Discovery of (2 r)- n-[3-[2-[(3-methoxy-1-methyl-pyrazol-4-yl)amino]pyrimidin-4-yl]-1 h-indol-7-yl]-2-(4-methylpiperazin-1-yl)propenamide (azd4205) as a potent and selective janus kinase 1 inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.9b01392

JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.

Full text of DOI:10.1021/acs.jmedchem.9b01392

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Article
Discovery of (2R)-N-[3-[2-[(3-methoxy-1-methyl-pyrazol-4-
yl)amino]pyrimidin-4-yl]-1H-indol-7-yl]-2-(4-methylpiperazin-1-yl)propenamide
(AZD4205) as a potent and selective Janus Kinase 1 (JAK1) inhibitor
Qibin Su, Erica Banks, Geraldine Bebernitz, Kirsten Bell, Cassandra F. Borenstein, Huawei
Chen, Claudio Chuaqui, Nanhua Deng, Andrew D. Ferguson, Sameer P. Kawatkar, Neil P.
Grimster, Linette Ruston, Paul Lyne, Jon A. Read, Xian You Peng, Xiaohui Pei, Stephen
Fawell, Zhanlei Tang, Scott Throner, Melissa Marie Vasbinder, Haoyu Wang, Jon Winter-
Holt, Richard Woessner, Allan Wu, Wenzhan Yang, Michael Zinda, and Jason Grant Kettle
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01392 • Publication Date (Web): 16 Apr 2020
Downloaded from pubs.acs.org on April 17, 2020
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of their duties.

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Discovery
of
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yl)amino]pyrimidin-4-yl]-1H-indol-7-yl]-2-(4-methylpiperazin-1-
yl)propenamide (AZD4205) as a potent and selective Janus Kinase 1
(JAK1) inhibitor
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Qibin Su*,1 Erica Banks,1 Geraldine Bebernitz,1 Kirsten Bell,1 Cassandra F. Borenstein,1 Huawei
Chen,1 Claudio E. Chuaqui,1 Nanhua Deng,1 Andrew D. Ferguson,2 Sameer Kawatkar,1 Neil P.
Grimster,1 Linette Ruston,1 Paul D. Lyne,1 Jon A. Read,3 Xianyou Peng,4 Xiaohui Pei,4 Stephen
Fawell,1 Zhanlei Tang,4 Scott Throner,1 Melissa M. Vasbinder,1 Haoyu Wang, 4 Jon Winter-
Holt,5 Richard Woessner,1 Allan Wu,2 Wenzhan Yang,6 Michael Zinda,1 Jason G. Kettle5
1Oncology R&D, AstraZeneca R&D, Waltham, MA 02451 USA. 2Discovery Sciences, R&D,
AstraZeneca R&D, Waltham, 02451 MA USA. 3Discovery Sciences, R&D, AstraZeneca R&D,
4
Cambridge, UK.
Pharmaron Beijing Co., Ltd., 6 Taihe Road, BDA, Beijing, 100176.
5Oncology R&D, AstraZeneca R&D, Cambridge, UK. 6Early Product Development,
Pharmaceutical Sciences, R&D, 02451 Boston, USA.
Abstract: JAK1 together with JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family.
They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT
pathways are associated with a wide variety of diseases. Particularly, pSTAT3 is
observed in response to the treatment with inhibitors of oncogenic signaling pathways
such as EGFR, MAPK and AKT, and is associated with resistance or poorer response
to agents targeting these pathways. Amongst the JAK family kinases, JAK1 has been
shown to be the primary driver of STAT3 phosphorylation and signaling, therefore selective
JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein,
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an account of the medicinal chemistry optimization from a promiscuous kinase screening hit 3
to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported.
Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced
antitumor activity in combination with approved EGFR inhibitor, osimertinib, in a preclinical
non-small cell lung cancer (NSCLC) xenograft NCI-H1975 model.
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Introduction
JAK1 together with JAK2, JAK3, and TYK2 belong to the JAK (Janus-associated kinase)
family of cytoplasmic tyrosine kinases that play important roles in cytokine and growth factor
mediated signal transduction.1 Consequently, the JAK family kinases are strongly associated
with both cancer2 and inflammatory3 diseases. Currently, there are several approved JAK
inhibitors to treat human diseases: tofacitinib (Xeljanz®, a pan-JAK inhibitor from Pfizer)4 for
rheumatoid arthritis, ruxolitinib (1, Jakafi®, a selective JAK1/2 inhibitor from Incyte)5 for
intermediate- or high-risk myeloproliferative neoplasms and baricitinib (Olumiant®, a JAK1/2
inhibitor)6-7 for the treatment of rheumatoid arthritis, together with peficitinib (Smyraf®, a pan-
JAK inhibitor)8-9 approved in Japan for treatment of RA. Both compounds have demonstrated
clinical benefit in a range of disease areas, but have additionally been associated with a number
of side effects. Specifically, tofacitinib’s adverse events have included non-serious infections,
reduction of leukocyte cell count number, and rare but severe incidences of perforation of the
stomach or intestine. With ruxolitinib treatment, thrombocytopenia and anemia were observed.10
These toxicities are believed to be associated with inhibition of JAK2. Thus, the pharmaceutical
industry has been motivated to evaluate second generation inhibitors, which possess increased
JAK isoform selectivity in the hope that treatment with such agents may maintain similar
efficacy to established therapies, but with fewer undesirable side effects. To this end, JAK1
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selective inhibitors (e.g. ABT49411, PF-0496584212, and filgotinib13) are currently being studied
in human clinical trials for the treatment of autoimmune or inflammatory diseases.14-15
We have a long-standing interest in studying JAK inhibition in the area of oncology.16-
22 There is growing evidence to suggest that disregulation of constitutively activated JAK/STAT
signaling is associated with a wide variety of malignancies.23 In particular, increased activation
of STAT3 through phosphorylation at Y705 (pSTAT3) is reported in up to 70% of human
tumors including ovarian, breast, liver, prostate, colorectal, head and neck, esophageal,
pancreatic, bladder, and non-small-cell lung cancers.24-31 In addition, such elevated pSTAT3
has also been observed in response to chemotherapy treatment, and is associated with resistance
or poor response to the treatment with agents targeting oncogenic signaling pathways such as
EGFR, MAPK and AKT.32-34 Amongst the JAK family kinases, JAK1 has been shown to be the
primary driver of STAT3 phosphorylation and signaling.35 JAK2, however, is essential for
signal transduction downstream of erythropoietin, thrombopoietin and other related receptors
that control erythrocyte and megakaryocyte expansion.36 Taken together, it is believed that a
JAK1 selective inhibitor may enable higher target coverage of the JAK1-STAT3 signaling by
sparing toxicities such as thrombocytopenia and anemia, associated with JAK2 inhibition,
thereby, maximizing the clinical benefit of such agents in oncology10 and other therapeutic
areas37. Herein, an account of the medicinal chemistry optimization from a promiscuous kinase
screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor,
is reported. Detailed biological and pharmacokinetic evaluation of this compound is also
included.
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Results and Discussion
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During the course of our discovery program, a patent application describing JAK1
selective inhibitors, such as compound 2, were reported.38 We synthesized and tested this
compound in house. To our surprise, although derived from the same chemical class as 1, a dual
JAK1/2 inhibitor (JAK1 IC50 = 0.02 M, no selectivity of JAK1 over JAK2), compound 2
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displayed a high degree of JAK1 selectivity (JAK1 IC50 = 0.02 M, 120-fold selectivity of JAK1
over JAK2). In order to rationalize the observed selectivity, an X-ray crystal structure of 2 bound
to the kinase domain of JAK1 was solved (Figure 1, Panel A).
Table 1. Biochemical potencies of 1, JAK1 selective inhibitor 2 and in-house screening hit 3
Cl
CN
N
CN
CN
H
N
N
N
N N
N
N
N
N
N
N
N
H
N
H
N
H
N
N
2
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Compound
JAK1
IC50 (M) a
JAK2
IC50 (M) a
Selectivity over
JAK2 (fold) b
1
2
3
0.02
0.02
1
120
5
0.02
2.4
0.07
0.38
aBiochemical potency was measured at 5 mM ATP concentration. bSelectivity expressed as the ratio of JAK2 IC50 over JAK1 IC50. All
experimental values represent the geometric mean of at least two independent experiments and have SEM within 0.2 log units. For experimental
details, see the supporting information of reference.22
Analysis of the X-ray structure revealed a bidentate interaction between
pyrrolopyrimidine moiety of 2 with hinge residues Glu957 and Leu959. The pyrrolopyrimidine
and the pyrazole ring form hydrophobic interactions with Leu881, Val889 and Leu1010. The
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nitrile group occupies a small pocket, formed by the isobutyl sidechain of Leu101 and Gly1020
and may stabilize the conformation of the pyrrolidine ring while possibly contributing to
desolvation of this hydrophobic region. However, most interestingly, a well-ordered p-loop
region with clear electron density is seen in the crystal structure. In general, the p-loop in kinases
is very flexible. And it is common to observe poor or no electron density visible for this region
even in high-resolution protein kinase structures.39 The presence of such a well-ordered p-loop
region suggests a stabilizing interaction between the terminal chloro-cyano-phenyl group of 2
and the hydrophobic pocket defined by the sidechains of protein residues Gly884, His885,
Phe886 and Gly1028 of JAK1. We hypothesized that this stabilization is contributing to the
JAK1 selectivity of 2, because, in this region, there is a productive π- π stacking interaction
between the chloro-cyano-phenyl group of 2 and the imidazole side chain of His885 in JAK1,
which is absent in JAK2, where the corresponding residue is an asparagine.
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B
A
Phe886
His885
Val889
Phe886
His885
Val889
Gly884
Gly884
Leu881
Gly1023
Asp1021
Leu881
Gly957
Gly1023
Asp1021
Gly1020
Gly1020
Gly957
Leu1010
Leu1010
Figure 1. (A) X-ray crystal structure of 2 (yellow carbon atoms) bound to JAK1 (PDB code:
6SM8) reveals a p-loop JAK1 selectivity pocket. Selected residues are shown as sticks.
Hydrogen bonds are shown as dotted lines. (B) Overlay of 3 (green carbon atoms) with x-ray
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crystal structure of JAK1 in complex with compound 2 suggested substitutions of 7-position of
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indole ring can reach the p-loop selectivity pocket.
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Our program started with a high through-put screen of the AstraZeneca compound
collection against JAK1 to generate three-point IC50s values. The resulting hits were
followed-up in a five-point IC50 dose response versus JAK1 and JAK2, at the corresponding
Km ATP concentrations (55 and 15 μM, respectively), in order to identify potent and
selective starting points. In the lead optimization phase, we increased the ATP concentration
to a more physiologically relevant level in our biochemical assays (5 mM) to assess the
inhibitors’ JAK1 potency and their selectivity towards JAK2. Herein, all the biochemical
potencies are reported under the 5 mM ATP conditions.
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From the screening campaign, compound 3 was identified as a potent JAK1 inhibitor
(Table 1, IC50 = 0.07 M) with moderate selectivity over JAK2 (JAK2 IC50 = 0.38 M, 5-fold
selectivity). Docking of 3 into the JAK1 protein showed that the indole NH binds the carboxylate
moiety of Asp1021, while the 2-amino pyrimidine forms interactions with the hinge residue
Leu959. Intriguingly, when superimposing40 the docked pose of 3 onto the X-ray structure of 2,
we postulated that the 7-position of the indole may direct substituents toward the p-loop, thereby
accessing the JAK1 selectivity pocket (Figure 1, Panel B). Consequently, we designed 7-
substituted indole analogs containing an appropriate linker to direct an aryl group to the
selectivity pocket and achieve the desired π-π interaction with His885, in the hopes of increasing
the isoform selectivity of newly generated compounds. To test this hypothesis, with a
consideration of ease of synthesis, 4, bearing an amido-linker, was synthesized and evaluated in
the JAK1 and JAK2 biochemical assays. As shown in Table 2, 4 displayed an increased JAK1
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potency (IC50 = 0.004 M), and more importantly with improved selectivity (50-fold) toward
JAK2, compared to 3 (5-fold).
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Table 2. SAR of JAK1 selective 4-pyrimidine indoles with varied linkers and solvent tails
Linker
N
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N
O
H
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NH
H
NH
H
N
O
O
H
N
H
H
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O
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H
Solvent tail
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N
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N
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5
Compound
JAK1
IC50 (M) a
JAK2
IC50 (M) a
Selectivity over
JAK2 (fold)
Solubility
(M)b
4
5
6
7
8
0.004
0.20
50
--
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2
0.018
2.2
122
58
0.043
2.5
0.004
0.56
140
115
0.1
0.1
0.26
> 30
a
Biochemical potency was measured at 5 mM ATP concentration. Selectivity expressed as the ratio of JAK2 IC50 over JAK1 IC50. All
experimental values represent the geometric mean of at least two independent experiments and have SEM within 0.2 log units. For experimental
details, see the supporting information.22 bSolubility was measured at pH = 7.4.
With this encouraging result, we then turned our attention to explore the SAR of solvent
tail groups (denoted as in 4, Table 2). It has been reported that ortho-substitution on similar
solvent tails may reduce JAK2 activity, thus improving selectivity between JAK1 and JAK2.22
Capitalizing on this observation, we designed and synthesized 5 with a 3-methoxy-1-methyl
amino-pyrazole solvent tail. Indeed, 5 retained a high level of JAK1 potency (IC50 = 0.018 M)
with increased JAK2 selectivity (122-fold). The cyclopropyl amide has also been shown to be a
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viable hinge binding motif for JAK113 and compound 6 exhibited favorable JAK1 potency (IC50
= 0.043 M) with an encouraging degree of selectivity over JAK2 (60-fold).
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9
We then explored the linkers while keeping the 2-methoxy-1-methyl-aminopyrazole
solvent tail group constant. Compound 7, with a benzylic amine linker, exhibited favorable JAK1
potency (IC50 = 0.004 M), but with low solubility (< 0.1 µM). With the reversed amide linker,
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compound 8 displayed diminished JAK1 potency (IC50 = 0.26 M). This suggested the amido-
linker in 5 is critical for achieving a high level of JAK1 potency. We therefore elected to use this
linker for further SAR exploration.
Having leads with promising selectivity between JAK1 and JAK2, we were keen to
further evaluate their broader kinase selectivity. To this end, 5 was tested as representative of
the chemical series in a panel of 293 kinases at a single point concentration (1 M). To our
disappointment, 5 promiscuously inhibited a broad set of kinases in this panel (80 out of 289
members tested with over 50% inhibitory effect at this concentration. For details, see SI Table
1).
To gain additional structural insights, an X-ray structure of 5-bound to the JAK1 kinase
domain was obtained. Shown in Figure 2 (6SMB), 5 displayed the expected binding mode: the
amino pyrimidine moiety forms a two-point binding interaction with the kinase hinge,
consistent with our docked pose for compound 3. Notably, a distinct bidentate hydrogen bond
interaction of the indole NH and amide moiety with the carboxylate sidechain of Asp1021 was
observed. Gratifyingly, the interaction between the pyridyl group in 5 and imidazole sidechain
of His885 was also observed and stabilized the p-loop region, with clear electron density in the
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X-ray structure, thus presumably rendering isoform selectivity in a similar manner to that
described for 2. In addition to the p-loop stabilization, the methoxy functionality on the pyrazole
solvent tail group, which also potentially contributes to this isoform selectivity, points towards
a small hydrophobic pocket defined by the phenyl ring of Phe958 (side chain) and backbone of
Pro960, Ser961 and Gly962 in JAK1. The corresponding pocket in JAK2 is formed by the side
chain of Tyr931 and the amide backbones of Pro933, Tyr 834 and Gly934. We reasoned that
the pocket with a tyrosine residue in JAK2 was more sterically demanding than the one defined
by phenylalanine in JAK1. The methoxy-pyrazole solvent tail of 5 is therefore less well
tolerated when bound to JAK2 protein, thus leading to diminished activity.
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Figure 2. X-ray structure of 5-JAK1 complex (6SMB). The aminopyrimidine moiety maintains
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9
the key hydrogen bonds to the hinge. The amido-indole forms a bidentate hydrogen bond
interaction with the side chain carboxylate of Asp1021. The methyl pyridine group forms
several hydrophobic interactions in addition to a cation-π interaction with Lys908. Hydrogen
bonds are shown as dotted lines. Compound 5 (yellow carbon atoms) overlays with x-ray crystal
structure of JAK1 in complex with compound 2 (green carbon atoms).
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With this structural information in hand, we set out to improve the broader kinome
selectivity of the series, while also attempting to identify leads with better physical properties
and improved metabolic stability. In protein kinases, the p-loop regions are flexible with highly
diversified structures.39 We reasoned that a selective kinase inhibitor might be obtained by
further exploiting the interaction in this area of the protein. We synthesized over 300 amide
analogs of 6, using a diverse reagent set, comprising aromatic and aliphatic carboxylic acids,
while keeping the cyclo-propyl amide solvent tail group constant.41 All compounds
synthesized were tested in our JAK1 and JAK2 biochemical assays, and in parallel their
solubility and human microsome clearance (CLint) was measured. Figure 3 shows the JAK1
biochemical potency versus human microsomal CLint, with points colored by the ratio of the
IC50s of JAK1 over JAK2, and sized by measured solubility at pH=7.4. While most of
compounds in the library displayed low solubility or low JAK2 selectivity, we found that
compound 9 (Table 3) had a favorable balance of JAK1 potency and selectivity (JAK1 IC50 =
180 nM and more than 166-fold selectivity), with good aqueous solubility (>1000 µM) and low
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human microsome CLint (4.4 μL/min/mg). Given the low kinome selectivity of lead compound
5, we next tested 9 in the kinase panel described previously. Remarkably, 9 displayed an
improved kinase selectivity (with only 6 out of 293 kinases tested over 50% inhibition at single
dose concentration of 1 μM, see complete kinome screen data in supporting information SI Table
2).
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Figure 3. Plot of JAK1 biochemical potency vs. human microsome Clint. Compound 9 was
identified with a favorable balance of potency and properties. Dots were colored by ratio of
IC50’s of JAK1 over JAK2 and sized by measured solubility at pH=7.4.
As compound 9 bears a significant structural difference from 5, an X-ray structure of 9-
bound JAK1 protein was obtained to elucidate its binding mode (Figure 3). The interaction at
the hinge and the bidentate interaction of the indole NH and amide NH towards Asp1021 of
JAK1 are all maintained. Interestingly, an additional salt-bridge interaction between the chiral
piperazine moiety and Asp1003 is also evident in the crystal structure. While the X-ray structure
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also shows a stabilized p-loop with clear electron density, the ligand does not form a direct π-π
5
6
interactions with the loop residues due to lack of an aromatic ring in this region. As Asp1003 is
7
8
9
actually conserved between JAK2 and JAK1, we believe it is the combination of the
10
11
12
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14
15
16
17
18
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21
22
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additional polar interaction, p-loop stabilization and the selective nature of the solvent tail group
that contributes to the favorable JAK1/2 selectivity of 9.
Figure 4. X-ray structure of 9-JAK1 complex (PDB code: 6GGH). In addition to the retained
interactions with the hinge and Aps1021, compound 9 also forms a salt-bridge interaction
between the piperazine moiety and Asp1003. Hydrogen bonds are shown as dotted lines. .
Compound 9 (orange carbon atoms) overlays with x-ray crystal structure of JAK1 in complex
with compound 5 (yellow carbon atoms).
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9
Encouraged by the profile of 9, we set out to explore the SAR around the chiral basic
amides. As shown in Table 3, the R configuration in 9 is preferred (JAK1 IC50 = 0.18 M), over
its enantiomer 10 (JAK1 IC50 = 1.1 M). Achiral compounds, such as 11 and 12, exhibited
weaker JAK1 activity (IC50 = 0.46 M and 1.7 M, respectively). As suggested in the crystal
structure, the distal basic center of the chiral amide in 9 is essential for JAK1 potency. Removal
of the basic centers significantly lowered JAK1 potency (13, IC50 = 2.4 M and 14, IC50 = 2.2
M). Gratifyingly, even though the analogs in Table 3 have various level of JAK1 potency,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
they consistently displayed high aqueous solubility, indicating the chemical series has
favorable physical properties.
Table 3. Structure, JAK enzymatic activity and solubility of compounds 9-14.
O
N
N
N
N
O
O
O
N
N
O
O
N
O
N
N
N
HN
HN
HN
HN
HN
HN
H
N
H
N
H
N
H
N
H
N
H
N
N
O
N
O
N
O
N
O
N
O
N
O
N
N
N
N
H
N
N
H
N
N
9H
N
N
H
N
N
H
H
14
10
12
13
11
Compound
JAK1
JAK2
Selectivity over
JAK2 (fold)
Solubility
IC50 (M)a
0.18
1.1
IC50 (M)a
(M)b
9
>30
>166
>27
> 65
>18
>12
> 13
>930
>1000
230
10
11
12
13
14
> 30
0.46
1.7
> 30
> 30
>1000
--
2.4
>30
2.2
>30
--
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aBiochemical potency was measured at 5 mM ATP concentration. Selectivity expressed as the ratio of JAK2 IC50 over JAK1 IC50. All
experimental values represent the geometric mean of at least two independent experiments and have SEM within 0.2 log units. For experimental
details, see the supporting information.22 bSolubility measured at pH 7.4.
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9
Establishing that the initial R-methyl piperazine was the preferred amine, we set out to
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21
22
23
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explore SAR of the solvent tail groups with this newly identified piperazine amide. Having
learned that a small ortho-substitution played a critical role in achieving a high level of JAK2
selectivity, we scanned additional ortho-substituted aromatic rings. A series of compounds with
a high level of JAK1 selectivity were obtained by deploying this tactic (Table 4). Compound
15 with a 2-methylpyridine solvent tail displayed a high degree of JAK1/2 selectivity, albeit at
the expense of JAK1 potency (15, JAK1 IC50 = 0.13 M). In contrast, installation of a less-
sterically demanding ortho-substituent, such as F, restored JAK1 potency while maintaining a
high JAK1 selectivity (16, JAK1 IC50 = 0.027 M, >1000-fold selectivity versus JAK2). As
previously described, the ortho-methoxy pyrazole solvent tail was well-tolerated (17, JAK1
IC50 = 0.010 M, 540-fold selectivity versus JAK2). Having high JAK1 biochemical potency,
these compounds were subsequently evaluated in a cellular assay, measuring the inhibition of
phosphorylation of STAT3 (pSTAT3), a downstream substrate of JAK1, in an NCI-H1975 cell
line. We found that 15 inhibited pSTAT3 with IC50 = 0.18 M. Compound 16 displayed a
promising JAK1 cellular potency (IC50 = 0.08 M), however, disappointingly, the compound
was found to inhibit the hERG ion channel (IC50 = 10 M). Compound 17 exhibited a potent
cellular activity (IC50 = 0.13 M) without any detectable hERG activity (IC50 >100 M).
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Table 4. Structure, JAK enzymatic and cellular activity and 15-17.
5
6
7
8
9
N
N
N
O
HN
O
O
HN
N
N
N
HN
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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H
N
H
H
N
N
N
N
N
N
N
N
N
N
N
H
N
N
H
N
N
H
O
F
15
17
16
Compound
JAK1
IC50 (M)a
JAK2
Selectivity over
JAK2 (folds)
pSTAT3
IC50 (M)b
hERG
IC50 (M)a
(M)
> 33
10
15
16
17
0.13
0.027
0.010
> 30
> 230
> 1111
540
0.18
0.08
0.13
> 30
5.4
> 100
aMeasured at 5 mM ATP concentration. Selectivity expressed as the ratio of JAK2 IC50 over JAK1 IC50. All values represent the geometric
mean of at least two independent experiments and have SEM within 0.2 log units. bRepresents cellular mode of action assay measuring
inhibition of pSTAT3 in NCI-H1975 cells. For experimental details, see the supporting information.22
We next examined the SAR of pyrimidine C5-subsituents. JAK1 possesses a methionine
gatekeeper residue, therefore hydrophobic substituents or hydrogen may be favorable at the C-
5 position. Shown in the Table 5, increasing hydrophobicity of the substituents led to higher
JAK1 potency (18, 19, and 20, IC50 = 7, 19, and 8 nM respectively), however, we also observed
the same trend with JAK2 potency. In addition, 18 and 20 exhibited improved cellular potencies
(IC50 = 0.06 M and 0.094 M, respectively), but also displayed moderate hERG ion channel
activities (IC50 = 22 M and 15 M, respectively). Overall, 21, with a C5-H substituent
exhibited the desired balance of favorable JAK1 potency, selectivity versus JAK2, and no
detectable hERG activity. We therefore selected it for further profiling.
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Table 5. Structure, JAK enzymatic and cellular activity of 18-21
5
6
7
8
9
N
N
N
N
O
O
O
N
N
O
N
N
HN
HN
HN
HN
H
N
H
N
H
N
H
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
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49
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H
Cl
N
N
N
N
Br
N
N
F
N
N
N
N
N
N
N
N
H
N
N
H
N
N
H
N
N
H
O
O
O
O
18
21
19
20
Compound
JAK1
JAK2
IC50 (M)a
Selectivity over
JAK2 (folds)
pSTAT3
hERG
(M)
IC50 (M)a
IC50 (M)b
0.060
0.12
18
19
20
21
0.007
0.019
0.008
0.070
1.80
257
347
22
> 33
15
6.60
1.57
196
0.094
0.130
>15
>214
> 100
aMeasured at 5 mM ATP concentration. Selectivity expressed as the ratio of JAK2 IC50 over JAK1 IC50. All values represent the geometric
mean of at least two independent experiments and have SEM within 0.2 log units. bRepresents cellular mode of action assay measuring
inhibition of pSTAT3 in NCI-H1975 cells. For experimental details, see the supporting information.22
Having 21 with high selectivity between JAK1 and JAK2, first we tested 21 in the
biochemical assays of other JAK family kinases with a high concentration of ATP and found
that 21 is inactive toward JAK3 (IC50 > 30 M) with only moderate activity toward TYK2 (IC50
= 2.8 M). Next, in a panel of 293 kinases at single point concentration (1 M), we found that
21 showed a high level of selectivity, only inhibiting one other kinase (FLT4) over 75% at this
concentration (for details, see supporting information SI Table 3).
Compound 21 displayed high metabolic stability in hepatocyte CLint assays across
species, with high solubility at pH=6.5 (> 1000 M) (Table 6). Compound 21 did not inhibit
any of the isoforms of human cytochrome P450s up to a concentration of 30 M. Compound
21 also displayed high permeability (Papp = 7.5 1x10-6 cm/s) with a low efflux ratio (2.6) in a
Caco-2 assay (Table 6). Compound 21 displayed favorable pharmacokinetic properties across
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species (Table 7), showing a low in vivo clearance (20 mL/min/kg) with a moderate half-life (6
hours) and excellent oral bioavailability (100%) in rats. We similarly observed that 21 has low
in vivo clearance (9 mL/min/kg) with prolonged half-life (9 hour) and high oral availability
(71%) in dogs.
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16
17
18
19
20
21
22
23
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Table 6. Preclinical ADME and calculated properties of 21
aMeasured using shake-flask methodology with a buffer:octanol volume ratio of 100:1. at pH 7.4. b High-throughput solubility using DMSO
c
stock solution was measured in pH 7.4 phosphate buffer at 37 °C. Intrinsic clearance measured from fresh hepatocytes from the indicated
species. d Permeability was measured in both the apical to basolateral (A to B) and basolateral to apical (B to A) direction. Efflux ratio (ER)
determined as the ratio of both permeabilities (B to A permeability divided by A to B permeability)
Table 7. Pharmacokinetic properties of 21 in rats and dogs
Preclinical
species
AUC
(M.hr)
CL(obs)
Vdss
(L/kg)
T1/2
(hr)
Route
F%
(mL/min/kg)
i.v.
p.o.
i.v.
1.2
1.7
3.1
5.3
20
8.7
6.3
6
7
9
9
Rata
100%
71%
9.0
Dogb
p.o.
a Han Wistar rat male; 10 mg/kg p.o. (0.1% HPMC); 5 mg/kg i.v. (40%DMA/40%PEG/20% saline). b Beagle dog male; 20 mg/kg p.o. (0.1%
HPMC, pH 2); 10 mg/kg i.v. (20% TEG/D5W).
With advanced compound 21 in hand, we were keen to evaluate its preclinical in vivo
antitumor activity, and furthermore, to test the hypothesis that inhibition of JAK1 could be a
viable therapeutic approach to enhance antitumor activity in combination with the approved
therapies targeting oncogenic signaling pathways. In order to see the additive effect, we
evaluated 21 (dosed at 50 mpk BID) in a mouse non-small cell lung cancer (NSCLC) xenograft
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NCI-H1975 model in combination with the approved third generation mutant EGFR selective
kinase inhibitor, osimertinib, 22 at a low dose of 2.5 mpk..42-43 NCI-H1975 cells were implanted
subcutaneously in female NCr nude mice (Taconic Laboratories). Ten days after cell
implantation, mice were randomized into groups of 6 to 8 mice (average tumor volume 189
mm3, range 152 to 250 mm3), and were dosed orally with either vehicle (20% captisol), 21, 22,
or combinations of 21 plus 22, at the doses and schedules indicated in Figure 4 for 18 days. In
the combination treated groups, the addition of 21 enhanced the antitumor activity of 22 (green
diamonds), compared to treatment with 22 alone (red squares). When compound 21 was
administered as a single agent (blue triangles), it had only weak antitumor activity relative to
vehicle control treatment (black circles). On the last day of treatment, the inhibition of tumor
growth of the combination treatments was statistically significant (p < 0.05), compared to single
agent 22 alone. All treatments were well tolerated, with no significant weight loss or other
outward signs observed over the course of treatment (data not shown).
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A satellite group of the tumor bearing mice were treated for a single day with these
compounds for pharmacodynamic analysis. We measured pSTAT3 (Y705) levels in tumor
lysates as an in vivo biomarker of JAK1 kinase inhibition as described previously22 and pEGFR
(Y1173) levels as the biomarker of EGFR (T790M) inhibition.42-43 A single 2.5 mg/kg dose of
22 achieved a complete inhibition of pEGFR levels up to 24 h. With a 50 mg/kg dose of 21,
approximately 80% of the pSTAT3 inhibition was maintained up to 8 hours after dosing (Figure
5). The combined doses of 21 and 22 resulted in a robust knockdown of pSTAT3 and pEGFR,
measured at 8 hours after the dose. This correlated well with the observed enhanced antitumor
activity in the mice xenograft efficacy study. Taken together, the increased antitumor activity
of 21 plus 22 in combination compared to single agent 22, and the correlation with pSTAT3
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knockdown by 21, is consistent with a role for STAT3 signaling in escape from, or resistance
to, EGFR inhibition in this model. The results support the hypothesis that inhibition of STAT3
signaling can enhance the antitumor activity of an EGFR inhibitor in T790M EGFR mutant
NSCLC. As such, compound 21 displayed the best overall profile, and was thus selected as the
drug candidate to progress into the clinical development.
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21
22
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Figure 4. Compound 21 enhances the antitumor activity of low dose 22 (osimertinib) in an NCI-
H1975 (T790M) NSCLC xenograft model.
Vehicle
Vehicle
23, 2.5 mg/kg QD
22, 50 mg/kg BID
21, 50 mg/kg BID
22, 2.5 mg/kg QD
1000
750
22 (2.5 mg/kg) QD +
23 + 22
21 (50 mg/kg) BID
500
N
O
NH
O
N
N
N
N
H
N
250
22, osimertinib
100
10
15
20
25
Days after implant
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Figure 5. Plot of pSTAT3 and pEGFR signals in an NCI-H1975 mouse xenograft model with
5
6
the treatment of 21 and 22.
7
8
9
200.0
Dosing schedule:
22, 2.5 mg/kg
21, 50 mg/kg
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22
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22 (2.5 mg/kg) + 21 (50 mg/kg)
150.0
100.0
pSTAT3
pEGFR
50.0
0.0
9291 +
9291 +
Incyte JAK1 9291 + 0289 50 Incyte J1, 2
AZD9291 8
hr
Incyte J1 9291 + 0289 Incyte J1 8
200 8 hr
0289 50 24
hr
Incyte J1 929
200 24 hr 50
veh2 hr
23, 2 hr
22, 2 hr
200 2 hr
2 hr
hr
veh 8 hr
0289 50 8 hr
50 8 hr
hr
veh 24 hr
9291 24 hr
-50.0
Conclusion
we have described our efforts toward the development of a JAK1 selective inhibitor.
Starting from a non-kinome selective screening hit 3, structure based-design and a parallel
synthesis approach were used to identify the potent and selective lead 9. Further optimization
led to the discovery of candidate drug 21 (AZD4205) with a high degree of JAK1 potency and
favorable DMPK properties. The compound displayed an enhanced antitumor activity in
combination with approved EGFR inhibitor, osimertinib, in a preclinical NSCLC model. These
findings warrant the further investigation of 21 in human clinical trials.
Chemistry
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Suzuki reactions were used to couple the 7-nitro-indole boronic ester (SI3) to 2,4-
dichloropyrimidines, differentially substituted at the C-5 position, to furnish 2-chloro-4-indole
pyrimidines, i.e SI5. Subsequently an acid-mediated aniline displacement yielded the desired
nitro-intermediates. Reduction of the nitro group under mild conditions was followed by
amidation to produce the desired compounds (for an example, the synthesis of 21 is described in
Scheme 1. For the synthesis of other compounds, see supporting information).
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21
22
23
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Scheme 1. Example synthesis of compound 21a
N
O
N
HN
O2N
NO2
Tos
H
N
H
N
N
Cl
N
a.
c.
N
+
B
N
N
N
N
N
O
N
N
N
N
O
Cl
O
b.
N
N
H
d.
N
N
N
H2N
H
O
O
HO
O
(SI3)
(SI5)
21
o
o
aReagents and conditions: (a) (i) Pd(dppf)2, K2CO3; 83 %; (ii) NaOH, 50 C, 81%; (b) TsOH.H2O, 130 C, 75%; (c) Fe, NH4Cl, 74%; (d)
HATU, iPrEt2N, 48%.
EXPERIMENTAL
General information
All solvents used were commercially available in anhydrous grade. Reagents were utilized
without further purification unless otherwise stated. Evaporation of solvent was carried out using
a rotary evaporator under reduced pressure at a bath temperature of up to 60 ºC. Temperatures
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are given in degrees Celsius (ºC), and operations were carried out at room or ambient temperature,
that is, at a temperature in the range of 18-30 ºC. In general, the course of reactions was followed
by thin layer chromatography or mass spectroscopy and reaction times are given for illustration
only; where a synthesis is described as being analogous to that described in a previous example,
the amounts used are the millimolar ratio equivalents to those used in the previous example.
NMR data is in the form of delta values for major diagnostic protons, given in parts per million
(ppm) relative to tetramethylsilane (TMS) as an internal standard, determined at 300 MHz or
400 MHz using deuterated solvent. Analytical mass spectra were run with an electron energy of
70 eV in the chemical ionization (CI) mode using a direct exposure probe; where indicated
ionization was effected by electron impact (EI), electrospray (ESP), or atmospheric pressure
chemical ionization (APCI); values for m/z are given; generally, only ions which indicate the
parent mass are reported. Unless otherwise indicated, all final compounds were purified to ≥95%
purity, as assessed by analytical HPLC using an Agilent 1100 equipped with Waters columns
(Atlantis T3, 2.1 × 50 mm, 3 μm or Atlantis C18, 2.1 × 50 mm, 5 μm) eluted for >10 min with a
gradient mixture of water and acetonitrile with either formic acid or ammonium acetate added
as a modifier, monitored at wavelengths of 220, 254, and 280 nm.
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All experimental activities involving animals were carried out in accordance with AstraZeneca
animal welfare protocols, which are consistent with The American Chemical Society
Publications rules and ethical guidelines. For details of synthesis, please see supporting
information.
ASSOCIATED CONTENT
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Supporting information is available free of charge via the Internet at http://pubs.acs.org
including compound synthesis, characterization, crystallographic information, and kinase panel
selectivity data, additional DMPK data, compound purity data, NMR spectra of selected
compounds and molecular formula strings of the compounds.
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Author information:
Corresponding Author:
*Tel.: + +1 781-839-4175. E-mail: qibin.su@astrazeneca.com.
Author Contributions:
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Acknowledgement:
Authors would like to thank Dr Brian Aquila for insightful discussion, Dr. Michelle Lamb for
proofreading the manuscript, Dr. Kun Song for preparing structure pictures, Sharon Tentarelli
for LC/MS support and Holger Steuber for obtaining the x-ray crystal structures.
Notes:
The authors declare no competing financial interest.
Abbreviations:
Jak2, Janus kinase 2; Jak1, Janus kinase 1; Jak3, Janus kinase 3; MF, idiopathic myelofibrosis;
mpk, mg/kg; MPNs, myeloproliferative neoplasms; PAGE, Polyacrylamide gel electrophoresis;
PEG, polyethylene glycol; PV, polycythemia vera; SDS, sodium dodecyl sulfate; STAT, signal
transducers and activators of transcription; pSTAT, phosphorylated signal transducers
and activators of transcription; tSTAT, total signal transducers and activators of
transcription; TGI, tumor growth inhibition; Tyk2, tyrosine kinase 2.
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TOC
N
O
N
H
N
HN
H
N
N
N
N
H
N
N
N
N
H
N
N
N
H
O
3
21 (AZD4205)
JAK1 IC50 = 0.07 M
JAK2 IC50 = 0.38 M
JAK1 IC50 = 0.07 M
JAK2 IC50 > 15 M
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