3-amido pyrrolopyrazine JAK kinase inhibitors: Development of a JAK3 vs JAK1 selective inhibitor and evaluation in cellular and in vivo models

Article abstract of DOI:10.1021/jm301646k

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cycl

Full text of DOI:10.1021/jm301646k

Article
pubs.acs.org/jmc
3‑Amido Pyrrolopyrazine JAK Kinase Inhibitors: Development of a
JAK3 vs JAK1 Selective Inhibitor and Evaluation in Cellular and in
Vivo Models
Michael Soth,*,† Johannes C. Hermann,† Calvin Yee,† Muzaffar Alam,† Jim W. Barnett,‡ Pamela Berry,†
Michelle F. Browner,‡ Karl Frank,‡ Sandra Frauchiger,‡ Seth Harris,‡ Yang He,†
Mohammad Hekmat-Nejad,‡ Than Hendricks,‡ Robert Henningsen,‡ Ramona Hilgenkamp,†
Hoangdung Ho,‡ Ann Hoffman,† Pei-Yuan Hsu,† Dong-Qing Hu,† Andrea Itano,‡ Saul Jaime-Figueroa,†
Alam Jahangir,‡ Sue Jin,† Andreas Kuglstatter,† Alan K. Kutach,‡ Cheng Liao,† Stephen Lynch,†
John Menke,† Linghao Niu,† Vaishali Patel,† Aruna Railkar,† Douglas Roy,† Ada Shao,‡ David Shaw,‡
Sandra Steiner,† Yongliang Sun,† Seng-Lai Tan,† Sandra Wang,‡ and Minh Diem Vu†
†Hoffmann-La Roche, 340 Kingsland Street, Nutley, New Jersey 07110, United States
‡Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, California 94304, United States
S
* Supporting Information
ABSTRACT: The Janus kinases (JAKs) are involved in multiple signaling networks relevant to
inflammatory diseases, and inhibition of one or more members of this class may modulate
disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-
cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including
selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this
analogue in cellular and in vivo models confirmed functional selectivity for modulation of a
JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6
stimulated pathway.
INTRODUCTION
■
The Janus kinase family members (JAK3, JAK2, JAK1, and
TYK2) play critical and cooperative roles in transmitting many
signaling cascades in response to inflammatory and proliferative
signals. For instance, JAK3 acts in concert with JAK1 to relay
cytokine signaling through the cytokine receptor common γ
chain, also known as CD132.1,2 Null mutations in either JAK3 or
CD132 are associated with severe combined immunodeficiency
(SCID) disorder.3−5 JAK2 is important in the signaling of
Figure 1. Structures of Tofacitinib (1), profiling hit 2, and cyclopropyl
analogue 3a.
hormones and growth factors such as erythropoietin (EPO),
through the EPO receptor, as well as granulocyte-macrophage
colony stimulating factor (GM-CSF) via its receptor and the
common β chain IL-3 receptor.6,7 JAK1, JAK2, and TYK2 are
involved in IL-6 signaling.8−10 Across the pharmaceutical
industry, there is high interest in targeting the JAK family of
kinases for both oncology and inflammation indications.11−15
Within the inflammation arena, Pfizer’s Tofacitinib, 1 (Figure 1),
a JAK3/2/1 inhibitor initially reported as a selective JAK3
inhibitor, has shown promising efficacy results in clinical trials for
rheumatoid arthritis (RA).16,17
However, it is unclear if blockade of all JAK isozymes is
required for optimal efficacy without compromising the risk/
benefit profile or if a more isozyme-selective inhibitor would
suffice. There are divergent opinions in the literature as to the
functional consequences of selective inhibition of JAK3 over
JAK1. Recent papers from the Novartis group have questioned
whether this type of selectivity profile will be sufficient to
efficiently block immunologically relevant pathways mediated by
CD132 cytokines.18,19 The Novartis group reached this
conclusion on the basis of cellular data on a highly JAK3-
selective compound compared to Tofacitinib 1, coupled with
extensive biological studies dissecting the roles of JAK3 and JAK1
in IL-2-induced STAT5 phosphorylation (a CD132-dependent
process). However, an earlier paper from the Pharmacopeia/
Wyeth/Pfizer group concluded that JAK1 inhibition is not
required for potent inhibition of IL-2 signaling, and with a JAK3-
selective tool compound they demonstrated potent, selective
inhibition of IL-2 signaling over cellular pathways requiring JAK-
family enzymes other than JAK3.20 Thus, it would be important
Received: November 7, 2012
Published: December 9, 2012
© 2012 American Chemical Society
345
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
to resolve this discrepancy using structurally distinct scaffolds of
JAK inhibitors.
source of leads for new kinase programs.21 The “hit” that we
decided to pursue was pyrrolopyrazine 2, which had been
profiled by Kinomescan (now a division of DiscoveRx, formerly a
division of Ambit Biosciences)22 and showed binding to JAK3,
JAK2 and JAK1; however, it showed poor kinome selectivity.23
Exploration of trimethoxyphenyl replacements afforded multiple
subseries with varying kinome selectivities. This article describes
the cyclopropyl subseries.
Replacement of the trimethoxyphenyl group of 2 with a
cyclopropyl group to afford 3a resulted in a reasonably potent
JAK inhibitor (Table 1, first entry). We modeled 3a into the
ATP-binding site of JAK3 and looked for promising vectors to
improve potency and possibly kinome selectivity (Figure 2). As
both methyls of the amide isopropyl group point toward
apparent pockets, we focused our SAR work on further
derivatization of these two vectors (Table 1). During our efforts
on this program, the crystal structure of 1 was obtained (and later
published by others24), showing space-filling of these pockets.
SAR studies around 1 confirmed that these pockets are indeed
excellent areas of the protein to fill for potency.25 For the
examples in this pyrrolopyrazine series, potency trends are
similar across JAK isozymes, with many examples showing some
preference for inhibiting JAK3 versus JAK2 and JAK1.
As shown in Figure 2, the “lower” pocket is defined in part by
leucine (Leu956) and alanine (Ala966) residues. We therefore
targeted this region with small hydrophobic groups off of the
isopropyl amide (Entries 3b−3i). A significant boost in potency
was gained by simply replacing the lower methyl group with an
ethyl (entry 3b vs 3a). Further elaboration to isopropyl and
racemic cyclopropyl analogues (3c and 3d) did not improve
potency further; however, tert-butyl analogue 3e showed an
additional potency boost, bringing the potency down to a 1 nM
compound. Replacing one methyl of the tert-butyl group with a
hydroxyl group (3f) resulted in a significant potency loss; a nitrile
replacement (3h) was more favorable. A cyclohexyl analogue
(3i) was tolerated but less potent than the other examples from
this exercise to fill the lower pocket. A crystal structure of
analogue 3e in JAK3 showed excellent space-filling of the lower
pocket by the tert-butyl group (Figure 3).
Our JAK medicinal chemistry program strategy was to explore
a diverse range of series with varying JAK family selectivity
profiles as tool compounds to dissect the relative role of each JAK
member in inflammatory disease indications. Here, we describe
the optimization of a 3-amido-5-cyclopropyl pyrrolopyrazine
series to potent JAK inhibitors, culminating in an example with
selectivity for JAK3 over JAK1 and properties conducive to oral
dosing and testing in an acute PK/PD mouse model. Our
optimized compound shows both cellular and in vivo selectivity
for inhibition of an IL-2 signaling pathway, most notably against
an IL-6 receptor mediated signaling pathway. In addition, the
structure−activity relationships (SAR) around close analogues
indicate that improved selectivity for JAK3 over JAK1 does not
result in a decrease in potency for inhibition of the IL-2 signaling
pathway.
RESULTS AND DISCUSSION
■
Chemistry. The general synthetic scheme for the preparation
of 3-amido-5-cyclopropyl pyrrolopyrazines is shown in Scheme
1. Commercially available 5-bromopyrrolopyrazine was con-
a
Scheme 1. General Synthesis of Pyrrolopyrazines 3a−t
Beyond potency considerations, the other noteworthy effect of
filling the lower pocket is an improved selectivity for JAK3 versus
JAK1. The larger groups (tert-butyl analogues and cyclohexyl)
show 10−50-fold selectivity.
a
Reagents and conditions: (i) (a) formaldehyde, sodium hydroxide,
1,4-dioxane/water, (b) sodium hydroxide, tetrahydrofuran/water, (c)
Jones reagent, acetone, (d) 2-silylethoxymethyl chloride, sodium
hydride, N,N-dimethylformamide, 0 °C→rt, (42%); (ii) cyclo-
propylboronic acid, palladium diacetate, tricyclohexylphosphine,
potassium phosphate, toluene/water, 100 °C (81%); (iii) sodium
hypochlorite, potassium dihydrogen phosphate, sulfamic acid, 1,4-
dioxane/water (87%); (iv) (a) R1-NH2, amide coupling reagent,
solvent, (b) trifluoroacetic acid, dichloromethane, (c) ethylene
diamine/dichloromethane or sodium acetate/ethanol or triethyl-
amine/MeOH/H2O (17−87%).
Interestingly, the hydroxyl-containing enantiomers 3f and 3g
showed similar potencies against JAK3 (Table 1). Crystal
structures for both of these compounds bound to JAK3 were
obtained and showed a similar space-filling, i.e., the ligands were
free to shift in the pocket to obtain optimal binding (Figure 4).
For the second vector, leading to the “upper pocket”, a
modeling exercise highly prioritized a bis-amide motif as having
low energy conformations most likely to fill the space available.
The first simple examples 3j−m did not show potency
improvements over the parent 3a; however, a crystal structure
of compound 3k (Figure 5) confirmed the expected binding
motif and encouraged us to try further substitution, specifically
with nitrile side chains, as a nitrile group in this pocket was
observed to be particularly beneficial for potency by the Pfizer
group in their development of compound 1.25 The nitrile group
is also beneficial for our series (examples 3n, 3p). Comparison of
methyl analogue 3o to cyano analogue 3n suggests that the
potency boost is not due to simple space-filling. A recent paper
from the Pfizer group attempts to explain the potency benefits of
a nitrile group in this area of the protein.24 For our scaffold, the
verted in a four-step process to silylethoxymethyl (SEM)-
protected aldehyde 4. Aldehyde 4 was subjected to Suzuki
conditions to afford cyclopropyl analogue 5. Oxidation of the
aldehyde moiety afforded acid 6, which was coupled to a variety
of amines followed by SEM deprotection to afford final
compounds 3a−t.
Initial Hit and Optimization. The chemical starting point
for this work came from a scan of our internal collection of kinase
inhibitors for which we have kinome selectivity information.
Inhibitor sets annotated by selectivity profiles can be a fruitful
346
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
a
Table 1. Enzyme Potencies
Figure 2. Modeled structure of 3a in JAK3. Carbon atoms of 3a are in
cyan, carbon atoms of the JAK3 protein are in orange, oxygen atoms are
in red, and nitrogen atoms are colored in blue. Red-colored protein
surface indicates protein areas identified for potential exploration and
optimization of interactions. All visualizations generated with MOE.26.
Figure 3. X-ray crystal structure of 3e in JAK3 (PDB accession no.
4HVD, 1.85 Å resolution), demonstrating filling of the lower pocket by
the tert-butyl group. Carbon atoms of 3e are in yellow, carbon atoms of
the JAK3 protein are in orange, oxygen atoms are in red, and nitrogen
atoms are colored in blue. Molecular surface of 3e is colored cyan.
most promising side chains from this second exercise combined
well with the most promising side chains of the first exercise
(examples 3q−t), yielding highly potent inhibitors. The
potencies of these compounds were comparable to Tofacitinib
1. Importantly, while compounds 3q−t all show similar
potencies against JAK3, the tert-butyl example 3q is >10-fold
selective against JAK1 while the cyclopropyl examples 3r−t are
not selective against JAK1. This selectivity discrepancy among
very closely related compounds allows for a useful interpretation
of cellular data later in the manuscript.
Kinome Selectivity. We profiled our compounds by Caliper
screening against a 48 kinase panel (Table 2). Caliper profiling
measures percent inhibition of phosphorylation of a peptide
substrate; the data in Table 2 is represented by heat maps,
assigning <50% inhibition as completely green and >95%
inhibition as completely red. We pick six examples here to
a
Inhibition of phosphorylation of a biotinylated synthetic peptide
catalyzed by JAK1−3; see Experimental Section for details. All enzyme
reactions were run at adenosine triphosphate (ATP) concentrations of
1.5 μM. Kms of these enzymes for ATP under our experimental
conditions were determined to be 1.5 μM (JAK3), 6 μM (JAK2), and
20 μM (JAK1). Mean SEM (standard error of the mean), n ≥ 3
except for 3g and 3t (n = 2).
b
347
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
It is more difficult to interpret the selectivity data for entry 3p,
which presumably fills the upper pocket with the cyanoazetidine
amide group, because its JAK3 potency lies between that of 3a
and 3e/h and so it is difficult to decide which analogue is the
appropriate comparator. However, the upper pocket is mostly
defined by a main-chain β-sheet, with side chains pointing away
and unlikely to be key determinants of selectivity. What is clear is
that the combination of the tert-butyl side chain with the
cyanoazetidine amide is optimal for kinome selectivity (entry
3q). The added selectivity of 3q over 3e does not necessarily
mean that the cyanoazetidine group introduces specific binding
interactions that favor JAK3. The added selectivity of 3q over 3e
may simply be due to a tighter fit of a ligand optimized to JAK3
versus other kinases, i.e., the presence of the cyanoazetidine
group restricts freedom of movement for the ligand within kinase
pockets, disfavoring alternative ligand arrangements within other
kinases. This idea is supported by the earlier noted observation
that smaller ligands have freedom of movement within these
pockets (Figure 4). Freedom of ligand movement could also
explain the reduced selectivity of tertiary alcohol 3f compared to
3e and 3h. The polar hydroxyl group of 3f is likely more
amenable to alternative, water-exposed binding conformations.
Cellular Activity and Biopharmaceutical Properties.
Select examples were tested in cell-based assays (Table 3). These
assays measure the inhibition of phosphorylation of downstream
signal transducer and activator of transcription (STAT) proteins,
either in human peripheral blood mononuclear cells (PBMCs) or
in human whole blood (HWB), upon treatment with varying
stimuli (Table 3). The different cellular stimuli used induce
phosphorylation of STATs by either dual JAK3/1 (IL-2 stimulus,
STAT5a phosphorylation in T-cells), JAK2 (GM-CSF stimulus,
STAT5a phosphorylation in monocytes), or dual JAK2/1 (IFN-γ
stimulus, STAT1 phosphorylation in monocytes) containing
pathways.
Figure 4. X-ray crystal structures of 3f (PDB accession no. 4HVG, 2.75
Å resolution) and 3g (4HVH, 2.3 Å) in JAK3, superimposed. Carbon
atoms of 3f are in cyan, carbon atoms of 3g are in orange, oxygen atoms
are in red, and nitrogen atoms are colored in blue. The assignment of
methyl and hydroxyl in the tertiary alcohol moiety is tentative at the
current resolution. Meshes are colored according to ligand carbon atom
color and show the molecular surface for each ligand.
Only examples with high enzyme potency (10 nM or better)
against at least one relevant isoform achieved sub-μM cellular
potencies. Many of the compounds tested show cellular
potencies comparable to those seen for compound 1, although
HWB potencies are less, presumably because of higher protein
binding. For example, in our protein binding assays (human),
compound 1 showed a free fraction of 99% while compound 3q
showed a free fraction of only 3%. Cellular selectivities within this
panel are also comparable to those observed for 1, with moderate
selectivities seen for inhibition of IL-2 signaling versus GM-CSF
and IFN-γ signaling.
Figure 5. X-ray crystal structure of 3k in JAK3 (PDB accession no.
4HVI, 2.4 Å resolution), demonstrating filling of the upper pocket by the
tertiary amide. Carbon atoms of 3k are in yellow, carbon atoms of the
JAK3 protein are in orange, oxygen atoms are in red, and nitrogen atoms
are colored in blue. Molecular surface of 3k is colored cyan. Gray-
colored mesh shows the molecular surface and shape of the binding site.
Importantly, the examples with higher selectivity for JAK3
versus JAK1 still show potent inhibition of IL-2 signaling.
Compound 3q was equally potent in the IL-2 assay compared to
its close analogue 3t, which has a similar JAK3 enzyme potency
but no selectivity against JAK1. These results cannot be explained
by the cellular permeabilities of these compounds, which are
comparable in our Caco-2 permeability assay (data not shown).
We conclude that for these close comparators, loss of JAK1
potency did not affect potency in the IL-2 cellular assay and that a
more selective inhibition of JAK3 catalytic function can be
realized while still efficiently attenuating IL-2-induced STAT5a
phosphorylation.
The in vitro biopharmaceutical properties for compounds in
this series varied depending on the amide side chain. Our early
screening funnel focused on solubilities and microsomal
stabilities, which both tended toward low for purely aliphatic
side chains and improved for more polar examples. At various
stages in the optimization process, select compounds were more
demonstrate the key selectivity learnings. For purposes of fair
comparison, the concentration at which compounds were
profiled reflects their JAK3 potency: for the less potent entries
3a and 3f, profiling was performed at 10 μM; for the more potent
entries 3e, 3h, and 3q, profiling was performed at 1 μM. For entry
3p, which has an intermediate potency, we include both 10 and 1
μM data.
Entries 3e and 3h, which fill the lower pocket with either the
tert-butyl group or the closely related cyano analogue, show
selectivity improvements relative to parent 3a. These selectivity
improvements are easily explainable by analysis of the protein in
the areas filled. The lower pocket is in part defined by Ala966,
which is a variable residue across the kinome and is often larger
for other kinases, i.e., for other kinases there is not room for an
optimal fit of the tert-butyl group. Indeed, the profiled kinases
that are most strongly “hit” by entries 3e and 3h all contain
alanine in this position.
348
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
a
Table 2. Kinome Selectivity Profiles for Select Examples
a
% inhibition of phosphorylation of substrates at the indicated test compound concentrations, using a ProfilerPro Kit 1 (P/N 760373) supplied by
Caliper Lifesciences. In these heat maps, <50% inhibition is assigned as completely green and >95% inhibition is assigned as completely red.
deeply characterized. In vitro properties and in vivo rat
pharmacokinetics (PK) for compounds 3f, 3h, and 3q are
shown in Table 4. Tertiary alcohol 3f was an early example with
good in vitro properties translating to reasonable in vivo rat
exposures, indicating that the core 3-amido pyrrolopyrazine is a
drug-like scaffold and encouraging further optimization. Nitrile
3h and bis-amide 3q were designed and synthesized later in the
optimization exercise and were two of our best examples with
349
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
a
Table 3. Cellular and HWB Potencies for Select Compounds
b
b
b
b
compd
PBMC (IL-2) IC50 (μM)
PBMC (GM-CSF) IC50 (μM)
PBMC (IFN-γ) IC50 (μM)
HWB (IL-2) IC50 (μM)
3e
3f
0.211 0.069
2.23 0.56
5.48 0.96
>30
3.38 0.47
13.2 0.3
2.46 0.34
15.7 2.9
3h
3n
3p
3q
3r
3t
1
0.214 0.081
0.553 0.048
0.328 0.054
0.031 0.003
0.030 0.002
0.028 0.002
0.028 0.001
1.42 0.33
2.03 0.32
8.90 2.16
0.188 0.059
0.206 0.022
0.321 0.104
0.184 0.019
2.57 0.47
1.09 0.29
2.26 0.084
2.54 0.15
5.02 0.005
0.805 0.241
1.008 0.125
0.511 0.047
1.15 0.27
0.291 0.079
0.141 0.015
0.102 0.011
0.170 0.012
0.054 0.003
a
Inhibition of phosphorylation of STAT5a or STAT1 in PBMCs or HWB. The IL-2 readouts are gated to CD3-expressing T-cells; the GM-CSF and
b
IFN-γ readouts are gated to CD14-expressing monocytes. Mean SEM (standard error of the mean), n ≥ 3 except for 3n HWB value (n = 2).
Table 4. In Vitro and in Vivo Properties of Select Compounds
in vitro
a
b
c
solubility
Caco
protein binding
(% free)
d
compd
(μg/mL)
AB/BA
microsomal stability
3f
55.7
11.3/16.4
11.6
Clint (human) 32.0
Clint (rat) 38.2
3h
3q
<1
17.8/15.3
2.1/24.6
6.6
Clint (human) 27.7
Clint (rat) 50.3
32.7
3.4
Clint (human) 40.1
Clint (rat) 51.5
e,f
in vivo rat (Hanover−Wistar)
compd
t1/2 (h)
Vdss (mL/kg)
1010
Cl (mL/kg min)
7.4
% F
AUC (ng·h/mL)
Cmax (ng/mL)
3f
4.4
24
1090
429
0.5 mg/kg IV
2 mg/kg PO
3h
3q
0.66
3.8
1221
1063
26.1
14.6
33
25
506
200
398
0.5 mg/kg IV
2 mg/kg PO
1195
1 mg/kg IV
5 mg/kg PO
a
b
LYSA (Lyophilized solubility assay). Permeability in measured Caco-2 cells AB (apical to basolateral) and BA (basolateral to apical) movement of
c
10 μM test compound (50 μM test compound for 3q) in 7 days cultured Caco-2 cells (cm/s × 10−6), pH 7.4 on both sides. Equilibrium dialysis,
male and female human plasma. Liver microsomal intrinsic clearance (μL/min/mg protein). Doses were IV (intravenous) and PO (per os); mean
values of 2−3 rats per sample time. Cmax, AUC and % F were determined after the oral dose and Cl, Vdss, and t1/2 were determined from the IV dose.
d
e
f
a
Table 5. Further Cellular Profiling of 3q, 3t, and 1
b
b
PBMC (IL-6) IC50 (μM)
PBMC (IL-6) IC50 (μM)
b
b
b
PBMC (IL-2) IC50 (μM) PBMC (GM-CSF) IC50 (μM) PBMC (IFN-γ) IC50 (μM)
(CD3)
(CD14)
c
3q
3t
1
0.031 0.003
0.028 0.002
0.028 0.001
0.188 0.059
0.321 0.104
0.184 0.019
0.291 0.079
0.102 0.011
0.170 0.012
1.2 0.6
0.864 0.206
c
c
0.255 0.135
0.175 0.065
c
0.38 0.20
0.238 0.07
a
Inhibition of phosphorylation of STAT5a, STAT1, or STAT3 in PBMCs. The IL-2 readouts are gated to CD3-expressing T-cells; the GM-CSF and
IFN-γ readouts are gated to CD14-expressing monocytes. The IL-6 readouts are gated to either CD3-expressing T-cells or to CD14-expressing
b
c
monocytes, as noted. Mean SEM (standard error of the mean), n ≥ 3 except for the IL-6 PBMC data noted with footnote c. n = 2 data.
respect to the balance of properties with high enzyme and cellular
potencies as well as kinome selectivities. Nitrile 3h had poor
solubility, and bis-amide 3q had lower permeability and higher
efflux in our Caco-2 assay (a common issue for the bis-amides);
however, both compounds showed oral bioavailability in rat.
Compound 3q was chosen for further study, as its superior
solubility was likely to be more conducive to increasing exposures
with higher doses in in vivo experiments.
selectivity for inhibition of JAK3 versus JAK1, had one of the
cleanest Caliper selectivity profiles tested for this series, and
appeared suitable for in vivo studies. We chose this compound
for further characterization, including expanded kinome
selectivity profiling. Compound 3q was sent to Kinomescan to
test for compound binding against their entire kinase panel. The
Kinomescan platform measures percent displacement of bait
ligands by test compound. At 1 μM test compound
concentration, compound 3q displaces >90% of ligand for only
41 out of 385 wild-type kinases tested (profiling data is included
in the Supporting Information). Kinomescan also determined
Further Functional Characterization of Compound 3q.
As described above, compound 3q was highly potent in both our
JAK3 enzyme and IL-2 stimulated cellular assays, showed
350
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
Kds for 3q against the kinase domains of JAK3 (Kd 0.12 nM),
JAK2 (Kd 0.18 nM), JAK1 (Kd 12 nM), and Tyk2 (Kd 16 nM). In
these assays, compound 3q shows very strong binding to JAK3
(and JAK2), with high selectivity (100x) against both JAK1 and
Tyk2.
plasma concentrations of 3q for each dose. Compound 3q
prevented IL-2-driven STAT5 phosphorylation in a dose- and
concentration-dependent manner, with approximately 50%
inhibition observed at the 10 mg/kg dose (plasma concentration
∼480 nM). Complete inhibition of IL-2-driven STAT5
phosphorylation was achieved at the 30 mg/kg dose (plasma
concentration ∼2087 nM). Compound 3q also inhibited GM-
CSF-driven STAT5 phosphorylation in a dose- and concen-
tration-dependent manner, but a complete inhibition of STAT5
phosphorylation in blood monocytes was not achieved with the
maximum dose tested of 100 mg/kg (plasma concentration
∼6597 nM). Lastly, when whole blood from mice treated with
compound 3q was stimulated with IL-6 ex vivo, only 15%
inhibition was observed of IL-6-driven pSTAT3 phosphorylation
at the maximum tested dose of 100 mg/kg. Thus, the PK/PD
results clearly show that target engagement can be achieved after
oral administration of compound 3q and that compound 3q is
JAK3 selective in vivo, as judged by higher potency inhibiting
JAK1/JAK3- vs JAK2- or JAK1/JAK2/TYK2-driven signaling in
whole blood assays.
Discussion and Exploratory siRNA Studies. We were able
to optimize a new chemical series, 3-amidopyrrolopyrazines, into
highly potent JAK inhibitors with reasonable kinome selectivity
profiles. Structurally related examples with similar potencies
against JAK3 but differing potencies against JAK1 show similar
cellular potencies in an IL-2 induced STAT5 phosphorylation
assay. Our results suggest that JAK3 plays an essential role in IL-2
induced STAT5 phosphorylation. At first glance, this conclusion
appears to contradict that reached by the Novartis group, which
showed that selective depletion of JAK1 by siRNA in T-cells is
more detrimental to IL-2 signaling than depletion of JAK3.19 We
found similar results in our own siRNA work (see below).
Novartis also published their own JAK3-selective compound,
which shows poor activity in a similar IL-2 dependent cellular
assay. Novartis’s JAK3-selective compound is significantly more
selective than compound 3q against JAK1 at the enzyme level
(based on comparison with Tofacitinib as control) and, unlike
compound 3q, also shows high selectivity against JAK2. The
compound published by the Pharmacopeia group, with which
they concluded that JAK1 inhibition is not required for potent
inhibition of IL-2 signaling, has enzyme selectivities similar to
compound 3q. These observations suggest at least two possible
explanations for compound 3q’s retained potency for inhibiting
IL-2 signaling. One possibility is that 3q’s level of selectivity
against JAK1 is simply not high enough to have a strong
differential effect in this particular cellular context. A second
possibility is that JAK2 may also play a role in IL-2 signaling.27
We performed siRNA knockdown studies on JAK3, JAK2, and
JAK1 in primary T-cells, looking for effects on IL-2 induced
STAT5 phosphorylation (Figure 7). Our findings for JAK1
(strong involvement in STAT5 phosphorylation) and JAK3
(weaker involvement in STAT5 phosphorylation) qualitatively
agree with the findings of the Novartis group. The Novartis
group did not include JAK2 in their published studies; in our
work, we found no effect of JAK2 depletion on IL-2 induced
STAT5 phosphorylation at the siRNA concentrations tested.
Thus, inhibition of JAK2 by compound 3q is unlikely to be the
driver for activity in our IL-2 cellular assays.
We expanded our human PBMC assays panels for compounds
3q, 3t, and Tofacitinib 1 (Table 5). We added two variations
stimulating with IL-6, examining STAT3 phosphorylation in T-
cells and monocytes, which is known to depend on JAK1, JAK2,
and Tyk2. Notably, compound 3q showed a 40-fold selectivity
for inhibition of the IL-2 readout versus the IL-6 readout in T-
cells and a 30-fold selectivity for inhibition versus the IL-6
readout in monocytes. Compounds 3t and Tofacitinib 1 also
showed selectivity for inhibition of the IL-2 readout but to a
lesser degree against IL-6 for either readout. Our conclusion is
that increased functional selectivity to inhibit IL-2 signaling
versus IL-6 signaling is possible with increased enzyme-level
selectivity to inhibit JAK3 versus JAK1.
We also examined the functional effects of compound 3q on
human T-cell response. Compound 3q inhibits the proliferation
of human CD4 and CD8 T cells in a dose-dependent manner
upon stimulation by anti-CD3/anti-CD28 antibody-coated
beads partially mimicking the activation signals brought to a T-
cell by an antigen-presenting cell (see Supporting Information
for details). Thus, compound 3q is active in both mechanistic and
functional cell-based assays using T-cells, one of the major cell
types in which JAK3 is potentially relevant.12
Finally, an acute pharmacokinetic/pharmacodynamic (PK/
PD) mouse model was developed to measure the inhibition of
JAK family dependent signaling in vivo. As a bridging study
between this model and our human cellular assays, we first
evaluated compound 3q in mouse whole blood (MWB) assays
(Table 6). Compound 3q potently inhibits IL-2 stimulated
Table 6. Mouse Whole Blood (MWB) Potencies for Selected
a
Compounds
MWB
MWB (IL-2)
b
(GM-CSF) IC50 MWB (IFN-γ)
MWB (IL-6)
b
b
b
IC50 (μM)
(μM)
IC50 (μM)
IC50 (μM)
3q
3t
1
0.18 (0.02)
0.14 (0.02)
0.05 (0.01)
1.1 (0.24)
0.85 (0.09)
2.04 (0.39)
0.17 (0.03)
0.11 (0.01)
0.18 (0.04)
9.9 (1.0)
2.01 (0.16)
1.72 (0.11)
a
Inhibition of phosphorylation of STAT5a, STAT1 or STAT3 in
b
MWB. Mean (SEM (standard error of the mean)), n ≥ 4.
STAT5 phosphorylation in mouse whole blood (MWB), with
selectivity against GM-CSF and IL-6 stimulated readouts, as
observed in human PBMCs. In our MWB assays, compound 3q
did not show selectivity for inhibiting the IL-2 readout versus the
IFN-γ readout, suggesting a potential for species difference of
IFN-γ signaling in human and mouse systems. For comparison
purposes, we also evaluated compound 3t and Tofacitinib 1 in
the MWB assays. All three compounds showed selectivity for
inhibition of the IL-2 assay vs the IL-6 assay, with the rank order
of selectivity corresponding to the rank order of selectivity for
JAK3 versus JAK1 (i.e., 3q is more selective than 1 and 3t).
In the acute PK/PD mouse model, animals were orally treated
with different doses of compound 3q (0.3 to 100 mg/kg) and
whole blood was collected 2 h later, ex vivo stimulated with IL-2,
GM-CSF, or IL-6, and the appropriate STAT phosphorylation
was measured by flow cytometry. Figure 6 shows response versus
dose curves for the three different stimulations plus the measured
We cannot discount the possibility that the diminished JAK1
inhibitory activity of compound 3q remains a significant driver of
potency against IL-2 signaling. However, our results do confirm
that improved selectivity for IL-2 signaling over IL-6 signaling,
with retention of IL-2 cellular potency, is achievable for JAK3
351
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
Figure 6. Inhibition of STAT phosphorylation in mice treated with compound 3q in an acute PK/PD model.
inhibitors with improved selectivity against JAK1. Importantly,
the cellular potencies and selectivities for our most highly
optimized compound 3q was translated to in vivo potency and
selectivity in an acute PK/PD mouse model. Compound 3q may
therefore be an excellent tool compound for further dissecting
the roles of these different pathways in other in vivo models.
EXPERIMENTAL SECTION
■
Chemistry. Reagents and solvents were obtained from commercial
suppliers and were used without further purification. 1H NMR
measurements were recorded at 300 MHz using a Bruker AMX 300
instrument. Chromatographic purifications were performed with
automated chromatography systems using prepacked silica gel cartidges
supplied by Thomson Instrumental Co. or by TeleDyne Isco Inc.
Melting points recorded were uncorrected. Purity and characterization
of compounds were established by a variety of elemental analysis, LC/
MS, and NMR analytical techniques, and purities were >95% for all final
products.
Figure 7. siRNA depletion studies on JAK1−3 in primary T-cells.
Human primary T cells were transfected with siRNAs targeting JAK1,
JAK2, JAK3, or scramble siRNA (Ctrl RNA) by Amaxa electrophoresis,
and IL-2 induced phosphorylation of STAT5 was determined by flow
cytometric analysis. The intensity of pSTAT5 (%) was normalized to
control scramble siRNA treatment group represented as percentage
(%). The experiment was repeated in three independent experiments on
three different donors. Gene knockdown confirmation studies are
included in the Supporting Information.
2-Bromo-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo-
[2,3-b]pyrazine-7-carbaldehyde (4). To a partial suspension of 2-
bromo-5H-pyrrolo[2,3-b]pyrazine (5.0 g, 25 mmol) in 100 mL of 1,4-
dioxane was added 2 M aqueous sodium hydroxide solution (25 mL, 50
mmol) and 37% aqueous formaldehyde solution (19 mL, 250 mmol).
The dark homogeneous reaction mixture was stirred at 25 °C for 16 h,
and then the organic solvent was removed under reduced pressure. The
352
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
aqueous residue was adjusted to pH 7 with 1 M aqueous hydrochloric
acid and then extracted twice with ethyl acetate. The combined organic
phases were concentrated to afford 2.60 g of (2-bromo-5-hydrox-
ymethyl-pyrrolo[2,3-b]pyrazin-7-yl)-methanol as an orange solid. Upon
standing, a thick brown precipitate formed in the aqueous phase. The
precipitate was collected by filtration and dried. The brown solid was
extracted with three 200 mL portions of hot 10% methanol/ethyl
acetate. The extracts were combined and evaporated to provide an
additional 3.05 g of (2-bromo-5-hydroxymethyl-pyrrolo[2,3-b]pyrazin-
7-yl)-methanol as an orange solid. The overall yield was 5.65 g (87%) of
crude (2-bromo-5-hydroxymethyl-pyrrolo[2,3-b]pyrazin-7-yl)-metha-
then allowed to cool and filtered through a pad of Celite 545, rinsing
with ethyl acetate. The filtrate was concentrated under reduced pressure,
and the residue was purified by silica gel chromatography (10% ethyl
acetate/hexanes) to afford 0.240 g (81%) of 2-cyclopropyl-5-(2-
trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carbalde-
hyde (5) as a yellow powder. 1H NMR (300 MHz, CDCl3) δ ppm 10.38
(s, 1 H), 8.26 (s, 1 H), 8.22 (s, 1 H), 5.68 (s, 2 H), 3.53−3.61 (m, 2 H),
2.26−2.36 (m, 1 H), 1.09−1.24 (m, 4 H), 0.89−0.97 (m, 2 H), −0.04 (s,
9 H). MS (EI/CI) m/z 318 (M + H).
2-Cyclopropyl-5-(2-trimethylsilanyl-ethoxymethyl)-5H-
pyrrolo[2,3-b]pyrazine-7-carboxylic Acid (6). To a mixture of 2-
cyclopropyl-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]-
pyrazine-7-carbaldehyde (0.24 g, 0.75 mmol) in 10 mL of 1,4-dioxane
and 2 mL of water at 0 °C was added sulfamic acid (0.44 g, 4.5 mmol)
and then dropwise a solution of 80% sodium chlorite (0.09 g, 0.8 mmol)
and potassium dihydrogen phosphate (1.22 g, 9.00 mmol) in 6 mL of
water. The reaction mixture was allowed to warm to 25 °C and stirred
for 2 h and then partitioned between water and ethyl acetate. The
organic phase was washed with a saturated aqueous sodium chloride
solution, dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The residue was triturated with hexanes to obtain 0.22
g (87%) of 2-cyclopropyl-5-(2-trimethylsilanyl-ethoxymethyl)-5H-
pyrrolo[2,3-b]pyrazine-7-carboxylic acid as a light-yellow powder (6).
1H NMR (300 MHz, CDCl3) δ ppm 8.37 (s, 1 H), 8.28 (s, 1 H), 5.65−
1
nol, which was carried directly to the next step. H NMR (300 MHz,
DMSO-d6) δ ppm 8.42 (s, 1 H), 7.97 (s, 1 H), 6.91 (br s, 1 H), 5.59 (br s,
2 H), 5.13 (br s, 1 H), 4.65 (br s, 2 H). MS (EI/CI) m/z: 258, 260 (M +
H).
To a suspension of the above-prepared (2-bromo-5-hydroxymethyl-
pyrrolo[2,3-b]pyrazin-7-yl)-methanol (5.65 g, 21.9 mmol) in 150 mL of
tetrahydrofuran was added a solution of 2 M aqueous sodium hydroxide
solution (33 mL, 66 mmol). The mixture was stirred for 16 h, and then
the organic layer was removed under reduced pressure. The aqueous
residue was adjusted to pH 4 with 1 M aqueous hydrochloric acid. The
resulting precipitate was collected via filtration and rinsed with water to
afford 3.68 g of (2-bromo-5H-pyrrolo[2,3-b]pyrazin-7-yl)-methanol as a
yellow solid. The filtrate was extracted twice with ethyl acetate, and the
combined organic phases were concentrated under reduced pressure to
provide an additional 0.92 g of (2-bromo-5H-pyrrolo[2,3-b]pyrazin-7-
yl)-methanol as a yellow solid. The overall yield was 4.60 g (92%) of
crude (2-bromo-5H-pyrrolo[2,3-b]pyrazin-7-yl)-methanol, which was
carried directly to the next step. 1H NMR (300 MHz, DMSO-d6) δ ppm
12.20 (br s, 1 H), 8.35 (s, 1 H), 7.87 (s, 1 H), 4.98 (t, J = 5.3 Hz, 1 H),
4.63 (d, J = 4.9 Hz, 2 H). MS (EI/CI) m/z: 228, 230 (M + H).
A stock solution of Jones reagent (2.67 M) was prepared by carefully
adding concentrated sulfuric acid (2.3 mL) to chromium trioxide (2.67
g) and then diluting with water to a final volume of 10 mL. To a partial
suspension of the above-prepared (2-bromo-5H-pyrrolo[2,3-b]pyrazin-
7-yl)-methanol (4.60 g, 20.1 mmol) in 300 mL of acetone was slowly
added the Jones reagent (9.0 mL, 24 mmol). During the addition, the
starting material gradually dissolved and a thick green precipitate
formed. The reaction mixture was stirred for 15 min and then quenched
with 2 mL of 2-propanol and filtered through Celite 545, rinsing with
acetone. The filtrate was concentrated to provide 4.76 g (“104%”) of
crude 2-bromo-5H-pyrrolo[2,3-b]pyrazine-7-carbaldehyde as a yellow−
orange solid, which was carried directly to the next step. 1H NMR (300
MHz, DMSO-d6) δ ppm 13.36 (br s, 1 H), 10.04 (s, 1 H), 8.80 (s, 1 H),
8.55 (s, 1 H). MS (EI/CI) m/z: 226, 228 (M + H).
5.70 (m, 2 H), 3.52−3.63 (m, 2 H), 2.20−2.32 (m, 1 H), 1.13−1.22 (m,
4 H), 0.88−0.98 (m, 2 H), −0.03 (s, 9 H). MS (EI/CI) m/z 334 (M +
H).
2-Cyclopropyl-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic Acid
[(R)-1-(3-Cyano-azetidine-1-carbonyl)-2,2-dimethyl-propyl]-
amide (3q). A solution of [(R)-1-(3-cyano-azetidine-1-carbonyl)-2,2-
dimethyl-propyl]-carbamic acid tert-butyl ester (see preparation below)
(0.093 g, 0.32 mmol) in 3 mL of dichloromethane at 0−5 °C was treated
with 1 mL of trifluoroacetic acid. The reaction solution was allowed to
warm to 25 °C and stirred for 3 h and then concentrated to afford crude
(R)-1-(3-cyano-azetidine-1-carbonyl)-2,2-dimethyl-propyl-ammonium
trifluoroacetate as a colorless glass, which was used directly in the next
step.
A solution of 2-cyclopropyl-5-(2-trimethylsilanyl-ethoxymethyl)-5H-
pyrrolo[2,3-b]pyrazine-7-carboxylic acid (0.070 g, 0.21 mmol) in 2 mL
of acetonitrile was treated with O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate (0.081 g, 0.25 mmol), the
above-prepared crude (R)-1-(3-cyano-azetidine-1-carbonyl)-2,2-di-
methyl-propyl-ammonium trifluoroacetate, and N,N-diisopropylethyl-
amine (0.13 mL, 0.74 mmol). The reaction mixture was stirred at 25 °C
for 16 h and then was diluted with water and extracted with ethyl acetate.
The organic phase was washed with a saturated aqueous sodium chloride
solution, dried over sodium sulfate, filtered, and concentrated. The
residue was purified by silica gel chromatography (0−5% methanol/
dichloromethane) to afford 0.092 g (85%) of 2-cyclopropyl-5-(2-
trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic
acid [(R)-1-(3-cyano-azetidine-1-carbonyl)-2,2-dimethyl-propyl]-
To a solution of the above-prepared crude 2-bromo-5H-pyrrolo[2,3-
b]pyrazine-7-carbaldehyde (4.7 g) in 50 mL of N,N-dimethylformamide
at 0 °C was added sodium hydride (60% in mineral oil, 1.2 g, 30 mmol).
The reaction mixture was stirred at 25 °C for 30 min and then cooled to
0 °C. The mixture was then treated with 2-(trimethylsilyl)ethoxymethyl
chloride (4.3 mL, 24 mmol). The reaction mixture was allowed to warm
to 25 °C, stirred for 1 h, and then quenched with water and extracted
three times with ethyl acetate. The combined organics were sequentially
washed with three portions of water and a saturated aqueous sodium
chloride solution and then dried over magnesium sulfate, filtered, and
concentrated. The residue was purified by silica gel chromatography
(20−30% ethyl acetate/hexanes) to afford 3.82 g (51%; 42% over four
steps) of 2-bromo-5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo-
1
amide as a colorless semisolid. H NMR (300 MHz, CDCl3) δ ppm
8.93 (d, J = 9.1 Hz, 1 H), 8.30 (s, 1 H), 8.17 (s, 1 H), 5.59−5.68 (m, 2 H),
4.74−4.97 (m, 1 H), 4.16−4.58 (m, 4 H), 3.45−3.58 (m, 3 H), 2.16−
2.26 (m, 1 H), 1.32−1.41 (m, 1 H), 1.07−1.20 (m, 12 H), 0.86−0.95
(m, 2 H), −0.09 to −0.02 (m, 9 H). MS (EI/CI) m/z 511 (M + H).
A solution of the above-prepared 2-cyclopropyl-5-(2-trimethylsilanyl-
ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-carboxylic acid [(R)-1-(3-
cyano-azetidine-1-carbonyl)-2,2-dimethyl-propyl]-amide (0.092 g, 0.18
mmol) in 2 mL of dichloromethane was treated with 0.6 mL of
trifluoroacetic acid. The reaction solution was stirred at 25 °C for 2 h and
was then neutralized by addition of a saturated aqueous sodium
bicarbonate solution. The resultant mixture was extracted three times
with ethyl acetate, and the combined organic phases were washed with
saturated sodium chloride solution, dried over sodium sulfate, filtered,
and concentrated. The residue was redissolved in 8 mL of ethanol, and
the resulting solution was treated with sodium acetate (0.30 g, 3.6
mmol). The mixture was stirred at 50 °C for 20 h, allowed to cool, and
then treated with water and ethyl acetate. Layers were separated, the
aqueous phase was extracted twice with ethyl acetate, and the combined
1
[2,3-b]pyrazine-7-carbaldehyde (4) as a yellow solid. H NMR (300
MHz, DMSO-d6) δ ppm 10.07 (d, J = 0.8 Hz, 1 H), 8.97 (s, 1 H), 8.65 (s,
1 H), 5.71 (s, 2 H), 3.53−3.61 (m, 2 H), 0.79−0.88 (m, 2 H), −0.09 (s, 9
H). MS (EI/CI) m/z: 356, 358 (M + H).
2-Cyclopropyl-5-(2-trimethylsilanyl-ethoxymethyl)-5H-
pyrrolo[2,3-b]pyrazine-7-carbaldehyde (5). A mixture of 2-bromo-
5-(2-trimethylsilanyl-ethoxymethyl)-5H-pyrrolo[2,3-b]pyrazine-7-car-
baldehyde (0.330 g, 0.930 mmol), cyclopropylboronic acid (0.120 g,
1.40 mmol), tricyclohexylphosphine (0.026 g, 0.09 mmol), palladium-
(II) acetate (0.010 g, 0.046 mmol), potassium phosphate tribasic (0.630
g, 2.97 mmol), 4 mL of toluene, and 0.5 mL of water was flushed with
argon for 5 min. The reaction mixture was stirred at 100 °C for 18 h and
353
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
organic layers were washed with a saturated aqueous sodium chloride
solution, dried over sodium sulfate, filtered, and concentrated. The
residue was purified by silica gel chromatography (0−5% methanol/
dichloromethane) to afford 0.052 g (75%) of 2-cyclopropyl-5H-
pyrrolo[2,3-b]pyrazine-7-carboxylic acid [(R)-1-(3-cyano-azetidine-1-
carbonyl)-2,2-dimethyl-propyl]-amide as an off-white solid; mp 181−
183 °C. 1H NMR (300 MHz, DMSO-d6) δ ppm 12.63 (br s, 1 H), 8.63−
8.73 (m, 1 H), 8.40−8.44 (m, 1 H), 8.26−8.34 (m, 1 H), 4.41−4.68 (m,
2 H), 4.26−4.36 (m, 1 H), 3.98−4.24 (m, 2 H), 3.74−3.91 (m, 1 H),
2.26−2.39 (m, 1 H), 1.14−1.26 (m, 1 H), 1.04 (s, 11 H), 0.92−0.99 (m,
1 H). MS (EI/CI) m/z 381 (M + H). Analysis Calculated for
C20H24N6O2·(0.16H2O): C, 62.67; H, 6.39; N, 21.92. Found: C, 62.65;
H, 6.39; N, 21.63. [α]28D (c = 0.5, methanol) −149.6 o.
Use Committee (IACUC), and performed according to guidelines
established by the Guide for the Care and Use of Laboratory Animals by
National Research Council and by Association for the Assessment and
Accreditation of Laboratory Animal Care International (AAALAC), and
American Veterinary Medical Association Guidelines on Euthanasia
(2007). Animals used for the present studies were adult female C57BL/
6 mice (Jackson Laboratory, Bar Harbor, Maine) and female Wistar/
Han rats (Charles River, Hollister, CA).
Inhibition of STAT Phosphorylation in Mouse Whole Blood (MWB).
Whole blood from healthy C57BL/6 mice (n = 20) was collected in
heparinized tubes and pooled then incubated with compound 3q at
different concentrations (0.003 μM to 100 μM) for 30 min at 37 °C.
Blood cells were stimulated for 20 min at 37 °C with recombinant mouse
IL-2, GM-CSF or IL-6 (eBioscience) at EC90 concentration, which was
previously determined using that respective cytokine batch. Red blood
cells were then lysed with 1X Lyse/Fix solution (BD Bioscience) for 20
min at room temperature. Cells were pelleted, resuspended in Phosflow
Perm Buffer III (BD Bioscience), and left on ice for 30 min. Cells were
washed twice using 1× Perm/Wash buffer then resuspended in a volume
of 100 μL prior to staining with fluorochrome-conjugated antimouse
antibodies (BD Bioscience): staining A = anti-TCRβ PE, anti-CD4
FITC, anti-CD25 PerCP-Cy5.5, and anti-pSTAT5 AF647; staining B =
anti-CD11b FITC, anti-TCRβ PE, anti-B220 PerCP-Cy5, and anti-
pSTAT5 AF647; staining C = anti-CD11b FITC, anti-TCRβ PE, anti-
B220 PerCP-Cy5, and anti-pSTAT3 AF647. Flow cytometry sample
acquisition and data analysis were performed using an LSRII instrument
and Flowjo software, respectively. IC50 values were generated using
ActivityBase software.
Microsomal Stability. Microsome preparations prepared from male
and female rat or human livers were obtained from BD Biosciences
(Woburn, MA). Test compounds were diluted from 10 mM stock
solutions in DMSO; incubations contained <1% residual DMSO.
Incubations were performed in 96-well deep-well plates with a final
incubation volume of 600 μL. Incubations contained 1−2 μM test
compound, 0.5 mg/mL liver microsomes, and NADPH regenerating
system. Aliquots of 50 μL were removed at time points from 1 to 45 min,
quenched in acetonitrile containing internal standard, and cooled prior
to centrifugation and analysis by LC-MS/MS. The natural log of the
ratio of the test compound peak area to the internal standard peak area
was plotted against incubation time, and the slope of a linear fit of the
initial rate of compound disappearance was used to calculate the intrinsic
clearance.
[(R)-1-(3-Cyano-azetidine-1-carbonyl)-2,2-dimethyl-propyl]-
carbamic Acid tert-Butyl Ester. A solution of N-tert-butoxycarbonyl-
D-tert-leucine (0.500 g, 2.16 mmol), O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate (0.83 g, 2.6 mmol), and 3-
cyanoazetidinium chloride (0.270 g, 2.28 mmol) in 22 mL of
dichloromethane was treated with N,N-diisopropylethylamine (0.94
mL, 5.4 mmol), and the reaction mixture was stirred at 25 °C for 16 h.
The mixture was then treated with water and extracted twice with ethyl
acetate. The combined organic layers were washed with a saturated
aqueous sodium chloride solution, dried over sodium sulfate, filtered,
and concentrated. The residue was purified by silica gel chromatography
(20% ethyl acetate/hexanes) to afford 0.39 g (61%) of [(R)-1-(3-cyano-
azetidine-1-carbonyl)-2,2-dimethyl-propyl]-carbamic acid tert-butyl
1
ester as a white solid. H NMR (300 MHz, DMSO-d6) δ ppm 6.75−
6.89 (m, 1 H), 4.28−4.57 (m, 2 H), 3.93−4.21 (m, 2 H), 3.67−3.88 (m,
2 H), 1.31−1.45 (m, 9 H), 0.91 (d, J = 3.3 Hz, 9 H). MS (EI/CI) m/z
318 (M + Na).
Biological Methods. JAK1−3 Kinase Inhibition. The ability of
compounds to inhibit Janus kinase enzymes 1, 2, or 3 was determined by
a kinase filtration assay. JAK1 was purchased from Invitrogen and JAK2
and 3 proteins were prepared in house. Briefly, 6 nM JAK1 (866−1154
amino acids), 1 nM JAK2 (JAK2-SF21c 828−1132), or 0.1 nM of JAK3
enzyme (810−1124 amino acids) were preincubated with compound at
room temperature for 10 min in 50 mM HEPES buffer and 10 mM
magnesium chloride (MgCl2) that was supplemented with 1 mM
dithiothreitol (DTT) and 1 mg/mL bovine serum albumin (BSA). The
kinase reactions for all of the enzymes were initiated by the addition of a
substrate cocktail that contained 1.5 μM adenosine triphosphate (ATP),
0.025 μCi ATP-γ-P33, and 0.2 mM of biotinylated synthetic peptide
(Biomer Technology: Biotin-KAIETDKEYYTVKD) at a final volume
of 40 μL. The reaction was carried out at room temperature for 30 min
for JAK2 and JAK3, and for 1 h for JAK1. Reactions were quenched by
transferring 25 μL to a filter plate (Millipore MABVN0B50, 1.2 μM pore
size) containing 100 μL of prepared 10% streptavidin beads in 1× PBS
(Mg and Ca2+ free) and 100 mM ethylene diamine tetra-acetic acid
(EDTA). The bead/terminated solution was mixed and incubated for 30
min at room temperature. Using a vacuum manifold, plates were washed
three times with 240 μL of 2 M aqueous sodium chloride solution, three
times with 2 M aqueous sodium chloride +1% phosphoric acid solution,
and twice with water. The plates were then dried in a 60 °C oven for 1 h.
Finally, 70 μL of Microscint-20 was added and sealed. Resulting P33
incorporation was determined by counting the plate in a Packard
TopCount.
Inhibition of STAT Phosphorylation in Peripheral Blood Mono-
nuclear Cells (PBMCs) or Human Whole Blood (HWB). PBMCs or
HWB isolated from healthy volunteers were incubated with compounds
for 30 min at 37 °C. The PBMCs or HWB were then treated with either
100 ng/mL IL-2 or 20 pg/mL GM-CSF or 100 ng/mL IFNγ or 100 ng/
mL IL-6, and the PBMCs or HWB were incubated for an additional 20
min at 37 °C. PBMCs or HWB were lysed and/or fixed and
permeabilized (perm buffer III, BD Bioscience) and then stained with
anti-CD3 FITC and anti-CD14 PE and either anti-pSTAT5 or anti-
pSTAT1 or anti-pSTAT3. Flow cytometry analysis was performed using
an LSRII instrument (BD Bioscience). Data analysis was done using
Flowjo software. IC50 values were generated using ActivityBase software.
Experimental Animals. All procedures using animals and in vivo
procedures were approved by Roche’s Institutional Animal Care and
Clint (μL/min/mg = −slope (min−1) × 1000
)
/[protein concentration (mg/mL)]
Pharmacokinetics. Male Wistar/Han (CRL:WI) rats (Charles River
Laboratories, Hollister, CA or Raleigh, NC) used in these studies
weighed between 180 and 300 g and were surgically implanted with
cannulas in the jugular and/or femoral veins. All studies were conducted
under IACUC approved protocols, and animals were allowed free access
to food and water. Single intravenous bolus doses were administered
into the tail vein or into the jugular cannula of dual-cannulated animals;
the remaining cannulas were used for blood collection. Single oral doses
were administered by gavage. Blood was collected into tubes containing
EDTA at typically 0.08 (IV only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after
dosing. Plasma concentrations were determined by a liquid chromatog-
raphy/tandem mass spectrometry method (LC/MS/MS) following
protein precipitation with acetonitrile.
In Vivo Mouse PK/PD Model. Adult (10−12 weeks old) C57BL/6
mice were randomized into experimental groups (n = 5/group)
including control vehicle group and compound 3q treated groups (0.3,
1, 3, 10, 30, and 100 mg/kg). According to their respective group, mice
received a single oral suspension dose of compound 3q prepared in
aqueous vehicle or vehicle alone containing 0.9% NaCl, 0.5% sodium
carboxymethyl cellulose, 0.4% polysorbate 80, and 0.9% benzyl alcohol.
Two hours after treatment by oral gavage, mice were euthanized with
CO2 asphyxiation for collecting whole blood in heparinized tubes.
Whole blood from the respective mice was then ex vivo stimulated with
IL-2, GM-CSF, or IL-6 at EC90 concentration. The subsequent
354
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
experimental steps (from red blood cell lysis step to flow cytometry
analysis) were similar to that used in the MWB assay, as previously
described in this manuscript.
Notes
The authors declare no competing financial interest.
Human T Cell siRNA Transfection and Activation Assay. Primary
human T cells were enriched from PBMC by using Pan T Cell Isolation
Kit II (Miltenyi Biotec GmbH, Germany) and resuspended in PRMI-
1640 culture medium supplemented with 10% FBS (Invitrogen,
Carlsbad, CA). T cells were transfected with siRNAs targeting JAK1,
JAK2, JAK3, or scramble control siRNA (Invitrogen) with Amaxa P3
Primary Cell 4D-Nucleofector X kit (Lonza Cologne GmbH, Germany)
according to manufacturer’s protocol using program “FI-115” in 4D-
Necleofector (Lonza). Transfected cells were transferred into 12-well
plate and allowed 48 h of incubation at 37 °C followed by stimulation
with IL2 (100 ng/mL, R&D Systems) for 20 min. Cells were fixed with
1.5% formaldehyde for 10 min at room temperature and then
permeabilized with BD Phosflow Perm Buffer III at 4 °C overnight.
Fixed and permeabilized cells were washed with FACS staining buffer
(PBS with 2% FBS) twice, stained with FITC antihuman CD3 and Alexa
Fluor 647 anti-STAT5 (pY694), and quantitated for pSTAT5
fluorescence intensity gated on CD3+ T cell population.
REFERENCES
■
(1) Takeshita, T.; Asao, H.; Ohtani, K.; Ishii, N.; Kumaki, S.; Tanaka,
N.; Munakata, H.; Nakamura, M.; Sugamura, K. Cloning of the γ chain
of the human IL-2 receptor. Science 1992, 257 (5068), 379−382.
(2) Rochman, Y.; Spolski, R.; Leonard, W. J. New insights into the
regulation of T cells by γc family cytokines. Nature Rev. Immunol. 2009, 9
(7), 480−490.
(3) Macchi, P.; Villa, A.; Giliani, S.; Sacco, M. G.; Frattini, A.; Porta, F.;
Ugazio, A. G.; Johnston, J. A.; Candotti, F.; O’Shea, J. J.; Vezzoni, P.;
Notarangelo, L. D. Mutations of Jak-3 gene in patients with autosomal
severe combined immune deficiency (SCID). Nature 1995, 377 (6544),
65−68.
(4) Russell, S. M.; Tayebi, N.; Nakajima, H.; Riedy, M. C.; Roberts, J.
L.; Aman, M. J.; Migone, T. S.; Noguchi, M.; Markert, M. L.; Buckley, R.
H.; O’Shea, J. J.; Leonard, W. J. Mutation of Jak3 in a patient with SCID:
essential role of Jak3 in lymphoid development. Science 1995, 270
(5237), 797−800.
Protein Crystallography. JAK3 kinase domain (amino acids 811−
1124, C1040S/C1048S) at 10−15 mg/mL in 50 mM HEPES (pH 8.0),
0.5 M NaCl, 20% glycerol, 5 mM DTT, 0.05% n-octyl-β-D-glucoside was
incubated on ice for 1 h with 50 mM DTT, 0.75 mM inhibitor in DMSO,
and 0.26% phenylurea (originally identified from Hampton Research
Silver Bullet F10, later also described by others24). For crystallization by
vapor diffusion in hanging drops at 20 °C, 700 nL of JAK3−inhibitor
complex solution was mixed with 300 nL of 24−34% PEG 3350, 0.1 M
MES (pH 6.0), 0.2 M MgCl2, and 150 nL of crystal seed solution.
Crystals typically appeared overnight as rods or bipyramidal prisms.
They were cryoprotected by gradually adding glycerol to 20% and flash-
cooled in liquid nitrogen. Synchrotron data collection was provided by
Reciprocal Space Consulting, LLC (Oakland, California) at beamline
9−2 of the Stanford Synchrotron Radiation Laboratory (Palo Alto,
California) and beamline 5.0.2 of the Advanced Light Source (Berkeley,
California) using ADSC Quantum 315 CCD detectors. The diffraction
images were processed with DENZO, and the intensities were scaled
with SCALEPACK.28 The structure of the first JAK3−ligand complex
was solved with the molecular replacement program PHASER29 using
the crystal structure of JAK3 kinase domain in complex with a
staurosporine analogue30 (PDB accession no. 1YVJ) as search model.
The solution found was a JAK3 monomer in space group P21212 with
the unit cell dimensions α = 47 Å, β = 76 Å and γ = 90 Å. The same
crystal form has also been obtained for a similar JAK3 kinase domain
variant.24 The model was refined against the experimental data, and
electron density maps were calculated using REFMAC5.31 Subsequent
JAK3−ligand structures were determined by rigid body refinement. The
structure models were built with the graphics software COOT.32
(5) Noguchi, M.; Yi, H.; Rosenblatt, H. M.; Filipovich, A. H.; Adelstein,
S.; Modi, W. S.; McBride, O. W.; Leonard, W. J. Interleukin-2 receptor
gamma chain mutation results in X-linked severe combined
immunodeficiency in humans. Cell 1993, 73 (1), 147−157.
(6) Witthuhn, B. A.; Quelle, F. W.; Silvennoinen, O.; Yi, T.; Tang, B.;
Miura, O.; Ihle, J. N. JAK2 associates with the erythropoietin receptor
and is tyrosine phosphorylated and activated following stimulation with
erythropoietin. Cell 1993, 74 (2), 227−236.
(7) Quelle, F. W.; Sato, N.; Witthuhn, B. A.; Inhorn, R. C.; Eder, M.;
Miyajima, A.; Griffin, J. D.; Ihle, J. N. JAK2 associates with the beta c
chain of the receptor for granulocyte-macrophage colony-stimulating
factor, and its activation requires the membrane-proximal region. Mol.
Cell. Biol. 1994, 14 (7), 4335−4341.
(8) Lutticken, C.; Wegenka, U. M.; Yuan, J.; Buschmann, J.; Schindler,
̈
C.; Ziemiecki, A.; Harpur, A. G.; Wilks, A. F.; Yasukawa, K.; Taga, T.;
et al. Association of transcription factor APRF and protein kinase Jak1
with the interleukin-6 signal transducer gp130. Science 1994, 263
(5143), 89−92.
(9) Narazaki, M.; Witthuhn, B. A.; Yoshida, K.; Silvennoinen, O.;
Yasukawa, K.; Ihle, J. N.; Kishimoto, T.; Taga, T. Activation of JAK2
kinase mediated by the interleukin 6 signal transducer gp130. Proc. Natl.
Acad. Sci. U. S. A. 1994, 91 (6), 2285−2289.
(10) Guschin, D.; Rogers, N.; Briscoe, J.; Witthuhn, B.; Watling, D.;
Horn, F.; Pellegrini, S.; Yasukawa, K.; Heinrich, P.; Stark, G. R.; Ihle, J.
N.; Kerr, I. M. A major role for the protein tyrosine kinase JAK1 in the
JAK/STAT signal transduction pathway in response to interleukin-6.
EMBO J. 1995, 14 (7), 1421−1429.
(11) Ghoreschi, K.; Laurence, A.; O’Shea, J. J. Janus kinases in immune
cell signaling. Immunol Rev. 2009, 228 (1), 273−287.
ASSOCIATED CONTENT
* Supporting Information
■
(12) Pesu, M.; Laurence, A.; Kishore, N.; Zwillich, S. H.; Chan, G.;
O’Shea, J. J. Therapeutic targeting of Janus kinases. Immunol Rev. 2008,
223, 132−142.
S
Experimental procedures and analytical data for final compounds
3a−3p and 3r−3t, Kinomescan selectivity data for compound
3q, CD4 and CD8 cell proliferation studies, response versus
plasma concentration curves for the mouse acute PK/PD model
studies, and gene knockdown confirmation studies for the siRNA
study. This material is available free of charge via the Internet at
http://pubs.acs.org.
(13) Vijayakrishnan, L.; Venkataramanan, R.; Gulati, P. Treating
inflammation with the Janus kinase inhibitor CP-690,550. Trends
Pharmacol. Sci. 2011, 32 (1), 25−34.
(14) Tefferi, A.; Pardanani, A. JAK inhibitors in myeloproliferative
neoplasms: rationale, current data and perspective. Blood Rev. 2011, 25
(5), 229−237.
́
(15) Quintas-Cardama, A.; Kantarjian, H.; Cortes, J.; Verstovsek, S.
Accession Codes
Janus kinase inhibitors for the treatment of myeloproliferative neoplasias
and beyond. Nature Rev. Drug Discovery 2011, 10 (2), 127−140.
(16) Kremer, J. M.; Bloom, B. J.; Breedveld, F. C.; Coombs, J. H.;
Fletcher, M. P.; Gruben, D.; Krishnaswami, S.; Burgos-Vargas, R.;
Wilkinson, B.; Zerbini, C. A.; Zwillich, S. H. The safety and efficacy of a
JAK inhibitor in patients with active rheumatoid arthritis: Results of a
double-blind, placebo-controlled phase IIa trial of three dosage levels of
CP-690,550 versus placebo. Arthritis Rheum. 2009, 60 (7), 1895−1905.
(17) Fleischmann, R.; Cutolo, M.; Genovese, M. C.; Lee, E. B.; Kanik,
K. S.; Sadis, S.; Connell, C. A.; Gruben, D.; Krishnaswami, S.;
Protein crystal structure coordinates and structure factors are
deposited in the RCSB Protein Data Bank with the accession
numbers 4HVD, 4HVG, 4HVH, and 4HVI.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 973-235-2717. Fax: 973-235-6263. E-mail: michael.
soth@roche.com.
355
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356

Journal of Medicinal Chemistry
Article
Wallenstein, G.; Wilkinson, B. E.l; Zwillich, S. H. Phase IIb dose-ranging
study of the oral JAK inhibitor tofacitinib (CP-690,550) or adalimumab
monotherapy versus placebo in patients with active rheumatoid arthritis
with an inadequate response to disease-modifying antirheumatic drugs.
Arthritis Rheum. 2012, 64 (3), 617−629.
(31) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.
(32) Emsley, P.; Cowtan, K. Coot: Model-building tools for molecular
graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 2126−
2132.
(18) Thoma, G.; Nuninger, F.; Falchetto, R.; Hermes, E.; Tavares, G.
A.; Vangrevelinghe, E.; Zerwes, H.-G. Identification of a potent Janus
kinase 3 inhibitor with high selectivity within the Janus kinase family. J.
Med. Chem. 2011, 54 (1), 284−288.
(19) Haan, C.; Rolvering, C.; Raulf, F.; Kapp, M.; Druckes, P.; Thoma,
̈
G.; Behrmann, I.; Zerwes, H.-G. Jak1 has a dominant role over Jak3 in
signal transduction through γc-containing cytokine receptors. Chem.
Biol. 2011, 18 (3), 314−323.
(20) Lin, T. H.; Hegen, M.; Quadros, E.; Nickerson-Nutter, C. L.;
Appell, K. C.; Cole, A. G.; Shao, Y.; Tam, S.; Ohlmeyer, M.; Wang, B.;
Goodwin, D. G.; Kimble, E. F.; Quintero, J.; Gao, M.; Symanowicz, P.;
Wrocklage, C.; Lussier, J.; Schelling, S. H.; Hewet, A. G.; Xuan, D.;
Krykbaev, R.; Togias, J.; Xu, X.; Harrison, R.; Mansour, T.; Collins, M.;
Clark, J. D.; Webb, M. L.; Seidl, K. J. Selective functional inhibition of
JAK-3 is sufficient for efficacy in collagen-induced arthritis in mice.
Arthritis Rheum. 2010, 62 (8), 2283−2293.
(21) Goldstein., D. M.; Gray, N. S.; Zarrinkar, P. P. High-throughput
kinase profiling as a platform for drug discovery. Nature Rev. Drug
Discovery 2008, 7 (5), 391−397.
(22) Fabian, M. A.; Biggs, I. I. I.; William, H.; Treiber, D. K.; Atteridge,
C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.;
Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld,
R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.;
Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H.
K.; Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase interaction
map for clinical kinase inhibitors. Nature Biotechnol. 2005, 23 (3), 329−
336.
(23) Out of 293 wild-type kinases tested at Kinomescan, compound 2
at 10 μM showed >90% bait ligand displacement for 156 kinases,
including JAK1−3 (kinase domains) at the percentages indicated: JAK1
(100%), JAK2 (99%), and JAK3 (98%).
(24) Chrencik, J. E.; Patny, A.; Leung, I. K.; Korniski, B.; Emmons, T.
L.; Hall, T.; Weinberg, R. A.; Gormley, J. A.; Williams, J. M.; Day, J. E.;
Hirsch, J. L.; Kiefer, J. R.; Leone, J. W.; Fischer, H. D.; Sommers, C. D.;
Huang, H.-C.; Jacobsen, E. J.; Tenbrink, R. E.; Tomasselli, A. G.;
Benson, T. E. Structural and thermodynamic characterization of the
TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-
6. J. Mol. Biol. 2010, 400 (3), 413−433.
(25) Flanagan, M. E.; Blumenkopf, T. A.; Brissette, W. H.; Brown, M.
F.; Casavant, J. M.; Chang, S.-P.; Doty, J. L.; Elliott, E. A.; Fisher, M. B.;
Hines, M.; Kent, C.; Kudlacz, E. M.; Lillie, B. M.; Magnuson, K. S.;
McCurdy, S. P.; Munchhof, M. J.; Perry, B. D.; Sawyer, P. S.; Strelevitz,
T. J.; Subramanyam, C.; Sun, J.; Whipple, D. A.; Changelian, P. S.
Discovery of CP-690,550: a potent and selective Janus kinase (JAK)
inhibitor for the treatment of autoimmune diseases and organ transplant
rejection. J. Med. Chem. 2010, 53 (24), 8468−8484.
(26) Molecular Operating Environment (MOE), 2011.10; Chemical
Computing Group Inc.: 1010 Sherbooke St. West, Suite #910, Montreal,
QC, Canada, H3A 2R7, 2011.
(27) Tanaka, N.; Asao, H.; Ohbo, K.; Ishii, N.; Takeshita, T.;
Nakamura, M.; Sasaki, H.; Sugamura, K. Physical association of Jak1 and
Jak2 tyrosine kinases with the interleukin 2 receptor β and γ chains. Proc.
Natl. Acad. Sci U. S. A. 1994, 91, 7271−7275.
(28) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. In Macromolecular Crystallography, Part A;
Carter, C. W. J., Sweet, R. M., Eds.; Academic Press: New York, 1997; pp
307−326.
(29) McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J.
Likelihood-enhanced fast translation functions. Acta Crystallogr., Sect. D:
Biol. Crystallogr. 2005, 61, 458−464.
(30) Boggon, T. J.; Li, Y.; Manley, P. W.; Eck, M. J. Blood 2005, 106,
996−1002.
356
dx.doi.org/10.1021/jm301646k | J. Med. Chem. 2013, 56, 345−356