Design, synthesis and preliminary biological evaluation of N-hydroxy-4-(3-phenylpropanamido)benzamide (HPPB) derivatives as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.06.010

A novel series of N-hydroxy-4-(3-phenylpropanamido)benzamide (HPPB) derivatives comprising N-hydroxybenzamide group as zinc-chelating moiety were designed, synthesized and evaluated for their ability to inhibit histone deacetylases. These compounds posses

Full text of DOI:10.1016/j.ejmech.2009.06.010

European Journal of Medicinal Chemistry 44 (2009) 4470–4476
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and preliminary biological evaluation of N-hydroxy
-4-(3-phenylpropanamido)benzamide (HPPB) derivatives as novel histone
deacetylase inhibitors
*
Jie Jiao, Hao Fang, Xuejian Wang, Peng Guan, Yumei Yuan, Wenfang Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, 250012 Ji’nan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel series of N-hydroxy-4-(3-phenylpropanamido)benzamide (HPPB) derivatives comprising
Received 26 February 2009
Received in revised form
29 May 2009
Accepted 8 June 2009
Available online 17 June 2009
N-hydroxybenzamide group as zinc-chelating moiety were designed, synthesized and evaluated for their
ability to inhibit histone deacetylases. These compounds possessed inhibitory activity against the
enzymes with IC50 values as low as 4.0
(IC50 ¼ 0.3
m
M. Among them, the thiophene substituted derivative 5j
M) exhibited good antiproliferative
m
M) and benzo[d][1,3]dioxole derivative 5t (IC50 ¼ 0.4
m
activity against the growth of human colon carcinoma cell line HCT116 and non-small cell lung cancer
cell (NSCLC) line A549. In addition, they were found to potently induce cell-cycle arrest at G2 phase.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
HPPB
N-Hydroxybenzamide
Histone deacetylase inhibitors
Anticancer agents
Cell-cycle arrest
1. Introduction
apoptotic cell death of transformed cells in vitro and vivo [4,5].
Some HDACi have been developed into clinical trials for therapy of
Histone deacetylases (HDACs) take charge of modification
and remodeling of chromosomal histones by removing acetyl
groups from 3-NH2 of lysine residues in histones through zinc-
various solid tumors and hematological malignancy [6–8]. One
inhibitor, Vorinostat (ZolinzaÒ, SAHA), has been approved by FDA
for treatment of cutaneous T-cell lymphoma (CTCL) in 2006 and
became the first HDACi therapeutic agent [9]. Vorinostat is
a representative linear fatty chain HDACi, and the common
features of this class of HDACi have been summarized by Manfred
and Soon (Fig. 1) [10,11]. There are two rules to the structure–
activity relationship (SAR) of Vorinostat-like derivatives: 1) intro-
duction of hydrophobic groups to the para position of the benzene
ring results in a higher level of activity, and 2) the optimal chain
length between the benzene ring and zinc-binding group (ZBG) is
7–8 atoms. As reported in the literature [8,12], three structural
units are essential for an HDACi, an aromatic group (Ar), a zinc-
binding group (ZBG) and a spacer linking Ar and ZBG. N-Hydroxy-
4-(3-phenylpropanamido)benzamide (HPPB) [13], another HDACi
which contains N-hydroxybenzamide as zinc-chelating moiety
could provide a molecular framework for the design of novel
HPPB-like HDACi. However, so far the structure–activity relation-
ship (SAR) information for HPPB-like derivatives was not reported.
In this paper, the design, synthesis and biological evaluation of
a novel series of HPPB derivatives as potent HDACi has been
described.
dependent hydrolysis [1]. Acetylation results in the positive charge
density on the N-termini of nucleosomal histone increases, which
strengthens the interaction with the negative-charged DNA chain
and blocks the access of RNA polymerase and transcription factor
(TF) to DNA [2].
In many cancer cell lines, HDACs are over-expressed resulting in
excessive deacetylated histones being tightly packed with DNA to
form an abnormal ‘‘compact structure’’ of chromatin. In this
process, expression of the cell-cycle inhibitor p21WAF1 is inhibited
and activity of the onco-suppressor p53 is down-regulated,
whereas tumor activators HIF-1 and VEGF are up-regulated.
Therefore, the inhibition of HDAC activity is considered as
a potential strategy for cancer therapy [3]. HDAC inhibitors
(HDACi) become epigenetic anticancer agents due to their capacity
to achieve significant biological effects in preclinical models of
cancer. They can induce cell growth arrest, differentiation and
* Corresponding author. Tel./fax: þ86 531 88382264.
E-mail address: xuwenf@sdu.edu.cn (W. Xu).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.010

J. Jiao et al. / European Journal of Medicinal Chemistry 44 (2009) 4470–4476
4471
Optimal chain length 7~8atoms
spacer
R
ZBG
Introduction of hydrophobic
groups is better
8 atoms
O
H
N
NHOH
O
Vorinostat
O
Zn2+ chelating
moiety
O
HN
O
R
NHOH
8 atoms
O
O
O
HN
O
O
HN
O
NHOH
NHOH
O
N-hydroxybenzamide
HPPB
HPPB derivatives
Fig. 1. Structural features of HPPB derivatives.
2. Chemistry
one is n-butyl substituted derivative 5e which is still less potent
than the benzylether derivative 5a. Compound 5j, the thiophene
analogue of 5a, was 1-fold more potent than 5a, whereas
compound 5i, the pyridine analogue of 5a, showed very poor
inhibitory potency. Compounds 5k–s are various substituted
derivatives of the lead compound 5a. Among them, ortho bromine
substituted benzylether derivative 5n was the most potent one and
nearly as potent as Vorinostat. Shifting the Br from ortho position
into the para (5p) or meta (5o) position resulted in a little loss of
inhibitory potency, while the chlorine substituted benzylether
derivatives (5q–s) and methyl substituted benzylether derivatives
(5k–m) were all less potent than 5n. In addition, benzo[d][1,3]-
dioxole derivative 5t exhibited higher inhibitory potency than
Vorinostat. In general, this series of compounds showed potent
inhibitory activities against HDACs. Among them, compounds 5a,
5j, 5n, 5o, 5p and 5t were found to be more potent than the
paradigmatic hydroxamate HDACi-HPPB, furthermore 5j and 5t
exhibited higher inhibitory activity than Vorinostat. Therefore,
compounds 5a, 5j, 5n and 5t were chosen as the representative
active ones for evaluation of their antiproliferative activities against
the growth of cancer cells. Human colon carcinoma cell line HCT116
was the primary antiproliferative model in vitro because HDACs
express very highly in this cell line. Besides it, non-small cell lung
cancer cell line A549 is a minor model in vitro due to its low
expressed level of HDACs. The difference of the expressed level of
HDACs between the two cancer cell lines was utilized for better
elucidation of anticancer activities of the compounds. The anti-
proliferative activities were determined by MTT assays and the
results have been summarized in Table 2. These compounds were
found to have potent antiproliferative activities compared with
Vorinostat. Compound 5j greatly inhibits the carcinoma cells’
growth with IC50 values of 0.03 mM (HCT116) and 0.08 mM (A549),
5t was a little less potent than 5j with IC50 values of 0.06 mM
(HCT116) and 0.5 mM (A549). They are both more potent than
Vorinostat with IC50 values of 0.08 mM (HCT116) and 0.6 mM
(A549). Compound 5a and its bromine analogue 5n were nearly as
potent as Vorinostat. Further study was made on the effects of 5j
and 5t upon the cell-cycle by flow cytometric analysis. It has been
known that many HDACi including Vorinostat can potently induce
cell-cycle arrest at G1 phase or G2 phase which is associated with
Twenty compounds selected for biological evaluation were
prepared as described in Scheme 1. The synthesis of compound 5a
has been described in our previous paper [14], and the synthetic
route has been modified based on chemical experimental details of
this paper. 3-(4-(Benzyloxy)-3-methoxyphenyl)propanoic acid (1)
was coupled with methyl 4-aminobenzoate in the presence of
dimethylpropyl ethyl carbodiimide hydrochloride (EDCI) to obtain
the coupled product (2a). Deprotection of 2a using 10% Pd/C in
MeOH yielded the corresponding phenol product (3). Etherification
of compound 3 with various alkyl bromides and benzyl chloride
generated phenolic ether derivatives 2b–s. Deprotection of methyl
group in compound 3 with boron tribromide produced diphenol
derivative (4), which was closed to form a benzo[d][1,3]dioxole ring
containing derivative (2t) with diiodomethane. Compounds 2a–t
were hydrolyzed with sodium hydroxide to give carboxylic acids,
followed by condensation with isobutylchloroformate (ClCOOBu-i)
and then reaction with hydroxylamine in THF yields the final
hydroximic acid compounds 5a–t.
3. Results and discussion
In vitro bioactivity evaluation of compounds 5a–t was per-
formed by HDAC activity assays using an HDAC colorimetric activity
assay kit (AK501, Biomol Research Laboratories). The source of
HDACs was HeLa nuclear extracts comprising HDAC1 and HDAC2
(the major contributors to HDAC activity in HeLa nuclear extracts),
and the substrate was a type of [3H]acetylated histone peptide.
HDAC1 and HDAC2 are known to be the key nucleus enzymes in
charge of deacetylation of histones [15]. Assays were performed
according to kit instructions. The test results of compounds 5a–t for
inhibitory activity against HDACs in vitro have been presented in
Table 1. Vorinostat was used as positive control and IC50 values for
both HPPB and its derivatives have been determined. These
compounds showed potent inhibitory activities against HDACs
with IC50 values as low as 4.0
(5b–h) with IC50 values ranging from 1.6
the order of increasing activity of R as: n-butyl > n-propyl > s-
butyl > isopentyl z ethyl > methyl > cyclopentyl. The most potent
m
M. The alkyl substituted derivatives
m
M to 4.0 M displayed
m

4472
J. Jiao et al. / European Journal of Medicinal Chemistry 44 (2009) 4470–4476
CONHOH
COOCH3
O
O
COOH
O
a
f
O
O
N
N
BnO
H
H
BnO
BnO
2a
1
5a
b
COOCH3
CONHOH
COOCH3
O
O
O
O
e
f
O
O
N
N
H
N
H
H
RO
RO
HO
3
2b-2s
5b-5s
c
CONHOH
COOCH3
COOCH3
O
O
O
d
f
HO
HO
O
O
O
O
N
H
N
H
N
H
4
2t
5t
Scheme 1. Reagents and conditions: a) EDCI, Et3N, THF, methyl 4-aminobenzoate, r.t.; b) H2, 10% Pd–C, CH3OH, r.t. c) BBr3, CH2Cl2, ꢁ40 ꢂC; d) K2CO3, CH2I2, acetone, reflux; e) KOH,
RBr, DMF, 50 ꢂC; f) 2 mol/L NaOH, EtOH, 75 ꢂC (for two steps); ClCOOBu-s, Et3N, THF, NH2OH$HCl, r.t.
activation of cyclin-dependent kinase (CDK) inhibitor p21 expres-
sion or apoptotic cell death pathway [14]. The effects of 5j and 5t on
the cell-cycle were measured against human HCT116 cells at the
5.1.2. Methyl 4-(3-(4-(benzyloxy)-3-methoxyphenyl)
propanamido)benzoate (2a)
To a solution of 3-(4-(benzyloxy)-3-methoxyphenyl)propanoic
acid 1 (9.0 g, 32 mmol) in dry THF (200 mL) were added EDCI (7.4 g,
38.4 mmol) and Et3N (9.0 mL, 64 mmol) at room temperature. The
mixture was stirred for 2 h and then a solution of methyl 4-amino-
benzoate (4.8 g, 32 mmol) in dry THF (100 mL) was added into this
mixture. After stirring for 6 h, the reacted mixture was concentrated
under reduced pressure. To the residue was added 1 mol/L HCl
(100 mL), and then extracted by ethyl acetate (3 ꢀ100 mL), washed
with water (3 ꢀ100 mL) and dried over Na2SO4. After filtration and
evaporation of the solvent in vacuo, methyl 4-(3-(4-(benzyloxy)-3-
methoxyphenyl)propanamido)benzoate 2a was obtained as white
concentration of 1 mmol/L. The analytic results have been shown in
Table 3. The population proportion of HCT116 cells in each phase of
cell-cycle has been calculated. The two compounds were found to
strongly induce cell-cycle arrest at G2 phase which is associated
with p21-independent apoptotic cell death pathway and the anti-
cancer mechanism of these two compounds is worthy of further
scrutiny in the future.
4. Conclusion
In conclusion, we have synthesized a novel series of HPPB
derivatives with N-hydroxybenzamide as zinc-chelating moiety to
inhibit HDACs. Several HPPB derivatives exhibited subnanomolar
crystal. (9.6 g, 72.5%). 1H NMR (DMSO-d6, 300 MHz)
d 10.25 (s, 1H),
7.92–7.71 (dd, 4H), 7.43–7.31 (m, 5H), 6.94–6.71 (m, 3H), 5.02 (s, 2H),
3.82 (s, 3H), 3.73 (s, 3H), 2.85 (t, 2H, J ¼ 7.5 Hz), 2.64 (t, 2H, J ¼ 7.5 Hz).
ESI-MS m/z 420.4 (MHþ).
inhibitory activities against HDACs with IC50 values below 1
this series, the thiophene substituted derivative 5j (IC50 ¼ 0.3
M) were the
m
M. In
mM)
and benzo[d][1,3]dioxole derivative 5t (IC50 ¼ 0.4
m
5.1.3. Methyl 4-(3-(4-hydroxy-3-methoxyphenyl)
propanamido)benzoate (3)
most active and notably inhibited the growth of human carcinoma
cells HCT116 and A549. In addition, they can potently induce cell-
cycle arrest in G2 phase which exhibits their good anticancer
bioactivities. They can be good lead compounds for the develop-
ment of novel anticancer agents in future.
To a solution of methyl 4-(3-(4-(benzyloxy)-3-methoxyphenyl)
propanamido)benzoate 2a (5.0 g, 12 mmol) in MeOH (100 mL) was
added 10% wt. Pd–C (0.5 g). The reaction mixture was stirred for
10 h under H2 pressure at room temperature, filtered with Celite,
the solvent was removed in vacuo to give methyl 4-(3-(4-hydroxy-
3-methoxyphenyl)propanamido)benzoate 3 as a white solid (3.5 g,
5. Experimental procedure
88.7%). 1H NMR (DMSO-d6, 300 MHz)
d 10.24 (s, 1H), 8.70 (s, 1H),
7.92–7.70 (dd, 4H), 6.80–6.60 (m, 3H), 3.82 (s, 3H), 3.72 (s, 3H), 2.81
5.1. Chemistry
(t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz). ESI-MS m/z 330.3 (MHþ).
5.1.1. General
5.1.4. Methyl 4-(3-(3,4-dihydroxyphenyl)propanamido)
benzoate (4)
All materials and reagents used in this work are analytical
reagents. All reactions were monitored by thin-layer chromatog-
raphy (TLC) on 0.25 mm silica gel plates (60GF254) and visualized
with UV light. 1H NMR spectra were obtained on a Brucker Avance
300 spectrometer using TMS as an internal standard in DMSO-d6
To a solution of methyl 4-(3-(4-hydroxy-3-methoxyphenyl)
propanamido)benzoate 3 (3.0 g, 9 mmol) in dry CH2Cl2 (150 mL) was
added added KOH powder (0.34 g, 6.0 mmol). at ꢁ40 ꢂC. The mixture
was stirred for 1 h at ꢁ40 ꢂC and then the solvent was removed in
vacuo. To the residue was added 1 mol/L Na2CO3 (100 mL), then
extracted by ethyl acetate (3 ꢀ 100 mL), washed with water
(3 ꢀ 50 mL) and dried over MgSO4. After filtration and evaporation of
the solvent in vacuo, methyl 4-(3-(3,4-dihydroxyphenyl)propana
mido)benzoate 4 was obtained as a yellow solid (2.4 g, 83.6%). 1H
solutions. Chemical shifts were reported in ppm (d) downfield from
TMS. All the coupling constants (J) are in hertz. IR spectra were
recorded on a Perkin–Elmer FTIR 1600 spectrometer. ESI-MS
were determined on an API 4000 spectrometer. Melting points
were determined on an electrothermal melting point apparatus
and without correction. All reported yields are for purified
products.
NMR (DMSO-d6, 300 MHz)
d 10.23 (s, 2H), 8.71 (s, 1H), 7.90–7.72

J. Jiao et al. / European Journal of Medicinal Chemistry 44 (2009) 4470–4476
4473
Table 1
The inhibitory activity results of HPPB derivatives against HDACs in vitro.
O
O
NHOH
O
N
H
R
O
Compd
R
HDACs
Compd
R
HDACs
IC50
(
m
M)
IC50
(mM)
5a
0.7
5k
1.8
5b
5c
CH3–
3.3
2.9
5l
2.6
1.5
C2H5–
5m
Br
5d
CH3(CH2)2–
1.7
5n
0.6
Br
Br
5e
5f
CH3(CH2)3–
1.6
2.2
5o
5p
0.9
0.8
CH3CH2(CH3)CH–
Cl
5g
(CH3)2CH(CH2)2–
2.9
5q
1.2
Cl
Cl
5h
5i
4.0
3.9
5r
5s
1.6
1.4
N
O
5j
S
0.3
5t
0.4
O
HPPB
Vorinostat
1.1
0.5
(dd, 4H), 6.79–6.58(m, 3H), 3.83 (s, 3H), 2.80 (t, 2H, J ¼ 7.5 Hz), 2.61 (t,
2H, J ¼ 7.5 Hz). ESI-MS m/z 316.3 (MHþ).
stirred for 1 h and then CH3I (1.3 g, 9.0 mmol) was added at room
temperature. The mixture was heated to 50 ꢂC and stirred for 5 h,
then was added H2O (50 mL) and then extracted by ether
(3 ꢀ 50 mL). The organic layer was merged and washed with water
(3 ꢀ 50 mL) and dried over MgSO4. After filtration and evaporation
of the solvent in vacuo, methyl 4-(3-(3,4-dimethoxyphenyl)
propanamido)benzoate 2b was obtained as a yellow solid (0.9 g,
5.1.5. Methyl 4-(3-(3,4-dimethoxyphenyl)propanamido)
benzoate (2b)
To a solution of methyl 4-(3-(4-hydroxy-3-methoxyphenyl)
propanamido)benzoate 3 (1.0 g, 3 mmol) in DMF (10 mL) was
added KOH powder (0.34 g, 6.0 mmol). The reaction mixture was
87.4%). 1H NMR (DMSO-d6, 300 MHz)
d 8.70 (s, 1H), 7.90–7.68

4474
J. Jiao et al. / European Journal of Medicinal Chemistry 44 (2009) 4470–4476
Table 2
Table 3
Growth inhibitory results of HDACi against HCT116 and A549 cell lines (IC50, mM).
The effects of 5j and 5t on the cell-cycle were measured against human HCT116 cells
at the concentration of 1 mol/L, and the population proportion of HCT116 cells in
each phase of cell-cycle has been calculated.
m
Compd
HCT116
A549
5a
5j
5n
5t
0.12
0.03
0.10
0.06
0.08
0.6
0.08
0.6
0.5
0.6
Compd concentration (1
m
mol/L)
Proportion of HCT116 cells (%)
G1 phase
S phase
G2 phase
5j
5t
Control
28.51
17.09
34.60
24.33
34.11
46.55
47.16
48.80
18.85
Vorinostat
(dd, 4H), 6.79–6.63 (m, 3H), 3.83 (s, 3H), 3.73 (s, 6H), 2.80 (t, 2H,
J ¼ 7.5 Hz), 2.65 (t, 2H, J ¼ 7.5 Hz). ESI-MS m/z 344.4 (MHþ).
5.1.7.4. N-Hydroxy-4-(3-(3-methoxy-4-propoxyphenyl)propanamido)-
benzamide (5d). Yield: 19.2%. M.p. 182–183 ꢂC. IR (KBr): (cmꢁ1
3306, 1671, 1639, 1610, 1592, 1514, 1258. 1H NMR (DMSO-d6,
300 MHz) 11.10 (s, 1H), 10.13 (s, 1H), 8.94 (s, 1H), 7.72–7.62 (dd,
4H), 6.85–6.71 (m, 3H), 3.84 (t, 2H, J ¼ 6.6 Hz), 3.72 (s, 3H), 2.85 (t,
2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz), 1.75–1.63 (m, 2H), 0.95 (t, 3H,
J ¼ 7.5 Hz). ESI-MS m/z 373.2 (MHþ).
n
)
5.1.6. General procedure for the synthesis of ethers 2c–t
To a solution of methyl 4-(3-(4-hydroxy-3-methoxyphenyl)
propanamido)benzoate 3 (1.0 g, 3 mmol) in DMF (10 mL) was
added KOH powder (0.34 g, 6.0 mmol). The reaction mixture was
stirred for 1 h and then corresponding alkyl halide (9.0 mmol) was
added at room temperature. The mixture was heated to 50 ꢂC and
stirred for 5 h, then H2O was added (50 mL) and extracted by ether
(3 ꢀ 50 mL). The organic layer was merged and washed with water
(3 ꢀ 50 mL) and dried over MgSO4. After filtration and evaporation
of the solvent in vacuo, the ethers 2c–s were obtained.
d
5.1.7.5. 4-(3-(4-Butoxy-3-methoxyphenyl)propanamido)-N-hydroxy-
benzamide (5e). Yield: 28.2%. M.p. 163–165 ꢂC. IR (KBr):
n
(cmꢁ1
d 11.10 (s, 1H),
)
3302, 1668, 1514, 1255. 1H NMR (DMSO-d6, 300 MHz)
10.13 (s, 1H), 8.95 (s, 1H), 7.71–7.62 (m, 4H), 6.85–6.82 (m, 2H), 6.72
(d, 1H, J ¼ 8.1), 3.88 (t, 2H, J ¼ 6.6), 3.71 (s, 3H), 2.84 (t, 2H,
J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz), 1.70–1.61 (m, 2H), 1.47–1.35
(m, 2H), 0.91 (t, 3H, J ¼ 7.5 Hz). ESI-MS m/z 387.4 (MHþ).
5.1.7. General procedure for the synthesis of hydroxamic acids 5a–t
from methyl esters 2a–t
The corresponding methyl esters 2a–t (5.0 mmol) were dis-
solved in EtOH (50 mL), then 2 mol/L NaOH (10 mL) was added. The
solution was stirred at 75 ꢂC for 5–6 h, and then concentrated
under reduced pressure. To the residue was added 1 mol/L HCl
(50 mL), then the solution was filtered to get the sediment which
was washed with water for several times. The sediment was dried
and dissolved in dry THF (50 mL), then Et3N (1.0 g, 10 mmol) was
added. The solution was stirred under room temperature and
ClCOOBu-i (0.7 g, 5.3 mmol) was dropped into the solution. After
1–3 min, NH2OH/MeOH (5 mL, 2 mmol/mL) was dropped into the
reaction mixture. After stirring for 5 h, the reaction mixture was
filtered and the filtrate was evaporated under reduced pressure. To
the residue was added 1 mol/L HCl (30 mL) and extracted by EtOAc
(3 ꢀ 20 mL) for 3 times. The organic layer was merged and washed
with water (3 ꢀ 20 mL) and then dried with MgSO4. The solution
was filtered and evaporated to give a crude product which was
purified by column chromatography.
5.1.7.6. 4-(3-(4-sec-Butoxy-3-methoxyphenyl)propanamido)-N-hydroxy-
benzamide (5f). Yield: 38.4%. M.p. 156–158 ꢂC. IR (KBr):
n )
(cmꢁ1
3261, 1664, 1626, 1608, 1512, 1259. 1H NMR (DMSO-d6, 300 MHz)
11.09 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H), 7.71–7.62 (dd, 4H), 6.85–
d
6.70 (m, 3H), 4.25–4.19 (m, 1H, J ¼ 6.0 Hz), 3.71 (s, 3H), 2.84 (t, 2H,
J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz), 1.67–1.46 (m, 2H), 1.16 (d, 3H,
J ¼ 6.0 Hz), 0.90 (t, 3H, J ¼ 7.5 Hz). ESI-MS m/z 387.4 (MHþ).
5.1.7.7. N-Hydroxy-4-(3-(4-(isopentyloxy)-3-methoxyphenyl)prop-
anamido)benzamide (5g). Yield: 38.4%. M.p. 171–172 ꢂC. IR (KBr):
n
(cmꢁ1) 3301, 1670, 1643, 1609, 1590, 1515, 1255. 1H NMR (DMSO-
d6, 300 MHz)
d 11.08 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H), 7.71–7.62
(dd, 4H), 6.87–6.71 (m, 3H), 3.91 (t, 2H, J ¼ 6.6 Hz), 3.71 (s, 3H),
2.84 (t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz), 1.81–1.72 (m, 1H),
1.61–1.54 (q, 2H, J ¼ 6.6 Hz), 0.92 (s, 3H), 0.90 (s, 3H). ESI-MS m/z
401.5 (MHþ).
5.1.7.1. 4-(3-(4-(Benzyloxy)-3-methoxyphenyl)propanamido)-N-hydroxy-
5.1.7.8. 4-(3-(4-(Cyclopentyloxy)-3-methoxyphenyl)propanamido)-N-
benzamide (5a). Yield: 28.6%. M.p. 193–195 ꢂC. IR (KBr):
n
(cmꢁ1
10.09 (s,1H),
)
hydroxybenzamide (5h). Yield: 77.0%. M.p. 188–190 ꢂC. IR (KBr):
3266, 1674,1513,1256. 1H NMR (DMSO-d6, 300 MHz)
d
n
(cmꢁ1) 3309, 1670, 1641, 1609, 1593, 1511, 1256. 1H NMR (DMSO-
10.12 (s, 1H), 8.94 (s, 1H), 7.71–7.62 (m, 4H), 7.44–7.29 (m, 5H), 6.92
(d,1H, J ¼ 8.1 Hz), 6.88 (s,1H, J ¼ 8.1 Hz), 6.73 (d,1H, J ¼ 8.1 Hz), 5.02
(s, 2H), 3.73 (s, 3H), 2.85 (t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz).
ESI-MS m/z 421.4 (MHþ).
d6, 300 MHz) d 11.11 (s, 1H), 10.13 (s, 1H), 8.99 (s, 1H), 7.72–7.63
(dd, 4H), 6.84–6.71 (m, 3H), 4.70 (s, 1H), 3.70 (s, 3H), 2.83 (d, 2H,
J ¼ 7.5 Hz), 2.63 (d, 2H, J ¼ 7.5 Hz), 1.81–1.54 (m, 8H). ESI-MS m/z
399.4 (MHþ).
5.1.7.2. 4-(3-(3,4-Dimethoxyphenyl)propanamido)-N-hydroxybenzamide
5.1.7.9. N-Hydroxy-4-(3-(3-methoxy-4-(pyridin-2-ylmethoxy)phenyl)-
(5b). Yield: 19.0%. M.p. 178–179 ꢂC. IR (KBr):
n
(cmꢁ1) 3302, 1662,
propanamido)benzamide (5i). Yield: 57.9%. M.p. 205–206 ꢂC. IR
1608, 1592, 1515, 1255. 1H NMR (DMSO-d6, 300 MHz)
d
11.10 (s, 1H),
(KBr):
(DMSO-d6, 300 MHz)
n
(cmꢁ1) 3290, 1655, 1641, 1603, 1592, 1518, 1257. 1H NMR
10.13 (s, 1H), 8.95 (s, 1H), 7.75–7.62 (dd, 4H), 6.86–6.73 (m, 3H), 3.71
(s, 3H), 3.70 (s, 3H), 2.85 (t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz).
ESI-MS m/z 345.3 (MHþ).
d 11.09 (s, 1H), 10.12 (s, 1H), 8.92 (s, 1H), 8.55
(s, 1H), 7.83 (t, 1H, J ¼ 7.5 Hz), 7.71–7.62 (dd, 4H), 7.50 (d, 1H,
J ¼ 7.8 Hz), 7.33 (s, 1H), 6.93–6.71 (m, 3H), 5.10 (s, 2H), 3.76 (s, 3H),
2.83 (d, 2H, J ¼ 7.5 Hz), 2.63 (d, 2H, J ¼ 7.5 Hz). ESI-MS m/z 422.4
(MHþ).
5.1.7.3. 4-(3-(4-Ethoxy-3-methoxyphenyl)propanamido)-N-hydroxy-
benzamide (5c). Yield: 57.6%. M.p. 179–180 ꢂC. IR (KBr):
n
(cmꢁ1
11.10
)
3299, 1660, 1609, 1515, 1258. 1H NMR (DMSO-d6, 300 MHz)
d
5.1.7.10. N-Hydroxy-4-(3-(3-methoxy-4-(thiophene-2-ylmethoxy)phenyl)-
(s, 1H), 10.13 (s, 1H), 8.95 (s, 1H), 7.75–7.62 (dd, 4H), 6.85–6.70
(m, 3H), 3.94 (d, 2H, J ¼ 6.9 Hz), 3.71 (s, 3H), 2.84 (s, 2H), 2.63 (d, 2H,
J ¼ 7.2 Hz), 1.29 (t, 3H, J ¼ 6.9 Hz). ESI-MS m/z 359.5 (MHþ).
propanamido)benzamide (5j). Yield: 19.4%. M.p. 169–170 ꢂC. IR
(KBr):
(DMSO-d6, 300 MHz)
n
(cmꢁ1) 3305, 1674, 1638, 1609, 1589, 1512, 1255. 1H NMR
d
11.09 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H),

J. Jiao et al. / European Journal of Medicinal Chemistry 44 (2009) 4470–4476
4475
7.71–7.62 (dd, 4H), 7.53 (d, 1H, J ¼ 4.8 Hz), 7.15 (s, 1H), 7.01 (t, 1H,
J ¼ 4.2 Hz), 6.97–6.72 (m, 3H), 5.20 (s, 2H), 3.73 (s, 3H), 2.85 (t, 2H,
J ¼ 7.5 Hz), 2.63 (t, 2H, J ¼ 7.5 Hz). ESI-MS m/z 427.5 (MHþ).
5.1.7.19. 4-(3-(4-(4-Chlorobenzyloxy)-3-methoxyphenyl)propanamido)-
N-hydroxybenzamide (5s). Yield: 17.6%. M.p. 240–242 ꢂC. IR (KBr):
n
(cmꢁ1) 3307,1671,1641,1607,1592,1514,1256. 1H NMR (DMSO-d6,
300 MHz)
d 11.09 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H), 7.71–7.65 (m,
5.1.7.11. 4-(3-(4-(2-Methylbenzyloxy)-3-methoxyphenyl)propanamido)-
4H), 7.44 (s, 4H), 6.91 (d, 2H, J ¼ 8.7 Hz), 6.73 (d, 1H, J ¼ 7.8 Hz), 5.03
(s, 2H), 3.74 (s, 3H), 2.86 (t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz).
ESI-MS m/z 455.3 (MHþ).
N-hydroxybenzamide (5k). Yield: 48.3%. M.p. 173–175 ꢂC. IR (KBr):
n
(cmꢁ1) 3215, 1667, 1608, 1515, 1257. 1H NMR (DMSO-d6, 300 MHz)
d
11.09 (s, 1H), 10.12 (s, 1H), 8.96 (s, 1H), 7.71–7.62 (dd, 4H), 7.38–
7.22 (m, 4H), 6.98–6.76 (m, 3H), 5.00 (s, 2H), 3.73 (s, 3H), 2.86 (t, 2H,
J ¼ 7.5 Hz), 2.63 (t, 2H, J ¼ 7.5 Hz), 2.31 (s, 3H). ESI-MS m/z 435.5
(MHþ).
5.1.7.20. 4-(3-(Benzo[d][1,3]dioxol-5-yl)propanamido)-N-hydroxy-
benzamide (5t). Yield: 19.0%. M.p. 230–232 ꢂC. IR (KBr):
n )
(cmꢁ1
3280, 1658, 1608, 1534, 1501, 1255. 1H NMR (DMSO-d6, 300 MHz)
11.09 (s, 1H), 10.10 (s, 1H), 8.93 (s, 1H), 7.71–7.61 (dd, 4H),
d
5.1.7.12. 4-(3-(4-(3-Methylbenzyloxy)-3-methoxyphenyl)propanamido)-
6.83–6.69 (m, 3H), 5.95 (s, 2H), 2.83 (t, 2H, J ¼ 7.5 Hz), 2.60 (t, 2H,
N-hydroxybenzamide (5l). Yield: 19.4%. M.p. 178–179 ꢂC. IR (KBr):
J ¼ 7.5 Hz). ESI-MS m/z 329.4 (MHþ).
n
(cmꢁ1) 3308, 1673, 1643, 1610, 1590, 1513, 1257. 1H NMR (DMSO-
d6, 300 MHz) d 10.99 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H), 7.71–7.62 (dd,
5.2. HDAC inhibitory activity assays
4H), 7.29–7.13 (m, 4H), 6.93–6.71 (m, 3H), 4.97 (s, 2H), 3.71 (s, 3H),
2.85 (t, 2H, J ¼ 7.5 Hz), 2.63 (t, 2H, J ¼ 7.5 Hz), 2.31 (s, 3H). ESI-MS m/
z 435.4 (MHþ).
We performed assays according to kit instructions. The source of
HDACs was HeLa nuclear extracts including HDAC1 and HDAC2 (the
major contributors to HDAC activity in HeLa nuclear extracts), and
the substrate was a type of [3H]acetylated histone peptide. Both
HDAC1 and HDAC2 are known to be nucleus proteins in charge of the
deacetylation of histones. The compound samples and the control
drug were diluted to various concentrations. On the 96-well plate,
5.1.7.13. 4-(3-(4-(4-Methylbenzyloxy)-3-methoxyphenyl)propanamido)-
N-hydroxybenzamide (5m). Yield: 58.0%. M.p. 216–218 ꢂC. IR (KBr):
n
(cmꢁ1) 3314, 1675,1592, 1513, 1254. 1H NMR (DMSO-d6, 300 MHz)
d
11.09 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H), 7.87–7.62 (m, 4H), 7.31–7.16
HDACs (5
concentrations of samples and 25
30 min, Color de Lys Developer (50
m
L/well) were incubated at 37 ꢂC with 10
L of substrate. After reacting for
L/well) was added. Then, after
mL of various
(dd, 4H), 6.92–6.70 (m, 3H), 4.97 (s, 2H), 3.73 (s, 3H), 2.85 (t, 2H,
J ¼ 7.5 Hz), 2.64 (t, 2H, J ¼ 7.5 Hz), 2.30 (s, 3H). ESI-MS m/z 435.5
(MHþ).
m
m
15 min the ultraviolet absorption of the wells was measured on
a microtiter-plate reader at 405 nm. The % inhibition was calculated
fromthe ultraviolet absorption readings of inhibitedwells relative to
those of control wells. Finally, the IC50 values were determined using
a regression analysis of the concentration/inhibition data.
5.1.7.14. 4-(3-(4-(2-Bromobenzyloxy)-3-methoxyphenyl)propanamido)-
N-hydroxybenzamide (5n). Yield: 16.2%. M.p. 162–164 ꢂC. IR (KBr):
n
(cmꢁ1) 3246, 1661, 1595, 1514, 1558. 1H NMR (DMSO-d6, 300 MHz)
11.07 (s,1H),10.11 (s,1H), 8.93 (s,1H), 7.72–7.29 (m, 8H), 6.94–6.74
d
(m, 3H), 5.05 (s, 2H), 3.75 (s, 3H), 2.87 (s, 2H), 2.64 (s, 2H). ESI-MS
m/z 499.3/501.3 (MHþ).
5.3. Antiproliferative activity evaluation of HDAC inhibitors
by MTT assays
5.1.7.15. 4-(3-(4-(3-Bromobenzyloxy)-3-methoxyphenyl)propanamido)-
N-hydroxybenzamide (5o). Yield: 16.2%. M.p. 200–202 ꢂC. IR (KBr):
HCT116 cells were maintained in McCoy’s 5a medium with
10% fetal bovine serum (FBS) while A549 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS. Appropriate numbers of cells (2.0 ꢀ 105/mL) were
n
(cmꢁ1) 3285, 1671, 1611, 1512, 1256. 1H NMR (DMSO-d6, 300 MHz)
d
11.09 (s, 1H),10.12 (s, 1H), 8.93 (s,1H), 7.71–7.62 (m, 5H), 7.53–7.32
(m, 3H), 6.91 (d, 2H, J ¼ 8.7 Hz), 6.73 (d, 1H, J ¼ 8.1 Hz), 5.04 (s, 2H),
3.75 (s, 3H), 2.85 (t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz). ESI-MS
499.3/501.3 (MHþ).
inoculated into 96-well plates (50 mL/well). After 4 h, compounds
of various concentrations were dosed, and the cells were
cultured for 2 days. Then 0.5% MTT (10 L/well) was added into
m
5.1.7.16. 4-(3-(4-(4-Bromobenzyloxy)-3-methoxyphenyl)propanamido)-
each well. After additional 4 h incubation, OD570 and OD630 as
references were measured, and the IC50 values were calculated
according to a regression analysis of the concentration/inhibition
data.
N-hydroxybenzamide (5p). Yield: 16.2%. M.p. 208–210 ꢂC. IR (KBr):
n
(cmꢁ1) 3313,1674,1637, 1607,1591, 1514, 1256. 1H NMR (DMSO-d6,
300 MHz)
d 11.09 (s, 1H), 10.12 (s, 1H), 8.93 (s, 1H), 7.71–7.56 (m,
6H), 7.38 (d, 2H, J ¼ 7.8 Hz), 6.90 (d, 1H, J ¼ 8.4 Hz), 6.74 (s, 1H), 5.01
(s, 2H), 3.74 (s, 3H), 2.85 (t, 2H, J ¼ 7.5 Hz), 2.62 (t, 2H, J ¼ 7.5 Hz).
ESI-MS m/z 499.2/501.2 (MHþ).
Acknowledgments
This work was supported by National ‘‘863’’ Foundation (No.
2007AA02Z314) of PR China and the National Natural Foundation
Research Grant (Grant Nos. 30772654; 36072541).
5.1.7.17. 4-(3-(4-(2-Chlorobenzyloxy)-3-methoxyphenyl)propanamido)-
N-hydroxybenzamide (5q). Yield: 35.0%. M.p. 206–208 ꢂC. IR (KBr):
n
(cmꢁ1) 3253, 1663, 1609, 1594, 1514, 1259. 1H NMR (DMSO-d6,
300 MHz)
d 11.09 (s, 1H), 10.12 (s, 1H), 8.93 (s, 1H), 7.71–7.56 (m,
References
4H), 7.52–7.35 (m, 4H), 6.95–6.70 (m, 3H), 5.08 (s, 2H), 3.74 (s, 3H),
2.86 (t, 2H, J ¼ 7.5 Hz), 2.61 (t, 2H, J ¼ 7.5 Hz). ESI-MS m/z 455.3
(MHþ).
[1] S. Minucci, P.G. Pelicci, Nat. Rev. Cancer 6 (2006) 38–51.
[2] G. Legube, D. Trouche, EMBO Rep. 4 (2003) 944–947.
[3] D.H. Kim, M. Kim, H.J. Kwon, J. Biochem. Mol. Biol. 36 (2003) 110–119.
[4] J.S. Carew, F.J. Giles, S.T. Nawrocki, Cancer Lett. 269 (2008) 7–17.
[5] P.A. Marks, V.M. Richon, R.A. Rifkind, J. Natl. Cancer Inst. 92 (2000)
1210–1216.
[6] M.R. Acharya, A. Sparreboom, J. Venitz, W.D. Figg, Mol. Pharmacol. 68 (2005)
917–932.
[7] C. Monneret, Eur. J. Med. Chem. 40 (2005) 1–13.
[8] M. Paris, M. Porcelloni, M. Binaschi, D. Fattori, J. Med. Chem. 51 (2008)
1505–1529.
5.1.7.18. 4-(3-(4-(3-Chlorobenzyloxy)-3-methoxyphenyl)propanamido)-
N-hydroxybenzamide (5r). Yield: 35.0%. M.p. 238–240 ꢂC. IR (KBr):
n
(cmꢁ1) 3260, 1671, 1644, 1610, 1591, 1513, 1256. 1H NMR (DMSO-d6,
300 MHz) d 11.09 (s, 1H), 10.12 (s, 1H), 8.94 (s, 1H), 7.71–7.62 (m, 4H),
7.49–7.39 (m, 3H), 6.93–6.72 (m, 3H), 5.05 (s, 2H), 3.74 (s, 3H), 2.86 (t,
2H, J ¼ 7.5 Hz), 2.63 (t, 2H, J ¼ 7.5 Hz). ESI-MS m/z 455.3 (MHþ).

4476
J. Jiao et al. / European Journal of Medicinal Chemistry 44 (2009) 4470–4476
[9] D. Madeleine, V. Jenny, Expert Opin. Investig. Drugs 16 (2007) 1111–1120.
[10] M. Jung, G. Brosch, D. Kolle, H. Scherf, C. Gerhauser, P. Loidl, J. Med. Chem. 42
(1999) 4669–4679.
[12] T.A. Miller, D.J. Witter, S. Belvedere, J. Med. Chem. 46 (2003) 5097–5116.
[13] Q. Lu, D.S. Wang, C.S. Chen, Y.D. Hu, C.S. Chen, J. Med. Chem. 48 (2005)
5530–5535.
[11] S.H. Woo, S. Frechette, E.A. Khalil, G. Bouchain, A. Vaisburg, N. Bernstein,
O. Moradei, S. Leit, M. Allan, M. Fournel, M. Trachy-Bourget, Z. Li,
J.M. Besterman, D. Delorme, J. Med. Chem. 45 (2002) 2877–2885.
[14] J. Jiao, Q. Wang, H.W. Zhu, F. Hao, W.F. Xu, Chin. Chem. Lett. 19 (2008)
673–675.
[15] L. Pan, J. Lu, B. Huang, Cell. Mol. Immunol. 4 (2007) 337–343.