Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.07.045

Histone deacetylases (HDACs) have proven to be promising targets for the development of anti-cancer drugs. In this study, we reported a series of novel chalcone based tubulin and HDAC dual-targeting inhibitors. Three compounds inhibited the activities of

Full text of DOI:10.1016/j.bmcl.2019.07.045

Accepted Manuscript
Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting in-
hibitors
Baolei Wang, Xuehong Chen, Jianjun Gao, Li Su, Li Zhang, Hongwei Xu,
Yepeng Luan
PII:
S0960-894X(19)30502-5
https://doi.org/10.1016/j.bmcl.2019.07.045
BMCL 26586
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
24 April 2019
21 July 2019
23 July 2019
Please cite this article as: Wang, B., Chen, X., Gao, J., Su, L., Zhang, L., Xu, H., Luan, Y., Anti-tumor activity
evaluation of novel tubulin and HDAC dual-targeting inhibitors, Bioorganic & Medicinal Chemistry Letters (2019),
doi: https://doi.org/10.1016/j.bmcl.2019.07.045
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Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting inhibitors
a,#
c,#
b
a
a
c
a,*
Baolei Wang , Xuehong Chen , Jianjun Gao , Li Su , Li Zhang , Hongwei Xu , Yepeng Luan
a
Department of Medicinal Chemistry, School of Pharmacy, Qingdao University, Qingdao,
Shandong, China
b
Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, Shandong,
China
c
Department of Pharmacology, College of Basic Medicine, Qingdao University, Qingdao,
Shandong, China
⁎
Corresponding Author
Yepeng Luan, Mobile: (+86)15265260855; E-mail: luanqdu@sina.com.
#
These authors contributed equally to this work.
1

ABSTRACT
Histone deacetylases (HDACs) have been proven to be promising targets for the development of
anti-cancer drugs. In this study, we reported a series of novel chalcone based tubulin and HDAC
dual-targeting inhibitors. Three compounds inhibited the activities of HDAC and tubulin
polymerization simultaneously and displayed anti-proliferative activities toward eleven human
tumor cell lines. Compound 8a remarkably induced growth inhibition, apoptosis and G2/M phase
arrest of A549 tumor cells. Finally, the inhibitory activities of 8a against HDAC6 and tubulin
were rationalized by molecular docking studies.
ARTICLE INFO
Keywords:
Histone deacetylase
Tubulin polymerization
Dual-targeting
Chalcone,
Inhibitor
Antitumor
2

In epigenetics, histone deacetylases (HDACs) are a class of enzymes responsible for catalyzing
the removal of acetyl groups from the lysine residues of histones, consequently promote the
1
transcription. They play essential roles in many biological processes including cell proliferation,
2
angiogenesis, migration, differentiation, and metastasis. Accordingly, it is not surprising that they
play a highly important role in different diseases and are the targets of drugs. Overexpression of
HDACs is observed in various types of human tumors, including colorectal, gastric, liver, breast,
3
and lung cancers. The known HDACs are subdivided into four classes based on their sequence
homology: class I HDACs include HDAC1, 2, 3, and 8; class II HDACs include classes IIa
(HDAC4, 5, 7, and 9) and IIb (HDAC6 and 10); class III HDACs, known as sirtuins (sirt1−7);
4
class IV HDAC (HDAC11). Silencing or inhibiting HDACs has been shown to affect cell cycle,
cell growth, chromatin decondensation, cell differentiation, apoptosis, and angiogenesis in several
5
cancer cell types. Therefore, inhibition of HDACs has been approved as a successful strategy in
fighting with cancers. Five HDAC inhibitors have reached the market as anti-tumor drugs
including romidepsin (2004), vorinostat/SAHA (2006), belinostat (2014), panobinostat (2015),
and chidamide (2015). In general, HDAC inhibitors share the common three-motif pharmacophore
model consists of a cap group, a zinc-binding group (ZBG) and a linker with SAHA as a good
example. Over the past several years, a large number of clinical trials have been carried out and
some are ongoing involving HDAC inhibitors as a single therapy or in combination with other
anti-cancer drugs to magnify their anti-tumor activities.6
3

Fig. 1. Structures of five approved HDAC inhibitors
In cancer, because of the complicated network and the involvement of different proteins and
enzymes, selective single target drugs exert limited anti-tumor effect, and most curative cancer
7
treatments are mostly based on the combination of drugs acting on different targets. However, the
drug combination approach often needs to take into account the side effects, complicated drug-
drug interactions, poor patient compliance, etc.
Alternatively, another rational strategy of improving anti-cancer efficacy is design of hybrid
molecules whose structures are merged with pharmacophores of two or more drugs or agents
acting on different anti-tumor targets. These hybrid molecules usually bring about a superior
therapeutic effects and less side effects due to that these hybrid molecules are able to modulate
more than one targets or pathways simultaneously without the involvement of drug-drug
8
interactions. Accordingly, these hybrid molecules have gained considerable interests due to their
4

advantages in the treatment of complex human diseases including cancers in the past decade.
Out of all bioactive hybrid molecules, hybrid HDAC inhibitors or termed as multi-targeting
HDAC inhibitors are an important branch. Since HDAC inhibitors are capable to synergistically
enhance the inhibitory effects of other anti-tumor drugs or agents. Additionally, the “cap” part of
the HDAC inhibitors can tolerate extensive structure modification, that triggers many medicinal
chemists to devote their efforts to develop multi-targeting HDAC inhibitors based on the
9
10, 11
hormone receptors,12, 13 and
structures of inhibitors of kinases, topoisomerases (I or II),
1
4
tubulin inhibitors, etc. To the best of our knowledge, four hybrid HDAC inhibitors, CUDC-101,
CUDC-907, Tinostamustine, and Domatinostat, have entered different clinical trial phases against
different kinds of cancers.
Fig. 2. Four hybrid HDAC inhibitors undergoing clinical trials
The main challenge in the development of hybrid HDAC inhibitors is finding an appropriate
target, inhibition of which could synergize with the HDAC inhibitors. Considering that tumor cells
propagate much faster than normal ones and microtubule plays important roles in tumor cell
division and it is also the main component of the cytoskeleton, it makes tubulin a desirable target
for the development of chemotherapeutic agents.15-19 The tubulin heterodimer includes three
5

distinct drug binding sites: the paclitaxel, vinblastine, and colchicine.20 Studies have shown that
synergic anti-tumor effects can be achieved by combination of microtubule-destabilizing agents
and HDAC inhibitors in vitro and in vivo,21, 22 suggesting that it should be a reasonable strategy to
design tubulin/HDAC hybrid inhibitors to fight against cancers with more powers. Actually,
several groups have reported their works in design and synthesize tubulin/HDAC hybrid inhibitors
which indeed manifested potent anti-tumor activities, such as the hybrid inhibitors with the
colchicine as the structural core reported by Zhang et al23 as well as the iso-combretastatin
derivatives equipped with HDAC inhibitory activities reported by Lamaa et al,24 all of these just
validated this anti-tumor drug design strategy.
In this work, we designed and synthesized a series of 10 novel tubulin/HDAC hybrid inhibitors
with compounds A and B as the key skeletons just as showed in Fig.3. Compounds A and B are
two combretastatin-like chalcones displaying potent microtubule-destabilizing activities via acting
on the colchicine binding site.25, 26 Their hydrophobic structures make them possible to act as the
“
cap” part of HDAC inhibitor. The ZBG we used was hydroxamic acid due to its high affinity to
2
+
Zn . The linkers with different length (3 to 7 carbons) were utilized in order to test their effects
on the inhibition of dual targets. HDAC inhibition activity, isoform selectivity, tubulin
polymerization inhibitory activity and in vitro anti-proliferative activity of representative
compounds were all evaluated. Finally, a lead compound displaying promising anti-tumor activity
was obtained.
6

Fig. 3. The design strategy of novel tubulin and HDAC dual-targeting inhibitors.
The preparation route for all 10 target compounds was outlined in Scheme 1. Following the
method previously described,26 coupling of isovanillin with 3,4,5-trimethoxyacetophenone or 1-
(3,4,5-trimethoxyphenyl)propan-1-one gave A and B, respectively. Compound A and B were
further condensed with different terminal-substituted bromo-acid methyl ester to yield the
intermediates 1a-1e and 5a-5e. The methyl ester intermediates were hydrolyzed under basic
condition to give the carboxyl products 2a-2e and 6a-6e which went further to condense with
tetrahydropyran protected hydroxylamine (NH OTHP) to give the intermediates 3a-3e and 7a-7e.
2
After the deprotection mediated by HCl in methanol, all final products 4a-4e and 8a-8e could be
smoothly obtained.
O
O
H CO
OH
3
H CO
R
OHC
OH
3
a or b
c
+
R
H CO
OCH3
3
H CO
OCH3
3
OCH3
A: R=H
OCH3
B: R=CH3
O
O
O
O
H CO
O
H CO
O
3
d
3
e
O
OH
n
n
R
R
H CO
OCH3
H CO
OCH3
3
3
OCH3
OCH3
1a to 1e: R=H
2a to 2e: R=H
5a to 5e: R=CH3
6a to 6e: R=CH3
O
O
O
O
H CO
O
OH
H CO
O
3
3
f
N
NHOTHP
n
n
H
R
n=1-5
R
H CO
OCH3
H CO
OCH3
3
3
OCH3
OCH3
4a to 4e: R=H
3a to 3e: R=H
8
a to 8e: R=CH3
7
a to 7e: R=CH3
Scheme 1. Reagents and conditions: (a for R=H) KOH, MeOH, H O, RT, 16 h, then 1M HCl,
2
7

neutralized, 55%; (b for R=CH ) piperidine, AcOH, MeOH, 4 Å molecular sieve, anhydrous
3
EtOH, reflux, 56%; (c) bromo acid methyl esters, K CO , DMF, 25℃, 4 h, 98-100%; (d) 1M
2
3
LiOH, THF, H O, 25℃, overnight, 90-95%; (e) NH OTHP, HOBT, EDCI, TEA, anhydrous
2
2
DCM, RT, 18 h, 66-70%; (f) HCl, MeOH, RT, 96-98%.
After the preparation of all 10 target compounds, we first screened the inhibitory activity of
them against HeLa cell nucleus extracts whose main component is class I HDACs. Single
concentration (1 μM) was used and the inhibition rate (%) was calculated. The results was showed
in Fig. 4. All target compounds (4a-4e and 8a-8e) displayed moderate to significant inhibitory
activities against HDACs. The linker length seemed to be a key factor affecting the activity with
the 6-8 carbons as the desirable length. Notably, the inhibition rates of compounds 4d and 8e were
8
7.35% and 92.28%, respectively, comparable to and better than that of SAHA (90.75%).
Fig. 4. Inhibitory activities of all 10 target compounds towards HDACs
With this result in mind, the IC50 values of compounds 4d and 8e against HDACs were
determined as 132.6 and 71.5 nM, respectively as showed in Table 1, the SAHA’s was 73.2 nM.
8

Table 1
IC values of compounds 4d and 8e against HDACs from HeLa cell nucleus extract
5
0
Compd
IC (nM) to HDACs
50
4
d
132.6±6.6
71.5±2.2
73.2±3.1
8
e
SAHA
Since the hydroxamic acid based compounds (4a-4e and 8a-8e) displayed the inhibitory
activities to HDACs as we envisioned. So, we went further to test their anti-proliferative activities
against three human tumor cell lines, A549, HeLa, and SGC-7901. The result was summarized in
Table 2.
Table 2
Anti-proliferative activities of ten target compounds against A549, HeLa, and SGC-7901 tumor
cell lines.
a
Tumor cell, IC (μM)
5
0
Compd
CLog Pb
A549
12.25
14.32
9.40
HeLa
13.26
25.93
24.57
21.39
37.16
0.89
SGC-7901
17.37
>50
4
a
1.64
1.55
2.08
2.61
3.14
1.95
1.86
4
b
4
c
25.20
24.54
>50
4
d
7.15
4
e
13.33
0.55
8
a
1.06
8
b
1.45
3.15
9
13.91

8
c
13.60
11.51
20.03
2.61
10.39
16.61
19.44
1.17
14.02
19.99
28.31
1.03
2.39
2.92
3.45
0.99
2.78
2.62
8
d
8
e
SAHA
A
B
1.02
0.44
1.13
1.51
>50
2.06
aAssays were performed in triplicate, the SD values are < 10% of the mean.
bClogP values were produced by Chemdraw 2014 software.
Beyond our expectation, compound 8a with lowest inhibition rate to mixed HDACs displayed
strongest anti-proliferative activities against all three human tumor cell lines we used. Compound
4
d and 8e did not showed expected anti-proliferative activities, one magnitude weaker than 8a.
Compounds 4d and 8e with good HDACs inhibitory rates and compound 8a with highest
cytotoxicity to three human tumor cell lines were then evaluated for the IC values against three
5
0
HDAC isoforms 1, 6 and 8 to test whether they had isoform selectivity. Consistent with the results
of HDACs inhibition, compounds 4d and 8e showed high activities against HDAC1 and 6 with the
IC50 values all in nanomolar range, more potent than SAHA (Table 3). While, compound 8a
displayed moderate potency with the IC50 values of 996, 470, and 1700 nM against HDAC1, 6,
and 8, respectively.
Table 3
Inhibitory activities of 4d, 8a and 8e towards three HDACs.
a
IC (nM)
5
0
Compd
HDAC1
HDAC6
HDAC8
1
0

4
d
36.3
996
15.2
42
4.6
470
13.6
28
3880
1700
4910
4800
8
a
8
e
SAHA
aAssays were performed in duplicate, the SD values are < 10% of the mean.
Because the structures of compounds we designed were based on compounds A and B which
are potent microtubule-destabilizing agents, that promoted us to test the microtubule-destabilizing
activities of compounds 4d, 8a and 8e with the results showed in Table 4. Compounds A and CA-
4
were reference compounds.
Table 4
Inhibitory activities of representative compounds toward tubulin polymerization.
4
d
8a
8e
A
CA-4
IC50a (μM)
10.4±0.3
54.5±2.1
5.0±0.1
4.0±0.2
1.2±0.1
aAssays were all performed in triplicate. Tubulin concentration was 1 mg/mL, IC50 concentration
of drug to inhibit 50% of tubulin assembly.
Compounds 8e and 4d displayed potent tubulin assembly inhibition activities with the IC50
values of 5.0 and 10.4 μM, respectively, comparable to A and weaker than CA-4. While, 8a
displayed moderate tubulin assembly inhibition activity with the IC value of 54.5 μM.
5
0
Considering that compounds 4d and 8e possessed potent HDAC and tubulin assembly
inhibitory activities and compound 8a displayed highest cytotoxicity to three human tumor cells,
these three compounds were selected to evaluated for their anti-proliferative activities against
eight more human tumor cell lines with the results showed in Table 5. From the result we noticed
1
1

that 8a was still the most potent compound with the IC50 values all in low micromolar range. In
NCI-H460 and BGC-823 tumor cells, the growth inhibition activity of 8a was even stronger than
the reference compounds SAHA and A. Compound 8a did not showed most potent inhibitory
activity towards HDACs and tubulin assembly, but it possessed the highest anti-proliferative
activities toward 11 human tumor cell lines we selected, the mechanism still needs to be
investigated.
Table 5
Anti-proliferative activities of 4d, 8a and 8e against another eight human tumor cell lines.
a
Compd, IC (μM)
5
0
Tumor cells
4
d
8a
8e
A
SAHA
2.15
4.23
3.04
8.33
5.43
3.31
3.51
2.94
LoVo
HCT-116
NCI-H460
NCI-H226
MCF-7
12.59
29.89
13.22
15.65
17.92
15.21
10.14
11.47
4.88
7.46
2.95
7.54
7.33
6.95
3.66
2.40
9.93
3.99
2.85
4.98
4.38
8.60
4.28
8.33
7.58
10.72
20.19
23.25
11.97
11.84
16.40
15.21
MDA-MB-231
MGC80-3
BGC-823
a
IC values are the mean of at least two experiments. Standard deviation is <20% of the IC .
5
0
50
To determine whether the anti-proliferative effect of compound 8a is accompanied by
enhancing apoptosis, A549 cells were incubated with different concentrations of 8a for 48 h.
FITC-Annexin V/propidium iodide (PI) staining and flow cytometry assay were performed. As
1
2

shown in Fig. 5, 8a dose-dependently induced apoptosis in A549 cells and the apoptosis
percentage at 10 μM was slightly higher than that of SAHA.
Fig. 5. Flow cytometry analysis on A549 cells with 8a. Compounds 8a mediated A549 cells
apoptosis in a concentration dependent manner.
In order to further explore the cell cycle effect, 8a was evaluated for cell cycle arrest against
A549 cells. The results were shown in Table 6.
Table 6
Cell cycle arrest activity of 8a towards A549 cells.
Proportion of A549 cells (%)
Compd
Control
G /G
S
G /M
0
1
2
72.51
68.44
46.23
16.66
22.09
39.96
10.83
9.47
8
a (0.1μM)
8
a (0.3 μM)
13.81
1
3

8
8
a (1 μM)
a (3 μM)
16.57
9.13
1.57
9.61
20.54
10.18
16.55
39.53
62.89
80.69
81.88
50.86
8
a (10 μM)
SAHA (10 μM)
A549 cells were evidently arrested in G /M phase by the treatment of 8a in a concentration-
2
dependent manner as shown in Fig. 6. It is clearly observed that the percentage of cells in G /M
2
phase treated with 8a increased from 9.47% (0.1 μM) to 81.88% (10 μM) whereas the reference
drug SAHA was to 50.86% (10 μM).
1
4

Fig. 6. Cell cycle analysis on A549 cells with 8a. Compounds 8a arrested A549 cells in G /M
2
phase with a concentration dependent manner.
As reported, HDAC6 plays roles in migration of tumor cells, in this work, we also tested the
anti-migration activity of compounds 8a which possessed inhibitory activity toward HDAC6.
To investigate the anti-migration effect of 8a, the in vitro wound healing assay was performed
1
5

using A549 tumor cells. Result was showed in Fig. 7 which indicated that treatment with 8a at
concentrations of 5 and 10 μM both remarkably suppressed the migratory capability of A549
tumor cells compared to that of the blank control group at 12 h after wound creation, superior to
SAHA at the same concentration. The IC value of 8a against A549 cells was low, so, besides the
5
0
direct inhibition of HDAC6, the anti-proliferative activity of 8a should also contribute to its anti-
migration activity.
Fig. 7. Wound healing assay. Compounds 8a effectively suppressed the migratory capability of
A549 tumor cells.
Finally, in order to better explaining inhibitory activity of compound 8a towards HDAC6 and
tubulin, the docking assays (LeDock, www.lephar.com) were performed. Firstly, the binding
model between 8a and HDAC6 (PDB code: 5WGK) was predicted as shown in Fig. 8A.
Compound 8a and HPB shared the same binding mode. The hydroxamic acid of 8a could chelate
with the Zn2+ and could form another hydrogen bond with HIS574 residue in the active site of
1
6

HDAC6. Meanwhile, the phenyl of 8a could also form a π-π stacking interaction with PHE643
residue. And the chalcone part of 8a fitted well with the hydrophobic surface region of HDAC6.
All of these proposed interactions could preliminarily rationalize the inhibitory activity of 8a
towards HDAC6. Additionally, the binding model between 8a and tubulin was also predicted
using the tubulin/CA-4 complex as the template (PDB code: 5LYJ). The result was showed in Fig.
8
B. The binding position of 8a almost overlapped with that of CA-4 (green compound). The
hydroxamic acid of 8a could form three hydrogen bonds with ALA180, VAL181 and ASN349
residues.
1
7

Fig. 8. (A) Proposed binding mode of compound 8a with HDAC6 (PDB#5WGK). (B) Proposed
binding mode of compound 8a with tubulin (PDB#5LYJ).
In the field of novel HDAC inhibitor design and discovery, dual or multi-targeting HDAC
inhibitors have demonstrated promising potentials due to synergic effect resulted from the
inhibition of more than one anti-tumor targets. Based on previous researches of other groups,
compounds A and B which were two chalcone-based tubulin polymerization inhibitors were
utilized in this work to design and synthesize a series of tubulin/HDAC dual-targeting inhibitors.
1
8

Most of newly synthesized compounds with hydroxamic acid as the ZBG displayed potent
inhibitory activities against HDACs. And several compounds exhibited anti-proliferative activities
towards tumor cell lines we selected, with an IC50 values in μM or sub-μM range, the anti-
proliferative activity of compound 8a was better than SAHA and A against certain tumor cells.
Furthermore, 8a could significantly induce apoptosis and G /M arrest of A549 cells in a dose
2
dependent manner. In addition, 8a could effectively suppress the migratory capability of A549
cells, more potent than SAHA at the same concentration. Finally, the high potency of compound
8
a towards HDAC6 and tubulin polymerization could be interpreted by molecular docking study.
Taken together, these tubulin/HDAC dual-targeting inhibitors could be identified as promising
agents for cancer treatment.
DECLARATION OF INTEREST
None
Acknowledgment
We gratefully acknowledge the financial support from the National Science Foundation for
Young Scientists of China to Y. L. (NSFC no. 81602947).
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derivatives as antimitotic and antivascular agents with potent antitumor activity. Bioorg Med
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Anti-tumor activity evaluation of novel tubulin and HDAC dual-targeting inhibitors
a,#
c,#
b
a
a
c
a,*
Baolei Wang , Xuehong Chen , Jianjun Gao , Li Su , Li Zhang , Hongwei Xu , Yepeng Luan
a
Department of Medicinal Chemistry, School of Pharmacy, Qingdao University, Qingdao,
Shandong, China
b
Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, Shandong,
China
c
Department of Pharmacology, College of Basic Medicine, Qingdao University, Qingdao,
Shandong, China
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⁎
Corresponding Author:
Yepeng Luan, Mobile: (+86)15265260855; E-mail: luanqdu@sina.com.
#
These authors contributed equally to this work.
Design of multi-targeting histone deacetylases inhibitors have proven to be promising strategy in
anti-cancer drugs development. In this study, we reported a series of 20 novel chalcone based
tubulin and HDAC dual-targeting inhibitors. Out of them, compound 8a remarkably induced
growth inhibition, apoptosis and G2/M phase arrest of A549 tumor cells. The inhibitory activities
of 8a against HDAC6 and tubulin were rationalized by molecular docking studies.
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