Structure-based optimization of a series of selective BET inhibitors containing aniline or indoline groups

Article abstract of DOI:10.1016/j.ejmech.2018.02.070

Recently, several kinase inhibitors were found to also inhibit bromodomains, providing a new strategy for the discovery of bromodomain inhibitors. Along this line, starting from PLK1-BRD4 dual inhibitor BI-2536, we discovered a new series of dihydroquinoxalin-2(1H)-one with aniline and indoline WPF binders as selective BRD4 inhibitors. They showed better BRD4-BD1 potency and negligible PLK1 kinase activity comparing with BI-2536. Additionally, dihydroquinoxalin-2(1H)-ones containing indoline group showed profound activities in molecular and cellular based assays. Throughout the study, compounds 9, 28 and 37 showed significant inhibitory activity for c-Myc or PD-L1 protein expression and mRNA transcription both at concentration of 0.2 and 1 μM. Compound 9 was found possessing the best balance of binding affinity, in vitro metabolic stability and in vivo pharmacokinetic properties. Therefore, it was selected for in vivo pharmacological study. By using MM.1S cell derived xenograft model, we confirmed compound 9 showed comparable in vivo tumor inhibition to phase II investigation drug I-BET762, which, together with the novel WPF binder, further indicated the utility of this series of BRD4 inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.02.070

Accepted Manuscript
Structure-based optimization of a series of selective BET inhibitors containing aniline
or indoline groups
Jianping Hu, Yingqing Wang, Yanlian Li, Danyan Cao, Lin Xu, ShanShan Song,
Mohammadali Soleimani Damaneh, Jian Li, Yuelei Chen, Xin Wang, Lin Chen,
Jingkang Shen, Zehong Miao, Bing Xiong
PII:
S0223-5234(18)30207-1
10.1016/j.ejmech.2018.02.070
EJMECH 10247
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 December 2017
Revised Date: 29 January 2018
Accepted Date: 21 February 2018
Please cite this article as: J. Hu, Y. Wang, Y. Li, D. Cao, L. Xu, S. Song, M.S. Damaneh, J. Li, Y. Chen,
X. Wang, L. Chen, J. Shen, Z. Miao, B. Xiong, Structure-based optimization of a series of selective BET
inhibitors containing aniline or indoline groups, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.02.070.
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ACCEPTED MANUSCRIPT
Graphic Abstract

ACCEPTED MANUSCRIPT
Structure-based Optimization of a Series of Selective BET Inhibitors
Containing Aniline or Indoline Groups
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Jianping Hu , Yingqing Wang , Yanlian Li , Danyan Cao , Lin Xu , ShanShan Song ,
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Mohammadali Soleimani Damaneh , Jian Li , Yuelei Chen , Xin Wang , Lin Chen , Jingkang
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Shen , Zehong Miao , Bing Xiong
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Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
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Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
201203, China
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University of Chinese Academy of Sciences, NO.19A Yuquan Road, Beijing 100049, China
#
These authors contributed equally
*
Corresponding authors. Tel: +86 21 50806600 ext. 5412 fax: +86 21 50807088. Email: (B. X.)
bxiong@simm.ac.cn; (Z. M.) zhmiao@simm.ac.cn
Abbreviations
BCPs, Bromodomain-containing proteins;
BET, bromodomain and extra-terminal;
BRD4-BD1, first bromodomain of BRD4;
CREB, cyclic-AMP response element binding protein;
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FA , fluorescence anisotropy;
FBDD, fragment-based drug discovery;
HATs, histone acetyltransferases;
HDACs, histone deacetylases;
Kac, acetylated lysine;
NUT, nuclear protein in testis;
PLK1, Polo-like Kinase 1;
P-TEFb, positive transcription elongation factor B;
PTMs, post-translational modifications;
SARs, structure- activity relationships;
SSRF, Shanghai Synchrotron Radiation Facility.
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Abstract
Recently, several kinase inhibitors were found to also inhibit bromodomains,
providing a new strategy for the discovery of bromodomain inhibitors. Along this line,
starting from PLK1-BRD4 dual inhibitor BI-2536, we discovered a new series of
dihydroquinoxalin-2(1H)-one with aniline and indoline WPF binders as selective
BRD4 inhibitors. They showed better BRD4-BD1 potency and negligible PLK1
kinase activity comparing with BI-2536. Additionally, dihydroquinoxalin-2(1H)-ones
containing indoline group showed profound activities in molecular and cellular based
assays. Throughout the study, compounds 9, 28 and 37 showed significant inhibitory
activity for c-Myc or PD-L1 protein expression and mRNA transcription both at
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concentration of 0.2 and 1 µM. Compound 9 was found possessing the best balance of
binding affinity, in vitro metabolic stability and in vivo pharmacokinetic properties.
Therefore, it was selected for in vivo pharmacological study. By using MM.1S cell
derived xenograft model, we confirmed compound 9 showed comparable in vivo
tumor inhibition to phase II investigation drug I-BET762, which, together with the
novel WPF binder, further indicated the utility of this series of BRD4 inhibitors.
Keywords: BRD4; BI2536; indoline; WPF binders; c-Myc; PD-L1
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Introduction
Epigenetics is the study of the changes in gene expression pattern or heritable
,
phenotypes without altering the sequence of genes.[1] [2] The regulatory mechanisms
of epigenetics mainly consist of methylation of DNA and RNA, non-coding RNA
regulation, as well as the post-translational modifications (PTMs) of histones. One of
the most extensively studied histone PTMs is lysine acetylation,[3] which is balanced
,
by the action of histone acetyltransferases (HATs)[4] [5] and histone deacetylases
(HDACs). Bromodomain, a protein module recognizing acetylated lysine (Kac) in
histones, transduces the state of lysine acetylation to regulate specific gene
expression.[6] In the human genome there are 61 bromodomains distributed in 46
diversified proteins,[7] and these bromodomains can be classified into eight
subfamilies.[8] The function of BET (bromodomain and extra-terminal) subfamily
(including four members: BRD2, BRD3, BRD4, and BRDT) was extensively
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studied.[9] [10] The primary function of the BET proteins is to recruit transcriptional
regulatory complexes to acetylated chromatin, thereby controlling specific gene
networks involved in cell proliferation and cell cycle progression. For example,
BRD4 and BRDT can regulate transcriptional elongation, mainly through the
interaction with the positive transcriptional elongation factor (P-TEFb), resulting in
,
RNA polymerase II activation.[11] [12] BET family proteins, especially BRD4, are
,
associated with many diseases, such as cancer, inflammation, etc.[9] [13] This makes
the BET proteins attractive drug targets for cancer treatment.
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The discovery of the first potent BET inhibitors (+)-JQ-1 (1)[14] and I-BET762 (2)
[15] were reported in 2010 (Figure 1). (+)-JQ1 was widely used as chemical probe to
study the function of BET proteins in physiology as well as in many diseases. Similar
to (+)-JQ1, I-BET-762 is also a selective BET inhibitor and now in a phase II clinical
trial for nuclear protein in testis (NUT) midline carcinoma. Since the first reports of
BET inhibitors, the field of BET inhibitor development evolved rapidly, as many new
scaffolds were discovered to interact with acetylated lysine binding site. To name one
,
example,
PFI-1
(3,
Figure
1)
[16] [17]
containing
3-methyl-3,4-dihydroquinazolin-2(1H)-one ring was reported as a selective BET
inhibitor for BRD4 with IC of 0.22 µM in a cell-free assay and an EC of 1.89 µM
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for the inhibition of IL6 production from human blood mononuclear cells stimulated
by LPS. Examining the cocrystal structure of PFI-1 bound to BRD4 (PDB code: 4E96)
revealed that the urea group forms two critical hydrogen bonds with conserved
residue ASN140 in the first bromodomain of BRD4. Also, our group previously
reported that compound 4 with a novel scaffold of dihydrothiazolone had IC value
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of 140 nM for BRD4(1).[18] Interestingly, some kinase inhibitors also have been
identified to interact with bromodomains, indicating another method to discover new
selective inhibitors of bromodomains.[19] In cocrystal structure with BRD4 (PDB
code: 4O74), PLK1 inhibitor BI2536 (5) utilizes the amide oxygen in the core
structure to interact with the residue ASN140. Recently, Chen et al. optimized the
substitution on the 2-amino-dihydropteridinone ring and the linking
group—2-methoxylbenzene to find the selective bromodomain inhibitors.[20] Several
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strategies were tested to increase BRD4 selectivity, including replacement of
cyclopentyl with 3-bromobenzyl and substitution of the pyrimidine NH with an O
atom. However, the representative compound only showed weak selectivity and
reduced BRD4 activity comparing to the original BI2536, with IC value of 90 nM
5
0
for BRD4 and IC value of 240 nM for PLK1.
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Bearing the same purpose of Chen et al. in mind, based on the different binding
pattern of BI2536 with BRD4 and PLK1, we proposed a different strategy by
modifying the pyrimidine ring, as this is an essential hydrogen acceptor for binding to
the kinase hinge part of PLK1. Therefore, to design selective BET inhibitors, we
preserved the essential hydrogen bonding interactions with ASN140 of BRD4 while
switching the pyrimidine of BI2536 with phenyl or pyridine group to remove the
hydrogen bonding interactions with the hinge of PLK1. Previously, based on this
design we reported a series of dihydroquinoxalin-2(1H)-ones with sulfonamides or
amines WPF binder as selective BET inhibitors.[21] Some of them had good
molecular and cellular activity. However, these compounds showed instability in vitro
liver microsome stability assays. In order to improve the metabolic stability of this
chemotype of compounds, we further explored novel side chain which supported to
enter into the WPF pocket.
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In the previous study, compound 6 (Figure 1) attracted our attention because it had an
amide linker with the nitrogen atom attaching to the scaffold and moderate molecular
activity. We thought amide linker may have better metabolic stability than
sulfonamide linker. So, we firstly reversed the direction of the amide linker of
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compound 6 with the purpose of improving activity and metabolic stability.
Fortunately, compound 7 (Figure 1) was found having a better inhibitory activity
against BRD4 than compound 6 (Table 1). Meanwhile, compound 7 (Table 4)
demonstrated good stability in vitro liver microsome stability assays. These results
had fascinated our great interest. Therefore, we focused on this new amide linker to
explore the structure-activity relationships (SARs) of WPF binder.
N
N
O
N
N
H
O
N
Cl
I-BET762, 2
7
8
9
Figure 1. Structures of selected BET bromodomain inhibitors, including (+)-JQ1 (1),
I-BET762 (2), PFI-1 (3), our previously reported BET bromodomain ligand 4[18],
dual kinase-bromodomain inhibitors BI2536 (5), and designed two compounds 6 and
7.
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By extensively exploring the SARs of the side chain, we were able to obtain two
series of potent BRD4 inhibitors bearing novel aniline and indoline side chain.
Among them, compound 37 containing 5-methylindoline showed profound activities
in molecular and cellular based assays, providing an exceptional binding motif for
other chemotypes of BRD4 inhibitors. Finally, we found compound 9 had the
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favorable balance of activity, in vitro and in vivo druggability properties. In an in vivo
MM.1S cell derived xenograft model compound 9 demonstrated similar efficacy to
the phase II compound I-BET762, further reinforcing the utility of current reported
BET inhibitors.
Results and Discussion
Initially, we performed minor modifications on the basis of compound 7. As indicated
in Table 1, we found that removing para-methyl from the phenyl side chain reduced
the inhibitory activity by 3.3 fold (compare 8 with 7), while adding another methyl
group to ortho position of the phenyl side chain of compound 7 increased binding
affinity about 2.7 fold (compare 9 with 7), probably because addition of a methyl
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group could improve interaction with hydrophobic WPF pocket. Expanding R group
from cyclopropyl to cyclopentyl gave compounds with similar inhibitory activity
(compare 7 with 10 or 9 with 11). In accord with the results from compounds 7 and 9,
3
compound 11 with R as 2,4-dimethylphenyl group had slightly better inhibitory
3
activity than compound 10 with R as 4-methylphenyl group. However, changing
1
racemic methyl of R group to racemic ethyl lowered the potency about 3 times
(compare 12 with 11), indicating this position could not tolerate a larger group than a
methyl group. Ligand efficiency (LE) is a valuable metric to measure the ratio of
affinity over heavy atoms, and could guide the ligand optimization to prevent from
growing into a “fat” moelcule. From the Table 1, our inhibitors showed high LE value
than three positive BRD4 inhibitors, which imply our inhibitors have better druglike
properties. Therefore, we followed the LE value during the optimization and try to
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maintain the efficiency of the inhibitors.
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Table 1. Effects of R , R , and R groups in 1-methyl-3,4-dihydroquinoxalin-2(1H)-
a
one derivatives on inhibition of BRD4-BD1 from fluorescence anisotropy assay.
4
1
2
3
Cpd
R
-
R
-
R
BRD4(I) IC (nM)
LE
5
0
(
+)-JQ-1
-
28.5±1.5
93.4±13.6
59.3±8.5
99.9±11.0
0.39
0.37
0.30
0.43
I-BET762
BI-2536
6
-
-
-
-
-
-
Me
Me
Me
Me
Me
Me
Et
7
8
9
46.4±8.4
152.5±24.5
17.2±2.1
31.3±2.8
20.9±1.1
73.8±1.1
0.45
0.49
0.45
0.43
0.42
0.38
10
11
12
a
5
6
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SE. The fluorescence compound used in the assay was JQ1-FITC (The
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synthesis route was provided in our group previous work).[22]
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According to the preliminary exploration, we preferred R as racemic methyl and R
as cyclopropyl or cyclopentyl. Next, compounds 13-19 were prepared to investigate
the effects of the type and position of substituents at side chain phenyl group (Table 2).
Comparison of 7 with 13-15 indicated that replacing para-methyl with other
substituents lowered the binding affinity, irrelevant to electronic properties of the
substitution. Shifting para-methyl (7) to ortho-methyl (16) led to a 3.6-fold reduction
in potency. However, changing ortho-methyl to ortho-fluoro or ortho-methoxy
maintained the inhibitory activity (compounds 16-18). The addition of a meta-fluoro
to the side chain phenyl (compound 19) resulted in slightly decreased affinity when
compared to compound 7. Replacing the side chain phenyl with cyclopentyl (20),
cyclohexyl (21), para-methylcyclohexyl (22), or trimethylpyrazolyl (23), or extending
the side chain phenyl with a linker group including methylene (24), sulfone (25) and
amine (26) generally deteriorated the potency, possibly because WPF pocket was not
large enough to tolerate benzyl substituent. Especially, compound 25 almost had a
24-fold drop in activity (compare 25 with 7), implying its sulfonamide oxygen may
repulse with WPF pocket. Overall, compounds with hydrophobic phenyl side chain
showed better inhibitory activity than compounds with aliphatic ring side chain and
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the para substitution of side chain phenyl was superior to the ortho substitution.
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Table
2.
Effects
of
R
group
in
4-cyclopropyl-1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one
derivatives
on
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a
1
2
inhibition of BRD4-BD1 from fluorescence anisotropy assay.
4
4
Cpd
R
BRD4(I)
IC (nM)
LE
Cpd
R
BRD4(I)
LE
IC (nM)
5
0
50
(
+)-JQ-1
-
-
-
28.5±1.5
93.4±13.6
59.3±8.5
0.39
0.37
0.30
0.47
19
20
21
22
62.3±1.4
357.0±17.0
285.5±24.5
389.0±30.0
0.43
0.49
0.48
0.45
I-BET762
BI-2536
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1
1
3
4
5
184.5±14.5
107.5±8.5
124.5±5.5
0.48
0.46
23
24
320.0±28.0
189.5±2.5
0.44
0.45
1
6
168.0±15.0
0.47
25
26
1108.5±27.5
370.0±17.0
0.
4
3
1
1
7
8
177.5±30.5
175.8±13.8
0.47
0.45
0.45
a
3
4
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SE.
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Figure 2. The cocrystal structures of BRD4-BD1 bound with inhibitors. A)
superimpose the cocrystal structure of BRD4-BD1 bound with compound 11
(showing in yellow color, PDB entry: 5YOV) to BRD4-BD1 bound with JQ1
(showing in cyan, PDB entry: 3MXF). B) superimpose the cocrystal structure of
compound 28 (PDB entry: 5YOU, colored in green) to cocrystal structure of
compound 11 (colored in yellow).
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To examine the binding mode of this series of BRD4 inhibitors, we solved the
cocrystal structure of compound 11 bound with BRD4-BD1 (Figure 2A). Inspecting
the structure revealed that the 3,4-dihydroquinoxalin-2(1H)-one scaffold situates at
the KAc binding pocket and forms one hydrogen bond with the conserved residue
ASN140. Also, 2,4-dimethylphenyl group reached into WPF pocket. Comparing the
cocrystal structure of compound 11 with cocrystal structure of JQ1 bound with
BRD4-BD1, the most striking difference is the orientation of xylene group, not like
the chlorobenzene group in JQ1, it sticks downward at the WPF shelf and forms Van
der Waals interaction with the methylene of the ASP145 side chain, which explained
why substitution of methyl group with chloro (14) decreased binding affinity about 5
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fold. Since the linker-amide group pointed upward and reached to the top of the
binding site.
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Then, we explored the substitution effect on the nitrogen atom of the amide linker
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(Table 3). No matter if R was phenyl, cyclopentyl or cyclohexyl, addition of a methyl
group to the amide nitrogen could significantly increase the potency (compare 27 with
8, 28 with 7, 30 with 20, 31 with 21 or 33 with 22). Enlarging the methyl group to
ethyl (compare 29 with 28 or 32 with 31) was tolerable and showed similar potency,
implying that the methyl or ethyl at the nitrogen atom of the amide linker may lead to
a more favorable conformation in which the benzene ring could interact well with
WPF subpocket. To verify this hypothesis, we solved the cocrystal structure of
BRD4-BD1 bound with compound 28. As illustrated in Figure 2B, the compound
bound to the binding pocket as expected and overlapped well with compound 11,
which validated our optimization strategy.
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Table 3. Effects of R and R substituents in 4-cyclopropyl-1,3-dimethyl-3,4-dihydro-
quinoxalin-2(1H)-one derivatives on inhibition of BRD4-BD1 from fluorescence
a
anisotropy assay.
1
7
4
5
Cpd
R
R
BRD4(I) IC (nM)
LE
5
0
(
+)-JQ-1
-
-
28.5±1.5
0.39
0.37
I-BET762
-
-
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BI-2536
-
-
59.3±8.5
10.3±1.7
0.30
0.48
27
28
29
30
31
32
33
Me
Me
Et
10.6±0.7
10.5±1.2
125.5±11.5
27.4±5.4
37.9±1.9
13.6±1.8
0.47
0.45
0.50
0.46
0.44
0.46
Me
Me
Et
Me
a
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2
3
4
5
6
7
8
9
0
1
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SE.
Based on binding affinity and structural diversity, several compounds were selected
for in vitro metabolic stability evaluation. As listed in Table 4, compounds 27-29 and
31-33 with a methyl or ethyl group at the nitrogen atom of amide linker dramatically
decreased the in vitro liver microsomal metabolic stability when being compared with
compounds 7, 9-11, and 19, which may be due to N-dealkylation. Comparing
compound 19 or compound 9 with compound 7, we found that placing a fluoro
substituent at the meta or a methyl substituent at the ortho of side chain phenyl only
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affected stability slightly. Replacement of cyclopropyl with cyclopentyl of R group
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also had little effect on the in vitro liver microsomal metabolic stability (compare 7
with 10 or 9 with 11). Five cytochrome P450 enzymes (3A4, 2D6, 2C9, 1A2 and
2C19) commonly metabolizing exogenous chemicals were selected to test the direct
inhibition of these compounds. As showed in Table 4, most of these compounds
generally showed weak inhibition on CYP450 enzymes even at 10 µM concentration.
Several exceptions were found, including compound 11 exhibiting a 74% inhibition of
2C9 enzyme, compounds 19 and 28 exhibiting > 50% inhibition of 2C19 enzyme, and
compound 33 exhibiting about 60% inhibition of 3A4 enzyme. Time-dependent
inhibition of these compounds were also examined against five tested CYP450
enzymes, and all compounds did not show time-dependent inhibition for CYP450
enzymes except for compound 11, which showed time-dependent inhibition for 3A4
enzyme.
1
1
1
1
1
Table 4. In Vitro liver microsome stability assays and cytochrome P450 enzymes
inhibition assays.
a
b
c
HLM
mLM
DI
TDI
Cpd.
d
e
Stab.
t1/2
32
18
29
15
29
3
Stab.
92
t1/2
3A4
2D6 2C9 1A2 2C19 kobs
7
9
43
77
15
12
14
14
13
1
6/11
5/6
14
1
17
22
48
74
23
1
7
36
45
41
47
58
37
no
no
no
117
102
98
no
14
20
18
no
1
0
48
15/30
24/38
6/17
12/3
15
16
12
0
11
94
3A4
no
1
2
9
7
49
111
1073
525
no
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756
512
1462
1321
1275
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218
210
110
114
162
582
50
2
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1565
1040
1309
1360
1856
394
1
1
1
1
1
4
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7
12/5
25/28
37/33
22/7
no
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no
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55
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no
no
no
no
no
no
no
no
no
no
no
no
no
no
1
4
1
no
4
no
27
12
6
1
60/64
23/17
31/24
33/22
28/17
12/9
13
33
32
38
42
15
44
65
26
14
25
37
63
57
77
36
96
59
71
32
12
6
7
580
6
7
404
9
11
2
13
12
8
452
no
no
11
14
3
310
no
12
18
18
6
276
35/27
44/43
14/9
2
928
28
19
122
74
204
16/11
no
a
1
2
3
4
5
6
7
8
HLM presents human liver microsome, the unit of stability data is µL/min/mg protein and the
b
c
unit of half-life is min; mLM presents mice liver microsome; CYPs direct inhibition, sometimes
referred as reversible inhibition, is assessed by measurement of an enzyme (CYP) activity in the
d
presence of increasing concentration of inhibitor without a preincubation step; 3A4 column
contains two numbers, which indicates two methods used in the experiments. First one uses the
e
midazolam as the substrate and the second uses the testosterone as the substrate. TDI stands for
time-dependent inhibition, which is referring to a change in enzyme inhibition during an in vitro
-4
incubation and means an irreversible inactivation of CYPs, the unit of TDI is 10 /min.
1
6

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9
0
1
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9
0
1
2
As aforementioned, the addition of a methyl or ethyl group to the nitrogen atom of
amide linker improved the potency but significantly reduced the metabolic stability in
human or mouse liver microsomes. We conjectured the alkyl group on the nitrogen
atom of amide linker was easily removed via oxidation. So, together with the analysis
of cocrystal structure of 28 bound to BRD4, we adopted a strategy by cyclizing the
linker amide nitrogen with side chain phenyl to generate compounds 34-40. Most of
these new compounds showed potent BRD4 inhibition except for compound 39,
maybe due to the similar reason for the decreased activity of compound 14 that
replacement of the methyl group with a fluorine group attenuated the Van der Waals
interaction between methyl with the methylene of the ASP145 side chain. Comparing
compounds 35-39 with compound 34, it was found that the introduction of a methyl
group to C4 or C5 position or both introduction of a methyl group to C2 and C5
position of the indoline slightly affected the binding affinity, while addition of a
methyl group to C2 position or a fluoro to C5 position of the indoline led to a drop of
potency by almost 2 fold or 4 fold, respectively. These results were in line with the
hypothesis that indoline side chain reached into WPF pocket which was a
hydrophobic pocket and addition of a methyl group to the side chain phenyl could
1
1
1
1
1
1
1
1
1
1
2
2
2
2
enhance their interaction with WPF pocket. Furthermore, expanding the R group of
compound 34 from cyclopropyl to cyclopentyl gave compound 40 with similar
potency; removing the nitrogen atom from indoline of compound 34 produced
compound 41 with dramatically reduced activity by almost 8 fold; and reducing the
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9
0
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8
amide carbonyl of linker group of compound 34 to methylene resulted in compound
42 with a severely reduced potency, implying that amide carbonyl of linker group may
make key hydrogen bond interaction with BDR4(1) or form a more efficient
conformation allowing for a better binding of indoline to WPF pockets. To assess
druglike property of this structure type, compounds 34-40 were measured for
metabolic stability and all showed better stability than compounds 27-29 or 31-33
with a methyl or ethyl group at the nitrogen atom of amide linker, indicating the
cyclized modification can slow down the metabolic rate in liver microsomes.
However, the metabolic stability of 34-40 was still less than compounds 7, 9-11 and
19. In table 4, compounds 34-40 did not exhibit time-dependent inhibition for
CYP450 enzymes. However, except for 34 and 38, most of these compounds
demonstrated > 50% inhibition of 2C19 enzyme, indicating CYP450 enzymes may be
the major metabolic enzymes of these compounds. In short, compounds 34-40
maintained high potency but reduced in vitro liver microsomal metabolic stability
when being compared with compound 9.
1
1
1
1
1
1
1
1
1
2
6
Table 5. Effects of R substituent and R substituent of side chain indoline in
1,3-dimethyl-3,4-dihydroquinoxalin-2(1H)-one derivatives on inhibition BRD4-BD1
a
from fluorescence anisotropy assay.
1
9
2
6
Cpd
R
R
BRD4(I) IC (nM)
LE
5
0
1
8

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(
+)-JQ-1
-
-
-
-
-
28.5±1.5
93.4±13.6
59.3±8.5
20.4±5.2
0.39
0.37
0.30
0.45
I-BET762
BI-2536
-
34
35
36
37
38
39
40
41
H
2-Me
4-Me
36.2±7.0
20.2±5.3
15.9±2.7
22.3±3.3
81.7±2.7
17.4±1.7
168.0±1.0
0.42
0.43
0.44
0.42
0.40
0.42
0.44
5-Me
2,5-dimethyl
5-F
H
42
1574.5±506.5
0.47
a
1
2
3
4
5
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SE.
In order to further improve metabolic stability of these compounds with indoline WPF
binder,
we
replaced
the
dihydroquinoxalin-2(1H)-one
scaffold
by
dihydropyrido[2,3-b]pyrazin-2(1H)-one scaffold (compounds 43-46, Table 6).
1
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7
8
9
However, all new compounds showed reduced potency. Nevertheless, two more
potent compounds 45 and 46 were subjected to in vitro liver microsomal metabolic
stability test (Table 4). They showed better metabolic stability in human or mouse
liver microsomes than the analogs 37-38, especially compound 45, which emphasized
that the metabolism depends on whole molecule and slight changes on compounds
can resulted substantial difference in metabolic stability.
6
Table 6. Effects of
R
substituent of side chain indoline in
dihydropyrido[2,3-b]pyrazin-2(1H)-one derivatives on inhibition of BRD4-BD1 from
a
fluorescence anisotropy assay.
1
0
6
6
Cpd
R
BRD4(I)
IC (nM)
LE
Cpd
R
BRD4(I)
LE
IC (nM)
5
0
50
(
+)-JQ-1
-
-
-
28.5±1.5
93.4±13.6
59.3±8.5
0.39
0.37
0.30
0.45
44
45
46
4-Me
5-Me
199.0±14.0
54.6±13.0
0.44
0.41
0.40
I-BET762
BI-2536
2,5-dimethyl 60.7±11.0
4
3
H
238.5±16.5
a
1
1
1
1
1
1
2
3
4
5
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SE.
Bromodomain Selectivity Profile
Bromodomains are conserved module in evolution. Although they share a common
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1
2
3D structure pattern of one short helix and three long helices and the acetylated lysine
binding pocket at the top of the bromodomains, they usually have different roles in
cell context. As selectivity is critical for the safety issue, based on molecular activity
and in vitro metabolic stability, compound 9 was selected to profile the binding
specificity across 32 bromodomains in the BROMOscan assay. As shown in Figure 3,
compound 9 exhibited high selectivity towards BET family over other 23 screened
bromodomains, except weak inhibitory effect on TAF1 (The detailed number was
provided as Table S5 in supporting material). However, compound 9 was short of
selectivity for the BET subfamily and may cause potential toxicity.
1
1
1
1
1
1
1
1
1
1
2
2
2
Rooney et al. had reported
a
series of inhibitors containing
dihydroquinoxalin-2(1H)-one scaffold as CREB (cyclic-AMP response element
binding protein) binding protein (CBP) inhibitor.[23] Although the scaffold is similar
to current reported inhibitors, the low CBP activity of compound 9 indicated that our
compounds have different binding mode from the inhibitors discovered by Rooney et
al., which also can be identified from the comparison of cocrystal structures of two
chemtypes (PDB entry: 4NYW versus 5YOV, see the supporting information about
the structural comparison). By comparing these two structures, the binding
interactions of dihydroquinoxalin-2(1H)-one scaffold in CBP inhibitor showed
dramatic difference, which use the amide group to interaction with the conserved
asparagine residue. Also the CBP protein does not have tryptophan residue at the WPF
part. Instead, it has the leucine at similar position, which enlarges the subpocket.
Together, these two differences may result low affinity of compound 9 towards CBP
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1

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bromodomain.
2
3
Figure 3. Selectivity assessment of compound 9 against the BRD families in the BROMOscan
panel. The 32 screened targets are labeled in black on the phylogenetic tree of the BRD family;
BRDs that were not part of the screening panel are in gray. Compound 9 was screened at 1 µM
concentration, and results for primary screen binding interactions are reported as 'Percent Control',
where lower numbers indicate stronger hits in the matrix.
4
5
6
7
8
9
PLK1 Kinase Selectivity Profile
1
1
1
1
0
1
2
3
We aimed to discover a series of selective BRD4 inhibitors by starting with
PLK1-BRD4 dual inhibitor BI-2536. Therefore, considering the collected information,
we chose four compounds to profile their PLK1 kinase activities, along with positive
control compounds BI-2536 and a pan ATP competitive kinase inhibitor staurosporine.
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3
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From Table 7, as expected, BI-2536 showed a significant inhibitory effect on PLK1
kinase at 10 µM or 1 µM. While compounds 9, 28, 37 and 45 demonstrated only
negligible inhibitory activity on PLK1 kinase at 10 µM or 1 µM. Thus, modifying the
pyrimidine ring of BI-2536 to remove the essential hydrogen acceptor for kinase
hinge binding is a effective way to improve the selectivity towards BET proteins.
6
Table 7. PLK1 kinase activity of compounds 9, 28, 37 and 45
Cpd.
Inhibition
Inhibition
(%, 1 µM)
58
Cpd.
Inhibition
Inhibition
(
%, 10 µM)
(%, 10 µM)
(%, 1 µM)
Staurosporine
95
100
5
28
37
45
8
-2
0
1
3
8
BI-2536
9
101
10
7
8
9
0
1
2
3
4
5
6
7
Cellular antiproliferation activity
Based on their binding activities in FA assay and in vitro liver microsomal metabolic
stability, some compounds were selected to examine their cellular antiproliferative
effects. In Table 8, compounds 9, 27-29, and 37 were found with better potency than
(+)-JQ-1 both in MM.1S and TY-82 cells, consisting with their molecular activities.
Comparing compounds 27-29, or 37 with compound 9, it was found that addition of a
methyl or ethyl group to the linker amide NH or cyclization of NH with the side chain
phenyl increased the cell viability both in MM.1S and TY-82 cell lines. However,
further adding a methyl group to C2 position of the indoline of compound 37
(compound 38), changing the methyl group of the indoline of compound 37 to a
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fluoro substituent (compound 39) or modifying the scaffold to
3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-one (compare 45 with 37 or 46 with 38)
dramatically decreased the cellular antiproliferative activity.
Table 8. Effects of Some Compounds on Antiproliferative Effects against Cell Lines
a
MM.1S and TY-82
Cpd
MM.1S
TY-82
Cpd
MM.1S
TY-82
IC (nM)
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
50
(
+)-JQ-1
69.1±18.0
349.2±31.0
153.7±43.7
54.9±2.0
80.8±73.2
304.3±165.6
90.7±26.1
28
29
36
37
38
39
45
46
2.8±2.4
6.1±5.7
1.5±0.9
3.5±2.0
I-BET762
7
9
101.8±34.1
33.7±9.9
33.4±9.65
8.1±4.3
58.9±69.5
1
0
2584.8±403.2
609.5±187.0
1901.9±330.4
39.1±11.1
1610.5±393.2
213.3±14.8
1382.3±290.0
25.4±16.2
383.0±157.6
352.9±61.1
290.8±27.7
154.8±3.5
149.6±146.6
147.7±141.1
153.1±86.4
54.0±34.7
11
1
2
9
7
a
6
7
8
9
0
1
2
The IC50 in the table was calculated from three independent experimental measurements and
expressed as mean ± SD.
Effects on c-Myc Protein and mRNA Expression.
1
1
1
Delmore et al. discovered the BET bromodomain proteins as regulatory factors for
oncogene c-Myc.[24] The BRD4 inhibitor (+)-JQ1 induces an antiproliferative effect
associated with the down-regulation of c-Myc transcription.[14] Moreover, Hogg et al.
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0
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found that BET inhibitors promoted anti-tumor immune responses through
transcriptional repression of immune checkpoint ligand PD-L1 in genetically diverse
tumor models and in response to inflammatory stimuli.[25] To further assess our
dihydroquinoxalin-2(1H)-one series of BRD4 inhibitors, we performed the western
blotting experiment and quantitative real-time PCR (RT-qPCR) to study the cellular
effect related to c-Myc and PD-L1. We chose compounds 9, 28, 37 and the positive
controls (+)-JQ-1 and I-BET762 to evaluate their effects on protein and mRNA levels
of c-Myc and PD-L1. As showed in Figure 4A and 4B, all three compounds showed
significant inhibitory activity for c-Myc protein and mRNA expression, especially
compound 28, which displayed better inhibitory activity than (+)-JQ-1 and I-BET762
at concentrations of 0.2 and 1 µM. From Figure 4C, we found all these compounds
showed inhibitory activity for PD-L1 protein expression when compared to blank
control, especially (+)-JQ-1 and compound 28. Moreover, all three compounds
demonstrated inhibitory activity for PD-L1 RNA expression at concentrations of 0.2
and 1 µM, indicating these three compounds may be used to promote anti-tumor
immune responses through transcriptional repression of immune checkpoint ligand
PD-L1.
1
1
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2
5

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2
Figure 4. (A) Inhibition of compounds 9, 28 and 37 on the expression of c-Myc protein. TY-82
cells were treated with compounds 9, 28 and 37 (0.2 µM or 1 µM), I-BET762 (0.2 µM or 1 µM)
and (+)-JQ1 (0.2 µM or 1 µM) for 24 h, respectively. Cells were collected and lyzed for Western
blotting. The level of c-Myc protein was detected and GAPDH protein was chosen as the loading
control. The experiments were repeated three times. (B) Inhibition of compounds 9, 28 and 37 on
the expression of c-Myc mRNA. TY-82 cells were treated as in (A). Then, total RNA was isolated
and reverse-transcribed for RT-qPCR analyses. The data were expressed as mean ± SD,
representing the relative levels of c-Myc mRNA from three independent experiments. (C)
Inhibition of compounds 9, 28 and 37 on the expression of PD-L1 protein. NCI-H1299 cells were
treated as in (A). Cells were collected and lyzed for Western blotting. The level of PD-L1 protein
was detected and GAPDH protein was chosen as the loading control. (D) Inhibition of compounds
9, 28 and 37 on the expression of PD-L1 mRNA. NCI-H1299 cells were treated as in (A). Then,
3
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1
2
3
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total RNA was isolated and reverse-transcribed for RT-qPCR analyses. The data were expressed as
mean ± SD, representing the relative levels of PD-L1 mRNA from three independent experiments.
In Vivo PK Study
On the basis of the molecular, cellular activities, structural diversity and in vitro
metabolic stability of these compounds, we selected compounds 9, 37 and 45 for
further in vivo pharmacokinetics (PK) study. When injecting compounds 9, 37 and 45
(1% Tween80/water formulation) with specific dosage to male ICR (CD-1) mice, it
was found that compound 9 displayed higher plasma exposure than compounds 37
and 45 (Table 9), indicating the in vitro metabolic stability test could be used to guide
the lead optimization. After dosing at 20 mg/kg, compound 9 reached a maximum
plasma concentration of 1775.9 ng/mL at 0.3 h. Compound 9 possessed a moderate
half-life (1.14 h) and a reasonable plasma exposure of 2655.8 ng×h/mL, supporting
further in vivo pharmacology study.
1
1
1
1
1
1
a
Table 9. In Vivo PK Data for Compounds 9, 37 and 45
Tmax
Cmax
AUC0-t
h/mL)
T1/2
Route of
administration
p.o.
dose
(mg/kg)
20
CL
Cpd.
9
(h)
(ng/mL) (ng
×
(h) (L/h/kg)
0.3
1775.9
335.3
189.0
2655.8
188.7
382.8
1.14
0.55
1.51
7.7
3
4
7
5
p.o.
20
0.25
0.33
110.12
40.64
p.o.
15.4
a
1
1
6
7
1% Tween 80/water was used as vehicle.
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9
0
In Vivo Pharmacological Study
According to above investigation, we chose compound 9 for evaluating the
therapeutic effect in in vivo human MM.1S xenograft model of SCID mice.
Tumor-bearing mice were treated with 9 and I-BET762 at two doses of 80 mg/kg or
20 mg/kg daily. The results showed that treatment of compound 9 significantly
decreased the growth of tumor volume at dosage of 80 mg/kg daily (Figure 5). When
being compared to the positive control BRD4 inhibitor I-BET762, 9 showed marginal
improvement on tumor inhibition with RTV of 622% than I-BET762 with RTV of
797%. These data illustrated the therapeutic potential of compound 9 for tumor
treatment.
1
1
1
1
2
Figure 5. Relative tumor volume (RTV) of human MM.1S xenografts in SCID mice after
treatments. Formulation: 9 and I-BET762: 0.5% Tween80 and 0.5% Methylcellulose aqueous
solution.
1
1
1
1
1
1
3
4
5
6
7
8
Chemistry
The syntheses of novel dihydroquinoxalin-2(1H)-one derivatives were depicted in
schemes 1-4. As illustrated in scheme 1, aromatic nucleophilic substitutions (S Ar) of
N
2
8