Tetrahydroindazoles as interleukin-2 inducible T-cell kinase inhibitors. Part II. Second-generation analogues with enhanced potency, selectivity, and pharmacodynamic modulation in vivo

Article abstract of DOI:10.1021/jm501998m

The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

Full text of DOI:10.1021/jm501998m

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Article
Tetrahydroindazoles as Interleukin-2 Inducible T-Cell Kinase
Inhibitors. Part II. Second Generation Analogs with Enhanced
Potency, Selectivity, and Pharmacodynamic Modulation in Vivo
Jason Burch, Kathy Barrett, Yuan Chen, Jason DeVoss, Charles Eigenbrot, Richard
Goldsmith, Hicham Ismaili, Kevin Lau, Zhonghua Lin, Daniel Fred Ortwine, Paul
McEwan, John Barker, Claire Ellebrandt, Daniel Kordt, Daniel B Stein, Xiaolu Wang,
Yong Chen, Baihua Hu, Xiaofeng Xu, Po-wai Yuen, Yamin Zhang, and Zhonghua Pei
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501998m • Publication Date (Web): 06 Apr 2015
Downloaded from http://pubs.acs.org on April 11, 2015
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Tetrahydroindazoles as Interleukinꢀ2 Inducible Tꢀ
Cell Kinase Inhibitors. Part II. Second Generation
Analogs with Enhanced Potency, Selectivity, and
Pharmacodynamic Modulation in Vivo
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Jason D. Burch,† Kathy Barrett, † Yuan Chen,† Jason DeVoss,† Charles Eigenbrot,† Richard
Goldsmith, † M. Hicham A. Ismaili,† Kevin Lau,† Zhonghua Lin, † Daniel F. Ortwine,† Ali A.
Zarrin,† Paul A. McEwan,⊥ John J. Barker,⊥ Claire Ellebrandt,‡Daniel Kordt,‡ Daniel B. Stein,‡
Xiaolu Wang,‡ Yong Chen, § Baihua Hu,§ Xiaofeng Xu, § Po-Wai Yuen,§ Yamin Zhang,§ and
Zhonghua Pei†,*
†Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, United States
⊥Evotec (UK) Ltd., 114 Milton Park, Abingdon, Oxfordshire, OX14 4RZ, United Kingdom
‡Evotec AG, Manfred Eigen Campus, Essener Bogen 7, Dꢀ22419, Hamburg, Germany
§Pharmaron Beijing Ltd. Co., No. 6 TaiHe Rd. BDA, 100176, Beijing, P. R. China
ABSTRACT: The medicinal chemistry community has directed considerable efforts toward the
discovery of selective inhibitors of interleukinꢀ2 inducible Tꢀcell kinase (ITK) given its role in Tꢀ
cell signaling downstream of the Tꢀcell receptor (TCR), and the implications of this target for
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inflammatory disorders such as asthma. We have previously disclosed a structureꢀ and propertyꢀ
guided lead optimization effort which resulted in the discovery of a new series of
tetrahydroindazoleꢀcontaining selective ITK inhibitors. Herein we disclose further optimization
of this series that resulted in further potency improvements, reduced offꢀtarget receptor binding
liabilities and reduced cytotoxicity. Specifically, we have identified a correlation between the
basicity of solubilizing elements in the ITK inhibitors and offꢀtarget antiꢀproliferative effects,
which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized
analogs were shown to reduce ILꢀ2 and ILꢀ13 production in vivo following oral dosing in mice.
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INTRODUCTION
The Tec family kinases include ILꢀ2 inducible T cell kinase (ITK), resting lymphocyte kinase
(RLK), Bruton’s tyrosine kinase (Btk), tyrosine kinase expressed in hepatocellular carcinoma
(Tec) and bone marrow expressed kinase (Bmx). ITK is a tyrosine kinase that is involved in T
cell development, differentiation and effector function.1 While ITK, RLK and Tec are all
expressed in T cells, the prominent role for ITK and RLK in T cell receptor signaling (TCR),
phospholipase gammaꢀ1 (PLCγ1) phosphorylation and subsequent Ca2+ mobilization has been
established through murine knockꢀout studies.2 Several groups have shown that ITK deficiency
results in selective impairment of T helper 2 (Th2) cell function relative to other T cell subtypes.3
Furthermore, following ovalbumin challenge, ITKꢀ/ꢀ mice demonstrate reduced lung
inflammation, eosinophil infiltration and mucous production.4a Studies using an ITK kinaseꢀdead
transgenic mouse model suggest that the ITK kinase activity is required for the control of Th2
responses and the development of allergic asthma. 4b
The wealth of preclinical evidence supporting the role of ITK in allergic asthma and other
inflammatory disorders has prompted intense research effort from the pharmaceutical community
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toward the discovery of selective inhibitors of this kinase.5 Preꢀclinical in vivo activity using
ITK inhibitors has been reported, resulting in the reduction of ILꢀ2 production in one example,5f
and the reduction of ILꢀ4 production and lung inflammation in another.5b
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We have previously disclosed the evolution of an indazole series of ITK inhibitors discovered
from an HTS campaign5m using propertyꢀ and structureꢀbased methods to a new series of
tetrahydroindazole (THI) inhibitors, ultimately resulting in the potent and selective inhibitor
GNEꢀ9822 (1, Figure 1).5q As we continued to profile this lead compound toward in vivo
efficacy studies, we uncovered offꢀtarget receptor binding and undesirable antiꢀproliferative
effects in hepatocytes and Jurkat cells (vide infra). Herein we disclose further optimization
efforts that simultaneously eliminated these liabilities while further improving ITK inhibitory
potency, and ultimately resulted in inhibitors capable of modulating Tꢀcell receptor signaling in
vivo.
Figure 1. Previously disclosed indazole and tetrahydroindazole series of ITK inhibitors
CHEMISTRY
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Compounds were prepared in a manner analogous to that disclosed previously (Scheme 1).5q
Racemic nitropyrazoles 2i-ix were reduced to aminopyrazoles 3i-ix with palladium on carbon
under an atmosphere of hydrogen, then the requisite inhibitors 17-30 were obtained by HATUꢀ
mediated coupling with tetrahydroindazole carboxylic acids 4-6. Single stereoisomers were
obtained by supercritical fluid chromatography (SFC) on chiral solid supports when necessary.
Scheme 1. General Synthesis of inhibitors 17-30a
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a
NO2
NH2
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2i-ix
3i-ix
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NH
N
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17, 19-28
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S O
aReagents and conditions: (a) H2 (1 atm), 10% Pd/C, EtOH, rt; (b) 4-6, HATU, iPr2NEt, DMF,
rt.
The synthesis of tetrahydroindazole carboxylic acids 4 and 5 has been disclosed previously;5q
synthesis of carboxylic acid 6 is shown in Scheme 2. Difluorocyclopropanation6 of protected
cyclohexanone 7 yielded cyclopropane 8, which was deprotected under acidic conditions to
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provide ketone 9. Claisen condensation with diethyl oxalate mediated by potassium tertꢀ
butoxide7 provided αꢀketo ester 10, which was subsequently converted to carboxylic acid 6 via
cyclization with hydrazine followed by ester hydrolysis.
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Scheme 2. Synthesis of tetrahydroindazole carboxylic acid 6a
aReagents and conditions: (a) TMSCF3, NaI, THF, 65 °C; (b) pꢀMeC6H4SO3H, acetone/water,
50 °C; (c) diethyl oxalate, tꢀBuOK, THF, ꢀ70 °C; (d) hydrazine, AcOH, 120 °C; (e) NaOH,
EtOH, water, 50 °C.
While the synthesis of aminopyrazoles 3i-iii was presented in our previous publication,5q
preparation of nitropyrazoles 2iv-ix is shown in Scheme 3. Addition of phenyl magnesium
bromide to aldehydes 11v-vii provided benzylic alcohols 12v-vii, which were subsequently
converted to nitropyrazole sulfones by a Mitsunobu reaction8with nitropyrazole followed by
mCPBAꢀmediated oxidation. Nitropyrazole 2ix at the sulfoxide oxidation state could be
obtained by careful control of the equivalents of the oxidizing agent. Finally, the lithium anion of
tetrahydrothiopyran sulfoxide (13) was added to benzaldehyde to provide benzyl alcohol 14 as a
mixture of diastereomers.9 Mitsunobu reaction and oxidation provided nitropyrazole 2viii.
Scheme 3. Synthesis of nitropyrazoles 2iv-ixa
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aReagents and conditions: (a) PhMgBr, THF, 0 °C ꢀ rt; (b) 4ꢀnitropyrazole, PPh3, DIAD,
THF, 0 °C ꢀ rt; (c) mCPBA, DCM, 0 °C (excess for 2v-viii; 1.0 equiv. for 2ix).
For inhibitor 18, a slightly modified protocol (Scheme 4) was necessary due to issues with
elimination of the requisite nitropyrazole. 2ꢀ(Methylthio)ꢀ1ꢀphenylethanꢀ1ꢀol (15) was converted
to the aminopyrazole 16 via a Mitsonobu reaction without oxidation to the sulfone followed by
reduction of the nitro group. Amide bond formation with SEMꢀprotected indazole acid 4b,5q
oxidation to the sulfone with mCPBA, and then SEM deprotection provided 18.
Scheme 4. Synthesis of inhibitor 18a
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aReagents and conditions: (a) 4ꢀnitropyrazole, PPh3, DIAD, THF, 0 °C ꢀ rt; (b) 10% Pd/C, H2,
MeOH, rt; (c) 4b, HATU, Pr2NEt, DMF, rt; (d) mCPBA, DCM, 0 °C; (e) TFA, Pr3SiH, DCM,
rt.
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RESULTS and DISCUSSION
Our previous publication highlighted the identification of an outꢀofꢀplane selectivity pocket
near the phenylalanine “gatekeeper” (Phe435) of ITK, occupation of which resulted in a dramatic
improvement in broad kinase selectivity.5q That effort resulted in the identification of the
inhibitor GNEꢀ9822 (1), which exhibited strong ITK inhibitory activity (c.f. Figure 1) and
reasonable kinase selectivity (12 out of 310 kinases were inhibited >50% at 0.1 ꢀM). This
promising in vitro profile prompted further profiling of this compound, during which undesirable
in vitro toxicity liabilities were uncovered. When subjected to the CEREP receptor panel, 1
bound strongly (96%) to the 5ꢀHT2A receptor, dopamine and 5ꢀHT transporters (102%) and the
Ca2+ channel (88%), and also exhibited strong inhibition (88%) of the hERG channel (patch
clamp assay) at 10 ꢀM.10 Compound 1 exhibited antiproliferative effects in hepatocytes (IC50 =
15 ꢀM), where ITK is not expressed. In retrospect, receptor promiscuity might have been
expected, given that the structure of 1 shares the pharmacophore features of binding to hERG
and other receptors: strongly basic amine (calculated pKa ~ 9) tethered to a lipophilic,
polyaromatic core.11 On the other hand, the undesired broad cellular cytotoxicity was somewhat
surprising, given that kinase selectivity had improved dramatically relative to our earlier
chemical matter. This cytotoxicity would make 1 unsuitable for asthma indication. We thus
chose to explore the reduction of cytotoxicity associated with this series of compounds.
We developed and implemented a proliferation assay of Jurkat cells with no TCR stimulation
by the measurement of ATP production, which allowed us to gauge the offꢀtarget cytotoxicity of
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the compounds. Jurkat cells were selected for this investigation as this was the same cell type
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used for determination of cellular ITK activity, where inhibition of phosphorylation of PLCγꢀ1
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was measured.
When comparing Jurkat cell proliferation versus inhibition of PLCγ
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phosphorylation (Figure 2), we were encouraged by the lack of correlation between cellular ITK
inhibition and antiproliferative effects (cytotoxicity), which suggests that the cytotoxicity is not
caused by ITK inhibition.
Figure 2. Inhibition of Jurkat cell proliferation versus inhibition of phosphorylation of PLCγꢀ1
for all THI inhibitors colorꢀcoded by calculated most basic pKa (<7.5, green; >7.5, red). The
potencies are expressed as –log10(IC50) where IC50 values are in nM unit.
We then invested significant effort to identify offꢀtarget(s) responsible for this cytotoxicity.
After extensive kinase profiling of a number of compounds, we were unable to identify an offꢀ
target kinase or combination of kinases, inhibition of which correlated with observed
cytotoxicity in Jurkat cells.12 Instead, a plot of amine basicity within the THI series showed that
reduced basicity (calculated most basic pKa <7.513) was associated with reduced cytotoxicity
(Figure 2). Intrigued by the possibility that reduced basicity might also improve the receptor
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binding profile of the compounds (vide supra), we implemented a criteria that all new designed
molecules must have a calculated most basic pKa < 7.5 to increase the probability that these
inhibitors would have an improved in vitro safety profile.
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Figure 3. Coꢀcrystal structure of 1 with the kinase domain of ITK.5q Favorable interactions
between proteinꢀligand are denoted by dashed lines with cylinders. The solvent accessible
surface of the protein is color coded by lipophilic potential (pink is polar; green is hydrophobic).
Figure was created with MOE (www.chemcomp.com).
Using this strategy, we identified alkyl sulfones and sulfoxides as replacements for the basic
amine side chain of 1, and the structureꢀactivity relationships for this subꢀgroup of analogs is
shown in Table 1. Linear sulfones 17 and 18 showed similar biochemical potency and
significant reduction in Jurkat cytotoxicity relative to 1, but unfortunately ITK cellular potency
was also compromised. Gratifyingly, ITK enzymatic and cellular potencies were rescued
without increased cytotoxicity by introduction of cyclic sulfones (19, 21-23) or sulfoxides (20).
Cyclization of the appended chain may have a variety of effects; the most likely is to alter the
rotamer energy bias which presumably enhances the active rotamer population in this case. It
may however also add some lipophilic binding energy. These potential effects may explain the
increased potency of the cyclic analogs. Sulfoxide 20 (GNEꢀ7056) is especially noteworthy
since good kinetic solubility was maintained in addition to reduced cytotoxicity and improved
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cellular potency relative to 1. Although some analogs showed reduced solubility in phosphate
buffer solution (where no plasma protein was present) to the extent of below the cytotoxicity
IC50 compared to compound 1, solubility in the Jurkat proliferation assay buffer conditions,
where 10% FBS was present, was higher than the observed proliferation IC50s in all cases (data
not shown).
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Table 1. SAR of THI analogs containing nonꢀbasic benzylic side chains a
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aAll values are the mean of two or more independent assays; Stereochemistry of 1 was
assigned by xꢀray crystallography (Figure 3). Stereochemistry of the benzylic carbon of the rest
of the compounds was assigned by analogy: the more active enantiomer is presumed to have the
same stereochemistry as in 1. The relative stereochemistry of 20-23 is unknown, and only the
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two most potent diastereomers of the four possible are shown; Inhibition of PLCꢀγ
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phosphorylation in Jurkat cells with TCR stimulation; Antiꢀproliferation effect on Jurkat cells
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without TCR stimulation; eSolubility in aqueous phosphate buffer solution (pH 7.4).
Having identified chemical matter with improved cytotoxicity profiles, we were interested in
further improving ITK inhibitory potency. We postulated that further optimization of the
interactions between the ligand and the lipophilic pocket near Phe435 (c.f. Figure 3) could be a
source of improving potency, and Table 2 highlights our effort in this regard. Replacement of
the gemꢀdimethyl substituent present in earlier analogs with a cyclopropyl methyl moiety (25)
resulted in a significant improvement in cellular potency relative to the series progenitor 24.5q An
additional order of magnitude improvement was achieved by replacement of the methylene
moiety in the cyclopropane ring by a difluoromethylene (26).
Table 2. Optimization of the tetrahydroindazole subunita
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NH
N
LCK Ki
( M)
[fold over ITK]
H
PLC IC50
( M)
ITK Ki
( M)
N
N
N
O
R
Compoundb
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NH
N
H
N
24
0.0053
5.2
1.8 [340]
O
NH
NH
N
N
H
N
25
25'
0.0030
0.20
0.41
ꢀꢀꢀ
1.4 [460]
ꢀꢀꢀ
O
O
H
N
0.0003
>0.4
0.032
ꢀꢀꢀ
0.60 [1800]
ꢀꢀꢀ
26
26'
F
F
aAll values are the mean of two or more independent assays; bAbsolute stereochemistry of the
more potent isomers of 25 and 26 was inferred from the xꢀray crystal structure of 30 bound to
ITK (Figure 7).
Combining the pertinent discoveries summarized in Tables 1 and 2, we were able to discover
two additional noteworthy compounds: 27 (GNEꢀ6688) and 28 (GNEꢀ4997), with 28 achieving
cellular potency of 4 nM (Figure 4). These compounds, in addition to 20, were selected for inꢀ
depth profiling, and the results are summarized in Table 3 in comparison to our earlier lead 1
(GNEꢀ9822). All compounds showed improved potency relative to 1, with comparable kinase
selectivity. Compound 28 is especially noteworthy in this regard, demonstrating a 10ꢀfold
improvement in ITK enzymatic and cellular potency while improving fold of kinase selectivity at
0.1 ꢀM (a concentration that is >1,000ꢀfold over ITK Ki) compared to compound 1. The in vitro
cytotoxicity window was improved from 85ꢀfold for 1 to 3,000ꢀfold for 28. All compounds also
exhibited reduced receptor binding, hERG inhibition and hepatotoxicity (Table 3 and Table S1 in
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supporting information). Compounds 20, 27 and 28 do, however, have lower solubility than
compound 1.
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NH
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N
O
N
S
27 (GNE-6688)
ITK Ki = 0.0002
PLC IC50 = 0.039
O
O
M
M
Jurkat Prolif IC50 = 29
M
NH
N
H
N
O
O
N
N
S
O
F
F
28 (GNE-4997)
ITK Ki = 0.00009
PLC IC50 = 0.004
M
M
Jurkat Prolif IC50 = 12
M
Figure 4. Structures and potencies of compounds 27 and 28.
Table 3. Profile of inhibitors 1, 20, 27 and 28a
Compound
28
1
27
20
ITK Ki
0.0007
0.045
4.7
85x
0.34
470x
6/286
68
0.0002
0.028
30
1070x
1.6
8000x
10/285
45
3.6
92
1.9
94
0.0002
0.039
29
740x
0.26
1600x
9/225
6.5
3.5
109
4.4
97
0.00009
0.004
12
3000x
0.46
5000x
7/225
3.9
2.8
109
1.5
97
5.6
1/40
6.8
b
PLCg IC50
Jurkat Prolif. IC50
cytotox windowc
LCK Ki
Select. foldd
Kinase Sel.e
Solubility
logD7.4
TPSA
2.8
78
MDCK Perm. f
hPPB (%)
4.6
93
HHep Cl g
5.0
5/40
88
13
0/40
3.6
13
0/40
34
CEREP panelh
hERG %inhi
Hepatotox IC50
6.2
83
>100
>100
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aKi, IC50 and solubility have the unit of micormolar (ꢀM). bInhibition of PLCꢀγ
c
phosphorylation in Jurkat cells with TCR stimulation; Cytotoxicity window was calculated by
d
dividing Jurkat Prolif IC50 by PLCg IC50; Selectivity fold was calculated by Lck Ki divided by
ITK Ki; eKinase selectivity is gauged by the number of kinases inhibited >70% at 0.1 ꢀM out of
the total number of kinases in the kinase panel at Invitrogen; fPermeability in MDCK cell line in
g
h
the unit of 10ꢀ6 cm/s; Clearance in human hepatocytes in the unit of ml/min/kg; Number of
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i
receptors in the CEREP receptor panel showing >70% binding at 10 ꢀM; Percentage of hERG
inhibition at a compound concentration of 10 ꢀM.
We were able to obtain an xꢀray crystal structure of 28 bound to ITK (Figure 5). The binding
mode is consistent with that of 1 (Figure 3).5q The inhibitor amide NH engages the carbonyl of
Met438 and the pyrazole participates in a donorꢀacceptor pair with the NH of Met438 and the
carbonyl of Glu436, thus anchoring the inhibitor to the hinge region. The cyclic sulfone serves
to orient the benzyl phenyl ring for an edgeꢀtoꢀface interaction with Phe437. The
difluoromethylene group of the cyclopropane occupies a lipophilic pocket adjacent to Phe435
(the “gatekeeper” residue). The observed increase in potency for these difluorocyclopropane
inhibitors is likely due to more efficient spaceꢀfilling of this pocket.
Figure 5. Coꢀcrystal structure of 28 complexed with the kinase domain of ITK (PBD code
4rfm). The two fluorine atoms occupy a hydrophobic pocket within ITK adjacent to the
gatekeeper Phe435. Interactions and surface depictions are the same as in Figure 3.
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Having demonstrated improved cellular potency and improved in vitro safety, we tested the
compounds in an in vivo pharmacodynamics (PD) model. Crosslinking of the T cell receptor
(TCR) with an antibody against CD3 (145ꢀ2C11) resulted in the release of interleukinꢀ2 (ILꢀ2)
and other cytokines in a doseꢀ and timeꢀdependent fashion (data not shown). Mice were first
dosed with ITK inhibitors at different dose levels. One hour later, antiꢀCD3 was administered (10
ꢀg, IV) and serum ILꢀ2 and ILꢀ13 levels were measured by Meso Scale Discovery (MSD) after
1.5 hours. We chose to measure ILꢀ2 and ILꢀ13 levels as these two cytokines are affected by
ITKꢀmediated TCR signaling.1 As shown in Figure 7, ILꢀ2 levels were decreased in a doseꢀ
dependent manner, reaching a maximum 98% reduction at the 200 mg/kg of compound 20. We
observed a similar trend with regard to ILꢀ13 levels, reaching 100% reduction at the 200 mg/kg
dose. Compound concentration measured at 2.5 hr of the study increased as dose increased,
reaching a plasma concentration of ~ 80 ꢀM at the highest dose.14
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8000
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55%
*
* 90%
98%
Compound 20 (mg/kg)
anti-CD3
2000
1500
1000
500
0
*
62%
*
95%
*
100%
Compound 20 (mg/kg)
anti-CD3
Figure 7. Reduction of serum ILꢀ2 and ILꢀ13 following IP dosing of ITK inhibitor 20 in mice.
In conclusion, we have further optimized a tetrahydroindazole series of ITK inhibitors,
discovering second generation analogs with increased potency and decreased offꢀtarget in vitro
toxicity. Specifically, we have demonstrated an interesting relationship between cellular toxicity
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and compound basicity in the absence of significant differences in kinase selectivity. Taking
advantage of this observation, we were able to design compounds with decreased cytotoxicity.
Furthermore, we have now demonstrated in vivo pathway modulation with our optimized
inhibitors.
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EXPERIMENTAL SECTION
Chemistry General. All commercially available reagents and solvents were used as received
without further purification or drying unless otherwise stated. Reactions using airꢀ or moistureꢀ
sensitive reagents were performed under an atmosphere of nitrogen using freshly opened EMD
DriSolv solvents. Reaction progress was monitored by TLC and/or HPLC. Flash
chromatography was performed with Isco CombiFlash Companion systems using prepacked
silica gel columns (40−60 ꢁm particle size RediSep or 20−40 ꢁm spherical silica gel RediSep
Gold columns, or similar columns from other vendors). Preparative HPLC purifications were
performed on a Varian Prostar instrument, using a Phenomenex GeminiꢀNX Cꢀ18 (0.3 cm × 5
cm; 5 ꢁm particle size) stationary phase, with 0.1% aqueous formic acid/ acetonitrile (for acidic
or neutral compounds) or 0.1% aqueous ammonium hydroxide/acetonitrile (for basic
compounds) gradients as the mobile phase (typically 5−85% acetonitrile over 10 min) with a
flow rate of 60 mL/min. Preparative SFC separations were performed on a PIC Solutions
instrument, with conditions indicated in the Experimental Section. Proton NMR spectra were
measured on Bruker 300 or 400 MHz spectrometers, and chemical shifts are reported in ppm
downfield from TMS (0 ppm). Highꢀresolution mass spectrometry of final compounds were
performed on a Thermo UHPLC/QE with a ThermoꢀQ Exactive mass spectrometry detector
using ESI ionization, after elution on a Acquity BEH C18 (2.1 mm × 50 mm; 1.7 ꢁm particle
size) stationary phase using a gradient of water/acetonitrile (3−97% over 7 min; 0.1% formic
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acid in both phases). The purity of final compounds 14−32 was verified by HPLC on an Agilent
1200 instrument with an Agilent SB Cꢀ18 (2.1 mm × 30 mm; 1.8 ꢁm particle size) stationary
phase, and a gradient of water/acetonitrile (5−95% over 6 min; 0.05% TFA in both phases) at a
flow rate of 0.4 mL/min. Quantification of target and impurities was done by UV detection at
254 nm, and is >95% in all cases.
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Compounds 17, 24, 25 and 25’ were reported as examples 30, 17 and 26, respectively, in the
previous paper.5q
6,6-Dimethyl-N-(1-(2-(methylsulfonyl)-1-phenylethyl)-1H-pyrazol-4-yl)-4,5,6,7-tetrahydro-
1H-indazole-3-carboxamide (18)
To a solution of 2ꢀmethylsulfanylꢀ1ꢀphenylꢀethanone (452 mg, 2.7190 mmol, 452 mg) in
methanol (5 mL, 5 mL) and THF (5 mL) was added sodium borohydride (1.5 equiv., 4.07 mmol,
157.4 mg) and the mixture was stirred at rt for 90 minutes. Excess hydride was quenched with
~10 mL sat. NH4Cl(aq), then the mixture was diluted with 50 mL of brine and extracted with 50
mL of EtOAc. The organic extracts were dried (Na2SO4) and concentrated in vacuo to provide
2ꢀmethylsulfanylꢀ1ꢀphenylꢀethanol (447 mg, 97.7% yield) of sufficient purity to be used directly
in the next step.
To a solution of 2ꢀmethylsulfanylꢀ1ꢀphenylꢀethanol (447 mg, 2.65 mmol, 447 mg) and 4ꢀ
nitroꢀ1Hꢀpyrazole (1.1 equiv., 2.92 mmol, 330.4 mg) in THF (10 mL) was added
triphenylphosphine (1.1 equiv., 2.92 mmol, 766.5 mg) and diisopropyl azodicarboxylate (1.1
equiv., 2.92 mmol, 0.605 mL). The mixture was stirred overnight at rt. After in vacuo
concentration, the residue was purification by CombiFlash (40 g; 100:0 to 50:50 heptane:EtOAc
over 20 minutes) to provide provide 1ꢀ(2ꢀmethylsulfanylꢀ1ꢀphenylꢀethyl)ꢀ4ꢀnitroꢀpyrazole (666
mg, 2.529 mmol, 95.2% yield) containing small amounts of other impurities by NMR, but which
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was used without further purification.
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To a solution of 1ꢀ(2ꢀmethylsulfanylꢀ1ꢀphenylꢀethyl)ꢀ4ꢀnitroꢀpyrazole (666 mg, 2.53
mmol) in methanol (10 mL) was added palladium on carbon (0.1 equiv., 0.253 mmol, 538.4 mg)
and the mixture was stirred under an atmosphere of H2 (balloon) for 2 hours. LCꢀMS shows
complete consumption of starting material. The catalyst was deactivated by the addition of
CH2Cl2 then the mixture was filtered through Celite (CH2Cl2 and MeOH rinse). The filtrate was
concentrated in vacuo to provide 1ꢀ(2ꢀmethylsulfanylꢀ1ꢀphenylꢀethyl)pyrazolꢀ4ꢀamine of
sufficient purity to be used directly.
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General procedure Aꢀꢀꢀ amide coupling: To a solution of 1ꢀ(2ꢀmethylsulfanylꢀ1ꢀphenylꢀ
ethyl)pyrazolꢀ4ꢀamine (2.53 mmol, 590 mg) and 6,6ꢀdimethylꢀ1ꢀ(2ꢀtrimethylsilylethoxymethyl)ꢀ
5,7ꢀdihydroꢀ4Hꢀ indazoleꢀ3ꢀcarboxylic acid (1.0 equiv., 2.53 mmol, 821 mg) in
dimethylformamide (10 mL) was added HATU (1.0 equiv., 2.53 mmol, 992 mg) and N,N'ꢀ
diisopropylethylamine (1.5 equiv., 3.80 mmol, 0.668 mL) and the mixture was stirred overnight
at rt. The mixture was diluted with 100 mL of EtOAc and washed with 100 mL of sat.
NaHCO3(aq) and 2 x 100 mL of 1:1 H2O:brine. The organic extracts were dried (Na2SO4) and
concentrated in vacuo. The product was purification by CombiFlash (40 g; dry load; 100:0 to
50:50 heptane:EtOAc over 20 minutes) to give 6,6ꢀdimethylꢀNꢀ[1ꢀ(2ꢀmethylsulfanylꢀ1ꢀphenylꢀ
ethyl)pyrazolꢀ4ꢀyl]ꢀ1ꢀ(2ꢀtrimethylsilylethoxymethyl)ꢀ5,7ꢀdihydroꢀ4Hꢀindazoleꢀ3ꢀcarboxamide
(1.31 g, 2.43 mmol, 96% yield).
To a solution of 6,6ꢀdimethylꢀNꢀ[1ꢀ(2ꢀmethylsulfanylꢀ1ꢀphenylꢀethyl)pyrazolꢀ4ꢀyl]ꢀ1ꢀ (2ꢀ
trimethylsilylethoxymethyl)ꢀ5,7ꢀdihydroꢀ4Hꢀindazoleꢀ3ꢀcarboxamide (300 mg, 0.556 mmol) in
tetrahydrofuran (5 mL) was added 3ꢀchloroperbenzoic acid (2.2 equiv., 1.22 mmol, 274 mg) and
the mixture was stirred for 60 minutes at rt. The mixture was diluted with 50 mL EtOAc and
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washed with 50 mL sat. aq. NaHCO3 and 50 mL brine. The organic extracts were dried
(Na2SO4) and concentrated in vacuo. Purification by CombiFlash (24 g; dry load; 70:30 to 30:70
heptane: EtOAc over 20 minutes) provided 6, 6ꢀdimethylꢀNꢀ[1ꢀ(2ꢀmethylsulfonylꢀ1ꢀ phenylꢀ
ethyl)pyrazolꢀ4ꢀyl]ꢀ1ꢀ(2ꢀtrimethylsilylethoxymethyl)ꢀ5,7ꢀdihydroꢀ4Hꢀindazoleꢀ3ꢀcarboxamide
(90 mg, 0.157 mmol, 28% yield).
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General Procedure Bꢀꢀꢀremoval of SEM group: To a solution of 6,6ꢀdimethylꢀNꢀ[1ꢀ(2ꢀ
methylsulfonylꢀ1ꢀphenylꢀethyl)pyrazolꢀ4ꢀyl]ꢀ 1ꢀ(2ꢀtrimethylsilylethoxymethyl)ꢀ5,7ꢀdihydroꢀ4Hꢀ
indazoleꢀ3ꢀcarboxamide (90.0 mg, 0.157 mmol) in trifluoroacetic acid (2 mL) was added
triisopropylsilane (5 equiv., 0.787 mmol, 0.163 mL) and the mixture was stirred for 90 minutes
at rt. After in vacuo concentration, the residue was purified by reverseꢀphase HPLC, followed by
SFC on a chiral stationary phase to provide the title compounds as single enantiomers. SFC
conditions: Lux Celluloseꢀ3 (4.6x50 mm, 5 ꢀm particle size) at 30% methanol w/ 0.1% NH4OH;
5 mL/min, 120 bars, 40 °C. The fast eluting isomer is the more potent inhibitor of ITK and the
1
stereochemistry was assigned by analogy. H NMR (400 MHz, DMSOꢀd6) δ 12.85 – 12.62 (s,
1H), 10.22 – 9.97 (s, 1H), 8.29 – 8.24 (s, 1H), 7.75 – 7.70 (s, 1H), 7.46 – 7.40 (m, 2H), 7.40 –
7.27 (m, 3H), 6.02 – 5.95 (dd, J = 9.7, 3.9 Hz, 1H), 4.58 – 4.47 (dd, J = 14.9, 9.7 Hz, 1H), 3.94 –
3.83 (dd, J = 14.9, 4.0 Hz, 1H), 2.71 – 2.62 (m, 5H), 2.40 – 2.36 (s, 2H), 1.52 – 1.43 (t, J = 6.4
Hz, 2H), 0.99 – 0.92 (s, 6H); MS: m/z = 442 (M + H); SFC retention time: 0.47 min.
4-((4-Amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran 1,1-dioxide
Into a 2ꢀL 3ꢀnecked roundꢀbottom flask purged and maintained with a nitrogen
atmosphere, was placed tetrahydroꢀ2Hꢀthiopyranꢀ4ꢀcarbaldehyde (65.0 g, 499.2 mmol, 1.00
equiv) and tetrahydrofuran (300 mL). PhMgBr (1M, 750 mL, 1.50 equiv) was added dropwise to
o
the stirred solution at 0 C. The resulting solution was then stirred for 12 h at room temperature.
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The reaction progress was monitored by TLC with PE/DCM=2/1. The reaction was then
quenched by the addition of 500 mL of saturated NH4Cl and extracted with 3x500 mL of ethyl
acetate. The combined organic was washed with 3x500 mL of brine, dried over anhydrous
sodium sulfate and concentrated under vacuum. The residue was purified by silica gel column
chromatography (1/20 of acetate/petroleum ether) to provide 27.0 g (26%) of phenyl(tetrahydroꢀ
2Hꢀthiopyranꢀ4ꢀyl)methanol as a yellow oil.
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DIAD (18.9 g, 93.47 mmol, 1.50 equiv) was added to the solution of phenyl(tetrahydroꢀ
2Hꢀthiopyran ꢀ4ꢀyl)methanol (13.0 g, 62.4 mmol, 1.00 equiv), 4ꢀnitroꢀ1Hꢀpyrazole (8.5 g, 75.17
mmol, 1.20 equiv), and PPh3 (24.6 g, 93.8 mmol, 1.50 equiv) in tetrahydrofuran (300 mL)
dropwise at 0oC and stirred for 12 h at room temperature under nitrogen. The reaction solution
was concentrated under vacuum. The residue was purified by a silica gel column eluting with
ethyl acetate/petroleum ether (1/10) to afford 4ꢀnitroꢀ1ꢀ[phenyl(tetrahydroꢀ2Hꢀthiopyran ꢀ4ꢀ
yl)methyl]ꢀ1Hꢀpyrazole (13.0 g, 69%) as a white solid.
A solution of mꢀCPBA (23.90 g, 138.4 mmol, 3.00 equiv) in AcOEt (50 mL) was added
to the solution of 4ꢀnitroꢀ1ꢀ[phenyl(tetrahydroꢀ2Hꢀthiopyranꢀ4ꢀyl)methyl]ꢀ1Hꢀpyrazole (14.0 g,
46.15 mmol, 1.00 equiv) in dichloromethane (200 mL) dropwise at 5 °C and stirred for 30 min at
5 °C. The resulting solution was diluted with 300ml of DCM, washed with 3x300 ml of saturated
solution of Na2CO3, dried over anhydrous Na2SO4, and concentrated under vacuum to give 4ꢀ((4ꢀ
nitroꢀ1Hꢀpyrazolꢀ1ꢀyl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyranꢀ1,1ꢀdioxide (13.0 g, 84%) as a
white solid.
A mixture of 4ꢀ((4ꢀnitroꢀ1Hꢀpyrazolꢀ1ꢀyl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyran 1,1ꢀ
dioxide (8.00 g, 23.8 mmol, 1.00 equiv), methanol (500 mL), AcOEt (800 mL), and Raney Ni
(6.0 g) was stirred for 12 h at room temperature under an atmosphere of hydrogen. The solids
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were filtered out. The filtrate was concentrated under vacuum to afford 4ꢀ((4ꢀaminoꢀ1Hꢀpyrazolꢀ
1ꢀyl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyranꢀ1,1ꢀdioxide (6.5 g, 89%) as a light brown solid.
This material was used in the next step without purification.
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N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)(phenyl)methyl)-1H-pyrazol-4-yl)-6,6-
dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (19)
Prepared by following General procedure A & B. Chiral HPLC (ChiralPak IBꢀ3 (4.6x50
mm, 3 ꢀm particle size); eluent = Hex:EtOH 60:40; 1.0 ml/min, 3.5 MPA, 25 °C ) purification
1
gave 19 as the fastꢀeluting isomer. HꢀNMR (CDCl3, 300 MHz): δ 8.62 (s, 1H), 8.14 (s, 1H),
7.56 (s, 1H), 7.43ꢀ7.30 (m, 5H), 4.83 (d, J = 10.5Hz, 2H), 3.03ꢀ2.91 (m, 4H), 2.86ꢀ2.73 (m, 3H),
2.41 (s, 2H), 1.95ꢀ1.79 (m, 4H), 1.56 (t, J = 6.3Hz, 2H), 1.01 (s, 6H); MS: m/z = 482 (M + H);
HPLC retention time: 1.97 min.
4-((4-Amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran 1-oxide
A solution of mꢀCPBA (570 mg, 3.30 mmol, 1.00 equiv) in AcOEt (5 ml) was added
dropwise to a stirred solution of 4ꢀnitroꢀ1ꢀ[phenyl(tetrahydroꢀ2Hꢀthiopyran ꢀ4ꢀyl)methyl]ꢀ1Hꢀ
pyrazole (1.0 g, 3.30 mmol, 1.00 equiv) in dichloromethane (50 mL) at 0 °C. After 30 minutes
the resulting solution was diluted with 250 mL of AcOEt and washed with 3x150 mL of
saturated solution of sodium carbonate and 3x150 mL of brine. The organic was dried over
anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica
gel column with ethyl acetate/petroleum ether (1/2 to 2/1). This resulted in 500 mg (47%) and
400 mg (38%) of the two diastereomers (stereochemistry unassigned) of 4ꢀnitroꢀ1ꢀ[phenyl(1ꢀ
oxoꢀ tetrahydroꢀ2Hꢀthiopyran ꢀ 4ꢀyl)methyl]ꢀ 1Hꢀpyrazole.
General procedure Cꢀꢀꢀ reduction of nitro group: Each diastereomer was then reduced
individually: Hydrogen gas was introduced into a mixture of 4ꢀnitroꢀ1ꢀ[phenyl(1ꢀoxoꢀ
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tetrahydroꢀ2Hꢀthiopyran ꢀ4ꢀyl)methyl]ꢀ1Hꢀpyrazole (500 mg, 1.57 mmol, 1.00 equiv) and
palladium on carbon (500 mg) in methanol (50 mL). After 30 min at room temperature the solids
were filtered out. The filtrate was concentrated under vacuum. This resulted in 425 mg (94%) of
4ꢀaminoꢀ1ꢀ[phenyl(1ꢀoxoꢀ tetrahydroꢀ2Hꢀthiopyranꢀ4ꢀyl)methyl]ꢀ1Hꢀpyrazole as a light yellow
solid.
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6,6-Dimethyl-N-(1-((1-oxidotetrahydro-2H-thiopyran-4-yl)(phenyl)methyl)-1H-pyrazol-4-
yl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (20 and 20’)
Prepared by following General procedure A. Each diastereomer was reacted
independently, then resolved into their constituent stereoisomers by chiral LCMS yielding the 4
diastereomeric products.
Chiral HPLC Conditions: ChiralPak IAꢀ3 (4.6x50 mm, 3 ꢀm particle size); eluent = Hex (0.1%
Et3N):EtOH 50:50; 1.0 ml/min, 5.8 MPA, 25 °C
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20: H NMR (300 MHz, CDCl3) δ 8.85 (s, 1H), 8.14 (s, 1H), 7.56 (s, 1H), 7.41 (d, J = 1.5Hz,
2H), 7.39ꢀ7.26 (m, 3H), 4.96 (d, J = 10.8Hz, 1H), 3.03ꢀ2.94 (m, 2H), 2.85 (t, J = 6.3Hz, 2H),
2.72ꢀ2.60 (m, 1H), 2.52ꢀ2.34 (m, 4H), 2.27ꢀ1.97 (m, 2H), 1.56 (t, J = 6.4Hz, 2H), 1.49ꢀ1.38 (m,
2H), 1.00 (s, 6H); MS: m/z = 466 (M + H); HPLC retention time: 2.71 min.
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20’: H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 8.14 (s, 1H), 7.56 (s, 1H), 7.41 (d, J = 1.5Hz,
2H), 7.39ꢀ7.26 (m, 3H), 4.96 (d, J = 10.8Hz, 1H), 3.03ꢀ2.93 (m, 2H), 2.85 (t, J = 6.3Hz, 2H),
2.72ꢀ2.60 (m, 1H), 2.51ꢀ2.34 (m, 4H), 2.27ꢀ1.97 (m, 2H), 1.56 (t, J = 6.3Hz, 2H), 1.48ꢀ1.38 (m,
2H), 1.00 (s, 6H); MS: m/z = 466 (M + H); HPLC retention time: 5.89 min.
Chiral HPLC Conditions for the other two less potent isomers: ChiralPak IB (4.6x250 mm, 5 ꢀm
particle size); eluent = Hex (0.1% Et3N):EtOH 70:30; 1.0 ml/min, 5.9 MPA, 25 °C
HPLC retention time: 9.74 min and 15.63 min, respectively.
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N-(1-((1,1-Dioxidotetrahydrothiophen-3-yl)(phenyl)methyl)-1H-pyrazol-4-yl)-6,6-dimethyl-
4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (21 and 21’)
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Prepared in an analogous manner to compound 19. The stereoisomers were separated by
preparative chiral HPLC instead of SFC (Chiral HPLC Conditions: Lux Celluloseꢀ4 (4.6x150
mm, 3 ꢀm particle size); eluent = Hex:EtOH 60:40; 1.0 ml/min, 4.2 MPA, 25 °C).
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21: H NMR (300MHz, CDCl3) δ 8.62 (s, 1H), 8.14 (s, 1H), 7.57 (s, 1H), 7.43ꢀ7.31 (m, 5H),
5.06 (d, J = 10.5Hz, 1H), 3.69ꢀ3.66 (m, 1H), 3.24ꢀ2.98 (m, 3H), 2.86ꢀ2.82 (t, J = 6.3Hz, 2H),
2.78ꢀ2.70 (m, 1H), 2.43 (s, 2H), 2.24ꢀ2.22 (m, 1H), 2.01ꢀ1.94 (m, 1H), 1.59ꢀ1.55 (t, J = 6.3Hz,
2H), 1.02 (s, 6H); MS: m/z = 468 (M + H); HPLC retention time: 12.3 min.
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21’: H NMR (300MHz, CDCl3) δ 8.68 (s, 1H), 8.10 (s, 1H), 7.57 (s, 1H), 7.40ꢀ7.32 (m, 5H),
5.04 (d, J = 10.5Hz, 1H), 3.67ꢀ3.65 (m, 1H), 3.32ꢀ2.91 (m, 4H), 2.86 (t, J = 6 Hz, 2H), 2.44 (s,
2H), 2.02ꢀ1.86 (m, 2H), 1.59ꢀ1.55 (t, J = 6.3Hz, 2H), 1.02 (s, 6H); MS: m/z = 468 (M + H);
HPLC retention time: 15.1 min.
N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-3-yl)(phenyl)methyl)-1H-pyrazol-4-yl)-6,6-
dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (22 and 22’)
Prepared in an analogous manner to compound 19. The stereoisomers were separated by
preparative chiral HPLC instead of SFC (Chiral HPLC Conditions: ChiralPak IA (4.6x250 mm,
3 ꢀm particle size); eluent = (Hex+0.1% Et3N):EtOH 50:50; 1.0 ml/min, 4.2 MPA, 25 °C).
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22: H NMR (300MHz, CDCl3) δ 9.00 (s, 1H), 8.14 (s, 1H), 7.61 (s, 1H), 7.44ꢀ7.29 (m, 5H),
4.99 (d, J =9.3Hz, 1H), 3.35 (d, J =4.5Hz, 1H), 3.08ꢀ3.02 (m, 1H), 2.93ꢀ2.84 (m, 4H), 2.72ꢀ2.58
(m, 1H), 2.58 (s, 2H), 2.13ꢀ2.05 (m, 2H), 2.01 (s, 1H), 1.72ꢀ1.67 (m, 1H), 1.59 (t, J = 7.5Hz,
2H), 1.32ꢀ1.26 (m, 1H), 1.03 (s, 6H); MS: m/z = 482 (M + H); HPLC retention time: 15.5 min.
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22’: H NMR (300MHz, CDCl3) δ 8.92 (s, 1H), 8.11 (s, 1H), 7.68 (s, 1H), 7.39ꢀ7.29 (m, 5H),
5.07 (d, J = 9.3Hz, 1H), 3.27ꢀ3.21 (m, 1H), 3.06ꢀ2.84 (m, 6H), 2.72 (s, 2H), 2.11ꢀ2.03 (m, 2H),
1.77ꢀ1.72 (m, 1H), 1.55 (t, J = 7.5Hz, 2H), 1.25ꢀ1.20 (m, 1H), 1.03 (s, 6H); MS: m/z = 482 (M +
H); HPLC retention time: 13.2 min.
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2-((4-amino-1H-pyrazol-1-yl)(phenyl)methyl)tetrahydro-2H-thiopyran 1,1-dioxide
Butyllithium (84.2 mL, 1.20 equiv) was added to the solution of tetrahydroꢀ2Hꢀ
thiopyranꢀ1ꢀoxide (20.7 g, 175.13 mmol, 1.00 equiv) in tetrahydrofuran (200 mL) dropwise at ꢀ
78oCand stirred for 1 h at ꢀ78oC under nitrogen. To this was added benzaldehyde (18.6 g, 175.27
mmol, 1.00 equiv) dropwise at ꢀ78oC and stirred for 6 h at ꢀ78oC. The reaction was then
quenched by 500 mL of saturated NH4Cl, extracted with 3x500 mL of ethyl acetate, and
concentrated under vacuum. The residue was purified by a silica gel column eluting with
DCM:MeOH (20/1) to afford 2ꢀ(hydroxy(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyran 1ꢀoxide (30.0
g, 76%) as a light yellow solid.
DIAD (39.6 g, 196.0 mmol, 1.50 equiv) was added to the solution of 2ꢀ
(hydroxy(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyranꢀ1ꢀoxide (29.3 g, 130.6 mmol, 1.00 equiv), 4ꢀ
nitroꢀ1Hꢀpyrazole (17.7 g, 156.53 mmol, 1.20 equiv) and PPh3 (51.4 g, 196.18 mmol, 1.50
equiv) in tetrahydrofuran (700 mL) dropwise at 0 oC and stirred overnight at 25 oC under
nitrogen. The reaction was then quenched by 1 L of water, extracted with 3x1000 ml of ethyl
acetate, and concentrated under vacuum. The residue was purified by a silica gel column eluting
with ethyl acetate/petroleum ether (1/1) to afford 2ꢀ((4ꢀnitroꢀ1Hꢀpyrazolꢀ1ꢀyl)(phenyl)methyl)ꢀ
tetrahydroꢀ2Hꢀthiopyran 1ꢀoxide (40 g, crude) as a yellow oil.
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mꢀCPBA was added to the solution of 2ꢀ((4ꢀnitroꢀ1Hꢀpyrazolꢀ1ꢀ
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yl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyranꢀ1ꢀoxide (62.4 g, 195.4 mmol, 1.00 equiv) in
dichloromethane (700 mL) at 0oC under stirred for 4 h at 25oC. The reaction was then quenched
by the addition of 1000 ml of sodium carbonate/H2O, washed with 2x500 mL water and 2x500
mL of brine, and concentrated under vacuum. The residue was purified by a silica gel column
eluting with ethyl acetate/petroleum ether (1/3) to afford 2ꢀ((4ꢀnitroꢀ1Hꢀpyrazolꢀ1ꢀ
yl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyran 1,1ꢀdioxide (22 g, crude) as a yellow solid.
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A mixture of 2ꢀ((4ꢀnitroꢀ1Hꢀpyrazolꢀ1ꢀyl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyran 1,1ꢀ
dioxide (12 g, 35.78 mmol, 1.00 equiv), 10% palladium on carbon (6.0 g), methanol (300 mL),
and ethyl acetate (300 ml) was stirred for 3 h at room temperature under hydrogen. The solids
were filtered out. The filtrate was concentrated under vacuum to afford 2ꢀ((4ꢀaminoꢀ1Hꢀpyrazolꢀ
1ꢀyl)(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyran 1,1ꢀdioxide (11.0 g, crude) as a light red solid.
N-(1-((1,1-Dioxidotetrahydro-2H-thiopyran-2-yl)(phenyl)methyl)-1H-pyrazol-4-yl)-6,6-
dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (23 and 23’)
Prepared in an analogous manner to compound 19 starting with 2ꢀ
(hydroxy(phenyl)methyl)tetrahydroꢀ2Hꢀthiopyran 1ꢀoxide. The stereoisomers were separated by
preparative chiral HPLC instead of SFC (Chiral HPLC Conditions: ChiralPak IBꢀ3 (4.6x150
mm, 3 ꢀm particle size); eluent = Hex:EtOH 80:20; 1.0 ml/min, 4.2 MPA, 25 °C).
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23: H NMR (300MHz, CDCl3) δ 8.68 (s, 1H), 8.12 (s, 1H), 7.62ꢀ7.57 (m, 3H), 7.37ꢀ7.30 (m,
3H), 5.55 (d, J = 9.6Hz, 1H), 4.13ꢀ4.05 (m, 2H), 3.06ꢀ2.82 (m, 4H), 2.43 (s, 2H), 2.04ꢀ2.02 (m,
2H), 1.89ꢀ1.70 (m, 3H), 1.59ꢀ1.50 (m, 3H), 1.01 (s, 6H); MS: m/z = 482 (M + H); HPLC
retention time: 1.4 min.
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23’: H NMR (300MHz, CDCl3) δ 8.91 (s, 1H), 8.06 (s, 1H), 7.63 (s, 1H), 7.44ꢀ7.41 (m, 2H),
7.32ꢀ7.26 (m, 3H), 5.71 (d, J = 8.7Hz, 1H), 4.43ꢀ4.35 (m, 1H), 3.11ꢀ2.95 (m, 4H), 2.82ꢀ2.70 (m,
2H), 2.40 (s, 2H), 2.03ꢀ1.98 (m, 2H), 1.84ꢀ1.70 (m, 2H), 1.64ꢀ1.47 (m, 4H), 0.99 (s, 6H); MS:
m/z = 482 (M + H); HPLC retention time: 5.7 min.
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7-methyl-1,4-dioxaspiro[4.5]dec-7-ene (7)
A solution of 1ꢀmethoxyꢀ3ꢀmethylbenzene (11 g, 90.04 mmol, 1.00 equiv) in ether (60
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mL) was added dropwise to liquid ammonia (150 mL) at ꢀ78 C. tꢀButyl alcohol (60 mL) was
added dropwise to the above solution at ꢀ78 oC, then sodium (5.20 g, 226 mmol, 2.50 equiv) was
added in portions. The resulting solution was warmed to ꢀ35 oC and stirred at ꢀ35 oC for 2 h. The
resulting solution was diluted with 200 mL of pentane, quenched with 100 mL of water
(carefully and very slowly), extracted with 2x100 mL of pentane, dried over anhydrous sodium
sulfate, and concentrated under vacuum to give 9.2 g (82%) of 1ꢀmethoxyꢀ5ꢀmethylcyclohexaꢀ
1,4ꢀdiene as colorless oil.
A solution of 1ꢀmethoxyꢀ5ꢀmethylcyclohexaꢀ1,4ꢀdiene (4.6 g, 37.04 mmol, 1.00 equiv),
dichloromethane (100 mL), ethaneꢀ1,2ꢀdiol (11.5 g, 185 mmol, 5.00 equiv), 4ꢀmethylbenzeneꢀ1ꢀ
sulfonic acid (277 mg, 1.61 mmol, 0.05 equiv) was stirred at room temperature overnight. The
reaction mixture was washed with 2x50 mL of saturated sodium bicarbonate solution and 3x50
mL of water. The organic layer was dried over anhydrous sodium sulfate, and concentrated under
vacuum. The residue was purified by silica gel column chromatography eluting with petroleum
ether to give 2.8 g (49%) of 7ꢀmethylꢀ1,4ꢀdioxaspiro[4.5]decꢀ7ꢀene as a colorless oil.
7,7-difluoro-1-methylbicyclo[4.1.0]heptan-3-one (8)
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NaI (680 mg, 4.53 mmol, 0.50 equiv), tetrahydrofuran (28 mL), 7ꢀmethylꢀ1,4ꢀ
dioxaspiro[4.5]decꢀ7ꢀene (1.4 g, 9.08 mmol, 1.00 equiv.), and TMSCF3 (3.23 g, 22.75 mmol,
2.51 equiv) were weighed into a 100ꢀmL sealed tube. The tube was purged and maintained with
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an inert atmosphere of nitrogen. The reaction was stirred at 65 C for 12 h and then quenched
with 20 mL of water. The resulting solution was extracted with ethyl acetate. The combined
organic layer was washed with saturated Na2S2O3 and brine, dried over anhydrous sodium sulfate
and then concentrated under vacuum. The residue was purified by silica gel column
chromatography eluting with ethyl acetate/petroleum ether (1/100) to give 1.6 g (86%) of 7,7ꢀ
difluoroꢀ1ꢀmethylspiro[bicyclo[4.1.0]heptaneꢀ3,2ꢀ[1,3]dioxolane] as a colorless oil.
A solution of 7,7ꢀdifluoroꢀ1ꢀmethylspiro[bicyclo[4.1.0]heptaneꢀ3,2ꢀ[1,3]dioxolane] (1.60
g, 7.83 mmol, 1.00 equiv.) and PTSA (135 mg, 0.78 mmol, 0.10 equiv) in acetone (25 mL)/water
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(5 mL) was stirred for 12 h at 50 C. The reaction mixture was diluted with 300 mL of diethyl
ether. The organic layer was washed with saturated sodium bicarbonate solution and brine, dried
over anhydrous sodium sulfate, and concentrated under vacuum to give 1.10 g (88%) of 7,7ꢀ
difluoroꢀ1ꢀ methylbicyclo[4.1.0]heptanꢀ3ꢀone as a light yellow oil.
5,5-difluoro-5a-methyl-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxylic acid (6)
Under nitrogen tꢀBuOK (3.1 mL, 1M in THF, 1.00 equiv) was added dropwise into a
solution of 7,7ꢀdifluoroꢀ1ꢀmethylbicyclo[4.1.0]heptanꢀ3ꢀone (500 mg, 3.12 mmol, 1.00 equiv)
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and diethyl oxalate (456 mg, 3.12 mmol, 1.00 equiv) in tetrahydrofuran (10 mL) at ꢀ70 C. The
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reaction mixture was stirred for 12 h at ꢀ70 C, quenched by 5 mL of saturated NH4Cl and then
extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated under vacuum. This resulted in 620 mg (76%) of ethyl 2ꢀ[7,7ꢀ
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