Novel amino acid-substituted diphenylpyrimidine derivatives as potent BTK inhibitors against B cell lymphoma cell lines

Article abstract of DOI:10.1016/j.bmc.2018.07.007

A new family of diphenylpyrimidine derivatives bearing an amino acid substituent were identified as potent BTK inhibitors. Among them, compound 7b, which features an L-proline substituent, was identified as the strongest BTK inhibitor, with an IC₅₀

Full text of DOI:10.1016/j.bmc.2018.07.007

Accepted Manuscript
Novel amino acid-substituted diphenylpyrimidine derivatives as potent BTK
inhibitors against B cell lymphoma cell lines
Changyuan Wang, Si Li, Qiang Meng, Xiuli Sun, Hua Li, Xiaohong Shu, Huijun
Sun, Kexin Liu, Zhihao Liu, Xiaodong Ma
PII:
S0968-0896(18)30826-5
https://doi.org/10.1016/j.bmc.2018.07.007
BMC 14444
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 April 2018
15 June 2018
5 July 2018
Please cite this article as: Wang, C., Li, S., Meng, Q., Sun, X., Li, H., Shu, X., Sun, H., Liu, K., Liu, Z., Ma, X.,
Novel amino acid-substituted diphenylpyrimidine derivatives as potent BTK inhibitors against B cell lymphoma
cell lines, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel amino acid-substituted diphenylpyrimidine derivatives as
potent BTK inhibitors against B cell lymphoma cell lines
Changyuan Wang a,1, Si Li a,1, Qiang Meng a, Xiuli Sun c, Hua Li a, Xiaohong Shu a, Huijun Sun a,
Kexin Liu a, Zhihao Liu a,b  and Xiaodong Ma a, 
a College of Pharmacy, College of laboratorymedicine, Dalian Medical University, Dalian
116044, PR China
bState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, P. R. China
cDepartment of Hematology, the First Affiliated Hospital of Dalian Medical University, Dalian
116011, PR China
1 These authors contributed equally to this work
 Corresponding author
E-mail address: xiaodong.ma@139.com (X. Ma), liuzhihao12399@126.com ( Z. Liu)
Abstract
A new family of diphenylpyrimidine derivatives bearing an amino acid
substituent were identified as potent BTK inhibitors. Among them, compound 7b,
which features an L-proline substituent, was identified as the strongest BTK inhibitor,
with an IC50 of 8.7 nM. Compound 7b also displayed similar activity against B-cell
lymphoma cell lines as ibrutinib. Moreover, 7b exhibited low cytotoxic activity
against normal PBMC cells. In addition, the acridine orange/ethidium bromide
(AO/EB) staining assay, Western blot analysis and flow cytometry analysis also
showed its effectiveness in interfering with B-cell lymphoma cell growth. The
molecular simulation performance showed that 7b forms additional strong hydrogen
bonds with the BTK protein. All these findings provided new clues about the
pyrimidine scaffold as an effective BTK inhibitor for the treatment of B-cell
lymphoma.
1

1. Introduction
Bruton's tyrosine kinase (BTK), a member of the Tec family of non-receptor
tyrosine kinases, plays a key role in the B-cell signaling pathway linking the cell
surface B-cell receptor (BCR) stimulation to downstream intracellular responses [1-3].
A number of B-cell-derived malignancies, including chronic lymphocytic leukemia
(CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL),
Waldenström’s macroglobulinemia (WM) and multiple myeloma (MM), are related to
the dysregulation of BTK kinase [4,5]. Ibrutinib is the first-in-class BTK inhibitor,
which demonstrated high efficacy in patients with CLL, MCL, WM, MM, etc. [6-8].
Additionally, a number of covalent and noncovalent BTK inhibitors, including
spebrutinib (2) (ClinicalTrials.gov identifier: NCT01744626) [9], and PLS-123 (3)
[10] have also been advanced to preclinical or clinical trial studies (Fig. 1). Recently,
our immense interest in finding effective BTK inhibitors also led to the identification
of a set of novel diphenylpyrimidine (DPPY) analogues, including Pho-DPPY (4)
[11], SFA-DPPY (5) [12] and Imi-DPPY (6) [13], which exhibited improved
anti-B-cell lymphoma activity compared with ibrutinib (Fig. 1). Their structure and
activity relationships (SARs) showed that the pyrimidine core, along with a C-4
N-phenylacrylamide functionality are essential for their high anti-BTK activity. The
co-crystal structure of the typical BTK inhibitor 1 with the BTK enzyme also revealed
that the acryloyl substituent binds strongly through a covalent bond to the Cys481
residue in the BTK enzyme, while the C-2 aniline side chain of the pyrimidine core is
in tight contact with the Ser538, Asp539 and Lys430 residues at the bottom of the
ATP-binding pocket. All these binding forces contribute to the strong inhibitory
potency of this BTK inhibitor [11-16].
2

H
N
N
F
N
N
NH
O
HN
O
N
O
NH
O
NH
NH
O
O
N
O
N
NH
NH
N
O
O
H2N
N
N
F3C
3
1
2
Cl
NH
N
Cl
NH
N
N
N
HN
N
N
HN
N
HN
N
O
NH
O
NH
NH
O
O
O
NH
O
O
S
O
O
O
O
N
N
P
O
O
4
6
5
F
Fig. 1 Structures of the novel BTK inhibitors.
O
O
Cys481
O
covalent bond
N
H
N
HN
O
Amino acid
Hydrogen bond
BTK protein
N
O
HN
HN
Cl
N
NH
OMe
N
HN
F3C
N
NH
N
Amino acid: Glycine, L-Alanine, L-proline
DPPYs 7a-d
Rociletinib
EGFRT790M Inhibitor
BTK inhibitors
Phase Ш
Fig. 2 Discovery strategy of the new DPPYs as potent BTK inhibitors.
Amino acid-substituted derivatives, especially the short synthetic and long-acting
peptide, are involved in a variety of physiological and pathological processes. Drugs
that bear an amino acid functionality play very important roles in modulating various
cell functions, and have been successfully applied to treat certain human diseases
[17,18]. For example, goserelin (a synthetic gonadotropin-releasing hormone analog,
3

marketed as Zoladex®) is applied to treat breast cancer and prostate cancer [19],
glatiramer acetate (a synthetic peptide with four amino acids, marketed as Copaxone®)
is used to treat multiple sclerosis [20], and exenatide (a synthetic glucagon-like
peptide-1 analog, marketed as Byetta®) is used to treat type 2 diabetes [21].
Accordingly, amino acid derivatives do favor forming hydrogen-bonds with proteins,
thus contributing to enhance the contact with target proteins [22,23]. Therefore,
various amino acid -substituted aniline side chains were installed on the C-2 position
of the pyrimidine scaffold to form strong hydrogen bond with BTK (Fig. 2). The
synthesis of these amino acid-substituted DPPY derivatives, and their biological
evaluation of their activity against the BTK enzyme and B-cell lymphoma cell lines
are described in this study.
2. Results and Discussion
2.1 Chemistry
The amino acid -substituted pyrimidine analogues were synthesized as illustrated
in Scheme 1. 3-Nitroaniline (8) was reacted with acryloyl chlorine in the NaHCO3
base to produce the intermediate N-acryloyl-4-nitrobenzamide (9), which was then
reduced by the Fe-NH4Cl reagent to form the 4-aminobenzamide derivative (10).
After regioselective substitution of the C-4 chlorine atom in the
2,4,5-trichloropyrimidine reagent with aniline 10, the key pyrimidine intermediate 11
was prepared. Generally, the C-2 aniline side chains 12a-d were conveniently
synthesized via nucleophilic substitution, dehydration reaction, and reduction reaction
according to our previously reported procedures [24,25]. Finally, through the coupling
reactions of 11 with aniline intermediates 12a-d under the action of trifluoroacetic
acid (TFA) at reflux temperature, the title molecules 7a-d were obtained with yield
ranging from 12 to 23% [26].
4

Cl
NH2
N
NO2
Cl
Cl
N
Cl
N
NH
NO2
Cl
N
c
NH
b
a
O
NH
O
N
O
NH2
H
9
11
8
10
O
O
H
N
HN
Amino acid
O
O
H2N
O
Amino acid
N
HN
Cl
N
NH
12a-d
H
N
d
7a-d
Amino acid: Glycine, L-Alanine, L-Proline
Scheme 1. Synthetic route of title compounds 7a-d. Reagents and conditions: (a) acryloyl chloride,
NaHCO3, CH3CN, rt, 0.5 h, 95%; (b) Fe-NH4Cl, MeOH-H2O, 2 h, 70 C, 72%; (c)
2,4,5-trichloropyrimidine, DIPEA, i-PrOH, 60C, 2 h, 83%;
intermediates 12a-d, 2-BuOH, 100 C, 12 h, 12% to 23%.
(d) trifluoroacetic acid,
Table 1. Biological activity of theamino acid -substituted diphenylpyrimidine derivatives 7a-d.
Cl
N
HN
O
N
NH
O
N
H
R
O
Enzymatic activity(IC50,
nM)
Cellular antiproliferative activity
Compd
.
(IC50, μM)a
R
NAMALWA
>40.0
JAK3
BTK
Ramos
Raji
NH
OH
O
>500.0
11.2
>40.0
>40.0
7a
7b
7c
7d
O
N
196.0
>500.0
496.0
8.7
8.7
6.5
5
4.8±1.1
29.3±2.6
16.9±3.1
19.7±3.2
40.8
6.7±1.2
25.2±1.5
18.0±2.1
O
NH
O
O
O
NH
O
20.9±3.3

Ibrutini
b
<100.0
0.4
19.6
5.14
19.5
a
Data represent the mean of at least three separate experiments. Dose-response curves were
determined at five concentrations. The IC50 values are the concentrations in nano- or micro-molar
needed to inhibit cell growth by 50%, as calculated using GraphPad Prism version 5.0.
2.2 Biological activity
All theamino acid-substitute pyrimidine analogues were evaluated for their
potency to inhibit the phosphorylation of BTK, EGFRT790M and the JAK3 enzymes
using the ADP-GloTM kinase assay system (Promega, USA) [27,28]. Moreover, their
antiproliferative activity against the B-cell lymphoma cell lines (Raji, Ramos,
NAMALWA) were also evaluated with the CCK-8 assay [29]. For comparison, the
representative anti-BTK agent ibrutinib was evaluated as well. The results of the
biological evaluations are presented in Table 1. These results showed that most of
these compounds effectively inhibited the BTK enzymatic activity at concentrations
ranging from 6.5 to 11.2 nM. Interestingly, these molecules are not active against both
EGFRT790M and the JAK3 enzymes (IC50 >100 nM), indicating their improved
selectivity against BTK kinase. According to the kinase-based test result, it clearly
can be seen that the ester derivatives, including the L-alanine methyl ester, L-proline
methyl ester, and glycine methyl ester, which was installed on the C-2 aniline side
chain is favorable. Nevertheless, the acid derivative 7a, which was obtained by
hydrolyzing the ester substituent in compound 7d, has slightly reduced anti-BTK
enzymatic activity. In addition, their activity against the B-cell lymphoma cells also
showed that theseamino acid -substituted molecules could significantly inhibited the
proliferation of B-cell lymphoma cells at concentrations in the range of 4.8 to 29.3
μM. Apparently, the molecule 7a bearing an L-alanine amino acid is less effective in
inhibiting B-cell lymphoma cells, with an IC50 value higher than 40 μM. Notably, the
L-proline-substituted pyrimidine 7b displayed remarkably improved anti-proliferative
activity against NAMALWA (IC50 = 4.8 μM) and Raji (IC50 = 6.7 μM) cells
compared with ibrutinib. Although L-alanine-substituted compound 7d and
glycine-substituted compound 7c are as potent as L-proline-substituted 7b in
inhibiting the BTK enzymatic activity, both of them are less active against the B-cell
6

lymphoma cell lines (NAMALWA, Ramos and Raji). Possibly, the slightly high
hydrophilicity of the tetrahydropyrrole substituent in compound 7b results in its
enhanced anti-cancer activity. Compared with the novel drug ibrutinib, compound 7b
also displayed approximately 4 times higher activity to interfere with the proliferation
of NAMALWA cells, and 3-times higher activity to inhibit Raji cells activity,
respectively. Owing to these excellent kinase- and cell-based biological property,
compound 7b was selected to perform the further biological evaluation. The
4',6-diamidino-2-phenylindole (DAPI) and AO/ EB staining assays for the potent
inhibitor 7b showed its higher pro-apoptotic activity in NAMALWA cells than that of
ibrutinib at similar concentration (5 μM) (Fig. 3). Furthermore, the effects of
compound 7b were also analyzed in normal peripheral blood mononuclear cells
(PBMC) using the AO/EB staining assay. The results of the analysis revealed its low
cytotoxicity at concentrations of 5, 10 and 20 μM (Fig. 4). Additionally, inhibitor 7b
also exerted significant effects on the viability of NAMALWA cells in a dose- and
time-dependent manner. Also, treatment with inhibitor 7b at the concentration of 2
μM for 72 h almost completely inhibited NAMALWA cells (Fig. 5).
Control
2 μM
1 μM
5 μM
5 μM (Ibrutinib)
Fig. 3 AO/EB staining assays for the potent compound 7b.
7

5 μM
10 μM
Control
20 μM
b
7
)
5
5 μM
10 μM
20 μM
0
7b
1
X
ibrutinib
(
s
r
b
e
i
b
n
i
t
m
u
u
r
b
N
I
l
l
e
C
l
M
o
M
M
r
μ
t
μ
μ
n
0
5
0
2
o
1
C
Fig. 4 Cell cytotoxicity of compound 7b against the normal PBMC cells.
Fig. 5 The effects of compound 7b on the cell viability of NAMALWA cells.
Apoptosis is a biologically important type of cell death. The effect of compound
7b, the most active BTK inhibitor, on apoptosis was evaluated by flow cytometry
analysis of NAMALWA cells incubated with 1, 2 and 5 µM of compound 7b for 48 h.
The analysis results shown in Fig. 6, revealled that inhibitor 7b induced apoptosis in
NAMALWA cells, increasing the apoptosis rate from 10.77 to 22.08%. Noteworthy,
even at a low concentration of 1 µM, compound 7b induced higher apoptosis rate
(140.77%) than the reference agent ibrutinib (9.81%) at a concentration of 5 µM.
8

1 μM
2 μM
0.82%
5 μM
1.67%
13.33%
8.75%
0.92%
7.43%
6.96%
5.83%
4.94%
Control
5 μM (Ibrutinib)
)
%
(
4.05%
5.75%
4.06%
0.0%
0.0%
0.73%
e
t
22.08
a
r
s
i
s
14.39
10.77
i
t
o
9.81
t
p
o
p
A
0.0
k
n
M
M
M
M
a
μ
l
μ
μ
μ
B
5
1
5
2
-
b
i
n
i
t
u
r
b
I
Concentrations (μM)
Fig. 6 Compound 7b induced NAMALWA cell apoptosis in vitro. The cells were incubated with
the indicated concentrations of 7b 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.
NAMALWA cell
I
b
7
r
u
b
t
i
n
i
b
1
5
control
10
(μM)
BTK
10
p-BTK
Actin
Fig. 7 Compound 7b and ibrutinib inhibited the activation of EGFR and downstream signaling in
NAMALWA cells harboring BTK protein.
To evaluate the effects of theamino acid-substituted pyrimidine derivatives on the
BTK activation and downstream signaling, the most active inhibitor 7b was tested for
its capability to interfere with the BTK activation and downstream signaling in
NAMALWA cells using ibrutinib as a control. The cells were treated with various
9

concentrations (1, 5, 10 µM) of inhibitor 7b for 48 h, and the expression and
activation of BTK and phosphorylated BTK (p-BTK) were determined by
immunoblotting analysis. The Western blot analysis results, presented in Fig. 7,
clearly showed that treatment with 7b inhibited the phosphorylation of BTK in a
dose-dependent manner. Additionally, compound 7b displayed as strong inhibitory
activity as that of ibrutinib, at the same drug concentration (10 µM).
a)
Tyr476
b)
Tyr476
Lys430
Gly480
Gly409
Lys430
Gly480
Gly409
Val416
Val416
Asp539
Asp539
Cys481
Cys481
H2O
Ser538
Ser538
Asn484
Asn484
d)
d)
Tyr476
c)
Tyr476
Lys430
Gly480
Gly409
Lys430
Gly480
Gly409
Val416
H2O
Val416
Asp539
Asp539
Cys481
Cys481
H2O
Ser538
Ser538
Asn484
Asn484
Fig. 8 Putative binding models of spebrutinib and the title molecules within BTK enzyme (PDB
code: 3GEN), a): spebrutinib, b): inhibitor 7b, c): inhibitor 7d, d): inhibitor 7a.
2.3 Molecular docking
Using computer molecular simulation technology, we also examined the putative
binding mechanism of theamino acid-substituted analogues with the BTK protein.
Two of the most active inhibitors, namely 7b and 7d, in parallel with a less potent
inhibitor, namely 7a, were separately docked into the binding pocket of the BTK
protein (PDB code: 3GEN) [30]. The optimal binding conformations were obtained,
using the Autodock 4.2 software and its default parameters [31,32], and are presented
in Figures 8a-d. As expected, the designed pyrimidine derivatives form a strong
10

hydrogen bond between the introducedamino acid functionality with the amino acid
Ser538 via a water molecule. However, for spebrutinib, this binding force is not
present. Moreover, although the two potent inhibitors 7b and 7d formed the important
covalent bond with the Cys481 residue as spebrutinib, the pyrimidine core in 7b and
7d moved farther away from the binding pocket than that in spebrutinib, thus resulting
in a significantly reduced anti-BTK activity (approximately 10 times lower). For the
less active inhibitor 7a, this movement also caused the loss of the important covalent
bond between acryoylamide with the Cys481 residue. Accordingly, compound 7a has
approximately 11-fold lower anti-BTK activity than compound 7b. In a word, these
binding modes exactly explain the anti-BTK activity data.
3. Conclusion
A class of diphenylpyrimidine derivatives bearing aamino acid functionality were
synthesized and biologically evaluated their biological activity against BTK kinase.
Notably, compound 7b, the most active inhibitor in this set of molecules, displayed
strong activity against BTK, with an IC50 of 8.7 nM. The cell-based activity assay also
showed that 7b had a similar level of potency to inhibit the proliferation of B-cell
lymphoma cells. In addition, its effects on normal PBMC cells showed its low
cytotoxicity. Other biological evaluations, including the AO/EB staining assay,
Western blot analysis and flow cytometry analysis also suggested the high potency of
this compound to block the proliferation of B-cell lymphoma cells. Moreover, the
molecular simulation analysis revealed that 7b was in tight contact with the BTK
enzyme by forming additional strong hydrogen bonds. Overall, this study provided a
new insight to improve the activity ofamino acid-substituted diphenylpyrimidine
derivatives against the BTK enzyme.
4. Experimental section4.1. General methods for chemistry
All reagents and solvents were obtained from commercial sources and used without
further purification. High resolution ESI-MS was performed on an
AB Sciex TripleTOF® 4600 LC/MS/MS system. 1H and 13C NMR datas were
obtained on a Bruker Avance at 400 and 100 MHz, respectively. Coupling constants
11

(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-d
Amino acid-substituted anilines 12a-d, and 2-chloropyrimidine 11were generally
synthesized according to the methods reported by us and others [24-26]. All these
intermediates were used without any purification and structural characterization. With
these intermediates in hand, the desired compounds were synthesized as described
below.
A flask was charged with compounds 11 (1.0 mmol), anilines 12a-d, (1.0 mmol),
TFA (342 mg. 3 mmol), and 2-BuOH (15 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 by using flash chromatography with dichloromethane/methanol (v/v, 12a:
15/1; 12c-d: 30/1) as eluents.
4.2.1. N-[3-[[5-Chloro-2-[4-[2-[(carbonylmethyl)amino]-2-oxoethoxy]phenylamino]-
4-pyrimidinyl]amino]phenyl]-2-acrylamide (7a):
1
1
Yield, 12%; off-white solid; H NMR (400 MHz, DMSO-d6): H NMR (400 MHz,
DMSO) δ 10.93(br, 1H), 10.15(s, 1H), 9.43(br, 1H), 8.45-8.48(m, 1H), 8.22(s, 1H),
7.67(s, 1H), 7.51-7.47(m, 2H), 7.32-7.22(m, 2H), 6.82(d, J =8.0 Hz, 2H), 6.77(dd, J
=16.8, 10.4 Hz, 1H), 6.23(dd, J = 16.8, 4.0 Hz, 1H), 5.72(dd, J =10.4, 4.0 Hz, 1H),
13
4.45(s, 2H), 3.83(s, 2H); C NMR (100 MHz, DMSO) δ 171.07, 168.41, 168.36,
168.00, 163.49, 156.72, 153.32, 139.59, 139.34, 138.24, 132.22, 128.68, 126.71,
121.68, 121.42 (2C), 114.95 (2C), 114.73, 114.35, 103.93, 67.19, 42.15; HRMS (ESI)
m/z calcd for C23H21ClN6O5, [M+H]+ 497.1335, found: 497.1414.
12

4.2.2.
N-[3-[[5-Chloro-2-[4-[2-[2-(methoxycarbonyl)-1-pyrrolidinyl]-2-oxoethoxy]
phenylamino]-4-pyrimidinyl]amino]phenyl]-2-acrylamide (7b): Yield, 22%; off-white
1
solid; H NMR (400 MHz, DMSO-d6): δ10.27(s, 1H), 9.21(s, 1H), 8.88(s, 1H),
8.10(s, 1H), 7.94(s, 1H), 7.51-7.49(m, 3H), 7.34-7.28(m, 2H), 6.71-6.69(d, J =8.4 Hz,
2H), 6.50(dd, J =16.8, 10.0 Hz, 1H), 6.26(dd, J =10.0, 2.0 Hz, 1H), 5.76-5.73(dd, J
=16.8, 2.0 Hz, 1H), 4.75-4.66(m, 1H), 4.65(s, 2H), 3.62-3.37(m, 2H), 3.37 (s, 3H),
13
1.94(m, 2H), 1.50(m, 2H); C NMR (100 MHz, DMSO) δ 171.82, 166.70, 163.62,
158.24, 156.52, 155.19, 153.21, 139.60, 139.39, 134.41, 132.42, 129.00, 127.31,
120.89(2C), 115.58, 115.40, 114.67, 114.48(2C), 103.85, 72.62, 66.77, 59.36, 45.97,
28.64, 19.64; HRMS (ESI) m/z calcd for C27H27ClN6O5, [M+H]+ 551.1804, found:
551.1886.
4.2.3.
(S)-N-[3-[[5-Chloro-2-[4-[2-[2-(methoxycarbonylethyl)amino]-2-oxoethoxy]
phenylamino]-4-pyrimidinyl]amino]phenyl]-2-acrylamide (7c): Yield, 18%; off-white
1
solid; H NMR (400 MHz, DMSO-d6): δ 10.22(s, 1H), 9.22(s, 1H), 8.95(s, 1H),
8.49(s, 1H), 8.16(s, 1H), 7.93(s, 1H), 7.56-7.54(m, 3H), 7.37(d, J =8.0 Hz, 2H),
6.80(d, J =8.0 Hz, 2H), 6.48(dd, J =16.8, 12.0 Hz, 1H), 6.33(dd, J =16.8, 4.0 Hz, 1H),
6.02-5.79(dd, J =12.0, 4.0 Hz, 1H), 4.46-4.42(m, 3H), 3.67(s, 3H), 1.38(d, J =8.0 Hz,
3H);13C NMR (100 MHz, DMSO) δ 172.43, 168.26, 163.61, 158.22, 156.57, 155.22,
152.92, 139.56, 139.37, 134.73, 132.37, 129.00, 127.38, 120.84(2C), 119.56, 115.83,
115.53, 114.91(2C), 103.89, 72.82, 67.54, 48.11, 17.35; HRMS (ESI) m/z calcd for
C25H25ClN6O3S, [M+H]+ 525.1648, found: 525.1733.
4.2.4.
(S)-N-[3-[[5-Chloro-2-[4-[2-[(methoxycarbonylmethyl)amino]-2-oxoethoxy]
phenylamino]-4-pyrimidinyl]amino]phenyl]-2-acrylamide (7d): Yield, 19%; off-white
1
solid; H NMR (400 MHz, DMSO) δ 10.26(s, 1H), 9.23(s, 1H), 8.91(s, 1H),
8.47-8.45(d, J =8.0 Hz, 1H), 8.12(s, 1H), 7.94(s, 1H), 7.53-7.51(m, 3H), 7.32(d, J =
8.0 Hz, 2H), 6.76(d, J =8.0 Hz, 2H), 6.52-6.46(dd, J =16.0, 12.0 Hz, 1H), 6.28-6.24(J
=16.0 Hz, 1H), 5.77-5.74(J =12.0 Hz, 1H), 4.42(s, 2H), 3.63(s, 3H), 3.37(s, 2H); 13C
NMR (100 MHz, DMSO) δ 172.76, 167.81, 163.15, 157.70, 156.10, 154.67, 152.41,
139.09, 138.89, 134.23, 131.90, 128.54, 126.91, 120.39(2C), 119.06, 115.35, 115.02,
13

114.46(2C), 103.40, 67.04, 51.96, 47.27; HRMS (ESI) m/z calcd for C24H23ClN6O5,
[M+H]+ 511.1491, found: 511.1421.
4.3. Kinase enzymatic assays
Enzymatic assays were tested by using the ADP-GloTM assay system (BTK:
Catalog. V2941, JAK3: Catalog. V3701, Promega). The experiments were performed
according to the instructions of the manufacturer. The detailed and complete
protocols,
https://cn.promega.com/products/cell-signaling/kinase-assays-and-kinase-biology/btk-
kinase-enzyme-system/?catNum=V2941 and
and
the
active
kinase
data
were
available
at:
https://cn.promega.com/products/cell-signaling/kinase-assays-and-kinase-biology/jak
3-kinase-enzyme-system/?catNum=V3701), respectively. 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 minutes, (3) add 5 μL of ADP-Glo™ reagent to
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 minutes, (5)
add 10 μL of kinase detection, (6) reagent to convert ADP to ATP andintroduce
luciferase and luciferin to detect ATP, (7) incubate at room temperature for 30
minutes, (8) plates 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.
4.4.Cell and reagents
4.4.1.Cellular activity assay
Ramos, Raji and NAMALWA cells were purchased from Fuheng Biology
Company (Shanghai, China). Cell viability assays were performed by using CCK-8
reagent (Biotool Company). The cells were seeded in 96-well plates at a density of
3,000 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
14

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 to 1.2 at 450 nm with a microplate reader
(Thermo Fisher, 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 Prism version 5.0.
4.4.2.Cytotoxic activity assay
Peripheral venous blood was collected in an ACD anticoagulant (Vblood: V anticoagulant
= 9:1) tube from the normal healthy adult 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 1.077g/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 200,000 to 250,000
cells/well. Cells were treated with inhibitors 7b and Ibrutinib at appropriate
concentrations (1, 2, 5 μM), with each concentration 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.
4.5.Flow cytometry assay
The NAMALWA cells (1 × 106 cells/well) incubated in 6-well plates were treated
with solvent control (DMSO), ibrutinib, or compounds 7b in medium containing 5%
FBS for 72 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).
4.6.Western blotting assay
15

The lysates from Namalwa cells in different groups were extracted and
centrifuged at 14,000 g for 15 min at 4 °C, then the total proteins were obtained. An
aliquot (50 μg 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
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.
4.7.Molecular docking study
Docking studies were carried out on AutoDock 4.2. The crystal structure (PDB:
3GEN) of the kinase domain of BTK bound to a pyrrolopyrimidine-containing
compound. 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, b, 7d 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. During the performance, the covalent bond between
the acrylamide group and the amino acid Cys481 was set as default when the distance
beween them lower than 2.82 Å (spebrutinib could form covalent bond in this
distance). The structure optimization was performed using a genetic algorithm, and
only the best-scoring ligand protein complexes were kept for analyses.
Acknowledgements
16

We are grateful to the National Natural Science Foundation of China (No.
81672945, 81603186), and the Research Fund of Higher Education of liaoning
province (No. LF2017008, LQ2017039) for the financial support of this research.
References
[1] ppers . Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer.
2005; 5: 251−62.
[2] Seiler T, Dreyling M. Bruton's tyrosine kinase inhibitors in B-cell lymphoma:
current experience and future perspectives. Expert Opin Investig Drugs. 2017; 26:
909−15.
[3] Adem J, Eray M, Eeva J, Nuutinen U, Pelkonen J. Advantages of targeting B cell
receptor complex to treat B-cell derived autoimmune diseases and lymphomas.
Mol Immunol. 2017; 88: 135−7.
[4] Corneth OB, Klein Wolterink RG, Hendriks RW. BTK signaling in B cell
differentiation and autoimmunity. Curr Top Microbiol Immunol. 2016; 393:
67−105.
[5] Robak P, Robak T. Novel synthetic drugs currently in clinical development for
chronic lymphocytic leukemia. Expert Opin Investig Drugs. 2017; 26: 1−17.
[6] Honigberg LA, Smith AM, Sirisawad M, Verner E, Loury D, Chang B, Li S, Pan
Z, Thamm DH, Miller RA, Buggy JJ. The Bruton tyrosine kinase inhibitor
PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune
disease and B-cell malignancy. Proc Natl Acad Sci U S A. 2010; 107: 13075−80.
[7] de Rooij MF, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ, Pals ST,
Spaargaren M. The clinically active BTK inhibitor PCI-32765 targets B-cell
receptor- and chemokine-controlled adhesion and migration in chronic
lymphocytic leukemia. Blood. 2012; 119: 2590−4.
[8] de Claro RA, McGinn KM, Verdun N, Lee SL, Chiu HJ, Saber H, Brower ME,
George Chang CJ, Pfuma E, Habtemariam B, Bullock J, Wang Y, Nie L, Chen
XH, Lu DR, Al-Hakim A, Kane RC, Kaminskas E, Justice R, Farrell AT, Pazdur
R. FDA Approval: Ibrutinib for patients with previously treated mantle cell
lymphoma and previously treated chronic lymphocytic leukemia. Clin Cancer
es. 2015; 21: 3586−90.
[9] Evans E, Ponader S, Karp R,Tester R, Sheets M, Aslanian S, Martin, TS, Nacht M,
Zhu Z, Chaturvedi P, Witowski S, Lounsbury H, Stiede K, Burger J, Petter R,
17

Singh J, Westlin WF. Covalent inhibition of Btk with clinical development
compound AVL-292 disrupts signaling that maintains the microenvironment
necessary for chronic lymphocytic leukemia growth. Cl Lymph Myelom Leuk.
2011; 11: S173−4.
[10]Li X, Zuo Y, Tang G, Wang Y, Zhou Y, Wang X, Guo T, Xia M, Ding N, Pan Z.
Discovery of a series of 2,5-diaminopyrimidine covalent irreversible inhibitors of
Bruton’s tyrosine kinase with in vivo antitumor activity. J Med Chem. 2014; 57:
5112−28.
[11]Ge Y, Yang H, Wang C, Meng Q, Li L, Sun H, Zhen Y, Liu K, Li Y, Ma X. Design
and synthesis of phosphoryl-substituted diphenylpyrimidines (Pho-DPPYs) as
potent Bruton's tyrosine kinase (BTK) inhibitors: Targeted treatment of B
lymphoblastic leukemia cell lines. Bioorg Med Chem. 2017; 25: 765−72.
[12]Liu H, Qu M, Xu L, Han X, Wang C, Shu X, Yao J, Liu K, Peng J, Li Y, Ma X.
Design and synthesis of sulfonamide-substituted diphenylpyrimidines
(SFA-DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors with improved
activity toward B-cell lymphoblastic leukemia. Eur J Med Chem. 2017; 135:
60−9.
[13]Ge Y, Jin Y, Wang C, Zhang J, Tang Z, Peng J, Liu K, Li Y, Zhou Y, Ma X.
Discovery of novel Bruton’s tyrosine kinase (BT ) inhibitors bearing a
N,9-diphenyl-9H-purin-2-amine scaffold. ACS Med Chem Lett. 2016; 7: 1050−5.
[14]Seiler T, Dreyling M. Bruton's tyrosine kinase inhibitors in B-cell lymphoma:
current experience and future perspectives. Expert Opin Investig Drugs. 2017; 26:
909−15.
[15]Zhao D, Huang S, Qu M, Wang C, Liu Z, Li Z, Peng J, Liu K, Li Y, Ma X, Shu X.
Structural optimization of diphenylpyrimidine derivatives (DPPYs) as potent
Bruton's tyrosine kinase (BTK) inhibitors with improved activity toward B
leukemia cell lines. Eur J Med Chem. 2017; 126: 444−55.
[16]Wang B, Deng Y, Chen Y, Yu K, Wang A, Liang Q, Wang W, Chen C, Wu H, Hu
C, Miao W, Hur W, Wang W, Hu Z, Weisberg EL, Wang J, Ren T, Wang Y, Gray
NS, Liu Q, Liu J. Structure-activity relationship investigation for
benzonaphthyridinone derivatives as novel potent Bruton's tyrosine kinase (BTK)
irreversible inhibitors. Eur J Med Chem. 2017; 137: 545−57.
[17]Kukowska M. Amino acid oramino acid conjugates of acridine/acridone and
quinoline/quinolone-containing drugs. A critical examination of their clinical
18

effectiveness within a twenty-year timeframe in antitumor chemotherapy and
treatment of infectious diseases. Eur J Pharm Sci. 2017; 109: 587−615.
[18]Chen SW, Van Regenmortel MH, Pellequer JL. Structure-activity relationships in
peptide-antibody complexes: implications for epitope prediction and development
of synthetic peptide vaccines. Curr Med Chem. 2009; 16: 953−64.
[19]Saphner T, Troxel AB, Tormey DC, Neuberg D, Robert NJ, Pandya KJ,
Edmonson JH, Rosenbluth RJ, Abeloff MD. Phase II study of goserelin for
patients with postmenopausal metastatic breast cancer. J Clin Oncol. 1993; 11:
1529−35.
[20]Bell C, Anderson J, Ganguly T, Prescott J, Capila I, Lansing JC, Sachleben R,
Iyer M, Fier I, Roach J, Storey K, Miller P, Hall S, Kantor D, Greenberg BM,
Nair K, Glajch J. Development of Glatopa® (Glatiramer Acetate): The first
FDA-Approved generic disease-modifying therapy for relapsing forms of
multiple sclerosis. J Pharm Pract. 2017; doi: 10.1177/0897190017725984.
[21]DeFronzo RA, Okerson T, Viswanathan P, Guan X, Holcombe JH, MacConell L.
Effects of exenatide versus sitagliptin on postprandial glucose, insulin and
glucagon secretion, gastric emptying, and caloric intake: a randomized,
cross-over study. Curr Med Res Opin. 2008; 24: 2943−52.
[22]Ludwig R, Khudozhitkov AE, Stange P, Golub B, Paschek D, Stepanov AG,
Kolokolov DI. Characterization of doubly ionic hydrogen bonds in protic ionic
liquids by NMR deuteron quadrupole coupling constants: differences to H-bonds
in amides, peptides, and proteins. Angew Chem Int Ed Engl. 2017; 56: 14310−4.
[23]Qing G, Lu Q, Li X, Liu J, Ye M, Liang X, Sun T. Hydrogen bond based smart
polymer for highly selective and tunable capture of multiply
phosphorylated peptides. Nat Commun. 2017; 8: 461.
[24]Song Z, Huang S, Yu H, Jiang Y, Wang C, Meng Q, Shu X, Sun H, Liu K, Li Y,
Ma X. Synthesis and biological evaluation of morpholine-substituted
diphenylpyrimidine derivatives (Mor-DPPYs) as potent EGFR T790M inhibitors
with improved activity toward the gefitinib-resistant non-small cell lung cancers
(NSCLC). Eur J Med Chem. 2017; 133: 329−39.
[25]Lu C, Kim BM, Lee D, Lee MH, Kim JH, Pyo HB, Chai KY. Synthesis of lipoic
acid-peptide conjugates and their effect on collagen and melanogenesis. Eur J
Med Chem. 2013; 69: 449−54.
[26]Ge Y, Wang C, Song S, Huang J, Liu Z, Li Y, Meng Q, Zhang J, Yao J , Liu
19

K, Ma X, Sun X. Identification of highly potent BTK and JAK3 dual inhibitors
with improved activity for the treatment of B-cell lymphoma. Eur J Med Chem.
2017, 143;1847-57.
[27]Goueli S, Hsiao K, Vidugiris G, Hennek J, Zegzouti H. ADP-GloTM: A one
platform ideal for assay development, screening and profiling of all
phosphotransferases. FASEB J. 2015; 29: 934.1.
[28]Zegzouti H, Zdanovskaia M, Hsiao K, Goueli SA. ADP-Glo: A bioluminescent
and homogeneous ADP monitoring assay for kinases. Assay Drug Dev Technol.
2009; 7: 560−72.
[29]Wu H, Wang W, Liu F, Weisberg EL, Tian B, Chen Y, Li B, Wang A, Wang B,
Zhao Z, McMillin DW, Hu C, Li H, Wang J, Liang Y, Buhrlage SJ, Liang J, Liu
J, Yang G, Brown JR, Treon SP, Mitsiades CS, Griffin JD, Liu Q, Gray NS,
Discovery of a potent, covalent BTK inhibitor for B-cell lymphoma, ACS Chem
Biol. 2014; 9: 1086−91.
[30]Marcotte DJ, Liu YT, Arduini RM, Hession CA, Miatkowski K, Wildes CP,
Cullen PF, Hong V, Hopkins BT, Mertsching E, Jenkins TJ, Romanowski MJ,
Baker DP, Silvian LF. Structures of human Bruton's tyrosine kinase in active and
inactive conformations suggest a mechanism of activation for TEC family kinases.
Protein Sci. 2010; 19: 429−439.
[31]Wildman SA, Zheng X, Sept D, Auletta JT, Rosenberry TL, Marshall GR.
Drug-like leads for steric discrimination between substrate and inhibitors of
human acetylcholinesterase. Chem Biol Drug Des. 2011; 78: 495−504.
[32]Goodsell DS, Morris GM, Olson AJ. Automated docking of flexible ligands:
applications of AutoDock. J Mol ecognit. 1996; 9: 1−5.
20

Highlights
 New diphenylpyrimidine derivative as potent BTK inhibitors.
 Compound 7b displayed similar activity against B-cell lymphoma cell lines as
ibrutinib.
 Compound 7b exhibited low cytotoxic activity against normal PBMC cells.
 Western blot analysis and flow cytometry analysis also showed its effectiveness
in interfering with B-cell lymphoma cell growth.
21

Graphic abstract
1
8
4
covalent bond
O
s
y
C
N
N
O
O
O
O
HN
HN
R(Amino acid)
Hydrogen bond
HN
HN
Cl
N
NH
OMe
BTK protein
HN
F3C
N
NH
N
N
BTK inhibitors
DPPY 7b R= Methyl L-prolinate
IC50 = 8.7 nM(BTK), 96.0 nM(JAK3)
Rociletinib
EGFRT790M Inhibitor
= 4.8 μM(NAMALWA cell), 6.7 μM(Raji cell)
Phase Ш
22