Property- and structure-guided discovery of a tetrahydroindazole series of interleukin-2 inducible t-cell kinase inhibitors

Article abstract of DOI:10.1021/jm500550e

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.

Full text of DOI:10.1021/jm500550e

Article
pubs.acs.org/jmc
Property- and Structure-Guided Discovery of a Tetrahydroindazole
Series of Interleukin‑2 Inducible T‑Cell Kinase Inhibitors
Jason D. Burch,*,† Kevin Lau,† John J. Barker,‡ Fred Brookfield,‡ Yong Chen,∥ Yuan Chen,†
Charles Eigenbrot,† Claire Ellebrandt,§ M. Hicham A. Ismaili,† Adam Johnson,† Daniel Kordt,§
Colin H. MacKinnon,‡ Paul A. McEwan,‡ Daniel F. Ortwine,† Daniel B. Stein,§ Xiaolu Wang,§
Dirk Winkler,§ Po-Wai Yuen,∥ Yamin Zhang,∥ Ali A. Zarrin,† and Zhonghua Pei†
†Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States
‡Evotec (U.K.) Ltd., 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, United Kingdom
§Manfred Eigen Campus, Evotec AG, Essener Bogen 7, D-22419, Hamburg, Germany
∥Pharmaron Beijing Co. Ltd., No. 6 Tai He Road, BDA, 100176, Beijing, China
S
* Supporting Information
ABSTRACT: Interleukin-2 inducible T-cell kinase (ITK), a
member of the Tec family of tyrosine kinases, plays a major
role in T-cell signaling downstream of the T-cell receptor
(TCR), and considerable efforts have been directed toward
discovery of ITK-selective inhibitors as potential treatments of
inflammatory disorders such as asthma. Using a previously
disclosed indazole series of inhibitors as a starting point, and
using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with
improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the
ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification
of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.
We have recently disclosed the discovery of an indazole
series of ITK inhibitors, which evolved from the moderately
potent and selective HTS hit 1 into the exceptionally potent
inhibitor 3 (Figure 1).5m,6 While the potency and selectivity of
indazole 3 compare favorably with previous inhibitors disclosed
in the literature, this series in general required improvement of
ADME properties such as solubility and permeability. We
attributed the solubility to high aromatic ring count (4−5) and
moderate lipophilicity (cLogD7.4 ∼ 2−3), while the suboptimal
permeability could be attributed to the relatively high
topological polar surface area (TPSA, typically >120) and H-
bond donor count (3) for our best molecules. Guided by
structure- and property-based optimization techniques, we
sought to improve both of these characteristics simultaneously
in a second round of optimization, which led to the discovery of
a saturated subseries: the “tetrahydroindazole” or THI series.
INTRODUCTION
■
IL-2 inducible T cell kinase (ITK) is a nonreceptor tyrosine
kinase belonging to the Tec family of kinases involved in T cell
development, differentiation, and effector function.1 Tec family
kinases include ITK, resting lymphocyte kinase (RLK),
Bruton’s tyrosine kinase (Btk), tyrosine kinase expressed in
hepatocellular carcinoma (Tec), and bone marrow expressed
kinase (Bmx). Except for Bmx, which is expressed in
endothelial cells, the Tec kinases are primarily expressed in
hematopoietic cells.1 T cells express ITK, RLK, and Tec,
however, analyses of knockout mice have established a
prominent role for ITK and RLK as two critical Tec kinases
that integrate T cell receptor signaling (TCR) to activate
phospholipase C gamma-1 (PLCγ1) and subsequently Ca2+
mobilization.2 Several groups have shown that ITK deficiency
results in defective T helper 2 (TH2) cell function.3 Specifically,
in ITK−/− mice lung inflammation, eosinophil infiltration and
mucous production are drastically reduced in response to
challenge with ovalbumin.4
Prompted by the wealth of preclinical evidence supporting
the role of ITK in allergic asthma and other inflammatory
disorders, intense pharmaceutical research has been directed
toward development of selective inhibitors of this kinase.5
Furthermore, preclinical in vivo activity using ITK inhibitors
has been achieved, resulting in the reduction of IL-2 production
in one example5f and the reduction of IL-4 production and lung
inflammation in another.5b
CHEMISTRY
■
Two reaction sequences with divergent regiochemical con-
sequences were imperative to our construction of tetrahy-
droindazole carboxylates 6, 8, and 10 (Scheme 1). For THIs
possessing substitution at the C5 position (8iv and 10), a
Claisen condensation between ketones 4iv or 97 and diethyl
Received: April 8, 2014
© XXXX American Chemical Society
A
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more interested in THIs possessing C6 substitution (vide infra),
and thus we required an alternative approach to convert
symmetric ketones 4 to the desired tetrahydraindazole
carboxylates 6. We were drawn to a rarely utilized trans-
formation disclosed by Padwa and co-workers in which
pyrazole carboxylic esters were formed by addition of the
lithium anion of ethyl diazoacetate to ketones, followed by
dehydration and thermal cycloisomerization.9 Although, to the
best of our knowledge, the only cyclic ketone to which this
reaction had been applied prior to our work was cyclo-
hexanone, we realized that mechanistically this reaction would
specifically afford the alternative regioisomer relative to the
Claisen chemistry, starting with the same ketone 4.10 We
successfully applied this methodology to a diverse set of 4-
substituted cyclohexanones 4, providing ethyl pyrazolyl
carboxylates 5i−x, which were subsequently converted to the
protected acids 6i−x as above.
Formation of the western fragments 12 was straightforward
and followed chemistry disclosed previously for the indazole
series (Scheme 2):5m 3-nitropyrazole can either be directly
Figure 1. Evolution of the indazole series of ITK inhibitors.
a
Scheme 2. Synthesis of Benzylated Nitropyrazoles 12
Scheme 1. Synthesis of Tetrahydroindazole Carboxylic Acids
a
6, 8, and 10
a
Reagents and conditions: (a) K2CO3, DMF, rt; (b) PPh3, DEAD,
THF, 0 °C → rt; (c) mCPBA, CH2Cl2, rt.
benzylated with benzyl bromides and sodium hydride, or
secondary benzylic alcohols can be merged with 3-nitropyrazole
using Mitsonobu conditions.11 For nitropyrazole 12vi contain-
ing a sulfone appendage, the Mitsonobu reaction was done at
the sulfide oxidation state, and a subsequent mCPBA-mediated
oxidation afforded the sulfone.
Completion of the synthesis of our target inhibitors is
exemplified by the endgame toward representative inhibitor 17
(Scheme 3). Hydrogenative reduction of nitropyazole 12ii
yielded aminopyrazole 13ii, which was then immediately
acylated without purification with THI carboxylic acid 6iv
using TBTU. Deprotection using an aqueous acid/aqueous
base protocol provided the target inhibitor 17. The additional
aqueous base operation is necessary to remove residual N-
hydroxymethyl pyrazole byproducts that remain in varying
quantities following SEM deprotection. For inhibitors possess-
ing stereogenic elements, racemic mixtures were resolved into
single isomers by supercritical fluid chromatography on chiral
stationary phases at the final stage.
a
Reagents and conditions: (a) ethyl diazoacetate, LDA, THF, −78 °C;
(b) POCl3, pyr, rt; (c) n-octane, 110 °C; (d) diethyl oxylate, NaOEt,
EtOH, rt; (e) hydrazine, AcOH, rt; (f) SEM-Cl, NaH, THF, 0 °C →
rt; (g) LiOH, THF/ACN/H2O, rt.
oxylate,8 followed by ring closure with hydrazine, provided the
desired ethyl pyrazolyl carboxylates. SEM protection and ester
hydrolysis of 7iv provided the requisite protected acid 8iv,
whereas pyrazole carboxylic acids 10 were obtained without
SEM protection, as we subsequently determined protection of
the pyrazole NH was unnecessary. In general, however, we were
RESULTS AND DISCUSSION
■
The ITK active site, represented by the X-ray cocrystal
structure with inhibitor 2 (Figure 2),5m contains four residues
B
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a
four polar and two nonpolar interactions contribute to the
binding efficiency of these inhibitors. A tridentate array of
hydrogen bonds with the hinge region (carbonyl of Glu436;
carbonyl and NH of Met438) and a hydrogen bond between
the pyrazole NH and the side chain of Lys391 account for the
polar interactions. An edge-to-face π-stacking interaction
between the ligand phenyl group and Phe437 and face-to-face
π-stacking between the ligand pyrazole and Phe435 are also
important. The latter Phe435 interaction coupled with the
Lys391 hydrogen bond is postulated to account for the 40-fold
potency improvement between 1 and 2 (ITK Ki 43 nM → 1.0
nM); the additional 10-fold potency increase between 2 and 3
(ITK Ki 1.0 nM → 0.1 nM) can be rationalized by reduction of
the rotational degrees of freedom which results through
introduction of the benzylic stereocenter, thereby reducing
the entropic penalty paid by the ligand in order to adopt the
appropriate conformation to interact with Phe437.5m
Scheme 3. Representative Completion of Inhibitor 17
a
Reagents and conditions: (a) H2 (1 atm), 10% Pd/C, EtOH, rt; (b)
6i, TBTU, Pr2NEt, DMF, rt; (c) HCl in dioxane, 60 °C; NaOH(aq),
i
EtOH, rt.
While privileged analogues from the indazole class
demonstrated promising in vitro profiles, suboptimal solubility
and permeability precluded their use as in vivo tool
compounds. Inhibitor 2, for example, had no measurable
kinetic solubility in aqueous buffer (<1 μM) and displayed poor
permeability in MDCK cells (Papp = 0.1 × 10−6 cm/s), which
translated into unmeasurable bioavailability when dosed orally
as a methocel suspension in rats, dogs, or mice, despite
demonstrating IV clearance well below liver blood flow.
Furthermore, inhibition of Aurora kinases proved problematic
for this series (e.g., Aurora A Ki = 72 nM for inhibitor 2), which
we deemed a significant safety liability given the role for this
family of kinases in mitotic regulation and broad cellular
proliferation.14
We chose to address solubility concerns as our top priority
and with an eye toward exploring structural changes that had
the possibility of addressing selectivity improvements simulta-
neously. In a recent publication by scientists from Glax-
oSmithKline, a simple metric termed “solubility forecast index”
(SFI) was introduced to help predict solubility during the
design phase and thus increase the probability that synthesized
inhibitors demonstrate acceptable solubility characteristics.15
We retrospectively applied this metric, calculated by the simple
sum of aromatic ring count and the calculated log D at pH 7.4
(clogD7.4),16 to analogues prepared in the indazole series (such
as those exemplified in ref 5m) and found that solubility
decreased with increasing SFI (Figure 3A). Although most
analogues in this series had solubility forecast index greater than
6 and poor solubility, it was gratifying to observe that the vast
majority of analogues with SFI less than 6 had acceptable
solubility characteristics. Unfortunately, within the indazole
structural class, the majority of analogues with SFI less than 6
showed poor ITK inhibition (Figure 3B). We were thus
inspired to investigate scaffold modifications that would reduce
the aromatic ring count, and thus the solubility forecast index,
with an eye toward increasing the probability of improved
solubility without sacrificing potency.
Figure 2. (A) Co-crystal structure of 2 with the kinase domain of
ITK.5m Protein−ligand hydrogen bonds and π−π stacking interactions
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).13 (B) Close-up view of ITK selectivity
pocket (vide infra).
Examination of the indazole crystal structure (Figure 2)
suggested that the indazole core would be the most promising
candidate for saturation, as this ring makes no direct contact
with the protein. While we were cognizant that this may disrupt
the trajectory of the 6-pyrazole toward Phe435 and Lys391, we
considered this an opportunity rather than a liability: we hoped
that through this rescaffolding effort we would identify an
alternative substituent to the pyrazole. While this substituent
was clearly beneficial for ITK potency (cf. Figure 1), a
which distinguish it from other kinases: two phenylalanine
residues (the “gatekeeper” Phe435 and Phe437), a cysteine
residue (Cys442), and a serine residue (Ser499). The
phenylalanine pair alone is fairly unique, with only 14 of
>450 kinases possessing this combination, and neither the Phe/
Phe/Cys nor the Phe/Phe/Ser triad are contained in any other
kinase in the kinome.12 For the indazole class of ITK inhibitors,
C
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Figure 4. Co-crystal structure of 2 with the kinase domain of Aurora
A. Interactions and surface depiction are the same as for Figure 2
carbon at the 6-position of the THI core, which affects the
ground-state torsion of the pyrazole ring relative to the core,
orienting the heterocycle in a less-than-optimal configuration
with respect to Phe435. We thus sought to explore nonaromatic
lipophilic substitutions. We chose to carry out these
investigations with simple benzyl substitution (instead of m-
cyanobenzyl) of the western pyrazole since our previous SAR
studies in the indazole series showed the cyano moiety offered
little to no benefit.5m Monomethyl substitution was tolerated
(inhibitors 16 and 16′) but offered no significant benefit over
the unsubstituted THI 14. Our first breakthrough occurred
when gem-dimethyl substitution was added at the 6-position,
yielding inhibitor 17 (ITK Ki = 5.3 nM) that was approximately
6-fold more potent than the unsubstituted analogue 14.19
Gratifyingly, this ITK potency increase was not accompanied by
significant increase in Aurora A inhibition. Verifying that this
potency increase was not simply the result of nonspecific
lipophilic interactions, gem-dimethyl substitution at the 5-
position (18) offers no benefit relative to 14. gem-Difluoro
substitution of the six-position was not well tolerated (19; ITK
Ki = 180 nM), and spiro-cyclopropyl substitution results in poor
Aurora A selectivity (20; Aur-A/ITK = 18). While the ITK
potency increase observed for 17 was clearly promising, we
were disheartened that solubility issues remained apparent, and
we hypothesized that this was the source of the large enzyme-
to-cell shift observed for this inhibitor, as measured by
inhibition of the phosphorylation of PLCγ, the direct substrate
of ITK (IC50 = 5200 nM). We believed, however, that this poor
solubility was driven predominantly by the high lipophilicity of
this inhibitor (log D7.4 = 4.3), rather than the high aromatic ring
count intrinsic to the indazole series. To address this concern,
we explored introduction of oxygenation in the region of the
gem-dimethyl group of 17. We speculated this strategy had the
possibility to offer selectivity benefits as well because there was
the potential of projecting a hydrogen bond donor or acceptor
in the region of Ser499 (cf. Figure 2), a residue relatively
unique to ITK. While oxygenation clearly reduced lipophilicity
and increased solubility (inhibitors 21−24 all showed kinetic
solubility >20 μM), only 6-methyl-6-hydroxymethyl substitu-
tion present in 22 was beneficial for ITK potency. Gratifyingly,
although this inhibitor was slightly less potent than the gem-
dimethyl inhibitor 17 (8.0 vs 5.3 nM), the cellular potency of
22 increased significantly (PLCγ IC50 = 530 nM), a presumed
consequence of the improved solubility. Finally, combining a
Figure 3. Relationship between solubility forecast index and kinetic
solubility (A) and ITK potency (B) for the indazole class of ITK
inhibitors.
significant portion of this potency was likely driven by the H-
bond interaction with Lys391, the catalytic lysine that is highly
conserved across the kinome, and thus is not likely optimal with
respect to selectivity. Further, while the face-to-face π-stacking
interaction with Phe435 might be considered beneficial for
selectivity given the relative scarcity of phenylalanine gate-
keeper residues, literature evidence suggests that pyrazole
substitution in this region is beneficial for kinases with
lipophilic nonaromatic gatekeeper residues, such as leucine
present in the Aurora family.17 Finally, the pyrazole moiety
contributed negatively to the overall pharmaceutical property
profile of our inhibitors, adding an aromatic ring, a hydrogen
bond donor, and 29 Å2 to the TPSA.
From a structural standpoint, we were most excited about the
potential to occupy a lipophilic pocket adjacent to Phe435,
evident in surface representations of all internal and published18
ITK crystal structures. This pocket (Figure 2B), formed by the
side chains of Phe435, Lys391, Val377, and Ala389, lies directly
above the 6-position of the indazole core. The aromatic nature
of the indazole provides no out-of-plane vector toward this
pocket, but we envisioned that saturation of this region would
introduce appropriate three-dimensionality to access this space.
Furthermore, we obtained a crystal structure of inhibitor 2
bound to Aurora A, and no such pocket was evident, supporting
the exploration of this region of space as a means to improve
kinase selectivity (Figure 4).
Table 1 summarizes the first round of optimization of the
tetrahydroindazole series, which focused on the SAR around
the THI core itself (eastern fragment). Inhibitor 14 (ITK Ki =
31 nM) served as an important proof-of-concept for this
strategy, in that enzymatic potency was maintained via
saturation of the six-membered ring of the indazole nucleus
when compared to the progenitor indazole 1 (ITK Ki = 43
nM). Gratifyingly, kinetic solubility also trended in the right
direction (14 vs 5.8 μM). As was the case for the indazole
series, 3-pyrazolyl substitution at the 6-position did result in a
potency increase (ITK Ki = 8.3 nM for enantiomer 15; Ki = 9.4
nM for enantiomer 15′), however, the increase was much less
dramatic than that observed for the indazole series (1 → 2).
We postulate that this difference is due to the sp3 nature of the
D
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a
Table 1. Structure−Activity Relationships for THI Eastern Fragment Optimization
a
b
All values are the mean of two or more independent assays (see Experimental Section for assay details). For chiral inhibitors, the enantiomeric
pairs are given the same number, with the less potent isomer given the “prime” distinction (e.g., 14 and 14′). SFI = clogD7.4 + aromatic ring count.13
c
d
e
Kinetic solubility. Measured log D7.4.
5−6 fused cyclopropane with a 6-methyl group (26 and 26′)
yielded an interesting overall profile, with one enantiomer
showing excellent potency and selectivity for ITK (26: ITK Ki
= 3.0 nM; Aur-A Ki = 440 nM), whereas the alternative
enantiomer was more potent for Aurora A than ITK (26′: ITK
Ki = 200 nM; Aur-A Ki = 54 nM). Intriguingly, the small
reduction in lipophilicity for 26 over 17 (log D7.4 = 3.8 vs 4.3)
translates to low but measurable solubility (1.3 μM) and a
concomitant increase in cellular potency (PLCγ IC50 = 410
nM).
In a second phase of optimization, we explored benzylic and
aromatic substitution of the western fragment (Table 2). To
limit stereochemical complexity, we chose to explore this
optimization using the 6-gem-dimethyl substitution on the
eastern THI fragment. As was observed previously in the
indazole series,5m introduction of a basic amine tail to the
E
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a
Table 2. Optimization of the Western Fragment of the Tetrahydroindazole Series
a
b
All values are the mean of two or more independent assays (see Experimental Section for assay details). Stereochemistry of 28 and 28′ assigned by
c
d
X-ray structure of 28 (vide infra); all others assigned by analogy (only more potent enantiomer shown). Kinetic solubility. Measured log D7.4.
benzylic substituent of the western fragment resulted in a
significant increase in ITK potency and solubility. This was true
for either a one-carbon (27; ITK Ki = 0.8 nM; solubility = 42
μM) or two-carbon tail (28; 0.7 nM; solubility = 68 μM). More
importantly, this increase in ITK potency did not result in a
significant increase in potency for Aurora A. Furthermore,
cellular potency is increased by an even larger margin (PLCγ
IC50 = 38 and 55 nM, respectively) relative to the unsubstituted
comparator 17, which we postulate is driven by solubility
because permeability is relatively unchanged.20 It is interesting
to note that the enantiomer of 28 (28′; ITK Ki = 15 nM) loses
potency for ITK and gains potency for Aurora A (Ki = 170 nM)
relative to 17. Taken together, these data reinforce the
hypothesis that the potency increase is driven by reinforcement
of the binding conformation for the more potent enantiomer,
positioning the phenyl group appropriately to interact with
Phe437. Increasing the size of the benzylic substituent, such as
the piperidyl analogue 29, results in a further boost in ITK
potency (ITK Ki = 0.2 nM), but this is not captured in
increased cellular potency (PLCγ IC50 = 870 nM), presumably
due to the poor permeability of this analogue (0.1 × 10−6 cm/
s). Nonbasic solubilizing groups such as sulfone 30 were also
tolerated, and while no ITK potency increase is evident
enzymatically (ITK Ki = 6.2 nM), cellular potency was
increased relative to 17 (PLCγ IC50 = 310 nM), driven by
the increase in solubility (14 μM). Small lipophilic substituents
were tolerated on the phenyl ring (e.g., 31 and 32) but offered
no significant benefit over the unsubstituted phenyl ring.
An X-ray crystal structure of inhibitor 28 bound to the active
site of ITK was obtained (Figure 5) and revealed that the
inhibitor binds to the enzyme as designed. The axial methyl
Figure 5. Co-crystal structure of 28 with the kinase domain of ITK.
Interactions and surface depictions are the same as for Figure 2
F
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a
Table 3. In Vivo Pharmacokinetic Parameters for Inhibitor 28 (GNE-9822)
dose volume
(mL/kg)
dose
(mg/kg)
Cmax
(μM)
Cl
T1/2
(h)
Vss
(L/kg)
species
route
vehicle
AUC (μM·h) (mL/min/kg)
%F
b
mouse (BALB/c)
IV
SEDDS
1.0
5.0
1.0
50
1.5
27
40
70
21
2.9
3.0
5.4
10
14
6.7
c
PO
Crem/Cit
3.8
0.2
0.9
36
40
rat (Sprague−Dawley)
IV
SEDDS
SEDDS
1.0
2.0
1.0
5.0
0.6
1.1
PO
dog (beagle)
IV
SEDDS
SEDDS
1.0
1.0
5.0
1.9
PO
0.5
10
100
a
b
All values are the mean from at least two animals. SEDDS = self-emulsifying drug delivery system (1 part [50% cremophor, 35% NMP, 15%
Miglyol], 3 parts water).22 Crem/Cit = 5% cremophor in 50% citric acid.
c
group of the THI core projects into the lipophilic pocket
adjacent to Phe435, and the equatorial methyl group also
makes lipophilic contact with the phenyl group of this residue.
As with the indazole series, the inhibitor maintains three
hydrogen bonds with the hinge region, and the benzylic
stereocenter orients the western phenyl group for an
appropriate edge-to-face π-stack with Phe437.
Table 4. Comparison of Overall Profiles of Indazole 3 and
THI 28
Given its promising preliminary profile, inhibitor 28 (also
known as GNE-9822) was subjected to additional in vitro and
in vivo characterization. Broad kinase selectivity of this
molecule was observed: only six of 286 off-target kinases21
were inhibited >70% when tested at 0.1 μM (>100X ITK Ki).
In addition to the good solubility and moderate permeability
disclosed above, other in vitro ADME properties were
promising, with 28 demonstrating low clearance in human
hepatocytes (ClHHep = 5.0 mL/min/kg) and moderate plasma
protein binding (hPPB = 93%). In vivo ADME properties
(Table 3) were acceptable, with good bioavailability and half-
lives observed in all preclinical species, albeit with moderate
(mouse, dog) to high (rat) clearance.
To demonstrate the advantages afforded by saturation of the
indazole nucleus, a collection of potency, selectivity, and
calculated and measured pharmaceutical properties and ADME
data are summarized in Table 4 for the top indazole 3 and
tetrahydroindazole 28. Although enzymatic and cellular
potency of 3 is marginally superior, the lower molecular weight
of 28 results in the two inhibitors showing equivalent ligand
efficiency.23 Kinase selectivity clearly favors 28, whether
measured as inhibition of our sentinel off-target Aurora A or
in terms of a broader kinase panel. Although both molecules
show acceptable kinetic solubility, permeability of 28 is greatly
improved, resulting in a dramatic improvement in bioavail-
ability.
In conclusion, we have evolved a previously disclosed
indazole series of inhibitors through saturation of a core
aromatic ring to yield a related but distinct tetrahydroindazole
inhibitor class. This new chemical matter demonstrates
improved selectivity and pharmaceutical properties, specifically
solubility and permeability, when compared to their progeni-
tors. Future publications will disclose further improvements on
this chemical matter in the areas of potency, selectivity, and
ADME properties.
a
Ligand efficiency = 1.4 × (−log10(ITK Ki))/(no. of heavy atoms).20
b
Number of kinases inhibited >70% at 0.1 μM in an Invitrogen panel.
c
d
Clearance in human hepatocytes. Dosed PO at 5 mg/kg as a
solution.
performed with Isco CombiFlash Companion systems using prepacked
silica gel columns (40−60 μm particle size RediSep or 20−40 μm
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 Gemini-NX C-18 (0.3
cm × 5 cm; 5 μM) stationary phase, with 0.1% aqueous formic acid/
acetonitrile or 0.1% aqueous ammonium hydroxide/acetonitrile
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. NMR spectra were measured on Bruker 300
or 400 MHz spectrometers, and chemical shifts are reported in ppm
downfield from TMS using residual nondeuterated solvent as internal
standards (CHCl3, 7.26 ppm; DMSO, 2.50 ppm; MeOH, 3.31 ppm).
High-resolution mass spectrometry of final compounds were
performed on a Thermo UHPLC/QE with a Thermo-Q Exactive
mass spectrometry detector using ESI ionization, after elution on a
Acquity BEH C18 (2.1 mm × 50 mm; 1.7 μm particle size) stationary
phase using a gradient of water/acetonitrile (3−97% over 7 min; 0.1%
formic acid in both phases). The purity of final compounds 14−32 was
verified by HPLC on an Agilent 1200 instrument with an Agilent SB
EXPERIMENTAL SECTION
■
Chemistry. General. All commercially available reagents and
solvents were used as received. Reactions using air- or moisture-
sensitive reagents were performed under an atmosphere of N2 using
freshly opened EMD DriSolv solvents. Reaction progress was
monitored by TLC and/or HPLC. Flash chromatography was
G
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C-18 (2.1 mm × 30 mm; 1.8 μm 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.
General Procedure A: Formation of Pyrazole Carboxylic Acids 8
or 10 via Claisen Condensation. Step 1. To a solution of ketone 4 or
9 (1.0 equiv) in EtOH (0.5 mL/mmol) was added NaOEt (21 wt %
solution in EtOH; 1.1 equiv) and diethyl oxylate (1.0 equiv) at room
temperature. After stirring overnight, the mixture was concentrated in
vacuo and used directly without purification.
Step 2. The residue from step 1 was diluted with acetic acid (0.5
mL/mmol), cooled to 0 °C, and hydrazine hydrate (1.1 equiv) was
added. The mixture was warmed to room temperature, stirred for 1 h,
and then was diluted with satd NaHCO3(aq) and extracted with 10%
MeOH/CH2Cl2. The organic extracts were dried (MgSO4) and
concentrated in vacuo. Purification by CombiFlash using a mixture of
heptane and EtOAc provided the intermediate ethyl pyrazole
carboxylates.
Step 3. The ethyl pyrazole carboxylate obtained in step 2 was
diluted with THF (20 mL/mmol) and cooled to 0 °C and sodium
hydride (60 wt %; 3.0 equiv) was added. After stirring for 1 h at 0 °C,
SEM-Cl (1.2 equiv) was added and the mixture was allowed to warm
to room temperature overnight. Excess hydride was quenched by the
careful addition of water, and then the mixture was diluted with
additional water and extracted three times with EtOAc. The combined
organic extracts were dried (MgSO4) and concentrated in vacuo.
Intermediate SEM protected ethyl pyrazole carboxylates were then
obtained by purification by CombiFlash using a mixture of EtOAc and
heptane. These esters were diluted with THF (6 mL/mmol),
acetonitrile (6 mL/mmol), and water (6 mL/mmol). Lithium
hydroxide monohydrate (7 equiv) was then added, and the mixture
stirred overnight at rt. After dilution with additional water, the mixture
was acidified to pH 3 with 1 N HCl(aq), and then extracted three
times with 10%MeOH/CH2Cl2. The combined organic extracts were
dried (MgSO4) and concentrated in vacuo to provide the title
compounds of sufficient purity to be used directly.
General Procedure B: Formation of Pyrazole Carboxylic Acids 6
via Diazoacetate Addition/Rearrangement. Step 1. A solution of
diisopropylamine (1.7 equiv) in THF (5 mL/mmol) was cooled to
−78 °C, and a solution of n-butyl lithium in hexanes (1.6 M; 1.5
equiv) was added. After stirring for 5 min, this mixture was added via
cannula to a −78 °C solution of ethyl diazoacetate (1.6 equiv) and
ketone 4 in THF (5.0 mL/mmol). After stirring for 1 h at −78 °C, the
reaction was quenched by the addition of satd NH4Cl(aq). The
mixture was warmed to room temperature, diluted with water, and
extracted twice with EtOAc. The combined organic extracts were dried
(MgSO4) and concentrated in vacuo. Purification by CombiFlash with
a mixture of heptane and EtOAc as the eluent provided the
intermediate ethyl 2-diazo-2-hydroxycyclohexanyl-acetates.
Step 2. The ethyl 2-diazo-2-hydroxycyclohexanyl-acetate from step
1 was diluted with pyridine (4 mL/mmol), and then POCl3 (4.0
equiv), and the mixture was stirred overnight at room temperature.
After in vacuo concentration, the mixture was poured onto ice and
extracted three times with EtOAc. The combined organic extracts were
dried (MgSO4) and concentrated in vacuo. This residue was diluted
with n-octane (2 mL/mmol) and heated to 110 °C overnight. After in
vacuo concentration, purification by CombiFlash with a mixture of
heptane and EtOAc provided the intermediate ethyl pyrazole
carboxylate.
concentrated in vacuo. Purification by CombiFlash with a mixture of
EtOAc and heptane provided the title compounds.
General Procedure D: Formation of Nitropyrazoles 12 via
Mitsonobu Reaction. A solution of benzylic alcohols 11iii−viii (1.0
equiv) in THF (2 mL/mmol) was cooled to 0 °C, and then 4-nitro-
1H-pyrazole (1.0 equiv), PPh3 (2.0 equiv), and diethyl azodicarbox-
ylate (2.0 equiv) were added sequentially. The mixture was allowed to
warm to room temperature overnight, and then the mixture was
concentrated in vacuo. The residue was purified by CombiFlash with a
mixture of heptane and EtOAc to provide the title compounds,
occasionally contaminated with triphenylphosphine oxide of varying
quantities. Yields are assumed to be quantitative for purpose of
downstream chemistry quantities, and this reagent is used in excess.
General Procedure E: Formation of Target Compounds 14 and
15. Step 1. To a solution of 3-((4-nitro-1H-pyrazol-1-yl)methyl)-
benzonitrile (12i) (1.0 equiv) in ethanol (7 mL/mmol) was added
NH4Cl (5.0 equiv) as a saturated solution in water then iron (5.0
equiv). The mixture was heated to 80 °C for 60 min and then cooled
to room temperature. The mixture was diluted with EtOAc and
washed with satd NaHCO3(aq) and brine. The organic extracts were
dried (Na2SO4) and concentrated in vacuo to provide 3-((4-amino-
1H-pyrazol-1-yl)methyl)benzonitrile (13i), which was used without
purification.
Step 2. To a solution of pyrazole carboxylic acid 6i or 6ii (1.0
equiv) and aminopyrazole 13i (1.5 equiv) in DMF (3 mL/mmol) was
added TBTU (1.4 equiv) and iPr2NEt (3.0 equiv), and the mixture
was stirred overnight at rt. After dilution with water, the mixture was
extracted three times with EtOAc, and then the combined organic
extracts were dried (MgSO4) and concentrated in vacuo. Purification
by CombiFlash with an EtOAc and heptane mixture provided the SEM
protected title compounds.
Step 3. The product of step 2 was diluted with 4 N HCl in dioxane
(15 mL/mmol) and heated to 60 °C for 1 h. After cooling to room
temperature and in vacuo concentration, the residue was diluted with
EtOH (30 mL/mmol) and 5 M NaOH(aq) (12 mL/mmol) and
stirred for an additional hour to remove an residual N-
hydroxymethylation of the pyrazole. The mixture was diluted with
water, extracted three times with 10% MeOH/CH2Cl2, dried
(MgSO4), and concentrated in vacuo. Mass directed reverse-phase
HPLC purification, followed by lyophilization, provided the title
compounds. For samples possessing stereogenecity, racemic mixtures
were resolved into single enantiomers using SFC purification (or
preparative HPLC) on chiral stationary phases as indicated.
General Procedure F: Formation of Target Compounds 16−32.
Step 1. To a solution of benzylated nitro pyrazoles 12 (1.0 equiv; may
contain OPPh3) in ethanol (3 mL/mmol) was added palladium on
carbon (10 wt %; 50 mg/mmol), and the mixture was stirred under an
atmosphere of hydrogen overnight. The sample was then purged with
nitrogen, the catalyst was deactivated by the addition of CH2Cl2, and
the mixture was filtered through Celite. Concentration in vacuo
provided the intermediate aminopyrazoles 13, which were used
without purification.
Steps 2 and 3. To a solution of pyrazole carboxylic acid 6, 8, or 10
(1.0 equiv) and aminopyrazole 13 (1.5 equiv; may contain OPPh3)
were reacted according to steps 2 and 3 of general procedure E to
provide the title compounds.
1-((2-(Trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-inda-
zole-3-carboxylic Acid (6i). Cyclohexanone (4i; commercial) was
reacted according to general procedure B to provide 6i in 10% yield
1
over the three steps. H NMR (400 MHz, DMSO) δ 12.45 (s, 1H),
5.36 (s, 2H), 3.51 (t, J = 7.9, 2H), 2.66−2.58 (m, 4H), 1.78−1.61 (m,
4H), 0.82 (t, J = 7.9, 2H), −0.05 (s, 9H).
Step 3. The ethyl pyrazole carboxylate obtained in step 2 was
reacted in a manner identical to step 3 of general procedure A above,
to provide the title compounds.
General Procedure C: Formation of Nitropyrazoles 12 via Benzyl
Bromide Alkylation. To a solution of 4-nitro-1H-pyrazole (1.0 equiv)
in DMF (6 mL/mmol) was added K2CO3 (1.2 equiv) and benzyl
bromide 11i or 11ii (1.0 equiv). After stirring overnight at room
temperature, the mixture was diluted with EtOAc and washed twice
with 1:1 H2O:brine. The organic extracts were dried (Na2SO4) and
1-((2-(Trimethylsilyl)ethoxy)methyl)-6-(1-((2-(trimethylsilyl)-
ethoxy)methyl)-1H-pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-indazole-3-
carboxylic Acid (6ii). 4-(1-((2-(Trimethylsilyl)ethoxy)methyl)-1H-
pyrazol-4-yl)cyclohexanone (4ii)7 was reacted according to general
procedure B, to provide 6ii in 43% yield over the three steps. 1H NMR
(400 MHz, CDCl3) δ 7.47−7.33 (m, 2H), 5.40−5.34 (m, 2H), 3.78−
3.51 (m, 4H), 3.18−1.49 (m, 9H), 0.96−0.65 (m, 4H), 0.02 − −0.14
(m, 18H).
H
dx.doi.org/10.1021/jm500550e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
6-Methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-
1H-indazole-3-carboxylic Acid (6iii). 4-Methylcyclohexanone (4iii;
commercial) was reacted according to general procedure B, to provide
6iii in 32% yield over the three steps. 1H NMR (400 MHz, CDCl3) δ
5.39 (s, 2H), 3.59−3.52 (m, 2H), 2.98−2.88 (m, 1H), 2.81 (dd, J =
16.2, 5.3, 1H), 2.70−2.59 (m, 1H), 2.23 (dd, J = 16.2, 9.6, 1H), 2.02
(s, 1H), 1.99−1.84 (m, 2H), 1.44−1.32 (m, 1H), 1.11 (d, J = 6.6, 3H),
0.93−0.85 (m, 2H), −0.02 (s, 9H).
(4iv; commercial) was reacted according to general procedure A to
provide 8iv in 46% yield over the three steps. H NMR (500 MHz,
1
CDCl3) δ 5.41 (s, 2H), 3.58−3.53 (m, 2H), 2.68 (t, J = 6.5, 2H), 2.57
(s, 2H), 1.60 (t, J = 6.5, 2H), 1.00 (s, 6H), 0.91−0.85 (m, 2H), −0.03
(s, 9H).
1,4,4a,5,5a,6-Hexahydrocyclopropa[f ]indazole-3-carboxylic Acid
(10xi). Bicyclo[4.1.0]heptan-3-one (9xi)25 was reacted according to
general procedure A, omitting the SEM protection step, to provide
10xi in 11% yield over the three steps. Isomeric 3,4,5,5a,6,6a-
hexahydrocyclopropa[e]indazole-1-carboxylic acid was also obtained
through this sequence and was separated from 10xi by preparative
HPLC. MS: m/z = 179 [M + H]+.
5a-Methyl-1,4,4a,5,5a,6-hexahydrocyclopropa[f ]indazole-3-car-
boxylic Acid (10xii). 1-Methylbicyclo[4.1.0]heptan-3-one (9xii)7 was
reacted according to general procedure A, omitting the SEM
protection step, to provide 10xii in 33% yield over the three steps.
1H NMR (300 MHz, DMSO) δ 12.86 (br, 2H), 3.15−3.09 (d, J = 17.1
Hz, 1H), 2.96−2.79 (m, 2H), 2.66−2.61 (d, J = 15.9 Hz, 1H), 1.20 (s,
3H), 1.08−1.00 (m, 1H), 0.32−0.28 (m, 1H), 0.07−0.05 (m, 1H).
3-((4-Nitro-1H-pyrazol-1-yl)methyl)benzonitrile (12i). m-Cyano-
benzyl bromide (11i; commercial) was reacted according to general
procedure C to provide 12i in 95% yield. 1H NMR (400 MHz,
CDCl3) δ 8.15 (s, 1H), 8.12 (s, 1H), 7.72−7.64 (m, 1H), 7.61−7.48
(m, 3H), 5.35 (s, 2H).
6,6-Dimethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahy-
dro-1H-indazole-3-carboxylic Acid (6iv). 4,4-Dimethylcyclohexanone
(4iv; commercial) was reacted according to general procedure B, to
1
provide 6iv in 50% yield over the three steps. H NMR (400 MHz,
CDCl3) δ 5.39 (s, 2H), 3.58−3.50 (m, 2H), 2.77 (t, J = 6.4, 2H), 2.45
(s, 2H), 2.03 (s, 1H), 1.54 (t, J = 6.4, 2H), 1.02 (s, 6H), 0.91−0.83 (m,
2H), −0.03 (s, 9H).
6,6-Difluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahy-
dro-1H-indazole-3-carboxylic Acid (6v). 4,4-Difluorocyclohexanone
(4v; commercial) was reacted according to general procedure B, to
1
provide 6v in 41% yield over the three steps. H NMR (400 MHz,
CDCl3) δ 5.43 (s, 2H), 3.59−3.52 (m, 2H), 3.24 (t, J = 13.2, 2H), 3.00
(t, J = 6.6, 2H), 2.29−2.16 (m, 2H), 2.03 (s, 1H), 0.92−0.86 (m, 2H),
−0.02 (s, 9H).
1′-((2-(Trimethylsilyl)ethoxy)methyl)-1′,4′,5′,7′-tetrahydrospiro-
[cyclopropane-1,6′-indazole]-3′-carboxylic Acid (6vi). Spiro[2.5]-
octan-6-one (4vi; commercial) was reacted according to general
procedure B to provide 6vi in 31% yield over the three steps. 1H NMR
(400 MHz, CDCl3) δ 5.37 (s, 2H), 3.60−3.51 (m, 2H), 2.83 (t, J =
6.2, 2H), 2.55 (s, 2H), 2.11 (s, 1H), 1.55 (t, J = 6.2, 2H), 0.91−0.84
(m, 2H), 0.51−0.46 (m, 2H), 0.45−0.40 (m, 2H), −0.03 (s, 9H).
6-Methoxy-6-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-
tetrahydro-1H-indazole-3-carboxylic Acid (6vii). 4-Methoxy-4-meth-
ylcyclohexanone (4vii)24 was reacted according to general procedure B
to provide 6vi in 43% yield over the three steps. 1H NMR (400 MHz,
CDCl3) δ 3.58−3.51 (m, 2H), 3.25 (s, 3H), 2.88 (d, J = 16.4 Hz, 1H),
2.80 (t, J = 6.3 Hz, 2H), 2.63 (d, J = 16.4 Hz, 1H), 2.12−1.98 (m, 4H),
1.73−1.63 (m, 1H), 1.32 (s, 3H), 0.92−0.84 (m, 2H), −0.01 − −0.04
(m, 9H).
1-Benzyl-4-nitro-1H-pyrazole (12ii). Benzyl bromide (11ii; com-
mercial) was reacted according to general procedure C to provide 12ii
1
in 97% yield. H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 8.04 (s,
1H), 7.45−7.36 (m, 3H), 7.33−7.27 (m, 2H), 5.31 (s, 2H).
N,N-Dimethyl-2-(4-nitro-1H-pyrazol-1-yl)-2-phenylethanamine
(12iii). 2-(Dimethylamino)-1-phenylethanol (11iii)26 was reacted
according to general procedure D to provide 12iii contaminated
with OPPh3 (yield assumed to be 100% for downstream chemistry).
1H NMR (400 MHz, Chloroform-d) δ 8.29 (s, 1H), 8.11 (s, 1H),
7.42−7.24 (m, 5H), 5.44 (dd, J = 10.2, 4.7 Hz, 1H), 3.40 (dd, J = 13.5,
10.2 Hz, 1H), 2.80 (dd, J = 13.5, 4.7 Hz, 1H), 2.30 (s, 6H).
N,N-Dimethyl-3-(4-nitro-1H-pyrazol-1-yl)-3-phenylpropan-1-
amine (12iv). 3-(Dimethylamino)-1-phenyl-propan-1-ol (11iv)27 was
reacted according to general procedure D to provide 12iv in 80% yield.
1H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 8.09 (s, 1H), 7.41−7.32
6-((tert-Butyldimethylsilyloxy)methyl)-6-methyl-1-((2-
(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-1H-indazole-3-
carboxylic Acid (6viii). 4-((tert-Butyldimethylsilyloxy)methyl)-4-
methylcyclohexanone (4viii)7 was reacted according to general
(m, 5H), 5.49 (dd, J = 8.5, 6.5, 1H), 2.65−2.54 (m, 1H), 2.34−2.24
(m, 1H), 2.20 (s, 6H), 2.19−2.14 (m, 2H).
1
procedure B to provide 6viii in 76% yield over the three steps. H
tert-Butyl 4-((4-Nitro-1H-pyrazol-1-yl)(phenyl)methyl)piperidine-
1-carboxylate (12v). tert-Butyl 4-(hydroxy(phenyl)methyl)piperidine-
1-carboxylate (11v)28 was reacted according to general procedure D to
NMR (400 MHz, CDCl3) δ 5.39 (s, 2H), 3.57−3.51 (m, 2H), 3.42−
3.35 (m, 2H), 2.83 (dt, J = 17.2, 5.5, 1H), 2.77−2.68 (m, 1H), 2.64 (d,
J = 16.6, 1H), 2.35 (d, J = 16.4, 1H), 2.03 (s, 1H), 1.73−1.64 (m, 1H),
1.54−1.44 (m, 1H), 0.94 (s, 3H), 0.91−0.85 (m, 11H), 0.04 (s, 3H),
0.03 (s, 3H), −0.03 (s, 9H).
1
provide 12v in 51% yield. H NMR (400 MHz, CDCl3) δ 8.17 (s,
1H), 8.10 (s, 1H), 7.47−7.32 (m, 5H), 4.76 (d, J = 10.7 Hz, 1H),
4.23−3.98 (m, 2H), 2.75−2.57 (m, 3H), 1.44 (s, 9H), 1.40−1.03 (m,
4H).
1-((2-(Trimethylsilyl)ethoxy)methyl)-1,4,5,7-tetrahydrospiro-
[indazole-6,3′-oxetane]-3-carboxylic Acid (6ix). 2-Oxaspiro[3.5]-
nonan-7-one (4ix; commercial) was reacted according to general
procedure B to provide 6ix in 31% yield over the three steps. The
dehydration step (step 2) was performed using modified conditions as
follows: To a solution of ethyl 2-diazo-2-(7-hydroxy-2-oxaspiro[3.5]-
nonan-7-yl)acetate (100 mg, 0.393 mmol) in CH2Cl2 (2.5 mL) was
added triethylamine (0.138 mL, 0.983 mmol) and trifluoroacetic
anhydride (0.111 mL, 0.787 mmol). The mixture was stirred for 15
min and then diluted with H2O and extracted with CH2Cl2. The
combined organic extracts were dried (MgSO4) and concentrated in
vacuo. The residue was used directly in step 3 without purification. 1H
NMR (400 MHz, CDCl3) δ 5.50 (s, 2H), 4.60 (d, J = 6.0, 2H), 4.48
(d, J = 6.0, 2H), 3.62−3.56 (m, 2H), 3.07 (s, 2H), 2.87 (t, J = 6.2, 2H),
2.15−2.06 (m, 3H), 0.92 (t, J = 8.2, 2H), −0.00 (s, 9H).
1-(3-(Methylsulfonyl)-1-phenylpropyl)-4-nitro-1H-pyrazole
(12vi). 1-(3-(Methylthio)-1-phenylpropyl)-4-nitro-1H-pyrazole was
formed by subjection of 3-(methylthio)-1-phenylpropan-1-ol (11vi)
to general procedure D, followed by the following sulfone oxidation
step: mCPBA (5.0 g, 29 mmol, 2.5 equiv) was added in several
portions to a stirred solution of 1-[3-(methylsulfanyl)-1-phenyl-
propyl]-4-nitro-1H-pyrazole (3.0 g, 10.8 mmol, 1.0 equiv) in
dichloromethane (100 mL) at room temperature. The solids from
the reaction were filtered out, and the filtrate was diluted with 300 mL
of dichloromethane. The resulting mixture was washed with 2 × 150
mL of saturated sodium carbonate, dried over anhydrous sodium
sulfate, and concentrated under vacuum to provide the title compound
in 79% yield from 11vi that was used without further purification. MS:
m/z = 310 [M + H]+.
N,N-Dimethyl-3-(4-nitro-1H-pyrazol-1-yl)-3-(m-tolyl)propan-1-
amine (12vii). 3-(Dimethylamino)-1-(m-tolyl)propan-1-ol (11vii)29
was reacted according to general procedure D to provide 12vii in 52%
yield. MS: m/z = 289 [M + H]+.
3-(3-Chlorophenyl)-N,N-dimethyl-3-(4-nitro-1H-pyrazol-1-yl)-
propan-1-amine (12viii). 1-(3-Chlorophenyl)-3-(dimethylamino)-
propan-1-ol (11viii)30 was reacted according to general procedure D
to provide 12viii in 40% yield. MS: m/z = 309 [M + H]+.
1′-((2-(Trimethylsilyl)ethoxy)methyl)-1′,4,4′,5,5′,7′-hexahydro-
2H-spiro[furan-3,6′-indazole]-3′-carboxylic Acid (6x). 2-Oxaspiro-
[4.5]decan-8-one (4x)7 was reacted according to general procedure B
1
to provide 6x in 38% yield over the three steps. H NMR (400 MHz,
CDCl3) δ 5.45−5.37 (m, 2H), 3.98−3.91 (m, 2H), 3.63−3.51 (m,
4H), 2.89−2.77 (m, 2H), 2.76−2.63 (m, 2H), 2.03 (s, 1H), 1.93−1.69
(m, 4H), 0.91−0.85 (m, 2H), −0.03 (s, 9H).
5,5-Dimethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahy-
dro-1H-indazole-3-carboxylic Acid (8iv). 4,4-Dimethylcyclohexanone
I
dx.doi.org/10.1021/jm500550e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
N-(1-(3-Cyanobenzyl)-1H-pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-in-
dazole-3-carboxamide (14). 6i and 12i were reacted according to
general procedure E to provide 14 in 49% yield. 1H NMR (400 MHz,
DMSO) δ 12.82 (s, 1H), 10.08 (d, J = 12.0, 1H), 8.16 (s, 1H), 7.77 (d,
J = 6.8, 1H), 7.67 (s, 2H), 7.60−7.52 (m, 2H), 5.35 (s, 2H), 2.67 (t, J
= 5.5, 2H), 2.61 (t, J = 5.8, 2H), 1.77−1.62 (m, 4H). HRMS m/z calcd
for C19H19ON6 [M + H]+ 347.1615; found 347.1628.
N-(1-Benzyl-1H-pyrazol-4-yl)-6-methoxy-6-methyl-4,5,6,7-tetra-
hydro-1H-indazole-3-carboxamide (21 and 21′). 6vii and 12ii were
reacted according to general procedure F to provide racemic 21. SFC
purification (Lux Cellulose-3 (4.6 mm × 50 mm, 5 μm particle size) at
20% MeOH w/0.1%NH4OH; 5 mL/min, 120 bar, 40 °C) provided 21
(21% yield) and 21′ (21% yield). 21: 1H NMR (400 MHz, DMSO) δ
12.80 (s, 1H), 10.07 (s, 1H), 8.07 (s, 1H), 7.64 (s, 1H), 7.38−7.19 (m,
5H), 5.27 (s, 2H), 3.13 (s, 3H), 2.78−2.54 (m, 4H), 1.95−1.84 (m,
1H), 1.68−1.57 (m, 1H), 1.22 (s, 3H); HRMS m/z calcd for
C20H24O2N5 [M + H]+ 366.1925; found 366.1941; SFC retention time
0.38 min. 21′: 1H NMR (400 MHz, DMSO) δ 12.80 (s, 1H), 10.07 (s,
1H), 8.07 (s, 1H), 7.64 (s, 1H), 7.38−7.20 (m, 5H), 5.27 (s, 2H), 3.13
(s, 3H), 2.77−2.55 (m, 4H), 1.95−1.85 (m, 1H), 1.68−1.57 (m, 1H),
1.22 (s, 3H); HRMS m/z calcd for C20H24O2N5 [M + H]+ 366.1925;
found 366.1938; SFC retention time 0.46 min.
N-(1-(3-Cyanobenzyl)-1H-pyrazol-4-yl)-5-(1H-pyrazol-4-yl)-
4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (15 and 15′). 6ii
and 12i were reacted according to general procedure E to provide
racemic 15. SFC purification (Chiralpak OJ (21.2 mm × 250 mm, 5
μm particle size) at 35% MeOH w/0.1%NH4OH; 70 mL/min, 100
bar, 40 °C) provided 15 (6% yield) and 15′ (9% yield). 15: 1H NMR
(400 MHz, DMSO) δ 12.88 (s, 1H), 12.58 (s, 1H), 10.14 (s, 1H),
8.17 (s, 1H), 7.77 (d, J = 6.9, 1H), 7.68 (s, 2H), 7.60−7.46 (m, 4H),
5.36 (s, 2H), 3.03−2.82 (m, 3H), 2.72−2.60 (m, 2H), 2.11−2.01 (m,
1H), 1.76−1.61 (m, 1H); HRMS m/z calcd for C22H21ON8 [M + H]+
413.1833; found 413.1850; SFC retention time 0.96 min. 15′: 1H
NMR (400 MHz, DMSO) δ 12.89 (s, 1H), 12.57 (s, 1H), 10.14 (s,
1H), 8.17 (s, 1H), 7.77 (d, J = 6.9, 1H), 7.68 (s, 2H), 7.60−7.46 (m,
4H), 5.36 (s, 2H), 3.03−2.82 (m, 3H), 2.73−2.57 (m, 2H), 2.12−1.99
(m, 1H), 1.76−1.61 (m, 1H); HRMS m/z calcd for C22H21ON8 [M +
H]+ 413.1833; found 413.1848; SFC retention time 1.22 min.
N-(1-Benzyl-1H-pyrazol-4-yl)-6-methyl-4,5,6,7-tetrahydro-1H-in-
dazole-3-carboxamide (16 and 16′). 6iii and 12ii were reacted
according to general procedure F to provide racemic 16. SFC
purification (Phenomenex Cellulose-4 (21.2 mm × 150 mm, 5 μm
particle size) at 40% MeOH w/0.1%NH4OH; 70 mL/min, 100 bar, 40
N-(1-Benzyl-1H-pyrazol-4-yl)-6-(hydroxymethyl)-6-methyl-
4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (22 and 22′). 6viii
and 12ii were reacted according to general procedure F to provide
racemic 22. SFC purification (Phenomenex Amylose-2 (21.2 mm ×
250 mm, 5 μm particle size) at 35% MeOH w/0.1%NH4OH; 40 mL/
min, 100 bar, 40 °C) provided 22 (24% yield) and 22′ (18% yield).
22: 1H NMR (400 MHz, DMSO) δ 12.61 (s, 1H), 10.05 (s, 1H), 8.07
(s, 1H), 7.64 (s, 1H), 7.37−7.25 (m, 3H), 7.25−7.20 (m, 2H), 5.27 (s,
2H), 4.63 (s, 1H), 3.21 (s, 2H), 2.78−2.65 (m, 1H), 2.63−2.48 (m,
2H), 2.26 (d, J = 16.0, 1H), 1.59−1.49 (m, 1H), 1.47−1.37 (m, 1H),
0.87 (s, 3H); HRMS m/z calcd for C20H24O2N5 [M + H]+ 366.1925;
1
found 366.1940; SFC retention time 0.61 min. 22′: H NMR (400
MHz, DMSO) δ 12.79 (s, 1H), 10.08 (s, 1H), 8.07 (s, 1H), 7.64 (s,
1H), 7.37−7.26 (m, 3H), 7.25−7.20 (m, 2H), 5.27 (s, 2H), 4.62 (t, J =
5.4, 1H), 3.21 (d, J = 5.4, 2H), 2.72 (dt, J = 16.7, 5.6, 1H), 2.63−2.48
(m, 2H), 2.26 (d, J = 16.1, 1H), 1.59−1.49 (m, 1H), 1.47−1.37 (m,
1H), 0.87 (s, 3H); HRMS m/z calcd for C20H24O2N5 [M + H]+
366.1925; found 366.1939; SFC retention time 0.49 min.
1
°C) provided 16 (4% yield) and 16′ (4% yield). 16: H NMR (400
MHz, DMSO) δ 10.07 (s, 1H), 8.36 (s, 1H), 8.07 (s, 1H), 7.64 (s,
1H), 7.37−7.25 (m, 3H), 7.25−7.20 (m, 2H), 5.27 (s, 2H), 2.87−2.78
(m, 1H), 2.73 (dd, J = 15.8, 5.2, 1H), 2.60−2.48 (m, 1H), 2.18 (dd, J =
15.9, 9.6, 1H), 1.90−1.73 (m, 2H), 1.38−1.25 (m, 1H), 1.04 (d, J =
6.6, 3H); HRMS m/z calcd for C19H22ON5 [M + H]+ 336.1819; found
336.1833; SFC retention time 0.49 min. 16b: H NMR (400 MHz,
DMSO) δ 12.79 (s, 1H), 10.07 (s, 1H), 8.07 (s, 1H), 7.64 (s, 1H),
7.37−7.25 (m, 3H), 7.25−7.20 (m, 2H), 5.27 (s, 2H), 2.87−2.77 (m,
1H), 2.73 (dd, J = 15.8, 5.2, 1H), 2.61−2.48 (m, 1H), 2.18 (dd, J =
15.9, 9.6, 1H), 1.89−1.73 (m, 2H), 1.38−1.24 (m, 1H), 1.04 (d, J =
6.6, 3H); HRMS m/z calcd for C19H22ON5 [M + H]+ 336.1819; found
336.1832; SFC retention time 0.57 min.
N-(1-Benzyl-1H-pyrazol-4-yl)-1,4,5,7-tetrahydrospiro[indazole-
6,3′-oxetane]-3-carboxamide (23). 6ix and 12ii were reacted
1
according to general procedure F to provide 23 in 58% yield. H
NMR (400 MHz, DMSO) δ 12.93 (s, 1H), 10.10 (s, 1H), 8.07 (s,
1H), 7.64 (s, 1H), 7.37−7.25 (m, 3H), 7.25−7.20 (m, 2H), 5.27 (s,
2H), 4.39 (d, J = 5.8, 2H), 4.32 (d, J = 5.8, 2H), 2.94 (s, 2H), 2.72 (t, J
= 6.1, 2H), 1.97 (t, J = 6.3, 2H). HRMS m/z calcd for C20H22O2N5 [M
+ H]+ 364.1768; found 364.1781.
N-(1-Benzyl-1H-pyrazol-4-yl)-1′,4,4′,5,5′,7′-hexahydro-2H-spiro-
[furan-3,6′-indazole]-3′-carboxamide (24 and 24′). 6x and 12ii
were reacted according to general procedure F to provide racemic 24.
SFC purification (Lux Cellulose-3 (4.6 mm × 50 mm, 5 μm particle
size) at 25% MeOH w/0.1%NH4OH; 5 mL/min, 120 bar, 40 °C)
N-(1-Benzyl-1H-pyrazol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-
indazole-3-carboxamide (17). 6iv and 12ii were reacted according to
general procedure F to provide 17 in 57% yield. 1H NMR (400 MHz,
DMSO) δ 12.77 (s, 1H), 10.04 (s, 1H), 8.07 (s, 1H), 7.64 (s, 1H),
7.37−7.20 (m, 5H), 5.27 (s, 2H), 2.66 (t, J = 6.3, 2H), 2.38 (s, 2H),
1.47 (t, J = 6.4, 2H), 0.96 (s, 6H). HRMS m/z calcd for C20H24ON5
[M + H]+ 350.1975; found 350.1990.
N-(1-Benzyl-1H-pyrazol-4-yl)-5,5-dimethyl-4,5,6,7-tetrahydro-1H-
indazole-3-carboxamide (18). 8ii and 12ii were reacted according to
general procedure F to provide 18 in 38% yield. 1H NMR (400 MHz,
DMSO) δ 12.84 (s, 1H), 10.07 (s, 1H), 8.05 (s, 1H), 7.64 (s, 1H),
7.37−7.26 (m, 3H), 7.25−7.20 (m, 2H), 5.27 (s, 2H), 2.60 (t, J = 6.4,
2H), 2.48 (s, 2H), 1.51 (t, J = 6.4, 2H), 0.94 (s, 6H). HRMS m/z calcd
for C20H24ON5 [M + H]+ 350.1975; found 350.1989.
N-(1-Benzyl-1H-pyrazol-4-yl)-6,6-difluoro-4,5,6,7-tetrahydro-1H-
indazole-3-carboxamide (19). 6v and 12ii were reacted according to
general procedure F to provide 19 in 19% yield. 1H NMR (400 MHz,
DMSO) δ 13.12 (s, 1H), 10.22 (s, 1H), 8.08 (s, 1H), 7.64 (s, 1H),
7.37−7.26 (m, 3H), 7.26−7.21 (m, 2H), 5.28 (s, 2H), 3.35−3.21 (m,
2H), 2.87 (t, J = 6.4, 2H), 2.28−2.14 (m, 2H). HRMS m/z calcd for
C18H18ON5F2 [M + H]+ 358.1474; found 358.1487.
1
provided 24 (29% yield) and 24′ (28% yield). 24: H NMR (400
MHz, DMSO) δ 12.88 (s, 1H), 10.09 (s, 1H), 8.07 (s, 1H), 7.64 (s,
1H), 7.39−7.18 (m, 5H), 5.27 (s, 2H), 3.80 (t, J = 7.1 Hz, 2H), 3.48
(d, J = 8.4 Hz, 1H), 3.42 (d, J = 8.4 Hz, 1H), 2.71 (t, J = 6.4 Hz, 2H),
2.61 (s, 2H), 1.82−1.60 (m, 4H); HRMS m/z calcd for C21H24O2N5
[M + H]+ 378.1925; found 378.1940; SFC retention time 0.36 min.
24′: 1H NMR (400 MHz, DMSO) δ 12.85 (s, 1H), 10.09 (s, 1H), 8.07
(s, 1H), 7.64 (s, 1H), 7.37−7.20 (m, 5H), 5.27 (s, 2H), 3.80 (t, J = 7.1
Hz, 2H), 3.48 (d, J = 8.4 Hz, 1H), 3.42 (d, J = 8.4 Hz, 1H), 2.71 (t, J =
6.4 Hz, 2H), 2.61 (s, 2H), 1.82−1.60 (m, 4H); HRMS m/z calcd for
C21H24O2N5 [M + H]+ 378.1925; found 378.1940; SFC retention time
0.51 min.
N - ( 1 - B e n z y l - 1 H - p y r a z o l - 4 - y l ) - 1 , 4 , 4 a , 5 , 5 a , 6 -
hexahydrocyclopropa[f ]indazole-3-carboxamide (25 and 25′). 10xi
and 12ii were reacted according to general procedure F, omitting step
3 (SEM deprotection), to provide racemic 25. Preparative chiral
HPLC (ChiralPak IC (4.6 mm × 250 mm, 5 μm particle size); eluent
= hexane(0.1% Et3N):EtOH 80:20; 1.0 mL/min, 5.2 MPA, 25 °C)
N-(1-Benzyl-1H-pyrazol-4-yl)-1′,4′,5′,7′-tetrahydrospiro-
[cyclopropane-1,6′-indazole]-3′-carboxamide (20). 6vi and 12ii
were reacted according to general procedure F to provide 20 in
1
provided 25 (5.1% yield) and 25′ (3.9% yield). 25: H NMR (400
1
44% yield. H NMR (400 MHz, DMSO) δ 12.89 (s, 1H), 10.13 (s,
MHz, DMSO) δ 12.84 (1H, s), 10.11 (1H, s), 8.09 (1H, s), 7.66 (1H,
s), 7.39−7.23 (5H, m), 5.29 (2H, s), 2.70−2.65 (1H, t), 2.36−2.22
(2H, m), 2.13−2.08 (1H, t), 1.73−1.67 (1H, m), 0.91−0.80 (1H, m),
0.50−0.40 (1H, m); HRMS m/z calcd for C19H20ON5 [M + H]+
1H), 8.07 (s, 1H), 7.65 (s, 1H), 7.38−7.26 (m, 3H), 7.26−7.21 (m,
2H), 5.28 (s, 2H), 4.38 (d, J = 5.7, 2H), 4.27 (d, J = 5.7, 2H), 2.99 (s,
2H), 2.69 (t, J = 6.3, 2H), 2.03 (t, J = 6.3, 2H). HRMS m/z calcd for
C20H22ON5 [M + H]+ 348.1819; found 348.1832.
1
334.1662; found 334.1676; HPLC retention time 16.23 min. 25′: H
J
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Journal of Medicinal Chemistry
Article
NMR (400 MHz, DMSO) δ 12.84 (1H, s), 10.11 (1H, s), 8.09 (1H,
s), 7.66 (1H, s), 7.39−7.23 (5H, m), 5.29 (2H, s), 2.70−2.65 (1H, t),
2.36−2.22 (2H, m), 2.13−2.08 (1H, t), 1.73−1.67 (1H, m), 0.91−0.80
(1H, m), 0.50−0.40 (1H, m); HRMS m/z calcd for C19H20ON5 [M +
H]+ 334.1662; found 334.1678; HPLC retention time 14.26 min.
N-(1-Benzyl-1H-pyrazol-4-yl)-5a-methyl-1,4,4a,5,5a,6-
hexahydrocyclopropa[f ]indazole-3-carboxamide (26 and 26′).
10xii and 12ii were reacted according to general procedure F,
omitting step 3 (SEM deprotection), to provide racemic 25.
Preparative chiral HPLC (ChiralPak AD-H (4.6 mm × 150 mm, 5
μm particle size); eluent = hexane(0.1% Et3N):EtOH 60:40; 1.0 mL/
min, 4.4 MPA, 25 °C) provided 26 (13.9% yield) and 26′ (14.4%
yield). 26: 1H NMR (300 MHz, CDCl3, ppm) δ 8.58 (s, 1H), 8.05 (s,
1H), 7.55 (s, 1H), 7.37−7.32 (m, 3H), 7.30−7.24 (m, 2H), 5.28 (s,
2H), 3.42−3.36 (m, 1H), 3.07−2.97 (m, 2H), 2.74−2.69 (m, 1H),
1.40−1.25 (m, 3H), 1.13−1.07 (m, 1H), 0.42−0.38 (m, 1H), 0.24−
0.22 (m, 1H); HRMS m/z calcd for C20H22ON5 [M + H]+ 348.1819;
× 250 mm, 5 μm particle size); eluent = hexane(0.1% Et3N):EtOH
80:20; 1.0 mL/min, 5.0 MPA, 25 °C) provided 30 (18% yield). H
1
NMR (300 MHz, CD3OD) δ 8.08 (s, 1H), 7.7 (s, 1H), 7.37−7.26 (m,
5H), 5.55−5.50 (m, 1H), 3.29−2.83 (m, 6H), 2.80−2.72 (m, 2H),
2.67−2.59 (m, 1H), 2.40 (s, 2H), 1.55−1.50 (t, J = 6.3, 2H), 0.99 (s,
6H). HRMS m/z calcd for C23H30O3N8S [M + H]+ 456.2064; found
456.2080. HPLC retention time 16.70 min (undesired isomer: 20.43
min).
(S)-N-(1-(3-(Dimethylamino)-1-(m-tolyl)propyl)-1H-pyrazol-4-yl)-
6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (31).
6iv and 12vii were reacted according to general procedure F to
provide racemic 31. Preparative chiral HPLC (Venusil chiral OD-H
(4.6 mm × 250 mm, 5 μm particle size); eluent = hexane(0.1%
Et3N):EtOH 90:10; 1.0 mL/min, 3.3 MPA, 25 °C) provided 31 (15%
1
yield). H NMR (300 MHz, CD3OD) δ 8.10 (s, 1H), 7.70 (s, 1H),
7.27−7.11 (m, 4H), 5.40−5.35 (m, 1H), 2.81−2.77 (t, J = 12.3, 2H),
2.64−2.61 (m, 1H), 2.57 (s, 5H), 2.54 (s, 7H), 1.59−1.32 (m, 2H),
1.04 (s, 6H). MS: m/z = 435 (M + H) (insufficient material for
HRMS analysis). HPLC retention time 14.10 min (undesired isomer:
21.73 min).
1
found 348.1834; HPLC retention time 9.29 min. 26′: H NMR (300
MHz, CDCl3, ppm) δ 8.58 (s, 1H), 8.05 (s, 1H), 7.55 (s, 1H), 7.37−
7.32 (m, 3H), 7.30−7.24 (m, 2H), 5.28 (s, 2H), 3.42−3.36 (m, 1H),
3.07−2.97 (m, 2H), 2.74−2.69 (m, 1H), 1.40−1.25 (m, 3H), 1.13−
1.07 (m, 1H), 0.42−0.38 (m, 1H), 0.24−0.22 (m, 1H); HRMS m/z
calcd for C20H22ON5 [M + H]+ 348.1819; found 348.1834; HPLC
retention time 6.55 min.
(S)-N-(1-(1-(3-Chlorophenyl)-3-(dimethylamino)propyl)-1H-pyra-
zol-4-yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxa-
mide (32). 6iv and 12viii were reacted according to general procedure
F to provide racemic 32. Preparative chiral HPLC (Venusil chiral OD-
H (4.6 mm × 250 mm, 5 μm particle size); eluent = hexane(0.1%
Et3N):EtOH 90:10; 1.0 mL/min, 3.3 MPA, 25 °C) provided 32 (12%
(R)-N-(1-(2-(Dimethylamino)-1-phenylethyl)-1H-pyrazol-4-yl)-
6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (27).
6iv and 12iii were reacted according to general procedure F to
provide racemic 27. SFC purification (Chiralpak AD (4.6 mm × 50
mm, 5 μm particle size) at 35% MeOH w/0.1%NH4OH; 5 mL/min,
120 bar, 40 °C) provided 27 (9% yield). 1H NMR (400 MHz,
DMSO) δ 12.91−12.72 (s, 1H), 10.20−9.97 (s, 1H), 8.17−8.15 (s,
1H), 7.64−7.61 (s, 1H), 7.38−7.23 (m, 5H), 5.68−5.46 (dd, J = 9.1,
5.8 Hz, 1H), 3.28−3.19 (dd, J = 12.7, 9.4 Hz, 1H), 2.82−2.74 (dd, J =
12.9, 5.7 Hz, 1H), 2.70−2.63 (t, J = 6.3 Hz, 2H), 2.42−2.37 (s, 2H),
2.21−2.16 (s, 6H), 1.50−1.44 (t, J = 6.3 Hz, 2H), 0.99−0.93 (s, 6H);
HRMS m/z calcd for C23H31ON6 [M + H]+ 407.2554; found
407.2569; SFC retention time 0.58 min (undesired isomer, 0.43 min).
(S)-N-(1-(3-(Dimethylamino)-1-phenylpropyl)-1H-pyrazol-4-yl)-
6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (28)
and (R)-N-(1-(3-(Dimethylamino)-1-phenylpropyl)-1H-pyrazol-4-
yl)-6,6-dimethyl-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide
(28′). 6iv and 12iv were reacted according to general procedure F to
provide racemic 28. SFC purification (Phenomenex Cellulose-4 (21.2
mm × 150 mm, 5 μm particle size) at 45% MeOH w/0.1%NH4OH;
70 mL/min, 100 bar, 40 °C) provided 28 (21% yield) and 28′ (24%
1
yield). H NMR (300mHz, CD3OD, ppm) δ 8.13 (s, 1H), 7.74 (s,
1H), 7.38−7.28 (m, 4H), 5.47−5.42 (m, 1H), 2.81−2.77 (t, J = 12.6,
2H), 2.69−2.61 (m, 1H), 2.58 (s, 1H), 2.44−2.35 (m, 2H), 2.34 (s,
9H), 1.59−1.55 (t, J = 12.9, 2H), 1.04 (s, 6H). HRMS m/z calcd for
C24H32ON6Cl [M + H]+ 455.2321; found 455.2341. HPLC retention
time 13.35 min (undesired isomer: 17.71 min).
Pharmaceutical and Biological Assay Protocols. Measured
log D7.4. Two buffers were used in the assay: a saturated 1-octanol/
phosphate buffer solution (20 mL of PBS pH 7.4 + 200 mL of 1-
octanol) and a saturated PBS/1-octanol solution (1 mL of 1-octanol +
200 mL of PBS pH 7.4). Each solution was freshly prepared, shaken
for 1 h, and allowed to stand for 24 h before use. Saturated 1-octanol/
PBS solution (250 μL) was dispensed into each well of a 96-deep well
plate and followed by 5 μL of a 10 mM test compound in DMSO
stock solution. The plates were sealed with aluminum sealing film and
shaken for 20 min at 1000 rpm. After shaking, 250 μL of saturated
PBS/1-octanol solution was added to each well. The plates were then
resealed, shaken for 1 h at 1000 rpm at room temperature, and
centrifuged at 3000 rpm for 10 min. Sample analyses were carried out
by transferring the octanol and PBS phases to 384-well plates, with 2
μL of 0.1 mM propranolol in acetonitrile solution added to each well
as an internal standard. A UPLC/MS/MS equipped with Shimadzu
LC and Sciex API4000 was used for quantification of octanol and PBS
samples. The quantification was performed by MRM (multiple
reaction monitor) transition analysis. With separate injections, 3 μL
for the octanol samples and 10 μL for the PBS samples, MRM peaks
were collected, processed, and integrated. The log D7.4 of the
compound was determined as the log of the ratio of MRM peaks in
octanol and PBS phases.
Kinetic Solubility. Kinetic solubility in PBS buffer at pH 7.4 was
determined using 10 mM DMSO stock solutions for each compound.
96-well Millipore filtration plates were used for this assay with each
well containing 196 μL of PBS buffer solution and 4 μL of DMSO
stock solution. The filter plate was sealed with aluminum sealing film
and was shaken for 24 h at 1000 rpm at room temperature. The
incubation solution was then filtered into a clean 96-well plate using a
vacuum manifold and diluted by 2 and 4 times (two different dilutions
used for internal quality control) with PBS/MeOH(1/1 v/v) mixture.
An Agilent 1290 HPLC and equipped with an Antek 8060 CLND was
used for solubility quantitation. The CLND was calibrated using
caffeine in a H2O/MeOH (1/1 v/v) solution as a standard at eight
different concentrations from 2 to 4500 μM. Then 5 μL of each
sample was injected onto a reverse phase C18 column and a 4 min LC
gradient from 98:2 to 2:98 A:B (A = 0.1% TFA in H2O; B = 0.1% TFA
1
yield). 28: H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 10.05 (s,
1H), 8.08 (s, 1H), 7.66 (s, 1H), 7.37−7.22 (m, 5H), 5.42 (dd, J = 9.3,
5.3, 1H), 2.66 (t, J = 6.0, 2H), 2.38 (s, 2H), 2.25−2.02 (m, 10H), 1.47
(t, J = 6.4, 2H), 0.96 (s, 6H); HRMS m/z calcd for C24H33ON6 [M +
H]+ 421.2710; found 421.2726; SFC retention time 0.37 min. 28′: 1H
NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 10.04 (s, 1H), 8.08 (s,
1H), 7.66 (s, 1H), 7.36−7.23 (m, 5H), 5.42 (dd, J = 9.2, 5.6, 1H), 2.66
(t, J = 6.2, 2H), 2.38 (s, 2H), 2.23−2.00 (m, 10H), 1.47 (t, J = 6.3,
2H), 0.96 (s, 6H); HRMS m/z calcd for C24H33ON6 [M + H]+
421.2710; found 421.2729; SFC retention time 0.59 min.
(S)-6,6-Dimethyl-N-(1-(phenyl(piperidin-4-yl)methyl)-1H-pyrazol-
4-yl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (29). 6iv and
12v were reacted according to general procedure F to provide racemic
29. SFC purification (Lux Cellulose-1 (4.6 mm × 50 mm, 5 μm
particle size) at 30% MeOH w/0.1%NH4OH; 5 mL/min, 120 bar, 40
°C) provided 29 (5% yield). 1H NMR (400 MHz, DMSO) δ 12.82 (s,
1H), 10.06 (s, 1H), 8.14 (s, 1H), 7.63 (s, 1H), 7.55−7.48 (m, 2H),
7.37−7.30 (m, 2H), 7.30−7.24 (m, 1H), 5.00 (d, J = 10.7 Hz, 1H),
2.97−2.84 (m, 2H), 2.70−2.62 (m, 2H), 2.55−2.36 (m, 6H), 1.46 (t, J
= 6.3 Hz, 2H), 1.21−1.00 (m, 4H), 0.96 (s, 6H). HRMS m/z calcd for
C25H33ON6 [M + H]+ 433.2710; found 433.2723; SFC retention time
0.43 min (undesired isomer: 0.58 min).
(S)-6,6-Dimethyl-N-(1-(3-(methylsulfonyl)-1-phenylpropyl)-1H-
pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-indazole-3-carboxamide (30).
6iv and 12vi were reacted according to general procedure F to
provide racemic 30. Preparative chiral HPLC (ChiralPak IB (4.6 mm
K
dx.doi.org/10.1021/jm500550e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
in MeOH) at a flow rate of 400 μL/min was used. The solubility was
determined by nitrogen response of CLND.
ASSOCIATED CONTENT
* Supporting Information
■
S
ITK Biochemical Assay. GST-ITK full-length enzyme was from
Invitrogen (PV3875) and the substrate was BLK peptide (Ac-
EFPIYDFLPAKKK-NH2). Reactions were carried out in a final
volume of 51 μL with 50 mM HEPES (pH 7.2), 15 mM MgCl2, 2 mM
DTT, 0.015% Brij-35, 1 nM ITK, 2 μM substrate, 20 μM ATP, and
test article in a final concentration of 2% DMSO. After 35 min
incubation at rt, reactions were stopped upon addition of 10 μL of
30% TCA. Samples were centrifuged (4350 rpm, 4 °C, 5 min) and
subjected to LC/MS analysis on a Waters Acquity UPLC/TQD
system equipped with a Waters Acquity UPLC BEH C18 (2.1 mm ×
50 mm) 1.7 μm column (injection volume, 5 μL; column temperature,
60 °C; flow rate, 1 mL/min; solvent A, 0.1% formic acid in LC/MS
grade water; solvent B, 0.1% formic acid in LC/MS grade ACN).
Analytes were separated by applying a gradient from 15% to 32%
solvent B within 0.7 min and detected in positive mode ESI-MS/MS
by MRM (multiple reaction monitoring) of transitions 819.8/84.8
(BLK substrate) and 859.0/84.8 as well as 859.0/120.7 (phosphory-
lated BLK product). Ki values were determined using the Morrison
tight-binding model31 modified to account for an ATP-competitive
mechanism of inhibition and for the concentration of active kinase
used (0.3 nM ITK). Using this approach, the lower limit of Ki
measurable is approximately 250-fold lower than the ITK concen-
tration, or 0.0012 nM.
Aurora A Biochemical Assay. HIS-tagged human AURKA full
length enzyme was purchased either from Invitrogen (PV3612) or
from Millipore (14−511), and substrate was a generic Kemptide
peptide (Ac-LRRASLG-NH2). Reactions were carried out in a final
volume of 51 μL with 50 mM HEPES (pH 7.2), 10 mM MgCl2, 2 mM
DTT, 0.015% Brij-35, 10 nM AURKA, 2 μM substrate, 100 μM ATP,
and 2% DMSO. After 60 min incubation at RT, reactions were
stopped upon addition of 10 μL of 30% TCA. Samples were
centrifuged (4350 rpm, 4 °C, 5 min) and subjected to LC/MS analysis
on a Waters Acquity UPLC/TQD system equipped with a Waters
Acquity UPLC BEH C18 (2.1 mm × 50 mm) 1.7 μm column
(injection volume, 5 μL; column temperature, 60 °C; flow rate, 1 mL/
min; solvent A, 0.1% formic acid in LC/MS grade water; solvent B,
0.1% formic acid in LC/MS grade ACN). Analytes were separated by
applying a gradient from 10 to 13% solvent B within 0.7 min and
detected in positive mode ESI-MS/MS by MRM (multiple reaction
monitoring) of transitions 813.3/86.2 (Kemptide substrate) and
893.3/86.2 as well as 893.3/795.5 (phosphorylated Kemptide
product). Ki values were calculated as above for the ITK assay.
PLCγ Cellular Assay. Jurkat T-cells (400 K cells/well) were
suspended in assay buffer (D-PBS + 0.1% glucose + 10% FBS + 0.22
μM sodium pyruvate), seeded in 96-well plate, treated with
compounds for 30 min, and stimulated with anti-CD3 Dynabeads
(Invitrogen, 4 × 106 beads/well) for 2 min at 37 °C. The cells were
pelleted by brief spinning, and after aspirating off media, 30 μL of 1×
lysis buffer (Cell Signaling Technology) was added to each well, and
the plate was incubated at 4 °C for 30 min while shaking. After beads
were quickly spun down, 25 μL of lysate from each well was
transferred to capture antibody (mouse monoclonal antibody to
human PLCγ1, from Abcam) coated MSD goat antimouse 96-well
plate (MesoScale Discovery) and incubated at 4 °C overnight with
shaking. After washing four times with 150 μL of MSD Tris buffer (50
mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.02% Tween-20), 25 μL of
detection antibodies (1:2000 dilution mixture of rabbit anti-pPLCγ1,
from Cell Signaling Technology and goat antirabbit SULFO-Tag from
MesoScale Discovery) were added and incubated for 2 h at room
temperature. After washing of plate four times with 150 μL of MSD
Tris buffer, 150 μL of 1× MSD read buffer T was added to each well,
and the plate was read out on SECTOR Imager 6000 (MesoScale
Discovery).
Procedures for the synthesis of ketones 4ii,viii−x, and 9xii;
crystallographic method, data refinement and statistics for all
disclosed crystal structures; full kinase selectivity panel for 28.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
The PDB ID code for 2 bound to Aurora-A is 4PRJ; the ID
code for 28 bound to ITK is 4PQN.
AUTHOR INFORMATION
Corresponding Author
*Phone: 650-467-6059. E-mail: burch.jason@gene.com.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Deven Wang for HRMS measurements, Dr. Baiwei
Lin for kinetic solubility and log D measurements, Chris
Hamman, Mengling Wong, and Michael Hayes for compound
purification, Allan Jaochico, Xiao Ding, and Justin Ly for in vivo
PK data, and Connie Chan for formulation work. Drs. Matthew
Volgraf, James Crawford, and Steven Staben are gratefully
acknowledged for their careful editing of this manuscript.
ABBREVIATIONS USED
■
ACN, acetonitrile; ADME, absorption−distribution−metabo-
lism−elimination; AUC, area under the curve; ATP, adenine
triphosphate; Aur-A, Aurora A; Cl, clearance; CLND,
chemiluminescence nitrogen detector; DEAD, diethyl azodi-
carboxylate; %F, bioavailability (fraction absorbed); HEPES, 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, high-
pressure liquid chromatography; hPPB, human plasma protein
binding; HTS, high-throughput screen; IL, interleukin; ITK,
interleukin-2 inducible T-cell kinase; LDA, lithium diisopropy-
lamide; LE, ligand efficiency; mCPBA, meta-chloroperbenzoic
acid; MDCK, Madin−Darby canine kidney; MOE, Molecular
Operating Environment; MRM, multiple reaction monitoring;
NMP, N-methyl pyrrolidone; PBS, phosphate buffer solution;
PLCγ, phosphoinositide phospholipase C gamma; pyr,
pyridine; rt, room temperature; SAR, structure−activity
relationships; SEDDS, self-emulsifying drug delivery system;
SEM, 2-(trimethylsilyl)ethoxymethyl; SFC, supercritical fluid
chromatography; SFI, solubility forecast index; T1/2, half-life;
TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
tetrafluoroborate; TCA, trichloroacetic acid; TCR, T-cell
receptor; TFA, trifluoroacetic acid; THI, tetrahydroindazole;
THF, tetrahydrofuran; TPSA, topological polar surface area;
Vss, volume of distribution at steady state
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