Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers, as potent JAK3 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.03.015

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.

Full text of DOI:10.1016/j.bmcl.2013.03.015

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2522–2526
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl
ethers, as potent JAK3 kinase inhibitors
a
a
b
a
Saul Jaime-Figueroa a, , Javier De Vicente , Johannes Hermann , Alam Jahangir , Sue Jin ,
Andreas Kuglstatter a,b, Stephen M. Lynch a, John Menke a, Linghao Niu a, Vaishali Patel a, Ada Shao b,
Michael Soth a, Minh Diem Vu a, Calvin Yee a
⇑
a Hoffmann-La Roche Inc., pRED, Pharma Research & Early Development, Small Molecule Research, Discovery Chemistry, 340 Kingsland Street, Nutley, NJ 07110-1199, United States
b Roche Palo Alto, 3401 Hillview Ave, Palo Alto, CA 94304, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the
5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed prom-
ising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crys-
tallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve
selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhib-
itors, demonstrating improved JAK family and kinase selectivity.
Received 15 January 2013
Revised 26 February 2013
Accepted 4 March 2013
Available online 14 March 2013
Keywords:
JAK
Ó 2013 Elsevier Ltd. All rights reserved.
JAK3
Janus kinase
Kinase inhibitors
Structure based drug design
The Janus kinases (JAKs) are a family of four non-receptor tyro-
sine kinases whose central role is to respond to cytokine or growth
factor receptor activation. Signals from more than 38 cytokines are
transduced through the four JAK family members and seven signal
transducers and activators of transcription proteins (STATs). The
members of the JAK family include the closely related isoforms
JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2). JAK family kinases
are essential for the signaling pathways of various cytokines impli-
cated in the pathogenesis of autoimmune and inflammatory dis-
eases.1,2 Selective JAK inhibitors may be useful as therapeutic
agents in the areas of oncology, organ transplantation, and autoim-
mune diseases.3–5 This expectation has been realized in the two re-
cent FDA approvals of ruxolitinib, a drug for the treatment of
myelofibrosis,5 and tofacitinib (CP690, 550), a pan-JAK inhibitor
that shows efficacy and acceptable safety for the treatment of
rheumatoid arthritis (RA)(Fig. 1).6–9
While JAK1, JAK2, and Tyk2 are ubiquitously expressed in verte-
brates, JAK3 is mainly limited to hematopoietic cells.4 Therefore,
selective targeting of JAK3 may offer therapeutic benefit while
minimizing potential liabilities associated with inhibition of
broader JAK signaling.10 Recently we have disclosed a new inhibi-
tor scaffold which demonstrated promising selectivity for JAK3
over JAK1.11 Herein we report a new phenyl ether-containing ser-
CN
N N
N
N
N
CN
O
N
N
N
H
N
H
N
Tofacitinib
Ruxolitinib
Figure 1. Structures of FDA approved JAK inhibitors.
ies of JAK3 selective inhibitors derived from our previous work
through a structure based drug design strategy.
Initially, we were attracted to the 5H-pyrrolo[2,3-b]pyrazine-2-
phenyl ethers 1a and 1h based on their promising JAK3 potency in
our enzyme assay (Fig. 2). However, these compounds were lim-
ited by their modest JAK family and kinome selectivity.12 To over-
come this selectivity challenge, we developed a strategy targeting a
cysteine residue (Cys909) rare to JAK3. Only 10 out of 518 protein
kinases possess a cysteine at this position, which is occupied by
serine in both JAK2 and JAK1. We hypothesized that hydrophobic
groups placed in this region of the binding pocket should experi-
⇑
Corresponding author. Tel.: +1 9732357643.
E-mail address: saul.jaime@roche.com (S. Jaime-Figueroa).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.015

S. Jaime-Figueroa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2522–2526
2523
the phenyl ether 1h bound to JAK3 confirmed that the phenyl ring
does indeed offer attractive vectors to reach Cys-909 (Fig. 3). Thus,
further computational and structure-based design resulted in the
syntheses of 12a, 12b, and 12d, which demonstrated potent JAK3
inhibition as well as JAK family and kinase selectivity.
Our first efforts to explore this series focused on meta-substitu-
tion which appeared to be the most attractive vector on the phenyl
ring to reach Cys 909. Thus, we designed and synthesized a series
of phenyl ethers around parent compounds 1a and 1h to optimize
selectivity for JAK3 over JAK1/2.
The syntheses of these compounds were achieved starting from
the bromo-aldehyde compound 2.14 Oxidation of the aldehyde 2
provided the corresponding carboxylic acid 3. Amidation of 3 with
iso-propylamine or 2-cyclopropyl-ethylamine afforded compounds
4a and 4b in good yield. These bromo-amide compounds were cru-
cial intermediates for the preparation of our first series of com-
pounds. Buchwald cross coupling15 of the bromo-compounds 4a
and 4b with the corresponding phenol gave the phenyl-ethers
5a–5i. Subsequent removal of the trimethylsilanyl-ethoxymethyl
group (SEM) provided the final products 1a–1i in this series
(Scheme 1). For phenyl ethers 1j and 1k a similar approach was fol-
lowed using the N-(tert-butoxycarbonyl)(Boc) protected interme-
diates 6a and 6b (Scheme 2).
O
O
N
H
HN
N
N
H
HN
N
N
O
N
O
(1a)
Figure 2. Structures of initial phenyl ethers.
(1h)
From the initial SAR on these compounds, the (S)-a-amino ind-
ano-ether 1k stood out based on its good JAK3 enzyme potency
and promising JAK family selectivity (Table 1). In an attempt to im-
prove both potency and kinase selectivity, we modeled 1h into the
binding site of JAK3 and looked for the best vectors from the iso-
propyl amide to optimize the filling of back pockets of the protein.
This led to the cyclopropyl-methyl amides 12a and 12b with a sig-
nificant potency boost while maintaining good selectivity com-
pared to 1j and 1k. The crystal structure of 12a bound to JAK3
(Fig. 4) showed the anticipated lipophilic interaction with Cys909
and a novel backbone interaction of the inhibitor with Leu828.
We rationalized that this intermolecular H-bond provides a rigidi-
fying element for selectivity and the cyclopropyl improves potency
due to space filling of a back pocket.
Figure 3. Compound 1h bound to JAK3 non-phosphorylated kinase domain (1.85 Å
resolution, PDB accesion number 416Q).13 The enantiomer bound to the protein
was clearly identified.
Next and based on previous knowledge11 we decided to prepare
larger bis-amide analogs in order to better fill the upper-back
pocket of the protein. For this exercise, we also used the bromo-
aldehyde compound 2 as starting material (Scheme 3). Buchwald
cross coupling15 of the bromo-compound 2 with the corresponding
ence fewer unfavorable interactions with cysteine in JAK3 than ser-
ine (and its water network) in other JAK family members, thus
leading to improved JAK family selectivity. The X-ray structure of
O
O
O
O
R'
R'
R'
CHO
N
OH
Br
N
H
N
H
N
H
a
b
c
d
N
N
N
SEM
N
HN
SEM
SEM
SEM
N
N
N
N
R
R
N
N
N
N
N
Br
O
Br
O
2
3
4a R' = -Me
5a - 5i
1a - 1i
4b R' = -CyPr
Scheme 1. Reagents and conditions: (a) sulfamic acid, NaClO2, KH2PO4, dioxane/water, 94%; (b) i-PrNH2 or 2-cyclopropyl-ethylamine, HATU, DMF, 80–94%; (c) Pd(OAc)2,
K3PO4, 2-di-t-butylphosphino-20-(N,N-dimethylamino)-biphenyl, Ar-OH, toluene 140 °C, 20–60%; (d) AcOH/HCl, at 65 °C, then EDA/MeOH/H2O, 20–65%.
O
O
O
O
N
N
N
H
N
H
H
H
N
a
b, c
SEM
d
N
HN
SEM
N
N
SEM
N
N
N
N
N
N
O
N
O
O
Br
NHAc
NHBoc
NHAc
6a-(S)
6b-(R)
7a-(S)
7b-(R)
1j-(S)
1k-(R)
4a
Scheme 2. Reagents and conditions: (a) (R)-t-butyl 6-hydroxy-2,3-dihydro-1H-inden-1-ylcarbamate, Pd(OAc)2, K3PO4, 2-di-t-butylphosphino-20-(N,N-dimethylamino)biphe-
nyl, toluene 140 °C, 53%; (b) AcCl, MeOH, quantitative yield; (c) Ac2O, pyridine, DCM, 59%; (d) AcOH/HCl, at 65 °C, then EDA/MeOH/H2O, 65%.

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S. Jaime-Figueroa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2522–2526
Table 1
Enzyme potenciesb for compounds prepared according to Schemes 1 and 2
O
R'
N
H
HN
N
N
O
R
IC50
(l
M)
Selectivity
Compound
R
R’
JAK3
JAK2
JAK1
JAK2/JAK3
JAK1/JAK3
1a
1b
1c
1d
1e
1f
–H
–Me
–Me
–Me
–Me
–Me
–Me
–Me
–CyPr
0.21 (0.06)c
0.28 (0.07)
0.47 (0.06)
0.40 (0.09)
0.97 (0.36)
0.42 (0.04)
1.38 (0.47)
0.25 (0.06)
0.41 (0.10)
1.21 (0.10)
1.00 (0.29)
1.25 (0.02)
0.90 (0.15)
2.32 (0.41)
0.07 (0.008)
0.84 (0.05)
2.15 (0.15)
4.16 (0.19)
8.22 (0.80)
3.19 (0.30)
3.47 (0.71)
5.09 (0.17)
0.22 (0.05)
1.2
1.4
2.6
2.5
1.3
2.1
1.7
2.1
3.9
7.5
8.8
20.2
3.3
8.2
3.7
6.5
m-Me
m-Et
3,5-di-OMe
m-CN
p-CN
o-Me
–H
1g
1h
0.03 (0.004)
O
N
HN
N
H
1i
0.37 (0.06)
0.58 (0.12)
3.32 (0.09)
1.5
8.8
N
O
O
N
H
HN
N
N
O
1ja
0.34 (0.05)
1.71 (0.10)
4.39 (0.87)
4.9
12.6
O
NH
O
N
H
HN
N
N
O
1ka
0.14 (0.04)
1.01 (0.10)
6.30 (0.51)
7.1
43.9
O
NH
a
These compounds were prepared according with the Scheme 2.
Inhibition of phosphorylation of a biotinylated synthetic peptide catalyzed by JAK1-3.11 All enzyme reactions were run at adenosine triphosphate (ATP) concentrations of
b
1.5
l
M. Km’s of these enzymes for ATP under our experimental conditions were determined to be 1.5
lM (JAK3), 6 lM (JAK2), and 20 lM (JAK1).
c
Mean [SEM(standard error of the mean)], n P3.
phenol gave the (R)-Boc-aminoindanephenyl ether 8. Cleavage of
the t-butoxycarbonyl (Boc) group on compound 8 followed by
acetylation gave the aldehyde 9 in good yields. Pinnick oxidation
of this compound provided the corresponding carboxylic acid 10
in excellent yields. Finally, amidation of 10 using regular peptide
coupling conditions with the corresponding amine afforded com-
pounds 11 in good yields. Further SEM-deprotection provided the
final amides 12c–12e in acceptable yields (Scheme 3). Comparison
of the methyl, cyclopropyl, and t-butyl analogs in this subseries
showed that cyclopropyl compound 12d had the best potency for
JAK3 while maintaining selectivity over JAK1 (Table 2). We also se-
lected this compound for a KinomeScan profile (Fig. 5). Compound
12d showed exceptional kinase selectivity over a panel of 451 ki-
nases (394 wild-type).
Selected examples were further tested in cellular assays of tar-
get modulation. These assays measured inhibition of phosphoryla-
tion of downstream STATs, in peripheral blood mononuclear cells
(PBMCs) (Table 3). These analogs experienced a pronounced shift
in potency in the cellular context. This was likely a consequence
Figure 4. Compound 12a bound to JAK3 non-phosphorylated kinase domain
(2.35 Å resolution, PDB accesion number 3ZEP).16

S. Jaime-Figueroa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2522–2526
2525
O
H
O
O
H
OH
O
N
SEM
a
N
b, c
d
SEM
N
N
SEM
N
N
2
N
N
O
N
O
NHAc
NHBoc
NHAc
8
9
10
e
O
N
H
12a = (S)-CyPr
12b = (R)-CyPr
HN
R
R
N
O
O
R'
R'
N
N
N
O
H
H
f
NHAc
N
HN
SEM
N
N
N
N
O
O
NHAc
NHAc
CN
12
11a-11e
R
N
O
12c = (R)-Me
N
12d = (R)-CyPr
12e = (R)-t-Bu
O
H
HN
N
N
O
NHAc
Scheme 3. Reagents and conditions: (a) (R)-t-butyl 6-hydroxy-2,3-dihydro-1H-inden-1-ylcarbamate, Pd(OAc)2, K3PO4, 2-di-t-butylphosphino-20-(N,N-dimethylamino)biphe-
nyl, toluene 90 or 140 °C/sealed tube, 37%; (b) AcCl, MeOH, quantitative; (c) Ac2O, pyridine, DCM, 62%; (d) sulfamic acid, NaClO2, KH2PO4, dioxane/water, 75%; (e) R-NH2,
HATU, DMF, 22–71%; (f) TFA, EDA, DCM or TBAF, EDA, THF, 18–69%.
Table 2
Enzyme potenciesa for compounds prepared according to Scheme 3
Compound
IC50
JAK2
(lM)
Selectivity
JAK3
JAK1
JAK2/JAK3
JAK1/JAK3
12a
12b
12c
12d
12e
Tofacitinib
0.022 (0.009)b
0.014 (0.002)
0.112 (0.050)
0.005 (0.002)
0.057 (0.017)
0.002 (0.6)
0.084 (0.028)
0.194 (0.034)
1.35 (0.436)
0.039 (0.013)
0.446 (0.046)
0.004 (0.7)
0.863 (0.082)
2.076 (0.466)
11.6 (1.663)
0.262 (0.079)
0.863 (0.082)
0.002 (0.0001)
3.7
38.3
140.4
103.1
47.4
42.2
0.7
13.2
12.0
7.1
7.7
1.8
a
Inhibition of phosphorylation of a biotinylated synthetic peptide catalyzed by JAK1-3.11 All enzyme reactions were run at adenosine triphosphate (ATP) concentrations of
1.5
l
M. Km’s of these enzymes for ATP under our experimental conditions were determined to be 1.5
l
M (JAK3), 6
lM (JAK2), and 20
l
M (JAK1).
b
Mean (SEM (standard error of the mean)), n P3.
Table 3
Cellular potencies for selected compounds
a
Compound
PBMC IC50
(l
M)
c
JAK1/3 (IL-2)b
JAK2 (GM-CSF)b
JAK1/2 (IFNc)
12a
12b
12d
Tofacitinib
0.70
1.75
7.41
0.03
3.61
14.40
>30
>30
16.22
23.30
0.19
0.17
a
b
c
IC50 values are the average of at least two experiments, except for 12d (n = 1).
Inhibition of phosphorylation of STAT5a in PBMCs.
Inhibition of phosphorylation of STAT1 in PBMCs. The IL-2 readouts are gated to
CD3-expressing T-cells; the GM-CSF and IFN-
expressing monocytes.
c readouts are gated to CD14-
measured enzyme and cellular selectivity ratios was not easily
defined. All tested compounds, including tofacitinib, showed selec-
tivity to inhibit an IL-2 stimulated STAT5a phosphorylation (a
Figure 5. Kinomescan dendrogram of 12d versus 451 kinases (394 wild-type) at
1 lM. JAK3 is shaded in blue.
JAK3/1-dependent process) versus GM-CSF and IFNc-stimulated
phosphorylations (JAK2 and JAK1/2-dependent processes).
Compared to tofacitinib, analogs from our series appeared to show
of both an expected potency shift moving to a high concentration
of ATP in the cellular environment, and the unoptimized physical
chemical properties of these compounds. The relationship between
improved selectivity inhibiting IL-2 signaling versus IFN
c signal-
ing; however, improvements versus GM-CSF signaling were mod-

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est or non-existent. Our compounds have improved selectivity
against JAK3 versus JAK2, but they also have improved selectivity
against JAK2 versus JAK1. Cellular selectivities between IL-2 and
GM-CSF signaling likely depend on both JAK2/3 and JAK2/1 selec-
tivities. Therefore, the observed modest cellular selectivities for
our compounds inhibiting IL-2 signaling versus GM-CSF signaling
may be due to the diminished relative potencies against JAK1,
and highlight the complexity of JAK/STAT signaling.
In summary, we discovered a series of phenyl ethers as potent
JAK3 inhibitors with good, JAK family and kinase selectivity. We
demonstrated by a structure-based approach that phenyl ethers
are good scaffolds for achieving JAK family selectivity. The ex-
pected lipophilic interaction with Cys909 and a novel polar back-
bone interaction of the inhibitor with the Gly-loop may explain
the excellent kinase selectivity observed within this series.
Supplementary data
12. Compound 1h showed very modest selectivity kinomescan profile S(65) = 203
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.015.
over a panel of 451 kinases (394 wild-type) at 10 lM.
13. The X-ray crystal structure was determined as described previously in Ref. 10.
14. Hendricks, R. T.; Hermann, J. C.; Jaime-Figueroa, S.; Kondru, R. K.; Lou, Y.;
Lynch, S. M.; Owens, T. D.; Soth, M.; Yee, C. W. Pyrrolo[2,3-b]pyrazine-7-
carboxamide derivatives as JAK and SYK inhibitors and their preparation and
use for the treatment of autoimmune and inflammatory diseases. Pat. Int. Appl.
2011, WO 2011144585 A1 20111124.
References and notes
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Am. Chem. Soc. 1999, 121, 4369.
16. The X-ray crystal structure was determined by Proteros Biostructures GmbH
(Maertinsried Germany).
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