Phenylalanine-containing hydroxamic acids as selective inhibitors of class IIb histone deacetylases (HDACs)

Article abstract of DOI:10.1016/j.bmc.2007.10.092

We synthesized biarylalanine-containing hydroxamic acids and tested them on immunoprecipitated HDAC1 and HDAC6 and show a subtype selectivity for HDAC6 that was confirmed in cells by Western blot (tubulin vs histones). We obtained an X-ray structure with

Full text of DOI:10.1016/j.bmc.2007.10.092

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2011–2033
Phenylalanine-containing hydroxamic acids as selective
inhibitors of class IIb histone deacetylases (HDACs)
Stefan Scha¨fer,a Laura Saunders,b Elena Eliseeva,c Alfredo Velena,c Mira Jung,c
Andreas Schwienhorst,d Anja Strasser,e Achim Dickmanns,e Ralf Ficner,e
Sonja Schlimme,f Wolfgang Sippl,f Eric Verdinb and Manfred Junga,*
aInstitute of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany
bGladstone Institute of Virology and Immunology, University of California, San Francisco, 1650 Owens Street,
San Francisco, CA 94158, USA
cDepartment of Radiation Medicine, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC, USA
dDepartment of Molecular Genetics and Preparative Molecular Biology, Institute for Microbiology and Genetics,
University of Goettingen, Grisebachstr. 8, 37077 Goettingen, Germany
eDepartment of Molecular Structural Biology, Institute for Microbiology and Genetics,
University of Goettingen, Grisebachstr. 8, 37077 Goettingen, Germany
fInstitute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeckstr. 4, 06120 Halle (Saale), Germany
Received 14 August 2007; revised 22 October 2007; accepted 30 October 2007
Available online 4 November 2007
Abstract—We synthesized biarylalanine-containing hydroxamic acids and tested them on immunoprecipitated HDAC1 and
HDAC6 and show a subtype selectivity for HDAC6 that was confirmed in cells by Western blot (tubulin vs histones). We
obtained an X-ray structure with a HDAC6-selective inhibitor with the bacterial deacetylase HDAH. Docking studies were car-
ried out using HDAC1 and HDAC6 protein models. Antiproliferative activity was shown on cancer cells for selected
compounds.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
HDACs are divided into three structural classes depend-
ing on their homology to yeast proteins. Class I includes
four subtypes (HDAC1, 2, 3 and 8) and shows homol-
ogy to the yeast protein rpd3. Class II consists of six
subtypes and is subdivided into two classes, class IIa
with subtypes HDAC4, 5, 7 and 9 and class IIb with
subtypes HDAC6 and 10. HDAC11 is referred to as
class IV. The enzymatic activity of class I and II HDACs
occurs in a Zn2+-dependent mechanism. In contrast, the
class III HDAC enzymes, also called sirtuins, operate by
a NAD+-dependent mechanism. There are seven sub-
types in humans (SIRT1-7) which show homology to
the yeast protein Sir2.3
Enzymes catalyzing the posttranslational modifications
of chromatin, for example, histone acetyltransferases
(HATs) and histone deacetylases (HDACs), have
emerged as new targets for cancer therapy. Since the
inhibition of HDACs leads to growth arrest, differentia-
tion or apoptosis of tumour cell lines, clinical studies of
HDAC inhibitors for the therapy of cancer are under
way. The reversible acetylation and deacetylation of his-
tones and non-histone proteins play important roles in
the regulation of chromatin structure and gene expres-
sion. Inhibiting the deacetylation of non-histone pro-
teins may be an important contributor to the
anticancer activity of HDAC inhibitors.1,2
A wide range of chemical structures including hydroxa-
mic acids, benzamides, electrophilic ketones, thiols and
mercaptoamides as well as small fatty acids showing
inhibitory potential for Zn2+-dependent HDACs (class
I and II) have been developed in the last 10 years.2,4–6
Our detailed structure–activity relationships led to the
Keywords: Histone deacetylase (HDAC); Class I and II HDAC inhi-
bitor; Hydroxamic acid; Subtype selectivity; Gold docking.
*
Corresponding author. Tel.: +49 0761 203 4896; fax: +49 0761 203
6321; e-mail: manfred.jung@pharmazie.uni-freiburg.de
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.092

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S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
development of various HDAC inhibitors including the
potent antiproliferative agent SW55 (3).7,8
2. Results
2.1. Chemistry
Limited information is available on the structural
requirements for HDAC subtype selective inhibition,
and especially on the biological significance of subtype
selective HDAC inhibitors. Several class I selective inhib-
itors are available,9–13 especially for HDAC1, but only a
few for specific class II enzymes (HDAC4 or 6).14,15 Tub-
acin was reported to be selective for tubulin acetylation16
but inhibits other subtypes in vitro as well.17 We set out
to obtain structure–activity relationships (SAR) on bia-
rylalanines 4 as analogues of 3, esp. with regard to sub-
type selectivity (see Fig. 1). Specifically, we investigated
the influence of the spacer length as well as that of the
second aryl ring. To elucidate the probable binding mode
of the most active inhibitors docking studies were carried
out using generated homology models of HDAC1 and
HDAC6. These studies are supported by a new X-ray
structure of a HDAC6-selective inhibitor with the bacte-
rial deacetylase HDAH.18
The synthesis of the new series of compounds was car-
ried out in a six-step synthesis from p-bromophenylala-
nine methyl ester (5) in analogy to previous work from
our group7,8 and is shown in Scheme 1. Compounds
8a, 9a and 10a have been described before, and by using
monomethylazelate (7) instead of suberoylchloride (6)
the respective homologues were obtained. 10a and 10b
were then coupled in a microwave-assisted Suzuki-cou-
pling reaction with arylboronic acids which led to the
protected biarylalanine-containing hydroxamates 11a–
m. The biaryls 11 and the bromophenylalanines 10a–b
were then deprotected and the desired inhibitors 4a–o
were obtained.
2.2. Enzyme inhibition
In a first test series the compounds 4a–o were assayed
using purified rat liver extract as a HDAC source and
an unselective small molecule substrate in a homoge-
neous assay (see Section 4) developed in our group.
The inhibitors CHAP15,10 M34419 and 37 were included
for comparisons. All compounds showed inhibitory po-
tency between 270 nM and 5.1 lM (Table 1). Most
compounds are active below 1 lM but with the bromo
derivatives 4n and 4o the potency was increased com-
pared to the lead structure 3 (see Table 1). Among the
biaryl analogues the most potent compound is the ter-
HDAC1 and 6 were chosen as representative subtypes
and served as sources of enzymatic activity. HDAC6 is
interesting to study because it is the only subtype that
has been shown to deacetylate tubulin, an important
anticancer target, and there is limited availability of
selective inhibitors for this subtype. Confirmation of
the in vitro selectivities in the cell was achieved by com-
paring histone and tubulin acetylation via Western blot.
Additionally, the antiproliferative properties of selected
inhibitors were compared.
H
O
O
O
O
O
N
H
H
N
OH
H
O
N
N
H
O
N
O
2
1
O
O
O
H
N
R
N
H
OH
O
O
O
HO
O
N
H
n
N
3
H
O
4
n=6, 7
R= -Br, -Aryl
Figure 1. HDAC inhibitors: TSA (1), trapoxin (2), SW55 (3), general structure for new inhibitors 4.

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2013
O
H2N
O
x HCl
Br
5
O
a)
O
H
N
Cl
O
O
O
O
O
8a
6
c)
d)
H
N
OH
O
n
O
O
b)
O
HO
H
O
N
9a-b
O
O
O
O
O
8b
7
O
O
O
H
H
H
H
e)
f) or g)
H
H
N
N
N
N
N
N
HO
n
O
O
O
n
O
O
n
O
O
O
O
O
O
4a-o
11a-m
10a-b
R
R
Br
Scheme 1. Reagents and conditions: (a) O-tritylhydroxylamine, DIPEA, DCM, 0 ꢁC; (b) O-tritylhydroxylamine, IBCF, NMM, THF, ꢀ10 ꢁC; (c)
LiOH, H2O2, THF; (d) 5, IBCF, NMM, THF, ꢀ10 ꢁC; (e) R-B(OH)2, tri-o-tolylphosphine, Pd(OAc)2, Na2CO3, DME, H2O, microwave: 5–10 min,
100 W; (f) TFA, TES, 0 ꢁC (4a–j, 4l–o); (g) TSMTF, TES, 0 ꢁC (4k).
Table 1. HDAC-inhibitory activity of 4a–o
IC50 SE (lM)
Enzyme
Rat liver extract
HDAC1
None
HDAC6
None
Compound
n=
R=
Subtype selectivity of substrate
None
HDAC1
HDAC6
CHAP15a
—
—
6
—
—
0.14 0.01
0.07 0.01
0.50 0.03
0.70 0.07
0.64 0.09
1.16 0.12
0.81 0.15
0.87 0.06
1.00 0.13
1.00 0.06
0.86 0.08
1.13 0.07
0.66 0.13
0.99 0.26b
1.07 0.06
5.12 0.74
0.27 0.04
0.43 0.06
0.021 0.003
0.24 0.04
0.44 0.13
1.68 0.47
0.57 0.07
2.55 0.47
0.59 0.10
3.01 0.19
7.53 0.85
2.44 0.23
3.18 0.65
3.03 0.31
0.13 0.01
8.76 1.26c
0.63 0.04
5.19 1.22d
3.19 0.52
2.11 0.31
0.23 0.02
0.08 0.01
0.58 0.06
6.10 0.42
0.58 0.03
0.84 0.08
0.50 0.04
0.81 0.02
6.76 0.41
0.47 0.10
2.25 0.20
0.68 0.04
0.78 0.05
6.76 0.83
0.91 0.05
3.76 0.59d
4.98 0.27
0.68 0.06
0.0004 0.0002
2.96 1.39
1.22 0.20
9.54 1.72
5.07 1.12
16.75 1.42
3.34 0.37
19.19 3.98
2.61 1.68
9.76 1.72
6.35 0.64
10.30 0.54
0.96 0.39
38.28 4.48
2.98 0.16
10.54 0.99
22.86 7.25
28.44 1.61
0.04 0.01
0.75 0.09
1.05 0.18
2.33 0.45
9.99 1.83
4.09 1.04
4.02 2.92
1.77 0.08
4.49 0.21
2.33 0.42
1.13 0.34
1.05 0.18
0.69 0.21
14.72 2.67
1.17 0.13
2.85 1.97
1.69 0.91
4.67 2.76
M344a
3
–Phenyl
–Phenyl
–2-Thienyl
–2-Thienyl
–3-Thienyl
–3-Thienyl
–2-Furyl
–2-Furyl
–3-Furyl
–3-Furyl
–Biphenyl
–Biphenyl
–2-Naphthyl
–2-Naphthyl
Br
4a
4b
4c
7
6
7
4d
4e
6
7
4f
6
4g
4h
4i
7
6
7
4j
6
4k
4l
7
6
4m
4n
4o
7
6
7
Br
a Data taken from lit.21
b Max. inhibition: 55%.
c Max. inhibition: 60%.
d Values determined using the trypsin assay.
phenyl 4j with a six-carbon spacer while the 2-naphthyl
substituent in 4l–m leads to a decrease in rat liver
HDAC inhibition (see Table 1).
All compounds were tested for the inhibition of immu-
noprecipitated FLAG-HDAC1 and 6. For these en-
zymes another small molecule substrate20 was used

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S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
which shows higher conversion rates with the isolated
subtypes as compared to the rat liver substrate and
has no subtype selectivity. Again, for both subtypes
the terphenyl 4j displayed the most potent inhibition
and it exceeded the lead structure 3 in its inhibitory
activity.
and 4i). The trend regarding the dependence of the
selectivity on the spacer length is reversed in the bromo
substituted compounds where the suberoyl analogue 4n
is more selective than the azelaic acid derivative 4o. The
highest affinity to one of the subtypes is seen with the
terphenylalanine 4j with the short spacer (700 nM for
HDAC6).
We have previously reported that HDAC-subtype selec-
tive substrates can be used in inhibition assays with puri-
fied rat liver extracts containing a mixture of HDAC
subtypes to get an indication of actual subtype selectiv-
ity.21 Therefore, we used our new set of inhibitors to test
the predictive power of these substrates on a larger set of
compounds. We compared the IC50-values obtained
with the subtype selective substrates and a mixture of
subtypes (rat liver extract) with the respective ratios ob-
tained with the subtypes and an unselective substrate
(see Table 2). A minimum factor of three was arbitrarily
chosen to state selectivity.
For confirmation of the observed selectivity we analyzed
the extent of histone vs. tubulin acetylation for selected
inhibitors 4d, 4e and 4n. HDAC1 activity is mostly cor-
related to histone deacetylation, whereas HDAC6 activ-
ity should be responsible for a preference in tubulin
deacetylation. We were able to demonstrate a hyperacet-
ylation in the low micromolar range with regard to
tubulin for the HDAC6 selective inhibitors 4e and 4n,
whereas the unselective 4d as well as the control TSA
(1) show a hyperacetylation of both tubulin and histones
(Fig. 2).
None of the inhibitors showed a pronounced selectivity
for HDAC1 although in two cases (4a and 4j) such a
selectivity was predicted. On the other hand, nine com-
pounds showed selectivity for HDAC6 of which five
cases were predicted. All biaryls predicted to be
HDAC6 selective indeed showed that selectivity but
the bromophenylalanine that was shown to be the most
selective compound (4n) was missed. Altogether, the
inhibitors 4e, 4h, 4i, 4n and 4o showed pronounced
(more than fivefold) selectivity for HDAC6. In com-
pounds with the same biaryl group the inhibitors with
a spacer length of 7 methylene groups displayed a high-
er selectivity for HDAC6 than the congeners with the
shorter spacer. The selectivity is the highest for heterob-
iaryls with the connecting bond in the 30-position (4e
2.3. Analysis of the HDAC binding sites and inhibitor
binding modes
The X-ray crystallographic analysis of bacterial histone
deacetylase-like
proteins
(HDLP
and
FB188
HDAH)18,22–24 as well as HDAC7 and HDAC818,25
complexes with 1 (see Fig. 1) or suberoylanilide
hydroxamic acid (SAHA) has shown that the HDAC
catalytic domain consists of a narrow tube-like pocket
spanning a length equivalent to a straight chain of four
to six carbons. Two conserved aromatic residues of
HDAC enzymes (Phe152 and Phe208 in FB188 HDAH,
Phe152 and Phe208 in HDAC8) located in the tube-like
pocket fix the position of the flexible alkyl spacer of the
inhibitors. The catalytic zinc ion buried near the bottom
of the active site is coordinated by His and Asp residues
and the hydroxamic acid function of the inhibitors. A
similar architecture and interaction pattern is assumed
for the related HDAC1 and HDAC6 enzymes.
Table 2. Selectivity indices
Compound
Substrates
HDAC1/HDAC6
Subtypes
HDAC1/HDAC6
Only for HDAC1 a few homology models have already
been published and used to explain the interaction of
TSA or SAHA analogues.26–28 However, they all were
based on the crystal structure of the bacterial homo-
logue HDLP which shows only moderate sequence iden-
tity to the human HDACs. In order to better consider
the specific architecture of human HDACs we initiated
a homology modelling approach using the very recently
solved X-ray structures of human HDAC8, HDAC7
and the bacterial FB188 HDAH.18,22,23,25 HDAC6 is un-
ique among deacetylases in having two HDAC catalytic
domains (HDAC6 CD I and HDCA6 CD II) sharing
46% sequence identity and 60% similarity, respectively.
The presence of two catalytic domains within HDAC6
makes it difficult to understand how these active sites
contribute to the biochemistry and cellular physiology.
There are conflicting results and interpretations in the
HDAC6 literature. Work from Zhang et al.29 suggested
that both domains are active as histone deacetylases. In
a more detailed study using natural and synthetic sub-
strates Zou et al. recently showed that the second cata-
lytic site is the major functional domain of HDAC6.30
They analyzed highly purified human recombinant
CHAP15a
0.09
3.00
0.76
0.28
0.98
3.04
1.18
3.72
1.11
5.19
1.41
4.46
0.17
1.30
0.69
1.38
0.64
3.10
0.01
3.95
1.16
4.09
0.51
4.10
0.83
10.84
0.58
4.19
5.62
9.81
1.39
2.60
2.55
3.70
13.53
6.09
M344a
3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
The predicted selectivities are obtained by dividing the IC50 values
obtained with rat liver HDAC and the subtype selective substrates
(HDAC1/HDAC6), respectively, the measured selectivities by dividing
the IC50 values obtained with immunoprecipitated subtypes and a non-
selective substrate (HDAC1/HDAC6).
a Data taken from lit.21

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2015
Figure 2. Western blots of HCT116 cells incubated with 4d, 4e, 4n and TSA (1).
HDAC6 and demonstrated that the inhibition of
HDAC6 by inhibitors can be solely attributed to the
interaction with the second binding domain. To address
the question whether both catalytic domains are poten-
tial binding sites for the hydroxamic acid derivatives,
protein models for both catalytic domains of HDAC6
(CD I and II) were generated and analyzed. A detailed
description of the protein modelling together with the
sequence alignment is given in Section 4 and in the Sup-
porting Material.
The derived HDAC1, HDAC6 CD I and HDAC6 CD II
protein models show high structural similarity in the cat-
alytic regions, whereas larger deviations can be observed
at the entrance region of the binding pocket (Fig. 3a–d).
In Figure 3a–d the HDAC1, HDAC6 CD I and HDAC6
CD II models are superimposed with the template struc-
tures used for the homology modelling, HDAC8 and
HDAC7, respectively. Especially the loop regions form-
ing the entrance of the binding cavity are dissimilar in
the different HDAC structures. On the other hand, the
Figure 3. (a) Superimposition of the HDAC8 X-ray structure (green ribbon) with the co-crystallized inhibitor TSA (1) (cyan) and the HDAC1
˚
homology model (red tube) (RMSD backbone atoms 1.8 A). The zinc ion is coloured magenta. (b) Superimposition of the HDAC7 X-ray structure
˚
(yellow ribbon) with the co-crystallized inhibitor TSA (1) (orange) and the HDAC6 CD I model (cyan ribbon). (RMSD backbone atoms 1.5 A.) The
zinc ion is coloured magenta. (c) Superimposition of the HDAC7 X-ray structure (yellow ribbon) with the co-crystallized inhibitor TSA (1)and the
˚
HDAC6 CD II model (magenta ribbon). (RMSD backbone atoms 2.9 A). The zinc ion is coloured magenta. (d) Superimposition of the HDAC6 CD
˚
I model (cyan ribbon) and the HDAC6 CD II model (magenta ribbon). (RMSD backbone atoms 2.2 A.) The zinc ion is coloured magenta.

2016
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
zinc binding site (Fig. 4a and b) shows high similarity
between the different HDAC structures.
docking studies. In advance of docking the novel inhib-
itors into the HDAC1 and HDAC6 homology models,
we first tested whether the docking program GOLD31
is able to reproduce the experimentally observed HDAC
inhibitor binding modes. For this purpose we took the
The generated HDAC1 and HDAC6 protein models as
well as FB188 HDAH were subsequently used for ligand
Figure 4. (a) Comparison of the HDAC8 X-ray structure (1T69.pdb, left site) with the co-crystallized inhibitor SAHA (coloured green) and the
HDAC1 homology model (right site) with the docked inhibitor 4n (coloured orange). Only the binding pocket residues are shown. Hydrogen bonds
are displayed as dashed line. (b) Comparison of the HDAC7 X-ray structure (2PQP.pdb, left site) with the co-crystallized inhibitor TSA (1) and the
HDAC6 CD I model (right site) with the docked inhibitor 4n (coloured orange). Only the binding pocket residues are shown. Hydrogen bonds are
displayed as dashed line.

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2017
Figure 5. Schematic representation of the interaction between 4n and HDAC1. Hydrogen bonds are displayed as green arrows, whereas the zinc
coordination is indicated by magenta dashed lines.
Figure 6. (a) GOLD docking solutions for HDAC1 and inhibitors with a C6-alkyl chain spacer. 4n is displayed for comparison in ball-and-stick
mode. The surface of the HDAC1 binding pocket is coloured according the hydrophilicity (brown = hydrophobic, blue = hydrophilic). (b) GOLD
docking solutions for HDAC1 and inhibitors with a C7-alkyl chain spacer. 4n is displayed for comparison in ball-and-stick mode. The surface of the
HDAC1 binding pocket is coloured according the hydrophilicity (brown = hydrophobic, blue = hydrophilic).

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S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
published X-ray crystallographic structures of HDACs
and FB188 HDAH from the Protein Database32 as well
as the novel solved X-ray structure of FB188 HDAH in
complex with 4n (see below). In case of the X-ray struc-
tures of FB188 HDAH complexes with SAHA (pdb
code 1ZZ1) and CypX (pdb code 1ZZ3) RMSD values
between the best docking solution and the crystal struc-
Phe205 sandwich the hydrophobic alkyl chain of the
inhibitor. Van der Waals interactions are also of great
importance in the inhibitor-HDAC recognition process,
especially for the accommodation of the cap group. The
cap group of 4n interacts with several aromatic and
hydrophobic residues at the entrance of the binding
pocket (His28, Pro29 and Pro101).
˚
ture were found to be below 1.5 A (for details, see Sup-
porting Material, Figs. A and B). Additionally, the two
observed different conformations of 4n in the FB188
HDAH crystal structure could be reproduced by GOLD
(RMSD values below 1.5 A, Figs. C and D, Supporting
Material).
Next we docked all other newly synthesized inhibitors
into the HDAC1 binding pocket and analyzed the bind-
ing mode and the obtained ChemScore values. Similar
docking poses observed for 4n could be obtained for
the other inhibitors. In this binding mode the cap group
interacts with an open cavity at the entrance of the bind-
ing pocket formed by several aromatic and hydrophobic
residues (His28, Pro29 and Pro101) (Fig. 6a and b). The
analysis of the derived ChemScore values showed that
they are in qualitative agreement with the experimen-
tally obtained IC50-values for the recombinant HDAC1.
The regression analysis for HDAC pIC50 and Chem-
Score values is shown in Figure 7.
˚
Next, the bromophenylalanine-containing hydroxamic
acid 4n was docked into the HDAC1 homology model
(Figs. 4a and 5). The docking of 4n as well as the other
developed inhibitors into HDAC1 showed that the
hydroxamic acid and the alkyl chain interact in a similar
way at the binding pocket. The hydroxamic acid func-
tion of the inhibitors is located in a polar pocket formed
by several residues capable of forming hydrogen bonds
(His140, His141 and Tyr303), whereas Phe150 and
In case of the HDAC6 CD I and CD II homology mod-
els the entrance of the binding pocket itself shows sev-
eral clearly defined cavities and offers additional
interaction possibilities for the hydrophobic cap groups
of the inhibitors (Fig. 3d). The analysis of the docking
solutions obtained for both HDAC6 homology models
showed a similar binding pattern for the hydroxamic
acid inhibitors. His215, His216, Tyr386 in HDAC6
CD I and His610, His611, Tyr782 in HDAC6 CD II
form hydrogen bonds to the hydroxamic acid function.
Phe283 and Tyr225 in HDAC6 CD I, as well as
Phe620 and Phe680 in HDAC6 CD II, sandwich the al-
kyl chain linker between their hydrophobic portions
(Figs. 4b and 8). Larger deviations were observed in
the interaction of the bulky cap groups. The HDAC6
CD II model shows a clearly defined hydrophobic bind-
ing cavity formed by several aromatic and hydrophobic
residues (His499, His560, Phe566, Ile569). For all
docked inhibitors the top-ranked pose (based on the
HDAC1 - ChemScore
33.00
28.00
23.00
R2 = 0.5444
6.00
18.00
4.00
5.00
pIC50 HDAC1
Figure 7. Correlation between obtained ChemScore values and pIC50
data for HDAC1.
Figure 8. Schematic representation of the interaction between 4n and HDAC6 CD II. Hydrogen bonds are displayed as green arrows, whereas the
zinc coordination is indicated by magenta dashed lines.

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2019
Figure 9. (a) GOLD docking solutions for HDAC6 inhibitors with a C6-alkyl chain spacer. 4n is displayed for comparison in ball-and-stick mode.
The surface of the HDAC6 binding pocket is coloured according the hydrophilicity (brown = hydrophobic, blue = hydrophilic). (b) GOLD docking
solutions for HDAC6 inhibitors with a C7-alkyl chain spacer. 4n is displayed for comparison in ball-and-stick mode. The surface of the HDAC6
binding pocket is coloured according the hydrophilicity (brown = hydrophobic, blue = hydrophilic).
ChemScore) places its hydrophobic cap group in this
binding groove (Fig. 9a and b). The highest docking
scores were obtained for the C6-spacer compounds con-
taining a bulky cap group (4j and 4l). In general, also for
HDAC6 - Chemscore
35.00
the docking into HDAC6 CD II, the ChemScore values
showed a moderate correlation with the experimental
30.00
biological data (Fig. 10). In case of the HDAC6 CD I
model, the docking solutions showed no clear preference
for this pocket and several energetically equal docking
poses were obtained for the individual compounds (data
25.00
R2 = 0.2654
not shown). In addition, no significant correlation was
observed between the ChemScore values and the biolog-
ical data. Therefore, based on the docking poses and
scores, only the HDAC6 CD II model is able to explain
the experimentally derived structure–activity relation-
ships. This observation supports the findings of Zou
20.00
4.50
5.00
5.50
6.00
6.50
pIC50 HDAC6
Figure 10. Correlation between obtained ChemScore values and pIC50
data for HDAC6.

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S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
Table 3. X-ray data
et al. that the second catalytic domain is the predomi-
nant binding site for HDAC6 inhibitors.
Data collection
Data set
Wavelength (A)
4n
0.8015
˚
The differences in biological activity observed for
HDAC1 and HDAC6 were partially clarified by the
inspection of the binding mode of the inhibitors and
the comparison of the docking scores. The distance be-
tween the catalytic centre and the rim of the gorge is
shorter in case of HDAC1 compared to HDAC6. Thus,
for high HDAC1 activity the six-carbon chain represents
the optimal distance between the hydroxamic acid and
cap group. This is reflected by the highest docking score
for the most active inhibitor 4j containing a C6-spacer.
The biphenyl moiety fits perfectly in the hydrophobic
pocket close to Phe566 (Fig. 9a). In case of the C7-
spacer compounds, the hydrophobic cap-group is less
favourably placed at the solvent-exposed entrance (i.e.,
more solvent exposed) of the cavity, resulting in de-
creased biological activity for these compounds. This ef-
fect is most dramatic in case of the bulky terphenyl
group of compounds 4j versus 4k. The HDAC6 binding
cavity is longer, resulting in favourable van-der-Waals
interactions of the hydrophobic cap group of both series
of compounds (C6- and C7-spacer). As a consequence
the HDAC6 inhibitory activities of corresponding com-
pounds (C6- and C7-spacer) are very similar. Only in
case of the large terphenyl group (4j vs 4k), the cap
group of the C7-spacer compound is more solvent ex-
posed, resulting in unfavourable interaction which is re-
flected by lower docking scores.
Resolution rangea (A)
117.8–1.9 (1.97–1.90)
P21
˚
Space group
˚
Cell dimensions (A/ꢁ)
a = 67.7
b = 93.6
c = 121.6
b = 104
323,438
2.9 (2.4)
96.0 (93.8)
8.5 (38.3)
11.2 (2.1)
0.6
Observed reflections
Redundancy
Completeness (%)
b
Rsym (%)
Average I/r(I)
Mosaicity (ꢁ)
Refinement statistics
Resolution rangea (A)
˚
c
Rcryst (%)/Rfree (%)
30–1.9
15.6/20.6
87.9
FOM
Coordinate errord (A)
˚
0.090
0.010
1.345
0.088
Bond lengths
Bond angles
Chiral
Ramachandran plote
Most favourable regions (%)
Additionally allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
rms deviations from ideality
90.0
9.6
0.4
0
˚
Bonds (A)/angles (ꢁ)
Average B values (A )
Residues/waters
0.010/1.344
16.3
1474/487
2
˚
a Numbers in parentheses refer to the highest resolution shell.
In general, the docking poses and scores show a qualita-
tive agreement with the in vitro data obtained for
HDAC1 and HDAC6. However, a correlation between
the measured selectivity and the structural data was only
obtained within the biaryl series.
P P
P
b Rsym = 100 Æ
ijIi(h) ꢀ hI(h)ij/ hI(h) where Ii(h) is the ith mea-
h
surement of the h reflection and hI(h)i is the average value of the
reflection intensity.
P
P
c Rcryst = 100 Æ kFoj ꢀ jFck/ jFoj, where Fo and Fc are the structure
factor amplitudes from the data and the model, respectively. Rfree is
Rcryst with 5% of test set structure factors.
2.4. Crystal structure
d Based on maximum likelihood.
e Calculated using PROCHECK.
In addition to the modelling studies we were able to ob-
tain a crystal structure of the bacterial FB188 HDAH in
complex with a phenylalanine-containing hydroxamic
acid which served to verify the predicted inhibitor bind-
ing mode. This structure with the bound inhibitor 4n
HDAH,18,24 HDLP22 or HDAC823,33 and have been
attributed to a high flexibility of the cap group.
˚
was determined at 1.9 A resolution. The inhibitor crys-
2.5. Antiproliferative activities
tals were in the monoclinic form (P21). The structure
was refined to a free R-value of 20.6% with very good
stereochemistry (Table 3). The overall structure of
FB188 HDAH is essentially the same as described else-
where.18 In the inhibitor–HDAH complex the hydroxa-
mate group of 4n is bound very similar to that of SAHA
or CypX.18 Briefly, the hydroxamate coordinates the
zinc ion in the active site through its carbonyl and hy-
droxyl groups. The hydroxamic acid also hydrogen-
bonds with His142 (inner charge-transfer relay) and
His143 and the Tyr312 hydroxyl group (see Fig. 11).
The two rings of Phe152 and Phe208, which line the ac-
tive site channel, are in a coplanar orientation and sand-
wich the spacer region of the inhibitor. In contrast to the
electron density of the hydroxamate and long spacer
moieties, the electron density of the cap group is rather
poorly defined. Similar observations have been reported
for other HDAC inhibitors in complexes with FB188
Finally, we tested the effect of the observed selectivities
on cytotoxicity in MCF7 breast cancer cells. This
screening revealed a correlation between HDAC6-selec-
tivity and a diminished cytotoxicity on the cells. Com-
pounds 4e and 4n that presented the highest HDAC6-
selectivity showed the least cytotoxicity in this test
series (Table 4) but the selective 4h was among the
more active compounds on MCF-7 cells. To verify this
correlation, we chose four selective (4a, 4e, 4i and 4n)
inhibitors as well as one unselective (4b) compound
and tested them on a panel of different cell lines (Table
5). 4b is the most toxic compound on all cell lines,
whereas the selective compounds again show reduced
antiproliferative properties. The Jurkat leukaemia cell
line is generally the most sensitive one towards all
HDAC inhibitors (Table 5).

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
Table 6. Elemental analyses of 4a–o
2021
Compound
Calculated
% H
Found
% H
% C
% N
% C
% N
4a
4b
4c
4d
4e
4f
68.16
61.09
61.86
61.09
61.86
63.45
64.17
63.45
64.17
71.69
72.07
70.57
71.00
50.36
51.48
7.32
6.52
6.77
6.52
6.77
6.78
7.02
6.78
7.02
6.82
7.02
6.77
6.99
5.87
6.14
6.36
6.48
6.27
6.48
6.27
6.73
6.51
6.73
6.51
5.57
5.42
5.88
5.71
6.53
6.32
68.21
61.07
61.87
61.06
61.78
63.10
64.18
63.16
63.79
71.02
72.07
70.56
70.92
50.59
51.63
7.40
6.58
6.83
6.67
6.83
7.01
7.19
7.05
6.99
6.98
7.03
6.85
7.11
6.09
6.18
6.11
6.31
6.13
6.29
6.00
6.48
6.37
6.64
6.43
6.42
5.30
5.71
5.62
6.44
6.14
4g
4h
4i
4j
4k
4l
4m
4n
4o
Figure 11. The active site residues of FB188 HDAH with bound
inhibitor 4n. Shown are the inhibitor 4n (C-atoms depicted as orange
sticks) and the amino acids of HDAH in close vicinity of the inhibitor
(C-atoms in light grey sticks), the bound zinc-ion (purple sphere) and
the amino acids proposed to take part in the charge transfer relay
systems (Asp184-His148 and Asn191-His149). The electron density
map is a 2jFo ꢀ Fcj map contoured at 1.0r. The distance between the
two phenylalanines (Phe158 and Phe214) sandwiching the inhibitor is
3. Conclusion
Structure–activity studies on biarylalanine inhibitors of
HDACs have led to the development of selective
HDAC6 inhibitors. We identified structural factors that
favour such selectivity (azelaic acid spacer, 3-heteroaryl
substituent) but the most selective compound was the
synthetic intermediate with a bromophenylalanine struc-
ture and a suberic acid spacer. We gained insight into
the structural requirements for the binding of inhibitors
to HDAC isoenzymes by docking the inhibitors into
homology models of HDAC1 and HDAC6. Com-
pounds having a seven-carbon spacer show decreased
inhibitory activity for HDAC1 compared to the com-
pounds with a six-carbon spacer. The reason for that
is the smaller entrance of the HDAC1 binding channel
and the diminished possibilities to bury the hydrophobic
cap group. In case of HDAC6 that has a wider entrance
of the binding pocket, formed by several non-polar res-
idues, compounds having a longer seven-carbon spacer
(e.g., 4e) are able to interact with the non-polar residues
at the entrance of the pocket. This observation is in
agreement with the solved X-ray structure of 4n in com-
plex with FB188 HDAH, where the bromophenyl cap
group interacts with the hydrophobic residues at the en-
trance of the binding pocket. Next to thiol based inhib-
itors that were published14 while we were finishing this
study, these are the most selective in vitro HDAC6
inhibitors developed so far.
˚
approximately 7.4 A.
Table 4. Cytotoxicity measurements by cell proliferation assay (MCF-
7 cell line)
Compound
IC50 (lM)
3
5.0
21.0
4.0
4a
4b
4c
4d
4e
4f
4.0
1.5
17.0
1.5
4g
4h
4i
18.0
1.5
32.0
0.8
4j
4k
4l
3.8
1.9
4m
4n
4o
4.0
22.0
0.9
In general, the calculated docking scores are in agree-
ment with the in vitro data obtained for HDAC1 and
HDAC6. The most active inhibitor 4j gave the highest
ChemScore values for HDAC1 and HDAC6. However,
a quantitative correlation between the measured selec-
tivity and the in vitro data was not obtained. The
homology models can interpret the activity of the biaryl-
alanines quite well but were limited in the case of the
bromophenylalanines which are quite similar. This
could not be foreseen. The variability of the biological
data adds to the uncertainty of the prediction in that
case. However, we have established these models so they
can be further probed and possibly refined with the data
Table 5. Cytotoxicity measurements by cell proliferation assay on
selected compounds
Cell line
IC50 (lM)
4a
4b
4e
4i
4n
25.0
SQ20B (adenocarcinoma)
HeLa (cervix cancer)
Jurkat (leukaemia)
33.0
7.0
6.0
3.0
0.4
8.0
37.0
10.0
2.8
35.0
21.0
4.0
15.0
3.0
2.7
42.0
MCF10A (non-malignant
breast epithelial cell)
PC3 (prostate cancer)
MCF7 (breast cancer)
32.0
44.0
>50
9.9
21.0
1.8
4.0
10.0
17.0
10.0
32.0
>50
22.0

2022
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
available from the literature and from further com-
pounds from our laboratory. Especially interesting is
the fact that those models can contribute to the ongoing
conflicting discussion about the relevance of the two cat-
alytic sites in HDAC6. Our data support the reports that
CD II is the one that has to be considered for further
inhibitor design. So our homology models can be added
to the set of tools available for HDAC inhibitor optimi-
zation but have to be inspected for their validity on a
case to case basis as is necessary with every other model.
was cooled down to ꢀ15 ꢁC and 3.5 mmol thionylchlo-
ride was added slowly. The amino acid (1 mmol) was
then added in portions. The mixture was warmed to
room temperature and stirred overnight. The solvent
was evaporated and the residue was suspended three
times in diethyl ether and evaporated again. The remain-
ing residue was suspended in diethyl ether, filtered,
washed with diethyl ether three times and dried in high
vacuum.
4.2.1.2. Method B. Amide formation from carboxylic
acid chlorides. 1 mmol carboxylic acid chloride was dis-
solved in anhydrous dichloromethane (1 ml/mmol car-
boxylic acid chloride) and cooled down to ꢀ15 ꢁC. A
mixture of 3 mmol N-ethyldiisopropylamine (DIPEA)
and 1 mmol O-tritylhydroxylamine in anhydrous dichlo-
romethane (2 ml/mmol carboxylic acid chloride) was
added slowly. The solution was stirred for 4 h at
ꢀ15 ꢁC and overnight at room temperature. The solvent
was evaporated and the residue was dissolved in ethyl
acetate. The solution was washed three times in each
case with hydrochloric acid (2 M), water, sodium bicar-
bonate solution (5%) and water. After washing with
brine and drying over sodium sulfate the product was
crystallized from petroleum ether (80–110 ꢁC). The crys-
tals were collected by filtration and the product was
dried in high vacuum.
The predictive power of subtype selective small molecule
substrates was investigated and in this structurally
biased series the prediction of HDAC6 activity did not
lead to false positives but several false negatives were de-
tected. Thus, if a larger number of presumably HDAC6
selective compounds were available, testing on isolated
subtypes could be reduced based on the filtering func-
tion of these assays. For selected compounds, the selec-
tivity was confirmed in cancer cells based on the
investigation of hyperacetylation of established protein
targets. Thus, immunoprecipitated HDACs 1 and 6
may be used as screening tools for compounds with
intracellular HDAC subtype selectivity. The new
HDAC6 inhibitors exhibit antiproliferative properties
on cancer cells with a tendency for reduced cytotoxicity.
Given the strong synergistic effects observed for a tubu-
lin selective HDAC inhibitor with the proteasome inhib-
itor bortezomib,34 our new compounds and further
analogues may be useful agents for combination chemo-
therapy with reduced systemic toxicity.
4.2.1.3. Method C. Amide formation using mixed
anhydride. 1 mmol carboxylic acid was dissolved in
anhydrous tetrahydrofuran (THF, 20 ml/mmol carbox-
ylic acid). 1 mmol N-methylmorpholine (NMM) was
added to the solution. The reaction mixture was then
cooled down to ꢀ15 ꢁC and stirred for 5 min. 1 mmol
isobutylchloroformate (IBCF) was added and the mix-
ture was stirred for another ten minutes at ꢀ15 ꢁC.
Afterwards 1 mmol of the amine and another 2 mmol
of NMM were added and the mixture was stirred for
15 min at ꢀ15 ꢁC and 2 h at room temperature. After-
wards the mixture was poured into hydrochloric acid
(2 M) and extracted three times with ethyl acetate.
The organic layers were pooled and washed three times
in each case with water, sodium bicarbonate solution
(5%) and water. After washing with brine and drying
over sodium sulfate the product was crystallized from
petroleum ether (80–110 ꢁC). The crystals were col-
lected by filtration and the product was dried in high
vacuum.
4. Experimental
4.1. General
Melting points are uncorrected and were determined by
using the melting point apparatus SMP2 (Stuart Scien-
tific). Elemental analyses were performed on a Vario EL
elemental analyzer (Elementaranalysensysteme GmbH)
(Table 6). ESI- and APCI-mass spectra were measured
with a LCQ-Advantage mass spectrometer, EI- and CI-
mass spectra with a TSQ700 mass spectrometer (Thermo-
electron). NMR spectra were recorded on a Unity 300
spectrometer (300 MHz) by Varian and an Avance
DRX 400 spectrometer (400 MHz) by Bruker, respec-
tively. Chemical shifts are presented in ppm relative to tet-
ramethylsilane. For the microwave syntheses we used a
Discover microwave synthesis system by CEM. The rat li-
4.2.1.4. Method D. Ester cleavage. 1 mmol of the ester
was dissolved in tetrahydrofuran (THF, 4 ml/mmol es-
ter). 4 mmol of a lithiumhydroxide solution (0.5 M)
and 1 ml hydrogen peroxide solution (30%) were added
and the solution was stirred overnight. Afterwards the
mixture was extracted three times with ethyl acetate.
The pH of the aqueous layer was adjusted to three with
hydrochloric acid (2 M) and extracted three times with
ethyl acetate. The organic layers were pooled and
washed three times with water and one time with brine.
The solution was dried over sodium sulfate and the
product was crystallized from petroleum ether (80–
110 ꢁC). The crystals were collected by filtration and
the product was dried in high vacuum.
¨
ver HDACwas purified with an Akta primechromatogra-
phy system by Amersham Biosciences. The fluorescent
assays were performed on microplate readers by BMG
(Fluostar Optima) and Molecular Devices (FlexStation),
respectively. M344,19 37 and small molecule sub-
strates20,21,35 were prepared according to the literature.
4.2. Synthesis of inhibitors
4.2.1. General procedures
4.2.1.1. Method A. Preparation of amino acid methyl
esters. Anhydrous methanol (10 ml/1 mmol amino acid)

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2023
2
3.24 (dd, 1H, J = 14.4 Hz, J = 6.8 Hz, CH–CH2–Ar),
3
4.2.1.5. Method E. Microwave-assisted Suzuki cou-
pling. All Suzuki-coupling reactions for compounds
11a–m were performed in a dried glass vessel under
nitrogen atmosphere. 1 mmol arylbromide, 1.5 mmol
boronic acid and 0.1 mmol tri-(o-tolyl)-phosphine were
dissolved in 14 ml 1,2-dimethoxyethane. 2.4 ml degassed
water, 0.05 mmol palladium(II)acetate and 2 mmol so-
dium carbonate were added and the mixture was heated
under reflux in a microwave reactor for 5–10 min with a
power of 100 W. The crude product was purified by
flash chromatography on silica gel using ethyl acetate
and cyclohexane (1:1) as mobile phase. The product
was crystallized afterwards from ethyl acetate and petro-
leum ether (80–110 ꢁC). The crystals were collected by
filtration and the product was dried in high vacuum.
2
3
3.20 (dd, 1H, J = 14.4 Hz, J = 6.8 Hz, CH–CH2–Ar);
13C NMR (100 MHz, CD3OD) d = 169.98 (COOCH3),
134.44 (10-C), 133.00 (30/50-C), 132.24 (20/60-C), 122.64
(40-C), 54.87, 53.65 (COOCH3, NH–CH–CO), 36.70
(CH–CH2–Ar).
4.2.3. 7-Trityloxycarbamoyl-heptanoic acid methyl ester
(8a). Compound 8a was synthesized by method B from
7-chlorocarbonyl-heptanoic acid methyl ester (4.8 g,
23.2 mmol), 60 ml anhydrous dichloromethane, DIPEA
(12.1 ml, 69.9 mmol) and O-tritylhydroxylamine (6.52 g,
23.2 mmol); yield: 8.37 g (81%) white powder; melting
point: 90 ꢁC; MS (ESI pos.) m/z = 468 [M+Na]+, 243
[C19H15]+; IR (ATR) m (cmꢀ1) = 1736 (COOCH3),
1660 (CO–NH–O–Trt); 1H NMR (400 MHz, CDCl3)
d = 7.74 (br s, 1H, Trt–O–NH–CO), 7.47–7.27 (m,
15H, Ar-H), 3.65 (s, 3H, COOCH3), 2.25 (t, 2H,
3J = 7.5 Hz, CO–CH2), 1.90–0.91 (m, 10H, (CH2)5);
13C NMR (100 MHz, CDCl3) d = 177.20, 174.26 (CO),
141.19, 129.20, 128.30 (Ar-C), 93.57 (Ph3-C), 51.83
(COOCH3), 34.36, 31.47 (CO–CH2), 29.11, 25.11,
23.64 (CH2).
4.2.1.6. Method F. Removal of trityl protecting groups
with trifluoro acetic acid. The trityl protected precursor
was dissolved in dichloromethane. The reaction mixture
was then cooled in an ice bath and trifluoro acetic acid
(TFA) and triethylsilane (TES) were added alternately
dropwise until the yellow colour disappeared. The sol-
vent was evaporated. The residue was dissolved in meth-
anol and evaporated until the smell of acetic acid
disappeared. The residue was then dissolved in methanol
and the product was crystallized from diethyl ether. The
crystals were collected by filtration and the product was
dried in high vacuum.
4.2.4. 8-Trityloxycarbamoyl-octanoic acid methyl ester
(8b). Compound 8b was synthesized by method C from
non-anedioic acid monomethyl ester (2.5 g, 12.3 mmol),
100 ml anhydrous THF, NMM (4.1 ml, 36.9 mmol),
IBCF (1.6 ml, 12.3 mmol) and O-tritylhydroxylamine
(3.5 g, 12.3 mmol); yield: 4.54 g (80%) white powder;
melting point: 101 ꢁC; MS (ESI pos.) m/z = 482
[M+Na]+, 243 [C19H15]+, 239 [C10H18NO4Na]+; IR
(ATR) m (cmꢀ1) = 1736 (COOCH3), 1663 (CO–NH–O–
4.2.1.7. Method G. Removal of trityl protecting groups
with trimethylsilyl trifluoromethanesulfonate. 1 mmol of
the trityl protected precursor was dissolved in dichloro-
methane (20 ml/mmol). 0.01 mmol trimethylsilyl trifluo-
1
romethanesulfonate
(TMSTF)
and
1.2 mmol
Trt); H NMR (400 MHz, CDCl3) d = 7.72 (br s, 1H,
triethylsilane (TES) were added and the solution was
stirred until the yellow colour disappeared. The solvent
was evaporated and the residue was dissolved in a mix-
ture of acetic acid and tetrahydrofuran (1:1). After stir-
ring for 30 minutes, an equal volume of water was added
and the mixture was extracted three times with ethyl ace-
tate. The organic layers were combined and extracted
three times with water and once with brine. The solvent
was dried over sodium carbonate, filtered and evapo-
rated. The residue was again dissolved in methanol
and evaporated until the smell of acetic acid disap-
peared. The crude product was purified by flash chroma-
tography on reversed phase material with acetonitrile
and water (1:1) as mobile phase. The product was crys-
tallized from methanol and diethyl ether. The crystals
were collected by filtration and the product was dried
in high vacuum.
Trt–O–NH–CO), 7.46–7.28 (m, 15H, Ar-H), 3.66 (s,
3H, COOCH3), 2.27 (t, 2H, 3J = 7.5 Hz, CO–CH2),
1.90–0.99 (m, 12H, (CH2)6); 13C NMR (100 MHz,
CDCl3) d = 177.07, 174.11 (CO), 140.98 128.98, 128.08
(Ar-C), 93.57 (Ph3-C) 51.61 (COOCH3), 34.21, 31.31
(CO–CH2), 29.06, 29.04, 25.03, 23.56 (CH2).
4.2.5. 7-Trityloxycarbamoyl-heptanoic acid (9a). Com-
pound 9a was synthesized by method D from 8a (8.3 g,
18.6 mmol), 50 ml THF, 100 ml lithiumhydroxide solu-
tion (0.5 M) and 16 ml hydrogen peroxide solution
(30%); yield: 6.4 g (80%) white powder; melting point:
142 ꢁC; MS (ESI pos.) m/z = 454 [M+Na]+, 243
[C19H15]+; IR (ATR) m (cmꢀ1) = 1737 (COOH), 1674
(CO–NH–O–Trt); 1H NMR (400 MHz, CDCl3)
3
d = 7.46–7.28 (m, 15H, Ar-H), 2.25 (t, 2H, J = 7.5 Hz,
CO–CH2), 1.86–0.99 (m, 10H, (CH2)5); 13C NMR
(100 MHz, CDCl3) d = 178.91, 177.69 (CO), 141.18,
129.22, 128.30 (Ar-C), 93.64 (Ph3-C), 34.20, 31.42 (CO–
CH2), 29.03, 24.83, 23.63 (CH2).
4.2.2. (S)-2-Amino-3-(4-bromo-phenyl)-propionic acid
methyl ester hydrochloride (5). Compound 5 was synthe-
sized by method A from L-bromo-phenylalanine (5 g,
20.1 mmol), 250 ml anhydrous methanol and thionyl-
chloride (5.2 ml, 71.8 mmol); yield: 5.82 g (96%) white
powder; melting point: 200 ꢁC; MS (CI(NH3) pos.) m/
z = 258/260 [C10H12NO2Br]+, 198/200 [C8H8NBr]+; IR
4.2.6. 8-Trityloxycarbamoyl-octanoic acid (7b). Com-
pound 9b was synthesized by method D from 8b (1.16 g,
2.4 mmol), 50 ml THF, 14 ml lithiumhydroxide solution
(0.5 M) and 7 ml hydrogen peroxide solution (30%); yield:
0.8 g (77%) white powder; melting point: 100 ꢁC; MS (ESI
pos.) m/z = 468 [M+Na]+, 243 [C19H15]+, IR (ATR) m
(cmꢀ1) = 1734 (COOH), 1698 (CO–NH–O–Trt); 1H
NMR (400 MHz, CDCl3) d = 7.46–7.28 (m, 15H,
(ATR)
m
(cmꢀ1) = 1740 (COOCH3), 1H NMR
(400 MHz, CD3OD) d = 7.53–7.50 (m, 2H, 30/50-H),
3
7.21–7.18 (m, 2H, 40/60-H), 4.33 (pt, 1H, J = 6.8 Hz,
3J = 6.8 Hz, NH–CH–CO), 3.80 (s, 3H, COOCH3),

2024
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
3
Ar-H), 2.32 (t, 2H, J = 7.4 Hz, CO–CH2), 2.30 (t, 2H,
3J = 7.5 Hz, CO–CH2), 1.65–1.53 (m, 4H, (CH2)2),
1.37–0.97 (m, 6H, (CH2)3); 13C NMR (100 MHz, CDCl3)
d = 179.28, 177.81 (CO), 141.18, 129.23, 128.28 (Ar-C),
93.67 (Ph3-C), 34.14, 31.22 (CO–CH2), 29.26, 29.24,
29.19, 25.00, 23.78 (CH2).
94 ꢁC; MS (ESI pos.) m/z = 705 [M+Na]+, 462
[C25H31N2O5Na]+, 243 [C19H15]+; IR (ATR)
m
(cmꢀ1) = 1745 (COOCH3), 1668, 1644 (CO–NH, CO–
1
NH–O–Trt); H NMR (300 MHz, CDCl3) d = 7.70 (br
s, 1H, Trt–O–NH–CO), 7.59–7.15 (m, 24H, Ar-H), 5.91
(d, 1H, 3J = 7.9 Hz, NH–CH–CO), 4.94 (ddd, 1H,
3J = 7.9 Hz, 3J = 5.8 Hz, 3J = 5.8 Hz, NH–CH–CO),
3.76 (s, 3H, COOCH3), 3.21 (dd, 1H, 2J = 14.0 Hz,
4.2.7. (S)-3-(4-Bromo-phenyl)-2-(7-trityloxycarbamoyl-
heptanoylamino)-propionic acid methyl ester (10a). Com-
pound 10a was synthesized by method C from 9a (1.0 g,
2.32 mmol), 50 ml anhydrous THF, NMM (759 ll,
6.94 mmol), IBCF (301 ll, 2.32 mmol) and 5 (678 mg,
2.32 mmol); yield: 1.22 g (78%) white powder; melting
point: 109 ꢁC; MS (ESI pos.) m/z = 693/695 [M+Na]+,
450/452 [C18H24N2O5Na]+, 243 [C19H15]+; IR (ATR) m
(cmꢀ1) = 1745 (COOCH3), 1644 (CO–NH, CO–NH–O–
2
3J = 5.8 Hz, CH–CH2–Ar), 3.14 (dd, 1H, J = 14.0 Hz,
3J = 5.8 Hz, CH–CH2–Ar), 2.16 (t, 2H, 3J = 7.6 Hz,
CO–CH2), 1.65–1.51 (m, 4H, (CH2)2), 1.32–1.09 (m, 8H,
(CH2)4); 13C NMR (75.5 MHz, CDCl3) d = 172.63,
172.20, 170.92 (CO), 140.60, 139.96, 134.93, 129.65,
128.75, 128.05, 127.90, 127.89, 127.21, 126.94 (Ar-C),
93.20 (Ph3-C), 52.88, 52.32 (COOCH3, NH–CH–CO),
37.54 (CH–CH2–Ar), 36.48, 31.45 (CO–CH2), 28.85,
28.38, 28.29, 25.38, 25.08 (CH2).
1
Trt); H NMR (400 MHz, CDCl3) d = 7.79 (br s, 1H,
Trt–O–NH), 7.46 (m, 2H, Ar-H), 7.40–7.38 (m, 2H, 30/
50-H), 7.37–7.26 (m, 13H, Ar-H), 6.96–6.94 (m, 2H, 40/
60-H), 5.92 (d, 1H, 3J = 7.5 Hz, CO–NH–CH), 4.88–
4.84 (m, 1H, NH–CH–CO), 3.72 (s, 3H, COOCH3),
4.2.10. (S)-3-(4-Thiophen-2-yl-phenyl)-2-(7-trityloxycar-
bamoyl-heptanoylamino)-propionic acid methyl ester
(11b). Compound 11b was synthesized by method E from
10a (360 mg, 0.54 mmol), 2-thienylboronic acid (134 mg,
0.87 mmol), tri-(o-tolyl)-phosphine (16 mg, 0.054 mmol),
7.4 ml 1,2-dimethoxyethane, 1.2 ml degassed water, pal-
ladium(II)acetate (6 mg, 0.027 mmol) and sodium car-
bonate (113 mg, 1.05 mmol); reaction time: five minutes;
yield 175 mg (48%) white powder; melting point:
166 ꢁC; MS (ESI pos.) m/z = 697 [M+Na]+, 454
2
3
3.12 (dd, 1H, J = 13.9 Hz, J = 5.7 Hz, CH–CH2–Ar),
3.03 (dd, 1H, J = 13.9 Hz, J = 5.7 Hz, CH–CH2–Ar),
2
3
3
2.12 (t, 2H, J = 7.4 Hz, CO–CH2), 1.91–1.77 (m, 2H,
CO–CH2), 1.64–1.45 (m, 4H, (CH2)2), 1.29–1.06 (m, 4H,
(CH2)2); 13C NMR (100 MHz, CDCl3) d = 172.45,
171.79 (CO), 141.27, 134.88, 131.59, 130.90, 129.00,
128.09, 121.09 (Ar-C), 52.88, 52.59 (COOCH3, NH–
CH–CO), 37.51 (CH–CH2–Ar), 36.52 (CO–CH2), 28.90,
25.47 (CH2).
[C22H27N2O5SNa]+, 243 [C19H15]+; IR (ATR)
m
(cmꢀ1) = 1748 (COOCH3), 1686, 1641 (CO–NH, CO–
1
NH–O–Trt); H NMR (300 MHz, CDCl3) d = 7.72 (br
s, 1H, Trt–O–NH–CO), 7.55–7.05 (m, 22H, Ar-H), 5.94
4.2.8. (S)-3-(4-Bromo-phenyl)-2-(8-trityloxycarbamoyl-
octanoylamino)-propionic acid methyl ester (10b). Com-
pound 10b was synthesized by method C from 9b
(2.82 g, 6.33 mmol), 125 ml anhydrous THF, NMM
(2.69 ml, 24.1 mmol), IBCF (801 ll, 5.88 mmol) and 5
(1.8 g, 6.11 mmol); yield: 2.11 g (49%) white powder;
melting point: 86 ꢁC; MS (ESI pos.) m/z = 707/709
[M+Na]+, 464/466 [C19H26BrN2O5Na]+, 243 [C19H15]+;
IR (ATR) m (cmꢀ1) = 1747 (COOCH3), 1643 (CO–
NH, CO–NH–O–Trt); 1H NMR (300 MHz, CDCl3)
d = 7.40–7.37 (m, 2H, 30/50-H), 7.36–7.27 (m, 15H, Ar-
H), 6.96–6.94 (m, 2H, 40/60-H), 5.91 (d, 1H, 3J = 7.8 Hz,
CO–NH–CH), 4.89–4.83 (m, 1H, NH–CH–CO), 3.71 (s,
3
(d, 1H, J = 7.7 Hz, NH–CH–CO), 4.94–4.88 (m, 1H,
NH–CH–CO), 3.74 (s, 3H, COOCH3), 3.17 (dd, 1H,
3
2J = 14.0 Hz, J = 5.8 Hz, CH–CH2–Ar), 3.09 (dd, 1H,
2J = 14.0 Hz, 3J = 5.8 Hz, CH–CH2–Ar), 2.13 (t, 2H,
3J = 7.4 Hz, CO–CH2), 1.89–0.96 (m, 10H, (CH2)5); 13
C
NMR (75.5 MHz, CDCl3) d = 172.50, 172.07 (CO),
143.89, 141.30, 135.16, 133.26, 129.73, 128.99, 128.05,
127.98, 125.98, 124.72, 123.00 (Ar-C), 52.83, 52.31
(COOCH3, NH–CH–CO), 37.56 (CH–CH2–Ar), 36.31
(CO–CH2), 28.68, 25.22 (CH2).
4.2.11. (S)-3-(4-Thiophen-2-yl-phenyl)-2-(8-trityloxycar-
bamoyl-octanoylamino)-propionic acid methyl ester
(11c). Compound 11c was synthesized by method E from
10b (400 mg, 0.58 mmol), 2-thienylboronic acid (148 mg,
1.16 mmol), tri-(o-tolyl)-phosphine (18 mg, 0.058 mmol),
8.3 ml 1,2-dimethoxyethane, 1.4 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.029 mmol) and sodium car-
bonate (124 mg, 1.16 mmol); reaction time: five minutes;
yield 317 mg (79%) white powder; melting point: 85 ꢁC;
MS (ESI pos.) m/z = 711 [M+Na]+, 468 [C23H29N2O5S-
2
3
3H, COOCH3), 3.11 (dd, 1H, J = 13.9 Hz, J = 5.9 Hz,
2
3
CH–CH2–Ar), 3.01 (dd, 1H, J = 13.9 Hz, J = 5.9 Hz,
CH–CH2–Ar), 2.13 (t, 2H, 3J = 7.7 Hz, CO–CH2), 1.62–
1.47 (m, 4H, (CH2)2), 1.28–1.06 (m, 8H, (CH2)4); 13C
NMR (75.5 MHz, CDCl3) d = 172.63, 171.94 (CO),
141.12, 134.92, 131.62, 130.94, 129.02, 128.06, 121.10
(Ar-C), 52.74, 52.39 (COOCH3, NH–CH–CO), 37.38
(CH–CH2–Ar), 36.42, 33.64 (CO–CH2), 28.84, 25.36
(CH2).
Na]+, 243 [C19H15]+; IR (ATR)
m
(cmꢀ1) = 1744
1
4.2.9. (S)-3-Biphenyl-4-yl-2-(8-trityloxycarbamoyl-octa-
noylamino)-propionic acid methyl ester (11a). Compound
11a was synthesized by method E from 10a (500 mg,
0.73 mmol), benzeneboronic acid (174 mg, 1.40 mmol),
tri-(o-tolyl)-phosphine (22 mg, 0.07 mmol), 10 ml 1,2-
dimethoxyethane, 1.7 ml degassed water, palla-
dium(II)acetate (8 mg, 0.035 mmol) and sodium
carbonate (150 mg, 1.4 mmol); reaction time: five min-
utes; yield: 228 mg (46%) white powder; melting point:
(COOCH3), 1675, 1644 (CO–NH, CO–NH–O–Trt); H
NMR (300 MHz, CDCl3) d = 7.75 (br s, 1H, Trt–O–
NH–CO), 7.54–7.05 (m, 22H, Ar-H), 5.93 (d, 1H,
3J = 7.9 Hz, NH–CH–CO), 4.95–4.88 (dpt, 1H,
3J = 7.9 Hz, 3J = 5.9 Hz, 3J = 5.9 Hz, NH–CH–CO),
3.74 (s, 3H, COOCH3), 3.18 (dd, 1H, 2J = 14.0 Hz,
2
3J = 5.9 Hz, CH–CH2–Ar), 3.10 (dd, 1H, J = 14.0 Hz,
3J = 5.9 Hz, CH–CH2–Ar), 2.15 (t, 2H, 3J = 7.4 Hz,
CO–CH2), 1.65–1.48 (m, 4H, (CH2)2), 1.32–1.08 (m, 8H,

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2025
(CH2)4); 13C NMR (75.5 MHz, CDCl3) d = 172.60,
172.09 (CO), 143.91, 141.10, 135.17, 133.26, 129.75,
129.00, 128.07, 127.99, 125.98, 124.74, 123.00 (Ar-C),
52.83, 52.33 (COOCH3, NH–CH–CO), 37.58 (CH–
CH2–Ar), 36.44 (CO–CH2), 28.85, 25.37 ((CH2)5).
1.20 mmol), tri-(o-tolyl)-phosphine (19 mg, 0.06 mmol),
8.4 ml 1,2-dimethoxyethane, 1.4 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.03 mmol) and sodium carbon-
ate (128 mg, 1.20 mmol); reaction time: five minutes; yield
261 mg (66%) white powder; melting point: 87 ꢁC; MS
(ESI pos.) m/z = 681 [M+Na]+, 438 [C22H27N2O6Na]+,
243 [C19H15]+; IR (ATR) m (cmꢀ1) = 1744 (COOCH3),
4.2.12. (S)-3-(4-Thiophen-3-yl-phenyl)-2-(7-trityloxycar-
bamoyl-heptanoylamino)-propionic acid methyl ester
(11d). Compound 11d was synthesized by method E from
10a (400 mg, 0.60 mmol), 3-thienylboronic acid (137 mg,
1.05 mmol), tri-(o-tolyl)-phosphine (17 mg, 0.053 mmol),
7.4 ml 1,2-dimethoxyethane, 1.2 ml degassed water, pal-
ladium(II)acetate (6 mg, 0.026 mmol) and sodium car-
bonate (113 mg, 1.05 mmol); reaction time: 5 min; yield
299 mg (74%) white powder; melting point: 173 ꢁC; MS
(ESI pos.) m/z = 697 [M+Na]+, 454 [C22H27N2O5SNa]+,
243 [C19H15]+; IR (ATR) m (cmꢀ1) = 1753 (COOCH3),
1670, 1642 (CO–NH, CO–NH–O–Trt); 1H NMR
(300 MHz, CDCl3) d = 7.69 (br s, 1H, Trt–O–NH–CO),
7.53–7.10 (m, 22H, Ar-H), 5.89 (d, 1H, 3J = 7.8 Hz,
1
1640 (CO–NH, CO–NH–O–Trt); H NMR (300 MHz,
CDCl3) d = 7.60–7.57 (m, 2H, 30/50-H), 7.44 (m, 1H,
500-H), 7.33 (m, 15H, Ar-H), 7.11–7.08 (m, 2H, 20/60-H),
6.62–6.61 (m, 1H, 300-H), 6.46–6.44 (m, 1H, 400-H), 5.86
3
(d, 1H, J = 7.8 Hz, NH–CH–CO), 4.92–4.86 (m, 1H,
NH–CH–CO), 3.73 (s, 3H, COOCH3), 3.16 (dd, 1H,
3
2J = 13.9 Hz, J = 5.9 Hz, CH–CH2–Ar), 3.08 (dd, 1H,
2J = 13.9 Hz, 3J = 5.9 Hz, CH–CH2–Ar), 2.12 (t, 2H,
3J = 7.6 Hz, CO–CH2), 1.81–0.96 (m, 10H, (CH2)5); 13
C
NMR (75.5 MHz, CDCl3) d = 172.48, 172.10 (CO),
153.67 (200-C), 142.03, 141.20, 134.99, 129.84, 129.57,
129.02, 128.07, 123.95 (Ar-C), 111.64 (300-C), 104.97 (400-
C), 52.89, 52.32 (COOCH3, NH–CH–CO), 37.73 (CH–
CH2–Ar), 36.37 (CO–CH2), 28.71, 25.26 (CH2).
3
3
NH–CH–CO), 4.91 (dpt, 1H, J = 7.8 Hz, J = 5.8 Hz,
3J = 5.8 Hz, NH–CH–CO), 3.74 (s, 3H, COOCH3), 3.18
(dd, 1H, J = 13.9 Hz, J = 5.8 Hz, CH–CH2–Ar), 3.10
2
3
4.2.15. (S)-3-(4-Furan-2-yl-phenyl)-2-(8-trityloxycarba-
moyl-octanoylamino)-propionic acid methyl ester (11g).
Compound 11g was synthesized by method E from 10b
(400 mg, 0.58 mmol), 2-furanboronic acid (131 mg,
1.16 mmol), tri-(o-tolyl)-phosphine (18 mg, 0.058 mmol),
8.2 ml 1,2-dimethoxyethane, 1.4 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.03 mmol) and sodium carbon-
ate (126 mg, 1.16 mmol); reaction time: 5 min; yield
324 mg (83%) white powder; melting point: 93 ꢁC; MS
(ESI pos.) m/z = 695 [M+Na]+, 452 [C23H29N2O6Na]+,
243 [C19H15]+; IR (ATR) m (cmꢀ1) = 1738 (COOCH3),
1668, 1644 (CO–NH, CO–NH–O–Trt); 1H NMR
(300 MHz, CDCl3) d = 7.71 (br s, 1H, Trt–O–NH–CO),
7.61–7.58 (m, 2H, 30/50-H), 7.44 (m, 1H, 500-H), 7.34
(m, 15H, Ar-H), 7.12–7.10 (m, 2H, 20/60-H), 6.63 (d,
2
3
(dd, 1H, J = 13.9 Hz, J = 5.8 Hz, CH–CH2–Ar), 2.12
(t, 2H, 3J = 7.5 Hz, CO–CH2), 1.65–1.47 (m, 4H,
(CH2)2), 1.28–1.00 (m, 6H, (CH2)3); 13C NMR
(75.5 MHz, CDCl3) d = 172.73, 172.37 (CO), 142.02,
141,35, 134.98, 134.91, 129.92, 129.24, 128.29, 126.76,
126.47, 126.39, 120.42 (Ar-C), 53.10, 52.56 (COOCH3,
NH–CH–CO), 37.81 (CH–CH2–Ar), 36.59 (CO–CH2),
28.93, 25.48 ((CH2)4).
4.2.13. (S)-3-(4-Thiophen-3-yl-phenyl)-2-(8-trityloxycar-
bamoyl-octanoylamino)-propionic acid methyl ester
(11e). Compound 11e was synthesized by method E from
10b (400 mg, 0.58 mmol), 3-thienylboronic acid (150 mg,
1.16 mmol), tri-(o-tolyl)-phosphine (18 mg, 0.058 mmol),
8.2 ml 1,2-dimethoxyethane, 1.3 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.029 mmol) and sodium car-
bonate (125 mg, 1.16 mmol); reaction time: 5 min; yield
337 mg (84%) white powder; melting point: 137 ꢁC; MS
(ESI pos.) m/z = 711 [M+Na]+, 468 [C23H29N2O5SNa]+,
243 [C19H15]+; IR (ATR) m (cmꢀ1) = 1742 (COOCH3),
4
1H, 3J = 3.3 Hz, J = 0.6 Hz, 300-H), 6.46 (d, 1H,
3J = 3.3 Hz, 3J = 1.8 Hz, 400-H), 5.90 (d, 1H,
3
3J = 7.8 Hz, NH–CH–CO), 4.91 (dpt, 1H, J = 7.8 Hz,
3J = 5.9 Hz, 3J = 5.9 Hz, NH–CH–CO), 3.74 (s, 3H,
COOCH3), 3.17 (dd, 1H, 2J = 13.9 Hz, 3J = 5.9 Hz,
2
3
CH–CH2–Ar), 3.10 (dd, 1H, J = 13.9 Hz, J = 5.9 Hz,
CH–CH2–Ar), 2.15 (t, 2H, 3J = 7.5 Hz, CO–CH2), 1.65–
1.49 (m, 4H, (CH2)2), 1.27–1.00 (m, 8H, (CH2)4); 13C
NMR (75.5 MHz, CDCl3) d = 172.54, 172.10 (CO),
153.65 (200-C), 142.02, 141,30, 134.99, 129.81, 129.56,
129.01, 128.05, 123.93 (Ar-C), 111.63 (300-C), 104.94 (400-
C), 52.86, 52.31 (COOCH3, NH–CH–CO), 37.72 (CH–
CH2–Ar), 36.45 (CO–CH2), 28.84, 25.38 (CH2).
1
1649 (CO–NH, CO–NH–O–Trt); H NMR (300 MHz,
CDCl3) d = 7.71 (br s, 1H, Trt–O–NH–CO), 7.54–7.51
3
3
(m, 2H, 30/50-H), 7.43 (pt, 1H, J = 2.1 Hz, J = 2.1 Hz,
200-H), 7.37–7.00 (m, 17H, Ar-H), 7.14–7.11 (m, 2H, 20/
60-H), 5.90 (d, 1H, 3J = 7.9 Hz, NH–CH–CO), 4.91
(dpt, 1H, 3J = 7.9 Hz, 3J = 5.8 Hz, 3J = 5.8 Hz, NH–
CH–CO), 3.75 (s, 3H, COOCH3), 3.19 (dd, 1H,
3
2J = 13.9 Hz, J = 5.8 Hz, CH–CH2–Ar), 3.11 (dd, 1H,
2J = 13.9 Hz, 3J = 5.8 Hz, CH–CH2–Ar), 2.16 (t, 2H,
4.2.16. (S)-3-(4-Furan-3-yl-phenyl)-2-(7-trityloxycarba-
moyl-heptanoylamino)-propionic acid methyl ester (11h).
Compound 11h was synthesized by method E from 10a
(400 mg, 0.60 mmol), 3-furanboronic acid (133 mg,
1.20 mmol), tri-(o-tolyl)-phosphine (19 mg, 0.06 mmol),
8.4 ml 1,2-dimethoxyethane, 1.4 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.03 mmol) and sodium carbon-
ate (128 mg, 1.20 mmol); reaction time: 5 min; 316 mg
(80%) white powder; melting point: 110 ꢁC; MS (ESI
pos.) m/z = 681 [M+Na]+, 438 [C22H27N2O6Na]+, 243
[C19H15]+; IR (ATR) m(cmꢀ1) = 1750 (COOCH3), 1666,
3J = 7.6 Hz, CO–CH2), 1.89–0.97 (m, 12H, (CH2)6); 13
C
NMR (75.5 MHz, CDCl3) d = 172.55, 172.14 (CO),
141.80, 141,19, 134.76, 134.68, 129.69, 129.00, 128.05,
126.52, 126.25, 126.15, 120.17 (Ar-C), 52.86, 52.31
(COOCH3, NH–CH–CO), 37.60 (CH–CH2–Ar), 36.46,
31.56 (CO–CH2), 28.85, 25.38 (CH2).
4.2.14. (S)-3-(4-Furan-2-yl-phenyl)-2-(7-trityloxycarba-
moyl-heptanoylamino)-propionic acid methyl ester (11f).
Compound 11f was synthesized by method E from 10a
(400 mg, 0.60 mmol), 2-furanboronic acid (133 mg,
1
1641 (CO–NH, CO–NH–O–Trt); H NMR (300 MHz,

2026
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2
CDCl3) d = 7.72–7.71 (m, 1H, 200-H), 7.46 (pt, 1H,
3J = 5.8 Hz, CH–CH2–Ar), 3.14 (dd, 1H, J = 13.9 Hz,
3J = 1.7 Hz, J = 1.7 Hz, 500-H), 7.42–7.40 (m, 2H, 30/50-
3J = 5.8 Hz, CH–CH2–Ar), 2.12 (t, 2H, 3J = 7.4 Hz,
CO–CH2), 1.65–0.93 (m, 10H, (CH2)5); 13C NMR
(75.5 MHz, CDCl3) d = 172.54, 172.16 (CO), 141.12,
140.63, 140.15, 139.49, 139.45, 135.07, 129.72, 129.00,
128.79, 128.04, 127.48, 127.34, 127.29, 127.10, 127.00
(Ar-C), 52.91, 52.33 (COOCH3, NH–CH–CO), 37.56
(CH–CH2–Ar), 36.35 (CO–CH2), 28.69, 25.24 (CH2).
4
H), 7.34 (m, 15H, Ar-H), 7.11–7.08 (m, 2H, 20/60-H),
4
6.68 (dd, 1H, 3J = 1.7 Hz, J = 0.8 Hz, 400-H), 5.90
(d,1H, 3J = 7.7 Hz, NH–CH–CO), 4.89 (dpt, 1H,
3J = 7.7 Hz, 3J = 5.9 Hz, 3J = 5.9 Hz, NH–CH–CO),
3.74 (s, 3H, COOCH3), 3.17 (dd, 1H, 2J = 14.0 Hz,
2
3J = 5.9 Hz, CH–CH2–Ar), 3.09 (dd, 1H, J = 14.0 Hz,
3J = 5.9 Hz, CH–CH2–Ar), 2.13 (t, 2H, 3J = 7.5 Hz,
CO–CH2), 1.90–1.00 (m, 10H, (CH2)5); 13C NMR
(75.5 MHz, CDCl3) d = 172.50, 172.13 (CO), 143.67 (500-
C), 141.02, 138.45, 134.59, 131.28, 129.68, 129.00,
128.05, 126.02, 125.97 (Ar-C), 108.70 (400-C), 52.88,
52.31 (COOCH3, NH–CH–CO), 37.60 (CH–CH2–Ar),
36.34 (CO–CH2), 28.68, 25.23 (CH2).
4.2.19. (S)-3-[1,10:40,100]Terphenyl-4-yl-2-(8-trityloxycar-
bamoyl-octanoylamino)-propionic acid methyl ester (11k).
Compound 11k was synthesized by method E from 10b
(400 mg, 0.58 mmol), 4-biphenylboronic acid (172 mg,
0.87 mmol), tri-(o-tolyl)-phosphine (18 mg, 0.058 mmol),
8.2 ml 1,2-dimethoxyethane, 1.4 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.03 mmol) and sodium carbon-
ate (124 mg, 1.16 mmol); reaction time: ten minutes; the
crude product was purified by flash chromatography on
silica gel using ethyl acetate, cyclohexane and dichloro-
methane (2:1:1) as mobile phase; yield 370 mg (84%) white
powder; melting point: 184 ꢁC; MS (ESI pos.) m/z = 781
[M+Na]+, 538 [C31H35N2O5Na]+, 243 [C19H15]+; IR
(ATR) m (cmꢀ1) = 1739 (COOCH3), 1669, 1647 (CO–
NH, CO–NH–O–Trt); 1H NMR (300 MHz, CDCl3)
4.2.17. (S)-3-(4-Furan-3-yl-phenyl)-2-(8-trityloxycarba-
moyl-octanoylamino)-propionic acid methyl ester (11i).
Compound 11i was synthesized by method E from 10b
(400 mg, 0.58 mmol), 3-furanboronic acid (131 mg,
1.16 mmol), tri-(o-tolyl)-phosphine (18 mg, 0.058 mmol),
8.2 ml 1,2-dimethoxyethane, 1.4 ml degassed water, pal-
ladium(II)acetate (7 mg, 0.03 mmol) and sodium carbon-
ate (126 mg, 1.16 mmol); reaction time: five minutes; yield
335 mg (86%) white powder; melting point: 143 ꢁC; MS
(ESI pos.) m/z = 695 [M+Na]+, 452 [C23H29N2O6Na]+,
243 [C19H15]+; IR (ATR) m (cmꢀ1) = 1741 (COOCH3),
1668, 1645 (CO–NH, CO–NH–O–Trt); 1H NMR
(300 MHz, CDCl3) d = 7.71 (dd, 1H, 4J = 1.7 Hz,
4J = 0.9 Hz, 200-H), 7.46 (pt, 1H, 3J = 1.7 Hz,
4J = 1.7 Hz, 500-H), 7.42–7.39 (m, 2H, 30/50-H), 7.34 (m,
15H, Ar-H), 7.11–7.08 (m, 2H, 20/60-H), 6.68 (dd, 1H,
3J = 1.7 Hz, 4J = 0.9 Hz, 400-H), 5.91 (d, 1H,
3
d = 7.66–7.16 (m, 28H, Ar-H), 5.93 (d, 1H, J = 7.7 Hz,
CO–NH–CH), 4.96 (dpt, 1H, J = 7.7 Hz, J = 5.9 Hz,
3
3
3J = 5.9 Hz, NH–CH–CO), 3.76 (s, 3H, COOCH3), 3.22
(dd, 1H, J = 13.9 Hz, J = 5.9 Hz, CH–CH2–Ar), 3.14
2
3
2
3
(dd, 1H, J = 13.9 Hz, J = 5.9 Hz, CH–CH2–Ar), 2.17
(t, 2H, 3J = 7.6 Hz, CO–CH2), 1.69–0.96 (m, 12H,
(CH2)6); 13C NMR (75.5 MHz, CDCl3) d = 172.59,
172.16 (CO), 141.12, 140.61, 140.15, 139.48, 139.43,
135.05, 129.73, 129.01, 128.80, 128.05, 127.49, 127.35,
127.28, 127.09, 127.00 (Ar-C), 93.34 (Ph3-C), 52.90,
52.34 (COOCH3, NH–CH–CO), 37.58 (CH–CH2–Ar),
36.47 (CO–CH2), 28.85, 25.39 (CH2).
3
3J = 7.8 Hz, NH–CH–CO), 4.89 (dpt, 1H, J = 7.8 Hz,
3J = 5.9 Hz, 3J = 5.9 Hz, NH–CH-CO), 3.74 (s, 3H,
COOCH3), 3.17 (dd, 1H, 2J = 13.9 Hz, 3J = 5.9 Hz,
2
3
CH–CH2–Ar), 3.10 (dd, 1H, J = 13.9 Hz, J = 5.9 Hz,
CH–CH2–Ar), 2.15 (t, 2H, 3J = 7.5 Hz, CO–CH2), 1.66–
1.53 (m, 4H, (CH2)2), 1.29–1.01 (m, 8H, (CH2)4); 13C
NMR (75.5 MHz, CDCl3) d = 172.08, 171.65 (CO),
143.19 (500-C), 140.59, 137.93, 134.09, 130.76, 129.19,
128.51, 127.58, 125.52, 125.47 (Ar-C), 108.21 (400-C),
52.37, 51.84 (COOCH3, NH–CH–CO), 37.10 (CH–
CH2–Ar), 35.96 (CO–CH2), 28.38, 24.89 (CH2).
4.2.20. (S)-3-(4-Naphthalen-2-yl-phenyl)-2-(7-trityloxyc-
arbamoyl-heptanoylamino)-propionic acid methyl ester
(11l). Compound 11l was synthesized by method E from
10a (400 mg, 0.60 mmol), 2-naphthylboronic acid
(181 mg, 1.06 mmol), tri-(o-tolyl)-phosphine (17 mg,
0.055 mmol), 7.4 ml 1,2-dimethoxyethane, 1.2 ml de-
gassed water, palladium(II)acetate (6 mg, 0.026 mmol)
and sodium carbonate (113 mg, 1.06 mmol); reaction
time: ten minutes; yield 334 mg (77%) white powder; melt-
ing point: 155 ꢁC; MS (ESI pos.) m/z = 741 [M+Na]+, 498
4.2.18. (S)-3-[1,10:40,100]Terphenyl-4-yl-2-(7-trityloxycar-
bamoyl-heptanoylamino)-propionic acid methyl ester
(11j). Compound 11j was synthesized by method E from
10a (400 mg, 0.60 mmol), 4-biphenylboronic acid
(176 mg, 0.90 mmol), tri-(o-tolyl)-phosphine (19 mg,
0.06 mmol), 8.4 ml 1,2-dimethoxyethane, 1.4 ml degassed
water, palladium(II)acetate (7 mg, 0.03 mmol) and so-
dium carbonate (128 mg, 1.20 mmol); reaction time: ten
minutes; the crude product was purified by flash chroma-
tography on silica gel using ethyl acetate, cyclohexane and
dichloromethane (2:1:1) as mobile phase; yield 285 mg
(64%) white powder; melting point: 205 ꢁC; MS (ESI
pos.) m/z = 767 [M+Na]+, 524 [C30H33N2O5Na]+, 243
[C19H15]+; IR (ATR) m (cmꢀ1) = 1737 (COOCH3), 1669,
[C28H31N2O5Na]+, 243 [C19H15]+; IR (ATR)
m
(cmꢀ1) = 1739 (COOCH3), 1670, 1645 (CO–NH, CO–
1
NH–O–Trt); H NMR (300 MHz, CDCl3) d = 8.02 (br
s, 1H, Trt–O–NH–CO), 7.92–7.19 (m, 26H, Ar-H), 5.94
(d, 1H, 3J = 7.8 Hz, CO–NH–CH), 4.96 (dpt, 1H,
3J = 7.8 Hz, 3J = 5.8 Hz, 3J = 5.8 Hz, NH–CH–CO),
3.77 (s, 3H, COOCH3), 3.23 (dd, 1H, 2J = 13.9 Hz,
2
3J = 5.8 Hz, CH–CH2–Ar), 3.16 (dd, 1H, J = 13.9 Hz,
3J = 5.8 Hz, CH–CH2–Ar), 2.15 (t, 2H, 3J = 7.6 Hz,
CO–CH2), 1.59–1.00 (m, 10H, (CH2)5); 13C NMR
(75.5 MHz, CDCl3) d = 172.54, 172.15 (CO), 141.13,
139.86, 137.92, 135.06, 133.66, 132.62, 129.76, 129.00,
128.42, 128.15, 128.05, 127.61, 127.48, 126.29, 125.94,
125.62, 125.34 (Ar-C), 93.50 (Ph3-C), 52.92, 52.35
(COOCH3, NH–CH–CO), 37.56 (CH–CH2–Ar), 36.37
(CO–CH2), 28.72, 25.25 (CH2).
1
1644 (CO–NH, CO–NH–O–Trt); H NMR (300 MHz,
CDCl3) d = 7.63–7.14 (m, 28H, Ar-H), 5.91 (d, 1H,
3J = 7.8 Hz, CO–NH–CH), 4.94–4.88 (m, 1H, NH–CH–
CO), 3.73 (s, 3H, COOCH3), 3.19 (dd, 1H, 2J = 13.9 Hz,

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2027
4.2.21. (S)-3-(4-Naphthalen-2-yl-phenyl)-2-(8-trityloxyc-
arbamoyl-octanoylamino)-propionic acid methyl ester
(11m). Compound 11m was synthesized by method E
from 10b (400 mg, 0.58 mmol), 2-naphthylboronic acid
(149 mg, 0.87 mmol), tri-(o-tolyl)-phosphine (18 mg,
0.058 mmol), 8.2 ml 1,2-dimethoxyethane, 1.4 ml de-
gassed water, palladium(II)acetate (7 mg, 0.029 mmol)
and sodium carbonate (124 mg, 1.16 mmol); reaction
time: ten minutes; yield 367 mg (86%) white powder; melt-
ing point: 97 ꢁC; MS (ESI pos.) m/z = 755 [M+Na]+,
512 [C29H33N2O5Na]+, 243 [C19H15]+; IR (ATR) m
(cmꢀ1) = 1744 (COOCH3), 1652, 1615 (CO–NH, CO–
3J = 8.0 Hz, 3J = 5.5 Hz, NH–CH–CO), 3.60 (s, 3H,
COOCH3), 3.03 (dd, 1H, 2J = 13.8 Hz, 3J = 5.5
Hz, CH–CH2–Ar), 2.91–2.84 (dd, 1H, 2J = 13.8 Hz,
3J = 9.5 Hz, CH–CH2–Ar), 2.03 (t, 2H, 3J = 7.3 Hz,
CO–CH2), 1.88 (t, 2H, 3J = 7.4 Hz, CO–CH2), 1.43–1.31
(m, 4H, (CH2)2), 1.21–1.04 (m, 4H, (CH2)2); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.23, 172.08, 169.04 (CO),
143.16, 136.76, 132.02, 129.74, 128.38, 125.80, 125.17,
123.36 (Ar-C), 53.20, 51.78 (COOCH3, NH–CH–CO),
36.28 (CH–CH2–Ar), 34.90, 32.18 (CO–CH2), 28.29,
28.14, 24.99, 24.93 (CH2).
1
NH–O–Trt); H NMR (300 MHz, CDCl3) d = 8.00 (br
4.2.24. (S)-2-(8-Hydroxycarbamoyl-octanoylamino)-3-(4-
thiophen-2-yl-phenyl)-propionic acid methyl ester (4c).
Compound 4c was synthesized by method F from 11c
(300 mg, 0.44 mmol), 3.0 ml dichloromethane, TFA and
TES; yield 152 mg (77%) grey powder; melting point:
142 ꢁC; MS (ESI pos.) m/z = 469 [M+Na]+, 447
[M+H]+; IR (ATR) m (cmꢀ1) = 1750 (COOCH3), 1639,
1625 (CO–NH, CO–NH–OH); 1H NMR (300 MHz,
DMSO-d6) d = 10.28 (s, 1H, HO–NH–CO), 8.61 (s, 1H,
s, 1H, Trt–O–NH–CO), 7.91–7.19 (m, 26H, Ar-H), 5.94
(d, 1H, 3J = 7.8 Hz, CO–NH–CH), 4.96 (dpt, 1H,
3J = 7.8 Hz, 3J = 5.8 Hz, 3J = 5.8 Hz, NH–CH–CO),
3.76 (s, 3H, COOCH3), 3.23 (dd, 1H, 2J = 13.9 Hz,
3J = 5.8 Hz, CH–CH2–Ar), 3.16 (dd, 1H, 2J = 13.9
Hz, 3J = 5.8 Hz, CH–CH2–Ar), 2.17 (t, 2H, 3J = 7.6 Hz,
CO–CH2), 1.59–0.98 (m, 12H, (CH2)6); 13C NMR
(75.5 MHz, CDCl3) d = 172.59, 172.16 (CO), 141.22,
139.85, 137.92, 135.07, 133.65, 132.61, 129.77, 129.00,
128.43, 128.15, 128.04, 127.61, 127.47, 126.30, 125.94,
125.61, 125.34 (Ar-C), 93.34 (Ph3-C), 52.91, 52.34
(COOCH3, NH–CH–CO), 37.58 (CH–CH2–Ar), 36.47
(CO–CH2), 28.85, 25.39 (CH2).
3
HO–NH–CO), 8.22 (d, 1H, J = 8.0 Hz, NH–CH–CO),
3
7.55–7.53 (m, 2H, 30/50-H), 7.49 (dd, 1H, J = 5.1 Hz,
4J = 1.1 Hz, 500-H), 7.45 (dd, 1H, 3J = 3.6 Hz,
4J = 1.1 Hz, 300-H), 7.25–7.22 (m, 2H, 20/60-H), 7.10 (dd,
3
1H, 3J = 5.1 Hz, J = 3.6 Hz, 400-H), 4.49 (ddd, 1H,
3J = 9.6 Hz, 3J = 8.0 Hz, 3J = 5.2 Hz, NH–CH–CO),
3.60 (s, 3H, COOCH3), 3.03 (dd, 1H, 2J = 13.7 Hz,
4.2.22. (S)-3-Biphenyl-4-yl-2-(8-hydroxycarbamoyl-octa-
noylamino)-propionic acid methyl ester (4a). Compound
4a was synthesized by method F from 11a (200 mg,
0.29 mmol), 2.0 ml dichloromethane, TFA and TES;
yield 88 mg (69%) grey powder; melting point: 130 ꢁC;
MS (ESI pos.) m/z = 463 [M+Na]+; IR (ATR) m
(cmꢀ1) = 1751 (COOCH3), 1640 (CO–NH, CO–NH–
2
3J = 5.2 Hz, CH–CH2–Ar), 2.86 (dd, 1H, J = 13.7 Hz,
3J = 9.6 Hz, CH–CH2–Ar), 2.02 (t, 2H, 3J = 7.3 Hz,
CO–CH2), 1.87 (t, 2H, 3J = 7.4 Hz, CO–CH2), 1.45–1.32
(m, 4H, (CH2)2), 1.21–1.05 (m, 6H, (CH2)3); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.24, 172.09, 169.07 (CO–
NH), 143.18, 136.76, 132.02, 129.74, 128.39, 125.30
125.16, 123.34 (Ar-C), 53.17, 51.79 (COOCH3, NH–
CH–CO), 36.29 (CH–CH2–Ar), 34.94, 32.20 (CO–CH2),
28.44, 28.29, 25.06 (CH2).
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.28 (s, 1H,
HO–NH–CO), 8.61 (s, 1H, HO–NH–CO), 8.25 (d, 1H,
3J = 7.9 Hz, NH–CH–CO), 7.64–7.28 (m, 9H, Ar-H),
4.55–4.47 (m, 1H, NH–CH–CO), 3.61 (s, 3H, COOCH3),
2
3
3.07 (dd, 1H, J = 13.8 Hz, J = 5.3 Hz, CH–CH2–Ar),
2
3
4.2.25. (S)-2-(7-Hydroxycarbamoyl-heptanoylamino)-3-
(4-thiophen-3-yl-phenyl)-propionic acid methyl ester
(4d). Compound 4d was synthesized by method F from
11d (280 mg, 0.41 mmol), 2.8 ml dichloromethane, TFA
and TES; yield 110 mg (62%) grey powder; melting point:
128 ꢁC; MS (ESI pos.) m/z = 455 [M+Na]+; IR (ATR) m
(cmꢀ1) = 1737 (COOCH3), 1645, 1630 (CO–NH, CO–
NH–OH); 1H NMR (300 MHz, DMSO-d6) d = 10.28
(s, 1H, HO–NH–CO), 8.62 (s, 1H, HO–NH–CO), 8.24
(d, 1H, 3J = 7.9 Hz, NH–CH–CO), 7.82 (dd, 1H,
2.91 (dd, 1H, J = 13.8 Hz, J = 9.8 Hz, CH–CH2–Ar),
2.04 (t, 2H, 3J = 7.2 Hz, CO–CH2), 1.88 (t, 2H,
3J = 7.4 Hz, CO–CH2), 1.46–1.34 (m, 4H, (CH2)2),
1.20–1.08 (m, 6H, (CH2)2); 13C NMR (75.5 MHz,
DMSO-d6) d = 172.27, 172.15, 169.05 (CO), 139.81,
138.27, 136.59, 129.59, 128.84, 126.41, 126.37, 125.81
(Ar-C), 53.25, 51.78 (COOCH3, NH–CH–CO), 36.24
(CH–CH2–Ar), 34.94, 32.19 (CO–CH2), 28.42, 28.29,
25.05 (CH2).
4
4J = 2.9 Hz, J = 1.3 Hz, 200-H), 7.63–7.60 (m, 2H, 30/50-
4.2.23. (S)-2-(7-Hydroxycarbamoyl-heptanoylamino)-3-
(4-thiophen-2-yl-phenyl)-propionic acid methyl ester
(4b). Compound 4b was synthesized by method F from
11b (160 mg, 0.24 mmol), 1.6 ml dichloromethane, TFA
and TES; yield 57 mg (55%) grey powder; melting point:
106 ꢁC; MS (ESI pos.) m/z = 433 [M+H]+; IR (ATR) m
(cmꢀ1) = 1736 (COOCH3), 1645, 1629 (CO–NH, CO–
NH–OH); 1H NMR (300 MHz, DMSO-d6) d = 10.28
(s, 1H, HO–NH–CO), 8.61 (s, 1H, HO–NH–CO), 8.24
3
H), 7.61–7.59 (m, 1H, 500-H), 7.52 (dd, 1H, J = 5.0 Hz,
4J = 1.3 Hz, 400-H), 7.25–7.22 (m, 2H, 20/60-H), 4.52–
4.44 (m, 1H, NH–CH–CO), 3.60 (s, 3H, COOCH3),
3.03 (dd, 1H, J = 13.8 Hz, J = 5.3 Hz, CH–CH2–Ar),
2
3
2
3
2.88 (dd, 1H, J = 13.8 Hz, J = 9.5 Hz, CH–CH2–Ar),
2.04 (t, 2H, 3J = 7.4 Hz, CO–CH2), 1.88 (t, 2H,
3J = 7.4 Hz, CO–CH2), 1.45–1.32 (m, 4H, (CH2)2),
1.21–1.06 (m, 4H, (CH2)2); 13C NMR (75.5 MHz,
DMSO-d6) d = 172.23, 171.15, 169.05 (CO–NH),
141.15, 136.16, 133.37, 129.51, 126.91, 126.02, 125.81,
120.49 (Ar-C), 53.28, 51.75 (COOCH3, NH–CH–CO),
36.30 (CH–CH2–Ar), 34.91, 32.18 (CO–CH2), 28.29,
28.15, 25.00, 24.94 (CH2).
3
(d, 1H, J = 8.0 Hz, NH–CH–CO), 7.57–7.54 (m, 2H,
3
4
30/50-H), 7.49 (dd, 1H, J = 5.1 Hz, J = 1.0 Hz, 500-H),
3
4
7.46 (dd, 1H, J = 3.7 Hz, J = 1.0 Hz, 300-H), 7.25–7.23
(m, 2H, 20/60-H), 7.12–7.09 (dd, 1H, 3J = 5.1 Hz,
3J = 3.7 Hz, 400-H), 4.49 (ddd, 1H, 3J = 9.5 Hz,

2028
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
4.2.26. (S)-2-(8-Hydroxycarbamoyl-octanoylamino)-3-(4-
thiophen-3-yl-phenyl)-propionic acid methyl ester (4e).
Compound 4e was synthesized by method F from 11e
(300 mg, 0.44 mmol), 3.0 ml dichloromethane, TFA
and TES; yield 157 mg (80%) grey powder; melting
point: 124 ꢁC; MS (ESI pos.) m/z = 447 [M+H]+; IR
(ATR) m (cmꢀ1) = 1750 (COOCH3), 1639 (CO–NH,
CO–NH–OH); 1H NMR (300 MHz, DMSO-d6)
d = 10.28 (s, 1H, HO–NH–CO), 8.61 (s, 1H, HO–NH–
COOCH3), 3.04 (dd, 1H, 2J = 13.8 Hz, 3J = 5.4 Hz,
CH–CH2–Ar), 2.87 (dd, 1H, J = 13.8 Hz, J = 9.7 Hz,
2
3
3
CH–CH2–Ar), 2.03 (t, 2H, J = 7.2 Hz, CO–CH2), 1.89
(t, 2H, 3J = 7.4 Hz, CO–CH2), 1.46–1.32 (m, 4H,
(CH2)2), 1.20–1.03 (m, 6H, (CH2)2); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.22, 172.11, 169.08 (CO),
152.96, 142.63, 136.58, 129.52, 128.67, 123.23, 111.94,
105.41 (Ar-C), 53.16, 51.77 (COOCH3, NH–CH–CO),
36.40 (CH–CH2–Ar), 34.93, 32.20 (CO–CH2), 28.45,
28.42, 28.29, 25.07, 25.05 ((CH2)5).
3
CO), 8.23 (d, 1H, J = 8.0 Hz, NH–CH–CO), 7.81 (dd,
4
4
1H, J = 2.8 Hz, J = 1.3 Hz, 200-H), 7.63–7.60 (m, 2H,
30/50-H), 7.61–7.59 (m, 1H, 500-H), 7.52 (dd, 1H,
4.2.29. (S)-3-(4-Furan-3-yl-phenyl)-2-(7-hydroxycarba-
moyl-heptanoylamino)-propionic acid methyl ester (4h).
Compound 4h was synthesized by method F from 11h
(310 mg, 0.47 mmol), 3.1 ml dichloromethane, TFA and
TES; yield 106 mg (54%) grey powder; melting point:
141 ꢁC; MS (ESI pos.) m/z = 439 [M+Na]+; IR (ATR) m
(cmꢀ1) = 1738 (COOCH3), 1623 (CO–NH, CO–NH–
3J = 5.0 Hz, J = 1.3 Hz, 400-H), 7.25–7.22 (m, 2H, 20/
4
60-H), 4.49 (ddd, 1H, 3J = 9.5 Hz, 3J = 8.0 Hz,
3J = 5.4 Hz, NH–CH–CO), 3.60 (s, 3H, COOCH3),
3.04 (dd, 1H, J = 13.7 Hz, J = 5.4 Hz, CH–CH2–Ar),
2
3
2
3
2.88 (dd, 1H, J = 13.7 Hz, J = 9.5 Hz, CH–CH2–Ar),
2.04 (t, 2H, 3J = 7.3 Hz, CO–CH2), 1.88 (t, 2H,
3J = 7.3 Hz, CO–CH2), 1.46–1.32 (m, 4H, (CH2)2),
1.21–1.05 (m, 6H, (CH2)2); 13C NMR (75.5 MHz,
DMSO-d6) d = 172.23, 172.14, 169.07 (CO), 141.16,
136.15, 133.36, 129.50, 126.91, 126.00 125.80, 120.47
(Ar-C), 53.25, 51.75 (COOCH3, NH–CH–CO), 36.30
(CH–CH2–Ar), 34.93, 32.19 (CO–CH2), 28.44, 28.42,
28.29, 25.06, 25.05 ((CH2)5).
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.31 (s, 1H,
HO–NH–CO), 8.65 (s, 1H, HO–NH–CO), 8.26 (d, 1H,
3J = 7.9 Hz, NH–CH–CO), 8.16 (dd, 1H, 4J = 1.7
4
Hz, J = 0.9 Hz, 200-H), 7.73 (pt, 1H, 2J = 1.7 Hz,
3J = 1.7 Hz, 500-H), 7.53–7.51 (m, 2H, 30/50-H), 7.23–7.21
3
4
(m, 2H, 20/60-H), 6.95 (dd, 1H, J = 1.7 Hz, J = 0.9 Hz,
400-H), 4.48 (ddd, 1H, 3J = 9.7 Hz, 3J = 7.9 Hz,
3J = 5.3 Hz, NH–CH–CO), 3.61 (s, 3H, COOCH3), 3.03
2
3
4.2.27. (S)-3-(4-Furan-2-yl-phenyl)-2-(7-hydroxycarba-
moyl-heptanoylamino)-propionic acid methyl ester (4f).
Compound 4f was synthesized by method F from 11f
(225 mg, 0.34 mmol), 2.2 ml dichloromethane, TFA and
TES; yield 75 mg (53%) grey powder; melting point:
137 ꢁC; MS (ESI pos.) m/z = 417 [M+H]+; IR (ATR) m
(cmꢀ1) = 1736 (COOCH3), 1646 (CO–NH, CO–NH–
(dd, 1H, J = 13.8 Hz, J = 5.3 Hz, CH–CH2–Ar), 2.87
(dd, 1H, J = 13.8 Hz, J = 9.7 Hz, CH–CH2–Ar), 2.04
2
3
3
3
(t, 2H, J = 7.3 Hz, CO–CH2), 1.90 (t, 2H, J = 7.4 Hz,
CO–CH2), 1.46–1.35 (m, 4H, (CH2)2), 1.20–1.08 (m, 4H,
(CH2)2); 13C NMR (75.5 MHz, DMSO-d6) d = 172.23,
172.14, 169.05 (CO), 144.14, 139.04, 135.94, 130.15,
129.44, 125.55, 125.31, 108.58 (Ar-C), 53.32, 51.75
(COOCH3, NH–CH–CO), 36.34 (CH–CH2–Ar), 34.91,
32.19 (CO–CH2), 28.30, 28.15, 25.00, 24.95 ((CH2)4).
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.29 (s, 1H,
HO–NH–CO), 8.62 (s, 1H, HO–NH–CO), 8.23 (d, 1H,
3J = 7.9 Hz, NH–CH–CO), 7.70 (m, 1H, 500-H), 7.61–
7.58 (m, 2H, 30/50-H), 7.26–7.24 (m, 2H, 20/60-H), 6.89–
6.88 (m, 1H, 300-H), 6.57–6.55 (m, 1H, 400-H), 4.52–4.45
(m, 1H, NH–CH–CO), 3.60 (s, 3H, COOCH3), 3.03 (dd,
4.2.30. (S)-3-(4-Furan-3-yl-phenyl)-2-(8-hydroxycarba-
moyl-octanoylamino)-propionic acid methyl ester (4i).
Compound 4i was synthesized by method G from 11i
(300 mg, 0.45 mmol), 8.9 ml dichloromethane, 1 ll
TMSTF and 85 ll TES; yield: 147 mg (76%) grey powder;
melting point: 138 ꢁC; MS (ESI pos.) m/z = 431 [M+H]+;
IR (ATR) m (cmꢀ1) = 1733 (COOCH3), 1646, 1623 (CO–
NH, CO–NH–OH); 1H NMR (300 MHz, DMSO-d6)
d = 10.28 (s, 1H, HO–NH–CO), 8.62 (s, 1H, HO–NH–
2
3
1H, J = 13.8 Hz, J = 5.3 Hz, CH–CH2–Ar), 2.88 (dd,
1H, 2J = 13.8 Hz, 3J = 9.6 Hz, CH–CH2–Ar), 2.03 (t,
2H, 3J = 7.2 Hz, CO–CH2), 1.88 (t, 2H, 3J = 7.3 Hz,
CO–CH2), 1.46–1.32 (m, 4H, (CH2)2), 1.20–1.06 (m, 4H,
(CH2)2); 13C NMR (75.5 MHz, DMSO-d6) d = 172.01,
171.84, 169.00 (CO), 152.89, 142.41, 136.45, 129.32,
128.57, 123.13, 111.71 105.18 (Ar-C), 53.03, 51.51
(COOCH3, NH–CH–CO), 36.34 (CH–CH2–Ar), 34.80,
32.04 (CO–CH2), 28.14, 27.99, 24.79 ((CH2)4).
3
CO), 8.22 (d, 1H, J = 8.0 Hz, NH–CH–CO), 8.12 (m,
3
200-H), 7.70 (pt, 1H, 2J = 1.7 Hz, J = 1.7 Hz, 500-H),
7.51–7.49 (m, 2H, 30/50-H), 7.22–7.19 (m, 2H, 20/60-H),
6.92 (dd, 1H, 3J = 1.7 Hz, 4J = 0.8 Hz, 400-H), 4.48 (ddd,
1H, 3J = 9.4 Hz, 3J = 8.0 Hz, 3J = 5.3 Hz, NH–CH–
CO), 3.60 (s, 3H, COOCH3), 3.02 (dd, 1H, 2J = 13.8 Hz,
4.2.28. (S)-3-(4-Furan-2-yl-phenyl)-2-(8-hydroxycarba-
moyl-octanoylamino)-propionic acid methyl ester (4g).
Compound 4g was synthesized by method F from 11g
(150 mg, 0.22 mmol), 1.5 ml dichloromethane, TFA and
TES; yield 59 mg (62%) grey powder; melting point:
135 ꢁC; MS (ESI neg.) m/z = 429 [MꢀH]ꢀ; IR (ATR) m
(cmꢀ1) = 1751 (COOCH3), 1639 (CO–NH, CO–NH–
2
3J = 5.3 Hz, CH–CH2–Ar), 2.86 (dd, 1H, J = 13.8 Hz,
3J = 9.4 Hz, CH–CH2–Ar), 2.03 (t, 2H, 3J = 7.2 Hz,
CO–CH2), 1.89 (t, 2H, 3J = 7.3 Hz, CO–CH2), 1.46–1.33
(m, 4H, (CH2)2), 1.19–1.04 (m, 6H, (CH2)3); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.25, 172.15, 169.09 (CO),
144.15, 139.03, 135.93, 130.14, 129.44, 125.55, 125.30,
108.57 (Ar-C), 53.28, 51.76 (COOCH3, NH–CH–CO),
36.34 (CH–CH2–Ar), 34.93, 32.20 (CO–CH2), 28.44,
28.42, 28.30, 25.05 ((CH2)5).
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.28 (s, 1H,
HO–NH–CO), 8.62 (s, 1H, HO–NH–CO), 8.23 (d, 1H,
3J = 8.1 Hz, NH–CH–CO), 7.71–7.70 (m, 1H, 500-H),
7.61–7.58 (m, 2H, 30/50-H), 7.26–7.24 (m, 2H, 20/60-H),
6.89–6.88 (m, 1H, 300-H), 6.56 (dd, 1H, 3J = 3.3 Hz,
3J = 1.8 Hz, 400-H), 4.49 (ddd, 1H, 3J = 9.7 Hz,
3J = 8.1 Hz, 3J = 5.4 Hz, NH–CH–CO), 3.60 (s, 3H,
4.2.31. (S)-2-(7-Hydroxycarbamoyl-heptanoylamino)-3-
[1,10:40,100]terphenyl-4-yl-propionic acid methyl ester (4j).

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2029
Compound 4j was synthesized by method F from 11j
(280 mg, 0.38 mmol), 2.8 ml dichloromethane, TFA and
TES; yield 107 mg (56%) grey powder; melting point:
176 ꢁC; MS (ESI pos.) m/z = 525 [M+Na]+; IR (ATR) m
(cmꢀ1) = 1737 (COOCH3), 1646 (CO–NH, CO–NH–
(CH2)2); 13C NMR (75.5 MHz, DMSO-d6) d = 172.30,
172.16, 169.05 (CO), 138.07, 137.13, 136.78, 133.30,
132.13, 129.69, 128.38, 128.11, 127.41, 126.68, 126.32,
125.98, 124.92, 124.90 (Ar-C), 53.33, 51.80 (COOCH3,
NH–CH–CO), 36.28 (CH–CH2–Ar), 34.92, 32.20 (CO–
CH2), 28.30, 28.16, 25.00, 24.96 ((CH2)4).
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.28 (s, 1H,
HO–NH–CO), 8.61 (s, 1H, HO–NH–CO), 8.27 (d, 1H,
3J = 8.0 Hz, NH–CH–CO), 7.73–7.29 (m, 13H, Ar-H),
4.49 (ddd, 1H, 3J = 9.5 Hz, 3J = 8.0 Hz, 3J = 5.3 Hz,
NH–CH–CO), 3.61 (s, 3H, COOCH3), 3.07 (dd, 1H,
4.2.34. (S)-2-(8-Hydroxycarbamoyl-octanoylamino)-3-(4-
naphthalen-2-yl-phenyl)-propionic acid methyl ester (4m).
Compound 4m was synthesized by method F from 11m
(300 mg, 0.41 mmol), 3.0 ml dichloromethane, TFA and
TES; yield 158 mg (79%) grey powder; melting point:
160 ꢁC; MS (ESI pos.) m/z = 491 [M+H]+, 513
[M+Na]+; IR (ATR) m (cmꢀ1) = 1744 (COOCH3), 1652,
1615 (CO–NH, CO–NH–OH); 1H NMR (300 MHz,
DMSO-d6) d = 10.28 (s, 1H, HO–NH–CO), 8.62 (s, 1H,
3
2J = 13.9 Hz, J = 5.3 Hz, CH–CH2–Ar), 2.92 (dd, 1H,
2J = 13.9 Hz, 3J = 9.5 Hz, CH–CH2–Ar), 2.05 (t, 2H,
3J = 7.4 Hz, CO–CH2), 1.88 (t, 2H, 3J = 7.4 Hz, CO–
CH2), 1.46–1.35 (m, 4H, (CH2)2), 1.19–1.09 (m, 4H,
(CH2)2); 13C NMR (75.5 MHz, DMSO-d6) d = 172.29,
172.16, 169.06 (CO), 139.57, 138.95, 138.78, 137.69,
136.74, 129.66, 128.93, 127.45, 127.12, 126.93, 126.50,
126.27 (Ar-C), 53.29, 51.79 (COOCH3, NH–CH–CO),
36.26 (CH–CH2–Ar), 34.93, 32.20 (CO–CH2), 28.31,
28.16, 25.00, 24.96 ((CH2)4).
3
HO–NH–CO), 8.28 (d, 1H, J = 8.0 Hz, NH–CH–CO),
8.19 (br s, 1H, 100-H), 8.00–7.96 (m, 2H, 050 00/800-H), 7.93–
7.90 (m, 1H, 400-H), 7.84–7.81 (m, 1H, 3 -H), 7.74–7.71
(m, 2H, 30/50-H), 7.56–7.47 (m, 2H, 600/700-H), 7.36–7.33
(m, 2H, 20/60-H), 4.54 (ddd, 1H, 3J = 9.5 Hz,
3J = 8.0 Hz, 3J = 5.4 Hz, NH–CH–CO), 3.63 (s, 3H,
COOCH3), 3.10 (dd, 1H, 2J = 13.8 Hz, 3J = 5.4 Hz,
4.2.32. (S)-2-(8-Hydroxycarbamoyl-octanoylamino)-3-
[1,10:40,100]terphenyl-4-yl-propionic acid methyl ester
(4k). Compound 4k was synthesized by method F from
11k (370 mg, 0.49 mmol), 3.7 ml dichloromethane, TFA
and TES; yield 216 mg (85%) grey powder; melting point:
193 ꢁC; MS (ESI pos.) m/z = 517 [M+H]+; IR (ATR) m
(cmꢀ1) = 1742 (COOCH3), 1653, 1616 (CO–NH, CO–
NH–OH); 1H NMR (300 MHz, DMSO-d6) d = 10.28 (s,
1H, HO–NH–CO), 8.62 (s, 1H, HO–NH–CO), 8.26 (d,
2
3
CH–CH2–Ar), 2.82 (dd, 1H, J = 13.8 Hz, J = 9.5 Hz,
3
CH–CH2–Ar), 2.06 (t, 2H, J = 7.3 Hz, CO–CH2), 1.88
(t, 2H, 3J = 7.3 Hz, CO–CH2), 1.46–1.36 (m, 4H,
(CH2)2), 1.20–1.10 (m, 6H, (CH2)3); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.28, 172.14, 169.03 (CO),
138.04, 137.12, 136.76, 133.28, 132.12, 129.68, 128.36,
128.08, 127.40, 126.65, 126.31, 125.96, 124.89 (Ar-C),
53.29, 51.78 (COOCH3, NH–CH–CO), 36.28 (CH–
CH2–Ar), 34.93, 32.17 (CO–CH2), 28.42, 28.40, 28.30,
25.05, 25.03 ((CH2)5).
3
1H, J = 8.0 Hz, NH–CH–CO), 7.73–7.29 (m, 13H, Ar-
H), 4.49 (ddd, 1H, 3J = 9.6 Hz, 3J = 8.0 Hz, 3J = 5.2 Hz,
NH–CH–CO), 3.61 (s, 3H, COOCH3), 3.07 (dd, 1H,
3
2J = 13.8 Hz, J = 5.2 Hz, CH–CH2–Ar), 2.91 (dd, 1H,
2J = 13.8 Hz, 3J = 9.6 Hz, CH–CH2–Ar), 2.05 (t, 2H,
3J = 7.2 Hz, CO–CH2), 1.88 (t, 2H, 3J = 7.4 Hz, CO–
CH2), 1.46–1.34 (m, 4H, (CH2)2), 1.20–1.08 (m, 6H,
(CH2)3); 13C NMR (75.5 MHz, DMSO-d6) d = 172.34,
172.18, 169.11 (CO), 139.59, 138.98, 138.80, 137.70,
136.75, 129.68, 128.95, 127.46, 127.13, 126.93, 126.52,
126.28 (Ar-C), 53.28, 51.81 (COOCH3, NH–CH–CO),
36.28 (CH–CH2–Ar), 34.97, 32.22 (CO–CH2), 28.46,
28.45, 28.32, 25.09, 25.08 ((CH2)5).
4.2.35. (S)-3-(4-Bromo-phenyl)-2-(7-hydroxycarbamoyl-
heptanoylamino)-propionic acid methyl ester (4n). Com-
pound 4n was synthesized by method F from 10a
(100 mg, 0.15 mmol), 1.0 ml dichloromethane, TFA and
TES; yield 33 mg (51%) grey powder; melting point:
150 ꢁC; (ESI pos.) m/z = 451/453 [M+Na]+; IR (ATR) m
(cmꢀ1) = 1736 (COOCH3), 1620 (CO–NH, CO–NH–
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.21 (s, 1H,
HO–NH–CO), 8.49 (s, 1H, HO–NH–CO), 8.10 (d, 1H,
3J = 8.1 Hz, NH–CH–CO), 7.47–7.42 (m, 2H, 30/50-H),
3
4.2.33. (S)-2-(7-Hydroxycarbamoyl-heptanoylamino)-3-
(4-naphthalen-2-yl-phenyl)-propionic acid methyl ester
(4l). Compound 4l was synthesized by method F from
11l (230 mg, 0.32 mmol), 2.3 ml dichloromethane, TFA
and TES; yield 107 mg (70%) grey powder; melting point:
130 ꢁC; MS (ESI pos.) m/z = 477 [M+H]+, 499 [M+Na]+;
IR (ATR) m (cmꢀ1) = 1742 (COOCH3), 1653, 1616 (CO–
NH, CO–NH–OH); 1H NMR (300 MHz, DMSO-d6)
d = 10.28 (s, 1H, HO–NH–CO), 8.61 (s, 1H, HO–NH–
CO), 8.28 (d, 1H, 3J = 8.0 Hz, NH–CH–CO), 8.18 (br s,
1H, 100-H), 7.99–7.96 (m, 2H, 500/800-H), 7.92–7.89 (m,
1H, 400-H), 7.83–7.80 (m, 1H, 300-H), 7.74–7.71 (m, 2H,
30/50-H), 7.55–7.46 (m, 2H, 600/700-H), 7.35–7.32 (m, 2H,
20/60-H), 4.54 (ddd, 1H, 3J = 9.5 Hz, 3J = 8.0 Hz,
3J = 5.3 Hz, NH–CH–CO), 3.62 (s, 3H, COOCH3), 3.09
7.18–7.13 (m, 2H, 20/60-H), 4.49 (ddd, 1H, J = 9.4 Hz,
3J = 8.1 Hz, 3J = 5.4 Hz, NH–CH–CO), 3.60 (s, 3H,
COOCH3), 3.01 (dd, 1H, 2J = 13.8 Hz, 3J = 5.4 Hz,
CH–CH2–Ar), 2.85 (dd, 1H, J = 13.8 Hz, J = 9.4 Hz,
2
3
3
CH–CH2–Ar), 2.03 (t, 2H, J = 7.3 Hz, CO–CH2), 1.93
(t, 2H, 3J = 6.8 Hz, CO–CH2), 1.50–1.34 (m, 4H,
(CH2)2), 1.24–1.07 (m, 4H, (CH2)2); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.00, 171.68, 168.95 (CO),
136.62, 131.07, 130.78, 119.46 (Ar-C), 52.79, 51.54
(COOCH3, NH–CH–CO), 35.88 (CH–CH2–Ar), 34.78,
32.07 (CO–CH2), 28.14, 27.96, 24.76 ((CH2)4).
4.2.36. (S)-3-(4-Bromo-phenyl)-2-(8-hydroxycarbamoyl-
octanoylamino)-propionic acid methyl ester (4o). Com-
pound 4o was synthesized by method F from 10b
(200 mg, 0.29 mmol), 2.0 ml dichloromethane, TFA and
TES; yield 115 mg (89%) grey powder; melting point:
140 ꢁC; (ESI pos.) m/z = 465/467 [M+Na]+; IR (ATR) m
(cmꢀ1) = 1736 (COOCH3), 1619 (CO–NH, CO–NH–
2
3
(dd, 1H, J = 13.8 Hz, J = 5.3 Hz, CH–CH2–Ar), 2.93
(dd, 1H, J = 13.8 Hz, J = 9.5 Hz, CH–CH2–Ar), 2.05
2
3
3
3
(t, 2H, J = 7.3 Hz, CO–CH2), 1.87 (t, 2H, J = 7.3 Hz,
CO–CH2), 1.46–1.35 (m, 4H, (CH2)2), 1.19–1.09 (m, 4H,

2030
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
1
OH); H NMR (300 MHz, DMSO-d6) d = 10.29 (s, 1H,
HO–NH–CO), 8.61 (s, 1H, HO–NH–CO), 8.21 (d, 1H,
3J = 8.0 Hz, NH–CH–CO), 7.46–7.43 (m, 2H, 30/50-H),
M2-agarose beads (Sigma) as previously described. We
used the purified subtypes to determine the inhibitory
potential using an unselective substrate (called
ZMAL)20 that was shown to be suitable for this test
system.
3
7.18–7.15 (m, 2H, 20/60-H), 4.47 (ddd, 1H, J = 9.8 Hz,
3J = 8.0 Hz, 3J = 5.3 Hz, NH–CH–CO), 3.59 (s, 3H,
COOCH3), 3.01 (dd, 1H, 2J = 13.9 Hz, 3J = 5.3 Hz,
CH–CH2–Ar), 2.82 (dd, 1H, J = 13.9 Hz, J = 9.8 Hz,
2
3
293T cell lines overexpressing FLAG-tagged HDAC1
and HDAC6 were cultured and lysed. M2 agarose was
used at 25 ll/ml cell lysate. The immunoprecipitated en-
zymes were washed in enzyme buffer, diluted (1:10) and
used as an enzyme source. Fifty microliters of beads
suspension was incubated with 5 ll of the inhibitor dilu-
tion and 5 ll of the substrate dilution at 37 ꢁC for
180 min. The reaction was stopped by adding 190 ll of
freshly prepared stop solution (3.3 lM TSA, borate buf-
fer, pH 9.5, and naphthalene-2,3-dicarboxaldehyde
(16 mM in methanol) in a ratio of 5:180:5). The samples
were measured at an excitation wavelength of 330 nm
and an emission wavelength of 390 nm.
3
CH–CH2–Ar), 2.01 (t, 2H, J = 7.2 Hz, CO–CH2), 1.92
(t, 2H, 3J = 7.4 Hz, CO–CH2), 1.47–1.31 (m, 4H,
(CH2)2), 1.19–1.02 (m, 6H, (CH2)3); 13C NMR
(75.5 MHz, DMSO-d6) d = 172.70, 172.45, 169.57 (CO),
137.27, 131.79, 131.47, 120.15 (Ar-C), 53.44, 52.31
(COOCH3, NH–CH–CO), 36.45 (CH–CH2–Ar), 35.43,
32.73 (CO–CH2), 28.94, 28.76, 25.58, 25.54 ((CH2)5).
4.3. Assays
4.3.1. Assays with rat liver extract. Enzyme inhibition of
the new inhibitors on HDACs was determined using
standard methods that have previously been reported.
Rat liver HDAC was purified by ammonium sulfate pre-
cipitation and chromatography on Q-Sepharose as de-
scribed. To get a first idea of the potential of the new
compounds we used this partially purified extract with
a fluorescent substrate (MAL)35 that was shown to be
unselective21 for different subtypes. In a second test ser-
ies we used the same extract with subtype selective sub-
strates previously discovered in our group21 in order to
discover compounds showing subtype selectivity. For
HDAC1 prediction Z-(propionyl)Lys-AMC and for
HDAC6 prediction Boc(Ac)Lys-(F3-AMC) were used.
4.3.3. Determination of IC50-values. The amount of
remaining substrate in the samples was calculated rela-
tive to two standards equivalent to 0% and 100% en-
zyme inhibition. For 100% inhibition we used 50 ll of
enzyme source, 5 ll of a 3.3 lM TSA dilution and 5 ll
of the substrate dilution. For 0% inhibition we used
50 ll of enzyme source, 5 ll of enzyme buffer and 5 ll
of the substrate dilution. The IC50-values were deter-
mined by measuring the enzyme inhibition of a dilution
series of the particular inhibitor in relation to the stan-
dards and following evaluation with the GraphPadP-
rism software.
Stock solutions of the inhibitors 4a–o were prepared at
12 mM in DMSO and further diluted with enzyme buf-
fer [1.4 mM NaH2PO4, 18.6 mM Na2HPO4, pH 7.9,
0.25 mM EDTA, 10 mM NaCl, 10% (v/v) glycerol and
10 mM 2-mercaptoethanol] to the desired concentration.
Stock solutions of the substrates were prepared at
12.6 mM in DMSO and also further diluted with en-
zyme buffer to a concentration of 126 lM and used for
the assay. Fifty microliters of rat liver extract was then
incubated with 5 ll of the inhibitor dilution and 5 ll
of the substrate dilution at 37 ꢁC. Incubation times were
90 min for the unselective substrates and 180 min for the
selective substrates, respectively. The reaction was
stopped by adding 190 ll of freshly prepared stop solu-
tion (TSA 3.3 lM, borate buffer, pH 9.5, and naphtha-
lene-2,3-dicarboxaldehyde (NDA, 16 mM in methanol)
in a ratio of 5:190:5). The samples were measured at
an excitation wavelength of 330 nm and an emission
wavelength of 390 nm except for HDAC6-selective sub-
strate (excitation 340 nm, emission 430 nm). For com-
pound 4m we had to use a different version of the
assay due to interferences with the test system by the
inhibitor. After incubation of the enzyme with the inhib-
itor instead of NDA trypsin was used in order to devel-
op the reaction according to the literature.36–38
4.3.4. Cell proliferation. For cell proliferation assays,
cells were seeded at 5000 per well in 80 ll of growing
medium in 96-well tissue culture plates. At 24 h after
seeding, diluted compounds or DMSO vehicle control
was added to each well to a total volume of 100 ll (three
replicates per concentration) and incubated for 48 h at
37 ꢁC. Growth inhibition was determined using One-
Aqueous Proliferation Assay (MTS, Promega, Madison,
WI) according to manufacturer’s instructions, and the
absorbance measured at 490 nm on a microplate reader.
The 50% growth inhibition (GI50) was calculated as the
compound concentration required to reduce cell number
by 50% compared with control using GraphPad Prism
4.0.
4.3.5. Western blot analysis. HCT116 human colon car-
cinoma cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM; Mediatech, Herndon, VA) supple-
mented with 10% foetal bovine serum (FBS; Gemini
Bio-products, Woodland, CA), 1% penicillin–strepto-
mycin and 2 mM L-glutamine (Gibco Invitrogen Corpo-
ration). The cells were treated with different
concentrations of the inhibitors 4d, 4e, 4n and TSA as
a control for 6 h. Cells were lysed in 50 mM Tris–HCl
(pH 7.5), 0.5 mM EDTA, 150 mM NaCl, 0.5% NP-40
and 1· complete protease inhibitors (Roche, Penzberg,
Germany), and protein concentration was determined
with the DC Protein Assay (Bio-Rad). Protein samples
were electrophoresed on 10% and 15% SDS–polyacryl-
amide gels and transferred to a nitrocellulose membrane
4.3.2. Immunoprecipitated FLAG-tagged HDAC1 and
HDAC6. To verify the data generated with the subtype
selective substrates in the rat liver extract test system, we
isolated FLAG-tagged HDAC1 and HDAC6 from 293T
cell lines overexpressing either HDAC1 or HDAC6 with

S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
2031
(Bio-Rad). Membranes were blocked with 5% non-fat
dried milk in TBS–Tween (10 mM Tris–HCl (pH 7.5),
150 mM NaCl and 0.1% Tween 20) and probed with
anti-acetylated a-tubulin (6-11B-1; Sigma) or anti-a-
tubulin (B-5-1-2; Sigma) at 1:2000, anti-acetylated his-
tone H3 (06-599; Upstate) and anti-acetylated histone
H4 (06-866; Upstate).
the default derivative convergence criterion of
˚
0.01 kcal/(mol A) was met.
4.5.1. Generation of homology models for HDAC1 and
HDAC6. The X-ray structures of HDAC8 and HDLP
were used as templates for the generation of a homology
model for HDAC1, whereas HDAC7 was used as tem-
plate for modelling both catalytic domains (CD) of
HDAC6. HDAC-6 is unique among deacetylases in hav-
ing two HDAC domains (HDAC6 CD I and HDCA6
CD II) sharing 41% and 49 % sequence identity with
HDAC7, respectively. Sequences were obtained from
the Uniprot database.45 Multiple sequence alignment
of human HDAC1, HDAC8, HDAC7, HDAC6 CD I
and CD II, bacterial HDLP and HDAH was carried
out using CLUSTALW46 using the BLOSUM matrices
for scoring the alignments. The sequence identity be-
tween the different HDACs and the resulting sequence
alignment are shown in Fig. F and Table A in the Sup-
porting Information. Coordinates of the template struc-
tures (HDAC8: 1T64.pdb; HDAC7: 2PQO.pdb; HDLP:
1C3R.pdb) were retrieved from the Protein Database.32
Homology models for human HDAC1 and HDAC6
were generated on the basis of the best-scored alignment
(Fig. E, Supplementary material) and using the COM-
POSER module as part of the Sybyl 7.2 program.47
The loop region Gln26-Pro32 in HDAC1 was modelled
on the basis of the HDLP structure, due to higher se-
quence identity (for details, see the sequence alignment
in the Supporting Information). The visualization of
the molecular surface was carried out using the MOL-
CAD module within Sybyl 7.2.
4.4. X-ray crystallography
4.4.1. Crystallization and data collection. Highly purified
HDAH was obtained as described before.18 Crystals
were obtained at 20 ꢁC after 3–4 days using the sitting
drop vapour diffusion method in Chryschem plates
(Hampton Research, Riverside, USA) sealed with clear
tape by mixing equal volumes of FB188 HDAH with
reservoir solution (inhibitor crystals: 600 mM NaCl,
200 mM Na-Cacodylate, pH 6.5). The size of the crys-
tals was increased by seeding crystals into a fresh drop
in the presence of 0.2 mM zinc-acetate and a fivefold ex-
cess of the inhibitor ST-17. 20–25% glycerol added to
the reservoir solution served as cryoprotectant. The
crystals are primitive monoclinic. X-ray data were col-
lected from cryo-cooled crystals at beamlines X13
˚
(k = 0.8015 A) at the EMBL Outstation Hamburg. The
data were processed with HKL2000 (HKL Research,
Charlottesville, USA). The unit cell parameters are
shown in Table 3. Systematic absences along the b axis
indicated that the monoclinic space group was P21.
3
˚
The Matthews coefficient was 2.5 A /Da suggesting for
four molecules in the asymmetric unit corresponding
to a solvent content of 50.8%, respectively.
4.4.2. Structure determination and refinement. The struc-
ture of the HDAH in complex with 4n was determined
by molecular replacement and refined using REF-
4.5.2. Molecular dynamics (MD) simulations. All models
were refined by minimization procedures and MD simu-
lation approaches using the GROMACS software.48
A
MAC539 with 5% of the reflections omitted in the refine-
solvent box with dimensions 5.6 · 5.4 · 6.0 nm was gen-
erated in order to perform simulations in an aqueous
environment. The protonation state of each amino acid
residue was adjusted according to the pKa values in a
medium of pH 7.4. Protonation states of histidines were
selected manually by considering the protein environ-
ment and potential hydrogen bond partners. Water mol-
ecules were represented with the simple point charge
(SPC) model.49 Na+ and Clꢀ counter-ions were added
by replacing water molecules to ensure the overall neu-
trality of the simulated system. The models were mini-
mized using 8000 iterations of steepest descent within
the GROMOS96 force field.47 Molecular dynamics
(MD) simulations with periodic boundary conditions
were performed at 310 K. The Particle-mesh Ewald
(PME) method was used for accurate determination of
long-range electrostatic interactions.50,51 van der Waals
interactions were considered applying a cut-off of
0.9 nm. The timestep for the simulations was set to
1 fs. To keep the system at constant temperature a Ber-
endsen thermostat was applied using a coupling time of
0.1 ps.52 Constant pressure was maintained by coupling
to an external bath with a reference value of 105 Pa,
with a coupling time of 0.5 ps and an isothermal com-
pressibility of 4.5 · 10–10 Paꢀ1. For the homology mod-
els 1 ns equilibration run with decreasing constraints on
backbone atoms (1000–250 kJ molꢀ1) were employed.
40
ment for the calculation of Rfree
.
Water molecules
were added automatically with the ARP/wARP func-
tion41 in REFMAC5 and afterwards examined manu-
ally for reasonable hydrogen bonding. The final
models consist of either residues 7–374 for one molecule
(A) or 8–375 for the other three molecules in the asym-
metric unit. The refinement statistics are summarized in
Table 1. The restraints for 4n used in the refinement
were created with PRODRG.42 The quality of the mod-
el was checked using PROCHECK43 and was found to
be good. 90% of the residues are located in the most
favourable region of the Ramachandran plot. Superpo-
sitions enabling comparison of the different structures
are performed in LSQMAN44 using the brute-force op-
tion. All figures are made in PYMOL (The PyMOL
Molecular Graphics System (2002), DeLano Scientific,
San Carlos, USA).
4.5. Molecular modelling
All calculations were performed on a Pentium 1.8 GHz
Linux cluster. The molecular structures of the inhibitors
were generated using the MOE modelling package
(Chemical Computing Group, Montreal, Canada). The
structures were energy minimized using the MMFF94s
force field and the conjugate gradient method, until

2032
S. Scha¨fer et al. / Bioorg. Med. Chem. 16 (2008) 2011–2033
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Free MD simulations were carried out for 3.15 ns.
Bonds between heavy atoms and corresponding hydro-
gen atoms were constrained to their equilibrium bond
lengths using the LINCS algorithm.53 The RMSD of
the backbone atoms was stabilized after 2 ns. Thus,
the average protein structure from a time frame of 2–
3.5 ns was calculated, and further energy-minimization
steps with the steepest descent method were performed
to create the final HDAC protein structures used in
the study. Heavy atom RMSD values between the final
protein structure and the starting structure of the MD
simulation were calculated to ensure that no large-scale
structural arrangements have taken place during the
MD simulation. The stereochemical quality of the
resulting models was evaluated using PROCHECK
and ProSa and compared with the X-ray structures.43,54
4.5.3. Docking study. Docking of the co-crystallized li-
gands, as well as the tested inhibitors, was carried out
using the HDAH X-ray structure as well as the HDAC1
and HDCAC6 homology models. Program GOLD 3.0
was taken for the ligand docking. All torsion angles in
each compound were allowed to rotate freely. For each
molecule, 30 docking runs were carried out. The result-
ing solutions were clustered on the basis of the heavy
˚
atom RMSD values (1 A). We tested several docking
setups in order to reproduce the experimentally derived
complexes of HDAC inhibitors. The best agreement be-
tween the available X-ray structures and docking poses
could be derived by applying the ChemScore scoring
function within GOLD. A hydrogen bond between the
active site histidines (His143 in HDAH, His141 in
HDAC1, His216 in HDAC6 CD I, His611 in HDAC6
CD II) was used as docking constraint. This docking set-
˚
up resulted in good reproduction (RMDS below 2.5 A)
of the experimentally derived structures of SAHA,
CypY and compound 4n which were co-crystallized with
HDAH (for details, see Figs. A–D, Supporting Informa-
tion). The top-ranked poses for each ligand were se-
lected and viewed graphically within the MOE program.
Acknowledgments
S. Scha¨fer and M. Jung thank the Hans and Gertie
Fischer-Foundation for funding and Sanofi-Aventis
for an [i]lab travel award. We thank the staff at the
EMBL-outstation Hamburg, especially Matthew
Groves for guidance during data collection at DESY
beamline X13.
22. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.;
Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P.
Nature 1999, 401, 188.
23. Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J.
D.; Jennings, A. J.; Luong, C.; Arvai, A.; Buggy, J. J.; Chi,
E.; Tang, J.; Sang, B. C.; Verner, E.; Wynands, R.; Leahy,
E. M.; Dougan, D. R.; Snell, G.; Navre, M.; Knuth, M.
W.; Swanson, R. V.; McRee, D. E.; Tari, L. W. Structure
2004, 12, 1325.
Supplementary data
24. Nielsen, T. K.; Hildmann, C.; Riester, D.; Wegener, D.;
Schwienhorst, A.; Ficner, R. Acta Crystallogr. Sect. F
Struct. Biol. Cryst. Commun. 2007, 63, 270.
25. Schuetz, A.; Min, J. R.; Allali-Hassani, A.; Loppnau, P.;
Kwiatkowski, N. P.; Mazitschek, R.; Edwards, A. M.;
Arrowsmith, C. H.; Vedadi, M.; Bochkarev, A.; Plotni-
kov, A. N. PDB 2NVR, in press.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.10.092.
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