Design, synthesis and structure-activity relationship study of aminopyridine derivatives as novel inhibitors of Janus kinase 2

Article abstract of DOI:10.1016/j.bmcl.2019.04.011

Janus Kinase 2 (JAK2) is a kind of intracellular non-receptor protein tyrosine kinase and has been certified as an important target for the treatment of myeloproliferative neoplasms and rheumatoid arthritis. However, the low selectivity and potential safety issues restrict the clinical applications of JAK2 inhibitors. Here we found that crizotinib showed good inhibitory activity against JAK2 by enzymatic assays (IC₅₀ = 27 nM). Then we carried out structure-based drug design and synthesized a series of compounds with an aminopyridine scaffold. Finally, compound 12k and 12l were identified as the promising inhibitors of JAK2, which exhibited high inhibitory activity (IC₅₀ = 6 nM and 3 nM, respectively) and selectivity for JAK2 over JAK1 and JAK3, and showed potent antiproliferative activities toward HEL human erythroleukemia cells. Moreover, 12k suppressed symptoms of the collagen-induced arthritis (CIA) model in rats.

Full text of DOI:10.1016/j.bmcl.2019.04.011

Accepted Manuscript
Design, synthesis and structure-activity relationship study of aminopyridine de-
rivatives as novel inhibitors of Janus Kinase 2
Wanqi Wang, Yanyan Diao, Wenjie Li, Yating Luo, Tingyuan Yang, Yuyu
Zhao, TianTian Qi, Fangling Xu, Xiangyu Ma, Huan Ge, Yingfan Liang,
Zhenjiang Zhao, Xin Liang, RuiWang, Lili Zhu, Honglin Li, Yufang Xu
PII:
S0960-894X(19)30220-3
https://doi.org/10.1016/j.bmcl.2019.04.011
BMCL 26378
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 March 2019
30 March 2019
6 April 2019
Please cite this article as: Wang, W., Diao, Y., Li, W., Luo, Y., Yang, T., Zhao, Y., Qi, T., Xu, F., Ma, X., Ge, H.,
Liang, Y., Zhao, Z., Liang, X., RuiWang, Zhu, L., Li, H., Xu, Y., Design, synthesis and structure-activity relationship
study of aminopyridine derivatives as novel inhibitors of Janus Kinase 2, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.04.011
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Compounds 12l potently and selectivity
inhibited the enzymatic activity of JAK2
Design, synthesis and structure-activity
relationship of aminopyridine derivatives as
novel inhibitors of Janus Kinase 2
WanqiWang,#,aYanyanDiao,#,aWenjie Li,#,aYatingLuo,aTingyuan Yang,a YuyuZhao,aTianTian Qi,a
FanglingXu,aXiangyuMa,aHuanGe,aYingfanLiang,a Zhenjiang Zhao,a Xin Liang,aRuiWang,a
LiliZhu,*,a Honglin Li *,a and Yufang Xu *,a

Bioorganic & Medicinal Chemistry Letters
Design, synthesis and structure-activity relationship study of aminopyridine
derivatives as novel inhibitors of Janus Kinase 2
Wanqi Wang,#,a Yanyan Diao,#,a Wenjie Li,#,a Yating Luo,a Tingyuan Yang,a Yuyu Zhao,a TianTian Qi,a
Fangling Xu,a Xiangyu Ma,a Huan Ge,a Yingfan Liang,a Zhenjiang Zhao,a Xin Liang,a RuiWang,a Lili
Zhu,*,a Honglin Li *,a and Yufang Xu ,a
aShanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science &
Technology, Shanghai 200237, China
#Authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Janus Kinase 2 (JAK2) is a kind of intracellular non-receptor protein tyrosine kinase and has
been certified as an important target for the treatment of myeloproliferative neoplasms and
rheumatoid arthritis. However, the low selectivity and potential safety issues restrict the clinical
applications of JAK2 inhibitors. Here we found that crizotinib showed good inhibitory activity
against JAK2 by enzymatic assays (IC50 = 27 nM). Then we carried out structure-based drug
design and synthesized a series of compounds with an aminopyridine scaffold. Finally,
compound 12k and 12l were identified as the promising inhibitors of JAK2, which exhibited
high inhibitory activity (IC50 =6 nM and 3 nM, respectively) and selectivity for JAK2 over JAK1
and JAK3, and showed potent antiproliferative activities toward HEL human erythroleukemia
cells. Moreover, 12k suppressed symptoms of the collagen-induced arthritis (CIA) model in rats.
Keywords:
JAK2
Rheumatoid arthritis
Crizotinib
2019 Elsevier Ltd. All rights reserved.
Molecular docking
Janus kinase is a family of soluble non-receptor protein
tyrosine kinases with four members, JAK1, JAK2, JAK3 and
tyrosine kinase 2 (TYK2).1-3 JAK1, JAK2 and TYK2 are widely
present in cells and tissues, while JAK3 is mainly expressed in
myeloid and lymphoid cells. Each of the four members of the
JAK family plays an individual role in the oncogenesis of the
immune system.4, 5 Cytokines stimulate cells through cytokines
receptors on the cell membrane, thereby activating downstream
signals and affecting the biological functions of cells.6 This
process is called cell signal transduction. Janus kinase, signal
transducers and activators of transcription constitute the JAK-
STAT pathway, which is a hot signal pathway in the current
cytokine research and also plays important roles in many
biological reactions in the body.7 It regulates cell proliferation,
differentiation and apoptosis, and also participates in the
physiological processes such as immunity and inflammatory
reactions of the body.8 The main process of JAK-STAT
signalling is described as follows: firstly, cytokines bind to their
receptors outside the cell and activate receptors, which dimerize
and phosphorylate JAKs on the receptor, and generate docking
sites for STATs; after that, the STAT is phosphorylated and
homo- or hetero-dimerization and multimerization, and then bind
to DNA and participate in the regulation of gene transcription
and expression.9-11
Abnormalities in the JAK-STAT pathway have been linked to
both inflammatory diseases and cancer, such as rheumatoid
arthritis (RA), psoriasis and myeloproliferative neoplasms
(MPNs).12-17 Rheumatoid arthritis is a chronic systemic
inflammatory disease that cocurs in 0.5-1% of adults worldwide.
Although it has a variety of treatment options, for instance, biotic
and abiotic disease-modifying anti-rheumatic disease drugs
(DMARDs), there is still a significant unmet medical need.
Therefore, new treatment methods are required to address the
needs of patients with RA.18 Studies have shown that the JAKs
and STATs are significantly activated by cytokines in synovial
cells and synovial tissues in RA patients, so inhibiting the JAK-
STAT pathway can reduce the activation and proliferation of
inflammatory cells, thereby relieving rheumatoid arthritis.
Therefore, the development of drugs acting on the JAK-STAT
pathway has become an important research direction for the
treatment of RA.19 For example, tofacitinib is a JAK1/3 inhibitor
from Pfizer and has been approved for the treatment of RA by the
dimerizes; ultimately, STATs enter the nucleus in the form of
———
 Corresponding author. Tel.: +86-21-64250213; fax: +86-21-64250213; email: zhulfl@ecust.edu.cn, hlli@ecust.edu.cn; yfxu@ecust.edu.cn

US Food and Drug Administration (FDA) in 2012.20,21 After that,
several JAK kinase inhibitors were developed for RA, such as
baricitinib and peficitinib. These JAK inhibitors have potential
application for treating RA.22, 23
recombinant protein by separation and purification
technologies.35 In order to identify new compounds binding to
JAK2, we performed SPR-based screening from our in-house
compound library containing 672 listed drugs. Ruxolitinib, which
is a reported compound targeting JAK2, was chosen as the
positive control for the binding assays. Initially, the compounds
from our library were screened at 50 μM to evaluate whether they
can bind to JAK2. After the initial screening, the dose-response
assays were performed to calculated the KD values. Finally, we
identified that the most potent compound crizotinib bound to
JAK2 with a KD value of 10.99 nM (Figure S1). This result
showed that crizotinib had a strong binding affinity with JAK2
protein. To further confirm their interaction, we performed
fluorescence-based thermal shift assay and found the melting
temperature of JAK2 was increased in the presence of crizotinib,
with an increase of 8℃(Table S1). Subsequently, we conducted
the Z’-LYTETM Kinase Assay to measure the kinase level of
inhibitory activity of crizotinib (IC50 = 27 nM for JAK2) and
obtained the selectivity for JAK2 over JAK1 and JAK3 (20- and
50-fold), respectively. In addition, the result of the Mobility shift
assay showed that crizotinib has a higher selectivity for JAK2
over TYK2, about 47-fold (Table 2).
MPNs are malignant hematological diseases that originate
from bone marrow hematopoietic stem cells. Polycythemiavera,
essential thrombocythemia and primary myelofibrosis are the
three most common myeloproliferative cancers.24 Studies show
that JAK2 V617F mutation are found in 80% of cancer patients
with MPNs. This mutation causes the excessive activation of
JAK2, which leads to the excessive proliferation of tumour
cells.25-27 The discovery of JAK2 V617F mutation in MPNs has
prompted the development of molecularly targeted therapy.
In order to further elucidate the mechanism of crizotinib at the
cellular level, we carried out cell proliferation assay and
investigated the effects of crizotinib on the JAK2-STAT
signaling pathway. We found that crizotinib had an inhibition
effect on HEL cells proliferation (IC50 = 4.725 μM) (Table 3).
Moreover, crizotinib had effects on inducing concentration
dependent inhibition of phosphorylation on STAT3 and STAT5,
and increasing JAK2 autophosphorylation level at the Y1007/8 in
HEL cells (Figure S2), which are general effects of JAK2
inhibitors36. Based on the above, we confirmed that crizotinib is a
new lead compound targeting JAK2.
Figure1. Structures of (1) Ruxolitinib, (2) Pacritinib, (3) AZD1480, and (4)
Crizotinib.
The strategies for the synthesis of compounds adopted in this
research are shown in Schemes 1.37 3-Hydroxy-5-bromopyridine
was used as a raw material to obtain compound 6 by nitration.
Mitsunobu reaction was then performed with the alcohol
substrate 8a-8c, 8e-8o to obtain intermediates of 9a-9c, 9e-9o.
The 9d was obtained by an electrophilic substitution reaction.
After being reduced with iron powder, a Suzuki reaction occurred
with R2-Boc to obtain intermediates of 11a-11u. Finally Boc is
removed under acidic conditions to give final product of 12a-12u
which were racemic compounds.
At present, some JAK2 inhibitors have entered clinical
research, such as ruxolitinib, pacritinib and AZD1480 (Figure
1).28-30 Ruxolitinib is the first JAK inhibitor approved by FDA,
targeting JAK2 kinase (IC50 = 2.8 nM) for treating primary
myelofibrosis.31 These JAK2 inhibitors have shown good efficacy
in a number of disease areas, but have known on-target toxicities,
such as increased risk of infection, anemia (platelets, red and
white blood cells), hypercholesterolemia, increased
transglutaminases and creatinine levels, which have led to
clinical safety issues.32-33 Therefore, the discovery of selective
JAK2 inhibitors is an area of high interest. In addition, due to
high homology amongst JAK family members, particularly in the
catalytic domain, the identification of selective JAK2 inhibitors
has represented a significant challenge. Herein, we screened our
compound library containing 672 listed drugs and tried to find
candidate inhibitors targeting JAK2.
Crizotinib is a previously reported c-MET/ALK inhibitor and
the crystal structures of crizotinib binding to the kinase domains
of these two kinases, respectively, have been resolved.38 In view
of the highly conserved nature of the ATP-binding pocket within
the kinase domain, we envisioned that crizotinib may adopt a
similar conformation when interacting with JAK2. After super
imposing the crystal structure of ALK in complex with crizotinib
(PDB code 2XP2) onto JAK2 in complex with NVP-BSK805
(PDB code 3KRR), it was found that the shared piperidinyl-
pyrazole moieties between the two crystallographic ligands
overlaid well with each other.39 To investigate the proposed
binding mode of crizotinib with JAK2 and explore significant
clues for further structural optimization, crizotinib was extracted
and merged into the ATP-binding pocket of JAK2 (Figure 2A).
Through surface plasmon resonance assays (Figure S1),
protein thermal shift assays (Table S1), and Z'-LYTETM
fluorescence assays, we finally obtained the lead compound,
crizotinib, has a good inhibitory effect on JAK2. Crizotinib was
reported to be a ALK/c-Met/ROS1 multitarget tyrosine kinase
inhibitor, and it was approved by FDA for the treatment of ALK-
positive locally advanced or metastatic non-small cell lung
cancer (NSCLC). 34
The 2-aminopyridine core of crizotinib putatively participates
in hydrogen bond interactions with the backbone atoms of
residues Gly930 and Leu932 in the hinge region (Figure 2B). The
3-benzyloxy moiety inserts into the hydrophobic pocket toward
the activation loop, however, it does not seem to fit the pocket
very well. On the one hand, the methyl group protrudes to the N-
terminal lobe, leading to steric clashes with the side chain of
In order to improve the activity and selectivity, a series of
crizotinib derivatives with an aminopyridine scaffold were
designed and synthesized.
Firstly, we used the Sf9 eukaryotic expression system to
express the kinase domain of JAK2 and obtained the purified

residue Val863. On the other hand, an unfavourable conflict
could happen between the 2-chlorine of the phenyl ring and the
oxygen on the backbone of residue Gly993, as the distance
between these two atoms is as short as 1.6 Å. Owing to the
formation of van der Waals effects between the 6-chlorine and
residues Leu855 and Gly856, enhanced inhibitory activity could
be pursued by introduction of larger groups to fulfill the
hydrophobic subpocket. At the other end of crizotinib, the
piperidinyl-pyrazole moiety extends to the hinge region near the
entrance of the ATP-binding pocket, where the nonconserved
residues may be critical for the design of compounds capable of
selectively discriminating within the JAK family.40
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, 0 oC to room temperature, 6 h; (b) HNO3, H2SO4, 0 oC to room temperature, 6 h; (c) DIAD, PPh3, THF,
0 oC to room temperature, 4 h; (d) TBAB, KI, Cs2CO3, CH3CN, 80 oC, 10 h; (e) Fe, AcOH/EtOH, reflux, 3 h; (f) BPin-R2-Boc, Pd(dppf)Cl2, Cs2CO3, Toluene, 80
oC, 12 h; (g) Trifluoroacetic acid, CH2Cl2, 0 oC to room temperature, 12 h.
Figure 2. (A) Overview of crizotinib in the ATP-binding site of JAK2 kinase domain (PDB code 3KRR). The receptor is shown in marine cartoon, and the ligand
is presented as a ball and stick model. (B) Detailed binding interactions of crizotinib against JAK2 with only key residues displayed for clarity. The hydrogen
bonds are indicated as blue dashed lines. The residues and atoms of the ligand involved in steric clashes and hydrophobic effects are highlighted in red and green,
respectively. Key residues which differ in the JAK family are also labeled.
On the basis of the putative binding mode, the essential 2-
aminopyridine core was fixed, and our initial efforts were
performed on the optimization of the linker between the pyridine
ring and the terminal 2,6-dichloro-3-fluorophenyl group.
Inconsistent with our model, the removal of the methyl group
yielded compound 12b, with 6-fold activity loss against JAK2
compared to the racemic crizotinib 12a (IC50 = 0.572 vs 0.092
μM). While the introduction of a larger ethyl group based on 12b
led to 5-fold enhanced potency of 12c, the IC50 value of which is
comparable to that of 12a. These unexpected results might be
ascribed to the plasticities of the ligand and the residues in the N-
terminal lobe, which may adjust their conformations to tolerate
each other. In addition, the replacement of -O- by -NH- did not
make obvious improvements in terms of inhibitory effect against
JAK2 (compound 12d, IC50= 0.126 μM).
Subsequently, an alternative strategy was carried out by
modification of the R2 group, which may significantly influence
the potency and selectivity against JAK2. Compounds 12e-12i,
bearing substituents with different sizes and electrostatic effects
in the R2 position, were therefore developed. Compound 12e,

containing a tetrahydropyran group, displayed slightly improved
potency against JAK2, but its IC50 value was still in the same
range as that of 12a (0.065 vs 0.092 μM). When the terminal
piperazinyl group was moved from 5- to 6-position of the
pyridin-3-yl moiety, up to 6-fold discrepancy with respect to
potency against JAK2 was found between compounds 12f and
12g (IC50= 0.119 vs 0.804 μM), implying that the region at the
entrance of the cavity is relatively sensitive to the substituents
Table 1
harbored in it. The removal of the piperazinyl ring resulted in a
substantial drop in potency as observed for compound 12h with a
single-digit micromolar IC50 value. Furthermore, compound 12i,
which had an electronically neutral phenyl ring attached to the 2-
aminopyridine core, almost suffered a complete loss of activity,
suggesting that polar basic groups are probably preferential at the
R2 position for better inhibitory potency against JAK2.
Structure and activity profiles of compounds 12a-12u against JAK2 kinase
Compd
R1
X
O
R2
R3
IC50 (μM)
0.092±0.007
12a
CH3
0.572±0.020
0.100±0.002
0.126±0.005
0.065±0.001
0.119±0.008
12b
12c
12d
12e
12f
H
O
C2H5
CH3
CH3
CH3
O
NH
O
O
0.804±0.009
3.049±0.204
12g
CH3
O
12h
12i
CH3
CH3
O
O
7.152%
(inhibition rate at1μM)
0.026±0.002
12j
CH3
CH3
CH3
O
0.006±0.001
0.003±0.001
12k
12l
O
O
0.442±0.003
0.215±0.012
12m
12n
CH3
CH3
O
O

Table 1 (continues)
Compd
R1
X
O
R2
R3
IC50 (μM)
3.400±0.431
12o
CH3
0.461±0.002
0.016±0.001
12p
12q
CH3
CH3
O
O
0.037±0.003
0.048±0.008
12r
12s
CH3
CH3
O
O
0.027±0.002
0.035±0.001
12t
CH3
CH3
O
O
12u
In accordance with our previous hypothesis, compared to
compound 12a, the introduction of a larger methoxy group at the
6-position of the phenyl ring led to about 3-fold potency increase
of compound 12j (IC50= 0.092 vs 0.026 μM). Meanwhile, the
removal of the 2-chlorine atom yielded a more potent compound
12k (IC50= 0.006 μM), which may be attributed to avoiding the
steric conflicts with Gly993. Although the structure of 12k has
been reported as a mutant ALK inhibtor, we have found its
potential to be a novel selective JAK2 inhibitor.35 On the basis of
compound 12k, the replacement of chlorine with methoxy group
generated the most potent JAK2 inhibitor (compound 12l, IC50=
0.003 μM) of this chemical series. To thoroughly delineate the
underlying SAR, compound 12l was docked to the ATP-binding
pocket of JAK2 (PDB code 3KRR) using Glide SP mode
(Maestro v10.1, Schrödinger Inc.). In the predicted docking pose,
Table 2
as shown in Figure S2, 12l displayed a very similar conformation
to that of crizotinib binding to ALK (PDB code 2XP2). The alkyl
linker tilts slightly toward the inside of the pocket, leaving
enough space for the methyl group to be away from the N-
terminal lobe to avoid the steric conflicts with Val863. Moreover,
the methoxy group on the phenyl ring putatively participates in
favorable hydrophobic interactions with around residues such as
Leu855 and Gly856. The 3-fluorine atom appears to be essential
to obtain potent inhibitory effect against JAK2, and its absence
gave rise to 4-fold diminished potency of compound 12m in
relative to 12a. Likewise, the smaller phenyl (12n) or cyclohexyl
(12o) group in the R3 position also did not make any
improvements for the inhibitory activity against JAK2.
Evaluation of the inhibitory activities of selected compounds against JAK1, JAK3 and TKY2.
Enzyme inhibitory activity (IC50/μM)
Enzyme Selectivity
Compd
JAK1/JAK2
ratio
JAK3/JAK2
ratio
TYK2/JAK2
ratio
JAK1
JAK2
JAK3
TYK2
12j
1.223±0.013
0.660±0.005
1.796±0.041
0.082±0.003
0.120±0.001
0.563±0.011
0.004±0.001
0.026±0.002
0.006±0.001
0.003±0.001
0.016±0.001
0.027±0.002
0.027±0.002
0.0006±0.0001
1.759±0.063
0.513±0.023
1.584±0.044
0.965±0.006
2.788±0.051
1.360±0.074
0.051±0.002
0.774±0.041
1.298±0.318
0.794±0.203
1.453±0.162
2.530±0.437
1.269±0.150
0.011±0.002
46
110
598
5
66
30
12k
86
216
264
91
12l
528
60
12q
12t
4
107
50
94
Crizotinib
Ruxolitinib
20
6
47
85
18
Based on the previously established proper 2-chloro-5-
fluorophenyl moiety, compounds 12p-12u possessing various R2
groups were synthesized, most of which exhibited IC50 values at
two-digit nanomolar level. It is noteworthy that compound 12p
retrieved the inhibitory ability against JAK2 in comparison with
12i (0.461 vs >10 μM). The addition of the piperidinyl or

piperazinyl increased the inhibitory potency, moreover, the para-
substituent was more favoured than the meta-substituent (12r vs
12t, 12s vs 12u). As illustrated in Figure S2, the -NH- group on
the piperazinyl moiety of compound 12t could form an extra
hydrogen bond with residue Asp939, which would contribute to
the enhanced potency against JAK2.
group appeared to significantly influence the selectivity of
compound 12l within the JAK family, as its introduction resulted
in reversed potency changes against JAK1 and JAK3. As
displayed in Figure S2, the methoxy group is located towards the
flexible glycine-rich loop. Although the non-conserved residues
in this region did not directly interact with the inhibitor, they may
contribute to the conformational plasticity and affect the
selectivities of inhibitors.
Five compounds with high inhibitory activities against JAK2
were selected from the obtained compounds, and selective
experiments of the JAK family were performed (Table2). The
results showed that compounds 12k and 12l dispalyed very good
selectivity for JAK2 over other JAKs, and 12l was 598-, 528-,
and 264-fold selective over JAK1, JAK3, and TYK2,
respectively. We speculated that the superior selectivity of
compound 12l, relative to the hit compound crizotinib (12a), was
partially ascribed to the removal of the chloride atom from the
phenyl ring at the 2-position. This group extended to the
activation loop which has a glycine residue in JAK2, JAK1 and
On the basis of enzymatic assays, we performed the
antiproliferative activity assays on the HEL human
erythroleukemia cell line, which is known to contain the JAK2
V617F mutation and proliferate activity is sensitive to the JAK2-
STAT signaling pathway. The results showed that crizotinib and
some of its derivatives showed potent inhibition on the
proliferation of HEL cells (Table 3 and Table S3). Among them,
crizotinib, 12k and 12l inhibited HEL cells proliferation with IC50
equal to 4.725 μM, 15.024 μM and 4.194 μM, respectively.
TYK2 but an alanine residue in JAK3. In addition, the methoxy
Table 3
The antiproliferative activities on HEL cells of 12k and 12l
Compd
Cellular Antiproliferative Activity (IC50/μM)
12k
15.024±0.032
4.194±0.056
4.725±0.049
7.639±0.363
12l
Crizotinib
Ruxolitinib
Based on the molecular level, 12k and 12l have comparable
activities (6 nM and 3nM). And compared with 12l, 12k is easier
to synthesize to obtain the larger amounts of material, so we
chose 12k for in vivo experiments. In order to evaluate the effect
of 12k in the collagen-induced arthritis (CIA) model in rats, all
rats were weighted every third day and their clinical scores were
recorded every day. Compared to the control group, the growth of
the weight in the RA group increased slowly after the onset of
disease due to serious illness, especially from day 10 post
immunization to the end of the experiment. The rats in
compared with RA group and had no disease phenotype.
Although treatment with 12k had no obvious effect on the growth
of body weight (Figure 4A), 12k markedly decreased the arthritis
swelling score (at dose of 50 mg/kg) and alleviated foot swelling
in a dose dependent manner (Figure 4B and 4C). The incidences
were lower in three 12k-treated groups than models (data were
not shown). All those data indicated that compounds 12k could
suppressed symptoms of the collagen-induced arthritis (CIA)
model in rats.
methotrexate group showed a significant increase in body weight
B
A
C
Figure 4. The effects of compound 12k on CIA rats in vivo. Arthritis was induced in Wistar rats by twice immunization with type II collagen on day 0 and day 8.
Compound 12k was orally administered at 10 mg/kg, 30 mg/kg and 50 mg/kg until the end of experiment. Methotrexate, as the positive control, was administered
at dose of 0.3 mg/kg. (A) Measurement of body weight was taken every three days. (B) Arthritis scores were evaluated every day. (C) The hind paws
representative photos of different treatment groups. All quantitative data were expressed as mean ± SEM. n=10/group. Statistic difference was indicated as * P <
0.05, ** P < 0.01, ***P <0.001 vs control; # P < 0.05 ## P < 0.01, ### P <0.001 vs RA. RA, Rheumatoid Arthritis.
In summary, we successfully identified crizotinib displaying
good inhibitory activity against JAK2 and synthesized a series of
derivatives with an aminopyridine scaffold as JAK2 inhibitors.
Among them, compound 12k and 12l had high inhibitory effects

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(IC50 = 6 nM and 3 nM, respectively) and selectivity for JAK2
over JAK1, JAK3 and TYK2. In conjunction with molecular
docking, we had extensively explored the structure-activity
relationships of this series of compounds. The pharmacodynamic
results in vivo showed that 12k had a certain preventive effect on
rheumatoid arthritis. In conclusion, our study indicated that a
new class of JAK2 inhibitors with an aminopyridine scaffold had
the potential for further development and application in the field
of rheumatoid arthritis. And drug metabolism and
pharmacokinetic properties of these compounds will be studied in
the future.
Acknowledgments
29. Pardanani, A.; Gotlib, G. R.; Jamieson, C.; Cortes, J. E.; Talpaz,
M.; Stone, R. M.; Silverman, M. H.; Gilliland, D. G.; Shorr, J.;
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Scherle, P. A.; Caulder, E.; Wen, X.; Li, Y.; Waeltz, P.; Rupar, M.;
Burn, T.; Lo, Y.; Kelley, J.; Covington, M.; Shepard, S.; Rodgers,
J. D.; Haley, P.; Kantarjian, H.; Fridman, J. S.; Verstovsek, S.
Blood. 2010, 115, 3109.
32. Grimster, N. P.; Anderson, E.; Alimzhanov, M.; Bebernitz, G.;
Bell, K.; Chuaqui, C.; Deegan, T.; Ferguson, A. D.; Gero, T.;
Harsch, A.; Huszar, D.; Kawatkar, A.; Kettle, J. G.; Lyne, P.;
Read, J. A.; Costa, C. R.; Ruston, L.; Schroeder, P.; Shi, J.; Su, Q.;
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5235.
The research is supported in part by the National Key
Research and Development Program (Grant 2016YFA0502304),
the National Natural Science Foundation of China (grant
81825020), the National Science & Technology Major Project
“Key New Drug Creation and Manufacturing Program”, China
(No. 2018ZX09711002), the Special Program for Applied
Research on Super Computation of the NSFC-Guangdong Joint
Fund (the second phase) under Grant No. U1501501. Honglin Li
is also sponsored by National Program for Special Supports of
Eminent Professionals and National Program for Support of Top-
Notch Young Professionals.
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