Discovery of highly potent, selective, covalent inhibitors of JAK3

Article abstract of DOI:10.1016/j.bmcl.2017.09.023

A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site cysteine residue. The design was based on crystal structure information and a comparative study of several electrophilic warheads.

Full text of DOI:10.1016/j.bmcl.2017.09.023

Accepted Manuscript
Discovery of highly potent, selective, covalent inhibitors of JAK3
James Kempson, Damaso Ovalle, Junqing Guo, Stephen T. Wrobleski, Shuqun
Lin, Steven H. Spergel, James J.-W. Duan, Bin Jiang, Zhonghui Lu, Jagabandhu
Das, Bingwei V. Yang, John Hynes Jr., Hong Wu, John Tokarski, John S. Sack,
Javed Khan, Gary Schieven, Yuval Blatt, Charu Chaudhry Luisa M. Salter-Cid,
Aberra Fura, Joel C. Barrish, Percy H. Carter, William J. Pitts
PII:
S0960-894X(17)30919-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.023
BMCL 25287
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 May 2017
6 September 2017
11 September 2017
Please cite this article as: Kempson, J., Ovalle, D., Guo, J., Wrobleski, S.T., Lin, S., Spergel, S.H., Duan, J.J., Jiang,
B., Lu, Z., Das, J., Yang, B.V., Hynes, J. Jr., Wu, H., Tokarski, J., Sack, J.S., Khan, J., Schieven, G., Blatt, Y.,
Salter-Cid, C.L.M., Fura, A., Barrish, J.C., Carter, P.H., Pitts, W.J., Discovery of highly potent, selective, covalent
inhibitors of JAK3, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.09.023
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Discovery of highly potent, selective, covalent inhibitors of JAK3
James Kempson,* Damaso Ovalle, Junqing Guo, Stephen T. Wrobleski, Shuqun
Lin, Steven H. Spergel, James J-W. Duan, Bin Jiang, Zhonghui Lu, Jagabandhu
Das, Bingwei V. Yang, John Hynes Jr., Hong Wu, John Tokarski, John S. Sack,
Javed Khan, Gary Schieven, Luisa M. Salter-Cid, Aberra Fura, Yuval Blatt, Charu
Chaudhry, Joel C. Barrish, Percy H. Carter and William J. Pitts.
Bristol-Myers Squibb Research and Development, Princeton, NJ 0854-4000, USA
A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site
cysteine residue. The design was based on crystal structure information and a comparative study of several
electrophilic warheads.
* Corresponding author. Tel.: +1-609-252-4539;
e-mail: james.kempson@bms.com

Discovery of highly potent, selective, covalent inhibitors of JAK3
James Kempson,† Damaso Ovalle, Junqing Guo, Stephen T. Wrobleski, Shuqun Lin, Steven H.
Spergel, James J-W. Duan, Bin Jiang, Zhonghui Lu, Jagabandhu Das, Bingwei V. Yang, John Hynes
Jr., Hong Wu, John Tokarski, John S. Sack, Javed Khan, Gary Schieven, Yuval Blatt, Charu Chaudhry
Luisa M. Salter-Cid, Aberra Fura, Joel C. Barrish, Percy H. Carter and William J. Pitts.
Bristol-Myers Squibb Research and Development, Princeton, NJ, 08543-4000, USA
Received Month XX, 2016; Accepted Month XX, 2016
Abstract— A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3
active site cysteine residue. The design was based on crystal structure information and a comparative study of
several electrophilic warheads.
©2016 Elsevier Science Ltd. All rights reserved
† Corresponding author. Tel.: +1-609-252-4539;
e-mail: james.kempson@bms.com

The Janus kinases (JAKs) play a central role in
regulating the immune system.1 The JAK family of
kinases is comprised of four family members, JAK1,
JAK2, JAK3 and tyrosine kinase 2 (TYK2), each of
which can bind to distinct cytokines and/or growth
factor receptors. More specifically, JAK3 is required
for signaling by cytokines which include interleukin 2
(IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21, which act
via receptors that contain the common gamma chain
(c) cytokine receptor subunit. JAK3 associates with
the c receptor in the cytoplasm and works in concert
with JAK1 to activate/phosphorylate the Signal
Transducers and Activators of Transcription (STAT)
proteins. Once phosphorylated, these STAT proteins
then dimerize and translocate to the nucleus to initiate
gene transcription which specifically activates T, B
and NK cell immune responses.
points to design selective JAK3 covalent inhibitors
based on their similar predicted binding orientations in
JAK3 from early modeling efforts.
Figure 2: Reversible inhibitors of JAK3
Docking7 of 3 to a published JAK3 X-ray structure8
led to a model that suggested placement of an
electrophile at the meta-position of the benzylamine
would provide a suitable vector to target Cys909
(Figure 3). Based on these considerations, we
designed and prepared compounds 9a, containing an
acrylamide group as the electrophile, and the
propanamide 9b as an isosteric, but unreactive
comparator.
Figure 1: FDA approved JAK inhibitors.
Significant drug discovery efforts have resulted in
the development of several JAK inhibitors including
tofacitinib2 (1, pan-JAK inhibitor) which has been
approved for the treatment of rheumatoid arthritis and
ruxolitinib3 (2, JAK1/2 inhibitor) which has been
approved for the treatment of myelofibrosis (Figure 1).
Several other JAK family inhibitors are currently in
clinical trials.4 Due to the highly conserved nature of
the enzyme active sites across all JAK family
members, the traditional ATP-competitive inhibitor
approach has found difficulty in obtaining a high
degree of selectivity within the family. However, the
active site of JAK3 is unique as it contains a cysteine
(Cys909) which if properly engaged could provide
access to JAK3 selective inhibitors. Recently there
have been several reports of selective covalent JAK3
inhibitors.5 Indeed, studies with one of these
inhibitors5c-d suggests a therapeutic potential for JAK3
selective inhibition as demonstrated by both potent
inhibition of in-vitro cellular signalling through the c
cytokines in addition to a high degree of efficacy in a
variety of in-vivo inflammatory disease models. Our
efforts to covalently target JAK3 are described below.
Figure 3 : Model of compound 3 docked in JAK3 kinase domain
showing a close-up of the binding site. Hydrogen bond interactions are
shown with dotted lines. JAK3 ribbon and carbons in green. Carbons of
3 in magenta. Yellow arrow indicates vector towards Cys909.
As part of our efforts to identify JAK1/3 inhibitors, we
identified two lead compounds (3, 4, Figure 2) which
were potent reversible inhibitors of JAK3 and showed
a degree of selectivity over other JAK family
members. We reasoned these would be good starting

This finding was confirmed using an in-house kinase
caliper screening panel which showed that 9a only
inhibited FMS (IC50 =180 nM, n=2), while inhibition
of BTK (IC50 > 2.5 M, n=3) and EGFR (IC50 > 10
M, n=1) was weak at the concentrations tested. As
IC50 data is less than ideal for determining selectivity
of covalent inhibitors, we proceeded to examine 9a
using a time-dependent assay in which BTK inhibition
was measured at four time points (Figure 4). EC50
values are noted in M and reveal the time-dependent
nature of inhibition against BTK with ~10-fold
increase in potency during the course of the
experiment as target (Cys) engagement takes place.
However, after 120 minutes, the EC50 for BTK
inhibition is still greater than 1 M.9 While irreversible
inhibitors are often characterized using IC50 values, these
measurements are time-dependent. The efficiency of
covalent inhibitors can be more accurately expressed as a
function of the kinact/Ki ratio.13 To accurately gauge
JAK3 vs BTK selectivity for Cmpd 9a, we determined
corresponding kinact/Ki values using a continuous
fluorescence assay. Consistent with predictions from our
preincubation experiment results, kinact/Ki for cmpd 9a
at JAK3 ( 0.3 uM-1 sec-1) was at at least 3 orders of
magnitude larger than at BTK (0.00005 uM-1 sec-1),
revealing a significant selectivity window for this
compound. Additionally 9a had an IC50 greater than
10M in a a BCR-stimulated CD69 expression assay in
human whole blood.14
9
Scheme 1: Synthetic route to 9a and 9b.
The synthesis of 9 (Scheme 1) was accomplished in 4
steps starting from intermediate 5 and the addition of
meta-nitro benzylamine to first give 6 in 88% yield.
Subsequent addition of aniline under acidic conditions
then produced 7 which was then subjected to
hydrogenolysis using Pd/C as catalyst. The resulting
aniline was then acylated with acroyl chloride to give
9a. Acylation of 8 with propionyl chloride provided
the comparator compound 9b.
Testing for JAK3 inhibition in a fixed time point
enzymatic assay,10 9a was found to be a highly potent
(JAK3 IC50 < 1 nM, n=3) inhibitor and exhibited time-
dependent inhibition (100% inhibition at 15 minutes).
In contrast 9b, was moderately potent (JAK3 IC50
=
100 nM, n=1) with no change in IC50 after pre-
incubation. The JAK family selectivity of 9a was
significantly improved compared to the reversible
leads 3, 4 (9a enzyme data: JAK2 IC50 = 1,300 nM,
n=3; JAK1 IC50 = 1,100 nM, n=3; TYK2 IC50 > 5000
nM, n=3) and exhibited good cellular potency (IL2
driven T-Cell proliferation IC50 = 22 nM, IL2 driven
pSTAT3 IC50 = 51 nM) and human whole blood
Figure 4: BTK pre-incubation enzyme assay 9a
potency (IL-2 driven IFNproduction hWB IC50
=
Pharmacokinetic evaluation revealed that 9a had low
490 nM). 9a also possesed a high degree of selectivity
over a JAK2 cellular endpoint (EPO driven pSTAT5
IC50 = 11 M) and a JAK1/TYK2 cellular endpoint
(IFN driven pSTAT3 IC50 = 4.9 M ) To assess
broad kinome selectivity, we evaluated 9a in an
external panel screen against >350 kinases which
showed excellent overall kinome selectivity (Kinases
< 10% of control at 1 M = JAK3, FMS, BMPR2).11
This selectivity result is somewhat surprising, as there
are ten other protein kinases with cysteines at this
location in the active site including the TEC family
(e.g. BTK) and several other kinases such as EGFR.12
bioavailability
(<30%)
and
high
clearance
(significantly greater than hepatic blood flow) in the
mouse. While high clearance desirable attribute for
covalent inhibitors, the plasma levels achieved were
only very briefly above the whole blood IC50 which
precluded advancement to in vivo studies. Thus we
sought to extend this proof-of-concept work in the
nicotinamide series to the closely related
pyrazolopyridazine series 4 where we had generated
analogs with an improved pharmacokinetic profile and
demonstrated efficacy in a mouse model of arthritis.15

Utilizing the same acrylamide probe within this series
provided 13a (Figure 5),
With the activity of 13a confirmed, our focus shifted
to exploration of alternative electrophilic groups16
(Table 1) utilizing similar chemistry to that depicted in
Scheme 2 for the synthesis of 13a.
Figure 5: Structure and in-vitro profile for compound 13a
Scheme 2. Synthetic route to 13a
Compounds were evaluated for their ability to inhibit
the JAK family of kinases and also evaluated in an IL-
2 driven cell T cell proliferation assay (IL-2 T-Cell).
The structure-activity relationships for the inhibition
of JAK family and cell based activity are summarized
in Table 1.
In comparison to 13a, addition of a beta-methyl
group to the acrylamide group (13b) resulted in
significant loss of JAK3 potency presumably due to
steric hinderance along the trajectory for interaction
with Cys909. The vinyl sulfone electrophile 13c
displayed similar enzymatic potency to 13a, but this
was not reflected in the cellular assay (IL-2 T-Cell =
6700 nM). Incorporation of nitrile functionality (13d)
in an attempt to reversibly engage Cys909 resulted in
a modest decrease in JAK3 potency compared to 13a.
Incorporation of larger electrophiles (13e-13f) led to a
decrease in JAK3 potency but exhibited excellent
selectivity against JAK1/2, and across the kinome. We
also explored the benzisothiazolone motif12b (13g)
which was more potent against JAK3 compared to
(13e,f) but given the similarity in size to 13f we were
somewhat surprised to see an erosion of selectivity
against JAK1/2 and TYK2.
Figure 6: X-ray co-crystal structure of JAK3 kinase domain bound
with compound 13a showing a close-up of the binding site. PDB code:
5WFJ. Hydrogen bond interactions are shown with dotted lines. JAK3
ribbon and carbons in green. Carbons of 13a in magenta. The electron
desnisty map around the inhibitor and Cys909 are shown in blue mesh
at a sigma level = 1.0. The density is continuous between the
acrylamide warhead and Cys909 indicating a covalent bond.
which was also found to give a selective JAK3
inhibition profile. We were able to confirm the
structural basis for achieving selectivity with this
inhibitor by solving the co-crystal structure of the
JAK3 kinase domain in complex with 13a at a
resolution of 2.9Å (Figure 6). In this structure, the
primary amide and pyrazolopyridazine core binds to
the hinge region with the benzylamine orientated
under the P-loop. Continuous electron density is
observed between the acrylamide group and Cys909,
suggestive of covalent bond formation.
Table 1... SAR with respect to JAK family inhibition.

Compd
13b
JAK3
IC50 nM
JAK2
IC50 nM
JAK1
IC50 nM
TYK2
IC50 nM
IL-2 T-Cell
IC50 nM
R
175
>2000
1422
>2000
26600
13c
13d
3.5
35
1270
658
974
264
>2000
2000
6700
8450
13e
13f
942
743
>2000
>2000
>2000
>2000
>2000
>2000
14400
15400
13g
30
745
196
240
5480
Research Center (BBRC) for the scale-up synthesis of
intermediates 5 and 10.
In summary, we have identified acrylamide 9a as a
potent, selective JAK3 covalent inhibitor which may be
a useful addition to the in vivo tool molecules and
clinical compounds reported by others.5,6 In a closely-
related pyrrolopyridazine series, we were able to
confirm a covalent interaction with Cys909 in the JAK3
active site, by both kinetic evaluation and x-ray
crystallography studies using 13a. As a result of these
studies, future efforts are currently focused on the
continued optimization of irreversible JAK inhibitors
within this novel series and additional results will be
reported in due course.
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