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

Article abstract of DOI:10.1016/j.ejmech.2017.04.037

A new series of diphenylpyrimidine derivatives (SFA-DPPYs) were synthesized by introducing a functional sulfonamide into the C-2 aniline moiety of pyrimidine template, and then were biologically evaluated as potent Bruton's tyrosine kinase (BTK) inhibitors. Among these molecules, inhibitors 10c, 10i, 10j and 10k displayed high potency against the BTK enzyme, with IC₅₀ values of 1.18?nM, 0.92?nM, 0.42?nM and 1.05?nM, respectively. In particular, compound 10c could remarkably inhibit the proliferation of the B lymphoma cell lines at concentrations of 6.49?μM (Ramos cells) and 13.2?μM (Raji cells), and was stronger than the novel agent spebrutinib. In addition, the inhibitory potency toward the normal PBMC cells showed that inhibitor 10c possesses low cell cytotoxicity. All these explorations indicated that molecule 10c could serve as a valuable inhibitor for B-cell lymphoblastic leukemia treatment.

Full text of DOI:10.1016/j.ejmech.2017.04.037

Accepted Manuscript
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
He Liu, Menghua Qu, Lina Xu, Xu Han, Changyuan Wang, Xiaohong Shu, Jihong
Yao, Kexin Liu, Jinyong Peng, Yanxia Li, Xiaodong Ma
PII:
S0223-5234(17)30301-X
10.1016/j.ejmech.2017.04.037
EJMECH 9386
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 September 2016
Revised Date: 4 April 2017
Accepted Date: 5 April 2017
Please cite this article as: H. Liu, M. Qu, L. Xu, X. Han, C. Wang, X. Shu, J. Yao, K. Liu, J. Peng, Y. Li,
X. Ma, 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,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.037.
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ACCEPTED MANUSCRIPT
Phe540/Ser538/Ile472
R3
Cys481
covalent bond
O
O
O
S
O
O
O
NH
HN
R2
HN
Anti-BTK activity(IC50
)
10c(1.18 nM): R1=Cl,R2=MeO,R3=F,X=NH
10i(0.92 nM): R1=F,R2=H,R3=MeO,X=NH
10j(0.42 nM): R1=Cl,R2=H,R3=MeO,X=O
10k(1.05 nM): R1=Cl,R2=Me,R3=Me,X=O
HN
N
NH
X
N
NH
N
N
R1
F
Met477
Spebrutinib
Phase
BTK Inhibitors
SFA-DPPYs 10a-k
10c: IC50(Ramos,6.49 µM; Raji,13.2 µM)
Improved anti-B-lymphoma cells activity

ACCEPTED MANUSCRIPT
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
He Liua, Menghua Qua, Lina Xua, Xu Hana, Changyuan Wanga, Xiaohong Shua, Jihong Yaoa,
Kexin Liua, Jinyong, Penga, Yanxia Lib and Xiaodong Maa,
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. Ma).
Abstract
A new series of diphenylpyrimidine derivatives (SFA-DPPYs) were synthesized
by introducing a functional sulfonamide into the C-2 aniline moiety of pyrimidine
template, and then were biologically evaluated as potent Bruton’s tyrosine kinase
(BTK) inhibitors. Among these molecules, inhibitors 10c, 10i, 10j and 10k displayed
high potency against the BTK enzyme, with IC50 values of 1.18 nM, 0.92 nM, 0.42
nM and 1.05 nM, respectively. In particular, compound 10c could remarkably inhibit
the proliferation of the B lymphoma cell lines at concentrations of 6.49 µM (Ramos
cells) and 13.2 µM (Raji cells), and was stronger than the novel agent spebrutinib. In
addition, the inhibitory potency toward the normal PBMC cells showed that inhibitor
10c possesses low cell cytotoxicity. All these explorations indicated that molecule 10c
could serve as a valuable inhibitor for B-cell lymphoblastic leukemia treatment.
Keywords: Leukemia; BTK; Inhibitor; Pyrimidine; Sulfonamide.
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
S
N
N
N
NH
HN
O
N
NH
HN
N
O
O
NH
NH
O
NH
NH
O
NH
O
N
N
O
O
O
F3C
4
5
6
Spebrutinib
HM71224
PLS-123
Fig. 1. Structures of the novel BTK inhibitors.
Bruton’s tyrosine kinase (BTK) is an essential component of the B-cell receptor
(BCR) signaling pathway regulating survival and proliferation of B lymphocytes [1–
3]. BTK inhibition has become an attractive strategy for treating autoimmune diseases,
B-cell leukemia and lymphomas [4,5]. Ibrutinib (1, Fig. 1) is the first selective
and covalent BTK inhibitor, approved by the US Food and Drug Administration (FDA)
for the treatment of mantle cell lymphoma in 2013. Subsequently, it was also approved for the
treatment of chronic lymphocytic leukemia in 2014, and for the treatment of Waldenström
macroglobulinemia, a form of non-Hodgkin s lymphoma in 2015 [6–9]. Untill now, a
number of novel BTK inhibitors have been progressed to preclinical or clinical studies,
including acalabrutinib (2 Fig. 1) (ClinicalTrials.gov identifier: NCT02387762) [10–
12], GS-4059 (3 Fig. 1) (ClinicalTrials.gov identifier: NCT02457559) [13,14],
spebrutinib (4
’s
’
,
,
,
Fig. 1) (ClinicalTrials.gov identifier: NCT01744626) [15], HM71224
2

ACCEPTED MANUSCRIPT
(5
,
Fig. 1) (ClinicalTrials.gov identifier: NCT01765478) [16], and PLS-123 (6, Fig. 1)
[17]. Generally, these BTK inhibitors shown in figure 1 possess a pyrimidine core,
along with a typical N-phenlyacrylamide functional group. It has been confirmed 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 pocket [7,18–20].
Cl
HO
O
C6F5
O
HN
NH
O
O
N
N
S
O S
S
O
O
O
O
O
O
S
O
NH2
NO2
9
8
KCN1
E7070
7
Fig. 2. Typical sulfonamide analogues as potential anticancer agents.
Phe540/Ser538/Ile472
R1
Cys481
covalent bond
O
O
O
S
O
O
O
NH
HN
HN
R2
HN
N
NH
HN
R1
N
NH
N
F
N
Spebrutinib
Phase Ⅲ
BTK Inhibitors
Met477
SFA-DPPYs 10a-k
Fig. 3. Designed strategy of the SFA-DPPYs as BTK inhibitors.
The sulfonamide functional group is present in many active compounds displaying,
for example, antibacterial, anti-carbonic anhydrase, diuretic, hypoglycemic, and
antithyroid properties [21]. Furthermore, it has been reported that a number of
sulfonamide derivatives possess promising antitumor properties (7-9, Fig. 2) [22–24].
E7070 (8) is a novel sulfonamide antitumor agent, which could affect cell cycle
progression and exhibit potent antitumor activity in vitro and in vivo [23]. 3,4-
3

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Dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-
phenylbenzenesulfonamide (KCN1, 9), could induce cell cycle arrest and inhibit
human pancreatic cancer cells growth in a dose-dependent manner in vitro, and KCN1
also showed in vivo anticancer efficacy in mice bearing Panc-1 or Mia Paca-2 tumor
xenografts [24]. In order to improve the binding affinity with BTK, a family of
sulfonamide-substituted diphenylpyrimidines (SFA-DPPYs, 10a-k) were designed
and synthesized to investigate their activity against the BTK kinase and the
B lymphoma cell lines (Fig. 3).
2. Chemistry
R3
R3
R3
H2N
NO2
O
R2
O
S
S
b
HN
HN
O
O
a
R2
R2
R1
X
O
N
S
Cl
O
HN
N
NH2
NO2
R3 = F, Me, MeO
11a-c
R2 = H, Me, MeO
R3 = F, Me, MeO
12a-f
R2 = H, Me, MeO
R3 = F, Me, MeO
13a-f
e
R2
NH
O
NH
S
O
O
R1
R1
N
X
N
X
Cl
NH
N
Cl
c
Cl
N
X
R1 = Cl, NO2, F
d
R3
R2 = H, Me, MeO
R3 = F, Me, MeO
X = O or NH
10a-k
NH2
O
NH
X = OH or NH2
O
X = OH or NH2
14a-b
15a-b
R1 = Cl, NO2, F
X = O or NH
16a-d
Scheme 1. Synthetic route of the title compounds 10a-k. Reagents and conditions: (a) NaHCO3,
CH3CN, rt., 0.5 h, 87–91%; (b) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 70–88%; (c) acryloyl chloride,
NaHCO3, CH3CN, rt., 0.5 h, 81–85%; (d) DIPEA, 1,4-dioxane, rt., 2 h, 85–91%; (e) TFA, 2-
BuOH, 100 °C, 12 h, 10–25%.
As depicted in Scheme 1, the title molecules 10a-k were synthesized according to
the general procedure [25,26]. Acylation of the 4-nitroaniline derivatives with
benzenesulfonyl chloride 11a-c in the presence of NaHCO3, and followed by the
reduction reaction of the nitro-substituted compounds 12a-f under Fe-NH4Cl
4

ACCEPTED MANUSCRIPT
conditions, conventionally prepared the N-(4-aminophenyl)benzenesulfonamides 13a-
f. On the other hand, acylation reaction of acryloyl chloride with anilines 14a-b under
the action of NaHCO3 produced the acrylamide-substituted intermediates 15a-b.
Compounds 15a-b were regioselectively coupled to the C-4 position of the 2,4-
dichloropyrimidine reagents in the presence of N,N-diisopropylethylamine (DIPEA)
to afford the pyrimidine intermediates 16a-d. With these key intermediates in hand,
coupling reactions of 16a-d with various anilines 13a-f were accomplished in the
presence of trifluoroacetic acid (TFA) under reflux conditions, offering the title
molecules SFA-DPPYs 10a-k with yield ranging from 10 to 25%.
3. Results and discussion
3.1.Biological activity
Table 1 Biological activity of the newly synthesized compounds 10a-k. a
R1
5
N
2
4
HN
N
X
1`
5`
R2
NH
4`
O
NH
S
1``
O
O
4``
R3
10a-k
Enzymatic activity
(IC50, nM) b
Antiproliferative
activity (IC50, µM) c
Compounds
X
R1
R2
R3
BTK
58.5
78.6
1.18
48.6
Ramos
>40.0
6.44
Raji
17.4
10.5
13.2
14.2
NH Cl
NH Cl
H
Me
F
10a
10b
10c
10d
Me
NH Cl MeO
NH NO2 Me
F
6.49
Me
38.0
5

ACCEPTED MANUSCRIPT
NH NO2 MeO Me
>100
85.4
9.14
23.8
0.92
0.42
1.05
0.72
0.34
>40.0
>40.0
18.8
34.1
22.4
15.5
18.7
36. 9
40.9
18.7
29.4
19.3
10e
10f
NH NO2 Me
F
NH Cl
H
MeO
MeO
MeO
MeO
Me
10g
NH NO2
H
H
H
>40.0
>40.0
>40.0
7.23
10h
NH
O
F
10i
Cl
Cl
10j
O
Me
10k
Spebrutinib
Ibrutinib
18.8
5.88
a
b
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 nanomolar needed to
c
inhibit cell growth by 50%, as calculated using GraphPad Prism version 5.0. Dose-response
curves were determined at five concentrations. The IC50 values are the concentrations in
nanomolar needed to inhibit cell growth by 50%, as calculated using GraphPad Prism version 5.0.
The activity against the BTK kinase of the title molecules was evaluated using
ADP-Glo™ Kinase Assay system [27–29]. Their potency to inhibit the proliferation
of B leukemia cells was performed according to the CCK-8 assay protocol [18]. For
this test, two typical B-cell lymphoblastic leukemia cell lines, Ramos and Raji, which
overexpressed BTK enzyme, were used. For comparison, two novel BTK inhibitors,
spebrutinib and ibrutinib were also tested as reference compounds. All these result
data are depicted in Table 1.
Among these inhibitors, four molecules, 10c, 10i, 10j and 10k could strongly
inhibit the BTK kinase activity at concentrations of 1.18 nM, 0.92 nM, 0.42 nM and
1.05 nM, respectively. In particular, the C-4 oxygen-substituted pyrimidine derivative
10j even displayed higher inhibitory ability than spebrutinib (IC50 = 0.72 nM). The
kinase-based test results confirm that an oxygen atom installed at the C-4 position of
the pyrimidine core (analogue 10j) is more favorable than the amino substituent
6

ACCEPTED MANUSCRIPT
(analogue 10g). While replacement of a chlorine atom (analogue 10g) at the C-5
pyrimidine position with a nitro group (analogue 10h) is clearly less beneficial. For
their activity against the B lymphoma cells, compounds 10c and 10k, which were
very potent to inhibit the BTK enzyme activity, also could effectively suppress the
proliferation of Ramos cells at concentrations of 6.49 µM and 7.23 µM, respectively.
Obviously, both compounds 10c and 10k exhibited stronger activity against Ramos
cells than spebrutinib (IC50 = 18.8 µM). Nevertheless, the Cˈ-5 methoxy-substituted
compounds 10i and 10j almost lost the activity against Ramos cells (IC50 > 40 µM) in
spite of their high capability to inhibit the BTK activity. According to our
explorations, the low water solubility probably was the major reason for their low
anti-B lymphoma cells capability. On the contrary, the C-5 fluoro-substituted
compound 10b which only possessed moderate activity against the BTK kinase (IC50
= 78.6 nM), was able to strongly inhibit the proliferation of Ramos cells with an IC50
of 6.44 µM. Apparently, compounds 10d-f bearing a nitro substituent at the C-5
pyrimidine position, were not active to interfere with Ramos cells growth (IC50 > 40
µM). As for their activity against the Raji cell line, compounds 10b, 10c and 10k
showed more powerful inhibitory activity than reference substances, with IC50 values
of 10.5 µM, 13.2 µM and 18.7 µM, respectively. The structure and activity
relationship (SAR) information yielded from these biological evaluations revealed
that introduction of a nitro group to the C-5 position of pyrimidine core was
unfavorable. In a word, this contribution eventually led to the identification of a
potent BTK inhibitor 10c, which could significantly inhibit B lymphoma cells growth
and proliferation.
Additionally, the effects of treating time and drug concentrations on the cell
viability in Ramos cells for compound 10c were also evaluated. The results showed
7

ACCEPTED MANUSCRIPT
that inhibitor 10c could inhibit the proliferation of Ramos cells in dose- and time-
dependent manners (Fig.4). It can be clearly seen that Ramos cells were almost
completely inhibited by treating with 10c at concentration of 10 µM for 72 h.
Inhibitor 10c
Activity against Ramos cells
150
24h
48h
72h
100
50
0
Control
20
10
5
Concentrations (µmol/L)
Fig. 4. The effects of treating time and drug concentrations for compound 10c on cell viability.
3.2. Cytotoxicity
A
B
E
H
C
D
F
G
Fig. 5. Morphological changes of the PBMC cells treated by inhibitor 10c (100 ×, final
magnification) at concentration of 5, 10, 20 µM for 24 h. Cell numbers were calculated by
8

ACCEPTED MANUSCRIPT
traditional manual method with an ordinary cell counting chamber. One representative experiment
is shown. A: Control, B: Ibrutinib, 5 µM, C: Ibrutinib, 10 µM, D: Ibrutinib, 20 µM, E: 10c, 5
µM, F: 10c, 10 µM, G: 10c, 20 µM, H: Living cell numbers represented by a histogram.
To investigate the cytotoxicity of the potent inhibitor 10c, the
AO/PI fluorescent dyes,
which
are impermeable through the
cell membranes of viable cells, and can be used as fluorescent indicators of dead cells,
were used to detect the effects on the normal peripheral blood mononuclear cells
(PBMC). The test result showed that compound 10c was not sensitive to PBMC cells
at concentration lower than 10 µM, while ibrutinib significantly inhibited the
proliferation of PBMC cells (approximately 50%) at this concentration (Fig.5). This
exploration revealed that compound 10c has lower cell cytotoxicity than the approved
drug ibrutinib.
3.3.Flow cytometry assays
B
A
C
E
D
F
Fig. 6. Compound 10c induced Ramos cells apoptosis in vitro. The cells were incubated with the 1
µM, 5 µM compound 10c for 48 h, respectively, then the cells were stained with annexin V/FTIC,
followed by flow cytometry analysis. One representative experiment is shown above. A: Control,
B: Spebrutinib, 10 µM, C: Ibrutinib, 10 µM, D: 10c, 1 µM, E: 10c, 5 µM, F: Apoptosis rates
represented by a histogram.
Moreover, flow cytometry assays by incubating Ramos cells with 1 µM, 5 µM
compound 10c for 48 h were also performed. The result, shown in figure 6, displayed
that inhibitor 10c induced Ramos cells apoptosis with rates increased from 27.6 to
9

ACCEPTED MANUSCRIPT
61.8%. Excitingly, even at a low concentration of 5 µM, compound 10c exhibited
higher apoptosis rates (61.8%) than the reference agents spebrutinib (33.6%) and
ibrutinib (45.1%) at a high drug concentration (10 µM).
3.4. Binding model analysis
Asn484
Asn484
C
B
Val478
Val478
Cys481
Cys481
Phe540
Phe540
Met477
Met477
Lys430
A
Lys430
Ser538
Leu488
Ser538
Leu488
Ile472
Ile472
Val458
Val458
Phe540
Ser538
Asn484
Cys481
E
D
Asn484
Cys481
Val478
Ile472
Val478
Phe540
Phe540
Met477
Lys430
Met477
Met477
Lys430
Ser538
Leu488
Cys481
Ser538
Leu488
Ile472
Ile472
Val458
Val458
Asn484
Cys481
F
Val478
Phe540
Ser538
Met477
Lys430
Leu488
Ile472
Val458
Fig. 7. Putative binding models of the typical SFA-DPPY molecules within BTK enzyme (PDB
code: 3GEN), A: spebrutinib, B: inhibitor 10c, C: inhibitor 10k, D: inhibitor 10f, E: inhibitor 10g,
F: inhibitor 10e.
To investigate the possible mechanism of the newly synthesized SFA-DPPYs with
the BTK enzyme, five representative compounds, containing two potent inhibitors 10c
and 10k, two moderately active inhibitors 10f and 10g, and one inactive inhibitor 10e
were docked into the ATP binding pocket of BTK (PDB: 3GEN) [30], respectively.
The program AutoDock 4.2 with its default parameters was used [31–33]. For
10

ACCEPTED MANUSCRIPT
comparison, the lead compound spebrutinib was also simulated using the same
protocol. The docking models are presented in Fig. 7.
Previous biological investigation showed that compounds 10c and 10k, which
have very low IC50 values (around 1 nM), were as potent as spebrutinib for inhibiting
the BTK activity. In accordance with the activity data, their docking models (Fig. 7a-c)
indicated that all of them formed tight contacts with the BTK enzyme in an identical
manner, mainly through: (1) covalent-bond forces between the acrylamide group with
the amino acid Cys481; (2) hydrogen-bond interactions formed by the introduced
sulfonamide substituent with the amino acid Val478; (3) strong contacts generated by
the benzenesulfonamide moiety with the amino acids Val458, Phe540, Ser538 and
Ile472, that consist of the bottom section of the ATP binding pocket. In the case of the
moderately active inhibitors 10f (Fig. 7d) and 10g (Fig. 7e), the covalent-bond
contacts between the acrylamide and the amino acid Cys481 still retained, but the
important hydrogen bond between the sulfonamide group and the amino acid Val478
disappeared. Moreover, the pyrimidine core in compound 10f was squeezed out of the
binding pocket after replacing the C-5 chlorine atom with a nitro substituent, which
produced the obvious loss of the interactions with the resides at the bottom of the
binding pocket. Therefore, inhibitor 10c is less potent than compounds 10c and 10k,
with an IC50 value of 85.4 nM. As for the inactive inhibitor 10e (IC50 > 100 nM) (Fig.
7f), the important hydrogen bonds and the covalent bonds for maintaining the anti-
BTK activity, almost lost. Taken together, the above models reasonably interpreted
the biological activity data.
4. Conclusion
In summary, a series of diphenylpyrimidine derivatives bearing a C-2
sulfonamide-substituted aniline moiety were synthesized as potent BTK inhibitors.
Among these newly obtained compounds, inhibitors 10c, 10i, 10j and 10k displayed
11

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strong activity against the BTK kinase, with IC50 values of 1.18 nM, 0.92 nM, 0.42
nM and 1.05 nM, respectively. In particular, compound 10c, which could suppress the
proliferation of two typical B-cell leukemia cell lines at concentrations of 6.49 µM
(Ramos cells) and 13.2 µM (Raji cells), displayed stronger inhibitory activity than the
lead compound spebrutinib. In addition, compound 10c also has low cell cytotoxicity
toward the normal PBMC cells. Overall, these studies provide a promising BTK
inhibitor 10c for the further development of drug for treatment of B-cell
lymphoblastic leukemia.
5. Experimental section
5.1. Chemistry
Unless otherwise noted, all solvents and reagents were obtained from commercial
supplies and were used as received. High resolution ESI-MS were performed on an
13
AB Sciex TripleTOF® 4600 LC/MS/MS system. 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
1
million (ppm) units relative to internal control (TMS). H NMR spectra are reported
in the following order: multiplicity, approximate coupling constant (J value) in hertz,
and number of protons; signals are characterized as s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quartet), m (multiplet) and br (broad signal). All
reactions were monitored by TLC, using silica gel plates with fluorescence GF254
(TLC Silica gel 60 GF254, Merck) and UV light visualization. Flash chromatography
separations were obtained on silica gel (300–400
dichloromethane/methanol as eluents.
mesh) using
5.2. General procedure for the synthesis of title molecules 10a-k [25,26].
5.2.1. Preparation of N-(4-aminophenyl)benzenesulfonamides 13a-f.
A solution of benzenesulfonyl chlorides 11a-c (0.020 mol) in acetone (10 mL)
was added dropwise to a mixture of 4-nitroaniline derivatives (0.020 mol), acetone
12

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(10 mL), sodium bicarbonate (0.040 mol) and water (1 mL) over 20 min, and the
resulting mixture was stirred at reflux temperature for 2 h. The reaction mixture was
then poured into water (50 mL), and filtered to give the nitro-substituted intermediates
12a-f. Subsequently, 12a-f (0.020 mol), ammonium chloride (0.050 mol）and iron
powder (0.080 mol) was mixed in methanol-water (50 mL, VMeOH: VH2O = 1: 1)
solution, and the mixture was heated at 70 ℃ for 6 h. After completion, the reaction
solution was filtrated, and the black reside was washed with DMSO (3 × 10 mL). The
combined filtration was poured into water (100 mL), filtered and washed with H2O (3
× 20 mL). The filter reside was then dried in a vacuum oven to give crude product
13a-f without further purification or characterization.
5.2.2. Preparation of 2-chloropyrimidine intermediates 16a-d.
A mixture of chemical reagents 14a-b (0.01 mol), acetone (30 mL) and sodium
bicarbonate (0.04 mol) was cooled to 0 °C in an ice bath, then acryloyl chloride (0.01
mol) was added dropwise over 10 min, and the resulting mixture was allowed to
warm to room temperature and stirred for another 30 min. The reaction mixture was
then poured into water (150 mL), filtered and washed with water to give the crude
product 15a-b as a beige solid.
To a solution of 2,4-dichloropyrimidines (0.002 mol) in 1,4-dioxane was added
intermediates 15a-b (0.002 mol) and DIPEA (0.003 mol). The reaction mixture was
stirred at 80 °C for 4 h. After being cooled to room temperature, the reaction mixture
was added to water (30 mL). The precipitate was filtered, and the filtered cake was
rinsed with additional cool water and dried in a vacuum oven to give the compounds
16a-b, which were used without further purification.
5.2.3.Preparation of the title molecules 10a-k.
13

ACCEPTED MANUSCRIPT
A flask was charged with compounds 13a-f (0.10 mmol), 11a-d (0.10 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 organic layer was dried over
anhydrous sodium sulfate and concentrated in in a vacuum oven to obtain a crude
product. The residue was purified by column chromatography on silica gel (elution
with 3% methanol /dichloromethane) to give the title product.
5.2.3.1.N-(3-((5-Chloro-2-(4-(4-methylbenzenesulfonicamide)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (10a). Yield: 15.5%; off-yellow solid; H
NMR (400 MHz, DMSO-d6): δ 2.32 (s, 3H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.28
(dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.49 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 6.83 (d, J = 8.0 Hz,
2H), 7.23 (d, J = 8.0 Hz, 2H), 7.28-732 (m, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.51 (d, J =
8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.88 (s, 1H), 8.10 (s, 1H), 8.93 (s, 1H), 9.26 (s,
1H), 9.87 (s, 1H), 10.24 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ 21.7, 104.5,
115.6, 116.0, 130.1, 120.3 (2C), 122.2 (2C), 127.5 (2C), 127.6, 129.2, 130.3 (2C),
131.7, 132.7, 137.5, 137.9, 139.6, 140.0, 143.7; 155.5, 156.9, 158.3, 164.0; HRMS
(ESI) for C26H23ClN6O3S, [M+H]+ calcd: 535.1314 ; found: 535.1274.
5.2.3.2. N-(3-((5-Chloro-2-(4-(3-(4-fluorinebenzenesulfonicamide)methyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (10b). Yield: 12.3%; off-
yellow solid; 1H NMR ( 400MHz, DMSO-d6): δ 1.92 (s, 3H), 5.75 (dd, J = 4.0 Hz, 8.0
Hz, 1H); 6.25 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.52 (dd, J = 8.0 Hz, 16.0 Hz, 2H), 6.66
(d, J = 8.0 Hz, 1H), 7.05-7.10 (m, 1H), 7.20-7.22 (m, 1H), 7.31-7.34 (m, 1H), 7.37-
7.50 (m, 3H), 7.66 (dd, J = 8.0 Hz, 1H), 7.95-8.04 (m, 2H), 8.14 (s, 1H), 8.71-8.77 (m,
1H), 8.92 (s, 1H), 9.40 (s, 1H), 10.36 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ 29.6,
55.6, 116.9 (d, J = 80.0 Hz, 2C), 120.1, 122.9, 125.5, 126.4, 127.2, 127.7, 128.5,
129.8 (d, J = 80.0 Hz, 2C), 132.6, 134.1, 135.5, 136.4, 137.5, 139.5 (d, J = 160.0 Hz,
1C), 140.3, 154.4, 156.5, 158.0, 158.8, 160.0, 163.4; HRMS (ESI) for
C26H22ClFN6O3S, [M+H]+ calcd: 553.1219; found: 553.1177.
5.2.3.3.N-(3-((5-Chloro-2-(4-(3-(4-fluorinebenzenesulfonicamide)methoxyl)
14

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phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (10c). Yield: 14.7%; off-
brown solid; 1H NMR (400 MHz, DMSO-d6): δ 3.14 (s, 3H), 5.76 (dd, J = 4.0 Hz, 8.0
Hz, 1H), 6.26 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.48 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 6.93
(d, J = 8.0 Hz, 1H), 7.17-7.25 (m, 4H), 7.34 (dd, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz,
1H), 7.66 (dd, J = 8.0 Hz, 2H), 7.90 (s, 1H), 8.15 (s, 1H), 8.95 (s, 1H), 9.30 (s, 1H),
13
9.35 (s, 1H), 10.25 (s, 1H); C NMR (400 MHz, DMSO-d6): δ 55.2, 102.7, 104.8,
110.9, 115.2, 115.7, 116.2 (d, J = 80.0 Hz, 2C), 118.0, 119.5, 127.4, 128.0, 129.0,
130.3 (d, J = 80.0 Hz, 2C), 132.4, 137.6, 139. 4 (d, J = 160.0 Hz, 1C), 140.6, 153.9,
155.1, 156.5, 157.9, 163.3, 163.6, 165.7; HRMS (ESI) for C26H22ClFN6O4S, [M+H]+
calcd: 569.1169; found: 569.1197.
5.2.3.4.N-(3-((5-Nitro-2-(4-(3-(4-methylbenzenesulfonicamide)methyl)phenylamino)
1
-4-pyrimidinyl)amino)phenyl)acrylamide (10d). Yield: 25.3%; off-brown solid; H
NMR (400MHz, DMSO-d6): δ 1.74 (s, 3H),2.38 (s, 3H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz,
1H), 6.27 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.45 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 6.73 (d, J
= 8.0 Hz, 1H), 7.22-7.28 (m, 3H), 7.34 (t, J = 8.0 Hz, 2H), 7.50-7.58 (m, 3H), 7.77 (d,
J = 8.0Hz, 2H), 9.09 (s,1H), 9.37 (s, 1H), 10.30 (s, 1H), 10.35 (s, 1H), 10.40 (s, 1H);
13C NMR (400 MHz, DMSO-d6): δ 18.1, 21.4, 113.9, 116.6, 118.3, 121.2, 127.1 (2C),
127.3, 127.6, 129.3, 130.0 (2C), 130.5, 132.2, 135.2, 137.2, 137.8, 138.2, 139.9, 140.1,
143.3, 143.8, 155.2, 158.2, 159.6, 163.7; HRMS (ESI) for C27H25N7O5S, [M+H]+
calcd: 560.1711; found: 560.1651.
5.2.3.5.N-(3-((5-Nitro-2-(4-(3-(4-methylbenzenesulfonicamide)methoxyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (10e). Yield: 16.96%; off-
yellow solid; 1H NMR (400 MHz, DMSO-d6): δ 2.39 (s, 3H), 3.19 (s, 3H), 5.81 (dd, J
= 4.0 Hz, 8.0 Hz, 1H), 6.30 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.50 (dd, J = 8.0 Hz, 16.0
Hz, 1H), 6.96 (s, 1H), 7.06 (s, 1H), 7.18-7.33 (m, 6H), 7.38 (d, J = 8.0 Hz, 2H), 7.58
13
(d, J = 8.0 Hz, 2H), 7.84 (s, 1H), 9.12 (s, 1H), 9.30 (s, 1H), 10.37 (s, 1H); C NMR
(400 MHz, DMSO-d6): δ 21.4, 55.5, 104.6, 112.8, 116.5, 117.1, 121.2, 125.9, 127.2
(2C), 127.4, 127.6, 129.3, 129.6 (2C), 129.9, 132.2, 137.4, 137.8, 138.2, 140.1, 143.1,
152.9, 155.1, 158.2, 159.7, 163.8; HRMS (ESI) for C27H25N7O6S, [M+H]+ calcd:
576.1660; found: 575.1561.
5.2.3.6.N-(3-((5-Nitro-2-(4-(3-(4-fluorinebenzenesulfonicamide)methyl)
phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (10f). Yield: 13.9%; off-
yellow solid; 1H NMR (400 MHz, DMSO-d6): δ 1.90 (s, 3H), 5.78 (dd, J = 4.0 Hz, 8.0
15

ACCEPTED MANUSCRIPT
Hz, 1H), 6.28 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.46 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 7.10
(d, J = 8.0 Hz, 2H), 7.18-7.23 (m, 5H), 7.33 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz,
1H), 7.61 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 9.05 (s, 1H), 10.21 (s, 1H), 10.31 (s, 1H),
13
10.38 (s, 1H); C NMR (400 MHz, DMSO-d6): δ 55.4, 114.0, 114.1, 115.9 (d, J =
80.0 Hz, 2C), 120.1, 121.4, 120.4, 123.1, 123.2, 127.6, 127.9, 129.4, 130.1 (d, J =
80.0 Hz, 2C), 132.2, 132.4, 137.9, 138.0 (d, J = 160.0 Hz, 1C), 140.0, 143.8, 155.0,
155.1, 157.9, 159.3, 163.7; HRMS (ESI) for C26H22FN7O5S, [M+H]+ calcd:
564.1460.2277; found:564.1389.
5.2.3.7.N-(3-((5-Chloro-2-(4-(4-methoxylbenzenesulfonicamide)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (10g). Yield: 10.5%; off-yellow solid; H
NMR (400 MHz, DMSO-d6): δ 3.78 (s, 3H), 5.77 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.27
(dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.50 (dd, J = 8.0 Hz, 16.0 Hz, 1H), 6.83 (d, J = 8.0 Hz,
2H), 7.03 (d, J = 8.0 Hz, 2H ), 7.21-7.31 (m, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.51 (d, J
= 8.0 Hz, 1H), 7.61 (dd, J = 8.0 Hz, 2H), 7.90 (s, 1H), 8.10 (s, 1H), 8.92 (s, 1H), 9.27
13
(s, 1H), 9.79 (s, 1H), 10.25 (s, 1H); C NMR (400 MHz, DMSO-d6): δ 56.0, 104.2,
114.7 (2C), 115.3, 115.6, 119.7, 119.9 (2C), 121.9 (2C), 127.3, 128.9, 129.4 (2C),
131.5, 131.7, 132.4, 137.6, 139.3, 139.7, 155.2, 156.6, 157.9, 162.7, 163.7; HRMS
(ESI) for C26H23ClN6O4S, [M+H]+ calcd: 551.1263; found: 551.1292.
5.2.3.8.
N-(3-((5-Nitro-2-(4-(4-methoxylbenzenesulfonicamide)phenylamino)-4-
1
pyrimidinyl)amino)phenyl)acrylamide (10h). Yield: 10.2%; off-yellow solid; H
NMR (400 MHz, DMSO-d6): δ 3.80 (s, 3H), 5.77 (d, J = 4.0 Hz, 8.0 Hz 1H), 6.27 (dd,
J = 4.0 Hz, 16.0Hz, 1H), 6.49 (dd, J = 12.0 Hz, 16.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 2H),
7.02 (d, J = 8.0 Hz, 2H), 7.11-7.24 (m, 6H), 7.37 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0
Hz, 2H), 7.82 (s, 1H), 9.05 (s, 1H), 10.32 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ
56.1, 114.7 (2C), 116.7, 117.1, 120.9, 121.5, 127.5, 128.3 , 129.2, 129.4 (2C), 126.6,
131.6, 132.3, 133.8, 135.1, 137.8, 140.1 (2C), 155.2; 158.2, 159.5, 162.8 (2C), 163.8;
HRMS (ESI) for C26H23N7O6S, [M+H]+ calcd: 562.1503, found: 562.1533.
5.2.3.9. N-(3-((5-Fluoro-2-(4-(4-methoxylbenzenesulfonicamide)phenylamino)-4-
pyrimidinyl)amino)phenyl)acrylamide (10i). Yield: 18.8%; off-brown solid; 1H
NMR (400 MHz, DMSO-d6): δ 3.37 (s, 3H), 5.76 (d, J = 4.0 Hz, 8.0 Hz 1H), 6.26 (dd,
J = 4.0 Hz, 16.0 Hz, 1H), 6.54 (dd, J = 12.0 H, 16.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H),
7.04 (d, J = 12.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.50-7.62 (m, 5H), 8.07 (d, J = 4.0
Hz, 2H), 9.22 (s, 1H), 9.42 (s, 1H), 9.81 (s, 1H), 10.35 (s, 1H); 13C NMR (400 MHz,
16

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DMSO-d6): δ 56.03, 113.3, 114.7 (2C), 114.9, 117.5, 119.5 (2C), 122.2 (2C), 127.2,
128.9, 129.4 (2C), 131.2 (2C), 131.7, 132.5, 138.2, 139.7 (d, J = 72.0 Hz, 1C), 140.9
(d, J = 32.0 Hz, 1C), 142.2, 150.2 (d, J = 44.0 Hz, 1C), 155.8, 162.7, 163.7; HRMS
(ESI) for C26H23FN6O4S, [M+H]+ calcd: 535.1558; found: 535.1598.
5.2.3.10. N-(3-((5-Chloro-2-(4-(3-(4-methylbenzenesulfonicamide)methyl)phenoxy)-
1
4-pyrimidinyl)yloxy]phenyl)-2-acrylamide (10j). Yield: 16.6%; off-white solid; H
NMR (400 MHz, DMSO-d6): 1H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3H), 5.79
(dd, J = 4.0 Hz, 8.0 Hz, 1H), 6.28 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.47 (dd, J = 12.0 Hz,
16.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H ), 7.23 (d, J = 8.0 Hz,
2H), 7.42 (d, J = 8.0 Hz, 2H), 7.60-7.71 (m, 4H), 8.44 (s, 1H), 9.69 (s, 1H), 9.85 (s,
1H), 10.41 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ 56.05, 113.0, 114.7 (2C),
116.9, 117.5, 119.9, 120.9, 121.5 (2C), 127.9, 129.3 (2C), 129.4, 130.3, 131.6, 132.1
(2C), 136.6, 140.9, 152.7, 157.9, 158.5, 162.7, 163,9, 164.2; HRMS (ESI) for
C26H22ClN5O5S, [M+H]+ calcd: 552.1103; found: 552.1137.
5.2.3.11. N-(3-((5-Chloro-2-(4-(3-(4-methylbenzenesulfonicamide)methyl)phenoxy)-
1
4-pyrimidinyl)amino)phenyl)acrylamide (10k). Yield: 15.5%; off-white solid; H
NMR (400 MHz, DMSO-d6): δ 1.69 (s, 3H), 2.37 (s, 3H), 5.79 (dd, J = 4.0 Hz, 8.0 Hz,
1H), 6.26 (dd, J = 4.0 Hz, 16.0 Hz, 1H), 6.43 (dd, J = 12.0 Hz, 16.0 Hz, 1H), 6.63 (d,
J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.47
(d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.70 (s, 1H), 8.47 (s,1H), 9.25 (s, 1H),
9.74 (s, 1H), 10.39 (s, 1H); 13C (400 MHz, DMSO-d6): δ 18.2, 21.5, 105.1, 112.9,
116.9, 117.4, 120.5, 127.1 (2C), 127.6, 127.9, 128.8, 129.9, 130.4 (2C), 132.1, 135.3,
138.3 (2C), 138.5, 140.9, 143.3, 152.7, 157.9, 158.6, 163.8, 164.3; HRMS (ESI) for
C27H24ClN5O4S [M+H]+ calcd: 550.1310; found: 550.1343.
5.3. In vitro kinase enzymatic assay
The BTK kinase enzyme assay system (Catalog: V9071, Promega) was used in
this test. 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 can be
seen 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, through the
steps below: (1) perform a 5 µL kinase reaction using 1× kinase buffer (e.g., 1×
17

ACCEPTED MANUSCRIPT
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. [27–29].
5.4. Cellular activity assay
5.4.1. 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. All the cell viability assays were performed using the CCK-8 method. The
Cell Counting Kit-8 (CCK-8) reagent was purchased from Biotool Company
(Switaerland). 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%
penicillin-streptomycin (Beyotime Company, China). The cells were maintained and
propagated as monolayer cultures at 37 °C in humidified 5% CO2 incubator.
5.4.2. Cellular activity assay
Ramos and Raji cells were seeded in 96-well plates at a density of 3,000 to 5,000
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.
5.4.3. Cytotoxicity 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
18

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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 approximately 2.5 × 105
cells/well. Cells were treated with inhibitors 10c and ibrutinib at appropriate
concentrations (5, 10, 20 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 cells were washed with PBS twice, and
subsequently incubated in AO/PI dye mix (1.0 µg/mL of AO and 1.0 µg/mL of PI in
PBS) at room temperature for 10 min. The apoptotic, necrotic, and live cells were
observed and counted under the fluorescent microscope (OLYMPUS, Tokyo, Japan),
and the number of living PBMC cells was calculated by traditional manual method
with an ordinary cell counting chamber.
5.5. Cell Apoptosis Assay
Ramos cells (3 × to 5 ×105 cells/well) incubated in 6-well plates were treated with
solvent control (DMSO), spebrutinib, ibrutinib, or compound 10c 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).
5.6. Molecular modeling
The crystal structure (PDB: 3GEN) of the kinase domain of BTK bound to a
pyrrolopyrimidine-containing compound was used for molecular simulation [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 10c, 10e, 10f, 10g, and 10k
were placed, respectively. The binding interaction energy was calculated, including
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ACCEPTED MANUSCRIPT
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, 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), and the Cultivation Project of Youth Techstars, Dalian, China
(No.2016RQ043) for the financial support of this research.
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ò A class of SFA-DPPYs were synthesized as potent BTK inhibitors.
ò Compounds 10c, 10i, 10j and 10k remarkably inhibited BTK activity with
IC50 valuses of 1.18, 0.92, 0.42,1.05 nM, respectively.
ò Compound 10c displayed stronger antiproliferative activity against B
lymphoma cells than spebrutinib.
ò Inhibitor 10c possesses low cytotoxicity toward the normal PBMC cells.