A hybrid of thiazolidinone with the hydroxamate scaffold for developing novel histone deacetylase inhibitors with antitumor activities

Article abstract of DOI:10.1039/c5ob02250a

A series of novel histone deacetylase (HDAC) inhibitors were designed, synthesized and evaluated based on the strategies of a hybrid of the classic pharmacophore of HDAC inhibitors with the thiazolidinone scaffold. Some of the compounds showed potent HDAC1 inhibition with nM IC₅₀ values, more importantly, compound 12i displayed much better anti-metastatic effects than vorinostat (SAHA) against migration of the A549 cell line. Further mechanism exploration implied that compound 12i may inhibit tumor metastasis via modulating the epithelial-mesenchymal transition (EMT) and upregulating the acetylation of α-tubulin.

Full text of DOI:10.1039/c5ob02250a

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DOI: 10.1039/C5OB02250A
Organic & Biomolecular Chemistry
ARTICLE
Hybrid of Thiazolidinone with Hydroxamate Scaffold for
Developing Novel Histone Deacetylase Inhibitors with Antitumor
Activities
Received 00th January 20xx,
Accepted 00th January 20xx
Feifei Yang,a,b,# Shihong Peng,a,#, Yunqi Li,a Liqiang Su,a Yangrui Peng,a Jing Wu,a Huang Chen,a
Mingyao Liu,a Zhengfang Yi,a,*, and Yihua Chen.a,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of novel histone deacetylase (HDAC) inhibitors were designed, synthesized and evaluated based on the strategies
of hybrid of classic pharmacophore of HDAC inhibitors with thiazolidione scaffold. Some of compounds showed potent
HDAC1 inhibition with nM IC50 values, More importantly, compound 12i displayed much better anti-metastatic effects than
Vorinostat (SAHA) against migration of A549 cell line. Further mechanism exploration implied that compound 12i may inhibit
tumor metastasis via modulating the epithelial-mesenchymal transition (EMT) and upregulating the acetylation of -tubulin.
nicotinamide adenine dinucleotide (NAD+) dependent.7, 13 Different
Introduction
HDACs contribute to numerous specific processes during
Epigenetic regulations, involving modifications of chromatin
structure, were one of the main causes of disordered gene expressions,
which may be responsible for the occurrence and development of
tumors, characterized with uncontrolled cell growth, proliferation,
migration and so on.1, 2 Histone deacetylases (HDACs) along with
histone acetyl transferases (HATs) have been proven to regulate the
acetylation states of histones and some other key non-histone
proteins.3, 4 For example, HDACs were involved in remodelling of
chromatin and down-regulating many gene expressions,5 while HATs
were involved in opening chromatin structure and activation of
transcription.6 Global hyper-deacetylation of histones is also
tumorigenesis and development.14 HDAC inhibitors, 1 (Vorinostat,
suberoylanilide hydroxamic acid, SAHA) and 2 (Romidepsin, FK-228)
have been approved by the FDA for the treatment of cutaneous T-cell
lymphoma (CTCL) in 2006 and 2009 respectively (Figure 1),15-17
Recently the third and fourth HDAC inhibitors, 3 (Belinostat) and 4
(Panobinostat), were also approved by the FDA for the treatment of
relapsed or refractory peripheral T-cell lymphoma (PTCL) and
multiple myeloma (MM) respectively.17 In addition, many other
HDAC inhibitors are in clinical trials, which validate that design and
discovery of novel HDAC inhibitors are still one of effective
strategies for cancer therapies.18-21
considered as a common hallmark of many kinds of cancers.7,
8
Various clinical trials of combining HDAC inhibitors with other
anticancer agents have been proven as an effective strategy for
broadening the spectrum in the treatment of solid tumor fields.22
Several dual or multi- action HDAC inhibitors (Figure 1) were
developed based on this successful strategy. For example, Compound
5 was equipped with HDAC inhibition as well as estrogen receptor
modulation.23 Compound 6 targeted HDAC and 3-hydroxy-3-methyl
glutaryl coenzyme A reductase (HMGR) for cancer treatment.24
Moreover, Compounds 7 and 8 were also reported by different
research groups.25, 26 These results indicated that conjugation of the
classic HDAC inhibitor moiety with other pharmacophores could
promote the potency of HDAC inhibitors in the treatment of solid
tumors. Tumor metastasis is regarded as a major contributor to the
mortality of cancer patients,27-29 therefore discovery of novel dual or
Therefore inhibition of HDACs activities has been largely validated
as one of the viable therapeutic strategies for cancer treatment.9, 10
Up to now, 18 human HDACs have been found and divided into
four distinct classes.11, 12 Class I (HDACs 1, 2, 3 and 8), II (HDACs 4,
5, 6, 7, 9 and 10), and IV (HDAC 11) are zinc-dependent
metalloproteinases, whereas class III HDACs (sirtuins 1–7) are
a.Shanghai Key Laboratory of Regulatory Biology, The Institute of Biomedical
Sciences and School of Life Sciences, East China Normal University, Shanghai,
200241, China.
b.School of biological science and technology, University of Jinan, Jinan, Shandong
Province, 250022, China
† Corresponding Authors: Dr. Yihua Chen or Dr. Zhengfang Yi, Phone: +86-21-2420-
6647;
zfyi@bio.ecnu.edu.cn.
# These authors contribute equally to this research work.
Fax:
+86-21-54344922;
E-mail:
yhchen@bio.ecnu.edu.cn
or
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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multi-functional HDAC inhibitors bearing antitumor growth as well
as metastasis activities is still our priority for solid tumor treatments.
(Figure 1 Insert here)
Compounds 11a–b were prepared following VitehweArtmicleetOhnolidnes
DOI: 10.1039/C5OB02250A
described in Scheme 2. The starting material 17 was synthesized in a
similar manner of step 1 of scheme 1, then the ester group was
hydrolyzed under basic condition to yield acid analogue 18, which
was coupled with amino acid esters (n = 3 or 5) and the target
compounds were achieved by transforming ester to hydroxamate
group.
The classic structural scaffold of HDAC inhibitors consists of
three distinct structural motifs,30 a recognition cap group (CAP), a
linker domain (Linker) and a zinc-binding group (ZBG, Figure 2).31-
34
The cap group presents a potential opportunity to discover more
potent HDAC inhibitors.32 One small molecule 9 (CK-548) and its
derivatives, with thiazolidinone scaffold, have been reported to
regulate the cellular movements and strongly inhibit tumor growth
and metastasis.35, 36 In addition, a series of novel hydroxmate-based
HDAC inhibitors was also designed and optimized in our previous
research work and displayed excellent antitumor growth and
metastasis activities,8 therefore one type of novel HDAC inhibitors
was herein designed and modified with hybrid of hydroxamate
scaffold and thiazolidinone structure as the CAP region (Figure 2).24,
Scheme 2. Synthesis of compounds 11a–b
O
H
N
O
H
N
O
O
COOCH
COOH
3
n
NHOH
OCH
n
3
b
S
N
S
S
S
c
a
N
N
N
O
3
O
3
O
O
3
OCH
OCH
OCH
3
OCH
17
18
19a n = 3
19b n = 5
11a n = 3
11b n = 5
Reagents and conditions: (a) LiOH•H2O, MeOH/H2O, 85%; (b)
NH2(CH2)nCOOCH3, EDC, HOBt, DMF, 60-80%; (c) NH2OH•HCl,
MeOH, KOH, 30-70%.
37-40
Their biological activities were further explored against tumor
cell proliferation and migration as well as possible mechanisms.
(Figure 2 Insert here)
Target compounds 12a–j were synthesized according to the
procedures described in Scheme 3. The intermediate 2,3-diaryl-4-
thiazolidinone derivatives 21a-j were constructed from aminophenol,
a series of aromatic aldehydes and thioglycolic acid, which was
alkylated with brominated fatty acid esters (n = 3-7) to yield esters
compounds 22a-j, and the ester groups were treated with NH2OH•HCl
in methanol to get the compounds 12a–j.
Chemistry
With the goal of discovering more novel HDAC inhibitors with
high potent antitumor activities against solid tumors, two conjugated
compounds 10a–b were firstly synthesized according to the
procedures described in Scheme 1. The intermediate 2-(3-nitrophenyl)
-3-(2-methoxyphenyl)-4-thiazolidinone 15 was constructed from 2-
methoxyaniline, 3-nitro-benzaldehyde and thioglycolic acid in
41
toluene,36,
which was then reduced to corresponding amine.
Scheme 3. Synthesis of compounds 12a–j
Condensation of this amine intermediate 15 with succinic or pimelic
acid anhydride in 1,4-dioxane and with subsequent esterification to
afford esters compounds, and then the ester groups were treated with
NH2OH•HCl in methanol to get the compounds 10a–b.42
Scheme 1. Synthesis of compounds 10a–ba
O
O
O
O
NH
2
Reagents and conditions: (a) ArCHO, thioglycolic acid, toluene,
reflux, 40-50%; (b) Br(CH2)nCOOCH3, K2CO3, DMF, 80-90%; (c)
NH2OH•HCl, MeOH, KOH. 30-70%
HN
HN
n
n
NHOH
OH
NO
2
S
a, b
CHO
d, e
c
S
S
N
N
N
O
O
OCH
O
3
OCH
OCH
3
3
The compounds 13a–b were synthesized according to the methods
described in Scheme 4. The starting compound 23 was firstly
protected by Boc group, and was converted to the 2,3-diaryl-4-
thiazolidinone compound 24, then the Boc group was cleaved in
acidic condition, and subsequent route was the same as step c-e in
Scheme 1.
14
15
16a n = 2
16b n = 5
10a n = 2
10b n = 5
aReagents and conditions: (a) o-anisidine, thioglycolic acid, toluene,
reflux, 82%; (b) Zn, CH3COOH, EtOH/H2O, 85%; (c) Succinic or
pimelic acid anhydride, 1,4-dioxane, reflux, 60-80%; (d) MeOH,
SOCl2, reflux, 80-90%; (e) NH2OH•HCl, MeOH, KOH, 30-70%.
Scheme 4. Synthesis of compounds 13a–b
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---
---
10 ± 3
View Article Online
1
DOI: 10.1039/C5OB02250A
On the other hand, the linker and ZBG region were transferred to
the ortho-position of B ring and a series of compounds 12a–e and
13a–b were synthesized with different linker length in order to further
demonstrate the structure activity relationship between the different
linker length and HDAC1 inhibitory activities. As shown in Table 2,
the activities of target compounds were improved with the elongation
of linker, for example, compound 12e, containing the longest linker
length, showed the most potent activity (IC50 = 29 ± 8 nM) with the n
= 7 in this series of analogues (12a–e). In addition, when the oxygen
atom was then replaced by amide, the inhibitory activities were found
to be similar to that with the same linker length (12d vs 13a and 12e
vs 13b).
Reagents and conditions: (a) (Boc)2O, NaHCO3, Et2O, 46%; (b) 3-
bromobenzaldehyde ，thioglycolic acid, toluene, reflux, 75%; (c)
TFA, DCM, 80%; (d) anhydride, 1,4-dioxane, reflux, 60-80%; (e)
MeOH, SOCl2, reflux; (f) NH2OH•HCl, MeOH, KOH, 30-70%.
Results and discussion
HDAC1 Inhibition Assay.
All the target compounds were firstly evaluated for their inhibitory
activities against HDAC1 considering that class I HDACs isozymes
are mainly involved in tumorigenesis and development. Compound 1
(Vorinostat) was selected as a positive control. In order to validate the
location of linker and ZBG region in the scaffold, compounds 10a–b
and 11a–b were synthesized with the linker and ZBG region located
on the meta-position of A ring. The results in Table 1 indicated that
there was a linker-length-dependent HDAC1 inhibition, compounds
10b and 11b containing five-methylene in the linker part showed
better activities than analogues with two or three-methylene (10a and
11a respectively), which was consistent with classical feature of
HDAC inhibitors. In addition, the inversion of amide bond listed in
the linker part seemed to show little difference against HDAC1
activity (10b vs 11b).
Table 2. IC50 values of HDAC1 Inhibition of compounds 12a–e and
13a–b
Br
A
S
N
B
O
X'
O
n
NHOH
X’
n
IC50 (nM)
Cpd.
O
O
3
4
1860 ± 110
530 ± 20
12a
12b
O
O
O
5
6
7
260 ± 5
50 ± 4
29 ± 8
12c
12d
12e
Table 1. IC50 values of HDAC1 inhibition of compounds 10a–b and
11a–b
-NHCOCH2-
-NHCOCH2-
---
4
5
29 ± 0
52 ± 7
10 ± 3
13a
13b
1
X
NHOH
n
---
O
A
S
N
Further modifications focused on A ring while keeping the linker
region with three-methylene linker length on the ortho–position of B
ring in order to maintain suitable molecular weights of target
compounds. The A ring was replaced with heterocycles for increasing
activities, which were also reported in our previous research work for
obtaining increased activities.8 Unfortunately, compounds 12g and
12h displayed decreased inhibitory activities. However, when the
linker and ZBG region were introduced to para–position of B ring,
the enzymatic inhibitory activities were similar to that of the
B
O
OCH3
X
n
1
4
2
4
IC50 (nM)
350 ± 70
17 ± 2
Cpd.
10a
10b
11a
11b
-NHCOCH2-
-NHCOCH2-
-CONHCH2-
-CONHCH2-
4930 ± 440
37 ± 1
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corresponding analogues (12i and 12j). The results suggested that
substituted positions of the linker and ZBG region of B ring impacted
the efficacy of compounds definitely. Inhibition activity of compound
12i with para–position substitution was approximately 20-fold better
than its counterpart compound 12g with ortho–position.
Table 4. Anti-proliferative activities of representative compounds
View Article Online
DOI: 10.1039/C5OB02250A
(The values are averaged from 3 independent experiments)
Anti-proliferation activities IC50 (M)
Cpd.
HeLa
MCF-7
LNCaP
A549
Table 3. IC50 values of HDAC1 Inhibition of compounds 12f–j
21 ± 3
21 ± 3
10 ± 2
> 50
> 50
10b
11b
12d
12e
12i
13a
1
7.3 ± 0.8
3.2 ± 0.4
6.4 ± 1.1
3.2 ± 0.5
2.1 ± 0.4
2.8 ± 0.2
27 ± 2
47 ± 3
R
1
S
N
4.1 ± 0.4
6.0 ± 0.6
2.1 ± 0.5
5.2 ± 0.8
1.3 ± 0.3
4.0 ± 0.7
31 ± 3
6.7 ± 0.2
>50
O
B
3'
O
4'
2'
O
HOHN
2.9 ± 0.3
2.5 ± 0.9
3.6 ± 1.0
4.6 ± 0.8
5.4 ± 0.5
7.2 ± 0.1
position
R1
IC50 (nM)
320 ± 70
Cpd.
2’-
4-Cl-Ph(CH2)2NHCOPh-
5-bromothiopheneyl
12f
12g
12h
2’-
2’-
1360 ± 230
1440 ± 190
5-(5-pyrimidinyl)thiopheneyl
Compounds 12i and 13a inhibited the colony formation of A549 cell
4’-
4’-
---
5-bromothiopheneyl
73 ± 14
91 ± 1
12i
12j
line.
5-(5-pyrimidinyl)thiopheneyl
---
10 ± 3
1
In order to further evaluate the efficacy of these compounds against
tumor cell proliferation and development, the colony formation assay
on A549 cell line was performed to confirm the anti-proliferative
activities of compounds 12i and 13a with compound 1 as the positive
control. As one of the gold standards for measuring the effects of
cytotoxic agents on cancer cells in vitro, it closely mimics the
pathological process from individual tumor cell development to
macroscopic cell clones in vivo.44, 45 As shown in Figure 3, the data
indicated that both of compounds 12i and 13a significantly inhibited
colony formation of A549 cells in a dose-dependent manner, at
meantime, they also showed better efficacy compared to 1 concerning
the number and size of colony. All of these results suggested that both
of 12i and 13a may partly block the in vivo growth and development
of A549 cancer cell line.
Anti-proliferative activities against four different HDAC1 highly-
expressed cell lines in vitro.
In order to evaluate the activities of those compounds against tumor
cells with high HDAC1 expression, compounds 10b, 11b, 12d–e, 12i
and 13a were selected based on their potent inhibition against HDAC1
enzyme to test their anti-proliferative effect against a panel of four
different tumor cell lines (HeLa, MCF-7, LNCaP and A549) through
SRB assay with their highly expression of HDAC1.43 And compound
1 was also chosen as the positive control, as listed in table 4. It is
surprising for us that although compounds (10b and 11b) with the
linker and ZBG region located on A ring showed potent HDAC1
inhibition with low nM IC50 values, both of them lost most of
proliferative effects against LNCaP and A549 cell lines compared to
the analogues with located in B ring or positive control 1. These
results indicated that the introduction of the linker and ZBG to A ring
may not be suitable for increasing their potential as novel anticancer
agents. However, compounds with the linker and ZBG to B ring
exhibited comparable activities as 1, especially for 12i and 13a, which
showed almost equivalent activities compared to 1 against four
different cancer cell lines, with the IC50 values ranging from 2.1 ± 0.5
to 5.4 ± 0.5 M.
(Figure 3 Insert here)
Compounds 12i and 13a induced the level of acetylated histone on
A549 cell line.
Growing evidences indicated
a
strong correlation among
overexpression of HDACs activities, deacetylation of the histone tails
(particularly histone H3 and H4 tails) and chromatin architecture in
tumors.46 Thus the anticancer effect of HDAC inhibitors could be also
assessed by their potency in inducing hyper-acetylation of histone H3
or H4,14 In order to confirm the HDAC inhibitory activity of these
compounds, 12i and 13a were chosen to examine their efficacy for
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ARTICLE
histone acetylation at two different concentrations (2.0 and 5.0 μM)
and with compound 1 as a positive control. As listed in Figure 4, the
results demonstrated that compounds 12i and 13a significantly
upregulated the level of acetylated histone H3 and H4 in a dose-
dependent manner as well as positive control 1, which also indicated
that both of 12i and 13a can increase the acetyl level of histone of
cancer cells through inhibiting the activities of HDACs.
epithelial markers (E-cadherin and -catenin) and a dVoiewwnArretigcluelOatniloinne
DOI: 10.1039/C5OB02250A
of a mesenchymal marker (vimentin) after treatment with 12i (Figure
6C). According to previous research, the regulation of tubulin
acetylation was closely related to cell motility.48 In our experiment，
compound 12i was found to induce the acetylation level of -tubulin
significantly (Figure 6D). These results implied that compound 12i
may inhibit tumor metastasis via modulating the EMT and
upregulating the acetylation of tubulin.
(Figure 4 Insert here)
Compounds 12i and 13a induced cell cycle arrest and enhanced
apoptosis on A549 cell line
(Figure 6 Insert here)
Conclusions
In summary, a series of hybrid of thiazolidinone and hydroxamic acid
derivatives were designed and synthesized as potent HDAC1
inhibitors. Some of them showed comparable enzymatic activities as
1 against HDAC1. Among them, compound 12i not only exhibited
potent anti-proliferative activities against four different cancer cell
lines and inhibited colony formation of cancer cells, but also showed
better anti-metastasis activity via modulating the EMT and
upregulating the acetylation of -tubulin against A549 cell line. In
addition, compound 12i was found to induce cell cycle arrest and
enhance cancer cell apoptosis. Preliminary mechanism studies also
indicated that 12i behaved according to multiple mechanisms
including HDAC inhibition. All of these data led to a new promising
direction of exploring novel HDAC inhibitors with dual functions.
Continued efforts are in progress in our laboratory to develop more
potent dual or multi-functional HDAC inhibitors.
Previous results of cellular cytotoxicity assay and colony formation
assay indicated that 12i and 13a inhibited cancer cell proliferation.
Next, whether these compounds affected the cancer cell cycle arrest
and induction of apoptosis was explored by flow cytometry. The
results showed that 12i and 13a caused cell apoptosis in A549 cell line
in a dose-dependent manner, and early apoptosis induction was
especially evident (Figure 5A). And 12i and 13a were also found to
increase cell numbers at G2/M phase after 24 h treatment with raising
concentration, accompanied with decreased cell numbers at S phase
and G1 phase in A549 cell line (Figure 5B). These results indicated
that 12i and 13a inhibited cell proliferation by inducing cell cycle
arrest and enhancing apoptosis on cell line.
(Figure 5 Insert here)
Compound 12i suppressed metastatic activities on A549 cell line.
Based on our initial strategies for developing novel HDAC
inhibitors with potent anti-proliferative and anti-migratory effects
against tumor cells, it is valuable to investigate their anti-migratory
effect against cancer cell line. Compounds 12i and 13a were also
chosen and the results were shown in Figure 6A and 6B. In a transwell
migration assay, compound 12i obviously decreased A549 cell
migration at the investigated concentration (2.0 and 5.0 M), with the
IC50 value about 3.2 M, which was about 6-7 fold more potent than
Figure legends:
Figure 1. FDA approved HDAC Inhibitors and representative
examples of dual-action HDAC inhibitors
Figure 2. Design of conjugated compounds based on HDAC and
Arp2/3 complex inhibitors
Figure 3. A. Compounds 12i and 13a inhibited the colony formation
of A549 cell lines. After treatment by various concentration of 12i and
13a in 6-well plates for 7 days, cells were fixed and stained with
1, with IC50 value about 20 M. These data suggested that compound crystal violet and the numbers of cell colonies were counted. ** p <
0.01, *** p < 0.001 versus the control group. B. Compounds 12i and
13a induced the level of acetylated histone in A549 cell lines.
Figure 4. Western blot analysis of the effects of compounds 12i at
different concentrations on tumor cell A549 acetylated histone levels.
Treatment resulted in upregulated expression of acetyl-histone H3 and
H4 levels in a dose-dependent manner.
12i possesses more anti-migration efficacy than 1 in the transwell
migration model. To study the possible mechanism of 12i on cell
migration, some potential downstream effectors of tumor metastasis
were chosen to be determined. The epithelial-mesenchymal transition
(EMT), as a highly conserved cellular program that allows polarized,
immotile epithelial cells to be converted to motile mesenchymal cells,
has been widely implicated in promoting carcinoma invasion and
metastasis.47 Western blot analysis displayed the upregulation of
Figure 5. A. 12i and 13a mediated A549 cells apoptosis in
concentration-dependent manner. Cells were treated with different
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13. R. W. Johnstone, Nat. Rev. Drug discovery, 2002V,iew, A2r8tic7le-2O9n9lin.e
concentration of 12i and 13a for 48 h. Then the percentage of
apoptosis cells was analyzed with flow cytometry by staining with PI
and annexin V-FITC. B. 12i and 13a induced cell cycle arrest of A549
cell line. Cells were stained with PI and analyzed by flow cytometry
after treatment with various concentration of 12i and 13a for 24 h.
Figure 6. A. Representive images of Compound 12i inhibiting A549
cell migration in the transwell assay different concentrations. B.
Statistical analysis result of cell migration of 12i, 13a and 1, ** p <
0.01, *** p < 0.001 versus the control group. C. and D. Compound
12i inhibits tumor metastasis via modulating the EMT and
upregulating the acetylation of tubulin. Cell lysates were extracted
after treating A549 with 12i or 1 for 48 h. Cell extracts were run on
SDS−polyacrylamide gels and probed using indicated antibodies.
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Acknowledgements
This work was partially supported by the grants from National
Basic Research Program of China (2012CB910404), National
Major Scientific and Technological Special Project for
"Significant New Drugs Development" (2013ZX09507001),
National Natural Science Foundation of China (81202407 and
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