Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the treatment of B-cell lymphoma

Article abstract of DOI:10.1016/j.ejmech.2017.10.080

The BTK and JAK3 receptor tyrosine kinases are two validated and therapeutically amenable targets in the treatment of B-cell lymphomas. Here we report the identification of several classes of pyrimidine derivatives as potent BTK and JAK3 dual inhibitors.

Full text of DOI:10.1016/j.ejmech.2017.10.080

European Journal of Medicinal Chemistry xxx (2017) 1e11
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Identification of highly potent BTK and JAK3 dual inhibitors with
improved activity for the treatment of B-cell lymphoma
,
b
,
,
Yang Ge a 1, Changyuan Wang b 1, Shijie Song b, Jiaxin Huang b, Zhihao Liu b,
Yongming Li b, Qiang Meng b, Jianbin Zhang b, Jihong Yao b, Kexin Liu b, Xiaodong Ma b
,
,
*
Xiuli Sun a
,
**
a Department of Hematology, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China
b College of Pharmacy, College of Basic Medical Sciences, Dalian Medical University, Dalian 116044, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The BTK and JAK3 receptor tyrosine kinases are two validated and therapeutically amenable targets in
the treatment of B-cell lymphomas. Here we report the identification of several classes of pyrimidine
derivatives as potent BTK and JAK3 dual inhibitors. Among these molecules, approximately two thirds
displayed strong inhibitory capacity at less than 10 nM concentration, and four compounds (7e, 7g, 7m
and 7n) could significantly inhibit the phosphorylation of BTK and JAK3 enzymes at concentrations lower
than 1 nM. Additionally, these pyrimidine derivatives also exhibited enhanced activity to block the
proliferation of B-cell lymphoma cells compared with the representative BTK inhibitor ibrutinib. In
particular, two structure-specific compounds 7b and 7e displayed stronger activity than reference agents
Received 24 August 2017
Received in revised form
24 October 2017
Accepted 30 October 2017
Available online xxx
Keywords:
Lymphoma
BTK
in cell-based evaluation, with IC50 values lower than 10 mM. Further biological studies, including flow
cytometric analysis, and a xenograft model for in vivo evaluation, also indicated their efficacy and low
toxicity in the treatment of B-cell lymphoma. These findings provide a new insight for the development
of novel anti-B-cell lymphoma drugs with multi-target actions.
JAK3
Inhibitor
Pyrimidine
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
Bruton's tyrosine kinase (BTK), a member of the Tec family of
protein tyrosine kinases (PTKs), is involved in signal transduction
B lymphocytes have historically contributed to the pathogenesis
of autoimmune disease through autoantibody production [1,2]. The
malignant growth of B-lymphocytes in the lymph system will lead
to B-cell lymphomas, which develop more frequently in older
adults and in immunocompromised individuals. B-cell lymphomas
include both Hodgkin's lymphomas and most non-Hodgkin's
lymphomas. B-cell lymphomas represent 80e90% of non-Hodg-
kin's lymphomas in the Western world, while in China, where
approximately 5e6 new lymphatic malignancy cases are diagnosed
per 100,000 people per year, B-cell lymphoma accounts for
approximately 66% of these patients [3]. Thus, development of
effective drugs for the treatment of this debilitating disease is ur-
gently needed.
pathways that regulate the growth, differentiation and survival of
B-lineage lymphoid cells [4,5]. Several studies revealed that BTK is
involved in the induction of macrophage effector functions. In
addition, BTK was found to be
a key element of the
lipopolysaccharide-induced tumor necrosis factor production in
monocytes [6,7]. Furthermore, BTK inhibition has become an
attractive strategy for treating B-cell leukemia and lymphomas
[8,9]. Ibrutinib (1) is the first-in-class potent BTK inhibitor,
approved by the US Food and Drug Administration (FDA) for the
treatment of mantle cell lymphoma, chronic lymphocytic leukemia,
€
Waldenstrom's macroglobulinemia, etc [10e12]. Also, spebrutinib
(2) (Phase Ⅱ, ClinicalTrials.gov identifier: NCT01744626) [13] and
PLS-123 (preclinical, 3) [14] are two of the novel BTK inhibitors that
form strong covalent-bond with BTK enzyme to exert their inhib-
itory effect (Fig. 1).
Janus kinase 3 (JAK3), another important PTK, was also found to
play an important role in cytokine-induced proliferation and sur-
vival of normal and leukemic B-cell precursors [15e17]. Besides the
activity of the antiapoptotic transcription factors signal transducer
* Corresponding author.
** Corresponding author.
E-mail addresses: xiaodong.ma@139.com (X. Ma), sunxl0411@163.com (X. Sun).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2017.10.080
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Y. Ge, et al., Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the
treatment of B-cell lymphoma, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.080

2
Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
H
N
N
F
N
N
NH
O
HN
O
N
NH
O
NH
NH
O
O
N
O
N
NH
NH
N
O
O
H2N
N
O
N
F3C
N
1
2
3
Cl
Cl
N
N
N
N
N
HN
N
O
NH
HN
O
HN
N
O
O
NH
NH
NH
O
NH
N
N
S
O
O
O
O
O
N
6
F
4
5
Fig. 1. Novel pyrimidine derivatives as potent protein tyrosine kinase inhibitors.
R1
NH
N
HN
N
Cl
O
N
N
Cyclization
N
N
R2
NH
HN
O
N
HN
N
Flexible
Conformational
constraint
R
O
O
O
O
NH
NH
Covalent bond
Cys481 and Cys909
O
N
N
O
O
O
N
N
X
N
X
WZ4002
EGFRT790M Inhibitor
Imi-DPPY
BTK inhibitors
DPPYs 7a-n
BTK and JAK3 dual inhibitors
R1 = Cl, F; R2 = H, Me, MeO
X = O, MeN, EtN
Fig. 2. Discovery strategy of the title molecules as BTK and JAK3 dual inhibitors.
and activator of transcription 3 (STAT3), STAT5 and activator pro-
tein 1, JAK3 also regulates the antiapoptotic PI3K-AKT pathway and
their downstream targets, some of which have been implicated as
important oncogenic proteins [18]. These findings have collectively
identified JAK3 as an attractive molecular target for disrupting the
constitutively active antiapoptotic STAT3 and STAT5 signaling
pathways in leukemia/lymphoma cells [19].
Diphenylpyrimidine derivatives (DPPYs), which possess broad
and strong biological activity, are currently used as novel anticancer
scaffold [20e25]. The well-known DPPY analogues include:
WZ4002 (4, Fig. 1), the first reported epidermal growth factor re-
ceptor (EGFR) inhibitor, that can efficiently overcome anoikis
resistance in EGFR-mutant lung adenocarcinomas [25]; and spe-
brutinib, a typical BTK inhibitor (Fig. 1) [13]. Generally, the acryl-
amide functional group is important for these DPPY analogues to
form covalent bond contacts with Cys481 in BTK, with Cys797 in
EGFRT790M and Cys909 in JAK3 [25e27]. According to the WZ4002
scaffold, our previous structural modification has unexpectedly led
to the identification of several classes of DPPY derivatives as potent
and selective BTK inhibitors, including Sul-DPPYs (5) and Imi-DPPYs
(6) [28,29]. Considering that a flexible molecule may favorably
interact with a flexible protein [30,31], we further synthesized a
new class of DPPY derivatives bearing various of the flexible C-2
aniline side chains according to the Imi-DPPY scaffold (Fig. 2).
Interestingly, these newly designed compounds exhibited strong
inhibitory activity against the BTK and JAK3 enzymes. In this paper,
we describe all these newly discovered BTK and JAK3 dual in-
hibitors, including their synthesis and biological studies of their
effects on B-cell lymphomas in vitro and in vivo.
Please cite this article in press as: Y. Ge, et al., Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the
treatment of B-cell lymphoma, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.080

Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
3
R1
NH
N
NO2
Cl
N
NH2
R1
R1
Cl
NO2
N
N
a
Cl
N
b
HN
N
NH
c
NH
NH
NH
NH2
O
O
O
11a R1= Cl
11b R1= F
R2
N
NH
i
10
8
9
O
O
O
NH2
NO2
NO2
NO2
NO2
f,g
h
e
d
O
R2
O
R2
R2
R2
O
7a-d
R1= Cl,F
O
OH
O
O
O
O
N
R2= H,Cl,MeO
N
O
O
OH
O
OMe
12
15a-c
13a-c
14a-c
16a-c
R2= H,Cl,MeO
R2= H,Cl,MeO
R2= H,Cl,MeO R2= H,Cl,MeO
Scheme 1. Synthetic route of title compounds 7a-d. Reagents and conditions: (a) acryloyl chloride, NaHCO3, CH3CN, 0 ꢀC, 0.5 h, 95%. (b) Fe-NH4Cl, MeOH-H2O, 2 h, 70 ꢀC, 81%. (c)
ArNH2, DIPEA, 1,4-dioxane, 60 ꢀC, 2 h, 82-91%. (d) ethyl bromoacetate, K2CO3, CH3CN, reflux, 5 h, 81-95%. (e) NaOH, H2O, 2 h, 50 ꢀC. (f) (COCl)2, DMF, 60 ꢀC, 2 h (g) morpholine,
NaHCO3, CH3CN, rt, 5 h, 62-73%. (h) Fe-NH4Cl, MeOH-H2O, 2 h, 70 ꢀC, 71-83%. (i) trifluoroacetic acid, 2-BuOH, 100 ꢀC, 12 h, 18-31%.
R1
N
NO2
O
NH2
NO2
2
4
NH
HN
N
NO2
R2
R2
R2
2`
a
2`
3`
R2
c
d
b
R2
HO
O
O
3`
NH
4` O
4`
N
N
X
X
O
N
Cl
18a-e
X
17a-e
19a-h
20a-h
7e-n
R2= Me, MeO, Cl, H (C-2` or C-3`);
OH (C-3` or C-4`)
R1= Cl, F;
R2= Me, MeO, Cl, H (C-2` or C-3`);
X= O, MeN, EtN
Scheme 2. Synthetic route of title compounds 7e-n. Reagents and conditions: (a) 1-bromo-3-chloropropane, K2CO3, CH3CN, reflux, 12 h, 77-89%. (b) morpholine or piperazine,
K2CO3, KI, DMF, 100 ꢀC, 5 h, 72-81%. (c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 ꢀC, 71-80%. (d) TFA, 2-chloropyrimidine analogues 11a,b, 2-BuOH, 100 ꢀC, 12 h, 12-27%.
2. Chemistry
chloro-3-bromopropane to give the chloro-substituted com-
pounds 18a-e, which were then subjected to the nucleophilic
substitution reaction with morpholine to form the intermediates
19a-h. After reducing the nitro-substituted analogue 19a-h to the
aniline analogue 20a-h, and subsequently substituting the C-2
chlorine atom in the pyrimidines 11a,b, the desired molecules 7e-n
were produced (Scheme 2).
The synthetic strategies of the title compounds 7a-n are
described in Schemes 1-2 [32e34]. Generally, to prepare these
molecules, the key intermediates 2-chloropyrimidines 11a,b were
synthesized first as follows. Commercially available material 3-
nitroaniline was reacted with acryloyl chloride under the action
of NaHCO3 base to form N-acryloyl-3-nitrobenzamide 9, which was
then reduced to the aniline derivative 10 under Fe-NH4Cl reduction
condition. Through regioselective substitution of the C-4 chlorine
atom in the 2,4-dicloropyrimidine reagent with aniline 10 in the
presence of N,N-diisopropylethylamine (DIPEA), the 2-
chloropyrimidines 11a,b were synthesized with overall yields of
41e52% for all three steps. The C-2 aniline side chains 16a-c were
generally synthesized via nucleophilic substitution, hydrolysis,
acylation reduction reactions starting from the material 4-
nitorphenol. Ultimately, the desired diphenylpyrimidine de-
rivatives 7a-d were synthesized by coupling morpholine-
substituted anilines 16a-c with 2-chloropyrimidines 11a,b
(Scheme 1) utilizing trifluoroacetic acid (TFA) reagent at the reflux
temperature.
3. Results and discussion
3.1. Biological activity
All the newly synthesized compounds were evaluated for their
activity against the BTK and the JAK3 enzymes by using the ADP-
Glo™ kinase assay system [35,36]. In addition, their anti-
proliferative activity against the B lymphoma cell lines (Ramos, Raji
and Namalwa) was also assessed using the CCK-8 assay [37,38]. For
comparison, the activity of two typical BTK inhibitors, namely
spebrutinib and ibrutinib, was also evaluated using the same pro-
cedure. The results from all these assays are presented in Table 1.
For preparation of another family of title molecules, namely the
7e-n, the nitrophenol reagents 17a-e were used to react with 1-
3.1.1. Kinase inhibitory activity
Clearly, most of these compounds exhibited strong inhibitory
Please cite this article in press as: Y. Ge, et al., Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the
treatment of B-cell lymphoma, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.080

4
Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
Table 1
Biological activity of the newly synthesized compounds 7a-n.a
R1
N
4
2
HN
1`
N
NH
2`
3`
R2
NH
R
O
Compounds
R
R2
R1
Enzymatic
activity
Antiproliferative activity
(IC50
M)c
,
m
(IC50, nM)b
BTK
1.00
JAK3
1.39
Ramos
25.56
Raji
Namalwa
4.42
7a
7b
7c
7d
H
Cl
Cl
Cl
F
5.68
O
N
N
N
N
O
O
O
O
O
Cl(30)
MeO(30)
MeO(30)
0.80
8.34
2.46
8.89
6.45
O
O
10.50
17.10
21.59
11.23
>40.00
>40.00
22.52
11.16
17.20
14.99
O
O
O
O
7e
7f
H
Cl
Cl
Cl
0.60
0.70
0.50
0.83
1.06
0.91
4.85
2.00
5.72
9.32
8.23
19.36
2.41
Cl(30)
Cl(30)
22.48
11.44
7g
7h
7i
MeO(30)
Me(20)
Cl
Cl
31.30
6.77
2.49
5.92
14.40
35.22
>100
>100
14.37
35.26
7j
Me(20)
F
90.0
>100
18.46
26.02
26.63
7k
7l
Me(20)
Me(20)
Cl
Cl
Cl
F
81.70
89.32
0.80
91.15
>100
0.69
22.61
18.74
7.31
25.00
25.47
3.86
24.64
27.96
5.17
7m
7n
MeO
MeO
(30)
(30)
0.70
0.95
11.32
13.92
3.17
Spebrutinib
Ibrutinib
0.60
0.30
22.50
16.10
14.20
6.62
25.30
20.88
3.42
10.45
a
Data represent the mean of at least three separate experiments, the exact concentrations for cytotoxic studies were 1.25
Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in nanomolar needed to inhibit cell growth by 50% as determined
m
M, 5
m
M, 10
m
M, 20
m
M and 40
m
M.
b
from these curves.
c
Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in micromolar needed to inhibit cell growth by 50% as determined
from these curves.
effect on the activity of the BTK and JAK3 enzymes at concentra-
tions around 10 nM. For the inhibitory activity against the BTK
enzyme, approximately one third of them (7b, 7e, 7f, 7g, 7m and
7n) were very effective, with IC50 values lower than 1 nM. However,
for the class of compounds 7i-l, which possess a C-20 methyl sub-
stituent, are clearly unfavorable (IC50 > 81.70 nM). Regarding their
activity against the JAK3 enzyme, compounds 7e, 7g, 7m and 7n
demonstrated high inhibitory potency, with IC50 values of 0.83,
0.91, 0.69, and 0.95 nM, respectively. Compared with the two
representative BTK inhibitors ibrutinib (IC50 ¼ 16.1 nM) and spe-
brutinib (IC50 ¼ 22.5 nM), the four strongest JAK3 inhibitors
showed a remarkably improved inhibitory activity (17- and 40-fold
higher). The test result also indicated that the analogues 7i-l
bearing a C-20 methyl substituent are inactive, with IC50 values of
more than 91.15 nM. In summary, this effective work led to the
identification of numerous potent BTK and JAK3 dual inhibitors,
Please cite this article in press as: Y. Ge, et al., Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the
treatment of B-cell lymphoma, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.080

Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
5
Fig. 3. The effects of treating time and drug concentration for compounds 7b and 7e on B lymphoma cells (Ramos cells) viability.
which possess great value to warrant further biological evaluation
and Namalwa cells at concentrations more than 14.37
mM. Further
in future work.
exploration of their effect of the treatment time and drug con-
centration for structure-specific compounds 7b and 7e on B lym-
phoma cells (Ramos cells) viability revealed that both of them
blocked the proliferation of Ramos cells in a dose- and time-
dependent manner, and almost completely inhibited the Ramos
3.1.2. Inhibitory activity against B lymphoma cells
Besides the above effects, most of these compounds also dis-
played remarkable capacity to interfere with the proliferations of B
(91%) at a concentration of 20 mM after 72 h (Fig. 3).
lymphoma cells at concentrations lower than 10
compound 7f (IC50 ¼ 2.00 M) was the strongest inhibitor against
Ramos cells, compound 7m (IC50 ¼ 3.86 M) was the most active
M) was
mM. In particular,
m
m
3.1.3. Cell cytotoxicity evaluation
inhibitor against Raji cells, and compound 7g (IC50 ¼ 2.41
m
The cytotoxicity of the two active compounds 7b and 7e against
the normal peripheral blood mononuclear cells (PBMC) were also
evaluated by the AO/PI staining assay (Fig. 4). After treatment with
inhibitors 7b and 7e for 24 h, it was found that both compounds 7b
and 7e did not produce apparent inhibition of the growth rate of
PBMC cells at a concentration lower than 10
inhibited the growth of PBMC cells by approximately 50% at this
concentration (Fig. 4). Based on this evaluation, it can be concluded
the most potent inhibitor against Namalwa cells. Nevertheless,
compounds 7c and 7d bearing a MeO (C-30) group almost lost their
inhibitory capability toward the Ramos cells (IC50 > 40
mM).
Generally, the less active inhibitors against BTK and JAK3 are also
not effective to interfere with proliferation of B lymphoma cells. For
instance, compounds 7i-l, which has IC50 values of more than
91.15 nM against BTK and JAK3 enzymes, could inhibit Ramos, Raji
mM, but ibrutinib
Fig. 4. Morphological changes of the PBMC cells treated by molecules 7b and 7e (100 ꢁ , final magnification) at concentrations of 5, 10, 20
mM for 24 h. Cell numbers were calculated
by traditional manual method with an ordinary cell counting chamber. One representative experiment is shown.
Please cite this article in press as: Y. Ge, et al., Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the
treatment of B-cell lymphoma, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.080

6
Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
that compounds 7b and 7e are less cytotoxic than the approved
agent ibrutinib. Taken together, these biological evaluations sug-
gested that our discovered potent BTK and JAK3 dual inhibitors are
effective against the B-cell lymphoma, and compounds 7b and 7e
represents a promising dual BTK and JAK3 inhibitor candidate to
perform further biological investigation on the treatment of B-cell
lymphoma.
evaluation by cell-based assays, compounds 7b and 7e exhibited
stronger activity than the reference agents, with IC50 values lower
than 10 mM. Additionally, normal PBMC cells are not sensitive to
inhibitors 7b and 7e, indicating their low cell cytotoxicity.
Furthermore, flow cytometric analysis and a xenograft model for
in vivo evaluation also indicated their efficiency and low toxicity for
the treatment of B-cell lymphoblastic leukemia. Overall, these
newly discovered BTK and JAK3 dual inhibitors are of great enough
value to warrant further investigation as potential agents for the
treatment of B-cell lymphomas.
3.1.4. Flow cytometry analysis
The effects of compounds 7b and 7e on the apoptosis rates of B-
cell lymphoma cancer cells were also investigated by flow cyto-
metric analysis. The results presented in Fig. 5 revealed that 7b
triggered apoptosis in Ramos cells in a dose- and time-dependent
manner, with apoptosis rates increasing from 61.8 to 79.5%, after
5. Experimental section
5.1. General methods for chemistry
treatment with drug concentrations of 5, 10 and 20
Likewise, 7e induced similar apoptosis rate (59.0e80.2%) under
these concentrations. At the identical concentration of 10 M, the
apoptosis rates induced by ibrutinib and spebrutinib are 47.9 and
41.5%, which are evidently lower than those of 7b (69.9%) and 7e
(77.2%). This analysis also proved their stronger biological activity
against B lymphoma cells.
mM for 72 h.
Unless otherwise indicated, all reagents and solvents were ob-
tained from commercial sources and used as received. 1H and 13C
NMR datas were obtained on a Bruker Avance at 400 and 100 MHz,
respectively. Coupling constants (J) are expressed in hertz (Hz).
m
Chemical shifts (d) of NMR are reported in parts per million (ppm)
units relative to internal control (TMS). High resolution ESI-MS was
performed on an AB Sciex TripleTOF® 4600 LC/MS/MS system. All
reactions were monitored by TLC, using silica gel plates with fluo-
rescence F254 and UV light visualization. Flash chromatography
separations were obtained on Silica Gel (300-400 mesh) using
dichloromethane/methanol as eluents.
3.1.5. Effects of the inhibitors on BTK and JAK3 activation and
downstream signaling
To gain a better insight into the mechanisms underlying the
activity of these BTK and JAK3 dual inhibitors, the inhibitory effects
of compounds 7b and 7e on the BTK and JAK3-STAT3 activation and
downstream signaling in Namalwa cells were examined using
ibrutinib as a control. The cells were treated with various concen-
5.2. General procedure for the synthesis of 7a-n
trations (1, 5, 10
m
M) of inhibitors for 48 h, and the expression and
Various 4-morpholine-substituted C-2 anilines 16a-c, 20a-h,
and the key intermediates N-(3-((2-chloropyrimidin-4-yl)amino)
phenyl)acrylamides 11a,b were synthesized according to our pre-
vious reported method [32-34] . All these intermediates were
directly used without any purification and structural character-
ization. With these intermediates in hand, the desired compounds
were synthesized as described below.
activation of BTK, p-BTK, and STAT3, p-STAT3 were determined by
immunoblotting analysis. The results, summarized in Fig. 6, indi-
cated that treatment with 7b and 7e inhibited the phosphorylation
of BTK and JAK3 in a dose-dependent manner, and compounds 7b
and 7e displayed stronger inhibitory activity, at the same drug
concentration (10 mM), than ibrutinib.
A flask was charged with compounds 11a,b (0.70 mmol), ani-
lines 16a-c, 20a-h, (0.70 mmol), TFA (1.05 mmol), and 2-BuOH
(10 mL). The slurry was heated to 100 ꢀC for 12 h. The reaction
mixture was allowed to cool to room temperature and was
neutralized with a saturated aqueous sodium bicarbonate solution.
The aqueous mixture was then extracted with CH2Cl2 (20 mL) three
times. The crude product was purified using flash chromatography
with dichloromethane/methanol (v/v, 30:1) as eluents.
3.1.6. In vivo antitumor activity
The antitumor efficacy of the two potent BTK inhibitors 7b and
7e were also evaluated in vivo using a BTK-driven human Ramos
cells xenograft mouse model (Fig. 7). Obviously, tumor growth was
dose-dependently inhibited by repeated oral administration of 7b
or 7e (qd) for 14 days. For compound 7b, at dosages of 60 and
30 mg/kg/day the tumor growth inhibition (TGI) values were 36.6%
and 24.45%, respectively, while for compound 7e, at similar dos-
ages, the TGI values were 47.48% and 36.60%, respectively (Fig. 7A-
C). In addition, it is clear that even at high doses of compounds 7b
and 7e, the body-weight change was within 5% (compared to their
initial body-weight), which suggests that the two novel therapeutic
agents do not affect body weight and have high safety margin
in vivo (Fig. 7D).
5.2.1. N-(3-((5-chloro-2-(4-((1-morpholino)acetoxyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7a)
Off-white solid (102 mg, 30.5%); MS (ESI) m/z 509.2 [MþH]þ; 1H
NMR (400 MHz, DMSO-d6): d 10.18 (s, 1H), 9.15 (s, 1H), 8.90 (s, 1H),
8.10 (s, 1H), 7.87 (s, 1H), 7.48 (d, J ¼ 8.8 Hz, 3H), 7.40e7.10 (m, 2H),
6.71 (d, J ¼ 8.8 Hz, 2H), 6.46 (dd, J ¼ 16.8, 10.0 Hz, 1H), 6.26 (dd,
J ¼ 16.8, 2.0 Hz, 1H), 5.76 (dd, J ¼ 10.0, 2.0 Hz, 1H), 4.70 (s, 2H),
3.67e3.52 (m, 4H), 3.50e3.41 (m, 4H); 13C NMR (100 MHz, DMSO-
4. Conclusion
d6):
d 166.17, 163.14, 157.79, 156.12, 154.79, 152.68, 139.05, 138.90,
Multi-target drugs possess several outstanding advantages in
the treatment of malignant tumors, including the improved ther-
apeutic efficacy, safety and resistance profiles by collective regu-
lations of a primary therapeutic target together with compensatory
elements and resistance activities. In this study, a number of potent
BTK and JAK3 dual inhibitors for the treatment of B-cell lymphoma
were identified and evaluated. Most of these compounds displayed
strong potency to block the phosphorylation of BTK and JAK3 ki-
nases, with IC50 values of less than 10 nM. In particular, four com-
pounds (7e, 7g, 7m and 7n) remarkably inhibited the activity of BTK
and JAK3 kinases at concentrations lower than 1 nM. In the
133.93, 131.89, 128.57, 126.96, 120.53 (2C), 119.21, 115.39, 115.17,
114.24 (2C), 103.33, 66.21, 66.15 (2C), 44.87, 41.62; HRMS (ESI) m/z
calcd for C25H25ClN6O4 [MþH]þ 509.1699, found 509.1706.
5.2.2. N-(3-((5-chloro-2-(2-chloro-4-((1-morpholino)acetoxyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7b)
Off-white solid (153 mg, 30.1%); MS (ESI) m/z 543.1 [MþH]þ; 1H
NMR (400 MHz, DMSO-d6): d 10.16 (s, 1H), 9.30 (s, 1H), 8.95 (s, 1H),
8.15 (s, 1H), 7.84 (s, 1H), 7.76 (s, 1H), 7.49 (d, J ¼ 7.2 Hz, 1H),
7.45e7.24 (m, 3H), 6.82 (d, J ¼ 9.2 Hz, 1H), 6.45 (dd, J ¼ 17.2, 10.4 Hz,
1H), 6.25 (dd, J ¼ 16.8, 2.0 Hz,1H), 5.75 (dd, J ¼ 10.0,1.2 Hz,1H), 4.82
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Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
7
Fig. 5. Compounds 7b and 7e induced Ramos cells apoptosis in vitro. The cells were incubated with the indicated concentrations of 7b and 7e for 72 h, and the cells were stained
with annexin V/FTIC, followed by flow cytometry analysis. One representative experiment is shown. p < 0.05.
Fig. 6. Compounds 7b and 7e inhibited the activation of BTK and JAK-STAT3 downstream singnaling in Namalwa cells.
(s, 2H), 3.62e3.55 (m, 4H), 3.51e3.42 (m, 4H); 13C NMR (100 MHz,
DMSO-d6): 165.68, 163.12, 157.51, 156.16, 154.73, 147.81, 139.25,
138.78, 134.70, 131.92, 128.80, 126.89, 120.78, 120.23, 119.09, 118.44,
115.47, 114.93, 113.85, 103.95, 66.84, 66.10 (2C), 44.90, 41.67; HRMS
d
Please cite this article in press as: Y. Ge, et al., Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the
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8
Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
Fig. 7. In vivo effects of compounds 7b and 7e on the tumor growth of ramos cell xenografts in nude mice. (A) Images of the tumor collected from the mice; (B) Tumor weight; (C)
Tumor volume; (D) Body weight. Data are presented as mean SD (n ¼ 5).
(ESI) m/z calcd for C25H24Cl2N6O4 [MþH]þ 543.1309, found
5.2.5. N-(3-((5-chloro-2-(4-((1-morpholino)propoxy)
543.1319.
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7e)
Off-white solid (121 mg, 26.8%); MS (ESI) m/z 509.2 [MþH]þ; 1H
NMR (400 MHz, DMSO-d6):
d 10.17 (s, 1H), 9.14 (s, 1H), 8.88 (s, 1H),
5.2.3. N-(3-((5-chloro-2-(2-methoxy-4-((1-morpholino)acetoxyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7c)
Yellow-white solid (86 mg, 26.3%); MS (ESI) m/z 539.2 [MþH]þ;
8.10 (s, 1H), 7.87 (s, 1H), 7.52e7.42 (m, 3H), 7.30 (d, J ¼ 5.2 Hz, 2H),
6.68 (d, J ¼ 8.4 Hz, 2H), 6.45 (dd, J ¼ 17.2, 10.0 Hz, 1H), 6.26 (dd,
J ¼ 17.2, 2.0 Hz, 1H), 5.75 (dd, J ¼ 10.0, 2.0 Hz, 1H), 3.88 (t, J ¼ 6.4 Hz,
2H), 3.56 (t, J ¼ 4.4 Hz, 4H), 2.37 (t, J ¼ 7.2 Hz, 6H), 1.86e1.77 (m,
1H NMR (400 MHz, DMSO-d6):
d 10.15 (s, 1H), 9.13 (s, 1H), 8.89 (s,
2H); 13C NMR (100 MHz, DMSO-d6):
d 163.10, 157.79, 156.10, 154.80,
1H), 8.12 (s, 1H), 7.84 (s, 1H), 7.47 (d, J ¼ 8.0 Hz, 1H), 7.38 (d,
J ¼ 8.0 Hz, 1H), 7.32e7.12 (m, 3H), 6.68 (d, J ¼ 8.8 Hz, 1H), 6.45 (dd,
J ¼ 16.8,10.0 Hz,1H), 6.26 (dd, J ¼ 17.2, 2.0 Hz,1H), 5.75 (dd, J ¼ 10.0,
2.4 Hz, 1H), 4.64 (s, 2H), 3.67e3.57 (m, 4H), 3.56 (s, 3H), 3.50e3.42
153.34, 139.01, 138.87, 133.47, 131.88, 128.56, 126.90, 120.54 (2C),
119.14, 115.39, 115.26, 114.05 (2C), 103.19, 66.21 (2C), 65.81, 54.95,
53.40 (2C), 25.96; HRMS (ESI) m/z calcd for C26H29ClN6O3 [MþH]þ
509.2062, found 509.2073.
(m, 4H); 13C NMR (100 MHz, DMSO-d6):
d 166.23, 163.11, 157.76,
156.01, 154.73, 148.91, 142.03, 139.12, 138.95, 134.95, 131.90, 128.59,
126.93, 118.97, 115.32, 114.83, 114.42, 110.98, 104.82, 103.65, 67.66,
66.13 (2C), 55.39, 45.07, 41.66; HRMS (ESI) m/z calcd for
5.2.6. N-(3-((5-chloro-2-(2-chloro-4-((1-morpholino)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7f)
Pale-yellow solid (79 mg,12.6%); MS (ESI) m/z 543.2 [MþH]þ; 1H
C
26H27ClN6O5 [MþH]þ 539.1804, found 539.1809.
NMR (400 MHz, DMSO-d6): d 10.19 (s, 1H), 9.32 (s, 1H), 8.93 (s, 1H),
8.14 (s, 1H), 7.87 (s, 1H), 7.71 (s, 1H), 7.54e7.45 (m, 2H), 7.31 (d,
J ¼ 5.2 Hz, 2H), 6.88 (d, J ¼ 8.8 Hz, 1H), 6.46 (dd, J ¼ 16.8, 10.0 Hz,
1H), 6.25 (dd, J ¼ 17.2, 1.6 Hz, 1H), 5.74 (dd, J ¼ 10.0, 2.0 Hz, 1H), 3.96
(t, J ¼ 6.0 Hz, 2H), 3.62e3.50 (m, 4H), 2.39 (t, J ¼ 7.2 Hz, 6H),
5.2.4. N-(3-((5-fluoro-2-(2-methoxy-4-((1-morpholino)acetoxyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7d)
Red-brown solid (67 mg, 18.9%); MS (ESI) m/z 523.2 [MþH]þ; 1H
NMR (400 MHz, DMSO-d6): d 10.12 (s, 1H), 9.40 (s, 1H), 8.98 (s, 1H),
1.88e1.80 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
d 163.10, 157.51,
8.08 (s, 1H), 7.90 (s, 1H), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.40 (d, J ¼ 8.0 Hz,
1H), 7.35e7.07 (m, 3H), 6.74 (d, J ¼ 8.8 Hz, 1H), 6.46 (dd, J ¼ 17.2,
10.4 Hz, 1H), 6.26 (dd, J ¼ 16.8, 1.6 Hz, 1H), 5.75 (dd, J ¼ 10.0, 3.6 Hz,
1H), 4.66 (s, 2H), 3.62 (s, 3H), 3.61e3.52 (m, 4H), 3.51e3.40 (m, 4H);
156.14, 154.73, 148.40, 139.23, 138.77, 134.44, 131.93, 128.78, 126.84,
121.17, 120.21, 118.99, 118.57, 115.45, 114.97, 114.05, 103.87, 67.11,
66.16 (2C), 54.76, 53.34 (2C), 25.80; HRMS (ESI) m/z calcd for
26H28Cl2N6O3 [MþH]þ 543.1673, found 543.1652.
13C NMR (100 MHz, DMSO-d6):
d 166.29, 163.10, 155.61 (d,
C
J ¼ 10.8 Hz), 149.78, 149.67, 148.99, 141.76 (d, J ¼ 42.0 Hz), 140.80,
140.60, 139.17 (d, J ¼ 25.2 Hz), 135.51, 131.94, 128.69, 126.89, 117.19,
114.63 (2C), 113.04, 110.58, 104.57, 67.78, 66.14 (2C), 55.44, 45.09,
41.67; HRMS (ESI) m/z calcd for C26H27FN6O5 [MþH]þ 523.2100,
found 523.2105.
5.2.7. N-(3-((5-chloro-2-(2-chloro-4-((1-ethylpiperazine)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7g)
Off-white solid (54 mg, 12.3%); MS (ESI) m/z 571.2 [MþH]þ; 1H
NMR (400 MHz, DMSO-d6): d 10.28 (s, 1H), 9.37 (s, 1H), 8.92 (s, 1H),
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8.14 (s, 1H), 7.94 (s, 1H), 7.73 (s, 1H), 7.56e7.47 (m, 2H), 7.30 (d,
J ¼ 4.8 Hz, 2H), 6.88 (d, J ¼ 8.8 Hz, 1H), 6.49 (dd, J ¼ 16.8, 10.0 Hz,
1H), 6.24 (dd, J ¼ 16.8, 2.0 Hz,1H), 5.74 (dd, J ¼ 10.0, 2.0 Hz,1H), 3.96
(t, J ¼ 6.0 Hz, 2H), 2.48e2.21 (m, 12H), 1.86e1.79 (m, 2H), 0.98 (t,
5H), 2.13 (s, 3H), 1.86e1.79 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
163.04, 159.33, 155.70, 155.58, 154.86, 139.16, 138.85, 134.19,
131.98, 130.82, 128.30, 127.23, 126.79, 117.67, 115.81, 114.67, 113.69,
111.51, 103.04, 65.80, 54.67 (2C), 54.41, 52.62 (2C), 45.62, 26.27,
18.26; HRMS (ESI) m/z calcd for C28H34ClN7O2 [MþH]þ 537.2574,
found 537.2622.
d
J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
d 163.11, 157.49,
156.07, 154.68, 148.40, 139.26, 138.80, 134.46, 131.98, 128.74, 126.75,
121.16, 120.19, 118.79, 118.56, 115.35, 114.78, 114.06, 103.88, 67.18,
54.28, 52.62 (2C), 52.26 (2C), 51.55, 26.18, 11.83; HRMS (ESI) m/z
calcd for C28H33Cl2N7O2 [MþH]þ 571.2185, found 571.2226.
5.2.12. N-(3-((5-chloro-2-(3-methyl-4-((1-morpholino)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7l)
Yellow-white solid (95 mg, 18.0%); MS (ESI) m/z 523.2 [MþH]þ;
5.2.8. N-(3-((5-chloro-2-(2-methoxy-4-((1-morpholino)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7h)
Yellow solid (128 mg, 25.2%); MS (ESI) m/z 539.2 [MþH]þ; 1H
1H NMR (400 MHz, DMSO-d6):
d 10.07 (s, 1H), 8.69 (s, 1H), 8.37 (s,
1H), 8.01 (s, 1H), 7.87 (s, 1H), 7.43e7.21 (m, 3H), 7.16e7.07 (m, 1H),
6.74 (d, J ¼ 2.4 Hz, 1H), 6.64 (dd, J ¼ 8.4, 2.4 Hz, 1H), 6.47 (dd,
J ¼ 16.8,10.0 Hz,1H), 6.26 (dd, J ¼ 17.2, 2.0 Hz,1H), 5.76 (dd, J ¼ 10.0,
1.6 Hz,1H), 3.94 (t, J ¼ 6.0 Hz, 2H), 3.60e3.55 (m, 4H), 2.45e2.26 (m,
6H), 2.13 (s, 3H), 1.90e1.78 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
NMR (400 MHz, DMSO-d6):
d 10.17 (s, 1H), 9.11 (s, 1H), 8.88 (s, 1H),
8.12 (s, 1H), 7.86 (s, 1H), 7.49 (d, J ¼ 7.6 Hz, 1H), 7.34 (d, J ¼ 7.2 Hz,
1H), 7.31e7.12 (m, 3H), 6.67 (d, J ¼ 8.0 Hz, 1H), 6.45 (dd, J ¼ 16.8,
10.0 Hz, 1H), 6.25 (d, J ¼ 16.8 Hz, 1H), 5.75 (dd, J ¼ 17.6, 7.6 Hz, 1H),
3.86 (t, J ¼ 6.0 Hz, 2H), 3.57 (s, 3H), 3.56e3.47 (m, 4H), 2.45e2.30
d
163.49, 159.79, 156.16, 156.02, 155.33, 139.60, 139.28, 134.63,
132.41, 131.28, 128.76, 127.69, 127.29, 118.16, 116.27, 115.12, 114.17,
111.97, 103.49, 66.68 (2C), 66.20, 55.36, 53.86 (2C), 26.38, 18.70;
HRMS (ESI) m/z calcd for C27H32ClN6O3 [MþH]þ 523.2219, found
523.2264.
(m, 6H),1.85e1.68 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
d 163.54,
158.24, 156.45, 155.20, 149.39,143.32, 139.55, 139.39, 134.73,132.37,
129.04, 127.34, 119.31, 115.76, 115.35, 114.24, 111.54, 105.31, 103.94,
67.55, 66.66 (2C), 55.80, 55.44, 53.85 (2C), 26.56; HRMS (ESI) m/z
calcd for C27H32ClN6O4 [MþH]þ 539.2168, found 539.2207.
5.2.13. N-(3-((5-chloro-2-(2-methoxy-5-((1-morpholino)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7m)
Yellow-white solid (103 mg, 19.6%); MS (ESI) m/z 539.2 [MþH]þ;
5.2.9. N-(3-((5-chloro-2-(3-methyl-4-((1-ethylpiperazine)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7i)
Off-white solid (59 mg, 13.8%); MS (ESI) m/z 550.3 [MþH]þ; 1H
1H NMR (400 MHz, DMSO-d6):
d 10.18 (s, 1H), 9.11 (s, 1H), 8.88 (s,
1H), 8.11 (s, 1H), 7.87 (s, 1H), 7.48 (d, J ¼ 7.6 Hz, 1H), 7.37e7.23 (m,
2H), 7.18 (d, J ¼ 9.2 Hz, 2H), 6.68 (d, J ¼ 8.4 Hz,1H), 6.45 (dd, J ¼ 16.8,
10.0 Hz, 1H), 6.24 (dd, J ¼ 16.8, 2.0 Hz, 1H), 5.75 (dd, J ¼ 10.0, 2.0 Hz,
1H), 3.68 (t, J ¼ 4.8 Hz, 2H), 3.65 (s, 3H), 3.57e3.51 (m, 4H), 2.33 (t,
J ¼ 7.2 Hz, 6H), 1.82e1.73 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
NMR (400 MHz, DMSO-d6): d 10.14 (s, 1H), 8.68 (s, 1H), 8.40 (s, 1H),
8.01 (s, 1H), 7.90 (s, 1H), 7.34 (d, J ¼ 5.2 Hz, 2H), 7.25 (d, J ¼ 8.4 Hz,
1H), 7.18e7.00 (m, 1H), 6.73 (d, J ¼ 2.0 Hz, 1H), 6.64 (d, J ¼ 8.8 Hz,
1H), 6.49 (dd, J ¼ 16.4, 9.2 Hz, 1H), 6.25 (dd, J ¼ 16.8, 9.2 Hz, 1H),
5.75 (d, J ¼ 10.0 Hz, 1H), 3.93 (t, J ¼ 6.0 Hz, 2H), 2.48e2.25 (m, 12H),
2.13 (s, 3H), 1.92e1.72 (m, 2H), 1.00 (t, J ¼ 7.2 Hz, 3H); 13C NMR
d
163.07, 157.75, 155.90, 154.78, 147.83, 143.82, 139.09, 138.91,
134.05, 131.90, 128.53, 126.86, 118.74, 115.15, 114.68, 112.27, 111.06,
105.76, 103.42, 66.26, 66.17 (2C), 55.88, 54.87, 53.34 (2C), 25.89;
HRMS (ESI) m/z calcd for C27H31ClN6O4P [MþH]þ 539.2168, found
539.2185.
(100 MHz, DMSO-d6):
d 163.50, 159.78, 156.15, 156.02, 155.31,
139.61, 139.32, 134.65, 132.45, 131.29, 128.75, 127.69, 127.23, 118.11,
116.27, 115.11, 114.12, 111.97, 103.49, 66.21, 54.79 (2C), 52.85 (2C),
52.56, 51.94, 26.65, 18.72, 12.09; HRMS (ESI) m/z calcd for
C
29H37ClN7O2 [MþH]þ 550.2692, found 550.2640.
5.2.14. N-(3-((5-fluoro-2-(2-methoxy-5-((1-morpholino)propoxy)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7n)
Off-white solid (107 mg, 18.6%); MS (ESI) m/z 523.2 [MþH]þ; 1H
5.2.10. N-(3-((5-fluoro-2-(3-methyl-4-((1-ethylpiperazine)
propoxy)phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide
(7j)
NMR (400 MHz, DMSO-d6): d 10.13 (s, 1H), 9.39 (s, 1H), 8.95 (s, 1H),
8.08 (s, 1H), 7.91 (s, 1H), 7.54 (d, J ¼ 8.0 Hz, 1H), 7.42 (d, J ¼ 8.0 Hz,
1H), 7.29e7.18 (m, 3H), 6.74 (d, J ¼ 8.4 Hz, 1H), 6.46 (dd, J ¼ 16.8,
10.0 Hz, 1H), 6.25 (dd, J ¼ 16.8, 2.0 Hz, 1H), 5.75 (dd, J ¼ 10.0, 2.0 Hz,
1H), 3.76 (t, J ¼ 6.0 Hz, 2H), 3.66 (s, 3H), 3.60e3.50 (m, 4H),
2.40e2.18 (m, 6H), 1.87e1.70 (m, 2H); 13C NMR (100 MHz, DMSO-
Yellow-white solid (52 mg, 12.5%); MS (ESI) m/z 534.3 [MþH]þ;
1H NMR (400 MHz, DMSO-d6):
d 10.09 (s, 1H), 9.28 (s, 1H), 8.20 (s,
1H), 7.96 (s, 2H), 7.50 (d, J ¼ 7.2 Hz, 1H), 7.28 (d, J ¼ 6.8 Hz, 2H),
7.13e7.00 (m, 1H), 6.78 (d, J ¼ 21.2 Hz, 1H), 6.67 (d, J ¼ 8.0 Hz, 1H),
6.48 (dd, J ¼ 16.4, 9.6 Hz, 1H), 6.25 (d, J ¼ 16.0 Hz, 1H), 5.75 (d,
J ¼ 10.0 Hz, 1H), 3.95 (t, J ¼ 6.0 Hz, 2H), 2.48e2.23 (m, 12H), 2.15 (s,
3H), 1.94e1.72 (m, 2H), 1.01 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz,
d6):
d
163.53, 156.11 (d, J ¼ 11.2 Hz), 150.09 (d, J ¼ 42.8 Hz), 148.37,
144.10, 142.06, 141.27 (d, J ¼ 79.2 Hz), 139.60 (d, J ¼ 14.8 Hz), 135.07,
132.39 (2C), 129.12, 127.31, 117.58, 115.04, 113.49, 112.92, 111.17,
106.05, 66.81, 66.64 (2C), 56.42, 55.34, 53.81 (2C), 26.37; HRMS
(ESI) m/z calcd for C27H32FN6O4 [MþH]þ 523.2464, found 523.2438.
DMSO-d6):
d
163.49, 157.62 (d, J ¼ 8.4 Hz), 155.85, 150.01 (d,
J ¼ 41.6 Hz), 141.92, 141.31 (d, J ¼ 77.6 Hz), 139.96, 139.42 (d,
J ¼ 43.2 Hz), 134.57, 132.46, 131.91, 128.88, 127.45, 127.20, 116.68,
116.31, 114.56, 112.65, 112.02, 66.23, 54.79 (2C), 52.81 (2C), 52.53,
51.93, 26.66, 18.76, 12.05; HRMS (ESI) m/z calcd for C29H37FN7O2
[MþH]þ 534.2987, found 534.2903.
5.3. Kinase enzymatic assays
All the enzymatic assays were tested applying with the ADP-
Glo™ assay system (BTK: Catalog. V2941, JAK3: Catalog. V3701),
were purchased from Promega Corporation (USA). The experiments
were performed according to the instructions of the manufacturer.
The detailed and complete protocols, and the active kinase data
were available at: https://cn.promega.com/products/cell-signaling/
kinase-assays-and-kinase-biology/btk-kinase-enzyme-system/?
5.2.11. N-(3-((5-chloro-2-(3-methyl-4-((1-methylpiperazine)
propoxy)phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide
(7k)
Off-white solid (62 mg, 17.4%); MS (ESI) m/z 537.3 [MþH]þ; 1H
NMR (400 MHz, DMSO-d6): d 10.11 (s, 1H), 8.68 (s, 1H), 8.39 (s, 1H),
8.01 (s, 1H), 7.90 (s, 1H), 7.39e7.23 (m, 3H), 7.10 (dd, J ¼ 16.0, 8.0 Hz,
1H), 6.73 (d, J ¼ 2.4 Hz, 1H), 6.63 (dd, J ¼ 8.8, 2.8 Hz, 1H), 6.48 (dd,
J ¼ 16.8,10.0 Hz,1H), 6.26 (dd, J ¼ 16.8,1.6 Hz,1H), 5.75 (dd, J ¼ 10.0,
2.0 Hz,1H), 3.92 (t, J ¼ 6.4 Hz, 2H), 2.47e2.31 (m, 8H), 2.24e2.14 (m,
catNum¼V2941
and
https://cn.promega.com/products/cell-
signaling/kinase-assays-and-kinase-biology/jak3-kinase-enzyme-
system/?catNum¼V3701), respectively. For all of the tested com-
pounds, concentrations consisting of suitable levels from 0.1 to
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10
Y. Ge et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
1000 nM were used. The test was performed in a 384-well plates,
including the main steps below: (1) perform a 5 L kinase reaction
using 1 ꢁ kinase buffer (e.g., 1 ꢁ reaction buffer A), (2) incubate at
room temperature for 60 min, (3) add 5 L of ADP-Glo™ reagent to
V-FITC (5 mL)/propidium iodide (5 mL), and analyzed by flow
cytometry assay (Becton-Dickinson, USA).
m
m
5.8. Western blotting assay
stop the kinase reaction and deplete theunconsumed ATP, leaving
only ADP and a very low background of ATP, (4) incubate at room
temperature for 40 min, (5) add 10 mL of kinase detection, (6) re-
The lysates from Namalwa cells in different groups were
extracted and centrifuged at 14,000 g for 15 min at 4 ꢀC, then the
agent to convert ADP to ATP andintroduce luciferase and luciferin
to detect ATP, (7) incubate at room temperature for 30 min, (8)
plates was measured on TriStar® LB942 Multimode Microplate
Reader (BERTHOLD) to detect the luminescence (Integration time
0.5e1 s). Curve fitting and data presentations were performed us-
ing GraphPad Prism version 5.0.
total proteins were obtained. An aliquot (50 mg protein) was loaded
onto a 8%-12% SDS-PAGE gels and separated electrophoretically.
Then the target proteins were transferred to a PVDF membrane
(Millipore, USA). After blocking the PVDF membrane in 5% dried
skim milk (Boster Biological Technology, China) for 2 h at room
temperature, the membrane was incubated overnight at 4 ꢀC with
primary antibodies for 12 h. Protein detection was performed based
on an enhanced chemiluminescence (ECL) method and photo-
graphed by using a BioSpectrum Gel Imaging System (HR410, UVP,
5.4. Cell and reagents
Ramos, Raji and Namalwa cells were purchased from Fuheng
Biology Company (Shanghai, China). Peripheral blood mononuclear
cells (PBMC) were obtained from a healthy adult male. The Cell
Counting Kit-8 (CCK-8) reagent was purchased from Biotool Com-
pany (Swizerland). The BTK enzyme and the ADP-Glo™ Kinase
Assay system that measures ADP formed from a kinase reaction
were purchased from Promega Corporation (USA).
USA). In order to eliminate the variations, data were adjusted to b-
Actin expression: IOD of objective protein versus IOD of
expression.
b-Actin
5.9. Mouse tumor xenograft efficacy study
The female CB17/SCID mice weighing 20-25 g in 5e6 week-old
were provided by the Model Animal Research Center of Nanjing
University (Nanjing, China) (SCXK: 2015-0001). All animals were
housed in a controlled environment at 23 2 ꢀC under a 12-h dark/
light cycle with free access to food and water. The animal mainte-
nance and experiments were performed in accordance with the
guidelines of the Animal Care and Use Committee. Ramos cells
(3 ꢁ 106) were suspended in 0.1 mL of PBS and injected subcuta-
neously into the right oxter region of the mice. Three days after
implantation, the mice were randomly divided into seven groups in
which the mice in control group were received by oral 25% PEG-
400, and the mice in other six groups were oral administrated
with 7b and 7e at the doses of 30 and 60 mg/kg for 14 days. In the
process, the side length of the tumor was measured to calculate the
tumor volume based on the formula: V ¼ A ꢁ B2/2, where A means
the lager perpendicular diameters and B is the smaller perpendic-
ular diameters. At the end of the test, the animals were sacrificed
and the tumors were obtained, photographed and weighted.
5.5. Cellular activity assay
All the cell viability assays were performed according to the
CCK-8 method. The cells were seeded in 96-well plates at a density
of 3000 cells/well and were maintained at 37 ꢀC in a 5% CO2
incubator in RPMI-1640 containing 10% fetal bovine serum (FBS,
Gibco) for 4 h. Cells were exposed to treatment for 72 h, and the
number of cells used per experiment for each cell lines was
adjusted to obtain an absorbance of 0.5e1.2 at 450 nm with a
microplate reader (Thermo Fisher, USA). Compounds were tested at
appropriate concentrations (1.25e40 mM), with each concentration
duplicated five times. The IC50 values were calculated using
GraphPad Prism version 5.0.
5.6. Cytotoxic activity assay
Peripheral venous blood was collected in an ACD anticoagulant
(Vblood: V
¼ 9:1) tube from the normal healthy adult
Acknowledgement
anticoagulant
male. The leucocyte-rich plasma was removed and the peripheral
blood mononuclear cells (PBMC) consisting of monocytes and the
lymphocytes were separated on Lymphoprep (density 1.077 g/ml,
Nyegaard & Co. As., Oslo, Norway). The cells collected from the
interface layer were washed three times with PBS buffered salt
solution and counted using cell counting chamber.
We are grateful to the National Natural Science Foundation of
China (No. 81402788), and the Cultivation Project of Youth Tech-
stars, Dalian, China (No. 2016RQ043) for the financial support of
this research.
PBMC cells were seeded in 24-well plates at a density of 200,000
to 250,000 cells/well. Cells were treated with inhibitors 7b and 7e
Appendix A. Supplementary data
at appropriate concentrations (5, 10, 20 mM), with each concen-
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2017.10.080.
tration duplicated five times. Aftering maintaining the cells at 37 ꢀC
in a 5% CO2 incubator in RPMI-1640 containing 10% fetal bovine
serum (FBS, Gibco) for 24 h, the morphology of the cells was
observed with a phase contrast microscope (Nikon, Japan), and the
number of living PBMC cells was calculated by traditional manual
method with an ordinary cell counting chamber.
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treatment of B-cell lymphoma, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.080