Synthesis of n-hydroxycinnamides capped with a naturally occurring moiety as inhibitors of histone deacetylase

Article abstract of DOI:10.1002/cmdc.200900494

Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc-chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate-based compounds as inhibitors of HDAC. Nine novel osthole-based N-hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9d, 9e, 9g exhibited inhibitory activities (IC₅₀=24.5, 20.0, 19.6 nm) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC ₅₀=24.5 nm), a potent inhibitor clinically used for the treatment of cutaneous T-cell lymphoma (CTCL). While compounds 9d and 9e showed SAHA-like activity towards HDAC1 and HDAC6, compound 9g was more selective for HDAC1. Compound 9d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either a-tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class-specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N-hydroxycinnamide-derived HDAC inhibitors.

Full text of DOI:10.1002/cmdc.200900494

MED
DOI: 10.1002/cmdc.200900494
Synthesis of N-Hydroxycinnamides Capped with a
Naturally Occurring Moiety as Inhibitors of Histone
Deacetylase
Wei-Jan Huang,[b] Ching-Chow Chen,[c] Shi-Wei Chao,[b] Shoei-Sheng Lee,[d] Fen-Lin Hsu,[b]
Yeh-Lin Lu,[b] Ming-Fang Hung,[a] and Chung-I Chang*[a]
Histone deacetylase (HDAC) inhibitors are regarded as promis-
ing therapeutics for the treatment of cancer. All reported
HDAC inhibitors contain three pharmacophoric features: a
zinc-chelating group, a hydrophobic linker, and a hydrophobic
cap for surface recognition. In this study we investigated the
effectiveness of osthole, a hydrophobic Chinese herbal com-
pound, as the surface recognition cap in hydroxamate-based
compounds as inhibitors of HDAC. Nine novel osthole-based
N-hydroxycinnamides were synthesized and screened for
enzyme inhibition activity. Compounds 9d, 9e, 9g exhibited
inhibitory activities (IC50 =24.5, 20.0, 19.6 nm) against nuclear
HDACs in HeLa cells comparable to that of suberoylanilide hy-
droxamic acid (SAHA; IC50 =24.5 nm), a potent inhibitor clini-
cally used for the treatment of cutaneous T-cell lymphoma
(CTCL). While compounds 9d and 9e showed SAHA-like activi-
ty towards HDAC1 and HDAC6, compound 9g was more selec-
tive for HDAC1. Compound 9d exhibited the best cellular
effect, which was comparable to that of SAHA, of enhancing
acetylation of either a-tubulin or histone H3. Molecular dock-
ing analysis showed that the osthole moiety of compound 9d
may interact with the same hydrophobic surface pocket ex-
ploited by SAHA and it may be modified to provide class-spe-
cific selectivity. These results suggest that osthole is an effec-
tive hydrophobic cap when incorporated into N-hydroxycinn-
amide-derived HDAC inhibitors.
Introduction
The nucleosome, which is the basic functional unit of chroma-
tin, consists of DNA wrapped around a histone octamer of
pairs of H2A, H2B, H3, and H4 proteins.[1–3] A variety of
enzyme-catalyzed epigenetic modifications of histones regu-
late the function of chromatin. Of these modifications, reversi-
ble acetylation of histones, balanced by histone acetyltransfer-
ases (HATs) and histone deacetylases (HDACs),[4] plays an im-
portant role in chromatin remolding and regulation of gene
expression.[5–8] Acetylation of cationic lysine tails in nucleo-
some-associated histones neutralizes charge and results in re-
laxation of chromatin and transcriptional expression, while de-
acetylation results in the formation of condensed chromatin
and transcriptional repression. Aberrant regulation of this epi-
genetic system can cause inappropriate gene expression asso-
ciated with the pathogenesis of many forms of malignancy.[9–11]
Moreover, HDACs are involved with acetylation of nonhistone
proteins such as tubulin, p21 and p53, suggesting that these
enzymes may also regulate a broad spectrum of cellular
events.[12,13] HDAC inhibitors have been shown to exhibit anti-
neoplastic effects in many types of tumor cell lines, as well as
in vivo models.[14–16] Hence, they are considered as promising
anticancer therapeutics.
(HDAC4, -5, -7, -9) and class IIb (HDAC6, -10).[17] Studies of se-
lective, gene knockdown by siRNA have demonstrated that
class I enzymes, in particular HDAC1 and HDAC3, are crucial for
proliferation and survival of cancer cells.[18,19] HDAC6, a repre-
sentative member of the class IIb enzymes, has been associat-
ed with acetylation of tubulin, microtubule and HSP 90, imply-
ing that it may be a target for treatment of neurodegenerative
diseases.[20]
Natural products such as trichostatin A (TSA),[21] cyclic tetra-
peptide trapoxin (TPX),[22] apicidin[23] and depsipeptide FK-
228,[24] as well as synthetic compounds including SAHA,[25] beli-
nostat (PXD101)[26] and benzamide MS-275,[27] exhibit strong
HDAC inhibitory activity. Some of them are currently in clinical
[a] M.-F. Hung, Prof. C.-I. Chang
Institute of Biological Chemistry, Academia Sinica
128 Academia Road, Sec. 2, Nankang, Taipei 11529 (Taiwan)
Fax: (+886)2-2788-9759
E-mail: chungi@gate.sinica.edu.tw
[b] Prof. W.-J. Huang, S.-W. Chao, Prof. F.-L. Hsu, Y.-L. Lu
Graduate Institute of Pharmacognosy, Taipei Medical University
250 Wu-Xing Street, Taipei 110 (Taiwan)
[c] Prof. C.-C. Chen
Department of Pharmacology, National Taiwan University
College of Medicine, 1 Ren-Ai Road, Taipei 100 (Taiwan)
The mammalian HDACs are divided into four classes based
on their sequence homology, subcellular distribution and cata-
lytic 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 1–7) enzymes require NAD+
for activity. Class II enzymes are subdivided into class IIa
[d] Prof. S.-S. Lee
School of Pharmacy, National Taiwan University College of Medicine
1 Ren-Ai Road, Taipei 100 (Taiwan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900494.
598
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 598 – 607

Histone Deacetylase Inhibitors
trials for single-agent therapy or for use as combination thera-
py with classical chemotherapeutic agents for treatment of
solid and hematologic malignancies (Figure 1).[28] Co-crystal
nieri. Osthole is hydrophobic in nature with a Clog P value
around 3.5.[42] The hydrophobic nature of osthole is compatible
with the chemical feature of the capping group in known
HDAC inhibitors, which serves to form hydrophobic
contact with the surface residues at the edge of the
active site. Moreover, the branched aliphatic side
chain in the chemical structure of osthole may be fur-
ther exploited to provide specific interactions with
different HDACs. Osthole reportedly exhibits a wide
variety of pharmacological activities, including anti-
hepatitic, antiproliferative and antiosteoporotic func-
tions.[36–41] With its multiple significant pharmacologi-
cal activities, osthole might offer combinatorial thera-
peutic advantages when incorporated into selected
hydrophobic scaffolds with defined targets. Herein,
we describe the introduction of an osthole group
into an N-hydroxycinnamide-derived scaffold with dif-
ferent chain lengths and substitution positions. All
synthesized compounds were screened for HDAC in-
hibition and growth inhibition against cancer cells.
Furthermore, we examined the expression of bio-
markers associated with HDAC inhibition, and carried
out molecular modeling studies on selected com-
pounds.
Figure 1. HDAC inhibitors currently in clinical trials.
structures of bacterial HDAC-like protein (HDLP; a class I homo- Results and Discussion
logue), human HDAC8 (class I), HDAC4 and HDAC7 (class IIa),
Chemistry
and a bacterial HDAC-like amidohydrolase (HDAH; a class IIb
homologue) with several HDAC inhibitors, including TSA and
SAHA, have revealed structural details about the active site of
these zinc-dependent HDACs and the binding interactions
with small-molecule inhibitors.[29–35] The active site consists of a
deep pocket spanning a length equivalent to four- to six-
carbon straight chain. A zinc ion bound at the bottom of the
pocket coordinates with two His–Asp charge-relay systems and
facilitates the deacetylation catalysis. Although the zinc-bind-
ing feature of the active-site pockets in these HDAC structures
is conserved, class I and IIb HDACs appear to have a narrow,
tube-like cavity, whereas class IIa enzymes contain an enlarged
pocket that is shaped by an outwardly-oriented histidine in
the active site, which is conserved in all class IIa HDACs.[29,33]
The structure of all HDAC inhibitors reported so far can be
divided into three functional subunits, each of which interacts
with a discrete region of the HDAC active site. The three sub-
units include: a zinc-binding motif (such as hydroxamic acid), a
hydrophobic cavity-binding linker with four to six carbon
atoms, and a hydrophobic surface recognition cap that may
confer class-specific recognition of the HDAC surface at the
rim of the active-site cavity (Figure 1). We have sought to de-
velop class-specific HDAC inhibitors by incorporating various
naturally occurring hydrophobic motifs into the compounds.
These hydrophobic moieties display structural complexity and
thus may identify lead compounds that bind to the enzymes
by exploiting class-specific active site features in HDACs.
The preparation of osthole-based building blocks was achieved
as described in Scheme 1. Atmospheric hydrogenation of ost-
hole (1) in the presence of palladium on carbon gave 2 in a
Scheme 1. Synthesis of 4a–c. Reagents and conditions: a) H2, EtOAc, RT, 16 h,
92%; b) BBr3, CH2Cl2, N2, 08C!RT, 2.5 h, 92%; c) K2CO3, acetone, N2, 568C,
16 h, 81–85%.
quantitative yield. Demethylation of 2 by using boron tribro-
mide etherate afforded 3 in high yield. Compound 3 reacted
with the appropriate alkyl bromide, such as 2-chloroethyl, 3-
chloropropyl and 4-chlorobutyl, to give 4a–c. The preparation
of N-hydroxycinnamides 9a–j was achieved as illustrated in
Scheme 2. Methyl esterification of ortho-, meta- and para-cou-
maric acids 5a–c gave the corresponding methyl coumarates
6a–c. Coupling of the methyl coumarates 6a–c with the previ-
Osthole (1), a prenylated coumarin derivative, is one of the
major active compounds in the Chinese herb Cnidium mon-
ChemMedChem 2010, 5, 598 – 607
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
599

C.-I. Chang et al.
MED
ortho-substituted compounds (9a–c) had much lower
activity, suggesting weaker binding to the active site,
perhaps due to a twisted conformation in the linker
region that these ortho compounds must adapt
when bound to the narrow, tube-like cavity. In the
meta-substituted series, compounds 9d and 9e were
equally potent, while 9 f was 12-fold less potent than
SAHA, indicating that a long linker chain with more
than three carbons weakens the binding affinity, per-
haps by preventing the osthole group from effective-
ly interacting with the potential binding pockets at
the rim of the active site.[43] Compound 9g was nine-
fold more potent than 9h, suggesting that for para-
substituted N-hydroxycinnamides, a linker with a
chain length of more than two carbons leads to
decreased binding affinity.
Scheme 2. Synthesis of 9a–j. Reagents and conditions: a) H2SO4, MeOH, RT, 16 h, 91–95%;
b) 4a–c, K2CO3, DMF, N2, RT, 16 h, 51–60%; c) LiOH, MeOH, N2, 638C, 16 h, 93–97%;
d) 1. HCO2Et, Et3N, THF, RT, 1 h; 2. NH2OH-HCl, KOH, MeOH, RT, 3 h, 52–64%.
Compounds 9a–j were also assessed for growth in-
ously prepared chlorides 4a–c provided 7a–j. Base-catalyzed
hydrolysis of 7a–j using lithium hydroxide gave the corre-
sponding cinnamic acids 8a–j in quantitative yields. Reaction
of cinnamic acids 8a–j with ethyl chloroformate generated the
corresponding mixed anhydride in situ, and subsequent treat-
ment with hydroxylamine gave N-hydroxycinnamides 9a–j. All
test compounds were estimated to be at least 95% pure as
judged by HPLC analysis.
hibition activity in human hepatoma HepG2 cells (Table 1). For
most of the compounds tested, the cell growth inhibition re-
sults were consistent with the HDAC inhibition profiles, except
for 9i. Compounds 9d, 9e, and 9g were further evaluated for
inhibitory activities towards HDAC1 and HDAC6 (Table 2). For
Table 2. IC50 value for the inhibition of HDACs 1 and 6 by compounds
9d–e, 9g and 9i.
HDAC1
IC50[a] [nm]
HDAC6
IC50[a] [nm]
Compd
Relative
potency[b]
Relative
potency[b]
Enzyme inhibition
All synthesized N-hydroxycinnamides 9a–j were tested for
their ability to inhibit HeLa nuclear HDACs (which consist of
mostly HDAC1–3), with SAHA as a reference compound.
Among them, compounds 9d, 9e and 9g exhibited potent
HDAC inhibition comparable to that of SAHA, suggesting that
either meta- or para- substitution of the N-hydroxycinnamide
has a positive contribution to enzyme inhibition (Table 1). All
9d
9e
9g
9i
66.83ꢀ1.75
64.70ꢀ1.61
83.22ꢀ0.47
203.71ꢀ2.43
98.13ꢀ5.76
1.47
1.52
1.18
0.48
1
6.31ꢀ1.55
12.14ꢀ1.56
55.61ꢀ4.67
82.62ꢀ6.47
7.90ꢀ0.59
1.25
0.65
0.14
0.10
1
SAHA
[a] Data are expressed as meanꢀSD of three determinations. [b] Potency
of a compound relative to SAHA. This value is calculated by dividing the
IC50 value of SAHA by the IC50 value of the compound in question.
Table 1. Inhibition of HeLa nuclear HDAC and growth inhibitory activity
against HepG2 cells by compounds 9a–j.
these assays, we chose to measure the relative potency of the
compounds with respect to SAHA, a pan HDAC inhibitor,
against commercial preparations of HDAC1 and HDAC6 be-
cause of limited enzyme purity (Experimental Section). The IC50
values of the compounds, including SAHA, for each HDAC
were obtained using the same batch of enzyme to avoid possi-
ble variations in IC50 values arising through differences in
purity among preparations. Compounds 9d and 9e showed
similar potency over SAHA for both HDAC1 and HDAC6. In
contrast, compound 9g was less potent than SAHA for HDAC6,
but exhibited SAHA-like potency for HDAC1. This result sug-
gests that 9g is less selective for HDAC6.
Compd
Substitution
position
Chain
length (n)
HeLa nuclear HDAC
HepG2
GI50 [mm]
IC50[a] [nm]
9a
9b
9c
9d
9e
9 f
9g
9h
9i
ortho
ortho
ortho
meta
meta
meta
para
2
3
4
2
3
4
2
3
4
1031.52ꢀ0.11
857.33ꢀ52.2
404.64ꢀ19.04
24.54ꢀ2.89
20.01ꢀ2.09
231.43ꢀ0.42
19.60ꢀ0.83
170.22ꢀ7.32
100.34ꢀ0.13
18.73ꢀ0.29
18.8
6.9
6.8
2.6
2.7
5.8
2.7
3.8
2.6
1.3
Hyperacetylation of a-tubulin and histone
para
para
Hyperacetylation of a-tubulin and histones is a common effect
induced by all known HDAC inhibitors. Therefore, we exam-
ined the effect of compounds 9d, 9e, and 9g on a-tubulin
and histone acetylation in cultured human hepatoma HepG2
SAHA
[a] Data are expressed as mean ꢀSD of three determinations.
600
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 598 – 607

Histone Deacetylase Inhibitors
cells at various concentrations, using SAHA as a reference com-
pound. Compound 9d induced the most hyperacetylation of
both biomarkers in a dose-dependant manner (see Figure 2
pocket with which the phenyl ring of SAHA binds (Figure 3a).
Interestingly, in the calculated structure, the dimethylpropyl
arm of osthole is oriented towards an adjacent surface pocket
Figure 2. Effect of compound 9d and SAHA on induction of a-tubulin acety-
lation and histone H3 acetylation in cultured human hepatoma cells. Cells
were incubated with the HDAC inhibitors at 0.5, 1.0, 2.5, 5 and 10 mm for
16 h. Then whole-cell lysates were analyzed for a-tubulin and histone H3
acetylation by SDS-PAGE and Western immunoblot with antibodies specific
for either acetylated histone H3 or a-tubulin.
and figure S1 in the Supporting information). Compound 9d
induced significant a-tubulin acetylation at 5 mm; the level was
similar to that achieved with SAHA at 1 mm suggesting that
SAHA is more potent than 9d for promoting a-tubulin acetyla-
tion. However, compound 9d at 5 mm increased the acetylation
level of histone H3, similar to the level induced by SAHA at
10 mm, demonstrating that 9d is a more active inducer of his-
tone H3 acetylation than SAHA.
Molecular modeling
To obtain insight into the possible molecular interactions be-
tween the osthole cap of 9d and the HDAC active site, we car-
ried out docking analysis of 9d on HDLP (PDB code: 1C3R), a
class I homologue, using Autodock4 and its accompanying
graphical user interface AutoDockTools.[44] For the docking
study, which was performed in a rigid receptor mode, the crys-
tal structure of HDLP was chosen as a preferred target over
that of HDAC8, a class I HDAC, because co-crystal structures of
the latter enzyme in complex with several hydroxamate inhibi-
tors have revealed significant conformational plasticity around
the active site. By contrast, the active sites of HDLP and
HDAC1–3 are believed to be more conformationally rigid.[34]
The residues around the zinc-binding site, and the hydropho-
bic residues around the tube-like channel of HDLP, are identical
with those in human HDAC1; therefore the crystal structure of
HDLP has been employed extensively to investigate the bind-
ing modes of various HDAC inhibitors.[43,45–47] The parameters
used in the docking calculations were as described previous-
ly.[43] Docking of SAHA into HDLP was also carried out to vali-
date the quality of the calculations.
Figure 3. Molecular modeling results of compound 9d (blue) and SAHA
(magenta) docked to a) HDLP (PDB code: 1C3R; surface representation) and
b) HDLP (PDB code: 1C3R; yellow ribbons) and HDAC7 (PDB code: 3C0Z;
green ribbons) superimposed. Silver sphere represents the catalytic zinc ion.
where an enlarged hydrophobic pocket would be in the corre-
sponding region of class IIa HDACs. This enlarged hydrophobic
pocket is shaped by a class IIa-specific histidine residue whose
side chain is oriented away from the active site channel (resi-
due 843 in HDAC7; Figure 3b). This histidine is replaced by an
inwardly oriented tyrosine in all class I and IIb HDACs. Conse-
quently, the aliphatic handle of osthole may be further modi-
fied to confer selectivity for class IIa enzymes. Co-crystallo-
graphic analysis of these osthole compounds complexed with
class I and II HDACs is needed to confirm this hypothesis.
The docked structure of 9d in the HDLP active site suggest
that the hydroxamate moiety binds to the catalytic zinc ion,
and that the coumarin ring interacts with the same surface
ChemMedChem 2010, 5, 598 – 607
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
601

C.-I. Chang et al.
MED
2H), 0.96 (s, 3H), 0.95 ppm (3H, s); 13C NMR (125 MHz, CDCl3): d=
169.4, 153.6, 150.6, 125.2, 118.2, 114.7, 111.0, 38.4, 29.5, 28.2, 23.5,
22.6, 21.2 ppm; IR (KBr): n˜ =3327, 2953, 2868, 1734, 1618, 1506,
1455, 1348, 1314, 1284, 1253, 1206, 1152, 1055, 997 cmꢁ1; MS
(ESI+) m/z: 235.2 [M+H]+.
Conclusions
In conclusion, we showed that, based on enzyme assays, as
well as assays for growth inhibition and acetylation of a-tubu-
lin and histone H3, the osthole-based N-hydroxycinnamide
compounds reported herein are potent HDAC inhibitors. Mo-
lecular modeling analysis suggests that the dimethylethyl
branch of the osthole group in our N-hydroxycinnamide series
might be replaced/modified with new functionalities that spe-
cifically interact with the surface residues at the class IIa-specif-
ic active site, which is uniquely shaped with an enlarged
pocket compared with other classes of HDACs. To this end, ob-
taining co-crystal structures of class IIa enzymes, such as
HDAC4 or HDAC7, with these compounds would be desirable
for the further development of class-specific HDAC inhibitors.
8-Isopentyl-7-(2-chloroethoxy)-chroman-2-one (4a): A solution of
3 (2.00 g, 8.55 mmol) and K2CO3 (2.95 g, 21.38 mmol) in acetone
(40 mL) was treated with 1-bromo-2-chloroethane (1.42 mL,
17.10 mmol) and stirred under N2 at 568C overnight. After filtration
to remove K2CO3, the filtrate was concentrated in vacuo. The resi-
due was diluted with EtOAc (50 mL), washed with distd H2O (3ꢁ
25 mL), dried (Na2SO4), filtered and concentrated in vacuo. The resi-
due was purified by flash chromatography (silica gel; EtOAc/
1
n-hexane, 1:8) to give 4a (2.05 g, 81%): H NMR (500 MHz, CDCl3):
d=6.94 (d, J=8.3 Hz, 1H), 6.57 (d, J=8.3 Hz , 1H), 4.22 (2H, t, J=
5.7 Hz), 3.82 (t, J=5.8 Hz, 2H), 2.92 (t, J=6.7 Hz, 2H), 2.74 (m, 2H),
2.71 (m, 2H), 1.60 (m, 2H), 1.40 (m, 2H), 0.96 (s, 3H), 0.94 ppm (s,
3H); 13C NMR (125 MHz, CDCl3): d=169.0, 155.9, 150.5, 125.0,
120.4, 115.7, 107.3, 68.3, 42.3, 38.5, 29.5, 28.4, 23.6, 22.6, 21.1 ppm;
MS (EI, 70 eV) m/z: 296.1 [M]+.
Experimental Section
Chemistry
8-Isopentyl-7-(3-chloropropoxy)-chroman-2-one (4b): Following
the procedure described for 4a, reaction of 3 (2.00 g, 8.55 mmol),
K2CO3 (2.95 g, 21.38 mmol) and 1-bromo-3-chloropropane (1.69 mL,
17.10 mmol) in acetone (40 mL) gave 4b (2.25 g, 85%): 1H NMR
(500 MHz, CDCl3): d=6.94 (d, J=8.2 Hz, 1H), 6.61 (d, J=8.2 Hz,
1H), 4.10 (t, J=5.7 Hz, 2H), 3.76 (t, J=6.2 Hz, 2H), 2.92 (t, J=
6.8 Hz, 2H), 2.78 (m, 2H), 2.68 (m, 2H), 2.25 (m, 2H), 1.58 (m, 2H),
1.37 (m, 2H), 0.95 (s, 3H), 0.93 ppm (s, 3H); 13C NMR (125 MHz,
CDCl3): d=169.1, 156.3, 150.4, 125.0, 120.3, 115.2, 107.1, 64.6, 41.5,
38.6, 32.3, 29.6, 28.5, 28.3, 23.6, 22.6, 21.2 ppm; MS (ESI+) m/z:
311.2 [M+H]+.
IR spectra (KBr disk) were recorded using a Jasco fourier transform
infrared spectrometer (Jasco FT/IR-410). H NMR spectra were ob-
1
tained on a Bruker AV400 or AV500 spectrometer using standard
pulse programs. Melting points were recorded on a Fisher–Johns
apparatus and are uncorrected. Mass spectrometry (MS) data were
measured on a JEOL JMX-HX110 mass spectrometer (HRMS-EI and
HRMS-FAB), JMS-SX102A mass spectrometer (MS-EI and MS-FAB),
and Finnigan Mat 95S mass spectrometer (HRMS-ESI and MS-ESI).
TLC analysis was carried out on silica gel plates (KG60-F254,
Merck). The microplate spectrophotometer Victor 2x (Perkin–Elmer,
Fremont, CA, USA) was used for fluorometric analysis. Unless other-
wise mentioned, all chemicals and materials were used as received
from commercial suppliers without further purification. CH2Cl2 was
distilled from CaH2 under N2. THF was distilled from Na and benzo-
phenone under N2. HPLC analysis was performed on an Ascentis
C18 column (150ꢁ4.6 mm) using an L-2130 pump (Hitachi) and a
UV-VIS L-2420 detector (Hitachi) with UV detection at 250 nm.
8-Isopentyl-7-(4-chlorobutoxy)-chroman-2-one (4c): Following
the procedure described for 4a, reaction of 3 (2.00 g, 8.55 mmol),
K2CO3 (2.95 g, 21.38 mmol) and 1-bromo-3-chlorobutane (1.98 mL,
17.10 mmol) in acetone (40 mL) gave 4c (2.33 g, 84%): 1H NMR
(500 MHz, CDCl3): d= 6.93 (d, J=8.3 Hz, 1H), 6.58 (d, J=8.3 Hz,
1H), 3.99 (t, J=5.7 Hz, 2H), 3.62 (t, J=6.0 Hz, 2H), 2.92 (t, J=
6.7 Hz, 2H), 2.73 (m, 2H), 2.68 (m, 2H), 1.99 (m, 4H), 1.58 (m, 2H),
1.37 (m, 2H), 0.95 (s, 3H), 0.94 ppm (s, 3H) ; 13C NMR (125 MHz,
CDCl3): d=169.1, 156.6, 152.6, 150.4, 124.9, 120.3, 115.0, 106.9,
67.3, 44.8, 38.5, 29.6, 28.5, 28.4, 26.8, 23.6, 22.6, 21.2 ppm; MS (EI,
70 eV) m/z: 324.2 [M]+.
8-Isopentyl-7-methoxy-chroman-2-one (2): A solution of 1 (8.00 g,
32.79 mmol) in EtOAc (250 mL) was treated with 10% Pd-C (1.60 g)
and stirred under H2 (1 atm) at RT overnight. After filtration
through celite pad, the EtOAc was removed in vacuo and the resi-
due was purified by flash chromatography (silica gel; EtOAc/
n-hexane, 1:8) to give 2 (7.32 g, 92%): 1H NMR (500 MHz, CDCl3):
d=6.95 (d, J=8.3 Hz, 1H), 6.60 (d, J=8.4 Hz, 1H), 3.82 (s, 3H), 2.92
(t, J=6.7 Hz, 2H), 2.73 (m, 1H), 2.69 (m, 1H), 1.58 (m, 2H), 1.37 (m,
2H), 0.95 (s, 3H), 0.94 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=
169.0, 157.4, 150.4, 124.9, 120.3, 115.0, 106.2, 55.9, 38.5, 29.6, 28.2,
23.6, 22.6, 21.1 ppm; IR (KBr): n˜ =1722, 1605, 1498, 1433, 1279,
1251, 1159, 1123, 1089, 1030 cmꢁ1; MS (ESI+) m/z: 249.2 [M+H]+.
Methyl 2-hydroxycinnamate (6a):
A solution of 5a (8.20 g,
50.00 mmol) in MeOH (20 mL) was treated dropwise with H2SO4
(10 mL) and stirred at RT overnight. The solution was diluted with
EtOAc (100 mL), washed with 10% aq NaHCO3 (2ꢁ50 mL) and
distd H2O (2ꢁ50 mL), dried (Na2SO4), filtered and concentrated in
1
vacuo to give 6a (8.46 g, 95%): mp: 119–1208C; H NMR (500 MHz,
CDCl3): d=8.02 (1H, d, J=16.1 Hz), 7.46 (1H, d, J=7.7 Hz), 7.23
(1H, d, J=7.9 Hz), 6.92 (1H, t, J=7.5 Hz), 6.84 (1H, d, J=8.1 Hz),
6.62 (1H, d, J=16.2 Hz), 3.82 ppm (s, 3H); 13C NMR (125 MHz,
CDCl3): d=167.0, 155.5, 141.0, 131.6, 129.3, 121.7, 120.8, 117.9,
116.5, 51.9 ppm; IR (KBr): n˜ =3388, 2952, 1695, 1625, 1498, 1457,
1436, 1328, 1259, 1199, 1176, 1094, 1017, 991 cmꢁ1; MS (EI, 70 eV)
m/z: 178.1 [M]+.
8-Isopentyl-7-hydroxy-chroman-2-one (3): A solution of 2 (7.00 g,
28.23 mmol) in anhyd CH2Cl2 (50 mL) was treated dropwise with
BBr3 (13.34 mL, 141.15 mmol) at 08C under N2. The resulting solu-
tion was then warmed to RT and stirred for an additional 2.5 h. The
reaction was diluted with CH2Cl2 (50 mL), cooled in an ice-bath and
slowly quenched with 10% aq NaHCO3. The organic phase was
washed with brine, dried (Na2SO4), filtered and concentrated in
vacuo. The residue was purified by flash chromatography (silica
gel; EtOAc/n-hexane, 1:4) to give 3 (6.08 g, 92%): mp: 115–1178C;
1H NMR (500 MHz, CDCl3): d=6.86 (d, J=8.1 Hz, 1H), 6.54 (d, J=
8.1 Hz, 1H), 4.90 (s, 1H), 2.91 (t, J=8.3 Hz, 2H), 2.73 (t, J=6.8 Hz,
2H), 2.68 (t, J=8.1 Hz, 2H), 1.63 (m, 1H), 1.42 (dd, J=7.0, 16.9 Hz,
Methyl 3-hydroxycinnamate (6b): Following the procedure de-
scribed for 6a, reaction of 5b (6.56 g, 40.00 mmol) in MeOH
(20 mL) with H2SO4 (8 mL) gave 6b (6.55 g, 92%): mp: 79–818C;
1H NMR (500 MHz, CDCl3): d=7.64 (d, J=16.0 Hz, 1H), 7.25 (d, J=
7.8 Hz, 1H), 7.08 (d, J=7.6 Hz, 1H), 7.01 (s, 1H), 6.88 (dd, J=1.7,
8.0 Hz, 1H), 6.40 (d, J=16.0 Hz, 1H), 3.81 ppm (s, 3H); 13C NMR
602
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 598 – 607

Histone Deacetylase Inhibitors
(125 MHz, CDCl3): d=168.1, 156.3, 145.2, 135.7, 130.2, 120.7, 117.8,
117.8, 114.7, 52.0 ppm; IR (KBr): n˜ =3336, 1685, 1635, 1593, 1450,
1333, 1257, 973 cmꢁ1; MS (EI, 70 eV) m/z: 178.1 [M]+.
Methyl
3-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)ethoxy]cinnamate (7d): Following the procedure described
for 7a, reaction of 6b (492 mg, 3.00 mmol), K2CO3 (1.04 g,
7.50 mmol) and 4a (888 mg, 3.00 mmol) gave 7d (670 mg, 51%):
mp: 95–968C; 1H NMR (500 MHz, CDCl3): d=7.65 (d, J=16.0 Hz,
1H), 7.29 (t, J=7.9 Hz, 1H), 7.14 (d, J=7.6 Hz, 1H), 7.07 (s, 1H),
6.97 (m, 2H), 6.65 (d, J=8.3 Hz, 1H), 6.42 (d, J=15.9 Hz, 1H), 4.34
(m, 4H), 3.81 (s, 3H), 2.93 (t, J=6.6 Hz, 2H), 2.74 (t, J=7.2 Hz, 2H),
2.69 (m, 2H), 1.56 (m, 1H), 1.39 (m, 2H), 0.90 (s, 3H), 0.88 ppm (s,
3H); 13C NMR (125 MHz, CDCl3): d=168.9, 167.4, 159.0, 156.3,
150.5, 144.7, 135.9, 130.0, 125.0, 121.2, 120.9, 118.2, 116.8, 115.6,
113.8, 107.5, 67.2, 66.6, 51.8, 38.5, 29.5, 28.2, 23.6, 22.6, 22.5,
21.1 ppm; IR (KBr): n˜ =2950, 1771, 1708, 1638, 1578, 1489, 1448,
1321, 1232, 1171, 1086, 985 cmꢁ1; MS (EI, 70 eV) m/z: 438.0 [M]+.
Methyl 4-hydroxycinnamate (6c): Following the procedure de-
scribed for 6a, reaction of 5c (6.56 g, 40.00 mmol) in MeOH
(20 mL) with H2SO4 (8 mL) gave 6c (6.48 g, 91%): mp: 124–1308C;
1H NMR (500 MHz, CDCl3): d=7.64 (d, J=15.9 Hz, 1H), 7.43 (d, J=
8.5 Hz, 2H), 6.86 (d, J=8.5 Hz, 1H), 6.30 (d, J=15.9 Hz, 1H),
3.80 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=168.4, 158.1, 145.1,
130.1, 127.0, 116.0, 115.0, 51.9 ppm; IR (KBr): n˜ =3379, 2952, 1683,
1633, 1600, 1515, 1433, 1328, 1283, 1198, 985 cmꢁ1; MS (EI, 70 eV)
m/z: 178.1 [M]+.
Methyl
2-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)ethoxy]cinnamate (7a): A solution of 6a (356 mg, 2.00 mmol)
and K2CO3 (690 mg, 5.00 mmol) in DMF (10 mL) was treated with
4a (592 mg, 2.00 mmol) and stirred at RT under N2 overnight. After
filtration to remove K2CO3, the filtrate was concentrated in vacuo.
The residue was diluted EtOAc (50 mL), washed with distd H2O (2ꢁ
25 mL), dried (Na2SO4), filtered and concentrated in vacuo. The resi-
due was purified by flash chromatography (silica gel; EtOAc/
Methyl 3-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)-
propoxy]cinnamate (7e): Following the procedure described for
7a, reaction of 6b (492 mg, 3.00 mmol), K2CO3 (1.04 g, 7.50 mmol)
and 4b (930 mg, 3.00 mmol) gave 7e (786 mg, 58%): mp: 66–
1
678C; H NMR (500 MHz, CDCl3): d=7.64 (d, J=16.0 Hz, 1H), 7.29
(d, J=7.9 Hz, 1H), 7.11 (d, J=7.6 Hz ,1H), 7.04 (s, 1H), 6.94 (d, J=
8.2 Hz, 1H), 6.62 (d, J=8.3 Hz, 1H), 6.41 (d, J=16.0 Hz, 1H), 4.21 (t,
J=6.0 Hz, 2H), 4.16 (t, J=5.9 Hz, 2H), 3.80 (s, 3H), 2.92 (t, J=
6.8 Hz, 2H), 2.73 (t, J=7.6 Hz, 2H), 2.69 (t, J=8.0 Hz, 2H), 2.30 (m,
2H), 1.57 (m, 1H), 1.36 (dd, J=7.1, 15.6 Hz, 2H), 0.92 (s, 3H),
0.91 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=169.0, 167.4, 159.2,
156.5, 150.4, 144.8, 135.8, 129.9, 125.0, 120.9, 120.3, 118.1, 116.5,
115.1, 113.7, 107.0, 64.7, 64.5, 51.8, 38.6, 38.5, 29.6, 29.4, 28.3, 23.6,
22.6, 21.2 ppm; IR (KBr): n˜ =2954, 1773, 1719, 1643, 1586, 1490,
1436, 1271, 1120, 1075, 980 cmꢁ1; MS (ESIꢁ) m/z: 451.5 [MꢁH]ꢁ.
1
n-hexane, 1:8) to give 7a (481 mg, 55%): H NMR (500 MHz, CDCl3):
d=7.99 (1H, d, J=16.2 Hz), 7.53 (dd, J=1.4, 7.6 Hz, 1H), 7.34 (t,
J=7.8 Hz, 1H), 7.00 (d, J=7.5 Hz, 1H), 6.97 (d, J=8.2 Hz, 2H), 6.68
(d, J=8.3 Hz, 1H), 6.51 (d, J=16.2 Hz, 1H), 4.40 (m, 4H), 3.76 (s,
3H), 2.93 (t, J=6.7 Hz, 2H), 2.74 (m, 2H), 2.68 (m, 2H), 1.52 (m,
1H), 1.36 (m, 2H), 0.86 (s, 3H), 0.85 ppm (s, 3H); 13C NMR
(125 MHz, CDCl3): d=179.1, 169.1, 168.0, 157.4, 156.3, 152.8, 150.5,
140.2, 131.5, 129.0, 127.4, 125.0, 123.7, 121.3, 118.5, 112.3, 107.5,
104.4, 67.2, 66.7, 51.6, 38.5, 28.3, 24.4, 22.5, 21.5 ppm; IR (KBr): n˜ =
2951, 1715, 1629, 1489, 1455, 1244, 1166, 655 cmꢁ1; MS (EI, 70 eV)
m/z: 438.0 [M]+.
Methyl 3-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)-
butoxy]cinnamate (7 f): Following the procedure described for 7a,
reaction of 6b (492 mg, 3.00 mmol), K2CO3 (1.04 g, 7.50 mmol) and
1
Methyl 2-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)-
propoxy]cinnamate (7b): Following the procedure described for
7a, reaction of 6a (445 mg, 2.50 mmol), K2CO3 (863 mg,
6.25 mmol) and 4b (778 mg, 2.50 mmol) gave 7b (588 mg, 52%):
mp: 70–718C; 1H NMR (500 MHz, CDCl3): d=8.00 (d, J=16.2 Hz,
1H), 7.51 (d, J=7.8 Hz, 1H), 7.33 (t, J=7.3 Hz, 1H), 7.00 (d, J=
7.5 Hz, 1H), 6.96 (m, 2H), 6.63 (d, J=8.3 Hz, 1H), 6.51 (d, J=
16.2 Hz, 1H), 4.26 (t, J=6.1 Hz, 2H), 4.19 (t, J=5.8 Hz, 2H), 3.79 (s,
3H), 2.91 (t, J=6.7 Hz, 2H), 2.74 (m, 2H), 2.68 (m, 2H), 2.35 (m,
2H), 1.58 (m, 1H), 1.36 (m, 2H), 1.09 (m, 2H), 0.92 (s, 3H), 0.91 ppm
(s, 3H); 13C NMR (125 MHz, CDCl3): d=169.0, 168.0, 157.5, 156.4,
150.4, 140.2, 131.6, 128.9, 125.0, 123.5, 121.0, 120.2, 118.2, 115.1,
112.1, 107.1, 64.9, 64.7, 51.6, 38.6, 29.6, 29.4, 28.3, 23.6, 22.7, 22.6,
21.2 ppm; IR (KBr): n˜ =2953, 1775, 1714, 1635, 1491, 1455, 1323,
1245, 1174, 1076, 987 cmꢁ1; MS (ESIꢁ) m/z: 451.5 [MꢁH]ꢁ.
4c (972 mg, 3.00 mmol) gave 7 f (755 mg, 54%): H NMR (500 MHz,
CDCl3): d=8.01 (d, J=16.2 Hz,1H), 7.51 (dd, J=1.34, 7.7 Hz, 1H),
7.31 (t, J=7.1 Hz, 1H), 6.97 (d, J=7.6 Hz, 1H), 6.92 (t, J=8.2 Hz,
1H), 6.91 (s, 1H), 6.60 (d, J=8.3 Hz, 1H), 6.53 (d, J=16.0 Hz, 1H),
4.14 (t, J=6.0 Hz, 2H), 4.04 (t, J=5.6 Hz, 2H), 3.79 (s, 3H), 2.92 (t,
J=6.6 Hz, 2H), 2.74 (t, J=6.7 Hz, 2H), 2.69 (m, 2H), 2.07 (m, 2H),
2.04 (m, 2H), 1.58 (m, 1H), 1.37 (dd, J=7.1, 15.6 Hz, 2H), 0.94 (s,
3H), 0.92 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=169.1, 167.5,
159.3, 156.7, 150.4, 144.9, 135.7, 129.9, 127.3, 124.9, 120.8, 118.0,
116.6, 114.9, 113.6, 107.0, 103.9, 67.8, 67.5, 51.8, 38.5, 29.6, 28.5,
28.3, 26.1, 26.0, 23.6, 22.6, 21.2 ppm; IR (KBr): n˜ =3430, 2949, 1716,
1630, 1247, 1170, 750 cmꢁ1; MS (EI, 70 eV) m/z: 466.0 [M]+.
Methyl
4-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)ethoxy]cinnamate (7g): Following the procedure described
for 7a, reaction of 6c (445 mg, 2.50 mmol), K2CO3 (863 mg,
5 mmol) and 4a (740 mg, 2.50 mmol) gave 7g (624 mg, 57%): mp:
Methyl 2-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)-
butoxy]cinnamate (7c): Following the procedure described for 7a,
reaction of 6a (445 mg, 2.50 mmol), K2CO3 (863 mg, 6.25 mmol)
and 4c (810 mg, 2.50 mmol) gave 7c (664 mg, 57%): mp: 60–
1
102–1048C; H NMR (500 MHz, CDCl3): d=7.65 (d, J=16.0 Hz, 1H),
7.48 (d, J=8.6 Hz, 2H), 6.96 (d, J=8.4 Hz, 1H), 6.94 (d, J=8.6 Hz,
2H), 6.64 (d, J=8.3 Hz, 1H), 6.32 (d, J=16.0 Hz, 1H), 4.36 (m, 2H),
4.32 (m, 2H), 3.80 (s, 3H), 2.93 (t, J=7.7 Hz, 2H), 2.73 (t, J=7.6 Hz,
2H), 2.69 (m, 2H), 1.54 (m, 1H), 1.37 (dd, J=7.2, 15.6 Hz, 2H), 0.89
(s, 3H), 0.88 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=168.9,
167.8, 160.4, 156.3, 150.5, 144.4, 129.8, 127.5, 125.0, 120.9, 115.6,
115.5, 114.9, 107.5, 67.1, 66.6, 51.7, 38.5, 29.5, 28.2, 23.6, 22.5,
21.1 ppm; IR (KBr): n˜ =2954, 1779, 1701, 1605, 1513, 1492, 1432,
1250, 1140, 1077, 987 cmꢁ1; MS (ESI+) m/z: 439.5 [M+H]+.
1
618C; H NMR (500 MHz, CDCl3): d=7.65 (d, J=16.0 Hz, 1H), 7.29
(t, J=7.8 Hz, 1H), 7.26 (d, J=8.2 Hz, 1H), 7.11 (d, J=7.5 Hz, 1H),
6.93 (m, 2H), 6.59 (d, J=8.2 Hz, 1H), 6.41 (d, J=16 Hz, 1H), 4.11 (t,
J=7.2 Hz, 2H), 4.05 (t, J=5.1 Hz, 2H), 3.81 (s, 3H), 2.92 (t, J=
6.7 Hz, 2H), 2.73 (m, 2H), 2.70 (m, 2H), 2.01 (m, 4H), 1.58 (m, 1H),
1.39 (m, 2H), 0.95 (s, 3H), 0.94 ppm (s, 3H); 13C NMR (125 MHz,
CDCl3): d=169.1, 168.0, 157.7, 156.6, 150.4, 140.3, 131.5, 129.1,
129.0, 124.9, 123.4, 120.8, 120.3, 118.2, 114.9, 112.0, 107.0, 68.0,
67.8, 51.6, 38.6, 29.6, 28.3, 26.3, 26.1, 23.6, 22.6, 21.2 ppm; IR (KBr):
n˜ =3430, 2949, 1716, 1630, 1247, 1170, 750 cmꢁ1; MS (EI, 70 eV)
m/z: 466.0 [M]+.
Methyl 4-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)-
propoxy]cinnamate (7h): Following the procedure described for
7a, reaction of 6c (445 mg, 2.50 mmol), K2CO3 (863 mg, 5 mmol)
ChemMedChem 2010, 5, 598 – 607
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
603

C.-I. Chang et al.
MED
and 4b (775 mg, 2.50 mmol) gave 7h (655 mg, 58%): mp: 99–
1008C; H NMR (500 MHz, CDCl3): d=7.64 (d, J=16.0 Hz, 1H), 7.46
2-[4-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)but-
oxy]cinnamic acid (8c): Following the procedure described for 8a,
reaction of 7c (400 mg, 0.86 mmol) in MeOH (15 mL) with LiOH
(206 mg, 8.60 mmol) gave 8c (377 mg, 97%): 1H NMR (500 MHz,
[D6]acetone): d=8.03(d, J=16.2 Hz, 1H), 7.66 (dd, J=1.5, 7.7 Hz,
1H), 7.36 (t, J=7.2 Hz, 1H), 7.10 (d, J=8.2 Hz, 1H), 6.98 (d, J=
7.5 Hz, 1H), 6.89 (d, J=8.4 Hz, 1H), 6.58 (d, J=16.3 Hz, 1H), 6.46
(d, J=8.3 Hz, 1H), 4.22 (t, J=6.3 Hz, 2H), 4.05 (t, J=6.1 Hz, 2H),
2.82 (t, J=7.0 Hz, 2H), 2.69 (t, J=8.0 Hz, 2H), 2.62 (t, J=6.9 Hz,
2H), 2.06 (m, 4H), 1.57 (m, 1H), 1.37 (m, 2H), 0.92 (s, 3H), 0.90 ppm
(s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=175.0, 168.4, 157.5,
156.2, 153.3, 139.3, 132.2, 129.1, 127.0, 125.9, 123.0, 121.1, 119.8,
118.9, 113.0, 107.7, 103.6, 68.2, 67.5, 38.8, 34.8, 29.3, 28.3, 26.2, 25.7,
23.0, 21.6 ppm; IR (KBr): n˜ =3444, 2953, 1685, 1622, 1490, 1455,
1246, 1120, 1077, 751 cmꢁ1; HRMS-EI: m/z [M]+ calcd for C27H32O6:
452.2199, found: 452.2193.
1
(d, J=8.7 Hz, 2H), 6.94 (d, J=8.2 Hz, 1H), 6.90 (d, J=8.6 Hz, 2H),
6.61 (d, J=8.3 Hz, 1H), 6.31 (d, J=16.0 Hz, 1H), 4.21 (t, J=6.1 Hz,
2H), 4.15 (t, J=6.0 Hz, 2H), 3.79 (s, 3H), 2.92 (t, J=6.7 Hz, 2H), 2.73
(m, 2H), 2.69 (m, 2H), 2.30 (m, 2H), 1.57 (m, 1H), 1.37 (dd, J=7.2,
15.6 Hz, 2H), 0.92 (s, 3H), 0.91 ppm (s, 3H); 13C NMR (125 MHz,
CDCl3): d=168.9, 167.8, 160.7, 156.5, 150.5, 144.5, 129.8, 127.2,
125.0, 120.3, 115.3, 115.1, 114.8, 107.0, 64.6, 64.5, 51.6, 38.6, 29.6,
29.3, 28.3, 23.6, 22.6, 21.2 ppm; IR (KBr): n˜ =2944, 1761, 1718, 1631,
1604, 1512, 1493, 1466, 1433, 1349, 1289, 1250, 1164, 1138, 1076,
1024, 983 cmꢁ1; MS (ESI+) m/z: 453.07 [M+H]+.
Methyl 4-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)-
butoxy]cinnamate (7i): Following the procedure described for 7a,
reaction of 6c (445 mg, 2.50 mmol), K2CO3 (863 mg, 5 mmol) and
4c (810 mg, 2.50 mmol) gave 7i (699 mg, 60%): mp: 90–918C;
1H NMR (500 MHz, CDCl3): d=7.65 (d, J=16.0 Hz, 1H), 7.46 (d, J=
8.7 Hz, 2H), 6.93 (d, J=8.2 Hz, 1H), 6.89 (d, J=8.7 Hz, 2H), 6.59 (d,
J=8.3 Hz, 1H), 6.31 (d, J=16.0 Hz, 1H), 4.07 (t, J=5.5 Hz, 2H), 4.02
(t, J=5.5 Hz, 2H), 3.79 (s, 3H), 2.92 (t, J=6.7 Hz, 2H), 2.73 (t, J=
7.7 Hz, 2H), 2.69 (t, J=5.5 Hz, 2H), 2.00 (m, 4H), 1.59 (m, 1H), 1.38
(dd, J=7.0, 15.8 Hz, 2H), 0.95 (s, 3H), 0.93 ppm (s, 3H); 13C NMR
(125 MHz, CDCl3): d=160.0, 167.8, 160.8, 156.6, 150.4, 144.6, 129.8,
127.0, 124.9, 120.3, 115.2, 115.0, 114.8, 106.9, 67.8, 67.6, 51.6, 38.6,
29.6, 28.3, 26.1, 26.0, 23.6, 22.6, 21.1 ppm; IR (KBr): n˜ =2950, 1768,
1714, 1604, 1512, 1433, 1245, 1140, 1078, 827 cmꢁ1; MS (ESIꢁ) m/z:
465.6 [MꢁH]ꢁ.
3-[2-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)eth-
oxy]cinnamic acid (8d): Following the procedure described for 8a,
reaction of 7d (450 mg, 1.03 mmol) in MeOH (15 mL) with LiOH
(247 mg, 10.30 mmol) gave 8d (415 mg, 95%): mp: 99–1018C;
1H NMR (500 MHz, [D6]acetone): d=7.64 (d, J=16.0 Hz, 1H), 7.35 (t,
J=8.0 Hz, 1H), 7.31 (s, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.05 (d, J=
8.2 Hz, 1H), 6.92 (d, J=8.4 Hz, 1H), 6.53 (d, J=16.1 Hz, 1H), 6.51
(d, J=8.8 Hz, 1H), 4.44 (m, 2H), 4.33 (m, 2H), 2.83 (t, J=7.1 Hz,
2H), 2.68 (m, 2H), 2.63 (t, J=7.0 Hz, 2H), 1.54 (m, 1H), 1.35 (dd, J=
7.2, 15.6 Hz, 2H), 0.86 (s, 3H), 0.85 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=174.9, 168.1, 159.3, 156.0, 153.4, 144.3, 136.2, 130.4,
127.0, 121.4, 121.3, 120.3, 119.0, 117.3, 113.9, 104.1, 67.2, 60.2, 38.6,
34.7, 28.2, 25.7, 22.9, 21.5, 21.2 ppm; IR (KBr): n˜ =2953, 1698, 1635,
1494, 1447, 1239, 1120, 775 cmꢁ1; HRMS-EI: m/z [M]+ calcd for
C25H28O6: 424.1886, found: 424.1880.
2-[2-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)eth-
oxy]cinnamic acid (8a): A solution of 7a (450 mg, 1.03 mmol) in
MeOH (20 mL) was treated with LiOH (247 mg, 10.30 mmol) and
stirred under N2 at 638C overnight. The reaction mixture was dilut-
ed with distd H2O (50 mL), acidified to pH 5~6 with 2n HCl and
extracted with EtOAc (3ꢁ50 mL). The combined organic layer was
dried (Na2SO4), filtered and concentrated in vacuo to give 8a
(414 mg, 95%): mp: 100–1058C; 1H NMR (500 MHz, [D6]acetone):
d=8.04 (d, J=16.2 Hz, 1H), 7.67 (dd, J=1.4, 7.7 Hz, 1H), 7.40 (t,
J=7.6 Hz, 1H), 7.16 (d, J=8.3 Hz, 1H), 7.01 (t, J=7.5 Hz, 1H), 6.92
(d, J=8.3 Hz, 1H), 6.58 (d, J=16.3 Hz, 1H), 6.54 (d, J=8.3 Hz, 1H),
4.48 (m, 2H), 4.39 (m, 2H), 2.82 (t, J=7.1 Hz, 2H), 2.68 (t, J=8.0 Hz,
2H), 2.63 (t, J=7.0 Hz, 2H), 1.50 (m, 1H), 1.36 (m, 2H), 0.82 (s, 3H),
0.81 ppm (s 3H); 13C NMR (125 MHz, Acetone): d=175.5, 167.4,
157.6, 156.1, 153.2, 139.7, 131.6, 128.6, 127.2, 123.4, 120.9, 120.8,
118.9, 118.8, 112.4, 103.9, 67.5, 66.8, 38.4, 34.5, 29.7, 28.1, 24.9, 22.0,
21.3 ppm; IR (KBr): n˜ =2953, 1658, 1626, 1487, 1446, 1209, 1121,
3-[3-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)prop-
oxy]cinnamic acid (8e): Following the procedure described for 8a,
reaction of 7e (678 mg, 1.50 mmol) in MeOH (20 mL) with LiOH
(360 mg, 15.00 mmol) gave 8e (611 mg, 93%): mp: 128–1308C;
1H NMR (500 MHz, [D6]acetone): d=7.63 (d, J=16.0 Hz, 1H), 7.33 (t,
J=7.9 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J=7.6 Hz 1H), 7.02 (d, J=
8.2 Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 6.51 (d, J=16.0 Hz, 1H), 6.47
(d, J=8.4 Hz, 1H), 4.30 (t, J=6.3 Hz, 2H), 4.15 (t, J=6.0 Hz, 2H),
2.81 (t, J=7.1 Hz, 2H), 2.68 (m, 2H), 2.61 (t, J=6.9 Hz, 2H), 2.27 (m,
2H), 1.55 (m, 1H), 1.36 (dd, J=7.2, 15.6 Hz, 2H), 0.90 (s, 3H),
0.88 ppm (s, 3H,); 13C NMR (125 MHz, [D6]DMSO): d=174.9, 168.1,
159.3, 156.0, 153.4, 144.3, 136.2, 130.4,127.0, 121.3, 121.1, 120.0,
118.6, 117.1, 113.8, 103.7, 64.9, 64.5, 38.8, 34.8, 29.3, 28.3, 25.7, 23.0,
21.6 ppm; IR (KBr): n˜ =2958, 1698, 1634, 1494, 1447, 1259, 1120,
1078, 811, 752 cmꢁ1 HRMS-EI: m/z [M]+ calcd for C25H28O6:
;
424.1886, found: 424.1885.
1077, 947 cmꢁ1 HRMS-ESI: m/z [M+H]+ calcd for C26H31O6:
;
439.2121, found: 439.2118.
2-[3-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)prop-
oxy]cinnamic acid (8b): Following the procedure described for 8a,
reaction of 7b (500 mg, 1.11 mmol) in MeOH (20 mL) with LiOH
(266 mg, 11.10 mmol) gave 8b (466 mg, 96%): mp: 148–1508C;
1H NMR (500 MHz, [D6]acetone): d=8.01 (d, J=16.1 Hz, 1H), 7.65
(d, J=7.9 Hz, 1H), 7.38 (d, J=7.3 Hz, 1H), 7.11 (d, J=8.3 Hz, 1H),
7.00 (t, J=7.5 Hz, 1H), 6.89 (d, J=8.4 Hz, 1H), 6.56 (d, J=16.2 Hz,
1H), 6.49 (d, J=8.4 Hz, 1H), 4.35 (t, J=6.3 Hz, 2H), 4.20 (t, J=
6.0 Hz, 2H), 2.81 (t, J=7.0 Hz, 2H), 2.68 (m, 2H), 2.61 (t, J=7.0 Hz,
2H), 2.34 (m, 2H), 1.54 (m, 1H), 1.35 (dd, J=7.2, 15.6 Hz, 2H), 0.90
(s, 3H), 0.88 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=174.9,
168.3, 157.5, 155.9, 153.4, 139.2, 132.2, 129.1, 127.0, 123.1, 121.3,
121.1, 119.8, 118.7, 112.9, 103.7, 65.4, 64.5, 38.8, 34.7, 29.3, 28.3,
25.7, 23.0, 21.6 ppm; IR (KBr): n˜ =2956, 1682, 1492, 1453, 1202,
1129, 986 cmꢁ1, 752; HRMS-ESI: m/z [M+OH]ꢁ calcd for C26H31O7:
455.2070, found: 455.2062.
3-[4-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)but-
oxy]cinnamic acid (8 f): Following the procedure described for 8a,
reaction of 7 f (700 mg, 1.50 mmol) in MeOH (25 mL) with LiOH
(360 mg, 15.00 mmol) gave 8 f (637 mg, 94%): mp: 137–1388C;
1H NMR (500 MHz, [D6]acetone): d=7.64 (d, J=16.0 Hz, 1H), 7.33 (t,
J=7.8 Hz, 1H), 7.26 (s, 1H), 7.22 (t, J=7.6 Hz, 1H), 7.00 (dd, J=2.2,
8.0 Hz, 1H), 6.90 (d, J=8.3 Hz, 1H), 6.52 (d, J=16.0 Hz, 1H), 6.45
(d, J=8.3 Hz, 1H), 4.16 (t, J=5.9 Hz, 2H), 4.03 (t, J=6.0 Hz, 2H),
2.82 (t, J=7.0 Hz, 2H), 2.69 (m, 2H), 2.63 (t, J=7.0 Hz, 2H), 2.08 (m,
2H), 2.02 (m, 2H), 1.58 (m, 1H), 1.36 (dd, J=7.1, 15.6 Hz, 2H), 0.93
(s, 3H), 0.92 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=174.9,
168.1, 159.4, 156.2, 153.3, 144.4, 136.1, 130.4, 126.9, 121.2, 120.9,
120.0, 118.6, 117.3, 113.8, 103.7, 67.7, 67.5, 38.8, 34.8, 28.4, 26.2,
26.1, 25.7, 23.0, 22.9, 21.6 ppm; IR (KBr): n˜ =3471, 2949, 1684, 1611,
1578, 1492, 1466, 1383, 1267, 1196, 1159, 1127, 1061, 981, 861,
604
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 598 – 607

Histone Deacetylase Inhibitors
781 cmꢁ1; HRMS-EI: m/z [M]+ calcd for C27H32O6: 452.2199, found:
centrated in vacuo. The residue was purified by flash chromato-
452.2208.
graphy (silica gel; EtOAc/n-hexane, 1:1!2:1) to give 9a (252 mg,
1
61%): H NMR (500 MHz, [D6]DMSO): d=10.65 (s, 1H), 10.44 (s, 1H),
4-[2-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)eth-
oxy]cinnamic acid (8g): Following the procedure described for 8a,
reaction of 7g (600 mg, 1.37 mmol) in MeOH (25 mL) with LiOH
(329 mg, 13.70 mmol) gave 8g (552 mg, 95%): mp: 159–1608C;
1H NMR (500 MHz, [D6]acetone): d=7.64 (d, J=8.6 Hz, 2H), 7.63 (d,
J=16.0 Hz, 1H), 7.05 (d, J=8.6 Hz, 2H), 6.93 (d, J=8.4 Hz, 1H),
6.52 (d, J=8.4 Hz, 1H), 6.38 (d, J=16.0 Hz, 1H), 4.44 (t, J=4.8 Hz,
2H), 4.33 (t, J=4.7 Hz, 2H), 2.81 (m, 2H), 2.68 (m, 2H), 2.63 (t, J=
7.1 Hz, 2H), 1.55 (m, 1H), 1.37 (dd, J=7.2, 15.6 Hz, 2H), 0.87 (s,
3H), 0.86 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=174.9,
168.3, 160.7, 156.0, 153.4, 144.2, 130.4, 127.4, 127.0, 121.3, 119.0,
117.0, 115.3, 104.1, 67.3, 67.1, 38.6, 34.7, 28.2, 25.7, 22.9, 21.5 ppm;
7.72 (d, J=15.9 Hz, 1H), 7.50 (d, J=7.6 Hz, 1H), 7.33 (t, J=7.5 Hz,
1H), 7.11 (d, J=8.3 Hz, 1H), 6.97 (t, J=7.5 Hz, 1H), 6.83 (d, J=
8.4 Hz, 1H), 6.47 (d, J=15.8 Hz, 1H), 6.46 (d, J=8.6 Hz, 1H), 4.35 (t,
J=6.3 Hz, 2H), 4.28 (t, J=6.0 Hz, 2H), 2.71 (t, J=7.0 Hz, 2H), 2.57
(m, 2H), 2.23 (t, J=7.1 Hz, 2H), 1.40 (m, 1H), 1.20 (m, 2H), 0.74 (s,
3H), 0.73 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=169.9,
163.6, 157.3, 156.0, 153.5, 133.8, 131.2, 128.1, 127.2, 124.0, 121.6,
121.3, 119.8, 119.1, 113.0, 104.1, 67.7, 67.0, 38.6, 33.4, 28.2, 25.4,
22.9, 22.7, 21.5 ppm; IR (KBr): n˜ =3647, 1651, 1488, 752 cmꢁ1
;
HRMS-ESI: m/z [MꢁH]ꢁ calcd for C25H28NO6: 438.1917, found:
438.1915.
IR (KBr): n˜ =2946, 1697, 1602, 1513, 1428, 1250, 1127, 827 cmꢁ1
;
N-Hydroxy-2-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)propoxy]cinnamide (9b): Following the procedure described
for 9a, reaction of 8b (400 mg, 0.91 mmol) in THF (10 mL) with
ethyl chloroformate (0.13 mL, 1.37 mmol) and Et3N (0.19 mL,
1.37 mmol) gave 9b (412 mg, 64%): mp: 92–978C; 1H NMR
(500 MHz, CD3OD): d=7.91 (d, J=15.9 Hz, 1H), 7.51 (d, J=7.4 Hz,
1H), 7.30 (t, J=7.6 Hz, 1H), 7.00 (d, J=8.3 Hz, 1H), 6.93 (t, J=
7.4 Hz, 1H), 6.83 (d, J=8.3 Hz, 1H), 6.50 (d, J=15.9 Hz, 1H), 6.44
(d, J=8.3 Hz, 1H), 4.26 (t, J=6.1 Hz, 2H), 4.13 (t, J=5.7 Hz, 2H),
2.82 (t, J=7.2 Hz, 2H), 2.61 (t, J=8.2 Hz, 2H), 2.36 (t, J=7.1 Hz,
2H), 2.29 (t, J=5.9 Hz, 2H), 1.52 (m, 1H), 1.30 (m, 2H), 0.88 (s, 3H),
0.87 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=169.8, 159.3,
156.0, 153.4, 138.7, 136.7, 130.5, 130.4, 127.2, 121.4, 120.3, 119.9,
118.7, 116.1, 113.6, 103.7, 64.8, 64.5, 38.8, 33.4, 29.3, 28.4, 25.4, 23.0,
21.6 ppm; IR (KBr): n˜ =3218, 2951, 1650, 1490, 1455, 1246, 1120,
1072, 751 cmꢁ1; HRMS-FAB: m/z [M+H]+ calcd for C26H31NO6:
453.2152, found: 453.2162.
HRMS-ESI: m/z [M+OH]ꢁ calcd for C25H29O7: 441.1913, found:
441.1905.
4-[3-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)prop-
oxy]cinnamic acid (8h): Following the procedure described for 8a,
reaction of 7h (600 mg, 1.33 mmol) in MeOH (25 mL) with LiOH
(320 mg, 13.30 mmol) gave 8h (553 mg, 95%): mp: 145–1468C;
1H NMR (500 MHz, [D6]acetone): d=7.62 (d, J=15.0 Hz,1H), 7.61 (d,
J=8.8 Hz, 2H), 7.01 (d, J=8.7 Hz, 2H), 6.90 (d, J=8.4 Hz, 1H), 6.47
(d, J=8.4 Hz, 1H), 6.37 (d, J=15.0 Hz, 1H), 4.30 (t, J=6.2 Hz, 2H),
4.14 (t, J=6.0 Hz, 2H), 2.80 (t, J=6.9 Hz, 2H), 2.67 (m, 2H), 2.62 (t,
J=6.9 Hz, 2H), 2.27 (m, 2H), 1.55 (m, 1H), 1.35 (dd, J=7.2, 15.6 Hz,
2H), 0.91 (s, 3H), 0.89 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO):
d=174.9, 168.3, 160.7, 156.0, 153.4, 144.2, 130.4, 127.3, 127.0,
121.1, 118.6, 117.0, 115.2, 103.7, 65.0, 64.4, 38.8, 34.7, 29.2, 28.3,
25.7, 23.0, 21.6 ppm; IR (KBr): n˜ =2942, 1697, 1632, 1602, 1573,
1511, 1494, 1466, 1427, 1247, 1211, 1172, 1121, 1068, 1032,
980 cmꢁ1; HRMS-ESI: m/z [M+H]+ calcd for C26H31O6: 439.2121,
found: 439.2117.
N-Hydroxy-2-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)butoxy]cinnamide (9c): Following the procedure described
for 9a, reaction of 8c (350 mg, 0.77 mmol) in THF (10 mL) with
ethyl chloroformate (0.11 mL, 1.16 mmol) and Et3N (0.16 mL,
1.16 mmol) gave 9c (216 mg, 60%): mp: 71–748C; 1H NMR
(500 MHz, CD3OD): d=7.92 (d, J=16.0 Hz, 1H), 7.51 (d, J=7.5 Hz,
1H), 7.31 (t, J=7.6 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.94 (t, J=
7.4 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H), 6.51 (d, J=15.9 Hz, 1H), 6.41
(d, J=8.3 Hz, 1H), 4.14 (t, J=6.1 Hz, 2H), 3.99 (t, J=6.0 Hz, 2H),
2.82 (t, J=7.2 Hz, 2H), 2.62 (m, 2H), 2.36 (t, J=7.1 Hz, 2H), 2.07 (m,
2H), 1.98 (m, 2H), 1.54 (m. 1H), 1.32 (dd, J=7.2, 15.6 Hz, 2H), 0.92
(s, 3H), 0.89 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=169.9,
163.6, 157.3, 156.2, 153.4, 134.0, 131.2, 128.5, 127.2, 123.8, 121.2,
119.9, 118.7, 113.0, 103.8, 68.1, 67.5, 38.8, 33.4, 28.3, 26.2, 26.0, 25.4,
23.1, 22.9, 21.9, 21.6 ppm; IR (KBr): n˜ =2953, 1652, 1489, 1246,
4-[4-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yloxy)but-
oxy]cinnamic acid (8i): Following the procedure described for 8a,
reaction of 7i (600 mg, 1.29 mmol) in MeOH (25 mL) with LiOH
(310 mg, 12.90 mmol) gave 8 f (554 mg, 95%): mp: 144–1458C;
1H NMR (500 MHz, [D6]acetone): d=7.63 (d, J=7.5 Hz, 2H), 7.62 (d,
J=16.0 Hz, 1H), 7.00 (d, J=8.6 Hz, 2H), 6.90 (d, J=8.3 Hz, 1H),
6.45 (d, J=8.4 Hz, 1H), 6.34 (d, J=15.9 Hz, 1H), 4.16 (t, J=5.9 Hz,
2H), 4.03 (t, J=6.1 Hz, 2H),2.82 (t, J=6.9 Hz, 2H), 2.69 (t, J=8.3 Hz,
2H), 2.36 (t, J=7.2 Hz, 2H), 2.23 (t, J=6.0 Hz, 2H), 1.52 (m, 1H),
1.30 (m, 2H), 0.88 (s, 3H), 0.87 ppm (s, 3H); 13C NMR (125 MHz,
[D6]DMSO): d=174.9, 168.3, 160.8, 156.2, 153.3, 144.3, 130.4, 127.2,
126.9, 120.9, 118.6, 116.9, 115.3, 103.7, 67.8, 67.5, 38.8, 34.8, 28.4,
26.1, 26.0, 25.7, 23.0, 21.6 ppm; IR (KBr): n˜ =2950, 1695, 1603, 1512,
1493, 1428, 1248, 1173, 1118, 1017, 945, 826, 803 cmꢁ1; HRMS-EI:
m/z [M]+ calcd for C27H32O6: 452.2199, found: 452.2153.
1118, 752 cmꢁ1
466.2230, found: 466.2238.
;
HRMS-ESI: m/z [MꢁH]ꢁ calcd for C27H32NO6
N-Hydroxy-3-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)ethoxy]cinnamide (9d): Following the procedure described
for 9a, reaction of 8d (400 mg, 0.94 mmol) in THF (10 mL) with
ethyl chloroformate (0.13 mL, 1.41 mmol) and Et3N (0.19 mL,
N-Hydroxy-2-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)ethoxy]cinnamide (9a): A solution of NH2OH·HCl (135 mg,
1.88 mmol) in MeOH (10 mL) was treated with KOH (105 mg,
1.88 mmol) and stirred in an ice-bath for 1 h. Filtration to remove
the white salt gave a solution of the free NH2OH in MeOH. A solu-
tion of 8a (400 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 NH2OH solution was then added to the reac-
tion and stirring continued for 3 h. The reaction was concentrated
in vacuo and the crude was diluted with distd H2O (50 mL), acidi-
fied to pH 2~3 with 1n HCl and extracted with EtOAc (3ꢁ25 mL).
The combined organic layer was dried (Na2SO4), filtered and con-
1
1.41 mmol) gave 9d (235 mg, 57%): H NMR (500 MHz, [D6]DMSO):
d=7.34 (d, J=15.8 Hz, 1H), 7.30 (t, J=7.8 Hz, 1H), 7.12 (m, 2H),
6.95 (d, J=8.2 Hz, 1H), 6.82 (t, J=8.4 Hz, 1H), 6.44 (d, J=15.8 Hz,
1H), 6.43 (d, J=7.8 Hz, 1H), 4.28 (t, J=6.1 Hz, 2H), 4.21 (t, J=
6.0 Hz, 2H), 2.69 (t, J=7.2 Hz, 2H), 2.51 (m, 2H), 2.22 (t, J=7.2 Hz,
2H), 1.54 (m, 1H), 1.24 (dd, J=7.2, 15.6 Hz, 2H), 0.79 (s, 3H),
0.77 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=169.8, 163.2,
159.3, 156.0, 153.5, 138.7, 136.7, 130.5, 127.2, 121.6, 120.3, 119.9,
119.1, 116.2, 113.8, 104.2, 67.6, 67.1, 38.6, 33.4, 28.2, 25.4, 22.9, 22.8,
21.6 ppm; IR (KBr): n˜ =3224, 1652, 1489, 1257, 1121, 1058,
ChemMedChem 2010, 5, 598 – 607
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
605

C.-I. Chang et al.
MED
784 cmꢁ1; HRMS-ESI: m/z [MꢁH]ꢁ calcd for C25H28NO6: 438.1917,
[D6]DMSO): d=169.8, 163.6, 160.1, 156.0, 153.4, 138.5, 130.4, 129.5,
127.9, 127.2, 121.4, 118.7, 117.0, 115.3, 103.7, 64.9, 64.4, 38.8, 33.4,
29.3, 28.4, 25.4, 23.0. 21.6 ppm; IR (KBr): n˜ =3205, 2948, 1644, 1604,
1513, 1492, 1467, 1258, 1176, 1119, 1062, 821 cmꢁ1; HRMS-ESI: m/z
[M+H]+ calcd for C26H32NO6: 454.2230, found: 454.2249.
found: 438.1921.
N-Hydroxy-3-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)propoxy]cinnamide (9e): Following the procedure described
for 9a, reaction of 8e (550 mg, 1.26 mmol) in THF (10 mL) with
ethyl chloroformate (0.17 mL, 1.89 mmol) and Et3N (0.5 mL,
1.89 mmol) gave 9e (319 mg, 56%): mp: 130–1358C; 1H NMR
(500 MHz, CD3OD): d=7.51 (d, J=15.8 Hz, 1H), 7.28 (t, J=8.0 Hz,
1H), 7.11 (d, J=7.6 Hz, 1H),7.09 (s, 1H), 6.95 (d, J=8.1 Hz, 1H),
6.84 (d, J=8.4 Hz, 1H), 6.43 (d, J=15.8 Hz, 1H), 6.42 (s, 1H), 4.21 (t,
J=6.1 Hz, 2H), 4.10 (t, J=5.9 Hz, 2H), 2.82 (t, J=7.2 Hz, 2H), 2.62
(t, J=8.3 Hz, 2H), 2.36 (t, J=7.2 Hz, 2H), 2.23 (t, J=6.0 Hz, 2H),
1.52 (m, H), 1.30 (m, 2H), 0.88 (s, 3H), 0.87 ppm (s, 3H); 13C NMR
(125 MHz, [D6]DMSO): d=169.9, 163.6, 157.3, 156.0, 153.4, 133.9,
131.2, 128.8, 127.2, 123.7, 121.3, 120.1, 118.7, 112.8, 103.7, 65.3,
64.5, 38.8, 33.4, 29.3, 28.4, 25.4, 23.1, 22.9, 21.6 ppm; IR (KBr): n˜ =
N-Hydroxy-4-[4-(3,4-Dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)butoxy]cinnamide (9i): Following the procedure described for
9a, reaction of 8i (500 mg, 1.11 mmol) in THF (10 mL) with ethyl
chloroformate (0.15 mL, 1.67 mmol) and Et3N (0.23 mL, 1.67 mmol)
gave 9i (306 mg, 59%): mp: 99–1048C; 1H NMR (500 MHz,
[D6]DMSO): d=7.51 (d, J=15.8 Hz, 1H), 7.47 (d, J=8.4 Hz, 2H),
6.92 (d, J=8.5 Hz, 2H), 6.83 (d, J=8.3 Hz, 1H), 6.40 (d, J=8.4 Hz,
1H), 6.31 (d, J=15.7 Hz, 1H), 4.08 (t, J=5.8 Hz, 2H), 3.97 (t, J=
5.8 Hz, 2H), 2.83 (t, J=7.2 Hz, 2H), 2.63 (t, J=7.9 Hz, 2H), 2.37 (t,
J=7.2 Hz, 2H), 1.98 (m, 2H), 1.94 (m, 2H), 1.55 (m, 1H), 1.34 (dd,
J=7.2, 9.8 Hz, 2H), 0.92 (s, 3H), 0.91 ppm (s, 3H); 13C NMR
(125 MHz, [D6]DMSO): d=169.9, 160.2, 156.2, 153.4, 138.6, 129.5,
127.8, 127.2, 121.2, 118.7, 116.9, 115.3, 103.8, 67.8, 67.5, 38.8, 33.4,
28.4, 26.1, 26.0, 25.4, 23.0, 21.6 ppm; IR (KBr): n˜ =3218, 1603, 1511,
1256, 1173, 1117, 1056, 823 cmꢁ1; HRMS-EI: m/z [M]+ calcd for
C27H33NO6: 467.2308, found: 467.2312.
3209, 2951, 1627, 1491, 1266, 1159, 1119, 1078, 1005, 776 cmꢁ1
;
HRMS-EI: m/z [M]+ calcd for C26H31NO6: 453.2152, found: 453.2162.
N-Hydroxy-3-[4-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)butoxy]cinnamide (9 f): Following the procedure described
for 9a, reaction of 8 f (600 mg, 1.33 mmol) in THF (10 mL) with
ethyl chloroformate (0.18 mL, 2.00 mmol) and Et3N (0.27 mL,
1
2.00 mmol) gave 9 f (379 mg, 61%): H NMR (500 MHz, CD3OD): d=
Biology
7.26 (d, J=15.7 Hz, 1H), 7.30 (t, J=7.8 Hz, 1H), 7.12 (m, 2H), 6.95
(d, J=8.2 Hz, 1H), 6.82 (t, J=8.4 Hz, 1H), 6.44 (d, J=15.8 Hz, 1H),
6.43 (d, J=7.8 Hz, 1H), 4.28 (t, J=6.1 Hz, 2H), 4.21 (t, J=6.0 Hz,
2H), 2.69 (t, J=7.2 Hz, 2H), 2.51 (m, 2H), 2.22 (t, J=7.2 Hz, 2H),
1.54 (m, 1H), 1.24 (dd, J=7.2, 15.6 Hz, 2H), 0.79 (s, 3H), 0.77 ppm
(s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=169.9, 163.6, 157.5,
156.3, 153.4, 144.4, 133.9, 131.2, 128.8, 126.9, 121.2, 120.1, 118.7,
117.3, 112.8, 103.7, 67.3, 67.5, 38.8, 34.8, 28.4, 26.1, 25.7, 23.0, 22.9,
HDAC activity assay: This assay was carried out as described previ-
ously.[48] Enzyme, inhibitors and substrate were diluted with HDAC
buffer (15 mm Tris HCl, pH 8.1, 0.25 mm EDTA, 250 mm NaCl, 10%
v/v glycerol). Briefly, 10 mL diluted HDAC, such as HeLa nuclear ex-
tract (Enzo), HDAC1 (Enzo; >50% purity by SDS-PAGE) and HDAC6
(Enzo; >75% purity by SDS-PAGE), and 50 mL test compound solu-
tion at different concentrations were added to each well of a 96-
well microtiter plate and pre-incubated at 308C for 5 min. The en-
zymatic reaction was started by addition of 40 mL substrate (Boc-
Lys(Ac)-AMC, Bachem) in HDAC buffer. After incubation at 308C for
30 min, the reaction was terminated by the addition of 100 mL
trypsin solution (10 mgmLꢁ1 trypsin in 50 mm Tris-HCl, pH 8,
100 mm NaCl, 2 mm SAHA). After incubation at 308C for a further
20 min, fluorescence was measured (extinction l=355 nm, emis-
sion l=460 nm). For the calculation of IC50 values, the fluores-
cence in wells without test compound (0.1% DMSO, negative con-
trol) was set as 100% enzymatic activity and the ratio of the fluo-
rescence in wells with 2 mm SAHA (positive control) were set at 0%
enzymatic activity. The IC50 values were calculated from linear re-
gression of the data. All experiments were carried out in triplicate.
21.6 ppm; IR (KBr): n˜ =3627, 3564, 1651, 1540, 1507, 1455 cmꢁ1
;
HRMS-ESI: m/z [M+H]+ calcd for C27H34NO6: 468.2386, found:
468.2381.
N-Hydroxy-4-[2-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)ethoxy]cinnamide (9g): Following the procedure described
for 9a, reaction of 8g (500 mg, 1.18 mmol) in THF (10 mL) with
ethyl chloroformate (0.16 mL, 1.77 mmol) and Et3N (0.24 mL,
1.77 mmol) gave 9g (277 mg, 52%): mp: 100–1058C; 1H NMR
(500 MHz, CD3OD): d=7.52 (d, J=16.9 Hz, 1H), 7.49 (d, J=8.9 Hz,
2H), 6.98 (d, J=8.6 Hz, 2H), 6.86 (d, J=8.4 Hz, 1H), 6.46 (t, J=
8.4 Hz, 1H), 6.32 (d, J=15.8 Hz, 1H), 4.33 (t, J=4.7 Hz, 2H), 4.25 (t,
J=4.2 Hz, 2H), 2.84 (t, J=7.2 Hz, 2H), 2.61 (m, 2H), 2.37 (t, J=
7.2 Hz, 2H), 1.50 (m, 1H), 1.32 (dd, J=7.1, 15.6 Hz, 2H), 0.85 (s,
3H), 0.84 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO): d=169.8,
160.1, 156.0, 153.5, 138.5, 129.5, 128.0, 127.2, 121.6, 119.1, 117.0,
115.3, 104.2, 67.2, 67.1, 38.6, 33.4, 28.2, 25.4, 22.9, 21.5 ppm; IR
The sulforhodamine B (SRB) growth inhibition assay: Cells were
seeded in 96-well plates in medium with 5% fetal bovine serum
(FBS). After 24 h, cells were fixed with 10% trichloroacetic acid
(TCA) to represent cell population at the time of N-hydroxycinna-
mides addition (T0). After additional incubation of vehicle (0.1%
DMSO) or N-hydroxycinnamides (all dissolved in media without ap-
parent precipitation) for 48 h, cells were fixed with 10% TCA and
SRB (0.4% (w/v) in 1% acetic acid) was added to stain cells. Un-
bound SRB was washed out using 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 absorb-
ance measurements: time zero (T0), control growth (C), and cell
growth in the presence of N-hydroxycinnamides (TX), the percent-
age growth was calculated for each of the compound concentra-
tions levels. Percentage growth inhibition was calculated as:
[1ꢁ(TXꢁT0)/(CꢁT0)] ꢁ 100% for concentrations where TX ꢂT0. The
GI50 value is defined as the drug concentration that results in 50%
inhibition of growth compared with control cells.
(KBr): n˜ =2951; 1601, 1511, 1256, 1176, 1127, 1069, 835 cmꢁ1
;
HRMS-ESI: m/z [M+OH]ꢁ calcd for C25H30NO7: 456.2022, found:
456.2016.
N-Hydroxy-4-[3-(3,4-dihydro-8-isopentyl-2-oxo-2H-chromen-7-yl-
oxy)propoxy]cinnamide (9h): Following the procedure described
for 9a, reaction of 8h (500 mg, 1.14 mmol) in THF (10 mL) with
ethyl chloroformate (0.15 mL, 1.71 mmol) and Et3N (0.23 mL,
1.71 mmol) gave 9h (325 mg, 63%): mp: 109–1108C; 1H NMR
(500 MHz, [D6]DMSO): d=7.48 (d, J=8.3 Hz, 2H), 7.39 (d, J=
15.7 Hz, 1H,), 7.03 (d, J=8.4 Hz, 1H), 6.96 (d, J=8.5 Hz, 2H), 6.74
(d, J=8.4 Hz, 1H), 6.30 (d, J=15.8 Hz, 1H), 4.18 (t, J=6.0 Hz, 2H),
4.11 (t, J=5.9 Hz, 2H), 2.86 (t, J=7.4 Hz, 2H), 2.70 (t, J=7.5 Hz,
2H), 2.57 (t, J=7.8 Hz, 2H), 2.18 (m, 2H), 1.45 (m, 1H), 1.27 (dd, J=
7.1, 15.6 Hz, 2H), 0.86 (s, 3H), 0.84 ppm (s, 3H); 13C NMR (125 MHz,
606
www.chemmedchem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 598 – 607

Histone Deacetylase Inhibitors
[24] H. Ueda, H. Nakajima, Y. Hori, T. Fujita, M. Nishimura, T. Goto, M. Oku-
hara, J. Antibiot. 1994, 47, 301–310.
[25] V. M. Richon, S. Emiliani, E. Verdin, Y. Webb, R. Breslow, R. A. Rifkind, P. A.
Marks, Proc. Natl. Acad. Sci. USA 1998, 95, 3003–3007.
[26] A. Monks, C. D. Hose, P. Pezzoli, S. Kondapaka, G. Vansant, K. D. Peters-
en, M. Sehested, J. Monforte, R. H. Shoemaker, Anticancer Drugs 2009,
20, 682–692.
[27] A. Saito, T. Yamashita, Y. Mariko, Y. Nosaka, K. Tsuchiya, T. Ando, T.
Suzuki, T. Tsuruo, O. Nakanishi, Proc. Natl. Acad. Sci. USA 1999, 96,
4592–4607.
[28] H. Wang, N. Yu, H. Song, D. Chen, Y. Zou, W. Deng, P. L. Lye, J. Chang,
M. Ng, E. T. Sun, K. Sangthongpitag, X. Wang, X. Wu, H. H. Khng, L.
Fang, S. K. Goh, W. C. Ong, Z. Bonday, W. Stunkel, A. Poulsen, M. Entzer-
oth, Bioorg. Med. Chem. Lett. 2009, 19, 1403–1408.
[29] M. J. Bottomley, P. Lo Surdo, P. Di Giovine, A. Cirillo, R. Scarpelli, F. Fer-
rigno, P. Jones, P. Neddermann, R. De Francesco, C. Steinkuhler, P. Galli-
nari, A. Carfi, J. Biol. Chem. 2008, 283, 26694–26704.
[30] N. P. Pavletich, M. S. Finnin, J. R. Donigian, A. Cohen, V. M. Richon, R. A.
Rifkind, P. A. Marks, R. Breslow, Nature 1999, 401, 188–193.
[31] T. K. Nielsen, C. Hildmann, A. Dickmanns, A. Schwienhorst, R. Ficner, J.
Mol. Biol. 2005, 354, 107–120.
Western blot analysis: After treatment with HDAC inhibitor for 24 h,
the cells were rapidly washed with phosphate buffered saline
(PBS), and then lysed with ice-cold lysis buffer (50 mm Tris-HCl,
pH 7.4, 1 mm EGTA, 150 mm NaCl, 1% Triton X-100, 1 mm PMSF,
5 mgmLꢁ1 leupeptin, 20 mgmLꢁ1 aprotinin, 1 mm NaF, and 1 mm
Na3VO4) as described previously.[49] The lysates were subjected to
SDS-PAGE by using 13% polyacrylamide gels. Protein bands were
transferred to a nitrocellulose membrane and immunoblot analysis
performed as described.[49] Briefly, the membrane was incubated
successively with 0.1% milk in tris-Tween buffered saline (TTBS) at
room temperature for 1 h with the following specific antibodies:
monoclonal antibody specific for b-actin (Santa Cruz Biotech-
nology), polyclonal antibody specific for Ac-histone H3 (Upstate)
and monoclonal antibody specific for Ac-a-tubulin (Sigma), and
then with horseradish peroxidase-labeled antirabbit second anti-
body for 30 min. After each incubation, the membrane was
washed extensively with TTBS and the immunoreactive bands
were detected by enhanced chemiluminescence (ECL) and devel-
oped with Hyperfilm-ECL (GE Healthcare).
[32] T. K. Nielsen, C. Hildmann, D. Riester, D. Wegener, A. Schwienhorst, R.
Ficner, Acta Crystallogr. Sect. F 2007, 63, 270–273.
[33] A. Schuetz, J. Min, A. Allali-Hassani, M. Schapira, M. Shuen, P. Loppnau,
R. Mazitschek, N. P. Kwiatkowski, T. A. Lewis, R. L. Maglathin, T. H.
McLean, A. Bochkarev, A. N. Plotnikov, M. Vedadi, C. H. Arrowsmith, J.
Biol. Chem. 2008, 283, 11355–11363.
Acknowledgements
We gratefully acknowledge financial support from Academia
Sinica and the National Science Council in Taiwan (grant num-
bers: NSC97-2320-B-038-010-MY3, NSC97-2323-B-002-015).
[34] J. R. Somoza, R. J. Skene, B. A. Katz, C. Mol, J. D. Ho, A. J. Jennings, C.
Luong, A. Arvai, J. J. Buggy, E. Chi, J. Tang, B. C. Sang, E. Verner, R. Wy-
nands, E. M. Leahy, D. R. Dougan, G. Snell, M. Navre, M. W. Knuth, R. V.
Swanson, D. E. McRee, L. W. Tari, Structure 2004, 12, 1325–1334.
[35] A. Vannini, C. Volpari, G. Filocamo, E. C. Casavola, M. Brunetti, D. Renzo-
ni, P. Chakravarty, C. Paolini, R. De Francesco, P. Gallinari, C. Steinkuhler,
S. Di Marco, Proc. Natl. Acad. Sci. USA 2004, 101, 15064–15069.
[36] L. You, S. Feng, R. An, X. Wang, Nat. Prod. Commun. 2009, 4, 297–302.
[37] R. L. Huang, C. C. Chen, Y. L. Huang, D. J. Hsieh, C. P. Hu, C. F. Chen, C.
Chang, Hepatology 1996, 24, 508–515.
[38] Q. Zhang, L. Qin, W. He, L. Van Puyvelde, D. Maes, A. Adams, H. Zheng,
N. De Kimpe, Planta Med. 2007, 73, 13–19.
[39] T. Fujioka, K. Furumi, H. Fujii, H. Okabe, K. Mihashi, Y. Nakano, H. Matsu-
naga, M. Katano, M. Mori, Chem. Pharm. Bull. 1999, 47, 96–100.
[40] P. L. Kuo, Y. L. Hsu, C. H. Chang, J. K. Chang, J. Pharmacol. Exp. Ther.
2005, 314, 1290–1299.
[41] A. Tosun, E. K. Akkol, E. Yesilada, Z. Naturforsch., C: J. Biosci. 2009, 64,
56–62.
[42] R. Wang, J. Kong, D. Wang, L. L. Lien, E. J. Lien, Chin. Med. 2007, 2, 5.
[43] D. F. Wang, O. Wiest, P. Helquist, H. Y. Lan-Hargest, N. L. Wiech, J. Med.
Chem. 2004, 47, 3409–3417.
[44] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Good-
sell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785–2791.
[45] Q. Lu, D. S. Wang, C. S. Chen, Y. D. Hu, J. Med. Chem. 2005, 48, 5530–
5535.
Keywords: epigenetics · histone deacetylase · inhibitors ·
molecular modeling · ostholes
[1] R. D. Kornberg, Science 1974, 184, 868–871.
[2] R. D. Kornberg, Y. Lorch, Cell 1999, 98, 285–294.
[3] T. J. Richmond, K. Luger, A. W. Mader, R. K. Richmond, D. F. Sargent,
Nature 1997, 389, 251–260.
[4] J. R. Davie, Curr. Opin. Genet. Dev. 1998, 8, 173–178.
[5] M. Grunstein, Nature 1997, 389, 349–352.
[6] T. Kouzarides, Curr. Opin. Genet. Dev. 1999, 9, 40–48.
[7] K. Struhl, Z. Moqtaderi, Cell 1998, 94, 1–4.
[8] A. P. Wolffe, D. Guschin, J. Struct. Biol. 2000, 129, 102–122.
[9] W. D. Cress, E. Seto, J. Cell. Physiol. 2000, 184, 1–16.
[10] P. Marks, R. A. Rifkind, V. M. Richon, R. Breslow, T. Miller, W. K. Kelly, Nat.
Rev. Cancer 2001, 1, 194–202.
[11] P. A. Wade, Hum. Mol. Genet. 2001, 10, 693–698.
[12] C. Hubbert, A. Guardiola, R. Shao, Y. Kawaguchi, A. Ito, A. Nixon, M.
Yoshida, X. F. Wang, T. P. Yao, Nature 2002, 417, 455–458.
[13] B. J. North, B. L. Marshall, M. T. Borra, J. M. Denu, E. Verdin, Mol. Cell
2003, 11, 437–444.
[14] C. M. Grozinger, S. L. Schreiber, Chem. Biol. 2002, 9, 3–16.
[15] R. W. Johnstone, Nat. Rev. Drug Discovery 2002, 1, 287–299.
[16] M. Jung, Curr. Med. Chem. 2001, 8, 1505–1511.
[17] I. V. Gregoretti, Y. M. Lee, H. V. Goodson, J. Mol. Biol. 2004, 338, 17–31.
[18] M. Curtin, K. Glaser, Curr. Med. Chem. 2003, 10, 2373–2392.
[19] K. B. Glaser, J. Li, M. J. Staver, R. Q. Wei, D. H. Albert, S. K. Davidsen, Bio-
chem. Biophys. Res. Commun. 2003, 310, 529–536.
[20] M. Paris, M. Porcelloni, M. Binaschi, D. Fattori, J. Med. Chem. 2008, 51,
1505–29.
[46] A. K. Oyelere, P. C. Chen, W. Guerrant, S. C. Mwakwari, R. Hood, Y.
Zhang, Y. Fan, J. Med. Chem. 2009, 52, 456–468.
[47] P. C. Chen, V. Patil, W. Guerrant, P. Green, A. K. Oyelere, Bioorg. Med.
Chem. 2008, 16, 4839–4853.
[48] D. Wegener, F. Wirsching, D. Riester, A. Schwienhorst, Chem. Biol. 2003,
10, 61–68.
[49] C. C. Chen, Y. T. Sun, J. J. Chen, Y. J. Chang, Mol. Pharmacol. 2001, 59,
493–500.
[21] M. Yoshida, M. Kijima, M. Akita, T. Beppu, J. Biol. Chem. 1990, 265,
17174–17179.
[22] M. Kijima, M. Yoshida, K. Sugita, S. Horinouchi, T. Beppu, J. Biol. Chem.
1993, 268, 22429–22435.
[23] J. W. Han, S. H. Ahn, S. H. Park, S. Y. Wang, G. U. Bae, D. W. Seo, H. K.
Kwon, S. Hong, H. Y. Lee, Y. W. Lee, H. W. Lee, Cancer Res. 2000, 60,
6068–6074.
Received: December 2, 2009
Revised: February 8, 2010
Published online on March 5, 2010
ChemMedChem 2010, 5, 598 – 607
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
607