In vitro and in vivo evaluation of 6-aminopyrazolyl-pyridine-3- carbonitriles as JAK2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.03.053

Synthesis and biological evaluation of a series of 6-aminopyrazolyl- pyridine-3-carbonitriles as JAK2 kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and pharmacodynamic effects.

Full text of DOI:10.1016/j.bmcl.2011.03.053

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2958–2961
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro and in vivo evaluation of 6-aminopyrazolyl-pyridine-3-carbonitriles as
JAK2 kinase inhibitors
⇑
,
Tao Wang a, , Stephanos Ioannidis a, , Lynsie Almeida a, Michael H. Block a, Audrey M. Davies a,
Michelle L. Lamb a, David A. Scott a, Mei Su a, Hai-Jun Zhang a, Marat Alimzhanov b, Geraldine Bebernitz b,
Kirsten Bell b, Michael Zinda b
a Department of Cancer Chemistry, Oncology Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
b Department of Cancer Bioscience, Oncology Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and biological evaluation of a series of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2
kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in
JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and
pharmacodynamic effects.
Received 22 February 2011
Revised 14 March 2011
Accepted 15 March 2011
Available online 21 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
JAK2 kinase inhibitors
Myeloproliferative neoplasms
Myeloproliferative neoplasms (MPNs) are a group of clonal
hematopoietic stem cell disorders that include polycythemia vera
(PV), essential thrombocythemia (ET), and idiopathic myelofibrosis
(IMF), all of which have the potential to transform to acute myeloid
leukemia (AML).1,2 Several research groups have independently
identified a single point mutation in the Janus-associated Kinase
2 (JAK2) encoding a valine-to-phenylalanine change (V617F).3–5
JAK2 belongs to a family that comprises four different cytoplasmic
protein tyrosine kinases: JAK1, JAK2, JAK3 and TYK2. JAKs play an
important role in cellular survival, proliferation, and differentia-
tion.6 JAK2 V617F is constitutively phosphorylated and able to acti-
vate downstream signaling in the absence of cytokine stimulation
when transfected into factor-dependent cell lines.7 This mutation
has been found in around 50% in ET and IMF patients, 95% in PV
patients.8–10
F
H
N
N
NH
N
F
N
HN
1a
Figure 1. 6-Aminopyrazolyl-pyridine-3-carbonitrile 1a.
propyl for compound 1a) on the pyrazole ring is packed against the
methionine gatekeeper of JAK2 kinase. As described previously,23
the steric bulk of this substituent affects the potency against
JAK2 and selectivity versus other kinases. As the data in Table 1
demonstrates, replacing the cyclopropyl group with larger lipo-
philic groups such as an OiPr group (1b) resulted in a drop in
JAK2 enzyme potency. An analogue with a smaller R1 group such
as methyl (1c, AZ960)25,26 showed less than 3 nM potency against
JAK2 enzyme. While analogues with the cyclopropyl and methyl
substituents at R1 showed comparable potencies at biochemical le-
vel (e.g., 1a vs c), in a TEL-JAK2 proliferation assay in Ba/F3 cells
where the fusion of oligomerization domain TEL with the kinase
domain of JAK2 resulted in constitutive kinase activity, 1c had po-
There are a number of inhibitors against JAK2 along with other
kinases11–18 and several JAK2 kinase inhibitors are currently in
clinical development against MPNs.19–21 In the context of our
interest in this target,22–24 a subset screening of the AstraZeneca
compound collection identified compound 1a (Fig. 1) as a JAK2
inhibitor (JAK2 kinase IC50: 0.003 lM). Compound 1a belongs to
the 6-aminopyrazolyl-pyridine-3-carbonitrile structural class.
With such scaffolds we anticipate that the pyrazol-3-ylamine
occupies the adenine binding-site while the C5 substituent (cyclo-
tency of 0.024 lM versus 0.17 lM for the cyclopropyl analogue 1a.
These compounds were 3- to 50-fold selective against JAK3 in the
enzyme assay.
⇑
Corresponding author. Tel.: +1 781 839 4734; fax: +1 781 839 4230.
E-mail address: tao.wang@astrazeneca.com (T. Wang).
These authors have contributed equally to the preparation of this manuscript.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.053

T. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2958–2961
2959
Table 1
Cl
Cl
N
N
O
6-Aminopyrazolyl-pyridine-3-carbonitriles structure–activity relationships
a
Cl
b
R2
H
NH
N
N
N
O
7
NH
6
N
8
R1
N
Cl
HN
Cl
Cl
Cl
c
d
Y
Cl
N
HO
N
1a-e
O
Cl
O
Cl
9
F
10
Compd R1/R2/Y
JAK2 IC50
JAK3 IC50
M)
TEL-JAK2b
M)
TEL-JAK3b
(lM)
a
a
Cl
Cl
(l
M)
(l
(l
Cl
Cl
f
e
1a
Cyclo-Pr/
F/CH
0.003
0.183
0.17
1.00
H2N
N
N
OiPr/F/CH
Me/F/CH
Me/F/N
Me/Cl/N
0.688
1.735
0.009
0.007
0.021
NTc
0.024
0.036
0.04
NTc
0.35
1.30
0.43
N
1b
1c
1d
1e
O
Cl
Cl
12
<0.003
<0.003
<0.003
11
Scheme 2. Preparation of 2,5,6-trichloropyridine-3-carbonitrile. Reagents and
conditions: (a) H2SO4, AcOH; (b) PCl5, 150 °C; (c) KMnO4, 2 days, HCl; (d) oxalyl
chloride, DCM; (e) NH4OH, dioxane, 0 °C; (f) POCl3, 90 °C.
a
b
c
At Km ATP concentration.
Protocols for the cellular assays in Ref. 22
Denotes not tested.
H2N
F/Cl
N
R2
R2
Table 2
Kinase selectivity data for compound 1da
NH
R1
H
N
N
NH
R1
Kinase
IC50
(lM)
Kinase
IC50 (lM)
3
a
N
N
JAK2
TrkA
JAK3
Ret
Flt3 (D835Y)
FGFR3
Pyk2
<0.001
0.003
0.015
0.019
0.082
0.115
0.129
CDK2/cyclinE
Lck
MuSK
EGFR
EphB2
InsR
0.174
0.215
0.224
>10
>10
>10
N
N
F/Cl
2
F/Cl
4
R2
H
N
H2N
HCl
N
NH
R1
N
F
Y
a
All kinases above are of human sequence.
N
HN
5
F
b
Y
was screened against a selected group of kinases.27 As the data in
Table 2 indicates, 1d has >170-fold selectivity against kinases such
as CDK2 and EGFR. Over 15-fold selectivity against JAK3 was again
observed. TrkA kinase was inhibited in this assay; however, in a
cellular context, the inhibitory activity of 1d against TrkA (MCF-
10A TrkA
Y = CH or N
1a-e
Scheme 1. Preparation of 6-aminopyrazolyl-pyridine-3-carbonitriles. Reagents and
conditions: (a) Et3N, CH3CN or THF, room temperature to 80 °C; (b) DIPEA, n-BuOH,
150 °C, microwave.
28
D
)
was found to be 0.276 lM. For those kinases whose
Replacement of the phenyl ring with a 2-pyridyl group gener-
ated compounds 1d–e that had equivalent activities at enzymatic
and cellular levels as compared to 1c (Table 1). A compound with
a 5-chloro substituent was as potent as the 5-fluoro analogue (1e
vs d).
Compounds depicted in Table 1 were prepared via the general
synthetic sequence shown in Scheme 1. Commercially available
2,6-dihalo-pyridine-3-carbonitriles 2 were reacted with pyrazol-
3-ylamines 3 in the presence of a base to give intermediates 4.
Nucleophilic aromatic substitutions of 4 with amines 5 (usually
as the hydrochloride salts) were achieved by heating under basic
conditions in microwave reactors to provide the target compounds
1a–e.
For the 5-chloropyridine core that was not readily available,
synthesis commenced from 2-methylpentanedinitrile 6. As shown
in Scheme 2, hydrolysis of 6 gave piperidine-dione 7 that was then
chlorinated to provide the corresponding 2,3,6-trichloropyridine 8.
Oxidation of the methyl group on 8 generated acid 9, which was
subsequently transformed to nitrile 12 via acid chloride 10 and
amide 11 intermediates.
IC50’s fall below 100 nM, it is difficult to extrapolate the selectivity
due to the inhibition of JAK2 enzyme below the detection limit of
the assay. The kinase selectivity of 1c has been previously re-
ported,25 and 1c and d shared some common kinase hits such as
FGFR and Flt family members. They also revealed some differences
in that 1d inhibited, for example, JAK3 and Ret, while 1c instead,
inhibited Aurora A, Ark and ALK.
Having achieved sub-100 nM in vitro enzyme activity and cell
potency against JAK2, we then evaluated the pharmacokinetic
properties of the lead compounds 1c–e. As shown in Table 3, these
compounds were found to have less than 7.5 mL/min/kg for in vivo
clearances and between 2 and 6 h for half-lives in rats when dosed
intravenously. A rough correspondence between rat microsomal
and in vivo clearance can be seen in Table 3, suggesting that the
in vivo metabolism of these compounds is mainly cytochrome
P450-mediated, at least in rats. Furthermore, these analogues were
orally bioavailable in rats (39–47%). In a dog pharmacokinetic
study, compound 1d had an in vivo clearance of 7.4 mL/min/kg, a
half life of 3.6 h and was 100% orally bioavailable. Regarding their
physical properties, compounds 1d and e exhibited aqueous solu-
To further investigate the overall kinase selectivity of the 6-
aminopyrazolyl-pyridine-3-carbonitrile scaffold, compound 1d
bilities of 303 and 100
lM, respectively, as compared to 13 lM
for 1c.29 The difference of aqueous solubilities could be attributed

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T. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2958–2961
Table 3
see whether these compounds are efficacious in a cell line that car-
ries such a mutation. Compound 1c was tested in a SET-2 cell line,
which is heterozygous to JAK2 V617F mutation, and did show
Rat microsomal stability and rat pharmacokinetic profile of lead compoundsa
Compd Clint (lL/min/mg) CL (mL/min/kg) T1/2 (h) Vdss (L/kg) F (%)
growth inhibition (GI50 = 0.033 l
M).25 The PK and PD coupled with
1c
1d
1e
17
5
7.5
4.8
2.3
5.7
2.2
2.7
2.6
0.84
0.52
44
39
47
the cellular potency shown above suggested that compounds such
as 1c (or 1d) might be useful probe compounds in disease models
with this key mutation.
5
a
Han Wistar rat male; 10 mg/kg po (0.1% HPMC); 3 mg/kg iv (DMA/PEG/
In summary, a series of 6-aminopyrazolyl-pyridine-3-carbonitr-
iles has been evaluated as JAK2 kinase inhibitors. Compounds such
as 1c–e offered the potential to test the modulation of JAK2 kinase
activity in vivo.
saline = 40:40:20).
Acknowledgments
The authors would like to thank Ethan Hoffmann for PK stud-
ies, David Ayres for PK sample analysis and Array BioPharma
team (John Josey, Yongxin Han, Bin Wang and Peter Mohr) for
synthetic support. Special thanks to Susan Ashwell and Dennis
Huszar for useful recommendations during the preparation of
this letter.
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lM, still
above its cellular GIC50 (0.036 M). This data suggested that the
l
10 mg/kg dose under an optimized dosing schedule would provide
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inhibition in mice.
As discussed previously, JAK2 V617F is the causative mutation
for a major population of MPNs, and it is therefore interesting to

T. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2958–2961
2961
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29. Solubility measurement described in Ref. 24
27. http://www.millipore.com/drugdiscovery/dd3/KinaseProfiler.