Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165

Article abstract of DOI:10.1021/acs.jmedchem.9b00444

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formi

Full text of DOI:10.1021/acs.jmedchem.9b00444

Article
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/jmc
Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the
Treatment of Autoimmune Diseases: Discovery of the Allosteric
Inhibitor BMS-986165
,
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Stephen T. Wrobleski,* Ryan Moslin,* Shuqun Lin, Yanlei Zhang, Steven Spergel,
‡
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James Kempson, John S. Tokarski, Joann Strnad, Adriana Zupa-Fernandez, Lihong Cheng,
David Shuster, Kathleen Gillooly, Xiaoxia Yang, Elizabeth Heimrich, Kim W. McIntyre,
Charu Chaudhry, Javed Khan, Max Ruzanov, Jeffrey Tredup, Dawn Mulligan, Dianlin Xie,
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∥
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⊥
§
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§
§
§
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Huadong Sun, Christine Huang, Celia D’Arienzo, Nelly Aranibar, Manoj Chiney,
Anjaneya Chimalakonda, William J. Pitts, Louis Lombardo, Percy H. Carter, James R. Burke,∥
#
†
†
†
and David S. Weinstein†
†
‡
§
Immunosciences Discovery Chemistry, Department of Discovery Synthesis, Molecular Structure and Design, Molecular
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Discovery Technologies, Immunosciences Discovery Biology, Leads Discovery and Optimization, Metabolism and
Pharmacokinetic Department, Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research & Development, P.O. Box
4
000, Princeton, New Jersey 08543, United States
*
S Supporting Information
ABSTRACT: Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune
diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase
family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2
pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water
displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor
of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with
minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11
appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed
therapeutic in clinical development as an oral treatment for autoimmune diseases.
INTRODUCTION
catalytic activity of the kinase by blocking ATP, downstream
phosphorylation, and resulting pathway signal transduction.
■
6
1
Tyrosine kinase 2 (TYK2) is a member of the Janus family of
kinases (JAK) that also includes JAK1, JAK2, and JAK3. The
JAK family of nonreceptor tyrosine kinases is known to be
critical in mediating the signaling of numerous cytokines that
This includes the first generation clinically approved inhibitors
1−4 as well as the second-generation inhibitors 5−10 currently
in development (Figure 1). In contrast, BMS-986165 (11) is
differentiated from previous JAK inhibitors due its unique
ability to selectively bind to the pseudokinase (JH2) domain of
TYK2 and inhibit its function through an allosteric mechanism.
This work will describe the late-stage discovery efforts that
2
−4
cause inflammation. As a result, small molecule inhibitors of
the JAK kinases offer promise as effective treatments for a
5
,6
variety of serious inflammatory and autoimmune diseases.
To date, all known small molecule JAK inhibitors that have
progressed into development are active site-directed inhibitors
that bind to the adenosine triphosphate (ATP) site of the
catalytic domain (also referred to as the JH1 or “Janus
Homology 1” domain) of the JAK protein, which prevents
Received: March 14, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.9b00444
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Journal of Medicinal Chemistry
Article
Figure 1. Structures of the first-generation clinically approved JAK inhibitors 1−4, the second-generation experimental JAK inhibitors 5−10, and
the TYK2-selective allosteric inhibitor 11 in the clinic for the treatment of chronic immunological disorders. Inhibitors 1−10 are conventional
active-site (JH1) inhibitors, whereas 11 is an allosteric (JH2) inhibitor.
ultimately led to the identification of 11, currently in phase III
clinical trials as a potential treatment for psoriasis.
baricitinib (3). Inhibitor 2 has been approved as a treatment
for myelofibrosis and is also being explored in phase III studies
as an oral treatment for steroid-refractory graft vs host disease
(GvHD) and as a topical treatment for atopic dermatitis
Because of the high homology of the ATP active site across
the kinome and especially within the JAK family, achieving
high selectivity for a specific JAK family member while also
maintaining selectivity within the kinome is a significant
challenge. As a result, many JAK inhibitors that have been
developed are pan-JAK inhibitors or are modestly selective for
one or more JAK family member. While these inhibitors have
shown encouraging results in treating autoimmune diseases,
undesirable side effects leading to a narrow therapeutic index
have been observed and suggests the need for improved
treatments. Tofacitinib (1), a pan-JAK inhibitor of JAK1,
JAK2, JAK3, and to a lesser extent TYK2, became the first
orally available small molecule kinase inhibitor to be approved
for the treatment of moderately to severely active rheumatoid
24−26
(AD).
Inhibitor 3 has demonstrated efficacy in RA and
PSO patients in phases III and IIb, respectively, and is also
being explored in SLE and atopic dermatitis in phase II
27−30
trials.
However, safety concerns remain as only the lower
2 mg dose of 3 was recently approved by the FDA to treat
moderate-to-severely active RA patients due to a safety
concern related to a potential thrombosis risk at the higher,
3
1
more efficacious 4 mg dose. The pan-JAK inhibitor
peficitinib (4) has also shown efficacy in RA patients in two
32,33
phase III trials and was recently approved in Japan.
Although these first-generation JAK inhibitors have established
proof-of-concept in treating serious diseases, achieving robust
efficacy while maintaining a safe therapeutic window, has been
a significant challenge in the long term treatment of chronic
diseases. Dose-limiting side effects such as anemia, neutrope-
nia, and increased infection risk and dyslipidemia have been
7
arthritis (RA) in 2012. However, only the lower 5 mg twice-
daily dose of 1 was approved by the Food and Drug
Administration (FDA) based on an unfavorable risk-to-benefit
ratio observed with the higher 10 mg twice-daily dose. Efficacy
was also achieved with 1 in late-stage phase III clinical trials for
the treatment of psoriasis (PSO), psoriatic arthritis (PsA), and
ulcerative colitis (UC) and recently gained FDA approval for
the treatment of active PsA and moderately to severely active
34−36
observed
and attributed to inhibition of JAK1 and JAK2,
the latter of which is well-known to be involved in
37
hematopoiesis. These safety concerns led to the development
of second-generation JAK inhibitors that have been reported to
be more selective over JAK2, including the reported JAK1-
8
−16
UC.
Despite these achievements, the FDA declined
approval of 1 for the treatment of PSO in 2015 based on
issues of clinical efficacy and long-term safety. It is currently
selective inhibitors filgotinib (5), upadacitinib (6), and
17
38−40
abrocitinib (7).
These inhibitors have recently advanced
in phase III studies for the treatment of ankylosing spondylitis
into phase III clinical trials with some encouraging safety and
tolerability results. Inhibitor 5 has been reported to show
efficacy in RA in combination with methotrexate or as a single
agent without the side effects such as anemia or changes in
(
AS), phase II studies for alopecia areata (AA) and
inflammatory eye disease, and phase I studies for systemic
discoid lupus erythematosus (DLE), systemic lupus eryth-
ematosus (SLE), diffuse cutaneous systemic sclerosis (dcSSc),
levels of lymphocyte, natural killer (NK) cells, or liver function
18−23
41−43
and treatment-refractory dermatomyositis.
Other first-
tests (LFT) observed with 1.
However, increased
generation JAK inhibitors approved include the JAK1/JAK2
selective inhibitors ruxolitinib (2) and the structurally related
creatinine levels and reduced neutrophil and platelet counts
were observed. Inhibitor 5 was also reported to show efficacy
B
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in Crohn’s disease (CD) by meeting phase II primary end
Table 1. Reported JAK Family Biochemical Potencies for
Clinical Inhibitors 1−10 Compared to 11
e
points in a 10-week interim analysis without any drug-related
44
significant adverse events (SAEs). More recently, the JAK1-
selective inhibitor 6 was disclosed to have met all primary and
ranked secondary end points in a phase III study in RA, yet
laboratory changes were shown to be very similar to those
assay IC50 (nM)
compd
JAK1
15
JAK2
77
JAK3
TYK2 (JH1/JH2)
a
1
55
487
787
489/nd
30/nd
61/nd
45
a
observed with 1. This included increased levels of liver
transaminases, creatine phosphokinase, LDL cholesterol, and
HDL cholesterol and decreased counts of NK cells,
lymphocytes, and neutrophils. Other reported more selective
inhibitors are in development, including the JAK1-selective
inhibitor 7, that has advanced to phase III studies in atopic
2
6
4
4
363
47
29
9
7
5
a
3
b
4
0.7
>10000
2304
>15000
33
6494
5/nd
a
5
2400
120
803
>10000
77
2600/nd
4690/nd
1250/nd
>10000/nd
23/nd
c
6
a
7
4
6
a
dermatitis and the dual JAK3/TEC family irreversible
8
>10000
17
383
>10000
47
a
inhibitor PF-06651600 (8)4 being investigated in phase II
9
8−50
a
studies in CD, UC, and AA.
Despite this progress, it is yet
10
74
>10000
>10000
>10000
17/nd
>10000/0.2
d
to be determined whether more selective JAK1 and/or JAK3
inhibition can achieve a superior therapeutic profile compared
to first-generation inhibitors in these complex diseases.
Selective inhibition of TYK2 has recently emerged as a
potential strategy for treating various autoimmune dis-
11
a
Assays run in the presence of 1 mM ATP according to ref 5 for 1−3
b
and 5, ref 40 for 7, ref 47 for 8, ref 54 for 9, and ref 58 for 10. Assays
c
run at the K for ATP according to ref 33. Assays run in the presence
m
d
of 0.1 mM ATP according to ref 39. Using homogeneous time-
5
1−53
54
resolved fluorescence (HTRF) binding assays measuring the displace-
eases.
A dual JAK1/TYK2 inhibitor PF-06700841 (9)
e
ment of a probe compound (please see Experimental Section). nd:
has shown efficacy in patients with plaque PSO in phase II
5
5,56
not determined.
studies
and is currently enrolled in additional phase II
4
8−50
studies for CD, UC, and AA.
Yet, at maximally efficacious
64
doses in PSO patients, 9 gave significant reductions in absolute
reticulocyte and neutrophil counts as well as reduced platelet
counts that was believed to be associated with JAK2 and JAK1
has recently shown encouraging efficacy in phase II studies in
treating patients with active PSO without incidences of adverse
effects that have commonly been observed with less selective
JAK inhibitors such as neutropenia, elevations in liver enzyme
levels or serum creatinine, or dyslipidemia. Furthermore, 11
has demonstrated robust efficacy in murine models of lupus
56
inhibition, respectively. The inhibitor PF-06826647 (10) has
recently been disclosed to be more selective for TYK2 and is
currently under evaluation in phase I studies for the treatment
5
7−59
63
of PSO.
nephritis and IBD. Herein, we disclose additional preclinical
studies with 11 that demonstrate robust efficacy in a murine
psoriasis-like disease model that further supports the
therapeutic potential of this agent as a treatment across
multiple autoimmune diseases.
Our goal was to identify a more selective TYK2 inhibitor in
the hope of establishing clinical efficacy in autoimmune
diseases while demonstrating an improved safety profile.
Toward that goal, we were the first to report the discovery
of highly selective allosteric TYK2 inhibitors that act by their
unique ability to bind to and stabilize the TYK2 pseudokinase
A Novel Allosteric Approach to Selective TYK2
Inhibition. Small molecule TYK2 inhibitors that are able to
block both the IL-23/IL-12 and the type I interferon (IFNα,
IFNβ) pathways may offer the potential to safely and more
efficaciously treat a broad spectrum of inflammatory and
autoimmune diseases. However, a significant challenge in this
regard is achieving the desired level of TYK2 selectivity to
avoid undesirable off-target effects. This includes ensuring
adequate selectivity over the other members of the JAK kinases
as well as against the entire kinome containing >500 kinases.
Despite the potential of TYK2 inhibition, identifying small
molecules that are selective is challenging. Our strategy relies
on small molecule allosteric inhibitors such as the N-
deuteromethyl pyridazine carboxamide 12 that potently inhibit
TYK2-dependent signaling with high specificity by binding to
60,61
(
JH2) domain to block TYK2 signaling.
On the basis of
these initial findings, we disclose in the preceding article our
hit-to-lead efforts that led to a new series of N-methylpyridine-
3
-carboxamides (nicotinamides) and N-methyl pyridazine-3-
carboxamides as allosteric TYK2 inhibitors that have
demonstrated in vivo proof-of-concept in CD40 agonist-
induced colitis, a murine disease model of inflammatory bowel
6
2
disease (IBD).
Herein, we report further optimization efforts within this
series that culminated in the discovery of BMS-986165 (11)
currently under clinical development as a potential treatment
for PSO, CD, and SLE (Clinicaltrials.gov identifiers
NCT03624127, NCT03611751, NCT03599622,
NCT03252587). Comparison of the TYK2 and other JAK
family biochemical potencies of 11 relative to the classic active-
site clinical JAK inhibitors 1−10 show a distinct selectivity
profile that is envisioned to permit maximal efficacy from
selective TYK2 inhibition but with decreased potential for
adverse side effects that have been observed with less selective
inhibitors (Table 1). As noted, 11 is a potent allosteric
inhibitor of TYK2 that acts by binding to the TYK2 JH2
domain with high affinity (IC50 = 0.2 nM) and achieves
remarkable selectivity over inhibition of catalytically active JH1
domains of the JAK family, a distinct profile that translates to
an observed high functional selectivity for TYK2 over JAK1,
60−64
and stabilizing its pseudokinase JH2 domain (Figure 2).
The JH2 domain directly precedes the catalytically active JH1
kinase domain containing the canonical ATP-binding site, a
65
structural feature that is unique to the JAK family. While the
TYK2 JH2 domain closely resembles its JH1 domain and
contains an ATP binding site much like the JH1 domain,
specific residue differences within JH2 precludes any catalytic
66
function. Although the precise mechanism of the allosteric
inhibition of TYK2 through the JH2 domain remains to be
elucidated, evidence suggests that small molecule ligand
binding to JH2 stabilizes autoinhibitory intramolecular
interactions between the JH2 domain and the JH1 active
site. These JH2−JH1 interactions are believed to limit the
63
JAK2, and JAK3. Consistent with this selectivity profile, 11
C
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efforts that provided in vivo proof-of-concept in a murine
62
model of IBD. As a result, further optimization was focused
on improving potency in human whole blood (hWB) within
this series. In addition, mitigation of a potential cardiovascular
liability was required because 12 was shown to be an inhibitor
of the human ether-a-
in vitro (patch clamp IC = 10.9 μM) that translated into an
́
go-go-related gene (hERG) ion channel
5
0
observed QT prolongation in an in vivo telemetrized rabbit
c
study (data not shown). Fortunately, emerging structure−
activity relationships (SAR) studies in replacing the 2-
aminopyridyl side chain in 12 with alkyl amide side chains,
in particular cyclopropyl amide, gave reduced hERG inhibition
and suggested a path forward, albeit with some decrease in
62
potency. Therefore, immediate optimization efforts were
focused on improving potency within the series to better
enable mitigation of the potential hERG liability while allowing
for a lower projected human dose. Because of the
aforementioned role of the deuteromethyl amide group in
providing high selectivity by binding to the atypical “alanine
pocket” of TYK2 JH2, this group was maintained and
optimization was instead focused on modifications around
the aryl methyl sulfone group of 12. These efforts were guided
by X-ray cocrystal structures of sulfone analogues such as 12
Figure 2. Schematic illustrating the general protein structure of TYK2
and depicting the N-deuteromethyl pyridazine carboxamide 12 that
potently inhibits TYK2 dependent signaling by binding to the TYK2
pseudokinase (JH2) domain with high specificity over the JAK family
kinase (JH1) domains.
conformational mobility of the JH1 active site that is required
62
67,68
for phosphotransfer catalysis.
A notable advantage of
bound to TYK2 JH2 (Figure 3). Notably, the methyl group
inhibiting TYK2 by binding to its JH2 domain is the potential
to achieve maximal efficacy while maintaining a high level of
selectivity over the kinome, especially with respect to the other
JAKs. To that end, we have previously demonstrated in vivo
proof-of-concept studies with the JH2 ligand 12 in a CD40
6
2
agonist-induced colitis murine disease model of IBD
Inhibitor 12 has been shown to be highly selective for TYK2
over JAK1, JAK2, and JAK3 by virtue of a key deuteromethyl
amide substituent. Importantly, this group provides high
selectivity by binding to a pocket created by a rare alanine
residue (“alanine pocket”) in the TYK2 JH2 ligand binding
domain. Furthermore, deuteration of the N-methyl group was
shown to block generation of a less selective primary amide
metabolite in vivo by suppressing an N-demethylation
metabolic pathway via deuterium kinetic isotope effect
6
2
(
DKIE).
In homogeneous time-resolved fluorescence
(
HTRF) biochemical affinity assays that measure the
60
inhibition of binding of fluorescein-tagged probe molecules,
Figure 3. X-ray crystal structure representation of 12 cocomplexed
with TYK2 JH2 (PDB 6NZR). Carbons of 12 in magenta. TYK2 JH2
ribbon and carbons in green. Key hydrogen bond interactions within
the ligand binding site are represented as dotted lines. Binding
regions, including the unique “alanine pocket”, and the observed
structural water are highlighted in blue text with other key residues
labeled. The P-loop region has been partially omitted for clarity.
1
2 binds to the TYK2 JH2 domain with high affinity (IC50
=
0
.5 nM) and specificity over binding to the TYK2, JAK1, JAK2,
and JAK3 catalytically active JH1 domains (IC values >10000
50
6
2
nM). In cell-based luciferase reporter assays in T-cells that
measure JAK family functional selectivity, 12 is a selective
inhibitor of IFNα-stimulated TYK2-dependent signaling (IC50
60
=
27 nM) and is highly selective over GM-CSF stimulated
JAK2-dependent signaling (∼270-fold) and IL-2 stimulated
JAK1/3-dependent signaling (∼210-fold). Furthermore, 12 is
also highly selective (>1000-fold) against >250 kinases in an
in-house panel using similar biochemical affinity assays with
the only exception being cKit (∼690-fold). On the basis of the
encouraging selectivity profile and preclinical proof-of-concept
studies of 12, efforts were focused on continued optimization
within this series of allosteric TYK2 inhibitors to identify
potential candidates for advancement into clinical develop-
ment.
of the methyl sulfone of 12 favorably occupies a shallow
lipophilic pocket in the C-terminal domain region that is
created by Pro694, the side chain of Leu741, and the methine
backbone of Asn739 at the bottom of the ligand binding site.
Additional favorable interactions between 12 and TYK2 JH2
include two hydrogen bonds to the conserved Lys642 from the
N-deuteromethyl amide carbonyl oxygen and one of the
sulfone oxygens as well as the presence of a structural water
molecule facilitating indirect hydrogen bond interactions
between the second sulfone oxygen of 12 with the C-terminal
domain Arg738 and the Gln597 of the P-loop region. On the
basis of these observations, and due to our previous success in
displacing a structural water resulting in improved potency in
RESULTS AND DISCUSSION
■
Program Objectives for Further Optimization. Inhib-
itor 12 was a promising analogue from our initial hit-to-lead
69
an early series of p38 kinase inhibitors, we were inspired to
D
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investigate ligand modifications that might displace the
observed structural water. This strategy was especially
attractive due to the possibility of improving potency through
displacement of an energetically disfavored water molecule
and potentially forming an additional hydrogen bond
interaction with the protein.
Gratifyingly, one of the initial analogues 15 containing a cyano
group at C3′ showed a significant improvement in potency
compared to the C3′ unsubstituted analogue 14, giving
comparable potency to the sulfone 13 in both the TYK2
JH2 and IFNα cellular assay. Although 15 was ∼3-fold less
potent in hWB vs 13, we were encouraged by this initial result
and expanded our SAR investigation to include C3′ amides.
The intermediate acid 16 was prepared and tested along with
the primary amide 17 and the N-methyl amide 18. All of these
analogues were found to be nearly equipotent to 13 in binding
and cellular potency including in hWB, with the exception of
acid 16, which did not show any activity in cells likely due to
poor permeability as measured in an in vitro Caco-2
70
A Water Displacement Strategy. Initial SAR studies to
explore the water displacement proposal were pursued within
the closely related pyridine-3-carboxamide series, exemplified
by 13, that contained the methyl sulfone group at the C2′
position, a nondeuteromethyl amide, and a 5-fluoro-2-amino-
62
pyridine side chain (Table 2). Of particular interest was a
Table 2. C4-Aminophenyl SAR Exploring C2′−C4′
Modifications
permeability assay (apical-to-basal P < 15 nm/s). Remarkably,
c
removal of the C2′ methoxy group while retaining the C3′
methyl amide in 19 results in a ∼60-fold loss in TYK2 JH2
potency, ∼100-fold loss in functional potency, and no
significant activity in hWB at the highest concentration tested,
thereby highlighting the importance of the C2′ methoxy group.
Transposition of the amide group from the C3′ position to the
adjacent C4′ position was also explored while maintaining the
C2′ methoxy group as in 20 and 21. These analogues were also
potent in the TYK2 JH2 assay but were noticeably less potent
in the cellular assays relative to their C3′ amide counterparts
1
7 and 18, possibly due to decreased permeability as observed
in the Caco-2 assay (apical-to-basal P = 50 nm/s for 18 vs <15
c
nm/s for 21).
To further explore the C3′ amide SAR, additional amides
were prepared in a closely related series containing a 4-methyl
substituent on the pyridyl side chain (Table 3). Previous SAR
efforts had shown this series to have improved selectivity over
cKit (data not shown), and modeling studies suggested that
larger C3′ amide substitutions would be well-tolerated due to
their projection into a large open pocket beyond the Arg738
Table 3. Expansion of C3′ Amide SAR
a
In vitro assays. Mean values are determined from at least three
b
experiments unless otherwise noted. Assay measuring TYK2-
dependent IFNα-induced STAT5 phosphorylation in human whole
c
d
blood according to refs 62, 63. According to ref 62. Mean values
determined from two test occasions.
finding where the methyl sulfone group in 13 was replaced
62
with a methoxy substituent in 14. Although 14 was ∼5-fold
less potent compared to 13, we envisioned that 14 may be a
better starting point to explore the water displacement strategy
2
due to its lower starting polar surface area (PSA = 88 vs 113 Å
for 13). A proposal of particular interest from our modeling
studies was incorporation of hydrogen bond accepting groups
ortho to C2′ methoxy (C3′ position) that might favorably
displace the structural water and form a direct hydrogen bond
interaction with the Arg738 side chain. With this idea in mind,
C3′ modifications were explored in the C2′ methoxy series.
a
In vitro assays. Mean values determined from at least three
b
experiments unless otherwise noted. Assay measuring TYK2-
dependent IFNα-induced STAT5 phosphorylation in human whole
blood according to ref 62,63. n = 1.
c
E
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Figure 4. X-ray cocrystal structure of analogue 29 in TYK2 JH2 (PDB 6NZQ) with surface representation of protein added and showing H-bond
to Arg738 (3.1 Å) and amide N-substituent occupying large, open pocket. The methoxy group forms an intramolecular H-bond with the amide N−
H while also occupying the shallow C-terminal pocket of TYK2 JH2.
(
vide infra). Consistent with these modeling results, many
Furthermore, the critical role of the C2′ methoxy group for
potency can also be rationalized from this structure by noting
its ability to (1) preorganize the C3′ amide conformation
required for hydrogen bonding to the Arg738 through an
intramolecular hydrogen bond to the C3′ amide N−H and (2)
favorably position the methyl group to occupy the shallow C-
terminal lipophilic pocket that was also occupied by the methyl
sulfone group of 12 (vide supra).
amide substitutions were prepared and found to be potent in
the TYK2 JH2 biochemical assay. As in the previous 4-des-
methyl pyridyl series, the primary amide (22) and methyl
amide (23) were both potent (TYK2 JH2 IC50 values = 0.5
nM) with very good translation into hWB (IC50 values = 62
and 140 nM, respectively), with many other amides being
nearly equipotent in the TYK2 JH2 biochemical assay.
Unfortunately, many of the additional amides showed a
decrease in potency in the IFNα cellular and/or hWB assays
relative to the primary amide 22. This included the ethyl amide
Having validated our original water displacement strategy,
subsequent efforts focused on further optimization to address
the metabolic instability and permeability issues associated
with the C3′ amides while also optimizing the interaction with
Arg738. Modeling studies with a variety of heterocycles as C3′
amide replacements appeared promising in this regard. In
particular, heterocycles that contained a heteroatom at the
ortho position to the ring connection appeared optimal in
mimicking the C3′ amides due to their ability to accept a
hydrogen bond from Arg738. With this in mind, a variety of
five-membered heterocycles were explored. At this point in our
optimization effort, advancing compounds into mouse in vivo
models for further proof-of-mechanism studies became a
priority with new analogues being routinely screened for
potency in a mouse whole blood (mWB) assay and for
metabolic stability using a mouse liver microsomal (MLM)
assay. As shown, many heterocycles in place of the C3′ amides
afforded picomolar biochemical potency, with some achieving
single-digit nanomolar potency in the IFNα cellular assay for
the first time (Table 4). The oxadiazoles 30 and 31 and the N-
methyl pyrazoles 32 and 33 gave cellular IFNα IC50 values of
15, 12, 9, and 70 nM, respectively, but showed poor translation
into whole blood, particularly mWB. In addition, these
analogues suffered from only moderate stability in the MLM
assay. Fortunately, exploring more polar 1,2,4-triazoles 34−38
resulted in improved potency in hWB. In particular, the 1-
methyl-1,2,4-triazole analogue 38 was noteworthy due its
potency in the IFNα cellular assay (IC = 4 nM) and in whole
2
4 as well as the dimethylamide 25 that were 4−12-fold less
potent in hWB relative to 22. Larger alkyl amides containing
polar groups such as the hydroxy- and morpholino-substituted
alkyl amides 27 and 28 and the 2-picolinamine-derived amide
2
9 were also notably potent in the TYK2 JH2 assay but were
still ∼3−5-fold less potent in hWB compared to 22.
In addition, attempts to progress the most potent analogues
in hWB were hampered by poor microsomal stability and/or
poor permeability. For example, when tested in rodent
microsomal assays, the C3′ N-alkyl substituted amides
commonly afforded <50% of parent remaining after a 10 min
incubation. This observed high rate of metabolism was
attributed to an N-dealkylation metabolism pathway of the
C3′ amides based on biotransformation studies, and any
attempt to block this metabolic pathway with substitutions,
such as the addition of fluorines, was unsuccessful (data not
shown). The primary amide 22 did show improved micro-
somal stability relative to the substituted amides, but 22
suffered from poor permeability and high efflux in the Caco-2
assay (Caco-2 apical-to-basal P < 15 nm/s vs basal-to-apical P
c
c
∼
260 nm/s). While further advancement of the C3′ amides
was not pursued for these reasons, we were able to validate our
water displacement strategy by solving an X-ray cocrystal
structure of the 2-picolinamine-derived amide 29 bound to
TYK2 JH2 (Figure 4). As designed, 29 bound to the hinge
region in accord with previous analogues but with the newly
incorporated C3′ amide forming a direct interaction with
Arg738 by displacing the structural water that had been
previously observed in the cocrystal structure of 12. A direct
hydrogen bond between the C3′ amide carbonyl oxygen of 29
and Arg738 side-chain N−H is observed (3.1 Å ON
distance) with the pendant 2-picolinamine group protruding
into a large pocket beyond the Arg738 as had been predicted
by our modeling studies. This structure is consistent with SAR
that show many different amide N-substitutions are tolerated.
5
0
blood (mWB and hWB IC50 values = 130 and 12 nM,
respectively). In addition, 38 gave improved stability in the
MLM assay with 80% of parent remaining after a 10 min
incubation time.
To assess as a possible candidate for advancement, 38 was
profiled more extensively for potential undesirable off-target
activities. From the outset of our effort in pursuing allosteric
TYK2 inhibitors, we had consistently observed very high
selectivity for TYK2 across a large part of the kinome due in
large part to the ability of these compounds in accessing the
F
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d
Table 4. C3′ Heterocycles as Amide Replacements
more polar heterocycles, such as the substituted pyrimidine 41,
was successful in reducing activity in the hERG flux assay (IC50
>
80 μM), however, unacceptably low Caco-2 permeability
(
apical-to-basal P < 15 nm/s) with high efflux was commonly
c
observed. Fortunately, replacement of the 2-aminopyridine
side chain with a cyclopropyl amide, as previously demon-
62
strated in the methyl sulfone series, afforded 42 which gave
reduced hERG inhibition (IC50 > 80 μM) and a measurable,
but low, permeability in the Caco-2 assay (apical-to-basal P =
c
2
3 nm/s). More surprisingly, potency in hWB for the
cyclopropyl amide 42 was maintained (IC50 = 16 nM), in
contrast to cyclopropyl amide-containing analogues in the
methyl sulfone series, which typically showed reduced potency
62
in hWB. Furthermore, 42 showed good in vitro mouse
microsomal stability (93% remaining after 10 min incubation).
Because 42 represented a promising profile for advancement,
the corresponding deuterated methyl amide 43 was prepared
to prevent in vivo metabolism of the N-methyl amide 42 by an
N-dealkylation pathway, a strategy that had been shown to be
62
successful in the methyl sulfone series. Unfortunately, oral
administration of 43 to C57BL/6 mice at 10 mg/kg as a
solution in a PEG-300 based vehicle afforded lower than
expected drug exposures due to a low initial maximum
concentration (Cmax = 310 nM). This finding was attributed to
low permeability rather than poor metabolic stability as 43
showed reduced permeability in the Caco-2 assay (apical-to-
basal P < 15 nm/s). To specifically address the permeability
c
issue, the pyridazine variant of 43 was prepared because
previous pyridazines within the series had routinely resulted in
62
improved permeability relative to their pyridine counterparts.
This modification resulted in the identification of 11, a
compound with significantly improved permeability (apical-to-
basal P = 70 nm/s) that translated to a 24-fold improvement
c
in Cmax relative to its pyridine counterpart 43 in mouse PK
studies (Cmax = 7.5 μM for 11 vs 310 nM for 43). In addition,
1
1 maintained excellent potency in human and mouse whole
blood (IC values = 13 and 100 nM, respectively) and showed
5
0
no significant hERG inhibition in the flux assay (IC50 > 80
μM). Having achieved a favorable potency and selectivity
profile with excellent PK properties and reduced hERG
inhibition, 11 was more fully characterized as a potential
candidate for advancement.
a
In vitro assays. Mean values determined from at least three
b
experiments unless otherwise noted. Mouse liver microsomal
stability as percent remaining after a 10 min incubation. Value
determined from one experiment. nd: not determined.
c
d
X-ray Structure Confirms Proposed Binding Mode of
C3′ Triazole 11. An X-ray structure of triazole 11 bound to
TYK2 JH2 was solved, confirming the proposed binding mode
(Figure 5a). Similar to previous X-ray cocrystal structures
within the series, the pyridazine core and pendant C6 amino
and C3 methyl amide side chains form a hydrogen bonding
triad to the hinge region of the protein. The cyclopropyl group
projects into the extended hinge region and the N-
deuteromethyl amide group occupies the “alanine pocket”
near Ala671, the binding feature critical for achieving high
6
2
unique “alanine pocket” of TYK2 JH2. Gratifyingly, 38 was
consistent with this trend, providing >1000-fold selectivity for
TYK2 JH2 when tested in HTRF binding assays against an in-
house kinase panel consisting of ∼260 diverse kinases,
including the JH1 domains of TYK2, JAK1, JAK2, and JAK3.
Unfortunately, additional off-target profiling of 38 in a high-
throughput ion channel flux assays revealed that it was a
modest inhibitor of the hERG ion channel (IC50 = 31 μΜ)
reminiscent of the initial lead 12, which had shown QTc
prolongation in telemetrized rabbits (vide supra). While the
62
TYK2 JH2 selectivity. The C2′ methoxy forms a hydrogen
bond with the conserved Lys642 with the methyl group of the
methoxy buried into the shallow C-terminal lipophilic pocket
proximal to Pro694 and Leu741 (vide supra). Remarkably, the
des-methoxy variant of 11 (not shown) was determined to be
∼100-fold less potent in TYK2 JH2 binding affinity compared
to 11 and was inactive in hWB (IC > 10 μM), illustrating the
∼
10-fold improvement in potency in hWB was anticipated to
improve the therapeutic window, we continued to explore SAR
in an attempt to further reduce the hERG inhibition. These
investigations focused on modifications of the C6 side chain
within the potent C3′ N-methyl triazole series (Table 5).
5
0
1
Differentially substituted 2-aminopyridine side chains (R )
contribution of the C2′ group to potency. A similar finding was
previously observed in comparing the C3′ amide 18 and its
des-methoxy counterpart 19 (Table 2). In this comparison, the
results are reminiscent of the methyl effect in protein−ligand
were explored such as in 39 and 40, however, a reduction in
hERG inhibition was not realized (hERG flux IC50 = 6.7 and
6 μM, respectively). More extensive modification to include
1
G
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Table 5. Select Analogues from C3′ N-Methyl Triazole Series
a
b
c
In vitro assays. Mean values determined from at least three experiments unless otherwise noted. Value determined from one experiment. Mouse
d
liver microsomal stability assay measuring percent remaining after a 10 min incubation period or half-life (T1/2) in minutes. Mean value
determined from two experiments.
Figure 5. (a) X-ray cocrystal structure of triazole 11 in TYK2 JH2 (PDB 6NZP) showing key interactions including hydrogen bonds to hinge
region of JH2 via N-methyl pyridazine amide, the d -N-methyl amide occupying the unique alanine pocket (Ala671) with the cyclopropylamide in
3
the extended hinge region, the key triazole moiety displacing structural water present in X-ray structure of 12 to form a direct hydrogen bond to
Arg738, and the orientation of the C2′ methoxy near Lys642 protruding into a shallow, C-terminal lobe pocket (displayed in gray surface
73
representation). (b) WaterMap simulation results superimposed with X-ray structure of 11 predict multiple unfavorable (ΔG > 2.2 kcal/mol)
waters W1−W5 that are likely displaced by C3′ triazole and C2′ methoxy groups of 11.
binding, sometimes referred to as the “magic methyl” effect,
where addition of a methyl in place of hydrogen results in
dramatic increase in potency due to preferred conformational
biasing and burial of the methyl group in a hydrophobic pocket
bond interaction with the protein (Lys642) that likely
contributes to the observed increase in potency. The crystal
structure also confirmed that displacement of the structural
water molecule observed in 12 by the newly installed C3′
triazole group of 11 was realized with the N-2 triazole nitrogen
engaging in a direct hydrogen bond with Arg738 as designed.
In an attempt to understand additional factors that may be
7
1,72
of the protein.
Although, in this instance, in addition to
providing favorable conformational control and hydrophobic
interactions, the C2′ methoxy also forms a direct hydrogen
H
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73
contributing to the potency of 11, a WaterMap simulation of
the TYK2 JH2 ligand binding domain was performed using the
X-ray structure of 11 with the ligand present but with the
methoxy group and triazole ring replaced with hydrogens. This
simulation predicted the presence of an energetically
unfavorable water molecule (W1) that overlapped with the
structural water molecule that was shown to be displaced by
the C3′ triazole of 11 (Figure 5b). In addition, several other
high energy waters were predicted in the ligand binding site.
Most notably are the presence of additional waters that are
likely displaced by the C3′ triazole group (W2−W4) and the
C2′ methoxy group (W5) of 11. This analysis is consistent
with SAR that highlights the importance of these groups for
potency and suggests that displacement of energetically
unfavorable waters contributes to the high affinity of 11 for
binding to the TYK2 JH2 domain.
>1000-fold selective against an in-house panel of 249 protein
and lipid kinases and pseudokinases, with the exception of
BMPR2 (IC = 193 nM) and JAK1 JH2 pseudokinase domain
5
0
(IC = 1 nM). Binding affinity assays for JAK2 and JAK3 JH2
5
0
domains were not available for evaluation. Despite its potent
affinity for JAK1 JH2, low functional activity in a JAK1/JAK3
dependent IL-2 stimulated cellular assay is observed as 11 is
∼300-fold less potent in the IL-2 assay (IC = 592 nM)
5
0
compared to the TYK2-dependent IFNα assay using the same
pSTAT5 end point. Similarly, weak potency was measured
against other JAK1-regulated pathways not dependent on
TYK2, including IL-6-stimulated phosphorylation of STAT3
63
and IL-13-stimulated STAT6 phosphorylation. JAK1/JAK3
functional potency of 11 in hWB was also determined using an
IL-2 stimulated STAT5 phosphorylation assay (IC50 = 1900
nM), which is ∼150-fold less potent than TYK2-dependent
IFNα pSTAT5 phosphorylation in hWB (IC = 13 nM). The
Potency and Selectivity Profile of 11. Because of its
high affinity approaching the lower limits of the binding assay,
we evaluated 11 using a Morrison titration by varying the
concentration of the fluorescent probe in the TYK2 JH2
5
0
JAK1/JAK2-mediated IL-6-stimulated pSTAT3 response in
hWB was similarly weak (IC = 609 nM). This high functional
5
0
selectivity was consistently observed within the series and
suggests that either allosteric binding to JAK1 JH2 imparts
decreased functional consequences relative to TYK2 JH2
binding or binding affinity determined in the JAK1 JH2 assay is
overpredictive (higher) relative to JAK1 JH2 binding in cells.
In addition to functional selectivity over JAK1, 11 also exhibits
a high degree of functional selectivity over JAK2 relative to
other reported JAK inhibitors. In a JAK2-dependent EPO-
stimulated STAT5A phosphorylation assay in isolated TF-1
cells, 11 does not show any significant inhibition up to a
concentration of 10 μM, indicating >5000-fold selectivity for
TYK2 over JAK2 signaling. High selectivity over JAK2
signaling was also demonstrated in hWB using a JAK2-
dependent TPO-stimulated STAT5 phosphorylation assay in
platelets (IC50 > 10 μM), establishing a functional selectivity
window of >770-fold at the highest concentration tested.
Triazole 11 Shows Minimal Profiling Liabilities,
Excellent PK Properties, and Is Highly Efficacious in
Inflammatory and Autoimmune Disease Models. Having
demonstrated excellent potency and functional selectivity for
inhibition of TYK2-dependent responses, 11 was further
profiled in vitro and showed minimal liabilities and acceptable
PK properties for further advancement (Table 7). Incubation
in liver microsomes shows excellent stability (T1/2 > 120 min)
across multiple species including human, mouse, rat, monkey,
dog, and rabbit. Good permeability is also observed in a Caco-
6
3
assay. This analysis gave results that were consistent with
competitive binding and determined a dissociation constant
63
(Ki) of 0.02 nM for 11. In addition, 11 was evaluated in both
binding and human cellular assays to determine selectivity
63
within the JAK family and across the kinome (Table 6).
Consistent with earlier analogues, 11 is highly specific for
binding to TYK2 JH2 and does not show any significant
binding affinity in the canonical TYK2, JAK1, JAK2, and JAK3
JH1 assays (IC50 values >10000 nM). Furthermore, 11 is
Table 6. Selectivity Profile of 11 in Binding and Human
Cellular Assays
binding or cellular
a
assay
stimulus (pathway)
IC , nM (selectivity)
50
TYK2 JH2
(TYK2)
(JAK1)
(TYK2)
(JAK1)
(JAK2)
0.2 ± 0.1
1.0 ± 0.1
>10000
>10000
>10000
>10000
JAK1 JH2
TYK2 JH1
JAK1 JH1
JAK2 JH1
JAK3 JH1
(JAK3)
in-house selectivity
panel of 249 kinases
(kinome selectivity)
all >1000 fold-selective
except BMPR2
(
∼960-fold)
b
PBMC
PBMC
TF-1
PBMC
PBMC
PBMC
IFNα (TYK2)
2
9
c
IL-23 (TYK2)
2
assay with moderate efflux (apical-to-basal P = 73 nm/s;
c
d
EPO (JAK2)
>10000 (>5000×)
592 (∼300×)
615 (∼310×)
basal to apical P = 740 nm/s; efflux ratio ∼10). Assessment of
c
b
IL-2 (JAK1/JAK3)
b
IL-6 (JAK1)
IL-13 (JAK1)
Table 7. Profiling Properties of Triazole 11
e
2091 (>1000×)
profiling assays
11
b
a
human whole blood
IFNα (TYK2)
TPO (JAK2)
13
liver microsomal T1/2 (min)
>120 all species
73 ± 7
740 ± 140
f
b
>10000 (>770×)
1900 (∼150×)
609 (∼47×)
Caco-2 P (nm/s), A-to-B
c
b
b
IL-2 (JAK1/JAK3)
IL-6 (JAK1/JAK2)
Caco-2 P (nm/s), B-to-A
c
g
CYP450 inhibition IC s (1A2, 2C9, 2C19, all >40 μM
50
2
D6, 3A4)
a
In vitro assays. Mean values determined from at least three separate
CYP Induction/PXR-TA EC50
hERG (% inhibition at 10 μM)
protein binding (%free)
>40 μM
26 ± 11
b
c
experiments unless otherwise noted. Measuring STAT5 phosphor-
ylation in CD3+ T-cells as end point. Measuring STAT3
phosphorylation in CD161+ CD3+ T-cells. Measuring STAT5A
c
13% human, 12% cyno,
15% mouse
d
e
d
phosphorylation in TF-1 cells (n = 2). Measuring STAT6
phosphorylation in mononuclear cells (n = 2). Measuring STAT5
phosphorylation in platelets. Measuring STAT3 phosphorylation in
aq solubility at pH 7.4
5.2 μg/mL
f
g
a
b
c
Human, rat, mouse, dog, cyno, and rabbit. A = apical, B = basal. n
+
d
CD3 T-cells.
= 7 in patch clamp assay. Crystalline free base.
I
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Table 8. In Vivo PK Summary for Triazole 11
a
b
iv PK parameters
po PK parameters
1
species
mouse
dog
CL (mL min− kg−1
)
Vss (L/kg)
T1/2 (h)
MRT (h)
dose (mg/kg)
Cmax (μM)
AUC (μM × h)
F (%)
13.2
6.8
4.8
2.9
2.3
2
4.2
4.6
5.3
3.6
5.5
7
10
10
10
7.5
6.9
5.9
36.4
73.6
75.7
122
128
87
c
c
monkey
a
b
IV administration at 1 mg/kg in 80% PEG 400/20% water unless otherwise noted. PO administration at 10 mg/kg as a solution in ethanol/
c
TPGS/PEG 300 (5:5:90). IV administration at 2 mg/kg.
74
Figure 6. (a) Dose−response of 11 showing inhibitory effect in an IL-23 induced psoriasis-like acanthosis mouse model compared to an anti-IL-
75
2
3 adnectin as a positive control. Values are presented as means ± SEM, eight mice per group. Statistical analysis was performed with one-way
ANOVA. Vehicle, 5:5:90, EtOH:TGPS:PEG300. (b) Dose−response of histological scores showing dose-dependent inhibition of epidermal
hyperplasia (acanthosis) and inflammatory cellular infiltration. (c) Inflammatory cytokine expression by quantitative PCR analysis of skin biopsies
showing dose-dependent inhibition.
the potential for drug−drug interactions (DDI) indicates a low
overall risk, as no significant inhibition of multiple cytochrome
P450 (CYP) isozymes (1A2, 2C9, 2C19, 2D6, and 3A4) or
induction of CYP3A4 is observed up to the highest
concentration tested (40 μM). Testing of 11 in the hERG
potassium channel patch clamp assay gives a low percent
inhibition (26 ± 11% at 10 μM), suggesting a low potential for
in vivo clearance rates in mouse, dog, and monkey of 13.2, 6.8,
−
1
−1
and 4.8 mL min
kg , respectively. Low volume of
distribution (V ) is also observed in the 2−3 L/kg range
ss
with moderate half-lives of ∼4−5 h across species. When
administered orally at 10 mg/kg, 11 is well absorbed with
excellent exposures and high bioavailability (%F > 85) in
mouse, dog, and monkey. Circulating primary amide
metabolite formation from N-dealkylation of the deutero-
methyl amide of 11 was near the lower limits of detection (<2
nM) in these studies, consistent with the effective blocking of
this metabolic pathway by deuteration as previously reported
QT prolongation-associated cardiovascular risk. Protein bind-
c
ing is in the moderate range (12−15% free) across species
including human, monkey, and mouse. Aqueous solubility of
the crystalline free base form of 11 is low at 5.2 μg/mL but was
deemed acceptable for advancement into preclinical studies.
Good overall PK parameters were observed with 11 in
preclinical studies across multiple species (Table 8).
Consistent with the observed low rate of metabolism in the
microsomal assays (T1/2 > 120 min), 11 affords low to modest
62
within the series.
In mice, 11 was evaluated in a skin inflammation (psoriasis-
like) model of IL-23-driven acanthosis whereby repetitive
intradermal injections of IL-23 into the ears of mice induces a
profound epidermal hyperplasia (acanthosis) and inflammatory
J
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cellular infiltration mediated by Th17 cells and IL-22, similar
Scheme 1. General Preparation of N-Methyl Nicotinamides
and N-Methyl Pyridazine-3-Carboxamides
74
a
to the underlying mechanisms in psoriasis (Figure 6). In this
model, 11 dose-dependently protects from IL-23-induced
acanthosis in mice, with the 15 mg/kg oral dose of 11
administered twice-daily for 9 days proving to be as effective as
75
an anti-IL-23 adnectin as a positive control (Figure 6a). The
0 mg/kg twice-daily oral dose is more effective than the anti-
3
IL-23 adnectin at providing protection. Histological evaluation
shows that the epidermal hyperplasia and the inflammatory
cellular infiltration is also inhibited in a dose-dependent
manner, with the high dose of 30 mg/kg twice daily providing
protection more effectively than the anti-IL-23 adnectin
positive control (Figure 6b). Quantitative polymerase chain
reaction (PCR) analysis of skin biopsies reveals 11 to be quite
effective at blocking inflammatory cytokine expression,
including IL-17A, IL-21, and subunits of IL-12 and IL-23
a
(Figure 6c). PK measurements on study animals shows that
Reagents and conditions: (a) NaHMDS or LiHMDS, ArNH , THF,
2
the 7.5, 15, and 30 mg/kg twice-daily doses provides drug
levels at or above the in vitro mouse whole blood IC value of
rt; 53−92%; (b) Pd (dba) , XantPhos, Cs CO DMA or 1,4-dioxane,
2
3
2
3,
130−145 °C, 1 h, 23−64%; (c) Pd(OAc)
or Pd (dba) BrettPhos or
CO or Cs CO or LiHMDS, 1,4-dioxane, 85−110 °C, 1 h,
2 3 2 3
5
0
2
2 3
1
00 nM (IFNα-induced pSTAT1) for 19, 21, and 24 h,
dppf, K
40−97%.
respectively. In addition to preclinical models of psoriasis, 11
has also been shown to be highly efficacious in murine models
of colitis and lupus. These results demonstrate in an anti-
CD40-induced colitis model in severe-combined-immunodefi-
cient-diseased (SCID) mice, 11 administered 50 mg/kg twice-
daily provides inhibition of peak weight loss (IL-12 driven) by
63
Specific substitutions (R′) were explored by either incorporat-
ing on the aniline before C-4 coupling to afford 45 or at a later
stage after C6-coupling depending on functional group
compatibility and/or ease of intermediate isolations. This
synthetic route proved to be highly versatile for both SAR
exploration as well as larger scale preparation of key
compounds for advancing through preclinical studies.
9
9% and inhibition of histological scores by 70% comparable
to an anti-p40 monoclonal antibody control. Furthermore,
when orally administered up to a maximum 30 mg/kg once-
daily dose in a three-month lupus disease model using NZB/W
lupus-prone mice, 11 is well-tolerated and highly efficacious in
protecting from nephritis. In this latter study, efficacy is well-
correlated with inhibition of type I IFN-dependent gene
expression in both whole blood and kidneys in study mice and
is at least as effective as a blocking anti-IFNαR antibody.
In summary, 11 is efficacious against both type I IFN-, IL-
As an example, the synthesis of 11 is highlighted (Scheme
2). Beginning with commercially available methyl-2-hydroxy-3-
nitrobenzoate (47), methylation followed by ammonolysis of
the intermediate methyl ester afforded amide 48. The triazole
group was readily installed by reaction of 48 with DMF-DMA
followed by condensation with hydrazine hydrate to afford 49.
Methylation of the triazole with methyl iodide in the presence
of potassium carbonate afforded a 2:1 mixture of the desired
regioisomer 50 and undesired regioisomer 51, respectively.
The regioisomers could be separated by supercritical fluid
chromatography (SFC) to afford the desired, isomerically pure
50 in 67% overall yield. Attempts to improve the alkylation
regioselectivity found that using potassium hexamethylsilazide
as the base in place of potassium carbonate and using THF in
place of DMF, as the solvent afforded an improved
regioselectivity of ∼8:1 in favor of the desired isomer 50.
This result obviated separation of the isomers by SFC
chromatography on a larger scale, as the crude product
enriched in the desired isomer could be crystallized to afford
isomerically pure 50. With pure 50 in hand, nitro group
reduction under standard palladium-catalyzed hydrogenation
conditions afforded aniline 52 in 92%, which was subsequently
1
2-, and IL-23-dependent pathobiology in preclinical studies in
mice. Efficacy from oral administration of 11 in all disease
models studied is well correlated with coverage of the whole
blood IC50 value over the dosing intervals and is at least as
effective as the blocking antibody controls used in these
studies.
Chemistry. Preparation of the N-methyl nicotinamide and
N-methyl pyridazine-3-carboxamide analogues in this work
62
utilized similar chemistry methods as previously reported.
The analogues were prepared from the 4,6-dichloro-N-
(
(
methyl)nicotinamide (X = C) or pyridazine-3-carboxamide
X = N) intermediate 44 as the nondeuteromethyl amide (R =
CH ) or deuteromethyl variant (R = CD ) (Scheme 1).
3
3
Various substituted anilines (ArNH ) were coupled under
2
basic conditions and in moderate to high yields using lithium
or sodium hexamethylsilazide in THF at ambient temperature
to give coupling at the C4 position exclusively to afford 45. A
second coupling to install the C6 amino substituents was
performed using the palladium-catalyzed Buchwald−Hartwig
62
coupled to 53 using lithium hexamethyldisilazide as the base
at ambient temperature to afford the penultimate intermediate
54 in 66% yield. Coupling of 54 with cyclopropyl amide (55)
under palladium-catalyzed Buchwald−Hartwig reaction con-
76,77
amination reaction
to afford the final compounds 46.
76,77
Initial C6 couplings were performed using XantPhos as the
palladium ligand under high temperatures (130−145 °C),
which resulted in low yields in some cases. However, more
efficient coupling for problematic cases could be performed at
lower temperatures (85−110 °C) using BrettPhos as the
palladium ligand or 1,1′-bis(dicyclohexylphosphino)ferrocene
ditions
using XantPhos as the palladium ligand afforded 11
in 46% yield. A significant improvement in this final coupling
reaction was identified by replacing the XantPhos as a ligand
with 1,1′-bis(dicyclohexylphosphino)ferrocene (dppf) and
using aqueous potassium triphosphate in place of cesium
carbonate as the base. These modified conditions permitted a
lower reaction temperature and afforded an improved 76%
(
dppf) as a ligand in the case of cyclopropyl amide couplings.
K
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Journal of Medicinal Chemistry
Article
a
Scheme 2. Preparation of the Clinical TYK2 Inhibitor 11
a
Reagents and conditions: (a) CH I, K CO , DMF, rt, 98%; (b) aq NH OH, NH in MeOH, rt, 86%; (c) (i) DMF-DMA, 95 °C, (ii) hydrazine
3
2
3
4
3
hydrate, EtOH, AcOH, rt, 69% over 2 steps; (d) CH I, K CO , DMF, rt, 91%, (∼2:1 mixture); (e) KHMDS, CH I, THF, rt, 58% of pure 50; (f)
3
2
3
3
5
% Pd-C, 1 atm H , EtOH, rt, 92%; (g) LiHMDS, THF, rt, 66%; (h) Pd (dba) , XantPhos, Cs CO , 1,4-dioxane, 130 °C, 46%; (i) Pd (dba) ,
2 2 3 2 3 2 3
dppf, K PO dioxane, 85 °C, 76%.
3
4,
yield in the preparation of multigram quantities of 11 for
advanced preclinical studies.
that deuterium was incorporated during the de novo design
and optimization process to shunt a metabolic pathway in vivo
and address a specific metabolite concern. Other notable
strategies in our efforts include heterocyclic core modifications
CONCLUSION
■
(
pyridine to pyridazine) to provide optimal permeability and
The discovery of the TYK2 inhibitor 11 as the first known
pseudokinase-specific ligand to enter clinical development has
been described. This work was achieved through optimization
of a series of highly selective N-methyl nicotinamides and N-
methyl pyridazine-3-carboxamides as a novel class of potent
allosteric inhibitors that act by binding to and stabilizing the
pseudokinase (JH2) domain of TYK2. As a result of this
unique mechanism, 11 is highly differentiated from all other
reported JAK/TYK2 inhibitors due to its ability to achieve an
unprecedented level of selectivity for TYK2, especially over
JAK1, JAK2, and JAK3. Notably, several key discoveries and
innovative strategies were important in identifying 11 as a
suitable candidate for clinical development. A chemogenomics
approach was used to find initial lead molecules that
allosterically inhibit TYK2-dependent IL-23 signaling by virtue
bioavailability and the use of a cyclopropyl carboxamide as a
replacement for the C6 amino heterocycles to reduce
62
undesirable hERG ion channel activity. The versatility of
the cyclopropane fragment in overcoming multiple roadblocks
79
in drug discovery has been previously reported. In this
instance, it is notable that the cyclopropyl carboxamide group
was found to be an effective replacement for the 2-
aminopyridine side chain and reduced hERG affinity, likely
3
80
due to decreased aromaticity and increased sp character.
Finally, an innovative structure-based drug design strategy
targeted at displacing a structural water molecule observed
within the TYK2 JH2 binding site led to C3′ substituted
analogues having significantly enhanced potency and ultimately
resulting in the identification of 11 containing a C3′ N-methyl
triazole group. These results highlight the potential of targeting
the displacement of water molecules from ligand binding sites
as a successful strategy for drug design and optimization and
suggests the application of computational methods to predict
hydration thermodynamics as potentially useful tools in
60
of their binding to the TYK2 pseudokinase domain. This key
discovery spawned a medicinal chemistry effort that identified
additional novel leads for optimization including the N-methyl
61,62
nicotinamide series.
A critical methyl amide group within
this series was discovered that surprisingly afforded a high level
of selectivity by accessing an atypical pocket created by a rare
alanine residue (“alanine pocket”) in the ligand binding
domain of TYK2 JH2. To preserve the selectivity of the methyl
amides in vivo, deuterium was incorporated into the methyl
group to block an N-demethylation metabolic pathway that
generated a less selective primary amide metabolite. While
incorporation of deuterium into drug molecules is not an
uncommon practice, the large majority of known examples
typically involve deuterium incorporation into already existing
drug developmental candidates or marketed drugs in an
attempt to decrease overall clearance rates and improve PK
81−85
directing medicinal chemistry efforts.
Additional allosteric
TYK2 inhibitors from our efforts will be reported in due
course. In summary, 11 has been identified as a highly potent
and selective allosteric TYK2 inhibitor having excellent PK
properties across species with minimal profiling liabilities and
is orally efficacious with dose-dependent activity in a murine
disease model of psoriasis. Significant activity has also been
observed with 11 in other murine autoimmune disease models
63
of colitis and lupus. On the basis of these findings, 11 has
been advanced into clinical studies as a potential treatment for
patients suffering from a spectrum of IFN- and IL-12/IL-23
driven inflammatory and autoimmune diseases.
7
8
properties. Our approach is unique from these strategies in
L
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
534 mg, 0.924 mmol), and cesium carbonate (4.01 g, 12.3 mmol)
EXPERIMENTAL SECTION
■
were added. The vessel was evacuated three times (backfilling with
nitrogen) and then sealed and heated to 130 °C using a preheated oil
bath for 140 min. The reaction was filtered through diatomaceous
earth (washing with ethyl acetate), and the resulting filtrate was
concentrated to obtain the crude product. This material was adsorbed
onto diatomaceous earth using dichloromethane and was purified
using automated chromatography (100% ethyl acetate) to provide 11
All animal research was conducted in accordance with institutional
guidelines as defined by Institutional Animal Care and Use
Committee for U.S. institutions and with the approval of the
Bristol-Myers Squibb Animal Care and Use Committee. Mice were
housed under a 12 h/12 h light/dark cycle and provided standard
access to rodent chow diet and fresh drinking water ad libitum.
All biochemical potencies and selectivities were determined using
homogeneous time-resolved fluorescence (HTRF) assays where
compounds were shown to compete with a fluorescent probe for
binding to human recombinant JAK1, JAK2, JAK3, and TYK2 JH1
domain proteins in addition to TYK2 and JAK1 JH2 protein domains.
Dose−response curves were generated to determine the concen-
tration required for inhibiting 50% of the HTRF signal (IC ) as
derived by nonlinear regression analysis. Cellular potencies and
selectivities were determined using stably integrated STAT-dependent
luciferase reporter assays in T-cells using IFNα-stimulation for
measuring TYK2/JAK1 dependent signaling and IL-23 stimulation
for measuring TYK2/JAK1 dependent signaling. JAK2 dependent
signaling was measured in TF-1 cells using GM-CSF stimulation.
Dose−response curves were generated to determine the concen-
tration required to inhibit 50% of cellular response (IC ) as derived
1
as a near-white solid (1.22 g, 46% yield). H NMR (500 MHz,
CDCl ) δ 10.99 (s, 1H), 9.81 (s, 1H), 8.23 (s, 1H), 8.11 (s, 1H), 8.03
3
(s, 1H), 7.80 (dd, J = 7.9, 1.6 Hz, 1H), 7.52 (dd, J = 7.9, 1.5 Hz, 1H),
7
1
.26 (t, J = 7.8 Hz, 1H), 4.00 (s, 3H), 3.81 (s, 3H), 1.88−1.82 (m,
13
H), 1.16−1.06 (m, 2H), 0.94−0.83 (m, 2H). C NMR (500 MHz,
CDCl ) δ 173.2, 166.8, 160.3, 155.8, 151.5, 146.1, 143.9, 134.9, 132.4,
3
5
0
1
27.0, 126.0, 124.5, 123.4, 97.9, 61.5, 36.4, 26.0−24.1 (m, J = 21.8
+
Hz, 1C), 15.9, 8.7 (s, 2C). LCMS (E+) m/z: 426.1 (MH ), R = 0.62
min. An analytically pure sample was obtained by SFC. Anal. Calcd
for C H D N O ·0.06H O·0.03CH OH: C 56.24; H 5.32; N 26.20.
Found: C 55.86; H 5.22; N 26.12. HRMS calcd for C H D N O [M
T
2
0
19
3
8
3
2
3
2
0
20
3
8
3
+
+
H] , 426.20759; found, 426.20734.
Alternative Coupling Conditions for Synthesis of 11. A mixture of
5
4 (17.52 g, 46.5 mmol) and cyclopropane carboxamide 55 (4.75 g,
5.8 mmol) were taken up in 1,4-dioxane (186 mL) and nitrogen
5
0
5
by nonlinear regression analysis. Potencies and selectivities for JAK-
dependent signaling were also measured in human and mouse whole
blood using specific cytokine stimulations and measuring the
phosphorylation of specific STAT proteins by cellular staining and
flow cytometry. Experimental details for all assays have been
bubbled through the slurry for about 10−15 min. Pd (dba) (1.06 g,
1
2
3
.16 mmol) and 1,1′-bis(dicyclohexylphosphino)ferrocene (1.34 g,
2
.32 mmol) were then added with a continued nitrogen flow for an
additional ∼5 min. An aqueous solution of potassium phosphate
tribasic (2 M, 58.1 mL, 116 mmol) was then added in one portion
and the reaction heated to 85 °C for 48 h. The reaction was allowed
to cool to room temperature before diluting with ethyl acetate (200
mL) and water (200 mL). The separated aqueous phase was further
extracted with ethyl acetate (3 × 200 mL), and the combined organic
layers were then dried (MgSO ) and concentrated under vacuum to
give the crude product. The crude product was then taken up in Me-
THF (400 mL) and ethyl acetate (400 mL), then N-acetyl cysteine
60,63
previously reported.
All compounds active in biological assays
were electronically filtered for structural attributes common to pan
assay interference compounds (PAINS) and were found to be
86
negative.
Synthesis. Chemistry General Methods and Compound
Characterization. All reagents and starting materials were obtained
from commercial suppliers and used without further purification
unless otherwise stated. Reactions were run under an atmosphere of
nitrogen and at ambient temperature unless otherwise noted.
Reaction progress was monitored using a variety of LC instruments
equipped with electrospray positive ionization detectors. Reported
liquid chromatography retention times (R ) were established using
the following conditions: Column, Waters Acquity UPLC BEH C18,
.1 mm × 50 mm, 1.7 μm particles. Mobile phase A: 2:98
acetonitrile:water with 10 mM ammonium acetate. Mobile phase B:
4
(
10% aqueous solution, 300 mL) was added and the biphasic mixture
was allowed to stir at room temperature overnight. The layers were
separated and the aqueous phase was extracted with ethyl acetate (3 ×
2
00 mL). The combined organics were washed with 10% ammonium
T
hydroxide solution (500 mL), and the organic layer then dried
MgSO ) and evaporated under vacuum to afford the crude product
(
2
4
which was purified by MPLC using a RediSep 750 g gold silica gel
column and eluting with a gradient of solvent A (dichloromethane)
and solvent B (20% methanol in dichloromethane) at a flow rate of
9
5
8:2 acetonitrile:water with 10 mM ammonium acetate. Temperature:
0 °C. Gradient: 0−100% B over 1 min, then a 0.5 min hold at 100%
3
00 mL/min. The desired product was collected at 30% A to B
B. Flow: 0.8 mL/min. Detection: MS and UV (220 nm). In some
cases, alternative liquid chromatography conditions (specified as
condition B or condition C) were used. Condition B = column:
Waters Acquity BEH C18, 2.1 mm× 50 mm, 1.7 μm particles. Mobile
phase A: water. Mobile phase B: acetonitrile with buffer, 0.05% TFA.
Temperature: 50 °C. Gradient range: 2%−98% B (0−1 min), 98% B
concentration and fractions were concentrated under vacuum to
afford 11 as a cream-colored solid (15.0 g, 76% yield).
4
-((3-Cyano-2-methoxyphenyl)amino)-6-((5-fluoropyridin-2-yl)-
amino)-N-methyl-nicotinamide (15). To a suspension of 4-((3-
carbamoyl-2-methoxyphenyl)amino)-6-((5-fluoropyridin-2-yl)-
amino)-N-methylnicotinamide (17, 21 mg, 0.051 mmol) in dichloro-
methane (0.2 mL) and THF (0.2 mL) was added Burgess reagent
(
to 1.5 min), then 98%−2% B (to 1.6 min). Flow: 1.11 mL/min.
Detection: MS and UV (220 nm). Condition C = column: Waters
Acquity UPLC BEH C18, 2.1 mm× 50 mm, 1.7 μm particles. Mobile
phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate.
Mobile phase B: 95:5 acetonitrile:water with 10 mM ammonium
acetate. Temperature: 50 °C. Gradient: 2−98% B over 1 min, then a
(
24.4 mg, 0.10 mmol) in one portion under nitrogen, and the
resulting mixture was allowed to stir overnight at room temperature.
HPLC and LCMS indicated only ∼15% conversion of starting
material to afford the desired product (observed MH+ of 393).
Therefore, the reaction was concentrated to remove the THF and
dichloromethane and acetonitrile (0.3 mL) was added followed by
additional Burgess reagent (24.4 mg, 0.10 mmol). After 4 h at room
temperature, the reaction mixture became a clear solution and HPLC
analysis indicated completed conversion of starting material to the
desired product. The reaction was concentrated, diluted with DMF (1
mL), filtered, and was purified by reverse phase preparative LCMS
with the following conditions: Column, Waters XBridge C18, 19 mm
0
(
.6 min hold at 98% B. Flow: 0.8 mL/min. Detection: MS and UV
1
220 nm). Proton ( H NMR) magnetic resonance spectra were
obtained in CDCl , CD OD, or DMSO-d at 400 MHz at 298 K
3
3
6
unless otherwise noted. The following abbreviations were utilized to
describe peak patterns when appropriate: br = broad, s = singlet, d =
doublet, q = quartet, t = triplet, and m = multiplet. All final
compounds used for testing in assays and biological studies had
purities that were determined to be >95% by HPLC or LCMS based
on ultraviolet detection at 220 nm.
×
200 mm, 5 μm particles. Mobile phase A: 5:95 acetonitrile:water
with 10 mM ammonium acetate. Mobile phase B: 95:5 acetonitrile:-
water with 10 mM ammonium acetate. Gradient: 15−100% B over 20
min, then a 5 min hold at 100% B. Flow: 25 mL/min. Fractions
containing the desired product were combined and dried via
Synthesis of 6-(Cyclopropanecarboxamido)-4-((2-methoxy-3-(1-
methyl-1H-1,2,4-triazol-3-yl)phenyl)-amino)-N-(methyl-d )-
3
pyridazine-3-carboxamide (11). A mixture of 54 (2.3 g, 6.2 mmol)
and cyclopropane carboxamide (55, 1.05 g, 12.3 mmol) was dissolved
in dioxane (62 mL), and Pd (dba) (564 mg, 0.616 mmol), XantPhos
1
centrifugal evaporation to afford 7.2 mg (35%) of 15. H NMR
2
3
M
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
=
7
7
500 MHz, DMSO-d ) δ 10.79 (s, 1H), 9.87 (br s, 1H), 8.58 (br d, J
3.1 Hz, 1H), 8.50 (s, 1H), 8.17 (d, J = 3.1 Hz, 1H), 7.87 (dd, J =
through a Millipore filter. The resulting solution was subjected to
purification by reverse-phase preparative LCMS using the following
conditions: Column, Waters XBridge C18, 19 mm × 200 mm, 5 μm
particles. Mobile phase A: 5:95 acetonitrile:water with 10 mM
ammonium acetate. Mobile phase B: 95:5 acetonitrile:water with 10
mM ammonium acetate. Gradient: 100% B over 20 min, then a 5 min
hold at 100% B. Flow: 25 mL/min. Fractions containing the desired
6
.9, 1.2 Hz, 1H), 7.77−7.58 (m, 3H), 7.50 (d, J = 7.9 Hz, 1H), 7.43−
.33 (m, 1H), 3.92 (s, 3H), 2.79 (d, J = 4.9 Hz, 3H). LCMS (E+) m/
+
z: 393.1 (MH ), R = 1.53 min.
T
3
-((2-((5-Fluoropyridin-2-yl)amino)-5-(methylcarbamoyl)-
pyridin-4-yl)amino)-2-methoxy-benzoic Acid (16). A solution of 4,6-
dichloro-N-methylnicotinamide (2 g, 9.75 mmol) and 3-amino-2-
62
product were combined and dried via centrifugal evaporation to afford
87
1
methoxybenzoic acid (1.96 g, 11.7 mmol) in 20 mL of DMA was
cooled in an ice bath, and a solution of lithium bis(trimethylsilyl)-
amide (1 M in THF, 39.0 mL, 39.0 mmol) was added dropwise via
syringe over 3−5 min. After the addition was complete, the ice bath
was removed and the dark-brown mixture was allowed to warm to
room temperature and stir for ∼30 min. LCMS analysis at this time
indicated the complete conversion to afford a single major component
that was consistent with the formation of the desired coupled
7.6 mg (48%) of 17. H NMR (500 MHz, DMSO-d ) δ 10.64 (s,
6
1H), 10.06 (s, 1H), 8.55 (br s, 1H), 8.45 (s, 1H), 8.21−8.11 (m, 1H),
7.75 (br s, 1H), 7.63 (br dd, J = 7.6, 1.5 Hz, 4H), 7.56 (br s, 1H),
7.36−7.30 (m, 1H), 7.29−7.24 (m, 1H), 3.73 (s, 3H), 2.78 (d, J = 4.3
+
Hz, 3H). LCMS (E+) m/z: 411.2 (MH ), R = 1.23 min.
T
6-((5-Fluoropyridin-2-yl)amino)-4-((2-methoxy-3-
(methylcarbamoyl)phenyl)-amino)-N-methylnicotinamide (18).
Prepared from 16 in a manner similar to 17 using methylamine in
+
1
intermediate (observed MH 336/338). The reaction was cooled in
place of ammonium chloride to afford 7.6 mg of 18. H NMR (500
an ice bath and was quenched by the slow addition of cold water
MHz, DMSO-d ) δ 10.64 (s, 1H), 9.78 (s, 1H), 8.51 (br d, J = 4.3 Hz,
6
(
∼150 mL), giving a dark-amber colored solution. The pH of the
1H), 8.45 (s, 1H), 8.28−8.19 (m, 1H), 8.14 (d, J = 3.1 Hz, 1H),
solution was made acidic (pH ∼ 1−2) by a dropwise addition of 6 N
aq HCl, causing a heavy tan precipitate to form. The resulting slurry
was allowed to stir for 1 h, then the solid was collected by vacuum
filtration and the solid was rinsed with additional water (∼50 mL).
The solid was allowed to partially air-dry in the funnel for 2−3 h and
then was slurried in methanol (∼50 mL). Ethyl acetate (∼100 mL)
was added initially, giving complete dissolution followed by
precipitation of a solid. The solid was collected by vacuum filtration
and dried in a funnel overnight to afford 1.0 g of an off-white solid.
The resulting filtrate was concentrated under vacuum to afford an
additional 2 g of additional solid (overall yield 3 g, 92%). MS (E+) m/
7.74−7.57 (m, 4H), 7.30−7.20 (m, 2H), 3.71 (s, 3H), 2.78 (dd, J =
+
8.2, 4.6 Hz, 6H). LCMS (E+) m/z: 425.2 (MH ), R = 1.17 min.
T
6-((5-Fluoropyridin-2-yl)amino)-4-((3-(methylcarbamoyl)-
phenyl)-amino)-N-methyl-nicotinamide (19). Prepared from 4,6-
62
dichloro-N-methylnicotinamide using similar procedures as de-
scribed for the preparation of 17 and using 3-amino-N-methyl-
benzamide in place of 3-amino-2-methoxybenzoic acid to afford 8.7
1
mg of 19. H NMR (500 MHz, DMSO-d
) δ 10.63 (s, 1H), 9.81 (s,
6
1H), 8.48 (s, 2H), 8.56−8.43 (m, 1H), 8.10 (d, J = 2.5 Hz, 1H), 7.78
(s, 1H), 7.76−7.72 (m, 1H), 7.68−7.59 (m, 2H), 7.57−7.53 (m, 1H),
7.49 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 2.81−2.76 (m, 6H).
4-(4-Carbamoyl-2-methoxyphenylamino)-6-(5-fluoropyridin-2-
ylamino)-N-methyl-nicotinamide (20). To a stirred solution of 4,6-
+
z: 336/338 (MH with chloride isotope pattern).
A reaction vial was charged with solid obtained from previous step
62
dichloro-N-methylnicotinamide (1.0 g, 4.9 mmol) in DMA (30 mL)
was added 4-amino-3-methoxybenzoic acid (1.22 g, 7.32 mmol)
followed by addition of a solution of sodium bis(trimethylsilyl)amide
(1 M in THF, 36.6 mL, 36.6 mmol). The reaction mixture was stirred
for 2 h, at which point the THF was removed in vacuo and HCl (1 M
aq) was added to adjust pH to ∼5. The resulting heterogeneous slurry
was filtered to collect the solid as crude 4-(2-chloro-5-
(methylcarbamoyl)pyridin-4-ylamino)-3-methoxybenzoic acid. The
filtrate was extracted with dichloromethane and washed with water
(3×), dried, concentrated, and purified by automated silica gel
chromatography (0−100% MeOH/DCM) to yield additional 4-(2-
(
130 mg, 0.39 mmol), 5-fluoropyridin-2-amine (60.8 mg, 0.54 mmol),
BrettPhos (8.31 mg, 0.015 mmol), and Pd (dba) (7.1 mg, 7.8 μmol),
2
3
and the contents were flushed with nitrogen. Dioxane (1.5 mL) and
DMA (0.5 mL) were then added, and the resulting slurry was sparged
with additional nitrogen for ∼1 min. A solution of lithium
bis(trimethylsilyl)amide (1 M in THF, 0.85 mL, 0.85 mmol) was
added, and the resulting dark-amber-colored solution was heated in a
preheated heating block at 110 °C for 2 h. The reaction mixture was
cooled to room temperature and was analyzed by LCMS, which
indicated formation of a major product that was consistent with the
desired product (observed MH+ 412). The reaction mixture was
diluted with water (10 mL) and made slightly acidic (pH ∼ 3) by
slowly adding 1N aq HCl dropwise, causing a solid to precipitate. The
slurry was allowed to stir at room temperature overnight, and then the
solid was collected by vacuum filtration and dried under vacuum to
chloro-5-(methylcarbamoyl)pyridin-4-ylamino)-3-methoxybenzoic
1
acid (0.87 g total, 53%). H NMR (500 MHz, DMSO-d
) δ 10.63 (s,
6
1H), 9.81 (s, 1H), 8.48 (s, 2H), 8.56−8.43 (m, 1H), 8.10 (d, J = 2.5
Hz, 1H), 7.78 (s, 1H), 7.76−7.72 (m, 1H), 7.68−7.59 (m, 2H),
7.57−7.53 (m, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H),
afford 128 mg (80%) of 16 as a beige solid. MS (E+) m/z: 412.1
+
+
(MH ). An analytical sample for testing was prepared by purification
2.81−2.76 (m, 6H). LCMS (E+) m/z: 336 (MH ).
by preparative LC/MS with the following conditions: Column,
Waters XBridge C18, 19 mm × 250 mm, 5 μm particles. Mobile phase
A: 5:95 acetonitrile:water with 10 mM ammonium acetate. Mobile
phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate.
Gradient: 0−100% B over 25 min, then a 5 min hold at 100% B. Flow:
To a mixture of 5-fluoropyridin-2-amine (217 mg, 1.94 mmol) and
4-(2-chloro-5-(methylcarbamoyl)pyridin-4-ylamino)-3-methoxyben-
zoic acid (500 mg, 1.49 mmol) was added DMA (10 mL) followed by
Pd (dba) (136 mg, 0.15 mmol), XantPhos (172 mg, 0.30 mmol),
2 3
and cesium carbonate (0.970 g, 2.98 mmol). The vessel was then
evacuated and backfilled with nitrogen three times and then heated to
145 °C for 2 h. The crude product was filtered and then concentrated
to afford an oil as the crude product. This material was adsorbed onto
silica gel, dried and then purified using automated chromatography
(0−100% MeOH/DCM) to provide 300 mg (49%) of 4-((2-((5-
fluoropyridin-2-yl)amino)-5-(methylcarbamoyl)pyridin-4-yl)amino)-
2
0 mL/min. Fractions containing the desired product were combined
1
and dried via centrifugal evaporation to afford pure 16. H NMR (500
MHz, DMSO-d ) δ 10.68 (s, 1H), 9.81 (s, 1H), 8.52 (q, J = 4.1 Hz,
6
1
7
3
0
H), 8.47 (s, 1H), 8.15 (d, J = 3.1 Hz, 1H), 7.74−7.67 (m, 3H),
.66−7.60 (m, 1H), 7.41−7.37 (m, 1H), 7.31−7.26 (m, 1H), 3.76 (s,
+
H), 2.78 (d, J = 4.3 Hz, 3H). LCMS (E+) m/z: 412.1 (MH ), R =
T
+
.75 min.
3-methoxybenzoic acid. LCMS (E+) m/z: 412 (MH ).
4
-((3-Carbamoyl-2-methoxyphenyl)amino)-6-((5-fluoropyridin-
To a DMF (1 mL) solution containing 4-((2-((5-fluoropyridin-2-
yl)amino)-5-(methylcarbamoyl)-pyridin-4-yl)amino)-3-methoxyben-
zoic acid (30 mg, 0.073 mmol), ammonium chloride (7.8 mg, 0.15
mmol), and N,N-diisopropylethylamine (51 μL, 0.29 mmol) was
added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU, 36 mg, 0.095 mmol) and the reaction
2
2
-yl)amino)-N-methyl-nicotinamide (17). 3-((2-((5-Fluoropyridin-
-yl)amino)-5-(methylcarbamoyl)pyridin-4-5-yl)-amino)-2-methoxy-
benzoic acid (16, 15 mg, 0.036 mmol), Hunig’s base (0.019 mL,
.109 mmol), and ammonium chloride (3.90 mg, 0.073 mmol) were
stirred in DMF at room temperature for a few minutes, then BOP
20.96 mg, 0.047 mmol) was added to the resulting slurry. After
stirring at room temperature for 1 h, methanol (0.1 mL) and DMF
1.0 mL) were successively added and the solution was filtered
0
(
stirred for 1 h. The reaction was filtered and purified by preparative
1
HPLC, providing 20 (12 mg, 40%). H NMR (500 MHz, DMSO-d )
6
(
δ 10.66 (s, 1H), 9.86 (s, 1H), 8.53−8.48 (m, 1H), 8.47 (s, 1H), 8.20
N
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
7
3
d, J = 2.0 Hz, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.69−7.63 (m, 2H),
1H), 0.48−0.42 (m, 2H), 0.28−0.21 (m, 2H). LCMS (E+) m/z:
+
.60−7.55 (m, 3H), 7.31 (br s, 1H), 3.91 (s, 3H), 2.77 (d, J = 4.5 Hz,
479.2 (MH ), R = 1.57 min.
T
+
H). LCMS (E+) m/z: 411.2 (MH ), R = 1.09 min.
6-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((3-((2-
hydroxyethyl)carbamoyl)-2-methoxy-phenyl)amino)-N-methylni-
cotinamide (27). Prepared from 3-(2-chloro-5-(methylcarbamoyl)-
pyridin-4-ylamino)-2-methoxybenzoic acid using similar procedures
as described for the preparation of 17 and using 2-amino-4-methyl-5-
fluoropyridine in place of 2-amino-5-fluoropyridine and 2-hydrox-
T
6
-((5-Fluoropyridin-2-yl)amino)-4-((2-methoxy-4-
(
methylcarbamoyl)phenyl)-amino)-N-methylnicotinamide (21).
62
Prepared from 4,6-dichloro-N-methylnicotinamide using similar
procedures as described for the preparation of 20 and using
methylamine in place ammonium chloride to afford 18.3 mg of 21.
1
yethylamine in place of ammonium chloride to afford 6.5 mg of 27.
H NMR (500 MHz, DMSO-d ) δ 10.69−10.57 (m, 1H), 9.86 (br s,
6
1
H NMR (500 MHz, DMSO-d ) δ 10.71 (s, 1H), 9.71 (br s, 1H),
1
H), 8.51 (br s, 1H), 8.45 (s, 1H), 8.40 (br d, J = 4.4 Hz, 1H), 8.18
6
8
8
.51 (br d, J = 4.3 Hz, 1H), 8.48 (s, 1H), 8.30 (br t, J = 5.5 Hz, 1H),
.05 (s, 1H), 7.70 (br s, 1H), 7.65 (br d, J = 6.7 Hz, 1H), 7.57 (br d, J
(
s, 1H), 7.82 (br s, 1H), 7.69−7.50 (m, 5H), 3.91 (s, 3H), 2.81 (br d,
J = 4.2 Hz, 3H), 2.77 (br d, J = 4.1 Hz, 3H). LCMS (E+) m/z: 425.2
+
= 4.9 Hz, 1H), 7.35−7.23 (m, 2H), 4.79 (t, J = 5.5 Hz, 1H), 3.73 (s,
(
MH ), R = 1.25 min.
T
3
2
H), 3.54 (q, J = 5.9 Hz, 2H), 3.36 (m, 2H), 2.78 (d, J = 4.3 Hz, 3H),
4
-((3-Carbamoyl-2-methoxyphenyl)amino)-6-((5-fluoro-4-meth-
ylpyridin-2-yl)amino)-N-methylnicotinamide (22). Prepared from 3-
2-chloro-5-(methylcarbamoyl)pyridin-4-ylamino)-2-methoxybenzoic
+
.24 (s, 3H). LCMS (E+) m/z: 469.2 (MH ), R = 1.18 min.
T
6
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-((2-
(
morpholinoethyl)-carbamoyl)phenyl)amino)-N-methylnicotina-
mide (28). Prepared from 3-(2-chloro-5-(methylcarbamoyl)pyridin-4-
ylamino)-2-methoxybenzoic acid using similar procedures as
described for the preparation of 17 and using 2-amino-4-methyl-5-
fluoropyridine in place of 2-amino-5-fluoropyridine and 2-morpholi-
acid as described for the preparation of 17 and using 2-amino-4-
methyl-5-fluoropyridine in place of 2-amino-5-fluoropyridine to afford
9
1
.1 mg of 22. H NMR (500 MHz, DMSO-d ) δ 10.66 (s, 1H), 9.69
6
(
(
3
4
s, 1H), 8.53−8.43 (m, 2H), 8.04 (s, 1H), 7.77−7.66 (m, 2H), 7.63
dd, J = 7.6, 1.5 Hz, 1H), 7.58−7.53 (m, 2H), 7.32−7.23 (m, 2H),
nylethylamine in place of ammonium chloride to afford 8.3 mg of 28.
.74 (s, 3H), 2.78 (d, J = 4.3 Hz, 3H), 2.23 (s, 3H). LCMS (E+) m/z:
1
H NMR (500 MHz, DMSO-d ) δ 10.68 (s, 1H), 9.71 (s, 1H), 8.54−
+
6
25.2 (MH ), R = 1.20 min.
T
8
7
.49 (m, 1H), 8.48 (s, 1H), 8.36−8.29 (m, 1H), 8.05−8.02 (m, 1H),
.69 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.59−7.54 (m, 1H), 7.36−7.32
6
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-
methylcarbamoyl)phenyl)-amino)-N-methylnicotinamide (23).
Prepared from 3-(2-chloro-5-(methylcarbamoyl)pyridin-4-ylamino)-
-methoxybenzoic acid using similar procedures as described for the
preparation of 18 using 2-amino-4-methyl-5-fluoropyridine in place of
(
(
3
2
m, 1H), 7.31−7.25 (m, 1H), 3.76 (s, 3H), 3.60−3.54 (m, 4H),
.45−3.38 (m, 5H), 2.78 (d, J = 4.3 Hz, 3H), 2.46−2.37 (m, 3H),
2
+
.24 (s, 3H). LCMS (E+) m/z: 538.2 (MH ), R = 1.32 min.
T
1
6-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-((pyr-
2
-amino-5-fluoropyridine to afford 7.6 mg of 23. H NMR (500 MHz,
idin-2-ylmethyl)-carbamoyl)phenyl)amino)-N-methylnicotinamide
DMSO-d ) δ 10.69 (s, 1H), 9.65 (br s, 1H), 8.55 (br s, 1H), 8.46 (s,
1
s, 1H), 7.26 (br d, J = 4.9 Hz, 2H), 3.72 (s, 3H), 2.83−2.75 (m, 6H),
2
6
(29). Prepared from 3-(2-chloro-5-(methylcarbamoyl)pyridin-4-yla-
H), 8.29−8.18 (m, 1H), 8.07 (s, 1H), 7.66−7.58 (m, 2H), 7.50 (br
mino)-2-methoxybenzoic acid using similar procedures as described
for the preparation of 17 and using 2-amino-4-methyl-5-fluoropyr-
idine in place of 2-amino-5-fluoropyridine and pyridin-2-ylmethan-
+
.24 (s, 3H). LCMS (E+) m/z: 439.2 (MH ), R = 1.29 min.
T
4
-((3-(Ethylcarbamoyl)-2-methoxyphenyl)amino)-6-((5-fluoro-4-
1
amine in place of ammonium chloride to afford 11.2 mg of 29. H
methylpyridin-2-yl)amino)-N-methylnicotinamide (24). Prepared
from 3-(2-chloro-5-(methylcarbamoyl)pyridin-4-ylamino)-2-methox-
ybenzoic acid using similar procedures as described for the
preparation of 17 and using 2-amino-4-methyl-5-fluoropyridine in
place of 2-amino-5-fluoropyridine and ethylamine hydrochloride in
place of ammonium chloride to afford 9.7 mg of 24. H NMR (500
MHz, DMSO-d ) δ 10.70 (s, 1H), 9.71 (br s, 1H), 8.54−8.48 (m,
1
NMR (500 MHz, DMSO-d ) δ 10.72 (s, 1H), 9.72 (br s, 1H), 9.02
6
(
br t, J = 5.8 Hz, 1H), 8.53 (br dd, J = 11.3, 4.6 Hz, 2H), 8.49 (s, 1H),
.05 (s, 1H), 7.83−7.78 (m, 1H), 7.70 (br s, 1H), 7.67 (br d, J = 7.9
Hz, 1H), 7.57 (br d, J = 4.9 Hz, 1H), 7.42 (d, J = 7.3 Hz, 1H), 7.37
br d, J = 6.7 Hz, 1H), 7.33−7.26 (m, 2H), 4.61 (d, J = 5.5 Hz, 2H),
.75 (s, 3H), 2.79 (d, J = 4.3 Hz, 3H), 2.24 (s, 3H). LCMS (E+) m/z:
8
(
1
3
+
6
516.2 (MH ), R = 1.44 min.
T
H), 8.48 (s, 1H), 8.29 (br s, 1H), 8.05 (s, 1H), 7.70 (br s, 1H), 7.62
br d, J = 7.4 Hz, 1H), 7.56 (br s, 1H), 7.32−7.14 (m, 2H), 3.72 (s,
H), 3.35 (m, 2H), 2.78 (br d, J = 4.0 Hz, 3H), 2.24 (s, 3H), 1.13 (br
Preparation of 6-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-
methoxy-3-(1,3,4-oxadiazol-2-yl)phenyl)amino)-N-methylnicotina-
mide (30). Intermediate 3-((2-((5-fluoropyridin-2-yl)amino)-5-
(methylcarbamoyl)pyridin-4−5-yl)amino)-2-methoxybenzoic acid
(60 mg, 0.141 mmol) from the preparation of 22, Hunig’s base
(0.074 mL, 0.423 mmol), and tert-butyl hydrazinecarboxylate (22.4
mg, 0.169 mmol) was stirred in DMF (0.6 mL) for a few minutes at
room temperature, then (benzotriazol-1- yloxy)tris(dimethylamino)-
phosphonium hexafluorophosphate (BOP, 81 mg, 0.183 mmol) was
added. After stirring the resulting slurry at room temperature for 1 h,
the reaction mixture was slowly diluted with water (∼3 mL), and the
resulting suspension was sonicated briefly and the precipitated solid
was collected by vacuum filtration and dried under vacuum to afford
64 mg (84%) of tert-butyl 2-(3-(2-(5-fluoro-4-methylpyridin-2-
ylamino)-5-(methylcarbamoyl)pyridin-4-ylamino)-2-methoxybenzo-
yl)-hydrazinecarboxylate as a light-tan solid. LCMS (E+) m/z: 540.2
(
3
+
t, J = 7.1 Hz, 3H). LCMS (E+) m/z: 453.2 (MH ), R = 1.56 min.
T
4
-((3-(Dimethylcarbamoyl)-2-methoxyphenyl)amino)-6-((5-fluo-
ro-4-methylpyridin-2-yl)-amino)-N-methylnicotinamide (25). Pre-
pared from 3-(2-chloro-5-(methylcarbamoyl)pyridin-4-ylamino)-2-
methoxybenzoic acid using similar procedures as described for the
preparation of 17 and using 2-amino-4-methyl-5-fluoropyridine in
place of 2-amino-5-fluoropyridine and dimethylamine in place of
ammonium chloride to afford 6.8 mg of 25. H NMR (500 MHz,
DMSO-d ) δ 10.67−10.54 (m, 1H), 9.71−9.63 (m, 1H), 8.54−8.42
1
6
(
7
2
m, 2H), 8.04 (s, 1H), 7.68 (br s, 1H), 7.62−7.46 (m, 2H), 7.29−
.15 (m, 1H), 6.92 (br d, J = 7.5 Hz, 1H), 3.68−3.65 (m, 3H), 2.81−
+
.75 (m, 9H), 2.23 (s, 3H). LCMS (E+) m/z: 453.2 (MH ), R
T
(
condition B) = 0.69 min.
-((3-((Cyclopropylmethyl)carbamoyl)-2-methoxyphenyl)-
+
4
(MH ).
amino)-6-((5-fluoro-4-methyl-pyridin-2-yl)amino)-N-methylnicoti-
namide (26). Prepared from 3-(2-chloro-5-(methylcarbamoyl)-
pyridin-4-ylamino)-2-methoxybenzoic acid using similar procedures
as described for the preparation of 17 and using 2-amino-4-methyl-5-
fluoropyridine in place of 2-amino-5-fluoropyridine and cyclo-
To a slurry of tert-butyl 2-(3-(2-(5-fluoro-4-methylpyridin-2-
ylamino)-5-(methylcarbamoyl)pyridin-4-ylamino)-2-methoxybenzo-
yl)-hydrazinecarboxylate (64 mg, 0.119 mmol) in dichloromethane
(0.5 mL) was added trifluoroacetic acid (0.18 mL, 2.4 mmol) to give
a clear solution This mixture was stirred at room temperature for 1 h
then was concentrated and redissolved in DCM (10 mL). The
solution was reconcentrated, and the process was repeated one
additional time to remove residual trifluoroacetic acid. The resulting
material was triturated with ether (2 × 5 mL) to give an oil, which
foamed and solidified under vacuum to afford 55 mg of 6-(5-fluoro-4-
methylpyridin-2-ylamino)-4-(3-(hydrazinecarbonyl)-2-methoxyphe-
propylmethylamine in place of ammonium chloride to afford 6.3
1
mg of 26. H NMR (500 MHz, DMSO-d ) δ 10.72 (s, 1H), 9.72 (br
6
s, 1H), 8.51 (br d, J = 3.7 Hz, 1H), 8.48 (s, 1H), 8.37 (t, J = 5.5 Hz,
1
(
6
H), 8.05 (s, 1H), 7.70 (br s, 1H), 7.64 (dd, J = 7.6, 2.1 Hz, 1H), 7.56
br d, J = 4.3 Hz, 1H), 7.33−7.17 (m, 2H), 3.74 (s, 3H), 3.16 (t, J =
.4 Hz, 2H), 2.78 (d, J = 4.3 Hz, 3H), 2.24 (s, 3H), 1.11−0.98 (m,
O
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
nylamino)-N-methylnicotinamide as a pale-yellow solid. LCMS (E+)
m/z: 440.2 (MH ).
flask was evacuated and supplied with hydrogen gas from a balloon for
3 h. After the reaction was complete, the hydrogen balloon was
removed and the flask was flushed with nitrogen before adding 50 mL
of additional ethanol. The mixture was filtered to remove the catalyst,
and the resulting clear filtrate was concentrated to afford 120 mg
(92%) of a ∼4:1 isomeric mixture of 2-methoxy-3-(1-methyl-1H-
pyrazol-3-yl)aniline and 2-methoxy-3-(1-methyl-1H-pyrazol-5-yl)-
+
A portion of 6-(5-fluoro-4-methylpyridin-2-ylamino)-4-(3-(hydra-
zinecarbonyl)-2-methoxyphenylamino)-N-methylnicotinamide from
the previous step (15 mg, 0.027 mmol) was dissolved in trimethoxy-
methane (144 mg, 1.36 mmol) and was heated to 105 °C for 2 h
using a preheated heating block. The reaction mixture was
concentrated to remove excess trimethoxymethane, diluted with
DMF (∼1 mL), filtered, and was purified by preparative reverse phase
LCMS with the following conditions: column, Waters XBridge C18,
+
aniline, respectively. LCMS (E+) m/z: 204 (MH ).
62
To a solution of 4,6-dichloro-N-methylnicotinamide (110 mg,
0.54 mmol) and a ∼4:1 isomeric mixture of 2-methoxy-3-(1-methyl-
1H-pyrazol-3-yl)aniline and 2-methoxy-3-(1-methyl-1H-pyrazol-5-yl)-
aniline (120 mg, 0.59 mmol) in DMA (1 mL) was added a solution of
lithium bis(trimethylsilyl)amide (1 M in THF, 1.34 mL, 1.34 mmol)
dropwise via syringe at room temperature over ∼5 min. After stirring
for ∼30 min at room temperature, additional lithium bis-
(trimethylsilyl)amide (1 M in THF, 0.6 mL, 0.6 mmol) was added
and the mixture was stirred for an additional 30 min. Water was then
added, and the resulting mixture was concentrated to remove most of
the volatiles. The resulting aqueous solution was acidified to a pH of
∼4 by slowly adding 1N aq HCl dropwise with stirring, causing a solid
to precipitate from solution. The resulting slurry was stirred at room
temperature for ∼1 h. The resulting solid was collected by vacuum
filtration, rinsed with water, and dried to afford 155 mg (78%) of a tan
solid as a ∼4:1 isomeric mixture of 6-chloro-4-(2-methoxy-3-(1-
methyl-1H-pyrazol-3-yl)phenylamino)-N-methylnicotinamide and 6-
chloro-4-(2-methoxy-3-(1-methyl-1H-pyrazol-5-yl)phenylamino)-N-
1
9 mm × 200 mm, 5 μm particles. Mobile phase A: 5:95
acetonitrile:water with 10 mM ammonium acetate. Mobile phase B:
9
1
5:5 acetonitrile:water with 10 mM ammonium acetate. Gradient:
5−100% B over 20 min, then a 5 min hold at 100% B. Flow: 25 mL/
mins. Fractions containing the desired product were combined and
dried via centrifugal evaporation to afford 7.2 mg (59%) of 30. H
1
NMR (500 MHz, DMSO-d ) δ 10.83 (s, 1H), 9.75 (br s, 1H), 9.41
6
(
s, 1H), 8.59−8.53 (m, 1H), 8.50 (s, 1H), 8.07 (s, 1H), 7.82 (d, J =
7
7
.4 Hz, 1H), 7.78−7.68 (m, 1H), 7.63 (br d, J = 7.4 Hz, 1H), 7.60−
.51 (m, 1H), 7.43 (t, J = 7.9 Hz, 1H), 3.79 (s, 3H), 2.79 (d, J = 4.4
+
Hz, 3H), 2.24 (s, 3H). LCMS (E+) m/z: 450.2 (MH ), R = 1.40
T
min.
6
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(5-
methyl-1,3,4-oxadiazol-2-yl)phenyl)amino)-N-methylnicotinamide
31). Prepared using similar procedures as described for the
(
preparation of 30 and using trimethoxyorthoacetate in place of
trimethoxymethane to afford 7.5 mg (59%) of 31. H NMR (500
1
+
methylnicotinamide, respectively. LCMS m/z: 204 (MH ) for both
MHz, DMSO-d ) δ 10.82 (s, 1H), 9.73 (s, 1H), 8.53 (br d, J = 4.4 Hz,
isomers.
6
1
1
3
H), 8.50 (s, 1H), 8.06 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.73 (s,
H), 7.57 (br d, J = 6.7 Hz, 2H), 7.41 (t, J = 8.1 Hz, 1H), 3.78 (s,
A ∼4:1 isomeric mixture of 6-chloro-4-(2-methoxy-3-(1-methyl-
1H-pyrazol-3-yl)phenylamino)-N-methylnicotinamide and 6-chloro-
4-(2-methoxy-3-(1-methyl-1H-pyrazol-5-yl)phenylamino)-N-methyl-
nicotinamide (25 mg, 0.067 mmol), 5-fluoro-4-methylpyridin-2-amine
(12.7 mg, 0.10 mmol), cesium carbonate (43.8 mg, 0.134 mmol), and
2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′,6′-triisopropyl-l, I′-bi-
phenyl (BrettPhos, 5.4 mg, 10.1 μmol) in dioxane (0.5 mL) was
sparged with nitrogen for 5 min, then Pd (dba) (9.2 mg, 10.1 μmol)
was added and the reaction was placed into a preheated 110 °C
heating block for 1 h. The reaction was cooled to room temperature,
diluted with DMSO, filtered, and subjected to purification by reverse
phase preparative LCMS with the following conditions: column,
XBridge C18, 19 mm × 200 mm, 5 μm particles. Mobile phase A:
H), 2.79 (d, J = 4.4 Hz, 3H), 2.60 (s, 3H), 2.24 (s, 3H). LCMS (E+)
+
m/z: 464.2 (MH ), R = 1.46 min.
T
6
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(1-
methyl-1H-pyrazol-3-yl)phenyl)amino)-N-methylnicotinamide (32)
and 6-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(1-
methyl-1H-pyrazol-5-yl)phenyl)amino)-N-methylnicotinamide
8
8
2
3
(
33). A slurry of l-(2-methoxy-3-nitrophenyl)ethanone (450 mg,
2.306 mmol) in Ν,Ν-dimethylformamide dimethyl acetal (DMF-
DMA, 8.15 g, 68.4 mmol) was heated to 80 °C, giving a clear solution.
After stirring at this temperature for ∼30 min, the reaction was
cooled, diluted with 100 mL of ethyl acetate, washed with water (3×),
then brine, dried over anhydrous sodium sulfate, filtered, and
concentrated to afford 432 mg of a tan oil. This material was
dissolved in ethanol (4.0 mL) and was cooled in an ice bath, then
hydrazine hydrate (0.22 mL, 6.9 mmol) was added dropwise with
good stirring. After the addition was complete, the reaction was
allowed to warm to room temperature and stirred overnight (∼16 h).
The resulting mixture was concentrated to remove the ethanol,
diluted with 100 mL of ethyl acetate, washed with water (3×), then
brine, dried over sodium sulfate, filtered, and concentrated to afford a
tan semisolid. To this material was added 4 mL of acetone and
potassium carbonate (956 mg, 6.92 mmol), and the resulting mixture
was stirred at room temperature for 10 min before adding
iodomethane (0.577 mL, 9.22 mmol) dropwise. After stirring the
reaction mixture at room temperature overnight, the mixture was
concentrated and was partitioned between ethyl acetate and water.
The layers were separated, and the organic portion was washed with
water (3×), dried over sodium sulfate, filtered, and concentrated
under vacuum to afford tan oil. This material was purified by flash
silica gel chromatography using hexanes/ethyl acetate mixtures as the
eluent. Fractions containing the major uv active component were
combined and concentrated under vacuum to afford 155 mg (29%
overall yield) of a ∼4:1 isomeric mixture of 3-(2-methoxy-3-
nitrophenyl)-1-methyl-1H-pyrazole and 5-(2-methoxy-3-nitrophen-
yl)-1-methyl-1H-pyrazole, respectively, which was used directly in
5
:95 acetonitrile:water with 10 mM ammonium acetate. Mobile phase
B: 95:5 acetonitrile:water with 10-mM ammonium acetate. Gradient:
2
5−65% B over 20 min, then a 0 min hold at 100% B. Flow: 20 mL/
min. Fractions containing the resolved isomeric products were
combined and dried via centrifugal evaporation to afford 13.2 mg
(
3
40%) of the major isomer 32 and 3.0 mg (9%) of the minor isomer
3.
1
Data for the Major Isomer 32. H NMR (500 MHz, DMSO-d ) δ
6
1
1
(
0.79−10.59 (m, 1H), 9.68 (s, 1H), 8.49 (d, J = 4.9 Hz, 1H), 8.48 (s,
H), 8.04 (s, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.72 (s, 1H), 7.63−7.55
m, 2H), 7.49 (d, J = 1.9 Hz, 1H), 7.23 (t, J = 1.9 Hz, 1H), 6.73 (d, J
=
2.4 Hz, 1H), 3.91 (s, 3H), 3.64−3.58 (m, 3H), 2.79 (d, J = 4.3 Hz,
+
3H), 2.24 (s, 3H). LCMS (E+) m/z: 462.2 (MH ), R = 1.66 min.
T
1
Data for the Minor Isomer 33. H NMR (500 MHz, DMSO-d ) δ
6
1
1
7
2
0.76 (s, 1H), 9.85 (br s, 1H), 8.56 (br s, 1H), 8.47 (s, 1H), 8.09 (s,
H), 7.67 (d, J = 8.5 Hz, 2H), 7.51 (s, 2H), 7.32 (t, J = 1.6 Hz, 1H),
.07 (d, J = 7.3 Hz, 1H), 6.38 (s, 1H), 3.69 (s, 3H), 3.47 (br s, 3H),
.78 (d, J = 3.7 Hz, 3H), 2.25 (s, 3H). LCMS (E+) m/z: 462.2
+
(
MH ), R = 1.65 min.
T
6
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(1H-
1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide (34). A
slurry of 22 (25 mg, 0.06 mmol) in DMF-DMA (1.5 mL, 11.2
mmol) was heated to 110 °C, giving a clear solution initially then
eventually becoming a heterogeneous slurry after stirring at this
temperature for 30 min. The resulting mixture was then cooled
slightly and concentrated to remove the DMF-DMA to afford a solid
which was dried further under vacuum. To this residue was added
acetic acid (0.12 mL) and ethanol (0.6 mL), giving a clear solution
which was immediately by cooled in a −10 °C brine/ice bath, giving a
+
the next reaction. LCMS (E+) m/z: 235 (MH ).
To a solution of a ∼4:1 isomeric mixture of 3-(2-methoxy-3-
nitrophenyl)-1-methyl-1H-pyrazole and 5-(2-methoxy-3-nitrophen-
yl)-1-methyl-1H-pyrazole (0.15 g, 0.643 mmol) in ethanol (10 mL)
was added 10% palladium on carbon (0.021 g, 0.019 mmol), and the
P
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
slurry. At this time, 60 μL (∼10 equiv) of hydrazine hydrate was
added dropwise via syringe with good stirring to afford light-pink
slurry. After addition was complete, the reaction was slowly heated to
7.28 (t, J = 7.9 Hz, 1H), 3.95 (s, 3H), 3.74 (s, 3H), 2.81−2.77 (m,
+
3H), 2.24 (s, 3H). LCMS (E+) m/z: 463.2 (MH ), R = 1.32 min.
T
6-((5-Cyanopyridin-2-yl)amino)-4-((2-methoxy-3-(1-methyl-1H-
1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide (39). Pre-
pared using similar procedures as described for the preparation of 38
by using 2-amino-5-cyanopyridine in place of 2-amino-4-methyl-5-
60 °C and stirring was continued for 2 h. The reaction mixture was
then cooled to room temperature and allowed to stir overnight. The
reaction mixture was diluted with ∼2 mL of DMSO and was
subjected to reverse phase preparative LCMS purification with the
following conditions: column, Waters XBridge C18, 19 mm × 200
mm, 5 μm particles. Mobile phase A: 5:95 acetonitrile:water with 10
mM ammonium acetate. Mobile phase B: 95:5 acetonitrile:water with
1
fluoropyridine to afford 4.2 mg of 39. H NMR (400 MHz, methanol-
d ) δ 8.54−8.51 (m, 2H), 8.45 (s, 1H), 7.91 (dd, J = 8.8, 2.4 Hz, 1H),
4
7
=
3
.78 (s, 1H), 7.71−7.70 (m, 1H), 7.68 (d, J = 1.1 Hz, 1H), 7.64 (dd, J
7.9, 1.5 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 4.06 (s, 3H), 3.77 (s,
+
H), 2.96 (s, 3H). LCMS (E+) m/z: 456.0 (MH ), R = 0.64 min
1
0 mM ammonium acetate. Gradient: 10−100% B over 25 min, then
T
(
condition C).
-((2-Methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-
N-methyl-6-((5-(trifluoromethyl)pyridin-2-yl)amino)nicotinamide
40). Prepared using similar procedures described for the preparation
of 38 by using 2-amino-5-trifluoromethylpyridine in place of 2-amino-
a 5 min hold at 100% B. Flow: 25 mL/min. Fractions containing the
4
major product were combined and dried via centrifugal evaporation to
1
afford 8.6 mg (33%) of 34. H NMR (500 MHz, DMSO-d ) δ 10.73
6
(
(
(
s, 1H), 9.71 (s, 1H), 8.54 (br d, J = 3.7 Hz, 1H), 8.49 (s, 1H), 8.29
br s, 1H), 8.04 (s, 1H), 7.70 (s, 1H), 7.68−7.61 (m, 3H), 7.58 (br d,
1
5
-fluoropyridine to afford 6.9 mg of 40. H NMR (400 MHz,
J = 4.9 Hz, 1H), 7.34 (br t, J = 7.9 Hz, 1H), 3.69 (s, 3H), 2.79 (br d, J
=
methanol-d ) δ 8.51 (s, 1H), 8.47−8.45 (m, 1H), 8.45 (s, 1H), 7.89
+
4
3.8 Hz, 3H), 2.24 (s, 3H). LCMS (E+) m/z: 449.2 (MH ), R =
T
(
dd, J = 8.7, 2.3 Hz, 1H), 7.82 (s, 1H), 7.74−7.62 (m, 3H), 7.34 (t, J
1
.25 min.
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(5-
methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide
35). Prepared from 22 using a similar procedure as described for the
=
7.9 Hz, 1H), 4.06 (s, 3H), 3.78 (s, 3H), 2.97 (s, 3H). LCMS (E+)
6
+
m/z: 499.0 (MH ). R = 0.73 min (condition C).
T
6
-((2,6-Dimethylpyrimidin-4-yl)amino)-4-((2-methoxy-3-(1-
methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide
41). Prepared using similar procedures described for the preparation
(
preparation of 34 and using N,N-dimethylacetamide dimethyl acetal
(
1
in place of DMF-DMA to afford 12.3 mg (34%) of 35. H NMR (400
of 38 by using 2,6-dimethylpyrimidin-4-amine in place of 2-amino-5-
fluoropyridine to afford 13 mg of 41. H NMR (400 MHz, methanol-
MHz, methanol-d ) δ 8.43 (s, 1H), 7.99 (s, 1H), 7.74 (d, J = 7.7 Hz,
1
4
2
2
H), 7.64 (br s, 1H), 7.44−7.31 (m, 2H), 3.79 (s, 3H), 3.00 (s, 3H),
d4) δ 8.50 (s, 1H), 8.49 (s, 1H), 7.72 (br d, J = 7.9 Hz, 1H), 7.60 (d, J
+
.54 (s, 3H), 2.34 (s, 3H). LCMS (E+) m/z: 463.4 (MH ), R = 0.66
T
7.9 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 4.02 (s, 3H), 3.73 (s, 3H),
min (condition C).
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(4-
methyl-4H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide
36). Prepared from 22 using a similar procedure as described for the
6
+
T
Preparation of 6-(Cyclopropanecarboxamido)-4-((2-methoxy-3-
(
(
1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotina-
preparation of 34 and using N,N-dimethylacetamide dimethyl acetal
mide (42). Prepared by a sequential coupling of 2-methoxy-3-(1-
methyl-1H-1,2,4-triazol-3-yl)aniline (52) and cyclopropylamide (55)
in place of DMF-DMA and N-methyl hydrazine in place of hydrazine
1
hydrate to afford 4.8 mg (10%) of 36. H NMR (400 MHz, methanol-
62
to 4,6-dichloro-N-methylnicotinamide using similar procedures as
d4) δ 8.40 (s, 1H), 7.99 (d, J = 1.1 Hz, 1H), 7.84 (dd, J = 8.1, 1.6 Hz,
described in the preparation of 11 to afford 17.8 mg (36% over 2
1
7
3
H), 7.74 (s, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.31 (d, J = 5.5 Hz, 1H),
1
steps) of 42 as an off-white solid. H NMR (400 MHz, methanol-d )
4
.26 (dd, J = 7.7, 1.6 Hz, 1H), 3.76 (s, 3H), 3.58 (s, 3H), 2.94 (s,
δ 8.54 (s, 1H), 8.38 (s, 1H), 7.83 (dd, J = 7.9, 1.5 Hz, 1H), 7.59 (dd, J
= 7.9, 1.5 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1Η), 6.94 (br s, 1H), 4.06 (d, J
+
H), 2.43 (s, 3H), 2.33 (s, 3H). LCMS (E+) m/z: 477.4 (MH ), R
T
=
0.68 min (condition C).
-((4,5-Dimethylpyridin-2-yl)amino)-4-((2-methoxy-3-(1-methyl-
H-1,2,4-triazol-5-yl)phenyl)amino)-N-methylnicotinamide (37).
=
0.4 Hz, 3H), 3.75 (s, 3H), 2.98 (s, 3H), 1.87−1.76 (m, 1H), 1.15−
+
6
1
.07 (m, 2H), 1.06−0.97 (m, 2H). LCMS (E+) m/z: 422.2 (MH ),
1
R = 0.57 min (condition C).
T
Prepared from 22 using a similar procedure as described for the
preparation of 34 and using N-methyl hydrazine in place of hydrazine
6
-(Cyclopropanecarboxamido)-4-((2-methoxy-3-(1-methyl-1H-
1
,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d -nicotinamide (43).
3
1
hydrate to afford 15 mg (42%) of 37. H NMR (400 MHz, methanol-
Prepared by a sequential coupling of 2-methoxy-3-(1-methyl-1H-
62
d4) δ 8.43 (s, 1H), 8.11 (s, 1H), 8.01 (br s, 1H), 7.88 (d, J = 7.7 Hz,
1,2,4-triazol-3-yl)aniline (52) and cyclopropylamide to 53 using
similar procedures as described in the preparation of 11 to afford 26.3
1
H), 7.78 (s, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 5.5 Hz, 1H),
1
7
.29 (d, J = 7.7 Hz, 1H), 3.86 (s, 3H), 3.59 (s, 3H), 2.97 (s, 3H), 2.35
s, 3H). LCMS (E+) m/z: 463.1 (MH ). R = 0.69 min (condition
mg (60% over 2 steps) of 43 as an off-white solid. H NMR (500
+
(
MHz, DMSO-d ) δ 10.71 (br s, 1H), 10.61 (br s, 1H), 8.58 (br s,
T
6
C).
1H), 8.52 (br s, 1H), 8.48 (br s, 1H), 8.03 (br s, 1H), 7.62−7.42 (m,
2H), 7.22 (t, J = 7.4 Hz, 1H), 3.93 (br s, 3H), 3.69 (br s, 3H), 2.01−
6
-((5-Fluoro-4-methylpyridin-2-yl)amino)-4-((2-methoxy-3-(1-
methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide
38). Prepared from 3-((2-chloro-5-(methylcarbamoyl)pyridin-4-yl)-
1
4
.88 (m, 1H), 0.85−0.69 (m, J = 4.4 Hz, 4H). LCMS (E+) m/z:
(
+
25.3 (MH ), R = 1.09 min.
T
amino)-2-methoxybenzoic acid by amide formation using a similar
procedure as described in the preparation of 17 to afford 4-((3-
carbamoyl-2-methoxyphenyl)amino)-6-chloro-N-methylnicotinamide.
Reaction of 4-((3-carbamoyl-2-methoxyphenyl)amino)-6-chloro-N-
methylnicotinamide with DMF-DMA followed by hydrazine hydrate
using a similar procedure as described for the preparation of 34
afforded 6-chloro-4-((2-methoxy-3-(1H-1,2,4-triazol-3-yl)phenyl)-
amino)-N-methylnicotinamide. Methylation of 6-chloro-4-((2-me-
thoxy-3-(1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylnicotinamide
using a similar procedure as described for the preparation of 50
afforded 6-chloro-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)-
phenyl)amino)-N-methyl-nicotinamide. Final coupling of 6-chloro-4-
2
-Methoxy-3-nitrobenzamide (48). To a solution of 10 g (51
mmol) of commercially available methyl 2-hydroxy-3-nitrobenzoate
47) in DMF (100 mL) at room temperature was added potassium
carbonate (14 g, 101 mmol) followed by addition of methyl iodide
6.34 mL, 101 mmol), and the resulting orange mixture was heated to
(
(
6
0 °C for 1 h. LCMS analysis at this time showed complete and clean
conversion to a major product consistent with the expected product
(observed MH 212). The reaction mixture was allowed to cool to
+
room temperature and crushed ice (∼100 mL) was added followed by
water to a total volume of ∼400 mL, causing a yellow solid to
crystallize from the solution. The solution was stirred for a few
minutes to give a slurry, then the solid was collected by vacuum
filtration. The resulting solid was rinsed with additional water (∼100
mL) to afford a white solid, which was partially air-dried then
transferred to a round-bottom flask and further dried under vacuum
overnight to afford 10.5 g (98%) of a pale-yellow solid as the
(
(2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-
methylnicotinamide with 2-amino-4-methyl-5-fluoropyridine using a
similar procedure as described for the preparation of 32 and 33
1
afforded 38. H NMR (500 MHz, DMSO-d ) δ 10.70 (s, 1H), 9.70
6
1
(br s, 1H), 8.55 (s, 1H), 8.51 (br s, 1H), 8.48 (s, 1H), 8.06 (s, 1H),
intermediate methyl 2-methoxy-3-nitrobenzoate. H NMR (400
7.78−7.68 (m, 1H), 7.65−7.57 (m, 2H), 7.53 (br d, J = 7.3 Hz, 1H),
MHz, CDCl ) δ 8.04 (dd, J = 7.9, 1.8 Hz, 1H), 7.92 (dd, J = 8.1,
3
Q
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
.8 Hz, 1H), 7.31−7.28 (m, 1H), 4.02 (s, 3H), 3.97 (s, 3H). LCMS
nitrogen at room temperature was added a 1 M solution of potassium
bis(trimethylsilyl)amide in THF (269 mmol) dropwise over 20 min.
During the addition, the reaction turned dark-red to ultimately dark-
brown in color. The temperature of the reaction at the end of addition
was 27 °C. The reaction was left to stir at room temperature for 4 h,
then methyl iodide (28.0 mL, 448 mmol) was added dropwise over a
period of 15 min. The reaction was then left to stir at room
temperature overnight. After 20 h, the reaction was checked by
LCMS, indicating complete conversion had occurred. The reaction
was then slowly quenched with water (350 mL) and ethyl acetate
(650 mL) was added. The layers were partitioned in a separatory
funnel. The organic layer was washed with brine (200 mL) and then
dried over solid magnesium sulfate. Filtration and concentrating
afforded 49 g (93%) of a crude yellow oil that solidified upon
standing. The crude solid was then suspended in 30−40 mL of
ethanol and warmed slowly to 45 °C with a heating mantle. Once all
the solid has dissolved, the heat was shut off and the solution was
allowed to slowly cool. When the solution reached 30 °C, it was
seeded to initiate crystallization. The mixture was stirred for 1 h at
room temperature, then the slurry was filtered through Whatman 2
filter paper to collect the crystalline solid. The filter cake was washed
with cold (−30 °C) ethanol and was dried under vacuum overnight to
afford 12 g (51.3 mmol, 23%) of the major isomer 50 as an off-white
solid. The mother liquor from the filtration was concentrated, and the
crystallization step was repeated with stirring overnight. Filtration and
drying the collected crystalline solid afforded an additional 8.38 g
(35.8 mmol, 16%) of the major isomer 50 as an off-white solid. The
mother liquor was once again concentrated to an oil (29.3 g) and
purified on an Isco RediSep Silica 1.5 kg Gold column. The isomers
were eluted with a gradient of 100% A for 1 column volume then to
18% B over 10 column volumes where A = dichloromethane and B =
80/20 dichloromethane/methanol to afford 20.38 g of an isomeric
mixture. The material was then purified by SFC using previously
described conditions to yield an additional 10 g (19%) of 50 as a
white solid.
+
(E+) m/z: 212 (MH ), R = 0.83 min.
T
Methyl 2-methoxy-3-nitrobenzoate (11 g, 52 mmol) was dissolved
in a cold solution of ammonia in methanol (7 N, 250 mL), and
concentrated aqueous ammonium hydroxide (100 mL) was added.
The flask was sealed, and the resulting solution was allowed to gently
stir at room temperature overnight (∼17 h). The reaction mixture was
concentrated in vacuo under mild warming using a water bath to yield
an aqueous slurry. This slurry was diluted with additional water
(
∼300 mL) and was briefly sonicated. The resulting yellow solid was
collected by vacuum filtration and was rinsed with additional water
∼100 mL) and air-dried in the funnel for several hours then under
(
vacuum to afford 7.12 g of 48 as a yellow solid. A second crop was
obtained by extracting the filtrate with ethyl acetate (3 × 100 mL),
followed by washing the extracts with brine, drying over anhydrous
sodium sulfate, decanting, and concentration under vacuum to afford
1.67 g of additional 48 as a yellow solid (86% overall combined yield).
+
LCMS (E+) m/z: 197 (MH ), R = 0.58 min.
T
3
-(2-Methoxy-3-nitrophenyl)-1H-1,2,4-triazole (49). A slurry of
4
8 (7.1 g, 36 mmol) in dimethylformamide dimethyl acetal (DMF-
DMA, 48.5 mL, 362 mmol) was heated to 95 °C. giving a clear, pale-
yellow solution. After heating for ∼30 min at this temperature, the
reaction was cooled and concentrated in vacuo. The resulting yellow
oil was azeotroped twice with 1,2-dichloroethane (40 mL portions) to
ensure complete removal of any residual DMF-DMA. The oil
containing the crude DMF-DMA adduct was dissolved in 35 mL of
ethanol and was immediately used in the following step. In a separate
flask was prepared a mixture of ethanol (150 mL) and acetic acid (35
mL), and the resulting solution was cooled in an ice bath. Once
cooled, hydrazine hydrate (17.6 mL, 362 mmol) was added dropwise,
followed by a dropwise addition of the previously prepared ethanol
solution of the crude DMF-DMA adduct via cannula over ∼15 min
with stirring. During the addition, a pale-yellow solid formed in the
solution. After the addition was complete, the resulting cloudy yellow
mixture was allowed to warm to room temperature and stir for ∼4 h.
The reaction mixture was concentrated in vacuo to remove the
ethanol, diluted with additional water, and filtered to collect the solid.
The solid was washed with additional portions of water and was air-
2-Methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)aniline (52). A sol-
ution of 50 (1.87 g, 7.98 mmol) in ethanol (50 mL) was sparged with
nitrogen for a few minutes and charged with 5% Pd-C (0.85 g, 0.40
mmol). The reaction mixture was then sparged with hydrogen from a
balloon for a few minutes and allowed to stir under a balloon of
hydrogen for 1.5 h at room temperature. The mixture was then
sparged with nitrogen to deactivate the catalyst and the mixture was
filtered through a pad of diatomaceous earth washing with additional
amounts of ethanol, and the resulting clear, colorless filtrate
containing the product was concentrated under vacuum to afford a
colorless oil. This material was azeotroped with two portions of dry
toluene (∼25 mL each) to afford an off-white solid which was dried
dried in the funnel, then under vacuum to afford 5.5 g (69%) of 49 as
a pale-yellow solid. LCMS (E+) m/z: 221 (MH ), R = 0.61 min.
+
T
3
-(2-Methoxy-3-nitrophenyl)-1-methyl-1H-1,2,4-triazole (50). A
solution of 49 (2.2 g, 10.1 mmol) in DMF (20 mL) was treated with
potassium carbonate (4.2 g, 30 mmol). After cooling, the resulting
mixture in an ice bath, and a solution of iodomethane (0.86 mL, 13.7
mmol) in DMF (5 mL) was slowly added dropwise by syringe over 2
min. After the addition was complete, the ice bath was removed and
the reaction mixture was allowed to warm to room temperature. After
stirring at room temperature for ∼4 h, LCMS analysis indicated
complete and clean conversion to a regioisomeric mixture of products
in ∼2:1 ratio. The reaction was cooled in an ice bath and was diluted
with water (∼ 50 mL), and the solution was extracted with ethyl
acetate (3 × 40 mL) and the combined extracts were washed with
further under vacuum to afford 1.5 g (92%) of 52 as a free-flowing
1
white solid. H NMR (400 MHz, CDCl ) δ 8.09 (s, 1H), 7.35 (dd, J =
3
7.8, 1.7 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.82 (dd, J = 7.8, 1.7 Hz,
1H), 4.00 (s, 3H), 3.94 (br s, 2H), 3.78 (s, 3H). LCMS (E+) m/z:
+
205 (MH ), R = 0.44 min.
T
1
0% aq lithium chloride (2 × 20 mL), water (20 mL), then brine
6-Chloro-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)-
before concentrating to afford 2.2 g (91%) of a yellow oil as the crude
product, which solidified to a yellow solid upon standing. This crude
material was combined with another batch of additional crude
product (∼0.45 g) obtained from a previous similar reaction using 49,
and the combined material was purified by SFC to resolve the isomers
phenyl)amino)-N-methylpyridazine-3-carboxamide (54). To a
62
solution of 52 (10.26 g, 50.2 mmol) and 53 (10.5 g, 50.2 mmol)
in THF (120 mL) was added a 1 M solution of lithium
bis(trimethylsilyl)amide in THF (151 mL, 151 mmol) in a dropwise
manner using a pressure equalized addition funnel. The reaction was
run for 10 min after the completion of the addition and then
quenched with HCl (1 M aq, 126 mL, 126 mmol). The reaction was
concentrated in vacuo until the majority of the THF was removed and
a precipitate prevailed throughout the vessel. Water (∼500 mL) was
then added, and the slurry was sonicated for 5 min and stirred for 15
min. The solid was collected by vacuum filtration, rinsed with water,
and then air-dried for 30 min. The powder was collected and
dissolved in dichloromethane. The organic layer was washed with
water and brine and then dried over sodium sulfate, filtered, and
[
°
conditions: column = chiral IC 3 cm 5 cm, 5 μm; column temp = 35
C; flow rate = 200 mL/min; mobile phase = CO /MeOH = 80/20;
2
injection program = stacked (2.3 min/cycle), 2.5 mL/per injection;
sampler conc (mg/mL), 60 mg/mL; detector wavelength = 220 nm]
to afford 1.87 g (66%) of the major isomer 50 as a pale-yellow solid
and 0.7 g (25%) of the minor isomer 51 as a pale-yellow oil. Data for
major (desired) isomer 50: H NMR (400 MHz, methanol-d : δ 8.50
1
4
(
7
2
s, 1H), 8.11 (dd, J = 7.9, 1.8 Hz, 1H), 7.85 (dd, J = 8.1, 1.8 Hz, 1H),
.38 (t, J = 8.0 Hz, 1H), 4.03 (s, 3H), 3.83 (s, 3H). LCMS (E+) m/z:
35 (MH ), R = 0.74 min.
+
1
concentrated to provide 12.5 g (66%) of 54 as a pale-yellow solid. H
T
Alternative Conditions for Synthesis of 50. To a yellow
suspension of 49 (49.3 g, 224 mmol) in THF (1000 mL) under
NMR (400 MHz, DMSO-d ) δ 11.11 (s, 1H), 9.36 (s, 1H), 8.56 (s,
1H), 7.72 (dd, J = 7.8, 1.6 Hz, 1H), 7.60 (dd, J = 7.9, 1.5 Hz, 1H),
6
R
DOI: 10.1021/acs.jmedchem.9b00444
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7
.29 (t, J = 7.9 Hz, 1H), 7.19 (s, 1H), 3.95 (s, 3H), 3.72 (s, 3H).
AUTHOR INFORMATION
+
■
LCMS (E+) m/z: 377 (MH ), R = 0.83 min.
T
Corresponding Authors
For S.T.W.: phone, (609)252-4873; E-mail, stephen.
wrobleski@bms.com.
For R.M.: E-mail, ryan.moslin@bms.com.
IL-23-Induced Acanthosis in Mice. Acanthosis was induced in
*
6−8-week-old C57BL/6 female mice (19−20 g average weight,
Jackson Laboratories) by intradermal injection of dual chain,
recombinant human IL-23 into the right ear. IL-23 injections were
administered every other day from day 0 through day 9 of the study.
Treatment groups consisted of eight mice per group. Compound 11
at 7.5, 15, and 30 mg/kg BID in vehicle (EtOH:TPGS:PEG300,
*
ORCID
Stephen T. Wrobleski: 0000-0001-7793-1530
Ryan Moslin: 0000-0002-0332-4778
Steven Spergel: 0000-0002-5190-3942
James Kempson: 0000-0002-9540-3886
Percy H. Carter: 0000-0002-5880-1164
Present Address
For D.W.: Vividion Therapeutics, Inc. 5820 Nancy Ridge
Drive, San Diego, CA 92121, United States.
5:5:90) and vehicle alone dosed BID by oral gavage, with the first
dose given the evening before the first IL-23 injection. An anti-IL-23
adnectin (3 mg/kg) and PBS control were administered subcuta-
neously approximately 1 h prior to the first IL-23 injection and then
twice a week thereafter. Ear thickness was measured using a Mitutoyo
(no. 2412F) dial caliper and calculated as the percent change in
thickness from the baseline measurement taken on day 0 before initial
IL-23 injections for each animal. At the end of the study, IL-23-
injected ears as well as naive control ears were collected from four
̈
Author Contributions
S.W. and R. M. contributed equally to this work.
animals per group for histological examination and gene expression
analyses. Terminal blood samples collected via the retro-orbital sinus
were used for PK determinations. Statistical analyses were performed
using Student’s t tests or ANOVA with Dunnett’s post test. At the end
of the study, ears were removed and fixed in 10% neutral-buffered
formalin for 24−48 h. The fixed ears were then cut longitudinally, and
two pieces were parallel embedded to make the paraffin blocks. The
paraffin blocks were then sectioned and placed on microscope slides
for H&E staining for histological evaluation. Severity of ear
inflammation was scored using an objective scoring system based
on the following parameters: extent of the lesion, severity of
hyperkeratosis, number and size of pustules, height of epidermal
hyperplasia (acanthosis, measured in interfollicular epidermis), and
the amount of inflammatory infiltrate in the dermis and soft tissue.
The latter two parameters, acanthosis and inflammatory infiltrate,
were scored independently on a scale from 0 to 4: 0, none; 1,
minimal; 2, mild; 3, moderate; 4, marked. The histological changes
were blindly evaluated by a pathologist. Statistical analyses was
performed using one-way ANOVA with Dunnett’s test for comparison
of each treatment versus the vehicle control.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by Bristol-Myers Squibb Company. We
thank Dr. Robert Borzilleri, Dr. Joseph Tino, and Dr. John
Hynes Jr., and Dr. Murali Dhar for helpful comments in the
writing of this manuscript, Dr. Phil Baran for useful
discussions, Dr. Andrew Tebben and Charlotte Raymond for
their assistance in the design of the proposed cover art
illustration, Sylwia Stachura for assistance in the preparation of
analogues, the Synthesis and Analysis Technology Team and
the Department of Discovery Synthesis (DDS) including Dr.
Dauh-Rurng Wu, Shiuhang Yip, Richard Rampulla, and the
Biocon Bristol-Myers Squibb Research and Development
Center (BBRC) DDS team for assistance in the synthesis
and purification of compounds. Finally, the we acknowledge
the beamline staff at the IMCA-CAT beamline 17-ID at the
Advanced Photon Source and the CLS 08ID at Canadian Light
Source for their support in diffraction data collection.
X-ray Crystallography. Protein production and purification of
60−62
TYK2 JH2 (575-869) was carried out as previously reported.
Cocrystals of TYK2 JH2 in complex with a previously reported BMS
60
compound was first grown under conditions similar to that reported
60−62
earlier.
These cocrystals were subsequently used for soaking
ABBREVIATIONS USED
■
inhibitors of interest. Compounds 11, 12, and 29 was soaked into the
TYK2 JH2 cocrystals (24 h soak) by adding 5 mM inhibitor (final
concentration) to a 10 μL well of harvest mother liquor. Crystals were
frozen from mother liquor solution containing 25% glycerol. Structure
TYK2, tyrosine kinase 2; JAK1, Janus kinase 1; JAK2, Janus
kinase 2; JAK3, Janus kinase 3; JH1, Janus homology 1; JH2,
Janus homology 2; STAT, signal transducer and activator of
transcription; pSTAT, phospho signal transducer and activator
of transcription; hWB, human whole blood; mWB, mouse
whole blood; IC, Inhibitory concentration; nM, nanomolar;
μM, micromolar; mM, millimolar; IFN, interferon; IL,
interleukin; GM-CSF, granulocyte-macrophage colony-stimu-
latory factor; EPO, erythropoietin; TPO, thrombopoietin;
ATP, adenosine triphosphate; FDA, Food and Drug
Administration; RA, rheumatoid arthritis; PSO, psoriasis;
PsA, psoriatic arthritis; UC, ulcerative colitis; AS, ankylosing
spondylitis; AA, alopecia areata; DLE, discoid lupus eryth-
ematosus; SLE, systemic lupus erythematosus; dcSSc, diffuse
cutaneous systemic sclerosis; CD, Crohn’s disease; SAEs,
significant adverse events; mg, milligram; g, gram; NK, natural
killer; LFT, liver function tests; LDL, low density lipoprotein;
HDL, high density lipoprotein; CD40, cluster of differentiation
40; compd, compound; HTRF, homogeneous time-resolved
fluorescence; SPA, scintillation proximity assay; Th1, T-helper
1; Th17, T-helper 17; SH2, Src homology 2; IBD,
inflammatory bowel disease; Pro, proline; Leu, leucine; Asn,
asparagine; Lys, lysine; Gln, glutamine; Thr, threonine;; Val,
60−62
determination was as described previously.
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.9b00444.
X-ray crystallographic data and refinement statistics for
compounds 11, 12, and 29 in TYK2 JH2 (PDF)
Molecular formula strings list (CSV)
Accession Codes
Atomic coordinates for the X-ray structures of compound 11
(
PDB 6NZP), 12 (PDB 6NZR), and 29 (PDB 6NZQ) in
TYK2 JH2 are available from the RCSB Protein Data Bank
www.rscb.org). Authors will release the atomic coordinates
upon article publication.
(
S
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