Design, synthesis and bioevalution of novel benzamides derivatives as HDAC inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.114

A series of novel benzamides derivatives was designed and synthesized as HDAC inhibitors. Exploration of the structure-activity relationships resulted in compounds that are potent in vitro. In addition, the best compound 1a exhibited an acceptable pharmacokinetic profile with bioavailability in rat of 81% and could be considered as a candidate compound for further development.

Full text of DOI:10.1016/j.bmcl.2012.10.114

Bioorganic & Medicinal Chemistry Letters 23 (2013) 179–182
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and bioevalution of novel benzamides derivatives
as HDAC inhibitors
Yanyang Li a,b, Yan Zhou b, Pengyu Qian c, Yongzhen Wang b, Falong Jiang b, Zhenglin Yao b,
Wenxiang Hu d, Yanjin Zhao b, Shuxin Li b,c,
⇑
a The General Hospital of Chinese People’s Liberation Army, Beijing 100853, China
b Institute of Radiation and Irradiation Medicine, Academy of Military Medical Science, Beijing 100850, China
c Department of Medicinal Chemistry, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China
d College of Life Science, Department of Chemistry, Capital Normal University, Beijing 100084, 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 novel benzamides derivatives was designed and synthesized as HDAC inhibitors. Exploration
of the structure–activity relationships resulted in compounds that are potent in vitro. In addition, the best
compound 1a exhibited an acceptable pharmacokinetic profile with bioavailability in rat of 81% and
could be considered as a candidate compound for further development.
Received 10 September 2012
Revised 25 October 2012
Accepted 29 October 2012
Available online 5 November 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Benzamide derivatives
HDAC
Antiproliferative activity
Bioavailability
O
H
N
OH
N
H
O
Histones deacetylases (HDACs) are a family of enzymes that cat-
alyze the deacetylation of lysine residues located in the NH2 termi-
nal tails of core histones.1 They play an important role in epigenetic
regulation of gene transcription and also control other cellular
functions, such as proliferation, cell death, and motility.2–4 There-
fore, HDACs are regarded as promising targets for cancer therapy.
There are currently 18 known mammalian deacetylase enzymes
which can be divided into two major groups: Zn2+-dependent
HDACs and II AD+-dependent sirtuins. The enzymes of class I
(HDACs 1–3 and 8), II (HDACs 4–7, 9 and 10) and IV (HDAC 11)
are all Zn2+-dependent metalloenzymes, and the class III HDACs
(sirtuins 1–7) are NAD+-dependent.5,6 Indeed, only Zn2+-dependent
HDACs, especially class I and class II isozymes are involved in pro-
moting tumor cells proliferation, angiogenesis and differentiation.7
Currently, several HDAC inhibitors (HDACi) have entered
clinical trials as anticancer treatments.8 The nonselective HDAC
inhibitors (pan-inhibitors), which have nanomolar and submi-
cromolar IC50s, include hydroxamic acid inhibitors, such as SAHA
(Vorinostat), or cyclic tetrapeptides (Romidepsin or depsipep-
tide).9,10 A benzamide HDAC inhibitor, MS-275, is reported to be
a selective HDAC inhibitor targeting class I HDACs.11,12 Although
there is still some argument, in general, pan-inhibitors may have
more side effects, a relatively selective profile would be preferred
(SAHA, ZolinzaTM
)
ZBG
Linker
O
Cap
O
N
H
NH2
H
N
N
O
(MS-275)
N
N
N
H
NH2
H
N
N
O
(MGCD0103)
X
N
Y
R1
N
R2
N
N
N
NH2
n
H
H
N
O
R3
R4
target compounds
⇑
Corresponding author. Tel./fax: +86 10 68214653.
E-mail address: lisx28@163.com (S. Li).
Figure 1. Structure of HDAC inhibitors: SAHA, MS-275, MGCD0103 and target
compounds.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.114

180
Y. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 179–182
X
X
X
N
N
N
Y
Y
Y
R1
R1
R1
N
N
N
R2
R2
b
R2
a
O
O
N
N
N
H
n
N
OH
1
2
3
O
X
N
Y
R1
N
c
N
N
N
H
NH2
n
H
N
O
1a-c
2a-c
3a
3b
N
d
N
N
N
N
N
H
NH2
H
N
4a
O
O
N
F
N
e
N
N
H
NH2
H
N
5a
F
N
N
f
Br
N
N
N
H
6a
NH2
H
N
O
1
1
1
3a:
3b:
1a:
1b:
2a:
X=H, Y=H, R =Me, n=2
X=H, Y=H, R =H, n=1
X=H, Y=H, R =Me, n=1
1
1
1
2b:
X=N, Y=H, R =H, n=2
X=N, Y=H, R =H, n=1
X=N, Y=H, R =Me, n=1
1c: X=H, Y=N, R1=H, n=1
2c: X=H, Y=N, R1=Me, n=1
Scheme 1. Reagent and conditions: (a) DMFDMA, reflux; (b) n-BuOH, K2CO3; (c) EDCI, HOBT, THF, TEA; (d) (1) EDCI, HOBT; (2) HCl; (e) CDI, CF3COOH; (f) EDCI, HOBT, THF,
TEA.
for the frequent of chronic diseases. A great number of HDAC inhib-
itors under development have a common general pharmacophore,
which consisting of a ‘capping region’ linked to a ‘zinc binding
group (ZBG)’ via a lipophilic linker (Fig. 1).13 MS-275 used N-(2-
aminophenyl) formamide as ZBG for the interaction with the
catalytic Zn2+ in HDACs’ active site, which is a moderately potent
HDAC inhibitor in phase II clinical trial. Although some benzamides
derivatives have developed and modified, many compounds re-
stricted the linkers’ part using more rigid group compared with
MS-275. Among them, MGCD0103 has a slight improvement
in vitro activity, but it is unfavorable.14 In addition, although the
oral bioavailability of MGCD0103 was 12% in mice and 47% in rats,
the coefficient of variation was 1–92% in dogs.15 Thus, there
remains a need to develop novel HDAC inhibitors with higher anti-
tumor activities and favorable pharmacokinetic profile.
activities in vitro. Bicyclic heterocyclic compounds are known
and widely used in medicinal chemistry and chemical biology
research for its unique physiochemical property and excellent
biocompatibility. In addition, bicyclic heterocycles and its
substituents were found to have significant effects on the pharma-
cokinetic properties and physicochemical properties of some
inhibitors. Therefore, we decided to develop a ligand-based phar-
macophore based on a set of active compounds by utilizing bicyclic
heterocycles as the group of capping region in our approach.
Based on the HDACi pharmacophore and our previous study, we
designed and synthesized a series of novel benzamides derivatives
as histone deacetylase inhibitors (Scheme 1).
All compounds with new chemical scaffolds mentioned above
were prepared as depicted in Scheme 1. Synthesis of compound
2 was obtained by refluxing intermediate 1 with DMFDMA. The
key intermediates 3 can be synthesized by reacting 2 with 4-(guan-
idinomethyl) benzoic acid or 4-(guanidinoethyl) benzoic acid in
n-BuOH. Compounds 1a–c, 2a–c, 3a, 3b and 6a were synthesized
The major objectives of our discovery efforts toward therapeu-
tically useful HDAC inhibitors were to achieve sufficient oral expo-
sure in vivo for bioevaluation and highly potent biological

Y. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 179–182
181
Table 1
Apparently, all the tested compounds exhibited a reasonable
inhibitory potency against HDAC1 protein and more or similar
potent antiproliferative activities against all cell lines in compari-
son with MGCD0103. Moreover, the best compound 1a (with IC50
Enzymatic and antiproliferative potency of the synthesized compounds
N
Ar
N
N
H
NH2
n
values = 0.082, 0.661 and 0.110
activities to MGCD0103 (with IC50 values = 0.327, 4.807 and
1.279 M) in HCT-116, MCF-7 and A549. Obviously, most of the
lM) exhibited superior antitumor
H
N
l
O
R3
prepared compounds exhibited superior activities against A549
and HCT-116 to MCF-7. These compounds provide valuable infor-
mation, which reflects selective behavior of the target compounds
to some extent. We hypothesized that the enhanced potency could
arise from a series of contributing factors, including size, electronic
property, and hydrogen-bond forming capability.
R4
Compound
IC50
(
l
M)a
MCF-7
HDAC1
HCT-116
A549
1a
1b
1c
2a
2b
2c
3a
3b
4a
5a
6a
0.053 0.011 0.082 0.013 0.661 0.156 0.110 0.016
0.035 0.012 0.183 0.021 0.856 0.271 0.427 0.054
0.110 0.037 0.214 0.086 2.178 0.801 0.509 0.163
0.108 0.014 0.203 0.011 2.088 0.626 0.439 0.077
0.127 0.025 0.189 0.027 4.111 1.186 0.857 0.104
0.097 0.021 0.264 0.015 3.741 1.265 0.694 0.169
0.131 0.013 0.304 0.048 4.119 0.579 0.851 0.033
0.154 0.012 0.256 0.062 1.723 0.893 0.724 0.012
0.097 0.026 0.270 0.081 1.004 0.265 0.176 0.011
0.135 0.016 0.244 0.060 2.400 0.934 0.649 0.035
0.125 0.024 0.301 0.017 2.359 0.148 0.501 0.047
An electron withdrawing group like fluoro atom was introduced
into the benzamide ring for investigation of structure–activity rela-
tionships (SAR). Compounds bearing a fluoro group at the para or
meta position of phenyl ring exhibited reasonable antitumor activ-
ities against the three cell lines (4a, 5a), which indicates that the
fluoro group rarely affects the antitumor activities. Systematic
evaluation of longer linker indicted that trifluoromethyl led to low-
er antitumor activities, suggesting the importance of the appropri-
ate linker in the inhibitors. For example, analogues 3a and 3b
which contained a longer linker exhibited weaker antitumor activ-
ities compared to analogues 2a and 1b. The analogues with an
appropriate length chain linker showed more HDAC inhibitory
and antiproliferative activities, because the appropriate chain
linker provides proper fitting into the active site of HDAC.17 All pre-
pared compounds exhibited higher or similar HDAC inhibition
against human cancer cell lines and HDAC1 inhibition than
MGCD0103 did, which confirmed that bicyclic heterocycles can
be utilized as the group of capping region. These compounds have
higher inhibition on HDAC1 than MGCD0103, which indicate the
introduction of the bicyclic heterocycles has higher affinity for
HDAC1 protein, improve HDAC inhibitory activity. Moreover, com-
pounds devoid of methyl on the bicyclic heterocycles (compounds
1a–c) exhibit higher antitumor activities and higher HDAC1 inhib-
itory potency. We hypothesized that the enhanced potency could
arise from a number of contributing factors, including differences
MGCD0103 0.184 0.023 0.327 0.026 4.807 0.181 1.279 0.054
a
Results are expressed as means SD (standard deviation) of three independent
experiments.
by acylation reaction of the intermediate 3 with benzene-1,2-dia-
mine, EDCI and HOBt in dry THF. Under similar condition reaction,
compound 4a was synthesized. Compound 5a was synthesized
utilizing intermediate 3 and 4-fluorobenzene-1,2-diamine in the
conventional way.
These prepared compounds were tested for their ability to inhi-
bit histone deacetylase activity of HDAC1 activity and antiprolifer-
ative activities by MTT assays,15 using MGCD0103 as the reference
standard. The HDAC1 protein was chosen since it is widely impli-
cated in both transcriptional repression and chromatin remodel-
ing. HCT116, MCF-7 and A549 cancer cell lines were chosen for
MTT assays and the IC50 values were summarized in Table 1.16
Imidazo[1,2-a]pyridine
favorable
X
N
Y
R1
N
R2
Amide moiety required
N
Amino group required
N
N
NH2
n
H
H
N
O
R3
F group rarely affects activities
R4
CH2-Group favorable
Ring substitution detrimental
Figure 2. Structure–activity relationships of prepared compounds.
Table 2
Pharmacokinetic parameter of compound 1a in ratsa
Compound
Dose iv (mg/Kg)
5
Dose po (mg/Kg)
25
T1/2, iv (h)
T1/2, po (h)
Tmax, po (h)
AUClast, po (hÁng/ml)
71850.04
AUCINF_obs, po (hÁng/ml)
72318.28
F (%)
81
1a
1.73
4.58
0.083
a
The dosing vehicles in rats were 0.1 N HCl in saline (both iv and po).

182
Y. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 179–182
in conformational energetic, parameters relating to exposed and
buried surface area, and hydrogen-bonding capabilities. Struc-
ture–activity relationships (SAR) of prepared compounds were
thoroughly investigated2,18 and summarized in Figure 2. For in-
stance, pharmacokinetic studies of orally administered 1a were
carried out in female SD rats (Table 2).19 The compound was
quickly absorbed, with a short Tmax across tested and the terminal
phase elimination half-life for 1a administered iv was 1.73 h in
rats. The results indicated that these novel bicyclic heterocyclic
compounds can be further investigated as HDAC inhibitors. The re-
sults have demonstrated our idea which will lead the design efforts
of novel structures.
In the case of pharmacokinetics, the best compound 1a dis-
played better bioavailability than MGCD0103,14 which suggesting
that bicyclic heterocycles can improve the compound generation
properties and the in vivo antitumor assay is currently under
investigation in our group.
2012ZX09102101-013) and National Natural Science Foundation
of China (Grant No. 30772628).
References and notes
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In conclusion, benzamides derivatives presented here are po-
tent HDAC inhibitors, which have been prepared and assessed for
their antiproliferative activities against HCT116, MCF-7 and A549
human cancer cell lines and against HDAC1 enzyme. All com-
pounds exhibited highly potent HDAC inhibitory activity and
antitumor activities, while the most potent compound 1a, which
showed a threefold increase in enzymatic potency to MGCD0103,
exhibit significant anticancer effect, particularly sevenfold
enhancement compared to MGCD0103 against MCF-7 and A549
human tumor cell lines. In addition, pharmacokinetic studies of or-
ally administered 1a showed that it has sufficient oral exposure
in vivo and it could be considered as a candidate compound for
further development. Moreover, we have also observed the mul-
ti-fold decrease in activities with methyl on the 2 substituted bicy-
clic heterocycles. The results suggested that bicyclic heterocycles
could be taken as the capping region and offered us much useful
information to lead future anticancer design efforts targeting
HDAC proteins. The in vivo anticancer activities and detailed SAR
of the compounds are currently under investigation in our labora-
tory and the results of such studies will be reported in due course.
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15. MTT assay: HCT-116, A549 and MCF-7 were cultured in RPMI-1640 medium,
supplemented with 10% fetal calf serum (FCS), and maintained in a 5% CO2, 95%
humidity atmosphere at 37 °C. In 96-well plates were seeded 5.0 Â 103 cells/
well and incubated for 24 h. The cells were then incubated for another 72 h
with various concentrations of papered compounds. Subsequently, 20 lL of
fresh MTT solution (5 mg/ml) was added to each well and incubated with cells
at 37 °C for additional 4 h. The supernatant was carefully removed from each
well and 100 lL of DMSO was added to each well to dissolve the formazan
crystals which were formed by the cellular reduction of MTT. After mixing with
a mechanical plate mixer, the absorbance of each well was measured by a plate
reader at a test wavelength of 570 nm. IC50 data were calculated by linear
regression analysis as described above.
16. The human cancer cell lines, HCT-116 (colon carcinoma), MCF-7 (breast
carcinoma) and A549 (non-small cell lung carcinoma) were from American
Type Culture Collection.
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19. Pharmacokinetic study: The evaluation of compound 1a were carried out using
female Sprague Dawley rats after single iv (5 mg/kg) and po (25 mg/kg)
administration. Blood was collected from rats at various time points, and
plasma samples were analyzed using an Agilent 1200 HPLC system coupled
with Agilent 6410 B triple quadruple mass spectrometer.
Acknowledgments
We gratefully acknowledge the financial support for National
Science and Technology Major Project (2009ZX09103017 and