Design and synthesis of novel 1-substituted 3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine analogs as selective BTK inhibitors for the treatment of mantle cell lymphoma

Article abstract of DOI:10.1016/j.bioorg.2019.103367

Ibrutinib (IBN), a first-in-class BTK-inhibitor, was approved by the FDA for the treatment of mantle cell lymphoma (MCL). Although IBN shows excellent performance as an anti-lymphoma agent, it has some undesirable side effects due to its off-target activities. Our studies yielded a novel series of 3-(6-phenoxypyridin-3-yl)-4-amine-1H-pyrazolo[3,4-d]pyrimidine derivatives capable of potent inhibition of Bruton's tyrosine kinase (BTK). Notably, compound 13e explained potent BTK inhibitory activity and could completely inhibit the phosphorylation of BTK and PLCγ2 in Z138 cells at low micromolar concentration. Compared with IBN, compound 13e improved anti-proliferative activities 3–40 folds in MCL cell lines with IC₅₀ values lower than 1 μM. Low micromolar doses of 13e could induce strong cell apoptosis in Jeko-1 and Z138 cells. In addition, compound 13e showed greater BTK selectivity and higher stability in human liver microsomes than IBN and potential safety improvement for the treatment of MCL.

Full text of DOI:10.1016/j.bioorg.2019.103367

Journal Pre-proofs
Design and synthesis of novel 1-substituted 3-(6-phenoxypyridin-3-yl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine analogs as selective BTK inhibitors for the
treatment of mantle cell lymphoma
Fansheng Ran, Yang Liu, Shengping Yu, Kaiwen Guo, Wendi Tang, Xin
Chen, Guisen Zhao
PII:
S0045-2068(19)30631-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103367
YBIOO 103367
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
23 April 2019
16 September 2019
14 October 2019
Please cite this article as: F. Ran, Y. Liu, S. Yu, K. Guo, W. Tang, X. Chen, G. Zhao, Design and synthesis of
novel 1-substituted 3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine analogs as selective BTK
inhibitors for the treatment of mantle cell lymphoma, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103367
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Design and synthesis of novel 1-substituted 3-(6-phenoxypyridin-3-
yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine analogs as selective BTK
inhibitors for the treatment of mantle cell lymphoma
a
b
a
a
a
a
†
Fansheng Ran †, Yang Liu , Shengping Yu , Kaiwen Guo , Wendi Tang , Xin Chen , Guisen
Zhaoa,*
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, PR China
b Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX
77030, USA
†These authors contributed equally to this work.
* Corresponding author: Guisen Zhao
E-mail: guisenzhao@sdu.edu.cn
Abstract
Ibrutinib (IBN), a first-in-class BTK-inhibitor, was approved by the FDA for the treatment of
mantle cell lymphoma (MCL). Although IBN shows excellent performance as an anti-lymphoma
agent, it has some undesirable side effects due to its off-target activities. Our studies yielded a novel
series of 3-(6-phenoxypyridin-3-yl)-4-amine-1H-pyrazolo[3,4-d]pyrimidine derivatives capable of
potent inhibition of Bruton’s tyrosine kinase (BTK). Notably, compound 13e explained potent BTK
inhibitory activity and could completely inhibit the phosphorylation of BTK and PLCγ2 in Z138
cells at low micromolar concentration. Compared with IBN, compound 13e improved anti-
proliferative activities 3-40 folds in MCL cell lines with IC50 values lower than 1 μM. Low
micromolar doses of 13e could induce strong cell apoptosis in Jeko-1 and Z138 cells. In addition,
compound 13e showed greater BTK selectivity and higher stability in human liver microsomes than
IBN and potential safety improvement for the treatment of MCL.

Keywords:
Mantle cell lymphoma; BTK; Docking; Anticancer; Selectivity
1. Introduction
Bruton’s tyrosine kinase (BTK), a member of the Tec kinase family, is expressed in many
hematopoietic cells, including myeloid cells and B cells, but not in T cells1-3. BTK plays a critical
role in B-cell receptor (BCR) signaling pathway, which regulates cell survival, proliferation,
activation, differentiation and maturation of B cells4-7. The deregulation of BTK has been observed
in numerous B-cell-derived malignancies, including mantle cell lymphoma (MCL), chronic
lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL)6, 8-9
.
MCL, a subtype of Non-Hodgkin's Lymphoma (NHL) with poor prognosis, is responsible for
7% of NHL10. BTK is commonly overexpressed in MCL, and the BCR signaling pathway is
implicated in the pathogenesis of MCL11-12. In 2013, the first-in-class BTK-inhibitor ibrutinib (IBN,
1) was approved by FDA for the treatment of MCL13-15. Although IBN shows excellent performance
as an anti-lymphoma agent, it has some undesirable side effects due to its unexpected off-target
toxicities resulting from potential binding to non-target locations16. In addition to targeting BTK,
IBN interacts with several kinases involved in other important signaling pathways, such as EGFR,
ErbB2, ErbB4, Itk, Lyn, Yes, Hck, and PTK517-19
.
We performed molecular docking using Sybyl X 2.0 to investigate the interactions between
IBN and BTK (PDB code: 5P9I). As shown in Fig 1A, the pyrazolopyrimidine core occupied the
ATP binding pocket, performing several important interactions with the hinge region. The primary
amine directly formed hydrogen bond with the backbone carbonyl of Glu475, and the N-3 nitrogen
of the pyrimidine ring formed a hydrogen bond interaction with the amine of Met477. The carbonyl
group formed a hydrogen bond with the amine of Cys481. The diphenyl ether group extended to the
hydrophobic pocket behind the gatekeeper Thr474 and the distal phenyl group displayed an edge-
to-face aromatic interaction with the phenyl of Phe540. Moreover, there were many hydrogen bond
donors around the ring A of IBN, such as the amino group of Lys430 with 2.70 Å distance and the
hydroxy group of Ser538 with 2.96 Å distance. If we introduce a hydrogen bond acceptor at the A
ring, potential hydrogen bond interactions may be formed with Ser538 or/and Lys430 with the

improvement of the selectivity on BTK20. Therefore, we introduced a nitrogen atom as a hydrogen
bond acceptor at the 3’-position of ring A and optimized the substituents at the N-1 position of
pyrazolopyrimidine to obtain a novel series of 3-(6-phenoxypyridin-3-yl) -4-amine-1H-
pyrazolo[3,4-d] pyrimidine BTK inhibitors.
O
O
3，
，
N
A
3
A
NH2
NH2
4
4
3
N
3
N
N
N
N
N
N
1
N
1
R
N
O
Target compounds
Ibrutinib, 1
A)
B)
Figure 1. (A) Molecular docking mode of IBN with BTK. PDB ID:5P9I. (B) Design of the
target compounds.
2. Results and discussion
2.1. Chemistry
The synthetic routes for these compounds are explained in Scheme 1. Condensation of various
substituted amines (2) with bromoacetyl bromide afforded compounds 3a-3j. Nucleophilic aromatic
substitution of compound 4 with phenol under the presence of sodium hydride provided the
compound 5. Subsequent coupling of compound 5 with bis(pinacolato)diboron catalyzed by
palladium acetate afforded compound 6. Bromination of compound 7 by N-bromosuccinimide (NBS)
provided the compound 8, which reacted with compound 6 through Suzuki reaction to provide
compound 9. N-alkylation of compound 9 with compounds 3a-3j gave the target compounds 10a-
10j. Compound 9 was transformed to racemic compounds 11 with racemic 1-Boc-3-
hydroxypiperidine by the Mitsunobu reaction, which were deprotected in HCl/1,4-dioxane and
subsequently reacted with different acyl chlorides or substituted acids to produce target compounds
13a-13e.

O
O
Br
NH2
N
n
n
a
H
N
R3
R1
R3
R1
NH2
N
R2
R3
R2
R2
N
R1
f
2
3a-3j
N
H
N
N
n
O
10a-10j
F
O
O
N
N
N
b
c
O
O
O
O
Br
4
B
Br
5
N
N
N
O
O
N
NH2
NH2
N
NH2
NH2
N
e
g
h
i
6
N
N
N
N
N
N
N
N
N
H
N
N
N
N
N
NH2
NH2
Br
9
R4
N
d
N
N
NH
HCl
N
N
Boc
N
N
N
N
N
O
H
H
11
12
13a-13e
7
8
Scheme 1. Synthetic route of series 10 and 13. Reagents and conditions: (a) NaHCO3,
bromcacetyl bromide, ethyl acetate:H2O = 1:1, r.t., 1h; (b) Phenol, NaH, THF, 80°C, 12h; (c)
Pd(OAc)2, X-PHOS, KOAc, bis(pinacolato)diboron, 1,4-dioxane, 90°C, 12h; (d) NBS, DMF, 90°C,
1h; (e) Pd(PPh3)4, K3PO4, 1,4-dioxane:H2O = 4:1, 120°C, 24h; (f) K2CO3, DMF, r.t., 5h; (g) 1-Boc-
3-hydroxypiperidine (racemate), PPh3, DIAD, 0°C to r.t., 5h; (h) HCl\1,4-dioxane, r.t., 5h; (i) Acyl
chlorides, Et3N, THF, 0°C to r.t., 5h or substituted acids, HBTU, Et3N, r.t., 8h.
2.2. BTK inhibition activity
The newly synthesized pyrazolopyrimidine analogs were evaluated for their activity against
BTK via a KinaseProfiler radiometric protein kinase assay. As shown in Table 1, series 10 remained
BTK residual activity more than 70% at 1μM. It was found that the inhibitory activities of series 10
were not increased with either extending the chain length (10a, 10b and 10c) or changing the groups
on the terminal benzene ring (10d-10j). It implied that introduction of a substituted acetamide at the
N-1 position of the pyrazolopyrimidine core might not be a preferred approach. For series 13, when
the group at R4 position was a hydroxyacetyl group, 2-hydroxypropionyl group, hydroxy tert-
butanoyl group or methoxypropionyl group respectively, these compounds (13a-13d) showed weak
inhibitory activities against BTK. Notably, compound 13e with a chloroacetyl group at R4 position
increased significantly the BTK inhibitory activity that is consistent with the finding reported21.
Table 1. The effects of series 10 and 13 on BTK activity.

BTK residue
activity (%)
at 1μM
BTK IC50
(nM)
Structure
Code
n
R1
R2
R3
R4
IBN
10a
-
-
-
-
-
-
-1
8
O
0
H
F
92
NDa
N
H
10b
10c
10d
10e
10f
10g
10h
10i
1
2
0
0
0
0
0
0
0
-
H
H
F
H
H
F
H
H
H
-
F
H
F
H
F
-
-
-
-
-
-
-
-
89
101
79
98
80
91
101
97
87
80
77
72
83
5
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
36
O
N
F
NH2
R2
R3
Cl
Cl
Cl
CF3
CF3
H
-
F
N
R1
N
H
N
N
CN
H
Br
CN
Me
-
N
n
O
10j
13a
13b
13c
13d
13e
-
O
OH
O
O
OH
OH
-
-
-
-
N
NH2
O
-
-
-
-
N
N
O
N
N
-
-
-
-
O
O
N
R4
Cl
-
-
-
-
NDa: not detected.
2.3. Cell viability activity
According to the CellTiter-Glo Luminescent cell viability assay, compound 13e was
determined for their anti-proliferative activity in MCL cell lines and primary MCL patient cells. The
tested MCL cell lines included Mino, Jeko-1, Z138, and Maver-1. As shown in Table 2, compound
13e showed potent effect against MCL cells with IC50 values lower than 1 μM, which is 3-39 folds
higher than IBN. As shown in Fig 2A, 13e also demonstrated more potent antiproliferative activity
in primary MCL patient cells than IBN. Compound 13e exhibited robust antiproliferative effects in
both MCL cell lines and primary patient tumor cells.
Table 2. Cell proliferation assay assessing the effects of compounds on MCL cell lines.
Cell viability assay, IC50 μM
Code
Mino Jeko-1
Z138
9.7
Maver-1
7.80
IBN
13e
15.7
0.4
1.1
0.4
0.4
0.4
2.4. Inhibitory effects on BTK signaling
To further examine the inhibitory effects of 13e on BTK signaling, immunoblotting was

performed to detect the phosphorylation of BTK and its downstream effector PLCγ2 in Z138 cells.
The results showed that 13e treatment for 16 h completely inhibited the phosphorylation of BTK
and PLCγ2 at 0.5 μM (Fig 2B). These results suggested that 13e behaved strong inhibitory effect
on BTK signaling pathway.
Figure 2. (A) Cell viability assay after 24 h treatment of 13e and IBN in two naïve MCL patient
cells. Statistical significance is indicated as **P < 0.01. (B) Effects of 13e on BTK signaling in
Z138 cells after 16 h treatment.
2.5. Cell apoptosis assay
We proceeded to test the effects of 13e on apoptosis in Jeko-1 and Z138 cells via an annexin
V-FITC/propidium iodide (PI) binding assay. As illustrated in Fig 3, after 24 h of treatment, 13e
triggered dose-dependent apoptosis of the cells with ratios of 35% and 69% at concentrations of 1
μM and 2 μM compared with 14% in non-treated Jeko-1 cells, and 19% and 82% at concentrations
of 1 μM and 2 μM compared with 17% in non-treated Z138 cells.

(A)
(B)
(C)
(D)
Figure 3. Cell apoptosis assay for 13e in Jeko-1 and Z138 cells. (A/C) Apoptosis assay of 13e
at indicated concentrations in Jeko-1 cells for 24 h; (B/D) Apoptosis assay of 13e at indicated
concentrations in Z138 cells for 24 h. The data shown are the mean of three independent experiments.
Statistical significance is indicated as *P < 0.05, **P < 0.01 compared with the DMSO control.
2.6. Selectivity over other kinases
We further tested the biochemical activity of compound 13e against a panel of 22 kinases. The
major off-target kinases of 13e were those with cysteine residue in the ATP binding pocket, which
could be irreversibly bound by the covalent reactive group. As shown in Table 3, 13e showed high
potency against Blk, Bmx, Tec, Txk, ErbB4 and EGFR with IC50 values of 10, 6, 36, 104, 147 and

292 nM, respectively. Compared to IBN, 13e showed much lower inhibitory effect against Brk, B-
raf, Csk, Fgr, Flt3, Fyn, Itk, Hck, Lck, Lyn, PTK5, Src, SRM and Yes, indicating improved BTK
selectivity and potential safety improvement for the treatment of MCL.
Table 3. Inhibition efficacy (%) among different kinases at 1 μM by 13e and IBN.
Kinases
Blk
13e
100% (10nM)
97% (6nM)
33%
IBN
100%
102%
92%
19%
87%
98%
73%
101%
92%
8%
Kinases
Hck
Itk
13e
16%
IBN
96%
86%
54%
99%
99%
91%
96%
100%
103%
97%
102%
Bmx
Brk
12%
JAK3
Lck
41%
B-raf
Csk
-4%
35%
7%
Lyn
-10%
EGFR
ErbB2
ErbB4
Fgr
77% (292nM)
69% (948nM)
91% (147nM)
16%
PTK5
Src
-8%
5%
SRM
Tec
19%
91% (36nM)
83% (104nM)
-28%
Flt3
-27%
Txk
Fyn
16%
97%
Yes
2.7. Stability in human liver microsomes
Compound 13e was further evaluated its stability in human liver microsomes (Table 4). Higher
metabolic stability was detected after a 60-min incubation, as evidenced by total remaining
percentage (6.7%) for 13e, while only 0.2% for IBN was left. Half-life (T1/2) and intrinsic clearance
(CLint(mic)) values were calculated. 13e showed longer half-life (16.8 min) and lower clearance (82.5
μL/min/mg) compared with IBN (T1/2 = 1.6 min, CLint(mic) = 846.7 μL/min/mg).
Table 4. Metabolic stability of IBN and 13e in human liver microsomes. R2 is the correlation
coefficient of the linear regression for the determination of kinetic constant, T1/2 is half-life and
CLint(mic) is the intrinsic clearance.
HLM 0.5
Sample
T1/2
(min)
1.6
CLint(mic)
(μL/min/mg)
846.7
CLint(liver)
(mL/min/kg)
762.0
Remaining
Remaining
R2
Name
(T=60 min) (*NCF=60 min)
IBN
13e
0.9975
0.9070
0.2%
6.7%
87.4%
62.8%
16.8
82.5
74.2
2.8. Molecular modeling
The molecular modeling results of compound 13e with BTK are shown in Fig 4. The
pyrazolopyrimidine core formed several important hydrogen bonds with hinge region through

Glu475, Met477 and Cys481. The 6-phenoxypyridine group extended to the hydrophobic pocket
behind the gatekeeper Thr474 displaying an edge-to-face aromatic interaction with Phe540. As
expected, N-1 nitrogen at pyridine ring formed a hydrogen bond with the amino of Lys430. This
interaction could play an important role in kinase selectivity for 13e.
O
N
NH2
N
N
N
N
Cl
N
O
13e
Figure 4. Docking pose of 13e with BTK. PDB ID:5P9I.
3. Conclusion
In summary, this study outlined the design, synthesis, and biological evaluation of novel 3-(6-
phenoxypyridin-3-yl)-4-amine-1H-pyrazolo[3,4-d]pyrimidine derivatives as potential agents for
treating MCL. Among these compounds, 13e exhibited potent BTK inhibitory activity and could
completely inhibit the phosphorylation of BTK and PLCγ2 in Z138 cells at 0.5 μM. Compared to
IBN, 13e exhibited robust antiproliferative effects in both mantle cell lymphoma cell lines and
primary patient tumor cells. 13e explained a dose-dependent apoptosis in Jeko-1 and Z138 cells. In
addition, compound 13e showed greater BTK selectivity and higher stability in human liver
microsomes than IBN and potential safety improvement for the treatment of MCL, indicating 13e
would be a promising candidate in cancer therapy.
4. Experimental section
4.1. Chemistry
All the chemical reagents and solvents were purchased from commercial sources and used
without further purification. Reactions were monitored using thin-layer chromatography (TLC)

performed on SGF254 plates. Column chromatography was carried out with the indicated solvents
using silica gel (60 Å, 200 - 300 mesh). Melting points were determined using Büchi capillary
melting point apparatus (Buchi Labortechnik AG, Switzerland) without correction. Using
tetramethylsilane (TMS) as an internal standard in DMSO-d6 or CDCl3, NMR spectra were recorded
at 400 MHz for 1H and 100 MHz for 13C on a Bruker Avance DRX-400 Spectrometer (Bruker,
Germany). The chemical shifts (δ) were reported in parts per million (ppm) using tetramethylsilane
as internal standard.
4.2. General synthesis of compounds
4.2.1. General procedure for the synthesis of 3a-3j
To a stirred solution of various substituted amines 2 (10 mmol) and NaHCO3 (3.10 g, 37 mmol)
in the mixed solution of ethyl acetate/water = 1/1 (20 mL), bromoacetyl bromide (3.84 g, 23 mmol)
was added at room temperature. The mixture was stirred at room temperature for 1 h. After
completion of the reaction, the reaction mixture was extracted with ethyl acetate. The organic layer
was dried over Na2SO4 and the solvent was evaporated in vacuo to give 3a-3j.
4.2.2. 5-Bromo-2-phenoxypyridine (5)
To a solution of phenol (2.82 g, 30 mmol) in anhydrous tetrahydrofuran (THF) (20 mL) was
slowly added 60% sodium hydride (0.96 g, 40 mmol). The mixture was stirred for 30 min at room
temperature. 5-Bromo-2-fluoropyridine (4) (3.52 g, 20 mmol) was added and the reaction was
heated to 80 °C for 12 h. After cooling down to room temperature, the mixture was poured into
water and extracted with ethyl acetate. The organic phase was washed with brine, dried on Na2SO4,
filtered, evaporated and the crude product was purified by silica gel column chromatography
(petroleum ether/ethyl acetate, 100/1) yielding intermediate 5 as a colorless oil (4.4 g, 88%). 1H
NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 2.4 Hz, 1H), 8.06 (dd, J = 8.7, 2.6 Hz, 1H), 7.47 - 7.39
(m, 2H), 7.23 (t, J = 7.4 Hz, 1H), 7.18 - 7.12 (m, 2H), 7.04 (d, J = 8.7 Hz, 1H).
4.2.3. 2-Phenoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (6)
A premixed and degassed solution of Pd(OAc)2 (154.60 mg, 0.69 mmol) and X-Phos (656.00
mg, 1.38 mmol) in dioxane (5 mL) that had been stirred for 20 minutes, was added to a stirred,

degassed mixture of bis(pinicolato)diboron (6.99 g, 27.50 mmol), potassium acetate (4.05 g, 41.30
mmol) and compound 5 (3.44 g, 13.77 mmol) in dioxane (50 mL). The mixture was stirred at 90 °C
for 12 h. Upon completion, the reaction mixture was filtered and concentrated in vacuo. The residue
was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 30/1) to afford
intermediate 6 as a white solid (3.06 g, 75%). 1H NMR (400 MHz, DMSO-d6) δ 8.36 (d, J = 1.5 Hz,
1H), 8.03 (dd, J = 8.3, 2.0 Hz, 1H), 7.43 (t, J = 7.9 Hz, 2H), 7.24 (t, J = 7.4 Hz, 1H), 7.14 (d, J =
7.7 Hz, 2H), 7.00 (d, J = 8.3 Hz, 1H), 1.30 (s, 12H).
4.2.4. 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (8)
N-bromosuccinimide (6.65 g, 37.35 mmol) was added to a stirred solution of 1H-pyrazolo[3,4-
d]pyrimidin-4-amine (7) (5.04 g, 37.35 mmol) in N, N-dimethylformamide (DMF) (30 mL). The
reaction was heated at 90 °C for 1 h. Upon completion, the reaction was quenched with water (400
mL). The suspension was filtered and the filter cake was washed completely with water to yield
compound 8 as a yellow solid (6.71 g, 84%). 1H NMR (400 MHz, DMSO-d6) δ 13.77 (s, 1H), 8.18
(s, 1H).
4.2.5. 3-(6-Phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9)
The Pd(PPh3)4 (0.48 g, 0.41 mmol) was added to a mixture of compound 6 (2.97 g, 10 mmol),
compound 8 (1.78 g, 8.33 mmol) and K3PO4 (3.53 g, 16.66 mmol) in the mixed solution of
dioxane/water = 4/1 (50 mL). The mixture was degassed and put under nitrogen atmosphere, then
was stirred at 120 °C for 24 h. After cooled to room temperature, the mixture was partitioned
between ethyl acetate and water. The organic layer was separated and the aqueous layer was
extracted. The combined organic phases were washed with brine, then dried and concentrated. The
crude product was purified by silica gel column chromatography (dichloromethane/methanol, 80/1)
to afford compound 9 as a white solid (1.92 g, 76%). 1H NMR (400 MHz, DMSO-d6) δ 13.67 (s,
1H), 8.38 (s, 1H), 8.23 (s, 1H), 8.08 (dd, J = 8.4, 1.8 Hz, 1H), 7.46 (t, J = 7.8 Hz, 2H), 7.27 - 7.15
(m, 4H), 6.94 (s, 2H).
4.2.6. General procedure for the synthesis of 10a-10j
To a mixture of compound 9 (1 mmol) and K2CO3 (3.1 g,1.5 mmol) in DMF (5 mL) was added
compounds 3a-3j (1.2 mmol) and the reaction mixture was stirred for 5 h at room temperature. The

reaction solution was poured into water (50 mL). The suspension was filtered and the crude product
was purified by silica gel column chromatography (dichloromethane/methanol, 150/1-60/1) to yield
target compounds 10a-10j.
4.2.6.1. 4-(2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetamido)-
2-fluoro-N-methylbenzamide (10a) White solid, yield 74%, Mp:208-210 °C, 1H NMR (400 MHz,
DMSO-d6) δ 10.86 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 8.28 (d, J = 10.6 Hz, 1H), 8.11 (s, 2H), 7.72 -
7.59 (m, 2H), 7.52 - 7.42 (m, 2H), 7.35 (t, J = 8.3 Hz, 1H), 7.31 - 6.92 (m, 6H), 5.29 (s, 2H), 2.77
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 166.31, 163.90, 163.49, 161.10, 158.74, 158.65, 156.54,
155.72, 154.32, 147.18, 142.45, 142.34, 141.58, 140.38, 131.45, 130.23, 125.11, 124.66, 121.60,
118.77, 118.63, 115.12, 112.27, 106.68, 106.39, 98.01, 50.25, 26.77. MS(ESI) m/z: 513.04 [M +
H]+
4.2.6.2.
2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(3-
1
fluorobenzyl)acetamide (10b) White solid, yield 80%, Mp:233-235 °C, H NMR (400 MHz,
DMSO-d6) δ 8.75 (t, J = 5.9 Hz, 1H), 8.39 (d, J = 2.2 Hz, 1H), 8.27 (s, 1H), 8.08 (dd, J = 8.5, 2.4
Hz, 1H), 7.46 (t, J = 7.9 Hz, 2H), 7.37 (q, J = 7.5 Hz, 1H), 7.30 - 6.93 (m, 9H), 5.10 (s, 2H), 4.34
(d, J = 5.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 167.09, 163.93, 163.43, 161.52, 158.71,
156.40, 155.54, 154.34, 147.17, 142.59, 142.52, 141.46, 140.34, 130.70, 130.62, 130.22, 125.09,
124.74, 123.58, 123.55, 121.56, 114.34, 114.12, 113.91, 112.24, 98.09, 49.77, 42.16. MS(ESI) m/z:
470.20 [M + H]+
4.2.6.3.
2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(4-
1
fluorophenethyl)acetamide (10c) White solid, yield 82%, Mp:194-196 °C, H NMR (400 MHz,
DMSO-d6) δ 8.38 (d, J = 2.1 Hz, 1H), 8.27 (s, 1H), 8.23 (t, J = 4.5 Hz, 1H), 8.07 (dd, J = 8.5, 2.3
Hz, 1H), 7.46 (t, J = 7.8 Hz, 2H), 7.37 - 6.97 (m, 10H), 4.97 (s, 2H), 3.30 (q, J = 6.8 Hz, 2H), 2.72
(t, J = 7.1 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 166.69, 163.43, 162.51, 160.11, 158.68,
156.40, 155.53, 154.33, 147.17, 141.37, 140.35, 135.89, 135.86, 130.98, 130.90, 130.22, 125.09,
124.76, 121.57, 115.52, 115.31, 112.23, 98.01, 49.55, 40.87, 34.48. MS(ESI) m/z: 484.30 [M + H]+
4.2.6.4.
2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(2,3,4-
1
trifluorophenyl)acetamide (10d) White solid, yield 78%, Mp:235-237 °C, H NMR (400 MHz,

DMSO-d6) δ 10.45 (s, 1H), 8.39 (d, J = 1.9 Hz, 1H), 8.27 (s, 1H), 8.09 (dd, J = 8.5, 2.2 Hz, 1H),
7.64 (q, J = 6.2 Hz, 1H), 7.46 (t, J = 7.8 Hz, 2H), 7.33 - 7.27 (m, 1H), 7.26 - 6.94 (m, 6H), 5.33 (s,
2H). 13C NMR (100 MHz, DMSO-d6) δ 166.38, 163.50, 158.74, 156.53, 155.72, 154.34, 148.74,
148.70, 148.62, 147.19, 146.28, 146.20, 146.18, 144.99, 144.88, 141.61, 140.98, 140.96, 140.37,
138.66, 138.52, 138.50, 138.36, 130.22, 125.10, 124.68, 124.06 124.03, 123.97, 123.94, 121.58,
119.07, 119.05, 119.02, 118.98, 118.96, 112.47, 112.44, 112.26, 98.03, 49.84. MS(ESI) m/z: 492.04
[M + H]+
4.2.6.5. 2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(3-chloro-4-
1
fluorophenyl)acetamide (10e) White solid, yield 86%, Mp:213-215 °C, H NMR (400 MHz,
DMSO-d6) δ 10.68 (s, 1H), 8.40 (d, J = 2.3 Hz, 1H), 8.27 (s, 1H), 8.09 (dd, J = 8.5, 2.4 Hz, 1H),
7.89 (dd, J = 6.8, 2.4 Hz, 1H), 7.48 - 7.38 (m, 4H), 7.26 - 6.99 (m, 6H), 5.26 (s, 2H). 13C NMR (100
MHz, DMSO-d6) δ 165.89, 163.47, 158.72, 156.51, 155.71, 154.94, 154.31, 152.53, 147.17, 141.55,
140.36, 136.26, 136.23, 130.22, 125.10, 124.66, 121.58, 121.11, 120.03, 119.97, 119.79, 119.61,
117.69, 117.47, 112.25, 98.00, 50.13. MS(ESI) m/z: 490.06 [M + H]+
4.2.6.6. 2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(3-chloro-4-
1
cyanophenyl)acetamide (10f) White solid, yield 74%, Mp:154-156 °C, H NMR (400 MHz,
DMSO-d6) δ 11.10 (s, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.27 (s, 1H), 8.09 (dd, J = 8.5, 2.4 Hz, 1H),
8.02 (d, J = 1.7 Hz, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.61 (dd, J = 8.6, 1.8 Hz, 1H), 7.46 (t, J = 7.9 Hz,
2H), 7.35 - 6.94 (m, 6H), 5.32 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 166.87, 163.49, 158.73,
156.56, 155.73, 154.30, 147.17, 144.23, 141.67, 140.36, 136.58, 135.83, 130.22, 125.11, 124.61,
121.59, 119.67, 118.35, 116.62, 112.26, 106.37, 97.99, 50.34. MS(ESI) m/z: 495.30 [M - H]-
4.2.6.7. 2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(3-chloro-2-
1
fluorophenyl)acetamide (10g)
White solid, yield 81%, Mp:214-216 °C, H NMR (400 MHz,
DMSO-d6) δ 10.42 (s, 1H), 8.40 (s, 1H), 8.28 (s, 1H), 8.10 (d, J = 8.6 Hz, 1H), 7.86 (t, J = 7.5 Hz,
1H), 7.46 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 8.4 Hz, 1H), 7.33 - 7.13 (m, 6H), 7.12 - 6.90 (m, 1H), 5.35
(s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 166.35, 163.47, 158.72, 156.53, 155.69, 154.30, 150.90,
148.43, 147.17, 141.57, 140.37, 130.22, 127.72, 127.62, 126.24, 125.54, 125.10, 124.66, 123.01,
121.59, 120.46, 120.30, 112.25, 97.99, 49.95. MS(ESI) m/z: 490.18 [M + H]+

4.2.6.8. 2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(4-bromo-3-
(trifluoromethyl)phenyl)acetamide (10h) White solid, yield 87%, Mp:118-120 °C, 1H NMR (400
MHz, DMSO-d6) δ 10.91 (s, 1H), 8.39 (d, J = 1.9 Hz, 1H), 8.27 (s, 1H), 8.18 (d, J = 2.0 Hz, 1H),
8.09 (dd, J = 8.5, 2.2 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.46 (t, J = 7.8 Hz,
2H), 7.34 - 7.00 (m, 6H), 5.28 (s, 2H). 13C NMR (100 MHz, DMSO-d6) 166.36, 163.49, 158.73,
156.53, 155.74, 154.32, 147.18, 141.62, 140.35, 138.91, 136.12, 130.21, 129.55, 129.25, 128.95,
128.64, 127.28, 125.09, 124.65, 124.56, 121.85, 121.57, 119.13, 118.66, 118.59, 112.42, 112.40,
112.26, 98.02, 50.24. MS(ESI) m/z: 584.00 [M + H]+
4.2.6.9. 2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(4-cyano-3-
(trifluoromethyl)phenyl)acetamide (10i) White solid, yield 80%, Mp:132-134 °C, 1H NMR (400
MHz, DMSO-d6) δ 11.27 (s, 1H), 8.40 (d, J = 2.2 Hz, 1H), 8.27 (s, 2H), 8.13 (d, J = 8.6 Hz, 1H),
8.09 (dd, J = 8.5, 2.4 Hz, 1H), 7.95 (dd, J = 8.6, 1.6 Hz, 1H), 7.46 (t, J = 7.9 Hz, 2H), 7.36 - 6.97
(m, 6H), 5.34 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 167.09, 163.50, 158.73, 156.58, 155.74,
154.30, 147.17, 143.52, 141.71, 140.36, 137.14, 132.45, 132.13 130.22, 125.11, 124.60, 124.19,
122.63, 121.59, 121.47, 117.09, 117.02, 116.97, 116.92, 116.97, 116.15, 112.26, 102.55, 98.00,
50.34. MS(ESI) m/z: 529.39 [M - H]-
4.2.6.10.
2-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-(p-
tolyl)acetamide (10j) White solid, yield 79%, Mp:238-240 °C, 1H NMR (400 MHz, DMSO-d6) δ
10.33 (s, 1H), 8.39 (d, J = 1.8 Hz, 1H), 8.26 (s, 1H), 8.09 (dd, J = 8.5, 2.2 Hz, 1H), 7.45 (t, J = 7.1
Hz, 4H), 7.26 - 7.11 (m, 8H), 5.22 (s, 2H), 2.25 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.25,
163.46, 158.73, 156.47, 155.69, 154.35, 147.18, 141.41, 140.37, 136.57, 133.01, 130.23, 129.69,
125.09, 124.74, 121.58, 119.65, 112.26, 98.01, 50.16, 20.91. MS(ESI) m/z: 452.30 [M + H]+
4.2.7. 1-Boc-3-(4-amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine
(11)
A mixture of compound 9 (1.83 g, 6.00 mmol), racemic 1-Boc-3-hydroxypiperidine (1.81 g,
6.00 mmol) and triphenylphosphine (PPh3) (4.72 g, 18.00 mmol) in absolute anhydrous THF (25
mL) was stirred for 30 min under ice-cold conditions. Diisopropyl azodiformate (DIAD) (3.64 g,
18.00 mmol) was added dropwise to the reaction mixture. The mixture was stirred for 5 h at the

room temperature. Upon completion, the reaction was quenched with water and extracted with ethyl
acetate. The combined organic phases were washed with water and brine, then dried and
concentrated. The crude product was purified by silica gel column chromatography (petroleum
ether/ethyl acetate, 6/1) to yield racemic compounds 11 as a white solid (1.40g, 48%).
4.2.8.
hydrochloride (12)
To a solution of racemic compounds 11 (0.49 g, 1 mmol) in THF (1 mL) was added 4M HCl
3-(6-Phenoxypyridin-3-yl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
in dioxane (5 mL) and the reaction mixture was stirred for 5 h at room temperature. The precipitate
was filtrated, washed with ethyl acetate (25 mL) afforded racemic compounds 12 as a white solid
(0.39 g, 92%).
4.2.9. General procedure for the synthesis of 13a-13e
To a solution of racemic compounds 12 (0.21 g, 0.5 mmol) and triethylamine (0.30 g, 1.5 mmol)
in THF (20 mL), different acyl chloride (0.6 mmol) was added under ice-cold conditions and the
mixture was stirred at room temperature for 5 h. Or to a solution of racemic compounds 12 (0.21 g,
0.5 mmol), HBTU (0.23g, 0.6 mmol) and triethylamine (0.30 g, 1.5 mmol) in THF (20 mL),
different substituted acids (0.6 mmol) was added under ice-cold conditions and the mixture was
stirred at room temperature for 8 h. Upon completion, the reaction was quenched with water and
extracted with ethyl acetate. The combined organic phases were washed with water and brine, then
dried and concentrated. The crude product was purified by silica gel column chromatography
(dichloromethane/methanol, 100/1-60/1) to afford compounds 13a-13e.
4.2.9.1. 1-(3-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
1
yl)-2-hydroxyethan-1-one (13a) White solid, yield 56%, Mp: 232-234 °C, H NMR (400 MHz,
DMSO-d6) δ 8.30 (s, 1H), 8.20 (d, J = 6.8 Hz, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.38 (t, J = 7.8 Hz,
2H), 7.31 - 6.74 (m, 6H), 4.72 (t, J = 9.6 Hz, 0.5H), 4.65 - 4.57 (m, 0.5H), 4.54 (s, 0.5H), 4.41 (d, J
= 11.7 Hz, 0.5H), 4.16 - 3.80 (m, 3H), 3.65 (d, J = 12.3 Hz, 0.5H), 3.55 - 3.45 (m, 0.5H), 3.12 (t, J
= 11.5 Hz, 0.5H), 3.01 (t, J = 13.0 Hz, 0.5H), 2.93 - 2.81 (m, 0.5H), 2.23 - 2.11 (m, 1H), 2.06 (s,
1H), 1.82 (d, J = 12.6 Hz, 1H), 1.64 - 1.44 (m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 170.78,
170.60, 163.45, 158.71, 156.23, 154.57, 154.49, 154.37, 147.29, 141.15, 141.07, 140.46, 130.21,

125.08, 124.82, 121.56, 112.23, 98.14, 60.71, 60.62, 52.98, 52.63, 48.27, 46.04, 44.08, 41.95, 29.97,
29.89, 24.82, 23.77. MS(ESI) m/z: 446.16 [M + H]+
4.2.9.2. 1-(3-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)-2-hydroxypropan-1-one (13b) White solid, yield 62%, Mp:165-168 °C, 1H NMR (400 MHz,
DMSO-d6) δ 8.38 (s, 1H), 8.27 (s, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.31 - 6.93
(m, 6H), 5.06 - 4.88 (m, 1H), 4.82 - 4.63 (m, 1H), 4.55 - 4.44 (m, 1H), 4.42 - 4.15 (m, 1.5H), 4.06 -
4.01 (m, 0.5H), 3.67 - 3.49 (m, 0.5H), 3.20 - 3.07 (m, 1H), 2.91 - 2.72 (m, 0.5H), 2.27 - 2.13 (m,
1H), 2.13 (s, 1H), 1.90 (s, 1H), 1.71 - 1.49 (m, 1H), 1.27 - 1.11 (m, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 172.92, 172.80, 172.49, 163.45, 158.73, 156.24, 154.56, 154.47, 154.37, 147.29,
141.13, 140.45, 130.21, 125.07, 124.83, 121.55, 112.22, 98.16, 65.04, 64.91, 64.73, 53.42, 53.15,
52.78, 52.66, 49.21, 49.20, 46.24, 45.09, 45.07, 44.95, 42.09, 42.06, 42.04, 30.20, 30.06, 29.94,
25.15, 25.02, 24.13, 24.09, 21.34, 21.25, 21.08, 20.89. MS(ESI) m/z: 460.28 [M + H]+
4.2.9.3. 1-(3-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
yl)-3-hydroxy-2,2-dimethylpropan-1-one (13c) White solid, yield 62%, Mp: 212-214 °C, 1H NMR
(400 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.19 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H),
7.32 - 6.77 (m, 6H), 4.64 (t, J = 10.6 Hz, 1H), 4.51 (t, J = 5.6 Hz, 1H), 4.39 (d, J = 12.2 Hz, 1H),
4.22 (d, J = 12.9 Hz, 1H), 3.36 (d, J = 5.6 Hz, 2H), 3.18 (t, J = 11.6 Hz, 1H), 2.89 (t, J = 12.0 Hz,
1H), 2.20 (q, J = 11.9 Hz, 1H), 2.04 (d, J = 9.9 Hz, 1H), 1.83 (d, J = 13.0 Hz, 1H), 1.53 (q, J = 12.5
Hz, 1H), 1.10 (d, J = 7.0 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 174.92, 163.44, 158.73, 156.23,
154.48, 154.35, 147.29, 141.08, 140.46, 130.22, 125.14, 124.84, 121.56, 112.22, 98.16, 69.43, 53.03,
48.80, 45.21, 44.16, 30.07, 24.81, 23.43, 23.16. MS(ESI) m/z: 487.91 [M + H]+
4.2.9.4. 2-Methoxyethyl 3-(4-amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)piperidine-1-carboxylate (13d) White solid, yield 58%, Mp: 152-154 °C, 1H NMR (400 MHz,
DMSO-d6) δ 8.38 (s, 1H), 8.27 (d, J = 9.7 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.8 Hz,
2H), 7.40 - 6.83 (m, 6H), 4.82 - 4.72 (m, 05H), 4.68 - 4.59 (m, 0.5H), 4.52 (d, J = 12.1 Hz, 0.5H),
4.21 (d, J = 12.8 Hz, 0.5H), 4.06 (d, J = 12.4 Hz, 0.5H), 3.92 (d, J = 13.2 Hz, 0.5H), 3.65 - 3.45 (m,
2.5H), 3.22 (d, J = 13.2 Hz, 3H), 3.16 - 3.08 (m, 1H), 2.87 (t, J = 11.0 Hz, 0.5H), 2.71 - 2.56 (m,
1.5H), 2.46 - 2.41 (m, 0.5H), 2.28 - 2.20 (m, 1H), 2.12 (s, 1H), 1.89 (t, J = 13.8 Hz, 1H), 1.71 - 1.45
(m, 1H). 13C NMR (100 MHz, DMSO-d6) δ 169.43, 169.30, 163.42, 158.72, 156.24, 156.22, 154.57,

154.44, 154.34, 147.28, 141.08, 140.98, 140.44, 130.20, 125.08, 124.83, 121.55, 112.20, 98.17,
98.10, 68.74, 58.45, 58.40, 53.20, 52.64, 45.73, 45.53, 33.13, 33.07, 30.05, 29.86, 25.09, 23.89.
MS(ESI) m/z: 474.06 [M + H]+
4.2.9.5. 1-(3-(4-Amino-3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-
1
yl)-2-chloroethan-1-one (13e) White solid, yield 68%, Mp: 182-184 °C, H NMR (400 MHz,
DMSO-d6) δ 8.37 (s, 1H), 8.27 (d, J = 7.0 Hz, 1H), 8.08 (d, J = 5.6 Hz, 1H), 7.46 (t, J = 7.8 Hz,
2H), 7.36 - 6.76 (m, 6H), 4.90 - 4.83 (m, 0.5H), 4.71 - 4.63 (m, 0.5H), 4.49 - 4.41 (m, 2H), 4.26 (d,
J = 12.9 Hz, 0.5H), 4.17 (d, J = 11.1 Hz, 0.5H), 4.04 (d, J = 13.6 Hz, 0.5H), 3.85 (d, J = 13.0 Hz,
0.5H), 3.78 - 3.69 (m, 0.5H), 3.24 - 3.18 (m, 1H), 3.00 - 2.91 (m, 0.5H), 2.30 - 2.07 (m, 2H), 1.96 -
1.82 (m, 1H), 1.78 - 1.69 (m, 0.5H), 1.61 - 1.51 (m, 0.5H). 13C NMR (100 MHz, DMSO-d6) δ 165.32,
165.28, 163.46, 158.72, 156.24, 154.62, 154.50, 154.37, 147.30, 147.26, 141.17, 141.11, 140.46,
140.40, 130.21, 125.07, 124.82, 121.55, 112.22, 98.20, 98.14, 52.97, 52.53, 49.72, 46.26, 45.91,
42.60, 42.47, 42.22, 30.02, 29.81, 24.84, 23.64. MS(ESI) m/z: 464.14 [M + H]+
4.3. In vitro BTK kinase activity assay and kinase selectivity assay
The in vitro BTK kinase activity and kinases selectivity activities were evaluated via a
radiometric protein kinase assay 21. All compounds were prepared to 50 X final assay concentration
in 100% DMSO. A portion of this working stock of the compounds were added to the assay well as
the first component in the reaction, followed by adding kinases dilution with specific buffer. The
reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room
temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%.
10 µL of the reaction was then spotted onto a P30 filtermat and washed four times for 4 minutes in
0.425% phosphoric acid and once in methanol prior to drying and scintillation counting. IC50 values
were calculated using the Graphpad prism 6 software.
Count퐶 ― Count퐵
activity rate % =
∗ 100%
Count푃 ― Count퐵
CountC: the counts of testing compounds.
CountP: the counts of positive control. The positive control wells contained all components of
the reaction, except the compound of interest, and DMSO (at a final concentration of 2%) was

included in these wells to control for solvent effects.
CountB: the counts of blank. The blank wells contained all components of the reaction with
staurosporine replacing the compound of interest. This abolished kinase activity and established the
baseline (0% kinase activity remaining).
4.4. Cell lines and primary MCL cells
MCL cell lines Mino, Jeko-1, Z138 and Maver-1 were purchased from the American Type
Culture Collection (ATCC). Peripheral blood was obtained from MCL patients who provided
informed consent. Mononuclear cells were separated by Ficoll-Hypaque density centrifugation, and
tumor cells were isolated using anti-CD19 antibody coated magnetic microbeads (Miltenyi Biotec).
Cells were cultured in RPMI-1640, supplemented with 10% heat-inactivated fetal bovine serum, 2%
HEPES buffer and 1% penicillin (10,000 units/mL; Sigma), streptomycin (10 mg/mL; Sigma).
4.5. Cell proliferation assay
Cell proliferation assay was performed on MCL cell lines and primary MCL patient cells with
the CellTiter-Glo Luminescent cell viability assay kit (Promega) following the manufacturer's
protocol. In short, 50 uL cells were plated in 96-well plates at a density of 1 × 104 cells/well for
MCL cell lines and 12.5 × 104 cells/well for primary MCL patient cells, then treated with DMSO
(control) and different concentrations of the synthesized compounds in triplicate and incubated in a
humidified atmosphere with 5% CO2 at 37 °C for 72 h on MCL cell lines and 24 h on primary
MCL patient cells. Cells were lysed with 30 uL Cell Titer-Glo Luminescent Cell Viability Assay
Reagent (Promega, Madison, WI, USA), and luminescence was quantified using the BioTek
Synergy HTX Multi-mode Micro Plate Reader (Winooski, VT, USA). Each triplicate experiment
was performed no less than three times to establish the cell survival curve. IC50 values were
calculated using the Graphpad prism 6 software.
4.6. Western blotting
Z138 cells were cultured with 0.5 μM and 1 μM of 13e and 1 μM of IBN for 16 h. Then cells
were harvested and lysed in a lysis buffer (Cell Signaling, Danvers, MA). The cell lysates were kept
on ice for 30 min and centrifuged at 12,000 × rpm for 20 min at 4 °C. The protein concentration
was determined by the Bradford assay (Bio-Rad, Hercules, CA). Twenty micrograms of sample

proteins were mixed with the 4 × loading buffer and separated by 10% SDS-PAGE. The proteins
were then transferred onto methanol equilibrated PVDF membrane (BIO-RAD Laboratories,
162e0177), which was blocked for 1 h in 5% nonfat dry milk in TBST (BD Bioscience, San Jose,
CA). The membranes were incubated with a primary antibody overnight at 4 °C. Secondary
antibodies were added for 1 h at room temperature. Finally, the membrane was visualized by ECL
(Perkin Elmer Life Sciences, NE104001EA). Antibodies against BTK, PLCγ2, p-BTK, p-PLCγ2
and GAPDH were obtained from Cell Signaling.
4.7. Cell apoptosis assay
Apoptosis was quantified by Annexin V/Propidium Iodide (PI)- binding assay. Cells were
seeded in 6-well plates with 1 μM and 2 μM of 13e for 24 h. Treated cells were washed twice with
cold phosphate-buffered saline (PBS) and then resuspended in100 μL binding buffer, to which 2 μL
of Annexin V-FITC and 5 μL of PI were added. The samples were gently vortexed and incubated
for 15 min at room temperature in the dark. After addition of 200 μL binding buffer, samples were
immediately analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San
Jose, CA). The number of apoptotic cells was determined using the Flowjo software.
4.8. Stability in human liver microsomes
In vitro metabolic stability in human liver microsomes was assessed using testosterone,
diclofenac, and propafenone as control. A DMSO solution of the tested compound (10 mm, 10
μl/well) and solution of microsome (80 μl/well) were added to a 96-well plate; this mixture was first
incubated at 37 °C for 10 min. Then potassium phosphate buffer (100 mm, 10 μl/well) was added,
and no cofactor (NCF) remaining was evaluated after a further incubation of 60 min. After this
prewarming process, NADPH regenerating system (10 μl/well) was added, and remaining of each
compound was tested at five time-points (5, 10, 20, 30 and 60 min). At each timepoint, stop solution
(including 100 ng/ml tolbutamide and 100 ng/ml labetalol, cold in 4 °C, 300 μl/well) was added to
terminate the reaction. The sampling plates were shaken for approximate 10 min, and then samples
were centrifuged at 1306 g for 20 min under 4 °C to afford the supernatant (100 μl) for LC/MS test.
Intrinsic clearance (CLint) and halflife (T1/2) values were then calculated. CLint(mic) = 0.693/ half-
life/mg microsome protein per ml. CLint(liver) = CLint(mic) × (45 mg microsomal protein/g liver
weight) × (20 g liver weight/kg body weight).

4.9. Molecular docking
Molecular docking was performed using the Sybyl 2.0 software and the BTK crystal structure
(PDB: 5P9I) was retrieved from the Protein Data Bank. Protein preparation was performed by
extracting the ligand, removing water molecules, adding hydrogen atoms and assigning AMBER7
FF99 charges to the protein. Compound 13e was docked into BTK and the hydrogen bonds and
hydrophobic interactions were observed in the model, the best conformation with the highest CScore
was selected for interaction analysis.
4.10. Statistical analysis
Student’s tests were performed for statistical analyses of 13e induced apoptotic effect, two-
way ANOVA analysis of variance was used to compare the IBN and 13e treatment in the primary
patient cells in vitro. P values of <0.05 were considered statistically significant.
Acknowledgements
This work was supported by the key research and development project of Shandong Province [No.
2017CXGC1401].
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Graphical abstract

Highlights:




13e explained potent BTK inhibitory activity.
13e obtained antiproliferative effects in primary patient tumor cells.
13e showed more potent antitumor effects in mantle cell lymphoma than ibrutinib.
13e could completely inhibit the phosphorylation of BTK and PLCγ2 in Z138 cells at low
micromolar concentration.
Low micromolar doses of 13e induced strong cell apoptosis in Jeko-1 and Z138 cells.
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