Structure-based design and synthesis of pyrimidine-4,6-diamine derivatives as Janus kinase 3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.03.009

Janus kinases (JAKs) play a key role in the proliferation, apoptosis and differentiation of immune cells, and JAKs are considered as an attractive target for the treatment of inflammatory and autoimmune diseases. Here we show the design and optimization of pyrimidine-4,6-diamine derivatives as selectivity JAK3 inhibitors. Compound 11e, which might interact with unique cysteine (Cys909) residue in JAK3, exhibited excellent JAK3 inhibitory activity (IC₅₀ = 2.1 nM) and high JAK kinase selectivity. In cellular assay, 11e showed moderate potency inhibiting IL-2-stimulated T cell proliferation. The data supports the further development of novel JAKs inhibitors.

Full text of DOI:10.1016/j.bmc.2019.03.009

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based design and synthesis of pyrimidine-4,6-diamine derivatives
as Janus kinase 3 inhibitors
Ru-Nan Yu , Cheng-Juan Chen , Lei Shu , Yuan Yin , Zhi-Jian Wang , Tian-Tai Zhangb,⁎,
a
b
a
a
a
a,⁎
Da-Yong Zhang
a
b
College of Science, China Pharmaceutical University, Nanjing, PR China
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Janus kinases (JAKs) play a key role in the proliferation, apoptosis and differentiation of immune cells, and JAKs
are considered as an attractive target for the treatment of inflammatory and autoimmune diseases. Here we show
the design and optimization of pyrimidine-4,6-diamine derivatives as selectivity JAK3 inhibitors. Compound
Janus kinase
JAK3 inhibitors
Pyrimidine-4,6-diamine derivatives
Cys909
1
1e, which might interact with unique cysteine (Cys909) residue in JAK3, exhibited excellent JAK3 inhibitory
activity (IC50 = 2.1 nM) and high JAK kinase selectivity. In cellular assay, 11e showed moderate potency in-
hibiting IL-2-stimulated T cell proliferation. The data supports the further development of novel JAKs inhibitors.
to a lack of erythropoiesis.1
0–12
TYK2 knockout mice are viable and
1
. Introduction
fertile but showed increased susceptibility to infections, decreased re-
sponses to lipopolysaccharide triggering and development of collagen-
The Janus kinases (JAKs) as non-receptor tyrosine kinases consist of
1
3
four known members in humans: JAK1, JAK2, JAK3, and tyrosine ki-
nase 2 (TYK2), which are critically important for signaling transduction
induced rheumatoid arthritis (RA).
From these insights in the role of JAK3 for the development and
functions of the immune system and its restricted expression in hema-
topoietic tissues, JAK3 has been pursued as a target for the treatment of
inflammation, autoimmune diseases, such as RA psoriasis, in-
flammatory bowel disease, and organ transplant rejection. Another
feature of JAK3 inhibitors is that toxic and side effects are avoided
1
pathways mediated by cytokines. JAKs transduce cytokines signaling
from membrane receptors to various signal transducer and activator of
transcription proteins (STATs), and the JAK-STAT signaling pathway
2
,3
has been extensively studied.
Upon binding of cytokine to the re-
ceptors, JAKs become activated and phosphorylate tyrosine residues in
the receptor cytoplasmic domains, which lead to recruitment and ac-
1
4
observed with current therapies. We therefore selected JAK3 as the
target for our research, with an immediate goal of understanding the
relationships between inhibitor structure and selectivity across the
Janus associated kinases, JAK 1–3 and TYK2.
4
,5
tivation of downstream signaling proteins such as STATs.
STATs
phosphorylation leads to their dimerization and translocation to the
nucleus where they regulate expression of specific genes involved in
6
,7
proliferation, apoptosis and differentiation.
Due to the high degree of structural conservation of the JAK ATP
binding pockets, it remains a challenging task, for medicinal chemists,
to develop highly selective inhibitors as pharmacological probes and as
clinical drugs. To date, only a few compounds with appropriate isoform
selectivity for JAK3 have been reported. Tofacitinib (1, Fig. 1) was, at
first, described as a selective JAK3 inhibitor and then subsequent stu-
dies revealed that it is a pan JAK inhibitor, and Ruxolitinib (2, Fig. 1) is
In humans, JAKs are nearly ubiquitously expressed, with the ex-
8
ception of JAK3, which is mainly expressed in hematopoietic cells.
Another feature of JAK3 is that it specifically associates with the
common gamma chain (γc) cytokines such as IL-2, IL-4, IL-7, IL-9, and
IL-15, which play important roles in T-cell differentiation, proliferation
1
8
and survival. Genetic analysis of severe combined immunodeficiency
1
5,16
(
SCID) patients showed genetic mutations of JAK3 and decreased ex-
accepted as a selective JAK1/2 inhibitor.
A US patent from Portola
9
pression of JAK3 protein expression. JAK3 knockout mice also exhibit
immunodeficiency, consistent with the mutational consequences found
in humans. On the other hand, JAK1 knockout mice were found to die
shortly after birth and JAK2 deficiency in mice is embryonic lethal due
describes 4 is at least 100-fold selective for JAK3 over JAK1/2. In 2016,
it was reported that 3 (PF-06651600) inhibited JAK3 kinase activity
with an IC50 of 33.1 nm but without activity against JAK1, JAK2 and
TYK2. Then they demonstrated that PF-06651600 form an irreversible
⁎
Corresponding authors. Tel.: +86 25 83271315 (D.-Y. Zhang), +86 10 63035779 (T.-T. Zhang).
E-mail addresses: ttzhang@imm.ac.cn (T.-T. Zhang), cpuzdy@163.com (D.-Y. Zhang).
https://doi.org/10.1016/j.bmc.2019.03.009
Received 9 January 2019; Received in revised form 28 February 2019; Accepted 3 March 2019
0968-0896/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Ru-Nan Yu, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2019.03.009

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
was treated with (3-nitrophenyl)methanamine or (4-nitrophenyl)me-
thanamine to afford 8a or 8a’. Then 8a or 8a’ was reacted with several
kinds of amines to give the N, N-substituted pyrimidine-4,6-diamine
compounds (9a-f, 9a’–e’) under acidic conditions. Treatment of 9a–f or
9
a’–e’ with 85% hydrazine hydrate under nitrogen and Pd/C catalysis
provided reduction products 10a–f or 10a’–e’.
The target compounds was prepared from 10a–f or 10a’–e’ as
shown in Scheme 2. Intermediates (10a–f, 10a’–e’) was treated with a
few kinds of acyl chlorides to give the desired compounds (11a–f,
1
1a’–e’, 12a–e, 12a’–e’, 13a–e and 13a’–e’) under basic conditions.
Scheme 3 shows the synthesis of the other target compounds.
Treatment of 10c-e or 10e’ with cyanoacetic acid and 2-(7-Aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(
HATU) gave 14c–e, 14e’. Intermediates (10c–e and 10e’) were reacted
with cyanogen bromide to give the targets 15c–e and 15e’. And 10d or
1
0d’ were treated with trifluoroacetic anhydride to give 16d, 16d’.
Scheme 4 shows the synthesis of several amines. Commercially
available 4-nitro-1H-pyrazole was treated with alkyl halide such as
Fig. 1. Chemical structures of JAK inhibitors.
iodomethane, 2-bromoethanol, 1-bromo-2-methoxyethane to give
1
7a–c, and followed by hydrogenation in the presence of Pd/C to give
amines (18a–c). Commercially available 4-fluoronitrobenzene was re-
acted with morpholine or 1-methylpiperazine to provide 19d, 19e.
Then 4-fluoronitrobenzene and 19d, 19e were, separately, treated with
hydrogen under Pd/C catalysis to give several amines (20d–f).
covalent bond with Cys909 in JAK3, a residue replaced by a serine in
1
0,17
19
the other three JAK isoforms.
In 2017, Michael Forster also re-
ported that acrylamide could react with Cys909 covalently via 1,4-
(
conjugate) addition (sulfa-Michael addition).
Here we present our initial studies toward selective JAK3 inhibitors.
In our laboratory, we aimed to exploit a compound which can interact
3
. Results and discussion
with Cys909. Based on the structure comparison of 3 and 4, compound
7
, a pyrimidine-4,6-diamine derivative, was designed with the strategy
3.1. In vitro enzymatic inhibitor activities
of scaffold hopping (Fig. 2). The molecular structure of pyrimidine-4,6-
diamine derivatives consist of four parts: core, side chain, tail and the
linkage between core and tail. The core was expected to interact with
hinge residues by two hydrogen bonds in similar binding way to that of
We evaluated the inhibitory activity of newly synthesized com-
pounds on human JAK1, JAK2 and JAK3 enzyme, and some compounds
(
JAK3 IC50 < 10 nM) on human TYK2 enzyme. After scaffold hopping,
3
. The side chain was considered to facilitate interaction with JAK3 at
the chemotype of target compounds was divided to four moieties, core,
the hydrophobic cavity, and have opportunities to improve pharma-
cokinetic profile. And benzylamine links the core and the tail. The tail,
as the key to both high isoform and kinome selectivity, tends to possess
electrophilic group, such as Michael acceptors, which can form a
side chain (R
1
), tail (R ) and the linkage between core and tail. Then
2
side chain and tail were varied sequentially.
The first series of analogues focused on modification of the side
chain (R ) (Table 1). Compound 7 showed low inhibitory activity, and
1
1
9
covalent bond with the cysteine thiol group. After further optimiza-
tion, our studies showed that the compound 11e, 11d are potent JAK3
inhibitors. Here, we report the design, synthesis and structure-activity
relationship (SAR) of pyrimidine-4,6-diamine JAK inhibitors.
when the 4-position of the aniline was substituted by fluorine, 11f had
increased the potency against JAK3 (IC50 = 589 nM). In consideration
of 4 possessing a long side chain, adding a morpholine ring at 4-position
of the aniline dramatically improved the kinase selectivity and potency.
Introduction of a piperazine ring 11e exhibited a similar or higher JAK3
inhibitory activity than 11d (11d, IC50 = 2.4 nM, 11e, IC50 = 2.1 nM).
Regarding the feature of JAK3-ATP binding site, side chains of 11d and
11e might effectively interact with hydrophobic cavity. To enhance
affinity for JAK3 in the pocket of the hydrophobic region, we in-
vestigated the conversion of side chain to a pyrazole ring (11a, 11b,
11c, IC50 = 12.3 nM, 7.2 nM, 7.0 nM, respectively) showed higher
2
. Chemistry
A series of pyrimidine-4,6-diamine derivatives were synthesized
according to pathways described in Schemes 1–4.
The key intermediates 10a–f and 10a’–e’ were synthesized ac-
cording to Scheme 1. Commercially available 4,6-dichloropyrimidine
Fig. 2. Design of the target compound.
2

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 1. Reagents and conditions: (i) Nitro-substituted benzyl amine, DIEA, Isopropanol, 82 °C, 6 h; (ii) amine, TFA, n-BuOH, 120 °C, 6 h; (iii) Pd/C, hydrazine
hydrate, EtOH, 100 °C, 2 h.
activity than 11f, and exhibited moderately selective JAK3 inhibition,
with about 50-fold, 150-fold, 110-fold, respectively, higher IC50 against
JAK1. Obviously the 2-methoxyethyl (11c) displayed more potent than
3.2. The docking study to human JAK3
Docking study of the pyrimidine-4,6-diamine derivatives with JAK3
kinase was investigated. In Fig. 3, this study showed that the structure
was found to be consistent with our expected model. The pyrilamine
core makes the expected bidentate hinge hydrogen bonds with Leu905,
and the unique Cys909 in human JAK3 have been successfully targeted
by acrylamide, as the distance between the sulfur atom of Cys909 and
acrylamide warhead in the 11e is 1.948 Å. The side chain of 11e ex-
tends toward the hydrophobic region. Furthermore, the phenyl group of
11e is in van der Waals contact with Leu956 and Leu828 in the ATP-
binding pocket. Moreover, the acrylamide forms a hydrogen bond with
the carbonyl of Arg953.
1
-methyl (11a), which means that the length of side chain plays an
important role in JAK3 inhibitory activity. In addition, some com-
pounds with acrylamide, such as 11b, 11c, 11d, 11e, exhibited poor
TYK2 inhibitory activity, which may be due to acrylamide not matching
TYK2 active site.
After optimization of the side chain, the SAR of the tail (R ) was
2
investigated (Tables 1 and 2). To investigate spatial tolerance around
the tail, the acrylamide was switched from meta- to para- on the phenyl
ring. The results (11a’–11e’) exhibited weaker both JAK3 inhibitory
potency and selectivity than analogues with meta-acrylamide
(
11a–11e). As shown in Table 2, replacing the acrylamide with a cro-
tonamide (12d, 12e) or propionamide (13d, 13e) resulted in over 40-
fold or 100-fold decrease of potency against JAK3. Incorporation of
cyanoacetamide (14e) maintained the potency with the JAK3 IC50 of
3.3. Rat T cell proliferation
2
1,22
3
.5 nM, while 14e’ did not, and as reported by H. Yamagishi et al.,
Due to its high kinase potency and selectivity, we selected com-
pounds 11c, 11d, 11e, 14e and 14d for further tests (Table 3). In the
cellular assay of IL-2-stimulated T cell proliferation, 11e exhibited ex-
cellent anti-proliferative activity of the T cell lines with IC50 value of
12 nM, and 11d (IC50 = 15 nM) showed lower inhibitory compared to
11e. Compounds 14d, 14e were demonstrated low potency for the T
cell lines, despite the fact that they have moderate potency against
JAK3. From this point of view, acrylamide plays a more important role
in inhibitors than cyanoacetamide.
they showed poor selectivity against JAK3. Meanwhile cyanoaceta-
mides showed higher TYK2 inhibitory activity than acrylamides. To our
surprise, 15c, 15d, 15e, with a cyan group, exhibited not JAK3 se-
lectivity but JAK1 selectivity. And 16d, 16d’, with
a
tri-
fluoroacetamide, exhibited JAK2 selectivity. In summary, the mod-
ification of R failed to further improve our JAK3 inhibitors, but found
some similar potent JAK3 inhibitors (14e, 14d).
2
Scheme 2. Reagents and conditions: (i) acyl chlorides, THF, 0 °C to room temperature (RT), 30 min.
3

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 3. Reagents and conditions: (i) Cyanoacetic acid, HATU, DIEA, THF, 16 h (for 14c–e, 14e’); (ii) cyanogen bromide, potassium acetate, THF, RT, 16 h (for
5c–e, 15e’); (iii) trifluoroacetic anhydride, THF, RT, 16 h (for 16d, 16d’).
1
4
. Conclusion
5.1.1. 6-Chloro-N-(3-nitrobenzyl)pyrimidin-4-amine (8a)
2
,6-Dichloropyrimidine (0.1 g, 0.68 mmol), (3-nitrophenyl)metha-
We designed and synthesized pyrimidine-4,6-diamine derivatives as
namine (0.11 g, 0.74 mmol), and N, N-diisopropylethylamine (DIEA,
0.22, 1.69 mmol) were combined in isopropanol (5 mL) and stirred
overnight at 82 °C. The mixture was then diluted with ethyl acetate and
novel JAK3 inhibitors. Among these analogues, compound 11d, 11e,
1
4e stood out with overall favorable inhibitory activity and selectivity.
And 11e is our most selective JAK3 inhibitor, with at least 400-fold
over other JAKs in vitro enzymatic assays. Primary studies demon-
strated that hinge hydrogen bonds, hydrophobic moiety and its specific
length are responsible for 11e’s interaction with JAK3. Furthermore,
acrylamide, reacting covalently with Cys909 same as reported by
washed with water and brine, dried over Na
2
SO , filtered, and con-
4
centrated. The crude product was purified by column chromatography
(hexane:ethyl acetate = 1:1) to yield 0.14 g (81%) of 8a as a white
solid. MS (ESI) m/z 265 [M+H]+
.
1
9,20
others,
significantly enhance both activity and selectivity against
5
.1.2. 6-Chloro-N-(4-nitrobenzyl)pyrimidin-4-amine (8a’)
JAK3. Our further studies will be aimed to exploiting the complicated
SARs, and the studies about metabolic stability and oral bioavailability
in rats will be planned in the future.
Compound 8a’ was prepared in 80% yield by a method similar to
that described for 8a. MS (ESI) m/z 265 [M+H]+
.
5
.1.3. N
4
-(1-Methyl-1H-pyrazol-4-yl)-N -(3-nitrobenzyl)pyrimidine-4,6-
6
diamine (9a)
5
. Experimental
To 8a (0.25 g, 0.95 mmol) and 1-methyl-1H-pyrazol-4-amine hy-
drochloride salt (0.14 g, 1.1 mmol) in n-butanol (5 mL) was added tri-
fluoroacetic acid (0.27 g, 2.4 mmol), and the mixture was stirred
overnight at 120 °C. The mixture was then concentrated, neutralized
with ammonia in methanol, and purified by column chromatography
5.1. Chemical synthesis
1
13
H NMR and C NMR spectra were recorded with Bruker ARX-300
spectrometer with TMS as the internal standard in CDCl
3
, DMSO-d .
6
(
dichloromethane: methanol = 60:1) to yield 0.26 g (86.7%) of 9a as a
+
Peak multiplicities are abbreviated: singlet, s; doublet, d; triplet, t;
quartet, q; multiplet, m; etc. ESI-MS was run on a Bruker micro-TOF-Q
mass spectrometer. Most chemicals were purchased from
Sigma–Aldrich and Fluka. All commercially available reagents were
used without further purification. The solvents used were all AR grade
and were redistilled under positive pressure of dry nitrogen atmosphere
in the presence of proper desiccant when necessary. Silica gel (200–300
mesh, Qingdao city, China) was used for column chromatography. The
progress of the reactions was monitored by analytical thin-layer chro-
matography (TLC) on HSGF254 precoated silica gel plates.
pale-yellow solid. MS (ESI) m/z 326 [M+H]
.
5
.1.4. 2-(4-((6-((3-Nitrobenzyl)amino)pyrimidin-4-yl)amino)-1H-
pyrazol-1-yl)ethan-1-ol (9b)
Compound 9b was prepared from 8a in 79% yield by a method
+
similar to that described for 9a. MS (ESI) m/z 356 [M+H]
.
5.1.5. N
pyrimidine-4,6-diamine (9c)
Compound 9c was prepared from 8a in 83% yield by a method
4
-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-N
6
-(3-nitrobenzyl)
Scheme 4. Reagents and conditions: (i) alkyl halide, K
methylpiperazine, K CO , DMSO, RT, 6 h(for 19e).
2
CO
3
, MeCN, ref., 6 h; (ii) Pd/C, H
2
, RT, 3 h; (iii) morpholine, K
2
3
CO , DMSO, 120 °C, 20 h (for 19d); (iv) 1-
2
3
4

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 1
SARs of side chains (R ).
1
Compd
7
R
1
IC50a(nM)
JAK3
JAK1/JAK3b
JAK2/JAK3c
TYK2/JAK3d
JAK2
JAK1
TYK2
NDe
730
> 3000
> 3000
–
–
–
–
–
–
1
1
1f
589
> 3000
> 3000
> 3000
656
NDe
NDe
1a
12.3
53.3
26.7
–
–
–
–
1
1a’
24.1
> 3000
643
NDe
11b
11b’
11c
7.2
9.4
7.0
> 3000
2098
1126
1240
774
2561
2175
2290
156.4
131.9
110.6
–
156.4
131.9
327.1
223.2
291.8
2043
1
1c’
9.3
NDe
852
2295
91.6
–
246.8
1
1
1
1
1d
1d’
1e
2.4
8.4
2.1
7.9
0.7
1096
916
1286
1159
> 3000
731
> 3000
> 3000
> 3000
> 3000
32.3
535.8
138.0
–
456.7
109
450
–
–
–
945
–
1e’
> 3000
0.5
92.5
2
–
Tofacitinib
a
b
c
d
e
1.4
0.7
46.1
IC50 values are the average of duplicate experiments.
Fold selectivity derived from JAK1/JAK3 enzyme IC50
.
Fold selectivity derived from JAK2/JAK3 enzyme IC50
.
Fold selectivity derived from TYK2/JAK3 enzyme IC50
ND = not determined.
.
similar to that described for 9a. MS (ESI) m/z 370 [M+H]+
.
5.1.8. N
9f)
-(4-fluorophenyl)-N -(3-nitrobenzyl)pyrimidine-4,6-diamine
4
6
(
Compound 9f was prepared from 8a in 82% yield by a method si-
5
.1.6. N
4
-(4-Morpholinophenyl)-N
6
-(3-nitrobenzyl)pyrimidine-4,6-
milar to that described for 9a. MS (ESI) m/z 340 [M+H]+
.
diamine (9d)
Compound 9d was prepared from 8a in 89% yield by a method
5
.1.9. N
4
-(1-Methyl-1H-pyrazol-4-yl)-N -(4-nitrobenzyl)pyrimidine-4,6-
6
+
similar to that described for 9a. MS (ESI) m/z 407 [M+H]
.
diamine (9a’)
To 8a’ (0.25 g, 0.95 mmol) and 1-methyl-1H-pyrazol-4-amine hy-
drochloride salt (0.14 g, 1.1 mmol) in n-butanol (5 mL) was added tri-
fluoroacetic acid (0.27 g, 2.4 mmol), and the mixture was stirred
overnight at 120 °C. The mixture was then concentrated, neutralized
with ammonia in methanol, and purified by column chromatography
5
.1.7. N
4
-(4-(4-Methylpiperazin-1-yl)phenyl)-N -(3-nitrobenzyl)
6
pyrimidine-4,6-diamine (9e)
Compound 9e was prepared from 8a in 81.5% yield by a method
+
similar to that described for 9a. MS (ESI) m/z 420 [M+H]
.
(
dichloromethane: methanol = 60:1) to yield 0.26 g (83.0%) of 9a’ as a
5

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 2
SARs of tails (R ).
2
Compd
R
1
R
2
IC50a(nM)
JAK3
JAK1/JAK3b
JAK2/JAK3c
TYK2/JAK3d
JAK2
945
JAK1
TYK2
11e
12e
13e
14e
2.1
> 3000
> 3000
–
450
–
–
89
NDe
NDe
1015
1821
NDe
923
NDe
NDe
1532
20.46
–
–
212
3.5
–
–
263
290
437.7
1
5e
> 3000
7.9
350
90
NDe
–
–
–
–
–
1
1e’
> 3000
731
> 3000
92.5
12e’
13e’
14e’
101
290
155
NDe
NDe
NDe
2610
NDe
NDe
NDe
NDe
25.8
–
–
–
–
–
–
–
1242
8
1
1
5e’
1d
> 3000
2.4
503
124
NDe
–
–
–
–
1096
1286
> 3000
535.8
456.7
12d
13d
14d
99
NDe
NDe
1444
2032
NDe
920
NDe
NDe
1564
20.52
–
–
–
278
4.84
–
–
190
298
323.1
1
5d
6d
> 3000
> 3000
345
366
92
NDe
NDe
–
–
–
–
–
–
1
> 3000
1
1
1
1
1
6d’
4c
> 3000
7.3
280
> 3000
1262
285
NDe
1740
NDe
NDe
NDe
–
–
–
–
–
–
–
NDe
2790
NDe
NDe
172.8
–
238.4
5c
> 3000
342
–
–
–
2a
2a’
1319
1586
3.9
4.1
389
(
continued on next page)
6

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 2 (continued)
Compd
R
1
R
2
IC50a(nM)
JAK3
JAK1/JAK3b
2
JAK2/JAK3c
0.7
TYK2/JAK3d
46.1
JAK2
0.5
JAK1
1.4
TYK2
32.3
Tofacitinib
a
0.7
IC50 values are the average of duplicate experiments.
Fold selectivity derived from JAK1/JAK3 enzyme IC50
b
c
d
e
.
Fold selectivity derived from JAK2/JAK3 enzyme IC50
.
Fold selectivity derived from TYK2/JAK3 enzyme IC50
ND = not determined.
.
5
.1.14. N
4
-(3-Aminobenzyl)-N -(1-methyl-1H-pyrazol-4-yl)pyrimidine-
6
4
,6-diamine (10a)
To 9a (100 mg, 0.30 mmol) and 10% Pd/C 20 mg in EtOH (20 mL)
was added drop by drop 85% hydrazine hydrate (0.36 g, 6.1 mmol) at
1
00 °C. The reaction mixture was stirred for 2 h under nitrogen. The
mixture was then filtered with Celite, and the filtrate was concentrated
and dried under vacuum to yield 86 mg (95%) of 10a as a white solid.
MS (ESI) m/z 296 [M+H]+
.
5
.1.15. 2-(4-((6-((3-Aminobenzyl)amino)pyrimidin-4-yl)amino)-1H-
pyrazol-1-yl)ethan-1-ol (10b)
Compound 10b was prepared from 9b in 95% yield by a method
+
similar to that described for 10a. MS (ESI) m/z 326 [M+H]
.
Fig. 3. The structure of JAK3 bound with compound 11e (yellow sticks) in the
ATP-binding site. Hydrogen bonds of 11e with JAK3 are indicated by green
dotted lines. The distance between the sulfur atom of Cys909 and acrylamide
warhead in the 11e is showed.
5.1.16. N
4
-(3-Aminobenzyl)-N -(1-(2-methoxyethyl)-1H-pyrazol-4-yl)
6
pyrimidine-4,6-diamine (10c)
Compound 10c was prepared from 9c in 96% yield by a method
+
similar to that described for 10a. MS (ESI) m/z 340 [M+H]
.
Table 3
5.1.17. N4-(3-Aminobenzyl)-N6-(4-morpholinophenyl)pyrimidine-4,6-
Cellular assay of pyrimidine-4,6-diamine derivatives.
diamine (10d)
T cell proliferation IC50a(nM)
Compound 10d was prepared from 9d in 93% yield by a method
Compound
+
similar to that described for 10a. MS (ESI) m/z 377 [M+H]
.
1
1
1
1
1
1c
1d
1e
4e
4d
41
15
12
50
64
2
5
.1.18. N4-(3-Aminobenzyl)-N6-(4-(4-methylpiperazin-1-yl)phenyl)
pyrimidine-4,6-diamine (10e)
Compound 10e was prepared from 9e in 98% yield by a method
+
Tofacitinib
a
similar to that described for 10a. MS (ESI) m/z 390 [M+H]
.
Inhibitory effect on IL-2-stimulated T cell proliferation using
5
.1.19. N
4
-(3-Aminobenzyl)-N -(4-fluorophenyl)pyrimidine-4,6-diamine
6
rats spleen cells.
(
10f)
pale-yellow solid. MS (ESI) m/z 326 [M+H]+
Compound 10f was prepared from 9f in 95% yield by a method
.
+
similar to that described for 10a. MS (ESI) m/z 310 [M+H]
.
5
.1.10. 2-(4-((6-((3-Nitrobenzyl)amino)pyrimidin-4-yl)amino)-1H-
5
.1.20. N
4
-(4-Aminobenzyl)-N -(1-methyl-1H-pyrazol-4-yl)pyrimidine-
6
pyrazol-1-yl)ethan-1-ol (9b’)
4
,6-diamine (10a’)
Compound 9b’ was prepared from 8a’ in 82% yield by a method
Compound 10a’ was prepared from 9a’ in 93% yield by a method
+
similar to that described for 9a’. MS (ESI) m/z 356 [M+H]
.
+
similar to that described for 10a. MS (ESI) m/z 296 [M+H]
.
5
.1.11. N
4
-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-N -(3-nitrobenzyl)
6
5
.1.21. 2-(4-((6-((4-Aminobenzyl)amino)pyrimidin-4-yl)amino)-1H-
pyrazol-1-yl)ethan-1-ol (10b’)
Compound 10b’ was prepared from 9b’ in 96% yield by a method
pyrimidine-4,6-diamine (9c’)
Compound 9c’ was prepared from 8a’ in 85% yield by a method
+
similar to that described for 9a’. MS (ESI) m/z 370 [M+H]
.
+
similar to that described for 10a. MS (ESI) m/z 326 [M+H]
.
5
.1.12. N
4
-(4-Morpholinophenyl)-N -(3-nitrobenzyl)pyrimidine-4,6-
6
5
.1.22. N -(4-Aminobenzyl)-N -(1-(2-methoxyethyl)-1H-pyrazol-4-yl)
4
6
diamine (9d’)
pyrimidine-4,6-diamine (10c’)
Compound 9d’ was prepared from 8a’ in 80% yield by a method
Compound 10c’ was prepared from 9c’ in 92% yield by a method
+
+
similar to that described for 9a’. MS (ESI) m/z 407 [M+H]
.
similar to that described for 10a. MS (ESI) m/z 340 [M+H]
.
5
.1.13. N
4
-(4-(4-Methylpiperazin-1-yl)phenyl)-N
6
-(3-nitrobenzyl)
5.1.23. N
4
-(4-Aminobenzyl)-N -(4-morpholinophenyl)pyrimidine-4,6-
6
pyrimidine-4,6-diamine (9e’)
diamine (10d’)
Compound 9e’ was prepared from 8a’ in 82% yield by a method
Compound 10d’ was prepared from 9d’ in 97% yield by a method
+
+
similar to that described for 9a. MS (ESI) m/z 420 [M+H]
.
similar to that described for 10a. MS (ESI) m/z 377 [M+H]
.
7

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
1
3
5
.1.24. N
4
-(4-Aminobenzyl)-N
6
-(4-(4-methylpiperazin-1-yl)phenyl)
5.72 (m, 2H), 4.41 (s, 1H), 4.27 (s, 1H), 3.77 (s, 3H); C NMR (300 M
pyrimidine-4,6-diamine (10e’)
DMSO-d ) δ 163.18, 162.59, 160.13, 157.82, 140.90, 139.18, 131.94,
6
Compound 10e’ was prepared from 9e’ in 94% yield by a method
131.02, 128.83, 128.78, 126.91, 122.20, 117.79, 117.70, 114.51,
+
82.44, 43.89, 38.73; MS (ESI) m/z 350 [M+H]+
.
similar to that described for 10a. MS (ESI) m/z 390 [M+H]
.
5
.1.25. N
4
-(3-nitrobenzyl)-N
6
-phenylpyrimidine-4,6-diamine (5)
5
.1.30. N-(3-(((6-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)
To 8a (0.25 g, 0.95 mmol) and aniline (0.09 g, 1 mmol) in n-butanol
amino)methyl)phenyl)but-2-enamide (12a)
(
5 mL) was added trifluoroacetic acid (0.27 g, 2.4 mmol), and the
Compound 12a was prepared from 10a in 73% yield by a method
mixture was stirred overnight at 120 °C. The mixture was then con-
centrated, neutralized with ammonia in methanol, and purified by
column chromatography (DCM: MeOH = 150:1) to yield 0.25 g
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.11
(
s, 1H), 8.94 (br, 1H), 8.34 (s, 1H), 7.83 (s, 1H),7.60 (s, 1H), 7.54 (d,
83.3%) of 5 as a pale-yellow solid. MS (ESI) m/z 322 [M+H]+
J = 6 Hz,1H),7.45 (s, 1H),7.24(m, 1H), 6.97 (d, J = 6 Hz, 1H),6.77(dd,
J = 15, 6 Hz, 1H) 6.20 (d, J = 15 Hz, 1H), 5.84 (s, 1H), 4.48 (s, 2H),
(
.
1
3
3
1
1
.81 (s, 3H),1.83(d, 3H); C NMR (300 M DMSO-d ) δ 164.00, 159.27,
6
5
.1.26. N
To 5 (100 mg, 0.31 mmol) and 10% Pd/C 20 mg in EtOH (20 mL)
was added drop by drop 85% hydrazine hydrate (0.36 g, 6.1 mmol) at
4
-(3-aminobenzyl)-N
6
-phenylpyrimidine-4,6-diamine (6)
57.85, 141.17, 139.99, 139.21, 130.75, 128.24, 126.59, 122.17,
21.48, 119.80, 119.08, 116.47, 83.62, 70.87, 44.76, 17.94; MS (ESI)
+
m/z 364 [M+H]
.
1
00 °C. The reaction mixture was stirred for 2 h under nitrogen. The
mixture was then filtered with Celite, and the filtrate was concentrated
and dried under vacuum to yield 76 mg (84%) of 6 as a white solid. MS
5
.1.31. N-(4-(((6-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)
+
(
ESI) m/z 292 [M+H]
.
amino)methyl)phenyl)acrylamide (11a’)
Compound 11a’ was prepared from 10a’ in 70% yield by a method
5
.1.27. N-(3-(((6-(phenylamino)pyrimidin-4-yl)amino)methyl)phenyl)
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.51
acrylamide (7)
(
s, 1H), 10.06 (br, 1H), 8.78 (s, 1H), 8.31 (s, 1H),7.81(s, 1H), 7.70(d,
To a solution of 6 (40 mg, 0.14 mmol) in DCM (5 mL) were added
DIEA (17.7 mg, 0.14 mmol) and acryloyl chloride (12.4 mg, 0.14 mmol)
at −5 °C. The resulting mixture was stirred for 30 min. Then it was
quenched by MeOH (2 mL), concentrated, and purified by silica gel
column chromatography (DCM: MeOH = 40:1) to afford the title
J = 9 Hz, 1H), 7.44 (s, 1H), 7.28 (d, J = 6 Hz, 2H), 7.09 (d, J = 9 Hz,
1
H), 6.55 (dd, J = 12,9 Hz, 1H), 6.23 (d, J = 18 Hz, 1H), 5.82 (s, 1H),
13
5
.71 (d, J = 9 Hz, 1H), 3.81 (s, 3H); C NMR (300 M DMSO-d
6
) δ
1
63.65, 159.54, 158.82, 150.94, 138.84, 133.32, 132.43, 128.89,
28.23, 127.06, 120.50, 119.90, 119.68, 81.31, 49.01, 44.63; MS (ESI)
1
1
compound 7 (24.6 mg, 52%) as a white solid. H NMR (300 MHz,
+
m/z 350 [M+H]
.
DMSO-d ) δ 10.28 (s, 1H), 8.78 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.60
6
(
d, J = 6 Hz, 2H), 7.44 (t, J = 9 Hz, 1H), 7.34 (t, J = 9 Hz, 2H), 7.26 (d,
5
.1.32. N-(4-(((6-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)
J = 9 Hz, 1H), 7.06 (d, J = 9 Hz, 1H), 7.00(t, J = 6 Hz, 1H), 6.46(dd,
J = 15 Hz, 12 Hz, 1H), 6.17(d, J = 12 Hz, 1H), 5.64 (s, 1H), 5.56(d,
amino)methyl)phenyl)but-2-enamide (12a’)
1
3
Compound 12a’ was prepared from 10a’ in 75% yield by a method
J = 12 Hz, 1H), 4.41(d, J = 6 Hz, 2H); C NMR (300 M DMSO-d
6
) δ
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.27
1
1
7
64.76, 161.72, 158.86, 156.75, 139.82, 138.95, 137.54, 129.91,
(
s,1H), 10.15(br, 1H),8.87 (s, 1H), 8.33 (s, 1H), 7.82(s, 1H),7.67 (d,
28.88, 128.01, 126.57, 124.25, 121.76, 119.28, 118.20, 117.75,
+
J = 9 Hz, 2H), 7.45 (s, 1H), 7.26 (d, J = 9 Hz, 2H), 6.76(q, 1H),6.22(d,
J = 15 Hz, 1H),5.85 (s, 1H), 4.46 (s, 2H), 3.81 (s, 3H),1.83(d, J = 6 Hz,
9.17, 44.75; MS (ESI) m/z 346 [M+H]
.
1
3
3
1
8
H); C NMR (300 M DMSO-d ) δ 163.98, 158.16, 157.72, 141.15,
6
5
.1.28. N-(3-(((6-((4-fluorophenyl)amino)pyrimidin-4-yl)amino)methyl)
39.99, 139.16, 128.90, 128.21, 126.59, 121.86, 121.13, 119.81,
phenyl)acrylamide (11f)
4.69, 44.66, 17.93; MS (ESI) m/z 364 [M+H]+
.
To a solution of 10f (40 mg, 0.13 mmol) in DCM (5 mL) were added
DIEA (16.7 mg, 0.13 mmol) and acryloyl chloride (11.7 mg, 0.13 mmol)
at −5 °C. The resulting mixture was stirred for 30 min. Then it was
quenched by MeOH (2 mL), concentrated, and purified by silica gel
column chromatography (DCM: MeOH = 40:1) to afford the title
5
.1.33. N-(3-(((6-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)
pyrimidin-4-yl)amino)methyl)phenyl)acrylamide (11b)
Compound 11b was prepared from 10b in 85% yield by a method
1
compound 5 (27.8 mg, 59%) as a white solid. H NMR (300 MHz,
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.36
DMSO-d
6
) δ 10.28 (s, 1H), 8.78 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.60
(
s, 1H), 10.23 (br, 1H), 9.08 (br, 1H), 8.34 (s, 1H), 7.87(s, 1H), 7.56 (s,
(
(
d, J = 6 Hz, 2H), 7.44 (t, J = 9 Hz, 1H), 7.26 (d, J = 9 Hz, 1H), 7.06
d, J = 9 Hz, 1H), 6.99(d, J = 9 Hz, 2H), 6.46(dd, J = 15 Hz, 12 Hz,
1
H), 7.44 (s, 1H), 7.37 (t, 1H), 7.25(d, J = 12 Hz, 1H), 7.00 (d,
J = 6 Hz, 1H), 6.70 (dd, J = 15, 9 Hz, 1H), 6.33 (d, J = 15 Hz, 1H),
1
4
1
1
3
H), 6.17(d, J = 12 Hz, 1H), 5.64 (s, 1H), 5.56(d, J = 12 Hz, 1H),
5
2
1
1
.89 (s, 1H), 5.77 (d, J = 9 Hz, 1H), 4.59 (s, 2H),4.11(t, 2H), 3.71(t,
13
.41(d, J = 6 Hz, 2H); C NMR (300 M DMSO‑d
6
) δ 163.58, 163.14,
13
H); C NMR (300 M DMSO-d
6
) δ 163.96, 162.06, 160.05, 155.99,
58.04, 139.62, 139.56, 132.35, 129.16, 127.18, 124.61, 124.37,
39.81, 138.13, 132.45, 130.36, 129.36, 128.93, 127.47, 122.88,
22.59, 118.27, 116.23, 115.66, 115.37, 78.83, 49.04; MS (ESI) m/z
22.44, 121.99, 120.56, 81.12, 60.38, 54.95, 49.00; MS (ESI) m/z 380
+
64 [M+H]
.
+
[
M+H]
.
5
.1.29. N-(3-(((6-((1-methyl-1H-pyrazol-4-yl)amino)pyrimidin-4-yl)
amino)methyl)phenyl)acrylamide (11a)
5.1.34. N-(4-(((6-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)
To a solution of 10a (40 mg, 0.13 mmol) in DCM (5 mL) were added
DIEA (16.7 mg, 0.13 mmol) and acryloyl chloride (11.7 mg, 0.13 mmol)
at −5 °C. The resulting mixture 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
pyrimidin-4-yl)amino)methyl)phenyl)acrylamide (11b’)
Compound 11b’ was prepared from 10b’ in 80% yield by a method
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.51
(s, 1H), 8.91 (br, 1H), 8.34 (s, 1H), 7.85 (s, 1H), 7.70(d, J = 6 Hz, 2H),
7.47 (s, 1H), 7.29 (d, J = 6 Hz, 2H), 6.54 (dd, J = 15,9 Hz, 1H), 6.23 (d,
J = 15 Hz, 1H), 5.89 (s, 1H), 5.71 (d, J = 9 Hz, 1H), 4.54 (s, 2H),4.11(t,
1
compound 11a (29.9 mg, 63.6%) as a white solid. H NMR (300 MHz,
1
3
DMSO-d
6
) δ 10.17 (s, 1H), 8.66 (s, 1H), 8.05 (s, 1H), 7.75 (s, 1H),7.62
2H), 3.71(t, 2H); C NMR (300 M DMSO-d ) δ 163.67, 162.77, 161.08,
6
(
d, J = 6 Hz, 1H), 7.56 (s, 1H),7.30 (s, 1H),7.24(t, 1H), 7.00 (d,
157.84, 138.87, 135.47, 132.40, 128.28, 127.07, 123.72, 119.92,
+
J = 9 Hz, 1H),6.95(t, 1H), 6.25 (dd, J = 15, 6 Hz, 1H), 5.76 (s, 1H),
117.58, 81.72, 60.41, 54.94, 48.99; MS (ESI) m/z 380 [M+H]
.
8

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
5
.1.35. N-(3-(((6-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)
4H), 7.15 (s, 2H), 6.59 (dd, J = 15, 9 Hz, 1H), 6.24 (d, J = 15 Hz, 1H),
pyrimidin-4-yl)amino)methyl)phenyl)acrylamide (11c)
5.88 (s, 1H), 5.72 (d, J = 9 Hz, 1H), 4.45 (s, 2H), 3.80 (s, 4H), 3.19 (s,
1
3
Compound 11c was prepared from 10c in 85% yield by a method
4H); C NMR (300 M DMSO-d ) δ 163.67, 162.17, 159.90, 157.43,
6
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ10.57
150.31, 139.24, 138.92, 132.49, 128.25, 127.00, 124.06, 119.60,
+
(
s, 1H), 10.35 (s, 1H), 9.07 (s, 1H), 8.18 (s, 1H), 7.97 (d, 2H), 7.85 (d,
117.50, 82.07, 65.95, 49.01, 44.76; MS (ESI) m/z 431 [M+H]
.
2
1
H), 7.67 (d, 2H), 7.30 (t, 1H), 6.98 (d, 1H), 6.55 (dd, J = 18, 9 Hz,
H), 6.22 (d, J = 15 Hz, 1H), 5.70 (d, J = 15 Hz, 1H), 4.41 (s, 4H), 3.57
5
.1.41. N-(3-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
1
3
(
s, 5H); C NMR (300 M DMSO-d
6
) δ 163.63, 157.32, 154.14, 153.58,
4
-yl)amino)methyl)phenyl)acrylamide (11e)
1
1
5
40.90, 139.82, 137.81, 136.57, 134.55, 132.29, 129.37, 127.11,
Compound 11e was prepared from 10e in 86% yield by a method
22.96, 122.07, 119.88, 118.98, 118.63 109.29, 81.12, 63.89, 54.49,
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 11.45
+
0.41; MS (ESI) m/z 394 [M+H]
.
(
s, 1H), 10.58 (s, 1H), 8.99 (s, 1H), 8.36 (s, 1H), 7.67 (d, 2H), 7.32 (m,
2
H), 7.20 (m, 2H), 7.01 (s, 2H), 6.57 (dd, J = 18, 9 Hz ,1H), 6.24 (d,
5
.1.36. N-(4-(((6-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)
J = 18 Hz, 1H), 5.87 (s, 1H), 5.74 (d, J = 9 Hz, 1H), 4.49 (s, 2H), 3.09
pyrimidin-4-yl)amino)methyl)phenyl)acrylamide (11c’)
13
(
s, 4H), 2.57 (s, 4H),2.30 (s, 3H); C NMR (300 M DMSO-d
6
) δ 163.71,
Compound 11c’ was prepared from 10c’ in 82% yield by a method
1
1
5
61.86, 158.13, 156.11, 150.32, 139.92, 138.42, 132.43, 129.44,
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.50
27.20, 125.03, 122.65, 118.87, 118.41, 117.04, 116.67, 80.12, 67.47,
(
s, 1H), 10.14 (br, 1H), 8.85 (s, 1H), 8.34 (s, 1H), 7.84 (s, 1H),7.70 (d,
2.37, 45.91, 42.31; MS (ESI) m/z 444 [M+H]+
.
J = 9 Hz, 2H), 7.49 (s, 1H),7.29 (d, J = 9 Hz, 2H), 6.54 (dd, J = 15,
9
4
Hz, 1H), 6.24 (d, J = 18 Hz, 1H), 5.85 (s, 1H), 5.73 (d, J = 9 Hz, 1H),
13
5.1.42. N-(3-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
.46 (s, 2H), 4.23 (s, 2H), 3.67 (s, 2H), 3.22 (s, 3H); C NMR (300 M
4
-yl)amino)methyl)phenyl)but-2-enamide (12e)
DMSO-d
6
) δ 163.64, 159.13, 157.96, 138.87, 138.40, 132.45, 128.25,
Compound 12e was prepared from 10e in 80% yield by a method
1
4
27.06, 126.26, 124.50, 122.88, 119.92, 83.17, 70.89, 58.44, 51.95,
+
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d ) δ 11.41
6
4.67; MS (ESI) m/z 394 [M+H]
.
(
br, 1H), 10.29 (s, 1H), 8.69 (s, 1H), 8.30 (s, 1H), 7.65 (m, 2H), 7.21 (m,
2
4
H), 6.99 (s, 2H), 6.79 (m, 2H), 6.23 (d, J = 15 Hz ,1H), 5.81 (s, 1H),
13
5
.1.37. N-(3-(((6-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)
.46 (s, 2H), 3.74 (s, 4H), 3.40 (s, 4H), 2.78 (s, 3H), 1.85 (s, 3H);
C
methyl)phenyl)acrylamide (11d)
NMR (300 M DMSO-d
6
) δ 147.43, 142.79, 140.14, 138.81, 130.20,
Compound 11d was prepared from 10d in 80% yield by a method
1
129.30, 126.57, 124.26, 122.29, 118.68, 118.30, 117.03, 81.27, 67.47,
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.50
5
2.41, 46.01, 42.32, 17.98; MS (ESI) m/z 458 [M+H]+
.
(
s,1H), 9.82(br, 1H),8.49 (s, 1H), 8.27 (s, 1H), 7.67 (s, 1H),7.30(t,
J = 6 Hz, 1H), 7.18 (d, J = 6 Hz, 2H), 7.07 (d, J = 6 Hz, 1H), 7.00(d,
J = 6 Hz, 1H),6.94(d, J = 6 Hz, 2H),6.56(dd, J = 18,12 Hz,1H),6.25(d,
5
.1.43. N-(3-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
-yl)amino)methyl)phenyl)propionamide (13e)
1
3
J = 15 Hz,1H),5.73 (d, 2H), 4.41 (d, 2H), 3.73 (s, 4H),3.07(s, 4H);
C
4
NMR (300 M DMSO-d
6
) δ 163.68, 162.17, 158.90, 158.13, 148.71,
Compound 13e was prepared from 10e in 81% yield by a method
1
1
39.85, 132.44, 129.81, 129.62, 129.33, 127.14, 124.07, 122.53,
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 11.37
18.65, 118.34, 116.13, 115.32, 114.98, 81.47, 66.52, 49.10, 44.85;
(
s,1H),10.30(s, 1H), 9.05 (br, 1H), 8.35 (s, 1H), 7.47 (d, 1H), 7.34 (s,
+
MS (ESI) m/z 431 [M+H]
.
3
H), 7.20 (s, 2H), 7.01 (d, J = 6 Hz, 2H), 5.83 (s, 1H), 4.57 (s, 2H), 3.79
(
s, 2H), 3.46 (d, J = 6 Hz, 2H), 3.15 (d, J = 6 Hz, 2H),2.79(s,
5
.1.38. N-(3-(((6-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)
13
3
1
1
4
H),2.32(q, 2H),1.06(t, 3H); C NMR (300 M DMSO-d
6
) δ 172.02,
methyl)phenyl)but-2-enamide (12d)
62.99, 161.16, 159.86, 150.43, 140.29, 139.61, 138.06, 132.99,
Compound 12d was prepared from 10d in 83% yield by a method
30.35, 127.09, 122.62, 122.18, 118.65, 117.08, 81.34, 52.43, 45.92,
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.17
2.36, 10.15; MS (ESI) m/z 446 [M+H]+
.
(
s,1H), 8.90(br, 1H),8.36 (s, 1H), 7.65 (d, J = 6 Hz, 2H),7.45(d,
J = 6 Hz, 2H), 7.34 (d, J = 6 Hz, 1H), 6.93 (d, J = 6 Hz, 2H), 6.78(dd,
J = 15,9 Hz,1H),6.18(d, J = 15 Hz,1H),5.81 (s, 1H), 4.48 (s, 2H), 3.78
5
.1.44. N-(4-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
1
3
4-yl)amino)methyl)phenyl)acrylamide (11e’)
(
s, 4H),3.16(s, 4H),1.85(d, 3H); C NMR (300 M DMSO-d ) δ 163.99,
6
Compound 11e’ was prepared from 10e in 83% yield by a method
1
1
1
61.39, 158.88, 158.03, 146.11, 140.26, 136.36, 136.10, 129.43,
1
similar to that described for 11a. H NMR (300 MHz, HCl salt, DMSO-
26.50, 122.26, 118.74, 118.23, 116.95, 116.59, 78.80, 66.17, 49.59,
+
d
6
) δ 10.34 (s,1H), 8.59 (s, 1H), 8.03 (s, 1H), 7.50 (d, J = 9 Hz, 2H),
7.97; MS (ESI) m/z 445 [M+H]
.
7
6
2
.24 (m, 4H), 6.86 (d, J = 9 Hz, 2H), 6.49(dd, J = 9 Hz, 9 Hz, 1H),
.16(d, J = 15 Hz, 1H), 5.65 (s, 1H), 5.59(d, J = 12 Hz, 1H), 4.37 (s,
5
.1.39. N-(3-(((6-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)
1
3
H), 3.09 (s, 4H), 2.57 (s, 4H), 2.30 (s, 3H); C NMR (300 M DMSO-d )
6
methyl)phenyl)propionamide (13d)
δ 163.67, 162.64, 158.20, 157.92, 147.26, 138.76, 132.49, 130.49,
Compound 13d was prepared from 10d in 89% yield by a method
1
128.81, 128.14, 124.10, 119.88, 118.56, 117.03, 81.66, 52.43, 46.06,
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.44
4
4.56, 42.34; MS (ESI) m/z 444 [M+H]+
.
(
br, 1H), 10.12(s, 1H), 8.99(br, 1H),8.38 (s, 1H), 7.61 (s, 1H),7.49(d,
J = 9 Hz, 1H), 7.35 (m, 3H), 7.19 (d, J = 6 Hz, 1H), 6.96(d,
J = 6 Hz,2H), 5.92 (s, 1H), 4.49 (s, 2H), 3.86 (s, 4H),3.26(s,
5.1.45. N-(4-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
1
3
4
1
1
2
H),2.32(d, 2H)1.05(t, 3H); C NMR (300 M DMSO-d
6
) δ 172.56,
4-yl)amino)methyl)phenyl)but-2-enamide (12e’)
65.49, 157.69, 157.18, 150.42, 147.06, 140.23, 132.76, 130.36,
Compound 12e’ was prepared from 10e in 83% yield by a method
1
29.33, 124.27, 122.12, 118.45, 118.09, 80.77, 65.63, 50.78, 45.09,
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.34
+
9.95; MS (ESI) m/z 433 [M+H]
.
(s, 1H), 8.59 (s, 1H), 8.03 (s, 1H), 7.50 (d, J = 9 Hz, 2H), 7.24 (m, 5H),
.85 (d, J = 9 Hz, 2H), 6.64 (q, 1H), 6.17 (d, J = 15 Hz, 1H), 5.65 (s,
1H), 4.37 (s, 2H), 3.09 (t, 4H), 2.57 (t, 4H), 2.30 (s, 3H), 1.88 (d,
6
5
.1.40. N-(4-(((6-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)
1
3
methyl)phenyl)acrylamide (11d’)
J = 6 Hz, 3H); C NMR (300 M DMSO-d ) δ 163.96, 162.35, 160.36,
6
Compound 11d’ was prepared from 10d’ in 88% yield by a method
159.64, 146.25, 139.89, 138.69, 132.39, 128.66, 127.92, 123.01,
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.61
119.72, 117.11, 115.48, 82.37, 52.53, 46.37, 44.38, 42.39, 17.96; MS
(
s, 1H), 10.37 (s, 1H), 8.98 (s, 1H), 8.36 (s, 1H),7.72 (s, 2H), 7.25 (m,
(ESI) m/z 458 [M+H]+
.
9

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
5
.1.46. N-(4-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
The reaction mixture was stirred at room temperature for 16 h. It was
quenched with water, and was evaporated to dryness. The crude mass
was purified by silica gel column chromatography (DCM:
4-yl)amino)methyl)phenyl)propionamide (13e’)
Compound 13e’ was prepared from 10e in 83% yield by a method
1
similar to that described for 11a. H NMR (300 MHz, DMSO-d
6
) δ 10.34
MeOH = 15:1) to afford compound 15c (52 mg, 61%) as a white solid.
1
(
s, 1H), 8.59 (s, 1H), 8.03 (s, 1H), 7.50 (d, J = 9 Hz, 2H), 7.24 (m, 4H),
H NMR (300 MHz, DMSO-d ) δ 10.25 (s, 1H), 8.60 (s, 1H), 8.04 (s,
6
6
4
.85 (d, J = 9 Hz, 2H), 5.65 (s, 1H), 4.37 (s, 2H), 3.09 (t, 4H), 2.57 (t,
1H), 7.90 (s, 1H), 7.74(s, 1H), 7.52 (d, J = 9 Hz, 1H), 7.40 (t, J = 6 Hz,
1
3
H), 2.30 (s, 3H), 2.13 (q, 2H), 1.09 (t, 3H); C NMR (300 M DMSO-d )
6
1H), 7.36(s, 1H), 7.25 (d, J = 6 Hz, 1H), 5.57 (s, 1H), 4.37 (d, J = 6 Hz,
1
3
δ 172.58, 166.89, 158.52, 157.33, 147.73, 139.18, 134.62, 128.86,
2H), 4.18 (t, J = 6 Hz, 2H), 3.65 (t, J = 6 Hz, 2H), 3.22 (s, 3H);
C
1
2
28.14, 122.23, 119.59, 116.99, 80.87, 52.42, 45.94, 44.67, 42.35,
NMR (300 M DMSO-d ) δ 164.35, 157.72, 157.59, 140.18, 139.08,
6
+
9.91, 10.16; MS (ESI) m/z 446 [M+H]
.
133.16, 129.87, 127.32, 118.49, 118.10, 117.66, 114.99, 80.77, 70.33,
5
8.22, 50.25, 45.02; MS (ESI) m/z 407 [M+H]+
.
5
.1.47. 2-cyano-N-(3-(((6-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)
pyrimidin-4-yl)amino)methyl)phenyl)acetamide (14c)
5.1.52. N-(3-(((6-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)
To a stirred solution of 10c (80 mg, 0.24 mmol) in THF (15 mL) was
added DIEA (76 mg, 0.74 mmol), HATU (220 mg, 0.59 mmol), and 2-
cyanoacetic acid (22 mg, 0.26 mmol) at 0 °C. The reaction mixture was
stirred at room temperature for 16 h. It was quenched with water and
extracted with ethyl acetate. The organic layer was washed with water
followed by brine, dried over anhydrous sodium sulfate, and was eva-
porated to dryness. The crude mass was purified by silica gel column
methyl)phenyl)cyanamide (15d)
Compound 15d was prepared from 10d in 68% yield by a method
1
similar to that described for 15c. H NMR (300 MHz, DMSO-d
6
) δ 10.28
(s, 1H), 8.78 (s, 1H), 8.07 (s, 1H), 7.54 (t, 1H), 7.47 (d, J = 9 Hz, 1H),
7.43 (s, 1H), 7.29 (t, J = 9 Hz, 1H), 7.23 (d, J = 6 Hz, 2H), 7.02 (d,
J = 9 Hz, 1H), 6.88 (d, J = 9 Hz, 2H), 5.64 (s, 1H),4.41 (d, J = 6 Hz,
1
3
2H), 3.73 (t, J = 6 Hz, 4H),3.03(t, J = 6 Hz, 4H); C NMR (300 M
chromatography (DCM: MeOH = 15:1) to afford compound 14c
DMSO-d ) δ 162.56, 160.52, 157.61, 146.55, 141.85, 138.74, 135.87,
6
1
(
50 mg, 53%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 10.25 (s,
132.56, 129.64, 121.72, 121.14, 115.73, 113.22, 112.06, 82.75, 66.12,
1
H), 8.60 (s, 1H), 8.04 (s, 1H), 7.90 (s, 1H), 7.74(s, 1H), 7.52 (d,
49.15, 43.47; MS (ESI) m/z 402 [M+H]+
.
J = 9 Hz, 1H), 7.40 (t, J = 6 Hz, 1H), 7.36(s, 1H), 7.25 (d, J = 6 Hz,
1
2
1
1
4
H), 5.57 (s, 1H), 4.37 (d, J = 6 Hz, 2H), 4.18 (t, J = 6 Hz, 2H), 3.86 (s,
5.1.53. N-(3-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
1
3
H), 3.65 (t, J = 6 Hz, 2H), 3.22 (s, 3H); C NMR (300 M DMSO-d
6
) δ
4-yl)amino)methyl)phenyl)cyanamide (15e)
63.16, 162.51, 150.18, 141.26, 139.18, 131.28, 128.96, 128.20,
Compound 15e was prepared from 10e in 69% yield by a method
1
20.30, 119.62, 117.07, 116.60, 115.28, 80.52, 60.39, 54.95, 49.01,
similar to that described for 15c. H NMR (300 MHz, DMSO-d
6
) δ 9.61
+
4.72, 29.91; MS (ESI) m/z 407 [M+H]
.
(s, 1H), 8.59 (s, 1H), 8.01 (s, 1H), 7.38(s, 1H), 7.33 (d, J = 9 Hz, 1H),
.28 (s, 1H), 7.22 (d, J = 6 Hz, 2H), 6.93(d, J = 9 Hz, 1H), 6.84(d,
J = 9 Hz, 2H), 5.63(s, 1H), 4.37(s, 2H), 3.64 (s, 4H), 3.06(s, 4H),2.24(s,
7
5
.1.48. 2-cyano-N-(3-(((6-((4-morpholinophenyl)amino)pyrimidin-4-yl)
1
3
amino)methyl)phenyl)acetamide (14d)
3H); C NMR (300 M DMSO-d ) δ 163.04, 158.09, 154.43, 146.95,
6
Compound 14d was prepared from 10d in 65% yield by a method
139.65, 139.31, 132.79, 130.12, 122.20, 117.14, 116.48, 114.99,
1
+
similar to that described for 14c. H NMR (300 MHz, DMSO-d
6
) δ 10.27
113.71, 79.58, 55.04, 52.00, 49.12, 46.05; MS (ESI) m/z 415 [M+H]
.
(
s,1H), 8.78 (s, 1H), 8.06 (s, 1H), 7.54 (s, 1H), 7.46 (d, J = 9 Hz, 1H),
7
.42 (s, 1H), 7.28 (t, J = 9 Hz, 1H), 7.23 (d, J = 9 Hz, 2H), 7.01 (d,
5.1.54. N-(4-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-
J = 9 Hz, 1H), 6.87 (d, J = 9 Hz, 2H), 5.64 (s, 1H),4.41 (d, 2H), 3.86 (s,
4-yl)amino)methyl)phenyl)cyanamide (15e’)
+
2
H), 3.73 (m, 4H),3.03(m, 4H). MS (ESI) m/z 444 [M+H]
.
Compound 15e’ was prepared from 10e’ in 65% yield by a method
1
similar to that described for 15c. H NMR (300 MHz, DMSO-d
6
) δ 10.34
5
.1.49. 2-cyano-N-(3-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)
(s, 1H), 8.59 (s, 1H), 8.03 (s, 1H), 7.76 (d, J = 9 Hz, 2H), 7.50 (d, 4H),
7.11 (d, J = 9 Hz, 2H), 5.65 (s, 1H), 4.37 (s, 2H), 3.09 (t, 4H), 2.57 (t,
pyrimidin-4-yl)amino)methyl)phenyl)acetamide (14e)
1
3
Compound 14e was prepared from 10e in 68% yield by a method
4H), 2.30 (s, 3H); C NMR (300 M DMSO-d ) δ 162.32, 160.08, 157.46,
6
1
similar to that described for 14c. H NMR (300 MHz, DMSO-d
6
) δ 10.26
149.39, 137.16, 136.73, 134.16, 128.47, 122.70, 118.96, 117.58,
+
(
s, 1H), 8.57 (s, 1H), 8.03 (s, 1H), 7.48 (d, J = 9 Hz, 1H), 7.41 (s, 1H),
114.85, 82.48, 58.45, 51.65, 46.59, 43.31; MS (ESI) m/z 415 [M+H]
.
7
2
2
1
1
4
.28 (d, 1H), 7.25(d, 3H),7.02(d, J = 6 Hz, 1H),6.86(d, J = 9 Hz,
H),5.64 (s, 1H), 4.40 (d, 2H), 3.86 (s, 2H), 3.09 (s, 4H), 2.57 (s, 4H),
5.1.55. 2,2,2-trifluoro-N-(3-(((6-((4-morpholinophenyl)amino)pyrimidin-
4-yl)amino)methyl)phenyl)acetamide (16d)
1
3
.31 (s, 3H); C NMR (300 M DMSO-d ) δ 163.07, 161.36, 161.03,
6
58.11, 146.79, 141.46, 138.88, 132.93, 129.25, 122.99, 118.08,
To a stirred solution of 10d (120 mg, 0.32 mmol) in THF (15 mL)
was added triethylamine (48 mg, 0.47 mmol) and trifluoroacetic an-
hydride (70 mg, 0.35 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 16 h. It was quenched with water, and was eva-
porated to dryness. The crude mass was purified by silica gel column
16.58, 116.34, 83.24, 54.82, 48.90, 45.68, 44.09, 27.14; MS (ESI) m/z
+
57 [M+H]
.
5
.1.50. 2-cyano-N-(4-(((6-((4-(4-methylpiperazin-1-yl)phenyl)amino)
pyrimidin-4-yl)amino)methyl)phenyl)acetamide (14e’)
chromatography (DCM: MeOH = 15:1) to afford compound 15c
1
Compound 14e’ was prepared from 10e’ in 68% yield by a method
(80 mg, 53%) as a white solid. H NMR (300 MHz, DMSO-d
6
) δ 11.25 (s,
1
similar to that described for 14c. H NMR (300 MHz, HCl salt, DMSO-
1H), 8.63 (s, 1H), 8.03 (s, 1H), 7.58(d, 2H), 7.36 (m, 2H), 7.24 (d,
d
6
7
) δ 10.34 (s, 1H), 8.59 (s, 1H), 8.03 (s, 1H), 7.50 (d, J = 9 Hz, 2H),
J = 9 Hz, 2H), 7.14 (d, J = 6 Hz, 1H), 6.86 (d, J = 9 Hz, 2H), 5.65 (s,
1
3
.25 (m, 4H), 6.85 (d, J = 9 Hz,2H), 5.65 (s, 1H), 4.37 (s, 2H), 3.89 (s,
1H), 4.43 (s, 2H), 3.73 (s, 4H),3.02(s, 4H); C NMR (300 M DMSO-d )
6
1
3
2
H), 3.09 (s, 4H), 2.57 (s, 4H), 2.30 (s, 3H); C NMR (300 M DMSO-d
6
)
δ 163.02, 160.96, 158.06, 155.19, 154.67, 147.45, 141.68, 140.00,
δ 163.06, 161.29, 161.02, 158.09, 146.78, 137.40, 135.99, 132.93,
136.83, 129.33, 124.70, 122.21, 120.57, 119.86, 116.52, 116.23,
+
1
4
27.98, 122.23, 119.75, 116.57, 116.38, 83.29, 54.78, 48.86, 45.64,
114.13, 83.26, 66.61, 49.64, 45.43; MS (ESI) m/z 473 [M+H]
.
+
3.84, 27.08; MS (ESI) m/z 457 [M+H]
.
5
.1.56. 2,2,2-trifluoro-N-(4-(((6-((4-morpholinophenyl)amino)pyrimidin-
5
.1.51. N-(3-(((6-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)
4-yl)amino)methyl)phenyl)acetamide (16d’)
pyrimidin-4-yl)amino)methyl)phenyl)cyanamide (15c)
Compound 16d’ was prepared from 10d’ in 65% yield by a method
1
To a stirred solution of 10c (80 mg, 0.29 mmol) in THF (5 mL) was
added KOAc (58 mg, 0.59 mmol) and BrCN (37 mg, 0.35 mmol) at 0 °C.
similar to that described for 16d. H NMR (300 MHz, HCl salt, DMSO-
d ) δ 11.23 (s, 1H), 8.63 (s, 1H), 8.03 (s, 1H), 7.60(d, J = 9 Hz, 2H),
6
10

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
7
4
1
1
8
.32 (d, 3H), 7.26 (d, J = 9 Hz, 2H), 6.86 (d, J = 6 Hz, 2H), 5.65(s, 1H),
5.2. Computational analysis
5.2.1. Docking calculation
1
3
.41 (s, 2H), 3.73 (s, 4H), 3.02(s, 4H); C NMR (300 M DMSO-d ) δ
6
63.04, 161.00, 158.11, 155.10, 154.61, 147.02, 137.94, 135.23,
33.08, 127.97, 122.20, 121.57, 118.20, 116.23, 114.98, 114.37,
We used the X-ray cocrystal structure of JAK3 in the Protein Data
Bank (PDB ID: 3PJC). The protein structure for the docking study was
prepared with Protein Preparation Workflow. All crystallographic wa-
ters were removed and chain A was kept. Hybridization states, charges,
and angles were assigned in the protein structure with missing bond
orders, and hydrogen atoms were added. The energy of the protein
structure was minimized in 100 steps of the smart minimize method.
Docking grids were generated and defined based on the centroid of
compound 11e in the ATP binding site incorporating hydrogen-bond
constraints to the hinge and hydrophobic regions. Ligands were pre-
pared using Discovery Studio 3.0 (DS 3.0). energy-minimized con-
formation of each ligands were used to docking calculation input mo-
lecules. Ligand-Fit and CDOCKER docking programs implemented in DS
+
3.30, 66.61, 49.64, 43.77; MS (ESI) m/z 473 [M+H]
.1.57. 1-methyl-4-nitro-1H-pyrazole (17a)
.
5
To a stirred solution of 4-nitro-1H-pyrazole (2 g, 17.7 mmol) in
acetonitrile (15 mL) was added potassium carbonate (2.7 g, 19.5 mmol)
and iodomethane (2.76 g, 19.5 mmol). The reaction mixture was stirred
at reflux temperature for 6 h. And it was evaporated to dryness. The
crude mass was purified by silica gel column chromatography (PE:
EA = 10:1) to afford compound 15c (2.2 g, 98%) as a white solid. MS
+
(
ESI) m/z 128 [M+H]
.
5.1.58. 2-(4-nitro-1H-pyrazol-1-yl)ethan-1-ol (17b)
3
.0 were used in this study. Other docking parameters were kept to the
Compound 17b was prepared from 4-nitro-1H-pyrazole in 99%
default values. The top-scoring pose was employed for discussions.
yield by a method similar to that described for 17a. MS (ESI) m/z 158
+
[
M+H]
.
5
.3. Bioactivity
5
.1.59. 1-(2-methoxyethyl)-4-nitro-1H-pyrazole (17c)
Compound 17c was prepared from 4-nitro-1H-pyrazole in 98% yield
5.3.1. JAK1-3 and TYK2 enzyme assay
by a method similar to that described for 17a. MS (ESI) m/z 172 [M
Human JAK1-3 and TYK2 kinases were obtained from Invitrogen
+
+
H]
.
and assays were performed using HTRF (Homogenous Time-Resolved
Fluorescence) detection technology. Briefly, the enzyme reaction was
run in reaction buffer, which consisted of 50 mM HEPES (pH7.0), 5 mM
5.1.60. 4-(4-Nitrophenyl)morpholine (19d)
In a 25 mL round-bottom flask equipped with a stirring bar, a
MgCl
2
, 1 mM DTT, 0.1% NaN and 0.1% orthovanadate. The assay was
3
mixture of 2.62 g (30 mmol) of morpholine, 4.24 g (30 mmol) of 4-
done by a 384-well plate (10 μL) assay format. A typical enzyme reac-
fluoronitrobenzene, and 4.14 g (30 mmol) of K
2
CO in 15 mL of DMSO
3
tion contains 1 ng/μL JAK1, 1 μM TK-substrate-biotin, 4 μM ATP;
was heated with stirring at 120 °C for 20 h. After cooling, the mixture
0
.004 ng/μL JAK2, 1 μM TK-substrate-biotin, 4 μM ATP; 0.012 ng/μL
was poured into 80 mL mixed solution of ethanol/water (1:1), and the
JAK3, 1 μM TK-substrate-biotin, 1.43 μM ATP; 0.012 ng/μL TYK2, 1 μM
TK-substrate-biotin, 1.43 μM ATP. Compounds were screened at serial
diluted concentration in the presence of 2% DMSO with a 5 min pre-
incubation of kinase and compounds. All reactions were started by the
addition of ATP and TK-substrate-biotin, incubated at 30 °C for 30 min
and quenched with the stop buffer containing 25 nM Strep-XL665 and
TK Ab-Cryptate. The plates were incubated for 1 h. Time-resolved
fluorescence was monitored with a Synergy H1 (Biotek) by excitation at
formed yellow crystals were collected by filtration to give 19d with an
+
yield of 6.08 g (97%). MS (ESI) m/z 209 [M+H]
.
5
.1.61. 1-Methyl-4-(4-nitrophenyl)piperazine (19e)
A solution of 4-fluoronitrobenzene (5 g, 35.5 mmol), and K
2
CO
3
(
4.9 g, 35.5 mmol) in DMSO (10 mL) was stirred at room temperature
for 0.5 h. Subsequently, 1-methylpiperazine (3.55 g, 35.5 mmol) was
added dropwise, and the resulting reaction mixture was stirred at room
temperature for 6 h. The mixture was then poured into ice-water. A
3
30 nm and emission donor at 620 nm or emission acceptor at 665 nm,
respectively. The files recorded by the Synergy H1 were read with Excel
and contained the acceptor and donor counts for each sample. And IC50
values were determined using the Graphpad Prism 5.0 Software.
yellow precipitate formed and was collected by filtration to give 19e
+
with a yield of 6.66 g (85%). MS (ESI) m/z 222 [M+H]
.
5
.1.62. 1-methyl-1H-pyrazol-4-amine (18a)
5
.3.2. Rat T cell proliferation
To 17a (1 g, 7.8 mmol) in EtOH (20 mL) was added 10% Pd/C
Spleen cells were suspended in RPMI1640 medium, supplemented
5
0 mg. The reaction mixture was stirred for 3 h under hydrogen at room
with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL strepto-
temperature. The mixture was then filtered with Celite, and the filtrate
6
mycin, and 50 μM 2-mercaptoethanol at a density of 1.5 × 10 cells/
was concentrated and dried under vacuum to yield 0.74 g (97%) of 18a
+
mL. Splenocytes were cultured with Concanavalin A for 24 h at 37 °C in
as a white solid. MS (ESI) m/z 98 [M+H]
.
5
% CO to induce IL-2 receptor expression and then incubated with IL-2
2
and test compounds at designated concentrations in 96-well tissue
culture plates. After incubation for 72 h, cell viability was monitored
using the CellTiter 96® AQueous One Solution Cell Proliferation Assay
5.1.63. 2-(4-amino-1H-pyrazol-1-yl)ethan-1-ol (18b)
Compound 18b was prepared from 17b in 98% yield by a method
+
similar to that described for 18a. MS (ESI) m/z 128 [M+H]
.
(
MTS) (Promega, Madison, WI) according to the manufacturer’s re-
commended protocols. Colorimetric assay of each well was read using
microplate reader synergy H1 (Biotek). Values were transformed to
percent inhibition relative to vehicle control, and IC50 curves were
fitted according to nonlinear regression analysis of the data using
PRISM GraphPad 5.0.
5.1.64. 1-(2-methoxyethyl)-1H-pyrazol-4-amine (18c)
Compound 18c was prepared from 17c in 97% yield by a method
+
similar to that described for 18a. MS (ESI) m/z 142 [M+H]
.
5.1.65. 4-morpholinoaniline (20d)
Compound 20d was prepared from 19d in 99% yield by a method
+
similar to that described for 18a. MS (ESI) m/z 179 [M+H]
.
Acknowledgments
5
.1.66. 4-(4-methylpiperazin-1-yl)aniline (20e)
The authors are grateful to financial supports from National Natural
science Foundation of China (81573445, 81172934) and the Beijing
Municipal Natural Science Foundation (7182115).
Compound 20e was prepared from 19e in 99% yield by a method
+
similar to that described for 18a. MS (ESI) m/z 192 [M+H]
.
11

R.-N. Yu, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
References
11. Yamaoka K, Saharinen P, Pesu M, et al. Genome Biol. 2004;5:253.
1
1
1
1
1
1
1
1
2. Yamaoka K, Min B, Zhou YJ, et al. Blood. 2005;106:3227–3233.
3. Kisseleva T, Bhattacharya S, Braunstein J, et al. Gene. 2002;285:1–24.
4. Changelian Paul S, et al. Science. 2003;302:875.
1
.
Musumeci Francesca, Greco Chiara, Giacchello Ilaria, Fallacara Anna Lucia, Ibrahim
Munjed M, Grossi Giancarlo, Brullo Chiara, Schenone Silvia. Curr Med Chem.
5. Flanagan ME, Blumenkopf TA, Brissette WH, et al. J Med Chem. 2010;53:8468.
6. Quintas-Cardama A, et al. Blood. 2010;115:3109–3117.
2
018;25:1.
2
3
4
5
.
.
.
.
Wrobleski Stephen T, Pitts William J. Ann Rep Med Chem. 2009;44:247.
O’Sullivan LA, Liongue C, Lewis RS, et al. Mol Immunol. 2007;44:2497–2506.
Ivashkiv Lionel B, Xiaoyu Hu. Arthritis Res Therapy. 2004;6:159.
Smirnova OV, Ostroukhova TY, Bogorad RL. World J Gastroenterol.
7. Telliez JB, et al. ACS Chem Biol. 2016;11:3442–3451.
8. Yin Yuan, Chen Cheng-Juan, Ru-Nan Yu, et al. Bioorg Med Chem. 2018;26:4774.
9. Forster Michael, Gehringer Matthias, Laufer Stefan A. Bioorg Med Chem.
2
017;27:4229.
2
007;13:6478–6491.
2
2
2
0. Kempson James, Ovalle Damaso, Guo Junqing, et al. Bioorg Med Chem.
017;27:4622.
1. Yamagishi Hiroaki, Inoue Takayuki, Nakajima Yutaka, et al. Bioorg Med Chem.
017;25:5311.
2. Yamagishi Hiroaki, Inoue Takayuki, Nakajima Yutaka, et al. Bioorg Med Chem.
015;23:4846.
6
7
8
.
.
.
O’Shea JJ, Gadina M, Schreiber RD. Cell. 2002;109:S121.
Ihle JN, Kerr IM. Trends Genet. 1995;11:69.
2
Menet Christel J, Van Rompaey Luc, Geney Raphaël. Progress Med Chem.
2
2
013;52:153.
9
.
Papageorgiou Anastassios C, Wikman Linnea EK. TRENDS Pharm Sci. 2004;25:558.
2
1
0. Dymock Brian W, Shang See Cheng. Expert Opin Ther Patents. 2013;23:449–501.
12