Characterization and histone deacetylase inhibitory activity of three novel fluorescent benzamide derivatives

Article abstract of DOI:10.1002/bkcs.10104

New fluorescent benzamide derivatives were developed by using four different aromatic groups with expanding sizes from thiophene to naphthalene, anthracene, and pyrene in an attempt to identify a novel histone deacetylase (HDAC) inhibitor. The compounds e

Full text of DOI:10.1002/bkcs.10104

Article
DOI: 10.1002/bkcs.10104
BULLETIN OF THE
I. S. Shin et al.
KOREAN CHEMICAL SOCIETY
Characterization and Histone Deacetylase Inhibitory Activity of Three
Novel Fluorescent Benzamide Derivatives
In Su Shin,† Dong Gyu Lee,† Jin Hee Lee,† Hwan-Jeong Jeong,‡ and Young Jun Seo†,§,
*
†Department of Chemistry, Chonbuk National University, Jeonju-si 561-756, South Korea.
*E-mail: yseo@jbnu.ac.kr
‡Molecular Imaging Therapeutic Medicine Research Center, Department of Nuclear Medicine, Chonbuk
National University Medical School & Hospital, Jeonju-si 561-712, South Korea
§Department of Bioactive Material Sciences, Chonbuk National University, Jeonju-si 561-756, South Korea
Received September 18, 2014, Accepted October 30, 2014
New fluorescent benzamide derivatives were developed by using four different aromatic groups with expand-
ing sizes from thiophene to naphthalene, anthracene, and pyrene in an attempt to identify a novel histone dea-
cetylase (HDAC) inhibitor. The compounds exhibited red shift absorption and emission dependent on the size
of the aromatic group as well as a high fluorescence efficiency with high quantum yields. However, the HDAC
binding affinity of the compounds with polyaromatic modification of the substituent was very low (IC50 ran-
ging from 46 to 101 μM) in contrast to that of the monoaromatic substituted compound (<0.12 μM). Likely, the
modification of the aromatic substituent results in the enzyme's inability to bind the inhibitors due to space
limitation at the binding site. Nevertheless, we believe that these results will be useful in the design of new
fluorescent HDAC inhibitors based on the benzamide scaffold with diverse applications as diagnostic tools
targeting the HDAC enzyme, which has an important role in the prevention of a number of diseases.
Keywords: HDAC enzyme, Benzamide drug, Inhibitor, Probe, Fluorophore
Introduction
Recently, we reported the synthesis of new types of benza-
mide HDAC inhibitors that were designed to target the brain
as they were able to penetrate the blood–brain barrier.13 These
new compounds showed a high binding affinity to HDAC, and
we were able to observe the specific localization of the inhibi-
torsinthespecificregionofinterest.13,14 Eventhoughmanydif-
ferent types of benzamide drugs have been developed, there are
noreportsofsize-expandedfluorescentbenzamides.Thedevel-
opment of a novel fluorescent benzamide-targeting HDAC
material could provide a useful tool for epigenetic research as
a specific marker of histone deacetylation in various diseases.
Fluorescence isspecialphoto-physicalproperty thatcouldbe
used for diverse probing systems and diagnostic tools for
in vitro as well as in vivo applications.15 Fluorescence probing
systems show remarkable resolution, a high sensitivity toward
target recognition, fully three-dimensional imaging, and highly
quantitative results and reproducibility.16 Thus, the develop-
ment of benzamide-based fluorescent compounds would be
valuable for diverse applications in epigenetic research.
To achieve this, we would have to modify the benzamide
structure with a highly fluorescent moiety that would provide
high quantum yields without interruption of the binding affin-
ity toward HDAC. As the benzamide scaffold is the essential
structure for binding to HDAC,14,17 we decided to modify the
substituentontheR1 positionandscrutinizethesizesensitivity
of the binding site (Scheme 2). To our knowledge, no rational
studies have been published in which the effect of the size of
the R1-substituent on HDAC inhibition has been investigated.
Epigenetics involves the study of changes in gene expression
that arenot caused by changes intheDNA sequence, butrather
by chemical modifications to DNA, including DNA methyla-
tion, post-transcriptional regulation with noncoding RNA,
histone acetylation, and deacetylation that controls the folding
and unfolding of DNA, chromatin remodeling, and telomere
regulation of the DNA and RNA secondary structure.1,2
Among these, recently discovered histone acetylase, which
induces acetylation of histones causing DNA unfolding as a
starting signal for gene expression, and histone deacetylase
(HDAC), which induces the deacetylation of histones resulting
in DNA folding to repress gene expression, are both essential
gene regulating processes.3 The disruption of these enzyme
functions is related to many different types of diseases such as
cancer, central nervous system disorders, and heart disease.4–7
Particularly, the deacetylation of histones with HDAC is a
crucial repression process that regulates gene repression,
apoptosis, angiogenesis, and cell cycle arrest during transcrip-
tion.8,9 Therefore, intensive research efforts have been under-
taken to develop HDAC inhibitors. In 2006, the FDA
approved the first HDAC inhibitor, SAHA, for the treatment
of T-cell lymphoma.10,11 First-generation inhibitors, such as
SAHA, were based on the hydroxamate structure. Currently,
other inhibitors are being developed and are in phase I–III clin-
ical trials (Scheme 1). The most promising of these second
generation inhibitors is a benzamide derivative.12
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Fluorescent Benzamide Drug Targeting HDAC Enzyme
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N
compound 4 (200 mg, 0.410 mmol) was dissolved in dry
tetrahydrofuran (2 mL), and 2-bromonaphthalene (95.13
mg, 0.370 mmol), Tetrakis(triphenylphosphine)palladium(0)
(4.62 mg, 0.004 mmol), and K2CO3 (161.97 mg, 1.172 mmol)
were added, the mixture was heated under reflux (80 ꢀC) for
3 h and cooled to room temperature. The resulting mixture
was extracted with EtOAc and water; the combined organic
phases were dried with Na2SO4, filtered, and evaporated to
dryness under reduced pressure. The crude product was puri-
fied through column chromatography using ethyl acetate:hex-
ane (10:30%) to obtain the title compound as a yellow-brown
O
O
H
N
OH
NH
N
H
N
H
H
3
O
N
NH2
O
O
MS-275
SAHA
HO
O
O
O
N
O
H
NH2
N
N
H
Trichostatin A (TSA)
Cl-994
1
Scheme 1. Known HDAC inhibitor drugs.
solid (68 mg, 34%). H NMR (400 MHz, chloroform-d1) δ
(ppm) 8.26 (s 1H, NH), 8.15–8.12 (t, 3H, J = 7.2 Hz, ArH),
8.01–7.97 (m, 3H, ArH, NH), 7.84–7.81 (dd, 1H, J = 1.6Hz,
ArH), 7.66–7.61 (m, 2H, ArH), 7.56–8.51 (m, 2H, ArH),
4.67 (s, 1H, Ph-CH2), 1.69–1.65 (m, 9H, C(CH3)3); 13C
NMR (100 MHz chloroform-d1) 165.071, 154.508,
141.020, 138.455, 136.908, 133.680, 133.360, 132.413,
130.758, 129.251, 129.004, 128.420, 128.173, 127.984,
127.646, 127.366, 127.259, 126.074, 125.753, 125.366,
125.045, 124.732, 124.584, 34.661, 28.075(2C); LCMS calcd
for C29H27ClN2O3 mol. wt.: 486.99 [M + H]+ m/z 487.28.
1
Compound 5–2: H NMR (400 MHz, chloroform-d1) δ
(ppm) 8.96–8.94 (dd, 2H, J = 8.4 and 8.4 Hz, ArH),
8.17–8.15(d, 4H, J = 8.4 Hz, ArH), 8.07–8.05 (d, 1H, J =
7.6 Hz, NH), 7.87–7.53 (m, 9H, ArH), 7.13 (s, 1H, ArH),
4.78 (s, 2H, Ph-CH2), 1.76–1.74 (d, 9H, C(CH3)3); 13C
NMR (100 MHz chloroform-d1) 165.864, 164.143,
162.999, 161.352, 160.858, 160.554, 160.043, 159.862,
159.615, 159.253, 158.199, 157.894, 157.631, 156.964,
156.660, 155.910, 155.688, 155.910, 155.688, 155.169,
152.024, 151.440, 147.068, 145.241, 89.308, 70.875,
63.111, 58.518, 57.834, 48.630, 42.225; LCMS calcd for
C33H29ClN2O3 mol. wt.: 537.05 [M + H]+ m/z 537.37.
Scheme 2. Designed fluorescent benzamide derivatives: EB1 (thio-
phene), EB2 (naphthalene), EB3 (anthracene), and EB4 (pyrene).
1
Compound 5–3: H NMR (400 MHz, chloroform-d1) δ
(ppm) 8.39–8.33 (m, 5H, ArH and NH), 8.27 (s, 2H, ArH),
8.22–8.19 (m, 3H, ArH), 8.14–8.12 (d, 3H, J = 7.6 Hz,
ArH), 7.65–7.64 (m, 2H, ArH), 7.56–7.54 (d, 2H, J = 8.4
Hz, ArH), 7.17 (s, 1H, ArH), 4.69 (s, 2H, Ph-CH2),
1.77–1.72 (d, 9H, C(CH3)3); 13C NMR (100 MHz chloro-
form-d1) 165.120, 154.755, 138.966, 136.315, 133.985,
131.433, 130.964, 130.676, 129.276, 128.609, 128.445,
128.313, 127.811, 127.671, 127.564, 127.465, 127.383,
125.991, 125.094, 124.880, 124.600, 124.419, 120.229,
81.660, 60.379, 45.239, 28.322; LCMS calcd for
C35H31ClN2O3 mol. wt.: 563.09 [M + H]+ m/z 563.39.
General Procedure for Amination at the Benzylic Position.
Compound 6–1: A solution of compound 5 (30 mg, 0.061
mmol) in dimethyl amine (10 mL) was stirred under nitrogen
atmosphere for 3 h at room temperature, while monitoring
using TLC. The solvent was evaporated, and purified through
column chromatography using ammonia solution in methyl
alcohol:dichloromethane:methanol (1:10:5%) to obtain the
title compound as a deep orange crystal (16.1 mg, 53%). 1H
NMR (400 MHz, chloroform-d1) δ (ppm) 8.33–8.31 (d,
1H, J = 1.6 Hz, NH), 8.20 (s, 1H, NH), 8.15–8.13 (d, 2H,
To examine the extent to which bulkiness would be tolerated
at the R1 position, four fluorescentcompoundsof differentsizes
were selected for attachment to the benzamide structure.
After establishing that a phenyl substituent in the R1 posi-
tion does not result in a fluorescent benzamide product, a
heterocyclic thiophene derivative of which the synthesis has
already been reported14 was selected as the smallest fluores-
cent moiety. Three poly-aromatic compounds of increasing
size were subsequently chosen, starting with naphthalene, fol-
lowed byanthracene, which is a well-known fluorescent mate-
rial, and finally, pyrene as the largest fluorescent moiety.
Pyrene is the most well-known fluorescent material and nor-
mally appears blue, but turns green when the excimer is
generated.18
Experimental
General Procedure for Coupling Aromatic Hydrocarbon
to the Phenyl Amine. Compound 5–1: A suspension of
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J = 8.4 Hz, ArH), 8.06–8.01 (m, 3H, ArH), 7.92–7.89 (dd, 1H,
J = 2 and 1.6 Hz, ArH), 7.71–7.63 (m, 3H, ArH), 7.59–7.55
(m, 3H, ArH), 7.21 (s, 1H, ArH), 3.66–3.65 (d, 2H, Ph-
CH2), 2.44 (s, 6H, N(CH3)2), 1.71 (s, 9H, C(CH3)3); 13C
NMR (100 MHz chloroform-d1) 166.026, 154.912,
143.378, 139.081, 137.583, 133.911, 133.285, 132.956,
131.474, 129.663, 129.507, 128.683, 128.527, 127.885,
127.737, 127.350, 126.559, 126.230, 125.967, 125.678,
125.160, 125.061, 124.839, 124.435, 64.166, 45.659(2C),
28.601(3C); LCMS calcd for C31H33N3O3 mol. wt.: 495.61
[M + H]+ m/z 496.17.
2H, J = 8.4 Hz, ArH), 7.83–7.71 (m, 2H, ArH and NH),
7.67–7.39 (m, 11H, ArH), 4.34–4.25 (s, 2H, Ph-CH2),
2.72–2.70 (s, 6H, (NCH3)2); 13C NMR (100 MHz, MeOH-
d4) 132.757, 132.247–132.074(8C), 131.827–131.456(4C),
130.403, 130.098, 129.835 129.168, 128.863, 128.114,
127,892, 127.373, 124.228, 123.644, 61.522(2C), 43.221,
43.081; LCMS calcd for C30H27N3O mol. wt.: 445.55 [M +
H]+ m/z 446.2.
Compound 7–3: 1H NMR (400 MHz, MeOH-d4) δ (ppm)
8.42–8.40 (t, 2H, J = 7.6 Hz, ArH), 8.17–8.08 (m, 6H, ArH
and NH), 8.03–7.90 (m, 9H, ArH), 4.33 (s, 2H, Ph-CH2),
2.81 (s, 6H, N(CH3)2); 13C NMR (100 MHz, MeOH-d4)
144.883, 137.120, 137.120, 134.774, 133.251, 132.930,
132.387, 131.917, 130.897, 129.958, 129.686, 129.431,
128.937, 128.526, 128.451, 128.402, 128.081, 127.357,
127.200, 126.533, 126.204, 126.130, 125.891, 125.825,
125.743, 61.620, 43.138; LCMS calcd for C32H27N3O mol.
wt.: 469.58 [M + H]+ m/z 470.2.
1
Compound 6–2: H NMR (400 MHz, chloroform-d1) δ
(ppm) 8.62–8.60 (d, 2H, J = 8.0 Hz, ArH), 8.53–8.51 (d,
2H, J = 8.0 Hz, ArH), 8.28–8.19 (m, 6H, ArH), 8.05–7.90
(m, 7H, ArH and NH), 7.83–7.81 (dd, 1H, J = 8.4 and 8.4
Hz, ArH), 3.84–3.83 (s, 2H, Ph-CH2), 3.22 (s, 6H,
N(CH3)2), 1.95–1.94 (m, 9H, C(CH3)3); 13C NMR (100
MHz, chloroform-d1) 163.199, 160.754, 160.243, 160.062,
159.453, 158.399, 158.094, 157.823, 157.156, 156.851,
156.110, 155.888, 155.369, 152.225, 151.640, 89.508,
71.075, 63.311, 48.830, 42.425; LCMS calcd for
C35H35N3O3 mol. wt.: 545.67 [M + H]+ m/z 546.3.
Results and Discussion
For the synthesis, a Boc-protected 2-nitro-4-bromophenyl
amine compound was used as starting material (Scheme 3).
A Suzuki coupling method was used for attaching a boronic
ester to the R1 position, which, after reduction of the nitro
group to give 3, was then coupled with chlorobenzoyl chloride
to form the benzamide scaffold 4, ready for the attachment of
the fluorescent substituents. The fluorescent materials were
subjected to Suzuki coupling to produce EB2, EB3, and
EB4, respectively (Scheme 1). An N-dimethyl group
was introduced at the R2 position to control the polarity, which
was hydrophilic, after which the amino group of 6 (Scheme 3)
was deprotected by removing the Boc-group to free the impor-
tant site that binds with the active pocket of the HDAC
enzyme.10
1
Compound 6–3: H NMR (400 MHz, chloroform-d1) δ
(ppm) 8.25–8.23 (d, 1H, J = 9.6 Hz, ArH), 8.19–8.14 (m,
3H, ArH), 8.08 (s, 2H, NH), 8.06–8.05 (d, 1H, J = 3.6 Hz,
ArH), 8.02–8.00 (m, 2H, ArH), 7.98–7.95 (m, 5H, ArH),
7.51–7.43 (m, 4H, ArH), 4.599–4.568 (d, 2H, Ph-CH2),
2.03 (s, 6H, N(CH3)2), 1.09–1.02 (m, 9H, C(CH3)3); 13C
NMR (100 MHz, chloroform-d1) 154.443, 140.958,
138.776, 136.027, 133.820, 131.137, 130.988, 13.750,
130.667, 130.429, 128.823, 128.387-128.156(6C), 128.008,
127.514, 127.336, 127.292, 127.169, 127.078, 125.798,
125.678, 124.798, 124.567, 124.312, 124.098, 81.413,
60.066, 44.976, 31.606(2C), 29.375–28.000(3C).
General Procedure for Boc Deprotection. Compound 7–1:
A suspension of compound 6 (10 mg, 0.201 mmol) was dis-
solved in dry dichloromethane (0.4 mL), and trifluoroacetic
acid (TFA) (0.59 mg, 0.523 mmol) was added under drop-
wise. The solution was stirred under room temperature. After
stirring for 2 h, the solvent was evaporated, and diethyl
ether was added to the product, and vigorously stirred for
1 h. The solvent was dried in a natural state to obtained yellow-
ish-brown solid product (7 mg, 84.9%) 1H NMR (400 MHz,
MeOH-d4) δ (ppm) 8.23–8.20 (d, 1H, J = 8.4 Hz, ArH),
8.12–8.01 (d, 2H, J = 8.4 Hz, ArH), 8.023–8.00 (d, 1H, J =
9.2 Hz, NH), 7.92–7.79 (m, 6H, ArH), 7.65–7.57 (m, 3H,
ArH), 7.46–7.38 (m, 3H, ArH), 4.39–4.36 (s, 2H, Ph-CH2),
2.86–2.83 (d, 6H, N(CH3)2); 13C NMR (100 MHz, chloro-
form-d1) 162.690, 133.053, 132.214, 129.917, 129.670,
129.250, 129.044, 128.666, 128.757, 127.488, 127.266,
127.143, 126.961, 126.665, 126.525, 126.344, 126.138,
125.998, 125.521, 119.865, 61.645, 61.522, 61.299, 44.760,
43.155; LCMS calcd for C26H25N3O mol. wt.: 395.50 [M +
H]+ m/z 396.1.
Next, the UV and fluorescence spectra of the synthesized
benzamide compounds were recorded. All compounds exhib-
ited a consistent red shift pattern in their UV spectra that
depended on the substituent size (EB1 < EB2 ≤ EB3 < EB4)
(Figure 1(a)). This observation was expected, because the
expansionofthearomatic ringresultsinanincrease intheelec-
tronic conjugation.
To analyze the fluorescent properties of the benzamide
compounds, the highest absorption wavelength of each com-
pound was used as an excitation wavelength. Neither the
unsubstituted benzamide derivative nor the simple phenyl
derivative (data not shown) exhibited any fluorescence. How-
ever, once the more conjugated systems were attached to the
R1 position, all these compounds exhibited fluorescence sig-
nals, starting with the two aromatic rings of naphthalene
and ending with anthracene and pyrene, all of which showed
strong fluorescence from 400 to 450 nm (Figure 1(b)). The
emission wavelength displayed a red shift that increased along
with an increase in the size of the conjugated aromatic system.
To characterize the efficiency of our new polyaromatic fluo-
rescent compounds, we measured the quantum yield (Φ) for
Compound 7–2: 1H NMR (400 MHz, MeOH-d4) δ (ppm)
8.70–8.62 (dd, 2H, J = 8.2 and 8.2 Hz, ArH), 8.04–8.02 (d,
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Fluorescent Benzamide Drug Targeting HDAC Enzyme
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O
O
O
O
Boc
B
O
O
B
HN
B
NO
O
4
2
c
a
b
N
H N
2
H
Cl
NH
O N
2
NH
3
Br
Boc
NH
2
Boc
Boc
1
R
R
1
1
R
1
O
O
7
O
5
e
d
N
H
f
N
H
N
H
N
NH
N
NH
2
Cl
NH
Boc
Boc
6
R1:
5-2, 6-2, 7-2
5-1, 6-1, 7-1
5-3, 6-3, 7-3
Scheme 3. (a) Pinacol diborane, Pd(dppf )Cl2, AcOK, 1,4-dioxane, 80 ꢀC, overnight; (b) 10% Pd/C, H2, 45 psi, EtOAc, room temperature (rt),
overnight; (c) 4-chloromethyl benzoyl chloride, Et3N, CH2Cl2, 0 ꢀC-rt, 4 h; (d) R2, Pd black, MeOH, reflux, 4 h; (e) (CH3)2NH2, rt, 36 h; (f )
TFA, CH2Cl2, rt, 3 h.
(a)
(b)
1.0
0.8
0.6
0.4
0.2
0.0
0.09
0.06
0.03
0.00
EB1
EB2
EB3
EB4
EB1
EB2
EB3
EB4
300
350
400
450
500
550
600
650
200
300
400
500
600
λ(nm)
λ(nm)
Figure 1. (a) UV absorption spectra of EB1, EB2, EB3, and EB4. (b) Normalized fluorescence spectra of EB1, EB2, EB3, and EB4 at room
temperature. All samples were measured in concentrations of 1.0 μM in MeOH at pH 7, and were excited at each UV absorption max.
each compound using diphenyl anthracene as a reference
material (Table 1). As expected, EB4 and EB1 showed the
high fluorescence yield (Φ = 0.3154) and (Φ = 0.5302), fol-
lowed by the anthracene derivative EB3, for which a high
quantum yield (Φ = 0.21042) was also obtained, while the
naphthalene derivative EB2 showed a low fluorescence yield
(Φ = 0.0870). Thus, the fluorescence properties of EB3 and
EB4 proved to be strong enough for these compounds to be
used to detect the HDAC enzyme, provided they are capable
of efficient binding with the enzyme.
Finally, each compound's binding affinity to the HDAC
enzyme was examined (Table 1, Figure 2). The attachment
of the larger polyaromatic groups to the R1 position signifi-
cantly decreased the binding affinity of the benzamide deriva-
tives compared to the monoaromatic groups (benzene and
thiophene), which showed a relatively high binding affinity
(under 120 nM scale within 1 h) as we reported previously.7
Of the polyaromatic groups, pyrene, the largest fluorophoric
substituent, showed the lowest binding affinity with IC50
=
105 μM, while anthracene and naphthalene also showed low
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Fluorescent Benzamide Drug Targeting HDAC Enzyme
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Table 1. Quantum yield, absorption, fluorescence, and binding affinity of the fluorescent benzamide derivatives.
Φa
in MeOH
λab
λem
IC50 (μM)b
(HDAC1)
Compound
in MeOH (nm)
in MeOH (nm)
EB1
EB2
EB3
EB4
0.5302
0.08074
0.2042
0.315
347
360
370
396
468
410
421
475
0.12
68
45
105
a Quantum yield (Φ) were determined using 2,5-diphenyl oxazole as a standard, 1 × 10−5 M in MeOH.
b To determine IC50, Promega HDAC-Glo I/II assay format was used.19 Compounds were tested in duplicate 10-dose IC50 mode with threefold serial
dilution starting at 100 μM against 1 HDAC. IC50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose–response
equation.
Figure 2. Binding affinity of (a) EB2 and EB3, (b) EB4, (HDAC1-Kinetic, IC50,1 h). Compounds were tested in duplicate 10-dose IC50 mode
with threefold serial dilution starting at 100 μM against 1 HDAC. IC50 values were calculated using the GraphPad Prism 4 program based on a
sigmoidal dose–response equation.
binding affinities of 68 and 45 μM, respectively. Therefore,
the addition of only one more aromatic ring to the R1 position
(EB2 IC50 = 68 μM) has the effect of considerably reducing
the binding affinity of the benzamide (ΔIC50 = 566) compared
Acknowledgments. This research was performed with
support from the Industry, University and Research Institute
Core Technology Development & Industrialized Supporting
Business in Jeonbuk province in 2014. We are grateful to
the “Leaders–Industry–University Cooperation” Project,
funded by the Ministry of Education, Science and Technology
(MEST, South Korea) for funding.
to that of the monoaromatic ring system (EB1, IC50
=
0.12 μM).
Conclusion
We designed, synthesized, and characterized new fluorescent
benzamide derivatives as potential fluorescent inhibitors of
HDAC. While it was possible to achieve high fluorescence
yields by increasing the conjugation of the aromatic fluoro-
phores, the binding affinity of the compounds decreased along
with an increase in the size of the fluorescent group. This was
thought to be most likely due to steric hindrance at the site at
which HDAC would be active. Based on these results, it
appears that the active site pocketof theHDAC enzyme is very
tight andsensitive tomodification attheR1 positionoftheben-
zamide inhibitors. These results are valuable in the future for
tuning efficient fluorescent benzamide HDAC inhibitors and
could be used as diverse diagnostic tools for HDAC enzyme
detection.
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Fluorescent Benzamide Drug Targeting HDAC Enzyme
KOREAN CHEMICAL SOCIETY
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© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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