Novel estrone mimetics with high 17β-HSD1 inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2010.03.065

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone into estradiol, which is the most potent estrogen in humans. Lowering intracellular estradiol concentration by inhibition of this enzyme is a promising new option for th

Full text of DOI:10.1016/j.bmc.2010.03.065

Bioorganic & Medicinal Chemistry 18 (2010) 3494–3505
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel estrone mimetics with high 17b-HSD1 inhibitory activity q
Alexander Oster, Tobias Klein, Ruth Werth, Patricia Kruchten, Emmanuel Bey, Matthias Negri,
,
*
Sandrine Marchais-Oberwinkler, Martin Frotscher, Rolf W. Hartmann
Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), PO Box 15 11 50, D-66041 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
17b-Hydroxysteroid dehydrogenase type 1 (17b-HSD1) catalyzes the reduction of estrone into estradiol,
which is the most potent estrogen in humans. Lowering intracellular estradiol concentration by inhibi-
tion of this enzyme is a promising new option for the treatment of estrogen-dependent diseases like
breast cancer and endometriosis. Combination of ligand- and structure-based design resulted in hetero-
cyclic substituted biphenylols and their aza-analogs as new 17b-HSD1 inhibitors. The design was based
on mimicking estrone, especially focusing on the imitation of the D-ring keto group with (substituted)
heterocycles. Molecular docking provided insights into plausible protein–ligand interactions for this class
of compounds. The most promising compound 12 showed an inhibitory activity in the high nanomolar
Received 4 February 2010
Revised 23 March 2010
Accepted 25 March 2010
Available online 29 March 2010
Keywords:
17b-Hydroxysteroid dehydrogenase 1 (17b-
HSD1) inhibitors
Ligand- and structure-based design
Estrone mimetics
range and very low affinity for the estrogen receptors
a and b. Thus, compound 12 is a novel tool for
the elucidation of the pharmacological relevance of 17b-HSD1 and might be a lead for the treatment
of estrogen-dependent diseases.
Endometriosis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
A more sophisticated approach is focused on the hormone con-
centration in the target cell (intracrine approach). This strategy has
Estrogens, especially the most active one estradiol (E2), are well
known to be responsible for the development of estrogen-depen-
dent diseases like breast cancer1 and endometriosis.2 Current
endocrine therapies for breast cancer are either focused on block-
ing the estrogen action at the receptor level by selective estrogen
receptor modulators (SERMs) and pure antiestrogens3 or on
decreasing the formation of estrogens by application of GnRH ana-
logs and aromatase inhibitors.4,5 In case of the latter compounds
intensive efforts in the last two decades6,7 resulted in therapeutics
which—according to the FDA guidelines—are first-line therapeutics
for the treatment of breast cancer.7 However, all of these therapeu-
tic approaches show disadvantages by causing side effects due to
their rather radical reduction of systemic estrogen concentration.
been pursued for androgen-dependent diseases for quite some
time. Steroidal8 and later non-steroidal8–12
5a-reductase inhibitors
have been developed and two of the former are used clinically to-
day.8 In case of estrogen-dependent diseases a promising target
came up, 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1).
This enzyme catalyzes the reduction of the weakly active estrone
(E1) to yield the highly active E2 using NAD(P)H as cofactor, a reac-
tion which represents the last step in E2 biosynthesis (Chart 1).
Although the subtypes 17b-HSD7 and 12 are also able to catalyze
this conversion, their main physiological roles are supposed to be
restricted to cholesterol biosynthesis13 and regulation of lipid bio-
synthesis,14 respectively.
17b-HSD1 mRNA is often overexpressed in breast cancer tis-
sues15 and endometriotic lesions.16 Therefore, the local reduction
of estrogen action by inhibiting this enzyme appears to be a prom-
ising therapy with less side effects than existing therapies. Proof of
concept for the treatment of breast cancer was recently reported in
different mouse-models by applying steroidal compounds intraper-
itoneally or subcutaneously.17,18 However, these compounds do not
seem to be appropriate for clinical application.
Abbreviations: 17b-HSD1, 17b-hydroxysteroid dehydrogenase type 1; 17b-
HSD2, 17b-hydroxysteroid dehydrogenase type 2; E1, estrone; E2, 17b-estradiol;
ER, estrogen receptor; SERM, selective estrogen receptor modulator; GnRH,
gonadotropin-releasing hormone; NADP(H), nicotinamide adenine dinucleotide
phosphate; NAD(H), nicotinamide adenine dinucleotide; RBA, relative binding
affinity; SAR, structure–activity relationship; PDB, protein databank; HPLC, high
performance liquid chromatography; CC, column chromatography; TLC, thin layer
chromatography; FDA, food and drug administration.
The type 2 enzyme in the 17b-hydroxysteroid dehydrogenase
family is responsible for the oxidation of E2 to E1 using NAD+ as
cofactor and acts as a biological counterpart of 17b-HSD1. Regarding
thetherapeuticconcept, potentialinhibitorsof17b-HSD1shouldnot
affect 17b-HSD2. Furthermore, intrinsic estrogenic effects should be
avoided, that is, potential inhibitors should have no or only little
q
Remark: For the sake of clarity, IUPAC nomenclature is not strictly followed
except for the experimental part where the correct IUPAC names are given.
* Corresponding author. Tel.: +49 681 302 70300; fax: +49 681 302 70308.
E-mail address: rwh@mx.uni-saarland.de (R.W. Hartmann).
http://www.pharmmedchem.de.
affinity to the estrogen receptors
a and b (ERa and ERb).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.065

A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
3495
keto-function by means of different heterocycles avoiding a second
OH-group. The distance between the two hydrophilic features of
E1 is approximately 11 Å and will be considered as a further com-
ponent in the drug design concept. In order to keep the lipophilic
character of the steroidal scaffold, the two hydrophilic anchor
points will be linked by a non-hydrophilic benzene moiety.
A similar approach was applied by Allan et al.22 who also inves-
tigated E1-mimetics. In their study phenylindanone C (Chart 2)
bearing a carbonyl function was found to be the most potent inhib-
O
OH
17 -HSD1, NAD(P)H
β
17 -HSD2, NAD+
β
HO
HO
Chart 1. Interconversion of estrone (E1) and estradiol (E2).
Over the last decade, several groups have been working on the
development of 17b-HSD1 inhibitors. Most of the latter are based
on the steroidal scaffold with expansions at the 6, 15, 16 and 17
positions.19,20 Only a few non-steroidal inhibitors have been pub-
lished so far. In 2006, Messinger et al.21 described thiophenepyri-
midinones as the first non-steroidal inhibitors, which selectively
inhibit 17b-HSD1. Mimicking the E1 skeleton, Allan et al.22 investi-
gated biphenyl ethanones containing side chains known from ste-
roidal inhibitors. Recently, we reported on the development of two
highly potent and selective, non-steroidal classes of 17b-HSD1
inhibitors, (hydroxyphenyl)naphthols23–25 (A, Chart 2) and
bis(hydroxyphenyl)heterocycles26–31 (B, Chart 2). In these investi-
gations molecular modelling studies with X-ray protein structures
were performed revealing that all the mentioned inhibitors are
likely to bind in the substrate binding site, except the bis(hydroxy-
phenyl)heterocycles. The latter most probably bind between the
cofactor and substrate binding site showing interactions with
NADPH.29
itor with an IC50-value of 1.7
compounds bearing a pyrazole E-ring, which showed also remark-
able inhibitory activity (compound D, IC50 = 0.18
M),33 indicating
lM. They also published steroidal
l
that the heterocycle is well tolerated by the enzyme.
In this study, compound 1 (Chart 3) was chosen as starting
point for the development of E1-mimetics as potential 17b-HSD1
inhibitors, with furane replacing the steroidal D-ring. Besides fur-
ane, 2-pyridone, morpholine and thiophene seemed to be appro-
priate moieties to investigate the suitability of further
heteroatoms.
2.2. Structure-based
Among the numerous published crystal structures34 there is no
structure of the enzyme cocrystallized with E1 but with the closely
related, well known 17b-HSD1 inhibitor equilin, an equine estro-
gen with a C7–C8 double bond and a carbonyl function in position
17. Therefore, the ternary complex of human 17b-HSD1 with equi-
lin and NADP+ (PDB-ID: 1EQU)35 appeared to be suitable for study-
ing the three-dimensional architecture of the enzyme. A substrate
binding site, a cofactor binding pocket and a highly flexible sub-
In both compound classes recently described by us (A and B),
the most active inhibitors exhibit two OH-groups in a distance of
about 11 Å.23–29 Regarding pharmacokinetics, an exchange of one
hydroxyphenyl moiety seems to be desirable because of the sus-
ceptibility of phenols to phase II metabolism.32
strate-entry loop (a
G0bF) structure can be defined. The steroid
Therefore herein we report on the design of new steroidomi-
metics bearing only one OH-group. The compounds were designed
using a combination of ligand- and structure-based approach. In
the following, synthesis and biological evaluation of new non-ste-
roidal 17b-HSD1 inhibitors of the heterocyclic substituted biphe-
nylol type and their aza-analogs are described. Molecular
modelling studies were performed in order to elucidate protein–li-
gand interactions in the active site.
binding site is almost exclusively hydrophobic except for two
hydrophilic ends: the 17-keto oxygen of equilin accepts protons
from Tyr155 and Ser142 at the catalytic end. At the other end,
the 3-hydroxy group of equilin establishes bifurcated hydrogen
bonds to His221 and Glu282. Closer analysis of the substrate bind-
ing pocket reveals three additional polar amino acids (Asn152,
Tyr218 and Ser222) in the hydrophobic area, which are not in-
volved in steroid binding. Introduction of heteroatoms in the ben-
zene core of a potential inhibitor might be appropriate to explore
supplementary interactions.
2. Design
2.1. Ligand-based
3. Chemistry
In order to reduce the risk of undesired side effects, novel com-
pounds should not have affinity to steroid receptors. Accordingly,
we focused on the design of non-steroidal inhibitors. In this field
we previously designed highly active inhibitors of 17b-HSD1 bear-
ing two OH-groups mimicking the two hydrophilic features of the
substrate.23,26 Interestingly, some inhibitors containing only one
OH-group showed still a notable activity.27 A prerequisite for their
activity is a meta OH-phenyl moiety. This substitution pattern will
play an important role in the design of the compounds presented in
this study.
The synthesis of benzene derivatives 1–15 is depicted in
Scheme 1. Compounds 1–3, 4i, 7–13 and 14i–15i were synthesized
via Suzuki reaction starting from the commercially available boro-
nic acids and the brominated benzene derivatives. Microwave as-
sisted Suzuki cross-coupling reactions were carried out as one-
pot syntheses using method A27 (Cs2CO3, DME/EtOH/water
(1:1:1), Pd(PPh3)4, MW (150 W, 150 °C, 15 bar, 15 min)). The ether
functions of the methylated hydroxyphenyl derivatives 5, 14 and
15 were cleaved according to method D27 (BBr3, CH2Cl2, ꢀ78 °C
to rt, 18 h). The pyridin-2(1H)-one derivative 4 was prepared by
demethylation of intermediate 4i with borontrifluoride dimethyl
sulfide complex by stirring the reaction mixture at rt for 20 h in
anhydrous dichloromethane.
We herein investigate E1, the natural substrate of 17b-HSD1 as
template for ligand-based design, with a meta OH-phenyl group
mimicking the steroidal A-ring and the replacement of the D-ring
R2
O
N
NH
Het
OH
OH
R2
R1
HO
R1
HO
HO
HO
A
B
C
D
Chart 2. Recently published compound classes from our group (hydroxyphenyl)naphthols (A), bis(hydroxyphenyl)heterocycles (B) and two known inhibitors (C, D).

3496
A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
inactive. The percent inhibition values of compounds 1–20 are
shown in Table 1. The IC50-values of selected compounds are de-
picted in Table 2.
R
R
HO
HO
Compound 1, bearing a furane moiety only showed marginal
inhibitory activity of 17% at a concentration of 1 lM. To get
1-10
11-15
more insight into the role of the heterocycles, we synthesized
compounds 2–5, 7 and 8 differing in the heteroatoms, the elec-
tronic and the geometric properties of the heterocycle. Com-
pounds 2–3, in which furane is replaced with non-aromatic
heterocycles, showed no activity, indicating that a lack of aroma-
ticity is counterproductive. The inactivity of pyridone 4 supports
this hypothesis since its aromatic character is less pronounced
compared to the methoxypyridine 5.36 Pyridine 5 and thiophenes
7, 8 showing moderate activity (58%, 41% and 36% inhibition,
respectively) seemed to be appropriate mimics of the steroidal
cmpd
1
R
cmpd
9
R
O
O
S
S
S
Cl
O
N
2
3
4
5
6
7
8
10
11
12
13
14
15
O
O
N
O
NH
S
S
Cl
O
N
O
keto-function. Regarding compound 5 (IC50 = 0.69 lM, Table 2),
S
S
the methoxy-group and the pyridine–nitrogen offer hydrogen
bond acceptor properties. This means that both the pyridine–
nitrogen and the methoxy–oxygen are able to form hydrogen
bonds with the catalytic amino acids. The slightly reduced activ-
ity of 7 and 8 might be explained by the lack of these hydrogen
bond interactions. Introduction of an electron withdrawing (9) as
well as an electron donating (10) substituent did not improve
potency significantly.
As the 1,4-disubstituted benzene derivatives 9 and 10 are larger
than 11 Å, the 1,3-disubstituted 11 and 12 were synthesized in or-
der to readjust the E1-derived requirements. While the activity of
the acetylthiophene derivative 11 decreased, the chlorine substi-
tuted thiophene 12 is the most potent inhibitor of 17b-HSD1 iden-
H
S
S
X
W
Y
Z
S
HO
R
16-20
W
CH CH
Cl CH CH
cmpd
16
17
18
19
R
H
X
Y
Z
tified in this study with an IC50 of 0.56 lM. Compounds 13 and 14
N
N
CH
CH
N
bearing H and CH3 instead of Cl and compound 15 with benzothio-
phene substituent, were synthesized as tools to evaluate the im-
pact of the chlorine atom on the activity and get insight into
possible inhibitor–protein interactions. They all turned out to be
less active than 12, highlighting the positive effect of chlorine for
enzyme affinity.
Cl CH CH CH
Cl
Cl CH
N
CH CH CH
CH CH
20
N
Chart 3. Title compounds.
As discussed above, insertion of a heteroatom, capable of acting
as hydrogen bond acceptor, in the central benzene core might lead
to additional interactions with Tyr218 and Ser222. For the sake of
clarifying this hypothesis, the benzene core was replaced by pyri-
dine, leading to isomeric compounds 16–20. None of the com-
pounds in this series enhances 17b-HSD1 inhibition, indicating
that the pyridine nitrogen is not able to target these hydrophilic
amino acids. In case of compounds 17, 18 and 20, the insertion
of a nitrogen even decreases inhibitory activity compared to 12.
The synthetic routes for the pyridine derivatives 16–20 are
shown in Scheme 2. Starting from the commercially available
dibrominated pyridines and boronic acid derivatives, compounds
17i and 19ii were obtained via Suzuki reaction following the con-
ditions of method B (K2CO3, toluene/EtOH/water (1:1:1), Pd(PPh3)4,
100 °C, 18 h) and method
C (NaHCO3, toluene/water (1:1),
Pd(PPh3)4, 100 °C, 18 h), respectively. Method A was used for the
coupling of 3-hydroxybenzene boronic acid with compound 17i
to yield compound 17. Compound 16 devoid of the chlorine substi-
tuent at the thiophene was isolated as a side product.
Nevertheless, the isomer 19 with an IC50-value of 0.85
nearly the same activity as compound 12.
lM showed
4.2. Selectivity: inhibition of human 17b-HSD2 and affinities for
ER and ERb
Cross-coupling of 5-chloro-2-thienyl boronic acid with bromi-
a
nated pyridine derivatives 18ii–20ii according to method
A
(Cs2CO3, DME/EtOH/water (1:1:1), Pd(PPh3)4, MW (150 W,
150 °C, 15 bar, 15 min)) and method B (K2CO3, toluene/EtOH/water
(1:1:1), Pd(PPh3)4, 100 °C, 18 h) led to compounds 19i (method A),
18i and 20i (method B). Compounds 18–20 were prepared by
cleaving the methoxy function with boron tribromide (method D:
BBr3, CH2Cl2, ꢀ78 °C to rt, 18 h).
Since 17b-HSD2 catalyzes the oxidative transformation of E2
into E1, inhibitory activity toward this enzyme must be avoided.
17b-HSD2 inhibition was determined using an assay similar to
the 17b-HSD1 test. Placental microsomes were incubated with tri-
tiated E2 in the presence of NAD+ and inhibitor. Quantification of
labelled product formed was performed by HPLC and subsequent
radio detection. Compounds showing less than 10% inhibition at
4. Biological results
1 lM were considered to be inactive. Percentage of inhibition
and IC50-values are shown in Tables 1 and 2. All compounds, which
did not inhibit 17b-HSD1 were also inactive on the type 2 enzyme.
Comparing the isomeric pyridines 17 and 19, it is striking that
the position of the nitrogen has a major impact on the selectivity.
Compound 17, bearing the pyridine nitrogen next to the thiophene,
4.1. Activity: inhibition of human 17b-HSD1
Placental enzyme isolated following a described procedure,31
was incubated with tritiated E1, cofactor and inhibitor. The separa-
tion of substrate and product was performed by HPLC. Compounds
showed an IC50 of 0.47
lM for 17b-HSD2, which demonstrates a 5
showing less than 10% inhibition at 1 lM were considered to be
fold higher inhibition than for the type 1 enzyme. Having the posi-

A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
3497
NH
OH
B
O
OH
R2
HO
b
4
Br
R1
X
R3
a
c
X
R3
n
R1
HO
B
1, 4i, 7
n
N
OH
4´
O
a
R2
3´
HO
5
a
4´ S
R1
R4
HO
S
R4
B
1i, 2, 3, 4ii,
c
HO
6, 11i, 14ii
S
3´
R1
OH
HO
S
8 to 13, 14i
14
a
B
HO
S
S
c
O
OH
15i
15
cmpd
R1
R2
R3
X
n
1i
1
2
OH
OH
OH
OH
OCH3
OCH3
H
4´-Br
H
O
0
4´-morpholino
4´-(2-morpholinoethoxy)
4´-Br
3
4ii
4i
6
OCH3
H
N
S
1
0
OH
7
OH
cmpd
8
9
10
11i
11
12
13
R1
R2
thienyl
R4
H
CO-CH3
Cl
OH
OH
OH
OH
OH
OH
OH
4´
4´
4´
3´-Br
3´
3´
3´
CO-CH3
Cl
H
14ii OCH3 3´-Br
14i OCH3
3´
CH3
Scheme 1. Synthesis of compounds 1–15. Reagents and conditions: (a) method A: Cs2CO3, Pd(PPh3)4, DME/EtOH/water (1:1:1), microwave conditions (150 W, 15 bar, 150 °C,
15 min); (b) BF3ꢁSMe2, CH2Cl2, rt, 20 h; (c) method D: BBr3, CH2Cl2, ꢀ78 °C, 20 h.
tion of the nitrogen next to the hydroxyphenyl ring (19), the selec-
tivity changed completely (4-fold higher affinity toward 17b-
HSD1). According to the position of the nitrogen, a 17b-HSD1
inhibitor can be transformed into a 17b-HSD2 inhibitor. Among
the compounds revealing 17b-HSD1 activity, however, the chloro-
thiophene 10 showed the best selectivity (>10-fold) toward 17b-
HSD2.
5. Molecular modelling
In order to gain deeper insight into plausible molecular interac-
tions between the synthesized steroidomimetics and 17b-HSD1,
docking experiments were performed using the monomer of the
X-ray structure 1EQU (PDB-ID).
Selected compounds were docked into 17b-HSD1 by means of
the docking software Autodock4.237 and GOLDv4.0.1.38 In Fig-
ure 1A, a plausible binding pose for 5 is shown as an example.
The compound is found in the steroidal binding mode. The meta
OH-group establishes hydrogen bonds with His221 and Glu282
(dO–N = 3.2 Å and dO–O = 2.7 Å) in a bifurcated fashion, mimicking
the OH-group of the steroidal A-ring. The methoxy substituted pyr-
idine ring points toward the catalytic region of the protein. The
pyridine nitrogen and the methoxy oxygen might be implicated
The therapeutic concept of 17b-HSD1 inhibition includes that
potential inhibitors should have no or low affinity toward ER
and ERb in order to avoid systemic effects. Binding affinities for
ER and ERb were measured for selected compounds and are
a
a
shown in Table 2. Using recombinant human protein, a competi-
tion assay applying tritium labelled E2 (RBA = 100%) was per-
formed. All evaluated compounds present very low binding
affinity to both estrogen receptors.

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A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
OH
B
OH
N
N
HO
B
S
Cl
Br
Br
Br
Br
HO
O
a
b
OH
B
OH
HO
X
Z
X
S
B
Cl
W
Y
W
HO
N
OH
S
Z
Br
R2
b, c
c
S
Br
Cl
O
O
R1
17i
18ii to 20ii
16, 17, 18i to 20i
d
X
W
S
Z
Cl
OH
18 to 20
middle ring = pyridine
16, 17: Y = N
18ii, 18i, 18: Z = N
19ii, 19i, 19: W = N
20ii, 20i, 20: X = N
cmpd
16
17
18i
19i
20i
R1
H
H
CH3
CH3
CH3
R2
H
Cl
Cl
Cl
Cl
method
c
c
b
c
b
Scheme 2. Synthesis of compounds 16–20. Reagents and conditions: (a) method C: NaHCO3, Pd(PPh3)4, toluene/water (1:1), 100 °C, 18 h; (b) method B: K2CO3, Pd(PPh3)4,
toluene/EtOH/water (1:1:1), 100 °C, 18 h; (c) method A: Cs2CO3, Pd(PPh3)4, DME/EtOH/water (1:1:1), microwave conditions (150 W, 15 bar, 150 °C, 15 min); (d) method D:
BBr3, CH2Cl2, ꢀ78 °C, 20 h.
Table 1
clear if the interactions established by the methoxypyridine are
relevant for the stabilization of 5 as the chlorothiophene moiety
of 10 is located in the same area and is not able to establish the
same H-bond interaction patterns.
Inhibition of human 17b-HSD1 and 17b-HSD2 by compounds 1–20
Cmpd
% Inhibitiona
17b-HSD1b
17b-HSD2c
Taking in account that compound 12 was designed to better fit
the E1-distance requirements, the same interactions as with the
linear molecules were expected for this compound.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
17
n.i.
n.i.
n.i.
58
n.i.
41
36
38
48
22
71
48
32
49
33
39
40
61
40
n.i.
n.i.
n.i.
n.i.
35
n.i.
36
22
10
n.i.
23
31
40
36
39
62
63
21
25
46
However, the results of the docking experiments indicate that
the chlorothiophene moiety of 12 is not directed toward the cata-
lytic centre (Fig. 1B). While the hydroxyphenyl group is still imitat-
ing the steroidal A-ring (dO–N = 2.7 Å and dO–O = 3.5 Å), the non-
linear scaffold enables the heterocycle to point to a rather lipo-
philic subpocket consisting of Leu95, Leu96, Asn152, Tyr155 and
Phe192. Compounds 13–15 were found in the same binding mode
as 12 (data not shown).
6. Discussion and conclusions
The biological results described herein provide evidence that
mimicking the chemical features of E1 is an appropriate approach
for the development of new 17b-HSD1 inhibitors. While in previ-
ous studies, the inhibitors bore two OH-groups, here only one
OH-group was retained, namely the meta OH-phenyl group imitat-
ing the A-ring. The study was mainly focused on the investigation
of (substituted) heterocycles as suitable D-ring mimics. It is shown
that compounds 2 and 3, with an aliphatic heterocycle replacing
the D-ring, showed no inhibitory activity. This indicates that flat-
n.i. = no inhibition (inhibition <10%).
a
Mean value of three determinations, standard deviation less than 10%, com-
pound concentration: 1 M.
Human placenta, cytosolic fraction, substrate [3H]-E1, 500 nM, cofactor NADH,
M.
Human placenta, microsomal fraction, substrate [3H]-E2, 500 nM, cofactor
NAD+, 1500
M.
l
b
500
l
c
l
ness and a delocalised
activity.
p-system are prerequisites for inhibitory
The interaction pattern of Ser142, Tyr155 and the 17b-OH group
of E2 has been described to be a triangle-shaped hydrogen bond
network.39 Until now, it was not clarified whether the hydrogen
bond donor function is a crucial component of this network. In this
in hydrogen bonds with Ser142 (dO–N = 4.0 Å) and Tyr155 (dO–
O = 3.8 Å), respectively. The docking studies also showed that the
equipotent 10 binds in the same area as compound 5 (Fig. 1), com-
parable to equilin cocrystallized with 17b-HSD1 (1EQU). It is not

A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
3499
Table 2
Inhibition of 17b-HSD1, 17b-HSD2 and binding affinities for the oestrogen receptors
a
and b by selected compounds
RBAd (%)
a
Cmpd
Structure
IC50
(l
M)
17b-HSD1b
17b-HSD2c
ER
a
ERb
N
O
5
0.69
2.97
0.001<RBA<0.01
0.01<RBA<0.1
<0.001
HO
HO
Cl
S
10
1.02
0.56
>10
0.001<RBA<0.01
<0.001
12
15
2.37
<0.01
<0.01
S
Cl
HO
S
1.37
1.94
<0.001
HO
HO
HO
N
17
18
19
2.38
1.39
0.85
0.47
7.11
3.64
0.001<RBA<0.01
0.001<RBA<0.01
<0.001
<0.001
<0.001
<0.001
S
S
Cl
Cl
N
N
S
Cl
HO
a
b
c
Mean value of three determinations, standard deviation less than 15%.
Human placenta, cytosolic fraction, substrate [3H]-E1, 500 nM, cofactor NADH, 500
Human placenta, microsomal fraction, substrate [3H]-E2, 500 nM, cofactor NAD+, 1500
l
M.
l
M.
d
RBA: relative binding affinity, E2: 100%, mean value of three determinations, standard deviation less than 10%.
study we were able to show that heterocycles like methoxypyri-
dine and chlorothiophene are able to unfold high inhibitory
activity.
and these residues should be in an appropriate range for hydrogen
bonding. However, none of these aza-analogs enhanced 17b-HSD1
inhibitory activity compared to 12. The nitrogens are obviously
neither able to interact with these amino acids nor can any addi-
tional interaction of the nitrogens be observed. The biological data
might be explained by differences in molecular electrostatic poten-
tial (MEP) distributions which were found to play an important
role in the inhibition of 17b-HSD1 by other compounds, too.27,29
For these it was shown that insertion of nitrogen and/or exchange
of its position have a high impact on the MEP distributions.27
The selectivity of compounds 17–20 is influenced by the posi-
tion of the nitrogen. As there is neither a crystal structure nor an
appropriate homology model of 17b-HSD2 available, protein–li-
gand interactions cannot be interpreted. Comparing the aza-com-
pounds it becomes apparent that only compound 17 showed a
significantly higher inhibitory activity toward 17b-HSD2. In this
compound, the nitrogen close to the chlorothiophene ring is
responsible for a more negative electrostatic potential in this part
of the structure (see MEP calculations in Supplementary data). This
distribution might be beneficial for 17b-HSD2 inhibition whereas a
negative MEP in the area of the meta hydroxyphenyl ring led to
higher inhibitory activity toward 17b-HSD1.
In summary, a new series of heterocyclic substituted biphenyl-
ols and their aza-analogs was designed as 17b-HSD1 inhibitors and
synthesized by combining a ligand- and structure-based approach.
Among these, a promising compound was discovered (12), which
showed high activity, selectivity over 17b-HSD2 and very low
affinity toward the ERs. The binding mode of the angulate 1,3-ben-
zene derivatives differed from that observed for the linear com-
pounds. The latter superimpose well to equilin which indicates
that an OH-group pointing into the catalytic centre is not necessary
for high inhibitory activity and can be taken over by other substit-
The results of the docking study showed that the 1,3-benzene
derivatives most likely do not exclusively cover the substrate bind-
ing area. Parts of them were found to reach into the rather lipo-
philic subpocket recently described by Mazumdar et al.40 As the
chlorothiophene moiety of compound 12 was found to bind into
this subpocket, compounds 13–15 were designed to validate this
finding. Since the benzothiophene moiety of 15 is too bulky to fit
into the area of the catalytic centre as observed for compounds 5
and 10, the only slightly reduced inhibitory activity of 15 com-
pared to 12 supports our hypothesis of these ligands binding to
the subpocket. The high to moderate inhibitory activity of com-
pounds 12 and 15 can be explained by hydrophobic and van der
Waals interactions within this subpocket. In order to investigate
the relevance of the chloro substituent for the activity, compounds
13 and 14 were synthesized replacing Cl by H (13) and by CH3 (14).
Compound 13 is a weaker inhibitor of 17b-HSD1 compared to the
parent compound (12), which might be due to its decreased lipo-
philicity. However, compound 14 shows less inhibition compared
to 12, although chloro and methyl exhibit very similar lipophilic
properties. This indicates that not only lipophilicity is responsible
for high affinity to this subpocket. In this context, the different
electronic characteristics of the substituents as well as the possible
formation of Cl–p
interactions41 might be involved in the increased
potency of 12.
With the aim of increasing activity and selectivity the middle
benzene ring was exchanged by a pyridine in order to target the
hydrophilic amino acids Tyr218 and Ser222 in the predominantly
hydrophobic binding site. According to the expected binding mode,
the distances between the pyridine nitrogens of compounds 16–20

3500
A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
d = 7.24 ppm (1H NMR) and d = 77 ppm
(
13C NMR), CD3OD:
d = 3.35 ppm (1H NMR) and d = 49.3 ppm (13C NMR), CD3COCD3:
d = 2.05 ppm (1H NMR) and d = 29.9 ppm (13C NMR), CD3SOCD3
d = 2.50 ppm (1H NMR) and d = 39.5 ppm (13C NMR)). Signals are
described as s, d, t, dd, ddd, m, dt, q for singlet, doublet, triplet,
doublet of doublets, doublet of doublets of doublets, multiplet,
doublet of triplets and quadruplet, respectively. All coupling con-
stants (J) are given in hertz (Hz).
Mass spectra (ESI and APCI) were recorded on a TSQ Quantum
(Thermo Finnigan) instrument.
Tested compounds are >95% chemical purity as measured by
HPLC. The methods for HPLC analysis and a table of data for all
tested compounds are provided in Supplementary data.
The following compounds were prepared according to
previously described procedures: 40-bromobiphenyl-3-ol (1i),42
40-bromo-3-methoxybiphenyl (4ii),43 biphenyl-3-ol (6),44 30-bro-
mobiphenyl-3-ol (11i),45 3-bromo-30-methoxybiphenyl (14ii),46
2-bromo-6-(3-methoxyphenyl)pyridine (18ii),47 3-bromo-5-(3-
methoxyphenyl)pyridine (20ii).48
7.1.1. General procedure for Suzuki coupling
7.1.1.1. Method A. A mixture of arylbromide (1 equiv), boronic
acid derivative (1.2 equiv), caesium carbonate (4 equiv) and tetra-
kis(triphenylphosphine) palladium (0.03 equiv) was suspended in
an oxygen-free DME/EtOH/water (1:1:1) mixture. The reaction
mixture was exposed to microwave irradiation (15 min, 150 W,
150 °C, 15 bar). After cooling to rt, water was added and the aque-
ous layer was extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate, filtered
and concentrated to dryness. The product was purified by CC, pre-
parative TLC or preparative HPLC, respectively.
Figure 1. Docking complex between 17b-HSD1 (PDB-ID: 1EQU) and compounds 5
(cyan), 10 (brown) overlay depicted in A and 12 (violet) depicted in B. Cofactor
NADPH (orange), interacting residues (green) and cartoon rendered tertiary
structure (grey) of the active site are shown. Hydrogen bonds are drawn in black
dashed lines. The surface of the subpocket is illustrated (light brown) in Figure 1B.
Figures generated with MOE (Chemical Computing Group Inc., Montreal, Canada).
7.1.1.2. Method B. A mixture of arylbromide (1 equiv), boronic
acid derivative (1.2 equiv), potassium carbonate (2 equiv) and tet-
rakis(triphenylphosphine) palladium (0.03 equiv) in an oxygen-
free toluene/EtOH/water (1:1:1) mixture was stirred at 100 °C for
18 h under nitrogen atmosphere. The reaction mixture was cooled
to rt. The aqueous layer was extracted with ethyl acetate. The com-
bined organic layers were washed with brine, dried over sodium
sulfate, filtered and concentrated to dryness. The product was puri-
fied by CC.
uents like methoxypyridine or chlorothiophene. This observation
might be important for the design of other classes of 17b-HSD1
inhibitors. Furthermore it seems feasible that by combination of
the two interaction patterns observed in this study (angulate-
shaped structures interacting with the subpocket and linear struc-
tures with the catalytic centre) a further increase of activity and
selectivity could be achieved leading to the development of clinical
candidates.
7.1.1.3. Method C. A mixture of aryldibromide (1 equiv), methoxy-
benzene boronic acid (1 equiv), sodium hydrogencarbonate
(2 equiv) and tetrakis(triphenylphosphine) palladium (0.03 equiv)
in an oxygen-free toluene/water (1:1) mixture was stirred at
100 °C for 18 h under nitrogen atmosphere. The reaction mixture
was cooled to rt. The aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
magnesium sulfate, filtered and concentrated in vacuo. The product
was purified by CC.
7. Experimental section
7.1. Chemical methods
Chemical names follow IUPAC nomenclature. Starting materials
were purchased from Aldrich, Acros, Lancaster, Roth, Combi Blocks,
Merck or Fluka and were used without purification.
Column chromatography (CC) was performed on silica gel (70–
200 lm) coated with silica, preparative thin layer chromatography
(TLC) on 1 mm SIL G-100 UV254 glass plates (Macherey-Nagel) and
reaction progress was monitored by TLC on Alugram SIL G UV254
(Macherey-Nagel).
7.1.2. General procedure for ether cleavage
7.1.2.1. Method D. To a solution of methoxybenzene derivative
(1 equiv) in anhydrous dichloromethane at ꢀ78 °C (dry ice/acetone
bath), boron tribromide in dichloromethane (1 M, 3 equiv) was
added dropwise. The reaction mixture was stirred for 20 h to rt un-
der nitrogen atmosphere. Water was added to quench the reaction,
and the aqueous layer was extracted with ethyl acetate. The com-
bined organic layers were washed with brine, dried over sodium
sulfate, evaporated to dryness under reduced pressure and purified
by CC, preparative TLC or preparative HPLC, respectively.
IR spectra were recorded on a Bruker Vector 33 spectrometer
(neat sample).
1H NMR and 13C NMR spectra were measured on a Bruker
AM500 spectrometer (500 MHz) at 300 K. Chemical shifts are re-
ported in d (parts per million: ppm), by reference to the hydroge-
nated residues of deuteriated solvent as internal standard (CDCl3:
7.1.3. General procedure for purification using preparative HPLC
All declared compounds were purified via an Agilent Technolo-
gies Series 1200-preparative HPLC using a linear gradient run (sol-

A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
3501
vents: acetonitrile, water) starting from 20% acetonitrile up to
100% in 36 min.
oride dimethyl sulfide complex (39 mmol, 75 equiv) was added
dropwise at rt. The reaction mixture was stirred for 20 h at rt.
Water was added to quench the reaction and the aqueous layer
was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over sodium sulfate, evaporated
to dryness under reduced pressure and purified by preparative
TLC (hexane/ethyl acetate 1:1); yield: 12% (17 mg); MS (ESI): 264
(M+H)+; 1H NMR (CD3SOCD3): 11.82 (s, 1H), 9.51 (s, 1H), 7.87
(dd, J = 2.9 Hz and J = 9.5 Hz, 1H), 7.75 (d, J = 2.9 Hz, 1H), 7.62 (s,
4H), 7.25 (t, J = 7.9 Hz, 1H), 7.10–7.08 (m, 1H), 7.04 (t, J = 2.1 Hz,
1H), 7.76 (ddd, J = 0.9 Hz and J = 2.5 Hz and J = 8.2 Hz, 1H), 6.45
(d, J = 9.5 Hz, 1H); 13C NMR (CD3SOCD3): 161.70, 157.80, 140.90,
139.80, 138.40, 135.10, 132.60, 129.90, 127.20, 126.90, 125.50,
122.80, 120.00, 117.20, 114.40, 113.20; IR: 3134, 2921, 1657,
7.1.4. Detailed synthesis procedure for all compounds
7.1.4.1. 40-(3-Furyl)biphenyl-3-ol (1). The title compound was
prepared by reaction of 40-bromobiphenyl-3-ol (1i) (150 mg,
0.60 mmol), furane-3-boronic acid (81 mg, 0.72 mmol), caesium
carbonate (785 mg, 2.40 mmol) and tetrakis(triphenylphosphine)
palladium (21 mg, 18 lmol) according to method A. The product
was purified by preparative TLC (hexane/ethyl acetate 7:3); yield:
3% (4 mg); MS (ESI): 237 (M+H)+; 1H NMR (CD3COCD3): 8.44 (s,
1H), 8.06 (dd, J = 0.9 Hz and J = 1.6 Hz, 1H), 7.69–7.67 (m, 2H),
7.65–7.63 (m, 3H), 7.28 (t, J = 8.1 Hz, 1H), 7.15–7.13 (m, 2H), 6.92
(dd, J = 0.9 Hz and J = 1.9 Hz, 1H), 6.84 (ddd, J = 0.9 Hz and
J = 2.5 Hz and J = 8.2 Hz, 1H); 13C NMR (CD3COCD3): 162.50,
146.10, 144.00, 141.10, 133.50, 131.80, 129.10, 128.10, 128.00,
119.90, 118.30, 118.10, 116.30, 115.50, 110.50; IR: 3452, 3413,
1584, 1205, 825, 779 cmꢀ1
.
7.1.4.6. 40-(6-Methoxypyridin-3-yl)biphenyl-3-ol (5). The title
compound was prepared by reaction of 2-methoxy-5-(30-methoxy-
biphenyl-4-yl)pyridine (4i) (30 mg, 0.10 mmol) and boron tribro-
mide (0.30 mmol) according to method D. The product was
purified by preparative TLC (hexane/ethyl acetate 1:1); yield: 55%
(16 mg); MS (ESI): 292 (M+H)+; 1H NMR (CD3COCD3): 8.49–8.47
(m, 2H), 7.99 (dd, J = 2.9 Hz and J = 8.9 Hz, 1H), 7.71 (s, 4H), 7.29
(t, J = 8.1 Hz, 1H), 7.17–7.16 (m, 2H), 6.87–6.85 (m, 2H), 3.94 (s,
3H); 13C NMR (CD3COCD3): 165.60, 159.90, 146.70, 143.80,
141.90, 139.20, 138.60, 131.90, 131.30, 129.30, 128.70, 119.90,
116.40, 115.50, 112.60, 54.70; IR: 3234, 3023, 2955, 1601, 1487,
3281, 1597, 1164, 835, 780 cmꢀ1
.
7.1.4.2. 40-Morpholin-4-ylbiphenyl-3-ol (2). The title compound
was prepared by reaction of 4-(4-bromophenyl)morpholine
(150 mg, 0.62 mmol), 3-hydroxybenzene boronic acid (103 mg,
0.74 mmol), caesium carbonate (807 mg, 2.48 mmol) and tetra-
kis(triphenylphosphine) palladium (22 mg, 19 lmol) according to
method A. The product was crystallized in ethyl acetate; yield:
27% (42 mg); MS (ESI): 256 (M+H)+; 1H NMR (CD3SOCD3): 7.01
(d, J = 8.8 Hz, 2H), 6.73 (t, J = 7.9 Hz, 1H), 6.55–6.53 (m, 3H), 6.50
(t, J = 2.2 Hz, 1H), 6.22 (ddd, J = 0.9 Hz and J = 2.2 Hz and
J = 7.9 Hz, 1H), 3.29 (t, J = 4.7 Hz, 4H), 2.69 (t, J = 4.7 Hz, 4H); 13C
NMR (CD3SOCD3): 149.10, 132.70, 122.20, 121.10, 118.40, 108.00,
106.60, 104.80, 104.00, 57.40, 39.90; IR: 3245, 2957, 1585, 1207,
1298, 822 cmꢀ1
.
7.1.4.7. 40-(3-Thienyl)biphenyl-3-ol (7). The title compound was
prepared by reaction of 40-bromobiphenyl-3-ol (1i) (150 mg,
0.60 mmol), thiophene-3-boronic acid (93 mg, 0.72 mmol), cae-
sium carbonate (785 mg, 2.40 mmol) and tetrakis(triphenylphos-
1110, 828, 785 cmꢀ1
.
7.1.4.3. 40-(2-Morpholin-4-ylethoxy)biphenyl-3-ol (3). The title
compound was prepared by reaction of 4-[2-(4-bromophen-
oxy)ethyl]morpholine (150 mg, 0.52 mmol), 3-hydroxybenzene
boronic acid (87 mg, 0.63 mmol), caesium carbonate (678 mg,
2.08 mmol) and tetrakis(triphenylphosphine) palladium (18 mg,
phine) palladium (21 mg, 18 lmol) according to method A. The
product was purified by preparative TLC (hexane/ethyl acetate
7:3); yield: 19% (28 mg); MS (ESI): 253 (M+H)+; 1H NMR
(CD3COCD3): 8.44 (s, 1H), 7.79–7.77 (m, 3H), 7.67 (d, J = 8.5 Hz,
2H), 7.57 (d, J = 2.2 Hz, 2H), 7.28 (t, J = 8.2 Hz, 1H), 7.17–7.15 (m,
2H), 6.85 (ddd, J = 1.3 Hz and J = 2.2 Hz and J = 7.9 Hz, 1H); 13C
NMR (CD3COCD3): 192.70, 159.80, 143.90, 143.60, 141.60, 136.90,
131.90, 129.20, 128.60, 128.50, 128.00, 122.40, 119.90, 116.30,
16 lmol) according to method A. The product was purified by CC
(dichloromethane/methanol 96:4) followed by preparative HPLC;
yield: 20% (32 mg); MS (ESI): 300 (M+H)+; 1H NMR (CD3COCD3):
8.19 (s, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.23 (t, J = 7.6 Hz, 1H), 7.07–
7.05 (m, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.78 (ddd, J = 1.3 Hz and
J = 2.5 Hz and J = 8.2 Hz, 1H), 4.18 (t, J = 5.7 Hz, 2H), 3.64 (t,
J = 4.7 Hz, 4H), 2.82 (t, J = 5.7 Hz, 2H), 2.60 (t, J = 4.7 Hz, 4H); 13C
NMR (CD3COCD3): 164.50, 160.40, 159.70, 144.00, 135.40, 131.70,
129.70, 119.50, 116.70, 115.60, 115.20, 68.20, 67.60, 59.20,
115.50; IR: 3479, 3394, 3096, 1596, 1200, 835, 775 cmꢀ1
.
7.1.4.8. 40-(2-Thienyl)biphenyl-3-ol (8). The title compound was
prepared by reaction of 40-bromobiphenyl-3-ol (1i) (150 mg,
0.60 mmol), thiophene-2-boronic acid (93 mg, 0.72 mmol), cae-
sium carbonate (785 mg, 2.40 mmol) and tetrakis(triphenylphos-
55.80; IR: 3405, 2868, 1607, 1477, 1116, 835, 783 cmꢀ1
.
phine) palladium (21 mg, 18 lmol) according to method A. The
product was purified by preparative TLC (hexane/ethyl acetate
7:3); yield: 22% (34 mg); MS (APCI): 251 (MꢀH)+; 1H NMR
(CD3COCD3): 8.47 (s, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.66 (d,
J = 8.6 Hz, 2H), 7.49 (d, J = 3.8 Hz, 1H), 7.45 (d, J = 3.8 Hz, 1H),
7.29 (t, J = 8.1 Hz, 1H), 7.16–7.12 (m, 3H), 6.87–6.85 (m, 1H); 13C
NMR (CD3COCD3): 159.90, 145.60, 143.60, 142.00, 135.40, 131.90,
130.20, 129.30, 127.90, 127.00, 125.30, 119.80, 116.40, 115.40;
7.1.4.4. 2-Methoxy-5-(30-methoxybiphenyl-4-yl)pyridine (4i). The ti-
tle compound was prepared by reaction of 40-bromo-3-methoxybiphenyl
(4ii) (300 mg, 1.14 mmol), 2-methoxy-5-pyridineboronic acid (209 mg,
1.37 mmol), caesium carbonate (1486 mg, 4.56 mmol) and tetrakis(tri-
phenylphosphine) palladium (40 mg, 34 lmol) according to method A.
The product was purified by CC (hexane/ethyl acetate 9:1); yield: 84%
(280 mg); 1H NMR (CDCl3): 8.45 (d, J = 2.2 Hz, 1H), 7.83 (dd, J = 2.5 Hz
and J = 8.5 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.38
(t, J = 7.9 Hz, 1H), 7.23–7.22 (m, 1H), 7.17 (t, J = 2.1 Hz, 1H), 6.93–6.91
(m, 1H), 6.84 (dd, J = 0.6 Hz and J = 8.5 Hz, 1H), 4.00 (s, 3H), 3.88 (s,
3H); 13C NMR (CDCl3): 163.70, 160.00, 144.90, 142.10, 140.10, 137.30,
137.00, 129.90, 129.60, 127.70, 127.00, 119.50, 112.80, 110.90, 55.30,
53.50.
IR: 3504, 3392, 1596, 1447, 1166, 821 cmꢀ1
.
7.1.4.9. 1-[5-(30-Hydroxybiphenyl-4-yl)-2-thienyl]ethanone (9). The
title compound was prepared by reaction of 40-bromobiphenyl-3-ol (1i)
(100 mg, 0.40 mmol), 5-acetylthiophene-2-boronic acid (82 mg,
0.48 mmol), caesium carbonate (521 mg, 1.60 mmol) and tetrakis(tri-
phenylphosphine) palladium (14 mg, 12 lmol) according to method A.
The product was purified by CC (dichloromethane/methanol 98:2) fol-
lowed by preparative HPLC; yield: 5% (6 mg); MS (APCI): 295 (M+H)+;
1H NMR (CD3COCD3): 8.47 (s, 1H), 7.87 (d, J = 4.1 Hz, 1H), 7.84 (d,
J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 4.1 Hz, 1H), 7.30 (t,
7.1.4.5. 5-(30-Hydroxybiphenyl-4-yl)pyridin-2(1H)-one (4). To a
solution of 2-methoxy-5-(30-methoxybiphenyl-4-yl)pyridine (4i)
(150 mg, 0.52 mmol, 1 equiv) in dry dichloromethane, borontriflu-

3502
A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
J = 8.1 Hz, 1H), 7.19–7.16 (m, 2H), 6.87 (ddd, J = 0.9 Hz and J = 2.5 Hz and
J = 7.9 Hz, 1H), 2.55 (s, 3H); 13CNMR(CD3COCD3): 191.70, 153.40, 145.30,
136.10, 132.00, 129.50, 128.40, 126.40, 119.90, 119.30, 115.50, 27.50; IR:
phenyl (14ii) (200 mg, 0.76 mmol), 5-methyl-2-thienyl boronic acid
(129 mg, 0.91 mmol), caesium carbonate (991 mg, 3.04 mmol) and tetra-
kis(triphenylphosphine) palladium (26 mg, 23 lmol) according to meth-
3331, 1634, 1599, 1451, 804 cmꢀ1
.
od A. The product was purified by CC (hexane/ethyl acetate 99:1); yield:
94% (200 mg); 1H NMR (CD3COCD3): 7.84 (t, J = 1.9 Hz, 1H), 7.58 (ddd,
J = 1.3 Hz and J = 1.9 Hz and J = 7.6 Hz, 1H), 7.55–7.53 (m, 1H), 7.46 (t,
J = 7.6 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.34 (d, J = 3.5 Hz, 1H), 7.25 (ddd,
J = 0.9 Hz and J = 1.6 Hz and J = 7.6 Hz, 1H), 7.23 (t, J = 2.1 Hz, 1H), 6.96
(ddd, J = 0.9 Hz and J = 2.6 Hz and J = 8.2 Hz, 1H), 6.82–6.80 (m, 1H),
3.88 (s, 3H), 2.50 (s, 3H); 13C NMR (CD3COCD3): 162.30, 143.70, 143.40,
141.50, 131.80, 131.70, 131.40, 128.50, 127.70, 126.20, 125.70, 125.50,
121.20, 115.00, 114.80, 114.50, 56.60, 16.30.
7.1.4.10. 40-(5-Chloro-2-thienyl)biphenyl-3-ol (10). The title com-
pound was prepared by reaction of 40-bromobiphenyl-3-ol (1i)
(100 mg, 0.40 mmol), 5-chlorothiophene-2-boronic acid (78 mg,
0.48 mmol), caesium carbonate (521 mg, 1.60 mmol) and tetrakis(tri-
phenylphosphine) palladium (14 mg, 12 lmol) according to method
A. The product was purified by CC (dichloromethane) followed by pre-
parative HPLC; yield: 18% (21 mg); MS (ESI): 287 (M+H)+; 1H NMR
(CD3COCD3): 8.46 (s, 1H), 7.67 (s, 4H), 7.35 (d, J = 4.1 Hz, 1H), 7.29
(t, J = 8.1 Hz, 1H), 7.15–7.14 (m, 2H), 7.06 (d, J = 3.8 Hz, 1H), 6.86
(ddd, J = 0.9 Hz and J = 2.2 Hz and J = 7.9 Hz, 1H); 13C NMR
(CD3COCD3): 159.90, 144.60, 143.40, 142.50, 134.40, 131.90, 130.10,
129.80, 129.40, 127.60, 124.90, 119.90, 116.60, 115.50; IR: 3484,
7.1.4.15. 30-(5-Methyl-2-thienyl)biphenyl-3-ol (14). The title
compound was prepared by reaction of 2-(30-methoxybiphenyl-
3-yl)-5-methylthiophene (14i) (200 mg, 0.71 mmol) and boron tri-
bromide (2.13 mmol) according to method D. The product was
purified by CC (hexane/ethyl acetate 99:1); yield: 95% (180 mg);
MS (ESI): 267 (M+H)+; 1H NMR (CD3COCD3): 8.42 (s, 1H), 7.80 (t,
J = 1.6 Hz, 1H), 7.57–7.56 (m, 1H), 7.51–7.49 (m, 1H), 7.45 (t,
J = 7.6 Hz, 1H), 7.33 (d, J = 3.5 Hz, 1H), 7.30 (t, J = 8.2 Hz, 1H),
7.16–7.15 (m, 2H), 6.86 (ddd, J = 0.9 Hz and J = 2.6 Hz and
J = 8.2 Hz, 1H), 6.81–6.80 (m, 1H), 2.50 (s, 3H); 13C NMR
(CD3COCD3): 159.80, 144.10, 143.80, 143.50, 141.40, 137.10,
131.90, 131.40, 128.50, 127.60, 126.10, 125.50, 125.40, 120.10,
3412, 1584, 1436, 1186, 779 cmꢀ1
.
0
7.1.4.11. 1-[5-(3 -Hydroxybiphenyl-3-yl)-2-thienyl]ethanone (11). The
title compound was prepared by reaction of 30-bromobiphenyl-3-ol
(11i) (150 mg, 0.60 mmol), 5-acetylthiophene-2-boronic acid (123 mg,
0.72 mmol), caesium carbonate (785 mg, 2.40 mmol) and tetrakis(tri-
phenylphosphine) palladium (21 mg, 18 lmol) according to method A.
The product was purified by CC (dichloromethane/methanol 98:2) fol-
lowed by preparative HPLC; yield: 27% (48 mg); MS (ESI): 295 (M+H)+;
1H NMR (CD3COCD3): 8.49 (s, 1H), 7.95 (t, J = 1.9 Hz, 1H), 7.84 (d,
J = 4.1 Hz, 1H), 7.71–7.70 (m, 1H), 7.64–7.62 (m, 2H), 7.52 (t, J = 7.6 Hz,
1H), 7.31 (t, J = 8.1 Hz, 1H), 7.19–7.17 (m, 2H), 6.89 (ddd, J = 0.9 Hz
and J = 2.5 Hz and J = 8.2 Hz, 1H), 2.54 (s, 3H); 13C NMR (CD3COCD3):
191.80, 159.80, 153.60, 145.40, 144.00, 143.60, 136.00, 135.80, 131.90,
131.60, 129.40, 127.00, 126.70, 126.40, 120.20, 116.70, 115.80, 27.50;
116.50, 115.80, 15.50; IR: 3486, 3375, 1575, 1184, 777, 692 cmꢀ1
.
7.1.4.16. 2-(30-Methoxybiphenyl-3-yl)-1-benzothiophene (15i). The
title compound was prepared by reaction of 30-bromo-3-methoxybi-
phenyl (14ii) (200 mg, 0.76 mmol), benzothiophene-2-boronic acid
(162 mg, 0.91 mmol), caesium carbonate (991 mg, 3.04 mmol) and tet-
rakis(triphenylphosphine) palladium (26 mg, 23 lmol) according to
IR: 3365, 3088, 1599, 1443, 1277, 780 cmꢀ1
.
method A. The product was purified by CC (hexane/ethyl acetate
9:1); yield: 47% (113 mg); 1H NMR (CD3COCD3): 8.05 (t, J = 1.8 Hz,
1H), 7.95–7.94 (m, 1H), 7.89 (s, 1H), 7.86 (dd, J = 1.9 Hz and J = 7.2 Hz,
1H), 7.77 (ddd, J = 0.9 Hz and J = 1.9 Hz and J = 7.5 Hz, 1H), 7.66 (ddd,
J = 1.3 Hz and J = 1.9 Hz and J = 7.9 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H),
7.43–7.35 (m, 3H), 7.31 (ddd, J = 0.9 Hz and J = 1.6 Hz and J = 7.5 Hz,
1H), 7.29 (t, J = 2.1 Hz, 1H), 6.98 (ddd, J = 0.9 Hz and J = 2.5 Hz and
J = 8.2 Hz, 1H), 3.89 (s, 3H); 13C NMR (CD3COCD3): 162.30, 143.80,
142.90, 141.20, 136.70, 131.90, 131.60, 130.70, 129.00, 127.30, 126.70,
126.60, 126.50, 125.70, 124.20, 122.20, 121.30, 115.20, 114.60, 56.70.
7.1.4.12. 30-(5-Chloro-2-thienyl)biphenyl-3-ol (12). The title
compound was prepared by reaction of 30-bromobiphenyl-3-ol
(11i) (100 mg, 0.40 mmol), 5-chlorothiophene-2-boronic acid
(78 mg, 0.48 mmol), caesium carbonate (521 mg, 1.60 mmol) and
tetrakis(triphenylphosphine) palladium (14 mg, 12 lmol) accord-
ing to method A. The product was purified by CC (dichlorometh-
ane) followed by preparative HPLC; yield: 9% (10 mg); MS (APCI):
287 (M+H)+; 1H NMR (CD3COCD3): 8.50 (s, 1H), 7.82–7.81 (m,
1H), 7.58–7.56 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H), 7.42 (d, J = 3.8 Hz,
1H), 7.30 (t, J = 8.2 Hz, 1H), 7.17–7.15 (m, 2H), 7.07 (d, J = 3.8 Hz,
1H), 7.89–7.86 (m, 1H); 13C NMR (CD3COCD3): 159.90, 144.80,
144.00, 143.80, 135.90, 131.90, 131.60, 130.30, 129.80, 128.50,
126.30, 126.20, 125.70, 125.30, 120.20, 116.60, 115.80; IR: 3535,
7.1.4.17. 30-(1-Benzothien-2-yl)biphenyl-3-ol (15). The title
compound was prepared by reaction of 2-(30-methoxybiphenyl-
3-yl)-1-benzothiophene (15i) (113 mg, 0.36 mmol) and boron tri-
bromide (1.08 mmol) according to method D. The product was
purified by preparative TLC (hexane/ethyl acetate 8:2); yield: 77%
(83 mg); MS (ESI): 303 (M+H)+; 1H NMR (CD3COCD3): 8.45 (s,
1H), 8.01 (t, J = 1.8 Hz, 1H), 7.95–7.94 (m, 1H), 7.89 (s, 1H), 7.87–
7.85 (m, 1H), 7.77 (ddd, J = 1.3 Hz and J = 1.9 Hz and J = 7.9 Hz,
1H), 7.62 (ddd, J = 0.9 Hz and J = 1.6 Hz and J = 7.6 Hz, 1H), 7.55
(t, J = 7.8 Hz, 1H), 7.41–7.31 (m, 3H), 7.22–7.20 (m, 2H), 6.90–
6.88 (m, 1H); 13C NMR (CD3COCD3): 171.90, 159.90, 143.90,
142.90, 141.20, 136.60, 131.90, 131.60, 128.90, 127.20, 126.70,
126.60, 126.50, 125.70, 124.20, 122.20, 120.20, 116.60, 115.80;
3403, 1590, 1188, 778 cmꢀ1
.
7.1.4.13. 30-(2-Thienyl)biphenyl-3-ol (13). The title compound
was prepared by reaction of 30-bromobiphenyl-3-ol (11i) (100 mg,
0.40 mmol), thiophene-2-boronic acid (62 mg, 0.48 mmol), caesium
carbonate (521 mg, 1.60 mmol) and tetrakis(triphenylphosphine)
palladium (14 mg, 12 lmol) according to method A. The product
was purified by preparative TLC (hexane/ethyl acetate 7:3) followed
by preparative HPLC; yield: 36% (36 mg); MS (ESI): 253 (M+H)+; 1H
NMR (CD3COCD3): 8.49 (s, 1H), 7.88 (t, J = 1.9 Hz, 1H), 7.66–7.64
(m, 1H), 7.56–7.54 (m, 2H), 7.50–7.47 (m, 2H), 7.30 (t, J = 7.9 Hz,
1H), 7.18–7.17 (m, 2H), 7.16–7.14 (m, 1H), 6.88 (ddd, J = 0.9 Hz and
J = 2.2 Hz and J = 7.9 Hz, 1H); 13C NMR (CD3COCD3): 159.90,
145.80, 144.00, 136.80, 131.90, 131.50, 130.20, 128.00, 127.10,
126.60, 126.00, 125.60, 120.10, 116.50, 115.80; IR: 3365, 1593,
IR: 3464, 3387, 1574, 1179, 779, 692 cmꢀ1
.
7.1.4.18. 3-[2-(2-Thienyl)pyridin-4-yl]phenol (16). Compound
16 was obtained as a side product of the reaction of compound
17; yield: 22% (17 mg); MS (ESI): 254 (M+H)+; 1H NMR (CD3OD):
8.42 (dd, J = 0.6 Hz and J = 5.4 Hz, 1H), 7.91 (dd, J = 0.6 Hz and
J = 1.6 Hz, 1H), 7.71 (dd, J = 0.9 Hz and J = 3.8 Hz, 1H), 7.46 (dd,
J = 1.3 Hz and J = 5.1 Hz, 1H), 7.40 (dd, J = 1.6 Hz and J = 5.4 Hz,
1H), 7.27 (t, J = 7.9 Hz, 1H), 7.17 (ddd, J = 0.9 Hz and J = 1.9 Hz
and J = 7.9 Hz, 1H), 7.11 (t, J = 2.1 Hz, 1H), 7.09 (dd, J = 3.5 Hz and
1456, 1186, 777, 692 cmꢀ1
.
7.1.4.14. 2-(30-Methoxybiphenyl-3-yl)-5-methylthiophene (14i). The
title compound was prepared by reaction of 30-bromo-3-methoxybi-

A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
3503
J = 5.1 Hz, 1H), 6.84 (ddd, J = 0.9 Hz and J = 2.5 Hz and J = 8.2 Hz,
1H); 13C NMR (CD3OD): 159.40, 154.40, 151.40, 150.70, 145.30,
140.50, 131.40, 129.30, 128.90, 126.70, 121.30, 119.30, 118.20,
tetrakis(triphenylphosphine) palladium (44 mg, 38
l
mol) accord-
ing to method C. The product was purified by CC (hexane/ethyl
acetate 96:4); yield: 44% (147 mg); 1H NMR (CD3COCD3): 8.54
(dd, J = 0.6 Hz and J = 5.4 Hz, 1H), 8.15–8.14 (m, 1H), 7.72–7.71
(m, 1H), 7.70–7.69 (m, 1H), 7.56 (dd, J = 1.9 Hz and J = 5.4 Hz,
1H), 7.41 (t, J = 7.8 Hz, 1H), 7.03 (ddd, J = 0.9 Hz and J = 2.5 Hz
and J = 8.2 Hz, 1H), 3.88 (s, 3H); 13C NMR (CD3COCD3): 162.20,
160.20, 152.50, 141.20, 134.90, 131.70, 127.40, 125.30, 121.10,
117.40, 114.00, 56.70.
117.50, 114.80; IR: 3075, 1599, 1202, 781, 722, 692 cmꢀ1
.
7.1.4.19. 4-Bromo-2-(5-chloro-2-thienyl)pyridine (17i). The ti-
tle compound was prepared by reaction of 2,4-dibromopyridine
(300 mg, 1.27 mmol), 5-chloro-2-thienyl boronic acid (206 mg,
1.27 mmol), potassium carbonate (350 mg, 2.54 mmol) and tetra-
kis(triphenylphosphine) palladium (44 mg, 38 lmol) according to
method B using 1 equiv boronic acid. The product was purified
by CC (hexane/ethyl acetate 99:1); yield: 24% (83 mg); 1H NMR
(CD3COCD3): 8.36 (dd, J = 0.6 Hz and J = 5.4 Hz, 1H), 8.07 (dd,
J = 0.6 Hz and J = 1.9 Hz, 1H), 7.70 (d, J = 4.1 Hz, 1H), 7.48 (dd,
J = 1.9 Hz and J = 5.4 Hz, 1H), 7.08 (d, J = 4.1 Hz, 1H); 13C NMR
(CD3COCD3): 154.80, 152.40, 144.40, 134.90, 134.40, 129.80,
127.40, 127.30, 122.90.
7.1.4.24. 4-(5-Chloro-2-thienyl)-2-(3-methoxyphenyl)pyridine
(19i). The title compound was prepared by reaction of 4-bro-
mo-2-(3-methoxyphenyl)pyridine (19ii) (147 mg, 0.56 mmol), 5-
chloro-2-thienyl boronic acid (109 mg, 0.67 mmol), caesium car-
bonate (730 mg, 2.24 mmol) and tetrakis(triphenylphosphine) pal-
ladium (20 mg, 17 lmol) according to method A. The product was
purified by CC (hexane/ethyl acetate 9:1); yield: 49% (83 mg); 1H
NMR (CD3COCD3): 8.65 (dd, J = 0.6 Hz and J = 5.1 Hz, 1H), 7.80
(dd, J = 0.6 Hz and J = 1.6 Hz, 1H), 7.77–7.76 (m, 1H), 7.75–7.73
(m, 1H), 7.72 (d, J = 3.8 Hz, 1H), 7.49 (dd, J = 1.6 Hz and J = 5.1 Hz,
1H), 7.41 (t, J = 7.9 Hz, 1H), 7.16 (d, J = 4.1 Hz, 1H), 7.02 (ddd,
J = 0.9 Hz and J = 2.5 Hz and J = 8.2 Hz, 1H), 3.88 (s, 3H); 13C NMR
(CD3COCD3): 162.20, 152.30, 143.10, 142.30, 142.00, 132.80,
130.10, 127.80, 121.00, 120.40, 117.60, 116.80, 114.20, 56.60.
7.1.4.20. 3-[2-(5-Chloro-2-thienyl)pyridin-4-yl]phenol (17). The title
compound was prepared by reaction of 4-bromo-2-(5-chloro-2-thie-
nyl)pyridine (17i) (83 mg, 0.30 mmol), 3-hydroxybenzene boronic acid
(50 mg, 0.36 mmol), caesium carbonate (391 mg, 1.20 mmol) and tetra-
kis(triphenylphosphine)palladium(11 mg, 9 lmol)accordingtomethod
A. The product was purified by preparative TLC (hexane/ethyl acetate
6:4) followed by preparative HPLC; yield: 49% (42 mg); MS (ESI): 288
(M+H)+; 1H NMR (CD3OD): 8.43 (dd, J = 0.9 Hz and J = 5.4 Hz, 1H), 7.90
(dd, J = 0.9 Hz and J = 1.9 Hz, 1H), 7.56 (d, J = 4.1 Hz, 1H), 7.43 (dd,
J = 1.9 Hz and J = 5.4 Hz, 1H), 7.28 (t, J = 7.9 Hz, 1H), 7.18 (ddd,
J = 0.9 Hz and J = 1.9 Hz and J = 7.9 Hz, 1H), 7.11 (t, J = 1.9 Hz, 1H), 6.97
(d, J = 3.8 Hz, 1H), 6.85 (ddd, J = 0.9 Hz and J = 2.5 Hz and J = 8.2 Hz,
1H); 13C NMR (CD3OD): 159.40, 151.40, 150.80, 144.60, 140.40, 131.40,
128.80, 125.80, 121.60, 119.30, 118.30, 117.80, 117.50, 117.30, 114.80;
7.1.4.25. 3-[4-(5-Chloro-2-thienyl)pyridin-2-yl]phenol (19). The ti-
tle compound was prepared by reaction of 4-(5-chloro-2-thienyl)-2-
(3-methoxyphenyl)pyridine (19i) (83 mg, 0.27 mmol) and boron tri-
bromide (0.81 mmol) according to method D. The product was purified
by CC (hexane/ethyl acetate 8:2) followed by preparative HPLC; yield:
47% (54 mg); MS (ESI): 288 (M+H)+; 1H NMR (CD3SOCD3): 8.63 (dd,
J = 0.9 Hz and J = 5.4 Hz, 1H), 8.03 (dd, J = 0.9 Hz and J = 1.9 Hz, 1H),
7.85 (d, J = 4.1 Hz, 1H), 7.57–7.56 (m, 2H), 7.52 (dd, J = 1.9 Hz and
J = 5.1 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.28 (d, J = 3.8 Hz, 1H), 6.86
(ddd, J = 0.9 Hz and J = 2.5 Hz and J = 8.2 Hz, 1H); 13C NMR (CD3SOCD3):
157.70, 154.70, 140.60, 139.40, 129.60, 126.70, 118.00, 117.50, 116.30,
IR: 3077, 1584, 1471, 799, 622 cmꢀ1
.
7.1.4.21. 2-(5-Chloro-2-thienyl)-6-(3-methoxyphenyl)pyridine
(18i). The title compound was prepared by reaction of 2-bro-
mo-6-(3-methoxyphenyl)pyridine (18ii) (250 mg, 0.95 mmol), 5-
chloro-2-thienyl boronic acid (185 mg, 1.14 mmol), potassium
carbonate (262 mg, 1.89 mmol) and tetrakis(triphenylphosphine)
115.40, 113.50; IR: 3045, 1580, 1310, 780, 693 cmꢀ1
.
palladium (33 mg, 28 lmol) according to method B. The product
was purified by CC (hexane/ethyl acetate 9:1); yield: 33%
(95 mg); 1H NMR (CD3COCD3): 7.97 (dd, J = 0.6 Hz and J = 7.6 Hz,
1H), 7.80 (t, J = 7.8 Hz, 1H), 7.66–7.64 (m, 2H), 7.54 (dd, J = 0.6 Hz
and J = 7.9 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.10 (d, J = 3.8 Hz, 1H),
7.05 (ddd, J = 0.9 Hz and J = 2.5 Hz and J = 8.2 Hz, 1H), 7.02 (d,
J = 4.1 Hz, 1H), 3.88 (s, 3H); 13C NMR (CD3COCD3): 162.20,
159.80, 143.50, 141.80, 136.80, 133.70, 130.30, 129.60, 129.40,
128.50, 125.80, 121.20, 120.90, 117.30, 114.00, 56.70.
7.1.4.26. 3-(5-Chloro-2-thienyl)-5-(3-methoxyphenyl)pyridine
(20i). The title compound was prepared by reaction of 3-bro-
mo-5-(3-methoxyphenyl)pyridine (20ii) (60 mg, 0.23 mmol), 5-
chloro-2-thienyl boronic acid (44 mg, 0.27 mmol), potassium car-
bonate (49 mg, 0.46 mmol) and tetrakis(triphenylphosphine) pal-
ladium (8 mg, 7 lmol) according to method B. The product was
purified by CC (hexane/ethyl acetate 8:2); yield: 68% (47 mg); 1H
NMR (CD3COCD3): 8.80 (t, J = 1.9 Hz, 2H), 8.18 (t, J = 2.2 Hz, 1H),
7.54 (d, J = 4.1 Hz, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.33–7.31 (m, 2H),
7.13 (d, J = 3.8 Hz, 1H), 7.03–7.01 (m, 1H), 3.89 (s, 3H); 13C NMR
(CD3COCD3): 169.00, 162.40, 149.30, 147.10, 140.50, 134.50,
133.00, 131.50, 131.30, 130.70, 129.90, 126.70, 121.30, 115.90,
114.60, 56.70.
7.1.4.22. 3-[6-(5-Chloro-2-thienyl)pyridin-2-yl]phenol (18). The title
compound was prepared by reaction of 2-(5-chloro-2-thienyl)-6-(3-
methoxyphenyl)pyridine (18i) (95 mg, 0.32 mmol) and boron tribromide
(0.96 mmol)accordingtomethodD. TheproductwaspurifiedbyCC(hex-
ane/ethyl acetate 8:2) followed by preparative HPLC; yield: 29% (27 mg);
MS (ESI): 288 (M+H)+; 1H NMR (CD3OD): 7.83 (t, J = 7.9 Hz, 1H), 7.71 (dd,
J = 0.6 HzandJ = 7.9 Hz, 1H), 7.68 (dd, J = 0.6 HzandJ = 7.9 Hz, 1H), 7.63–
7.62 (m, 1H), 7.57–7.55 (m, 1H), 7.54 (d, J = 4.1 Hz, 1H), 7.33 (t, J = 7.9 Hz,
1H), 7.02 (d, J = 4.1 Hz, 1H), 6.90 (ddd, J = 0.9 Hz and J = 2.5 Hz and
J = 8.2 Hz, 1H); 13C NMR (CD3OD): 159.00, 157.90, 152.70, 141.40,
139.00, 133.30, 133.20, 130.80, 129.80, 128.60, 125.10, 119.80, 119.10,
7.1.4.27. 3-[5-(5-Chloro-2-thienyl)pyridin-3-yl]phenol (20). The title
compound was prepared by reaction of 3-(5-chloro-2-thienyl)-5-(3-
methoxyphenyl)pyridine (20i) (120 mg, 0.40 mmol) and boron tribro-
mide (1.20 mmol) according to method D. The product was purified
by CC (hexane/ethyl acetate 8:2) followed by preparative HPLC; yield:
37% (42 mg); MS (ESI): 288 (M+H)+; 1H NMR (CD3OD): 8.79 (s, 1H),
8.75 (s, 1H), 8.27 (s, 1H), 7.51 (dd, J = 0.9 Hz and J = 3.8 Hz, 1H), 7.38
(t, J = 7.9 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.14 (s, 1H), 7.11 (dd,
J = 0.9 Hz and J = 3.8 Hz, 1H), 6.94–6.92 (m, 1H); 13C NMR (CD3OD):
159.50, 132.30, 131.50, 129.20, 126.50, 119.50, 117.00, 115.50, 115.10;
117.40, 114.70; IR: 3384, 1566, 1452, 1218, 775 cmꢀ1
.
7.1.4.23. 4-Bromo-2-(3-methoxyphenyl)pyridine (19ii). The ti-
tle compound was prepared by reaction of 2,4-dibromopyridine
(300 mg, 1.27 mmol), 3-methoxybenzene boronic acid (193 mg,
1.27 mmol), sodium hydrogencarbonate (213 mg, 2.54 mmol) and
IR: 3188, 1589, 1329, 1014, 776, 693 cmꢀ1
.

3504
A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
7.2. Biological methods
7.3. Computational chemistry
[2, 4, 6, 7-3H]-E2 and [2, 4, 6, 7-3H]-E1 were bought from Perkin
Elmer, Boston. Quickszint Flow 302 scintillator fluid was bought
from Zinsser Analytic, Frankfurt.
17b-HSD1 and 17b-HSD2 were obtained from human placenta
according to previously described procedures.31 Fresh human pla-
centa was homogenized and cytosolic fraction and microsomes
were separated by centrifugation. For the partial purification of
17b-HSD1, the cytosolic fraction was precipitated with ammonium
sulfate. 17b-HSD2 was obtained from the microsomal fraction.
7.3.1. Molecular modelling
All calculations and graphical manipulations were performed
on Intel(R) Core(TM)2 Duo CPU 3.00 GHz running Linux CentOS
5.3 while using the software packages AutoDock4.237 as imple-
mented through the graphical user interface AutoDockTools1.5.437
,
GOLDv4.0.138 and MOE2008.10 (Chemical Computing Group Inc.,
Montreal, Canada).
Atomic coordinates of 17b-HSD1 used during molecular model-
ling simulations were derived from the structure of the ternary
complex between 17b-HSD1, NADP+ and equilin (RCSB Protein
Data Bank entry 1EQU).
7.2.1. Inhibition of 17-HSD1
Inhibitory activities were evaluated by an established method
To set the initial coordinates for the docking studies, chain A
was isolated in the coordinate file and the cocrystallized equilin
as well as all water molecules were removed. Missing protein
atoms were added and correct atom types set. Ionization states
and hydrogen positions were assigned using the Protonate 3D util-
ity of MOE. Furthermore the crystal structure was energy mini-
mized with MOE applying MMFF94ꢂ force field and generalized
Born model, keeping the coordinates of protein backbone atoms
fixed.
with minor modifications.31 Briefly, the enzyme preparation was
incubated with NADH [500 lM] in the presence of potential inhib-
itors at 37 °C in a phosphate buffer (50 mM) supplemented with
20% of glycerol and EDTA (1 mM). Inhibitor stock solutions were
prepared in DMSO. The final concentration of DMSO was adjusted
to 1% in all samples. The enzymatic reaction was started by addi-
tion of a mixture of unlabelled- and [2, 4, 6, 7-3H]-E1 (final concen-
tration: 500 nM, 0.15 lCi). After 10 min, the incubation was
stopped with HgCl2 and the mixture was extracted with diethyl-
ether. After evaporation, the steroids were dissolved in acetonitrile.
E1 and E2 were separated using acetonitrile/water (45:55) as mo-
bile phase in a C18 reverse phase chromatography column (Nucle-
Inhibitors were energy minimized with MOE using the
MMFF94ꢂ force field and the atomic partial charges for the inhib-
itors and the cofactor were calculated using the AM1-BCC
approximation.
odur C18 Gravity, 3
l
m, Macherey-Nagel, Düren) connected to a
Two docking softwares, namely Autodock4.2 and GOLDv4.0.1,
were used to evaluate the binding mode of the inhibitors.
HPLC-system (Agilent 1100 Series, Agilent Technologies, Wald-
bronn). Detection and quantification of the steroids were per-
formed using a radioflow detector (Berthold Technologies, Bad
Wildbad). The conversion rate was calculated after analysis of
the resulting chromatograms according to the following equation:
7.3.2. Autodock4.2
The docking area has been defined by a box, centered on the ste-
roid binding site. Grid points of 86 ꢂ 80 ꢂ 80 with 0.250 Å spacing
were calculated around the docking area for all the ligand atom
types using AutoGrid4.2. For each inhibitor, 100 separate docking
calculations were performed. Each docking calculation consisted
of 25 ꢂ 105 energy evaluations using the Lamarckian genetic algo-
rithm local search (GALS) method. Each docking run was per-
formed with a population size of 250. A mutation rate of 0.02
and a crossover rate of 0.8 were used to generate new docking tri-
als for subsequent generations. The docking results from each of
the 100 calculations were clustered on the basis of root-mean-
square deviation (RMSD = 2.0 Å) between the Cartesian coordi-
nates of the ligand atoms and were ranked on the basis of the free
binding energy.
%E2
%conversion ¼ %E2þ%E1 ꢂ 100. Each value was calculated from at
least three independent experiments.
7.2.2. Inhibition of 17b-HSD2
The 17b-HSD2 inhibition assay was performed similarly to the
17b-HSD1 procedure. The microsomal fraction was incubated with
NAD+ [1500
l
M], test compound and a mixture of unlabelled- and
[2, 4, 6, 7-3H]-E2 (final concentration: 500 nM, 0.11
lCi) for 20 min
at 37 °C. Further treatment of the samples and HPLC separation
was carried out as mentioned above.
The conversion rate was calculated after analysis of the result-
ing chromatograms according to the following equation:
%E1
%conversion ¼ %E1þ%E2 ꢂ 100.
7.3.3. GOLDv4.0.1
Active site origin was set at the centre of the steroid binding site
and a radius of 15 Å was chosen. The automatic active-site detec-
tion was switched on. Further, GOLDscore38 fitness function was
used and genetic algorithm default parameters were set as sug-
gested by the GOLD authors. For each of the selected compounds
50 solutions were generated and compounds were ranked accord-
ing to GOLDscore.
Both programs performed in a similar way, supporting the here-
in suggested binding modes. The quality of the docked poses was
evaluated based mainly on visual inspection of the putative bind-
ing modes of the ligand, and secondly on the scoring functions.
7.2.3. ER affinity
The binding affinity of selected compounds to the ER
a and ERb
was determined according to Zimmermann et al.49 Briefly,
0.25 pmol of ER
a or ERb, respectively, were incubated with [2, 4,
6, 7-3H]-E2 (10 nM) and test compound for 1 h at room tempera-
ture. The potential inhibitors were dissolved in DMSO (5% final
concentration). Evaluation of non-specific-binding was performed
with diethylstilbestrol (10 lM). After incubation, ligand–receptor
complexes were selectively bound to hydroxyapatite (5 g/60 mL
TE-buffer). The complex formed was separated, washed and resus-
pended in ethanol. For radiodetection, scintillator cocktail (Quicks-
zint 212, Zinsser Analytic, Frankfurt) was added and samples were
measured in a liquid scintillation counter (Rack Beta Primo 1209,
Wallac, Turku). For determination of the relative binding affinity
(RBA), inhibitor and E2 concentrations required to displace 50%
of the receptor bound labelled E2 were determined. RBA values
7.3.4. MEP
For selected compounds ab initio geometry optimizations were
performed for the gas phase at the B3LYP/6-311++G (d,p) level of
density functional theory (DFT) by means of the Gaussian 03 soft-
ware, and the molecular electrostatics potential map (MEP) was
plotted using GaussView, version 3.0, the 3D molecular graphics
package of Gaussian.50 These electrostatic potential surfaces were
generated by mapping 6-311++G electrostatic potentials onto sur-
were calculaðtEe2dÞ according to the following equation:
IC50
IC50
RBA½%ꢃ ¼
ðcompoundÞ ꢂ 100. The RBA value for E2 was arbitrarily
set at 100%.

A. Oster et al. / Bioorg. Med. Chem. 18 (2010) 3494–3505
3505
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faces of molecular electron density (isovalue of 0.004 e/Å). The
MEP maps are colour-coded, where red stands for negative values
(ꢀ1.0 ꢂ 10ꢀ2 Hartree) and blue for positive ones (2.5 ꢂ 10ꢀ2
Hartree).
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Acknowledgements
We thank Jannine Ludwig and Beate Geiger for their help in per-
forming the in vitro tests (17b-HSD1, 17b-HSD2, ERs). Patricia
Kruchten is grateful to the European Postgraduate School 532
(DFG) for a scholarship.
Supplementary data
27. Bey, E.; Marchais-Oberwinkler, S.; Werth, R.; Negri, M.; Al-Soud, Y. A.;
Kruchten, P.; Oster, A.; Frotscher, M.; Birk, B.; Hartmann, R. W. J. Med. Chem.
2008, 51, 6725.
Supplementary data (methods for analytical HPLC; purity data
of all tested compounds, MEP calculations of compounds 17 and
19) associated with this article can be found, in the online version,
at doi:10.1016/j.bmc.2010.03.065.
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