Targeting epigenetic reader and eraser: Rational design, synthesis and in vitro evaluation of dimethylisoxazoles derivatives as BRD4/HDAC dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.04.034

The bromodomain protein module and histone deacetylase (HDAC), which recognize and remove acetylated lysine, respectively, have emerged as important epigenetic therapeutic targets in cancer treatments. Herein we presented a novel design approach for cancer drug development by combination of bromodomain and HDAC inhibitory activity in one molecule. The designed compounds were synthesized which showed inhibitory activity against bromodomain 4 and HDAC1. The representative dual bromodomain/HDAC inhibitors, compound 11 and 12, showed potent antiproliferative activities against human leukaemia cell line K562 and MV4-11 in cellular assays. This work may lay the foundation for developing dual bromodomain/HDAC inhibitors as potential anticancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2016.04.034

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Targeting epigenetic reader and eraser: Rational design, synthesis
and in vitro evaluation of dimethylisoxazoles derivatives as BRD4/
HDAC dual inhibitors
Zhimin Zhang a, Shaohua Hou a, Hongli Chen a, Ting Ran b, Fei Jiang a, Yuanyuan Bian a, Dewei Zhang a,
Yanle Zhi a, Lu Wang a, Li Zhang a, Hongmei Li a, Yanmin Zhang b, Weifang Tang a, Tao Lu b,c,
,
⇑
Yadong Chen b,
⇑
a Department of Organic Chemistry, School of Science, 639 Longmian Avenue, Nanjing 211198, China
b Laboratory of Molecular Design and Drug Discovery, School of Science, 639 Longmian Avenue, Nanjing 211198, China
c State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The bromodomain protein module and histone deacetylase (HDAC), which recognize and remove acety-
lated lysine, respectively, have emerged as important epigenetic therapeutic targets in cancer treatments.
Herein we presented a novel design approach for cancer drug development by combination of bromod-
omain and HDAC inhibitory activity in one molecule. The designed compounds were synthesized which
showed inhibitory activity against bromodomain 4 and HDAC1. The representative dual bromodomain/
HDAC inhibitors, compound 11 and 12, showed potent antiproliferative activities against human leukae-
mia cell line K562 and MV4-11 in cellular assays. This work may lay the foundation for developing dual
bromodomain/HDAC inhibitors as potential anticancer therapeutics.
Received 5 January 2016
Revised 30 March 2016
Accepted 14 April 2016
Available online xxxx
Keywords:
Epigenetic
Protein–protein interactions
Bromodomain
HDAC
Ó 2016 Elsevier Ltd. All rights reserved.
Antiproliferative activity
In the past few years, epigenetic modulators have emerged as
promising targets for therapeutic development.1 Targeting epige-
netic modifiers and readers, particularly histone deacetylases
(HDACs) and bromodomain-containing proteins (BCPs) have
recently set the foundation for a new generation of anti-cancer
drugs.2–4 Histone lysine acetylation (KAc) is an epigenetic mark
associated with gene activation. These acetyl groups are reversibly
maintained by histone acetyltransferases (HATs) and histone
deacetylases (HDACs).5–7 HDACs as epigenetic ‘eraser’ modules
catalyse the removal of acetyl groups from lysine residues on the
histone tail to induce a condensed chromatin structure, and
thereby inhibit transcription. HDACs play key roles in coordinating
the interaction of intracellular signaling pathways with chromatin
remodeling and transcription factor function to specifically medi-
ate gene expression during cellular activation, proliferation, and
differentiation.8,9 Mounting evidence points to a link between dys-
regulated HDAC activity and many oncologic and nononcologic dis-
eases.10–12 Several small molecule HDAC inhibitors have been
approved by FDA for the treatment of a variety of haematological
and solid tumors, such as the hydroxamate containing inhibitor
vorinostat (SAHA, 1)13 (Fig. 1) and more types have entered clinical
trials.14
Histone acetylation also promotes transcription by binding to
proteins involved in gene activation such as BCPs. The dysregula-
tion of the bromo and extraterminal (BET) family of BCPs, which
includes BRD2, BRD3, BRD4 and BRDT, has been implicated in the
development of cancers such as NUT midline carcinoma and
AML. Inhibitors of BET proteins (BETi) can block cancer cell prolif-
eration and induce apoptosis in a wide range of tumor types.15,16
To date, many effects of BETi have been attributed to transcrip-
tional suppression of genes like the MYC oncogene. Specifically,
BET inhibitors showed strong efficacy in AML models as well as a
nut midline carcinoma model. Several distinct chemotypes, such
as methyltriazolodiazepines (2)17 and 3,5-dimethylisoxazoles (3
and 4)18,19 (Fig. 1), have been identified.
Studies have suggested that BET and HDAC inhibitors induce
similar genetic and biological effects and synergize to kill Myc-
induced murine lymphoma.20 The synergistic and therapeutic
combinations by targeting the genetic link between BETi and
HDACi will be a feasible approach to cancer drug development.
Atkinson et al.21 reported a dual active BRD/HDAC small molecule
probe by the fusion of a BRD active tetrahydroquinoline (THQ) core
⇑
Corresponding authors. Tel.: +86 25 86185180; fax: +86 25 86185179 (T.L.);
tel.: +86 25 86185163; fax: +86 25 86185182 (Y.C.).
E-mail addresses: lutao@cpu.edu.cn (T. Lu), ydchen@cpu.edu.cn (Y. Chen).
http://dx.doi.org/10.1016/j.bmcl.2016.04.034
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang, Z.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.034

2
Z. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Linker
O
H
N
OH
N
H
O
N
O
N
3
ZBG
1
O
NH
Cap
HDAC IC50~10 nM
N
O
2
1
O
O
4
N
N
N
HO
5
O
N
6
N
O
N
S
4
3
(I-BET151)
BRD4(1/2) IC50 = 790 nM
BRD4(1) IC50 =544 nM
Cl
(+)-JQ1 2
BRD4(1) IC50 =77nM
Figure 1. Structures of representative HDAC inhibitors (1), BRD inhibitors (2, 3 and 4) with IC50 values.
with a hydroxamic acid HDACi motif. This demonstrated that dual
BET/HDACs inhibitors can be rationally designed for disease appli-
cations. However, obtaining potent, selective BET/HDACs dual inhi-
bitors still need further investigation.
atom forming a hydrogen bond with the side chain NH2 of N140
and the isoxazole nitrogen atom interacting with the conserved
tyrosine Y97 via the bridging water molecule. Moreover, the ben-
zoyl moiety of 4 well occupied the WPF shelf. It is notable that
the ZA channel, a hot spot region for the design of BRD4 inhibitors,
has not been fully occupied by the hydroxyl group of 4. The hydro-
xyl offers a vector projecting toward the ZA channel, where flexible
chain-like groups may be favorable. The long alkyl chain of SAHA
located in a long narrow tubular channel. Hydroxamate forms
three hydrogen bonds with Y297, H131 and H132, respectively.
The capping phenyl group binding to the hydrophobic part on
the protein surface. It is well established that the pharmacophore
of HDACi (Fig. 1, compound 1) consists of a capping group, an
appropriate linker and a zinc-binding group (ZBG). Generally,
ZBG plays a significant role in the binding efficiency between
HDACi and enzyme. Hydroxamate is one of the most potent zinc
ion chelating group among all types of ZBGs. Given the known
binding modes of the BET inhibitor 4 and SAHA, it was envisaged
that amalgamation of these pharmacophores would be a viable
strategy to single molecules retaining both activities. We postu-
lated that introduction of the linker and ZBG of HDACi SAHA at
Recently, dimethylisoxazole has been proved to be an effective
scaffold of BETi. I-BET151 (3), a representative of this series, dis-
plays outstanding pharmacokinetic properties, such as bioavail-
ability and half-life.22 This suggested that dimethylisoxazole
derivatives as a good starting point are worth further exploring.
Investigating structural requirements of BRD4 and HDAC inhibitors
led us to design and synthesize a series of novel 3,5-dimethylisox-
azole derivatives as BRD4/HDAC dual inhibitors. These compounds
were evaluated for inhibitory activities toward both HDAC1 and
BRD4 with selectivity versus other bromodomain-containing pro-
teins, and anti-proliferative activity against human chronic myel-
ogenous leukemia (CML) cell line K562.
The rationale for the dual-inhibitor design originated from our
deep insights into previously reported X-ray crystal structure of
4 bound to human BRD4 (Fig. 2A) and SAHA bound to a HDAC
homolog (Fig. 2B). The 3,5-dimethylisoxazole of 4 occupies the
KAc-binding pocket, acting as a KAc mimic, with isoxazole oxygen
Figure 2. Design of dual inhibitors of BRD4/HDAC (A) X-ray crystal structures of 4 bound to human BRD4 (PDB entry: 4J0S), (B) SAHA bound to a HDAC homolog (PDB entry:
1C3S). The proteins are shown as white surface or cartoon. 4 and SAHA are shown in sticks. Water molecule is shown as blue spheres, and the hydrogen bonds were denoted
by black dash lines. Figures were prepared using PyMOL.
Please cite this article in press as: Zhang, Z.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.034

Z. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
the position 1 of 4 would be compatible with HDAC inhibition
since that the ZA channel of Bromodomain and the tubular channel
of HDAC share considerable similarities (Fig. 2). In the meantime,
the linker and ZBG of SAHA may direct into the ZA channel to make
it well occupied which may increase the affinity to BRD4. The fused
molecules were expected to retain the essential interactions with
both proteins to exert desired biological functions.
The synthetic routes of 5–9 and 10–13 were outlined in
Schemes 1 and 2. Synthesis of intermediate 5–4 started with the
Friedel–Crafts acylation of benzene with 3-bromo-5-methoxyben-
zoic acid 5–1 to give benzophenone 5–2. The 3,5-dimethylisoxa-
zole moiety was introduced to 5–2 by coupling reaction to afford
5–3. Treatment of compound 5–3 with tribromoborane produced
the key intermediate 5–4. The refluxing of 5–4 with alkyl bromides
in the presence of anhydrous K2CO3 and tetrabutylammonium
iodide in acetone afforded 5a–5d. Finally, the ethyl ester groups
in compounds 5a–5d were treated with freshly prepared hydroxy-
lamine in methanol to produce target compounds 5–8. Similar
chemistry was applied to the synthesis of 10–13 in Scheme 2.
Hydrogenation of compound 10–3 using Pd/C under an
atmosphere of hydrogen yielded amine 10–4. Subsequent amida-
tion of amine 10–4 with the corresponding carboxylic acid
afforded compounds 10–13.
N
O
N
O
Br
Br
O
a
c
b
OH
O
O
HO
O
O
O
5-2
O
5-1
O
5-3
N O
5-4
N
d
e
H
N
O
HO
O
O
n
n
O
O
O
O
5-8
5a-5d
5a
5b
5c
5d
5 6 7 8
n=2; n=3; n=4; n=5
n=2;
n=3;
n=4;
n=5
N
O
f
7
H
N
HO
O
n
O
OH
9
n=4
Scheme 1. Synthesis of Compounds 5–9. Reaction conditions: (a) SOCl2, Benzene, AlCl3, 75%; (b) 3,5-dimethylisoxazole, PdCl2, AcOK, DMAc, 71%; (c) BBr3, CH2Cl2, 81%; (d)
tetrabutylammonium iodide, K2CO3, alkyl bromide, 68–75%; (e) MeOH, NH2OH in MeOH, 86–91%; (f) NaBH4, THF, 90%.
N O
N O
Br
Br
a
b
c
OH
O2N
O2N
H2N
O2N
O
O
10-1
10-2
N O
O
O
10-4
10-3
N O
e
O
O
O
O
d
HO
N
H
N
H
O
N
H
n
n
O
O
10a-c
10a n=2; 10b n=3; 10c n=4
10 n=2; 11 n=3; 12 n=4
N O
Br
O
O
b
c
d
e
HO
N
H
N
H
n
NO2
13 n=3
Scheme 2. Synthesis of Compounds 10–13. Reaction conditions: (a) SOCl2, Benzene, AlCl3, 69%; (b) 3,5-dimethylisoxazole, PdCl2, AcOK, DMAc, 94%; (c) H2, Pd/C, MeOH, 95%;
(d) EDCI, HOBT, carboxylic acid, 81–87%; (e) MeOH, NH2OH in MeOH, 82–86%.
Please cite this article in press as: Zhang, Z.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.034

4
Z. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
To study the biological activity, compounds 5–13 were evalu-
ated against BRD4 activity assays with JQ1 as a positive control
and HDAC1 enzyme assay with MS275 as a positive control. As
shown in Table 1, most compounds exhibited moderate to good
inhibitory activity against both BRD4 and HDAC1. The length of
the hydroxamic acid side chains of this series have marginal effects
on HDAC1 inhibition (6, 7 > 8 > 5) and the optimal carbon chain
length is five or six carbon atoms. Diversity length of the side
chains are tolerable to BRD4 inhibition due to the long ZA channel
in BRD4. The linker between the hydroxamic acid side chain and
the phenylisoxazole skeleton has relatively significant effect on
both BRD4 and HDAC1 inhibition. Compounds with an amide
group as the linker are more potent than those with oxygen atoms.
The activity against BRD4 declined when removing the benzoyl
moiety from 11. This is probably because there is no fragment in
13 to occupy the WPF shelf in BRD4. Among the compounds tested,
12 showed powerful inhibitory activity against HDAC1
IC50 values of compounds (n = 3) 5–13 against BRD4 and HDAC1
Compd
IC50(lM)
HDAC1
BRD4
5
6
7
8
5.45
1.49
1.43
1.74
0.97
0.76
0.27
0.15
0.36
0.14
n.d.a
1.49
1.38
1.05
2.99
0.77
0.78
0.85
0.67
3.73
n.d.a
0.09
9
10
11
12
13
MS275
JQ1
a
n.d. = not determined.
(IC50 = 0.15 lM) and BRD4 (IC50 = 0.67 lM).
Table 2
The temperature shifts (Tm) of compounds (n = 3) 5–13 against diverse BRDs under
25
The biology is still not well understood for many bromod-
omains; therefore, contamination of off-target bromodomain
activity can easily confound results. Consequently, gaining selec-
tivity against the BET bromodomains has been of utmost impor-
tance because inhibition of BET produces very strong and broad
phenotypes.23 Fragments in the ZA channel may contribute to
the selectivity of inhibitors due to less conservative residues at this
region. We selected all nine compounds to profile the binding
specificity in six representative bromodomain modules, including
BRD4, BRD9, CECR2, CREBBP, PCAF and ATAD2 by a thermal melt-
ing assay based on protein stability shift (Table 2). Of compounds
that produced substantial changes in melting temperature (Tm),
all displayed selectivity for BRD4.
l
M
Compd
Temperature shifts (
D
Tm
)
BRD4
BRD9
CECR2
CREBBP
PCAF
TAF1
5
6
7
8
5.89
7.54
7.41
4.91
7.53
6.21
6.37
7.49
3.58
9.23
À0.75
1.56
1.72
1.22
1.55
2.79
2.26
2.97
n.d.a
1.18
n.d.
2.06
1.32
1.65
1.52
1.96
0.64
1.14
À0.01
1.69
2.13
0.65
4.03
2.05
2.32
3.24
0.73
0.07
À7.9
0.28
1.04
À0.31
0.35
0.31
0.92
1.35
0.05
0.79
1.14
1.69
2.22
1.77
1.72
1.95
1.76
2.22
1.08
À0.05
9
10
11
12
13
JQ1
À0.13
À0.22
BET bromodomain and HDAC inhibitors have previously shown
antiproliferative effects in a variety of hematopoietic malignancies,
including myelogenous leukemia and multiple myeloma. Conse-
quently, three most potent compounds 10–12 were chosen as rep-
resentatives to evaluated antiproliferative activities against human
chronic myelogenous leukemia (CML) cell line K562 and human
acute myelogenous leukemia (AML) cell line MV4-11. The IC50 val-
ues of compounds 10–12 were shown in Table 3. As expected, com-
pounds 11 and 12 exhibited remarkable effects on antiproliferative
activities in vitro, and comparable to the positive control JQ1.
Compound 12, the most potent compound of this series, was
chosen as a representative for docking into the BRD4 (1) and
a
n.d. = not determined.
Table 3
Antiproliferative activities of 11 and 12
Compd
IC50
(lM)
K562
MV4-11
11
12
JQ1
SAHA
2.33
1.86
0.64
0.17
1.04
0.91
0.24
0.72
Figure 3. The docking model of 12 bound to human BRD4(1) (PDB entry 3MXF) and HDAC1 (PDB entry 1C3R). (A and C) The protein is shown as surface. 12 is shown in stick
with carbon atoms colored in green, oxygen atoms in red and nitrogen atoms in blue. Water molecules are shown as pink spheres, and the hydrogen bonds were denoted by
black dash lines. Figures were prepared using PyMOL.
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Z. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
HDAC1, revealing excellent shape complementarity between
ligand and the binding pocket. As shown in Figure 3, 12 binds to
the BRD4 (1) as expected. The 3,5-dimethylisoxazole of 12 acts
as an AcK mimic, with the methyl group occupying the hydropho-
bic pocket that recognizes the methyl group of AcK. The isoxazole
oxygen atom forms the expected hydrogen bond with N140, and
the isoxazole nitrogen atom interacts with the side chain Y97 via
a water molecule. The benzoyl moiety well occupied the WPF shelf.
The long alkyl chain oriented toward the ZA channel. The docking
mode of 12 in complex with HDAC1 reveals that this compound
binds in the HDAC1 pocket in a similar manner to SAHA and forms
same interactions as SAHA. The hydroxamic acid group anchors 12
into the active site by chelating the essential catalytic zinc ion.
Hydroxamate forms three hydrogen bonds with Y297, H131 and
H132, respectively. The long alkyl chain of 12 exits the reaction
centre along the long narrow tubular channel. This places the moi-
ety from 4 in an open environment, weakly contacting hydropho-
bic residues on the protein surface.
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.04.
034.
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Acknowledgments
We are grateful to Prof. Stefan Knapp from University of Oxford
for the biological determination of Bromodomain. This work was
financially supported by the National Natural Science Foundation
of China (NSFC81473077), a project funded by the Priority Aca-
demic Program Development of Jiangsu Higher Education Institu-
tions and QingLan Project with grant from Jiangsu Province.
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Supplementary data
Supplementary data (general biological methods and chemical
experimental procedures) associated with this article can be found,
Please cite this article in press as: Zhang, Z.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.034