Design and optimization of selective protein kinase C θ (PKCθ) inhibitors for the treatment of autoimmune diseases

Article abstract of DOI:10.1021/jm301465a

Protein kinase C θ (PKCθ) has a central role in T cell activation and survival; however, the dependency of T cell responses to the inhibition of this enzyme appears to be dictated by the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that while antiviral responses are PKCθ-independent, T cell responses associated with autoimmune diseases are PKCθ-dependent. Thus, potent and selective inhibition of PKCθ is expected to block autoimmune T cell responses without compromising antiviral immunity. Herein, we describe the development of potent and selective PKCθ inhibitors, which show exceptional potency in cells and in vivo. By use of a structure based rational design approach, a 1000-fold improvement in potency and 76-fold improvement in selectivity over closely related PKC isoforms such as PKCδ were obtained from the initial HTS hit, together with a big improvement in lipophilic efficiency (LiPE).

Full text of DOI:10.1021/jm301465a

Article
pubs.acs.org/jmc
Design and Optimization of Selective Protein Kinase C θ (PKCθ)
Inhibitors for the Treatment of Autoimmune Diseases
Juan-Miguel Jimenez,*,† Dean Boyall,† Guy Brenchley,† Philip N. Collier,† Christopher J. Davis,†
Damien Fraysse,† Shazia B. Keily,† Jaclyn Henderson,† Andrew Miller,† Francoise Pierard,† Luca Settimo,†
Heather C. Twin,† Claire M. Bolton,‡ Adam P. Curnock,‡ Peter Chiu,‡ Adam J. Tanner,‡
and Stephen Young†
‡
†Department of Chemistry and Department of Biology, Vertex Pharmaceuticals (Europe) Ltd., 88 Milton Park, Abingdon,
Oxfordshire OX14 4RY, U.K.
S
* Supporting Information
ABSTRACT: Protein kinase C θ (PKCθ) has a central role in T cell
activation and survival; however, the dependency of T cell responses to the
inhibition of this enzyme appears to be dictated by the nature of the antigen
and by the inflammatory environment. Studies in PKCθ-deficient mice have
demonstrated that while antiviral responses are PKCθ-independent, T cell
responses associated with autoimmune diseases are PKCθ-dependent. Thus,
potent and selective inhibition of PKCθ is expected to block autoimmune T
cell responses without compromising antiviral immunity. Herein, we
describe the development of potent and selective PKCθ inhibitors, which
show exceptional potency in cells and in vivo. By use of a structure based
rational design approach, a 1000-fold improvement in potency and 76-fold
improvement in selectivity over closely related PKC isoforms such as PKCδ
were obtained from the initial HTS hit, together with a big improvement in
lipophilic efficiency (LiPE).
because PKCθ-deficient mice are resistant to or show markedly
reduced symptoms in models of MS, IBD, arthritis, and
asthma.18−23
The Novartis pan-PKC inhibitor sotrastaurin (AEB071)
(Figure 1) is currently in phase II clinical studies for
INTRODUCTION
■
Protein kinase C θ (PKCθ) is a member of a large family of
serine/threonine kinases that are involved in diverse cellular
functions. There are 11 PKC isoforms that can be subdivided
into diacylglycerol- (DAG) and calcium-dependent classical
isoforms (PKCs α, βI, βII, and γ), DAG-dependent novel
isoforms (PKCs δ, ε, η, and θ), and DAG- and calcium-
independent atypical isoforms (PKCs λ, ι, and ζ).1 PKCθ
expression is relatively restricted. The phenotype of the
knockout mouse indicates a predominant role for this enzyme
in T cells where it appears to play a central role in their
activation by integrating signals from both the TCR (T cell
receptor) and the co-stimulatory CD28.2−4 During T cell
activation, PKCθ is recruited to the center of the immune
synapse (IS) where it is responsible for the activation of key
transcription factors (AP-1, NFκB, and NFAT) that drive
transcription of the IL-2 gene.3−8 PKCθ also provides potent
survival signals to T cells through the up-regulation of
expression of the antiapoptotic molecules Bcl-xL and Bcl-2, as
well as the inactivation of the proapoptotic molecule Bad.9−12
PKCθ-deficient mice are able to mount a normal T cell
response to certain pathogens, and their antiviral responses in
particular appear to remain intact. These observations have
obvious implications regarding the safety of drugs targeting this
enzyme.13−17 Interestingly, PKCθ appears to be essential for
the development of T cell mediated inflammatory diseases
Figure 1. Structure of pan PKC inhibitor from Novartis.
transplantation and psoriasis.24,25 Sotrastaurin is a potent
inhibitor of both novel and classical PKC isoforms (IC50:
PKCθ 1 nM, PKCδ 1.3 nM, PKCα 2.1 nM, PKCβI 2 nM).26 Its
potent inhibition of PKC isoforms involved in B cell and T cell
function (PKCα, PKCβ, and PKCθ) results in a level of
immunosuppression that makes it suitable for use in trans-
Received: October 10, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
suggesting that development candidates with high lipophilic
efficiency (LiPE of 5−7) are more likely to succeed.42 The
addition of a fluorine substitution in the 5-position of the
pyridine central ring was also well tolerated (4). Encouraged by
this data and the overall kinase selectivity offered by these lead
compounds (data not shown), we decided to initiate our lead
optimization campaign.
Scheme 1
a
CHEMISTRY
(i) Base, 110−190 °C, solvent; (ii) TFA, Et3SiH, DCM, rt.
■
Scheme 1 outlines the versatile synthetic sequence developed
to both fully explore the effects of substitution around the
piperazine ring and install a diverse set of substituted
piperazines as a final step on both the unsubstituted and F-
substituted central rings.
plantation.26,27 In the treatment of autoimmune diseases, it is
anticipated that selective inhibition of PKCθ will provide a
better balance of efficacy and safety by inhibiting autoimmune
T cell responses without causing overt immunosuppression.
Such an approach has found significant favor within the
pharmaceutical industry; however, while some companies have
reported promising preclinical data, there are no selective
PKCθ inhibitors reported to be in clinical trials.28−40
Pyridylpyrazolopyridines A were prepared (see Materials and
Methods and Supporting Information) and reacted with the
appropriate piperazine using a range of bases and solvents
under microwave conditions. In the case of the trityl protected
3-pyrazolopyridine and Boc protected piperazines, a final TFA
mediated deprotection afforded the desired products (3−20).
Schemes 2 and 3 highlight the synthetic routes used to
explore effects of the substitution in the central pyridine ring.
Trisubstituted pyridylpyrazolopyridines (D) were constructed
from either the Weinreb amide (B) or pyrazolopyridine (E) as
shown in Scheme 2. Nucleophilic addition of an anion derived
from a suitable pyridine derivative to Weinreb amide (B) gave
dipyridyl ketone (C) which was ring closed with hydrazine to
pyridylpyrazolopyridine (D). Alternatively, suitable protection
of 3-bromo-1H-pyrazolo[3,4-b]pyridine (E) and formation of
the pyrazolopyridine boronic ester (G) allowed the pyridine
central ring to be installed by a Suzuki cross-coupling to
provide pyridylpyrazolopyridine (D).
To access tetrasubstituted pyridine derivatives, it was
necessary to control the regiochemistry on a polysubstituted
pyridine ring. The route described above was therefore
modified to allow introduction of 3,5-substituted derivatives.
Deprotonation of 3-chloro-2,5-difluoropyridine (H) occurs
preferentially in the 4-position, and it was necessary to
introduce a transient substituent to block this position to
allow selective coupling of the pyridine with Weinreb amide
(B) at the 2-position. Scheme 3 illustrates this with the route to
the 3-chloro-5-fluoro derivatives. Deprotonation of 3-chloro-
2,5-difluoropyridine (H) and quenching with TBSCl intro-
duced a TBS group at the 4-position to provide 4-(tert-
As part of our own program to identify potent and selective
PKCθ inhibitors, we identified compound 1 as an interesting
hit from HTS of the Vertex corporate compound collection.41
Compound 1 has a low molecular weight (297.16), good
physicochemical properties with acceptable lipophilicity (cLogP
= 2), and good PKCθ potency, which result in good ligand and
lipophilic efficiency (LE = 0.3, LiPE = 4.8) (Figure 2).42 The
compound, however, displays poor selectivity over PKCδ, an
enzyme that when knocked out in mice causes a fatal B cell
autoimmunity syndrome.43,44 Compound 1 also showed
modest selectivity over PKCα as a representative isoform of
the classical subfamily. It was quickly established that
replacement of the diazepane with a piperazine ring (2)
improved PKCθ potency approximately 5-fold and improved
the lipophilic efficiency (LiPE = 5.5). This was desirable, since
substituted piperazines are both more commercially and
synthetically accessible and would allow us to explore the
SAR around the piperazine more easily. We also realized that
transformation of the aminopyridine ketone hinge-binding
motif into a 3-pyrazolo[3,4-b]pyridine (3) resulted in a trend
toward enhanced selectivity for PKCθ over both novel and
classical PKC isoforms while maintaining a good level of
lipophilic efficiency. Maintaining a good level of lipophilic
efficiency was deemed important, as kinase inhibitors are
notoriously bad in gaining potency through unspecific lipophilic
interactions45 and there is an increasing body of evidence
a
Scheme 2
a
(i) Pyridine derivative, base; (ii) NH2NH2, solvent; (iii) protection; (iv) bis(pinacolato)diboron, PdCl2(dppf)·DCM, KOAc, DMF, heat; (v)
Pd(PPh3)4, Na2CO3, toluene, ethanol, water, heat, pyridine derivative.
B
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2.
a
Scheme 3
Figure 3. Proposed binding mode of compounds 3 (yellow) and 4
(blue) in PKCθ (green) showing the surface defined in the ATP active
site. Within the C-terminus loop, there is a significant difference of a
cysteine in PKCθ to a larger tyrosine in PKCδ (brown). Phe664 (C-
terminus loop) and Leu386 (GRL) (green) create a bigger
hydrophobic pocket in PKCθ as compared with Phe633 and Leu355
(brown) in PKCδ.
a
(i) Diisopropylamine, n-BuLi, THF, −78 °C; (ii) TBSCl, −78 °C,
72%; (iii) diisopropylamine, n-BuLi, THF, −85 °C; (iii) Weinreb
amide, 95%; (iv) NH2NH2, dioxane, CaCO3, −10 °C to rt, 98%.
butyldimethylsilyl)-3-chloro-2,5-difluoropyridine (I). A second
deprotonation at the 2-position and reaction with Weinreb
amide (B) gave dipyridyl ketone (J), and the usual closure with
hydrazine provided pyrazolopyridine (K).
The synthesis of key intermediates D and K allowed easy
introduction of the piperazine moiety at a late stage and a series
of simple deprotections, as described in Materials and Methods
and Supporting Information, provided access to the com-
pounds as reported in Table 3.
minimization of our scaffold, we realized that we could access
this pocket from the piperazine ring. So we set out to exploit
this difference to affect δ/θ selectivity and PKCθ potency
(Table 1).
Table 1. Structures and Inhibitory Activity of Compounds
a
3−12
RESULTS AND DISCUSSION
■
Good selectivity over PKCδ was considered to be crucial, so we
aimed to address this early in the optimization process. Initial
analysis based upon amino acid sequence suggested that the
ATP active sites of both these isoforms are highly conserved
with a single residue difference (Tyr108 in PKCθ and Phe108
in PKCδ). However, analysis of the crystal structures of human
PKCθ, reported by Novartis, in complex with both
staurosporine and 3-(8-((dimethylamino)methyl)-6,7,8,9-
tetrahydropyrido[1,2-a]indol-10-yl)-4-(1-methyl-1H-indol-3-
yl)-1H-pyrrole-2,5-dione (PDB code 2JED) showed the C-
terminus in close contact with the ligand.46 A homology model
of PKCδ was built based upon this structure of PKCθ and
showed that within the C-terminus there is a significant
difference of a Cys661 in PKCθ to a larger Tyr630 in PKCδ
(Figure 3).46 The consequence of this residue difference is the
presence of a bigger hydrophobic pocket in PKCθ as compared
to PKCδ that might be exploitable to gain potency and
selectivity (see Figure 3). On the basis of docking and
b
b
compd
X
R1
PKCθ Ki (nM) PKCδ Ki (nM)
δ/θ
3
H
H
H
H
H
H
H
F
H
50
68
12
15
5
526
336
81
10
5
5
(R)-Me
(R)-Et
(S)-iPr
(R)-iBu
(S)-iBu
(R)-Ph
(R)-iBu
(S)-iBu
6
7
7
132
131
89
9
8
27
45
27
88
90
9
2
10
11
12
5
151
792
358
9
F
4
a
Functionalization at the 2-position of piperazine and central pyridine
ring. Mean from n ≥ 2 independent experiments.
b
C
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Structures and Inhibitory Activity of Piperazine Derivatives 12−20
a
Mean from n ≥ of 2 independent experiments.
To the unsubstituted pyridine central ring (3), the
introduction of simple lipophilic substituents in the 2-position
of the piperazine ring resulted in a size dependent increase in
PKCθ potency by up to 25-fold (9), which is consistent with
filling a hydrophobic pocket. There was no obvious preference
for the S-stereochemistry around the piperazine ring (8 vs 9).
As such, compounds bearing fluorine on the central ring were
considered the most attractive leads because they combined a
good PKCθ potency, high lipophilic efficiency, and enhanced
δ/θ selectivity (90-fold).
With this encouraging selectivity, we set out to explore the
SAR around the piperazine ring in more detail, with the view to
improving PKCθ potency while maintaining good selectivity
over PKCδ (Table 2).
2,6-Disubstituted piperazines did not provide an increase in
potency, were detrimental to selectivity, and demonstrated a
large difference in potency between the R- and S-methylated
piperazines (13 and 14). Introduction of a tertiary alcohol in
the side chain showed an increase in PKCθ enzyme inhibition,
providing a 200 pM Ki inhibitor of PKCθ (16). The significant
improvement in LiPE (6.8) highlighted the fact that the
i
The larger lipophilic groups (Ph and Bu) also provided better
selectivity over PKCδ (up to 45-fold), consistent with the
proposed model. Although it had little impact on potency,
introduction of fluorine into the central ring further enhanced
selectivity over PKCδ (11 and 12) up to 90-fold. This
enhancement is possibly a consequence of a greater torsion
angle between the central pyridine ring and the hinge binder,
which subtly repositions the lipophilic substituent on the
piperazine ring resulting in a greater clash in PKCδ (Figure 3).
D
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
increase in potency is being driven by improvements in key
polar interactions. In addition, there was a significant increased
selectivity over PKCδ (285-fold). This piperazine alcohol is
believed to form a bidentate hydrogen bond network with the
Asp508, which locks the molecule in a conformation that places
the isopropyl group in the hydrophobic pocket created by the
C-terminus and GRL (see Figure 4). The stereochemistry at
isopropyl upward into the lipophilic pocket. Methylation of the
alcohol (20, mixture of all four isomers) confirmed that the
alcohol was likely acting as a hydrogen bond donor and forming
an integral part of the bidentate H-bond network (0.2 nM
compared to 10 nM).
With the piperazine alcohol 16 providing very good potency
and selectivity, we turned our attention back to the pyridine
central ring because the model suggested that there was an
adjacent pocket that could be accessed to further improve
potency. However, substitutions bigger than fluorine in R1 was
not well tolerated and resulted in a significant loss in PKCθ
affinity (21 and 22) (Table 3). Similarly, substitution in
position 4 of the pyridine was not beneficial for potency or
selectivity (data not shown). Introduction of polarity at R2 had
either a detrimental effect on PKCθ potency (24) or selectivity
over PKCδ (23 and 25). The presence of a 3-fluoro-5-fluoro
and 3-chloro-5-fluoro substitution (26 and 27) provided a 100
and 80 pM PKCθ potency, respectively, with good selectivity
over PKCδ of approximately 200-fold. Compounds in this
series in general showed a good translation into potency in a
cell-based assay, as measured by inhibition of IL-2 in stimulated
PBMCs. In particular, compounds 16, 23, 25, 26, and 27
showed impressive potency in a cell-based assay in the low
nanomolar range (Table 3).
On the basis of the PKCθ potency, selectivity over PKCδ,
and excellent potency in a cell-based assay, compounds 16, 26,
and 27 were characterized further. The impressive potency in
our cell based IL-2 assay observed for these compounds
translated into good potency in the more physiologically
relevant whole blood IL-2 assay (WB IL-2) with values in the
range 100−200 nM (Table 4). Table 4 also summarizes their
pharmacokinetic properties in rat. Compounds 16 and 26
demonstrate a modest oral bioavailability despite a low plasma
clearance of compound 26. Compound 27 showed the best PK
Figure 4. Predicted binding mode of compound 16 in PKCθ showing
the proposed bidentate hydrogen-bonding network between the
secondary amine and hydroxyl group, with the main chain carbonyl
oxygen of Asp 508. Correct positioning of both the H-bond and
lipophilic isopropyl group is crucial to obtain exquisite PKCθ potency
and good selectivity over PKCδ.
both the piperazine ring and the tertiary alcohol center is
important to attain this exquisite potency and selectivity. The S-
stereochemistry at the piperazine (16 vs 19) and the R-
stereochemistry of the alcohol (16 vs 18) best orientate the
Table 3. Structures and Inhibitory Activity of Substituted Central Pyridine Derivatives 16 and 21−27
a
b
c
Mean from n ≥ 2 independent experiments. Data from n = 1. Racemic.
E
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 4. Overall Profile of Lead Compounds 16, 26, and 27
b
rat PK
compd R1 PKCθ Ki (nM) PKCδ Ki (nM) PKCα Ki (nM) IL-2 IC50 (nM) WB IL-2 IC50 (nM) CL (mL min−1 kg−1
a
a
a
a
a
)
t1/2 (h) F (%)
16
26
27
H
F
0.2
57
18
16
549
198
356
6
200
80
39
9.5
7
2.6
1.7
4.7
36
30
65
0.1
2
Cl
0.08
11
70
a
b
Mean from n ≥ 2 independent experiments. Rat PK reported (iv bolus 3 mg/kg and po dose of 10 mg/kg). Data reported as an average of three
animals.
Table 5. Selectivity Profile for Compound 27
a
Mean from n ≥ 2 independent experiments.
Figure 5. In vivo data for 27 in the SEB IL-2 model. Each column represents a mean SEM (n = 6−8). Statistical analysis of data was conducted
with a one-way ANOVA followed by a Dunnett’s post-test: (∗∗∗) p < 0.001, and NS denotes no significant effect compared to the vehicle control
group.
F
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
profile with a low clearance (7 mL min−1 kg−1), long half-life
(4.7 h), and good oral bioavailability (65%).
mass spectrometer operating in single MS mode with electrospray
ionization. Samples were introduced into the mass spectrometer using
chromatography. The melting points were determined on a Mettler
Toledo DSC1 STAR system, calibrated using indium. All final
products had a purity of >95%. The purity of final products was
determined by HPLC or combustion analyses. HPLC purity was
measured on an Agilent HP1100 HPLC instrument with a Waters
Quattro MS system equipped with an ACE C8 reverse phase column
(4.6 mm × 150 mm, 5 μm). The mobile phases were acetonitrile/
methanol (1:1) and water (20 mM, pH 7.0, Tris phosphate).
Combustion analyses were performed on a Control Equipment
Corporation 440 elemental analyzer.
Compound 27 possessed excellent overall characteristics and
was selected for further characterization. Importantly, this
compound did not show cross-reactivity against other proximal
TCR kinases such as Src kinases, Syk kinases, Tec kinases, or
MAP kinases (Table 5). Good selectivity was also seen against
other PKC family members, particularly classical isoforms
(>1000-fold except PKCβ I, 200-fold) and atypical isoforms
(>10000-fold). As anticipated, attaining selectivity over the
more closely related novel PKC family members was more
challenging, with a good 200-fold being achieved over PKCδ
(Table 5). This was perceived as especially important because
of the risk of B cell autoimmunity associated with inhibition of
the PKCδ isoform. Extended tests were conducted to examine
the cross-reactivity profiles of these compounds against a
diverse range of enzymes, receptors, and ion channels. Again,
excellent selectivity over non-kinase targets was seen (data not
shown). Because of the promising overall profile, compound 27
was selected for in vivo evaluation in a murine model of
staphylococcal enterotoxin B induced IL-2 release (SEB IL-2
model) (Figure 5 and experimental section for details). A single
dose of 27 was administered orally at 6.25, 12.5, 25, and 50 mg/
kg (e.g., at 25 mg/kg Cmax concentration 700 ng/mL) and
demonstrated potent dose dependent inhibition of IL-2
production (Figure 5).
3-(6-(Piperazin-1-yl)pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine
(3). Step 1. A solution of 2-(dimethylamino)ethanol (3.53 g, 3.98 mL,
39.63 mmol) in hexane (45 mL) cooled to −78 °C under N2 was
treated with n-BuLi (31.70 mL of 2.5 M, 79.26 mmol), and the
reaction mixture was allowed to stir for 20 min. A solution of 2-
chloropyridine (1.50 g, 13.21 mmol) in hexane (12 mL) was added
dropwise and the resultant orange solution stirred for a further 50 min.
The mixture was then treated dropwise with a solution of 2-fluoro-N-
methoxy-N-methylpyridine-3-carboxamide (9.25 g, 50.20 mmol) in
THF (15 mL) to give a deep-orange suspension. Stirring became
difficult at this stage, and a further 8 mL of THF was added. The
reaction mixture was kept at −78 °C for 1 h, quenched with saturated
aqueous NH4Cl, and then diluted with EtOAc. The mixture was
allowed to warm to room temperature. The aqueous layer was
extracted further with EtOAc, and the combined organics were washed
with saturated aqueous NaHCO3 solution followed by brine. The
mixture was dried over MgSO4 and concentrated to give an orange
residue, which was purified by column chromatography (ISCO
Companion, 80 g column, compound preabsorbed onto silica), eluting
with 5−100% EtOAc/petroleum ether to afford (6-chloropyridin-2-
yl)-(2-fluoropyridin-3-yl)methanone as an off-white solid (1.17 g,
CONCLUSION
■
In summary, the design and optimization of a novel class of
potent and selective PKCθ inhibitors based on the pyrazolo-
[3,4-b]pyridine scaffold have been described. Compound 27 is
a potent and selective inhibitor of the PKCθ enzyme with an
excellent overall profile, where a 1000-fold improvement in
potency and 76-fold improvement in selectivity over closely
related PKC isoforms such as PKCδ were obtained compared
with the initial HTS hit. In addition, a significant improvement
in lipophilic efficiency (LiPE) was achieved, indicating that our
lead candidate improves nonlipophilic interactions with the
PKCθ enzyme. Accessing these key interactions enabled us to
precisely position the molecule to capitalize on subtle
differences between closely related PKC isoforms that have
almost identical ATP binding sites. On the basis of its favorable
physicochemical and ADMET properties, compound 27 was
selected for further evaluation in disease models of MS and
IBD. These studies will be the subject of further communica-
tions from this laboratory.
1
37.4%). H NMR (CDCl3) δ 7.37 (1H, m), 7.57 (1H, t), 7.93 (1H,
m), 8.05 (1H, m), 8.21 (1H, t), 8.45 (1H, m). MS m/z: 236.9 (M +
H)+.
Step 2. A solution of (6-chloro-2-pyridyl)(2-fluoro-3-pyridyl)-
methanone (307 mg, 1.30 mmol) in THF (2.5 mL) was treated
with hydrazine (1.0 M in THF) (1.30 mL of 1 M, 1.30 mmol), and the
mixture was heated in the microwave at 100 °C for 60 min. The
reaction mixture was concentrated in vacuo to give 3-(6-chloropyridin-
2-yl)-1H-pyrazolo[3,4-b]pyridine as a yellow solid (288 mg, 86.6%).
1H NMR (DMSO-d6) δ 7.35 (1H, m), 7.52 (1H, m), 8.01 (1H, t),
8.17 (1H, m), 8.61 (1H, m), 8.81 (1H, m), 14.11 (NH). MS m/z:
230.9 (M + H)+.
Step 3. A mixture of 3-(6-chloropyridin-2-yl)-1H-pyrazolo[3,4-
b]pyridine (60 mg, 0.26 mmol) and piperazine (112 mg, 1.30 mmol)
was heated in a microwave at 190 °C for 90 min. The crude material
was purified on FractionLynx and freeze-dried to afford compound 3
bis-TFA salt as a yellow solid (75.5 mg, 57%). 1H NMR (DMSO-d6) δ
3.30 (4H, s), 3.84 (4H, t), 6.96 (1H, d), 7.31 (1H, dd), 7.58 (1H, d),
7.73−7.77 (1H, m), 8.58 (1H, dd), 8.79 (1H, dd), 8.82 (2H, s), and
13.89 (1H, s). MS m/z: 281.0 (M + H)+.
(R)-3-(3-Fluoro-6-(3-isobutylpiperazin-1-yl)pyridin-2-yl)-1H-
pyrazolo[3,4-b]pyridine (11). Step 1. A suspension of
diazabicyclo[2.2.2]octane (32.2 g, 0.29 mol) in dry TBME (365
mL) was cooled to −78 °C. n-BuLi (2.5 M in hexane, 0.29 mol) was
added dropwise to the suspension. The mixture was stirred for
approximately 30 min at −78 °C and then treated dropwise with 2,5-
difluoropyridine (30.0 g, 0.26 mol) in dry TBME (15 mL). After 1 h,
2-fluoro-N-methoxy-N-methylnicotinamide (52.8 g, 0.29 mol) in dry
TBME (30 mL) was added dropwise to the mixture at −78 °C. The
solution was stirred for 1 h, and the reaction was quenched with
saturated aqueous NH4Cl (300 mL). The mixture was diluted with
EtOAc (600 mL) and allowed to warm to room temperature
overnight. The aqueous phase was extracted further with EtOAc (3
× 300 mL), and the combined organic phases were washed with
saturated aqueous NaHCO3 (500 mL) and brine (500 mL), dried over
MgSO4, and concentrated under reduced pressure to afford (3,6-
MATERIALS AND METHODS
■
General Experimental Details. All commercially available
solvents and reagents were used as received. Microwave reactions
were carried out using a CEM Discovery microwave. Analytical thin
layer chromatography was carried out using glass-backed plates coated
with Merck Kieselgel 60 GF240. Plates were visualized using UV light
(254 or 366 nm) and/or by staining with potassium permanganate
followed by heating. Flash chromatography was carried out on an
ISCO Combiflash Companion system, eluting with a 0−100% EtOAc/
petroleum ether gradient. Samples were applied preabsorbed on silica.
Where stated, purification was performed using a Waters FractionLynx
HPLC mass directed instrument using an appropriate acetonitrile/
water gradient with either TFA or ammonia modifier. Where stated,
supercritical fluid chromatography (SFC) was performed on a Berger
Minigram SFC machine. 1H NMR and 19F NMR spectra were
recorded at 400 and 376 MHz, respectively, using a Bruker DPX 400
instrument. MS samples were analyzed on a MicroMass Quattro Micro
G
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
difluoropyridin-2-yl)(2-fluoropyridin-3-yl)methanone as a yellow/
orange solid (66.7 g, 97%).
(159.6 g, 78%). H NMR (CDCl3) δ 0.35 (6H, s), 0.88 (9H, s), 7.29
(1H, m), 8.13 (1H, m), 8.37 (1H, m); 19F NMR (decoupled) δ
−112.47, −106.7, −89.3, −61.95.
Step 2. To a solution of (3,6-difluoropyridin-2-yl)(2-fluoropyridin-
3-yl)methanone) (66.7 g, 0.26 mol) in THF (450 mL) was added
hydrazine hydrate (12.2 g, 0.38 mol). The mixture was stirred for 1 h
at room temperature and then treated with another batch of hydrazine
hydrate (12.2 g, 0.38 mol). The mixture was stirred overnight at room
temperature, diluted with H2O (200 mL), and stirred for 10 min. The
product was isolated by filtration, washed with Et2O, and dried under
reduced pressure to afford 3-(3,6-difluoropyridin-2-yl)-1H-pyrazolo-
[3,4-b]pyridine as a white solid (33.6 g, 55.5%). 1H NMR (DMSO-d6)
δ 7.33 (1H, dt), 7.36 (1H, dd), 8.15 (1H, td), 8.62 (1H, dd), 8.71 (1H,
brs), 9.15 (1H, brs). MS m/z: 233.8 (M + H)+.
Step 3. A mixture of 3-(3,6-difluoropyridin-2-yl)-1H-pyrazolo[3,4-
b]pyridine (100 mg, 0.43 mmol), (R)-2-isobutylpiperazine-bis-TFA
salt (202 mg, 0.55 mmol), and DIPEA (235 mg, 1.82 mmol, 2.5 mL)
was heated in a microwave at 185 °C for 40 min. The crude material
was purified on FractionLynx and freeze-dried to afford compound 11
as a white solid (20.0 mg, 13%). MS m/z: 355.1 (M + H)+.
Step 3. [4-[tert-Butyl(dimethyl)silyl]-3,5,6-trifluoro-2-pyridyl]-(2-
fluoro-3-pyridyl)methanone (159 g, 429.2 mmol) was dissolved in
dioxane (1.6 L), and calcium carbonate (85.91 g, 858.4 mmol) was
added to the solution. The reaction mixture was stirred in an ice bath,
and hydrazine hydrate (107.4 g, 104.4 mL, 2.15 mol) was added
dropwise over 30 min. The resultant mixture was stirred for 18 h,
gradually warming to room temperature. The reaction mixture was
filtered through a pad of Celite, washing with EtOAc/MeOH 7:1 (2 ×
500 mL) and water (100 mL). The filtrate was partitioned between
EtOAc (1 L) and water (500 mL). The organic phase was washed with
a saturated aqueous NaHCO3 solution (500 mL). The combined
aqueous layers were extracted with EtOAc (2 × 200 mL). The
combined organics were washed with brine (400 mL), dried (MgSO4),
and concentrated to give an orange solid. The solid was triturated with
DCM and filtered, washing with DCM and petroleum ether. The
filtrate was concentrated under reduced pressure, and the resulting
solid was again triturated and filtered, washing with more petroleum
ether. The solids were combined to afford 3-(4-(tert-butyldimethylsil-
yl)-3,5,6-trifluoropyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine as an off-
(R)-2-((S)-4-(3,5-Difluoro-6-(1H-pyrazolo[3,4-b]pyridin-3-yl)-
pyridin-2-yl)piperazin-2-yl)-3-methylbutan-2-ol (26). Step 1. A
solution of diisopropylamine (98.9 g, 136.9 mL, 976.9 mmol) in THF
(1.35 L) was cooled to −65 °C. n-BuLi (2.5 M in hexanes) (375.8 mL
of 2.5 M, 939.4 mmol) was added dropwise via cannula over 1 h at
such a rate as to maintain reaction temperature below −60 °C. Once
the addition was completed, the cooling bath was removed and the
reaction mixture was allowed to warm to 0 °C. The reaction mixture
was stirred for 15 min at 0 °C, then recooled to −78 °C. 2,3,5-
Trifluoropyridine (100.0 g, 751.5 mmol) was added dropwise via
cannula over 20 min at such a rate to maintain the reaction
temperature below −69 °C. The reaction mixture was stirred for 45
min at −78 °C during which time the solution turned orange/brown.
A solution of tert-butylchlorodimethylsilane (147.2 g, 976.9 mmol) in
THF (150 mL) was then added via cannula over 30 min. The reaction
mixture was stirred at −78 °C for 90 min during which time the
solution darkened. A saturated aqueous NH4Cl solution (300 mL) was
added, and the mixture was allowed to warm to room temperature.
The reaction mixture was diluted with water (100 mL) and extracted
with EtOAc (1.5 L, then 2 × 500 mL). The combined organics were
washed with saturated aqueous NaHCO3 (500 mL) and brine (400
mL). The crude mixture was partially concentrated in vacuo, dried
(MgSO4), and concentrated in vacuo. The material was purified by
flash chromatography (CombiFlash Companion XL, 1.5 kg column,
0−20% EtOAc in petroleum ether) to afford 4-(tert-butyldimethylsil-
1
white solid (140.5 g, 90%). H NMR (CDCl3) δ 0.55 (6H, s), 1.02
(9H, s), 7.21 (1H, m), 8.69 (1H, m), 8.91 (1H, s), 11.69 (1H, brs).
MS m/z: 365.2 (M + H)+.
Step 4. A 500 mL glass pressure vessel was charged with 3-(4-(tert-
butyldimethylsilyl)-3,5,6-trifluoropyridin-2-yl)-1H-pyrazolo[3,4-b]-
pyridine (26.80 g, 73.53 mmol), a mixture of (R)-3-methyl-2-((S)-
piperazin-2-yl)butan-2-ol and (R)-3-methyl-2-((R)-piperazin-2-yl)-
butan-2-ol (19.0 g, 110.3 mmol), THF (53 mL), and imidazol-1-
yltrimethylsilane (51.57 g, 53.72 mL, 367.7 mmol). An identical
experiment was run in parallel. The containers were sealed, and the
mixture was heated and stirred at 95 °C for 18 h. Analysis of the crude
reaction mixture showed a mixture consisting of 73% of the desired
(R,S)-diastereoisomer and 18% of the (R,R)-diastereoisomer. Both
reaction mixtures were cooled, combined, and diluted with EtOAc
(500 mL). The solution was washed with saturated aqueous NaHCO3
(2 × 200 mL). The combined aqueous layers were extracted three
times with EtOAc (200 mL). The combined organics were washed
with brine (200 mL), dried (MgSO4), and concentrated in vacuo to
give 163.8 g of crude product. The diastereoisomers were separated on
silica gel (10−95% EtOAc in petroleum ether with 2% triethylamine)
to afford 3-(4-(tert-butyldimethylsilyl)-3,5-difluoro-6-((S)-3-((R)-3-
methyl-2-(trimethylsilyloxy)butan-2-yl)piperazin-1-yl)pyridin-2-yl)-
1H-pyrazolo[3,4-b]pyridine as an orange foam (60.1 g, 69%). 1H
NMR (CDCl3) δ 0.01 (9H, s), 0.32 (6H, s), 0.76−0.84 (9H, m), 1.11
(4H, m), 1.45 (4H, m), 1.72 (1H, m), 1.90 (1H, s), 2.61 (1H, m), 2.74
(2H, m), 2.97 (1H, m), 3.11 (1H, m), 3.64 (1H, m), 3.82 (1H, m),
3.96 (1H, m), 7.09 (1H, m), 8.48 (1H, m), 8.74 (1H, m), 11.31 (1H,
brs); 19F NMR (decoupled) δ −113.45, −108.39. MS m/z: 589.2 (M
+ H)+.
Step 5. 3-(4-(tert-Butyldimethylsilyl)-3,5-difluoro-6-((S)-3-((R)-3-
methyl-2-(trimethylsilyloxy)butan-2-yl)piperazin-1-yl)pyridin-2-yl)-
1H-pyrazolo[3,4-b]pyridine (60 g, 101.9 mmol) was dissolved in THF
(216.0 mL) at room temperature. A solution of tetrabutylammonium
fluoride in THF (214.0 mmol. 214.0 mL of 1 M) was added dropwise
via cannula to the brown solution over 45 min. The reaction mixture
was stirred for 18 h at room temperature. The mixture was diluted
with EtOAc (600 mL) and washed with saturated aqueous NaHCO3
(100 mL, then 2 × 200 mL). The organic phase was further washed
with a 2:3 mixture of saturated brine/water (100 mL) until no
remaining Bu4N+ salts were observed. The organic phase was dried
(MgSO4) and concentrated in vacuo. The solid was triturated with
diethyl ether, filtered, and dried to give an orange-brown solid (34.5
g). The solid was recrystallized from isopropanol to afford compound
1
yl)-2,3,5-trifluoropyridine as a colorless oil (136.2 g, 73%). H NMR
(CDCl3) δ 0.34 (6H, s), 0.89 (9H, s), 7.73 (1H, s). MS m/z: 248.1 (M
+ H)+.
Step 2. A solution of diisopropylamine (66.78 g, 92.49 mL, 659.9
mmol) in THF (1.36 L) was cooled to −20 °C under nitrogen. n-BuLi
(2.5 M in hexane) (253.0 mL of 2.5 M, 632.4 mmol) was added
dropwise via cannula at such a rate as to maintain the internal
temperature below −15 °C. The solution was warmed to 0 °C, then
immediately recooled to −90 °C. tert-Butyldimethyl-(2,3,5-trifluoro-4-
pyridyl)silane (136 g, 549.9 mmol) was added dropwise via cannula at
such a rate as to maintain the internal temperature below −85 °C.
After complete addition, the reaction mixture was stirred at −85 °C for
1 h and then 2-fluoro-N-methoxy-N-methylpyridine-3-carboxamide
(116.5 g, 632.4 mmol) was added dropwise over 1 h, keeping the
internal temperature below −85 °C. The mixture was stirred at −85
°C for 45 min. The cooling bath was removed and the mixture
warmed to −50 °C. A saturated ammonium chloride aqueous solution
(300 mL) was added and the mixture allowed to warm to room
temperature. The mixture was diluted with EtOAc (2.5 L). The
aqueous phase was separated and extracted with EtOAc (500 mL).
The combined organics were washed with brine and partially
concentrated in vacuo. The solution was dried (MgSO4), filtered,
and concentrated in vacuo to yield a brown oil. The material was
purified on silica gel, eluting with 0−20% EtOAc in petroleum ether to
afford (4-(tert-butyldimethylsilyl)-3,5,6-trifluoropyridin-2-yl)(2-fluoro-
pyridin-3-yl)methanone as a yellow oil which solidified on standing
1
26 as a beige solid (28.3 g, 69%). H NMR (DMSO-d6) δ 0.84 (3H,
d), 0.90 (3H, d), 1.06 (3H, s), 1.83 (2H, m), 2.74 (2H, d), 2.85 (2H,
d), 3.09 (1H, d), 3.78 (1H, d), 4.04 (1H, d), 4.13 (1H, s), 7.30 (1H,
dd), 7.92 (1H, dd), 8.60 (1H, dd), 8.77 (1H, dd), 13.99 (1H, NH);
19F NMR (decoupled) δ −128.5, −124.1. MS m/z: 403.2 (M + H)+.
H
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Anal. Calcd for C20H24F2N6O: C, 59.69%; H, 6.01%; N, 20.87%.
Found: C, 59.69%; H, 5.96%; N, 20.95%.
extracted with DCM (2 × 400 mL). The combined organics were
dried (MgSO4), filtered, and concentrated in vacuo. The resulting solid
was slurried in petroleum ether, filtered, and dried (81 g). The Celite
was further washed with DCM/MeOH 7:1 (3 × 800 mL) and with 4 L
of DCM/MeOH 4:1 until no remaining compound was observed on
TLC. The filtrate was concentrated in vacuo. The residual solid was
triturated with petroleum ether as described above (185 g). The solids
were combined and dried at 40 °C under vacuum to afford 3-(4-(tert-
butyldimethylsilyl)-5-chloro-3,6-difluoropyridin-2-yl)-1H-pyrazolo-
[3,4-b]pyridine as an off-white solid (260.5 g, 89%). 1H NMR
(CDCl3) δ 0.63 (6H, d), 1.07 (9H, s), 7.44 (1H, m), 8.76 (1H, m),
9.07 (1H, m), 13.31 (1H, NH); 19F NMR (decoupled) δ −104.7,
−73.8. MS m/z: 381.1 (M + H)+.
(R)-2-((S)-4-(3-Chloro-5-fluoro-6-(1H-pyrazolo[3,4-b]pyridin-
3-yl)pyridin-2-yl)piperazin-2-yl)-3-methylbutan-2-ol (27). Step
1. A solution of diisopropylamine (78.52 g, 108.8 mL, 776.0 mmol) in
THF (1.21 L) was cooled to −20 °C. n-BuLi (2.5 M in hexanes)
(298.4 mL of 2.5 M, 746.1 mmol) was added dropwise via cannula
over 30 min at such a rate that the temperature was kept below −15
°C. Once the addition was completed, the cooling bath was removed
and the reaction mixture was allowed to warm to 0 °C. The reaction
mixture was stirred for 15 min at 0 °C and recooled to −78 °C. 3-
Chloro-2,5-difluoropyridine (95.96 g, 596.9 mmol) was added
dropwise via cannula over 20 min at such a rate that the temperature
was kept below −70 °C. The reaction mixture was stirred for 45 min at
−78 °C. A solution of tert-butylchlorodimethylsilane (117.0 g, 776.0
mmol) in THF (133.9 mL) was added via cannula at such a rate to
maintain the reaction temperature below −70 °C. The reaction
mixture was stirred at −78 °C for 90 min. A saturated aqueous NH4Cl
solution (300 mL) was added, and the reaction mixture was allowed to
warm to room temperature. The reaction mixture was diluted with
water (200 mL) and saturated aqueous NaHCO3 (500 mL) and
extracted with EtOAc (1.5 L and 500 mL). The combined organics
were washed with brine (400 mL), dried (MgSO4), and concentrated
in vacuo to an oil (187 g). The crude material was purified by flash
column chromatography (CombiFlash Companion XL, 1.5 kg silica
column, 0.5−10% EtOAc in petroleum ether) to afford 4-(tert-
butyldimethylsilyl)-3-chloro-2,5-difluoropyridine as a pale yellow oil
Step 4. 3-(4-(tert-Butyldimethylsilyl)-5-chloro-3,6-difluoropyridin-
2-yl)-1H-pyrazolo[3,4-b]pyridine (132.7 g, 348 mmol) was dissolved
in anhydrous THF (265 mL). Imidazol-1-yl(trimethyl)silane (254
mL) was added followed by a mixture of (R)-3-methyl-2-((S)-
piperazin-2-yl)butan-2-ol and (R)-3-methyl-2-((R)-piperazin-2-yl)-
butan-2-ol (90 g, 522 mmol). The mixture was divided into four
glass pressure vessels, and the sealed containers were heated and
stirred at 95 °C for 48 h. The crude mixtures were cooled, combined,
and diluted with EtOAc (1.7 L). The solution was washed with
saturated NaHCO3 (2 × 500 mL). The combined aqueous layers were
extracted with EtOAc (500 mL). The combined organics were washed
with brine (500 mL), dried (MgSO4), filtered, and concentrated in
vacuo. The diastereoisomers were separated by flash chromatography
(silica gel, 10−100% EtOAc containing 2% triethylamine in petroleum
ether) to afford 3-(4-(tert-butyldimethylsilyl)-5-chloro-3-fluoro-6-((S)-
3-((R)-3-methyl-2 (trimethyl silyl oxy)butan-2-yl)piperazin-1-yl)-
pyridin-2-yl)-1H-pyrazolo[3,4-b]pyridine as a yellow foam (125.3 g,
1
(113.8 g, 72%). H NMR (CDCl3) δ 0.46 (6H, d), 0.93 (9H, s), 7.84
(1H, d); 19F NMR (decoupled) δ −112.8, −74.6. MS m/z: 264.1 (M
+ H)+.
1
Step 2. A solution of diisopropylamine (52.41 g, 72.59 mL, 517.9
mmol) in THF (967.3 mL) was cooled to −20 °C. n-BuLi (2.5 M in
hexane) (198.5 mL of 2.5 M, 496.3 mmol) was added dropwise over
15 min via cannula, maintaining the internal temperature below −15
°C. The solution was warmed to 0 °C and then immediately recooled
to −90 °C. A solution of 4-(tert-butyldimethylsilyl)-3-chloro-2,5-
difluoropyridine (113.84 g, 431.6 mmol) in THF (85 mL) was added
dropwise over 20 min via cannula at such a rate that the internal
temperature was kept below −85 °C. The reaction mixture was stirred
at −85 °C for 1 h. A solution of 2-fluoro-N-methoxy-N-
methylpyridine-3-carboxamide (91.40 g, 496.3 mmol) in THF (85
mL) was added dropwise over 15 min via cannula at such a rate that
the internal temperature was kept below −85 °C. The mixture was
stirred at −85 °C for 90 min. The cooling bath was removed and the
mixture allowed to warm to −50 °C. A saturated aqueous NH4Cl
solution (400 mL) was added, and the mixture was allowed to warm to
room temperature. The mixture was diluted with EtOAc (1.5 L). The
aqueous layer was separated and extracted with more EtOAc (300
mL). The combined organics were washed with brine (300 mL), dried
(MgSO4), and concentrated in vacuo. The crude material was purified
by flash chromatography (CombiFlash Companion XL, 1.5 kg silica
gel, 0.5−10% EtOAc in petroleum ether, 0−50% EtOAc in petroleum
ether) to afford (4-(tert-butyldimethylsilyl)-5-chloro-3,6-difluoropyr-
idin-2-yl)(2-fluoropyridin-3-yl)methanone as a yellow oil which
solidified on standing (159.6 g, 95%). 1H NMR (CDCl3) δ 0.60
(6H, d), 1.07 (9H, s), 7.47 (1H, m), 8.33 (1H, m), 8.52 (1H, m); 19F
NMR (decoupled) δ −107.2, −72.4, −61.8. MS m/z: 387.1 (M + H)+.
Step 3. (4-(tert-Butyldimethylsilyl)-5-chloro-3,6-difluoropyridin-2-
yl)(2-fluoropyridin-3-yl)methanone (294.86 g, 762.2 mmol) was
dissolved in dioxane (2.2 L), and calcium carbonate (152.5 g, 1.52
mol) was added. The reaction mixture was cooled in an ice bath, and
hydrazine hydrate (190.8 g, 185.4 mL, 3.811 mol) was added dropwise
over 15 min via cannula to the yellow suspension, keeping the internal
temperature below 10 °C. The resulting mixture was allowed to warm
to ambient temperature and stirred at room temperature for 18 h. The
reaction mixture was filtered through a pad of Celite, washing with
DCM (2.5 L). Water (400 mL) was added to the filtrate followed by a
saturated solution of sodium bicarbonate (400 mL). The organic layer
was separated and washed again with a saturated aqueous sodium
bicarbonate solution (350 mL). The combined aqueous layers were
59%). H NMR (CDCl3) δ 0.01 (9H, s), 0.42 (6H, s), 0.78−0.89
(15H, m), 1.09−1.13 (3H, m), 1.74 (1H, m), 2.61 (1H, m), 2.80−2.99
(4H, m), 3.12 (1H, m), 3.45 (1H, d), 3.69 (1H, d), 7.12 (1H, m), 8.55
(1H, m), 8.81 (1H, m), 12.75 (1H, brs); 19F NMR (decoupled) δ
−108.9. MS m/z: 605.6 (M + H)+.
Step 5. 3-(4-(tert-Butyldimethylsilyl)-5-chloro-3-fluoro-6-((S)-3-
((R)-3-methyl-2-(trimethylsilyloxy)butan-2-yl)piperazin-1-yl)pyridin-
2-yl)-1H-pyrazolo[3,4-b]pyridine (125 g, 206.5 mmol) was dissolved
in THF (450 mL) at room temperature. Tetrabutylammonium
fluoride (433.6 mL of 1 M THF solution, 433.6 mmol) was added
dropwise via cannula to the yellow solution. The mixture was stirred
for 48 h. The mixture was diluted with EtOAc (1.5 L) and washed with
a 1:1 mixture of saturated aqueous NaHCO3 and water (3 × 600 mL).
The combined aqueous layers were extracted with EtOAc (2 × 600
mL), and the organic phase was washed with brine (600 mL), dried
(MgSO4) and partially concentrated in vacuo until the title product
began to precipitate. A few drops of methanol were added, and the
mixture was stirred at room temperature for 18 h. The product was
filtered, rinsed with a minimum of EtOAc and petroleum ether, and
dried under vacuum for 24 h (40 °C and 1.5 mbar) to afford
compound 27 as a white solid (73.1 g, 84%). Mp: 217−218 °C. [α]D19
−54.8 (c 1.0, EtOH). 1H NMR (DMSO-d6) δ 0.91 (3H, d), 0.94 (3H,
d), 1.10 (3H, s), 1.83 (2H, m), 2.72 (1H, t), 2.80−2.94 (3H, m),
3.14−3.17 (1H, m), 3.62 (1H, d), 3.88 (1H, d), 4.14 (1H, s), 7.37
(1H, dd), 8.16 (1H, d), 8.65 (1H, dd), 8.86 (1H, dd), 14.12 (1H,
NH); 19F NMR (decoupled) δ −127.9. MS m/z: 419.1 (M + H)+.
>98% ee (Minigram SFC, ODH 250 mm × 10 mm, 20% MeOH
(0.1% TEA), 5 mg/mL. Anal. Calcd for C20H24ClFN6O: C, 57.35%;
H, 5.77%; N, 20.06%. Found: C, 57.42%; H, 5.94%; N, 19.92%.
Kinase Inhibition Assays. Compounds were screened for their
ability to inhibit PKCθ in a radioactive-phosphate incorporation assay
using full-length recombinant PKCθ enzyme.
Assays were carried out in a 96-well plate containing 100 mM
HEPES (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 0.1 mM EDTA,
0.01% Brij, 2 mM DTT, 0.002% Triton X-100, 200 μg/mL
phosphatidylserine, 20 μg/mL diacylglycerol, 150 μM substrate
peptide (ERMRPRKRQGSVRRRV), 0.5 nM PKCθ (Invitrogen,
Madison, WI), and 240 μM ATP (ATP Km = 40 μM) in a final
volume of 100 μL. Assays were performed at 25 °C in the presence of
23 nCi/μL [γ-33P]ATP (Perkin-Elmer, Beconsfield, U.K.). Serial
I
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
dilutions of inhibitors were prepared in DMSO, and the reaction was
initiated by the addition of ATP.
2 in plasma samples were determined using a sandwich enzyme
immunoassay technique.
Statistical analysis of data was conducted with a one-way ANOVA
followed by a Dunnett’s post-test. Statistical significance was defined as
p < 0.05.
The reaction was stopped after 60 min by the addition of 100 μL of
100 mM phosphoric acid, and a phosphocellulose 96-well plate
(Millipore, catalog no. MAPHNOB) was washed with 200 μL of 100
mM phosphoric acid prior to the addition of the stopped reaction
mixture (120 μL). The plate was left to soak for at least 30 min prior
to washing (4 × 200 μL of 100 mM phosphoric acid). After it was
dried, 100 μL of Optiphase “SuperMix” liquid scintillation cocktail
(Perkin-Elmer) was added to the well. Initial rate data were
determined from the level of 33P incorporation measured using a
1450 MicroBeta liquid scintillation counter, Wallac.
Molecular Modeling. The crystal structure of human PKCθ in
complex with 3-(8-((dimethylamino)methyl)-6,7,8,9-
tetrahydropyrido[1,2-a]indol-10-yl)-4-(1-methyl-1H-indol-3-yl)-1H-
pyrrole-2,5-dione solved at a resolution of 2.32 Å was downloaded
from the Protein Data Bank (PDB code 2JED; Stark et al.,
unpublished study). This structure shows the C-terminus in close
contact with the ligand. When this structure was overlaid with other
kinase crystal structures, an unusual conformation of Phe664 was
observed (this conformation is most likely induced by the ligand
crystallized, and it is not usually observed among kinases structures).
Therefore, this structure, in absence of the ligand, was energy
minimized using the MMFF94x force field within MOE (version
2010.10, Chemical Computing Group, Montreal, Canada) and a
conformation for Phe664 similar to other kinases was obtained and
this new minimized structure was used for docking.
A protein model of PKCδ was created using the homology
modeling tool within MOE. In order to create this protein model, the
sequence of PKCδ was retrieved from UNIPROT (UNIPROT code
Q05655; The UniProt Consortium, 2011) and homology modeling
was carried out using as template the minimized PKCθ crystal
structure described above. Medium model refinements were applied
during the protein model generation. The sequence identity
percentage between human PKCθ and human PKCδ for the kinase
domain is approximately 72%.
Compounds were screened for their ability to inhibit PKCδ or
PKCα in a spectrophotometric assay using full-length, recombinant
enzyme.
Assays were carried out in a 384-well plate containing 100 mM
HEPES (pH 7.5), 10 mM MgCl2, 25 mM NaCl, 0.1 mM EDTA,
0.01% Brij, 100 μM CaCl2 (PKCα only), 2 mM DTT, 0.002% Triton
X-100, 200 μg/mL phosphatidylserine (100 μg/mL for PKCα), 20
μg/mL diacylglycerol, 150 μM substrate peptide (ERMRPRKR-
QGSVRRRV(PKCδ); RRRRRKGSFKRKA(PKCα)), 360 μM
NADH, 3 mM phosphoenolpyruvate, 70 μg/mL pyruvate kinase, 24
μg/mL lactate dehydrogenase with 46 nM PKCδ or 4.5 nM PKCα,
and 150 μM ATP (Km = 50 μM) or 130 μM ATP (Km = 45 μM),
respectively, in a final volume of 33 μL. Serial dilutions of inhibitors
were prepared in DMSO, and the reaction was initiated by the
addition of ATP.
Initial rate data were determined from the rate of change of
absorbance at 340 nM (corresponding to stoichiometric consumption
of NADH) using a Molecular Devices Spectramax plate reader
(Sunnyvale, CA) over 15 min at 25 °C.
Molecular docking was performed using “Extra Precision (XP)
Glide” (Schrodinger, New York, U.S.; Friesner et al., 2006). The
̈
docking algorithm performs a conformational analysis of the ligand in
the kinase active site. In particular, the XP scoring function considers
hydrophobic packing and presence of hydrogen bonds in hydrophobic
environments that are important features in predicting protein−ligand
interactions in the active sites of kinases.
Ki values were calculated by fitting initial rate data to the model for
competitive tight binding inhibition using the Prism software package
(Prism 4.0a, Graphpad Software, San Diego, CA).
PBMC T Cell IL-2 Assays. PBMCs were prepared from human
blood, washed, and adjusted to a cell density of 5 × 105 cells/mL in
complete medium, and an amount of 100 μL was added to wells of 96-
well Immunosorp plates (Nunc), containing immobilized anti-human
CD3 (1.5 μg/mL, Serotec, U.K.), anti-human CD28 (1 μg/mL, BD,
U.K.), and serial dilutions of PKCθ inhibitors or DMSO vehicle. The
cell assay plates were incubated for 24 h in a humidified incubator in
5% CO2 at 37 °C. Supernatants were collected and IL-2 levels
determined using a bead-based FLISA assay (Applied Biosystems,
U.K.). IC50 values were determined by nonlinear regression analysis in
Prism (Graphpad Software Inc.).
WB IL-2. Human whole blood (950 μL) was dispensed into a 96-
well cluster plate and 50 μL of serial dilutions of PKCθ inhibitor or
DMSO vehicle added and incubated for 1 h on a plate shaker. The
whole blood was treated with 25 μL/well PHA solution (Gibco, U.K.)
and incubated in a shaking incubator at 37 °C for 6 h. Cells were
pelleted by centrifuging the plates. The supernatant was collected, and
IL-2 levels were determined using a human IL-2 ELISA (R&D
Systems, U.K.).
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: + 44 (0) 1235 438800. E-mail: juan-miguel_jimenez@
vrtx.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
SEB-Induced IL-2 Release. All procedures described below were
performed in accordance with the Animals (Scientific Procedures) Act
1986 and under the authority of a UK Home Office Project License.
Female Balb/c mice (8 weeks old) were purchased from Harlan UK
Ltd. Animals were allowed to acclimatize for at least 1 week prior to
experimentation.
We thank Barbara Cipriani and Roya for support in cellular
assays, James Westcott for support with biochemical studies,
and the full PKCθ project team and Julian Golec and Don
Middleton for valuable discussions.
Compound 27 was formulated as a solution in 1% Tween 80/99%
sodium citrate buffer, pH 3. All treatments were given by oral gavage
and at a dose volume of 10 mL/kg. The number of animals per
treatment group ranged from six to eight. A single dose of compound
27 (6.25, 12, 25, 50 mg/kg) was administered to animals. A control
group received vehicle (1% Tween 80/99% sodium citrate buffer, pH
3). Cyclosporin A (15 mg/kg) was used as a reference compound.
One hour following the administration of vehicle, cyclosporin A, or
compound 27, animals were injected intraperitoneally with SEB (10
mg in 200 μL). At 2 h later, terminal blood samples were taken and
plasma was analyzed for IL-2 concentration. The concentrations of IL-
ABBREVIATIONS USED
■
LipE, lipophilic ligand efficiency; DAG, diacylglycerol; TCR, T
cell receptor; CD28, cluster of differentiation 28; AP-1,
activator protein 1; NFκB, nuclear factor κ light-chain enhancer
of activated B cells; NFAT, nuclear factor of activated T cells;
IL-2, interleukin 2; Bcl-xL, B-cell lymphoma, extra large; Bcl-2,
B-cell lymphoma 2; Bad, Bcl-2-associated death promoter; MS,
multiple sclerosis; GRL, glycine rich loop; PBMC, peripheral
blood mononuclear cell
J
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(18) Salek-Ardakani, S.; So, T.; Halteman, B. S.; Altman, A.; Croft,
M. Differential regulation of Th2 and Th1 lung inflammatory
responses by protein kinase C theta. J. Immunol. 2004, 173, 6440−
6447.
(19) Salek-Ardakani, S.; So, T.; Halteman, B. S.; Altman, A.; Croft,
M. Protein kinase Ctheta controls Th1 cells in experimental
autoimmune encephalomyelitis. J. Immunol. 2005, 175, 7635−7641.
(20) Tan, S. L.; Zhao, J.; Bi, C.; Chen, X. C.; Hepburn, D. L.; Wang,
J.; Sedgwick, J. D.; Chintalacharuvu, S. R.; Na, S. Resistance to
experimental autoimmune encephalomyelitis and impaired IL-17
production in protein kinase C theta-deficient mice. J. Immunol.
2006, 176, 2872−2879.
(21) Anderson, K.; Fitzgerald, M.; Dupont, M.; Wang, T.; Paz, N.;
Dorsch, M.; Healy, A.; Xu, Y.; Ocain, T.; Schopf, L.; Jaffee, B.;
Picarella, D. Mice deficient in PKC theta demonstrate impaired in vivo
T cell activation and protection from T cell-mediated inflammatory
diseases. Autoimmunity 2006, 39, 469−478.
(22) Healy, A. M.; Izmailova, E.; Fitzgerald, M.; Walker, R.;
Hattersley, M.; Silva, M.; Siebert, E.; Terkelsen, J.; Picarella, D.;
Pickard, M. D.; LeClair, B.; Chandra, S.; Jaffee, B. PKC-theta-deficient
mice are protected from Th1-dependent antigen-induced arthritis. J.
Immunol. 2006, 177, 1886−1893.
(23) Nagahama, K.; Ogawa, A.; Shirane, K.; Shimomura, Y.;
Sugimoto, K.; Mizoguchi, A. Protein kinase C theta plays a
fundamental role in different types of chronic colitis. Gastroenterology
2008, 134, 459−469.
(24) Skvara, H.; Dawid, M.; Kleyn, E.; Wolff, B.; Meingassner, J. G.;
Knight, H.; Dumortier, T.; Kopp, T.; Fallahi, N.; Stary, G.; Burkhart,
C.; Grenet, O.; Wagner, J.; Hijazi, Y.; Morris, R. E.; McGeown, C.;
Rordorf, C.; Griffiths, C. E.; Stingl, G.; Jung, T. The PKC inhibitor
AEB071 may be a therapeutic option for psoriasis. J. Clin. Invest. 2008,
118, 3151−3159.
(25) Budde, K.; Sommerer, C.; Becker, T.; Asderakis, A.; Pietruck, F.;
Grinyo, J. M.; Rigotti, P.; Dantal, J.; Ng, J.; Barten, M. J.; Weber, M.
Sotrastaurin, a novel small molecule inhibiting protein kinase C: first
clinical results in renal-transplant recipients. Am. J. Transplant. 2010,
10, 571−581.
(26) Wagner, J.; von Matt, P.; Sedrani, R.; Albert, R.; Cooke, N.;
Ehrhardt, C.; Geiser, M.; Rummel, G.; Stark, W.; Strauss, A.; Cowan-
Jacob, S. W.; Beerli, C.; Weckbecker, G.; Evenou, J. P.; Zenke, G.;
Cottens, S. Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-
yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective
inhibitor of protein kinase C isotypes. J. Med. Chem. 2009, 52, 6193−
6196.
REFERENCES
■
(1) Rosse, C.; Linch, M.; Kermorgant, S.; Cameron, A. J.; Boeckeler,
K.; Parker, P. J. PKC and the control of localized signal dynamics. Nat.
Rev. Mol. Cell Biol. 2010, 11, 103−112.
(2) Baier, G.; Telford, D.; Giampa, L.; Coggeshall, K. M.; Baier-
Bitterlich, G.; Isakov, N.; Altman, A. Molecular cloning and
characterization of PKC theta, a novel member of the protein kinase
C (PKC) gene family expressed predominantly in hematopoietic cells.
J. Biol. Chem. 1993, 268, 4997−5004.
(3) Sun, Z.; Arendt, C. W.; Ellmeier, W.; Schaeffer, E. M.; Sunshine,
M. J.; Gandhi, L.; Annes, J.; Petrzilka, D.; Kupfer, A.; Schwartzberg, P.
L.; Littman, D. R. PKC-theta is required for TCR-induced NF-kappaB
activation in mature but not immature T lymphocytes. Nature 2000,
404, 402−407.
(4) Pfeifhofer, C.; Kofler, K.; Gruber, T.; Tabrizi, N. G.; Lutz, C.;
Maly, K.; Leitges, M.; Baier, G. Protein kinase C theta affects Ca2+
mobilization and NFAT cell activation in primary mouse T cells. J.
Exp. Med. 2003, 197, 1525−1535.
(5) Manicassamy, S.; Sadim, M.; Ye, R. D.; Sun, Z. Differential roles
of PKC-theta in the regulation of intracellular calcium concentration in
primary T cells. J. Mol. Biol. 2006, 355, 347−359.
(6) Baier-Bitterlich, G.; Uberall, F.; Bauer, B.; Fresser, F.; Wachter,
H.; Grunicke, H.; Utermann, G.; Altman, A.; Baier, G. Protein kinase
C-theta isoenzyme selective stimulation of the transcription factor
complex AP-1 in T lymphocytes. Mol. Cell. Biol. 1996, 16, 1842−50.
(7) Coudronniere, N.; Villalba, M.; Englund, N.; Altman, A. NF-
kappa B activation induced by T cell receptor/CD28 costimulation is
mediated by protein kinase C-theta. Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 3394−3399.
(8) Lin, X.; O’Mahony, A.; Mu, Y.; Geleziunas, R.; Greene, W. C.
Protein kinase C-theta participates in NF-kappaB activation induced
by CD3-CD28 costimulation through selective activation of IkappaB
kinase beta. Mol. Cell. Biol. 2000, 20, 2933−40.
(9) Bertolotto, C.; Maulon, L.; Filippa, N.; Baier, G.; Auberger, P.
Protein kinase C theta and epsilon promote T-cell survival by a rsk-
dependent phosphorylation and inactivation of BAD. J. Biol. Chem.
2000, 275, 37246−37250.
(10) Villalba, M.; Bushway, P.; Altman, A. Protein kinase C-theta
mediates a selective T cell survival signal via phosphorylation of BAD.
J. Immunol. 2001, 166, 5955−5963.
(11) Barouch-Bentov, R.; Lemmens, E. E.; Hu, J.; Janssen, E. M.;
Droin, N. M.; Song, J.; Schoenberger, S. P.; Altman, A. Protein kinase
C-theta is an early survival factor required for differentiation of effector
CD8+ T cells. J. Immunol. 2005, 175, 5126−5134.
(12) Manicassamy, S.; Gupta, S.; Huang, Z.; Sun, Z. Protein kinase
C-theta-mediated signals enhance CD4+ T cell survival by up-
regulating Bcl-xL. J. Immunol. 2006, 176, 6709−6716.
(13) Berg-Brown, N. N.; Gronski, M. A.; Jones, R. G.; Elford, A. R.;
Deenick, E. K.; Odermatt, B.; Littman, D. R.; Ohashi, P. S. PKCtheta
signals activation versus tolerance in vivo. J. Exp. Med. 2004, 199,
743−752.
(14) Marsland, B. J.; Soos, T. J.; Spath, G.; Littman, D. R.; Kopf, M.
Protein kinase C theta is critical for the development of in vivo T
helper (Th)2 cell but not Th1 cell responses. J. Exp. Med. 2004, 200,
181−189.
(15) Giannoni, F.; Lyon, A. B.; Wareing, M. D.; Dias, P. B.; Sarawar,
S. R. Protein kinase C theta is not essential for T-cell-mediated
clearance of murine gammaherpesvirus 68. J. Virol. 2005, 79, 6808−
6813.
(27) Wagner, J.; von Matt, P.; Faller, B.; Cooke, N. G.; Albert, R.;
Sedrani, R.; Wiegand, H.; Jean, C.; Beerli, C.; Weckbecker, G.; Evenou,
J. P.; Zenke, G.; Cottens, S. Structure−activity relationship and
pharmacokinetic studies of sotrastaurin (AEB071), a promising novel
medicine for prevention of graft rejection and treatment of psoriasis. J.
Med. Chem. 2011, 54, 6028−6039.
(28) Subrath, J.; Wang, D.; Wu, B.; Niu, C.; Boschelli, D. H.; Lee, J.;
Yang, X.; Brennan, A.; Chaudhary, D. C-5 Substituted heteroaryl 3-
pyridinecarbonitriles as PKCtheta inhibitors: part I. Bioorg. Med. Chem.
Lett. 2009, 19, 5423−5425.
(29) Prashad, A. S.; Wang, D.; Subrath, J.; Wu, B.; Lin, M.; Zhang, M.
Y.; Kagan, N.; Lee, J.; Yang, X.; Brennan, A.; Chaudhary, D.; Xu, X.;
Leung, L.; Wang, J.; Boschelli, D. H. C-5 substituted heteroaryl-3-
pyridinecarbonitriles as PKCtheta inhibitors: part II. Bioorg. Med.
Chem. Lett. 2009, 19, 5799−5802.
(30) Cole, D. C.; Asselin, M.; Brennan, A.; Czerwinski, R.; Ellingboe,
J. W.; Fitz, L.; Greco, R.; Huang, X.; Joseph-McCarthy, D.; Kelly, M.
F.; Kirisits, M.; Lee, J.; Li, Y.; Morgan, P.; Stock, J. R.; Tsao, D. H.;
Wissner, A.; Yang, X.; Chaudhary, D. Identification, characterization
and initial hit-to-lead optimization of a series of 4-arylamino-3-
pyridinecarbonitrile as protein kinase C theta (PKCtheta) inhibitors. J.
Med. Chem. 2008, 51, 5958−5963.
(31) Dushin, R. G.; Nittoli, T.; Ingalls, C.; Boschelli, D. H.; Cole, D.
C.; Wissner, A.; Lee, J.; Yang, X.; Morgan, P.; Brennan, A.; Chaudhary,
D. Synthesis and PKCtheta inhibitory activity of a series of 4-
(16) Marsland, B. J.; Nembrini, C.; Schmitz, N.; Abel, B.; Krautwald,
S.; Bachmann, M. F.; Kopf, M. Innate signals compensate for the
absence of PKC-{theta} during in vivo CD8(+) T cell effector and
memory responses. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 14374−
14379.
(17) Marsland, B. J.; Nembrini, C.; Grun, K.; Reissmann, R.; Kurrer,
M.; Leipner, C.; Kopf, M. TLR ligands act directly upon T cells to
restore proliferation in the absence of protein kinase C-theta signaling
and promote autoimmune myocarditis. J. Immunol. 2007, 178, 3466−
3473.
K
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
indolylamino-5-phenyl-3-pyridinecarbonitriles. Bioorg. Med. Chem. Lett.
2009, 19, 2461−2463.
(32) Wu, B.; Boschelli, D. H.; Lee, J.; Yang, X.; Chaudhary, D.
Second generation 4-(4-methyl-1H-indol-5-ylamino)-2-phenylthieno-
[2,3-b]pyridine-5-carbonitrile PKCtheta inhibitors. Bioorg. Med. Chem.
Lett. 2009, 19, 766−769.
(33) Boschelli, D. H.; Wu, B.; Barrios Sosa, A. C.; Chen, J.; Asselin,
M.; Cole, D. C.; Lee, J.; Yang, X.; Chaudhary, D. Synthesis and
PKCtheta inhibitory activity of a series of 4-(indol-5-ylamino)thieno-
[2,3-b]pyridine-5-carbonitriles. Bioorg. Med. Chem. Let. 2008, 18,
2850−2853.
(34) Nathan Tumey, L.; Boschelli, D. H.; Lee, J.; Chaudhary, D. 2-
Alkenylthieno[2,3-b]pyridine-5-carbonitriles: potent and selective
inhibitors of PKCtheta. Bioorg. Med. Chem. Lett. 2008, 18, 4420−4423.
(35) Boschelli, D. H.; Wang, D.; Prashad, A. S.; Subrath, J.; Wu, B.;
Niu, C.; Lee, J.; Yang, X.; Brennan, A.; Chaudhary, D. Optimization of
5-phenyl-3-pyridinecarbonitriles as PKCtheta inhibitors. Bioorg. Med.
Chem. Lett. 2009, 19, 3623−3626.
(36) Boschelli, D. H.; Subrath, J.; Niu, C.; Wu, B.; Wang, Y.; Lee, J.;
Brennan, A.; Ho, M.; Deng, B.; Yang, X.; Xu, X.; Leung, L.; Wang, J.;
Atherton, J.; Chaudhary, D. Optimization of 5-vinylaryl-3-pyridine-
carbonitriles as PKCtheta inhibitors. Bioorg. Med. Chem. Lett. 2010, 20,
1965−1968.
(37) Niu, C.; Boschelli, D. H.; Tumey, L. N.; Bhagirath, N.; Subrath,
J.; Shim, J.; Wang, Y.; Wu, B.; Eid, C.; Lee, J.; Yang, X.; Brennan, A.;
Chaudhary, D. First generation 5-vinyl-3-pyridinecarbonitrile PKCthe-
ta inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 5829−5832.
(38) Shim, J.; Eid, C.; Lee, J.; Liu, E.; Chaudhary, D.; Boschelli, D. H.
Synthesis and PKCtheta inhibitory activity of a series of 5-vinyl phenyl
sulfonamide-3-pyridinecarbonitriles. Bioorg. Med. Chem. Lett. 2009, 19,
6575−6577.
(39) Cywin, C. L.; Dahmann, G.; Prokopowicz, A. S., 3rd; Young, E.
R.; Magolda, R. L.; Cardozo, M. G.; Cogan, D. A.; Disalvo, D.; Ginn, J.
D.; Kashem, M. A.; Wolak, J. P.; Homon, C. A.; Farrell, T. M.; Grbic,
H.; Hu, H.; Kaplita, P. V.; Liu, L. H.; Spero, D. M.; Jeanfavre, D. D.;
O’Shea, K. M.; White, D. M.; Woska, J. R., Jr.; Brown, M. L. Discovery
of potent and selective PKC-theta inhibitors. Bioorg. Med. Chem. Lett.
2007, 17, 225−230.
(40) Boschelli, D. H. Small molecule inhibitors of PKCTheta as
potential antiinflammatory therapeutics. Curr. Top. Med. Chem. 2009,
9, 640−654.
(41) Jimenez, J.-M.; Davis, J. C.; Boyall, D.; Fraysee, D.; Knegtel, R.;
Settimo, L.; Young, S.; Bolton, C.; Chiu, P.; Curnock, A.; Rasmussen,
R.; Tanner, A.; Ager, I. Structure-based optimization of amino-
pyridines as PKCθ inhibitors. Bioorg. Med. Chem. Lett. 2012, 22,
4645−4649.
(42) Leeson, P. D.; Springthorpe, B. The influence of drug-like
concepts on decision-making in medicinal chemistry. Nat. Rev. Drug
Discovery 2007, 6, 881−890.
(43) Mecklenbrauker, I.; Saijo, K.; Zheng, N. Y.; Leitges, M.;
Tarakhovsky, A. Protein kinase Cdelta controls self-antigen-induced B-
cell tolerance. Nature 2002, 416, 860−865.
(44) Miyamoto, A.; Nakayama, K.; Imaki, H.; Hirose, S.; Jiang, Y.;
Abe, M.; Tsukiyama, T.; Nagahama, H.; Ohno, S.; Hatakeyama, S.;
Nakayama, K. I. Increased proliferation of B cells and auto-immunity
in mice lacking protein kinase C delta. Nature 2002, 416, 865−869.
(45) Gill, A. L.; Verdonk, M.; Boyle, R. G.; Taylor, R. A comparison
of physicochemical property profiles of marketed oral drugs and orally
bioavailable anti-cancer protein kinase inhibitors in clinical develop-
ment. Curr. Top. Med. Chem. 2007, 7, 1408−1422.
(46) Rummel, G.; Stark, W.; Strauss, A.; Wagner, J. Three
Dimensional Structure of the Catalytic Domain of Protein C theta,
Methods and Use Thereof. PCT Int. Appl. WO2004076654, 2004.
L
dx.doi.org/10.1021/jm301465a | J. Med. Chem. XXXX, XXX, XXX−XXX