Zn2+-Chelating Motif-Tethered Short-Chain Fatty Acids as a Novel Class of Histone Deacetylase Inhibitors

Article abstract of DOI:10.1021/jm0303655

Among various classes of histone deacetylase (HDAC) inhibitors, short-chain fatty acids exhibit the least potency, with IC₅₀ in the millimolar range. We rationalized that this weak potency was, in part, attributable to their inability to access the zinc cation in the HDAC active-site pocket, which is pivotal to the deacetylation catalysis. We thus explored the structural optimization of valproate, butyrate, phenylacetate, and phenylbutyrate by coupling them with Zn²⁺-chelating motifs (hydroxamic acid and o-phenylenediamine) through aromatic ω-amino acid linkers. This strategy has led to a novel class of Zn²⁺-chelating, motif-tethered, short-chain fatty acids that exhibited varying degrees of HDAC inhibitory potency. One hydroxamatetethered phenylbutyrate compound, N-hydroxy-4-(4-phenylbutyrylamino)benzamide (HTPB), displayed nanomolar potency in inhibiting HDAC activity. Exposure of several cancer cell lines to HTPB at the submicromolar level showed reduced cell proliferation accompanied by histone hyperacetylation and elevated p21^WAF/CIP1 expression, which are hallmark features associated with intracellular HDAC inhibition.

Full text of DOI:10.1021/jm0303655

J . Med. Chem. 2004, 47, 467-474
467
Zn 2+-Ch ela tin g Motif-Teth er ed Sh or t-Ch a in F a tty Acid s a s a Novel Cla ss of
Histon e Dea cetyla se In h ibitor s
Qiang Lu,† Ya-Ting Yang,† Chang-Shi Chen,† Melanie Davis,‡ J ohn C. Byrd,‡ Mark R. Etherton,§
Asad Umar,§ and Ching-Shih Chen*,†
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and Division of Hematology-Oncology, The Arthur
J ames Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, and Laboratory of Biosystems &
Cancer, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892
Received J uly 31, 2003
Among various classes of histone deacetylase (HDAC) inhibitors, short-chain fatty acids exhibit
the least potency, with IC50 in the millimolar range. We rationalized that this weak potency
was, in part, attributable to their inability to access the zinc cation in the HDAC active-site
pocket, which is pivotal to the deacetylation catalysis. We thus explored the structural
optimization of valproate, butyrate, phenylacetate, and phenylbutyrate by coupling them with
Zn2+-chelating motifs (hydroxamic acid and o-phenylenediamine) through aromatic ω-amino
acid linkers. This strategy has led to a novel class of Zn2+-chelating, motif-tethered, short-
chain fatty acids that exhibited varying degrees of HDAC inhibitory potency. One hydroxamate-
tethered phenylbutyrate compound, N-hydroxy-4-(4-phenylbutyrylamino)benzamide (HTPB),
displayed nanomolar potency in inhibiting HDAC activity. Exposure of several cancer cell lines
to HTPB at the submicromolar level showed reduced cell proliferation accompanied by histone
hyperacetylation and elevated p21WAF/CIP1 expression, which are hallmark features associated
with intracellular HDAC inhibition.
In tr od u ction
fatty acids (e.g., butyrate, valproate, phenylacetate,
and phenylbutyrate),12-15 benzamide derivatives (e.g.,
MS-27-275),16,17 trichostatin A (TSA) and analogues,18-20
hybrid polar compounds [e.g., suberoylanilide hydrox-
amic acid (SAHA)],21,22 cyclic tetrapeptides (e.g.,
apicidin),23-26 and the depsipeptide FR901228.26 Among
these agents, short-chain fatty acids are the least potent
inhibitors with IC50 in the millimolar range, as com-
pared to micromolar or even nanomolar for other types
of HDAC inhibitors. Although the use of short-chain
fatty acids in cancer treatment has been reported, their
therapeutic efficacy has been limited by low antiprolif-
erative activity, rapid metabolism, and nonspecific mode
of action.
The acetylation status of core histones plays a pivotal
role in regulating gene transcription through the modu-
lation of nucleosomal packaging of DNA.1-3 In a hy-
poacetylated state, nucleosomes are tightly compacted,
resulting in transcriptional repression due to restricted
access of transcriptional factors to their targeted DNA.
Conversely, histone acetylation leads to relaxed nucleo-
somal structures, giving rise to a transcriptionally
permissive chromatin state. The level of this posttrans-
lational modification is maintained by a dynamic bal-
ance between the activities of histone acetyltransferases
(HATs) and histone deacetylases (HDACs), both of
which are recruited to target genes in complexes with
sequence-specific transcription activators. Aberrant regu-
lation of this epigenetic marking system has been shown
to cause inappropriate gene expression, a key event in
the pathogenesis of many forms of cancer.4-6 Moreover,
evidence demonstrates that inhibition of HDAC triggers
growth arrest, differentiation, and/or apoptosis in many
types of tumor cells by reactivating the transcription of
a small number of genes.7-11 These in vitro findings
have also been confirmed in xenograft models, suggest-
ing that modulation of HDAC’s function might be
targeted for the prevention and/or therapeutic interven-
tion of cancer.
Recently, X-ray crystallographic analysis of HDLP
(histone deacetylase-like protein), a bacterial HDAC
homologue, has revealed a distinctive mode of protein-
ligand interactions whereby TSA and SAHA mediate
enzyme inhibition.27 The HDAC catalytic domain con-
sists of a narrow, tubelike pocket spanning the length
equivalent to four- to six-carbon straight chains. A Zn2+
cation is positioned near the bottom of this enzyme
pocket, which, in cooperation with two His-Asp charge-
relay systems, facilitates the deacetylation catalysis.
Accordingly, the structures of TSA and SAHA might be
divided into three motifs, each of which interacts with
a discrete region of the enzyme pocket. These include a
Zn2+-chelating function (i.e., hydroxamic acid), an ali-
phatic chain as linker, and a polar, planar cap group
(i.e., dimethylaminophenyl and phenylamino functions,
respectively).27 To mediate the enzyme inhibition, the
long aliphatic chain facilitates the insertion of the
HDAC inhibitor into the tubelike enzyme pocket, per-
mitting the hydroxamate group to reach the polar
bottom of the pocket and coordinate with the Zn2+
To date, several structurally distinct classes of HDAC
inhibitors have been reported,7-11 including short-chain
* To whom correspondence should be addressed. Address: Parks
Hall, Rm 336, 500 West 12th Avenue, The Ohio State University,
Columbus, OH 43210-1291. Tel: (614) 688-4008. Fax: (614) 688-8556.
E-mail: chen.844@osu.edu.
† Division of Medicinal Chemistry and Pharmacognosy, The Ohio
State University.
‡ Division of Hematology-Oncology, The Ohio State University.
§ National Cancer Institute.
10.1021/jm0303655 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/11/2003

468 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Lu et al.
cation. Meanwhile, the polar cap group at the other end
of the spacer makes contacts at the pocket entrance,
thereby capping the pocket. This unique mode of mech-
anism also applies to other potent HDAC inhibitors,
while the structures of individual motifs vary. This
three-component concept has proven successful in de-
veloping structural analogues of TSA and trapoxin as
potent HDAC inhibitors.19,28
(2-propylpentanoyl)phenyl]acrylic acid with o-phenylene-
diamine and hydroxylamine, respectively, under typical
EDC coupling condition (Scheme 1B).
Resu lts
Zn 2+-Ch ela tin g, Motif-Teth er ed Va lp r oa te De-
r iva tives. The divergent conjugation of valproic acid
with five different aromatic linkers and subsequently
two Zn2+-binding motifs yielded compounds 1-10.
These tethered conjugates displayed varying degree of
HDAC inhibitory potency (Figure 2), with IC50 values
ranging from 5 µM (compound 8) to 80 µM (compound
5). This potency was a 5-80-fold improvement over that
of the parent molecule (IC50, 0.4 mM). Moreover, re-
moval of the valproyl moiety or the Zn2+-chelating motif
from any of these conjugates would completely abolish
the HDAC inhibitory activity (data not shown), indicat-
In light of this working model, we rationalized that
the weak potency of short-chain fatty acids in HDAC
inhibition was, in part, attributable to their inability
to access the Zn2+ cation in the active-site pocket, which
plays a pivotal role in the deacetylation catalysis. In the
present study, we explored the structural optimization
of short-chain fatty acids by tethering them with a Zn2+
-
chelating motif via an aromatic linker. This strategy has
led to the development of a novel class of Zn2+-chealting,
motif-tethered, short-chain fatty acids, some of which
show inhibition of HDAC activity and cancer cell
proliferation in nanomolar range, a 3 orders of magni-
tude improvement over their parent compounds.
ing the importance of the acyl function and the Zn2+
-
chelating motif in the protein-ligand interactions.
These data bore out our tethering strategy with short-
chain fatty acids as molecular platforms to develop a
new class of HDAC inhibitors.
Ch em istr y
Among the 10 tethered conjugates examined, com-
pounds 1, 2, 4, and 8 represented the optimal deriva-
tives (IC50, 5-8 µM), followed by 3, 9, and 10 (IC50, 10-
20 µM). Relatively, the hydroxamates (compounds 2, 4,
6, 8, and 10) were generally more potent than their
phenylenediamine counterparts (compounds 1, 3, 5, 7,
and 9). Moreover, the aromatic linker exhibited a subtle
effect on HDAC inhibitory activity. Among the five
aromatic ω-amino acids examined, (4-aminophenyl)-
acetate gave rise to conjugates with the least HDAC
inhibitory potency (5 and 6), while 4-(aminomethyl)-
benzoate appeared to be optimal. For further structural
modifications, compounds 1, 2, 4, and 8 were used as
leads, since all of them exhibited IC50 < 10 µM.
Str u ctu r a l Op tim iza tion . The finding that removal
of the valproyl group would completely abrogate the
inhibitory activity of the conjugate underscored the
importance of the acyl moiety in interacting with the
active-site pocket. We further examined the stereoelec-
tronic effect of substituting the valproyl group in
compounds 1, 2, 4, and 8 with a butyryl, phenylacetyl,
or phenylbutyryl function on HDAC inhibition (Figure
3). All these derivatives showed an improved potency
in HDAC inhibition as compared to the valproyl coun-
terparts. Among various acyl functions examined, the
relative potency was in the order of phenylbutyryl >
phenylacetyl > butyryl > valproyl when conjugated to
the same spacer and Zn2+-chelating motif. Of these 12
derivatives, compound 19 was especially noteworthy.
This hydroxamate-tethered phenylbutyrate (HTPB), i.e.,
compound 19, exhibited an IC50 of 44 nM, a 4 order of
magnitude improvement over phenylbutyrate. This
optimal compound was used to examine its effect on
HDAC activity in DU-145 prostate cancer cells.
In general, short-chain fatty acids exhibit HDAC
inhibitory and antiproliferative activities in the milli-
molar range irrespective of the structure of the acyl
chains.8-10 Here, we propose that these fatty acids
exerted HDAC inhibition through nonspecific hydro-
phobic interactions with surface residues located at the
enzyme pocket entrance and/or the hydrophobic region
inside the tubelike pocket. Accordingly, we examined
the working hypothesis that the HDAC inhibitory
potency of these short-chain fatty acids might be
enhanced by tethering to a Zn2+-chelating moiety through
a hydrophobic spacer. In this study, we used two types
of Zn2+-chealting motifs that could reversibly coordinate
with the zinc cation, i.e., hydroxamic acid and o-
phenylenediamine. With regard to the spacer, we chose
aromatic linkers in lieu of aliphatic chains for two
reasons: (1) to enhance the structural rigidity of the
conjugate and (2) to increase van der Waals contacts
with the tubelike hydrophobic region of the pocket to
improve binding affinity. The linkers used here included
4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, (4-
aminophenyl)acetic acid, 3-(4-aminophenyl)propionic
acid, and 3-(4-aminophenyl)acrylic acid. Among them,
4-(aminomethyl)benzoic acid has been used as the linker
for MS-27-275.17 These aromatic ω-amino acids exhib-
ited lengths equivalent to that of four- to six-carbon
straight chains, in line with the depth of the hydropho-
bic region. For the first series of structural modifica-
tions, we employed valproic acid as lead to synthesize
Zn2+-tethered conjugates (Figure 1) according to the
procedures depicted in Scheme 1 (A, compounds 1-8;
B, compounds 9 and 10). For compounds 1-8, valproic
acid was coupled with four different ω-amino acid
methyl ester spacers via EDC activation. The resulting
esters were cleaved to acids via alkaline hydrolysis.
Under typical peptide coupling conditions (BOPCl or
EDC), the resulting acids were treated with Bn-
protected hydroxylamine and Cbz-protected o-phenylene-
diamine to form, after hydrogenolysis, the respective
anilides and hydroxamic acids (Scheme 1A). Compounds
9 and 10 were synthesized by direct coupling of 3-[4-
Effect of HTP B on Histon e Acetyla tion a n d
p 21WAF /CIP 1 Exp r ession in DU-145 P r osta te Ca n cer
Cells. Histone hyperacetylation and increased expres-
sion of the cyclin-dependent kinase inhibitor p21WAF/CIP1
represent two hallmark features in association with
intracellular HDAC inhibition.6 Consequently, we ex-
amined the effect of HTPB vis-a`-vis TSA and phenyl-
butyrate on HDAC activity in DU-145 prostate cancer

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 469
F igu r e 1. Divergent conjugations of valproic acid with five aromatic ω-amino acids and two Zn2+-chelating moieties to generate
compounds 1-10.
cells by characterizing the status of histone acetylation
and p21WAF/CIP1 expression. DU-145 cells were exposed
to HTPB at 0.5, 1, 2.5 µM, TSA at 0.25 and 0.5 µM, or
phenylbutyrate at 1, 2.5, and 5 mM in 10% FBS-
supplemented RPMI 1640 medium for 24 h. Western
blot analysis of the cell lysates indicates that treatment
of these agents gave rise to elevated levels of acetylated
histones H3 and H4 and p21WAF/CIP1 (Figure 4). The
effect of 1 µM HTPB on these biomarkers approximated
that of 0.25 µM TSA or 2.5 mM phenylbutyrate. DU-
145 cells displayed small but significant amounts of
intrinsic p21WAF/CIP1, and the level increased substan-
tially after exposure to HTPB as low as 0.5 µM.
Together, these data confirmed that HTPB targeted
HDAC activity in DU-145 cells.
to HTPB, with IC50 in growth inhibition of approxi-
mately between 0.5 and 1 µM (Figure 5A). As evidenced
by DNA fragmentation, HTPB sensitized DU-145 cells
to apoptosis in a dose-dependent manner (Figure 5B).
As shown, extensive apoptosis occurred at 24 h when
the drug concentration exceeded 1 µM, indicating that
this cytotoxic effect was, at least in part, attributable
to the induction of apoptosis by HDAC inhibition. Other
cell lines examined including AN3CA endometrial
cancer cells and SW-48 and HCT-15 colorectal cancer
cells. These cancer cells were also susceptible to the
cytotoxic effect of HTPB with similar potency (data not
shown).
Discu ssion
Effect of HTP B on DU-145 Cell Via bility. Effect
of HTPB on cancer cell viability was assessed in DU-
145 cells in 10% FBS-supplemented RPMI-1640 me-
dium. These cells displayed a high degree of sensitivity
In this paper, we report the development of a novel
class of HDAC inhibitors, in which short-chain fatty
acids were tethered to a Zn2+-chelating moiety through
hydrophobic linkage. This structural optimization was

470 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Lu et al.
Sch em e 1a
a
Reagents: (a) EDC, THF; (b) KOH/MEOH, 80 °C; (c) NH2OBn‚HCl, BOPCl, Et3N; (d) (2-aminopheny)carbamic acid benzyl ester,
EDC, THF; (e) 10% PDC, H2, MeOH/THF; (f) NH2OH‚HCl, EDC, HOBT, Et3N; (g) o-phenylenediamine, EDC, THF.
HTPB is structurally distinct from existing HDAC
inhibitors, of which many have cap groups consisting
of polar, planar structures. For example, the cap groups
of TSA, SAHA, and MS-275 contain dimethylaminophe-
nyl, phenylamino, and pyridin-3-yl-methoxycarbonyl
groups, respectively. The present study suggests that
the active-site pocket exhibited a high degree of flex-
ibility in accommodating cap groups with different
stereoelectronic properties. Our data indicate that phe-
nylbutyryl and phenylacetyl were more effective than
aliphatic acyl moieties in facilitating the binding of the
conjugates to the active-site pocket. This discrepancy
might, in part, be due to differences in electron density
F igu r e 2. HDAC inhibitory potency of compounds 1-10. In
and/or steric hindrance imposed by the branched side
vitro HDAC assay was carried out by using a commercial
chain. With regard to the aromatic linker, 4-aminoben-
enzyme assay kit as described in the Experimental Section.
Data are the means ( SD (n ) 3).
zoate appeared to be optimal to tether phenylbutyryl
with hydroxamate, of which the length was sufficient
to make contacts at both ends of the pocket.
based on the working model provided by the unique
mode of HDAC inhibition by TSA and SAHA.27 This
novel tethering strategy led to the discovery of HTPB
(or compound 19) that displays HDAC inhibitory and
antiproliferative activities at submicromolar concentra-
tions, which are in line with that reported for SAHA.21
We obtained two lines of evidence that HTPB targeted
HDAC activity in several cancer cell lines. Specifically,
treatment of DU-145 prostate cancer cells with HTPB
at as low as 0.5 µM caused the hyperacetylation of
histones H-3 and H-4 in a dose-dependent manner.
Likewise, p21WAF/CIP1 expression was substantially up-
regulated in response to HTPB. In contrast, the parent
molecule phenylbutyrate required at least 2.5 mM to
achieve the same intracellular effects on histone acetyl-
ation and p21WAF/CIP1 expression.
Con clu sion
The efficacy of HTPB in HDAC inhibition demon-
strates that potent HDAC inhibitors can be designed
on the basis of the framework provided by the crystal
structures of HDLP-ligand complexes. The present
study presents a proof of principle that this tethering
strategy allows the generation of a large library of
compounds via the divergent combination of short-chain
fatty acids, ω-amino acids, and hydroxamate. Further
studies are under way to employ this strategy to develop
HDAC isozyme-specific inhibitors.
Exp er im en ta l Section
Chemical reagents and organic solvents were purchased
from Aldrich unless otherwise mentioned. Nuclear magnetic

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 471
F igu r e 3. Structures and HDAC inhibitory potency of compounds 11-22. Values are the means ( SD (n ) 3).
F igu r e 4. Effect of HTPB, TSA, and phenylbutyrate on
histone acetylation and p21WAF/CIP1 expression in DU-145 cells.
DU-145 cells were exposed to HTPB, TSA, and phenylbutyrate
at the indicated concentrations in 10% FBS-supplemented
RPMI 1640 medium for 24 h. An equivalent amount of protein
from individual lysates was electrophoresed and probed by
Western blot with respective antibodies. Actin was used as an
internal reference protein.
resonance spectra (1H NMR) were measured on Bruker 250
MHz. Chemical shifts (δ) are reported in parts per million
(ppm) relative to TMS peak. Electrospray ionization (ESI)
mass spectrometry analyses were performed with a 3-tesla
Finnigan FTMS-2000 Fourier transform mass spectrometer.
Elemental analyses were within (0.4% of calculated values.
Flash column chromatography was performed with silica gel
(230-400 mesh). The ω-amino acid methyl esters were pre-
pared from the commercially available acids using methanol/
TMSCl, and (2-aminophenyl)carbamic acid benzyl ester was
synthesized from o-phenylenediamine and benzyl chloride
formate according to standard procedures. Rabbit anti-acetyl-
histone H3 and H4 polyclonal antibodies were purchased from
Upstate Biotechnology (Lake Placid, NY), Rabbit anti-p21
antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA). Mouse monoclonal anti-actin was from ICN Biomedicals.-
(Irvine, CA). HRP-conjugated goat anti-rabbit IgG and HRP-
conjugated goat anti-mouse IgG were from J ackson Immu-
noResearch (West Grove, PA).
Compounds 1-8 and 11-22 were synthesized according to
methods a-e described as follows (Scheme 1A), and compounds
9 and 10 were prepared from 3-[4-(2-propylpentanoylamino)-
phenyl]acrylic acid by methods f and g, respectively (Scheme
1B), which are described separately under the title compounds.
Meth od a [1-(3-Dim eth yla m in op r op yl)-3-eth ylca r bo-
d iim id e Hyd r och lor id e (EDC) Cou p lin g]. To a solution of
individual short-chain fatty acids in dry THF (5-10 mmol/
F igu r e 5. Growth inhibitory effect of HTPB on DU-145 cells.
(A) Time course of the dose-dependent effect of HTPB on cell
viability. DU-145 cells were treated with 0-2.5 µM HTPB in
10% FBS-containing RPMI 1640 medium for the indicated
times. Viable cells were examined by the MTT assay. Data
are means ( SD (n ) 6). (B) Dose-dependent effect of HTPB
on the formation of nucleosomal DNA after 24-h exposure. The
formation of nucleosomes was quantitatively measured by cell
death detection ELISA with lysates equivalent to 2 × 103 cells
for each assay. Data are the average of two independent
determinations.
mL) under N2 was added various ω-amino acid methyl ester
(1 equiv), followed by EDC (1.3 equiv). After stirring overnight,
THF was removed under reduced pressure, and the residue
was dissolved in ethyl acetate (100 mL). The mixture was
washed consecutively with water (50 mL) twice and saturated

472 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Lu et al.
brine (50 mL). The organic layer was dried over Na2SO4 and
concentrated under vacuum. The resulting residue was puri-
fied by silica gel flash chromatography.
H), 9.33 (s, 1 H), 7.56 (d, J ) 8.5 Hz, 2 H), 7.26 (d, J ) 8.5 Hz,
2 H), 7.14 (d, J ) 7.9 Hz, 1 H), 6.90 (t, J ) 7.8 Hz, 1 H), 6.72
(d, J ) 7.9 Hz, 1 H), 6.53 (t, J ) 7.7 Hz, 1 H), 4.83 (s, 2 H),
2.41 (m, 1 H), 1.7-1.1 (m, 8 H), 0.89 (t, 6 H); HRMS exact
mass of (M + Na)+ 390.215195 amu; observed mass of (M +
Na)+, 390.21523 amu. Anal. (C22H29N3O2) C, H, N.
2-P r op ylp en ta n oic a cid (4-h yd r oxyp h en ylca r ba m oyl-
m eth ylp h en yl)a m id e (6): 1H NMR (DMSO-d6) δ 10.61 (s, 1
H), 9.82 (s, 1 H), 8.81 (s, 1 H), 7.52 (d, J ) 8.5 Hz, 2 H), 7.16
(d, J ) 8.5 Hz, 2 H), 3.22 (s, 2 H), 2.38 (m, 1 H), 1.7-1.1 (m,
8 H), 0.89 (t, 6 H); HRMS exact mass of (M + Na)+, 315.167911
amu; observed mass of (M + Na)+, 315.16751 amu. Anal.
(C16H24N2O3) C, H, N.
Meth od b (Ester Clea va ge). The resulting ester from
method a was dissolved in a 2 M KOH/MeOH solution. The
mixture was stirred at 80 °C for 1 h, cooled to 0 °C, acidified
with 2 N HCl to pH 3, and concentrated under vacuum, and
ethyl acetate (100 mL) and H2O (50 mL) were added. The
organic phase was separated, washed consecutively with water
and saturated brine (50 mL each), dried over Na2SO4, and
concentrated under vacuum. The resulting residue was puri-
fied by silica gel flash chromatography.
Meth od c [Bis(2-oxo-3-oxa zolid in yl)p h osp h or d ia m id ic
Ch lor id e (BOP -Cl) Cou p lin g]. To a solution of the resulting
acid from method b in dry THF (5-10 mmol/mL) was added
triethylamine (TEA, 1 equiv) under N2. The mixture was
stirred at room temperature for 10 min, and BOP-Cl (1.1
equiv), O-benzylhydroxylamine hydrochloride (1 equiv), and
TEA (3 equiv) were added. After stirring at room temperature
overnight, the solution was concentrated under vacuum, and
ethyl acetate (100 mL) was added, followed by 3% NaHCO3
(50 mL). The organic phase was separated, washed consecu-
tively with water and saturated brine (50 mL each), dried over
Na2SO4, and concentrated under vacuum. The resulting
residue was purified by silica gel flash chromatography.
Meth od d (EDC Cou p lin g). To a solution of individual
acids resulting from method b in dry THF (5-10 mmol/mL)
under N2 was added (2-aminophenyl)carbamic acid benzyl
ester (1 equiv), followed by EDC (1.3 equiv). After stirring
overnight, the mixture was concentrated under vacuum, and
ethyl acetate (100 mL) was added. The organic phase was
washed consecutively with water (50 mL) twice, followed by
saturated brine (50 mL), dried over Na2SO4, and concentrated.
The resulting residue was purified by silica gel flash chroma-
tography.
Meth od e (Hyd r ogen olysis). The N-benzyloxy or N-Cbz
derivative, resulting from method c or d, was dissolved in 1:1
methanol/THF (5-10 mmol/mL), and 10% palladium on
charcoal (10% w/w) was added. The mixture was treated with
hydrogen under atmospheric pressure for 2 h and filtered. The
solvent was evaporated and the residue was recrystallized with
ethyl acetate.
N-(2-Am in op h en yl)-4-[(2-p r op ylp en t a n oyla m in o)m e-
th yl]ben za m id e (1): 1H NMR (DMSO-d6) δ 9.63 (s, 1 H), 8.45
(t, J ) 6.1 Hz, 1 H), 7.94 (d, J ) 7.8 Hz, 2 H), 7.36 (d, J ) 8.3
Hz, 2 H), 7.16 (d, J ) 7.6 Hz, 1 H), 6.98 (t, J ) 7.5 Hz, 1 H),
6.78 (d, J ) 7.7 Hz, 1 H), 6.60 (t, J ) 7.6 Hz, 1 H), 4.90 (s, 2
H), 4.35 (d, J ) 5.9 Hz, 2 H), 2.24 (m, 1 H), 1.6-1.1 (m, 8 H),
0.87 (t, 6 H); HRMS exact mass of (M + Na)+, 390.215195 amu;
observed mass of (M + Na)+, 390.21793 amu. Anal. (C22H29N3O2)
C, H, N.
2-P ropylpentanoic acid {4-[2-(2-amino-phenylcarbamoyl)-
eth yl]p h en yl}a m id e (7): 1H NMR (DMSO-d6) δ 9.80 (s, 1
H), 9.15 (s, 1 H), 7.53 (d, J ) 8.4 Hz, 2 H), 7.17 (d, J ) 8.4 Hz,
2 H), 7.11 (d, J ) 7.8 Hz, 1 H), 6.89 (t, J ) 7.9 Hz, 1 H), 6.70
(d, J ) 7.9 Hz, 1 H), 6.53 (t, J ) 7.7 Hz, 1 H), 4.80 (s, 2 H),
2.86 (t, J ) 7.9 Hz, 2 H), 2.59 (t, J ) 8.1 Hz, 2 H), 2.38 (m, 1
H), 1.7-1.1 (m, 8 H), 0.87 (t, 6 H); HRMS exact mass of (M +
Na)+, 404.230845 amu; observed mass of (M + Na)+ 404.23043
amu. Anal. (C23H31N3O2) C, H, N.
2-P r op ylp en ta n oic a cid [4-(2-h yd r oxyp h en ylca r ba m -
oyl)eth yl)p h en yl]a m id e (8): 1H NMR (DMSO-d6) δ 10.38
(s, 1 H), 9.78 (s, 1 H), 8.70 (s, 1 H), 7.50 (d, J ) 8.5 Hz, 2 H),
7.10 (d, J ) 8.5 Hz, 2 H), 2.75 (t, J ) 7.3 Hz, 2 H), 2.40 (m, 1
H), 2.22 (t, J ) 7.4 Hz, 2 H), 1.7-1.1 (m, 8 H), 0.87 (t, 6 H);
HRMS exact mass of (M + Na)+, 329.183561 amu; observed
mass of (M + Na)+ 329.18295 amu. Anal. (C17H26N2O3) C, H,
N.
3-[4-(2-P r op ylp en ta n oyl)p h en yl]a cr ylic Acid . This com-
pound, a precursor to compounds 9 and 10, was synthesized
from 2-propylpentanoic acid (0.78 mL, 4.9 mmol) and 3-(4-
aminophenyl)acrylic acid methyl ester (0.86 g, 4.9 mmol)
according to methods a and b aforementioned: total yield, 1.05
g (70% for two steps); 1H NMR (CDCl3, 10% DMSO-d6) δ 9.49
(s, 1 H), 7.71 (d, J ) 8.5 Hz, 2 H), 7.55 (d, J ) 15.9 Hz, 1 H),
7.45 (d, J ) 8.4 Hz, 2 H), 6.31 (d, J ) 15.9 Hz, 1 H), 2.40 (m,
1 H), 1.7-1.1 (m, 8 H), 0.87 (t, 6 H).
N-(2-Am in oph en yl)-3-[4-(2-pr opylpen tan oylam in o)ph e-
n yl]a cr yla m id e (9). To a solution of 3-[4-(2-propylpentanoyl)-
phenyl]acrylic acid (200 mg, 0.7 mmol) in dry THF was added
benzene-1,2-diamine (450 mg, 4.2 mmol) under N2, followed
by EDC (180 mg, 0.9 mmol). After stirring overnight, the
mixture was concentrated under vacuum, ethyl acetate (50
mL) was added, and the reaction mixture was washed con-
secutively with water (30 mL) twice and saturated brine (30
mL). The organic layer was dried over Na2SO4 and concen-
trated under vacuum. The crude product was purified by flash
chromatography (ethyl acetate-hexane, 1:1), giving compound
9 (200 mg, 76% yield) as white solid: 1H NMR (DMSO-d6) δ
10.07 (s, 1 H), 9.35 (s, 1 H), 7.71 (d, J ) 8.6 Hz, 2 H), 7.56 (d,
J ) 8.5 Hz, 2 H), 7.51 (d, J ) 15.7 Hz, 1 H), 7.34 (d, J ) 6.6
Hz, 1 H), 6.92 (t, J ) 7.1 Hz, 1 H), 6.80 (d, J ) 15.5 Hz, 1 H),
6.75 (d, J ) 6.6 Hz, 1 H), 6.58 (t, J ) 7.3 Hz, 1 H), 4.96 (s, 2
H), 2.44 (m, 1 H), 1.7-1.1 (m, 8 H), 0.87 (t, 6 H); HRMS exact
mass of (M + Na)+, 402.215195 amu; observed mass of (M +
Na)+, 402.21448 amu. Anal. (C23H29N3O2) C, H, N.
N-Hyd r oxy-4-[(2-p r op ylp en ta n oyla m in o)m eth yl]ben -
1
za m id e (2): H NMR (DMSO-d6) δ 11.17 (s, 1 H), 9.00 (s, 1
H), 8.40 (t, J ) 5.9 Hz, 1 H), 7.70 (d, J ) 8.2 Hz, 2 H), 7 0.30
(d, J ) 8.2 Hz, 2 H), 4.31 (d, J ) 5.7 Hz, 2 H), 2.24 (m, 1 H),
1.6-1.1 (m, 8 H), 0.85 (t, 6 H); HRMS exact mass of (M + Na)+,
315.167911 amu; observed mass of (M + Na)+ 315.16755 amu.
Anal. (C16H24N2O3) C, H, N.
N-(2-Am in op h en yl)-4-(2-p r op ylp en t a n oyla m in o)b en -
za m id e (3): H NMR (DMSO-d6) δ 10.13 (s, 1 H), 9.57 (s, 1
N-Hyd r oxy-3-[4-(2-p r op ylp en ta n oyla m in o)p h en yl]a cr -
yla m id e (10). To a solution of 3-[4-(2-propylpentanoylamino)-
phenyl]acrylic acid (100 mg, 0.35 mmol) in dry DMF (3 mL)
was added EDC (79 mg, 0.53 mmol) and hydroxybenzotriazole
hydrate (HOBT) (62 mg, 0.46 mmol) under nitrogen. The
mixture was stirred for 1 h, and hydroxylamine hydrochloride
(27.4 mg, 0.39 mmol) and TEA (54 µL) were added. The
mixture was stirred for an additional 12 h and concentrated
under vacuum, and ethyl acetate (40 mL) and saturated
NaHCO3 solution (15 mL) were added. The organic phase was
separated and washed consecutively with water and saturated
brine (20 mL each). The organic layer was dried over Na2SO4
and concentrated under vacuum. The crude product was
purified by flash chromatography [ethyl acetate-MeOH (9:
1)], yielding compound 10 (45 mg, 40% yield) as white solid:
1H NMR (DMSO-d6) δ 10.69 (s, 1 H), 10.07 (s, 1 H), 9.00 (s, 1
1
H), 7.95 (d, J ) 8.7 Hz, 2 H), 7 0.74 (d, J ) 8.8 Hz, 2 H), 7.16
(d, J ) 7.8 Hz, 1 H), 6.98 (t, J ) 7.6 Hz, 1 H), 6.78 (d, J ) 8.0
Hz, 1 H), 6.60 (t, J ) 7.7 Hz, 1 H), 4.89 (s, 2 H), 2.44 (m, 1 H),
1.7-1.1 (m, 8 H), 0.89 (t, 6 H); HRMS exact mass of (M + Na)+,
376.199545 amu; observed mass of (M + Na)+, 376.19762 amu.
Anal. (C21H27N3O2) C, H, N.
N-Hydr oxy-4-(2-pr opylpen tan oylam in o)ben zam ide (4):
1H NMR (DMSO-d6) δ 11.07 (s, 1 H), 10.13 (s, 1 H), 8.94 (s, 1
H), 7.65 (m, 4 H), 2.42 (m, 1 H), 1.7-1.1 (m, 8 H), 0.8 (t, 6 H);
HRMS exact mass of (M + Na)+, 301.152261 amu; observed
mass of (M + Na)+, 301.15194 amu. Anal. (C15H22N2O3) C, H,
N.
2-P r opylpen tan oic acid {4-[(2-am in oph en ylcar bam oyl)-
m eth yl]p h en yl}a m id e (5): 1H NMR (DMSO-d6) δ 9.84 (s, 1

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 473
H), 7.67 (d, J ) 7.9 Hz, 2 H), 7.53 (d, J ) 8.4 Hz, 2 H), 7.39 (d,
J ) 15.6 Hz, 1 H), 6.39 (d, J ) 15.3 Hz, 1 H), 2.40 (m, 1 H),
1.7-1.1 (m, 8 H), 0.87 (t, 6 H); HRMS exact mass of (M + Na)+,
327.167911 amu; observed mass of (M + Na)+, 327.16809 amu.
Anal. (C17H24N2O3) C, H, N.
7.4 Hz, 3 H); HRMS exact mass of (M + Na)+, 273.120961 amu;
observed mass of (M + Na)+, 273.12080 amu. Anal. (C13H18N2O3)
C, H, N.
N-H yd r oxy-3-(4-p h en yla cet yla m in op h en yl)p r op ion -
1
a m id e (21): H NMR (DMSO-d6) δ 10.36 (s, 1 H), 10.14 (s, 1
N -(2-Am in o p h e n y l)-4-(b u t y r y la m in o m e t h y l)b e n z-
a m id e (11): 1H NMR (DMSO-d6) δ 9.63 (s, 1 H), 8.41 (t, J )
5.7 Hz, 1 H), 7.94 (d, J ) 8.1 Hz, 2 H), 7 0.36 (d, J ) 8.0 Hz,
2 H), 7.17 (d, J ) 7.6 Hz, 1 H), 6.98 (t, J ) 7.6 Hz, 1 H), 6.78
(d, J ) 8.1 Hz, 1 H), 6.60 (t, J ) 7.7 Hz, 1 H), 4.90 (s, 2 H),
4.35 (d, J ) 5.8 Hz, 2 H), 2.15 (t, J ) 7.3 Hz, 2 H), 1.60 (m, 2
H), 0.88 (t, J ) 7.3 Hz, 3 H); HRMS exact mass of (M + Na)+,
334.152595 amu; observed mass of (M + Na)+, 334.15221 amu.
Anal. (C18H21N3O2) C, H, N.
H), 8.70 (s, 1 H), 7.50 (d, J ) 8.3 Hz, 2 H), 7.2-7.4 (m, 5 H),
7.10 (d, J ) 8.3 Hz, 2 H), 3.62 (s, 2 H), 2.75 (t, J ) 7.5 Hz, 2
H), 2.22 (t, J ) 7.6 Hz, 2 H); HRMS exact mass of (M + Na)+
321.120961 amu; observed mass of (M + Na)+, 321.12040 amu.
Anal. (C17H18N2O3) C, H, N.
N-[4-(2-Hyd r oxyca r ba m oyleth yl)p h en yl]-4-p h en ylbu -
1
tyr a m id e (22): H NMR (DMSO-d6) δ 10.36 (s, 1 H), 9.80 (s,
1 H), 8.70 (s, 1 H), 7.52 (d, J ) 8.5 Hz, 2 H), 7.2-7.4 (m, 5 H),
7.10 (d, J ) 8.4 Hz, 2 H), 2.75 (t, J ) 7.4 Hz, 2 H), 2.62 (t, J
) 7.5 Hz, 2 H), 2.15-2.4 (m, 4 H), 1.8-2.0 (m, 2 H); HRMS
exact mass of (M + Na)+, 349.152261 amu; observed mass of
(M + Na)+, 349.15223 amu. Anal. (C19H22N2O3) C, H, N.
N-(2-Am in op h en yl)-4-(p h en yla cetyla m in om eth yl)ben -
za m id e (12): 1H NMR (DMSO-d6) δ 9.60 (s, 1 H), 8.66 (t, J )
6.1 Hz, 1 H), 7.92 (d, J ) 8.2 Hz, 2 H), 7 0.31 (m, 7 H), 7.16 (d,
J ) 7.0 Hz, 1 H), 6.98 (t, J ) 7.4 Hz, 1 H), 6.78 (d, J ) 6.6 Hz,
1 H), 6.60 (t, J ) 7.4 Hz, 1 H), 4.90 (s, 2 H), 4.35 (d, J ) 5.7
Hz, 2 H), 3.51 (s, 2 H); HRMS exact mass of (M + Na)+,
382.152595 amu; observed mass of (M + Na)+, 382.15228 amu.
Anal. (C22H21N3O2) C, H, N.
In Vitr o HDAC Assa y. HDAC activity was analyzed by
using a histone deacetylase assay kit (Upstate Biotechnology,
Lake Placid, NY) by following the manufacturer’s instruction
with slight modifications. This assay was based on the ability
of DU-145 nuclear extract, which is rich in histone deacetylase
activity, to mediate the deacetylation of biotinylated [3H]acetyl
histone H4 peptide that was bound to streptavidin agarose
beads. The release of [3H]acetate into the supernatant was
measured to calculate the HDAC activity. Sodium butyrate
(0.25-1 mM) was used as a positive control.
Cell Via bility Assa y. The effect of HTPB on cell viability
was assessed by the MTT {[3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide]} assay in 96-well, flat-
bottomed plates, in which 4000 DU-145 cells/well were seeded.
Cells were exposed to HTPB at the indicated concentrations
in 10% FBS-supplemented RPMI-1640 medium at 37 °C in 5%
CO2 for the indicated time. The medium was removed and
replaced by 150 µL of 0.5 mg/mL of MTT in RPMI-1640
medium, and cells were incubated in the CO2 incubator at 37
°C for 2 h. Supernatants were removed from the wells, and
the reduced MTT dye was solubilized with 200 µL/well DMSO.
Absorbance was determined on a plate reader at 570 nm. Each
treatment was repeated in six wells.
N-(2-Am in op h en yl)-4-[(4-p h en ylbu tyr yla m in o)m eth yl]-
ben za m id e (13): 1H NMR (DMSO-d6) δ 9.63 (s, 1 H), 8.43 (t,
J ) 6.1 Hz, 1 H), 7.94 (d, J ) 8.3 Hz, 2 H), 7.34 (d, J ) 8.3 Hz,
2 H), 7 0.21 (m, 6 H), 6.98 (t, J ) 7.2 Hz, 1 H), 6.78 (d, J )
8.11 Hz, 2 H), 6.61 (t, J ) 6.5 Hz, 1 H), 4.90 (s, 2 H), 4.34 (d,
J ) 6.1 Hz, 2 H), 2.59 (t, J ) 7.6 Hz, 2 H), 2.20 (t, J ) 7.7 Hz,
2 H), 1.84 (m, 2 H); HRMS exact mass of (M + Na)+,
410.183895 amu; observed mass of (M + Na)+, 410.18232 amu.
Anal. (C24H25N3O2) C, H, N.
4-(Bu tyr yla m in om eth yl)-N-h yd r oxyben za m id e (14): 1H
NMR (DMSO-d6) δ 11.15 (s, 1 H), 9.01 (s, 1 H), 8.36 (t, J ) 5.6
Hz, 1 H), 7.7 (d, J ) 8.1 Hz, 2 H), 7 0.32 (d, J ) 8.0 Hz, 2 H),
4.30 (d, J ) 5.7 Hz, 2 H), 2.15 (t, J ) 7.3 Hz, 2 H), 1.60 (m, 2
H), 0.88 (t, J ) 7.3 Hz, 3 H); HRMS exact mass of (M + Na)+,
259.10569 amu; observed mass of (M + Na)+, 259.10569 amu.
Anal. (C12H16N2O3) C, H, N.
N-H yd r oxy-4-(p h en yla cet yla m in om et h yl)b en za m id e
(15): 1H NMR (DMSO-d6) δ 11.2 (s, 1 H), 8.9 (s, 1 H), 8.6 (t, J
) 5.8 Hz, 1 H), 7.9 (d, J ) 8.3 Hz, 2 H), 7.28 (m, 7 H), 4.04 (d,
J ) 5.7 Hz, 2 H), 3.5 (s, 2 H); HRMS exact mass of (M + Na)+,
307.105311 amu; observed mass of (M + Na)+, 307.10512 amu.
Anal. (C16H16N2O3) C, H, N.
Ap op tosis Detection by An En zym e-Lin k ed Im m u n -
osor ben t Assa y (ELISA). Induction of apoptosis was as-
sessed by using a cell death detection ELISA (Roche Diagnos-
tics, Mannheim, Germany) by following the manufacturer’s
instruction. This test is based on the quantitative determina-
tion of cytoplasmic histone-associated DNA fragments in the
form of mononucleosomes and oligonucleosomes after induced
apoptotic death. In brief, 1 × 106 DU-145 cells were cultured
in a T-75 flask 24 h prior to the experiment. Cells were treated
with HTPB at the indicated concentrations in 10% FBS-
supplemented RPMI 1640 medium. Both floating and adherent
cells were collected, and cell lysates equivalent to 2 × 103 cells
were used in the ELISA.
Wester n Blot An a lysis. DU-145 cells (1 × 106) treated with
HTPB at the indicated concentrations in 10% FBS-supple-
mented RPMI 1640 medium for 24 h were collected and
sonicated. Protein concentrations of the lysates were deter-
mined by using a Bradford protein assay kit (Bio-Rad, Her-
cules, CA); equivalent amounts of proteins from each lysate
were resolved in 10% SDS-polyacrylamide gel and then
transferred onto Immobilon-nitrocellulose membranes (Milli-
pore, Bellerica, MA) in a semidry transfer cell. The transblot-
ted membrane was washed twice with Tris-buffered saline
(TBS) containing 0.1% Tween 20 (TBST). After blocking with
TBST containing 5% nonfat milk for 40 min, the membrane
was incubated with the primary antibody (1:1000 dilution) in
TBST-1% nonfat milk at 4 °C overnight. After treatment with
the primary antibody, the membrane was washed three times
with TBST for a total of 15 min, followed by goat anti-rabbit
or anti-mouse IgG-horseradish peroxidase conjugates (diluted
1:3000) for 1 h at room temperature and wash three times with
TBST for a total of 1 h. The immunoblots were visualized by
enhanced chemiluminescence.
N-H yd r oxy-4-[(4-p h en ylb u t yr yla m in o)m et h yl]b en z-
1
a m id e (16): H NMR (DMSO-d6) δ 11.2 (s, 1 H), 9.0 (s, 1 H),
8.4 (t, J ) 5.8 Hz, 1 H), 7.7 (d, J ) 8.0 Hz, 2 H), 7.21 (m, 7 H),
4.3 (d, J ) 5.8 Hz, 2 H), 2.58 (t, J ) 7.3 Hz, 2 H), 2.18 (t, J )
7.3 Hz, 2 H), 1.83 (m, 2 H); HRMS exact mass of (M + Na)+,
335.136611 amu; observed mass of (M + Na)+, 335.13716 amu.
Anal. (C18H20N2O3) C, H, N.
4-Bu tyr yla m in o-N-h yd r oxyben za m id e (17): 1H NMR
(DMSO-d6) δ 11.08 (s, 1 H), 10.09(s, 1 H), 8.94 (s, 1 H), 7.67
(m, 4 H), 2.31 (t, J ) 7.3 Hz, 2 H), 1.61 (m, 2 H), 0.92 (t, J )
7.4 Hz, 3 H); HRMS exact mass of (M + Na)+, 245.089661 amu;
observed mass of (M + Na)+, 245.08971 amu; difference <1.0
ppm. Anal. (C11H14N2O3) C, H, N.
N-Hyd r oxy-4-p h en yla cetyla m in oben za m id e (18): 1H
NMR (DMSO-d6) δ 11.1 (s, 1 H), 10.40 (s, 1 H), 8.94 (s, 1 H),
7.67 (m, 4 H), 7.33 (m, 5 H), 3.67 (s, 2 H); HRMS exact mass
of (M + Na)+, 293.089661 amu; observed mass of (M + Na)+,
293.08957 amu. Anal. (C15H14N2O3) C, H, N.
N-Hyd r oxy-4-(4-p h en ylbu tyr yla m in o)ben za m id e (19):
1H NMR (DMSO-d6) δ 11.02 (s, 1 H), 10.1 (s, 1 H), 8.94 (s, 1
H), 7.67 (m, 4 H), 7.27 (m, 5 H), 2.63 (t, J ) 7.5 Hz, 2 H), 2.35
(t, J ) 7.4 Hz, 2 H), 1.87 (m, 2 H); HRMS exact mass of (M +
Na)+, 321.120961 amu; observed mass of (M + Na)+, 321.11940
amu. Anal. (C17H18N2O3) C, H, N.
N-[4-(2-H yd r oxyca r b a m oylet h yl)p h en yl]b u t yr a m id e
1
(20): H NMR (DMSO-d6) δ 10.4 (s, 1 H), 9.8 (s, 1 H), 8.70 (s,
1 H), 7.50 (d, J ) 8.4 Hz, 2 H), 7.10 (d, J ) 8.4 Hz, 2 H), 2.75
(t, J ) 7.3 Hz, 2 H), 2.24 (m, 4 H), 1.61 (m, 2 H), 0.92 (t, J )

474 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
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