Discovery and optimization of 2-aminopyridine derivatives as novel and selective JAK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127048

Janus kinases (JAKs) including JAK1, JAK2, JAK3, and TYK2 are members of a family of intracellular nonreceptor tyrosine kinases, which have been demonstrated to be critical in the cell signaling pathway and involved in inflammatory diseases and cancer. V617F mutation in JAK2 has been implicated in polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis (MF). Here, we described the design, synthesis, and biological evaluation of a series of 2-aminopyridine derivatives. The results of enzymatic activity assays supported compound 16m-(R) as a potential and selective JAK2 inhibitor, which exhibited high inhibitory activity with an IC₅₀ of 3 nM against JAK2, and 85- and 76-fold selectivity over JAK1 and JAK3, respectively. Structure-activity relationships (SAR) and mechanistic analysis demonstrated that 16m-(R) might be a promising selective JAK2 inhibitor for further study.

Full text of DOI:10.1016/j.bmcl.2020.127048

Journal Pre-proofs
Discovery and optimization of 2-aminopyridine derivatives as novel and se‐
lective JAK2 inhibitors
Xiangyu Ma, Yanyan Diao, Huan Ge, Fangling Xu, Lili Zhu, Zhenjiang Zhao,
Honglin Li
PII:
S0960-894X(20)30122-0
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127048
BMCL 127048
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
5 December 2019
19 January 2020
16 February 2020
Please cite this article as: Ma, X., Diao, Y., Ge, H., Xu, F., Zhu, L., Zhao, Z., Li, H., Discovery and optimization
of 2-aminopyridine derivatives as novel and selective JAK2 inhibitors, Bioorganic & Medicinal Chemistry
Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127048
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
A novel 2-aminopyridine derivative 16m-(R)
Discovery and optimization of 2-aminopyridine
derivatives as novel and selective JAK2 inhibitors
was designed as a potent and selective JAK2
Xiangyu Ma#,a , Yanyan Diao#,a, Huan Ge#,a, Fangling Xua, Lili Zhua,*, Zhenjiang Zhaoa,*, Honglin Lia,
*
16m-(R)

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery and optimization of 2-aminopyridine derivatives as novel and selective
JAK2 inhibitors
Xiangyu Ma#,a , Yanyan Diao#,a, Huan Ge#,a, Fangling Xua, Lili Zhua,*, Zhenjiang Zhaoa,*, Honglin Lia,*
a Shanghai Key Laboratory of New Drug Design, 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 kinases (JAKs) including JAK1, JAK2, JAK3, and TYK2 are members of a family of
intracellular nonreceptor tyrosine kinases, which have been demonstrated to be critical in the cell
signaling pathway and involved in inflammatory diseases and cancer. V617F mutation in JAK2
has been implicated in polycythaemia vera (PV), essential thrombocythaemia (ET) and
myelofibroisis (MF). Here, we described the design, synthesis, and biological evaluation of a
series of 2-aminopyridine derivatives. The results of enzymatic activity assays supported
compound 16m-(R) as a potential and selective JAK2 inhibitor, which exhibited high inhibitory
activity with an IC50 of 3 nM against JAK2, and 85- and 76-fold selectivity over JAK1 and JAK3,
respectively. Structure-activity relationships (SAR) and mechanistic analysis demonstrated that
16m-(R) might be a promising selective JAK2 inhibitor for further study.
Keywords:
JAK2
Selectivity
SBDD
SAR
2019 Elsevier Ltd. All rights reserved.
myelofibrosis. The success of ruxolitinib provided a strong
Many cytokines exert their effects by employing the JAK-STAT
cell signaling pathway, which play critical role in the regulation of
cell growth, metabolism, differentiation, as well as cell migration
and survival.1 Dysregulation of the JAK-STAT pathway can result
in inflammation, immunodeficiency and cancer.2
impetus for the exploitation of other JAK2 inhibitors. For example,
pacritinib7, targeting both JAK2 and FLT3, has been proven to
become more effective than ruxolitinib for reducing splenomegaly
and symptoms in patients with myelofibrosis and
Janus kinases (JAKs) including JAK1, JAK2, JAK3, and TYK2
are members of a family of intracellular nonreceptor tyrosine
kinases. JAK2 is known to be associated with myeloproliferative
neoplasms (MPNs) including polycythaemia vera (PV), essential
thrombocythaemia (ET) and myelofibroisis (MF), owing to the
strong evidence provided by the identification of the activating
O
CN
N
N
O
O
NC
N
H
H
O
N
N
N
N
N
N
N
N
N
N
H
N
H
Momelotinib
3
O
Ruxolitinib (Jakafi)
1
Pacritinib
2
3
V617F mutation in the pseudo kinase domain (JH2) of JAK2.
Almost all patients with PV (97%), and in half with ET (57%), MF
(50%) have been found carrying V617F mutation in JAK2.4
V617F is proximal to the SH2-JH2 linker and the catalytically
active conformation of αC in JH1 is more stable in V617F than in
wild-type JAK2, which can help promote the process of
phosphorylation.5 The discovery ignite interests in the exploitation
of JAK2 inhibitors for targeted therapy since 2005, which
culminated in the discovery of ruxolitinib, a selective JAK
inhibitor, that inhibit JAK2 and JAK1, with IC50s of 5.7 nM and
5.9 nM respectively.6 In 2013, ruxolitinib was allowed by FDA for
the treatment of patients with intermediate- and high-risk
N
N
H
N
N
F
N
N
O
N
N
NH
t-BuHN
S
N
H
N
N
H
O
Fedratinib (Inrebic)
5
NS-018
4
Cl
H
N
N
NH2
N
HN
HN
HN
N
N
O
N
N
F
Cl
N
N
O
N
N
F
NH HN
O
HN
N
AZD1480
Cl
8
XL-019
7
6
JAK2 IC50: 27 nM
HEL cells IC50: 15.024 M
Figure 1. Structures of (1) Ruxolitinib, (2) Pacritinib, (3) Momelotinib, (4)
NS-018, (5) Fedratinib, (6) AZD1480, (7) XL-019, (8) Crizotinib.
*Corresponding author. Tel.: +86-21-64250213; fax: +86-21-64250213; email: zhulfl@ecust.edu.cn, zhjzhao@ecust.edu.cn, hlli@ecust.edu.cn;


N

O
N




16i: R=
16j: R=
16k: R=
N
N
16m R=
16n R=
16o: R=
16q: R=
16r R=
16e: R=
16f: R=
16a: R=
16b: R=
16c: R=
16d: R=
N
thrombocytopenia, currently in NDA (New Drug Application)
phase.8 Another JAK1 and JAK2 inhibitor, momelotinib, was
shown to be effective and well tolerated in patients with
myelofibrosis at an oral administration of 300 mg per day.9
N
O
N
H
N

N

N


N
O
N
N
N
O
N
N
N
N
H





N
N
16g: R=
16h: R=
N
O
N
N
N
N
H



O
N
N
N
N
16p: R=
16l: R=
Unfortunately, some of the discovered JAK2 inhibitors had to
be cancelled, owing to their severe side effects. For example, the
unexpected occurrence of neurological toxicity, especially central
nervous system (CNS) neurotoxicity, in AZD148010 and XL01911,
lead to the suspension in the early stage. The study of fedratinib
was ever suspended in phase 3 phase trial due to 3 cases of
Wernicke encephalopathy.12 The unexpected is that fedratinib has
been approved by FDA for the treatment of (intermediate-2/high-
risk) primary or secondary myelofibrosis. The redevelopment of
fedratinib gives us the courage to develop novel selective JAK2
inhibitors.
NH
N
N
Scheme 1. Reagents and Conditions: (a) NaBH4, MeOH, 0℃; (b) concentrated
H2SO4, HNO3, 0 ℃ ; (c) PPh3, DIAD, THF, 0 ℃ ; (d) Fe powder in
HOAc/C2H5OH 1:1 78℃; (e) Cs2CO3, Pd(dppf)Cl2, toluene, H2O, 80℃.
At the initial optimization stage, we designed several compounds
with simple aromatic rings at R position with the purpose of
providing special π-π interactions with Tyr931 at the JAK2 hinge
region. Compounds 16a-c were synthesized. However, these
compounds showed weaker activities against JAK2 than
crizotinib. The loss of potencies might indicate that the sizes of the
aromatic six-membered ring substituents were a little small and
couldn’t fit the pocket well, or in another case, the single aromatic
rings were probably too far away from Tyr931 to form π-π stack
interactions.
However, the process of discovering specific inhibitor against
JAK2 over other JAK family is proved to be a great challenge
because of the high homology in catalytic domain among the JAK
family, although amino acid differences in family members do
exist.13 In our previous work, a c-MET and ALK inhibitor
crizotinib (compound 8 in Figure 1) was found to have good
activity against JAK2 with an IC50 value of 27 nM. Then, structural
optimization from crizotinib had been carried out and some potent
compounds were obtained.14 In this study, we paid our attention to
modify the solvent exposed area (R group in Figure 2), where the
non-conserved residues are thought to be the main reason to
provide high selectivity.15 Therefore, we described the design,
synthesis and biological evaluation of a series of 2-aminopyridine
derivatives from crizotinib, finally leading to the discovery of
some potential JAK2 inhibitors (Figure2).
In order to verify our hypothesis, compounds 16d-j containing
five-fused-six membered heterocyclic rings were therefore
synthesized. As shown in table 1, compounds 16e and 16f (IC50
=
0.061 μM and 0.145 μM, respectively) displayed better activities
against JAK2 than other compounds in this series (except for
compound 16j). The improved potency may be ascribed to the
advantageous π-π stack effects between the pyrazolo[1,5-
a]pyridine or pyrazolo[1,5-a]pyrimidine ring and Tyr931.
Interestingly, compound 16i, with the introduction of a nitrogen
atom to the ortho-position of the benzene ring, suffered 10-fold
potency loss compared to compound 16g. The same tendency was
also observed between compounds 16e and 16f. Surprisingly,
when the nitrogen atom was moved from the ortho- to the meta-
position of the benzene ring, compound 16j showed 452-fold
increase in potency compared to compound 16i (IC50 = 0.022 μM
vs 9.963 μM). In the predicted binding mode of compound 16j to
JAK2 (Figure 3A), the 2-aminopyrimidine core participates in
hydrogen bond interactions with residues Glu930 and Leu 932 in
the hinge region. The added nitrogen atom possibly forms an
additional hydrogen bond to hinge residue Tyr931, offering a
possible explanation for the significant improvement in potency of
compound 16j.
N
NH2
O
R
F
Cl
16a-r
Figure 2. General molecular formula of 16a-r.
The general synthetic procedures for 2-aminopyridine derivatives
16a-r were described in Scheme 1. 1-(2-chloro-5-
fluorophenyl)ethanone 9 was reduced by sodium borohydride to
give 10. 3-Hydroxy-5-bromopyridine 11 was subjected to nitration
to obtain 12. Mitsunobu reaction was then conducted to gain
compound 13. The key intermediate 14 was then obtained via
reduction by Fe powder. In the end, Suzuki-coupling reaction
occurred between 14 and 15a-r to obtain the final products 16a-r.
After the second rounds of structural optimization mentioned
above, obvious improvement in terms of activity against JAK2,
comparing with crizotinib, still had not been made. The reason
may attribute to the steric clash which was caused by the terminal
six-membered ring of these substituents with adjacent residues
such as Gly935 and Tyr931. Therefore, an alternative optimization
strategy was adopted with different oriented di-heterocyclic R
substituents. Compounds 16k and 16l were synthesized and then
we evaluated their inhibitory activities against JAK2. The results
were consistent with our expectation. Compounds 16k and 16l
exhibited superior activity against JAK2 with the IC50 values of
0.051 μM and 0.071 μM, respectively. The increased activity of
16k and 16l compared to 16e could be explained in Figure 3B. The
predicted binding modes of compounds 16e and 16k docked in
JAK2 were superimposed to dissect the SAR. The distal five-
membered ring lies in a position farther away from the hinge
region, putatively leaving sufficient space to avoid the possible
steric conflicts with the surrounding amino acid residues.
O
OH
F
F
a
Cl
Cl
9
N
NO2
O
10
Br
c
Br
Br
b
F
N
N
HO
HO
Cl
13
NO2
12
11
N
NH2
O
d
Br
F
N
NH2
e
Cl
14
O
R
F
O
Cl
16a-r
R
B
O
15a-r


Although some of our present compounds displayed good JAK2
inhibitory activity, for example 16j showed a high activity with the
IC50 of 22nM at the JAK2 kinase level. Results were still not very
encouraging when these compounds were compared with JAK2
inhibitor fedratinib (IC50=3 nM). Therefore, we designed and
synthesized another series of compounds (16m-r) based on
crizotinib and our previous work. Encouragingly, this series
showed remarkable potency against JAK2, especially for
compound 16m with an IC50 value of 8 nM. Compound 16m was
docked to the ATP-binding pocket of JAK2. As expect, 16m
>10
16h
16i
NH
N

9.963±0.035
N
H
N

0.022±0.001
16j
N
H
shares
a similar binding profile with 16j, with the 2-

N
aminopyrimidine core forming critical hydrogen bond interactions
with the hinge region (Figure 3C). In addition, the R substituent of
16m could swing either to the left side of the solvent channel or
the right side at the bottom of the pocket. When the tail extends
toward the right side, there is no direct interaction between JAK2
and the inhibitor. However, the oxygen atom of the
tetrahydropyran moiety could form the third hydrogen bond with
Tyr931 when it pointed to the left side. Compounds 16n and 16o,
two analogues of 16m, also showed good potencies against JAK2,
with the IC50 values of 0.025 and 0.027μM, respectively. Based on
the above SAR, we inferred that oxygen atoms in rings played an
important role in the potency against JAK2. Subsequently, several
open-chained ethers substituted compounds were synthesized.
Compound 16r, in which the oxygen atom in alkyl chains formed
a hydrogen bond with Tyr931 (Figure 3D), displaying a
comparable JAK2 inhibitory activity (IC50 = 0.011 μM) to
compounds 16m. However, 16p showed a slight decrease in
potency, probably due to the unsuitable position of the oxygen
atom, which was not capable of forming the hydrogen bond
interaction.
0.051±0.001
0.071±0.001
16k
N

N
N
N
16l
O
0.008±0.001

N
16m
N


0.025±0.001
0.027±0.001
O
N
N
16n
16o
N
N
O

O
N
16p
0.157±0.031
0.015±0.001
N
N

Table 1. Structure and activity profiles of compounds 16a-16r against JAK2
kinase
16q
16r
N
N
NH2
O
O
O

R
F
N
0.011±0.001
N
Cl
JAK2
IC50/μM
Compd
16a
R
Because of the satisfying JAK2 inhibitory potency of compound
16m, its two optical isomers were obtained by chiral resolution.
The structure of 16m-(R) was determined through single crystal
X-ray diffraction (Figure 4). After the evaluation of the enzymatic
activity, the R-enantiomer was found to be the most potent
compound in our study (IC50 =3 nM) , while the S-enantiomer
showed poor activity (IC50 =1399 nM). Alignment of the proposed
binding poses of 16m-(R) and 16m-(S) (figure S2) indicate that
the methyl group of 16m-(S) lies in a position closer to residue
Asp994 than 16m-(R), and bad contacts were detected by the
measurement toolbar of Maestro v10.1 with default criteria.
Therefore, the less potent inhibitory activity of 16m-(S) against
JAK2 may be contributed to the steric conflicts between the
methyl group and Asp994.

0.461±0.002
0.090±0.002


16b
16c
N
N
0.566±0.029
0.695±0.002
N


16d
N
The fluorescence-based thermal shift assay is a general method
for identification of inhibitors of the target protein. The inhibitor
binding affinity can be assessed from the shift of the melting
temperature in the presence of the inhibitor relative to that
obtained in the absence of the inhibitor.16 The ability of racemate
16m and optical isomers 16m-(S), 16m-(R) increasing thermal
stability of the JAK2（JH1）were evaluated by fluorescence-
based thermal shift assays. The melting temperature (Tm) for
JAK2（JH1）alone was 45.1℃, and the present of 16m, 16m-(S)
and 16m-(R) with an increase of 11.9℃, 5.5℃ and 13.1℃,
respectively. These results declared that 16m-(R) had a stronger
ability to improve the thermal stability of JAK2 than 16m-(S),
16e
16f
N
0.061±0.007
0.145±0.010
N
N


N
N
0.925±0.102
16g
N
H

which corroborated 16m-(R) showing a higher activity against
JAK2 than 16m-(S).
Figure 3. Predicted binding poses of compounds 16j (A), 16e and 16k (B), 16m (C), and 16r (D) in the ATP binding pocket of JAK2 (PDB code 2XA4). The
docking process was implemented using Glide SP mode of Maestro v10.1 (Schrödinger Inc.). Key residues around the binding pocket are displayed as marine lines,
and the hydrogen bonds are highlighted as black dashed lines.
difference exists (JAK2/Gln 853, JAK1/Arg 868, JAK3/Ser 826),
contributing to the observed selectivity against JAK2 over JAK1
and JAK3. Then, 16m-(R) was evaluated its antiproliferative
activity against JAK2V617F-dependent cells, Human Erythroid
Leukemia cells (HEL cells). The result showed that compound
16m-(R) exhibited potent antiproliferative effects with an IC50 of
5.75 μM.
In order to verify the mechanism of 16m-(R) in the JAK2-STAT
signaling pathway, the phosphorylation levels of JAK2 and its
downstream substrates were evaluated. As shown in figure 6, the
treatment of the HEL cells with 16m-(R) or fedratinib led to the
increase of pJAK2 (Y1007/8) level. The same phenomenon was
also found in fedratinib, which are general effects of type I ATP
competitive inhibitors of JAK2, such as Gӧ6976, SB1518 and
Figure 4. X-ray diffraction structure of 16m-(R). CCDC NO. 1922072
18
ruxolitinib.17, Furthermore, JAK2 has been reported to have
other autophosphorylation sites for the exception of Y1007/8,
which is necessary for JAK2 full activated.19 In our experiment,
16m-(R) can effectively block the phosphorylation Y221 of JAK2,
and also inhibit STAT3 and STAT5 phosphorylation. Overall,
these results revealed that 16m-(R) down-regulated the JAK2-
STAT signaling pathway by inhibiting JAK2 kinase activity in
HEL cell.
Subsequently, 16m- (R) was selected to evaluate the selectivity
of JAK family (JAK1 and JAK3) inhibition, and fedratinib was
used as the positive control. The results were shown in table2.
16m-(R) displayed an IC50 of 3 nM against JAK2 and was
approximately 85-and 76-fold selectivity over JAK1 and JAK3.
The tetrahydropyran ring occupied the region where amino acid

Figure 5. Thermal melting fluorescence curves (up) and derivative curves of the denaturation data (down) of JAK2(JH1) in the present of DMSO (red line)、
racemate 16m (green line),optical isomers 16m-(S) (blue line) and 16m-(R) (black line ).
Table 2. Evaluation of the inhibitory activities of selected compounds against JAK1, JAK2 and JAK3, and antiproliferative effects in HEL cells.
Comd
Enzyme inhibitory activity (IC50/μM)
JAK1 JAK2 JAK3
Enzyme selectivity
JAK1/JAK2
HEL cell (IC50/μM)
JAK3/JAK2
ratio
85
32
ratio
76
88
16m-(R) 0.254±0.041 0.003±0.001 0.228±0.007
Fedratinib 0.096±0.001 0.003±0.001 0.263±0.002
5.754±0.038
1.519±0.023
16m-(R)
0.01 0.1
Fedratinib
μM
0
1
10
1
In conclusion, a series of 2-aminopyridine derivatives were
designed, synthesized and evaluated for their biological activity
against JAK2. Half of these compounds exhibited potent
biological activities against JAK2 in kinase level with IC50 values
in nanomolar range. Among them, compound 16m-(R) [(R)-3-(1-
(2-chloro-5-fluorophenyl)ethoxy)-5-(1-(tetrahydro-2H-pyran-4-
yl)-1H-pyrazol-4-yl)pyridin-2-amine] exhibited the most potent
inhibitory activity against JAK2, with IC50 of 3 nM and 85- and
76-fold selectivity over JAK1 and JAK3, respectively. In addition,
16m-(R) significantly suppressed JAK-STAT signaling pathways
in HEL cells. Hence, 16m-(R) might be selected as a promising
JAK2 inhibitor for further detailed investigation in the future.
(a)
p-JAK2(Y1007/8)
p-JAK2(Y221)
t-JAK2
p-STAT3(Y705)
t-STAT3
p-STAT5a(Y694)
t-STAT5
α-Tublin
(b)
p-JAK2(Y1007/8) /t-JAK2
p-JAK2(Y221) /t-JAK2
16m-(R)
16m-(R)
4
3
2
1
0
1.5
1.0
0.5
0.0
Fedratinib
Fedratinib
Acknowledgments
*
1
*
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.
k
an
k
1
1
0
1
1
10
01
0.
01
0.
0.1
0.1
an
l
l
b
b
Concentration(M)
Concentration(M)
p-STAT3(Y705)/t-STAT3
p-STAT5a(Y694)/t-STAT5
16m-(R)
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
16m-(R)
Fedratinib
Fedratinib
**
*
***
***
1
****
***
***
****
k
1
1
0
1
01
0.
1
1
0.
1
1
0.
an
0
0.
10
l
ank
b
l
b
Concentration(M)
Concentration(M)
Figure 6. 16m-(R) effectively blocks the JAK2-STAT signaling pathways in
HEL cells. (a) Western blot analysis of phosphorylated JAK2(Y1007/1008),
JAK2 (Y221), STAT3, STAT5. (b) Quantitative analysis of the intensity of
the bands of p-JAK2, p-STAT3 and p-STAT5 from western blot images (n =
References and notes
1.
Referen O'Shea, J. J.; Holland, S. M.; Staudt, L. M. N Engl J Med
2013, 368, 161.
3). Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P <
0.001, ****p < 0.0001.

2.
3.
4.
Kontzias, A.; Kotlyar, A.; Laurence, A.; Changelian, P.; O'Shea, J.
J. Curr Opin Pharmacol 2012, 12, 464.
Quintas-Cardama, A.; Kantarjian, H.; Cortes, J.; Verstovsek, S. Nat
Rev Drug Discov 2011, 10 (2), 127-40.
Baxter, E. J.; Scott, L. M.; Campbell, P. J.; East, C.; Fourouclas,
N.; Swanton, S.; Vassiliou, G. S.; Bench, A. J.; Boyd, E. M.;
Curtin, N.; Scott, M. A.; Erber, W. N.; Green, A. R. The Lancet.
2005, 365, 1054.
14. 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.; Wang, R.;
Zhu, L.; Li, H.; Xu, Y. Bioorg Med Chem Lett 2019, 29, 1507.
15. Wan, H.; Schroeder, G. M.; Hart, A. C.; Inghrim, J.; Grebinski, J.;
Tokarski, J. S.; Lorenzi, M. V.; You, D.; McDevitt, T.; Penhallow,
B.; Vuppugalla, R.; Zhang, Y.; Gu, X.; Iyer, R.; Lombardo, L. J.;
Trainor, G. L.; Ruepp, S.; Lippy, J.; Blat, Y.; Sack, J. S.; Khan, J.
A.; Stefanski, K.; Sleczka, B.; Mathur, A.; Sun, J. H.; Wong, M.
K.; Wu, D. R.; Li, P.; Gupta, A.; Arunachalam, P. N.; Pragalathan,
B.; Narayanan, S.; K, C. N.; Kuppusamy, P.; Purandare, A.V.ACS
Med Chem Lett 2015, 6, 850.
5.
6.
Nat Struct Mol Biol. 2014 July; 21(7): 579–584.
Fridman, J. S.; Scherle, P. A.; Collins, R.; Burn, T. C.; Li, Y.; Li,
J.; Covington, M. B.; Thomas, B.; Collier, P.; Favata, M. F.; Wen,
X.; Shi, J.; McGee, R.; Haley, P. J.; Shepard, S.; Rodgers, J. D.;
Yeleswaram, S.; Hollis, G.; Newton, R. C.; Metcalf, B.; Friedman,
S. M.; Vaddi, K. The Journal of Immunology. 2010, 184, 5298.
William, A. D.; Lee, A. C.; Blanchard, S.; Poulsen, A.; Teo, E. L.;
Nagaraj, H.; Tan, E.; Chen, D.; Williams, M.; Sun, E. T.; Goh, K.
C.; Ong, W. C.; Goh, S. K.; Hart, S.; Jayaraman, R.; Pasha, M. K.;
Ethirajulu, K.; Wood, J. M.; Dymock, B. W. J Med Chem 2011,
54 , 4638.
Mascarenhas, J.; Hoffman, R.; Talpaz, M.; Gerds, A. T.; Stein, B.;
Gupta, V.; Szoke, A.; Drummond, M.; Pristupa, A.; Granston, T.;
Daly, R.; Al-Fayoumi, S.; Callahan, J. A.; Singer, J. W.; Gotlib, J.;
Jamieson, C.; Harrison, C.; Mesa, R.; Verstovsek, S. JAMA Oncol
2018, 4 , 652.
16. Anal Biochem. 2004 Sep 1;332(1):153-9
17. Hart, S.; Goh, K. C.; Novotny-Diermayr, V.; Hu, C. Y.; Hentze,
H.; Tan, Y. C.; Madan, B.; Amalini, C.; Loh, Y. K.; Ong, L. C.;
William, A. D.; Lee, A.; Poulsen, A.; Jayaraman, R.; Ong, K. H.;
Ethirajulu, K.; Dymock, B. W.; Wood, J. W. Leukemia 2011, 25,
1751.
7.
8.
9.
18. Grandage, V. L.; Everington, T.; Linch, D. C.; Khwaja, A. Br J
Haematol 2006, 135, 303.
19. Meyer, S. C.; Keller, M. D.; Chiu, S.; Koppikar, P.; Guryanova,
O. A.; Rapaport, F.; Xu, K.; Manova, K.; Pankov, D.; O'Reilly, R.
J.; Kleppe, M.; McKenney, A. S.; Shih, A. H.; Shank, K.; Ahn, J.;
Papalexi, E.; Spitzer, B.; Socci, N.; Viale, A.; Mandon, E.; Ebel,
N.; Andraos, R.; Rubert, J.; Dammassa, E.; Romanet, V.;
Dolemeyer, A.; Zender, M.; Heinlein, M.; Rampal, R.; Weinberg,
R. S.; Hoffman, R.; Sellers, W. R.; Hofmann, F.; Murakami, M.;
Baffert, F.; Gaul, C.; Radimerski, T.; Levine, R. L. Cancer Cell
2015, 28, 15.
Pardanani, A.; Gotlib, J.; Roberts, A. W.; Wadleigh, M.; Sirhan,
S.; Kawashima, J.; Maltzman, J. A.; Shao, L.; Gupta, V.; Tefferi,
A. Leukemia 2018, 32 , 1035.
10. Pardanani, A.; Harrison, C.; Cortes, J. E.; Cervantes, F.; Mesa, R.
A.; Milligan, D.; Masszi, T.; Mishchenko, E.; Jourdan, E.;
Vannucchi, A. M.; Drummond, M. W.; Jurgutis, M.;
Kuliczkowski, K.; Gheorghita, E.; Passamonti, F.; Neumann, F.;
Patki, A.; Gao, G.; Tefferi, A. JAMA Oncol 2015, 1, 643.
11. Verstovsek, S.; Hoffman, R.; Mascarenhas, J.; Soria, J. C.;
Bahleda, R.; McCoon, P.; Tang, W.; Cortes, J.; Kantarjian, H.;
Ribrag, V. Leuk Res 2015, 39, 157.
12. Verstovsek, S.; Tam, C. S.; Wadleigh, M.; Sokol, L.; Smith, C. C.;
Bui, L. A.; Song, C.; Clary, D. O.; Olszynski, P.; Cortes, J.;
Kantarjian, H.; Shah, N. P. Leukemia Research 2014, 38, 316.
13. Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P.
M.; Wilks, A. F.; Fantino, E.; Rossjohn, J.; Lucet, I. S. Journal of
Molecular Biology 2009, 387, 219.
Supplementary Material
Supplementary material that may be helpful in the review
process should be prepared and provided as a separate electronic
file. That file can then be transformed into PDF format and
submitted along with the manuscript and graphic files to the
appropriate editorial office.
Graphical Abstract
To create your abstract, type over the instructions in the
template box below.
Fonts or abstract dimensions should not be changed or altered.
Declaration of interests
A novel 2-aminopyridine derivative 16m-(R)
Discovery and optimization of 2-aminopyridine
derivatives as novel and selective JAK2 inhibitors
was designed as a potent and selective JAK2
Xiangyu Ma#,a , Yanyan Diao#,a, Huan Ge#,a, Fangling Xua, Lili Zhua,*, Zhenjiang Zhaoa,*, Honglin Lia,
*

☒ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
*Corresponding author. Tel.: +86-21-64250213; fax: +86-21-64250213; email: zhulfl@ecust.edu.cn, zhjzhao@ecust.edu.cn, hlli@ecust.edu.cn;