Design, synthesis, and pharmacological evaluation of 4- or 6-phenyl-pyrimidine derivatives as novel and selective Janus kinase 3 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112148

As non-receptor tyrosine kinases, Janus kinases (JAKs) have become an attractive target for the treatment of autoimmune diseases and cancers. JAKs play a pivotal role in innate immunity, inflammation, and hematopoiesis by mediating the signaling of numerous cytokines, growth factors, and interferons (IFNs). Selective inhibitors of a variety of JAK members are expected to inhibit pro-inflammatory cytokine-mediated inflammation and immune responses, while preventing targeting other subtypes of JAKs. In this work, poorly selective compounds based on 4- or 6-phenyl-pyrimidine derivatives have been improved to highly potent and selective compounds by designing a covalent binding tether, which attaches to the unique cysteine (Cys909) residue in JAK3. Compound 12 exhibited potent JAK3 inhibitory activity (IC₅₀ = 1.7 nM) with an excellent selectivity profile when compared to the other JAK isoforms (>588-fold). In a cellular assay, compound 12 strongly inhibited JAK3-dependent signaling and T cell proliferation. Moreover, in vivo data revealed that compound 12 significantly suppressed oxazolone (OXZ)-induced delayed hypersensitivity responses in Balb/c mice. Compound 12 also displayed decent pharmacokinetic properties and was suitable for in vivo use. Taken together, these results indicated that compound 12 may be a promising tool compound as a selective JAK3 inhibitor for treating autoimmune diseases.

Full text of DOI:10.1016/j.ejmech.2020.112148

Journal Pre-proof
Design, synthesis, and pharmacological evaluation of 4- or 6-phenyl-pyrimidine
derivatives as novel and selective Janus kinase 3 inhibitors
Lei Shu, Chengjuan Chen, Xueting Huan, Hao Huang, Manman Wang, Jianqiu
Zhang, Yile Yan, Jianming Liu, Tiantai Zhang, Dayong Zhang
PII:
S0223-5234(20)30115-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112148
EJMECH 112148
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 November 2019
Revised Date: 13 February 2020
Accepted Date: 13 February 2020
Please cite this article as: L. Shu, C. Chen, X. Huan, H. Huang, M. Wang, J. Zhang, Y. Yan, J. Liu,
T. Zhang, D. Zhang, Design, synthesis, and pharmacological evaluation of 4- or 6-phenyl-pyrimidine
derivatives as novel and selective Janus kinase 3 inhibitors, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2020.112148.
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 Masson SAS.

Design, synthesis, and pharmacological evaluation of 4- or
-phenyl-pyrimidine derivatives as novel and selective Janus kinase 3
6
inhibitors
a,1
b,1
a
a
a
Lei Shu , Chengjuan Chen , Xueting Huan , Hao Huang , Manman Wang ,
a
a
a
b, ∗ ∗
a,*
Jianqiu Zhang , Yile Yan , Jianming Liu , Tiantai Zhang
, Dayong Zhang
a
Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing
2
10009, PR China
b
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, PR China
*
Corresponding author. Institute of Pharmaceutical Science, China Pharmaceutical
University, Nanjing 210009, PR China.
**
Corresponding author. State Key Laboratory of Bioactive Substance and Function
of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, China.
E-mail address: cpuzdy@163.com; ttzhang@imm.ac.cn (Tiantai Zhang).
1
These authors contributed equally to this work.

Abstract
As non-receptor tyrosine kinases, Janus kinases (JAKs) have become an attractive
target for the treatment of autoimmune diseases and cancers. JAKs play a pivotal role
in innate immunity, inflammation, and hematopoiesis by mediating the signaling of
numerous cytokines, growth factors, and interferons (IFNs). Selective inhibitors of a
variety of JAK members are expected to inhibit pro-inflammatory cytokine-mediated
inflammation and immune responses, while preventing targeting other subtypes of
JAKs. In this work, poorly selective compounds based on 4- or 6-phenyl-pyrimidine
derivatives have been improved to highly potent and selective compounds by
designing a covalent binding tether, which attaches to the unique cysteine (Cys909)
residue in JAK3. Compound 12 exhibited potent JAK3 inhibitory activity (IC = 1.7
5
0
nM) with an excellent selectivity profile when compared to the other JAK isoforms (>
88-fold). In a cellular assay, compound 12 strongly inhibited JAK3-dependent
5
signaling and T cell proliferation. Moreover, in vivo data revealed that compound 12
significantly suppressed oxazolone (OXZ)-induced delayed hypersensitivity responses
in Balb/c mice. Compound 12 also displayed decent pharmacokinetic properties and is
suitable for in vivo use. Taken together, these results indicated that compound 12 may
be a promising tool compound as a selective JAK3 inhibitor for treating autoimmune
diseases.
Keywords: Autoimmune diseases; Covalent JAK3 inhibitors; Cys909; Janus kinase;
4
- Or 6-phenyl-pyrimidine derivatives
1
. Introduction
Janus kinases (JAK1, JAK2, JAK3, and TYK2) are a small family of non-receptor
tyrosine kinases that are essential for mediating the signaling cascade that is
associated with multiple cytokines by activating signal transducer and activator
transcription (STAT) [1]. The JAK-STAT pathway is considered as an evolutionarily
conserved signaling pathway involved in the regulation of immune and inflammatory

disease [2]. Binding of cytokines with their distinct receptor is the trigger for
activation of JAKs, and upon activation STATs bind to phosphorylated cytokine
receptors and undergo phosphorylation by JAKs [3]. Subsequently, dimerized STATs
translocate to the nucleus and drive gene transcription, thereby regulating the immune
system and inflammatory responses [4]. With the first US Food and Drug
Administration (FDA)-approved pan-JAKs small molecule inhibitor tofacitinib
[
5,10-11] for the treatment of rheumatoid arthritis (RA), the development of small
molecule JAK inhibitors became attractive for treatment of autoimmune and
inflammatory diseases.
In recent years, significant research efforts have been performed towards the
discovery of inhibitors of JAK kinases. As shown in Fig. 1 and Table 1, several
small-molecule inhibitors are current in the clinical or preclinical stage for the
treatment of autoimmune diseases, various cancers, and myeloproliferative disorders
[
5-16]. For the first-generation of non-selective JAKs inhibitors that have been proven
safe and efficacious, their broad inhibiting spectrum for cytokines inevitably leads to
side effects by inhibiting many factors that can drive immunopathology. However, the
next generation selective JAKs inhibitors are expected to be developed and prevent
targeting other subtypes of JAKs. In August 2019, the selective JAK1 inhibitor
upadacitinib [12-14], as a second-generation JAKs inhibitor, was first approved by the
US FDA for the treatment of RA. PF-06651600 [15,16] has been reported as a
selective JAK3 inhibitor that has been used in clinical trials.

Fig. 1. Chemical structures of JAK inhibitors.
Table 1.
The selectivity of JAK inhibitors in a cell-free enzyme assay.
a
IC50 (nM)
b
c
Compound
JAK3/JAK2
JAK3/JAK1
JAK3
>400
>400
2304
0.71
1
JAK2
3
JAK1
3
Ruxolitinib [6]
Baricitinib [7]
Upadacitinib [8]
Peficitinib [9]
>133
>133
6
6
>67
>67
120
5.0
47
19
-
49
-
3.9
d
Tofacitinib
20
112
>10000
-
-
PF-06651600 [15]
33
>10000
-
-
a
IC against JAK3, JAK2, JAK1 in enzyme assay.
5
0
b
c
d
Fold selectivity derived from JAK3/JAK2 enzyme IC .
5
0
Fold selectivity derived from JAK3/JAK1 enzyme IC .
5
0
Tofacitinib is originally described as being selective for JAK3 [10], however it was later proven
to be a pan-JAK inhibitor [11].
Among members of the JAKs family, JAK3 exhibited predominant expression in
the hematopoietic system versus other JAKs, and were ubiquitously expressed in a
variety of cell types [17]. Another feature of JAK3 is that it is required for cytokine
signaling, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which act through
relative receptors that possess the common gamma chain (γc) cytokines receptor

subunit [18]. Cytokine signaling through the γc receptor is essential for normal
activity and function of the immune system [19]. In humans, loss of function mutation
leading to JAK3 deficiency in γc results in severe combined immunodeficiency
(SCID) [20]. A selective JAK3 inhibitor should only block common γc cytokine
signaling to resulting in much narrower spectrum of cytokine activity compared to
first-generation non-selective inhibitors and concomitant prevention of some the side
effects. Human JAK3 has a unique cysteine residue (Cys909), which is replaced with
a serine residue at the equivalent position in the other three isoforms of JAK1, JAK2,
and TYK2 [21]. In crystal structure, this feature gives the opportunity to design a
selective inhibitor for JAK3. Based on the difference of Cys909, targeting the Cys909
ATP-competitive binding site in a covalent manner would be an optimized strategy
[
22]. Recently, several selective JAK3 inhibitors have been reported to bind the
Cys909 site with a covalent bond formation, leading to irreversible inhibition and
high selectivity, including PF-06651600, which was reported to irreversibly
covalently bind Cys909 in JAK3 [16,23]. Based on the essential role in the immune
system as well as the crystal structure feature, JAK3 has been considered as a
potential target for the treatment of autoimmune and inflammatory diseases.
In our previous study, we reported a series of compounds, including the bicyclic
structure of pyrrolopyrimidine or pyrazolopyrimidine targeting JAKs, however, their
inhibitory potency and selectivity need to be further improved [24-27]. Herein, we
aimed to generate potent substituted pyrimidine-based inhibitors that exploit an
acrylamide electrophile to form a covalent bond with Cys909 in JAK3. In this study,
4
- Or 6-phenyl-pyrimidine derivatives as potent JAK3 inhibitors were designed and
synthesized (Fig. 2). Covalent JAK3 inhibitor (1) has excellent biological activities
against JAK3, both in vitro and in vivo [23]. Furthermore, guided by the structure of
CYT-387 [28,29] and N-(3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl) acrylamide (3)
[
15], new molecules (1-39) were designed by further optimization. Our studies
showed that compound 12 had significant potent JAK3 inhibitory activity and

selectivity. Therefore, in this study, we report the design, synthesis, and
structure-activity relationship (SAR) of 4- or 6-phenyl-pyrimidine JAK inhibitors.
Fig. 2. Design of selective JAK3 inhibitors.
2
. Results and discussion
2
.1. Chemistry
A series of 4- or 6-phenyl-pyrimidine derivatives and intermediates were
synthesized according to the pathways described in Schemes 1-4 (see Scheme 4,
supplementary material).

Scheme 1. Reagents and conditions: (i) bis(pinacolato)diboron, 1,4-dioxane/H O,
2
Pd(PPh ) , KOAc, 100 °C, 12 h; (ii) 1,4-dioxane/H O, Pd(PPh ) , K CO , 100 °C, 12
3
4
2
3 4
2
3
h; (iii) amine, DIEA, isopropanol, 82 °C, 6h; (iv) Pd/C, hydrazine hydrate, EtOH, 100
°
C, 2h.
As presented in Scheme 1, 3-bromo-nitrobenzene reacted with
bis(pinacolato)diboron, then commercially available 4,6-dichloropyrimidine was
treated with benzene pinacol borate to provide intermediate 40 at moderate yield
(74%). Next, intermediate 40 was treated with several types of amines that were
prepared as previously reported [24] for R to produce 6-phenyl-pyrimidine amine
1
4
1a-f under alkaline conditions. Treatment of compounds 41a-f with 85% hydrazine
hydrate under nitrogen and Pd/C catalysis provided compounds 42a-f.
Intermediates 43, 46, and 49 were prepared following a synthetic procedure of that
was similar to that of compound 40 (see Scheme 4, supplementary material).

Scheme 2. Reagents and conditions: (i) acyl chlorides, THF, 0 °C to room
temperature (RT), 30min; (ii) cyanoacetic acid, HATU, DIEA, THF, 16h.
Scheme 3. Reagents and conditions: (i) Boc-glycine, EDCI, HOBT, DIEA, DCM, 8h;
(ii) TFA, DCM, 5h; (iii) acyl chlorides, THF, 0 °C to room temperature (RT), 30min;
(iv) cyanoacetic acid, HATU, DIEA, THF, 16h.
As shown in Scheme 3, intermediates 42a-e were condensed with Boc-glycine to
produce compounds 52a-e. Subsequently, Boc deprotection using TFA in DCM
cleanly generate compounds 53a-e at near quantitative yield. The target compounds
were prepared as presented in Scheme 2 and 3, which involved two types of reactions.
First, intermediates were reacted with acyl chlorides to generate the desired

compounds under basic conditions. Subsequently, intermediates were treated with
cyanoacetic acid using standard HATU conditions to generate compounds at good
yield as previously reported [24].
2
.2. In vitro enzymatic inhibitor activities
All newly synthesized compounds were evaluated for their activity against JAK
enzymes using a Homogenous Time-Resolved Fluorescence (HTRF) Kin EASE-TK
assay system. For comparison, the activity of pan JAK inhibitor tofacitinib, was also
evaluated using the same procedure.
In this study, the inhibitory activity of newly synthesized compounds was evaluated
using human JAK1, JAK2, JAK3, and TYK2 enzymes. To approach this optimization
in a systematic fashion, this chemotype of target compounds was divided into four
moieties, core (4, 6- or 2, 4-disubstituted pyrimidine), side chain (R ), tail (R ) and the
1
2
linkage between core and tail. Furthermore, the side chain and tail varied sequentially.
In vitro, the preliminary screening indicated that some derivatives showed good JAK3
inhibitory activity and selectivity compared with positive controls. The IC values
5
0
obtained in JAK enzyme assays are summarized in Tables 2 and 3.
Table 2.
SARs of side chains (R1)
a
IC50 (nM)
JAK2
JAK1
TYK2
Compd
R1
JAK3
303
b
c
d
JAK2/JAK3
JAK1/JAK3
TYK2/JAK3
e
2
237
7]
2852
28]
657
[2]
ND
-
1
2
[
e
O
568
[6]
ND
-
N
N
101
[

e
e
e
e
2
043
11]
1107
[6]
ND
3
4
5
6
7
8
9
183
309
138
599
55
-
[
>
3000
731
[2]
ND
-
-
e
ND
840
ND
-
-
[
6]
e
2
588
4]
1286
ND
ND
-
-
[
O
O
O
1102
[20]
2119
[71]
1658
[25]
961
2871
[52]
2439
[30]
>3000
-
N
N
[23]
1796
[60]
30
>3000
-
N
N
67
e
1
569
14]
ND
-
1
0
112
9.1
1.7
0.9
[
[9]
1079
119]
000
588]
.5
1.7]
IC values are the average of duplicate experiments.
1562
[172]
1320
[777]
1.3
>3000
-
N
N
11
[
1
2783
[1637]
35.5
1
2
[
1
Tofacitinib
[
[1.1]
[39.4]
a
b
c
d
e
5
0
Fold selectivity derived from JAK2/JAK3 enzyme IC .
5
0
Fold selectivity derived from JAK1/JAK3 enzyme IC .
5
0
Fold selectivity derived from TYK2/JAK3 enzyme IC .
5
0
ND = not determined.
Table 3.
SARs of side chains (R2)
a
IC50 (nM)
JAK2
JAK1
TYK2
Compd
R1
R2
JAK3
b
c
d
JAK2/JAK3 JAK1/JAK3 TYK2/JAK3
e
2
761
1521
[3]
ND
-
13
567
[
5]

e
e
ND
-
854
6]
ND
-
14
15
16
145
29
[
2
580
89]
1286
[44]
322
[5]
>3000
-
[
5
87
2264
[35]
65
[
9]
2
129
61]
2876
[82]
>3000
-
17
35
[
e
7
34
1861
[11]
899
ND
18
19
20
21
22
23
170
67
-
[4]
e
1
886
28]
109
ND
-
[
[13]
e
e
1
ND
-
ND
-
92
[
2
[
12]
808
13]
143
e
2443
[11]
1542
[6]
ND
-
221
243
95
e
1
ND
[5]
-
e
6
9
89
ND
-
[1]
[1]
e
1
542
23]
56
ND
-
24
25
26
66
[
[1]
e
8
86
45
ND
-
108
103
[9]
[0.4]
e
>
3000
-
354
[3]
ND
-
e
e
ND
586
ND
27
28
29
30
31
32
33
34
375
253
127
33
-
-
[2]
e
3
09
1]
1432
[6]
ND
-
[
e
2
732
22]
788
85]
>3000
ND
-
[
-
e
2
[
ND
-
2653
[577]
>3000
-
2
16
2233
[15]
152
76
[
1]
e
e
ND
-
2764
ND
-
[36]
1
459
2677
[31]
321
[3]
1872
[22]
86
[17]
e
2
155
22]
ND
-
99
[

e
e
ND
-
528
7]
ND
-
3
3
3
3
3
5
6
7
8
9
78
14
24
6.1
15
[
1
981
1445
[103]
183
>3000
-
[142]
e
ND
2496
[104]
>3000
-
-
[8]
1412
1147
[188]
433
[232]
8
8
>3000
-
[
6]
1.5
[1.7]
[29]
1.3
Tofac
35.5
[39.4]
0
.9
itinb
[1.4]
a
IC values are the average of duplicate experiments.
5
0
b
c
d
e
Fold selectivity derived from JAK2/JAK3 enzyme IC .
5
0
Fold selectivity derived from JAK1/JAK3 enzyme IC .
5
0
Fold selectivity derived from TYK2/JAK3 enzyme IC .
5
0
ND = not determined.
N-methyl-6-(3-(substituted amino)phenyl)pyrimidin-4-amine derivatives (1-6,
3-29) were synthesized with changes in the side chain (R ) and tail (R ). Several
1
1
2
analogs using different ammonia groups (Table 2) were generated to develop stronger
JAK3 inhibitors, including compounds 1-6 that showed an IC50 of 100-600 nM
against JAK3. Next, to improve potency for JAK3, the SAR of the tail (R ) for
2
compounds 13-29 was investigated (Table 3). When butenamide groups were
introduced (compounds 15 and 17), both the inhibitory activities and selectivity for
JAK3 increased up to 3-6-fold, when compared with compounds 2 and 3. The
resulting increase in inhibitory activities might be attributed to the length of the tail
that covalently reacts with Cys909 in JAK3 kinases. Nevertheless, when the “length”
of amide in compounds 15 and 17 was enhanced, creating compounds 23-29, a
decrease in inhibitory activities against JAK3 was observed. Therefore,
para-substituted compounds 9-10 and 32-35 at the 4-position of the phenyl ring were
designed for further modifications. The results showed that compounds showed a
significant increase in inhibitory activities against JAK3.
Replacement of 4,6-substituted pyrimidine with 2,4-substituted pyrimidine
(compounds 7-8, 11-12, 30-31, and 36-39, Table 2 and 3) retained the activities
against JAK3 (inhibitory potency with an IC ranging between 1.7 nM to 55 nM) and
5
0

selectivity for JAK3 versus other kinases. Among this series, regarding the feature of
the hydrophobic cavity, we mainly explored the side chain is
1
-(2-methoxyethyl)-1H-pyrazol-4-amine or 4-morpholinoaniline. Compound 12
exhibited excellent inhibitory activity against JAK3 (IC50 = 1.7 nM) and
unprecedented selectivity (777-fold JAK1/3 and 588-fold JAK2/3), when compared to
other derivatives. The results revealed that 4-morpholinoaniline (compounds 12 and
3
8) have a higher potency compared to a pyrazole ring (11 and 36) at the 2-position of
the pyrimidine ring. Compounds 12 (ClogP = 3.77) and 38 (ClogP = 4.02) exhibited a
higher lipophilicity compared to compounds 11 and 36 (Table 4), respectively. The
results demonstrated that the hydrophobic aromatic amine group improved the JAK3
inhibitory activity, which can be confirmed by molecular docking study where the
hydrophobic aromatic amine group can create two σ-π interactions with amino acids
Leu828 and Gly908 (Fig. 3). After comparing the above-mentioned derivatives, we
concluded that compounds with a para-acrylamide on the phenyl ring showed the
greatest potency against JAK3 among others with meta-acrylamide. A similar trend
was observed among other analogues with their parent compounds in 4,6-substituted
pyrimidine derivatives. Based on the above-mentioned SAR, compounds 11, 12, and
3
8 were selected as tool compounds for additional studies.
2
.3. Molecular docking study
Based on the above discussion, compound 12 possessed the best inhibitory
activities and selectivity for JAK3. To investigate the possible binding mode of
compound 12 with JAK3 (PDB: 4Z16), computer simulation of molecular docking is
performed by Molecular Operating Environment (MOE) software. As shown in Fig. 3,
the anilinopyrimidine moiety of compound 12 makes the expected bidentate hinge
hydrogen bonds with Leu905, and continuous electron density was observed between
the acrylamide warhead and Cys909, indicative of covalent bond formation. The
major difference in JAK3 binding models is that two σ-π interactions are produced by
the morpholinoaniline of 12 with the amino acids Leu828 and Gly908 and a σ-π
interaction between the pyrimidine core of 12 with the amino acid Leu828.

Presumably, these interactions enhanced the activity of 12 against JAK3. Interestingly,
the thiol group and Asp912 formed a hydrogen bond after the acryl group of
compound 12 and the thiol group of residue Cys909 form a covalent bond, which
indicated that compound 12 had tight contact with the ATP-binding pocket in JAK3.
These docking observations reasonably explain the activity data.
Fig. 3. Compound 12 bound to JAK3 enzyme (PDB code: 4Z16). 3D interaction
diagram representing the docked conformation of compound 12 covalently binds to
Cys909 of JAK3 enzyme.
2
.4. Cell cytotoxicity evaluation
To evaluate the effects of active compounds 11, 12, and 38 on the cell viability,
resting mouse T cells, IL-4-treated activated T cells, and Raw 264.7 cells were used
for the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS assay). In
general, resting T cells and IL-4-treated activated T cells do not have the ability to
proliferate, but maintain normal survival. Raw 267.4 cells served as non-lymphocyte
controls, and were selected for cytotoxicity evaluation. Treatment of the three cell
types with compounds 11, 12, and 38 for 72 h at 10 µM, showed that three
compounds did not have any influence on the three cell types (Fig. 4). Taken together,

these results indicated that the compounds had no obvious cytotoxicity against these
cells when used at a concentration of 10 µM.
Fig. 4. The cell viability of 11, 12, and 38 was determined by the MTS method.
Resting mouse T cell (A), IL-4-treated activated T cell (B), and RAW264.7 cells (C)
were respectively treated with compounds 11, 12, and 38 at 10 µM for 72 h, and
cytotoxicity was expressed by the percentage of surviving cells over control cells.
2
.5. Compounds on T cell proliferation in vitro
Based on the inhibitory potency and selectivity of compounds for JAK3,
compounds 11, 12, and 38 were selected for further evaluation. In addition, their
anti-proliferative activity against mouse T cell lines was assessed using the MTS
assay. To analyze the immunosuppressive activity, mouse T cells were purified and
activated with or without anti-CD3/-CD28 or IL-2 in the presence or absence of
different concentrations of compounds 11, 12, and 38 for 72 h, then, the proliferation
of T cells was analyzed by the MTS assay. As shown in Table 4, compounds 11, 12,
and 38 significantly suppressed T cell proliferation when stimulated by
anti-CD3/CD28 with IC values of 6.49, 0.83, and 1.60 µM or IL-2 with IC value
5
0
50
of 7.5, 0.77, and 2.11 µM, respectively. Especially, compound 12 displayed a stronger
inhibition for T cell proliferation when compared to tofacitinib with an IC value of
5
0
0
.83 vs 1.38 (anti-CD3/CD28 stimulation) and 0.77 vs 1.54 (IL-2 stimulation),

respectively. Together, our data demonstrated that compound 12 showed obvious
significant immunosuppressive activity under selective inhibition of JAK3.
Table 4.
The effects of compounds inhibiting T cell proliferation after stimulation by
anti-CD3/-CD28 or IL-2.
Inhibition of T cell proliferation, IC (µM)
5
0
a
Compound
ClogP
anti-CD3/-CD28
IL-2
7.50
0.77
2.11
1.50
11
6.49
0.83
1.60
1.38
2.18
3.77
4.02
1.54
1
2
8
3
Tofacitinib
a
Calculated as miLogP using Molinspiration property engine version 2018.10
http://www.molinspiration.com).
(
2
.6. Compound 12 selectively inhibits JAK3-dependent signaling in live cells
To test whether the compounds were effective in a native cellular environment, we
evaluated the ability of compounds to inhibit STAT5 phosphorylation in live cells
using tofacitinib as a control. As shown in Fig. 5, IL-2 or IL-15-induced STAT5
phosphorylation was significantly inhibited when used at a concentration of 10 µM
after incubating mouse T cells with compounds 11, 12, and 38 for 1 h (Fig. 5A, B).
Compared to the other two compounds and tofacitinib, compound 12 exhibited better
inhibitory activity when used at the same concentration (10 µM). Further analysis
indicated that compound 12 almost completely abrogated IL-2 or IL-15-induced
phosphorylation of STAT5 at 1 µM treatment in a concentration-dependent manner
(Fig. 5C, D). Data obtained from the cellular signaling pathway level further
confirmed the selective activity of compound 12 on JAK3. Thus, these results
demonstrated that compound 12 had potent inhibitory activity and selectivity on
JAK3.

Fig. 5. Compounds potently and selectively inhibited JAK3-dependent signaling in
mouse T cells. After pre-incubating with compounds 11, 12, and 38 for 2 h at 10 µM,
T cells were stimulated with IL-2 (A) or IL-15 (B) for another 30 min. The levels of
p-STAT5 were determined by Western blot analysis. The concentration-dependent
expression of p-STAT5 was further determined by indicated concentrations of
compounds 11 and 12 for 2 h prior to IL-2 (C) or IL-15(D) stimulation to T cells. Tof
(tofacitinib) served as a positive agent in this assay.
2
.7. Kinase selectivity profiling of compound 12
To further understand the selective kinase profile, the inhibitory effect of compound
2 for tyrosine kinases other than JAK family isoforms were evaluated, including
1
BLK, BMX, BTK, EGFR, ERBB2, ERBB4, ITK, RLK, and TEC, which all carry a
thiol in a position analogous to that of Cys909 in JAK3. The results are presented in
Table 5, and show that compound 12 exhibited a potent inhibition rate of 100% for
JAK3 at a concentration of 100 nM. However, the inhibition rate of the other 9
kinases was much lower than that of JAK3. Taken together, our data indicated that
compound 12 exhibited a higher selective inhibition to JAK3 than other kinases with
a cysteine at a position compared to that of Cys909.

Table 5.
The inhibitory activity of compound 12 (100 nM) on other kinases.
Inhibition rate (%)
compd
EGFR
ErbB1) (HER2) (HER4)
1.2 19.2 8.0
ERBB2 ERBB4
JAK3 BLK BMK BTK
100 20.6 3.7 6.3
ITK RLK TEC
2.3 2.9 29.5
(
12
Biochemical assays were conducted using SelectScreen® Kinase Profiling by Z’-LYTE (TEC by
Lantha Screen) at the Km concentration of ATP.
2
.8. Compound 12 covalently binds to JAK3 and irreversibly inhibits JAK3
To confirm that compound 12 irreversibly interacted with JAK3, a time-dependent
inhibition assay was performed (Fig. 6A). For fair comparison, the concentration was
performed at 2 nM at which compounds were profiled reflects their JAK3 potency.
Extending the pre-treatment time of compound 12 with JAK3 improved the inhibitory
effect, whereas the reversible inhibitor tofacitinib did not exhibit such time-dependent
behavior (Fig. 6A). To further determine if compound 12 shared the features of
irreversible inhibitors, the dissociation kinetics of compound 12 were evaluated.
Compound 12 at an IC (500 nM) was mixed and incubated with JAK3 for 30
9
0
minutes to allow complete association. This mixture was then diluted with kinase
buffer 1000-fold to less than the IC , and the enzymatic activity was evaluated via
1
0
measuring the phosphorylated product by TR-FRET. The recovery rate of kinase
activity related to JAK3 that had not been exposed to compound 12 was measured.
Pre-treatment of JAK3 with compound 12 resulted in lack of any measurable JAK3
activity once the compound was diluted, suggesting that the inhibitor irreversibly
bound to JAK3 for more than 24 hours (Fig. 6B).

Fig. 6. (A) Time-dependent inhibition curves of compound 12 and Tofacitinib. (B)
JAK3 enzymatic activity was not restored up to 24 hours after diluting from an IC to
90
an IC concentration of compound 12 (blue) when compared to a DMSO control
1
0
(red).
2
.9. Pharmacokinetic property evaluation
Preliminary pharmacokinetic (PK) properties of compound 12 were performed in
male ICR (Institute of Cancer Research) mice following intravenous and oral
administration. The results are summarized in Table 6 and the corresponding
concentration-time curve was showed in Fig. S1 of supplementary material. Upon oral
administration of 30 mg/kg, compound 12, a reasonable PK profile was obtained with
a t1/2 of 1.52 h, area under curve (AUC) of 889.42 µg/L*h, and moderate
bioavailability of 23.82%. These data suggested that compound 12 may be a useful
probe for cellular and animal studies.
Table 6.
Preliminary pharmacokinetic data of compound 12 in male ICR Mice.
Compound 12
iv (10 mg/kg)
po (30 mg/kg)
a
AUC(0-t) (µg/L*h)
1244.41±77.83
889.42±48.32
AUC(0-
∞
) (µg/L*h)
1274.41±57.18
0.73±0.08
897.12±56.72
1.42±0.38
b
MRT (0-
∞
)(h)
c
Vz (L/kg)
8.36±1.83
220.42±24.71
97.14±20.87
1.52±0.34
d
CLz (L/h/kg)
8.15±1.21
e
t1/2 (h)
0.47±0.06
f
Cmax (µg/L)
8763.23±324.65
2008.21±189.44

g
Bioavailability(%)
23.82%
a
c
f
b
Area under the concentration time curve. Mean residence time
d
e
Volume in steady state. Plasma clearance. Terminal half-life.
g
Peak plasma concentrations. Bioavailability = AUC (po)/AUC (iv) × 100%
0
–t
0–t
2
2
.10. In vivo immunosuppressive activity
.10.1. Compound 12 inhibited oxazolone-induced delayed type hypersensitivity
reaction
Delayed type hypersensitivity (DTH) reaction is a T cell-mediated pathologic
response. To test the immunosuppressive activity of compounds 11, 12, and 38 in vivo,
the effects of these compounds on an oxazolone (OXZ)-induced DTH Balb/c mice
model [30] were determined using tofacitinib as the positive control [31] (Fig. 7A).
Following sensitization by OXZ, mice were administrated compounds or tofacitinib
by p.o. at a dose of 30 mg/kg. Ear swelling and spleen index were used to assess the
effect of drugs. Following 7 days of administration, only compound 12 displayed a
significant reduction of ear swelling by OXZ-induced DTH (Fig. 7B, C). In addition,
all agents showed a reduction in the spleen index, and compound 12 and tofacitinib
exhibited potent suppression (Fig. 7D). The spleen index is an important index to
evaluate immunosuppression, and our data indicated that compound 12 exhibited
obvious immunosuppressive activity.

Fig. 7. The effect of compounds 11, 12, and 38 on oxazolone (OXZ)-induced delayed
type hypersensitivity (DTH) mouse model. (A) Timeline for the process of DTH
development and treatment. Ear weight (B), the percentage of ear swelling (C), and
the spleen index (D) were presented to evaluate the effect of compounds 11, 12, and
3
8 on OXZ-induced DTH responses. Tofacitinib served as a positive agent for
compounds. Data are presented as the mean ± SEM. n = 5-6 per group for treated
#
##
*
**
mice and 3 for control mice. p < 0.001 versus control group; p < 0.05, p < 0.01,
***
and p < 0.001 versus vehicle group.
2
.10.2. Compound 12 inhibited dose-dependent OXZ-induced DTH responses
To further evaluate the effect of compound 12 on OXZ-induced DTH responses,
three doses of compound 12 were administered to OXZ-induced DTH mice. Our
results indicated that in OXZ-induced DTH mice, compound 12 reduced edema of ear
tissue in a dose-dependent manner (Fig. 8A, B). In addition, high doses of compound
1
2 and tofacitinib significantly inhibited the spleen index (Fig. 8C). Histopathological
changes and inflammatory cell infiltration were evaluated by eosin and hematoxylin
HE) staining. HE-staining showed less neutrophil leukocyte infiltration and
(

well-structured tissue architecture in the mice that were given compound 12 (30
mg/kg) when compared to the vehicle group (Fig. 8D), which showed significant
dense neutrophil leukocyte infiltration and increased ear thickness. Upon oral
administration of 30 mg/kg compound 12, the neutrophil leukocyte infiltration was
reduced and edema was decreased, and ear tissue was similar to that in control tissues.
In addition, no signs of toxicity were observed in compound-treated mice based on
body weight and microscopic examination of individual organs (data not shown).
Fig. 8. Immunosuppressive effects of compound 12 on oxazolone-induced delayed
type hypersensitivity mice. (A-C) Compound 12 (30, 10, and 3 mg/kg, p.o.) inhibited
oxazolone (OXZ)-induced delayed type hypersensitivity (DTH) responses in a
dose-dependent manner. (D) The effect of compound 12 on histopathological changes

and inflammatory cell infiltration were evaluated by HE-staining in ear tissue. Scale
bar 200 µm. Tofacitinib was positive agent and compared with compound 12. Data are
#
##
the mean ± SEM. n = 5-6 per group for treated mice and 3 for control mice. p <
*
**
***
0
.001 versus control group; p < 0.05, p < 0.01, and p < 0.001 versus vehicle
group.
3
. Conclusion
JAK is a confirmed pivotal target for immune and inflammatory diseases, and there
is continuous interest in the discovery of novel selective inhibitors that overcome the
side effects. In a recent study, novel derivatives were designed and synthesized based
on 4- or 6-phenyl-pyrimidine as novel selective JAK3 inhibitors. After primary SAR
analysis and enzymatic inhibitory activity in vitro, compound 12 showed a highly
potent and selective inhibition for JAK3 with an IC value of 1.7 nM, and exceeding
5
0
5
88-fold selectivity versus other enzymes in the JAK family. Molecular docking
studies suggested that compound 12 might covalently bind to the Cys909 position of
JAK3. T cell proliferation and kinase signal pathway experiments revealed that
compound 12 presented a high selectivity within the JAK family. Further in vivo
studies using an OXZ-induced DTH mouse model indicated that compound 12 had
significant anti-inflammatory and immunosuppressive activities. Moreover,
compound 12 possessed reasonable oral pharmacokinetic properties. Therefore, the
data obtained in the current study suggested that compound 12 may be a promising
tool compound for treating autoimmune diseases as a selective JAK3 inhibitor.
4
. Experimental procedures
4
.1. Chemistry and chemical methods
Unless specified otherwise, all starting materials, reagents, and solvents were
commercially available and used without further purification. All chemicals or
reagents were of analytical grade. Silica gel (200–300 mesh, Qingdao city, China) was
used for column chromatography. The progress of reactions was monitored by
analytical thin-layer chromatography (TLC) on silica gel plates (GF-254) and

visualized under UV light at 254 or 365 nm. Melting points of individual compounds
1
were determined using a Mel-TEMP II and melting point apparatus uncorrected. H
13
NMR and C NMR spectra were recorded using a Bruker ARX-300 spectrometer
with TMS as the internal standard in CDCl , DMSO-d6. Peak multiplicities were
3
abbreviated as follows: singlet, s; doublet, d; triplet, t; m, multiplet; dd, double
doublet. Mass spectrum (MS) was obtained on an Agilent 6120 quadrupole LC/MS
(ESI). High resolution mass spectrum (HRMS) was run on an Agilent Q-TOF mass
spectrometer.
4
.1.1 4-Chloro-6-(3-nitrophenyl)pyrimidine (40)
-Bromonitrobenzene (1 g, 5.0 mmol), bis(pinacolato)diboron (1.34 g, 2.6 mmol),
KOAc (1.48 g, 7.5 mmol), and Pd(PPh ) (0.11 g, 0.09 mmol) were combined in
3
3
4
1
1
,4-dioxane (50 mL). The reaction mixture was placed into an oil bath preheated to
00 ℃ , with stirring at this temperature for 12 h under argon. When completion of the
reaction showed TLC, cooled to room temperature, respectively added to
,6-dichloropyrimidine (1.07 g, 7.2 mmol), 2M K CO (2 g, 14.5 mmol) aqueous
2
2
3
solution and Pd(PPh ) (0.10 g, 0.09 mmol), heated to 100 °C in oil bath, stirred at
3
4
this temperature for 12 h under argon. The reaction mixture was filtered through a
Celite bed, and the filtrate was concentrated and purified by silica gel column
chromatography (hexane: ethyl acetate = 50:1) to afford 40 as a white solid (0.82 g,
1
total yield 68%). H NMR (300 MHz, CDCl ) δ 9.11 (s, 1H), 8.95 (s, 1H), 8.45 (d, J =
3
8
.0 Hz, 1H), 8.41 (d, J = 9.3 Hz, 1H), 7.85 (s, 1H), 7.74 (t, J = 8.0 Hz, 1H).
4
.1.2. 4-Chloro-6-(4-nitrophenyl)pyrimidine (43)
Compound 43 was prepared with the same procedures as for 40 as reported in 70%
1
total yield. H NMR (300 MHz, CDCl ) δ 9.11 (s, 1H), 8.38 (d, J = 8.8 Hz, 2H), 8.27
3
(d, J = 8.8 Hz, 2H), 7.83 (s, 1H).
4
.1.3. 2-Chloro-4-(3-nitrophenyl)pyrimidine (46)

Compound 46 was prepared with the same procedures as for 40 as reported in 81%
1
total yield. H NMR (300 MHz, CDCl ) δ 8.92 (s, 1H), 8.76 (d, J = 5.2 Hz, 1H), 8.48
3
(d, J = 7.9 Hz, 1H), 8.40 (d, J = 7.1 Hz, 1H), 7.76 – 7.73 (m, 2H).
4
.1.4. 2-Chloro-4-(4-nitrophenyl)pyrimidine (49)
Compound 49 was prepared with the same procedures as for 40 as reported in 76%
1
total yield. H NMR (300 MHz, CDCl ) δ 8.76 (d, J = 5.2 Hz, 1H), 8.37 (d, J = 8.9 Hz,
3
2
H), 8.28 (d, J = 8.9 Hz, 2H), 7.73 (d, J = 5.2 Hz, 1H).
4
.1.5. N-(1-methyl-1H-pyrazol-4-yl)-4-(3-nitrophenyl)pyrimidin-2-amine (41a)
To 40 (0.2 g, 0.85 mmol) and 1-methyl-1H-pyrazol-4-amine hydrochloride salt
(0.13 g, 0.94 mmol) in isopropyl alcohol (8 mL) was added N,N-
diisopropylethylamine (DIEA, 0.27g, 2.13 mmol), and the mixture was stirred
overnight at 110°C. The mixture was then concentrated, and purified by column
chromatography (DCM: MeOH = 40:1) to yield 0.21 g (82%) of 41a as a light yellow
1
solid. H NMR (300 MHz, DMSO) δ 8.82 (s, 1H), 8.71 (s, 1H), 8.44 (d, J = 7.8 Hz,
1
H), 8.35 (d, J = 8.2 Hz, 1H), 8.04 (s, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.54 (s, 1H), 7.21
(s, 1H), 3.84 (s, 3H).
Compounds 41b-f, 44b-c, 47b-c, and 50b-c were prepared following the similar
synthetic procedure of compound 41a. Detailed experimental procedure and spectral
data are presented in the supplementary material.
4
.1.6. 4-(3-Aminophenyl)-N-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (42a)
To 41a (0.28g, 0.95 mmol) and 10% Pd/C 0.1g in EtOH (50 mL) was added drop
by drop 85% hydrazine hydrate (0.47 g, 9.5 mmol) at 88 °C. The reaction mixture was
stirred for 2h under nitrogen. The mixture was then filtered with a Celite bed, the
filtrate was concentrated and dried under vacuum to yield 0.24 mg (95%) of 42a as a
1
white solid. H NMR (300 MHz, DMSO) δ 8.60 (s, 1H), 8.00 (s, 1H), 7.50 (s, 1H),
7
.26 (s, 1H), 7.12 (s, 2H), 6.97 (s, 1H), 6.80 – 6.56 (m, 1H), 3.83 (s, 3H).

Compounds 42b-f, 45b-c, 48b-c, and 51b-c were prepared following the similar
synthetic procedure of compound 42a. Detailed spectral data are presented in the
supplementary material.
4
.1.7. Tert-butyl(2-((3-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)
amino)-2-oxoethyl)carbamate (52a)
To a solution of N-((2,2-dimethylpropoxy)carbonyl)-glycine (0.2 g, 1.13 mmol) in
DCM (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI,
0
.36 g, 1.88 mmol), 1-hydroxybenzotriazole (HOBT, 0.25 g, 1.88 mmol), and DIEA
0.34 g, 2.6 mmol) were added, respectively. And the mixture was stirred for 30min at
℃ . 42a (0.2 g, 0.75 mmol) was added to the reaction mixture, then allowed to warm
(
0
to room temperature with stirring for 6h. After completion of the reaction, the mixture
was extracted with DCM, washed with brine, and the organic phase was dried over
Na SO . The solvent was removed and the residue was purified by flash column
2
4
chromatography on silica (DCM: MeOH = 30:1) to give a yellow solid (0.27g, 85%).
1
H NMR (300 MHz, CDCl ) δ 8.64 (s, 1H), 8.00 (s, 1H), 7.76 (s, 1H), 7.60 (s, 1H),
3
7
.58 (s, 1H), 7.55 (s, 1H), 7.34 – 7.27 (m, 1H), 6.77 (s, 1H), 3.98 (d, J = 5.6 Hz, 2H),
.90 (s, 3H), 1.46 (s, 9H).
3
Compounds 52b-c and 52e were prepared following the similar synthetic procedure
of compound 52a. Detailed spectral data are presented in the supplementary material.
4
.1.8.
2-Amino-N-(3-(2-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)
acetamide (53a)
To a solution containing 52a (0.5 g, 1.34 mmol) in 2 mL of dry DCM, cooled to
0
°C was slowly added TFA (2 mL, 13 mmol). After the consumption of the starting
material (5 h, monitored by TLC), the solvent was evaporated, and removed under
vacuum. Semi-solid was slurred with dry ether, and filtered to afford crude product
(0.36 g, 98%), which was used directly in the next step.
Compounds 53b-c and 53e were prepared following the similar synthetic procedure
of compound 53a. Detailed spectral data are presented in the supplementary material.

Method A: General procedure for the synthesis of targets 1-12, 13, 15, 17, 19-21,
4-27, 30, 32, 34, 36, and 38.
2
To a solution of 42a (0.1 g, 0.37 mmol) in THF (5 mL) were added DIEA (0.05g,
.37 mmol) and acryloyl chloride (0.03g, 0.37 mmol) at -5 °C. The resulting mixture
0
was stirred for 30 min. Then it was quenched by MeOH (2 mL), concentrated, and
purified by silica gel column chromatography (DCM: MeOH = 20:1) to afford the
title compounds.
4
.1.9. N-(3-(6-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)acrylamide
(1)
1
White solid (yield: 71%). m. p.: 214–216 ℃ . H NMR (300 MHz, DMSO) δ 8.65 (s,
1
H), 8.44 (s, 1H), 8.01 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.52
(s, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.07 (s, 1H), 6.47 (dd, J = 16.9, 10.0 Hz, 1H), 6.32 –
1
3
6
1
1
.26 (m, 1H), 5.80 – 5.75 (m, 1H), 3.84 (s, 3H); C NMR (75 MHz, DMSO) δ 163.35,
60.38, 158.48, 139.52, 137.60, 131.79, 130.66, 129.26, 127.01, 122.03, 121.44,
20.96, 117.41, 104.10, 100.75, 38.72; HRMS m/z (ESI): calcd for C H N O
17
17
6
+
[
M+H] 321.1461, found 321.1460.
4
.1.10. N-(3-(6-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)phenyl)
acrylamide (2)
1
White solid (yield: 72%). m. p.: 213–215 ℃ . 213.2–215.1℃ . H NMR (300 MHz,
DMSO) δ 8.69 (s, 1H), 8.48 (s, 1H), 8.06 (s, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.73 (d, J
=
7.5 Hz, 1H), 7.59 (s, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.10 (s, 1H), 6.49 (dd, J = 16.9,
1
2
1
1
0.0 Hz, 1H), 6.31 (d, J = 16.7 Hz, 1H), 5.78 (d, J = 10.6 Hz, 1H), 4.25 (t, J = 4.9 Hz,
1
3
H), 3.68 (t, J = 4.9 Hz, 2H), 3.23 (s, 3H); C NMR (75 MHz, DMSO) δ 163.31,
60.23, 158.52, 139.54, 137.56, 131.77, 130.61, 129.26, 127.03, 121.88, 121.38,
20.91, 117.34, 101.03, 70.65, 57.92, 51.33; HRMS m/z (ESI): calcd for C H N O
19
21
6
2
+
[
M+H] 365.1882, found 365.1880.

4
.1.11. N-(3-(6-((4-morpholinophenyl)amino)pyrimidin-4-yl)phenyl)acrylamide (3)
1
White solid (yield: 74%). m. p.: 224–225 ℃ . 223.7–225.1℃ . H NMR (300 MHz,
DMSO) δ 8.62 (s, 1H), 8.45 (s, 1H), 7.80 (d, J = 7.3 Hz, 1H), 7.69 (d, J = 7.0 Hz, 1H),
7
2
1
1
.53 (d, J = 8.2 Hz, 2H), 7.46 (t, J = 7.8 Hz, 1H), 7.15 (s, 1H), 6.95 (d, J = 8.8 Hz,
H), 6.50 (dd, J = 16.6, 10.2 Hz, 1H), 6.29 (d, J = 17.0 Hz, 1H), 5.78 (d, J = 10.1 Hz,
1
3
H), 3.74 (s, 4H), 3.06 (s, 4H); C NMR (75 MHz, DMSO) δ 163.82, 161.63, 161.06,
58.77, 147.52, 140.04, 138.10, 132.27, 129.75, 127.48, 122.07, 121.85, 121.43,
1
17.80, 116.14, 101.63, 66.60, 49.04; HRMS m/z (ESI): calcd for C H N O
23 24 5 2
+
[
M+H] 402.1926, found 402.1924.
4
.1.12. N-(3-(6-((1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)
phenyl)acrylamide (4)
1
White solid (yield: 65%). m. p.: 198–199 ℃ . H NMR (300 MHz, DMSO) δ 8.66 (s,
H), 8.47 (s, 1H), 8.06 (s, 1H), 7.80 (d, J = 6.4 Hz, 1H), 7.71 (d, J = 7.2 Hz, 1H), 7.55
1
(s, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.09 (s, 1H), 6.49 (dd, J = 16.7, 10.0 Hz, 1H), 6.30 (d,
J = 17.1 Hz, 1H), 5.78 (d, J = 10.0 Hz, 1H), 4.18 (s, 2H), 2.63 (t, J = 5.7 Hz, 2H),
13
2
.16 (s, 6H); C NMR (75 MHz, DMSO) δ 163.27, 160.01, 158.51, 149.03, 139.56,
1
37.54, 131.79, 129.27, 127.02, 121.32, 120.86, 117.27, 101.01, 58.71, 49.61, 45.11;
+
HRMS m/z (ESI): calcd for C H N O [M+H] 378.2044, found 378.2041.
2
0
24
7
4
4
.1.13. N-(3-(6-((1-(2-(methylamino)-2-oxoethyl)-1H-pyrazol-4-yl)amino)pyrimidin-
-yl)phenyl)acrylamide (5)
1
White solid (yield: 66%). m. p.: 244–245 ℃ . H NMR (300 MHz, DMSO) δ 8.68 (s,
1
H), 8.47 (s, 1H), 8.09 (s, 1H), 7.81 (d, J = 3.5 Hz, 1H), 7.72 (d, J = 7.0 Hz, 1H), 7.58
(s, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.11 (s, 1H), 6.50 (dd, J = 16.4, 9.7 Hz, 1H), 6.30 (d,
1
3
J = 16.9 Hz, 1H), 5.79 (d, J = 9.8 Hz, 1H), 4.77 (s, 2H), 2.62 (s, 2H); C NMR (75
MHz, DMSO) δ 167.17, 163.29, 160.19, 158.50, 154.37, 139.57, 137.52, 131.80,
1
29.27, 127.01, 121.35, 120.90, 117.30, 54.29, 25.56; HRMS m/z (ESI): calcd for
+
C H N NaO [M+Na] 400.1496, found 400.1498.
19
19
7
2