Histone deacetylase inhibitor prodrugs in nanoparticle vector enhanced gene expression in human cancer cells

Article abstract of DOI:10.1016/j.ejmech.2009.06.036

We developed histone deacetylase inhibitor (HDACI) prodrugs to enhance the expression of the external genes transfected into human cells with cationic nanoparticles (NPs). We synthesized five kinds of lipid-linked HDACI prodrugs in which n-dodecanoic acid

Full text of DOI:10.1016/j.ejmech.2009.06.036

European Journal of Medicinal Chemistry 44 (2009) 4603–4610
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Histone deacetylase inhibitor prodrugs in nanoparticle vector enhanced
gene expression in human cancer cells
,
Yuta Ishii a, Yoshiyuki Hattori b, Toshiharu Yamada a, Shinichi Uesato a, Yoshie Maitani b, Yasuo Nagaoka a
*
a Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680, Japan
b Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We developed histone deacetylase inhibitor (HDACI) prodrugs to enhance the expression of the external
genes transfected into human cells with cationic nanoparticles (NPs). We synthesized five kinds of lipid-
linked HDACI prodrugs in which n-dodecanoic acid or cholesterol is linked with a potent HDACI, K-182,
by an ester bond or a disulfide carbonate linker. The prodrugs were able to admix as a component of NPs,
Received 14 May 2009
Received in revised form
15 June 2009
Accepted 28 June 2009
Available online 4 July 2009
although the intact K-182 was not incorporated into NPs. Namely, NPs composed of cholesteryl-3b-
carboxyamidoethylene-N-hydroxyethylamine and Tween 80 with the 10 mol% K-182 prodrug were
prepared as a DNA vector to transfect plasmid DNAs into human prostate cancer cells, PC-3, or human
breast cancer cells, Sk-Br-3. The NPs containing K-182 prodrugs with n-dodecanoic acid exhibited two to
four times higher the gene expression than the original NPs. The enhancement of the gene expression
will be due to the hyperacetylation of core histones caused by intact K-182 degraded from the prodrug in
the vector incorporated into the cells.
Keywords:
Non-viral vector
Histone deacetylase inhibitor
Cationic nanoparticle
HDAC
Cationic cholesterol
Gene expression
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
overcome even though they have successfully intruded into the
cytoplasm [11,12]. We assumed that for the further development of
Development of an efficient and safe method for the delivery of
exogenous DNAs into mammalian cells is critical both for basic
biosciences and clinical applications, including iPS cell techniques
and gene therapy. Viral vectors have been used for this purpose
because of their high transfection efficiency, but disadvantages such
as pathogenicity, immunogenicity, mutational potential and low
capacity of gene carrying have led to the search of a non-viral
delivery system [1–4]. Among the non-viral systems, lipofection
using cationic cholesterol derivatives is considered to be safer since
it is less immunogenic and has low toxicity [5,6]. We reported that
more efficient NP vector, improvements have to be made to the
processes of intracellular DNA delivery and intranucleus DNA
transcription. Recently, it was reported that inhibition of histone
deacetylase 6 (HDAC6) increased transfection efficiency due to the
improved gene transfer in cytoplasm caused by hyperacetylation of
microtubules [13]. It is also known that many kinds of HDAC
inhibitors (HDACIs) augment the expression of genes because of the
hyperacetylation of subnuclear core histone proteins. We therefore
attempted to associate the function of NP with HDACI for the
development of highly efficient vectors.
cationic nanoparticles (NPs) composed of cholesteryl-3
b
-carbox-
There are three different chemical types in major HDACIs, i.e. the
fatty acid-type, benzamide-type and hydroxamate-type. Fatty acid-
type HDACIs, such as butylate, or valproate [14], may have affinity
to NPs, however, the HDACI activity of them is three orders of
magnitude lower than the other types. Since, the benzamide-type
HDACIs such as MS275 [15] and MGCD0103 [16] are selective
inhibitor of class I HDACs, the improvement of DNA transport in the
cytoplasm cannot be expected because the class II HDAC, namely
HDAC6, may not be inhibited by them [17]. On the other hand, the
yamidoethylene-N-hydroxyethylamine (OH-Chol, Fig. 1) [5] and
Tween 80 could deliver plasmid DNA into cells with high transfec-
tional efficiency [7]. Cationic NPs bind electrostatically to negatively
charged DNAs toform complexes called nanoplexes, which canenter
the cells via endocytosis [8,9] followed by the escape from the
endosomes into the cytoplasm and transfer into the nucleus [10].
In order to deliver DNAs efficiently to the nucleus and achieve
high levels of expression, there are several obstacles that must be
hydroxamate-type HDACIs such as trichostatin
A [18], sub-
eroylanilide hydroxamic acid (SAHA, vorinostat) [19] and our K-
series compounds [20–22] have potent inhibitory effects against
broad HDAC isoenzymes, including cytoplasmic HDAC6 and nuclear
* Corresponding author. Tel.: þ81 (0)6 6368 0884; fax: þ81 (0)6 6388 8609.
E-mail address: ynagaoka@ipcku.kansai-u.ac.jp (Y. Nagaoka).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.036

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Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
N
H
N
OH
OH
O
K-182
N
O
N
H
N
H
N
O
OH
OH
O
O
O
O
K-182-DD
DD-K-182
H
N
N
O
H
N
H
N
O
H
H
OH
O
O
O
O
OH
O
O
O
CS-K-182
O
H
H
K-182-CS
H
H
N
H
N
H
N
H
H
OH
HO
N
H
O
O
O
O
O
OH-Chol
S
DDTS-K-182
S
Fig. 1. Chemical structures of K-182, K-182 prodrugs and OH-Chol.
HDAC1. We therefore assessed the compatibility of the hydrox-
amate-type HDACIs as a component of NP formulation. However,
unsatisfactory results were obtained from admixed formulation of
the HDACIs and NP, namely a considerable segregation of HDACIs
occurred. Hence, we attempted to synthesize HDACI prodrugs
compatible to NP. Taking into consideration the diverse derivati-
zation, we selected one of the K-series compounds, K-182, (Fig. 1)
[20] which has two OH groups to tether other groups with
a biodegradable ester bond.
In this study, five kinds of K-182 prodrugs, DD–K-182, DDTS–K-
182, CS–K-182, K-182–DD and K-182–CS, (Fig. 1) were synthesized
to evaluate their compatibility to NP formulation and their ability to
enhance the expression of the transfected genes.
hydroxamic acid than in alcoholic OH in the hydroxyethyl group
(Scheme 1). On the other hand, coupling of n-dodecanoic acid to
a hydroxyethyl group was performed using a substrate in which OH
in a hydroxamic acid was protected with tetrahydropyranyl ether
(THP). After the coupling reaction, THP was deprotected with acetic
acid in THF–H2O at 60 ꢀC for 10 h to give a monoester DD–K-182
with an excellent yield (Scheme 2). Two kinds of cholesteryl
succinate monoesters of K-182, K-182–CS and CS–K-182, were
synthesized by coupling cholesteryl succinate in the same manner
as the coupling of n-dodecanoic acid (Scheme 2 and 3).
We also attempted to connect an aliphatic thiol to hydroxyethyl
moiety in K-182 via a disulfide carbonate linker which is designed to
release unmodified K-182 in the reductive cytosolic condition as
shown in the route in Scheme 3 [23]. The mixed disulfide, (2-pyr-
idinyldithio)ethyl carbonate 3, for the subsequent coupling with
aliphatic thiol was synthesized with the reaction of 1 and tri-
phosgene, followed by condensation of 2-(2-pyridinyldithio)
ethanol [24] to the acid chloride intermediate. Coupling of dodec-
anethiol to 3 followed by deprotection of THP afforded the target
molecule DDTS–K-182 (Scheme 4). Accordingly, five kinds of K-182
prodrugs were prepared (Fig. 1).
2. Chemistry
We tried to form NPs containing hydroxamate-type HDACIs, but
we decided against this because of the segregation of the
compounds during preparation. To overcome this problem, we
attempted to form HDACI prodrugs which have affinity to OH-Chol,
the major component of NP. Regarding the activity and feasibility of
derivatization, we selected K-182 as a substrate of the prodrugs.
K-182 has two hydroxyl (OH) groups that can link with other
functional groups via ester bonds. We designed K-182 prodrugs in
which K-182 is tethered to an aliphatic fatty acid or cholesteryl
group via biodegradable bonds or linkers. When non-protected K-
182 was treated with equimolar n-dodecanoic acid (lauric acid)
with the coupling regent, DCC–DMAP, n-dodecanoic acid was
selectively coupled to an OH group in hydroxamic acid to form
mono-dodecanoate, K-182–DD, due to the higher acidity of OH in
3. Results and discussion
3.1. Optimization of concentration of K-182 prodrugs in NP and of
concentration of the NPs in the medium affected to PC-3-Gluc cells
In order to optimize the formulation, NPs containing K-182–DD
with different proportions were prepared using the modified
ethanol injection method as previously described [7] (Table 1). The
a) n-Dodecanoic acid
b) Cholesteryl succinate
N
N
H
N
H
N
R
OH
DCC,DMAP
O
OH
K-182
O
OH
K-182-DD (a:R=n-dodecanoyl
K-182-CS (b:R=cholestenyl-succinyl)
O
CH2Cl2 ,rt, 5hrs
Scheme 1.

Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
4605
a) n-Dodecanoic acid
b) Cholesteryl succinate
DCC, DMAP
N
N
H
N
H
N
O
O
O
O
CH2Cl2, rt, 5 hrs
O
OH
O
O
R
1
2a (R = n-dodecanoyl)
2b (R = cholestenyl-succinyl)
CH3CO2H-THF-H2O
reflux, 10 hrs
N
H
N
OH
O
O
R
DD-K-182 (a: R = R = n-dodecanoyl)
CS-K-182 (b: R = cholestenyl-succinyl)
Scheme 2.
formulations were based on the original NP, yet 5, 10 or 20 mol% of
OH-Chol in the original was displaced by K-182–DD (NP-5K, NP-10K
and NP-20K, respectively). Subsequently, the resulting water
suspension of the NPs (1 mg/mL) was incubated with plasmid DNAs
for 10 min at room temperature to form nanoplexes (DNA/NP
complexes). According to our previous study, the ratio between
DNA and total NP is fixed so as to form nanoplexes at a charge ratio
(þ/ꢁ) of 3/1 of cationic NP to plasmid DNA pGL3-basic encoding
luciferase gene without promoter, corresponding to a concentra-
3.2. Effect of K-182 prodrugs contained in NPs on expression
of externally transfected genes
Nanoplexes were formed by the incubation of NPs with plasmid
pCMV-Gluc control encoding secretable Gaussia luciferase (Gluc)
under the control of the CMV promoter. According to the optimi-
zation mentioned above, nanoplexes were diluted to form the
transfection medium of 20 mM NPs, containing 2 mM K-182 pro-
drugs at a charge ratio (þ/ꢁ) of 3/1 of cationic NP to plasmid DNA,
and were transfected to PC-3 cells. As shown in Fig. 3, NPs con-
taining 10 mol% DD–K-182, K-182–DD and DDTS–K-182 (NP–DD–K-
182, NP–K-182–DD and NP–DDTS–K-182) augmented the luciferase
expression 2–3.5 times more than control NP. However, NPs con-
taining 10 mol% cholesteryl ester, CS–K-182 and K-182–CS (NP–CS–
K-182 and NP–K-182–CS) did not increase the expression. It is
tion ratio of NPs (mM) to DNA (mg/mL) of 10 throughout this
experiment [25]. The nanoplexes were diluted with a cell growth
medium to prepare the transfection medium containing 5, 10, 20
and 40 mM NPs with twofold serial dilution of the initial 40 mM NPs.
In this optimization, PC-3 cells stably expressing Gaussia luciferase
(PC-3-Gluc cells) were used to evaluate the effect of K-182–DD in
each NP on the gene expression, excluding the factor of trans-
fection. The cells were incubated with each medium for 24 or 48 h
and the increased expression of the Gluc gene was evaluated using
luciferase assay (Fig. 2).
interesting that the addition of 2 mM K-182 to the medium con-
taining control NP did not potentiate the expression of transfected
genes. Similar results were obtained when Sk-Br-3 cells were used
as a host for the transfection with NP–DD–K-182, NP–K-182–DD
and NP–DDTS–K-182, i.e. they enhanced the expression about three
times more than control NP. The addition of external K-182 did not
increase the expression, in these cells. The simultaneous incorpo-
ration of DNA and K-182 prodrugs into the cells with NPs will be
critical for the activation of gene expression.
Increased expression of the Gluc gene was observed significantly
in the cells incubated with the medium containing 5–20
10K and NP-20K both for 24 h and 48 h incubation times. (Fig. 2A
and B) NP formulations of 40 M, however, did not show the
mM NP-
m
augmentation effect of K-182–DD, probably because of the higher
cytotoxicity caused by higher concentration of OH-Chol and K-182–
DD. Although 20
m
M
NP-20K seems optimum for the gene
3.3. Effect of NPs composed of K-182 prodrugs on acetylation
of histone H3 in human prostate tumor PC-3 cells
expression, the cytotoxicity in that condition was more significant
(cell viability: 19.6 ꢂ 1.4% of total cells as mean ꢂ S.D.) compare to
that of control NP or NP-10K (cell viability: 49.4 ꢂ 2.2% and
34.3 ꢂ 2.5%, respectively) at the same concentration (P < 0.01). We
Histone H3 is one of the core histone proteins in the chromatin
of eukaryotic cells. Hyperacetylation of lysine residues, i.e. lysine 9,
in the N-terminal tails of histone H3 loosens the histone–DNA
binding and activates gene transcription. On the other hand,
deacetylation of acetylated lysine residues leads to tight histone–
DNA binding, which restricts the access of transcriptional factors.
Histone acetyltransferases (HATs) and HDACs play a crucial role in
this reversible acetylation and deacetylation of histones regulating
gene expression. Inhibition of HDACs, therefore, induces histone
hyperacetylation and activates gene transcription [26–28].
therefore chose the transfection media containing 20 mM NPs and
we prepared NPs with 10 mol% of DD–K-182, CS–K-182, K-182–CS,
and DDTS–K-182, as well as K-182–DD in further experiments.
Their average particle sizes and z-potentials are in the range of
137.9–176.7 nm (polydispersity index: 0.1–0.2) and 64.0–63.0 mV,
respectively and those of control NPs are 165.3 nm (polydispersity
index: 0.02) and 48.8 mV, respectively. Replacement of 10 mol% of
OH-Chol to K-182 prodrugs increased the surface potential of NPs,
probably because of a tertiary amine, that can be positively charged,
in K-182 moiety exposed on the surface of NPs.
Prior to examine the acetylation of cellular histone H3, we tested
the effect of intact K-182 prodrugs, K-182–DD, DD–K-182 and
O
N
N
H
H
N
N
+
O
S
K-182
OH
OH
O
O
O
O
O
O
O
S
O
DDTS-K-182
S
SH
Scheme 3. Disulfide mediated release of K-182 from the carbonate linker.

4606
Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
O
N
O
HO
S
N
N
Cl3C
triphosgene
CCl3
H
N
H
N
S
O
O
DMAP
O
O
O
O
O
OH
1
O
CH2Cl2, rt, 15min
CH2Cl2, rt, 7h, 37%
3
S
N
S
N
H
Dodecanethiol
N
OR
DMSO, rt, 2.5h
94%
O
O
O
O
S
S
4 (R=tetrahydropyran-2-yl)
DDTS-K-182 (R=H)
CH3COOH/H2O
THF, 60 °C, 6h
76%
Scheme 4.
DDTS–K-182 on HDAC activity. As shown in Table 2, K-182 prodrugs
are inactive (IC50 > 10 M), although K-182 exhibited IC50 of
0.90 M. The activity of HDACI was recovered when DDTS–K-182
human breast cancer cells, Sk-Br-3. These NPs exhibited expression
of the genes two to four times more efficiently than those with the
original NP. The enhancement of the gene expression will be due to
the hyperacetylation of core histones caused by K-182 derived from
K-182 prodrugs in the NP formulation.
m
m
was treated with dithiothreitol, indicating the release of K-182 by
degradation of disulfide carbonate linker with reductive conditions.
We tested the effect of nanoplexes containing K-182 prodrugs
on the acetylation of core histone H3 protein in human prostate
tumor cells, PC-3, using western blot analysis detecting the acety-
lation on Lys-9 side chains. Incubation of the cells with nanoplexes
formed with NP–DDTS–K-182, NP–DD–K-182 or NP–K-182–DD
resulted in increased levels of acetylated histone H3 protein in the
A 400
(24 hrs)
Control NP
NP-5K
NP-10K
NP-20K
350
cells to a similar extent as those increased with 2 mM K-182 or
*
300
*
*
another type of HDACI K-32 [22] in the medium with or without the
presence of NP–pGL3-basic nanoplexes (Fig. 4). This observation,
together with the result that intact prodrugs are inactive in HDAC
inhibition, suggesting that free K-182 is efficiently released from
the complexes of prodrugs in the cells and inhibits HDAC in the
nucleus resulting in the hyperacetylation of histone H3.
It remains controversial that addition of K-182 externally to the
medium did not increase the expression of transfected genes in PC-
3 cells as shown in Fig. 3, even though the hyperacetylation of
histone H3 in the cells was observed in the similar condition (Fig. 4)
and the fact that addition of K-182 to PC-3-Gluc cells, on the other
hand, increased the expression of Gluc gene stably expressed in the
cells (Fig. 5). The simultaneous delivery of K-182 prodrugs and
genes into the cells with the formulation of nanoplexes is probably
important to enhance the outer gene expression.
*
250
200
150
100
50
*
*
0
5
10
20
40
Concentration of NPs (µM)
B
400
350
300
250
200
150
100
50
Control NP
NP-5K
(48 hrs)
*
NP-10K
NP-20K
4. Conclusion
*
*
*
*
In conclusion, cationic NPs composed of 10 mol% K-182 pro-
drugs, K-182–DD, DD–K-182 and DDTS–K-182, with 85 mol% OH-
Chol and 5 mol% Tween 80 were prepared as a DNA vector to
transfect plasmid DNA into human prostate cancer cells, PC-3, or
*
**
Table 1
0
Formulae of NPs containing K-182–DD.
5
10
20
40
NP formulationa
Components (mol%)
OH-Chol
Concentration of NPs (µM)
Tween 80
K-182–DD
Fig. 2. Effect of concentration of K-182–DD in NPs on increased expression of Gluc
gene stably expressed in PC-3-Gluc cells at a charge ratio (þ/ꢁ) of 3/1 of cationic NP to
Control NP
NP-5K
NP-10K
NP-20K
95
90
85
75
5
5
5
5
0
5
10
20
plasmid DNA (corresponding to a concentration ratio of NPs (mM) to DNA (mg/mL) of
10). PC-3-Gluc cells were incubated for 24 h (A) or 48 h (B) with cell growth media
containing nanoplexes formed with 5, 10 and 20 mol% K-182–DD in NPs; NP-5K, NP-
10K or NP-20K. Each column represents the mean ꢂ S.D. (n ¼ 3). *P < 0.01, significantly
different from control.
a
NPs were prepared with a cationic cholesterol (OH-Chol), Tween 80 and K-182–
DD using modified ethanol injection method [7] as 1 mg/mL suspension in water.

Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
4607
A 450
PC-3 cells
16.2
*
400
350
300
250
200
150
100
50
10.2
kDa
*
*
Fig. 4. Acetylation of core histone H3 protein in PC-3 cells induced by HDACIs (K-32
and K-182) and NPs composed of K-182 prodrugs. The cells were treated with
a medium containing (a–f) or not containing (g, h) NP–pGL3-basic nanoplexes in the
presence (b–h) or absence (a) of the HDACIs. The medium containing the nanoplexes
0
(a–f) was formed with pGL3-basic plasmid DNA (2 mg/mL) and 20 mM NP (a–c), NP–
DDTS–K-182 (d), NP–DD–K-182 (e) or NP–K-182–DD (f). After 24 h incubation of the
medium, cellular proteins were isolated from the cells and were resolved on a 12%
SDS–PAGE (each 3 mg protein in a well), followed by Western blot analysis for acety-
B
lated histone H3 protein.
400
350
300
250
200
150
100
50
Sk-Br-3 cells
*
*
*
NMR and 100.4 MHz for 13C NMR) instruments using tetrame-
thylsilane as an internal standard. Analytical and preparative TLC
were performed using Silica gel 60 F254 (Merck, 0.25 and 0.5 mm,
respectively) glass plates. Column chromatography was performed
using Silica Gel 60 (70–230 mesh ASTM).
0
5.1.1. N-(Dodecanoyloxy)-4-{[(2-hydroxyethyl)(2-naphthylmethyl)-
amino]methyl}benzamide (K-182–DD)
To a solution of K-182 (33.2 mg, 94.6
were added n-dodecanoic acid (19.0 mg, 94.6
94.6 mol) and DMAP (11.6 mg, 94.6 mol). After being stirred for
mmol) in CH2Cl2 (4.0 mL)
mmol), DCC (19.5 mg,
m
m
5 h at rt, the precipitate was filtered off and the filtrate was
concentrated in vacuo. Purification of the resulting residue by silica
gel column chromatography (eluent: ethylacetate/hexane ¼ 1/1)
Fig. 3. Effects of 10 mol% K-182 prodrugs contained in NPs on the expression of genes
transfected to PC-3 cells (A) and Sk-Br-3 cells (B). PC-3 cells or Sk-Br-3 cells were
incubated for 24 h in the transfection medium containing nanoplexes formed with
gave K-182–DD (12.2 mg, 23.0
IR (CHCl3)
773; 1H NMR (CDCl3)
mmol, 24.3% yield) as a colorless oil.
20
m
M NPs and 2
m
g/mL plasmid DNA, pCMV-Gluc. For the control, the media con-
M K-182 were used for transfection with the control NP.
g
/cmꢁ1: 3341, 3010, 2927, 2854, 1780, 1697, 14,111, 1141,
taining or not containing 2
m
Each column represents the mean ꢂ S.D. (n ¼ 3). *P < 0.01, significantly different from
d
: 0.88 (t, 3H, J ¼ 7.2 Hz, CH3CH2), 1.0–1.4 (m,
control.
16H, –(CH2)8–), 1.60–1.75 (m, 2H, COCH2CH2), 2.55 (t, 2H, J ¼ 7.6 Hz,
COCH2), 2.71 (t, 2H, J ¼ 5.2 Hz, NCH2CH2), 3.62 (t, 2H, J ¼ 5.2 Hz,
HOCH2CH2), 3.70 (s, 2H, NCH2–naph), 3.78 (s, 2H, NCH2Ph), 7.41–
7.52 (m, 5H, Ar–H), 7.71–7.84 (m, 6H, Ar–H); 13C NMR (CDCl3)
d:
5. Experimental
14.10, 22.66, 24.70, 24.90, 25.59, 28.99, 29.14, 29.31, 29.37, 29.56,
5.1. Synthesis of K-182 prodrugs
Melting points were determined on a Yanagimoto MP-32
micromelting point apparatus and are uncorrected. IR spectra were
recorded on Shimadzu FTIR-8400 infrared spectrophotometer. FAB-
MS spectrawere measured on a JEOL JMS-HX 100 instrument.1H and
13C NMR spectra are recorded on JEOL EX-400 (399.7 MHz for 1H
600
*
500
400
Table 2
300
Inhibitory effect of HDACIs and K-182 prodrugs on HDAC.
*
IC50 (m
M)a,b
*
Compounds
TSAc
200
0.012
0.90
K-182
K-182–DD
DD–K-182
DDTS–K-182
DDTS–K-182 þ DTTd
>10
>10
>10
100
0
2.7
cont
0.01
0.1
1
a
Inhibition of crude HDACs from nuclear extract of HeLa cells provided in CycLex
HDAC Assay Kit.
Concentration of K-182 (µM)
b
Assays were performed in duplicate.
Trichostatin A (TSA) as positive control of HDACI.
DDTS–K-182 was preincubated with equimolar dithiothreitol (DTT) in DMSO for
Fig. 5. Effect of K-182 on the expression of genes stably expressed in PC-3-Gluc cells.
PC-3-Gluc cells were incubated for 24 h with cell growth media with or without K-182.
Each column represents the mean ꢂ S.D. (n ¼ 3). *P < 0.01, significantly different from
control.
c
d
2 h before addition to the assay buffer.

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Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
31.78, 31.89, 33.90, 55.11, 58.03, 58.61, 58.77, 125.85, 126.19, 126.86,
127.63, 127.67, 127.71, 127.80, 128.37, 129.29, 129.78, 132.84, 133.29,
135.86, 144.22, 172.20; HR-FAB-MS m/z: 533.3370 (calcd for
C33H45N2O4, 533. 3379).
(t, 2H, J ¼ 7.6 Hz, COCH2), 2.77 (t, 2H, J ¼ 5.6 Hz, NCH2CH2), 3.64–
3.68 (1H, m, –CHa–O of THP), 3.72 (s, 2H, NCH2Ph), 3.78 (s, 2H,
NCH2), 3.95–4.10 (m, 1H, –CHb–O of THP), 4.20 (t, 2H, J ¼ 6.0 Hz,
OCH2CH2N), 5.08 (m,1H, OCHO), 7.43–7.52 (m, 5H, Ar–H), 7.70–7.83
(m, 6H, Ar–H); 13C NMR (CDCl3)
d: 14.09, 18.66, 22.66, 24.91, 25.02,
5.1.2. Cholest-5-en-3-yl 4-{[(4-{[(2-hydroxyethyl)(2-
naphthylmethyl)amino]methyl}benzoyl) amino]oxy}-4-
oxobutanoate (K-182–CS)
25.60, 28.06, 29.18 29.28, 29.31, 29.46, 29.59, 31.86, 33.91, 34.34,
51.91, 58.31, 58.89, 62.05, 62.69, 102.71, 125.62, 126.00, 126.92,
127.20, 127.30, 127.61, 127.65, 128.01, 128.88, 130.75, 132.80, 133.27,
136.56, 144.06, 173.74; HR-FAB-MS m/z: 617.3960 (calcd for
C38H53N2O5, 617.3954).
To a solution of K-182 (21.2 mg, 60.5
were added cholesteryl succinate (29.5 mg, 60.5
(12.5 mg, 60.5 mol) and DMAP (7.4 mg, 60.5 mol). After being
m
mol) in CH2Cl2 (2.0 mL)
mmol), DCC
m
m
stirred for 6 h at rt, the precipitate was filtered off and the filtrate
was concentrated in vacuo. Purification of the resulting residue by
preparative SiO2 TLC (eluent: ethylacetate/hexane ¼ 4/5) gave K-
5.1.5. 1-Cholest-5-en-3-yl 4-{2-[(2-naphthylmethyl)(4-{[(tetrahydro-
2H-pyran-2-yloxy) amino]carbonyl}benzyl)amino]ethyl} succinate (2b)
To a solution of 1 (48.6 mg, 112
added cholesteryl succinate (54.4 mg, 112
111.6 mol) and DMAP (13.6 mg, 111.6 mol). After being stirred for
mmol) in CH2Cl2 (2.0 mL) were
182–CS (10.5 mg,12.8
mmol, 21.2% yield) as a colorless oil. IR (CHCl3)
mmol), DCC (23.0 mg,
g
/cmꢁ1: 3320, 2949, 2868, 1759, 1724, 1466, 1364, 1105, 667; 1H
m
m
NMR (CDCl3)
d
: 0.67 (s, 3H, 180-CH3), 0.85 (d, 3H, J ¼ 1.6 Hz, 260 or
4 h at rt, the precipitate was filtered off and the filtrate was
concentrated in vacuo. Purification of the resulting residue by
preparative SiO2 TLC (eluent: ethylacetate/hexane ¼ 1/2) gave 2b
270-CH3), 0.87 (d, 3H, J ¼ 1.6 Hz, 260 or 270-CH3), 0.90–2.05 (m, 26H),
0.91 (d, 3H, J ¼ 6.4 Hz, 210-CH3), 1.01 (s, 3H, 190-CH3), 2.32 (d, 2H,
J ¼ 7.6 Hz, 40-CH2), 2.71 (m, 4H, COCH2CH2CO), 2.87 (t, 2H,
J ¼ 6.8 Hz, NCH2CH2), 3.62 (t, 2H, J ¼ 5.6 Hz, HOCH2CH2), 3.70 (s, 2H,
NCH2–naph), 3.78 (s, 2H, NCH2–Ph), 4.63 (m, 1H, 30-CH), 5.36 (m,
1H, 60-CH]C), 7.41–7.48 (m, 5H, Ar–H), 7.70–7.83 (m, 6H, Ar–H);
(66.3 mg, 73.4 mmol, 65.8% yield) as a colorless oil. IR (CHCl3) g/
cmꢁ1: 3008, 2947, 2854, 1728, 1683, 1456, 1363, 1112, 761; 1H NMR
(CDCl3)
d
: 0.67 (s, 3H, 180-CH3), 0.85 (d, 3H, J ¼ 1.6 Hz, 260 or 270-
CH3), 0.87 (d, 3H, J ¼ 1.6 Hz, 260 or 270-CH3), 0.9–2.1 (m, 32H), 0.91
(d, 3H, J ¼ 6.4 Hz, 210-CH3), 1.01 (s, 3H, 190-CH3), 2.30 (d, 2H,
J ¼ 8.0 Hz, 40-CH2), 2.57 (m, 4H, COCH2CH2CO), 2.76 (t, 2H, J ¼ 5.6 Hz,
NCH2CH2), 3.63–3.67 (1H, m, –CHa–O of THP), 3.69 (s, 2H, NCH2Ph),
3.78 (s, 2H, NCH2), 3.95–4.10 (m, 1H, –CHb–O of THP), 4.20 (t, 2H,
J ¼ 5.6 Hz, OCH2CH2N), 4.62 (m, 1H, 30-CH), 5.08 (m, 1H, OCHO of
THP), 5.35 (s, 1H, 60-CH]C), 7.43–7.53 (m, 5H, Ar–H), 7.70–7.82 (m,
13C NMR (CDCl3)
d: 11.85, 18.70, 19.28, 21.02, 22.54, 22.80, 23.83,
24.27, 27.05, 27.69, 28.00, 28.21, 29.14, 31.84, 31.89, 35.78, 36.18,
36.58, 36.94, 38.00, 39.52, 39.73, 42.32, 50.01, 55.17, 56.16, 56.69,
58.07, 58.65, 58.82, 74.79, 122.77, 125.84, 126.19, 126.87, 127.64,
127.68, 127.71, 127.79, 128.36, 129.29, 129.61, 132.86, 133.30, 135.87,
139.49, 144.29, 171.06; HR-FAB-MS m/z: 819.5320 (calcd for
C52H71N2O6, 819.5234).
6H, Ar–H); 13C NMR (CDCl3)
d: 11.83, 18.63, 18.70, 19.27, 21.00, 22.54,
22.80, 23.81, 24.26, 24.91, 25.04, 25.60, 27.71, 27.99, 28.07, 28.21,
29.41, 31.83, 31.88, 33.91, 35.76, 36.17, 36.55, 36.92, 38.04, 39.50,
39.71, 42.29, 49.99, 51.96, 56.12, 56.67, 58.31, 59.01, 62.44, 62.61,
74.43, 102.63, 122.71, 125.63, 126.02, 126.94, 127.24, 127.34, 127.62,
127.66, 128.06, 128.87, 130.82, 132.81, 133.27, 136.52, 144.01, 171.74,
172.17; HR-FAB-MS m/z: 903.5882 (calcd for C57H79N2O7: 903.5887).
5.1.3. 4-{[(2-Hydroxyethyl)(2-naphthylmethyl)amino]methyl}-N-
(tetrahydro-2H-pyran-2-yloxy) benzamide (1)
To a mixture of 4-{[(2-naphthylmethyl)amino]methyl}-N-(tet-
rahydro-2H-pyran-2-yloxy) benzamide [20] (1.0 g, 0.94 mmol) and
Et3N 0.71 mL (5.12 mmol) in CH3CN (20 mL) was added 2-bromoe-
thanol (0.36 mL,5.12 mmol). After stirring for 6 h at 60 ꢀC, the
mixture was evaporated to remove the solvent. Purification of the
resulting residue by SiO2 column chromatography (eluent: ethyl-
acetate/hexane ¼ 3/2) gave 1 as colorless amorphous (650 mg, 58.5%
5.1.6. 2-[{4-[(Hydroxyamino)carbonyl]benzyl}(2-
naphthylmethyl)amino]ethyl laurate (DD–K-182)
To a solution of 2a (16.5 mg, 26.8 mmol) in THF (1.0 mL) were
yield). IR (KBr)
g
/cmꢁ1: 3221, 2936,1719,1653,1275,1204,1051, 905;
added acetic acid (2.0 mL) and H2O (0.5 mL). After refluxing for
10 h, the solution was concentrated in vacuo. Purification of the
residue by preparative SiO2 TLC (eluent: ethylacetate/hexane ¼ 1/2)
1H NMR (CDCl3)
d: 1.62–1.86 (6H, m, CH2 ꢃ 3 of THP), 2.71 (2H, t,
J ¼ 5.6 Hz, N–CH2–CH2), 3.62 (2H, t, J ¼ 5.6 Hz, CH2–CH2–OH), 3.64–
3.68 (1H, m, –CHa–O of THP), 3.69 (2H, s, N–CH2–Ar), 3.78 (2H, s,
naph–CH2–N), 4.00 (1H, ddd, J ¼ 11.2, 9.2, 2.8 Hz, –CHb–O of THP)
5.07 (1H, t, J ¼ 2.8 Hz, O–CH–O of THP), 7.38–7.84 (11H, m, Ar–H),
gave DD–K-182 (11.5 mg, 21.6
IR (CHCl3)
NMR (CDCl3)
–(CH2)8–), 1.57–1.70 (m, 2H, COCH2CH2), 2.27 (t, 2H, J ¼ 7.6 Hz,
COCH2), 2.76 (t, 2H, J ¼ 5.6 Hz, NCH2CH2O), 3.70 (s, 2H, naph–
CH2N), 3.78 (s, 2H, NCH2–Ph), 4.18 (t, 2H, J ¼ 5.6 Hz, NCH2CH2O),
7.42–7.50 (m, 5H, Ar–H), 7.68–7.82 (m, 6H, Ar–H); 13C NMR (CDCl3)
mmol, 80.6% yield) as a brownish oil.
g
/cmꢁ1: 3405, 3210, 2927, 2854,1728,1612,1454, 763; 1H
d
: 0.87 (t, 3H, J ¼ 6.8 Hz, –CH2CH3), 1.03–1.25 (m, 16H,
8.81 (1H, s, CO–NH–O); 13C NMR (CDCl3)
d: 18.61, 24.99, 28.03,
54.98, 57.97, 58.54, 58.71, 62.63, 102.65, 125.79, 126.14, 126.83,
127.37, 127.59, 127.67, 127.73, 128.29, 129.09, 130.97, 132.79, 133. 24,
135.91, 143.33; HR-FAB-MS m/z: 435.2289 (calcd for C26H31N2O4,
435.2284).
d: 14.09, 22.66, 24.90, 24.93, 25.58, 29.18, 29.28, 29.32, 29.46, 29.60,
31.89, 33.87, 34.36, 52.04, 58.36, 59.00, 62.08, 125.66, 126.03,
126.87, 126.92, 127.34, 127.62, 127.66, 128.04, 129.01, 129.37, 132.83,
133.30, 136.54, 144.41, 173.78; HR-FAB-MS m/z: 533.3373 (calcd for
C33H45N2O4, 533.3379).
5.1.4. 2-[(2-Naphthylmethyl)(4-{[(tetrahydro-2H-pyran-2-
yloxy)amino]carbonyl}benzyl)amino] ethyl laurate (2a)
To a solution of 1 (26.2 mg, 60.3
m
mol) in CH2Cl2 (2.0 mL) were
mol), N,N-dicyclohex-
mol) and N,N-dimethyl-4-
mol). After being stirred for
added n-dodecanoic acid (12.1 mg, 60.3
m
ylcarbodiamide (DCC) (12.4 mg, 60.3
aminopyridine (DMAP) (7.37 mg, 60.3
m
m
5.1.7. 1-Cholest-5-en-3-yl 4-{2-[{4-[(hydroxyamino)
carbonyl]benzyl}(2-naphthylmethyl)
5 h at rt, the precipitate was filtered off and the filtrate was
concentrated in vacuo. Purification of the resulting residue by silica
gel column chromatography (eluent: ethylacetate/hexane ¼ 1/4)
amino]ethyl} succinate (CS–K-182)
To a solution of 2b (33.0 mg, 36.6 mmol) in THF (1.0 mL) were
added acetic acid (2.0 mL) and H2O (0.5 mL). After refluxing for 7 h,
the solution was concentrated in vacuo. Purification of the residue
by preparative SiO2 TLC (eluent: ethylacetate/hexane ¼ 2/3) gave
gave 2a (22.4 mg, 36.3
(CHCl3)
NMR (CDCl3)
–(CH2)8–), 1.43–1.58 (m, 6H), 1.84–1.92 (m, 2H, COCH2CH2), 2.28
mmol, 60.2% yield) as a colorless oil. IR
g
/cmꢁ1: 3010, 2927, 1728, 1683, 1456, 1112, 777, 669; 1H
: 0.87 (t, 3H, J ¼ 7.2 Hz, –CH2CH3), 1.25–1.28 (m, 16H,
d
CS–K-182 (18.4 mg, 22.5
IR (CHCl3)
mmol, 61.5% yield) as a brownish oil.
g
/cmꢁ1: 3421, 2935, 2854,1728,1612,1465,1365,1164; 1H

Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
4609
NMR (CDCl3)
d
: 0.67 (s, 3H, 180-CH3), 0.85 (d, 3H, J ¼ 1.6 Hz, 260 or
5.1.10. 2-(Dodecyldisulfanyl)ethyl 2-[{4-[(hydroxyamino)carbonyl]-
benzyl}(2-naphthylmethyl) amino]ethyl carbonate (DDTS–K-182)
To a solution of 4 (29.0 mg, 39.0 mol) in THF (0.4 mL) were
270-CH3), 0.87 (d, 3H, J ¼ 1.6 Hz, 260 or 270-CH3), 0.9–2.1 (m, 26H),
0.91 (d, 3H, J ¼ 6.4 Hz, 210-CH3), 1.01 (s, 3H,190-CH3), 2.30 (m, 2H, 40-
CH2), 2.56 (m, 4H, COCH2CH2CO), 2.74 (m, 2H, NCH2CH2), 3.68 (s, 2H,
NCH2–naph), 3.80 (s, 2H, NCH2Ph), 4.19 (t, 2H, J ¼ 4.8 Hz,
OCH2CH2N), 4.61 (m, 1H, 30-CH), 5.34 (s, 1H, 60-CH]C), 7.44–7.54
m
added acetic acid (0.8 mL) and H2O (0.2 mL). After refluxing for 6 h,
the solution was concentrated in vacuo. Purification of the residue
by preparative SiO2 TLC (eluent: ethylacetate/hexane ¼ 1/2) gave
(m, 5H) 7.69–7.80 (m, 6H); 13C NMR (CDCl3)
d: 11.85, 18.71, 19.28,
DDTS–K-182 (19.0 mg, 76.0% yield) as a brownish oil. IR (neat)
cmꢁ1: 3300, 3026, 2928, 2855, 1730, 1653, 705; 1H NMR (CDCl3)
g
d
/
:
21.02, 22.55, 22.80, 23.83, 24.27, 27.72, 28.00, 28.21, 29.18, 29.41,
29.68, 31.85, 31.88, 35.78, 36.19, 36.56, 36.91, 38.03, 39.52, 39.72,
42.32, 49.98, 52.22, 56.15, 56.67, 58.37, 59.26, 62.42, 74.64, 122.77,
125.68, 126.05, 126.94, 127.39, 127.63, 127.67, 128.09, 128.97, 132.84,
133.300, 136.50, 139.50, 144.36, 171.99, 172.15; HR-FAB-MS m/z:
819.5304 (calcd for C52H71N2O6, 819.5234).
0.88 (t, 3H, J ¼ 7.2 Hz, CH2(CH2)10CH3), 1.15–1.45 (m, 20H,
CH2(CH2)10CH3), 2.69 (t, 2H, J ¼ 7.2 Hz, SS–CH2(CH2)10CH3), 2.81 (t,
2H, J ¼ 5.2 Hz, NCH2CH2O), 2.90 (t, 2H, J ¼ 5.6 Hz, OCH2CH2–SS),
3.73 (s, 2H, NCH2–naph), 3.80 (s, 2H, PhCH2N), 4.24 (t, 2H,
J ¼ 5.2 Hz, NCH2CH2OCO), 4.34 (t, 2H, J ¼ 5.6 Hz, OCH2CH2–SS),
7.47–7.81 (m, 11H, Ar–H); 13C NMR (CDCl3)
d: 14.06, 14.14, 20.99,
5.1.8. 2-[(2-Naphthylmethyl)(4-{[(tetrahydro-2H-pyran-2-
yloxy)amino]carbonyl}benzyl)amino] ethyl 2-(2-
pyridinyldisulfanyl)ethyl carbonate (3)
22.64, 28.47, 29.10, 29.18, 29.30, 29.46, 29.55, 29.59, 29.60, 29.65,
31.88, 36.82, 39.20, 51.91, 58.39, 59.03, 60.41, 65.83, 65.90, 125.66,
126.02, 126.87, 127.37, 127.63, 128.07, 128.99, 132.82, 133.28, 136.32,
144.05, 154.91; HR-FAB-MS m/z: 654.3243 (calcd for C36H51N2O5S2
[M þ Hþ], 655.3239).
To a solution of 1 (100 mg, 0.230 mmol) in CH2Cl2 (2.0 mL)
were added DMAP (167 mg, 1.38 mmol) and triphosgene (23.9 mg,
0.0805 mmol). After being stirred for 15 min at rt, to the solution
was added 2-(2-pyridinyldisulfanyl)ethanol [24,29] (43.0 mg,
0.230 mmol) and the solution was stirred for another 7 h at rt. The
reaction mixture was diluted with CHCl3 (20 mL), washed with
water, dried over Na2SO4 and concentrated in vacuo. Purification
of the resulting residue by preparative SiO2 TLC (eluent: ethyl-
acetate/hexane ¼ 3/2) gave 3 (55.0 mg, 36.9% yield) as a colorless
5.2. Preparation of nanoparticles and nanoplexes
OH-Chol was synthesized as previously described [7]. Tween 80
was obtained from NOF Co. Ltd. (Tokyo, Japan). NPs were prepared
by a modified ethanol injection method as previously described [7].
For example, in the case of NP–K-182–DD, OH-Chol:Tween 80:K-
182–DD ¼ 85:5:10 molar ratio (¼10:1.42:1.17, weight) was dis-
solved in about 5 mL of ethanol, then the ethanol was removed
with a rotary evaporator till 1–2 mL was left. Next, a constant
volume of water was added to the ethanol solution. NPs formed
instantly after further evaporation of the residual ethanol. The
concentration of OH-Chol was adjusted to 1 mg/mL in the final NP
suspension with drop of water. Then the nanoparticle suspension
oil. IR (neat)
703; 1H NMR (CDCl3)
g
/cmꢁ1: 3400, 3027, 3010, 2950, 2824, 1743, 1684,
d
:1.60–1.86 (m, 6H, CH2 ꢃ 3 of THP), 2.79 (t,
2H, J ¼ 6 Hz, CH2CH2–SS), 3.05 (t, 2H, J ¼ 3.2 Hz, NCH2CH2), 3.64
(m, 1H, –CHa–O of THP), 3.72 (s, 2H, NCH2–naph), 3.80 (s, 2H,
PhCH2N), 3.99 (ddd, 1H, J ¼ 11.2, 9.2, 2.8 Hz, –CHb–O of THP), 4.23
(t, 2H, J ¼ 3.2 Hz, CH2CH2OCO), 4.35 (t, 2H, J ¼ 6.0 Hz, OCOO–CH2–
CH2), 5.08 (m, 1H, OCHO of THP), 7.07–7.81 (m, 15H, Ar–H), 8.45
(m, 1H, Ar–H); 13C NMR (CDCl3)
d: 18.65, 24.99, 28.07, 37.00, 51.79,
was filtered through 0.45-mm Millex-HA filters (Millipore, Cork,
58.33, 58.86, 62.61, 65.34, 65.84, 76.68, 77.00, 77.31, 102.60, 119.88,
120.88, 125.63, 125.99, 126.87, 127.24, 127.32, 127.62, 128.04, 128.82
132.78, 133.25, 136.32, 137.05, 143.73, 149.68, 154.76, 159.47; HR-
FAB-MS m/z: 648.2205 (calcd for C34H38N3O6S2 [M þ Hþ],
648.2202).
Ireland) to sterilize it. The particle size distributions were measured
by the dynamic light scattering method (ELS-Z2, Otsuka Electronics
Co., Ltd., Osaka, Japan), at 25 ꢀC after the dispersion was diluted to
an appropriate volume with water.
5.3. Cell culture
5.1.9. 2-(Dodecyldisulfanyl)ethyl 2-[(2-naphthylmethyl)(4-
{[(tetrahydro-2H-pyran-2-yloxy)
PC-3 cells were supplied by the Cell Resource Center for
Biomedical Research, Tohoku University (Miyagi, Japan). The cells
were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% heat-inactivated fetal bovine serum (FBS)
(Invitrogen) and kanamycin (100
humidified atmosphere.
For the preparation of PC-3 cells stably expressing Gaussia
luciferase (Gluc), PC-3 cells were plated on 35-mm culture dishes.
Twenty-four hours later, the cells were transfected with 2 mg of
pCMV-Gluc (New England Biolabs, MA, USA) using lipofectamine
2000 reagents (Invitrogen). The transfected cells were selected in
amino]carbonyl}benzyl)amino]ethyl carbonate (4)
To a solution of 3 (55 mg, 0.085 mmol) in DMSO (1.5 mL) was
added dodecanethiol (0.02 mL, 0.085 mmol). After being stirred for
2.5 h at rt, the reaction mixture was diluted with CHCl3 (10 mL),
washed with water, dried over Na2SO4 and concentrated in vacuo.
Purification of the resulting residue by preparative SiO2 TLC (eluent:
ethylacetate/hexane ¼ 3/2) gave 4 (59.0 mg, 93.9% yield) as a color-
m
g/mL) at 37 ꢀC in a 5% CO2
less oil. IR (neat)
NMR (CDCl3)
: 0.88 (3H, t, J ¼ 7.2 Hz, CH2 (CH2)9CH3) 1.15–1.35 (18H,
g
/cmꢁ1: 3400, 3030, 2927, 2855, 1744, 1683; 1H
d
m, CH2(CH2)9CH3), 1.35 (2H, t, J ¼ 7.2 Hz, CH2(CH2)9CH3), 1.62–1.86
(6H, m, CH2 ꢃ 3 of THP), 2.69 (2H, t, J ¼ 7.2 Hz, SS–
CH2CH2(CH2)9CH3), 2.81 (2H, t, J ¼ 6.0 Hz, N–CH2–CH2), 2.90 (2H, t,
J ¼ 6.8 Hz, OCH2CH2–SS), 3.64 (1H, m, CH2CHa–O of THP), 3.73 (2H, s,
NCH2–naph), 3.80 (2H, s, naph–CH2N), 4.00 (1H, ddd, J ¼ 11.2, 9.2,
2.8 Hz, CH2CHb–O of THP), 4.25 (2H, t, J ¼ 6.0 Hz, NCH2CH2–OCO),
4.34 (2H, t, J ¼ 6.8 Hz, COOCH2CH2S), 5.07 (1H, m, OCHO of THP),
medium with 800 mg/mL G418 sulfate for 2 weeks. G418-resistant
colonies were subcultured and established as a permanent cell line
transduced with pCMV-Gluc (PC-3-Gluc) and were used for
subsequent experiments.
7.45–7.93 (11H, m, Ar–H); 13C NMR (CDCl3)
d: 14.05, 18.68, 22.63,
5.4. Transfection
24.99, 28,06. 28.46, 29.09, 29.16, 29.28, 29.44, 29.53, 29.58, 31.86,
36.82, 39.09, 39.19, 41.18, 51.82, 58.35, 58.91, 60.35, 62.70, 65.81,
65.87, 76.68, 77.00, 77.31, 102.72, 125.61, 125.98, 126.86, 127.22,
127.32, 127.62, 128.04, 128.58, 128.87, 129.71, 130.78, 132.80, 133.27,
136.34, 143.83, 154.87. HR-FAB-MS m/z: 738.3816 (calcd for
C41H59N2O6S2 [M þ Hþ], 739.3815).
Based on a preliminary experiment [25], the optimized ratio
(þ/ꢁ) of cationic lipid to plasmid DNA was determined as 3:1. The
nanoplex at a charge ratio (þ/ꢁ) of 3/1 was formed by addition of
each NP (9.5 mL) to plasmid DNA with gentle shaking and leaving at
room temperature for 10 min. For transfection, each nanoplex was

4610
Y. Ishii et al. / European Journal of Medicinal Chemistry 44 (2009) 4603–4610
diluted in 1 mL of medium supplemented with 10% FBS and then
incubated with the cells for 24 or 48 h.
protocol. The IC50 values represent the molar concentrations (
required to inhibit the HDACs by 50%.
mM)
5.5. Luciferase assay
Acknowledgments
PC-3 and PC-3-Gluc cells were plated on 96-well culture dishes.
For transfection of pCMV-Gluc, each nanoplex of pCMV-Gluc was
diluted in 1 mL of medium supplemented with 10% FBS and then
incubated with PC-3 cells for 24 h. For transfection of pGL3-basic
(Promega, Madison, WI, USA), each nanoplex of pCMV-basic was
diluted in 1 mL of medium supplemented with 10% FBS and then
incubated with PC-3-Gluc cells for 24 h or 48 h. The level of Gluc
activitywasevaluatedbyluciferaseactivityinthemedium, whichwas
measured as counts per sec (cps)/culture medium (mL) using
a Gaussia Luciferase Assay Kit (New England BioLabs, Inc., MA, USA).
Glucactivity(%)wascalculatedasrelativetotheGlucactivity(cps/mL)
of NP.
This research was supported by a Grant-in-Aid for Scientific
Research (C) 19590110 and ‘‘Strategic Project to Support the
Formation of Research Bases at Private Universities’’: Matching
Fund Subsidy from MEXT (Ministry of Education, Culture, Sports,
Science and Technology), 2008–2012.
References
[1] I.M. Verma, N. Somia, Nature 389 (1997) 239–242.
[2] H. Chong, R.G. Vile, Gene Ther. 3 (1996) 624–629.
[3] E. Otto, A. Jones-Trower, E.F. Vanin, K. Stambaugh, S.N. Mueller, W.F. Anderson,
G.J. McGarrity, Human Gene Ther. 5 (1994) 567–575.
5.6. Immunoblotting
[4] E.S. Song, V. Lee, C.D. Surh, A. Lynn, D. Brumm, D.J. Jolly, J.F. Warner, S. Chada,
Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1943–1948.
[5] S. Hasegawa, N. Hirashima, M. Nakanishi, Bioorg. Med. Chem. Lett. 12 (2002)
1299–1302.
[6] M. Nakanishi, Curr. Med. Chem. 10 (2003) 1289–1296.
[7] Y. Hattori, H. Kubo, K. Higashiyama, Y. Maitani, J. Biomed. Nanotechol. 1 (2005)
176–184.
[8] I.S. Zuhorn, R. Kalicharan, D. Hoekstra, J. Biol. Chem. 277 (2002) 18021–18028.
[9] Y. Hattori, A. Hagiwara, W. Ding, Y. Maitani, Bioorg. Med. Chem. Lett. 18 (2008)
5228–5232.
PC-3 cells were seeded in a 35-mm culture dish and incubated
overnight. The cells at 30% confluency were transfected with
nanoplexes containing pGL3-basic in the presence or absence of the
HDAC inhibitor and then incubated for 24 h. The cells were sus-
pended in lysis buffer (1% Triton-X 100 in phosphate-buffered
saline pH 7.4 (PBS)), and then centrifuged at 15,000 rpm for 10 min.
The supernatants (3
mg protein) were resolved on a 12% sodium
dodecyl sulfate–polyacrylamide gel by electrophoresis (SDS–PAGE)
and transferred to a polyvinylidene difluoride (PVDF) membrane
(FluoroTransÒ W, PALL Gelman Laboratory, Ann Arbor, MI, USA).
Acetylated histone H3 was detected by rabbit anti-human acetyl
histone H3 antibody (Sigma Chemical Co., St. Louis, MO, USA). The
goat anti-rabbit IgG peroxidase conjugate (Santa Cruz Biotech-
nology, Inc., Santa Cruz, CA, USA) was used as secondary antibody.
These proteins were detected with peroxidase-induced chem-
iluminescence (Super Signal West Pico Chemiluminescent
Substrate, Pierce, Rockford, IL, USA).
[10] I.S. Zuhorn, J.B.F.N. Engberts, D. Hoekstra, Eur. Biophys. J. 36 (2007) 349–362.
[11] J. Zabner, A.J. Fasbender, T. Moninger, K.A. Poellinger, M.J. Welsh, J. Biol. Chem.
270 (1995) 18997–19007.
[12] E. Dauty, A.S. Verkman, J. Biol. Chem. 280 (2005) 7823–7828.
[13] E.E. Vaughan, R.C. Geiger, A.M. Miller, P.L. Loh-Marley, T. Suzuki, N. Miyata,
D.A. Dean, Mol. Ther. 16 (2008) 1841–1847.
[14] M. Goettlicher, S. Minucci, P. Zhu, O.H. Kramer, A. Schimpf, S. Giavara,
J.P. Sleeman, F. Lo-Coco, C. Nervi, P.G. Pelicci, T. Heinzel, EMBO J. 20 (2001)
6969–6978.
[15] A. Saito, T. Yamashita, Y. Mariko, Y. Nosaka, K. Tsuchiya, T. Ando, T. Suzuki,
T. Tsuruo, O. Nakanishi, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 4592–4597.
[16] A. Kalita, C. Bonfils, C. Maroun, M. Fournel, G. Rahil, T.P. Yan, A. Lu, G.P. Reid,
J.M. Besterman, Z. Li, Abstract of AACR-NCI-EORTC Conference, Philadelphia,
USA, 2005. C216.
5.7. Cytotoxicity
[17] T. Beckers, C. Burkhardt, H. Wieland, P. Gimmnich, T. Ciossek, T. Maier,
K. Sanders, Int. J. Cancer 121 (2007) 1138–1148.
[18] M. Yoshida, M. Kijima, M. Akita, T. Beppu, J. Biol. Chem. 265 (1990)
17174–17179.
Cytotoxicity upon transfection using NP–K-182–DD was evalu-
ated with a cell proliferation assay kit (Dojindo, Kumamoto, Japan).
PC-3 cells were placed in a 96-well plate in medium containing 10%
FBS, and were transfected at various concentrations of nanoplex.
After 24 h of incubation, the medium was removed, and the cells
were treated with a WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
salt) solution (10 mL) in medium containing serum (100 mL) for
30 min. Cell viability was expressed as relative to the absorbance at
450 nm of untransfected cells.
[19] V.M. Richon, S. Emiliani, E. Verdin, Y. Webb, R. Breslow, R.A. Rifkind,
P.A. Marks, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 3003–3007.
[20] Y. Nagaoka, T. Maeda, Y. Kawai, D. Nakashima, T. Oikawa, K. Shimoke,
T. Ikeuchi, H. Kuwajima, S. Uesato, Eur. J. Med. Chem. 41 (2006) 697–708.
[21] T. Maeda, Y. Nagaoka, Y. Kawai, N. Takagaki, C. Yasuda, S. Yogosawa, Y. Sowa,
T. Sakai, S. Uesato, Biol. Pharm. Bull. 28 (2005) 849–853.
[22] T. Maeda, Y. Nagaoka, H. Kuwajima, C. Seno, S. Maruyama, M. Kurotakid,
S. Uesato, Bioorg. Med. Chem. 12 (2004) 4351–4360.
[23] W.A. Henne, D.D. Doorneweerd, A.R. Hilgenbrink, S.A. Kularatne, P.S. Low,
Bioorg. Med. Chem. Lett. 16 (2006) 5350–5355.
[24] C.P. Leamon, J.A. Reddy, I.R. Vlahov, M. Vetzel, N. Parker, J.S. Nicoson, L.C. Xu,
E. Westrick, Bioconjugate Chem. 16 (2005) 803–811.
[25] Y. Hattori, W. Ding, Y. Maitani, J. Controlled Release 120 (2007) 122–130.
[26] C.A. Hassig, S.L. Schreiber, Curr. Opin. Chem. Biol. 1 (1997) 300–308.
[27] T. Kouzarides, Curr. Opin. Genet. Dev. 9 (1999) 40–48.
[28] B.D. Strahl, C.D. Allis, Nature (London) 403 (2000) 41–45.
[29] L.R. Jones, E.A. Goun, R. Shinde, J.B. Rothbard, C.H. Contag, H. Christopher,
P.A. Wender, J. Am. Chem. Soc. 128 (2006) 6526–6527.
5.8. Evaluation of histone deacetylase inhibitory activities
The IC50 values were measured by using CycLex HDAC Assay Kit
(CycLex Co. Ltd, Nagano, Japan) according to the manufacturer’s