Effects of E/Z configuration of fluoroalkene-containing HDAC inhibitors on selectivity for HDAC isoforms

Article abstract of DOI:10.1246/cl.130243

Histone deacetylase (HDAC) inhibitors belong to a new class of potential anticancer agents. It may be possible to reduce some of the toxicity by specifically targeting only the HDAC isoform. Here, stereoisomeric HDAC inhibitors containing fluoroalkene were analyzed for their specificity toward HDAC isoforms. Z-Form 1(Z) showed high affinity to HDACs whereas E-isoform 1(E) had lower affinity to HDAC1 and HDAC4. These data suggested that introduction of alkene with E/Z configuration to HDAC inhibitor can be a new strategy to develop the isoform-selective HDAC inhibitors.

Full text of DOI:10.1246/cl.130243

doi:10.1246/cl.130243
Published on the web May 29, 2013
833
Effects of E/Z Configuration of Fluoroalkene-containing HDAC Inhibitors
on Selectivity for HDAC Isoforms
Yoshiro Chuman,1 Mariko Ueyama,1 Satoshi Sano,2 Fei Wu,1,³ Yuhei Kiyota,1
Takayoshi Higashi,1 Satoshi Osada,*2 and Kazuyasu Sakaguchi*1
1Department of Biological Chemistry, Division of Chemistry, Graduate School of Science,
Hokkaido University, Kita 8 Nishi 5, Sapporo, Hokkaido 060-0808
2Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering,
Saga University, 1 Honjo, Saga 840-8502
(Received March 21, 2013; CL-130243; E-mail: kazuyasu@sci.hokudai.ac.jp)
Histone deacetylase (HDAC) inhibitors belong to a new
class of potential anticancer agents. It may be possible to reduce
some of the toxicity by specifically targeting only the HDAC
isoform. Here, stereoisomeric HDAC inhibitors containing
fluoroalkene were analyzed for their specificity toward HDAC
isoforms. Z-Form 1(Z) showed high affinity to HDACs whereas
E-isoform 1(E) had lower affinity to HDAC1 and HDAC4.
These data suggested that introduction of alkene with E/Z
configuration to HDAC inhibitor can be a new strategy to
develop the isoform-selective HDAC inhibitors.
isozyme-selective inhibitors.2 Recently, we developed HDAC
inhibitors containing a sulfanylmethyl group attached with
fluoroalkene. As the fluoroalkene is known for nonhydrolyzable
amide mimetic, it is stable with respect to hydrolytic cleavage.4
The thiol group may interact with zinc ion in a chelating
manner5 (Figure 1). The Z-isomer 1(Z) compound showed the
stronger general HDAC inhibitory activity using HeLa extract
than SAHA.6 Here, we designed and synthesized the stereo-
isomer, E-isomer 1(E) to control the special configuration of
thiol group. The effects of E- and Z-isomers on HDAC
inhibitory activity and selectivity were analyzed against various
HDAC-isoforms.
Histone deacetylases (HDACs) have crucial roles in numer-
ous biological processes. There are eighteen encoded human
HDACs, which are classified as class I (HDAC1, -2, -3, and -8),
class II (HDAC4, -5, -6, -7, -9, and -10), class III (SIRT1-7), and
class IV (HDAC11) enzymes. Recent studies of knockout mice
lacking HDAC genes have revealed highly specific functions for
individual HDAC isoforms.1 These facts emphasize the impor-
tance of developing HDAC-isoform specific inhibitors.
A number of HDAC inhibitors have been reported to date,
and they were used as anticancer drugs and reagents for serial
animal cloning without clone-specific abnormalities.2,3 HDAC
inhibitors, including SAHA, typically consist of a zinc-binding
group that coordinates to the catalytic metal atom within the
HDAC active site, a linker that accommodates the tubular access
of the active site, and a capping group that interacts with the
residues at the entrance of the active site. It is very important to
have moieties to enhance isoform selectivity to reduce side
effects. Replacement of hydroxamic acid as the zinc-binding
group has been carried out to produce new types of HDAC
The synthesis of 1(E) is outlined in Scheme 1. As outlined
in Scheme 1, synthesis of the complementary 1(E) was started
from bromide A, which was used for preparation of Z-isomer.7
F
NH
SH
1(Z)
1(E)
2
O
H
SH
F
NH
NH
O
O
H
H
F
Br
Spacer
O
OH
NH
NH
SAHA
O
H
Zinc-binding group
Cap
Figure 1. Structure of HDAC inhibitors.
COOEt
F
NH
Br
NH
OH
NH
O
NH
( )
( )
( )
( )
5
5
5
5
a
b
c
O
O
O
O
A
B
C
D
OH
F
S
F
O
SH
NH
NH
CH3
NH
( )
( )
( )
d
e
f
5
5
5
F
O
O
O
E
F
1(E)
Scheme 1. Reagents and conditions: (a) (i) AcOK, 18-crown-6, CH3CN, (ii) NaOH, H2O-MeOH, 82%; (b) DMSO, (COCl)2, Et3N,
CH2Cl2, 87%; (c) (EtO)2P(O)CHFCOOEt, n-BuLi, THF, 87%; (d) NaBH4, LiCl, THF-EtOH, 62%; (e) (i) MsCl, Et3N, THF, (ii) AcSK,
EtOH, 82%; (f) K2CO3, MeOH, 88%.
Chem. Lett. 2013, 42, 833-835
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

834
Table 1. HDAC inhibitor and their inhibitory activitiesa
IC50/¯M
Class I
Class IIa Class IIb
HeLa
extract
HDAC1 HDAC8 HDAC4 HDAC6
p21
1(Z)
1(E)
2
0.4
0.8
N.A.
1.1
2.9
8.6
N.D.
5.5
1.9
4.5
N.D.
5.8
1.3
9.1
N.D.
6
0.8
2.3
N.D.
1.7
actin
SAHA
aN.A.: not active, N.D.: not determined.
Figure 2. Induction of p21 in HeLa cells by HDAC inhibitors
(25 ¯M).
At first, bromide A was converted to acetate by treating with
potassium acetate, and the acetate was saponified to give alcohol
B.8 The obtained alcohol B was converted to aldehyde C by the
Swern oxidation, and the aldehyde C was subjected to the
Horner-Wadsworth-Emmons (HWE) olefination.9 In contrast to
the syn-elimination of sulfoxide to form fluoroalkene with Z-
selectivity,4 the kinetically controlled HWE reaction by the
lithium salt of triethyl 2-fluoro-2-phosphonoacetate resulted in
the formation of fluoroalkene D with E-selectivity. Further
transformations to construct a thiol moiety followed procedures
described for Z-isomer.10 Thus, E-fluoroalkene D was reduced to
the allyl alcohol E using in situ formed LiBH4;11 the alcohol E
was activated as mesylate, and treatment of E with AcSK gave
thioacetate F.12 Finally, alkaline hydrolysis gave the desired
thiol 1(E) in satisfactory yield.13 Purities of 1(E) and 1(Z) were
After 24 h of incubation, induction of p21 was observed with
the treatment of 1(Z)- or 1(E)-treated HeLa cells, whereas no
induction was observed with compound 2 (Figure 2). 1(Z)
showed stronger induction of p21 than 1(E). This data was well
consistent with that of their in vitro HDAC inhibitory activity.
The compound 1(Z) with Z-configuration showed signifi-
cantly higher activities to HDACs, including HDAC6 compared
to SAHA. On the other hand, E-form 1(E) decreased the
inhibitory activities to HDAC1 and especially to HDAC4,
resulting in preference of E-form 1(E) to HDAC6. Although
SAHA showed higher affinity to HDAC6 as previously reported,
the HDAC6-selectivity over HDAC4 by 1(E) is higher than that
by SAHA.16,17 These suggested that the configuration of thiol
and/or fluorine in 1(E) is suitable for HDAC6 but not HDAC4.
The zinc-binding region connected with the linker region in 1(E)
is estimated to form a wide bended structure by E-configuration,
although 1(Z) forms a thin straight structure by Z-configuration
(Figure 1). Butler et al. reported that a homology model of
HDAC6 showed a wider channel rim than that of HDAC1 did.18
The crystal structure of HDAC4 (PDB: 2VQM) also showed that
the channel rim of HDAC4 was narrower than those of HDAC1
and HDAC6.19 These suggested that 1(E) with wide and bended
structure might provide a possible binding conformation to
HDAC6 with a wider channel rim, resulting in HDAC6
selectivity by 1(E).
1
confirmed to be more than 95% by H NMR analysis.
In order to evaluate the effect of stereoisomerization on
HDAC inhibitory activity, synthetic compounds were analyzed
for HDAC inhibitory activity in vitro using a HeLa nuclear
extract as described.6 The inhibitory activity of 1(E) was very
strong and comparable with that of SAHA. 1(Z) showed almost
threefold stronger activity than SAHA (Table 1). No inhibitory
activity was detected in compound 2, in which the bromo group
was substituted for thiol group in 1(Z), indicating that the thiol
group, not the bromine group, was essential for HDAC
inhibitory activity. These data suggested that the difference of
stereoisomer of fluoroalkene affected the inhibitory activity for
HDACs.
The stereoisomeric inhibitors were analyzed for their
specificity to various subtypes of HDACs (HDAC1 and HDAC8
for class I, HDAC4 for class IIa, and HDAC6 for class IIb). For
all subtypes, 1(Z) showed much stronger inhibitory activity than
SAHA (Table 1). However, the selectivity of 1(Z) among the
subtypes was very low. 1(E) also showed strong activity for the
subtypes analyzed. Interestingly, inhibitory activities of 1(E)
against HDAC1 and HDAC4 decreased compared with that
of 1(Z), suggesting that the inhibitory activity against HDAC1
and HDAC4 was effectively decreased by E-isomer. SAHA is
known to take a stick form when it binds with class-II-type
HDAC.14 These data suggested that the particular configuration
of sulfur and fluorine should be important for chelating with
Zn2+ ion.
In summary, the introduction of the sulfanylmethyl group
with E- or Z-form into HDAC inhibitors should be a useful
strategy to develop the isoform-selective HDAC inhibitors
through the different configuration of zinc-binding position in
inhibitors. Although several HDAC6 selective inhibitors has
been reported,17,20 further optimization of the compound might
lead to the development of useful HDAC6-specific inhibitors as
not only anticancer drugs but also antidepression drugs.
References and Notes
³
Present address: College of Food Sciences, Northeast Agricultural
University, Harbin 150030, P. R. China
1
M. Haberland, R. L. Montgomery, E. N. Olson, Nat. Rev. Genet.
2009, 10, 32.
2
3
H.-J. Kim, S.-C. Bae, Am. J. Transl. Res. 2011, 3, 166.
S. Wakayama, T. Kohda, H. Obokata, M. Tokoro, C. Li, Y. Terashita,
E. Mizutani, V. T. Nguyen, S. Kishigami, F. Ishino, T. Wakayama,
Cell Stem Cell 2013, 12, 293.
Next, we analyzed the effects of the compounds on HDAC
inhibitory activity in the cells. The HDAC inhibitors were
known to induce the cell cycle arrest and apoptosis through the
induction of p21.15 In order to evaluate the biological functions
of the stereoisomer, the compounds were applied to HeLa
human cervical cancer cells to analyze the induction of p21.
4
5
6
A. Niida, K. Tomita, M. Mizumoto, H. Tanigaki, T. Terada, S. Oishi,
A. Otaka, K.-i. Inui, N. Fujii, Org. Lett. 2006, 8, 613.
A. P. Kozikowski, Y. Chen, A. Gaysin, B. Chen, M. A. D’Annibale,
C. M. Suto, B. C. Langley, J. Med. Chem. 2007, 50, 3054.
S. Osada, S. Sano, M. Ueyama, Y. Chuman, H. Kodama, K.
Sakaguchi, Bioorg. Med. Chem. 2010, 18, 605.
Chem. Lett. 2013, 42, 833-835
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

835
7
Melting points (uncorrected) were recorded on a Yanako MPS3
micro-melting point apparatus. All commercial reagents were used
without further purification unless otherwise noted. 1H NMR,
13C NMR, and 19F NMR spectra were obtained using a JEOL AL-
300 spectrometer (300, 75, and 283 MHz, respectively). All chemical
¤ ¹123.7 (d, J = 22 Hz); HRMS (FAB) m/z: (M + H)+ calcd for
C17H23FNO3 308.1662, found 308.1658.
11 NaBH4 (0.25 g, 6.6 mmol) was slowly added to a solution of LiCl
(0.28 g, 6.6 mmol) dissolved in EtOH (3.7 mL). A solution of D
(0.40 g, 1.3 mmol) dissolved in THF (1.5 mL) was added to this
suspension at 0 °C, and this mixture was stirred for 48 h at room
temperature. The reaction was quenched with aqueous NH4Cl and
the solution was evaporated to remove EtOH. The residue was
extracted with AcOEt, and the organic extracts were successively
washed with saturated NaHCO3 solution and brine. After drying the
organic phase over Na2SO4, solvent was removed in vacuo and the
residue was purified by flash chromatography (hexane/ethyl ace-
tate = 1/1) yielding E as colorless crystals (0.22 g, 62%). Mp 60-
61 °C; 1H NMR: ¤ 7.47 (d, J = 7.8 Hz, 2H), 7.32-7.27 (m, 3H), 7.08
(t, J = 7.4 Hz, 1H), 5.11 (dt, J = 21, 8.3 Hz, 1H), 4.19 (dd, J = 21,
5.6 Hz, 2H), 2.36-2.31 (m, 3H), 2.09 (t, J = 7.2 Hz, 2H), 1.70 (quin,
J = 7.4 Hz, 2H), 1.44-1.32 (m, 4H); 13C NMR: ¤ 172.2, 156.2 (d,
J = 249 Hz), 138.0, 128.8, 124.1, 120.0, 108.6 (d, J = 19 Hz), 56.5
(d, J = 31 Hz), 37.2, 29.0, 27.9, 24.9, 24.4; 19F NMR: ¤ ¹115.0 (dt,
J = 21, 21 Hz); HRMS (FAB) m/z: (M + H)+ calcd for C15H21NO2F
266.1556, found 266.1559.
12 To a solution of E (0.21 g, 0.79 mmol) and Et3N (0.18 mL, 1.3 mmol)
dissolved in THF (7.9 mL) was slowly added MeSO2Cl (0.10 mL,
1.3 mmol) at 0 °C. After the addition, the mixture was stirred for 6 h
at room temperature. The precipitate was filtered off and the filtrate
was concentrated in vacuo. The residue was dissolved in ethanol
(3.2 mL), AcSK (0.35 g, 3.1 mmol) was added to the solution, and the
mixture was stirred for 13 h at room temperature. This mixture was
diluted with AcOEt, washed with water and brine. After drying the
organic phase over Na2SO4, solvent was removed in vacuo and the
residue purified by flash chromatography (hexane/ethyl acetate =
3/2) yielding F (0.25 g, 96%) as a white powder. Mp 61-62 °C;
1H NMR: ¤ 7.46 (d, J = 7.8 Hz, 2H), 7.27-7.22 (m, 3H), 7.03 (t,
J = 7.5 Hz, 1H), 5.11 (dt, J = 20, 8.1 Hz, 1H), 3.72 (d, J = 22 Hz,
2H), 2.31-2.26 (m, 5H), 1.99 (q, J = 6.9 Hz, 2H), 1.71-1.66 (m, 2H),
1.36-1.32 (m, 4H); 13C NMR: ¤ 194.8, 171.4, 152.9 (d, J = 245 Hz),
138.0, 128.9, 124.1, 119.8, 108.8 (d, J = 20 Hz), 37.4, 30.3, 29.1,
28.4, 26.5, 26.1, 25.2; 19F NMR: ¤ ¹106.8 (dt, JH-F = 21, 21 Hz);
HRMS (FAB) m/z: (M + H)+ calcd for C17H23NO2FS 324.1434,
found 324.1437.
shifts are reported in ppm as
¤ values relative to internal
tetramethylsilane (1H and 13C) or benzotrifluoride (19F) in CDCl3
unless otherwise noted. Multiplicities are described using the
abbreviations s, singlet; d, doublet; t, triplet; q, quartet; and m,
multiplet. HRMS spectra were acquired using a JEOL GCmateII
mass spectrometer or a JEOL JMS-700 mass spectrometer. Tetra-
hydrofuran was distilled from sodium benzophenone ketyl prior
to use. All the manipulations with air-sensitive reagents were
performed under a dry argon atmosphere. Analytical TLC was
performed using Merck Silica Gel 60 F254 plates (0.25 mm on glass).
Flash column chromatography was performed using either Wakogel
C-300 (45-75 mm).
8
To a solution of compound A (4.16 g, 14.6 mmol) dissolved in
CH3CN (70 mL) was added 18-crown-6 (0.20 g, 0.76 mmol) and
AcOK (5.74 g, 58.5 mmol). The mixture was refluxed for 20 h, then
cooled to rt. Solvent was removed in vacuo and the residue was
dissolved in AcOEt. The organic solution was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
dissolved in MeOH (140 mL), 1 M aqueous NaOH solution (20 mL)
was slowly added. After stirring for 30 min, the mixture was
concentrated and extracted with AcOEt. The organic solution was
washed with water and brine, and dried over Na2SO4. Solvent was
removed in vacuo to give B as white crystals, which is pure enough
for the next reaction (2.64 g, 82%). An analytical sample was
chromatographically purified. Mp 72-74 °C; 1H NMR: ¤ 7.51 (d,
J = 7.8 Hz, 2H), 7.32-7.27 (m, 3H), 7.08 (t, J = 7.5 Hz, 1H), 3.63 (t,
J = 6.5 Hz, 2H), 2.34 (t, J = 7.4 Hz, 2H), 1.73 (quin, J = 7.1 Hz,
2H), 1.63 (br s, 1H), 1.56 (quin, J = 6.6 Hz, 2H), 1.42-1.37 (m, 4H);
13C NMR: ¤ 172.0, 138.1, 128.8, 124.1, 119.9, 62.5, 37.3, 32.3, 28.8,
25.5, 25.3; HRMS (FAB) m/z: (M + H)+ calcd for C13H20NO2
222.1494, found 222.1486.
9
A solution of DMSO (1.2 g, 15 mmol) in CH2Cl2 (2.5 mL) was added
to a solution of oxalyl chloride (0.74 mL, 8.0 mmol) in CH2Cl2
(10 mL) at ¹78 °C. The reaction mixture was kept for 15 min at
¹78 °C, and a solution of compound B (1.0 g, 4.6 mmol) in CH2Cl2
(20 mL) was slowly added over 50 min. The reaction mixture was
stirred for 15 min at ¹60 °C. Et3N (4.5 mL, 32 mmol) was then added
at ¹60 °C, the cooling bath was removed, and the reaction mixture
was stirred for 2 h at rt. The reaction mixture was quenched by the
addition of 1 M aqueous HCl solution (10 mL), and the aqueous
solution was extracted with CH2Cl2. The combined organic phases
were washed with aqueous NaHCO3, water, and brine. After drying
the organic phase over Na2SO4, solvent was removed in vacuo and
the residue was purified by flash chromatography (hexane/ethyl
acetate = 1/1) to give C as white crystals (0.88 g, 87%). Mp 51-
13 To a solution of compound 1(E) (0.14 g, 0.40 mmol) dissolved in
MeOH (4 mL) was added K2CO3 (290 mg, 0.21 mmol), and this
mixture was stirred for 1 h at room temperature. The mixture was
diluted with AcOEt, washed with water and brine, and dried over
Na2SO4. Solvent was removed in vacuo and the residue was purified
by flash chromatography (hexane/ethyl acetate = 3/2), yielding
1
1(E) (0.10 g, 88%) as a white solid. Mp 47-48 °C; H NMR: ¤ 7.48
(d, J = 7.8 Hz, 2H), 7.30 (t, J = 7.8 Hz, 2H), 7.11-7.06 (m, 3H), 5.01
(dt, J = 20, 8.1 Hz, 1H), 3.23 (dd, J = 22, 7.8 Hz, 2H), 2.34 (t,
J = 8.0 Hz, 2H), 1.95 (q, J = 7.2 Hz, 2H), 1.83 (t, J = 8.0 Hz, 1H),
1.73 (quin, J = 7.2 Hz, 2H), 1.41-1.38 (m, 4H); 13C NMR: ¤ 171.2,
155.8 (d, J = 245 Hz), 137.8, 128.9, 124.2, 119.8, 106.6 (d, J =
21 Hz), 37.5, 29.4, 28.6, 25.3, 21.3, 20.9; 19F NMR: ¤ ¹109.9
(dt, J = 21, 21 Hz); HRMS (FAB) m/z: (M + H)+ calcd for
C15H21NOFS 282.1328, found 282.1319.
1
52 °C; H NMR analysis is consistent with published data.3
10 n-Butyllithium (2.5 mL of 1.54 M solution in hexane, 3.8 mmol) was
added slowly to a stirred solution of triethyl 2-fluoro-2-phospho-
noacetate (0.71 mL, 3.5 mmol) in THF (11 mL) at ¹78 °C. The
reaction mixture was then stirred at the same temperature for further
20 min. A solution of compound C (0.70 g, 3.2 mmol) in THF (8 mL)
was added to the above reaction mixture, stirred at ¹78 °C for
30 min, allowed to warm slowly to rt, and maintained for 2 h. The
reaction mixture was quenched by the addition of 1 M aqueous HCl
solution (10 mL), and the aqueous solution was extracted with
AcOEt. The organic extracts were washed with aqueous NaHCO3,
water, and brine. After drying the organic phase over Na2SO4,
solvent was removed in vacuo and the residue was purified by
flash chromatography (hexane/ethyl acetate = 3/2) to give D as a
colorless oil (0.86 g, 87%). 1H NMR: ¤ 7.54 (br s, 1H), 7.46 (d,
J = 7.5 Hz, 2H), 7.22 (t, J = 7.7 Hz, 2H), 7.01 (t, J = 7.4 Hz, 1H),
5.87 (dt, J = 22, 8.3 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.48-2.40 (m,
2H), 2.27 (t, J = 7.5 Hz, 2H), 1.66 (quin, J = 7.2 Hz, 2H), 1.42-1.23
(m, 4H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR: ¤ 171.4, 160.8 (d,
J = 36 Hz), 145.3 (d, J = 252 Hz), 138.0, 128.8, 124.1, 123.3 (d,
J = 18 Hz), 119.8, 61.3, 37.3, 28.7, 28.5, 25.2, 25.1, 14.0; 19F NMR:
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Chem. Lett. 2013, 42, 833-835
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/