JAK3 inhibitors based on thieno[3,2-d]pyrimidine scaffold: design, synthesis and bioactivity evaluation for the treatment of B-cell lymphoma

Article abstract of DOI:10.1016/j.bioorg.2019.103542

JAK3 is predominantly expressed in hematopoietic cells and has been a promising therapeutic target for the treatment of B-cell lymphoma. In this study, a new class of thieno[3,2-d]pyrimidines harboring acrylamide pharmacophore were synthesized as potent covalent JAK3 inhibitors (IC₅₀ < 10 nM). Among them, 9a and 9 g displayed the strongest inhibitory potency against JAK3 kinase activity, with IC₅₀ values of 1.9 nM and 1.8 nM, respectively. Furthermore, compared with the reference agents, Spebrutinib and Ibrutinib, 9a not only demonstrated enhanced antiproliferative activity against B lymphoma cells, but also showed very weak proliferative inhibition against normal peripheral blood mononuclear cells (PBMCs) at a concentration of 20 μM. Analysis of the mechanism revealed that 9a could induce the obvious apoptosis in B lymphoma cells and prevent JAK3-STAT3 cascade as well as BTK pathway. Taken together, 9a may be served as a potential new JAK3 inhibitor for the treatment of B-cell lymphoma.

Full text of DOI:10.1016/j.bioorg.2019.103542

BioorganicChemistry95(2020)103542
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
JAK3 inhibitors based on thieno[3,2-d]pyrimidine scaffold: design, synthesis
and bioactivity evaluation for the treatment of B-cell lymphoma
Fuyun Chia,1, Lixue Chena,b,1, Changyuan Wanga, Lei Lia, Xiuli Sunc, Youjun Xub, Tengyue Mad,
⁎
⁎
Kexin Liua, Xiaodong Maa, , Xiaohong Shua,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China
b School of Pharmaceutical Engineering, and Key Laboratory of Structure-Based Drug Design & Discovery (Ministry of Education), Shenyang Pharmaceutical University,
Shenyang 110016, PR China
c Department of Hematology, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China
d Dalian Buyun Biotechnology Co., Ltd. 116085, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
JAK3 is predominantly expressed in hematopoietic cells and has been a promising therapeutic target for the
treatment of B-cell lymphoma. In this study, a new class of thieno[3,2-d]pyrimidines harboring acrylamide
pharmacophore were synthesized as potent covalent JAK3 inhibitors (IC50 < 10 nM). Among them, 9a and 9 g
displayed the strongest inhibitory potency against JAK3 kinase activity, with IC50 values of 1.9 nM and 1.8 nM,
respectively. Furthermore, compared with the reference agents, Spebrutinib and Ibrutinib, 9a not only de-
monstrated enhanced antiproliferative activity against B lymphoma cells, but also showed very weak pro-
liferative inhibition against normal peripheral blood mononuclear cells (PBMCs) at a concentration of 20 μM.
Analysis of the mechanism revealed that 9a could induce the obvious apoptosis in B lymphoma cells and prevent
JAK3-STAT3 cascade as well as BTK pathway. Taken together, 9a may be served as a potential new JAK3
inhibitor for the treatment of B-cell lymphoma.
B-cell lymphoma
JAK3 inhibitor
Thieno[3,2-d]pyrimidines
1. Introduction
an essential role in T lymphocyte development, proliferation and dif-
ferentiation and its deficiency causes impaired T lymphocyte immunity,
Janus Kinase (JAK) is a family of non-receptor tyrosine kinases that
transduce signal from transmembrane receptors to the nucleus and
further modulate the transcription of target genes [1–3]. In mamma-
lian, JAK family has four members: JAK1, JAK2, JAK3 and Tyk2 [4].
Unlike other JAKs that are ubiquitously expressed and associated with
diverse cytokine receptors, JAK3 is predominantly expressed in hema-
topoietic cells and is induced by cytokine receptors that contain a
common γ chain, such as receptors for interleukin2 (IL2), IL4, IL7, and
IL21 [5,6]. After phosphorylation by a JAK, STAT forms hetero- or
homo-dimers and is translocated to the cell nucleus, where it induces
the transcription of target genes [7,8]. The JAK-STAT signaling
pathway conveys the extracellular signals from various peptides, in-
cluding many cytokines and chemokines as well as growth factors and
hormones, directly to the nucleus to induce a variety of cellular re-
sponses, which is of fundament importance in innate immunity, in-
flammation, and hematopoiesis, and dysregulation is frequently ob-
served in immune disease and cancer. JAK3 also has been shown to play
leading to severe combined immunodeficiency [9].
Various JAK inhibitors have been evaluated in clinical trials, in-
cluding Tofacitinib (1) [10], Ruxolitinib (2) [11], Pacritinib (3) [12],
CYT-387 (4) [13], AZD1480 (5) [14] and others [15] (Fig. 1). Recent
studies have shown that the expression of JAK3 pathway genes are
upregulated in different diagnostic classes of B-lineage lymphoid ma-
lignancies and are associated with the overexpression of genes for
JAK3-activating cytokines [16,17]. It is different from the other JAKs, a
unique cysteine residue (Cys909) locates in the gatekeeper-plus-7
(GK + 7) position of JAK3, which makes it possible to develop covalent
binding inhibitors [18]. 6 [19], 7 [20] and 8 [21] have been reported as
selective covalent JAK3 inhibitors. These findings have collectively
identified JAK3 as an attractive molecular target for disrupting the
constitutively active anti-apoptotic STAT3 and STAT5 signaling path-
ways in lymphoma cells.
In our continuous effort to discover potent EGFRT790M and BTK
inhibitors, we have identified several classes of pyrimidine derivatives
⁎ Corresponding authors.
E-mail addresses: xiaodong.ma@139.com (X.D. Ma), xiaohong_shu@dlmedu.edu.cn (X.H. Shu).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.103542
Received 30 September 2019; Received in revised form 2 December 2019; Accepted 21 December 2019
Availableonline24December2019
0045-2068/©2019PublishedbyElsevierInc.

F. Chi, et al.
BioorganicChemistry95(2020)103542
Fig. 1. The structures of potent JAK inhibitors.
as potential anticancer agents in our previous work [22–26]. Notably,
the residue is Cys909 in human JAK3, and it is structurally similar to
cysteine residues in EGFR (Cys797) and BTK (Cys481) that have been
successfully targeted by covalent kinase inhibitors which are now ap-
proved drugs. Considering the structural similarity of the catalytic re-
gions of these kinases, skillful structural transformation for the EGFR or
BTK inhibitors may be a good protocol to discover new JAK3 inhibitors.
The docking suggested that introduction of thiazole ring could come
into closer contact with the gatekeeper Met902 in JAK3 to give en-
hanced interaction. We surmised that was a critical point for the ex-
ploration of selective JAK3 inhibitors. To investigate more effective
JAK3 inhibitors, in this work, BTK inhibitor Spebrutinib as the lead, a
family of thieno[3,2-d]pyrimidines harboring acrylamide pharmaco-
phore was synthesized and systematical structural modification was
carried out here (see Fig. 2). In addition, biological activity against
JAK3 and B lymphoma cells in vitro were also investigated in detail in
this study.
2. Results and discussion
2.1. Chemistry
The synthetic routes of the title compounds 9a–k were depicted in
Schemes 1–3. The commercially available starting material 3-amino-
phenol (10) was reacted with acryloyl chloride to produce 11 which in
the next step substituted the C-4 chloride atom in 2,4-di-
chlorothiopheno [3,2-d]pyrimidine to give the key intermediate 12.
Additionally, 13 was reacted with 1-bromo-3-chloropropane to syn-
thesize 14. Intermediate 14 was reacted with morpholine and then
reduced by Fe-NH4Cl reagent to get aniline 16. The nucleophilic sub-
stitution reaction of the chlorine atom in 12 with 16 was occurred
under the action of trifluoroacetic acid (TFA) to obtain the desired 9a
and 9b. For the synthesis of 9c–i, a condensation reaction was per-
formed in the first step using the EDCI and HOBT; then, the desired 9c–i
was conveniently prepared via reduction and nucleophilic substitution
reaction. The preparation of 9j–k using the method below: 4-
Fig. 2. Discovery strategy of the title molecules as JAK3 inhibitors.
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F. Chi, et al.
BioorganicChemistry95(2020)103542
Scheme 1. The synthetic route of 9a, b.
nitroaniline was reacted with bromoacetyl bromide to prepare the
bromo-substituted 21; then, through a nucleophilic substitution reac-
tion with morpholine and a reduction reaction using Fe-NH4Cl reagents,
the aniline 23 was synthesized; additionally, in the presence of TFA, 23
was conveniently converted to the title molecules 9j–k by reacting with
12.
into the C-3 aniline, the produced 9f and 9k showed about 38 and 250
times lower activity than that of 9d and 9j, respectively. On the other
hand, the deletion of a methylene group in the aniline side chain of 9i
was beneficial to the inhibitory activity against JAK3. Overall, these
work led to the successful identification of several thieno[3,2-d]pyr-
imidines with improved anti-JAK3 activity compared with reference
agents.
Reagents and conditions: (a) Acryloylchloride, NaHCO3, CH3CN,
0 °C, 0.5 h, 95%; (b) 2,4-dichlorothiopheno [3,2-d]pyrimidine, K2CO3,
KI, DMF, 81%; (c) 1-bromo-3-chloropropane, K2CO3, CH3CN, reflux,
12 h, 77–82%; (d) Morpholine, K2CO3, KI, DMF, 100 °C, 5 h, 72–85%;
(e) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 66–70%; (f) Trifluoroacetic acid,
2-BuOH, 100 °C, 12 h, 9.3–21%.
These newly obtained thieno[3,2-d]pyrimidines were also evaluated
for their capability to inhibit the proliferation of B lymphoma cell lines
(Ramos, Raji and Namalwa). The results shown in Table 1 indicated
that most of these compounds interfered with the proliferation of B
lymphoma cells with IC50 values lower than 20 μM. The representative
9b displayed the strongest activity against both Ramos (IC50, 9.7 μM)
and Raji cells (IC50, 5.3 μM), despite its moderate activity against JAK3
(IC50, 95.2 nM). 9k was the most potent inhibitor against Namalwa cells
(IC50, 6.2 μM), but only exhibited very weak activity against JAK3
(IC50, 568.9 nM), suggesting that it may block the cell proliferation
through a signaling pathway independent of JAK3. 9d not only dis-
played strong activity against JAK3 (IC50, 2.3 nM), but also effectively
interfered with the proliferation of B lymphoma cells with IC50 values
lower than 15.1 μM. The cell-based test results showed that the short
linker between the morpholine and phenyl was unfavorable for the
inhibition of B lymphoma cells. The prototypical 9h and 9i showed
reduced anti-proliferative activity against B lymphoma cells in this fa-
mily of inhibitors. The results presented in Fig. 3 clearly indicated that
9a exhibited a dose-and time-dependent inhibition of Ramos and Raji
cells with concentrations ranging from 2.5 to 20 μM. In addition, the
anti-proliferative activity of 9a against B lymphoma cells (Ramos cells
and Raji cells) was also evaluated by the acridine orange/ethidium
bromide (AO/EB) staining assay (Fig. 4). After treatment with 9a for
Reagents and conditions: (a) Morpholine, EDCI, HOBT, rt, 12 h,
47–71%; (b) Fe-NH4Cl, MeOH-H2O,
2 h, 70 °C, 71–83%; (c)
Trifluoroacetic acid, 2-BuOH, 100 °C, 12 h, 9–34%.
Reagents and conditions: (a) Bromoacetyl bromide, NaHCO3,
CH3CN, rt, 5 h, 93–95%; (b) Morpholine, K2CO3, CH3CN, reflux, 5 h,
85–90%; (c) Fe-NH4Cl, MeOH-H2O,
2 h, 70 °C, 73–81%; (d)
Trifluoroacetic acid, 2-BuOH, 100 °C, 12 h, 12.6–21%.
2.2. Biological activity
2.2.1. The inhibitory evaluation of kinase and cells proliferation
All these title molecules were evaluated for their inhibitory effects
on JAK3. As shown in Table 1, most of the target compounds sig-
nificantly inhibited the enzymatic activity with an IC50 ranging from
1.8 to 568.9 nM among which 9a (IC50, 1.9 nM) and 9g (IC50, 1.8 nM)
showed the strongest activity against JAK3. Regarding the structure and
activity relationships, most of the introduced groups were favorable for
the inhibition of JAK3. However, when a chlorine atom was introduced
Scheme 2. The synthetic route of 9c–i.
3

F. Chi, et al.
BioorganicChemistry95(2020)103542
Scheme 3. The synthetic route of 9j, k.
Table 1
Inhibitory activity against BTK, JAK3 and B lymphoma cells of 9a-k.a
b
Compounds
R1
R2
Enzymatic activity (IC50, nM)
Antiproliferative activity (IC50, μM)c
BTK
JAK3
Ramos
Raji
Namalwa
L-02
9a
9b
9c
2′-CH3
3′-OCH3
H
> 100.00
90.14
1.89
95.21
7.31
0.04
10.44
9.61
0.14
0.44
9.43
5.32
18.57
0.14
0.13
9.92
0.30
23.44
10.27
10.98
0.08
0.09
0.11
0.04
0.02
0.13
14.17
14.39
0.37
0.53
80.32
0.08
13.51
0.40
0.26
0.29
0.24
0.11
0.31
0.41
0.27
9d
9e
9f
H
> 100.00
33.60
2.27
0.04
15.11
19.00
18.57
19.66
29.08
28.22
7.54
0.22
0.24
9.73
0.43
18.26
19.56
11.93
18.28
16.51
14.88
0.30
0.25
0.13
0.19
0.38
0.07
2′-CH3
3′-Cl
3′-OCH3
H
0.04
0.09
0.28
20.61
87.02
1.78
0.05
0.16
0.06
0.13
0.09
0.07
9.72
19.53
14.63
18.91
22.63
12.37
0.16
0.56
0.13
0.45
0.22
25.28
12.13
9.90
0.31
0.11
9g
9h
9i
19.96
> 100.00
> 100.00
2.13
> 40.00
28.92
H
460.20
0.46
9j
H
24.05
0.47
8.77
20.36
18.14
0.67
0.30
0.40
19.76
18.66
0.11
0.20
14.51
6.23
0.42
3.10
6.46
0.06
0.12
9k
3′-Cl
> 100.00
568.90
0.03
0.04
0.53
0.41
Spebrutinib
Ibrutinib
0.72
0.34
0.04
0.03
66.54
16.11
14.62
5.01
19.14
10.42
0.09
0.14
18.72
9.63
< 2.50
10.87
0.07
0.11
0.38
0.15
a
b
Data represent the mean of at least three separate experiments, the exact concentrations for cytotoxic studies were 1.25 µM, 5 µM, 10 µM, 20 µM and 40 µM.
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 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.
48 h, the proliferation of B lymphoma cells was markedly blocked at
concentration below 20 μM, which was not the case with Spebrutinib.
than that of Spebrutinib. Taken together, these findings indicated that
9a could serve as a promising JAK inhibitor for the treatment of B-cell
lymphoma after further biological evaluations.
2.2.2. Cell cytotoxicity evaluation
The effects of 9a on the viability of Ramos, Raji, Namalwa cells and
L-02 cells were evaluated by cck-8 method (Fig. 5). With the increasing
of the concentrations of 9a, the viability of Ramos, Raji, Namalwa cells
decreased suddenly. When 9a inhibited B lymphoma cells by a half, L-
02 still remained high viability. Meanwhile, the cell cytotoxicity of 9a
against normal peripheral blood mononuclear cells (PBMCs) was also
evaluated by the AO/EB staining assay (Fig. 6). Clearly, 9a did not
significantly inhibit the proliferation of PBMCs at a concentration of
20 μM as well as the approved kinase inhibitor Spebrutinib. The results
of cytotoxic evaluation showed that 9a possessed less effect on cells line
2.2.3. Flow cytometry analysis
In addition, to further investigate the antiproliferative mechanism
of these inhibitors against B lymphoma cells, the effects of the most
active inhibitor 9a on apoptosis and on the cell cycle in Ramos cell line
were also examined using flow cytometry analysis. For comparison,
Spebrutinib was also evaluated as the reference compound. The results
presented in Fig. 7 revealed that 9a induced apoptosis in Ramos cells in
a dose- and time-dependent manner. Additionally, the apoptosis rate
increased from 19.1 to 49.2% after 48 h with increasing concentration
from 5 to 10 μM. Treatment with identical concentration (20 μM) of
4

F. Chi, et al.
BioorganicChemistry95(2020)103542
Fig. 3. The effects of treating time and concentration of 9a against B lymphoma cells (Ramos cells and Raji cells). *p < 0.05, ** p < 0.01, *** p < 0.001 compared
with control group.
Spebrutinib and 9a for 48 h, Ramos cells obtained 44.1 and 78.2% loss
of cell viability, respectively. The effects of 9a on the cell-cycle pro-
gression of Romas cells were shown in Fig. 8. Incubation with 9a at
concentrations from 5 to 20 μM for 48 h, compared with the control
group, the percentage of Ramos cells in the G2/M phase increased from
20.4 to 38.9%, and those in the G0/G1 decreased from 51.9 to 35.5%.
Apparently, the newly synthesized 9a blocked the cell cycle in Ramos
cells at the G2/M phase.
molecular docking performance. As depicted in Fig. 10 A-D, all the four
potent JAK3 inhibitors could form a potential covalent bond between
the acrylamide and Cys909 in the JAK3 protein. However, for the
weaker inhibitors 9k (Fig. 10 E) and 9i (Fig. 10 F), this important bond
disappeared due to the long distance (> 10.10 Å) between the acryla-
mide group and the Cys909 residue. Clearly, the acrylamide group and
morpholine substrate in both 9k and 9i were in the opposite position in
the JAK3 binding pocket, thus resulting in a long distance. Notably, for
9a (Fig. 10 D) and 9g (Fig. 10 C), the newly introduced thiazole ring
could come into closer contact with Met902 than that in Spebrutinib,
which possibly led to the higheranti-JAK3 activity. In short, these
binding models reasonably explained their activity against JAK3.
2.2.4. Effects of the inhibitors on BTK and JAK3 activation and
downstream signaling
To gain a better insight into the underlying mechanism of JAK3
inhibitors, the inhibitory effects of 9a on the JAK-STAT3 activation and
downstream signaling were examined in Namalwa cells using
Spebrutinib as a comparison control. In addition, considering that BTK
protein was also highly expressed in Namalwa cells, the effects of 9a on
BTK activation were also evaluated. The cells were treated with dif-
ferent concentrations (5, 10, 20 μM) of inhibitors for 24 h, and the
levels of STAT3, p-STAT3, BTK and p-BTK was determined by immune
blotting analysis. The results summarized in Fig. 9 revealed that the
phosphorylation of STAT3 which located downstream of the signaling
pathway was inhibited by 9a in Namalwa cells in a dose-dependent
manner. 9a was also found to clearly downregulated the expression of
p-BTK. These results indicated that 9a could block the phosphorylation
of STAT3 and then restrain the activation of the signaling pathway.
3. Conclusion
JAK3 is an important therapeutic target for the treatment of B-cell
lymphoma. Targeting JAK3 may result in several outstanding ad-
vantages in the treatment of malignant tumors, including improved
therapeutic efficacy, safety and resistance profiles by collective reg-
ulations of a primary therapeutic target together with compensatory
elements and resistance activities. In this study, several potent JAK3
inhibitors were identified and evaluated for the treatment of B-cell
lymphoma. Most of these compounds displayed strong inhibitory effects
against JAK3 at concentrations below 10 nM. In particular, 9a and 9g
were the strongest JAK3 inhibitors in this class of kinase inhibitors,
with IC50 values less than 2 nM. In addition, the cell-based assay results
revealed that 9a exhibited enhanced anticancer activity compared with
the reference agents. Additionally, the normal PBMCs were found to be
not sensitive to 9a, indicating its low cell cytotoxicity. Furthermore,
analysis of the mechanism of these compounds revealed that 9a in-
duced significant apoptosis in Romas cells by arresting the cell cycle at
G2/M stage and blocked JAK3-STAT3 cascade. Overall, these newly
discovered JAK3 inhibitors are of great therapeutic value and warrant
further study as potential agents for the treatment of B-cell lymphoma.
2.2.5. Molecular modeling analysis
To determine the relationship between the inhibitory effect of the
title molecules against JAK3 and their binding models with JAK3, four
representative compounds, namely 9a, 9g, 9k and 9i, were individually
docked into the binding pocket of the JAK3 protein. Similarly,
Spebrutinib was also docked into the binding pocket. The crystal
structure of the prototypical molecule 8 with JAK3 protein was used for
comparison. The AutoDock 4.2 software (The Scripps Research
Institute, La Jolla, CA, USA) and its default parameters were used for
5

F. Chi, et al.
BioorganicChemistry95(2020)103542
Fig. 4. Morphological changes of the Ramos, Raji cells treated by 9a (200×, final magnification) at concentrations of 5, 10, 20 μM for 48 h.
4. Experimental section
4.2. General procedure for the synthesis of 9a–k
4.1. General methods for chemistry
4-Morpholine-substituted aniline intermediates 12, 16, 19, 23 were
generally prepared referencing the procedures showed in literatures
[22–26]. All these intermediates were directly used without any pur-
ification and structural characterization. With these intermediates in hand,
the desired compounds were synthesized as described below. A flask was
charged with 12 (0.70 mmol), anilines 16, 19, 23 (0.70 mmol), TFA
(1.05 mmol), and 2-BuOH (10 mL). The slurry was heated to 100 °C for
5 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 di-
chloromethane/methanol (v/v, 30:1) as eluents.
Unless otherwise indicated, all reagents and solvents were obtained
from commercial sources and used as received. 1H and 13C NMR data
were obtained on a Bruker Avance at 400 and 100 MHz, respectively.
Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ)
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
Triple TOF® 4600 LC/MS/MS system. All reactions were monitored by
TLC, using silica gel plates with fluorescence F254 and UV light vi-
sualization. Flash chromatography separations were obtained on Silica
Gel (300–400 mesh) using dichloromethane/methanol as eluents.
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F. Chi, et al.
BioorganicChemistry95(2020)103542
(m, 2H); 13C NMR (DMSO-d6): δ 165.1, 164.0, 163.9, 158.3, 152.7,
149.5, 142.7, 140.9, 137.3, 135.5, 132.1, 130.4, 127.8, 123.7, 117.3,
117.1, 115.0, 113.2, 111.2, 108.1, 105.0, 67.3, 64.4 (2C), 55.8, 54.6,
52.1 (2C), 24.4; HRMS (ESI) for
562.2119, found: 562.2127.
C
28H27N5O6S, [M+H]+ calcd:
4.2.3. N-(3-(2-(4-((1-morpholino)acetylthio)phenylamino)thieno[3,2-d]
pyrimidin-4-yloxy)phenyl) acrylamide(9c)
Yield 15.6%; off-white solid. 1H NMR (DMSO-d6): δ 10.38 (s, 1H),
9.61 (s, 1H), 8.33 (d, J = 5.4 Hz, 1H), 7.75 (t, J = 2.3 Hz, 1H), 7.62 (d,
J = 8.1 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.49 (t, J = 8.1 Hz, 1H),
7.39 (d, J = 5.4 Hz, 1H), 7.21 (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 8.1,
2.3 Hz, 1H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.27 (dd, J = 17.0,
2.0 Hz, 1H), 5.79 (dd, J = 10.1, 2.0 Hz, 1H), 3.80 (s, 2H), 3.55–3.50
(m, 4H), 3.45–3.39 (m, 4H); 13C NMR (DMSO-d6): δ 167.1, 165.1,
164.2, 163.8, 157.9, 152.7, 140.8, 140.3, 137.6, 132.1, 131.8 (2C),
130.5, 127.8, 125.9, 123.7, 119.5 (2C), 117.6, 117.2, 113.4, 108.4,
66.5 (2C), 46.6, 42.2, 37.2; HRMS (ESI) for C27H25N5O4S2, [M+H]+
calcd: 548.1421, found: 548.1411.
Fig. 5. The effects of treating for 72 h by 9a on B lymphoma cells (Ramos, Raji
and Namalwa cells) and human normal liver cells (L-02 cells). *p < 0.05, **
p < 0.01, *** p < 0.001 compared with L-02 group.
4.2.4. N-(3-(2-(4-((1-morpholino)acetoxyl)phenylamino)thieno[3,2-d]
pyrimidin-4-yloxy)phenyl)acrylamide(9d)
4.2.1. N-(3-(2-(4-(3-morpholinopropoxy)-2-methylphenylamino)thieno
[3,2-d]pyrimidin-4-yloxy) phenyl)acrylamide (9a)
Yield 23.1%; Pale-yellow solid. 1H NMR (DMSO-d6): δ 10.36 (s, 1H),
9.31 (s, 1H), 8.28 (d, J = 5.4 Hz, 1H), 7.73 (t, J = 2.2 Hz, 1H), 7.61 (d,
J = 8.1 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.45 (t, J = 8.1 Hz, 1H),
7.34 (d, J = 5.4 Hz, 1H), 7.09 (dd, J = 8.1, 2.2 Hz, 1H), 6.73 (d,
J = 8.6 Hz, 2H), 6.45 (dd, J = 17.0, 10.1 Hz, 1H), 6.28 (dd, J = 17.0,
2.0 Hz, 1H), 5.79 (dd, J = 10.1, 2.0 Hz, 1H), 4.72 (s, 2H), 3.63–3.54
(m, 4H), 3.49–3.44 (m, 4H); 13C NMR(DMSO-d6): δ 166.7, 165.3, 164.1,
163.8, 158.3, 153.1, 152.7, 140.8, 137.3, 134.6, 132.1, 130.4, 127.8,
123.7, 120.7 (2C), 117.6, 117.1, 114.8 (2C), 113.4, 107.7, 66.7, 66.5
Yield 9.3%; off-white solid. 1H NMR(DMSO-d6): δ 10.33 (s, 1H),
8.50 (s, 1H), 8.20 (d, J = 5.4 Hz, 1H), 7.71 (t, J = 2.0 Hz, 1H), 7.54 (d,
J = 8.2 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.22 (d, J = 5.4 Hz, 1H),
7.19 (d, J = 8.7 Hz, 1H), 7.05 (dd, J = 8.2, 2.0 Hz, 1H), 6.73 (d,
J = 2.6 Hz, 1H), 6.60 (dd, J = 8.7, 2.6 Hz, 1H), 6.46 (dd, J = 17.0,
10.1 Hz, 1H), 6.28 (dd, J = 17.0, 1.9 Hz, 1H), 5.79 (dd, J = 10.1,
1.9 Hz, 1H), 3.95 (t, J = 6.8 Hz, 2H), 2.58 (t, J = 4.6 Hz, 4H), 2.42 (t,
J = 6.8 Hz, 2H), 2.40–2.32 (m, 4H), 2.13 (s, 3H), 1.85 (qui, J = 6.8 Hz,
2H); 13C NMR (DMSO-d6): δ 165.6, 164.0, 163.8, 160.1, 156.0, 152.6,
140.7, 136.9, 134.8, 132.1, 131.3, 130.3, 127.8, 127.4, 123.6, 117.4,
116.9, 116.3, 113.2, 112.0, 107.3, 66.7 (2C), 66.2, 55.3, 53.8 (2C),
26.4, 18.8; HRMS (ESI) for C29H31N5O4S, [M+H]+ calcd: 546.2170,
found: 546.2155.
(2C), 45.3, 42.1; HRMS (ESI) for
532.1649, found: 532.1635.
C
27H25N5O5S, [M+H]+ calcd:
4.2.5. N-(3-(2-(4-((1-morpholino)acetoxyl)-2-methylphenylamino)thieno
[3,2-d]pyrimidin-4-yloxy) phenyl)acrylamide(9e)
Yield 9.3%; off-yellow solid. 1H NMR (DMSO-d6): δ 10.33 (s, 1H),
8.52 (s, 1H), 8.20 (d, J = 5.4 Hz, 1H), 7.77 (t, J = 2.3 Hz, 1H), 7.54 (d,
J = 8.1 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.24 (d, J = 5.4 Hz, 1H),
7.20 (d, J = 8.7 Hz, 1H), 7.07 (dd, J = 8.1, 2.3 Hz, 1H), 6.76 (d,
J = 3.0 Hz, 1H), 6.63 (dd, J = 8.7, 3.0 Hz, 1H), 6.45 (dd, J = 16.9,
10.1 Hz, 1H), 6.28 (dd, J = 16.9, 2.0 Hz, 1H), 5.79 (dd, J = 10.1,
2.0 Hz, 1H), 3.66–3.56 (m, 4H), 3.50–3.44 (m, 4H), 2.13 (s, 3H); 13C
NMR (DMSO-d6): δ 166.6, 165.6, 164.1, 163.8, 160.1, 155.4, 152.6,
140.7, 136.9, 134.8, 132.1, 131.8, 130.3, 127.8, 127.4, 123.6, 117.4,
116.9, 116.4, 113.2, 112.2, 107.4, 66.5 (2C), 66.5, 45.3, 42.1, 18.8;
4.2.2. N-(3-(2-(4-(3-morpholinopropoxy)-3-methoxylphenylamino)thieno
[3,2-d]pyrimidin-4-yloxy) phenyl)acrylamide(9b)
Yield 23.3%; Pale-yellow solid. 1H NMR (DMSO-d6): δ 10.46 (s, 1H),
9.29 (s, 1H), 8.28 (d, J = 5.3 Hz, 1H), 7.73 (s, 1H), 7.60 (d, J = 8.1 Hz,
1H), 7.44 (t, J = 8.1 Hz, 1H), 7.38 (s, 1H), 7.35 (d, J = 5.3 Hz, 1H),
7.19 (d, J = 8.6 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.76 (d, J = 8.6 Hz,
1H), 6.47 (dd, J = 16.8, 10.1 Hz, 1H), 6.24 (dd, J = 16.8, 2.0 Hz, 1H),
5.77 (dd, J = 10.1, 2.0 Hz, 1H), 3.94 (t, J = 5.72 Hz, 2H), 3.84–3.73
(m, 4H), 3.61 (s, 3H), 3.42–3.36 (m, 2H), 3.16–3.05 (m, 4H), 2.10–1.98
Fig. 6. Morphological changes of the PBMC cells treated by 9a (200×, final magnification) at concentrations of 5, 10, 20 μM for 24 h.
7

F. Chi, et al.
BioorganicChemistry95(2020)103542
Fig. 7. 9a induced Ramos cells apoptosis in vitro. The cells were incubated with the indicated concentrations of 9a for 48 h, and the cells were stained with annexin
V/FITC, followed by flow cytometry analysis. One representative experiment was shown. ** p < 0.01 compared with Spebrutinib group.
Fig. 8. Cell cycle arrest of the cells induced by 9a was determined by flow cytometric analysis. One representative experiment is shown. *p < 0.05 compared with
control group.
8

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BioorganicChemistry95(2020)103542
Fig. 9. (A) 9a inhibited the activation of BTK and JAK-STAT3 signaling in Namalwa cells. (B) Gray intensity analysis of the Western blots, *p < 0.05, ** p < 0.01,
*** p < 0.001 compared with control group.
HRMS (ESI) for C28H27N5O5S, [M+H]+ calcd: 546.1806, found:
546.1790.
2.2 Hz, 1H), 6.44 (dd, J = 16.8, 10.1 Hz, 1H), 6.27 (dd, J = 16.8,
2.0 Hz, 1H), 5.79 (dd, J = 10.1, 2.0 Hz, 1H), 3.64–3.54 (m, 4H),
3.54–3.37 (m, 4H); HRMS (ESI) for C26H23N5O4S, [M+H]+ calcd:
502.1544, found: 502.1533.
4.2.6. N-(3-(2-(4-((1-morpholino)acetoxyl)-3-chlorophenylamino)thieno
[3,2-d]pyrimidin-4-yloxy) phenyl)acrylamide(9f)
Yield 15.1%; off-white solid. 1H NMR (DMSO-d6): δ 10.35 (s, 1H),
9.48 (s, 1H), 8.31 (d, J = 5.3 Hz, 1H), 7.76 (s, 1H), 7.73 (t, J = 2.2 Hz,
1H), 7.60 (dd, J = 8.1, 2.2 Hz, 1H), 7.47 (t, J = 8.1 Hz, 1H), 7.45 (d,
J = 9.1 Hz, 1H), 7.38 (d, J = 5.3 Hz, 1H), 7.09 (dd, J = 8.1, 2.2 Hz,
1H), 6.86 (d, J = 9.1 Hz, 1H), 6.44 (dd, J = 17.0, 10.1 Hz, 1H), 6.27
(dd, J = 17.0, 2.0 Hz, 1H), 5.78 (dd, J = 10.1, 2.0 Hz, 1H), 4.84 (s,
2H), 3.64–3.56 (m, 4H), 3.49–3.43 (m, 4H); 13C NMR (DMSO-d6): δ
166.2, 165.0, 164.1, 163.8, 158.0, 152.6, 148.3, 140.9, 137.5, 135.3,
132.1, 130.5, 127.8, 123.7, 121.2, 120.5, 118.7, 117.4, 117.3, 114.4,
113.1, 108.3, 67.3, 66.5 (2C), 45.3, 42.1; HRMS (ESI) for
4.2.9. N-(3-(2-(4-(morpholinoacetyl)phenylamino)thieno[3,2-d]
pyrimidin-4-yloxy)phenyl)acrylamide(9i)
Yield 29.8%; off-white solid. 1H NMR (DMSO-d6): δ 10.37 (s, 1H),
9.49 (s, 1H), 8.31 (d, J = 5.4 Hz, 1H), 7.74 (t, J = 2.2 Hz, 1H), 7.61
(dd, J = 8.1, 2.2 Hz, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 8.1 Hz,
1H), 7.38 (d, J = 5.4 Hz, 1H), 7.10 (d, J = 8.1, 2.2 Hz, 1H), 6.97 (d,
J = 8.2 Hz, 2H), 6.44 (dd, J = 16.9, 10.1 Hz, 1H), 6.27 (dd, J = 16.9,
2.0 Hz, 1H), 5.79 (dd, J = 10.1, 2.0 Hz, 1H), 3.59 (s, 2H), 3.54–3.49
(m, 4H), 3.47–3.42 (m, 4H); 13C NMR (DMSO-d6): δ 169.7, 165.2,
164.1, 163.8, 158.2, 152.7, 140.8, 139.3, 137.4, 132.1, 130.4, 129.1
(2C), 128.6, 127.8, 123.7, 119.1 (2C), 117.6, 117.1, 113.3, 108.0, 66.5
(2C), 46.4, 42.1, 39.2; HRMS (ESI) for C27H25N5O4S, [M+H]+ calcd:
516.1700, found: 516.1692.
C
27H24ClN5O5S, [M+H]+ calcd:566.1259, found: 566.1265.
4.2.7. N-(3-(2-(4-((1-morpholino)acetoxyl)-3-methoxylphenylamino)
thieno[3,2-d]pyrimidin-4-yloxy)phenyl)acrylamide(9g)
Yield 33.3%; Pale-yellow solid. 1H NMR (DMSO-d6): δ 10.35 (s, 1H),
9.29 (s, 1H), 8.29 (d, J = 5.4 Hz, 1H), 7.71 (t, J = 2.2 Hz, 1H), 7.59
(dd, J = 8.1, 2.2 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.38 (s, 1H), 7.37 (d,
J = 5.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.09 (dd, J = 8.1, 2.2 Hz,
1H), 6.72 (d, J = 8.8 Hz, 1H), 6.44 (dd, J = 16.8, 10.1 Hz, 1H), 6.27
(dd, J = 16.8, 2.0 Hz, 1H), 5.79 (dd, J = 10.1, 2.0 Hz, 1H), 4.67 (s,
2H), 3.63 (s, 1H), 3.61–3.55 (m, 4H), 3.51–3.43 (m, 4H); 13C NMR
(DMSO-d6): δ 166.7, 165.1, 164.0, 163.8, 158.3, 152.7, 149.4, 142.4,
140.8, 137.3, 135.6, 132.1, 130.4, 127.8, 123.8, 117.4, 117.1, 115.1,
113.1, 111.1, 108.1, 105.1, 68.2, 66.6 (2C), 55.9, 45.5, 42.1; HRMS
(ESI) for C28H27N5O6S, [M+H]+ calcd: 562.1755, found: 562.1755.
4.2.10. N-(3-(2-(4-((1-morpholino)acetylamino)-phenylamino)thieno
[3,2-d]pyrimidin-4-yloxy) phenyl)acrylamide(9j)
Yield 20.1%; Pale-yellow solid. 1H NMR (DMSO-d6): δ 10.34 (s, 1H),
9.52 (s, 1H), 9.41 (s, 1H), 8.29 (d, J = 5.3 Hz, 1H), 7.72 (s, 1H), 7.59
(d, J = 8.2 Hz, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.46 (t, J = 8.2 Hz, 1H),
7.37–7.36 (m, 3H), 7.09 (d, J = 8.2 Hz, 1H), 6.43 (dd, J = 16.9,
10.1 Hz, 1H), 6.26 (dd, J = 16.9, 2.0 Hz, 1H), 5.77 (dd, J = 10.1,
2.0 Hz, 1H), 3.63 (t, J = 4.3 Hz, 4H), 3.07 (s, 2H), 2.53–2.48 (m, 4H);
13C NMR(DMSO-d6): δ 167.9, 165.2, 164.1, 163.8, 158.1, 152.7, 140.8,
137.4, 136.8, 132.7, 132.1, 130.4, 127.8, 123.7, 120.3 (2C), 119.3
(2C), 117.6, 117.0, 113.2, 108.0, 66.6 (2C), 62.5, 53.7 (2C); HRMS
(ESI) for C27H25N5O5S, [M+H]+ calcd: 553.1628, found: 553.1641.
4.2.8. N-(3-(2-(4-(morpholinoformyl)phenylamino)thieno[3,2-d]
pyrimidin-4-yloxy)phenyl)acrylamide(9h)
4.2.11. N-(3-(2-(4-((1-morpholino)acetylamino)-3-chlorophenylamino)
thieno[3,2-d]pyrimidin-4-yloxy)phenyl)acrylamide(9k)
Yield 24.3%; White solid. 1H NMR (DMSO-d6): δ 10.38 (s, 1H), 9.79
(s, 1H), 8.35 (d, J = 5.4 Hz, 1H), 7.78 (t, J = 2.2 Hz, 1H), 7.65 (d,
J = 8.6 Hz, 2H), 7.61 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 8.1 Hz, 1H),
7.41 (d, J = 5.4 Hz, 1H), 7.18 (d, J = 8.6 Hz, 2H), 7.11 (d, J = 8.1,
Yield 12.6%; off-gray solid.1H NMR(DMSO-d6): δ 10.37 (s, 1H), 9.71
(s, 1H), 9.65 (s, 1H), 8.34 (d, J = 5.4 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H),
7.86 (s, 1H), 7.79 (t, J = 2.2 Hz, 1H), 7.59 (dd, J = 8.1, 2.2 Hz, 1H),
9

F. Chi, et al.
BioorganicChemistry95(2020)103542
Fig. 10. Proposed binding models of the typical inhibitors with JAK3 (PDB code:4Z16), A: 8, B: Spebrutinib, C: inhibitor 9g, D: inhibitor 9a, E: inhibitor 9k, F:
inhibitor 9i.
7.53–7.44 (m, 2H), 7.41 (d, J = 5.4 Hz, 1H), 7.10 (dd, J = 8.1, 2.2 Hz,
1H), 6.43 (dd, J = 16.8, 10.0 Hz, 1H), 6.25 (dd, J = 16.8, 2.0 Hz, 1H),
5.77 (dd, J = 10.0, 2.0 Hz, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.14 (s, 2H),
2.60–2.53 (m, 4H); 13C NMR (DMSO-d6): δ 168.2, 165.0, 164.2, 163.8,
157.8, 152.6, 141.0, 138.2, 137.8, 132.1, 130.5, 128.2, 127.8, 124.1,
123.7, 122.6, 118.7, 118.0, 117.4, 117.3, 113.1, 108.6, 66.9 (2C), 62.0,
53.6 (2C); HRMS (ESI) for C27H25ClN6O4S, [M+H]+ calcd: 565.1419,
found: 565.1410.
purchased from Promega Corporation (USA). The experiments were
performed according to the instructions of the manufacturer. The de-
tailed 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/?catNum=V2941
and
https://cn.promega.com/products/cell-signaling/kinase-assays-and-
kinase-biology/jak3-kinase-enzyme-system/?catNum=V3701), respec-
tively. For all of the tested compounds, concentrations consisting of
suitable levels from 0.1 to 1000 nM were used. The test was performed
in a 384-well plates, including the main steps below: 1) perform a 5 μL
kinase reaction using 1 × kinase buffer (e.g., 1 × reaction buffer A), 2)
incubate at room temperature for 60 min, 3) add 5 μL of ADP-Glo™
reagent to stop the kinase reaction and deplete the unconsumed ATP,
4.3. Kinase enzymatic assays
All the enzymatic assays were tested applying with the ADP-GloTM
assay system (BTK: Catalog. V2941, JAK3: Catalog. V3701), were
10

F. Chi, et al.
BioorganicChemistry95(2020)103542
leaving only ADP and a very low background of ATP, 4) incubate at
room temperature for 40 min, 5) add 10 μL of kinase detection, 6) re-
agent to convert ADP to ATP and introduce luciferase and Luciferin to
detect ATP, 7) incubate at room temperature for 30 min, 8) plates was
measured on TriStar® LB942 Multimode MicroplateReader (BERTH-
OLD) to detect the luminescence (Integration time0.5–1 s). Curve fitting
and data presentations were performed using GraphPad Prism version
8.0.
medium containing 5% FBS for 48 h. Then, collected and fixed with
70% ethanol at 4 °C overnight. After being fixed with 70% ethanol at
4 °C, the cells were stained with Annexin V-FITC (5 μL)/propidium
iodide (5 μL), and analyzed by flow cytometry assay (Becton-Dickinson,
USA). For cell cycle analysis, Ramos cells at a density of approximately
2 × 105 cells/well were incubated in 6-well plates, treated with dif-
ferent concentrations of inhibitors for 48 h, collected and fixed with
70% ethanol at 4 °C overnight. After fixation, the cells were washed
with PBS and stained with propidium iodide (PI) for 30 min under
subdued light. Stained cells were analyzed flow cytometry assay
(Becton-Dickinson, USA).
4.4. Cells and reagents
Ramos, Raji, Namalwa and L-02 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 Company
(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).
4.9. Western blotting assay
The lysates from Namalwa cells in different groups were extracted
and centrifuged at 14,000g for 15 min at 4 °C, then the total proteins
were obtained. An aliquot (40 μg protein) was loaded onto a 8–12%
SDS-PAGE gels and separated electrophoretically. Then the target pro-
teins 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 photographed by using a BioSpectrum Gel Imaging
System (HR410, UVP, USA). In order to eliminate the variations, data
were adjusted to β-Actin expression: IOD of objective protein versus
IOD of β-Actin expression.
4.5. Cellular activity assay
Ramos, Raji, Namalwa cells viability assays were performed ac-
cording to the CCK-8 method. The cells were seeded in 96-well plates at
a density of 3000, 8000, 7000 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 0.5–1.2 at 450 nm with a micro-
plate reader (Thermo Fisher, USA). Compounds were tested at appro-
priate concentrations (1.25–40 μM), with each concentration dupli-
cated five times. The IC50 values were calculated using GraphPad Prism
version 8.0.
4.10. Molecular docking study
The AutoDock 4.2 software was used to perform the docking ex-
ploration. Detailed tutorials that guide users through basic AutoDock
usage, docking with flexible rings, and virtual screening with AutoDock
may be found at: http://autodock.scripps.edu/faqs-help/tutorial.
Generally, the crystal structure (PDB: 4Z16) of the kinase domain of
JAK3 bound to inhibitor 9a was used in the docking studies. The en-
zyme preparation and the hydrogen atoms adding was performed in the
prepared process. The whole JAK3 enzyme was defined as a receptor
and the site sphere was selected on the basis of the binding location of
Spebrutinib. By moving Spebrutinib and the irrelevant water, molecule
9a. The binding interaction energy was calculated to include Van der
Waals, electrostatic, and torsional energy terms defined in the TRIPOS
force field. The structure optimization was performed using a genetic
algorithm, and only the best-scoring ligand protein complexes were
kept for analyses.
4.6. AO/EB staining assay
Approximately 2 × 105 cells/well of Ramos and Raji cells in 6-well
plates were incubated in an incubator for 48 h, then treated with dif-
ferent concentrations of inhibitors for 48 h. Then, total of 20 μL of the
solution containing the AO/EB dye mix (1.0 μg/mL of AO and 1.0 μg/
mL of EB in PBS) was added to the cells. The apoptotic, necrotic, and
live cells were observed under the fluorescent microscope (OLYMPUS,
Tokyo, Japan).
4.7. Cytotoxic activity assay
Peripheral venous blood was collected in an ACD anticoagulant
(Vblood: V
= 9:1) tube from the normal healthy adult male.
4.11. Statistical analysis
The leucoacnytitceo-argiuclhantplasma was removed and the peripheral blood
mononuclear cells (PBMC) consisting of monocytes and the lympho-
cytes 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.
All data were displayed as a mean
SD. The statistical difference
between two groups was tested by ANOVA test and Tukey’s multiple
comparison test. p < 0.05 was considered statistically significant.
Declaration of Competing Interest
PBMC cells were seeded in 24-well plates at a density of 200,000 to
250,000 cells/well. Cells were treated with inhibitors 9a at appropriate
concentration (5, 10, 20 μM), with each concentration duplicated five
times. After maintaining the cells at 37 °C, in a 5% CO2 incubator in
RPMI-1640 containing 10% fetal bovineserum (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.
We want to submit the revised manuscript in the category of re-
search articles titled “JAK3 inhibitors based on thieno[3,2-d]pyrimidine
scaffold: design, synthesis and bioactivity evaluation for the treatment
of B-cell lymphoma”. There are no concerns about third-party copyright
or conflicts of interest or online supplementary material.
Acknowledgements
4.8. Flow cytometry assay
We are grateful to the National Natural Science Foundation of China
(No. 81672945), the “Xing Liao” Talents Project of Liaoning province,
China (XLYC1807011), the Research Fund of Higher Education of
Liaoning province (LZ2019004), the Natural Science Foundation of
The Ramos cells (2 × 105 cells/well) incubated in 6-well plates
were treated with solvent control (DMSO), Spebrutinib or 9a in
11

F. Chi, et al.
BioorganicChemistry95(2020)103542
Liaoning province (20180530066), the Cultivation Project of Youth
Tech stars, Dalian, China (No. 2016RQ043), and the Foundation of
Science and Technology Innovation of Dalian, China (2019J13SN76)
for the financial support of this research.
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