Discovery of potent and highly selective thienopyridine janus kinase 2 inhibitors

Article abstract of DOI:10.1021/jm200911r

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity. (Figure presented)

Full text of DOI:10.1021/jm200911r

Article
pubs.acs.org/jmc
Discovery of Potent and Highly Selective Thienopyridine Janus
Kinase 2 Inhibitors
Laurie B. Schenkel,*,† Xin Huang,‡ Alan Cheng,‡ Holly L. Deak,† Elizabeth Doherty,† Renee Emkey,§
Yan Gu,‡ Hakan Gunaydin,‡ Joseph L. Kim,‡ Josie Lee,§ Robert Loberg,⊥ Philip Olivieri,† Jeanne Pistillo,∥
Jin Tang,‡ Qian Wan,† Hui-Ling Wang,† Shen-Wu Wang,⊥ Mary C. Wells,# Bin Wu,† Violeta Yu,§
Liqin Liu,∥ and Stephanie Geuns-Meyer†
†
‡
§
∥
⊥
Departments of Medicinal Chemistry, Molecular Structure, Lead Discovery, Hematology/Oncology Research, Molecular
#
Sciences and Computational Biology, and Pharmacokinetics and Drug Metabolism, Amgen, Inc., 360 Binney Street, Cambridge,
Massachusetts 02142, 1120 Veterans Boulevard, South San Francisco, California 94080, and One Amgen Center Drive, Thousand
Oaks, California 91320, United States
S
* Supporting Information
ABSTRACT: Developing Janus kinase 2 (Jak2) inhibitors has
become a significant focus for small molecule drug discovery
programs in recent years due to the identification of a Jak2
gain-of-function mutation in the majority of patients with
myeloproliferative disorders (MPD). Here, we describe the
discovery of a thienopyridine series of Jak2 inhibitors that
culminates with compounds showing 100- to >500-fold
selectivity over the related Jak family kinases in enzyme
assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood
mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided
structure−activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1
kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.
family kinases, although amino acid differences do exist,3 and
INTRODUCTION
■
compounds with varying levels of Jak2 selectivity have been
The Janus kinases (Jak) are members of a family of intracellular
reported.2c,e,4 Planning to exploit the subtle sequence differ-
tyrosine kinases that play important roles in cytokine receptor-
ences in the active sites of the Jak family kinases via a
mediated signal transduction via activation of downstream
combination of structure−activity relationships (SAR) and
signal transducers and activators of transcription (STAT),
structure-based design, we initiated a program to develop highly
phosphatidylinositol 3-kinase (PI3K), and mitogen-activated
selective Jak2 inhibitors. Here, we describe a series of potent
protein kinase (MAPK) pathways. There are four kinases in the
thienopyridine Jak2 inhibitors that demonstrated high Jak
Jak family [Jak1, Jak2, Jak3, and tyrosine kinase 2 (Tyk2)], and
family selectivity in enzyme and cell assays. X-ray cocrystal
Jak2 has emerged in recent years as a potential therapeutic
structures of small molecules bound to the Jak2 and Jak1 kinase
target. A significant proportion of patients with myeloprolifer-
domains helped guide SAR efforts and enabled the formation of
ative disorders (MPD), which include polycythemia vera (PV),
hypotheses for the observed high selectivity.
essential thrombocythemia (ET), and primary myelofibrosis
(MF), have been shown to carry a mutation in the
pseudokinase domain of Jak2 (V617F), which renders the
CHEMISTRY
■
All thienopyridine final compounds were prepared from key inter-
mediate 1 (Scheme 1).5 The primary amide 3 was synthesized via
hydrolysis of 1 with sulfuric acid at room temperature to provide
iodide 2, followed by Suzuki reaction with 4-morpholinophenylbo-
ronic acid. Treatment of 1 with concentrated HCl at 95 °C provided
in good yield the carboxylic acid intermediate 4, from which
analogues bearing diverse amide and aryl group substitutions could
be accessed. Secondary amides 8−13 and 21−26 were prepared
via cross-coupling of 4 with either 4-morpholinophenylboronic acid
kinase constitutively active and suggests that inhibition of Jak2
may be an effective approach for the treatment of MPD.1
Several inhibitors of Jak family kinases showing efficacy in both
preclinical models and in the clinic have been reported.2
However, because a chronic dosing regimen may be anticipated
for the treatment of PV and ET patients, an inhibitor that is
selective for Jak2 over the broader kinome and the other Jak
family kinases may be desirable to avoid unwanted
immunosuppression and other off-target side effects. The
discovery of Jak2 inhibitors having Jak family selectivity has
posed a significant challenge due to high homology in the
adenosine triphosphate (ATP) binding pocket among the Jak
Received: July 11, 2011
Published: November 16, 2011
© 2011 American Chemical Society
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a
Scheme 1. Synthesis of Thienopyridines
a
Reagents and conditions: (a) H2SO4, 23 °C, 84%. (b) Pd(PPh3)4, aqueous Na2CO3, ArB(OH)2, DMF, 100 °C, or Pd(dppf)Cl2, aqueous Na2CO3,
ArB(OH)2, dioxane, 100 °C, 10−93%. (c) Concentrated HCl, 95 °C, 84%. (d) EDC-HCl, HOBt hydrate, Hunig's base, R2R3NH, DMF, 25−58%.
or 4-tert-butylsulfonamidophenylboronic acid to give intermedi-
ates 5 and 6, respectively, followed by amide couplings with the
appropriate amines using 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDC)/N-hydroxybenzotriazole (HOBt). Con-
version of 4 to the methyl amide intermediate 7 followed by
Suzuki reaction with the appropriate aryl boronic acid furnished
compounds 14−19.
panel of 100 human kinases and had promising kinome
selectivity, as only seven other kinases showed >90% inhibition
(Table 1).6
Because of its kinome selectivity and the modular nature of
its synthesis, compound 3 was selected for additional studies.
Analogues were prepared to determine if changes to either side
of the molecule would impact Jak family selectivity. Efforts to
establish SAR around the thienopyridine core began with
preparation of secondary amides 8−13 (Table 2). The methyl-,
ethyl-, phenyl-, and benzyl-amides 8−11 were only moderately
less potent than 3 in a Jak2 enzyme assay, while the sterically
bulky tert-butyl- and N,N-dimethyl amides 12 and 13 were
poorly tolerated. Compounds 8−11 also showed significantly
improved Jak family selectivity profiles, particularly over Jak3
and Tyk2. Methyl amide 8 was noteworthy, as it showed 43-
fold selectivity against both Jak3 and Tyk2 and 7-fold selectivity
against Jak1.
RESULTS AND DISCUSSION
■
To identify suitable chemical matter from which selective Jak2
inhibitors could be developed, a high-throughput screen (HTS)
of Amgen's compound collection was performed using a Jak2
enzymatic assay. Thienopyridine 3 was a representative primary
amide hit (Table 1). Although it showed good potency in the
Table 1. HTS Hit 3 in Jak Family and Other Kinase Assays
An X-ray cocrystal structure of thienopyridine 8 with the
kinase domain of Jak2 was solved (Figure 1a). Compound 8
binds Jak2 in the ATP pocket and makes several key
interactions with the protein. The pyridine nitrogen forms a
hydrogen bond with the backbone NH of hinge residue
Leu932, while the amide NH in 8 forms an additional hydrogen
bond with the carbonyl of Leu932. The NH2 of the
aminopyridine moiety binds the protein through two hydrogen
bonds, one with the backbone carbonyl of Glu930 along the
hinge and the other through a water-mediated hydrogen bond
to the backbone carbonyl of Gly993. Gly993 is the residue
immediately preceding the conserved DFG motif and is present
in Jak2, Jak1, and Tyk2 but is instead an alanine in Jak3, which
results in a unique conformation at this site in Jak3 relative to
the other three Jak family members.7 This conformational
difference does not readily explain the selectivity gains observed
over Jak3, as the relatively unselective screening hit 3 should
share the same hydrogen-bonding pattern as 8. Jak1 and Tyk2
have sequences that are identical with Jak2 in the DFG region,
Jak family
ab
,
b
kinases
other kinases
kinase
kinase
IC50
(μM)
inhibition
(%)
Jak2
Jak1
0.019
0.039
Jak2
96
Tyk2, YSK4, TAK1, KIT,
MEK5, GAK, IRAK4
>90
Jak3
0.018
0.066
8 kinases
70−85
<70
Tyk2
84 kinases
Measured at the apparent ATP Km.7 Each value represents the
average of ≥2 independent experiments, where each experiment
consisted of a single determination.
a
b
Jak2 enzyme assay, 3 showed little selectivity over Jak1, Jak3,
and Tyk2. Compound 3 was tested at 1 μM against a custom
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Table 2. Data in Jak Enzymatic Assays for 3 and 8−13
a
enzyme: IC50 (μM) (selectivity vs Jak2, x-fold)
no.
R1
R2
Jak2
Jak1
Jak3
Tyk2
3
8
H
H
0.019
0.031
0.048
0.072
0.100
1.17
0.039 (2)
0.211 (7)
0.133 (3)
0.406 (6)
0.307 (3)
8.82 (8)
0.018 (1)
1.34 (43)
0.967 (20)
2.87 (40)
>125 (>250)
10.3 (9)
0.066 (3)
1.33 (43)
1.07 (22)
1.99 (28)
2.60 (26)
6.98 (6)
H
Me
9
H
Et
10
11
12
13
H
Ph
H
CH2Ph
tBu
H
Me
Me
1.17
3.10 (3)
1.66 (1)
5.15 (4)
a
Measured at the apparent ATP Km.7 Each IC50 value represents the average of ≥2 independent experiments, where each experiment consisted of a
single determination.
occupies a pocket beneath the conserved glycine-rich loop.
Replacement of the morpholine group with a hydrogen atom
provided compound 14, which was slightly less potent against
Jak2 and showed diminished selectivity over Jak3 and Tyk2
relative to 8 (Table 3). This suggested that 4-substitution of the
phenyl ring may enhance both Jak2 potency and selectivity, and
a set of compounds containing a range of 4-substituents was
prepared (Table 3).
Compounds 3, 8, and 14 were also tested in a panel of high-
throughput Jak family cellular assays consisting of Ba/F3 cell
lines expressing translocated ETS leukemia (TEL) fusions of
each Jak kinase domain, which confer constitutive kinase
activity.8 Whereas 8 was relatively potent (IC50 = 0.031 μM) in
the Jak2 enzyme assay and showed promising Jak family
selectivity, a significant enzyme to cell shift was observed (IC50
= 3.94 μM, 127-fold) in the TEL-Jak2 cell assay. Compounds 3
and 14 also showed large potency shifts in the TEL-Jak2 cell
assay. Discrepancies between enzyme and cell assay data have
been well-documented for several kinase targets,9 and large
enzyme to cell shifts have also been reported for other Jak
kinase inhibitors.4b−d Numerous factors, including cell
permeability and efflux, may play a role in enzyme to cell
assay shifts. The data for a subset of thienopyridines show that
the majority of the compounds have high average apparent
permeability and are likely efflux substrates.10 Despite the
potency shifts observed between enzyme and cell assays, further
development of thienopyridine SAR was expedited by the TEL-
Jak panel, which provided a facile method to rapidly assess
cellular potency and selectivity for all analogues. Importantly,
these assays enabled us to study relationships between
selectivity in enzyme assays and selectivity in cells for each of
the individual Jak family kinases. The enzyme and TEL-Jak cell
data for compound 8 illustrated a general trend that was
present across the thienopyridine and several other series.11
While enzyme to cell shifts existed for all of the Jak kinases, the
magnitude of the shift for each kinase was different, with Jak1
showing a significantly smaller shift than Jak2. Thus 8, which
showed 7-fold selectivity for Jak2 over Jak1 in enzyme assays,
showed no selectivity for Jak2 over Jak1 in cells. On the basis of
these data, we hypothesized that achieving a higher threshold of
selectivity over Jak1 in enzyme assays than that observed for 8
may be required to obtain significant selectivity over Jak1 in the
TEL-Jak cell assay.
Figure 1. (a) X-ray cocrystal structure (PDB ID: 3TJC) of 8 bound to
the ATP pocket of the Jak2 kinase domain at 2.4 Å resolution. (b)
Overlay of the X-ray cocrystal structures of 8 (blue) at 2.4 Å resolution
and 19 (green) (PDB ID: 3TJD) at 2.9 Å resolution bound to the
ATP pocket of Jak2 (yellow for 8/Jak2 and tan for 19/Jak2).
so the selectivity of 8 for Jak2 over Jak1 and Tyk2 also cannot
be rationalized by the observed hydrogen-bonding interactions.
Although a structural rationale for the improved selectivity
profile of 8 for Jak2 was not readily apparent, SAR studies were
conducted around the 4-morpholinophenyl moiety, which
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Table 3. Jak Family Enzyme and Cellular Data for 3, 8, and 14−19
a
Measured at the apparent ATP Km.7 Each IC50 value represents the average of ≥2 independent experiments, where each experiment consisted
b
of a single determination. Each IC50 value represents the average of ≥2 independent experiments, where each experiment consisted of two
replicates. Percent inhibition at 25 μM. The IC50 could not be calculated because complete inhibition was not achieved in the concentration range
c
tested.
SAR studies at the 4-position of the phenyl ring continued
with replacement of the morpholine ring in 8 with a methyl
group (15) or a tert-butyl group (16), which provided
compounds having comparable Jak2 potency but with slightly
diminished selectivity over Jak3 and Tyk2 (Table 3).
Trifluoromethoxy substitution (17) was equipotent with tert-
butyl substitution (16) in the Jak2 enzyme assay; yet, it was also
active in the TEL-Jak2 assay with a similar selectivity profile to
8 in cell assays. While nitrile 18 and sulfonamide 19 achieved
additional improvements in enzyme and cellular potency, the
bulkier tert-butylsulfonamide (19) provided a significant
breakthrough in Jak2 enzyme selectivity. Compound 19 was
potent against Jak2 (IC50 = 0.001 μM) and was 30-fold
selective over Jak1 and approximately 200-fold selective over
both Jak3 and Tyk2 in enzyme assays. Although it showed
improved selectivity over Jak1 enzyme, Jak1 cell selectivity in
the TEL-Jak assays was not achieved. Compound 19 appeared
comparable to 18 in terms of cellular potency and selectivity,
but its relatively high selectivity for Jak2 in enzymatic assays
warranted further investigation.
same set of key hydrogen bonds with the protein. The
heteroatoms of the sulfonamide do not form any additional
hydrogen bonds with Jak2. Comparison of the structures of 8
and 19 suggests that the improved potency, Jak3 selectivity, and
Tyk2 selectivity of 19 may be due to the orientation of the tert-
butanesulfonamide moiety beneath the glycine-rich loop, which
is shifted in the Jak2/19 structure to accommodate the bulky
tert-butyl group. This movement of the glycine-rich loop,
coupled with the shift of the phenylsulfonamide portion of 19
downward in the active site versus that of 8, may be better
tolerated in Jak2 and Jak1 than in Jak3 and Tyk2.
In an effort to better understand and improve selectivity over
Jak1, a study of the Jak1 active site was undertaken. Data from
two published3 Jak1 X-ray cocrystal structures showed that the
ATP pocket of Jak1 is smaller than that of Jak2. The average
distance between the two Jak1 structures, measured from
Pro960 of the hinge region to Asp1021 of the DFG motif, is
14.9 ± 0.1 Å, while the same distance in the Jak2/19 structure
is 15.4 ± 0.1 Å. An overlay of the published cocrystal structure
of the pan-Jak inhibitor CP-6905502a (20) with the Jak1 kinase
domain and the Jak2/19 structure (Figure 2) illustrates the
subtle difference in active site size between Jak1 and Jak2.
These data suggested that increased steric bulk along the hinge
region might be better tolerated in Jak2 than Jak1 and could
An X-ray cocrystal structure of sulfonamide 19 in the kinase
domain of Jak2 was solved, and an overlay with the Jak2/8
structure is shown in Figure 1b. Compound 19 binds Jak2 in
the same orientation as 8 in the ATP pocket and forms the
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maintained the >400-fold selectivity over Jak3 and Tyk2
observed for 22. Consistent with the charge−charge clash
hypothesis, morpholine 25, which is the least basic among 23−
25, was the most potent of the three compounds against Jak1,
and piperidine 24, which is the most basic among 23−25, was
the most selective for Jak2 over Jak1. Consistent with their
improved Jak1 selectivity versus compound 22 in enzyme
assays, compounds 23 and 24 showed >10-fold selectivity for
Jak2 over Jak1 in the TEL-Jak assays, and 25 also showed
promising levels of cellular selectivity.12 Compound 26, which
contains an additional methylene between the core and the
morpholine, showed diminished potency and selectivity versus
25. Although several compounds have been reported to achieve
selectivity for Jak2 over Jak3 in TEL-Jak assays,4 22−25
represent a compound class that is one of few reported to
achieve selectivity for Jak2 over all of the Jak kinases in TEL-Jak
cell assays.2d
Compound 25 was profiled against the same 100 human
kinases as HTS hit 3 and showed improved selectivity versus
the broader kinome. Against Jak2 at 1 μM, 25 showed 100%
inhibition, and GAK (98%) and YSK4 (91%) were the only
other kinases tested that showed >90% inhibition. Five
additional kinases were inhibited at 65−80%,13 and 92 kinases
returned values of <50% inhibition.
Figure 2. (a) Overlay of the X-ray cocrystal structures of 19 (PDB ID:
3TJD) bound to Jak2 (tan) and 20 (PDB ID: 3EYG) bound to Jak1
(blue). (b) Structure of 20.
Selected thienopyridines were next tested in primary cell
assays in which cytokine stimulation results in activation of
full-length endogenous Jak kinases. These assays served to
establish functional activity and selectivity in a more
physiologically relevant context than the TEL-Jak cell assays.
Purified peripheral blood mononuclear cells (PBMC) and
human whole blood (WB) were stimulated with either
thrombopoietin (TPO)14 or interleukin-2 (IL-2)15 to
activate the Jak2 and Jak1/Jak3 pathways, respectively.
Because inhibition of Jak1 and Jak3 is required for blockade
of signaling through γC-containing cytokines, which leads to
downstream inhibition of STAT5 phosphorylation,16 only
compounds that are selective for Jak2 over both Jak1 and
Jak3 should be selective in these assays. As expected, 3, which
showed no selectivity for Jak2 over the other Jak kinases, also did
not exhibit any selectivity for the Jak2-driven pathway in
PBMC and was slightly more potent in the IL-2-stimulated
assay (Table 5).
therefore further improve selectivity over Jak1. This hypothesis
is consistent with the observed increase in Jak1 selectivity upon
replacement of the primary amide in 3 with the methyl amide
in 8. Further increasing bulk in this region also had the
potential to exploit an active site residue difference between
Jak1 (Arg879) and Jak2 (Gln853) (Figure 2a). Arg879 of Jak1
engages in a water-mediated hydrogen bond with the hinge
Pro960. The pyrrolopyrimidine moiety of 20 participates in this
hydrogen-bonding network in Jak1, which may contribute to its
Jak1 potency.3 It was hypothesized that incorporation of a
tertiary amine into the thienopyridine scaffold, which would be
protonated at physiological pH, could disrupt this hydrogen
bond and introduce a charge−charge clash with Arg879 of Jak1
that would not occur in Jak2, thereby providing increased
selectivity.
A similar phenomenon has been reported in the literature for
the pan-Jak kinase inhibitor 20, which is equipotent against
Jak1, Jak2, and Jak3 in enzyme assays but is more potent in
cellular assays driven by a combination of Jak1 and Jak3
activities than those driven by Jak2 alone.2a,16 Compound 20
was approximately 5-fold more potent in our IL-2-stimulated
assays than our TPO-stimulated assays (Table 5), which is in
general agreement with reported values that show good
selectivity for inhibition of Jak1/Jak3- versus Jak2-mediated
cytokine signaling in WB.17 Experiments reported in the
literature with 20 and a Jak3 selective inhibitor also showed that
Jak1 plays a dominant role over Jak3 in signal transduction
through γC-containing cytokines,18 which suggests that high
selectivity over Jak1 in particular may be necessary to observe
significant Jak2 selectivity in physiologically relevant assays.
Likewise, the Jak2/Jak1 inhibitor CYT387, which is 9-fold
selective for Jak2 over Jak3 in enzyme assays, shows only
marginal selectivity (<2-fold) for a Jak2-driven cellular assay
over a combined Jak1/Jak3-driven cellular assay.4c This
suggests that its lack of cellular selectivity is driven by its
Jak1 potency. Although it was obtained from a variety of
To test these structure-based hypotheses for improving Jak1
selectivity, a series of 4-sulfonamides with a variety of amide
moieties was prepared (Table 4). Data from the enzyme and
TEL-Jak cell assays for 20 are included for comparison. As
previously reported, 20 shows good potency against all of the
Jak family members in enzyme assays,2a with cell shifts that are
comparable to those of the thienopyridines and compounds
representing other series.11 Alcohol 21 was less selective over
Jak1 than 19 in enzyme assays, but capping the alcohol with a
methyl group to give methoxy amide 22 provided increased
Jak1 selectivity relative to 19 with concomitant improvements
in Jak3 and Tyk2 selectivity. Furthermore, its 44-fold selectivity
for Jak2 over Jak1 in enzyme assays appeared to be sufficient for
translation into Jak1 cell selectivity, as 22 was 7-fold more
potent in the TEL-Jak2 cell assay than in the TEL-Jak1 assay.
Introduction of a tertiary amine moiety provided additional
improvement in Jak1 selectivity. Compounds 23−25, which
incorporate both larger amide substituents and tertiary amines
along the hinge region of the ATP pocket, approached and
exceeded 100-fold selectivity for Jak2 over Jak1 and also
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Table 4. Jak Family Enzyme and Cellular Data for 20−26
a
Measured at the apparent ATP Km.7 Each IC50 value represents the average of ≥2 independent experiments, where each experiment consisted of a
b
single determination. Each IC50 value represents the average of ≥2 independent experiments, where each experiment consisted of two replicates.
See the Experimental Section for details. Percent inhibition at 25 μM. The IC50 could not be calculated because complete inhibition was not
c
achieved in the concentration range tested.
Table 5. Activity of Selected Thienopyridines and 20 in Cytokine-Stimulated PBMC and WB Assays
a
a
PBMC (IC50, μM)
WB (IC50, μM)
b
compd
TPO
IL-2
fold selectivity
TPO
IL-2
fold selectivity
human Fu (%)
TPO-stimulated WB IC50/PBMC IC50
d
3
19
20
22
23
25
0.761
0.048
0.155
0.135
0.515
0.148
0.548
0.625
0.033
4.45
<1
13
>25
1.48
0.682
3.95
7.83
2.19
>25
>25
ND
>33
37.5
4.4
>17
<1
1.2
c
<1
0.143
>25.0
>25.0
>25.0
24
33
>6
2.3
29.3
15.2
14.8
>25.0
7.30
>49
49
>3
6.4
>11
10.1
a
Each IC50 value represents the average of ≥2 independent experiments, where each experiment consisted of two replicates. Measures inhibition of
b
phosphorylation of STAT5 using intracellular phosflow staining and analysis. Fraction unbound. Binding to human plasma proteins was determined
by equilibrium dialysis. Reported value, determined using by equilibrium dialysis. See ref 2a. Not determined.
c
d
different cell assay formats, data for these previously reported
inhibitors and compound 3 taken together illustrate the
difficulty in identifying Jak2 selective inhibitors using enzyme
assays alone, and it highlights the importance of establishing
relationships between apparent Jak2 selectivity in enzyme
assays with that in downstream cellular assays.
Interestingly, compound 19, which was highly selective for
Jak2 over Jak3 in enzyme and TEL-Jak assays but showed
discordant Jak1 enzyme (30-fold selectivity for Jak2) and Tel-
Jak1 (equipotent with Jak2) data, was 13-fold selective for the
Jak2 driven pathway in the PBMC assay. Compound 19 is an
outlier in that its selectivity over Jak1 and Jak3 in TEL-Jak
assays was not predictive of selectivity in the PBMC assays. As
expected, compounds that showed good cellular selectivity in
the TEL-Jak2 assay over all of the Jak kinases, such as 22, 23,
and 25, were more selective than 19 for TPO versus IL-2
stimulation in the PBMC assays. Compounds 23 and 25 are
particularly noteworthy as they were ≥49-fold more potent
in the TPO-stimulated assay than in the IL-2 stimulated
assay, thus demonstrating high selectivity for the Jak2-driven
pathway.
In WB assays, 19, 22, 23, and 25 returned micromolar IC50
values with TPO stimulation. Although multiple factors could
contribute to shifts between the IC50 values in the TPO-
stimulated PBMC and the TPO-stimulated WB assays, the
values obtained in the WB assay for 19, 20, 22, 23, and 25
(Table 5) were all less than or equal to PBMC assay IC50 values
after normalization for human plasma free fraction (TPO
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4-Amino-2-iodothieno[3,2-c]pyridine-7-carboxamide (2). A vial
was charged with 1 (300 mg, 0.712 mmol) and neat sulfuric acid (1.42
mL), and the dark red mixture was stirred at room temperature (RT)
for 1 h. Ice was added, and the resulting precipitate was filtered,
washed with water, and dried. The solids were dissolved in DMSO/
MeOH and passed through an SCX column, which was washed with
MeOH. The product was eluted with 2.0 M NH3/MeOH, and the
filtrate was concentrated to provide 2 (190 mg, 84%) as a tan solid.
MS: m/z 319.6 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.44
(s, 1H), 7.94 (s, 2H), 7.29 (br s, 1H), 7.22 (s, 2H).
PBMC IC50/Fu). Notably, despite its potency shift into WB,
compound 19 still showed good selectivity for the TPO- over
the IL-2 stimulated pathway (>17-fold). Because 22, 23, and 25
showed higher selectivity than 19 in PBMC assays, it was
reasonable to anticipate that they would also show >17-fold
selectivity in WB. However, accurate determination of IC50
values in the IL-2-stimulated assays was not possible in the
concentration ranges tested.
CONCLUSION
4-Amino-2-(4-morpholinophenyl)thieno[3,2-c]pyridine-7-carbox-
amide (3). To a solution of 2 (80.0 mg, 0.251 mmol) in
dimethylformamide (DMF) (3.0 mL) and Na2CO3 (2.0 M, 0.5 mL)
was added 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
morpholine (83.4 mg, 0.288 mmol). The solution was degassed, and
Pd(PPh3)4 (11.6 mg, 10.0 μmol) was added. The reaction was heated
at 100 °C for 1 h and was cooled to RT. The mixture was filtered and
washed with dichloromethane (DCM) and water to give 3 (60.0 mg,
■
Here, we have described the identification of a highly selective
thienopyridine series of Jak2 inhibitors. SAR developed around
the phenyl and amide moieties combined with structure-based
design led to the discovery of compounds possessing 100-fold
selectivity for Jak2 over Jak1, >400-fold selectivity over Jak3,
and >500-fold selectivity over Tyk2 in enzyme assays. Use of a
high-throughput panel of TEL-Jak cellular assays enabled us to
determine that very high selectivity for Jak2 in enzyme assays
was required to observe selectivity in both the engineered TEL-
Jak and the more biologically relevant cytokine-stimulated
PBMC and WB assays. Despite large potency shifts between
enzyme and cellular assays for the compounds in this series,
two of the most Jak2 selective inhibitors in enzyme and TEL-
Jak cell assays (23 and 25) approached and exceeded 50-fold
selectivity for the TPO-stimulated over the IL-2-stimulated
pathway in PBMC assays. These thienopyridines represent two
of only a small number of Jak2 inhibitors reported to show
significant selectivity in cells. In addition to serving as valuable
tools for understanding Jak family selectivity, the thienopyr-
idines provided novel insight about the active site of Jak2
kinase. Additional chemical series designed to take advantage of
information learned from this work will be reported in future
publications.
1
68%) as a light green solid. MS: m/z 355.1 ([M + H]+). H NMR
(400 MHz, DMSO-d6): δ 8.44 (s, 1H), 7.86 (s, 1H), 7.57 (d, J = 8.8
Hz, 2H), 7.01−7.10 (m, 4H), 3.71−3.79 (m, 4H), 3.15−3.22 (m, 4H).
HRMS for C18H18N4O2S [M + H]+: calcd, 355.1223; found, 355.1224.
4-Amino-2-iodothieno[3,2-c]pyridine-7-carboxylic Acid (4). A
pressure bottle was charged with 1 (5.00 g, 11.87 mmol), concentrated
HCl (23.74 mL, 11.87 mmol), and DMSO (11.9 mL). The bottle was
sealed, and the reaction was heated at 95 °C overnight. After they were
cooled to RT, the solids were filtered, washed with water, and air-dried
to provide 4 as the hydrochloride salt (3.57 g, 84%) as a tan solid. MS:
m/z 320.6 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.90 (br s,
2H), 8.35 (s, 1H), 8.32 (s, 1H).
4-Amino-2-(4-morpholinophenyl)thieno[3,2-c]pyridine-7-carbox-
ylic Acid (5). A vial was charged with Pd(dppf)Cl2 (7.70 mg, 10.52
μmol), 4-(morpholino)phenylboronic acid (65.3 mg, 0.316 mmol), 4
(75 mg, 0.210 mmol), aqueous Na2CO3 (2.0 M, 316 μL, 0.631 mmol),
and dioxane (841 μL). The vial was sealed, and the mixture was heated
at 100 °C for 2 h. After it was cooled to RT, the mixture was passed
through an SCX column, which was washed with MeOH. The product
was eluted with 2.0 M NH3/MeOH, and the filtrate was concentrated
to provide 5 (52 mg, 70%) as a tan solid. MS: m/z 356.0 ([M + H]+).
1H NMR (400 MHz, DMSO-d6): δ 8.40 (s, 1H), 7.90 (s, 1H), 7.58 (d,
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise noted, all materials were obtained
from commercial suppliers and used without further purification.
Anhydrous solvents were obtained from Aldrich and used directly.
Strong cation exchange chromatography was performed using Biotage
Isolute SCX-2 columns. Silica gel chromatography was performed
using medium-pressure liquid chromatography (MPLC) on a
CombiFlash Companion (Teledyne Isco) with RediSep normal-
phase silica gel (35−60 μm) columns and UV detection at 254 nm.
Preparative reversed-phase high-pressure liquid chromatography
(HPLC) was performed on a Gilson (215 liquid handler), YMC-
Pack Pro C18, 150 mm × 30 mm i.d. column, eluting with a binary
solvent system A and B using a gradient elusion [A: H2O with 0.1%
trifluoroacetic acid (TFA); B: CH3CN with 0.1% TFA] with UV
detection at 254 nm. All final compounds were purified to ≥95%
purity as determined by Agilent 1100 Series HPLC with UV detection
at 254 nm (method A: Zorbax SB-C8, 4.6 mm × 150 mm, 15 min; 1.5
mL/min flow rate; 0−100% 0.1% TFA in CH3CN/0.1% TFA in H2O;
method B: Phenomenex Synergi, 2 mm × 50 mm, 3 min, 1.0 mL/min
flow rate, 5−95% 0.1% formic acid in CH3CN/0.1% formic acid in
H2O). Low-resolution mass spectral (MS) data were determined on an
Agilent 1100 Series LCMS with UV detection at 254 nm and a low
resonance electrospray mode (ESI). High-resolution mass spectra
(HRMS) were obtained on an Agilent 1100 HPLC/MS time-of-flight
mass spectrometer. Nuclear magnetic resonance (NMR) spectra were
acquired with either a Bruker AVANCE-400 (at 400.13 MHz) or a
Bruker AVANCE III-500 (at 500.34 MHz) spectrometer operating at
298 K. Chemical shifts are reported in ppm relative to the residual
proton signal in the solvent [dimethylsulfoxide (DMSO)-d6 at 2.50
ppm]. Data are reported as follows: chemical shift, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, br = broad, and m =
multiplet), coupling constants (in Hz), and number of protons.
J = 8, 2H), 7.24 (br s, 2H), 7.05 (d, J = 8, 2H), 3.76 (m, 4H), 3.19 (m,
4H).
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)thieno[3,2-c]-
pyridine-7-carboxylic Acid (6). Following the procedure described
for 5, 4-(N-tert-butylsulfamoyl)phenylboronic acid and 4 provided the
title compound as the hydrochloride salt (660 mg, 93%) as a brown
solid. MS: m/z 405.8 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ
8.46 (s, 1H), 8.28 (s, 1H), 7.91 (m, 4H), 7.59 (m, 3H), 1.13 (s, 9H).
4-Amino-2-iodo-N-methylthieno[3,2-c]pyridine-7-carboxamide
(7). A round-bottom flask was charged with 4 (2.65 g, 7.43 mmol),
Hunig's base (2.86 mL, 16.35 mmol), HOBt hydrate (1.707 g, 11.15
mmol), EDC-HCl (2.137 g, 11.15 mmol), DMF (29.7 mL), and
methylamine (2.0 M in THF, 5.57 mL, 11.15 mmol). The reaction
mixture was stirred at RT for 15 h. After completion, the mixture was
concentrated. The crude material was purified by silica gel
chromatography, 10−100% 90/10/1 DCM/MeOH/NH4OH in
DCM to provide 7 (1.4 g, 56.5%) as a tan solid. MS: m/z 333.8
1
([M + H]+). H NMR (400 MHz, DMSO-d6): δ 8.37 (m, 2H), 7.92
(s, 1H), 7.09 (br s, 2H), 2.77 (d, J = 4, 3H).
4-Amino-N-methyl-2-(4-morpholinophenyl)thieno[3,2-c]-
pyridine-7-carboxamide (8). Following the procedure described for
7, methylamine and 5 provided the title compound (62 mg, 40%). MS:
1
m/z 369.3 ([M + H]+). H NMR (400 MHz, DMSO-d6): δ 8.24 (s,
1H), 8.05 (s, 1H), 7.60 (d, J = 8, 2H), 7.05 (d, J = 8, 2H), 3.77−3.73
(m, 4H), 3.18−3.14 (m, 4H), 2.81 (s, 3H). HRMS for C19H20N4O2S
[M + H]+: calcd, 369.1380; found, 369.1377.
4-Amino-N-ethyl-2-(4-morpholinophenyl)thieno[3,2-c]pyridine-
7-carboxamide (9). Following the procedure described for 7,
ethylamine and 5 provided the title compound (83 mg, 52%). MS:
1
m/z 383.2 ([M + H]+). H NMR (400 MHz, DMSO-d6): δ 8.40
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Journal of Medicinal Chemistry
Article
(t, J = 4. 1H), 8.37 (s, 1H), 7.82 (s, 1H) 7.58 (d, J = 8, 2H), 7.03
(d, J = 8, 2H), 3.75−3.72 (m, 4H), 3.29 (dq, J = 4, J = 8, 2H), 3.17−
3.15 (m, 4H), 1.12 (t, J = 8, 3H). HRMS for C20H22N4O2S [M + H]+:
calcd, 383.1536; found, 383.1536.
4-Amino-2-(4-morpholinophenyl)-N-phenylthieno[3,2-c]-
pyridine-7-carboxamide (10). Following the procedure described for
7, aniline and 5 provided the title compound (47 mg, 26%). MS: m/z
431.0 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H),
7.91 (s, 1H), 7.79 (d, J = 8, 2H), 7.59 (d, J = 12, 2H), 7.35 (t, J = 8,
2H), 7.24 (s, 2H), 7.11−7.05 (m, 3H), 7.09−7.04 (m, 4H), 3.77−3.76
(m, 4H), 3.20−3.16 (m, 4H). HRMS for C24H22N4O2S [M + H]+:
calcd, 431.1536; found, 431.1540.
4-Amino-N-benzyl-2-(4-morpholinophenyl)thieno[3,2-c]pyridine-
7-carboxamide (11). Following the procedure described for 7,
benzylamine and 5 provided the title compound (25 mg, 13%). MS:
m/z 445.0 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.97 (t, J
= 4, 1H), 8.51 (s, 1H), 7.87 (s, 1H), 7.56 (d, J = 8, 2H), 7.34−7.31 (m,
4H), 7.26−7.24 (m, 1H), 7.09−7.04 (m, 4H), 4.95 (d, J = 4, 2H),
3.76−3.74 (m, 4H), 3.20−3.17 (m, 4H). HRMS for C18H18N4O2S [M
+ H]+: calcd, 445.1693; found, 445.1692.
4-Amino-N-tert-butyl-2-(4-morpholinophenyl)thieno[3,2-c]-
pyridine-7-carboxamide (12). Following the procedure described for
7, tert-butylamine and 5 provided the title compound (40 mg, 23%).
MS: m/z 411.0 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.45
(s, 1H), 8.37 (s, 1H), 7.85 (s, 1H) 7.65 (s, 1H), 7.57 (d, J = 8, 2H),
7.04 (d, J = 8, 2H), 7.06 (s, 2H), 3.76−3.74 (m, 4H), 3.18−3.17 (m,
4H), 1.40 (s, 9H). HRMS for C22H26N4O2S [M + H]+: calcd,
411.1849; found, 411.1847.
4-Amino-N,N-dimethyl-2-(4-morpholinophenyl)thieno[3,2-c]-
pyridine-7-carboxamide (13). Following the procedure described for
7, dimethylamine and 5 provided the title compound (35 mg, 22%).
MS: m/z 383.1 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 7.89
(s, 1H), 7.82 (s, 1H), 7.55 (d, J = 8, 2H), 7.01 (d, J = 8, 2H), 3.73−
3.71 (m, 4H), 3.16−3.13 (m, 4H), 3.03 (s, 6H). HRMS for
C20H22N4O2S [M + H]+: calcd, 383.1536; found, 383.1532.
4-Amino-N-methyl-2-phenylthieno[3,2-c]pyridine-7-carboxa-
mide (14). Following the procedure described for 5, phenylboronic
acid and 7 provided the title compound (35 mg, 41%). MS: m/z 284.0
([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.36 (q,
J = 4, 1H), 8.05 (s, 1H), 7.72−7.66 (m, 2H), 7.53−7.44 (m, 2H),
7.40−7.36 (m, 1H), 7.12 (br s, 2H), 2.81 (d, J=4, 3H). HRMS for
C15H13N3OS [M + H]+: calcd, 284.0852; found, 284.0852.
4-Amino-N-methyl-2-p-tolylthieno[3,2-c]pyridine-7-carboxamide
(15). Following the procedure described for 5, 4-methylphenylboronic
acid and 7 provided the title compound (33 mg, 46%). MS: m/z 297.8
([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.42 (s, 1H), 8.35 (q,
J = 4, 1H), 7.99 (s, 1H), 7.59 (d, J=8, 2H), 7.30 (d, J = 8, 2H), 7.08 (br
s, 2H), 2.81 (d, J = 4, 3H), 2.34 (s, 3H). HRMS for C16H15N3OS [M +
H]+: calcd, 284.0852; found, 284.0852. HRMS for C18H18N4O2S [M +
H]+: calcd, 298.1009; found, 298.1007.
4-Amino-2-(4-tert-butylphenyl)-N-methylthieno[3,2-c]pyridine-7-
carboxamide (16). Following the procedure described for 5, 4-tert-
butylphenylboronic acid and 7 provided the title compound (8 mg,
8%). MS: m/z 340.0 ([M + H]+). 1H NMR (400 MHz, DMSO-d6): δ
8.42 (s, 1H), 8.37 (q, J = 4, 1H) 7.99 (s, 1H), 7.63 (d, J = 8, 2H), 7.51
(d, J = 8, 2H), 7.09 (br s, 2H), 2.81 (d, J = 4, 3H), 1.32 (s, 9H).
4-Amino-N-methyl-2-(4-(trifluoromethoxy)phenyl)thieno[3,2-c]-
pyridine-7-carboxamide (17). Following the procedure described for
5, 4-(trifluoromethoxy)phenylboronic acid and 7 provided the title
compound (31 mg, 35%). MS: m/z 367.8 ([M + H]+). 1H NMR (400
MHz, DMSO-d6): δ 8.45 (s, 1H), 8.37 (q, J = 4, 1H) 8.09 (s, 1H),
7.81 (d, J = 8, 2H), 7.50 (d, J = 8, 2H), 7.16 (br s, 2H), 2.81 (d, J = 4,
3H). HRMS for C16H18N3O2SF3 [M + H]+: calcd, 368.0675; found,
368.0673.
4-Amino-2-(4-(2-cyanopropan-2-yl)phenyl)-N-methylthieno[3,2-
c]pyridine-7-carboxamide (18). Following the procedure described
for 5, 4-(2-cyanopropan-2-yl)phenylboronic acid and 7 provided the
title compound (28 mg, 33%). MS: m/z 350.8 ([M + H]+). 1H NMR
(400 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.37 (q, J = 4, 1H) 8.08
(s, 1H), 7.75 (d, J = 8, 2H), 7.64 (d, J = 8, 2H), 7.14 (br s, 2H), 2.81
(d, J = 4, 3H), 1.73 (s, 6H). HRMS for C19H18N4OS [M + H]+: calcd,
351.1274; found, 351.1275.
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-methylthieno-
[3,2-c]pyridine-7-carboxamide (19). Following the procedure de-
scribed for 5, 4-(N-tert-butylsulfamoyl)phenylboronic acid and 7
provided the title compound (50 mg, 50%). MS: m/z 418.8 ([M +
H]+). 1H NMR (400 MHz, DMSO-d6): δ 8.46 (s, 1H), 8.39 (q, J = 4,
1H) 8.20 (s, 1H), 7.91 (d, J = 8, 2H), 7.86 (d, J = 8, 2H), 7.57, (s,
1H), 7.19 (br s, 2H), 2.82 (d, J = 4, 3H), 1.13 (s, 9H). HRMS for
C19H22N4O3S2 [M + H]+: calcd, 419.1206; found, 429.1209.
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-(2-hydroxyethyl)-
thieno[3,2-c]pyridine-7-carboxamide (21). Following the procedure
described for 7, 2-aminoethanol and 6 provided the title compound
1
(10 mg, 11%). MS: m/z 448.7 ([M + H]+). H NMR (500 MHz,
DMSO-d6): δ ppm 8.53 (s, 1 H), 8.40 (t, J = 5.60, 1 H), 8.21 (s, 2 H),
7.90−7.93 (m, 2 H), 7.85−7.89 (m, 2 H), 7.57 (s, 1 H), 7.21 (s, 2 H),
4.72 (t, J = 5.59, 1 H), 3.54 (q, J = 6.19, 2 H), 3.36 (q, J = 6.02, 2 H),
1.13 (s, 9 H).
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-(2-
methoxyethyl)thieno[3,2-c]pyridine-7-carboxamide (22). Following
the procedure described for 7, 2-methoxyethanamine and 6 provided
1
the title compound (29 mg, 32%). MS: m/z 462.8 ([M + H]+). H
NMR (500 MHz, DMSO-d6): δ ppm 8.53 (s, 1 H), 8.48 (t, J = 5.34
Hz, 1 H), 8.22 (s, 1 H), 7.85−7.95 (m, 4 H), 7.57 (s, 1 H), 7.22 (br s,
2 H), 3.42−3.51 (m, 4 H), 3.28 (s, 3 H), 1.13 (s, 9 H). HRMS for
C21H26N4O4S2 [M + H]+: calcd, 464.1496; found, 464.1497.
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-(2-
(dimethylamino)ethyl)thieno[3,2-c]pyridine-7-carboxamide (23).
Following the procedure described for 7, N1,N1-dimethylethane-1,2-
diamine and 6 provided the title compound (26 mg, 28%). MS: m/z
475.8 ([M + H]+). 1H NMR (500 MHz, DMSO-d6): δ ppm 8.50 (s, 1
H), 8.36 (t, J = 5.60 Hz, 1 H), 8.21 (s, 1 H), 7.90−7.94 (m, 2 H),
7.84−7.89 (m, 2 H), 7.57 (s, 1 H), 7.21 (br s, 2 H), 3.39 (q, J = 6.61
Hz, 2 H), 2.43 (t, J = 6.99 Hz, 2 H), 2.20 (s, 6 H), 1.13 (s, 9 H).
HRMS for C22H29N5O3S2 [M + H]+: calcd, 476.1785; found,
476.1788.
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-(2-(piperidin-1-
yl)ethyl)thieno[3,2-c]pyridine-7-carboxamide (24). Following the
procedure described for 7, 2-(piperidin-1-yl)ethanamine and 6
provided the title compound (40 mg, 39%). MS: m/z 515.8 ([M +
H]+). 1H NMR (400 MHz, DMSO-d6): δ ppm 8.49 (s, 1 H), 8.42 (m,
1 H), 8.22 (s, 1 H), 7.90−7.95 (m, 2 H), 7.82−7.90 (m, 2 H), 7.58 (s,
1 H), 7.24 (br s, 2 H), 3.44 (m, 2 H), 2.67−2.33 (m, 6H), 1.54 (m, 4
H), 1.41 (m, 2 H), 1.13 (s, 9 H). HRMS for C25H33N5O3S2 [M + H]+:
calcd, 516.2098; found, 516.2103.
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-(2-
morpholinoethyl)thieno[3,2-c]pyridine-7-carboxamide (25). Fol-
lowing the procedure described for 7, 2-morpholinoethanamine and
6 provided the title compound (42 mg, 41%). MS: m/z 517.8 ([M +
H]+). 1H NMR (400 MHz, DMSO-d6): δ ppm 8.49 (s, 1 H), 8.38 (t,
J = 5.77 Hz, 1 H), 8.21 (s, 1 H), 7.89−7.95 (m, 2 H), 7.82−7.89 (m,
2 H), 7.57 (s, 1 H), 7.22 (br s, 2 H), 3.53−3.61 (m, 4 H), 3.37−3.47
(m, 2 H), 2.54−2.52 (m, 2H), 2.38−2.46 (m, 4 H), 1.13 (s, 9 H).
HRMS for C24H31N5O4S2 [M + H]+: calcd, 518.1890; found, 518.1896.
4-Amino-2-(4-(N-tert-butylsulfamoyl)phenyl)-N-(3-
morpholinopropyl)thieno[3,2-c]pyridine-7-carboxamide (26). Fol-
lowing the procedure described for 7, 3-morpholinopropan-1-amine
and 6 provided the title compound (32 mg, 30%). MS: m/z 531.8 ([M
+ H]+). 1H NMR (400 MHz, DMSO-d6): δ ppm 8.50 (s, 1 H), 8.45 (t,
J = 5.48 Hz, 1 H), 8.21 (s, 1 H), 7.89−7.94 (m, 2 H), 7.83−7.90 (m, 2
H), 7.57 (s, 1 H), 7.22 (br s, 2 H), 3.58 (m, 4 H), 3.61−3.51 (m, 2 H),
3.34−3.31 (m, 2H), 2.31−2.40 (m, 6 H), 1.71 (m, 2 H), 1.13 (s, 9 H).
HRMS for C25H33N5O3S2 [M + H]+: calcd, 532.2047; found,
532.2049.
TR-FRET Jak Enzyme Assays. The Jak Homology 1 (JH1, catalytic
kinase) domain of each human Jak (Jak1, Jak2, Jak3, and Tyk2) was
expressed in Sf-9 cells as an N-terminal GST fusion. The kinase
activity of the purified recombinant Jaks was assessed using a Lance
TR-FRET assay, which measured the phosphorylation of a Tyk2-based
peptide substrate (Biotin-LC-EQEDEPEGDYFEWLE, Biopeptide,
San Diego, CA). Kinase assays were performed in black 384-well
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assay plates (Costar) using a reaction buffer that contained 5 mM
MgCl2, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), pH 7.5, 4 mM dithiothreitol (DTT), and 0.05% bovine
serum albumin (BSA). The enzyme reactions (40 μL) contained 0.5
μM peptide, apparent Km concentrations of ATP (19 μM for Jak1,
10 μM for Jak2, 1.8 μM for Jak3, and 15 μM for Tyk2), and either 625
pM Jak1, 4 pM Jak2, 4 pM Jak3, or 75 pM Tyk2. Reactions were
incubated for 90 min at RT with compound [in a 22 pt (1:2) titration
series with final concentrations ranging from 62.5 μM to 29.8 pM].
Reactions were then stopped with 40 μL of a detection mix containing
400 pM Lance Eu-PT66 Antibody (Perkin-Elmer), 6.6 μg/mL
streptavidin allophycocyanin (SA-APC) (Perkin-Elmer), 40 mM
ethylenediaminetetraacetic acid (EDTA), 100 mM HEPES, pH 7.5,
100 mM NaCl, 0.1% BSA, and 0.5% Tween 20. Plates were incubated
for 30 min at RT before reading the TR-FRET signal on the RUBYstar
(BMG Labtek, Cary, NC) instrument using an excitation setting of
320 nm and an emission collection at both 615 and 665 nm.
Phosphorylation of the peptide resulted in an increased emission at
665 nm; the ratio of signal generated at 665 nm over 615 nm was used
in calculations. Automation was accomplished with a Mulitdrop 384
bulk liquid dispenser equipped with a 160-plate Titan stacker (Thermo
Electron Corporation, Waltham, MA) for all additions (enzyme,
substrate, and detection mix).
AlphaScreen pSTAT5 Detection Assay. TEL-Jak Ba/F3 cells
were washed 1× with assay buffer (HBSS, 0.1% BSA, and 5 mM
HEPES, pH 7.0) and then resupended to a density of 5 × 106 cells/mL
(7.2 × 106 cells/mL for TEL-Jak2 Ba/F3). Three microliters of cell
suspension was added to a 384-well low volume white-walled
polystyrene Proxiplate (Perkin-Elmer, Downers Grove, IL), which
contained 1 μL of compound [in a 22 pt (1:2) titration series with
final concentrations ranging from 25.0 μM to 11.9 pM] in 2% DMSO
(98% assay buffer) per well. The low control contained a final
concentration of 10 μM pan Jak standard inhibitor (at least 10× the
cellular IC50 of each Jak). Cells were incubated with compound (final
DMSO concentration of 0.5%) at RT for 60 min before proceeding
with pSTAT5 detection. Measurement of pSTAT5 was accomplished
with the SureFire pSTAT5 Assay kit (Perkin-Elmer). Cells were lysed
with 1 μL of 5 × lysis buffer and incubated at RT for 10 min. Next, 4.3
μL of a solution containing reaction buffer, activation buffer, and
acceptor beads prepared as per manufacturer's protocol was added.
Plates were incubated overnight in the dark at RT before adding 1.8
μL of a mixture containing dilution buffer and donor beads also
prepared as per manufacturer's protocol. After a final incubation of 1 h
at RT, TEL-Jak plates were read on the EnVision (Perkin-Elmer) using
the AlphaScreen setting. Automation of cell, lysis, and detection
reagent additions was performed with a FlexDrop (Perkin-Elmer) bulk
liquid dispenser.
conjugated mouse antiphospho-STAT5 (Y694) (BD Biosciences, San
Jose, CA) according to the manufacturer's suggestion and incubated at
37 °C for 30 min. Cells were then washed and resuspended in 150 μL
of wash buffer (0.5% BSA in DPBS). Events were acquired on the
FACS Calibur flow cytometer (BD Biosciences) equipped with a
Cytek Development AMS 96-well plate loader using side and forward
scatter properties. Greater than 10000 events were captured for each
sample.
Cytokine-Stimulated WB Assays. WB assays were performed
similar to PBMC assays with the following exceptions: WB, about 3−4
× 105 cells were preincubated with compounds; final rHuIL-2
concentrated used for WB stimulation was 60 ng/mL; only half of
the cells were subjected to Ab staining after fixation and
permeabilization.
Data Analysis. AlphaScreen and TR-FRET data were analyzed
with the mFit nonlinear regression algorithm and the Screener
(Genedata AG, Basel, Switzerland) data analysis software. Phosflow
IC50 determination was performed using a Levenberg−Marquardt
nonlinear regression algorithm and with Activity base Global fit
software (IDBusiness Solution Limited, Emeryville, CA).
Cell Permeability. Data were obtained from MDR1-LLC-PK1
cells, pig kidney cells expressing human MDR1, at 5 μM in the
presence of 0.1% BSA.19
Plasma Protein Binding. Data were obtained by equilibrium
dialysis.20
ASSOCIATED CONTENT
* Supporting Information
■
S
Permeability data for key compounds; enzyme to cell shift data
for the thienopyridines, compound 20, and a larger set of Jak
kinase inhibitors; and statistical data for key compounds in the
enzyme, TEL-Jak cell, PBMC, and WB assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
PDB ID for the 8-Jak2 complex: 3TJC; PDB ID for the 19-Jak2
complex: 3TJD.
AUTHOR INFORMATION
Corresponding Author
*Tel: 617-444-5244. E-mail: laurie.schenkel@amgen.com.
■
ACKNOWLEDGMENTS
■
We thank Brett Janosky for conducting permeability experi-
ments, Loren Berry for conducting plasma protein binding
experiments, and Liyue Huang and Min-Hwa Jasmine Lin for
scientific input.
Cytokine-Stimulated pSTAT5 PBMC Assays. Peripheral blood
samples were obtained from healthy donors, and PBMC were isolated
via density gradient separation using Ficoll-Paque PREMIUM (GE
Healthcare, Uppsala, Sweden). PBMC (300000−400000 per well) in
36 μL of Dulbecco's phosphate-buffered saline (DPBS) were
dispensed into a 96-well (deep well, v-bottom) polypropylene plate
(Costar, Corning, NY) containing 4 μL of 10 × compound [in a 10 pt
(1:3) titration series with final concentrations ranging from 25.0 μM to
1.27 nM] in 10% DMSO (90% DPBS) per well. Cells were pretreated
with compound for 30 min at 37 °C followed by stimulation with EC90
concentrations of either TPO at 10 ng/mL or IL-2 at 10 ng/mL for a
further 15 min at 37 °C (R&D Systems, Minneapolis, MN). The low
control was defined by vehicle in buffer only (1% DMSO in DPBS).
PBMCs were fixed with 800 μL of Lyse Fix buffer (BD, Franklin
Lakes, NJ) for 15 min at 37 °C. Next, cells were pelleted, resuspended,
and permeabilized with 600 μL of 100% MeOH (JT Baker,
Phillipsburg, NJ) and stored at −40 °C in MeOH for at least 1 h
before staining.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; BSA, bovine serum albumin;
DPBS, Dulbecco's phosphate-buffered saline; DCM, dichloro-
methane; DMF, dimethylformamide; DMSO, dimethylsulfoxide;
DTT, dithiothreitol; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; EDTA, ethylenediaminetetraacetic acid; ESI,
electrospray ionization; ET, essential thrombocythemia;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
HOBt, N-hydroxybenzotriazole; HPLC, high-pressure liquid
chromatography; HRMS, high-resolution mass spectrometry;
HTS, high-throughput screen; IL-2, interleukin-2; Jak, Janus
kinase; MAPK, mitogen-activated protein kinase; MF, primary
myelofibrosis; MPD, myeloproliferative disorder; MPLC,
medium-pressure liquid chromatography; NMR, nuclear
magnetic resonance; PBMC, peripheral blood mononuclear
cells; PI3K, phosphatidylinositol 3-kinase; PV, polycythemia
vera; RT, room temperature; SA-APC, streptavidin allophycocya-
nin; SAR, structure−activity relationship; STAT, signal transducers
For intracellular pSTAT5 staining, permeabilized cells were
centrifuged and resuspended in 200 μL of blocking buffer [1%
mouse serum (Sigma-Aldrich) in Stain buffer (BD)] and incubated for
20 min at RT. After the blocking buffer was removed by centrifugation
followed by aspiration, cells were stained with Alexa Fluor 647
8448
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Journal of Medicinal Chemistry
Article
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and activators of transcription; TEL, translocated ETS
leukemia; TFA, trifluoroacetic acid; Tyk2, tyrosine kinase 2;
TPO, thrombopoietin; WB, whole blood
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