Design and synthesis of novel substituted benzyl pyrrolopyrimidine derivatives as selective BTK inhibitors for treating mantle cell lymphoma

Article abstract of DOI:10.1016/j.bioorg.2021.104968

Ibrutinib, a potent irreversible Bruton's tyrosine kinase (BTK) inhibitor, was approved by the FDA for treating mantle cell lymphoma (MCL). Although ibrutinib exhibited excellent antitumor activity, it was associated with certain adverse reactions, with o

Full text of DOI:10.1016/j.bioorg.2021.104968

Bioorganic Chemistry 112 (2021) 104968
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of novel substituted benzyl pyrrolopyrimidine
derivatives as selective BTK inhibitors for treating mantle cell lymphoma
a
,
c
b
a
a
a
a
Fansheng Ran , Yang Liu , Xin Chen , Huijun Zhuo , Changqing Xu , Yuxia Li ,
a
a,*
Xiaoming Duan , Guisen Zhao
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, Jinan 250012, Shandong, PR China
b
Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
c
School of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ibrutinib, a potent irreversible Bruton’s tyrosine kinase (BTK) inhibitor, was approved by the FDA for treating
mantle cell lymphoma (MCL). Although ibrutinib exhibited excellent antitumor activity, it was associated with
certain adverse reactions, with off-target effects against EGFR, Itk and Src family kinases. Our studies yielded a
novel series of substituted benzyl pyrrolopyrimidine derivatives capable of potent inhibition of BTK. Compared
with ibrutinib, compound 15c exhibited potent BTK inhibitory activity and enhanced antiproliferative activity, a
Mantle cell lymphoma
BTK inhibitor
Apoptosis
Antitumor
Selectivity
1
2–24-fold increase, against MCL cell lines, with IC50 values lower than 1
μ
M. Low micromolar doses of 15c
inhibited the BCR signaling pathway and strongly induced the apoptosis of Z138 cells. Ibrutinib and 15c induced
autophagy in a dose-dependent manner in Z138 cells. Moreover, compound 15c induced the production of
reactive oxygen species (ROS), which may be a reason for its potent antiproliferative activity. Importantly,
compound 15c showed greater BTK selectivity than ibrutinib, indicating a potentially safer treatment of MCL.
1
. Introduction
malignancies. BTK is activated by upstream Src family kinases and then
phosphorylates downstream phospholipase-Cγ2 (PLCγ2), leading to
2
+
Mantle cell lymphoma (MCL), a type of non-Hodgkin lymphoma, is
Ca mobilization and activation of the NF-κB and MAP kinase pathways
[4]. These proximal signaling events facilitate the expression of genes
related to cell survival and proliferation. BTK is commonly overex-
pressed in MCL. In recent years, BTK inhibitors have been investigated
as potential treatments for MCL.
characterized by involvement of the lymph nodes, blood, bone marrow
and spleen. The remission duration of standard therapies against MCL is
short, and the median overall survival (OS) is 4–5 years [1]. The mo-
lecular hallmark of MCL is chromosomal translocation t(11;14)(q13;
q32), leading to the overexpression of cyclin D1 [2]. The B-cell receptor
In 2013, ibrutinib (1), a first-generation irreversible BTK inhibitor,
was approved by the FDA to treat MCL, chronic lymphocytic leukemia
(CLL), Waldenstr o¨ m’s macroglobulinemia (WM) and chronic graft-
versus-host disease (cGVHD) [7–10]. However, side effects, including
rash, diarrhea, bleeding and atrial fibrillation, have been reported, and
these are believed to be associated with off-target effects on EGFR and
SRC family proteins [5,11]. Nearly 30% of patients receiving ibrutinib
therapy ultimately discontinue treatment due to related adverse events,
highlighting the importance of developing more-selective BTK inhibitors
[12].
(
BCR) signaling pathway plays a key role in the development of B-cell
malignancies. Constitutive or aberrant activation of the BCR signaling
cascade has been implicated in the proliferation, survival, differentia-
tion, maturation, and apoptosis of B-cell malignancies [3].
Bruton tyrosine kinase (BTK), a cytoplasmic protein tyrosine kinase,
was initially implicated in the pathogenesis of X-linked agammaglobu-
linemia [4]. The BTK gene, which is located on the X chromosome in the
region Xq21.3-22.1, was isolated and identified in 1993 [5]. BTK, with
6
59 amino acid residues, is expressed in all hematopoietic lineage cells
except for T cells [6]. BTK is a key effector of the BCR signaling pathway
and plays significant roles in the initiation and progression of B-cell
Several other BTK inhibitors have also made breakthroughs in clin-
ical studies. Acalabrutinib (2) and zanubrutinib (3), second-generation
*
Corresponding author.
E-mail address: guisenzhao@sdu.edu.cn (G. Zhao).
https://doi.org/10.1016/j.bioorg.2021.104968
Received 26 January 2021; Received in revised form 19 April 2021; Accepted 4 May 2021
Available online 7 May 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
BTK inhibitors, were approved by the FDA for the treatment of MCL in
produced compound 10. Compound 11 reacted with various substituted
benzyl bromides to give compound 12, which reacted with compound
10 via a Suzuki reaction to provide target compound 13. Reduction of
2
017 and 2019, respectively [13–15]. Recently, orelabrutinib (4) was
approved by CFDA for the treatment of MCL and CLL/SLL [16]. More-
over, a considerable number of clinical studies of BTK inhibitors,
including tirabrutinib (5, phase 2) and evobrutinib (6, phase 3), are
ongoing (Fig. 1A) [17,18].
4
the nitro groups on compound 13 in the presence of Fe-NH Cl yielded
compound 14. Following condensation of compounds 14 and acryloyl
chloride or chloroacetyl chloride afforded the final product, compound
It has been reported that the pharmacophore model of BTK inhibitors
consists of large hydrophobic groups, aromatic heterocyclic nuclei,
linkers, and warheads or terminal groups. The aromatic core structure
with a hydrogen bond donor and acceptor occupies the hinge region of
BTK. A large hydrophobic group extending to the hydrophobic pockets
adjacent to the hinge region mediates hydrophobic interactions. The
linker connects the warhead or terminal group extending to Cys481 or
the solvent region in the enzyme (Fig. 1B) [14,19–22]. The 4-phenoxy-
phenyl group and pyridine-2-benzamide group are common hydropho-
bic groups in reported BTK inhibitors. In our previous study, we found
that compound 7 with a benzyl linker showed moderate BTK inhibitory
activity (IC50 = 229 nM) [22]. In this work, we made additional struc-
tural modifications to compound 7. Specifically, we retained the back-
bone of lead compound 7, replaced the 4-phenoxyphenyl hydrophobic
group with a pyridine-2-benzamide group, and introduced different
terminal groups or warheads on the benzyl linker to produce reversible
or irreversible BTK inhibitors for further evaluation of the structur-
e–activity relationship. Herein, a series of substituted benzyl pyrrolo-
pyrimidine derivatives are reported as novel potent BTK inhibitors
15.
2
.2. BTK inhibitory activity
The newly synthesized compounds were evaluated for their activity
against BTK using a kinase profiler radiometric protein kinase assay. The
initial screening was performed at a concentration of 1 M for each
compound. Compounds exhibiting BTK inhibition rates ≥ 80% at 1
were selected for determination of IC50 values. As shown in Table 1,
most compounds exhibited moderate BTK inhibitory activity at 1 M.
μ
μM
μ
Compared with chlorine and bromine substituents, the fluorine sub-
stituent exhibited the best BTK inhibitory activity (13a vs 13b & 13c,
1
3g vs 13h & 13i, 13m vs 13n & 13o). When the R groups were Br, Cl,
NO , or NH , introduction of a trifluoromethyl group at the R position
2
2
1
produced an obvious improvement in BTK inhibition compared to the
introduction of a methyl group and hydrogen (13o vs 13i & 13c, 13n vs
1
1
3h & 13b, 13q vs 13k & 13e, 14i vs 14f & 14c). Analyzing compounds
4g, 14h and 14i, we found that amino substituents at the para-position
produced more-potent BTK inhibitory activity than those at the meta-
position or ortho-position (14i IC50 = 47 ± 5 nM, 14 h IC50 = 87 ± 9 nM,
(
Fig. 1C).
1
4g IC50 = 296 ± 11 nM). Compared to 15b and 15d with a acrylamide
2
. Results and discussion
group, 15a and 15c with a chloroacetamide group exhibited greater
efficacy against BTK, which was consistent with previously reported
findings [22]. Among these new compounds, 15c, an irreversible in-
hibitor, showed the highest BTK inhibitory activity.
2
.1. Chemistry
The synthetic routes for these compounds are illustrated in Scheme
1
. Condensation of various substituted 2-aminopyridine (8) and 4-bro-
2
.3. Antiproliferative activity against MCL cell lines
mobenzoic acid afforded compound 9. Subsequent coupling of com-
pound 9 with bis(pinacolato)diboron catalyzed by palladium acetate
We selected potent BTK inhibitors (IC50 < 100 nM) to investigate
Fig. 1. (A) Representative chemical structures of the novel BTK inhibitors; (B) Common features of BTK inhibitors; (C) Design of target compounds.
2

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
Scheme 1. Synthetic routes of compounds 13, 14 and 15. Reagents and conditions: (a) 4-bromobenzoic acid, HBTU, Et
3
N, DMF, r.t., 12 h; (b) Pd(OAc)
2
, X-PHOS,
◦
◦
KOAc, bis(pinacolato)diboron, 1,4-dioxane, 90 C, 12 h; (c) substituted benzyl bromide, K
2
CO
3
, DMF, r.t., 5 h; (d) Pd(PPh
3
)
4
, K
3
PO
4
, 1,4-dioxane/H
2
O = 4/1, 120 C,
2
4 h; (e) Fe, NH
4
Cl, EtOH/H
2
O = 3/1, 90 ◦C, 6 h; (f) acryloyl chloride or chloroacetyl chloride, Et
3
N, THF, 0 C to r.t., 5 h.
◦
Table 1
The inhibitory effects of series 13, 14 and 15 on BTK activity.
a
a
Code
R
1
R
BTK inhibition (%) at 1
μ
M
BTK IC50 (nM)
Code
R
1
R
BTK inhibition (%) at 1
μ
M
BTK IC50 (nM)
Ibrutinib
–
–
101
67
56
53
49
8
8 ± 0.6
13p
13q
13r
14a
14b
14c
14d
14e
14f
CF
CF
CF
H
3
3
3
2-NO
3-NO
4-NO
2-NH
3-NH
4-NH
2-NH
3-NH
4-NH
2-NH
3-NH
4-NH
2
2
2
2
2
2
2
2
2
2
2
2
91
80
65
44
29
64
63
71
71
81
95
98
99
47
100
75
127 ± 10
444 ± 18
b
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
H
2-F
ND
b
b
H
2-Cl
2-Br
2-NO
3-NO
4-NO
2-F
ND
ND
b
b
H
ND
ND
b
b
H
2
2
2
ND
H
ND
b
b
H
ND
H
ND
b
b
H
32
87
75
72
76
26
48
90
81
82
ND
Me
Me
Me
ND
b
3g
3h
3i
Me
Me
Me
Me
Me
Me
109 ± 8
ND
b
b
2-Cl
2-Br
2-NO
3-NO
4-NO
2-F
ND
ND
b
ND
14g
14h
14i
CF
CF
CF
CF
CF
CF
CF
3
296 ± 11
87 ± 9
47 ± 5
52 ± 3
b
3j
2
2
2
ND
3
3
3
3
3
3
b
3k
3l
ND
b
ND
15a
15b
15c
15d
3-NHCOCH
3-NHCOCHCH
4-NHCOCH Cl
4-NHCOCHCH
2
Cl
b
3m
3n
3o
CF
CF
CF
3
146 ± 5
217 ± 9
200 ± 7
2
2
ND
3
3
2-Cl
2-Br
2
45 ± 1
b
ND
a
b
All IC50 values were obtained by testing in triplicate ND = Not detected
their antiproliferative activity against four MCL cell lines using a Cell
Titer-Glo luminescent cell viability assay. As illustrated in Table 2,
compared with ibrutinib, amino-substituted compounds 14h and 14i
exhibited poor antiproliferative activity in MCL cells, whereas com-
pounds 15a and 15c demonstrated excellent antiproliferative activity in
MCL cell lines. Compound 15c was the most potent antitumor agent of
immunoblotting was performed to assess the phosphorylation of BTK
and its downstream effector PLCγ2 in Z138 cells. As shown in Fig. 2A,
after 24 h of treatment with 15c, phosphorylation of BTK and PLCγ2 was
significantly inhibited, suggesting that 15c strongly inhibits the BTK
signaling pathway.
MCL cells, with IC50 values lower than 1
lower than ibrutinib.
μM, which was 12–24-fold
2
.5. Cell apoptosis assay
We continued to test the effects of 15c on the apoptosis of Z138 cells
2
.4. Inhibitory effects on BTK signaling
To further evaluate the inhibitory effects of 15c on BTK signaling,
using the annexin V-FITC/propidium iodide (PI) binding assay. As
illustrated in Fig. 2B, compound 15c demonstrated strong apoptosis
induction of Z138 cells in a dose-dependent manner. Treating Z138 cells
with 1
cells.
μM 15c for 24 h resulted in the apoptosis of more than 95% of the
Table 2
Cell proliferation assay assessing the effects of target compounds on MCL cell
a
lines.
2
.6. Inducing autophagy in MCL cells
Autophagy is considered a mechanism of cell death induced by death
a
Code
Cell viability assay, IC50
(μM)
Rec-1
Mino
Maver-1
Z138
programs. Muqbil et al. reported that ibrutinib treatment caused auto-
phagic cell death of Hodgkin’s lymphoma cells [23]. In another study,
Wang et al. showed that the antitumor mechanism of ibrutinib involves
the activation of autophagy pathways in a glioblastoma model [24]. We
therefore investigated the induction of autophagy by ibrutinib and 15c
in Z138 cells. As shown in Fig. 2C, the Western blotting results showed
Ibrutinib
8.6 ± 1.2
45.2 ± 5.8
8.6 ± 0.9
1.0 ± 0.08
0.7 ± 0.05
4.9 ± 0.8
6.6 ± 0.6
>60
9.7 ± 0.5
>60
1
1
1
1
4 h
4i
34.7 ± 2.2
16.3 ± 1.7
0.4 ± 0.06
0.4 ± 0.02
>60
40.5 ± 6.1
0.8 ± 0.04
0.4 ± 0.01
5a
5c
0.4 ± 0.03
0.4 ± 0.01
a
All IC50 values were obtained by testing in triplicate.
3

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
Fig. 2. (A) Effect of 15c on BTK signaling in Z138 cells after 24 h of treatment. (B) Apoptosis assay with 15c at the indicated concentrations in Z138 cells treated for
4 h. (C) Autophagy induction by 15c and ibrutinib in Z138 cells. Western blotting results showing expression of LC3B-II and β-actin in Z138 cells treated with
different concentrations of 15c and ibrutinib. Significance is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the DMSO control.
2
that expression of LC3B-II, a critical component in regulating the for-
mation of autophagosomes, significantly increased in a dose-dependent
manner after ibrutinib and 15c treatment of Z138 cells.
2.7. Regulation of reactive oxygen species levels
Reactive oxygen species (ROS) play important roles in the genesis,
progression, and metastasis of tumors. The proliferation, angiogenesis,
Fig. 3. ROS levels in Z138 cells after 12 h or 24 h of treatment with 1 M ibrutinib or 15c. The DCFH-DA probe is hydrolyzed to DCFH in cells, which reacts with
μ
ROS, generating DCF with green fluorescence. The intensity of the green fluorescence reflects the level of ROS.
4

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
and metastasis of cancer cells are promoted under low levels of oxidative
stress. However, high levels of ROS can cause irreversible damage to
lipids and DNA, ultimately resulting in the apoptosis of cancer cells [25].
It has been reported that BTK negatively regulates the production of ROS
and stimulation-induced apoptosis in human neutrophils [26]. Galicia-
Vazquez et al. found that ibrutinib enhanced ROS levels in CLL [27].
We evaluated the effect of ibrutinib and 15c on ROS levels in MCL via a
DCFH-DA probe. As demonstrated in Fig. 3, ibrutinib and 15c enhanced
ROS production in Z138 cells in a time-dependent manner. Compared
with the ibrutinib groups, the 15c groups showed higher ROS levels at
low micromolar concentrations, which may be a reason for their higher
antiproliferative activity.
activity against MCL cell lines, with IC50 values lower than 1 M, sup-
μ
porting further evaluation and development of the promising candidate
15c in cancer therapy.
4. Experimental section
4.1. Chemistry
All chemical reagents and solvents were purchased from commercial
sources and used without further purification. Reactions were moni-
tored 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 a Büchi capillary melting point apparatus (Buchi
Labortechnik AG, Switzerland) without correction. Using tetrame-
2
.8. Selectivity compared with other kinases
6 3
thylsilane (TMS) as an internal standard in DMSO‑d or CDCl , NMR
We further tested the kinase selectivity of 15c against a panel of 20
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) are reported in parts per million (ppm) using tetramethylsilane
as an internal standard.
kinases. As shown in Table 3, ibrutinib exhibited potent inhibitory ac-
tivity against 19 kinases, showing poor selectivity. Compound 15c
exhibited no inhibitory activity against 16 kinases, including EGFR, Itk,
and Src family kinases. Ibrutinib, which inhibits EGFR and Src family
kinases, can induce gastrointestinal adverse effects, dramatic cutaneous
toxicity and high bleeding risk [11]. Moreover, ibrutinib inhibited Itk in
NK cells, which could reduce the effect of rituximab-induced antibody-
dependent cell-mediated cytotoxicity (ADCC), which would be a po-
tential drawback in combination regimens [28–29]. Compound 15c
exhibited greater BTK selectivity than ibrutinib and had the potential for
better safety in the treatment of MCL.
4
4
.2. General synthesis of compounds
.2.1. Preparation of compound 9
A mixture of 4-bromobenzoic acid (10 mmol), HBTU (12 mmol) and
triethylamine (30 mmol) in DMF (30 mL) was stirred under ice-cold
conditions for 30 min. Then, compound 8 (10 mmol) was added, and
the mixture was stirred at room temperature for 12 h. Upon completion,
the reaction solution was poured into water (300 mL), and the suspen-
sion was filtered to give compound 9.
2
.9. Molecular modeling
The binding mode of compound 15c with BTK (PDB ID: 5fbo) as
4
.2.2. Preparation of compound 10
determined by molecular docking analysis is shown in Fig. 4. The pyr-
azolopyrimidine core occupies the hinge region, forming several
important hydrogen bonds with Glu475 and Met477. The hydrophobic
group extends to the hydrophobic pocket behind the gatekeeper,
Thr474, forming an edge-to-face aromatic interaction with Phe540.
Furthermore, the acylamide in the hydrophobic group forms a hydrogen
bond with the carbonyl of Ser538, which may be the essential interac-
tion for the kinase selectivity of 15c.
2
A premixed and degassed solution of Pd(OAc) (0.23 mmol) and X-
PHOS (0.46 mmol) in dioxane (5 mL) that had been stirred for 20 min
was added to a stirred, degassed mixture of bis(pinacolato)diboron (9.2
mmol), potassium acetate (13.8 mmol) and compound 9 (4.6 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
(
dichloromethane/methanol, 200/1–60/1) to afford intermediate 10.
3
. Conclusion
4
.2.3. Preparation of compound 12
Substituted benzyl bromide (1.2 mmol) was added to a mixture of
In this study, we described the design, synthesis and results of bio-
2 3
compound 11 (1 mmol) and K CO (1.5 mmol) in DMF (5 mL), and the
logical evaluations of novel substituted benzyl pyrrolopyrimidine de-
rivatives as potential agents for treating MCL. Among these compounds,
reaction mixture was stirred for 5 h at room temperature. The reaction
solution was poured into water (60 mL). The suspension was filtered to
yield compound 12.
1
5c showed potent BTK inhibitory activity and high enzyme selectivity.
In kinase screening with a panel of 20 kinases, 15c demonstrated a
promising selectivity profile toward 16 kinases. Compound 15c induced
a high apoptosis rate of Z138 cells in a dose-dependent manner at low
micromolar concentrations. Compound 15c also enhanced ROS pro-
duction in Z138 cells in a time-dependent manner. Compared with
ibrutinib, 15c exerted 12- to 24-fold improvement in antiproliferative
4
.2.4. Preparation of compound 13
Pd(PPh (0.05 mmol) was added to a mixture of compound 12 (1.2
mmol), compound 10 (1 mmol) and K PO (2 mmol) in 4/1 dioxane/
3 4
)
3
4
water mixture (15 mL). The mixture was degassed and placed under a
◦
nitrogen atmosphere and then stirred at 120 C for 24 h. After cooling to
room temperature, the mixture was extracted using ethyl acetate and
water. The organic layer was separated, and the aqueous layer was
extracted. The combined organic phases were washed with brine, dried
and concentrated. The crude product was purified by silica gel column
chromatography (dichloromethane/methanol, 150/1–50/1) to afford
target compound 13.
Table 3
Inhibition efficacy (%) among different kinases at 1 M by 15c and ibrutinib.
μ
Kinases
15c
Ibrutinib
Kinases
15c
Ibrutinib
Blk
98
94
37
ꢀ 9
26
37
72
13
20
ꢀ 4
100
102
92
Itk
29
48
32
8
86
Bmx
Brk
Jak3
Lck
Lyn
Ptk5
Src
54
99
4
-(4-amino-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
Csk
87
99
yl)-N-(pyridin-2-yl)benzamide (13a)
EGFR
ErbB2
ErbB4
Fgr
98
16
12
27
84
85
8
91
White solid, yield 67%, Mp: 204–207 ℃, 1H NMR (400 MHz,
73
96
101
92
Srm
Tec
Txk
Yes
100
103
97
DMSO‑d
6
) δ 10.90 (s, 1H, NH), 8.41 (d, J = 3.9 Hz, 1H, Ar-H), 8.33 (s,
1H, Ar-H), 8.21 (t, J = 9.0 Hz, 3H, Ar-H), 7.86 (t, J = 7.7 Hz, 1H, Ar-H),
.79 (d, J = 8.1 Hz, 2H, Ar-H), 7.44–6.97 (m, 7H, Ar-H and NH ), 5.65
) δ 166.02, 160.34 (d, J =
Fyn
97
7
2
Hck
96
102
13
(
2 6
s, 2H, CH ). C NMR (100 MHz, DMSO‑d
5

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
Fig. 4. Molecular docking mode of 15c with BTK. (PDB ID: 5fbo).
2
1
1
1
1
45 Hz, 1C), 158.69, 156.61, 155.13, 152.61, 148.46, 143.96, 138.64,
36.38, 134.33, 130.66 (d, J = 3 Hz, 1C), 130.35 (d, J = 8 Hz, 1C),
29.31, 128.61, 125.14 (d, J = 3 Hz, 1C), 124.22 (d, J = 15 Hz, 1C),
20.38, 115.94 (d, J = 21 Hz, 1C), 115.26, 97.88, 44.10 (d, J = 4 Hz,
White solid, yield 69%, Mp: 225–228 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 10.86 (s, 1H, NH), 8.41 (d, J = 3.9 Hz, 1H, Ar-H), 8.34 (s,
1H, Ar-H), 8.23–8.20 (m, 4H, Ar-H), 8.16 (d, J = 8.1 Hz, 1H, Ar-H), 7.86
(t, J = 8.7 Hz, 1H, Ar-H), 7.81 (d, J = 8.3 Hz, 2H, Ar-H), 7.75 (d, J = 7.7
Hz, 1H, Ar-H), 7.65 (t, J = 7.9 Hz, 1H, Ar-H), 7.54 – 6.49 (m, 3H, Ar-H
+
C). MS(ESI) m/z: 440.27 [M + H]
1
3
4
-(4-amino-1-(2-chlorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
2 2 6
and NH ), 5.77 (s, 2H, CH ). C NMR (100 MHz, DMSO‑d ) δ 166.01,
3
-yl)-N-(pyridin-2-yl) benzamide (13b)
158.75, 156.77, 155.19, 152.61, 148.46, 148.32, 144.25, 139.68,
White solid, yield 70%, Mp: 224–226 ℃, 1H NMR (400 MHz,
138.64, 136.29, 134.78, 134.40, 130.80, 129.33, 128.62, 123.17,
+
DMSO‑d
6
) δ 10.90 (s, 1H, NH), 8.42 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H),
122.80, 120.39, 115.26, 97.98, 49.52. MS(ESI) m/z: 467.13 [M+H] .
8
.24–8.20 (m, 3H, Ar-H), 7.92–7.76 (m, 3H, Ar-H), 7.64–6.75 (m, 7H,
4-(4-amino-1-(4-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(pyridin-2-yl)benzamide (13f)
1
3
2 2 6
Ar-H and NH ), 5.69 (s, 2H, CH ). C NMR (100 MHz, DMSO‑d ) δ
1
1
1
66.01, 158.74, 156.65, 155.37, 152.62, 148.46, 144.14, 138.64,
White solid, yield 68%, Mp: 247–249 ℃, 1H NMR (400 MHz,
36.37, 134.78, 134.35, 132.38, 129.96, 129.91, 129.85, 129.32,
DMSO‑d
6
) δ 10.86 (s, 1H, NH), 8.41 (d, J = 4.6 Hz, 1H, Ar-H), 8.32 (s,
28.62, 128.01, 120.38, 115.26, 97.90, 47.92. MS(ESI) m/z: 456.14
1H, Ar-H), 8.28 – 8.18 (m, 5H, Ar-H), 7.90 – 7.79 (m, 3H, Ar-H), 7.54 (d,
J = 8.6 Hz, 2H, Ar-H), 7.42 – 6.49 (m, 3H, Ar-H and NH ), 5.76 (s, 2H,
) δ 166.01, 158.74, 156.75, 155.24,
+
[
M+H] .
2
1
3
4
-(4-amino-1-(2-bromobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
2 6
CH ). C NMR (100 MHz, DMSO‑d
3
-yl)-N-(pyridin-2-yl) benzamide (13c)
152.61, 148.46, 147.42, 145.08, 144.28, 138.64, 136.28, 134.39,
White solid, yield 61%, Mp: 240–242 ℃, 1H NMR (400 MHz,
129.32, 129.17, 128.63, 124.32, 120.39, 115.26, 97.97, 49.72. MS(ESI)
+
DMSO‑d
6
) δ 10.90 (s, 1H, NH), 8.41 (d, J = 4.5 Hz, 1H, Ar-H), 8.31 (s,
m/z: 467.11 [M+H] .
1
H, Ar-H), 8.24–8.20 (m, 3H, Ar-H), 7.89–7.84 (m, 1H, Ar-H), 7.81 (d, J
4-(4-amino-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-methylpyridin-2-yl) benzamide (13g)
=
8.2 Hz, 2H, Ar-H), 7.68 (d, J = 7.8 Hz, 1H, Ar-H), 7.42–7.13 (m, 4H,
13
White solid, yield 71%, Mp: 212–215 ℃, 1H NMR (400 MHz,
Ar-H and NH
2
), 7.12–6.69 (m, 2H, Ar-H and NH
) δ 166.02, 158.74, 156.66, 155.41, 152.61,
48.46, 144.18, 138.64, 136.37, 134.37, 133.17, 130.16, 129.68,
2
), 5.66 (s, 2H, CH
2
).
C
NMR (100 MHz, DMSO‑d
6
DMSO‑d
6
) δ 10.80 (s, 1H, NH), 8.32 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
1
1
1H, Ar-H), 8.19 (d, J = 8.2 Hz, 2H, Ar-H), 8.08 (s, 1H, Ar-H), 7.78 (d, J =
8.2 Hz, 2H, Ar-H), 7.37 (q, J = 6.1 Hz, 1H, Ar-H), 7.26–7.14 (m, 4H, Ar-
29.31, 128.63, 128.55, 122.45, 120.39, 115.26, 97.92, 50.34. MS(ESI)
+
m/z: 500.22 [M+H] .
H and NH
2
), 7.11–6.63 (m, 2H, Ar-H and NH
3 6
2
), 5.65 (s, 2H, CH
2
), 2.37
1
3
4
-(4-amino-1-(2-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
(s, 3H, CH ). C NMR (100 MHz, DMSO‑d
) δ 165.92, 160.34 (d, J =
yl)-N-(pyridin-2-yl)benzamide (13d)
245 Hz, 1C), 158.69, 156.60, 155.13, 152.65, 149.33, 148.07, 143.97,
136.34, 134.39, 130.66 (d, J = 4 Hz, 1C), 130.44 (d, J = 8 Hz, 1C),
129.25, 128.60, 125.14 (d, J = 3 Hz, 1C), 124.23 (d, J = 15 Hz, 1C),
121.40, 115.94 (d, J = 21 Hz, 1C), 115.64, 97.87, 44.10 (d, J = 4 Hz,
White solid, yield 59%, Mp: 236–238 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 10.85 (s, 1H, NH), 8.41 (d, J = 3.9 Hz, 1H, Ar-H), 8.29 (s,
1
H, Ar-H), 8.26–8.18 (m, 3H, Ar-H), 8.13 (d, J = 8.0 Hz, 1H, Ar-H), 7.86
t, J = 8.7 Hz, 1H, Ar-H), 7.79 (d, J = 8.2 Hz, 2H, Ar-H), 7.68 (t, J = 7.4
Hz, 1H, Ar-H), 7.59 (t, J = 7.6 Hz, 1H, Ar-H), 7.47–6.59 (m, 4H, Ar-H
+
(
1C), 21.42. MS(ESI) m/z: 454.10 [M+H] .
4-(4-amino-1-(2-chlorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
3-yl)-N-(4-methylpyridin-2-yl) benzamide (13h)
1
3
and NH
2
), 5.97 (s, 2H, CH
2 6
). C NMR (100 MHz, DMSO‑d ) δ 166.01,
1
1
1
58.77, 156.74, 155.54, 152.62, 148.45, 148.18, 144.40, 138.63,
White solid, yield 73%, Mp: 226–228 ℃, 1H NMR (400 MHz,
36.27, 134.65, 134.43, 132.24, 129.86, 129.57, 129.32, 128.62,
DMSO‑d
6
) δ 10.80 (s, 1H, NH), 8.31 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
+
25.46, 120.38, 115.26, 97.98, 47.42. MS(ESI) m/z: 467.05 [M+H] .
1H, Ar-H), 8.19 (d, J = 8.3 Hz, 2H, Ar-H), 8.09 (s, 1H, Ar-H), 7.79 (d, J =
8.3 Hz, 2H, Ar-H), 7.52 (d, J = 7.8 Hz, 1H, Ar-H), 7.40–7.26 (m, 3H, Ar-
4
-(4-amino-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(pyridin-2-yl)benzamide (13e)
H and NH
2
), 7.04–7.01 (m, 3H, Ar-H and NH
2 2
), 5.69 (s, 2H, CH ), 2.37
6

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
(
s, 3H, CH
3
). 13C NMR (100 MHz, DMSO‑d
55.37, 152.65, 149.33, 148.08, 144.14, 136.32, 134.78, 134.42,
6
) δ 165.92, 158.73, 156.65,
1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.22 (d, J = 8.2 Hz, 2H, Ar-H), 7.82 (d, J =
8.2 Hz, 2H, Ar-H), 7.56 (d, J = 4.4 Hz, 1H, Ar-H), 7.51 (d, J = 7.8 Hz, 1H,
1
1
1
32.38, 129.96, 129.92, 129.86, 129.26, 128.62, 128.01, 121.40,
Ar-H), 7.44–7.15 (m, 3H, Ar-H and NH ), 7.12–6.66 (m, 2H, Ar-H and
2
+
13
15.64, 97.89, 47.92, 21.43. MS(ESI) m/z: 470.14 [M+H] .
2 2 6
NH ), 5.69 (s, 2H, CH ). C NMR (100 MHz, DMSO‑d ) δ 166.68,
4
-(4-amino-1-(2-bromobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
158.70, 156.60, 155.37, 153.72, 150.42, 144.08, 138.82 (q, J = 31 Hz,
1C), 136.71, 134.76, 133.83, 132.39, 129.97, 129.92, 129.88, 129.47,
128.68, 128.01, 123.43 (q, J = 272 Hz, 1C), 115.70 (q, J = 3 Hz, 1C),
3
-yl)-N-(4-methylpyridin-2-yl) benzamide (13i)
White solid, yield 66%, Mp: 224 – 226 ℃, 1H NMR (400 MHz,
+
DMSO‑d
6
) δ 10.80 (s, 1H), 8.30 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz, 1H, Ar-
110.30 (q, J = 4 Hz, 1C), 97.92, 47.94. MS(ESI) m/z: 524.08 [M+H] .
H), 8.19 (d, J = 8.3 Hz, 2H, Ar-H), 8.08 (s, 1H, Ar-H), 7.80 (d, J = 8.3 Hz,
H, Ar-H), 7.68 (d, J = 7.8 Hz, 1H, Ar-H), 7.41 – 7.22 (m, 3H, Ar-H and
NH ), 5.66 (s, 2H, CH ), 2.37 (s, 3H,
), 7.09 – 6.88 (m, 3H, Ar-H and NH
CH ) δ 170.82, 165.91, 158.72, 156.64,
4-(4-amino-1-(2-bromobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
3-yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (13o)
Yellow solid, yield 65%, Mp: 218–220 ℃, 1H NMR (400 MHz,
2
2
2
2
1
3
3
). C NMR (100 MHz, DMSO‑d
6
DMSO‑d
6
) δ 11.41 (s, 1H, NH), 8.71 (d, J = 5.1 Hz, 1H, Ar-H), 8.58 (s,
1
1
5
55.39, 149.33, 148.07, 144.17, 136.36, 136.30, 134.41, 133.16,
30.17, 129.68, 129.25, 128.62, 128.56, 122.44, 121.40, 115.63, 97.90,
1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.22 (d, J = 8.2 Hz, 2H, Ar-H), 7.83 (d, J =
8.2 Hz, 2H, Ar-H), 7.69 (d, J = 7.8 Hz, 1H, Ar-H), 7.57 (d, J = 5.1 Hz, 1H,
+
13
0.33, 21.43. MS(ESI) m/z: 513.97 [M+H] .
Ar-H), 7.39–6.63 (m, 5H, Ar-H and NH
2 2
), 5.67 (s, 2H, CH ). C NMR
4
-(4-amino-1-(2-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
6
(100 MHz, DMSO‑d ) δ 166.68, 158.74, 156.66, 155.42, 153.71, 150.41,
yl)-N-(4-methylpyridin-2-yl) benzamide (13j)
144.11, 138.81 (q, J = 33 Hz, 1C), 136.71, 136.35, 133.83, 133.17,
130.17, 129.69, 129.47, 128.68, 128.55, 123.42 (q, J = 272 Hz, 1C),
122.46, 115.68 (q, J = 4 Hz, 1C), 110.30 (q, J = 4 Hz, 1C), 97.93, 50.35.
White solid, yield 72%, Mp: 228–231 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 10.80 (s, 1H, NH), 8.29 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
+
1
8
H, Ar-H), 8.20 (d, J = 8.3 Hz, 2H, Ar-H), 8.14 (d, J = 7.7 Hz, 1H, Ar-H),
.09 (s, 1H, Ar-H), 7.79 (d, J = 8.2 Hz, 2H, Ar-H), 7.68 (t, J = 7.3 Hz, 1H,
),
) δ
MS(ESI) m/z: 568.02 [M+H] .
-(4-amino-1-(2-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (13p)
White solid, yield 55%, Mp: 105–108 ℃, 1H NMR (400 MHz,
Ar-H), 7.59 (t, J = 7.5 Hz, 1H, Ar-H), 7.41–6.52 (m, 4H, Ar-H and NH
2
1
3
5
1
1
1
4
2 3 6
.97 (s, 2H, CH ), 2.37 (s, 3H, CH ). C NMR (100 MHz, DMSO‑d
65.91, 158.76, 156.74, 155.53, 152.66, 149.32, 148.18, 148.07,
44.40, 136.22, 134.66, 134.48, 132.23, 129.85, 129.57, 129.26,
28.61, 125.46, 121.39, 115.63, 97.95, 47.40, 21.42. MS(ESI) m/z:
DMSO‑d
6
) δ 11.39 (s, 1H, NH), 8.70 (d, J = 5.0 Hz, 1H, Ar-H), 8.58 (s,
1H, Ar-H), 8.30 (s, 1H, Ar-H), 8.23 (d, J = 7.8 Hz, 2H, Ar-H), 8.14 (d, J =
8.1 Hz, 1H, Ar-H), 7.82 (d, J = 7.8 Hz, 2H, Ar-H), 7.68 (t, J = 7.5 Hz, 1H,
+
13
81.15 [M+H] .
Ar-H), 7.61–6.52 (m, 5H, Ar-H and NH
2 2
), 5.98 (s, 2H, CH ). C NMR
4
-(4-amino-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
6
(100 MHz, DMSO‑d ) δ 166.66, 158.76, 156.74, 155.55, 153.72, 150.39,
yl)-N-(4-methylpyridin-2-yl) benzamide (13k)
148.18, 144.32, 138.82 (q, J = 33 Hz, 1C), 136.62, 134.64, 133.89,
132.21, 129.85, 129.57, 129.47, 128.66, 125.45, 123.42 (q, J = 272 Hz,
1C), 115.66 (q, J = 3 Hz, 1C), 110.29 (q, J = 4 Hz, 1C), 97.98, 47.42. MS
White solid, yield 72%, Mp: 210–213 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 10.76 (s, 1H, NH), 8.34 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
ꢀ
1
H, Ar-H), 8.21–8.15 (m, 4H, Ar-H), 8.08 (s, 1H, Ar-H), 7.80 (d, J = 8.3
Hz, 2H, Ar-H), 7.75 (d, J = 7.7 Hz, 1H, Ar-H), 7.65 (t, J = 7.9 Hz, 1H, Ar-
H), 7.50 – 6.36 (m, 3H, Ar-H and NH ), 5.77 (s, 2H, CH ), 2.37 (s, 3H,
) δ 165.91, 158.75, 156.76, 155.19,
(ESI) m/z: 533.23 [Mꢀ H] .
4-(4-amino-1-(3-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (13q)
White solid, yield 69%, Mp: 248–251 ℃, 1H NMR (400 MHz,
2
2
1
3
3 6
CH ). C NMR (100 MHz, DMSO‑d
1
1
4
52.65, 149.32, 148.32, 148.07, 144.25, 139.68, 136.24, 134.78,
DMSO‑d
6
) δ 11.38 (s, 1H, NH), 8.69 (d, J = 4.7 Hz, 1H, Ar-H), 8.57 (s,
34.46, 130.79, 129.27, 128.62, 123.16, 122.80, 121.39, 115.64, 97.98,
1H, Ar-H), 8.34 (s, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.22 (s, 2H, Ar-H), 8.15
(d, J = 7.9 Hz, 1H, Ar-H), 7.83 (d, J = 7.8 Hz, 2H, Ar-H), 7.74 (d, J = 7.4
Hz, 1H, Ar-H), 7.64 (t, J = 7.8 Hz, 1H, Ar-H), 7.54 (d, J = 4.4 Hz, 1H, Ar-
+
9.52, 21.42. MS(ESI) m/z: 481.11 [M+H] .
4
-(4-amino-1-(4-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
1
3
yl)-N-(4-methylpyridin-2-yl) benzamide (13l)
H), 7.44–6.20 (s, 2H, NH
2 2
), 5.77 (s, 2H, CH ). C NMR (100 MHz,
White solid, yield 71%, Mp: 222–225 ℃, 1H NMR (400 MHz,
DMSO‑d ) δ 166.64, 158.75, 156.75, 155.21, 153.71, 150.35, 148.30,
6
DMSO‑d
6
) δ 10.80 (s, 1H, NH), 8.32 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
144.18, 139.64, 138.81 (q, J = 33 Hz, 1C), 136.64, 134.75, 133.87,
130.74, 129.48, 128.66, 123.41 (q, J = 271 Hz, 1C), 123.12, 122.80,
115.62 (q, J = 3 Hz, 1C), 110.29 (q, J = 4 Hz, 1C), 98.01, 49.54. MS(ESI)
1
H, Ar-H), 8.22–8.19 (m, 4H, Ar-H), 8.08 (s, 1H, Ar-H), 7.80 (d, J = 8.2
Hz, 2H, Ar-H), 7.70–6.29 (m, 5H, Ar-H and NH ), 5.76 (s, 2H, CH ), 2.37
) δ 165.91, 158.74, 156.75,
55.24, 152.64, 149.33, 148.07, 147.42, 145.07, 144.29, 136.24,
2
2
1
3
ꢀ
(
s, 3H, CH
3
). C NMR (100 MHz, DMSO‑d
6
m/z: 533.40 [Mꢀ H] .
1
1
2
4-(4-amino-1-(4-nitrobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (13r)
34.45, 129.26, 129.16, 128.63, 124.31, 121.40, 115.64, 97.97, 49.71,
+
White solid, yield 68%, Mp: 224–227 ℃, 1H NMR (400 MHz,
1.42. MS(ESI) m/z: 481.18 [M+H] .
4
-(4-amino-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
DMSO‑d
6
) δ 11.40 (s, 1H, NH), 8.64 (d, J = 47.2 Hz, 2H, Ar-H),
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (13m)
8.33–8.22 (m, 6H, Ar-H), 7.84 (s, 2H, Ar-H), 7.55 (s, 3H, Ar-H and
White solid, yield 73%, Mp: 248–250 ℃, 1H NMR (400 MHz,
NH
), 7.05 (br, s, 1H, NH
) δ 166.65, 158.75, 156.75, 155.27, 153.72, 150.37, 147.43,
13
2
2 2
), 5.77 (s, 2H, CH ). C NMR (100 MHz,
DMSO‑d
6
) δ 11.41 (s, 1H, NH), 8.70 (d, J = 5.1 Hz, 1H, Ar-H), 8.58 (s,
DMSO‑d
6
1
H, Ar-H), 8.32 (s, 1H, Ar-H), 8.22 (d, J = 8.3 Hz, 2H, Ar-H), 7.81 (d, J =
145.04, 144.22, 138.82 (q, J = 33 Hz, 1C), 136.65, 133.86, 129.47,
129.16, 128.68, 124.29, 123.42 (q, J = 272 Hz, 1C), 115.64 (q, J = 3 Hz,
1C), 110.30 (q, J = 5 Hz, 1C), 98.01, 49.73. MS(ESI) m/z: 533.57
8
.3 Hz, 2H, Ar-H), 7.57 (d, J = 5.0 Hz, 1H, Ar-H), 7.40–7.34 (m, 1H, Ar-
13
H), 7.32–6.73 (m, 5H, Ar-H and NH
MHz, DMSO‑d
2 2
), 5.65 (s, 2H, CH ). C NMR (100
ꢀ
6
) δ 166.67, 160.34 (d, J = 244 Hz, 1C), 158.70, 156.61,
[Mꢀ H] .
1
1
1
55.16, 153.72, 150.39, 143.89, 138.81 (q, J = 33 Hz, 1C), 136.74,
33.80, 130.65 (d, J = 4 Hz, 1C), 130.41 (d, J = 10 Hz, 1C), 129.46,
28.65, 125.12 (d, J = 4 Hz, 1C), 124.27 (d, J = 4 Hz, 1C), 123.43 (q, J
4.2.5. Preparation of compounds 14
Compound 13 with a nitro group (1.00 mmol) was added to a
=
272 Hz, 1C), 115.93 (d, J = 21 Hz, 1C), 115.66 (q, J = 3 Hz, 1C),
mixture of Fe (3 mmol) and NH
4
Cl (3 mmol) in 3/1 ethanol/water
◦
1
5
10.30 (q, J = 4 Hz, 1C), 97.91, 44.11 (d, J = 4 Hz, 1C). MS(ESI) m/z:
mixture (20 mL). Then, the mixture was stirred at 90 C for 6 h. The
reaction mixture was hot filtered, and the filtrate was concentrated
under reduced pressure. The crude product was purified by silica gel
column chromatography (dichloromethane/methanol, 120/1–30/1) to
afford target compound 14.
+
08.09 [M+H] .
4
-(4-amino-1-(2-chlorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
3
-yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (13n)
Yellow solid, yield 71%, Mp: 210–212 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 11.40 (s, 1H, NH), 8.70 (d, J = 5.1 Hz, 1H, Ar-H), 8.58 (s,
4-(4-amino-1-(2-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-
7

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
3
-yl)-N-(pyridin-2-yl)benzamide (14a)
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (14g)
White solid, yield 69%, Mp: 240–243 ℃, 1H NMR (400 MHz,
DMSO‑d
White solid, yield 63%, Mp: 250–253 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 10.90 (s, 1H, NH), 8.41 (d, J = 4.1 Hz, 1H, Ar-H), 8.33 (s,
6
) δ 11.40 (s, 1H, NH), 8.70 (d, J = 5.1 Hz, 1H, Ar-H), 8.58 (s,
1
H, Ar-H), 8.24–8.20 (m, 3H, Ar-H), 7.86 (t, J = 7.3 Hz, 1H, Ar-H), 7.80
), 5.42 (s, 2H,
). C NMR (100 MHz, DMSO‑d ) δ 166.02,
1H, Ar-H), 8.34 (s, 1H, Ar-H), 8.23 (d, J = 7.8 Hz, 2H, Ar-H), 7.82 (d, J =
(
d, J = 8.1 Hz, 2H, Ar-H), 7.34–6.44 (m, 7H, Ar-H and NH
2
7.9 Hz, 2H, Ar-H), 7.56 (d, J = 5.0 Hz, 1H, Ar-H), 7.45–6.32 (m, 6H, Ar-
1
3
13
CH
2
), 5.39 (s, 2H, NH
2
6
H and NH
2 2 2
), 5.42 (s, 2H, CH ), 5.40 (s, 2H, NH ). C NMR (100 MHz,
1
1
1
58.74, 156.49, 154.69, 152.62, 148.46, 146.90, 143.54, 138.63,
DMSO‑d ) δ 166.69, 158.75, 156.49, 154.71, 153.73, 150.41, 146.90,
6
36.40, 134.31, 130.21, 129.32, 129.22, 128.57, 120.52, 120.38,
143.47, 138.81 (q, J = 33 Hz, 1C), 136.76, 133.78, 130.21, 129.48,
129.22, 128.62, 123.43 (q, J = 272 Hz, 1C), 120.52, 116.70, 116.70,
16.70, 115.74, 115.27, 97.78, 47.63. HRMS(ESI) m/z calcd for
+
C
24
H
20
N
8
O [M + H] 437.1833, found 437.1833.
115.75, 115.67 (q, J = 3 Hz, 1C), 110.31 (q, J = 4 Hz, 1C), 97.80, 47.64.
+
4
-(4-amino-1-(3-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
HRMS(ESI) m/z calcd for C25
H
19
F N
3 8
O [M + H] 505.1707, found
yl)-N-(pyridin-2-yl)benzamide (14b)
505.1725.
White solid, yield 61%, Mp: 226–229 ℃, 1H NMR (400 MHz,
4-(4-amino-1-(3-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (14h)
DMSO‑d
6
) δ 10.89 (s, 1H, NH), 8.41 (d, J = 3.9 Hz, 1H, Ar-H), 8.31 (s,
1
H, Ar-H), 8.24–8.20 (m, 3H, Ar-H), 7.87 (t, J = 7.8 Hz, 1H, Ar-H), 7.80
d, J = 8.3 Hz, 2H, Ar-H), 7.54–6.65 (m, 4H, Ar-H and NH ), 6.47–6.44
). C NMR (100 MHz,
) δ 166.03, 158.66, 156.48, 154.93, 152.63, 149.36, 148.46,
43.46, 138.63, 138.08, 136.57, 134.22, 129.49, 129.30, 128.59,
White solid, yield 73%, Mp: 240–242 ℃, 1H NMR (400 MHz,
(
(
2
DMSO‑d
6
) δ 11.32 (s, 1H, NH), 8.63 (d, J = 4.8 Hz, 1H, Ar-H), 8.51 (s,
1
3
m, 3H, Ar-H), 5.42 (s, 2H, CH
2
), 5.10 (s, 2H, NH
2
1H, Ar-H), 8.25 (s, 1H, Ar-H), 8.16 (d, J = 7.9 Hz, 2H, Ar-H), 7.76 (d, J =
7.9 Hz, 2H, Ar-H), 7.48 (d, J = 4.5 Hz, 1H, Ar-H), 7.31–6.65 (m, 3H, Ar-
DMSO‑d
6
1
3
1
1
H and NH
2
), 6.40 (s, 3H, Ar-H), 5.35 (s, 2H, CH
2
), 5.03 (s, 2H, NH
2
).
C
20.37, 115.46, 115.27, 113.61, 113.21, 97.88, 50.68. HRMS(ESI) m/z
NMR (100 MHz, DMSO‑d ) δ 166.70, 158.67, 156.48, 154.95, 153.73,
6
+
calcd for C24
H
20
N
8
O [M + H] 437.1833, found 437.1833.
150.40, 149.36, 143.39, 138.81 (q, J = 33 Hz, 1C), 138.07, 136.93,
133.70, 129.49, 129.46, 128.65, 123.43 (q, J = 271 Hz, 1C), 115.65 (q,
4
-(4-amino-1-(4-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(pyridin-2-yl)benzamide (14c)
J = 3 Hz, 1C), 115.48, 113.63, 113.23, 110.31 (q, J = 4 Hz, 1C), 97.91,
White solid, yield 72%, Mp: 224–227 ℃, 1H NMR (400 MHz,
50.69. HRMS(ESI) m/z calcd for C25
H
19
O [M + H] 505.1707,
+
3
F N
8
DMSO‑d
6
) δ 10.89 (s, 1H, NH), 8.41 (d, J = 4.7 Hz, 1H, Ar-H), 8.31 (s,
found 505.1712.
1
H, Ar-H), 8.24–8.19 (m, 3H, Ar-H), 7.86 (t, J = 8.6 Hz, 1H, Ar-H), 7.78
4-(4-amino-1-(4-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (14i)
(
d, J = 8.2 Hz, 2H, Ar-H), 7.52–6.60 (m, 5H, Ar-H and NH ), 6.49 (d, J =
2
1
3
White solid, yield 75%, Mp: 266–269 ℃, 1H NMR (400 MHz,
8
.3 Hz, 2H, Ar-H), 5.38 (s, 2H, CH
MHz, DMSO‑d ) δ 166.03, 158.62, 156.38, 154.61, 152.63, 148.79,
48.46, 143.23, 138.63, 136.63, 134.17, 129.43, 129.29, 128.56,
2 2
), 5.07 (s, 2H, NH ). C NMR (100
6
DMSO‑d
6
) δ 11.38 (s, 1H, NH), 8.69 (d, J = 5.0 Hz, 1H, Ar-H), 8.58 (s,
1
1
1H, Ar-H), 8.32 (s, 1H, Ar-H), 8.22 (d, J = 7.5 Hz, 2H, Ar-H), 7.80 (d, J =
7.6 Hz, 2H, Ar-H), 7.55 (d, J = 5.0 Hz, 1H, Ar-H), 7.37–6.74 (m, 4H, Ar-
24.30, 120.37, 115.27, 114.14, 97.92, 50.38. HRMS(ESI) m/z calcd for
+
C
24
H
20
N
8
O [M + H] 437.1833, found 437.1831.
H and NH
2
), 6.50 (d, J = 7.5 Hz, 2H, Ar-H), 5.38 (s, 2H, CH
2
), 5.07 (s,
1
3
4
-(4-amino-1-(2-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
2 6
2H, NH ). C NMR (100 MHz, DMSO‑d ) δ 166.74, 158.63, 156.39,
yl)-N-(4-methylpyridin-2-yl)benzamide (14d)
154.64, 153.94, 150.40, 148.79, 143.18, 138.76 (q, J = 33 Hz, 1C),
White solid, yield 68%, Mp: 205–208 ℃, 1H NMR (400 MHz,
136.94, 133.79, 129.45, 129.43, 128.60, 123.45 (q, J = 272 Hz, 1C),
DMSO‑d
6
) δ 10.76 (s, 1H, NH), 8.33 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
124.30, 115.54 (q, J = 3 Hz, 1C), 114.15, 110.35 (q, J = 4 Hz, 1C),
+
1
8
5
1
1
1
H, Ar-H), 8.19 (d, J = 8.3 Hz, 2H, Ar-H), 8.08 (s, 1H, Ar-H), 7.79 (d, J =
.3 Hz, 2H, Ar-H), 7.36–6.45 (m, 7H, Ar-H and NH ), 5.41 (s, 2H, CH ),
). C NMR (100 MHz, DMSO‑d ) δ
97.94, 50.40. HRMS(ESI) m/z calcd for C25
H
19
F
N
3 8
O [M + H]
2
2
505.1707, found 505.1707.
1
3
.37 (s, 2H, NH
2
), 2.37 (s, 3H, CH
3
6
65.91, 158.75, 156.48, 154.69, 152.66, 149.30, 148.06, 146.91,
4.2.6. Preparation of compounds 15
43.54, 136.36, 134.38, 130.23, 129.28, 129.22, 128.57, 121.38,
3
To a solution of 14 (0.5 mmol) and Et N (3 mmol) in THF (10 mL),
20.54, 116.70, 115.76, 115.65, 97.78, 45.84, 21.41. HRMS(ESI) m/z
acryloyl chloride or chloroacetyl chloride (1.2 mmol) was added under
ice-cold conditions. The mixture was stirred at room temperature for 5 h.
Upon completion, the mixture was extracted with ethyl acetate. The
combined organic phases were washed with water and brine, then dried
and concentrated. The crude product was purified by column chroma-
tography (dichloromethane/methanol, 80/1–40/1) yielding target
compounds 15.
+
calcd for C25
H
22
N
8
O [M+H] 451.1989, found 451.1986.
4
-(4-amino-1-(3-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-methylpyridin-2-yl)benzamide (14e)
White solid, yield 68%, Mp: 224–226 ℃, 1H NMR (400 MHz,
DMSO‑d
6
) δ 10.81 (s, 1H, NH), 8.32 (s, 1H, Ar-H), 8.26 (d, J = 4.8 Hz,
1
8
5
1
1
1
H, Ar-H), 8.20 (d, J = 7.9 Hz, 2H, Ar-H), 8.09 (s, 1H, Ar-H), 7.80 (d, J =
.0 Hz, 2H, Ar-H), 7.46–6.24 (m, 7H, Ar-H and NH ), 5.42 (s, 2H, CH ),
). C NMR (100 MHz, DMSO‑d ) δ
2
2
4-(4-amino-1-(3-(2-chloroacetamido)benzyl)-1H-pyrazolo[3,4-
d]pyrimidin-3-yl)-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide
(15a)
1
3
.11 (s, 2H, NH
2
), 2.37 (s, 3H, CH
3
6
65.94, 158.66, 156.47, 154.92, 152.66, 149.36, 149.32, 148.07,
43.46, 138.08, 136.53, 134.28, 129.50, 129.24, 128.59, 121.40,
White solid, yield 60%, Mp: 166–168 ℃, 1H NMR (400 MHz,
15.65, 115.47, 113.61, 113.22, 97.87, 50.67, 21.43. HRMS(ESI) m/z
DMSO‑d
6
) δ 11.40 (s, 1H, NH), 10.32 (s, 1H, NH), 8.70 (d, J = 4.6 Hz,
+
calcd for C25
H
22
N
8
O [M+H] 451.1989, found 451.1978.
1H, Ar-H), 8.58 (s, 1H, Ar-H), 8.34 (s, 1H, Ar-H), 8.23 (d, J = 7.7 Hz, 2H,
Ar-H), 7.83 (d, J = 7.6 Hz, 2H, Ar-H), 7.57 (s, 2H, Ar-H), 7.48 (s, 1H, Ar-
4
-(4-amino-1-(4-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
yl)-N-(4-methylpyridin-2-yl)benzamide (14f)
H), 7.40–6.67 (m, 4H, Ar-H and NH
2 2
), 5.58 (s, 2H, CH ), 4.22 (s, 2H,
Yellow solid, yield 58%, Mp: 188–192 ℃, 1H NMR (400 MHz,
13
2 6
CH ). C NMR (100 MHz, DMSO‑d ) δ 166.69, 165.13, 158.50, 156.33,
DMSO‑d
6
) δ 10.80 (s, 1H, NH), 8.31 (s, 1H, Ar-H), 8.26 (d, J = 5.0 Hz,
154.96, 153.73, 150.42, 143.81, 139.23, 138.81 (q, J = 33 Hz, 1C),
138.21, 136.76, 133.80, 129.64, 129.48, 128.68, 123.66, 123.43 (q, J =
271 Hz, 1C), 119.07, 118.75, 115.69 (q, J = 4 Hz, 1C), 110.31 (q, J = 3
1
8
5
1
1
1
H, Ar-H), 8.19 (d, J = 8.3 Hz, 2H, Ar-H), 8.08 (s, 1H, Ar-H), 7.77 (d, J =
.3 Hz, 2H, Ar-H), 7.43–6.30 (m, 7H, Ar-H and NH ), 5.37 (s, 2H, CH ),
). C NMR (100 MHz, DMSO‑d ) δ
2
2
1
3
.11 (s, 2H, NH
2
), 2.37 (s, 3H, CH
3
6
Hz, 1C), 97.92, 50.38, 44.01. HRMS(ESI) m/z calcd for C27
H
20ClF
3
8
N O
2
+
65.93, 158.61, 156.38, 154.61, 152.66, 149.30, 148.80, 148.08,
[M + H] 581.1423, found 581.1423.
43.23, 136.58, 134.23, 129.42, 129.24, 128.55, 124.28, 121.39,
4-(1-(3-acrylamidobenzyl)-4-amino-1H-pyrazolo[3,4-d]pyr-
15.65, 114.13, 97.90, 50.37, 21.43. HRMS(ESI) m/z calcd for
imidin-3-yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (15b)
+
1
C
25
H
22
N
8
O [M + H] 451.1989, found 451.1998.
White solid, yield 62%, Mp: 278–281 ℃, H NMR (400 MHz,
DMSO‑d
) δ 11.36 (s, 1H, NH), 10.17 (s, 1H, NH), 8.69 (d, J = 5.0 Hz,
4
-(4-amino-1-(2-aminobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
6
8

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
◦
1
H, Ar-H), 8.57 (s, 1H, Ar-H), 8.50 (d, J = 8.4 Hz, 2H, Ar-H), 8.46 (s, 1H,
Ar-H), 8.12 (d, J = 8.4 Hz, 2H, Ar-H), 7.81 – 7.44 (m, 4H, Ar-H and
NH
), 7.43 – 6.87 (m, 3H, Ar-H and NH
H, CH), 6.24 (d, J = 16.9 Hz, 1H, CH ), 5.74 (d, J = 10.0 Hz, 1H, CH
). C NMR (100 MHz, DMSOd ) δ 166.69, 163.66,
58.68, 156.58, 155.02, 153.72, 150.40, 143.70, 139.81, 138.80 (q, J =
3 Hz, 1C), 138.10, 136.83, 133.76, 132.31, 129.50, 129.47, 128.68,
27.33, 123.42 (q, J = 272 Hz, 1C), 123.30, 119.08, 118.79, 115.66 (q,
for 30 min and centrifuged at 12,000 × rpm for 20 min at 4 C. The
protein concentration was determined by Bradford assay (Bio-Rad,
Hercules, CA). Twenty micrograms of sample protein was mixed with 4
× loading buffer and separated by 10% SDS-PAGE. The proteins were
then transferred onto methanol-equilibrated PVDF membranes (Bio-Rad
Laboratories, 162e0177), which were blocked for 1 h in 5% nonfat dry
milk in TBST (BD Bioscience, San Jose, CA). The membranes were
2
2
), 6.41 (dd, J = 17.0, 10.1 Hz,
1
5
1
3
1
2
2
),
1
3
.61 (s, 2H, CH
2
6
◦
incubated with a primary antibody overnight at 4 C. Secondary anti-
J = 3 Hz, 1C), 110.30 (q, J = 4 Hz, 1C), 97.94, 50.42. HRMS(ESI) m/z
bodies were added for 1 h at room temperature. Finally, the membrane
was visualized by ECL (Perkin Elmer Life Sciences, NE104001EA). An-
tibodies against BTK, PLCγ2, p-BTK, p-PLCγ2, LC3B-II, β-actin and
GAPDH were obtained from Cell Signaling.
+
calcd for C28
H
21
F
3
N
8
O
2
[M + H] 559.1812, found 559.1816.
4
-(4-amino-1-(4-(2-chloroacetamido)benzyl)-1H-pyrazolo[3,4-
d]pyrimidin-3-yl)-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide
15c)
Gray solid, yield 59%, Mp: 140–143 ℃, 1H NMR (400 MHz,
DMSO‑d
) δ 11.40 (s, 1H, NH), 10.34 (s, 1H, NH), 8.70 (d, J = 4.9 Hz,
H, Ar-H), 8.58 (s, 1H, Ar-H), 8.33 (s, 1H, Ar-H), 8.23 (d, J = 8.0 Hz, 2H,
(
4.6. Cell apoptosis assay
6
1
Ar-H), 7.83 (d, J = 8.0 Hz, 2H, Ar-H), 7.56 (d, J = 7.2 Hz, 3H, Ar-H),
Apoptosis was quantified by Annexin V/PI-binding assay. Cells were
seeded in 6-well plates containing 15c at concentrations of 250 nM, 500
nM, or 1000 nM and incubated for 24 h. Treated cells were washed twice
with cold phosphate-buffered saline (PBS) and then resuspended in 100
7
.32 (d, J = 8.2 Hz, 2H, Ar-H), 7.26–6.20 (s, 2H, NH
2
), 5.56 (s, 2H,
1
3
CH
2
), 4.24 (s, 2H, CH
2
). C NMR (100 MHz, DMSO‑d ) δ 166.68,
6
1
3
1
65.09, 158.62, 156.47, 154.91, 153.73, 150.40, 143.65, 138.81 (q, J =
3 Hz, 1C), 138.40, 136.83, 133.75, 132.86, 129.46, 128.84, 128.66,
23.43 (q, J = 272 Hz, 1C), 120.01, 115.64 (q, J = 3 Hz, 1C), 110.31 (q,
μ
L of binding buffer, to which 2
added. The samples were gently vortexed and incubated for 15 min at
room temperature in the dark. After 200 L of binding buffer was added,
μ
L of Annexin V-FITC and 5 L of PI were
μ
J = 4 Hz, 1C), 97.96, 50.06, 43.99. HRMS(ESI) m/z calcd for
μ
+
C
27
H
20ClF
3
N
8
O
2
[M + H] 581.1423, found 581.1425.
the 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 FlowJo software.
4
-(1-(4-acrylamidobenzyl)-4-amino-1H-pyrazolo[3,4-d]pyr-
imidin-3-yl)-N-(4-(trifluoromethyl) pyridin-2-yl)benzamide (15d)
1
White solid, yield 65%, Mp: 125–128 ℃, H NMR (400 MHz,
DMSO‑d
6
) δ 11.40 (s, 1H, NH), 10.19 (s, 1H, NH), 8.70 (d, J = 4.6 Hz,
4
.7. ROS assay
1
H, Ar-H), 8.59 (s, 1H, Ar-H), 8.34 (s, 1H, Ar-H), 8.23 (d, J = 7.8 Hz, 2H,
Ar-H), 7.83 (d, J = 7.7 Hz, 2H, Ar-H), 7.65–7.55 (m, 4H, Ar-H),
.42–6.82 (m, 3H, Ar-H and NH ), 6.47–6.40 (m, 1H, CH), 6.25 (d, J
16.9 Hz, 1H, CH ), 5.56 (s, 2H, CH ).
), 5.75 (d, J = 10.0 Hz, 1H, CH
C NMR (100 MHz, DMSO‑d ) δ 166.68, 163.58, 158.69, 156.55,
Intracellular ROS levels were measured by detecting the conversion
7
2
of DCFH-DA to fluorescent DCF. Z138 cells were seeded in 6-well plates
and treated with 1 M test compound (ibrutinib or 15c) for different
=
2
2
2
1
3
μ
6
times (12 h or 24 h). Then, the cells were collected and washed with PBS
1
1
1
54.92, 153.73, 150.39, 143.60, 138.97, 138.78 (q, J = 29 Hz, 1C),
36.86, 133.73, 132.49, 132.27, 129.46, 128.79, 128.66, 127.37,
23.43 (q, J = 272 Hz, 1C), 119.95, 115.65 (q, J = 3 Hz, 1C), 110.31 (q,
three times. After washing, the cells were incubated with DCFH-DA at
◦
3
7 C for 20 min. Then, the DCF fluorescence distribution was observed
under a confocal laser scanning microscope (Leica TCS SP8) using an
J = 4 Hz, 1C), 97.97, 50.10. HRMS(ESI) m/z calcd for C28
21 3 8 2
H F N O [M
+
excitation wavelength of 488 nm and an emission wavelength of 525
nm.
+
H] 559.1812, found 559.1806.
4
.3. In vitro kinase activity assay
4
.8. Molecular docking analysis
The in vitro BTK kinase activity and kinases selectivity activities were
evaluated via a radiometric protein kinase assay following the protocol
as previously described [30].
Molecular docking analysis was performed using Sybyl 2.0 software,
and the BTK crystal structure (PDB: 5fbo) 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 15c was docked into
BTK, hydrogen bonds and hydrophobic interactions were observed in
the model, and the best conformation with the highest CScore was
selected for interaction analysis.
4
.4. Cell antiproliferation assay
MCL cell lines Mino, Rec-1, Maver-1 and Z138 were purchased from
the American Type Culture Collection (ATCC). Cell antiproliferation
assays were performed on the MCL cell lines using a CellTiter-Glo
luminescent cell viability assay kit (Promega) following a protocol
previously described [31]. Briefly, cells were plated in 96-well plates at
4
a density of 1 × 10 cells per well and then treated with different con-
Declaration of Competing Interest
centrations of target compounds and DMSO (control) and incubated in a
◦
humidified atmosphere with 5% CO
with 30
2
at 37 C for 72 h. Cells were lysed
The authors declare that there is no conflict of interest.
μ
L of Cell Titer-Glo luminescent cell viability assay reagent, and
the luminescence was quantified using a BioTek Synergy HTX multi-
mode microplate reader. Each triplicate experiment was performed no
less than three times to establish the cell survival curve. IC50 values were
calculated with GraphPad Prism 6 software.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (No. 82073708).
4
.5. Western blotting
Appendix A. Supplementary material
Z138 cells were cultured with different concentrations of 15c and
ibrutinib for 24 h. Then, cells were harvested and lysed in lysis buffer
Cell Signaling, Danvers, MA). The cell lysates were maintained on ice
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104968.
(
9

F. Ran et al.
Bioorganic Chemistry 112 (2021) 104968
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