Design, synthesis, structure-selectivity relationship, and effect on human cancer cells of a novel series of histone deacetylase 6-selective inhibitors

Article abstract of DOI:10.1021/jm7009217

To uncover novel histone deacetylase 6 (HDAC6)-selective inhibitors and to elucidate the structural requirements for their inhibitory activity, we designed and prepared a series of thiolate analogues based on the structure of an HDAC6-selective substrate and evaluated their properties by Western blotting and enzyme assays. Several thiolate analogues were found to be potent and selective HDAC6 inhibitors. Study of the structure-selectivity relationship revealed that the presence of a bulky alkyl group and tert-butylcarbamate group in these compounds is important for HDAC6-selective inhibition. Compounds 16b and 20b, the most selective and active compounds in this series, exerted a synergistic inhibition of cancer cell growth in combination with paclitaxel. They also blocked the growth of estrogen receptor α-positive breast cancer MCF-7 cells that had been treated with estrogen. These findings suggested that HDAC6-selective inhibitors have potential as anticancer agents.

Full text of DOI:10.1021/jm7009217

J. Med. Chem. 2007, 50, 5425-5438
5425
Design, Synthesis, Structure-Selectivity Relationship, and Effect on Human Cancer Cells of a
Novel Series of Histone Deacetylase 6-Selective Inhibitors
Yukihiro Itoh,† Takayoshi Suzuki,*,† Akiyasu Kouketsu,† Nobuaki Suzuki,† Satoko Maeda,‡ Minoru Yoshida,‡,§
Hidehiko Nakagawa,† and Naoki Miyata*,†
Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan,
Chemical Genetics Laboratory, RIKEN, Saitama 351-0198, Japan, and CREST Research Project, Japan Science and Technology Agency,
Saitama 332-001, Japan
ReceiVed July 30, 2007
To uncover novel histone deacetylase 6 (HDAC6)-selective inhibitors and to elucidate the structural
requirements for their inhibitory activity, we designed and prepared a series of thiolate analogues based on
the structure of an HDAC6-selective substrate and evaluated their properties by Western blotting and enzyme
assays. Several thiolate analogues were found to be potent and selective HDAC6 inhibitors. Study of the
structure-selectivity relationship revealed that the presence of a bulky alkyl group and tert-butylcarbamate
group in these compounds is important for HDAC6-selective inhibition. Compounds 16b and 20b, the most
selective and active compounds in this series, exerted a synergistic inhibition of cancer cell growth in
combination with paclitaxel. They also blocked the growth of estrogen receptor R-positive breast cancer
MCF-7 cells that had been treated with estrogen. These findings suggested that HDAC6-selective inhibitors
have potential as anticancer agents.
Introduction
Although a large number of HDAC inhibitors have been
found to date,9 including trichostatin A (TSA, 1),10 suberoyla-
nilide hydroxamic acid (SAHA, vorinostat, 2),11 trapoxin B
(TPX B, 3),12 and MS-275 (4;13 Chart 1), most lack HDAC6
selectivity. Most hydroxamate HDAC inhibitors, such as 1 and
2, exhibit little or no preference for HDAC6, whereas most
nonhydroxamate inhibitors, such as 3 and 4, do not inhibit
HDAC6.5b,c,14 It has been reported that HDAC6 is selectively
inhibited by tubacin (5; Chart 1), which was discovered by the
screening of a 7392 small molecule library.15 However, there
is only limited information on HDAC6-selective inhibitors, and
therefore, there is a need to develop novel candidates and to
then elucidate their structural requirements for inhibiting
HDAC6.
In the course of our study on nonhydroxamate HDAC
inhibitors,14e,16 we found a new class of small molecule thiol-
based analogues including NCH-26 (6) and NCH-31 (7;16b Chart
2). Thiols are presumed to inhibit HDACs by coordinating the
zinc ion, which is required for deacetylation of the acetylated
lysine substrate. Furthermore, the S-isobutyryl prodrugs NCH-
47 (8) and NCH-51 (9;17 Chart 2), which are thought to be
hydrolyzed to their free thiol forms within cells, showed potent
inhibition of cancer cell growth. Following these findings, we
recently performed further investigation of thiolate analogues
to uncover those that were HDAC6-selective, and discovered
highly potent and selective HDAC inhibitors.18 In the present
report, we describe in full detail the design, synthesis, structure-
selectivity relationship, and cellular activity of thiol-based
HDAC6-selective inhibitors.
Reversible protein acetylation is an important post-transla-
tional modification that regulates the function of histones and
many nonhistone proteins.1 Acetylation of histone lysine
residues is controlled by histone acetyltransferases and histone
deacetylases (HDACsa) and is closely connected with gene
expression and cell cycle progression.2 The inhibition of HDACs
causes histone hyperacetylation and leads to the transcriptional
activation of genes such as p21WAF/CIP1 and Gadd 45, which
are associated with growth arrest and apoptosis in tumor cells.3
Thus far, 18 HDAC family members have been identified and
categorized into two groups, namely, zinc-dependent enzymes
(HDAC1-11) and NAD+-dependent enzymes (SIRT1-7).2a,4
Among these, HDAC6, one of the zinc-dependent HDAC family
members, is unique in that it deacetylates nonhistone
proteins, such as R-tubulin, HSP90, and cortactin, and is
involved in microtubule stabilization, molecular chaperone
activity, and cell motility.5 Furthermore, recent studies have
revealed that HDAC6 is associated with several disease states.
It has been reported by Hideshima et al. that the inhibition of
HDAC6 causes growth inhibition of multiple myeloma cells
without affecting noncancerous cells,6 and Saji et al. reported
that expression of HDAC6 is induced by estrogen stimulation
of estrogen receptor R (ERR)-positive breast cancer cells.7 In
addition, HDAC6 inhibition has been reported to be strongly
involved in neuroprotection.8 Therefore, HDAC6-selective
inhibitors are of great interest not only as tools for probing the
biological functions of the isoform, but also as therapeutic agents
having few side effects.
Chemistry
* To whom correspondence should be addressed. Tel. and fax: +81-
52-836-3407. E-mail: suzuki@phar.nagoya-cu.ac.jp (T.S.); miyata-n@
phar.nagoya-cu.ac.jp (N.M.).
The compounds prepared for this study are shown in Figures
1 and 3 and Table 1. Syntheses were carried out as outlined in
Schemes 1-4. The route for synthesis of compounds 11b-
25b is described in Scheme 1. (S)-2-amino-7-bromoheptanoic
acid 4219 was treated with (Boc)2O to yield N-Boc compound
43. Condensation of acid 43 with an appropriate amine afforded
amides 11c-25c. Bromides 11c-25c were treated with thio-
† Graduate School of Pharmaceutical Sciences, Nagoya City University.
‡ Chemical Genetics Laboratory, RIKEN.
§ CREST Research Project.
a Abbreviations: HDAC, histone deacetylase; SIRT, sirtuin; ERR,
estrogen receptor R; TSA, trichostatin A; SAHA, suberoylanilide hydrox-
amic acid; TPX B, trapoxin B; PTX, paclitaxel; E2, 17â-estradiol.
10.1021/jm7009217 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/11/2007

5426 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Chart 1. Examples of HDAC Inhibitors
Itoh et al.
Chart 2. Thiolate HDAC Inhibitors
40a by Boc deprotection of 16d, treatment of amine 26d with
an appropriate acid chloride, carboxylic acid, chloroformate,
isocyanate, or thioisocyanate, and subsequent hydrolysis of
thioacetates 27d, 28d, 31d, 33d, 35d-38d, and 40d.
Results and Discussion
In designing novel HDAC6-selective inhibitors, we focused
initially on small molecule HDAC6-selective substrate 10 (Chart
3), which was discovered by Jung and co-workers.20 According
to their report, compound 10 is selectively deacetylated by
HDAC6 in preference to HDAC1 and HDAC3, and the presence
of N-Boc and trifluoromethyl coumaryl amide in this compound
was found to be important for HDAC6 selectivity. This indicated
that the structure of N-Boc and trifluoromethyl coumaryl amide
of compound 10 is selectively recognized by HDAC6, and so,
we considered that compound 11a, in which the acetamide of
10 is replaced by a thiol, might selectively inhibit HDAC6
(Chart 3).
isobutyric acid under alkaline conditions to yield the desired
thioesters 11b-25b.
Compounds 26b-41b were synthesized from thioester 16b
by the route shown in Scheme 2. Removal of the Boc group of
16b gave amine 26b. Amine 26b was allowed to react with the
corresponding acid chloride, carboxylic acid, chloroformate,
isocyanate or thioisocyanate to give 27b-41b.
Compounds 13a, 15a-20a, and 26a were prepared from the
corresponding bromides 13c and 15c-20c (Scheme 3). Treat-
ment of the bromides with potassium thioacetate and subsequent
hydrolysis of the thioesters gave thiols 13a and 15a-20a.
Deprotection of the Boc group of 16a gave amine 26a.
Thiols 27a, 28a, 31a, 33a, 35a-38a, and 40a were synthe-
sized from thioester 16d as outlined in Scheme 4. Thioester
16d was converted to thiols 27a, 28a, 31a, 33a, 35a-38a, and
Because HDAC6 has been reported as an R-tubulin deacety-
lase, inhibition of HDAC6 and that of other HDACs can be
assessed according to the accumulation of acetylated R-tubulin
and acetylated histones, respectively, using Western blot
analysis. We initially evaluated the histone H4/R-tubulin acety-
lation selectivity of compounds 2, 8, 9, and 11b, the S-isobutyryl
prodrug of 11a, using Western blot analysis (Figure 2). As
expected, compound 11b seemed to induce the accumulation
Scheme 1a
a Reagents and conditions: (a) (Boc)2O, Et3N, THF, H2O, rt, 95%; (b) R1NH2, POCl3, pyridine, -15 °C, 10-48%; (c) R1NH2, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole hydrate (HOBt‚H2O), THF, rt, 69-93%; (d) thioisobutyric acid, Et3N, EtOH, rt, 19-
100%.

Series of Histone Deacetylase 6-SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5427
Scheme 2a
a Reagents and conditions: (a) HCl, AcOEt, rt, 100%; (b) R2COCl, Et3N, CH2Cl2, rt, 11-74%; (c) (R2CO)2O, 4-dimethylaminopyridine (DMAP), Et3N,
CH2Cl2, rt, 52%; (d) R2COOH, EDCI, HOBt‚H2O, DMF, 28-62%; (e) R2NCX, Et3N, CH2Cl2, rt, 24-76%.
Scheme 3a
a Reagents and conditions: (a) KSAc, EtOH, rt, 57-93%; (b) NaOH, H2O, EtOH, rt, 53-77%; (c) HCl, AcOEt, rt, 94%.
Scheme 4a
a Reagents and conditions: (a) HCl, AcOEt, rt, 100%; (b) R2COCl, Et3N, CH2Cl2, rt, 26-91%; (c) (R2CO)2O, DMAP, Et3N, CH2Cl2, rt, 52%; (d)
R2COOH, EDCI, HOBt‚H2O, DMF, 60%; (e) R2NCX, Et3N, CH2Cl2, rt, 39-95%; (f) NaOH, H2O, EtOH, rt, 63-100%.
Chart 3. Design of HDAC6-Selective Inhibitors
of acetylated R-tubulin as compared with 2, 8, and 9, which
did not have this effect. To obtain highly more selective
compounds, the coumarin structure of 11b was converted to
analogs with various functional groups (12b-25b; Figure 1),
and these were examined for their selectivity. Compounds 12b-
15b and 21b, with an aromatic ring, compounds 22b-24b, with
a smaller hydrophilic group, and compound 25b, with a tertiary
amide group, did not show high selectivity, whereas compounds
16b-20b, in which R1 ) a bulky alkyl group, induced a dose-
dependent increase in R-tubulin acetylation but no major
increase in acetylated histone H4 (Figure 2). These results
indicated that compounds 16b-20b selectively inhibit HDAC6
in preference to nuclear HDACs in cells.
Having investigated the requirements for the R1 group
(Figures 1 and 2), we next turned our attention to the
replacement of the N-Boc group (Figures 3 and 4). We employed
the cyclopentyl group as the R1 group and determined the effect
of replacement of the N-Boc group with other functional groups.
We initially evaluated amine 26b in which the Boc group of
16b is removed, however, 26b induced neither histone H4
acetylation nor R-tubulin acetylation. Next, we tested aromatic
amides (27b-29b). Among these, phenyl amide 27b induced
the acetylation of R-tubulin, but its selectivity seemed to
decrease when compared with that of the lead compound 16b.
We also examined aliphatic amides (30b-34b). Although
hydroxymethyl compound 30b was found to be a highly potent
R-tubulin acetylating agent, its selectivity seemed to be low as
compared with 16b. Compounds 31b and 32b caused the
accumulation of both acetylated histone H4 and acetylated
R-tubulin, whereas compounds 33b and 34b only displayed a
weak acetylation activity. It is remarkable that neopentyl amide
34b did not show any R-tubulin acetylating activity or selectivity
at all. In other words, replacement of the oxygen atom of the
Boc group with a methylene group significantly decreased both
potency and selectivity. This suggests that the carbamate group
is important for R-tubulin acetylation selectivity. To confirm
the importance of the carbamate structure, we replaced the
carbamate group of 16b with urea (35b) and thiourea (36b) and

5428 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Itoh et al.
Figure 1. Structures of 11b-25b.
Figure 2. Western blot detection of acetylated R-tubulin and acetylated histone H4 levels in HCT116 cells after 8 h treatment with 2, 8, 9, and
11b-25b.

Series of Histone Deacetylase 6-SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5429
Figure 3. Structures of 26b-41b.
Figure 4. Western blot detection of acetylated R-tubulin and acetylated histone H4 levels in HCT116 cells after 8 h treatment with 26b-41b.
evaluated these two compounds. While compounds 35b and 36b
sustained R-tubulin acetylation activity, selectivity was signifi-
cantly reduced. These results highlighted the importance of the
carbamate group in R-tubulin/histone acetylation selectivity.

5430 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Itoh et al.
Table 1. In Vitro HDAC1-, HDAC4-, and HDAC6-Inhibitory Activities of 13a, 15a-20a, 26a-28a, 31a, 33a, 35a-38a, and 40aa
IC50 (nM)
HDAC4
selectivity
HDAC1/HDAC6 HDAC4/HDAC6
0.26 0.42
4b 4b
cmpd
R1
R2
HDAC1
HDAC6
1
5
7
21
NDc
48
62
51
1210
1270
900
3000
3800
52 300
560
1430
1980
1430
460
34
ND
32
38
33
1030
1140
840
1900
4200
31 200
280
650
970
730
310
220
1160
580
81
ND
41
54
32
29
36
23
71
1.2
1.2
1.6
0.78
0.70
1.0
13a
15a
16a
17a
18a
19a
20a
26a
27a
28a
30a
33a
35a
36a
37a
38a
40a
3-biphenyl
3-quinolinyl
cyclopentyl
cyclohexyl
cycloheptyl
-t-Bu
-Ot-Bu
-Ot-Bu
-Ot-Bu
-Ot-Bu
-Ot-Bu
-Ot-Bu
-Ot-Bu
42
36
35
39
42
46
14
32
37
26
51
1-adamantyl
82
3860
190
510
150
430
100
170
120
120
78
8.1
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
-Ph
3.0
2.8
13
3.3
4.6
2.4
19
8.6
1.7
1.5
1.3
6.5
1.7
3.1
1.3
9.7
4.8
1.7
4-chlorophenyl
-CH2OH
-t-Bu
-NHt-Bu
400
cyclopentyl
cyclopentyl
cyclopentyl
-OMe
2320
1030
130
cyclohexyloxy
-OBn
130
a Values are means of at least three experiments. b Taken from the literature (ref 22). c ND ) No data.
and 15a did not discriminate among HDAC1, HDAC4, and
HDAC6. The strong HDAC1 inhibitory activity of 13a and 15a
is consistent with the results observed in similar aromatic group-
containing HDAC inhibitors.21 The HDAC6 inhibitory activity
of compounds 16a-20a, in which R1 ) a bulky alkyl group,
was similar to or greater than that of 1 (IC50 of 81 nM, 16a 29
nM, 17a 36 nM, 18a 23 nM, 19a 71 nM, 20a 82 nM).
Furthermore, while 1 inhibited HDAC1 and HDAC4 rather than
HDAC6 (HDAC1 IC50/HDAC6 IC50 ) 0.26; HDAC4 IC50/
HDAC6 IC50 ) 0.42), compounds 16a-20a efficiently inhibited
HDAC6 in preference to HDAC1 and HDAC4 (HDAC1 IC50/
HDAC6 IC50 ) 35-46; HDAC4 IC50/HDAC6 IC50 ) 26-51).
The HDAC6 selectivity of compounds 16a-20a was much
higher than that of 5, which showed only about a 4-fold
selectivity for HDAC6 over HDAC1 and HDAC 4 in enzyme
assays.22 In addition, in the case of compounds with various R2
groups (26a-28a, 30a, 33a, 35a-38a, and 40a), a reasonable
correlation between cellular and enzyme assay data was
observed. Amine 26a did not show strong HDAC inhibition,
and aromatic amides 27a and 28a and aliphatic amides 30a and
33a were nonselective inhibitors as compared with 16a. Conver-
sion of the carbamate of 16a to urea (35a) and thiourea (36a)
significantly reduced the HDAC6 selectivity. As for carbamate
derivatives, methyl carbamate 37a exerted a moderate level of
HDAC6 selectivity (HDAC1 IC50/HDAC6 IC50 ) 19; HDAC4
IC50/HDAC6 IC50 ) 9.7), whereas cyclopentyl 38a and benzyl
40a showed a decrease in HDAC6 selectivity as compared with
that of 16a and 37a. As a result, compounds 16a-20a, in which
R1 ) a bulky alkyl group and R2 ) a tert-butoxy group, were
the most potent and selective HDAC6 inhibitors.
Figure 5. Structure-selectivity relationship.
Next, we converted the tert-butyl group of 16b to other
functional groups (37b-41b). While methyl group (37b)
retained the R-tubulin acetylation activity and selectivity to some
extent, larger alkyl groups (38b, 40b, and 41b) and a phenyl
group (39b) caused a decrease in either selectivity or potency.
Medium-sized alkyl groups such as the tert-butyl group seem
to be preferred as the substituents attached to the carbamate.
As a result, the tert-butoxy group (16b) was the best R2 group
choice for the selective accumulation of acetylated R-tubulin
in cells.
To confirm HDAC6 selectivity and to examine the structure-
selectivity relationship for this series of inhibitors, we performed
in vitro enzyme assays using HDAC1, HDAC4, and HDAC6.
A selected set of thiols was prepared and evaluated for enzyme
inhibition activity. The results of the enzyme assays are shown
in Table 1. Compound 1 was chosen as a reference compound.
We initially tested compound 7 and compounds with various
R1 groups (13a, 15a, and 16a-20a). Consistent with the results
of Western blotting, compound 7 and aromatic compounds 13a
As shown in Figure 5, the results of Western blotting and
enzyme assays clarified the structural requirements for HDAC6-
selective inhibition. For R1, bulky alkyl groups such as

Series of Histone Deacetylase 6-SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5431
Figure 6. Cell growth inhibition of HCT116 cells using different
combinations of HDAC6 inhibitor 16b or 20b and paclitaxel (PTX).
*P < 0.05; ANOVA and Bonferroni-type multiple t test results indicated
differences between PTX (0.03 µM) and a combination of PTX (0.03
µM) with 16b (5 µM) and between PTX (0.03 µM) and a combination
of PTX (0.03 µM) with 20b (5 µM).
Figure 7. Cell growth inhibition of MCF-7 cells stimulated with
estradiol (E2) using HDAC6 inhibitor 16b or 20b. **P < 0.01; ANOVA
and Bonferroni-type multiple t test results indicated differences between
E2 (1 nM) and a combination of E2 (1 nM) with 16b (5 µM) and
between E2 (1 nM) and a combination of E2 (1 nM) with 20b (5 µM).
adamantyl and cycloalkyl are preferred. The carbamate structure
is also important for HDAC6-selective inhibition. For R3,
medium-sized alkyl groups such as tert-butyl are suitable.
Because there is no information at present on the three-
dimensional structure of HDAC6, the reason for the high
HDAC6 selectivity of compounds 16-20 is unclear, but it is
likely that HDAC6 has at least two hydrophobic pockets where
a bulky R1 group and a medium-sized R3 group could be placed,
as well as an amino acid residue, which could interact with the
carbamate group, whereas the other HDAC isoforms lack the
pockets and amino acid residue.
To explore the potential of HDAC6-selective inhibitors as
anticancer drugs, we first tested compounds 16b and 20b, the
most selective and active compounds in this series, by means
of a cancer cell growth inhibition assay using human colon
cancer HCT116 cells. However, they were found to be inactive
up to a concentration of 5 µM above which they displayed
distinct R-tubulin acetylation on Western blot analysis (Figure
2). We next evaluated whether our HDAC6-selective inhibitors
could act synergistically with paclitaxel (PTX) in growth
inhibition assays using HCT116 cells. Because PTX exerts its
anticancer effect by stabilizing microtubules, where acetylated
R-tubulins are most abundant,5b,c,23 we considered that the
combination of our HDAC6-selective inhibitors and PTX might
cause a synergistic inhibition of cancer cell growth. As shown
in Figure 6, 5 µM of 16b or 20b did not show any activity as
a single agent, whereas treatment with 0.03 µM of PTX reduced
the cell growth by approximately 50%. As expected, however,
a combination of 5 µM of 16b or 20b and 0.03 µM of PTX
reduced cell growth by approximately 70%, suggesting that
HDAC6-selective inhibitors have potential as drug candidates
when used in combination with PTX.
We also evaluated the effect of HDAC6-selective inhibitors
on ERR-positive breast cancer MCF-7 cells. It has been reported
that estrogen stimulation of MCF-7 cells increases expression
of HDAC6 and enhances cell motility.7 This report suggested
to us that HDAC6 may be associated with the growth of ERR-
positive breast cancer cells treated with estrogen. We therefore
examined whether HDAC6-selective inhibitors could inhibit
growth of MCF-7 cells stimulated with this hormone (Figure
7). As with HCT116 cells, the growth of MCF-7 cells was not
influenced by treatment with 5 µM of 16b or 20b, whereas
treatment with 1 nM of 17â-estradiol (E2)-induced cell growth
by approximately 40%. Interestingly, treatment with 5 µM of
16b or 20b significantly blocked the growth of MCF-7 cells
stimulated with 1 nM of E2. These results suggested that
HDAC6 is involved in the growth of ERR-positive breast cancer
cells and that HDAC6-selective inhibitors may be useful in the
treatment of ERR-positive breast cancer.
Conclusion
We have identified novel HDAC6-selective inhibitors whose
designs were based on the structure of HDAC6-selective
substrate 10. Compounds 16-20 showed high HDAC6 selectiv-
ity in both cellular and enzyme assays and investigation of the
structure-selectivity relationship revealed that the presence of
a bulky alkyl group, such as adamantyl and cycloalkyl, and a
tert-butylcarbamate group in these compounds is important for
HDAC6-selective inhibition. In biological experiments, the
combination of compound 16b or 20b and PTX caused a
synergistic inhibition of cancer cell growth. The synergistic
effect of these HDAC6-selective inhibitors may allow for the
reduction of the PTX dosage with consequently fewer side
effects. Compounds 16b and 20b also showed growth inhibition
of MCF-7 cells stimulated by estrogen. Inhibition of ERR-
positive breast cancer cell growth by HDAC6-selective inhibitors
suggests that these inhibitors may be effective as antibreast
cancer drugs. We believe that the findings presented here will
provide a basis for constructing new tools for probing the
biology of HDAC6 and uncovering new strategies for cancer
treatments.
Experimental Section
Chemistry. Melting points were determined using a Yanagimoto
micromelting point apparatus or a Bu¨chi 545 melting point
apparatus and were left uncorrected. Proton nuclear magnetic
resonance spectra (1H NMR) and carbon nuclear magnetic reso-
nance spectra (13C NMR) were recorded JEOL JNM-LA500, JEOL
JNM-A500 or BRUKER AVANCE600 spectrometer in solvent as
indicated. Chemical shifts (δ) are reported in parts per million
relative to the internal standard tetramethylsilane. 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) and fast atom bombardment (FAB)
were recorded on a JEOL JMS-SX102A mass spectrometer. 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.

5432 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Itoh et al.
(S)-S-6-(tert-Butoxycarbonyl)-7-oxo-7-[2-oxo-4-(trifluoromethyl)-
2H-chromen-7-ylamino]heptyl 2-Methylpropanethioate (11b).
Step 1: Preparation of (S)-7-Bromo-2-tert-butoxycarbonylami-
noheptanoic Acid (43). To a solution of (S)-2-amino-7-bromo-
heptanoic acid (42; 2.01 g, 8.97 mmol) and Et3N (5 mL, 67.7 mmol)
in THF/H2O (20 mL/40 mL) was added a solution of (Boc)2O (3.90
g, 17.9 mmol) in THF (20 mL), and the mixture was stirred
overnight at room temperature. The reaction mixture was poured
into 2 N aqueous NaOH (15 mL), and the whole mixture was
washed with CHCl3. To the separated aqueous layer was added
10% aqueous citric acid (15 mL) and was extracted with AcOEt.
The organic layer was washed with water and brine and dried over
Na2SO4. Filtration and concentration in vacuo gave 2.77 g (95%)
of 43 as a colorless oil: 1H NMR (CDCl3, 500 MHz, δ, ppm) 5.02
(1H, d, J ) 7.0 Hz), 4.31 (1H, s), 3.40 (2H, t, J ) 6.7 Hz), 1.87
(1H, m), 1.72-1.65 (1H, m), 1.87 (2H, quintet, J ) 7.1 Hz), 1.52-
1.43 (13H, m).
Step 2: Preparation of (S)-tert-Butyl 7-Bromo-1-oxo-1-(2-oxo-
4-(trifluoromethyl)-2H-chromen-7-ylamino)heptan-2-ylcarbam-
ate (11c). To a solution of 7-amino-4-(trifluoromethyl)coumarin
(688 mg, 3.00 mmol) and 43 (973 mg, 3.00 mmol) obtained above
in dry pyridine (18 mL) was added phosphoryl chloride (750 µL,
2.97 mmol) at -15 °C, and the solution was stirred at -15 °C for
15 min. The solution was poured into water, and the solution was
extracted with AcOEt. The AcOEt layer was separated, washed
with saturated aqueous NaHCO3, 10% aqueous citric acid, and brine,
and dried over Na2SO4. Filtration, concentration in vacuo, and
purification by silica gel flash column chromatography (AcOEt/n-
hexane ) 1/3) gave 922 mg (57%) of 11c as a colorless solid: 1H
NMR (CDCl3, 500 MHz, δ, ppm) 9.02 (1H, s), 7.80 (1H, d, J )
1.8 Hz), 7.62 (1H, d, J ) 7.9 Hz), 7.46 (1H, d, J ) 9.1 Hz), 6.67
(1H, s), 4.97 (1H, s), 4.19 (1H, s), 3.41 (2H, t, J ) 6.7 Hz), 2.02-
1.96 (1H, m), 1.88 (2H, quintet, J ) 7.0 Hz), 1.71-1.60 (1H, m),
1.48 (9H, s), 1.52-1.35 (4H, m).
(6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.45,
170.26, 129.00, 124.38, 119.87, 77.22, 43.12, 29.36, 28.31, 28.25,
25.13, 19.42; MS (EI) m/z 422 (M+); HRMS calcd for C22H34O4N2S,
422.224; found, 422.225.
(S)-S-7-(Biphenyl-3-ylamino)-6-(tert-butoxycarbonyl)-7-oxo-
1
heptyl 2-Methylpropanethioate (13b). Yield 6%; yellow oil; H
NMR (CDCl3, 500 MHz, δ, ppm) 8.24 (1H, broad s), 7.79 (1H, s),
7.58 (2H, d, J ) 7.6 Hz), 7.48 (1H, d, J ) 7.9 Hz), 7.44-7.34
(5H, m), 4.98 (1H, s), 4.18 (1H, s), 2.84 (2H, t, J ) 7.3 Hz), 2.72
(1H, quintet, J ) 7.0 Hz), 2.00-1.95 (1H, m), 1.71-1.65 (1H,
m), 1.60-1.54 (2H, m), 1.45-1.40 (13H, m), 1.18 (6H, d, J ) 7.0
Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.43, 170.44, 142.12,
140.66, 129.35, 128.73, 127.47, 127.20, 123.16, 118.68, 81.28,
77.24, 43.13, 29.36, 28.33, 28.26, 25.16, 19.43; MS (EI) m/z 498
(M+); HRMS calcd for C28H38O4N2S, 498.255; found, 498.255.
(S)-S-6-(tert-Butoxycarbonyl)-7-oxo-7-(4-phenylthiazol-2-ylami-
no)heptyl 2-Methylpropanethioate (14b). Yield 28%; yellow oil;
1H NMR (CDCl3, 500 MHz, δ, ppm) 9.97 (1H, broad s), 7.82 (2H,
d, J ) 7.0 Hz), 7.41 (2H, t, J ) 7.6 Hz), 7.32 (1H, d, J ) 7.3 Hz),
7.14 (1H, s), 5.05 (1H, d, J ) 7.5 Hz), 4.37 (1H, s), 2.83 (2H, t, J
) 7.3 Hz), 2.73 (1H, quintet, J ) 7.0 Hz), 2.00-1.95 (1H, m),
1.71-1.65 (1H, m), 1.60-1.54 (2H, m), 1.48 (9H, s), 1.45-1.40
(4H, m), 1.18 (6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 600 MHz,
δ, ppm) 204.42, 170.36, 157.27, 150.08, 134.34, 128.72, 128.05,
126.12, 107.95, 80.94, 77.24, 54.61, 43.12, 31.75, 29.29, 28.30,
28.20, 25.00, 19.42; MS (EI) m/z 505 (M+); HRMS calcd for
C25H35O4N3S2, 505.207; found, 505.214.
(S)-S-6-(tert-Butoxycarbonyl)-7-oxo-7-(quinolin-3-ylamino)-
1
heptyl 2-Methylpropanethioate (15b). Yield 3%; yellow oil; H
NMR (CDCl3, 500 MHz, δ, ppm) 8.76 (1H, s), 8.74 (2H, s), 8.03
(1H, d, J ) 8.8 Hz), 7.88 (1H, d, J ) 7.9 Hz), 7.62 (1H, d, J ) 7.2
Hz), 7.53 (1H, d, J ) 7.3 Hz), 5.01 (1H, s), 4.25 (1H, s), 2.85 (2H,
t, J ) 7.5 Hz), 2.73 (1H, quintet, J ) 7.0 Hz), 2.04-1.95 (1H, m),
1.71-1.65 (1H, m), 1.60-1.54 (2H, m), 1.50-1.45 (13H, m), 1.19
(6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.59,
171.08, 143.97, 131.40, 128.94, 128.29, 128.17, 127.74, 127.25,
123.88, 77.22, 43.14, 29.33, 28.31, 28.18, 25.13, 19.43; MS (EI)
m/z 473 (M+); HRMS calcd for C25H35O4N3S, 473.235; found,
473.234.
(S)-S-6-(tert-Butoxycarbonyl)-7-(cyclopentylamino)-7-oxohep-
tyl 2-Methylpropanethioate (16b). Step 1: Preparation of (S)-
tert-Butyl 7-Bromo-1-(cyclopentylamino)-1-oxoheptan-2-ylcar-
bamate (16c). To a solution of 43 (2.49 g, 7.68 mmol) obtained
above in THF (12 mL) were added 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDCI; 2.22 g, 11.6 mmol), 1-hydroxy-1H-
benzotriazole monohydrate (HOBt‚H2O; 1.77 g, 11.6 mmol), and
cyclopentylamine (781 µL, 7.92 mmol), and the mixture was stirred
overnight at room temperature. The reaction mixture was poured
into water and was extracted with AcOEt. The AcOEt layer was
separated, washed with 10% aqueous citric acid, water, saturated
NaHCO3, and brine, and dried over Na2SO4. Filtration, concentra-
tion in vacuo, and purification by silica gel flash column chroma-
tography (AcOEt/n-hexane ) 2/5) gave 2.28 g (76%) of 16c as a
colorless solid: 1H NMR (CDCl3, 500 MHz, δ, ppm) 5.97 (1H, d,
J ) 7.6 Hz), 4.98 (1H, s), 4.18 (1H, sextet, J ) 7.0 Hz), 3.95 (1H,
s), 3.40 (2H, t, J ) 6.7 Hz), 1.97 (2H, m), 1.90-1.78 (3H, m),
1.71-1.53 (5H, m), 1.49-1.30 (15H, m).
Step 2: Preparation of (S)-S-6-(tert-Butoxycarbonyl)-7-(cy-
clopentylamino)-7-oxoheptyl 2-Methylpropanethioate (16b). To
a solution of sodium hydrosulfide (426 mg, 7.60 mmol) in EtOH
(12 mL) was added iso-butyryl chloride (820 µL, 7.62 mmol)
dropwise at 0 °C, and the mixture was stirred at room temperature
for 1 h. Then, to the mixture was added a solution of 16c (296 mg,
0.756 mmol) in EtOH (7 mL) and Et3N (1 mL, 13.6 mmol), and
the resulting mixture was stirred overnight at room temperature.
The reaction mixture was poured into water and was extracted with
AcOEt. The AcOEt layer was separated, washed with brine, and
dried over Na2SO4. Filtration, concentration in vacuo, and purifica-
tion by silica gel flash column chromatography (AcOEt/n-hexane
) 1/4) gave 314 mg (100%) of 16b as a yellow oil: 1H NMR
(CDCl3, 500 MHz, δ, ppm) 5.97 (1H, d, J ) 7.6 Hz), 4.97 (1H, s),
Step 3: Preparation of (S)-S-6-(tert-Butoxycarbonyl)-7-oxo-
7-[2-oxo-4-(trifluoromethyl)-2H-chromen-7-ylamino]heptyl 2-m-
ethylpropanethioate (11b). To a solution of sodium hydrosulfide
(4.29 g, 77.9 mmol) in MeOH (50 mL) was added iso-butyryl
chloride (4 mL, 37.2 mmol) and stirred at 0 °C for 1 h. Then, the
solution was stirred at room temperature for 2 h. After that, the
reaction mixture was poured into water and extracted with AcOEt.
The extract was washed with 1 N aqueous HCl and brine and dried
over Na2SO4. Filtration and evaporation in vacuo gave 2.29 g (59%)
of thioisobutyric acid as a yellow oil. To a solution of 11c (443
mg, 0.827 mmol) obtained above in EtOH (10 mL) was added a
solution of thioisobutyric acid (455 mg, 4.27 mmol) in EtOH (5
mL) and triethylamine (1 mL, 13.6 mmol), and the mixture was
stirred overnight at room temperature. The reaction mixture was
poured into water, and the mixture was extracted with AcOEt. The
AcOEt layer was separated, washed with 10% aqueous citric acid
and brine, and dried over Na2SO4. Filtration, concentration in vacuo,
purification by silica gel flash column chromatography (AcOEt/n-
hexane ) 1/4), and recrystallization from AcOEt/n-hexane gave
1
250 mg (54%) of 11b as colorless crystals: mp 163-165 °C; H
NMR (CDCl3, 500 MHz, δ, ppm) 9.07 (1H, s), 7.81 (1H, s), 7.62
(1H, d, J ) 7.9 Hz), 7.41 (1H, d, J ) 7.3 Hz), 6.68 (1H, s), 5.06
(1H, s), 4.18 (1H, broad s), 2.84 (2H, t, J ) 7.8 Hz), 2.73 (1H,
quintet, J ) 6.9 Hz), 1.99-1.95 (1H, m), 1.70-1.65 (1H, m), 1.60-
1.54 (2H, m), 1.47 (9H, s), 1.45-1.40 (4H, m), 1.18 (6H, d, J )
7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.59, 171.00,
159.10, 155.24, 142.34, 128.50, 125.83, 122.40, 116.18, 113.84,
109.36, 107.51, 81.26, 77.24, 43.14, 29.32, 28.31, 28.20, 25.12,
19.42; Anal. (C26H33F3N2O6S‚1/4H2O) C, H, N.
Compounds 12b-15b were prepared from 43 and an appropriate
amine using the procedure described for 11b.
(S)-S-6-(tert-Butoxycarbonyl)-7-oxo-7-(phenylamino)heptyl
1
2-Methylpropanethioate (12b). Yield 9%; yellow oil; H NMR
(CDCl3, 500 MHz, δ, ppm) 8.22 (1H, s), 7.52 (2H, d, J ) 8.0 Hz),
7.31 (2H, t, J ) 7.8), 7.10 (1H, t, J ) 7.5 Hz), 5.04 (1H, s), 4.17
(1H, s), 2.84 (2H, t, J ) 7.3 Hz), 2.73 (1H, quintet, J ) 7.0 Hz),
1.96-1.92 (1H, m), 1.65-1.55 (3H, m), 1.50-1.45 (13H, m), 1.19

Series of Histone Deacetylase 6-SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5433
4.18 (1H, sextet, J ) 6.9 Hz), 3.94 (1H, s), 2.83 (2H, t, J ) 7.2
Hz), 2.73 (1H, septet, J ) 6.9 Hz), 1.97-1.94 (2H, m), 1.83-1.78
(1H, m), 1.68-1.53 (5H, m), 1.44 (9H, s), 1.47-1.37 (8H, m),
1.19 (6H, t, J ) 6.7 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm)
204.29, 170.59, 155.72, 77.23, 50.37, 43.11, 35.04, 34.95, 29.40,
28.43, 28.32, 28.30, 27.99, 25.06, 24.36, 19.42; Anal. (C21H38N2O4S‚
1/3H2O) C, H, N.
J ) 6.7 Hz), 1.84 (1H, m), 1.60-1.31 (16H, m), 1.18 (6H, d, J )
7.0 Hz); 13C NMR (CDCl3, 500 MHz, δ, ppm) 204.37, 174.46,
155.74, 80.23, 54.03, 43.13, 32.10, 29.38, 28.33, 28.26, 25.94,
19.33; MS (FAB) m/z 347 (MH+); Anal. (C16H30N2O4S‚1/3H2O)
C, H, N.
(S)-S-6-(tert-Butoxycarbonyl)-7-oxo-7-(pyrrolidin-1-yl)hep-
1
tyl 2-Methylpropanethioate (25b). Yield 74%; colorless oil; H
Compounds 17b and 19b-25b were prepared from 43 and an
appropriate amine using the procedure described for 16b.
(S)-S-6-(tert-Butoxycarbonyl)-7-(cyclohexylamino)-7-oxohep-
tyl 2-Methylpropanethioate (17b). Yield 19%; yellow oil; 1H
NMR (CDCl3, 500 MHz, δ, ppm) 5.89 (1H, d, J ) 8.2 Hz), 4.99
(1H, s), 3.94 (1H, s), 3.75 (1H, s), 2.83 (2H, t, J ) 7.4 Hz), 2.73
(1H, quintet, J ) 7.0 Hz), 1.90-1.78 (3H, m), 1.72-1.68 (2H,
m), 1.65-1.50 (4H, m), 1.47-1.28 (15H, m), 1.21-1.10 (9H, m);
13C NMR (CDCl3, 600 MHz, δ, ppm) 204.35, 170.87, 155.69,
77.23, 48.13, 43.11, 33.04, 29.39, 28.41, 28.32, 25.49, 25.03, 24.72,
19.42; MS (EI) m/z 428 (M+); HRMS calcd for C22H40O4N2S,
428.270; found, 428.276.
(S)-S-6-(tert-Butoxycarbonyl)-7-(tert-butylamino)-7-oxohep-
tyl 2-Methylpropanethioate (19b). Yield 33%; yellow oil; 1H
NMR (CDCl3, 500 MHz, δ, ppm) 5.80 (1H, s), 4.97 (1H, s), 3.88
(1H, s), 2.83 (2H, t, J ) 7.3 Hz), 2.73 (1H, septet, J ) 6.9 Hz),
1.81-1.74 (1H, m), 1.59-1.50 (2H, m), 1.48-1.40 (11H, m),
1.39-1.34 (11H, m), 1.18 (6H, d, J ) 6.7 Hz); 13C NMR (CDCl3,
600 MHz, δ, ppm) 204.29, 171.11, 77.23, 51.32, 43.11, 32.33,
29.41, 28.71, 28.46, 28.31, 25.02, 19.42; Anal. (C20H38N2O4S‚2/
3H2O) C, H, N.
(S)-S-7-(Adamant-1-ylamino)-6-(tert-Butoxycarbonyl)-7-oxo-
heptyl 2-Methylpropanethioate (20b). Yield 66%; colorless oil;
1H NMR (CDCl3, 500 MHz, δ, ppm), 5.62 (1H, s), 4.99 (1H, m),
3.88 (1H, m), 2.83 (2H, t, J ) 7.3 Hz), 2.72 (1H, septet, J ) 6.7
Hz), 2.07 (3H, s), 1.98 (6H, s), 1.77 (1H, sextet, J ) 7.0 Hz), 1.67
(6H,s), 1.56-1.30 (16H, m), 1.18 (6H, d, J ) 6.7 Hz); 13C NMR
(CDCl3,500 MHz, δ, ppm), 204.27, 170.87, 155.69, 79.90, 55.01,
43.12, 41.58, 36.33, 36.20, 32.51, 29.55, 29.50, 29.43, 28.49, 28.34,
25.00, 19.42; MS (FAB) m/z 481 (MH+); Anal. (C26H44N2O4S) C,
H, N.
(S)-S-6-(tert-Butoxycarbonyl)-7-(2,3-dihydro-1H-inden-2-ylami-
no)-7-oxoheptyl 2-Methylpropanethioate (21b). Yield 87%;
colorless oil; 1H NMR (CDCl3, 500 MHz, δ, ppm) 7.23-7.14 (4H,
m), 6.24 (1H, d, J ) 7.6 Hz), 4.94 (1H, m), 4.71 (1H, m), 3.94
(1H, m), 3.30 (2H, m), 2.76 (4H, m), 2.69 (2H, septet, J ) 6.7
Hz), 1.79 (1H, m), 1.58-1.51 (3H, m), 1.48-1.51 (13H, m), 1.18
(6H, d, J ) 6.7 Hz); 13C NMR (CDCl3, 500 MHz, δ, ppm) 204.32,
171.76, 155.68, 140.71, 126.81, 124.79, 80.09, 54.54, 50.57, 43.11,
40.09, 39.99, 32.24, 29.70, 28.37, 28.28, 25.05, 19.41; MS (FAB)
m/z 463 (MH+); Anal. (C25H38N2O4S‚1/2 H2O) C, H, N.
(S)-S-6-(tert-Butoxycarbonyl)-7-(2-hydroxyethylamino)-7-oxo-
heptyl 2-Methylpropanethioate (22b). Yield 46%; colorless oil;
1H NMR (CDCl3, 500 MHz, δ, ppm) 6.43 (1H, m), 4.98 (1H, m),
3.99 (1H, m), 3.72 (2H, q, J ) 4.8 Hz), 3.43 (2H, m), 2.61 (1H,
broad s) 1.82 (1H, m), 1.57-1.30 (16H, m), 1.18 (6H, d, J ) 6.7
Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.68, 173.06, 155.57,
80.37, 61.96, 54.89, 43.13, 42.34, 32.12, 29.35, 28.32, 28.26, 28.39,
28.18, 24.95, 19.42; MS (FAB) m/z 391 (MH+); Anal. (C18H34N2O5S‚
1/2H2O) C, H, N.
NMR (CDCl3, 500 MHz, δ, ppm) 5.32 (1H, d, J ) 8.5 Hz), 4.40
(1H, m), 3.63 (1H, m), 3.53 (1H, m), 3.41 (2H, m), 2.82 (2H, t, J
) 7.3 Hz), 2.72 (2H, quintet, J ) 6.7 Hz), 1.97 (2H, quintet, J )
6.7 Hz), 1.87 (2H, m), 1.66 (1H, m), 1.60-1.29 (16H, m), 1.18
(6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 500 MHz, δ, ppm) 170.86,
155.58, 79.48, 51.84, 46.45, 45.94, 33.83, 33.11, 28.40, 28.10,
26.07, 24.48, 24.17; MS (FAB) m/z 401 (MH+); MS (EI) m/z 400
(M+); HRMS calcd for C20H36N2O4S, 400.240; found, 400.240.
(S)-S-6-(tert-Butoxycarbonyl)-7-(cycloheptylamino)-7-oxohep-
tyl 2-Methylpropanethioate (18b). Compound 18b was prepared
from 43 using the procedure described for 11b (step 2) and 16b
(step 2) in 63% yield: yellow oil; 1H NMR (CDCl3, 500 MHz, δ,
ppm) 5.98 (1H, d, J ) 8.2 Hz), 4.98 (1H, s), 3.95-3.91 (2H, m),
2.83 (2H, t, J ) 7.3 Hz), 2.73 (1H, septet, J ) 7.0 Hz), 1.88-1.87
(2H, m), 1.81-1.78 (1H, m), 1.61-1.36 (26H, m), 1.18 (6H, d, J
) 7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.31, 171.16,
77.23, 60.40, 50.37, 43.11, 32.27, 29.40, 28.32, 28.30, 27.99, 25.06,
24.03, 21.06, 19.42, 14.21; Anal. (C23H42N2O4S‚1/2H2O) C, H, N.
(S)-S-6-Amino-7-(cyclopentylamino)-7-oxoheptyl 2-Methyl-
propanethioate Hydrochloride (26b‚HCl). To a solution of 16b
(962 mg, 2.32 mmol) in AcOEt (12 mL) was added 4 N HCl/AcOEt
(6 mL), and the mixture was stirred at room temperature for 2 h.
Then, the solvent was removed in vacuo to give 813 mg (100%)
of 26b‚HCl as a colorless oil: 1H NMR (DMSO-d6, 500 MHz, δ,
ppm) 8.59 (1H, d, J ) 7.3 Hz), 8.19 (3H, broad s), 4.03 (1H, sextet,
J ) 6.7 Hz), 3.67 (1H, m), 2.81 (2H, t, J ) 7.3 Hz), 2.73 (1H,
septet, J ) 6.7 Hz), 1.85-1.76 (2H, m), 1.69-1.58 (4H, m), 1.52-
1.20 (10H, m), 1.10 (6H, t, J ) 7.0 Hz); 13C NMR (DMSO-d6,
500 MHz, δ, ppm) 203.17, 167.70, 52.14, 50.55, 42.40, 32.32,
31.83, 30.87, 28.82, 27.61, 27.59, 23.60, 23.38, 23.33, 19.09; MS
(FAB) m/z 315 (MH+ - HCl); Anal. (C16H30N2O2S‚HCl‚H2O) C,
H, N.
(S)-S-6-Benzamido-7-(cyclopentylamino)-7-oxoheptyl 2-Methyl-
propanethioate (27b). To a suspension of 26b‚HCl (250 mg, 0.712
mmol) and Et3N (1 mL, 13.6 mmol) in CH2Cl2 (2 mL) was added
a solution of benzoyl chloride (248 µL mg, 2.13 mmol) in CH2Cl2
(3 mL) dropwise. The reaction mixture was poured into water and
was extracted with AOEt. The AcOEt layer was separated, washed
with 10% aqueous citric acid and brine, and dried over Na2SO4.
Filtration, evaporation of the solvent in vacuo, and purification by
flash column chromatography (AcOEt/n-hexane ) 1/4 to 1/2) gave
a crude solid. The solid was recrystallized from AcOEt/n-hexane
and collected by filtration to give 208 mg (70%) of 27b as colorless
1
crystals: mp 116-117 °C; H NMR (CDCl3, 500 MHz, δ, ppm)
7.78 (2H, m), 7.51 (1H, t, J ) 7.3 Hz), 7.43 (2H, t, J ) 7.6 Hz),
6.89 (1H, d, J ) 7.9 Hz), 6.24 (1H, d, J ) 7.6 Hz), 4.57 (1H, q,
J ) 7.3 Hz), 4.19 (1H, sextet, J ) 7.0 Hz), 2.83 (2H, m), 2.72
(1H, septet, J ) 6.7 Hz), 2.02-1.90 (3H, m), 1.80-1.65 (7H, m),
1.45-1.35 (6H, m), 1.18 (6H, dd, J ) 6.9, 1.2 Hz); 13C NMR
(CDCl3, 500 MHz, δ, ppm) 204.36, 170.98, 167.24, 133.95, 131.73,
128.57, 127.06, 53.50, 51.34, 43.09, 33.10, 32.94, 32.57, 29.35,
28.35, 28.20, 24.85, 23.75, 23.72, 19.40; MS (EI) m/z 418 (M+);
HRMS calcd for C23H34N2O3S, 418.229; found, 418.229; Anal.
(C23H34N2O3S) C, H, N.
(S)-S-6-(tert-Butoxycarbonyl)-7-(2-methoxyethylamino)-7-
oxoheptyl 2-Methylpropanethioate (23b). Yield 72%; colorless
1
oil; H NMR (CDCl3, 500 MHz, δ, ppm) 6.30 (1H, s), 4.98 (1H,
m), 4.02 (1H, m), 3.45 (4H, m), 3.35 (3H, m), 2.83 (2H, t, J ) 7.3
Hz), 2.72 (1H, septet, J ) 6.9 Hz), 1.82 (1H, sextet, J ) 7.0 Hz),
1.60-1.55 (2H, m), 1.50-1.29 (14H, m), 1.18 (6H, d, J ) 7.0
Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.28, 172.02, 155.61,
80.02, 71.06, 58.77, 54.59, 43.12, 39.20, 32.57, 29.42, 28.43, 28.32,
25.03, 19.41; MS (FAB) m/z 495 (MH+); Anal. (C19H36N2O5S‚1/
3H2O) C, H, N.
Compounds 28b and 29b were prepared from 26b‚HCl and an
appropriate acid chloride using the procedure described for 27b.
(S)-S-6-(4-Chlorobenzamido)-7-(cyclopentylamino)-7-oxohep-
tyl 2-Methylpropanethioate (28b). Yield 95%; mp 119-121 °C;
1H NMR (CDCl3, 500 MHz, δ, ppm) 7.74 (2H, d, J ) 8.5 Hz),
7.40 (2H, d, J ) 8.5 Hz), 6.89 (1H, d, J ) 7.9 Hz), 6.04 (1H, d,
J ) 7.6 Hz), 4.51 (1H, q, J ) 7.0 Hz), 4.20 (1H, sextet, J ) 7.0
Hz), 2.82 (2H, m), 2.72 (1H, septet, J ) 7.0 Hz), 2.04-1.87 (3H,
m), 1.80-1.53 (9H, m), 1.40 (6H, m), 1.18 (6H, dd, J ) 7.0, 1.5
Hz); 13C NMR (CDCl3, 500 MHz, δ, ppm) 204.48, 170.88, 166.13,
(S)-S-7-Amino-6-(tert-butoxycarbonyl)-7-oxoheptyl 2-Methyl-
1
propanethioate (24b). Yield 36%; yellow oil; H NMR (CDCl3,
500 MHz, δ, ppm) 6.04 (1H, broad s), 5.36 (1H, broad s), 4.97
(1H, m), 4.10 (1H, m), 2.83 (2H, t, J ) 7.6 Hz), 2.72 (1H, septet,

5434 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Itoh et al.
138.03, 132.32, 128.85, 128.55, 53.37, 51.44, 43.13, 33.13, 32.97,
32.64, 29.64, 28.25, 28.13, 24.76, 23.75, 23.72, 19.43, 19.41; Anal.
(C23H33ClN2O3S) C, H, N.
chromatography (AcOEt/n-hexane ) 3/1 to AcOEt only) gave a
crude solid. The solid was recrystallized from AcOEt/n-hexane and
collected by filtration to give 54 mg (52%) of 31b as colorless
1
crystals: mp 130-131 °C; H NMR (CDCl3, 500 MHz, δ, ppm)
(S)-S-7-(Cyclopentylamino)-6-(furan-2-carboxamido)-7-oxo-
heptyl 2-Methylpropanethioate (29b). Yield 37%; mp 88-89 °C;
1H NMR (CDCl3, 500 MHz, δ, ppm) 7.46 (1H, d, J ) 0.9 Hz),
7.11 (1H, d, J ) 3.6 Hz), 6.88 (1H, d, J ) 7.9 Hz), 6.50 (1H, dd,
J ) 3.5, 1.8 Hz), 6.03 (1H, d, J ) 7.0 Hz), 4.46 (1H, q, J ) 7.6
Hz), 4.19 (1H, sextet, J ) 6.7 Hz), 2.84 (2H, td, J ) 7.5, 2.1 Hz),
2.72 (1H, septet, J ) 7.0 Hz), 2.02-1.87 (3H, m), 1.74-1.53 (7H,
m), 1.45-1.34 (6H, m), 1.18 (6H, dd, J ) 7.0, 0.9 Hz); 13C NMR
(CDCl3, 600 MHz, δ, ppm) 204.39, 170.66, 158.20, 147.48, 144.28,
114.66, 112.15, 52.83, 51.39, 43.13, 33.13, 32.98, 32.43, 29.37,
28.35, 28.26, 24.93, 23.76, 23.73, 19.42; MS (EI) m/z 408 (M+);
HRMS calcd for C21H32N2O4S, 408.208; found, 408.209; Anal.
(C21H32N2O4S) C, H, N.
6.10 (1H, d, J ) 8.2 Hz), 5.94 (1H, d, J ) 7.6 Hz), 4.28 (1H, q,
J ) 7.3 Hz), 4.17 (1H, sextet, J ) 7.3 Hz), 2.82 (2H, m), 2.73
(1H, septet, J ) 6.7 Hz), 2.02-1.93 (5H, m), 1.80 (1H, m), 1.74-
1.49 (7H, m), 1.46-1.29 (6H, m), 1.18 (6H, dd, J ) 7.0, 0.9 Hz);
13C NMR (CDCl3, 500 MHz, δ, ppm) 204.46, 170.02, 170.02,
53.17, 51.30, 43.14, 33.12, 32.97, 32.34, 29.32, 28.26, 28.16, 24.77,
23.73, 23.71, 23.26, 19.43; MS (EI) m/z 356 (M+); HRMS calcd
for C18H32N2O3S, 356.213; found, 356.213; Anal. (C18H32N2O3S)
C, H, N.
(S)-S-6-(3-tert-Butylureido)-7-(cyclopentylamino)-7-oxohep-
tyl 2-Methylpropanethioate (35b). To a solution of 26b‚HCl (99
mg, 0.282 mmol) and Et3N (400 µL, 5.44 mmol) in CH2Cl2 (4
mL) was added tert-butyl isocyanate (132 µL, 1.14 mmol), and
the mixture was stirred at room temperature for 6 h. The reaction
mixture was poured into water and was extracted with AcOEt. The
AcOEt layer was separated, washed with brine, and dried over Na2-
SO4. Filtration, evaporation of the solvent in vacuo, and purification
by silica gel flash column chromatography (AcOEt/n-hexane ) 1/1)
gave 89 mg (76%) of 35b as a colorless oil: 1H NMR (CDCl3,
500 MHz, δ, ppm) 6.27 (1H, d, J ) 7.9 Hz), 4.81 (1H, d, J ) 7.9
Hz), 4.48 (1H, s), 4.16 (1H, sextet, J ) 6.7 Hz), 4.07 (1H, q, J )
5.8 Hz), 2.92-2.70 (3H, m), 1.94 (2H, m), 1.76 (1H, m), 1.72-
1.55 (7H, m), 1.43-1.25 (15H, m), 1.18 (6H, dd, J ) 6.7, 0.9 Hz);
13C NMR (CDCl3, 500 MHz, δ, ppm) 204.82, 172.39, 157.10,
53.85, 51.12, 50.57, 43.14, 33.08, 33.00, 32.07, 29.43, 29.22, 28.21,
28.12, 24.78, 23.73, 23.69, 19.46, 19.44; MS (EI) m/z 413 (M+);
HRMS calcd for C21H39N3O3S, 413.271; found, 413.273.
Compounds 30b and 32b-34b were prepared from 26b‚HCl and
an appropriate carboxylic acid using the procedure described for
16b (step 1).
(S)-S-7-(Cyclopentylamino)-6-(2-hydroxyacetamido)-7-oxo-
heptyl 2-Methylpropanethioate (30b). Yield 28%; colorless oil;
1H NMR (CDCl3, 500 MHz, δ, ppm) 6.97 (1H, d, J ) 8.2 Hz),
5.99 (1H, d, J ) 7.3 Hz), 4.34 (1H, m), 4.21-4.13 (3H, m), 2.91
(1H, broad s), 2.83 (2H, m), 2.74 (1H, septet, J ) 7.0 Hz), 1.98
(2H, m), 1.85 (1H, m), 1.71-1.53 (7H, m), 1.44-1.31 (6H, m),
1.18 (6H, dd, J ) 7.0, 1.4 Hz); 13C NMR (CDCl3, 500 MHz, δ,
ppm) 204.91, 171.85, 170.86, 62.21, 52.78, 51.38, 43.14, 33.08,
32.92, 32.25, 29.28, 29.24, 29.06, 28.04, 24.73, 23.74, 19.44, 19.42;
MS (EI) m/z 372 (M+); HRMS calcd for C18H32N2O4S, 372.208;
found, 372.208.
(S)-S-6-(Cyclohexanecarboxamido)-7-(cyclopentylamino)-7-
oxoheptyl 2-Methylpropanethioate (32b). Yield 62%; colorless
oil; 1H NMR (CDCl3, 500 MHz, δ, ppm) 6.34 (2H, m), 4.29 (1H,
q, J ) 6.7 Hz), 4.16 (1H, sextet, J ) 7.0 Hz), 2.82 (2H, m), 2.73
(1H, septet, J ) 7.0 Hz), 2.10 (1H, tt, J ) 7.0, 3.3 Hz), 1.94 (2H,
m), 1.90-1.75 (5H, m), 1.70-1.56 (9H, m), 1.49-1.22 (10H, m),
1.18 (6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm)
204.39, 176.22, 171.14, 52.75, 51.22, 45.42, 43.13, 33.07, 33.01,
32.03, 29.70, 29.35, 28.33, 28.22, 25.72, 25.68, 25.64, 24.87, 23.74,
19.42; MS (EI) m/z 424 (M+); HRMS calcd for C23H40N2O3S,
424.276; found, 424.275.
(S)-S-7-(Cyclopentylamino)-7-oxo-6-pivalamidoheptyl 2-Methyl-
propanethioate (33b). Yield 62%; colorless oil; 1H NMR (CDCl3,
500 MHz, δ, ppm) 6.21 (1H, d, J ) 7.6 Hz), 6.08 (1H, d, J ) 7.3
Hz), 4.29 (1H, q, J ) 6.7 Hz), 4.17 (1H, sextet, J ) 7.0 Hz), 2.82
(2H, m), 2.72 (1H, septet, J ) 7.0 Hz), 1.96 (2H, m), 1.83 (1H,
m), 1.70-1.53 (7H, m), 1.42-1.29 (6H, m), 1.20 (9H, m), 1.18
(6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 204.34,
178.62, 171.17, 52.89, 51.22, 43.13, 38.75, 33.07, 31.98, 29.37,
28.36, 28.23, 27.48, 24.90, 23.74, 23.69, 19.41; MS (EI) m/z 398
(M+); HRMS calcd for C21H38N2O3S, 398.260; found, 398.261.
(S)-S-7-(Cyclopentylamino)-6-(3,3-dimethylbutanamido)-7-
oxoheptyl 2-Methylpropanethioate (34b). Yield 59%; colorless
oil; 1H NMR (CDCl3, 500 MHz, δ, ppm) 6.21 (1H, d, J ) 7.3 Hz),
6.05 (1H, d, J ) 8.2 Hz), 4.30 (1H, q, J ) 6.7 Hz), 4.16 (1H,
sextet, J ) 6.9 Hz), 2.81 (2H, m), 2.73 (1H, septet, J ) 6.7 Hz),
2.07 (2H, m), 1.96 (2H, m), 1.80 (1H, m), 1.70-1.52 (7H, m),
1.42-1.30 (6H, m), 1.18 (6H, d, J ) 6.7 Hz), 1.02 (9H, s); 13C
NMR (CDCl3, 600 MHz, δ, ppm) 204.41, 171.89, 171.10, 53.02,
52.94, 51.23, 50.46, 43.13, 33.09, 32.97, 32.34, 31.90, 30.99, 29.85,
29.33, 28.31, 28.19, 23.75, 23.73, 19.43, 19.42; MS (EI) m/z 412
(M+); HRMS calcd for C22H40N2O3S, 412.276; found, 412.276.
(S)-S-6-Acetamido-7-(cyclopentylamino)-7-oxoheptyl 2-Methyl-
propanethioate (31b). To a solution of 26b‚HCl (102 mg, 0.291
mmol) and a catalytic amount of DMAP in CH2Cl2 (3 mL) were
added acetic acid anhydride (55 µL, 0.582 mmol) and Et3N (200
µL, 2.72 mmol), and the mixture was stirred at room temperature
for 4 h. The reaction mixture was poured into water and was
extracted with AcOEt. The AcOEt layer was separated, washed
with brine, and dried over Na2SO4. Filtration, evaporation of the
solvent in vacuo, and purification by silica gel flash column
(S)-S-6-(3-tert-Butylthioureido)-7-(cyclopentylamino)-7-oxo-
heptyl 2-Methylpropanethioate (36b). Compound 36b was pre-
pared from 26b‚HCl and tert-thioisocyanate using the procedure
1
described for 35b in 24% yield: colorless oil; H NMR (CDCl3,
500 MHz, δ, ppm) 6.53 (1H, d, J ) 7.0 Hz), 6.09 (1H, m), 5.97
(1H, d, J ) 7.0 Hz), 4.85 (1H, m), 4.19 (1H, sextet, J ) 7.0 Hz),
2.82 (2H, m), 2.73 (1H, septet, J ) 7.0 Hz), 1.98 (3H, m), 1.78-
1.52 (7H, m), 1.48-1.29 (15H, m), 1.19 (6H, dd, J ) 7.0, 1.5 Hz);
13C NMR (CDCl3, 600 MHz, δ, ppm) 204.47, 179.78, 170.93,
58.73, 52.94, 51.53, 43.12, 33.10, 32.94, 32.64, 29.46, 29.34, 28.33,
28.15, 24.21, 23.69, 23.64, 19.44, 19.42; MS (EI) m/z 429 (M+);
HRMS calcd for C21H39N3O2S2, 429.248; found, 429.248.
(S)-S-7-(Cyclopentylamino)-6-(methoxycarbonyl)-7-oxohep-
tyl 2-Methylpropanethioate (37b). To a solution of 26b‚HCl (177
mg, 0.504 mmol) in CH2Cl2 (3 mL) were added methyl chloro-
formate (39 µL, 0.505 mmol) and Et3N (500 µL, 6.80 mmol), and
the mixture was stirred at room temperature for 15 min. The reaction
mixture was poured into water and was extracted with AcOEt. The
AcOEt layer was separated, washed with brine, and dried over Na2-
SO4. Filtration, evaporation of the solvent in vacuo, and purification
by silica gel flash column chromatography (AcOEt/n-hexane ) 1/2
to AcOEt only) gave a crude solid. The solid was recrystallized
from AcOEt/n-hexane and collected by filtration to give 138 mg
(74%) of 37b as colorless crystals: mp 88-89 °C; 1H NMR
(CDCl3, 500 MHz, δ, ppm) 5.90 (1H, d, J ) 6.7 Hz), 5.24 (1H,
broad s), 4.18 (1H, sextet, J ) 7.0 Hz), 4.02 (1H, m), 3.68 (3H, s),
2.82 (2H, td, J ) 7.6, 2.4 Hz), 2.73 (1H, septet, J ) 7.0 Hz), 1.89
(2H, m), 1.81 (1H, m), 1.73-1.53 (7H, m), 1.42-1.31 (6H, m),
1.18 (6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm)
204.38, 171.08, 156.86, 54.99, 52.38, 51.29, 43.13, 33.12, 33.01,
32.61, 29.71, 29.36, 28.30, 28.23, 24.82, 23.74, 19.42; MS (EI)
m/z 372 (M+); HRMS calcd for C18H32N2O4S, 372.208; found,
372.208; Anal. (C18H32N2O4S) C, H, N.
Compounds 39b-41b were prepared from 26b‚HCl and an
appropriate chloroformate using the procedure described for 37b.
(S)-S-7-(Cyclopentylamino)-7-oxo-6-(phenoxycarbonyl)hep-
1
tyl 2-Methylpropanethioate (39b). Yield 17%; colorless oil; H
NMR (CDCl3, 500 MHz, δ, ppm) 7.36 (2H, t, J ) 7.9 Hz), 7.20
(1H, t, J ) 7.6 Hz), 7.12 (2H, d, J ) 7.9 Hz), 5.85 (1H, d, J ) 7.0
Hz), 5.66 (1H, d, J ) 8.2 Hz), 4.22 (1H, sextet, J ) 7.3 Hz), 4.09

Series of Histone Deacetylase 6-SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5435
(1H, q, J ) 7.3 Hz), 2.83 (2H, td, J ) 7.0, 2.4 Hz), 2.73 (1H,
septet, J ) 7.0 Hz), 1.99 (2H, m), 1.86 (1H, m), 1.72-1.53 (7H,
m), 1.46-1.36 (6H, m), 1.18 (6H, d, J ) 7.0 Hz); 13C NMR (CDCl3,
600 MHz, δ, ppm) 204.42, 170.69, 154.82, 150.92, 129.33, 125.47,
121.54, 55.06, 51.42, 43.14, 33.15, 33.02, 32.71, 29.37, 28.26,
28.21, 24.76, 23.74, 19.42; MS (FAB) m/z 435 (MH+); Anal.
(C23H34N2O4S) C, H, N.
16d (300 mg, 0.778 mmol) obtained above in EtOH (5 mL) was
added 2 N aqueous NaOH (2 mL, 4.00 mmol), and the solution
was stirred at room temperature for 10 min. The reaction mixture
was poured into water and was extracted with AcOEt. The AcOEt
layer was separated, washed with water and brine, and dried over
Na2SO4. Filtration, concentration in vacuo, and purification by silica
gel flash column chromatography (AcOEt/n-hexane ) 1/4) gave
200 mg (74%) of 16a as a colorless solid. The solid (106 mg) was
recrystallized from AcOEt/n-hexane to give 60 mg of 16a as
(S)-S-6-(Benzyloxycarbonyl)-7-(cyclopentylamino)-7-oxohep-
1
tyl 2-Methylpropanethioate (40b). Yield 24%; colorless oil; H
1
colorless crystals: mp 108-110 °C; H NMR (CDCl3, 500 MHz,
NMR (CDCl3, 500 MHz, δ, ppm) 7.35 (5H, m), 5.89 (1H, m), 5.31
(1H, m), 5.10 (2H, s), 4.17 (1H, sextet, J ) 6.7 Hz), 4.04 (1H, m),
2.82 (2H, td, J ) 7.2, 2.1 Hz), 2.72 (1H, septet, J ) 7.0 Hz), 1.94
(2H, s), 1.81 (1H, m), 1.70-1.51 (7H, m), 1.42-1.23 (6H, m),
1.17 (6H, d, J ) 7.0 Hz); 13C NMR (CDCl3, 500 MHz, δ, ppm)
204.39, 170.98, 156.19, 136.27, 128.56, 128.22, 128.06, 67.04,
55.00, 51.28, 43.12, 33.09, 32.99, 32.52, 29.36, 28.30, 28.23, 24.84,
23.71, 19.42; MS (EI) m/z 448 (M+); HRMS calcd for C24H36N2O4S,
448.240; found, 448.239; Anal. (C24H36N2O4S) C, H, N.
(S)-S-6-[{(9H-Fluoren-9-yl)methoxy}carbonyl]-7-(cyclopen-
tylamino)-7-oxoheptyl 2-Methylpropanethioate (41b). Yield
11%; colorless oil; 1H NMR (CDCl3, 500 MHz, δ, ppm) 7.76 (2H,
d, J ) 7.6 Hz), 7.58 (2H, d, J ) 7.6 Hz), 7.40 (2H, t, J ) 7.6 Hz),
7.31 (2H, td, J ) 7.7, 0.9 Hz), 5.82 (1H, m), 5.34 (1H, d, J ) 7.7
Hz), 4.40 (2H, m), 4.23-4.15 (1H, m), 4.02 (1H, m), 2.84 (2H,
m), 2.73 (1H, septet, J ) 6.7 Hz), 1.97 (2H, m), 1.82 (1H, m),
1.68-1.52 (7H, m), 1.45-1.25 (6H, m), 1.18 (6H, d, J ) 7.0 Hz);
13C NMR (CDCl3, 600 MHz, δ, ppm) 204.38, 170.95, 156.19,
143.80, 141.35, 127.75, 127.10, 125.04, 120.02, 120.00, 67.01,
54.99, 51.32, 47.21, 43.14, 33.12, 33.02, 32.59, 29.37, 28.28, 28.23,
24.80, 23.74, 23.72, 19.42; MS (FAB) m/z 537 (MH+); Anal.
(C31H40N2O4S‚1/3H2O) C, H, N.
(S)-S-6-(Cyclohexyloxycarbonyl)-7-(cyclopentylamino)-7-oxo-
heptyl 2-Methylpropanethioate (38b). To a solution of triphosgene
(654 mg, 2.20 mmol) in dry Et2O (4 mL) was added dropwise a
mixture of cyclohexanol (682 µL, 6.46 mmol) and pyridine (626
µL, 7.74 mmol) at -78 °C. The mixture was stirred for 1 h at -78
°C and then 1.5 h at room temperature. The reaction mixture was
poured into 1 N aqueuos HCl and was extracted with AcOEt,
washed with brine, and dried over Na2SO4. Filtration and evapora-
tion in vacuo gave a colorless oil. To a solution of 26b‚HCl (94
mg, 0.268 mmol) and Et3N (300 µL, 4.08 mmol) in CH2Cl2 (4
mL) was added the colorless oil, and the mixture was stirred at
room temperature for 13 h. The reaction mixture was poured into
water and was extracted with AcOEt. The AcOEt layer was
separated, washed with brine, and dried over Na2SO4. Filtration,
evaporation of the solvent in vacuo, and purification by silica gel
flash column chromatography (AcOEt/n-hexane ) 1/3) gave 30
mg (26%) of 38b as a colorless oil: 1H NMR (CDCl3, 500 MHz,
δ, ppm) 5.91 (1H, d, J ) 7.3 Hz), 5.08 (1H, m), 4.61 (1H, m),
4.18 (1H, sextet, J ) 7.6 Hz), 3.98 (1H, m), 2.82 (2H, td, J ) 7.0,
1.8 Hz), 2.73 (1H, septet, J ) 7.0 Hz), 1.98 (2H, m), 1.84 (3H,
m), 1.75-1.50 (11H, m), 1.37 (10H, m), 1.18 (6H, d, J ) 7.0 Hz);
13C NMR (CDCl3, 600 MHz, δ, ppm) 204.38, 171.20, 63.46, 54.80,
51.20, 43.11, 33.11, 33.03, 32.41, 31.92, 29.37, 28.34, 28.25, 25.36,
24.92, 23.72, 23.70, 23.69, 19.42; MS (FAB) m/z 441 (MH+); MS
(EI) m/z 440 (M+); HRMS calcd for C23H40N2O4S, 440.271; found,
440.270.
(S)-tert-Butyl 1-(Cyclopentylamino)-7-mercapto-1-oxoheptan-
2-ylcarbamate (16a). Step 1: Preparation of (S)-S-6-(tert-
Butoxycarbonyl)-7-(cyclopentylamino)-7-oxoheptyl Ethaneth-
ioate (16d). A solution of 16c (410 mg, 1.05 mmol) obtained above
and KSAc (183 mg, 1.60 mmol) in EtOH (3 mL) was stirred
overnight at room temperature. The reaction mixture was evaporated
in vacuo. The residue was subjected to silica gel flash column
chromatography (AcOEt/n-hexane ) 1/4) to give 303 mg (75%)
of 16d as a yellow solid: 1H NMR (CDCl3, 500 MHz, δ, ppm)
5.97 (1H, d, J ) 7.3 Hz), 4.97 (1H, s), 4.18 (1H, m), 3.94 (1H, m),
2.85 (2H, t, J ) 7.3 Hz), 2.32 (3H, s), 1.97-1.96 (2H, m),
1.81-1.80(1H,m),1.67-1.55(8H,m),1.44(9H,s),1.39-1.36(5H,m).
Step 2: Preparation of (S)-tert-Butyl 1-(Cyclopentylamino)-
7-mercapto-1-oxoheptan-2-ylcarbamate (16a). To a solution of
δ, ppm) 5.93 (1H, d, J ) 7.0 Hz), 4.95 (1H, s), 4.19-4.16 (1H,
m), 3.94 (1H, s), 2.52 (2H, q, J ) 7.3 Hz), 1.98-1.96 (2H, m),
1.85-1.80 (1H, m), 1.67-1.52 (7H, m), 1.44 (9H, s), 1.42-1.31
(7H, m); 13C NMR (CDCl3, 600 MHz, δ, ppm) 171.89, 77.23, 48.04,
36.96, 33.79, 33.31, 28.65, 28.04, 25.68, 25.56, 24.88, 24.56; Anal.
(C17H32N2O3S) C, H, N.
Compounds 13a, 15a, and 17a-20a were prepared from the
corresponding bromide using the procedure described for 16a.
(S)-tert-Butyl 1-(Biphenyl-3-ylamino)-7-mercapto-1-oxohep-
tan-2-ylcarbamate (13a). Yield 34%; colorless oil: 1H NMR
(CDCl3, 500 MHz, δ, ppm) 8.23 (1H, broad s), 7.79 (1H, s), 7.58
(2H, d, J ) 7.3 Hz), 7.48 (1H, d, J ) 7.9 Hz), 7.44-7.33 (5H, m),
4.97 (1H, s), 4.18 (1H, s), 2.53 (2H, q, J ) 7.3 Hz), 2.00-1.96
(1H, m), 1.71-1.59 (3H, m), 1.47-1.43 (13H, m), 1.33 (1H, t, J
) 7.8 Hz); 13C NMR (CDCl3, 600 MHz, δ, ppm) 170.39, 156.27,
142.14, 140.64, 138.12, 129.35, 128.73, 127.48, 127.18, 123.18,
118.65, 118.61, 80.71, 55.27, 33.70, 30.93, 28.33, 27.99, 25.25,
24.46; MS (EI) m/z 428 (M+); HRMS calcd for C24H34N2O3S,
428.213; found, 428.213.
(S)-tert-Butyl 7-Mercapto-1-oxo-1-(quinolin-3-ylamino)hep-
tan-2-ylcarbamate (15a). Yield 39%; yellow oil; 1H NMR (CDCl3,
500 MHz, δ, ppm) 8.79 (1H, broad s), 8.74 (2H, s), 8.03 (1H, d,
J ) 8.5 Hz), 7.78 (1H, d, J ) 7.9 Hz), 7.62 (1H, t, J ) 7.0 Hz),
7.52 (1H, t, J ) 7.8 Hz), 5.00 (1H, d, J ) 6.7 Hz), 4.26 (1H, m),
2.53 (2H, q, J ) 7.6 Hz), 2.00 (1H, m), 1.73-1.63 (3H, m), 1.50-
1.36 (13H, m), 1.34 (1H, t, J ) 7.9 Hz); 13C NMR (CDCl3, 600
MHz, δ, ppm) 171.17, 145.18, 143.89, 131.37, 128.97, 128.20,
128.07, 127.68, 127.14, 123.66, 81.13, 77.23, 33.65, 30.94, 28.35,
27.97, 25.35, 24.43; MS (EI) m/z 403 (M+); HRMS calcd for
C21H29N3O3S, 403.193; found, 403.194.
(S)-tert-Butyl 1-(cyclohexylamino)-7-mercapto-1-oxoheptan-
2-ylcarbamate (17a). Yield 72%; mp 125-127 °C; 1H NMR
(CDCl3, 500 MHz, δ, ppm) 5.85 (1H, d, J ) 7.9 Hz), 4.96 (1H, s),
3.95 (1H, s), 3.75 (1H, s), 2.51 (2H, q, J ) 7.4 Hz), 1.90-1.78
(3H, m), 1.72-1.68 (2H, m), 1.64-1.56 (4H, m), 1.44 (9H, s),
1.43-1.31 (7H, m), 1.19-1.13 (3H, m); 13C NMR (CDCl3, 600
MHz, δ, ppm) 170.84, 77.23, 48.13, 33.74, 33.08, 32.93, 30.94,
28.33, 27.99, 25.49, 25.07, 24.71, 24.46; MS (EI) m/z 358 (M+);
HRMS calcd for C18H34O3N2S, 358.229; found, 358.229; Anal.
(C18H34N2O3S) C, H, N.
(S)-tert-Butyl 1-(Cycloheptylamino)-7-mercapto-1-oxoheptan-
2-ylcarbamate (18a). Yield 62%; mp 76-78 °C; 1H NMR (CDCl3,
500 MHz, δ, ppm) 5.92 (1H, d, J ) 7.6 Hz), 4.96 (1H, s), 3.95-
3.91 (2H, m), 2.52 (2H, q, J ) 7.3 Hz), 1.88-1.79 (3H, m), 1.62-
1.58 (7H, m), 1.54-1.33 (20H, m); 13C NMR (CDCl3, 600 MHz,
δ, ppm) 170.55, 77.21, 54.60, 50.38, 35.06, 34.96, 33.74, 32.34,
28.33, 27.99, 25.08, 24.47, 24.02; Anal. (C18H34N2O3S) C, H, N.
(S)-tert-Butyl 1-(tert-Butylamino)-7-mercapto-1-oxoheptan-2-
ylcarbamate (19a). Yield 58%; mp 90-91 °C; 1H NMR (CDCl3,
500 MHz, δ, ppm) 5.78 (1H, s), 4.97 (1H, m), 3.89 (1H, m), 2.52
(2H, q, J ) 7.6 Hz), 1.79 (1H, sextet, J ) 8.2 Hz), 1.66-1.50
(3H, m), 1.49-1.38 (11H, m), 1.36-1.30 (12H, m); 13C NMR
(CDCl3, 500 MHz, δ, ppm) 171.05, 155.69, 79.90, 54.91, 51.26,
33.68, 32.36, 28.66, 28.26, 27.94, 24.96, 24.39; MS (FAB) m/z
333 (MH+); Anal. (C16H32N2O3S) C, H, N.
(S)-tert-Butyl 1-(Adamant-1-ylamino)-7-mercapto-1-oxohep-
tan-2-ylcarbamate (20a). Yield 41%; colorless oil; 1H NMR
(CDCl3, 500 MHz, δ, ppm) 5.60 (1H, s), 4.97 (1H, m), 3.88 (1H,
m), 2.81 (2H, q, J ) 7.3 Hz), 2.07 (3H, s), 1.98 (6H, d, J ) 2.7
Hz), 1.78 (1H, sextet, J ) 7.8 Hz), 1.67 (3H, s), 1.65-1.30 (17H,
m); 13C NMR (CDCl3, 500 MHz, δ, ppm) 170.87, 155.71, 79.93,

5436 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Itoh et al.
54.98, 41.60, 36.32, 33.76, 32.60, 30.92, 29.55, 29.43, 29.22, 28.35,
28.02, 25.00, 24.47; MS (FAB) m/z 411 (MH+); Anal. (C22H38N2O3S‚
1/4H2O) C, H, N.
(CDCl3, 500 MHz, δ, ppm) 6.48 (1H, d, J ) 7.6 Hz), 6.06 (1H, s),
5.85 (1H, d, J ) 7.6 Hz), 4.87 (1H, m), 4.19 (1H, sextet, J ) 7.0
Hz), 2.50 (2H, q, J ) 7.0 Hz), 2.02 (3H, m), 1.76-1.58 (7H, m),
1.48-1.36 (16H, m); 13C NMR (CDCl3, 600 MHz, δ, ppm) 179.92,
171.32, 58.04, 53.03, 51.49, 33.71, 33.10, 32.87, 32.74, 29.42,
28.08, 24.50, 24.45, 23.70, 23.66; MS (EI) m/z 359 (M+); HRMS
calcdforC17H33N3O2S,359.207;found,359.205;Anal.(C17H33N3O2S)
C, H, N.
Compounds 37a and 40a were prepared from 16d using the
procedure described for 26b‚HCl, 37b, and 16a (step 2).
(S)-Methyl 1-(Cyclopentylamino)-7-mercapto-1-oxoheptan-
2-ylcarbamate (37a). Yield 89%; mp 81-82 °C; 1H NMR (CDCl3,
500 MHz, δ, ppm) 5.79 (1H, d, J ) 7.0 Hz), 5.18 (1H, m), 4.18
(1H, sextet, J ) 6.7 Hz), 4.02 (1H, m), 3.68 (3H, s), 2.51 (2H, q,
J ) 7.6 Hz), 1.98 (2H, m), 1.81 (1H, m), 1.71-1.53 (7H, m), 1.46-
1.30 (7H, m); 13C NMR (CDCl3, 500 MHz, δ, ppm) 171.02, 54.97,
52.41, 51.30, 33.68, 33.14, 33.03, 32.73, 27.93, 24.91, 24.45, 23.71;
MS (EI) m/z 302 (M+); HRMS calcd for C14H26N2O3S, 302.166;
found, 302.169; Anal. (C14H26N2O3S) C, H, N.
(S)-Benzyl 1-(Cyclopentylamino)-7-mercapto-1-oxoheptan-2-
ylcarbamate (40a). Yield 77%; mp 119-122 °C; 1H NMR (CDCl3,
500 MHz, δ, ppm) 7.35 (5H, m), 5.80 (1H, m), 5.27 (1H, m), 5.11
(2H, s), 4.17 (1H, q, J ) 6.7 Hz), 4.03 (1H, m), 2.50 (2H, q, J )
7.3 Hz), 1.96 (2H, s), 1.84 (1H, m), 1.70-1.53 (7H, m), 1.45-
1.26 (7H, m); 13C NMR (CDCl3, 500 MHz, δ, ppm) 170.98, 156.18,
136.22, 128.57, 128.24, 128.06, 67.04, 54.17, 51.26, 33.68, 33.11,
32.98, 32.66, 27.93, 24.92, 24.44, 23.71; MS (EI) m/z 378 (M+);
HRMS calcd for C20H30N2O3S, 378.198; found, 378.197; Anal.
(C20H30N2O3S‚1/3H2O) C, H, N.
(S)-2-Amino-N-cyclopentyl-7-mercaptoheptanamide Hydro-
chloride (26a‚HCl). Compound 26a‚HCl was prepared from 16a
using the procedure described for 26b‚HCl in 94% yield: colorless
oil; 1H NMR (DMSO-d6, 500 MHz, δ, ppm) 8.43 (1H, d, J ) 7.3
Hz), 8.13 (3H, broad s), 4.03 (1H, sextet, J ) 6.7 Hz), 3.65 (1H,
t, J ) 6.7 Hz), 2.46 (2H, q, J ) 7.3 Hz), 2.28 (1H, t, J ) 7.6 Hz),
1.82 (2H, m), 1.66 (4H, m), 1.58-1.23 (10H, m); 13C NMR
(DMSO-d6, 500 MHz, δ, ppm) 167.65, 52.08, 50.46, 32.89, 32.24,
31.75, 30.85, 27.08, 23.50, 23.41, 23.30, 23.24; MS (FAB) m/z
245 (MH+ - HCl); Anal. (C12H24N2OS‚HCl‚3/4H2O) C, H, N.
Compounds 27a and 28a were prepared from 16d using the
procedure described for 26b‚HCl, 27b, and 16a (step 2).
(S)-N-(1-(Cyclopentylamino)-7-mercapto-1-oxoheptan-2-yl)-
benzamide (27a). Yield 69%; mp 171-172 °C; 1H NMR (CDCl3,
500 MHz, δ, ppm) 7.79 (2H, m), 7.52 (1H, t, J ) 7.3 Hz), 7.44
(2H, t, J ) 7.6 Hz), 6.79 (1H, d, J ) 8.2 Hz), 6.01 (1H, d, J ) 7.0
Hz), 4.54 (1H, q, J ) 7.0 Hz), 4.19 (1H, sextet, J ) 6.7 Hz), 2.51
(2H, q, J ) 7.6 Hz), 1.98 (3H, m), 1.77-1.56 (7H, m), 1.48-1.35
(6H, m), 1.32 (1H, t, J ) 7.6 Hz); 13C NMR (CDCl3, 600 MHz, δ,
ppm) 170.94, 167.23, 133.94, 131.81, 128.65, 127.04, 53.50, 51.41,
33.71, 33.17, 33.00, 32.76, 28.04, 25.01, 24.46, 23.75, 23.72; MS
(EI) m/z 348 (M+); HRMS calcd for C19H28N2O2S, 348.187; found,
348.187; Anal. (C19H28N2O2S) C, H, N.
(S)-4-Chloro-N-[1-(cyclopentylamino)-7-mercapto-1-oxohep-
tan-2-yl]benzamide (28a). Yield 54%; mp 178-180 °C; 1H NMR
(CDCl3, 500 MHz, δ, ppm) 7.73 (2H, d, J ) 8.5 Hz), 7.41 (2H, d,
J ) 8.5 Hz), 6.84 (1H, d, J ) 7.6 Hz), 5.95 (1H, d, J ) 7.3 Hz),
4.53 (1H, q, J ) 7.0 Hz), 4.20 (1H, sextet, J ) 6.7 Hz), 2.51 (2H,
q J ) 7.0 Hz), 1.96 (3H, m), 1.74-1.54 (7H, m), 1.41 (6H, m),
1.32 (1H, t, J ) 7.9 Hz); 13C NMR (CDCl3, 500 MHz, δ, ppm)
170.84, 166.10, 138.08, 132.29, 128.88, 128.51, 53.55, 51.44, 33.66,
33.17, 32.96, 32.91, 28.01, 24.94, 24.45, 23.74, 23.71; Anal.
(C19H27ClN2O2S) C, H, N.
Compounds 30a and 33a were prepared from 16d using the
procedure described for 26b‚HCl, 16b (step 1), and 16a (step 2).
(S)-N-Cyclopentyl-2-(2-hydroxyacetamido)-7-mercaptohep-
tanamide (30a). Yield 55%; mp 85-86 °C; 1H NMR (CDCl3, 500
MHz, δ, ppm) 6.93 (1H, d, J ) 8.5 Hz), 5.90 (1H, d, J ) 6.7 Hz),
4.34 (1H, q, J ) 7.9 Hz), 4.21-4.10 (3H, m), 2.69 (1H, t, J ) 5.7
Hz), 2.51 (2H, q, J ) 7.3 Hz), 1.97 (2H, m), 1.83 (1H, m), 1.72-
1.53 (7H, m), 1.46-1.30 (7H, m); 13C NMR (CDCl3, 500 MHz, δ,
ppm) 171.82, 170.99, 62.16, 52.97, 51.40, 33.65, 33.10, 32.89,
32.48, 27.93, 25.02, 24.45, 23.73; MS (EI) m/z 302 (M+); HRMS
calcdforC14H26N2O3S,302.166;found,302.167;Anal.(C14H26N2O3S)
C, H, N.
(S)-N-Cyclopentyl-7-mercapto-2-pivalamidoheptanamide (33a).
Yield 60%; mp 134-139 °C; 1H NMR (CDCl3, 500 MHz, δ, ppm)
6.20 (1H, d, J ) 7.9 Hz), 6.08 (1H, d, J ) 7.3 Hz), 4.30 (1H, q,
J ) 7.3 Hz), 4.17 (1H, sextet, J ) 6.7 Hz), 2.51 (2H, q, J ) 7.3
Hz), 1.96 (2H, m), 1.83 (1H, m), 1.71-1.55 (8H, m), 1.45-1.25
(6H, m), 1.20 (9H, s); 13C NMR (CDCl3, 500 MHz, δ, ppm) 178.60,
171.13, 52.83, 51.22, 38.75, 33.70, 33.08, 33.03, 32.13, 28.01,
27.47, 24.97, 24.45, 23.73, 23.68; MS (EI) m/z 328 (M+); HRMS
calcdforC17H32N2O2S,328.218;found,328.219;Anal.(C17H32N2O2S)
C, H, N.
(S)-Cyclohexyl 1-(Cyclopentylamino)-7-mercapto-1-oxohep-
tan-2-ylcarbamate (38a). Compound 38a was prepared from 16d
using the procedure described for 26b‚HCl, 38b, and 16a (step 2)
1
in 78% yield: mp 110-111 °C; H NMR (CDCl3, 500 MHz, δ,
ppm) 5.85 (1H, m), 5.06 (1H, m), 4.62 (1H, m), 4.18 (1H, sextet,
J ) 7.0 Hz), 3.99 (1H, m), 2.51 (2H, q, J ) 7.0 Hz), 1.97 (2H, m),
1.85 (3H, m), 1.74-1.46 (11H, m), 1.45-1.30 (11H, m); 13C NMR
(CDCl3, 600 MHz, δ, ppm) 171.21, 156.06, 73.63, 54.75, 51.20,
33.70, 33.12, 33.03, 32.53, 31.91, 29.70, 27.96, 25.35, 24.99, 24.46,
23.76, 23.71, 23.39; MS (FAB) m/z 371 (MH+); Anal. (C19H34N2O3S‚
1/4H2O) C, H, N.
Biology. Western Blot Analysis. Human colon cancer HCT116
cells were purchased from American Type Culture Collection
(ATCC, Manassas, VA) and cultured in McCoy5A culture medium
containing penicillin and streptomycin, which was supplemented
with fetal bovine serum as described in the ATCC instructions.
HCT-116 cells (5 × 105) were treated for 8 h with samples at the
indicated concentrations in 10% FBS supplemented with McCoy’s
5A medium and were then collected and extracted with SDS buffer.
Protein concentrations of the lysates were determined using a
Bradford protein assay kit (Bio-Rad Laboratories) with which
equivalent amounts of protein from each lysate were resolved in
15% SDS-polyacrylamide gels and then transferred onto nitrocel-
lulose membranes (Bio-Rad Laboratories). After blocking for 30
min with Tris-buffered saline (TBS) containing 3% skimmed milk,
the transblotted membranes were incubated overnight at 4 °C with
hyperacetylated histone H4 antibody (Upstate Biotechnology;
1:4000 dilution), acetylated R-tubulin antibody (SIGMA; 1:4000
dilution), or â-actin antibody (Abcam; 1:500 dilution) in TBS
containing 3% skimmed milk. After probing with the primary
antibody, the membrane was washed twice with water and then
incubated with goat, antirabbit, or antimouse IgG-horseradish
peroxidase conjugates (diluted 1:5000) for 2 h at room temperature
and washed twice more with water. The immunoblots were
visualized by enhanced chemiluminescence.
Compounds 35a and 36a were prepared from 16d using the
procedure described for 26b‚HCl, 35b, and 16a (step 2).
(S)-1-tert-Butyl-3-[1-(cyclopentylamino)-7-mercapto-1-oxo-
1
heptan-2-yl]urea (35a). Yield 71%; mp 188-191 °C; H NMR
(CDCl3, 500 MHz, δ, ppm) 6.19 (1H, d, J ) 6.7 Hz), 4.92 (1H, d,
J ) 8.5 Hz), 4.49 (1H, s), 4.16 (1H, sextet, J ) 7.3 Hz), 4.09 (1H,
q, J ) 7.6 Hz), 2.52 (2H, q, J ) 7.6 Hz), 1.94 (2H, m), 1.76 (1H,
m), 1.72-1.50 (7H, m), 1.46-1.22 (16H, m); 13C NMR (CDCl3,
500 MHz, δ, ppm) 172.45, 157.02, 53.83, 51.16, 50.57, 33.75,
33.10, 32.98, 32.64, 29.44, 28.07, 25.20, 24.47, 23.72, 23.67; Anal.
(C17H33N3O2S‚1/6H2O) C, H, N.
Enzyme Assays. The inhibitory activities of the test compounds
against partially purified HDAC1, HDAC4, and HDAC6 were
assayed according to a method reported in ref 14c.
Cell Growth Inhibition Assay. Human colon cancer HCT116
cells and ERR-positive breast cancer MCF-7 cells, which were
purchased from American Type Culture Collection (ATCC, Ma-
nassas, VA), were cultured in McCoy5A and Dulbecco’s Modified
(S)-1-tert-Butyl-3-[1-(cyclopentylamino)-7-mercapto-1-oxo-
heptan-2-yl)thiourea (36a). Yield 25%; mp 125-127 °C; 1H NMR

Series of Histone Deacetylase 6-SelectiVe Inhibitors
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which was supplemented with fetal bovine serum as described in
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plated in 96-well plates at initial densities of 5000 (HCT116) or
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Acknowledgment. This research was partly supported by
Grants-in-Aid for Young Scientists (B) from the Ministry of
Education, Science, Culture, Sports, Science, Technology, Japan,
and grants from Takeda Science Foundation and the Program
for the Promotion of Fundamental studies in Health Science of
the National Institute of Biomedical Innovation (NIBIO), Japan.
We thank the Screening Committee of Anticancer Drugs,
supported by a Grant-in-Aid for Scientific Research on Priority
Area “Cancer” from the Ministry of Education, Culture, Sports,
Science and Technology of Japan for HDAC1 inhibition assay
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Supporting Information Available: Results of the elemental
analysis of 11b, 16b, 18b-24b, 26b-29b, 31b, 37b, 39b-41b,
16a-20a, 26a-28a, 30a, 33a, 35a-38a, and 40a are reported. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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