2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.09.080

A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core structure is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substituent of the purinone, compound 24 was identified incorporating a chroman-based functional group. Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-γ (INF-γ) production.

Full text of DOI:10.1016/j.bmcl.2009.09.080

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6788–6792
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Benzimidazolyl-9-(chroman-4-yl)-purinone derivatives as JAK3 inhibitors
*
Andrew G. Cole , Adolph C. Bohnstedt, Vidyadhar Paradkar, Celia Kingsbury, Jorge G. Quintero,
Haengsoon Park, Yingchun Lu, Ming You, Irina Neagu, David J. Diller, Jeffrey J. Letourneau, Yuefei Shao,
Ray A. James, Christopher M. Riviello, Koc-Kan Ho, Tsung H. Lin, Bojing Wang, Kenneth C. Appell,
Matthew Sills, Elizabeth Quadros, Earl F. Kimble, Michael H. J. Ohlmeyer, Maria L. Webb
Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core struc-
ture is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substi-
tuent of the purinone, compound 24 was identified incorporating a chroman-based functional group.
Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in
Received 16 August 2009
Accepted 22 September 2009
Available online 25 September 2009
an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-
(INF- ) production.
c
Keywords:
JAK3
Janus kinase
Rheumatoid arthritis
Purinone
c
Ó 2009 Elsevier Ltd. All rights reserved.
Benzimidazole
Chroman
The Janus kinase (JAK) family of cytoplasmic protein tyrosine
kinases consists of four members (JAK1, JAK2, JAK3 and TYK2) that
help regulate cellular functions in the lympho-hematopoietic system
thatarecriticalforcellproliferation, survivalanddifferentiation.JAK3
has limited tissue distribution, specifically expressed in T cells and
other hematopoietic lineages, whereas the other JAK’s are ubiqui-
tously expressed in both hematopoietic and non-hematopoietic lin-
eages. JAK3 interacts with cytokine receptors that contain the
highly homologous to JAK3. JAK2 plays a critical role in signaling via
many cytokines, such as erythropoietin, granulocyte-macrophage
colony-stimulating factor and thrombopoietin. Inhibition of JAK2
may result in anemia, granulocytopenia and thrombocytopenia. A
number of JAK3 inhibitors have been reported,5 including CP-
690,550 (Fig. 1), which is currently in phase III clinical trials.6 The
aim of this research was to identify novel and potent JAK3 inhibitors
with selectivity over JAK2 to avoid potential side effects.
gamma common chain (
c
c), the receptor subunit for interleukins
Compound 5a (Table 1) was identified as an early JAK3 inhibi-
tor, exhibiting an IC50 value of 115 nM in a JAK3 kinase assay
and approximately 28-fold selectivity versus JAK2.7 Preparation
of 5a was achieved through regioselective arylation of 4-aminotet-
rahydropyran with 2,4-dichloro-5-nitropyrimidine8 (1) to give 2
(Scheme 1). Displacement of the 2-position chlorine of 2 with
benzimidazole provided 4 (R = H) which was then followed by
reduction of the nitro group9 and generation of the purinone via
cyclization with 1,10-carbonyldiimidazole.10
(IL)-2, -4, -7, -9, -15 and -21.1 Interleukin binding to the
cc cytokine
receptor initiates heterodimerization of JAK3 and JAK1 resulting in
JAK activation, which in turn, phosphorylates and activates signal
transducers and activators of transcription (STAT). STATdimerization
leads to migration to the nucleus where they regulate gene expres-
sion. Dysfunction of the cc-JAK3 signaling results in severe combined
immunodeficiency(SCID)phenotype.2,3 Currentimmunosuppressive
drugs have narrow therapeutic indices as a consequence of toxicity
related to the ubiquitous distribution of their targets. For example,
the molecular target of the immunosuppressants Tacrolimus and
Cyclosporin is calcineurin which is widely expressed in all tissues.
Thus, the development of a selective JAK3 inhibitor may provide a no-
vel immunosuppressive agent that gives an optimal therapeutic win-
dow for the treatment of immune cell-mediated diseases.4 JAK2 is
H
N
N
N
N
O
CN
N
* Corresponding author. Tel.: +1 609 452 3653; fax: +1 609 452 3671.
E-mail address: acole@ligand.com (A.G. Cole).
In 2008, Pharmacopeia, Inc. was acquired by Ligand Pharmaceuticals, Inc.
CP-690,550
Figure 1. JAK3 Inhibitor CP-690,550.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.080

A. G. Cole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6788–6792
6789
Table 1
Table 2
R1
O
R2
O
N
N
N
N
N
O
N
N
N
H
N
N
H
Compound
R
JAK3 IC50 (nM)
JAK2 IC50 (nM)
3250
Compound
R1
R2
JAK3 IC50 (nM)
JAK2 IC50 (nM)
N
6a
6b
6c
6d
6e
6f
6g
6h
6i
CN
CN
CN
CN
CN
CN
F
CN
F
CN
CN
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2,3-Difluorophenyl
2,3-Difluorophenyl
2,6-Difluorophenyl
2,6-Difluorophenyl
2,6-Dichlorophenyl
76a
245a
1450a
25a
38a
70a
108
17
930a
4000a
3200a
260a
1000a
230a
1000
270
5a
5b
115
N
N
N
245
68
1800
560
N
F3C
N
5c
5d
5e
5f
70
6
320
28
1550
F3C
6j
6k
N
220
N
38
14
7
3400
393
a
Tested as a mixture of 5- and 6-substituted benzimidazoles.
F
N
N
Difluorinated analogs (6g–6j) provided a further increase in po-
tency, with the 2,6-difluorobenzyl analog 6j displaying an IC50 va-
lue of 6 nM.
Cl
N
Based on the inhibitory activity of the 6-substituted benzimid-
azole analogs, a regiospecific process whereby the benzimidazole
was constructed via arylation of a suitably protected substituted
dianiline was investigated (Schemes 2–4). The devised synthesis
would ideally allow late incorporation of the N-9 substituent to al-
low further exploration of the SAR related to this position.
For generation of the 6-cyano-substituted benzimidazole, inter-
mediate 9 was synthesized in two steps (Scheme 2) via arylation of
2,4-dimethoxybenzylamine with 4-fluoro-3-nitrobenzonitrile (7)
followed by nitro group reduction.
For generation of the 6-fluoro, 6-chloro and 6-trifluoromethyl-
substituted benzimidazoles, a similar monoprotected dianiline
intermediate (12) was synthesized from the corresponding ortho-
aminonitrobenzene 10 (Scheme 3).
Boc protection of 10 using dimethylaminopyridine (DMAP) re-
sulted in the generation of a bis-boc intermediate, which was par-
tially deprotected with stoichiometric trifluoroacetic acid to
provide 11. Subsequent nitro group reduction of 11 provided 12.
Construction of the benzimidazole moiety at the 2-position of
the purinone core required manipulation of pyrimidine precursor
1 to suppress reactivity at the 4-position. Consequently, reaction
of 1 with sodium thiocyanate provided 13, which reacts preferen-
N
470
NC
Reaction of a 5-monosubstituted benzimidazole 3 with 2 pro-
vided a mixture of the 5- and 6-position regioisomers 4, which
were separated by flash chromatography. A two to 15-fold increase
in kinase activity was observed on incorporation of electron with-
drawing substituents in the 6-position of the benzimidazole (5c–f),
with the 6-cyano analog 5e exhibiting an IC50 value of 7 nM in the
kinase assay. The 5-trifluoromethyl-substituted benzimidazole
derivative 5b was approximately fourfold weaker than the corre-
sponding 6-position isomer 5c. Of the initial analogs synthesized,
5d and 5f demonstrated the highest level of selectivity versus
JAK2 (89- and 67-fold, respectively).
Concurrent studies incorporating a substituted benzyl group at
N-9 of the purinone core using a similar procedure to that outlined
in Scheme 1 demonstrated that halogenated benzylic N-9 substit-
uents also provided compounds with good JAK3 inhibitory activity.
Compounds were either tested as a mixture of 5- and 6-substituted
benzimidazoles, or where possible, separated to provide the 6-
substituted analogs (Table 2). Monofluorinated benzylic substitu-
ents at the N-9 position (6d–f) provided increased potency in com-
parison to the corresponding monochloro analogs (6a–c).
O
O
O
O
NH
NH
F
O
b
a
R
N
NC
NH2
NO2
NC
NO2
NC
9
N
H
Cl
N
Cl
Cl
N
NH
7
8
a
b,
3
N
N
Scheme 2. Reagents and conditions: (a) 2,4-dimethoxybenzylamine, iPr2NEt, THF,
25 °C; (b) Na2S2O4, NaHCO3, THF, H2O, MeOH, 25 °C.
NO2
NO2
2
1
O
O
N
N
R = F, Cl or CF3
Boc
NH
Boc
NH
N
N
N
N
NH
N
NH2
c,d
O
c
a, b
N
N
R
R
N
H
NO2
R
NO2
R
NO2
R
NH2
5
4
10
12
11
Scheme 1. Reagents and conditions: (a) 4-aminotetrahydropyran, iPr2NEt, THF,
ꢀ78 °C; (b) iPr2NEt, THF, 25 °C; (c) Na2S2O4, NaHCO3, THF, H2O, MeOH, 25 °C; (d)
1,10-carbonyldiimidazole, CH2Cl2, 25 °C.
Scheme 3. Reagents and conditions: (a) ditertbutyldicarbonate, DMAP, CH2Cl2,
25 °C; (b) TFA, CH2Cl2, 25 °C; (c) Na2S2O4, NaHCO3, THF, H2O, MeOH, 25 °6C.

6790
A. G. Cole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6788–6792
Table 3
Pg
NH
Chroman-based JAK3 inhibitors
H
N
N
SCN
NO2
Cl
N
Cl
Cl
N
SCN
NO2
a
b
N
N
N
NO2
R1
N
13
14
1
N
N
N
(Pg = Protecting group)
O
N
N
H
N
R2
N
N
e
Compound
R
JAK3 IC50 (nM)
95
JAK2 IC50 (nM)
170
N
SCN
NO2
N
N
NH
c,d
N
N
O
O
NO2
R1
R1
21a
15
16
N
R2
N
f, g
N
N
21b
48
28
97
83
O
N
N
H
R1
F
17
O
O
O
Scheme 4. Reagents and conditions: (a) sodium thiocyanate, EtOH, 0 °C; (b) 9 or 12,
K2CO3, CH3CN, 25 °C; (c) TFA, Et3SiH, CH2Cl2, 25 °C (Pg = dimethoxybenzyl) or TFA,
CH2Cl2, 25 °C (Pg = Boc); (d) trimethylorthoformate, MeOH, 25 °C; (e) R2–NH2, THF,
iPr2NEt, 25 °C; (f) Na2S2O4, NaHCO3, THF, H2O, MeOH, 25 °C; (g) 1,10-carbonyldi-
imidazole, THF, 25 °C.
21c
F
21d
365
260
30
500
95
Cl
F
O
O
O
N
O
21e
a
b
R
R
R
F
Cl
F
NH2
HO
O
O
O
O
19
18
20
21f
285
840
333
7000
Scheme 5. Reagents and conditions: (a) hydroxylamine hydrochloride, NaOAc,
EtOH, 25 °C; (b) H2, Raney Ni, EtOH, 25 °C.
21g
170
28
tially at the 2-position with the protected dianiline (9 or 12) to give
14 (Scheme 4). Protecting group removal under acidic conditions
followed by reaction with trimethylorthoformate provided 15,
effectively controlling the regiochemistry of the 6-substituted
benzimidazole.
Incorporation of the N-9 substituent can subsequently be
achieved by thiocyanate displacement of 15 with a primary amine
(R2–NH2) to provide 16, which is converted to the purinone ana-
logs 17 via nitro reduction and ring formation with 1,1’-carbonyl-
diimidazole. Generation of analogs incorporating the N-9
substituent as tetrahydropyran confirmed the regiochemical
assignment of 5c–f as the 6-substituted benzimidazole.
A study was initiated whereby the structural features of the THP
functionalized analogs 5 and the substituted benzyl analogs 6 were
combined, resulting in the incorporation of a series of substituted
chromans at N-9 of the purinone. 4-Aminochroman synthesis
was achieved via catalytic hydrogenation of an oxime 19 generated
from the corresponding chromanone 18 to provide racemic 20
(Scheme 5).
(R)-21f
(S)-21f
F
540
The stereochemical assignment of the preferred (R)-enantiomer
was made following resolution of 8-fluoro-4-aminochroman using
Lipase B (Scheme 6).11 Enzymatic acylation of the (R)-enantiomer
of 22 allowed separation of the components to give (S)-22 and sub-
sequent acid mediated hydrolysis of 23 provided (R)-22. Incorpo-
F
F
F
The unsubstituted chroman analog 21a, incorporating benz-
imidazole at the 2-position, showed moderate JAK3 potency (Table
3), comparable to that of the corresponding THP derivative 5a.
JAK2 selectivity, however, was significantly reduced in comparison
to 5a. Fluorination at the 5- (21b), 6- (21c) and 8-positions (21f) of
the chroman showed a slight increase in inhibitory activity,
whereas the 7-fluorochroman analog 21e was approximately
threefold weaker. As with the substituted benzylic analogs 6, chlo-
rine substitution (21d and 21g) was found to be inferior to both
fluorine and hydrogen. Of the substituted chroman-based analogs,
21f provided the best balance between JAK3 activity and JAK2
selectivity (ꢁ10-fold), and subsequent resolution of the stereoiso-
mers of 21f demonstrated a preference for the (R)-enantiomer
((R)-21f) of the N-9 chromanyl purinone.
O
O
O
a
+
NH2
HN
NH2
OMe
(S)-22
22
23
O
b
F
O
NH2HCl
(R)-22
Scheme 6. Reagents and conditions: (a) novozyme 435 (Candida antarctica),
tBuOMe, methoxymethylacetate, 25 °C; (b) HCl, EtOH, 25 °C.

A. G. Cole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6788–6792
6791
-
Table 4
(R)-8-fluorochroman-based JAK3 inhibitors
F
600
400
200
O
N
N
O
N
N
-
H
Compound
R
JAK3 IC50 (nM)
28
JAK2 IC50 (nM)
333
N
0
(R)-21f
N
N
N
23
24
25
15
3
56
26
5
Cl
F
N
Figure 2. Efficacy of 24 in an acute mechanistic IL-2 mouse model.
N
dependent reduction in IFN-c was observed resulting in an ED50 of
5 mpk (n = 2) (Fig. 2).
N
N
1
In summary, key structural features associated with 2-benzim-
idazolyl-9-(chroman-4-yl)-purinone derivatives as potent JAK3
inhibitors have been identified. The development of a regioselec-
tive synthesis has allowed the substitution pattern of the benz-
imidazole moiety to be explored. In addition, combination of SAR
related to N-9 substitution has led to extremely potent JAK3 inhib-
itors with excellent pharmacokinetic properties, allowing the dem-
onstration of in vivo efficacy in an acute mechanistic mouse IL-2
model. Further optimization of this series of inhibitors with respect
to JAK3 potency, JAK2 selectivity and in vivo characterization will
be reported elsewhere.
NC
Table 5
Pharmacokinetic parameters for 24 in female Balb/c mice at 10 mpk po and 2 mpk iv
F
F
O
N
N
N
N
O
N
N
H
References and notes
24
AUCpo
Fpo (%)
13,224 ng h/mL
100
5472
14.6
1.41
0–1
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A significant increase in potency was observed for the chloro-,
fluoro- and cyano-substituted benzimidazoles, in comparison to
(R)-21f, with 24 and 25 showing a 10- and 30-fold increase in
JAK3 kinase activity, respectively. Moderate JAK2 selectivity was
observed for 24 and 25 (nine and fivefold, respectively).
The 6-fluorobenzimidazole analog 24 demonstrated good JAK3
kinase activity and approximately ninefold selectivity over JAK2
in the kinase assay, and was selected for further characterization.
Oral dosing in mice showed that 24 was orally bioavailable and
had an excellent PK profile (Table 5).
The favorable PK profile exhibited by 24 supported further
in vivo characterization of the compound in an acute mechanistic
mouse model whereby IL-2-induction of IFN-c production in mice
is determined. Compound 24 was administered orally to female
Balb/c mice and after 1 h, IL-2 (100,000 IU/mouse) or its carrier
PBS was administered ip. After an additional 3 h, the mice were
sacrificed and the blood was collected by cardiac puncture. The
generated using
a baculovirus expression system. Test compounds were
incubated with JAK3 or JAK2, the peptide substrate biotinylated poly Glu-Ala-
Tyr, and ATP for 60 min at room temperature. The tyrosine phosphorylation of
biotinylated substrate was detected using Europium (LANCE) labeled anti-
amount of INF-
24 was efficacious in reducing the IFN-
c
in serum was determined by ELISA. Compound
response to IL-2. A dose-
c

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