Design, synthesis, and SAR study of highly potent, selective, irreversible covalent JAK3 inhibitors

Article abstract of DOI:10.1007/s11030-017-9803-2

Here, we report the design and synthesis of pyrimidinyl heterocyclic compounds containing terminal electrophiles as irreversible covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of the structure–activity relationship utilizing kinase assays resulted in the identification of potent and selective JAK3 inhibitors such as T1, T8, T15, T22, and T29. Among them, T29 was verified as a promising JAK3 irreversible inhibitor that possessed the best bioactivity and selectivity against JAKs and kinases containing a cysteine in the residue analogous to Cys909 in JAK3, suggesting that covalent modification of this Cys residue allowed the identification of a highly selective JAK3 inhibitor. Moreover, T29 also displayed a significant anti-inflammatory effect in ICR mice through the inhibition of increased paw thickness, which is worth further optimization to increase its potency and medicinal properties.

Full text of DOI:10.1007/s11030-017-9803-2

Molecular Diversity
https://doi.org/10.1007/s11030-017-9803-2
ORIGINAL ARTICLE
Design, synthesis, and SAR study of highly potent, selective,
irreversible covalent JAK3 inhibitors
Linhong He1,2 · Mingfeng Shao1 · Taijin Wang1 · Tingxuan Lan1 · Chufeng Zhang1 · Lijuan Chen1
Received: 29 April 2017 / Accepted: 13 December 2017
© Springer International Publishing AG, part of Springer Nature 2018
Abstract
Here, we report the design and synthesis of pyrimidinyl heterocyclic compounds containing terminal electrophiles as
irreversible covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of the structure–
activity relationship utilizing kinase assays resulted in the identification of potent and selective JAK3 inhibitors such as T1, T8,
T15, T22, and T29. Among them, T29 was verified as a promising JAK3 irreversible inhibitor that possessed the best bioac-
tivity and selectivity against JAKs and kinases containing a cysteine in the residue analogous to Cys909 in JAK3, suggesting
that covalent modification of this Cys residue allowed the identification of a highly selective JAK3 inhibitor. Moreover, T29
also displayed a significant anti-inflammatory effect in ICR mice through the inhibition of increased paw thickness, which is
worth further optimization to increase its potency and medicinal properties.
Keywords JAK3 · Covalent inhibitor · Selectivity · Structure–activity relationship (SAR) · Docking
Abbreviations
nal transducer and activator of transcription (STAT) family
of transcription factors, which ultimately regulate target gene
expression that governs cell proliferation and survival [4,5].
The JAK/STAT signaling pathway is involved in a variety of
biological processes, including the regulation of immune and
inflammatory responses [5–7].
JAK
Janus kinase
STAT Signal transducer and activator of transcription
Cys
Cysteine
SAR
Structure–activity relationship
Biochemical and genetic studies have identified that JAK3
is restrictedly expressed in lymphoid tissue and is only acti-
vated by cytokines of γc subfamily (IL-2, IL-4, IL-7, IL-9,
IL-15, and IL-21), while the other JAK family members
widely express and are activated by numerous cytokines [8–
10]. Although JAK3 deficiency in humans or mice can result
in severe combined immunodeficiency (SCID) due to the
complete abrogation of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-
21 signaling in T cells and NK cells, these cases are only
limited to immune cells [5,11]. Moreover, heterozygous par-
ents of SCID patients do not exhibit immune-compromised
phenotypes, suggesting that partial inhibition of JAK3 can
be used to modulate immune responses [6,8]. This also has
been applied in several important clinical practices, such as
the prevention of transplant rejection and the treatment of
various autoimmune disorders [6,8]. Taking these findings
together, selective targeting of JAK3 has long been viewed
as a potent strategy for the treatment of immune system indi-
cations while avoiding unexpected side effects caused by
inhibition of other JAKs [8,12,13].
Introduction
The Janus kinases (JAKs) are a family of four cytoplas-
mic nonreceptor tyrosine kinases (JAK1, JAK2, JAK3, and
TYK2) that play important roles in the signaling of var-
ious cytokines [1–3]. Upon activation through cytokine
signaling, JAKs subsequently activate the downstream sig-
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11030-017-9803-2) contains supplementary
material, which is available to authorized users.
Lijuan Chen
clijuan125@163.com
B
1
Cancer Center, West China Hospital, Sichuan University and
Collaborative Innovation Center, # 17, Section 3, Ren Min
Nan Lu, Chengdu 610041, People’s Republic of China
2
Department of Pharmacology, School of Pharmacy, Guangxi
Medical University, # 22 Shuangyong Road, Nanning
530021, Guangxi, People’s Republic of China
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Molecular Diversity
Table 1 Sequence conservation of human Janus kinase active sites
Position (in JAK3)
904
905
906
907
908
909
912
940
945
947
948
953
954
956
965
966
JAK3
JAK1
JAK2
TKY2
Y
F
L
L
L
V
P
P
P
P
S
S
Y
L
G
G
G
G
C
S
S
S
D
E
L
L
L
L
C
Y
Y
Y
H
H
H
H
R
R
R
R
R
R
R
R
N
N
N
N
L
L
L
L
I
I
I
I
A
G
G
G
Y
Y
D
D
Given that the four JAKs share 50–60% sequence identity
over the entire kinase domain and more than 80% sequence
identity in the catalytic domain, selective targeting of JAK3
with traditional ATP-competitive inhibitors has proven to be
difficult [2,6,14]. Tofacitinib, initially described as a selec-
tive JAK3 inhibitor, has been approved by the US Food
and Drug Administration for the treatment of rheumatoid
arthritis [16,17]. However, tofacitinib has recently been ver-
ified to be a pan-JAK inhibitor (Ki = 0.68, 0.99, 0.24,
and 4.39 nM for JAK1, JAK2, JAK3, and TYK2, respec-
tively), which may lead to undesirable side effects caused
by JAK1 and JAK2 inhibition, such as dyslipidemia and
suppression of hematopoiesis, respectively [15–20]. Another
JAK3 inhibitor, decernotinib, has also been identified as
a pan-JAK inhibitor (Ki = 11, 13, 2.5, and 13 nM for
JAK1, JAK2, JAK3, and TYK2, respectively) [4]. What this
means is that no true JAK3-selective inhibitors have been
promoted into clinical trials to date [4,20]. Further improv-
ing the understanding of the structure of JAK3, researchers
have found that JAK3 has a unique cysteine residue at posi-
tion 909 (Cys909) in the binding pocket (Table 1). This is
different from the other JAKs and could be used to form
a covalent interaction with a specific inhibitor to achieve
high selectivity [8,10,14]. Employing this mechanism, some
irreversible covalent JAK3 inhibitors have been recently pub-
lished (Fig. 1) [4,11,13,21,22].
Excitingly, most JAK3 covalent inhibitors have been
identified as highly potent and selective biochemical and
cellular inhibitors of JAK3 [4,11,13,21,22]. Among them,
PF-06651600, a true JAK3 inhibitor, exhibits overall proper-
ties suitable for both preclinical assessment and advance-
ment to human clinical trials [23]. Bioisosterism, a well-
established concept in drug discovery and structure optimiza-
tion, can be used to improve the potency, selectivity, and
physicochemical properties of bioactive compounds [24].
Therefore, we designed and synthesized a novel series of
JAK3 irreversible covalent inhibitors by taking tofacitinib
as a lead structure: cyclizing (3R,4R)-(4-methylpiperidin-
3-yl)methylamine and replacing it with aniline substituents
as linkers in the lipophilic pocket of JAK3; generation of
7H-pyrrolo[2,3-d]pyrimidine bioisosteres as scaffolds in
the hinge region of JAK3; and extending side chains with
more electrophilic warheads to form covalent bonds with
Cys909 in JAK3 (Fig. 2). Furthermore, the results from
kinase assays for all the synthesized compounds showed a
clear structure–activity relationship (SAR). The representa-
tive compound T29 displayed the best enzymatic potency
and kinase selectivity not only among all the synthesized
compounds in this study, but also among the examples in
Fig. 1.
Results and discussion
Chemistry and kinase inhibition assays
To investigate the SAR (scaffolds, linkers, and electrophilic
warheads) of these designed JAK3 covalent inhibitors, differ-
ent synthetic approaches and protective group strategies were
necessary. At the beginning of our research, compounds T1–
T14, derived from the purine scaffold, were prepared through
the five steps as shown in Scheme 1. The common reagent
6-chloropurine (1) was protected with a tetrahydropyran
(THP) group to produce adduct2, whose improved solu-
bility allowed further coupling. 6-Phenyl-substituted purine
derivatives 3-1 and 3-2 were obtained via Suzuki coupling by
reacting the adduct with (3-nitrophenyl)boronic acid and (4-
methyl-3-nitrophenyl)boronic acid, respectively. Reduction
of the nitro group using Pd/C and ammonium formate gave
the respective key anilines intermediates 4-1 and 4-2. Sev-
eral kinds of electrophilic warheads were introduced into the
anilines, and subsequently target compounds T1–T14 were
obtained by removing the protecting groups in acidic condi-
tions.
The inhibitory activities of the prepared compounds were
first evaluated against JAK3 kinase in vitro at 0.5 and 5 μM
in the presence of Km ATP. Compounds that inhibited JAK3
over 50% at 0.5 μM were further tested to determine their
IC50 values for JAK3 and inhibition rates for other JAKs at
0.5 and 5 μM (Sup. Table S1). The enzymatic potencies of
compounds T1–T14 are summarized in Table 2. Compounds
with acrylamide (T1, T8) and α-haloketones (T5, T6, T12,
T13) as electrophilic warheads selectively inhibited JAK3
at low nanomolar concentrations but did not inhibit other
JAKs (IC50 > 0.5 μM), showing that using a covalent inter-
action with cysteine 909 could lead to inhibitors with high
123

Molecular Diversity
Fig. 1 Structures of tofacitinib, decernotinib, and reported JAK3 irreversible covalent drugs
Fig. 2 Design strategy for JAK3 covalent compounds
Scheme 1 General synthesis of purine derivatives T1–T14. Reagents and conditions: a TsOH, AcOEt, reflux, 2 h; b Pd(dppf)Cl2, K2CO3,
PhMe/EtOH, reflux, 3–4 h; c Pd/C, NH4COOH, MeOH, reflux, 2 h; d DIEA, dry DMF, r.t., overnight; e HCl in dioxane, MeOH, reflux, 1 h
JAK3 selectivity versus other JAKs. Unfortunately, modi-
fications of acrylamide or α-haloketones (eg., T2, T7, T9,
T14) resulted in a dramatic loss of inhibitory activity, prob-
ably because of the substituents blocking the electrophilic
warheads from forming a covalent bond with cysteine. Mod-
ifying the phenyl group with R’ substituents led to no obvious
influence on inhibitory activity against JAKs, as shown by
comparing T1 with T8, T5 with T12, and T6 with T13.
123

Molecular Diversity
Table 2 Structures and enzyme potencies of compounds T1–T14a,b
Comp.
T1
Rꢀ
R
JAK1 ICa50 (μM)
JAK2 ICa50 (μM)
> 5
JAK3 ICb50
TYK2 ICa50 (μM)
> 5
–
> 5
2.6 0.13 nM
T2
T3
T4
T5
T6
T7
–
–
–
–
–
–
> 5
NT
NT
> 5
> 5
NT
> 5
NT
496 2.21 nM
> 5 μM
> 5
NT
NT
> 5
> 5
NT
NT
> 5 μM
> 5
> 0.5
NT
2.6 0.09 nM
2.3 0.11 nM
> 0.5 μM
T8
T9
4-CH3
4-CH3
> 5
> 5
3.6 0.07 nM
> 5
NT
NT
> 0.5 μM
NT
T10
T11
T12
T13
T14
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
NT
NT
> 5
> 5
NT
NT
NT
> 5
> 5
NT
> 5 μM
NT
NT
> 5
> 5
NT
> 5 μM
2.5 0.10 nM
2.5 0.15 nM
> 0.5 μM
NT not tested
aCompounds were measured at concentrations of 5 and 0.5 μM with KmATP (30 nM JAK1 and 90 μM ATP; 2 nM JAK2 and 20 μM ATP; 15 nM
TYK2 and 16 μM ATP). Mean standard error of the mean (SEM), n = 2
b
IC50 values for JAK3 were an average of at least two independent dose–response curves at KmATP (4 nM JAK3 and 6.2 μM ATP)
Based on the above results, we selected meta-nitroaniline
as the linker and focused on the study of a scaffold.
Compounds derived from 7H-pyrrolo[2,3-d]pyrimidine, a
bioisostere of purine and pharmacophore of tofacitinib,
were prepared as described in Scheme 2. 4-Chloro-7H-
123

Molecular Diversity
Scheme 2 General synthesis of pyrrolo[2,3-d]pyrimidine derivatives T15–T28. Reagents and conditions: a DMAP, THF, r.t., 0.5 h; b
Pd(dppf)Cl2, K2CO3, PhMe/EtOH, reflux, 3–4 h; c Pd/C, NH4COOH, MeOH, reflux, 2 h; d DIEA, dry DMF, r.t., overnight; e HCl in diox-
ane, MeOH, reflux, 1 h
pyrrolo[2,3-d]pyrimidine (5) reacted with di-tert-butyl dicar-
bonate to produce Boc-protected intermediate 6, which was
then coupled with nitrophenylboronic acid to obtain com-
pound 7. Reduction of the nitro group with Pd/C gave two
products, 8 and 9, and the former was protected again with
a Boc group. Final compounds T15–T28 were obtained
by introducing electrophilic warheads to the correspond-
ing key intermediates 10 and 9, and removing protecting
groups.
Ultimately, compounds T29–T33, which are based on the
1H-pyrrolo[2,3-b]pyridine scaffold, another bioisostere of
purine and the core of decernotinib, were synthesized as illus-
trated in Scheme 3. Removal of the tosyl protecting group
As expected, acrylamide (T15 and T22) and α-haloke-
tones (T19, T20, T26, and T27) analogues retained excellent
inhibitory activities against JAK3 (Table 3). In
comparison with the corresponding purine derivatives, 7H-
in compound 11 and 14 was achieved under alkaline condi-
pyrrolo[2,3-d]pyrimidine derivatives significantly increased
the inhibitory potency, while 6,7-dihydro-5H-pyrrolo[2,3-
d]pyrimidine derivatives slightly decreased potency. It is
worth mentioning that compounds T15, T16, T19, and T20
JAK3 over 50% at 0.5 μM (Sup. Table S1), and their IC50
notably inhibited JAK2, (> 70% at 5 μM, see Sup. Table S1),
whichhasthehighesthomologytoJAK3[2,6,14]. Theresults
above suggest that compounds bearing a 7H-pyrrolo[2,3-
d]pyrimidine scaffold increase the inhibition of JAK3, as
ing electrophilic warheads. More importantly, those 1H-
well as JAK2.
tions, while the other reactions were similar to those shown
in Scheme 2.
Surprisingly, both compounds T30 and T33 inhibited
values were also more potent than 100 nM (Table 4). The
other three compounds (T29, T31 , and T32) exhibited better
inhibition of JAK3 than any other cores with the correspond-
pyrrolo[2,3-b]pyridine analogues all could not efficaciously
inhibit JAK2, with IC50 values greater than 5 μM. Taken
123

Molecular Diversity
Table 3 Structures and enzyme potencies of compounds T15–T28a,b
Comp.
R
JAK1 ICa50 (μM)
JAK2 ICa50 (μM)
> 0.5
JAK3 ICb50
TYK2 ICa50 (μM)
> 5
T15 [21]
> 5
0.77 0.04 nM
T16
> 5
> 0.5
23 0.11 nM
> 5
T17
T18
T19
T20
T21
NT
NT
> 5
> 5
NT
NT
> 0.5 μM
NT
NT
> 5
> 5
NT
NT
> 5 μM
> 0.5
> 0.5
NT
1.02 0.02 nM
0.93 0.01 nM
> 0.5 μM
T22
T23
> 5
> 5
> 5
> 5
3.5 0.09 nM
> 5
> 5
> 0.5 μM
T24
T25
T26
T27
T28
NT
NT
> 5
> 5
NT
NT
NT
> 5
> 5
NT
> 5 μM
NT
NT
> 5
> 5
NT
> 5 μM
2.7 0.10 nM
2.5 0.21 nM
> 0.5 μM
NT not tested
aCompounds were measured at concentrations of 5 and 0.5 μM with KmATP (30 nM JAK1 and 90 μM ATP; 2 nM JAK2 and 20 μM ATP; 15 nM
TYK2 and 16 μM ATP). Mean standard error of the mean (SEM), n = 2
b
IC50 values of JAK3 were an average of at least two independent dose–response curves at KmATP (4 nM JAK3 and 6.2 μM ATP)
together, these results suggest that compounds derived from
the 1H-pyrrolo[2,3-b]pyridine pharmacophore possessed
favorable inhibition potencies for JAK3 and high selectiv-
ity over other JAKs. Therefore, the SAR of pyrimidinyl
heterocyclic derivatives in suppressing JAK3 can be summa-
rized as follows: For the scaffold, the order of potency was
1H-pyrrolo[2,3-b]pyridine > 7H-pyrrolo[2,3-d]pyrimidine
> 9H-purine > 6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine;
for the linker, no significant bioactivity changes are observed,
whether there were substituents on the phenyl group or
123

Molecular Diversity
Scheme 3 General synthesis of 1H-pyrrolo[2,3-b]pyridine derivatives
T29–T33. Reagents and conditions: a aq NaOH, ACE, r.t., 0.5 h; b
Pd(dppf)Cl2, K2CO3, PhMe/EtOH, reflux, 3–4 h; c Pd/C, NH4COOH,
MeOH, reflux, 2 h; d MeOH/THF, Cs2CO3, r.t., overnight; e DMAP,
THF, r.t., 0.5 h; f DIEA, dry DMF, r.t., overnight; g HCl in dioxane,
MeOH, reflux, 1 h
Table 4 Structures and enzyme potencies of compounds T29–T33a,b
Comp.
T29
R
JAK1 ICa50 (μM)
JAK2 ICa50 (μM)
> 5
JAK3 ICb50 (nM)
0.14 0.02
TYK2 ICa50 (μM)
> 5
> 5
T30
> 5
> 5
14 0.15
> 5
T31
T32
T33
> 5
> 5
> 5
> 5
> 5
> 5
0.21 0.09
0.18 0.09
82 0.03
> 5
> 5
> 5
aCompounds were measured at concentrations of 5 and 0.5 μM with KmATP (30 nM JAK1 and 90 μM ATP; 2 nM JAK2 and 20 μM ATP; 15 nM
TYK2 and 16 μM ATP). Mean standard error of the mean (SEM), n = 2
bIC50 values of JAK3 were an average of at least two independent dose–response curves at KmATP (4 nM JAK3 and 6.2 μM ATP)
not; for the electrophilic warhead, the order of potency
was acrylamide ≈ α-haloketone > methacrylamide > 2-
chloropropanamide > (E)-but-2-enamide and 3-methylbut-
2-enamide.
Cys kinome profiling for selected compounds
JAK3 inhibitors T1, T15, and T29 were selected to con-
duct further investigations for kinase selectivity on the basis
of their structure features, bioisosteric scaffolds, and clas-
sical electrophilic warhead (acrylamide). Given that these
compounds were designed to form covalent bonds with
Cys909 in JAK3, kinase selectivity assays were carried out
for another nine protein kinases containing a cysteine in the
residueanalogoustoCys909inJAK3(Blk, Bmx, Btk, EGFR,
123

Molecular Diversity
Table 5 Cys kinome profiling
for select compounds
(IC50/nM)a,b
Comp. JAK3
Blk
Btka Bmx
EGFRa HER2a HER4a Itk
Tec
0.454b > 1a
0.890b 0.640b 1.000b
> 1a 0.466b > 1a
Rlk
T1
0.0026
> 1a
> 1
0.105b > 1
> 1
> 1
> 1
> 1
> 1
> 1
> 1a
T15
T29
0.00077 0.911b > 1
0.00014 > 1a
> 1
0.024b > 1
0.240b > 1
aEach enzymatic assay was performed in the presence of an ATP concentration at or close to the Km for ATP
bIC50 values were an average of two independent dose–response curves
Fig. 3 Inhibition of
carrageenan-induced paw edema
in ICR mice. Results are
expressed as the mean SD
(n = 6)
HER2, HER4, Itk, Rlk, Tec, see Sup. Table S2) [14,25]. As
summarized in Table 5, compounds T1 and T29 could not
potently inhibit enzymes that may be good targets for oncol-
ogy (EGFR, HER2, Blk) and immunological diseases (Btk,
Itk), with IC50 values greater than 1 μM (Sup. Table S3).
Although both compounds displayed similar inhibitory activ-
ities against Bmx and Tec, T29 possessed higher selectivity
for JAK3 (over 1700-fold) compared to T1 (over 40-fold).
Compound T15 inhibited more than half of the kinases of
the Cys kinome, especially Bmx, whose IC50 value was only
31-fold that for JAK3. Among these synthesized compounds,
T29 proved to be the optimal compound that possessed not
only the best inhibitory activity against JAK3, but also the
highest selectivity for other JAKs and kinases belonging to
the Cys kinome.
tion potency, T29 showed the best ability to inhibit increased
paw thickness, showing a 23.78% (p < 0.001) paw size
increase at the endpoint. T1 showed the worst inhibition of
edema formation with 36.21%, while T15 and the positive
control tofacitinib displayed 30.78% (p < 0.01) and 30.11%
(p < 0.01), respectively. Therefore, T29 is the most promis-
ing compound of the series with good anti-inflammatory
activity in vivo.
Covalent docking for selected compounds
To better understand the interaction models for selected com-
pounds in the JAK3 active site, we performed covalent in
silico docking of the inhibitors in the JAK3 kinase domain.
In the modeling of T29 bound to JAK3 (Fig. 4a), the acry-
lamidemoietyofT29wasfoundtoformaclearcovalentbond
with the sulfur atom in Cys909 of JAK3 as expected, which
is thought to make T29 highly selective against other JAKs.
In addition, T29 can also form two hydrogen bonds with
Glu903 and Leu905 in the hinge region. Similar results were
observed in Fig. 4b, and the overlay of selected inhibitors
T1, T15, and T29 covalently docked into JAK3 in the same
manner. Additionally, in the further analysis of the interac-
tion between JAK3 and T29, we found that T29 extended
into or near the protein’s hydrophobic region, possibly mak-
ing hydrophobic interactions with Ala853, Met902, Leu905,
Val884, and Leu828 residues (Fig. 4c). Consistent with the
order of inhibition potency, the hydrophobicity of scaffold
Evaluation of anti-inflammatory activity in ICR mice
Edema is an important and effective symptom of acute
inflammation used to evaluate potential compounds with
anti-inflammatory activities. In a carrageenan-induced paw
edema test (Fig. 3), the inflammatory response was quantified
by measuring the increase in paw size after carrageenan was
injected. The paw size in model group mice quickly increased
after injecting carrageenan and achieved a maximum at 3 h.
Following the oral administration of JAK3 inhibitors (dose:
30 mg/kg, 1 h), paw size began to decrease, in contrast to
model mice. Consistent with the order of enzyme inhibi-
123

Molecular Diversity
Fig. 4 Top view of selected compounds (T1, T15, and T29) binding
in the JAK3 kinase domain. a Compound T29 covalently docked into
JAK3. b Overlay of selected compounds covalently docked into JAK3
(T1: cyan; T15: green; T29: magenta). c T29 covalently docked into
JAK3, and analysis of the hydrophobic interaction
1H-pyrrolo[2,3-b]pyridine (T29) is higher than that of 7H-
pyrrolo[2,3-d]pyrimidine (T15), while 9H-purine (T1) has a
hydrophilic structure, explaining why T29 achieves a higher
degree of efficacy in inactivating JAK3 than T15 or T1. The
same explanation applies to other compounds with the same
electrophilic warhead and different scaffolds, such as T2,
T16, and T30.
Reversibility of T29 binding
To confirm that the optimal compound T29 was an irre-
versible inhibitor, jump dilution analysis [13,26] was used
to monitor the conversion of active JAK3 after it was
pre-incubated with the inhibitor and the equilibrium was
subsequently shifted by dilution to a state favoring inhibitor
123

Molecular Diversity
Sichuan, P.R. China, and they were directly used without any
purification. TLC was performed on 0.20 mm Silica gel 60
F254 plates (Qingdao Ocean Chemical Factory, Shandong,
China). Hydrogen nuclear magnetic resonance(1H NMR)
spectra were recorded at 400 MHz while carbon nuclear
magnetic resonance(13C NMR) spectra were recorded at
101 MHz on a Varian spectrometer (Varian, Palo Alto, CA)
model Gemini 400 and reported in parts per million. Chem-
ical shifts (δ) are quoted in ppm relative to tetramethylsilane
(TMS) as an internal standard, where (δ) TMS = 0.00 ppm.
The multiplicity of the signal is indicated as s, singlet; brs,
broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet,
defined as all multipeak signals where overlap or com-
plex coupling of signals makes definitive descriptions of
peaks difficult. High-resolution mass spectra (HRMS) were
measured by MALDI Q-ToF Premier mass spectrometer
(Micromass, Manchester, UK). Room temperature is within
the range 20–25◦C. The purity was analyzed by HPLC sys-
tem (Waters 2695, separations module) with a photodiode
array detector (Waters 2996, Milford, MA, USA), and the
chromatographic column was a reversed phase C18 column
(Waters, 150 mm × 4.6 mm, i.d. 5 μm). All compounds were
supplied in HPLC degree methanol with 10 μL, which was
injected into a partial loop, with isocratic elution with 70%
methanol and 30% water at a flow rate of 1 mL/min. The
purity of all tested compounds was ≥ 95% according to our
analytical HPLC method.
Fig. 5 T29 irreversibly inhibited JAK3. T29 with pre-incubation: com-
pound T29 was pre-incubated with AK3 for 30 min in the absence of
ATP, thendilutedwithkinasebufferthatcontainspeptidesubstrate, ATP,
and cofactors to the final concentration for reaction, and conversion data
were read on a caliper 20 times for 1 h
release. As displayed in Fig. 5, no active JAK3 was detected
after it was pre-incubated with T29 at each time point, iden-
tical to the situation in which there is essentially no enzyme,
suggesting that JAK3 was completely inhibited by T29.
Without being pre-incubated with T29, JAK3 was observed
to be active with a similar increasing trend to conversion with
no inhibitor compound. Taken together, T29 was identified
as an irreversible inhibitor.
General procedure for the preparation of compound 2
A suspension of 6-chloro-9H-purine (1, 7.73 g, 50 mmol)
and 4-methylbenzenesulfonic acid (130 mg, 0.75 mmol) in
EtOAc (100 mL) was treated with 3,4-dihydro-2H-pyran
(5.05 g, 60 mmol). The mixture was stirring and heating
at 90 ◦C, then the solid slowly dissolved over 2 h. The flask
was removed from the oil bath and the cloudy yellow reac-
tion mixture was filtered, the filtrate extracted with water
and brine in sequence, and the combined organic layers were
dried over anhydrous Na2SO4. The organic layer was con-
centrated in vacuo to afford desired product as yellow oil
Conclusion
In this study, we have described the identification and charac-
terization of a novel series of JAK3 inhibitors with different
bioisosteric scaffolds and electrophilic warheads. Biolog-
ical evaluation of these compounds confirmed that they
were selective JAK3 inhibitors; this finding, together with
the covalent docking results, revealed a clear SAR. Among
the compounds, T29 proved to be a promising irreversible
covalent JAK3 inhibitor that not only possessed the high-
est potency of inhibition for JAK3 and selectivity against
JAKs and kinases containing a cysteine in the residue anal-
ogous to Cys909 in JAK3, but also showed significant
anti-inflammatory activity in ICR mice by inhibiting the
increase in paw thickness. In conclusion, as a potent and
selective JAK3 inhibitor, T29 has the potential to be an effi-
cacious treatment for inflammatory diseases and is worth
further optimizing to improve its potency and medicinal
properties.
1
in a good yield of 92.3% (11.02 g). H NMR (400 MHz,
CDCl3 −d1)δ: 8.69 (s, 1H), 8.28 (s, 1H), 5.73 (dd, J = 10.4,
2.3 Hz, 1H), 4.16–4.09 (m, 1H), 3.73 (td, J = 11.6, 2.8 Hz,
1H), 2.11 (d, J = 10.8 Hz, 1H), 2.06–1.96 (m, 2H), 1.78–
1.67 (m, 2H), 1.62 (d, J = 8.0 Hz, 1H).
General procedure for the preparation of compounds 6 and
10
A mixture of 5 or 9 (1 eq) in THF was added DMAP (0.02
eq) and di-tert-butyl dicarbonate (1.1 eq) in sequence. The
reaction mixture was continued to stir at ambient temperature
for about 1 h and then evaporated in vacuo. The residue was
diluted with water and extracted with EtOAc. The combined
Experimental section
Chemistry Chemistry reagents of analytical grade were
purchased from Changzheng Chemical Factory, Chengdu,
123

Molecular Diversity
organic layers were washed with brine, dried over anhydrous
Na2SO4, and evaporated in vacuo. The crude product was
used in the next procedure without further purification.
J = 11.8 Hz, 1H), 3.76 (m, 1H), 2.38 (d, J = 12.1 Hz, 1H),
2.31 (s, 3H), 2.03 (t, J = 13.7 Hz, 2H), 1.77 (m, 1H), 1.63
(d, J = 11.4 Hz, 2H).
Tert-butyl
4-chloro-7H-pyrrolo[2,3-d]pyrimidine-7-
Tert-butyl
4-(3-nitrophenyl)-7H-pyrrolo[2,3-d]
1
carboxylate (6). White solid. Yield 94.8%. H NMR (400
MHz, CDCl3-d1)δ: 8.78 (s, 1H), 7.64 (d, J = 4.1 Hz,
1H),6.60 (d, J = 4.1 Hz, 1H), 1.62 (s, 9H).
pyrimidine-7-carboxylate (7). Yellow solid. Yield 67.7%. 1H
NMR (400 MHz, DMSO-d6)δ: 9.13 (s, 1H), 8.87 (s, 1H),
8.57 (d, J = 7.7 Hz, 1H), 8.44 (d, J = 8.1 Hz, 1H), 8.04
(d, J = 4.1 Hz, 1H), 7.92 (t, J = 8.0 Hz, 1H), 7.19 (d,
J = 4.1 Hz, 1H), 1.65 (s, 9H).
Tert-butyl
4-(3-aminophenyl)-7H-pyrrolo[2,3-d]
pyrimidine-7-carboxylate (10). White solid. Yield 72.3%.
1H NMR (400 MHz, DMSO-d6)δ: 8.99 (s, 1H), 7.92 (d,
J = 4.1 Hz, 1H), 7.36 (s, 1H), 7.23 (d, J = 4.9 Hz, 2H),
7.04 (d, J = 4.1 Hz, 1H), 6.75 (dd, J = 7.3, 3.8 Hz, 1H),
5.39 (s, 2H), 1.64 (s, 9H).
4-(3-Nitrophenyl)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine
(13). Yellowsolid. Yield60.8%. 1HNMR(400MHz, CDCl3-
d1)δ: 8.56 (s, 1H), 8.46 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H),
8.13 (d, J = 7.7 Hz, 2H), 7.93 (d, J = 7.2 Hz, 1H), 7.85
(d, J = 3.7 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.31 (d,
J = 8.0 Hz, 3H), 6.73 (d, J = 3.7 Hz, 1H), 2.39 (s, 3H).
General procedure for the preparation of compound 12
At about 0 ◦C, NaOH (2 mol/L in water, 4.8 g, 120 mmol)
was added to a solution of 4-chloro-1H-pyrrolo[2,3-b]
pyridine (15.2 g, 100 mmol) and 4-methylbenzene-1-sulfony
chloride (19.95 g, 105 mmol) in acetone. The resulting solu-
tion was stirred at ambient temperature for about 6 h. The
solids were collected by filtration and washed with ace-
tone/water to give the title product as white solid in a yield of
General procedure of nitro reduction for the preparation of
compounds 4, 8, 9, and 14
To the Suzuki coupling product (1 eq) and ammonium for-
mate (3 eq) in MeOH was added 10% Pd/C (0.5%w). Then,
the mixture was stirred under nitrogen at 80 ◦C for 2 h. The
mixture was filtered with celite and washed with MeOH. The
filtrate was concentrated in vacuo. The residue was dissolved
in water, extracted with EtOAc, brine, and dried over anhy-
drous Na2SO4. The organic layer was evaporated in vacuo,
and the residue was purified on a silica gel column to give
the corresponding product.
1
88.1% (26.92 g). H NMR (400 MHz, DMSO-d6) δ: 8.35
(d, J = 5.2 Hz, 1H), 8.04 (d, J = 4.0 Hz, 1H), 8.01
(d, J = 8.1 Hz, 2H), 7.47 (d, J = 5.2 Hz, 1H), 7.43 (d,
J = 8.1 Hz, 2H), 6.88 (d, J = 4.0 Hz, 1H), 2.35 (s, 3H).
General procedure of Suzuki coupling for the preparation of
compounds 3, 7, and 13
3-(7-(Tetrahydro-2H-pyran-2-yl)-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)aniline (4-1), Yellow solid. Yield 62.5%. 1H
NMR (400 MHz, DMSO-d6)δ: 8.95 (s, 1H), 8.84 (s, 1H),
8.06 (s, 1H), 8.04 (d, J = 1.1 Hz, 1H), 7.22 (t, J = 7.9 Hz,
1H), 6.79*–6.71 (m, 1H), 5.82 (d, J = 9.5 Hz, 1H), 5.32 (s,
2H), 4.05 (d, J = 9.0 Hz, 1H), 3.74 (td, J = 11.3, 4.1 Hz,
1H), 2.42–2.30 (m, 1H), 2.08–1.97 (m, 2H), 1.84–1.71 (m,
1H), 1.63 (d, J = 11.8 Hz, 2H).
Toluene/EtOH (7/3, v/v) was added to an nitrogen-purged
flask containing the halide intermediate (1 eq), PaCl2(dppf)
(0.05 eq), K2CO3 (3 eq), and appropriate boronic acid (1.1
eq), and then the mixture was stirred under nitrogen at
◦
80 C for 3–4 h. After cooling to ambient temperature, the
mixture was filtered with celite and concentrated in vacuo.
The residue was dissolved in water, extracted with EtOAc,
washed with brine, and dried over anhydrous Na2SO4. The
organic layer was evaporated in vacuo, and the residue was
purified on a silica gel column to give desired product.
6-(3-Nitrophenyl)-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine (3-1). Yellow solid. Yield 65.7%. 1H NMR (400 MHz,
CDCl3-d1)δ: 9.74 (d, J = 1.8 Hz, 1H), 9.22 (d, J = 7.9 Hz,
1H), 9.07 (s, 1H), 8.41 (s, 1H), 8.39–8.35 (m, 1H), 7.75 (t,
J = 8.0 Hz, 1H), 5.88 (dd, J = 10.3, 2.5 Hz, 1H), 4.05 (d,
J = 9.0 Hz, 1H), 3.74 (td, J = 11.3, 4.1 Hz, 1H), 2.42–
2.30 (m, 1H), 2.08–1.97 (m, 2H), 1.84–1.71 (m, 1H), 1.63
(d, J = 11.8 Hz, 2H).
6-(4-Methyl-3-nitrophenyl)-9-(tetrahydro-2H-pyran-2-
1
yl)-9H-purine (4-2). Yellow solid. Yield 45.7%. H NMR
(400 MHz, DMSO-d6)δ: 8.92 (s, 1H), 8.83 (s, 1H), 8.07
(d, J = 8.9 Hz, 2H), 7.12 (d, J = 7.7 Hz, 1H), 5.81 (d,
J = 10.6 Hz, 1H), 5.13 (s, 2H), 4.04 (d, J = 11.9 Hz, 1H),
3.74 (td, J = 11.4, 4.3 Hz, 1H), 2.35 (dd, J = 17.6, 6.4 Hz,
1H), 2.31 (s, 3H), 2.02 (d, J = 10.8 Hz, 2H), 1.77 (m, 1H),
1.63 (d, J = 11.5 Hz, 2H).
3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)aniline (8). White
solid. Yield 30.1%. 1H NMR (400 MHz, DMSO-d6)δ: 12.16
(s, 1H), 8.77 (s, 1H), 7.95 (s, 1H), 7.44 (s, 1H), 7.32 (d,
J = 7.5 Hz, 1H), 7.20 (t, J = 7.7 Hz, 1H), 6.85 (d,
6-(4-Methyl-3-nitrophenyl)-9-(tetrahydro-2H-pyran-2-yl) J = 2.3 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 5.29 (s, 2H).
-9H-purine (3-2). Yellow solid. Yield 63.2%. 1H NMR
(400 MHz, DMSO-d6)δ: 9.47 (d, J = 2.0 Hz, 1H), 9.13
(dd, J = 8.6, 2.0 Hz, 1H), 9.10 (s, 1H), 9.03 (s, 1H), 8.06
(d, J = 8.5 Hz, 1H), 5.87 (d, J = 10.9 Hz, 1H), 4.07 (d,
Tert-butyl4-(3-aminophenyl)-5H-pyrrolo[2,3-d]pyrimid-
1
ine-7(6H)-carboxylate (9). White solid. Yield 37.4%. H
NMR (400 MHz, DMSO-d6)δ: 8.69 (s, 1H), 7.20 (s, 1H),
7.15 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 6.67
123

Molecular Diversity
(d, J = 7.4 Hz, 1H), 5.29 (s, 2H), 3.95 (t, J = 8.4 Hz, 2H),
3.28 (t, J = 8.4 Hz, 2H), 1.51 (s, 9H).
N-(3-(9H-Purin-6-yl)phenyl)acrylamide (T1). White
solid. HPLC purity97.8%. Yield42.8%. 1HNMR(400MHz,
DMSO-d6)δ: 10.49 (s, 1H), 8.99 (s, 2H), 8.71 (s, 1H), 8.58
(d, J = 6.7 Hz, 1H), 8.00 (d, J = 7.3 Hz, 1H), 7.56 (t,
J = 7.7 Hz, 1H), 6.53 (dd, J = 16.4, 9.8 Hz, 1H), 6.31
(d, J = 16.7 Hz, 1H), 5.79 (d, J = 9.9 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6) δ: 163.84, 154.59, 151.77, 146.21,
139.93, 135.66, 132.35, 129.60, 127.49, 125.33, 122.59,
120.59. HRMS (ESI), m/z: 266.1034 [M + H]+.
3-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)aniline(14).
1
White solid. Yield 56.9%. H NMR (400 MHz, CDCl3 −
d1)δ: 8.44 (d, J = 4.9 Hz, 1H), 8.10 (d, J = 8.1 Hz, 2H),
7.74 (d, J = 3.9 Hz, 1H), 7.29 (s, 3H), 7.20 (d, J = 4.8 Hz,
1H), 7.00 (d, J = 7.5 Hz, 1H), 6.93 (s, 1H), 6.81 (d,
J = 7.9 Hz, 1H), 6.78 (d, J = 3.9 Hz, 1H), 2.37 (s, 3H).
N-(3-(9H-Purin-6-yl)phenyl)methacrylamide(T2). White
solid. HPLC purity98.3%. Yield42.2%. 1HNMR(400MHz,
DMSO-d6)δ: 10.07 (s, 1H), 9.01 (s, 1H), 8.99 (s, 1H), 8.72
(s, 1H), 8.59 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H),
7.55 (t, J = 7.9 Hz, 1H), 5.90 (s, 1H), 5.56 (s, 1H), 1.99 (s,
3H). HRMS (ESI), m/z: 302.1019 [M + Na]+.
General Procedure for the preparation of compound 15
A solution of 14 (1.09 g, 3 mmol) in MeOH/THF (3/1, v/v,
26 mL) was added Cs2CO3 (1.47 g, 4.5 mmol) under stirring
at ambient temperature, and then the reaction mixture was
stirred overnight. The solvent was removed by evaporation.
TheresiduewasdilutedwithwaterandextractedwithEtOAc.
The combined organic layers were washed with brine, dried
over anhydrous Na2SO4, and evaporated in vacuo. The crude
intermediate was directly used to react with di-tert-butyl
dicarbonate, just like the preparation for compound 6, and
ultimately giving target compound 15 as white solid in a yield
of40.5%(375. 7mg). 1HNMR(400MHz, DMSO-d6)δ:8.44
(d, J = 4.9 Hz, 1H), 7.74 (d, J = 3.9 Hz, 1H), 7.29 (s, 1H),
7.20 (d, J = 4.8 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.93 (s,
1H), 6.81 (d, J = 7.9 Hz, 1H), 6.78 (d, J = 3.9 Hz, 1H),
1.64 (s, 9H).
(E)-N-(3-(9H-Purin-6-yl)phenyl)but-2-enamide (T3).
1
White solid. HPLC purity 98.6%. Yield 38.6%. H NMR
(400 MHz, DMSO-d6)δ: 10.24 (s, 1H), 8.93 (s, 1H), 8.89
(s, 1H), 8.67 (s, 1H), 8.47 (d, J = 7.5 Hz, 1H), 7.91 (d,
J = 7.7 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 6.78 (dd,
J = 14.9, 6.9 Hz, 1H), 6.16 (d, J = 15.1 Hz, 1H), 1.83
(d, J = 6.5 Hz, 3H). HRMS (ESI), m/z: 302.1013 [M +
Na]+.
N-(3-(9H-Purin-6-yl)phenyl)-3-methylbut-2-enamide
1
(T4). White solid. HPLC purity 96.2%. Yield 39.7%. H
NMR (400 MHz, DMSO-d6)δ: 10.12 (s, 1H), 8.97 (s, 1H),
8.95 (s, 1H), 8.68 (s, 1H), 8.55 (d, J = 7.7 Hz, 1H), 7.91
(d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 5.95 (s, 1H),
2.19 (s, 3H), 1.89 (s, 3H). HRMS (ESI), m/z: 316.1173 [M
+ Na]+.
General Procedure for the preparation of compounds T1–
T33
A solution of amino intermediate (1 eq) and DIEA (3 eq) in
anhydrous DMF was added dropwise to acyl chloride deriva-
tive (1.2 eq) under stirring at ambient temperature, and then
the reaction mixture was stirred overnight. The reaction mix-
ture was quenched with water and extracted with EtOAc.
The organic extract was washed with water and brine, dried
over anhydrous Na2SO4. The solvent was removed in vacuo
to yield the crude product for the next step without further
purification.
N-(3-(9H-Purin-6-yl)phenyl)-2-chloroacetamide (T5).
White solid. HPLC purity 96.2%. Yield 35.1%. H NMR
(400 MHz, DMSO-d6)δ: 13.66 (s, 1H), 10.57 (s, 1H), 8.97
(s, 2H), 8.67 (s, 2H), 7.89 (d, J = 8.1 Hz, 1H), 7.56 (t,
J = 8.0 Hz, 1H), 4.31 (s, 2H). HRMS (ESI), m/z: 310.0473
[M + Na]+.
1
N-(3-(9H-Purin-6-yl)phenyl)-2-bromoacetamide (T6).
1
Light yellow solid. HPLC purity 96.5%. Yield 34.8%. H
NMR (400 MHz, DMSO-d6)δ: 10.64 (s, 1H), 9.01 (s, 1H),
8.98 (s, 1H), 8.72 (s, 1H), 8.60 (d, J = 7.9 Hz, 1H), 7.89 (d,
J = 7.1 Hz, 1H), 7.56 (d, J = 7.1 Hz, 1H), 4.32 (s, 2H).
HRMS (ESI), m/z: 354.0277 [M + Na]+.
General procedure for THP-deprotection and Boc-
deprotection
N-(3-(9H-Purin-6-yl)phenyl)-2-chloropropanamide
(T7). White solid. HPLC purity 97.1%. Yield 35.1%. H
1
4M HCl in 1,4-dioxane (3 eq) was added to a stirred solution
of appropriate intermediate (1 eq) in MeOH at amb◦ient tem-
perature. The resulting solution was heated to 75 C under
stirring for about 1 h, and the solvent was removed under
reduced pressure. The residue was dissolved in water and
slowly basified with 10% NaOH solution to pH = 7, then
extracted with EtOAc, washed with water and brine, and
dried with anhydrous Na2SO4. The solvent was removed in
vacuo, and the crude product was purified by silica gel col-
umn to give target compound.
NMR (400 MHz, DMSO-d6)δ: 13.66 (s, 1H), 10.59 (s, 1H),
8.98 (d, J = 9.1 Hz, 2H), 8.68 (s, 2H), 7.90 (d, J = 8.0 Hz,
1H), 7.57 (t, J = 7.9 Hz, 1H), 4.75 (dd, J = 13.2, 6.6 Hz,
1H), 1.65 (d, J = 6.6 Hz, 3H). HRMS (ESI), m/z: 302.0830
[M + H]+.
N-(2-Methyl-5-(9H-purin-6-yl)phenyl)acrylamide (T8).
1
White solid. HPLC purity 98.9%. Yield 35.7%. H NMR
(400 MHz, DMSO-d6)δ: 9.75 (s, 1H), 8.94 (s, 1H), 8.92
(s, 1H), 8.65 (s, 1H), 8.62 (d, J = 8.1 Hz, 1H), 7.46 (d,
123

Molecular Diversity
J = 8.1 Hz, 1H), 6.59 (dd, J = 17.1, 10.5 Hz, 1H), 6.29
(dd, J = 17.0, 2.0 Hz, 1H), 5.79 (dd, J = 10.2, 1.9 Hz, 1H),
2.31 (s, 3H). HRMS (ESI), m/z: 302.1017 [M + Na]+.
N-(2-Methyl-5-(9H-purin-6-yl)phenyl)methacrylamide
N-(3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)
methacrylamide (T16). White solid. HPLC purity 98.1%.
1
Yield 32.2%. H NMR (400 MHz, DMSO-d6)δ: 12.95 (s,
1H), 10.18 (s, 1H), 9.01 (s, 1H), 8.61 (s, 1H), 7.93 (d,
J = 6.5 Hz, 2H), 7.85 (d, J = 7.9 Hz, 1H), 7.61 (t,
J = 8.0 Hz, 1H), 7.11 (s, 1H), 5.90 (s, 1H), 5.60 (s, 1H),
1.99 (s, 3H). HRMS (ESI), m/z: 301.1014 [M + Na]+.
(E)-N-(3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)but-
2-enamide (T17). White solid. HPLC purity 98.9%. Yield
1
(T9). White solid. HPLC purity 98.1%. Yield 36.0%. H
NMR (400 MHz, DMSO-d6)δ: 9.60 (s, 1H), 8.94 (s, 1H),
8.74 (s, 1H), 8.66 (d, J = 6.0 Hz, 2H), 7.47 (d, J = 8.1 Hz,
1H), 5.92 (s, 1H), 5.54 (s, 2H), 2.28 (s, 3H), 2.00 (s, 3H).
HRMS (ESI), m/z: 332.1417 [M + K]+.
(E)-N-(2-Methyl-5-(9H-purin-6-yl)phenyl)but-2-enami-
de (T10). White solid. HPLC purity 97.9%. Yield 33.8%. 1H
NMR (400 MHz, DMSO-d6)δ: 9.53 (s, 1H), 8.93 (s, 1H),
8.90 (s, 1H), 8.65 (s, 1H), 8.60 (d, J = 7.9 Hz, 1H), 7.44 (d,
J = 8.1 Hz, 1H), 6.85–6.79 (m, 1H), 6.27 (d, J = 15.6 Hz,
2H), 2.30 (s, 3H), 1.89 (d, J = 6.8 Hz, 3H). HRMS (ESI),
m/z: 316.1170 [M + Na]+.
1
31.4%. H NMR (400 MHz, DMSO-d6)δ: 13.30 (s, 1H),
10.60 (s, 1H), 9.08 (s, 1H), 8.65 (s, 1H), 8.02 (d, J = 2.4 Hz,
1H), 7.91 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H),
7.63 (t, J = 7.9 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 6.87 (dq,
J = 13.8, 6.8 Hz, 1H), 6.25 (dd, J = 15.2, 1.5 Hz, 1H), 1.90
(dd, J = 6.8, 1.1 Hz, 2H). HRMS (ESI), m/z: 279.0935 [M
+ H]+.
3-Methyl-N-(2-methyl-5-(9H-purin-6-yl)phenyl)but-2-
enamide (T11). White solid. HPLC purity 98.0%. Yield
33.5%. 1H NMR (400 MHz, DMSO-d6)δ: 9.44 (s, 1H), 8.99
(s, 1H), 8.85 (s, 1H), 8.74 (s, 1H), 8.55 (d, J = 7.9 Hz, 1H),
7.45 (d, J = 8.0 Hz, 1H), 6.04 (s, 1H), 2.31 (s, 3H), 2.17 (s,
3H), 1.89 (s, 3H). HRMS (ESI), m/z: 330.1387 [M + Na]+.
2-Chloro-N-(2-methyl-5-(9H-purin-6-yl)phenyl)
N-(3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-3-
methylbut-2-enamide (T18). White solid. HPLC purity
99.0%. Yield 35.0%. 1H NMR (400 MHz, DMSO-d6)δ:
13.32 (s, 1H), 10.40 (s, 1H), 9.08 (s, 1H), 8.59 (d, J =
16.9 Hz, 1H), 8.05–8.00 (m, 1H), 7.88 (d, J = 8.1 Hz, 1H),
7.79 (d, J = 7.9 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.14
(d, J = 2.2 Hz, 1H), 5.98 (s, 1H), 2.19 (s, 3H), 1.90 (s, 3H).
HRMS (ESI), m/z: 293.1214 [M + H]+.
acetamide (T12). White solid. HPLC purity 95.7%. Yield
1
30.0%. H NMR (400 MHz, DMSO-d6)δ: 13.63 (s, 1H),
N-(3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-2-
chloroacetamide (T19). White solid. HPLC purity 97.1%.
9.89 (s, 1H), 8.92 (s, 1H), 8.88 (s, 1H), 8.69 (d, J = 7.9 Hz,
1H), 8.63 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 4.36 (s, 2H),
2.30 (s, 3H). HRMS (ESI), m/z: 324.0633 [M + Na]+.
2-Bromo-N-(2-methyl-5-(9H-purin-6-yl)phenyl)
1
Yield 29.7%. H NMR (400 MH, DMSO-d6)δ: 12.30 (s,
1H), 10.56 (s, 1H), 8.84 (s, 1H), 8.54 (s, 1H), 7.94 (d,
J = 7.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.70 (s, 1H),
7.55 (t, J = 7.9 Hz, 1H), 6.94 (s, 1H), 4.32 (s, 2H). HRMS
(ESI), m/z: 309.0512 [M + Na]+.
acetamide (T13). White solid. HPLC purity 97.0%. Yield
1
30.7%. H NMR (400 MHz, DMSO-d6)δ: 13.62 (s, 1H),
9.89 (s, 1H), 8.92 (s, 1H), 8.87 (d, J = 9.8 Hz, 1H), 8.69 (d,
J = 8.2 Hz, 1H), 8.63 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H),
4.36 (s, 2H), 2.30 (s, 3H). HRMS (ESI), m/z: 368.0125 [M
+ Na]+.
N-(3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-2-
bromoacetamide (T20). Light yellow solid. HPLC purity
95.4%. Yield 29.7%. 1H NMR (400 MHz, DMSO-d6)δ:
12.31 (s, 1H), 10.57 (s, 1H), 8.84 (s, 1H), 8.78 (s, 1H), 8.55
(s, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H),
7.71 (s, 1H), 7.55 (t, J = 7.9 Hz, 1H), 6.95 (s, 1H), 4.32 (s,
2H). HRMS (ESI), m/z: 353.0007 [M + Na]+.
2-Chloro-N-(2-methyl-5-(9H-purin-6-yl)phenyl)
propanamide (T14). White solid. HPLC purity 96.9%. Yield
1
27.4%. H NMR (400 MHz, DMSO) 13.63 (s, 1H), 9.94
δ
(s, 1H), 8.93 (s, 1H), 8.82 (s, 1H), 8.67 (d, J = 22.0 Hz,
2H), 7.47 (d, J = 7.8 Hz, 1H), 4.82 (q, J = 6.6 Hz, 1H),
2.30 (s, 3H), 1.68 (d, J = 6.7 Hz, 3H). HRMS (ESI), m/z:
338.0782 [M + Na]+.
N-(3-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)-2-
chloropropanamide (T21). White solid. HPLC purity 97.2%.
Yield 30.2%. H NMR (400 MHz, DMSO-d6)δ: 12.35 (s,
1H), 10.63 (s, 1H), 8.90 (s, 1H), 8.64 (s, 1H), 8.01 (d,
J = 7.9 Hz, 1H), 7.87–7.80 (m, 1H), 7.76 (s, 1H), 7.61
(s, 1H), 7.01 (dd, J = 3.5, 1.6 Hz, 1H), 4.78 (q, J = 6.6 Hz,
1H), 1.72 (d, J = 6.7 Hz, 3H). HRMS (ESI), m/z: 301.1402
[M + H]+.
1
N-(3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)
acrylamide (T15). White solid. HPLC purity 99.1%. Yield
1
32.5%. H NMR (400 MHz, DMSO-d6)δ: 12.28 (s, 1H),
10.39 (s, 1H), 8.84 (s, 1H), 8.64 (s, 1H), 7.92 (d, J = 7.8 Hz,
1H), 7.82 (d, J = 8.1 Hz, 1H),7.75–7.67 (m, 1H), 7.54
(t, J = 7.9 Hz, 1H), 6.96 (dd, J = 3.5, 1.7 Hz, 1H),
6.48 (dd, J = 17.0, 10.1 Hz, 1H), 6.32 (dd, J = 17.0,
2.0 Hz, 1H), 5.80 (dd, J = 10.0, 1.9 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6)δ: 164.16, 152.86, 151.83, 146.76,
140.37, 132.22, 130.33, 127.86, 124.58, 123.09, 120.61,
115.06, 102.73. HRMS (ESI), m/z: 287.0907 [M + Na]+.
N-(3-(6,7-Dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)
phenyl)acrylamide (T22). White solid. HPLC purity 98.3%.
1
Yield 33.4%. H NMR (400 MHz, DMSO-d6)δ: 10.79 (s,
1H), 9.83 (s, 1H), 8.60 (s, 1H), 8.30 (s, 1H), 7.82 (d,
J = 8.0 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.49 (d,
J = 7.8 Hz, 1H), 6.56 (dd, J = 17.0, 10.2 Hz, 1H), 6.30
(dd, J = 17.0, 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 1.8 Hz, 1H),
123

Molecular Diversity
3.86 (t, J = 8.3 Hz, 2H), 3.36 (t, J = 8.3 Hz, 2H). HRMS
(ESI), m/z: 267.0638 [M + H]+.
8.35 (d, J = 4.9 Hz, 1H), 8.29 (s, 1H), 7.81–7.76 (m, 1H),
7.66–7.61 (m, 1H), 7.59–7.51 (m, 2H), 7.25 (d, J = 4.9 Hz,
1H), 6.76 (dd, J = 3.4, 1.8 Hz, 1H), 6.53 (dd, J = 17.0,
10.1 Hz, 1H), 6.36 (dd, J = 17.0, 2.0 Hz, 1H), 5.85 (dd,
J = 10.1, 2.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
δ: 168.68, 163.83, 149.67, 143.38, 140.43, 139.9, 139.34 (s,
1H), 129.94, 127.14, 123.50, 119.47, 117.64, 114.41, 99.55.
HRMS (ESI), m/z: 264.1132 [M + H]+.
N-(3-(6,7-Dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)
phenyl)methacrylamide (T23). White solid. HPLC purity
98.3%. Yield 33.7%. 1H NMR (400 MHz, DMSO-d6)δ:
10.13 (s, 1H), 9.72 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 7.83
(d, J = 8.1 Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 7.47
(d, J = 7.8 Hz, 1H), 5.86 (s, 2H), 5.59 (s, 1H), 3.85 (t,
J = 8.1 Hz, 2H), 3.34 (t, J = 8.1 Hz, 2H), 1.97 (s, 3H).
HRMS (ESI), m/z: 319.1315 [M + K]+.
(E)-N-(3-(6,7-Dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-
yl)phenyl)but-2-enamide (T24). White solid. HPLC purity
98.5%. Yield 32.9%. 1H NMR (400 MHz, DMSO) δ: 10.63
(s, 1H), 9.88 (s, 1H), 8.60 (s, 1H), 8.29 (s, 1H), 7.82 (d,
J = 7.8 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.47 (d,
J = 7.8 Hz, 1H), 6.84 (dd, J = 15.2, 6.9 Hz, 1H), 6.25
(dd, J = 15.2, 1.5 Hz, 1H), 3.86 (t, J = 8.1 Hz, 2H), 3.35
(t, J = 8.2 Hz, 2H), 1.90–1.84 (m, 3H). HRMS (ESI), m/z:
281.1402 [M + H]+.
N-(3-(6,7-Dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)
phenyl)-3-methylbut-2-enamide (T25). White solid. HPLC
purity 98.0%. Yield 31.5%. 1H NMR (400 MHz, DMSO-d6)
δ: 10.27 (s, 1H), 9.82 (s, 1H), 8.62 (s, 1H), 8.24 (s, 1H), 7.71
(d, J = 8.1 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.41 (d,
J = 8.0 Hz, 1H), 5.92 (s, 1H), 3.86 (t, J = 8.4 Hz, 2H),
3.33 (t, J = 8.3 Hz, 2H), 2.17 (s, 3H), 1.89 (s, 3H). HRMS
(ESI), m/z: 295.0978 [M + H]+.
N-(3-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)
methacrylamide (T30). White solid. HPLC purity 98.7%.
1
Yield 34.8%. H NMR (400 MHz, DMSO-d6)δ: 11.86 (s,
1H), 10.03 (s, 1H), 8.34 (d, J = 4.8 Hz, 1H), 8.28 (s, 1H),
7.83 (d, J = 7.1 Hz, 1H), 7.63 (s, 1H), 7.59–7.49 (m, 2H),
7.25 (d, J = 4.8 Hz, 1H), 6.78 (s, 1H), 5.91 (s, 1H), 5.61 (s,
1H), 2.04 (s, 3H). HRMS (ESI), m/z: 278.1296 [M + H]+.
N-(3-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)-2-
chloroacetamide (T31). White solid. HPLC purity 97.0%.
1
Yield 35.0%. H NMR (400 MHz, DMSO-d6) δ: 11.83 (s,
1H), 10.51 (s, 1H), 8.30 (d, J = 4.9 Hz, 1H), 8.14 (s, 1H),
7.66 (d, J = 7.1 Hz, 1H), 7.59–7.56 (m, 1H), 7.53–7.50 (m,
2H), 7.20 (d, J = 4.9 Hz, 1H), 6.71–6.66 (m, 1H), 4.31 (s,
2H). HRMS (ESI), m/z: 286.0750 [M + H]+.
N-(3-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)-2-
bromoacetamide (T32). White solid. HPLC purity 96.3%.
1
Yield 30.9%. H NMR (400 MHz, DMSO-d6) δ: 11.79 (s,
1H), 10.51 (s, 1H), 8.29 (d, J = 4.9 Hz, 1H), 8.19 (s,
1H), 7.74 (d, J = 6.6 Hz, 1H), 7.59–7.53 (m, 1H), 7.48
2-Chloro-N-(3-(6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- (d, J = 6.3 Hz, 2H), 7.18 (d, J = 4.9 Hz, 1H), 6.71 (s, 1H),
4-yl)phenyl)acetamide (T26). White solid. HPLC purity
4.31 (s, 2H). HRMS (ESI), m/z: 330.0240 [M + H]+.
N-(3-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)-2-
1
96.1%. Yield 31.5%. H NMR (400 MHz, DMSO-d6) δ:
10.55 (s, 1H), 8.23 (s, 1H), 7.41 (s, 1H), 7.19 (s, 1H), 7.11
(t, J = 7.5 Hz, 1H), 7.00 (d, J = 7.1 Hz, 1H), 6.63 (d, J =
7.0 Hz, 1H), 5.20 (s, 2H), 3.56 (t, J = 8.3 Hz, 2H), 3.25 (t,
J = 8.3 Hz, 2H). HRMS (ESI), m/z: 311.1133 [M + Na]+.
chloropropanamide (T33). White solid. HPLC purity 96.9%.
Yield 29.1%. H NMR (400 MHz, DMSO-d6) δ: 11.82 (s,
1H), 10.58 (s, 1H), 8.30 (s, 0H), 8.16 (s, 0H), 7.68 (s, 1H),
1
7.58 (s, 1H), 7.51 (d, J = 5.5 Hz, 1H), 7.20 (s, 0H), 6.69 (s,
2-Bromo-N-(3-(6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin- 0H), 4.73 (s, 1H), 1.64 (d, J = 6.2 Hz, 1H). HRMS (ESI),
4-yl)phenyl)acetamide (T27). White solid. HPLC purity
m/z: 322.0721 [M + Na]+.
1
95.4%. Yield 28.5%. H NMR (400 MHz, DMSO-d6) δ:
Enzymatic assays The enzymatic activities against JAK1,
JAK2, JAK3, and TYK2 were assessed using the Caliper
Mobility Shift Assay in the presence of Km ATP (ChemPart-
ner, Shanghai, China). The assay protocol guide is commer-
cially available from ChemPartner. The inhibitory activities
against cysteine kinome were tested by Eurofins Cerep
(CEREP, Celle l’Evescault, France). The protocols are avail-
able from http://www.cerep.fr/Cerep/Users/index.asp. The
assay protocol guide can be accessed at www.eurofins.com/
discovery.
10.55 (s, 1H), 8.23 (s, 1H), 7.39 (s, 1H), 7.18 (s, 1H), 7.10
(t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.62 (d,
J = 9.5 Hz, 1H), 5.18 (s, 2H), 3.55 (t, J = 8.3 Hz, 2H),
3.25 (t, J = 8.3 Hz, 2H). HRMS (ESI), m/z: 333.0384 [M
+ H]+.
2-Chloro-N-(3-(6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-
4-yl)phenyl)propanamide (T28). White solid. HPLC purity
1
96.7%. Yield 24.9%. H NMR (400 MHz, DMSO-d6) δ:
10.53 (s, 1H), 8.27 (d, J = 4.2 Hz, 2H), 7.71 (d, J = 8.0 Hz,
1H), 7.65 (d, J = 7.9 Hz, 1H), 7.54 (s, 1H), 7.45 (t,
J = 7.9 Hz, 1H), 4.71 (q, J = 6.6 Hz, 1H), 3.60 (t,
J = 8.4 Hz, 2H), 3.31 (d, J = 8.5 Hz, 2H), 1.63 (d,
J = 6.6 Hz, 3H). HRMS (ESI), m/z: 325.0836 [M + Na]+.
N-(3-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)acrylamide
Evaluation of anti-inflammatory activity in ICR mice ICR
mice were randomized into five groups (n = 6) and labeled
with picric acid. Mice in treatment groups were orally admin-
istered with corresponding inhibitors at a single dose of
30 mg/kg. After 30 min, all mice were injected carrageenan
and this time point was recorded as 0 h. The data of paw size
1
(T29). White solid. HPLC purity 99.3%. Yield 33.9%. H
NMR (400 MHz, DMSO-d6)δ: 11.87 (s, 1H), 10.40 (s, 1H),
123

Molecular Diversity
were measured and collected at 0, 1, 2, 3, 4, and 5 h, and then
the data were described as a figure.
3. Jasuja H, Chadha N, Kaur M, Silakari O (2014) Dual inhibitors
of Janus kinase 2 and 3 (JAK2/3): designing by pharmacophore-
and docking-based virtual screening approach. Mol Divers 18:253–
267. https://doi.org/10.1007/s11030-013-9497-z
JAK3 reversibility experiments Jump dilution [13,26] was
used to assess the reversibility of T29 binding to JAK3. Pre-
pare enzyme mixes in 1× kinase buffer (50 Mm HEPES,
pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 Mm DTT) and
incubate enzyme mix at r.t. for 30 min: Enzyme mix 1 con-
tains 75X JAK3 (300 nM) of final concentration in reaction;
enzyme mix 2 contains 75X JAK3 (300 nM) and compound
T29 at high concentration 20X IC50 (2.8 nM). Prepare sub-
strate mix in 1× kinase buffer: Substrate mix 1 contains
peptide substrate, ATP, and cofactors at the final concentra-
tion for reaction; substrate mix 2 contains peptide substrate,
ATP, and cofactors at the concentration for reaction with
extremely low concentration 20/74X IC50 of compound T29
(0.037 nM). Transfer 1 μL enzyme mix and 74 μL substrate
mix to the 384-well assay plate: no compound (enzyme mix
1 + substrate mix 1); T29 with pre-incubation (enzyme mix
2 + substrate mix 1); T29 without pre-incubation (enzyme
mix 1 + substrate mix 2); no enzyme (buffer + substrate
mix 1). Then, continually read each well for 20 times for 1
h on caliper, collect JAK3 conversion data, and describe as a
figure.
4. Farmer LJ, Ledeboer MW, Hoock T, Arnost MJ, Bethiel RS, Ben-
nani YL, Black JJ, Brummel CL, Chakilam A, Dorsch WA, Fan
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Covalent in silico docking The crystal structure of the JAK3
kinase domain was obtained from the Protein Data Bank
(PDB code 1YVJ) [27]. Water molecules were removed,
and hydrogen atoms were added to the structure. Three-
dimensional structure of the compounds were generated and
optimized by the Discovery Studio 3.1 package (Accelrys,
San Diego, CA, USA). The small molecule was docked into
the JAK3 kinase domain using the Covalent Dock Cloud
server (http://docking.sce.ntu.edu.sg) [28]. Figure 4 was gen-
erated using Pymol 1.7.
11. Flanagan ME, Blumenkopf TA, Brissette WH, Brown MF, Casa-
vant JM, Shang-Poa C, Doty JL, Elliott EA, Fisher MB, Hines
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C, Sun J, Whipple DA, Changelian PS (2010) Discovery of CP-
690,550: a potent and selective Janus kinase (JAK) inhibitor for the
treatment of autoimmune diseases and organ transplant rejection.
J Med Chem 53:8468–8484. https://doi.org/10.1021/jm1004286
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Nelles L, Smets B, Lepescheux L, Christophe T, Conrath K, Van-
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13. Kulagowski JJ, Blair W, Bull RJ, Chang C, Deshmukh G, Dyke
HJ, Eigenbrot C, Ghilardi N, Gibbons P, Harrison TK, Hewitt PR,
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Kohli P, Maxey RJ, Mendonca R, Mortara K, Murray J, Narukulla
R, Shia S, Steffek M, Ubhayakar S, Ultsch M, van Abbema A,
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55:5901–5921. https://doi.org/10.1021/jm300438j
Acknowledgements The authors greatly appreciate the financial sup-
portfromtheNationalNaturalScienceFoundationofChina(81373260).
Compliance with ethical standards
Conflict of interest The authors have declared no conflict of interest.
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