R*,2S*,4S*)-
norbornan-2-yl]-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-
6-one 61f (84.3 mg, 0.344 mmol) in 1,4-dioxane (1.4 mL)
was added DDQ (117 mg, 0.515 mmol) and the mixture
was heated at 100 °C for 2 h. After cooling to rt the mixture
was concentrated under reduced pressure. The residue
was suspended in sat. NaHCO3 solution (10 mL) and the
mixture was extracted with DCM:MeOH 9:1 (3 × 20 mL).
The combined organic phases were washed with brine (10
mL), dried over Na2SO4, and volatiles were evaporated. The
residue was purified by column chromatography (silica gel
column, DCM:MeOH 96:4 → 95:5) to afford 3-methyl-4-
afforded
4-cyclohexyl-3-ethyl-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61h (120 mg,
86%) as a white solid.
To
a
suspension of 4-cyclohexyl-3-ethyl-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61h (20 mg,
0.081 mmol) in 1,4-dioxane (1.0 mL) was added DDQ (128
mg, 0.562 mmol) and the mixture was heated at 100 °C for
15 min. After cooling to rt the resulting white precipitate
was collected by filtration and washed with methanol. The
ACS Paragon Plus Environment

Page 21 of 33
Journal of Medicinal Chemistry
1
crude product was purified by HPLC (acidic method) to
afford 4-cyclohexyl-3-ethyl-2,7-dihydropyrazolo[3,4-
a white solid. H NMR (300 MHz, DMSO-d6) δ 12.70 (br s,
1H), 11.46 (br s, 1H), 4.49 (s, 2H), 3.08 (br s, 1H), 2.55 (br
s, 3H), 1.91 – 1.56 (m, 7H), 1.47 – 1.21 (m, 3H). Very broad
peaks, probably due to slow interconversion of tautomers.
UPLC tR = 1.86 min, Purity UV254 94%. HRMS (ESI) m/z: [M
+ H]+ Calcd for C14H20N3O2 262.1550; Found 262.1529.
1
2
3
4
5
6
7
8
1
b]pyridin-6-one 15 (4.1 mg, 21%). H NMR (600 MHz,
DMSO-d6) δ 12.73 (br s, 1H), 11.33 (br s, 1H), 5.82 (s, 1H),
2.85 (q, J = 7.6 Hz, 2H), 2.72 (s, 1H), 1.89 – 1.77 (m, 4H),
1.76 – 1.69 (m, 1H), 1.47 – 1.32 (m, 4H), 1.27 (t, J = 7.6 Hz,
3H), 1.26 – 1.21 (m, 1H). UPLC tR = 2.02 min, Purity UV254
>99%. HRMS (ESI) m/z: [M + H]+ Calcd for C14H20N3O
246.1601; Found 246.1614.
4-Cyclohexyl-5-(2-hydroxyethyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (18). A mixture of 6-
chloro-4-cyclohexyl-3-methyl-1H-pyrazolo[3,4-b]pyridine-
5-carbaldehyde 62 (0.30 g, 1.1 mmol), 4.4 M NaOMe
solution in MeOH (2.0 mL, 8.8 mmol) and MeOH (10 mL)
was heated in the microwave at 120 °C for 40 min. After
cooling to rt, the reaction was quenched with AcOH (1 mL)
and volatiles were evaporated. The residue was dissolved
in ether (25 mL) and washed with brine (10 mL). The
organic layer was dried over Na2SO4 and evaporated to
dryness to afford 4-cyclohexyl-6-methoxy-3-methyl-1H-
pyrazolo[3,4-b]pyridine-5-carbaldehyde 63 (298 mg,
99%) which was used without further purification.
4-Cyclohexyl-3-cyclopropyl-2,7-dihydropyrazolo[3,4-
9
b]pyridin-6-one
(16).
To
a
solution
of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
cyclohexanecarbaldehyde 59a (100 mg, 0.892 mmol) in
ethanol (2.7 mL) was added Meldrum’s acid 60 (129 mg,
0.892 mmol). The reaction mixture was stirred at rt for 5
min followed by the addition of 3-cyclopropyl-1H-pyrazol-
5-amine 43e (110 mg, 0.892 mmol) and the resulting
mixture was stirred at 60 °C overnight. The reaction
mixture was cooled, concentrated to half its volume, and
ether (5 mL) was added to afford a white precipitate. The
white solid was collected by filtration and was washed
with ether. Drying under high vacuum afforded 4-
cyclohexyl-3-cyclopropyl-2,4,5,7-tetrahydropyrazolo[3,4-
b]pyridin-6-one 61i (144 mg, 62%) as a white solid.
A 2.5 M solution of n-BuLi in heptanes (0.29 mL, 0.73
mmol) was added slowly to
methyl(triphenyl)phosphonium bromide (261 mg, 0.732
mmol) in dry THF (10 mL) at 0 °C under argon. The
resulting orange solution was stirred for 30 min at 0 °C. A
a
suspension of
To a suspension of 4-cyclohexyl-3-cyclopropyl-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-6-one 61i (20 mg, 0.077
mmol) in 1,4-dioxane (1.0 mL) was added DDQ (202 mg,
0.892 mmol) and the mixture was heated at 100 °C for 15
min. After cooling to rt the resulting white precipitate was
collected by filtration and washed with methanol. Vacuum
drying afforded 4-cyclohexyl-3-cyclopropyl-2,7-dihydro-
solution
of
4-cyclohexyl-6-methoxy-3-methyl-1H-
pyrazolo[3,4-b]pyridine-5-carbaldehyde 63 (100 mg,
0.366 mmol) in dry THF (5 mL) was added and the mixture
was stirred for 6 h at rt. The reaction was quenched with
AcOH (0.5 mL), diluted with water (10 mL) and extracted
with DCM (2 × 10 mL). The combined organic layers were
dried over Na2SO4 and evaporated. The residue was
purified by chromatography (ISCO CombiFlash, silica gel
column, heptane:EtOAc) to afford 4-cyclohexyl-6-methoxy-
3-methyl-5-vinyl-1H-pyrazolo[3,4-b]pyridine 65 (90 mg,
91%) as a clear oil.
1
pyrazolo[3,4-b]pyridin-6-one 16 (4.4 mg, 22%). H NMR
(600 MHz, DMSO-d6) δ 12.47 (br s, 1H), 11.36 (br s, 1H),
5.83 (s, 1H), 3.04 (br t, 1H), 2.15 – 2.07 (m, 1H), 1.98 – 1.90
(m, 2H), 1.86 – 1.78 (m, 2H), 1.76 – 1.69 (m, 1H), 1.46 –
1.31 (m, 4H), 1.30 – 1.21 (m, 1H), 1.02 – 0.95 (m, 2H), 0.93
– 0.88 (m, 2H). UPLC tR = 2.05 min, Purity UV254 >99%.
HRMS (ESI) m/z: [M + H]+ Calcd for C15H20N3O 258.1601;
Found 258.1624.
A 0.50 M solution of 9-BBN in THF (1.3 mL, 0.65 mmol)
was added to a solution of 4-cyclohexyl-6-methoxy-3-
methyl-5-vinyl-1H-pyrazolo[3,4-b]pyridine 65 (90.0 mg,
0.332 mmol) in dry THF (4 mL) and the mixture was
stirred at rt for 3 h. A solution of 50% H2O2 (0.123 mL, 1.99
mmol) in 4M NaOH (1 mL) was added carefully and the
resulting mixture was evaporated. The residue was
purified by HPLC to afford 2-(4-cyclohexyl-6-methoxy-3-
methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)ethanol 66 (45 mg,
47%) as a clear oil.
4-Cyclohexyl-5-(hydroxymethyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (17). A mixture of 6-
chloro-4-cyclohexyl-3-methyl-1H-pyrazolo[3,4-b]pyridine-
5-carbaldehyde 62 (0.15 g, 0.54 mmol) and 4.4 M NaOMe
in MeOH (1.0 mL, 4.4 mmol) in MeOH (2 mL) was heated in
the microwave at 120 °C for 40 min. After cooling to rt,
NaBH4 (10 mg, 0.27 mmol) was added and stirred until the
yellow color disappeared and a clear solution was
obtained (ca. 30 min). The reaction was quenched by
adding AcOH, volatiles were evaporated, the residue was
triturated with water and the solid was collected by
filtration and dried in vacuo to afford (4-cyclohexyl-6-
methoxy-3-methyl-1H-pyrazolo[3,4-b]pyridin-5-
A mixture of pyridine hydrochloride (500 mg) and 2-(4-
cyclohexyl-6-methoxy-3-methyl-1H-pyrazolo[3,4-
b]pyridin-5-yl)ethanol 66 (45 mg, 0.16 mmol) was stirred
at 90 °C for 45 min. Water (0.5 mL) was added, the mixture
was purified by HPLC and pure fractions combined and
freeze dried to afford 4-cyclohexyl-5-(2-hydroxyethyl)-3-
methyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-one 18 (1.9
yl)methanol 64 (120 mg, 81%) as an off-white solid that
was used directly in the following step.
1
mg, 4.4%) as a white solid. H NMR (600 MHz, DMSO-d6) δ
A
mixture of (4-cyclohexyl-6-methoxy-3-methyl-1H-
12.84 (br s, 1H), 11.73 (br d, 1H), 5.62 (br s, 0.4H), 5.44 (d,
J = 10.5 Hz, 0.6H), 5.26 (br s, 0.4H), 5.05 (br s, 0.6H), 3.50
(br s, 0.4H), 3.07 (br s, 0.6H), 2.66 (br s, 1H), 2.53 (br s,
2H), 1.92 – 1.23 (m, 13H). Two sets of broad peaks, ca. 4:6,
probably due to two slowly interconverting tautomers.
UPLC tR = 1.97 min, Purity UV254 >99%. HRMS (ESI) m/z:
[M + H]+ Calcd for C15H22N3O2 276.1706; Found 276.1700.
pyrazolo[3,4-b]pyridin-5-yl)methanol 64 (22.0 mg, 0.0799
mmol) and 5 M HBr in AcOH (0.50 mL, 2.5 mmol) was
stirred at 40 °C for 30 min. After cooling to rt, water (0.50
mL) was added, the mixture was purified by HPLC and
pure fractions combined and freeze dried to afford 4-
cyclohexyl-5-(hydroxymethyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one 17 (4.9 mg, 23%) as
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Journal of Medicinal Chemistry
Page 22 of 33
4-Cyclohexyl-3-methyl-6-oxo-2,7-dihydropyrazolo[3,4- (50 mg, 0.09 mmol) in TFA (4 mL) was prepared at 0 °C,
1
2
3
4
5
6
7
8
b]pyridine-5-carbonitrile (19). A mixture of 3-methyl-1H-
pyrazol-5-amine 43c (1.18 g, 12.2 mmol) and methyl (Z)-2-
cyano-3-cyclohexyl-prop-2-enoate 67 (2.35 g, 12.2 mmol)
in MeOH (15 mL) was stirred at 60 °C for 18 h. Volatiles
were evaporated and the residue was purified by column
chromatography (ISCO CombiFlash, silica gel column,
DCM:MeOH) to afford 4-cyclohexyl-3-methyl-6-oxo-
2,4,5,7-tetrahydropyrazolo[3,4-b]pyridine-5-carbonitrile
68 (1.47 g, 47%) as a pale orange solid (mixture of
diastereomers).
and was then refluxed for 6 h. On completion, the reaction
mixture was concentrated under reduced pressure. The
crude product was diluted with MeOH and basified with
Amberlyst A21 free base. The reaction mixture was
filtered, the filtrate was concentrated under reduced
pressure and the crude product was purified by HPLC to
afford
3-[trans-4-(3-methyl-6-oxo-2,7-dihydro-
pyrazolo[3,4-b]pyridin-4-yl)cyclohexyl]propanenitrile 22
1
(6.9 mg, 25%) as an off-white solid. H NMR (600 MHz,
9
DMSO-d6) δ 12.73 (br s, 1H), 11.29 (br s, 1H), 5.78 (br s,
1H), 2.76 – 2.66 (br m, 1H), 2.54 (s, 2H), 2.46 (s, 3H), 1.92 –
1.82 (m, 4H), 1.53 (q, J = 7.2 Hz, 2H), 1.44 – 1.33 (m, 3H),
1.17 – 1.06 (m, 2H). UPLC tR = 1.88 min, Purity UV254 >99%.
HRMS (ESI) m/z: [M + H]+ Calcd for C16H21N4O 285.1710;
Found 285.1729.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To a solution of 4-cyclohexyl-3-methyl-6-oxo-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridine-5-carbonitrile 68 (62.0
mg, 0.240 mmol) in MeCN (5 mL) was added NBS (42.7 mg,
0.240 mmol) at rt. The mixture was stirred for 30 min at rt
after which DBU (72 µL, 0.48 mmol) was added and
stirring was continued for an additional 10 min at rt.
Volatiles were evaporated and the residue was purified by
trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexanecarbonitrile (23). To a solution
of trans-4-formylcyclohexanecarbonitrile 59k33 (212 mg,
1.55 mmol) in ethanol (8 mL) was added Meldrum’s acid
60 (223 mg, 1.55 mmol). The reaction mixture was stirred
at rt for 5 min followed by the addition of 3-methyl-1H-
pyrazol-5-amine 43c (150 mg, 1.55 mmol) and the
resulting mixture was stirred at 60 °C for 2 h. After cooling
to rt, the mixture was concentrated to half its volume
under reduced pressure and ether (10 mL) was added. The
solid thus obtained was collected by filtration, washed
with ether and dried to afford 4-(3-methyl-6-oxo-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-4-yl)cyclohexanecarbo-
nitrile 61k (341 mg, 85%) which was used without
purification.
HPLC
to
afford
4-cyclohexyl-3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridine-5-carbonitrile 19 (5.6 mg,
1
9.1%) as a white solid. H NMR (600 MHz, DMSO-d6) δ
13.08 (br s, 1H), 12.51 (br s, 1H), 3.13 (br t, J = 12.6 Hz,
1H), 2.57 (s, 3H), 2.14 – 1.95 (m, 2H), 1.91 – 1.69 (m, 5H),
1.45 – 1.34 (m, 2H), 1.32 – 1.18 (m, 1H). UPLC tR = 1.98
min, Purity UV254 >99%. HRMS (ESI) m/z: [M + H]+ Calcd
for C14H17N4O 257.1397; Found 257.1392.
2-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]acetonitrile (21). To a solution of
2-(trans-4-formylcyclohexyl)acetonitrile 59j (500 mg, 3.31
mmol) in ethanol (16.5 mL) was added Meldrum’s acid 60
(477 mg, 3.31 mmol). The reaction mixture was stirred at
rt for 5 min followed by the addition of 3-methyl-1H-
pyrazol-5-amine 43c (321 mg, 3.31 mmol) and the
resulting mixture was stirred at 60 °C overnight. After
cooling to rt the mixture was concentrated to half its
volume under reduced pressure and ether (5 mL) was
added. The solid thus obtained was collected by filtration,
washed with ether and dried under vacuum to afford 2-[4-
(3-methyl-6-oxo-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-
4-yl)cyclohexyl]acetonitrile 61j (764 mg, 85%) which was
used without purification.
To
a
suspension of 4-(3-methyl-6-oxo-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-4-yl)cyclohexanecarbo-
nitrile 61k (341 mg, 1.32 mmol) in 1,4-dioxane (10 mL)
was added DDQ (300 mg, 1.32 mmol) and the mixture was
heated at 100 °C for 15 min. After cooling to rt the
resulting white precipitate was collected by filtration and
washed with 1,4-dioxane. It was triturated with MeOH to
afford trans-4-(3-methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexanecarbonitrile as a light brown
solid 23 (79 mg, 23%). A sample (7 mg) was further
purified using HPLC (acidic method) to afford an
To
a
suspension of 2-[4-(3-methyl-6-oxo-2,4,5,7-
1
analytically pure sample. H NMR (600 MHz, DMSO-d6) δ
tetrahydropyrazolo[3,4-b]pyridin-4-
12.80 (br s, 1H), 11.40 (br s, 1H), 5.75 (s, 1H), 2.87 – 2.78
(m, 1H), 2.77 (tt, J = 12.3, 3.7 Hz, 1H), 2.47 (s, 3H), 2.16 –
2.09 (m, 2H), 1.91 – 1.83 (m, 2H), 1.76 – 1.66 (m, 2H), 1.47
– 1.36 (m, 2H). UPLC tR = 1.75 min, Purity UV254 >99%.
HRMS (ESI) m/z: [M + H]+ Calcd for C14H17N4O 257.1397;
Found 257.1414.
yl)cyclohexyl]acetonitrile 61j (100 mg, 0.367 mmol) in
1,4-dioxane (8 mL) was added DDQ (83.4 mg, 0.367 mmol)
and the mixture was heated at 100 °C for 30 min. After
cooling to rt the resulting white precipitate was collected
by filtration and washed with 1,4-dioxane. The crude solid
was purified using HPLC (acidic method) to afford 2-
[trans-4-(3-methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]acetonitrile 21 (57 mg, 57%). 1H
NMR (600 MHz, DMSO-d6) δ 12.73 (br s, 1H), 11.32 (br s,
1H), 5.81 (br s, 1H), 2.81 – 2.66 (m, 1H), 2.50 (d, J = 6.5 Hz,
2H), 2.47 (s, 3H), 1.94 – 1.84 (m, 4H), 1.76 – 1.65 (m, 1H),
1.49 – 1.38 (m, 2H), 1.32 – 1.19 (m, 2H). UPLC tR = 1.80
min, Purity UV254 97%. HRMS (ESI) m/z: [M + H]+ Calcd for
C15H19N4O 271.1553; Found 271.1574.
General procedure for amide formation in library format.
One amine, multiple acids. A stock solution of 4-(4-trans-
aminocyclohexyl)-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one 32 (45 mg, 0.18 mmol) in DMF (3.6 mL)
was prepared, and HATU (69 mg, 0.18 mmol) and DIPEA
(94 µL, 0.54 mmol) were added. To each acid (20 µmol) in
the library was added an aliquot of the stock solution (0.40
mL) and the resulting mixture was shaken at rt for 15 min.
The reaction mixtures were purified by HPLC (acidic
method) without prior work-up to afford the
corresponding amides.
3-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]propanenitrile (22). A solution of
3-[4-[1,7-bis[(4-methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]propanenitrile 74
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Journal of Medicinal Chemistry
N-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
dihydropyrazolo[3,4-b]pyridin-4-yl)cyclohex-
1
2
b]pyridin-4-yl)cyclohexyl]cyclopropanecarboxamide (24).
anecarboxamide 25 (2.3 mg, 9.4%) as the pure trans
1
1
Prepared using the general procedure. H NMR (600 MHz,
isomer. H NMR (600 MHz, DMSO-d6) δ 12.74 (br s, 1H),
DMSO-d6) δ 12.75 (br s, 1H), 11.31 (br s, 1H), 7.95 (d, J =
7.8 Hz, 1H), 5.80 (br s, 1H), 3.66 – 3.55 (m, 1H), 2.78 – 2.66
(br m, 1H), 2.48 (s, 3H), 1.96 – 1.83 (m, 4H), 1.55 – 1.40 (m,
3H), 1.40 – 1.30 (m, 2H), 0.69 – 0.58 (m, 4H). UPLC tR =
1.72 min, Purity UV254 98%. HRMS (ESI) m/z: [M + H]+
Calcd for C17H23N4O2 315.1816; Found 315.1820.
11.32 (br s, 1H), 7.80 (br d, J = 4.3 Hz, 1H), 5.78 (br s, 1H),
2.81 – 2.68 (br m, 1H), 2.64 – 2.58 (m, 1H), 2.47 (s, 3H),
2.11 (tt, J = 12.0, 3.7 Hz, 1H), 1.93 – 1.85 (m, 2H), 1.84 –
1.77 (m, 2H), 1.60 – 1.49 (m, 2H), 1.42 – 1.31 (m, 2H), 0.61
– 0.56 (m, 2H), 0.39 – 0.35 (m, 2H). UPLC tR = 1.68 min,
Purity UV254 >99%. HRMS (ESI) m/z: [M + H]+ Calcd for
C17H23N4O2 315.1816; Found 315.1823.
3
4
5
6
7
8
9
N-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]cyclopentanecarboxamide
(26).
[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Prepared using the general procedure. H NMR (600 MHz,
DMSO-d6) δ 12.74 (br s, 1H), 11.31 (br s, 1H), 7.62 (d, J =
7.8 Hz, 1H), 5.80 (br s, 1H), 3.58 (tdt, J = 11.7, 8.0, 4.0 Hz,
1H), 2.80 – 2.63 (br m, 1H), 2.53 – 2.50 (m, 1H), 2.47 (s,
3H), 1.94 – 1.83 (m, 4H), 1.76 – 1.67 (m, 2H), 1.65 – 1.55
(m, 4H), 1.54 – 1.41 (m, 4H), 1.38 – 1.29 (m, 2H). UPLC tR =
1.86 min, Purity UV254 >99%. HRMS (ESI) m/z: [M + H]+
Calcd for C19H27N4O2 343.2129; Found 343.2133.
b]pyridin-4-yl)cyclohexyl]
(1S)-2,2-difluorocyclopropane-
carboxylate (29). A mixture of 4-(4-trans-hydroxycyclo-
hexyl)-3-methyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-one
33 (100 mg, 0.404 mmol), (1S)-2,2-difluorocyclo-
propanecarboxylic acid, HATU (154 mg, 0.404 mmol) and
DMAP (74 mg, 0.607 mmol) in DMF (2 mL) was stirred at
rt for 15 h. The mixture was purified without workup by
HPLC to afford [trans-4-(3-methyl-6-oxo-2,7-dihydro-
pyrazolo[3,4-b]pyridin-4-yl)cyclohexyl] (1S)-2,2-difluoro-
cyclopropanecarboxylate 29 (18 mg, 13%) as a white solid.
1H NMR (600 MHz, DMSO-d6) δ 12.75 (br s, 1H), 11.33 (br
s, 1H), 5.80 (br s, 1H), 4.83 – 4.75 (m, 1H), 3.39 – 3.36 (m,
1H), 2.83 – 2.74 (m, 1H), 2.48 (s, 3H), 2.09 – 1.95 (m, 4H),
1.93 – 1.87 (m, 2H), 1.63 – 1.48 (m, 4H). UPLC tR = 1.98
min, Purity UV254 95%. HRMS (ESI) m/z: [M + H]+ Calcd for
C17H20F2N3O3 352.1467; Found 352.1482.
(1S)-2,2-Difluoro-N-[trans-4-(3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-yl)cyclo-
hexyl]cyclopropanecarboxamide (27). Prepared using the
1
general procedure. H NMR (600 MHz, DMSO-d6) δ 12.75
(br s, 1H), 11.31 (br s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 5.81 (br
s, 1H), 3.64 (tdt, J = 11.6, 7.9, 3.7 Hz, 1H), 2.79 – 2.67 (m,
1H), 2.55 – 2.51 (m, 1H), 2.48 (s, 3H), 1.98 – 1.92 (m, 2H),
1.92 – 1.85 (m, 3H), 1.85 – 1.77 (m, 1H), 1.55 – 1.43 (m,
2H), 1.42 – 1.31 (m, 2H). UPLC tR = 1.78 min, Purity UV254
97%. HRMS (ESI) m/z: [M + H]+ Calcd for C17H21F2N4O2
351.1627; Found 351.1639.
General procedure for sulfonamide formation in library
format. A stock solution of 4-(4-trans-aminocyclohexyl)-3-
methyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-one 32 (80
mg, 0.32 mmol) in DMSO (4.0 mL) was prepared, and
DIPEA (288 µL, 1.65 mmol) was added. To each sulfonyl
chloride (0.12 mmol) in the library was added an aliquot of
the stock solution (0.50 mL) and the resulting mixture was
shaken at rt for 15 min. The reaction mixtures were
purified by HPLC (acidic method) without prior work-up to
afford the corresponding sulfonamides.
(1R)-2,2-Difluoro-N-[trans-4-(3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-yl)cyclo-
hexyl]cyclopropanecarboxamide (28). Prepared using the
1
general procedure. H NMR (600 MHz, DMSO-d6) δ 12.74
(br s, 1H), 11.32 (br s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 5.82 (br
s, 1H), 3.64 (tdt, J = 11.6, 7.9, 4.1 Hz, 1H), 2.82 – 2.68 (br m,
1H), 2.55 – 2.51 (m, 1H), 2.48 (s, 3H), 2.00 – 1.92 (m, 2H),
1.92 – 1.85 (m, 3H), 1.85 – 1.77 (m, 1H), 1.54 – 1.43 (m,
2H), 1.42 – 1.31 (m, 2H). UPLC tR = 1.78 min, Purity UV254
>99%. HRMS (ESI) m/z: [M + H]+ Calcd for C17H21F2N4O2
351.1627; Found 351.1632.
N-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]cyclopropanesulfonamide
(30).
1
Prepared using the general procedure. H NMR (600 MHz,
DMSO-d6) δ 12.30 (br s, 1H), 11.78 (br s, 1H), 7.09 (br s,
1H), 5.82 (br s, 1H), 3.22 (tt, J = 11.0, 4.1 Hz, 1H), 2.75 –
2.67 (m, 1H), 2.60 – 2.54 (m, 1H), 2.47 (s, 3H), 2.09 – 2.02
(m, 2H), 1.91 – 1.84 (m, 2H), 1.54 – 1.37 (m, 4H), 0.98 –
0.90 (m, 4H). UPLC tR = 1.73 min, Purity UV254 >99%. HRMS
(ESI) m/z: [M + H]+ Calcd for C16H23N4O3S 351.1485; Found
351.1513.
N-Cyclopropyl-trans-4-(3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-yl)cyclohex-
anecarboxamide (25). To a solution of 4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-pyrazolo[3,4-
b]pyridin-4-yl]cyclohexanecarboxylic acid 77 (40 mg,
0.078 mmol) in DMF (1 mL) was added HATU (38 mg, 0.10
mmol), cyclopropylamine (6.6 mg, 0.12 mmol) and DIPEA
(40.5 µL, 0.233 mmol). The reaction was stirred at room
temperature for 5 min. It was poured into 1 N HCl (10 mL)
and the product was extracted with EtOAc (3 × 10 ml). The
combined organic layers were washed with sat. NaHCO3
(10 mL), brine (10 mL), dried over MgSO4, filtered and
1-Cyclobutyl-N-[trans-4-(3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-yl)cyclohexyl]methane-
sulfonamide (31). Prepared using the general procedure.
1H NMR (600 MHz, DMSO-d6) δ 12.75 (s, 1H), 11.31 (s, 1H),
7.01 (d, J = 7.4 Hz, 1H), 5.78 (s, 1H), 3.19 – 3.13 (m, 1H),
3.12 (d, J = 7.2 Hz, 2H), 2.73 – 2.63 (m, 2H), 2.46 (s, 3H),
2.15 – 2.06 (m, 2H), 2.03 – 1.96 (m, 2H), 1.91 – 1.74 (m,
6H), 1.52 – 1.34 (m, 4H). UPLC tR = 1.89 min, Purity UV254
>99%. HRMS (ESI) m/z: [M + H]+ Calcd for C18H27N4O3S
379.1798; Found 379.1825.
evaporated
to
afford
4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-pyrazolo[3,4-
b]pyridin-4-yl]-N-cyclopropyl-cyclohexanecarboxamide
(41 mg, 90%) as a solid. A portion of this material (27 mg,
0.049 mmol) was dissolved in TFA (1 mL) and the solution
was heated in a microwave synthesizer at 80 °C for 1.5 h.
The reaction mixture was purified by HPLC (acidic
method) to afford N-cyclopropyl-4-(3-methyl-6-oxo-2,7-
4-(4-trans-Aminocyclohexyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (32). To a solution of
tert-butyl N-(4-formylcyclohexyl)carbamate 59l (1.17 g,
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Journal of Medicinal Chemistry
Page 24 of 33
5.15 mmol) in ethanol (13 mL) was added Meldrum’s acid 1H), 2.68 – 2.57 (m, 1H), 2.56 – 2.51 (m, 1H), 2.11 (s, 3H),
1
2
3
4
5
6
7
8
60 (371 mg, 2.57 mmol). The reaction mixture was stirred
at rt for 5 min followed by the addition of 3-methyl-1H-
pyrazol-5-amine 43c (250 mg, 2.57 mmol) and the
resulting mixture was stirred at 60 °C overnight. After
cooling to rt, the reaction mixture was reduced to half its
initial volume by evaporation and ether (10 mL) was
added. The resulting white precipitate was collected by
filtration and washed with ether to afford tert-butyl N-[4-
(3-methyl-6-oxo-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-
4-yl)cyclohexyl]carbamate 61l (502 mg, 56%) as a white
1.82 – 1.73 (m, 2H), 1.67 – 1.47 (m, 2H), 1.28 – 1.21 (m,
1H), 1.16 – 1.07 (m, 2H), 1.02 – 0.92 (m, 2H), 0.86 (s, 9H),
0.02 (s, 6H).
To a solution of 4-[4-[tert-butyl(dimethyl)silyl]oxycyclo-
hexyl]-3-methyl-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-
6-one 61m (170 mg, 0.468 mmol) in 1,4-dioxane (5 mL)
was added DDQ (106 mg, 0.468 mmol) and the mixture
was stirred at 100 °C for 30 min. After cooling to rt, the
precipitate was collected by filtration, washed with 1,4-
dioxane and dried under vacuum to afford 4-[4-[tert-
butyl(dimethyl)silyl]oxycyclohexyl]-3-methyl-2,7-dihydro-
pyrazolo[3,4-b]pyridin-6-one 33a (100 mg, 59%) as an off-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
solid that was used without purification. H NMR (600
MHz, DMSO-d6) δ 11.71 (s, 1H), 10.05 (s, 1H), 6.63 (d, J =
8.0 Hz, 1H), 3.11 – 3.01 (m, 1H), 2.61 (t, J = 6.5 Hz, 1H),
2.53 – 2.49 (m, 1H), 2.35 (d, J = 16.8 Hz, 1H), 2.11 (s, 3H),
1.80 – 1.69 (m, 2H), 1.68 – 1.62 (m, 1H), 1.55 – 1.47 (m,
1H), 1.35 (s, 9H), 1.22 – 1.14 (m, 1H), 1.11 – 1.00 (m, 2H),
1.00 – 0.90 (m, 2H).
1
white solid. H NMR (500 MHz, DMSO-d6) δ 12.85 (s, 1H),
11.40 (s, 1H), 5.78 (br s, 1H), 3.75 – 3.65 (m, 1H), 2.47 (s,
3H), 1.93 – 1.91 (m, 2H), 1.87 – 1.81 (m, 2H), 1.50 – 1.39
(m, 4H), 0.87 (s, 9H), 0.06 (s, 6H).
To a solution of 4-[4-[tert-butyl(dimethyl)silyl]oxycyclo-
hexyl]-3-methyl-2,7-dihydropyrazolo[3,4-b]pyridin-6-one
33a (100 mg, 0.275 mmol) in DCM (5 mL) at 0 °C was
added 1 M TBAF in THF (0.55 mL, 0.55 mmol) at 0 °C. The
resulting reaction mass was stirred at 40 °C for 16 h.
Volatiles were evaporated and the residue was triturated
with saturated NaHCO3 solution to give a solid. The solid
was filtered, washed with ether and dried under vacuum to
To a suspension of tert-butyl N-[4-(3-methyl-6-oxo-
2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-4-
yl)cyclohexyl]carbamate 61l (502 mg, 1.44 mmol) in 1,4-
dioxane (8 mL) was added DDQ (327 mg, 1.44 mmol) and
the mixture was heated at 100 °C for 30 min. After cooling
to rt the precipitated solid was collected by filtration and
washed with ether to afford tert-butyl N-[4-(3-methyl-6-
oxo-2,7-dihydropyrazolo[3,4-b]pyridin-4-
afford
4-(4-trans-hydroxycyclohexyl)-3-methyl-2,7-
yl)cyclohexyl]carbamate 32a (498 mg, 99%) as a white
1
dihydropyrazolo[3,4-b]pyridin-6-one 33 (56 mg, 40%). H
NMR (500 MHz, DMSO-d6) δ 12.73 (s, 1H), 11.31 (s, 1H),
5.78 (s, 1H), 4.61 (d, J = 4.4 Hz, 1H), 3.51 – 3.41 (m, 1H),
2.77 – 2.62 (m, 1H), 2.47 (s, 3H), 1.98 – 1.89 (m, 2H), 1.87 –
1.77 (m, 2H), 1.48 – 1.21 (m, 4H). UPLC tR = 1.54 min,
Purity UV254 98%. HRMS (ESI) m/z: [M + H]+ Calcd for
C13H18N3O2 248.1394; Found 248.1382.
1
solid that was used without purification. H NMR (600
MHz, DMSO-d6) δ 12.74 (s, 1H), 11.31 (s, 1H), 6.77 (d, J =
7.9 Hz, 1H), 5.81 (s, 1H), 2.75 – 2.59 (m, 1H), 2.52 – 2.49
(m, 1H), 2.46 (s, 3H), 1.96 – 1.81 (m, 4H), 1.51 – 1.40 (m,
2H), 1.39 (s, 9H), 1.36 – 1.27 (m, 2H).
The intermediate tert-butyl N-[4-(3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-yl)cyclohexyl]carbamate
32a (498 mg, 1.44 mmol) was dissolved in 4 M HCl in 1,4-
dioxane and the mixture was stirred at rt for 15 min.
General procedure for amide formation in library format.
One acid, multiple amines. A stock solution of 2-[trans-4-(3-
methyl-6-oxo-2,7-dihydropyrazolo[3,4-b]pyridin-4-
Evaporation
to
dryness
afforded
4-(4-trans-
yl)cyclohexyl]acetic acid 78 (95.0 mg, 0.328 mmol) in DMF
(4.75 mL) was prepared, and an aliquot (0.25 mL, 0.017
mmol) was added to each amine (0.0346 mmol) in the
library. A stock solution of HATU (124 mg, 0.326 mmol) in
DMF (4.75 mL) was prepared, and an aliquot (0.25 mL,
0.017 mmol) was added to each of the reaction mixtures.
Finally, DIPEA (9.0 µL, 0.052 mmol) was added to each of
the reaction mixtures, which were then shaken at rt for 15
min. The reaction mixtures were purified by HPLC without
prior work-up to afford the corresponding amides.
aminocyclohexyl)-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one 32 as the hydrochloride salt (350 mg,
1
86%). H NMR (600 MHz, DMSO-d6) δ 8.11 (br s, 3H), 5.86
(s, 1H), 3.13 – 3.01 (m, 1H), 2.80 – 2.71 (m, 1H), 2.48 (s,
3H), 2.14 – 2.03 (m, 2H), 1.97 – 1.86 (m, 2H), 1.57 – 1.44
(m, 4H). Not all exchangeable protons are visible. UPLC tR =
0.87 min, Purity UV254 95%. HRMS (ESI) m/z: [M + H]+
Calcd for C13H19N4O 247.1553; Found 247.1576.
4-(4-trans-Hydroxycyclohexyl)-3-methyl-2,7-
dihydropyrazolo[3,4-b]pyridin-6-one (33). To solution of
trans-4-[tert-butyl(dimethyl)silyl]oxycyclohexanecarb-
aldehyde 59m34 (20 g, 83 mmol) in ethanol (1.4 L) was
added Meldrum’s acid 60 (5.6 g, 58 mmol). The resulting
mixture was stirred at rt for 15 min followed by the
addition of 3-methyl-1H-pyrazol-5-amine 43c (8.3 g, 58
mmol) and the resulting mixture was stirred at 90 °C for 3
h. After cooling to rt, the mixture was concentrated under
reduced pressure and the solid thus obtained was collected
by filtration, washed with water and diethyl ether, and
dried under vacuum to afford 4-[4-[tert-butyl(dimethyl)-
silyl]oxycyclohexyl]-3-methyl-2,4,5,7-
1-[2-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]acetyl]azetidine-3-carbonitrile
(34). Prepared using the general procedure. H NMR (600
1
MHz, DMSO-d6) δ 12.72 (br s, 1H), 11.29 (br s, 1H), 5.78 (br
s, 1H), 4.40 (t, J = 8.8 Hz, 1H), 4.32 (dd, J = 8.4, 6.0 Hz, 1H),
4.13 (t, J = 9.3 Hz, 1H), 3.98 (dd, J = 9.4, 6.1 Hz, 1H), 3.76 (tt,
J = 9.1, 6.0 Hz, 1H), 2.75 – 2.64 (m, 1H), 2.45 (s, 3H), 2.03 –
1.93 (m, 2H), 1.90 – 1.79 (m, 4H), 1.78 – 1.68 (m, 1H), 1.46
– 1.32 (m, 2H), 1.21 – 1.08 (m, 2H). UPLC tR = 1.72 min,
Purity UV254 81%. HRMS (ESI) m/z: [M + H]+ Calcd for
C19H24N5O2 354.1925; Found 354.1943.
1-[2-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]acetyl]pyrrolidine-3-carbonitrile
(35). Prepared using the general procedure. Two rotamers
tetrahydropyrazolo[3,4-b]pyridin-6-one 61m (10 g, 45%)
as an off-white solid. 1H NMR (500 MHz, DMSO-d6) δ 11.71
(s, 1H), 10.05 (s, 1H), 3.50 – 3.42 (m, 1H), 3.40 – 3.34 (m,
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Page 25 of 33
Journal of Medicinal Chemistry
around the amide bond are visible in the NMR spectrum. 10.25 – 9.98 (m, 1H), 5.34 – 5.20 (m, 1H), 3.17 (s, 1H), 2.66
1H NMR (600 MHz, DMSO-d6) δ 12.73 (br s, 1H), 11.29 (br
s, 1H), 5.78 (br s, 1H), 3.78 (dd, J = 10.3, 7.2 Hz, 0.5H), 3.67
(dd, J = 10.3, 6.0 Hz, 0.5H), 3.62 (dd, J = 11.8, 7.4 Hz, 0.5H),
3.59 – 3.35 (m, 3.5H), 2.75 – 2.64 (br m, 1H), 2.46 (s, 3H),
2.33 – 2.26 (m, 0.5H), 2.24 – 2.14 (m, 3H), 2.11 – 2.03 (m,
0.5H), 1.90 – 1.74 (m, 5H), 1.47 – 1.33 (m, 2H), 1.23 – 1.10
(m, 2H). UPLC tR = 1.76 min, Purity UV254 >99%. HRMS
(ESI) m/z: [M + H]+ Calcd for C20H26N5O2 368.2081; Found
368.2093.
– 2.53 (m, 2H), 2.26 (br s, 3H), 2.17 (d, J = 4.4 Hz, 3H), 2.00
(br d, J = 17.6 Hz, 1H), 1.56 – 1.18 (m, 3H), 1.20 – 1.08 (m,
2H).
1
2
3
4
5
6
7
8
To
a solution of 2-[(1S*,4S*,5R*)-5-(6-oxo-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-4-yl)norbornan-2-
ylidene]acetonitrile 61o (0.700 g, 2.48 mmol) in 1,4-
dioxane (20 mL) was added DDQ (0.845 g, 3.73 mmol). The
resulting mixture was stirred at 100 °C for 3 h. After
cooling to rt, the mixture was concentrated under reduced
pressure and the residue was purified by column
chromatography (silica gel column, DCM:MeOH 95:5) to
afford 2-[(1S*,4S*,5R*)-5-(6-oxo-2,7-dihydropyrazolo[3,4-
9
4-[trans-4-[2-(3-Benzylazetidin-1-yl)-2-oxo-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ethyl]cyclohexyl]-3-methyl-2,7-dihydropyrazolo[3,4-b]-
pyridin-6-one (36). Prepared using the general procedure.
1H NMR (600 MHz, DMSO-d6) δ 12.72 (br s, 1H), 11.32 (br
s, 1H), 7.33 – 7.26 (m, 2H), 7.24 – 7.17 (m, 3H), 5.80 (br s,
1H), 4.15 (t, J = 7.9 Hz, 1H), 3.88 (t, J = 8.6 Hz, 1H), 3.81 (dd,
J = 8.3, 4.6 Hz, 1H), 3.55 (dd, J = 9.5, 4.8 Hz, 1H), 2.89 – 2.81
(m, 3H), 2.75 – 2.67 (m, 1H), 2.46 (s, 3H), 1.95 (d, J = 6.9
Hz, 2H), 1.88 – 1.77 (m, 4H), 1.76 – 1.67 (m, 1H), 1.44 –
1.34 (m, 2H), 1.18 – 1.09 (m, 2H). UPLC tR = 2.04 min,
Purity UV254 97%. HRMS (ESI) m/z: [M + H]+ Calcd for
C25H31N4O2 419.2442; Found 419.2459.
b]pyridin-4-yl)norbornan-2-ylidene]acetonitrile
39a
1
(0.500 g, 71%) as an off-white solid. H NMR (300 MHz,
DMSO-d6) δ 13.03 – 12.61 (m, 1H), 11.39 (br d, J = 9.2 Hz,
1H), 5.97 – 5.77 (m, 1H), 5.49 – 5.23 (m, 1H), 3.23 (br d, J =
3.7 Hz, 1H), 3.07 (br d, J = 4.0 Hz, 1H), 2.54 – 2.39 (m, 3H),
2.18 – 1.77 (m, 2H), 1.72 – 1.41 (m, 4H), 1.31 – 1.10 (m,
1H).
A
suspension
of
2-[(1S*,4S*,5R*)-5-(6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-yl)norbornan-2-
3-Methyl-4-[trans-4-[2-oxo-2-(3-phenylpyrrolidin-1-
yl)ethyl]cyclohexyl]-2,7-dihydropyrazolo[3,4-b]pyridin-6-
one (37). Prepared using the general procedure. Two
rotamers around the amide bond are visible in the NMR
ylidene]acetonitrile 39a (0.500 g, 2.14 mmol) and 5% Pt/C
(0.500 g, 50% of moisture) in methanol (30 mL) was
stirred under H2 (1 atm.) at rt for 16 h. The reaction
mixture was filtered through a celite pad and the pad was
washed with methanol. The filtrate was concentrated
under reduced pressure and the residue was subjected to
chiral SFC purification (Column: Chiralpak AD-H 30 × 250
mm, 5 µm; CO2:MeOH 35:65 wt/wt; total flow: 90.0 g/min;
back pressure: 100 bar; detection: UV 296 nm; stack time:
12.5 min; load per injection: 12.5 mg dissolved in 1.25 mL
MeOH; number of injections: 45). Compounds 41 and 42
were isolated as the pure enantiomers, but compounds 39
and 40 were obtained as a mixture. The enantiomeric
purities of 41 and 42 were determined by analytical chiral
SFC (Column: Chiralpak AD-H 4.6 × 250 mm, 5 µm;
CO2:MeOH 60:40 wt/wt, column temperature: 25 °C, total
flow: 4 g/min). The mixture of compounds 39 and 40 was
subjected to a second round of chiral SFC purification
(Column: Chiralpak IC 30 × 250 mm, 5 µm; CO2:co-solvent
60:40 wt/wt, where the co-solvent is 0.5% DEA in MeOH;
total flow: 90.0 g/min; back pressure: 100 bar; detection:
UV 214 nm; stack time: 16.5 min; load per injection: 5.04
mg dissolved in MeCN + MeOH; number of injections: 20),
which afforded compounds 39 and 40 as the pure
enantiomers. The enantiomeric purities of 39 and 40 were
determined by analytical chiral SFC (Column: Chiralpak IC
4.6 × 250 mm, 5 µm; CO2:co-solvent 60:40 wt/wt, where
the co-solvent is 0.5% DEA in MeOH, column temperature:
30 °C, total flow: 4 g/min). The relative stereochemistry of
1
spectrum. H NMR (600 MHz, DMSO-d6) δ 12.73 (br s, 1H),
11.30 (br s, 1H), 7.38 – 7.28 (m, 4H), 7.26 – 7.20 (m, 1H),
5.79 (br s, 1H), 3.93 (dd, J = 9.4, 7.1 Hz, 0.5H), 3.84 (dd, J =
11.5, 7.6 Hz, 0.5H), 3.70 – 3.64 (m, 0.5H), 3.64 – 3.58 (m,
0.5H), 3.52 (td, J = 9.8, 6.8 Hz, 0.5H), 3.46 – 3.40 (m, 0.5H),
3.40 – 3.34 (m, 0.5H), 3.20 (dd, J = 11.4, 9.3 Hz, 0.5H), 2.78
– 2.64 (m, 1H), 2.47 (s, 1.5H), 2.45 (s, 1.5H), 2.33 – 2.26 (m,
0.5H), 2.24 – 2.17 (m, 3H), 2.06 – 1.97 (m, 0.5H), 1.96 –
1.77 (m, 6H), 1.47 – 1.33 (m, 2H), 1.26 – 1.10 (m, 2H).
UPLC tR = 2.00 min, Purity UV254 94%. HRMS (ESI) m/z: [M
+ H]+ Calcd for C25H31N4O2 419.2442; Found 419.2460.
4-[trans-4-[2-(4,4-Dimethyl-1-piperidyl)-2-oxo-
ethyl]cyclohexyl]-3-methyl-2,7-dihydropyrazolo[3,4-
b]pyridin-6-one (38). Prepared using the general
procedure. 1H NMR (600 MHz, DMSO-d6) δ 12.73 (br s, 1H),
11.29 (br s, 1H), 5.78 (br s, 1H), 3.47 – 3.38 (m, 4H), 2.76 –
2.63 (m, 1H), 2.46 (s, 3H), 2.23 (d, J = 6.9 Hz, 2H), 1.89 –
1.80 (m, 4H), 1.79 – 1.70 (m, 1H), 1.45 – 1.33 (m, 2H), 1.32
– 1.27 (m, 2H), 1.24 – 1.19 (m, 2H), 1.19 – 1.10 (m, 2H),
0.94 (s, 6H). UPLC tR = 2.04 min, Purity UV254 >99%. HRMS
(ESI) m/z: [M + H]+ Calcd for C22H33N4O2 385.2598; Found
385.2609.
Norbornane acetonitriles 39 – 42. To a solution of 2-
[(1S*,4S*,5R*)-5-formylnorbornan-2-ylidene]acetonitrile
59o (0.750 g, 4.61 mmol) in ethanol (30 mL) was added
Meldrum’s acid 60 (0.670 g, 4.6 mmol), the mixture was
stirred at rt for 15 min, and 3-methyl-1H-pyrazol-5-amine
43c (0.450 g, 4.6 mmol) was added. The resulting mixture
was stirred at 80 °C for 3 h. After cooling to rt, the mixture
was concentrated under reduced pressure and the residue
was washed with ether. The solid thus obtained was dried
under vacuum to afford 2-[(1S*,4S*,5R*)-5-(6-oxo-2,4,5,7-
tetrahydropyrazolo[3,4-b]pyridin-4-yl)norbornan-2-
1
compounds 39 to 42 was assigned from H NMR NOE
experiments, see supporting information for details.
Absolute configurations were not determined.
[(1S,2S,4S,5R)-5-(3-Methyl-6-oxo-1,2,7-triaza-2,7-
dihydroinden-4-yl)-2-norbornanyl]acetonitrile (39). Yield
1
30 mg, 6.0%. H NMR (600 MHz, DMSO-d6) δ 12.05 (br s,
2H), 5.78 (br s, 1H), 2.93 (br t, J = 7.3 Hz, 1H), 2.53 – 2.49
(m, 1H), 2.48 (s, 3H), 2.43 (dd, J = 16.9, 7.7 Hz, 1H), 2.31 (br
d, J = 4.0 Hz, 1H), 2.16 (br s, 1H), 1.95 – 1.89 (m, 1H), 1.79 –
1.73 (m, 1H), 1.72 – 1.62 (m, 2H), 1.44 – 1.40 (m, 1H), 1.35
ylidene]acetonitrile 61o (0.7 g, 53%) as an off-white solid.
1H NMR (300 MHz, DMSO-d6) δ 11.77 – 11.52 (m, 1H),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
– 1.30 (m, 1H), 1.22 (dt, J = 12.5, 4.5 Hz, 1H). Chiral SFC tR = cyclohexyl-3-methyl-2,4,5,7-tetrahydropyrazolo[3,4-
Page 26 of 33
1
2
3
4
5
6
7
8
14.28 min, 94% ee. UPLC tR = 1.82 min, Purity UV254 95%.
HRMS (ESI) m/z: [M + H]+ Calcd for C16H19N4O 283.1553;
Found 283.1566.
b]pyridin-6-one 61a (854 mg, 3.66 mmol) in DMF (7 mL)
was slowly added POCl3 (0.716 mL, 7.69 mmol) at rt
(exothermic) in a closed microwave vial. The mixture
became yellow and homogenous. It was stirred at 100 °C
for 3 h to produce a dark red solution. After cooling to rt,
volatiles were evaporated and the residual thick syrup was
stirred in water (20 mL) overnight. The resulting yellow
precipitate was filtered off and dried. The so obtained
crude intermediate was suspended in MeCN (50 mL) and
MnO2 (3 g) was added. The mixture was stirred at rt for 2
hours, filtered and evaporated. The residue was purified by
chromatography (ISCO CombiFlash, silica gel column,
heptane:EtOAc) to afford 6-chloro-4-cyclohexyl-3-methyl-
1H-pyrazolo[3,4-b]pyridine-5-carbaldehyde 62 (150 mg,
[(1R,2R,4R,5S)-5-(3-Methyl-6-oxo-1,2,7-triaza-2,7-
dihydroinden-4-yl)-2-norbornanyl]acetonitrile (40). Yield
1
11 mg, 2.2%. H NMR (600 MHz, DMSO-d6) δ 12.72 (br s,
1H), 11.30 (br s, 1H), 5.78 (br s, 1H), 2.92 (br s, 1H), 2.53 –
2.49 (m, 1H), 2.48 (s, 3H), 2.43 (dd, J = 17.0, 7.7 Hz, 1H),
2.31 (br d, J = 3.6 Hz, 1H), 2.16 (br d, J = 2.7 Hz, 1H), 1.95 –
1.89 (m, 1H), 1.79 – 1.73 (m, 1H), 1.72 – 1.62 (m, 2H), 1.44
– 1.40 (m, 1H), 1.35 – 1.30 (m, 1H), 1.22 (dt, J = 12.5, 4.5
Hz, 1H). Chiral SFC tR = 10.28 min, >99% ee. UPLC tR = 1.81
min, Purity UV254 96%. HRMS (ESI) m/z: [M + H]+ Calcd for
C16H19N4O 283.1553; Found 283.1556.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
15%) as a white solid. H NMR (300 MHz, CDCl3) δ 10.93
[(1S,2R,4S,5R)-5-(3-Methyl-6-oxo-1,2,7-triaza-2,7-
(br s, 1H), 10.65 (s, 1H), 3.62 (br t, J = 12.5 Hz, 1H), 2.79 (s,
3H), 2.09 – 1.87 (m, 4H), 1.87 – 1.72 (m, 3H), 1.52 – 1.31
(m, 3H).
dihydroinden-4-yl)-2-norbornanyl]acetonitrile (41). Yield
1
80 mg, 16%. H NMR (400 MHz, DMSO-d6) δ 12.70 (br s,
1H), 11.29 (br s, 1H), 5.77 (br s, 1H), 2.94 (br s, 1H), 2.67
(dd, J = 16.8, 8.1 Hz, 1H), 2.56 (dd, J = 16.9, 8.0 Hz, 1H),
2.50 (s, 3H), 2.32 (br s, 1H), 2.28 – 2.15 (m, 2H), 2.01 – 1.91
(m, 1H), 1.90 – 1.82 (m, 1H), 1.62 – 1.53 (m, 1H), 1.52 –
1.45 (m, 1H), 1.38 – 1.31 (m, 1H), 1.05 – 0.96 (m, 1H).
Chiral SFC tR = 4.38 min, >99% ee. UPLC tR = 1.81 min,
Purity UV254 94%. HRMS (ESI) m/z: [M + H]+ Calcd for
C16H19N4O 283.1553; Found 283.1567.
2-[4-[1,7-bis[(4-Methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]acetonitrile (70). To
a
solution of 2-[4-(3-methyl-6-oxo-2,4,5,7-tetrahydro-
pyrazolo[3,4-b]pyridin-4-yl)cyclohexyl]acetonitrile 61j
(4.00 g, 14.7 mmol) in DMF (20 mL) was added Cs2CO3
(14.4 g, 44.1 mmol) followed by 4-methoxybenzyl chloride
(4.3 mL, 32 mmol). The resulting reaction mixture was
stirred at rt for 16 h. The mixture was then diluted with
water and extracted with EtOAc (2 × 100 mL). The
combined organic layers were dried over anhydrous
Na2SO4 and concentrated under reduced pressure to afford
2-[4-[1,7-bis[(4-methoxyphenyl)methyl]-3-methyl-6-oxo-
4,5-dihydropyrazolo[3,4-b]pyridin-4-
[(1R,2S,4R,5S)-5-(3-Methyl-6-oxo-1,2,7-triaza-2,7-
dihydroinden-4-yl)-2-norbornanyl]acetonitrile (42). Yield
1
80 mg, 16%. H NMR (400 MHz, DMSO-d6) δ 11.96 (br s,
2H), 5.79 (br s, 1H), 2.96 (br t, J = 7.2 Hz, 1H), 2.67 (dd, J =
16.9, 8.0 Hz, 1H), 2.56 (dd, J = 16.8, 8.0 Hz, 1H), 2.50 (s,
3H), 2.32 (br s, 1H), 2.28 – 2.15 (m, 2H), 2.01 – 1.91 (m,
1H), 1.90 – 1.82 (m, 1H), 1.62 – 1.53 (m, 1H), 1.52 – 1.45
(m, 1H), 1.38 – 1.31 (m, 1H), 1.05 – 0.96 (m, 1H). Chiral SFC
tR = 3.65 min, >99% ee. UPLC tR = 1.82 min, Purity UV254
>99%. HRMS (ESI) m/z: [M + H]+ Calcd for C16H19N4O
283.1553; Found 283.1555.
yl]cyclohexyl]acetonitrile 69 (4.0 g, 53%), which was used
without purification.
To
a
solution of 2-[4-[1,7-bis[(4-methoxyphenyl)-
methyl]-3-methyl-6-oxo-4,5-dihydropyrazolo[3,4-
b]pyridin-4-yl]cyclohexyl]acetonitrile 69 (4.0 g, 7.8 mmol)
in 1,4-dioxane (30 mL) was added DDQ (1.8 g, 7.8 mmol).
The resulting mixture was stirred at 100 °C for 16 h. The
mixture was concentrated under reduced pressure and the
residue was dissolved in 1,4-dioxane and filtered. The
filtrate was concentrated under reduced pressure. The
crude product was purified by column chromatography
(silica gel column, hexane:EtOAc 100:0 → 50:50) to afford
2-[4-[1,7-bis[(4-methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]acetonitrile 70 (2.0
Methyl 4-(3-methyl-6-oxo-2,4,5,7-tetrahydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexanecarboxylate (61n). Meldrum’s
acid 60 (423 mg, 2.94 mmol) and methyl trans-4-
formylcyclohexanecarboxylate 59n (500 mg, 2.94 mmol)
were mixed in pyridine (5 mL). Piperidine (29 µL, 0.29
mmol) was added and the reaction mixture was stirred at
room temperature for 3 h. To the reaction mixture was
added ethanol (10 mL) and 3-methyl-1H-pyrazol-5-amine
43c (285 mg, 2.94 mmol). The reaction mixture was
heated at 70 °C for 1 h. After cooling to rt, volatiles were
evaporated and the residue was purified by
chromatography (ISCO CombiFlash, 40 g silica gel column,
EtOAc:MeOH 100:0 → 90:10) to give methyl 4-(3-methyl-6-
oxo-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-4-
1
g, 50%) as a brown solid. H NMR (500 MHz, DMSO-d6) δ
7.29 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.9 Hz, 2H), 6.94 – 6.86
(m, 2 H) 6.83 – 6.78 (m, 2H), 5.96 (s, 1H), 5.32 (s, 2H), 5.09
(s, 2H), 3.72 (s, 3H), 3.71 – 3.68 (m, 3H), 2.77 (br t, J = 11.8
Hz, 1H), 2.52 (s, 3H), 2.50 (s, 2H), 1.86 (br d, J = 11.0 Hz,
4H), 1.68 (ddd, J = 8.8, 5.7, 3.1 Hz, 1H), 1.49 – 1.38 (m, 2H),
1.30 – 1.22 (m, 2H).
yl)cyclohexanecarboxylate 61n (200 mg, 23%) as a yellow
1
solid (mixture of isomers, cis:trans ca. 20:80). H NMR for
4-[4-(2-Hydroxyethyl)cyclohexyl]-1,7-bis[(4-
the trans isomer (600 MHz, DMSO-d6) δ 11.71 (s, 1H),
10.05 (s, 1H), 3.56 (s, 3H), 2.68 – 2.59 (m, 1H), 2.54 – 2.51
(m, 1H), 2.38 – 2.31 (m, 1H), 2.21 – 2.14 (m, 1H), 2.11 (s,
3H), 1.93 – 1.83 (m, 2H), 1.74 – 1.65 (m, 1H), 1.60 – 1.54
(m, 1H), 1.32 – 1.18 (m, 3H), 1.03 – 0.92 (m, 2H).
methoxyphenyl)methyl]-3-methyl-pyrazolo[3,4-b]pyridin-6-
one (72). A solution of 2-[4-[1,7-bis[(4-methoxyphenyl)-
methyl]-3-methyl-6-oxo-pyrazolo[3,4-b]pyridin-4-
yl]cyclohexyl]acetonitrile 70 (600 mg, 1.18 mmol) in
toluene (10 mL) was cooled to 0 °C, 1.0 M DIBAL-H in
hexane (2.35 mL, 2.35 mmol) was added via syringe and
the mixture was stirred for 2 h at 0 °C. The reaction was
6-Chloro-4-cyclohexyl-3-methyl-1H-pyrazolo[3,4-
b]pyridine-5-carbaldehyde (62). To a suspension of 4-
ACS Paragon Plus Environment

Page 27 of 33
Journal of Medicinal Chemistry
quenched with sat. potassium tartrate and extracted with concentrated under reduced pressure. The residue was
1
2
3
4
5
6
7
8
EtOAc (2 × 50 mL). The combined organic layers were
dried over Na2SO4 and concentrated under reduced
purified by column chromatography (silica gel column,
DCM:MeOH 100:0 → 98:2) to afford 3-[4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-pyrazolo[3,4-
b]pyridin-4-yl]cyclohexyl]propanenitrile 74 (70 mg, 66%)
pressure
to
afford
2-[4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-pyrazolo[3,4-
b]pyridin-4-yl]cyclohexyl]acetaldehyde 71 (500 mg, 82%)
as a colorless oil that was used without purification.
1
as a pale yellow viscous oil. H NMR (300 MHz, CDCl3) δ
7.52 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.84 (d, J =
8.8 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.14 (s, 1H), 5.27 (d, J =
4.4 Hz, 4H), 3.79 (s, 3H), 3.76 (s, 3H), 2.65 (br t, J = 12.1 Hz,
1H), 2.42 (s, 3H), 2.37 (t, J = 7.3 Hz, 2H), 1.93 (d, J = 11.0
Hz, 4H), 1.68 – 1.59 (m, 2H), 1.52 – 1.41 (m, 3H), 1.15 –
1.03 (m, 2H).
To
a
solution of 2-[4-[1,7-bis[(4-methoxyphenyl)-
methyl]-3-methyl-6-oxo-pyrazolo[3,4-b]pyridin-4-
yl]cyclohexyl]acetaldehyde 71 (3.5 g, 6.8 mmol) in THF
(70 mL) was added NaBH4 (1.89 g, 50.0 mmol) slowly at 0
°C. The resulting mixture was stirred at rt for 2 h. The
reaction was quenched with sat. NH4Cl at 0 °C and
extracted with EtOAc (2 × 100 mL). The combined organic
layers were dried over Na2SO4, concentrated under
reduced pressure and the residue was purified by column
chromatography (silica gel column, petroleum ether:EtOAc
80:20) to afford 4-[4-(2-hydroxyethyl)cyclohexyl]-1,7-
bis[(4-methoxyphenyl)methyl]-3-methyl-pyrazolo[3,4-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methyl 4-[1,7-bis[(4-methoxyphenyl)methyl]-3-methyl-6-
oxo-4,5-dihydropyrazolo[3,4-b]pyridin-4-
yl]cyclohexanecarboxylate (75). To a solution of 4-(3-
methyl-6-oxo-2,4,5,7-tetrahydropyrazolo[3,4-b]pyridin-4-
yl)cyclohexanecarboxylate 61n (540 mg, 1.85 mmol) in
DMF (25 mL) was added Cs2CO3 (1.51 g, 4.63 mmol)
followed by 4-methoxybenzyl chloride (578 µL, 4.26
mmol). The reaction was stirred at rt for 3 h. A second
portion of 4-methoxybenzyl chloride (58 mg, 0.37 mmol)
was added and the reaction was stirred overnight at rt.
Water (40 mL) was added and the product was extracted
with EtOAc (4 × 20 mL). The combined organic layers were
washed with brine (20 mL), dried over MgSO4, filtered and
evaporated. The residue was purified by chromatography
(ISCO CombiFlash, 40 g silica gel column, heptane:EtOAc
1
b]pyridin-6-one 72 (2.5 g, 71%) as a light yellow oil. H
NMR (400 MHz, DMSO-d6): δ 7.29 (d, J = 8.8 Hz, 2H), 7.15
(d, J = 8.7 Hz, 2H), 6.89 (s, 2H), 6.81 (d, J = 8.8 Hz, 2H), 5.94
(s, 1H), 5.31 (s, 2H), 5.09 (s, 2H), 3.72 (s, 3H), 3.70 (s, 3H)
3.48 – 3.45 (m, 1H), 2.80 – 2.71 (m, 1H), 2.55 (s, 3H), 2.43 –
2.41 (m, 2H), 1.83 – 1.82 (m, 4H), 1.40 – 1.33 (m, 4H), 1.14
– 1.05 (m, 3H).
2-[4-[1,7-bis[(4-Methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]ethyl
100:0
→
0:100) to afford methyl 4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-4,5-
dihydropyrazolo[3,4-b]pyridin-4-
methanesulfonate (73). To
a
solution of 4-[4-(2-
hydroxyethyl)cyclohexyl]-1,7-bis[(4-methoxyphenyl)-
methyl]-3-methyl-pyrazolo[3,4-b]pyridin-6-one 72 (150
mg, 0.29 mmol) in DCM (10 mL) was added triethylamine
(0.13 mL, 0.87 mmol) at 0 °C. After 5 min, methanesulfonyl
chloride (33 µL, 0.43 mmol) was added. The resulting
mixture was stirred at rt for 30 min. The reaction was
quenched with sat. NaHCO3 and extracted with DCM (2 ×
50 mL). The combined organic layers were washed with
water (30 mL), brine (50 mL), dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The
residue was purified by column chromatography (silica gel
column, DCM:MeOH 100:0 → 98:2) to afford 2-[4-[1,7-
bis[(4-methoxyphenyl)methyl]-3-methyl-6-oxo-
yl]cyclohexanecarboxylate 75 (903 mg, 92%) as a mixture
of isomers, cis:trans ca. 20:80. 1H NMR for the trans isomer
(600 MHz, CDCl3) δ 7.42 – 7.37 (m, 2H), 7.03 – 6.99 (m,
2H), 6.86 – 6.81 (m, 2H), 6.80 – 6.73 (m, 2H), 5.12 – 5.05
(m, 3H), 4.90 – 4.84 (m, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.63
(s, 3H), 2.73 – 2.65 (m, 2H), 2.59 – 2.55 (m, 1H), 2.11 – 2.02
(m, 4H), 1.90 – 1.80 (m, 2H), 1.65 – 1.59 (m, 1H), 1.54 –
1.47 (m, 1H), 1.34 – 1.17 (m, 3H), 0.95 – 0.77 (m, 2H).
Methyl 4-[1,7-bis[(4-methoxyphenyl)methyl]-3-methyl-6-
oxo-pyrazolo[3,4-b]pyridin-4-yl]cyclohexanecarboxylate
(76). To
a
solution of methyl 4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-4,5-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]ethyl
dihydropyrazolo[3,4-b]pyridin-4-
methanesulfonate 73 (120 mg, 69%) as a pale yellow
viscous oil. H NMR (300 MHz, CDCl3) δ 7.52 (d, J = 8.8 Hz,
yl]cyclohexanecarboxylate 75 (794 mg, 1.49 mmol) in 1,4-
dioxane (50 mL) was added DDQ (576 mg, 2.54 mmol) and
the reaction mixture was heated at 100 °C for 1 h. After
cooling to rt, volatiles were removed by rotary evaporation
and the residue was purified by chromatography (ISCO
CombiFlash, 40 g silica gel column, heptane:EtOAc 100:0 →
1
2H), 7.09 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.41 Hz, 2H), 6.79
(d, J = 8.8 Hz, 2H), 6.18 (s, 1H), 5.27 (d, J = 4.8 Hz, 4H), 4.29
(t, J = 6.8 Hz, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.01 (s, 3H),
2.65 (br t, J = 12.1 Hz, 1H), 2.42 (s, 3H), 1.92 (br d, J = 11.3
Hz, 4H), 1.71 (q, J = 6.6 Hz, 2H), 1.43 (br d, J = 10.6 Hz, 3H),
1.18 – 1.02 (m, 2H).
0:100)
to
afford
methyl
4-[1,7-bis[(4-
methoxyphenyl)methyl]-3-methyl-6-oxo-pyrazolo[3,4-
b]pyridin-4-yl]cyclohexanecarboxylate 76 (810 mg, 87%)
as a semi-solid. 1H NMR (600 MHz, CDCl3) δ 7.53 – 7.48 (m,
2H), 7.11 – 7.06 (m, 2H), 6.86 – 6.82 (m, 2H), 6.80 – 6.76
(m, 2H), 6.17 (s, 1H), 5.29 (s, 2H), 5.27 (s, 2H), 3.79 (s, 3H),
3.76 (s, 3H), 3.68 (s, 3H), 2.75 – 2.67 (m, 1H), 2.44 (s, 3H),
2.38 (tt, J = 12.2, 3.7 Hz, 1H), 2.16 – 2.09 (m, 2H), 2.00 –
1.93 (m, 2H), 1.62 – 1.51 (m, 2H), 1.50 – 1.39 (m, 2H).
3-[4-[1,7-bis[(4-Methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]propanenitrile (74).
To a solution of 2-[4-[1,7-bis[(4-methoxyphenyl)methyl]-
3-methyl-6-oxo-pyrazolo[3,4-b]pyridin-4-yl]cyclohexyl]-
ethyl methanesulfonate 73 (120 mg, 0.202 mmol) in DMF
(10 mL) was added NaCN (11.8 mg, 0.241 mmol) at rt. The
resulting reaction mixture was stirred at 90 °C for 16 h.
The mixture was cooled to rt, diluted with water and
extracted with EtOAc (2 × 30 mL). The combined organic
layers were dried over anhydrous Na2SO4 and
4-[1,7-bis[(4-Methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexanecarboxylic
acid
.
(77). A solution of LiOH H2O (29.7 mg, 0.709 mmol) in
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Journal of Medicinal Chemistry
Page 28 of 33
water (1 mL) was added to a solution of methyl 4-[1,7-
chromatography (silica gel column, petroleum ether:EtOAc
90:10) to afford (1S*,2R*)-2-
1
2
3
4
5
6
7
8
bis[(4-methoxyphenyl)methyl]-3-methyl-6-oxo-
pyrazolo[3,4-b]pyridin-4-yl]cyclohexanecarboxylate
76
methylcyclohexanecarbaldehyde 59d (0.600 g, 30%) as a
1
(289 mg, 0.545 mmol) in THF (5 mL). The reaction mixture
was stirred overnight at rt and then at 40 °C for 3 days. The
reaction mixture was poured into water (20 mL) and the
aqueous phase was washed with EtOAc (3 × 10 mL). The
aqueous phase was then acidified with 4 M HCl (pH = 3)
and the product was extracted with EtOAc (3 × 10 mL).
The combined organic layers were washed with brine (10
mL), dried over MgSO4, filtered and evaporated to afford 4-
[1,7-bis[(4-methoxyphenyl)methyl]-3-methyl-6-oxo-
light yellow oil. H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H),
2.40 (m, 1H), 2.21 (m, 1H), 1.85 (m, 1H), 1.79 – 1.45 (m,
6H), 1.35 (m, 1H), 0.90 (m, 3H).
Isopropyl 5-norbornene-2-exo-carboxylate (82) and
isopropyl 5-norbornene-2-endo-carboxylate (83). Adapted
from Ricks et al.31 To a solution of 5-norbornene-2-
carboxylic acid 81 (commercial grade, endo/exo mixture,
1.38 g, 10.0 mmol) in 1,2-dichloroethane (10 mL) was
added DMAP (367 mg, 3.00 mmol), 2-propanol (0.91 mL,
12 mmol) and EDCI (2.30 g, 12.0 mmol). The resulting
white suspension was stirred at rt (slightly exothermic) for
2 h at which point a clear solution was obtained and TLC
showed complete consumption of the starting material.
The solution was poured into water/sat. NaHCO3 solution
1:1 (70 ml) and the mixture was extracted with DCM (2 ×
80 mL). The combined organic phases were washed with
brine (70 mL), dried over Na2SO4, and volatiles were
evaporated to afford a turbid, pale yellow oil. The endo and
exo isomers were separated by chromatography in two
stages. First, the crude product was purified using a Grace
Reveleris system (silica gel column, heptane:EtOAc 100:0
→ 90:10) to afford one fraction containing a ~4:1 endo/exo
mixture (1.38 g, colorless liquid) followed by a second
fraction containing the pure endo isomer 83 (311 mg). The
first fraction was then purified again by column
chromatography (silica gel column, heptane:diisopropyl
ether 98:2) to afford one fraction containing the pure exo
isomer 82 (353 mg, 14%, colorless liquid, contains ~25%
heptane), followed by a second fraction containing the
pure endo isomer 83 (878 mg). This was combined with
the pure endo fraction from the first chromatographic
separation to afford the pure endo isomer 83 (1.05 g,
55%).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pyrazolo[3,4-b]pyridin-4-yl]cyclohexanecarboxylic acid 77
(142 mg, 50%), which was used without purification in the
1
next step. H NMR (600 MHz, DMSO-d6) δ 12.06 (br s, 1H),
7.31 – 7.27 (m, 2H), 7.17 – 7.13 (m, 2H), 6.90 – 6.86 (m,
2H), 6.83 – 6.79 (m, 2H), 5.95 (s, 1H), 5.32 (s, 2H), 5.09 (s,
2H), 3.72 (s, 3H), 3.70 (s, 3H), 2.83 – 2.76 (m, 1H), 2.52 (s,
3H), 2.27 (tt, J = 12.0, 3.6 Hz, 1H), 2.02 – 1.95 (m, 2H), 1.90
– 1.83 (m, 2H), 1.55 – 1.37 (m, 4H).
2-[trans-4-(3-Methyl-6-oxo-2,7-dihydropyrazolo[3,4-
b]pyridin-4-yl)cyclohexyl]acetic acid (78). To a stirred
solution
of
2-[trans-4-(3-methyl-6-oxo-2,7-
dihydropyrazolo[3,4-b]pyridin-4-
yl)cyclohexyl]acetonitrile 21 (450 mg, 1.66 mmol) in 1,4-
dioxane (4.5 mL) was added conc. HCl (1 mL). The
resulting reaction mixture was stirred at 100 °C for 24 h.
The mixture was concentrated under reduced pressure.
The residue was diluted with water (5 mL) and the
resulting precipitate was filtered, washed with water and
dried under vacuum to afford 2-[trans-4-(3-methyl-6-oxo-
2,7-dihydropyrazolo[3,4-b]pyridin-4-yl)cyclohexyl]acetic
acid 78 (400 mg, 83%) as a pale brown solid. 1H NMR (400
MHz, DMSO-d6) δ 12.00 (br s, 3H), 5.80 (s, 1H), 2.71 (t,
J=11.6 Hz, 1H), 2.45 (s, 3H), 2.15 (d, J = 6.8 Hz, 2H), 1.85 (d,
J = 10.4 Hz, 4H), 1.75 - 1.73 (m, 1H), 1.40 (q, J = 12.8 Hz,
2H), 1.16 (q, J = 11.2 Hz, 2H).
1
Isopropyl 5-norbornene-2-exo-carboxylate 82: H NMR
[(1S*,2R*)-2-methylcyclohexyl]methanol (80). To
a
(300 MHz, CDCl3) δ 6.16 – 6.07 (m, 2H), 5.01 (hept, J = 6.3
Hz, 1H), 3.02 (br s, 1H), 2.91 (br s, 1H), 2.22 – 2.14 (m, 1H),
1.96 – 1.86 (m, 1H), 1.56 – 1.49 (m, 1H), 1.42 – 1.29 (m,
2H), 1.24 (dd, J = 6.3, 1.0 Hz, 6H).
Isopropyl 5-norbornene-2-endo-carboxylate 83: 1H NMR
(300 MHz, CDCl3) δ 6.18 (dd, J = 5.7, 3.0 Hz, 1H), 5.92 (dd, J
= 5.7, 2.8 Hz, 1H), 4.94 (hept, J = 6.3 Hz, 1H), 3.20 (s, 1H),
2.96 – 2.84 (m, 2H), 1.94 – 1.81 (m, 1H), 1.48 – 1.37 (m,
2H), 1.34 – 1.23 (m, 2H), 1.20 (dd, J = 6.3, 1.8 Hz, 6H).
solution of methyl (1S*,2R*)-2-
methylcyclohexanecarboxylate 79 [1] (3.00 g, 19.2 mmol)
in THF (30 mL) was added 1 M LiAlH4 in THF (38.4 mL,
38.4 mmol) slowly at −30 °C. The resulting mixture was
stirred at −30 °C for 2 h and was then warmed to 0 °C. The
reaction was quenched with NH4Cl (sat) and the mixture
was extracted with diethyl ether (2 × 100 mL). The
combined organic layers were dried over Na2SO4,
concentrated under reduced pressure and the residue was
purified by column chromatography (silica gel column,
DCM) to afford [(1S*,2R*)-2-methylcyclohexyl]methanol
5-Norbornene-2-endo-methanol (84). Adapted from Ricks
et al.31 To a solution of isopropyl 5-norbornene-2-endo-
carboxylate (500 mg, 2.77 mmol) in THF (6 mL) was added
1 M LiAlH4 solution in THF (1.75 mL, 1.75 mmol) over 2
min at rt. The resulting pale yellow solution was stirred for
1 h at 40 °C and was then cooled to rt. The reaction was
quenched by the sequential addition of water (70 μL), 15%
aq. NaOH (70 μL) and water (210 μL). The solid precipitate
formed was removed by filtration and the filter cake was
washed with diethyl ether (30 mL). The filtrate was
washed with brine (30 mL) and the aqueous layer was
extracted with diethyl ether (30 mL). The combined
organic phases were dried over Na2SO4 and volatiles were
evaporated to afford 5-norbornene-2-endo-methanol 84 as
a colorless oil (408 mg) that was used without purification.
1
80 (2.04 g, 83%) as a light yellow oil. H NMR (400 MHz,
CDCl3) δ 3.56 – 3.44 (m, 2H), 2.39 – 2.37 (m, 3H), 1.95 (m,
1H), 1.65 (m, 2H), 1.46 – 1.36 (m, 6H), 0.80 (m, 3H).
(1S*,2R*)-2-methylcyclohexanecarbaldehyde (59d). To a
solution of [(1S*,2R*)-2-methylcyclohexyl]methanol 80
(2.04 g, 15.9 mmol) in DCM (60 mL) was added NaHCO3
(1.31g, 15.9 mmol) followed by Dess-Martin periodinane
(9.94 g, 23.4 mmol) slowly at 0 °C. The resulting mixture
was stirred at rt for 3 h. On completion, the reaction
mixture was diluted with DCM (100 mL) and filtered
through celite. The filtrate was concentrated under
reduced pressure and the residue was purified by column
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Journal of Medicinal Chemistry
1H NMR (300 MHz, CDCl3) δ 6.15 (dd, J = 5.8, 3.0 Hz, 1H),
NaHCO3, dried over MgSO4, filtered and evaporated to
1
2
3
4
5
6
7
8
5.96 (dd, J = 5.8, 2.9 Hz, 1H), 3.40 (dd, J = 10.3, 6.5 Hz, 1H),
3.26 (dd, J = 10.5, 8.8 Hz, 1H), 2.93 (br s, 1H), 2.81 (br s,
1H), 2.37 – 2.22 (m, 1H), 1.82 (ddd, J = 11.6, 9.2, 3.8 Hz,
1H), 1.50 – 1.41 (m, 1H), 1.30 – 1.22 (m, 1H), 0.53 (ddd, J =
11.6, 4.5, 2.6 Hz, 1H).
afford methyl trans-4-(methylsulfonyloxymethyl)cyclo-
hexanecarboxylate 89 (1.21 g, 99%) which was used
1
without purification. H NMR (300 MHz, DMSO-d6) δ 4.01
(d, J = 6.2 Hz, 2H), 3.59 (s, 3H), 3.15 (s, 3H), 2.25 (tt, J =
12.2, 3.6 Hz, 1H), 2.00 – 1.87 (m, 2H), 1.85 – 1.72 (m, 2H),
1.72 – 1.56 (m, 1H), 1.43 – 1.25 (m, 2H), 1.21 – 0.95 (m,
2H).
Norbornane-2-endo-methanol (85). To a solution 5-
norbornene-2-endo-methanol 84 (~90% purity, 185 mg,
1.34 mmol) in EtOAc (4.6 mL) was added 10% Pd/C (18.5
mg, 0.0174 mmol). The reaction was hydrogenated for 4 h
10 min at rt and ambient pressure (complete conversion
by TLC). The catalyst was removed by filtration and the
filtrate was evaporated to afford norbornane-2-endo-
methanol 85 (~90% purity, 181 mg, 96%) as a colorless
Methyl
(90).
(methylsulfonyloxymethyl)cyclohexanecarboxylate
trans-4-(cyanomethyl)cyclohexanecarboxylate
A
solution
of
methyl
trans-4-
9
89
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(1.22 g, 4.87 mmol) in DMSO (2 mL) was added to a
suspension of KCN (635 mg, 9.75 mmol) in DMSO (2 mL)
and the mixture was stirred at rt for 30 min. The mixture
was diluted with water (20 mL) and sat. NaHCO3 (3 mL). It
was extracted with ether (3 × 20 mL), the combined
extracts were washed with brine, dried over MgSO4,
filtered and evaporated to dryness to afford methyl trans-
4-(cyanomethyl)cyclohexanecarboxylate 90 (720 mg,
1
oil that was used without purification. H NMR (300 MHz,
CDCl3) δ 3.70 – 3.43 (m, 2H), 2.27 (br s, 1H), 2.20 (br s, 1H),
2.14 – 1.98 (m, 1H), 1.72 (ddd, J = 12.0, 4.7, 2.9 Hz, 1H),
1.61 – 1.44 (m, 2H), 1.41 – 1.24 (m, 3H), 1.14 – 1.01 (m,
1H), 0.63 (ddd, J = 12.2, 5.2, 2.2 Hz, 1H).
1
81%) which was used without purification. H NMR (300
Norbornane-2-endo-carbaldehyde (59f). To a solution of
oxalyl chloride (0.16 mL, 1.9 mmol) in DCM (8.5 mL) at
−75 °C was added a solution of DMSO (0.183 mL, 2.58
mmol) in DCM (0.45 mL) via syringe over 4 min while
maintaining the temperature between −75 °C and −70 °C.
The resulting colorless solution was stirred for 10 min at
−75 °C. Then a solution of norbornane-2-endo-methanol
85 (163 mg, 1.29 mmol) in DCM (1.5 mL) was added via
syringe over 3 min while maintaining the temperature
between −75 °C and −70 °C. After 30 min at −75 °C,
triethylamine (0.600 mL, 4.30 mmol) was added via
syringe over 3 min at −75 °C to −68 °C. The viscous
colorless solution was stirred for a further 20 min at −75
°C. The cooling bath was then removed, and the mixture
was warmed to rt over 30 min. After a further 30 min at rt,
the resulting white turbid mixture was washed with water
(30 ml). The aqueous phase was extracted with DCM (30
mL) and the combined organic phases were washed with
brine, dried over Na2SO4, and concentrated under
evaporation at max. 30 °C / 50 mbar to afford a yellow
turbid oil. The crude product was purified by column
chromatography (silica gel column, petroleum ether:TBME
99:1 → 98:2) to afford norbornane-2-endo-carbaldehyde
(115 mg, 68%) as a colorless oil. 1H NMR (300 MHz, CDCl3)
δ 9.77 (d, J = 1.1 Hz, 1H), 2.77 – 2.66 (m, 2H), 2.31 (br t,
1H), 1.73 – 1.47 (m, 3H), 1.47 – 1.33 (m, 2H), 1.32 – 1.18
(m, 2H), 0.93 – 0.80 (m, 1H).
MHz, DMSO-d6) δ 3.58 (s, 3H), 2.44 (d, J = 6.5 Hz, 2H), 2.24
(tt, J = 12.2, 3.6 Hz, 1H), 1.98 – 1.85 (m, 2H), 1.84 – 1.71 (m,
2H), 1.69 – 1.49 (m, 1H), 1.44 – 1.25 (m, 2H), 1.17 – 0.97
(m, 2H).
2-[trans-4-(Hydroxymethyl)cyclohexyl]acetonitrile (91).
To
a
solution
of
methyl
trans-4-
(cyanomethyl)cyclohexanecarboxylate 90 (720 mg, 3.97
mmol) in dry THF (20 mL) was added 2.0 M LiBH4 solution
in THF (4.0 mL, 8.0 mmol) and the resulting colorless
solution was stirred at rt for 2 h and then at 40 °C
overnight. The reaction was quenched by the addition of
brine (30 mL) and extracted with EtOAc (3 × 30 mL). The
organic phase was dried over MgSO4, filtered and
concentrated
in
vacuo
to
afford
2-[trans-4-
(hydroxymethyl)cyclohexyl]acetonitrile 91 (571 mg, 84%)
as a clear oil which was used without purification. 1H NMR
(600 MHz, DMSO-d6) δ 4.36 (t, J = 5.3 Hz, 1H), 3.22 – 3.17
(m, 2H), 2.42 (d, J = 6.5 Hz, 2H), 1.81 – 1.70 (m, 4H), 1.57 –
1.47 (m, 1H), 1.32 – 1.22 (m, 1H), 1.06 – 0.97 (m, 2H), 0.94
– 0.82 (m, 2H).
2-(trans-4-Formylcyclohexyl)acetonitrile (59j). A solution
of oxalyl chloride (0.47 mL, 5.6 mmol) in DCM (25 mL)
under Ar was cooled to −70 °C. A solution of DMSO (529
µL, 7.45 mmol) in DCM (2 mL) was added slowly over 5
min, such that the temperature was kept between −65 and
−70 °C. The reaction mixture was stirred at −70 °C for 15
min. A solution of 2-[trans-4-(hydroxymethyl)cyclohexyl]-
acetonitrile 91 (571 mg, 3.73 mmol) in DCM (5 mL) was
added slowly, such that the temperature was kept between
−75 and −70 °C. The reaction mixture was stirred at −75 °C
for 30 min after which Et3N (1.73 mL, 12.4 mmol) was
added dropwise, such that the temperature was kept
between −74 °C and −68 °C. The resulting solution was
stirred at −75 °C for 30 min. The cooling bath was removed
and the temperature was raised to room temperature over
30 min. After a further 5 min at room temperature, the
clear yellow solution was quenched with 150 ml water.
The phases were separated and the aqueous phase was
extracted with DCM (2 × 100 mL). The combined organic
phases were washed with brine, dried over Na2SO4, filtered
and concentrated by evaporation to afford 2-(trans-4-
Norbornane-2-exo-carbaldehyde (59e). Prepared using
1
the method described above for the endo isomer 59f. H
NMR (300 MHz, CDCl3) δ 9.63 (d, J = 1.5 Hz, 1H), 2.61 –
2.53 (m, 1H), 2.38 – 2.27 (m, 2H), 1.95 – 1.84 (m, 1H), 1.69
– 1.47 (m, 3H), 1.40 – 1.19 (m, 3H), 0.94 – 0.78 (m, 1H).
Methyl
trans-4-(methylsulfonyloxymethyl)cyclohexane-
carboxylate (89). To a solution of methyl trans-4-
(hydroxymethyl)cyclohexanecarboxylate 88 (836 mg,
4.85 mmol) in DCM (10 mL) was added Et3N (1.01 mL, 7.28
mmol). The solution was cooled in an ice bath and MsCl
(451 µL, 5.83 mmol) was added dropwise over 5 min. The
mixture was stirred at 0 °C for 20 min after which the
reaction was quenched by the addition of water (20 mL), 4
N HCl (1.8 mL, 7.3 mmol) and DCM (20 mL). The layers
were separated, the organic layer was washed with sat.
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