Synthesis and antitumor activity of novel diaryl ether hydroxamic acids derivatives as potential HDAC inhibitors

Article abstract of DOI:10.1007/s12272-012-1003-0

A series of diaryl ether hydroxamic acids were synthesized for the first time and evaluated for the HDAC biology and antiproliferative activity. The structures of these new hydroxamic acids derivatives were confirmed by IR, ¹H-NMR and mass spectrum. Some of these compounds showed micro molar activity in the HDAC inhibitory assay and against four cancer cell lines.

Full text of DOI:10.1007/s12272-012-1003-0

Arch Pharm Res Vol 35, No 10, 1723-1732, 2012
DOI 10.1007/s12272-012-1003-0
Synthesis and Antitumor Activity of Novel Diaryl Ether Hydroxamic
Acids Derivatives as Potential HDAC Inhibitors
Yong Zhu, Xin Chen, Zeng Wu, Yixuan Zheng, Yadong Chen, Weifang Tang, and Tao Lu
Department of Organic Chemistry, China Pharmaceutical University, Nanjing 210009, China
(Received November 16, 2011/Revised March 23, 2012/Accepted May 14, 2012)
A series of diaryl ether hydroxamic acids were synthesized for the first time and evaluated for
the HDAC biology and antiproliferative activity. The structures of these new hydroxamic acids
1
derivatives were confirmed by IR, H-NMR and mass spectrum. Some of these compounds
showed micro molar activity in the HDAC inhibitory assay and against four cancer cell lines.
Key words: HDAC inhibitors, Diaryl ether, Hydroxamic acid, Design, Synthesis, Antiprolif-
erative activity
and Sinclair, 2007). Class I (HADC1, 2, 3, and 8), class
II (HDAC4, 5, 6, 7, 9 and 10), and class IV (HDAC11)
INTRODUCTION
Epigenetic regulation of gene expression is partly HDACs are zinc-dependent enzymes, while class III
controlled by chromatin remodeling, which is initiated HDACs are sirtuin related proteins and require the
either by DNA methylation (Abreu et al., 2008) or cofactor NAD+ for their deacetylase function (Bolden
post-translational modification of histone proteins et al., 2006). Of these known HDAC enzymes, it is com-
(Grunstein, 1997; Wolffe and Guschin, 2000). Several monly accepted that class I serves a critical function
different modifications that occur in histone amino in cell proliferation and survival. Several natural pro-
termini are defined as acetylation, methylation, ubiq- ducts, including trapoxin, herbimycin, depudecin, apici-
uitinylation, and glycosylation. Histone acetylation- din, FR 901228 and a few small molecule HDAC inhib-
deacetylation is a reversible phenomenon and is tight- itors, such as suberoylanilide hydroxamic acid (SAHA),
ly regulated by two competing enzymes, known as his- MS-275, VX-563, Valproic acid and Oxamflatin (Fig.
tone acetyl transferases (HATs) and histone deacety- 1A) (Bolden et al., 2006; Butler and Kozikowski, 2008;
lases (HDACs) (Marks et al., 2000). In general, HATs Paris et al., 2008) have been found to have promising
catalyse the acetylation of N-terminal lysine amino antitumor activities in clinical studies and in 2006,
groups in nuclear histones, resulting in neutralization the US FDA approved SAHA for the treatment of a
of the positive charges on the histones and a more rare cancer, cutaneous T-cell lymphoma (CTCL) (Marks,
open, transcriptionally active chromatin structure, 2007). While many of the compounds in clinical devel-
while the HDACs catalyze the reverse reaction and opment have been shown to have better activity, they
suppress transcription. Altered HDAC activity is found typically appear to have limitations, including low po-
to be associated with many cancers, validating HDACs tency, undesirable safety profiles, such as cardiovascular
as promising targets for cancer therapy (Seo, 2012). concerns, and potential drug-drug interactions via cyto-
Currently, there are 18 HDAC known in humans, which chrome P450 inhibition (Vassiliou et al., 1999; Minucci
are classified into four main phylogenetic classes (de and Pelicci, 2006; Glaser, 2007). Hence, there remains
Ruijter et al., 2003; Gregoretti et al., 2004; Michan a significant clinical opportunity for the discovery of
effective and specific HDAC inhibitors that are safe and
well tolerated. It is promising to develop potentially
superior HDAC inhibitors, based on SAHA, since de-
tailed crystallographic data of their binding interac-
Correspondence to: Tao Lu and Weifang Tang, Department of
Organic Chemistry, China Pharmaceutical University, Nanjing
210009, China
tions with the active binding sites of several zinc-de-
pendent isozymes (HDAC7, HDAC8, HDLP) have been
Tel: 86-25-8618-5180, Fax: 86-25-8618-5179
Email: lutao@cpu.edu.cn (T. Lu), twf@cpu.edu.cn (W. Tang)
1723

1724
Y. Zhu et al.
Fig. 2. Structure of diaryl ether hydroxamic acids.
sign, synthesis and HDAC inhibitory data for a series
of the diaryl ether hydroxamic acids (Fig. 2), which con-
tain various substituted aryl and ureido functionality
as the capping group, a diaryl ether spacer linker and
hydroxamic acid terminal group capable of binding to
the zinc residue, contained within an active site of
HDAC enzymes. Hydroxamate moiety lied in the para
or meta position of diaryl ether oxygen, as to discuss
the effect of different locations on the activity.
Fig. 1. Small molecule HDAC inhibitors. (
A
) Selected ex-
amples of acyclic HDAC inhibitors; (B) pharmacophore
model of HDAC inhibitors.
reported and pharmacophoric model of HDAC inhibi-
tors has been studied, too (Fig. 1B) (Finnin et al., 1999;
Somoza et al., 2004; Nielsen et al., 2005; Schuetz et
al., 2008; Chen et al., 2009).
MATERIALS AND METHODS
Chemistry
All chemicals were reagent grade and purchased from
The active site of an HDAC enzyme consists of a tu- commercial suppliers. Melting points were determined
bular pocket leading to a zinc (II) and two aspartate- in open capillaries on a WRS-1A digital melting point
1
histidine charge relay systems; class I and II HDACs apparatus (Shenguang). The H-NMR and 13C-NMR
share this common feature of a zinc (II) species present spectra were recorded on a Bruker Avance 300 or Bruker
at the enzymatic catalytic site. Consequently, reversible Avance 500 (Bruker), operating at 300 MHz or 500 MHz,
HDAC inhibitors reported to date have a chelating using TMS as the internal standard. The chemical
group that forms a complex with the zinc (II) and a shifts are reported in ppm (
δ) and coupling constants
hydrophobic spacer linked to the chelating group that
(
J
) values are given in Hertz (Hz). Signal multiplicities
fits into the tubular pocket. Therefore, options for the are represented by: s (singlet), d (doublet), dd (double
structural modifications of the chelating group and doublet), and m (multiplet). The IR spectra were per-
the hydrophobic spacer are limited (Marks, 2007).
formed on a FTIR-8400S (Shimadzu) in KBr pellets;
Several groups have reported the synthesis and bio- the frequencies are expressed in cm−1. Mass spectra
assay of novel HDAC inhibitors, based on SAHA with were obtained from Agilent 1100 LC/MSD (Agilent) or
varying chain length, aryl substitution, and aryl-chain Q-tof micro MS (Micromass).
connection with functionality that includes ketones,
alkenes, oximes, and etc. Of these analogs, those pos-
sessing a hydroxamic acid metal binding group have Synthesis of ethyl 4-(4-nitrophenoxy)benzoate (3)
shown HDAC inhibition IC50 in the low nanomolar
K2CO3 (55.2 g, 0.4 mol) was suspended in dry CH3CN
Synthesis
range. And some inhibitors with an aryl-chain moiety solution (50 mL), ethyl 4-hydroxybenzoate (16.6 g, 0.1
in the linker region adjacent to the hydroxamic acid mol) was added, and the reaction mixture was heated
group have better metabolic stability (Sternson et al., to refluxing temperature, then to this stirred suspension,
2001; Vickerstaffe et al., 2003). These studies led us to a solution of 1-fluoro-4-nitrobenzene (15.5 g, 0.11 mol)
initiate structural modifications of the terminal residue in freshly distilled CH3CN (50 mL) was added over a
(‘Cap’), while retaining the hydroxamic acid group. period of 0.5 h from a dropping funnel. After the addi-
Meanwhile, the traditional chain in the connection area tion was completed, the reaction mixture was refluxed
was substituted with diaryl ether. It is postulated that and stirred overnight. After filtering, the solvent was
structural features of the molecule in the ‘cap’ region evaporated to give a grown powder, which was washed
interact at the entrance of the HDAC pocket and pro- several times with water. The product was purified by
vides opportunities to discover potent and possibly more crystallization with hexane/ethyl acetate to give
selective HDAC inhibitors.
pale yellow crystals (24.3 g, 84.4%); m.p. 68~70oC (Lit.
In this context, we now report our results on the de- mp 67~68oC) (Consiglio et al., 2000).
3 as

Design and Synthesis of New HDAC Inhibitors
1725
Synthesis of ethyl 4-(4-aminophenoxy)benzoate DMSO-d6),
δ
, ppm: 9.05 (s, 1H, NHCONH), 8.82 (s,
(4)
1H, NHCONH), 7.77 (d, J = 8.7 Hz, 2H, ArH), 7.51 (d,
To a solution (water 200 mL) of NH4Cl (3.7 g, 70 mmol) J = 8.4 Hz, 2H, ArH), 7.37 (d, J = 8.6 Hz, 2H, ArH),
and EtOH (10 mL), ethyl 4-(4-nitrophenoxy)benzoate 7.02 (d, J = 8.1 Hz, 2H, ArH), 6.99 (d, J = 8.7 Hz, 2H,
3
(20.0 g, 69.6 mmol) was added and heated to reflux- ArH), 6.88 (d, J = 8.4 Hz, 2H, ArH), 4.27 (q,
J
J
= 7.3
= 7.3
ing temperature, then iron powder (37 g, 700 mmol) Hz, 2H, OCH2CH3), 3.72 (s, 3H, OCH3), 1.34 (t,
was added slowly over a period of 0.5 h. Refluxing was Hz, 3H, OCH2CH3).
continued for a period of 4 h, after which, the reaction
mixture was filtered off and the solvent evaporated at Synthesis of methyl 4-(4-(3-(4-chlorophenyl)ure-
reduced pressure. The oily residue was purified by col- ido)phenoxy)benzoate(5d)
1
umn chromatography (SiO2; cyc1ohexane:ethyl acetate Yield: 56.1%; mp: 164~166oC; H-NMR (300 MHz,
= 1:1 ) to give
4
(15.7 g, 87.2%) (Consiglio et al., 2000).
DMSO-d6),
1H, NHCONH), 7.79 (d, J = 8.3 Hz, 2H, ArH), 7.59~
General Synthetic Procedure for methyl 4-(4-(3- 7.51 (m, 4H, ArH), 7.33 (d, J = 8.0 Hz, 2H, ArH), 7.04
arylureido)phenoxy)benzoate (5a-e) (d, J = 8.1 Hz, 2H, ArH), 6.99 (d, J = 8.4 Hz, 2H, ArH),
To a stirred dry CH2Cl2 solution (5 mL) of CDI (0.58 4.33 (q, = 7.3 Hz, 2H, OCH2CH3), 1.43 (t, = 7.3 Hz,
δ, ppm: 9.17 (s, 1H, NHCONH), 9.09 (s,
J
J
g, 3.6 mmol), amine (3 mmol) dissolved in 3 mL of dry 3H, OCH2CH3).
CH2Cl2 was added slowly. The mixture was allowed to
be stirred at room temperature. After 6 h, ethyl 4-(4- Synthesis of methyl 4-(4-(3-(4-bromophenyl)ure-
amino phenoxy)benzoate (0.93 g, 3.6 mmol), dissolv- ido)phenoxy)benzoate (5e)
ed in 15 mL of dry CH2Cl2, was added to this reaction Yield: 90.5%; mp: 250~252oC; H-NMR (300 MHz,
mixture. The reaction mixture was stirred at room DMSO-d6), , ppm: 9.17 (s, 1H, NHCONH), 8.99 (s,
4
1
δ
temperature for one day and filtered. The solid obtained 1H, NHCONH), 7.75 (d, J = 8.3 Hz, 2H, ArH), 7.52 (d,
was washed with CH2Cl2 and purified by crystalliza- J = 7.8 Hz, 2H, ArH), 7.48~7.38 (m, 4H, ArH), 7.04 (d,
tion from ethylacetate/THF.
J = 7.8 Hz, 2H, ArH), 6.97 (d, J = 8.4 Hz, 2H, ArH),
4.33 (q, = 7.3 Hz, 2H, OCH2CH3), 1.42 (t, = 7.3 Hz,
J
J
Synthesis of ethyl 4-(4-(3-pyridin-2-ylureido)phen- 3H, OCH2CH3).
oxy)benzoate (5a)
1
Yield: 58.7%; mp: 190~192oC; H-NMR (300 MHz, Synthesis of 4-(4-(3-arylureido)phenoxy)benzoic
DMSO-d6),
NHCONH), 8.30 (d,
8.8 Hz, 2H, ArH), 7.85 (d,
(d, = 8.8 Hz, 2H, ArH), 7.52 (d,
7.24 (d, = 9.0 Hz, 2H, ArH), 7.03 (dt,
1.3 Hz, 1H, ArH), 6.99 (d,
= 8.8 Hz, 2H, ArH), 4.29 mixture was cooled to 0oC and acidified to pH 4 by
(q, = 7.3 Hz, 2H, OCH2CH3), 1.38 (t,
OCH2CH3).
δ
, ppm: 9.56 (s, 1H, NHCONH), 8.98 (s, 1H, acid (6a-e)
= 2.9 Hz, 1H, ArH), 8.17 (d, Ethyl 4-(4-(3-arylureido)phenoxy)benzoate
= 8.5 Hz, 1H, ArH), 7.71 mmol) and EtOH (2 mL) was added to the solution of
= 8.2 Hz, 1H, ArH), NaOH (0.12 g, 3 mmol) in water (10 mL). The reaction
= 7.5 Hz and mixture was refluxed and stirred for 4 h. Then the
J
J
=
5 (1.5
J
J
J
J
J
J
J
J
= 7.3 Hz, 3H, dropwise addition of 6 N hydrochloric acid. The solid
was filtered off and washed with water.
Synthesis of methyl 4-(4-(3-(3-methoxyphenyl) Synthesis of 4-(4-(3-pyridin-2-ylureido)phenoxy)
ureido)phenoxy)benzoate (5b) benzoic acid (6a)
Yield: 59.9%; mp: 203~205oC; H-NMR (300 MHz, Yield: 94.2%; mp: 243~245oC; H-NMR (500 MHz,
DMSO-d6), , ppm: 9.54 (s, 1H, NHCONH), 8.98 (s, DMSO-d6), , ppm: 12.16 (br, 1H, COOH), 9.59 (s, 1H,
1H, NHCONH), 8.28 (d, J = 3.8 Hz, 1H, ArH), 7.80 (d, NHCONH), 9.21 (s, 1H, NHCONH), 8.33 (d, = 2.9
J = 8.8 Hz, 2H, ArH), 7.81 (dt, J = 8.8 Hz and 1.9 Hz, Hz, 1H, ArH), 8.19 (d, = 8.8 Hz, 2H, ArH), 7.85 (d,
1H, ArH), 7.60 (d, J = 8.9 Hz, 2H, ArH), 7.49 (d, J = = 8.5 Hz, 1H, ArH), 7.80 (d, = 8.8 Hz, 2H, ArH), 7.52
1
1
δ
δ
J
J
J
J
8.2 Hz, 1H, ArH), 7.08 (d, J = 8.9 Hz, 2H, ArH), 7.03 (d, J = 8.5 Hz, 1H, ArH), 7.33 (d, J = 9.0 Hz, 2H, ArH),
(dt, J = 6.5 Hz and 1.3 Hz, 1H, ArH), 6.98 (d, J = 8.8 7.02 (dt, J = 7.6 Hz and 1.3Hz, 1H, ArH), 6.99 (d, J =
Hz, 2H, ArH), 4.30 (q,
(s, 3H, OCH3), 1.39 (t,
J
J
= 7.3 Hz, 2H, OCH2CH3), 3.82 8.8 Hz, 2H, ArH).
= 7.3 Hz, 3H, OCH2CH3).
Synthesis of 4-(4-(3-(3-methoxyphenyl)ureido)phe-
Synthesis of methyl 4-(4-(3-(4-methoxyphenyl) noxy)benzoic acid (6b)
ureido)phenoxy)benzoate (5c)
Yield: 93.3%; mp: 380~382oC; H-NMR (300 MHz,
Yield: 62.0%; mp: 180~181oC; H-NMR (300 MHz, DMSO-d6),
, ppm: 12.10 (br, 1H, COOH), 9.57 (s, 1H,
1
1
δ

1726
Y. Zhu et al.
NHCONH), 9.01 (s, 1H, NHCONH), 8.29 (d, J = 3.8 Hz, Synthesis of
N-hydroxy-4-(4-(3-pyridin-2-ylureido)
1H, ArH), 7.80 (d, J = 8.8 Hz, 2H, ArH), 7.75 (dt, J = 8.8 phenoxy)benzamide (1a)
Hz and 1.9 Hz, 1H, ArH), 7.61 (d, J = 8.9 Hz, 2H, ArH), Yield: 52.2%; mp: 220~222oC; H-NMR (500 MHz,
7.50 (d, J = 8.2 Hz, 1H, ArH), 7.09 (d, J = 8.9 Hz, 2H, DMSO-d6), , ppm: 11.12 (s, 1H, C(O)NHOH), 10.56 (s,
ArH), 7.03 (dt, J = 6.5, 1.3 Hz, 1H, ArH), 6.98 (d, J = 8.8 1H, C(O)NHOH), 9.44 (s, 1H, NHCONH), 8.94 (s, 1H,
1
δ
Hz, 2H, ArH), 3.83 (s, 3H, OCH3).
NHCONH), 8.28 (d, J = 3.8 Hz, 1H, ArH), 7.78 (d, J =
8.8 Hz, 2H, ArH), 7.75 (dt, J = 8.8, 1.9 Hz, 1H, ArH),
Synthesis of 4-(4-(3-(4-methoxyphenyl)ureido)phe- 7.59 (d, J = 8.9 Hz, 2H, ArH), 7.49 (d, J = 8.2 Hz, 1H,
noxy)benzoic acid (6c) ArH), 7.07 (d, J = 8.9 Hz, 2H, ArH), 7.02 (dt, J = 6.5,
Yield: 95.0%; mp: 270~272oC; H-NMR (300 MHz, 1.3 Hz, 1H, ArH), 6.98 (d, J = 8.8 Hz, 2H, ArH); 13C-
DMSO-d6), , ppm: 12.04 (s, 1H, COOH), 8.17 (s, 1H, NMR (300 MHz, DMSO-d6), , ppm: 166.02, 162.36,
NHCONH), 8.90 (s, 1H, NHCONH), 7.79 (d, J = 8.7 152.93, 147.17, 140.02, 138.84, 136.25, 134.27, 131.58,
Hz, 2H, ArH), 7.62 (d, J = 8.4 Hz, 2H, ArH), 7.48 (d, 127.61, 124.01, 123.87, 121.65, 116.42, 115.63; ESI-
8.6 Hz, 2H, ArH), 7.12 (d, J = 8.1 Hz, 2H, ArH), 7.03 MS m/z: 365.2 ([M+H]+); IR (KBr, cm−1): 3310, 1647,
1
δ
δ
J
=
(d, J = 8.7 Hz, 2H, ArH), 6.96 (d, J = 8.4 Hz, 2H, ArH), 1568, 1499, 1383, 1254, 1163, 844, 683, 519.
3.76 (s, 3H, OCH3); ESI-MS m/z: 379.1 ([M+H]+).
Synthesis of 4-(4-(3-(3-methoxyphenyl)ureido)phe-
Synthesis of 4-(4-(3-(4-chlorophenyl)ureido)phe- noxy)benzoic acid (1b)
1
noxy)benzoic acid (6d)
Yield: 90.6%; mp: 385~387oC; H-NMR (300 MHz, DMSO-d6),
DMSO-d6), , ppm: 12.46 (s, 1H, COOH), 9.23 (s, 1H, 1H, C(O)NHOH), 8.75 (s, 1H, NHCONH), 8.71 (s, 1H,
Yield: 56.0%; mp: 210~212oC; H-NMR (300 MHz,
1
δ, ppm: 11.11 (s, 1H, C(O)NHOH), 8.94 (s,
δ
NHCONH), 9.14 (s, 1H, NHCONH), 7.78 (d, J = 8.3 NHCONH), 7.75 (d, J = 8.9 Hz, 2H, ArH), 7.51 (d, J =
Hz, 2H, ArH), 7.65~7.55 (m, 4H, ArH), 7.35 (d, J = 8.0 9.0 Hz, 2H, ArH), 7.20~7.15 (m, 2H, ArH), 7.05~6.92
Hz, 2H, ArH), 7.08 (d, J = 8.1 Hz, 2H, ArH), 6.99 (d,
8.4 Hz, 2H, ArH); ESI-MS m/z: 383.1 ([M+H]+), ArH), 3.73 (s, 3H, OCH3); 13C-NMR (300 MHz, DMSO-
405.1 ([M+Na]+).
d6), , ppm: 165.97, 162.33, 162.21, 154.19, 149.77,
136.97, 134.38. 130.08, 124.05, 123.87, 121.66, 116.73,
Synthesis of 4-(4-(3-(4-bromophenyl)ureido)phe- 116.42, 115.68, 113.95, 110.22, 55.84; ESI-MS m/z:
noxy)benzoic acid (6e)
394.2 ([M+H]+); IR (KBr, cm−1): 3310, 1651, 1607,
Yield: 90.5%; mp: 250~252oC; H-NMR (300 MHz, 1566, 1504, 1232, 1032, 831, 692, 538.
DMSO-d6), , ppm: 12.39 (s, 1H, COOH), 9.20 (s, 1H,
J
(m, 5H, ArH), 6.55 (dd, J = 8.0 Hz and 1.9 Hz, 1H,
=
δ
1
δ
NHCONH), 9.08 (s, 1H, NHCONH), 7.76 (d, J = 8.3 Synthesis of 4-(4-(3-(4-methoxyphenyl)ureido)phe-
Hz, 2H, ArH), 7.52 (d, J = 7.8 Hz, 2H, ArH), 7.55~7.46 noxy)benzoic acid (1c)
1
(m, 4H, ArH), 7.05 (d, J = 7.8 Hz, 2H, ArH), 6.97 (d,
8.4 Hz, 2H, ArH).
J
Yield: 56.2%; mp: 205~207oC; H-NMR (300 MHz,
=
DMSO-d6),
1H, C(O)NHOH), 8.99 (s, 1H, NHCONH), 8.79 (s, 1H,
Synthesis of N-hydroxy-4-(4-(3-arylureido)phe- NHCONH), 7.75 (d, J = 8.7 Hz, 2H, ArH), 7.50 (d, J =
noxy)benzamide (1a-e) 8.4 Hz, 2H, ArH), 7.36 (d, J = 8.6 Hz, 2H, ArH), 7.02
To a solution of the synthesized 4-(4-(3-arylureido) (d, J = 8.1 Hz, 2H, ArH), 6.96 (d, J = 8.7 Hz, 2H, ArH),
phenoxy)benzoic acid (1 mmol) in dry THF (10 mL) 6.86 (d, J = 8.4 Hz, 2H, ArH), 3.71 (s, 3H, OCH3); ESI-
at
5oC, ethylchloroformate (0.13 g, 1.2 mmol) and tri- MS m/z: 394.2 ([M+H]+); IR (KBr, cm−1): 3300, 1647,
δ, ppm: 11.09 (s, 1H, C(O)NHOH), 9.05 (s,
6
−
ethylamine (0.17 mL, 1.2 mmol) were added and the 1568, 1499, 1383, 1254, 1163, 844, 683, 519.
mixture was stirred for 30 min. The solid was filtered
off and the filtrate was added to freshly prepare hy- Synthesis of 4-(4-(3-(4-chlorophenyl)ureido)phe-
droxylamine (0.05 g, 1.5 mmol) in methanol (Gediya et noxy)benzoic acid (1d)
1
al., 2005). The reaction mixture was stirred at room Yield: 50.3%; mp: 302~304oC; H-NMR (300 MHz,
temperature for 30 min. The solvent was evaporated DMSO-d6), δ, ppm: 11.06 (s, 1H, C(O)NHOH), 9.22 (s,
and the residue was purified by a silica gel column 1H, NHCONH), 9.12 (s, 1H, NHCONH), 9.05 (s, 1H,
chromatography (CH2Cl2:CH3OH = 40:1) to obtain N- NHCONH), 7.76 (d, J = 8.3 Hz, 2H, ArH), 7.53~7.49
hydroxy- 4-(4-(3-arylureido)phenoxy)benzamide 1a
-e
(m, 4H, ArH), 7.31 (d, J = 8.0 Hz, 2H, ArH), 7.03 (d, J
=
8.1 Hz, 2H, ArH), 6.96 (d, J = 8.4 Hz, 2H, ArH); ESI-
MS m/z: 398.1 ([M+H]+); IR (KBr, cm−1): 3300, 1645,
1607, 1566, 1232, 1092, 837, 675, 646, 519.

Design and Synthesis of New HDAC Inhibitors
1727
Synthesis of 4-(4-(3-(4-bromophenyl)ureido)phe- Synthesis of methyl 3-(4-(3-(3-methoxyphenyl)ure-
noxy)benzoic acid (1e) ido)phenoxy)benzoate (9b)
Yield: 49.7%; mp: 308~310oC; H-NMR (300 MHz, Yield: 61.2%; mp: 178~179oC; H-NMR (300 MHz,
DMSO-d6), , ppm: 11.09 (s, 1H, C(O)NHOH), 9.15 (s, CDCl3), , ppm: 8.96 (br, 1H, NHCONH), 8.70 (br, 1H,
1H, C(O)NHOH), 9.13 (s, 1H, NHCONH), 8.95 (s, 1H, NHCONH), 7.55 (d, 1H, = 8.5 Hz, ArH ), 7.51-7.41 (m,
NHCONH), 7.76 (d, J = 8.3 Hz, 2H, ArH), 7.51 (d, J = 3H, ArH), 7.39 (s, 1H, ArH), 7.20 (d, 1H, = 8.5 Hz,
7.8 Hz, 2H, ArH), 7.46~7.40 (m, 4H, ArH), 7.04 (d, J = ArH), 7.17-7.14 (m, 2H, ArH), 7.03 (d, 2H, J = 8.4 Hz,
7.8 Hz, 2H, ArH), 6.97 (d, J = 8.4 Hz, 2H, ArH); ESI- ArH), 6.95 (dd, 1H, = 8.5 Hz and 2.5 Hz), 6.57 (dd,
MS m/z: 464.1 ([M+Na]+); IR (KBr, cm−1): 3302, 1647, 1H,
= 8.5 Hz and 2.5 Hz), 3.90 (s, 3H, OCH3), 3.73
1605, 1562, 1495, 1231, 903, 835, 658, 515. (s, 3H, OCH3).
1
1
δ
δ
J
J
J
J
Synthesis of methyl 3-(4-nitrophenoxy)benzoate Synthesis of methyl 3-(4-(3-(4-methoxyphenyl)
(7) ureido)phenoxy)benzoate (9c)
K2CO3 (55.2 g, 0.4 mol) was suspended in dry Yield: 58.5%; mp: 201~203oC; H-NMR (300 MHz,
CH3CN solution (50 mL), methyl 3-hydroxybenzoate CDCl3), , ppm: 8.93 (s, 1H, NHCONH), 8.70 (s, 1H,
1
δ
(15.2 g, 0.1 mol) was added, then the reaction mixture NHCONH), 7.72-7.65 (m, 3H, ArH), 7.58 (dd, 2H, J =
was heated to refluxing temperature and to this stirred 9.0, 2.5 Hz, ArH), 7.37 (dd, 2H, J = 8.6, 2.5 Hz, ArH),
suspension, a solution of 1-fluoro-4-nitrobenzene (15.5 7.19 (d, 2H, J = 9.0 Hz, ArH), 7.10 (d, 1H, J = 8.5 Hz,
g, 0.11 mol) in freshly distilled CH3CN (50 mL) was ArH), 6.87 (d, 2H, J = 8.6 Hz, ArH), 3.92 (s, 3H, OCH3),
added over a period of 0.5 h from a dropping funnel. 3.73 (s, 3H, OCH3); ESI-MS m/z: 492.3 ([M+H]+).
After the addition was completed, the reaction mixture
was refluxed, stirred overnight and filtered, then the Synthesis of methyl 3-(4-(3-(4-chlorophenyl)ureido)
solvent was evaporated to give a grown oil, which was phenoxy)benzoate (9d)
1
purified by silica gel column chromatography (cyclohex- Yield: 55.5%; mp: 211~213oC; H-NMR (500 MHz,
ane:ethyl acetate = 3:1) to give 1 as yellow oil (24.3 g, DMSO-d6), δ, ppm: 8.99 (br, 1H, NHCONH), 8.93 (br,
85.3%) (Consiglio et al., 2000).
1H, NHCONH), 7.58-7.49 (m, 6H, ArH), 7.36-7.32 (m,
3H, ArH), 7.26-7.19 (m, 3H, ArH), 3.90 (s, 3H, OCH3).
Synthesis of methyl 3-(4-aminophenoxy)benzoate
(8)
Synthesis of methyl 3-(4-(3-(4-bromophenyl)ure-
Compounds
same manner as compounds
(300 MHz, DMSO-d6), , ppm: 7.72 (d, 1H,
ArH), 7.60 (s, 1H, ArH), 7.34 (t, 1H, = 7.9 Hz, ArH), 1H, NHCONH), 7.55-7.53 (m, 3H, ArH), 7.50-7.44 (m,
7.10 (d, 1H, = 7.9 Hz, ArH), 6.88 (d, 2H, = 8.9 Hz, 5H, ArH), 7.35-7.33 (m, 1H, ArH), 7.16-7.15 (m, 1H,
ArH), 6.70 (d, 2H, = 8.9 Hz, ArH), 3.93 (s, 3H, ArH), 7.10-7.06 (m, 2H, ArH), 3.89 (s, 3H, OCH3).
OCH3), 3.63 (br, 2H, NH2).
8
was obtained with compound
. Yield: 85.3%; 1H-NMR Yield: 56.8%; mp: 241~243oC; H-NMR (500 MHz,
= 7.9 Hz, DMSO-d6), , ppm: 8.97 (br, 1H, NHCONH), 8.90 (br,
7 in the ido)phenoxy)benzoate (9e)
1
4
δ
J
δ
J
J
J
J
Synthesis of 3-(4-(3-arylureido)phenoxy)benzoic
Synthesis of methyl 3-(4-(3-arylureido)phenoxy) acid (10a~e)
benzoate (9a~e) Compounds 10a~e were obtained in the same manner
Compounds 9a were obtained in the same manner as compounds 6a~e
as compounds 5a
~e
.
~e.
Synthesis of 3-(4-(3-(4-chloro-3-(trifluoromethyl)
Synthesis of methyl 3-(4-(3-(4-chloro-3-(trifluoro- phenyl)ureido)phenoxy)benzoic acid (10a)
1
methyl)phenyl)ureido)phenoxy) benzoate (9a)
Yield: 94.3%; mp: 210~211oC; H-NMR (500 MHz,
Yield: 67.8%; mp: 189~191oC; H-NMR (500 MHz, DMSO-d6),
, ppm: 12.40 (br, 1H, COOH), 9.08 (br,
DMSO-d6), , ppm: 9.13 (br, 1H, NHCONH), 8.91 (br, 1H, NHCONH), 8.93 (br, 1H, NHCONH), 8.15 (d, 1H,
1H, NHCONH), 8.12 (d, 1H, = 2.3 Hz, ArH), 7.66 = 2.4 Hz, ArH), 7.67 (dd, 1H, = 8.8, 2.4 Hz, ArH),
(dd, 1H, = 8.8, 2.3 Hz, ArH), 7.60 (d, 1H, = 8.5 Hz, 7.61 (d, 1H, = 8.8 Hz, ArH), 7.52-7.55 (m, 2H, ArH),
ArH), 7.55-7.47 (m, 4H, ArH), 7.33 (t, 1H, = 8.5 Hz, 7.43-7.49 (m, 2H, ArH), 7.32-7.33 (m, 1H, ArH), 7.11-
= 8.7 Hz 7.13 (m, 1H, ArH), 7.03 (dd, 1H, = 8.6, 2.4 Hz 2H,
ArH); ESI-MS m/z: 451.6 ([M+H]+).
1
δ
δ
J
J
J
J
J
J
J
ArH), 7.13-7.17 (m, 1H, ArH), 7.06 (dd, 2H,
and 2.4 Hz, ArH), 3.90 (s, 3H, OCH3).
J
J

1728
Y. Zhu et al.
Synthesis of 3-(4-(3-(3-methoxyphenyl)ureido) 123.37, 123.12, 121.35, 121.56, 121.05, 118.86, 117.97,
phenoxy)benzoic acid (10b)
116.24; ESI-MS m/z: 466.7 ([M+H]+); IR (KBr, cm−1):
Yield: 92.7%; mp: 224~226oC; H-NMR (500 MHz, 3305, 1645, 1589, 1556, 1502, 1303, 1226, 1072, 833,
DMSO-d6), , ppm: 12.12 (s, 1H, COOH), 8.88 (br, 1H, 696.
NHCONH), 8.76 (br, 1H, NHCONH), 7.60-7.50 (m,
1
δ
4H, ArH), 7.40 (s, 1H, ArH), 7.29 (d,
ArH), 7.25-7.17 (m, 2H, ArH), 7.11 (d,
J
J
= 7.9 Hz, 1H, Synthesis of
= 8.9 Hz, 2H, ureido)phenoxy)benzamide (2b)
N-hydroxy-3-(4-(3-(3-methoxyphenyl)
1
ArH), 7.02 (d,
Hz and 2.4 Hz, 1H, ArH), 3.73 (s, 3H, OCH3).
J = 7.9 Hz, 1H, ArH), 6.75 (dd, J
= 8.9 Yield: 61.1%; mp: 203~205oC; H-NMR (500 MHz,
DMSO-d6), , ppm: 11.21 (br, 1H, C(O)NHOH), 9.03
(br, 1H, C(O)NHOH), 8.68 (br, 1H, NHCONH), 8.66
Synthesis of 3-(4-(3-(4-methoxyphenyl)ureido) (br, 1H, NHCONH), 7.50-7.40 (m, 4H, ArH), 7.30 (s,
phenoxy)benzoic acid (10c) 1H, ArH), 7.18 (d, = 7.9 Hz, 1H, ArH), 7.14-7.10 (m,
Yield: 91.0%; mp: 247~249oC; H-NMR (500 MHz, 2H, ArH), 7.02 (d, J = 8.0 Hz, 2H, ArH), 6.93 (d, J =
δ
J
1
DMSO-d6), δ, ppm: 12.10 (s, 1H, COOH), 8.69 (br, 1H, 8.1 Hz 1H, ArH), 6.55 (dd, J = 2.5, 8.8 Hz, 1H, ArH),
NHCONH), 8.55 (br, 1H, NHCONH), 7.50 (d, 2H, J = 3.73 (s, 3H, OCH3); ESI-MS m/z: 394.1 ([M+H]+), 392.5
8.0 Hz, ArH), 7.49-7.40 (m, 2H, ArH), 7.39-7.32 (m, ([M-H]-); IR (KBr, cm−1): 3309, 1647, 1604, 1504, 1498,
3H, ArH), 7.14-7.10 (m, 3H, ArH), 7.03 (d, 2H, J = 7.8 1307, 1230, 854, 511.
Hz, ArH), 2.30 (s, 3H, OCH3).
Synthesis of
N-hydroxy-3-(4-(3-(4-methoxyphenyl)
Synthesis of 3-(4-(3-(4-chlorophenyl)ureido) ureido)phenoxy)benzamide (2c)
1
phenoxy)benzoic acid (10d)
Yield: 92.5%; mp: 255~257oC; H-NMR (500 MHz, DMSO-d6),
DMSO-d6), , ppm: 12.15 (s, 1H, COOH), 9.10 (br, 1H, (br, 1H, C(O)NHOH), 8.63 (br, 1H, NHCONH), 8.52
NHCONH), 9.02 (br, 1H, NHCONH), 7.53-7.43 (m, (br, 1H, NHCONH), 7.48 (d, 2H, J = 8.0 Hz, ArH),
Yield: 63.6%; mp: 241~243oC; H-NMR (500 MHz,
1
δ, ppm: 11.21 (br, 1H, C(O)NHOH), 9.00
δ
6H, ArH), 7.36-7.28 (m, 4H, ArH), 7.12 (d,
2H, ArH).
J
= 8.6 Hz, 7.47-7.41 (m, 2H, ArH), 7.34-7.30 (m, 3H, ArH), 7.12-
7.11 (m, 1H, ArH), 7.10-7.07 (m, 2H, ArH), 7.01 (d,
2H, J = 8.0 Hz, ArH), 2.24 (s, 3H, OCH3); ESI-MS m/
Synthesis of 3-(4-(3-(4-bromophenyl)ureido)phe- z: 394.1 ([M+H]+); IR (KBr, cm−1): 3303, 2802, 1650,
noxy)benzoic acid (10e)
Yield: 91.5%; mp: 261~263oC; H-NMR (500 MHz,
DMSO-d6), , ppm: 12.15 (s, 1H, COOH), 8.99 (br, 1H, Synthesis of 3-(4-(3-(4-chlorophenyl)ureido)phe-
1564, 1504, 1307, 1230, 854, 511.
1
δ
NHCONH), 8.90 (br, 1H, NHCONH), 7.52-7.51 (m, noxy)-N-hydroxybenzamide (2d)
1
3H, ArH), 7.50-7.48 (m, 5H, ArH), 7.35-7.34 (m, 1H, Yield: 59.1%; mp: 253~255oC; H-NMR (500 MHz,
ArH), 7.14-7.12 (m, 1H, ArH), 7.05-7.02 (m, 2H, ArH). DMSO-d6), δ, ppm: 11.22 (br, 1H, C(O)NHOH), 9.02
(br, 1H, C(O)NHOH), 8.99 (br, 1H, NHCONH), 8.92
-hy- (br, 1H, NHCONH), 7.50-7.40 (m, 6H, ArH), 7.33-7.10
(m, 4H, ArH), 7.02 (d, J = 8.0 Hz, 2H, ArH); 13C-NMR
Synthesis of 3-(4-(3-arylureido)phenoxy)-
droxybenzamide (2a~e)
N
Compounds 2a
as compounds 1a
~e were obtained in the same manner (300 MHz, DMSO-d6), δ, ppm: 166.15, 158.91, 152.89,
~e.
148.93, 137.56, 133.38, 132.87, 132.57, 129.07, 128.39,
123.11, 121.53, 121.05, 121.03, 117.95, 116.24; ESI-
Synthesis of 3-(4-(3-(4-chloro-3-(trifluoromethyl) MS m/z: 398.2 ([M+H]+), 420.1 ([M+Na]+); IR (KBr,
phenyl)ureido)phenoxy)-
N
-hydroxy benzamide cm−1): 3301, 2846, 1645, 1562, 1500, 1402, 1307, 1228,
(2a)
841, 509.
1
Yield: 64.6%; mp: 199~201oC; H-NMR (500 MHz,
DMSO-d6),
(br, 1H, C(O)NHOH), 9.01 (br, 1H, NHCONH), 8.88 noxy)-
(br, 1H, NHCONH), 8.11 (d, 1H,
= 2.4 Hz, ArH), Yield: 56.4%; mp: 256~257oC; H-NMR (500 MHz,
7.65 (dd, 1H, = 8.8, 2.4 Hz, ArH), 7.60 (d, 1H, = 8.8 DMSO-d6), , ppm: 11.21 (br, 1H, C(O)NHOH), 9.01 (br,
δ
, ppm: 11.22 (br, 1H, C(O)NHOH), 9.17 Synthesis of 3-(4-(3-(4-bromophenyl)ureido)phe-
N
-hydroxybenzamide (2e)
1
J
J
J
δ
Hz, ArH), 7.52-7.50 (m, 2H, ArH), 7.49-7.42 (m, 2H, 1H, C(O)NHOH), 8.87 (br, 1H, NHCONH), 8.80 (br,
ArH), 7.31 (m, 1H, ArH), 7.13-7.11 (m, 1H, ArH), 7.03 1H, NHCONH), 7.50-7.49 (m, 3H, ArH), 7.48-7.41 (m,
(dd,
DMSO-d6),
143.03, 134.36, 132.92, 129.56, 129.38, 129.03, 128.41, ([M+H]+), 464.1 ([M+Na]+); IR (KBr, cm−1): 3301, 2921,
J
= 8.8, 2.4 Hz, 2H, ArH); 13C-NMR (300 MHz, 5H, ArH), 7.31-7.30 (m, 1H, ArH), 7.12-7.10 (m, 1H,
δ, ppm: 166.17, 158.97, 152.92, 149.01, ArH), 7.03-7.00 (m, 2H, ArH); ESI-MS m/z: 444.1

Design and Synthesis of New HDAC Inhibitors
2850, 1647, 1562, 1502, 1230, 838, 505.
1729
staining and a colorimetric mitochondrial activity
using MTS.
Pharmacology
Histone deacetylase assay
RESULTS
The activity of HDAC inhibitors in vitro was assayed,
using a BML-AK501, according to the instructions from
the manufacturer (Biomolecular Research Laboratories).
Chemistry
The synthesis of compounds 1a~e is shown in Scheme
The assay is automated and performed by a Tecan 1. Nucleophilic substitution reaction of 4-hydroxyben-
Freedom EVO work station. The samples were incu- zoate with 1-fluoro-4-nitrobenzene and potassium car-
bated for 15 min, at room temperature (RT), and then bonate gave ethyl 4-(4-nitro phenoxy)benzoate (
exposed to a developer (10 min at RT). In this last duction with iron powder in water/ethanol gave re-
step, a fluorophore was produced, whose fluorescence quired amine ( ). Compound reacted with several
was measured using an excitation wavelength of 355 aromatic amines and CDI to give urea (
), which was
hydrolyzed with alkaline solution to give the corres-
ponding acid . Compound was aminolysized in two
steps, initial intermediate mixed anhydride, using
3), re-
4
4
5
nm and an emission at 460 nm.
6
6
Cytotoxicity assay (MTT method)
Human acute lymphoblastic leukemia cells (MOLT- ethyl chlorformate, followed by fresh hydroxylamine
4), hepatoma carcinoma cell (SMMC7721), Chronic to form compound
myeloid leukemia cells (K562) and Burkitt's lymphoma Scheme 2 shows the synthesis of compounds 2a~e
1.
.
cells (Raji) were obtained from Institute of Biochem- Firstly, nucleophilic substitution reaction of 3-hydroxy-
istry and Cell Biology, SIBS, CAS. Cultures were grown benzoate with 1-fluoro-4-nitrobenzen gave compound
in EMEM, containing Lglutamine (5 mM), sodium pyru-
8. Then Compounds 2a~e were obtained in the same
vate (1 mM), sodium bicarbonate (1500 mg/L) and step as compound
1.
10% fetal bovine serum, and were maintained at 37oC
in an atmosphere of 5% CO2. SAHA, and all other syn-
thesized potential HDAC inhibitors, were dissolved in
Pharmacology
The HDAC inhibition of all synthesized compounds
DMSO at a stock concentration of 10 mM, and were was evaluated by using a BML-AK501. The results
stored at
−
80oC. Cultures were passaged and allowed are summarized in Table I. The results suggested that
to grow for 24 h prior to the addition of compounds. some HDAC inhibitory abilities of diaryl ether
Compounds were diluted to the appropriate concen- hydroxamic acids derivatives were potent. Though to
tration in a new medium, and were incubated with cul- a lesser extent than SAHA, some compounds showed
tures for 72 h. Viability was assessed by Trypan Blue better activity with inhibition ratio in the region of
Scheme 1. Synthetic pathways of
N-hydroxy-4-(4-(3-arylureido)phenoxy)benzamide (1a~e)

1730
Y. Zhu et al.
Scheme 2. Synthetic pathways of 3-(4-(3-arylureido)phenoxy)-N-hydroxybenzamide (2a~e).
micromole (Table I).
Table I. In vitro HDAC inhibitory activity
HDAC inhibitors have been implicated to have
potent antitumor competence in cancer cells (Takai et
al., 2004; Feng et al., 2008; Conti et al., 2010). We then
examined the effects of all synthesized compounds on
the growth and proliferation of various cancer cells by
using the MTT assay. As shown in Table II, growth of
MOLT-4, K562 and Raji cells IC50 was markedly sup-
pressed in the presence of 2c and 2d, in a dose-de-
pendent manner. In addition, 2d could restrain the
growth of SMMC7721. These data suggested that 2c
and 2d may potentially be promising antitumor drugs.
Inhibition
0.1 M (%)
Inhibition
M (%)
Inhibition
10 M (%)
Compds
µ
1
µ
µ
1a
1b
1c
1d
1e
2a
2b
2c
2d
48.32
0
0
69.20
57.08
56.03
50.83
61.16
21.54
8.85
15.27
19.67
10.12
90.76
84.56
63.18
72.94
80.79
80.64
46.40
11.95
25.33
29.74
25.25
98.51
16.84
7.21
17.87
0
12.27
15.45
7.87
51.49
2e
SAHA
DISCUSSION
The new synthesized compounds were submitted to
biological evaluation to identify the molecules endowed
with HDAC inhibitory activity. The assays were carried
out using a commercially available HDAC assay kit,
with HeLa cell nuclear extracts as the enzyme source,
and a fluorogenic acetylated histone peptide fragment
(Fluor de Lys) as a substrate. As shown in Table I, the
Table II. Cell growth inhibitory data for compound in
human tumor cell lines
MOLT-4 SMMC7721
K562
Raji
M)
Compd
IC50 M) IC50 M) IC50
(µ
(µ
(µM) IC50 (µ
1a
1b
1c
1d
1e
2a
2b
2c
2d
81.0
80.0
>100
95.3
55.5
70.0
>100
1.1
>100
>100
>100
>100
>100
>100
>100
>100
7.1
>100
>100
>100
>100
>100
87.0
>100
7.0
>100
>100
>100
>100
>100
>100
>100
2.1
compound 1a
ent moderate biochemical activity (percentage of in-
hibition in a range between 50.83 and 69.20% at 1
and between 63.18 and 83.56% at 10 M). However,
~e demonstrated to have a dose depend-
µM
µ
the inhibitory activity was lower than that measured
for the reference compound SAHA, especially, when
measured at low concentrations, except for compound
1.5
5.7
1.9
2e
SAHA
>100
5.3
>100
19.5
>100
8.2
>100
3.9
1a (percentage of inhibition 48.32% at 0.1
µM). In com-
parison, compound 2a showed almost no HDAC
~e

Design and Synthesis of New HDAC Inhibitors
1731
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inhibition activity. The reason for this could be that
the meta substituted hydroxamic group cannot interact
with zinc ion.
Then, we evaluated the antiproliferation effect in
cancer cells in vitro. Compound 1a~e with HDAC in-
hibition showed no cytotoxicity. Interestingly, compound
2c and 2d showed significant cell growth inhibition in
various cancer cell lines with IC50 values at the micro-
molar levels. These data suggested that 2c and 2d may
effect other targets or antitumor mechanisms. Mean-
while, based on the structure of compound 2c and 2d
,
we could introduce some groups to the meta position
of oxygen atoms in compound 1a, the cytotoxicity of
which may be increased.
The results of the present study demonstrate that
diaryl ether-based linker groups, which could be used
instead of the aliphatic or aryl chain in the design of
structurally diverse hydroxamate HDAC inhibitors,
were not more effective, although diaryl ether was
long enough to make the hydroxamic group into the
zinc binding pocket, and form a complex with the zinc.
However, compound 2c and 2d showed highly anti-
proliferative effect in cancer cells, which would help
discussion of the other targets or antitumor mechani-
sms. Meanwhile, structure optimization of compound
1a~e, including modification of 3-sustituted diaryl
ether for improving the cytotoxicity could lead to the
development of a new type of hydroxamate HDAC
inhibitors.
ACKNOWLEDGEMENTS
This research was supported by the Fundamental
Research Funds for the Central Universities (Program
No. JKQ2011002). The authors also thank C&O Pharm-
aceutical Technology (Holdings) Limited and Center
for Instrumental Analysis of China Pharmaceutical
University for assistance with pharmacological data
and characterization of the compounds.
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