Design, synthesis, and biological evaluation of novel histone deacetylase 1 inhibitors through click chemistry

Article abstract of DOI:10.1016/j.bmcl.2013.03.102

We report the design, synthesis, and biological evaluation of a new series of HDAC1 inhibitors using click chemistry. Compound 17 bearing a phenyl ring at meta-position was identified to show much better selectivity for HDAC1 over HDAC7 than SAHA. The compond 17 also showed better in vitro anticancer activities against several cancer cell lines than that of SAHA. This work could serve as a foundation for further exploration of selective HDAC inhibitors using the compound 17 molecular scaffold.

Full text of DOI:10.1016/j.bmcl.2013.03.102

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3295–3299
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of novel histone
deacetylase 1 inhibitors through click chemistry
Qiao Sun a, Yiwu Yao a, Chunping Liu a, Hua Li a, Hequan Yao b, Xiaowen Xue b, Jinsong Liu a,
Zhengchao Tu a, , Sheng Jiang a,
⇑
⇑
a Laboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, PR China
b School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis, and biological evaluation of a new series of HDAC1 inhibitors using click
chemistry. Compound 17 bearing a phenyl ring at meta-position was identified to show much better
selectivity for HDAC1 over HDAC7 than SAHA. The compond 17 also showed better in vitro anticancer
activities against several cancer cell lines than that of SAHA. This work could serve as a foundation for
further exploration of selective HDAC inhibitors using the compound 17 molecular scaffold.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 10 December 2012
Revised 11 March 2013
Accepted 26 March 2013
Available online 4 April 2013
Keywords:
HDAC inhibitors
Suberoylanilide hydroxamic acid (SAHA)
Structure–activity relationship
Click chemistry
Molecular docking
Epigenetics is classically defined as reversible heritable changes
in gene expression that occur without a change in DNA sequence.
DNA methylation and histone acetylation are two well known epi-
genetic chromatin modifications, although ethyl, phosphoryl, ubiq-
uitylation and other modifications of histones have been
described.1,2 The steady state of histone acetylation was balanced
by histone deacetylases (HDACs) and histone acetyltransferases
(HATs). HATs add acetyl groups to lysine residues of histone tails
causing localized relaxation of chromatin and transcriptional acti-
vation of nearby genes, while HDACs remove the acetyl groups of
acetylated histones leading to transcriptional repression.3 HDACs
play important roles in the upstream control of gene transcription,
cell cycle progression, and apoptosis. It has been widely recognized
that HDACs are promising targets for cancer therapy. Thus far, 18
Mammalian HDAC subtypes have been discovered and accordingly
classified into four classes based upon their size, number of cata-
lytic active sites, subcellular localization, and sequence homology
to yeast models.4,5 Classes I HDACs (1, 2, 3, and 8), class IIa HDACs
(4, 5, 7, and 9), class IIb HDACs (6 and 10), and the class IV HDAC
(11) are Zn2+-dependent metallohydrolases, whereas class III
HDACs (sirtuins 1–7) are NAD+-dependent Sir2-like deacetylases.5
HDAC isoforms are highly expressed in various cancers, including
gastric cancer, pancreatic cancer, colorectal cancer, prostate cancer
and hepatocellular carcinoma.6 HDACs are part of a transcriptional
corepressor complex that plays a critical role in a variety of tran-
scriptional regulatory pathways involving various tumor suppres-
sor genes. HDAC inhibitors have been shown to induce cell
growth arrest, differentiation and/or apoptosis in different cancer
cell lines. HDAC inhibitors are emerging as a new class of chemo-
therapeutic agents.7
Since the suberoylanilide hydroxamic acid (SAHA, Vorinostat)
has been licensed for the treatment of cutaneous T cell lymphoma
treatment (CTCL) on 2006, more than 11 new HDACi are in various
stages of clinical development for therapy of multiple cancer
types.8 Although HDAC inhibitors showed potent antitumor ef-
fects, they have exhibited side effects that might limit their clinical
potential.9 Therefore, looking for new HDAC inhibitors that are
specific-inhibition to one kind HDAC subtype is extremely urgent
and necessary.
Although a lot of HDAC inhibitors have been investigated, few
class selective or isoform selective HDACi was found.10 Not only
the discovery of novel isozyme selective inhibitors are likely to
provide more effective chemotherapy compared to pan-inhibitors,
but also are useful as tools for probing the biology of the enzyme.
Compound 1 is a HDAC inhibitor reported by Oyelere et al in 2008,
which showed good binding affinity against HDACs.11 Compound 1
are composed by three parts: zinc binding group (ZBG), linker and
capping group (Fig. 1). Molecular modeling of the complex struc-
ture of HDAC1 with compound 1 indicated that the capping group
region of compound 1 plays important role for HDAC isoform
⇑
Corresponding authors. Fax: +86 20 32290606.
E-mail addresses: tu_zhengchao@gibh.ac.cn (Z. Tu), jiang_sheng@gibh.ac.cn
(S. Jiang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.102

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Q. Sun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3295–3299
Table 1
Surface recognition
group
Zinc binding
IC50 Values for HDAC1 and HDAC7 Inhibition (lM)
group (ZBG)
O
O
2
N
N
N
N
NHOH
NHOH
1
R
4
N
N
1
Linker group
O
Sructure
IC50(lM)
N
N
NHOH
HDAC1
HDAC7
38.9
R
N
H
2
N
NHOH
O
4
0.131
O
O
Figure 1. Design of new class I histone deacetylase inhibitors using a scaffold-
hopping strategy.
SAHA
N
N
N
NHOH
5.13
1.68
18.7
NA
selectivity. We envisioned that changing the position of substitu-
ents on the triazole ring would increase the selectivity for HDAC1.
In this paper, we report a series of triazole-based compounds built
by Click chemistry (Huisgen 1,3-dipolar cycloaddition).
1
O
3
N
N
NHOH
The triazole analogues 2–29 in table 1 were synthesized by
using the procedure described in Figure 2. The N-hydroxyhept-6-
ynamide was easily synthesized from the commercially available
6-heptynoic acid.12 Then all compounds were prepared by a conve-
nient one-pot procedure from a variety of readily available substi-
tuted benzyl bromide, sodium azide and N-hydroxyhept-6-
ynamide in 27% ꢀ 68% yields.
N
2
Br
F
O
3
N
N
NHOH
NHOH
3.58
NA
N
3
The HDAC inhibition activity of the compounds reported herein
were examined based on fluorescent-based HDAC biochemical
activity assay using recombinant human HDACs. It is noteworthy
that this set of structures show promising selectivity for HDAC1,
as they are no activity in the inhibition of HDAC7. As shown in ta-
O
3
N
N
1.36
1.05
2.21
1.60
1.85
0.601
0.440
NA
NA
NA
NA
NA
NA
NA
N
4
O
3
ble 1, compound 2 bound to the HDAC1 with an IC50 of 1.68 lM,
which is three times more potent than compound 1. The position
of substitution might have great impact on the effectiveness of
the set of compounds against HDAC1. It is observed that ortho-po-
sition substitution in the phenyl ring did not improve the inhibi-
tion activity (3–5). For instance, when a bromide group was
introduced at ortho-position of compound 2, the inhibitory activity
N
N
NHOH
O
N
5
N
N
NHOH
3
N
against HDAC1 was decreased two times with an IC50 = 3.58 lM in
Cl
6
comparison with original compound 2. Interestingly, different
functional groups at the para-position of phenyl ring show differ-
ent inhibition activities (6–15). When a variety of functional
groups such as tert-butyl, methyl, trifluoromethyl, fluoro, CN, phe-
nyloxy or phenyl groups were introduced at para-position of com-
pound 2, respectively, the inhibition activities of HDAC1 were
significantly increased (9–15). Among them, the compound 13
O
3
N
N
NHOH
NHOH
NHOH
N
Br
7
O
N
with phenoxy at para-position displayed IC50 value of 0.122 lM
3
N
N
against HDAC1, which was about 13.7 times more potent than
the original compound 2. However, it was disappointing that
chloro, bromo and methoxy substitutions did not improve the po-
tency (6–8). To our delight, the functional groups at the meta-posi-
tion of phenyl ring could significantly increase inhibition activities,
which suggested that meta-position might be a feasible position for
further optimization (16 and 17). For example, when a phenyl
group was introduced at meta-position of compound 2, the inhib-
itory activity against HDAC1 was obviously improved and the
O
8
O
3
N
N
N
9
O
3
N
N
NHOH
O
N
resulting compound 17 displayed IC50 value of 0.058 lM, which
10
was about 29 times more potent than the compound 2. Molecular
modeling indicates that the phenyl ring adopted a coplanar geom-
etry in order to fit within hydrophobic pocket (Fig. 3). However,
these improved interactions can be overweighed by increasing ex-
posed hydrophobic surface area under some circumstances. For
N
N
NHOH
3
0.440
NA
N
F
F
11
F

Q. Sun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3295–3299
3297
Table 1 (continued)
Table 1 (continued)
Sructure
IC50
(lM)
Sructure
IC50(lM)
HDAC1
HDAC7
NA
HDAC1
HDAC7
O
3
O
3
O
N
N
N
N
NHOH
O
NHOH
0.645
0.252
NA
N
N
F
25
12
O
O
Cl
F
N
N
NHOH
N
N
NHOH
NHOH
3
0.122
NA
0.649
0.862
NA
NA
3
Ph
N
N
O
13
26
O
3
O
O
N
N
N
N
NHOH
0.701
0.202
0.396
0.058
0.107
2.57
NA
NA
NA
NA
NA
NA
NA
NA
3
N
N
O
Br
NC
27
14
O
3
O
3
N
N
N
N
NHOH
NHOH
NHOH
NHOH
NHOH
N
N
Ph
Br
O
0.807
NA
15
N
N
NHOH
O
3
3
N
N
N
28
N
Br
O
3
16
N
NHOH
NA
NA
O
3
N
N
Br
Br
Ph
N
N
29
N
aSAHA was used as a positive control. Values are means of three experiments, and
standard error of the IC50 was generally less than 10%.
17
bNA, no activity observed at 10
l
M concentration tested.
O
N
3
N
N
18
O
3
N
NHOH
O
N
N
N
19
O
Br
OH
N
N
H
NaN3
N
NHOH
R
0.246
1.22
3
N
30
N
31-58
20
O
.
F
O
3
OH
CuSO4 5H2O
N
N
N
H
N
N
NHOH
N
R
H2O / t-BuOH (1/1)
N
66 oC, 27%-68%
2-29
F
21
O
Figure 2. General procedure for the synthesis of compound 2–29.
N
N
NHOH
O
instance, the binding affinity of the resulting compound 18
0.269
0.159
1.90
NA
NA
NA
3
N
(IC50 = 0.246
lM) was decreased when diphenyl was substituted
22
with -2-naphthylalanine (2-Nal). Incorporation of nitrogen into
L
the phenyl ring did not improve the potency of the compound 2.
When the phenyl group was replaced with quinoline ring, the
resulting compound 22 showed IC50 value of 0.246 lM, which
O
N
N
NHOH
3
N
Br
was about 6.8 times more potent than the compound 2. The impact
of the di-substituted or tri-substituted compounds was also inves-
tigated, and the results indicated that the methyl group (22) or
methoxy group (23, 25) caused dramatic increase of the potency
against HDAC1 (21–29). To our surprise, the 2,4,6-tribromo substi-
tuted compound 29 completely lost its inhibitory activities against
HDAC1. Molecular modeling indicates that the ortho-position
substituted bromo atom could not fit within hydrophobic pocket
due to its large size.
23
O
O
O
N
NHOH
3
N
N
24

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Q. Sun et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3295–3299
A
B
C
Figure 3. Plausible binding mode of SAHA, 15 and 17 to HDAC1.
To further investigate the antiproliferative activities of these
derivatives, some of these compounds were selected to test their
effects on the human tumor cell lines, such as Hela (human cervical
cancer cell line), BGC823 (human stomach cancer cell line), A549
(human lung cancer cell line), HT1080 (human sarcoma cell line),
A431 (human epithelial carcinoma cell line), and DU-145 (human
prostate cancer cell line), with SAHA as the positive control. The re-
sults are summarized in table 2. Compounds 7, 9, 13, 15, 17–20, 23
and 25 showed very good GI50 values in the micromolar range
against DU145 cell lines. It is remarkable that compounds 15 and
17, especially in view of their higher HDAC1 activities, have much
better anti-cancer activities than that of SAHA against several can-
cer cell lines. In contrast, compound 3, the poor inhibitor of HDAC1,
shows much weaker activity against those cell lines. Thus, taken
together, the results of the enzyme and cell-based assays verify
the high correlation between the in vitro HDAC1 inhibitory activity
and cellular cytotoxicity of HDAC inhibitors.
against HDAC1. The compond 17 showed promising in vitro anti-
cancer activities against several cancer cell lines. Biological results
demonstrate those compounds which demonstrated a high degree
of HDAC1 inhibition (15, 17 and SAHA) also exhibited high levels of
potency in the cell-based assay. In contrast, the weaker inhibitors
of HDAC1 also generally exhibited reduced potency against cancer
cells. Inhibitory studies on metastatic tumors in animal models are
currently in progress in our laboratory.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation (Grant Nos. 21172220 and 20972160), and the National Ba-
sic Research Program of China (2009CB940900).
Supplementary data
In summary, we have developed one-pot click chemistry ap-
proach to design a series of HDAC inhibitors, which show features
of high potency and selectivity of HDAC1, as well as ability to inhi-
bit several cancer cell growth. We identified a representative lead
from this series, 17, which is several folds more potent than SAHA
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
102.
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1.19
7.23
4.06
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