Carbonyl- and sulfur-containing analogs of suberoylanilide hydroxamic acid: Potent inhibition of histone deacetylases

Article abstract of DOI:10.1016/j.bmc.2005.12.047

Suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase, is used in clinical trials for a variety of advanced cancers. Twelve new analogs of SAHA were synthesized and tested as in vitro inhibitors of isolated histone deacetylases (HDACS) and in vivo inhibitors of interferon regulated transcriptional responses (a marker for HDAC activity). The analogs containing an α-mercaptoketone or an α-thioacetoxyketone were more potent than SAHA in both assays.

Full text of DOI:10.1016/j.bmc.2005.12.047

Bioorganic & Medicinal Chemistry 14 (2006) 3320–3329
Carbonyl- and sulfur-containing analogs of suberoylanilide
hydroxamic acid: Potent inhibition of histone deacetylases
Wenxin Gu,a Inna Nusinzon,b,c Ronald D. Smith, Jr.,b Curt M. Horvathb,c
and Richard B. Silvermana,b,d,
*
aDepartment of Chemistry, Northwestern University, Evanston, IL 60208-3113, USA
bDepartment of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA
cDepartment of Medicine, Evanston Northwestern Healthcare, Evanston, IL 60201-1718, USA
dCenter for Drug Discovery and Chemical Biology, Northwestern University, Chicago, IL 60611-3093, USA
Received 20 September 2005; revised 21 December 2005; accepted 22 December 2005
Available online 24 January 2006
Abstract—Suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase, is used in clinical trials for a variety of
advanced cancers. Twelve new analogs of SAHA were synthesized and tested as in vitro inhibitors of isolated histone deacetylases
(HDACS) and in vivo inhibitors of interferon regulated transcriptional responses (a marker for HDAC activity). The analogs
containing an a-mercaptoketone or an a-thioacetoxyketone were more potent than SAHA in both assays.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
results in altered chromatin structure and gene expres-
sion, and has been shown to induce growth arrest, cell
differentiation, and apoptosis of tumor cells.3 Further-
more, they suppress cell proliferation in a variety of
transformed cells in culture and in tumor, bearing ani-
mals, and have shown great promise as new anticancer
drugs.4 Several structurally diverse HDAC inhibitors
now in clinical trials have demonstrated encouraging
antitumor activity in a variety of cancer types, enhanc-
ing the rationale for continued development of new
HDAC inhibitors.5
Epigenetic regulation of gene expression is mediated by
several mechanisms, including DNA methylation,
ATP-dependent chromatin remodeling, and post-
]translational modifications of histones, such as methyl-
ation, phosphorylation, ubiquitinylation, or acetylation.
Dynamic acetylation of e-amino groups of lysine resi-
dues on core histone tails is regulated by two opposing
enzymes, histone acetyltransferase (HAT) and histone
deacetylase (HDAC).1 Transcriptional regulation is con-
trolled by HAT enzymes, which add acetyl groups to ly-
sine residues of histones and non-histone proteins, and
HDACs, which remove these modifications, thereby
mediating chromatin remodeling and gene expression.2
Pharmacological HDAC inhibitors comprise a novel
class of cancer chemotherapeutics in clinical develop-
ment that target HDAC enzymatic activity thereby
inducing hyperacetylation of HDAC substrates, which
Suberoylanilide hydroxamic acid (SAHA) (Fig. 1) is one
of the early HDAC inhibitors,6 which inhibits cell
growth, induces terminal differentiation in tumor cells,7
prevents the formation of malignant tumors in mice,8
and is currently in phase III clinical trials.9 Crystal struc-
tures of a hyperthermophilic bacterium HDAC10 and of
human HDAC811 with SAHA bound show that the
Abbreviations: HDAC, histone deacetylase; SAHA, suberoylanilide
hydroxamic acid; HAT, histone acetyltransferase; ACE, angiotensin-
converting enzyme; IFN, interferon; ISG, IFN-stimulated gene; TSA,
trichostatin A.
Keywords: Histone deacetylase; Inhibitors; a-Mercaptoketone; Sube-
roylanilide hydroxamic acid; SAHA; a-Thioacetoxyketone.
A
B
OH
O
H
N
N
H
O
Suberoylanilide Hydroxamic Acid (SAHA)
*
Corresponding author. Tel.: +1 847 491 5653; fax: +1 847 491
7713; e-mail: Agman@chem.northwestern.edu
Figure 1. Structure of suberoylanilide hydroxamic acid (SAHA).
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.047

W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
3321
hydroxamic acid moiety coordinates to the active-site
zinc ion. Hydroxamates, however, have been found to
exhibit unfavorable pharmacokinetic behavior resulting
from glucuronidation and sulfation,12 and from meta-
bolic hydrolysis,13 all of which result in short in vivo
half-lives of the hydroxamic acid group. Consequently,
new potent inhibitors have been sought. Non-hydroxa-
mate HDAC inhibitors discovered to date, however,
have reduced potency compared to hydroxamates and
have not shown any metabolic advantage.14
12-membered library of SAHA analogs was designed
(Fig. 2). The type of carbonyl moiety (A) was compared
(Fig. 2, 1 vs 2, 3, 6,and 11). To compare the effectiveness
of a sulfur atom versus an oxygen atom, compounds in
which a sulfur or oxygen at the same position were syn-
thesized (Fig. 2, 1 vs 2; 3 vs 4 and 5; 6 vs 7; 12 vs 13). The
group at B was investigated as well (Fig. 2, 4 vs 9; 7 vs 8
and 10). While this research was being carried out, com-
pounds 3, 4, and 9 were reported.17
SAHA was synthesized as previously reported.18 The
syntheses of 2–13 are shown in Schemes 1–6. Although
3, 4, and 9 have already been reported, we accomplished
their syntheses by different routes. The synthesis of 2
was carried out by treating SAHA directly with Lawes-
son’s reagent in THF at room temperature. Surprisingly,
the expected target molecule 2 was obtained as a single
product. The regioselectivity between the amide carbon-
yl and the hydroxamic carbonyl groups presumably de-
rived from the hydroxamate hydroxyl group, which may
form a strong bond with the phosphorus atom in Lawes-
son’s reagent, favoring that carbonyl group (Scheme 2).
Although hydroxamates bind to zinc ions, they are not
necessarily the optimal coordinators. Sulfur ligands are
well known to bind tightly to zinc-containing enzymes.15
This was clearly demonstrated by the invention of cap-
topril, a thiol-containing inhibitor of the zinc-dependent
enzyme angiotensin-converting enzyme (ACE).16
We have designed a series of SAHA analogs as potential
inhibitors of HDACs, in which the hydroxamic acid was
replaced by sulfur-containing moieties, which may have
enhanced binding affinities for zinc. The optimal ligand
from this series can be utilized as the preferred ligand for
other HDAC inhibitor compounds.
Compounds 6, 7, 8, and 10 were synthesized from com-
mon intermediate 12, which was tested as an inhibitor of
HDACs as well. As shown in Scheme 3, compound 14,
which was prepared from 8-bromo-1-octene,19 was
treated with DIC and HOBt, and then coupled with ani-
line to afford 15. The double bond of 15 was oxidized
with mCPBA to give epoxide 12, which was treated with
potassium bromide in acetic acid and water to afford 16.
Compound 16 was oxidized with Jones reagent to
2. Results and discussion
2.1. Chemistry
The hydroxamate group of SAHA was separated into
two moieties: A and B, as shown in Figure 1, and a
S
O
H
H
N
N
OH
N
OH
N
H
H
O
O
SAHA
1
2
O
H
O
S
H
N
N
SH
N
OH
SH
SH
N
H
H
O
O
O
O
O
4
3
5
O
O
H
N
H
N
OH
N
H
6
H
N
O
H
N
SAc
O
8
7
9
O
O
H
N
H
N
SAc
OH
S
N
H
CH3
O
O
O
O
10
H
H
OH
O
S
H
N
N
N
O
12
13
11
Figure 2. SAHA analogs synthesized.

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W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
O
S
H
N
H
N
H
O
OH
N
OH
a
b
NHBoc
O
a
N
H
N
H
BocHN
OH
O
O
O
O
20
21
SAHA (1)
2
H
H
N
N
Scheme 1. Reagents and condition: (a) Lawesson’s reagent, THF, rt,
62%.
SAc
c
NH2
N
H
O
22
9
d
e
HN
O
O
S
H
N
H
H
H
S
OH
N
SH
O
O
O
N
N
N
H
H
Lawesson's reagent
OH
N
H
P
N
O
O
H
3
O
O
4
1
O
MeO
f
HN
O
S
S
S
H
S
H
H
N
H
N
N
OH
SH
N
H
N
H
P
O
O
2
5
O
MeO
Scheme 5. Reagents and conditions: (a) aniline, DIC, HOBt, DIPEA,
DMF, rt, 92%; (b) TFA, methylene chloride, rt, 99%; (c) AcS-
CH2COOH, PyBOP, DIPEA, DMF, rt, 88%; (d) glycolic acid, PyBOP,
DIPEA, DMF/methylene chloride (1:9), rt, 80%; (e) NaOH (aq, 3M)/
acetone (1:1), rt, 80%; (f) Lawesson’s reagent, THF, rt, 57%.
Scheme 2. Possible mechanism for regioselective thionation of SAHA.
a
b
Br
HOOC
14
H
N
O
H
S
H
H
O
N
N
N
a
c
d
O
O
O
O
12
13
15
12
OH
O
H
H
Scheme 6. Reagents and condition: (a) thiourea, methanol, rt, 88%.
N
Br
N
Br
e
O
O
16
17
f
Several different oxidation conditions were attempted,
but all of them failed to give the expected ketone 6
directly from 11.20 Compound 11 was then protected
selectively with TBSCl to afford 18, which was treated
under mild oxidation conditions (KMnO4, CuSO4ÆH2O)21
to generate 19. Deprotection of 19 afforded 6.
g
O
O
H
H
N
SAc
SH
N
S
O
O
8
10
h
O
H
N
O
As shown in Scheme 5, compounds 3, 4, 5, and 9 were
synthesized from the same intermediate (22). Commer-
cially available 20 was coupled with aniline using DIC
and HOBt as the coupling reagents to afford 21, which
was deprotected to generate 22. Compound 22 was cou-
pled with acetyl thiolacetic acid using PyBOP as the cou-
pling reagent to produce 9. Hydrolysis of 9 gave 4.
Compound 4 was treated under the same conditions as
was 2 using Lawesson’s reagent, thereby producing the
corresponding thiocarboxamide compound (5). Com-
pound 22 also was converted to 3 in one step by cou-
pling with glycolic acid. According to earlier work,17
3, 4, and 9 were synthesized in four steps, six steps,
and five steps, respectively. The syntheses described here
are each one step shorter. Scheme 6 shows the conver-
sion of 12 to 13 by treatment with thiourea at room
temperature.
7
Scheme 3. Reagents and conditions: (a) two steps, 60%, Ref. 19; (b)
aniline, DIC, HOBt, DIPEA, DMF, rt, 92%; (c) mCPBA, methylene
chloride, rt, 85%; (d) KBr, AcOH, H2O, rt, 71%; (e) Jones reagent, rt,
68%; (f) KSAc, DMF, rt, 86%; (g) KSCH3, DMF, rt, 80%; (h) NaOH
(aq, 3M)/acetone (1:1), rt, 83%.
OH
OH
H
N
H
N
OTBS
OH
a
b
15
O
O
11
18
O
O
H
N
H
OTBS
N
OH
d
c
O
O
19
6
Scheme 4. Reagents and conditions: (a) OsO4, NMO, tBuOH/H2O
(9:1), 0 ꢁC to rt, 77%; (b) TBSCl, imidazole, DMF, rt, 90%; (c)
KMnO4, CuSO4ÆH2O, benzene, rt, 52%; (d) TBAF, THF, rt, 89%.
2.2. Enzyme inhibition results
generate a-bromoketone 17. Potassium thioacetate was
employed as the nucleophile to give 8, which was then
hydrolyzed to generate 7. Compound 17 was treated
with sodium thiomethoxide in DMF at room tempera-
ture to afford 10.
SAHA and its analogs were tested in vitro with commer-
cially available HDAC assay kits, one for HeLa nuclear
extract containing a mixture of HDACs and one with
purified human recombinant HDAC8 (Table 1).22
Compounds 11 and 6 were synthesized via 15. As shown
in Scheme 4, 15 was oxidized with OsO4 to generate 11.
It is apparent from Table 1 that 7 and 8 are more potent
in vitro inhibitors of HDACs and of HDAC8 than is

W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
3323
Table 1. IC50 valuea of analogs of SAHA against HDACs and
HDAC8
possible lability of the thiol group, modifications were
made to it. As discussed above, if the thiol group of 4
is converted to a thioacetoxyl group (9), the potency
decreases by a factor of about 8, but if the same modifi-
cation is made to the corresponding a-mercaptoketone 7
to give the a-thioacetoxyketone 8, there is an almost 2-
fold increase in potency. It appears that the ketone car-
bonyl is synergistic in binding with the a-thioacetoxyl
group. S-Methylation of a-mercaptoketone 7 to give a-
methylthioketone 10 results in a >250-fold decrease in
potency.
Entry
Inhibitors
IC50 (lM)
(HDACs)
(HDAC8)
1
2
SAHA (1)
0.37
>10
>40
2.44
13.3
>40
0.15
0.081
20.1
>40
>40
>40
>40
0.82
>10
>40
3.89
>40
>40
0.69
0.19
>40
>40
>40
>40
>40
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
2.3. Effect of HDAC inhibitors on interferon transcrip-
tional responses
10
11
12
13
10
11
12
13
Further biological tests were made in cell culture.
Although HDAC activity has been conventionally
associated with transcriptional repression, recent
findings demonstrated that HDACs can function as
transcriptional coactivators in the interferon (IFN) sys-
tem.25 In response to IFN, the IFN-stimulated gene
(ISG) mRNAs rapidly and transiently accumulate in
cells, but HDAC inhibitors such as trichostatin A
(TSA), sodium butyrate, or SAHA prevent ISG
transcription.23a,b
a Values are means of at least three experiments.
SAHA (1); others have reported a similar observation in
other series of compounds.23 The similarity in potencies
of 7 and 8 is unexpected because thiols are strong
ligands to Zn2+, so 7 should have been more effective
than 8. Given the similar IC50 values for these two
inhibitors, it was thought that possibly 8 was hydrolyzed
to 7. However, mass spectrometry after inhibition
showed that 8 was intact and no 7 was formed. These
results are different from those with 4 and 9, which hold
the same relationship as 7 and 8, respectively. In this
case, thioacetate 9 is considerably less potent than
thiol 4. Additional studies into these differences are
underway.
The ability of SAHA and its analogs to inhibit IFN
transcriptional responses was tested. Human fibrosar-
coma cell line 2fTGH was used in an IFN-dependent
luciferase reporter gene assay in the absence or pres-
ence of HDAC inhibitor compounds (Fig. 3). In this
assay, both the IFN-responsive luciferase construct
and a control Renilla luciferase were transfected into
cells, and cells were stimulated with IFNa for 6 h prior
to analysis. Compounds 7 and 8 were able to prevent
IFN-inducible reporter gene activity, in agreement
with their abilities to function as general HDAC
inhibitors.
In general, the potency of the compounds toward the
mixture of HDACs is greater than that for HDAC8. A
change in the hydroxamate to an a-hydroxyamide (3)
or to an a-hydroxyketone (6) resulted in a sharp de-
crease in potency (>100-fold for HDACs or HDAC8);
conversion to the corresponding thiohydroxamate (2)
decreased the potency by >25 times. Reduction of the
ketone carbonyl to an alcohol (11) also gave poor poten-
cy. Because of the superior inhibition of gelatinases (ma-
trix metalloproteinases) by a thiirane relative to the
corresponding oxirane,24 thiirane 13 was prepared and
compared to oxirane 12. Both were found to be weak
inhibitors of HDACs, too weak to make an accurate
comparison.
3. Conclusions
An a-mercaptoketone and a-thioacetoxylketone are im-
proved zinc-binding ligands relative to hydroxamate in
The effect of modifying the B moiety (the a-hydroxyl
group) also was dramatic. In the reverse amide series,
replacement of the a-hydroxyl group of 3 with an a-mer-
capto group (4) resulted in a >15-fold increase in poten-
cy; conversion of the amide to a thioamide along with
substitution of the a-hydroxyl group by an a-mercapto
group (5) lowered the potency 5-6 fold relative to 4. It
is apparent that a thiol group is beneficial for binding,
but conversion of the carbonyl oxygen to sulfur is detri-
mental to binding. Another example of this phenome-
non is the conversion of the a-hydroxyl group of
ketone 6 to an a-mercapto group (7), which results in
about a 250-fold increase in potency. Because of the
Figure 3. Inhibition of IFN-induced reporter gene expression by
SAHA derivatives. Luciferase assays were performed in human
fibrosarcoma 2fTGH cells using an IFN-dependent luciferase reporter
and Renilla luciferase for normalization. Cells were treated with IFNa
and simultaneous HDAC inhibitors (HDI) as shown.

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W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
this study and should be valuable groups to attach to
molecules that target not only histone deacetylase, but
other zinc-dependent enzymes.
4.3. 6-(2-Hydroxyacetylamino)-N-phenylhexanamide (3)
To a stirred solution of glycolic acid (114 mg, 1.5 mmol)
in a mixed solvent system of anhydrous DMF and anhy-
drous methylene chloride (1:9, 5 mL) were added
PyBOP (781 mg, 1.5 mmol) and DIPEA (0.78 mL,
4.5 mmol) at room temperature. Then 22 hydrochloric
acid salt (365 mg, 1.5 mmol) was added to the reaction
mixture in one portion. The reaction mixture was stirred
overnight. The solvent was removed under vacuum, and
the residue was dissolved in ethyl acetate. The organic
phase was washed with 0.5 N HCl solution, saturated
NaHCO3, and brine. The organic phase was then dried
over Na2SO4 and evaporated in vacuo. The crude
product was recrystallized from EtOH/hexanes to give
4. Experimental
Conventional organic solvents were purchased from
Fisher. All of the reagents were purchased from Aldrich
Chemical Co. and were used without further purification
unless stated otherwise. Methylene chloride was distilled
under N2 from calcium hydride. Flash chromatography
was performed with Merck silica gel (230–400 mesh).
TLC plates (silica gel 60-F254) were purchased from
VWR Scientific. All 1H NMR spectra were recorded
on Varian Gemini 300 MHz, Mercury 400, or Inova
500 spectrometers (75, 100, or 125 MHz for 13C NMR
spectra). Chemical shifts (d) are reported downfield from
tetramethylsilane (Me4Si) in parts per million (ppm).
Compounds were visualized with a ninhydrin spray re-
agent or a UV/vis lamp. Mass spectra were recorded
either on a VG Instrument VG70-250SE high-resolution
mass spectrometer (ESI) or on a Micromass Quattro II
spectrometer (APCI).
2
(317 mg, 80%). 1H NMR (CD3OD):
d 1.42
(t, J = 8.0 Hz, 2H), 1.59 (p, J = 7.1 Hz, 2H), 1.73
(p, J = 7.2 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 3.26
(t, J = 7.2 Hz, 2H), 3.95 (s, 2H), 7.08 (t, J = 7.6 Hz,
1H), 7.29 (t, J = 8.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H);
13C NMR (CD3OD): d 26.7, 27.7, 30.4, 37.9, 39.9,
48.5, 62.7, 121.4, 125.3, 129.9, 140.0, 174.6, 175.3. EI-
MS (M+): 264.2, 233.1, 135.1, 93.1. HRMS Calcd for
C14H20N2O3 264.1468, found 264.1464. Anal. Calcd
for C14H20N2O3Æ0.2H2O: C, 62.76; H, 7.67; N, 10.46.
Found: C, 62.89; H, 7.62; N, 10.31.
4.1. Suberoylanilide hydroxamic acid (1)
Compound 1 (SAHA) was synthesized by a literature
method.ꢀ 1H NMR (DMSO-d6): d 1.27 (s, 4H), 1.48
(t, J = 6.0 Hz, 2H), 1.56 (br s, 2H), 1.94 (t, J = 7.2 Hz,
2H), 2.28 (t, J = 7.2 Hz, 2H), 7.01 (t, J = 7.2 Hz, 1H),
7.27 (t, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 8.67
(s, 1H), 9.86 (s, 1H), 10.34 (s, 1H); 13C NMR
(DMSO-d6): d 25.1, 28.4, 32.3, 36.4, 38.9, 119.0, 122.9,
128.6, 139.4, 169.1, 171.2. EI-MS (M+): 264.2, 248.2,
230.2, 190.2, 148.1, 135.1, 93.1. HRMS Calcd for
C14H20N2O3 264.1468, found 264.1466. Anal. Calcd
for C14H20N2O3Æ0.05H2O: C, 63.40; H, 7.64; N, 10.56.
Found: C, 63.14; H, 7.67; N, 10.27.
4.4. 6-(2-Mercaptoacetylamino)-N-phenylhexanamide (4)
Reagent grade acetone (5 mL) and 3 N NaOH (5 mL) in a
25 mL flame-dried flask were deoxygenated with argon
for 20 min at 0 ꢁC. Under positive Ar pressure, the crude
compound 9 (32 mg, 0.1 mmol) was added as a solid to the
flask. This reaction mixture was stirred under Ar for 6 h.
A solution of 1 M HCl was added to neutralize the solu-
tion. The aqueous fraction was extracted three times with
ethyl acetate. The combined extracts were washed with
brine (10 mL), dried over Na2SO4, and evaporated in vac-
uo, and the residue was purified by flash chromatography
eluting with EtOAc followed by recrystallization from
1
4.2. Thiosuberoylanilide hydroxamic acid (2)
95% EtOH to give 4 (22 mg, 80%) as a white solid: H
NMR (CD3OD): d 1.44 (d, J = 7.1 Hz, 2H), 1.57
(p, J = 7.2 Hz, 2H), 1.72 (p, J = 7.0 Hz, 2H), 2.38
(t, J = 7.2 Hz, 2H), 3.12 (s, 2H), 3.20 (t, J = 6.6 Hz, 2H),
7.08 (t, J = 7.2 Hz, 1H), 7.29 (t, J = 7.6 Hz, 2H), 7.54 (d,
J = 7.6 Hz, 2H), 8.11 (s, 1H); 13C NMR (CDCl3): d
26.7, 27.6, 28.4, 30.2, 37.9, 40.7, 121.4, 125.2, 129.9,
140.0, 173.4, 174.6. EI-MS (M+): 280.1, 248.2, 207.2,
188.1, 178.1, 135.1, 114.1, 93.1. HRMS Calcd for
C14H20N2O2S 280.1240, found 280.1236. Anal. Calcd
for C14H20N2O2SÆ0.2H2O: C, 59.21; H, 7.24; N, 9.86; S,
11.29. Found: C, 59.23; H, 6.96; N, 9.72; S, 11.16.
Compound 1 (SAHA) (0.26 g, 1.00 mmol) was dissolved
in anhydrous THF (5 mL) followed by the addition of
2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-
2,4-disulfide (Lawesson’s reagent, 0.20 g, 0.50 mmol)
portionwise. The reaction mixture was covered with alu-
minum foil and stirred for 4 h at room temperature. The
solvent was removed under vacuum, and the residue
was purified by flash chromatography eluting with hex-
ane/EtOAc (1:1) to give the desired thiosuberoylanilide
1
hydroxamic acid 2 (174 mg, 62%). H NMR (CD3OD):
d 1.40 (s, 4H), 1.70 (m, 4H), 2.36 (t, J = 7.2 Hz, 2H),
2.58 (t, J = 7.2 Hz, 2H), 7.06 (t, J = 7.2 Hz, 1H), 7.28 (t,
J = 8.0 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H); CI-HRMS
Calcd for C14H20N2O2S 281.1318, found 281.1317. Anal.
Calcd for C14H20N2O2SÆ0.2H2O: C, 59.21; H, 7.24; N,
9.86; S, 11.29. Found: C, 59.23; H, 6.96; N, 9.72; S, 11.16.
4.5. 6-(2-Mercapto[thioacetylamino])-N-phenylhexan-
amide (5)
Compound 4 (28 mg, 0.1 mmol) was dissolved in
anhydrous THF (2 mL) followed by the portion-
wise addition of 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,
4-diphosphetane-2,4-disulfide (Lawesson’s reagent,
20 g, 0.05 mmol). The reaction mixture was covered with
aluminum foil and stirred for 4 h at room temperature.
The solvent was removed under vacuum, and the residue
ꢀ
Stowell, J. C.; Huot, R. I.; Voast L. V. The synthesis of N-hydroxy-
N0-phenyloctanediamide and its inhibitory effect on proliferation of
AXC rat prostate cancer cells. J. Med. Chem. 1995, 38, 1411–1413.

W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
3325
was purified by flash chromatography eluting with
hexane/EtOAc (1:1) to give 5 (16.9 mg, 57%). 1H
NMR (CD3OD): d 1.42 (p, J = 7.0 Hz, 2H), 1.56
(p, J = 7.0 Hz, 2H), 1.71 (p, J = 7.0 Hz, 2H), 2.36
(t, J = 7.0 Hz, 2H), 3.22 (t, J = 7.0 Hz, 2H), 3.29
(s, 2H), 6.92 (d, 9.0 Hz), 7.06 (t, J = 7.0 Hz, 1H), 7.27
(t, J = 7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.86
(m, 1H). ESI-MS (M++1): 297.2, 207.1, 188.1, 190.1,
135.1, 114.1, 93.1.
(10 mL), dried over Na2SO4, and evaporated in vacuo,
and the residue was purified by flash chromatography
eluting with hexane/EtOAc (1:1) to give 8 (276 mg,
1
86%) as a white solid: H NMR (CDCl3): d 1.36 (m,
4H), 1.62 (p, J = 7.0 Hz, 2H), 1.73 (t, J = 7.0 Hz,
2H), 2.34 (t, J = 7.0 Hz, 2H), 2.39 (s, 3H), 2.56 (t,
J = 7.0 Hz, 2H), 3.74 (s, 2H), 7.09 (t, J = 7.0 Hz, 1H),
7.31 (t, J = 7.5 Hz, 2H), 7.43 (s, 1H), 7.52 (d,
J = 7.5 Hz, 2H); 13C NMR (CDCl3): d 23.7, 25.5,
28.8, 29.0, 30.4, 37.7, 39.3, 41.8, 120.0, 124.4, 129.2,
138.3, 171.6, 194.9, 204.3. EI-MS (M+): 321.2, 279.2,
232.0, 135.1, 93.1. HRMS Calcd for C17H23NO3S
321.1393, found 321.1392. Anal. Calcd for
C17H23NO3S: C, 63.52; H, 7.21; N, 4.36; S, 9.98.
Found: C, 63.46; H, 7.33; N, 4.37; S, 9.93.
4.6. 9-Hydroxy-8-oxo-N-phenylnonanamide (6)
To a solution of 19 (10 mg, 0.027 mmol) in 0.5 mL THF
was added TBAF (0.05 mL, 1.0 M in THF, 0.05 mmol)
at room temperature. The reaction was monitored by
TLC until it was complete. The reaction mixture was then
evaporated in vacuo, and the residue was purified by flash
chromatography eluting with hexane/EtOAc (1:1) to give
6 (6.3 mg, 89%). 1H NMR (CDCl3): d 1.39 (br s, 4H), 1.67
(p, J = 7.2 Hz, 2H), 1.75 (t, J = 7.2 Hz, 2H), 2.36 (t,
J = 7.2 Hz, 2H), 2.43 (t, J = 8.0 Hz, 2H), 3.09 (br s, 1H),
4.24 (d, J = 3.6 Hz), 7.11 (t, J = 7.2 Hz, 1H), 7.33 (t,
J = 8.0 Hz, 2H), 7.43 (s, 1H), 7.52 (d, J = 8.0 Hz, 2H);
13C NMR (CDCl3): d 23.6, 25.4, 27.0, 29.0, 37.8, 38.5,
68.3, 119.9, 124.5, 129.3, 171.3, 210.0. EI-MS (M+):
263.2, 232.0, 135.1, 93.1. HRMS Calcd for C15H21NO3
263.1516, found 263.1513.
4.9. 6-(2-Acetylthio[acetylamino])-N-phenylhexanamide
(9)
To a stirred solution of (acetylthio)acetic acid (200 mg,
1.5 mmol) in anhydrous DMF (5 mL) were added
PyBOP (781 mg, 1.5 mmol) and DIPEA (0.78 mL,
4.5 mmol) at room temperature. Then 22 hydrochloric
acid salt (365 mg, 1.5 mmol) was added to the reaction
mixture in one portion. The reaction mixture was stirred
overnight. The solvent was removed under vacuum, and
the residue was dissolved in ethyl acetate. The organic
phase was washed with 0.5 N HCl solution, saturated
NaHCO3 and brine. The organic phase was then dried
over Na2SO4, and evaporated in vacuo. The crude
product was recrystallized from chloroform to give
4.7. 9-Mercapto-8-oxo-N-phenylnonanamide (7)
Reagent grade acetone (5 mL) and 3 N NaOH (5 mL) in
a 25 mL flame-dried flask were deoxygenated with argon
for 20 min at 0 ꢁC. Under positive Ar pressure, crude 8
(32 mg, 0.1 mmol) was added to the flask as a solid. This
reaction mixture was stirred under Ar for 6 h. A solution
of 1 M HCl was added to neutralize the solution. The
aqueous fraction was extracted three times with ethyl
acetate. The combined extracts were washed with brine
(10 mL), dried over Na2SO4, and evaporated in vacuo,
and the residue was purified by flash chromatography
eluting with hexane/EtOAc (1:2) and recrystallized from
9
(425 mg, 88%). 1H NMR (CDCl3):
d
1.35
(t, J = 7.0 Hz, 2H), 1.50 (t, J = 7.0 Hz, 2H), 1.72
(t, J = 7.0 Hz, 2H), 2.35 (t, J = 7.0 Hz, 2H), 2.37 (s,
3H), 3.21 (t, J = 6.5 Hz, 2H), 3.53 (s, 2H), 6.50 (br s,
1H), 7.07 (t, J = 7.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 2H),
7.56 (d, J = 8.0 Hz, 2H), 8.14 (s, 1H); 13C NMR
(CDCl3): d 26.4, 29.2, 30.4, 33.2, 37.4, 39.6, 46.5,
120.0, 124.2, 129.0, 138.4, 168.4, 171.7, 196.0. EI-MS
(M+): 322.4, 280.3, 188.3, 114.2, 93.2. HRMS Calcd
for C16H22N2O3S 322.1352, found 322.1350.
1
95% EtOH to give 7 (23 mg, 83%) as a white solid: H
NMR (CDCl3): d 1.37 (m, 4H), 1.62 (p, J = 7.2 Hz,
2H), 1.73 (t, J = 7.0 Hz, 2H), 2.35 (t, J = 7.2 Hz, 2H),
2.57 (t, J = 7.2 Hz, 2H), 3.34 (d, J = 7.2 Hz, 2H), 7.10
(t, J = 7.2 Hz, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.37 (s,
1H), 7.52 (d, J = 7.2 Hz, 2H); 13C NMR (CDCl3): d
23.9, 25.5, 28.9, 29.1, 34.5, 37.8, 41.0, 119.9, 124.3,
129.1, 138.1, 171.4, 205.7. EI-MS (M+): 279.2, 247.2,
232.0, 190.2, 135.1, 94.1. HRMS Calcd for C15H21NO2S
279.1288, found 279.1283. Anal. Calcd for
C15H21NO2S: C, 64.48; H, 7.58; N, 5.01; S, 11.47.
Found: C, 64.39; H, 7.53; N, 4.77; S, 11.17.
4.10. 9-Methylthio-8-oxo-N-phenylnonanamide (10)
To a solution of bromomethylketone 17 (32.6 mg,
0.1 mmol) in 2 mL DMF was added sodium thiometh-
oxide (7.7 mg, 0.11 mmol). The solution was stirred
overnight at room temperature and then diluted with
10 mL of water. The aqueous fraction was extracted
three times with ethyl acetate. The combined extracts
were washed with brine (10 mL), dried over Na2SO4,
and evaporated in vacuo, and the residue was purified
by flash chromatography eluting with hexane/EtOAc
(2:1) to give 10 (23.4 mg, 80%) as a white solid. 1H
NMR (CDCl3): d 1.38 (m, 4H), 1.63 (t, J = 6.0 Hz,
2H), 1.75 (d, J = 8.0 Hz, 2H), 2.07 (s, 3H), 2.36 (t,
J = 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 3.17 (s, 2H),
7.10 (m, 1H), 7.25 (br s, 1H), 7.32 (t, J = 8.0 Hz, 2H),
7.52 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3): d 15.9,
23.8, 25.5, 28.9, 29.0, 37.8, 40.2, 43.1, 119.9, 124.4,
129.2, 138.2, 171.4, 205.9. EI-MS (M+): 293.2, 232.1,
200.1, 135.1, 93.1. HRMS Calcd for C16H23NO2S
293.1444, found 293.1443. Anal. Calcd for
4.8. 9-Acetylthio-8-oxo-N-phenylnonanamide (8)
To a solution of bromomethylketone 17 (326 mg,
1 mmol) in 5 mL DMF was added potassium thioace-
tate (240 mg, 2.1 mmol). The cloudy orange-yellow
solution was stirred overnight at room temperature
and then diluted with 10 mL of water. The aqueous
fraction was extracted three times with ethyl acetate.
The combined extracts were washed with brine

3326
W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
C16H23NO2S: C, 65.49; H, 7.90; N, 4.77; S, 10.93.
Found: C, 65.27; H, 7.73; N, 4.67; S, 10.73.
temperature. Most of the DMF was evaporated with a
high vacuum rotary evaporator, and the residue was dis-
solved to 500 mL of ethyl acetate. The organic phase
was washed with saturated NH4Cl solution (3· 50 mL)
followed by brine (3· 50 mL) and dried over anhydrous
Na2SO4. The solvent was removed under vacuum, and
the residue was purified by flash chromatography elut-
ing with hexane/EtOAc (5:1) to give 15 (4.27 g, 92%)
4.11. 8,9-Dihydroxy-N-phenylnonanamide (11)
To a solution of 15 (1.0 g, 4.33 mmol) in tert-butanol
(18 mL) and water (2 mL) at 0 ꢁC were added N-meth-
ylmorpholine N-oxide (NMO) (608 mg, 5.20 mmol)
and osmium tetroxide (33 mg, 0.13 mmol). The mixture
was stirred at 0 ꢁC for 1 h and then overnight at room
temperature. Aqueous sodium thiosulfate was added,
and the mixture was stirred at room temperature for
another 0.5 h. The aqueous fraction was extracted three
times with ethyl acetate. The combined extracts were
washed with brine (10 mL), dried over Na2SO4, and
evaporated in vacuo, and the residue was purified by
flash chromatography eluting with hexane/EtOAc (1:1)
1
as a white solid. H NMR (CDCl3): d 1.28 (m, 6H),
1.65 (t, J = 7.0 Hz, 2H), 1.98 (t, J = 7.0 Hz, 2H), 2.34
(t, J = 7.5 Hz, 2H), 4.90-4.98 (2H), 5.74 (m, 1H), 7.00
(t, J = 7.5 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 9.41 (s,
1H); 13C NMR (CDCl3): d 25.6, 28.5, 28.6, 28.9, 33.5,
37.1, 114.0, 120.2, 123.7, 128.3, 138.2, 138.3, 172.6.
CI-MS (M++1): 232.1, 139.1, 93.1. HRMS Calcd for
C15H22NO 232.1696, found 232.1695. Anal. Calcd for
C15H21NO: C, 77.88; H, 9.15; N, 6.05. Found: C,
77.59; H, 9.27; N, 5.92.
1
to give 11 (884 mg, 77%) as a white solid: H NMR
(CDCl3): d 1.26 (m, 8H), 1.58 (m, 2H), 2.23 (t,
J = 7.2 Hz, 2H), 3.27 (dd, J = 8.0 Hz, 11.2 Hz, 1H),
3.43 (dd, J = 4.8 Hz, 12.8 Hz, 1H), 3.50 (br d,
J = 3.2 Hz, 1H), 6.96 (t, J = 7.2 Hz, 1H), 7.18 (t,
J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 8.42 (m,
1H); EI-MS (M+): 265.2, 234.2, 135.1, 93.1. HRMS
Calcd for C15H23NO3 265.1672, found 265.1669. Anal.
Calcd for C15H23NO3: C, 67.90; H, 8.74; N, 5.28.
Found: C, 67.92; H, 8.66; N, 5.26.
4.14. 9-Bromo-8-hydroxy-N-phenylnonanamide (16)
A solution of 12 (1.0 g, 4.0 mmol) in peroxide-free THF
(5 mL) was added dropwise to a stirred mixture of
AcOH/THF/saturated aq KBr (2/1/1, 40 mL) at 0 ꢁC un-
der argon. After 10 h, the reaction mixture was reduced in
vacuo to one-quarter volume, diluted with H2O (50 mL),
and extracted three times with Et2O. The combined ethe-
real extracts were washed with 10% aqueous NaHCO3
solution, H2O, brine, dried over Na2SO4, and evaporated
to dryness in vacuo, and the residue was used for the next
step directly without further purification.
4.12. N-Phenyl-8-nonenamide oxide (12)
To a stirred solution of 15 (2.31 g, 10 mmol) in 100 mL
of methylene chloride was added 77% mCPBA (3.36 g,
15 mmol) at 0 ꢁC and then the reaction mixture was stir-
red overnight at 0 ꢁC. The reaction was monitored by
TLC and was quenched by the addition of a saturated
NaHCO3 solution. The mixture was extracted with ethyl
acetate (3· 100 mL), and the combined organic extracts
were washed with brine and dried over anhydrous
Na2SO4. The solvent was removed under vacuum, and
the residue was purified by flash chromatography elut-
ing with hexane/EtOAc (3:1) to give 12 (2.10 g, 85%)
4.15. 9-Bromo-8-oxo-N-phenylnonanamide (17)
The above mixture of bromohydrins 16 (900 mg,
2.74 mmol) in diethyl ether (4 mL) was added dropwise
to a 0 ꢁC solution of 30 mL of Jones reagent in 15 mL of
diethyl ether. After 5 h, the dark green mixture was
diluted with 50 mL of water, and the aqueous fraction
was extracted three times with Et2O. The combined
ethereal extracts were washed with brine (10 mL), dried
over Na2SO4, and evaporated in vacuo, and the residue
was purified by flash chromatography eluting with hex-
ane/EtOAc (2:1) to give 17 (607 mg, 68%) as a white
solid: 1H NMR (CDCl3): d 1.36 (m, 4H), 1.62
(p, J = 7.0 Hz, 2H), 1.73 (p, J = 7.0 Hz, 2H), 2.35
(t, J = 7.5 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H), 3.88 (s,
2H), 7.10 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 8.0 Hz, 2H),
7.52 (d, J = 7.0 Hz, 2H); 13C NMR (CDCl3): d 23.7,
25.5, 28.8, 29.0, 34.5, 37.4, 39.9, 120.0, 124.4, 129.2,
138.1, 171.4, 202.4. EI-MS (M+): 327.1, 232.0, 135.1,
93.1. HRMS Calcd for C15H20NO2Br 325.0672 and
327.0651, found 325.0674 and 327.0649.
1
as a white solid. H NMR (CDCl3): d 1.33 (m, 4H),
1.43 (m, 2H), 1.49 (d, J = 4.8 Hz, 2H), 1.69 (br s, 2H),
2.33 (t, J = 7.2 Hz, 2H), 2.45 (dd, J = 2.4 Hz, 4.8 Hz,
1H), 2.73 (d, J = 4.4 Hz, 1H), 2.89 (br s, 1H), 7.06 (t,
J = 6.8 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.55 (d,
J = 8.0 Hz, 2H), 8.42 (m, 1H); 13C NMR (CDCl3): d
25.6, 25.9, 28.5, 29.2, 32.4, 37.4, 47.1, 52.5, 120.0,
123.9, 128.7, 138.3, 172.0. EI-MS (M+): 247.2, 135.1,
93.1. HRMS Calcd for C15H21NO2 247.1567, found
247.1566. Anal. Calcd for C15H21NO2: C, 72.84; H,
8.56; N, 5.66. Found: C, 72.77; H, 8.62; N, 5.62.
4.13. N-Phenyl-8-nonenamide (15)
4.16. 9-(tert-Butyldimethylsilanyloxy)-8-hydroxy-N-
phenylnonanamide (18)
To a solution of the 8-nonenoic acid19 (14, 3.2 g,
0.02 mol) in dry DMF (100 mL) was carefully added
DIC (3.41 mL, 0.022 mol) at 0 ꢁC followed by HOBt
(2.97 g, 0.022 mol) and DIPEA (11.5 mL, 0.06 mol).
After being stirred for 10 min a solution of aniline
(2.01 mL, 0.022 mol) in 10 mL DMF was added drop-
wise to the reaction mixture at 0 ꢁC. Stirring was contin-
ued for 1 h at 0 ꢁC and then overnight at room
To a solution of 11 (265 mg, 1.0 mmol) in anhydrous
DMF (2 mL) at 0 ꢁC were added imidazole (134 mg,
2.0 mmol) and then tert-butyldimethylsilyl chloride
(165 mg, 1.1 mmol). The mixture was stirred overnight
at room temperature and then aqueous sodium thiosul-
fate was added, and the mixture was stirred at room

W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
3327
temperature for another 0.5 h. The aqueous fraction was
extracted three times with ethyl acetate. The combined
extracts were washed with brine (10 mL), dried over
Na2SO4, and evaporated in vacuo, and the residue was
purified by flash chromatography eluting with hexane/
EtOAc (1:1) to give 11 (884 mg, 77%) as a white solid.
1H NMR (CDCl3): d 0.08 (s, 6H), 0.91 (s, 9H), 1.36
(m, 8H), 1.72 (m, 2H), 2.34 (t, J = 7.2 Hz, 2H), 3.38 (t,
J = 8.4 Hz, 1H), 3.62 (d, J = 10.8 Hz, 2H), 7.09 (t,
J = 7.2 Hz, 1H), 7.31 (t, J = 8.0 Hz, 2H), 7.47 (s, 1H),
7.53 (d, J = 7.6 Hz, 2H); 13C NMR (CDCl3): d ꢀ5.3,
18.5, 25.7, 25.8, 26.1, 26.2, 29.3, 29.6, 32.9, 37.9, 67.4,
72.0, 119.9, 124.3, 129.1, 138.2, 171.6. EI-MS: 322.4,
306.4, 230.5, 93.2. HRMS Calcd for C21H38NO3Si
380.2615, found 380.2612.
124.3, 129.1, 138.3, 156.3, 171.6. EI-MS (M+): 306.4,
250.3, 233.2, 187.4, 135.2, 93.2. HRMS Calcd for
C17H26N2O3 306.1938, found 306.1940.
4.19. 6-Amino-N-phenylhexanamide (22)
To a solution of 21 (3.06 g, 10 mmol) in 50 mL of meth-
ylene chloride was added 50 mL TFA in one portion at
room temperature. The reaction mixture was stirred for
another 1 h and then was evaporated under vacuum to
remove the solvent. The crude 22 TFA salt was then dis-
solved in 10 mL of ethanol containing 0.5 mL of HCl
acid. The mixture was evaporated under vacuum again
to form 22 HCl salt (2.41 g, 99%), which was used to
make 3 and 9 without further purification.
4.17. 9-(tert-Butyldimethylsilanyloxy)-8-oxo-N-phenyl-
nonanamide (19)
4.20. 7-Oxiranyl-heptanoic acid phenylamide (13)
To a stirred solution of 12 (100 mg, 0.40 mmol) in 2 mL
of methanol was added thiourea (62 mg, 0.80 mmol) at
room temperature then the reaction mixture was stirred
overnight. The solvent was evaporated, the resulting res-
idue partitioned between ethyl acetate (50 mL) and
water (10 mL), and the organic phase washed with brine
and dried over anhydrous Na2SO4. The solvent was re-
moved under vacuum, and the residue was purified by
flash chromatography eluting with hexane/EtOAc (5:1)
to give 13 (92 mg, 88%) as a white solid: 1H NMR
(CDCl3): d 1.36 (m, 6H), 1.50 (m, 2H), 1.80 (m, 2H),
2.13 (d, J = 6.0 Hz, 1H), 2.34 (t, J = 7.2 Hz, 2H), 2.49
(d, J = 6.4 Hz, 1H), 2.85 (m, 1H), 7.05 (t, J = 6.4 Hz,
1H), 7.26 (t, J = 7.2 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H),
7.75 (s, 1H); 13C NMR (CDCl3): d 25.6, 26.1, 29.1,
29.3, 36.2, 36.6, 37.7, 120.0, 124.3, 129.0, 138.2, 171.8.
HRMS (ESI) calcd for C15H22NOS 264.1422, found
264.1417. Anal. Calcd for C15H21NOS: C, 68.40; H,
8.04; N, 5.32; S, 12.17. Found: C, 68.14; H, 8.10; N,
5.21; S, 12.05.
A mixture of 1.0 g of KMnO4 and 0.5 g of CuSO4Æ5H2O
was ground to a fine powder and added to 18 (38 mg,
0.1 mmol) in 3 mL of benzene at room temperature.
The reaction mixture was then heated to 70 ꢁC for two
days. The solid was filtered and washed with benzene.
The combined filtration was evaporated in vacuo, and
the residue was purified by flash chromatography elut-
ing with hexane/EtOAc (1:1) to give 19 (20 mg, 52%).
1H NMR (CDCl3): d 0.10 (s, 6H), 0.93 (s, 9H), 1.38
(br s, 4H), 1.60 (m, 2H), 1.75 (t, J = 7.2 Hz, 2H), 2.35
(t, J = 7.6 Hz, 2H), 2.50 (t, J = 7.6 Hz, 2H), 4.17 (s,
2H), 7.11 (t, J = 7.6 Hz, 1H), 7.22 (br s, 1H), 7.32 (t,
J = 8.0 Hz, 2H), 7.43 (s, 1H), 7.53 (d, J = 7.6 Hz, 2H);
13C NMR (CDCl3): d ꢀ5.3, 18.5, 23.2, 25.5, 26.0, 29.0,
29.1, 37.8, 38.4, 69.5, 119.9, 124.4, 129.2, 138.2, 171.5,
211.4. HRMS Calcd for C21H36NO3Si 378.2459, found
378.2468.
4.18. 6-(tert-Butyloxycarbonylamino)-N-phenylhexan-
amide (21)
4.21. HDACs and HDAC8 enzyme assays
To a solution of the 6-(tert-butoxycarbonylamino)hexa-
noic acid (20) (4.6 g, 0.02 mol) in dry DMF (200 mL)
was carefully added DIC (3.41 mL, 0.022 mol) at 0 ꢁC
followed by HOBt (2.97 g, 0.022 mol) and DIPEA
(11.5 mL, 0.06 mol). After being stirred for 10 min, a
solution of aniline (2.01 mL, 0.022 mol) in 10 mL
DMF was added dropwise to the reaction mixture at
0 ꢁC. Stirring was continued for 1 h at 0 ꢁC and then
overnight at room temperature. Most of the DMF was
evaporated with a high vacuum rotary evaporator, and
the residue was dissolved in 500 mL of ethyl acetate.
The organic phase was washed with saturated NH4Cl
solution (3· 50 mL) followed by brine (3· 50 mL) and
dried over anhydrous Na2SO4. The solvent was removed
under vacuum, and the residue was purified by flash
chromatography eluting with hexane/EtOAc (5:1) to
give 21 (5.63 g, 92%) as a white solid. 1H NMR (CDCl3):
d 1.38 (t, J = 7.0 Hz, 2H), 1.44 (s, 9H), 1.50 (t,
J = 7.0 Hz, 2H), 1.73 (t, J = 7.5 Hz, 2H), 2.35 (t,
J = 7.0 Hz, 2H), 3.11 (d, J = 6.0 Hz, 2H), 4.65 (br s,
1H), 7.08 (t, J = 7.0 Hz, 1H), 7.30 (t, J = 7.5 Hz, 2H),
7.54 (d, J = 7.5 Hz, 2H), 7.76 (s, 1H); 13C NMR
(CDCl3): 23.7, 25.3, 26.5, 28.6, 29.9, 37.6, 40.7, 120.0,
The in vitro HDAC inhibition assay was performed using
the HDAC fluorescent histone deacetylase activity assay
kit (Biomol Research Laboratories, Plymouth Meeting,
PA). A HeLa cell nuclear extract, which contains a num-
ber of HDAC isozymes and other nuclear factors, was
used as the source of the HDAC activity. The final sub-
strate concentration in the assay mixture was 50 lM.
The reaction was allowed to proceed for 30 min at
37 ꢁC before stopping the reaction. Test compounds were
prepared as 1 mM stock solutions in DMSO (molecular
biology grade, Sigma–Aldrich Co., St. Louis, MO) and
stored at 0 ꢁC. The final DMSO concentration per well
was no more than 2%. Assays were performed in white
polystyrene 96-well half-area assay plates (Corning,
NY) and measured on an EM Fluorescence/Chemilumi-
nescence Plate Reader (Molecular Devices Spectramax
Gemini fluorometric detection device) with an excitation
wavelength of 355 nm and an emission wavelength of
460 nm using SOFTmax PRO software.
The HDAC8 inhibition assay was performed using the
HDAC8 fluorescent activity assay kit (Biomol Research

3328
W. Gu et al. / Bioorg. Med. Chem. 14 (2006) 3320–3329
Laboratories, Plymouth Meeting, PA). The final sub-
strate concentration in the assay mixture was 25 lM.
The reaction was allowed to proceed for 45 min at
37 ꢁC before stopping the reaction with the Fleur de
LysTM Developer II and then was incubated for 10 min
at room temperature. The absorbance was determined
as above for the mixture of HDACs. Test compounds
were prepared as 1 mM stock solutions in DMSO
(molecular biology grade, Sigma–Aldrich Co., St. Louis,
MO) and stored at 0 ꢁC. The final DMSO concentration
per well was no more than 2%. Assays were performed
as described above.
R41 CA116971 to R.B.S. and R01 GM063872 to
C.M.H.).
References and notes
1. Wang, A. H.; Bertos, N. R.; Vezmar, M.; Pelletier, N.;
Crosato, M.; Heng, H. H.; Th’ng, J.; Han, J.; Yang, X. J.
Mol. Cell. Biol. 1999, 19, 7816–7827.
2. Grozinger, C. M.; Schreiber, S. L. Chem. Biol. 2002, 9,
3–16.
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during HDAC inhibition
Compound 8 was incubated with HeLa cell nuclear ex-
tract for 30 min at 37 ꢁC before stopping the reaction with
Fleur de LysTM developer. The combined buffer solution
(100 lL) was then extracted with ethyl acetate (1·
3 mL). The organic phase was combined and washed with
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simultaneously with IFNa. Sodium butyrate (Upstate
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at a final concentration of 5 mM. SAHA and its deriva-
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box and the firefly luciferase ORF. Cells were treated
with IFN and HDAC inhibitors 24 h after transfection.
Data represent means SD of triplicate samples.
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Acknowledgments
The authors are grateful to the National Institutes of
Health for financial support of this research (Grant

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