Development of new pyrrolopyrimidine-based inhibitors of Janus kinase 3 (JAK3)

Article abstract of DOI:10.1016/j.bmcl.2006.12.018

A new class of bicyclic pyrrolopyrimidine-based Janus kinase 3 (JAK-3) inhibitors are described. Many of these inhibitors showed low nanomolar activity against JAK-3.

Full text of DOI:10.1016/j.bmcl.2006.12.018

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1250–1253
Development of new pyrrolopyrimidine-based
inhibitors of Janus kinase 3 (JAK3)
Michael P. Clark,b,* Kelly M. George,a Roger G. Bookland,a Jack Chen,a
Steven K. Laughlin,a Kumar D. Thakur,a Wenlin Lee,a Jan R. Davis,a
Ed J. Cabrera,a Todd A. Brugel,a John C. VanRens,a Matthew J. Laufersweiler,a
Jennifer A. Maier,a Mark P. Sabat,a Adam Golebiowski,a Vijay Easwaran,a
Mark E. Webster,a Biswanath Dea and George Zhanga
aProcter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 45040, USA
bMetastatix, Inc., 1860 Montreal Road, Tucker, GA 30084, USA
Received 7 November 2006; revised 2 December 2006; accepted 4 December 2006
Available online 9 December 2006
Abstract—A new class of bicyclic pyrrolopyrimidine-based Janus kinase 3 (JAK-3) inhibitors are described. Many of these inhibitors
showed low nanomolar activity against JAK-3.
Ó 2006 Elsevier Ltd. All rights reserved.
Janus kinases (JAK), including JAK1, JAK2, Tyk2, and
JAK3, are a small family of cytoplasmic protein tyrosine
kinases which play pivotal roles in the initiation of cyto-
kine-triggered signaling events by activators of
transcription (STAT) proteins via tyrosine phosphoryla-
tion.1–3 Upon phosphorylation, the STAT proteins
dimerize and translocate to the nucleus where they can
then induce transcription of the corresponding cyto-
kine-responsive genes. This association with relevant
cytokine signaling pathways makes JAK3 an important
target for therapeutic intervention in the treatment of
autoimmune disorders, inflammatory diseases, and
organ transplant rejection. An important feature of
JAK3 is that it specifically associates with the common
cytokine receptor gamma chain (cc) which is a shared
component of the receptors for IL-2, IL-4, IL-7, IL-9,
and IL-15. Unlike the other JAK family members, that
are more widely expressed, JAK3 expression seems to be
mainly limited to hematopoietic cells. The unique inter-
action with the cytokine receptor gamma chain, along
with its limited expression, makes JAK3 an attractive
therapeutic target relative to the other JAK family
members.
In the relatively young field of Janus kinase inhibition,
Pfizer has already progressed their JAK3 inhibitor,
CP-690,550, into Phase II clinical trials for acute
rejection in kidney transplant patients4 (Fig. 1). Other
JAK3 inhibitors have been reported, such as the non-se-
lective (within JAK family) but very potent tetracyclic
pyridone 2, reported by Merck.5 Aventis has published
an oxindole inhibitor, 3, that is one of the only
published JAK3 inhibitors that shows good enzymatic
selectivity vs JAK2.6 However, this oxindole inhibitor
also shows very strong inhibition vs a panel of CDK
kinases.
Our search for a new structural class of JAK3 inhibitors
resulted in the identification of two novel classes of com-
pounds, a previously published trisubstituted pyrimidine
class7 and a new potent pyrrolopyrimidine class of com-
pounds, exemplified by 4.
Synthesis of the bicyclic pyrrolopyrimidine 4 was
accomplished in seven steps from readily available
Keywords: JAK3; Janus Kinase; Tyrosine kinases; Cytokines;
Pyrrolopyrimidine.
*
dichloropyrimidine carboxaldehyde 5 (Scheme 1).
Displacement of the first chloride with sarcosine
Corresponding author. Tel.: +1 404 863 4275; e-mail:
mclark@metastatix.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.018

M. P. Clark et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1250–1253
1251
two regioisomers was accomplished via meticulous
silica gel chromatography to yield the desired 6-aza-
benzimidazole analog. Standard manipulations of the
ester functionality provided final amides, such as
pyrrolopyrimidine 4.
H
N
H
N
N
O
N
F
N
O
N
N
HN
NC
Table 1 summarizes a survey of bicyclic pyrrolopyrim-
idine analogs. All compounds were tested for inhibi-
tion of JAK3 and JAK2 using the Kinase-Glo
Luminescent Assay.8 We found a remarkable differ-
ence in potency between the 6-azabenzimidazole and
the corresponding 5-azabenzimidazole regioisomers
(data not shown). The 6-azabenzimidazole analogs
typically showed a 4-fold to 15-fold increase in poten-
cy (both JAK3 and JAK2) relative to the 5-azabenz-
imidazoles. In most cases, the most potent analogs
in this series show either equipotency between JAK3
and JAK2 (compounds 12, 13, and 15) or in some
cases, a stronger potency for JAK2 than JAK3 (com-
pounds 4, 10, 11, 14, and 16). Interestingly, the Pfizer
inhibitor CP-690,550, reported to possess 20:1 selectiv-
ity favoring JAK3 over JAK2,1 shows virtually no
selectivity in either the Kinase-Glo assay (Table 1),
or in a separate radiometric filter binding assay.9
While the analogs substituted at the 4-position with
a (S)-a-methylbenzylamino substituent showed stron-
ger potency, it comes at the expense of selectivity ver-
sus JAK2. Analogs that incorporate an alkylamino
substituent at the 4-position of the pyrrolopyrimidine
core show hints of JAK3:JAK2 enzymatic selectivity,
exemplified by 17 and 19–21. Analysis of the 2-amido
substituent SAR shows that it has a less significant
role in potency, and provides a good opportunity
for fine tuning selectivity, along with other properties,
such as solubility.
2
CP-690,550
N
H
N
N
H
N
O
N
N
H
N
N
N
N
N
O
N
3
4
Figure 1. JAK3 inhibitors.
tert-butyl ester, followed by base induced cyclization
of the resulting aminoaldehyde, gave the core bicy-
clic pyrrolopyrimidine ring system 6. Oxidation of
the thiomethyl group with m-CPBA, followed by
reaction of the resulting sulfoxide with 5-azabenzim-
idazole under basic conditions at elevated tempera-
ture, yielded a mixture of 5-azabenzimidazole and
6-azabenzimidazole analogs, typically favoring the
5-azabenzimidazole regioisomer. Separation of the
Cl
O
We then utilized a TF-1 cellular assay10 to further
measure the responses through either the JAK3 (IL-
4 induced proliferation) or the JAK2 (IL-3 induced
proliferation) signaling pathways, respectively (Table
2). The Pfizer benchmark, CP-690,550, shows a 10:1
selectivity in favor of JAK3 in the TF-1 cellular as-
says. Our pyrrolopyrimidine analogs typically showed
moderate selectivity in the 2- to 5-fold range in favor
of JAK3. The most promising analogs screened in
the TF-1 cell proliferation assay, pyrrolopyrimidines
18 and 19 show a 4-fold and 10-fold selectivity in fa-
vor of JAK-3, respectively. Once again, the 4-alkyla-
mino substituted analogs generally showed improved
selectivity versus JAK2 than the 4-benzylamino
analogs.
Cl
c
O
O
N
H
a, b
N
N
MeS
S
N
Cl
MeS
N
5
6
N
H
N
N
N
H
N
N
N
N
d, e
N
N
N
O
7
O
O
O
8
N
+ 5-azabenzimidazole regioisomer (9)
N
H
N
N
N
N
We have reported a novel series of bicyclic pyrrolo-
pyrimidine Janus Kinase inhibitors. Many of the
pyrrolopyrimidine inhibitors showed strong potency
in the JAK3 enzymatic assay below the 100 nM
level. Several of these analogs showed promising
selectivity for JAK3 relative to JAK2. One of the
pyrrolopyrimidines, 19, showed comparable potency
and selectivity in the TF-1 cell proliferation assay
to the Pfizer benchmark CP-690,550. Further optimi-
zation of this novel scaffold may be reported in
future publications.
f, g
N
N
O
N
4
Scheme 1. Reagents and conditions: (a) sarcosine-tert-butyl ester-HCl,
Et3N, THF, 75%; (b) NaH, DMF, 0 °C, 58%; (c) (S)-a-methyl
benzylamine, iPr2NEt, DMF, 90 °C, 86%; (d) m-CPBA, CH2Cl2, 0 °C;
(e) 5-azabenzimidazole, K2CO3, DMF, 140 °C, 74%; (f) CF3CO2H,
i
CH2Cl2; (g) 1-methylpiperazine, EDAC, HOBt, Pr2NEt, THF, 68%
—2 steps.

1252
M. P. Clark et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1250–1253
Table 1. JAK3 and JAK2 data for bicyclic pyrrolopyrimidine analogs
N
N
N
R1
N
N
N
R2
O
R1
—
R2
—
a
JAK3 IC50 (nM)
a
JAK2 IC50 (nM)
Compound
CP-690,550
13
11
H
N
N
4
22
11
N
N
H
N
NH
O
10
11
12
13
14
15
16
17
18
19
20
21
22
18
6
H
N
NH
34
19
H
N
SO2Me
42
55
N
H
H
N
NH
46
42
N
N
O
H
N
N
49
21
O
H
N
NH
67
53
N
H
N
NH
N
77
25
O
N
N
N
H
N
126
142
142
143
200
430
222
132
209
359
323
298
O
H
N
N
O
Cl
H
N
N
OH
H
N
SO2Ph
N
H
H
N
N
N
N
N
H
N
H
N
N
23
3050
1860
N
F
a S.D. for enzyme assays were typically 30% of the mean or less.

M. P. Clark et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1250–1253
1253
Table 2. Inhibition of proliferation of TF-1 cells, induced by either
IL-3 or IL-4, by pyrrolopyrimidine analogs
Thurairatnam, S.; Vine, M.; Wyman, B. Bioorg. Med.
Chem. Lett 2003, 13, 3105.
7. Chen, J. J.; Thakur, K.; Clark, M. P.; Laughlin, S. K.;
George, K. M.; Davis, J. R.; Cabrera, E. J.; Easwaran, V.; De,
B.; Zhang, G. Y. Bioorg. Med. Chem. Lett. 2006, 16, 5633.
8. Promega’s Kinase-GloTM Luminescent Kinase Assay is a
homogeneous non-radioactive method of determining the
activity of purified kinases by quantifying the amount of
ATP remaining in solution following a kinase reaction.
The assay procedure involves addition of a single reagent
(Kinase-GloÒ Reagent) directly to a completed kinase
reaction. This addition results in the generation of a
luminescent signal correlated with the amount of ATP
present and inversely proportional to the amount of
kinase activity.
Compound Inhibition of IL-4
induced TF-1 cell
Inhibition of IL-3
induced TF-1 cell
a
proliferation IC50 (nM) proliferation IC50 (nM)
b
CP-690,550
4
80b
80
800b
90
10
11
13
14
17
18
19
450
740
460
910
1950
2790
1400
4790
2200
1420
670
1160
490
140
9. Kinase ProfilerTM Assays (radiometric filter binding
assays) were run by Upstate Cell Signaling Solutions
(JAK3 IC50 ¼ 5 nM , JAK2 IC50 ¼ 6 nM).
a S.D. for enzyme assays were typically 25% of the mean or less.
b Mean, n > 10.
10. All compounds were tested using an assay developed using
TF-1 (1, 2) cells (CRL-2003 from ATCC). These cells are
unique because they respond to multiple cytokines. They
proliferate in response to GM-CSF, IL-3 (3), and IL-4.
They were maintained as recommended by the ATCC. IL-3
(Human recombinant Sigma #I 1646) or IL-4 (Human
recombinant. Sigma #I 4269) at 1 nM was used to
stimulate the cells. Proliferation of the TF-1 cells was used
to measure the responses through JAK2 (IL-3) or JAK3
(IL-4) and the inhibition of such response by specific
compounds. Five replicates per compound were seeded at a
concentration of 5000 cells/well in a 100-ll volume. Five
different concentrations of experimental compounds (five
replicates each) were used to determine their EC50. After 4
days of incubation, cell proliferation was measured during
the last 18 h of incubation, using an ELISA (BrdU,
chemiluminescence) kit (Roche Diagnostics, Indianapolis,
IN). The relative luminescence units (RLU) were measured
using a Packard Fusion instrument (Perkin-Elmer). The
RLU were used to calculate EC50s (Prism4 software).
References and notes
1. Thomis, D.; Berg, L. Curr. Opin. Immunol. 1997, 9, 541.
2. O’Shea, J. J.; Pesu, M.; Borie, D. C.; Changelian, P. S.
Nat. Rev. Drug Disc. 2004, 3, 555.
3. Notarangelo, L.; Giliani, S.; Mazza, C. Immunol. Rev.
2000, 178, 39.
4. Changelian, P. S.; Flanagan, M. E.; Ball, D. J. Science
2003, 302, 875.
5. Thompson, J. E.; Cubbon, R. M.; Cummings, R. T.;
Wicker, L. S.; Frankshun, B. R.; Cunningham, B. R.;
Cameron, P. M.; Meinke, P. T.; Liverton, N.; Weng, Y.;
DeMartino, J. A. Bioorg. Med. Chem. Lett. 2002, 12, 1219.
6. Adams, C.; Aldous, D. J.; Amendola, S.; Bamborough, P.;
Bright, C.; Crowe, S.; Eastwood, P.; Fenton, G.; Foster,
M.; Harrison, T. K. P.; King, S.; Lai, J.; Lawrence, C.;
Letallec, J.-P.; McCarthy, C.; Moorcroft, N.; Page, K.;
Rao, S.; Redford, J.; Sadiz, S.; Smith, K.; Souness, J. E.;