Structure-Based Discovery and Development of a Series of Potent and Selective Bromodomain and Extra-Terminal Protein Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01094

BRD4 has recently emerged as a promising drug target. Therefore, identifying novel inhibitors with distinct properties could enrich their use in anticancer treatment. Guided by the cocrystal structure of hit compound 4 harboring a five-membered-ring linker motif, we quickly identified lead compound 7, which exhibited good antitumor effects in an MM.1S xenograft model by oral administration. Encouraged by its high potency and interesting scaffold, we performed further lead optimization to generate a novel potent series of bromodomain and extra-terminal (BET) inhibitors with a (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one structure. Among them, compound 19 was found to have the best balance of activity, stability, and antitumor efficacy. After confirming its low brain penetration, we conducted comprehensive preclinical studies, including a multiple-species pharmacokinetics profile, extensive cellular mechanism studies, hERG assay, and in vivo antitumor growth effect testing, and we found that compound 19 is a potential BET protein drug candidate for the treatment of cancer.

Full text of DOI:10.1021/acs.jmedchem.9b01094

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Article
Structure-based Discovery and Development of a Series of Potent
and Selective Bromodomain and Extra-Terminal Protein Inhibitors
Jianping Hu, Changqing Tian, Mohammadali Soleimani Damaneh, Yanlian Li, Danyan Cao,
kaikai Lv, Ting Yu, Tao Meng, Danqi Chen, Xin Wang, lin Chen, jian li, Shanshan Song,
Xiajuan Huan, Lihuai Qin, Jingkang Shen, Yingqing Wang, Ze-Hong Miao, and Bing Xiong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01094 • Publication Date (Web): 06 Sep 2019
Downloaded from pubs.acs.org on September 6, 2019
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of their duties.

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Structure-based Discovery and Development of a Series of Potent
and Selective Bromodomain and Extra-Terminal Protein Inhibitors
Jianping Hu1,4,#, Changqing Tian2,4,#, Mohammadali Soleimani Damaneh , Yanlian Li , Danyan
2,4
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Cao , Kaikai Lv , Ting Yu , Tao Meng , Danqi Chen , Xin Wang , Lin Chen , Jian Li,
Shanshan Song , Xiajuan Huan , lihuai Qin , Jingkang Shen , Yingqing Wang2,4,*, Zehong
2
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Miao2,4,*, Bing Xiong1,4,*
1Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
2Division 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
3Center for Chemical Biology and Drug Discovery, Department of Pharmacological Sciences,
Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY10029, United
States
4University 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; (Y. W.) yqwang@simm.ac.cn
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ABSTRACT
BRD4 has recently emerged as a promising drug target. Therefore, identifying novel
inhibitors with distinct properties could enrich their use in anticancer treatment.
Guided by the cocrystal structure of hit compound 4 harboring a five-member-ring
linker motif, we quickly identified lead compound 7, which exhibited good anti-tumor
effects in a MM.1S xenograft model by oral administration. Encouraged by its high
potency and interesting scaffold, we performed further lead optimization to generate a
novel potent series of bromodomain and extraterminal (BET) inhibitors with a
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(1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one structure. Among them,
compound 19 was found to have the best balance of activity, stability, and anti-tumor
efficacy. After confirming its low brain penetration, we conducted comprehensive
preclinical studies, including a multiple species pharmacokinetics profile, extensive
cellular mechanism studies, hERG assay, and in vivo anti-tumor growth effect testing,
and we found that compound 19 is a potential BET protein drug candidate for the
treatment of cancer.
Keywords: Bromodomain; (1,2,4-triazol-5-yl)-3,4-dihydroquinoxalin-2(1H)-one;
blood-brain barrier penetration; five-membered-ring linker
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INTRODUCTION
Bromodomain-containing proteins (BCPs) can decipher the acetylation code of
proteins via binding to their acetylated lysine (KAc) residues, which further
modulates acetylation signal transduction and produces diverse physiological
functions.1-3 In many diseases, and best studied in cancers, BCPs are found to be
aberrantly expressed, and they further enhanced the expression of oncogenes and
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anti-apoptotic proteins.4, 5 Thus, BCPs have emerged as attractive epigenetic
therapeutic targets for cancer.6, 7
There are 61 bromodomains distributed in 46 distinct proteins encoded in the
8
human genome, and these bromodomains can be divided into 8 subfamilies. Among
them, the most studied is the Bromodomain and Extra-Terminal domain (BET)
subfamily, which includes the following four members: BRD2, BRD3, BRD4, and
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BRDT. Since the first BET inhibitor, (+)-JQ1 (1), was discovered, diverse BET
inhibitors have arisen, with some of them entering into clinical trials, including
OTX-015 (2),11, 12 I-BET762 (3),13, 14 CPI0610 and ABBV-075,
15
16, 17
to name a few.
However, the cellular functions of BET proteins, specifically BRD4, are more
complex than previously thought.18 As an epigenetic reader, BRD4 was initially
believed to recognize the acetylated lysine in H4 with its two N-terminal
bromodomains, further recruiting the transcription elongation factor b (p-TEFb)
cyclin T1/CDK9 complex to phosphorylate RNA polymerase II (RNA pol II) to
activate gene transcription.19 The profound downstream effect of this event was
initially attributed to a reduction in the expression of the oncogene Myc, which
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encodes the multipurpose Myc protein related to cell growth and differentiation.20
Since directly targeting Myc protein was very difficult, targeting BRD4 to interrupt
p-TEFb binding to histones was considered as a surrogate for Myc-related cancer
treatment.
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Recently, with the advance of sequencing technologies, many other cellular roles
of BRD4 have been revealed from transcriptome and epigenome studies. In addition
to binding to acetylated H4, BRD4 can also act as a critical component of the
superenhancer complex by binding to many transcription factors, including Myc,
TWIST,21 CEBPA, and CEBPB,22 and the transcription-related protein RelA.23
Meanwhile, new downstream effects have been discovered; for example, BET protein
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4
inhibition can promote anti-tumor immunity by suppressing PD-L1 expression. As
nontranscriptional roles, BRD4 has also been shown to control DNA damage
25
checkpoint activation and repair as well as telomere maintenance. Using (+)-JQ1 as
a chemical probe, BRD4 was identified to have an essential cognitive function in the
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brain, and BRD4 inhibition in the brain could impair memory abilities. These recent
studies on the different functions of BRD4 reinforced the argument of Andrieu et al.
that clinical trials for BET inhibitors should design with caution, as currently we are
still in the primary phase of understanding the roles of BRD4.27
N
N
N
O
N
O
N
N
S
S
O
NH
N
N
OH
Cl
+)JQ-1, 1
Cl
(
OTX-015, 2
Figure 1. Structures of typical BET bromodomain inhibitors including (+)-JQ1
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(1), OTX-015 (2), I-BET762 (3), and hit compound 4.
Nevertheless, the important role of BRD4 in cancer biology and partial successes
of clinical trials with BET inhibitors28, 29 have spurred researchers to pursue new
inhibitors with novel scaffolds, as they may bring distinct values with different
pharmacological effects or tissue distribution. Researchers recently discovered
previous patents that described a series of dihydroquinoxalin-2(1H)-ones with
substituted or unsubstituted nitrogen or oxygen linkers as BET inhibitors.30, 31
Moreover, researchers such as Conway et al. revealed a series of potent CREBBP
bromodomain inhibitors with a dihydroquinoxalinone scaffold, and they emphasized
the importance of the internal hydrogen bond, which is formed from the scaffold
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amine NH with the side-chain carbonyl. Meanwhile, we previously incrementally
mapped out some novel BRD4-selective inhibitors with
a
dihydroquinoxalin-2(1H)-one scaffold that were derived from the dual PLK1-BRD4
inhibitor BI-2536.33-35 However, they did not possess comprehensive drug-like
properties. Furthermore, we found that others had also tried to tune the selectivity and
potency of BI-2536; for example, Watts et al. designed a series of anaplastic
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lymphoma kinase (ALK) and BRD4 dual inhibitors from BI-2536 and Liu et al.
described a series of compounds with different selectivity between PLK1 and BRD4
using BI-2536 as starting point.37
Now, in this research, by preserving the core dihydroquinoxalin-2(1H)-ones
modified from BI2536, we explored new side-chains and discovered novel hit
compound 4 (Figure 1). Guided by crystal structures, we were able to discover a
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potent and selective inhibitor (19) as a potential drug candidate. The extensive profile
of this compound showed that it displayed high selectivity for the BET subfamily
over other bromodomains and more than twenty kinases from different families,
possessed favorable metabolic stability in multiple species, showed a low blood-brain
barrier penetration ratio and good PK properties in rodents and dog, and demonstrated
excellent in vivo antitumor efficacy. Hereinafter, we report on the optimization of this
potential drug candidate as well as comprehensive profiles of its in vitro and in vivo
properties.
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RESULTS AND DISCUSSION
Hit to Lead
Based on the BET-selective inhibitors reported in our previous work,33, 34 we
wanted to further improve their in vitro and in vivo properties to develop a drug
candidate for clinical research. We initially preserved the core
dihydroquinoxalin-2(1H)-one and screened for new side chains, finding that
compound 4 with a pyrrole ring had moderate activity (IC = 303.0 nM) and a low
5
0
molecular weight that could be used as a starting point. Superimposition of the
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cocrystal structures of a reported BET inhibitor (5) in BRD4-BD2 to our crystal
structure of 4 bound to BRD4-BD1 (Figure 2) found that there was a large space for
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modification around the pyrrole ring. From our previous study, we believed that the
ortho position could be extended to reach the WPF subpocket to improve the potency
and selectivity. Hence, we added a hydrophobic group (e.g., phenyl or p-methyl
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phenyl) to this position and synthesized different five-membered ring linkers to
generate a series of dihydroquinoxalin-2(1H)-one compounds (6-12, Table 1). The FP
binding assay showed that they all had excellent potency, indicating this hydrophobic
group may enter the WPF subpocket.
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Figure 2. Superimposition of the cocrystal structure of BRD4 BD1 bound with 4
(PDB entry: 6JI3, green color) and BRD4-BD2 bound with compound 5 (PDB entry:
4Z93, gray color).
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As listed in Table 1, we kept R as a racemic methyl or (R)-methyl, R as
cyclopropyl or cyclopentyl and R3 as methyl or hydrogen to explore the
five-membered ring linker. A comparison of compound 6 with 7 found that the
addition of a methyl group to the triazole could enhance the molecular and cellular
activity by 1.5-fold and 2.0-fold, respectively. By substituting the triazole group with
a tetrazole ring, we found that the molecular binding activities were only slightly
decreased (6 versus 8 and 10 versus 11); however, the cellular anti-proliferative
activities were dramatically reduced, indicating that the more hydrophilic tetrazole
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may decrease the membrane permeability of the compounds. Replacement of the
triazole with thiazolidinone or methylisoxazole resulted in a significant loss of
activity (6 versus 9 or 10 versus 12). These results indicated that the methyl position
of the five-member ring may be surrounded by small hydrophobic residues and could
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not tolerate the polar nitrogen or carbonyl group. Only by changing R cyclopropyl
group to a cyclopentyl group could maintain the molecular activity; in contrast, the
cellular activity was dependent on the overall molecule properties and could be
retained (7 and 10) or weakened (8 and 11). These results indicated that the nature of
2
the five-member ring linker and R group significantly affected their activities; in the
future, perhaps we can change the linker to a pyrazole, pyrrole, or six-member linker
such as phenyl or pyridine to explore more potent compounds. In short, compounds 6,
7
and 10 were found to have good molecular and cellular activities with IC values
50
below 10 nM, which were worth further exploration.
Table 1. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of
compounds 6-12 on BRD4 (I).
N
O
Linker
N
R2
R1
R3
Linker
Cpd
R1
R2
R3
BRD4(I)
MM.1S
a
b
IC (nM)
IC (nM)
50
50
(+)-JQ-1
-
-
-
-
28.5 ± 1.5
29.8 ± 16.6
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OTX-015
6
-
-
-
-
34.3 ± 6.5
8.6 ± 1.0
6.3 ± 13.5
6.7 ± 0.3

Me
Me
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


7
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9
Me
Me
Me
Me
Me
Me
5.7 ± 0.2
14.4 ± 0.9
142.0 ± 3.0
3.3 ± 1.2
47.7 ± 25.1
N.D.c




1
1
1
0
1
2
Me
Me
H
5.2 ± 1.6
9.5 ± 1.2
3.9 ± 3.0
1228.7 ± 376.0
N.D.c
Me

100.4 ± 6.4
a
The IC50 values are the mean of two independent experiments performed in duplicate with
standard error of the mean (SEM) values. b The IC50 values are the mean of three independent
c
experiments performed in triplicate with standard error of the mean (SEM) values. N.D. means
not determined.
According to the molecular and cellular activities, compounds 6, 7 and 10 were
selected for in vitro metabolic stability evaluation (Table S2). We found that
compounds 6 and 7 had better metabolic stabilities than compound 10 in both human
and mouse liver microsomes. At 10 μM, compound 6 showed evident inhibition of
most of the tested CYP450 enzymes; in contrast, compounds 7 and 10 exhibited a
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weak inhibitory effect on the CYP450 enzymes, except that compound 10 showed a
more than 50% inhibition ratio for the 2C9 enzyme. Additionally, none of the
compounds exhibited time-dependent inhibition of CYP450 enzymes, indicating less
vulnerability of these compounds during drug-drug interactions.
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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
Considering the liver microsomal metabolic stability, we chose compounds 6 and
7 for in vivo pharmacokinetics (PK) study (Table 2). Pharmacokinetic tests
demonstrated that compound 7 indeed possessed better plasma exposure after oral
administration at a 3 mg/kg dosage than compound 6 at a 10 mg/kg dosage,
suggesting that compound 7 could be advanced to an in vivo pharmacological study.
Table 2. In vivo PK data for compounds 6 and 7 in male ICR mice.a
Route of
Dose
Tmax
(h)
Cmax
AUC0-t
AUC0-inf
T1/2
(h)
Cpd.
administration
(mg/kg)
(ng/mL)
(ng·h/mL)
(ng·h/mL)
6
7
p.o.
p.o.
10
3
0.3
0.3
163
265
161
243
164
244
1.5
0.7
a1% DMSO/0.5% HPMC was used as the vehicle.
The in vivo therapeutic effects of compound 7 were evaluated in a human MM.1S
xenograft model. Tumor-bearing SCID mice were treated with 7 or the phase II BET
inhibitor I-BET762 by oral administration at a dosage of 20 mg/kg daily. The results
showed that treating with compound 7 demonstrated good efficacy on tumor growth
inhibition with a T/C value of 67.4% (Figure S1). These encouraging results
demonstrated that this new series of compounds bearing a novel triazole linker
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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
deserved further optimization.
Lead to Candidate
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
The good in vivo antitumor efficacy of lead compound 7 indicated that this class
of compounds should be advanced for further exploitation. However, the moderate
plasma exposure of compound 7 and its instability in mouse liver microsomes limited
its subsequent development. To confirm the binding mode and guide further
optimization, we solved the cocrystal structure of compound 7 bound to BRD4-BD1.
As illustrated in Figure 3, the methyl-benzene group indeed stretched into the WPF
shelf and contributed favorable VDW interactions to increase the binding affinity.
Meanwhile, it was also found that the position adjacent to the methyl group on the
triazole ring pointed to the solvent-accessible part, which could be utilized for
improving the drug-like properties. Considering its fast metabolic rate and bad
solubility (Table S3), we intended to introduce a hydrophilic group into this position
to reduce the binding potential to metabolism enzymes and improve the solubility.
Figure 3. The crystal structure of compound 7 bound to BRD4-BD1 (PDB entry:
6JI4) is shown in cartoon mode and surface mode.
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We preserved the core structure of compound 7 or compound 10, which also
displayed excellent molecular and cellular potency, and added a hydrophilic
piperazine group to the five-member ring linker to generate compounds 13 and 14
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 3), respectively, finding that their molecular and cellular activities could be
retained. Hence, we evaluated the drug-like properties of compounds 13 and 14.
Table 3. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of
compounds 13 and 14 on BRD4 (I).
Cpd
R1
R2
R3
BRD4(I)
IC (nM)
MM.1S
a
b
IC (nM)
5
0
50
(
+)-JQ-1
-
-
-
-
-
28.5 ± 1.5
34.3 ± 6.5
9.4 ± 0.6
29.8 ± 16.6
6.3 ± 13.5
8.0 ± 6.1
OTX-015
-

1
3
Me


1
4
5.4 ± 1.2
26.1 ± 12.3
aThe IC50 values are the mean of two independent experiments performed in duplicate with
b
standard error of the mean (SEM) values. The IC values are the mean of three independent
5
0
experiments performed in triplicate with standard error of the mean (SEM) values.
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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
We first evaluated the in vitro metabolic stabilities of compounds 13 and 14
Table 4). Comparison of compound 13 or 14 with compound 7 demonstrated that
(
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
compounds 13 and 14 had remarkably improved metabolic stability in mouse liver
microsomes, indicating that the addition of a hydrophilic group could decrease the
hydrophobic properties of the compounds to reduce their binding probability with
metabolic enzymes. Moreover, they did not show evident direct or time-dependent
inhibition on five selected CYP450 enzymes, revealing the potential defect in the
drug-drug interaction.
Table 4. In vitro liver microsome intrinsic clearance data and cytochrome P450
enzyme inhibition data.
HLMa
Clearance
(μL/min/mg) (min) (μL/min/mg) (min)
mLMb
DI (%) / TDI (k *10 )
c
-4 d
obs
Cpd
T1/2
Clearance
T1/2
3A4 2D6
2C9
1A2
2C19
7
44
22
61
31.5
62
460
13
3.0
107
41
3/no no/no 10/no
no/no
2/41
41/no
17/no
1
1
3
4
12/19 no/14
31/55 3/no
6/19
23
34
30/74
no/51 19/141
a
b
c
HLM represents the human liver microsome. mLM represents the mouse liver microsome. DI,
which indicated direct inhibition and is sometimes referred to reversible inhibition, is assessed by
measuring enzyme (CYP) activity in the presence of increasing concentrations of inhibitor without
a preincubation step. DI < 20% means no direct inhibition, 20% < DI < 50% means weak direct
inhibition, 50% < DI < 70% means moderate direct inhibition, and DI > 70% means strong direct
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d
inhibition, TDI stands for the time-dependent inhibition, which refers to a change in enzyme
inhibition during in vitro incubation and means irreversible inactivation of CYPs. The unit of TDI
-4
is 10 /min, and TDI < 200 means no time-dependent inhibition. When the TDI value was close to
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
zero, it was considered as none.
Encouraged by this result, we further investigated the in vivo pharmacokinetics
properties of compounds 13 and 14 (Table 5). Pharmacokinetic tests showed that
compound 14 had much better plasma exposure than compound 13 at the same 10
mg/kg dosage; when compared to compound 7, both compounds improved the plasma
exposure considerably, indicating that the addition of a hydrophilic group to the
triazole ring could enhance the solubility to increase oral absorption.
Table 5. In vivo PK data for compounds 13 and 14 in male ICR mice.a
Route of
Dose
Tmax
(h)
Cmax
AUC0-t
AUC0-inf
T1/2
(h)
Cpd.
administration
(mg/kg)
(ng/mL)
(ng·h/mL)
(ng·h/mL)
1
1
3
4
p.o.
p.o.
10
10
0.3
0.3
1231
2742
2764
1.7
0.6
42904
58132
58146
a1%DMSO/0.5%HPMC was used as the vehicle.
Hence, we further explored the structure-activity relationships of the hydrophilic
group based on the core structure of 14. Initially, we explored the SAR of the
hydrophilic piperazine position. As elucidated in Table 6, opening the
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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
1
1
2
N-methylpiperazine to an N ,N ,N -trimethylethane-1,2-diamine group (15);
replacing the N-methylpiperazine methyl group with ethyl, isopropyl or cyclopropyl
(
16-18); or substituting the N-methylpiperazine with (2S,6R)-2,6-dimethylpiperazine
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
(19) or larger groups, such as 1-methyl-1,4-diazepane (20) or a bridged ring, spiro
ring or fused ring (21-27), could maintain the molecular activity, suggesting that the
hydrophilic N-methylpiperazine group might be exposed to the solvent moiety of the
protein and did not make direct interactions with the protein. We attempted to resolve
the crystal structures of compounds 14, 15, 19 and 22 with BRD4-BD1 protein to
verify this conjecture but had no success. However, a later crystal structure study did
confirm this hypothesis (discussed in below part). Although their molecular activities
were similar, compounds 15, 16 and 19-23 had better activities in the MM.1S cell
viability test, which may be due to the subtle structural changes that affect compound
permeability.
Table 6. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of
compounds 14-27 on BRD4 (I).
N
N
O
O
N
R4
N
N
Cpd
R4
BRD4(I)
IC (nM)
MM.1S
Cpd
R4
BRD4(I)
MM.1S
a
b
a
b
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
50
(
+)-JQ-1
-
28.5 ± 1.5
29.8 ± 16.6
20
4.6 ± 0.1
8.0 ± 5.1
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OTX-015
14
-
34.3 ± 6.5
6.3 ± 13.5
21
4.6 ± 0.2
10.1 ± 3.9
5.4 ± 1.2
7.0 ± 1.3
26.1 ± 12.3
16.2 ± 3.5
22
23
6.6 ± 0.2
8.7 ± 0.6
7.9 ± 1.8
6.4 ± 2.1
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
1
1
5
6
5.8 ± 0.8
4.8 ± 0.6
5.9 ± 0.9
5.3 ± 0.4
25.1 ± 7.6
125.0 ± 70.3
87.5 ± 21.8
2.3 ± 0.9
24
25
26
27
27.2 ± 1.8
7.9 ± 0.8
9.6 ± 0.5
6.1 ± 0.8
2403 ± 1940
484.8 ± 803.4
484.6 ± 171.3
43.5 ± 8.8
17
18
1
9
aThe IC50 values are the mean of two independent experiments performed in duplicate with
b
standard error of the mean (SEM) values. The IC values are the mean of three independent
50
c
experiments performed in triplicate with standard error of the mean (SEM) values. N.D. means
not determined.
We also explored the SAR of the side-chain benzene ring (Table 7). By
comparing compounds 28 and 31 with compound 14, we found that removal of the
para-methyl group or replacement of the methyl with a chlorine substituent slightly
reduced the molecular activity slightly. However, the introduction of a methyl group
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9
1
1
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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
at the ortho position or a fluorine substituent at the meta position could slightly
improve the molecular and cellular activities, indicating that the addition of another
small hydrophobic group such as a methyl or fluoro group could enhance the VDW
interaction of the side-chain with the WPF pocket.
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 7. Inhibitory activity and MM.1S cell proliferation inhibitory activity results of
compounds 28-31 on BRD4(I).
N
N
O
O
N
N
N
N
N
R3
Cpd
R3
BRD4(I)
IC (nM)
MM.1S
Cpd
R3
BRD4(I)
MM.1S
a
b
a
b
IC (nM)
IC (nM)
IC (nM)
5
0
50
50
50
(
+)-JQ-1
-
-
28.5 ± 1.5
34.3 ± 6.5
29.8 ± 16.6
6.3 ± 13.5
29
30
2,4-dimethyl
3-fluoro-4
-methyl
4.5 ± 0.4
4.2 ± 0.2
17.8 ± 7.3
13.0 ± 1.4
OTX-015
1
2
4
8
Me
H
5.4 ± 1.2
7.1 ± 0.6
26.1 ± 12.3
60.1 ± 18.0
31
4-chloro
6.8 ± 0.6
48.5 ± 10.4
aThe IC50 values are the mean of two independent experiments performed in duplicate with
b
standard error of the mean (SEM) values. The IC values are the mean of three independent
5
0
c
experiments performed in triplicate with standard error of the mean (SEM) values. N.D. means
not determined.
We attempted to solve the crystal complex structures of these compounds with the
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BRD4-BD1 protein, and successfully obtained the crystal structure of compound 29
with BRD4-BD1 protein (Figure 4). As shown in Figure 4, the N-methyl piperazine
group reached into the solvent region around the protein and the amide group oxygen
formed a hydrogen bond with the residue Lys91. This binding mode was consistent
with our presumption that this N-methyl piperazine position could be utilized for
drug-like optimization and will not significantly affect the inhibitors’ binding affinity.
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 4. The crystal structure of BRD4-BD1 bound with compound 29 (PDB entry:
6
JI5).
Based on the molecular and cellular activities, compounds 14, 15, 19-23 and 30
were selected to assess their in vivo pharmacokinetics properties in mice (Table 8 lists
the PK properties for compounds 14, 19 and 30; less promising compounds 15 and
2
0-23 are listed in Table S4). In addition to compound 14, compounds 19 and 30 were
also found to have good PK properties. When compared to compound 14, compounds
1
9 and 30 had lower plasma exposure but a longer half-life; in particular, compound
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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
19 had a half-life of 2.1 hours. Moreover, compound 19 had superior cellular activity
compared with compounds 14 and 30. Considering the cellular activities and PK
properties, we selected compound 19 as a preclinical candidate for subsequent
comprehensive evaluation.
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 8. In vivo PK data for compounds 14, 19 and 30 in male ICR mice.a
Route of
Dose
Tmax
(h)
Cmax
AUC0-t
AUC0-inf
T1/2
(h)
Cpd.
administration
(mg/kg)
(ng/mL)
(ng·h/mL)
(ng·h/mL)
1
1
3
4
9
0
p.o.
p.o.
p.o.
10
10
10
0.3
1.1
0.3
42904
1898
4967
58132
7383
8154
58146
7386
8365
0.6
2.1
1.5
a1% DMSO/0.5% HPMC was used as the vehicle.
Comprehensive Profile of Compound 19
To comprehensively understand the drug-like properties of compound 19, we
performed a series of evaluations on compound 19, including examining its selectivity
and cellular activities, cellular mechanism studies, a hERG study, in vitro metabolic
stability studies, in vivo pharmacokinetics studies, and an in vivo antitumor study.
Bromodomains Selectivity
Bromodomains are evolutionarily conserved protein-protein interaction modules
that are found in a wide range of proteins with diverse catalytic and scaffolding
functions. They recognize acetylated lysine by forming several hydrogen bonds with
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the conserved asparagine (ASN140 in BRD4-BD1) residue at the top of
bromodomains. Given that selectivity is critical for the success of drug discovery, we
screened the binding specificities of compound 19 across 32 bromodomains using the
DiscoveRx BROMOScan platform of Eurofins company at 1 μM concentration. As
depicted in Figure 5, 19 exhibited inherent selectivity for BET family members over
most tested bromodomains, except for moderate inhibitory effect on TAF1(2) (Details
are provided in Table S5).
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
We also examined the inhibition of compound 19 as well as other potent
compounds, including 7, 10, 14-15, 20-23, and 30 on BRD4-BD2, BRD2-BD1,
BRD2-BD2, and EP300, to examine the impact of the core structure on selectivity
(Table S6). The results showed that these compounds also demonstrated good potency
on other BET members (BRD4-BD2, BRD2-BD1, and BRD2-BD2), though not as
good as on BRD4-BD1. Importantly, none of them displayed any potency on EP300,
indicating that the scaffold may be the dominant factor for the BET selectivity.
Moreover, we utilized isothermal titration calorimetry (ITC) as another binding
assay method to test the binding of compound 19 to BRD4-BD1, and the results
confirmed that compound 19 had excellent binding affinity (K = 15.2 nM) (Figure S2
d
and Table S7).
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Figure 5. Selectivity assessment of compound 19 against the BRD family members
using a BROMOscan panel. The 32 screened targets are labeled in black on the BRD
family phylogenetic tree. BRDs that were not part of the screening panel are in gray.
Compound 19 was screened at 1 μM concentration, and the results for primary screen
binding interactions are reported as the ‘Percent Control’, with lower numbers
indicating stronger hits in the matrix.
Kinase Selectivity
As the original core structure stems from the PLK1-BRD4 dual inhibitor BI-2536,
we also tested the inhibition of compound 19 on the PLK1 kinase and 24 other
kinases distributed in different subfamilies in the kinome (Table S8). We found that
BI-2536 displayed a significant inhibitory effect on PLK1 kinase at 1 μM; however,
compound 19 only demonstrated negligible inhibitory activity for PLK1 kinase at 10
μM and 1 μM (less than 10% inhibition ratio). This is consistent with our previous
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findings that changing the pyrimidine nitrogen atoms in BI-2536 to carbon atoms will
eliminate the essential hydrogen bonding with the PLK1 kinase hinge part. Moreover,
compound 19 did not show obvious inhibition of other tested kinases, though it
displayed moderate inhibition of Src, Abl, and IGF1R at 10 μM. Given the nanomolar
activity of BET proteins, these results reinforced that compound 19 is a selective BET
inhibitor.
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Cellular Activities and Mechanism Studies
To further evaluate the therapeutic potential of compound 19, we tested its
anti-proliferative activities against seventeen different cell lines, including leukemia,
multiple myeloma, thymic carcinoma, hepatocellular carcinoma, lung cancer, breast
cancer, colorectal cancer, gastric adenocarcinoma, ovarian cancer, etc. (Figure 6 and
Table S9). As shown in Figure 6, compound 19 was found to exhibit better inhibitory
activities on all of these cell lines than (+)-JQ-1, and show better inhibitory activities
than OTX-015 in several sensitive cell lines, such as MV4-11 and Ty-82. Overall, the
MV4-11, MM.1S, Ty-82, KG-1 and NCI-H1299 cell lines were sensitive to
compound 19 treatment, implying that, besides leukemia, it may also provide
potential therapeutic opportunities for treating multiple myeloma, thymic carcinoma,
and lung cancer, which is worth further exploration.
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Figure 6. The inhibitory activities of compound 19 against seventeen cell lines.
According to the above results, among the tested cell lines, MV4-11 is the most
sensitive to compound 19. Therefore, we selected it to explore the anti-proliferative
activity mechanism. As we know, Myc is an oncogene and its deregulation is
40
frequently involved in human oncogenesis. It was reported that Myc expression is
reduced after BRD4 inhibition, which leads to the inhibition of cell proliferation.41
Hence, we performed Western blotting and real-time quantitative PCR (RT-qPCR)
assays to study the inhibitory effects of compound 19 on c-Myc mRNA and protein
expression. Figure 7A shows that compound 19 displayed profound inhibitory effects
on c-Myc protein expression, even at the 10 nM, while OTX-015 did not show
obvious inhibition at that concentration. Moreover, Figure 7B showed that compound
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9 demonstrated a better inhibitory effect on c-Myc mRNA expression at 10 nM and
100 nM than OTX-015, which was consistent with changes in protein levels (Figure
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7A). Therefore, we concluded that the inhibitory effects of compound 19 on MV4-11
cell proliferation is partially related to the downregulation of c-Myc expression.
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Figure 7. (A) MV4-11 cells were treated with compound 19 or OTX-015 at the
indicated doses for 24 h and examined for c-Myc and GAPDH protein levels by
Western blotting. (B) Similar to (A), but examining c-Myc mRNA levels by
RT-qPCR. p < 0.05, *. Error bars represent the SD from three independent
experiments.
Previous studies suggest that BET inhibitors can induce apoptosis in tumor cells.42
Thus, we examined the effect of compound 19 on apoptosis in MV4-11 cells. The
results showed that compound 19 could induce apoptosis in a dose-dependent manner
(Figure 8A). At the same dose, compound 19 could induce more apoptosis than
OTX-015 (Figure 8A). The changes in cleaved PARP, an apoptotic marker, were
consistent with the above apoptotic phenomena (Figure 8B). Next, we found that
compound 19 and OTX-015 caused a dose-dependent increase in apoptotic initiators
(cleaved caspase-8 and cleaved caspase-9), accompanied by a decrease in full-length
caspase-8 (Figure 8C). Additionally, apoptotic executors (cleaved caspase-3 and
cleaved caspase-7) also showed a dose-dependent increase (Figure 8C).
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Overexpression of pro-survival BCL-2 family regulator proteins inhibits apoptosis,
so we further investigated whether compound 19 induced apoptosis by reducing
BCL-2 family levels. The results showed that compound 19 and OTX-015 reduced
the levels of BCL-2, p-BCL2 (Thr56), p-BCL2 (Ser70) and BCL-xL in a
dose-dependent manner, as well as the pro-apoptotic proteins PUMA, BID and BIM
(Figure 8D). Moreover, compound 19 and OTX-015 reduced the protein levels of the
inhibitors of apoptosis family proteins (IAP) XIAP, c-IAP1 and c-IAP2 in a
dose-dependent manner (Figure 8D). RT-qPCR results showed that changes in BCL-2
mRNA expression were in line with that of BCL-2 at the protein level (Figures 8D
and 8E). Based on the above results, we concluded that compound 19 reduced the
level of many anti-apoptotic proteins and apoptotic inhibitors, thereby activating
caspase family proteins and inducing more apoptosis than OTX-015 at the same
doses.
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BRD4 is also necessary for cell cycle progression, and inhibition of BRD4 leads to
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G1 arrest. Our results showed that at the same dose, compound 19 caused more G1
phase arrest than OTX-015 (Figure S3), which was consistent with the increased
proliferation inhibitory activity of compound 19.
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Figure 8. (A) MV4-11 cells were treated with compound 19 or OTX-015 at the
indicated doses for 24 h, and apoptosis was measured by Annexin V Apoptosis assay.
p < 0.05, *; 0.01, **. (B) Similar to (A), but examined for cleaved PARP and GAPDH
protein expression by Western blotting. (C) Similar to (A), but examined for
caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved
caspase-8, caspase-9, cleaved caspase-9, and GAPDH protein expression by Western
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blotting. (D) Similar to (A), but examined for BCL-2, p-BCL-2(Thr56),
p-BCL-2(Ser70), BCL-xL, MCL-1, XIAP, c-IAP1, c-IAP2, BAK, BAX, PUMA,
BID, BIM and GAPDH protein expression by Western blotting. (E) Similar to (A),
but examined for BCL-2 mRNA expression by RT-qPCR. p < 0.05, *; 0.01, **. Error
bars represent the SD of three independent experiments.
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In Vitro Metabolic Stability Studies
Next, we examined the metabolic stability of 19 in liver microsomes from four
different species, including human, mouse, rat, and dog. As depicted in Table 9,
compound 19 showed good stability in these liver microsome assay. We also
evaluated the effects of compound 19 on five common cytochrome P450 enzymes,
finding that compound 19 displayed negligible direct or time-dependent inhibition on
these enzymes, which indicated that it has less risk of drug-drug interactions (Table
9).
Table 9. In vitro liver microsome intrinsic clearance data and cytochrome P450
enzyme inhibition data.
Liver
Microsome
HLMa
Clearance
T1/2
(min)
27
CYP450
Direct Inhibition (%)e
TDI
kobsf
8
(μL/min/mg)
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7
3A4
2D6
2C9
1A2
23/28
4
mLMb
193
49
no
RLMc
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77
28
no
DLMd
18
no
no
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2C19
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no
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HLM represents the human liver microsome; mLM represents the mouse liver microsome; RLM
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represents the rat liver microsome; and DLM represents the dog liver microsome. CYP direct
inhibition (DI), which is sometimes referred to as reversible inhibition, is assessed by measuring
enzyme (CYP) activity in the presence of increasing concentrations of inhibitor without a
preincubation step. DI < 20% means no direct inhibition, 20% < DI < 50% means weak direct
inhibition, 50% < DI < 70% means moderate direct inhibition, and DI > 70% means strong direct
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f
inhibition; TDI stands for time-dependent inhibition, which refers to a change in enzyme
inhibition during in vitro incubation and indicates irreversible CYP inactivation. The unit of TDI
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is 10 /min. TDI < 200 means no time-dependent inhibition. When the TDI value was close to
zero, it was considered as none.
hERG Study
In cardiomyocytes, human ether-a-go-go related gene (hERG) encodes a
potassium channel that mediates a delayed rectifier potassium current (IKr), and the
inhibition of IKr is the most important mechanism for drug-induced prolongation of
QT.44 Thus, we wanted to examine the effects of compound 19 and the positive
control Cisapride on hERG before we advance the compound into in vivo studies. As
illustrated in Table S10, compound 19 had no evident effects on hERG, even at 40
μM, indicating that it has a low potential for side effects on the heart.
In Vivo PK Study
(
+)-JQ1 was reported to have a high rate of brain penetration, which could cause
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