Discovery and optimization of indazoles as potent and selective interleukin-2 inducible T cell kinase (ITK) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.04.023

There is evidence that small molecule inhibitors of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signaling cascade, could represent a novel asthma therapeutic class. Moreover, given the expected chronic dosing regimen of any asthma treatment, highly selective as well as potent inhibitors would be strongly preferred in any potential therapeutic. Here we report hit-to-lead optimization of a series of indazoles that demonstrate sub-nanomolar inhibitory potency against ITK with strong cellular activity and good kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of the complexes.

Full text of DOI:10.1016/j.bmcl.2014.04.023

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2448–2452
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of indazoles as potent and selective
interleukin-2 inducible T cell kinase (ITK) inhibitors
⇑
Richard M. Pastor , Jason D. Burch, Steven Magnuson, Daniel F. Ortwine, Yuan Chen, Kelly De La Torre,
Xiao Ding, Charles Eigenbrot, Adam Johnson, Marya Liimatta, Yichin Liu, Steven Shia, Xiaolu Wang,
Lawren C. Wu, Zhonghua Pei
Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 February 2014
Revised 6 April 2014
Accepted 7 April 2014
Available online 16 April 2014
There is evidence that small molecule inhibitors of the non-receptor tyrosine kinase ITK, a component of
the T-cell receptor signaling cascade, could represent a novel asthma therapeutic class. Moreover, given
the expected chronic dosing regimen of any asthma treatment, highly selective as well as potent inhib-
itors would be strongly preferred in any potential therapeutic. Here we report hit-to-lead optimization of
a series of indazoles that demonstrate sub-nanomolar inhibitory potency against ITK with strong cellular
activity and good kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the
X-ray crystal structures of the complexes.
Keywords:
SBDD
ITK
Ó 2014 Elsevier Ltd. All rights reserved.
Indazole
X-ray crystallography
p-stacking
Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec
H
N
kinase family of non-receptor tyrosine kinases, plays a major role
in T-cell signaling.1 Along with other T-cell specific tyrosine
kinases (including LCK and ZAP-70), ITK serves to amplify the sig-
nal associated with the T-cell receptor (TCR) cascade. Aberrant sig-
naling behavior in this cascade is known to lead to autoimmune
disorders and inflammation.2 In studies with ITK knockout mice,
levels of Th2 cytokines such as IL-4, IL-5, and IL-13 have been
reduced.3 Moreover, immunological symptoms of allergic asthma
were attenuated in these mice and lung inflammation, eosinophil
infiltration and mucous production were drastically reduced in
response to challenge with the allergen ovalbumin.4 These and
other reports suggest that selective inhibition of ITK could repre-
sent a novel therapy for the treatment of asthma.5
N
ITK Ki = 43 nM
LE = 0.397
O
LogD7.4 = 3.0 (ref. 9)
>50% inhibition against
18/75 kinases in kinase panel (ref.10)
> 85% inhibition against
6/75 kinases including JAK2
and FLT310
HN
N
N
N
1
Figure 1. Initial indazole high-throughput screening hit. (See above-mentioned
references for further information).
In order to identify novel ITK inhibitors, a high throughput
screen of our internal small molecule screening collection was con-
ducted.6 From this screen, 1 (Fig. 1) was identified as a suitable
starting point for chemistry optimization. This compound was syn-
thesized starting from methyl-1H-indazole-3-carboxylate (2) as
described in Scheme 1. SEM protection of indazole 2 and subse-
quent hydrolysis of the methyl ester gave SEM protected indazole
acid 3. In parallel, 3-nitropyrazole (4) was reacted with 3-cyanob-
enzyl alcohol under standard Mitsunobu conditions7 and thereaf-
ter reduced to obtain 3-amino pyrazole 5. Carboxylic acid 3 and
amino-pyrazole 5 were then coupled to give amide 6, which upon
SEM deprotection afforded indazole 1.
With 1 in hand, attempts to co-crystallize 1 with the kinase
domain of ITK were taken and proved to be successful (Fig. 2).
Examination of the structure revealed the central ligand-receptor
interaction to consist of 3 hydrogen bonds between the hinge of
the kinase domain and the indazole-carboxamide component of
1. This then positioned the pyrazole and cyanobenzyl moieties to
form
p–p stacking interactions with Phe-437 in face to face and
⇑
Corresponding author. Tel.: +1 650 225 8114.
E-mail address: pastor.richard@gene.com (R. M. Pastor).
edge to face manners, respectively.8 In addition to the interactions
http://dx.doi.org/10.1016/j.bmcl.2014.04.023
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

R. M. Pastor et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2448–2452
SEM SEM
2449
H
N
H
N
R
a, b
Br
a,b
N
N
N
N
N
N
O
O
O
O
N
HN
O
2
3
HN
HO
N
N
N
NO2
NH2
N
e
c, d
N
N
N
N
NH
4
N
5
N
Reagents and Conditions: a) RB(OH)2, PdCl2(Dppf),
1M Na2CO3, ACN, 120 °C; b) TFA, DCM
SEM
H
N
N
N
N
Scheme 2. Parallel synthesis of indazole C6 substituted analogs.
f
O
O
N
HN
HN
Table 1
SAR of Indazole C6 substituents
1
N
6
N
N
H
N
R
N
O
N
N
HN
Reagents and Conditions: a) SEM-Cl, NaH, THF;
N
b) 2M LiOH, THF; c) 3-Cyanobenzyl alcohol, PPh3, DIAD, DCM;
d) Fe, NH4Cl, H2O, EtOH, 70 °C for 1 hr; e) 2, HATU, DIPEA, DMF;
f) TFA, DCM.
N
N
Scheme 1. Synthesis of inhibitor 1.
Example
7
R
Ki (nM)
580
N
8
9
59
10
N
H2N
N
10
11
1.4
0.8
N
HN
H
N
N
12
7.8
Figure 2. X-ray crystal structure of 1 with the kinase domain of ITK (resolution
2.6 Å, PDB code 4PP9). Protein–ligand hydrogen bonds are denoted by dashed lines
with cylinders. Figure was generated using MOE (www.chemcomp.com).
10 resulted in a 6-fold increase in potency relative to 9. A pyrazole
was also appended to indazole C6 in both regioisomeric configura-
tions. 4-Pyrazolyl substitution (11) resulted in a Ki of 0.8 nM, while
the 3-pyrazolyl substituted analog (12) demonstrated a Ki of
7.8 nM. Upon obtaining a co-crystal structure of 11, two key inter-
actions involving the pyrazole were revealed (Fig. 3). In addition to
elucidated by the crystal structure, we were also encouraged by
the proximal location of the indazole-C6 to the gatekeeper Phe-
435 side chain. This orientation presented an opportunity for mak-
the expected face-to-face
H-bond with the sidechain of Lys-391 was also evident. In order
to optimize the -stacking interaction, the pyrazole rotates 28°
out of plane with the indazole ring and calculations show this to
be a low energy torsion.12 Calculations also show regioisomer 12
would prefer a co-planar arrangement of the pyrazole and indazole
rings. Adopting a 28 degree out of plane orientation to maximize a
p-stacking interaction with Phe-435, an
ing additional interactions with the protein in the form of
stacking interactions with this residue.
Based on the crystal structure, exploration of substitution at the
indazole C6 position was undertaken. Desired analogs were syn-
thesized via parallel Suzuki couplings according to Scheme 2.11
Five and six-membered aromatic and heteroaromatic rings were
p–p
p
explored to examine the potential of forming face-to-face
p stack-
p
-stacking interaction with Phe-435 would place 12 in a higher
ing interactions with the gatekeeper (Table 1). Appending a phenyl
group (7) resulted in a 10-fold loss in activity relative to 1. How-
ever, substitution with a 4-pyridyl moiety (8) led to an equipotent
analog, while a 3-pyridyl ring addition (9) resulted in a 4-fold
potency increase. Addition of an amine to give 4-amino-3-pyridine
energy conformation, consistent with an 8-fold loss in potency.13
Focus then turned to optimization of the cyanobenzyl-
substituted pyrazole moiety. Based on the existing crystal structures,
the pyrazole and cyanobenzyl components
p stack with Phe-437

2450
R. M. Pastor et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2448–2452
analogs containing an aromatic CH with a partially positively
charged hydrogen atom at this position could participate in a
nontraditional H-bonding interaction with this carbonyl oxygen,
while compounds with an imino nitrogen or oxygen would incur
lone pair repulsions, driving this sidechain into an unfavorable
binding geometry. While a sulfur atom at this location might be
expected to exhibit a favorable interaction with the Met438
carbonyl oxygen, the longer bond length and shorter valence angle
of the sulfur would be expected to result in an altered bond vector
to the cyanobenzyl group, effectively moving it away from a close
edge-to-face stacking interaction with Phe437. Additionally, the
negatively charged imino ring nitrogen on 15 would be expected
to electrostatically clash with the neighboring amide carbonyl
oxygen in the amide linker if the molecule is placed in a similar
conformation as observed with 1, driving the inhibitor into a
conformation not tolerated by the protein active site.
Modifications of the cyanobenzyl moiety were then prepared
using Mitsunobu couplings as described in Scheme 3 (Table 3).14
Mono-substitution of the methylene carbon was well tolerated.
Potency was somewhat sensitive to chirality of the benzyl substi-
tuent, as the S enantiomer (19) proved 5-fold more potent than
the R (18). Disubstitution resulted in a 30-fold loss in potency
(compare 17 and 19). Changes on the cyanophenyl ring were well
tolerated. Removing the cyano moiety or moving it to the para
position proved to be potency neutral. Replacement with a chlorine
or dimethylaminomethyl moiety also resulted in equipotent ana-
logs. On one hand, this is not surprising given the projection of this
ring into a solvent accessible region of the protein. However, given
the apparent edge to face interaction of this ring with Phe-437
observed in the X-rays, sensitivity to electron density might have
been expected.8
Prompted by the high potency of 19, further exploration of
methylene substitution was initiated in the context of the
6-pyrazolo-indazole subseries (Table 4). While ethyl substitution
was well tolerated, polar substituents such as tertiary amines
and tertiary alcohols (27–30) were preferred, with the most potent
compound, S enantiomer 28, demonstrating a Ki of 0.1 nM. Potency
was again dependent on chirality as the R antipode 27 was 10-fold
less potent. Inspection of the crystal structures of 27 and 28 (Fig. 4)
shows that in both cases, the N,N-dimethylaminoethyl group
projected into solvent, consistent with the preference for polar
substituents at the benzylic position. For the S enantiomer 28, this
Figure 3. Co-crystal structure of 11 with the kinase domain of ITK (Resolution
2.7 Å, PDB code 4PPA). Protein–ligand hydrogen bonds and
tions are denoted by dashed lines with cylinders.
p–p stacking interac-
in face to face and edge to face manners, respectively. The methy-
lene group acts as the linker connecting these components,
enabling the requisite interaction geometries. In order to assess
the spatial and electronic details of these interactions, a variety
of heterocyclic replacements of the pyrazole were tested, with sig-
nificant decreases in potency observed in all cases (Table 2). The
2,5-disubstituted pyrazole 13 and 3,5-disubstituted thiazole 14
demonstrated roughly 100 fold losses in potency relative to 1. Even
larger reductions in potency were observed with thiazole 15 and
oxazole 16, both of which had Ki values of >4 lM. Among the more
potent compounds, an aromatic CH group is present at one of the
ring positions ortho to the amide substituent, while in the less
potent or inactive compounds, heteroatoms are present at both
ortho positions. Examination of the crystal structure of 1 reveals
that one of the ortho-amide positions on the pyrazole ring is direc-
ted towards the backbone carbonyl oxygen of Met-438. Thus,
Table 2
SAR of pyrazole heterocyclic replacements
H
N
enforces the benzyl geometry required for edge to face
p stacking
N
with Phe-437. However, for the R-enantiomer 27 to project the
dimethylaminoethyl group out to solvent, the pyrazole ring rotates
180° relative to the S-enantiomer, moving the benzyl moiety
O
HN
beyond
p stacking distance with Phe-437 into a hydrophobic pack-
R
ing interaction with Ile-369. The difference in potency between the
enantiomers is likely a result of the difference in free energy
between these two interactions.
Cellular potencies of selected compounds was measured using a
Jurkat line in which phosphorylated phospholipase C-
c1 (PLC c-1)
Example
13
R
Ki (nM)
levels were quantitated after treatment with compound (Table 5).14
N
N
3.4
SEM
H
N
N
a,b
N
S
N
14
15
16
1.7
>4
N
O
O
HN
HN
S
N
N
N
N
N
R
N
H
O
>4
Reagents and Conditions: a) ROH, PPh3, DEAD, DCM; b) TFA, DCM
Scheme 3. Parallel synthesis of cyanobenzyl modifications.

R. M. Pastor et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2448–2452
2451
Table 3
Table 4
SAR of cyanobenzyl replacements
SAR of benzylic substituents
H
N
H
N
H
N
N
N
N
O
O
N
HN
HN
N
N
R
N
R
Example
17
R
Ki (nM)
Example
R
Ki (nM)
381
53
24
25
1.0
0.56
HO
HO
N
26
27
6.6
18
19
20
0.93
28
29
0.1
0.8
N
11
HO
HO
36
30
0.23
21
22
27
35
N
Cl
N
23
44
In general, cell-based and biochemical potencies were well corre-
lated. For instance, the 20-fold increase in biochemical activity of
11 versus 1 translated to a 20-fold increase in cellular potency.
Comparison of 30 and 11 showed a similar effect, as a 4-fold differ-
ence in biochemical potency tracked with a 5-fold difference in cel-
lular potency. An exception was 28, which is effectively equipotent
to 11 in the cell assay despite being 8-fold more potent
biochemically.
As the series progressed, compounds were selected for kinase
selectivity assessment and in vivo pharmacokinetic studies. Selec-
tivity testing for 1 and 28 were conducted at Invitrogen10 by exam-
ining compounds at a single concentration corresponding to
roughly 100ꢀ ITK Ki against a panel of 75 kinases. The initial HTS
hit 1 showed >50% inhibition against 18 kinases and >85% inhibi-
tion against JAK2, FLT3, MSK, PAK4, RET, and TRK. Compound 28,
which was 100-fold more potent than 1 against ITK, demonstrated
>50% inhibition against 7 kinases and >85% inhibition against only
FLT3. Representative compounds were also screened against LCK, a
TEC kinase upstream of ITK in the T-cell receptor signaling path-
way. This series consistently demonstrated >100-fold selectivity
against LCK, increasing our confidence that cellular pathway inhi-
bition was being driven by ITK interference.
Figure 4. Co-crystal structures of 27 (cyan, resolution 2.8 Å, PDB code 4PPC) and 28
(orange, resolution 2.9 Å, PDB code 4PPB) with the kinase domain of ITK. For clarity,
only residues from the co-crystal structure of 27 are shown. A solvent accessible
surface from this complex is included. Protein–ligand hydrogen bonds are denoted
by dashed lines with cylinders. Distances (Å) between the 30 methyl of Ile-369 and
the closest benzyl phenyl carbons of less potent enantiomer 27 are shown in green.
Table 5
Cellular potencies of selected indazoles
Example
Ki (nM)
pPLC
4.6
0.231
0.173
0.044
c
IC50
(lM)
MDCK Papp (A:B/B:A)
LogD 7.4
1
15
6.7/13.6
0.7/10
0.3/7
3.0
3.2
1.9
3.4
11
28
30
0.8
0.1
0.23
2.4/14
115 mL/min/kg and no oral bioavailability. Both 11 and 28 had
much lower IV clearance relative to 1, but demonstrated no oral
bioavailability, presumably because of their low permeabilities.
Pharmacokinetic measurements of 1, 11, 28, and 30 were taken
in rat (Table 6). Compound 1 displayed a high in vivo clearance of

2452
R. M. Pastor et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2448–2452
Table 6
Pharmacokinetic parameters of selected indazoles
Example
1
Route (dose)
AUC (h
l
M)
t1/2 (h)
Cl (mL/min/kg)
%F
MDCK Papp (A:B/B:A)
IV (1 mg/kg)
PO (5 mg/kg)
IV (1 mg/kg)
PO (5 mg/kg)
IV (1 mg/kg)
PO (5 mg/kg)
IV (1 mg/kg)
PO (5 mg/kg)
0.42
—
1.8
—
0.837
—
2.5
—
0.26
—
1.3
—
5.7
—
1.8
2.6
115
—
20
—
44
—
11
—
—
0
—
0
—
0
—
9
6.7/13.6
11
28
30
0.7/10
0.3/7
2.4/14
H.; Pullen, S. S.; Woska, J. R., Jr.; Raymond, E. L.; Sellati, R.; Cywin, C. L.; Snow, R.
J.; Kashem, M. A.; Wolak, J. P.; King, J.; Kaplita, P. V.; Liu, L. H. Bioorg. Med. Chem.
Lett. 2008, 18, 5541; (f) Riether, D.; Zindell, R.; Kowalski, A., et al Bioorg. Med.
Chem. Lett. 2009, 19, 1588; (g) Lo, H. Y.; Bentzien, J.; Fleck, R. W.; Pullen, S. S.;
Khine, H. H.; Woska, J. R.; Kugler, S. Z.; Kashen, M. A.; Takahashi, H. Bioorg. Med.
Chem. Lett. 2008, 18, 6218; (h) Charrier, J. D.; Miller, A.; Kay, D. P.; Brenchley, G.;
Twin, H. C.; Collier, P. N.; Ramaya, S.; Keily, S. B.; Durrant, S. J.; Knegtel, R. M.;
Tanner, A. J.; Brown, K.; Curno, A. P.; Jimenez, J. M. J. Med. Chem. 2011, 54, 2341;
(i) Herdemann, M.; Weber, A.; Jonveaux, J.; Jonveaux, J.; Schwoebel, S.; Heit, I.
Bioorg. Med. Chem. Lett. 2011, 21, 1852; (j) McLean, L. R.; Zhang, Y.; Zaidi, N.; Bi,
X.; Wang, R.; Dharanipragada, R.; Jurcak, J. G.; Gillespy, T. A.; Zhao, Z.; Musick,
K. Y.; Choi, Y. M.; Barrague, M.; Peppard, J.; Smicker, M.; Duguid, M.; Parkar, A.;
Fordham, J.; Kominos, D. Bioorg. Med. Chem. Lett. 2012, 22, 3296; (k) Zapf, C. W.;
Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han,
S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R.
M.; Seth, N.; Medley, Q. G. J. Med. Chem. 2012, 55, 10047; (l) Alder, C.; Ambler,
M.; Campbell, A.; Champigny, A.; Deakin, A.; Harling, J.; Harris, C.; Longstaff, T.;
Lynn, S.; Maxwell, A.; Mooney, C.; Scullion, C.; Singh, O.; Smith, I.; Somers, D.;
Tame, C.; Wayne, G.; Wilson, C.; Woolven, J. ACS Med. Chem. Lett. 2013, 4, 948;
(m) Harling, J.; Deakin, A.; Campos, S.; Grimley, R.; Chaudry, L.; Nye, C.;
Polyakova, O.; Bessant, C.; Barton, N.; Somers, D.; Barrett, J.; Graves, R.; Hanns,
L.; Kerr, W.; Solari, R. J. Biol. Chem. 2013, 288, 28195; For a recent review on ITK
inhibitors, see: (n) Charrier, J.-D.; Knegtel, R. M. A. Expert Opin. Drug Discov.
2013, 8, 369.
In contrast, 30 demonstrated moderate MDCK permeability and
this translated to low, but measureable, oral exposure.
By pursuing a structure-based design approach, an HTS-derived
indazole hit was evolved into an ITK inhibitor series that demon-
strated sub-nanomolar biochemical and low nanomolar cellular
potencies, with good kinase selectivity. However, optimized com-
pounds consistently demonstrated poor pharmacokinetic proper-
ties. Lead optimization efforts to improve pharmacokinetics will
be discussed in future communications.
Acknowledgments
Mengling Wong, Michael Hayes and Chris Hamman are grate-
fully acknowledged for the purification of all compounds described
in this manuscript, and Dr. Baiwei Lin is acknowledged for the
determination of logD for all compounds.
Supplementary data
6. Biochemical assay conditions: The enzyme reaction was carried out in a final 4-
l
L reaction volume in 1536-well black MAKO COP plates (Aurora
Supplementary data (full torsion profiles of 6-(1H-pyrazol-4-
yl)-1H-indazole and both tautomeric forms of 6-(1H-pyrazol-3-
yl)-1H-indazole. Experimentals for Schemes 1–3. Experimentals
for X-ray crystallography.) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2014.04.023.
Biotechnologies). Phosphorylation of the peptide substrate biotin-
EQEDEPEGIYGVLF-NH2 (American Peptide Company, Sunnyvale CA) was
initiated by the addition of 2
into 2 L 2 nM full-length ITK (Invitrogen) with library compounds in kinase
reaction buffer (50 mM HEPES pH 7.2, 15 mM MgCl2, 0.005% Brij-35, 2 mM
DTT). The reaction mixture was incubated for 1 h at ambient temperature prior
lL 800 nM peptide substrate and 40 lM ATP
l
to the addition of 2 lL detection reagent containing 6 nM Europium cryptate-
labeled PT66 antibody and 200 nM streptavidin XL-665 (Cisbio) in detection
buffer (50 mM HEPES pH 7.2, 0.1% BSA, 0.5 M KF, 40 mM EDTA). The final
reaction mixture was incubated at ambient temperature for 2 h. The signal
References and notes
measurement was performed using
a ViewLux reader (Perkin Elmer) via
excitation wavelength of 320 nm and emission wavelengths of 618 and
671 nm. The HTRF ratio was determined using the following equation:
Em671/Em618x10,000.
1. Felices, M.; Falk, M.; Kosaka, Y.; Berg, L. J. Adv. Immunol. 2007, 93, 145.
2. Sakaguchi, S.; Benham, H.; Cope, A. P.; Thomas, R. Immunol. Cell Biol. 2012, 90,
277.
7. Mitsunobu, O.; Yamada, M. Bull. Chem. Soc. Jpn. 1967, 40, 2380.
8. Castellano, R. K.; Diederich, F.; Meyer, E. A. Angew. Chem., Int. Ed. 2003, 42, 11.
9. Lin, B.; Pease, J. H. Comb. Chem. High Throughput Screen. 2013, 16, 817.
10. Selectivity screening was performed by SelectScreenÒ Kinase Profiling Services
(Invitrogen-Life Technologies, Madison, USA).
3. Mueller, C.; August, A. J. Immunol. 2003, 170, 5056.
4. Schaeffer, E. M.; Debnath, J.; Yap, G.; McVicar, D.; Liao, X. C.; Littman, D. R.;
Sher, A.; Varmus, H. E.; Lenardo, M. J.; Schwartzberg, P. L. Science 1999, 284,
638.
5. For small-molecule inhibitors of ITK, see: (a) Das, J.; Liu, C.; Moquin, R. V.; Lin,
J.; Furch, J. A.; Spergel, S. H.; McIntyre, K. W.; Shuster, D. J.; O’Day, K. D.;
Penhallow, B.; Huang, C.-Y.; Kanner, S. B.; Lin, T.-A.; Dodd, J. H.; Barrish, J. C.;
Wityak, J. Bioorg. Med. Chem. Lett. 2006, 16, 2411; (b) Lin, T. A.; Mcintyre, K. W.;
Das, J.; Liu, C.; O’Day, K. D.; Penhallow, C.-Y.; Whitney, G. S.; Shuster, D. J.; Xia,
X.; Towsend, R.; Postelnek, J.; Spergel, S. H.; Lin, J.; Moquin, R. V.; Furch, J. A.;
Kamath, A. V.; Zhang, H.; Marathe, P. H.; Perez-Villar, J. J.; Doweyko, A.; Killar,
L.; Dodd, J. H.; Barrish, J. C.; Wityak, J.; Kanner, S. B. Biochemistry 2004, 43,
11056; (c) Snow, R. J.; Abeywardane, A.; Campbell, S.; Lord, J.; Kashem, M. A.;
Khine, H. H.; King, J.; Kowalski, J. A.; Pullen, S. S.; Roma, T.; Roth, G. P.; Sarko, C.
R.; Wilson, N. S.; Winter, M. P.; Wolak, J. P.; Cywin, C. L. Bioorg. Med. Chem. Lett.
2007, 17, 3660; (d) Moriarty, K. J.; Winters, M.; Qiao, L.; Ryan, D.; DesJarlis, R.;
Robinson, D.; Cook, B. N.; Kashen, M. A.; Kaplita, P. V.; Liu, L. H.; Farrell, T. M.;
Khine, H. H.; King, J.; Pullen, S. S.; Roth, G. P.; Magolda, R.; Takahashi, H. Bioorg.
Med. Chem. Lett. 2008, 18, 5537; (e) Winters, K. M. P.; Robinson, D. J.; Khine, H.
11. Miyaura, Norio; Suzuki, Akira Chem. Rev. 1995, 95, 7.
12. 6-(1H-Pyrazol-4-yl)-1H-indazole was minimized in the gas phase using Jaguar
(6-31 g⁄⁄, DFT B3LYP basis set) using 3° increments with minimization at each
step. A dihedral angle of 27° was found to be the global minimum. See
Supporting information (Fig. S.1) for the full torsion profile.
13. Tautomeric indazoles 6-(1H-pyrazol-3-yl)-1H-indazole and 6-(1H-pyrazol-5-
yl)-1H-indazole were minimized using Jaguar (same specifications as Ref. 12).
6-(1H-Pyrazol-3-yl)-1H-indazole was found to be the more stable tautomer
(DDH = 1.4 kcal/mol), and this tautomer preferred a co-planar dihedral angle.
See Supporting information (Figs. S.2 and S.3) for the full torsion profile of both
tautomers.
14. The ability of compounds to inhibit phosphorylation of PLCc1 in Jurkat cells
was determined using an immuno-detection assay that will be described in
detail in a separate communication.