Discovery of Benzo[ cd]indol-2(1 H)-ones and Pyrrolo[4,3,2- de]quinolin-2(1 H)-ones as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with Selectivity for the First Bromodomain with Potential High Efficiency against Acute Gouty Arthritis

Article abstract of DOI:10.1021/acs.jmedchem.9b01010

The bromodomain and extra-terminal domain (BET) family of proteins are readers which specifically recognize histone-acetylated lysine residues. Each BET bromodomain protein contains two highly homologous domains: the first bromodomain (BD1) and the second bromodomain (BD2). Pan-BET bromodomain inhibition is a potential therapy for various cancers and immune-inflammatory diseases, but only few reported inhibitors show selectivity within the BET family. Herein, we identified a series of benzo[cd]indol-2(1H)-ones and pyrrolo[4,3,2-de]quinolin-2(1H)-ones with good selectivity for BET BD1. Through structure-based optimization, highly active and selective compounds are ultimately obtained. The representative compounds are the first reported inhibitors with selectivity more than 100-fold for BRD4(1) over BRD4(2). Among them, we further show that 68 (LT052) mediates BRD4/NF-κB/NLRP3 signaling inflammatory pathways with comparable protein expression and significantly improves symptoms of gout arthritis in a rat model. Therefore, selective pharmacological modulation of individual bromodomains could represent a strategy for the treatment of acute gouty arthritis.

Full text of DOI:10.1021/acs.jmedchem.9b01010

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Article
Discovery of Benzo[cd]indol-2(1H)-ones and Pyrrolo[4,3,2-
de]quinolin-2(1H)-ones as Bromodomain and Extra-Terminal
Domain (BET) Inhibitors with Selectivity for the First Bromodomain
with Potential High Efficiency Against Acute Gouty Arthritis
fei jiang, Qing-Hua Hu, Zhimin Zhang, Hongmei Li, Huili Li, Dewei Zhang, Hanwen Li, Yu Ma, Jingjing Xu,
Haifang Chen, Yong Cui, Yanle Zhi, Yanmin Zhang, Junyu Xu, jiapeng zhu, Tao Lu, and Yadong Chen
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 30 Nov 2019
Downloaded from pubs.acs.org on November 30, 2019
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course of their duties.

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Discovery of Benzo[cd]indol-2(1H)-ones and
Pyrrolo[4,3,2-de]quinolin-2(1H)-ones as
Bromodomain and Extra-Terminal Domain
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(BET) Inhibitors with Selectivity for the First
Bromodomain with Potential High Efficiency
Against Acute Gouty Arthritis
Fei Jiang†,⋚,⊥, Qinghua Hu† ,⊥, Zhimin Zhang, , Hongmei Li , Huili Li , Dewei Zhang , Hanwen Li ,
†,ǁ
†,⋚
†
†
†
†
‡
†
†
†
†
†
Yu Ma , Jingjing Xu , Haifang Chen , Yong Cui , Yanle zhi , Yanmin Zhang , Junyu Xu , Jiapeng
Zhu* , Tao Lu* , Yadong Chen*,†,⋚
,
‡
,†,⋚
.
†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.
ǁKey Laboratory of Neuropsychiatric Drug Research of Zhejiang Province, Institute of Materia
Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China.
‡School of Medicine and Life Sciences, State Key Laboratory Cultivation Base for TCM Quality and
Efficacy, Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia
Medica, Nanjing University of Chinese Medicine , Nanjing 210023 , China.
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ABSTRACT
The bromodomain and extra-terminal domain (BET) family of proteins are readers which
specifically recognize histone acetylated lysine residues. Each BET bromodomain protein contains
two highly homologous domains: the first bromodomain (BD1) and the second bromodomain
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(
BD2), respectively. Pan-BET bromodomain inhibition is a potential therapy for various cancers
and immune-inflammatory diseases, but only few reported inhibitors show selectivity within the
BET family. Herein, we identified series of benzo[cd]indol-2(1H)-ones and
a
pyrrolo[4,3,2-de]quinolin-2(1H)-ones with good selectivity for BET BD1. Through structure-based
optimization, highly active and selective compounds are ultimately obtained. The representative
compounds are the first reported inhibitors with selectivity more than 100-fold for BRD4(1) over
BRD4(2). Among them, we further show that 68 (LT052) mediates BRD4/NF-κB/NLRP3 signaling
inflammatory pathways with comparable protein expression and significantly improves
symptoms of gout arthritis in a rat model. Therefore, selective pharmacological modulation of
individual bromodomains could represent a strategy for the treatment of acute gouty arthritis.
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INTRODUCTION
Gout is the most common type inflammatory arthritis with increasing prevalence in recent
years, which is characterized by the precipitation of monosodium urate (MSU) crystals in the
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joint . Pro-inflammatory cytokine IL-1β is a critical manifestation in response to MSU crystal
attack. IL-1β secretion is dependent on a thoroughly studied inflammasome, nucleotide-binding
oligomerization domain-like receptor, pyrin domain containing 3 (NLRP3)2-5. The activation of
NLRP3 involves a two-step process. The first signal triggers NF-κB-dependent pro-IL1β and NLRP3
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expression . The second signal triggers the formation of inflammasome multimers by microbial
molecules or dangerous signals (Such as MSU) of different structures. NLRP3 induces
self-cleavage and activation of caspase-1. Activated caspase-1 promotes the hydrolysis and
maturation of IL-1β and IL-18, and the formation of pores on the cell membrane and triggers
proinflammatory cell death, pyroptosis7, 8
.
In recent years, Bromodomain-containing proteins (BRDs) have been found to play an
important role in the development and progression of inflammation and cancer as epigenetic
targets. Bromodomain-containing proteins (BRDs) are epigenetic reader proteins of histone
acetylation involved in chromatin remodeling and transcriptional regulation, which recruit
transcriptional regulator complexes to chromatin and binding to acetylated lysine (KAc) residues
on the N-terminal tails of histones9-11. So far, 61 bromodomains have been identified in 46
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human proteins, which are classified into eight families . The second family of bromodomain
and extraterminal (BET) includes BRD2, BRD3, BRD4, and BRDT, each of which contains two
bromodomains (BD1 and BD2). The BD2 domains in these four BET proteins display a high degree
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of structural similarity, as do the four BD1 domains.
BET family members recruit different partners. BRD4 recruits positive transcriptional
elongation factor complex (P-TEFb), which plays an essential role in the regulation of
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transcription by RNA polymerase II (RNA Pol II) in eukaryotes . BET proteins facilitate the
transcription of tumor-growth-promoting and antiapoptotic genes, including c-Myc, and their
dysfunction plays critical roles in a variety of human diseases14-16. Small-molecule BET inhibitors
have shown efficacy against a range of cancers, and several inhibitors have entered clinical trials
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in oncology . BET inhibition has been shown to potently modulate inflammatory autoimmune
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+
gene expression . BET bromodomains control the differentiation of CD4 T cells to inflammatory
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Th17 cells through genomic localization and their effects on cytokine regulation . Jiang Yafei et
al20 reported that BRD4 ligands have dual effects on the HMGB1 and NF-κB signalling pathways
and BRD4 is a potential therapeutic target for osteoarthritis. Meanwhile, BRD4 proteins regulate
+
gene transcription during lineage-specific differentiation of naïve CD4 T cells to produce mature
T-helper cells. Pan-BET bromodomain (BRD) inhibitors have distinct immune functions, thus
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limiting their therapeutic potential . In human monocytes, BET inhibition suppressed the
transcriptional response of HMGB1 and NF-κB pathway to cytokine stimulation, and
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downregulates pro-inflammatory cytokine production in macrophages , dendritic cells , and
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rheumatoid arthritis synovial fibroblasts . In vivo, BET inhibitors exhibit therapeutic effects in a
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variety of anti-inflammatory disease models, such as colitis , arthritis , psoriasis , and
pathologic bone loss26.
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N
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GSK340
RVX-208
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MS611
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Figure 1. Representative BET bromodomain family selective inhibitors
In the past decade, multiple structural cores that mimic acetylated lysine (KAc) binding have
been discovered. Among BET inhibitors, the triazolodiazepine chemotype has been extensively
explored. With (+)-JQ1 (1)27 as a representative widely used in biological mechanism research, its
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derivatives have been advanced into clinical trials, including i-BET762 , OTX-015 , TEN-10 and
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CPI-0610 . Except RVX-208 (2) , these clinical-stage molecules exhibit little or no selectivity
between the eight bromodomains of the BET family. Given the fact that eight bromodomains in
BET family are highly homologous, it is still a challenge to obtain highly selective inhibitors. It’s
3
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still not clear if side effects and toxicity were related to their selectivity in biological function .
Therefore, the development of highly selective inhibitors is urgently needed.
Currently, only a few selective BET inhibitors were reported. Among them, RVX-208 (2) is a
compound with high degree of selectivity for BET BD2 and good response in the treatment of
34
metabolic diseases in clinical trials . Based on AlphaScreen data, RVX-208 (2) has about 170-fold
selectivity towards BD2 over BD2 (IC50 values of 87±10 μM and 0.510±0.041 μM for BD1 of BRD3
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and BD2 of BRD3, respectively) . The sequence alignment (Figure 2A-C) shows that the BC
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loop-specific DDI of the first Bromodomain of the four BET proteins is different from the BC loop
HDV or HEV sequence of the second Bromodomain. RVX-208 (2) selectively binds to the second
Bromodomain by utilizing the fine structural differences of the BC loop-specific histidine in the
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two Bromodomains . Compound 3 selectively binds to the second bromodomain, and His433,
which is unique to BRD4 BD2, is utilized to form hydrogen bond interactions to increase
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selectivity . Weak (∼10-fold) BRD4 BD1 bias is also observed for the cyclopent-2-en-1-one
+
inhibitor MS402 (4), which blocked Th17 maturation from mouse naive CD4 T cells, with limited
37
or no effects on Th1, Th2, or Treg cells . Experimental models of mouse colitis induced by T cell
transfer have been observed to potentially treat inflammatory bowel diseases. The diazobenzene
MS611 (5) was reported to be moderately active for BRD4 BD1 with K = 0.41 μM and 100-fold
d
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selectivity over BRD4 BD2, where the phenol acts as KAc mimetic .
Figure 2. (A) Sequence alignment of BET bromodomains with the residues highlighted. (B)
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Comparison of BRD4 BD1 (3MXF, green) and BD2 (5uf0, gray) with (+)-JQ1 (1). The cartoons and
lines show the binding pockets of BD1 and BD2, and the sticks are shown to highlight key
differential residues. (C) Comparison of BRD4 BD1 (3MXF, green) and BD2 (5uf0, gray) with
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(+)-JQ1 (1). The surfaces and lines show the binding pockets of BD1 and BD2, and the color
surface is shown to highlight the volume of the space differential residues.
There are currently very few small molecule inhibitors that selectively bind to a single
bromodomain of the BET family proteins. It remains a challenge to identify highly potent and
selective-bromodomain small-molecule BET inhibitors. Importantly, Ke Ding et al39 reported a
novel chemical core of 1-ethylbenzo[cd]indol-2(1H)-one based on virtual screening which
showed promising BRD4 binding activity. Subsequently, Hua Chen et al40 also reported that this
core has excellent BRD4 activity. Although derivatives of this core only displayed moderate
anti-cancer cell proliferation activity in vitro compared to triazole derivatives (e.g., JQ1), they
show great potential for the development of anti-cancer therapeutics. Further investigation on
benzo[cd]indol-2(1H)-ones may stimulate discovery of selective-bromodomain BET inhibitors .
A
previous fragment-based screen carried out in our laboratories identified
benzo[cd]indol-2(1H)-one 6 (Figure 1), which bound weakly but with unique structural features
to BRD4 BD1 (IC50 = 15.53 μM and ΔTm = 2.23 ℃). Through a series of structural modifications,
we obtained a class of inhibitors that specifically bind to BRD4 BD1. Among them, we identified a
lead compound with nanomolar potency towards BD1, and more importantly, it displayed more
than 100-fold selectivity towards BD1 over BD2. The lead compound has a potential in the
treatment of BRD4/NF-κB/NLRP3-mediated gout arthritis, providing a very useful tool for the
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disease phenotype of subsequent selective-bromodomain BET inhibitors.
CHEMISTRY
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The synthesis of compound 9 and 12-34 was shown in Scheme 1. Benzo[cd]indol-2(1H)-one
was used as the starting material and compound 9 was prepared through methylation,
electrophilic aromatic substitution with chlorosulfonic acid and nucleophilic addition with aniline.
Compounds 11a-11c are synthesized by alkylation, nitration reaction followed by hydrogenation.
With starting from the intermediate 11a, the title compounds 12-14 were prepared through the
nucleophilic aromatic substitution with appropriate halides or benzoyl chlorides. Further, the
title compounds 15-34 were prepared by appropriate benzenesulfonyl chlorides with 11a-11b in
the presence of a small amount of pyridine.
Scheme 1. Synthetic routes of compounds 9 and 12-14
O
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~
74%
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75%~90%
54%
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7b: R1 = ethyl
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13: R = 1-phenylethyl;
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g
h
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10b, 11b: R1 = methyl
O
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5%~65%
0c, 11c: R1 = ethyl
56%
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Ar
5 ~ 25, 27 ~ 34
O
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15-28 as in table 1
9-34 as in table 2
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Reagents and conditions: (a) appropriate Iodide, 60% NaH, DMF; (b) CISO H, 0 °C; (c) aniline,
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pyridine, DCM, r.t; (d) 69% HNO , AcOH, r.t; (e) H , 10% Pd/C, MeOH, THF, r.t; (f) appropriate
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bromide or benzoyl chloride, 60% NaH, DMF, r.t; (g) appropriate sulfonyl chlorides, pyridine,
DCM, r.t; (h) CH I, 60% NaH, DMF.
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In scheme 2, benzo[cd]indol-2(1H)-one derivatives were prepared (Table 3 and Table 4). The
synthesis of intermediate 39 is no longer described in the same way as reported by Sheng-Wei
Yang et al41 Starting from inexpensively available 4-chloroindoline-2,3-dione intermediate was
nitrated with nitric acid sulfuric acid system from -25 °C to -10 °C to afford compound 40.
Intermediates 43 and 44 were synthesized from 39 by hydrogenation and dechlorination.
Meanwhile, 43a-43n was prepared from 40 with different amines by nucleophilic reaction
followed by hydrogenation of nitro group and 43o-43v was prepared from 40 upon a
Suzuki−Miyaura cross coupling reaction with different boronic acids/esters, followed by
hydrogenation of nitro group. Finally, the title compounds 46-70 were prepared with different
benzene sulfonyl chloride in the presence of a small amount of pyridine.
Scheme 2. Synthetic routes of compounds 46-70
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
Boc
Boc
Cl
Cl
O
NH
NH
O
O
c
a
b
O
O
O
O
N
O
N
79%
N
91%
N
84%
N
H
3
5
36
37
O
N
O
O
O
N
N
N
N
d
e
f
g,h
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
80%
N
R3
83%
Cl
i, j
75%
Cl
N
H
O
4
0%~95%
NO2
NO2
38
39
40
4
1a ~ 41r
30%~80%
O
N
O
N
N
j
k
R3
R3
4
5%~90%
30%~70%
N
O
4
4
3: R3 = H
4: R3 = Cl
NH2
HN
46-48 as in Table 2
49-66 as in Table 3
67-73 as in Table 4
S
Ar
5~70
O
4
4
3,44, 45a~45r
a: R3 = tert-butylamino
b: R3 = cyclopropylamino
h: R3 = morpholino
i: R3 = 4-methylpiperazin-1-yl
j: R3 = (hydroxymethyl)piperidin-1-yl
c: R3 = (1-methylpiperidin-4-yl)methylamino
d: R3 = (tetrahydro-2H-pyran-4-yl)methylamino
e: R3 = 2-(piperidin-1-yl)ethylamino
f: R3 = (2-(4-methylpiperazin-1-yl)ethyl)amino
g: R3 = (R)-(1-(pyridin-2-yl)ethyl)amino
k: R3 = 4-(2-ethoxy-2-oxoethyl)piperidin-1-yl
l: R3 = 4-(tert-butoxycarbonyl)piperazin-1-y
m: R3 = furan-2-yl
o: R3 = 1H-imidazol-1-yl
p: R3 = 4-methyl-1H-imidazol-1-yl
q: R3 = 1-methyl-1H-pyrazol-4-yl
n: R3 = furan-3-yl
r: R3 = 1-cyclopropyl-1H-pyrazol-4-yl
Reagents and conditions: (a) CH I, 60% NaH, DMF; (b) BocNH , Pd(AcO) , x-PHOS, K CO ,
3
2
2
2
3
5
anhydrous 1,4-dioxane, 80 °C, 10 h; (c) tert-butyl 2-(triphenyl-λ -phosphanylidene)acetate, DMF,
r.t; (d) TFA, 100 °C; (e) POCl , 100 °C ;(f) H SO , HNO , -25 - -10 °C; (g) appropriate boronic ester,
3
2
4
3
Pd(PPh ) , 1,4-dioxane, N , 80 °C; (h) appropriate amine derivatives, anhydrous 1,4-dioxane,
3 4
2
1
00 °C; (i) K CO , 10% P/C, H , THF; (j) 10% P/C, H , THF, MeOH; (K) appropriate sulfonyl
2 3 2 2
chlorides, pyridine, DCM, r.t.
In scheme 3, benzo[cd]indol-2(1H)-one derivatives were prepared (Table 4). Starting from
inexpensively available 1,8-Naphthalic anhydride, intermediate 71 was synthesized through
halogenation reaction with bromine and silver sulfate in concentrated sulfuric acid. Two key
steps, nucleophilic substitution and strong base decarboxylation, were carried out in one pot in
acceptable yields to obtain 72. Subsequently, 75a-75g were synthesized through a three-step
reaction including methylation with methyl iodide, nitrification under acetic acid-nitric acid
conditions and Suzuki coupling reaction with different boric acid or borate esters. The
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intermediates were subjected to hydrogenation and were used directly for a next step without
further purification. Finally, the title compounds 77-90 were prepared with different benzene
sulfonyl chlorides and 76a-76g in the presence of a small amount of pyridine.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthetic routes of compounds 77-90
O
O
HN
O
O
O
O
O
O
HN
c
b
a
Br
2
0%~35%
83%
~
90%
Br
O
Br
NO2
7
1
72
73
O
O
O
N
N
N
N
e
d
f
g
R3
R3
R3
81%
Br 40%~80%
30%~70%
O
HN
NO2
NH2
76a~76g
NO2
75a~75g
S
Ar
7~90
O
7
74
a: R3 = 1-methyl-1H-pyrazol-4-yl
b: R3 = 1-ethyl-1H-pyrazol-4-yl
c: R3 = 1-cyclopropyl-1H-pyrazol-4-yl
d: R3 = 5-methylfuran-2-yl
e: R3 = 1-benzyl-1H-pyrazol-4-yl
77-90 as in Table 5
Reagents and conditions: (a) Br , H SO , Ag SO , 60 °C; (b) i: NH OH HCl, TsCl, pyridine,
2
2
4
2
4
2
9
0 °C; ii:NaOH, HCl, EtOH, H O; (c) HNO3, AcOH, 60 °C; (d) CH I, 60% NaH, DMF; (e) appropriate
2 3
boronic este, Pd(PPh ) , 1,4-dioxane, N , 80 °C; (f) Fe, NH Cl, EtOH, 80 °C; (g) appropriate sulfonyl
3
4
2
4
chlorides, pyridine, DCM, r.t.
RESULTS AND DISCUSSION
Optimization of the Linker to the WPF Shelf
To improve the potency of benzo[cd]indol-2(1H)-one-based BET bromodomain inhibitors
such as 6, we explored interactions between the inhibitor core and the conserved Asn140
residue and the WPF shelf of the BET protein. Compounds were evaluated using differential
scanning fluorimetry (DSF) and amplified luminscent proximity homogeneous assay
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(AlphaScreen). The DSF binding assay and AlphaScreen assay were used to determine the
thermal stability (ΔTm) and the affinities (IC ) of compounds for a construct containing the two
5
0
bromodomains of BRD4. All derivatives were tested for their inhibitory potency toward
recombinant human BRD4 BD1 His and BRD4 BD2 His.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Binding mode analysis of compound 6 and 15 to BRD4 BD1 protein (PDB code 3MXF).
(A) Compound 6 (shown as blue) docked into BRD4 BD1 overlaid JQ1 (shown as green) with
BRD4(1) protein. (B) Compound 6 (shown as blue) and 15 (shown as purple) docked into BRD4
BD1 overlaid JQ1 (shown as green) with BRD4(1) protein (displayed as gray surface).
From the binding mode of 6 with BRD4 BD1 (Figure 3A), we can see that the carbon-based
oxygen of the benzo[cd]indol-2(1H)-one forms a molecular key hydrogen bond with the amino
group of Asn140 and indirectly forms another molecular key hydrogen bond with the water
molecule and Tyr97. The phenylmethyl group of compound 6 extended to the WPF shelf via a
sulfonamide linker, not in the WPF hydrophobic center region. To better fit the WPF shelf, we
examined a series of linkers in compounds 9, 12-15, including methylene, ethylene, amide, and
back-linked sulfonamides. As shown in Table 1, methylene, ethylene, and amide did not achieve
desired effect and instead had a large increase in the non-BET family. It is very exciting that the
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value of ΔTm of 15 was elevated by 3.53 ℃ over the forward sulfonyl linker in compound 9, still
remaining selective for the BET family. The binding mode (Figure 3B) showed that the benzene
ring of 15 has a high degree of fit with the space of the benzene ring of JQ1 (1) at the center of
the WPF shelf.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Structures and biological evaluation of target compounds
O
O
O
O
O
O
N
N
N
N
N
O
HN
HN
O S O
NH
HN
HN
S
O
15
9
13
14
12
Tm (℃)a
△
Compounds
9
BRD4(1)
2.19
BRD9(1)
0.47
CECR2(1) CREBBP(1)
-0.03
1.92
0.13
2.17
-0.31
0.53
1.60
0.55
0.93
1.51
1
1
2
3
2.70
-0.90
4.33
3.15
14
2.17
4.10
1
5
5.72
1.93
a
using the differential scanning fluorimetry (DSF) at 10 μM concentration
Optimization of the WPF Shelf and the KAc pocket
With these encouraging results, the benzene ring moiety of 15 was investigated with a
suitable aromatic ring and a substituted ortho or para-monosubstituted benzene ring (Table 2)
while maintaining the sulfonamide linker of 15. From the comparison of BRD4 BD1 protein and
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
BRD4 BD2 sequence (Figure 1A), it was found that the amino acids (Tyr, Pro and Phe) constituting
the WPF region were significantly different in terms of space volume, electrical property and
flexibility. First, the spatial volume and electrical properties of the WPF shelf were explored using
five-membered thiophene in 16, electron-deficient pyridine in 17 and bulkier quinoline ring in 18.
Among them, the IC50 values of 15 are 709.1±4.5 nM and 2621±10 nM for BD1 and BD2,
respectively, suggesting about 3.7-fold selectivity (Table 2). The selectivity was reduced by the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
increase in the size of R and the activity of the methyl group (19) appears to be the best.
Changes in methylation of 22, selectivity and binding were not significant compared to 15.
Table 2. Structures and biological evaluation of target compound 15-28.
R1
O
N
O
N
R2
S
Ar
O
BRD4 IC50
BRD4(1)
Selectivity
(% protein activity at 2 μM)
1
2
Cpd. R R
Ar
b
△Tm (℃)a
BD1 (nM)d
BD2 (nM) BD2/BD1
1
1
5 Me H
5.72
4.02
709.1±4.5
2621±10
3.7
---
nd. c
6 Me H
3724±46
S
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1
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7 Me H
8 Me H
9 Me H
2.3
2436±290
328.6±21.3
167.1±14.3
13675±1029
260.3±15.9
2717±401
5.6
0.8
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4.13
N
1
2
2
6.5
16
20.1
11.4
4.1
3.9
---
Cl
Cl
Cl
0
H H
3.73
6.36
7.36
5.37
5.1
769.2±114.3 15502±125
1 Et H
237.4±9.3
545.2±23.4
1529±41
2717±401
2283±132
6048±67
nd.
22 Me H
2
2
2
2
3 Me H
4 Me H
5 Me H
6 Me Me
Cl
708.7±9.1
49.5±10.3
(90.39±1.3%)
3888±130
8.72
1.06
4.73
2479±170
nd.
50.5
---
O
O
27 Me H
nd.
---
CF3
O
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
8 Me H
5.51
172.1±12.8
1640±68
9.5
O
a
b
using the differential scanning fluorimetry (DSF) at 10 μM concentration; Using AlphaScreen
c
d
assay, the values are the mean ± SD from two independent experiments. nd: not determined.
n = 2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To investigate the effect of ortho substituents on the potency and selectivity, we designed
chloro, methyl, ethoxy trifluoromethoxy and ethoxy substituents. The increase in the potency is
very limited with the 2-position benzene ring replaced with electron-withdrawing chlorine (23),
trifluoromethoxy (27), and electron-donating methyl (24) and ethoxy groups (28). Surprisingly, a
breakthrough level in the selectivity and the affinity was found in compound 25 (IC , 49.5 ± 10.3
5
0
nM and 2479 ± 170 nM for BD1 and BD2, respectively thus about 50-fold selectivity) with a
methoxy group at the ortho-phenyl.
Table 3. Structures and biological evaluation of target compound 29-28.
O
N
X
O
HN
S
Ar
O
BRD4 IC50
BRD4(1)
Tm (℃)
Selectivity
Cpd.
X
Ar
(% protein activity at 2 μM)
△
BD1 (nM) d
BD2 (nM) BD2/BD1
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Cl
F
2
3
3
9
0
1
C
C
C
2.75
3.73
4.83
(91.12±3.3%)
nd.
nd.
nd.
---
---
---
Cl
F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9065±14
1696±33
F
Cl
32
C
C
4.32
6.23
7.1
810.4±17.4
91.3±3.3
nd.
---
42
F
F
3
3
4
4
3
4
5
6
3828±48
3468±37
nd.
Cl
C
299.3±23.6
(87.36±0.45%)
3466±89
11.5
---
Cl
O
N
N
N
N
2.31
6.53
5.12
7.64
Cl
Cl
F
20540±258
6623±76
5.9
4.2
22.4
47
1569±55
4
8
205.2±22.4
4599±53
O
a
b
using the differential scanning fluorimetry (DSF) at 10 μM concentration; Using AlphaScreen
c
d
assay, the values are the mean ± SD from two independent experiments. nd: not determined.
n = 2.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We further designed a series of derivatives with multiple substitutions on the phenyl ring
and examined their effects on activity and selectivity of BD1 and BD2 as shown in Table 3. The
introduction of 2,6-dichloro (29) and 2,6-difluoro (30) substitutions in WPF shelf has a greater
decreasing effect on the activity , while the activity of the 2,3-substituents (31) on the same side
of the benzene ring is restored. In addition, it was found that 2,4-substituent groups (32-34) are
more selective to BRD4 BD1. Among them, the IC50 values of 33 were 91.3±3.3 nM and 3828±48
nM for BD1 and BD2, respectively, thus about 42-fold selectivity (Table 3). Taking into account
the physical and chemical properties, we have moderately decreased the activity and selectivity
of the 5-position C of the benzofluorene ring with N in compound 48, and its physical and
chemical properties (48 predicted tPSA: 88 and CLogP: 1.29; 25 predicted tPSA: 75 and CLogP:
2.03) are improved.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. The conformation of compound 25 in the BRD4 BD1 and BRD4 BD2 proteins. (A) X-ray
crystal structure of compound 25 bound to BRD4 BD1 (PDB ID: 6JJB, Authors will release the
atomic coordinates and experimental data upon article publication). The ligands are shown as
sticks models, and the protein is shown as surface and a cartoon with line model. (B) X-ray crystal
structure of 25 (Surface and line, PDB ID: 6JJB) bound to BRD4 BD1, overlaid with BRD4 BD2 (stick
and line, PDB ID: 5uf0).
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To clarify the outstanding activity and selectivity of compound 25, we determined crystal
structure of compound 25 with BRD4 BD1 as shown in Figure 4A. The phenyl group of compound
2
1
5 differs from the conformation of the phenyl group of JQ1 (1) or other above derivatives (6 and
5, the binding mode in Figure 3) and does not completely occupy the WPF shelf, but is more
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
adjacent to the region between Asp144 and Lue92. As shown in Figure 4B, steric hindrance may
exist between the 2-methoxyphenyl of 25 and the imidazole of BD2-specific His437 (with a
distance of around 3.2 Å from 25), which suggests a very unfavorable conformation binding to
BRD4 BD2. The 2-position methoxy group may play a crucial role in the selective mechanism, and
this hypothesis was verified by the selection and activity in Table 2. The 2-methoxyphenyl
module was moved forward in further optimization and new modules were added to further
improve selectivity and activity.
Optimization of the BC loop solvent region
The BC loop solvent region formed by B loop and C loop was found to have a large
difference in residues between the two domains BD1 and BD2, although the two domains are
highly homologous in the BRD4 protein. The greatest differences lie in the BC loop, with Asp144
in BD1 changed to His437 in BD2 and an aspartic to glutamic acid switch of the adjacent residue.
It was suspected that the introduction of appropriate hydrophilic groups in this region may be
beneficial for activity and selectivity. Therefore, a series of derivatives in Table 3 were
synthesized and evaluated for the binding potency of BRD4 BD1 and BRD4 BD2. First, 49 and 50
moderately decreased BRD4 BD1 efficacy compared to 5-position chlorine substitutions in 46 and
4
8. Cyclic amine had good potency in different volumes of amino groups including
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tert-butylamine, and cyclopropylamine.
Inspired by this structure-activity relationship, the difference in binding potency tolerance
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
between BD1 and BD2 was examined using groups (different chain lengths and hydrophilic
3
contributions at the 5-position R ). The IC values from the AlphaScreen data showed that these
50
derivatives had approximately a 5-fold change in potency (except 57) for BRD4 BD1, but their
binding potency to BRD4 BD2 was greatly affected. The methylpiperidine of 53 was more suitable
for the solvent region of BRD4 BD1 than for the tetrahydro-2H-pyran of 54. Interestingly, subtle
changes of nitrogen atoms in 55 – 57 could obtain unexpected results. Among them, the IC50
values of 55 are 328 ±7.8 nM and 7068±69 nM for BD1 and BD2, respectively, thus about 21-fold
selectivity. In contrast, the introduction of moderately hydrophobic pyridine 57 was nearly
20-fold lower for BD1 on potency. In order to further explore the characteristics of BD1 and BD2
in the solvent accessible region of BC, we designed a larger volume of six-membered cyclic
hydrophilic fragments directly linked to the pyridine 2-position. As shown in the AlphaScreen
assay of Table 4, the morpholine ring and the nitrogen methylpiperazine ring exhibited good
potency against BD1 over BD2. However, the N-methyl group of N-methylpiperazine (60 - 61)
didn’t show improvement when it was replaced by other groups.
Table 3. Optimization of the hydrophilic group in the solvent accessible region
O
N
R3
O
HN
S
Ar
O
2
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8
9
BRD4 IC50
(% protein activity at 2 μM)a
Selectivity
BD2/BD1
--
Cpd.
Ar
R3
BD1 (nM)c
BD2 (nM)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
5
9
0
1
Cl
Cl
2598±49
nd. b
Cl
655.80±84.6
573.1±35.8
271.2±22.8
113.0±1.8
503.2±15.1
328 ±7.8
nd.
---
3.6
2.4
11.4
---
O
O
O
O
O
O
2085±47
660.2±22.6
1284±62
nd.
N
H
52
N
H
N
H
5
5
5
3
4
5
N
O
H
N
7068±69
21.5
N
N
H
N
N
5
5
6
7
N
223±15.3
1520±61
6.8
H
O
N
N
2226±46
nd.
---
H
O
O
58
N
O
95.2±12.3
808.4±26.9
8.5
2
1
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
6
6
9
0
1
N
O
217.1±32.5
100.0 ±6.8
391.3±7.9
169.1±27.8
nd.
2117±42
nd.
---
21.1
---
Cl
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
N
Cl
N
62
29638.1
17.5
OH
O
O
O
N
O
6
3
709.4±73.2
334.2±7.8
nd.
---
O
64
N
N Boc
2167±45
6.5
a
Using AlphaScreen assay, the values are the mean ± SD from two independent experiments.
b
c
nd: not determined. n = 2.
To better understand the mechanism of selective binding of compound 60 to BRD4 BD1
over BRD4 BD1, we determined a co-crystal structure of compound 60 with BRD4 BD1. In Figure
A, the 2-methoxyphenyl group of 60 differs from the selective dominant conformation of 25,
5
with deflection fully entering the WPF shelf. Therefore, the selectivity decrease of 60 is due to
3
the fact that R N-methylpiperazinyl changes the original conformation of 2-oxyphenyl group,
resulting in reduced steric hindrance to imidazole H437 in BD2 (Figure 5B) without affecting the
binding of BD1. Although the selective contribution of 2-methoxyphenyl was weakened in 60, a
new steric hindrance effect was obtained between N-methylpiperazinyl and imidazole H437 in
2
2
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BD2 (the closest distance of 1.5 Å, Figure 5B). As shown in Table 3, the selectivity of 60 was
2
1-fold lower than 25 for BD1 over BD2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. The conformation of compound 60 in the BRD4 BD1 and BRD4 BD2 proteins. (A) X-ray
crystal structure of compound 60 bound to BRD4 BD1 (PDB ID: 6JJ3, Authors will release the
atomic coordinates and experimental data upon article publication). The ligands are shown as
sticks models, and the protein is shown as surface and a cartoon with line model. (B) X-ray crystal
structure of 60 (stick) bound to BRD4 BD1 (line and cartoon, PDB ID: 6JJ3), overlaid with BRD4
BD2 (line and cartoon, PDB ID: 5uf0).
Based on the structure-activity relationship, we made an assumption that an appropriate
3
rigid group substitution in the solvent region of R may render a ligand with a high selectivity for
BRD4 BD1 over BRD4 BD2. The rigid ring is more likely to have a repulsive effect with the
imidazole ring of the His437 residue in BD2 and reduce the affinity for BD2. We designed
multiple rigid five-membered heterocycles to validate this hypothesis. The activity evaluation
results showed excellent protein binding ability and selectivity (Table 3). When X is a quinoline
3
core and R is unsubstituted 2-furan (65) and 3-furan (66), the IC50 values for BD1 are 35.1±2.2
nM and 37.0±1.3 nM, respectively. Next, we introduced an imidazole in 67 and a
2
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methylimidazole ring in 68 with IC50 values of 889.0±32.9 nM and 87.7±4.9 nM for BD1,
respectively. Excitingly, the IC50 values of Compound 68 are 87.7±4.9 nM and 12130±308 nM for
BD1 and BD2, respectively, thus about 138-fold selectivity. The methyl group of 68 resulted in a
significant steric hindrance effect between the methylimidazole (68) and the H437 residue of
BD2, which in turn increased selectivity without affecting the binding of BD1. Compound 69
merely changes the position of the nitrogen atom compared to compound 68 resulting in a loss
of selectivity of more than 100-fold. However, when 5-methylfuran-2-yl was introduced in 79,
the IC50 values suggested 857-fold selectivity (IC50 54.8±6.7 nM and 47000±542 nM for BD1 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
BD2). The hetero five-membered ring and a methyl group at R in (68, 69, 77 and 79) showed
good selectivity and activity. This also shows that the volume is favorable for the steric hindrance
effect of the His437 of BRD4(2). In addition, due to the imidazole electron-deficient ring of
3
His437, the hetero atom position of R has a great contribution to the selectivity (68 and 79). The
affinity of 80 for BD1 was reduced by more than 3-fold by the introduction of 3-furyl. When Ar is
3
an 2-methoxyphenyl group in the WPF shelf and R is a methyl (69 and 77), propyl (82),
cyclopropyl (70 and 84) and benzyl (90) substituted pyrazole, its IC50 value for BD1 was between
14.4±5.7 nM and 205.2±16.2 nM, its selectivity being within 25-fold. Furthermore, when Ar in the
WPF shelf is substituted by other substituted phenyl in 85-89 (except
2
-methoxy-4-chlorophenyl), the activity is drastically reduced or even lost.
Table 3. Optimization of the hydrophilic group in the solvent accessible area
2
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9
O
N
X
R3
O
HN
S
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ar
O
BRD4 IC50
(% protein activity at 2 μM)
Selectivity
BD2/BD1
27.3
Cpd.
X
Ar
R3
BD1 (nM)c
BD2 (nM)
O
6
6
6
5
6
7
N
N
N
N
N
N
C
35.1±2.2
981.7±42.7
O
O
O
O
O
O
O
37.0±1.3
889.0±32.9
87.7±4.9
12.6±2.7
16.6±9.3
17.4±5.6
348.2±1.1
458.7±52.5
4110±56
12130±308
289.3±53.6
107.1±8.5
434.9±7.3
nd.
12.4
4.6
O
N
N
N
68
138.3
22.9
6.5
N
N
6
7
7
7
9
0
7
8
N
N
N
N
25.0
---
N
N
C
N
Cl
2
5
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
7
9
C
54.8±6.7
47000±542
857.2
O
O
O
O
8
8
8
0
1
2
C
C
C
163.7±19.3
(56.59±1.31 %)
43.7±12.6
nd.
nd.
---
---
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
N
N
265.6±7.9
6.2
O
N
N
8
3
C
C
C
C
C
C
C
C
106.7±8.5
14.4±5.7
320.2±46.7
237.6±7.4
nd.
3.0
16.9
---
Cl
O
84
N
N
O
8
8
8
5
6
7
N
N
665.7±25.7
37.5±6.8
Cl
N
N
434.9±72.6
nd.
11.6
---
Cl
O
N
N
(65.17±3.27%)
(64.88± 0.54%)
(61.84±0.23 %)
205.2±16.2
Cl
F
Cl
88
N
N
nd.
---
O
8
9
9
0
N
N
nd.
---
Cl
Cl
N
N
nd.
---
O
a
Using AlphaScreen assay, the values are the mean ± SD from two independent experiments.
2
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9
b
c
nd: not determined. n = 2.
In vitro evaluation of selected compounds on anti-inflammatory activity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To investigate the role of single Bromodomains in the development of inflammatory diseases,
we evaluated the selective-bromodomain BET inhibitors and pan-BET inhibitors to inhibit NF-κB
activity in HUVECs cells and inhibit nitric oxide (NO) production in RAW264.7 cells. Detection of
NO in macrophages is widely used in the screening of anti-inflammatory drugs42, 43. Ana S. Leal etl
etc.44 reported that pan-BRD4 inhibitors play an important role in the tumor microenvironment.
These drugs suppressed the production of NO in RAW264.7 cells. Therefore, we selected 17
compounds at 1 μM to evaluate NO inhibition rate treated with LPS 1 μg/mL in macrophages.
From the results shown (Table 4), most of the compounds showed significant activity to reduce
the concentration of NO production. The inhibition rate of 7 compounds was greater than 90%
and the selectivity of compounds 33 and 68 was more than 40-fold. Remarkably, the NO
inhibition rate of RVX-208 with selective binding to BD2 was significantly lower than the NO
inhibition rate of inhibitors with high activity for BD1 (JQ1 and other compounds). In addition,
the highly selective compounds 25 and 79 failed to decrease in NO production which may be due
to poor permeability (79 with low permeability 0.060 nM/s and 68 with moderate permeability
1
.172 nM/s in Table S2).
Table 4. In RAW264.7 cells the downregulate NO production activities of selected compounds at
μM treated with LPS 1 μg/mL
1
Cpd.
BRD4 IC ( nM )
Sel.
At 1 μM
5
0
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NO Inhibition rate
(%)a
BD1
BD1
BD2/BD1
1
9
167±14
49.5±10.3
172.1±12.8
91.3±3.3
2717±401
2479±170
1640±68
3828±48
4599±53
1284±62
7068±69
1520±61
16.2
50.1
9.5
15.09±8.64%
51.37±4.13%
101.89±0.05%
98.11±3.27%
94.34±3.26%
47.17±18.20%
43.40±8.65%
39.62±1.05%
100.00±3.28%
92.45±3.22%
64.86±8.14%
66.28±2.98%
101.89±1.01%
101.89±5.66%
58.49±17.29%
105.66±3.27%
83.73±1.10%
47.56±0.55%
9.17±0.41%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25
2
8
33
41.9
22.4
11.4
21.5
6.8
4
5
5
5
5
6
8
3
5
6
8
0
205.2±22.4
113.0±1.8
328 ±7.8
223±15.3
95.2±12.3 808.4±26.9
8.5
100.0 ±6.8
35.1±2.2
37.0±1.3
87.7±4.9
17.4±5.6
54.8±6.7
43.7±12.6
14.4±5.7
2117±42
981.7±42.7
458.7±52.5
12130±308
434.9±7.3
47000±542
265.6±7.9
237.6±7.4
21.1
27.3
12.37
138.3
24.99
857.2
6.2
65
6
6
68
7
7
79
8
8
2
4
16.9
0.5
JQ1
42.24±5.08 24.71±1.01
1800 40
RVX-208 b
0.02
a
Using Griess reagents method, the values are the mean ± SDs from three independent
experiments, n = 3. NO inhibition rate % = (model group NO concentration – Compound group
NO concentration) / (model group NO concentration– control group NO concentration) ×
2
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b
45
1
00%. The data was assayed by TR-FRET .
The dual luciferase reporter gene assay data (Figure 7 and Table S3) showed that the
selective BD1 inhibitors and pan-BET inhibitor (JQ1) were significantly more potent in
down-regulating the NF-κB activity than the selective BD2 inhibitor (RVX-208). Compound 68 of
NF-κB transfection activity was superior to compounds 65 and 79, comparable to JQ1 inhibitory
activity. Obviously, these results suggest that inhibitors selectively binding to BD1 have a better
anti-inflammatory activity than inhibitors selectively binding to BD2 in vitro.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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In the evaluation of in vitro inflammatory activity, the selective BET BD1 inhibitor 68
(
inhibition rate: 101.89±1.01%) was significantly superior to the pan-BET inhibitor JQ1 (inhibition
rate: 47.56±0.55%) in inhibiting the NO production of macrophage RAW264.7. The NO
production is regulated by multiple inflammatory signaling pathways, and NF-κB signaling
pathway is one of the important pathways regulating NO production. Selective BET BD1 inhibitor
6
8 of NF-κB transfection activity was comparable to pan-BET inhibitor JQ1 inhibitory activity. It is
demonstrated that the selective BET BD1 inhibitor (68 BRD4(1): 87.7±4.9 nM; BRD4(2):
2130±308 nM) still maintains comparable or better anti-inflammatory activity than the pan-BET
1
inhibitor (JQ1 BRD4(1): 42.24±5.08 nM; BRD4(2): 24.71±1.01 nM) compared to the protein weak
activity. Based on our analysis, compound 68 was considered for further studies in biological
mechanisms and diseases.
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