Identification of a potent janus kinase 3 inhibitor with high selectivity within the janus kinase family

Article abstract of DOI:10.1021/jm101157q

We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on

Full text of DOI:10.1021/jm101157q

284 J. Med. Chem. 2011, 54, 284–288
DOI: 10.1021/jm101157q
Identification of a Potent Janus Kinase 3 Inhibitor with High Selectivity within the
Janus Kinase Family†
Gebhard Thoma,* Francois Nuninger, Rocco Falchetto, Erwin Hermes, Gisele A. Tavares, Eric Vangrevelinghe, and
€
Hans-Gunter Zerwes
Novartis Institutes for BioMedical Research, Forum 1, Novartis Campus, CH-4002 Basel, Switzerland
Received September 7, 2010
We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the
discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a
rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1
bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor
CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular
assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ
chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and
cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for
signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine
signal transduction required for strong immunosuppression.
Introduction
Janus kinase 3 (Jak3) is essential for signal transduction of
cytokines utilizing the common γ chain (CD132/γC) and its
absence results in immunodeficiency. Thus, it has been as-
sumed that specific inhibition of Jak3 kinase function would
have therapeutic potential as a selective and safe immunosup-
pressive principle.1 However, signaling through γC cytokines
Figure 1. Structure of CP-690,550 = 2.
leading to the phosphorylation of the adaptor protein STAT5a
rationale for the selectivity of aryl-indolyl maleimides within
also requires Jak1 in addition to Jak3.2 The relative contri-
the Jak kinases. Surprisingly, compared to the pan Jak inhibitor
butions of the kinase function of Jak1 and Jak3 are not fully
2, compound 1 was found to be significantly less potent in
understood. The Pfizer Jak inhibitor CP-690,5503 (2, see
cellular assays measuring STAT5 phosphorylation despite
Figure 1) proved to be active in preclinical models4 and is
similar permeability and cellular uptake for both compounds.
currently being clinically assessed in multiple indications.5
Although originally described as being selective for Jak3,3 2
Chemistry
also potently inhibits other Jak kinases including Jak1.6 Thus,
Commercially available phenylacetic acid 2 was converted
into the corresponding amide 3 (Scheme 1). Reaction with
indolyl-oxo-acetic acid ester 4 led to the versatile intermediate
5 allowing for the late stage modification of phenyl-indolyl
maleimides. Treatment of 5 with amines 6 furnished com-
pounds 7-10. Coupling of 7 with acid chloride 11 followed by
deprotection gave Jak3 inhibitor 1.
the effects observed with 2 cannot be assigned solely to the
specific inhibition of Jak3 kinase function and do not neces-
sarily validate the assumption that specific inhibition of Jak3
kinase function will efficiently block signal transduction
through γC-receptors leading to immunosuppression.
Setting out to explore the effects of specific Jak3 inhibition
on signal transduction, we identified maleimide 1 exhibiting
very high selectivity within the Jak kinase family. Here we
describe its synthesis via late stage modification of a pre-
formed maleimide. Furthermore, on the basis of the cocrystal
structure of a related analogue with Jak3, we provide a
Results and Discussion
The maleimides 1, 5, and 7-10 were tested in enzymatic
assays measuring Jak kinase activity. Compound 1 was highly
potent on Jak3 (IC50 = 8.0 nM) and showed an unprecedented
selectivity within the Jak kinases (IC50>1000 nM on Jak1,
Jak2, and Tyk2).7 The selectivity for Jak3 seems to be general as
indicated by compounds 7-10, which were also most active on
Jak3. Compound 5 was inactive on all Jak kinases, indicating
the importance of the substituent of the phenyl group.
†Structure deposited in the RCSB Protein Data Bank under PDB ID:
3PJC.
*To whom correspondence should be addressed. Phone: þ41 61
3243342. Fax: þ41 61 3246735. E-mail: gebhard.thoma@novartis.com.
a Abbreviations: CDI, carbonyldiimidazole; DBU, diazabicycloun-
decene; DMAP, 4-dimethylaminopyridine; UPLC, ultra performance
liquid chromatography; STAT5, signal transducer and activator of
transcription 5; PKC, protein kinase C; GSK3, glycogen synthase kinase
3; TCR, T-cell receptor.
The X-ray structure of the related maleimide 128 cocrys-
tallized with the Jak3 kinase domain furnished a rationale for
r
pubs.acs.org/jmc
Published on Web 12/14/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 285
Scheme 1. Synthesis of Phenyl-indolyl Maleimidesa
a Reagents and conditions: (a) (1) CDI, DMF, 25 °C, 0.5 h, (2) NH3 (7M) in MeOH, 25 °C, 1 h; (b) (1) tBuOK, THF, 10 °C, 2 h, (2) DBU, DMF, 85 °C,
0.5 h; (c) DMSO, microwave, 170 °C, 1 h; (d) (1) DMAP, CH2Cl2, 25 °C, 2 h, (2) MeONa, MeOH, 25 °C, 0.5 h.
Table 1. Enzymatic Activitiesa
compd
Jak1
Jak2
Jak3
Tyk2
1
1017 ( 117
2550 ( 326
8.0 ( 2
8055 ( 589
5
>10000
>10000
>10000
>10000
7
737 ( 137
4700 ( 173
754 ( 166
4700 ( 326
9150 ( 50
1444 ( 455
133 ( 45 >10000
423 ( 20 >10000
8
9
36 ( 8
5040 ( 682
10
12
2
6500 ( 1305 >10000
460 ( 154 >10000
253 ( 66
6.1 ( 2
330 ( 68
12 ( 2
27 ( 7
4333 ( 841
176 ( 25
8.0 ( 2
a The final ATP concentration used in the assays corresponds to the
individually determined Km ATP for the respective enzyme. IC50 values
are reported in nM as the average ( SEM of g3 experiments.
reported structures of Jak1 and Jak2, this backbone amide
adopts a different conformation which would not favor the
water-mediated interaction with the maleimide ring observed
in Jak3.10 This different conformation of both the Jak1 and
the Jak2structure canbeexplainedbythe presence of a glycine
adjacent to the conserved Asp967. Because Tyk2 also has a
glycine in this position, Jak3 is the only Jak kinase with an
alanine (Ala966) adjacenttoAsp967. The side chainof Ala966
restrains the backbone amide into a conformation favorable
to a water-mediated interaction, with the maleimide ring
explaining the observed selectivity.
Figure 2. Detailed view of the X-ray crystal structure of compound
˚
12 (carbons in orange) bound to the ATP-binding site of Jak3 (2.5 A
resolution). Hydrogen bonds between the ligand and the protein are
represented by dashed magenta lines.
Maleimide 1 was tested against a panel of 40 kinases and
found to only inhibit Pkc and Gsk3β with IC50 values below
1000 nM. The Pfizer Jak inhibitor 2 was also tested and found
to be highly selective against non-Jak kinases. However,
within the Jak kinase family, compound 2 was unselective,
inhibiting Jak1, Jak2, and Jak3, with IC50 values between 6.1
and 13 nM (Table 1). Compounds 2 and 1 were equally potent
on Jak3 (IC50 = 8 nM). Compound 1 was tested in cellular
assays measuring STAT5 phosphorylation triggered by cyto-
kine stimulation (IL-2 in CTLL cells; IL-15 in MO7 cells) and
found to be >20-fold less potent compared to 2 (Table 2).
The weak activity of compound 1 in these cellular assays
was not caused solely by a poor permeability of 1, as permea-
tion through artificial membranes and flux through Caco2
the selectivity of compounds 1 and 7-10 within the Jak kinase
family (Figure 2, see Table 1 for activity of 12 in enzymatic Jak
assays).9 The maleimide ring binds to the hinge part of the
kinase through two hydrogen bonds with adjacent backbone
amides (Glu903, Leu905) and makes hydrophobic contacts
with the side chains of Ala853 and Leu956. Additional favor-
able hydrophobic contacts are observed between the indole of
12 and the side chains of Leu828 and Val836. The trifluoro-
methyl group optimally fits into a small pocket delimited by
the side chains of Val836, Ala853, Lys855, and Met902. Most
important for the observed selectivity is a water-mediated
H-bond between an oxygen atom of the maleimide and the
NHof the backbone amide of the catalytic Asp967. Inrecently

286 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Table 2. Cellular Activitya
Thoma et al.
Further work to better understand the individual roles of Jak
kinases in STAT phosphorylation is in progress.15
pSTAT5/IL2
(CTLL cells)
pSTAT5/IL15
(MO7 cells)
TCR/CD288
(Jurkat cells)
compd
1
2
1294 ( 259
48 ( 2
525 ( 182
24 ( 1
689 ( 125
>5000
Experimental Section
General. All reactions were carried out under an atmosphere
of dry argon. Commercially available absolute solvents were used.
The NMR spectra were recorded on a Bruker Avance DPX 400
spectrometer at room temperature. The HR-MS spectra were
obtained on Finnigan MAT 900 S or Bruker Daltronics 9.4T
APEX-III FT-MS mass spectrometers. LC/MS was performed
on a Waters Acquity UPLC linked to a Waters ZQ 2000 mass
spectrometer using a Waters BEH C18 1.7 μm 2.1 mm ꢀ 50 mm
column (flow = 0.7 mL/min; detection 240-350 nM DAD;
solvent A, water þ 0.1% formic acid; solvent B, acetonitrile;
method for compound 7, t = 0 min, 95% A, 5% B; t = 1 min
90% A, 10% B; t = 4.0 min 10% A, 90% B; t = 4.1 min 100% B;
t = 4.5 min, 100% B; method for compounds 1, 8, 9, 10, 12: t =
0 min, 80% A, 20% B; t = 1 min 75% A, 25% B; t = 4.2 min 5%
A, 95% B; t = 4.3 min 100% B; t = 4.5 min, 100% B). The
purity of all test compounds was >95%.
2-(5-Fluoro-2-trifluoromethyl-phenyl)-acetamide (3). CDI
(18.8 g, 110 mmol) was added to a solution of (5-fluoro-2-
trifluoromethyl-phenyl)-acetic acid (2) in DMF (60 mL). After
stirring for 0.5 h at 25 °C, NH3 (60 mL of a 7 M solution in
MeOH) was added and stirring continued for 1 h. Water was
added (200 mL) and the precipitate was filtered off, washed with
water, and dried to give 3 (16.5 g, 74%), which was used without
further purification. 1H NMR (400 MHz, DMSO-d6) δ = 7.75
(m, 1 H), 7.50 (s, 1 H), 7.30 (m, 2 H), 7.00 (s, 1 H), 3.65 (s, 2 H).
MS/ESI 222 (M þ 1)þ.
a IC50 values are reported in nM as the average (SEM of g3
experiments.
Table 3. Compound Amounts in Whole Cell Extracts
compd 2
whole cell extract
[pmol/50000 cells]
compd 1
whole cell extract
[pmol/50000 cells]
cell
type
analyte in growth
medium [μM]
Jurkat
0.33
1.0
1.04 ( 0.08
3.0 ( 0.08
8.09 ( 0.58
20.7 ( 2.24
2.4 ( 0.38
7.88 ( 1.85
23.48 ( 2.91
55.58 ( 6.91
3.3
10.0
CTLL
0.33
1.0
1.19 ( 0.17
3.05 ( 0.73
10.41 ( 0.31
22.14 ( 4.23
3.12 ( 1.51
10.97 ( 1.92
31.27 ( 4.71
64.33 ( 5.02
3.3
10.0
cells were only moderately inferior for compound 1 compared
to 2 (compound 1: Log PAMPA11 = -5.5, PappA-BCaco
=
3 ꢀ 106 cm/s; 2: Log PAMPA = -5.2, PappA-BCaco = 10 ꢀ
106 cm/s). In addition to Jak3, compound 1 inhibits PkcR,
Pkcθ, and GSK3β, with IC50 values of 13, 68, and 3 nM,
respectively.12 To probe its ability to block kinase function in
cells, compound 1 was tested in a reporter gene assay measur-
ing TCR/CD28-mediated T cell activation12 (Jurkat cells)
which is dependent on PkcR and Pkcθ (but not on Jak kinases
or Gsk3β).13 Compound 1 showed moderate activity in this
cellular assay (IC50=689 nM, Table 2), which is in good
agreement with its enzymatic Pkc activity.14 As expected, the
pan Jak inhibitor 2 was inactive (Table 2). Next, we measured
amounts of compounds 1 and 2 in whole cell extracts of
both CTLL cells (IL-2 pSTAT5 assay) and Jurkat cells (T-cell
activation assay). We found that compound amounts asso-
ciated with whole cells (the sum of membrane-associated
amount and intracellular amount) were independent of the
cell type and similar for compounds 1 and 2 (Table 3).
However, cytosolic concentrations which might be even more
relevant for the inhibition of Jak3 in the cell could not be
determined. Nevertheless, our data suggest that compound 1
is present in cells and can block intracellular processes trig-
gered by PKC. Thus, because Jak3 and Jak1 always cooperate
for signaling through γC-receptors, the inferior cellular po-
tency of the selective Jak3 inhibitor 1 compared to the pan Jak
inhibitor 2 could indicate that selective inhibition of Jak3 is
not sufficient to achieve strong immunosuppressive effects.
3-(5-Fluoro-2-trifluoromethyl-phenyl)-4-(1H-indol-3-yl)-pyrrole-
2,5-dione (5). At 0 °C, tBuOK (370 mL 1 M in THF) was added
to a solution of 3 (16.5 g, 74.6 mmol) and 4 (18.2 g, 85.1 mmol) in
THF (350 mL). The solution was stirred for 2 h at 10 °C, HCl
(125 mL 4N) was added, the mixture was extracted with ethyl
acetate, and the organic phase was dried with Na2SO4. The
solvent was removed and the residue dried. DBU (17 g) was
added to a solution of this residue in DMF (50 mL) and stirred
for 0.5 h at 85 °C. The mixture was cooled to 25 °C, diluted with
ethyl acetate, and the pH adjusted to 1 with HCl (4N). The
organic phase was separated, dried with Na2SO4, the solvent
removed, and the residue purified by chromatography (SiO2,
cyclohexane/acetone gradient) to give 5 (25.6 g, 91%) as a yellow
powder. 1H NMR (400 MHz, DMSO-d6) δ = 12.0 (s, 1 H), 11.2
(s, 1 H), 8.02 (m, 1 H), 7.96 (m, 1 H), 7.54 (m, 1 H), 7.42 (m, 2 H),
7.06 (m, 1 H), 6.74 (m, 1 H), 6.45 (m, 1 H). MS/ESI 375 (M þ 1)þ.
3-(1H-Indol-3-yl)-4-(5-piperazin-1-yl-2-trifluoromethyl-phenyl)-
pyrrole-2,5-dione (7). A mixture of 5 (1.0 g, 2.67 mmol), piper-
azine (1.0 g, 11.6 mmol), and DMSO (5 mL) was heated in a
microwave oven for 1 h at 150 °C. The mixture was diluted with
ethyl acetate and tert-butyl-methylether and the precipitate fil-
tered off. Compound 7 (1.15 g, 97%) was isolated as a yellow
powder. 1H NMR (400 MHz, DMSO-d6) δ = 11.85 (s, 1 H), 11.2
(broad m, 1 H), 7.95 (s, 1 H), 7.60 (m, 1 H), 7.28 (m, 1 H), 7.08
(m, 2 H), 6.90 (m, 1 H), 6.71 (m, 2 H), 3.08 (m, 4 H), 2.68 (m, 4 H).
The signal for the NH of the piperazine could not be detected
most likely due to line broadening. HR-MS [M þ 1]þ observed =
441.1534, estimated = 441.1533.
Conclusion
We have described the discovery of the phenyl-indolyl
maleimide 1, which is a potent Jak3 inhibitor with high
selectivity within the Jak kinase family. Surprisingly, 1 was
found to be significantly less potent in cellular STAT5 phos-
phorylation assays compared to the pan Jak inhibitor 2 which
is equally potent on Jak1 and Jak3. Cellular permeability and
compound amounts associated with whole cells were found to
be similar for compounds 1 and 2. In addition, compound 1
(which also inhibits PKC) was as potent as expected in a
cellular assay dependent on PKC. Thus, our data suggest that
selectiveinhibition of Jak-3 is not sufficient to efficiently block
immunologically relevant pathways mediated by γC cytokines.
3-{5-[4-(2-Hydroxy-2-methyl-propionyl)-piperazin-1-yl]-2-tri-
fluoromethyl-phenyl}-4-(1H-indol-3-yl)-pyrrole-2,5-dione (1).
Compound 11 (0.83 g, 5.0 mmol) was slowly added to a suspen-
sion of 7 (0.881 g, 2.0 mmol) and N,N-dimethylpyridine-4-amine
(0.204 g, 2.0 mmol) in THF (20 mL) and the mixture stirred for
2 h at 25 °C. The mixture was diluted with ethyl acetate, washed
with HCl and brine, and dried with Na2SO4. The solvent was
removed and the residue subjected to chromatography (SiO2,
cyclohexane/acetone gradient) to give 1 (0.50 g, 45%) as a yellow
1
powder. H NMR (400 MHz, DMSO-d6) δ=11.87 (s, 1 H),
11.08 (s, 1 H), 7.95 (s, 1 H), 7.62 (m, 1 H), 7.38 (m, 1 H), 7.14 (m,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 287
1 H), 7.05 (m, 1 H), 6.95 (s, 1 H), 6.72 (m, 1 H), 6.65 (m, 1 H), 5.43
(s, 1 H), 3.95 (m, 2 H), 3.50 (m, 2 H), 3.20 (m, 4 H), 1.30 (2s, 6 H).
HR-MS M þ 1þ observed = 527.1904, estimated = 527.1901.
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(7) Very recently a compound has been described as a selective Jak3
inhibitor. However, this compound inhibited all Jak kinases with
low nanomolar IC50 values and did not substantially differ from the
pan Jak inhibitor 2 when directly compared in cellular assays. Data
from enzymatic Jak kinase assays allowing for a direct comparison
of this compound and 2 have not been provided. Lin, T. H.; Hegen,
M.; Quadros, E.; Nickerson-Nutter, C. L.; Appell, K. C.; Cole,
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(8) Generally, phenyl-indolyl maleimides and corresponding pyrimidyl-
indolyl maleimides showed similar activites and selectivities within
the Jak kinases. Compound 12 was prepared in analogy to proce-
dures previously disclosed. Albert, R.; Cooke, N. G.; Cottens, S.;
Ehrhardt, C.; Evenou, J.-P.; Sedrani, R.; Von Matt, P.; Wagner, J.;
Zenke, G. Preparation of indolylmaleimide derivatives as protein
kinase c inhibitors. PCT Int. Appl. WO 2002/038561 A1, 2002.
(9) Structure deposited in the RCSB Protein Data Bank under PDB
ID: 3PJC.
Acknowledgment. We thank the Analytical Sciences unit
of the Novartis Institutes for BioMedical Research for their
support in the structure verification of the compounds de-
scribed in this paper and gratefully acknowledge the expert
€
technical assistance of M. Schafer, J. Peter, and V. Caballero.
Supporting Information Available: Analytical data for com-
pounds and 8, 9, 10, and 12, details of enzymatic and cellular
assays as well as procedures for the measurement of compound
amounts in whole cells. This material is available free of charge
via the Internet at http://pubs.acs.org.
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(12) Details of assays have been described in the following reference.
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Zenke, G.; Cottens, S. Discovery of 3-(1H-Indol-3-yl)-4-[2-(4-
methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione(AEB071),
a Potent and Selective Inhibitor of Protein Kinase C Isotypes.
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Misener, E.; Briere, D.; O’Toole, J. C.; Porter, W. J.; Queener, S.;
Reel, J. K.; Owens, R. A.; Brier, R. A.; Eessalu, T. E.; Wagner,
J. R.; Campbell, R. M.; Vaughn, R. Substituted 3-Imidazo[1,2-a]-
pyridin-3-yl-4-(1,2,3,4-tetrahydro-[1,4]diazepino-[6,7,1-hi]-
indol-7-yl)pyrrole-2,5-diones as Highly Selective and Potent Inhibitors
of Glycogen Synthase Kinase-3. J. Med. Chem. 2004, 47, 3934–3937.
(14) Reference 12 describes PKC inhibitors which were also tested in the
TCR/CD28-mediated T cell activation assay. Compounds with enzy-
matic activities comparable to maleimide 1 showed similar potencies in
the cellular assay.
€
(15) Haan, C.; Rolvering, C.; Raulf, F.; Kapp, M.; Druckes, P.; Thoma,
G.; Behrmann, I.; Zerwes, H.-G. unpublished results.