Development of Selective Covalent Janus Kinase 3 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b00710

The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 ? cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00710

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Article
Development of Selective Covalent JAK3 Inhibitors
Li Tan, Koshi Akahane, Randall McNally, Kathleen M.S.E Reyskens, Scott B Ficarro, Suhu Liu, Grit S. Herter-
Sprie, Shohei Koyama, Michael J. Pattison, Katherine M. Labella, Liv Johannessen, Esra A Akbay, Kwok-Kin
Wong, David A. Frank, Jarrod A. Marto, A Thomas Look, Simon Arthur, Michael J. Eck, and Nathanael S Gray
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00710 • Publication Date (Web): 10 Aug 2015
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Development of Selective Covalent Janus Kinase 3 Inhibitors
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Li Tan1,2,†, Koshi Akahane3,†, Randall McNally1,2, Kathleen M. S. E. Reyskens4, Scott B.
Ficarro1,2, Suhu Liu5, Grit S. HerterꢀSprie5, Shohei Koyama5, Michael J. Pattison4, Katherine
Labella5, Liv Johannessen1,2, Esra A. Akbay5, KwokꢀKin Wong5, David A. Frank5, Jarrod A.
Marto1,2, Thomas A. Look3, J. Simon C. Arthur4, Michael J. Eck1,2, Nathanael S. Gray1,2,*
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, MA 02115 (USA); 2Department of Cancer Biology, 3Department of Pediatric Oncology,
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5Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA 02215 (USA);
Division of Cell Signaling and Immunology, College of Life Sciences, University of Dundee,
Dundee DD1 5EH (UK). †These authors contributed equally.
*Correspondence: nathanael_gray@dfci.harvard.edu (NSG)
Abstract
The Janus Kinases (JAKs) and their downstream effectors Signal Transducer and Activator of
Transcription proteins (STATs) form a critical immune cell signaling circuit, which is of
fundamental importance in innate immunity, inflammation and hematopoiesis and dysregulation
is frequently observed in immune disease and cancer. The high degree of structural conservation
of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists
seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs.
Here we report the discovery and optimization of 2,4ꢀsubstituted pyrimidines as covalent JAK3
inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structureꢀ
activityꢀrelationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted
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in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å coꢀ
crystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9
exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors
provide a set of useful tools to pharmacologically interrogate JAK3ꢀdependent biology.
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Key words: JAK3; Covalent kinase inhibitors; Structureꢀbased design; Structureꢀactivity
relationship; Drug discovery
Introduction
The Janus Kinases (JAKs) and their downstream effectors Signal Transducer and
Activator of Transcription proteins (STATs) form a critical immune cell signaling circuit.1 There
are four JAK family members in mammals, JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2),
each of which can bind to distinct cytokines and/or growth factor receptors. As the receptors that
act upstream of JAKs are dimeric or multimeric, more than one JAK molecule is recruited to
each receptor. Ligand binding to the receptor results in a conformation change that brings two
JAK molecules close together allowing them to transꢀphosphorylate and activate each other.
Once active, JAKs catalyze the phosphorylation of tyrosine residues in the receptor allowing the
recruitment of specific STATs which are in turn phosphorylated by the JAK. This promotes
STAT dimerization which induces translocation to the nucleus and activation of gene regulatory
programs.2 JAK/STAT signaling is of fundamental importance in innate immunity, inflammation
and hematopoiesis and dysregulation is frequently observed in immune disease and cancer.3 The
JAKs have been the subject of extensive drug discovery efforts and numerous small molecule
ATPꢀcompetitive inhibitors have been developed including ruxolitinib (1, JAK1/2 inhibitor)4
approved as an antiꢀmyelofibrosis drug, tofacitinib (2, panꢀJAK inhibitor)5 approved for
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rheumatoid arthritis, and several others that are currently undergoing clinical investigation.6 The
high degree of structural conservation of the JAK ATP binding pockets has posed a considerable
challenge to medicinal chemists seeking to develop highly selective inhibitors as
pharmacological probes and as clinical drugs.7
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Among the JAK family JAK3 exhibits predominant expression in the hematopoietic
system in contrast to other JAKs which are broadly expressed in a variety of cell types.2b JAK3 is
required for signaling by cytokines, including interleukin 2 (ILꢀ2), ILꢀ4, ILꢀ7, ILꢀ9, ILꢀ15 and
ILꢀ21, that act via receptors that contain the common gamma chain (γc) cytokine receptor
subunit.8 In addition to the γc subunit, most of these receptors contain a second unique α subunit
that determines cytokine specificity. In these receptors, JAK3 binds to the γc subunit and JAK1
to the α subunit. The ILꢀ2 Receptor (ILꢀ2R) and ILꢀ15R are exceptions to this as they can form
trimeric complexes through the recruitment of α and β chains. In this case the β subunit binds
JAK1; the α subunit, while involved in cytokine binding, does not directly bind to a JAK.9
Signaling via the γc receptors is essential for the normal development and function of the
immune system. Lossꢀofꢀfunction mutations in either γc or JAK3 in humans results in severe
combined immunodeficiency caused by defective development of Tꢀ and NK cells. The role for
JAK3 in T and natural killer (NK) cell development was subsequently confirmed by deletion of
Jak3 in mice; JAK3 knockouts also lacked normal numbers of B cells indicating a more
important role for JAK3 in B cell development in mice than in humans.2b, 10 On the basis of
JAK3’s essential function in immune signaling and its restricted expression in hematopoietic
tissues, JAK3 has been pursued as a target for the treatment of autoimmune and inflammatory
diseases. JAK3 has also been explored as a potential antiꢀcancer target due to aberrant activation
in several lymphoproliferative disorders;11 for example, mutations in JAK3 have been found in
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leukemias such as Tꢀcell acute lymphoblastic leukemia (TꢀALL) and Tꢀcell prolymphocytic
leukemia (TꢀPLL).12 The A572V activating mutation in the kinase domain of JAK3 was
discovered in acute megakaryoblastic leukemia and natural killer/Tꢀcell lymphoma, and has been
demonstrated to be capable of forming tumors in mice.13 Recently JAK3 mutations were also
found to promote programmed death ligand 1 (PDꢀL1) induction in lung cancer cells an in the
tumor immune microenvironments. 14
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The most obvious strategy to develop smallꢀmolecule JAK3 inhibitors is to target the
catalytic ATPꢀbinding site of the JAK3 kinase domain (JH1). Numerous ATPꢀcompetitive JAK3
inhibitors have been developed (Figure 1A).15 Tofacitinib (2) was developed as a specific JAK3
inhibitor to prevent organꢀtransplant rejection but subsequent studies revealed that it is also a
potent inhibitor of JAK1 and JAK2.5, 16 The combination of compound 2’s excellent potency,
selectivity and pharmacological properties have made it a favored inhibitor to interrogate JAK
kinase activity in numerous biological models.13b, 17 In our efforts to develop a more selective
Jak3 inhibitor, we noted that among JAK family members JAK3 is unique in having a cysteine
residue at the gatekeeperꢀplusꢀ7 (GK+7) position.18 This residue is Cys909 in human JAK, and it
is structurally equivalent to cysteine residues in epidermal growth factor receptor (EGFR) and
Bruton’s tyrosine kinase (BTK) that have been successfully targeted by covalent kinase
inhibitors that are now approved drugs. Afatinib19 (6) targets Cys797 in EGFR and ibrutinib20 (7)
targets Cys481 in BTK. We and others have also developed mutantꢀselective inhibitors of the
drugꢀresistant EGFR T790M that react covalently with Cys797. WZ400221 (8) is a pyrimidineꢀ
based inhibitor with an acrylamide “warhead”22 that potently inhibits EGFR T790M. Recently
three other groups have also reported the development of covalent JAK3 inhibitors. In 2013
Winssinger et al. reported a covalent probe which selectively labeled JAK3, EGFR and
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ERBB2.23 Taunton et.al reported the development of ‘reversibleꢀcovalent’ JAK3 inhibitors
incorporating a cyanoacrylamide warhead which inhibited JAK3 in a biochemical kinase assay
with an IC50 of 100 nM while sparing other JAKs up to a concentration of 10 µM.24 Goedken
et.al reported several tricyclic covalent JAK3 inhibitors with optimized inhibitors exhibiting
biochemical IC50’s of 7 nM, good kinome selectivity and confirmed covalent binding to Cys909
using kinetic and massꢀspectrometry studies.25 Here we report a medicinal campaign to derive
potent disubstituted pyrimidineꢀbased inhibitors that exploit an acrylamide electrophile to form a
covalent bond to Cys909. Optimization was guided by cellular assays using Translocation ETS
Leukemia protein (Tel)ꢀJAK26 fusion kinase transformed Ba/F3 cells that provided an efficient
and reliable measure of the ability of compounds to inhibit JAK signaling in cellular context.
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Results
Identification of Compound 9 as a Potent JAK3 Inhibitor.
Amongst the 11 kinases that
possess a cysteine at the GK+7 position only JAK3 and mitogenꢀactivated protein kinase kinase
7 (MAP2K7) have a methionine gatekeeper, which is also present in the drugꢀresistant
EGFRT790M that is targeted by the pyrimidineꢀbased inhibitor WZ4002 (8). Compound 8 is a
much less potent inhibitor of cellular JAK3 kinase activity (JAK3 Kd = 150 nM, TELꢀJAK3
Ba/F3 IC50 = 2.82 µM) compared to T790M EGFR (EGFRL858R/T790M Ba/F3 IC50 = 8 nM).21
Through the syntheses of a small collection of analogs of 8 we discovered that relatively subtle
changes could result in a dramatic improvement in JAK3 inhibitory potency relative to
EGFRT790M. For example, 9 where the ether linkage of 8 is replaced with an aminomethylene (ꢀ
NHCH2ꢀ) linkage resulted in a great improvement in JAK3 inhibition in a fixed timeꢀpoint Z’ꢀ
lyte enzymatic assay (Life Technology, SelectScreen, IC50 = 4.8 nM) (Scheme 1A).
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To broadly assess its kinase selectivity, 9 was profiled against a diverse panel of 456
kinases (DiscoveRX, KinomeScan27) using an in vitro ATPꢀsite competition binding assay at a
concentration of 1.0 ꢁM. Compound 9 exhibited good overall kinase selectivity with an S(5)
selectivity score, defined as the percentage of kinases with scores less than 5 (S(5))27b, of 0.02.
The results suggested that 9 most potently inhibits JAK3 and identified fmsꢀrelated tyrosine
kinase 3 (FLT3) and several tyrosine protein kinase (TEC)ꢀfamily kinases as being potential offꢀ
targets (Figure 2). Enzymatic assays using the Z’ꢀlyte or LanthaScreen28 formats confirmed
enzymatic inhibition of FLT3 (IC50 = 13 nM), TTK protein kinase (TTK, IC50 = 49 nM), BLK
protoꢀoncogene (BLK, IC50 = 157 nM) and tyrosine protein kinase TXK (TXK, IC50 = 36 nM).
Compound 9 showed very low inhibition scores for other JAKs and wildꢀtype (WT) EGFR,
which is consistent with the over 180ꢀfold higher IC50s against JAK1, JAK2, TYK2 and
EGFRWT (IC50s = 896, 1050, > 10000 and 409 nM respectively). As BTK and ITK have
important functions in Bꢀcell and Tꢀcell signaling pathways, we confirmed that compound 9
possesses over 165ꢀfold higher IC50s for BTK or ITK (IC50s = 794 and 1070 nM respectively)
(Table S1).
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As enzymatic potencies sometimes do not translate into cellular inhibition, the ability of 9
to inhibit the proliferation of kinaseꢀtransformed Ba/F3 cells was evaluated. Ba/F3 cells are a
murine preꢀB cell that can readily be transformed with activated kinases to allow for growth in
the absence of ILꢀ3, and are frequently used to evaluate the activity of compounds against
kinases of interest in a cellular context.29 We utilized JAK1, JAK2 and JAK3 dependent Ba/F3
cell lines, where the JH1 domain of each JAK is fused with the oligomerization domain of TEL
which results in constitutive tyrosine kinase activity and confers ILꢀ3 independent proliferation.26
In addition, we engineered a TYK2 Ba/F3 cell line whose proliferation is driven by
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constitutively activated TYK2 (TYK2E957D).30 As a further control, we also used a Ba/F3 cell line
transformed by TELꢀ Abelson murine leukemia viral oncogene homolog (ABL). To enable a
direct comparison with the commonly used JAK inhibitors we profiled reported compounds 1ꢀ
5,4ꢀ5, 15b, 15g, 31 and 1232 against this panel of Ba/F3 cells. Compound 1 exhibited the most potent
inhibition of JAK1 and JAK2 Ba/F3 cells, 2 exhibited most potent inhibition of JAK3 Ba/F3
cells and 3 exhibited most selective inhibition of JAK3 (Table S2). Overall the potency and
selectivity of these inhibitors are consistent with their reported properties. Consistent with the
biochemical assays, 9 selectively inhibited the proliferation of JAK3ꢀdependent Ba/F3 cells (IC50
= 69 nM) relative to other JAKꢀdependent Ba/F3 cells, for which there was no antiproliferative
effect at concentrations below 3.0 µM (Table 2). The general antiproliferative activity that
appears at concentrations of approximately 3.0 µM could be due to inhibition of other kinases
such as TTK (aka Mps1, IC50 = 49 nM) as inhibition of this kinase has been reported to decrease
cancer cell viability.33
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Co-crystal Structure of JAK3-Compound 9 Complex. To investigate the structural basis for
achieving selectivity for JAK3 we solved the coꢀcrystal structure of the JAK3 kinase domain in
complex with 9 at a resolution of 2.9 Å (Figure 3). In this structure (PDB 4Z16), the
anilinopyrimidine moiety of 9 makes the expected bidentate hinge hydrogen bonds with Leu905,
and continuous electron density is observed between the acrylamide warhead and Cys909,
indicative of covalent bond formation. The kinase exhibits an active conformation, with the
DFGꢀmotif in the inward position and both tyrosines 980 and 981 in the activation loop
phosphorylated. The orientation and interactions of the anilinopyrimidine portion of the
compound closely resembles the interactions observed in the EGFRT790Mꢀ8 coꢀcrystal structure
(PDB 3IKA)21 (Figure S1). In addition to the hinge hydrogen bonds, the chlorine is directed
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towards the gatekeeper methionine (Met902 in the present structure) and the methoxy substituent
extends toward the sidechain of Tyr904 in the hinge region. Interestingly, the “linker” segment
that attaches the acrylamide adopts a distinctly different conformation as compared with the
corresponding region of 8 in complex with EGFRT790M. The phenyl group in the linker pivots
down and is in van der Waals contact with Leu956 in the floor of the ATPꢀbinding cleft. This
orientation is likely enabled by the longer aminomethylene moiety in 9, as compared with the
ether linkage in compound 8. In addition, the acrylamide amide is positioned to hydrogen bond
with the carbonyl of Arg953, also in the floor of the binding pocket.
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Structure-Activity Relationship (SAR) Studies Based on Compound 9. The syntheses of 9
was readily achieved with highꢀoverallꢀyield in four steps starting from 2,4,5ꢀtrichloropyrimidine.
The 4ꢀchloride group was substituted with a 3ꢀnitrobenzyl amine under basic conditions,
followed by the substitution of the 2ꢀchloride with a 2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀ
yl)aniline under acidic conditions to give 11. The nitro group of 11 was reduced using
hydrogenation and the resulted aniline was acrylated to afford 9 (Scheme 1B). In order to
elucidate the structural requirements to achieve potent and selective inhibition of JAK3 we
prepared approximately 70 analogs. To approach this optimization in a systematic fashion, this
chemotype was divided into three moieties: tail (R1), arm (R2) and core (R3), and each of these
moieties were varied sequentially. The 2,4ꢀdisubstited aniline tail moiety of 9 was replaced with
different anilines, aliphatic amines or 4ꢀaminopyrazoles; the 4ꢀacrylamidobenzyl arm of 9 was
substituted with functional groups at the double bond, phenyl ring or benzyl position; the
pyrimidine core of 9 was modified at 5ꢀ or 6ꢀposition, or was replaced with a variety of bicyclic
cores. All the analogs were profiled with the same JAK3 enzymatic assay and JAKs and ABL
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dependent Ba/F3 cell line assays. Additionally, the in vitro metabolic stability of some analogs
was measured using mouse liver microsomes (MLM) to calculate halfꢀlives (T1/2).34
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The first series of analogs focused on modification of the tail moiety (R1) (Table 2).
Substitutions at the 5ꢀpostion of the aniline (14, 15) resulted in a dramatic loss of potency. In
contrast, removing the 2ꢀmethoxy group (16) enhanced potency over 5ꢀfold against JAK3,
however, it also increased the cytotoxicity toward other transformed Ba/F3 cell lines.
Substitutions at the 3ꢀposition did not mitigate the cytotoxicity (17ꢀ19), nor did introduction of a
fluorine at the 2ꢀposition (20). Compound 21 with a 4ꢀmorpholinylaniline tail potently inhibited
JAK3 despite not having the 2ꢀmethoxy substitution. Furthermore, 21 showed comparable
cellular selectivity and MLM T1/2 as 9 (T1/2 of 4.7 min.). However, 21 only showed moderate
selectivity in biochemical assays, with 10~20ꢀfold higher IC50s against JAK1, JAK2 or TTK
(Table 1). Adding a 2ꢀ or 3ꢀfluoro (22, 23) did not improve the kinase selectivity of 21 and also
did not improve the MLM stability. 24 which contains two fluorine substituents at the 2ꢀ and 5ꢀ
positions showed good selectivity but decreased potency against JAK3. 25, with 3,5ꢀdifluorine
substitutions, exhibited improved potency compared to 24 with good selectivity in cells, even
though it showed moderate selectivity in enzymatic assays. Based on 25 several analogs (26ꢀ28)
were made and exhibited increased potency, but none of them were as selective as 25. Aliphatic
tails resulted in complete loss of potency (29ꢀ31). 32 with a 1ꢀmethylꢀ1Hꢀpyrazolꢀ3ꢀamine tail
showed increased potency, similar selectivity and MLM stability compared with 9; it also
showed a 300ꢀfold selectivity window over TTK. Addition of a 4ꢀmethyl group to the pyrazole
ring (33) enhanced the MLM stability but decreased the potency over 10ꢀfold. The isoxazolꢀ4ꢀ
amine tail in 34 is disfavored in terms of both potency and stability. Modifications were then
focused on the 1ꢀmethyl group of the pyrazole tail. Replacement of the 1ꢀmethyl with a 1ꢀ
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difluoromethyl (35) or a 1ꢀdifluoroethyl (36) maintained the potency and enhanced the MLM
stability. However, bulky and hydrophobic substituents resulted in decreased MLM stability (37,
38). 39 with a 2ꢀsubstituted Nꢀmethylacetamide exhibited noticeably improved MLM stability
(T1/2 of 14.3 min) and favorable selectivity albeit with only moderate potency against JAK3.
Nevertheless, other 2ꢀsubstituted Nꢀalkylacetamide groups (40ꢀ42) failed to improve the potency
or stability. Several analogs elaborated with a 2ꢀsubstituted ethyl group (43ꢀ45) showed good
potency but decreased MLM stability. To our surprise, 45 with a 2ꢀmethoxyethyl exhibited an
IC50 of 19 nM against JAK3 Ba/F3 cells, and over 390ꢀfold selectivity over other Ba/F3 cell lines.
45 showed excellent selectivity with JAK3 IC50 of less than 0.5 nM in fixed end point enzymatic
assay, and over 70ꢀfold higher IC50 against JAK1 and over 100ꢀfold higher IC50s against JAK2,
TYK2, TTK, BTK or ITK (Table 1, S1). 45 was profiled against a diverse panel of 468 kinases
(DiscoveRX, KinomeScan27) at a concentration of 100 nM; the compound exhibited excellent
overall kinase selectivity with selectivity scores, defined as the percentage of kinases with scores
less than 5 (S(5))27b, of 0.01 (Figure 4). The results suggested 45 inhibits JAK3 most potently,
and aurora A (AURKA) was likely to be the only offꢀtarget which we confirmed to be inhibited
by 45 with IC50 of 43 nM. 45 showed relatively poor MLM stability with T1/2 of 3.3 min. Thus,
several analogs with different 2ꢀalkoxyethyl groups (46ꢀ48) were made to solve this problem. To
our disappointment, all changes did not improve the MLM stability. Dimethyl substitution of the
2ꢀmethoxyethyl (49) is disfavored with respect to MLM stability as well as potency and
selectivity. 50, with a hydroxyl group in the tail, showed 3ꢀfold longer MLM T1/2 compared with
45. Although 50 was not as selective as 45 in Ba/F3 cell lines, it was more potent than 9 with
comparable selectivity in Ba/F3 cells. Further modification of 50 failed to improve the selectivity
but led to decreased potency and MLM stability (51). In this series of analogs, 45 and 50 stood
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out with good selectivity or MLM stability, furthermore, we found 50 was actually one of the
MLM metabolites of 45 (Figure S2).
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After optimization of the tail moiety, the SAR of the arm (R2) was investigated (Table 3).
Replacing the acrylamide with a propionamide (52) resulted in over an 80ꢀfold decrease of
potency against JAK3. Installation of a methyl group at αꢀposition of the acrylamide (53)
resulted in significant loss of potency and a shorter MLM T1/2. Polar substitutions at the βꢀ
position of the acrylamide (54ꢀ56) also resulted in loss of potency. Addition of a fluorine ortho to
the acrylamide in the phenyl ring (57) also decrease the potency dramatically. Switching the
acrylamide from metaꢀ to paraꢀ on the phenyl ring decreased the potency considerably in
combination with the aniline tail of 9 (58), but did not affect the potency when used in
conjunction with other tails (59ꢀ62). This change did however decrease the selectivity in some
cases (59, 62). Analogs with a paraꢀacrylamide generally showed comparable or diminished
MLM stability. Finally, adding a methyl at the benzyl position (63, 64) reduced both potency and
selectivity. In summary, the modifications of R2 failed to further improve our JAK3 inhibitors.
Several reported inhibitors with similar acrylamide warheads (6, 7, 8 and AZD929135 (65)) were
also profiled and compared, none of them showed good inhibition against JAK3 Ba/F3 cells with
IC50s over 1.0 µM (Table S2).
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Simultaneously we investigated the SAR of the core moiety (R3) (Table 4). We found
that a thioether linkage at the 4ꢀpostion (66, 67) slightly increased the MLM stability but
decreased the potency, and a tertiary amine linkage (ꢀNMeꢀ) decreased the potency even more
dramatically (68). Next we investigated the effects of various substituents at the 5ꢀposition. We
evaluated substituents that possessed electron withdrawing groups such as chlorine (69ꢀ72),
electron donating groups (73ꢀ75) or simply a hydrogen atom (76). However, only 70 with a 5ꢀ
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bromo group showed good potency among these analogs. A 4,6ꢀdisubstituted pyrimidine core
(77) also led to substantial loss of potency. Next we tried to cyclize the 4,5ꢀsubstituents into a
fused 5ꢀmembered or 6ꢀmembered ring (78ꢀ82), and found that only 78 with a pyrrolo[2,3ꢀ
d]pyrimidine core showed good potency against JAK3, but with a slightly decreased MLM
stability. 81 with a pyrimido[4,5ꢀd]pyrimidinone core possessed a good IC50 against JAK3 in the
biochemical assay, but mediocre potency in the TELꢀJAK3ꢀtransformed Ba/F3 cells. In the end,
modifications at the 6ꢀposition of the pyrimidine core (83, 84) were fairly disfavored. These
results suggested that 5ꢀchlorine is the most suitable, and the secondary amine (ꢀNHꢀ) is a
satisfactory linkage at 4ꢀpostion; some bicyclic cores did improve the inhibitor modestly, and
modifications at 6ꢀposition are not tolerated.
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Validation of the Target-Engagement and the Covalent Binding. Based on the SAR results
above, we selected 9, 45 and 50 as our optimal JAK3 inhibitors, and investigated their inhibitory
activity on JAK3ꢀdependent signaling. As STAT5 has been reported as a direct substrate of TELꢀ
JAK3 in Ba/F3 cells,26 we examined the effect of our JAK3 inhibitors on STAT5
phosphorylation in this context. As we expected, STAT5 was constitutively phosphorylated in
the cells treated with DMSO (Figure S3). Compound 2 at 300 nM almost fully inhibited the
phosphorylation of STAT5 (pꢀSTAT5), while 9 at 300 nM completely abolished pꢀSTAT5, and
so did 45 or 50 at 100 nM. These results are consistent with the IC50s in JAKꢀtransformed Ba/F3
proliferation assays. In order to confirm that 45 and 50 are bonafide covalent inhibitors in
analogy to compound 9 we performed cellular ‘washꢀout’ experiments. JAK3 Ba/F3 cells were
treated with these four inhibitors at various concentrations for 3 h, the cells were washed
extensively with PBS and then allowed to recover for 4 h. Western blots of the cellular lysates
revealed that 9, 45 or 50 were capable of sustained inhibition of pꢀSTAT5 after the washout,
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whereas the reversible inhibitor 2 was not (Figure 5A). To monitor the degree of JAK3 ‘target
engagement’, a biotinylated version of 45 (85) was designed with a biotin tethered via a flexible
PEG linker at the tip of 45’s tail moiety (Figure 5C). We confirmed that 85 maintained similar
biochemical potency and selectivity for JAK3 (Table 1). 85 showed selective but weak potency
against JAK3 Ba/F3 cells with an IC50 of 1.3 µM (Table S2) presumably due to poor membrane
permeability. We demonstrated that streptavidin mediated pulldown of 85 in cell lysates allowed
for efficient recovery of JAK3 as assessed by western blotting. Consistent with the washꢀout
results, 85 strongly labeled JAK3 when cells were treated with the reversible inhibitor 2 but not
when cells were treated with acrylamide modified inhibitors 9, 45 or 50 (Figure 5B). Compounds
9, 45 and 50 were further demonstrated to be covalent inhibitors using electrospray ionization
mass spectrometry, where incubation of recombinant JAK3 kinase domain with inhibitor resulted
in addition of the expected molecular weight (Figure 6). Subsequent protease digestion and
LC/MS2 experiments identified a single modified peptide LVMEYLPSGC*LR (C*, cysteine
labeled by 9, 45 or 50, JAK3 residues 900ꢀ911) and localized the site of modification to Cys909
(Figure S4), in agreement with the JAK3/9 coꢀcrystal structure. Cumulatively these results
provide strong evidence that 9, 45 and 50 are all irreversible, covalent inhibitors and that Cys909
of JAK3 is the only labeled site.
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Validation in Cells with Endogenous JAK3. Compounds 9, 45 and 50 were further evaluated
for their ability to inhibit JAK3 kinase activity in a variety of other cell types. We evaluated the
ability of 9, 45 or 50 to inhibit signaling following stimulation of primary murine bone marrow
derived macrophage (BMDMs) with a panel of 4 different cytokines based on their requirement
for different cytokines (Figure 7). Among these cytokines, only signaling via ILꢀ4, whose
receptor binds JAK1 and JAK3,36 is dependent on JAK3. In contrast, granulocyteꢀmacrophage
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colonyꢀstimulating factor (GMꢀCSF), which signals via JAK2, and ILꢀ10 and interferon beta
(IFNβ), which signal via JAK1 and TYK2, are JAK3 independent.37 9, 45 or 50 completely
inhibited ILꢀ4 induced pꢀSTAT6 at a concentration of 500 nM and only partially inhibited IFNβꢀ
induced pꢀSTAT1 at a concentration of 5.0 µM. None of our inhibitors detectably reduced the pꢀ
STAT5 level stimulated by GMꢀCSF or pꢀSTAT3 induced by ILꢀ10 at a concentration of 5.0 µM.
While demonstrating excellent selectivity in Ba/F3 cellular assays, these inhibitors showed
decreased but still decent selectivity in primary cells. The washout experiments with 45 or 50 in
BMDMs cells exhibited consistent results as in TELꢀJAK3 Ba/F3 cells, and suggested that the
inhibitory effect lasted for at least 24 h after the washout (Figure S5). In a similar way, 9, 45 and
50 exhibited consistent potency and selectivity for JAK3 in leukemia cancer cell lines: TꢀALL1
and OCLꢀAML5 (Figure 8). In TꢀALL1 cells, ILꢀ2 induced STAT5 phosphorylation was
completely inhibited by 9 at a concentration of 1.0 µM or by 45 or 50 at a concentration of 100
nM (Figure 8A). However, in OCLꢀAML5 cells the GMꢀCSF induced STAT5 phosphorylation
was maintained in the presence of the inhibitors up to a concentration of 10 µM (Figure 8B). In
contrast, previously reported panꢀJAK inhibitors 1 and 2 could abolish pꢀSTAT5 in both
stimulated cell lines at 1.0 µM. Based on these results compounds 9, 45 and 50 are capable of
efficiently inhibiting JAK3 and specifically blocking the JAK3ꢀdependent signaling pathway in
human cells at subꢀmicromolar concentrations.
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Mouse Pharmacokinetic Properties and In Vivo Studies. We evaluated the pharmacokinetic
properties of 9 in mice following intravenous and oral delivery. Compound 9 demonstrated
reasonable pharmacokinetic properties, with moderate T1/2 of 1.4 h, area under the curve (AUC)
value of 795 ng*hr/mL following a 10 mg/Kg oral dose and good oral bioavailability of 66%
(Table 5). This suggests that 9 may be a suitable probe for future murine efficacy studies. We
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evaluated the ability of compound 9 to alter immune cell numbers in genetically engineered
models (GEM) of lung adenocarcinoma in mice. Immune cell numbers in mice harboring tumors
driven by KrasG12D;Lkb1L/L–38 were assessed in tumorꢀassociated (lung) as well as general
immune cell populations (spleen) upon treatment with compound 9. After oral administration
with 9 (75 mpk, QD) for 8 days, the numbers of B or T lymphocytes in the tumorꢀbearing lungs
and spleens of treated mice was not affected, however, the number of NK cells was reduced
(Figure S6). Similarly, the number of tumorꢀassociated macrophages and neutrophils in the lung
of treated mice were not affected. During the short duration of this study, we did not detect any
effect on the tumor growth.
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Discussion
Building on our covalent T790M EGFR inhibitor 8 we designed and synthesized new
analogs aiming to target the analogous cysteine residue in JAK3. Among those analogs
compound 9 was identified as a potent and selective JAK3 inhibitor compared with current
reversible JAK inhibitors. We determined a 2.9 Å coꢀcrystal structure of JAK3 with 9, which
revealed that 9 covalently binds to Cys909 of JAK3 as expected. With 9 as a lead, we designed
and synthesized dozens of analogs to intensively study the SAR with regard to each moiety of
this scaffold. These analogs were tested in enzymatic assays for their potency against JAK3 and
profiled against a panel of transformed Ba/F3 cell lines, for their potency and selectivity in a
cellular context. Based on these studies we discovered that a 2ꢀmethoxy group in the aniline tail
of 9 is disfavored by JAK3 presumably due to a disfavored interaction with the bulky side chain
of Tyr904 located at the GK+2 position. Hydrophobic or less polar tails are better for selectivity
for JAK3 presumably because they have weaker nonꢀcovalent affinity for the kinase thereby
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allowing covalent bond formation to be a more important contributor to the potency. The
acrylamidobenzyl arm moiety of 9 is significant for the potency against JAK3 and sensitive to
changes, its specific length and flexibility are critical to enable the inhibitors to efficiently form a
covalent bond with Cys909 of JAK3. The Hꢀbond between the acrylamide of 9 and the carbonyl
of Arg953 fixed the conformation of the acrylamide and left no space to accommodate
substituents. The chlorine atom in the pyrimidine core is responsible for both potency and
selectivity with its interaction with Met902 of JAK3. Modifications at the 6ꢀposition of the
pyrimidine core were fairly disfavored as they collided with the carbonyl of Glu903 3.3Å away.
Some of these analogs were also evaluated for their MLM stability, the results indicated that
more lipophilic analogs with aliphatic nonꢀpolar substituents tend to have a shorter halfꢀlife,
maybe resulting from their enhanced binding with phospholipid in microsomes.39
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Among these analogs 9, 45 and 50 stood out with overall favorable potency, selectivity
and MLM stability. They all exhibited efficient and selective inhibition of JAKꢀdependent
signaling within different contexts. So far 45 is our most selective JAK3 inhibitor with at least
70ꢀfold selectivity over other JAKs in biochemically assays, at least 390ꢀfold selectivity in JAKꢀ
transformed Ba/F3 cells, and decent selectivity in primary cells. In addition to the evidence
provided by JAK3ꢀ9 coꢀcrystal structure, all three inhibitors were demonstrated to covalently
modify Cys909 of JAK3 based upon washꢀout and pullꢀdown experiments and mass
spectrometry. We developed a biotinylated probe 85 which was demonstrated to be a useful
reagent for establishing “targetꢀengagement” in cellular assays. We also found that a shortꢀterm
treatment with high dose of compound 9 in mice with lung tumors resulted in a decrease in the
number of NK cells suggesting that the compounds are active in vivo. Further investigations will
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be required to elucidate the basis for this phenomena and to assess the activity of covalent JAK3
inhibitors in relevant disease models.
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In conclusion, the covalent JAK3 inhibitors 9, 45, 50, and the biotinylated probe 85
provide a set of useful tools to pharmacologically interrogate JAK3ꢀdependent biology.
Compound 45 is a highly potent and selective biochemical and cellular inhibitor of JAK3 and is
an ideal reagent for cell biological studies. Compound 9 combines favorable target profile with
good pharmacokinetic properties and is ready to be used in future animal studies. Finally, the
structureꢀactivity relationships with respect to JAK3 combined with the coꢀcrystal structure will
serve as an excellent foundation for future JAK3 inhibitor development efforts.
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Experimental Section.
Chemistry. Unless otherwise noted, reagents and solvents were obtained from commercial
1
suppliers and were used without further purification. H NMR spectra were recorded on 600
MHz (Varian AS600), and chemical shifts are reported in parts per million (ppm, δ) downfield
from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are
described as s (singlet), br (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
Mass spectra were obtained on a Waters Micromass ZQ instrument. Preparative HPLC was
performed on a Waters Sunfire C18 column (19 x 50 mm, 5µM) using a gradient of 15ꢀ95%
methanol in water containing 0.05% trifluoroacetic acid (TFA) over 22 min (28 min run time) at
a flow rate of 20 mL/min. Purities of assayed compounds were in all cases greater than 95%, as
determined by reverseꢀphase HPLC analysis.
2,5-Dichloro-N-(3-nitrobenzyl)pyrimidin-4-amine (10). 2,4,5ꢀTrichloropyrimidine (112 µL,
1.0 mmol), (3ꢀnitrophenyl)methanamine hydrochloride salt (227 mg, 1.2 mmol), and N,Nꢀ
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diisopropylethylamine (DIEA, 530 µL, 3.0 mmol) were combined in dioxane (5 mL) and stirred
overnight. The mixture was then diluted with ethyl acetate and washed with water and brine,
dried over Na2SO4, filtered and concentrated. The crude product was purified by column
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chromatography (hexane:ethyl acetate = 1:1) to yield 270 mg (80%) of 10 as a white solid. H
NMR (600 MHz, CDCl3) δ 8.21 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.10 (s, 1H), 7.71 (d, J = 7.8
Hz, 1H), 7.56 (dd, J = 7.8, 7.8 Hz, 1H), 5.94 (br, 1H), 4.84 (d, J = 6.0 Hz, 1H). MS (ESI) m/z
299 (M+H)+.
5-Chloro-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(3-nitrobenzyl)pyrimidine-
2,4-diamine (11). To 10 (150 mg, 0.5 mmol) and 2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)aniline
(166 mg, 0.75 mmol) in secꢀbutanol (5 mL) was added trifluoroacetic acid (57 µL, 0.75 mmol)
and the mixture was stirred overnight at 100 °C. The mixture was then concentrated, neutralized
with ammonia in methanol and purified by column chromatography (dichloromethane:methanol
= 10:1) to yield 184 mg (76%) of 11 as a paleꢀyellow solid. 1H NMR (600 MHz, CD3OD) δ 8.23
(s, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.84 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H),
7.54 (dd, J = 7.8, 7.8 Hz, 1H), 6.60 (d, J = 3.0 Hz, 1H), 6.36 (dd, J = 9.0, 3.0 Hz, 1H), 4.75 (s,
1H), 3.83 (s, 3H), 3.15 (m, 4H), 2.64 (m, 4H), 2.36 (s, 3H). MS (ESI) m/z 484 (M+H)+.
N-(3-(((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-
yl)amino)methyl)phenyl)acrylamide (9). To 11 (97 mg, 0.2 mmol) in MeOH (20 mL) was
added 1 mL Raney nickel suspension in MeOH. The reaction mixture was stirred for 3 h under 1
atm of hydrogen. The mixture was then filtered with Celite, and the filtrate was concentrated and
dried under vacuum to give a crude product as a white solid. To the obtained white solid in DMF
(2 mL) was added DIEA (53 µL, 0.3 mmol), the stirred mixture was then cooled to ꢀ60 °C, and
acryloyl chloride (17.8 µL, 0.22 mmol) was added dropwise. The reaction mixture was stirred at
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ꢀ60 °C for 10 min, allowed to recover to RT (room temperature) gradually in 30 min, and
purified by reverse phase HPLC to give 88 mg (TFA salt, 71% for 2 steps) of 9 as a white solid.
1H NMR (600 MHz, DMSOꢀd6) δ 10.07 (s, 1H), 7.82 (s, 1H), 7.73 (m, 1H), 7.59 (s, 1H), 7.52 (d,
J = 8.4 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.29 (s, 1H), 7.18 (dd, J = 7.8, 7.8 Hz, 1H), 6.90 (d, J =
7.2 Hz, 1H), 6.48 (s, 1H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.31 (d, J = 8.4 Hz, 1H), 6.25 (d, J =
16.8 Hz, 1H), 5.75 (d, J = 10.2 Hz, 1H), 4.56 (d, J = 6.0 Hz, 2H), 3.78 (s, 3H), 3.04 (m, 4H),
3.01 (m, 4H), 2.22 (s, 3H). MS (ESI) m/z 508 (M+H)+.
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Compound 14ꢀ64, 66ꢀ85 were synthesized with same procedures as 9, 3 and 8 were synthesized
as reported.19, 31 Compound 1, 2, 6, 7, 12, 13 and 65 were from Selleckchem, 4 was from Santa
Cruz, and 5 was from Millipore.
Enzymatic assays. The enzymatic activities against JAK1, JAK2, JAK3, TYK2, FLT3, BLK,
TXK, BTK, ITK and AURKA were tested in Z’ꢀLyte assays with ATP concentrations of Km for
each kinases, the activities against TTK were tested in LanthaScreen binding assays. All the
protocols are available from Life Technologies.40
Protein expression and purification. Human JAK3 kinase domain (residues 811ꢀ1124) was
expressed and purified as described previously41 except that JAK3 was coꢀexpressed with human
cꢀSRC (residues 86ꢀ536) to achieve more consistent phosphorylation of JAK3.
Crystallization and structure determination. Crystals of JAK3 in complex with 9 were
prepared using the hanging drop vapor diffusion method; JAK3 at a concentration of 5 mg/mL
was incubated for 30 min with 500 ꢁM compound 9, then added to an equal volume of well
solution (0.1 M bisꢀtris pH 6.5, 0.2 M ammonium sulfate, 16% PEG 3350, 5 mM TCEP) and
equilibrated over well solution at 4 °C. Crystals were looped into cryoprotectant solution (0.1 M
bisꢀtris pH 6.5, 0.2 M ammonium sulfate, 20 % PEG 3350, 15% glycerol, 5 mM TCEP) for 30ꢀ
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60 sec, then flashꢀfrozen in liquid nitrogen. Xꢀray diffraction data were collected at APS
beamline 24ꢀIDꢀE (NEꢀCAT), processed with XDS,42 and scaled with Scala.43 The structure was
solved by molecular replacement with Phaser44 using the JAK3 kinase domain as a search model
(PDB 1YVJ).41 Manual refitting of the crystallographic model was performed with Coot45 and
refinement was performed with Phenix46 and BUSTER.47 Topology and parameter files for
compound 9 were generated with PRODRG.48
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Mass spectrometry. JAK3 protein (5 µg) was labeled with a 10ꢀfold excess of inhibitor or
DMSO for 30 minutes at 4 ºC. After labeling, proteins were desalted using 0.5 mL Zeba spin
desalting columns (Thermo Fisher Scientific). Analysis of intact protein was performed
essentially as described.49 Briefly, ~5 µg protein was injected onto a selfꢀpacked reversedꢀphase
column (500 µm inner diameter, 5 cm of POROS 50R2 resin). After washing to remove salts,
protein was eluted with an HPLC gradient (0%–100% B in 1 min, A = 0.2 M acetic acid in water,
B = 0.2 M acetic acid in acetonitrile, flow rate = 10 µL/min) into a linear ion trap mass
spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA). Data were acquired in profile
mode scanning m/z 300ꢀ2000. Mass spectra were deconvoluted using MagTran software (version
1.03b2).50 Sites of covalent modification were identified using a “bottomꢀup” strategy. Desalted
proteins were reduced with tris(2ꢀcarboxyethyl)phosphine (10 mM, 10 min room temperature),
alkylated with iodoacetamide (20 mM, 30 min room temperature in dark), and digested with
trypsin for 4 h at 37 ºC. Digests were analyzed by nanoLCꢀESIꢀMS as described with
modifications.51 Peptides were loaded onto a selfꢀpacked preꢀcolumn (4 cm POROS10R2),
resolved on an analytical column (30 µm I.D., packed with 12 cm C18) and eluted into the mass
spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) using an HPLC gradient (Waters
NanoAcquity, Milford, MA; 0%–35% B in 60 min, A = 0.2 M acetic acid in water, B = 0.2 M
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acetic acid in acetonitrile, flow rate = ~30 nL/min). The instrument was operated in data
dependent mode such that the top 10 most abundant precursors were subjected to MS/MS
(electron multiplier detection, relative collision energy 35%, q = 0.25). Raw data files were
converted to .mgf using inꢀhouse software52 and searched using Mascot 2.2.1 against a forwardꢀ
reverse human refseq database. Search parameters specified variable oxidation of methionine,
variable inhibitor modification (9, 45, or 50) of cysteine, and fixed carbamidomethylation of
cysteine (i.e. cysteines are considered carbamidomethylated or inhibitor labeled). To confirm
labeling sites, internally calibrated HCD spectra (image current detection, resolution at m/z 200 =
15,000, relative collision energy 35%) were acquired using an Orbitrap Fusion mass
spectrometer (Thermo Fisher Scientific). For these experiments, LC parameters were similar to
those described above, except that the analytical column was packed with 50 cm of C18 and
peptides were eluted with a gradient of 0ꢀ35% B in 90 min.
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Mouse liver microsomal stability. The MLM assays were previously reported and are
commercially available from Scripps Florida.34
Ba/F3 cell viability assays. Ba/F3 derivatives expressing various oncogenic fusion kinases,
TELꢀJAK1, TELꢀJAK2, TELꢀJAK3 and TELꢀABL were described previously.26 Ba/F3 cells
transformed by TYK2E957D were generated as previously described.30 These cells were
maintained in RPMIꢀ1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS)
(SigmaꢀAldrich) and penicillin/streptomycin (Invitrogen). For cell viability assays, the cells were
plated at a density of 10,000 cells per well in a 96ꢀwell white plate and incubated with DMSO or
increasing concentrations of drugs. At 72 h after the initiation of treatment, the relative cell
viability was determined using the Cell Titer Glo assay (Promega) and reported as a percentage
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of the DMSO control. Concentration values for 50% inhibition (IC50) of cell viability were
determined with GraphPad Prism software.
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Immunoblotting analysis and washout experiment. Ba/F3 cells transformed by TELꢀJAK3
were incubated with JAK3 inhibitors or DMSO for 3 h. Then wholeꢀcell lysates were prepared in
RIPA buffer (Cell Signaling) with FOCUSTM, ProteaseArrestTM (GꢀBiosciences) and
Phosphatase Inhibitor Cocktail Set II (EMD Millipore). Western blotting analyses were
conducted after separation by SDSꢀPAGE electrophoresis and transfer to nitrocellulose
membranes. Immunoblotting was performed with each of specific antibodies to STAT5,
phosphoꢀSTAT5 (Tyr694), αꢀtubulin (Cell Signaling Tech) and JAK3 (Santa Cruz, #scꢀ513). For
washout experiment, cells were incubated with JAK3 inhibitors or DMSO for 3 h, washed with
PBS three times, maintained in RPMIꢀ1640 medium with 10% of FBS for 4 h, and then
harvested and the resulting lysates were analyzed. Primary bone marrow derived macrophages
(BMDMs) were prepared as described.53 Cells were maintained on bacterial grade DMEM
supplemented with 10% heatꢀinactivated fetal bovine serum, 2 mM Lꢀglutamine, 100 U/ml of
penicillin G, 100 ꢁg/ml of streptomycin, 0.25 ꢁg/ml of amphotericin (Invitrogen), and 5 ng/ml of
macrophage colonyꢀstimulating factor (MꢀCSF; PreProTech) for 7 days. Cells were then replated
onto tissue culture grade plastic and used the following day. Following stimulation cells were
lsyed into SDS sample buffer and run on 10% polyacrylamide gels according to standard
techniques. Proteins were transferred onto nitrocellulose membranes and blotted with antibodies
against total ERK1/2, pꢀY641 STAT6, pꢀY701 STAT1, pY701 STAT3 or pY694 STAT5 (all
from Cell Signaling Tech). The experiments in cancer cell lines were done with similar
procedures.
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Streptavidin pulldown experiment. Ba/F3 cells transformed by TELꢀJAK3 were incubated
with JAK3 inhibitors or DMSO for 3 h, and then lysed with Pierce IP lysis buffer (Thermo
Scientific) with HaltTM protease inhibitor cocktail (Thermo Scientific). The lysates were treated
with 85 (5 µM) at 4 ºC overnight, and then further incubated at room temperature for 3 h. The
solution was mixed with Streptavidin beads (25 µL, Sigma) and incubated with rotation at 4 ºC
for 2 h. The beads were washed five times using the lysis buffer with 4 M urea (1 mL), and then
boiled in the presence of Lammli sample buffer with 2ꢀmercaptoethanol. The eluted proteins
were immunoblotted with each of specific antibodies.
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In Vivo Pharmacokinetic Studies. Male Swiss albino mice were dosed via tail vein
(intravenous, 0.1% v/v Tween 80, 0.5% w/v NaCMC in water at a dose of 10 mg/kg) or via oral
gavage (suspensions in 5% NMP, 5% solutol HS in normal saline intravenously via tail vein at a
dose of 2 mg/kg). Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8 and 24 h (i.v.) and at
Predose, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h (p.o.). Plasma samples were separated by centrifugation
of whole blood and stored below ꢀ70ºC until bioanalysis. All samples were processed for
analysis by protein precipitation using acetonitrile and analyzed with fitꢀforꢀpurpose LC/MS/MS
method (LLOQ, 1.06 ng/mL). Pharmacokinetic parameters were calculated using the nonꢀ
compartmental analysis tool of WinNonlin Enterprise software (version 6.3).
Mouse treatment studies
KrasG12D;Lkb1L/L mice were intrathoracically injected with adenovirus expressing Cre
recombinase (2.5x107 PFU/10 µl) at 6 weeks of age to initiate tumor formation.38b All
experimental mice were maintained on a mixed genetic background (C57Bl/6, Balbꢀc, and S129).
MR imaging and tumor quantification were performed as described previously. 38b Compound 9
and vehicle controls were given per os once daily at a 75 mpk dose.
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Total mouse lung cell and tumor infiltrating immune cell characterization was performed as
previously described.54 Splenocytes were isolated similarly without collagenase treatment. The
antibodies used for immune analysis are listed in supplementary methods. Acquisition of eight
color samples was performed on a BD Canto II cytometer equipped with Diva software and
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analyzed using Flowjo. All in vivo experiments performed in the PK studies and this study were
approved by the Animal Care and Use Committee of the DanaꢀFarber Cancer Institute.
Acknowledgements. We thank Dr. CaiꢀHong Yun for his valuable help with creating topology
and parameter files for the refinement of the JAK3ꢀ9 coꢀcrystal structure. The TELꢀJAK1, TELꢀ
JAK2, TELꢀJAK3 and TELꢀABL plasmids used in this studies were generously provided by Dr.
Richard Moriggl. G.S.H.ꢀS. was supported by the Deutsche Forschungsgemeinschaft (HE
6897/1ꢀ1). We thank Dr. Ying Li for data analysis and Dr. Sara Buhrlage for the proofꢀreading.
Supporting Information Available: Figure S1ꢀ6; Table S1ꢀ4; Structures and spectral data of 14ꢀ
64 and 66ꢀ85; Complete KinomeScan profiling data of 9 and 45. This material is available free
of charge via the Internet at http://pubs.acs.org. Xꢀray coordinates and structure factors have
been deposited in the Protein Data Bank. PDB accession code: 4Z16.
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