Rapid discovery of highly potent and selective inhibitors of histone deacetylase 8 using click chemistry to generate candidate libraries

Article abstract of DOI:10.1021/jm300837y

To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-selective inhibitors including C149 (IC₅₀ = 0.070 μM), which was more potent than PCI-34058 (6) (IC₅₀ = 0.31 μM), a known HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI₅₀ = 3-80 μM). These findings suggest that HDAC8-selective inhibitors have potential as anticancer agents.

Full text of DOI:10.1021/jm300837y

Article
pubs.acs.org/jmc
Rapid Discovery of Highly Potent and Selective Inhibitors of Histone
Deacetylase 8 Using Click Chemistry to Generate Candidate Libraries
Takayoshi Suzuki,*,†,‡ Yosuke Ota,§ Masaki Ri,∥ Masashige Bando,⊥ Aogu Gotoh,†,§ Yukihiro Itoh,†
Hiroki Tsumoto,§ Prima R. Tatum,§ Tamio Mizukami,# Hidehiko Nakagawa,§ Shinsuke Iida,∥
Ryuzo Ueda,∥ Katsuhiko Shirahige,⊥ and Naoki Miyata*,§
†Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 13 Taishogun Nishitakatsukasa-Cho, Kita-ku, Kyoto
403-8334, Japan
‡PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
§Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
∥Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan
⊥Institute of Molecular and Cellular Biosciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
#Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga, 526-0829,
Japan
S
* Supporting Information
ABSTRACT: To find HDAC8-selective inhibitors, we designed a library of HDAC inhibitor candidates, each containing a zinc-
binding group that coordinates with the active-site zinc ion, linked via a triazole moiety to a capping structure that interacts with
residues on the rim of the active site. These compounds were synthesized by using click chemistry. Screening identified HDAC8-
selective inhibitors including C149 (IC50 = 0.070 μM), which was more potent than PCI-34058 (6) (IC50 = 0.31 μM), a known
HDAC8 inhibitor. Molecular modeling suggested that the phenylthiomethyl group of C149 binds to a unique hydrophobic
pocket of HDAC8, and the orientation of the phenylthiomethyl and hydroxamate moieties (fixed by the triazole moiety) is
important for the potency and selectivity. The inhibitors caused selective acetylation of cohesin in cells and exerted growth-
inhibitory effects on T-cell lymphoma and neuroblastoma cells (GI50 = 3−80 μM). These findings suggest that HDAC8-selective
inhibitors have potential as anticancer agents.
cell lymphomas.8 In addition, Oehme et al. suggested that
INTRODUCTION
■
HDAC8 is implicated in neuroblastoma tumorigenesis.9
Reversible acetylation/deacetylation of the ε-amino groups of
Therefore, HDAC8-selective inhibitors are of great interest,
not only as tools for probing the biological functions of this
isozyme but also as candidate anticancer agents having few side
effects.
lysine residues on histones and nonhistone proteins, catalyzed
by histone acetyltransferases and histone deacetylases
(HDACs), is involved in various life phenomena such as
gene expression and cell cycle regulation.1 Thus far, 18 HDAC
family members have been identified and categorized into two
groups, namely zinc-dependent enzymes (HDAC1−11) and
NAD+-dependent sirtuins (SIRT1−7).2 Among them, HDAC8
is expressed tissue specifically and is localized in the nucleus3 or
cytoplasm.4 It is responsible for genetic repression in acute
myeloid leukemia5 and is associated with the actin cytoskeleton
in smooth muscle cells.6 Furthermore, recent studies have
revealed that HDAC8 is associated with several disease states.
Gallimari et al. reported that siRNA targeting HDAC8 showed
antitumor effects in cell culture.7 Balasubramanian et al.
reported that inhibition of HDAC8 induced apoptosis in T-
Although many HDAC inhibitors have been found so far,10
including trichostatin A (TSA, 1),11 vorinostat (2),12 and MS-
275 (3)13 (Chart 1), most lack HDAC8 selectivity. It has been
reported that HDAC8 is selectively inhibited by SB-379278A
(4),14 linkerless hydroxamic acid 5,15 PCI-34051 (6),8
azetidinone 7,16 and A8B4 (8) (Chart 2).17 Among these
compounds, compound 6 is a representative HDAC8-selective
Received: June 14, 2012
© XXXX American Chemical Society
A
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Chemistry. The routes used for the synthesis of the
compounds prepared for this study are shown in Schemes
1−11.
Chart 1. Examples of HDAC Inhibitors
a
Scheme 1. Synthesis of Alkyne A1
a
Reagents and conditions: (a) NH2OTHP, DCC, CH2Cl2, 0 °C to rt,
57%; (b) p-TsOH·H2O, MeOH, rt, 70%.
inhibitor that shows high potency and selectivity for HDAC8
over HDAC1, 2, 3, 6, and 10.
The preparation of alkyne A1 is shown in Scheme 1.
Propiolic acid 9 was reacted with NH2OTHP in the presence of
DCC to give the corresponding O-THP hydroxamate 10.
Deprotection of the THP group of 10 by p-TsOH afforded A1.
The preparation of alkyne A2 (Scheme 2) started with 5-
bromothiophene-2-carboxylic acid 11, which was esterified and
subjected to Sonogashira cross coupling with trimethylsilylace-
tylene to give 13. Hydrolysis and EDCI/HOBt-mediated
coupling with NH2OTHP afforded the corresponding hydrox-
amate 15, which provided A2 upon removal of the THP group
with p-TsOH. Compounds A3 and A4 were prepared from
carboxylic acids 16a and 16b by using the same procedure
described for the synthesis of A2 (Scheme 3). o-Aminoanilides
A5−A8 were obtained by the condensation of 1,2-phenyl-
enediamine with an appropriate carboxylic acid in the presence
of condensing reagents (Scheme 4).
Chart 2. HDAC8-Selective Inhibitors
Azides B2−B9, B11, B12, and B14 were prepared by azide
displacement (NaN3, DMSO) of the corresponding alkyl
halides 21 (Scheme 5). Scheme 6 shows the synthesis of
azides B13 and B15 from alcohols 22a and 22b via the
tosylates 23a,b. Azide B10 was accessed from carboxylic acid 24
by LiAlH4 reduction and alcohol-to-azide conversion using
DPPA and DBU (Scheme 7). Phenethyl azide analogues B16−
B34 and B36 were synthesized by azide displacement of the
corresponding alkyl bromides 26 (Scheme 8). Scheme 9 shows
the preparation of azides B35 and B37−B43 with an ethylene
chain from alcohols 27 via the mesylate, bromide, or tosylates
28. 1-Phenylethanone-2-azide B45 and (E)-(2-azidovinyl)-
benzene B46 were prepared by azide displacement of the
bromide 29 and the boronic acid 30, respectively (Scheme 10).
Cu-catalyzed coupling of alkyne A3 and azide B1, B2, B37,
or B44 provided triazoles C31, C32, C142, and C149 (Scheme
11).
RESULTS AND DISCUSSION
■
Click Chemistry Approach. Fragment-based assembly is a
drug discovery approach that enables high-throughput identi-
fication of small-molecular inhibitors using a minimal number
of compounds as building blocks.20 It allows medicinal chemists
to explore n × m possibilities with n + m combinations. Click
chemistry is a powerful fragment-based assembly method and
was shown to be highly versatile and effective for rapid
synthesis/assembly of libraries of small molecules directed
against a number of enzymes.21 As reported before, triazole
synthesis by Cu(I)-catalyzed azide−alkyne cycloaddition
(CuAAC),22 a key reaction in click chemistry, can provide a
library of triazole-containing compounds, which are pure
enough to be evaluated by means of in vitro assay without
purification.
Various strategies have been employed to identify HDAC8-
selective inhibitors to date. Compound 4 was identified by the
evaluation of a chemical compound collection using an HDAC8
enzyme-based high-throughput screening,14 and compounds
5−7 were discovered by drug design based on the crystal
structure of HDAC8.8,15,16 Here, we report the rapid
identification of highly potent and selective inhibitors of
HDAC8 by the use of Cu (I)-catalyzed azide−alkyne
cycloaddition, a representative reaction in click chemistry,18
to generate a library of candidates. Although several groups
have used click chemistry to find HDAC inhibitors,19 there has
been no previous report on the use of click chemistry for the
discovery of isozyme-selective HDAC inhibitors.
B
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a
Scheme 2. Synthesis of Alkyne A2
a
Reagents and conditions: (a) conc H2SO4, MeOH, reflux, 96%; (b) trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI, Et2NH, rt, 81%; (c) NaOH, H2O,
MeOH, rt, 97%; (d) NH2OTHP, EDCI, HOBt·H2O, DMF, rt, 90%; (e) p-TsOH·H2O, MeOH, rt, 77%.
a
a
Scheme 3. Synthesis of Alkynes A3 and A4
Scheme 6. Synthesis of Azides B13 and B15
a
Reagents and conditions: (a) TsCl, pyridine, rt, 68% for 23a, 84% for
23b; (b) NaN3, DMSO, 80 °C, 26% for B13, 84% for B15.
a
Scheme 7. Synthesis of Azide B10
a
Reagents and conditions: (a) conc H2SO4, MeOH, reflux, 96% for
17a, 99% for 17b; (b) trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI,
Et3N, THF, rt, 90% for 18a, 64% for 18b; (c) 2 N NaOH, MeOH, rt,
99% for 19a, 92% for 19b; (d) NH2OTHP, EDCI, HOBt·H2O, DMF,
rt, 93% for 20a, 86% for 20b; (e) p-TsOH·H2O, MeOH, rt, 86% for
A3, 94% for A4.
a
Reagents and conditions: (a) LiAlH4, THF, 0−80 °C, 87%; (b)
a
DPPA, DBU, DMF, rt, 30%.
Scheme 4. Synthesis of Alkynes A5−A8
a
Scheme 8. Synthesis of Azides B16−B34 and B36
a
Reagents and conditions: (a) 1,2-phenylenediamine, DCC, CH2Cl2, 0
°C to rt, 44% for A5; (b) 1,2-phenylenediamine, EDCI, HOBt·H2O,
DMF, rt, 79% for A6, 91% for A7, 86% for A8.
a
Scheme 5. Synthesis of Azides B2−B9, B11, B12, and B14
a
Reagents and conditions: (a) NaN3, DMSO, rt or 80 °C, 62−100%.
inhibitor, consists of hydroxamic acid (ZBG), anilide (cap), and
alkyl chain (linker). Accordingly, we designed a library of
candidate HDAC inhibitors in which the ZBG and the cap
group are connected by a triazole-containing linker (Figure
1).19a We designed various alkynes with hydroxamic acid (A1−
A4) or o-aminoanilide (A5−A8) as the ZBG and azides with
various cap structures (B1−B15) as suitable building blocks for
click assembly to construct a structurally diverse chemical
library that might provide a high hit rate. We then prepared
eight alkynes A1−A8 and 15 alkyl azides B1−B15 as shown in
Schemes 1−7. Using the CuAAC reaction, a 120-member
HDAC inhibitor library was assembled in microtiter plates.
Each of the eight alkynes was mixed with each of the 15 azides
in DMSO solution, followed by the addition of catalytic
amounts of CuSO4, sodium ascorbate, and tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]amine (TBTA). The disappearance of
the starting materials and the quantitative formation of the
triazole products were confirmed by TLC and LC-MS
a
Reagents and conditions: (a) NaN3, DMSO, rt or 80 °C, 21−92%.
In general, the pharmacophore for HDAC inhibitors consists
of a zinc-binding group (ZBG) that coordinates with the zinc
ion in the active site, a capping structure that interacts with
residues on the rim of the active site, and a linker connecting
the cap part and the ZBG with an appropriate separation. For
example, compound 2 (Chart 1), a clinically used HDAC
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a
Scheme 9. Synthesis of Azides B35 and B37−B43
a
Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, rt; (b) TsCl, pyridine, rt; (c) CBr4, PPh3, CH2Cl2, rt; (d) NaN3, DMSO, rt or 80 °C, 16−86%
(two steps).
a
To identify HDAC8-selective inhibitors more potent than
compound 6, we further designed a C32-based library of
compounds, which could be prepared from alkyne A3 and
azides B16−B46 (Schemes 8−10) (Figure 4) using the same
click chemistry approach. The CuAAC reaction between alkyne
A3 and azides B16−B46 gave 31 triazoles C121−C151, which
are structurally related to compound C32 (Supporting
Information Figure S3). The 31 triazole compounds were
screened against both HDAC8 and total HDACs from HeLa
nuclear extracts. Two crude compounds, C142 and C149,
showed more potent inhibition than C32 at the concentration
of 0.3 μM (Figure 5) while displaying weak inhibition toward
HDACs from HeLa nuclear extracts at the concentration of 3
μM, indicating high selectivity for HDAC8 (Figure 6).
Isozyme Selectivity. Compounds C31, C32, C142, and
C149 were resynthesized and purified by column chromatog-
raphy and recrystallization (Scheme 11). The pure compounds
C31, C32, C142, and C149 were evaluated for inhibitory
effects on HDAC1, HDAC2, HDAC4, HDAC6, and HDAC8
as well as total HDACs from nuclear extracts. The results of the
enzyme assays are shown in Table 1. In these assays, compound
6 inhibited HDAC8 with an IC50 of 0.31 μM and showed
selectivity for HDAC8 over HDAC1, HDAC2, HDAC4, and
HDAC6.
Compounds C31, C32, C142, and C149 all showed potent
HDAC8-inhibitory activity. The HDAC8-inhibitory activity of
compounds C31 and C32 was greater than that of compound 2
and comparable to that of compound 6 (IC50 of 2 1.5 μM, 6
0.31 μM, C31 0.35 μM, C32 0.18 μM). Compounds C142 and
C149, which are derivatives of C32, showed more potent
HDAC8-inhibitory activity than compound 6 (IC50 of C142
0.10 μM, C149 0.070 μM). Furthermore, while compound 2
inhibited HDAC1, HDAC2, and HDAC6 more potently than
HDAC8, compounds C31, C32, C142, and C149 inhibited
HDAC8 in preference to the other isozymes, like compound 6.
Thus, C31, C32, C142, and C149 are potent and selective
inhibitors of HDAC8.
Scheme 10. Synthesis of Azides B45 and B46
a
Reagents and conditions: (a) NaN3, DMSO, rt, 53%; (b) NaN3,
CuSO4, MeOH, rt, 40%.
(Supporting Information Figure S1). The generated triazole
compounds C1−C120 are shown in Supporting Information
Figure S2. The 120 triazoles could be directly screened for
HDAC-inhibitory activity in the microtiter plates without
further purification. To find HDAC8-selective inhibitors,
compounds C1−C120 were tested for activity against both
HDAC8 at 0.3 μM and total HDACs from HeLa nuclear
extracts at 3 μM. We chose total HDACs from HeLa nuclear
extracts for primary screening because these extracts are rich in
the activity of HDAC1 and HDAC2, which are major isozymes
of the HDAC family.23 In this HDAC8 assay, the HDAC8-
inhibitory activity of compound 2 at 0.3 μM in the presence of
10 mol % Cu(I) was almost the same as that in the absence of
Cu(I) (31.9 0.12% inhibition in the presence of Cu(I), 35.0
0.72% inhibition in the absence of Cu(I)), suggesting that
Cu(I) does not affect the activity of compounds C1−C120. As
shown in Figures 2 and 3, two hits emerged from these screens.
The HDAC8-inhibitory activity of these two crude compounds
C31 and C32 was comparable to that of compound 6 at 0.3 μM
(Figure 2). Furthermore, at the concentration of 3 μM, crude
C31 and C32 showed weak inhibitory activity against total
HDACs from HeLa nuclear extract (Figure 3).
a
Scheme 11. Synthesis of HDAC8-Selective Inhibitors C31, C32, C142, and C149
a
Reagents and conditions: (a) CuSO4·5H2O, sodium ascorbate, TBTA, MeOH, H2O, rt, 92−100%.
D
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Figure 1. Design of alkynes and azides.
software packages Glide 3.5 and MacroModel 8.1 (Figures 7
and 8).
In the simulated HDAC8/compound 6 complex, the
hydroxamate group of compound 6 coordinates in a bidentate
fashion to the zinc ion and forms three hydrogen bonds with
His 142, His 143, and Tyr 306 in the active center of HDAC8
(Figure 7). The 4-methoxybenzyl group of compound 6 is
located in the unique hydrophobic pocket of HDAC8, where
the CH of the methoxy group can interact with Tyr 154 via
CH−π interaction. It is also suggested that the indole ring of
compound 6 orients the hydroxamate group and 4-methox-
ybenzyl group into the appropriate geometry.
Inspection of the simulated HDAC8/compound C149
complex showed that the hydroxamic acid group coordinates
the zinc ion through its CO and OH groups in a similar way to
compound 6. It is also suggested that the hydroxamate group of
C149 forms a hydrogen bond between its CO moiety and His
142 and also forms another hydrogen bond between its OH
moiety and Tyr 306 (Figure 8). Unlike compound 6, it is
suggested that C149 has a U-shaped conformation in the active
site of HDAC8, and most importantly, the phenylthiomethyl
group of compound C149 binds to a hydrophobic pocket
formed by Trp 141, Ile 34, and Pro 35, which is thought to be
unique to HDAC8 and where the methylthio group can interact
with Trp 141 and the phenyl group can bind to Pro 35 and Tyr
306 through hydrophobic interactions. The three nitrogens of
the triazole ring of C149 do not appear to interact with any
amino acid residues of HDAC8, but the triazole ring can
interact with the methylene group of Phe 152 through CH−π
or hydrophobic interactions (the distance between the CH2 of
Phe 152 and the triazole is 3.2 Å). The triazole ring is also
considered to be important to fix the orientation of the zinc-
binding hydroxamate and the hydrophobic pocket-binding
phenylthiomethyl group appropriately, thereby contributing to
the potent HDAC8-inhibitory activity and HDAC8-selectivity
of compound C149. Comparison of the simulated HDAC8/
inhibitor complex structures suggests that compound C149 can
bind to HDAC8 more tightly than can compound 6 through
efficient hydrophobic interactions.
Figure 2. HDAC8 activity in the presence of 0.3 μM C1−C120.
Figure 3. Total HDACs activity in the presence of 3 μM C1−C120.
Molecular Modeling. Although several X-ray crystal
structures of HDAC8 have been reported,16,23 we chose the
crystal structure of HDAC8 complexed with CRA-A (PDB
code 1VKG) for the molecular modeling study on compounds
6 and C149 with HDAC8. Unlike the other structures, the
HDAC8−CRA-A complex structure has a hydrophobic pocket
which is thought to be unique to HDAC8.15,23a It is therefore
reasonable to assume that HDAC8-selective inhibitors bind to
this unique pocket. The lowest energy conformations of 6, a
previously reported HDAC8-selective inhibitor, and C149, the
most active HDAC8-selective inhibitor in the present series,
were obtained when they were docked into a model based on
the crystal structure of HDAC8 (PDB code 1VKG), using the
In addition, we attempted to carry out a docking study of
compound C149 to HDAC2 (PDB code 3MAX),24 another
HDAC isozyme. However, compound C149 could not be
docked in the active site of HDAC2 because of a steric clash
E
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Figure 4. Design of a C32-based library of compounds.
dose-dependent increase of cohesin acetylation without a major
increase in acetylated H4. These results indicate that
compounds C142 and C149 potently and selectively inhibit
HDAC8 in preference to other HDACs in cells.
Because it has been reported that inhibition of HDAC8
induces apoptosis in T-cell lymphomas8 and HDAC8 is
implicated in neuroblastoma tumorigenesis,9 compounds 6,
C32, and C149 were tested in cell growth-inhibition assays
using human T-cell lymphoma and neuroblastoma cell lines.
The results are shown in Table 2. These HDAC8-selective
inhibitors showed clear growth-inhibitory effects on both T-cell
lymphoma and neuroblastoma cell lines, including Jurkat, HH,
MT2, MT4, NB-1, and LA-N-1. In particular, the growth-
inhibitory activity against T-cell lymphoma cells of compound
C149 was greater than that of compound 6. It should be noted
that these inhibitors did not affect the growth of healthy donor
peripheral blood mononuclear cells (PBMCs) (IC50 values
>100 μM), suggesting that the cytotoxicity of HDAC8-selective
inhibitors is cell-type-specific. These results suggest that
HDAC8 is involved in the growth of T-cell lymphoma and
neuroblastoma cells and that our HDAC8-selective inhibitors
may be useful in the treatment of T-cell lymphoma and
neuroblastoma.
Figure 5. HDAC8 activity in the presence of 0.3 μM C121−C151.
CONCLUSION
■
In summary, we used click chemistry with the reliable CuAAC
reaction initially to prepare a triazole library of 120 compounds,
among which two potent HDAC8 inhibitors C31 and C32
were identified. For further structural optimization, we next
prepared a 31-member library based on the structure of
compound C32. Screening led to the identification of two
highly potent and selective HDAC8 inhibitors, C142 and
C149. A molecular modeling study of the HDAC8/C149
complex suggested that the orientation of the hydrophobic
pocket-binding phenylthiomethyl group and the zinc-binding
hydroxamate, fixed by the triazole ring, is important for both
HDAC8-inhibitory activity and HDAC8-selectivity. Com-
pounds C32 and C149 also showed in-cell HDAC8 selectivity
and potent T-cell lymphoma cell growth-inhibitory activity.
Thus, we have rapidly identified a novel series of HDAC8-
selective inhibitors by using click chemistry to generate libraries
of candidate molecules. It should be possible to obtain even
more potent and selective HDAC8 inhibitors by means of
further structural development. HDAC8-selective inhibitors are
thought to have considerable potential both for the develop-
Figure 6. Total HDACs activity in the presence of 3 μM C121−C151.
between the phenylthiomethyl group of C149 and the rim of
the HDAC2 active site. These calculation results are consistent
with the experimental fact that compound C149 inhibits
HDAC8 in preference to HDAC2 (Table 1).
Cellular Assays. Because HDAC8 is a cohesin deacety-
lase,25 inhibition of HDAC8 and that of other HDACs can be
assessed by evaluating accumulation of acetylated cohesin and
acetylated histone, respectively, using Western blot analysis.
Therefore, we examined the effects of compound 6, C142, and
C149 on accumulation of acetylated cohesin and histone H4
(Figure 9). In accordance with the results of enzyme assays,
compound 6 and compounds C142 and C149 produced a
F
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a
Table 1. HDAC-Inhibitory Activity of Compounds 2, 6, C31, C32, C142, and C149
IC50 (μM)
HDACs
class I
class IIa
HDAC8
class IIb
compd
(nuclear extract)
HDAC1
HDAC2
HDAC4
HDAC6
2
0.17
>100
37
0.27
>100
41
0.78
>100
65
1.5
>10
>100
30
0.21
9.3
7.9
3.2
1.1
2.4
6
0.31
0.35
0.18
0.10
0.070
C31
C32
C142
C149
23
>100
>100
38
76
>100
>100
44
44
>100
>100
54
a
Values are means of at least three experiments.
Figure 7. The lowest energy conformation of 6 (ball-and-stick representation) in the HDAC8 catalytic core. Compound 6 was docked into a model
based on the crystal structure of HDAC8 (PDB code 1VKG), using the software packages Glide 3.5 and MacroModel 8.1. (top left) Surface view of
6 docked in the HDAC8 catalytic pocket. (top right) View of the catalytic center of HDAC8 complexed with 6. Residues within 3 Å of the zinc ion
are displayed. (bottom) Schematic representation of 6 binding with HDAC8.
Corporation). 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.
ment of novel therapeutic agents and as tools for biological
research.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a Yanagimoto
micro melting point apparatus or a Buchi 545 melting point apparatus
■
Propynoic Acid Hydroxyamide (A1). Step 1: Preparation of
Propynoic Acid Tetrahydropyran-2-yloxyamide (10). To a solution
of propynoic acid (9, 300 mg, 4.28 mmol) in CH2Cl2 (6 mL) and
NH2OTHP (552 mg, 4.71 mmol) was added dropwise a solution of
DCC (972 mg, 4.71 mmol) in CH2Cl2 (9 mL) at 0 °C. The reaction
mixture was stirred for 3 h at room temperature. Filtration,
concentration in vacuo, and purification of the residue by silica gel
flash chromatography (AcOEt/n-hexane = 2/3) gave 416 mg (57%) of
̈
and were left uncorrected. Proton nuclear magnetic resonance spectra
(1H NMR) and carbon nuclear magnetic resonance spectra (13C
NMR) were recorded in the indicated solvent on a JEOL JNM-LA500,
JEOL JNM-A500, or BRUKER AVANCE600 spectrometer. 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, confirming >95% purity. High-
resolution mass spectra (HRMS) and fast atom bombardment (FAB)
mass spectra were recorded on a JEOL JMS-SX102A mass
spectrometer. GC-MS analyses were performed on a Shimadzu
GCMS-QP2010. IR spectra were measured on a Shimadzu FTIR-
8400S spectrometer. LCMS was performed with a Waters instrument
equipped with Unison US-C18 (2 mm × 5 mm/2 × 150 mm, Imtakt
1
10 as a pale-yellow oil. H NMR (CDCl3, 500 MHz, δ; ppm): 8.76
(1H, s), 5.01 (1H, s), 3.96 (1H, t, J = 10.2 Hz), 3.96−3.65 (1H, m),
2.89 (1H, s), 1.94−1.78 (3H, m), 1.71−1.61 (3H, m).
Step 2: Preparation of Propynoic Acid Hydroxyamide (A1). To a
solution of 10 (416 mg, 2.46 mmol) obtained above in MeOH (21
mL) was added TsOH·H2O (47 mg, 0.246 mmol). The reaction
mixture was stirred for 30 h at room temperature. After removal of the
solvent, the residue was purified by silica gel flash column
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Figure 8. The lowest energy conformation of C149 (ball-and-stick representation) in the HDAC8 catalytic core. Compound C149 was docked into
a model based on the crystal structure of HDAC8 (PDB code 1VKG), using the software packages Glide 3.5 and MacroModel 8.1. (top left) Surface
view of C149 docked in the HDAC8 catalytic pocket. (top right) View of the catalytic center of HDAC8 complexed with C149. Residues within 3 Å
of the zinc ion are displayed. (bottom) Schematic representation of C149 binding with HDAC8.
Figure 9. Western blot detection of acetylated structural maintenance of chromosome 3 (SMC3), a subunit of cohesin, and H4 levels in HeLa cells
after 4 h treatment with compounds 6, C142, and C149. Values of Ac SMC3/SMC3 ratio determined by optical density measurement of the blots
are shown below the uppermost photograph.
chromatography (AcOEt/n-hexane = 3/1) to give 147 mg (70%) of
A1 as a white solid. The solid was recrystallized from AcOEt/n-hexane
to give 70 mg of A1 as colorless crystals; mp 69−71 °C. H NMR
Step 2: Preparation of 5-Trimethylsilanylethynylthiophene-2-
carboxylic Acid Methyl Ester (13). To a solution of 12 (1.03 g, 4.66
mmol) obtained above, PdCl2(PPh3)2 (32.7 mg, 46.6 μmol), and CuI
(13.3 mg, 69.9 μmol) in diethylamine (15.5 mL) was added
trimethylsilylacetylene (687 mg, 6.99 mmol). The mixture was stirred
for 18 h at room temperature. After removal of the solvent, the residue
was diluted with Et2O and the Et2O solution was washed with 1 N
aqueous HCl, saturated NaHCO3, and brine, and then dried over
Na2SO4. Filtration, concentration in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/20) gave 895 mg (81%) of 13 as a brown solid. H NMR (CDCl3,
500 MHz, δ; ppm): 7.63 (1H, d, J = 4.0 Hz), 7.16 (1H, d, J = 4.0 Hz),
3.88 (3H, s), 0.257 (9H, s).
Step 3: Preparation of 5-Ethynylthiophene-2-carboxylic Acid
(14). To a solution of 13 (895 mg, 3.75 mmol) obtained above in
MeOH (10 mL) was added 2 N aqueous NaOH (3.75 mL, 7.50
mmol) at 0 °C. The reaction mixture was stirred for 5 h at room
temperature. The mixture was acidified with 2 N aqueous HCl and
1
(DMSO-d6, 500 MHz, δ; ppm): 11.2 (1H, broad s), 9.28 (1H, s), 4.18
(1H, s). 13C NMR (DMSO-d6, 150 MHz, δ; ppm): 149.15, 77.32,
76.03. HRMS (EI) Calcd. for C3H3O2N 85.0160, Found 85.0159.
5-Ethynylthiophene-2-carboxylic Acid Hydroxyamide (A2).
Step 1: Preparation of 5-Bromothiophene-2-carboxylic Acid Methyl
Ester (12). To a solution of 5-bromo-2-thiophenecarboxylic acid (11,
1.00 g, 4.83 mmol) in MeOH (30 mL) was added concentrated H2SO4
(1.00 mL). The reaction mixture was refluxed for 30 h. After removal
of the solvent, the residue was poured into water and extracted with
AcOEt. The organic layer was separated, washed with brine, and dried
over Na2SO4. Filtration, concentration in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/10) gave 1.03 g (96%) of 12 as a white solid. 1H NMR (CDCl3, 500
MHz, δ; ppm): 7.55 (1H, d, J = 4.0 Hz), 7.07 (1H, d, J = 4.0 Hz), 3.88
(3H, s).
1
H
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J = 7.9 Hz), 6.55 (1H, t, J = 7.6 Hz), 4.91 (2H, s), 4.32 (1H, s). 13C
NMR (DMSO-d6, 125 MHz, δ; ppm): 149.94, 141.94, 126.59, 125.52,
121.65, 116.01, 115.96, 78.43, 76.61. MS (EI) m/z 160 (M+). Anal.
(C9H8N2O) C, H, N.
Table 2. Growth Inhibition of Various Cancer Cells by
Compounds 6, C32, and C149
a
GI50 (μM)
Compounds A6−A8 were prepared from 1,2-phenylenediamine and
the corresponding carboxylic acids 14 and 189a,b using the same
procedure described for A2 (step 4).
cell type
T-cell
cell line
6
C32
C149
Jurkat
HH
11
>100
15
11
79
19
30
7.6
23
5-Ethynylthiophene-2-carboxylic Acid 2-Aminophenylamide
1
MT2
MT4
11
(A6). Yield 79%; colorless crystals; mp 143 °C. H NMR (DMSO-
d6, 500 MHz, δ; ppm): 9.88 (1H, broad s), 7.98 (1H, d, J = 4.0 Hz),
7.51 (1H, d, J = 4.0 Hz), 7.17 (1H, d, J = 7.9 Hz), 7.07−7.04 (1H, m),
6.86−6.84 (1H, m), 6.68−6.64 (1H, m), 5.01 (2H, s), 4.83 (1H, s).
13C NMR (CDCl3, 125 MHz, δ; ppm): 159.04, 143.46, 141.22, 133.93,
128.97, 127.03, 126.98, 125.45, 122.13, 116.17, 116.01, 87.04, 76.42.
MS (EI) m/z 242 (M+). Anal. (C7H5NO2S·1/4H2O) C, H, N.
N-2-Aminophenyl-3-ethynylbenzamide (A7). Yield 91%; colorless
25
15
neuroblastoma cell
healthy PBMC cell
NB-1
14
39
27
12
LA-N-1
3.9
6.4
PB-O
PB-N
97
>100
>100
>100
>100
>100
1
crystals; mp 194−195 °C. H NMR (DMSO-d6, 500 MHz, δ; ppm):
a
Values are means of at least three experiments.
9.73 (1H, broad s), 8.09 (1H, broad s), 7.99 (1H, d, J = 7.9 Hz), 7.67
(1H, d, J = 7.6 Hz), 7.53 (1H, t, J = 7.8 Hz), 7.15 (1H, d, J = 7.9 Hz),
6.97 (1H, m), 6.78 (1H, dd, J = 1.2, 7.9 Hz), 6.59 (1H, t, J = 7.3 Hz),
4.93 (2H, broad s), 4.30 (1H, s). 13C NMR (DMSO-d6, 150 MHz, δ;
ppm): 164.47, 143.28, 135.09, 134.35, 130.88, 128.85, 128.38, 126.86,
126.69, 122.96, 121.76, 116.20, 116.06, 82.92, 81.43. MS (EI) m/z:
236 (M+). Anal. (C15H12N2O·1/3H2O) C, H, N.
concentrated in vacuo. The residue was diluted with AcOEt and the
AcOEt solution was washed brine and dried over Na2SO4. Filtration
and concentration in vacuo gave 552 mg (97%) of 14 as a brown solid.
1H NMR (DMSO-d6, 500 MHz, δ; ppm): 7.64 (1H, d, J = 4.0 Hz),
7.40 (1H, d, J = 4.0 Hz), 4.80 (1H, s).
N-2-Aminophenyl-4-ethynylbenzamide (A8). Yield 86%; colorless
Step 4: 5-Ethynylthiophene-2-carboxylic Acid Tetrahydropyran-
2-yloxyamide (15). To a solution of 14 (150 mg, 0.986 mmol), EDCI
(226 mg, 1.18 mmol), and HOBt·H2O (159 mg, 1.18 mmol) in DMF
(3 mL) was added NH2OTHP (173 mg, 1.48 mmol), and the mixture
was stirred at room temperature for 24 h. The reaction mixture was
poured into water and extracted with AcOEt. The organic layer was
separated, washed with saturated NaHCO3 and brine, and dried over
Na2SO4. Filtration, concentration in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/30) gave 224 mg (90%) of 15 as a yellow solid. 1H NMR (DMSO-
d6, 500 MHz, δ; ppm): 11.9 (1H, broad s), 7.62 (1H, d, J = 4.0 Hz),
7.39 (1H, d, J = 4.0 Hz), 4.96 (1H, s), 4.77 (1H, s), 4.05−4.01 (1H,
m), 3.55−3.53 (1H, m), 1.73−1.71 (3H, m), 1.55−1.53 (3H, m).
Step 5: Preparation of 5-Ethynylthiophene-2-carboxylic Acid
Hydroxyamide (A2). To a solution of 15 (224 mg, 0.891 mmol)
obtained above in MeOH (10 mL) was added TsOH·H2O (16.9 mg,
0.0891 mmol). The reaction mixture was stirred for 12 h at room
temperature. After removal of the solvent, the residue was purified by
silica gel flash column chromatography (AcOEt/n-hexane = 1/1) to
give 147 mg (77%) of A2 as a brown solid; mp 146−147 °C. 1H NMR
(DMSO-d6, 500 MHz, δ; ppm): 11.4 (1H, broad s), 9.25 (1H, broad
s), 7.53 (1H, d, J = 4.0 Hz), 7.36 (1H, d, J = 4.0 Hz), 4.74 (1H, s). 13C
NMR (CDCl3, 125 MHz, δ; ppm): 158.32, 138.73, 133.83, 127.36,
124.66, 86.93, 76.20. MS (EI) m/z 167 (M+); Anal. Calcd for
C7H5NO2S·1/5H2O: C, 49.23; H, 3.19; N, 8.20. Found: C, 49.10; H,
3.18; N, 8.07.
1
crystals; mp 181−183 °C. H NMR (DMSO-d6, 500 MHz, δ; ppm):
9.73 (1H, broad s), 7.99 (2H, d, J = 7.9 Hz), 7.61 (2H, d, J = 8.5 Hz),
7.15 (1H, d, J = 7.3 Hz), 6.99−6.96 (1H, m), 6.78−6.77 (1H, m),
6.61−6.58 (1H, m), 4.92 (2H, s), 4.41 (1H, s). 13C NMR (DMSO-d6,
125 MHz, δ; ppm): 164.41, 143.13, 134.62, 131.47, 127.96, 126.67,
126.52, 124.45, 122.90, 116.06, 115.95, 82.82, 82.79. MS (EI) m/z 236
(M+). Anal. (C15H12N2O·1/5H2O) C, H, N.
General Procedure for the Synthesis of Azides B2−B9, B11,
B12, B14, B16−B34, B36, and B45. To a 0.5 M solution of NaN3
(1.1 equiv) in DMSO was added an appropriate alkyl halide (1.0
equiv), and the mixture was stirred at room temperature or 80 °C and
periodically monitored by TLC. When the reaction was completed, the
mixture was quenched with water and stirred until it cooled to room
temperature and then extracted with AcOEt or Et2O. The organic
layer was separated, washed with water and brine, and dried over
Na2SO4. Filtration, concentration in vacuo, and purification of the
residue by silica gel flash column chromatography gave the
corresponding alkyl azide.
Phenethyl Azide (B2).26 Yield 76%; a pale-yellow oil. H NMR
1
(CDCl3, 500 MHz, δ; ppm): 7.33−7.21 (5H, m), 3.50 (2H, t, J = 7.3
Hz), 2.89 (2H, t, J = 7.31 Hz). 13C NMR (CDCl3, 150 MHz, δ; ppm):
137.99, 128.72, 128.62, 126.76, 52.44, 35.33. FTIR (neat, cm−1) 2090.
MS (EI) m/z 119 (M+ − 28).
3-Phenylpropyl Azide (B3).26 Yield 55%; a pale-yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.31−7.17 (5H, m), 3.28 (2H, t, J
= 6.9 Hz), 2.69 (2H, t, J = 7.6 Hz), 1.91 (2H, m). 13C NMR (CDCl3,
150 MHz, δ; ppm): 140.85, 128.51, 128.45, 126.14,50.64, 32.76, 30.43.
FTIR (neat, cm−1) 2090. MS (EI) m/z 133 (M+ − 28).
Compounds A3 and A4 were prepared from 16a and 16b,
respectively, using the same procedure described for A2.
3-Ethynyl-N-hydroxybenzamide (A3). Yield 68%; colorless crys-
tals; mp 165−166 °C. 1H NMR (DMSO-d6, 500 MHz, δ; ppm): 11.3
(1H, broad s), 9.14 (1H, broad s), 7.82 (1H, s), 7.78 (1H, d, J = 7.9
Hz), 7.62 (1H, d, J = 7.6 Hz), 7.49 (1H, t, J = 7.8 Hz), 4.28 (1H, s).
13C NMR (DMSO-d6, 150 MHz, δ; ppm): 163.07, 134.07, 133.08,
4-Phenylbutyl Azide (B4).27 Yield 58%; a pale-yellow oil. 1H NMR
(CDCl3, 500 MHz, δ; ppm): 7.30−7.17 (5H, m), 3.28 (2H, t, J = 6.9
Hz), 2.65 (2H, t, J = 7.5 Hz), 1.74−1.68 (2H, m), 1.66−1.60 (2H, m).
13C NMR (CDCl3, 125 MHz, δ; ppm): 141.80, 128.37, 128,35, 125.89,
51.34, 35.35, 28.42. FTIR (neat, cm−1) 2090.
129.81, 128.89, 127.35, 121.75, 82.65, 81.40. MS (EI) m/z: 161 (M+).
Anal. (C9H7NO2) C, H, N.
1
5-Phenylpentyl Azide (B5). Yield 75%; a pale-yellow oil. H NMR
(CDCl3, 500 MHz, δ; ppm): 7.29−7.16 (5H, m), 3.25 (2H, t, J = 7.0
Hz), 2.65 (2H, t, J = 7.6 Hz), 1.68−1.59 (4H, m), 1.44−1.38 (2H, m).
13C NMR (CDCl3, 125 MHz, δ; ppm): 142.26, 128.34, 128.29, 125.73,
51.34, 35.74, 30.94, 28.72, 26.34. FTIR (neat, cm−1) 2090. MS (EI)
m/z 133 (M+ − 28).
4-Ethynyl-N-hydroxybenzamide (A4). Yield 47%; colorless crys-
tals; mp 166−167 °C. 1H NMR (DMSO-d6, 500 MHz, δ; ppm): 11.3
(1H, broad s), 9.12 (1H, broad s), 7.75 (2H, d, J = 8.2 Hz), 7.55 (2H,
d, J = 8.2 Hz), 4.37 (1H, s). 13C NMR (DMSO-d6, 150 MHz, δ; ppm):
163.24, 132.77, 131.61, 127.04, 124.25, 82.72, 82.66. MS (EI) m/z 161
(M+). Anal. (C9H7NO2·1/10H2O) C, H, N.
4-Methoxybenzyl Azide (B6).28 Yield 60%; a pale-yellow oil. H
1
Propynoic Acid 2-Aminophenylamide (A5). Compound A5 was
prepared as colorless crystals in 44% yield from 8 and 1,2-
phenylenediamine, using the same procedure described for A1 (step
1); mp 127−128 °C. H NMR (DMSO-d6, 500 MHz, δ; ppm): 9.97
(1H, s), 7.15 (1H, d, J = 7.9 Hz), 6.93 (1H, t, J = 7.6 Hz), 6.73 (1H, d,
NMR (CDCl3, 500 MHz, δ; ppm): 7.24 (2H, d, J = 8.8 Hz), 6.91 (2H,
d, J = 8.5 Hz), 4.26 (2H, s), 3.81(2H, s). 13C NMR (CDCl3, 150 MHz,
δ; ppm): 159.60, 129.72, 127.36, 114.16, 55.26, 54.36. FTIR (neat,
cm−1) 2090. MS (EI) m/z 163 (M+). HRMS (EI) calcd for C8H9ON3,
163.0758; found, 163.0761.
1
I
dx.doi.org/10.1021/jm300837y | J. Med. Chem. XXXX, XXX, XXX−XXX

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1
3-Phenylbenzyl Azide (B7). Yield 78%; colorless crystals. H NMR
(CDCl3, 500 MHz, δ; ppm): 7.59−7.52 (4H, m), 7.37−7.34 (1H, m),
7.46−7.42 (3H, m), 7.29 (1H, d, J = 7.6 Hz), 4.39 (2H, s). 13C NMR
(CDCl3, 125 MHz, δ; ppm): 141.95, 140.66, 135.94, 129.28, 128.84,
127.56, 127.20, 127.14, 127.00, 126.98, 54.87. FTIR (neat, cm−1)
2094. MS (EI) m/z 209 (M+). HRMS (EI) calcd for C13H11N3,
209.0957; found, 209.0960.
4-Methoxyphenethyl Azide (B21). Yield 100%; a pale-yellow oil.
1H NMR (CDCl3, 500 MHz, δ; ppm): 7.14 (2H, d, J = 8.8 Hz), 6.86
(2H, d, J = 8.5 Hz), 3.80 (3H, s), 3.46 (2H, t, J = 7.2 Hz), 2.84 (2H, t,
J = 7.2 Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 158.50, 130.06,
129.73, 114.09, 55.28, 52.73, 34.50. FTIR (neat, cm−1) 2090. HRMS
(EI) calcd for C9H11ON3, 177.0904; found, 177.0903.
2-Chlorophenethyl Azide (B22). Yield 90%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.37 (1H, d, J = 7.0 Hz), 7.26−
7.21 (3H, m), 3.53 (2H, t, J = 7.3 Hz), 3.03 (2H, t, J = 7.3 Hz). 13C
NMR (CDCl3, 125 MHz, δ; ppm): 135.57, 134.07, 131.11, 129.71,
128.39, 127.02, 50.63, 33.29. FTIR (neat, cm−1) 2090. HRMS (EI)
calcd for C8H8N3Cl, 181.0405; found, 181.0404.
1
4-Phenylbenzyl Azide (B8). Yield 21%; a pale-yellow oil. H NMR
(CDCl3, 500 MHz, δ; ppm): 7.62−7.59 (4H, m), 7.45 (2H, t, J = 7.6
Hz), 7.40−7.35 (3H, m), 4.39 (2H, s). 13C NMR (CDCl3, 125 MHz,
δ; ppm): 141.31, 140.54, 134.37, 128.84, 128.67, 127.58, 127.52,
127.13, 54.57. FTIR (neat, cm−1) 2090. MS (EI) m/z 209 (M+).
HRMS (EI) calcd for C13H11N3, 209.0952; found, 209.0953.
3-Phenoxybenzyl Azide (B9). Yield 86%; a pale-yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.37−7.32 (3H, m), 7.12 (1H, t, J
= 7.5 Hz), 7.05−7.01 (3H, m), 6.96−6.89 (2H, m), 4.30 (2H, s). 13C
NMR (CDCl3, 125 MHz, δ; ppm): 157.79, 156.78, 137.29, 130.19,
129.85, 123,60, 122.77, 119.15, 118.48, 118.27, 54.42. FTIR (neat,
cm−1) 2090. MS (EI) m/z 225 (M+). HRMS calcd for C13H11ON3,
225.0901; found, 225.0902.
1
3-Chlorophenethyl Azide (B23). Yield 80%; a pale-yellow oil. H
NMR (CDCl3, 500 MHz, δ; ppm): 7.25−7.22 (3H, m), 7.11 (1H, d, J
= 6.7 Hz), 3.51 (2H, t, J = 7.2 Hz), 2.87 (2H, t, J = 7.2 Hz). 13C NMR
(CDCl3, 125 MHz, δ; ppm): 140.06, 134.43, 129.90, 128.92, 127.04,
126.97, 52.13, 35.03. FTIR (neat, cm−1) 2090. MS (EI) m/z 153 (M+
− 28).
4-Chlorophenethyl Azide (B24). Yield 92%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.29 (2H, d, J = 8.2 Hz), 7.15 (2H,
d, J = 8.2 Hz), 3.49 (2H, t, J = 7.2 Hz), 2.86 (2H, t, J = 7.2 Hz). 13C
NMR (CDCl3, 125 MHz, δ; ppm): 136.52, 132.66, 130.11, 128.79,
52.27, 34.72. FTIR (neat, cm−1) 2090. HRMS (EI) calcd for
C8H8N3Cl, 181.0406; found, 181.0404.
1-Azidomethylnaphthalene (B11). Yield 91%; a pale-yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 8.01 (1H, d, J = 8.2 Hz), 7.88−
7.83 (2H,m), 7.58− 7.50 (2H, m), 7.46−7.42 (2H, m), 4.74 (2H, s).
13C NMR (CDCl3, 125 MHz, δ; ppm): 133.87, 131.32, 130.93, 129.39,
128.77, 127.23, 126.69, 126.12, 125.17, 123.442, 52.97. FTIR (neat,
cm−1) 2094. MS (EI) m/z 183 (M+). HRMS (EI) calcd for C11H9N3,
183.0804; found, 183.0802.
2-Fluorophenethyl Azide (B25). Yield 82%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.26−7.22 (2H, m), 7.11−7.03
(2H, m), 3.52 (2H, t, J = 7.0 Hz), 2.94 (2H, t, J = 7.0 Hz). 13C NMR
(CDCl3, 150 MHz, δ; ppm): 161.24, 131.15, 128.69, 124.90, 124.23,
115.45, 51.08, 29.00. FTIR (neat, cm−1) 2090. MS (EI) m/z 137 (M+
− 28).
2-Azidomethylnaphthalene (B12).29 Yield 83%; colorless crystals.
1H NMR (CDCl3, 500 MHz, δ; ppm): 7.88−7.84 (3H, m), 7.78 (1H,
s), 7.53−7.49 (2H, m), 7.43 (1H, dd, J = 1.5, 8.2 Hz), 4.51 (2H, s).
13C NMR (CDCl3, 125 MHz, δ; ppm): 133.28, 133.11, 132.83, 128.79,
127.96, 127.77, 127.19, 126.48, 126.35, 125,86, 55.05. FTIR (neat,
cm−1) 2090. MS (EI) m/z 183 (M+). HRMS (EI) calcd for C11H9N3,
183.0803; found. 183.0803.
3-Fluorophenethyl Azide (B26). Yield 95%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.31−7.26 (1H, m), 7.01−6.93
(3H, m), 3.52 (2H, t, J = 7.3 Hz), 2.89 (2H, t, J = 7.3 Hz). 13C NMR
(CDCl3, 125 MHz, δ; ppm): 162.96, 140.54, 130.12, 124.42, 115.69,
113.75, 52.13, 35.09. FTIR (neat, cm−1) 2090. MS (EI) m/z 137 (M+
− 28).
1
Azidomethylcyclohexane (B14). Yield 92%; a pale-yellow oil. H
NMR (CDCl3, 500 MHz, δ; ppm): 3.11 (2H, d, J = 6.7 Hz), 1.78−
1.67 (5H, m), 1.59−1.51 (1H, m), 1.29−1.11 (3H, m), 1.01−0.92
(2H, m). 13C NMR (CDCl3, 125 MHz, δ; ppm): 58.08, 38.09, 30.67,
26.27, 25.76. FTIR (neat, cm−1) 2094.
4-Fluorophenethyl Azide (B27). Yield 95%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.19−7.17 (2H, m), 7.01 (2H, t, J
= 8.7 Hz), 3.49 (2H, t, J = 7.3 Hz), 2.86 (2H, t, J = 7.3 Hz). 13C NMR
(CDCl3, 125 MHz, δ; ppm): 161.83, 133.72, 130.22, 115.49, 52.51,
34.58. FTIR (neat, cm−1) 2090. HRMS (EI) calcd for C8H8N3Cl,
165.0702; found, 165.0701.
2-Methylphenethyl Azide (B16). Yield 95%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.16 (4H, s), 3.47 (2H, t, J = 7.6
Hz), 2.91 (2H, t, J = 7.6 Hz), 2.34 (3H, s). 13C NMR (CDCl3, 125
MHz, δ; ppm): 136.20, 136.07, 130.48, 129.34, 126.96, 126.25, 51.44,
32.62, 19.31. FTIR (neat, cm−1) 2090. MS (EI) m/z 133 (M+ − 28).
3-Methylphenethyl Azide (B17). Yield 81%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.21 (1H, t, J = 7.5 Hz), 7.07−7.01
(3H, m), 3.49 (2H, t, J = 7.3 Hz), 2.86 (2H, t, J = 7.3 Hz), 2.34 (3H,
s). 13C NMR (CDCl3, 125 MHz, δ; ppm): 138.28, 137.94, 129.55,
128.54, 127.52, 125.73, 52.50, 35.27, 21.38. FTIR (neat, cm−1) 2090.
MS (EI) m/z 133 (M+ − 28).
2-(Trifluoromethyl)phenethyl Azide (B28). Yield 88%; a colorless
1
oil. H NMR (CDCl3, 500 MHz, δ; ppm): 7.66 (1H, d, J = 7.6 Hz),
7.51 (1H, t, J = 7.6 Hz), 7.39− 7.35 (2H, m), 3.51 (2H, t, J = 7.3 Hz),
3.08 (2H, t, J = 7.3 Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm):
131.93, 131.73, 128.99, 127.00, 126.29, 125.56, 123.38, 52.08, 32.30.
FTIR (neat, cm−1) 2094. MS (EI) m/z 187 (M+ − 28).
3-(Trifluoromethyl)phenethyl Azide (B29). Yield 87%; a colorless
1
oil. H NMR (CDCl3, 600 MHz, δ; ppm): 7.52 (1H, d, J = 7.8 Hz),
7.48 (1H, s), 7.46− 7.41 (2H, m). 13C NMR (CDCl3, 125 MHz, δ;
ppm): 139.01, 132.22, 129.11, 125.52, 123.74, 52.11, 35.16. FTIR
(neat, cm−1) 2094. MS (EI) m/z 153 (M+ − 28).
4-Methylphenethyl Azide (B18). Yield 85%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.13 (2H, d, J = 8.2 Hz), 7.11 (2H,
d, J = 8.2 Hz), 3.48 (2H, t, J = 7.3 Hz), 2.86 (2H, t, J = 7.3 Hz), 2.33
(3H, s). 13C NMR (CDCl3, 125 MHz, δ; ppm): 136.36, 134.92,
129.33, 128.62, 52.59, 34.92, 21.05. FTIR (neat, cm−1) 2090;. HRMS
(EI) calcd for C9H11N3, 161.0950; found, 161.0947.
4-(Trifluoromethyl)phenethyl Azide (B30). Yield 90%; a pale-
yellow oil. 1H NMR (CDCl3, 600 MHz, δ; ppm): 7.58 (2H, d, J = 8.1
Hz), 7.34 (2H, d, J = 8.0 Hz), 3.55 (2H, t, J = 7.1 Hz), 2.95 (2H, t, J =
7.1 Hz). 13C NMR (CDCl3, 150 MHz, δ; ppm): 142.16, 129.13,
125.60, 125.08, 123.27, 52.02, 35.16. FTIR (neat, cm−1) 2094. MS
(EI) m/z 153 (M+ − 28).
2-Methoxyphenethyl Azide (B19). Yield 72%; a pale-yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 7.24 (1H, dt, J = 1.8, 5.9 Hz), 7.16
(2H, dd, J = 1.5, 5.8 Hz), 6.91 (1H, dt, J = 1.2, 6.2 Hz), 6.86 (1H, d, J
= 8.2 Hz), 3.83 (3H, s), 3.47 (2H, t, J = 7.6 Hz), 2.92 (2H, t, J = 7.6
Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 157.57, 130.61, 128.17,
126.23, 120.56, 110.35, 55.24, 50.97, 30.34. FTIR (neat, cm−1) 2090.
HRMS (EI) calcd for C9H11ON3, 177.0901; found, 177.0903.
1
2-Nitrophenethyl Azide (B31). Yield 91%; a yellow oil. H NMR
(CDCl3, 500 MHz, δ; ppm): 7.99 (1H, d, J = 8.2 Hz), 7.58 (1H, t, J =
7.5 Hz), 7.45−7.41 (2H, m), 3.64 (2H, t, J = 6.9 Hz), 3.18 (2H, t, J =
6.9 Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 149.34, 133.28,
133.13, 132.86, 128.14, 125.14, 51.48, 33.00. FTIR (neat, cm−1) 2090,
1519, 1342. MS (EI) m/z 164 (M+ − 28).
1
3-Methoxyphenethyl Azide (B20). Yield 89%; a colorless oil. H
NMR (CDCl3, 500 MHz, δ; ppm): 7.24 (1H, t, J = 7.9 Hz), 6.82−6.76
(3H, m), 3.81 (3H, s), 3.50 (2H, t, J = 7.3 Hz), 2.87 (2H, t, J = 7.3
Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 159.83, 139.61, 129.66,
121.07, 114.62, 112.05, 55.20, 52.38, 35.40. FTIR (neat, cm−1) 2090.
HRMS (EI) calcd for C9H11ON3, 177.0909; found, 177.0910.
1
3-Nitrophenethyl Azide (B32). Yield 62%; a yellow oil. H NMR
(CDCl3, 500 MHz, δ; ppm): 8.13−8.11 (2H, m), 7.58 (1H, d J = 7.6
Hz), 7.51 (1H, t J = 7.8 Hz), 3.60 (2H, t, J = 6.9 Hz), 3.00 (2H, t, J =
6.9 Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 148.47, 140.15,
J
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135.09, 129.58, 123.70, 122.01, 51.86, 34.98. FTIR (neat, cm−1) 2090,
(2-Azidoethyl)-1-adamantane (B43). Yield 79%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 3.27 (2H, t, J = 7.9 Hz), 1.96 (3H,
s), 1.72−1.62 (6H, m), 1.51 (6H, d, J = 2.1 Hz), 1.40 (2H, t, J = 7.9
Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 46.61, 42.42, 42.34,
36.98, 31.80, 30.93, 28.55. FTIR (neat, cm−1) 2090.
1523, 1346. MS (EI) m/z 164 (M+ − 28).
1
4-Nitrophenethyl Azide (B33). Yield 93%; a yellow oil. H NMR
(CDCl3, 500 MHz, δ; ppm): 8.19 (2H, d, J = 8.5 Hz), 7.40 (2H, d, J =
8.5 Hz), 3.59 (2H, t, J = 6.9 Hz), 3.00 (2H, t, J = 6.9 Hz). 13C NMR
(CDCl3, 125 MHz, δ; ppm): 147.05, 145.77, 129.70, 123.89, 51.72,
35.19. FTIR (neat, cm−1) 2090, 1516, 1342. MS (EI) m/z 164 (M+ −
28).
4-Phenoxybenzyl Azide (B10). Step 1: 4-Phenoxyphenylmethanol
(25). A solution of 4-phenoxybenzoic acid (24, 2.00 g, 9.34 mmol) in
THF (45 mL) was added dropwise to a suspension of LiAlH4 (1.06 g,
28.0 mmol) in THF (65.0 mL) with cooling by an ice-bath. The
mixture was heated at reflux for 7 h. After cooling of the reaction
mixture, water (1.00 mL), 15% aqueous NaOH (1.00 mL), and water
(3.00 mL) were successively added, and the slurry was filtered. The
solid was washed with THF, the combined filtrates were concentrated
in vacuo, and the residue was purified by silica gel flash column
chromatography (AcOEt/n-hexane = 1/3) to give 1.63 g (87%) of 25
1
1-(2-Azidoethyl)naphthalene (B34). Yield 91%; a yellow oil. H
NMR (CDCl3, 500 MHz, δ; ppm): 8.00 (1H, d, J = 8.5 Hz), 7.89−
7.87 (1H, m), 7.77 (1H, d, J = 8.2 Hz), 7.56−7.38 (4H, m), 3.64 (2H,
t, J = 7.6 Hz), 3.38 (2H, t, J = 7.6 Hz). 13C NMR (CDCl3, 150 MHz,
δ; ppm): 133.93, 133.86, 131.70, 129.01, 127.68, 127.00, 126.27,
125.72, 125.55, 123.14, 51.74, 32.49. FTIR (neat, cm−1) 2090. HRMS
(EI) calcd for C12H11N3, 197.0953; found, 197.0954.
1
3-(2-Azidoethyl)indole (B36). Yield 92%; a pale-yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 8.01 (1H, s), 7.59 (1H, dd, J = 1.2,
7.9 Hz), 7.37 (1H, dt, J = 0.9, 8.2 Hz), 7.21 (1H, m), 7.14 (1H, m),
7.07 (1H, d, J = 2.4 Hz), 3.57 (2H, t, J = 7.2 Hz), 3.07 (2H, dt, J = 0.9,
7.0 Hz). 13C NMR (CDCl3, 150 MHz, δ; ppm): 136.25, 127.13,
122.24, 122.21, 119.56, 118.52, 112.37, 111.27, 51.66, 25.09. FTIR
(neat, cm−1) 2090. HRMS (EI) calcd for C10H10N4, 186.0906; found,
186.0904.
as a white solid. H NMR (CDCl3, 500 MHz, δ; ppm): 7.35−7.31
(4H, m), 7.10 (1H, t, J = 7.5 Hz), 7.02−7.00 (4H, m), 4.67 (2H, d, J =
5.8 Hz).
Step 2: Preparation of 4-Phenoxybenzyl Azide (B10). To a
mixture of 25 (300 mg, 1.50 mmol) obtained above and DPPA (495
mg, 1.80 mmol) in dry DMF (2.7 mL) was added neat DBU (274 mg,
1.80 mmol) at 0 °C under Ar. The reaction mixture was stirred at
room temperature for 24 h and then poured into water and extracted
with AcOEt. The AcOEt layer was separated, washed with water, 2 N
aqueous HCl, and brine, and dried over Na2SO4. Filtration,
concentration in vacuo, and purification of the residue by silica gel
flash column chromatography (AcOEt/n-hexane = 1/50) gave 103 mg
(30%) of B10 as a colorless oil. 1H NMR (CDCl3, 500 MHz, δ; ppm):
7.37−7.31 (2H, m), 7.10 (1H, t, J = 7.5 Hz), 7.02−7.00 (4H, m), 2H
(d, J = 5.8 Hz). 13C NMR (CDCl3, 125 MHz, δ; ppm): 157.49, 156.81,
130.01, 129.81, 123.57, 119.15, 118.89, 54.30. FTIR (neat, cm−1)
2090. MS (EI) m/z 225 (M+). HRMS (EI) calcd for C13H11ON3,
225.0903; found, 225.0900.
2-(2-Azidoethyl)naphthalene (B35). To a solution of 2-naphtha-
leneethanol (27a, 1.00 g, 5.81 mmol) in CH2Cl2 (4 mL) were added
triethylamine (705 mg, 6.97 mmol) and MsCl (798 mg, 6.97 mmol) at
0 °C. The mixture was stirred for 3 h at room temperature and then
poured into saturated aqueous NaHCO3 and extracted with CHCl3.
The organic layer was washed with water and brine and dried over
Na2SO4. Filtration and concentration in vacuo gave the crude
mesylate. To a 0.5 M solution of NaN3 in DMSO (13.9 mL, 6.97
mmmol) was added the mesylate, and the mixture was stirred at 80 °C
for 2 h and then poured into water and extracted with AcOEt. The
AcOEt layer was washed with water and brine and dried over Na2SO4.
Filtration, concentration in vacuo, and purification of the residue by
silica gel flash column chromatography (AcOEt/n-hexane = 1/50)
gave 906 mg (79%, 2 steps) of B35 as a white solid. The solid was
recrystallized from AcOEt/n-hexane to give 319 mg of B35 as a white
solid. 1H NMR (CDCl3, 500 MHz, δ; ppm): 7.83−7.79 (3H, m), 7.68
(1H, s), 7.49−7.43 (2H, m), 7.35 (1H, dd, J = 1.8, 8.2 Hz), 3.60 (2H,
t, J = 7.3 Hz), 3.06 (2H, t, J = 7.3 Hz). 13C NMR (CDCl3, 125 MHz,
δ; ppm): 135.49, 133.58, 132.38, 128.33, 127.69, 127.55, 127.31,
126.98, 126.19, 125.65, 52.41, 35.52. FTIR (neat, cm−1) 2075. HRMS
(EI) calcd for C12H11N3, 197.0950; found, 197.0952.
1
2-Azidoacetophenone (B45). Yield 53%; a yellow oil. H NMR
(CDCl3, 500 MHz, δ; ppm): 7.93−7.90 (2H, m), 7.65−7.61 (1H, m),
7.52−7.49 (2H, m), 4.57 (2H, s). 13C NMR (CDCl3, 150 MHz, δ;
ppm): 193.19, 134.41, 134.15, 129.00, 127.95, 54.90. FTIR (neat,
cm−1) 2098, 1693. MS (EI) m/z 133 (M+ − 28).
Azidomethylcyclopentane (B13). Step 1: Preparation of Cyclo-
pentylmethyl Tosylate (23a). To a solution of cyclopentanemethanol
(22a, 500 mg, 4.99 mmol) in pyridine (5 mL) was added TsCl (1.43 g,
7.49 mmol) at 0 °C. The reaction mixture was stirred for 36 h at room
temperature. After removal of the solvent, the residue was diluted with
AcOEt. The AcOEt solution was washed with saturated NaHCO3 and
brine and dried over Na2SO4. Filtration, concentration in vacuo, and
purification of the residue by silica gel flash column chromatography
(AcOEt/n-hexane = 1/50) gave 864 mg (68%) of 23a as a colorless
1
oil. H NMR (CDCl3, 500 MHz, δ; ppm): 7.79 (2H, d, J = 8.2 Hz),
7.35 (2H, d, J = 7.9 Hz), 3.89 (2H, d, J = 7.3 Hz), 2.50 (3H, s), 2.24−
2.15 (1H, m), 1.74−1.68 (2H, m), 1.57−1.49 (4H, m), 1.22−1.15
(2H, m).
Step 2: Preparation of Azidomethylcyclopentane (B13). To a 0.5
M solution of NaN3 in DMSO (26.6 mL, 13.3 mmmol) was added 23a
(1.13 g, 13.3 mmmol) obtained above, and the mixture was stirred at
80 °C for 5 h. The reaction mixture was quenched with water and
stirred until it cooled to room temperature, then extracted with
AcOEt. The AcOEt layer was separated, washed with water and brine,
and dried over Na2SO4. Filtration and concentration in vacuo gave 147
1
mg (26%) of B13 as a yellow oil. H NMR (CDCl3, 500 MHz, δ;
ppm): 3.20 (2H, d, J = 7.3 Hz), 2.19−2.10 (1H, m), 1.83−1.77 (2H,
m), 1.64−1.55 (4H, m), 1.29−1.21 (2H, m). 13C NMR (CDCl3, 125
MHz, δ; ppm): 56.51, 39.64, 30.33, 25.28; FTIR (neat, cm−1) 2098.
Azides B15 and B41−B43 were prepared from the corresponding
alcohols using the same procedure described for B13.
1-Azidomethyladamantane (B15).30 Yield 45%; a colorless oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 2.95 (2H, s), 1.99 (3H, s), 1.72
(3H, d, J = 12 Hz), 1.64 (3H, d, J = 12 Hz), 1.52 (6H, d, J = 2.4 Hz).
13C NMR (CDCl3, 125 MHz, δ; ppm): 64.37, 40.09, 36.85, 34.78,
28.21. FTIR (neat, cm−1) 2094. MS (EI) m/z 191 (M+). HRMS (EI)
calcd for C11H17N3, 191.1423; found, 191.1424.
Azides B37, B39, and B40 were prepared from the corresponding
alcohols using the same procedure described for B35.
3-(2-Azidoethyl)thiophene (B37). Yield 84%; a colorless oil. H
1
NMR (CDCl3, 500 MHz, δ; ppm): 7.29 (1H, m), 7.06 (1H, d, J = 1.8
Hz), 6.98 (1H, d, J = 4.3 Hz), 3.51 (2H, t, J = 7.0 Hz), 2.93 (2H, t, J =
7.0 Hz). 13C NMR (CDCl3, 150 MHz, δ; ppm): 138.26, 127.97,
125.95, 121.79, 51.79, 29.81. FTIR (neat, cm−1) 2086. MS (EI) m/z
125 (M+ − 28).
(2-Azidoethyl)cyclopentane (B41). Yield 53%; a pale-yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 3.27 (2H, t, J = 7.3 Hz), 1.87−1.78
(3H, m), 1.64−1.62 (4H, m), 1.60−1.49 (2H, m). 13C NMR (CDCl3,
150 MHz, δ; ppm): 50.83, 37.41, 34.92, 32.48, 25.05. FTIR (neat,
cm−1) 2090.
5-(2-Azidoethyl)-4-methylthiazole (B39). Yield 86%; a colorless
oil. 1H NMR (CDCl3, 500 MHz, δ; ppm): 8.62 (1H, s), 3.51 (2H, t, J
= 6.9 Hz), 3.04 (2H, t, J = 6.9 Hz), 2.43 (3H, s). 13C NMR (CDCl3,
125 MHz, δ; ppm): 150.06, 150.02, 127.01, 52.06, 26.27, 14.95. FTIR
(neat, cm−1) 2090. HRMS (EI) calcd for C6H8N4S, 168.0473; found,
198.0475.
1
(2-Azidoethyl)cyclohexane (B42). Yield 55%; a pale-yellow oil. H
NMR (CDCl3, 500 MHz, δ; ppm): 3.29 (2H, t, J = 7.2 Hz), 1.72−1.61
(5H, m), 1.50 (2H, q, J = 7.1 Hz), 1.38−1.37 (1H, m), 1.28−1.11
(3H, m), 0.951 (2H, m). 13C NMR (CDCl3, 125 MHz, δ; ppm):
49.20, 36.05, 35.00, 33.02, 26.44, 26.14. FTIR (neat, cm−1) 2090.
4-(2-Azidoethyl)morpholine (B40). Yield 50%; a yellow oil. 1H
NMR (CDCl3, 500 MHz, δ; ppm): 3.73 (4H, t, J = 4.7 Hz), 3.35 (2H,
K
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t, J = 5.9 Hz), 2.60 (2H, t, J = 5.9 Hz), 2.51 (4H, t, J = 4.6 Hz). 13C
NMR (CDCl3, 125 MHz, δ; ppm): 66.91, 57.63, 53.62, 47.94. FTIR
(neat, cm−1) 2094.
NMR (DMSO-d6, 600 MHz, δ; ppm): 11.3 (1H, broad s), 9.08 (1H,
s) 8.59 (1H, s), 8.20 (1H, d, J = 1.6 Hz), 7.93 (1H, d, J = 7.7 Hz), 7.68
(1H, dt, J = 1.2, 6.5 Hz), 7.52 (1H, t, J = 7.7 Hz), 7.30−7.20 (5H, m),
4.68 (2H, t, J = 7.3 Hz), 3.23 (2H, t, J = 7.3 Hz). 13C NMR (DMSO-
d6, 125 MHz, δ; ppm): 163.98, 145.53, 137.56, 133.49, 130.96, 128.99,
128.65, 128.41, 127.54, 126.58, 126.03, 123.68, 121.90, 50.65, 35.49.
MS (FAB) m/z 309 (MH+). Anal. (C17H16N4O2·H2O) C, H, N.
N-Hydroxy-3-[1-(2-thiophen-3-yl-ethyl)-1H-[1,2,3]triazol-4-yl]-
benzamide (C142). Yield 100%; colorless crystals; mp 164−166 °C.
1H NMR (DMSO-d6, 500 MHz, δ; ppm): 11.3 (1H, broad s), 9.09
(1H, s) 8.59 (1H, s), 8.21 (1H, s), 7.94 (1H, d, J = 7.6 Hz), 7.68 (1H,
d, J = 7.9 Hz), 7.52 (1H, t, J = 7.6 Hz), 7.48−7.46 (1H, m), 7.24 (1H,
s), 7.00 (1H, d, J = 4.8 Hz), 4.67 (2H, t, J = 7.3 Hz), 3.25 (2H, t, J =
7.3 Hz). 13C NMR (DMSO-d6, 150 MHz, δ; ppm): 163.88, 145.47,
137.68, 133.40, 130.89, 128.93, 128.11, 127.48, 126.12, 125.95, 123.62,
122.01, 121.59, 49.94, 30.08. MS (FAB) m/z 315 (MH+). Anal.
(C15H14N4O2S) C, H, N.
N-Hydroxy-3-(1-phenylsulfanylmethyl-1H-[1,2,3]triazol-4-yl)-
benzamide (C149). Yield 92%; colorless crystals; mp 160−161 °C. 1H
NMR (DMSO-d6, 500 MHz, δ; ppm): 11.3 (1H, broad s), 9.09 (1H,
s) 8.63 (1H, s), 8.21 (1H, t, J = 1.5 Hz), 7.96 (1H, d, J = 7.9 Hz), 7.70
(1H, dt, J = 1.2, 7.6 Hz), 7.52 (1H, t, J = 7.9 Hz), 7.45−7.43 (2H, m),
7.37−7.30 (3H, m), 6.01 (2H, s). 13C NMR (DMSO-d6, 125 MHz, δ;
ppm): 163.87, 146.10, 133.47, 132.27, 130.66, 130.52, 129.31, 129.06,
127.79, 127.70, 126.35, 123.71, 121.37, 52.02. MS (FAB) m/z 327
(MH+). Anal. (C16H14N4O2S) C, H, N.
Biology. Enzyme Assays. The HDAC activity assay was performed
using an HDACs/HDAC8 deacetylase fluorometric assay kit (CY-
1150/CY-1158, Cyclex Company Limited), HDAC1/HDAC6 fluo-
rescent activity drug discovery kit (AK-511/AK-516, BIOMOL
Research Laboratories), fluorescent SIRT1 activity assay/drug
discovery kit (AK-555, BIOMOL Research Laboratories), or
fluorogenic HDAC class 2α assay kit (BPS Bioscience Incorporated)
with HDACs (CY-1150, Cyclex Company Limited), HDAC1 (SE-456,
BIOMOL Research Laboratories), HDAC2 (SE-500, BIOMOL
Research Laboratories), HDAC4 (BPS Bioscience Incorporated),
HDAC6 (SE-508, BIOMOL Research Laboratories), and HDAC8
(CY-1158, Cyclex Company Limited), according to the supplier’s
instructions. 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 a compound that results in 50% inhibition
(IC50) was determined by plotting log[Inh] versus the logit function of
% inhibition. IC50 values were determined by regression analysis of the
concentration/inhibition data. For determination of IC50 values,
various concentrations (0.0001, 0.001, 0.01, 0.1, 1.0, 10, and 100
μM) of inhibitors were used.
2-(2-Azidoethyl)pyridine (B38). To a solution of 2-pyridineethanol
(27c, 1.10 g, 8.93 mmol) and CBr4 (3.26 g, 9.83 mmol) in CH2Cl2 (10
mL) was added a solution of PPh3 (2.58 g, 9.83 mmol) in CH2Cl2 (5
mL) at 0 °C, and the mixture was stirred for 1 h at room temperature
and then concentrated in vacuo. Purification of the residue by silica gel
flash column chromatography (AcOEt/n-hexane = 1/4) gave 596 mg
(36%) of the bromide as a colorless oil. To a 0.5 M solution of NaN3
in DMSO (7.68 mL, 3.84 mmmol) was added the bromide (596 mg,
3.20 mmol), and the mixture was stirred at room temperature for 2 h.
The reaction mixture was poured into water and extracted with AcOEt.
The AcOEt layer was washed with water and brine and dried over
Na2SO4. Filtration, concentration in vacuo, and purification of the
residue by silica gel flash column chromatography (AcOEt/n-hexane =
1/3) gave 207 mg (44%) of B38 as a pale-yellow oil. 1H NMR
(CDCl3, 500 MHz, δ; ppm): 8.57 (1H, d, J = 4.3 Hz), 7.64 (1H, dt, J =
1.5, 7.6 Hz), 7.21−7.16 (2H, m), 3.72 (2H, t, J = 7.0 Hz), 3.06 (2H, t,
J = 7.0 Hz). 13C NMR (CDCl3, 150 MHz, δ; ppm): 158.04, 149.60,
136.55, 123.57, 121.84, 50.70, 37.52. FTIR (neat, cm−1) 2090. MS
(EI) m/z 119 (M+ − 28).
(E)-(2-Azidovinyl)benzene (B46). To a solution of CuSO4 (118 mg,
0.737 mmol) and NaN3 (575 mg, 8.84 mmol) in MeOH (20 mL) was
added (E)-2-phenylvinylboronic acid (30, 1.09 g, 7.37 mmol), and the
mixture was stirred for 30 h at room temperature. After removal of the
solvent, the residue was diluted with AcOEt. The AcOEt solution was
washed water and brine and dried over Na2SO4. Filtration,
concentration in vacuo, and purification of the residue by silica gel
flash column chromatography (AcOEt/n-hexane = 1/10) gave 425 mg
1
(40%) of B46 as a yellow oil. H NMR (CDCl3, 500 MHz, δ; ppm):
7.32−7.20 (5H, m), 6.61 (1H, d, J = 14 Hz), 6.27 (1H, d, J = 14 Hz).
13C NMR (CDCl3, 125 MHz, δ; ppm): 135.02, 128.76, 127.39, 126.68,
125.82, 119.80. FTIR (neat, cm−1) 2090, 1635. HRMS (EI) calcd for
C8H7N3, 145.0644; found, 145.0645.
Construction of a 120-Member Triazole Library (C1−C120)
and a 31-Member Triazole Library (C121−C151). To a solution
of an alkyne (25 mM, 20 μL), an azide (30 mM, 20 μL), and TBTA (5
mM, 10 μL) in DMSO was added an aqueous solution of
CuSO4·5H2O (2 mM, 25 μL) on a 96-well plate. To the resulting
mixture was added an aqueous solution of sodium ascorbate (10 mM,
25 μL), and the mixture was shaken for 1−3 days at room
temperature. The reaction was monitored by TLC and LCMS.
When the reaction was completed, DMSO (150 μL) was added to the
mixture, in which the concentration of the triazole is assumed to be 2
mM. The crude triazoles were diluted to a desired concentration for
enzyme assays.
3-(1-Benzyl-1H-[1,2,3]triazol-4-yl)-1-carboxylic Acid Hydroxya-
mide (C31). To a solution of A3 (43.2 mg, 0.268 mmol), B1 (51.8
mg, 0.322 mmol), and TBTA (14.2 mg, 10 mol %) in MeOH (5 mL)
was added a solution of CuSO4·5H2O (6.69 mg, 10 mol %) and
sodium ascorbate (26.5 mg, 50 mol %) in water (5 mL). The reaction
mixture was stirred for 24 h at room temperature and then poured into
water and extracted with AcOEt. The AcOEt layer was separated,
washed with water and brine, and dried over Na2SO4. Filtration,
concentration in vacuo, and purification of the residue by silica gel
flash column chromatography (AcOEt/n-hexane = 2/1) gave 82.2 mg
(q.y.) of C31 as a pale-yellow solid. The solid was recrystallized from
MeOH to give 40.9 mg of C31 as colorless crystals; mp 154−155 °C.
1H NMR (DMSO-d6, 600 MHz, δ; ppm): 11.3 (1H, broad s), 9.08
(1H, s) 8.69 (1H, s), 8.23 (1H, t, J = 1.6 Hz), 7.98 (1H, d, J = 7.7 Hz),
7.69 (1H, dt, J = 1.4, 7.9 Hz), 7.52 (1H, t, J = 7.7 Hz), 7.42−7.34 (5H,
m), 5.67 (2H, s). 13C NMR (DMSO-d6, 125 MHz, δ; ppm): 163.93,
146.07, 135.89, 133.45, 130.80, 128.99, 128.79, 128.18, 127.93, 127.63,
126.18, 123.73, 121.90, 53.06. MS (FAB) m/z 295 (MH+). Anal.
(C16H14N4O2·H2O) C, H, N.
Compounds C32, C142, and C149 were prepared from A3 and the
corresponding azides using the same procedure described for C31.
3-(1-Phenethyl-1H-[1,2,3]triazol-4-yl)-1-carboxylic Acid Hydrox-
yamide (C32). Yield 92%; colorless crystals; mp 168−170 °C. 1H
Cell Growth Inhibition Assay. The cells were plated at the initial
density of 2 × 105 cells/well (50 μL/well) in 96-well plates in medium
culture and exposed to inhibitors for 72 h at 37 °C in 5% CO2
incubator. A solution of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-
thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was
then added (20 μL/well), and incubation was continued for 2 h. The
solubilized dye was quantified by colorimetric reading at 490 nm using
a reference wavelength of 650 nm. The absorbance values of control
wells (C) and test wells (T) were measured. The absorbance of the
test wells (T0) was also measured at time 0 (addition of compounds).
Using these measurements, cell growth inhibition (percentage of
growth) by a test inhibitor at each concentration used was calculated
as: % growth = 100[(T − T0)/(C − T0)], when T > T0 and % growth
= 100[(T − T0)/T], when T < T0. Computer analysis of the % growth
values afforded the 50% growth inhibition parameter (GI50). The GI50
was calculated as 100[(T − T0)/(C − T0)] = 50.
Western Blot Analysis. The cohesin- or H4-acetylating activities of
the test compounds were assayed according to the method reported in
ref 24.
Molecular Modeling. The X-ray structure of HDAC8 (PDB code
1VKG) was used as the target structure for docking. Protein
preparation, receptor grid generation, and ligand docking were
L
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Journal of Medicinal Chemistry
Article
performed using the software Glide 3.5. Compounds C149 and 6 were
docked into the ligand binding site of HDAC8. The standard precision
mode of Glide was used to determine favorable binding poses, which
allowed the ligand conformation to be flexibly explored while holding
the protein as a rigid structure during docking. Then, the predicted
complex structure was fully energy-minimized, with both the protein
and the ligand allowed to move, using Macromodel 8.1 software. The
conformation of compounds C149 and 6 in the ligand binding site was
minimized by MM calculation based on the OPLS-AA force field with
the following parameter set: solvent, water; method, LBFGS; max no.
of iterations, 10000; converge, gradient; convergence threshold, 0.05.
Pharm. 2005, 338, 347−357. (e) Itoh, Y.; Suzuki, T.; Miyata, N.
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Zhang, G. F.; Johanson, K.; Sung, C. M.; Liu, R.; Winkler, J. Cloning
and characterization of a novel human class I histone deacetylase that
functions as a transcription repressor. J. Biol. Chem. 2000, 275, 15254−
15264. (b) Buggy, J. J.; Sideris, M. L.; Mak, P.; Lorimer, D. D.;
McIntosh, B.; Clark, J. M. Cloning and characterization of a novel
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ASSOCIATED CONTENT
* Supporting Information
■
S
(4) Waltregny, D.; De Leval, L.; Glen
́
isson, W.; Ly Tran, S.; North, B.
Results of LCMS analysis of representative compounds, the
120-member triazole library for HDAC inhibitors obtained by
the combination of alkynes A1−A8 and azides B1−B15, the
31-member C32-based library for HDAC8-selective inhibitors
obtained by the combination of alkyne A3 and azides B16−
B46, and results of elemental analysis of A3−A8, C31, C32,
C142, and C149 are reported. This material is available free of
charge via the Internet at http://pubs.acs.org.
J.; Bellahcene, A.; Weidle, U.; Verdin, E.; Castronovo, V. Expression of
̀
histone deacetylase 8, a class I histone deacetylase, is restricted to cells
showing smooth muscle differentiation in normal human tissues. Am. J.
Pathol. 2004, 165, 553−564.
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Hiebert, S. W. The inv(16) fusion protein associates with corepressors
via a smooth muscle myosin heavy-chain domain. Mol. Cell. Biol. 2003,
23, 607−619.
́
(6) Waltregny, D.; Glenisson, W.; Ly Tran, S.; North, B. J.; Verdin,
E.; Colige, A.; Castronovo, V. Histone deacetylase HDAC8 associates
with smooth muscle alpha-actin and is essential for smooth muscle cell
contractility. FASEB J. 2005, 19, 966−968.
AUTHOR INFORMATION
Corresponding Author
*Phone/Fax: +81-52-836-3407. E-mail: suzukit@koto.kpu-m.
ac.jp (T.S.); miyata-n@phar.nagoya-cu.ac.jp (N.M.).
■
(7) Gallinari, P.; Di Marco, S.; Jones, P.; Pallaoro, M.; Steinkuhler, C.
̈
HDACs, histone deacetylation and gene transcription: from molecular
biology to cancer therapeutics. Cell Res. 2007, 17, 195−211.
(8) Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner,
E.; Buggy, J. J. A novel histone deacetylase 8 (HDAC8)-specific
inhibitor PCI-34051 induces apoptosis in T-cell lymphomas. Leukemia
2008, 22, 1026−1034.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mie Tsuchida for her technical support. This work
was supported in part by JST PRESTO program (T.S.), a
Grant-in-Aid for Scientific Research from the JapanSociety for
the Promotion of Science (T.S.), Foundation NAGASE Science
Technology Development (T.S.), and the Shorai Foundation
for Science and Technology (T.S.).
(9) Oehme, I.; Deubzer, H. E.; Wegener, D.; Pickert, D.; Linke, J. P.;
Hero, B.; Kopp-Schneider, A.; Westermann, F.; Ulrich, S. M.; von
Deimling, A.; Fischer, M.; Witt, O. Histone deacetylase 8 in
neuroblastoma tumorigenesis. Clin. Cancer Res. 2009, 15, 91−99.
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from bench to clinic. J. Med. Chem. 2008, 51, 1505−1529. (c) Suzuki,
T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.; Maruyama, S.; Kurotaki,
M.; Nakagawa, H.; Miyata, N. Novel inhibitors of human histone
deacetylases: Design, synthesis, enzyme inhibition, and cancer cell
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ABBREVIATIONS USED
■
HDAC, histone deacetylase; SIRT, sirtuin; TSA, trichostatin A;
CuAAC, Cu(I)-catalyzed azide−alkyne cycloaddition; ZBG,
zinc-binding group; TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]amine; SMC, structural maintenance of chromo-
some; PBMC, peripheral blood mononuclear cell
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