Targeted selective degradation of Bruton’s tyrosine kinase by PROTACs

Article abstract of DOI:10.1007/s00044-020-02526-3

Bruton’s tyrosine kinase (BTK) is critical for B-cell receptor signaling and related to many types of human cancers. However, the drug resistance and off-target effect of present traditional BTK inhibitors occurred over time. As a new strategy for drug development, the proteolysis targeting chimera (PROTAC) has been proved to target varieties of proteins. Here SPB5208 (4-(2-(2-(2-(2-(3-(4-amino-3-(4-phenoxy phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethoxy)ethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione) was synthesized as a new PROTAC degradation agent of BTK by linking Ibrutinib and Thalidomide. In vitro study indicated that SPB5208 reduced the BTK enzyme activity with high selectivity and inhibited effectively cancer cell proliferation. More than that, SPB5208 induced BTK protein degradation through a proteasome- and CRBN- dependent manner in JeKo-1 cells. In addition, SPB5208 was also confirmed to induce significantly BTK protein degradation in vivo. Thus, this study provides a new pharmacological tool for further study of new BTK PROTAC and their mechanism of action in physiological environment.

Full text of DOI:10.1007/s00044-020-02526-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02526-3
ORIGINAL RESEARCH
Targeted selective degradation of Bruton’s tyrosine kinase
by PROTACs
1
Shaodong Liu1 Yang Da1 Feng Wang2 Renjie Yan2 Yongzhi Shu2 Pei Lin1 Jun Lin
●
●
●
●
●
●
Received: 28 November 2019 / Accepted: 20 February 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Bruton’s tyrosine kinase (BTK) is critical for B-cell receptor signaling and related to many types of human cancers.
However, the drug resistance and off-target effect of present traditional BTK inhibitors occurred over time. As a new
strategy for drug development, the proteolysis targeting chimera (PROTAC) has been proved to target varieties of proteins.
Here SPB5208 (4-(2-(2-(2-(2-(3-(4-amino-3-(4-phenoxy phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethoxy)
ethoxy)ethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione) was synthesized as a new PROTAC degradation
agent of BTK by linking Ibrutinib and Thalidomide. In vitro study indicated that SPB5208 reduced the BTK enzyme activity
with high selectivity and inhibited effectively cancer cell proliferation. More than that, SPB5208 induced BTK protein
degradation through a proteasome- and CRBN- dependent manner in JeKo-1 cells. In addition, SPB5208 was also confirmed
to induce significantly BTK protein degradation in vivo. Thus, this study provides a new pharmacological tool for further
study of new BTK PROTAC and their mechanism of action in physiological environment.
●
●
Keywords PROTAC Bruton’s tyrosine kinase Targeted degradation
Abbreviations
PROTAC Proteolysis targeting chimera
cell lymphoma (Michael et al. 2013), follicular lymphoma
(Radford et al. 2013). Most of cancers above-mentioned
were mainly caused by the overexpression of BTK, there-
fore, the development of drug molecules targeting it has
become an attractive therapeutic approach (Miao et al.
2013; Xin et al. 2019).
BTK
Bruton’s tyrosine kinase
Introduction
With the deepening of BTK research, many research
groups have devoted great efforts to develop its inhibitors
(Satterthwaite and Witte 2000). Ibrutinib, as the first-in-
class BTK inhibitor, is an orally available and highly
potent small-molecule inhibitor with subnanomolar
activity (Kim 2019). Although Ibrutinib has remarkable
achievement in clinical therapy, there were still many
patients developed resistance to it after treated with
Ibrutinib for 1–2 years. One reason is the critical mutation
during tumorigenesis, and the other reason is its off-target
activity (Jing et al. 2016). The limitation of Ibrutinib led
to active developments of better selective second-
generation BTK inhibitors, such as Acalabrutinib. How-
ever, the drug resistance and side effect still exist. Hence,
it is very urgent to develop the new drug molecules tar-
geted BTK or its mutant.
Bruton’s tyrosine kinase (BTK) is a nonreceptor kinase of
the TEC kinase family which is a crucial integrant of B-cell
receptor signaling and functions as a key regulator of cell
proliferation and survival in B-cell malignancies (George
et al. 2016; Khan 2001). B-cell malignancies include a
series of hematologic malignancies, such as chronic lym-
phocytic leukemia (Sachdeva and Dhingra 2015), mantle
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02526-3) contains supplementary
material, which is available to authorized users.
* Jun Lin
junlin@jiangnan.edu.cn
1
Currently, a new technology named proteolysis targeting
chimera (PROTAC) is aimed at utilizing the ubiquitin‐
protease system to target a specific protein and induce its
degradation (Gadd et al. 2017). Typically, PROTAC
School of Pharmaceutical Science, Jiangnan University, 1800 Lihu
Avenue, Wuxi 214122, China
2
Shanghai Meizer Pharmaceuticals Co., Ltd, 797 Puxing Road,
Shanghai 201114, China

Medicinal Chemistry Research
contains an E3 ligase targeting moiety, which is connected
to a ligand for the target of interest via a linker. The chimera
recruits E3 ubiquitin ligases to target proteins for their
ubiquitination and proteasome-mediated degradation.
Compared with the traditional enzyme inhibitors, PRO-
TACs induce the degradation of the enzyme rather than
inhibit it. Till now, the PROTACs can be used to target
varieties of proteins, including transcription factors, skele-
ton proteins and regulatory proteins (Ottis and Crews 2017).
Various PROTACs were reported to degrade specific
kinases, such as CDK9 (Olson et al. 2017), BTK (Huang
et al. 2017), epidermal growth factor receptors (EGFR), and
TBK1 (Cheng et al. 2018).
13C, 100 MHz) spectrometer using DMSO-d6 as solvent
and TMS (Tetramethyl silane) as internal standard. All
chemical shifts were reported in ppm. The mass spectra
were carried out on Thermo Scientific Quantum Access max
LC-MS spectrometer.
Cell culture
JeKo-1 cells were kindly provided by Stem Cell Bank,
Chinese Academy of Sciences. Cells were cultured in
RPMI-1640 medium supplemented with 20% fetal bovine
serum and 1% penicillin and streptomycin. Cells were
cultured at 37 °C with 5% CO2.
Targeted degradation of BTK is explored as a new
strategy for developing antineoplastic drugs. Consistently,
PROTAC technology is aimed at developing a model
system for the efficient targeted protein degradation
(Sakamoto et al. 2001). PROTAC made use of the cell’s
own ubiquitin-proteasome mechanism to degrade targeted
proteins. As a well BTK inhibitor, Ibrutinib is a good
ligand for targeting BTK. More than that, it is reported
that the type of linker is vital for the successful devel-
opment of some PROTACs and small-molecule inhibitors
(Ravichandiran et al. 2014). On the other, many studies
shown that some small-molecule compounds have good
solubility and cell activity with a piperidine group extent
linker (Ravichandiran et al. 2014). So, we designed a
series of PROTACs and chose the most potential com-
pound by linking Ibrutinib and Thalidomide with a PEG
linker and a piperidine group extent linker. The effects of
SPB5208 on BTK protein degradation were investigated
in vitro and in vivo.
Cell proliferation assay
Effects of compounds on cell viability were determined by
using cell counting kit-8 (CCK-8, Meilun MA0218). JeKo-
1 cells were seeded in 96-well plates at a density of 104 cells
per well in triplicate. After the treatment of SPB5208 for
72 h, 10 μL CCK-8 was added and incubated for 4 h. The
absorbance was read at 450 nm using a micro plate reader
(TECAN Infinite M200pro Switzerland). Each concentra-
tion was analyzed in triplicate.
Western blot assay
Briefly, equivalent amounts of protein for each sample
was separated by 8% SDS-PAGE gels and transferred to
the polyvinylidene difluoride membranes. Then, mem-
brane was blocked in 5% BSA (Albumin from bovine
serum) in the TBST for 1 h, incubated with a primary
antibody overnight at 4 °C and treated with secondary
antibody for 1 h at room temperature. The band signals
were imaged with extreme hypersensitivity ECL chemi-
luminescence kit. The total gray of each band was quan-
tified via the ImageJ software.
Materials and methods
Chemicals and antibodies
The triethylene glycol and bis[2-(2-hydroxyethoxy)ethyl]
ether was purchased from Energy Chemical (Shanghai,
China). All organic solvents were purchased from Sigma
Aldrich. The Ibrutinib was from Taiwo (Shandong, China).
The antibodies included β-actin (4970 s) and BTK (3533 S)
were purchased from Cell Signaling Technology.
Kinases selectivity
All kinases, peptide FAM-P2, ATP, staurosporine and some
labwares were bought from Carna and Sangon. The com-
pound was transferred in tubes to one well on 96-well
storage plate and serially diluted for a series of concentra-
tions. Then, 90 μL of 1× kinase buffer was added to each
well of the intermediate plate. Transfer 5 μL of each well
from the 96-well intermediate plate to a 384-well plate in
duplicates. At last, prepare enzyme solution, peptide solu-
tion and 5 μL of compound in 10% DMSO for the Kinase
reaction. Incubate at room temperature for 10 min and stop
the reaction by adding 25 μL of stop buffer. Detect them
with a micro plate reader and fit the data in XL fit excel to
obtain IC50 values.
Chemical analysis methods
The purities of the compounds were monitored by thin layer
chromatography (Silica gel 60 F 254 aluminum from Merck
Germany). The mobile phase was ethyl acetate, hexane,
dichloromethane or methanol (95:5–80:20). The detection
1
was made using UV light. H NMR and 13C NMR spectra
were recorded on a Varian-Mercury 400 (1H, 400 MHz;

Medicinal Chemistry Research
In vivo degradation study
bis[2-(2-hydroxyethoxy) ethyl] ether (2a). Then, the lin-
ker 2b reacted with the 3-hydroxy thalidomide by the
Mitsunobu reaction to afford the intermediate 2c (Lai and
Crews 2016). Followed by the TBS deprotection with
diluted HCl, the intermediate 2c was transformed to the
intermediate 2d and reacted with Ibrutinib intermediate
(2e) by oxidation and reductive amination (Ravichandiran
et al. 2015) to get the target compound SPB5208. The
The ICR mice were purchased from Beijing Vital River
Laboratory Animal Technology Co., Ltd (IACUC Issue:
JN. No20190530i0150610). SPF-grade ICR male mice
were housed in constant temperature (20–26 °C) and
humidity (40–60%). The blank control group was intra-
peritoneally injected with 0.1 mL/10 g of Vehicle every day.
The SPB5208 group was intraperitoneally injected with
15 mg/kg or 50 mg/kg of SPB5208 once a day for 5 days.
After the last administration, the mice were anesthetized
with 1% pentobarbital sodium (0.1 mL/10 g) and the
spleens were harvested and frozen at −80 °C for further
analysis. The tissue samples were analyzed through WB
detection.
1
structure of compound SPB5208 was confirmed by H
NMR, 13C NMR spectroscopy and liquid chromatograph
mass spectrometer. As the most important component, the
type and length of linkers are vital for the successful
development of PROTAC (Gadd et al. 2017). The piper-
idinyl group is solvent exposed relatively by analyzing the
X-ray crystal structure of BTK in complex with Ibrutinib
(PDBID: 5P9J) (Bender et al. 2017). Based on these
studies, SPB5208 was developed with a PEG linker and a
piperidine group extent linker.
Statistical analysis
Data were analyzed using GraphPad Prism. The data are
presented as mean standard deviation of three independent
experiments. Student’s t tests were performed to compare
individual treatment to the control, and comparisons
between different treatments were analyzed by one-way
analysis of variance with a significance level of p < 0.05.
SPB5208 effectively inhibit the proliferation of
Jeko-1 cells
First, the JeKo-1 cells were used to evaluate the effects of
SPB5208 on cell proliferation. The result suggested that the
inhibition of SPB5208 on the proliferation of JeKo-1 cells
was concentration-dependent manner (Fig. 2a). Although its
activity was lower than the positive control (Ibrutinib),
SPB5208 still showed good potential on inhibiting cell
proliferation. It is well known that BTK is overexpressed in
many human cancer cells (Sehn and Gascoyne 2015).
Moreover, BTK protein is critical for proliferation and
survival of leukemic cells in many B-cell malignancies
(Qian et al. 2012). These researches indicated that it is a
Results and discussion
Synthesis of SPB5208
As shown in Fig. 1, SPB5208 was synthesized by a
common synthetic route. The mono-TBS protected linker
2b was obtained from tert-butyldimethylsilyl chloride and
Fig. 1 Approach to develop SPB5208. Reagents and conditions: (1) TBS-Cl, Imidazle, DCM, 0 °C, overnight. (2) PPh3, DIAD, THF, RT, 8 h. (3)
HCl (aq), CH3OH, RT, 2 h. (4) Dess-Martin periodinane, DCE, RT, 2 h. (5) HOAc, NaHB(OAc)3, RT, overnight

Medicinal Chemistry Research
Fig. 2 The inhibition of SPB5208 and Ibrutinib on the proliferation of
JeKo-1 cells (a). SPB5208 degraded the BTK protein in the JeKo-1
cells with a concentration-dependent manner. JeKo-1 cells were trea-
ted with DMSO or different concentrations of SPB5208 for 24 h (b).
SPB5208 degraded the BTK protein in the JeKo-1 cells with a time-
dependent manner. JeKo-1 cells were treated with SPB5208 for a
series of times and Western blot analysis with specific antibodies
toward BTK (d). Western blotting of JeKo-1 cells treated with
SPB5208 with different time and concentrations gradients. The band
intensities were quantified by densitometry, normalized to the level of
β-actin, and calculated as the percentage of the basal response (c, e).
*p < 0.05; **p < 0.01; ***p < 0.001, compared with the control

Medicinal Chemistry Research
Fig. 3 The degradation of BTK
protein induced by SPB5208 can
be rescued. Cells were pretreated
with DMSO, Thalidomide
(50 µM), or MLN2238 (100 nM)
for 2 h, and then were treated
with the 100 nM SPB5208 for
24 h
valid strategy to inhibit the proliferation of Jeko-1 cells for
some B-cell malignancies.
Table 1 The inhibition IC50 (nM) of compounds on 5 kinases
JAK3
ITK
EGFR
BLK
BTK
SPB5208 effectively induce the degradation of BTK
in Jeko-1 cells
SPB5208
>10,000
160
>10,000
238
4356
12
459
1.5
2.4
127
2.4
Ibrutinib
Staurosporine
0.35
5.3
221
152
Next, the effect of SPB5208 on BTK protein was investi-
gated. The result indicated that SPB5208 significantly
reduced the BTK protein level in the JeKo-1 cell with a
concentration-dependent manner (Fig. 2b). After treated
with SPB5208 (500 nM) for 24 h, more than 70% of BTK
protein was degraded (Fig. 2c). As expected, Ibrutinib did
not change the BTK protein level under the investigated
concentration (Fig. S2).
Based on the above results, the time course studies of
SPB5208 induced BTK degradation in JeKo-1 cells were
investigated. The result showed that the BTK protein began
to degrade after treated with SPB5208 (500 nM) for 2 h.
SPB5208 could degrade BTK protein with a time-
dependent manner (Fig. 2d). The maximum degradation
of BTK protein, over 70% degradation, was observed after
24 h treatment with SPB5208 (Fig. 2e).
Generally, traditional small chemical inhibitors were
extensively synthesized to bind specific proteins and aimed
to inhibit the activity of the protein. In recent years, many
researches showed that the degradation of targeted protein is
more efficient than inhibition, which is more likely to
overcome drug resistance and some side effects (Matyskiela
et al. 2016). Significantly, SPB5208 degraded BTK protein
directly in vitro with a proteasome dependent manner.
inhibitor) for 2 h. Then, it increased BTK protein levels
significantly after treated with SPB5208 for 24 h. Both of
two diminished the degradation effect of SPB5208 (Fig. 3).
It indicated that SPB5208 degraded BTK proteins through a
CRBN- and proteasome- dependent mechanism. We will try
to obtain crystal structures of ternary complexes of BTK-
SPB5208-CRBN and explore the concrete mechanism of
the degradation on next work.
SPB5208 exhibits high selectivity on BTK
Then, to investigate the kinase selectivity of SPB5208,
in vitro inhibition activity of SPB5208 on five kinases was
detected. Staurosporine and Ibrutinib were used as the
positive control. As shown in Table 1, the SPB5208 showed
no inhibitory activity against inducible T-cell kinase (ITK
IL-2), EGFR, B-lymphoid tyrosine kinase (BLK) and
Janus kinase 3 (JAK3). On the other, its inhibition IC50
was 127 nM, which showed the high selectivity on BTK
(Table 1).
In recent researches, drug resistance, off-target and side
effects occurred when a small-molecule inhibitor was con-
tinually used (Jing et al. 2016). For example, Rao’s study
showed that EGFR, ITK, and TEC family kinases can also
be inhibited by low concentrations of Ibrutinib, resulting in
serious side effects or off-target in patients (Yong et al.
2018). However, SPB5208 inhibited the BTK with high
selectivity and almost showed no inhibitory activity against
ITK or EGFR, which indicates that SPB5208 will unlikely
lead to the side effects of Ibrutinib.
SPB5208 recruits CRBN E3 ligase to degrade BTK
protein
To explore the mechanism of BTK degradation by
SPB5208, a rescued experiment was designed. First, the
JeKo-1 cells were pretreated with an excess amount of the
Thalidomide (the E3 ubiquitin ligase CRBN) and
MLN2238 (a potent and selective reversible proteasome

Medicinal Chemistry Research
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Bender AT, Gardberg A, Pereira A, Johnson T, Wu Y, Grenningloh R
(2017) Ability of Bruton’s tyrosine kinase inhibitors to sequester
y551 and prevent phosphorylation determines potency for inhi-
bition of fc receptor but not B-cell receptor signaling. Mol Pharm
91(3):208–219
Cheng WZ, Xiao RX, Xiao BY, Bin J, Jin J (2018) Proteolysis tar-
geting chimeras (protacs) of anaplastic lymphoma kinase (ALK).
Eur J Med Chem 150:301–314
George VD, Qing S, Qing JL, Douglas GB, Myra BB, Rick R, Arvind
M, Lorell D, Celia DA, Jun D, Mary O, Rodney V, Ying RZ,
Zheng Y, Punit M, Andrew JT, Jodi K, Chiehying JC, Hui PZ,
Kathleen G, Tracy T, Mark AP, Stacey S, Daniel WK, Kim WM,
Luisa SC, Aberra F, James RB, Joel CB, Percy HC, Joseph AT
(2016) Small molecule reversible inhibitors of Bruton’s tyrosine
kinase (BTK). J Med Chem 59(3):7915–7935
Gadd MS, Testa A, Lucas X, Chan KH, Chen W, Lamont DJ (2017)
Structural basis of PROTAC cooperative recognition for selective
protein degradation. Nat Chem Biol 13(5):514–521
Fig. 4 SPB5208 reduced the BTK protein level in the spleen of mice
with different concentration groups. The ICR mice were intraper-
itoneally injected with the SPB5208 or Vehicle once a day for 5 days.
Western blot analysis was carried out on whole cell lysate from tissue
samples of spleens in mice. The band intensities were quantified by
densitometry, normalized to the level of β-actin, and calculated as the
percentage of the basal response, compared with the control
Huang HT, Dobrovolsky D, Paulk J, Yang G, Gray NS (2017) A
chemo proteomic approach to query the degradable kineme using
a multi-kinase degrader. Cell Chem Biol 25(1):88–97
Jing JW, Christina L, Tsui ST, Dong L (2016) Second-generation
inhibitors of Bruton tyrosine kinase. J Clin Hematol Oncol
9(1):80–91
Khan WN (2001) Regulation of B lymphocyte development and
activation by bruton’s tyrosine kinase. Immunol Res 23
(2–3):147–156
SPB5208 effectively induce the degradation of BTK
in vivo
At last, the in vivo degradation of SPB5208 on the BTK
protein was investigated. The ICR mice were treated with
SPB5208 by intraperitoneal injection once a day for 5 days.
For the concentration of 15 mg/kg and 50 mg/kg, the result
suggested that BTK protein could be degraded by SPB5208
in vivo (Fig. S3). Moreover, there was no obvious
abnormality of viscera in mice and over 50% of spleen BTK
protein was degraded by SPB5208 (Fig. 4).
Kim HO (2019) Development of BTK inhibitors for the treatment of
B-cell malignancies. Arch Pharmacal Res 3:171–181
Lai AC, Crews CM (2016) Induced protein degradation: an emerging
drug discovery paradigm. Nat Rev Drug Discov 16(2):101–114
Michael L, Brad S, Ranjana H, Michael E, Jorge E (2013) Targeting
BTK with ibrutinib in relapsed or refractory mantle-cell lym-
phoma. Boston Med Surg J 369(6):507–516
Miao X, Hou Y, Sheng L, Peng J (2013) Therapeutic effects of tha-
lidomide in hematologic disorders: a review. Front Med 7
(3):290–300
Conclusion
Regulating protein function through targeted degradation as
opposed to more conventional target inhibition has emerged
as a new modality for the development of new therapeutics.
In conclusion, SPB5208, a new PROTAC compound, was
designed and synthesized in this study. SPB5208 could
inhibit the growth of the JeKo-1 cells and induce BTK
protein degradation in vitro and in vivo. Besides,
SPB5208 suppressed BTK enzyme activity with high
selectivity. Therefore, these results indicated that SPF5208
could be a potential pharmacological research tool in the
development of antineoplastic drugs.
Matyskiela ME, Lu G, Ito T, Pagadian B, Lu CC, Miller K (2016) A
novel cereblon modulator recruits GSPT1 to the CRL4CRBN
ubiquitin ligase. Nature 535(7611):252–7
Ottis P, Crews CM (2017) Proteolysis-targeting chimeras: induced
protein degradation as a therapeutic strategy. ACS Chem Biol 12
(4):892–898
Olson CM, Jiang B, Erb MA, Liang Y, Doctor ZM, Zhang Z (2017)
Pharmacological perturbation of cdk9 using selective CDK9
inhibition or degradation. Nat Chem Biol 14(2):163–170
Qian K, Bori ID, Chen CH, Huang L, Lee KH (2012) Anti-aids agents
90. novel c-28 modified bevirimat analogues as potent hiv
maturation inhibitors. J Med Chem 55(18):8128–8136
Radford J, Davies A, Cartron G, Morschhauser F, Vitolo U (2013)
Obinutuzumab (GA101) plus CHOP or FC in relapsed/refractory
follicular lymphoma: results of the GAUDI study (BO21000).
Blood 122(7):390–400
Ravichandiran P, Jegan A, Premnath D, Periasamy VS, Muthusu-
bramanian S, Vasanthkumar S (2014) Synthesis, molecular
docking and cytotoxicity evaluation of novel 2-(4-amino-benzo-
sulfonyl)-5h-benzo[b]carbazole-6,11-dione derivatives as histone
deacetylase (HDAC8) inhibitors. Bioorg Chem 53(4):24–36
Ravichandiran P, Jegan A, Premnath D, Periasamy VS, Vasanthkumar
S (2014) Design, synthesis, molecular docking as histone
Acknowledgements This work was supported by the Fundamental
Research Funds for the Central Universities (JUSRP11961).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

Medicinal Chemistry Research
deacetylase (HDAC8) inhibitors, cytotoxicity and antibacterial
evaluation of novel 6-(4-(4-aminophenylsulfonyl) phenylamino)-
5h-benzo[a]Phenoxazine-5-one derivatives. Med Chem Res 24
(1):197–208
Sakamoto KM, Kim KB, Kumagai A, Mercurio F, Crews CM,
Deshaies RJ (2001) Protacs: chimeric molecules that target pro-
teins to the Skp1-Cullin-F box complex for ubiquitination and
degradation. Proc Natl Acad Sci USA 98(15):8554–8559
Sehn LH, Gascoyne RD (2015) Diffuse large B-cell lymphoma:
optimizing outcome in the context of clinical and biologic het-
erogeneity. Blood 125(1):22–32
Xin YL, Bin S, Yu T, Yu C, Hui ZY, Jiu RL, Lian JW, Si YH, Qi DY,
Hua X (2019) Design, synthesis and biological evaluation of
novel 2-phenyl pyrimidine derivatives as potent Bruton’s tyrosine
kinase (BTK) inhibitors. MedChemComm 10:421–428
Yong HS, Xing WZ, Ning D, Hong YG, Yue W, Yi QY (2018)
Protac-induced BTK degradation as a novel therapy for mutated
BTK c481s induced ibrutinib-resistant B-cell malignancies. Cell
Res 28(7):779–781
Ravichandiran P, Athinarayanan J, Premnath D, Periasamy VS,
Alshatwi AA, Vasanthkumar S (2015) Synthesis, molecular
docking and biological evaluation of novel 6-(4-(4-aminophe-
nylsulfonyl) phenylamino)-5h-benzo[a]phenothiazin-5-one deri-
vative. Spectrochim Acta, Part A 139(1):477–487
Sachdeva M, Dhingra S (2015) Obinutuzumab: an FDA approved
monoclonal antibody in the treatment of untreated chronic lym-
phocytic leukemia. Int J Appl Basic Med Res 5(1):54–57
Satterthwaite AB, Witte ON (2000) The role of Bruton’s tyrosine
kinase in B-cell development and function: a genetic perspective.
Immunol Rev 175:120–127