Search for novel histone deacetylase inhibitors. Part II: Design and synthesis of novel isoferulic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2014.03.019

Previously, we described the discovery of potent ferulic acid-based histone deacetylase inhibitors (HDACIs) with halogeno-acetanilide as novel surface recognition moiety (SRM). In order to improve the affinity and activity of these HDACIs, twenty seven isoferulic acid derivatives were described herein. The majority of title compounds displayed potent HDAC inhibitory activity. In particular, IF5 and IF6 exhibited significant enzymatic inhibitory activities, with IC₅₀ values of 0.73 ± 0.08 and 0.57 ± 0.16 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against human cancer cells. Especially, IF6 displayed promising profile as an antitumor candidate with IC₅₀ value of 3.91 ± 0.97 μM against HeLa cells. The results indicated that these isoferulic acid derivatives could serve as promising lead compounds for further optimization.

Full text of DOI:10.1016/j.bmc.2014.03.019

Bioorganic & Medicinal Chemistry 22 (2014) 2707–2713
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Search for novel histone deacetylase inhibitors. Part II: Design
and synthesis of novel isoferulic acid derivatives
⇑
Wen Lu , Fang Wang , Tao Zhang, Jinyun Dong, Hongping Gao, Ping Su, Yaling Shi, Jie Zhang
School of Pharmacy, Health Science Center, 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:
Previously, we described the discovery of potent ferulic acid-based histone deacetylase inhibitors (HDA-
CIs) with halogeno-acetanilide as novel surface recognition moiety (SRM). In order to improve the affinity
and activity of these HDACIs, twenty seven isoferulic acid derivatives were described herein. The majority
of title compounds displayed potent HDAC inhibitory activity. In particular, IF5 and IF6 exhibited signif-
Received 25 February 2014
Revised 10 March 2014
Accepted 11 March 2014
Available online 22 March 2014
icant enzymatic inhibitory activities, with IC50 values of 0.73 0.08 and 0.57 0.16
Furthermore, these compounds showed moderate antiproliferative activity against human cancer cells.
Especially, IF6 displayed promising profile as an antitumor candidate with IC50 value of 3.91 0.97
lM, respectively.
Keywords:
l
M
Histone deacetylase
HDAC inhibitor
Isoferulic acid
Anticancer
against HeLa cells. The results indicated that these isoferulic acid derivatives could serve as promising
lead compounds for further optimization.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
developed by Novartis for the treatment of various cancers is a
non-selective HDACI. Entinostat is an oral benzamide HDACI
Histone deacetylase (HDAC) inhibition is a clinically validated
therapeutic strategy for cancer treatment. HDAC are involved in
remodelling of chromatin and play an essential role in cell prolifer-
ation, cell-cycle regulation and apoptosis.1 An aberrant activity of
HDACs has been documented in several human cancers leading
to development of histone deacetylase inhibitors (HDACIs) as
anticancer agents.2 Therefore, HDAC is becoming a prominent ther-
apeutic target for the treatment of cancer. Research of HDACIs is
becoming an interesting field in anticancer agent design.
Classical HDACs are zinc-dependent enzymes bearing a highly
conserved catalytic domain with a zinc ion.3 So far, a wide range
of natural and synthetic derivatives have been identified as potent
HDACIs. They are structurally diverse group compounds with
attractive antitumor properties. Majority of them consists of a zinc
binding group (ZBG) interacting with the zinc ion. Moreover, HDA-
CIs share other common features such as a linker domain, which
occupies the narrow channel; a connect unit (CU), which connects
SRM and linker; and a surface recognition moiety (SRM), which
interacts with residues on the rim of active site (Fig. 1).
undergoing clinical trials for the treatment of various cancer.4
In the course of search for novel HDACIs with novel structure
and higher potency, we have developed several ferulic acid deriv-
atives with potent HDAC inhibitory activity and antiproliferative
property.5 It was validated that phenyl-substituted ethylene could
serve as rigid linker of HDACIs. Moreover, halogeno-acetanilides
were also confirmed to be suitable as SRM of HDACIs. However,
in the molecular docking study of ferulic acid derivatives with
HDAC, we found that acetanilide did not form any hydrogen bind-
ing with SRM as we initially supposed.
On the basis of these observations, we aimed to enhance the
structural diversity and affinity with the SRM of HDAC. Rearrange-
ment of acetanilide to meta-position was performed to afford cor-
responding isoferulic acid derivatives. In this study, ferulic acid
was replaced by isoferulic acid as rigid linker of HDACIs. The halo-
geno-acetanilide was incorporated at meta-position instead of
para-position. We supposed that para-position might be more suit-
able for interaction with SRM. These novel HDACIs comprised com-
mon hydroxamic acid or 2-aminobenzamide group as ZBG as
previously reported (Fig. 2). The conformations of these com-
pounds were more similar to that of SAHA.
Vorinostat (SAHA) is the first HDACI approved by the FDA for
the treatment of CTCL in 2006. Belinostat has been granted orphan
drug and fast track designation by the FDA. Panobinostat
As part of our ongoing effort to develop HDACIs with higher
affinity and activity, we developed two series of isoferulic acid
derivatives bearing halogeno-acetanilide at meta-position. The
structures of these compounds are quite consistent with common
pharmacophore of HDACIs. The binding mode of the most potent
⇑
Corresponding author. Tel./fax: +86 29 82655451.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.03.019
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2708
W. Lu et al. / Bioorg. Med. Chem. 22 (2014) 2707–2713
K2CO3 to afford corresponding 2-aminobenzamide-containing
O
H
N
derivatives IF10–IF27.10
OH
N
H
Vorinostat (SAHA)
O
3. Results and discussion
SRM
Linker
ZBG
CU
O
All the synthesized compounds were tested for their HDAC
inhibitory activity with SAHA as positive control. It was indicated
from Table 1 that majority of them displayed moderate to high
inhibitory activity against HDAC. Some of the title compounds
were shown to inhibit HDAC with IC50 values below the micromo-
lar range. In general, hydroxamic acids were found to be more
potent than benzamides. IF6 was the most potent HDAC inhibitor
H
N
O
O
O
N
N
S
OH
N
H
N
H
N
NH2
O
H
N
Belinostat (PXD101)
Mocetinostat (MGCD0103)
O
N
OH
N
H
with an IC50 value of 0.57 0.16
played potent HDAC inhibitory activities with IC50 values of
0.94 0.26 M and 0.73 0.08 M, respectively. The variety and
lM, while IF4 and IF5 also dis-
H
N
N
NH2
O
H
HN
HN
O
l
l
Entinostat (MS275)
Panobinostat (LBH589)
position of halogen-substitution on aniline played important role
in biological activity. Benzamides IF15 and IF11 were active at
Figure 1. Structures and pharmacophore features of HDACIs.
higher doses with IC50 values of 15.60 3.28
22.16 2.37 M, respectively. In contrast, six benzamide deriva-
tives were inactive at 200 M. According to the results, both sub-
l
M
and
l
Rearragement
l
stitution on aniline and structure of ZBG played important role in
potency. It was indicated that compounds bearing substituents like
fluorine, bromine and trifluoromethyl on aniline possessed potent
anticancer activity. For ZBG, hydroxamic acid was more suitable for
these HDACIs than benzamide.
O
O
ZBG
Linker
H
N
O
R
R
O
O
ZBG
N
H
O
SRM CU
Linker
SRM CU
To further investigate their antiproliferative activity, six com-
pounds (five of hydroxamic acids and one of benzamide) were se-
lected to test for their antiproliferative potential by MTT method.
The test compounds were evaluated for their anticancer potency
against HeLa and MDA-MB-231 cell lines. The results were de-
scribed in Table 2. It was found that they displayed potent antipro-
Figure 2. Design strategy and structures of isoferulic acid derivatives.
compound with HDAC was also established in order to analysis the
interaction of acetanilide with SRM. Herein, we describe the dis-
covery and evaluation of isoferulic acid-based HDACIs.
liferative activities with IC50 values ranging from 1.90 0.04
lM to
20.51 1.36 M. Some of them exhibited promising antiprolifera-
l
tive activity against MDA-MB-231. IF3 displayed the most potent
antiproliferative potency against MDA-MB-231 comparable with
SAHA. Moreover, IF6 exhibited the most potent growth inhibition
against HeLa cell line. The antiproliferative results were consistent
with the HDAC inhibitory assays. It was indicated that inhibition of
HDAC might be one of the basis for anticancer activity.
2. Chemistry
The synthetic routes of the title compounds were outlined in
Schemes 1 and 2. An efficient synthesis of hydroxamic acids was
developed in 5-step reaction sequence (Scheme 1). Isoferulic acid
2 was prepared from isovanillin 1 utilizing classical Knoevenagel
condensation reaction conditions.6 Isoferulic acid was esterified
in the presence of concentrated H2SO4 to afford isoferulic acid
methylester 3. The hydroxyl group at meta-position of (2) was
etherified with various substituted acetanilides in anhydrous ace-
tone in the presence of K2CO3 to afford corresponding intermedi-
ates 4a–4i.7 These resulting esters 4a–4i were treated with
methanolic NH2OK at room temperature to yield corresponding
hydroxamic acid derivatives IF1–IF9.8
Here again, we have developed a new two-step procedure for
preparation of isoferulic acid derivatives bearing 2-aminobenzam-
ide as ZBG (Scheme 2). Isoferulic acid was converted into
corresponding imidazolide derivative by reaction with N,N0-car-
bonyldiimidazole (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.9 The hydroxyl group in 5a–5b was etherified
with haloacylanilines in anhydrous acetone in the presence of
The most potent inhibitor IF6 was selected for computational
studies. Docking studies were carried out to understand interaction
between inhibitors and HDAC. IF6 was docked into active site of
HDAC (PDB ID: 3F07) by SYBYL-X 2.0. Molecular insights based
on molecular docking indicated favorable binding mode of IF6 with
HDAC (Fig. 3). The results suggested that hydroxamic group was
bonded to zinc ion as ZBG. Hydroxamate OH made two hydrogen
bond interactions with His142 and His143 with distance of
1.96 Å and 2.41 Å, respectively. N–H could also form a hydrogen
bond to His143 with distance of 1.79 Å. Carbonyl group accepted
a hydrogen bond from Tyr306 with distance of 1.75 Å. The distance
between zinc ion and two oxygen atoms were 1.89 Å and 2.17 Å,
respectively.11 Based on these results, the binding mode of isofer-
ulic acid-based HDACI was the same as ferulic acid-based HDACI.
However, the direction of binding with surface of active site was
totally difference (Fig. 4). Moreover, there was an additional hydro-
gen bond between oxygen atom of acetyl and Gly151 on the surface.
This interaction might contribute to affinity and activity of isoferulic
O
O
O
O
O
O
CHO
O
OH
H
N
H
N
H
N
R1
a
b
c
d
O
OH
O
O
O
O
O
O
O
OH
OH
OH
IF1-IF9
1
2
3
4a-4i
Scheme 1. Preparation of compounds IF1–IF9. Reagents and conditions: (a) malonic acid, DBU, pyridine; (b) CH3OH, H2SO4; (c) K2CO3, acetone; (d) NH2OK, NH2OH, DMF.

W. Lu et al. / Bioorg. Med. Chem. 22 (2014) 2707–2713
2709
R2
O
O
O
NH2
H
N
H
N
R1
OH
f
e
N
H
O
O
NH2
O
O
O
R2
OH
2
OH
IF10-IF27
5a-5b
Scheme 2. Preparation of compounds IF10–IF27. Reagents and conditions: (e) CDI, THF, O-phenylenediamine or 3,4-diaminotoluene, TFA; (f) K2CO3, acetone.
Table 1
The structures and inhibitory activity of isoferulic acid derivatives (IC50
,
lM)
O
O
H
N
R2
R1
O
O
R1
R2
IC50
(l
M)
R1
R2
IC50 (lM)
H
NH2
NH2
NH2
NH2
H
N
N
OH
OH
OH
OH
OH
OH
OH
OH
OH
IF1
3-Cl-4-F
5.82 0.61
2.96 0.52
1.37 0.07
0.94 0.26
0.73 0.08
0.57 0.16
2.81 0.03
4.91 0.13
9.04 0.29
66.52 3.86
22.16 2.37
>200
IF15
IF16
IF17
IF18
IF19
IF20
IF21
IF22
IF23
IF24
IF25
IF26
IF27
2-F
15.60 3.28
24.01 0.35
>200
H
N
H
N
IF2
3-CF3
3-F
H
N
H
N
IF3
3,4-Cl
4-OCH3
4-Br
3,5-CF3
3-CF3-5-Br
3-Cl-4-F
3-CF3
H
N
H
N
IF4
>200
H
N
NH2
H
N
IF5
31.32 6.51
>200
H
N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
N
IF6
2-F
H
N
H
N
IF7
3-F
3,4-Cl
4-OCH3
4-Br
>200
H
N
H
N
IF8
3,5-CF3
3-CF3-5-Br
3-Cl-4-F
3-CF3
164.61 6.52
58.24 1.78
29.50 5.44
71.02 21.09
56.71 7.65
124.67 42.81
H
N
H
N
IF9
NH2
H
N
H
N
IF10
IF11
IF12
IF13
2-F
NH2
NH2
NH2
NH2
H
N
H
N
3-F
H
N
H
N
3,4-Cl
4-OCH3
4-Br
3,5-CF3
3-CF3-5-Br
H
N
H
N
49.30 11.50
>200
H
N
IF14
SAHA
0.26 0.08
The bold values mean the lowest IC50 value in each series.
acid-based HDACIs. The results indicated that these two kinds of
HDACIs had similar binding mode and orientation as APHA in the
pocket of HDAC. IF6 did not only chelate zinc ion but also form four
hydrogen bonds with His142, His143, Tyr306, and Gly151.

2710
W. Lu et al. / Bioorg. Med. Chem. 22 (2014) 2707–2713
Table 2
4. Conclusion
Growth inhibition of HDACIs against a panel of cancer cells (IC50, lM)
O
In summary, we proved that ferulic acid and isoferulic acid
derivatives with novel SRM as potent HDACIs. All the title com-
pounds displayed varied degree of anticancer potency and some
of them exhibited excellent HDAC inhibitory activity comparable
to positive control. The results indicated that hydroxamic acid
was more suitable for these HDACIs than benzamide. Hydroxamic
acid could interact tightly with zinc ion of HDAC. Therefore,
hydroxamates were somewhat more potent than benzamides.
Moreover, it was confirmed that meta-position of styrene was
more favorable for incorporation of SRM. Both ferulic acid and isof-
erulic acid could serve as molecular scaffold and rigid linker. Three
compounds (IF4, IF5, IF6) exhibited significant HDAC inhibitory
H
N
R2
O
R1
O
O
R1
R2
HeLa
MDA-MB-231
1.90 0.04
3.81 0.86
5.87 0.81
15.63 3.52
3.41 1.11
H
N
IF3
IF4
IF5
IF6
IF7
3,4-Cl
4-OCH3
4-Br
2-F
13.35 2.83
4.87 2.52
5.39 2.03
3.91 0.97
6.48 0.04
OH
H
N
OH
OH
OH
OH
H
N
H
N
activities, with IC50 values below 1 lM. Among them, IF6 exhibited
H
N
potency comparable with SAHA. Moreover, some of them exhibited
potent antiproliferative activities against MDA-MB-231 and HeLa
cell lines. Molecular docking results indicated that isoferulic acid
derivatives could interact more tightly with the active site of HDAC
than ferulic acid derivatives. In summary, these isoferulic 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.
3-F
NH2
H
N
IF15
2-F
20.51 1.36
15.51 9.56
SAHA
0.72 0.55
1.29 0.21
The bold values mean the lowest IC50 value in each series.
5. Experimental
5.1. Chemistry: general procedure
All solvents and reagents were purified according to standard
procedure. All reactions except those in aqueous media were
carried out by standard techniques for exclusion of moisture.
Petroleum ether used refers to the fraction boiling in the range
60–90 °C. Anhydrous reactions were performed over dried glass-
ware under nitrogen atmosphere. Reactions were monitored by
TLC on 0.25-mm silica gel plates (60GF-254) and visualized with
UV light. Melting points were determined on electrothermal melt-
ing point apparatus and are uncorrected. 1H NMR spectra were
measured on Bruker Advance (400 MHz) in CDCl3 or DMSO-d6
and reference to TMS. Mass spectra were obtained on Shimadzu
HPLC–MS-QP2010 instrument.
5.1.1. Preparation of isoferulic acid (2) from isovanillin (1)
through Knoevenagel condensation reaction
To a solution of isovanillin (1) (22.81 g, 150 mmol) and malonic
acid (62.44 g, 600 mmol) in pyridine (150 mL) was added
1,8-diazabicyclo(5.4.0)undec-7-ene (DBU, 34.26 g). The stirred
Figure 3. Molecular modeling of IF6 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.
Figure 4. Molecular modeling of isoferulic acid-based HDACIs showing additional hydrogen bond with surface of active site.

W. Lu et al. / Bioorg. Med. Chem. 22 (2014) 2707–2713
2711
reaction solution was heated at reflux for 4 h, then cooled to room
temperature. The cooled reaction mixture was poured into a solu-
tion (500 mL) of concentrated HCl and ice (v/v = 1:1) and the
resulting precipitate was filtered and washed with acidified water
to give a crude product. The crude product was dissolved in aque-
ous NaOH (1 M, 200 mL) and extracted with CH2Cl2) (3 ꢀ 50 mL).
The aqueous solution was acidified to pH = 1 and the resultant pre-
cipitate washed with acidified water to afford 22.56 g (77.2%) of
isoferulic acid as white solid. Mp: 228–230 °C.
5.1.4.2. (2E)-N-Hydroxy-3-[4-methoxy-3-(2-oxo-2-{[3-(trifluo-
romethyl)phenyl]amino}ethoxy)phenyl]acrylamide
(IF2).
Mp: 158–161 °C. EI-MS (m/z): 411.0 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 8.14 (s, 1H) 7.86 (d, J = 4 Hz, 1H), 7.61–
7.57 (m, 1H), 7.45 (d, J = 4 Hz, 1H), 7.39 (d, J = 8 Hz, 1H), 7.22 (s,
1H), 7.21 (d, J = 4 Hz, 1H), 7.06 (d, J = 4 Hz, 1H), 6.31 (d, J = 8 Hz,
1H), 4.78 (s, 2H), 3.84 (s, 3H).
5.1.4.3.
(2E)-3-(3-{2-[(3,4-Dichlorophenyl)amino]-2-oxoeth-
oxy}-4-methoxyphenyl)-N-hydroxyacrylamide (IF3).
Mp:
5.1.2. Methyl (2E)-3-(3-hydroxy-4-methoxyphenyl)acrylate (3)12
Isoferulic 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
aqueous solution of NaHCO3 until neutral pH. The organic layer
was then washed with distilled water and dried over anhydrous
Na2SO4. The solvent was removed under vacuum to afforded (3)
(6.65 g, 91.2%) as white solid. Mp: 74–76 °C.
153–154 °C. EI-MS (m/z): 394.0 [MꢁOH]+. 1H NMR (400 MHz,
(CD3)2SO) d 8.05 (d, J = 4 Hz, 1H), 7.61 (d, J = 6 Hz, 1H), 7.57 (d,
J = 2 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 7.21 (s, 2H), 7.06 (d, J = 4 Hz,
1H), 6.30 (d, J = 8 Hz, 1H), 4.75 (s, 2H), 3.84 (s, 3H).
5.1.4.4.
(2E)-N-hydroxy-3-(4-methoxy-3-{2-[(4-methoxyphenyl)
amino]-2-oxoethoxy}phenyl)acrylamide (IF4). Mp: 160–162 °C.
EI-MS (m/z): 372.1 (M+). 1H NMR (400 MHz, (CD3)2SO) d 7.54 (d,
J = 4 Hz, 2H), 7.37 (d, J = 8 Hz, 1H), 7.22 (s, 1H), 7.21 (d, J = 6 Hz, 1H),
7.06 (d, J = 6 Hz, 1H), 6.91 (d, J = 6 Hz, 2H), 6.31 (d, J = 8 Hz, 1H), 4.70
(s, 2H), 3.84 (s, 3H), 3.73 (s, 3H).
5.1.3. Methyl (2E)-3-(3-{2-[3,5-bis(trifluoromethyl)phenyl]
amino]-2-oxoethoxy}-4-methoxyphenyl)acrylate (4h)13
To a suspension of (3) (1.67 g, 8 mmol) in dehydrated acetone
(100 mL) was added anhydrous potassium carbonate (3.31 g,
24 mmol). The mixture was stirred at room temperature for
30 min and then 2-chloro-N-[3,5-bis(trifluoromethyl)]acetamide
(2.69 g, 8.8 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-
leum/AcOEt = 5:1 to 1:1) to yield (4h) as white solid (1.98 g,
71.7%). Mp: 180–182 °C. EI-MS (m/z): 476.9 (M+). 1H NMR
(400 MHz, CDCl3) d 8.15 (s, 2H), 7.67 (s, 1H), 7.63 (d, J = 8 Hz,
1H), 7.29 (d, J = 6 Hz, 1H), 7.20 (s, 1H), 7.00 (d, J = 4 Hz, 1H), 6.35
(d, J = 8 Hz, 1H), 4.73 (s, 2H), 4.02 (s, 3H), 3.83 (s, 3H).
5.1.4.5. (2E)-3-(3-{2-[(4-Bromophenyl)amino]-2-oxoethoxy}-4-
methoxyphenyl)-N-hydroxyacrylamide (IF5).
Mp: 175–
177 °C. EI-MS (m/z): 421.9 (M+). 1H NMR (400 MHz, (CD3)2SO) d
7.62 (d, J = 4 Hz, 2H), 7.52 (d, J = 4 Hz, 2H), 7.37 (d, J = 8 Hz, 1H),
7.21 (s, 1H), 7.20 (d, J = 4 Hz, 1H), 7.05 (d, J = 4 Hz, 1H), 6.30 (d,
J = 8 Hz, 1H), 4.74 (s, 2H), 3.84 (s, 3H).
5.1.4.6. (2E)-3-(3-{2-[(2-Fluorophenyl)amino]-2-oxoethoxy}-4-
methoxyphenyl)-N-hydroxyacrylamide (IF6).
Mp: 149–
151 °C. EI-MS (m/z): 360.0 (M+). 1H NMR (400 MHz, (CD3)2SO) d
7.95 (d, J = 2 Hz, 1H), 7.38 (d, J = 8 Hz, 1H), 7.33–7.28 (m, 1H),
7.25 (s, 1H), 7.22–7.19 (m, 1H), 7.20 (d, J = 2 Hz, 1H), 7.19 (d,
J = 2 Hz, 1H), 7.07 (d, J = 4 Hz, 1H), 6.33 (d, J = 8 Hz, 1H), 4.81 (s,
2H), 3.85 (s, 3H).
5.1.4.7. (2E)-3-(3-{2-[(3-Fluorophenyl)amino]-2-oxoethoxy}-4-
Compounds (4a–4l) were prepared by using the same proce-
dure described above.
methoxyphenyl)-N-hydroxyacrylamide (IF7).
Mp: 151–
152 °C. EI-MS (m/z): 344.1 [MꢁOH]+. 1H NMR (400 MHz, (CD3)2SO)
d 7.63 (d, J = 6 Hz, 1H), 7.39 (d, J = 2 Hz, 1H), 7.36 (s, 1H), 7.35 (d,
J = 4 Hz, 1H), 7.21 (s, 1H), 7.20 (d, J = 2 Hz, 1H), 7.06 (d, J = 4 Hz,
1H), 6.95–6.90 (m, 1H), 6.31 (d, J = 6 Hz, 1H), 4.75 (s, 2H), 3.84 (s,
3H).
5.1.4. (2E)-3-[3-(2-{[3,5-Bis(trifluoromethyl)phenyl]amino}-2-
oxoethoxy)-4-methoxyphenyl]-N-hydroxyacrylamide (IF8)14
NH2OK/NH2OH solution was prepared as previously described.
To
a solution of (4h) (1.70 g, 3.50 mmol) in 10 mL of
anhydrous DMF was added NH2OK/NH2OH solution (7 mL). The
mixture was stirred at room temperature for one day. The reac-
tion mixture was taken up in dilute aqueous HCl (pH = 2), ex-
tracted with EtOAc (120 mL), dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chro-
matography (AcOEt/MeOH = 20:1) to give hydroxamic acid IF8
as white solid (0.85 g, 49.7%). Mp: 158–160 °C. EI-MS (m/z):
478.0 (M+). 1H NMR (400 MHz, (CD3)2SO) d 8.37 (s, 2H), 7.82
(s, 1H), 7.38 (d, J = 8 Hz, 1H), 7.23 (s, 1H), 7.22 (d, J = 4 Hz,
1H), 7.07 (d, J = 4 Hz, 1H), 6.32 (d, J = 8 Hz, 1H), 4.81 (s, 2H),
3.84 (s, 3H).
5.1.4.8. (2E)-3-[3-(2-{[3-Bromo-5-(trifluoromethyl)phenyl]ami-
no}-2-oxoethoxy)-4-methoxyphenyl]-N-hydroxyacrylamide
(IF9). Mp: 151–153 °C. EI-MS (m/z): 474.9 [MꢁCH3]+. 1H NMR
(400 MHz, (CD3)2SO) d 8.21 (s, 1H), 8.07 (s, 1H), 7.68 (s, 1H),
7.38 (d, J = 8 Hz, 1H), 7.22 (s, 2H), 7.07 (d, J = 6 Hz, 1H), 6.31
(d, J = 8 Hz, 1H), 4.78 (s, 2H), 3.84 (s, 3H).
5.1.5. (2E)-N-(2-Aminophenyl)-3-(3-hydroxy-4-methoxyphenyl)-
acrylamide (5a)
To a solution of isoferulic 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
acylimidazole followed by addition of 1,2-phenylenediamine
(25.96 g, 240 mmol) and TFA (3.49 g, 30 mmol). The reaction mix-
ture 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 (6.95 g, 81.5%). Mp: 171–173 °C. EI-MS
(m/z): 284.1 (M+). 1H NMR (400 MHz, CDCl3) d 7.41 (d, J = 8 Hz,
1H), 7.33 (d, J = 4 Hz, 1H), 7.05 (s, 1H), 7.04 (d, J = 4 Hz, 1H), 6.97
Compounds (IF1–IF9) were synthesized following the same
procedure described above.
5.1.4.1. (2E)-3-(3-{2-[(3-Chloro-4-fluorophenyl)amino]-2-oxoeth-
oxy}-4-methoxyphenyl)-N-hydroxyacrylamide (IF1). Mp: 139–
141 °C. EI-MS (m/z): 395.0 (M+). 1H NMR (400 MHz, (CD3)2SO) d
7.97 (d, J = 2 Hz, 1H), 7.55 (d, J = 2 Hz, 1H), 7.42 (d, J = 6 Hz, 1H),
7.37 (d, J = 4 Hz, 1H), 7.21 (s, 2H), 7.06 (d, J = 4 Hz, 1H), 6.30 (d,
J = 8 Hz, 1H), 4.74 (s, 2H), 3.84 (s, 3H).

2712
W. Lu et al. / Bioorg. Med. Chem. 22 (2014) 2707–2713
(d, J = 4 Hz, 1H), 6.92–6.88 (m, 1H), 6.75 (d, J = 4 Hz, 1H), 6.68 (d,
J = 6 Hz, 1H), 6.60–6.56 (m, 1H), 3.81 (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).
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).
5.1.6.6.
(2E)-N-(2-Aminophenyl)-3-(3-{2-[(2-fluorophenyl)
(IF15).
5.1.6. (2E)-N-(2-Aminophenyl)-3-[3-(2-{[3-bromo-5-(trifluoro-
methyl)phenyl]amino}-2-oxoethoxy)-4-methoxyphenyl]acryl-
amide (IF18)
amino]-2-oxoethoxy}-4-methoxyphenyl)acrylamide
Mp: 193–194 °C. EI-MS (m/z): 435.1 (M+). 1H NMR (400 MHz,
(CD3)2SO) d 7.98 (d, J = 8 Hz, 1H), 7.49 (d, J = 8 Hz, 1H), 7.35–7.28
(m, 4 H), 7.20 (d, J = 2 Hz, 1H), 7.18 (d, J = 2 Hz, 1H), 7.11 (d,
J = 4 Hz, 1H), 6.93 (d, J = 2 Hz, 1H), 6.77 (d, J = 4 Hz, 1H), 6.74 (s,
1H), 6.58 (d, J = 8 Hz, 1H), 4.83 (s, 2H), 3.87 (s, 3H).
To a suspension of 5a (0.85 g, 3 mmol) in dehydrated acetone
(70 mL) was added anhydrous potassium carbonate (1.30 g,
9 mmol). The mixture was stirred at room temperature for
30 min and then 2-chloro-N-(3-bromo-5(trifluoromethyl)acetam-
ide (3.33 g, 15 mmol) was added. The reaction mixture was re-
fluxed 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 re-
moved. The crude product was purified by silica gel column chro-
matography (PE/AcOEt = 3:1 to 1:3) to yield (IF18) as white solid
(0.98 g, 58.0%). Mp: 193–195 °C. EI-MS (m/z): 563.0 (M+). 1H
NMR (400 MHz, (CD3)2SO) d 8.22 (s, 1H), 8.08 (s, 1H), 7.69 (s,
1H), 7.49 (d, J = 8 Hz, 1H), 7.33–7.28 (m, 2H), 7.29 (d, J = 4 Hz,
1H), 7.11 (d, J = 4 Hz, 1H), 6.91 (d, J = 6 Hz, 1H), 6.76 (s, 1H), 6.74
(d, J = 2 Hz, 1H), 6.58 (d, J = 8 Hz, 1H), 4.80 (s, 2H), 3.86 (s, 3H).
Compounds (IF10–IF27) were prepared following the proce-
dure described above.
5.1.6.7. (2E)-N-(2-Aminophenyl)-3-(3-{2-[(3-fluorophenyl)ami
no]-2-oxoethoxy}-4-methoxyphenyl)acrylamide
(IF16). Mp:
199–201 °C. EI-MS (m/z): 435.1 (M+). 1H NMR (400 MHz, (CD3)2-
SO) d 7.65 (d, J = 6 Hz, 1H), 7.48 (d, J = 8 Hz, 1H), 7.39 (d, J = 2 Hz,
1H), 7.39–7.37 (m, 1H), 7.33 (d, J = 4 Hz, 1H), 7.28 (d, J = 4 Hz,
1H), 7.27 (s, 1H), 7.10 (d, J = 4 Hz, 1H), 6.94–6.90 (m, 2H), 6.76
(s, 1H), 6.73 (d, J = 4 Hz, 1H), 6.58 (d, J = 8 Hz, 1H), 4.77 (s,
2H), 3.86 (s, 3H).
5.1.6.8.
methyl)phenyl]amino}-2-oxoethoxy)-4-methoxyphenyl]acrylam-
ide (IF17).
Mp: 231–233 °C. EI-MS (m/z):553.3 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 8.38 (s, 2H), 7.83 (s, 1H), 7.49 (d, J = 8 Hz,
1H), 7.33–7.29 (m, 2H), 7.30 (d, J = 4 Hz, 1H), 7.12 (d, J = 4 Hz, 1H),
6.91 (d, J = 8 Hz, 1H), 6.76 (d, J = 4 Hz, 1H), 6.76 (s, 1H), 6.57 (d,
J = 8 Hz, 1H), 4.83 (s, 2H), 3.86 (s, 3H).
(2E)-N-(2-Aminophenyl)-3-[3-(2-{[3,5-bis(trifluoro-
5.1.6.1.
(2E)-N-(2-Aminophenyl)-3-(3-{2-[(3-chloro-4-fluoro-
phenyl)amino]-2-oxoethoxy}-4-methoxyphenyl)acrylamide
(IF10). Mp: 191–193 °C. EI-MS (m/z): 469.1. (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.99 (d, J = 6 Hz, 1H), 7.91 (d, J = 4 Hz,
1H), 7.59–7.55 (m, 1H), 7.50 (s, 1H), 7.42 (d, J = 4 Hz, 1H),
7.40 (d, J = 6 Hz, 1H), 7.21–7.19 (m, 1H), 6.91 (d, J = 6 Hz, 1H),
6.76 (s, 1H), 6.73 (d, J = 4 Hz, 1H), 6.58 (d, J = 6 Hz, 1H), 4.94
(s, 2H), 3.86 (s, 3H).
5.1.6.9. (2E)-N-(2-Amino-4-methylphenyl)-3-(3-{2-[(3-chloro-4-
fluorophenyl)amino]-2-oxoethoxy}-4-methoxyphenyl)
amide (IF19).
NMR (400 MHz, (CD3)2SO)
J = 4 Hz, 1H), 7.40 (d, J = 6 Hz, 1H), 7.26 (s, 2H), 7.17 (d, J = 4 Hz,
1H), 7.10 (d, J = 4 Hz, 1H), 6.75 (d, J = 2 Hz, 1H), 6.65 (d, J = 4 Hz,
1H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz, 1H), 4.76 (s, 2H), 3.86 (s, 3H),
2.16 (s, 3H).
acryl-
Mp: 170–171 °C. EI-MS (m/z): 483.1 (M+). 1H
d 7.98 (d, J = 4 Hz, 1H), 7.44 (d,
5.1.6.2. (2E)-N-(2-Aminophenyl)-3-[4-methoxy-3-(2-oxo-2-{[3-
(trifluoromethyl)phenyl]amino}ethoxy)phenyl]acrylamide
(IF11). Mp: 188–189 °C. EI-MS (m/z): 485.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 8.15 (s, 1H), 7.87 (d, J = 4 Hz, 1H), 7.61–
7.57 (m, 1H), 7.49 (d, J = 8 Hz, 1H), 7.46 (d, J = 6 Hz, 1H), 7.33 (d,
J = 2 Hz, 1H), 7.29 (s, 1H), 7.28 (d, J = 2 Hz, 1H), 7.11 (d, J = 4 Hz,
1H), 6.93–6.89 (m, 1H), 6.77–6.73 (m, 2H), 6.57 (d, J = 6 Hz, 1H),
4.80 (s, 2H), 3.86 (s, 3H).
5.1.6.10. (2E)-N-(2-Amino-4-methylphenyl)-3-[4-methoxy-3-(2-
oxo-2-{[3-(trifluoromethyl)phenyl]amino}ethoxy)phenyl] acryl-
amide (IF20).
NMR (400 MHz, (CD3)2SO) d 8.15 (s, 1H), 7.87 (d, J = 4 Hz, 1H), 7.59
(d, J = 8 Hz, 1H), 7.47 (d, J = 4 Hz, 1H), 7.45 (d, J = 4 Hz, 1H), 7.28 (s,
1H), 7.27 (d, J = 4 Hz, 1H), 7.17 (d, J = 4 Hz, 1H), 7.10 (d, J = 4 Hz,
1H), 6.76–6.70 (m, 1H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz, 1H), 4.79 (s,
2H), 3.86 (s, 3H), 2.17 (s, 3H).
Mp: 192–194 °C. EI-MS (m/z): 499.1 (M+). 1H
5.1.6.3. (2E)-N-(2-Aminophenyl)-3-(3-{2-[(3,4-dichlorophenyl)
amino]-2-oxoethoxy}-4-methoxyphenyl)acrylamide (IF12).
Mp: 209–210 °C. EI-MS (m/z): 485.0 (M+). 1H NMR (400 MHz,
(CD3)2SO) d 8.06 (s, 1H), 7.61 (d, J = 4 Hz, 1H), 7.59 (d, J = 8 Hz,
1H), 7.51 (d, J = 4 Hz, 1H), 7.33 (d, J = 6 Hz, 1H), 7.28 (d,
J = 4 Hz, 1H), 7.27 (s, 1H), 7.10 (d, J = 4 Hz, 1H), 6.93–6.89 (m,
2H), 6.73 (d, J = 4 Hz, 1H), 6.57 (d, J = 6 Hz, 1H), 4.77 (s, 2H),
3.86 (s, 3H).
5.1.6.11. (2E)-N-(2-Amino-4-methylphenyl)-3-(3-{2-[(3,4-dichlo-
rophenyl)amino]-2-oxoethoxy}-4-methoxyphenyl)acrylamide
(IF21).
(400 MHz, (CD3)2SO) d 8.05 (s, 1H), 7.61 (d, J = 4 Hz, 1H), 7.59
(d, J = 6 Hz, 1H), 7.46 (d, J = 8 Hz, 1H), 7.27 (d, J = 4 Hz, 1H), 7.26
(s, 1H), 7.17 (d, J = 4 Hz, 1H), 7.10 (d, J = 4 Hz, 1H), 6.72 (d,
J = 4 Hz, 1H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz, 1H), 4.77 (s, 2H),
3.86 (s, 3H), 2.17 (s, 3H).
Mp: 178–180 °C. EI-MS (m/z): 500.3 (M+). 1H NMR
5.1.6.4. (2E)-N-(2-Aminophenyl)-3-(4-methoxy-3-{2-[(4-meth
oxyphenyl)amino]-2-oxoethoxy}phenyl)acrylamide
(IF13).
Mp: 190–191 °C. EI-MS (m/z): 447.1 (M+). 1H NMR (400 MHz,
(CD3)2SO) d 7.56 (d, J = 4 Hz, 2H), 7.48 (d, J = 8 Hz, 1H), 7.33 (d,
J = 4 Hz, 1H), 7.28 (s, 1H), 7.27 (d, J = 4 Hz, 1H), 7.10 (d, J = 4 Hz,
1H), 6.93 (d, J = 2 Hz, 1H), 6.91 (d, J = 4 Hz, 2H), 6.77–6.73 (m,
2H), 6.58 (d, J = 8 Hz, 1H), 4.72 (s, 2H), 3.86 (s, 3H), 3.73 (s, 3H).
5.1.6.12. (2E)-N-(2-Amino-4-methylphenyl)-3-(4-methoxy-3-{2-
[(4-methoxyphenyl)amino]-2-oxoethoxy}phenyl)acrylamide
(IF22).
Mp: 184–186 °C. EI-MS (m/z): 461.2 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.56 (d, J = 6 Hz, 2H), 7.49 (d, J = 2 Hz, 1H),
7.27 (s, 1H), 7.26 (d, J = 4 Hz, 1H), 7.18 (d, J = 4 Hz, 1H), 7.10 (d,
J = 6 Hz, 1H), 6.74 (d, J = 6 Hz, 1H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz,
1H), 4.71 (s, 2H), 3.87 (s, 3H), 3.73 (s, 3H), 2.17 (s, 3H).
5.1.6.5. (2E)-N-(2-Aminophenyl)-3-(3-{2-[(4-bromophenyl)ami-
no]-2-oxoethoxy}-4-methoxyphenyl)acrylamide
(IF14).
Mp:
217–219 °C. EI-MS (m/z): 497.1 (M+). 1H NMR (400 MHz, (CD3)2SO)

W. Lu et al. / Bioorg. Med. Chem. 22 (2014) 2707–2713
2713
5.1.6.13. (2E)-N-(2-Amino-4-methylphenyl)-3-(3-{2-[(4-bromo-
phenyl)amino]-2-oxoethoxy}-4-methoxyphenyl)acrylamide
(IF23).
Mp: 198–200 °C. EI-MS (m/z): 509.0 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.64 (d, J = 6 Hz, 2H), 7.55 (d, J = 6 Hz,
2H), 7.46 (d, J = 8 Hz, 1H), 7.26 (s, 1H), 7.17 (d, J = 4 Hz, 1H),
7.10 (d, J = 4 Hz, 1H), 6.73 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz,
1H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz, 1H), 4.75 (s, 2H), 3.86 (s,
3H), 2.17 (s, 3H).
5.3. Cell growth inhibitory activity in cancer cells16
The six potent compounds were tested for their antiprolifera-
tive potency against breast cancer cell line (MDA-MB-231) and
cervical cancer cell (HeLa) using MTT method. Exponentially grow-
ing cells were harvested and plated in 96-well plates at a concen-
tration of 1 ꢀ 104 cells/well, and then incubated for 24 h at 37 °C.
The cells in the wells were treated with the title 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. Absorbance values were determined by a microplate reader
(Bio-Rad Instruments) at 570 nm. The IC50 values were calculated
according to inhibition ratios.
5.1.6.14. (2E)-N-(2-Amino-4-methylphenyl)-3-(3-{2-[(2-fluoro-
phenyl)amino]-2-oxoethoxy}-4-methoxyphenyl) acrylamide
(IF24).
Mp: 184–186 °C. EI-MS (m/z): 449.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.97 (d, J = 8 Hz, 1H), 7.47 (d, J = 8 Hz,
1H), 7.31 (d, J = 4 Hz, 2H), 7.30 (s, 2H), 7.28 (d, J = 2 Hz, 1H),
7.21 (d, J = 2 Hz, 1H), 7.20 (d, J = 2 Hz, 1H), 7.11 (d, J = 4 Hz, 1H),
6.78–6.62 (m, 2H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz, 1H), 4.83 (s,
2H), 3.87 (s, 3H), 2.17 (s, 3H).
5.4. Molecular docking modeling17
Molecule docking was carried out 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.
Compound (IF6) was depicted with Sybyl/Sketch module (Tripos
Inc.) and optimized applying Powell’s method with Tripos force
field with convergence criterion set at 0.05 kcal/(Å mol), and
assigned with Gasteiger–Hückel method. Other docking parame-
ters were kept at default.
5.1.6.15. (2E)-N-(2-Amino-4-methylphenyl)-3-(3-{2-[(3-fluoro-
phenyl)amino]-2-oxoethoxy}-4-methoxyphenyl)
(IF25).
acrylamide
Mp: 162–164 °C. EI-MS (m/z): 450.1 (M+). 1H NMR
(400 MHz, (CD3)2SO) d 7.65 (d, J = 6 Hz, 1H), 7.58 (d, J = 6 Hz, 1H),
7.46 (d, J = 8 Hz, 1H), 7.38 (s, 1H), 7.37 (d, J = 4 Hz, 1H), 7.27 (s,
1H), 7.17 (d, J = 4 Hz, 1H), 7.10 (d, J = 4 Hz, 1H), 6.93–6.90 (m,
1H), 6.73 (d, J = 2 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).
5.1.6.16.
(2E)-N-(2-Amino-4-methylphenyl)-3-[3-(2-{[3,5-bis
(trifluoromethyl)phenyl]amino}-2-oxoethoxy)-4-methoxyphenyl]
Acknowledgments
acrylamide (IF26).
Mp: 225–227 °C. EI-MS (m/z): 567.1 (M+).
1H NMR (400 MHz, (CD3)2SO) d 8.38 (s, 2H), 7.82 (s, 1H), 7.47 (d,
J = 8 Hz, 1H), 7.29 (d, J = 2 Hz, 1H), 7.28 (s, 1H), 7.17 (d, J = 4 Hz,
1H), 7.11 (d, J = 4 Hz, 1H), 6.72 (d, J = 8 Hz, 1H), 6.55 (s, 1H), 6.39
(d, J = 2 Hz, 1H), 4.82 (s, 2H), 3.86 (s, 3H), 2.17 (s, 3H).
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 81302737 and 81302641), and
Natural Science Basic Research Plan in Shaanxi Province of China
(Program No. 2011K15-05-02).
5.1.6.17. (2E)-N-(2-Amino-4-methylphenyl)-3-[3-(2-{[3-bromo-
Supplementary data
5-(trifluoromethyl)phenyl]amino}-2-oxoethoxy)-4-methoxy-
phenyl]acrylamide (IF27).
Mp: 229–231 °C. EI-MS (m/z):
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.019.
579.0 (M+). 1H NMR (400 MHz, (CD3)2SO) d 8.22 (s, 1H), 8.07
(s, 1H), 7.69 (s, 1H), 7.46 (d, J = 8 Hz, 1H), 7.29 (d, J = 4 Hz, 1H),
7.27 (s, 1H), 7.17 (d, J = 4 Hz, 1H), 7.11 (d, J = 6 Hz, 1H), 6.72 (d,
J = 8 Hz, 1H), 6.56 (s, 1H), 6.39 (d, J = 4 Hz, 1H), 4.79 (s, 2H),
3.86 (s, 3H), 2.17 (s, 3H).
References and notes
1. Venugopal, B.; Evans, T. T. Curr. Med. Chem. 2011, 18, 1658.
2. Petrella, A.; Fontanella, B.; Carratù, A.; Bizzarro, V.; Rodriquez, M.; Parente, L.
Mini-Rev. Med. Chem. 2011, 11, 519.
3. Pontiki, E.; Hadjipavlou-Litina, D. Med. Res. Rev. 2012, 32, 1.
4. Zhang, L.; Lei, J.; Shan, Y.; Yang, H.; Song, M.; Ma, Y. Mini-Rev. Med. Chem. 1999,
2013, 13.
5.2. HDAC inhibitory activity assays15
The HDAC inhibitory activity assay was carried out using Color-
de-Lys™ HDAC colorimetric activity assay kit (Enzo Life Sciences,
Inc.) according to the manufacturer’s instructions. 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 color intensity at 405 nm.
The title compounds and positive control were diluted in buffer
5. Wang, F.; Lu, W.; Zhang, T.; Dong, J.; Gao, H.; Li, P.; Wang, S.; Zhang, J. Bioorg.
Med. Chem. 2013, 21, 6973.
6. Simpson, C. J.; Fitzhenry, M. J.; Stamford, N. P. J. Tetrahedron Lett. 2005, 54,
5211.
7. Zhang, J.; Zhang, Y.; Zhang, S.; Wang, S.; He, L. Bioorg. Med. Chem. Lett. 2010, 20,
718.
8. Zhou, M.; Ning, C.; Liu, R.; He, Y.; Yu, N. Bioorg. Med. Chem. Lett. 2013, 23, 3200.
9. Gediya, L. K.; Belosay, A.; Khandelwal, A.; Purushottamachar, P.; Njar, V. C.
Bioorg. Med. Chem. 2008, 16, 3352.
10. Zhang, J.; Zhang, Y.; Pan, X.; Wang, C.; Hu, Z.; Wang, S.; He, L. Med. Chem. 2012,
8, 145.
11. Dowling, D. P.; Gantt, S. I.; Gattis, S. G.; Fierke, C. A.; Christianson, D. W.
Biochemistry 2008, 47, 13554.
12. Foti, M. C.; Daquino, C.; Geraci, C. J. Org. Chem. 2004, 69, 2309.
13. Zhang, J.; Zhang, Y.; Shan, Y.; Li, N.; Ma, W.; He, L. Eur. J. Med. Chem. 2010, 45,
2798.
14. Hanessian, S.; MacKay, D. B.; Moitessier, N. J. Med. Chem. 2001, 44, 3074.
15. Guan, P.; Sun, F.; Hou, X.; Wang, F.; Yi, F.; Xu, W.; Fang, H. Bioorg. Med. Chem.
2012, 20, 3865.
to various concentrations (20
mL, 0.032 g/mL). HDACs (5
10 L of compounds and 25 L of substrate on 96-well plates. After
incubation for 30 min, Color de Lys™ developer (50 L/well) was
lg/mL, 4
lg/mL, 0.8 lg/mL, 0.16 lg/
l
lL) were incubated at 37 °C with
l
l
l
added to stop HDAC reactions. Incubate plate at 37 °C for 15 min
and read plate in microtiter-plate reader at 405 nm. The inhibition
rates were calculated from ultraviolet absorption readings of inhib-
ited wells related to those of control wells. The IC50 values were
calculated according to inhibition ratios.
16. Wang, C.; Gao, H.; Dong, J.; Zhang, Y.; Su, P.; Shi, Y.; Zhang, J. Bioorg. Med. Chem.
2014, 22, 277.
17. Zhang, Y.; Feng, J.; Liu, C.; Zhang, L.; Jiao, J.; Fang, H.; Su, L.; Zhang, X.; Zhang, J.;
Li, M.; Wang, B.; Xu, W. Bioorg. Med. Chem. 2010, 18, 1761.