Functional differences in epigenetic modulators - Superiority of mercaptoacetamide-based histone deacetylase inhibitors relative to hydroxamates in cortical neuron neuroprotection studies

Article abstract of DOI:10.1021/jm070178x

We compare the ability of two structurally different classes of epigenetic modulators, namely, histone deacetylase (HDAC) inhibitors containing either a hydroxamate or a mercaptoacetamide as the zinc binding group, to protect cortical neurons in culture from oxidative stress-induced death. This study reveals that some of the mercaptoacetamide-based HDAC inhibitors are fully protective, whereas the hydroxamates show toxicity at higher concentrations. Our present results appear to be consistent with the possibility that the mercaptoacetamide-based HDAC inhibitors interact with a different subset of the HDAC isozymes [less activity at HDAC1 and 2 correlates with less inhibitor toxicity], or alternatively, are interacting selectively with only the cytoplasmic HDACs that are crucial for protection from oxidative stress.

Full text of DOI:10.1021/jm070178x

3054
J. Med. Chem. 2007, 50, 3054-3061
Functional Differences in Epigenetic ModulatorssSuperiority of Mercaptoacetamide-Based
Histone Deacetylase Inhibitors Relative to Hydroxamates in Cortical Neuron Neuroprotection
Studies
Alan P. Kozikowski,*,† Yufeng Chen,† Arsen Gaysin,† Bin Chen,† Melissa A. D’Annibale,‡ Carla M. Suto,§ and
Brett C. Langley‡,|
Drug DiscoVery Program, Department of Medicinal Chemistry and Pharmacognosy, UniVersity of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612, Burke Medical Research Institute, White Plains, New York 10605, Amphora DiscoVery Corporation,
800 Capitola DriVe, Durham, North Carolina 27713, and Department of Neurology and Neuroscience, Weill Medical College of Cornell
UniVersity, New York, New York 10021
ReceiVed February 15, 2007
We compare the ability of two structurally different classes of epigenetic modulators, namely, histone
deacetylase (HDAC) inhibitors containing either a hydroxamate or a mercaptoacetamide as the zinc binding
group, to protect cortical neurons in culture from oxidative stress-induced death. This study reveals that
some of the mercaptoacetamide-based HDAC inhibitors are fully protective, whereas the hydroxamates
show toxicity at higher concentrations. Our present results appear to be consistent with the possibility that
the mercaptoacetamide-based HDAC inhibitors interact with a different subset of the HDAC isozymes [less
activity at HDAC1 and 2 correlates with less inhibitor toxicity], or alternatively, are interacting selectively
with only the cytoplasmic HDACs that are crucial for protection from oxidative stress.
Introduction
transcription and expression of genes, including tumor suppres-
sor genes. HDAC inhibitors also enhance the cytotoxic effects
of therapeutic agents used in cancer treatment, including
radiation and chemotherapeutic drugs.10,11
To date, considerable research activity has focused on
understanding the “histone code” and, in particular, on the design
of histone deacetylase (HDACa) inhibitors as novel therapeutics
for the treatment of a wide range of disorders including cancer,
as well as neurodegenerative diseases, and even malaria.1 These
compounds owe their action to their ability to reactivate silenced
genes by modulating the condensation status of DNA. The post-
translational acetylation status of chromatin is determined by
the competing activities of two classes of enzymes, histone
acetyltransferases (HATs) and HDACs, which control the
acetylation of lysine residues making up the histones. In general,
HATs function to acetylate lysine groups in nuclear histones,
resulting in neutralization of the charges on the histones and a
more open, transcriptionally active chromatin structure, while
the HDACs function to deacetylate and suppress transcription.
A shift in the balance of acetylation on chromatin may result
in changes in the regulation of patterns of gene expression.2-5
Because many cancers are associated with aberrant transcrip-
tional activity and HDACs can affect transcription factors and
gene regulation, these enzymes have been identified as attractive
targets for cancer therapy. Indeed, chemical inhibitors of HDACs
have been shown to inhibit tumor cell growth and induce
differentiation and cell death.6 Several such inhibitory agents,
including suberoylanilide hydroxamic acid (SAHA) and dep-
sipeptide (FR901228) have reached clinical trials,7-9 and SAHA
has been approved by the FDA for use in cutaneous T-cell
lymphoma (CTCL). HDAC inhibitors work by allowing the
In addition to their applications in oncology, several of the
known HDAC inhibitors have been found to be protective in
different cellular and animal models of acute and chronic
neurodegenerative injury and disease, for example, ischemic
stroke,12-14 multiple sclerosis,15 and polyglutamine-expansion
diseases, such as Huntington’s disease16-20 and spinal and bulbar
muscular atrophy (SBMA).21 Thus, the administration of SAHA
in drinking water using cyclodextrin as a carrier and sodium
butyrate administered intraperitoneally have been shown to
significantly improve rotarod performance and to decrease
neuronal atrophy in R6/2 mice. Sodium butyrate treatment
significantly extended survival in R6/2 mice by 20%. Consistent
with the idea that HDAC inhibition relieves transcriptional
repression and that protection is downstream of mutant htt,
neither inhibitor was found to decrease mutant htt expression
or aggregates.17 The use of sodium butyrate as a therapy has
also been explored in a transgenic mouse model of SBMA. Oral
administration of this compound increased histone acetylation
in spinal cord tissue and was found to reduce the functional
and histopathological deficits associated with SBMA. Moreover,
Nestler has recently highlighted the therapeutic potential of
HDAC inhibitors (HDAC5) in depression.22
The utility of HDAC inhibitors in medicine would thus appear
to be tremendous, but the translation of these ideas to the clinic
will ultimately require the design of isoform selective molecules
to minimize side effect issues. The HDAC inhibitors now in
the clinic do not show any significant selectivity for the
individual HDAC isoforms of which there are now 11 that
operate by zinc (or possibly iron23) dependent mechanisms (class
I includes HDACs 1, 2, 3, and 8, class II includes 4, 5, 6, 7, 9,
and 10, and class IV includes HDAC 11).24,25 While we were
still investigating complete details of isoform selectivity of our
designed HDAC inhibitors, we deemed it important to examine
* To whom correspondence should be addressed. Phone: 312-996-7577.
Fax: 312-996-7107. E-mail: kozikowa@uic.edu.
† University of Illinois at Chicago.
‡ Burke Medical Research Institute.
§ Amphora Discovery Corporation.
| Weill Medical College of Cornell University.
a Abbreviations: HDAC, histone deacetylase; ZBG, zinc binding group;
HAT, histone acetyltransferase; SAHA, suberoylanilide hydroxamic acid;
TSA, trichostatin A; CTCL, cutaneous T-cell lymphoma; SBMA, spinal
and bulbar muscular atrophy; HCA, homocysteate; MTT, 3-[4,5-dimeth-
ylthiazol-2-yl]2,5-diphenyltetrazolium bromide.
10.1021/jm070178x CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/01/2007

Functional Differences in Epigenetic Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3055
Table 1. List of Compounds Studied in the Neuroprotection Assays Using Cortical Neurons
a The CLogP values were calculated from website http://www.syrres.com/esc/est_kowdemo.htm. b An accurate estimate of standard error on the IC50
could not be determined due to the poor fit of the curve.
possible differences in the neuroprotective effects of our
mercaptoacetamide-based inhibitors relative to the more com-
monly explored hydroxamates such as TSA or SAHA. In
particular, as will be shown below, some of the mercapto-
acetamides exhibit a superior neuroprotection profile in an in
vitro model of oxidative-stress-induced death.
Results and Discussion
Chemistry. Previously, we have reported on the synthesis
of some mercaptoacetamide-based HDAC inhibitors that owe
their action to the ability of the thiol and carbonyl groups to
interact with the zinc atom present in the catalytic gorge of the
HDACs.26 We have also found that some of these compounds

3056 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Kozikowski et al.
Scheme 1. Synthesis of Mercaptoacetamides 2, 6, 7, and 11-13
Scheme 2. Synthesis of Hydroxamates 1, 3, 4, 5, and 8-10
are very potent in blocking prostate tumor xenograft growth in
rodents. As we were aware of the fact that the hydroxamate
group is associated with potential toxicity,27 we believed that it
was important to compare these two classes of HDAC inhibitors
head-to-head for their effects on neurons. Thus, 13 mercap-
toacetamides and hydroxamates with CLogP values ranging from
0.78 to 2.86 were chosen from our HDAC inhibitors library,
and their structures and inhibitory activities toward HDACs 1,
2, 8, 10, and 6 are provided in Table 1. The synthetic procedures
used for the preparation of the mercaptoacetamides (2, 6, 7,
and 11-13) from trityl-protected mercaptoacetic acid and its
methyl ester are outlined in Scheme 1. Other hydroxamates (1,
4, 5, and 8-10) were prepared from their carboxylic acid
precursors using general literature methods (Scheme 2).28
HDAC Inhibitors Protect Cortical Neurons from Oxida-
tive Stress-Induced Death. The possible effects of our hy-
droxamic acid-based or mercaptoacetamide-based HDAC in-
hibitors were assessed in comparison with the commercially
available hydroxamate-based inhibitors, TSA and Scriptaid,
using an in vitro model of oxidative stress-induced neurode-
generation in primary cortical neurons. In this model, neuro-
degeneration is induced by the presence of a 5 mM concentration
of the glutamate analog, homocysteate (HCA),28 which depletes
the cellular antioxidant glutathione by the competitive inhibition
of cyst(e)ine uptake at the level of the plasma membrane cystine/
glutamate antiporter system xc-. Because cysteine is required
for synthesis of glutathione, the inhibition of its uptake results
in glutathione depletion. Cellular redox homeostasis, therefore,
becomes disrupted with the accumulation of endogenously
produced and unopposed oxidants resulting in neuronal degen-
eration over an approximately 24 hour period of time. Impor-
tantly, primary neurons at this early developmental stage lack
ionotropic and metabotropic receptors and are not susceptible
to excitotoxicity, rather death is induced by accumulation of
unopposed free radicals and the neurons exhibit a number of
apoptotic features.29 Moreover, the commercially available
HDAC inhibitors such as TSA, SAHA, and sodium butyrate
have previously been shown to offer protection in this model.30
Here, in addition to TSA, we also include the commercially
available HDAC inhibitor, Scriptaid, and the inactive hy-
droxamate control, Nullscript. Nullscript, which is structurally
similar to Scriptaid, lacks sufficient length in the linker region
to allow the hydroxamic acid moiety to coordinate the zinc atom
at the catalytic core of the HDAC enzyme.
The results of our preliminary neuroprotection studies are
shown in Figure 1, where the data are presented as bar graphs
of cell survival in the presence and absence of HCA after 48 h,
which is well beyond the time for neuronal degeneration in this
model to occur (Figure 1). Neuron survival was quantified by
the MTT assay, a colorimetric assay in which the amount of
yellow MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazo-
lium bromide, Promega, Madison, WI) is reduced to purple
formazan by active mitochondrial reductase enzymes in viable
cells. These data demonstrate that the most effective neuropro-

Functional Differences in Epigenetic Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3057
shaped curves when tested in the presence of HCA for 24 h,
with the greatest protection being observed at approximately 1
µM. At concentrations of 10 µM and above, the toxicity from
the hydroxamate-based HDAC inhibitors themselves mask much
of the beneficial effect that the HDAC inhibitors may have
against oxidative stress. In contrast, the mercaptoacetamides 6
and 13 cause no toxicity at any of the concentrations tested
and show a steadily increasing concentration-dependent ability
to protect from HCA toxicity, with 100% protection beginning
at approximately 10 µM. Similarly, the mercaptoacetamides 2
and 7 are not toxic and protect against oxidative stress at 10
µM. However, unlike mercaptoacetamide 6 and 13, at higher
concentrations, these inhibitors do exhibit some toxicity. It is
also possible that the greater toxicity of the hydroxamate HDAC
inhibitors compared to the mercaptoacetamide inhibitors reflects
some isoform specificity for HDAC1. As is apparent from the
data presented in Table 1, the hydroxamates all generally exhibit
good inhibition for HDAC1. Based on a recent publication
investigating the role of HDACs in cerebella granule neurons,
in which HDAC1 was found to be essential for survival,31 it is
possible that the inhibition of HDAC1 could result in neuronal
toxicity in this model. Again, this underscores the need to move
from pan-HDAC inhibitors such as TSA or Scriptaid to isoform-
specific HDAC inhibitors when considering neuroprotective
therapies. In contrast to the hydroxamate-based HDAC inhibi-
tors, the mercaptoacetamide-HDAC inhibitors, like compound
13, show some degree of HDAC6 selectivity. Concurrent with
this selectivity is the ability of these inhibitors to protect neurons
from oxidative stress-induced death without any inhibitor-
associated toxicity. It is possible that some of this protection in
the mercaptoacetamide-based inhibitors may, at least in part,
be due to radical trapping by the sulfur group. However, our
observation that phenylethanethiol, a simple thiol-containing
compound that, much like the inactive hydroxamate Nullscript,
we would predict to be ineffective at inhibiting HDAC activity,
is only protective at high concentrations >500 µM (Figure 3),
which argues that we are well below the concentration required
for a simple antioxidant effect. Indeed, thiol antioxidants that
show therapeutic efficacy in this model, such as lipoic acid and
N-acetylcysteine, are used at concentrations of 100 µM-1 mM,
while doses lower than 100 µM do not protect.32 Nonetheless,
the idea of a bifunctional compound that could both inhibit
HDAC activity and have antioxidant activity would hold
significant appeal in the clinical setting.
Figure 1. Survival of cortical neurons upon exposure to various HDAC
inhibitors in the absence (blue bar) or presence (red bar) of HCA. Data
are presented as percent of control ( SEM.
tection is provided by the mercaptoacetamide-based inhibitors,
which protected the cortical neurons to approximately untreated
control levels. While many of the hydroxamates exhibit protec-
tion against the HCA-induced oxidative stress, in all cases, the
inhibitors themselves, at the 10 µM concentrations used,
displayed toxicity. Thus, compared to control cells with no
inhibitor present, the viability of the hydroxamate-HDAC
inhibitor-treated cells is less. Consistent with Nullscript lacking
sufficient length in the linker region to allow HDAC inhibition,
it had little protective effect. However, it is interesting to note
that unlike the other hydroxamates, Nullscript by itself resulted
in no death to the cortical neurons, suggesting that the
hydroxamate group is not inherently toxic.
Additionally, we present cell micrographs for the control and
HCA-treated cells together with the micrographs for TSA-,
hydroxamate 1-, and mercaptoacetamide 2-treated cells (Figure
2). The cells were visualized using a two-color fluorescence
live-dead assay (Molecular Probes, Eugene, OR). Two-color
fluorescence live-dead staining was generated by incubating
unfixed cells for 20 min at 37 °C with a solution of acetoxy-
methyl ester calcein (2 M) and ethidium homodimer-1 (1 M).
Calcein acetoxymethyl ester is hydrolyzed by esterase activity
in living cells to membrane-impermeant calcein (green fluo-
rescence), while the ethidium homodimer-1 binds to the nucleic
acids of damaged and dead cells (red fluorescence). Again, these
cell micrographs show a striking difference between the
hydroxamate- and the mercaptoacetamide-based HDAC inhibi-
tors.
To obtain a more precise picture of the effects of HDAC
inhibitor concentration on the extent of neuroprotection in the
HCA model, dose response curves were obtained for eight of
the compounds reported herein. These are displayed in Figure
3. As is apparent, the hydroxamates show approximate bell-
Acetylation of Histone Protein Accompanies HDAC In-
hibition. To investigate whether the neuroprotective concentra-
tions of the HDAC inhibitors used result in global histone
acetylation in neurons and, thus, potentially, changes in gene
Figure 2. Cell micrographs of cortical neurons treated with HDAC inhibitor in the presence or absence of HCA. The cells were visualized using
a two-color fluorescence live-dead assay (Molecular Probes, Eugene, OR).

3058 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Kozikowski et al.
repression at this promoter by inhibiting HDAC1 would result
in increased levels of the pro-apoptotic c-Jun and neuronal death.
Although further work is required to understand these differ-
ences, including more extensive studies of isozyme selectivity,
our results are consistent with the possibility that the mercap-
toacetamide-based HDAC inhibitors interact with a different
subset of the HDAC isozymes, or alternatively, are interacting
selectively with only the cytoplasmic HDACs that are crucial
for protection from oxidative stress. Further experiments are
underway to investigate these and other putative mechanisms
of mercaptoacetamide-based HDAC inhibitor neuroprotection.
Summary
The present research findings suggest that it will be important
to take into consideration the nature of the ZBG that is present
in the HDAC inhibitors that are being used to accomplish
specific therapeutic endpoints. We note that the dose-dependent
toxicity associated with the hydroxamate-based inhibitors is
likely due to the inhibition of pro-survival or the activation of
pro-death or toxic (in a neuronal context) gene(s) rather than
the hydroxamate group per se. As in our experiments, we see
no toxicity using the hydroxamate-containing HDAC inhibitor
control, Nullscript. Thus, in treating neurodegenerative diseases,
some of the mercaptoacetamide-based inhibitors may prove
worthy of further consideration, although ultimately, it will be
necessary to carefully assess these inhibitors in other models
of neurodegeneration in their ability to penetrate to the blood-
brain barrier and for any other toxicological or metabolic
liabilities they might have. Nonetheless, we believe these
findings are significant, for they chart a possible course forward
in the development of HDAC inhibitor-based therapies for
neurodegenerative disorders. The isoform data presented for the
compounds disclosed herein, while still incomplete, are in fact
more thorough than the data generally available in the literature.
These data thus provide structural information relevant to
identifying more selective molecules. Efforts are also being
made to obtain measurements of IC50 values against the missing
HDAC isoforms, which will be reported in due course.
Figure 3. Dose-dependent neuroprotection for eight of the HDAC
inhibitors and phenylethanethiol in the HCA-cortical neuron model.
Figure 4. Acetylation levels of histone H4 after treatment with some
of the HDAC inhibitors. (A) Effect of different HDAC inhibitors at 10
µM. (B) Effect of different HDAC inhibitors at 50 µM.
transcription, the relative acetylation levels of histone H4 were
evaluated after treatment with the HDAC inhibitor compounds
(Figure 4A). Histone proteins were acid-precipitated from cell
nuclear extracts obtained from rat primary cortical neurons
treated with TSA, Scriptaid, Nullscript, and the new ligands at
10 µM for 8 h. Western blot analysis using acetyl-histone
H4-specific antibodies (Upstate Cell Signaling Solutions,
Charlottesville, VA) demonstrates that acetylation of H4 histone
proteins is promoted by some but not all of the HDAC inhibitors.
It is, therefore, clear that the extent of H4 acetylation does not
correlate with the degree of cellular protection. In fact, for the
most part, there is a good correlation between the level of H4
acetylation and neuronal toxicity. Because some of the mer-
captoacetamide-based HDAC inhibitors showed toxicity at
higher concentrations, we examined whether this toxicity was
reflected by histone H4 acetylation. Histone proteins were acid-
precipitated from cell nuclear extracts obtained from rat primary
cortical neurons treated with several different mercaptoacet-
amide-based inhibitors at 50 µM for 8 h. Immunoblot analysis
of acetyl-H4 demonstrated that with the mercaptoacetamides
that showed toxicity at this concentration (compounds 2 and
7), there was a marked increase in H4 acetylation, whereas
inhibitors that showed no toxicity at this concentration (com-
pounds 6 and 13) induced no increased H4 acetylation (Figure
4B). These findings suggest that the HDAC or HDACs that are
the target for neuroprotection against oxidative stress do not
play a predominant role in global transcription in the nucleus.
This result would also be consistent with our findings that
toxicity correlated with the ability of our ligands, particularly
the hydroxamates, to inhibit HDAC1 and HDAC2. As previ-
ously discussed, HDAC1 activity has been shown to be required
for neuronal survival. The mechanism of this survival has been
shown to be through histone deacetylation and suppression of
the c-Jun promoter within the nucleus.31 Thus, the loss of
Experimental Section
1
Chemistry. H NMR and 13C NMR spectra were recorded on
Bruker spectrometer at 300/400 MHz and 75/100 MHz, respec-
tively, with TMS as an internal standard. HRMS experiment was
performed on Q-TOF-2TM (Micromass). TLC was performed with
Merck 60F254 silica gel plates. Preparative TLC was performed with
analtech 1000 mm silica gel GF plates. Column chromatography
was performed using Merck silica gel (40-60 mesh). HPLC was
carried out on an ACE AQ column (100 × 4.6 mm and 250 × 10
mm), with detection at 210, 240, 254, 280, and 300 nm on a
Shimadzu SPD-10A VP detector; flow rate ) 2.0-3.5 mL/min;
from 10% acetonitrile in water to 100% acetonitrile with 0.05%
TFA. Detailed methods for preparation of intermediates 14-27 can
be found in the Supporting Information together with their HPLC
data and conditions for the tested compounds.
General Procedure for the Synthesis of Mercaptoacetamides
by Trityl Group Deprotection. To a solution or suspension of
compound 14 (1 mmol) in dichloromethane (10 mL) was added
trifluoroacetic acid (1.0 mL), followed by the addition of triethyl-
silane (1.1 mmol). After the mixture was stirred at room temperature
for 2 h, saturated sodium bicarbonate (15 mL) was added slowly,
and the mixture was stirred for 30 min. The organic layer was
separated, and the aqueous layer was extracted with chloroform
several times (followed by TLC). The combined organic layers were
dried over sodium sulfate and concentrated in vacuo. The crude
product was purified by column chromatography to give mercap-
toacetamides 2 (248 mg, 75%).

Functional Differences in Epigenetic Modulators
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3059
6-(2-Mercaptoacetylamino)-hexanoic Acid Quinolin-8-yl-
amide (2). H NMR (CDCl3, 300 MHz) δ (ppm) 9.81 (br s, 1H),
123.8, 121.2, 119.8, 118.6, 117.4, 114.7, 110.9, 106.1, 99.5, 53.5,
36.0, 31.9, 28.1, 28.0, 26.9, 24.9,24.8. ESI-HRMS calcd for
[C31H35N5O4 + H]+, 542.2761; found, 542.2762.
1
8.80 (dd, J ) 4.2, 1.5 Hz, 1H), 8.77 (dd, J ) 6.9 and 1.8 Hz, 1H),
8.16 (dd, J ) 8.4 and 1.5 Hz, 1H), 7.56-7.43 (m, 3H), 6.81 (br s,
1H), 3.32 (dt, J ) 6.6 and 6.3 Hz, 2H), 3.21 (d, J ) 9.0 Hz, 2H),
2.58 (t, J ) 7.2 Hz, 2H), 1.90 (t, J ) 9.0 Hz, 1H), 1.85 (m, 2H),
1.62 (m, 2H), 1.48 (m, 2H). 13C NMR (CDCl3, 75 MHz) δ (ppm)
171.5, 169.1, 148.2, 138.3, 136.4, 134.4, 127.9, 127.4, 121.6, 121.5,
116.4, 39.6, 37.8, 29.1, 28.3, 26.4, 25.0. ESI-HRMS calcd for
[C17H21N3O2S + H]+, 332.1432; found, 332.1429.
Octanedioic Acid [2′-(2-Amino-3-phenylpropionylamino)-
1
biphenyl-4-yl]amide hydroxyamide (1, Yield 90%). H NMR
(DMSO-d6, 400 MHz) δ (ppm) 10.34 (s, 1H), 9.96 (s, 1H), 8.66
(br s, 1H), 8.22 (d, J ) 8.1 Hz, 1H), 7.64 (d, J ) 8.4 Hz, 2H),
7.35-7.14 (m, 10 H), 3.52 (m, 1H), 3.34 (m, 2H), 3.01 (dd, J )
4.0 and 13.0 Hz, 1H), 2.75 (dd, J ) 8 and 13 Hz, 1H), 2.31 (t, J
) 7.0 Hz, 2H), 1.94 (t, J ) 7.0 Hz, 2H), 1.59 (m, 2H), 1,49 (m,
2H), 1.23 (m, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm) 173.1,
171.7, 169.5, 139.2, 138.6, 135.2, 132.6, 132.5, 130.6, 129.8, 129.7,
128.1, 126.7, 124.5, 121.5, 119.5, 55.9, 36.8, 32.7, 28.8, 25.4. ESI-
HRMS calcd for [C29H34N4O4 + H]+, 503.2658; found, 503.2648.
Octanedioic Acid {2′-[2-Amino-3-(4-hydroxy-phenyl)pro-
pionylamino]biphenyl-4-yl}amide Hydroxyamide (9, Yield 52%).
1H NMR (CD3OD, 300 MHz) δ (ppm) 7.90 (d, J ) 7.8 Hz, 1H),
7.61 (d, J ) 8.2 Hz, 2H), 7.40-7.10 (m, 6H), 7.02 (d, J ) 7.2 Hz,
2H), 6.74 (d, J ) 8.2 Hz, 2H), 3.65 (t, J ) 5.8 Hz, 1H), 3.00-2.90
(m, 1H), 2.82-2.70 (m, 1H), 2.39 (t, J ) 7.1 Hz, 2H), 2.11 (t, J )
7.1 Hz, 2H), 1.75-1.30 (m, 8H). 13C NMR (CD3OD, 75 MHz) δ
(ppm) 173.7, 173.2, 171.9, 156.5, 138.4, 135.1, 134.3, 134.3, 130.5,
130.4, 129.6, 127.9, 127.5, 123.9, 115.5, 120.3, 56.6, 39.2, 36.8,
32.7, 28.9, 28.8, 25.9, 25.7, 15.7. ESI-HRMS calcd for [C29H34N4O5
+ H]+, 519.2602; found, 519.2595.
2-Mercapto-N-[6-(3-phenyl-ureido)-hexyl]acetamide (6, Yield
1
78%). H NMR (DMSO-d6, 300 MHz) δ (ppm) 8.37 (br s, 1H),
7.97 (br t, J ) 5.1 Hz, 1H), 7.37 (d, J ) 7.5 Hz, 2H), 7.20 (t, J )
7.5 Hz, 2H), 6.87 (t, J ) 7.5 Hz, 1H), 6.10 (br t, J ) 5.4 Hz, 1H),
3.07 (d, J ) 7.8 Hz, 2H), 3.04 (t, J ) 6.9 Hz, 2H), 2.71 (t, J ) 7.8
Hz, 1H), 1.41 (m, 4H), 1.28 (m, 4H). 13C NMR (DMSO-d6, 75
MHz) δ (ppm) 169.4, 155.2, 140.6, 128.6, 120.9, 117.5, 38.96,
38.79, 29.7, 29.0, 27.1, 26.1 (2C). ESI-HRMS calcd for [C15H23N3O2S
+ Na]+, 332.1409; found, 332.1407.
4-Dimethylamino-N-[6-(2-mercaptoacetylamino)hexyl]ben-
zamide (7, Yield 91%). 1H NMR (CDCl3, 300 MHz) δ (ppm) 7.68
(d, J ) 9.0 Hz, 2H), 6.89 (br s, 1H), 6.66 (d, J ) 9.0 Hz, 2H), 6.18
(br t, 1H), 3.43 (dt, J ) 6.9 and 6.0 Hz, 2H), 3.27 (dt, J ) 6.6 and
6.0 Hz, 2H), 3.24 (d, J ) 9.0 Hz, 2H), 3.01 (s, 6H), 1.94 (t, J )
9.0 Hz, 1H), 1.60 (m, 2H), 1.54 (m, 2H), 1.39 (m, 4H). 13C NMR
(CDCl3, 75 MHz) δ (ppm) 169.3, 167.6, 152.4, 128.3, 121.4, 111.1,
40.1, 39.4, 39.3, 29.7, 29.2, 28.3, 26.1, 26.0. ESI-HRMS calcd for
[C17H27N3O2S + Na]+, 360.1722; found, 360.1721.
6-(3-Adamantan-1-yl-ureido)hexanoic Acid Hydroxyamide (3,
1
Yield 72%). H NMR (CD3OD, 300 MHz) δ (ppm) 3.04 (t, J )
7.2 Hz, 2H), 2.09 (t, J ) 7.5 Hz, 2H), 2.03 (m, 3H), 1.96 (m, 6H),
1.70 (m, 6H), 1.62 (m, 2H), 1.45 (m, 2H), 1.34 (m, 2H). 13C NMR
(CD3OD, 75 MHz) δ (ppm) 173.0, 160.5, 51.5, 43.6, 40.5, 37.7,
33.8, 31.19, 31.14, 27.5, 26.6. ESI-HRMS calcd for [C17H29N3O3S
+ Na]+, 346.2106; found, 346.2098.
4-Dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]ben-
1
zamide (11, Yield 86%). H NMR (CDCl3, 300 MHz) δ (ppm)
7.68 (d, J ) 9.0 Hz, 2H), 6.88 (br s, 1H), 6.66 (d, J ) 9.0 Hz, 2H),
6.19 (br t, 1H), 3.44 (dt, J ) 6.9, 6.0 Hz, 2H), 3.29 (dt, J ) 6.6,
6.0 Hz, 2H), 3.20 (d, J ) 9.0 Hz, 2H), 3.02 (s, 6H), 1.90 (t, J )
9.0 Hz, 1H), 1.61 (m, 4H), 1.41 (m, 2H). 13C NMR (CDCl3, 75
MHz) δ (ppm) 169.5, 167.7, 152.4, 128.3, 121.2, 111.1, 40.1, 39.6,
39.2, 29.4, 28.7, 28.3, 23.7. ESI-HRMS calcd for [C16H25N3O2S
+ Na]+, 346.1565; found, 346.1559.
8-[3-(4-Dimethylaminobenzyl)ureido]octanoic Acid Hydroxy-
1
amide (4, Yield 41%). H NMR (DMSO-d6, 300 MHz) δ (ppm)
10.33 (br s, 1H), 8.66 (br s, 1H), 7.05 (d, J ) 8.4 Hz, 2H), 6.67 (d,
J ) 8.4 Hz, 2H), 6.05 (t, J ) 5.7 Hz, 1H), 5.81 (t, J ) 5.7 Hz,
1H), 4.05 (d, J ) 6.0 Hz, 2H), 2.97 (dt, J ) 6.6 and 6.0 Hz, 2H),
2.85 (s, 6H), 1.93 (t, J ) 7.5 Hz, 2H), 1.47 (m, 2H), 1.34 (m, 2H),
1.24 (m, 6H). 13C NMR (DMSO-d6, 75 MHz) δ (ppm) 169.1, 158.0,
149.5, 128.4, 128.0, 112.4, 42.5, 40.4, 39.2, 32.3, 30.0, 28.59, 28.51,
26.3, 25.1. ESI-HRMS calcd for [C18H30N4O3 + Na]+, 373.2215;
found, 373.2198.
4-Dimethylamino-N-[4-(2-mercaptoacetylamino)butyl]ben-
1
zamide (12, Yield 84%). H NMR (CDCl3, 300 MHz) δ (ppm)
7.69 (d, J ) 9.0 Hz, 2H), 7.05 (br s, 1H), 6.66 (d, J ) 9.0 Hz, 2H),
6.31 (br s, 1H), 3.47 (dt, J ) 6.3 and 6.3 Hz, 2H), 3.33 (dt, J )
6.6 and 6.0 Hz, 2H), 3.24 (d, J ) 8.7 Hz, 2H), 3.02 (s, 6H), 1.94
(t, J ) 8.7 Hz, 1H), 1.64 (m, 4H). 13C NMR (CDCl3, 75 MHz) δ
(ppm) 169.7, 167.7, 152.4, 128.4, 121.1, 111.0, 40.1, 39.5, 39.3,
28.3, 27.2, 26.6. ESI-HRMS calcd for [C15H23N3O2S + Na]+,
332.1409; found, 332.1404.
7-[3-(4-Dimethylaminophenyl)-ureido]heptanoic Acid Hy-
1
droxyamide (5, Yield 34%). H NMR (DMSO-d6, 300 MHz) δ
(ppm) 10.34 (br s, 1H), 8.64 (br s, 1H), 7.98 (br s, 1H), 7.17 (d, J
) 9.0 Hz, 2H), 6.65 (d, J ) 9.0 Hz, 2H), 5.91 (t, J ) 5.7 Hz, 1H),
3.03 (dt, J ) 6.6 and 6.0 Hz, 2H), 2.79 (s, 6H), 1.94 (t, J ) 7.5
Hz, 2H), 1.48 (m, 2H), 1.39 (m, 2H), 1.25 (m, 4H). 13C NMR
(DMSO-d6, 75 MHz) δ (ppm) 169.1, 155.6, 145.9, 130.7, 119.6,
113.3, 40.9, 39.0, 32.2, 29.8, 28.4, 26.2, 25.1. ESI-HRMS calcd
for [C16H26N4O3 + H]+, 323.2083; found, 323.2088.
4-Dimethylamino-N-[3-(2-mercaptoacetylamino)propyl]ben-
1
zamide (13, Yield 93%). H NMR (CDCl3, 300 MHz) δ (ppm)
7.74 (d, J ) 9.0 Hz, 2H), 7.37 (br t, 1H), 6.85 (br t, 1H), 6.68 (d,
J ) 9.0 Hz, 2H), 3.48 (dt, J ) 6.3 and 6.0 Hz, 2H), 3.38 (dt, J )
6.3 and 6.0 Hz, 2H), 3.25 (d, J ) 9.0 Hz, 2H), 3.02 (s, 6H), 1.96
(t, J ) 9.0 Hz, 1H), 1.74 (m, 2H). 13C NMR (CDCl3, 75 MHz) δ
(ppm) 170.3, 168.4, 152.4, 128.4, 121.0, 111.1, 40.2, 36.4, 36.0,
29.8, 28.5. ESI-HRMS calcd for [C14H21N3O2S + Na]+, 318.1252;
found, 318.1248.
Synthesis of Hydroxamate through the Mixed Anhydride. To
a solution of acid 23 (0.100 g, 0.26 mmol) in dry THF was added
Et3N (0.18 mL, 1.3 mmol) under nitrogen, and the solution was
stirred for 5 min. The solution was cooled to -15 °C and stirred
for another 5 min. Then iso-butyl chloroformate (67 µL, 0.52 mmol)
was added dropwise, and the mixture was stirred for 15 min. The
solid was filtered off. The filtrate was cooled to 0 °C, and a 50%
aqueous solution (1 mL) of NH2OH was added over 10 min. The
reaction mixture was diluted with EtOAc, washed with saturated
aqueous NH4Cl and brine, and then dried over Na2SO4. The solvent
was removed by rotary evaporation. The crude solid was purified
by HPLC to give compound 10 (0.027 g, 26.4%).
General Synthesis of Hydroxamate by Hydrogenation. A
suspension of the O-benzyl-protected compound (26; 0.031 g, 0.049
mmol) and 10% Pd/C (0.010 g) in methanol (5 mL) was stirred
under a hydrogen atmosphere at rt for 4 h. The catalyst was removed
by filtration through a pad of Celite, and the residue was thoroughly
washed with MeOH. The solvent was evaporated in vacuo, and
the residue was crystallized from methanol/ether, 5:95, to give
hydroxamate 8 (0.008 g, 30%).
Octanedioic Acid {2′-[2-Amino-3-(1H-indol-3-yl)propionyl-
amino]biphenyl-4-yl}amide Hydroxyamide (8). 1H NMR (CD3-
OD, 300 MHz) δ (ppm) 7.74 (d, J ) 7.7 Hz, 1H), 7.67-7.52 (m,
3H), 7.48-7.23 (m, 5H), 7.22-7.09 (m, 4H), 7.08-6.96 (m, 1H),
4.05-3.96 (m, 1H), 3.35-3.25 (m, 1H), 3.15-3.02 (m, 1H), 2.37
(d, J ) 7.0 Hz, 2H), 2.10 (d, J ) 7.0 Hz, 2H), 1.78-1.30 (m, 8H).
13C NMR (DMSO-d6, 75 MHz) δ (ppm) 173.1, 171.3, 167.5, 137.5,
136.4, 136.0, 133.8, 132.7, 129.8, 128.7, 127.2, 126.4, 126.1, 125.2,
Octanedioic Acid Hydroxyamide [4-(3-Nitro-phenyl)thiazol-
2-yl]amide (10). 1H NMR (DMSO-d6, 300 MHz) δ (ppm) 12.3 (s,
1H), 10.3 (s, 1H), 8.72 (s, 1H), 8.67 (br s, 1H), 8.34 (d, J ) 7.7
Hz, 1H), 8.17 (dd, J ) 8.1 and 1.5 Hz, 1H), 7.91 (s, 1H), 7.73 (t,
J ) 7.9 Hz, 1H), 2.45 (t, J ) 7.2 Hz, 2H), 1.94 (t, J ) 7.2 Hz,
2H), 1.60 (br s, 2H), 1.50-1.35 (m, 4H), 1.50 (t, J ) 6.1 Hz, 2H).
13C NMR (DMSO-d6, 100 MHz) δ (ppm) 172.1, 169.5, 158.8,
148.7, 146.7, 136.2, 132.1, 130.8, 122.7, 120.4 110.8, 35.3, 32.6

3060 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Kozikowski et al.
28.7, 25.4, 24.9. ESI-HRMS calcd for [C17H20N4O5S + H]+,
393.1227; found, 393.1227.
inhibited by incubation in Tris-buffered saline with Tween 20
(TBST: 50 mM Tris-HCl, pH 8.0, 0.9% NaCl, and 0.1% Tween
20) containing 5% nonfat dried milk for at least 1.5 h. Primary
antibodies against acetylated histone H4 or total histone H4
(Upstate) were diluted 1:1000 or 1:4000, respectively, in TBST
containing 5% milk and incubated with the membrane for 3 h at
room temperature, followed by incubation with antirabbit horse-
radish peroxidase-conjugated secondary antibodies for 2 h at room
temperature. Acetyl histone H4 and total histone H4 immuno-
reactivity was detected according to the enhanced chemiluminescent
protocol (Amersham Biosciences).
Biological Methods. HDAC Inhibition Assay. The inhibitory
effects of compounds on HDAC activity were determined using a
fluorescence-based assay with electrophoretic separation of substrate
and product carried out using a microfluidic system followed by
quantitation of fluorescence intensity in the substrate and product
peaks. The assays were performed using isolated HDAC isoforms
that had been expressed as 6 × His-tagged fusion proteins in a
baculovirus expression system in Sf9 cells. HDACs 1, 2, 3, 6, and
8 were expressed as full length fusion proteins. The HDAC10 fusion
protein was expressed as a carboxy-terminal deletion of 38 amino
acids (residues 632-669). HDAC3 was coexpressed with a
fragment of the SMRT gene (residues 395-489) to generate
enzymatically active protein. Purified proteins were incubated with
1 µM carboxyfluorescein (FAM)-labeled acetylated peptide sub-
strate and test compound for 17 h at 25 °C in HDAC assay buffer
containing 100 mM HEPES (pH 7.5), 25 mM KCl, 0.1% BSA,
and 0.01% Triton X-100. Reactions were terminated by the addition
of buffer containing 0.078% SDS for a final SDS concentration of
0.05%. Substrate and product were separated electrophoretically
using a Caliper LabChip 3000 system with blue laser excitation
and green fluorescence detection (CCD2). The fluorescence intensity
in the substrate and product peaks was determined using the Well
Analyzer software on the Caliper system. The reactions were
performed in duplicate for each sample. IC50 values were automati-
cally calculated using the IDBS XLFit version 4.2.1 plug-in for
Microsoft Excel and the XLFit 4 Parameter Logistic Model
(sigmoidal dose-response model): (A + ((B - A)/1 + (C/x)D)),
where x is compound concentration, A is the estimated minimum,
B is the estimated maximum of % inhibition, C is the inflection
point, and D is the Hill slope of the sigmoidal curve. The standard
errors of the IC50s were automatically calculated using the IDBS
XLFit version 4.2.1 plug-in for Microsoft Excel and the formula
xf4_FitResultStdError( ).
Acknowledgment. This work was supported in part by gift
money (A.P.K.) and the Goldsmith Foundation (B.L.). We also
wish to thank the HighQ Foundation for providing support for
the HDAC assays.
Supporting Information Available: Methods for preparation
of intermediates 14-27 and the HPLC data of tested compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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