Development of novel ferulic acid derivatives as potent histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.09.021

Histone deacetylase inhibitors (HDACIs) offer a promising strategy for cancer therapy. The discovery of potent ferulic acid-based HDACIs with hydroxamic acid or 2-aminobenzamide group as zinc binding group was reported. The halogeno-acetanilide was introduced as novel surface recognition moiety (SRM). The majority of title compounds displayed potent HDAC inhibitory activity. In particular, FA6 and FA16 exhibited significant enzymatic inhibitory activities, with IC₅₀ values of 3.94 and 2.82 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against a panel of human cancer cells. FA17 displayed promising profile as an antitumor candidate. The results indicated that these ferulic acid derivatives could serve as promising lead compounds for further optimization.

Full text of DOI:10.1016/j.bmc.2013.09.021

Bioorganic & Medicinal Chemistry 21 (2013) 6973–6980
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel ferulic acid derivatives as potent histone
deacetylase inhibitors
⇑
Fang Wang , Wen Lu , Tao Zhang, Jinyun Dong, Hongping Gao, Pengfei Li, Sicen Wang, Jie Zhang
School of Medicine, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylase inhibitors (HDACIs) offer a promising strategy for cancer therapy. The discovery of
potent ferulic acid-based HDACIs with hydroxamic acid or 2-aminobenzamide group as zinc binding
group was reported. The halogeno-acetanilide was introduced as novel surface recognition moiety
(SRM). The majority of title compounds displayed potent HDAC inhibitory activity. In particular, FA6
Received 15 August 2013
Revised 7 September 2013
Accepted 7 September 2013
Available online 19 September 2013
and FA16 exhibited significant enzymatic inhibitory activities, with IC50 values of 3.94 and 2.82 lM,
respectively. Furthermore, these compounds showed moderate antiproliferative activity against a panel
of human cancer cells. FA17 displayed promising profile as an antitumor candidate. The results indicated
that these ferulic acid derivatives could serve as promising lead compounds for further optimization.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase
HDAC inhibitor
Ferulic acid
Anticancer
1. Introduction
all shared common pharmacophore composed of four portions:
(a) zinc binding group (ZBG), which chelates zinc ion at the bottom
Histone deacetylases (HDACs) are zinc metalloenzyme which
remove acetyl group of lysine residues located on nucleosomal his-
tones. Histone acetylation and deacetylation are essential in mod-
ulation of chromatin topology and gene transcription. HDACs are
critical in the epigenetic regulation of gene expression.1 They could
cause condensation of chromatin resulting in transcriptional
repression. Therefore, HDACs play an essential role in cell prolifer-
ation, cell-cycle regulation and apoptosis.2 Aberrant activity of
HDACs has been found in several human cancers leading to devel-
opment of histone deacetylase inhibitors (HDACIs). As clinically
validated cancer targets, their inhibition has been proven to be suc-
cessful strategy for the development of novel anticancer agents.3
Moreover, HDACIs exhibit attractive antitumor properties by
inducing transcriptional events involved in growth arrest, cell pro-
liferation, and apoptosis.4 There are numbers of HDACIs emerging
as an exciting novel class of antitumor agents (Fig. 1). Suberoylan-
ilide hydroxamic acid (Vorinostat, SAHA) is the first HDACI ap-
proved by FDA in 2006. Belinostat and Panobinostat are also
hydroxamate HDACIs which induce acetylation of histone at nano-
molar concentrations. Entinostat is an oral benzamide HDACI with
limited cardiac toxicity in preclinical studies.5
of pocket, (b) linker (scaffold), usually hydrophobic which occupies
the narrow channel, (c) connect unit (CU), which connects SRM and
linker, (d) surface recognition moiety (SRM), which interacts with
residues on the rim of active site (Fig. 1). The common linkers
are aliphatic chain, aromatic chain and vinyl-aromatic chain. The
most common ZBGs are hydroxamic acid and 2-aminobenzamide.7
In the past two decades, a number of HDACIs have been developed
by modifying of SRM, linker and ZBG. However, recent studies have
focused on varying SRM or linker portion. Based on the common
pharmacophore, we designed and synthesized a series of ferulic
acid-based HDACIs.
Ferulic acid (Fig. 2) is a natural product isolated from many sta-
ple foods, including fruits, vegetables, cereals, and coffee.8 Ferulic
acid and its derivatives displayed broad range of therapeutic ef-
fects, with applications including anticancer, antidiabetic, cardio
protective, neuroprotective, and antiinflammatory activity. Herein,
we introduced rigid ferulic acid as linker of HDACIs.
In our earlier work, structural optimization of natural alkaloid
taspine afforded a novel antitumor agent (HMQ1611, Fig. 2).9 It
displayed antiproliferative activity against several human cancer
cell lines. Moreover, numbers of acetanilides with halogen substit-
uents were prepared and exhibited potent anticancer activity.10
We supposed that halogeno-acetanilide might be suitable as SRM
of HDACIs. We focused our attention on searching for novel ferulic
acid derivatives with halogeno-acetanilide as SRM. It provides
opportunities to develop potent HDACIs. These novel HDACIs com-
prising common hydroxamic acid or 2-aminobenzamide group as
ZBG (Fig. 2).
HDACIs are classified into different classes depending on their
structures namely aliphatic acids, hydroxamic acids, 2-aminoani-
lides, cyclic peptides and electrophilic ketones.6 These structures
⇑
Corresponding author. Tel./fax: +86 29 82655451.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
Both authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.021

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F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
Figure 1. Structures and pharmacophore features of HDACIs.
Figure 2. Design strategy and structures of target compounds.
As part of our ongoing effort to develop antitumor agents, we
developed in 4-step reaction sequence (Scheme 1). Ferulic acid
was esterified in the presence of concentrated H2SO4 to afford feru-
lic acid methylester 2. Various substituted anilines were acylated
with chloroacetyl chloride in polar solvent to provide intermedi-
ates 3a–3i. The hydroxyl group in 2 was etherified with haloacy-
lanilines 3a–3i in anhydrous acetone in the presence of K2CO3 to
afford corresponding intermediates 4a–4i.11 The resulting esters
4a–4i were treated with methanolic NH2OK at room temperature
to yield corresponding hydroxamic acid derivatives FA1–FA9.12
Scheme 2 exhibited synthetic route of ferulic acid derivatives
bearing 2-aminobenzamide. Ferulic acid was converted into its
imidazolide derivative by reaction with N,N0-carbonyldiimidazole
developed two series of ferulic acid derivatives bearing halogeno-
acetanilide as SRM. Various anilines were used to investigate the
role of R1 substituent. The structures of these compounds are quite
consistent with common pharmacophore of HDACIs (Fig. 2). The
binding mode of the two most potent compounds with HDAC
was also established.
2. Chemistry
All the title compounds were prepared from commercial avail-
able ferulic acid. An efficient synthesis of hydroxamic acids was
O
O
O
O
a
O
O
OH
O
O
O
O
O
OH
N
H
O
HO
HO
d
c
H
N
H
N
R1
R1
2
H
N
H2N
Cl
Cl
O
O
b
R1
Cl
+
R1
O
4a-4i
FA1-FA9
O
3a-3i
Scheme 1. Preparation of compounds FA1–FA9. Reagents and conditions: (a) CH3OH, H2SO4; (b) CH2Cl2, Et3N; (c) K2CO3, acetone; (d) NH2OK, NH2OH, DMF.

F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
6975
(CDI) in THF at room temperature. This was further reacted with
benzene-1,2-diamine or 4-methylbenzene-1,2-diamine in the
presence of trifluoroacetic acid to afford key intermediates 5a–
5b.13 The hydroxyl group in 5a–5b was etherified with haloacylan-
ilines 3a–3i in anhydrous acetone in the presence of K2CO3 to af-
ford corresponding 2-aminobenzamide-containing derivatives
FA10–FA27.14
interaction might contribute to affinity and activity of benzamides.
The distance between zinc ion and oxygen atoms was 3.30 Å while
distance with nitrogen of amino was 2.49 Å. The results indicated
that these two compounds had similar binding mode and orienta-
tion as APHA in active site of HDAC. Both of them did not only che-
late zinc ion but also form four hydrogen bonds with His142,
His143 and Tyr306. In summary, docking results suggested that
FA6 and FA16 fit comfortably inside active site of HDAC.
3. Results and discussion
4. Conclusion
These compounds were initially evaluated for their inhibitory
activity against HDAC with SAHA as positive control. From the re-
sults listed in Table 1, majority of them displayed moderate to high
inhibitory activity against HDAC. FA6 was the most potent in
In conclusion, twenty-seven ferulic acid derivates with HDAC
inhibitory property and antiproliferative activity were developed.
These ferulic acid-based HDACIs comprised hydroxamic acid or
2-aminobenzamide group as ZBG. Ferulic acid was served as
molecular scaffold and rigid linker. Both series displayed moderate
to potent HDAC inhibitory activity, together with good levels of
antiproliferative activity on several cancer cell lines. Benzamides
were somewhat more potent than hydroxamates. Eight com-
pounds (FA5, FA6, FA7, FA12, FA13, FA16, FA17, FA27) displayed
significant enzymatic inhibitory activities, with IC50 values ranging
hydroxamate series with an IC50 value of 3.94
FA7 showed moderate inhibitory activities with IC50 values of
8.43 and 9.47 M, respectively. The positions of halogen-substitu-
lM, while FA5 and
l
tion on aniline may be critical for activity. Benzamide series were
afforded by introduce of 2-aminobenzamide as ZBG. Six com-
pounds of them exhibited promising HDAC inhibitory activity.
FA16 displayed the highest inhibitory activity with IC50 value of
2.82
lM.
from 2.82 lM to 10.3 lM. Among them, FA16 exhibited potency
Two compounds (FA6 and FA16) exhibited promising activity
comparable to that of SAHA. According to the results, both substi-
tution on aniline and structure of ZBG played important role in po-
tency. It was indicated that compounds bearing substituents like
fluorine, bromine and trifluoromethyl on aniline possessed potent
anticancer activity.
comparable with SAHA. Moreover, FA17 bearing 2-aminobenzam-
ide exhibited potent antiproliferative activities against MCF-7,
MDA-MB-231 and HeLa cell lines. In summary, these ferulic acid
derivatives had strong potential to be further developed as novel
HDACIs. Further structural optimization of these promising anti-
cancer agents will be reported in due course.
To further investigate their antiproliferative activity, eight com-
pounds were selected to test their anticancer potential by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method. The results were described in Table 2. It was found that
majority of them displayed potent antiproliferative activities with
5. Experimental
5.1. Chemistry: general procedure
IC50 values ranging from 0.76 to 100 lM. Some of them exhibited
Solvents and reagents were purified according to standard pro-
cedure. All reactions except those in aqueous media were carried
out by standard techniques for exclusion of moisture. Anhydrous
reactions were carried out under nitrogen atmosphere. Reactions
were monitored by thin layer chromatography on 0.25-mm silica
gel plates (60GF-254) and visualized with UV light. Melting points
were determined on electrothermal meliting point apparatus and
are uncorrected. 1H NMR spectra were measured at 400 MHz on
Bruker Advance AC 400 instrument. Mass spectra were obtained
on Shimadzu HPLC-MS-QP2010 instrument.
promising antiproliferative activity against MDA-MB-231. FA5
and FA7 showed potency comparable with SAHA in term of cancer
cell growth inhibition.
Docking studies were carried out to understand interaction be-
tween inhibitors and HDAC. The most potent HDACIs in two series,
FA6 and FA16, were docked into active site of HDAC (PDB ID:
3F07). Molecular insights based on molecular docking indicated
favorable binding models of them with HDAC (Fig. 3). The results
suggested that hydroxamic group and 2-aminobenzamide group
were bonded to zinc ion as ZBG. For FA6 (left), hydroxamate OH
made two hydrogen bond interactions with His142 and His143
with distance of 2.12 Å and 2.71 Å, respectively. N–H could also
bind to His143 with distance of 1.79 Å. Carbonyl group accepted
a hydrogen bond from Tyr306 with distance of 1.82 Å. The distance
between zinc ion and two oxygen atoms were 1.88 Å and 3.08 Å,
respectively.15 For FA16 (right), binding mode was consistent with
FA6. One hydrogen atom of 2-amine formed two hydrogen bonds
with His143 with distances of 1.90 Å and 2.74 Å, respectively. An-
other hydrogen atom could also bind to His143 with distance of
2.13 Å. Carbonyl group was identified to be involved in hydrogen
bond with Tyr306 with distance of 2.07 Å. There was an additional
hydrogen bond between oxygen atom of acetyl and Lys202. This
5.1.1. Methyl (2E)-3-(4-hydroxy-3-methoxyphenyl)acrylate (2)16
Ferulic acid (6.80 g, 35 mmol) was dissolved in methanol
(80 mL) containing catalytic of 2 mL concentrated H2SO4, and then
solution was heated at reflux for about 4 h. After cooling at room
temperature, solvent was evaporated under vacuum. The residue
was taken up in EtOAc (150 mL) and washed with saturated aque-
ous solution of NaHCO3 until neutral pH. The organic layer was
then washed with distilled water and dried over anhydrous Na2-
SO4. The solvent was removed under vacuum to afforded (2)
(6.54 g, 89.7%) as white solid. mp: 62–63 °C.
O
R'
NH2
O
O
H
N
O
O
R'
N
H2N
O
e
O
f
H
N
Cl
H
OH
R1
N
+
+
NH2
R1
H
O
NH2
HO
R'
HO
O
FA10-FA27
5a-5b
3a-3i
Scheme 2. Preparation of compounds FA10–FA27. Reagents and conditions: (e) CDI, THF, O-phenylenediamine or 3,4-diaminotoluene, TFA; (f) K2CO3, acetone.

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F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
Table 1
The structures and inhibitory activity of ferulic acid derivatives (IC50
)
O
O
O
R2
R1
H
N
O
Compd
R1
R2
IC50
(
lM)
Compd
R1
R2
IC50 (lM)
FA-1
3-Cl-4-F
49.3
FA-15
2-F
73.7
FA-2
3-CF3
12.5
43.0
10.8
8.43
3.94
9.47
10.5
67.8
11.5
49.6
10.3
8.95
12.8
FA-16
FA-17
FA-18
FA-19
FA-20
FA-21
FA-22
FA-23
FA-24
FA-25
FA-26
FA-27
3-F
2.82
7.80
24.2
15.4
15.1
64.5
118
FA-3
3,4-Cl
4-OCH3
4-Br
3,5-CF3
3-CF3-5-Br
3-Cl-4-F
3-CF3
FA-4
FA-5
FA-6
2-F
FA-7
3-F
3,4-Cl
4-OCH3
4-Br
FA-8
3,5-CF3
3-CF3-5-Br
3-Cl-4-F
3-CF3
FA-9
16.4
11.6
66.6
71.4
7.43
2.59
FA-10
FA-11
FA-12
FA-13
FA-14
2-F
3-F
3,4-Cl
4-OCH3
4-Br
3,5-CF3
3-CF3-5-Br
SAHA
5.1.2. Methyl (2E)-3-(4-{2-[(3-chloro-4-fluorophenyl)amino]-2-
oxoethoxy}-3- methoxyphenyl)acrylate (4a)17
To a suspension of 2 (2.08 g, 10 mmol) in dehydrated acetone
(70 mL) was added anhydrous potassium carbonate (4.14 g,
30 mmol). The mixture was stirred at room temperature for
30 min and then 2-chloro-N-(3-chloro-4-fluorophenyl)acetamide
(2.44 g, 11 mmol) was added. The reaction mixture was refluxed
for another 10 h. Filtration and evaporation of acetone was done
in vacuum. The residue was extracted with EtOAc (120 mL). The
combined layers were washed with water, 2 M NaOH, 2 M HCl
and brine, dried over Na2SO4 and solvent was removed. The crude
product was purified by silica gel column chromatography (Petro-

F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
6977
Table 2
flux. The solution was cooled to room temperature, and a solution
of KOH (0.56 g, 9.98 mmol) in methanol (1.4 mL) was added in one
portion to afford NH2OK/NH2OH solution.
Growth inhibition of HDACIs against a panel of cancer cells (IC50, lM)
To a solution of 4a (1.97 g, 5 mmol) in 7 mL of anhydrous DMF
was added NH2OK/NH2OH solution (10 mL). The mixture was stir-
red at room temperature for 3 days. The reaction mixture was ta-
ken up in dilute aqueous HCl (pH 2), extracted with EtOAc
(150 mL), dried over Na2SO4, filtered and concentrated in vacuo.
The residue was purified by column chromatography (AcOEt:-
MeOH = 20:1) to give hydroxamic acid FA1 as white solid (0.71 g,
35.34%). Mp: 177–179 °C. EI-MS (m/z):394.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.95 (d, J = 4 Hz, 1H), 7.54 (d, J = 4 Hz, 1H),
7.41 (d, J = 4 Hz, 1H), 7.38 (s, 1H), 7.22 (s, 1H), 7.11 (d, J = 4 Hz,
1H), 6.97 (d, J = 4 Hz, 1H), 6.37 (d, J = 8 Hz, 1H), 4.74(s, 2H), 3.84
(s, 3H).
Compd
R1
R2
MCF-7
HeLa
MDA-MB-231
FA5
FA6
FA7
4-Br
2-F
73.6
>100
39.5
>100
11.07
34.50
0.76
8.30
1.45
Compounds (FA2–FA9) were synthesized following the same
procedure described above.
3-F
5.1.3.1. (2E)-N-Hydroxy-3-[3-methoxy-4-(2-oxo-2-{[3-(trifluo-
FA12
FA13
FA16
FA17
3,4-diCl
4-OCH3
3-F
>100
>100
>100
43.1
72.09
>100
28.09
20.82
83.4
48.5
>100
25.3
romethyl)phenyl]amino}ethoxy)phenyl]acrylamide
(FA2).
Mp: 206–208 °C. EI-MS (m/z): 410.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 8.12 (s, 1H) 7.83 (d, J = 4 Hz, 1H), 7.59 (d,
J = 4 Hz, 1H), 7.52 (d, J = 8 Hz, 1H), 7.45 (d, J = 2 Hz, 1H), 7.38 (s,
1H), 7.23–7.19(m, 1H), 6.96 (d, J = 4 Hz, 1H), 6.48 (d, J = 8 Hz, 1H),
4.79(s, 2H), 3.86 (s, 3H).
5.1.3.2.
(2E)-3-(4-{2-[(3,4-Dichlorophenyl)amino]-2-oxoeth-
oxy}-3-methoxyphenyl)-N-hydroxyacrylamide (FA3).
Mp:
223–225 °C. EI-MS (m/z): 394.0[M-OH]+. 1H NMR (400 MHz,
(CD3)2SO) d 7.96(d, J = 4 Hz, 1H), 7.54(d, J = 4 Hz, 1H), 7.41(d,
J = 4 Hz, 1H), 7.38(s, 1H), 7.22(s, 1H), 7.11(d, J = 4 Hz, 1H), 6.97(d,
J = 4 Hz, 1H), 6.37(d, J = 8 Hz, 1H), 4.74(s, 2H), 3.84(s, 3H).
3,5-diCF3
3-CF3-5-Br
FA27
92.1
60.2
84.33
9.69
>100
0.16
SAHA
5.1.3.3.
phenyl)amino]-2-oxoethoxy}phenyl)acrylamide
(FA4).
Mp: 165–167 °C. EI-MS (m/z): 372.1 (M+). 1H NMR
(2E)-N-Hydroxy-3-(3-methoxy-4-{2-[(4-methoxy-
(400 MHz, (CD3)2SO) d 7.53(d, J = 8 Hz, 3H), 7.15(s, 1H), 7.13(d,
J = 2 Hz, 1H), 6.97(d, J = 4 Hz, 1H), 6.94(d, J = 8 Hz, 1H), 6.91(d,
J = 4 Hz, 3H), 4.71(s, 2H), 3.86(s, 3H), 3.72(s, 3H).
leum:AcOEt = 5:1 to 1:1) to yield 4a as white solid (3.37 g, 85.65%).
Mp: 156–158 °C. EI-MS (m/z): 393.1 (M+). 1H NMR (400 MHz,
CDCl3) d 7.80 (d, J = 4 Hz, 1H), 7.66 (d, J = 8 Hz, 1H), 7.43 (d,
J = 6 Hz, 1H), 7.29 (s, 1H), 7.17 (s, 1H), 7.14 (d, J = 4 Hz, 1H), 6.98
(d, J = 4 Hz, 1H), 6.39 (d, J = 8 Hz, 1H), 4.69 (s, 2H), 4.00 (s, 3H),
3.84 (s, 3H).
5.1.3.4. (2E)-3-(4-{2-[(4-Bromophenyl)amino]-2-oxoethoxy}-3-
methoxyphenyl)-N-hydroxyacrylamide (FA5).
Mp: 215–
217 °C. EI-MS (m/z): 420.0 (M+). 1H NMR (400 MHz, (CD3)2SO) d
7.61(d, J = 6 Hz, 2H), 7.53(s, 1H), 7.52(d, J = 6 Hz, 2H), 7.40(d,
J = 8 Hz, 1H), 7.20(d, J = 6 Hz, 1H), 7.11(d, J = 4 Hz, 1H), 6.95(d,
J = 4 Hz, 1H), 4.65(s, 2H), 3.84(s, 3H).
Compounds (4b–4l) were prepared by using the same proce-
dure described above.
5.1.3. (2E)-3-(4-{2-[(3-Chloro-4-fluorophenyl)amino]-2-
oxoethoxy}-3-methoxy phenyl)-N-hydroxyacrylamide (FA1)18
Preparation of NH2OK/NH2OH solution: NH2OHꢀHCl (0.476 g,
6.85 mmol) was solubilized in methanol (2.4 mL) by heating to re-
5.1.3.5. (2E)-3-(4-{2-[(2-Fluorophenyl)amino]-2-oxoethoxy}-3-
methoxyphenyl)-N-hydroxyacrylamide (FA6).
Mp: 204–
Figure 3. Molecular modeling of FA6 (left) and FA16 (right) binding to the active site of HDAC. The original ligands are shown in green. Metal coordination and hydrogen
bond interactions are shown as dotted yellow lines.

6978
F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
207 °C. EI-MS (m/z): 345.1[M-CH3]+. 1H NMR (400 MHz, (CD3)2SO)
d 7.95(s, 1H), 7.54 (d, J = 8 Hz, 1H), 7.42(d, J = 8 Hz, 1H), 7.30–
7.28(m, 2H), 6.99(d, J = 6 Hz, 1H), 6.49(d, J = 8 Hz, 1H), 6.38(d,
J = 8 Hz, 1H), 4.83(s, 2H), 3.86(s, 3H).
5.1.5.1. (2E)-N-(2-Aminophenyl)-3-[3-methoxy-4-(2-oxo-2-{[3-
(trifluoromethyl)phenyl]amino}ethoxy)phenyl]acrylamide
(FA11).
Mp: 212–214 °C. EI-MS (m/z): 485.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.54(d, J = 8 Hz, 2H), 7.48(s, 1H), 7.35(d,
J = 8 Hz, 1H), 7.28(s, 1H), 7.18(d, J = 12 Hz, 1H), 7.00(d, J = 8 Hz,
1H), 6.91(d, J = 8 Hz, 3H), 6.82–6.74(m, 2H), 6.60–6.56(m, 1H),
4.72(s, 2H), 3.88(s, 3H).
5.1.3.6. (2E)-3-(4-{2-[(3-Fluorophenyl)amino]-2-oxoethoxy}-3-
methoxyphenyl)-N-hydroxyacrylamide (FA7).
Mp: 176–
178 °C. EI-MS (m/z):344.1[M-OH]+. 1H NMR (400 MHz, (CD3)2SO)
d 7.62(d, J = 6 Hz, 1H), 7.40(d, J = 6 Hz, 3H), 7.36(s, 1H), 7.22(s,
1H), 7.11(d, J = 4 Hz, 1H), 6.96–6.92(m, 1H), 6.37(d, J = 8 Hz, 1H),
4.75 (s, 2H), 3.85(s, 3H).
5.1.5.2.
phenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA12).
Mp: 195–197 °C. EI-MS (m/z):485.0 (M+). 1H NMR
(2E)-N-(2-Aminophenyl)-3-(4-{2-[(3,4-dichloro-
(400 MHz, (CD3)2SO) d 8.03(s, 1H), 7.61(d, J = 4 Hz, 1H), 7.56(d,
J = 4 Hz, 1H), 7.51(d, J = 8 Hz, 1H), 7.35(d, J = 4 Hz, 1H), 7.29(s,
1H), 7.17(d, J = 4 Hz, 1H), 6.99(d, J = 4 Hz, 1H), 6.94–6.90(m, 1H),
6.80(d, J = 8 Hz, 1H), 6.76(d, J = 4 Hz, 1H), 6.60–6.57(m, 1H),
4.78(s, 2H), 3.87(s, 3H).
5.1.3.7. (2E)-3-[4-(2-{[3,5-Bis(trifluoromethyl)phenyl]amino}-2-
oxoethoxy)-3-methoxyphenyl]-N-hydroxyacrylamide
(FA8).
(400 MHz, (CD3)2SO) d 8.34(s, 2H), 7.83 (s, 1H), 7.41(d, J = 8 Hz,
1H), 7.23(s, 1H), 7.11(d, J = 4 Hz, 1H), 6.99(d, J = 6 Hz, 1H), 6.37(d,
J = 8 Hz, 1H), 4.81(s, 2H), 3.85(s, 3H).
Mp: 183–185 °C. EI-MS (m/z): 478.1 (M+). 1H NMR
5.1.5.3.
methoxyphenyl)amino]-2-oxoethoxy}phenyl)acrylamide
(FA13).
Mp: 189–191 °C. EI-MS (m/z): 447.1(M+). 1H NMR
(2E)-N-(2-Aminophenyl)-3-(3-methoxy-4-{2-[(4-
5.1.3.8.
(2E)-3-[4-(2-{[3-Bromo-5-(trifluoro-
methyl)phenyl]amino}-2-oxoethoxy)-3-methoxyphenyl]-N-
(400 MHz, (CD3)2SO) d 7.54(d, J = 6 Hz, 2H), 7.51(d, J = 6 Hz, 1H),
7.35(d, J = 4 Hz, 1H), 7.28(s, 1H), 7.18(d, J = 4 Hz, 1H), 7.01–
6.95(m, 2H), 6.91(d, J = 4 Hz, 2H), 6.80(d, J = 8 Hz, 2H), 6.65(d,
J = 4 Hz, 1H), 4.72(s, 2H), 3.88(s, 3H), 3.73(s, 3H).
hydroxyacrylamide (FA9).
Mp: 195–198 °C. EI-MS (m/
z):471.9[M-OH]+. 1H NMR (400 MHz, (CD3)2SO) d 8.17(s, 1H),
8.04(s, 1H), 7.69(s, 1H), 7.41(d, J = 8 Hz, 1H), 7.23(s, 1H), 7.11(d,
J = 4 Hz, 1H), 6.96(d, J = 4 Hz, 1H), 6.37(d, J = 8 Hz, 1H), 4.78(s,
2H), 3.84(s, 3H).
5.1.5.4.
nyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA14).
Mp: 194–195 °C. EI-MS (m/z): 495.1(M+). 1H NMR
(2E)-N-(2-Aminophenyl)-3-(4-{2-[(4-bromophe-
5.1.4. (2E)-N-(2-Aminophenyl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (5a)
(400 MHz, (CD3)2SO) d 7.62(d, J = 6 Hz, 2H), 7.52(d, J = 4 Hz, 2H),
7.50(d, J = 4 Hz, 1H), 7.35(d, J = 4 Hz, 1H), 7.28(s, 1H), 7.17(d,
J = 4 Hz, 1H), 6.99(d, J = 4 Hz, 1H), 6.94–6.90(m, 1H), 6.80(d,
J = 8 Hz, 1H), 6.76(d, J = 4 Hz, 1H), 6.61–6.57(m, 1H), 4.76(s, 2H),
3.87(s, 3H).
To a solution of ferulic acid (5.83 g, 30 mmol) in anhydrous THF
(120 mL) was added CDI (5.35 g, 33 mmol) portionwise at room
temperature. The reaction mixture was stirred for 1 h to form acy-
limidazole followed by addition of 1,2-phenylenediamine (25.96 g,
240 mmol)and TFA (3.49 g, 30 mmol). The reaction mixture was
stirred at room temperature for another 16 h. The mixture was
then filtered to give crude product which was purified by silica
gel column chromatography (Petroleum:AcOEt = 3:1 to 1:3) to
yield 5a as white solid (6.87 g, 80.53%). Mp: 164–166 °C. EI-MS
(m/z):284.1 (M+). 1H NMR (400 MHz, CDCl3) d 7.70(d, J = 8 Hz,
1H), 7.35(s, 1H), 7.12–7.09(m, 2H), 7.08(d, J = 6 Hz, 1H), 6.95(d,
J = 4 Hz, 1H), 6.86(d, J = 2 Hz, 2H), 6.49(d, J = 4 Hz, 1H), 3.96(s, 3H).
Compound 5b was prepared following the same procedure as
5a. Then it was purified by silica gel column chromatography
(Petroleum:AcOEt = 3:1 to 1:3).
5.1.5.5.
phenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA15).
Mp: 194–195 °C. EI-MS (m/z):435.0 (M+). 1H NMR
(2E)-N-(2-Aminophenyl)-3-(4-{2-[(2-fluoro-
(400 MHz, (CD3)2SO) d 7.52(d, J = 8 Hz, 1H), 7.36–7.30(m, 3H),
7.18(d, J = 2 Hz, 4H), 7.03(d, J = 4 Hz, 1H), 6.94–6.92(m, 1H),
6.81(d, J = 8 Hz, 2H), 6.63(s, 1H), 4.76(s, 2H), 3.87(s, 3H).
5.1.5.6.
phenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA16).
Mp: 177–178 °C. EI-MS (m/z): 435.1(M+). 1H NMR
(2E)-N-(2-Aminophenyl)-3-(4-{2-[(3-fluoro-
(400 MHz, (CD3)2SO) d 7.63(d, J = 6 Hz, 1H), 7.55(d, J = 6 Hz, 1H),
7.38(d, J = 6 Hz, 1H), 7.36(s, 1H), 7.35(d, J = 4 Hz, 1H), 7.29 (s, 1H),
7.17(d, J = 4 Hz, 1H), 6.99(d, J = 4 Hz, 1H), 6.93(d, J = 4 Hz, 1H),
6.91(d, J = 4 Hz, 1H), 6.82–6.75(m, 2H), 6.61–6.57(m, 1H), 4.78(s,
2H), 3.88(s, 3H).
5.1.5. (2E)-N-(2-Aminophenyl)-3-(4-{2-[(3-chloro-4-
fluorophenyl)amino]-2-oxoethoxy}-3-
methoxyphenyl)acrylamide (FA10)
To a suspension of 5a (1.43 g, 5 mmol) in dehydrated acetone
(70 mL) was added anhydrous potassium carbonate (2.07 g,
15 mmol). The mixture was stirred at room temperature for
30 min and then 2-chloro-N-(3-chloro-4-fluorophenyl)acetamide
(3.33 g, 15 mmol) was added. The reaction mixture was refluxed
for another 10 h. Filtration and evaporation of acetone was done
in vacuum. The residue was extracted with EtOAc (150 mL). The
combined layers were washed with water, 2 M NaOH, 2 M HCl
and brine, dried over Na2SO4 and solvent was removed. The crude
product was purified by silica gel column chromatography (CH2-
Cl2:MeOH = 80:1 to 20:1) to yield FA10 as white solid (1.33 g,
56.45%). Mp: 177–178 °C. EI-MS (m/z): 469.0(M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.90(d, J = 4 Hz, 1H), 7.51(d, J = 8 Hz, 2H),
7.42(d, J = 4 Hz, 1H), 7.39–7.34(m, 1H), 7.29(s, 1H), 7.17(d,
J = 4 Hz, 1H), 6.99(d, J = 4 Hz, 1H), 6.82(s, 1H), 6.77(d, J = 4 Hz,
1H), 6.63–6.59(m, 1H), 6.49(d, J = 4 Hz, 1H), 4.77(s, 2H), 3.87(s, 3H).
Compounds (FA11–FA27) were prepared following the proce-
dure described above.
5.1.5.7.
methyl)phenyl]amino}-2-oxoethoxy)-3-methoxyphenyl]acryl-
amide (FA17).
Mp: 190–192 °C. EI-MS (m/z):553.0 (M+). 1H
(2E)-N-(2-Aminophenyl)-3-[4-(2-{[3,5-bis(trifluoro-
NMR (400 MHz, (CD3)2SO) d 8.35(s, 2H), 7.83(s, 1H), 7.51(d,
J = 8 Hz, 1H), 7.35(d, J = 4 Hz, 1H), 7.30(s, 1H), 7.17(d, J = 4 Hz,
1H), 7.02(d, J = 4 Hz, 1H), 6.94–6.90(m, 1H), 6.81(d, J = 8 Hz, 1H),
6.76(d, J = 4 Hz, 1H), 6.61–6.57(m, 1H), 4.84(s, 2H), 3.88(s, 3H).
5.1.5.8. (2E)-N-(2-Aminophenyl)-3-[4-(2-{[3-bromo-5-(trifluo-
romethyl)phenyl]amino}-2-oxoethoxy)-3-methoxy-
phenyl]acrylamide (FA18).
Mp: 209–211 °C. EI-MS (m/z):
563.0(M+). 1H NMR (400 MHz, (CD3)2SO) d 8.18(s, 1H), 8.05(s,
1H), 7.69(s, 1H), 7.51(d, J = 8 Hz, 1H), 7.32(d, J = 8 Hz, 2H), 7.16(s,
1H), 7.01–6.92(m, 2H), 6.79(d, J = 8 Hz, 2H), 6.58(d, J = 2 Hz, 1H),
4.81(s, 2H), 3.87(s, 3H).

F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
6979
5.1.5.9. (2E)-N-(2-Amino-4-methylphenyl)-3-(4-{2-[(3-chloro-4-
J = 6 Hz, 1H), 7.02(d, J = 4 Hz, 1H), 6.78(d, J = 8 Hz, 1H), 6.56(s,
1H), 6.39(d, J = 4 Hz, 1H), 4.83(s, 2H), 3.87(s, 3H), 2.17(s, 3H).
fluorophenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylam-
ide (FA19).
Mp: 187–188 °C. EI-MS (m/z): 483.2(M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.96(d, J = 4 Hz, 1H), 7.52(d, J = 4 Hz, 1H),
7.47(s, 1H), 7.42(d, J = 4 Hz, 1H), 7.28(s, 1H), 7.20 (d, J = 4 Hz, 1H),
7.17(d, J = 6 Hz, 1H), 6.99(d, J = 4 Hz, 1H), 6.78(d, J = 8 Hz, 1H),
6.58(s, 1H), 6.43(d, J = 2 Hz, 1H), 4.76(s, 2H), 3.87(s, 3H), 2.18(s,
3H).
5.1.5.17. (2E)-N-(2-Amino-4-methylphenyl)-3-[4-(2-{[3-bromo-
5-(trifluoromethyl)phenyl]amino}-2-oxoethoxy)-3-methoxy-
phenyl]acrylamide (FA27).
Mp: 193–195 °C. EI-MS (m/z):
579.0(M+). 1H NMR (400 MHz, (CD3)2SO d 8.18(s, 1H), 8.05(s, 1H),
7.69(s, 1H), 7.49(d, J = 8 Hz, 1H), 7.28(s, 1H), 7.20(d, J = 4 Hz, 1H),
7.16(d, J = 4 Hz, 1H), 7.00(d, J = 4 Hz, 1H), 6.78(d, J = 8 Hz, 1H),
6.56(s, 1H), 6.39(d, J = 4 Hz, 1H), 4.80(s, 2H), 3.87(s, 3H), 2.17(s,
3H).
5.1.5.10. (2E)-N-(2-Amino-4-methylphenyl)-3-[3-methoxy-4-(2-
oxo-2-{[3-(trifluoromethyl)phenyl]amino}ethoxy)phenyl]acryl-
amide (FA20).
Mp: 200–202 °C. EI-MS (m/z): 499.1(M+). 1H
NMR (400 MHz, (CD3)2SO) d 7.84(d, J = 4 Hz, 1H), 7.77(d, J = 4 Hz,
1H), 7.60 (d, J = 4 Hz, 1H), 7.58(d, J = 4 Hz, 1H), 7.51(s, 1H), 7.28(s,
1H), 7.17(d, J = 6 Hz, 1H), 7.08–7.05(m, 1H), 7.00(d, J = 4 Hz, 1H),
6.78(d, J = 8 Hz, 1H), 6.56(s, 1H), 6.39(d, J = 4 Hz, 1H), 4.76(s, 2H),
3.85(s, 3H), 2.17(s, 3H).
5.2. HDAC inhibitory activity assays19
The HDAC inhibitory activities of title compounds were mea-
sured using Color-de-Lys™ HDAC colorimetric activity assay kit
(Enzo Life Sciences, Inc.) according to the manufacturer’s instruc-
tions. The kit is useful for inhibitors screening using HDAC from
HeLa nuclear extract. The Color de Lys™ substrate which comprises
an acetylated lysine is incubated with sample containing HDAC
activity. Deacetylation of substrate sensitizes the substrate. The
mixing with the Color de Lys™ developer causes an increase in col-
or intensity at 405 nm.
5.1.5.11.
dichlorophenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acryl-
amide (FA21).
Mp: 207–209 °C. EI-MS (m/z):499.1 (M+). 1H
(2E)-N-(2-Amino-4-methylphenyl)-3-(4-{2-[(3,4-
NMR (400 MHz, (CD3)2SO) d 8.03(s, 1H), 7.61(d, J = 6 Hz, 1H),
7.56(d, J = 4 Hz, 1H), 7.49 (d, J = 8 Hz, 1H), 7.28(s, 1H), 7.20(d,
J = 4 Hz, 1H), 7.16 (d, J = 4 Hz, 1H), 6.99(d, J = 4 Hz, 1H), 6.78(d,
J = 8 Hz, 1H), 6.56(s, 1H), 6.39(d, J = 4 Hz, 1H), 4.78(s, 2H), 3.87(s,
3H), 2.17(s, 3H).
The title compounds and SAHA were diluted in buffer to various
concentrations (20
HDACs (5 L) were incubated at 37 °C with 10
and 25 L of substrate on 96-well plates. After incubation for
30 min, Color de Lys™ developer (50 L/well) was added to stop
lg/mL,
4
lg/mL, 0.8
lg/mL, 0.16
lg/mL).
l
lL of compounds
l
5.1.5.12. (2E)-N-(2-Amino-4-methylphenyl)-3-(3-methoxy-4-{2-
l
[(4-methoxyphenyl)amino]-2-oxoethoxy}phenyl)acrylamide
(FA22).
HDAC reactions. Incubate plate at 37 °C for 15 min and read plate
in microtiter-plate reader at 405 nm. The inhibition rates were cal-
culated from ultraviolet absorption readings of inhibited wells re-
lated to those of control wells. The IC50 values were calculated
according to inhibition ratios.
Mp: 192–194 °C. EI-MS (m/z):461.2 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.54(d, J = 4 Hz, 2H), 7.49(d, J = 8 Hz, 1H),
7.27(s, 1H), 7.20(d, J = 4 Hz, 1H), 7.18 (d, J = 6 Hz, 1H), 7.00(d,
J = 4 Hz, 1H), 6.91(d, J = 4 Hz, 2H), 6.78(d, J = 8 Hz, 1H), 6.57(s,
1H), 6.41(d, J = 4 Hz, 1H), 4.72(s, 2H), 3.87(s, 3H), 3.73(s, 3H),
2.17(s, 3H).
5.3. Cell growth inhibitory activity in cancer cells
5.1.5.13. (2E)-N-(2-Amino-4-methylphenyl)-3-(4-{2-[(4-bromo-
The eight potent compounds were evaluated against breast can-
cer cell lines (MCF-7, MDA-MB-231) and cervical cancer cell (HeLa)
using MTT assay to assess cell proliferation. Exponentially growing
cells were harvested and plated in 96-well plates at a concentra-
tion of 1 ꢁ 104 cells/well, and then incubated for 24 h at 37 °C.
The cells in wells were treated with target compounds respectively
at various concentrations for 48 h. Then, 20 mL MTT (5 mg/mL)
was added to each well and incubated for 4 h at 37 °C. Supernatant
was discarded, and 150 mL DMSO was added to each well. Absor-
bance values were determined by microplate reader at 570 nm.
The IC50 values were calculated according to inhibition ratios.
phenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA23).
Mp: 184–186 °C. EI-MS (m/z): 509.0(M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.61(d, J = 4 Hz, 2H), 7.52(d, J = 4 Hz, 2H),
7.27(s, 1H), 7.20(d, J = 6 Hz, 1H), 7.17 (d, J = 6 Hz, 1H), 6.99(d,
J = 4 Hz, 1H), 6.80(d, J = 2 Hz, 1H), 6.75(d, J = 6 Hz, 1H), 6.56(s,
1H), 6.39(d, J = 4 Hz, 1H), 4.76(s, 2H), 3.87(s, 3H), 2.17(s, 3H).
5.1.5.14. (2E)-N-(2-Amino-4-methylphenyl)-3-(4-{2-[(2-fluoro-
phenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA24).
Mp: 187–189 °C. EI-MS (m/z): 449.1(M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.96(s, 1H), 7.49(d, J = 8 Hz, 2H), 7.33–
7.28(m, 2H), 7.20(d, J = 4 Hz, 2H), 7.18(d, J = 4 Hz, 1H), 7.03 (d,
J = 4 Hz, 1H), 6.79(d, J = 8 Hz, 1H), 6.56(s, 1H), 6.39(d, J = 4 Hz,
1H), 4.83(s, 2H), 3.88(s, 3H), 2.17(s, 3H).
5.4. Molecular docking modeling20
Molecule docking was performed using Sybyl/Surflex-dock
based on crystal structures of HDAC (PDB ID: 3F07). Hydrogen
was added and minimized using Tripos force field and Pullman
charges. All the waters were removed as well as all ions except
for catalytic zinc ion. The residues in a radius 5.0 Å around APHA
(ligand of HDAC in crystal complex) were selected as active site.
Compounds FA6 and FA16 were depicted with Sybyl/Sketch mod-
ule (Tripos Inc.) and optimized applying Powell’s method with Tri-
pos force field with convergence criterion set at 0.05 kcal/(Åmol),
and assigned with Gasteiger-Hückel method. Other docking
parameters were kept at default.
5.1.5.15. (2E)-N-(2-Amino-4-methylphenyl)-3-(4-{2-[(3-fluoro-
phenyl)amino]-2-oxoethoxy}-3-methoxyphenyl)acrylamide
(FA25).
Mp: 205–207 °C. EI-MS (m/z):449.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.63(d, J = 6 Hz, 1H), 7.58(d, J = 6 Hz, 1H),
7.49(d, J = 8 Hz, 1H), 7.38(d, J = 4 Hz, 1H), 7.37(s, 1H), 7.28(s, 1H),
7.20(d, J = 6 Hz, 1H), 7.17(d, J = 4 Hz, 1H), 6.99 (d, J = 4 Hz, 1H),
6.80–6.76(m, 1H), 6.58(s, 1H), 6.41(d, J = 4 Hz, 1H), 4.77(s, 2H),
3.87(s, 3H), 2.18(s, 3H).
5.1.5.16.
(2E)-N-(2-Amino-4-methylphenyl)-3-[4-(2-{[3,5-
bis(trifluoromethyl)phenyl]amino}-2-oxoethoxy)-3-methoxy-
Acknowledgments
phenyl]acrylamide (FA26).
Mp: 204–206 °C. EI-MS (m/z):
567.1(M+). 1H NMR (400 MHz, (CD3)2SO) d 8.35(s, 2H), 7.83(s,
1H), 7.49(d, J = 8 Hz, 1H), 7.29(s, 1H), 7.19(d, J = 2 Hz, 1H), 7.17(d,
This work was supported by the National Natural Science Foun-
dation of China (Grant Nos. 81302641 and 81302737), and Natural

6980
F. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6973–6980
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