Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of ((S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841)

Article abstract of DOI:10.1021/acs.jmedchem.8b00917

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the -common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

Full text of DOI:10.1021/acs.jmedchem.8b00917

Article
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/jmc
Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune
Diseases: Discovery of ((S)‑2,2-Difluorocyclopropyl)((1R,5S)‑3-(2-((1-
methyl‑1H‑pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-
diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841)
,†
‡
‡
‡
‡
Andrew Fensome,* Catherine M. Ambler, Eric Arnold, Mary Ellen Banker, Matthew F. Brown,
‡
§
†
†
‡
Jill Chrencik, James D. Clark, Martin E. Dowty, Ivan V. Efremov, Andrew Flick,
,†
†
‡
§
Brian S. Gerstenberger,* Ariamala Gopalsamy, Matthew M. Hayward, Martin Hegen,
Brett D. Hollingshead, Jason Jussif, John D. Knafels, David C. Limburg, David Lin, Tsung H. Lin,
Betsy S. Pierce, Eddine Saiah, Raman Sharma, Peter T. Symanowicz, Jean-Baptiste Telliez,
∥
§
‡
‡
‡
§
‡
†
‡
§
§
‡
‡
‡
†
†
†
‡
John I. Trujillo, Felix F. Vajdos, Fabien Vincent, Zhao-Kui Wan, Li Xing, Xiaojing Yang, Xin Yang,
and Liying Zhang†
†
Medicine Design, Pfizer Inc., 1 Portland Street, Cambridge, Massachusetts 02139, United States
‡
Medicine Design, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340, United States
§
Inflammation and Immunology, Pfizer Inc., 1 Portland Street, Cambridge, Massachusetts 02139, United States
∥
Drug Safety Research and Development, Pfizer Inc., 1 Portland Street, Cambridge, Massachusetts 02139, United States
*
S Supporting Information
ABSTRACT: Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines
signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for
the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN
signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has
demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies,
leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while
managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally
constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841
(
compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and
NCT02974868).
1
INTRODUCTION
(STAT) pathway and several oral small molecule JAK
■
2
inhibitors are now approved or in late stage development.
Autoimmune diseases are generally characterized by a
dysregulation of cytokine signaling. Targeting individual
cytokines or their receptors with mAbs or other injectable
biologics has shown efficacy in multiple autoimmune diseases
including rheumatoid arthritis (RA), psoriasis (PSO), inflam-
matory bowel disease (IBD), systemic lupus erythematosus
Across the industry, the most advanced compounds, either
marketed or in Phase III clinical development, are potent JAK1
inhibitors including tofacitinib 1, which inhibits JAK1, JAK2,
JAK3, and, to a lesser extent, TYK2. Baricitinib 2 and
ruxolitinib 3 both inhibit JAK1 and JAK2, while upadacitinib 4
(
SLE), and more recently atopic dermatitis (AD). Many of the
pathogenic cytokines in these diseases signal via the Janus
kinase (JAK)/signal transducer and activator of transcription
Received: June 8, 2018
©
XXXX American Chemical Society
A
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Article
Figure 1. JAK inhibitors on the market and in late stage clinical development.
Figure 2. Basic mechanism of JAK/STAT mediated cytokine signaling.
and filgotinib 5 are reported to be JAK1 selective inhibitors
The JAK kinases are intracellular protein tyrosine kinases
comprised of four members: JAK1, JAK2, JAK3, and TYK2.
Binding of a cytokine causes the formation of a dimer or higher
order complex of receptor subunits, which allows for
recruitment of a pair of JAK enzymes to the receptor (Figure
2). When in close proximity, the JAKs become phosphorylated
and, in turn, phosphorylate tyrosine residues on the cytokine
receptor intracellular domain. These phosphorylated receptor
residues serve as binding sites for STAT proteins, which are
then activated by JAK phosphorylation, enabling STAT
dimerization and translocation to the nucleus where they
3
−7
(Figure 1).
Compounds 1 and 2 are currently approved for
rheumatoid arthritis, while compound 1 is also approved for
the treatment of psoriatic arthritis (PSA) and is in registration
for ulcerative colitis (UC). The JAK1/2 inhibitor 3 is approved
for myelofibrosis, and the JAK1 selective inhibitors 4 and 5
(filgotinib and upadacitinib) are in Phase III studies for RA. In
addition compounds 2, 4, and PF-04965842 6 (JAK1
selective) are in clinical development for the treatment of
atopic dermatitis (AD). Compound 4 has also presented
8
results for Crohn’s disease (CD). Notably, compounds 1 and
2
2
are predicted to have a modest direct effect on IL-12 and IL-
serve as transcription factors for gene expression. JAK1 is the
2
3 signaling via inhibition of JAK2 and weak inhibition of
isoform most broadly found in these signaling pairs, and
therefore JAK1 inhibitors will inhibit JAK1/JAK3 dependent γ-
common chain cytokines, the JAK1/JAK2 dependent IFNγ
and IL-6 and other gp130 cytokines, the JAK1/TYK2
3
TYK2. Recently, Bristol Myers-Squibb advanced a selective
TYK2 inhibitor, BMS-986165 7, into Phase II studies for PSO
and SLE.
9
B
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dependent type I interferons, and the IL-10 family of
EPO signaling. Further, we related the clinical PK:PD
relationships back to in vitro human whole blood cytokine
inhibition using a battery of standardized assays, allowing us to
develop in vitro/in vivo correlations.
1
,3
cytokines. TYK2 is important for signaling associated with
a subset of cytokines, with a significant role for JAK1/TYK2
dependent type I interferons and JAK2/TYK2 dependent IL-
1
,10
1
2 and IL-23.
JAK2 uniquely forms a homodimer, which is
From infusion pump studies in the mouse collagen induced
arthritis (mCIA) model, it was demonstrated that the efficacy
of 1 was correlated with the in vitro whole blood IC50
inhibition values for the JAK1 heterodimer signaling pathways
important in hematopoiesis via signal transduction associated
with erythropoietin (EPO), thrombopoietin (TPO), and IL-3.
Although increasing dose with a JAK inhibitor generally
increases clinical efficacy in multiple diseases, dose-dependent
changes in clinical laboratory parameters may also occur. For
example, hematological changes such as reduction in
hemoglobin (Hgb) due to inhibition of JAK2 homodimer/
erythropoietin signaling have been observed in Phase II studies
which have informed Phase III doses which maximize efficacy,
while minimizing the risk of a meaningful reduction in Hgb
18
(IL6 and IL15). Using data from oral once daily (q.d.) and
twice daily (b.i.d.) dosing paradigms, it was observed that
efficacy was best explained by the 24 h average drug
concentration (C ) and not C
or Cmin values. Clinical
av
max
support for C levels as an efficacy driver were obtained from a
av
19
Phase II study of 1 in RA patients. Furthermore, comparison
of compound 1 q.d. and b.i.d. doses at the same total daily
dose (20 mg) showed similar efficacy for the two regimens.
Under these two dosing regimens, AUC24 was similar, while
Cmin and Cmax values were different, allowing us to conclude
6
,11,12
levels.
The IFNα receptor antibody Anifrolumab has recently
13
shown efficacy in a Phase II trial in SLE. Ustekinumab, which
inhibits the shared P40 subunit of IL-12 and IL-23, is approved
for PSO, PSA, and CD and has recently reported efficacy in a
that average daily inhibition (C ) can be used to derive PK:PD
av
relationships. The 5 mg b.i.d. Cav concentration for 1 was
found to be equivalent to the human whole blood potency
values for IL-6 and the γ-common chain cytokine signaling
pathways represented by IL-15, helping to establish a lower
1
4,15
Phase II SLE trial.
Anti-IL-23 antagonists have shown
14
efficacy in CD and also in PSO. These results support
targeting TYK2 to capitalize on the inhibition of IL-12 and IL-
3
2
3 signaling. Human genetics further support targeting TYK2.
A common nonsynonymous polymorphism in TYK2 P1104A
RS34536443) leads to an estimated 80% reduction in activity
boundary for potency. Further support was recently published
for 5 in RA patients whereby similar efficacy was observed for
20
(
b.i.d. and q.d. dosing regimens of the same total daily dose.
in homozygous carriers. Individuals with one hypomorphic
allele are at reduced odds of developing PSO, RA, SLE, CD,
Type I diabetes, ankylosing spondylitis, multiple sclerosis,
juvenile idiopathic arthritis, and primary biliary cholangitis,
while homozygous carriers are at reduced odds for UC.
Bacterial, viral (including VZV), and fungal infections that
have been observed in IL-12/23, type I interferon, and IL-17
deficiencies were not elevated in TYK2 hypomorphic allele
Having established that drug response (at least in RA and
models of RA) is driven by Cav exposure and that efficacy
benefited from at least IC50 Cav target coverage, we next
needed to determine the upper boundary for EPO signaling
inhibition. In Phase II dose ranging studies in RA and PSO
patients, the effect of 1 on HgB was monitored. In RA (study
NCT00147498) where HgB levels are suppressed due to
inflammation, HgB trended higher with 5 mg b.i.d. and
10
21
carriers relative to wild type individuals.
decreased in a dose dependent manner at 15 and 30 mg b.i.d.
In a subset of diseases with a strong interferon and/or IL-12
or IL-23 component to the pathogenesis of disease, we may
therefore be able to enhance the activity of a JAK1 inhibitor by
simultaneously targeting TYK2. Such a compound would be
postulated to possess the efficacy of a JAK1 inhibitor driven
through γ-common chain cytokines and IL-6 signaling, with
the added ability to mimic the effect of an anti-P40 inhibitor
blocking IL-12 and IL-23 signaling, through TYK2 inhibition.
Type I interferon signaling would be suppressed through both
JAK1 and TYK2 blockade. Selectivity against JAK2 and thus
JAK2 driven cytokine signaling (e.g., EPO) would in turn spare
undesirable hematopoietic changes.
In a PSO Phase IIb study (NCT00678210), HgB levels were
not statistically different from placebo at 5 mg b.i.d., but at 15
mg b.i.d. were associated with a significant reduction (−0.52 g/
22
dL vs −0.14 g/dL for placebo) at week 4. Across two large
Phase III PSO trials (OPT1 and OPT2), which are not
complicated by anemia associated with the disease, 741
patients received 1 at 10 mg b.i.d. The median HgB change
from baseline was −0.3 g/dL in OPT1 and −0.4 g/dL in
OPT2; neither was statistically significant. The proportion of
23
patients with Hgb < 10 g/dL was 0.3%. In long-term
extension studies in RA patients treated over 48 months with 1
at a dose of 10 mg b.i.d. on a background of methotrexate
(
standard of care), less than 3% of patients had a >2 g/dL
24
RESULTS AND DISCUSSION
■
change in HgB.
18
Program Objective. In establishing our program objec-
tives, it was important to determine how selectivity between
JAK isoforms and, more importantly, the relative level of
inhibition of different cytokines at a given concentration or
dose, would be defined. The relationship between pharmaco-
kinetics and pharmacodynamics (PK:PD) are key parameters
underlying these target coverage relationships. To help us with
this task, we used the large body of preclinical and clinical data
that we have published from the tofacitinib 1 program. Similar
data have also been made available in publications for
Utilizing the global RA PK model published for 1, a new
model was developed to determine the relationship of plasma
levels of 1 to inhibition of EPO signaling. Based upon this
model, the daily C EPO inhibition (IC ) for compound 1 at
5 and 10 mg b.i.d. doses was determined to be IC20 and IC33,
respectively.
From the data discussed above in aggregate, we established
target values for the program with efficacy to be driven by Cav
IC coverage of the primary TYK2 and JAK1 pathway (IFNα)
av
xx
8
0
and C ≤ IC for the JAK2 pathway (EPO). As an expedient
av
30
16,17
compounds 2 and 5.
Most importantly, we used these
for routine SAR, we calculated an IC * value (IC * =
xx xx
data to define the level of inhibition required to provide robust
efficacy, defined by IFNα (JAK1/TYK2) blockade, and the
level of JAK2 selectivity required to minimize inhibition of
100*((IC80 IFNα)/ (IC80 IFNα + IC EPO)) to determine
50
+
selectivity for EPO (CD34 cells spiked into human whole
blood (HWB); see Methods) inhibition as a function of the
C
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Table 1. Enzymatic Potencies for Compounds 8−10 Determined against TYK2, JAK1, JAK2, and JAK3 at the K for ATP for
m
Each Kinase
a
Compounds were assayed at least twice, and the IC50 reported as the geometric mean. ATP concentration at the apparent K for each kinase.
m
b
c
d
Shake-flask LogD measured between octanol/water phosphate buffered to pH 7.4. LipE = pIC − LogD. ND = Not Determined.
5
0
IFNα HWB IC . For advanced compounds, a human PK
Cell potencies were determined in human whole blood
assays, where blockade of STAT phosphorylation, following
cytokine stimulation, was monitored by flow cytometry. As the
potency is determined in whole blood, comparisons between
compounds can only be made when taking into account
unbound concentrations. Therefore, an estimation of the
unbound potency was made from the total HWB potency, the
human plasma free fraction, and red blood partitioning ratio,
Table S1. Where measured data were unavailable, a calculated
human plasma free fraction value was used, based upon an
8
0
model was developed from in vitro and in vivo data, which
allowed us to determine the predicted dose to cover the IFNα
IC . Target coverage IC values for EPO and other inhibited
8
0
xx
cytokines were derived from this model.
Assays and Data Reporting. Compounds were tested for
enzymatic potency in a Caliper assay (PerkinElmer) with the
ATP concentration at either the K for each of the JAK targets,
m
or at a pseudophysiological concentration of 1 mM. At higher
ATP concentrations the ability to identify weak lead matter can
be compromised. During the course of our work, we have
found that correlations between enzymatic potencies and
cellular activities are best determined with the ATP
concentration under pseudophysiological conditions at 1
26
internal in silico model.
Lead Chemical Matter. Initial work focused upon
identifying lead chemical matter which demonstrated promis-
ing potency, inhibiting both TYK2 and JAK1 kinases with
evidence of selectivity over JAK2 and JAK3. A series of 2,4-
diamino pyrimidines provided the interesting chiral diaza-
bicyclo[2.2.1]octane derivative 8 and its enantiomer 9 (Table
1). A clear stereochemical preference is evident from the
biochemical activity with 8 and 9 displaying TYK2 IC50 = 23
2
5
mM. Moreover, under these conditions, selectivity ratios
between the JAK kinases are more appropriately determined,
as opposed to using the Km for ATP which varies for each
target. For example, the ATP Km for JAK3 has been
determined in our system to be 4 μM, whereas the Km for
JAK1 is 40 μM. The K for TYK2 and JAK2 were determined
nM and 2690 nM respectively, determined at the K for ATP.
m
m
to be 12 and 4 μM, respectively. Lipophilic efficiencies (LipE)
were calculated from the TYK2 IC50 measured with 1 mM
ATP and the measured LogD value.
The role of the methylene bridge in the eutomer was apparent
by comparison with the less potent piperazine analog 10,
TYK2 IC50 = 368 nM.
D
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Figure 3. X-ray crystal structure of compound 8 in complex with TYK2, resolved to 2.0 Å (PDB code 6DBK).
The S,S-stereochemistry of the eutomer was determined
from a crystal structure obtained of compound 8 bound to the
TYK2 kinase domain (Figure. 3). Based on the crystal
structure, we suggest that the bridged piperazine is serving
two roles: first, to provide a conformationally rigid structure
that projects the pendant cyanoacetamide toward the P-loop of
TYK2, where the nitrile makes further interactions with Gly-
9
06 and, second, to provide an additional lipophilic interaction
by filling a pocket at the bottom of the binding site (Figure 3).
Compound 8 was further profiled against the JAK isoforms
under the pseudophysiological conditions with [ATP] = 1
mM. At this ATP concentration, the compound demonstrated
weaker inhibition of TYK2 consistent with Michaelis−Menten
behavior (TYK2 IC = 774 nM), and similarly weaker activity
5
0
against JAK1, JAK2, and JAK3 (JAK1 IC50 = 3714 nM, JAK2
IC50 > 9972 nM and JAK3 IC50 > 10 000 nM). Throughout
the remainder of this manuscript, enzymatic potencies will be
reported with the ATP concentration set at 1 mM.
Figure 5. Structures for compounds 11−15.
The modular nature of substituents around the pyrimidine
1
core allowed dissection of the molecule into four R-groups: R
2
Table 2. Enzymatic Potencies for Bridged Diamines 11−15
Determined against TYK2, JAK1, JAK2, and JAK3
the bridged piperazine moiety, R a hydrogen atom or fluorine
adjacent to the gatekeeper Met-978, R the solvent exposed
aromatic substituent, and the R functionality to interact with
3
4
IC50 (nM)
the P-loop of the kinase (Figure 4).
a
a
a
a
b
c
compd no. TYK2
JAK1
JAK2
JAK3
sfLogD
LipE
1
1
1
1
1
1
2
3
4
5
1738
148
201
32
>9524
748
3473
122
>10 000
2782
7851
329
>10 000
>10 000
>10 000
>10 000
3078
1.96
2.16
2.15
1.86
2.88
4.10
4.97
4.85
5.94
d
19
63
306
ND
a
Compounds were assayed at least twice, and the IC was reported as
5
0
b
the geometric mean. ATP concentration = 1 mM. Shake-flask LogD
measured between octanol/water phosphate buffered to pH 7.4.
c
d
LipE = pIC50 − LogD. ND = not determined.
Figure 4. R-group nomenclature for 2,4-diamino pyrimidines 8−25.
compounds in the series, presumably due to improved
hydrophobic interactions with the residues at the bottom of
the binding site as illustrated in Figure 3. The S,S-diaza-
bicyclo[2.2.2]octane 12 gained substantially in potency (TYK2
IC = 148 nM), as did the [3.1.1] system 13 (IC = 201 nM).
1
We first chose to evaluate alternate bridged diamines R to
explore the role on potency and selectivity (Figure 5 and Table
2
). In this instance, the compounds were prepared as
5
0
50
cyclopropyl amides. The S,S-cyclopropyl amide 11 showed
similar potency to the cyanoacetamide homologue 8 (TYK2
IC50 = 1738 and 774 nM, respectively) and served as a good
reference point. Potency against TYK2 improved for the other
The 3,8-diazabicyclo[3.2.1]octane system 14 was clearly the
most potent and efficient in the series, while maintaining the
desired JAK1 activity (IC50 = 32 and 122 nM respectively for
TYK2 and JAK1, TYK2 LipE = 5.94). Compound 14 also
E
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Article
showed interesting selectivity against both JAK2 and JAK3
case with the 2-pyridyl system 19 whereby to accommodate
both requirements of planarity and internal ligand strain; the
pyridyl nitrogen needs to be in close proximity with the
backbone carbonyl oxygen atom of Val-981, an energetically
unfavorable interaction.
Data from compounds in this series were important to
understand the translation of enzymatic to cellular potencies,
(
IC50 = 329 and >10 000 nM, respectively).
During the course of the program, we explored the R
2
substituent at the 5-position of the pyrimidine. From a
comparison with compound 15, the 5-fluoro analog of
compound 14 in the 3.2.1 series, it can be seen that the two
compounds are within 2-fold in terms of their TYK2 potency.
Furthermore, the fluorine adds approximately 1 unit of
lipophilicity (shake flask LogD (sfLogD) = 1.86 and 2.88 for
and corresponding selectivity (IC *) over EPO inhibition.
xx
The pyrazoles 16 and 17 exhibited good potency in the IFNα
HWB assay (IC50 (free) = 75 (70) and 144 (84) nM
respectively). The free IFNα potencies showed good
concordance with the TYK2 and JAK1 enzymatic activity. In
the JAK2 driven EPO HWB assay these compounds achieved
1
4 and 15, respectively), which consequently reduces the
lipophilic efficiency of the molecule (TYK2 LipE = 5.94 and
.13 for 14 and 15 respectively). As a result, although we
5
studied SAR within the 5-fluoropyrimidine series, the 5-H
analogs were generally preferred.
good selectivity (EPO IC * = 10 and 8 for 16 and 17,
xx
We elected to study the 3.2.1 series further, the initial
respectively). The pyridine 18 behaved similarly (IFNα HWB
3
priority was given to explore the R vector and identify
IC50 (free) = 385 (91) nM, EPO IC * = 11).
xx
3
replacement functionality for the embedded aniline moiety to
The R N-methyl pyrazole functionality was elected for
27
remove the structural alert.
further study due to its high lipophilic efficiency (15 TYK2
LipE = 5.45) and reduced risk of Phase II metabolism relative
to the N-ethanol derivative 14.
A small array of compounds, in this instance with a fluorine
at position 5 of the pyrimidine ring, was prepared to assess
SAR close to the solvent front of the ATP binding site of
TYK2 (Figure 6 and Table 3). It is apparent that potency can
A series of acylated derivatives, amides and ureas, were
prepared using the N-methyl pyrazole platform, in this instance
2
with hydrogen as R (Figure 8 and Table 4). The most active
compound in this series was the cyanomethylene urea 20 PF-
06730129 (TYK2 IC50 = 16 nM). Similar activity was also
observed for the trifluoroethyl urea 21, both of which were
more potent than the simple ethyl analog 22 (TYK2 IC = 28
5
0
and 71 nM, for 21 and 22 respectively). The trifluoroethyl urea
1 was more potent in the IFNα HWB assay than was the
2
cyanomethylene urea 20 (IFNα HWB IC50 (free) = 51 (26)
and 120 (73) nM, respectively). Compound 20 is the most
polar compound in this series (LogD 0.97) and as a result
suffers from relatively low permeability in the RRCK assay (A-
B mean PAPP 0.6 × 10 cm/s), which may explain its
surprisingly low potency in HWB. The other compounds
presented in Table 5 have good permeability in the RRCK
assay (mean PAPP 5.6−18.8 *10−6 cm/s), despite relatively
high polarity (sfLogD 1.40−1.72).
−
6
Figure 6. Structures of compounds 16−19.
be modulated by structural modifications close to this solvent
exposed vector, whereby key interactions need to be
maintained with the protein. Functionalities such as the
pyrazoles 16 and 17 and 3-pyridyls 18 are readily
accommodated by the protein (TYK2 IC50 = 55, 53, and 48
nM respectively). However, simple analogous systems such as
the 2-pyridine 19 are not accommodated (TYK2 IC50 = 7202
nM). This difference in activity can be explained through
structural analysis (Figure. 7). The planarity of the heteroaryl-
In the amide series, the 1,1-difluoro-cyclopropanes proved to
be particularly effective. The S-enantiomer 23 was found to be
the eutomer, with a balanced TYK2 and JAK1 inhibition
profile (TYK2 and JAK1 IC50 = 23 and 17 nM, respectively).
Compound 23 inhibited JAK2 and JAK3 with IC50 = 77 and
6494 nM, respectively. In the IFNα human whole blood assay
the compound showed good potency (IFNα HWB IC50 (free)
= 30 (13) nM), with EPO selectivity well within our program
2
-amino pyrimidine moiety is made necessary by the presence
of Leu-903 above the plane of the molecule, and Leu-1030 and
Gly-984 below the plane of the molecule. This planar
arrangement necessitates elimination of intramolecular stereo-
electronic interactions to reduce ligand strain. This is not the
objective (EPO IC * = 18). The unsubstituted cyclopropane
xx
24 was approximately 3-fold less potent against TYK2 and
somewhat unbalanced with respect to JAK1 (TYK2 and JAK1
IC50 = 76 and 199 nM, respectively), while the distomer 25,
Table 3. Enzymatic and Cellular Potencies for 16−19
a
e
IC50 (nM)
HWB IC50 (nM) (free)
b
b
b
b
c
d
b
b
compd no.
TYK2
JAK1
81
76
JAK2
JAK3
sfLogD
LipE
IFNα
EPO
EPO ICxx*
1
1
1
1
6
7
8
9
55
53
48
260
488
692
2531
2878
4962
1.78
2.13
3.21
2.71
5.78
5.45
4.41
2.73
75 (70)
144 (84)
385 (91)
2,596
6,285
12,166
ND
10
8
11
ND
234
f
7202
>10 000
>10 000
>10 000
ND
a
b
c
ATP concentration = 1 mM. Compounds were assayed at least twice, and the IC reported as the geometric mean. Shake-flask LogD measured
5
0
d
e
between octanol/water phosphate buffered to pH 7.4. LipE = pIC − LogD. Free IC = (IFNα HWB IC50 * human plasma free fraction)/
human red blood cell to plasma ratio. ND = not determined.
5
0
50
f
F
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Figure 7. Compounds 16 and 18 modeled in the ATP binding site of TYK2, based upon APO protein PDB code 6DBK..
Figure 8. Structures of acylated 3,8-diazabicyclo[3.2.1]octane derivatives 20−25.
Table 4. Enzymatic Potencies, Efficiency, and Human Whole Blood Activity for IFNa and EPO for Compounds 1, 20−25
d
IC50 (nM)
HWB IC50 (nM) (free)
a
a
a
a
b
c
compd no.
TYK2
JAK1
JAK2
JAK3
sfLogD
TYK2 LipE
IFNα
EPO
EPO ICxx*
3
3
3
3
e
e
1
489
15
77
67
55
1.13
0.97
1.72
1.44
1.61
1.52
1.55
5.17
7.13
6.13
6.01
6.31
5.90
4.91
38 (19)
120 (73)
51 (26)
116 (60)
30 (13)
119 (91)
328
33
f
2
2
2
2
2
2
0
1
2
3
4
5
16
28
71
23
76
702
13
14
104
17
199
842
2477
6872
>10 000
6494
>10 000
>10 000
1229
1160
2866
577
3737
5655
28
15
14
18
11
37
146
538
77
684
3148
823 (399)
a
b
Compounds were assayed at least twice, and the IC50 reported as the geometric mean. ATP concentration = 1 mM. Shake-flask LogD measured
c
d
between octanol/water phosphate buffered to pH 7.4. LipE = pIC − LogD. Compounds were assayed at least twice, and the IC reported as
5
0
50
e
the geometric mean. Free IC50 = (IFNα HWB IC * human plasma free fraction)/human red blood cell to plasma ratio. ICxx calculated at 10 mg
50
18 f
BID using an unbound Cav = 83 nM. Free IC50 = IFNα HWB IC50 * calculated human plasma free fraction.
bearing the R-1,1-difluorocyclopropane, was significantly less
and W3 respectively), their displacement would be predicted
active (TYK2 and JAK1 IC = 702 and 842 nM, respectively).
to increase the binding affinity for the molecule. W1, which we
considered a “neutral water” (ΔH = −0.54 and ΔG = 1.68),
would also be displaced by the inhibitor. For reference the
most energetically stable predicted water from the simulation
(W14 not shown) has calculated ΔH = −3.08 and ΔG =
−0.82. We built a model of the distomer 25, wherein the
overall binding mode of the compound is similar to that for 23;
however, in this case the difluoromethylene group projects
down into a region of negative electrostatic potential formed
by the side chain carbonyls of Asp-1041 and Asn-1028, an
energetically unfavorable binding mode (Figure 9c). This may
account for the loss in activity for this enantiomer.
50
To further understand the TYK2 potency of compound 23
we obtained a crystal structure with the protein (Figure 9).
The compound adopts the expected binding mode, forming
donor and acceptor hydrogen bonds between the amino
pyrimidine moiety and Val-981. The ethylene bridge of the
[
3.2.1]-diazabicycle projects into the lower hydrophobic
portion of the binding site as depicted in Figure 9, while the
difluoromethylene of the cyclopropyl amide projects up into
the P-loop of the ATP binding site. Overlaid onto the structure
are simulated APO waters (Hydrosite), colored by ΔH (Figure
2
8
9
a). Proximal to the cyclopropyl amide in the model are
three simulated waters W1, W3, and W12. W12 and W3 are
considered high energy waters (ΔH = 5.28 and 2.09 for W12
and W3 respectively, and ΔG = 7.78 and 3.11 for waters W12
A crystal structure of compound 23 was also obtained with
JAK1 (Figure 10). As expected, the compound binds to the
protein in approximately the same pose as in TYK2. The
G
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Table 5. Human Whole Blood Potencies and IC for
reference value for establishing C . 23 potently inhibits
eff
xx
Compounds 1 and 23 with Different Cytokine Stimuli
TYK2/JAK2 mediated IL-12/pSTAT4 and IL-23/pSTAT3
(
HWB IC50 = 65 and 120 nM, respectively). The compound
a
IC50 (nM)
ICxx
+
has good potency against IL6/pStat1 in the CD3 cellular
JAK
signaling
complex
subset (IC50 = 81 nM), but lower inhibition of IL6/pSTAT3,
+
4
1
b
c
again in the CD3 cellular subset (IC = 641 nM). The
Cytokine/pStat
1
23
1
23
50
mechanistic basis for the difference in potency in IL6 signaling
between pStat1 and pStat3 is presently unknown. The
compound also inhibits the JAK1/JAK3 driven γ-common
chain cytokines, represented by IL-15/pStat5 and IL-21/
pSTAT3 with reasonable potency (HWB IC50 = 238 and 204
nM, respectively). Compound 23 inhibits EPO/pSTAT5
IFNα induced pSTAT3
TYK2/
JAK1
38
30
81
76
75
23
36
34
74
76
40
76
33
80
29
60
16
29
65
34
38
50
59
18
(
lymphocytes)
+
IFNγ induced pSTAT1 (CD14 JAK1/JAK2
cells)
IL-6 induced pSTAT1 (CD3
cells)
IL-6 induced pSTAT3 (CD3
cells)
52
298
81
+
JAK1/JAK2
JAK1/JAK2
54
+
367
291
315
58
641
305
65
+
(JAK2 homodimer) in HWB spiked with CD34 progenitor
IL-10 induced pSTAT3
TYK2/
JAK1
JAK2/
TYK2
cells (IC50 = 577 nM). IL10/pSTAT3 (TYK2/JAK1) and
IL27/pSTAT3 (JAK1/JAK2/TYK2) are also inhibited by the
compound with IC50 = 305 nM and 86 nM, respectively.
Compound 23 was evaluated against a panel of 306 kinases
at the apparent Km for ATP for each kinase (Carna
Biosciences). A subset of 157 kinases was additionally assayed
at an ATP concentration of 1 mM. The test concentration of
PF-06700841 was 1 μM, and the data were expressed as %
inhibition. Under the screening conditions for ATP at the Km
for each kinase, 21 kinases out of 307 (7%) targets displayed
(
lymphocytes)
IL-12 induced pSTAT4
lymphocytes)
(
IL-15 induced pSTAT5
lymphocytes)
JAK1/JAK3
238
204
120
86
(
IL-21 induced pSTAT3
lymphocytes)
JAK1/JAK3
51
(
IL-23 induced pSTAT3
lymphocytes)
JAK2/
TYK2
JAK1/JAK2
249
51
(
IL-27 induced pSTAT3
lymphocytes)
(
>
50%: TYK2 (101%), JAK1 (101%), JAK2 (101%), AurA/
EPO induced pSTAT5 (spiked JAK2/JAK2
328
577
+
CD34 progenitor cells)
TPX2 (97%), TNK1 (94%), JAK3 (92%), QIK (91%), ACK
(82%), PDGFRα (Τ674I) (78%), SIK (77%), PDGFRα
(70%), MAP2K5 Cascade (70%), MNK2 (68%), PDGFRβ
(66%), ERK5 (63%), AurA (62%), PDGFRa (V561D) (60%),
Nuak1 (60%), FMS (59%), MARK4 (59%), and MARK3
a
Compounds were assayed at least twice, and the IC50 is reported as
b
the geometric mean. IC calculated at 10 mg BID using an unbound
C = 83 nM. ICxx calculated from predicted C exposure to cover
xx
18 c
av
av
IFNα IC .
80
(
50%). Under the higher ATP concentration of 1 mM, 4
kinases out of 157 (4%) displayed inhibition above 50%: TYK2
(
98%), JAK1 (96%), JAK2 (94%), and TNK1 (64%).
Compound 23 was also profiled in vitro against a broad
panel of receptors, ion channels, and transporters in the
CEREP Wide Ligand Profile screen at a single concentration of
interactions with both enzymes. Examination of the simulated
waters shows a similar picture as found in TYK2, with high
energy waters W1 and W6 proximal to the difluorocyclopropyl
amide (ΔH = 5.14 and 2.12, and ΔG = 6.21 and 3.19 for W1
and W6 respectively). In addition, a second high energy water
is present proximal to the cyclopropyl moiety (W5), and an
interesting unstable water (W2) is found in the hydrophobic
portion of the binding site, proximal to the ethylene bridge of
the ligand (ΔH = 2.37 and 3.38, and ΔG = 4.09 and 4.22 for
W1 and W6 respectively). As with TYK2, a relatively stable
water (W29) would also be expected to be displaced by the
compound (ΔH = −1.03 and ΔG = 0.26).
The [3.2.1] series was found, in general, to be in good
property space for oral absorption (Table S2). LogD values are
aligned with the in vitro clearance values as expected. For
example, human liver microsome stability was good (HLM
Clint ≤12 uL/min/mg) for all compounds, except for 18,
which had the highest LogD (3.21). Kinetic solubility was also
poor for compound 18 (14 μM at pH 6.5) and for compound
1
0 μM. Less than 50% inhibition of functional, binding, or
enzyme activity was observed against all targets except for
kinase insert domain receptor (KDR) (VEGFR2) (IC = 1600
nM). This in vitro VEGFR2 inhibitory activity did not translate
in to a functional response in a cell-based assay for VEGFR2
signaling (NovaScreen), where its IC50 was determined to be
5
0
>
30 μM, demonstrating that the screening data from kinase
assays conducted at enzymatic Km ATP concentrations can
translate poorly to functional cellular settings with higher
physiologic ATP conditions.
The physical properties of compound 23 were favorable for
oral dosing. The conjugate acid of the compound has a
measured pKa of 6.33 and was converted into the p-
toluenesulfonic acid salt, with a melting point of 275 °C.
The salt form had good solubility in buffered water (4.84 mg/
mL at pH 7.64 in phosphate buffered saline), as well as in
simulated gastric fluids FASSIF and FESSIF (>7 mg/mL).
Compound 23 was also shown to have high passive
permeability, RRCK (mean PAPP 18.8 × 10 cm/s). The
pharmacokinetics of 23 PF-06700841 were studied in
Sprague−Dawley rats following intravenous and oral admin-
istration (1 and 3 mg/kg respectively) of the tosylate salt,
where the compound showed a plasma clearance of 31 mL/
min/kg, a volume of distribution of 2.0 L/kg, and oral
bioavailability of 83%. Following the 3 mg/kg oral dose, the
Cmax was 774 ng/mL and the AUC∞ was 1340 ng·h/mL. The
high oral bioavailability indicated high absorption from the gut,
−
6
1
5 (40 μM at pH 6.5). Low LogD (sfLogD ≤ 1.52) for
compounds 20, 22, and 24 correlated with reduced
permeability in the RRCK assay (mean PAPP ≤ 6.3 × 10
cm/s)).
−
6
Compound 23 was selected for further characterization. The
compound was assayed in a human whole blood panel to
evaluate inhibition across the JAK/STAT cytokine signaling
families (Table 5). As previously discussed the IFNα/pSTAT3
HWB potency for the compound is 30 nM, which serves as the
H
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Figure 9. (a) Crystal structure of 23 with TYK2 (2.37 Å, PDB code 6DBM); spheres represent simulated apo waters, colored by ΔH: blue
represents negative ΔH stable water, red represents positive ΔH unstable water. (b) End view of 23 looking toward the hinge of the protein,
showing proximity to key simulated water molecules (spheres) W1, W3, and W12, colored by ΔH as in Figure 9a. (c) Model of 25 bound to TYK2
based upon PDB code 6DBM, showing proximity of fluorine atoms to electrostatically negative (red) region of the binding pocket formed by
carbonyl oxygens of Asp-1041 and Asn-1028.
Figure 10. (a) 23 bound to JAK1 (2.48 Å, PDB code 6DBN). Spheres represent simulated waters. colored by ΔH: blue represents negative ΔH
stable water, and red represents positive ΔH unstable water. (b) End view showing proximity of simulated waters, and associated energy values, to
the amide moiety of 23 bound to JAK1.
consistent with its in vitro passive permeability properties and
high solubility.
The in vitro metabolism of 23 was studied in human liver
(fraction unbound (f ) 0.69) and human (f 0.61), with no
u u
significant blood to plasma partitioning (human Crbc/Cp 1.2).
Oxidative metabolites of compound 23 accounted for the
primary routes of biotransformation in rat, monkey, and
human in vitro hepatic systems, consistent with CYP450 as the
primary clearance route. Metabolic pathways included
microsomes (Clint,u < 10 μL/min/mg protein) and hepatoctyes
(
Clint,u < 0.6 μL/min/million cells), which indicated low
metabolic turnover. Plasma protein binding was low in rat
I
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oxidation of the N-methyl pyrazole (primary), N-demethyla-
tion, and N-dealkylation (loss of pyrazole). The clearance
mechanisms of 23 were determined to be mediated primarily
by CYP450 metabolism (primarily through CYP3A4), with
limited renal and biliary clearance expected. Human
pharmacokinetic predictions were made using human liver
microsomes, hepatocytes, and recombinant CYP3A4, as well as
standard single species allometric scaling techniques (Table 6).
volume was significantly lower and dose-dependent in the
compound 23 q.d. (3, 10, and 30 mg/kg) treated groups. The
plasma concentrations in animals dosed with 23 at peak (30
min) and trough (24 h) time intervals post final dose
respectively were as follows (mean ± SD): 3 mg/kg, 3.54 ±
0.60 μM, 0.0221 ± 0.0139 μM; 10 mg/kg, 10.95 ± 2.06 μM,
0
.06 ± 0.0576 μM; and 30 mg/kg, 23.89 ± 4.12 μM, 0.06 ±
.029 μM. Pharmacodynamics modeling of the data fit an
0
Emax model, Figure S1.
Table 6. Summary of Predicted Human Blood
Pharmacokinetics of 23 from in Vivo and in Vitro Systems
a
CHEMISTRY
■
The synthesis strategy utilized key functionalized diamine
cores, to which we could introduce the acetylated group
amides or ureas) or solvent exposed ring system of the
diaminopyrimidine in either order to maximize synthetic
efficiency. To that end, diamine intermediates 28 were
obtained from the S Ar reaction of a 2,4-dichloropyrimidine
27) and a Boc-protected bridged piperazine 26 (Scheme 1).
At room temperature, the reaction was highly selective for
displacement at the pyrimidyl 4-chloride. Pyrimidine 5-
position functionality (H or F) had minimal effect on the
reaction except for an increase in yield in the case of the 5-
fluoro derivatives, presumably due to the higher reactivity of
this dichloropyrimidine intermediate. The Boc protecting
group was removed under standard acidic conditions followed
by amide coupling using HATU to provide acetylated
intermediates 29. The formation of the diaryl amine hinge-
binding moiety was completed to provide 8, 9, 10, and 17
Human Prediction
Method
Cl (mL/min/kg)
b
Vss (L/kg)
(
Human Liver Microsomes
Human Hepatocytes
rCYP3A4
<3.9
<0.8
4.5
N
Rat Single Species Scaling
5.8
1.7
(
a
CL : blood clearance. V : volume of distribution at steady state.
b
ss
Fraction unbound plasma protein binding in rat and human were 0.69
and 0.61 respectively. Human clearance predictions from in vitro
systems were scaled based on the well-stirred model or single species
42,43
scaling from allometry.
The overall cytokine inhibition profile (IC ) was calculated
xx
for 23 based upon the modeled C exposure to cover the IC
av
80
of IFNα (64 nM, unbound) (Figure 11). The IL-12 and IL-23
under acidic S Ar conditions and 13 and 19 via Buchwald
N
coupling conditions. Inversely the aryl group could be
introduced first on the Boc-protected diamine core 28 using
similar S Ar or Buchwald conditions. The S Ar conditions
N
N
concurrently hydrolyzed the protecting group to give the
amines 30, whereas the Buchwald conditions required a
subsequent acid deprotection step to afford 30. Amide
formation via standard HATU conditions provided 11, 12,
1
4, 15, 16, 18, 24, and 25. Using preformed imidazole-1-
carboxamides under basic conditions with 30 provided ureas
2
0, 21, and 22.
The synthesis of the 3,8-diazabicyclo[3.2.1]octane 23 is
described in Scheme 2. Starting from commercial racemic-31,
esterification with (S)-1-phenylethan-1-ol and DCC, followed
by diastereomeric separation via chiral column chromatog-
raphy, provided the single diastereomer 32. Ester hydrolysis
using sodium hydroxide in methanol provided the single
Figure 11. Radial plot of cytokine inhibition ICxx relative to the IFNα
IC80 for compound 23, derived from potency in human whole blood
assays and predicted average daily human plasma drug concentration.
enantiomer-33 in good yield. S Ar using commercial Boc
N
protected [3.2.1] diamine 34 with 2,4-dichloropyrimidine
under basic conditions provided selective 4-position sub-
stituted product 35 in 71% yield. The pyrazole-2-amino
target coverage is predicted to be high, achieving IC for IL-12
65
and IC50 for IL-23. IL-6 inhibition of pSTAT1 signaling
differentiates from that of IL6 pSTAT3 (IC60 and IC16,
respectively). Contribution to the profile from the γ-common
chain cytokines, represented by IL-15 and IL-21, are lower at
pyrimidine moiety was constructed via a second S Ar under
N
acidic conditions using 35 and 1-methyl-1H-pyrazol-4-ylamine.
The reaction conditions also resulted in loss of the Boc group
on the [3.2.1] diaminobicycle to provide 36 in excellent yield.
Amide formation between 36 and 33 under standard HATU
conditions provided 31 in 60% yield. The material was
converted to the tosylate salt in acetonitrile using 2 M p-
IC38 and IL , respectively. IL-27 and IL-10 target coverage is
34
anticipated to be at IC59 and IC , respectively. Importantly,
29
the compound is expected to spare JAK2 driven EPO signaling
(
IC ).
18
Compound 23 was studied in vivo under a therapeutic
dosing paradigm in the rat adjuvant induced arthritis (AIA)
following oral dosing of 23 p-tosylate salt (Figure 12).
toluenesulfonic acid in H O followed by collection of salt by
2
filtration in 76% yield.
29
Female Lewis rats immunized with complete Freund’s adjuvant
were dosed orally with 3, 10, or 30 mg/kg/day of 23 or vehicle
for 7 consecutive days after disease onset as measured by hind
paw volume using a plethysmograph. The increase in paw
CONCLUSION
■
In summary, we have described the rationale underpinning the
design of a subtype selective JAK inhibitor which was designed
J
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Figure 12. Activity of compound 23 in the rat AIA model following a therapeutic dosing paradigm. Immunized rats were enrolled into groups and
dosed with either: 30, 10, or 3 mg/kg 23 or vehicle PO. Compared to vehicle (left panel), treatment with PF-06700841 significantly reduced
disease severity as measured by plethysmograph in the 30 mg/kg (Day 1 to 7), 10 mg/kg (Day 1 to 7), and 3 mg/kg (Day 2 to 7) dose groups (p ≤
0
.05, t test).
a
Scheme 1. General Synthesis of Diamine Analogs 8−25
a
Reagents and conditions: (a) TEA or DIPEA, 10−50 °C, 1−18 h, 47−96%; (b) (i) 4 N HCl, 10−20 °C, 15−16 h, (ii) cyclopropanecarboxylic
acid or 2-cyanoacetic acid, HATU, DIPEA or TEA, rt, 15−60 h, 26−100%; (c) aryl amine, i-PrOH or dioxane, 1−16 h, 85−140 °C, 38−65%; (d)
(
i) aryl amine, Pd cat, Cs CO or NaOtBu, 100−145 °C, 0.5−4 h, 21−95%; (ii) 4 N HCl, 20−25 °C, 1−18 h, 86−99% (e) for amides, carboxylic
2
3
acid, DIPEA or TEA, HATU, rt, 1−18 h, 18−89%; (f) for ureas, alkyl-1H-imidazole-1-carboxamide, EtOH, 60 °C, 3 h, 59−70%.
to take advantage of inhibition of cytokines mediated by the
TYK2 isoform, in particular IL-12 and IL-23, as well as those
mediated by JAK1. In setting the objectives for this program,
we used published clinical and preclinical data from tofacitinib
dual TYK2 and JAK1 potency with appropriate in-family
selectivity against JAK2 and JAK3. The lead compound from
this series, compound 23, has an excellent off-target
polypharmacology profile and ADME profile consistent with
once daily dosing in humans. Compound 23 (PF-06700841)
has completed a Phase 1 clinical study in healthy volunteers
1
and other JAK inhibitors. This allowed us to establish
potency and selectivity targets in human whole blood assays,
which we believe predict both disease and certain safety end
points (e.g., EPO modulation).
30
and psoriasis patients and is currently being studied in
Early bicyclic leads identified from the Pfizer compound
collection were developed into a series of 3,8-diazabicyclo-
multiple indications in Phase II trials [NCT02969018,
NCT02958865, NCT03395184, and NCT02974868].
[3.2.1]octane substituted pyrimidines, which delivered potent
K
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Journal of Medicinal Chemistry
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a
Scheme 2. Synthesis of 3,8-Diazabicyclo[3.2.1]octane 23
a
Reagents and conditions: (a) (S)-1-phenylethan-1-ol (1.2 equiv), DCC (1.2 equiv), DMAP (0.1 equiv), rt, 18 h, 72%; (b) 1 N NaOH (3 equiv),
MeOH, rt, 1.5 h, 76%; (c) 2,4-dichloropyrimidine (1.1 equiv), TEA (1.3 equiv), MeOH, rt, 18 h, 71%; (d) 1-methyl-1H-pyrazol-4-ylamine
hydrochloride (1.1 equiv), i-PrOH, MW 140 °C, 1 h, 99%; (e) 33 (1.2 equiv) HATU (1.2 equiv), DIPEA (2 equiv), DCM, rt, 18 h, 60%; (f) 2 N
TsOH in H O, MeCN, rt, overnight, 71%.
2
CDCl , CD OD, or DMSO-d at 400 MHz at 298 K unless otherwise
noted. The following abbreviations were utilized to describe peak
patterns when appropriate: br = broad, s = singlet, d = doublet, and m
EXPERIMENTAL SECTION
3
3
6
■
All activities involving laboratory animals were carried out in
accordance with federal, state, local, and institutional guidelines
governing the use of laboratory animals in research and were reviewed
and approved by Pfizer (or other) Institutional Animal Care and Use
Committee. Pfizer animal care facilities that supported this work are
fully accredited by AAALAC International.
=
multiplet. Optical rotations were measured on an Autopol Iv
Automatic Polarimeter at ambient temperature and reported as an
average of three runs unless otherwise noted. High-resolution mass
measurements were obtained on an Agilent 6200 or 6500 series ToF
or Thermo Scientific Q Exactive mass spectrometer. Compounds
used in biological studies had purities that were >95%, determined by
HPLC, UPLC, or LC/MS based on ultraviolet detection at 220 or
The human Janus kinase (JAK) activity was determined by using a
microfluidic assay to monitor phosphorylation of a synthetic peptide
by the recombinant human kinase domain of each of the four
members of the JAK family, JAK1, JAK2, JAK3, and TYK2. Reaction
mixtures contained 1 μM of a fluorescently labeled synthetic peptide
and ATP at either a level equal to the apparent Km for ATP or at 1
mM ATP. Each assay condition was optimized to obtain a conversion
rate of 20%−30% phosphorylated peptide product, and reactions were
terminated by the addition of stop buffer containing EDTA. Utilizing
mobility shift technology (Caliper Life Science), each assay reaction
was sampled to determine the level of phosphorylation. Experimental
2
54 nm. The synthesis of compounds 8−22, 24, and 25 is provided in
the Supporting Information.
Synthesis of 23 ((S)-2,2-Difluorocyclopropyl)((1R,5S)-3-(2-((1-
methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo-
[3.2.1]octan-8-yl)methanone. (S)-1-Phenylethyl (S)-2,2-Difluoro-
cyclopropane-1-carboxylate 32. To a solution of 2,2-difluorocyclo-
propanecarboxylic acid (4.02 g, 33 mmol) in DCM (140 mL) was
added DCC (8.16 g, 39 mmol) followed by DMAP (403 mg, 3.30
mmol), and the reaction was stirred for 10 min. (S)-1-Phenylethan-1-
ol (4.83 g, 39 mmol) was added, and the reaction was stirred at room
temperature for 72 h. The reaction was filtered, concentrated in
vacuo, and purified using silica gel column chromatography eluting
with 0−5% EtOAc to afford the title compounds as a mixture of
diastereomers. The diastereomers were separated using chiral
chromatography as described below: Kromasil Silica 10μm, 4.6 mm
× 250 mm; run time 14 min; 1.0 mL/min; eluting with 3% MTBE in
heptanes. The (S,S) enantiomer 32 was the first eluting peak. Peak 1:
25
details are as previously reported.
Human Whole Blood Assays. In this study, the cellular potency
and selectivity of compounds within the JAK/STAT signaling
pathways was evaluated in human whole blood by measuring
phosphorylation of signal transducer and activator of transcription
(
STAT) proteins, in response to specific cytokines challenges, using
intracellular staining and flow cytometry. Human whole blood was
collected from healthy donors via vein puncture into Vacutainer
collection tubes containing sodium heparin in accordance with Pfizer
protocols (Protocol No. GOHW RDP-01) approved by the Shulman
Institutional Review Board. Assays were optimized to run at the
R = 5.34 min; (S)-1-phenylethyl (S)-2,2-difluorocyclopropane-1-
t
1
carboxylate 32 H NMR (400 MHz, CDCl ): δ ppm 1.61 (d, 3H),
3
1.74−1.79 (m, 1H), 2.06−2.12 (m, 1H), 2.45−2.53 (m, 1H), 5.95−
approximate EC90 − EC of each cytokine evaluated. Full
6.00 (m, 1H), 7.33−7.42 (m, 5H). Peak 2: R = 6.22 min; (S)-1-
95
t
experimental details are available in the Supporting Information.
Synthesis. Chemistry Synthetic Methods and Compound
Characterization. Reactions were carried out under nitrogen at
ambient temperature with reagents and solvents typically obtained
from Sigma-Aldrich unless otherwise noted. Thin-layer chromatog-
raphy (TLC) was visualized with ultraviolet (UV) light on 60 Å F254
glass plates precoated with a 0.25 mm thickness of silica gel purchased
from EMD Chemicals Inc. Silica gel column chromatography refers to
normal phase chromatography using a biotage or ISCO system using
prepacked commercial columns under medium pressure. Low-
resolution mass spectroscopy was carried out using liquid
chromatography−mass spectrometry (LC/MS) on a Waters Acquity
phenylethyl (R)-2,2-difluorocyclopropane-1-carboxylate.
(S)-2,2-Difluorocyclopropane-1-carboxylic Acid 33. To a solution
of (S)-1-phenylethyl (S)-2,2-difluorocyclopropane-1-carboxylate 32
(3.67 g, 16.2 mmol) in MeOH (48 mL) was added 1 N NaOH (48
mL, 48 mmol), and the reaction was stirred at room temperature for
1.5 h. The reaction was concentrated in vacuo, acidified to pH = 5.8
using 12 N HCl (aq), and extracted into n-BuOH. The organic layer
was collected and concentrated in vacuo to afford the title compound
1
(1.5 g, 76%). H NMR (400 MHz, DMSO-d ): δ ppm 1.90 (m, 2H),
6
2.65 (m, 1H).
tert-Butyl (1R,5S)-3-(2-Chloropyrimidin-4-yl)-3,8-diazabicyclo-
[3.2.1]octane-8-carboxylate 35. To a solution of tert-butyl
(1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (11 g, 52 mmol,
Combi-Blocks, CAS: 149771-44-8) in MeOH (500 mL) were added
2,4-dichloropyrimidine (8.4 g, 57 mmol) and TEA (6.7 g, 66 mmol)
UPLC instrument using atmospheric pressure chemical ionization
APCI) or electrospray ionization (ESI). Proton ( H NMR) magnetic
resonance spectra were obtained on a Varian or Bruker instrument in
1
(
L
DOI: 10.1021/acs.jmedchem.8b00917
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
at 0 °C. The reaction was stirred at room temperature for 18 h before
concentrating in vacuo. The residue was purified by silica gel column
chromatography eluting with 20% EtOAc in petroleum ether to afford
volume of the immunized rats was measured via plethysmograph
(Buxco Inc.). The rats were monitored daily for signs of arthritis
including change in body weight and hind paw volume measurement.
When individual hind paw volume measurements indicated an
increase of 0.2 mL (or greater) in a single hind paw, animals were
randomly assigned to a treatment group. Daily treatment with
compound 23 was administered via oral gavage. Treatment groups
were 30, 10, and 5 mg/kg of 23 p-tosic acid salt or vehicle (2% Tween
1
the title compound as a white solid (12 g, 71%). H NMR (400 MHz,
CDCl ): δ ppm 1.48 (s, 9H), 1.63−1.74 (m, 2H), 1.97 (br s, 2H),
3
3
8
.18 (br s, 2H), 3.38−3.74 (m, 1H), 4.36 (br s, 3H), 6.35 (d, 1H),
+
.06 (d, 1H). MS m/z 325 [M + H] .
4
-((1R,5S)-3,8-Diazabicyclo[3.2.1]octan-3-yl)-N-(1-methyl-1H-
pyrazol-4-yl)pyrimidin-2-amine Hydrochloride 36). A solution of
tert-butyl (1R,5S)-3-(2-chloropyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]-
octane-8-carboxylate 35 (2.25 g, 6.93 mmol) and 1-methyl-1H-
pyrazol-4-ylamine hydrochloride (1.02 g, 7.62 mmol) in i-PrOH (30
mL) was heated to 140 °C under microwave irradiation for 1 h. The
reaction was concentrated in vacuo to afford the title compound as
80/0.5% methylcellulose/deionized H O). Dosing began once
2
individuals were enrolled into respective groups. Treatment continued
for 7 days. Animals were euthanized after 7 days of dosing. At the
conclusion of the study, whole blood was taken at 15 min postdose
(peak concentration in plasma) for analysis of STAT phosphorylation,
and plasma was taken at peak (0.25 h) and trough (24 h) time points
for exposure PK.
+
the hydrochloride salt (2.2 g, 99%). MS m/z 286 [M + H] .
(
(S)-2,2-Difluorocyclopropyl)((1R,5S)-3-(2-((1-methyl-1H-pyrazol-
X-ray Crystallography. The cloning and expression of TYK2 has
31
4
-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-
been described previously. TYK2 cell pellets were resuspended in
binding buffer (50 mM Tris HCl, 1 mM TCEP, 60 mM imidazole, 0.4
M NaCl, 10% glycerol, pH 8.2) supplemented with Roche Complete
EDTA-free protease inhibitor tablets and Benzonase (EMD
Millipore), lysed with a Microfluidizer (Microfluidics Corp.), and
clarified by centrifuging at 30 000 × G. The supernatant was batch
bound to Nickel Sepharose HP resin (GE Healthcare Life Sciences)
overnight at 4 °C before being packed into a column. The resin was
washed and eluted with binding buffer containing 250 mM imidazole.
TYK2 was buffer exchanged into SEC buffer (25 mM Tris HCl, 0.25
mM TCEP, 0.25 M NaCl, 10% glycerol, pH 8.2) using a HiPrep 26/
10 desalting column (GE Healthcare Life Sciences) and loaded
unconcentrated onto a HiLoad 16/60 Superdex200 SEC column (GE
Healthcare Life Sciences) equilibrated in SEC buffer. Monomeric
TYK2 was pooled, and 10 μM tofacitinib 1 was added before
concentration. TYK2 was concentrated to 5−8 mg/mL and frozen in
aliquots at −80 °C. TYK2 was incubated with 1 mM CP-690550 and
crystallized by sitting drop vapor-diffusion at 20 °C against well
solution containing 0.1 M bis-tris pH 5.5, 0.25 M NaCl, 10 mM
TCEP, and 27−33% PEG-3350. The crystallization drops consisted of
0.3 μL TYK2:1 complex, 0.1 μL well solution, and 0.1 μL of TYK2
seed stock. Crystals appeared in 2−3 weeks and grew to full size in 2
additional weeks. TYK2 crystals were soaked for 4 days in 0.1 M bis-
tris pH 5.5, 0.25 M NaCl, 10 mM TCEP, 38% PEG-3350, and 2 mM
of either 8 or 23 and flash cooled in liquid nitrogen.
methanone 23. To a solution of (S)-2,2-difluorocyclopropane-1-
carboxylic acid 33 (318 mg, 2.61 mmol) in DCM (20 mL) were
added 4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-N-(1-methyl-
1
H-pyrazol-4-yl)pyrimidin-2-amine hydrochloride 36 (700 mg, 2.17
mmol), HATU (1.02 g, 2.61 mmol), and DIPEA (0.76 mL, 4.34
mmol), and the reaction was stirred at room temperature for 18 h.
The reaction was diluted with DCM and saturated aqueous
ammonium chloride solution. The organic layer was separated,
washed with further ammonium chloride solution, and concentrated
in vacuo. The residue was purified using silica gel column
chromatography eluting with 0−12% MeOH and 1% NH OH in
DCM. The residue was dissolved in DCM and further washed with
saturated aqueous ammonium chloride solution three times. The
4
organic layer was collected, concentrated in vacuo, and dried to afford
the title compound (500 mg, 60%). H NMR (400 MHz, DMSO-d ):
1
6
δ ppm 1.58−2.06 (m, 6H), 2.82−2.96 (m, 1 H), 2.98−3.26 (m, 2H),
3
2
8
(
+
.09−3.27 (m, 1H), 3.80 (s, 3H), 4.14 (br s, 2H), 4.55−4.74 (m,
H), 6.07−6.19 (m, 1H), 7.44 (s, 1H), 7.74 (br s, 1H), 7.93 (d, 1H),
+
.90 (br s, 1H); LCMS m/z 390 [M + H] HPLC 98.09% at 254 nM
XBridge RP18 50 mm × 2.1 mm × 5 μm) R = 3.55 min; HRMS [M
H] for C H F N O, calcd, 390.1848; found, 390.1850; [α]
0.1 (c 1.27, EtOH). Analytical chiral SFC EE = 98.25%, R = 7.695
min, 220 nM Chiralcel OJ-H 250 mm × 4.6 mm × 5 μm, 5−40%
methanol (0.05% DEA) in CO flow rate 2.35 mL/min.
t
2
0
18 22 2 7 D
5
t
2
32
(
(S)-2,2-Difluorocyclopropyl)((1R,5S)-3-(2-((1-methyl-1H-pyrazol-
JAK1 was cloned, expressed, and purified as described previously.
4
-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-
JAK1 was further purified by buffer exchanging into 25 mM Tris, 1
methanone 4-methylbenzenesulfonate (00110596-3088, 23-Ts).
mM TCEP, 10 mM MgCl , 1 mM ATP, pH 8.0 using a HiPrep 26/10
2
To a flask containing 23 (19.25 g, 49.43 mmol) was added acetonitrile
40 mL) followed by the addition of 2 M p-toluenesulfonic acid in
desalting column. Desalted JAK1 was applied to a HiTrap Q
Sepharose HP column (GE Healthcare Life Sciences) and eluted with
a linear gradient to 250 mM NaCl. Mass spectrometry was used to
pool Jak1 by phosphorylation state. JAK1 was concentrated to 10 mg/
mL and frozen in aliquots at −80 °C. Doubly phosphorylated JAK1
was diluted to 6 mg/mL and incubated with freshly added 1 mM ATP
and 5 mM TCEP before being crystallized by hanging drop vapor
diffusion at 10 °C. Well solutions consisted of a grid of 0.1 M Tris pH
8.5, 6−9% MPD, and 24−29% PEG-1500 and streak-seeded at setup.
JAK1 crystals were soaked in 0.1 M Tris pH 8.5, 4% MPD, 30% PEG-
1500, 20 mM EDTA, and 5 mM 23 for 3 h and frozen in liquid
nitrogen.
(
H O (28.4 mL, 56.8 mmol). The mixture was stirred at rt overnight.
2
The resulting white solid was collected by filtration. The filter cake
was washed with acetonitrile and then dried under vacuum over 2
days to provide the title compound (21.0 g, 76%). QNMR
determined the parent molecule at 70.30% and p-toluenesulfonic
2
0
1
acid 29.70%. Mp = 275 °C; [α] = 29.8° (c = 0.885 MeOH); H
NMR (400 MHz, DMSO-d6): δ ppm 1.57−1.66 (m, 1H), 1.69−1.76
(
3
(
(
m, 1H), 1.88−2.09 (m, 4H), 2.30 (s, 3H), 3.14−3.26 (m, 2H),
.26−45 (br m, 1 H), 3.86 (s, 3H), 3.92−4.19 (br m, 1H), 4.40−4.60
m, 1 H), 4.62−4.76 (m, 2H), 6.28 (s, 2H), 6.54−6.57 (m, 1 H), 7.15
d J = 7.8 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.55 (s, 1 H), 7.85 (s, 2
Data collection for all complexes was conducted at 100 K at the
Industrial Macromolecular Crystallography Association Collaborative
Access Team (IMCA-CAT) (Advanced Photon Source, Argonne
National Laboratory, Argonne, Il, USA), beamline 17ID outfitted with
a Pilatus 6 M detector (Dectris Ltd., Switzerland). Data processing
H). Analytical Chiral SFC EE = 100%, R = 7.416 min (TsOH R =
.413 min), 210 nM Lux Cellulose-1 250 mm × 4.6 mm × 5 μm, 5−
0% methanol (0.2% NH ) in CO flow rate 3.0 mL/min.
t
t
9
6
+
4
2
Rat Adjuvant Induced Arthritis Model. The effect of JAK1
33
34
inhibition by 23 was evaluated in vivo using a therapeutic dosing
paradigm in a rat adjuvant-induced arthritis. The efficacy of this
molecule was evaluated in two separate studies using successively
lower doses. Arthritis was induced by immunization of female Lewis
rats (8−10 weeks old; Charles River Laboratories, Portage, MI) via
intradermal injection at the base of the tail with complete Freund
adjuvant with three 50 μL injections (15 mg/mL Mycobacterium
tuberculosis (Difco) in incomplete Freund adjuvant (Sigma-Aldrich)).
Seven days after the initial immunization, the baseline hind paw
was performed using AUTOPROC and XDS. All other data
35
manipulations utilized the CCP4 suite of programs. A common set
(5%) of test reflections was used throughout refinement for cross-
validation. Structures were determined by rigid body and all-atom
36
positional refinement using REFMAC and an isomorphous model
from which all waters and bound ligands had been removed. Binding
of the compound of interest was confirmed visually, and the
37
compound was fit automatically using the program RHOFIT.
Iterative rounds of automatic refinement using the program
M
DOI: 10.1021/acs.jmedchem.8b00917
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3
8
AUTOBUSTER were interspersed with manual model building
scription; IL, Interleukin; IFN, Interferon; mAb, monoclonal
antibody; RA, rheumatoid arthritis; SLE, systemic lupus
erythematosus; AD, atopic dermatitis; PSA, psoriatic arthritis;
UC, ulcerative cholitis; CD, Crohn’s disease; PSO, psoriasis;
gp130, glycoprotein 130; EPO, erythropoietin; TPO, throm-
bopoietin; Hgb, hemoglobin; VZV, varicella zoster virus;
mCIA, mouse collagen induced arthritis; Cmax, maximum
39
using COOT. Crystallographic data and refinement statistics are
reported in Table S2.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00917.
■
*
S
concentration; Cmin, minimum concentration; C , average
av
concentration; HgB, hemoglobin; IC , % average daily
xx
inhibition; IC *, calculated % inhibition based upon human
(
(
(
1) Human whole blood assay experimental conditions;
2) Physicochemical properties for compounds 14−25;
3) Exposure and activity data for compound 23 from
xx
whole blood potency: IC * = 100*((IC IFNα)/(IC IFNα
xx
80
80
+
IC50 EPO)); lipe, lipophilic efficiency; HWB, human whole
blood; P-loop, fingerprint ATP binding motif Gly-X-X-Gly-X-
Gly-Lys-Thr/Ser; RRCK, Russ-Ralph canine kidney cell-line
permeability assay; PAPP, apparent permeability; APO,
unbound protein; W, water; HLM, human liver microsome;
Clint, intrinsic clearance; CD, cluster of differentiation;
pSTAT, phospho STAT; AURA, aurora A kinase; TPX2,
microtubule nucleation factor; TNK1, tyrosine kinase, non-
receptor 1; QIK, salt inducible kinase 2; ACK, activated
CDC42 kinase; PDGFRα, platelet-derived growth factor
receptor alpha; SIK, serine threonine protein kinase;
MAP2K2, dual specificity mitogen activated protein kinase
kinase 2; MNK2, MAP kinase-interacting serine/threonine-
protein kinase 2; FMS, feline McDonough sarcoma kinase;
NUAK1, NUAK family SNF1-like kinase 1; MARK, micro-
tubule affinity-regulating kinase; KDR, kinase insert domain
receptor; vascular endothelial growth factor receptor kinase;
FASSIF, fasted state simulated intestinal fluid; FESSIF, fed
state simulated intestinal fluid; Clint,u, intrinsic unbound
clearance; Crbc/Cp, red blood cell concentration/plasma
concentration; AIA, adjuvant induced arthritis; Emax,
sigmoidal Emax; HATU, hexafluorophosphate azabenzotria-
zole tetramethyl uronium.
the rat AIA model; (4) Syntheses for compounds 8−22,
24, and 25; (5) Thermodynamic solubility determi-
nation for compound 23; (6) Crystallographic data and
refinement statistics for compounds 8 and 23 in TYK2
and compound 23 in JAK1 (PDF)
Molecular formula strings (CSV)
Accession Codes
Atomic coordinates for the X-ray structure of compound 8 in
TYK2 (PDB code 6DBK) and compound 23 in TYK2 (PDB
code 6DBM) and JAK1 (PDB code 6DBN) are available from
the RCSB Protein Data Bank (www.rcsb.org). Authors will
release the atomic coordinates upon article publication.
AUTHOR INFORMATION
Corresponding Authors
A.F.: phone, 617-674-7237; e-mail, Andrew.Fensome@Pfizer.
■
*
com.
*
B.G.: phone, 860-686-3019; e-mail, Brian.Gerstenberger@
Pfizer.com.
ORCID
Andrew Fensome: 0000-0001-8916-1895
Notes
REFERENCES
■
The authors declare the following competing financial
interest(s): The authors were all employees of Pfizer while
this work was completed and declare no competing interests.
All of the compounds tested in biological assays were
electronically filtered for structural attributes consistent with
classification as pan-assay interference compounds (PAINS)
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0
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(
0
(
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(
ACKNOWLEDGMENTS
J.; Covington, M. B.; Thomas, B.; Collier, P.; Favata, M. F.; Wen, X.;
Shi, J.; McGee, R.; Haley, P. J.; Shepard, S.; Rodgers, J. D.;
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■
This work was funded by Pfizer, Inc. The authors thank Dr.
David Hepworth for helpful comments during the writing of
this manuscript. This research used resources at the Industrial
Macromolecular Crystallography Association Collaborative
Access Team (IMCA-CAT) beamline 17-ID, supported by
the companies of the Industrial Macromolecular Crystallog-
raphy Association through a contract with Hauptman-Wood-
ward Medical Research Institute. This research used resources
of the Advanced Photon Source, a U.S. Department of Energy
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TYK2, Tyrosine kinase 2; JAK1, Janus kinase 1; JAK, Janus
kinase; STAT, Signal Transducer and Activator of Tran-
■
N
DOI: 10.1021/acs.jmedchem.8b00917
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Journal of Medicinal Chemistry
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