Synthesis and evaluation of aliphatic-chain hydroxamates capped with osthole derivatives as histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.06.002

Our previous studies have demonstrated that osthole, a Chinese herbal compound, could be incorporated into the hydroxycinnamide scaffold of LBH-589, a potent HDAC inhibitor, as an effective hydrophobic cap; the resulting compounds showed significant poten

Full text of DOI:10.1016/j.ejmech.2011.06.002

European Journal of Medicinal Chemistry 46 (2011) 4042e4049
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of aliphatic-chain hydroxamates capped with osthole
derivatives as histone deacetylase inhibitors
Wei-Jan Huang a, Ching-Chow Chen b, Shi-Wei Chao a, Chia-Chun Yu c, Chen-Yui Yang d, Jih-Hwa Guh c,
,
Yun-Chieh Lin b, Chiao-I Kuo d, Ping Yang a, Chung-I Chang d
*
a Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan
b Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
c School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan
d Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Our previous studies have demonstrated that osthole, a Chinese herbal compound, could be incorporated
into the hydroxycinnamide scaffold of LBH-589, a potent HDAC inhibitor, as an effective hydrophobic cap;
the resulting compounds showed significant potency against several HDAC isoforms. Here, we presented
a series of osthole derivatives fused with the aliphatic-hydroxamate core of suberoylanilide hydroxamic
acid (SAHA), a clinically-approved HDAC inhibitor. Several compounds showed potent activity against
nuclear HDACs. Further assays against individual HDAC isoforms revealed that some compounds showed
not only SAHA-like activity towards HDAC1, -4 and -6, they inhibited HDAC8 by log difference than SAHA
and thus exhibited a broader HDAC inhibition spectrum. Among them, compound 6g showed potent
antiproliferative effect on several human cancer cell lines.
Received 9 February 2011
Received in revised form
7 May 2011
Accepted 1 June 2011
Available online 12 June 2011
Keywords:
Histone deacetylase
SAHA
Ó 2011 Elsevier Masson SAS. All rights reserved.
LBH-589
Osthole
Molecular modeling
1. Introduction
Mammalian HDACs consist of four classes, which are classified
by their sequence homology, sub-cellular distribution and catalytic
Epigenetic modifications such as DNA methylation, post-
translational modification of histone proteins, and chromatin
remolding regulate gene expression through change of chromatin
structure without change of gene sequence [1e4]. Of these epige-
netic processes, acetylation of the chromatin histones has been
widely studied. Acetylation of cationic lysine tails in nucleosome-
associated histones neutralizes charge and results in relaxed
chromatin and transcriptional expression. On the other hand,
deacetylation results in formation of condensed chromatin and
transcriptional repression. This dynamic system is controlled by the
balance of the activity between histone acetyltransferases (HATs)
and histone deacetylases (HDACs) [5]. Deregulation of the process
may cause various forms of malignancy [6e8]. Studies have also
revealed that deacetylation of some non-histone proteins such as
tubulin, p21, and p53 are controlled by HDACs; hence, HDACs may
also be involved in cell signaling events regulated by these mole-
cules [9,10].
activity. Class I (HDAC1, -2, -3, -8), class II (HDAC4, -5, -6, -7, -9, -10),
and class IV (HDAC11) enzymes are zinc-dependent HDACs,
whereas class III (Sirtuins 1e7) enzymes require NADþ for activity.
Class II enzymes are subdivided into class IIa (HDAC4, -5, -7, -9) and
class IIb (HDAC6, -10) [11]. Studies using knockout and transgenic
mice, as well as RNA interference (RNAi) have helped to delineate
the physiological role of HDAC [12,13]. For example, class I enzymes
(HDAC1, -2, -3, -8) have been implicated in differentiation, growth
inhibition and apoptosis in cancer cells [14,15]. In contrast to other
class I enzymes, HDAC8 is somewhat unique in that it does not exert
any effect on acetylation level of histone and
a-tubulin [16].
However, the enzyme has been shown to be associated with actin
and thus is involved in regulating the contractile capacity [17].
HDAC4, a representative of class IIa enzymes, has been shown to be
a regulator of chondrocyte hypertrophy and is crucial for skeleto-
genesis [18], as well as for neuronal death [19]. HDAC6, a repre-
sentative of class IIb enzymes, is associated with acetylation of
tubulin, microtubule [9,20] and HSP90 [21], suggesting that it may
be a target for treatment of neurodegenerative diseases [22].
Structurally diverse HDAC inhibitors currently in clinical
studies on solid and hematologic malignancies can be categorized
* Corresponding author. Tel.: þ886 2 2785 5696; fax: þ886 2 2788 9759.
E-mail address: chungi@gate.sinica.edu.tw (C.-I. Chang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.002

W.-J. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 4042e4049
4043
into four groups: hydroxamates-based compounds such as SAHA
[23], LBH-589 [24], SB-939 [25], PCI-24781 [26], JNJ26481585 [27]
and ITF-2357 [28]. Short-chain fatty acids such as butyrate and
valproate [29], cyclic tetrapeptides including FK-228 [30], tra-
poxin (TPX) [31], and apicidin [32] and 2-aminobenzamides
including MS-275 [33] and MGCD0103 [34]. These compounds
inhibit a number of HDAC isoforms and are regarded as pan HDAC
inhibitors [35]. To date, SAHA and FK-228 have gained approval
by FDA for treatment of refractory cutaneous T-cell lymphoma
(CTCL).
Cocrystal structures of a bacterial HDAC-like protein (HDLP;
a class I homolog), human HDAC8 (class I), ꢀ4, ꢀ7 (both belong to
class IIa), and a bacterial HDAC-like amidohydrolase (HDAH; a class
IIb homolog) with several HDAC inhibitors including TSA and SAHA
have revealed structural details on the active site of these zinc-
dependent HDACs and the binding interactions with small-
molecule inhibitors [36e42]. The active site consists of a deep
pocket spanning the length equivalent to four- to six-carbon
straight chain. A zinc ion bound at the bottom of the pocket
cooperates with two HiseAsp charge-relay systems and facilitates
the deacetylation catalysis. The chemical structures of HDAC
inhibitors reported so far can be divided into three functional
groups, each of which interacts with a discrete region of the HDAC
active site. These groups include a zinc-binding motif, a hydro-
phobic cavity-binding linker, and a surface recognition cap which is
also hydrophobic and acts to interact with HDAC surface located at
the rim of the active site cavity (Fig. 1). We have sought to develop
class-specific and selective-spectrum HDAC inhibitors with
a recognition cap effectively exploiting such surface region and
have hypothesized that such cap could be identified by searching
for hydrophobic motifs existing in natural products with structural
complexity [43].
Previously we have found that osthole, a naturally occurring
compound isolated from the Chinese herb Cnidium monnieri, could
be employed as a hydrophobic surface recognition cap when
incorporated into the hydroxycinnamide scaffold of LBH-589,
which is among the most potent pan-HDAC inhibitors. The result-
ing series of compounds showed potent inhibitory activity against
HDACs [43]. Molecular docking analysis further suggested that the
branched structure of osthole could potentially provide isoforms
selectivity by exploiting non-conserved surface region at the rim of
the active site pocket of HDACs [43]. In the present work, we have
combined osthole derivative with SAHA-liked hydroxamates made
by replacing the aryl connection unit in our original hydrox-
ycinnamide series with an aliphatic linker to see if using a flexible
linker to present the osthole-derived cap would allow us to identify
specific HDAC isoforms which the cap recognizes effectively.
Synthesized compounds were screened for enzyme inhibitory
activity and evaluated for cellular effects on cancer cells. We found
that in addition to inhibitory activity towards HDAC1, -4 and -6,
several compounds were more than 10-fold potent than SAHA
against HDAC8, confirming that osthole derivatives could be placed
in an HDAC inhibitory scaffold to offer selectivity to specific spec-
trum of isoforms.
Surface
Linker
Zinc binding
recognition
O
O
O
O
H
N
OH
S
OH
N
H
N
N
H
H
O
PXD101
SAHA
O
O
OH
OH
N
N
H
N
H
H
N
N
HN
N
SB-939
O
LBH-589
O
OH
N
H
N
O
O
N
H
OH
N
H
N
H
N
O
O
O
ITF-2357
PCI-24781
N
H
N
N
N
H
N
N
OH
O
JNJ-26481585
Fig. 1. Hydroxamates-based compounds currently in clinical trials.

4044
W.-J. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 4042e4049
2. Chemistry
3. Results and discussion
The synthesis of osthole-based building block is shown in
Scheme 1. Atmospheric hydrogenation of osthole (1) using catalytic
amount of palladium on carbon gave 2a [43]. Oxidation of 2a with
palladium on carbon under high temperature condition generated
2b in C3eC4 unsaturated form [44]. Demethylation of 2aeb in the
presence of boron tribromide etherate gave 3a and b, respectively
[44]. Reaction of compound 3a with appropriate bromo-substituted
methyl aliphatic acid esters with five to eight carbons chain length
by using K2CO3 as well as catalytic amount of 18-crow-6 and TBAI
yielded corresponding 4aed. Under similar condition reaction,
compound 3b upon replacing K2CO3 with NaH gave corresponding
4eeh. Saponification of 4aeh in the presence of LiOH produced
5aeh, respectively. Synthesis of hydroxamates (6ael) is illustrated
in Scheme 2 where compounds 5aed were first activated as reac-
tive mixed anhydrides in situ with ethyl chloroformate prior to
conversion into hydroxamates. During this synthetic step, we found
that after treating with 8 equiv of hydroxylamine, reactive inter-
mediates were converted into corresponding bishydroxamates
6aed instead of monohydroxamates. These unexpected products,
verified by high-resolution mass spectra, were generated as the
lactone ring attracts nucleophilic attack from excess amount of
hydroxylamine. Nevertheless, the bishydroxamate compounds
were moved forward for activity screening as Marks et al. have
previously reported a series of bishydroxamates with SAHA-like
HDAC inhibition potency, which could induce differentiation and
apoptosis of transformed cells [23]. Finally, monohydroxamate
compounds 6eel were produced after reaction with 2 equiv of
hydroxylamine. Details on the synthesis, isolation and character-
ization of compounds 2e5 can be found in the Supplementary
material.
Three series of compounds incorporated with osthole deriva-
tives, including bishydroxamates 6aed (Table 1), mono-
hydroxamates with unsaturated C3eC4 6eeh, and with saturated
C3eC4 6iel (Table 2) were tested for inhibition of HeLa nuclear
HDACs using SAHA as a reference. In spite of three different
recognition cap structures, bishydroxamates 6aed and mono-
hydroxamates 6eeh, 6iel showed similar structureeactivity
relationship (SAR) concerning chain length of the linker, namely
a six-carbon linker yielded optimal potency whereas the poorest
performers were those with the short four-carbon linker.
Compounds from all series possessing a six-carbon linker 6c, 6g
and 6k showed excellent inhibition potency against nuclear HDACs.
In the assay, these compounds were two-fold more potent than
SAHA, indicating that these osthole-derived caps, when presented
by an aliphatic linker with optimal chain length, could offer better
surface binding than the phenylamide cap of SAHA. The inhibitory
activities against nuclear HDACs of compounds 6eeh correlated
well with their antiproliferative activity in three human cancer cell
lines, including prostate cancer PC-3 cells, lung cancer A549 cells
and hepatoma Hep 3B cells; in particular, in these cells compound
6g showed up to two fold greater potency than SAHA (Table 1 and
Table 2). Although bishydroxamates 6aed showed enzyme inhibi-
tory potency comparable to that of hydroxamates 6eeh, they
showed only mild cancer cell cytotoxicity (Table 1). Similarly, when
compounds 6g and 6c were further evaluated for ability to promote
acetylation of histone and
a-tubulin in cellular assays, only
compound 6g (Fig. 2) but not 6c (data not shown) induced hyper-
acetylation of both substrates comparable to SAHA in a dose-
dependant manner. These results may be due to poor penetration
of the bishydroxamates across the cell membrane, which needs to
a
b
MeO
O
O
MeO
O
O
MeO
O
O
1
2a
2b
c
c
d
O
HO
O
O
HO
O
O
O
O
O
n
O
O
Br
O
n
n=4-7
3a
3b
4a-d
O
n
Br
d
e
O
n=4-7
e
O
HO
HO
O
n
O
O
O
O
O
O
O
O
n
n
O
O
O
4e-h
5a-d
5e-h
Scheme 1. Reagents and conditions: a) H2, Pd-C, EtOAc, RT, 16 h, 92%; b) Pd-C, mesitylene, Δ, 16 h, 81%; c) BBr3, CH2Cl2, N2, 0 ꢁC to RT, 2.5 h, 89e92%; d) 3a, K2CO3, 18-Crow-6, TBAI,
acetone, Δ, 2 h, 81e89%; 3b, 60% NaH, 18eCrow-6, TBAI, DMF, RT, 74e85%; e) LiOH, MeOH, Δ, 2 h, 91e95%.

W.-J. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 4042e4049
4045
O
OH
H
N
N
H
b
HO
a
H
N
HO
O
O
O
n
O
O
O
n
HO
O
OH
n
O
O
O
6i-l
5a-d
6a-d
H
HO
N
b
HO
O
O
O
O
n
O
O
n
O
O
5e-h
6e-h
Scheme 2. Reagents and conditions: a) 1. ClCO2Et, Et3N, THF, RT, 1 h; 2) 8 equiv NH2OH, KOH, MeOH, RT, 3 h, 59e65%; b) 1. ClCO2Et, Et3N, THF, RT, 1 h; 2. 2 equiv NH2OH, KOH,
MeOH, RT, 3 h, 38e65%.
be confirmed by cell-permeability analysis in order for solubility
optimization. Monohydroxamates 6iel and 6eeh differ only in
C3eC4 bonds; however, compounds 6iel exhibited low cytotox-
HDAC1, with compound 6g being 2-fold morepotent. Particularly for
HDAC8, monohydroxamates 6g and 6k exhibited 6- and 3-fold,
respectively, greater potency than SAHA. Similarly, osthole-derived
bishydroxamate 6c (as well as 6d) was 12-fold more potent. These
results demonstrated that osthole is an effective surface recognition
cap for HDAC8, whose cocrystal structures have revealed two flex-
ible active-site surface loops which could change conformation to
accommodate different inhibitors [41]. Thus it is possible that the
unique malleable feature of the HDAC8 active-site surface makes
these compounds with branched hydrophobic caps particularly
effective against this particular class I enzyme. Compounds 6c, 6d,
and 6g showed SAHA-like activity for HDAC6, while compound 6k
was less potent. By contrast, compounds 6c, 6d, 6g, and 6k together
icity in all three cell lines (GI50 > 30 mM) despite their potent HDAC
inhibition activity (IC50 ¼ 24e712 nM). We suspected that the
saturated C3eC4 connecting the lactone carbonyl of these
compounds may be liable to nucleophilic attacks; indeed, these
compounds were unstable and degraded within 6 h in culture
medium (detected by LC-MS; data not shown).
Although SAHA is inhibitory to a number of HDAC isoforms, the
compound is a weak inhibitor of HDAC8 (w3
mM in our assay
system). We performed molecular docking analysis of HDAC8 with
compounds 6c and 6g, both were most potent against nuclear
extract HDACs, using Autodock and its graphical user interface
AutoDockTools (ADT) [45]. In comparison with binding interaction
of SAHA to HDAC8 (PDB code 1T69) both 6c and 6g were found to
interact with the same surface region; however, the osthole-derived
caps are able to make potential additional hydrophobic contacts
with their dimethyl propyl groups (Fig. 3), possibly rendering the
compounds better HDAC8-binders than SAHA. Totestwhether these
hydroxamates with osthole-derived caps are potent HDAC8 inhibi-
tors, as well as to evaluate their HDAC isoform inhibition,
compounds 6c, 6d, 6g and 6k were chosen for in vitro assays against
enzymes of class I (HDAC1, -8), class IIa (HDAC4) and class IIb
(HDAC6) (Table 3). All of them showed potency superior to SAHA for
with SAHA were all inactive towards HDAC4 (IC50 > 10
mM), the
reason for such weak inhibitory activity of these hydroxamate
compounds is not clear. However, class IIa enzymes such as HDAC4
has been shown to possess an enlarged hydrophobic pocket shaped
by a class IIa-specific histidine, whose side chain is pointed away
from the active site tunnel [36].
In conclusion, we have developed SAHA-like hydroxamates
incorporated with three types of osthole derivative and showed
their SAR using HeLa nuclear HDAC assays. Three potent
compounds 6c, 6d, and 6g showed SAHA-comparable activity
Table 2
Inhibition of HeLa nuclear HDACa (IC50, nM) and cytotoxicity (GI50
, mM) against
various cancer cell lines by compounds 6eel.
Table 1
4
Inhibition of HeLa nuclear HDACa (IC50, nM) and cytotoxicity (GI50
,
mM) against
3
various cancer cell lines by compounds 6aed.
H
N
HO
O
O
O
O
2
n
1
OH
N
O
H
N
H
HO
O
OH
n
Compound C3eC4
Chain length Nuclear
(n)
GI50
O
HDACs
PC-3
A549 Hep3B
6e
6f
6g
6h
6i
6j
6k
6l
unsaturation 4
unsaturation 5
unsaturation 6
unsaturation 7
523.2
128.2
22.2
1.94
2.76
0.87
0.66
2.19
1.31
0.80
0.75
3.58
0.74
0.52
1.73
Compound
Chain length (n)
NuclearHDACs
GI50
PC-3
A549
Hep3B
39.0
6a
6b
6c
6d
4
5
6
7
426.3
209.9
24.6
28.9
41.7
14.41
2.52
4.73
1.73
1.02
10.02
5.41
6.16
2.67
0.79
14.07
1.98
4.72
1.27
1.21
saturation
saturation
saturation
saturation
4
5
6
7
711.6
425.5
23.7
111.7
41.7
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
SAHA
SAHA
1.02
0.79
1.21
a
a
Data were presented by mean value of three determinations.
Data were presented by mean value of three determinations.

4046
W.-J. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 4042e4049
Table 3
IC50a value for the inhibition of Class I and Class II HDACs by compounds 6ced, 6g
and 6k.
Compound Class I
HDAC1 (nM) HDAC8 (nM)
Class II
HDAC4 (
mM) HDAC6 (nM)
6c
6d
6g
6k
42.92 ꢃ 2.08
41.46 ꢃ 3.88
32.27 ꢃ 0.16
267.85 ꢃ 4.50
279.50 ꢃ 56.15
553.45 ꢃ 46.57
>10
>10
>10
>10
15.93 ꢃ 0.29
14.16 ꢃ 0.24
15.24 ꢃ 1.05
39.97 ꢃ 1.03
19.80 ꢃ 3.84
52.73 ꢃ 1.89 1075.12 ꢃ 7.13
SAHA
62.90 ꢃ 2.35 3295.01 ꢃ 109.62 >10
a
Data are expressed as mean ꢃ SD of three determinations.
JMX-HX110 mass spectrometer (HREIMS and HRFABMS), JMS-
SX102A mass spectrometer (EIMS and FABMS), and Finnigan Mat
TSQ-7000 mass spectrometer (ESIMS). TLC analyses were carried
out on silica gel plates (KG60-F254, Merck). The microplate spec-
trophotometer Victor 2x (Perkin Elmer, Fremont, CA, USA) was used
for fluorometric analysis. Unless otherwise mentioned, all chem-
icals and materials were used as received from commercial
suppliers without further purification. Dichloromethane was
distilled from calcium hydride under nitrogen. Tetrahydrofuran was
distilled from sodium and benzophenone under N2.
Fig. 2. Effect of compound 6g and SAHA on induction of
a-tubulin acetylation and
histone H3 acetylation in cultured human prostate cancer cells. Cells were incubated
with the HDAC inhibitors at 1, 5 and 10 M for 16 h. The whole-cell lysates were then
analyzed for histone H3 and -tubulin acetylation by SDSePAGE and Western blot for
either acetylated histone H3 or -tubulin.
m
a
a
towards HDAC1, -4 and -6. Importantly, they exhibited superior
anti-HDAC8 potency. Co-crystallography of these compounds with
HDAC8 is underway to find out the detailed binding modes of these
hydroxamates with osthole-derived caps. In this study, bishy-
droxamates are highly potent against nuclear HDACs but showed
only mild cancer cell cytotoxicity. Future work is needed to
understand whether the loss of efficacy in cell culture was due to
poor cell permeability of the compounds. Further modification of
the present series of structures for developing a broader pan-HDAC
inhibitor is being undertaken.
4.2. N-Hydroxy-5-(3-hydroxy-4-(2-hydroxycarbamoylethyl)-2-
isopentyl-phenoxy) pentanamide (6a)
To a solution of NH2OHeHCl (540 mg, 7.52 mmol) in MeOH
(15 mL) was added potassium hydroxide (420 mg, 7.52 mmol). The
reaction mixture was stirred in an ice-bath for 1 h. Filtration to
remove the white salt gave a solution of the free NH2OH in MeOH. A
solution of 5a (314 mg, 0.94 mmol) in freshly distd THF (10 mL) was
treated with ethyl chloroformate (0.13 mL, 1.41 mmol) and Et3N
(0.20 mL,1.41 mmol) and the resulting solution was stirred at RT for
1 h. The prepared free NH2OH solution was then added to the
reaction. Stirring was continued at RT for 3 h. The reaction was
concentrated in vacuo and the crude was diluted with distd H2O
(50 mL), acidified to pH 2e3 with 1N HCl and extracted with EtOAc
(25 mL ꢂ 3). The combined organic layer was dried over Na2SO4 and
the solvent removed in vacuo. The residue was purified by silica gel
chromatography (EtOAc: n-Hexane ¼ 1:1e2:1) to give 6a (233 mg,
65%) as a white solid. Mp 105e110 ꢁC. IR (KBr) 3221, 2953, 2868,
1746, 1645, 1492, 1469, 1383, 1365, 1273 cmꢀ1. 1H NMR (500 MHz,
4. Experimental
4.1. General procedures
IR spectra (KBr disk) were recorded using a Jasco Fourier
Transform infrared spectrometer (Jasco FT/IR-410). 1H NMR spec-
trum was obtained on a Bruker AV400 or AV500 spectrometer using
standard pulse programs. Melting point was recorded on Fish-
ereJohns apparatus (uncorrected). MS data were measured on JEOL
Acetone-d6):
d
7.59 (1br s, H), 6.88 (d, J ¼ 8.4 Hz, 1H), 6.43 (d,
J ¼ 8.4 Hz,1H), 3.95 (t, J ¼ 5.3 Hz, 2H), 2.80 (t, J ¼ 6.5 Hz, 2H), 2.65 (m,
2H), 2.47 (t, J ¼ 6.3 Hz, 2H), 2.19 (t, J ¼ 7.6 Hz, 2H), 1.78 (m, 4H), 1.57
(m, 1H), 1.34 (m, 2H), 1.37 (m, 2H), 0.92 (s, 3H), 0.91 (s, 3H). 13C NMR
(125 MHz, DMSO-d6): d 171.5, 171.3, 156.3, 152.7, 126.7, 120.3, 119.0,
103.4, 67.1, 38.4, 33.1, 32.0, 28.7, 28.2, 25.3, 22.2, 21.8, 21.1. HR-ESI-
MS m/z: (M ꢀ H)ꢀ calcd for C19H29N2O6 381.2026, found 381.2024.
4.3. N-Hydroxy-6-(3-hydroxy-4-(2-hydroxycarbamoylethyl)-2-
isopentyl-phenoxy) hexanamide (6b)
Following the procedure described for 6a, reaction of 5b
(348 mg,1 mmol) in THF (10 mL) with ethyl chloroformate (0.14 mL,
1.50 mmol) and Et3N (0.21 mL, 1.50 mmol) gave 6b (241 mg, 61%) as
a white solid. Mp 110e115 ꢁC. IR (KBr) 3212, 2951, 2867, 1643, 1492,
1469, 1383, 1365 cmꢀ1. 1H NMR (500 MHz, DMSO-d6):
d 10.51 (br s,
1H), 10.36 (br s, 1H), 8.69 (br s, 1H), 8.42 (br s, 1H), 6.79 (d,
J ¼ 8.4 Hz,1H), 6.35 (d, J ¼ 8.4 Hz,1H), 3.83 (t, J ¼ 6.1 Hz, 2H), 2.70 (t,
J ¼ 7.2 Hz, 2H), 2.54 (t, J ¼ 7.9 Hz, 2H), 2.23 (t, J ¼ 7.2 Hz, 2H), 1.95 (t,
J ¼ 7.5 Hz, 2H), 1.67 (q, J ¼ 6.3 Hz, 2H), 1.53 (m, 3H), 1.40 (q,
J ¼ 8.0 Hz, 2H), 1.26 (q, J ¼ 6.9 Hz, 2H), 0.90 (s, 3H), 0.89 (s, 3H). 13C
Fig. 3. Molecular modeling results of compounds 6c (green) and 6g (magenta) docked
to HDAC8 (PDB code 1T69 in surface representation). SAHA is shown in gray sticks.

W.-J. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 4042e4049
4047
NMR (125 MHz, DMSO-d6):
d
171.5, 156.4, 152.7, 126.7, 120.2, 118.9,
1.50 mmol) and Et3N (0.21 mL, 1.50 mmol) gave 6f (235 mg, 65%) as
a white solid. Mp 105e112 ꢁC. IR (KBr) 3484, 3232, 2951, 2867, 1711,
103.4, 67.4, 38.4, 33.2, 32.4, 28.9, 28.2, 25.5, 25.3, 25.2, 21.8, 21.1. HR-
ESI-MS m/z: (M ꢀ H)ꢀ calcd for C20H31N2O6, 395.2182, found
395.2187.
1654, 1606, 1561, 1497, 1466, 1402 cmꢀ1 1H NMR (500 MHz,
.
Acetone-d6):
d
10.02 (br s, 1H), 7.81 (d, J ¼ 9.4 Hz, 1H), 7.41 (d,
J ¼ 8.6 Hz,1H), 6.95 (d, J ¼ 8.6 Hz,1H), 6.14 (d, J ¼ 9.4 Hz,1H), 4.08 (t,
J ¼ 6.3 Hz, 2H), 2.76 (t, J ¼ 7.9 Hz, 2H), 2.10 (t, J ¼ 7.3 Hz, 2H), 1.81 (q,
J ¼ 7.4 Hz, 2H), 1.64 (q, J ¼ 7.4 Hz, 2H), 1.54 (m, 3H), 1.39 (dd, J ¼ 7.4,
15.4 Hz, 2H), 0.93 (s, 3H), 0.91 (s, 3H). 13C NMR (125 MHz, DMSO-
4.4. N-Hydroxy-7-(3-hydroxy-4-(2-hydroxycarbamoylethyl)-2-
isopentyl-phenoxy) heptanamide (6c)
Following the procedure described for 6a, reaction of 5c
(362 mg,1 mmol) in THF (10 mL) with ethyl chloroformate (0.14 mL,
1.50 mmol) and Et3N (0.21 mL, 1.50 mmol) gave 6c (258 mg, 63%) as
a white solid. Mp 105e110 ꢁC. IR (KBr) 3218, 2937, 2866, 1644, 1492,
d6): d 162.3, 160.0, 152.7, 145.0, 126.6, 118.4, 112.8, 111.5, 108.3, 68.2,
38.0, 32.3, 28.7, 28.1, 25.4, 25.1, 21.6, 20.3. HR-EI-MS m/z: (M)þ calcd
for C20H27NO5 361.1889, found 361.1894.
1467 cmꢀ1. 1H NMR (500 MHz, DMSO-d6):
d 10.46 (s, 1H), 10.30 (s,
1H), 8.78 (s, 1H), 8.66 (s, 1H), 8.43 (s, 1H), 6.78 (d, J ¼ 8.4 Hz, 1H),
6.34 (d, J ¼ 8.4 Hz,1H), 3.83 (t, J ¼ 6.1 Hz, 2H), 2.68 (t, J ¼ 7.2 Hz, 2H),
2.53 (m, 2H), 2.21 (t, J ¼ 7.2 Hz, 2H), 1.93 (t, J ¼ 7.3 Hz, 2H), 1.66 (m,
2H),1.50 (m, 3H),1.42 (m, 2H),1.26 (m, 4H), 0.90 (s, 3H), 0.88 (s, 3H).
4.8. N-Hydroxy-7-(8-isopentyl-2-oxo-2H-chromen-7-yloxy)
heptanamide (6g)
Following the procedure described for 6e, reaction of 5g
(360 mg,1 mmol) in THF (10 mL) with ethyl chloroformate (0.14 mL,
1.50 mmol) and Et3N (0.21 mL, 1.50 mmol) gave 6g (218 mg, 58%) as
a white solid. Mp 73e80 ꢁC. IR (KBr) 3246, 2932, 1711, 1606, 1666,
13C NMR (125 MHz, DMSO-d6):
d 171.6, 171.5, 156.4, 152.7, 126.7,
120.2, 118.9, 103.4, 67.5, 38.4, 33.2, 32.4, 29.1, 28.6, 28.2, 25.7, 25.4,
25.3, 21.8, 21.1. HR-ESI-MS m/z: (M ꢀ H)ꢀ calcd for C21H33N2O6
409.2339, found 409.2336.
1284 cmꢀ1. 1H NMR (400 MHz, CDCl3):
d
7.60 (d, J ¼ 9.4 Hz, 1H), 7.24
(d, J ¼ 8.5 Hz, 1H), 6.78 (d, J ¼ 8.6 Hz, 1H), 6.22 (d, J ¼ 9.4 Hz, 1H),
4.00 (t, J ¼ 6.3 Hz, 2H), 2.81 (t, J ¼ 7.2 Hz, 2H), 1.82 (t, J ¼ 6.9 Hz, 2H),
1.68 (m, 2H), 1.60 (q, J ¼ 6.6 Hz, 1H), 1.52 (q, J ¼ 7.0 Hz, 2H), 1.40 (dd,
J ¼ 6.8, 15.6 Hz, 4H), 0.95 (s, 3H), 0.94 (s, 3H). 13C NMR (125 MHz,
4.5. N-Hydroxy-8-(3-hydroxy-4-(2-hydroxycarbamoylethyl)-2-
isopentyl-phenoxy) octanamide (6d)
Following the procedure described for 6a, reaction of 5d
(376 mg, 1 mmol) in THF (10 mL) with ethyl chloroformate (0.14 mL,
1.50 mmol) and Et3N (0.21 mL,1.50 mmol) gave 6d (250 mg, 59%) as
DMSO-d6): d 171.5, 162.2, 160.0, 152.6, 145.0, 126.6, 118.3, 112.7,
111.5, 108.3, 68.2, 38.0, 32.4, 28.9, 28.5, 28.1, 25.6, 25.4, 21.6, 20.3.
HR-ESI-MS m/z: (M þ H)þ calcd for C21H30NO5 376.2124, found,
376.2146.
a colorless liquid. IR (KBr) 3235, 2934, 2866, 1644, 1470, 1383 cmꢀ1
.
1H NMR (500 MHz, Acetone-d6):
d 10.29 (br s, 1H), 10.12 (br s, 1H),
8.40 (br s,1H), 6.82 (d, J ¼ 8.3 Hz,1H), 6.37 (d, J ¼ 8.3 Hz,1H), 3.88 (t,
J ¼ 6.2 Hz, 2H), 2.81 (br s, 2H), 2.66 (t, J ¼ 7.9 Hz, 2H), 2.47 (br s, 2H),
2.11 (t, J ¼ 7.9 Hz, 2H), 1.74 (q, J ¼ 6.4 Hz, 2H), 1.57 (m, 3H), 1.48 (q,
J ¼ 6.4 Hz, 2H), 1.36 (m, 6H), 0.93 (s, 3H), 0.92 (s, 3H). 13C NMR
4.9. N-Hydroxy-8-(8-isopentyl-2-oxo-2H-chromen-7-yloxy)
octanamide (6h)
(125 MHz, DMSO-d6):
d
171.6, 156.5, 152.7, 126.7, 120.2, 118.9, 103.4,
Following the procedure described for 6e, reaction of 5h
(374 mg,1 mmol) in THF (10 mL) with ethyl chloroformate (0.14 mL,
1.50 mmol) and Et3N (0.21 mL,1.50 mmol) gave 6h (237 mg, 61%) as
a white solid. Mp 55e60 ꢁC. IR (KBr) 3244, 2935, 2866, 1710 1654,
67.6, 38.4, 33.2, 32.4, 29.3, 28.8, 28.3, 25.9, 25.4, 21.8, 21.1. HR-ESI-
MS m/z: (M ꢀ H)ꢀ calcd for C22H35N2O6 423.2495, found 423.2490.
4.6. N-Hydroxy-5-(8-isopentyl-2-oxo-2H-chromen-7-yloxy)
1606,1561,1496 cmꢀ1 1H NMR (500 MHz, DMSO-d6):
d 10.30 (s,1H),
pentanamide (6e)
8.62 (s, 1H), 7.94 (d, J ¼ 9.4 Hz, 1H), 7.50 (d, J ¼ 8.6 Hz, 1H), 7.00 (d,
J ¼ 8.6 Hz,1H), 6.24 (d, J ¼ 9.4 Hz,1H), 4.06 (t, J ¼ 6.2 Hz, 2H), 2.70 (t,
J ¼ 7.9 Hz, 2H),1.93 (t, J ¼ 7.3 Hz, 2H),1.73 (q, J ¼ 6.9 Hz,1H),1.53 (m,
1H), 1.48 (m, 2H), 1.44 (m, 2H), 1.35 (m, 2H), 1.29 (m, 2H), 0.92 (s,
To a solution of NH2OHeHCl (144 mg, 2.00 mmol) in MeOH
(10 mL) was added potassium hydroxide (112 mg, 2.00 mmol). The
reaction mixture was stirred in an ice-bath for 1 h. Filtration to
remove the white salt gave a solution of the free NH2OH in MeOH. A
solution of 5e (332 mg, 1 mmol) in THF (10 mL) was treated with
ethyl chloroformate (0.14 mL, 1.50 mmol) and Et3N (0.21 mL,
1.50 mmol) and the resulting solution was stirred at RT for 1 h. The
prepared free NH2OH solution was then added to the reaction.
Stirring was continued at RT for 3 h. Following the work-up
procedure described for 6a gave 6e (191 mg, 55%) as a white
solid. Mp 90e98 ꢁC. IR (KBr) 3499, 3223, 2952, 1708 1656, 1606,
3H), 0.91 (s, 3H). 13C NMR (125 MHz, DMSO-d6):
d 169.5, 160.9159.8,
152.8, 145.3, 127.5, 117.8, 112.9, 112.6, 109.0, 68.7, 60.4, 38.2, 32.7,
29.1, 28.9, 28.2, 26.0, 25.5, 22.9, 20.6. HR-ESI-MS m/z: (M ꢀ H)ꢀ
calcd for C21H30NO5 388.2124, found 388.2116.
4.10. N-Hydroxy-5-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-
7-yloxy)pentanamide (6i)
1496 cmꢀ1. 1H NMR (500 MHz, CDCl3):
(d, J ¼ 6.6 Hz, 1H), 6.76 (d, J ¼ 6.6 Hz, 1H), 6.22 (d, J ¼ 9.4 Hz, 1H),
4.04 (br s, 2H), 2.80 (t, J ¼ 8.0 Hz, 2H), 2.27 (br s, 2H), 1.88 (m, 4H),
1.60 (m, 1H), 1.40 (m, 2H), 0.95 (s, 3H), 0.94 (s, 3H). 13C NMR
d
7.59 (d, J ¼ 9.3 Hz,1H), 7.23
Following the procedure described for 6e, reaction of 5a
(167 mg, 0.5 mmol) in THF (10 mL) with ethyl chloroformate
(0.07 mL, 0.75 mmol) and Et3N (0.11 mL, 0.75 mmol) gave 6i (73 mg,
42%) as a light brown liquid. IR (KBr) 3446, 2955, 2870, 2363, 1747,
1712, 1665, 1616, 1493, 1451 cmꢀ1. 1H NMR (500 MHz, DMSO-d6):
(125 MHz, DMSO-d6): d 162.2, 159.9, 152.6, 144.9, 126.6, 118.4, 112.8,
111.6, 108.3, 67.9, 38.0, 31.9, 28.4, 28.1, 22.0, 21.6, 20.3. HR-EI-MS m/
d
9.49 (br s, 1H), 7.24 (s, 1H), 7.02 (d, J ¼ 8.3 Hz, 1H), 6.71 (d,
z: (M)þ calcd for C19H25NO5 347.1733, found 347.1725.
J ¼ 8.3 Hz,1H), 3.94 (t, J ¼ 5.7 Hz, 2H), 2.87 (t, J ¼ 6.6 Hz, 2H), 2.70 (t,
J ¼ 7.7 Hz, 2H), 2.57 (q, J ¼ 7.9 Hz, 2H), 2.09 (t, J ¼ 7.8 Hz, 2H), 1.66
(m, 4H),1.51 (m,1H),1.31 (q, J ¼ 7.0 Hz, 2H), 0.90 (s, 3H), 0.89 (s, 3H).
4.7. N-Hydroxy-6-(8-isopentyl-2-oxo-2H-chromen-7-yloxy)
hexanamide (6f)
13C NMR (125 MHz, DMSO-d6)
d 169.3, 168.9, 156.4, 150.4, 125.8,
118.9, 115.8, 107.7, 68.0, 38.7, 32.3, 31.1, 29.3, 28.8, 28.1, 23.1, 22.9,
22.2, 21.0. HR-ESI-MS m/z: (M ꢀ H)ꢀ calcd for C19H26NO5 348.1811,
found 348.1809.
Following the procedure described for 6e, reaction of 5f
(346 mg,1 mmol) in THF (10 mL) with ethyl chloroformate (0.14 mL,

4048
W.-J. Huang et al. / European Journal of Medicinal Chemistry 46 (2011) 4042e4049
4.11. N-Hydroxy-6-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-
4.15. HDAC activity assay
yloxy)hexanamide (6j)
The HDAC activity assay was carried out as described previously
[46]. Enzyme, inhibitors and substrate were diluted with HDAC
buffer (15 mM TriseHCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10%
Following the procedure described for 6e, reaction of 5b
(174 mg, 0.5 mmol) in THF (10 mL) with ethyl chloroformate
(0.07 mL, 0.75 mmol) and Et3N (0.11 mL, 0.75 mmol) gave 6j (85 mg,
47%) as a colorless liquid. IR (KBr) 3402, 3213, 2948, 2867, 2362,
1746, 1665, 1619, 1495, 1413 cmꢀ1. 1H NMR (500 MHz, DMSO-d6):
v:v glycerol). Briefly, 10
extract (Enzo), HDAC1 (Enzo), HDAC6 (Enzo), HDAC4 and HDAC8
and 50 L test compound solution at different concentrations were
mL diluted HDAC such as HeLa nuclear
m
d
7.22 (br s, 1H), 7.02 (d, J ¼ 8.4 Hz, 1H), 6.70 (d, J ¼ 8.4 Hz, 1H), 6.67
added to each well of a 96-well microtiter plate and pre-incubated
(br s, 1H), 3.93 (t, J ¼ 6.2 Hz, 2H), 2.87 (t, J ¼ 6.7 Hz, 2H), 2.70 (t,
J ¼ 7.6 Hz, 2H), 2.57 (t, J ¼ 7.8 Hz, 2H), 2.04 (t, J ¼ 7.3 Hz, 2H), 1.70 (q,
J ¼ 7.1 Hz, 2H),1.52 (m, 3H),1.42 (m, 2H),1.30 (q, J ¼ 7.0 Hz, 2H), 0.91
at 30 ꢁC for 5 min. The enzymatic reaction was started by addition
of 40
mL substrate such as Boc-Lys(Ac)-AMC (Bachem) for HeLa
nuclear extract and HDAC6, Boc-Lys(TFA)-AMC (Bachem) for
HDAC4, -8 and KI 177 (Enzo) for HDAC1 in HDAC buffer. After
incubation at 30 ꢁC for 30 min, the reaction was terminated by
(s, 3H), 0.90 (s, 3H). 13C NMR (125 MHz, DMSO-d6):
d 177.6, 169.7,
156.6,150.2,124.9,119.3,115.1,107.0, 67.8, 38.4, 35.0, 28.9, 28.1, 25.6,
25.2, 22.9, 21.6, 20.6. HR-ESI-MS m/z: (M ꢀ H)ꢀ calcd for C20H28NO5
362.1967, found 362.1988.
addition of 100
TriseHCl pH 8, 100 mM NaCl, 2
for further 20 min, fluorescence was measured (extinction
m
L trypsin solution (10 mg/mL trypsin in 50 mM
m
M SAHA). After incubation at 30 ꢁC
l
¼ 355 nm, emission ¼ 460 nm). For the calculation of IC50 values
l
4.12. N-Hydroxy-7-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-
7-yloxy)heptanamide (6k)
the fluorescence in wells without test compound (0.1% DMSO,
negative control) was set as 100% enzymatic activity and the fluo-
rescence in wells with 2 mM SAHA (positive control) were set at 0%
enzymatic activity. All experiments were carried out in triplicates.
Following the procedure described for 6e, reaction of 5c
(181 mg, 0.5 mmol) in THF (10 mL) with ethyl chloroformate
(0.07 mL, 0.75 mmol) and Et3N (0.11 mL, 0.75 mmol) gave 6k
(72 mg, 38%) as a colorless liquid. IR (KBr) 2935, 2867, 2369, 1711,
4.16. Molecular docking analysis
1658, 1619, 1493, 1468 cmꢀ1. 1H NMR (500 MHz, DMSO-d6):
d 10.30
Docking study of compounds 6c and 6g was performed using
a crystal structure of HDAC8 (PDB code 1T69; bound SAHA in the
coordinates was removed prior to docking calculations) as a rigid
receptor with Autodock4.2 and the graphical user interface Auto-
DockTools (ADT) [45]. The docking parameters, grid box and algo-
rithm settings were as described in [47]. Docking of SAHA into
HDAC8 was carried out separately as a control to validate the
quality of the docking settings. Best docking poses of the
compounds were with the lowest energy and offered chemical
match and charge complementarity with the receptor.
(s, 1H), 8.61 (s, 1H), 6.88 (d, J ¼ 8.4 Hz, 1H), 6.70 (d, J ¼ 8.4 Hz, 1H),
3.93 (t, J ¼ 6.1 Hz, 2H), 2.87 (t, J ¼ 6.7 Hz, 2H), 2.70 (t, J ¼ 7.7 Hz, 2H),
2.57 (t, J ¼ 7.8 Hz, 2H), 1.93 (q, J ¼ 6.6 Hz, 2H),1.70 (q, J ¼ 6.4 Hz, 2H),
1.51 (m, 3H), 1.42 (m, 2H), 1.28 (m, 6H), 0.90 (s, 3H), 0.89 (s, 3H). 13C
NMR (125 MHz, DMSO-d6): d 169.5, 169.0, 156.4, 150.4, 125.9, 118.9,
115.7, 107.7, 68.2, 38.7, 32.7, 29.3, 29.2, 28.8, 28.1, 25.8, 25.6, 23.1,
22.9, 21.0. HR-ESI-MS m/z: (M ꢀ H)ꢀ calcd for C21H30NO5 376.2124,
found 376.2125.
4.13. N-Hydroxy-8-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-
7-yloxy)octanoate (6l)
4.17. The sulforhodamine B (SRB) growth inhibition assay
Cells were seeded in 96-well plates in medium with 5% FBS. After
24 h, cells of T0 group were fixed with 10% trichloroacetic acid (TCA)
to represent cell population at the time of hydroxamates addition
(T0). After additional incubation of vehicle (0.1% DMSO) or
hydroxamates (all dissolved in media without apparent precipita-
tion) for 48 h, cells were fixed with 10% TCA and SRB at 0.4% (w/v) in
1% acetic acid was added to stain cells. Unbound SRB was washed
out by 1% acetic acid and SRB bound cells were solubilized with
10 mM Trizma base. The absorbance was read at a wavelength of
515 nm. Using the following absorbance measurements, such as
time zero (T0), control growth (C), and cell growth in the presence of
hydroxamates (Tx), the percentage growth was calculated at each of
the compound concentrations levels. Percentage growth inhibition
was calculated as: [1ꢀ(TxꢀT0)/(CꢀT0)] ꢂ 100% for concentrations
for which Tx ꢄ T0. Growth inhibition of 50% (GI50) is determined at
the drug concentration which results in 50% reduction of total
protein increase in control cells during the compound incubation.
Following the procedure described for 6e, reaction of 5d
(188 mg, 0.5 mmol) in THF (10 mL) with ethyl chloroformate
(0.07 mL, 0.75 mmol) and Et3N (0.11 mL, 0.75 mmol) gave 6l (88 mg,
45%) as a colorless liquid. IR (KBr) 2950, 2868, 2363, 1712, 1652,
1607, 1496, 1468 cmꢀ1 1H NMR (500 MHz, DMSO-d6):
. d 10.30 (d,
J ¼ 4.1 Hz,1H), 8.61 (s,1H), 7.02 (d, J ¼ 8.4 Hz,1H), 6.70 (d, J ¼ 8.4 Hz,
1H), 3.93 (t, J ¼ 6.0 Hz, 2H), 2.87 (t, J ¼ 6.7 Hz, 2H), 2.70 (t, J ¼ 7.7 Hz,
2H), 2.57 (t, J ¼ 7.7 Hz, 2H),1.93 (q, J ¼ 6.6 Hz, 2H),1.70 (p, J ¼ 6.4 Hz,
2H),1.51 (m, 2H),1.42 (m, 2H), 1.31 (m, 4H), 0.91 (s, 3H), 0.89 (s, 3H).
13C NMR (125 MHz, DMSO-d6):
d 169.5, 169.0, 156.4, 150.4, 125.9,
118.9, 115.7, 107.7, 68.2, 38.7, 32.7, 29.3, 29.2, 28.8, 28.1, 25.8, 25.6,
23.1, 23.0, 22.9, 21.0. HR-ESI-MS m/z: (M þ H)þ calcd for C22H34NO5
392.2437, found 392.2448.
4.14. Preparation of HDAC4 and HDAC8
Genes encoding HDAC4 (residues 648e1057) and HDAC8 (resi-
dues 1e377) flanked with NdeI and EcoRI sites at the 50- and 30-
ends, respectively, were synthesized by GenScript Corporation (NJ,
USA) and subcloned into expression vectors pET-28a(þ) and pET-
24b(þ), respectively. Proteins were expressed in BL21(DE3) cells
by induction with IPTG (1 mM) at 20e25 ꢁC for overnight and
purified from cleared cell lysates by sequential chromatography on
Ni-Sepharose 6 fast flow, Mono Q 5/50 GL, and Superdex 75 10/
300 GL columns (GE Healthcare). Protein concentrations were
quantified with Bradford Reagent (Bio-Rad).
4.18. Western blot analysis
After treatment with compounds for 24 h, the cells were rapidly
washed with PBS, and then lysed in ice-cold lysis buffer (50 mM
TriseHCl, pH 7.4, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM
PMSF, 5 mg/mL leupeptin, 20 mg/mL aprotinin, 1 mM NaF, and 1 mM
Na3VO4). The lysates were subjected to SDS-PAGE by using 13%
polyacrylamide gels; protein bands were transferred to a nitrocel-
lulose membrane and immunoblot analysis performed as described

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Acknowledgments
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Appendix. Supplementary material
Supplementary material related to this article can be found
online at doi:10.1016/j.ejmech.2011.06.002.
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