Design, synthesis and docking studies on benzamide derivatives as histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.06.001

A series of benzamide derivatives including two scaffolds were designed and synthesized as potential histone deacetylase inhibitors. Most of synthesized compounds showed moderate enzymatic potency at the same order of magnitude, and compound 12b possessed better potency to the positive control (3.8 μM vs 13.0 μM). It also showed a 50-fold increase in vitro anticancer activity against DU-145 cell-lines. Molecular docking studies were carried out and used to explain the structure-activity relationships observed in vitro. Then we found that the cavity surrounded by ASP104, HIS33, PRO34 and PHE155 may be crucial for the inhibitors' activity. The docking results provide some useful information for future design of more potent inhibitors.

Full text of DOI:10.1016/j.bmcl.2011.06.001

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4924–4927
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and docking studies on benzamide derivatives as histone
deacetylase inhibitors
⇑
Aijun Lu , Hongpeng Luo, Minfeng Shi, Gang Wu, Yunxia Yuan, Jian Liu, Feng Tang
JiangSu Simcere Pharmaceutical R&D Co., Ltd, and Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18 Xuan Wu Avenue, Nanjing 210042, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzamide derivatives including two scaffolds were designed and synthesized as potential his-
tone deacetylase inhibitors. Most of synthesized compounds showed moderate enzymatic potency at the
Received 24 January 2011
Revised 21 May 2011
Accepted 1 June 2011
Available online 16 June 2011
same order of magnitude, and compound 12b possessed better potency to the positive control (3.8
lM vs
13.0 M). It also showed a 50-fold increase in vitro anticancer activity against DU-145 cell-lines. Molec-
l
ular docking studies were carried out and used to explain the structure–activity relationships observed
in vitro. Then we found that the cavity surrounded by ASP104, HIS33, PRO34 and PHE155 may be crucial
for the inhibitors’ activity. The docking results provide some useful information for future design of more
potent inhibitors.
Keywords:
Histone deacetylase
Inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
Anticancer
Molecular docking
Histone deacetylases (HDACs) and the counteracting histone
acetyl transferases (HATs) play an important role in the regulation
of gene expression. HDACs catalyze the deacetylation of the acety-
in phase II clinical trial. Other companies also pursued new potent
HDAC inhibitors usually by modifying linker or CAP, and preserving
the same ZBG shown in Figure 1. MGCD-0103 and CS-055 are now
both in phase II, and WO2004035525-Ii is the most potent example
in a patent.4 We found that these compounds restricted their link-
ers’ conformation using more rigid fragment compared with
MS-275, which is commonly used in drug design to discover more
potent and specific compound. Among them, MGCD-01035 has a
slight improvement on in vitro activity, and CS-0556 only retains
the similar potency in vitro, but WO2004035525-Ii4 shows reduced
potency. Meanwhile, WO2004035525-Ii possesses the most rigid
linker, so probably it is beneficial for inhibition that the linker
makes a compromise between certain extent rigidity and flexibility.
Based on this, we integrated amide in MS-275 and CS-055, and
phencarbonyl in WO2004035525-Ii into a novel linker (Fig. 1),
together with other portions, to buildup a new chemical scaffold
1. After altering amide of scaffold 1 to sulfonamide, we gained
another chemical scaffold 2.
Then the compounds 11a–e, 12a–c with new chemical scaffolds
mentioned above were prepared as depicted in Scheme 1. The com-
mon intermediate 2-(4-(2-nitrophenylcarbamoyl)phenyl)ethana-
minium chloride 8 was synthesized in four steps.
Using Phosphorus oxychloride and catalytic pyridine, com-
pound 4 was acylated by 3 to provide 5, followed by bromination
to give 6 as yellow powder which was further aminated by methe-
namine to obtain white solid 7, and then hydrolyzed in strongly
acidic conditions to give the common intermediate 8. Compound
8 was dissolved in the mixture of ethyl acetate and potassium car-
bonate solution, and then acylated by various acylchlorides to form
compounds 9a–e and 10a–c which were reduced by iron powder
lated e
-amino groups of specific histone lysine residues,1 and then
the deacetylated histones acquire a net positive charge. Because
deacetylated histones interact strongly with the negatively
charged DNA, DNA becomes tightly wrapped around the nucleo-
some core, and then gene expression is impeded. That is to say, in-
creased levels of histone acetylation are associated with increased
transcriptional activity, whereas decreased levels of acetylation are
associated with repression of gene expression.2 Association be-
tween acetylation and gene expression has stimulated the study
of HDACs on the aberrant gene expression, which is often observed
in cancer. Many studies show that inhibition of HDAC elicits anti-
cancer activities in several tumors cell lines,3 which make HDAC an
attractive target for the development of new anticancer drugs.
More than 10 HDAC inhibitors are advancing in different clinical
trial phases. Currently, several HDAC inhibitors have been approved
by FDA, such as SAHA and FK-228 for the treatment of cutaneous T-
cell lymphoma. In addition, HDAC inhibitor’s pharmacophore is
characterized by three portions: a zinc-binding group (ZBG), a
hydrophobic group (CAP) for protein surface recognition, and a lin-
ker between ZBG and CAP as exemplified by MS-275 (Fig. 1). MS-
275 uses N-(2-aminophenyl) formamide as ZBG for the interaction
with the catalytic Zn in HDACs’ active site. MS-275 is a moderately
potent HDAC inhibitor with micromole enzymatic activity, which is
⇑
Corresponding author. Tel.: +86 25 85560000 3145.
E-mail addresses: luaijun@simcere.com, luaj520@gmail.com (A. Lu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.001

A. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4924–4927
4925
Figure 1. Known HDAC inhibitors classified as benzamides and their novel scaffolds.
Scheme 1. Reagents and conditions: POCl3, Py, DCM, room temp (70%); (b) Br2, DCM, room temp (74%); (c) C6H12N4, DCM, 30 °C (93%); (d) HCl, MeOH; (e) R1ArXCl, EA, H2O,
K2CO3, rt À40 °C (37–86%); (f) Fe, HAc, EtOH, H2O, 70 °C (20–75%).
in acid condition to provide target compounds 11a–e and 12a–c,
respectively.
all compounds bearing sulfonamide group showed higher activity
than those with scaffold 1. Though compound 12a is the least po-
tent inhibitor, it has showed comparative potency to MS-275
The synthesized compounds were tested in enzyme assay for
HDAC inhibition.7 The results are summarized in Table 1. As for
chemical scaffold 1, the first notable observation was lack of inhi-
bition activity for compound with naked phenyl CAP (11a). After
being substituted by amino regardless in meta- or para-position
of phenyl, the analogues showed improved activity. Meanwhile,
compounds containing pyridine (11b, 11c) showed certain level
of HDAC inhibitory effects, but compound substituted by amino
showed no improvement, not behaving as compounds bearing
with phenyl CAP (11d, 11e). Obviously, pyridine CAP was better
than phenyl CAP for HDAC inhibition, which was consistent with
the trend found in MS-275 analogs.8 As for chemical scaffold 2,
(22.4
group in compound 12a, compound 12b dramatically improved
its potency to 3.8 M, which showed 6-fold increase to compound
lM vs 13 lM). using amino group replacing para-methyl
l
12a, and 4-fold increase to MS-275. Compound 12c with halogen
substituted on CAP showed only similar potency to MS-275, which
resulted in a 3-fold loss of activity compared to compound 12b.
Pyridine based inhibitor Compound 12c was 2-fold more active
than compound 12a, indicating again that pyridine CAP was better
than phenyl CAP for HDAC inhibition. However it was 3-fold less
active than compound 12b, suggesting that p-NH2 substituent is
the most favored.

4926
A. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4924–4927
Table 1
In vitro HDAC enzyme, cell line assay of selected compounds
Ar
R
Hdac (
lM)
Cell (lM)
HCT-116
MDA-MB-231
DU-145
A2780
11a
11b
11c
11d
11e
12a
12b
–Phenyl
H
H
>100
30.2
42.6
70.7
50.2
22.4
3.8
Pyridine-2-yl
pyridine-4-yl
–Phenyl
–Phenyl
–Phenyl
2-NH2
m-NH2
p-NH2
p-Me
p-NH2
2-Cl
18.7
NTb
18.4
NT
37.7
0.16
>100
5.7
–Phenyl
pyridine-3-yl
12c
11.1
13.0
MS-275a
3.1
2.8
7.9
5.7
a
IC50 value obtained for MS-275 under our experimental conditions.
Not tested.
b
Figure 2. Docked compounds and MS-275 in the active site of HDAC2. (a) Compound 12b (depicted by ball and stick, H-bonds as yellow dotted lines) and MS-275 (cyan) in
HDAC2 active site, the surface is created in SYBYL. (b) The interaction between compound 11a (violet), 11c (light blue), 12a (maroon), 12b (yellow), 12c (cyan) and HDAC2.
The Zn is represented by a CPK sphere; The picture is prepared in Benchware 3D Explorer 2.3 (Tripos, St. Louis, MO).
Based on the HDAC potency, compounds 12a–b were selected for
anticancer activity test in vitro cancer cell-line assay based on cell
viability using dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT)9, and the results are presented in Table 1.
Compound 12a showed moderate potency against 3 cell lines
(HCT-116, MDA-MB-231 and DU-145), with growth inhibitions of
After running Surflex–Dock, the scores of 10 docked conformers
were ranked in a molecular spread sheet. Because of being
positioned outside the cavity, CAP of each conformer has certain
flexibility on the surface of the protein. We selected the conformer
with highest score for speculating the detailed binding patterns. As
we expected, MS-275 was docked into the binding site perfectly,
using the 3-methylpyridine moiety to occupy the small groove
(Fig. 2a) which is surrounded by ASP104, HIS33, PRO34 and
PHE155 (labeled in Fig. 2b). At the same time, compounds 12a–c
also were well accommodated within this cavity, with almost iden-
tical conformations and orientations (Fig 2b). From the HDLP co-
crystal structures15 (1C3S and 1C3R), we found that TSA and SAHA
also occupied the corresponding site which is in the vicinity of
ASP92, HIS21, PRO22 and PHE144. It is most likely that occupying
this site can improve inhibitors’ enzyme activity. Furthermore, the
para-amino on CAP make a comfortable hydrogen bond to Glu103
surrounding the cavity (Fig. 2), which amazingly enhances potency
of compound 12b. The N-(2-aminophenyl) formamide fragment of
each compound acting as ZBG group aligns very well with the one
on the native ligand in this co-crystal complex. Meanwhile, it
forms two H-bonds with protein involved in HIS145 and Gly154
using ortho-NH2 group and amide group (Fig. 2b), whereas the
nitrogen of ortho-NH2 group and the carbonyl oxygen chelate the
zinc (Fig. 2b), which is consistent with previous report.13 In
accord with literature precedent for aryl sulfonamides16, both the
nitrogen lone pair and the carbon aromatic p-orbital bisect the
18.7, 18.4, and 37.7
The result of compound 12b against DU-145 cell was very exciting,
with 50-fold improvement compared to MS-275 (0.16 vs 7.9 M). At
the same time, it showed identical anti-proliferative effects against
A2780 compared to MS-275 (5.7 M).
lM, which is one-fifth cell potency of MS-275.
l
l
In order to study the differences in potency of these com-
pounds, their binding mode in the catalytic site of the HDAC was
studied by molecular modeling. It is reported that benzamide com-
pounds such as MS-275 and MGCD0103 appear to be HDAC-class-
I-specific HDAC inhibitors10, which show HDAC1-HDAC3 inhibition
activity from 10À8 to 10À6 M. In HDAC class I, three members
(HDAC1-HDAC3) show high sequence homology. Human HDAC1
and HDAC2 proteins share 85% identity11, while human HDAC2
and HDAC3 share 52% identity12, so it is reasonable to use human
HDAC2 crystal structure as a representative template to investigate
the binding mode of these benzamide compounds. Here, we devel-
oped a model for the active site of HDAC2, based on the recently
published structure of a benzamide inhibitor/HDAC2 complex
(PDB code: 3max).13 All compounds were prepared using the li-
gand in crystal structure as template, and then minimized in SYBYL
environment. After the protein was prepared and the protomol file
was generated, all compounds were docked into the binding site
using Surflex-dock in SYBYL7.3.14
O@S@O angle resulting in
a conformation that places the
aryl-sulfonyl torsion angle at 90°,which allows the CAP groups of
compounds 12a–c being positioned into the cavity mentioned

A. Lu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4924–4927
4927
above. This minimum energy conformation is not readily accessi-
References and notes
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(Fig. 2b), and therefore prevents the CAP group from getting into
the cavity (compound 11a, Fig. 2b). This explains the lower
potency of these analogs. Because of the H-bond interaction be-
tween amide group in CAP and ASP104 in protein, CAP of com-
pound 11c can move close into the cavity mentioned above, but
not deeper than compounds 12a–c, which improves its potency
but still weaker than compounds with scaffold 2.
In conclusion, a series of benzamides including two scaffolds
were designed, synthesized and evaluated for their inhibition of
HDAC. According to the HDAC potency, compounds 12a–b were se-
lected for anticancer activity test. Compound 12a showed moderate
potency against 3 cell lines, while the most potent compound 12b,
which showed a 4-fold increase enzymatic potency to MS-275,
exhibited significant anticancer effect, particularly 50-fold
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Acknowledgments
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This research was supported by the Natural Science Foundation
of Jiangsu Province of China (BK2008514).
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We thank Dr. Huan Ming Chen, Institute of Discovery Chemis-
try, JiangSu Simcere Pharmaceutical R&D Co., Ltd, for his manu-
script reading.