Design, synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target inhibitors for the treatment of multiple myeloma

Article abstract of DOI:10.1016/j.bmcl.2018.12.052

Multiple myeloma (MM) is the second most common haematological malignancy. Almost all patients with MM eventually relapse, and most recommended treatment protocols for the patients with relapsed refractory MM comprise a combination of drugs with different mechanisms of action. Therefore novel drugs are in urgent need in clinic. Bcl-2 inhibitors and HDAC inhibitors were proved their anti-MM effect in clinic or under clinical trials, and they were further discovered to have synergistic interactions. In this study, a series of Bcl-2/HDAC dual-target inhibitors were designed and synthesized. Among them, compounds 7e–7g showed good inhibitory activities against HDAC6 and high binding affinities to Bcl-2 protein simultaneously. They also displayed good growth inhibitory activities against human MM cell line RPMI-8226, which proved their potential value for the treatment of multiple myeloma.

Full text of DOI:10.1016/j.bmcl.2018.12.052

Accepted Manuscript
Design, synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target
inhibitors for the treatment of multiple myeloma
Ruolan Zhou, Shaoyu Fang, Minmin Zhang, Qingsen Zhang, Jian Hu, Mingping
Wang, Chongqing Wang, Ju Zhu, Aijun Shen, Xin Chen, Canhui Zheng
PII:
S0960-894X(18)30998-3
https://doi.org/10.1016/j.bmcl.2018.12.052
BMCL 26221
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
10 November 2018
17 December 2018
21 December 2018
Please cite this article as: Zhou, R., Fang, S., Zhang, M., Zhang, Q., Hu, J., Wang, M., Wang, C., Zhu, J., Shen, A.,
Chen, X., Zheng, C., Design, synthesis, and bioactivity evaluation of novel Bcl-2/HDAC dual-target inhibitors for
the treatment of multiple myeloma, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/
j.bmcl.2018.12.052
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Design, synthesis, and bioactivity evaluation of novel Bcl-
/HDAC dual-target inhibitors for the treatment of multiple
myeloma
2
Ruolan Zhoua , b, †, Shaoyu Fanga, b, † , Minmin Zhangc, † , Qingsen Zhang , Jian Hu ,
b
b
Mingping Wang , Chongqing Wang , Ju Zhu , Aijun Shen *, Xin Chena, * and
b
b
b
c,
Canhui Zhengb, *
a
School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan
4
30023, China
bSchool of Pharmacy, Second Military Medical University, Shanghai 200433, China
c
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
Abstract: Multiple myeloma (MM) is the second most common haematological
malignancy. Almost all patients with MM eventually relapse, and most recommended
treatment protocols for the patients with relapsed refractory MM comprise a
combination of drugs with different mechanisms of action. Therefore novel drugs are
in urgent need in clinic. Bcl-2 inhibitors and HDAC inhibitors were proved their anti-
MM effect in clinic or under clinical trials, and they were further discovered to have
synergistic interactions. In this study, a series of Bcl-2/HDAC dual-target inhibitors
were designed and synthesized. Among them, compounds 7e - 7g showed good
inhibitory activities against HDAC6 and high binding affinities to Bcl-2 protein
*
Corresponding authors.
E-mail addresses: canhuizheng@smmu.edu.cn (C. Zheng), chenxin_0001@126.com (X. Chen),
shenaj@simm.ac.cn (A. Sheng).
†
These authors contributed equally.

simultaneously. They also displayed good growth inhibitory activities against human
MM cell line RPMI-8226, which proved their potential value for the treatment of
multiple myeloma.
Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells,
and is the second most common haematological malignancy. Along with autologous
stem cell transplantation, the use of new drugs such as proteasome inhibitors and
immunomodulatory drugs has increased response rates and survival substantially in
the past decade.1, 2 However, almost all patients with MM eventually relapse. As a
result, novel drugs are in urgent need in clinic. Histone deacetylase (HDAC) inhibitor
panobinostat (Fig. 1) has been approved recently for the treatment of
3
4
relapsed/refractory MM. Several other novel drugs are being explored under clinical
5
trials and seem to be promising, such as kinesin spindle protein inhibitors, nuclear
6
7
8
export protein inhibitors, histone deacetylase 6 inhibitors and Bcl-2 inhibitors.
Anti-apoptotic Bcl-2 proteins have become attractive targets for anti-cancer agents
development, because they are associated with progression of a wide variety of
human cancers.9-11 The small-molecular Bcl-2 inhibitor venetoclax (ABT-199) (Fig.
1
) has been approved recently for the treatment of patients with chronic lymphocytic
leukemia who have a chromosomal abnormality called 17p deletion.8, 12 At present,
8
ABT-199 is under clinical trials for the treatment of MM. HDACs, which are
frequently dysregulated in cancer, can be subdivided into 4 classes: classes I, II and
IV (zinc dependent enzymes), and class III (nicotinamide adenine dinucleotide

(NAD)-dependent). Five HDAC inhibitors vorinostat (SAHA), romidpesin, belinostat,
chidamide, and panobinostat have been approved as anti-cancer agents.13, 14 The class
IIb HDAC, HDAC6 is distinguished by its ability to deacetylate α-tubulin and HSP90,
in addition to the modification of histone.14, 15 A selective HDAC6 inhibitor ACY-
1
215 (Fig. 1) is currently under clinical trials for the treatment of MM.7
O
HN
O
NHOH
N
HN S
H
N
N
O
O
NO2
Panobinostat
O
Cl
NH
N
N
N
HN
H
N
N
NHOH
O
Venetoclax
O
O
(ABT-199)
ACY-1215
Figure 1. Chemical structures of representative HDAC inhibitors and Bcl-2 inhibitors
Most recommended treatment protocols for the patients with relapsed refractory MM
comprise a combination of drugs with different mechanisms of action, such as
lenalidomide or bortezomib in combination with dexamethasone, or even triplet
regimens.1, 16 HDAC inhibitors and Bcl-2 inhibitors were in combination with above
approved chemotherapeutic agents in clinic or clinical trials for the treatment of MM,
4
, 8
and showed synergistic effects with them.17-20 Additionally, Bcl-2 inhibitors and
HDAC inhibitors were discovered to have synergistic interactions in MM cell lines, at
least in part, through Bim upregulation.21, 22 The combination of Bcl-2 inhibitors and
HDAC inhibitors were further proved to overcome adaptive bortezomib resistance in

vitro.23 Above research provide the reasonability of the therapeutic strategies
combining these two type of agents for the treatment of MM.
Figure 2. Design of title compounds. Three structural features of HDAC inhibitors a
cap region, a zinc-binding group (ZBG), and a linker region were labeled on the
structure of SAHA. The binding modes of ABT-199 (purple sticks) in complex with
Bcl-2 (using crystal structure PDB ID: 4MAN as templet) was displayed in the black
box. Five pockets of the binding groove of Bcl-2 were showed as orange circles
labeled as P1, L, P2, P3 and P4. The place where the hydroxamic acid group and
linkers were attached (blue arrow) was exposed to the solvent.
Considered the more predictable pharmacokinetic profile, less drug-drug interactions,
the improved convenience, the potential superior efficacy and lower toxicity, drugs
with optimal multi-target activities may represent a valuable complement or even
alternative to therapeutic regimens based on drug combinations.24, 25 In this study, a
series of Bcl-2/HDAC dual-target inhibitors were designed. It is well known that the

HDAC inhibitors have a general structure consisting of a cap region, a zinc-binding
group (ZBG), and a linker region which connects the two (Fig. 2). Hydroxamic acid
group is the most common used ZBG. It chelates the zinc ion in the active site and is
critical for the inhibitory activity. However, the cap region, the so-called surface
recognition domain, occludes the entrance of the active site pocket. It is
predominantly responsible for selectivity and tolerates a large change.13, 26 In addition,
the docking binding mode of ABT-199 and Bcl-2 protein (Fig. 2) was obtained using
2
7
the complex crystal structure of its analog as template (PDB ID: 4MAN). Based on
the three-dimensional pharmacophore we constructed before,28-30 the groups binding
in the P1, L and P2 pockets of binding groove played major roles in the interaction
with Bcl-2 protein. While the tetrahydropyranyl methyl group binding in the P3
pocket exposed to the solvent and had less effect on the binding. As a result, Bcl-
2
/HDAC dual-target inhibitors were designed by the replacement of the
tetrahydropyranyl methyl group of ABT-199 by hydroxamic acid group with different
linkers (Fig. 2).
The synthetic routes of title compounds were shown in Scheme 1. Intermediate 1 was
obtained by electrophilic substitution reaction of 1-fluoro-2-nitrobenzene under the
condition of chlorosulfonic acid, and then intermediate 2 was obtained under the
condition of aqueous ammonia in ice bath. Using different amino acid as start
material, esterification reaction was carried out under the condition of thionyl chloride
and ethanol to produce the intermediates 3a - 3g. Intermediates 4a -4g was yielded
from intermediates 2 and intermediates 3a - 3g by nucleophilic substitution reaction

under the condition of DIEA in DMSO. Compound 5 was synthesized by the method
2
7
reported. Then intermediates 4a -4g and 5 were treated with condensation agent
EDCI to obtain intermediate 6a – 6g. It was ammoniated in methanol solution of
hydroxylamine hydrochloride in which KOH had been dissociated, title compounds
7
a – 7g was obtained finally.
SO Cl
2
SO NH2
2
a
b
SO NH2
2
NO2
NO2
NO2
OH
F
F
F
1
d
2
NO2
HN
n n=1-7
O
O
c
n
H N
2
H N
2
n
O
O
O
n=1-7
3a - 3g
4a - 4g
OH
N
N
O
O
HN S
O
N
N
O
Cl
O
NO2
N
O
Cl
NH
5
HN
N
n n=1-7
O
O
HN
e
6a - 6g
O
HN S
f
N
N
O
O
NO2
O
Cl
NH
N
n
n=1-7
HOHN
O
HN
7a - 7g
Reagents and conditions: a) HSO Cl， reflux; b) NH OH, 0℃; c) SOCl , EtOH, rt; d) DIEA,
3
4
2
DMSO, 80℃; e) EDCI, DMAP, DCM; f) NH OH, MeOH, KOH, rt.
2
Scheme 1. Synthesis of title compounds.

The inhibitory activities against HDAC1 and HDAC6 of title compounds were
evaluated in a fluorescent assay using SAHA and ACY-1215 as positive control. The
results were displayed in Fig. 3A. Compounds 7e - 7g with linker containing 5-7
carbons showed good inhibitory activities against HDAC6. They also displayed
obvious selectivity to HDAC6 over HDAC1 (selectivity index >ꢀ10), which was
comparable with that of ACY-1215, the selective HDAC6 inhibitor under clinical
trials. Additionally, these results were consistent with the biological effect in cells
based on the western blot analysis (Table 1). After incubation in human MM cell line
RPMI-8226 at 10 μM for 24 h, compound 7e - 7g increased the level of Ac-tubulin
significantly, and only compound 7g increased the level of Ac-H3 obviously.
Figure 3. A. Inhibitory activities against HDAC1 and HDAC6 of title compounds. B.
Western blot analysis of the level of Ac-H3 and Ac-tubulin in RPMI-8226 cell line
after incubating with title compounds at 10 μM for 24 h. Protein expression was
detected by immunoblot analysis with the specific antibody.
Then, binding affinities to Bcl-2 protein of compounds 7e - 7g were further evaluated

by a fluorescence polarization (FP)-based method using ABT-199 as positive control
Table 1). All of the compounds showed high binding affinities, which was
(
comparable with that of ABT-199. It indicated that the replacement of the
tetrahydropyranyl methyl group of ABT-199 by hydroxamic acid group with different
aliphatic chains had little effect on the binding to Bcl-2 protein. Based on the above
results, compounds 7e - 7g were potent Bcl-2/HDAC dual-target inhibitors. They
were evaluated the growth inhibitory activities against human MM cell lines RPMI-
8
226 and U266 (Table 1), and ABT-199, ACY-1215 and SAHA were used as positive
controls. RPMI-8226 cell line seems to be sensitive to these compounds than U266
cell line. On RPMI-8226 cell line, these compounds displayed obviously better
growth inhibitory activities than positive controls. It indicated that the Bcl-2/HDAC
dual-target inhibitors show potential value for the treatment of MM.
Table 1
Binding affinities to Bcl-2 protein and growth inhibitory activities against human MM
cell lines RPMI-8226 and U266 of 7e - 7g
Bcl-2 (IC , μM)
RPMI-8226 (IC , μM)
U266 (IC , μM)
5
0
50
50
7
e
0.23 ± 0.01
0.24 ± 0.02
0.25 ± 0.01
0.23 ± 0.06
/
0.3 ± 0.0
0.3 ± 0.1
0.2 ± 0.0
2.7± 0.3
27.1± 4.2
9.8± 2.2
7
f
7
g
8.7± 1.3
ABT-199
ACY-1215
SAHA
94.0 ± 10.5
21.2 ± 2.4
42.6 ± 3.8
11.0 ± 1.7
10.6 ± 2.1
/

In summary, encouraged by the synergistic interactions between Bcl-2 inhibitors and
HDAC inhibitors, a series of Bcl-2/HDAC dual-target inhibitors were designed and
synthesized in this study. Among them, compounds 7e - 7g showed good inhibitory
activities against HDAC6 and high binding affinities to Bcl-2 protein simultaneously.
They displayed good growth inhibitory activities against human MM cell line RPMI-
8
1
2
226, obviously better than Bcl-2 inhibitor ABT-199 and HDAC inhibitors ACY-
215 and SAHA. The studies presented here prove the potential value of Bcl-
/HDAC dual-target inhibitors for the treatment of multiple myeloma, and provide a
new structural type for the development of novel antitumor agents.
Acknowledgments
We thank the Shanghai Rising-Star Program (16QA1404800), the State Key
laboratory of Drug Research (SIMM1601KF-01), and the National Natural Science
Foundation of China (Grant No. 21572266 and 81673472) for financial support.
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Graphical Abstracts
Highlights
A series of Bcl-2/HDAC dual-target inhibitors were designed and synthesized.
Compounds 7e - 7g showed good inhibitory activities against HDAC6 and Bcl-2
protein.

Compounds 7e - 7g displayed good inhibitory activities against RPMI-8226 cell line.