Structural optimization of diphenylpyrimidine derivatives (DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors with improved activity toward B leukemia cell lines

Article abstract of DOI:10.1016/j.ejmech.2016.11.047

A new series of diphenylpyrimidine derivatives (DPPYs) bearing various aniline side chains at the C-2 position of pyrimidine core were synthesized as potent BTK inhibitors. Most of these inhibitors displayed improved activity against B leukemia cell lines compared with lead compound spebrutinib. Subsequent studies showed that the peculiar inhibitor 7j, with IC₅₀values of 10.5 μM against Ramos cells and 19.1 μM against Raji cells, also displayed slightly higher inhibitory ability than the novel agent ibrutinib. Moreover, compound 7j is not sensitive to normal cells PBMC, indicating low cell cytotoxicity. In addition, flow cytometry analysis indicated that 7j significantly induced the apoptosis of Ramos cells, and arrested the cell cycle at the G0/G1 phase. These explorations provided new clues to discover pyrimidine scaffold as more effective BTK inhibitors.

Full text of DOI:10.1016/j.ejmech.2016.11.047

Accepted Manuscript
Structural optimization of diphenylpyrimidine derivatives (DPPYs) as potent Bruton's
tyrosine kinase (BTK) inhibitors with improved activity toward B leukemia cell lines
Dan Zhao, Shanshan Huang, Menghua Qu, Changyuan Wang, Zhihao Liu, Zhen Li,
Jinyong Peng, Kexin Liu, Yanxia Li, Xiaodong Ma, Xiaohong Shu
PII:
S0223-5234(16)30985-0
10.1016/j.ejmech.2016.11.047
EJMECH 9080
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2016
Revised Date: 20 November 2016
Accepted Date: 21 November 2016
Please cite this article as: D. Zhao, S. Huang, M. Qu, C. Wang, Z. Liu, Z. Li, J. Peng, K. Liu, Y. Li,
X. Ma, X. Shu, Structural optimization of diphenylpyrimidine derivatives (DPPYs) as potent Bruton's
tyrosine kinase (BTK) inhibitors with improved activity toward B leukemia cell lines, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.047.
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ACCEPTED MANUSCRIPT
Cys481
Phe540/Ser538/Ile472
covalent bond
Various side chains
O
O
O
O
O
or
N
=
O
O
HN
R2
HN
HN
R1
N
NH
HN
N
NH
N
N
Met477
F
Spebrutinib
Phase Ш
DPPYs 7a-l
BTK inhibitors
Improved activity

ACCEPTED MANUSCRIPT
Structural optimization of diphenylpyrimidine derivatives
(DPPYs) as potent Bruton’s tyrosine kinase (BTK) inhibitors
with improved activity toward B leukemia cell lines
Dan Zhao a, Shanshan Huang a, Menghua Qu a, Changyuan Wang a, Zhihao Liu a, Zhen Li a,
Jinyong Peng a, Kexin Liu a, Yanxia Li b, Xiaodong Ma a, , and Xiaohong Shu a,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
b Department of Respiratory Medicine, the First Affiliated Hospital of Dalian Medical University,
Dalian 116011, PR China.
E-mail address: xiaodong.ma@139.com (X.D. Ma), xiaohong_shu@dmu.edu.cn (X.H. Shu).
Abstract
A new series of diphenylpyrimidine derivatives (DPPYs) bearing various aniline side
chains at the C-2 position of pyrimidine core were synthesized as potent BTK
inhibitors. Most of these inhibitors displayed improved activity against B leukemia
cell lines compared with lead compound spebrutinib. Subsequent studies showed that
the peculiar inhibitor 7j, with IC50 values of 10.5 µM against Ramos cells and 19.1
µM against Raji cells, also displayed slightly higher inhibitory ability than the novel
agent ibrutinib. Moreover, compound 7j is not sensitive to normal cells PBMC,
indicating low cell cytotoxicity. In addition, flow cytometry analysis indicated that 7j
significantly induced the apoptosis of Ramos cells, and arrested the cell cycle at the
G0/G1 phase. These explorations provided new clues to discover pyrimidine scaffold
as more effective BTK inhibitors.
Keywords: Leukemia; BTK; Pyrimidine; Synthesis; Inhibitor.
1

ACCEPTED MANUSCRIPT
1. Introduction
O
N
HN
O
O
O
N
N
N
O
O
N
O
N
N
N
N
N
H2N
H2N
N
H2N
N
N
N
N
1
2
3
Ibrutinib
Acalabrutinib
GS-4059
H
N
N
F
N
S
N
N
NH
O
HN
O
N
NH
HN
N
O
O
NH
NH
NH
NH
O
NH
O
O
N
N
O
O
F3C
PLS-123
Fig. 1. Structures of the novel BTK inhibitors.
4
6
5
Spebrutinib
HM71224
Bruton’s tyrosine kinase (BTK), a key member of the Tec family of non-receptor
tyrosine kinases, plays an important role in the B-cell signaling pathway linking cell
surface B-cell receptor (BCR) stimulation to downstream intracellular responses [1-3].
It has been reported that numerous B-cell-derived malignancies, such as acute
lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin’s
lymphoma (NHL), mantle cell lymphoma (MCL), Waldenström’s Macroglobunemia
(WM) and multiple myeloma (MM), are related to the deregulation of BTK kinase
[4,5]. Till now, considerable efforts focused on searching for effective agents against
various B-cell-derived tumors have brought promising prospect for cancer patients [6].
Inspiringly, a clinically advanced irreversible BTK kinase inhibitor, ibrutinib, which
has demonstrated efficacy in patients with CLL, MCL, WM and MCL, has been
broadly used in clinical [6-9]. Recently, a number of novel covalent and noncovalent
BTK inhibitors, including acalabrutinib (2) (ClinicalTrials.gov identifier:
NCT02717611) [10-12], GS-4059 (3) (ClinicalTrials.gov identifier: NCT02457559)
[13,14], spebrutinib (4) (ClinicalTrials.gov identifier: NCT01744626) [15], HM71224
2

ACCEPTED MANUSCRIPT
(5) (ClinicalTrials.gov identifier: NCT01765478) [16], and PLS-123 (6) [17], have
been advanced to preclinical or clinical trials (Fig. 1).
Cys481
Phe540
Ser538
Met477
Ile472
Fig. 2. Binding mode of spebrutinib with BTK enzyme (PDB: 3GEN).
Cys481
Phe540/Ser538/Ile472
covalent bond
Various side chains
O
O
O
O
or
N
=
O
O
O
HN
R2
HN
HN
R1
N
NH
HN
N
NH
N
N
Met477
F
Spebrutinib
Phase Ш
DPPYs 7a-l
BTK inhibitors
Improved activity
Fig. 3. Designed strategy of new DPPYs as potent BTK inhibitors.
It is clear that these novel BTK inhibitors possess a pyrimidine core, along with a
typical N-phenlyacrylamide functionality. Previous structure and activity relationships
(SARs) explorations showed that the acryloyl group was essential to form a covalent
bond with the amino acid Cys481 of BTK, while a C-4'-substituted aniline side chain
installed on the C-2 position of pyrimidine core was also beneficial to form contacts
with the resides Phe540, Ile472, Ser538, Lys430 at the bottom of the ATP-binding
3

ACCEPTED MANUSCRIPT
pocket (Fig. 2) [7,18-20]. To improve the binding affinity with BTK, herein, a series
of new dipenylpyrimidine derivatives (DPPYs) 7a-l bearing various C-2 aniline side
chains were synthesized and biologically evaluated for their activity against BTK
kinase and B lymphoma cell lines (Fig. 3). Accordingly, our molecule simulations
showed in Fig. 7 indicated that the newly designed molecules tightly contact with
BTK, and which would enhance the activity against BTK.
2. Results and Discussion
2.1 Chemistry
The title molecules 7a-l were synthesized as depicted in Scheme 1 [21,22].
Starting from 3-nitroaniline (8), acylation reaction using acryloyl chlorine under basic
conditions (NaHCO3) was furnished to produce N-acryloyl-4-nitrobenzamide (9).
After reduction of the nitro group of compound 9 under Fe-NH4Cl conditions, the 4-
aminobenzamide derivative (10) was conveniently synthesized. Compound 10 was
regioselectively coupled to the C-4 position of 2,4-dichloropyrimidine template to
afford the key intermediate 11a-b. As indicated in Scheme 2-4, various aniline side
chains 12a-h were prepared according to general synthetic methods [23,24], including
aromatic nucleophilic substitution , acylation, and reduction reactions, etc. Coupling
reactions of 11a-b with aniline intermediates 12a-h were accomplished in the
presence of trifluoroacetic acid (TFA) under reflux conditions, offering the title
molecules 7a-i and 7j-l with yield ranging from 31% to 45%.
4

ACCEPTED MANUSCRIPT
R1
N
NH2
Cl
R1
N
NH
NO2
N
NO2
Cl
N
c
Cl
NH
b
NH
a
NH
O
O
O
NH2
11a-b
9
8
10
O
O
R2
O
O
O
O
N
O
N
H2N
HN
R2
N
HN
R2
N
d,
12a,c-e,g,h
R2
O
or
HN
R1
NH
HN
R1
NH
H2N
or
N
N
12b,f
7j
R1= Cl, F
R2 = H, Cl, MeO
－
l
7a－
i
Scheme 1. Synthetic route of title compounds 7a-l. Reagents and conditions: (a) acryloyl chloride
,
NaHCO3, CH3CN, rt, 0.5 h, 95%; (b) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 72%; (c) ArNH2,
DIPEA, 1,4-dioxane, 60°C, 2 h, 91%; (d) trifluoroacetic acid, intermediates 12a-h, 2-BuOH, 100
°C, 12 h, 31% to 45%.
NO2
NH2
b
HN
O
O
O
O
O
O
N
N
NO2
14
12a
a
a
NH2
NO2
HO
F
13
b
O
O
O
O
O
15
12b
Scheme 2. Synthetic route of intermediates 12a,b .Reagents and conditions: (a) K2CO3, CH3CN,
80 °C, 12 h, 63-85%; (b) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 60-87%.
5

ACCEPTED MANUSCRIPT
NH2
NO2
NO2
NO2
NH2
R
R c
a
b
R
O
O
N
NH
O
O
NH
O
O
O
N
NH
R
Br
18a-c
16a-c
17a-c
12c-e
R = H, Me, MeO
Scheme 3. Synthetic route of intermediates 12c-e. Reagents and conditions: (a) bromocacetyl
bromide, NaHCO3, CH3CN, rt, 0.5 h, 93%; (b) bis(2-methoxyethyl)amine, K2CO3, CH3CN, 80 °C,
12 h, 71-88%; (c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 63-82%.
NO2
NH2
HO
O
e
O
O
O
O
NO2
O
O
12f
NO2
OH
O
O
21
c,d
a
b
R
NO2
NH2
R
c,d
O
HN
O
O
e
O
20a,b
HO
R
N
R
N
19a,b
O
O
O
O
O
O
O
O
22a,b
12g,h
R = H, Cl
Scheme 4. Synthetic route of intermediates 12f-h. Reagents and conditions: (a) ethyl
bromoacetate, K2CO3, CH3CN, rt, 5 h, 91%; (b) KOH, MeOH-H2O, 50 °C, 1 h, 62-78%; (c)
(COCl)2, 50 °C, 1 h; (d) K2CO3, CH3CN, 80 °C, 12 h, 55-78%; (e) Fe-NH4Cl, MeOH-H2O, 2 h, 70
°C, 61-72%.
2.2. Biological activity
All these compounds were evaluated for their activity against BTK enzyme using
ADP-Glo™ Kinase Assay [25-27]. Cell viability was determined by CCK-8 assay
[18], and the cell cycle and apoptosis were analyzed by flow cytometer. Two typical
B-cell lymphoblastic leukemia cell lines, Ramos and Raji, which overexpressed BTK
enzyme, were used for the cell proliferation evaluations. For comparison, two novel
6

ACCEPTED MANUSCRIPT
BTK inhibitors, ibrutinib and spebrutinib were also tested as reference compounds.
All these result data are depicted in Table 1.
Table 1 Biological activity of the newly synthesized DPPYs.a
5
R1
N
2
4
HN
N
NH
1`
3`
R2
NH
4`
R
O
7a-l
Enzymatic activity Antiproliferative activity
(IC50, nM)
(IC50, µM)b
Compound 5-R1 3ˈ-R2 4ˈ-R
BTK
Ramos
13.0
18.1
18.1
>40.0
16.9
14.8
35.6
11.0
19.6
10.5
22.3
>40
Raji
O
O
N
Cl
F
H
4.94
7.12
2.83
27.2
>100
22.6
91.3
>100
20.0
12.1
22.4
0.23
0.72
30.5
23.8
20.1
36.0
19.3
19.9
36.9
20.9
35.6
19.1
38.6
9.4
7a
O
N
H
7b
O
O
Cl
F
H
7c
O
O
O
O
O
N
N
O
O
O
O
O
H
7d
Cl
Cl
F
Cl
H
7e
O
O
N
N
H
N
7f
O
O
O
O
O
H
N
O
O
O
H
7g
N
N
N
O
O
O
H
N
Cl
Cl
Cl
F
OMe
Me
H
7h
H
N
7i
O
O
O
O
7j
H
7k
O
O
O
Cl
H
7l
O
Spebrutinib
20.9
29.4
7

ACCEPTED MANUSCRIPT
Ibrutinib
0.34
12.6
19.3
b
aData represent the mean of at least three separate experiments. Dose-response curves were
determined at five concentrations. The IC50 values are the concentrations in micromolar needed to
inhibit cell growth by 50% as calculated using GraphPad Prism version 5.0.
2.5 µM
Control
5 µM
12
10
8
6
10 µM
20 µM
40 µM
4
2
control
20 40
2.5 5 10
Concentrations (µM)
Fig. 4. Morphological changes of the PBMC cells treated by molecule 7j (100 ×, final
magnification) at concentration of 2.5, 5, 10, 20, 40 µM for 24 h. Cell numbers were calculated by
traditional manual method with an ordinary cell counting chamber. One representative experiment
is shown.
As for the kinase-based evaluation results shown in Table 1, these compounds
effectively inhibit BTK enzyme at the concentrations less than 100 nmol/L, and four
of them (7a-c, 7l) displayed strong anti-BTK activity with IC50 values lower than 7.12
nM. In particular, compound 7l with IC50 of 0.23 nM, is more potent than ibrutinib
(IC50 = 0.34 nM) and spebrutinib (IC50 = 0.72 nM). SAR analysis revealed that
substituents, such as chloro (7e), methoxy (7h), methyl (7i), were inappropriate to be
installed on the C-3ˈ position of the C-2 aniline side chain. Chloro substituent at the
C-5 position of pyrimidine core is more favorable than the fluoro atom. Prolonging
the C-2 aniline side chain is beneficial. The example analogues 7c and 7l bearing a
long side chain possess very low effective inhibitory concentrations, with IC50 values
of 2.83 nM and 0.23 nM, respectively.
To confirm whether compound 7j forms irreversible inhibition or not with BTK
enzyme, the kinase activity determined by the luminescence value was detected after
treating the inhibitor-linked BTK with the ADP-Glo™ Kinase Assay system (Table
2). Compared with the normal BTK (luminescence value = 9976), the inhibitor-linked
8

ACCEPTED MANUSCRIPT
BTK made the luminescence value (383) significantly reduced, which indirectly
proved that compound 7j irreversibly contacts with BTK target, and thus led to the
remarkable loss of the kinase activity.
Table 2 The luminescence values produced by treating BTK, and inhibitor-linked
BTK with ADP-Glo™ Kinase Assay system.
Times
Controla
Normal BTKb
9702
Inhibitor-linked BTKc
1
2
166
430
412
308
383
180
10652
Luminescence values
3
208
9574
Ave.
185
9976
a3 µL 1×buffer, 2 µL (0.5 µg/µL substrate, 125 µM ATP); b1 µL 1×buffer, 2 µL (0.5 µg/µL substrate,125 µM ATP),
2 µL 2.5 ng/µL enzyme; c1 µL 500 nmol/L drug, 2 µL (0.5 µg/µL substrate, 125 µM ATP), 2 µL 2.5 ng/µL enzyme.
In cell-based assays, a large part of these molecules strongly inhibit the
proliferations of Ramos and Raji cells at concentrations ranging from 10 to 20 µmol/L.
Inhibitor 7j, possessing an IC50 value of 12.1 nM in kinase-based activity, exhibited
slightly stronger inhibitory activity against Ramos (IC50 = 10.5 µM) and Raji (IC50 =
19.1 µM) cells than reference compounds. Interestingly, molecule 7l, the strongest
inhibitor within these newly obtained DPPYs, could remarkably inhibit Raji cell line
(IC50 = 9.4 µM), but was not sensitive to Ramos cells (IC50 > 40 µM). The analogues
7e and 7h also showed strong activity against the two B lymphoma cell lines, but they
showed high IC50 values (>100 nM) for targeting BTK kinase, suggesting that a new
action mechanism for them to be produced to interfere with B-cell lymphoblastic
leukemia cells. Fortunately, compound 7j is not active to interfere with the normal
PBMC cells at concentrations of less than 20 µM, suggesting low cell cytotoxicity
(Fig. 4). Overall, these biological explorations provided a new series of pyrimidine
derivatives to inhibit B-cell lymphoblastic leukemia cells by targeting BTK.
2.3. Flow cytometry
In addition, to further investigate the antiproliferative mechanism of these
inhibitors on B-cell leukemia cells, the most active inhibitor 7j, was also examined
9

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the effects on apoptosis and on the cell cycle in Ramos cell line using flow
cytometry analysis [28,29]. For comparison, spebrutinib and ibrutinib were also
evaluated as the reference compounds. As shown in Fig. 5, inhibitor 7j could
significantly induce the Ramos cells apoptosis, and the apoptosis rates were 38.0%,
46.8% and 83.6% at concentrations of 5 µmol/L, 10 µmol/L, and 20 µmol/L inhibitor
7j, respectively. Compared to spebrutinib (41.5%) and iburtinib (47.9%), 7j could
inhibit the proliferations of Ramos cell line and induce the similar cell apoptosis rates
at the low concentrations of 10 µmol/L. After incubation with 5 µmol/L, 10 µmol/L
and 20 µmol/L inhibitor 7j for 48h, the DNA contents of the Ramos cells were
53.18%, 63.1% and 70.8% in G0/G1 phase, and were 46.8%, 37.0% and 28.4% in S
phase, respectively (Fig. 6). Apparently, the newly synthesized molecule 7j blocked
Ramos cells at the G0/G1 phase, while ibrutinib (G2 phase) and spebrutinib (S phase)
arrested the cell cycle at the late phase.
Ibrutinib(10 µΜ)
Spebrutinib(10 µΜ)
Blank
100
80
60
40
20
0
7j(5 µΜ)
7j(10 µΜ)
7j(20 µΜ)
7
j
B
7
j
7
j
S
p
I
b
l
(
(
(
a
5
µ
1
2
0
r
n
k
e
b
u
0
µ
M
t
i
µ
r
n
M
M
)
u
i
t
b
)
)
i
n
(
1
i
b
0
µ
(
1
0
M
)
µ
M
)
Fig. 5. Compound 7j induced Ramos cell apoptosis in vitro. The cells were incubated with the
indicated concentrations of 7j for 48 h, and the cells were stained with annexin V/FTIC, followed
by flow cytometry analysis. One representative experiment is shown. p < 0.05.
10

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Ibrutinib(10µM)
Blank
Spebrutinib(10µM)
G0/G1: 46.35%
S: 53.50%
G2/M: 0.15%
G0/G1: 31.02%
S: 47.90%
G2/M: 20.91%
G0/G1: 56.19%
S: 42.23%
G2/M: 1.58%
7j(5µM)
7j(10µM)
7j(20µM)
G0/G1: 53.18%
S: 46.82%
G2/M: 0.00%
G0/G1: 63.00%
S: 37.00%
G2/M: 0.00%
G0/G1: 70.75%
S: 28.39%
G2/M: 0.86%
G0/G1
S
G2/M
100
70.8
63.0
56.2
80
53.2
46.8
53.5
48.4
47.9
42.2
37.0
40
20
0
31.0
28.4
20.9
1.58
Blank
7j
7j
7j
Ibrutinib Spebrutinib
(10µM)
(5µM) (10µM)
(20µM) (10µM)
Fig. 6. Effects of spebrutinib, ibrutinib and 7j on Ramos cell cycle arrest detected by flow
cytometry assay. Results are representative of three separate experiments, dates are expressed as
the mean±standard deviation, p < 0.05.
2.4. Molecular modeling calculations
In order to investigate the interaction mechanism of the newly synthesized DPPYs
with BTK enzyme, three potent BTK inhibitors 7a, 7c, 7l, and two less active
inhibitors 7e, 7h, in parallel with the lead compound spebrutinib were docked into the
ATP binding pocket of BTK enzyme (PDB: 3GEN), respectively [30]. The program
AutoDock 4.2 with its default parameters were used [31-33]. The molecular
simulations were depicted in Fig. 7. By analyzing the binding model of spebrutinib
11

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with BTK (Fig. 7a), it was found that several important interaction forces were
produced, including: (1) covalent bond forces between the essential acrylamide
functionality of spebrutinib with the amino acid Cys481; (2) Hydrogen-bond
interactions formed by the carbonyl group of acrylamide substituent with the amino
acid Lys430 through a water molecule; (3) strong contacts between the C-2 aniline
side chain of pyrimidine core with Ser538, Phe540, Ile472, which consist of the
bottom section of the ATP binding pocket; (4) hydrophobic interactions generated
from the C-5 chlorine atom of pyrimidine core with the mutant gatekeeper residue
Met477. Compared with spebrutinib, the three potent inhibitors 7a (Fig. 7b), 7c (Fig.
7d) and 7l (Fig. 7c), retained the important covalent bond formed by the acrylamide
substituent with Cys481. But the less active inhibitors 7e (Fig. 7e) and 7h (Fig. 7f)
lost these important interactions. Beyond the hydrogen bond between the carbonyl
group in acrylamide functionality with Lys430, compounds 7a, 7c and 7l, produced
additional strong hydrogen-bond contacts, including forces between oxygen atom in
C-2 aniline side chain of 7a with Phe540 through a water molecule, forces between
the carbonyl group in C-2 aniline side chain of 7c with Lys430 through a water
molecule, and forces between carbonyl group in C-2 aniline side chain with Ser538.
Clearly, 7a firmly occupied the ATP-binding pocket, maintaining the strong
interactions generated from the C-5 chlorine atom of pyrimidine ring with the residue
Met477. In terms of compounds 7c and 7l, the pyrimidine core was squeezed out of
the pocket, and directly reaches into the reside Asn484, thus forming new
supplementary binding forces. In contrast, the less active inhibitors 7e and 7h almost
lost these important interactions, and thus were not effective to interfere with BTK.
Overall, these binding modes actually explained their activity data.
3. Conclusion
12

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A new series of DPPYs bearing various aniline side chains at the C-2 position of
pyrimidine core were synthesized as potent BTK inhibitors. Most of them exhibited
equivalent capacity as lead compound (spebrutinib and ibrutinib) in both kinase-based
and cell-based assays. It was significant that the most active inhibitor 7l, which were
able to interfere with BTK kinase at concentrations of 0.23 nM, was discovered in this
contribution. Notably, the novel molecule 7j with IC50 values of 10.5 µM against
Ramos cells and 19.1 µM against Raji cells, showed stronger activity than the
available BTK inhibitors (spebrutinib and ibrutinib). In conclusion, these findings
expanded the structural diversity of pyrimidine derivatives as inreversible inhibitors
of BTK, and provided new insight to discover more effective BTK inhibitors.
a)
b)
Phe540
Ser538
Phe540
Met449
H2O
Met449
Ser538
H2O
H2O
Lys430
Lys430
Asn484
Asn484
Ile472
Leu460
Met477
Ile472
Leu460
Cys481
Cys481
Met477
Leu488
Leu488
d)
c)
Phe540
Ser538
Phe540
Ser538
Met449
Met449
Lys430
Lys430
H2O
Asn484
Ile472
Asn484
Ile472
Cys481
Cys481
Leu460
Met477
Leu460
Met477
Leu488
Leu488
e)
f)
Phe540
Ser538
Phe540
Ser538
Met449
Met449
Lys430
Lys430
Asn484
Asn484
Ile472
Ile472
Leu460
Met477
Cys481
Cys481
Leu460
Met477
Leu488
Leu488
13

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Fig. 7. a) Putative binding mode of spebrutinib within BTK (PDB code: 3GEN); b) Putative
binding mode of inhibitor 7a within BTK (PDB code: 3GEN); c) Putative binding mode of
inhibitor 7l within BTK (PDB code: 3GEN); d) Putative binding mode of inhibitor 7c within BTK
(PDB code: 3GEN); e) Putative binding mode of inhibitor 7e within BTK (PDB code: 3GEN); f)
Putative binding mode of inhibitor 7h within BTK (PDB code: 3GEN).
4. Experimential section
4.1. General Methods and Chemistry
Solvents and reagents were obtained from commercial supplies and were used
without further purification. High resolution ESI-MS were performed on an
13
AB Sciex TripleTOF® 4600 LC/MS/MSsystem. 1H NMR and C NMR spectra on a
Brucker AV 400 MHz spectrometer were recorded in [d]DMSO. 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). All reactions were monitored
by TLC, using silica gel plates with fluorescence F254 and UV light visualization.
Flash chromatography separations were obtained on Silica Gel (300–400 mesh) using
dichloromethane/methanol as eluents.
4.2. General procedure for the synthesis of 7a–l [21-24]
4-Morpholine-substituted aniline intermediates 12a-h were prepared according to
the literatures 23 and 24. While the intermediates N-(3-((2-chloro-5-
substitutedpyrimidin-4-yl)oxy)phenyl)acrylamides 11a-b were synthesized using the
procedures reported in references 21 and 22. All these intermediates were directly
used without any purification and structural characterization. With these intermediates
in hand, the newly obtained compounds were synthesized as described below.
A flask was charged with compounds 11a-b (0.70 mmol), 12a-h (0.70 mmol), TFA
(0.08 mL, 1.05 mmol), and 2-BuOH (10 mL). The slurry was heated to 100 °C for 12
h. Then,the reaction mixture was allowed to cool to room temperature and was
neutralized with a saturated aqueous sodium bicarbonate solution. The aqueous
14

ACCEPTED MANUSCRIPT
mixture was extracted with CH2Cl2 three times (20 mL × 3). The crude product was
purified using flash chromatography with dichloromethane/methanol (v/v, 1:1) as
eluents.
4.2.1.
N-(3-((5-Chloro-2-(4-(bis(2-methoxyethyl)amino))phenylamino-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7a). Yield: 15.50%; off-yellow solid; H
NMR (400MHz, DMSO-d6): δ 3.21 (s, 6H), 3.38 (s, 8H), 5.73 (dd, J = 4.0 Hz, 8.0 Hz,
1H), 6.24 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.44 (dd, J = 8.0 Hz, 16.0 Hz, 3H), 7.21−7.31
(m, 4H), 7.46 (d, J = 4.0 Hz, 1H), 7.85 (s, 1H), 8.02 (s, 1H), 8.76 (s, 1H), 8.89 (s, 1H),
10.14 (s, 1H); 13CNMR (400 MHz, DMSO-d6): δ 50.65 (2C), 58.63 (2C), 70.13 (2C),
103.00, 111.89 (2C), 115.19, 115.43, 119.22, 121.84 (2C), 127.31, 128.87, 129.76,
132.28, 139.38, 139.43, 143.42, 155.15, 156.36, 158.44, 163.48; HRMS (ESI) for
C25H29ClN6O3, (M+H)+ calcd: 497.2062; found: 497.2044.
4.2.2.
N-(3-((5-Fluorine-2-(4-(bis(2-methoxyethyl)amino))phenylamino-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7b). Yield: 12.34%; off-yellow solid; H
NMR (400MHz, DMSO-d6): δ 3.34 (s, 6H), 3.42 (s, 8H), 5.75 (dd, J = 4.0 Hz, 8.0 Hz,
1H), 6.25 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.44−6.55 (m, 3H), 7.19−7.42 (m, 4H),
7.51(d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 8.01 (d, J = 4.0 Hz, 1H), 8.75 (s, 1H), 9.31 (s,
1H), 10.15 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ 51.01 (2C), 58.94 (2C), 70.46
(2C), 112.36 (2C), 113.73, 115.12, 117.79, 122.01 (2C), 127.59, 129.28, 130.67,
132.63, 139.61, 139.76, 139.95 (J = 76.0 Hz), 141.46, 141.65 (J = 76.0 Hz), 142.05,
143.62, 150.28, 150.39 (J = 44.0 Hz), 156.78, 163.79; HRMS (ESI) for C25H29FN6O3,
(M+H)+ calcd: 481.2358; found: 480.2279.
4.2.3.
N-(3-((5-Chloro-2-(4-(2-(bis(2-methoxyethyl)amino)-2-
oxoethoxy)phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7c). Yield:
1
14.72%; off-brown solid; H NMR (400 MHz, DMSO-d6): δ 3.24 (s, 3H), 3.29 (s,
3H), 3.44 (dd, J = 8.0 Hz,16.0 Hz, 4H), 3.51 (s, 4H), 4.75 (s, 2H), 5.75 (dd, J = 4.0Hz,
8.0 Hz, 1H), 6.27 (dd, J = 4.0Hz, 16.0 Hz, 1H), 6.47 (dd, J = 8.0 Hz, 16.0 Hz, 1H),
6.68 (d, J = 8.0 Hz, 2H), 7.28−7.49 (m, 5H), 7.87 (s, 1H), 8.10 (s, 1H), 8.89 (s, 1H),
13
9.13 (s, 1H), 10.19 (s, 1H); CNMR (400 MHz, DMSO-d6): δ 44.88, 46.81, 58.09,
58.42, 69.67, 65.80, 70.10, 103.32, 114.08 (2C), 115.00, 115.32, 119.11, 120.62 (2C),
126.96, 128.54, 131.89, 133.69, 138.92, 139.06, 152.95, 154.79, 156.09, 157.83,
163.15, 167.79; HRMS (ESI) for C27H31ClN6O5, (M+H)+ calcd: 555.2117; found:
555.2105.
4.2.4.
N-(3-((5-Fluoro-2-(4-(2-(bis(2-methoxyethyl)amino)-2-
oxoethoxy)phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7d). Yield:
25.25%; off-brown solid; 1H NMR (400 MHz, DMSO-d6): δ 3.25 (s, 3H), 3.30(s, 3H),
3.45 (dd, J = 4.0 Hz, 16,0Hz, 4H), 3.52−3.53 (br, 4H), 4.78 (s, 2H), 5.78 (dd, J = 4.0
15

ACCEPTED MANUSCRIPT
Hz, 8.0 Hz, 1H), 6.29 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.49 (dd, J = 8.0 Hz, 16.0 Hz,
1H), 6.75 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.53
(d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.94 (s, 1H), 8.08 (d, J = 4.0 Hz, 1H),
13
8.99 (s, 1H), 9.41 (s, 1H), 10.16 (s, 1H); C NMR (400 MHz, DMSO-d6): δ 45.25,
47.19, 58.47, 58.80, 66.23, 70.05, 70.48, 113.50, 114.53 (2C), 115.01, 117.64, 120.73
(2C), 127.27, 129.00, 132.31, 134.58, 139.45, 139.54 (J = 36.0 Hz), 141.07, 141.26 (J
= 76.0 Hz), 141.98, 150.05, 150.16 (J = 44.0 Hz), 153.17, 156.11, 156.13, 163.49,
168.22; HRMS (ESI) for C27H31FN6O5, (M+H)+ calcd: 539.2413; found: 539.2407.
4.2.5. N-(3-((5-Chloro-2-(3-chloro-4-(2-(bis(2-methoxyethyl)amino)-2-oxoethoxy)-
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (7e). Yield: 16.96%; off-
yellow solid; 1H NMR (400 MHz, DMSO-d6): δ 3.25 (s, 3H), 3.30 (s, 3H), 3.45 (dd, J
= 4.0 Hz, 16,0Hz, 4H), 3.53 (s, 4H), 4.89 (s, 2H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz, 1H),
6.27 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.47 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 6.77 (d, J = 9.0
Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.39 (dd, J = 4.0 Hz, 8.0Hz, 2H), 7.50 (d, J = 8.0
Hz, 1H), 7.77 (s, 1H), 7.86 (s, 1H), 8.16 (s, 1H), 8.95 (s, 1H), 9.30 (s, 1H), 10.19 (s,
1H); 13C NMR (400 MHz, DMSO-d6): δ 45.25, 47.16, 58.48, 58.80, 66.75, 70.02,
70.41, 104.34, 113.99, 115.13, 115.78, 118.84, 119.37, 120.72, 121.05, 127.27,
129.14, 132.32, 134.84, 139.19, 139.65, 148.52, 155.11, 156.50, 157.93, 163.52,
167.70; HRMS (ESI) for C27H30Cl2N6O5, (M+H)+ calcd: 589.1727, found: 589.1714.
4.2.6.
N-(3-((5-Chloro-2-(4-(2-(bis(2-
methoxyethyl)amino)acetylamino)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7f). Yield: 13.89%; off-yellow solid; H
NMR (400 MHz, DMSO-d6): δ 2.79 (t, J = 4.0 Hz, 4H), 3.26 (s, 8H), 3.43 (t, J = 4.0
Hz, 4H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.25-6.31 (m, 1H), 6.45-6.51 (m, 1H),
7.26-7.43 (m, 4H), 7.54 (dd, J = 8.0 Hz, 16.0 Hz, 3H), 7.90 (s, 1H), 8.15 (s, 1H), 8.95
13
(s, 1H), 9.30 (s, 1H), 9.64 (s, 1H), 10.21 (s, 1H); C NMR (400 MHz, DMSO-d6): δ
54.74 (2C), 58.51 (2C), 59.36, 70.56 (2C), 103.97, 110.81, 115.02, 115.35, 115.63,
119.24, 119.60, 127.23, 128.86, 129.42, 132.28, 132.72, 136.38, 139.29, 139.59,
155.14, 156.54, 158.03, 163.54, 169.55; HRMS (ESI) for C27H32ClN7O4, (M+H)+
calcd: 554.2277; found: 554.2269.
4.2.7.
N-(3-((5-Fluoro-2-(4-(2-(bis(2-
methoxyethyl)amino)acetylamino)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7g). Yield: 15.5%; off-yellow solid; H
NMR (400 MHz, DMSO-d6): δ 2.77 (t, J = 8.0 Hz, 4H), 3.25 (s, 8H), 3.42 (t, J = 4.0
Hz, 4H), 5.75 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.26 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.47
(dd, J = 8.0 Hz, 16.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.44
(d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.95 (s, 1H),
13
8.10 (d, J = 4.0 Hz, 1H), 9.14 (s, 1H), 9.43 (s, 1H), 9.63 (s, 1H), 10.17 (s, 1H); C
16

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NMR (400 MHz, DMSO-d6): δ 54.81 (2C), 58.58 (2C), 59.43, 70.65 (2C), 113.59,
115.05, 117.85, 119.37 (2C), 119.39, 127.26, 129.04, 132.40, 132.56, 136.95, 139.58,
139.65 (J = 28.0 Hz), 139.70, 141.11, 141.30 (J = 76.0 Hz), 142.15, 150.17, 150.28 (J
= 44.0 Hz), 155.94, 155.97, 163.60,169.59; HRMS (ESI) for C27H32FN7O4, (M+H)+
calcd: 538.2573; found: 538.2563.
4.2.8.
N-(3-((5-Chloro-2-(3-methoxy-4-(2-(bis(2-
methoxyethyl)amino)acetylamino)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7h). Yield: 10.2%; off-yellow solid; H
NMR (400 MHz, DMSO-d6): δ 2.81 (t, J = 4.0 Hz, 4H), 3.24-3.28 (m, 8H), 3.47 (t, J
= 4.0 Hz, 4H), 3.65 (s, 3H), 5.79 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.29 (dd, J = 4.0 Hz,
16.0 Hz, 1H), 6.49 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 7.32 (t, J =
8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 8.03 (d, J
= 9.0 Hz, 1H), 8.20 (s, 1H), 8.97 (s, 1H), 9.30 (s, 1H), 9.60 (s, 1H), 10.20 (s, 1H); 13C
NMR (400 MHz, DMSO-d6): δ 55.23 (2C), 55.83, 58.43 (2C), 60.44, 71.00 (2C),
102.86, 104.27, 110.96, 115.06, 115.60, 119.36, 121.67, 127.20, 128.93, 132.28,
136.92, 139.34, 139.62, 148.47, 148.72, 155.11, 156.39, 158.06, 163.50, 169.16;
HRMS (ESI) for C28H34ClN7O5, (M+H)+ calcd: 584.2383, found; 584.2379.
4.2.9.
N-(3-((5-Chloro-2-(3-methyl-4-(2-(bis(2-
methoxyethyl)amino)acetylamino)phenylamino)-4-
pyrimidinyl)amino)phenyl)acrylamide (7i). Yield: 18.80%; off-brown solid; 1H
NMR (400 MHz, DMSO-d6): δ 2.05 (s, 3H), 2.76 (t, J = 4 Hz, 4H), 3.17 (s, 6H), 3.23
(s, 2H), 3.41 (t, J = 4.0 Hz, 4H), 5.73 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.23 (dd, J = 4.0
Hz, 16.0 Hz, 1H), 6.43 (dd, J = 9.0 Hz,16.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 3H), 7.52 (d,
J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.85 (s, 1H), 8.13 (s, 1H), 8.91 (s, 1H), 9.23
13
(s, 1H), 9.38 (s, 1H), 10.18 (s, 1H); C NMR(400 MHz, DMSO-d6): δ 55.17 (2C),
58.38 (3C), 59.67, 70.65 (2C), 103.92, 115.33, 115.64, 117.10, 119.63, 120.72,
121.70, 127.22, 128.85, 129.05, 130.50, 132.27, 136.91, 139.34, 139.66, 155.16,
156.53, 158.08, 163.51, 169.43; HRMS (ESI) for C28H34ClN7O4, (M+H)+ calcd:
568.2434; found: 568.2410.
4.2.10.
N-(3-((5-Chloro-2-(4-((tetrahydro-2-furanyl)methoxy)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7j). Yield: 16.56%; off-white solid; H
NMR (400 MHz, DMSO-d6): δ 1.61−1.68 (m, 1H), 1.81−1.90 (m, 2H), 1.95−2.01 (m,
1H), 3.67−3.70 (m, 1H), 3.75−3.82 (m, 3H), 4.09−4.12 (m, 1H), 5.75 (dd, J = 4.0 Hz,
8.0 Hz, 1H), 6.27 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.47 (dd, J = 8.0 Hz,16.0 Hz, 1H),
6.70 (d, J = 8.0 Hz, 2H), 7.32 (t, J = 8.0 Hz, 2H), 7.50 (t, J = 8.0 Hz, 3H), 7.90 (s,
1H), 8.11 (s, 1H), 8.91 (s, 1H), 9.17 (s, 1H), 10.22 (s, 1H); 13C NMR (400 MHz,
DMSO-d6): δ 25.59, 28.13, 67.83, 70.61, 76.91, 103.58, 114.40 (2C), 115.68, 115.82,
119.53, 120.85 (2C), 127.33, 128.99, 132.26, 133.99, 139.25, 139.41, 153.66, 155.19,
17

ACCEPTED MANUSCRIPT
156.51, 158.15, 163.51; HRMS (ESI) for C24H24ClN5O3, (M+H)+ calcd: 466.1640;
found: 466.1630.
4.2.11.
N-(3-((5-Fluoro-2-(4-((tetrahydro-2-furanyl)methoxy)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (7k). Yield: 12.80%; off-white solid; H
NMR (400 MHz, DMSO-d6): δ 1.54−1.63 (m, 1H), 1.76−1.84 (m, 2H), 1.89−1.97 (m,
1H), 3.59−3.64 (m, 1H), 3.69−3.78 (m, 3H), 4.03−4.09 (m, 1H), 5.70 (dd, J = 4.0 Hz,
16.0 Hz, 1H), 6.21 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.42 (dd, J = 8.0 Hz, 16 Hz, 1H),
6.70 (d, J = 9.0 Hz, 2H), 7.23 (t, J = 8.0 Hz, 1H), 7.40 (dd, J = 8.0 Hz,16.0 Hz, 2H),
7.48 (d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 8.02 (d, J = 4.0 Hz, 1H), 8.95 (s, 1H), 9.34 (s,
1H), 10.12 (s, 1H); 13C NMR(400 MHz, DMSO-d6): δ 26.20, 28.73, 68.43, 71.25,
77.54, 114.41, 115.09 (2C), 115.75, 118.32, 121.20 (2C), 127.88, 129.66, 132.90,
135.09, 140.02, 140.07 (J = 20.0 Hz), 141.71, 141.90 (J = 76.0 Hz), 142.53, 150.67,
150.78 (J = 44.0 Hz), 154.10, 156.65, 156.68, 164.09; HRMS (ESI) for C24H24FN5O3,
(M+H)+ calcd: 450.1936; found:. 450.1932.
4.2.12.
(Tetrahydro-2-furanyl)methyl,2-(4-((5-chloro-4-((3-((1-oxo-2-propen-1-
(7l). Yield:
yl)amino)phenyl)amino)-2-pyrimidinyl)amino)phenoxy)acetate
12.86%; off-yellow solid; 1H NMR (400 MHz, DMSO-d6): δ 1.49−1.56 (m, 4H), 3.68
(d, J = 8.0 Hz, 1H), 4.58 (d, J = 4.0 Hz, 1H), 4.61−4.66 (m, 3H), 4.79−4.84 (m, 2H),
5.24 (s, 1H), 6.53 (d, J = 8.0 Hz, 2H), 6.71 (dd, J = 4.0 Hz, 8.0Hz, 2H), 7.27 (dd, J =
8.0 Hz, 16.0Hz, 2H), 7.49 (d, J = 8.0 Hz, 3H), 7.80 (s, 1H), 8.09 (s, 1H), 8.87 (s, 1H),
9.17 (s, 1H), 9.99 (s, 1H); 13C NMR(400 MHz, DMSO-d6): δ 19.96, 28.88, 65.69,
66.29, 73.12, 73.27, 104.06, 113.75, 114.84, 116.25, 121.04, 127.66, 129.09, 132.54,
134.90, 139.49, 139.56, 139.74, 139.96, 144.13, 149.81, 152.97, 155.41, 156.82,
158.41, 163.83; HRMS (ESI) for C26H26ClN5O5, (M+H)+ calcd: 524.1695; found:
524.1684.
4.3 Biological evaluation
4.3.1 Cell culture and reagents
Ramos and Raji cells were purchased from Fuheng Biology Company (Shanghai,
China). Peripheral blood mononuclear (PBMC) cells were obtained from a healthy
adult male. The Cell Counting Kit-8 (CCK-8) reagent was purchased from Biotool
Company (Switaerland). The BTK enzym and the ADP-Glo™ Kinase Assay system
that measures ADP formed from a kinase reaction were purchased from Promega
Corporation (USA). The Annexin V-FITC Apoptosis Detection Kit and Cell Cycle
Assay were purchased from Beyotime Company (China). All cell lines were grown in
RPMI-1640 (Gibco®, USA) supplemented with 10% FBS (Gibco®, USA), 1%
18

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penicillin-streptomycin (Beyotime Company, China). The cells were maintained and
propagated as monolayer cultures at 37 °C in humidified 5% CO2 incubator.
4.3.2. In vitro kinase enzymatic assay
The BTK kinase enzyme system (Catalog. V9071) was purchased from Promega
Corporation (USA). Concentrations consisting of suitable levels from 0.1 to 100 nM
were used for all of the tested compunds. The experiments were performed according
to the instructions of the manufacturer. The more detailed and complete protocols, see
the
ADP-GloTM
kinase
Assay
Technical
Manual
available
at:
http://cn.promega.com/resources/protocols/product-information-sheets/n/btk-kinase-
enzyme-system-protocol/. The test was performed in a 384-well plate, and includes
the major 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 minutes, (3) add 5 µL
of ADP-Glo™ Reagent to stop the kinase reaction and deplete the unconsumed ATP,
leaving only ADP and a very low background of ATP, (4) incubate at room
temperature for 40 minutes, (5) add 10 µL of Kinase Detection, (6) reagent to convert
ADP to ATP and introduce luciferase and luciferin to detect ATP, (7) incubate at
room temperature for 30 minutes, (8) plate was measured on TriStar® LB942
Multimode Microplate Reader (BERTHOLD) to detect the luminescence (Integration
time 0.5-1 second). Curve fitting and data presentations were performed using
GraphPad Prism version 5.0. 25-27
The procedure to detect whether compound 7j forms irreversible inhibition or not
with BTK enzyme based on the ADP-Glo™ Kinase Assay system. Briefly, the
procedure includes: 1) Add inhibitor 7j (1µL, 50 µmol/ L) to 1×buffer (99 µL) to
afford 100 µL drug solution (500 nmol/L); 2) Dilute enzyme (10 µL) with 1×buffer
(190 µL) to provide 200 µL (2.5 ng/µL) enzyme solution; 3) Mix the drug solution
(100 µL) and the diluted enzyme solution (200 µL) at room temperature for 60 min,
then extract with dichloromethane two times (2×300 µL), the water solution
containing the inhibitor-linked BTK was retained; 4) Add substrate (100 µL) and ATP
(2.5µL) to1×buffer (97.5 µL) to afford the substrate/ATP mixture (200 µL); 5)
Combine the substrate/ATP mixture (2 µL) prepared in step 4 with the BTK-
19

ACCEPTED MANUSCRIPT
contained solution (3 µL), then incubate at room temperature for 60 min; 6) Add 5 µL
of ADP-Glo™ Reagent, and incubate at room temperature for 40 minutes to stop the
kinase reaction and deplete the unconsumed ATP; 6) Add 10 µL of Kinase Detection,
and incubate at room temperature for 30 minutes; 7) Record luminescence
(Integration time 0.5-1 second).
4.3.3 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 2,000 to 3,000 cells/well and were
maintained at 37 °C in a 5% CO2 incubator in RPMI1640 containing 10% fetal bovine
serum (FBS, Gibco) for one day. Cells were esposed to treatment for 48 h, and the
number of cells used per experiment for each cell lines was adjusted to obtain an
absorbance of 0.5 to 1.2 at 450 nm with a microplate reader (Thermo, USA).
Compounds were tested at appropriate concentrations (1.25 to 40 µM), with each
concentration duplicated five times. The IC50 values were calculated using GraphPad
Prim version 5.0.
4.3.4 Cytotoxic activity assay
Peripheral venous blood was collected in an ACD anticoagulant (Vblood: V
= 9:1) tube from the normal healthy adult male. The leucocyte-rich plasma
anticoagulant
was removed and the peripheral blood mononuclear cells (PBMC) consisting of
monocytes and the lymphocytes were separated on Lymphoprep (density 1.077g/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.
PBMC cells were seeded in 24-well plates at a density of 8,000 to 10,000
cells/well. Cells were treated with inhibitor 7j at appropriate concentrations (2.5, 5, 10,
20, 40 µM), with each concentration duplicated five times. Aftering maintaining the
cells at 37 °C in a 5% CO2 incubator in RPMI1640 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.
20

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4.3.5 Flow cytometry assay
The Ramos and Raji cells (1 to 5 ×105 cells/well) incubated in 6-well plates were
treated with solvent control (DMSO), spebrutinib, ibrutinib, or compound 7j in
medium containing 5% FBS for 48 h. Then, collected and fixed with 70% ethanol at
4 °C overnight. After being fixed with 75% ethanol at 4 °C for 24 h, 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 and Raji
cells at a density of approximately 2×105 cells/well were incubated in 6-well plates,
treated with different 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 10 min under subdued light. Stained cells were
analyzed flow cytometry assay (Becton-Dickinson, NJ, USA), and the results were
performed
using
FCS
Express
flow
cytometry
analysis
software
(ModFit LT 3.1).[28,29]
4.4 Molecular docking study
The crystal structure (PDB: 3GEN) of the kinase domain of BTK bound to a
pyrrolopyrimidine-containing compound.[30] The enzyme preparation and the
hydrogen atoms adding was performed in the prepared process. The whole BTK
enzyme was defined as a receptor and the site sphere was selected on the basis of the
binding location of pyrrolopyrimidine inhibitor. By moving the inhibitor and the
irrelevant water, molecules 7a, 7c, 7l, 7e, 7h and spebrutinib were placed,
respectively. The binding interaction energy was calculated to include van der Waals,
electrostatic, and torsional energy terms defined in the tripos force field. Ten docking
poses were generated and clustered, and compounds with predicted binding affinities
better than 7.0 kcal/mol in each of the three docking runs were selected for further
analysis and visual inspection. The structure optimization was performed using a
genetic algorithm, and only the best-scoring ligand protein complexes were kept for
analyses.[31-33] The WWW site also includes many resources for use of AutoDock,
21

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including detailed Tutorials that guide users through basic AutoDock usage, docking
with flexible rings, and virtual screening with AutoDock. Tutorials may be found at:
http://autodock.scripps.edu/faqs-help/tutorial.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (No.
81402788), the project of family noninvasive mechanical ventilation system
construction and promotion for AECOPD patients: Health and Family Planning
Commission of Liaoning Province (No. 2015B004), the Ph.D. Start-up Fund of
Natural Science Foundation of Liaoning Province (No. 20141115), and the
Cultivation Project of Youth Techstars, Dalian (2016RQ043) for the financial support
of this research.
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Fig. 1.
O
N
HN
O
O
O
N
N
N
O
O
N
O
N
N
N
N
N
H2N
H2N
N
H2N
N
N
N
N
1
2
3
Ibrutinib
Acalabrutinib
GS-4059
H
N
N
F
N
S
N
N
NH
O
HN
O
N
NH
HN
N
O
O
NH
NH
NH
NH
O
NH
O
O
N
N
O
O
F3C
PLS-123
4
6
5
Spebrutinib
HM71224
Fig. 2.
Cys481
Phe540
Ser538
Met477
Ile472

ACCEPTED MANUSCRIPT
Fig. 3.
Cys481
Phe540/Ser538/Ile472
covalent bond
Various side chains
O
O
O
O
O
or
N
=
O
O
HN
R2
HN
HN
R1
N
NH
HN
N
NH
N
N
Met477
F
Spebrutinib
Phase Ш
DPPYs 7a-l
BTK inhibitors
Improved activity
Fig. 4.
2.5 µM
Control
5 µM
12
10
8
6
10 µM
20 µM
40 µM
4
2
control
20 40
2.5 5 10
Concentrations (µM)
Fig. 5.
Ibrutinib(10 µΜ)
Spebrutinib(10 µΜ)
Blank
100
80
60
40
20
0
7j(5 µΜ)
7j(10 µΜ)
7j(20 µΜ)
7
j
B
7
j
7
j
S
p
I
b
l
(
(
(
a
5
µ
1
2
0
r
n
k
e
b
u
0
µ
M
t
i
µ
r
n
M
M
)
u
i
t
b
)
)
i
n
(
1
i
b
0
µ
(
1
0
M
)
µ
M
)