Design, synthesis, and biological activity of boronic acid-based histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm900125m

Guided by the proposed catalytic mechanism of histone deacetylases (HDACs), we designed and synthesized a series of boronic acid-based HDAC inhibitors bearing an R-amino acid moiety. In this series, compounds (S)-18, 20, and 21 showed potent HDAC-inhibito

Full text of DOI:10.1021/jm900125m

J. Med. Chem. 2009, 52, 2909–2922
2909
Design, Synthesis, and Biological Activity of Boronic Acid-Based Histone Deacetylase Inhibitors
Nobuaki Suzuki,† Takayoshi Suzuki,*,† Yosuke Ota,† Tatsuya Nakano,‡ Masaaki Kurihara,§ Haruhiro Okuda,§ Takao Yamori,#
Hiroki Tsumoto,† Hidehiko Nakagawa,† and Naoki Miyata*,†,§
Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan, DiVision
of Medicinal Safety Science, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan, DiVision of Organic
Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan, and DiVision of Molecular
Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan
ReceiVed February 2, 2009
Guided by the proposed catalytic mechanism of histone deacetylases (HDACs), we designed and synthesized
a series of boronic acid-based HDAC inhibitors bearing an R-amino acid moiety. In this series, compounds
(S)-18, 20, and 21 showed potent HDAC-inhibitory activity, highlighting the significance of the (S)-amino
acid moiety. In cancer cell growth inhibition assays, compounds (S)-18, 20, and 21 exerted strong activity,
and the values of the ratio of the concentration causing 50% growth inhibition (GI50) to the concentration
causing 50% enzyme inhibition (IC50), i.e., GI50/IC50, were low. The potency of these compounds was similar
to that of clinically used suberoylanilide hydroxamic acid (SAHA) (2). The results of Western blot analysis
indicated that the cancer cell growth-inhibitory activity of compounds (S)-18, 20, and 21 is the result of
HDAC inhibition. A molecular modeling study suggested that the hydrated boronic acid interacts with zinc
ion, Tyr residue, and His residue in the active site of HDACs. Our findings indicate that these boronic acid
derivatives represent an entry into a new class of HDAC inhibitors.
Introduction
Consequently, there are many ongoing research programs to
find more potent and selective HDAC inhibitors.11
The acetylation status of lysine residues in nucleosomal
histones is tightly controlled by two counteracting enzyme
families, the histone acetyl transferases and the histone deacety-
lases (HDACsa).1 The latter family can be divided into two
categories: Zn2+-dependent enzymes and NAD+-dependent
enzymes.2 The Zn2+-dependent HDACs are closely connected
with control of gene expression and cell cycle progression.3 The
inhibition of HDACs causes histone hyperacetylation and leads
to transcriptional activation of genes such as p21WAF/CIP1, Gadd
45, FAS, and caspase-3, which are associated with growth arrest
and apoptosis in tumor cells.4 Indeed, HDAC inhibitors, such
as trichostatin A (TSA, 1),5 suberoylanilide hydroxamic acid
(SAHA, also known as vorinostat, 2),6 PXD-101 (3),7 and MS-
275 (4)8 (Chart 1), have been reported to inhibit cell growth,
induce terminal differentiation in tumor cells, and prevent the
formation of malignant tumors, and they have been developed
as antitumor agents. Among them, SAHA has recently been
approved by the FDA for treatment of cutaneous T-cell
lymphoma. In addition, there are several lines of evidence
indicating that HDAC inhibitors are effective as therapeutic
agents for other diseases, including inflammation and neuro-
degenerative diseases.9 Moreover, it has recently been reported
that HDAC inhibitors, such as TSA (1), SAHA (2), and valproic
acid, improve the efficiency of induction of pluripotent stem
cells without introduction of the oncogene c-Myc, suggesting
the importance of HDAC inhibitors in regenerative medicine.10
To date, X-ray crystal structures of human HDAC4,12
HDAC7,13 HDAC8,14 and archaebacterial HDAC-like protein
(HDLP)15 have been published. A number of potent and
selective inhibitors have been identified by structure-based drug
design using these crystal structures.11 Recently, the catalytic
mechanism of the deacetylation of acetylated lysine substrates
by HDACs was proposed, based on a density functional theory
QM/MM study of HDLP,16 and this also offers many clues for
the design of HDAC inhibitors. Herein we report the synthesis
and biological activity of boronic acid-based HDAC inhibitors,
which were designed on the basis of the proposed catalytic
mechanism of HDACs.
Chemistry
Compounds 5-26 prepared for this study are shown in Tables
1-3. The routes used for the synthesis of the compounds are
shown in Schemes 1-5. Scheme 1 shows the preparation of
R-amino acid derivatives 5-10. These compounds were syn-
thesized from diethyl aminomalonate 27. Compound 27 was
treated with (Boc)2O to yield N-Boc compound 28. Compound
28 was allowed to react with 5-bromopent-1-ene in the presence
of NaOEt in refluxing EtOH to give C-alkylated compound 29.
Hydrolysis of the ethyl ester of 29 with an equivalent amount
of LiOH gave monocarboxylic acid 30, and subsequent decar-
boxylation reaction afforded R-amino acid derivative 31.
Compound 31 was hydrolyzed with LiOH followed by treatment
with an appropriate aromatic amine in the presence of EDCI
and HOBt to give amides 33-38. The hydroboration reaction
of amides 33-38 with pinacolborane using [Ir(cod)Cl]2 catalyst
and dppm or dppe ligand gave terminal boronic esters 39-44.17
Hydrolysis of the boronic esters 39-44 using NH4OAc and
NaIO4 in acetone-water yielded boronic acids 5-10.18
* To whom correspondence should be addressed. For T.S.: phone and
fax, +81-52-836-3407; e-mail, suzuki@phar.nagoya-cu.ac.jp. For N.M.:
phone and fax, +81-52-836-3407; miyata-n@phar.nagoya-cu.ac.jp.
†
Nagoya City University.
Division of Medicinal Safety Science, National Institute of Health
‡
Sciences.
§
Division of Organic Chemistry, National Institute of Health Sciences.
Japanese Foundation for Cancer Research.
#
a Abbreviations: HDAC, histone deacetylase; TSA, trichostatin A; SAHA,
suberoylanilide hydroxamic acid; HDLP, HDAC-like protein.
Scheme 2 shows the preparation of boronic acids 11-16, in
which the R-amino moiety of compounds 5-10 was removed.
10.1021/jm900125m CCC: $40.75
2009 American Chemical Society
Published on Web 04/14/2009

2910 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Chart 1. Examples of HDAC Inhibitors
Suzuki et al.
Scheme 1a
workers.19 Compound (RS)-31 was subjected to the action of
subtilisin from Bacillus licheniformis in a mixture of DMF and
water to yield (S)-32 as a white solid. The recovered (R)-31
was hydrolyzed with LiOH to afford (R)-32. The synthesis of
(S)- and (R)-18, 20, 21 was accomplished from (S)-32 or (R)-
32 using the procedure described for the synthesis of R-amino
acid derivatives (shown in Schemes 1 and 3). The enantiomeric
excess of (S)- and (R)-18, 20, 21 was determined to be >95%
by chiral column chromatography.
Results and Discussion
Drug Design. In 1999, Finnin et al. reported X-ray crystal
structures of HDLP complexed with inhibitors.15 The enzyme
contains a zinc ion at the bottom of the active site, and the active
center consists of a tyrosine, two aspartic acids, and three
histidines. In 2006, Corminboeuf et al. carried out density
functional theory QM/MM studies on the deacetylation reaction
catalyzed by HDLP.16 The proposed mechanism suggested by
the calculation results is depicted in Figure 1a. In this mecha-
nism, the carbonyl oxygen of the substrate binds the zinc ion
and is located adjacent to a water molecule. The electrophilicity
of the carbonyl carbon is increased by coordination to the zinc
ion, and so the carbonyl carbon is attacked by the water
molecule activated by His 140 and His 141 (HDAC1 number-
ing). This nucleophilic attack results in a tetrahedral transition
state, which is stabilized by a zinc-oxygen interaction and by
hydrogen bonds with Tyr 303 and His 140. In the final step,
proton transfer from His 141 to the nitrogen of the intermediate
triggers scission of the carbon-nitrogen bond to afford the
acetate and lysine products.
In designing selective HDAC inhibitors, we focused on the
proposed deacetylation mechanism. On the basis of this mech-
anism, we designed R-amino acid derivatives A (Figure 1b) in
which the acetamide of the acetylated lysine substrate is replaced
by boronic acid. Because the boron atom of the boronic acids
A has a vacant p-orbital, it could be attacked by a water
molecule in the active site. The generated boronic acid-H2O
“ate” complex could act as a transition state analogue of the
deacetylation of acetylated lysine substrates. That is, the
hydrated boronic acid would bind the zinc ion and form two
hydrogen bonds with Tyr 303 and His 140, which would lead
to HDAC inhibition.
a Reagents and conditions: (a) (Boc)2O, Et3N, THF, room temp, 81%;
(b) NaOEt, 5-bromopent-1-ene, EtOH, reflux, 89%; (c) LiOH ·H2O, EtOH,
H2O, 0 °C, 94% for 32, 99% for 30; (d) toluene, reflux, 94%; (e) R1-NH2,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzo-
triazole hydrate (HOBt ·H2O), DMF, room temp, 67-95%; (f) [Ir(cod)Cl]2,
bis(diphenylphosphino)methane (dppm) or 1,2-bis(diphenylphosphino)ethane
(dppe), pinacolborane, CH2Cl2, room temp, 32-93%; (g) NH4OAc, NaIO4,
acetone, H2O, room temp, 10-92%.
These compounds were synthesized from hept-6-enoic acid 45
using the same synthetic approach as described for compounds
5-10.
Scheme 3 shows the preparation of R-amino acid derivatives
17-24, in which the Boc group of 6 was replaced with various
carbonyl moieties. These compounds were prepared from 40
in three steps: deprotection of the Boc group, condensation with
an appropriate aromatic carboxylic acid, and hydrolysis of the
boronic esters.
Scheme 4 shows the preparation of malonic acid derivatives
25 and 26, in which the R-aminocarbonyl moiety of compounds
18 and 20 was replaced with the corresponding carboxamide.
These compounds were synthesized from tert-butyl ethyl
malonate 66. Treatment of 66 with 5-bromopent-1-ene in the
presence NaH in THF yielded compound 67. The ethyl ester of
67 was hydrolyzed with an equivalent amount of LiOH,
followed by condensation with biphenyl-3-ylamine to give
amide 69. Hydroboration, deprotection of tert-butyl ester using
TFA, condensation with an appropriate aromatic amine, and
hydrolysis of the boronic ester gave the malonic acid derivatives
25 and 26.
Enzyme Assays. In our exploratory study on boronic acid-
based HDAC inhibitors, we initially focused on the R1 group
(Table 1 and Supporting Information Figure S1). We chose
various aromatic rings as the R1 group because we had already
discovered that thiolate compounds bearing aromatic groups
such as phenyl, 3-biphenyl, and 3-quinolinyl potently inhibited
HDACs both in enzyme assays and in cellular assays.11 Boronic
Scheme 5 shows the preparation of optically active R-amino
acid derivatives (S)- and (R)-18, 20, 21. These compounds were
synthesized using the procedure reported by Nishino and co-

Boronic Acid-Based Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2911
Scheme 2a
a Reagents and conditions: (a) R2-NH2, EDCI, HOBt, DMF, room temp, 72-95%; (b) [Ir(cod)Cl]2, dppm or dppe, pinacolborane, CH2Cl2, room temp,
13-74%; (c) NH4OAc, NaIO4, acetone, H2O, room temp, 35-94%.
Scheme 3a
a Reagents and conditions: (a) HCl, AcOEt, CHCl3, room temp, 100%; (b) R3-COOH, EDCI, HOBt, DMF, room temp, 11-100%; (c) R3-COCl, Et3N,
CH2Cl2, N,N-dimethyl-4-aminopyridine (DMAP), room temp, 41%; (d) NH4OAc, NaIO4, acetone, H2O, room temp, 19-82%.
Scheme 4a
a Reagents and conditions: (a) NaH, 5-bromopent-1-ene, THF, reflux, 52%; (b) LiOH ·H2O, tert-BuOH, H2O, 0 °C, 100%; (c) biphenyl-3-ylamine, EDCI,
HOBt, DMF, room temp, 76%; (d) [Ir(cod)Cl]2, dppm, pinacolborane, CH2Cl2, room temp, 70%; (e) TFA, room temp, 100%; (f) (i) oxalyl dichloride, DMF,
CH2Cl2, 0 °C; (ii) R4-NH2, Et3N, CH2Cl2, 0 °C, 29% for 71, 51% for 72; (g) NH4OAc, NaIO4, acetone, H2O, room temp, 39% for 26, 71% for 25.
acids 5-10, in which R1 is various aromatic groups and R2 is
-NHBoc, were initially tested with an in vitro assay using HeLa
nuclear extract with high HDAC activity (Table 1 and Figure
S1). Compounds 5-10 showed HDAC-inhibitory activity at a
concentration of 100 µM, while the 3-biphenyl 6, 3-quinolinyl
7, and benzthiazole 8 compounds were more potent (78-86%
inhibition at 100 µM). Compounds 6, 7, and 8 inhibited HDAC
activity dose-dependently with IC50 values of 12, 16, and 23
µM, respectively; these values are much lower than those of
the other aromatic analogues 5, 9, and 10. We also tested
compounds 11-16, which lack the NH-Boc group of com-
pounds 5-10, and they were found to be much less potent
inhibitors than compounds 5-10 (Table 1 and Figure S1),
suggesting the significance of the R-amino acid moiety.
To find more potent HDAC inhibitors, we next focused on
the tert-butoxy group of compound 6, the most potent inhibitor

2912 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Suzuki et al.
Scheme 5a
a Reagents and conditions: (a) subtilisin, H2O/DMF (1:3), 37 °C, 42% for (R)-31, 50% for (S)-32; (b) LiOH ·H2O, EtOH, H2O, room temp, 100%; (c)
biphenyl-3-ylamine, EDCI, HOBt, DMF, room temp, 56-65%; (d) [Ir(cod)Cl]2, dppm, pinacolborane, CH2Cl2, room temp, 79-82%; (e) HCl, AcOEt,
CHCl3, room temp, 100%; (f) R5-COOH, EDCI, HOBt, DMF, room temp, 64-90%; (g) NH4OAc, NaIO4, acetone, H2O, room temp, 4-87%.
Figure 1. (a) Proposed mechanism for the deacetylation of acetylated lysine substrates. (b) Proposed mechanism of inhibition of HDACs by
boronic acids A.
in Table 1. We initially changed the Boc group of compound 6
to a Cbz group (compound 17) (Table 2 and Supporting
Information Figure S2). Compound 17 exhibited about 3-fold
higher potency than compound 6. We also examined the activity
of compound 18, in which the benzyloxy group of compound
17 was replaced with a simple benzene ring, and the activity of
18 (IC50 ) 2.0 µM) was about 2-fold higher than that of
compound 17. By the mimicking of the partial structure of TSA
(1), we introduced the dimethylamino group at the 4-position
of the phenyl ring of compound 18, but compound 19 displayed
a 6.5-fold decrease in potency compared to the parent compound
18. Next, we converted the phenyl ring of compound 18 to
various heteroaryl rings. The 2-indole 23 and 2-quinoline 24
compounds exhibited HDAC-inhibitory activity less potent than
that of the phenyl compound 18, whereas the 3-pyridine 20,
5-thiazole 21, and 4-thiazole 22 derivatives showed potencies
similar to or greater than that of the phenyl compound 18 (IC50
values: 20 ) 2.0 µM, 21 ) 1.4 µM, 22 ) 4.7 µM), highlighting
the importance of small ring size.
To confirm the importance of R-amino acid structure, we
examined the activity of malonic acid derivatives 25 and 26 in
which the R-aminocarbonyl moiety of compounds 18 and 20 is
replaced by the corresponding carboxamide (Table 2 and Figure
S2). Compounds 25 and 26 showed about 15- and 25-fold
decreases in potency compared with the parent compounds 18
and 20, respectively.

Boronic Acid-Based Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2913
Table 2. HDAC Inhibitory Activity of 17-26a
Table 1. HDAC Inhibitory Activity of Boronic Acids 5-16a
a Values are the mean of at least three experiments.
Table 3. HDAC Inhibitory Activity of (S)- and (R)-18, 20, 21a
IC50 (µM)
class I
HDAC
(nuclear extract)
class II,
HDAC6
a Values are the mean of at least three experiments. IC50 of SAHA (2)
is 0.28 µM.
compd
HDAC1 HDAC2 HDAC8
2
0.28
1.6
17
1.4
15
0.35
>10b
>100
2.8
>100
2.2
0.25
3.6
>100
0.83
99
0.53
>100
1.7
0.028
1.1
24
0.32
14
0.11
5.6
(S)-18
(R)-18
(S)-20
(R)-20
(S)-21
(R)-21
>100
>100
23
>100
6.6
Next we examined the influence of optical activity. Com-
pounds (R)-18, 20, 21 and (S)-18, 20, 21 were prepared and
evaluated for their inhibitory effects on HDAC1, HDAC2,
HDAC6, and HDAC8, as well as total HDACs from nuclear
extracts. As shown in Table 3 and Supporting Information
Figure S3, in all cases, (S)-18, 20, and 21 were at least 10-fold
more active than (R)-18, 20, and 21, respectively. Since the
stereochemistry of (S)-18, 20, and 21 is the same as that of
natural lysine, these results suggest that these boronic acids act
in the active site of HDACs. Among (S)-18, 20, and 21, (S)-21
was found to be the most potent inhibitor. Compound (S)-21
inhibited all HDACs with IC50 values in the micromolar or
submicromolar range. Although (S)-21 was less potent than
SAHA (2), the isoform inhibition profile of (S)-21 was similar
to that of 2, which is the only HDAC inhibitor currently used
in clinical practice.
0.92
24
>100
>100
a Values are the mean of at least three experiments. b 38% inhibition at
10 µM.
Table 4. Growth-Inhibitory Activity on MKN45 Cells and GI50/IC50
Values of SAHA (2) and (S)-18, 20, 21a
GI50/IC50
compd
GI50 (µM)
HDAC
HDAC1
HDAC2
2
7.3
9.3
5.5
3.5
26
21
29
(S)-18
(S)-20
(S)-21
5.8
3.9
3.8
<0.93
2.6
6.6
6.6
2.0
1.6
a Values are the mean of two separate experiments.
Cellular Assays. To examine the effectiveness of boronic
acid-based HDAC inhibitors as anticancer drugs and tools for
biological research, compounds (S)-18, 20, and 21, as well as
SAHA (2), were tested in a cancer cell growth inhibition assay.
For initial screening, we used stomach cancer MKN45 cells
because HDAC inhibitors have been reported to inhibit the cell
growth of MKN45 cells.20 The results are summarized in Ta-
ble 4.
SAHA (2) and its derivatives have been reported to show
potent HDAC-inhibitory activity in enzyme assays and cancer
cell growth inhibition assays.21 However, a relatively large shift
in potency between enzyme assay and cellular assay (GI50/IC50,
abbreviated as potency shift in this paper) was observed with
those hydroxamate HDAC inhibitors.21 Indeed, as shown in
Table 4, SAHA (2) exhibited a large potency shift in the cancer

2914 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Suzuki et al.
Table 5. Growth Inhibition of Various Cancer Cells Using SAHA (2)
and (S)-21a
exerted potent growth inhibition against various human cancer
cells, with GI50 values ranging from 0.73 to 9.1 µM, and these
inhibitory activities were similar to those of SAHA (2) (average
GI50 of (S)-21 is 4.0 µM, that of SAHA is 6.0 µM). In addition,
compound (R)-21 did not show strong activity against these
cancer cell lines (Table S1 and Figure S4 in Supporting
Information) with GI50s values ranging from 13 to 21 µM,
suggesting the responsibility of HDAC inhibition for cancer cell
growth repression.
GI50 (µM)
cell
2
(S)-21
HBC-5
breast cancer
central nervous system
colon cancer
lung cancer
melanoma
ovarian cancer
renal cancer
stomach cancer
prostate cancer
17
16
6.5
9.1
0.73
2.4
1.3
2.2
3.2
3.5
6.7
SNB-78
HCT116
DMS114
LOX-IMVI
SK-OV-3
RXF-631 L
MKN45
PC-3
0.58
2.9
1.3
2.5
2.0
7.3
4.6
Next, we performed Western blot analysis to examine the
HDAC-inhibitory effects of boronic acid derivatives in cells.
Since nuclear HDACs such as HDAC 1 and HDAC2 catalyze
the deacetylation of histones,3d the acetylation level of histone
H4 in human colon cancer HCT116 cells was analyzed after
treatment of the cells with compounds (S)-18, 20, and 21. As
can be seen in Figure 2, the level of acetylated histone H4 was
elevated dose-dependently. The level of acetylated R-tubulin
was also investigated because (S)-18, 20, and 21 displayed potent
inhibition of HDAC6, which is reported to catalyze the
deacetylation of R-tubulin.23 As expected, compounds (S)-18,
20, and 21 caused a dose-dependent increase in acetylation of
R-tubulin. These results suggest that the cancer cell growth
inhibition by boronic acids (S)-18, 20, and 21 was the result of
HDAC inhibition.
mean
6.0
4.0
a Values are the mean of two separate experiments.
cell growth inhibition assay using MKN45 cells.22 The reason
for the large potency shift of SAHA (2) is unclear, but it seems
reasonable to assume that it is at least partly due to poor
membrane permeability resulting from the highly polar character
of hydroxamic acid. Using Advanced Chemistry Development
(ACD/Laboratories) software, version 8.14 for Solaris, the log D
(pH 7) values of methylhydroxamic acid (CH3CONHOH) and
methylboronic acid (CH3B(OH)2) were calculated to be -1.59
and 1.0, respectively. The calculation results suggest that boronic
acid is more lipophilic than hydroxamic acid under physiological
conditions, and boronic acid derivatives might permeate through
the cell membrane more efficiently than hydroxamates; there-
fore, they might not show such large potency shifts as
hydroxamates like SAHA (2). We then evaluated the growth-
inhibitory activity of (S)-18, 20, and 21 on MKN45 cells. As
we expected, (S)-18, 20, and 21 inhibited the growth of MKN45
cells and showed smaller potency shift values than SAHA (2).
Among (S)-18, 20, and 21, compound (S)-21 showed the highest
activity, being more potent than SAHA (2). Next, we evaluated
growth inhibition by SAHA (2) and compound (S)-21 against
nine human cancer cell lines (Table 5). Compound (S)-21
Molecular Modeling. Since the results of the enzyme assays
(Table 3) suggested that boronic acids act within the active
center of HDACs, we studied the binding mode of hydrated
(S)-21 within this site. We calculated the low energy conforma-
tion of the hydrated (S)-21 complex docked in a model based
on the crystal structure of HDAC8 (PDB code 1T67) using
Macromodel 9.6 software (Figure 3). An inspection of the
complex shows that one of the oxygen atoms of the hydrated
boronic acid can coordinate to the zinc ion (distance between
oxygen and zinc, 2.04 Å) (Figure 3, left). In addition, the
hydrogen of the OH group of Tyr 306 is located 1.95 and 2.20
Å from the two oxygen atoms of the hydrated boronic acid,
Figure 2. Western blot detection of acetylated R-tubulin and histone H4 levels in HCT116 cells after 8 h treatment with (S)-18, 20, 21 and
reference compound 2.
Figure 3. View of the conformation of (S)-21 (ball-and-stick) docked in the HDAC8 catalytic core. Residues within 5 Å from the zinc ion are
displayed in the tube graphic (left), and the surface of the enzyme is displayed in gray (right).

Boronic Acid-Based Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2915
Tokyo Kasei Kogyo, Wako Pure Chemical Industries, and Kanto
Kagaku and used without purification. Flash column chromatog-
raphy was performed using silica gel 60 (particle size 0.046-0.063
mm) supplied by Merck.
which suggests that Tyr 306 can form two hydrogen bonds with
the hydrated boronic acid. Moreover, a short distance (2.22 Å)
between one of the hydrogens of the hydrated boronic acid and
the nitrogen of His 142 was also observed. This indicates that
hydrated boronic acids inhibit HDACs by interacting with the
zinc ion, Tyr residue, and His residue in the active center. On
the surface of HDAC8, the biphenyl ring is located in the
hydrophobic region formed by the benzene rings of Tyr 100
and Phe 152 and the alkyl chain of Lys 33, and the thiazole
ring lies in the hydrophobic area formed by the benzene ring
of Tyr100 and the methylene groups of Asp 101, Gly 151, and
Phe 208 (Figure 3, right). It is also suggested that two hydrogen
bonds can be formed between the two NH groups and the
carboxylate anion of Asp 101 (the distances are 1.72 and 1.74
Å). The observed interactions between (S)-21 and HDAC8 on
the surface of the enzyme suggest the importance of the amino
acid structure and the (S)-configuration of the inhibitor.
6-tert-Butoxycarbonylamino-7-oxo-7-phenylaminoheptylbo-
ronic Acid (5). Step 1: Preparation of Diethyl 2-tert-Butoxy-
carbonylaminomalonate (28). To a suspension of diethyl ami-
nomalonate (27; 10.0 g, 47.2 mmol) and Et3N (32.9 mL, 236 mmol)
in THF (20 mL) was added a solution of (Boc)2O (20.6 g, 94.4
mmol) in THF (10 mL), and the solution was stirred overnight at
room temperature. 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 and concentration in
vacuo and purification by silica gel flash column chromatography
(AcOEt/n-hexane ) 1/5) gave 10.5 g (81%) of 28 as a colorless
oil: 1H NMR (CDCl3, 500 MHz, δ, ppm) 5.56 (1H, d, J ) 6.7 Hz),
4.95 (1H, d, J ) 7.6 Hz), 4.27 (4H, m), 1.45 (9H, s), 1.30 (6H, t,
J ) 7.3 Hz).
Step 2: Preparation of Diethyl 2-tert-Butoxycarbonylamino-
2-(penten-4-yl)malonate (29). To a solution of 28 (10.2 g, 37.2
mmol) obtained above in EtOH (15 mL) was added NaOEt (2.78
g, 40.9 mmol) under Ar gas at 0 °C. The reaction mixture was
stirred at 0 °C for 7 min. After that, to the solution was added
5-bromopent-1-ene (8.32 g, 55.8 mmol), and the mixture was
refluxed for 10 h. It was then poured into water and extracted with
AcOEt. The AcOEt layer was washed with water and brine and
dried over Na2SO4. Filtration and concentration in vacuo and
purification by silica gel flash column chromatography (AcOEt/n-
hexane ) 1/6) gave 11.3 g (89%) of 29 as a colorless oil: 1H NMR
(CDCl3, 500 MHz, δ, ppm) 5.94 (1H, broad s), 5.76 (1H, ddt, J )
17, 10, 6.4 Hz), 5.00 (1H, dd, J ) 15, 1.8 Hz), 4.95 (1H, d, J ) 10
Hz), 4.26-4.18 (4H, m), 2.28 (2H, m), 2.07 (2H, q, J ) 7.3 Hz),
1.43 (9H, s), 1.30-1.19 (8H, m).
Conclusion
On the basis of the proposed catalytic mechanism for the
deacetylation of acetylated lysine substrates by HDACs, we
designed a series of boronic acids bearing an R-amino acid
moiety as candidate mechanism-based HDAC inhibitors. Among
the synthesized compounds, compounds (S)-18, 20, and 21
displayed potent HDAC-inhibitory activities, suggesting that
these boronic acids act in the active site of HDACs. Compounds
(S)-18, 20, and 21 also showed cancer cell growth-inhibitory
activities as potent as SAHA (2). Intracellular HDAC inhibition
by compounds (S)-18, 20, and 21 was confirmed by Western
blot analysis of acetylated histone H4 and acetylated R-tubulin.
A molecular modeling study of the HDAC8/(S)-21 complex
suggested that the hydrated boronic acid interacts with zinc ion,
Tyr residue, and His residue in the active site of HDACs.
Thus, we have identified a novel lead structure from which
it should be possible to develop more potent HDAC inhibitors.
Although boronic acids have been used as inhibitors for various
hydrolytic enzymes such as serine protease,24 arginase,25 and
proteasome,26 this is the first report of HDAC inhibitors
containing a boronic acid moiety. The results obtained in this
study suggest that boronic acid-based inhibitors of HDACs have
considerable potential for the development of novel therapeutic
agents and tools for biological research.
Step 3: Preparation of 2-tert-Butoxycarbonylamino-2-(ethoxy-
carbonyl)hept-6-enoic Acid (30). To a solution of 29 (11.3 g, 33.0
mmol) obtained above in EtOH/H2O (20 mL/10 mL) was added
LiOH·H2O (1.52 g, 36.3 mmol), and the solution was stirred
overnight at 0 °C. The reaction mixture was neutralized with 10%
citric acid and extracted with AcOEt. The AcOEt layer was washed
with brine and dried over Na2SO4. Filtration and concentration in
1
vacuo gave 10.3 g (99%) of 30 as a yellow oil: H NMR (CDCl3,
500 MHz, δ, ppm) 5.80-5.71 (2H, m), 5.01 (1H, dd, J ) 17, 1.5
Hz), 4.97 (1H, dd, J ) 10, 1.8 Hz), 4.31-4.18 (2H, m), 2.22 (2H,
m), 2.07 (2H, m), 1.44 (9H, s), 1.39-1.22 (5H, m).
Step 4: Preparation of Ethyl 2-tert-Butoxycarbonylamino-
hept-6-enoate (31). A solution of 30 (10.3 g, 32.6 mmol) obtained
above in toluene (40 mL) was refluxed for 9 h. The reaction mixture
was concentrated and purified by flash column chromatography
(AcOEt/n-hexane ) 1/6) to give 8.51 g (94%) of 31 as a yellow
Experimental Section
1
oil: H NMR (CDCl3, 500 MHz, δ, ppm) 5.77 (1H, ddt, J ) 17,
Chemistry. Melting points were determined using a Yanagimoto
micro melting point apparatus or a Bu¨chi 545 melting point
apparatus and were left uncorrected. Proton nuclear magnetic
resonance spectra (1H NMR) and carbon nuclear magnetic resonance
spectra (13C NMR) were recorded with a JEOL JNM-LA500, JEOL
JNM-A500, or Bruker Avance 600 spectrometer in solvents 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, which
indicates >95% purity of the tested compounds. Fast atom
bombardment (FAB) mass spectra were recorded on a JEOL JMS-
SX102A mass spectrometer. In positive-mode MS analysis using
3-nitrobenzyl alcohol (NBA) as a matrix, boronic acids are esterified
with NBA and the molecular weight detected corresponds to (M
+ 2NBA - 2H2O + H)+ or (M + 2NBA - 2H2O)+. In negative-
mode analysis using glycerol (Gly) as a matrix, the molecular
weight detected corresponds to (M + Gly - 2H2O - H)-. HPLC
was performed with a Shimazu instrument equipped with a
CHIRALPAK IA column (4.6 mm × 250 mm, Daicel Chemical
Industries), and the samples eluted at 1 mL/min with ethanol and
n-hexane. Reagents and solvents were purchased from Aldrich,
10, 6.7 Hz), 5.04-4.94 (3H, m), 4.28 (1H, m), 4.19 (2H, m), 2.07
(2H, m), 1.81 (1H, m,), 1.63 (1H, m), 1.53-1.37 (11H, m), 1.28
(3H, t, J ) 7.3 Hz).
Step 5: Preparation of 2-tert-Butoxycarbonylaminohept-6-
enoic Acid (32). To a solution of 31 (8.34 g, 30.8 mmol) obtained
above in EtOH/H2O (20 mL/10 mL) was added LiOH ·H2O (1.25
g, 29.8 mmol), and the solution was stirred overnight at 0 °C. The
reaction mixture was neutralized with 10% citric acid and extracted
with AcOEt. The AcOEt layer was washed with brine and dried
over Na2SO4. Filtration and concentration in vacuo gave 7.01 g
(94%) of 32 as a white solid: 1H NMR (CDCl3, 500 MHz, δ, ppm)
5.78 (1H, ddt, J ) 17, 10, 6.7 Hz), 5.06-4.94 (3H, m), 4.32 (1H,
m), 2.09 (2H, m), 1.87 (1H, m), 1.68 (1H, m), 1.57-1.37 (11H,
m).
Step 6: Preparation of tert-Butyl 1-(Phenylaminocarbonyl)
hex-5-en-1-ylcarbamate (33). To a solution of 32 (1.00 g, 4.11
mmol) and aniline (560 µL, 6.17 mmol) in DMF (15 mL) were
added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-
ride (EDCI; 1.18 g, 6.17 mmol) and 1-hydroxy-1H-benzotriazole
monohydrate (HOBt·H2O; 834 mg, 6.17 mmol), and the mixture
was stirred overnight at room temperature. The reaction mixture

2916 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Suzuki et al.
was poured into water and extracted with AcOEt. The AcOEt layer
was washed with water and brine and dried over Na2SO4. Filtration
and concentration in vacuo and purification by silica gel flash
column chromatography (AcOEt/n-hexane ) 1/7) gave 875 mg
(67%) of 33 as a white solid: 1H NMR (CDCl3, 500 MHz, δ, ppm)
8.26 (1H, broad s), 7.51 (2H, d, J ) 7.6 Hz), 7.31 (2H, t, J ) 7.6
Hz), 7.10 (1H, t, J ) 7.3 Hz), 5.77 (1H, ddt, J ) 17, 10, 6.7 Hz),
5.08 (1H, broad s), 5.01 (1H, dd, J ) 17, 1.8 Hz), 4.96 (1H, d, J
) 10 Hz), 4.20 (1H, m), 2.10 (2H, m), 1.95 (1H, m), 1.64 (1H,
m), 1.57-1.40 (11H, m).
7-(Benzothiazol-2-ylamino)-6-tert-butoxycarbonylamino-7-
oxoheptylboronic Acid (8). Yield 27% (three steps) (615 mg);
colorless crystals; mp 128-130 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 12.42 (1H, s), 7.98 (1H, d, J ) 7.9 Hz), 7.75 (1H, d, J )
7.9 Hz), 7.44 (1H, t, J ) 7.6 Hz), 7.36 (2H, s), 7.31 (1H, t, J ) 7.6
Hz), 7.22 (1H, d, J ) 7.3 Hz), 4.21 (1H, q, J ) 7.0 Hz), 1.71-1.54
(2H, m), 1.46-1.16 (15H, m), 0.56 (2H, t, J ) 7.6 Hz); 13C NMR
(DMSO-d6, 500 MHz, δ, ppm) 172.76, 157.84, 155.56, 148.56,
131.46, 126.14, 123.57, 121.72, 120.53, 78.28, 54.59, 31.72, 31.39,
28.20, 27.89, 25.50, 24.10; MS (FAB) m/z 692 (M + 2NBA -
2H2O+H)+, 476(M+Gly-2H2O-H)-. Anal. (C19H28BN3O5S·1/
2H2O) C, H, N.
Step 7: Preparation of tert-Butyl 1-(Phenylaminocarbonyl)-
6-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)hexylcarbam-
ate (39). A solution of [Ir(cod)Cl]2 (88.7 mg, 0.132 mmol) and
bis(diphenylphosphino)methane (dppm; 100 mg, 0.264 mmol) in
CH2Cl2 (10 mL) was stirred at room temperature for 5 min under
Ar gas. After that, to the solution was added pinacolborane (300
µL, 2.00 mmol) and 33 (420 mg, 1.32 mmol) obtained above in
CH2Cl2 (10 mL) in that order, and the mixture was stirred at room
temperature for 2 days. The reaction mixture was concentrated and
purified by silica gel flash column chromatography (AcOEt/n-
6-tert-Butoxycarbonylamino-7-oxo-7-(3-phenoxyphenylami-
no)heptylboronic Acid (9). Yield 15% (three steps) (142 mg);
colorless crystals; mp 89-91 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.00 (1H, s), 7.40 (2H, t, J ) 7.6 Hz), 7.36-7.26 (5H,
m), 7.15 (1H, t, J ) 7.3 Hz), 7.03 (2H, d, J ) 7.9 Hz), 6.97 (1H,
d, J ) 7.6 Hz), 6.70 (1H, d, J ) 7.9 Hz), 3.98 (1H, m), 1.62-1.46
(2H, m), 1.44-1.14 (15H, m), 0.55 (2H, t, J ) 7.6 Hz); 13C NMR
(DMSO-d6, 500 MHz, δ, ppm) 171.65, 157.07, 156.35, 155.45,
140.55, 130.04, 123.59, 118.92, 113.92, 113.14, 108.90, 77.98,
55.19, 31.83, 28.18, 25.57, 24.14; MS (FAB) m/z 727 (M + 2NBA
- 2H2O + H)+, 511 (M + Gly - 2H2O - H)-. Anal. (C24H33BN2-
O6) C, H, N.
1
hexane ) 1/5) to give 397 mg (67%) of 39 as a white solid: H
NMR (CDCl3, 500 MHz, δ, ppm) 8.15 (1H, broad s), 7.51 (2H, d,
J ) 7.6 Hz), 7.32 (2H, t, J ) 7.9 Hz), 7.10 (1H, t, J ) 7.6 Hz),
4.98 (1H, broad s), 4.13 (1H, m), 1.92 (1H, m), 1.64 (1H, m),
1.50-1.30 (15H, m), 1.24 (12H, s), 0.77 (2H, t, J ) 7.3 Hz).
Step 8: Preparation of 6-tert-Butoxycarbonylamino-7-oxo-7-
phenylaminoheptylboronic Acid (5). To a solution of 39 (213
mg, 0.478 mmol) in acetone/H2O (6 mL/3 mL) were added NaIO4
(307 mg, 1.43 mmol) and NH4OAc (110 mg, 1.43 mmol), and the
suspension was stirred at room temperature for 2 days. 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 and concentration in vacuo and purification by
silica gel flash column chromatography (AcOEt/n-hexane ) 1/1)
gave 161 mg (92%) of 5 as a white solid. The solid was
recrystallized from AcOEt to give 20.5 mg of 5 as colorless crystals:
6-tert-Butoxycarbonylamino-7-oxo-7-(4-phenoxyphenylami-
no)heptylboronic Acid (10). Yield 9% (three steps) (81 mg);
colorless crystals; mp 94-96 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 9.96 (1H, s), 7.61 (2H, d, J ) 8.8 Hz), 7.39-7.30 (4H,
m), 7.10 (1H, t, J ) 7.3 Hz), 7.03-6.90 (5H, m), 4.02 (1H, q, J )
6.9 Hz), 1.68-1.50 (2H, m), 1.46-1.14 (15H, m), 0.56 (2H, t, J
) 7.6 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.32,
157.44, 155.47, 151.63, 134.98, 129.95, 122.92, 120.87, 119.53,
117.78, 78.00, 55.15, 31.99, 31.88, 28.22, 28.04, 25.58, 24.18; MS
(FAB) m/z 726 (M + 2NBA - 2H2O)+, 511 (M + Gly - 2H2O -
H)-. Anal. (C24H33BN2O6) C, H, N.
1
mp 114-117 °C; H NMR (DMSO-d6, 500 MHz, δ, ppm) 9.92
Compounds 11-16 were prepared from hept-6-enoic acid (45)
and an appropriate amine using the procedure described for 5 (steps
6-8). For the synthesis of 54 and 55, dppe was used instead of
dppm (step 7).
(1H, s), 7.59 (2H, d, J ) 7.6 Hz) 7.34 (2H, s), 7.30 (2H, t, J ) 7.6
Hz), 7.04 (1H, t, J ) 7.3 Hz), 6.98 (1H, d, J ) 7.6 Hz), 4.02 (1H,
m), 1.66-1.50 (2H, m), 1.38-1.16 (15H, m), 0.55 (2H, t, J ) 7.6
Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.43, 155.43,
139.00, 128.66, 123.14, 119.15, 77.95, 55.15, 32.37, 31.97, 31.85,
28.18, 25.55, 24.14; MS (FAB) m/z 635 (M + 2NBA - 2H2O +
H)+, 419 (M + Gly - 2H2O - H)-. Anal. (C18H29BN2O5) C,
H, N.
7-Oxo-7-phenylaminoheptylboronic Acid (11). Yield 42%
(three steps) (178 mg); colorless crystals; mp 100-102 °C; 1H NMR
(DMSO-d6, 500 MHz, δ, ppm) 9.84 (1H, s), 7.58 (2H, d, J ) 7.6
Hz), 7.36 (2H, s), 7.28 (2H, t, J ) 7.6 Hz), 7.01 (1H, t, J ) 7.3
Hz), 2.28 (2H, t, J ) 7.6 Hz), 1.57 (2H, quintet, J ) 7.6 Hz),
1.35-1.20 (6H, m), 0.57 (2H, t, J ) 7.6 Hz); 13C NMR (DMSO-
d6, 500 MHz, δ, ppm) 171.32, 139.33, 128.62, 122.90, 119.03,
36.49, 31.90, 28.64, 25.16, 24.11; MS (FAB) m/z 520 (M + 2NBA
- 2H2O + H)+, 304 (M + Gly - 2H2O - H)-. Anal. (C13H20-
BNO3) C, H, N.
7-(Biphenyl-3-yl)amino-7-oxoheptylboronic Acid (12). Yield
28% (three steps) (184 mg); colorless crystals; mp 105-107 °C;
1H NMR (DMSO-d6, 500 MHz, δ, ppm) 9.94 (1H, s), 7.92 (1H,
s), 7.60 (2H, d, J ) 7.3 Hz), 7.57 (1H, d, J ) 8.8 Hz), 7.47 (2H,
t, J ) 7.9 Hz), 7.39-7.30 (5H, m), 2.31 (2H, t, J ) 7.6 Hz), 1.59
(2H, quintet, J ) 7.0 Hz), 1.37-1.21 (6H, m), 0.58 (2H, t, J ) 7.9
Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.36, 140.59,
140.13, 139.82, 129.15, 128.84, 127.41, 126.50, 121.22, 117.96,
117.22, 36.45, 31.81, 28.56, 25.05, 24.02; MS (FAB) m/z 596 (M
+ 2NBA - 2H2O + H)+, 380 (M + Gly - 2H2O - H)-. Anal.
(C19H24BNO3) C, H, N.
7-Oxo-7-(quinolin-3-yl)aminoheptylboronic Acid (13). Yield
65% (three steps) (72 mg); colorless crystals; mp 146-148 °C; 1H
NMR (DMSO-d6, 500 MHz, δ, ppm) 10.35 (1H, s), 8.89 (1H, d, J
) 2.4 Hz), 8.71 (1H, d, J ) 2.4 Hz), 7.94 (1H, d, J ) 8.2 Hz),
7.90 (1H, d, J ) 7.3 Hz), 7.63 (1H, t, J ) 7.0 Hz), 7.56 (1H, t, J
) 7.0 Hz), 7.35 (2H, s), 2.39 (2H, t, J ) 7.6 Hz), 1.63 (2H, quintet,
J ) 7.3 Hz), 1.38-1.21 (6H, m), 0.58 (2H, t, J ) 7.6 Hz); 13C
NMR (DMSO-d6, 500 MHz, δ, ppm) 172.11, 144.38, 143.97,
132.88, 128.41, 127.78, 127.52, 126.90, 121.64, 119, 36.29, 31.79,
Compounds 6-10 were prepared from 32 and an appropriate
amine using the procedure described for 5. For the synthesis of 41
and 42, dppe was used instead of dppm (step 7).
7-(Biphenyl-3-ylamino)-6-tert-butoxycarbonylamino-7-oxo-
heptylboronic Acid (6). Yield 41% (three steps) (223 mg); colorless
crystals; mp 140-143 °C; 1H NMR (DMSO-d6, 500 MHz, δ, ppm)
10.03 (1H, s), 7.91 (1H, s), 7.61 (3H, m), 7.48 (2H, t, J ) 7.9 Hz),
7.41-7.33 (5H, m), 7.00 (1H, d, J ) 7.0 Hz), 4.05 (1H, q, J ) 7.2
Hz), 1.70-1.50 (2H, m), 1.44-1.18 (15H, m), 0.56 (2H, t, J )
7.3 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.85, 155.67,
140.93, 140.24, 139.65, 129.54, 129.14, 127.75, 126.75, 121.85,
118.45, 117.67, 78.30, 60.81, 55.41, 32.46, 32.10, 31.97, 28.34,
25.68, 25.34, 2.28; MS (FAB) m/z 711 (M + 2NBA - 2H2O)+,
495 (M + Gly - 2H2O - H)-. Anal. (C24H33BN2O5) C, H, N.
6-tert-Butoxycarbonylamino-7-oxo-7-(quinolin-3-ylamino)hep-
tylboronic Acid (7). Yield 31% (three steps) (632 mg); colorless
crystals; mp 125-127 °C; 1H NMR (DMSO-d6, 500 MHz, δ, ppm)
10.45 (1H, s), 8.93 (1H, s), 8.71 (1H, s), 7.96 (1H, d, J ) 8.5 Hz),
7.93 (1H, d, J ) 7.9 Hz), 7.65 (1H, t, J ) 7.3 Hz), 7.58 (1H, t, J
) 7.0 Hz), 7.36 (2H, s), 7.12 (1H, d, J ) 7.6 Hz), 4.11 (1H, m),
1.75-1.53 (2H, m), 1.48-1.19 (15H, m), 0.57 (2H, t, J ) 7.6 Hz);
13C NMR (DMSO-d6, 500 MHz, δ, ppm) 172.35, 155.46, 144.43,
144.06, 132.60, 128.40, 127.75, 127.70, 127.57, 127.00, 122.11,
78.03, 55.17, 31.75, 31.71, 28.12, 25.51, 24.06; MS (FAB) m/z
686 (M + 2NBA - 2H2O + H)+, 470 (M + Gly - 2H2O - H)-.
Anal. (C21H30BN3O5 ·1/2H2O) C, H, N.

Boronic Acid-Based Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2917
28.54, 24.95, 24.01; MS (FAB) m/z 571 (M + 2NBA - 2H2O +
H)+, 355 (M + Gly - 2H2O - H)-. Anal. (C16H21BN2O3 ·H2O)
C, H, N.
(2H, m), 1.46-1.19 (6H, m), 0.56 (2H, t, J ) 7.6 Hz); 13C NMR
(DMSO-d6, 500 MHz, δ, ppm) 171.36, 156.09, 140.74, 140.12,
139.52, 137.01, 129.34, 128.95, 128.32, 127.77, 127.68, 127.56,
126.60, 121.66, 118.23, 117.49, 79.14, 65.39, 55.59, 31.90, 31.82,
25.57, 24.59, 24.15; MS (FAB) m/z 745 (M + 2NBA - 2H2O +
H)+, 529 (M + Gly - 2H2O - H)-. Anal. (C27H31BN2O5) C,
H, N.
7-(Benzothiazol-2-yl)amino-7-oxoheptylboronic Acid (14).
Yield 7% (three steps) (28 mg); colorless crystals; mp 121-124
1
°C; H NMR (DMSO-d6, 500 MHz, δ, ppm) 12.29 (1H, s), 7.96
(1H, d, J ) 7.6 Hz), 7.73 (1H, d, J ) 7.6 Hz), 7.43(1H, t, J ) 7.3
Hz), 7.35 (2H, s), 7.30 (1H, t, J ) 7.9 Hz), 2.48 (2H, t, J ) 7.6
Hz), 1.61 (2H, quintet, J ) 7.3 Hz), 1.37-1.20 (6H, m), 0.57 (2H,
t, J ) 7.9 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 172.24,
157.80, 148.44, 131.32, 125.93, 123.31, 121.53, 120.33, 35.10,
31.69, 28.40, 24.48, 23.95; MS (FAB) m/z 577 (M + 2NBA -
2H2O + H)+, 361 (M + Gly - 2H2O - H)-. Anal. (C14H19BN2O3S)
C, H, N.
6-(Benzenecarbonylamino)-7-(biphenyl-3-ylamino)-7-oxohep-
tylboronic Acid (18). Step 1: Preparation of 1-(Biphenyl-3-
ylaminocarbamoyl)-6-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-
2-yl)hexylbenzamide (59). To a solution of 2-amino-N-(biphenyl-
3-yl)-7-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)heptanamide
(240 mg, 0.568 mmol) obtained above and benzoic acid (104 mg,
0.852 mmol) in DMF (10 mL) were added EDCI (163 mg, 0.852
mmol) and HOBt ·H2O (130 mg, 0.852 mmol), and the mixture
was stirred overnight at room temperature. The reaction mixture
was poured into water and extracted with AcOEt. The AcOEt
layer was washed with water and brine and dried over Na2SO4.
Filtration and concentration in vacuo and purification by silica gel
flash column chromatography (AcOEt/n-hexane ) 1/3) gave 192
7-Oxo-7-(3-phenoxyphenyl)aminoheptylboronic Acid (15).
Yield 21% (three steps) (84 mg); colorless crystals; mp 85-87 °C;
1H NMR (DMSO-d6, 500 MHz, δ, ppm) 9.91 (1H, s), 7.39 (2H, t,
J ) 7.6 Hz), 7.36-7.26 (5H, m), 7.15 (1H, t, J ) 7.3 Hz), 7.02
(2H, d, J ) 8.5 Hz), 6.67 (1H, d, J ) 7.6 Hz), 2.25 (2H, t, J ) 7.3
Hz), 1.53 (2H, quintet, J ) 7.6 Hz), 1.34-1.18 (6H, m), 0.56 (2H,
t, J ) 7.6 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.43,
156.96, 156.43, 140.85, 129.99, 129.93, 123.48, 118.80, 113.78,
112.91, 108.85, 36.47, 31.83, 28.59, 25.00, 24.06; MS (FAB) m/z
612 (M + 2NBA - 2H2O + H)+, 396 (M + Gly - 2H2O - H)-.
Anal. (C19H24BNO4) C, H, N.
1
mg (64%) of 59 as a white solid: H NMR (CDCl3, 500 MHz, δ,
ppm) 8.73 (1H, s), 7.84-7.80 (3H, m), 7.59-7.51 (4H, m),
7.47-7.31 (7H, m), 6.81 (1H, d, J ) 8.2 Hz), 4.83 (1H, q, J ) 7.3
Hz), 2.08 (1H, m), 1.83 (1H, m), 1.51-1.31 (6H, m), 1.22 (12H,
s), 0.76 (2H, t, J ) 7.9 Hz).
7-Oxo-7-(4-phenoxyphenyl)aminoheptylboronic Acid (16).
Yield 37% (three steps) (270 mg); colorless crystals; mp 132 °C;
1H NMR (DMSO-d6, 500 MHz, δ, ppm) 9.86 (1H, s), 7.60 (2H, d,
J ) 8.8 Hz), 7.36 (2H, t, J ) 7.9 Hz), 7.33 (2H, s),7.09 (1H, d, J
) 7.3 Hz), 6.99-6.92 (4H, m), 2.27 (2H, t, J ) 7.6 Hz), 1.57 (2H,
quintet, J ) 7.3 Hz), 1.36-1.20 (6H, m), 0.57 (2H, t, J ) 7.9 Hz);
13C NMR (DMSO-d6, 600 MHz, δ, ppm) 171.00, 157.36, 151.31,
135.25, 129.82, 122.78, 120.60, 119.35, 117.65, 79.06, 36.33, 31.80,
28,56, 25.11, 24.03; MS (FAB) m/z 612 (M + 2NBA - 2H2O +
H)+, 396 (M + Gly - 2H2O - H)-. Anal. (C19H24BNO4) C, H, N.
6-(Benzyloxycarbonylamino)-7-(biphenyl-3-ylamino)-7-oxo-
heptylboronic Acid (17). Steps 1 and 2: Preparation of Benzyl
1-(Biphenyl-3-ylaminocarbonyl)-6-(4,4,5,5,-tetramethyl-1,3,2-di-
oxaborolan-2-yl)hexylcarbamate (58). To a solution of 40 (600
mg, 1.15 mmol) in CHCl3 (3 mL) was added 4 N HCl/AcOEt (3
mL), and the mixture was stirred at room temperature for 2 h. The
reaction mixture was poured into 2 N aqueous NaOH and extracted
with CH2Cl2. The CH2Cl2 layer was washed with brine and dried
over Na2SO4. Filtration and concentration in vacuo gave 498 mg
(100%) of 2-amino-N-(biphenyl-3-yl)-7-(4,4,5,5,-tetramethyl-1,3,2-
Step 2: Preparation of 6-(Benzenecarbonylamino)-7-(biphe-
nyl-3-ylamino)-7-oxoheptylboronic Acid (18). Compound 18 was
prepared from 59 using the procedure described for 5 (step 8) in
79% yield (127 mg): colorless crystals; mp 112-113 °C; 1H NMR
(DMSO-d6, 500 MHz, δ, ppm) 10.21 (1H, s), 8.59 (1H, d, J ) 7.6
Hz), 7.95 (1H, s), 7.92 (2H, d, J ) 7.0 Hz), 7.64-7.59 (3H, m),
7.55 (1H, t, J ) 7.3 Hz), 7.48 (2H, t, J ) 7.3 Hz), 7.47 (2H, t, J
) 7.9 Hz), 7.43-7.32 (5H, m), 4.57 (1H, q, J ) 7.4 Hz), 1.82
(2H, q, J ) 7.6 Hz), 1.51-1.23 (6H, m), 0.57 (2H, t, J ) 7.0 Hz);
13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.30, 166.60, 140.74,
140.13, 139.61, 134.03, 131.31, 129.33, 128.95, 128.17, 127.56,
121.63, 118.24, 117.50, 54.59, 31.64, 25.87, 24.18; MS (FAB) m/z
715 (M + 2NBA - 2H2O + H)+, 499 (M + Gly - 2H2O - H)-.
Anal. (C26H29BN2O4) C, H, N.
Compounds 19-24 were prepared from 2-amino-N-(biphenyl-
3-yl)-7-(4,4,5,5,-tetramethyl-1,3,2-dioxaborolan-2-yl)heptanamide
and an appropriate carboxylic acid using the procedure described
for 18.
7-(Biphenyl-3-ylamino)-6-(N,N-dimethyl-4-aminobenzenecar-
bonylamino]-7-oxoheptylboronic Acid (19). Yield 11% (three
steps) (51 mg); colorless crystals; mp 178-180 °C; 1H NMR
(DMSO-d6, 500 MHz, δ, ppm) 10.15 (1H, s), 8.16 (1H, d, J ) 7.9
Hz), 7.95 (1H, s), 7.80 (2H, d, J ) 9.1 Hz), 7.64-7.58 (3H, m),
7.47 (2H, t, J ) 7.9 Hz), 7.42-7.31 (5H, m), 6.71 (2H, d, J ) 8.8
Hz), 4.53 (1H, q, J ) 7.3 Hz), 2.98 (6H, s), 1.79 (2H, q, J ) 7.0
Hz), 1.48-1.22 (6H, m), 0.57 (2H, t, J ) 8.5 Hz); 13C NMR
(DMSO-d6, 600 MHz, δ, ppm) 171.65, 166.31, 152.09, 140.64,
140.58, 139.57, 129.22, 128.85, 127.45, 126.51, 121.46, 120.50,
118.09, 117.35, 110.55, 54.31, 31.83, 31.64, 30.59, 25.78, 24.10;
MS (FAB) m/z 757 (M + 2NBA - 2H2O)+, 542 (M + Gly -
2H2O - H)-. Anal. (C28H34BN3O4) C, H, N.
7-(Biphenyl-3-ylamino)-7-oxo-6-(pyridine-3-carbonylamino)-
heptylboronic Acid (20). Yield 62% (three steps) (162 mg);
colorless crystals; mp 134-135 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.24 (1H, s), 9.07 (1H, d, J ) 1.5 Hz), 8.85 (1H, d, J )
7.6 Hz), 8.72 (1H, dd, J ) 4.5, 1.8 Hz), 8.26 (1H, dt, J ) 8.2, 1.8
Hz), 7.95 (1H, s), 7.64-7.57 (3H, m), 7.52 (1H, dd, J ) 7.8, 4.9
Hz), 7.47 (2H, t, J ) 7.9 Hz), 7.43-7.32 (5H, m), 4.58 (1H, q, J
) 7.6 Hz), 1.82 (2H, m), 1.52-1.20 (6H, m), 0.57 (2H, t, J ) 7.3
Hz); 13C NMR (DMSO-d6, 600 MHz, δ, ppm) 171.04, 165.26,
148.71, 140.77, 140.12, 139.56, 135.31, 129.55, 129.36, 128.97,
127.58, 126.62, 123.38, 118.31, 117.56, 54.61, 31.88, 31.62, 25.84,
24.17; MS (FAB) m/z 716 (M + 2NBA - 2H2O + H)+, 500 (M
+ Gly - 2H2O - H)-. Anal. (C25H28BN3O4) C, H, N.
1
dioxaborolan-2-yl)heptanamide as a yellow oil: H NMR (CDCl3,
500 MHz, δ, ppm) 9.57 (1H, s), 7.85 (1H, s), 7.63-7.58 (3H, m),
7.45-7.30 (5H, m), 3.49 (1H, m), 1.97 (1H, m), 1.63-1.30 (9H,
m), 1.24 (12H, s), 0.78 (2H, t, J ) 7.6 Hz).
To a solution of 2-amino-N-(biphenyl-3-yl)-7-(4,4,5,5,-tetram-
ethyl-1,3,2-dioxaborolan-2-yl)heptanamide (250 mg, 0.592 mmol)
obtained above and a catalytic amount of N,N-dimethyl-4-ami-
nopyridine (DMAP) in CH2Cl2 (3 mL) were added benzyloxycar-
bonyl chloride (202 µL, 1.18 mmol) and Et3N (0.5 mL, 3.58 mmol),
and the mixture was stirred at room temperature for 3 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 and concentration in vacuo and purification by
silica gel flash column chromatography (AcOEt/n-hexane ) 1/3)
1
gave 134 mg (41%) of 58 as a white solid: H NMR (CDCl3, 500
MHz, δ, ppm) 8.20 (1H, s), 7.76 (1H, s), 7.57 (2H, d, J ) 7.3 Hz),
7.49 (1H, m), 7.42 (2H, t, J ) 7.3 Hz), 7.38-7.24 (8H, m), 5.35
(1H, broad s), 5.13 (2H, s), 4.26 (1H, m), 1.94 (1H, m), 1.69 (1H,
m), 1.45-1.15 (18H, m), 0.76 (2H, t, J ) 7.0 Hz).
Step 3: Preparation of 6-(Benzyloxycarbonylamino)-7-(bi-
phenyl-3-ylamino)-7-oxoheptylboronic Acid (17). Compound 17
was prepared from 58 using the procedure described for 5 (step 8)
in 32% yield (84 mg): colorless crystals; mp 141-142 °C; 1H NMR
(DMSO-d6, 500 MHz, δ, ppm) 10.12 (1H, s), 7.92 (1H, s),
7.64-7.55 (4H, m), 7.48 (2H, t, J ) 7.3 Hz), 7.43-7.28 (10H,
m), 5.04 (2H, d, J ) 2.1 Hz), 4.13 (1H, q, J ) 7.4 Hz), 1.72-1.54

2918 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Suzuki et al.
7-(Biphenyl-3-ylamino)-7-oxo-6-(thiazole-5-carbonylamino)-
heptylboronic Acid (21). Yield 54% (three steps) (201 mg);
colorless crystals; mp 145-147 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.24 (1H, s), 9.24 (1H, s), 8.89 (1H, d, J ) 7.6 Hz), 8.66
(1H, s), 7.94 (1H, s), 7.64-7.57 (3H, m), 7.47 (2H, t, J ) 7.9 Hz),
7.43-7.30 (5H, m), 4.56 (1H, q, J ) 7.4 Hz), 1.81 (2H, m),
1.50-1.24 (6H, m), 0.57 (2H, t, J ) 7.9 Hz); 13C NMR (DMSO-
d6, 500 MHz, δ, ppm) 170.71, 159.97, 158.00, 144.01, 140.67,
140.01, 139.38, 135.06, 129.26, 128.85, 127.46, 121.66, 121.66,
118.20, 117.47, 54.37, 31.73, 31.57, 25.66, 24.04; MS (FAB) m/z
722 (M + 2NBA - 2H2O + H)+, 506 (M + Gly - 2H2O - H)-.
Anal. (C23H26BN3O4S·1/2H2O) C, H, N.
7-(Biphenyl-3-ylamino)-7-oxo-6-(thiazole-4-carbonylamino)-
heptylboronic Acid (22). Yield 31% (three steps) (207 mg);
colorless crystals; mp 96-97 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.28 (1H, s), 9.22 (1H, d, J ) 2.1 Hz), 8.38 (1H, d, J )
1.8 Hz), 8.28 (1H, d, J ) 8.2 Hz), 7.93 (1H, s), 7.63-7.57 (3H,
m), 7.47 (2H, t, J ) 7.9 Hz), 7.44-7.31 (5H, m), 4.66 (1H, q, J )
7.4 Hz), 1.83 (2H, m), 1.42-1.22 (6H, m), 0.55 (2H, t, J ) 7.6
Hz); 13C NMR (DMSO-d6, 600 MHz, δ, ppm) 170.40, 160.02,
158.01, 150.05, 140.71, 139.95, 139.20, 129.31, 128.87, 127.50,
126.53, 124.52, 121.80, 118.24, 117.51, 53.37, 32.38, 31.74, 25.21,
24.04; MS (FAB) m/z 722 (M + 2NBA - 2H2O + H)+, 506 (M
+ Gly - 2H2O - H)-. Anal. (C23H26BN3O4S) C, H, N.
was neutralized with 10% citric acid and extracted with AcOEt.
The AcOEt layer was washed with brine and dried over Na2SO4.
Filtration and concentration in vacuo gave 1.80 g (100%) of 68 as
1
a yellow oil: H NMR (CDCl3, 500 MHz, δ, ppm) 5.78 (1H, ddt,
J ) 17, 10, 6.7 Hz), 5.03 (1H, dd, J ) 17, 1.5 Hz), 4.98 (1H, d, J
) 10 Hz), 3.29 (1H, t, J ) 7.3 Hz), 2.09 (2H, q, J ) 7.3 Hz), 1.91
(2H, m), 1.51-1.40 (11H, m).
Step 3: Preparation of tert-Butyl 2-(Biphenyl-3-ylaminocar-
bonyl)hept-6-enoate (69). Compound 69 was prepared from 68
using the procedure described for 5 (step 6) in 76% yield: a white
solid; 1H NMR (CDCl3, 500 MHz, δ, ppm) 8.86 (1H, s), 7.78 (1H,
s), 7.60 (2H, d, J ) 7.3 Hz), 7.55 (1H, d, J ) 7.6 Hz), 7.46-7.30
(5H, m), 5.78 (1H, ddt, J ) 17, 10, 6.7 Hz), 5.02 (1H, dd, J ) 17,
1.5 Hz), 4.98 (1H, d, J ) 10 Hz), 3.27 (1H, t, J ) 7.3 Hz), 2.11
(2H, q, J ) 7.0 Hz), 1.99 (2H, m), 1.52-1.48 (11H, m).
Step 4: Preparation of tert-Butyl 2-(Biphenyl-3-ylaminocar-
bonyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hep-
tanoate (70). Compound 70 was prepared from 69 using the
procedure described for 5 (step 7) in 70% yield: a white solid; 1H
NMR (CDCl3, 500 MHz, δ, ppm) 8.86 (1H, s), 7.78 (1H, s), 7.60
(2H, d, J ) 7.3 Hz), 7.56 (1H, d, J ) 8.3 Hz), 7.46-7.31 (5H, m),
3.26 (1H, t, J ) 7.6 Hz), 1.96 (2H, m), 1.50 (9H, s), 1.46-1.30
(6H, s), 1.23 (12H, s), 0.77 (2H, t, J ) 7.9 Hz).
7-(Biphenyl-3-ylamino)-6-(1H-indole-2-carbonylamino)-7-
oxoheptylboronic Acid (23). Yield 44% (three steps) (123 mg);
colorless crystals; mp 141-142 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 11.59 (1H, s), 10.26 (1H, s), 8.60 (1H, d, J ) 7.3 Hz),
7.95 (1H, s), 7.68-7.58 (4H, m), 7.50-7.30 (9H, m), 7.19 (1H, t,
J ) 7.0 Hz), 7.04 (1H, t, J ) 7.0 Hz), 4.62 (1H, q, J ) 7.4 Hz),
1.82 (2H, m), 1.50-1.25 (6H, m), 0.57 (2H, t, J ) 8.2 Hz); 13C
NMR (DMSO-d6, 500 MHz, δ, ppm) 171.21, 161.19, 140.76,
140.12, 139.56, 136.47, 131.23, 129.35, 128.95, 127.56, 127.03,
126.61, 123.40, 121.69, 121.56, 119.71, 118.29, 117.55, 112.25,
103.61, 79.15, 54.09, 31.89, 31.82, 25.78, 24.19; MS (FAB) m/z
754 (M + 2NBA - 2H2O + H)+, 538 (M + Gly - 2H2O - H)-.
Anal. (C28H30BN3O4 ·1/2H2O) C, H, N.
7-(Biphenyl-3-ylamino)-7-oxo-6-(quinoline-2-carbonylamino)-
heptylboronic Acid (24). Yield 56% (three steps) (165 mg);
colorless crystals; mp 127-128 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.40 (1H, s), 8.88 (1H, d, J ) 8.2 Hz), 8.61 (1H, d, J )
8.5 Hz), 8.22 (1H, d, J ) 8.8 Hz), 8.19 (1H, d, J ) 8.5 Hz), 8.11
(1H, d, J ) 7.6 Hz), 7.97 (1H, s), 7.90 (1H, d, J ) 7.3 Hz), 7.75
(1H, t, J ) 7.9 Hz), 7.65-7.57 (3H, m), 7.50-7.32 (7H, m), 4.78
(1H, q, J ) 7.4 Hz), 1.91 (2H, m), 1.45-1.26 (6H, m), 0.56 (2H,
t, J ) 7.3 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 170.43,
163.48, 149.42, 145.93, 140.82, 140.03, 139.26, 138.13, 130.69,
129.42, 129.29, 128.96, 128.25, 128.11, 127.60, 126.63, 121.96,
118.54, 118.39, 117.67, 53.62, 32.77, 31.87, 25.26, 24.12; MS
(FAB) m/z 766 (M + 2NBA - 2H2O + H)+, 550 (M + Gly -
2H2O - H)-. Anal. (C29H30BN3O4) C, H, N.
Steps 5 and 6: Preparation of N-Biphenyl-3-yl-N′-phenyl-2-
[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)]pentylmalona-
mide (71). A solution of 70 (637 mg, 1.26 mmol) in TFA (5 mL)
was stirred at room temperature for 3 h. The reaction mixture was
concentrated in vacuo to give 580 mg (100%) of 2-(biphenyl-3-
ylaminocarbonyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
heptanoic acid as a yellow oil: 1H NMR (CDCl3, 500 MHz, δ, ppm)
8.31 (1H, s), 7.77 (1H, s), 7.59 (2H, d, J ) 7.0 Hz), 7.51 (1H, d,
J ) 7.0 Hz), 7.47-7.34 (5H, m), 3.40 (1H, t, J ) 7.3 Hz), 2.06
(2H, m), 1.52-1.30 (6H, m), 1.24 (12H, d, J ) 1.8 Hz), 0.80 (2H,
t, J ) 7.6 Hz).
To a solution of 2-(biphenyl-3-ylaminocarbonyl)-7-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)heptanoic acid (357 mg, 0.790
mmol) obtained above in CH2Cl2 (5 mL) were added ethanedioxalyl
dichloride (151 µL, 1.19 mmol) and a catalytic amount of DMF,
and the mixture was stirred at room temperature for 3 h. It was
then concentrated in vacuo, and the residue was dissolved in CH2Cl2
(5 mL). To a solution of aniline (111 mg, 1.19 mmol) and Et3N (2
mL, 14.3 mmol) in CH2Cl2 (5 mL) was added a solution of the
acid chloride in CH2Cl2 (5 mL) at 0 °C, and the mixture was stirred
at room temperature for 2 h. The reaction mixture was poured into
water and extracted with CH2Cl2. The CH2Cl2 layer was washed
with water and brine and dried over Na2SO4. Filtration and
concentration in vacuo and purification by silica gel flash column
chromatography (AcOEt/n-hexane ) 1/5) gave 122 mg of 71 (29%)
1
as a white solid: H NMR (CDCl3, 500 MHz, δ, ppm) 8.82 (1H,
s), 8.67 (1H, s), 7.81 (1H, s), 7.62-7.54 (5H, m), 7.46-7.31 (7H,
m), 7.14 (1H, t, J ) 7.3 Hz), 3.31 (1H, t, J ) 7.3 Hz), 2.09 (2H,
q, J ) 7.6 Hz), 1.52-1.34 (6H, m), 1.24 (12H, s), 0.77 (2H, t, J )
7.6 Hz).
7-(Biphenyl-3-ylamino)-7-oxo-6-(phenylaminocarbonyl)hep-
tylboronic Acid (25). Step 1: Preparation of tert-Butyl Ethyl
2-(Pent-4-enyl)malonate (67). To a solution of tert-butyl ethyl
malonate (66; 5.11 g, 27.1 mmol) in dry THF (30 mL) was added
NaH (50%, 1.43 g, 29.9 mmol) under Ar gas at 0 °C. The reaction
mixture was stirred at 0 °C for 5 min. After that, 5-bromopent-1-
ene was added (4.45 g, 29.9 mmol), and the mixture was refluxed
for 10 h. It was then poured into water and extracted with AcOEt.
The AcOEt layer was washed with water and brine and dried over
Na2SO4. Filtration and concentration in vacuo and purification by
silica gel flash column chromatography (AcOEt/n-hexane ) 1/100
Step 7: Preparation of 7-(Biphenyl-3-ylamino)-7-oxo-6-(phe-
nylaminocarbonyl)heptylboronic Acid (25). Compound 25 was
prepared from 71 using the procedure described for 5 (step 8) in
1
71% yield (73 mg): colorless crystals; mp 99-100 °C; H NMR
(DMSO-d6, 500 MHz, δ, ppm) 10.03 (1H, s), 9.95 (1H, s), 7.94
(1H, s), 7.65-7.56 (5H, m), 7.52-7.26 (9H, m), 7.06 (1H, t, J )
6.7 Hz), 3.49 (1H, t, J ) 8.4 Hz), 1.91 (2H, m), 1.45-1.22 (6H,
m), 0.57 (2H, t, J ) 7.6 Hz); 13C NMR (DMSO-d6, 500 MHz, δ,
ppm) 168.08, 167.89, 140.76, 144.04, 139.42, 138.82, 129.36,
128.95, 128.71, 127.57, 126.60, 123.47, 121.84, 119.40, 118.37,
117.65, 55.21, 32.02, 29.74, 27.15, 24.10; MS (FAB) m/z 715 (M
+ 2NBA - 2H2O + H)+, 499 (M + Gly - 2H2O - H)-. Anal.
(C26H29BN2O4) C, H, N.
1
to 1/50) gave 4.02 g (52%) of 67 as a colorless oil: H NMR
(CDCl3, 500 MHz, δ, ppm) 5.79 (1H, ddt, J ) 17, 10, 6.7 Hz),
5.02 (1H, dd, J ) 17, 1.5 Hz), 4.96 (1H, d, J ) 10 Hz), 4.19 (2H,
m), 3.22 (1H, t, J ) 7.6 Hz), 2.08 (2H, q, J ) 7.0 Hz), 1.86 (2H,
q, J ) 7.6 Hz), 1.48-1.38 (11H, m), 1.27 (3H, t, J ) 7.0 Hz).
Step 2: Preparation of 2-tert-Butoxycarbonylhept-6-enoic
Acid (68). To a solution of 67 (2.00 g, 7.80 mmol) in tert-BuOH/
H2O (10 mL/5 mL) was added LiOH ·H2O (360 mg, 8.58 mmol),
and the solution was stirred overnight at 0 °C. The reaction mixture
Compound 26 was prepared from 2-(biphenyl-3-ylaminocarbo-
nyl)-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)heptanoic acid
and a pyridin-3-ylamine using the procedure described for 25.

Boronic Acid-Based Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2919
7-(Biphenyl-3-ylamino)-7-oxo-6-(pyridin-3-ylaminocarbonyl)
heptylboronic Acid (26). Yield 20% (two steps) (112 mg); colorless
crystals; mp 128-129 °C; 1H NMR (DMSO-d6, 500 MHz, δ, ppm)
10.24 (1H, s), 10.15 (1H, s), 8.84 (1H, d, J ) 2.1 Hz), 8.34 (1H,
d, J ) 4.3 Hz), 8.14 (1H, d, J ) 8.5 Hz), 8.02 (1H, s), 7.71-7.66
(3H, m), 7.55 (2H, t, J ) 7.6 Hz), 7.51-7.40 (6H, m), 3.61 (1H,
t, J ) 7.6 Hz), 2.00 (2H, m), 1.45-1.30 (6H, m), 0.65 (2H, t, J )
7.3 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm) 168.39, 167.78,
144.32, 144.01, 140.91, 140.67, 139.33, 135.44, 129.28, 128.87,
127.50, 126.33, 123.53, 121.78, 117.57, 55.05, 31.93, 29.53, 27.10,
24.01; MS (FAB) m/z 716 (M + 2NBA - 2H2O + H)+, 500 (M
+ Gly - 2H2O - H)-. Anal. (C25H28BN3O4 ·1/2H2O) C, H, N.
(R)-6-(Benzenecarbonylamino)-7-(biphenyl-3-ylamino)-7-oxo-
heptylboronic Acid ((R)-18). Step 1: Preparation of (S)-2-tert-
Butoxycarbonylaminohept-6-enoic Acid ((S)-32) and (R)-Ethyl
2-tert-Butoxycarbonylaminohept-6-enoate ((R)-31). Compound
(RS)-31 (10.8 g, 39.8 mmol) was suspended in a mixture of H2O
(750 mL) and DMF (150 mL) (1/3, v/v) solvent system at 37 °C,
and the pH was adjusted to about 7-8 by adding 1 M aqueous
ammonia solution. Then, subtilisin (39.8 mg, 1 mg of enzyme per
mmol of substrate) was added and the pH was maintained at 7-8
by continuous addition of 1 M aqueous ammonia solution. The
reaction was completed within 5 h. The solvents were evaporated,
and the residue was poured into 2 N aqueous NaOH and extracted
with diethyl ether. The diethyl ether layer was washed with water
and brine and dried over Na2SO4. Filtration and concentration in
vacuo gave 5.28 g (49%) of (R)-31 as a yellow oil. Then, the
aqueous solution was acidified and extracted with AcOEt. The
AcOEt layer was washed with brine and dried over Na2SO4.
Filtration and concentration in vacuo gave 5.92 g of (S)-32 (50%)
7.95 (1H, s), 7.65-7.58 (3H, m), 7.53 (1H, dd, J ) 7.6, 4.6 Hz),
7.48 (2H, t, J ) 7.9 Hz), 7.44-7.33 (5H, m), 4.59 (1H, q, J ) 7.3
Hz), 1.83 (2H, m), 1.52-1.25 (6H, m), 0.59 (2H, t, J ) 7.0 Hz);
13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.06, 165.30, 151.98,
148.71, 140.80, 140.14, 139.56, 135.34, 129.60, 129.38, 128.99,
127.60, 126.63, 123.41, 121.76, 118.37, 117.62, 54.66, 31.88, 31.65,
25.85, 24.18; MS (FAB) m/z 716 (M + 2NBA - 2H2O + H)+,
500 (M + Gly - 2H2O - H)-. Anal. (C25H28BN3O4 ·1/2H2O) C,
H, N.
(R)-7-(Biphenyl-3-ylamino)-7-oxo-6-(thiazole-5-carbonylami-
no)heptylboronic Acid ((R)-21). Yield 25% (>99% ee) (five steps)
(351 mg); colorless crystals; HPLC tR ) 13.25 min (ethanol/n-
hexane ) 10/90); mp 138-139 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.33 (1H, s), 9.31 (1H, s), 8.97 (1H, d, J ) 7.3 Hz), 8.73
(1H, s), 8.01 (1H, s), 7.71-7.65 (3H, m), 7.54 (2H, t, J ) 7.6 Hz),
7.51-7.40 (5H, m), 4.63 (1H, q, J ) 7.3 Hz), 1.88 (2H, m),
1.57-1.32 (6H, m), 0.65 (2H, t, J ) 7.6 Hz); 13C NMR (DMSO-
d6, 600 MHz, δ, ppm) 170.91, 160.17, 158.20, 144.16, 140.84,
140.15, 139.52, 135.21, 129.45, 129.04, 127.65, 126.67, 121.85,
118.37, 117.62, 54.56, 31.89, 31.70, 25.82, 24.20; MS (FAB) m/z
722 (M + 2NBA - 2H2O + H)+, 506 (M + Gly - 2H2O - H)-.
Anal. (C23H26BN3O4S·1/2H2O) C, H, N.
(S)-6-(Benzenecarbonylamino)-7-(biphenyl-3-ylamino)-7-oxo-
heptylboronic Acid ((S)-18). Compound (S)-18 was prepared from
(S)-32 using the procedure described for 5 (steps 6 and 7), and 18
(steps 1 and 2) in 23% yield (99% ee) (five steps) (647 mg). In
this case, biphenyl-3-ylamine was used instead of aniline: colorless
crystals; HPLC tR ) 10.97 min (ethanol/n-hexane ) 7/93); mp
102-104 °C; 1H NMR (DMSO-d6, 500 MHz, δ, ppm) 10.28 (1H,
s), 8.64 (1H, d, J ) 7.6 Hz), 8.01 (1H, s), 7.99 (2H, d, J ) 7.6
Hz), 7.71-7.65 (3H, m), 7.62 (1H, t, J ) 7.6 Hz), 7.55 (2H, t, J
) 7.6 Hz), 7.54 (2H, t, J ) 7.6 Hz), 7.50-7.39 (5H, m), 4.63 (1H,
q, J ) 7.3 Hz), 1.89 (2H, q, J ) 7.6 Hz), 1.58-1.30 (6H, m), 0.65
(2H, t, J ) 7.9 Hz); 13C NMR (DMSO-d6, 500 MHz, δ, ppm)
171.42, 166.81, 140.86, 140.20, 139.64, 134.09, 131.45, 129.46,
129.06, 128.1731, 127.67, 127.63, 126.69, 121.79, 118.39, 117.64,
54.72, 36.24, 31.96, 31.27, 25.93, 24.26; MS (FAB) m/z 715 (M
+ 2NBA - 2H2O + H)+, 499 (M + Gly - 2H2O - H)-. Anal.
(C26H29BN2O4 ·1/2H2O) C, H, N.
(S)-7-(Biphenyl-3-ylamino)-7-oxo-6-(pyridine-3-carbonylami-
no)heptylboronic Acid ((S)-20). Yield 32% (95% ee) (five steps)
(533 mg); colorless crystals; HPLC tR ) 14.47 min (ethanol/n-
hexane ) 15/85); mp 138-140 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.24 (1H, s), 9.07 (1H, d, J ) 1.5 Hz), 8.85 (1H, d, J )
7.3 Hz), 8.73 (1H, dd, J ) 4.5, 1.8 Hz), 8.26 (1H, d, J ) 8.2 Hz),
7.95 (1H, s), 7.65-7.58 (3H, m), 7.53 (1H, dd, J ) 7.6, 4.9 Hz),
7.48 (2H, t, J ) 7.9 Hz), 7.44-7.33 (5H, m), 4.58 (1H, q, J ) 7.5
Hz), 1.83 (2H, m), 1.52-1.25 (6H, m), 0.58 (2H, t, J ) 7.0 Hz);
13C NMR (DMSO-d6, 500 MHz, δ, ppm) 171.09, 165.34, 152.00,
148.71, 140.82, 140.16, 139.56, 135.37, 129.62, 129.41, 129.02,
127.63, 126.66, 123.45, 121.80, 118.40, 117.65, 54.68, 31.90, 31.66,
25.85, 24.20; MS (FAB) m/z 716 (M + 2NBA - 2H2O + H)+,
500 (M + Gly - 2H2O - H)-. Anal. (C25H28BN3O4 ·1/2H2O) C,
H, N.
1
as a white solid: H NMR (CDCl3, 500 MHz, δ, ppm) 5.79 (1H,
ddt, J ) 17, 10, 6.7 Hz), 5.08-4.92 (3H, m), 4.32 (1H, m), 2.09
(2H, m), 1.87 (1H, m), 1.69 (1H, m), 1.56-1.35 (11H, m).
Compound (R)-31 (5.28 g, 19.5 mmol) was further resolved with
5.00 mg of subtilisin using the same procedure as described above
to give pure (R)-31 (4.52 g, 42%) as a yellow oil: 1H NMR (CDCl3,
500 MHz, δ, ppm) 5.77 (1H, ddt, J ) 17, 10, 6.7 Hz), 5.03-4.95
(3H, m), 4.28 (1H, m), 4.23-4.15 (2H, m), 2.07 (2H, m), 1.81
(1H, m), 1.62 (1H, m), 1.53-1.35 (11H, m), 1.28 (3H, t, J ) 7.3
Hz).
Step 2: Preparation of (R)-2-tert-Butoxycarbonylaminohept-
6-enoic Acid ((R)-32). Compound (R)-32 was prepared from (R)-
31 using the procedure described for 5 (step 5) in 100% yield: a
white solid; 1H NMR (CDCl3, 500 MHz, δ, ppm) 5.78 (1H, ddt, J
) 17, 10, 6.7 Hz), 5.06-4.94 (3H, m), 4.32 (1H, m), 2.09 (2H,
m), 1.87 (1H, m), 1.69 (1H, m), 1.57-1.35 (11H, m).
Steps 3-7: Preparation of (R)-6-(Benzenecarbonylamino)-
7-(biphenyl-3-ylamino)-7-oxoheptylboronic Acid ((R)-18). Com-
pound (R)-18 was prepared from (R)-32 using the procedure
described for 5 (steps 6 and 7) and 18 (steps 1 and 2) in 2% yield
(>99% ee) (five steps) (21 mg). In this case, biphenyl-3-ylamine
was used instead of aniline: colorless crystals; HPLC tR ) 7.98
min (ethanol/n-hexane ) 7/93); mp 100-102 °C; 1H NMR (DMSO-
d6, 500 MHz, δ, ppm) 10.21 (1H, s), 8.58 (1H, d, J ) 7.6 Hz),
7.95 (1H, s), 7.93 (2H, d, J ) 7.3 Hz), 7.65-7.58 (3H, m), 7.55
(1H, t, J ) 7.3 Hz), 7.48 (2H, t, J ) 7.6 Hz), 7.48 (2H, t, J ) 7.3
Hz), 7.43-7.32 (5H, m), 4.57 (1H, q, J ) 7.4 Hz), 1.82 (2H, q, J
) 7.6 Hz), 1.51-1.24 (6H, m), 0.58 (2H, t, J ) 7.9 Hz); 13C NMR
(DMSO-d6, 600 MHz, δ, ppm) 171.39, 166.71, 140.82, 140.18,
139.65, 134.06, 131.40, 129.41, 129.02, 128.26, 127.61, 126.67,
121.72, 118.31, 117.56, 54.67, 31.95, 31.69, 25.93, 24.24; MS
(FAB) m/z 715 (M + 2NBA - 2H2O + H)+, 499 (M + Gly -
2H2O - H)-. Anal. (C26H29BN2O4 ·1/2H2O) C, H, N.
Compounds (R)-20 and (R)-21 were prepared from (R)-32 and
an appropriate amine using the procedure described for (R)-18.
(R)-7-(Biphenyl-3-ylamino)-7-oxo-6-(pyridine-3-carbonylami-
no)heptylboronic Acid ((R)-20). Yield 34% (>99% ee) (five steps)
(463 mg); colorless crystals; HPLC tR ) 10.93 min (ethanol/n-
hexane ) 15/85); mp 142-143 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.24 (1H, s), 9.07 (1H, d, J ) 1.5 Hz), 8.84 (1H, d, J )
7.3 Hz), 8.73 (1H, dd, J ) 4.5, 1.8 Hz), 8.26 (1H, d, J ) 7.9 Hz),
(S)-7-(Biphenyl-3-ylamino)-7-oxo-6-(thiazole-5-carbonylami-
no)heptylboronic Acid ((S)-21). Yield 26% (>99% ee) (five steps)
(435 mg); colorless crystals; HPLC tR ) 17.72 min (ethanol/n-
hexane ) 10/90); mp 140-142 °C; 1H NMR (DMSO-d6, 500 MHz,
δ, ppm) 10.27 (1H, s), 9.24 (1H, s), 8.92 (1H, d, J ) 7.3 Hz), 8.67
(1H, s), 7.95 (1H, s), 7.67-7.56 (3H, m), 7.48 (2H, t, J ) 7.6 Hz),
7.45-7.30 (5H, m), 4.56 (1H, q, J ) 7.3 Hz), 1.82 (2H, m),
1.53-1.22 (6H, m), 0.59 (2H, t, J ) 7.3 Hz); 13C NMR (DMSO-
d6, 600 MHz, δ, ppm) 170.94, 160.20, 158.22, 144.18, 140.86,
140.16, 139.54, 135.23, 129.47, 129.06, 127.67, 126.69, 121.87,
118.40, 117.65, 54.59, 31.90, 31.72, 25.84, 24.22; MS (FAB) m/z
722 (M + 2NBA - 2H2O + H)+, 506 (M + Gly - 2H2O - H)-.
Anal. (C23H26BN3O4S·1/2H2O) C, H, N.
Biology. Enzyme Assays. Assays of total HDAC activity and
HDAC1, 2, 6, and 8 activities were performed using SIRT1
fluorescence activity assay/drug discovery kits (AK-500 and AK-
555) with HeLa nuclear extract total (HDACs) or human recom-

2920 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Suzuki et al.
binant HDAC1, 2, 6, and 8 (SE-456, SE-500, SE-508, and SE-
145, respectively) produced by BIOMOL Research Laboratories,
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 test
compound that results in 50% inhibition was determined by plotting
log [Inh] versus the logit function of the % inhibition. IC50 values
were determined using regression analysis of the concentration/
inhibition data.
Cell Growth Inhibition Assay. The details of cell growth
inhibition measurement are described elsewhere.27 Briefly, the cells
were plated at appropriate density in 96-well plates in RPMI 1640
with 5% fetal bovine serum and allowed to attach overnight. The
cells were exposed to drugs for 48 h. Then, the cell growth was
determined by means of sulforhodamine B assay, as described by
Skehan et al.28 Calculations were made according to the method
described previously.27a Absorbance values of control wells (C)
and test wells (T) were measured at 525 nm. Moreover, absorbance
of the test wells (T0) was also measured at time 0 (addition of drugs).
By use of these measurements, cell growth inhibition (percentage
of growth) by a test drug 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. By use of the
computer to process % growth values, the 50% growth inhibition
parameter (GI50) was determined. The GI50 was calculated as 100
× [(T - T0)/(C - T0)] ) 50.
Supporting Information Available: Results of elemental analy-
sis of 5-26, (S)-18, 20, 21, and (R)-18, 20, 21; Figures S1-S4
showing in vitro activities and cell growth inhibition; Table S1
listing growth inhibition results. This material is available free of
charge via the Internet at http://pubs.acs.org.
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