Discovery of novel small molecule induced selective degradation of the bromodomain and extra-terminal (BET) bromodomain protein BRD4 and BRD2 with cellular potencies

Article abstract of DOI:10.1016/j.bmc.2019.115181

The BET proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation and can be degraded by proteolysis-targeting chimeras (PROTACs) for BET proteins. However, the lack of intra-BET proteins selectivity limits the scope of current degraders as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We describe herein the design, synthesis, and evaluation of PROTAC BET degraders, based on the BET inhibitor with selectivity for the first Bromodomain benzo[cd]indole-2-one, alkylamide linker and cereblon ligand thalidomide. Compound 15 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 and BRD2 over BRD3, which not only effectively inhibits cell growth in human acute leukemia cell lines, but also very effective in inhibiting solid tumors with low cytotoxic effect in the cell profiles of NCI 60 cell lines. Remarkable dependency on linker length was observed for BRD4-degrading and c-Myc-driven antiproliferative activities in acute myeloid leukemia cell line MV4-11. The small-molecular 15 represents a novel, potent, and selective class of BRD4 and BRD2 degraders for the development of therapeutics to treat cancers.

Full text of DOI:10.1016/j.bmc.2019.115181

Journal Pre-proofs
Discovery of Novel Small Molecule Induced Selective Degradation of the
Bromodomain and Extra-Terminal (BET) Bromodomain Protein BRD4 and
BRD2 with Cellular Potencies
Fei Jiang, Qingyun Wei, Huili Li, Hongmei Li, Yong Cui, Yu Ma, Haifang
Chen, Peng Cao, Tao Lu, Yadong Chen
PII:
S0968-0896(19)31558-5
https://doi.org/10.1016/j.bmc.2019.115181
BMC 115181
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
11 September 2019
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24 October 2019
Please cite this article as: F. Jiang, Q. Wei, H. Li, H. Li, Y. Cui, Y. Ma, H. Chen, P. Cao, T. Lu, Y. Chen,
Discovery of Novel Small Molecule Induced Selective Degradation of the Bromodomain and Extra-Terminal
(
BET) Bromodomain Protein BRD4 and BRD2 with Cellular Potencies, Bioorganic & Medicinal Chemistry
(
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Bioorganic & Medicinal Chemistry
Discovery of Novel Small Molecule Induced Selective Degradation of the
Bromodomain and Extra-Terminal (BET) Bromodomain Protein BRD4 and BRD2
with Cellular Potencies
a,b
c,d
a
a
a
a
a
c,d,
Fei Jiang , Qingyun Wei , Huili Li , Hongmei Li , Yong Cui , Yu Ma , Haifang Chen , Peng Cao *,
a,b,
b,
Tao Lu *, Yadong Chen *.
a
School of Sciences, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, PR China.
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China.
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, PR China.
Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, PR China.
b
c
d
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
2009 Elsevier Ltd. All rights reserved.
The BET proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation
and can be degraded by proteolysis-targeting chimeras (PROTACs) for BET proteins. However,
the lack of intra-BET proteins selectivity limits the scope of current degraders as probes for target
validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We describe
herein the design, synthesis, and evaluation of PROTAC BET degraders, based on the BET
inhibitor with selectivity for the first Bromodomain benzo[cd]indole-2-one, alkylamide linker and
cereblon ligand thalidomide. Compound 15 potently and rapidly induces reversible, long-lasting,
and unexpectedly selective removal of BRD4 and BRD2 over BRD3, which not only effectively
inhibits cell growth in human acute leukemia cell lines, but also very effective in inhibiting solid
tumors with low cytotoxic effect in the cell profiles of NCI 60 cell lines. Remarkable dependency
on linker length was observed for BRD4-degrading and c-Myc-driven antiproliferative activities
in acute myeloid leukemia cell line MV4-11. The small-molecular 15 represents a novel, potent,
and selective class of BRD4 and BRD2 degraders for the development of therapeutics to treat
cancers.
Available online
Keywords:
BRD4 and BRD2
PROTAC degraders
Selectivity
Cancers.
1
. Introduction
Bromodomain-containing proteins contain domains that
institutions, a large number of non-selective first and second
1
3
bromodomain BET inhibitors were discovered. The 1 ((+)JQ1)
with a triazole core structure is represented, and its derivatives
1
specifically recognize histone acetylation, named "reader".
Among them, the bromodomain and extra-terminal domain (BET)
family of proteins are the focus of current drug research, which
consist of four proteins BRD2, BRD3, BRD4 and BRDT . BET
family proteins are nuclear proteins and recruit transcriptional
regulatory complexes to acetylated chromatin, which involves in
a number of DNA-centered processes including regulation of gene
expression. BRD4 recruits a positive transcription elongation
factor complex (P-TEFb) , which plays a role in the regulation of
transcription by RNA polymerase Ⅱ(RNA Pol Ⅱ) in eukaryotes.
14
15
include OTX-015 , I-BET762 and other inhibitors are in clinical
trials as a new treatment of human cancers. There are reports of
BET inhibitors that selective for the second bromodomains,
2
16
17
18
including 2 (BY27) , 3 (RVX-208) , 4 (GSK340) and 5
19
(
ABBV-744) . Among them, 3 (RVX-208), a selective BET
3
17
inhibitor of BD2, is in phase Ⅲclinical trial .for the treatment of
cardiovascular diseases. Besides, 5 (ABBV-744) developed by
Abbvie is currently in phase Ⅰclinical trials for the treatment of
in acute myeloid leukemia (AML) and androgen receptor (AR)
4
19
positive prostate cancer .
BET family proteins not only regulate the expression of several
important oncogenes (e.g. MYC and BCL2) , but also cytokines
5
,6
The concept of the proteolysis-targeting chimeras
7
,9
20
(e.g. IL-17a, TNF-α and IL-6) . Therefore, BET proteins have
(PROTACS) was proposed by Deshaies and Crews in 2001 .
been implicated in a number of human diseases including cancer,
inflammation, cardiovascular diseases, human immunodeficiency
Great attention has been paid for PROTAC strategy because of its
prospects in discovering and developing new types of small
molecule therapies. The PROTAC molecule usually consists of
three parts: a target protein ligand, an E3 ubiquitinated ligase
ligand and a ligation module. Therefore, the molecule not only
binds to the target protein but also recruits the E3 ligase complex
and degrades the target protein after ubiquitination. In recent years,
many laboratories have developed many BET protein degradation
1
0,12
virus (HIV) Infection and other human diseases
.
Each member of the BET protein contains two
bromodomains (the first Bromodomain: BD1 and and the second
Bromodomain: BD2) that specifically bind to the acetylated lysine
residues of the histone tail to regulate gene transcription. Along
with the rapid push of pharmaceutical companies and research

agents using PROTAC technology as a new drug development
in leukemia cells at picomolar concentrations and shows
prominent growth inhibition in the RS4;11 and MV4;11 xenograft
models in mice. In comparison, 8 (ARV-771) was designed by
Arvinas based on the von Hippel−Lindau E3 ubiquitin ligase
ligand and the BET inhibitor 1 ((+)JQ1). This ligand can suppress
both AR signaling and AR levels and leads to tumor regression in
a CRPC mouse. 11 (A1874) is firstly reported that the E3 ligase
ligand and targeting warhead combine to exert a synergistic anti-
proliferative effect.
2
1
22
23
strategy, including 7 (dBET1) , 8 (ARV-771) , 9 (QCA570) , 10
2
4
25
(
BETd-260) and 11 (A1874) as shown in Figure 1. In 2015,
Winter GE et al. reported the first BET family protein degradation
agent, 7 (dBET1), which induced highly selective cereblon-
dependent BET protein degradation in vitro and in vivo and
delayed leukemia progression in mice. In addition, 9 (QCA570)
and 10 (BETd-260), designed and developed by Dr. Shaomeng
Wang, contains the cereblon ligand similar to the design of 7
2
5
(dBET1). QCA570 induces complete degradation of BET protein
Figure 1. Chemical structures of representative BET inhibitors and five representative previously reported BET degraders.
Table 1. Binding Affinities of BET Inhibitors
The BET proteins have achieved great success in treating
tumors. However, pan-BET inhibitor with the lack of intra-BET
selectivity limits the scope of current drugs as probes for target
validation and could lead to unwanted side effects or toxicity in a
therapeutic setting. Benzoindole derivatives have been reported to
2
6-27
have potential therapeutic cancers
.
In the previous
experimental work, our laboratory obtained compound 6 with
selectivity for BRD4 BD1. The IC50 values of 6 tested using
AlphaScreen assay were 49.5±10.3 nM and 2479±170 nM for
BRD4 BD1 and BRD4 BD2, respectively, thus about 50-fold
selectivity.
a
Binding affinities IC50 (nM)
1
Compound
R
BRD4 BD1
49.5±10.3
BRD4 BD2
2479±170
6
H
Herein, we describe the design, synthesis, and evaluation of
novel PROTAC degraders, basing on selective-BD1 BET inhibitor
1
2
365.3±17.3
62.57±5.83
3690±145
6
. We identified degrader which is highly potent against leukemia
b
JQ1
---
39.09±10.35
cell line MV4-11 and a variety of solid tumor cells in the NCI cell
bank, including, MDA-MB-231, MDA-MB-468, SK-MEL-5 and
SNB-75. The BET degrader was able to induce a degraduation of
BRD4 protein in a time-dependent and dose-dependent manner.
Moreover, representative compound showed depletion selectivity
for BRD4 and BRD2 over BRD3 with cellular potencies, low
cytotoxicity and induction of cell apoptosis for treating cancers.
a
b
IC50 values were obtained from two separate experiments; Used as a
positive control.
To explore the interaction with proteins, we predicted the
binding mode of compound 6 with BRD4 BD1 (Figure 2C). The
binding conformation of the 1-methylbenzo[cd]indol-2(1H)-one
moiety to Asn140 of the KAc pocket interacts with key hydrogen
2
7
bonds, consistent with similar reported conformations .
Additional hydrogen bonding interactions are formed between the
oxygen atom of the sulfonamide and the WPF water molecule. It
is a significant difference that the para position of the ortho-
methoxyphenyl group of compound 6 points to the Asp144 residue,
and the ortho-methoxy group points to Leu92 (Figure 2C). This is
the key to highlighting the selective binding of BD1 over BD2.
The steric effect was performed between the para-position of the
methoxy group on the benzene ring and the imidazole of His437
on BD2. Obviously, the binding to BD2 is unfavorable with
AlphaScreen. The 4-position of 1-methylbenzo[cd]indol-2(1H)-
one is in the BC loop solvent region and has a very large space. As
2
. Results and discussion
To further evaluate the selectivity of 6 to the Bromodomain
family, we used the differential scanning fluorimetry (DSF) to test
the ΔT value of 48 bromodomains at a concentration of 10 μM
Figure 2B and Figure S1 in Supporting Information). The results
of compound 6 vs. bromodomains showed that not only the BET
family protein was selected to have a ΔT value greater than 4.5 °C,
but also the binding of BD1 was better than BD2 in the BET family.
m
(
m
m
Furthermore, it exhibits moderate ΔT values for BRPF1B and
TAF1B in the non-BET family.

shown in Table 1, there was a slight decrease in the IC50 and
selectivity of compound 12. Therefore, the 4-position of 1-
methylbenzo[cd]indol-2(1H)-one is suitable for the introduction of
a pharmacodynamic fragment having the characteristics of
PROTAC.
for an E3 ligase complex for the design of PROTAC BET
degraders. Cereblon and Von Hippel-Lindau (VHL) are adaptor
protein for the cullin 4A RING E3 ligase complex. Thalidomide,
lenalidomide and VHL-based peptidic are widely used in the
design of novel PROTACs. We used thalidomide as a ligand for
cereblon / cullin 4A and 6 as a BET inhibitor for the design of new
PROTAC BET degraders.
2
8
Our model of 6 docking (Figure 2C) with BRD4 BD1 protein
suggested that the 4-position of the 1-methylbenzo[cd]indole-
2
(1H)-ketone core was exposed to the solvent environment
adjacent to the BC loop, making it a suitable site to link to a ligand
Figure 2. (A) Structure of selective-BD1 BET inhibitor 6 and its protein activities. (B) The bromodomains selectivity profile of 6 was shown by the bromodomains
family tree vs. 48 bromodomains at 10 μM. Heat map shows the relative ΔTm values. Red indicates large ΔTm, and green indicates small ΔTm. (c) Binding mode
analysis of compound 6 with BRD4 BD1 protein (protein shown as cartoon, PDB code 3MXF).
We first synthesized compound 13-17 as a potential BET
degrading agent, carrying the propionic acid group of intermediate
compound (the 4 position of compound 6 was replaced by a propyl
group) and connecting the C4 atom of the isoindolin ring of
thalidomide through a different carbon chain (Figure 3A). BET
degraders had been reported to be sensitive to most leukemia cell
lines such as MV4-11 and Molm-13. Therefore, we evaluated the
antiproliferative activity of the designed BET protein degradation
against these two cell lines (Figure 3B-C). As a control, we also
evaluated the IC50 values of compound 6 against MV4-11 and
Molm-13 (0.370 μM and 3.369 μM, respectively)
Figure 3. Structure and antiproliferative activity of designed PROTAC BET degraders: (A) Chemical structure designed PROTAC BET degraders (red shown as
BET inhibitor, blue shown as linker and black shown as Cereblon inhibitor); (B-C) MV4-11 and Molm-13 cells treated with PROTACs and their corresponding
BET targeting ligands for 48 h prior to quantitation of cell viability.
There are many factors affecting the efficiency of PROTAC
molecular degradation, including protein ligand affinity, ligand
type of E-ubiquitinated ligase, site of linker, length and type of
linker, and overall physical and chemical properties of the
molecule. The BET proteins are a "reader" key nuclear protein
involved in gene transcription. The James E. Bradner team
identified the relationship between their compound degradation
ability and treatment time. Figure 3B-C shows that the length of
2
9

degrader linker is closely related to their cellular activity. Among
them, the carbon chain length of 7 to 9 carbon chain length is
superior in two leukemia cell lines (14, 15 and 16 in Figure 3).
Compounds 14, 15 and 16 are performed better anti-proliferation
activity with IC50 values of 25.23 nM, 12.25 nM and 32.18 nM for
MV4-11 cells, respectively. Simultaneously, 15 has IC50 values of
cell lines. Compound 15 with optimal cell viability was used to
accurately identify the appropriate treatment time. We designed six
different time gradients to further determine the degradation-effect
relationship of the degradation agent (Figure. 4A-B). Treatment
of MV4-11 cells with compound 15 for 1 h, 2 h and 4 h failed to
show BRD4 degradation at 100 nM. When treatment time of ≥ 8
hours with compound 15 at a concentration of 100 nM, the BRD4
protein was reduced very efficiently. Therefore, during the
subsequent western blotting analysis, the incubation time of the
experimental design compounds was greater than 8 h.
5
1.96 nM against Molm-13 cell lines. In order to examine
treatment time, we investigated the degradation efficiency-time
dependence. First, we selected compounds 15 with better anti-
proliferation activity with IC50 values of 12.25 nM for MV4-11
Figure 4. Western blotting analysis of BRD4 proteins and c-Myc in MV4-11. (A-B) Immunoblot for BRD4, c-Myc and GAPDH after a 1 hours, 2 hours, 4 hours,
hours, 16 hours, or 24 hours of treatment with DMSO and 15 (1 μM) in MV4-11 and quantified relative to GAPDH. (C-D) Immunoblot for BRD4, c-Myc and
8
GAPDH after a 18-hour treatment with DMSO, 13-17 (1 μM), JQ1(1) and 6 (1 μM) in MV4-11 cells and quantified relative to GAPDH.
Figure 5. Western blotting analysis of BRD4 proteins and c-Myc in MV4-11 leukemia cells treated with BET degrader 14 and 15. (A-B) Immunoblot for BRD4,
c-Myc and GAPDH after treatment with 14 for 18 hours at the indicated concentration gradients (10 nM, 50 nM, 100 nM, 500 nM or 1000 nM), respectively and
quantified relative to GAPDH. (C-D) Immunoblot for BRD4, c-Myc and GAPDH after treatment with 15 for 18 hours at the indicated concentration gradients (10
nM, 50 nM, 100 nM, 500 nM or 1000 nM), respectively and quantified relative to GAPDH.

Figure 6. Western blotting analysis of 15: Immunoblot for BRD4, BRD3, BRD2 and c-Myc in MV4-11 leukemia cells treated with BET degrader 15 for 18
hours at the indicated concentration gradients (1 nM, 10 nM, 50 nM, 100 nM or 500 nM), respectively (A) and quantified relative to GAPDH (B).
The length of the linker is a very important factor. Therefore,
we designed compounds to to find the optimal length of linker.
Consistent with result of Figure 4C-D, 1((+)-JQ1) and 6 are
competitive BET inhibitors that down-regulate the expression of
the downstream Myc gene without altering the BRD4 protein.
Compound 13 with a short linker failed to reduce the BRD4
protein, consistent with its cell activity. Compared to compound
Table 2. The IG50 value of 15 against the NCI60 cancer cell lines
GI50
μM)
GI50
(μM)
Panel
Cell line
Panel
Cell line
a
(
CCRF-CEM 0.028
HL-60(TB) 0.022
LOX IMVI
3.97
MALME-3M 0.161
M14 0.937
MDA-MB-435 0.819
K-562
0.422
0.031
Leukemia
MOLT-4
1
3, compound 14 was designed to add a carbon, whose cell activity
increased 65-fold over 13 and the degradation activity was also
greatly enhanced. We found that compounds 14-16 with suitable
length linker are excellent in both anti-tumor cell proliferation
level and its western blotting analysis molecular level. Since the
linker was too long, the IC50 of compound 17 against MV4-11 was
reduced to 3429 nM. Its cell activity was reduced by more than
RPMI-8226 0.683 Melanoma SK-MEL-2
SR 0.122 SK-MEL-5
A549/ATCC 5.26
1.12
1.21
SK-MEL-28 0.891
b
EKVX
HOP-62
HOP-92
7.48
0.416
0.026
UACC-257
UACC-62
IGROV1
ND.
5.45
0.145
1.83
0.259
2.74
0.36
1
00-fold compared to compound 14-16. In summary, the length of
linker in the range of 8-10 atoms length can be very efficient in its
degradation efficiency. Among them, compound 15 with a 9 atoms
length linker, is the best.
To further study the concentration dependence manner,
compounds 14 and 15 with different concentration gradients were
set up to investigate the degradation capacity of BRD4 protein
Non-Small
Cell Lung NCI-H226 0.456
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
Cancer
NCI-H23
37.5
Ovarian
Cancer
NCI-H322M 0.355
NCI-H460
NCI-H522
ND.
2.74
(Figure 5). First, BRD4 protein was gradually degraded and c-
NCI/ADR-RES ND.
MYC was down-regulated in MV4-11 cells at a treatment
concentration of compound 14 of 50 nM for 18 h. As the
concentration of compound 14 increased to 500 nM, the gray scale
of the BRD4 protein band almost completely disappeared
consistent with the background. Desirably, the BRD4 protein was
degraded by treatment with compound 15 only at a concentration
of 10 nM. Complete degradation of the BRD4 protein can be
achieved at a concentration of 50 nM.
To confirm degradation activities of compound 15 basing on
the selective-BD1 BET inhibitor, we examined the impact of BET
proteins degradation and c-Myc levels. Interestingly, Figure 6
showed that compound 15 performed depletion selectivity for
BRD4 and BRD2 over BRD3, with good selectivity intra-BET
proteins selectivity. The selectivity of compound 15 reduces the
risk of potential toxicity and off-target effects. It is very difficult
that degraders acquire degradation activities profiles due to the
technical characteristics of PROTACS technology. Therefore, the
results of homologous protein selection are very necessary and
meaningful.
COLO 205 0.495
HCC-2998 0.316
SK-OV-3
MCF7
1.63
0.303
HCT-116
3.66
MDA-MB-231 0.032
Colon
Cancer
HCT-15
HT29
ND.
3.77
Breast
Cancer
HS 578T
BT-549
T-47D
0.186
0.078
0.034
SW-620
KM12
0.675
0.249
0.366
3.22
MDA-MB-468 0.020
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
786-0
A498
2.11
ND.
0.752
0.475
0.089
ND.
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
ND.
CNS
Cancer
2.63
Renal
Cancer
0.864
0.447
PC-3
0.248
5.91
0.895
Prostate
Cancer
Encouraged by the efficient protein degradation efficiency of
compound 15 and excellent blood tumor cell activity, we
submitted 15 to the Developmental Therapeutics Program at NCI
DU-145
ND.
a
GI50 Growth inhibition of 50 %: calculated from [(T
i
-T
z
)/(C-T
z
)] × 100 = 50;
b
(http://dtp.nci.nih.gov) to test their cell activity against the NCI 60
not tested
cancer cell lines. Compound 15 exhibit potent inhibit activity on
hematological tumors and solid tumors (Table 2 and Figure S2 in
Supporting Information).
From Table 2, compound 15 also exhibited excellent anti-
proliferative activity in 6 leukemia cell lines of NCI. Among them,
the GI50 values of the three leukemia cell lines were lower than 50
nM (GI50 is the concentration of test drug where 100 × [(T
i z
-T )/(C-
z
T )] = 50, the growth inhibitory power of the test com

pounds), consistented with the activity of leukemia MV4-11 cells.
Table 3. The IC50 value of 15 against multiple cancer cell lines
The safety considerations of compounds are important
research aspects of BET protein degraders. For the great majority
of the cell lines, LC50 (which signifies a cytotoxic effect, is the
IC 50
IC 50
(μM)
Panel
Cell line
Panel Cell line
(μM)
i z z
concentration of drug where [(T -T )/T ] × 100 = 50, the control
optical density is not used in the calculation of LC50) was >100 μM
(Figure S2 in Supporting Information). It is indirectly indicated
that the compound 15 with selective degradation for BRD4 and
BRD2 over BRD3 has good targeting and safety, since the data
aggregates specific cell lines, rather than extensive inhibitory
activity against all cell lines.
Subsequently, we used flow cytometry to study the ability of
15 to induce MV4-11 apoptosis lines, and found that 15 has a
strong induction effect in a concentration-dependent manner
(Figure 7A and Figure S4). In the MV4-11 cell line for 24 hours,
compared to the DMSO, it induced >80% of cells to undergo
apoptosis treatment at concentrations as low as 100 nM. The
induction apoptosis rate of negative control 6 was 86.07% at a high
concentration of 10 μM for 24 hours. Meanwhile, when the
treatment time was extended to 48 hours, the apoptosis rate of 15
significantly increased to 86.86% at a concentration as low as 30
nM (Figure 7B and Figure S4).
MV-4-11
RPMI8226
A375
0.009797 Colon colo-205 0.1557
Leukemia
Melanoma
0.4395
cancer
DU145
TT
A549
H1975 0.4588
HCC827 0.5802
H1650
H358
3.66
0.03745
13.74
a
2.435 Thyroid
0.081
Non-
2
2RV1
LNCap clone FGC 0.62
Prostate
Cancer
Small Cell
Lung
Cancer
LNCap
PC-3
Vcap
0.9095
0.932
1.332
1.381
0.2504
a
Human thyroid cancer cell line.
Excitingly, compound 15 also showed outstanding anti-cell
proliferation activity against multiple solid tumor cell lines.
Mainly concentrated in breast cancer cell lines, the GI50 values of
triple-negative cell lines MDA-MB-231 and MDA-MB-468 were
2 nM and 20 nM, respectively. Other breast cancer cell lines have
GI50 values less than 0.5 μM. In addition, other various solid tumor
cell lines can be inhibited by compound 15, such as GI50 values of
3
2
6 nM and 89 nM for non-small cell lung cancer HOP-92 and CNS
cancer SNB-75, respectively. Furthermore, we further increased
the cancer cell spectrum. As shown in Table 3 and Figure S3 in
Supporting Information, the cell activity results showed that the
compound 15 had good anti-proliferation activity including
prostate Cancer (22RV1 IC50: 0.081 μM), colon cancer(colo-205
IC50: 0.1557μM) and thyroid cancer(TT IC50: 0.037451 μM).
In addition, we used flow cytometry analysis (Figure 7C and
Figure S5) to investigate the effect of 15 on cell cycle in MV4-11.
Compound 15 was found to block the cell cycle at 100 nM
(71.37% of G1 phase) comparable to compound 6 at 10 μM.
Significantly higher than the G1 phase of the control was 59.66%.
As the concentration increases ≥ 300 nM, the tumor cells are
completely blocked in the G1 phase.
Figure 7. Flow cytometry analysis of apoptosis induction and cell cycle arrest by degrader 15 and inhibitor 6 in MV4-11 leukemia cells. (A-B), different
concentrations of gradient degrading agent 15 and inhibitor 6, the drug treatment time was 24 h (A) and 48 h (B) of apoptosis induction, respectively. (C), different
concentrations of gradient degrading agent 15 and inhibitor 6, the drug treatment time was 24 h of cell cycle arrest.
Heck coupling reaction with methyl acrylate. The intermediates
3
. Chemistry
were reduced to hydrogenation used for a next step. Finally, the
title compound 24 was prepared with 2-methoxybenzenesulfonyl
chloride. The synthesis of the BET Degraders 13-17 were shown
in Scheme 2. Starting from 4-fluoroisobenzofuran-1,3-dione and
The synthesis of the BET inhibitor 24 is shown in Scheme 1.
Starting from inexpensively available 1,8-Naphthalic anhydride,
intermediate 18 was synthesized through halogenation reaction
with bromine and silver sulfate in concentrated sulfuric acid. Two
crucial steps, a nucleophilic substitution reaction sulfonylation and
strong base decarboxylation, were carried out one pot, showing
moderate yields obtained 19. Subsequently, 22 was synthesized
through a three-step reaction including methylation with methyl
iodide, nitrification under acetic acid-nitric acid conditions and
Scheme 2. Compound 24
3
-aminopiperidine-2,6-dione hydrochloride, the intermediate 25
was synthesized under heating with acetic acid and acetic acid.
Subsequently, 29a-29e were prepared by two steps including
nucleophilic amination and trifluoroacetic acid Boc reaction.
Finally, the title compounds 13-17 were obtained by condensation
reaction of the intermediates 29a-29e with 24, respectively.

Reagents and conditions: (a) Br
I, 60% NaH, DMF; (e) methyl acrylate, Pd(PPh
DCM, r.t; (h) NaOH, HCl, H O, r.t.
2
, H
2
SO
4
, Ag
2
SO
4
, 60 °C; (b) i: NH
2
OH HCl, TsCl, pyridine, 90 °C; ii:NaOH, HCl, EtOH, H
2 3
O; (c) HNO , AcOH, 60 °C; (d)
CH
3
3
)
4
, 1,4-dioxane, N , 80 °C; (f) Pd/C, H
2
2
, THF, MeOH, r.t.; (g) 2-methoxybenzenesulfonyl chloride, pyridine,
2
Scheme 2. Compounds 13-17.
a
2
Reaction conditions: (a) AcONa, AcOH, 105 °C; (b) Proper amine, 1,4-dioxane, N , 80 °C; (c) TFA, DCM, rt. (d) Compound 24, HATU, DIPEA, DMF, rt..
(
Merck Kieselgel GF254) and spots were visualized with UV light
4
. Conclusion
or iodine.
We describe a BET inhibitor, which binds to BRD4 BD1
5.1.1 General Procedure for Synthesis of Compound 2 in Scheme
with an affinity of 50-fold over BRD4 BD2 and is selected for
1.
.1.1.1
5-bromo-1H,3H-benzo[de]isochromene-1,3-dione (18). To
binding to the BET family compared to other Bromodomains. The
binding conformation of compound 6 to BRD4(1) protein was
described, and novel PROTAC degraders of BET proteins were
designed by linking the Thalidomide with a linker. The structure-
activity relationship between length of the linker and anti-tumor
activity was explored and summarized. When the linker is 8
carbon-based distances, the degrader 15 has an efficient inhibition
of blood tumor and solid tumor cell growth in cell profiles. In
addition, compound 15 exhibited a very low cytotoxic effect in the
NCI60 cell lines. The study found that BRD4 proteins in MV4-11
was completely degraded by compound 15 for 8 h and dose-
dependent tests showed that compound 15 was still effective as
low as 10 nM. Besides, this molecule could induce apoptosis and
cell cycle arrest. We have demonstrated that BET ligands with
selectivity for bromodomains could be designed to achieve
selective degraders of intra-BET proteins, providing insights into
the design of selective degradation intra-BET proteins and low
toxicity degraders. More generally, we provide efficiently novel
PROTAC degraders of BRD4 and BRD2 proteins as a promising
drug for the treatment of cancer.
5
a solution of 1,8-Naphthalic anhydride (10.0 g, 50.5 mmol, 1
equiv) and silver sulfate (7.9 g, 25.25 mmol, 0.50 equiv) in
concentrated sulfuric acid (200 mL) were stirred at room
temperature. Bromine (3.2 mL, 63.0 mmol, 1.26 equiv) was added
over 30 min, and then heated to 60 °C for 8-10 h, before cooling
back to 20 °C. The solid silver bromide by product was filtered off
at a water pump to give a clear orange solution. This mixture
reaction was added dropwise to ice water mixture (1 L). An off-
white solid precipitates was filtered. The filter cake was washed
once by displacement with water (50 mL) and twice with cold
ethanol (100 mL each) and dried in vacuo at 60 °C to constant
weight to yield the title compound 18 as white solid (12.54 g, 45.5
+
1
mmol, 90% yield). [M+H] : 277.1. H NMR (300 MHz, DMSO-
) δ 8.83 (s, 1H), 8.57 – 8.44 (m, 3H), 7.93 (q, J = 9.4, 8.6 Hz,
1H).
d
6
5
.1.1.2
4
-bromobenzo[cd]indol-2(1H)-one (19). To a solution of 5-
5
. Materials and methods
bromo-1H,3H-benzo[de]isochromene-1,3-dione 18 (10 g, 36.2
mmol, 1 equiv) and hydroxylamine hydrochloride (2.5 g, 36.2
mmol, 1 equiv) in pyridine (70 mL) was conducted under reflux
for 2 h, followed by cooling to 80 °C. Then ptoluenesulfonyl
chloride (13.8 g, 72.4 mmol, 2 equiv) was added to the reaction
system. After addition, the temperature was raised and the reaction
was carried out under reflux for 2 h, followed by cooling. The
reaction mixture was poured into 0.40 L of water and stirred to
precipitate crystals, which were collected by filtration. The
crystals were transferred to a beaker and washed successively with
5
.1. Chemistry General Methods.
Unless otherwise specified, reagents were purchased from
commercial suppliers and used without further purification.
Melting points were determined by X-4 digital display micro-
melting point apparatus (Beijing Tech Instrument Co., Ltd.); NMR
spectra were recorded on Bruker AVANCE AV-600 spectrometer
1
3
(
400 or 300 MHz for 1H, 200 or 150 MHz for C); Mass spectra
were obtained on the Agilent 1100 LC / MSD mass spectrometer
Agilent, USA) and Q-tofmicro MS (micromass company). The
HPLC traces of title compounds were provided on the Agilent
260 HPLC (Agilent, USA). All reactions were monitored by TLC
0
3
.5 L of a NaHCO aqueous solution and 0.5 L of water, followed
(
by filtration. The crystals were washed with water and dried to
give an intermediate for further reaction. The whole amount of the
1

intermediate, 40 ml of ethanol and 50 ml of water were put in a
reactor and stirred. Then 130 ml of a 1.4 mol/L aqueous solution
of NaOH was added dropwise to the mixture. Thereafter, the
mixture was heated to refluxing temperature, at which the reaction
was carried out for 3 h while distilling off ethanol. After
completion of the reaction, the reaction mixture was cooled to 75
Methyl-3-(6-((2-methoxyphenyl)sulfonamido)-1-methyl-2-
oxo-1,2-dihydrobenzo[cd]indol-4-yl)propanoate (12). To a
solution of compounds 22 (380 mg, 1.21 mmol, 1 equiv) in 20 mL
EtOH was added 100 mg the Raney-Nickel catalyst (with about
50% water). The mixture reaction was flushed thrice with argon
then thrice with hydrogen. The flask was charged with hydrogen
gas, and reaction mixture was left to stir for 5 h. The reaction was
completely detected by LTC. The reaction mixture was filtered
through celite, and filtered and evaporated. The maroon rude was
used as a next step. To a solution of the maroon rude in 20 mL
DCM was added 2-methoxybenzenesulfonyl chloride (301 mg,
1.46 mmol, 1.2 equiv) and pyridine 0.5 mL. The reaction solution
was stirred at room temperature overnight. The reaction was
completed by TLC and then concentrated. Purification by column
chromatography (30% EA/PE) yielded the title compound 12 as
yellow solid (287. mg, 0.63 mmol, 53% yield). m.p 103-105 °C.
°
C, and 60 ml of concentrated HCl was added dropwise. In the
meantime, crystals precipitated at 60 °C. After completion of the
dropwise addition, the mixture was further cooled. The
precipitated crystals were collected by filtration, washed with ion-
exchanged water, and dried to give the title compound 19 as white
+
1
solid (2.76 g, 11.22 mmol, 31% yield). [M+H] : 248.1. H NMR
300 MHz, DMSO-d ) δ 10.92 (s, 1H), 8.44 (s, 1H), 8.10 (s, 1H),
.58 (d, J = 3.5 Hz, 2H), 7.01 (dd, J = 5.9, 1.8 Hz, 1H).
(
6
7
5
.1.1.3
-bromo-6-nitrobenzo[cd]indol-2(1H)-one (20). To
solution of 4-bromo-6-nitrobenzo[cd]indol-2(1H)-one 19 (7 g,
8.5 mmol) in AcOH (15 mL) was added 69% HNO (2 mL, 42.75
mmol) heat to 50 °C - 65 °C. The mixture reaction was stirred for
h. When TLC analysis showed complete conversion of the
1
4
a
HPLC analysis: retention time = 3.359 min; peak area, 97.75%. H
NMR (300 MHz, Chloroform-d) δ 7.99 (s, 1H), 7.90 (s, 1H), 7.68
(dd, J = 7.7, 1.7 Hz, 1H), 7.51 (dd, J = 15.9, 1.8 Hz, 1H), 7.16 (s,
1H), 7.10 (d, J = 7.8 Hz, 2H), 6.95 (t, J = 7.7 Hz, 1H), 6.63 (d, J =
7.5 Hz, 1H), 4.13 (s, 3H), 3.68 (s, 3H), 3.34 (s, 3H), 3.19 (t, J =
2
3
4
+
starting material, the reaction mixture was poured into water
filtrated through Cellit and the filtrate was concentrated in
vacuum. The crude product was purified by silica gel column
chromatography (PE/DCM) to yielded the title compound 20 as
7.6 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H). HRMS-EI m/z [M+H] calcd
+
for C H N O S : 455.1271, found: 455.1271.
2
3
23
2
6
5
.1.1.6
3-(6-((2-methoxyphenyl)sulfonamido)-1-methyl-2-oxo-1,2-
+
1
yellow solid (6.9 g, 23.65 mmol, 83% yield). [M+H] : 293.1. H
NMR (300 MHz, DMSO-d ) δ 8.91 (s, 1H), 8.67 (d, J = 8.1 Hz,
H), 8.31 (s, 1H), 7.32 (d, J = 8.1 Hz, 1H), 3.38 (s, 3H).
6
dihydrobenzo[cd]indol-4-yl)propanoic acid (24). To a solution
of compounds 12 (250 mg, 0.55 mmol, 1 equiv) in 15mL MeOH
was added 1 mL sodium hydroxide solution (2 mmol/mL). The
reaction solution was stirred at room temperature for 1 hour. The
reaction was completely detected by LTC. The methanol in the
reaction was distilled off under reduced pressure. This mixture was
extracted with AcOEt (50 mL) and water (30 mL × 3). The
aqueous layer was acidified with a solution of 2N HCl (1.5 mL)
and extracted with methylene chloride. The organic layer was
1
5
.1.1.4
-bromo-1-methyl-6-nitrobenzo[cd]indol-2(1H)-one (21).
4
To a solution of 4-bromo-1-methyl-6-nitrobenzo[cd]indol-2(1H)-
one 20 (6.5 g, 22.26 mmol, 1 equiv) in anhydrous DMF (100 mL)
was stirred at 0 °C. The solution was added 60% NaH (1.34 g,
3
3.35 mmol, 1.5 equiv) in 3 batches and stirring for 30 minutes.
Methyl iodide (2.06 mL, 33.35 mmol, 1 equiv) was added
dropwise to the mixture, and the whole was stirred at room
temperature for 3 h and then was poured into ice water. This
mixture was extracted with AcOEt. The organic layer was washed
dried over MgSO and concentrated under vacuum to give the title
compound 24 as yellow solid (220 mg, 0.5 mmol, 91% yield)
requiring no further purification.
4
5
.1.2 General Procedure for Synthesis of Compound 13-17.
2 4
with H O and brine for 4 times, dried (MgSO ), concentrated and
purified by silica gel column chromatography to afford the title
5.1.2.1
compound 21 as yellow solid (5.52 g, 18.04 mmol, 81% yield).
2
-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione
+
1
[
M+H] : 306.9. H NMR (300 MHz, DMSO-d
6
) δ 8.91 (s, 1H),
(
25). To a solution of 4-fluoroisobenzofuran-1,3-dione (2 g, 12.04
mmol, 1 equiv) in 20 mL AcOH was added 3-aminopiperidine-
,6-dione hydrochloride (2.18 g, 13.24 mmol, 1.1 equiv) and
8
3
.67 (d, J = 8.1 Hz, 1H), 8.31 (s, 1H), 7.32 (d, J = 8.1 Hz, 1H),
.38 (s, 3H).
2
5
.1.1.4
AcOK (2.36 g, 24.08 mmol, 2 equiv). The solution was then stirred
at 60 °C for 12 h. After cooling to room temperature, the solution
was extracted with AcOEt. The organic layer was washed with
H O and brine for 4 times, dried (MgSO ), concentrated and
2 4
purified by silica gel column chromatography to afford the title
Methyl(E)-3-(1-methyl-6-nitro-2-oxo-1,2-
dihydrobenzo[cd]indol-4-yl)acrylate (22). To mixture of 4-
bromo-1-methyl-6-nitrobenzo[cd]indol-2(1H)-one 20 (500 mg,
1
.63 mmol, 1 equiv), Pd(AcO)
tri-o-tolylphosphane (49 mg, 0.16 mmol, 0.1 equiv) and K
675 mg, 4.88 mmol, 3 equiv) was dissolved in anhydrous 1,4-
dioxane. The reaction mixture was added methyl acrylate (293 mL,
.88 mmol, 2 equiv). The reaction system was exchanged by N
2
(18 mg, 0.08 mmol, 0.05 equiv),
compound 25 as a white solid (2.13 g, 7.71 mmol, 64% yield).
2
CO
3
+
1
[
M+H] : 277.2. H NMR (300 MHz, DMSO-d
6
) δ 11.16 (s, 1H),
(
7
1
–
.95 (td, J = 7.8, 4.4 Hz, 1H), 7.84 – 7.68 (m, 2H), 5.17 (dd, J =
2.9, 5.4 Hz, 1H), 2.99 – 2.81 (m, 1H), 2.67 – 2.52 (m, 2H), 2.15
2.00 (m, 1H).
4
2
,
then, heated to 80 °C for 7 h. When TLC analysis showed complete
conversion of the starting material, the mixture was extracted with
AcOEt and water. The organic layer was washed with H
brine, dried (MgSO ), concentrated and purified by silica gel
column chromatography to afford the title compound 22 as yellow
5.1.2.2
tert-butyl(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)amino)ethyl)carbamate (26). To a solution
of 4-fluoroisobenzofuran-1,3-dione (300 mg, 1.09 mmol, 1 equiv)
in 15 mL anhydrous 1,4-dioxane was added tert-butyl (2-
aminoethyl)carbamate (226 mg, 1.41 mmol 1.3 equiv). The
solution was then stirred at 80 °C for 12 h. After cooling to room
temperature, the solution was extracted with AcOEt (50 mL × 3).
2
O and
4
+
1
solid (381 mg, 1.22 mmol,75% yield). [M+H] : 313.2. H NMR
300 MHz, DMSO-d ) δ 8.96 (s, 1H), 8.70 – 8.56 (m, 2H), 7.98 (d,
J = 16.1 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 6.98 (d, J = 16.1 Hz,
(
6
1
H), 3.78 (s, 3H), 3.40 (s, 3H).
5
.1.1.5
The organic layer was washed with H
2
O and brine for 4 times,
dried (MgSO ), concentrated and purified by silica gel column
4

chromatography (EA/PE) to afford the title compound 26 as a light
yellow solid (199 mg, 0.48 mmol, 44% yield). [M+H] : 417.2. H
NMR (300 MHz, Chloroform-d) δ 8.32 (s, 1H), 7.50 (dd, J = 8.5,
in 5 mL DCM was added 1 mL TFA. The solution was stirred at
room temperature for 1 h. The reaction was completely detected
by LTC. The trifluoroacetic acid in the solution was concentrated
under reduced pressure by an oil pump. The light yellow rude (135
mg, 94% yield) was used as a next step. To a solution of light
yellow rude (56 mg, 0.18 mmol, 1.3 equiv) in 20 mL anhydrous
DMF was added 24 (60 mg, 0.136 mmol, 1 equiv), HATU (76 mg,
+
1
7
6
2
.1 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H),
.40 (s, 1H), 4.93 (q, J = 5.3 Hz, 2H), 3.50 – 3.30 (m, 4H), 2.94 –
.70 (m, 3H), 2.12 (dt, J = 8.8, 3.1 Hz, 1H), 1.44 (s, 9H).
5
.1.2.3
0
.20 mmol, 1.5 equiv) and DIPEA (52 mg, 0.40 mmol, 3 equiv).
tert-butyl(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)amino)propyl)carbamate (27). Compound
7 was prepared according to 26 on 1.09 mmol scale, starting from
The reaction was completed by TLC. The reaction solution was
poured into water and extracted with AcOEt. The organic layer
was washed with H O and brine for 4 times, dried (MgSO ),
2
2
4
tert-butyl (3-aminopropyl)carbamate and 25. Purification by
column chromatography (EA/PE) yielded the title compound 27
concentrated and purified by silica gel column chromatography
(EA/PE) to afford the title compound 13 as a light yellow solid (74
mg, 0.10 mmol, 74% yield). m.p 183-185 °C. HPLC analysis:
+
as a light yellow solid (182 mg, 0.42 mmol, 39% yield). [M+H] :
1
1
4
31.3. H NMR (300 MHz, Chloroform-d) δ 8.59 (s, 1H), 7.49 (t,
retention time = 3.254 min; peak area, 98.47%. H NMR (300
J = 7.9 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H),
MHz, Chloroform-d) δ 8.82 (s, 1H), 8.06 (s, 1H), 7.84 (s, 1H), 7.65
(s, 2H), 7.49 (t, J = 7.9 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.05 (d,
J = 7.6 Hz, 3H), 6.91 (t, J = 7.7 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H),
6.55 (d, J = 7.5 Hz, 1H), 6.22 (s, 2H), 4.97 (s, 1H), 4.09 (s, 3H),
6
2
.34 (s, 1H), 4.95 (t, J = 5.9 Hz, 1H), 4.81 (s, 1H), 3.29 (dt, J =
9.0, 6.7 Hz, 4H), 2.96 – 2.63 (m, 3H), 2.19 – 2.05 (m, 1H), 1.85
(
p, J = 7.1 Hz, 2H), 1.46 (s, 9H).
.1.2.4
tert-butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)amino)butyl)carbamate (28a). Compound
8a was prepared according to 26 on 1.09 mmol scale, starting
3
1
.45 (s, 2H), 3.28 (s, 3H), 3.31 -3.11 (m, 6H), 2.83 (dt, J = 22.3,
5
+
0.3 Hz, 4H), 2.58 (d, J = 7.7 Hz, 2H). HRMS-EI m/z [M+H]
+
13
35 6 9
calcd for C37H N O S : 739.2181, found: 739.2175. C NMR
(101 MHz, Chloroform-d) δ 172.36 , 171.91 , 169.48 , 169.44 ,
2
1
1
1
1
3
67.98 , 167.68 , 156.37 , 146.44 , 142.22 , 138.09 , 136.06 ,
35.03 , 132.39 , 130.73 , 126.90 , 126.78 , 126.50 , 126.44 ,
25.71 , 125.61 , 124.36 , 123.96 , 120.54 , 116.52 , 112.04 ,
11.35 , 109.82 , 104.45 , 77.26 , 56.32 , 48.93 , 39.91 , 37.94 ,
1.43 , 28.68 , 26.27 , 22.79 .
from tert-butyl (4-aminobutyl)carbamate and 25. Purification by
column chromatography (EA/PE) yielded the title compound 28a
+
as a light yellow solid (248 mg, 0.57 mmol, 53% yield). [M+H] :
1
4
7
6
45.3. H NMR (300 MHz, Chloroform-d) δ 8.36 (s, 1H), 7.53 –
.45 (m, 1H), 7.09 (d, J = 7.1 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H),
.24 (s, 1H), 4.92 (dd, J = 11.9, 5.4 Hz, 1H), 4.63 (s, 1H), 3.30 (t,
5
.1.2.8
J = 6.7 Hz, 2H), 3.17 (d, J = 6.2 Hz, 2H), 2.94 – 2.63 (m, 4H), 2.12
dt, J = 8.9, 3.3 Hz, 1H), 1.77 – 1.50 (m, 5H).
N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)propyl)-3-(6-((2-methoxyphenyl)sulfonamido)-1-
(
methyl-2-oxo-1,2-dihydrobenzo[cd]indol-4-yl)propanamide
5
.1.2.5
tert-butyl(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)amino)pentyl)carbamate
(
14). Compound 14 was prepared according to 13 in two steps,
respectively, starting from 27 (180 mg, 0.42 mmol, 1 equiv) and
4 (60 mg, 0.136 mmol, 1 equiv). Purification by column
(28b).
2
Compound 28b was prepared according to 26 on 1.09 mmol scale,
starting from tert-butyl (5-aminopentyl)carbamate and 25.
Purification by column chromatography (EA/PE) yielded the title
compound 28b as a light yellow solid (229 mg, 0.50 mmol, 46%
chromatography (EA/PE) yielded the title compound 14 as a light
yellow solid (66 mg, 0.088 mmol, 65% yield). m.p 161-163 °C.
1
HPLC analysis: retention time = 3.317 min; peak area, 97.01%. H
NMR (300 MHz, Chloroform-d) δ 8.59 (s, 1H), 8.05 (s, 1H), 7.89
+
1
yield). [M+H] : 459.23. H NMR (300 MHz, Chloroform-d) δ 8.54
s, 1H), 7.48 (dd, J = 8.6, 7.1 Hz, 1H), 7.08 (d, J = 7.1 Hz, 1H),
(s, 1H), 7.72 (s, 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.43 – 7.38 (m, 1H),
(
7
1
3
3
.16 – 6.99 (m, 3H), 6.91 (t, J = 7.6 Hz, 1H), 6.70 (d, J = 8.5 Hz,
H), 6.60 (d, J = 7.6 Hz, 1H), 5.86 (s, 2H), 4.95 (s, 1H), 4.13 (s,
H), 3.31 (s, 3H), 3.34 – 3.31 (m, 2H), 3.18 (d, J = 7.0 Hz, 2H),
.08 (s, 2H), 2.89 – 2.72 (m, 4H), 2.59 (t, J = 7.2 Hz, 2H), 2.13 (d,
6
1
2
2
.87 (d, J = 8.5 Hz, 1H), 6.24 (t, J = 5.6 Hz, 1H), 4.97 – 4.87 (m,
H), 4.64 (s, 1H), 3.26 (q, J = 6.6 Hz, 2H), 3.13 (q, J = 6.6 Hz,
H), 2.91 – 2.72 (m, 3H), 1.67 (q, J = 7.1 Hz, 2H), 1.57 – 1.49 (m,
H), 1.44 (s, 9H), 1.26 (d, J = 3.5 Hz, 3H).
+
+
J = 8.5 Hz, 2H). HRMS-EI m/z [M+H] calcd for C38
H
37
N
6
O
9
S :
53.2337, found: 753.2325. C NMR (101 MHz, Chloroform-d)
δ 172.48 , 171.78 , 169.43 , 169.35 , 168.06 , 167.65 , 156.37 ,
1
3
7
5
.1.2.6
tert-butyl(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-
-yl)amino)hexyl)carbamate (28c). Compound 18c was prepared
146.44 , 142.18 , 138.10 , 136.08 , 135.03 , 132.40 , 130.74 ,
126.96 , 126.79 , 126.56 , 126.46 , 125.74 , 125.68 , 124.38 ,
124.00 , 120.57 , 116.52 , 112.06 , 111.39 , 109.87 , 104.53 , 77.25
, 56.35 , 48.93 , 39.98 , 37.97 , 32.44 , 31.43 , 28.67 , 26.31 , 22.80
4
according to 26 on 1.09 mmol scale, starting from tert-butyl(6-
aminohexyl)carbamate and 25. Purification by column
chromatography (EA/PE) yielded the title compound 28c as a light
.
+
1
yellow solid (240 mg, 0.51 mmol, 47% yield). [M+H] : 473.2. H
5
.1.2.9
NMR (300 MHz, Chloroform-d) δ 8.53 (s, 1H), 7.48 (dd, J = 8.5,
7
6
1
.1 Hz, 1H), 7.08 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H),
.24 (t, J = 5.7 Hz, 1H), 4.92 (dd, J = 11.8, 5.4 Hz, 1H), 4.60 (s,
H), 3.25 (q, J = 6.6 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H), 2.93 – 2.69
N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)butyl)-3-(6-((2-methoxyphenyl)sulfonamido)-1-
methyl-2-oxo-1,2-dihydrobenzo[cd]indol-4-yl)propanamide
15). Compound 15 was prepared according to 13 in two steps,
respectively, starting from 28a (240 mg, 0.56 mmol, 1 equiv) and
4 (60 mg, 0.136 mmol, 1 equiv). Purification by column
(
m, 3H), 2.12 (dt, J = 8.8, 3.1 Hz, 1H), 1.65 (q, J = 7.2 Hz, 2H),
.54 – 1.47 (m, 2H), 1.44 (s, 9H), 1.43 – 1.35 (m, 4H), 1.26 (d, J
3.4 Hz, 1H).
(
1
=
2
chromatography (EA/PE) yielded the title compound 15 as a light
5
.1.2.7
N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yellow solid (69 mg, 0.092 mmol, 68% yield). m.p 155-157 °C.
1
HPLC analysis: retention time = 2.734 min; peak area, 98.09%. H
yl)amino)ethyl)-3-(6-((2-methoxyphenyl)sulfonamido)-1-
methyl-2-oxo-1,2-dihydrobenzo[cd]indol-4-yl)propanamide
NMR (300 MHz, Chloroform-d) δ 8.46 (s, 1H), 8.06 (s, 1H), 7.88
(s, 1H), 7.69 – 7.55 (m, 2H), 7.48 (q, J = 9.0, 8.6 Hz, 2H), 7.18 –
(13). To a solution of compounds 26 (190 mg, 0.45 mmol, 1 equiv)

6
6
3
2
C
.99 (m, 3H), 6.91 (t, J = 7.6 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H),
.57 (d, J = 7.5 Hz, 1H), 5.92 (s, 2H), 4.93 (s, 1H), 4.12 (s, 3H),
BRD4 protein was added to a buffer solution containing 500 nM
NaCl, 10 nM HEPES, pH 7.5, and finally formulated into a 2
μM/20 μL solution in a 96-well plate. The compound 6 was added
to the solution at a concentration of 10 μM while adding the
fluorescent probe SYPRO Orange at a ratio of 1:1000. The
excitation and emission wavelengths of SYPRO Orange are set at
465 nm and 590 nm, respectively. The temperature is raised from
25 °C to 96 °C by 3 °C per minute and fluorescence readings are
taken at each interval. Then, according to the theoretical
conversion formula between fluorescence intensity and
temperature change, melting temperatures (Tm) were calculated
by fitting the sigmoidal melt curve to the Boltzmann equation
using GraphPad Prism. ΔTm is the difference in Tm values
calculated for reactions with and without compounds. The larger
the ΔTm value, the stronger the binding ability of the compound
to the protein, on the contrary, the weaker the binding force.
The AlphaScreen assay (Amplified Luminescent Proximity
Homogeneous Assay Screen) is used the binding assay of the
readers, which was performed by Reaction Biology Corp.
(Malvern PA) as described previously. The biotinylated peptide
binding to the reader domain of His-tagged protein is monitored
by the singlet oxygen transfer from the Streptavidin-coated donor
beads to the AlphaScreen Ni-chelate acceptor beads. Reaction
Procedure: 1. Deliver 2.5X BRD in wells of reaction plate except
No BRD control wells. Add buffer instead. 2. Deliver compounds
in 100% DMSO into the BRD mixture by Acoustic technology
(Echo550; nanoliter range). Spin down and pre-incubation for 30
min. 3. Deliver 5X Ligand. Spin and shake. 4. Incubate for 30 min
at room temperature with gentle shaking. IC values and curve fits
.27 (s, 3H), 3.26 - 3.20 (m, 2H), 3.08 (s, 2H), 2.94 – 2.67 (m, 4H),
+
.13 -2.09 (m, 2H). HRMS-EI m/z [M+H]
39
calcd for
+
13
39 6 9
H N O S : 767.2494, found: 767.2490. C NMR (101 MHz,
Chloroform-d) δ 171.57 , 171.37 , 169.56 , 168.81 , 167.97 ,
1
1
1
1
3
67.68 , 156.32 , 146.92 , 142.46 , 138.53 , 136.18 , 135.04 ,
32.44 , 130.72 , 126.92 , 126.81 , 126.50 , 126.44 , 126.06 ,
25.77 , 124.42 , 124.22 , 120.66 , 116.73 , 112.18 , 111.41 ,
09.78 , 104.20 , 77.26 , 56.51 , 48.90 , 42.46 , 39.34 , 38.20 ,
1.43 , 29.28 , 28.81 , 26.47 , 22.82 .
5
.1.2.10
N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)pentyl)-3-(6-((2-methoxyphenyl)sulfonamido)-1-
methyl-2-oxo-1,2-dihydrobenzo[cd]indol-4-yl)propanamide
(
16). Compound 15 was prepared according to 13 in two steps,
respectively, starting from 28b (220 mg, 0.48 mmol, 1 equiv) and
4 (60 mg, 0.136 mmol, 1 equiv). Purification by column
2
chromatography (EA/PE) yielded the title compound 16 as a light
yellow solid (67 mg, 0.085 mmol, 63% yield). m.p 151-153 °C.
HPLC analysis: retention time = 3.933 min; peak area, 97.41%. H
NMR (300 MHz, Chloroform-d) δ 8.36 (s, 1H), 8.05 (s, 1H), 7.89
1
(s, 1H), 7.66 (dd, J = 7.8, 1.7 Hz, 2H), 7.54 – 7.41 (m, 3H), 7.14 –
7
6
.02 (m, 3H), 6.92 (t, J = 7.6 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H),
.59 (d, J = 7.5 Hz, 1H), 6.13 (s, 1H), 5.67 (s, 1H), 4.91 (d, J = 5.4
Hz, 1H), 4.12 (s, 3H), 3.31 (s, 3H), 3.20 -3.11 (m, 6H), 2.87 – 2.76
m, 2H), 2.55 (t, J = 7.3 Hz, 2H), 1.53 (t, J = 7.3 Hz, 2H), 1.44 –
(
+
1
.37 (m, 2H), 1.33 – 1.16 (m, 4H). HRMS-EI m/z [M+H] calcd
5
0
+
13
for C40
H
41
N
6
O
9
S : 781.2650, found: 781.2648. C NMR (101
were obtained by Prism (GraphPad Software).
5.3. The Cell Apoptosis Assay
MHz, Chloroform-d) δ 171.81 , 171.61 , 169.54 , 169.06 , 167.99
,
167.68 , 156.35 , 146.84 , 142.46 , 138.35 , 136.14 , 135.06 ,
32.39 , 130.72 , 126.87 , 126.81 , 126.48 , 125.93 , 125.78 ,
24.30 , 124.11 , 120.62 , 116.75 , 112.15 , 111.39 , 109.74 ,
04.29 , 77.27 , 56.45 , 48.88 , 42.27 , 39.18 , 38.16 , 32.57 , 31.40
29.05 , 28.55 , 26.31 , 23.95 , 22.77 .
The apoptosis of MV4-11 cells was determined by Annexin
V-FITC/PI assay. Annexin V binds to phosphatidylserine, The
externalization of phosphatidylserine is one of the leading
indicators of apoptosis, which is one of the earliest indicators of
cellular apoptosis located on the cell membrane. Propidium iodide
1
1
1
,
5
.1.2.11
(PI) is a popular red-fluorescent nuclear and chromosome
N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)hexyl)-3-(6-((2-methoxyphenyl)sulfonamido)-1-
methyl-2-oxo-1,2-dihydrobenzo[cd]indol-4-yl)propanamide
17). Compound 17 was prepared according to 13 in two steps,
respectively, starting from 28c (240 mg, 0.50 mmol, 1 equiv) and
4 (60 mg, 0.136 mmol, 1 equiv). Purification by column
chromatography (EA/PE) yielded the title compound 17 as a light
yellow solid (63 mg, 0.081 mmol, 59% yield). m.p 142-144 °C.
HPLC analysis: retention time = 4.511 min; peak area, 96.29%. H
NMR (300 MHz, Chloroform-d) δ 8.63 (s, 1H), 8.03 (s, 1H), 7.85
counterstain. Since propidium iodide is not permeant to live cells,
it is also commonly used to detect dead cells in apopulation. It can
be used to differentiate necrotic, apoptotic and normal cells. Cells
5
(
(2 × 10 ) were seeded in 6-well plate and were treated with varying
concentrations of compounds for 24 h and 48 h. MV4-11 cells
were collected and incubated with 5 μL of FITC-conjugated
Annexin V (Nanjin keyGen Biotech Company). The nuclei were
then counterstained with 5 μL of Propidium Iodide. After the dual
staining, the cells were screened by a FAC Scan flow cytometer
(FACS Calibun, Becton Dickinson). The upper left corner of the
quadrant represents debris, lower left are live cells, upper right are
advanced apoptotic or necrotic cells and lower right are apoptotic
cells.
2
1
(
2
(
s, 1H), 7.75 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 8.0 Hz,
H), 7.07 (dd, J = 10.9, 7.9 Hz, 3H), 6.90 (t, J = 7.7 Hz, 1H), 6.83
d, J = 8.6 Hz, 1H), 6.59 (d, J = 7.6 Hz, 1H), 6.15 (s, 1H), 5.82 (d,
J = 6.2 Hz, 1H), 4.94 (s, 1H), 4.11 (s, 3H), 3.30 (s, 3H), 3.17 (t, J
5
.4. The Cell Cycle Assay
=
–
7.7 Hz, 6H), 2.90 – 2.70 (m, 3H), 2.55 (t, J = 7.6 Hz, 2H), 2.20
The Leukemia cells were treated with test compounds at
2.09 (m, 1H), 1.48 (t, J = 7.2 Hz, 2H), 1.32 (dd, J = 19.2, 11.8
different concentrations for 24 h. The prepared single cell
suspension was fixed with a volume fraction of 70% ethanol for 2
hours (or overnight), stored at 4 °C and the fixative was washed
away with PBS before staining (if necessary, the cell suspension
was filtered once with a 200 mesh screen). Add 100 microliter
RNase A 37 ° C water bath for 30 min; Add 400 microliter of PI
stain and mix well, avoiding light for 30 min at 4 °C. On-machine
detection, the red fluorescence was analyzed by flow cytometry
using a 488 nm laser (Cell Lab Quanta SC, Beckman Coulter). Cell
+
Hz, 4H), 1.15 (d, J = 7.5 Hz, 2H). HRMS-EI m/z [M+H] calcd
for C41
MHz, Chloroform-d) δ 171.68 , 171.31 , 169.51 , 168.83 , 167.95
+
13
H
43
N
6
O
9
S : 795.2807, found: 795.2797. C NMR (101
,
167.59 , 156.29 , 146.74 , 142.44 , 138.62 , 136.17 , 135.04 ,
32.38 , 130.72 , 126.94 , 126.73 , 126.52 , 126.44 , 126.20 ,
25.85 , 124.59 , 124.28 , 120.74 , 116.73 , 112.19 , 111.53 ,
09.86 , 104.16 , 77.25 , 56.54 , 48.90 , 41.97 , 38.87 , 38.20 ,
2.54 , 31.44 , 29.72 , 26.91 , 26.30 , 26.18 , 22.80 .
1
1
1
3
5
.2. Protein Inhibition Assay
cycle analysis was made using
FACSCalibun, Becton Dickinson).
.5. The Cell Growth Inhibition Assay
a FACScan cytometer
(
5
The differential scanning fluorimetry (DSF) was tested on
a Mx3005p Real Time PCR machine (Stratagene) device. The

2
3
.
.
C. Dhalluin, J.E. Carlson, L. Zeng, C. He, A.K. Aggarwal, M.-
M. Zhou, Structure and ligand of a histone acetyltransferase
bromodomain, Nature, 399 (1999) 491.
H. Fukazawa, A. Masumi, The conserved 12-amino acid stretch
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The human AML cell line MV4-11 was cultured in IMDM
media (Corning, USA) with 10% FBS and supplemented with 2%
L-glutamine and 1% pen/strep. The MV4-11 cell line was
maintained in culture media at 37 °C with 5% CO . The effects of
2
target compounds on MV4-11 proliferation were performed by
Nanjing Anacon Biotechnology Co., Ltd. Cells were cultured in
4
5
.
.
Q. Zhou, T. Li, D.H. Price, RNA polymerase II elongation
control, Annu Rev Biochem. 81 (2012) 119-143.
9
6-well culture plates (10000/well). The compounds of various
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determined after treatment with compounds for 72 h. Cell viability
was measured using the CellTiter-Glo assay (Promega, USA)
according to the manufacturer’s instructions, and luminescence
was measured in a multilabel reader (Envision2014, PerkinElmer,
USA). Data were normalized to control groups (DMSO) and
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9
25.
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5
.0 (GraphPad Software, San Diego, CA).
5
.6. The Western blotting
6
For Western blot analysis, 1.5 - 2 × 10 cells/well of MV4-11
cells were plated in 12-well plates and treated with compounds at
the preset concentrations and times. Cells were collected, washed
with cold PBS, and lysed in cold RIPA buffer containing Halt
protease inhibitors. 20 μL lysate were run in each lane of 4−20%
or 4−12% Novex gels and blotted into polyvinylidene difluoride
membranes. The antibodies used for immunoblotting were BRD4
7
.
K. Klein, P.A. Kabala, A.M. Grabiec, R.E. Gay, C. Kolling, L.L.
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suppressed by BET bromodomain inhibitor in mice through
RORC/IL-17A pathway modulation, Pharmacol Res. 99 (2015)
(
BRD4 (E2A7X) Rabbit mAb #13440), c-Myc (Myc-Tag (71D10)
Rabbit mAb #2278) and Santa Cruz Biotechnologies' GAPDH
GAPDH (D16H11) XP® Rabbit mAb #5174) purchased from
Cell Signaling Technology.
.7. Docking studies
8
.
(
5
2
48-257.
3
D structures of compounds 6 and 15 were built and
9
.
C.J. Ott, N. Kopp, L. Bird, R.M. Paranal, J. Qi, T. Bowman, S.J.
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minimized by the Ligand Preparation module in Schrodinger.
Crystal structures of BRD4 (PDB ID: 3MXF) were downloaded
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essential waters were deleted and the hydrogen atoms were added
to the protein, the protein was prepared using Protein Preparation
Wizard in the Schrodinger suite. The Glide module with extra
precision (XP) was selected for molecular docking. Compound 6
and 15 best conformations were minimized by a post docking
program. Figures of docking result were presented by Pymol
software (http://www.pymol.org).
2
852.
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1
0. A.C. Belkina, G.V. Denis, BET domain co-regulators in obesity,
inflammation and cancer, Nat. Rev. Cancer, 12 (2012) 465.
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11.
1
1
2. C.-W. Chung, Small molecule bromodomain inhibitors:
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Acknowledgments
We are grateful to the National Natural Science Foundation of
China (Program No. 81673301) for financial support. This work
was financially supported by the "Double First-Class" University
project from China Pharmaceutical University (Program No.
CPU2018GF02). A Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions
1
067.
1
1
1
1
4. M.-M. Coudé, T. Braun, J. Berrou, M. Dupont, S. Bertrand, A.
Masse, E. Raffoux, R. Itzykson, M. Delord, M.E. Riveiro, BET
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(
Integration of Chinese and Western Medicine), Jiangsu Province's
Outstanding Leader Program of Traditional Chinese Medicine
SLJ0212). We also express our gratitude to the National Cancer
(
Institute (Department of Health and Human Services, USA), Dr Ji
Yu (KeyGen BioTech, Nanjing, China) and Dr Jamie Planck
(RBC, Pennsylvania, USA) for their helpful support in biological
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Discovery of Novel Small Molecule Induced
Selective Degradation of the Bromodomain
and Extra-Terminal (BET) Bromodomain
Protein BRD4 and BRD2 with Cellular
Potencies
a,b
c,d
a
a
a
a
a
c,d,
Fei Jiang , Qingyun Wei , Huili Li , Hongmei Li , Yong Cui , Yu Ma , Haifang Chen , Peng Cao *, Tao
a,b,
b,
Lu *, Yadong Chen *.
a
School of Sciences, China Pharmaceutical University, 639 Longmian Avenue, Nanjing 211198, PR China.
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
2
10009, PR China.
c
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese
Medicine, Nanjing 210028, PR China.
d
Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine,
Nanjing 210028, PR China.

Declaration of interests
The authors declare that they have no known
☒
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐
The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: