Design, synthesis and biological evaluation of novel 3-substituted pyrazolopyrimidine derivatives as potent Bruton's tyrosine kinase (BTK) inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.03.017

A series of 3-substituted pyrazolopyrimidine derivatives as BTK inhibitors were designed by structure-based drug design and they were synthesized, evaluated by enzyme-based assay and anti-proliferation against Ramos and Raji cells. Most of them displayed good inhibitory activities against both BTK and B-cell lymphoblastic leukemia lines in vitro. Among them, compound 8a exhibited excellent potency (IC₅₀ = 7.95 nM against BTK enzyme, 8.91 μM against Ramos cells and 1.80 μM against Raji cells), with a better hydrophilicity (ClogP = 3.33). These explorations provided new clues to discover 3-substituted pyrazolopyrimidine derivatives as novel anti-tumor agents.

Full text of DOI:10.1016/j.bmc.2018.03.017

Accepted Manuscript
Design, synthesis and biological evaluation of novel 3-substituted pyrazolopyr-
imidine derivatives as potent Bruton's tyrosine kinase (BTK) inhibitors
Nan Zheng, Jing Pan, Qun Hao, Yingxia Li, Weicheng Zhou
PII:
S0968-0896(18)30182-2
https://doi.org/10.1016/j.bmc.2018.03.017
BMC 14255
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 January 2018
5 March 2018
10 March 2018
Please cite this article as: Zheng, N., Pan, J., Hao, Q., Li, Y., Zhou, W., Design, synthesis and biological evaluation
of novel 3-substituted pyrazolopyrimidine derivatives as potent Bruton's tyrosine kinase (BTK) inhibitors,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.03.017
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Design, synthesis and biological evaluation of novel
-substituted pyrazolopyrimidine derivatives as
potent Bruton's tyrosine kinase (BTK) inhibitors
3
a,b
a,b
a
b,*
a,*
Nan Zheng , Jing Pan , Qun Hao , Yingxia Li , Weicheng Zhou
a
Shanghai Key Lab. of Anti-infectives, State Key Lab. of New Drug and Pharmaceutical Process,
Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry,
285 Gebaini Road, Shanghai 201203, PR China
b
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China
*
Corresponding authors.
E-mail addresses: liyx417@fudan.edu.cn (Y. Li), zhouweicheng58@163.com (W. Zhou).
Abstract
A series of 3-substituted pyrazolopyrimidine derivatives as BTK inhibitors were
designed by structure-based drug design and they were synthesized, evaluated by
enzyme-based assay and anti-proliferation against Ramos and Raji cells. Most of them
displayed good inhibitory activities against both BTK and B-cell lymphoblastic
leukemia lines in vitro. Among them, compound 8a exhibited excellent potency
(
IC50=7.95 nM against BTK enzyme, 8.91 μM against Ramos cells and 1.80 μM
against Raji cells), with a better hydrophilicity (ClogP = 3.33). These explorations
provided new clues to discover 3-substituted pyrazolopyrimidine derivatives as novel
anti-tumor agents.
Keywords
BTK inhibitors
3-Substituted pyrazolopyrimidine
Structure-based drug design
Inhibitory activity
ClogP

1
. Introduction
Bruton's tyrosine kinase (BTK) is a key member of the Tec family of
non-receptor tyrosine kinases, it is mostly expressed in hematopoietic cells and plays
an important role in multiple cellular signaling pathways including B cell receptor
1
-3
(
BCR) and Fc receptor (FCR) signaling cascades. The deregulation of BTK has
been observed in many B-cell-derived malignancies such as acute lymphoblastic
leukemia (ALL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL),
non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM) and Waldenstrom’s
4
,5
Macroglobulinemia (WM). Thus, BTK is considered as a promising target for
treating autoimmune diseases and cancer.
Until now, many small-molecule inhibitors of BTK have been reported, which
can be divided into two classes, irreversible and reversible ones. Irreversible BTK
6
7
8
inhibitors, such as ibrutinib (1), acalabrutinib (2, ACP-196), GS-4059 (3),
9
10
spebrutinib (4, CC-292, AVL-292) and HM71224 (5) , contain a Michael addition
receptor moiety in the structures which can form a covalent bond with a noncatalytic
cysteine residue (Cys481) of BTK enzyme, and some of them have been advanced to
11
TM
the clinical stages. Ibrutinib (Imbruvica ) was approved by FDA as a first-in-class
irreversible inhibitor for the treatment of B-cell-derived malignancies, including MCL,
6
12
TM
CLL , and WM etc. Recently, acalabrutinib (Calquence ) was also approved into
1
3
14,15
16
market. Some reversible inhibitors including RN-486 (6)
and GDC-0834 (7)
are reported to come through preclinical and phase I with the purpose of autoimmune
diseases therapy.

Figure 1. Representative chemical structures of the novel BTK inhibitors
The binding mode of ibrutinib with BTK was disclosed by the available crystal
1
7
protein structure (PDB: 5P9J). Generally, the backbone of pyrazolopyrimidine
occupies the ATP binding pocket and makes several important interactions with the
hinge. The 4-amine directly forms two hydrogen bonds with the hydroxyl of
gatekeeper Thr474 and the backbone carbonyl of Glu475, the N-3 nitrogen
participates in a hydrogen bond with the amine of Met477. The distal phenyl group
forms a face-to-edge pi-stacking interaction with the phenyl of Phe540. The
acrylamide moiety participates in a hydrogen bond with the backbone amine of
Cys481 and a covalent bond with the mercapto group of Cys481 by Michael addition
reaction. These interactions are critical for BTK binding activity. The phenoxy group
of ibrutinib connects the distal phenyl group with pyrazolopyrimidine and twists plane
of the phenylether, such that the distal phenyl group enters a hydrophobic pocket, but
the phenoxy group does not form any significant interactions with BTK. On the other
hand, since ibrutinib showed low aqueous solubility, high hydrophobic property and
1
8,19
poor bioavailability , ibrutinib is clinically used in high dosages, so that it would
bring many unexpected side effects and toxicities including lymphopenia and
2
0
neutropenia . Therefore, in this research, the backbone and groups which are
necessary for activity of ibrutinib were retained, the phenyl ring was replaced by
three-carbon groups such as propanyl, allyl and propynyl, and four atoms including O,
C, N, S as the connection of the three-carbon group with the distal phenyl were
investigated in order to maintain the bioactive conformation of the distal phenyl group
and to decrease some hydrophobic property of ibrutinib. Herein, we report the
synthesis and biological evaluation of a series of 3-substituted 4-amino-1H-pyrazolo
[
3,4-d]pyrimidine derivatives bearing C-3 alkyl side chains as novel potent BTK
inhibitors.

Figure 2. Binding mode of ibrutinib with BTK enzyme (PDB: 5P9J).
Figure 3. Designed strategy of 3-substituted derivatives as novel potent BTK inhibitors
2
. Results and discussion
2
.1. Chemistry
As summarized in Table 1, the novel 3-substituted pyrazolopyrimidine
derivatives were designed and synthesized. The synthetic routes for these compounds
are illustrated in Scheme 1 and 2.
Iodination of 1H-pyrazolo[3,4-d]pyrimidin-4-amine (9) by N-iodosuccinimide
afforded 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10), which was transformed
by the Mitsunobu reaction to (R)-tert-butyl 3-(4-amino-3-iodo-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidine-1-carboxylate (11), followed by the Sonogashira coupling
reaction with 12a to afford compound 13a. Then 13a was stirred with 4 M HCl to
produce the deprotected intermediate 14a. Compound 14a was then reduced under
different conditions including hydrogenation on 10% Pd/C, or on 10% Pd-BaSO
LiAlH to provide the compounds 14a1-14a3, respectively. Finally 14a1-14a3 were
treated with acryloyl chloride at -10℃ to afford the targeted compounds 8a1-8a3
Scheme 1).
4
or
4
(

Scheme 2 depicted the synthesis of compounds 8a-8o. Compound 11 was
subsequently treated with different alkyne (12a-12o) under palladium catalysis to give
the Sonogashira coupling products 13a-13o. Deprotection of 13a-13o with 4 M HCl
and acylation with acryloyl chloride at -10℃ afforded the targeted compounds 8a-8o.
Scheme 1. Synthetic route of the compounds 8a1-8a3. Reagents and conditions: (a) NIS, DMF,
9
0℃, 4h, 80%; (b) (S)-1-Boc-3-hydroxypiperidine, DIAD, PPh
CuI, Et N, DMF, 80℃, 13h, 74%; (d) 4 M HCl, 1,4-dioxane, rt, 8h, 80%; (e
Methanol, 55℃, 24h, 80%; (e ) 10% Pd-BaSO , H , Methanol, 45℃, 6h, 70%; (e
rt, 12h, 60%; (f) acryloyl chloride, DIPEA, DCM, -10℃, 15min, 40%-75%.
3
, THF, rt, 12h, 51%; (c) Pd(PPh
3 4
) ,
3
1
) 10% Pd/C, H ,
2
2
4
2
3 4
) LiAlH , THF,

Scheme 2. Synthetic route of the compounds 8a-8o. Reagents and conditions: (a) Pd(PPh
3 4
) , CuI,
3
Et N, DMF, 80℃, 8h-13h, 60%-80%; (b) 4 M HCl, 1,4-dioxane, rt, 6-8h, 60%-80%; (c) acryloyl
chloride, DIPEA, DCM, -10℃, 15min, 40%-75%.
Table 1
In vitro BTK enzyme inhibition for 3-substituted pyrazolopyrimidine derivatives.
BTK enzymatic activity
Compound
L
1
L
2
R
a
IC50 [nM]
8
a
-C≡C-
2
-CH CH -
-CH
-CH
-CH
-CH
2
2
2
2
O-
O-
O-
O-
H
H
7.95
22.18
23.20
11.30
39.80
102.40
15.90
18.10
10.95
49.23
20.85
26.89
38.17
9.84
8
8
8
a1
a2
a3
2
-CH=CH-(Z)
-CH=CH-(E)
-C≡C-
H
H
8
b
c
d
e
-CH
-CH
-CH CH
-CH NH-
-CH
2
CH
2
O-
H
8
-C≡C-
2
-
H
8
8
-C≡C-
2
2
-
H
-C≡C-
2
H
8
f
-C≡C-
2
S-
H
8
8
g
h
-C≡C-
-CH
-CH
-CH
-CH
-CH
-CH
-CH
-CH
-CH
2
O-
2
O-
2
O-
2
O-
2
O-
2
O-
2
O-
2
O-
2
O-
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
3-Cl
-C≡C-
8
8
i
j
-C≡C-
-C≡C-
8k
-C≡C-
8
l
-C≡C-
89.62
18.11
209.20
45.35
5.49
8
m
-C≡C-
8n
-C≡C-
3-CF
3
8o
-C≡C-
3-CH(CH )
3 2
Ibrutinib
a
Data represented the mean of at least two separate experiments. The IC50 values were calculated
using GraphPad Prism version 5.0.
2
.2 Biological evaluation

2
.2.1 In vitro BTK enzymatic assay
All designed compounds were evaluated in vitro for their ability to inhibit BTK
enzyme activity. For comparison, ibrutinib was also tested as reference compound.
The resulting IC50 values are summarized in Table 1.
Among the synthesized C3-substituted pyrazolopyrimidine derivatives, most
could effectively inhibit BTK enzyme at the concentration less than 100 nmol/L, and
two of them (8a, 8k) displayed strong anti-BTK activity with IC50 values lower than
1
0 nM. The phenyl ring of ibrutinib was replaced by three-carbon groups such as
propanyl (8a1), allyl (8a2, 8a3) and propynyl (8a) resulted in moderate to high
potency against BTK, and 8a showed the highest inhibition, with the IC50 value of
7
.95 nM, equivalent to that of ibrutinib. Prolongation of the side chain with one
carbon or removal the oxygen, compounds 8b and 8c gave the reduced activity
approximately 5-fold or 13-fold lower than 8a), which suggested the suitable
(
distance between the distal phenyl and pyrazolopyrimidine was important to keep the
pi-stacking interaction with Phe540 of BTK. Although the replacement of oxygen
with C, N and S (8d-8f) did not lead to better potency, the S-derivative 8f gave a
similar activity to 8a. Meanwhile a range of groups with different substitution
position and electronic nature on the phenyl ring were explored (8g-8o), it seemed
that the m-substituted analogues in this set appeared to be slightly more potent than
the corresponding o-substituted or p-substituted derivatives (8h vs 8g or 8i, 8k vs 8j
or 8l). And the m-substituted electron-rich compound 8k was more potent than the
electron-deficient chloro or trifluoromethyl analogue 8m or 8n. The bulky
(
iso-propyl-substituted) compound 8o exhibited the activity loss compared with
methyl- and methoxyl-substituted compounds 8h and 8k. These results further
supported the hypothesis that the phenyl group formed a face-to-edge pi-stacking
interaction with the Phe540 of BTK.
2
.2.2 Cellular activity assay
To further validate the antiproliferative activity of these compounds, the
molecules with IC50 values below 20 nM were chosen to examine the effect in cancer
cells. Two typical B-cell lymphoblastic leukemia lines (Ramos and Raji), which
overexpressed BTK enzyme were used for the cellular inhibitory activity by CCK-8
assay. The results are depicted in Table 2.
In cell-based assays, most of these molecules showed inhibition on the

proliferations of Ramos or Raji cells at concentrations ranging from 1 to 20 μmol/L.
Compound 8a with the best kinase-based activity, exhibited the strongest inhibitory
activity against both Ramos and Raji cells. The analogues 8d, 8f, 8k and 8m could
remarkably inhibit Raji cell line (IC50 ca. 2 μM). Overall, these biological
explorations provided a new series of 3-substituted pyrazolopyrimidine derivatives to
inhibit B-cell lymphoblastic leukemia cells by targeting BTK.
Table 2
Anti-proliferative activities of compounds against cancer cells.
a
Antiproliferative activity IC50 [μM]
Compound
Ramos
8.91
Raji
1.80
>40
1.62
>40
2.72
1.69
2.28
1.49
8a
8a2
14.63
>40
8d
8e
18.18
37.18
>40
8f
8k
8
m
>40
Ibrutinib
8.26
a
Data represented the mean of at least three separate experiments. Dose-response curves were
determined at five concentrations. The IC50 values are calculated using GraphPad Prism version
.0.
5
Furthermore, in order to evaluate the hydrophobic property of the target
compounds, the ClogP values of compound 8a and ibrutinib were calculated by ACD
labs (Version 6.0). 8a showed an improved hydrophilicity (ClogP = 3.33), compared
to ibrutinib (ClogP = 4.1) and was considered as the good lead candidate for further
evaluation.
2
.3 Molecular modeling calculations
In order to investigate the binding mode of the newly synthesized 3-substituted
pyrazolopyrimidine derivatives with BTK enzyme, the potent BTK inhibitor 8a and
the lead compound ibrutinib were docked into the ATP binding pocket of BTK
enzyme (PDB:5P9J), respectively. Glide and covalent dock programs in Schrodinger
Suite Maestro 10.1 with their default parameters were used. The molecular
simulations were depicted in Fig 4. As predicted, it was found that compound 8a
interacted with the active site in a fashion similar to ibrutinib, several important

interaction forces were produced, including: (1)hydrogen bonds: the primary amine of
pyrazolopyrimidine interacted with the Thr474 hydroxyl and the backbone carbonyl
of Glu475, the N-3 of the pyrimidine ring accepted a hydrogen bond from the
backbone NH of Met477, the carbonyl of acrylamide moiety accepted an H-bond
from the backbone NH of Cys481 (2)pi-stacking: the distal phenyl ring formed a
face-to-edge pi-stacking interaction with the Phe540 of BTK (3)covalent bond: the
acrylamide moiety bounded covalently to the mercapto group of Cys481 of BTK by
Michael addition reaction. Taken together, the docking results provided some possible
explanations for the observed BTK inhibitory activities for the 3-substituted
pyrazolopyrimidines.
Figure 4. Modeled binding mode of inhibitor 8a within BTK (PDB code: 5P9J).
3
. Conclusion
In summary, a new series of 3-substituted pyrazolopyrimidine derivatives were
designed as BTK inhibitors by reasonable structure-based drug design strategy.
Eighteen compounds were synthesized and evaluated. They displayed good inhibitory
activities against BTK in vitro. Compound 8a, with a better hydrophilicity, displayed
a similar activity to ibrutinib both in BTK inhibition and in antitumor activities
against Ramos cells and Raji cells, it could be considered as an ideal lead candidate
for further evaluation and optimization.
4
. Experimental section
4
.1 General methods and chemistry
The starting materials, solvents and reagents were purchased from commercial
suppliers and were used without further purification. Melting points were determined
1
on a Buchi Melting Point M-565 apparatus and uncorrected. H NMR spectra (400

MHz) data were recorded in CDCl
3
or DMSO-d on a Bruker AV400 spectrometer.
6
Internal standard for all NMR spectra was TMS. Mass spectra were recorded with a
Q-TOF mass spectrometer using electrospray positive ionization (ESI+). Specific
rotations were determined on a Rudolph Research Analytical automatic polarimeter IV.
All reactions were monitored by TLC, which were carried out on silica gel GF254.
Column chromatography was carried out on silica gel (200-300 mesh) purchased from
Qindao Ocean Chemical Company of China. Ibrutinib was synthesized according to
2
1
the reference literature .
4
.2 General procedure for the synthesis of compounds 8a-8o and 8a1-8a3
The intermediate 11 and various side chains of alkynes 12a-12o were
2
2-25
synthesized according to the reported methods.
synthesized as described below.
The new compounds were
A mixture of compound 11 (10.4 mmol), 12a-12o (52 mmol), Pd(PPh
3
4
) (0.78
mmol), CuI (2.08 mmol), triethylamine (20.8 mmol) and DMF (40 ml) was stirred
under nitrogen atmosphere at 80℃ for 10h-13h. Then the reaction mixture was
allowed to cool to room temperature and a mixture of ethyl acetate and water was
added, the organic layer was separated and extracted with saturated brines, dried over
Na
2
SO and concentrated under reduced pressure. The residues were purified by silica
4
gel column chromatography eluting at a gradient of 30-50% ethyl acetate in petroleum
ether, giving the crude products 13a-13o, which were used for next step without
purification (Yield of crude: 60%-80%). To a stirred solution of compound 13a-13o
(
10.25 mmol) in ethyl acetate (20 mL) was added 4 M HCl in dioxane (25 mL), and it
was stirred at room temperature for 6-8 h. Then, the solid was collected by filtration
and was dried on vaccum to afford 14a-14o. The solid was directly used for the next
step as HCl salt (Yield: 60%-80%).
A mixture of compound 14a (2.6mmol), 10% Pd/C (400mg) and MeOH (30 ml)
was stirred under hydrogen atmosphere at 55℃ for 24 h. The reaction mixture was
filtered, and the filtrate was concentrated under reduced pressure. The residue 14a1
was used to next step without purification (Yield of crude: 80%).
A mixture of compound 14a (2.3 mmol), 10% Pd-BaSO
30 ml) was stirred under hydrogen atmosphere at 45℃ for 6 h. The reaction mixture
was filtered, and the filtrate was concentrated under reduced pressure. The residue
4
(260 mg) and MeOH
(

1
4a2 was used to next step without purification (Yield of crude: 70%).
To a stirred solution of compound 14a (2.04 mmol) in THF (50 mL) was added
LiAlH
the reaction mixture was poured into ethyl acetate (50 ml), and the solution was
adjusted to pH 9 with 15% NaOH solution and dried over Na SO . The yellow oil
4a3 was collected by filtration and used to next step without purification (Yield of
crude: 60%).
4
(6.12 mmol) at -15℃, and it was stirred at room temperature for 12h. Then,
2
4
1
To a stirred solution of compound 14a-14o, 14a1-14a3 (2 mmol) in DCM (30
mL) was added DIPEA (4mmol) at -10℃, then acryloyl chloride (2mmol) was added
dropwise to the reaction mixture at -10℃ for 15 min. The reaction was quenched
with ice water and washed with 5wt% citric acid (aq.), saturated NaHCO
3
and brine,
dried over NaSO and was evaporated to dryness. The crude material was purified by
4
silica gel column chromatography eluting at a gradient of 0-20% petroleum ether in
ethyl acetate, giving the target compound 8a-8o, 8a1-8a3 (Yield: 40%-75%).
4
.2.1. (R)-1-(3-(4-amino-3-(3-phenoxyprop-1-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-1
-
yl)piperidin-1-yl)prop-2-en-1-one (8a)
2
D
5
1
Yield: 26.3%; White foam; mp: 68.9~71.4℃; [α]
=-140.0° (c=0.1, CH OH); H
3
NMR (400 MHz, CDCl
3
): δ 8.26 (s, 1H), 7.37 (t, J = 8 Hz, 2H), 7.07~7.04 (m, 3H),
.63~6.50 (m, 1H), 6.31~6.26 (m, 1H), 5.94 (brs, 2H), 5.72~5.68 (m, 1H), 5.04 (s,
H), 4.81~4.59 (m, 2H), 4.15~4.01 (dd, J = 12 Hz, J = 44 Hz, 1H), 3.68 (t, J = 12
= 12 Hz, J = 40 Hz, 1H), 2.84 (t, J = 12 Hz, 0.5H),
6
2
1
2
Hz, 0.5H), 3.28~3.12 (dt, J
1
2
2
.31~2.19 (m, 2H), 1.99~1.94 (m, 1H), 1.74~1.65 (m, 1H); MS (ES+) m/z: 403.18
+
[
M+H] .
4
.2.2. (R)-1-(3-(4-amino-3-(3-phenoxypropyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)
piperidin-1-yl)prop-2-en-1-one (8a1)
2
D
5
Yield: 24.9%; Off-white foam; mp: 57.5~60.3℃; [α]
=-130.0° (c=0.1, CH OH);
3
1
H NMR (400 MHz, CDCl
3
): δ 8.27 (s, 1H), 7.33 (t, J = 8 Hz, 2H), 7.01 (t, J = 8 Hz,
H), 6.92~6.90 (m, 2H), 6.64~6.51 (m, 1H), 6.30~6.26 (m, 1H), 6.12 (brs, 2H),
.71~5.63 (m, 1H), 4.81~4.56 (m, 2H), 4.13~4.00 (dd, J = 12 Hz, J = 44 Hz, 1H),
.10 (t, J = 5.6 Hz, 2H), 3.70 (t, J = 12 Hz, 0.5H), 3.31~3.15 (dt, J = 12 Hz, J = 40
1
5
4
1
2
1
2
Hz, 1H), 3.17 (t, J = 8 Hz, 2H), 2.91 (t, J = 12 Hz, 0.5H), 2.29~2.15 (m, 4H),

+
1
.99~1.94 (m, 1H), 1.75~1.64 (m, 1H); MS (ES+) m/z: 407.18 [M+H] .
4
.2.3. (R,Z)-1-(3-(4-amino-3-(3-phenoxyprop-1-en-1-yl)-1H-pyrazolo[3,4-d]pyrimidin
-
1-yl)piperidin-1-yl)prop-2-en-1-one (8a2)
2
D
5
1
Yield: 21.8%; White foam; mp: 69.1~71.2℃; [α]
=-146.0° (c=0.1, CH OH); H
3
NMR (400 MHz, CDCl
3
): δ 8.32 (s, 1H), 7.29 (t, J = 8 Hz, 2H), 6.97 (t, J = 8 Hz, 1H),
.91~6.89 (m, 2H), 6.69~6.65 (dt, J = 2 Hz, J = 12 Hz, 1H), 6.60~6.49 (m, 1H),
.29~6.26 (m, 2H), 5.95 (brs, 2H), 5.72~5.63 (m, 1H), 5.12~5.01 (m, 2H), 4.89~4.81
= 12 Hz, J = 60 Hz, 1H), 3.71
= 60 Hz, 1H), 2.97 (t, J = 12 Hz,
.5H), 2.33~2.25 (m, 2H), 2.04~1.99 (m, 1H), 1.78~1.68 (m, 1H); MS (ES+) m/z:
6
6
1
2
(
(
m, 1.5H), 4.56 (d, J = 12 Hz, 0.5H), 4.18~4.10 (dd, J
1
2
t, J = 12 Hz, 0.5H), 3.37~3.17 (dt, J = 12 Hz, J
1
2
0
4
+
27.15 [M+Na] .
4
.2.4. (R,E)-1-(3-(4-amino-3-(3-phenoxyprop-1-en-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8a3)
Yield: 18.7%; White foam; mp: 69.4~71.9℃; [α]
2
D
5
1
=-144.0° (c=0.1, CH OH); H
3
NMR (400 MHz, CDCl
3
): δ 8.27 (s, 1H), 7.34 (t, J = 8 Hz, 2H), 7.01~6.90 (m, 3H),
.67~6.52 (m, 2H), 6.30~6.04 (m, 2H), 5.72~5.64 (m, 1H), 4.80~4.79 (m, 3H),
.62~4.56 (m, 0.5H), 4.15~4.01 (dd, J = 12 Hz, J = 40 Hz, 1.5H), 3.73 (t, J = 12 Hz,
.5H), 3.31~3.13 (dt, J = 12 Hz, J = 60 Hz, 1H), 2.92 (t, J = 12 Hz, 0.5H),
6
4
0
2
1
2
1
2
.35~2.22 (m, 2H), 2.00~1.96 (m, 1H), 1.74~1.66 (m, 1H); MS (ES+) m/z: 405.24
+
[
M+H] .
4
.2.5. (R)-1-(3-(4-amino-3-(4-phenoxybut-1-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)piperidin-1-yl)prop-2-en-1-one (8b)
2
D
5
Yield: 28.5%; Off-white foam; mp: 62.2~64.9℃; [α]
=-130.0° (c=0.1, CH OH);
3
1
H NMR (400 MHz, CDCl
3
): δ 8.26 (s, 1H), 7.34 (t, J = 8 Hz, 2H), 7.03 (t, J = 8 Hz,
H), 6.95~6.93 (m, 2H), 6.63~6.51 (m, 1H), 6.31~6.27 (m, 1H), 6.30 (brs, 2H),
.72~5.65 (m, 1H), 4.86~4.61 (m, 2H), 4.26 (t, J = 6 Hz, 2H), 4.16~4.01 (dd, J = 12
= 44 Hz, 1H), 3.69 (t, J = 12 Hz, 0.5H), 3.28~3.11 (dt, J = 12 Hz, J = 44 Hz,
H), 3.02 (t, J = 6 Hz, 2H), 2.83 (t, J = 12 Hz, 0.5H), 2.37~2.21 (m, 2H), 1.99~1.94
1
5
1
Hz, J
2
1
2
1
+
(
m, 1H), 1.74~1.65 (m, 1H); MS (ES+) m/z: 417.33 [M+H] .

4
.2.6. (R)-1-(3-(4-amino-3-(3-phenylprop-1-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)piperidin-1-yl)prop-2-en-1-one (8c)
2
D
5
Yield: 27.2%; Light-yellow foam; mp: 73.6~76.4℃; [α]
=-135.0° (c=0.1,
1
CH
3
OH); H NMR (400 MHz, CDCl
3
): δ 8.26 (s, 1H), 7.37 (t, J = 8 Hz, 2H),
7
.07~7.04 (m, 3H), 6.63~6.50 (m, 1H), 6.31~6.26 (m, 1H), 5.94 (brs, 2H), 5.72~5.68
m, 1H), 4.81~4.59 (m, 2H), 4.15~4.01 (dd, J = 12 Hz, J = 44 Hz, 1H), 3.68 (t, J =
= 12 Hz, J = 40 Hz, 1H), 2.84 (t, J = 12
(
1
2
1
2 Hz, 0.5H), 3.42 (s, 2H), 3.28~3.12 (dt, J
1
2
Hz, 0.5H), 2.31~2.19 (m, 2H), 1.99~1.94 (m, 1H), 1.74~1.65 (m, 1H); MS (ES+) m/z:
+
3
87.32 [M+H] .
4
.2.7. (R)-1-(3-(4-amino-3-(4-phenylbut-1-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)
piperidin-1-yl)prop-2-en-1-one (8d)
2
D
5
1
Yield: 26.6%; White foam; mp: 62.7~65.4℃; [α]
=-143.0° (c=0.1, CH OH); H
3
NMR (400 MHz, CDCl
.30~6.26 (m, 1H), 5.71~5.65 (m, 1H), 5.54 (brs, 2H), 4.84~4.62 (m, 2H), 4.15~4.01
dd, J = 12 Hz, J = 44 Hz, 1H), 3.68 (t, J = 12 Hz, 0.5H), 3.27~3.10 (dt, J = 12 Hz,
= 40 Hz, 1H), 3.01 (t, J = 8 Hz, 2H), 2.92 (t, J = 12 Hz, 2H), 2.81 (t, J = 12 Hz,
.5H), 2.36~2.19 (m, 2H), 1.98~1.93 (m, 1H), 1.74~1.64 (m, 1H); MS (ES+) m/z:
3
): δ 8.26 (s, 1H), 7.37~7.25 (m, 5H), 6.63~6.50 (m, 1H),
6
(
1
2
1
J
2
0
4
+
01.26 [M+H] .
4
.2.8. (R)-1-(3-(4-amino-3-(3-(phenylamino)prop-1-yn-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8e)
25
Yield: 30.5%; Light-yellow foam; mp: 117.0~119.4℃; [α] =-135.0° (c=0.1,
D
1
CH
3
OH); H NMR (400 MHz, CDCl
3
): δ 8.24 (s, 1H), 7.29 (t, J = 8 Hz, 2H),
6
.88~6.79 (m, 3H), 6.62~6.50 (m, 1H), 6.30~6.26 (m, 1H), 5.91 (brs, 2H), 5.72~5.64
m, 1H), 4.79~4.59 (m, 2H), 4.29 (s, 2H), 4.03~4.00 (dd, J = 1.6 Hz, J = 10 Hz,
= 40 Hz, 2H), 2.83 (t, J =
(
1
2
0
1
.5H), 3.66 (t, J = 12 Hz, 1H), 3.26~3.10 (dt, J
1
= 12 Hz, J
2
2 Hz, 0.5H), 2.27~2.18 (m, 2H), 1.99~1.93 (m, 1H), 1.73~1.63 (m, 1H); MS (ES+)
+
m/z: 402.30 [M+H] .
4
.2.9. (R)-1-(3-(4-amino-3-(3-(phenylthio)prop-1-yn-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8f)
2
D
5
Yield: 24.2%; Light-yellow foam; mp: 66.1~68.9℃; [α]
=-127.5° (c=0.1,

1
CH
.38~7.28 (m, 3H), 6.64~6.51 (m, 1H), 6.31~6.27 (m, 1H), 6.07~5.65 (m, 1H), 5.54
brs, 2H), 4.83~4.61 (m, 2H), 4.15~4.01 (m, 1H), 3.95 (s, 2H), 3.67 (t, J = 12 Hz,
3
OH); H NMR (400 MHz, CDCl
3
): δ 8.27 (s, 1H), 7.51 (d, J = 8 Hz, 2H),
7
(
0
2
.5H), 3.27~3.10 (dt, J
1
= 12 Hz, J = 40 Hz, 1H), 2.83 (t, J = 12 Hz, 0.5H),
2
.26~2.07 (m, 2H), 1.98~1.93 (m, 1H), 1.74~1.67 (m, 1H); MS (ES+) m/z: 419.0
+
[
M+H] .
4
.2.10. (R)-1-(3-(4-amino-3-(3-(o-tolyloxy)prop-1-yn-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8g)
2
D
5
Yield: 22.9%; Off-white foam; mp: 66.8~69.6℃; [α]
=-148.0° (c=0.1, CH OH);
3
1
H NMR (400 MHz, CDCl
3
): δ 8.27 (s, 1H), 7.21~7.19 (m, 2H), 7.04 (d, J = 8 Hz,
H), 6.97 (t, J = 8 Hz, 1H), 6.63~6.50 (m, 1H), 6.30~6.26 (m, 1H), 5.71~5.65 (m, 3H),
.05 (s, 2H), 4.81~4.61 (m, 2H), 4.15~4.01 (dd, J = 12 Hz, J = 40 Hz, 1H), 3.68 (t, J
12 Hz, 0.5H), 3.28~3.12 (dt, J = 12 Hz, J = 40 Hz, 1H), 2.83 (t, J = 12 Hz, 0.5H),
1
5
1
2
=
1
2
2
.27 (s, 3H), 2.21~2.18 (m, 2H), 1.98~1.95 (m, 1H), 1.74~1.64 (m, 1H); MS (ES+)
+
m/z: 417.25 [M+H] .
4
.2.11. (R)-1-(3-(4-amino-3-(3-(m-tolyloxy)prop-1-yn-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8h)
2
D
5
Yield: 25.8%; Off-white foam; mp: 65.6~68.1℃; [α]
=-128.0° (c=0.1, CH OH);
3
1
H NMR (400 MHz, CDCl
3
): δ 8.27 (s, 1H), 7.25 (t, J = 8 Hz, 1H), 6.87~6.85 (m, 3H),
.67~6.50 (m, 1H), 6.30~6.26 (m, 1H), 5.71~5.68 (m, 3H), 5.02 (s, 2H), 4.81~4.62 (m,
H), 4.16~4.01 (dd, J = 12 Hz, J = 40 Hz, 1H), 3.67 (t, J = 12 Hz, 0.5H), 3.28~3.12
= 12 Hz, J
6
2
1
2
(
dt, J
1
2
= 40 Hz, 1H), 2.84 (t, J = 12 Hz, 0.5H), 2.36 (s, 3H), 2.31~2.20 (m,
+
2
H), 1.99~1.94 (m, 1H), 1.74~1.65 (m, 1H); MS (ES+) m/z: 417.12 [M+H] .
4
.2.12. (R)-1-(3-(4-amino-3-(3-(p-tolyloxy)prop-1-yn-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8i)
2
D
5
Yield: 23.4%; Off-white foam; mp: 72.6~75.1℃; [α]
=-136.0° (c=0.1, CH OH);
3
1
H NMR (400 MHz, CDCl
3
): δ 8.28 (s, 1H), 7.15 (d, J = 8 Hz, 2H), 6.96 (d, J = 8 Hz,
H), 6.63~6.50 (m, 1H), 6.30~6.26 (m, 1H), 5.71~5.65 (m, 3H), 5.00 (s, 2H),
.81~4.61 (m, 2H), 4.16~4.01 (dd, J = 12 Hz, J = 44 Hz, 1H), 3.68 (t, J = 12 Hz,
.5H), 3.28~3.12 (dt, J = 12 Hz, J = 44 Hz, 1H), 2.84 (t, J = 12 Hz, 0.5H), 2.31 (s,
2
4
0
1
2
1
2

3
4
H), 2.29~2.15 (m, 2H), 2.00~1.93 (m, 1H), 1.74~1.65 (m, 1H); MS (ES+) m/z:
+
17.07 [M+H] .
4
.2.13. (R)-1-(3-(4-amino-3-(3-(2-methoxyphenoxy)prop-1-yn-1-yl)-1H-pyrazolo
[
3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8j)
Yield: 22.5%; Off-white foam; mp: 65.7~68.3℃; [α]
2
D
5
=-125.0° (c=0.1, CH OH);
3
1
H NMR (400 MHz, CDCl
3
): δ 8.27 (s, 1H), 7.14~7.12 (m, 1H), 7.07~7.02 (m, 1H),
6
.97~6.93 (m, 2H), 6.63~6.51 (m, 1H), 6.30~6.26 (m, 1H), 5.80 (brs, 2H), 5.72~5.65
(
m, 1H), 5.11 (s, 2H), 4.81~4.61 (m, 2H), 4.16~4.01 (dd, J
1
= 12 Hz, J
2
= 40 Hz, 1H),
= 40 Hz, 1H),
3
2
.90 (s, 3H), 3.67 (t, J = 12 Hz, 0.5H), 3.28~3.11 (dt, J
1
= 12 Hz, J
2
.83 (t, J = 12 Hz, 0.5H), 2.29~2.20 (m, 2H), 1.99~1.94 (m, 1H), 1.74~1.65 (m, 1H);
+
MS (ES+) m/z: 433.48 [M+H] .
4
.2.14. (R)-1-(3-(4-amino-3-(3-(3-methoxyphenoxy)prop-1-yn-1-yl)-1H-pyrazolo
[
3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8k)
2
D
5
Yield: 35.3%; Off-white foam; mp: 66.2~69.0℃; [α]
H NMR (400 MHz, CDCl ): δ 8.29 (s, 1H), 7.26~7.22 (m, 1H), 6.67~6.64 (m, 1H),
.61~6.53 (m, 3H), 6.30~6.26 (m, 1H), 5.72~5.65 (m, 3H), 5.01 (s, 2H), 4.82~4.61 (m,
H), 4.16~4.01 (dd, J = 12 Hz, J = 40 Hz, 1H), 3.81 (s, 3H), 3.68 (t, J = 12 Hz,
.5H), 3.28~3.12 (dt, J = 12 Hz, J = 40 Hz, 1H), 2.84 (t, J = 12 Hz, 0.5H),
=-135.0° (c=0.1, CH
3
OH);
1
3
6
2
0
2
1
2
1
2
.33~2.20 (m, 2H), 2.00~1.94 (m, 1H), 1.74~1.65 (m, 1H); MS (ES+) m/z: 433.43
+
[
M+H] .
4
.2.15. (R)-1-(3-(4-amino-3-(3-(4-methoxyphenoxy)prop-1-yn-1-yl)-1H-pyrazolo
[
3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8l)
Yield: 27.6%; Off-white foam; mp: 67.6~70.3℃; [α]
2
5
D
=-135.0° (c=0.1, CH
= 4 Hz, J = 12 Hz, 2H),
= 12 Hz, 2H), 6.63~6.51 (m, 1H), 6.30~6.26 (m, 1H),
.78 (brs, 2H), 5.72~5.65 (m, 1H), 4.98 (s, 2H), 4.81~4.60 (m, 2H), 4.16~4.01 (dd, J
12 Hz, J = 40 Hz, 1H), 3.78 (s, 3H), 3.68 (t, J = 12 Hz, 0.5H), 3.28~3.12 (dt, J
2 Hz, J
3
OH);
1
H NMR (400 MHz, CDCl
3
): δ 8.28 (s, 1H), 7.02~6.97 (dt, J
1
2
6
5
.90~6.85 (dt, J
1
= 4 Hz, J
2
1
=
2
1
=
1
1
2
= 40 Hz, 1H), 2.84 (t, J = 12 Hz, 0.5H), 2.33~2.19 (m, 2H), 1.99~1.94 (m,
+
H), 1.75~1.65 (m, 1H); MS (ES+) m/z: 433.25 [M+H] .

4
.2.16. (R)-1-(3-(4-amino-3-(3-(3-chlorophenoxy)prop-1-yn-1-yl)-1H-pyrazolo[3,4-d]
pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8m)
2
D
5
Yield: 17.2%; Light-yellow foam; mp: 69.8~71.6℃; [α]
=-123.0° (c=0.1,
1
CH
3
OH); H NMR (400 MHz, CDCl
3
): δ 8.30 (s, 1H), 7.29 (t, J = 8 Hz, 1H),
7
.05~7.02 (m, 2H), 6.96~6.93 (m, 1H), 6.63~6.50 (m, 1H), 6.31~6.27 (m, 1H), 5.86
brs, 2H), 5.72~5.66 (m, 1H), 5.02 (s, 2H), 4.83~4.60 (m, 2H), 4.16~4.01 (dd, J = 12
= 40 Hz,
(
1
Hz, J
2
= 44 Hz, 1H), 3.68 (t, J = 12 Hz, 0.5H), 3.28~3.12 (dt, J
1
= 12 Hz, J
2
1
1
H), 2.85 (t, J = 12 Hz, 0.5H), 2.33~2.19 (m, 2H), 2.00~1.95 (m, 1H), 1.75~1.65 (m,
+
H); MS (ES+) m/z: 437.32 [M+H] .
4
.2.17. (R)-1-(3-(4-amino-3-(3-(3-(trifluoromethyl)phenoxy)prop-1-yn-1-yl)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8n)
2
D
5
Yield: 16.8%; Off-white foam; mp: 68.5~71.1℃; [α]
H NMR (400 MHz, CDCl ): δ 8.30 (s, 1H), 7.49 (t, J = 8 Hz, 1H), 7.32~7.27 (m, 2H),
= 8 Hz, 1H), 6.63~6.50 (m, 1H), 6.31~6.27 (m, 1H), 5.88 (brs,
H), 5.72~5.66 (m, 1H), 5.07 (s, 2H), 4.82~4.60 (m, 2H), 4.17~4.01 (dd, J = 12 Hz,
= 44 Hz, 1H), 3.68 (t, J = 12 Hz, 0.5H), 3.29~3.12 (dt, J = 12 Hz, J = 44 Hz, 1H),
=-130.0° (c=0.1, CH
3
OH);
1
3
7
2
.24 (dd, J
1
= 4 Hz, J
2
1
J
2
1
2
2
.86 (t, J = 12 Hz, 0.5H), 2.33~2.20 (m, 2H), 2.01~1.95 (m, 1H), 1.75~1.65 (m, 1H);
+
MS (ES+) m/z: 471.35 [M+H] .
4
.2.18. (R)-1-(3-(4-amino-3-(3-(3-isopropylphenoxy)prop-1-yn-1-yl)-1H-pyrazolo
[
3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (8o)
2
D
5
Yield: 32.6%; Off-white foam; mp: 59.9~62.7℃; [α]
H NMR (400 MHz, CDCl ): δ 8.28 (s, 1H), 7.29 (t, J = 8 Hz, 1H), 6.93~6.91 (m, 2H),
.89~6.86 (m, 1H), 6.63~6.51 (m, 1H), 6.30~6.26 (m, 1H), 5.72 (brs, 2H), 5.67~5.65
m, 1H), 5.03 (s, 2H), 4.84~4.61 (m, 2H), 4.16~4.02 (dd, J = 12 Hz, J = 44 Hz, 1H),
= 40 Hz, 1H), 2.94~2.85 (m,
=-132.0° (c=0.1, CH
3
OH);
1
3
6
(
1
2
3
1
1
.68 (t, J = 12 Hz, 0.5H), 3.28~3.11 (dt, J
1
= 12 Hz, J
2
H), 2.84 (t, J = 12 Hz, 0.5H), 2.32~2.20 (m, 2H), 2.00~1.94 (m, 1H), 1.74~1.65 (m,
+
H), 1.26 (d, J = 8 Hz, 6H); MS (ES+) m/z: 445.06 [M+H] .
4
4
.3 Biological evaluation
.3.1 In vitro kinase enzymatic assay
The HTRF kinase assay (components supplied as kit by Cisbio) was chosen for

BTK enzyme assays. It uses time resolved fluorescence resonance energy transfer
TR-FRET) to detect production of a phosphorylated substrate. A peptide substrate is
(
labeled with a biotin that can bind to XL665 labeled streptavidin, and the
+
anti-phosphoresidue antibody is labeled with Eu . Upon phosphorylation of the
substrate, the antibody binds to phosphorylated substrate that enables TR-FRET
detection in homogenous assay format. All the reagents used for the BTK kinase
assays including their resources were BTK kinase (Carna), HTRF kinEASE-TK kit
(
Cisbio Bioassays), ATP(Sigma), DTT(Sigma), MgCl
composed of 5 mM MgCl , 1 mM DTT, 10.5 nM SEB, 50 mM HEPES (pH 7.0),
.02% NaN , 0.01% BSA and 0.1 mM orthovanadate. The HTRF assays were
2
(Sigma). The assay buffer was
2
0
3
preformed according to the manual in the kit. The test was performed in a 384-well
plate, all reagents were dispensed into each well plate according to the orders as
follow: (1) each compound as well as control: 0.5 nM-3 μM, 4 μl; (2) reagent: 28.2
μM ATP and 1 μM substrate, 2 μl respectively; (3) BTK enzyme: 0.2 ng/μl, 2 μl; (4)
incubation: ambient, at room temperature, 50 min; (5) reagent: TK antibody and
SA-XL665, 10 μl. Then following 1 h incubation at room temperature. Fluorescence
was measured on the PHERAStar FS microplate reader (BMG Lab Technologies),
signal was expressed in terms of HTRF ratio (fluorescence intensity at 665
nm/fluorescence intensity at 620 nm), curve fitting and data presentations were
performed using GraphPad Prism version 5.0.
4
.3.2 Cellular activity assay
All the cell viability assays were performed according to the CCK-8 method. The
cells were seeded in 96-well plates at a density of 2000 to 3000 cells/well and were
maintained at 37℃ in a 5% CO incubator for 24h. Each compound as well as variant
concentration was added to the culture plates, which were maintained in incubator for
8 h, then 10 μl CCK-8 solution was added to each hole, the culture plates were
2
4
maintained in incubator for another 1h-4h. The number of cells used per experiment
for each cell lines was adjusted to obtain an absorbance of 0.5-1.2 at 450 nm with a
microplate reader. Compounds were tested at appropriate concentrations (1.25-40 μM),
with each concentration duplicated three times. The IC50 values were calculated using
GraphPad Prim version 5.0.
4
.4 Molecular docking study

The published crystal structure (PDB code: 5P9J) of the kinase domain of BTK
bound to ibrutinib was used for the modeling of binding mode of 8a. The protein
preparation was performed in the Protein preparation wizard of Schrodinger Maestro
1
0.1. The waters were eliminated from the protein and the polar hydrogen was added.
Receptor grids were generated using Receptor Grid Generation. Ibrutinib and 8a were
processed by Ligand preparation wizard. The Glide docking and Covalent docking
protocols were used to model potential binding modes between ligands and protein.
The docking results of ibrutinib and 8a were further validated by comparing with the
crystal structure of ibrutinib in the protein. The figures were generated using Pymol.
Conflicts of interest: none
Acknowledgments
This research work was financially supported by Shanghai Rising-Star Program
(
Grant No. 16QB1403900).
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Highlights
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3-Substituted pyrazolopyrimidine derivatives were designed as BTK inhibitors.
They displayed good inhibitory activities.
Compound 8a could inhibit BTK activity at nanomolar concentrations.
Compound 8a exhibited a potent activity against B-cell leukemia lines.

Graphical abstract