Novel inhibitors of human histone deacetylases: Design, synthesis, enzyme inhibition, and cancer cell growth inhibition of SAHA-based non-hydroxamates

Article abstract of DOI:10.1021/jm049207j

To find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) was designed and synthesized. In this series, compound 7, in which the hydroxamic acid of SAHA is replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA. In cancer cell growth inhibition assay, S-isobutyryl derivative 51 showed strong activity, and its potency was comparable to that of SAHA. The cancer cell growth inhibitory activity was verified to be the result of histone hyperacetylation and subsequent induction of p21^WAF1/CIP1 by Western blot analysis. Kinetical enzyme assay and molecular modeling suggest the thiol formed by enzymatic hydrolysis within the cell interacts with the zinc ion in the active site of HDACs.

Full text of DOI:10.1021/jm049207j

J. Med. Chem. 2005, 48, 1019-1032
1019
Novel Inhibitors of Human Histone Deacetylases: Design, Synthesis, Enzyme
Inhibition, and Cancer Cell Growth Inhibition of SAHA-Based
Non-hydroxamates
Takayoshi Suzuki,*,† Yuki Nagano,† Akiyasu Kouketsu,† Azusa Matsuura,† Sakiko Maruyama,‡
Mineko Kurotaki,‡ Hidehiko Nakagawa,† and Naoki Miyata*,†
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya,
Aichi 467-8603, Japan, and Evaluation Group, Drug Research Department, R. & D. Division, Pharmaceuticals Group,
Nippon Kayaku Co., Ltd., 31-12, Shimo 3-chome, Kita-ku, Tokyo 115-8588, Japan
Received October 1, 2004
To find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds
modeled after suberoylanilide hydroxamic acid (SAHA) was designed and synthesized. In this
series, compound 7, in which the hydroxamic acid of SAHA is replaced by a thiol, was found
to be as potent as SAHA, and optimization of this series led to the identification of HDAC
inhibitors more potent than SAHA. In cancer cell growth inhibition assay, S-isobutyryl
derivative 51 showed strong activity, and its potency was comparable to that of SAHA. The
cancer cell growth inhibitory activity was verified to be the result of histone hyperacetylation
and subsequent induction of p21WAF1/CIP1 by Western blot analysis. Kinetical enzyme assay
and molecular modeling suggest the thiol formed by enzymatic hydrolysis within the cell
interacts with the zinc ion in the active site of HDACs.
Chart 1
Introduction
The reversible acetylation of the ꢀ-amino groups of
specific histone lysine residues by histone deacetylases
(HDACs) and histone acetyl transferases is an impor-
tant regulatory mechanism of gene expression.1 When
HDACs are inhibited, histone hyperacetylation occurs.
The disruption of the chromatin structure by histone
hyperacetylation leads to the transcriptional activation
of a number of genes.2 One important outcome of the
activation is induction of the cyclin-dependent kinase
inhibitory protein p21WAF1/CIP1, which causes cell cycle
arrest.3 Indeed, HDAC inhibitors such as trichostatin
A (TSA) and suberoylanilide hydroxamic acid (SAHA)
(Chart 1) have been reported to inhibit cell growth,
induce terminal differentiation in tumor cells,4 and
prevent the formation of malignant tumors in mice.5
Therefore, HDACs have emerged as attractive targets
in anticancer drug development, and HDAC inhibitors
have also been viewed as useful tools to study the
function of these enzymes.
Many groups have ongoing research programs to find
nonpeptide small-molecule inhibitors of HDACs, and
these efforts have led to the identification of several
classes of inhibitors.6 Most previously reported HDAC
inhibitors belong to hydroxamic acid derivatives, typi-
fied by TSA and SAHA, which are thought to chelate
the zinc ion in the active site in a bidentate fashion
through its CO and OH groups.7 However, hydroxamic
acids occasionally have been associated with problems
such as poor pharmacokinetics and severe toxicity.8
Thus, it has become increasingly desirable to find
replacements that possess strong inhibitory action
against HDACs. In addition, in terms of biological
research, the discovery of novel zinc-binding groups
(ZBGs) may lead to a new type of HDAC isozyme-
selective inhibitors which are useful as tools for probing
the biology of the enzyme.9 Thus far, o-aminoanilide,9,10
electrophilic ketones,11 and N-formyl hydroxylamine12
have been reported as ZBGs in small-molecule HDAC
inhibitors. However, most of them have reduced potency
as compared to hydroxamic acid, and unfortunately,
HDAC inhibitors bearing electrophilic ketones11 have
a metabolic disadvantage in that they are readily
reduced to inactive alcohols in vivo, even within cells.
We therefore initiated a search for replacement groups
for hydroxamic acid with the goal of drug discovery as
well as finding new tools for biological research, and
found some potent non-hydroxamate small-molecule
HDAC inhibitors.13 We now present a full account of
our study reporting the design, synthesis, HDAC inhibi-
tion, cancer cell growth inhibition, and binding mode
analysis of non-hydroxamates based on the structure
of SAHA.
Chemistry
The compounds prepared for this study are shown in
Tables 1-5. The routes used for synthesis of the
compounds are shown in Schemes 1-4. Scheme 1 shows
the preparation of compounds 4, 5, 10, 12-17, and 18.
Compounds 4 and 5 were synthesized from pimelic acid
56. The condensation of pimelic acid 56 with an equiva-
* To whom correspondence should be addressed. Tel and fax:
+81-52-836-3407; e-mail: miyata-n@phar.nagoya-cu.ac.jp; suzuki@
phar.nagoya-cu.ac.jp
† Graduate School of Pharmaceutical Sciences, Nagoya City Uni-
versity.
‡ Nippon Kayaku Co., Ltd.
10.1021/jm049207j CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/25/2005

1020 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Suzuki et al.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) Na2SO3, EtOH, H2O, reflux; (b)
SOCl2, DMF, toluene, reflux; (c) O-(2-tetrahydropyranyl)hydrox-
ylamine, 4-(dimethylamino)pyridine, pyridine, CH2Cl2, rt; (d) 2N
aq NaOH, EtOH, rt; (e) aniline, EDCI, HOBt, DMF, rt; (f) TFA,
CH2Cl2, 60 °C; (g) LiOH‚H2O, EtOH, THF, H2O, rt; (h) (COCl)2,
DMF, CH2Cl2, rt; (i) ArNH2 (63), Et3N, CH2Cl2, rt; (j) PhB(OH)2,
Pd(PPh3)4, NaHCO3, 1-methyl-2-pyrrolidinone, H2O, 80 °C; (k)
AcSK, EtOH, rt; (l) 15% aq NaSMe, EtOH, rt; (m) m-chloroper-
oxybenzoic acid, CH2Cl2, rt.
a Reagents and conditions: (a) aniline, 180 °C; (b) diphen-
ylphosphoryl azide (DPPA), Et3N, toluene, reflux, and then O-(2-
tetrahydropyranyl)hydroxylamine, reflux; (c) TsOH, MeOH, rt; (d)
DPPA, Et3N, benzene, reflux, and then hydrazine monohydrate,
reflux; (e) DPPA, Et3N, benzene, reflux, and then BnOH, reflux;
(f) H2, 5%Pd-C, MeOH, rt; (g) MsCl, pyridine, rt; (h) HOCH2COOH
or BocNHCH2COOH, EDCI, HOBt, DMF, rt; (i) TFA, CHCl3, rt;
(j) BrCH2COBr, Et3N, THF, rt; (k) AcSK, EtOH, rt; (l) K2CO3,
MeOH, rt; (m) propargyl bromide, K2CO3, MeOH, rt.
subsequent reaction with thionyl chloride. The sulfonyl
chloride 60 was treated with O-THP hydroxylamine to
give O-THP hydroxysulfonamide, after which hydrolysis
of the ester under alkaline conditions and subsequent
amide formation with aniline gave compound 61. Re-
moval of the THP group of compound 61 by treatment
with trifluoroacetic acid gave hydroxysulfonamide 6.
Compounds 7-9, 11, 19-21, 24-32, and 37 were
synthesized from the corresponding acid chlorides 62
(62a (n ) 4) and 62b (n ) 5) are commercially available)
by the route shown in Scheme 2. 62c (n ) 6) was
prepared from ester 59 by hydrolysis of the ethyl ester
and a subsequent reaction with oxalyl chloride, and 62d
(n ) 7) was obtained in the same way as 62c. The amino
group of aromatic amines 63 was acylated with an
appropriate acid chloride 62 to give the amides 64.
Suzuki coupling14 of bromobenzene 64a with phenylbo-
ronic acid provided the biphenyl 64b. Bromides 64 were
treated with potassium thioacetate to give compound
8, after which hydrolysis of the thioacetates under
alkaline conditions gave the desired compounds 7, 19-
21, 24-31, and 32, and disulfide 37 was obtained as a
byproduct when thiol 7 was synthesized. Sulfide 9 was
prepared by the alkylation of methylmercaptan with
bromide 64c (Ar ) Ph, n ) 6). Oxidation of 9 with 2
equiv of m-chloroperoxybenzoic acid provided the sul-
fone 11.
lent amount of aniline gave mono-anilide 57. Curtius
rearrangement of the acyl azide prepared from carbox-
ylic acid 57 using diphenylphosphoryl azide provided
the isocanates, which on treatment with O-tetrahydro-
pyranyl (THP) hydroxylamine or hydrazine gave O-THP
hydroxyurea and semicarbazide 5. Deprotection of the
THP group of the O-THP hydroxyurea under acidic
conditions afforded hydroxyurea 4.
Compounds 10, 12-17, and 18 were prepared from
carboxylic acid 57 obtained above via amine 58 by the
procedure outlined in Scheme 1. Carboxylic acid 57 was
converted to amine 58 with a three-step sequence:
Curtius rearrangement of the acyl azide prepared from
carboxylic acid 57, treatment of the resulting isocyan-
ates with benzyl alcohol, and removal of the Z group by
hydrogenation. Coupling between amine 58 and meth-
anesulfonyl chloride afforded sulfonamide 10. The reac-
tion of amine 58 with N-Boc glycine in the presence of
EDCI and HOBt in DMF was followed by treatment
with trifluoroacetic acid to give aminoacetamide 12.
Hydroxyacetamide 13 was obtained in one step using
the procedure described for 12. The amino group of 58
was acylated with bromoacetyl bromide to yield bro-
moacetamide 18. Bromide 18 was treated with potas-
sium thioacetate to give thioacetate 15, after which
deacetylation of the thioacetate in the presence of K2-
CO3 in MeOH gave mercaptoacetamide 14. The amine
58 was allowed to react with propargyl bromide in the
presence of K2CO3 to give mono- and di-alkylated
compounds 16 and 17.
Thiols 22, 23, 33-35, and 36 were prepared from
alcohol 65 or 66 by the procedure outlined in Scheme
3. Treatment of bromide 65 with phenol in the presence
of K2CO3 gave ether 67, and condensation of amine 66
with an appropriate aromatic carboxylic acid 69 afforded
amides 68. Alcohols 67 and 68 were converted to thiols
22, 23, 33-35, and 36 in three steps by conversion of
the alcohols to bromides, treatment of the bromides with
potassium thioacetate, and hydrolysis of the resulting
thioacetates.
Compounds 6-9, 11, 19-21, 24-32, and 37 were
prepared from another starting material, 59 (Scheme
2). The preparation of hydroxysulfonamide 6 was
achieved via sulfonyl chloride 60. Bromide 59 was
converted to sulfonyl chloride 60 by sulfation and by a
The preparation of S-chemically modified compounds
38-54, and 55 is shown in Scheme 4. Thiols 7, 26, 28-
32, 34, 35, and 36 were coupled with the corresponding

Inhibitors of Human Histone Deacetylases
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1021
Scheme 3a
a Reagents and conditions: (a) Phenol, K2CO3, DMF, 80 °C; (b)
ArCOOH (69), EDCI, HOBt, DMF, rt; (c) CBr4, PPh3, CH2Cl2, 0
°C; (d) AcSK, EtOH, rt; (e) 2N aq NaOH, EtOH, THF, rt.
Figure 1. The transition state proposed for HDACs (a), and
models for the binding of sulfone derivatives (b).
Scheme 4a
HDACs under reductive conditions.17 Surprisingly, al-
though the inhibitory ability of monodentate ZBGs such
as thiol was thought to be less than that of bidentate
ZBGs such as hydroxamate, N-formyl hydroxylamine,
and hydrated electrophilic ketones,16 the activity of thiol
7 was far greater than expected. A pronounced inhibi-
tory effect (IC50 ) 0.21 µM) was observed with thiol 7,
which was much more active than previously reported
non-hydroxamates such as o-aminoanilide, N-formyl
hydroxylamine, and trifluoromethyl ketone,11a and as
potent as R-ketoamide 2 and SAHA (entry 8). To confirm
that the thiol group plays an important role in anti-
HDAC activity, thioacetate 8a and methyl sulfide 9 were
tested. As expected, thiol transformation into thioac-
etate and methyl sulfide led to an inhibitor that was
about 30-fold less potent and a compound devoid of anti-
HDAC activity, respectively (entries 9 and 10). These
results suggest that thiolate anion generated under
physiological conditions has an intimate involvement in
the interaction with the zinc ion in the active site.
The crystal structures of the HDLP/hydroxamates
and HDAC8/hydroxamates complexes have led to a solid
understanding of not only the three-dimensional struc-
ture of the active site of HDACs but also the catalytic
mechanism for the deacetylation of acetylated lysine
substrate.7 It has been proposed that the carbonyl
oxygen of this substrate could bind the zinc, and the
carbonyl could be attacked by a zinc-chelating water
molecule (Figure 2a), which would result in the produc-
tion of deacetylated lysine via a tetrahedral carbon-
containing transition state (Figure 1a). On the basis of
the proposed catalytic mechanism, we attempted to
design non-hydroxamate HDAC inhibitors. First, we
designed transition-state (TS) analogues. The TS of
HDAC deacetylation was estimated to include a tetra-
hedral carbon (Figure 1a) as with other zinc proteases.18
We focused attention on sulfone derivative TS analogues
because it has been suggested that the sulfonamide
moiety has strong similarity with the TS of amide bond
hydrolysis, both from a steric and an electronic point of
view.19 Compounds 10 and 11, in which a hydroxamic
acid of SAHA is replaced by a sulfonamide and a
sulfone, respectively, were designed and synthesized as
TS analogues (Figure 1b). Of these two TS analogues,
sulfone 11 showed anti-HDAC activity and the IC50
value was 230 µM (entries 11 and 12). However, sulfone
11 was approximately 820-fold less effective than SAHA.
a Reagents and conditions: (a) RCOCl (70), 4-(dimethylami-
no)pyridine, pyridine, CH2Cl2, rt; (b) NaH, chloromethyl pivalate,
DMF, 0 °C to room temperature.
acyl chloride 70 to give thioesters 38-45, 47-54, and
55. Alkylation of thiol 7 with chloromethyl pivalate in
the presence of sodium hydride in DMF afforded com-
pound 46.
Results and Discussion
Enzyme Assays. The compounds synthesized in this
study were tested with an in vitro assay using a HeLa
nuclear extract rich in HDAC activity. The results are
summarized in Tables 1-3.
The IC50 values of SAHA and o-aminoanilide 1 were
0.28 µM and 120 µM, respectively (entries 1 and 2).
R-Ketoamide 2 and N-formyl hydroxylamine 3 were
reported previously to inhibit HDACs with an IC50 of
0.34 µM and 2.8 µM, respectively (entries 3 and 4).11b,12
The crystal structures of an archaebacterial HDAC
homologue (HDAC-like protein, HDLP)/hydroxamates
and HDAC8/hydroxamates complexes made it clear that
the hydroxamic acid group coordinates the zinc ion in
the active site through its CO and OH groups and also
forms three hydrogen bonds between its CO, NH, and
OH groups and Tyr 306, His 143, and His 142 (HDAC8
numbering), respectively.7 From these data, hydroxy-
urea 4, semicarbazide 5, and hydroxysulfonamide 6
were synthesized and tested as HDAC inhibitors be-
cause it is possible for them to chelate zinc ion and form
hydrogen bonds with Tyr and His like SAHA. Among
these three compounds, hydroxyurea 4 and semicarba-
zide 5 showed anti-HDAC activity and the IC50 values
were comparable to that of o-aminoanilide 1 (entries 5,
6, and 7). However, they were much less effective than
SAHA.
Thiols seemed to be reasonable targets for hydroxamic
acid replacements, because zinc ion is highly thiophilic
and thiol derivatives have been reported to inhibit zinc-
dependent enzymes such as angiotensin converting
enzyme15 and matrix metalloproteinases.16 Further-
more, macrocyclic compounds bearing a disulfide group
such as FK228 have been reported recently to inhibit
Our next approach was based on the proposed deacety-
lation mechanism whereby a zinc-chelating water mol-
ecule activated by His142 and His 143 (HDAC8 num-
bering) makes a nucleophilic attack on the carbonyl
carbon of an acetylated lysine substrate (Figure 2a).

1022 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Suzuki et al.
Table 2. Effect of Linker Variation on HDAC Inhibitory
Activity of Thiolsa
entry
compd
X
n
IC50 (µM)
1
2
3
4
5
6
7
-NHCO-
-NHCO-
-NHCO-
-NHCO-
-O-
6
7
5
4
6
6
0.21
1.5
0.37
6.2
11
0.36
19
20
21
22
23
-CONH-
a Values are means of at least three experiments.
Figure 2. The mechanism proposed for the deacetylation of
acetylated lysine substrate (a), and a model for the binding of
heteroatom-containing substrate analogues to zinc ion (b).
Table 3. Effect of Aromatic Group Variation on HDAC
Inhibitory Activity of Thiolsa
Table 1. HDAC Inhibition Data for SAHA and SAHA-based
Non-hydroxamtesa
a Values are means of at least three experiments. b Prepared
as described in ref 26. c Prepared as described in ref 9a. d Data
taken from the literature (ref 11b). e Data taken from the literature
(ref 12). f Trifluoroacetic acid salt. ND ) No data.
a Values are means of at least three experiments.
With this mechanism, if the water molecule is forcibly
removed from the zinc ion, the HDACs would suppos-
edly be inhibited. We then designed and synthesized
heteroatom-containing substrate analogues 12, 13, and
14. These analogues would be recognized as substrates
by HDACs and would be easily taken into the active
site where they could force the water molecule off the
zinc ion and the reactive site for the deacetylation by
chelation of the heteroatom to the zinc ion, and might
behave as HDAC inhibitors (Figure 2b). As shown in
Table 1 (entries 13, 14, and 15), potent inhibition was
observed with mercaptoacetamide 14, while 12 and 13
did not possess HDAC inhibitory activities. Mercap-
toacetamide 14 exhibited an IC50 of 0.39 µM, and its
activity largely surpassed those of o-aminoanilide 1 and
N-formyl hydroxylamine 3 and was comparable to those
of R-ketoamide 2 and SAHA. As expected, thiol trans-
formation into thioacetate (15) led to a 55-fold less
potent inhibitor. This result suggests the ease of ioniza-
tion of thiol is an important factor for HDAC inhibition
like the case of thiol 7.
We turned our attention to irreversible HDAC inhibi-
tors. TPX B is an irreversible HDAC inhibitor,20 and
finding more specific and simpler irreversible HDAC
inhibitors is useful for the isolation and cloning of an
HDAC.2 As described above, the crystal structures of
the HDLP/hydroxamates and HDAC8/hydroxamates
complexes revealed that the hydroxamic acid group
forms three hydrogen bonds with Tyr 306, His 143, and
His 142, and furthermore, zinc ion is coordinated by His
180, Asp 178, and Asp 267 (HDAC8 numbering). Since
the phenol group of Tyr, the imidazole group of His, and
the carboxyl group of Asp are able to react with
electrophiles, we prepared analogues bearing propargyl

Inhibitors of Human Histone Deacetylases
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1023
Table 4. Cell Growth Inhibition Data on NCI-H460 Cells for
Compound 7 and Its S-Modified Prodrugsa
Table 5. Cell Growth Inhibition Data on NCI-H460 Cells for
Compound 40 and Its Derivativesa
a Values are means of at least two experiments. b 34% inhibition
at 50 µM. c 10% inhibition at 50 µM. d 42% inhibition at 50 µM.
amino (16, 17) and bromoacetamide (18) which could
form covalent bonds with Tyr, His, and Asp of the
enzyme, and evaluated their anti-HDAC activities.
While propargyl amino compounds 16 and 17 did not
possess HDAC inhibitory activities, more potent inhibi-
tion was observed with bromoacetamide 18 (entries 17,
18, and 19). Bromoacetamide 18 exhibited an IC50 of
17 µM and its activity was about 9-fold as strong as that
of o-aminoanilide 1, but much weaker than that of
SAHA.
With the results shown in Table 1, we were encour-
aged to study further the structure-activity relation-
ship (SAR) and structural optimization. We selected
thiol 7 for further study.21 First, we examined the effect
of linker parts of thiol 7. The results are summarized
in Table 2. HDAC inhibition was distinctly dependent
on chain length, with n ) 7 (19) and n ) 4 (21) resulting
in less potent inhibitors. However, compound 20, in
which n ) 5, showed essentially the same potency as
compound 7, in which n ) 6 (entries 1-4). The similar
SAR between thiols and hydroxamates, with n ) 6
optimal,22 indicates that thiols inhibit HDACs in a
binding mode similar to that of hydroxamates. As for
the group attaching the phenyl moiety, ether 22 dis-
played moderate activity, whereas the activity of the
reversed amide 23 was maintained (entries 5 and 6).
Next, the aromatic group was examined (Table 3). In
the amide-linked series (entries 1-10), 4-substituted
phenyl compounds tended to decrease the potency.
Specifically, compounds 24 (Ar ) 4-NMe2-Ph), 25 (Ar
) 4-biphenyl), and 27 (Ar ) 4-PhO-Ph) showed about a
3- to 6-fold decrease in potency when compared to the
parent thiol 7 (entries 2, 3, and 5). On the other hand,
compound 26, in which a phenyl group was introduced
at the 3-position of the phenyl group of 7, showed 3-fold
increased inhibitory activity (IC50 of 0.075 µM, entry 4).
In addition, 3-phenoxy compound 28 was equipotent
with compound 7 (entry 6). We investigated the effect
of the replacement of the phenyl group of compound 7
with heteroaryl rings (entries 7, 8, 9, and 10). Changing
the benzene ring to a 3-pyridine ring (29), 4-phenyl-2-
thiazole ring (31), and 2-benzothiazole ring (32) sus-
a Values are means of at least two experiments.
Table 6. Growth Inhibition of Various Cancer Cells Using
SAHA and Compound 51a
SAHA,
EC50 (µM)
51,
cell
EC50 (µM)
MDA-MB-231
SNB-78
breast cancer
central nervous
system
1.5
16
2.3
9.1
HCT116
NCI-H226
LOX-IMVI
SK-OV-3
RXF-631L
St-4
colon cancer
lung cancer
0.58
2.6
1.3
2.5
2.0
5.2
1.6
3.0
2.6
1.1
4.5
2.4
5.0
4.5
melanoma
ovarian cancer
renal cancer
stomach cancer
prostate cancer
DU-145
mean
3.7
3.8
a Values are means of at least two experiments.
tained or slightly reduced the activity, whereas quino-
line 30 had improved activity (IC50 of 0.072 µM), and
turned out to be the most potent compound in this
series. The reverse amide-linked series (entries 11-15)
exhibited potencies similar to or greater than the parent
thiol 23, with the exception of 33 (Ar ) 4-NMe2-Ph),
which resulted in a slightly less potent inhibitor. In
particular, the reversed amides 34 with a naphthalene
substituent and 35 with a benzofuran substituent
exhibited about 3-fold increases in potency (IC50s of
0.085 µM and 0.079 µM, respectively). As a result, IC50s
in the double-digit nanomolar range were observed with
3-biphenyl 26, quinoline 30, naphthalene 34, and ben-
zofuran 35, which were approximately 3- to 4-fold more
potent than SAHA.
Cancer Cell Growth Inhibition Assay. To confirm
the effectiveness of thiol-based HDAC inhibitors as
anticancer drugs and tools for biological research, thiol
7 was initially tested in a cancer cell growth inhibition

1024 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Suzuki et al.
seemed to be attractive targets, because it has been
reported that the disulfide bond of macrocyclic com-
pounds bearing a disulfide group such as FK228 is
reduced in the cellular environment, releasing the free
thiol analogue as the active species.17 However, contrary
to our expectation, disulfide 37 failed to exhibit a growth
inhibitory effect on NCI-H460 cells (entry 2). Next, we
examined the activity of acetyl compound 8a. Acetyl
compound 8a proved to be relatively potent compared
with thiol 7 and disulfide 37 (EC50 of 36 µM) (entry 3).
This result suggests that 8a permeates the cell mem-
brane more efficiently than thiol 7, and is converted to
thiol 7 by enzymatic hydrolysis within the cell.23 En-
couraged by this finding, we prepared other S-acyl
compounds (38-45) and evaluated their activities (en-
tries 4-11). This series of compounds exhibited greater
potency than acetyl compound 8a, except for pivaloyl
compound 41, which was a less potent inhibitor. In
particular, isobutyryl compound 40 showed about a
2-fold increase in activity when compared to acetyl
compound 8a (EC50 of 20 µM). The compound bearing
a (pivaloyloxy)methyl group24 (46) was slightly less
active than isobutyryl compound 40 (entry 12).
With the results shown in Table 4, a selected set of
active compounds from the enzymatic assay was S-
isobutyrylated and tested as cancer cell growth inhibi-
tors (Table 5). Much to our satisfaction, changing the
phenyl group of compound 40 to other aromatic groups
led to positive results. Isobutyryl analogues 47-55 were
generally more potent than the parent compound 40;
the sole exception is 48 (Ar ) 3-OPh-Ph) which was
slightly less active than compound 40 (entry 3). Notably,
3-biphenyl (47), 3-pyridinyl (49), and 4-phenyl-2-thi-
azolyl (51) analogues showed strong activity in inhibit-
ing the growth of NCI-H460 cells, with EC50s of 2-3
µM. Furthermore, we evaluated cancer cell growth
inhibition by SAHA and 51, the most potent compound
in this study, against nine other human cancer cell lines
Figure 3. Western blot analysis of histone hyperacetylation
and p21WAF1/CIP1 induction in HCT 116 cells produced by
compound 51 and by reference compound SAHA.
Figure 4. Reciprocal rate vs reciprocal acetylated lysine
substrate concentration in the presence of 0.3 (b), 0.1 (2), 0.03
(9), and 0 (O) µM of 7.
assay using human lung cancer NCI-H460 cells against
which it was found to be only weakly potent, although
7 was highly active in an enzymatic assay (Table 4,
entry 1). The reason for the weak activity of thiol 7 is
unclear, but it is reasonable to assume that it was due
to poor membrane permeability resulting from the
highly polar character of this compound, and that a
transient masking of the sulfhydryl group could improve
its permeability and its ability to inhibit cancer cell
growth. Therefore, we investigated the possibility of
improving the inhibition using the prodrug approach.
In the search for a suitable prodrug of thiols, disulfides
Figure 5. Superposition of the low energy conformations of 7 (tube) and SAHA (wire) (left). The HDAC8 pocket is not shown for
the sake of clarity. View of the conformation of 7 (ball-and-stick) docked in the HDAC8 catalytic core (right). Residues around the
zinc ion and a water molecule are displayed as wires and tubes, respectively.

Inhibitors of Human Histone Deacetylases
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1025
(Table 6). Compound 51 exerted potent growth inhibi-
tion against various human cancer cells, with EC50
values ranging from 1 to 10 µM, and these inhibitory
activities were comparable to those of SAHA (average
EC50 of 51 3.8 µM, SAHA 3.7 µM) which is currently
being evaluated in clinical trials for use in the treatment
of cancer.
R-ketoamide 2 and SAHA. At present, thiol is one of the
most active ZBG among small-molecule HDAC inhibi-
tors. Optimization of thiol derivatives led to the iden-
tification of inhibitors more effective than SAHA (com-
pounds 26, 30, 34, and 35). We have also identified a
potent cancer cell growth inhibitor, compound 51, by the
prodrug formation of thiol-based HDAC inhibitors. Thiol
7 exhibits strong competitive inhibition of an acetylated
lysine substrate, and molecular modeling suggests that
the thiol interacts with zinc, Tyr 306, and His 142
(HDAC8 numbering) in the active site.
In conclusion, we have identified several new lead
structures including thiol, from which more potent
HDAC inhibitors can be developed. As far as we could
determine, this is the first systematic study of ZBGs for
HDAC inhibitors. We believe that the findings of this
study should be of value in future studies for the
development of ideal anticancer drugs and tools for
biological research such as HDAC isozyme-selective
inhibitors.
By Western blot analysis, cancer cell growth inhibi-
tion with compound 51 was verified to be the result
of inhibition of HDACs (Figure 3). Treatment of HCT
116 cells with compound 51 gave rise to elevated and
dose-dependent levels of acetylated histone H4 and
p21WAF1/CIP1
.
Inhibitory Mechanism Study. Since the results of
cancer cell growth inhibition and Western blot analysis
have suggested that thiols generated from S-acyl pro-
drugs by enzymatic hydrolysis within the cell inhibit
intracellular HDACs, we next studied the mechanism
by which thiols inhibit HDACs in greater detail. Al-
though the sulfhydryl group of thiol derivatives was
designed as a ZBG, it is possible that thiols inhibit
HDACs by forming a covalent disulfide bond with
cysteine residues on these enzymes. We examined this
possibility using a double reciprocal plot of 1/V versus
1/[substrate] at varying concentrations of inhibitor 7
(Figure 4), and the data from this study established that
thiol 7 engages in competitive inhibition versus acety-
lated lysine substrate, with an inhibition constant (Ki)
of 0.11 µM. Since cysteine is not a component in the
construction of the active site of HDACs, the sulfhydryl
group of 7 likely interacts with the zinc in the active
site.
Binding Mode Study. Since thiol 7 proved to be a
competitive inhibitor and to act within the active center
of HDACs, we studied its binding mode within this site.
The low energy conformations of 7 and SAHA were
calculated when docked in the model based on the
crystal structure of HDAC8 (PDB code 1T64, 1T67,
1T69, and 1VKG) using Macromodel 8.1 software.25 The
anilide group and alkyl chain of 7 and SAHA were
essentially superimposed in the binding pocket, and the
binding mode of 7 was found to be similar to that of
SAHA (Figure 5, left). An inspection of the HDAC8/7
complex shows that the sulfur atom of 7 was located
2.35 Å from the zinc ion, 2.24 Å from the OH group of
Tyr 306, and 2.66 Å from a water molecule which forms
a hydrogen bond with the imidazole group of His142
(Figure 5, right). This suggests that thiols strongly
inhibit HDACs by interacting directly with zinc ion, Tyr
306, and His 142 via a water molecule.
Experimental Section
Chemistry. Melting points were determined using a
Yanagimoto micro melting point apparatus or a Bu¨chi 545
melting point apparatus and were left uncorrected. Proton
nuclear magnetic resonance spectra (1H NMR) were recorded
on a JEOL JNM-LA400 or JEOL JNM-LA500 spectrometer
in solvent as indicated. Chemical shifts (δ) are reported in
parts per million relative to the internal standard tetrameth-
ylsilane. Elemental analysis was performed with a Yanaco
CHN CORDER NT-5 analyzer, and all values were within
(0.4% of the calculated values. High-resolution mass spectra
(HRMS) were recorded on a JEOL JMS-SX102A mass spec-
trometer. GC-MS analyses were performed on a Shimadzu
GCMS-QP2010. Reagents and solvents were purchased from
Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Industries,
and Kanto Kagaku and used without purification. Flash
column chromatography was performed using silica gel 60
(particle size 0.046-0.063 mm) supplied by Merck.
6-(3-Hydroxyureido)hexanoic Acid Phenylamide (4).
Step 1: Preparation of 6-Phenylcarbamoylhexanoic Acid
(57). A mixture of aniline (5.80 g, 62.3 mmol) and pimeric acid
(56, 10.0 g, 62.4 mmol) was stirred at 180 °C for 1 h. After
cooling, the mixture was diluted with AcOEt-THF and the
slurry was filtered. The filtrate was washed with saturated
aqueous NaHCO3, and the aqueous layer was acidified with
concentrated HCl. The precipitated crystals were collected by
filtration to give 7.11 g (49%) of 57 as a white solid: 1H NMR
(DMSO-d6, 400 MHz, δ; ppm) 11.97 (1H, broad s), 9.83 (1H,
s), 7.58 (2H, d, J ) 7.8 Hz), 7.27 (2H, t, J ) 7.9 Hz), 7.01 (1H,
t, J ) 7.4 Hz), 2.67 (2H, t, J ) 7.4 Hz), 2.21 (2H, t, J ) 7.3
Hz), 1.62-1.49 (4H, m), 1.34-1.27 (2H, m).
Steps 2 and 3: Preparation of 6-(3-Hydroxyureido)-
hexanoic Acid Phenylamide (4). To a suspension of 57 (958
mg, 4.07 mmol) obtained above and triethylamine (744 mg,
7.35 mmol) in toluene (10 mL) was added diphenylphosphoryl
azide (1.75 g, 6.34 mmol), and the mixture was heated at reflux
temperature for 1h. Next, O-(2-tetrahydropyranyl)hydroxyl-
amine (380 mg, 3.11 mmol) was added, and the reaction
mixture was stirred at reflux temperature for 18h. It was then
concentrated in vacuo, and the residue was dissolved in AcOEt.
The AcOEt solution was washed with water, saturated aque-
ous NaHCO3, and brine and was dried over Na2SO4. Filtration
and concentration in vacuo and purification by silica gel flash
chromatography (n-hexane/AcOEt ) 1/2) gave 988 mg (69%)
of the O-(2-tetrahydropyranyl)hydroxyurea as a white solid:
1H NMR (CDCl3, 500 MHz, δ; ppm) 7.53 (2H, d, J ) 7.9 Hz),
7.32 (2H, t, J ) 7.8 Hz), 7.26 (1H, broad s), 7.10 (1H, t, J ) 7
Hz), 7.05 (1H, broad s), 6.06 (1H, broad s), 4.75 (1H, d, J )
3.6 Hz), 3.93 (1H, m), 3.57 (1H, m), 3.33-3.26 (2H, m), 2.38
(2H, t, J ) 7.5 Hz), 1.82-1.77 (4H, m), 1.61-1.55 (6H, m),
1.44 (2H, quintet, J ) 7.3 Hz).
Conclusion
We have designed and prepared a series of SAHA-
based compounds as (i) hydroxamic acid mimics by
structure-based drug design (compounds 4-6), (ii) thiol-
based analogues (compounds 7-9), (iii) transition-state
analogues (compounds 10 and 11), (iv) heteroatom-
containing substrate analogues by mechanism-based
drug design (compounds 12-15), and (v) irreversible
inhibition-oriented compounds (compounds 16-18), and
evaluated their inhibitory effect on HDACs. In this
series, thiol 7 and mercaptoacetamide 14 were found
to be much more potent HDAC inhibitors than previ-
ously reported non-hydroxamates, and as potent as

1026 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Suzuki et al.
To a solution of the O-(2-tetrahydropyranyl)hydroxyurea
(185 mg, 0.53 mmol) obtained above in MeOH (2 mL) was
added 4-toluenesulfonic acid monohydrate (15 mg, 0.079
mmol). The solution was stirred overnight at room tempera-
ture, and the precipitated crystals were collected by filtration
to give 46 mg (32%) of 4 as a white solid. The solid was
recrystallized from MeOH-AcOEt and collected by filtration
7.4 Hz), 1.33 (2H, quintet, J ) 7.4 Hz); MS (EI) m/z: 284 (M+);
Anal. (C13H20N2O3S) C, H, N.
6-(2-Hydroxyacetylamino)hexanoic Acid Phenylamide
(13). To a solution of 58 (198 mg, 0.96 mmol) and glycolic acid
(81 mg, 1.07 mmol) in DMF (6 mL) were added 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (254 mg,
1.32 mmol) and 1-hydroxy-1H-benzotriazole monohydrate (244
mg, 1.59 mmol), and the mixture was stirred overnight at room
temperature. The reaction mixture was poured into water and
extracted with AcOEt. The AcOEt layer was separated, washed
with saturated aqueous NaHCO3 and brine, and dried over
Na2SO4. Filtration and concentration in vacuo gave 251 mg
(99%) of 13 as a crude solid. The solid was recrystallized from
AcOEt to give 155 mg of 13 as a colorless crystal: mp 109-
1
to give 34 mg of 4 as a colorless crystal: mp 148-149 °C; H
NMR (DMSO-d6, 500 MHz, δ; ppm) 9.93 (1H, s), 8.58 (1H, s),
8.29 (1H, s), 7.65 (2H, d, J ) 8 Hz), 7.35 (2H, t, J ) 7.9 Hz),
7.08 (1H, t, J ) 7.3 Hz), 6.75 (1H, t, J ) 6 Hz), 3.10 (2H, q, J
) 6.7 Hz), 2.36 (2H, t, J ) 7.5 Hz), 1.65 (2H, quintet, J ) 7.5
Hz), 1.50 (2H, quintet, J ) 7.2 Hz), 1.34 (2H, quintet, J ) 7.6
Hz); Anal. (C13H19N3O3) C, H, N.
6-(3-Aminoureido)hexanoic Acid Phenylamide (5). Com-
pound 5 was prepared from 57 obtained above by using the
procedure described for 4 (step 2) in 52% yield. In this case,
hydrazine monohydrate was used instead of O-(2-tetrahydro-
pyranyl)hydroxylamine: mp 146-147 °C; 1H NMR (DMSO-d6,
400 MHz, δ; ppm) 9.83 (1H, s), 7.58 (2H, d, J ) 7.8 Hz), 7.27
(2H, t, J ) 7.9 Hz), 7.01 (1H, t, J ) 7.3 Hz), 6.83 (1H, broad
s), 6.28 (1H, broad s), 4.03 (2H, broad s), 3.01 (2H, q, J ) 6.7
Hz), 2.29 (2H, t, J ) 7.4 Hz), 1.60-1.57 (2H, m), 1.40-1.38
(2H, m), 1.32-1.28 (2H, m); MS (EI) m/z: 264 (M+); Anal.
(C13H20N4O2) C, H, N.
6-Methanesulfonylaminohexanoic Acid Phenylamide
(10). Steps 1 and 2: Preparation of 6-Aminohexanoic
Acid Phenylamide (58). To a suspension of 57 (1.11 g, 4.73
mmol) obtained above and triethylamine (699 mg, 6.90 mmol)
in benzene (3 mL) was added diphenylphosphoryl azide (1.83
g, 6.64 mmol), and the mixture was heated at reflux temper-
ature for 1 h. Next, benzyl alcohol (1.20 mL, 11.6 mmol) was
added, and the reaction mixture was stirred at reflux temper-
ature for 24 h. It was then concentrated in vacuo and the
residue was dissolved in AcOEt. The AcOEt solution was
washed with 0.4 N aqueous HCl, water, saturated aqueous
NaHCO3, and brine and was dried over Na2SO4. Filtration and
concentration in vacuo and purification by recrystallization
from CHCl3-n-hexane gave 1.01 g (63%) of (6-phenylcarbam-
oylpentyl)carbamic acid benzyl ester as a colorless needle: 1H
NMR (DMSO-d6, 400 MHz, δ; ppm) 9.81 (1H, s), 7.57 (2H, d,
J ) 7.8 Hz), 7.37-7.22 (8H, m), 7.00 (1H, t, J ) 7.4 Hz), 4.99
(2H, s), 2.99 (2H, q, J ) 6.5 Hz), 2.28 (2H, t, J ) 7.4 Hz), 1.58
(2H, quintet, J ) 7.6 Hz), 1.43 (2H, quintet, J ) 7.1 Hz), 1.32
(2H, quintet, J ) 7.8 Hz); MS (EI) m/z: 340 (M+).
A solution of (6-phenylcarbamoylpentyl)carbamic acid ben-
zyl ester (1.00 g, 2.95 mmol) obtained above in MeOH (50 mL)
was stirred under H2 (atmospheric pressure) in the presence
of 5% Pd/C (106 mg) at room temperature for 7 h. The catalyst
was removed by filtration through Celite, and the filtrate was
concentrated in vacuo. The residue was purified by silica gel
flash chromatography (CHCl3/MeOH/iPrNH2 ) 19/1/1) to give
584 mg (96%) of 58 as a white solid: 1H NMR (DMSO-d6, 400
MHz, δ; ppm) 9.83 (1H, s), 7.58 (2H, d, J ) 7.6 Hz), 7.27 (2H,
t, J ) 7.9 Hz), 7.01 (1H, t, J ) 7.3 Hz), 2.55 (2H, m), 2.29 (2H,
t, J ) 7.4 Hz), 1.59 (2H, quintet, J ) 7.4 Hz), 1.37-1.30 (4H,
m).
1
113 °C; H NMR (DMSO-d6, 500 MHz, δ; ppm) 9.92 (1H, s),
7.79 (1H, broad s), 7.65 (2H, d, J ) 7.6 Hz), 7.35 (2H, t, J )
7.9 Hz), 7.08 (1H, t, J ) 7.3 Hz), 5.51 (1H, t, J ) 5.8 Hz), 3.84
(2H, d, J ) 5.8 Hz), 3.16 (2H, q, J ) 6.8 Hz), 2.36 (2H, t, J )
7.5 Hz), 1.65 (2H, quintet, J ) 7.5 Hz), 1.51 (2H, quintet, J )
7.3 Hz), 1.35 (2H, quintet, J ) 7.9 Hz); MS (EI) m/z: 264 (M+);
Anal. (C14H20N2O3) C, H, N.
6-(2-Aminoacetylamino)hexanoic Acid Phenylamide
Trifluoroacetic Acid Salt (12‚TFA). Step 1: Preparation
of [(5-Phenylcarbamoylpentylcarbamoyl)methyl]car-
bamic Acid tert-Butyl Ester. This compound was prepared
from 58 and N-(tert-butoxycarbonyl)glycine using the proce-
dure described for 13 in 70% yield: 1H NMR (CDCl3, 400 MHz,
δ; ppm) 7.53 (2H, d, J ) 7.8 Hz), 7.34 (2H, t, J ) 7.6 Hz), 7.10
(1H, t, J ) 7.6 Hz), 6.14 (1H, broad s), 5.07 (1H, broad s), 3.75
(2H, d, J ) 6 Hz), 3.30 (2H, q, J ) 6.5 Hz), 2.37 (2H, t, J ) 7.4
Hz), 1.76 (2H, quintet, J ) 7.4 Hz), 1.58-1.26 (13H, m).
Step 2: Preparation of 6-(2-Aminoacetylamino)hex-
anoic Acid Phenylamide Trifluoroacetic Acid Salt (12‚
TFA). To a solution of [(5-phenylcarbamoylpentylcarbamoyl)-
methyl]carbamic acid tert-butyl ester (147 mg, 0.40 mmol)
obtained above in CHCl3 (4 mL) was added trifluoroacetic acid
(1 mL), and the mixture was stirred overnight at room
temperature. The reaction mixture was concentrated in vacuo,
and the residue was triturated in diethyl ether to give 131
mg (84%) of 12‚TFA as a white solid. The solid was recrystal-
lized from AcOEt-MeOH to give 120 mg of 12‚TFA as colorless
1
crystals: mp 149-151 °C; H NMR (DMSO-d6, 500 MHz, δ;
ppm) 10.00 (1H, s), 8.43 (1H, t, J ) 5.2 Hz), 8.10 (3H, broad
s), 7.71 (2H, d, J ) 8.2 Hz), 7.41 (2H, t, J ) 7.9 Hz), 7.14 (1H,
t, J ) 7.3 Hz), 3.25 (2H, q, J ) 6.4 Hz), 2.43 (2H, t, J ) 7.3
Hz), 1.72 (2H, quintet, J ) 7.5 Hz), 1.58 (2H, quintet, J ) 7.2
Hz), 1.44 (2H, quintet, J ) 7.5 Hz); Anal. (C14H21N3O2‚TFA‚
1/10H2O) C, H, N.
6-(2-Bromoacetylamino)hexanoic Acid Phenylamide
(18). To a solution of 58 (70 mg, 0.340 mmol) and triethylamine
(0.40 mL, 2.88 mmol) in THF (2 mL) was added a solution of
bromoacetyl bromide (319 mg, 1.58 mmol) in THF (1 mL)
dropwise with cooling in an ice-water bath. The mixture was
stirred at room temperature for 30 min. The reaction mixture
was diluted with CHCl3, washed with aqueous saturated
NaHCO3, water, and brine, and dried over Na2SO4. Filtration
and concentration in vacuo and purification by silica gel flash
chromatography (CHCl3/MeOH ) 150/1) gave 25 mg (23%) of
Step 3: Preparation of 6-Methanesulfonylaminohex-
anoic Acid Phenylamide (10). To a solution of 58 (500 mg,
2.06 mmol) obtained above in pyridine (5 mL) was added
methanesulfonyl chloride (160 µL, 2.07 mmol) dropwise with
cooling in an ice-water bath. The solution was stirred for 30
min at room temperature. The mixture was concentrated and
diluted with AcOEt. The solution was washed with 2 N
aqueous HCl, water, and brine and was dried over Na2SO4.
Filtration and concentration in vacuo and purification by silica
gel flash chromatography (n-hexane/AcOEt ) 1/3) gave 418
mg (71%) of 10 as a crude solid. The solid was recrystallized
from AcOEt to give 10 (214 mg) as colorless crystals: mp 136-
1
18 as a white solid: H NMR (CDCl3, 400 MHz, δ; ppm) 7.52
(2H, d, J ) 8.1 Hz), 7.32 (2H, t, J ) 7.9 Hz), 7.19 (1H, broad
s), 7.10 (1H, t, J ) 7.6 Hz), 6.56 (1H, broad s), 3.87 (2H, s),
3.32 (2H, q, J ) 6.6 Hz), 2.38 (2H, t, J ) 7.3 Hz), 1.80-1.76
(2H, m), 1.63-1.59 (2H, m), 1.46-1.44 (2H, m); MS (EI) m/z:
326 (M+); Anal. (C14H19BrN2O2) C, H, N.
Thioacetic acid S-[(6-Phenylcarbamoylpentylcarbam-
oyl)methyl] Ester (15). To a suspension of 18 (187 mg, 0.57
mmol) obtained above in EtOH (2 mL) was added potassium
thioacetate (236 mg, 2.07 mmol), and the mixture was stirred
at room temperature for 16 h. The reaction mixture was
diluted with AcOEt and THF, washed with water and brine,
and dried over Na2SO4. Filtration and concentration in vacuo
and purification by silica gel flash chromatography (n-hexane/
AcOEt ) 1/1) gave 163 mg (89%) of 15 as a white solid. The
solid was recrystallized from n-hexane-AcOEt to give 48 mg
1
137 °C; H NMR (DMSO-d6, 500 MHz, δ; ppm) 9.85 (1H, s),
7.58 (2H, d, J ) 7.7 Hz), 7.28 (2H, t, J ) 7.4 Hz), 7.01 (1H, t,
J ) 7.4 Hz), 6.93 (1H, t, J ) 6.5 Hz), 2.92 (2H, q, J ) 6.5 Hz),
2.87 (3H, s), 2.30 (2H, t, J ) 7.6 Hz), 1.59 (2H, quintet, J )
7.6 Hz), 1.59 (2H, quintet, J ) 7.6 Hz), 1.48 (2H, quintet, J )

Inhibitors of Human Histone Deacetylases
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1027
of 15 as a colorless crystal: mp 130-133 °C; 1H NMR (CDCl3,
400 MHz, δ; ppm) 7.53 (2H, d, J ) 7.3 Hz), 7.32 (2H, t, J )
7.9 Hz), 7.25 (1H, broad s), 7.10 (1H, t, J ) 7.2 Hz), 6.25 (1H,
broad s), 3.51 (2H, s), 3.25 (2H, q, J ) 6.6 Hz), 2.39 (3H, s),
2.37 (2H, t, J ) 7.4 Hz), 1.75 (2H, quintet, J ) 7.6 Hz), 1.55
(2H, quintet, J ) 7.1 Hz), 1.39 (2H, quintet, J ) 7.3 Hz); MS
(EI) m/z: 322 (M+); Anal. (C16H22N2O3S) C, H, N.
a mixture of O-(2-tetrahydropyranyl)hydroxylamine (251 mg,
2.14 mmol), a catalytic amount of 4-(dimethylamino)pyridine,
pyridine (1 mL), and CH2Cl2 (10 mL) was added a solution of
60 (500 mg, 1.95 mmol) obtained above in CH2Cl2 (10 mL),
and the mixture was stirred at room temperature for 5 h. The
reaction mixture was poured into water and extracted with
AcOEt. The AcOEt layer was separated, washed with water,
saturated aqueous NaHCO3 and brine, and dried over Na2-
SO4. Filtration and concentration in vacuo and purification by
silica gel flash chromatography (n-hexane/AcOEt ) 2/1) gave
618 mg (94%) of the sulfonamide as a crude oil.
To a solution of the sulfonamide(615 mg, 1.82 mmol)
obtained above in EtOH (3 mL) was added 2 N aqueous NaOH
(3.0 mL, 6.0 mmol). The mixture was stirred overnight at room
temperature. The solvent was removed by evaporation in
vacuo, and water was added to the residue. The mixture was
neutralized with 2 N aqueous HCl (3.0 mL, 6.0 mmol) with
cooling in an ice-water bath, and the mixture was extracted
with AcOEt. The AcOEt layer was separated, washed with
water and brine, and dried over Na2SO4. Filtration and
concentration in vacuo gave 482 mg (86%) of the carboxylic
acid as a white solid: 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.40
(1H, broad s), 5.08 (1H, m), 3.93 (1H, m), 3.66 (1H, m), 3.21
(2H, m), 2.37 (2H, t, J ) 7.3 Hz), 1.90-1.35 (14H, m).
Compound 61 was prepared from the carboxylic acid ob-
tained above and aniline using the procedure described for 13
in 88% yield: 1H NMR (DMSO-d6, 400 MHz, δ; ppm) 10.04
(1H, broad s), 9.85 (1H, broad s), 7.58 (2H, d, J ) 8 Hz), 7.28
(2H, t, J ) 7.8 Hz), 7.01 (1H, t, J ) 7.6 Hz), 4.88 (1H, m), 3.81
(1H, m), 3.52 (1H, m), 3.19-3.09 (2H, m), 2.30 (2H, t, J ) 7.3
Hz), 1.80-1.25 (14H, m).
Step 6: Preparation of 7-Hydroxysulfamoylheptanoic
Acid Phenylamide (6). Compound 6 was prepared from 61
obtained above using the procedure described for 12 (step 2)
in 61% yield: mp 137-139 °C; 1H NMR (DMSO-d6, 400 MHz,
δ; ppm) 9.85 (1H, broad s), 9.51 (1H, d, J ) 3.2 Hz), 9.13 (1H,
d, J ) 3.2 Hz), 7.58 (2H, d, J ) 8 Hz), 7.28 (2H, t, J ) 7.8 Hz),
7.01 (1H, t, J ) 7.3 Hz), 3.09 (2H, t, J ) 7.6 Hz), 2.30 (2H, t,
J ) 7.3 Hz), 1.68 (2H, quintet, J ) 8 Hz), 1.59 (2H, quintet, J
) 7.6 Hz), 1.41 (2H, quintet, J ) 7.8 Hz), 1.32 (2H, quintet, J
) 7.1 Hz); Anal. (C13H20N2O4S‚1/20H2O) C, H, N.
Thioacetic acid S-(6-phenylcarbamoylhexyl) Ester
(8a). Steps 1, 2, and 3: Preparation of 7-Bromoheptanoic
Acid Phenylamide (64c). 7-Bromoheptanoic acid was pre-
pared from 59 using the procedure described for 6 (step 4) in
99% yield. In this case, LiOH was used instead of NaOH: 1H
NMR (CDCl3, 400 MHz, δ; ppm) 3.41 (2H, t, J ) 6.8 Hz), 2.37
(2H, t, J ) 7.3 Hz), 1.87 (2H, quintet, J ) 6.8 Hz), 1.66 (2H,
quintet, J ) 7.6 Hz), 1.54-1.32 (4H, m).
To a suspension of 7-bromoheptanoic acid (2.64 g, 12.6
mmol) obtained above in CH2Cl2 (30 mL) were added oxalyl
chloride (1.65 mL, 18.9 mmol) and a catalytic amount of DMF.
The mixture was stirred at room temperature for 2 h. The
solvent was removed by evaporation in vacuo to give acid
chloride 62c.
To a solution of aniline (3.50 g, 37.6 mmol) and triethyl-
amine (5.30 mL, 38.1 mmol) in CH2Cl2 (40 mL) was added a
solution of 62c obtained above in CH2Cl2 (10 mL) dropwise
cooling in an ice-water bath. The mixture was stirred at room
temperature for 1 h. It was diluted with AcOEt and washed
with aqueous saturated NaHCO3, water, and brine, before
being dried over MgSO4. Filtration and concentration in vacuo
and purification by silica gel flash chromatography (n-hexane/
AcOEt ) 3/1) gave 3.13 g (87%) of 64c: 1H NMR (CDCl3, 400
MHz, δ; ppm) 7.51 (2H, d, J ) 8.1 Hz), 7.32 (2H, t, J ) 7.6
Hz), 7.15 (1H, broad s), 7.10 (1H, t, J ) 7.6 Hz), 3.41 (2H, t, J
) 6.8 Hz), 2.36 (2H, t, J ) 7.3 Hz), 1.87 (2H, quintet, J ) 7.1
Hz), 1.75 (2H, quintet, J ) 7.3 Hz), 1.49 (2H, quintet, J ) 7.6
Hz), 1.41 (2H, quintet, J ) 6.8 Hz).
6-(2-Mercaptoacetylamino)hexanoic Acid Phenylam-
ide (14). To a solution of 15 (190 mg, 0.59 mmol) obtained
above in MeOH (5 mL) was added K2CO3 (141 mg, 1.02 mmol),
and the mixture was stirred at room temperature for 1 h. The
reaction mixture was diluted with AcOEt and THF, washed
with water and brine, and dried over Na2SO4. Filtration and
concentration in vacuo and purification by silica gel flash
chromatography (CHCl3/MeOH ) 20/1) gave 103 mg (62%) of
14 as a white solid. The solid was recrystallized from CHCl3-
MeOH to give 38 mg of 14 as a colorless crystal: mp 171-173
1
°C; H NMR (DMSO-d6, 500 MHz, δ; ppm) 9.84 (1H, s), 8.08
(1H, broad s), 7.57 (2H, d, J ) 8.2 Hz), 7.27 (2H, t, J ) 7.9
Hz), 7.00 (1H, t, J ) 7.3 Hz), 3.90 (1H, s), 3.44 (2H, s), 3.07
(2H, q, J ) 6.5 Hz), 2.28 (2H, t, J ) 7.5 Hz), 1.59 (2H, quintet,
J ) 7.3 Hz), 1.45 (2H, quintet, J ) 7 Hz), 1.31 (2H, quintet, J
) 7.5 Hz); MS (EI) m/z: 280 (M+); Anal. (C14H20N2O2S) C, H,
N.
6-(2-Propynylamino)hexanoic Acid Phenylamide Hy-
drochloride Salt (16‚HCl) and 6-(2-Dipropynylamino)-
hexanoic Acid Phenylamide (17). To a solution of 58 (230
mg, 1.12 mmol) obtained above and K2CO3 (39 mg, 0.28 mmol)
in MeOH (1 mL) was added propargyl bromide (38 mg, 0.32
mmol), and the mixture was stirred overnight at room tem-
perature. The reaction mixture was concentrated in vacuo, and
the residue was purified by silica gel flash chromatography
(CHCl3/MeOH ) 15/1) to give 40 mg (51%) of 16 as a pale
yellow oil and 12 mg (23%) of 17 as a pale yellow solid. To a
solution of 16 in MeOH was added 1 N aqueous HCl (0.5 mL),
and the solution was concentrated in vacuo. The residue was
recrystallized from MeOH-AcOEt to give 16 mg of 16‚HCl as
1
colorless needles: mp 161-165 °C; H NMR (DMSO-d6, 400
MHz, δ; ppm) 9.91 (1H, broad s), 9.12 (2H, broad s), 7.59 (2H,
d, J ) 7.6 Hz), 7.28 (2H, t, J ) 7.9 Hz), 7.01 (1H, t, J ) 7.3
Hz), 3.89 (2H, d, J ) 3.4 Hz), 3.70 (1H, t, J ) 2.6 Hz), 2.94
(2H, t, J ) 7.8 Hz), 2.32 (2H, t, J ) 7.3 Hz), 1.64-1.56 (4H,
m), 1.36-1.25 (2H, m); MS (EI) m/z: 244 (M--HCl); Anal.
(C15H20N2O‚HCl‚1/8H2O) C, H, N.
The crude solid of 17 was recrystallized from CHCl3-n-
hexane to give 12 mg of 17 as colorless needles: mp 56-57
1
°C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.51 (2H, d, J ) 8.3
Hz), 7.32 (2H, t, J ) 7.8 Hz), 7.11-7.10 (2H, m), 3.43 (4H, d,
J ) 2.4 Hz), 2.55 (2H, t, J ) 7.3 Hz), 2.37 (2H, t, J ) 7.4 Hz),
2.22 (2H, t, J ) 2.3 Hz), 1.79-1.75 (2H, m), 1.54-1.52 (2H,
m), 1.45-1.43 (2H, m); MS (EI) m/z: 281 (M+); Anal.
(C18H22N2O) C, H, N.
7-Hydroxysulfamoylheptanoic Acid Phenylamide (6).
Steps 1 and 2: Preparation of 7-Chlorosulfonylhept-
anoic Acid Ethyl Ester (60). To an aqueous solution (7 mL)
of anhydrous sodium sulfite (2.03 g, 16.1 mmol) was added a
solution of 7-bromoheptanoic acid ethyl ester (59, 2.0 g, 8.43
mmol) in EtOH (5 mL), and the solution was boiled under
reflux with stirring for 2 h. The solution was evaporated to
dryness, and the solid was dried in vacuo at 60 °C. This white
solid was placed in a flask, toluene (30 mL) was added followed
by a catalytic amount of DMF, and then thionyl chloride (6.2
mL, 85.0 mmol) was added dropwise. The mixture was boiled
under reflux with stirring for 5 h, diluted with AcOEt, washed
with aqueous saturated cold water and brine, and dried over
MgSO4. Filtration and concentration in vacuo and purification
by silica gel flash chromatography (n-hexane/AcOEt ) 4/1)
gave 2.02 g (93%) of 60: 1H NMR (CDCl3, 400 MHz, δ; ppm)
4.13 (2H, q, J ) 7.1 Hz), 3.66 (2H, t, J ) 7.8 Hz), 2.31 (2H, t,
J ) 7.3 Hz), 2.06 (2H, quintet, J ) 7.8 Hz), 1.66 (2H, quintet,
J ) 7.3 Hz), 1.53 (2H, quintet, J ) 7.8 Hz), 1.41 (2H, quintet,
J ) 7.1 Hz), 1.26 (2H, quintet, J ) 7.1 Hz).
Step 4: Preparation of Thioacetic acid S-(6-Phenyl-
carbamoylhexyl) Ester (8a). Compound 8a was prepared
from 64c obtained above using the procedure described for 15
in 98% yield: mp 80-81 °C; 1H NMR (CDCl3, 400 MHz, δ;
ppm) 7.51 (2H, d, J ) 8 Hz), 7.32 (2H, t, J ) 7.3 Hz), 7.22
Steps 3, 4, and 5: Preparation of 7-(2-Tetrahydropy-
ranyloxysulfamoyl)heptanoic Acid Phenylamide (61). To

1028 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Suzuki et al.
(1H, broad s), 7.10 (1H, t, J ) 7.3 Hz), 2.86 (2H, t, J ) 7.1
Hz), 2.35 (2H, t, J ) 7.3 Hz), 2.32 (3H, s), 1.73 (2H, quintet, J
) 7.1 Hz), 1.59 (2H, quintet, J ) 7.1 Hz), 1.40 (4H, m); MS
(EI) m/z: 279 (M+); Anal. (C15H21NO2S) C, H, N.
m), 1.32 (1H, t, J ) 7.6 Hz); MS (EI) m/z: 329 (M+); Anal.
(C19H23NO2S) C, H, N.
7-Mercaptoheptanoic acid 3-pyridinylamide (29): mp
1
74-76 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 8.54 (1H, d, J
) 2.4 Hz), 8.35 (1H, d, J ) 4.4 Hz), 8.19 (1H, d, J ) 8.3 Hz),
7.31 (1H, broad s), 7.28 (1H, dd, J ) 4.4, 8.3 Hz), 2.53 (2H, q,
J ) 7.1 Hz), 2.40 (2H, t, J ) 7.3 Hz), 1.75 (2H, quintet, J )
7.6 Hz), 1.64 (2H, quintet, J ) 7.1 Hz), 1.50-1.36 (4H, m),
1.33 (1H, t, J ) 7.6 Hz); MS (EI) m/z: 237 (M+); Anal.
(C12H18N2OS) C, H, N.
7-Mercaptoheptanoic Acid Phenylamide (7) and 7-(6-
Phenylcarbamoylhexyldisulfanyl)heptanoic Acid Phen-
ylamide (37). Compounds 7 and 37 were prepared from 8a
using the procedure described for 6 (step 4) in 87% and 4%
yield, respectively.
1
7: mp 88-89 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.51
7-Mercaptoheptanoic acid 3-quinolinylamide (30): mp
(2H, d, J ) 8 Hz), 7.32 (2H, t, J ) 7.6 Hz), 7.12 (1H, broad s),
7.10 (1H, t, J ) 7.1 Hz), 2.53 (2H, q, J ) 7.3 Hz), 2.36 (2H, t,
J ) 7.6 Hz), 1.74 (2H, quintet, J ) 7.1 Hz), 1.63 (2H, quintet,
J ) 7.1 Hz), 1.42 (4H, m), 1.33 (1H, t, J ) 7.8 Hz); MS (EI)
m/z: 237 (M+); Anal. (C13H19NOS) C, H, N.
1
75-76 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 8.79 (1H, d, J
) 2.7 Hz), 8.72 (1H, d, J ) 2.7 Hz), 8.04 (1H, d, J ) 8.3 Hz),
7.80 (1H, d, J ) 8.3 Hz), 7.64 (1H, t, J ) 7.1 Hz), 7.54 (1H, t,
J ) 7.1 Hz), 7.50 (1H, broad s), 2.54 (2H, q, J ) 7.1 Hz), 2.47
(2H, t, J ) 7.3 Hz), 1.80 (2H, quintet, J ) 7.3 Hz), 1.64 (2H,
quintet, J ) 7.3 Hz), 1.53-1.37 (4H, m), 1.34 (1H, t, J ) 7.8
Hz); MS (EI) m/z: 288 (M+); Anal. (C16H20N2OS) C, H, N.
7-Mercaptoheptanoic acid (4-phenyl-2-thiazolyl)am-
ide (31): mp 149-150 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm)
10.36 (1H, broad s), 7.83 (2H, d, J ) 7.1 Hz), 7.43 (2H, t, J )
7.3 Hz), 7.16 (1H, s), 2.49 (2H, q, J ) 7.1 Hz), 2.14 (2H, t, J )
7.6 Hz), 1.65-1.50 (4H, m), 1.32 (1H, t, J ) 7.6 Hz), 1.30 (2H,
quintet, J ) 7.3 Hz), 1.15 (2H, quintet, J ) 7.1 Hz); MS (EI)
m/z: 320 (M+); Anal. (C16H20N2OS2‚1/10H2O) C, H, N.
7-Mercaptoheptanoic acid 2-benzothiazolylamide (32):
mp 141-142 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 10.71 (1H,
broad s), 7.86 (1H, d, J ) 7.9 Hz), 7.77 (1H, d, J ) 8 Hz), 7.46
(1H, t, J ) 8.3 Hz), 7.34 (1H, t, J ) 8.3 Hz), 2.49 (2H, t, J )
7.1 Hz), 2.48 (2H, q, J ) 7.3 Hz), 1.72 (2H, quint, J ) 7.6 Hz),
1.57 (2H, quint, J ) 7.3 Hz), 1.40-1.25 (5H, m); MS (EI) m/z:
294 (M+); Anal. (C14H18N2OS2) C, H, N.
37: mp 105-107 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm)
7.51 (4H, d, J ) 8 Hz), 7.41 (2H, broad s), 7.30 (4H, t, J ) 7.8
Hz), 7.09 (2H, t, J ) 7.3 Hz), 2.68 (4H, t, J ) 7.3 Hz), 2.36
(4H, t, J ) 7.6 Hz), 1.74 (4H, quintet, J ) 7.3 Hz), 1.69 (4H,
quintet, J ) 7.1 Hz), 1.50-1.34 (8H, m); MS (EI) m/z: 472
(M+); Anal. (C26H36N2O2S2) C, H, N.
Compounds19-21, 24, 26-31, and 32 were prepared from
62 and an appropriate aromatic amine using the procedure
described for 8a and 7.
8-Mercaptooctanoic acid phenylamide (19): mp 84-
1
86 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.51 (2H, d, J ) 8
Hz), 7.32 (2H, t, J ) 7.6 Hz), 7.14 (1H, broad s), 7.10 (1H, t, J
) 7.3 Hz), 2.52 (2H, q, J ) 7.3 Hz), 2.35 (2H, t, J ) 7.6 Hz),
1.73 (2H, quintet, J ) 7.3 Hz), 1.61 (2H, quintet, J ) 7.1 Hz),
1.46-1.34 (6H, m), 1.33 (1H, t, J ) 7.8 Hz); MS (EI) m/z: 251
(M+); Anal. (C14H21NOS) C, H, N.
6-Mercaptohexanoic acid phenylamide (20): mp 84-
85 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.51 (2H, d, J ) 8.1
Hz), 7.32 (2H, t, J ) 7.6 Hz), 7.16 (1H, broad s), 7.11 (1H, t, J
) 7.8 Hz), 2.55 (2H, q, J ) 7.1 Hz), 2.37 (2H, t, J ) 7.3 Hz),
1.75 (2H, quintet, J ) 7.8 Hz), 1.68 (2H, quintet, J ) 7.6 Hz),
1.56-1.40 (2H, m), 1.35 (1H, t, J ) 7.8 Hz); MS (EI) m/z: 223
(M+); Anal. (C12H17NOS) C, H, N.
7-Mercaptoheptanoic Acid 4-Biphenylylamide (25).
Step 1: Preparation of 7-Bromoheptanoic Acid (4-Bro-
mophenyl)amide (64a). Compound 64a was prepared from
62c and 4-bromoaniline using the procedure described for 8a
(step 3) in 86% yield: 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.42
(4H, s), 7.14 (1H, broad s), 3.41 (2H, t, J ) 6.6 Hz), 2.36 (2H,
t, J ) 7.6 Hz), 1.87 (2H, quintet, J ) 7.1 Hz), 1.74 (2H, quintet,
J ) 7.3 Hz), 1.49 (2H, quintet, J ) 7.3 Hz), 1.40 (2H, quintet,
J ) 6.8 Hz).
Step 2: Preparation of 7-Bromoheptanoic Acid 4-Bi-
phenylylamide (64b). To a suspension of 64a (500 mg, 1.38
mmol) obtained above in 1-methyl-2-pyrrolidinone (8 mL) and
water (4 mL) were added phenylboronic acid (252 mg, 2.07
mmol), tetrakis(triphenylphosphine)palladium(0) (160 mg, 0.14
mmol), and NaHCO3 (235 mg, 2.80 mmol). The mixture was
heated at 80 °C for 1 h. The solution was diluted with AcOEt,
washed with saturated aqueous NaHCO3, water, and brine,
and dried over Na2SO4. Filtration and concentration in vacuo
and purification by silica gel flash chromatography (n-hexane/
AcOEt ) 3/1) gave 91 mg (18%) of 64b as a white solid: 1H
NMR (CDCl3, 400 MHz, δ; ppm) 7.65-7.50 (6H, m), 7.43 (2H,
t, J ) 7.6 Hz), 7.33 (1H, t, J ) 7.1 Hz), 7.20 (1H, broad s),
3.42 (2H, t, J ) 6.6 Hz), 2.39 (2H, t, J ) 7.3 Hz), 1.88 (2H,
quintet, J ) 7.1 Hz), 1.77 (2H, quintet, J ) 7.3 Hz), 1.50 (2H,
quintet, J ) 7.1 Hz), 1.43 (2H, quintet, J ) 6.4 Hz).
Steps 3 and 4: Preparation of 7-Mercaptoheptanoic
Acid 4-Biphenylylamide (25). Compound 25 was prepared
from 64b obtained above using the procedure described for 15
and 6 (step 4) in 48% yield: mp 114-115 °C; 1H NMR (CDCl3,
400 MHz, δ; ppm) 7.64-7.52 (6H, m), 7.43 (2H, t, J ) 7.6 Hz),
7.33 (1H, t, J ) 7.3 Hz), 7.17 (1H, broad s), 2.54 (2H, q, J )
7.4 Hz), 2.39 (2H, t, J ) 7.3 Hz), 1.76 (2H, quintet, J ) 7.3
Hz), 1.64 (2H, quintet, J ) 7.3 Hz), 1.52-1.37 (4H, m), 1.34
(1H, t, J ) 7.6 Hz); MS (EI) m/z: 313 (M+); Anal. (C19H23NOS‚
1/5H2O) C, H, N.
7-Methylsulfanylheptanoic Acid Phenylamide (9). To
a solution of 64c (300 mg, 1.06 mmol) in EtOH (10 mL) was
added methylmercaptan sodium salt (15% in water, 1.50 g,
3.21 mmol), and the solution was stirred at room temperature
for 5 h. The reaction mixture was diluted with AcOEt, washed
with water and brine, and dried over MgSO4. Filtration and
concentration in vacuo and purification by silica gel flash
5-Mercaptopentanoic acid phenylamide (21): mp 120-
1
121 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.51 (2H, d, J )
7.6 Hz), 7.33 (2H, t, J ) 8 Hz), 7.16 (1H, broad s), 7.11 (1H, t,
J ) 7.8 Hz), 2.58 (2H, q, J ) 6.4 Hz), 2.39 (2H, t, J ) 6.8 Hz),
1.85 (2H, quintet, J ) 7.8 Hz), 1.71 (2H, quintet, J ) 7.6 Hz),
1.39 (1H, t, J ) 8 Hz); MS (EI) m/z: 209 (M+); Anal. (C11H15-
NOS‚1/12H2O) C, H, N.
7-Mercaptoheptanoic acid (4-dimethylaminophenyl)-
1
amide (24): mp 121-122 °C; H NMR (CDCl3, 400 MHz, δ;
ppm) 7.51 (2H, d, J ) 9 Hz), 6.96 (1H, broad s), 6.70 (2H, d, J
) 9 Hz), 2.91 (6H, s), 2.53 (2H, q, J ) 7.3 Hz), 2.32 (2H, t, J
) 7.3 Hz), 1.73 (2H, quintet, J ) 7.4 Hz), 1.63 (2H, quintet, J
) 7.6 Hz), 1.50-1.35 (4H, m), 1.33 (1H, t, J ) 7.8 Hz); MS
(EI) m/z: 280 (M+); Anal. (C15H24N2OS) C, H, N.
7-Mercaptoheptanoic acid 3-biphenylylamide (26): mp
91-92 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.78 (1H, s),
7.59 (2H, d, J ) 7.6 Hz), 7.49 (1H, d, J ) 7.4 Hz), 7.47-7.30
(5H, m), 7.18 (1H, broad s), 2.53 (2H, q, J ) 7.3 Hz), 2.39 (2H,
t, J ) 7.3 Hz), 1.76 (2H, quintet, J ) 7.1 Hz), 1.64 (2H, quintet,
J ) 7.3 Hz), 1.50-1.37 (4H, m), 1.33 (1H, t, J ) 7.6 Hz); MS
(EI) m/z: 313 (M+); Anal. (C19H23NOS) C, H, N.
7-Mercaptoheptanoic acid (4-phenoxyphenyl)amide
1
(27): mp 87-89 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.47
(2H, d, J ) 8.8 Hz), 7.32 (2H, t, J ) 7.8 Hz), 7.12 (1H, broad
s), 7.08 (1H, t, J ) 7.3 Hz), 6.98 (4H, d, J ) 8.8 Hz), 2.53 (2H,
q, J ) 7.3 Hz), 2.36 (2H, t, J ) 7.6 Hz), 1.75 (2H, quintet, J )
7.1 Hz), 1.64 (2H, quintet, J ) 7.1 Hz), 1.50-1.37 (4H, m),
1.33 (1H, t, J ) 7.8 Hz); MS (EI) m/z: 329 (M+); Anal. (C19H23-
NO2S) C, H, N.
7-Mercaptoheptanoic acid (3-phenoxyphenyl)amide
1
(28): mp 68-69 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.34
(2H, t, J ) 7.6 Hz), 7.30-7.18 (3H, m), 7.16 (1H, broad s), 7.11
(1H, t, J ) 7.2 Hz), 7.02 (2H, d, J ) 8.5 Hz), 6.74 (1H, s), 2.52
(2H, q, J ) 7.3 Hz), 2.33 (2H, t, J ) 7.3 Hz), 1.71 (2H, quintet,
J ) 7.3 Hz), 1.62 (2H, quintet, J ) 7.1 Hz), 1.50-1.34 (4H,

Inhibitors of Human Histone Deacetylases
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1029
chromatography (n-hexane/AcOEt ) 2/1) gave 262 mg (99%)
of 9 as a crude solid. The solid was recrystallized from
n-hexane-AcOEt and collected by filtration to give 217 mg of
s), 3.47 (2H, q, J ) 6.4 Hz), 2.54 (2H, q, J ) 7.6 Hz), 1.68-
1.58 (4H, m), 1.50-1.36 (4H, m), 1.52-1.37 (4H, m), 1.34 (1H,
t, J ) 7.8 Hz); MS (EI) m/z: 237 (M+); Anal. (C13H19NOS‚1/
6H2O) C, H, N.
1
9 as a colorless crystal: mp 50-51 °C; H NMR (CDCl3, 400
MHz, δ; ppm) 7.51 (2H, d, J ) 8 Hz), 7.32 (2H, t, J ) 7.8 Hz),
7.16 (1H, broad s), 7.10 (1H, t, J ) 7.6 Hz), 2.49 (2H, t, J )
7.1 Hz), 2.36 (2H, t, J ) 7.3 Hz), 2.09 (3H, s), 1.74 (2H, quintet,
J ) 7.3 Hz), 1.61 (2H, quintet, J ) 7.3 Hz), 1.42 (4H, m); MS
(EI) m/z: 251 (M+); Anal. (C14H21NOS) C, H, N.
4-Dimethylamino-N-(6-mercaptohexyl)benzamide (33):
mp 103-104 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.66 (2H,
t, J ) 8.8 Hz), 6.67 (2H, d, J ) 8.8 Hz), 5.95 (1H, s), 3.43 (2H,
q, J ) 6.4 Hz), 3.02 (1H, s), 2.53 (2H, q, J ) 7.2 Hz), 1.67-
1.54 (4H, m), 1.49-1.36 (4H, m), 1.32 (1H, t, J ) 8 Hz); MS
(EI) m/z: 280 (M+); Anal. (C15H24N2OS) C, H, N.
7-Methanesulfonylheptanoic Acid Phenylamide (11).
To a solution of 9 (80 mg, 0.32 mmol) in CH2Cl2 (3 mL) was
added 3-chloroperoxybenzoic acid (65%, 180 mg, 0.68 mmol).
The mixture was stirred overnight at room temperature. Next,
saturated aqueous NaHCO3 and saturated aqueous Na2S2O3
were added, and the mixture was stirred at room temperature
for 1 h. It was then poured into water and extracted with
CHCl3. The CHCl3 layer was separated, washed with water
and brine, and dried over Na2SO4. Filtration and concentration
in vacuo and separation by silica gel flash chromatography
(n-hexane/AcOEt ) 1/3) gave 63 mg (70%) of 11 as a crude
solid. The solid was recrystallized from n-hexane-AcOEt and
collected by filtration to give 50 mg of 11 as a colorless
crystal: mp 121-123 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm)
7.51 (2H, d, J ) 7.8 Hz), 7.32 (2H, t, J ) 7.6 Hz), 7.17 (1H,
brs), 7.11 (1H, t, J ) 7.3 Hz), 3.01 (2H, t, J ) 7.8 Hz), 2.89
(3H, s), 2.37 (2H, t, J ) 7.3 Hz), 1.88 (2H, quint, J ) 7.6 Hz),
1.76 (2H, quint, J ) 7.6 Hz), 1.60-1.35 (4H, m); MS (EI) m/z:
283 (M+); Anal. (C14H21NO3S) C, H, N.
6-Phenoxy-1-hexanethiol (22). Step 1: Preparation of
6-Phenoxy-1-hexanol (67). To a solution of phenol (2.10 g,
22.31 mmol) and 6-bromo-1-hexanol (65, 2.00 g, 11.05 mmol)
in DMF (30 mL) was added K2CO3 (3.10 g, 22.4 mmol), and
the mixture was stirred at 80 °C for 1 h. The reaction mixture
was diluted with AcOEt and washed with water and brine,
before being dried over Na2SO4. Filtration and concentration
in vacuo and purification by silica gel flash chromatography
(n-hexane/AcOEt ) 2/1) gave 2.06 g (96%) of 67 as a white
solid: 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.28 (2H, t, J ) 7.8
Hz), 6.93 (1H, t, J ) 7.3 Hz), 6.89 (2H, d, J ) 8.6 Hz), 3.96
(2H, t, J ) 6.6 Hz), 3.67 (2H, m), 1.80 (2H, quintet, J ) 6.8
Hz), 1.61 (2H, quintet, J ) 7.3 Hz), 1.56-1.36 (4H, m), 1.27
(1H, m).
Step 2: Preparation of (6-Bromohexyloxy)benzene. To
a solution of 67 (1.75 g, 9.01 mmol) obtained above and carbon
tetrabromide (3.00 g, 9.05 mmol) in CH2Cl2 (50 mL) was added
triphenylphosphine (2.60 g, 9.91 mmol) with cooling in an ice-
water bath. The solution was stirred at room temperature for
1 h and concentrated in vacuo. To the residue was added
n-hexane (30 mL), and the slurry was filtered. After the solid
was washed with n-hexane (10 mL), the combined filtrates
were concentrated in vacuo. The residue was purified by silica
gel flash chromatography (n-hexane/AcOEt ) 1/30) to give 1.45
g (63%) of (6-bromohexyloxy)benzene as a colorless oil: 1H
NMR (CDCl3, 400 MHz, δ; ppm) 7.28 (2H, t, J ) 7.6 Hz), 6.93
(1H, t, J ) 7.3 Hz), 6.89 (2H, d, J ) 8.5 Hz), 3.96 (2H, t, J )
6.3 Hz), 3.43 (2H, t, J ) 6.8 Hz), 1.90 (2H, quintet, J ) 6.8
Hz), 1.80 (2H, quintet, J ) 6.4 Hz), 1.56-1.46 (4H, m).
Steps 3 and 4: Preparation of 6-Phenoxy-1-hexanethi-
ol (22). Compound 22 was prepared from (6-bromohexyloxy)-
benzene obtained above using the procedure described for 15
and 6 (step 4) in 45% yield: colorless oil; 1H NMR (CDCl3,
400 MHz, δ; ppm) 7.28 (2H, t, J ) 7.3 Hz), 6.93 (1H, t, J ) 7.6
Hz), 6.89 (2H, d, J ) 7.8 Hz), 3.96 (2H, t, J ) 6.4 Hz), 2.54
(2H, q, J ) 7.1 Hz), 1.79 (2H, quintet, J ) 6.6 Hz), 1.65 (2H,
quintet, J ) 6.8 Hz), 1.54-1.44 (4H, m), 1.34 (1H, t, J ) 7.8
Hz); MS (EI) m/z: 210 (M+); HRMS calcd for C12H18OS 210.108,
found 210.108.
Naphthalene-2-carboxylic acid (6-mercaptohexyl)am-
1
ide (34): mp 76-78 °C; H NMR (CDCl3, 400 MHz, δ; ppm)
8.28 (1H, s), 7.94-7.85 (3H, m), 7.82 (1H, d, J ) 6.8 Hz), 7.58-
7.53 (2H, m), 6.27 (1H, s), 3.52 (2H, q, J ) 6.8 Hz), 2.54 (2H,
q, J ) 7.6 Hz), 1.70-1.62 (4H, m), 1.52-1.36 (4H, m), 1.34
(1H, t, J ) 7.8 Hz); MS (EI) m/z: 287 (M+); Anal. (C17H21NOS)
C, H, N.
Benzofuran-2-carboxylic acid (6-mercaptohexyl)am-
1
ide (35): mp 72-73 °C; H NMR (CDCl3, 400 MHz, δ; ppm)
7.68 (1H, d, J ) 8 Hz), 7.50 (1H, d, J ) 8.4 Hz), 7.46 (1H, s),
7.41 (1H, t, J ) 8.4 Hz), 7.30 (1H, t, J ) 8 Hz), 6.64 (1H, s),
3.49 (2H, q, J ) 7.2 Hz), 2.54 (2H, q, J ) 7.2 Hz), 1.72-1.58
(4H, m), 1.52-1.38 (4H, m), 1.34 (1H, t, J ) 7.8 Hz); MS (EI)
m/z: 277 (M+); Anal. (C15H19NO2S) C, H, N.
Indole-2-carboxylic acid (6-mercaptohexyl)amide (36):
mp 128-130 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 9.12 (1H,
broad s), 7.65 (1H, d, J ) 8 Hz), 7.44 (1H, d, J ) 8.4 Hz), 7.29
(1H, t, J ) 8 Hz), 7.14 (1H, t, J ) 6.6 Hz), 6.82 (1H, s), 6.13
(1H, broad s), 3.49 (2H, q, J ) 6.8 Hz), 2.54 (2H, q, J ) 7.6
Hz), 1.70-1.60 (4H, m), 1.45-1.40 (4H, m), 1.34 (1H, t, J )
7.8 Hz); MS (EI) m/z: 276 (M+); Anal. (C15H20N2OS) C, H, N.
Thiopropionic Acid S-(6-Phenylcarbamoylhexyl) Es-
ter (38). To a solution of 7 (200 mg, 0.84 mmol) and a catalytic
amount of 4-(dimethylamino)pyridine in CH2Cl2 (2 mL) and
pyridine (0.5 mL) was added propionyl chloride (220 µL, 2.53
mmol). The mixture was stirred at room temperature for 30
min and then diluted with AcOEt. The solution was washed
with water and brine and dried over Na2SO4. Filtration and
concentration in vacuo and separation by silica gel flash
chromatography (n-hexane/AcOEt ) 3/1) gave 238 mg (96%)
of 38 as a crude solid. The solid was recrystallized from
n-hexane-AcOEt and collected by filtration to give 184 mg of
38 as a colorless crystal: mp 54-55 °C; 1H NMR (CDCl3, 500
MHz, δ; ppm) 7.52 (2H, d, J ) 7.9 Hz), 7.32 (2H, t, J ) 7.9
Hz), 7.21 (1H, broad s), 7.10 (1H, t, J ) 7.3 Hz), 2.86 (2H, t, J
) 7.4 Hz), 2.57 (2H, q, J ) 7.7 Hz), 2.35 (2H, t, J ) 7.6 Hz),
1.74 (2H, quintet, J ) 7.3 Hz), 1.59 (2H, quintet, J ) 7.3 Hz),
1.46-1.33 (4H, m), 1.18 (3H, t, J ) 7.7 Hz); MS (EI) m/z: 293
(M+); Anal. (C16H23NO2S) C, H, N.
Compounds 39-45, 47-54, and 55 were prepared from the
corresponding thiols and an appropriate acid chloride using
the procedure described for 38.
Thiobutyric acid S-(6-phenylcarbamoylhexyl) ester
1
(39): mp 45-46 °C; H NMR (CDCl3, 500 MHz, δ; ppm) 7.52
(2H, d, J ) 8 Hz), 7.32 (2H, t, J ) 7.6 Hz), 7.21 (1H, broad s),
7.10 (1H, t, J ) 7.3 Hz), 2.86 (2H, t, J ) 7 Hz), 2.52 (2H, t, J
) 7.3 Hz), 2.35 (2H, t, J ) 7.4 Hz), 1.73 (2H, quintet, J ) 7.4
Hz), 1.69 (2H, sextet, J ) 7.7 Hz), 1.59 (2H, quintet, J ) 7.4
Hz), 1.48-1.33 (4H, m), 0.95 (3H, t, J ) 7.3 Hz); MS (EI) m/z:
307 (M+); Anal. (C17H25NO2S) C, H, N.
Thioisobutyric acid S-(6-phenylcarbamoylhexyl) ester
1
(40): mp 44-45 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.52
(2H, d, J ) 8 Hz), 7.32 (2H, t, J ) 7.8 Hz), 7.22 (1H, broad s),
7.10 (1H, t, J ) 7.3 Hz), 2.85 (2H, t, J ) 7.3 Hz), 2.73 (1H,
septet, J ) 7 Hz), 2.35 (2H, t, J ) 7.3 Hz), 1.73 (2H, quintet,
J ) 7.3 Hz), 1.59 (2H, quintet, J ) 7.3 Hz), 1.46-1.36 (4H,
m), 1.19 (6H, d, J ) 7.6 Hz); MS (EI) m/z: 307 (M+); Anal.
(C17H25NO2S) C, H, N.
Compounds23, 33-35, and 36 were prepared from an
appropriate aromatic carboxylic acid and 6-amino-1-hexanol
(66) using the procedure described for 13, 22 (step 2), 15, and
6 (step 4).
2,2-Dimethylthiopropionic acid S-(6-phenylcarbam-
oylhexyl) ester (41): mp 57-59 °C; 1H NMR (CDCl3, 400
MHz, δ; ppm) 7.52 (2H, d, J ) 8.1 Hz), 7.32 (2H, t, J ) 7.6
Hz), 7.20 (1H, broad s), 7.10 (1H, t, J ) 7.6 Hz), 2.82 (2H, t, J
) 7.3 Hz), 2.35 (2H, t, J ) 7.3 Hz), 1.73 (2H, quintet, J ) 7.3
N-(6-Mercaptohexyl)benzamide (23): mp 43-44 °C; 1H
NMR (CDCl3, 400 MHz, δ; ppm) 7.77 (2H, d, J ) 7.2 Hz), 7.50
(1H, t, J ) 7.2 Hz), 7.43 (2H, t, J ) 6.8 Hz), 6.20 (1H, broad

1030 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Suzuki et al.
Hz), 1.58 (2H, quintet, J ) 7.3 Hz), 1.46-1.36 (4H, m), 1.23
(9H, s); MS (EI) m/z: 321 (M+); Anal. (C18H27NO2S) C, H, N.
Cyclopropanecarbothioic acid S-(6-phenylcarbamoyl-
hexyl) ester (42): mp 64-65 °C; 1H NMR (CDCl3, 500 MHz,
δ; ppm) 7.52 (2H, d, J ) 8.3 Hz), 7.32 (2H, t, J ) 7.6 Hz), 7.22
(1H, broad s), 7.10 (1H, t, J ) 7.3 Hz), 2.89 (2H, t, J ) 7.3
Hz), 2.35 (2H, t, J ) 7.3 Hz), 2.01 (1H, m), 1.73 (2H, quintet,
J ) 7 Hz), 1.59 (2H, quintet, J ) 7.3 Hz), 1.45-1.35 (4H, m),
1.15 (2H, m), 0.94 (2H, m); MS (EI) m/z: 305 (M+); Anal.
(C17H23NO2S) C, H, N.
Cyclopentanecarbothioic acid S-(6-phenylcarbamoyl-
hexyl) ester (43): mp 59-60 °C; 1H NMR (CDCl3, 500 MHz,
δ; ppm) 7.52 (2H, d, J ) 7.9 Hz), 7.32 (2H, t, J ) 7.9 Hz), 7.21
(1H, broad s), 7.10 (1H, t, J ) 7.3 Hz), 2.97 (1H, quintet, J )
8 Hz), 2.85 (2H, t, J ) 7.4 Hz), 2.35 (2H, t, J ) 7.7 Hz), 1.93-
1.67 (8H, m), 1.63-1.52 (4H, m), 1.47-1.33 (4H, m); MS (EI)
m/z: 333 (M+); Anal. (C19H27NO2S) C, H, N.
Thiobenzoic acid S-(6-phenylcarbamoylhexyl) ester
(44): mp 107-109 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.97
(2H, d, J ) 7.3 Hz), 7.57 (1H, t, J ) 7.3 Hz), 7.52 (2H, d, J )
7.8 Hz), 7.45 (2H, t, J ) 7.8 Hz), 7.31 (2H, t, J ) 7.6 Hz), 7.21
(1H, broad s), 7.10 (1H, t, J ) 7.3 Hz), 3.07 (2H, t, J ) 7.3
Hz), 2.36 (2H, t, J ) 7.3 Hz), 1.75 (2H, quintet, J ) 7.3 Hz),
1.70 (2H, quintet, J ) 7.3 Hz), 1.54-1.36 (4H, m); MS (EI)
m/z: 341 (M+); Anal. (C20H23NO2S) C, H, N.
4-Nitrothiobenzoic acid S-(6-phenylcarbamoylhexyl)
ester (45): mp 117-118 °C; 1H NMR (CDCl3, 400 MHz, δ;
ppm) 8.30 (2H, d, J ) 8.8 Hz), 8.11 (2H, d, J ) 8.6 Hz), 7.51
(2H, d, J ) 8.1 Hz), 7.32 (2H, t, J ) 7.8 Hz), 7.16 (1H, broad
s), 7.10 (1H, t, J ) 7.3 Hz), 3.12 (2H, t, J ) 7.3 Hz), 2.37 (2H,
t, J ) 7.3 Hz), 1.76 (2H, quintet, J ) 7.6 Hz), 1.72 (2H, quintet,
J ) 7.3 Hz), 1.54-1.38 (4H, m); MS (EI) m/z: 386 (M+); Anal.
(C20H22N2O4S) C, H, N.
Thioisobutyric acid S-[6-(3-biphenylylcarbamoyl)-
hexyl] ester (47): mp 73-74 °C; 1H NMR (CDCl3, 500 MHz,
δ; ppm) 7.79 (1H, s), 7.59 (2H, d, J ) 7.4 Hz), 7.50 (1H, d, J )
8.3 Hz), 7.43 (2H, t, J ) 7.3 Hz), 7.39 (1H, t, J ) 8 Hz), 7.35
(1H, t, J ) 7.3 Hz), 7.34 (1H, d, J ) 7.3 Hz), 7.28 (1H, broad
s), 2.85 (2H, t, J ) 7.3 Hz), 2.73 (1H, septet, J ) 6.8 Hz), 2.38
(2H, t, J ) 7.3 Hz), 1.75 (2H, quintet, J ) 7.6 Hz), 1.58 (2H,
quintet, J ) 7.3 Hz), 1.49-1.35 (4H, m), 1.18 (6H, d, J ) 7.1
Hz); MS (EI) m/z: 390 (M+); Anal. (C23H29NO2S) C, H, N.
Thioisobutyric acid S-[6-(3-phenoxyphenylcarbam-
oyl)hexyl] ester (48): colorless oil; 1H NMR (CDCl3, 500
MHz, δ; ppm) 7.34 (2H, t, J ) 7.6 Hz), 7.30-7.15 (4H, m), 7.11
(1H, t, J ) 7.4 Hz), 7.02 (2H, d, J ) 7.6 Hz), 6.74 (1H, d, J )
7.3 Hz), 2.84 (2H, t, J ) 7.3 Hz), 2.73 (1H, septet, J ) 7 Hz),
2.32 (2H, t, J ) 7.3 Hz), 1.71 (2H, quintet, J ) 7.4 Hz), 1.57
(2H, quintet, J ) 7.4 Hz), 1.45-1.33 (4H, m), 1.18 (6H, d, J )
7 Hz); MS (EI) m/z: 399 (M+); HRMS calcd for C23H29NO3S
399.187, found 399.191.
Thioisobutyric acid S-[6-(3-pyridinylcarbamoyl)hexyl]
ester (49): mp 47-48 °C; 1H NMR (CDCl3, 500 MHz, δ; ppm)
8.55 (1H, d, J ) 2.8 Hz), 8.34 (1H, d, J ) 4.6 Hz), 8.21 (1H, d,
J ) 8.5 Hz), 7.56 (1H, broad s), 7.28 (1H, dd, J ) 4.6, 8.3 Hz),
2.85 (2H, t, J ) 7 Hz), 2.74 (1H, septet, J ) 7 Hz), 2.39 (2H,
t, J ) 7.6 Hz), 1.75 (2H, quintet, J ) 7.4 Hz), 1.59 (2H, quintet,
J ) 7.1 Hz), 1.45-1.35 (4H, m), 1.19 (6H, d, J ) 6.8 Hz); MS
(EI) m/z: 308 (M+); Anal. (C16H24N2O2S) C, H, N.
J ) 7.3 Hz), 1.25 (2H, quintet, J ) 7.6 Hz), 1.19 (6H, d, J ) 7
Hz), 1.13 (2H, quintet, J ) 7.3 Hz); MS (EI) m/z: 383 (M+);
Anal. (C20H26N2O2S2) C, H, N.
Thioisobutyric acid S-[6-(2-benzothiazolylcarbamoyl)-
hexyl] ester (52): mp 106-107 °C; 1H NMR (CDCl3, 500
MHz, δ; ppm) 10.41 (1H, broad s), 7.85 (1H, d, J ) 7.4 Hz),
7.77 (1H, d, J ) 7.9 Hz), 7.46 (1H, dt, J ) 1.2, 7.1 Hz), 7.34
(1H, dt, J ) 1, 7.3 Hz), 2.81 (2H, t, J ) 7.4 Hz), 2.73 (1H,
septet, J ) 7.1 Hz), 2.47 (2H, t, J ) 7.7 Hz), 1.72 (2H, quintet,
J ) 7.3 Hz), 1.53 (2H, quintet, J ) 7.1 Hz), 1.38-1.27 (4H,
m), 1.18 (6H, d, J ) 7 Hz); MS (EI) m/z: 364 (M+); Anal.
(C18H24N2O2S2) C, H, N.
Thioisobutyric acid S-{6-[(2-naphthalenecarbonyl)-
amino]hexyl} ester (53): mp 70-71 °C; 1H NMR (CDCl3,
500 MHz, δ; ppm) 8.29 (1H, s), 7.93 (1H, d, J ) 7.1 Hz), 7.90
(1H, d, J ) 7.3 Hz), 7.88 (1H, d, J ) 7.3 Hz), 7.84 (1H, d, J )
7 Hz), 7.57 (1H, t, J ) 6.7 Hz), 7.54 (1H, t, J ) 6.7 Hz), 6.36
(1H, broad s), 3.51 (2H, q, J ) 6.4 Hz), 2.87 (2H, t, J ) 7.3
Hz), 2.73 (1H, septet, J ) 6.7 Hz), 1.67 (2H, quintet, J ) 7.1
Hz), 1.60 (2H, quintet, J ) 6.7 Hz), 1.50-1.38 (4H, m), 1.18
(6H, d, J ) 6.8 Hz); MS (EI) m/z: 357 (M+); Anal. (C21H27-
NO2S) C, H, N.
Thioisobutyric acid S-{6-[(2-benzofurancarbonyl)ami-
no]hexyl} ester (54): mp 67-68 °C; 1H NMR (CDCl3, 500
MHz, δ; ppm) 7.67 (1H, d, J ) 7.7 Hz), 7.50 (1H, d, J ) 7.6
Hz), 7.46 (1H, d, J ) 1 Hz), 7.41 (1H, dt, J ) 1.2, 7.3 Hz), 7.29
(1H, t, J ) 7.6 Hz), 6.66 (1H, broad s), 3.48 (2H, q, J ) 7 Hz),
2.86 (2H, t, J ) 7.4 Hz), 2.73 (1H, septet, J ) 7.1 Hz), 1.66
(2H, quintet, J ) 7 Hz), 1.59 (2H, quintet, J ) 7 Hz), 1.48-
1.37 (4H, m), 1.18 (6H, d, J ) 6.7 Hz); MS (EI) m/z: 347 (M+);
Anal. (C19H25NO3S) C, H, N.
Thioisobutyric acid S-{6-[(1H-2-indolecarbonyl)ami-
no]hexyl} ester (55): mp 142-143 °C; 1H NMR (CDCl3, 500
MHz, δ; ppm) 9.37 (1H, broad s), 7.65 (1H, d, J ) 7.3 Hz),
7.44 (1H, d, J ) 7.6 Hz), 7.29 (1H, t, J ) 7 Hz), 7.14 (1H, t, J
) 7.9 Hz), 6.86 (1H, s), 6.30 (1H, broad s), 3.49 (2H, q, J ) 6.1
Hz), 2.87 (2H, t, J ) 7.1 Hz), 2.74 (1H, septet, J ) 7 Hz), 1.65
(2H, quintet, J ) 7 Hz), 1.60 (2H, quintet, J ) 7 Hz), 1.50-
1.36 (4H, m), 1.19 (6H, d, J ) 7 Hz); MS (EI) m/z: 346 (M+);
Anal. (C19H26N2O2S) C, H, N.
2,2-Dimethylpropionic Acid 6-Phenylcarbamoylhex-
ylsulfanylmethyl Ester (46). To a suspension of sodium
hydride (60%, 40.0 mg, 1.00 mmol) in DMF (2 mL) was added
a solution of 7 (200 mg, 0.84 mmol) in DMF (3 mL) dropwise
with cooling in an ice-water bath. The mixture was stirred
for 30 min at 0 °C, and a solution of chloromethyl pivalate
(134 µL, 0.93 mmol) in DMF (2 mL) was added at 0 °C. The
solution was stirred at room temperature for 1 h. The reaction
mixture was poured into ice-water and extracted with AcOEt.
The AcOEt layer was separated, washed with water and brine,
and dried over Na2SO4. Filtration and concentration in vacuo
and purification by silica gel flash chromatography (n-hexane/
AcOEt ) 4/1) gave 93 mg (32%) of 46 as a colorless oil: 1H
NMR (CDCl3, 400 MHz, δ; ppm) 7.51 (2H, d, J ) 7.8 Hz), 7.32
(2H, t, J ) 7.6 Hz), 7.16 (1H, broad s), 7.10 (1H, t, J ) 7.3
Hz), 5.41 (2H, s), 2.65 (2H, t, J ) 7.3 Hz), 2.36 (2H, t, J ) 7.6
Hz), 1.74 (2H, quintet, J ) 7.1 Hz), 1.66 (2H, quintet, J ) 7.1
Hz), 1.50-1.36 (4H, m), 1.21 (9H, s); MS (EI) m/z: 351 (M+);
HRMS calcd for C19H29NO3S 351.187, found 351.189.
Thioisobutyric acid S-[6-(3-quinolinylcarbamoyl)-
hexyl] ester (50): mp 67-68 °C; 1H NMR (CDCl3, 500 MHz,
δ; ppm) 8.81 (1H, s), 8.73 (1H, d, J ) 2.8 Hz), 8.03 (1H, d, J )
8.6 Hz), 7.80 (1H, d, J ) 8.2 Hz), 7.70 (1H, broad s), 7.63 (1H,
t, J ) 7.1 Hz), 7.54 (1H, t, J ) 7.3 Hz), 2.86 (2H, t, J ) 7.3
Hz), 2.74 (1H, septet, J ) 7 Hz), 2.46 (2H, t, J ) 7.6 Hz), 1.79
(2H, quintet, J ) 7.3 Hz), 1.60 (2H, quintet, J ) 7.3 Hz), 1.50-
1.35 (4H, m), 1.19 (6H, d, J ) 6.7 Hz); MS (EI) m/z: 358 (M+);
Anal. (C20H26N2O2S) C, H, N.
Thioisobutyric acid S-[6-(4-phenyl-2-thiazolylcarbam-
oyl)hexyl] ester (51): mp 127-128 °C; 1H NMR (CDCl3, 500
MHz, δ; ppm) 10.48 (1H, broad s), 7.83 (2H, d, J ) 7.3 Hz),
7.43 (2H, t, J ) 7.3 Hz), 7.34 (1H, t, J ) 7.4 Hz), 7.16 (1H, s),
2.81 (2H, t, J ) 7.3 Hz), 2.74 (1H, septet, J ) 7 Hz), 2.11 (2H,
t, J ) 7.6 Hz), 1.56 (2H, quintet, J ) 7.6 Hz), 1.50 (2H, quintet,
Biology. Enzyme Assays. The assay of HDAC activity was
performed using an HDAC fluorescent activity assay/drug
discovery kit (AK-500, BIOMOL Research Laboratories). HeLa
nuclear extracts (0.5 µL/well) were incubated at 37 °C with
25 µM of Fluor de Lys substrate and various concentrations
of samples. Reactions were stopped after 30 min by adding
Fluor de Lys Developer with trichostatin A which stops further
deacetylation. Then, 15 min after addition of this developer,
the fluorescence of the wells was measured on a fluorometric
reader with excitation set at 360 nm and emission detection
set at 460 nm, and the % inhibition was calculated from the
fluorescence readings of inhibited wells relative to those of
control wells. The concentration of compound which results
in 50% inhibition was determined by plotting the log[Inh]
versus the logit function of the % inhibition. IC50 values are

Inhibitors of Human Histone Deacetylases
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 1031
determined using a regression analysis of the concentration/
inhibition data.
Sports, Science and Technology of Japan, for cancer cell
growth inhibition assay results.
Lineweaver-Burk Double-Reciprocal Plot Analysis.
The assay of HDAC activity was performed using an HDAC
fluorescent activity assay/drug discovery kit (AK-500, BIOMOL
Research Laboratories). HeLa nuclear extracts (0.5 µL/well)
were incubated at 37 °C with Fluor de Lys substrate (50, 100,
200, or 400 µM) in the presence of 0, 0.03, 0.1, or 0.3 µM of
compound 7. Reactions were stopped after 10 min by adding
Fluor de Lys Developer with trichostatin A which stops further
deacetylation. Then, 15 min after addition of this developer,
the fluorescence of the wells was measured on a fluorometric
reader with excitation set at 360 nm and emission detection
set at 460 nm.
Monolayer Growth Inhibition Assay. Cancer cells were
plated in 96-well plates at initial densities of 1500 cells/well
and incubated at 37 °C. After 24 h, cells were exposed to test
compounds at various concentrations in 10% FBS-supplement-
ed RPMI-1640 medium at 37 °C in 5% CO2 for 48 h. The
medium was removed and replaced with 200 µL of 0.5 mg/mL
of Methylene Blue in RPMI-1640 medium, and cells were
incubated at room temperature for 30 min. Supernatants were
removed from the wells, and Methylene Blue dye was dissolved
in 100 µL/well of 3% aqueous HCl. Absorbance was determined
on a microplate reader (BioRad) at 660 nm.
Western Blot Analysis. HCT-116 cells (purchased from
ATCC) (1 × 106) treated for 8 h with SAHA and compound 51
at the indicated concentrations in 10% FBS-supplemented
McCoy’s 5A medium were collected and sonicated. Protein
concentrations of the lysates were determined by using a
Bradford protein assay kit (Bio-Rad Laboratories); equivalent
amounts of proteins from each lysate were resolved in 15%
SDS-polyacrylamide gel and then transferred onto nitrocel-
lulose membranes (Bio-Rad Laboratories). After blocking with
Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST)
containing 3% skim milk for 30 min, the transblotted mem-
brane was incubated with hyperacetylated histone H4 antibody
(Upstate Biotechnology) (1:2000) or p21WAF1/CIP1 antibody
(Medical and Biological Laboratories) (1: 200) in TBST
containing 3% skim milk at 4 °C overnight. After treatment
with the primary antibody, the membrane was washed twice
with water for anti- hyperacetylated histone H4, or three times
with TBS for anti-p21WAF1/CIP1, then incubated with goat anti-
rabbit or anti-mouse IgG-horseradish peroxidase conjugates
(1:10000 or 1:5000) for 1.5 h at room temperature and washed
twice with water for anti- hyperacetylated histone H4, or three
times with TBS for anti-p21WAF1/CIP1. The immunoblots were
visualized by enhanced chemiluminescence.
Molecular Modeling. Docking and subsequent scoring
were performed using Macromodel 8.1 software. Coordinates
of HDAC8 complexed with MS344 were taken from the
Brookhaven Protein Data Bank (PDB code 1T67) and hydrogen
atoms were added computationally at appropriate positions.
The structures of SAHA and compound 7 bound to HDAC8
were constructed by molecular mechanics (MM) energy mini-
mization. The starting positions of SAHA and compound 7
were determined manually: the benzene ring and the linker
parts were superimposed in the active site onto its crystal-
lographic MS344 counterpart. The conformations of SAHA and
compound 7 in the active site were minimized by a MM
calculation based upon the OPLS-AA force field with each
parameter set as follows: solvent: water, method: LBFGS,
max. no. iterations: 10 000, converge on: gradient, conver-
gence threshold: 0.05.
Supporting Information Available: Results of the ele-
mental analysis of 4-21, 23-45, 47, 49-54, and 55 are
reported. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Acknowledgment. This work was supported in part
by grants from the Health Sciences Foundation of the
Ministry of Health, Labor and Welfare of Japan, and
the Mochida Memorial Foundation for Medical and
Pharmaceutical Research. We thank the Screening
Committee of New Anticancer Agents, supported by a
Grant-in-Aid for Scientific Research on Priority Area
“Cancer” from the Ministry of Education, Culture,

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