Design and Synthesis of a Highly Selective JAK3 Inhibitor for the Treatment of Rheumatoid Arthritis

Article abstract of DOI:10.1002/ardp.201700194

Selective inhibition of Janus kinase 3 (JAK3) has been identified as an important strategy for the treatment of autoimmune disorders. Based on the unique cysteine 909 residue (Cys909) of JAK3 at the gatekeeper position, we have developed a new irreversible covalent inhibitor, III-4, which is highly potent and selective in targeting JAK3. Importantly, III-4 selectively inhibited JAK3 (IC₅₀ = 57 ± 1.21 nM) over other JAKs (IC₅₀ > 10 μM) and Cys909 kinome members (IC₅₀ > 1 μM). A cellular selectivity study also confirmed that III-4 preferentially inhibited JAK3 over JAK1 in JAK/STAT signaling. Moreover, the fact that III-4 covalently modified the Cys909 residue in JAK3 was clearly validated by mass spectrometry and covalent docking analysis. Based on the favorable target profiles, the pharmacokinetic properties and its low toxicity, III-4 exhibited better efficacy than tofacitinib in impeding disease progression in CIA mice, without any significant adverse effects. Taken together, III-4 is a potent, selective, and durable inhibitor of JAK3 and has the potential for the treatment of inflammatory disorders and autoimmune diseases, such as rheumatoid arthritis.

Full text of DOI:10.1002/ardp.201700194

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Full Paper
Design and Synthesis of a Highly Selective JAK3 Inhibitor for the
Treatment of Rheumatoid Arthritis
Linhong Heꢀ, Heying Peiꢀ, Tingxuan Lan, Minghai Tang, Chufeng Zhang, and Lijuan Chen
Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu,
Sichuan, P. R. China
Selective inhibition of Janus kinase 3 (JAK3) has been identified as an important strategy for the
treatment of autoimmune disorders. Based on the unique cysteine 909 residue (Cys909) of JAK3 at
the gatekeeper position, we have developed a new irreversible covalent inhibitor, III-4, which is
highly potent and selective in targeting JAK3. Importantly, III-4 selectively inhibited JAK3 (IC50
57 ꢁ 1.21 nM) over other JAKs (IC50 > 10 mM) and Cys909 kinome members (IC50 > 1 mM). A cellular
selectivity study also confirmed that III-4 preferentially inhibited JAK3 over JAK1 in JAK/STAT
signaling. Moreover, the fact that III-4 covalently modified the Cys909 residue in JAK3 was clearly
¼
validated by mass spectrometry and covalent docking analysis. Based on the favorable target profiles,
the pharmacokinetic properties and its low toxicity, III-4 exhibited better efficacy than tofacitinib in
impeding disease progression in CIA mice, without any significant adverse effects. Taken together,
III-4 is a potent, selective, and durable inhibitor of JAK3 and has the potential for the treatment of
inflammatory disorders and autoimmune diseases, such as rheumatoid arthritis.
Keywords: Covalent inhibitor / JAK3 / Rheumatoid arthritis / Selectivity
Received: June 18, 2017; Revised: September 2, 2017; Accepted: September 5, 2017
DOI 10.1002/ardp.201700194
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
target owing to its essential functions and immunosuppres-
sive effects in cytokine signaling networks and evidences by
Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory joint
disease accounting for 1% of the world population, which is
characterized by circulating autoantibodies, synovial inflam-
mation, pannus formation, cartilage and bone damage, and
eventually leads to substantial loss of functioning and
mobility if not adequately treated [1, 2]. During the multi-
step pathogenesis of RA, any factor like immune effector cells,
pro-inflammatory cytokines, chemokines and proteinases,
could be considered as a potential treatment paradigm for
RA [1, 2]. Among them, Janus kinase 3 (JAK3) is an attractive
tofacitinib, which presents good treatments for patients with
moderate to severe RA with an inadequate response to or
intolerance of methotrexate [3, 4].
JAK3 is a member of the JAK family of non-receptor
tyrosine kinases, which contains JAK1, JAK2, and TYK2 [5–9].
JAK3, expressing in hematopoietic tissues, is solely activated
by type I cytokine receptors featuring a common g-chain (gc)
subunit that was activated by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-
21, then leads to the phosphorylation and activation of signal
transducer and activator of transcription (STAT) proteins, and
regulates target gene expression that governs cell develop-
ment, survival, differentiation, and proliferation in immune
system [6, 7]. Deficiency of JAK3 is associated with a specific
severe combined immunodeficiency (SCID) phenotype that
Correspondence: Dr. Lijuan Chen, Cancer Center, West China
Hospital, Sichuan University and Collaborative Innovation Center,
Chengdu, Sichuan 610041, P. R. China.
E-mail: chenlj125@163.com
Fax: þ86-28-85164060
ꢀThese two authors contributed equally to this work.
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manifests as depletion of T cells, NK cells and impairment of B
cells [3, 6, 7]. Given that JAK3-SCID patients have defects but
only limited to immune cells, and JAK3 deletion in mice
manifests as only in defective immune function, selective
Goedken et al. [18] (Fig. 1 ). Moreover, Pfizer also has reported
a series of irreversibly covalent JAK3-selective inhibitors and
identified PF-06651600 as the best one with overall properties
suitable for clinical trial [19, 20]. Encouraged by those
achievements, we focused structural modifications on
tofacitinib in an attempt to improve its kinase selectivity
and pharmacodynamics properties. Here, we describe com-
pound III-4, of which acrylamide binds to JAK3 irreversibly by
targeting the Cys909 residue in the ATP binding site via
covalent bond formation (Fig. 2). Similarly, III-4 also can form
two key hydrogen bond interactions with Glu-903 and Leu-
905 in the hinge region, respectively. Importantly, III-4 not
only preferentially inhibits JAK3 over the other JAKs, but also
exhibits favorable pharmacokinetic properties making it
suitable for oral dosing and excellent anti-rheumatic effect
in collagen-induced arthritis (CIA) mice model, suggesting
that III-4 had the potential to be an efficacious treatment
for RA.
targeting of JAK3 has been identified as
a potential
mechanism to treat RA [3, 4, 10].
As JAKs share high homology (50–60%) in the entire kinase
domain, particularly in the enzyme active sites (JAK3/JAK1
ꢂ 84%, JAK3/JAK2 ꢂ 87%, JAK3/TYK2 ꢂ 80%), selective tar-
geting on JAK3 with traditional ATP-competitive inhibitors
has been proven to be difficult [11, 12]. Tofacitinib, a first-
in-class JAK3 inhibitor (IC50 ¼ 1 nM), was approved by the US
Food and Drug Administration (FDA) for treatment of
rheumatoid arthritis in 2012 [13]. However, it still shows
limited selectivity for JAK1 and JAK2 with IC50 values of 112
and 20 nM, respectively, which accounts for the correspond-
ing side effects during clinical treatment [10, 14]. Another
JAK3 inhibitor, decernotinib, has been promoted into the
clinical trial of RA in Phase 2/3, also being identified as a pan-
JAK inhibitor (Ki ¼ 11, 13, 2.5, and 13 nM for JAK1, JAK2, JAK3,
and TYK2, respectively) [15]. That is to say, there are rarely
highly selective JAK3 inhibitors in clinical stages to date [20].
Thus, to alleviate the undesirable side effects of JAK1 and
JAK2 inhibition such as dyslipidemia and suppression of
hematopoiesis, exploration of highly selective JAK3 inhibitors
would still be in urgent need [3, 10, 16].
Results and discussion
Chemistry
Comparison to the complex synthetic procedures of tofaci-
tinib [21] and PF-06651600 [19], III-4 was simply prepared
according to five common reactions in
a good yield
Different from other JAK family members, JAK3 is unique in
having a cysteine residue (Cys909) at the ATP binding
pocket [11, 17, 18]. Cys909 can be used to confer selectivity
JAK3 versus JAK1, JAK2, and TYK2 by targeting and forming
of a covalent binding. Employing this mechanism, several
researchers have disclosed their studies on JAK3 irreversible
inhibitors, such as Smith et al. [17], Tan et al. [11], and
(Scheme 1). Commercially available 6-chloro-9H-purine (1)
was first protected by 3,4-dihydro-2H-pyrane to give 2, which
was allowed to react with (R)-tert-butyl-3-aminopiperidine-1-
carboxylate completely to provide 3 in a high yield about
90%. The secondary arylamino group of 3 was methylated
with methyl iodide to offer 4, whose THP protecting group
was removed in an acidic condition, giving the key
Figure 1. Examples of reported JAK3
inhibitors.
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Figure 2. Structural modifications from tofacitinib based on the interaction with JAK3 protein. This chart illustrates the interaction
of JAK3 with tofacitinib (left) and III-4 (right).
intermediate 5 as hydrochloride form. Final target compound
III-4 was obtained by reacting 5 with acryloyl chloride in dry
DMF.
to the concentration of 10000 nM, indicating that III-4 owned
high kinase selectivity within JAKs, which was better than
tofacitinib.
Biological evaluation
Enzyme potencies
Intact mass spectrometry to validate the covalent binding
position of III-4 and JAK3
Compound III-4 was firstly assayed with enzyme potencies
against JAKs at a series of concentrations. As illustrated in
Fig. 3, III-4 significantly inhibited JAK3 with an IC50 of
57 ꢁ 1.21 nM, but did not potently suppress the other JAKs up
To further pin down the exact binding site of JAK3 and III-4
based on their covalent docking result (Fig. 2), recombinant
JAK3 kinase was incubated with III-4, subsequently protease
digested and the resulting mixture was subjected to LC-MS/
Scheme 1. General synthesis of compound III-4. Reagents and conditions: (a) TsOH, AcOEt, reflux, 2 h; (b) DMF, Et3N, 80°C, 5 h; (c)
CH3I, Cs2CO3, KOH, DMF, 80°C, 5 h; (d) HCl in 1,4-dioxane, r.t., overnight; (e) acryloyl chloride, DIEA, dry DMF, r.t., 2 h.
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Figure 3. Dose–response curves showing
effects of III-4 on different JAKs. IC50 values
were an average of at least two independent
dose–response curves at Km ATP for each JAK
isoform (30 nM JAK1 and 90 mM ATP; 2 nM
JAK2 and 20 mM ATP; 4 nM JAK3 and 6.2 mM
ATP; 15 nM TYK2 and 16 mM ATP).
MS analysis. The m/z of the Cys909-containing peptide
LVMEYLPSGCLR (JAK3 residues 900–901) showed a mass
increase of 286.02 Da, which was consistent with the
calculated value for the addition of one III-4 molecule to
the peptide (Fig. 4). Further fragmentation of this peptide
produced a series of b/y-ion fragments, and only the Cys909-
containing fragments (y3–y10, marked in red) displayed a
286.02 Da mass shift, suggesting Cys909 is the covalently
modified site. Cumulatively, these results above strongly
demonstrated that compound III-4 was an irreversible
covalent inhibitor, and Cys909 of JAK3 was its only labeled
site.
enzymes that may be good targets for immunological diseases
(Btk, Itk, and Tec). By contrast to the IC50 of JAK3, the
selectivity ratios against these kinases might approach to or
be greater than 20-fold for compound III-4. That is to say,
cysteine targeting by covalent binding allows a JAK3 inhibitor
to achieve favorable selectivity within the JAK family
members as well as Cys909 kinome.
Cellular selectivity assay for III-4
Several cellular assays set-ups were applied to elucidate III-4’s
potency of inhibiting JAK and selectivity profile in a cellular
environment. III-4 firstly tested the inhibition of phosphor-
ylation of downstream signal STAT proteins in human
peripheral blood mononuclear cells (PBMCs) [24]. The
different cellular stimuli used induce phosphorylation of
STATs (pSTAT) by either dual JAK1/3 (IL-2 stimulus, STAT5
phosphorylation), JAK2 (GM-CSF stimulus, STAT5 phosphor-
ylation), or multiple JAK1/JAK2/TYK2 (IL-6 stimulus, STAT3
phosphorylation) containing pathways [6, 7, 25]. As exhibited
in Table 2, III-4 showed a 14.7-fold selectivity for inhibition of
the IL-2 readout (IC50 ¼ 40.19 nM) versus the IL-6 readout
Cys909 kinome profiling for III-4
Given that III-4 was identified to covalently bind with Cys909
in JAK3, the inhibitory activity of III-4 to other tyrosine kinases
(Blk, Btk, Bmx, Itk, Tec, EGFR, ErbB2, ErbB4, and Rlk) that
contain a modifiable cysteine residue at an analogous
position to Cys909 of JAK3 should be assessed [22, 23]. As
shown in Table 1, III-4 displayed poor potencies against these
kinases with no inhibition exceeding 40% at 1 mM, including
Figure 4. Mass spectra identified covalent binding site between JAK3 peptide and III-4. LC-MS/MS analysis of the Cys909-containing
tryptic peptide after recombinant JAK3 was incubated with III-4, followed by trypsin digestion. The asterisk indicates the residue
bound by III-4; red labels show ions that display a mass shift corresponding to III-4.
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Table 1. The inhibition % of other kinases that carry homologous cysteine residue to Cys909 in JAK3 with III-4a).
Comp.
III-4
JAK3
Blk
Btk
Bmx
Itk
Tec
EGFR
ErbB2
ErbB4
Rlk
99.9 ꢁ 1.3 24.4 ꢁ 3.5 15.6 ꢁ 3.1 39.3 ꢁ 2.0 20.8 ꢁ 1.8 31.9 ꢁ 1.1 12.4 ꢁ 2.9 2.0 ꢁ 0.9 32.5 ꢁ 2.8 34.8 ꢁ 4.0
a) III-4 was measured at the concentration of 1 mM in the presence of 10 mM ATP, and all inhibition data were an average of at
least two test values, mean ꢁ SD.
(IC50 ¼ 591.6 nM), and a 12.2-fold selectivity for inhibition of
the GM-CSF readout (IC50 ¼ 492 nM). Tofacitinib displayed
better potencies but lower selectivity than III-4 of inhibiting
the relevant phosphorylation of STATs in the parallel assays.
Hence, compound III-4 preferentially inhibited JAK3 over
JAK1 in JAK/STAT signaling pathway in PBMC.
Sprague-Dawley rats [26]. Besides, the oral bioavailability of
tofacitinib (F% ¼ 27%) is also not very favorable [26], which is
almost the half of the III-4’s. Although III-4 less potently
inhibits JAK3 than tofacitinib, we believe that its favorable
oral bioavailability and half-life will contribute to enhance its
efficacy in vivo.
In order to re-confirm the selectivity of III-4 for JAK3 versus
JAK1, it was further to examine the inhibition of STAT
phosphorylation induced by varying cytokines in THP-1 and
TF-1 cells. Comparing the results measured in the IL-6/pSTAT3
and IL-4/pSTAT6 assays, III-4 displayed better potent inhibition
of JAK1/JAK3 than tofacitinib in THP-1 cells, but less of JAK1 in
TF-1 cells (Fig. 5). Additionally, we also test the inhibition of
JAK auto-phosphorylation by III-4 in those two cell lines. As
expected, III-4 inhibited p-JAK1 in a dose-dependent manner
upon stimulations by IL-4 and IL-6, but showed a higher
degree against p-JAK3 for IL-4 readout (Fig. 6). Taking the
results of PBMC, III-4 could be concluded that its inhibition of
JAK/STAT signaling was more inclined to JAK3 inhibition.
Since III-4 was designed as a covalent inhibitor, the
toxicity assessment cannot be ignored or avoided. III-4
cannot potently inhibit the proliferation of LO2 cells
(IC50 ¼ 259.1 ꢁ 10.3 mM) and HEK293 cells (IC50 ¼ 154.3 ꢁ 7.3
mM). Similar results were observed from tofacitinib with
IC50 values of 287.7 ꢁ 9.5 mM and 173.8 ꢁ 8.1 mM, respec-
tively. In brief, III-4 exhibited extremely low hepatotoxicity
and nephrotoxicity in cellular tests. Apart from cytotoxicity
assays, acute toxicity test of III-4 also had to be carried out
to find the oral LD50 value. According to the kinase potency
of III-4, five oral single-dose concentrations were set up as
200, 400, 600, 800, and 1000 mg/kg. The mortality increased
with the increasing III-4 dose and treatment time. After the
oral administration III-4 of 800 mg/kg, six female mice died
at about 36 h and two male mice died before 48 h, and all
mice died after administration of 1000 mg/kg III-4 during
48 h. Although mice with oral dose of 600 mg/kg III-4 still
survived at the end study period (7 d), abnormal behaviors
still were observed, such as hair-raising, body weight loss,
and trembling. The other two doses, all mice were active
and healthy, with no signs of toxicity, adverse pharmaco-
logical effects or abnormal behaviors. Those results
concluded the maximum tolerated dose (MTD) of III-4
was about 600 mg/kg, suggesting that III-4 had a reasonable
safety treatment window.
Pharmacokinetic properties and toxicity assay
Subsequently, III-4 was continued to investigate its pharma-
cokinetic properties by LC-MS. As expected, III-4 exhibited
moderate clearance (33.83 L/h/kg), long half-life (11.05 h), and
a favorable oral bioavailability (56.97%), which met with
features of covalent inhibitors. The oral peak time Tmax was
0.23 h (13.8 min), indicating that III-4 could quickly reach the
peak concentration Cmax (273.38 mg/L) and produce thera-
peutic effects (Table 3). As reported, intravenous dosing
of tofacitinib results in a high plasma clearance (CL ¼ 6.2 mL/
min/kg) and an extremely short half-life (t1/2 ¼ 0.6 h) in
Table 2. Potency and selectivity determination of III-4 in human PBMCa).
IC50/nM
Selectivity
Tofacitinib
JAKs involved
Trigger
Readout
III-4
Tofacitinib
III-4
JAK1/JAK3
JAK1/JAK2/TYK2
JAK2
IL-2
IL-6
GM-CSF
pSTAT5
pSTAT3
pSTAT5
40.19
591.6
492
24.66
35.48
227
–
–
14.7
12.2
1.44
9.2
a) IC50 values in human PBMC were determined by plotting the compound concentration versus the effect on the readouts using
flow cytometry. Each value represented the average of ꢃ2 independent experiments, and each experiment consisted of two
replicates. (JAK1/JAK2/TYK2)/(JAK1/JAK3), JAK2/(JAK1/JAK3) selectivity were determined by comparing potencies measured in
the IL-6/pSTAT3 and IL-2/pSTAT5 assays, GM-CSF/pSTAT5 and IL-6/pSTAT3 assays, respectively.
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Figure 5. Dose-dependent inhibitions of cytokine-induced STAT phosphorylation by III-4 and tofacitinib were determined by
measuring STAT phosphorylation by Quantity One software based on the stripe gray value of Western blot in THP-1 (A and B) and
TF-1 (C and D) cells.
Figure 6. Dose-dependent inhibitions of cytokine-induced JAK auto-phosphorylation by III-4 in THP-1 (A) and TF-1 (B) cells.
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Table 3. Pharmacokinetic parameters of III-4 determined in Sprague-Dawley ratsa).
Dose
(mg/kg)
Cmax
(mg/L)
AUC(0-1)
CLz/F
(L/h/kg)
Vz/F
(L/kg)
Tmax (h)
(mg/Lꢀh)
t1/2 (h)
F (%)
i.v.
p.o.
5
10
614.03
273.38
0.08
0.23
278.19
308.99
9.05
11.05
18.49
33.83
230.07
559.04
56.97
a) Pharmacokinetics measured by analysis of plasma concentrations at indicated time points. Data were mean concentration in
plasma (n ¼ 5) following a single 5.0 mg/kg intravenous dose and 10 mg/kg oral dose.
Anti-arthritic activity evaluation of III-4 in CIA model
To characterize the effect of III-4 on the treatment of
autoimmune disease, we established CIA model in mice to
assess its anti-arthritic activity. On the basis of the pharmaco-
kinetic profiling of III-4 in rats, CIA mice were oral adminis-
tered 30 mg/kg III-4 per day, which was much lower than its
LD50 value. After immunization with collagen, the first clinical
signs of arthritis were apparent on day 21. All vehicle control
mice developed serious arthritis by day 35 and reached a
clinical score of 8.6 ꢁ 0.70. As shown in Fig. 7A, treatment with
tofacitinib and III-4 at respective dose of 30 mg/kg both
suppressed arthritis development without any significantly
changes of body weight (Supporting Information Fig. S2).
Notably, III-4 more significantly improved pathology form of
joints and completely abrogated disease development with a
clinical score of 4.3 ꢁ 0.21 in comparison to tofacitinib of
6.0 ꢁ 0.33. Similar results were seen from the pictures of hind
paws (Fig. 7B, upper).
synovium and periarticular tissue with inflammatory cells,
extensive cartilage damage (chondrocyte loss and collagen
matrix destruction), presence of osteoclasts, and bone
irreversible damage. In contrast, treatment with III-4 and
tofacitinib showed significant alleviation in inflammation,
cartilage, and bone damage. Notably, mice were almost
recovered to normal state after treatment with III-4 as
exhibited by H&E staining and Safranin-O staining (Fig. 7B).
Since III-4 could not potently inhibit enzymes that may be
good targets for immunological diseases, such as JAK1, Btk,
and Itk, the excellent anti-arthritic effect of III-4 was definitely
associated with its covalent features.
Although III-4 did not show better efficacies in inhibiting
JAK3 in vitro than tofacitinib and PF-06651600, it displayed a
significant and promising therapeutic effect in CIA mice in
vivo owing to its covalent features, which was even better
than tofacitinib. Particularly worth mentioning was that III-4
was simply prepared from commercially available materials
according to five common reactions that were easy to
industrialize.
Histopathological analysis of the untreated mice ankle
revealed severe changes, including massive infiltration of the
Figure 7. III-4 improves clinical symptoms in developing disease in a mice CIA model. Arthritis was induced in the mice immunization
with type II collagen. The animals were randomized according to body weight, and oral treatment with III-4 (30 mg/kg) or tofacitinib
(30 mg/kg) was initiated on day 36; n ¼ 8 for all group. (A) Clinical arthritic scores in each group, III-4 treatment leads to a significant
lower arthritis score than recorded for the model group. ꢀP < 0.05; ꢀꢀP < 0.01; ꢀꢀꢀP < 0.001 versus model control. (B) Hind paws of each
group of mice after treatment with III-4 and tofacitinib; histological analysis of mice ankle was performed with H&E staining and
Safranin-O staining.
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rate of 1 mL/min. The purity of all tested compounds was
ꢃ95% according to our analytical HPLC method.
Conclusion
We have identified III-4 as an irreversible covalent inhibitor
that was highly potent and selective targeting for JAK3. III-4
selectively inhibited JAK3 with an IC50 of 57 ꢁ 1.21 nM, but
not other JAKs to a concentration of 10 mM and Cys909
kinome to a concentration of 1 mM. Cellular selectivity study
also confirmed that III-4 preferentially inhibited JAK3 rather
than JAK1 in JAK/STAT signaling. Although the selectivity of
III-4 was obtained by its irreversible covalent binding with
JAK3’s Cys909, their interaction mode was clearly verified by
mass spectrum and covalent docking software. Moreover,
III-4 revealed low cytotoxicity in LO2 and HEK293 cells, as
well as a reasonable MTD and safety treatment window in
mice. Based on the favorable target profiles and pharmaco-
kinetic properties, III-4 exhibited wonderful therapeutic
effect of reducing the disease progression, paw swelling,
bone and cartilage degradation in CIA mice, and almost
recovered to normal state. Besides, no adverse effects were
General procedure for the preparation of compound 2
A suspension of 6-chloro-9H-purine (1, 7.73g, 50 mmol, eq) and
4-methylbenzenesulfonic acid (130mg, 0.75 mmol, 0.01eq) in
EtOAc (100 mL) was treated with 3,4-dihydro-2H-pyran (5.05 g,
60 mmol, 1.eq). The mixture was heated at 90°C and the solid
slowly dissolved over 2 h. The flask was removed from the oil
bath and the cloudy yellow solution was filtered, and the
filtrate was water and brine, dried over anhydrous Na2SO4. The
organic layer wasconcentratedin vacuo to afford the product 2
as yellow solid in a good yield of 91.0%.
1H NMR (400 MHz, CDCl3-d1): d 8.69 (s, 1H), 8.28 (s, 1H), 5.73
(dd, J ¼ 10.4, 2.3 Hz, 1H), 4.16–4.09 (m, 1H), 3.73 (td, J ¼ 11.6,
2.8 Hz, 1H), 2.11 (d, J ¼ 10.8 Hz, 1H), 2.06–1.96 (m, 2H),
1.78–1.67 (m, 2H), 1.62 (d, J ¼ 8.0 Hz, 1H). MS (ESI), m/z:
239.20 [MþH]þ.
observed during the treatment period. As
a
durable
General procedure for the preparation of compound 3
irreversible covalent inhibitor, III-4 is a potent, selective
JAK3 inhibitor and has the potential to be an efficacious
treatment for RA by oral administration, which is different
from pan-JAK inhibitors with undesirable side effects caused
by inhibiting JAK1 or/and JAK2. Furthermore, III-4 is ready to
be investigated in other animal modes of autoimmunity/
inflammation diseases.
A mixture of 2 (3.81 g, 16 mmol, 1 eq), (R)-tert-butyl
3-aminopiperidine-1-carboxylate (3.84 g, 19.2 mmol, 1.2 eq),
and DIEA (5.3 mL, 32 mmol, 2 eq) in 30 mL DMF was heated at
80 °C for 5 h. The DMF was removed by in vacuo and the
residue was diluted with water, extracted with ethyl acetate.
The organic layer was collected and washed with brine, dried
over anhydrous Na2SO4, and concentrated under vacuum to
give yellow oil. Target compound 3 was purified by column
chromatography on silica gel with gradient PE/EtOAc (80:20
to 20:80, v/v) eluent.
Experimental
1H NMR (400 MHz, DMSO-d6): d 8.36 (s, 1H), 8.23 (s, 1H), 7.61
(s, 1H), 5.63 (d, J ¼ 10.4 Hz, 1H), 4.00 (d, J ¼ 12.5 Hz, 1H),
3.72–3.62 (m, 1H), 2.81 (s, 1H), 2.27 (dd, J ¼ 26.5, 15.2 Hz, 2H),
2.00–1.88 (m, 3H), 1.80–1.53 (m, 6H), 1.38–1.33 (d, J ¼ 19.7 Hz,
13H). MS (ESI), m/z: 403.13 [MþH]þ.
Chemistry
General
Chemistry reagents of analytical grade were purchased from
Changzheng Chemical Factory, Chengdu, Sichuan, P. R. China.
TLC was performed on 0.20 mm Silica Gel 60 F254 plates
(Qingdao Ocean Chemical Factory, Shandong, China). Hydro-
gen nuclear magnetic resonance (1H NMR) spectra were
recorded at 400 MHz on a Varian spectrometer (Varian, Palo
Alto, CA) model Gemini 400 and reported in parts per million.
Chemical shifts (d) are quoted in ppm relative to tetrame-
thylsilane (TMS) as an internal standard, where (d) TMS ¼ 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 multi peak signals where overlap
or complex coupling of signals makes definitive descriptions
of peaks difficult. Mass spectra (MS) were measured by Q-TOF
Premier mass spectrometer (Micromass, Manchester, UK).
Room temperature is within the range 20–25°C. The purity
was analyzed by HPLC system (Waters 2695, separations
module) with a photodiode array detector (Waters 2996,
Milford, MA), and the chromatographic column was a
reversed phase C18 column (Waters, 150 mm ꢄ 4.6 mm, i.d.
5 mm). All compounds were supplied in HPLC degree metha-
nol with 10 mL, which was injected on a partial loop, with
isocratic elution with 70% methanol and 30% water at a flow
General procedure for the preparation of compound 4
A mixture of 3 (4.02 g, 10 mmol, 1 eq), KOH (0.84 g, 15 mmol,
1.5 eq), and Cs2CO3 (4.87 g, 15 mmol, 1.5 eq) in 40 mL DMF was
stirred at ice bath. Potassium iodide was slowly added
dropwise over 30 min. Then the mixture was heated at
40°C for another 5 h and cooled to room temperature. Water
was added to dilute the mixture, and then extracted with
ethyl acetate. The organic layer was collected and washed
with brine, dried over anhydrous Na2SO4, and concentrated
under vacuum to give yellow oil. Target compound 4 was
purified by column chromatography on silica gel with
gradient PE/EtOAc (80:20 to 20:80, v/v) eluent.
1H NMR (400 MHz, DMSO-d6): d 8.37 (s, 1H), 8.26 (s, 1H),
5.70–5.62 (m, 1H), 4.00 (dd, J ¼ 22.3, 13.0 Hz, 3H), 3.68 (td,
J ¼ 11.3, 4.5 Hz, 1H), 3.37 (s, 3H), 3.00 (s, 1H), 2.69 (d,
J ¼ 12.1 Hz, 1H), 1.92 (d, J ¼ 15.6 Hz, 2H), 1.86–1.70 (m, 4H),
1.58 (s, 2H), 1.38 (s, 12H). 1H NMR (400 MHz, CDCl3-d1): d 8.38
(s, 1H), 7.94 (s, 1H), 5.74 (dd, J ¼ 10.5, 2.1 Hz, 1H), 4.16 (dd,
J ¼ 11.5, 1.7 Hz, 2H), 3.78 (td, J ¼ 11.6, 2.4 Hz, 1H), 3.44 (s, 3H),
2.91 (t, J ¼ 10.7 Hz, 1H), 2.64 (t, J ¼ 11.2 Hz, 1H), 2.11–2.04 (m,
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2H), 1.99 (dd, J ¼ 14.3, 3.7 Hz, 2H), 1.81–1.73 (m, 4H), 1.45 (d,
J ¼ 1.3 Hz, 12H). MS (ESI), m/z: 417.20 [MþH]þ.
human IL-6 (400 ng/mL; R&D Systems), recombinant human
IL-2 (20 ng/mL; R&D Systems), recombinant human GM-CSF
(100 ng/mL; Pepro-Tech), or vehicle (PBS plus 0.1% [w/v]
BSA) at 37°C for 20 min and treated with pre-warmed
13 lysis/fix buffer (BD Biosciences) to lyse RBCs and
fix leukocytes. Cells were permeabilized with 100%
MeOH and incubated with anti-pSTAT3 and anti-CD4 (IL-
6-triggered samples), anti-pSTAT5 and anti-CD4 Abs
(IL-2-triggered samples), or anti-pSTAT5 Abs (GM-
CSF-triggered samples) (all Abs were from BD Biosciences)
at 4°C for 30 min, washed once with PBS, and analyzed on a
FACS Canto II flow cytometer.
General procedure for the preparation of compound 5
HCl (4 M) in 1,4-dioxane (3 eq) was added to a stirred
solution of 4 (1 eq) in 1,4-dioxane at ambient temperature
overnight. The solvent was removed under reduced
pressure and gave the crude product as hydrochloride,
which was used in the next step without any purification.
MS (ESI), m/z: 233.18 [MþH]þ.
General procedure for the preparation of compound III-4
A solution of 5 (102.0 mg, 0.3 mmol, 1 eq) and DIEA (149 mL,
0.9 mmol, 3 eq) in anhydrous DMF was added dropwise
acryloyl chloride (25.8 mg, 0.28 mmol, 0.95 eq) at ice bath with
stirring 1 h. The reaction mixture was quenched with water
and extracted with DCM. The organic extract was washed
with brine, dried over anhydrous Na2SO4, and concentrated
under vacuum to give yellow oil. Target compound III-4 was
purified by column chromatography on silica gel with
gradient DCM/MeOH (100:0 to 94:6, v/v) eluent.
STAT6 phosphorylation induced by IL-4 in THP-1 cells: THP-1
cells (ATCC TIB-202) were preincubated with a series concen-
trations of III-4 (0.1, 0.4, 1, 4, and 10 mM) at 37°C for 1 h,
followed incubated with IL-4 (10 ng/mL) at 37°C for another
1 h. Cells were collected and processed for Western blot,
pSTAT6 was detected with mouse anti-pSTAT6 Ab (BD
Biosciences). The quantitative analysis of pSTAT6 was used
Quantity One software based on the stripe gray value of
Western blot.
(R)-1-(3-(Methyl(9H-purin-6-yl)amino)piperidin-1-yl)prop-
2-en-1-one (III-4)
STAT3 phosphorylation induced by IL-6 in TF-1 cells: TF-1 cells
were cultured in media for 18 h without GM-CSF prior to
being plated in 24-well plates. III-4 with a series concen-
trations (0.1, 0.4, 1, 4, and 10 mM) were preincubated with
cells at 37°C for 30 min, followed incubated with IL-6 (400 ng/
mL) at 37°C for another 30 min. Cells were collected and
processed for Western blot, pSTAT3 was detected with mouse
anti-pSTAT3 Ab (BD Biosciences). The quantitative analysis of
pSTAT3 was used Quantity One software based on the stripe
gray value of Western blot.
White solid. Yield 81.9%. M.p.: 183.0–183.3°C. HPLC purity
98.8%; 1H NMR (400 MHz, DMSO-d6) d 13.03 (s, 1H), 8.22 (s,
1H), 8.11 (s, 1H), 6.88–6.77 (m, 1H), 6.10 (dd, J ¼ 16.6, 6.6 Hz,
1H), 5.65 (dd, J ¼ 29.7, 10.2 Hz, 1H), 4.46 (d, J ¼ 10.9 Hz, 1H),
4.07 (d, J ¼ 10.7 Hz, 1H), 3.34 (s, 3H), 3.01 (t, J ¼ 13.2 Hz, 1H),
2.92 (t, J ¼ 10.4 Hz, 1H), 2.56 (t, J ¼ 14.0 Hz, 1H), 1.99–1.82 (m,
3H), 1.55–1.44 (m, 1H). 1H NMR (400 MHz, CDCl3-d) d 8.38 (s,
1H), 7.91 (s, 1H), 6.80–6.59 (m, 1H), 6.30 (t, J ¼ 15.5 Hz, 1H),
5.68 (dd, J ¼ 38.1, 8.5 Hz, 1H), 4.75 (d, J ¼ 11.9 Hz, 1H), 4.19 (d,
J ¼ 11.3 Hz, 1H), 3.48 (s, 3H), 3.16 (t, J ¼ 7.3 Hz, 1H), 3.08–2.86
(m, 1H), 2.58 (t, J ¼ 12.4 Hz, 1H), 1.93 (d, J ¼ 13.4 Hz, 3H),
1.85–1.72 (m, 1H). MS (ESI), m/z: 287.18 [MþH]þ. The InChI
code of III-4 is provided in the Supporting Information.
Pharmacokinetics in Sprague-Dawley rats
Two groups (n ¼ 5) of male and female Sprague-Dawley rats
were fasted overnight and received III-4 as an intravenous
dose (5 mg/kg) or by oral gavage (10 mg/kg). Blood samples
(0.4 mL) were obtained from jugular vein bleeding at 5, 15,
30 min, 1, 2, 4, 6, 8, 10, 12, and 24 h post-dose for the p.o.
group. At each time point, three mice were bled resulting in a
composite pharmacokinetic profile. The tubes were inverted
several times to ensure mixing and placed on ice. The blood
samples were centrifuged to obtain the plasma fraction. The
plasma samples were deproteinized with acetonitrile con-
taining an internal standard. After centrifugation, the
supernatant was diluted and centrifuged again. The com-
pound concentrations in the supernatant were measured by a
high performance liquid chromatography-tandem mass
spectrometry (LC/MS/MS), and the Cmax is the average of
maximum compound concentration of five mice in each
group. The Tmax is the average of the time with Cmax of five
mice in each group. Other obtained pharmacokinetic
parameters are processed by the software DAS 2.0 through
inputting each time point and its corresponding compound’s
concentration.
Biological assays
Enzymatic assays
The enzymatic activities against JAK1, JAK2, JAK3, and TYK2
were tested by Caliper Mobility Shift Assay with ATP
concentration at Km (ChemPartner; Shanghai; China). The
assay protocol guide is commercially available from Chem-
Partner. The activities against Cys909-containing kinases were
tested by EurofinsCerep (CEREP, Celle l’Evescault, France). The
protocols are available from http://www.cerep.fr/Cerep/Users/
index.asp. The assay protocol guide can be visited from www.
eurofins.com/discovery services.
JAK cellular assays
Human peripheral blood mononuclear cells (PBMCs): PBMCs
were collected from healthy volunteers, who gave informed
consent, into sodium heparin vacutainer tubes by venipunc-
ture. After incubation with compound III-4 at 37°C for
30 min, blood was triggered with either recombinant
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Acute cytotoxicity test of III-4 in Balb/c mice
LC-MS/MS analysis: Peptides were dissolved in solvent A
(0.1% FA in 2% ACN), directly loaded onto a reversed-phase
pre-column (Acclaim PepMap 100, Thermo Scientific). Pep-
tide separation was performed using a reversed-phase
analytical column (Acclaim PepMap RSLC, Thermo Scientific)
with alinear gradient of 8–35% solvent B (0.1% FA in 98%
ACN) for 12 min, 35–80% solvent B for 4 min at a constant
flow rate of 320 nL/min on an EASY-nLC1000 UPLC system.
The resulting peptides were analyzed by Q ExactiveTM Plus
hybrid quadrupole-Orbitrap mass spectrometer (Thermo
Fisher Scientific). The peptides were subjected to NSI source
Six groups (n ¼ 6) of male and female half Balb/c mice (25–
30 g) were fasted overnight. In acute toxicity test, mice were
orally administrated III-4 with several doses 200, 400, 600, 800,
and 1000 mg/kg, and observed their behavioral or physiologi-
cal signs for 7 days. Serum levels of markers, liver and kidney
were analyzed according to the health ones after all animals
were sacrificed.
Collagen-induced arthritis model in the mice
The collagen-induced arthritis (CIA) model shares a number of
pathologic, genetic, and immunologic features with RA.
Therefore, the CIA mouse model was used to evaluate the
effects of oral III-4 (30 mg/kg p.o.) on joint inflammation and
histopathology. Arthritis was induced in 10-weekend male
DBA/1J mice by immunizations with a 1:1 emulsion of bovine
type II collagen (Chondrex) and Freund’s complete adjuvant
(Sigma–Aldrich) on day 0 and a 1:1 emulsion of bovine type II
collagen in Freund’s incomplete adjuvant (Sigma–Aldrich) on
day 21. Treatment was initiated when >20% of mice
demonstrated signs of disease. On the day treatment was
initiated, the mice were randomly assigned to controls (no
collagen injection plus vehicle; n ¼ 8), collagen plus vehicle
(n ¼ 8), collagen plus III-4 30 mg/kg (n ¼ 8), and collagen plus
tofacitinib 30 mg/kg (n ¼ 8). Mice were scored for signs of
arthritis daily for 33 days. CIA development was inspected
three times per week and inflammation of the four paws was
graded from 0 to 4 (grade 0, paws with no swelling and focal
redness; grade 1, paws with swelling of finger joints; grade 2,
paws with mild swelling of ankle or wrist joints; grade 3, paws
with severe inflammation of the entire paw; and grade 4,
paws with deformity or ankylosis). Each paw was graded and
the four scores were totaled so that the possible maximum
score per mouse was 16. Hematoxylin and eosin-stained tissue
sections from all paws from each of the eight mice per group
were evaluated microscopically using methods modified from
McKew et al. Kruskal–Wallis one way analysis of variance
nonparametric tests were used to determine statistical
significance among the histopathology groups, with signifi-
cance set at P ꢅ 0.05.
followed by tandem mass spectrometry (MS/MS) in
Q
ExactiveTM Plus (Thermo) coupled online to the UPLC. Intact
peptides were detected in the Orbitrap at a resolution of
70000. Peptides were selected for MS/MS using 30% NCE; ion
fragments were detected in the Orbitrap at a resolution of
17500.
A
data-dependent procedure that alternated
between one MS scan followed by 10 MS/MS scans was
applied for the top 20 precursor ions above a threshold ion
count of 5000 in the MS survey scan with 5.0 s dynamic
exclusion. The electrospray voltage applied was 2.0 kV.
Automatic gain control (AGC) was used to prevent overfilling
of the ion trap; 5E4 ions were accumulated for generation of
MS/MS spectra. For MS scans, the m/z scan range was 350–
1800. Database search: The resulting MS/MS data were
processed using Mascot search engine (v.2.3.0). Tandem mass
spectra were searched against JAK3 database. Trypsin/P was
specified as cleavage enzyme allowing up to two missing
cleavages. Mass error was set to 10 ppm for precursor ions
and 0.02 Da for fragment ions. Carbamidomethyl on Cys is
specified as fixed modification and Cys909 was found to be
the only covalently modified site by III-4.
Covalent 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 hydrogens were added to the
structure. 3D structure of the compound III-4 was generated
and optimized by the Discovery Studio 3.1 package (Accelrys,
San Diego, CA). The small molecule was docked into the JAK3
kinase domain by the program Covalent Dock Cloud (http://
docking.sce.ntu.edu.sg), which was used to perform the
covalent docking simulations [28]. Figure 2 was generated
though the professional graphics software Pymol 1.7.
Mass spectrometry experiments
Incubation of JAK3 proteins with III-4: A total of 67 mL HEPES
buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM DTT) was
added to 33 mL JAK3 proteins liquid (0.213 mg/mL, Abnova),
and incubated with 0.5 mL III-4 (1 mM in DMSO) at 20°C for
3 h. The mixture was freeze-dried after removing the
excessive compound by an ultrafiltration method at 4°C.
Supplementary materials
Trypsin digestion: The protein was reduced with 10 mM DTT
for 1 h at 56°C and alkylated with 20 mM iodoacetamide (IAA)
for 45 min at room temperature in darkness. Then, trypsin was
added at 1:50 trypsin-to-protein mass ratio for the first
digestion overnight and 1:100 trypsin-to-protein mass ratio
for a second 4 h digestion. The digested peptides were dried
with a SpeedVac (Thremo) for LC-MS/MS analysis.
Figures mean body weight of each group mice in CIA model,
mass and 1H NMR spectra of III-4.
The authors greatly appreciate the financial support from
National Natural Science Foundation of China (81373260).
The authors have declared no conflict of interest.
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L. F. Nelms, S. G. Des Etages, L. S. Hayes, T. T. Kawabata,
D. Finco-Kent, D. L. Baker, M. Larson, M. S. Si,
R. Paniagua, J. Higgins, B. Holm, B. Reitz, Y. J. Zhou,
R. E. Morris, J. J. O’Shea, D. C. Borie DC, Science 2003, 302,
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