Identification of 2-Imidazopyridine and 2-Aminopyridone Purinones as Potent Pan-Janus Kinase (JAK) Inhibitors for the Inhaled Treatment of Respiratory Diseases

Article abstract of DOI:10.1021/acs.jmedchem.9b00533

Janus kinases (JAKs) have a key role in regulating the expression and function of relevant inflammatory cytokines involved in asthma and chronic obstructive pulmonary disease. Herein are described the design, synthesis, and pharmacological evaluation of a series of novel purinone JAK inhibitors with profiles suitable for inhaled administration. Replacement of the imidazopyridine hinge binding motif present in the initial compounds of this series with a pyridone ring resulted in the mitigation of cell cytotoxicity. Further systematic structure-activity relationship (SAR) efforts driven by structural biology studies led to the discovery of pyridone 34, a potent pan-JAK inhibitor with good selectivity, long lung retention time, low oral bioavailability, and proven efficacy in the lipopolysaccharide-induced rat model of airway inflammation by the inhaled route.

Full text of DOI:10.1021/acs.jmedchem.9b00533

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Identification of 2‑Imidazopyridine and 2‑Aminopyridone Purinones
as Potent Pan-Janus Kinase (JAK) Inhibitors for the Inhaled
Treatment of Respiratory Diseases
†
†
†
†,#
‡
Jordi Bach,*,† Paul Eastwood, Jacob Gonzalez, Elena Gomez, Juan Antonio Alonso,
́
́
Silvia Fonquerna,† Estrella Lozoya,† Adela Orellana,†,∇ Monica Maldonado, Elena Calaf,
†
́
Joan Albertí,‡,○ Juan Perez, Ana Andres,
Neus Prats,∥,¶ Cristina Carren
̃
o,⊥ Elena Calama,⊥
§
∥,◆
́
́
Jorge De Alba,⊥,⋈ Marta Calbet,⊥ Montserrat Miralpeix,⊥,⧓ and Isabel Ramis⊥,⧖
‡
§
∥
†Medicinal Chemistry & Screening, Pharmacokinetics & Metabolism, Early Galenic Development, Pathology & Predictive
⊥
̀
́
Toxicology, and Respiratory Therapeutic Group, Almirall R&D, Crta. Laurea Miro 408-410, Sant Feliu de Llobregat, 08980
Barcelona, Spain
S
* Supporting Information
ABSTRACT: Janus kinases (JAKs) have a key role in regulating the expression and function of relevant inflammatory cytokines
involved in asthma and chronic obstructive pulmonary disease. Herein are described the design, synthesis, and pharmacological
evaluation of a series of novel purinone JAK inhibitors with profiles suitable for inhaled administration. Replacement of the
imidazopyridine hinge binding motif present in the initial compounds of this series with a pyridone ring resulted in the
mitigation of cell cytotoxicity. Further systematic structure−activity relationship (SAR) efforts driven by structural biology
studies led to the discovery of pyridone 34, a potent pan-JAK inhibitor with good selectivity, long lung retention time, low oral
bioavailability, and proven efficacy in the lipopolysaccharide-induced rat model of airway inflammation by the inhaled route.
respiratory diseases such as asthma and chronic obstructive
pulmonary disease (COPD) has been far less explored. To the
best of our knowledge, there are only three inhaled JAK
inhibitors in preclinical studies: R256 from AstraZeneca/Rigel,
a JAK1/3 inhibitor for moderate to severe asthma,11,12 and two
pan-JAK inhibitors from Pfizer (PF-06263276)13 and Vectura
(VR588)14 for COPD and severe asthma, respectively.
Most of the proinflammatory cytokines that play a critical
role as orchestrators of the persistent inflammation observed in
asthma and COPD such as interleukins IL-2, IL-3, IL-4, IL-5,
IL-6, IL-9, IL-12, IL-13, IL-22, and IL-23, granulocyte-
macrophage colony-stimulating factor (GM-CSF), and inter-
feron γ signal through JAKs (JAK1, 2, and 3).15−17 Therefore,
a pan-JAK inhibitor blocking multiple cytokine signaling has
the potential to be a broad anti-inflammatory therapy for the
treatment of these respiratory diseases. Substantial exper-
imental evidence supports a role for JAK−STAT signaling in
airway inflammation. In several animal models of allergic
asthma and COPD, it has been shown that different JAK
inhibitors are efficacious reducing the airway inflammation by
INTRODUCTION
■
Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases
associated with cytokine receptors. Following cytokine
stimulation, JAKs phosphorylate signal transducers and
activators of transcription (STAT), a family of transcription
factors that dimerize, translocate to the nucleus, and activate
gene transcription of inflammatory mediators. Four JAKs
[namely, JAK1, 2, and 3, and tyrosine kinase 2 (Tyk2)] and
seven STATs have been identified in the JAK−STAT pathway
where different cytokine receptors are associated with specific
combinations of paired JAKs (i.e., JAK1 and JAK3; JAK2 and
JAK2) and STAT proteins.1−7
As JAKs are implicated in cell signaling processes that have a
role in cancer as well as autoimmune and inflammatory
diseases, enormous efforts have been devoted to the develop-
ment of JAK inhibitors. As a consequence, several JAK
inhibitors have progressed into clinical trials for the oral and
topical treatment of myeloproliferative and autoimmune
diseases and three of them, ruxolitinib (Incyte/Novartis),
tofacitinib (Pfizer), and baricitinib (Incyte/Lilly), have reached
the market for the oral treatment of myelofibrosis and
rheumatoid arthritis.8−10 On the other hand, the potential
use of JAK inhibitors for the treatment of inflammatory
Received: March 27, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 1. Novel Purinone JAK Inhibitor 2 Bearing an Imidazo[1,2-a]pyridine Ring as Hinge Binder Motif
both the oral and inhaled routes.11,12,14,18−21 Moreover, in
human studies, it has been reported that overexpression of
pSTAT3 in COPD lung tissue relates to the degree of airway
inflammation and that total STAT-6 expression is increased in
the bronchial epithelium of asthma patients compared to
controls.16,22−24
benzimidazole moiety would play a key role for the binding of
1 into the adenosine triphosphate (ATP) binding site of JAK
enzymes. This structural motif would act as hinge binder
through two interactions with two specific amino acids from
the hinge region that are conserved among the different
members of the JAK family: a classical hydrogen bond between
the benzimidazole nitrogen and backbone NH of a leucine and
a noncanonical interaction between a heteroaromatic hydrogen
atom and the oxygen of the carbonyl group of a glutamic acid
(Scheme 1). The presence of electron-withdrawing groups
(i.e., a fluorine atom) attached to the benzimidazole core was
contemplated as a way to improve the nonclassical interaction
by increasing heteroaromatic H atom acidity. The chromane
ring of 1 was postulated to fill a lipophilic pocket in the active
site, whereas the purinone core would act as an optimal spacer
keeping the appropriate distance between the hinge and
lipophilic pocket moieties. The purinone ring might also have
additional hydrophobic interactions with alkylic residues from
amino acids located at the binding site. Nevertheless, the
presence of the benzimidazole hinge binder was perceived as a
concern for the chemical (and metabolic) stability of this
compound as it could potentially act as leaving group via initial
attack of a nucleophile on the purinone ring. Changing the
hinge binder motif to an imidazo[1,2-a]pyridine ring as in
compound 2 would prevent such potential instability.29−31
Initial hit 2 showed a similar profile of JAK inhibition to
benzimidazole 1, with potency against JAK2 and JAK3 in the
low nanomolar range and somewhat less potent against JAK1
(IC50 = 24 nM), demonstrating that the proposed hinge binder
replacement was well tolerated in terms of JAK activities.
Unfortunately, compound 2 also proved to be cytotoxic in the
cytotoxicity assay performed in Chinese hamster ovary (CHO)
cells [IC50 (CHO) = 4 μM]. Based on these findings, two main
goals were initially defined to improve the overall profile of 2:
(1) to increase JAK1 potency to identify truly pan-JAK
inhibitors and (2) to reduce cytotoxicity to the desired level for
a clinical candidate [IC50 (CHO) > 30 μM].
Over the past few years, we have been interested in the
development of inhaled pan-JAK inhibitors for the treatment
of respiratory diseases with the main goal of developing a novel
anti-inflammatory therapy with a favorable efficacy/safety ratio.
Pan-JAK inhibition may have an additive effect on anti-
inflammatory activity compared to JAK1/3 selective inhibitors
as several relevant inflammatory mediators signal through
JAK2 (i.e., IL-3, IL-5, and GM-CSF). On the other hand,
systemic inhibition of JAK2 is associated with adverse effects
such as thrombocytopenia, anemia, and hemoglobin reduction.
In this sense, we selected the inhaled route of administration to
avoid systemic exposure and limit the reported adverse effects
observed in humans with oral JAK inhibitors.25 Moreover, this
strategy was supported by the fact that the majority of the
target cells of JAK inhibitors are at the site of inflammation and
that we and others have demonstrated that administration of
JAK inhibitors by the inhaled route is also efficacious in animal
models of asthma and COPD.11,14,20 As a result, our efforts
have been devoted to the search for pan-JAK inhibitors with a
suitable profile for inhaled administration. It has been reported
that physicochemical properties such as basicity, polarity, and/
or aqueous solubility can be used to modulate retention times
in the lung and assist in finding the right balance between lung
and systemic exposure.26 In this context, many inhaled drugs
have a long duration period in the lung due to their low
aqueous solubility, which causes inhaled particles to slowly
dissolve and be absorbed into the lung tissue.
Following a low dissolution rate strategy for lung retention,
in this paper, we describe the design, synthesis, and
pharmacological evaluation of a series of novel purinone JAK
inhibitors bearing an imidazopyridine or pyridone moiety as
hinge binder. This work has resulted in the identification of
pyridone 34, a potent pan-JAK inhibitor with a long lung
retention time, low oral bioavailability, and proven efficacy in
the lipopolysaccharide (LPS)-induced rat model of airway
inflammation by the inhaled route.
Expansion toward the solvent-exposed region was first
explored to improve JAK1 potency by targeting new
interactions with amino acids in this area. Docking studies
performed with 2 led to the identification of glutamic acid 966
(Glu966) in JAK1 as an ideal target amino acid due to its
proximity to the imidazolone ring and potential for forming
ionic and hydrogen-bond interactions through the carboxylic
acid side chain. As the equivalent amino acid in both JAK2 and
JAK3 is aspartic acid (Asp939 and Asp912 respectively), this
approach could also be employed to improve JAK2 and JAK3
potencies as well. Docking studies also suggested that Glu966
RESULTS AND DISCUSSION
■
The design of an imidazo[1,2-a]pyridine ring as hinge binder
was inspired by PS-020613 (1), an early JAK3 inhibitor from
Pharmacopeia (now Ligand Pharma) reported in the
literature.27 Docking studies with JAK328 suggested that the
B
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Imidazopyridine Analogues: Solvent-Exposed Region SAR
could be easily accessed through functionalization of the
imidazolone NH, with a two-carbon chain as optimal spacer
for the introduction of heteroatoms to reach the targeted
interactions with the carboxylic acid. To test the hypothesis, a
simple tetrahydropyran ring was used as a lipophilic pocket
filling group and several functionalities with potential for
forming hydrogen-bond interactions were introduced onto the
imidazolone core, as shown in Table 1. In this initial
structure−activity relationship (SAR) study, replacement of
the fluorine atom of the hinge binder moiety by a more bulky
electron-withdrawing cyano group was also evaluated.
Unsubstituted analogues 3 and 4 were first synthesized
revealing that replacement of fluorine by a nitrile group had no
impact on JAK inhibition as both molecules showed a similar
profile of moderate JAK potencies. Interestingly, the
introduction of a methyl group onto the imidazolone core
(compound 5) was well tolerated in terms of JAK2 and JAK3
potencies but resulted in a dramatic loss of JAK1 potency with
respect to unsubstituted compound 3. This loss of JAK1
potency was partially recovered while maintaining JAK2 and
JAK3 potencies when a hydroxyethyl side chain was
introduced (compounds 6 and 7). JAK1 potency was lost
again when 6 was converted into the corresponding methyl
ether 8, suggesting a role for the hydroxyl group in JAK1
inhibition. Further modifications of 6 such as introduction of
an additional hydroxyl group (compounds 9 and 10) or
replacement of the primary alcohol by a tertiary alcohol
(compound 11) failed to improve JAK potencies. The scope of
solvent-exposed groups was then expanded to basic side chains,
but, unfortunately, tertiary amines 12 and 13 showed poor
inhibitory potency against JAK2 and JAK3 and were inactive
against JAK1. In summary, imidazolone functionalization to
target new interactions with solvent-exposed amino acids
(JAK1 Glu966, JAK2 Asp939, and JAK3 Asp912) failed to
improve potency against JAK2 and JAK3 and was detrimental
to JAK1 potency.
C
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. X-ray crystallographic structure of human JAK3 binding site in complex with 6 determined at 2.5 Å resolution [Protein Data Bank (PDB)
entry 6HZV]. For clarity, only interacting amino acids are shown.
Inhibition of STAT-6 phosphorylation induced by IL-4 in
human Ramos cells was used to assess JAK1/3 cellular potency
of selected compounds from this series. Although both
molecules were equipotent in JAK enzymatic assays, fluoro
derivative 4 proved to be 3-fold more potent than the
corresponding cyano analogue 3 (IC50 = 350 and 1100 nM,
respectively) in this cellular assay.
removal of the aromatic ring of 2 to yield 4 resulted in an
important loss of JAK potencies and extrusion of the oxygen
atom of the tetrahydropyran (THP) ring improved potency
(compound 15), alcohol 17 was designed and synthesized, and
proved to be equipotent to 2. Keeping in mind that the co-
crystal structure of JAK3 with 6 unveiled that the
tetrahydropyran oxygen is not involved in any key interactions
with amino acids in the lipophilic pocket, a series of more
flexible monocyclic compounds were explored (compounds
18−21). Phenylethyl analogues 18 and 19 highlighted the
importance of the absolute configuration of the chiral center
on JAK potencies as 19, having the same (R)-configuration as
chromane 2, proved to be at least 100-fold more potent against
all JAKs than its enantiomer 18. Results obtained with 19, a
molecule with a similar JAK inhibition profile to 2, confirmed
that the chromane oxygen is not essential for JAK potency.
Encouraged by these results, an exhaustive SAR exploration of
the aromatic ring was performed (data not shown) leading to
the identification of pyridine 20 and pyrimidine 21 as both
potent and balanced pan-JAK inhibitors. Both compounds
fulfilled the defined goal in terms of JAK potencies, but
cytotoxicity observed with these two molecules prevented
them from further progression.
Since all potent analogues synthesized up to this point
proved to be cytotoxic [IC50 (CHO) < 10 μM], further SAR
expansion around the lipophilic pocket was not perceived as an
approach that would solve this issue. Varying the hinge binder
motif was thus explored as a possible way to mitigate
cytotoxicity. In this context, cytotoxicity could be overcome
without dramatically affecting JAK potencies when a more
polar aminopyridone scaffold was introduced as a hinge
binding group. This scaffold was conceptualized as a truncated
analogue of the imidazopyridine ring that would keep key
interactions with amino acids Leu and Glu in the hinge region
through two classical hydrogen bonds, as shown in Scheme 2.
The pyridone carbonyl group could act as hydrogen-bond
acceptor with the backbone NH of leucine, whereas the
pyridone NH could act as hydrogen-bond donor with the
backbone carbonyl of the glutamic acid.
To understand the results obtained, compound 6 was
selected for structural biology studies. At this time point in the
project, it was not feasible to obtain the X-ray crystallographic
structure of human JAK1 with 6, but we were successful in
solving the co-crystal structure of this inhibitor with JAK3 at a
resolution of 2.5 Å (Figure 1). The postulated binding mode
was confirmed for this series of inhibitors with the
tetrahydropyran ring occupying the lipophilic pocket and the
imidazopyridine moiety acting as hinge binder through a
classical hydrogen bond with backbone NH of Leu905 and a
noncanonical interaction with the backbone carbonyl oxygen
of Glu903. As expected, the purinone core acts as a spacer
between the hinge and lipophilic pocket moieties and has no
hydrogen-bond interactions with the enzyme. The co-crystal
structure also confirmed the targeted interaction between the
hydroxylated side chain of 6 and the carboxylic acid chain of
solvent-exposed Asp912. Moreover, the hydroxyl group of 6
also showed an additional hydrogen-bond interaction with the
backbone NH of cysteine 909. Despite these two additional
interactions with solvent-exposed amino acids, no increase in
JAK3 potency compared to unsubstituted compound 3 was
seen. Regarding JAK1, it seems feasible that the targeted
interaction with Glu966 could be achieved with molecules
bearing a hydroxyl group (compounds 6, 7, and 9−11 in Table
1), but the dramatic loss of potency observed in molecules
devoid of hydrogen-bond donors (compounds 5, 8, 12, and
13) could not be explained at this point.
A more successful approach to improve JAK1 potency was
based on expanding the SAR in the lipophilic pocket (Table
2). For this SAR expansion exercise, 6-fluoroimidazopyridine
was fixed as hinge binder due to the higher cellular potency
observed for fluoro derivative 4 compared to cyano analogue 3.
Initial efforts were focused on modifying the tetrahydropyran
ring of 4 to improve JAK activities. Although dimethyl
substitution (compound 14) and extrusion of the oxygen
atom to give secondary and tertiary alcohols 15 and 16
improved on results obtained with 4, the JAK potency profile
of chromane 2 was not recovered. Taking into account that
Analogues of potent imidazopyridine derivatives 2, 17, and
20 bearing an unsubstituted aminopyridone hinge binder were
first synthesized (compounds 22, 27, and 28) revealing a
remarkable improvement in cytotoxicity for all cases, as shown
in Table 3. Compared to the corresponding imidazopyridine
analogues, chromane 22 proved to be equipotent, whereas a
D
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
introduction of small groups at the 5-position of the pyridone
ring was explored. Regarding chromane derivatives, an
improvement in JAK enzymatic and cellular potencies while
keeping cytotoxicity at the desired level was achieved with the
incorporation of electron-withdrawing groups such as halogen
atoms (compounds 23 and 24) at this position. On the other
hand, an important decrease of JAK1 potency was observed for
the methyl and methoxy derivatives 25 and 26. A similar effect
on JAK potencies was observed when halogen atoms were
introduced at the pyridone hinge binder of molecules bearing
the chiral fluoropyridine instead of the chromane ring
(compounds 29 and 30). For both lipophilic pocket groups,
the presence of a chlorine atom in the 5-position of the
pyridone ring provided the best results in terms of cellular
potency in the JAK1/3 IL-4/STAT-6 Ramos assay. At this
point, SAR studies varying the lipophilic pocket moiety were
expanded taking advantage of reported co-crystal structures of
tofacitinib with the four members of the JAK family.32,33 In this
sense, compounds 31 and 34 were designed to position a
cyano group toward the glycine-rich loop region, mimicking
tofacitinib’s cyanoacetamide (Figure 2). Although both
molecules proved to be potent and noncytotoxic pan-JAK
inhibitors, they exhibited a different behavior when tested in
the cellular assay. While cyclohexane derivative 34 was the
most potent compound identified in this series, chiral
piperidine 31 suffered from poor cellular potency. Dramatic
differences in permeability observed for these two compounds
in the parallel artificial membrane permeability (PAMPA)
assay (Papp = 0.03 × 10−6 cm/s for 31; 0.61 × 10−6 cm/s for
34) likely account for the difference seen in the cellular assay.
The corresponding enantiomer of 31 was synthesized
(compound 33), which resulted in a drop of potency in all
JAKs. With the aim of increasing cellular potency, a chlorine
atom was now introduced onto the pyridone ring of 31 and 34.
Unfortunately, chlorinated compounds 32 and 35 failed to
improve the overall profile of JAK enzymatic and cellular
potencies compared to the corresponding unsubstituted
analogues.
Table 2. Imidazopyridine Analogues: Lipophilic Pocket SAR
Among the most potent pyridones identified, compound 34
was selected for structural biology studies with JAK1.
Compound 34 was successfully co-crystallized with human
JAK1 at a 2.5 Å resolution (Figure 3), and the relevant
structural information obtained from these studies provided a
better understanding of JAK1 SAR. The expected binding
mode for this family of inhibitors was confirmed with the
pyridone ring acting as a hinge binding motif through two
classical hydrogen-bond interactions with the backbone
carbonyl group of Glu957 and the backbone NH of Leu959.
Interestingly, the co-crystal structure unveiled an unexpected
hydrogen-bonding interaction between the imidazolone NH
and the carboxylic acid side chain of solvent-exposed Glu966,
the amino acid that was selected as a target for possible
interactions in early SAR studies (vide supra). This additional
interaction would be lost in molecules where the imidazolone
ring is substituted (e.g., methyl derivative 5), resulting in the
observed loss of JAK1 potency, which can be partially
recovered in compounds that bear a hydrogen-bond donor
as part of the solvent-exposed group. As expected, the cyano
group present in 34 does not have any additional hydrogen-
bond interaction but is filling a cavity in the lipophilic pocket
region, in proximity to the glycine loop.
decrease in JAK potencies was observed for the other two
molecules: moderate for alcohol 27, more pronounced for
fluoropyridine 28. The impact of pyridone substitution on JAK
potencies was then evaluated. Based on docking studies,
Due to a balanced profile of JAK enzymatic potencies and
remarkable cellular potency in the JAK1/3 assay, compound
E
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 2. Novel Purinone JAK Inhibitors Bearing an Aminopyridone Ring as Hinge Binder
34 was selected for further profiling. Regarding additional
enzymatic assays, 34 was found to be less potent against Tyk2
(IC50 = 130 nM) and showed promising selectivity in a panel
of 36 human kinases, which included targets commonly
inhibited by JAK inhibitors (Figure 4).34 In this study,
performed at Millipore, only Aurora-A, FGFR1, Flt4, Ret,
and TrkA were inhibited >75% at a compound concentration
of 10 μM and were identified as potential off-target activities.
JAK2/2 cellular activity of 34 was assessed by measuring the
inhibition of phosphorylation of STAT-5 in monocytes
isolated from human peripheral blood mononuclear cells
stimulated with GM-CSF and resulted in a 5-fold drop of
potency compared to JAK1/3 cellular activity (IC50 = 219
nM). In the search for nonkinase off-target activities,
compound 34 showed weak activity against potassium
(hERG), sodium (Nav1.5), and calcium (Cav1.2) ion channels
(less than 15% inhibition when tested at a 10 μM
concentration), indicating a low potential to cause cardiovas-
cular side effects. In addition, 34 also proved to be clean when
tested at a 10 μM concentration against a panel of 55 diverse G
protein-coupled receptors, transporters, and ion channels
(Cerep ExpresSProfile). Furthermore, 34 was not genotoxic
in exploratory Ames and GreenScreen HC testing.35
In terms of physicochemical properties, compound 34 has
moderate lipophilicity (Log D7.4 = 2.1) and excellent chemical
stability at all pHs tested (pH = 1.2, 5.5, 7.4, and 9.0). Despite
its poor solubility at physiological pH (pH=7.4; 0 mg/mL), 34
showed sufficient solubility in various formulations used for in
vivo testing and its high melting point (319−332 °C) allows
this molecule to be micronized for potential use in a dry
powder inhaler.
The absorption, distribution, metabolism, and excretion
properties of 34 were also investigated and are summarized in
Table 4. In vitro, 34 showed moderate permeability in the
PAMPA assay (Papp = 0.61 × 10−6 cm/s), moderate plasma
protein binding (PPB, 79% in rat; 83% in human), and low
metabolism in liver microsomes (17% rat; 21% human). To
evaluate the suitability of 34 for inhaled delivery in the in vivo
animal model, both oral and intratracheal (it) pharmacokinetic
(PK) studies were performed in rat. Interestingly, a low oral
bioavailability was observed for 34 as very low levels of
compound were found in plasma after per os (po)
administration of a 30 mg/kg dose (AUCinf = 32 ng h/mL;
Cmax = 7 ng/mL). Moreover, 34 was well retained in the lung
when administered intratracheally as a nanosuspension at a
0.41 mg/kg dose (t1/2 = 16.3 h) and showed an excellent lung/
plasma partition (11 364) ideal for inhaled administration
(Figure 5).
Due to a favorable pharmacokinetic profile for inhaled
administration, compound 34 was further progressed to an
acute model of inhaled lipopolysaccharide (LPS)-induced
airway inflammation in rats. In this model, rats were exposed to
an aerosol of LPS that induces an increase of neutrophils in
bronchoalveolar lavage fluid (BALF) that peaks after 4 h of
LPS challenge. Further study of BALF shows an increase of
several cytokines that signal through the JAK−STAT pathway,
such as IL-6.21 Compound 34 was administered intratracheally
as an aerosolized nanosuspension, using phosphate-buffered
saline containing 0.2% Tween 80 and 0.3% methylcellulose as
vehicle, at doses of 0.01, 0.1, 1, and 3 mg/kg 1 h before rats
were exposed to the LPS challenge. Neutrophils were counted
and IL-6 levels were measured in BALF samples collected 4 h
after LPS exposure. Two additional groups of rats were also
challenged with aerosolized LPS: one was administered only
with vehicle to determine the maximum effect of LPS challenge
on neutrophil count, whereas the other was dosed intra-
tracheally with fluticasone (0.1 mg/kg) and used as positive
control. Compound 34 exhibited a dose-dependent inhibition
of LPS-induced neutrophilia with a maximum effect at the 3
mg/kg dose (56% inhibition, Figure 6). At the same dose, a
significant reduction of IL-6 levels in BALF (62%) was
observed. Intratracheal pharmacokinetic studies performed
with 34 at the efficacious dose of 3 mg/kg confirmed the high
lung/plasma partition observed in previous studies (AUClung
14 557 μg h/g, AUCplasma: 406 ng h/mL, AUClung/AUCplasma
:
:
35 856), with lung levels of compound well above and
unbound plasma levels [Cmax (plasma): 57 ng/mL] below
the cellular IC50 up to 24 h post administration, suggesting that
the observed efficacy is due to a local inhibition of JAK kinases
in the lung. The results obtained with 34 in the LPS-induced
airway inflammation model support the potential use of
inhaled JAK inhibitors for the treatment of respiratory
inflammatory diseases.
CHEMISTRY
■
Initial imidazopyridine compounds 2−4 were synthesized as
shown in Scheme 3. A regioselective nucleophilic aromatic
substitution (SNAr) reaction between the corresponding
amines and activated dichloropyrimidine 36 furnished
compounds 37 and 38. Catalytic hydrogenation of the nitro
group in the presence of zinc(II) bromide led to dianilines 39
and 40, which were treated with carbonyldiimidazole (CDI) to
form purinone intermediates 41 and 42, respectively. After 2-
(trimethylsilyl)ethoxymethyl (SEM) protection of the imida-
zolone NH, hinge binder motifs were introduced in one step
from commercial imidazopyridines 45 and 46 through a
palladium-catalyzed heteroaryl Heck reaction using tetrakis-
F
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Compounds synthesized to target new interactions in the
solvent-exposed region were prepared from common inter-
mediate 42, as described in Scheme 4. Compounds 5 and 11−
13 were obtained through alkylation of the imidazolone NH of
42 with the corresponding halo derivatives followed by a
heteroaryl Heck reaction to introduce the cyanoimidazopyr-
idine hinge binder moiety. The same sequence was used to
synthesize alcohols 6 and 7 and diols 9 and 10. For these
molecules, a final deprotection step under acidic conditions
was needed as both the alcohol and diol functionalities were
introduced protected as tetrahydropyranyl ethers and iso-
propylidene acetals, respectively. In the particular case of
compound 8, this molecule was synthesized in one step by
alkylation of 3 with 1-bromo-2-methoxyethane.
Table 3. Aminopyridones: Hinge Binder and Lipophilic
Pocket SAR
Imidazopyridines synthesized to explore lipophilic pocket
SAR were prepared following an alternative synthetic approach
based on intermediate 63 (Scheme 5). This approach would
allow variation of groups that would ultimately reside in the
lipophilic pocket at the last steps of the synthesis. Treatment of
aminopyridine 57 with 3-methoxyacrylonitrile 58 in the
presence of N-bromosuccinimide (NBS) furnished cyanoimi-
dazopyridine 59, which was converted into amidine 60 by
reaction with catalytic sodium methoxide in methanol followed
by addition of ammonium chloride. Cyclization of 60 with
nitroacrylate 61 provided pyridone 62, which was transformed
into chloropyrimidine 63 by treatment with phosphorus
oxychloride.
To explore SAR in the lipophilic pocket, a diverse set of
groups were introduced by SNAr reaction of the corresponding
amines with 63, as shown in Scheme 6. Nitro reduction with
tin(II) chloride in ethanol or by catalytic hydrogenation
followed by cyclization with carbonyldiimidazole yielded
compounds 14, 16, and 18−21.
Following the same synthetic approach, tert-butyldiphenyl-
silyl (TBDPS)-protected amino alcohols 76 and 80 were used
for the synthesis of compounds 15 and 17 (Scheme 7). After
nitro reduction and carbonyldiimidazole cyclization, the
TBDPS-protecting group was removed by treatment with
TBAF or cesium fluoride to yield alcohols 15 and 17,
respectively.
The majority of purinones bearing an aminopyridone hinge
binder motif were prepared as described in Scheme 8. SNAr
reaction of the corresponding amines with activated pyrimidine
36 furnished 37 and 84−85. Elaboration of these inter-
mediates to the corresponding SEM-protected purinones 43
and 90−91 proceeded using the same methods as described
above (Scheme 3). In the key step of these syntheses, a
palladium-catalyzed Buchwald−Hartwig amination reaction of
anilines 92−96 with the SEM-protected chloropurinones
yielded advanced intermediates 97−105. Finally, SEM
deprotection of these molecules by treatment with either
TBAF or trifluoroacetic acid followed by deprotection of the
methoxy groups by reaction with trimethylsilyl chloride
(TMSCl) and sodium iodide at 80 °C provided compounds
22−26, 28−30, and 35.
The synthesis of aminopyridines 93−96, which are key
intermediates utilized in the synthesis of pyridones 22−35
along with commercially available 2-methoxy-3-aminopyridine
92, is outlined in Scheme 9. The reaction of chloropyridines
115 and 116 with sodium methoxide followed by reduction of
the nitro group via catalytic hydrogenation afforded amino-
pyridines 94 and 95, where the R group is a chloro atom and a
methyl group, respectively. Oxidation of commercially
(triphenylphosphine)palladium(0) as catalyst and potassium
acetate as base at 150 °C. Finally, SEM removal using tetra-n-
butylammonium fluoride (TBAF) or under aqueous acidic
conditions afforded compounds 2−4.
G
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Overlapping studies with compounds 31 (pink), 34 (blue), and tofacitinib (green) showing similar arrangement of nitrile group in all of
the cases and also carbonyl disposition of amides in 31 and tofacitinib.
Figure 3. X-ray crystallographic structure of human JAK1 binding site complexed with compound 34 determined at 2.2 Å resolution (PDB entry
6HZU). For clarity, only interacting amino acids are shown.
Figure 4. Heat map showing the kinase inhibition profile of 34 dosed at 10 μM against a panel of 38 kinases at [ATP] = Km. Colors correspond to
% inhibition of a given kinase as shown in the legend.
Table 4. In Vitro and in Vivo Profiles of Compound 34
in vitro
% PPB
in vivo
assay
value
PAMPA
Papp = 0.61 × 10−6 cm/s
% metabolism (LM) po PK (rat), 30 mg/kg
it PK (rat), 0.41 mg/kg nanosuspension
79 (R)
83 (H)
17 (R)
21 (H)
AUCinf = 32 ng h/mL
Cmax = 7 ng/mL
t1/2 = 16.3 h; AUClung = 1341 μg h/g
AUCplasma = 118 ng h/mL; AUClung/AUCplasma = 11 364
available boronate 117 with hydrogen peroxide resulted in
hydroxypyridine 118, which was alkylated with methyl iodide
to yield dimethoxypyridine 124. Reduction of the nitro group
of 124 by catalytic hydrogenation as described above provided
aminopyridine 96. For the particular case where R is a fluorine
atom, nitration of hydroxypyridine 119 yielded nitro
intermediate 120. Selective O-alkylation of 120 was achieved
using methyl iodide as alkylating agent and silver carbonate as
base at room temperature to give 121, which was converted to
aminopyridine 93 as described above.
from commercial amino alcohol 125 (Scheme 10). The amino
group of 125 was selectively protected by reaction with 1 equiv
of di-tert-butyl dicarbonate, and the resultant alcohol 126 was
converted into tosylate 127 by reaction with p-toluenesulfonyl
chloride. The reaction of 127 with sodium cyanide followed by
removal of the tert-butyloxycarbonyl (Boc) group under acidic
conditions afforded aminonitrile 129.
Aminopyridones 31−34 were prepared following an
alternative synthetic approach in which the lipophilic pocket
group was introduced after the hinge binder moiety, as
illustrated in Scheme 11. In this approach, pyrimidine 36 was
first converted into thiocyanate 130 to reverse pyrimidine
Aminonitrile 129, a key intermediate for the synthesis of
compounds 34 and 35, was synthesized in four steps starting
H
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
bioisosteric hinge binder replacement was well tolerated in
terms of JAK potencies giving rise to 2, a compound with
potencies against JAK2 and JAK3 in the low nanomolar range
but 20-fold less potent against JAK1. In our search for a potent
pan-JAK inhibitor, systematic SAR efforts driven by structural
biology studies were performed to improve the JAK1 potency
of 2. Initial studies which attempted to target new interactions
with the solvent-exposed amino acid Glu966 in JAK1 were
unsuccessful. On the other hand, compounds with adequate
JAK potencies were identified by varying the groups in the
lipophilic pocket, but unfortunately, the cytotoxicity observed
with these molecules prevented them for further progression.
This cytotoxicity could be overcome without dramatically
affecting JAK potencies by the introduction of an amino-
pyridone ring as the hinge binding motif. Further, SAR
expansion in the lipophilic pocket region led to the discovery
of pyridone 34, a potent pan-JAK inhibitor with good
selectivity, long lung retention time, low systemic exposure,
and proven efficacy in the LPS model of airway inflammation
by the intratracheal route. Furthermore, 34 was successfully co-
crystallized with JAK1, confirming the expected binding mode
for this series of inhibitors and providing a better under-
standing of JAK1 SAR. Pyridone 34 underwent further
characterization to assess the potential as a preclinical
development candidate.
Figure 5. Pharmacokinetic profile of 34 after intratracheal
administration as a nanosuspension.
reactivity and direct hinge binder introduction to the 2-
position. SNAr reaction of aminopyridines 92 and 94 with
pyrimidine 130 at 0 °C proceeded with moderate yields but
excellent regioselectivity to the desired thiocyanates 131 and
132. The reaction of these thiocyanates with the corresponding
amines yielded intermediates 133−136. Nitro reduction by
catalytic hydrogenation followed by cyclization with carbon-
yldiimidazole afforded purinones 141−144, in which both the
hinge binder and the lipophilic moieties have been introduced
by SNAr reactions. For purinones 141−143, where the
purinone N-substituent is Boc-protected piperidine, a one-
pot, double deprotection was performed under acidic
conditions to give piperidines 145−147, which were further
converted into cyanoacetamides 31−33 by reaction with 2,5-
dioxopyrrolidin-1-yl 2-cyanoacetamide. For the particular case
of purinone 144, with a cyclohexylacetonitrile N-substituent,
methoxy deprotection under standard conditions provided
compound 34.
Synthesis of compound 27 is outlined in Scheme 12. The
reaction of pyrimidine 36 with amine 80 provided the desired
regioisomer 148, which was elaborated as detailed above to
provide compound 153. The SEM and TBDPS groups were
simultaneously removed by treatment of 153 with TBAF in
tetrahydrofuran, and subsequent deprotection of the benzyl
group by catalytic hydrogenation yielded the desired alcohol
27.
EXPERIMENTAL SECTION
■
General Experimental. The care of animals was undertaken in
compliance with the European Committee Directive 2010/63/EU
and the Spanish and autonomous Catalan laws (Real Decreto 53/
2013 and Decret 214/1997). Experimental procedures were reviewed
by the Animal Welfare Body of Almirall and approved by the
competent authority.
All test compounds have purity >95%. The purity of compounds
was determined by high-performance liquid chromatography (HPLC)
or ultraperformance liquid chromatography (UPLC) analysis. HPLC
analyses were performed using a Waters 2795 system equipped with a
Symmetry C-18 (2.1 × 100 mm, 3.5 μM) column. The mobile phases
were (B): formic acid (0.4 mL), ammonia (0.1 mL), methanol (500
mL), and acetonitrile (500 mL) and (A): formic acid (0.5 mL),
ammonia (0.125 mL), and water (1000 mL). A gradient between 0
and 95% of B was used. The flow rate was 0.4 mL/min. The run time
was 30 min. The injection volume was 5 μL. A Waters 2996 diode
array was used as a UV detector. Chromatograms were processed at
210 nm. Mass spectra of the chromatograms were acquired using
positive and negative electrospray ionization (ESI) in a Waters ZQ
detector coupled to the HPLC. UPLC analyses were performed using
a Waters Acquity UPLC system coupled to an SQD mass
spectrometer detector. The system was equipped with an ACQUITY
UPLC BEH C-18 (2.1 × 50 mm, 1.7 mm) column. The mobile
CONCLUSIONS
■
Herein are described the discovery and progression of a series
of novel purinone pan-JAK inhibitors with the potential for
treatment of respiratory diseases by the inhaled route.
Replacement of the benzimidazole in Pharmacopeia’s JAK3
inhibitor PS-020613 by an imidazo[1,2-a]pyridine ring was
designed as a way to improve potential instability issues. This
Figure 6. Effects of intratracheal administration of compound 34 on (A) neutrophil count in BALF; n = 14−16/group and (B) IL-6 levels in BALF;
n = 9−12/group, in the rat LPS challenge model. *** P < 0.001, ** P < 0.01 treated vs LPS group, analysis of variance + Dunnett’s test.
I
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of Initial Imidazopyridine Compounds 2−4
a
Reagents and conditions: (a) R−NH2, N,N-diisopropylethylamine (DIEA), CH2Cl2, −78 °C to room temperature (rt), 91−95%; (b) H2, Pd/C,
ZnBr2, EtOAc, rt, 97−99%; (c) CDI, THF or CH3CN, 80 °C, 65−80%; (d) NaH, SEM−Cl, dimethylformamide (DMF), 0 °C to rt, 86−90%; (e)
Pd(PPh3)4, KOAc, dimethylacetamide (DMA), 150 °C, 35−68%; (f) TBAF, CH2Cl2, rt to 70 °C or HCl 4 N, 70 °C, 32−83%.
a
Scheme 4. Imidazopyridines: Synthesis of N-Alkylated Purinones
a
Reagents and conditions: (a) MeI, NaH, DMF, 0 °C or R−Cl, K2CO3, DMF, rt to 90 °C, 45−88%; (b) 46, Pd(PPh3)4, KOAc, DMA, 150 °C,
11−40%; (c) 1-bromo-2-methoxyethane, K2CO3, DMF, rt, 60%; (d) 2-(2-bromoethoxy)tetrahydro-2H-pyran, K2CO3, DMF, 90 °C, 90%; (e) 45
or 46, Pd(PPh3)4, KOAc, DMA, 150 °C then 2 N HCl, rt, 14−77%; (f) (R)- or (S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane, K2CO3, DMF, 90
°C, 52−67%; (g) 46, Pd(PPh3)4, KOAc, DMA, 150 °C then 4 N HCl, rt to 70 °C, 43−47%.
phases were (B): formic acid (0.4 mL), ammonia (0.1 mL), methanol
(500 mL), and acetonitrile (500 mL) and (A): formic acid (0.5 mL),
ammonia (0.125 mL), and water (1000 mL). A gradient between 5
and 95% of B was used. The flow rate was 0.65 mL/min. The run time
was 5 min. The injection volume was 0.5 μL. Chromatograms were
processed at 210 nm. Mass spectra of the chromatograms were
acquired using positive and negative electrospray ionization. Reaction
products were purified, when necessary, by flash chromatography on
silica gel (40−63 μm) with the solvent system indicated. Purifications
in reverse phase were made in a Biotage SP1 automated purification
system equipped with a C-18 column and using a standard gradient of
water−acetonitrile/MeOH (1:1) (0.1% v/v ammonium formate both
phases) from 0 to 100% acetonitrile/MeOH (1:1) in 80 column
volumes. Other conditions are stated explicitly in the text. Preparative
HPLC−mass spectrometry (MS) was performed on a Waters
instrument equipped with a 2767 injector/collector, a 2525 binary
gradient pump, a 2996 PDA detector, a 515 pump as a make-up
pump, and a ZQ4000 mass spectrometer detector. 1H nuclear
magnetic resonance (NMR) spectra were recorded on a Gemini 200
MHz, Gemini-2000 300 MHz, Varian Mercury plus 400 MHz, or an
Agilent VNMRS-DD2 600 MHz spectrometer. Tetramethylsilane was
used as reference. Chemical shifts δ are in parts per million (ppm);
J
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 5. Imidazopyridines: Synthesis of Common Intermediate 63
a
Reagents and conditions: (a) NBS, dioxane, H2O, 0−60 °C, 79%; (b) NaOMe, NH4Cl, MeOH, reflux, 90%; (c) Et3N, EtOH, 90 °C, 60%; (d)
POCl3, 90 °C, 57%.
a
Scheme 6. Imidazopyridines: Synthesis of 9-Substituted Purinones
a
Reagents and conditions: (a) R−NH2, DIEA, THF, rt, 72−93%; (b) SnCl2, EtOH, 80 °C or H2, Pd/C, EtOH, 51−97%; (c) CDI, CH3CN, 80 °C,
30−74%.
a
Scheme 7. Imidazopyridines: Synthesis of Alcohols 15 and 17
a
Reagents and conditions: (a) DIEA, THF, rt, 81−87%; (b) H2, Pd/C, EtOH, 75−82%; (c) CDI, CH3CN, 80 °C, 67−91%; (d) TBAF, THF, rt,
55%; (e) CsF, DMF, 60 °C, 47%.
the following abbreviations are used: singlet (s), doublet (d), triplet
(t), quartet (q), double doublet (dd), quintet (quin), multiplet (m),
broad signal (br s), apparent signal (app). 13C Nuclear magnetic
resonance spectra were recorded on an Agilent VNMRS-DD2 600
MHz spectrometer equipped with a cold probe. Spectra were
referenced to the DMSO residual signal at δ = 39.9 ppm. Mass
spectra (m/z) were recorded on Micromass ZMD or Waters ZQ mass
spectrometer using ESI ionization. Isotopic distributions are quoted
for the mass ion of the lowest-molecular-weight isotope (35Cl and
79Br) and the number of isotopes detected. High-resolution mass
spectra (HRMS) were obtained from 1 μL of a 0.05 mg/mL
methanolic solution of each analyzed compound injected in a UHPLC
1290 Infinity (Agilent Technologies) coupled to a 6220 Accurate-
Mass time-of-flight (TOF) mass spectrometer (Agilent Technolo-
gies). A dual ESI in positive mode was used as ionization source. TOF
acquisition conditions were: scan acquisition mode (70−3000 Da,
scan rate: 1.00 spec/s), gas temp: 300 °C, gas flow: 8 L/min,
nebulizer: 40 psi, V cap: 2000 V, fragmentor: 150 V, skimmer 1: 65 V,
octopole radio frequency peak: 250 V, and reference masses: m/z
121.050873 and 922.009798.
Chemistry: Synthetic Procedures to 34. Additional exper-
imental procedures for all novel compounds prepared during the
course of this work can be found in the Supporting Information.
tert-Butyl (1r,4r)-4-(Hydroxymethyl)cyclohexylcarbamate (126).
Di-tert-butyl dicarbonate (3.04 g, 13.9 mmol) was added to a stirred
solution of ((1r,4r)-4-aminocyclohexyl)methanol (1.50 g, 11.6 mmol)
in tetrahydrofuran (20 mL). After stirring overnight at ambient
temperature, the solvent was evaporated and the residue partitioned
K
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 8. Pyridones: Early Introduction of Lipophilic Pocket Group
a
Reagents and conditions: (a) R−NH2, DIEA, CH2Cl2, −78 °C, 56−99%; (b) H2, Pt/C, ZnBr2, EtOAc, rt [R = (R)-8-fluorochromane, 75%]; H2,
Pd/C, EtOH, rt [R = (R)-2-ethyl-5-fluoropyridine, 30%] or SnCl2, EtOH, reflux [R = trans-2-cyclohexylacetonitrile, 70%]; (c) CDI, CH3CN, 80
°C, 63−99%; (d) NaH, SEM−Cl, DMF, 0 °C, 58−83%; (e) Pd2(dba)3, Xanthphos, Cs2CO3, dioxane, 110 °C, 33−81%; (f) TBAF, THF, 70 °C or
trifluoroacetic acid, NH3, MeOH, 60 °C, 56−97%; (g) TMSCl, NaI, CH3CN, 80 °C, 45−96%.
a
Scheme 9. Synthesis of Aminopyridines 93−96
a
Reagents and conditions: (a) NaOMe, MeOH, reflux, 78−87%; (b) H2, Pd/C, EtOH, rt, 87−98% or H2, Pt/C, ZnBr2, EtOAc, rt (R = Cl, 99%);
(c) H2O2, CH2Cl2, rt, 97%; (d) NaH, MeI, DMF, 0 °C to rt, 38%; (e) H2SO4, HNO3, 0−85 °C, 43%; (f) MeI, Ag2CO3, CHCl3, rt, 55%.
between ethyl acetate and water. The organic layer was separated,
washed with water and brine, dried (MgSO4), and the solvent was
evaporated. The residue was treated with hexanes, and the suspension
was filtered to give the title compound (2.11 g, 79%) as a white solid.
Low-resolution mass spectrometry (LRMS) (m/z): 228 (M − 1)+. 1H
NMR δ (300 MHz, DMSO-d6): 0.84−0.95 (m, 2H), 1.05−1.18 (m,
2H), 1.20−1.29 (m, 2H), 1.40 (s, 9H), 1.71−1.80 (m, 3H), 3.14 (m,
1H), 3.21 (t, J = 5.8 Hz, 2H), 4.41 (t, J = 5.3 Hz, 1H), 6.73 (d, J = 8.2
Hz, 1H).
((1r,4r)-4-(tert-Butoxycarbonylamino)cyclohexyl)methyl 4-Meth-
ylbenzene-sulfonate (127). A solution of 4-methylbenzene-1-sulfonyl
chloride (2.28 g, 11.96 mmol) in dichloromethane (10 mL) was
L
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 10. Synthesis of Aminonitrile 129
a
Reagents and conditions: (a) Boc2O, THF, rt, 79%; (b) TsCl, CH2Cl2, Et3N, rt, 83%; (c) NaCN, dimethyl sulfoxide (DMSO), 55 °C, 72%; (d) 4
N HCl, dioxane, rt, 89%.
a
Scheme 11. Pyridones: Early Introduction of the Hinge Binder Motif
a
Reagents and conditions: (a) KSCN, AcOH, 0 °C to rt, 63%; (b) 92, Et3N, EtOH, 0 °C (49%) or 94, DIEA, THF, 0 °C (19%); (c) DIEA, THF,
50 °C, 21−99%; (d) H2, Pd/C, EtOH, 88−99%; (e) CDI, CH3CN, 70 °C, 21−85%; (f) 4 N HCl, dioxane, rt, 98−99%; (g) 2,5-dioxopyrrolidin-1-
yl 2-cyanoacetamide, Et3N, DMF, rt, 20−54%; (h) NaI, TMSCl, CH3CN, 70 °C (81%).
added to a solution of tert-butyl (1r,4r)-4-(hydroxymethyl)-
cyclohexylcarbamate 126 (2.11 g, 9.2 mmol) and triethylamine
(1.59 mL, 11.4 mmol) in dichloromethane (50 mL), and the resulting
mixture was stirred overnight at ambient temperature. The mixture
was washed with 1 M aqueous sodium hydroxide solution, and the
organic layer was dried (MgSO4), evaporated, and the residue was
purified by flash chromatography (diethyl ether/hexanes) to give the
title compound (2.91 g, 83%) as a white solid. LRMS (m/z): 382 (M
was evaporated. The residue was purified by flash chromatography
(100% dichloromethane to 95:5 dichloromethane/methanol) to give
the title compound (0.450 g, 72%) as a white solid. LRMS (m/z): 239
(M + 1)+. 1H NMR δ (300 MHz, CDCl3): 4.59−4.12 (m, 1H), 3.51−
3.13 (m, 1H), 2.36−0.90 (m, 20H).
2-((1r,4r)-4-Aminocyclohexyl)acetonitrile Hydrochloride (129). A
mixture of tert-butyl (1r,4r)-4-(cyanomethyl)cyclohexylcarbamate
128 (0.348 g, 1.46 mmol) and 4 M hydrogen chloride solution in
dioxane (3.65 mL) was stirred overnight at ambient temperature. The
solvent was evaporated in vacuo, and the residue was treated with
diethyl ether. The resultant suspension was filtered to give the title
compound (0.226 g, 89%) as a white solid. LRMS (m/z): 139 (M +
1)+. 1H NMR δ (300 MHz, DMSO-d6): 8.08 (br s, 3H), 2.95 (d, J =
4.9 Hz, 1H), 2.50 (d, J = 6.5 Hz, 2H), 2.00 (d, J = 11.6 Hz, 2H), 1.83
(d, J = 12.3 Hz, 2H), 1.68−1.52 (m, 1H), 1.46−1.28 (m, 2H), 1.23−
1.07 (m, 2H).
1
− 1)+. H NMR δ (300 MHz, CDCl3): 0.90−1.12 (m, 4H), 1.43 (s,
3H), 1.87−1.68 (m, 2H), 2.10−1.90 (m, 2H), 3.34 (m, 1H), 3.48−
3.41 (m, 1H), 3.81 (d, J = 6.5 Hz, 2H), 4.37 (m, 1H), 7.34 (d, J = 8.2
Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H).
tert-Butyl (1r,4r)-4-(Cyanomethyl)cyclohexylcarbamate (128).
Sodium cyanide (0.38 g, 7.8 mmol) was added to a solution of
((1r,4r)-4-(tert-butoxycarbonylamino)cyclohexyl)methyl 4-methyl-
benzene sulfonate 127 (1.00 g, 2.6 mmol) in dimethyl sulfoxide
(10 mL), and the mixture was heated to 55 °C and stirred for 20 h.
After cooling to room temperature, the reaction mixture was diluted
with ethyl acetate; washed with saturated aqueous potassium
carbonate solution, water, and brine; dried (MgSO4); and the solvent
2-Chloro-5-nitro-4-thiocyanatopyrimidine (130). Potassium thi-
ocyanate (2.10 g, 21.6 mmol) was added in portions over 2 h to a
stirred solution of 2,4-dichloro-5-nitropyrimidine 36 (4.00 g, 20.6
mmol) in glacial acetic acid (25 mL) cooled to 10−15 °C using an
M
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 12. Pyridones: Synthesis of Compound 27
a
Reagents and conditions: (a) DIEA, CH2Cl2, −78 °C, 95%; (b) SnCl2, EtOH, 80 °C, 99%; (c) CDI, CH3CN, 70 °C, 26%; (d) NaH, SEM−Cl,
DMF, 0 °C, 71%; (e) Pd2(dba)3, Xanthphos, Cs2CO3, dioxane, 110 °C, 79%; (f) TBAF, THF, 70 °C, 45%; (g) H2, Pd/C, THF/EtOAc, 88%.
ice−water bath. The mixture was then stirred at ambient temperature
for 1 h, then diluted with water, and the precipitate was filtered,
washed with water and ice-cold diethyl ether, and dried to give the
title compound (2.82 g, 63%) as a white solid. 1H NMR δ (300 MHz,
CDCl3): 9.40 (s, 1H).
N-(2-Methoxypyridin-3-yl)-5-nitro-4-thiocyanatopyrimidin-2-
amine (131). 2-Methoxypyridin-3-amine 92 (0.100 g, 0.8 mmol) was
added portionwise to a stirred, cooled (ice-bath) suspension of 2-
chloro-5-nitro-4-thiocyanatopyrimidine 130 (0.174 g, 0.8 mmol) in
ethanol (3 mL). Triethylamine (0.170 mL, 1.2 mmol) was then added
dropwise, and the mixture was stirred for 30 min at 0 °C. The
precipitate was filtered and dried to give the title compound (0.135 g,
49%) as a yellow solid. LRMS (m/z): 305 (M + 1)+. 1H NMR δ (300
MHz, DMSO-d6): 3.95 (s, 3H), 7.03 (br s, 1H), 8.07 (br s, 1H), 9.24
(br s, 1H), 10.62 (s, 1H).
cyclohexyl)acetonitrile 140 (0.096 g, 0.27 mmol) and 1,1′-carbon-
ylbis-1H-imidazole (0.088 g, 0.54 mmol) in acetonitrile (1.5 mL) was
stirred and heated to 85 °C in a sealed tube. After 2 h, the solvent was
evaporated and water was added to the residue. After stirring for 1 h
at ambient temperature, the suspension was filtered and the solid was
washed with water and dried to give the title compound (0.084 g,
1
74%) as an off-white solid. LRMS (m/z): 380 (M + 1)+. H NMR δ
(300 MHz, DMSO-d6): 1.23 (m, 6H), 1.83 (m, 2H), 2.04 (m, 2H),
2.42 (m, 1H), 4.01 (s, 3H), 4.08 (m, 1H), 7.05 (m, 1H), 7.83 (m,
1H), 7.88 (m, 1H), 8.03 (m, 1H), 8.58 (m, 1H), 11.13 (br s, 1H).
2-((1r,4r)-4-(8-Oxo-2-(2-oxo-1,2-dihydropyridin-3-ylamino)-7H-
purin-9(8H)-yl) cyclohexyl)acetonitrile (34). Sodium iodide (2.43 g,
16.2 mmol) and trimethylsilyl chloride (2.05 mL, 16.2 mmol) were
added to a suspension of 2-((1r,4r)-4-(2-(2-methoxypyridin-3-
ylamino)-8-oxo-7H-purin-9(8H)-yl) cyclohexyl)acetonitrile 144
(2.05 g, 5.4 mmol) in acetonitrile (50 mL), and the mixture was
stirred and heated to 85 °C in a sealed tube. After 2 h, the mixture was
concentrated and treated with saturated aqueous sodium thiosulfate
solution. After stirring for 30 min, the precipitate was filtered, washed
with water and diethyl ether, and dried. The resulting solid was
treated with hot ethanol, filtered, and dried to give the title compound
(1.60 g, 81%) as a white solid. LRMS (m/z): 366 (M + 1)+. HRMS
2-((1r,4r)-4-(2-(2-Methoxypyridin-3-ylamino)-5-nitropyrimidin-
4-ylamino)cyclohexyl)acetonitrile (136). A mixture of N-(2-methox-
ypyridin-3-yl)-5-nitro-4-thiocyanatopyrimidin-2-amine 131 (0.178 g,
0.58 mmol), 2-((1r,4r)-4-aminocyclohexyl)acetonitrile hydrochloride
129 (0.123 g, 0.70 mmol), and N,N-diisopropylethylamine (0.62 mL,
3.5 mmol) in tetrahydrofuran (10 mL) was stirred and heated to 50
°C. After stirring overnight, the mixture was partitioned between
water and ethyl acetate and the organic extract was washed with brine,
dried (MgSO4), and evaporated. The residue was purified by flash
chromatography (0−50% ethyl acetate in hexanes) to give the title
compound (0.109 g, 49%) as a white solid. LRMS (m/z): 384 (M +
1
calcd for C18H20N7O2: 366.16730. Found [M + H]+: 366.16744. H
NMR δ (300 MHz, DMSO-d6): 1.38−1.21 (m, 2H), 2.05−1.68 (m,
5H), 2.46−2.30 (m, 2H), 4.26−4.12 (m, 1H), 6.29−6.19 (m, 1H),
6.93 (d, J = 5.8 Hz, 1H), 8.01−7.89 (m, 2H), 8.29 (d, J = 7.1 Hz,
1H), 10.77 (br s, 1H), 11.61 (br s, 1H). 13C NMR δ (151 MHz,
DMSO-d6): 23.4 (CH2), 28.7 (CH2), 31.1 (CH2), 33.7 (CH), 51.2
(CH), 106.2 (CH), 116.1 (C), 117.7 (CH), 120.0 (C), 124.6 (CH),
130.9 (C), 133.8 (CH), 151.1 (C), 153.1 (C), 153.7 (C), 157.7 (C).
1
1)+. H NMR δ (300 MHz, CDCl3): 1.27−1.48 (m, 5H), 1.82 (m,
1H), 2.05 (d, J = 9.4 Hz, 2H), 2.28 (m, 2H), 2.41 (d, J = 6.0 Hz, 2H),
4.08 (s, 3H), 6.96 (dd, J = 7.7, 5.2 Hz, 1H), 7.90 (d, J = 4.9 Hz, 1H),
7.96 (m, 1H), 8.43 (m, 1H), 8.65 (d, J = 8.0 Hz, 1H), 9.08 (s, 1H).
2-((1r,4r)-4-(5-Amino-2-(2-methoxypyridin-3-ylamino)pyrimidin-
4-ylamino)cyclohexyl)acetonitrile (140). 10% palladium on carbon
(0.048 g) was added to a solution of 2-((1r,4r)-4-(2-(2-methoxypyr-
idin-3-ylamino)-5-nitropyrimidin-4-ylamino)cyclohexyl)acetonitrile
136 (0.109 g, 0.28 mmol) in ethanol (15 mL), and the reaction
mixture was stirred at ambient temperature under a hydrogen
atmosphere. After 5 h, the mixture was then filtered through Celite
and the filter cake was washed with ethanol. The combined filtrate
and washings were concentrated to give the title compound (0.096 g,
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.9b00533.
Molecular formula strings (CSV)
1
96%) as an off-white solid. LRMS (m/z): 354 (M + 1)+. H NMR δ
(300 MHz, CDCl3): 1.19−1.43 (m, 4H), 1.76 (m, 1H), 2.06−1.95
(m, 2H), 2.30−2.17 (m, 2H), 2.35 (d, J = 6.5 Hz, 2H), 2.62 (s, 2H),
3.89−3.99 (m, 1H), 4.03 (s, 3H), 5.05 (d, J = 7.2 Hz, 1H), 6.87 (dd, J
= 7.8, 5.0 Hz, 1H), 7.63 (s, 1H), 7.70 (dd, J = 5.0, 1.6 Hz, 1H), 8.69
(dd, J = 7.8, 1.6 Hz, 1H).
2-((1r,4r)-4-(2-(2-Methoxypyridin-3-ylamino)-8-oxo-7H-purin-
9(8H)-yl)cyclohexyl)acetonitrile (144). A mixture of 2-((1r,4r)-4-(5-
amino-2-(2-methoxypyridin-3-ylamino)pyrimidin-4-ylamino)-
General chemistry and biology experimental procedures
(PDF)
Accession Codes
PDB codes are the following: 6HZV for 6; 6HZU for 34. The
authors will release the atomic coordinates and experimental
data upon article publication.
N
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
interleukin; JAK, Janus kinase; LPS, lipopolysaccharide;
LRMS, low-resolution mass spectrometry; NMR, nuclear
magnetic resonance; PAMPA, parallel artificial membrane
permeability assay; PK, pharmacokinetics; PPB, plasma protein
binding; SAR, structure−activity relationship; SEM, 2-
(trimethylsilyl)ethoxymethyl; SNAr, nucleophilic aromatic
substitution; STAT, signal transducer and activator of
transcription; TBAF, tetra-n-butylammonium fluoride;
TBDPS, tert-butyldiphenylsilyl; Tyk2, tyrosine kinase 2;
UPLC, ultraperformance liquid chromatography
AUTHOR INFORMATION
Corresponding Author
*E-mail: jordi.bach@almirall.com.
ORCID
■
Jordi Bach: 0000-0002-0449-0161
Present Addresses
#Premiumlab S. L., Carretera Santa Creu de Calafell 49-B, Sant
Boi de Llobregat, 08830 Barcelona, Spain (J.A.A.).
∇
́
̀
Research Center, Fundacio ACE, Institut Catala de Neuro-
̀
̀
ciencies Aplicades, C/ Marques de Sentmenat 57, 08029
Barcelona, Spain (A.O.).
REFERENCES
○Admetra, C/General Ramon Despuig 17, 07013 Palma de
Mallorca, Spain (J.A.).
■
(1) Leonard, W. J.; O’Shea, J. J. JAKS and STATs: Biological
implications. Annu. Rev. Immunol. 1998, 16, 293−322.
(2) Imada, K.; Leonard, W. J. The JAK-STAT pathway. Mol.
Immunol. 2000, 37, 1−11.
(3) Yamaoka, K.; Saharinen, P.; Pesu, M.; Holt, V. E. T.;
Silvennoinen, O.; O’Shea, J. J. The Janus kinases (Jaks). Genome
Biol. 2004, 5, No. 253.
◆Innoqua Toxicology Consultants, C/ Sant Antoni Maria
Claret 272 2-2, 08041 Barcelona, Spain (A.A.).
¶IRB Barcelona-Institute for Research in Biomedicine, Parc
́
Cientific de Barcelona, C/Baldiri Reixac 10, 08028 Barcelona,
Spain (N.P.).
⋈Peptinnovate, Stevenage Bioscience Catalyst, Gunnels Wood
Road, Stevenage SG1 2FX, United Kingdom (J.D.A.).
(4) O’Shea, J. J.; Pesu, M.; Borie, D. C.; Changelian, P. S. A new
modality for immunosuppression: Targeting the JAK/STAT pathway.
Nat. Rev. Drug Discovery 2004, 3, 555−564.
⧓
́
Montserrat Miralpeix Pharma Consulting, Parc Cientific de
(5) Wilks, A. F. The JAK kinases: Not just another kinase drug
discovery target. Semin. Cell Dev. Biol. 2008, 19, 319−328.
(6) Ghoreschi, K.; Laurence, A.; O’Shea, J. J. Janus kinases in
immune cell signaling. Immunol. Rev. 2009, 228, 273−287.
(7) O’Shea, J. J.; Plenge, R. JAK and STAT signaling molecules in
immunoregulation and immune-mediated disease. Immunity 2012, 36,
542−550.
(8) O’Shea, J. J.; Schwartz, D. M.; Villarino, A. V.; Gadina, M.;
McInnes, I. B.; Laurence, A. The JAK-STAT pathway: Impact on
human disease and therapeutic intervention. Annu. Rev. Med. 2015,
66, 311−328.
(9) Clark, J. D.; Flanagan, M. E.; Telliez, J.-B. Discovery and
development of Janus kinase (JAK) inhibitors for inflammatory
diseases. J. Med. Chem. 2014, 57, 5023−5038.
(10) Tam, C. S.; Verstovsek, S. Investigational Janus kinase
inhibitors. Expert Opin. Invest. Drugs 2013, 22, 687−699.
(11) Taylor, V.; Siu, S.; Young, C.; Ste Marie, L.; Ren, H.; Leusink-
Muis, T.; Van Ark, I.; Fokerts, G.; Park, G.; Pine, P.; Li, H.; Masuda,
E. R256 is a potent and selective IL-13/4 receptor signaling inhibitor
that reduces inflammation in a mouse model of chronic asthma. Am. J.
Respir. Crit. Care Med. 2012, 185, No. A2843.
(12) Ashino, S.; Takeda, K.; Pine, P. R.; Gelfand, E. W. Janus kinase
(JAK) 3 inhibitor, R256, attenuates allergen-induced airway hyper-
responsiveness and airway inflammation in mice. Am. J. Respir. Crit.
Care Med. 2012, 185, No. A2766.
(13) Jones, P.; Storer, R. I.; Sabnis, Y. A.; Wakenhut, F. M.;
Whitlock, G. A.; England, K. S.; Mukaiyama, T.; Dehnhardt, C. M.;
Coe, J. W.; Kortum, S. W.; Chrencik, J. E.; Brown, D. G.; Jones, R.
M.; Murphy, J. R.; Yeoh, T.; Morgan, P.; Kilty, I. Design and synthesis
of a pan-Janus kinase inhibitor clinical candidate (PF-06263276)
suitable for inhaled and topical delivery for the treatment of
inflammatory diseases of the lungs and skin. J. Med. Chem. 2017,
60, 767−786.
Barcelona, C/ Baldiri Reixac 4-8, 08028 Barcelona, Spain
(M.M.).
⧖
́
́
Centro de Investigacion Biomedica en Red de Diabetes y
Enfermedades Metabolicas Asociadas (CIBERDEM), Hospital
Duran i Reynals, Gran Via 199, 08908 L’Hospitalet de
́
Llobregat, Barcelona, Spain (I.R.).
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
The authors were all full-time employees of Almirall S. A. at
the time this work was completed and all funding for the work
was provided by Almirall S. A.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Andreas Griessner, Alfred Lammens,
Michael Blaesse, and Anja Jestel from Proteros Biostructures
GmbH for X-ray structure determination of human JAK3 and
JAK1 in complex with compounds 6 and 34 (PDB codes:
6HZV and 6HZU). They also acknowledge Eva Codesido,
́
́
́
́
́
Raul Fernandez, Gloria Fra, Marıa Jose Roo y Pablo Veiga
from GalChimia S. A. for their contribution to the synthesis
and scale-up of several of the compounds described in this
article. They also thank Sonia Espinosa and Josep Maria
Huerta for their support on physicochemical properties
analysis and structural characterization of compounds, Mireia
́
Verdu for her contribution to the evaluation of the activity of
(14) Wiegman, C. H.; Russell, K. E.; Seiffert, J.; Rossios, C.; Adcock,
I. M.; Rothaul, A.; Main, M.; Morgan, F. The selective pan-Janus
kinase (JAK) inhibitor VR588 demonstrates potent anti-inflammatory
activity in a murine chronic house dust mite (HDM) model of
asthma. Am. J. Respir. Crit. Care Med. 2015, 191, No. A6435.
(15) Murray, P. J. The JAK-STAT signaling pathway: input and
output integration. J. Immunol. 2007, 178, 2623−2629.
(16) Yew-Booth, L.; Birrell, M. A.; Lau, M. S.; Baker, K.; Jones, V.;
Kilty, I.; Belvisi, M. G. JAK-STAT pathway activation in COPD. Eur.
Respir. J. 2015, 46, 843−845.
́
34 in ion channels, and Elena Barcelo for performing HRMS
analysis of all tested compounds.
ABBREVIATIONS
■
ATP, adenosine triphosphate; AUC, area under the curve;
BAL, bronchoalveolar lavage; Boc, tert-butyloxycarbonyl;
CHO, Chinese hamster ovary; COPD, chronic obstructive
pulmonary disease; GM-CSF, granulocyte-macrophage colony-
stimulating factor; HPLC, high-performance liquid chromatog-
raphy; HRMS, high-resolution mass spectrometry; IL,
(17) Vale, K. Targeting the JAK-STAT pathway in the treatment of
‘Th2-high’ severe asthma. Future Med Chem. 2016, 8, 405−419.
O
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(18) Kudlacz, E.; Perry, B.; Sawyer, P.; Conklyn, M.; McCurdy, S.;
Brissette, W.; Flanagan, A. M.; Changelian, P. The novel JAK-3
inhibitor CP-690550 is a potent immunosuppressive agent in various
murine models. Am. J. Transplant. 2004, 4, 51−57.
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, 413−433.
(33) Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P.
M.; Wilks, A. F.; Fantino, E.; Rossjohn, J.; Lucet, I. S. Dissecting
specificity in the Janus kinases: the structures of JAK-specific
inhibitors complexed to the JAK1 and JAK2 protein tyrosine kinase
domains. J. Mol. Biol. 2009, 387, 219−232.
(19) Ashino, S.; Takeda, K.; Li, H.; Taylor, V.; Joetham, A.; Pine, P.
R.; Gelfand, E. W. Janus kinase 1/3 signaling pathways are key
initiators of TH2 differentiation and lung allergic responses. J. Allergy
Clin. Immunol. 2014, 133, 1162−1174.
̀
(20) Ramis, I.; Calama, E.; Domenech, A.; Carreno, C.; Calaf, E.;
̃
(34) This screening selectivity panel was mainly formed by kinases
inhibited by JAK inhibitors (29) and completed with a selection of
diverse representative kinases from the whole kinome (7). Kinases
inhibited by JAK inhibitors have been identified through literature
analysis and internal profiling of competitors’ compounds. List of
selected kinases from the kinome includes EGFR, IGF-1R, IKKα,
JNK1α1, JNK2α2, MEK1 and TAK1. JAK inhibitors described in this
article have shown poor activity when tested against kinases not
included in this panel.
(35) Compound 34 was negative for genotoxicity with and without
metabolic activation in the GreenScreen HC assays performed at
Gentronix (highest concentration tested: 39 μg/mL). In the Ames II,
Ames MPF and Ames MPF E. coli Combo mutagenicity assay
performed at Xenometrix, 34 proved to be negative in all strains at the
concentrations tested (highest concentration tested: 1581 μg/mL).
Cordoba, M.; Albertí, J.; De Alba, J.; Bach, J.; Prats, N.; Miralpeix, M.
New inhaled pan-JAK inhibitor A is efficacious at reducing allergen-
induced airway inflammation in rat. Am. J. Respir. Crit. Care Med.
2014, 189, No. A4217.
̀
(21) Calama, E.; Ramis, I.; Domenech, A.; Carreno, C.; De Alba, J.;
̃
Prats, N.; Miralpeix, M. Tofacitinib ameliorates inflammation in a rat
model of airway neutrophilia induced by inhaled LPS. Pulm.
Pharmacol. Ther. 2017, 60−67.
(22) Ruwanpura, S. M.; McLeod, L.; Miller, A.; Jones, J.; Vlahos, R.;
Ramm, G.; Longano, A.; Bardin, P. G.; Bozinovski, S.; Anderson, G.
P.; Jenkins, B. J. Deregulated stat3 signaling dissociates pulmonary
inflammation from emphysema in gp130 mutant mice. Am. J. Physiol.:
Lung Cell. Mol. Physiol. 2012, 302, L627−L639.
(23) Mullings, R. E.; Wilson, S. J.; Puddicombe, S. M.; Lordan, J. L.;
Bucchieri, F.; Djukanovic, R.; Howarth, P. H.; Harper, S.; Holgate, S.
T.; Davies, D. E. Signal transducer and activator of transcription 6
(STAT-6) expression and function in asthmatic bronchial epithelium.
J. Allergy Clin. Immunol. 2001, 108, 832−838.
(24) Christodoulopoulos, P.; Cameron, L.; Nakamura, Y.; Lemiere,
C.; Muro, S.; Dugas, M.; Boulet, L. P.; Laviolette, M.; Olivenstein, R.;
Hamid, Q. TH2 cytokine-associated transcription factors in atopic
and nonatopic asthma: evidence for differential signal transducer and
activator of transcription 6 expression. J. Allergy Clin. Immunol. 2001,
107, 586−591.
(25) Schwartz, D. M.; Kanno, Y.; Villarino, A.; Ward, M.; Gadina,
M.; O’Shea, J. J. JAK inhibition as a therapeutic strategy for immune
and inflammatory diseases. Nat. Rev. Drug Discovery 2017, 16, 843−
862.
(26) Cooper, A. E.; Ferguson, D.; Grime, K. Optimisation of DMPK
by the inhaled route: challenges and approaches. Curr. Drug Metab.
2012, 13, 457−473.
(27) Cole, A. G.; Bohnstedt, A. C.; Paradkar, V.; Kingsbury, C.;
Quintero, J. G.; Park, H.; Lu, Y.; You, M.; Neagu, I.; Diller, D. J.;
Letourneau, J. J.; Shao, Y.; James, R. A.; Riviello, C. M.; Ho, K. K.;
Lin, T. H.; Wang, B.; Appell, K. C.; Sills, M.; Quadros, E.; Kimble, E.
F.; Ohlmeyer, M. H.; Webb, M. L. 2-Benzimidazolyl-9-(chroman-4-
yl)-purinone derivatives as JAK3 inhibitors. Bioorg. Med. Chem. Lett.
2009, 19, 6788−6792.
(28) Wu, G.; Robertson, D. H.; Brooks, C. L., III; Vieth, M. Detailed
analysis of grid-based molecular docking: A case study of CDOCKER
- A CHARMm-based MD docking algorithm. J. Comput. Chem. 2003,
24, 1549−1562.
(29) This series of purinone JAK inhibitors bearing an imidazo[1,2-
a]pyridine hinge binder has been covered by the following patent
́
́
application: Eastwood, P. R.; Gonzalez, J.; Gomez, E.; Bach, J.
Heteroaryl Imidazolone Derivatives as JAK Inhibitors. WO2011/
157397, 2011.
(30) A similar approach using a pyrazolo[1,5-a]pyridine ring as
hinge binder motif has been reported by Palau Pharmax: Almansa, C.;
Salas, J.; Soliva, R.; Rodríguez, S.; Sicre, M. C. N-Containing
Heteroaryl Derivatives as JAK3 Kinase Inhibitors. WO2011/051452,
2011.
(31) After the publication of Almirall’s patent WO2011/157397,
Ligand pharma also reported the use of an imidazo[1,2-a]pyridine
ring as hinge binder motif for their JAK inhibitors: Cole, A. G.; James,
R. A.; Shao, Y.; Letourneau, J. J.; Quintero, J. G.; Riviello, C. M.; Zhi,
L. Janus Kinase Inhibitor and Methods. WO2013/025628, 2013.
(32) 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,
P
DOI: 10.1021/acs.jmedchem.9b00533
J. Med. Chem. XXXX, XXX, XXX−XXX