Design, synthesis and biological evaluation of bis(hydroxyphenyl) azoles as potent and selective non-steroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) for the treatment of estrogen-dependent diseases

Article abstract of DOI:10.1016/j.bmc.2008.04.073

The 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyses the reduction of the weakly active estrone (E1) into the most potent estrogen, 17β-estradiol (E2). E2 stimulates the growth of hormone-dependent diseases via activation of the estrogen receptors (ERs). 17β-HSD1 is often over-expressed in breast cancer cells. Thus, it is an attractive target for the treatment of mammary tumours. The combination of a ligand- and a structure-based drug design approach led to the identification of bis(hydroxyphenyl) azoles as potential inhibitors of 17β-HSD1. Different azoles and hydroxy substitution patterns were investigated. The compounds were evaluated for activity and selectivity with regard to 17β-HSD2, ERα and ERβ. The most potent compound is 3-[5-(4-hydroxyphenyl)-1,3-oxazol-2-yl]phenol (18, IC₅₀ = 0.31 μM), showing very good selectivity, high cell permeability and medium CaCo-2 permeability.

Full text of DOI:10.1016/j.bmc.2008.04.073

Bioorganic & Medicinal Chemistry 16 (2008) 6423–6435
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of bis(hydroxyphenyl) azoles as
potent and selective non-steroidal inhibitors of 17b-hydroxysteroid
dehydrogenase type 1 (17b-HSD1) for the treatment of estrogen-dependent
diseases
Emmanuel Bey, Sandrine Marchais-Oberwinkler, Patricia Kruchten, Martin Frotscher, Ruth Werth,
*
Alexander Oster, Oztekin Algül , Alexander Neugebauer, Rolf W. Hartmann
8.2 Pharmaceutical and Medicinal Chemistry, Saarland University, 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:
The 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1) catalyses the reduction of the weakly active
estrone (E1) into the most potent estrogen, 17b-estradiol (E2). E2 stimulates the growth of hormone-
dependent diseases via activation of the estrogen receptors (ERs). 17b-HSD1 is often over-expressed in
breast cancer cells. Thus, it is an attractive target for the treatment of mammary tumours. The combina-
tion of a ligand- and a structure-based drug design approach led to the identification of bis(hydroxy-
phenyl) azoles as potential inhibitors of 17b-HSD1. Different azoles and hydroxy substitution patterns
were investigated. The compounds were evaluated for activity and selectivity with regard to 17b-
HSD2, ERa and ERb. The most potent compound is 3-[5-(4-hydroxyphenyl)-1,3-oxazol-2-yl]phenol (18,
IC50 = 0.31 lM), showing very good selectivity, high cell permeability and medium CaCo-2 permeability.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 14 March 2008
Revised 24 April 2008
Accepted 30 April 2008
Available online 3 May 2008
Keywords:
17b-HSD1
17b-HSD2
Non-steroidal inhibitor
ER
Estrogen-dependent diseases
Bis(hydroxyphenyl) azoles
1. Introduction
estrogen action at the receptor level whilst aromatase inhibitors
and GnRH-analogues restrain the formation of estrogens. This
The naturally occurring steroidal hormones, estrone (E1) and
17b-estradiol (E2), are responsible for the development and differ-
entiation of estrogen-sensitive tissues. It is well known, however,
that E2, the most active estrogen, also plays a pivotal role in the
growth of estrogen-dependent breast cancer,1 and is involved in
the pathophysiology of endometriosis.2
Until now three endocrine therapies have been established for
the treatment of breast cancer.3,4 Selective estrogen receptor mod-
ulators (SERMs) and pure antiestrogens5 like fulvestrant block the
strong reduction of systemic estrogen action is a rather radical
approach. A softer therapy could be the inhibition of the enzyme
catalysing the last step of the E2 biosynthesis: 17b-hydroxysteroid
dehydrogenase type 1 (17b-HSD1).
17b-HSD1 is a member of the 17b-hydroxysteroid dehydroge-
nase family which is responsible for the intracellular regulation
of steroidal sex hormones activities.6,7 Until now, fourteen mem-
bers of this enzyme class are known.8 In humans, eleven of them
regulate the concentration of active androgens and estrogens in a
tissue-specific manner.8,9
17b-HSD1 (EC1.1.1.62) is a cytosolic enzyme, which converts
the weakly active estrone (E1) into the highly potent E2 using
NAD(P)H as cofactor (Chart 1). The enzyme is expressed in differ-
ent organs like ovaries, placenta, breast, endometrium10 and often
over-expressed in many breast cancer tissues.11–14 Inhibition of
17b-HSD1 should be a good strategy to selectively reduce the E2
level in diseased tissues, and might therefore be a new therapeutic
approach with probably less side effects for the treatment of estro-
gen-dependent diseases.
Abbreviations: 17b-HSD1, 17b-hydroxysteroid dehydrogenase type 1; 17b-
HSD2, 17b-hydroxysteroid dehydrogenase type 2; E1, estrone; E2, 17b-estradiol;
ER, estrogen receptor; PDB-ID, protein data bank identification code; Papp, apparent
permeability coefficient; RBA, relative-binding affinity; SAR, structure–activity
relationship; CC, column chromatography; MES, 2-(morpholino)ethanesulfonic
acid; EDTA, ethylene diaminetetraacetate; TE, Tris–EDTA; FCS, foetal calf serum;
DMEM, Dulbecco’s modified Eagle’s medium; AUC, area under the curve
* Corresponding author. Tel.: +49 681 302 3424; fax: +49 681 302 4386.
E-mail address: rwh@mx.uni-saarland.de (R.W. Hartmann).
URL: http://www.pharmmedchem.de
Present address: Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Mersin University, 33169 Mersin, Turkey.
As
a biological counterpart, the membrane-bound 17b-
hydroxysteroid dehydrogenase type 2 (17b-HSD2) catalyses the
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.073

6424
E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
His221/Glu282 and Ser142/Tyr155, respectively. This area also
OH
O
contains a mainly apolar region, corresponding to the B/C ring of
the steroid (Leu149, Val225, Phe226, Phe259). Interestingly, two
polar amino acids (Tyr218, Ser222) are located in the same domain
(Fig. 1).
17β-HSD1, NAD(P)H
17β-HSD2, NAD+
HO
HO
The catalytic centre of the enzyme is formed by a tetrad of ami-
no acids45 (Asn114, Ser142, Tyr155 and Lys159) which stabilises
the steroid and the nicotinamide moiety during hydride transfer.
The active site is limited by a flexible loop (amino acids 188–
201) which is not well resolved in all of the X-ray structures.
E1
E2
Chart 1. Interconversion of estrone (E1) to 17b-estradiol (E2).
NAD+-dependent oxidation of E2 into E1 (Chart 1). This enzyme
should not be affected by potential inhibitors of 17b-HSD1, as it
might protect the cell from excessively high concentrations of ac-
tive estrogen.15 Nor should 17b-HSD1 inhibitors show affinity to
the estrogen receptors a and b (ERa and ERb) to avoid intrinsic
estrogenic effects.
Over the last decade, several groups have reported on 17b-HSD1
inhibitors, most of them showing steroidal structures.6,16–30 Until
now, only three classes of non-steroidal compounds have been de-
scribed.31–34
2.2. Design of steroidomimetics
In the design process, the substrate-binding site was defined as
a binding region for the potential inhibitors as a lack of selectivity
is expected for the compounds targeting only the cofactor-binding
pocket: this area consists of Rossmann fold motifs which are highly
conserved in the majority of dehydrogenases. We focused on non-
steroidal structures as it is known that steroidal compounds often
show side effects which are caused by agonistic or antagonistic ef-
fects at steroid receptors. As the compounds have to mimic the ste-
roidal substrate, they should contain two hydroxyphenyl (or two
methoxyphenyl) moieties to imitate the A-ring (interaction with
His221/Glu282) and the D-ring (interaction with Ser142/Tyr155)
of the steroid. Additionally, the O–O distance should be in the same
range as observed for the steroid (d = 11 Å). Different substitution
patterns concerning the position of the phenolic OH groups were
therefore investigated to find out the one which fits best into the
active site (Chart 2). To mimic the flat shape of the substrate, the
two substituted benzenes should be linked by an additional aro-
matic ring. The five-membered heterocycles imidazole, triazole,
pyrazole, isoxazole and oxazole seemed to be especially suitable
as their heteroatoms might be able to establish additional interac-
tions with the amino acids Tyr218 and/or Ser222, which are lo-
cated close to the C6 position of the steroidal B-ring. Position,
number and nature of the heteroatoms in the five-membered ring
were varied in order to identify the most appropriate heterocycle.
1,2- and 1,3-bis(hydroxyphenyl) and tris(hydroxyphenyl) azoles
have been described by Fink et al.46 as novel ER ligands. The fact
In the following, we will report on the design, synthesis and bio-
logical evaluation of potent and selective non-steroidal inhibitors
of 17b-HSD1 that are appropriate for drug development.
2. Design of the inhibitors
2.1. Characterisation of the binding site
Sixteen crystal structures35–43 with different steroidal ligands
are available from the Protein Data Bank.44 The three dimensional
architecture of the enzyme was investigated using the ternary
complex of 17b-HSD1 with E2 and NADP+ (PDB-ID: 1FDT).35 A sub-
strate-binding site, a cofactor-binding pocket and an entry channel
can be defined. The former is a narrow hydrophobic tunnel show-
ing a high degree of complementarity to the steroid. Two polar re-
gions can be identified at each extremity of the substrate-binding
site corresponding to the binding positions of the 3- and 17-hydro-
xy group of E2. Each of them establish two hydrogen bonds with
Figure 1. Schematic presentation of the active site of 17b-HSD1 containing E2 (PDB-ID: 1FDT). Orange labels denote polar amino acids and white labels stand for lipophilic
amino acids. Hydrogen bonds are marked in red and cofactor in purple.

E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
6425
X
Y
N
X
Z
N
R2
R2
Y
R1
R1
1-8
9-19
compound
X
Y
Z
R1
R2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
SH
SH
SH
H
H
H
C
C
C
C
C
C
N
N
NH
NH
NH
CH
CH
CH
CH
CH
O
4-OH
4-OH
3-OH
4-OH
4-OH
3-OH
4-OH
3-OH
4-OH
4-OH
3-OH
4-OH
4-OH
4-OH
4-OH
3-OH
4-OH
3-OH
3-OH
3-OH
4-OH
4-OH
3-OH
4-OH
4-OH
3-OH
4-OH
3-OH
4-OH
4-OH
3-OH
4-OH
3-OH
4-OH
4-OH
4-OH
4-OH
4-OH
N
N
N
NH
NH
O
O
O
N
N
CH
CH
CH
N
N
N
N
N
CH
CH
CH
O
N
O
Chart 2. Title compounds.
that the compounds bearing two hydroxyphenyl moieties did not
show any binding affinity to the ERs46 is supportive of our design
concept. In the following, we describe the synthesis of compounds
1–19 (Chart 2) and their biological evaluation using human 17b-
HSD1, 17b-HSD2, ERa and ERb as well as T-47D and CaCo-2 cell
lines.
to the route presented in Scheme 1. Intermediates 1i–3i were pre-
pared by nucleophilic substitution followed by cyclisation with
potassium thiocyanate as described by Prakash et al.47 The sulfur
removal (4i–6i) was performed under strong acidic conditions
according to Dodson and Ross.48 Compounds 1i–6i were submitted
to ether cleavage with boron trifluoride dimethyl sulfide complex46
as reagent.
Synthesis of the 1,2,3-triazoles 7 and 8 was performed accord-
ing to the synthetic pathway shown in Scheme 2. Aromatic nucle-
ophilic substitution of iodophenol derivatives by sodium azide led
to intermediates 7i and 8ii. The resulting azides were converted to
3. Chemistry
The synthesis of the title compounds is depicted in Schemes 1–
4. 1,4-Bis(hydroxyphenyl) imidazoles were synthesised according
R
N
R
O
R2
N
NH2
OH
3
a, b
d
N
R2
N
+
4
R1
Br
R1
HO
R = SH 1i to 3i
R = H 4i to 6i
R = SH 1 to 3
R = H 4 to 6
c
compound
R1
R2
1i
2i
3i
4i
5i
6i
4-OMe 3-OMe
4-OMe 4-OMe
3-OMe 4-OMe
4-OMe 3-OMe
4-OMe 4-OMe
3-OMe 4-OMe
Scheme 1. Synthesis of compounds 1–6. Reagents and conditions: (a) NEt3, DMF, rt, 7 h; (b) KSCN, cat. HCl, MeOH, reflux, 18 h; (c) HNO3, NaNO2, glacial acetic acid, 0 °C,
20 min; (d) BF3ꢀSMe2, CH2Cl2, rt, 20 h.

6426
E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
N
N
N
R2
I
b
N3
a
R2
+
HO
HO
OH
7i: 4-OH
8ii: 3-OH
7: 4-OH, R2= 3-OH
8i:3-OH, R2= 4-OMe
c
8: 3-OH, R2= 4-OH
Scheme 2. Synthesis of compounds 7 and 8. Reagents and conditions: (a) NaN3, CuI,
24 h; (c) BF3ꢀSMe2, CH2Cl2, rt, 20 h.
L
-proline, NaOH, DMSO, 60 °C, 10 h; (b) Na-ascorbate, CuSO4, H2O/t-BuOH (1:1), 60 °C,
O
R1
Cl
NH2
R2
O
N
a
R2
N
H
O
N
R1
R1
b
c
9i to 11i
d
e
OH
N
H
X
R2
O
HO
N
O
9ii to 11ii
X = NH 9 to 11
X= O 17 and 18
R2
R1
17i and 18i
compound
9ii
R1
R2
compound
R1
R2
4-OMe 3-OMe
4-OMe 4-OMe
3-OMe 4-OMe
9i
4-OMe 3-OMe
4-OMe 4-OMe
3-OMe 4-OMe
4-OMe 4-OMe
3-OMe 4-OMe
10ii
11ii
10i
11i
17i
18i
Scheme 3. Synthesis of compounds 9–11, 17 and 18. Reagents and conditions: (a) NEt3, rt, 30 min.; (b) NH4OAc, AcOH, reflux, 2 h; (c) POCl3, pyridine, reflux, 8 h; (d)
BF3ꢀSMe2, CH2Cl2, rt, 20 h; (e) for 17i: pyridinium hydrochloride, 220 °C, 18 h, for 18i: BBr3, CH2Cl2, ꢁ78 °C to rt, 20 h.
compounds 7 and 8i in the presence of the corresponding phenyl
acetylene derivative using the method of Sharpless.49 The methoxy
group of 8i was cleaved with boron trifluoride dimethyl sulfide
complex.
pounds 14i–16i, the a,b-unsaturated ketones 12ii, 13ii and 16ii
were first activated with bromine and cyclised to isoxazole with
hydroxylamine hydrochloride (Method B). Ether cleavage was per-
formed with boron tribromide to yield 12–16.
The synthesis of the 2,5-bis(hydroxyphenyl) imidazoles and
2,5-bis(hydroxyphenyl) oxazoles is presented in Scheme 3. The
commercially available amine derivatives were N-acylated with
two different acid chlorides to build the key intermediates 9ii–
11ii. The latter were cyclised to the 2,4-disubstituted imidazoles
(9i–11i) according to Suzuki et al.50 and to the 2,5-disubstituted
oxazoles (17i and 18i) following Nicolaou et al.51 Ether cleavage
was successful with boron trifluoride dimethyl sulfide complex
for the imidazole compounds 9–11, pyridinium hydrochloride for
17 and boron tribromide for 18.
The 2, 4 oxazole 19i was prepared in a one-pot synthesis following
the procedure of Lee et al.53 Briefly, 4-methoxyacetophenone, [hydro-
xy(2,4-dinitrobenzensulfonyloxy)iodo]benzene and 3-methoxybenz-
amide were heated under reflux in acetonitrile for 10 h to yield
the intermediate 19i. The ether groups of 19i were cleaved with
boron tribromide.
4. Biological results
4.1. Inhibition of human 17b-HSD1
The pyrazoles 12 and 13 and the isoxazoles 14 and 16 were syn-
thesised according to the route shown in Scheme 4. Key intermedi-
ates 12ii, 13ii and 16ii were prepared via Claisen condensation of
the commercially available methoxylated acetophenone deriva-
tives with the appropriate benzaldehydes under strong basic con-
ditions. The cyclisation step for the pyrazoles52 12i and 13i was
carried out with hydrazine monohydrate (Method A). For com-
Placental enzyme was isolated following a described proce-
dure.54 Tritiated E1 was incubated with 17b-HSD1, cofactor and
inhibitor. The amount of formed E2 was quantified by HPLC. All
methoxy compounds and para–paradi (hydroxylated) derivatives
are inactive (data not shown). In contrast, some of the unsymmet-
rically substituted compounds were active (Table 1). Interestingly

E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
6427
H
R2
O
R1
O
a
O
X
N
N
X
b
c
OH
R2
R2
R1
R1
HO
X = NH 12i and 13i
X = O 14i to 16i
12ii and 13ii
X = NH 12 and 13
X = O 14 to 16
16ii
compound
R1
R2
method
12i
13i
14i
15i
16i
4-OMe 3-OMe
4-OMe 4-OMe
4-OMe 3-OMe
4-OMe 4-OMe
3-OMe 4-OMe
A
A
B
B
B
Scheme 4. Synthesis of compounds 12–16. Reagents and conditions: (a) EtOH, Na, rt, 30 min; (b) Method A: hydrazine monohydrate, AcOH, EtOH, reflux, 24 h; Method B:
bromine, diethylether, 0 °C, 1 h, hydroxylamine hydrochloride, abs. EtOH, reflux 24 h; (c) BBr3, CH2Cl2, ꢁ78 °C to rt, 20 h.
the para–meta disubstituted isoxazole 14 is inactive whilst its
meta–para analogue 16 (IC50 = 1.61 lM) shows inhibitory activity.
This exemplifies the importance of the positions of the OH groups.
The triazole 7, isoxazole 16 and 2,4-disubstituted oxazole 19 are
only weak inhibitors of 17b-HSD1, whilst derivatives 8 and 18
show IC50 values in the nanomolar range. The 2,5-disubstituted
oxazole 18 was the most potent compound identified with an
IC50 value of 0.31 lM.
It is striking that only compounds containing a heterocycle
which can function as a hydrogen bond acceptor (nitrogen and/or
oxygen) are active. The position of the heteroatoms in the hetero-
cyclic skeleton is also a decisive criterion for inhibitory activity.
Table 1
Inhibition of human 17b-HSD1 and 17b-HSD2 by selected inhibitors
Selectivity factorsd
a
Compound
Structure
IC50 (lM)
17b-HSD1b
17b-HSD2c
N
N
N
N
OH
OH
OH
OH
OH
7
1.32
8.12
6
HO
HO
HO
HO
HO
N
N
8
16
18
0.84
1.61
0.31
1.85
7.28
0.27
17.5
0.25
9
0.2
56
0.1
N O
N
O
O
N
19
a
Mean value of three determinations, standard deviation less than 10%.
Human placental, cytosolic fraction, substrate E1, 500 nM, cofactor NADH, 500 lM.
Human placental, microsomal fraction, substrate E2, 500 nM, cofactor NAD+, 1500 lM.
IC50 HSD2/IC50 HSD1.
b
c
d

6428
E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
The 2,5-disubstituted oxazole (18, IC50 = 0.31 lM) showed a stron-
ger inhibition of the enzyme as its 2,4-disubstituted oxazole (19,
IC50 = 1.85 lM) and 3,5-disubstituted isoxazole (16, IC50 = 1.61 lM)
analogues.
was observed after application of compound 18 even at a concen-
tration 1000-fold higher compared to E2.
Compound 18 was additionally evaluated using the same cell
line which expresses both 17b-HSD1 and 17b-HSD2. The oxazole
18 inhibits the formation of E2 after incubation with labelled E1
exhibiting an IC50 value of 0.38 lM. As this value is very similar
to the one observed in the cell free assay (0.31 lM, see Table 1) it
can be concluded that the compound is capable of unfolding its
activity in intact cells.
Further investigations were performed using CaCo-2 cells.
These cells exhibit morphological and physiological properties of
the human small intestine55 and are generally accepted to be an
appropriate model for the prediction of peroral absorption.
Depending on the Papp data obtained, compounds are usually clas-
sified as low (Papp (10ꢁ6 cm/s) < 1), medium (1 < Papp < 10) or
highly permeable (Papp > 10). Compound 18, showing a Papp value
of 7.9 10ꢁ6 cm/s, is a medium cell permeator like for example
acetyl salicylic acid.
4.2. Selectivity
Since 17b-HSD2 catalyses the inactivation of E2 into E1, inhibi-
tory activity towards this enzyme must be avoided. The 17b-HSD2
inhibition was determined using an assay similar to the 17b-HSD1
test. Placental microsomes were incubated with tritiated E2 in the
presence of NAD+ and inhibitor. Quantification of labelled product
(E1) was performed by HPLC and following radio detection. IC50
values and selectivity factors (IC50 HSD2/ IC50 HSD1) are presented
in Table 1.
Compound 18 is only a weak inhibitor of the type 2 enzyme
(IC50 = 17.5 lM) with a selectivity factor of 56, whilst the isoxazole
16 (IC50 = 0.27 lM) and the oxazole 19 (IC50 = 0.25 lM) isomers
show stronger inhibition for the type 2 versus type 1 enzyme
(selectivity factors: 0.2 and 0.1, respectively).
5. Molecular modelling
Furthermore, as 17b-HSD1 inhibitors should have no or low
affinity to ERa and ERb, binding affinity was measured for selected
compounds. Using recombinant human protein, a competition as-
say applying tritium labelled E2 (RBA = 100%) and hydroxyapatite
was performed. The triazoles 7 and 8, isoxazole 16 and oxazole
19 show very low binding affinity to both ERs, whereas for the
most interesting compound, the 2,5-disubstituted oxazole 18, a
very low affinity to ERa (0.01 < RBA(%) < 0.1) and a marginally
higher affinity to ERb (RBA = 0.5%, Table 2) is observed.
In order to get a better insight into the molecular interactions
between the most potent steroidomimetic 18 and 17b-HSD1, the
compound was docked into the active site of the protein (PDB-
ID: 1FDT, E2 removed) using the docking software Gold 3.0 (rigid
protein, flexible ligand).
Two different binding modes can be expected because of the
pseudo-symmetry of our steroidomimetic: each hydroxyphenyl
group could mimic the A-ring of the steroid. The energetically most
favourable pose is depicted in Figure 2. The para-hydroxyphenyl
substituent and the heterocycle are in the same plane, whilst the
meta-hydroxyphenyl moiety is rotated 32° out of this plane. This
conformation allows the inhibitor to establish hydrogen bond
interactions with His221/Glu282 (para-hydroxyphenyl moiety)
and Ser142/Tyr155 (meta-hydroxyphenyl 0 substituent). Interest-
ingly, the Noxazole–OTyr218 distance is 2.89 ÅA making an additional
hydrogen bond interaction very likely. On the other hand, an inter-
4.3. Further biological evaluation of compound 18 using T-47D
and CaCo-2 cell lines
The most promising inhibitor of this series, compound 18 was
evaluated for estrogenic effects on the ER-positive, mammary tu-
mour T-47D cell line. No agonistic, that is no stimulatory effect,
Table 2
Binding affinities for the estrogen receptors a and b by selected compounds
Compound
Structure
RBAa (%)
ERa
ERb
N
N
N
OH
OH
N
7
<0.01
<0.01
HO
HO
HO
HO
HO
N
N
N
8
16
18
0.01 < RBA < 0.1
0.01 < RBA < 0.1
0.01 < RBA < 0.1
0.01 < RBA < 0.1
0.01 < RBA < 0.1
O
N
OH
OH
<0.01
0.5
O
O
OH
19
0.01 < RBA < 0.1
N
a
RBA: relative-binding affinity, E2: 100%, mean value of three determinations, standard deviation less than 10%.

E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
6429
Figure 2. 17b-HSD1-binding pocket (green amino acids) with docked compound 18 (yellow). Hydrogen bonding interactions are marked by violet lines. All distances are
expressed in Å. For clarity, only selected amino acids are represented.
action with the OSer222 cannot be observed as in the rigid protein
structure the CH2OH moiety is turned away from the heterocycle.
However, it cannot be excluded that there is a conformational
change in that functional group after binding of this ligand. Addi-
tionally, it is likely that hydrophobic interactions (Van der Waals
and p–p stacking) are also involved in the binding of 18 into the ac-
tive site.
pared to the oxazole. This is obviously due to the electronegative
effect of the oxygen. It can therefore be assumed that only in case
of compound 18, hydrogen bond interactions with Tyr218 and
Ser222 (Fig. 2) are possible making the latter compound a potent
inhibitor. These explanations are supported by the fact that the
oxazole 19 shows a weak inhibitory activity.
The insertion of a third nitrogen (triazoles 7 and 8) in the
heterocycle increases the inhibitory potency of the correspond-
ingly substituted imidazoles 4, 6 and imidazoles-2-thiol 1, 3 which
are inactive. Obviously, this nitrogen plays the same role for the
interaction with the amino acid residues in the binding site as
observed for compound 18.
The most interesting compound of the present study is 18
showing a high 17b-HSD1 inhibition and a good selectivity to-
wards 17b-HSD2. In addition, compound 18 exhibits very low
affinity to ERa and ERb. It is striking that the high-binding affinity
of similar ER ligands like the tris(hydroxyphenyl) pyrazoles
described by Katzenellenbogen’s group46,58–60 depends on the
presence of three hydroxyphenyl moieties. No polar amino acids
in the B/C ring region of E2 can be identified in the X-ray structures
of the ERs as groups to establish interactions with the heterocycle.
Obviously, the pyrazole is playing a passive role in this class of ER
ligands. In case of our bis(hydroxyphenyl) substituted azoles the
combination of at least two parameters is implicated in the
17b-HSD1 inhibitory potency: an optimal OH substitution pattern
and an appropriate heterocycle.
Activation of the ERs would be detrimental for the treatment of
estrogen-dependent diseases. Therefore, agonism must be avoided.
Antagonistic activity would be less critical. The diseased cells cer-
tainly could benefit from compounds with dual activity. However,
antiestrogens would also exert systemic effects in other healthy ste-
roidogenic tissues leading to unwanted effects. Consequently, we
focused on the discovery of compounds without affinity to the ERs.
The fact that the 2,5-disubstituted oxazole 18 inhibits the cellu-
lar formation of E2 with an IC50 value in the nanomolar range
shows that this compound is able to enter the cell and to be active
at the target enzyme. The fact that the inhibitor shows a good per-
meation is supported by the CaCo-2 data. These results are indica-
tive of a sufficient intestinal absorption.
6. Discussion and conclusion
Looking at the SARs of the synthesised compounds, it becomes
apparent that the positions of the OH groups at the hydroxyphenyl
moieties are crucial for the activities of the corresponding deriva-
tives: all para–para substituted compounds are inactive, whilst
several of the meta–para/para–meta isomers are active. This sug-
gests that 17b-HSD1 is not as flexible to adjust its geometry to
the two OH groups as it is reported for the ERs,56 and is in agree-
ment with the non-flexibility already observed for 17b-HSD1
inhibitors in the class of 6-(hydroxyphenyl)naphthalenes.34 The
fact that not all meta–para/para–meta bis (hydroxyphenyl) deriva-
tives (with O–O distances comparable to E2) are active, indicates
that the nature of the heterocycle is also playing an important role
for the inhibitory potencies of the compounds.
Obviously hydrogen bond donors are unfavourable for activity
(e.g., imidazoles 9–11, pyrazoles 12 and 13), whilst several com-
pounds with only hydrogen bond acceptor groups show reasonable
activities (e.g., triazoles 7 and 8, isoxazole 16, oxazoles 18 and 19).
It is interesting to have a closer look at the inactive isoxazole 14
(para–meta) and its highly active isomer, the oxazole 18 (para–
meta). Provided that both compounds interact with the active site
in the same manner, for example, the para-hydroxyphenyl moiety
establishes hydrogen bond contacts with His221/Glu282, the
structure of the inhibitors only differ by the position of the oxygen
on the heterocycle. It has been described57 that only the nitrogen of
isoxazole and oxazole (and not the oxygen) is able to establish
hydrogen bond interaction. The position of the oxygen on the azole
structure seems to be determinant for the ability of the compound
for forming hydrogen bonds involving the nitrogen: in case of the
isoxazole, the electronic density on the nitrogen is reduced com-

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E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
Compound 18 might therefore be a good lead compound for the
development of a clinically applicable therapeutic for the treat-
ment of estrogen-dependent diseases.
7.1.1.1. 4-(3-Methoxyphenyl)-1-(4-methoxyphenyl)-1H-imidazol-
2-thiol (1i). The title compound was prepared by reaction of para-
methoxyaniline (751 mg, 6.11 mmol), 3-methoxyphenacyl bro-
mide (1.38 g, 6.11 mmol), triethylamine (0.89 mL, 6.11 mmol)
and potassium thiocyanate (593 mg, 6.11 mmol) according to the
procedure reported above. The product was purified by CC (hex-
ane/ethyl acetate 9:1); yield: 28% (500 mg); yellow powder; mp:
248 °C; 1H NMR (CDCl3): 7.36 (d, J = 9.40 Hz, 2H), 7.30–7.26 (m,
2H), 7.10 (d, J = 7.80 Hz, 2H), 6.84 (m, 1H), 6.81 (d, J = 8.80 Hz,
2H), 3.82 (s, 3H, OMe), 3.72 (s, 3H, OMe); 13C NMR (CDCl3):
175.45, 160.05, 159.65, 129.70, 127.55, 117.45, 114.15 (2C),
113.95, 110.00, 55.45, 55.40. IR: 1626, 1514, 1222, 1037,
824 cmꢁ1; MS (APCI): 313 (M+H)+.
7. Experimental Section
7.1. Chemical methods
Chemical names follow IUPAC nomenclature. Starting materials
were purchased from Aldrich, Acros, Lancaster, Roth, Merck or Flu-
ka 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).
7.1.1.2. 1-(3-Methoxyphenyl)-4-(4-methoxyphenyl)-1H-imidazol-
2-thiol (3i). The title compound was prepared by reaction of meta-
methoxyaniline (356 mg, 2.90 mmol), 4-methoxyphenacyl bro-
mide (658 mg, 2.90 mmol) and triethylamine (0.37 mL,
2.90 mmol), potassium thiocyanate (282 mg, 2.90 mmol) according
to the procedure reported above. The product was purified
by CC (hexane/ethyl acetate 9:1); yield: 16% (140 mg); white
powder; mp: 250 °C; 1H NMR (CDCl3): 7.51 (d, J = 8.50 Hz, 2H),
7.38 (t, J = 7.80 Hz, 1H), 7.27 (s, 1H), 7.18 (d, J = 7.80 Hz, 1H), 7.02
(m, 1H), 6.97 (dd, J = 2.50 and 8.50 Hz, 1H), 6.89 (d, J = 8.50 Hz,
2H), 3.84 (s, 3H, OMe), 3.79 (s, 3H, OMe); 13C NMR (CDCl3):
188.00, 160.00, 129.95, 126.50, 118.00, 114.65 (2C), 111.80,
55.60, 55.35; IR: 3055, 1601, 1455, 1181, 825 cmꢁ1; MS (ESI):
313 (M+H)+.
Melting points were measured on a Mettler FP1 melting point
apparatus and are uncorrected. IR spectra were recorded on a Bru-
ker 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:
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, m, dt, q for singlet, doublet, triplet, doublet
of doublets, multiplet, doublet of triplets and quadruplet, respec-
tively. All coupling constants (J) are given in hertz (Hz).
7.1.2. 4-(4-Methoxyphenyl)-1-(3-methoxyphenyl)-1H-
imidazole (6i)
Mass spectra (ESI and APCI) were recorded on a TSQ Quantum
(Thermo Finnigan) instrument. Elemental analyses were per-
formed at the Department of Instrumental Analysis and Bioanaly-
sis, Saarland University.
The title compound was prepared by reaction of sodium ni-
trite (11 mg, 0.16 mmol, 1 equiv), nitric acid (152 lL, 87 lmol,
0.5 equiv) and 1-(3-methoxyphenyl)-4-(4-methoxyphenyl)-1H-
imidazol-2-thiol (3i) (150 mg, 0.48 mmol, 3 equiv) mixed in gla-
cial acetic acid (15 mL) at 0 °C and stirred for 20 min. The reac-
tion was quenched with ammonium hydroxide (20 mL) and the
resulting precipitate was filtered, dried overnight in a desicca-
tor. The crude product was purified by CC (ethyl acetate/meth-
anol 98:2); yield: 48% (22 mg); yellow powder; mp: 246 °C; 1H
NMR (CDCl3): 8.90 (s, 1H) 7.60 (s, 1H), 7.53 (s, 1H), 7.48 (m,
3H), 7.32 (t, J = 7.80 Hz, 1H), 7.02 (d, J = 8.50 Hz, 2H), 6.99 (dd,
J = 1.80 and 8.20 Hz, 1H), 3.95 (s, 3H, OMe), 3.85 (s, 3H,
OMe); 13C NMR (CDCl3): 161.15, 160.55, 136.15, 133.10,
130.20, 127.20, 123.85, 117.90, 117.15, 115.85, 110.50, 56.25,
55.60; IR: 3200, 2966, 1520, 1255, 855 cmꢁ1; MS (ESI): 281
(M+H)+.
The following compounds were prepared according to previously
described procedures: 1,4-bis(4-methoxyphenyl)-1H-imidazol-2-
thiol (2i),47 4-(3-methoxyphenyl)-1-(4-methoxyphenyl)-1H-imid-
azole (4i),61 1,4-bis(4-methoxyphenyl)-1H-imidazole (5i),62 4-azid-
ophenol (7i),63 3-[1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl]-
phenol (7),63 3-azidophenol (8ii),63 3-[4-(4-hydroxyphenyl)-1H-
1,2,3-triazol-1-yl]phenol (8),63 3-methoxy-N-[2-(4-methoxyphenyl)-
2-oxo-ethyl]benzamide (9ii),64 4-methoxy-N-[2-(4-methoxy-
phenyl)-2-oxo-ethyl]benzamide
(10ii),65
2,5-bis(4-methoxy-
phenyl)-1H-imidazole (10i),66 3-methoxy-N-[2-(4-methoxyphenyl)-
2-oxo-ethyl]-benzamide (11ii),67 (2E)-1-(3-methoxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (12ii),68 (2E)-1,3-bis(4-methoxy-
phenyl)prop-2-en-1-one (13ii),68 3,5-bis(4-methoxyphenyl)-1H-pyra-
zole (13i),69 4,40-(1H-pyrazol-3,5-diyl)diphenol (13),46 3-(3-methoxy-
phenyl)-5-(4-methoxyphenyl)isoxazole (14i),70 3-[5-(4-hydroxy-
phenyl)isoxazol-3-yl]phenol (14),71 3,5-bis(4-methoxyphenyl)-isox-
azole (15i),71 4,40-(isoxazol-3,5-diyl)diphenol (15),71 (2E)-3-(3-methoxy-
phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (16ii),72 3-[3-(4-
hydroxyphenyl)isoxazol-5-yl]phenol (16),71 2,5-bis(4-methoxy-
phenyl)-1,3-oxazole (17i).64
7.1.3. 3-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]phenol (8i)
3-Azidophenol (8ii) (500 mg, 3.70 mmol, 1 equiv) and 1-ethy-
nyl-4-methoxybenzene (489 mg, 3.70 mmol, 1 equiv) were stirred
in a mixture of water/tert-butanol (1:1, 20 mL) for 2 min. Cop-
per(II)sulfate (9.24 mg, 0.04 mmol, 0.01 equiv) was suspended in
a freshly prepared solution of sodium ascorbate (1 M, 73.7 mg,
0.37 mmol, 0.1 equiv) and added dropwise to the latter mixture.
The reaction mixture was stirred at 60 °C for 24 h. After cooling
to rt, the mixture was washed with water and the aqueous layer
washed with ethyl acetate. The combined organic layers were
dried over sodium sulfate and solvent was removed under reduced
pressure. The crude product was purified by CC (ethyl acetate/
methanol 9:1); yield: 37% (350 mg); yellow powder; 1H NMR
(CD3COCD3): 8.80 (s, 1H) 7.91 (d, J = 8.80 Hz, 2H), 7.46 (s, 1H),
7.41 (d, J = 7.90 Hz, 2H), 7.04 (d, J = 8.80 Hz, 2H), 6.96 (m, 1H),
3.85 (s, 3H, OMe); 13C NMR (CD3COCD3): 170.90, 159.45, 131.60,
127.80 (2C), 118.50, 116.30, 115.10 (2C), 111.80, 108.10, 55.65;
7.1.1. General synthesis procedure for compounds 1i–3i
A mixture of methoxyaniline (1 equiv), methoxyphenacyl bro-
mide (1 equiv) and triethylamine (1 equiv) was stirred at rt in
2 mL DMF for 7 h. The crude material was poured into ice water.
The resulting precipitate was filtered and dried overnight in a desic-
cator. Toa stirredsolutionofthe precipitate(1 equiv)in 20 mL meth-
anol, potassium thiocyanate (1 equiv) and 60 lL concentrated
chlorhydric acid were added. The resulting mixture was refluxed
for 18 h. After cooling to rt, the precipitate was filtered off and dried
overnight in a desiccator. The crude product was purified by CC.
IR: 3148, 2928, 1614, 1499, 1257, 833 cmꢁ1
.

E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
6431
7.1.4. General synthesis procedure for compounds 9i–11i
Benzamides 9ii–11ii (1 equiv) were refluxed in acetic acid
(10 mL) with ammonium acetate (8 equiv) for 2 h. Solvent was
evaporated under reduced pressure and the crude product was
7.1.6.1. 3-(4-Methoxyphenyl)-5-(3-methoxyphenyl)-1H-
pyrazole (12i). The title compound was prepared by reaction of
(2E)-1-(3-methoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one
(12ii) (250 mg, 0.93 mmol) and hydrazine monohydrate (212 lL,
3.72 mmol) according to described Method A. The product was
purified by CC (hexane/ethyl acetate 5:5) followed by preparative
TLC (dichloromethane/methanol 99:1); yield: 25% (60 mg); yellow
oil; 1H NMR (CDCl3): 7.55 (d, J = 8.80 Hz, 2H), 7.21–7.19 (m, 2H),
7.18 (t, J = 7.80 Hz, 1H), 6.78–6.75 (m, 3H), 6.62 (s, 1H), 3.74 (s,
3H, OMe), 3.62 (s, 3H, OMe); 13C NMR (CDCl3): 159.85, 159.55,
129.70, 126.85, 126.85, 118.10, 114.15 (2C), 114.10, 110.50,
99.35, 55.20, 55.05; IR: 2933, 2837, 1601, 1439, 1250, 1033,
suspended in
a
mixture of ethanol/water/dichloromethane
(1:1:1). The dichloromethane layer was separated, washed with
brine, dried over magnesium sulfate, evaporated under reduced
pressure and purified by CC.
7.1.4.1. 2-(4-Methoxyphenyl)-5-(3-methoxyphenyl)-1H-
imidazole (9i). The title compound was prepared by reaction of 3-
methoxy-N-[2-(4-methoxyphenyl)-2-oxo-ethyl]benzamide
(9ii)
(917 mg, 3.20 mmol) and ammonium acetate (1.90 g, 25.6 mmol)
according to the procedure reported above. The product was puri-
fied by CC (hexane/ethyl acetate 5:5); yield: 25% (224 mg); white
powder; mp: 198 °C; 1H NMR (CD3SOCD3): 8.07 (d, J = 2.50 Hz,
1H), 7.98 (d, J = 8.50 Hz, 2H), 7.78 (d, J = 8.50 Hz, 1H), 7.57 (s,
1H), 7.37 (s, 1H), 7.12–7.08 (m, 2H), 6.75 (s, 1H), 3.83 (s, 3H,
OMe), 3.82 (s, 3H, OMe); 13C NMR (CD3SOCD3): 162.10, 160.85,
151.40, 137.50, 128.65 (2C), 127.25, 125.40, 123.35, 120.75,
115.75 (2C), 113.70, 56.80, 56.50; IR: 3070, 2950, 1578, 1242,
742 cmꢁ1; MS (ESI): 281 (M+H)+.
834 cmꢁ1
.
7.1.7. General synthesis procedure for compounds 14i–16i
(Method B)
Propenone intermediate (12ii, 13ii and 16ii, 1 equiv) was stir-
red at 0 °C in 5 mL dry diethylether and bromine (1 equiv) was
added dropwise. After 1 h at 0 °C, the reaction was warmed up to
rt, precipitate was filtered and washed with diethylether. The
resulting di-brominated propenone was obtained in quantitative
yield and used without further purification. The latter (1 equiv)
was refluxed in 10 mL absolute ethanol with 1 equiv hydroxyl-
amine hydrochloride and 1 equiv potassium hydroxide for 24 h.
After cooling to rt, the mixture was poured into a cold water solu-
tion. The resulting precipitate was filtered, washed with cold
water, dried overnight in a desiccator and purified by CC.
7.1.4.2. 2-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-1H-
imidazole (11i). The title compound was prepared by reaction
of 3-methoxy-N-[2-(4-methoxyphenyl)-2-oxo-ethyl]benzamide
(11ii) (917 mg, 3.20 mmol) and ammonium acetate (1.90 g,
25.6 mmol) according to the procedure reported above. The prod-
uct was purified by CC (hexane/ethyl acetate 5:5); yield: 6%
(54 mg); white powder; mp: 202 °C; 1H NMR (CDCl3): 8.04 (s,
1H), 7.85 (d, J = 8.20 Hz, 2H), 7.28–7.24 (m, 3H), 6.88 (d,
J = 8.20 Hz, 2H), 6.78 (dq, J = 1.50 and 7.60 Hz, 1H), 3.79 (s, 3H,
OMe), 3.77 (s, 3H, OMe); 13C NMR (CDCl3): 164.00, 132.35 (2C),
130.15, 120.60, 119.85, 113.75, 112.65, 55.60, 55.50; IR: 3077,
2965, 1678, 1468, 1240, 1031, 742 cmꢁ1; MS (ESI): 281 (M+H)+.
7.1.7.1. 5-(3-Methoxyphenyl)-3-(4-methoxyphenyl)isoxazole
(16i). The title compound was prepared by reaction of (2E)-3-(3-
methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
(16ii)
(275 mg, 1.03 mmol), bromine (52 lL, 1.03 mmol), hydroxylamine
hydrochloride (71.6 mg, 1.03.mmol) and potassium hydroxide
(57.8 mg, 1.03 mmol) according to Method B. The product was
purified by CC (hexane/ethyl acetate 9:1) followed by preparative
TLC (dichloromethane/methanol 1%); yield: 45% (130 mg); yellow
powder; mp: 161 °C; 1H NMR (CDCl3): 7.20 (m, 2H), 6.75 (dd,
J = 2.00 and 7.50 Hz, 1H), 6.80 (d, J = 7.50 Hz, 1H), 6.76 (s, 1H),
6.74 (s, 1H), 6.70 (m, 1H), 6.45 (d, J = 7.50 Hz, 2H), 3.72 (s, 3H,
OMe), 3.56 (s, 3H, OMe); 13C NMR (CDCl3): 171.65, 164.15,
162.40, 161.25, 131.80, 131.20, 128.70 (2C), 121.60, 120.60,
117.35, 115.70, 113.00, 97.50; IR: 2925, 2853, 1602, 1248,
7.1.5. 5-(4-Methoxyphenyl)-2-(3-methoxyphenyl)-1,3-oxazole
(18i)
The title compound was prepared by reaction of 3-methoxy-N-
[2-(4-methoxyphenyl)-2-oxo-ethyl]benzamide
(9ii)
(347 mg,
1.16 mmol, equiv) and phosphorous oxychloride (12 mL,
1
0.89 mmol, 0.8 equiv) in pyridine (20 mL) and refluxed for 8 h.
After cooling to rt, ethyl acetate (40 mL) was added and the crude
material was poured into a saturated sodium hydrogencarbonate
solution. The aqueous layer was extracted two times with ethyl
acetate. The combined organic layers were washed with brine,
dried over magnesium sulfate, evaporated under reduced pres-
sure and purified by CC (hexane/ethyl acetate 5:5); yield: 36%
(117 mg); yellow oil; 1H NMR (CD3COCD3): 7.79 (d, J = 8.80 Hz,
2H), 7.70 (dt, J = 1.00 and 8.80 Hz, 1H), 7.64 (q, J = 1.00 Hz, 1H),
7.53 (s, 1H, H-oxazole), 7.44 (t, J = 7.90 Hz, 1H), 7.08 (m, 3H),
3.90 (s, 3H, OMe), 3.86 (s, 3H, OMe); 13C NMR (CD3COCD3):
161.05, 160.95, 152.40, 130.95, 129.85, 126.65, 123.15, 121.65,
119.15, 116.95, 115.40, 111.85, 55.75, 55.70; IR: 2937, 1612,
746 cmꢁ1
.
7.1.8. 4-(4-Methoxyphenyl)-2-(3-methoxyphenyl)-1,3-oxazole
(19i)
4-Methoxyacetophenone (500 mg, 3.33 mmol, 1 equiv) was re-
fluxed for 2 h together with [hydroxy(2,4-dinitrobenzensulfonyl-
oxy)-iodo]benzene (1.88 g, 3.99 mmol, 1.2 equiv) in acetonitrile
(20 mL). After cooling to rt, 3-methoxybenzamide (1.52 g,
9.99 mmol, 3 equiv) was added and the reaction mixture was re-
fluxed for 10 h. Acetonitrile was evaporated under reduced pres-
sure. The crude product was suspended in dichloromethane. The
resulting organic layer was washed with a saturated sodium bicar-
bonate solution and dried over magnesium sulfate. Solvent was re-
moved under reduced pressure and the product was purified by CC
(hexane/ethyl acetate 7:3); yield 50% (465 mg); white powder;
mp: 165 °C; 1H NMR (CD3COCD3): 8.23 (s, 1H, H-oxazole), 7.90
(d, J = 9.20 Hz, 2H), 7.32 (m, 2H), 7.20 (t, J = 7.50 Hz, 1H), 6.93 (d,
J = 9.20 Hz, 2H), 6.76 (m, 1H), 3.73 (s, 3H, OMe), 3.70 (s, 3H,
1253, 1010, 872 cmꢁ1
.
7.1.6. General synthesis procedure for compounds 12i and 13i
(Method A)
A solution of hydrazine monohydrate (4 equiv) in glacial acetic
acid (4 equiv) was added dropwise to the propenone intermediate
12ii and 13ii (1 equiv). The reaction mixture was heated at reflux
for 24 h. After cooling to rt, the precipitate was filtered off. A mix-
ture of water/ethyl acetate (1:1) was added to the filtrate.
The combined organic layers were washed with brine, dried over
magnesium sulfate, evaporated under reduced pressure and
purified by CC.
OMe); IR: 3015, 2925, 1625, 789 cmꢁ1
.
7.1.9. Ether cleavage—General procedure for compounds 1–6
and 9–11
To a solution of bis (methoxyphenyl) derivative (1 equiv) in dry
dichloromethane was added dropwise boron trifluoride dimethyl

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E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
sulfide complex (75 equiv). The reaction mixture was stirred at rt
for 20 h. To quench the reaction, water was added and the aqueous
layer was washed with ethyl acetate. The combined organic layers
were washed with brine, dried over sodium sulfate, evaporated un-
der reduced pressure and purified by preparative TLC.
7.74 (dd, J = 2.20 and 8.80 Hz, 2H), 7.71 (dd, J = 2.20 and 8.80 Hz,
2H), 7.10 (dd, J = 2.20 and 8.80 Hz, 2H), 7.08 (dd, J = 2.20 and
8.80 Hz, 2H); 13C NMR (CD3COCD3): 160.40, 128.70 (2C), 125.30,
117.70 (2C), 117.45 (2C), 117.25; IR: 3563, 3155, 1684, 1048,
931, 836 cmꢁ1; MS (ESI): 253 (M+H)+; Anal. Calcd C15H12N2O2: C,
71.42; H, 4.79; N, 11.10. Found: C, 71.43; H, 4.85; N, 11.11.
7.1.9.1. 3-[1-(4-Hydroxyphenyl)-2-sulfanyl-1H-imidazol-4-yl]-
phenol (1). The title compound was prepared by reaction of 4-(3-
methoxyphenyl)-1-(4-methoxyphenyl)-1H-imidazol-2-thiol (1i)
(100 mg, 0.32 mmol) according to the procedure reported above.
The product was purified by preparative TLC (ethyl acetate);
yield: 61% (55 mg); orange powder; mp: 265 °C; 1H NMR
(CD3SOCD3): 12.76 (s, 1H, SH), 7.64 (s, 1H), 7.39 (d, J = 8.50 Hz,
2H), 7.19–7.15 (m, 2H), 7.09 (s, 1H), 6.84 (d, J = 8.50 Hz, 2H),
6.71–6.69 (m, 1H); 13C NMR (CD3SOCD3): 162.30, 157.60,
156.85, 129.90, 129.20, 128.95, 127.15, 116.15, 115.10 (2C),
114.85, 111.10; IR: 3214, 1604, 1514, 1395, 1101, 833,
750 cmꢁ1; MS (APCI): 284 (M)+; Anal. Calcd C15H12N2O2S: C,
63.36; H, 4.25; N, 9.85. Found: C, 63.20; H, 3.99; N, 9.80.
7.1.9.6. 3-[4-(4-Hydroxyphenyl)-1H-imidazol-4-yl]phenol (6). The
title compound was prepared by reaction of 4-(4-methoxyphenyl)-1-
(3-methoxyphenyl)-1H-imidazole (6i) (100 mg, 0.36 mmol) accord-
ing to the procedure reported above. The product was purified by pre-
parative TLC (ethyl acetate); yield: 26% (24 mg); yellow oil; 1H NMR
(CD3COCD3): 9.50 (d, J = 1.50 Hz, 1H), 8.40 (d, J = 1.50 Hz, 1H), 7.77
(m, 2H), 7.50 (t, J = 8.20 Hz, 1H), 7.36–7.34 (m, 2H), 7.16 (dd, J = 2.20
and 8.80 Hz, 1H), 7.04 (dt, J = 2.20 and 8.20 Hz, 2H); 13C NMR
(CD3COCD3): 137.45, 132.50, 128.80 (2C), 118.35, 117.50, 114.35,
110.70; IR: 3542, 3160, 2955, 1699, 1630, 1062, 841 cmꢁ1; MS (ESI):
253 (M+H)+; Anal. Calcd C15H12N2O2: C, 71.42; H, 4.79; N, 11.10.
Found: C, 71.59; H, 4.92; N, 10.97.
7.1.9.2. 4,40-(2-Sulfanyl-1H-imidazol-1,4-diyl)diphenol (2). The
title compound was prepared by reaction of 1,4-bis(4-methoxy-
phenyl)-1H-imidazol-2-thiol (2i) (100 mg, 0.32 mmol) according
to the procedure reported above. The product was purified by pre-
parative TLC (ethyl acetate); yield: 36% (35 mg); white powder;
mp: 268 °C; 1H NMR (CD3OD): 7.49 (d, J = 8.80 Hz, 2H) 7.42 (d,
J = 8.80 Hz, 2H), 7.34 (s, 1H), 6.92 (d, J = 8.80 Hz, 2H), 6.87 (d,
J = 8.80 Hz, 2H); 13C NMR (CD3OD): 162.00, 159.05, 158.80,
131.20, 131.10, 131.00, 128.55, 127.30, 120.55, 116.90, 116.50,
115.95; IR: 3135, 2469, 2072, 1511, 1116, 973, 836 cmꢁ1; MS
(APCI): 284 (M)+, 285 (M+H)+; Anal. Calcd C15H12N2O2S: C, 63.36;
H, 4.25; N, 9.85. Found: C, 63.40; H, 4.24; N, 9.95.
7.1.9.7. 3-[2-(4-Hydroxyphenyl)-1H-imidazol-5-yl]phenol
(9). The title compound was prepared by reaction of 2-(4-
methoxyphenyl)-5-(3-methoxyphenyl)-1H-imidazole (9i) (40 mg,
0.14 mmol) according to the procedure reported above. The
product was purified by preparative TLC (hexane/ethyl acetate
5:5); yield: 45% (15 mg); yellow powder; 1H NMR (CD3COCD3):
8.58 (s, 1H), 7.42 (t, J = 7.80 Hz, 2H), 7.40 (m, 1H), 7.33 (m, 1H),
7.27 (t, J = 7.80 Hz, 2H), 7.05 (dd, J = 0.90 and 1.50 Hz, 1H), 6.90
(dd, J = 0.90 and 1.50 Hz, 1H), 6.47 (s, 1H, NH); 13C NMR
(CD3COCD3): 168.95, 168.90, 158.25, 137.90, 136.95, 130.10 (2C),
119.40, 119.10, 118.70, 115.25; IR: 3450, 2950, 1604, 1580,
785 cmꢁ1. MS (ESI): 253 (M+H)+; Anal. Calcd C15H12N2O2: C,
71.42; H, 4.79; N, 11.10. Found: C, 71.23; H, 4.64; N, 10.98.
7.1.9.3. 3-[4-(4-Hydroxyphenyl)-2-sulfanyl-1H-imidazol-1-yl]-
phenol (3). The title compound was prepared by reaction of 1-(3-
methoxyphenyl)-4-(4-methoxyphenyl)-1H-imidazol-2-thiol (3i)
(100 mg, 0.32 mmol) according to the procedure reported above.
The product was purified by preparative TLC (ethyl acetate); yield:
37% (40 mg); yellow powder; 1H NMR (CD3SOCD3): 12.75 (s, 1H,
SH), 7.62 (s, 1H), 7.40 (d, J = 8.50 Hz, 2H), 7.18–7.13 (m, 2H),
7.07 (s, 1H), 6.83 (d, J = 8.50 Hz, 2H), 6.69–6.66 (m, 1H); 13C
NMR (CD3SOCD3): 162.35, 157.65, 156.95, 129.85, 129.00,
128.90, 127.20, 116.20 (2C), 115.05, 114.90, 111.25; IR: 3213,
1600, 1514, 1392, 1100, 845, 750 cmꢁ1; MS (APCI): 284 (M)+; Anal.
Calcd C15H12N2O2S: C, 63.36; H, 4.25; N, 9.85. Found: C, 63.12; H,
4.22; N, 9.84.
7.1.9.8. 4,40-(1H-Imidazol-2,5-diyl)diphenol (10). The title com-
pound was prepared by reaction of 2,5-bis(4-methoxyphenyl)-
1H-imidazole (10i) (82 mg, 0.29 mmol) according to the procedure
reported above. The product was purified by preparative TLC
(dichloromethane/methanol 99:1); yield: 17% (12 mg); yellow
powder; 1H NMR (CD3OD): 7.83 (d, J = 8.70 Hz, 2H), 7.62 (d,
J = 8.70 Hz, 2H), 7.60 (s, 1H), 7.03 (d, J = 8.70 Hz, 2H), 6.92 (d,
J = 8.70 Hz, 2H); 13C NMR (CD3OD): 131.30, 129.15, 120.15 (2C),
119.80 (2C), 115.55 (2C), 114.75 (2C), 114.30; IR: 2590, 1645,
1488, 1114, 841 cmꢁ1
15H12N2O2: C, 71.42; H, 4.79; N, 11.10. Found: C, 71.30; H, 4.52;
N, 11.00.
;
MS (ESI): 253 (M+H)+; Anal. Calcd
C
7.1.9.4. 3-[1-(4-Hydroxyphenyl)-1H-imidazol-4-yl]phenol (4). The
title compound was prepared by reaction of 4-(3-methoxyphenyl)-1-
(4-methoxyphenyl)-1H-imidazole (4i) (100 mg, 0.36 mmol) accord-
ing to the procedure reported above. The product was purified by pre-
parative TLC (ethyl acetate); yield: 28% (25 mg); yellow oil; 1H NMR
(CD3COCD3): 9.32 (d, J = 1.20 Hz, 1H), 8.33 (d, J = 1.20 Hz, 1H), 7.70
(dd, J = 2.20 and 8.80 Hz, 2H), 7.36–7.33 (m, 2H), 7.29 (t, J = 1.90 Hz,
1H), 7.06 (dd, J = 2.20 and 8.80 Hz, 2H), 6.99 (m, 1H); 13C NMR
(CD3COCD3): 159.70, 158.95, 134.95, 131.60, 129.10, 128.10, 124.95,
118.25, 117.95, 117.80, 117.35, 113.35; IR: 3563, 1684, 1629, 1048,
836 cmꢁ1; MS (ESI): 253 (M+H)+; Anal. Calcd C15H12N2O2: C, 71.42;
H, 4.79; N, 11.10. Found: C, 71.11; H, 4.62; N, 11.01.
7.1.9.9. 3-[5-(4-Hydroxyphenyl)-1H-imidazol-2-yl]phenol
(11). The title compound was prepared by reaction of 2-(3-
methoxyphenyl)-5-(4-methoxyphenyl)-1H-imidazole
(11i)
(40 mg, 0.14 mmol) according to the procedure reported above.
The product was purified by preparative TLC (hexane/ethyl acetate
5:5); yield: 45% (15 mg); yellow powder; 1H NMR (CD3COCD3):
8.56 (s, 1H), 7.40 (t, J = 7.80 Hz, 2H), 7.39 (m, 1H), 7.37 (m, 1H),
7.25 (t, J = 7.80 Hz, 2H), 6.99 (dd, J = 0.90 and 1.50 Hz, 1H), 6.97
(dd, J = 0.90 and 1.50 Hz, 1H), 6.47 (s, 1H, NH); 13C NMR
(CD3COCD3): 168.95, 168.90, 158.25, 137.95, 136.90, 130.15 (2C),
119.30 (2C), 118.95, 115.40; IR: 3350, 3045, 2922, 1664, 1582,
760 cmꢁ1; MS (ESI): 253 (M+H)+; Anal. Calcd C15H12N2O2: C,
71.42; H, 4.79; N, 11.10. Found: C, 71.42; H, 4.89; N, 11.08.
7.1.9.5. 4,40-bis(1H-Imidazol-1,4-diyl)-diphenol (5). The title
compound was prepared by reaction of 1,4-bis(4-methoxy-
phenyl)-1H-imidazole (5i) (100 mg, 0.36 mmol) according to the
procedure reported above. The product was purified by preparative
TLC (ethyl acetate), yield: 26% (24 mg); yellow powder; 1H NMR
(CD3COCD3): 9.43 (d, J = 1.50 Hz, 1H), 8.32 (d, J = 1.50 Hz, 1H),
7.1.10. Ether cleavage—General synthesis for compounds 12, 18
and 19
To a solution of bis(methoxyphenyl) derivative (1 equiv) in dry
dichloromethane at ꢁ78 °C (dry ice/acetone bath), boron tribro-
mide (1 M in dichloromethane, 6 equiv) was added dropwise and

E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
6433
the reaction mixture was stirred for 20 h. To quench the reaction,
water was added and the aqueous layer was washed with ethyl
acetate. The combined organic layers were washed with brine,
dried over sodium sulfate, evaporated under reduced pressure
and purified by preparative TLC.
17b-HSD1 and 17b-HSD2 were obtained from human placenta
according to previously described procedures.40,73 Fresh human
placenta was homogenised and the enzymes were separated by
centrifugation. For the purification of 17b-HSD1, the cytosolic frac-
tion was precipitated with ammonium sulfate. 17b-HSD2 was ob-
tained from the microsomal fraction.
7.1.10.1. 3-[3-(4-Hydroxyphenyl)-1H-pyrazol-5-yl]phenol (12). The
title compound was prepared by reaction of 3-(4-methoxyphenyl)-5-
(3-methoxyphenyl)-1H-pyrazole (12i) (82 mg, 0.29 mmol) according
to the procedure reported above. The product was purified by prepara-
tive TLC (dichloromethane/methanol 92:8); yield: 55% (40 mg); orange
powder; mp: 262 °C; 1H NMR (CD3OD): 7.65 (d, J = 8.50 Hz, 2H), 7.22
7.2.1. Inhibition of 17b-HSD1
Inhibitory activities were evaluated by an established method
with minor modifications.54,74,75 Briefly, the enzyme preparation
was incubated with NADH [500 lM] in the presence of potential
inhibitors 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 ad-
justed to 1% in all samples. The enzymatic reaction was started by
addition of a mixture of unlabelled- and [2,4,6,7-3H]-E1 (final con-
centration: 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-
odur C18 Gravity, 3 lm, Macherey-Nagel, Düren) connected to a
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 following equation:
(m, 1H), 6.83 (d, J = 8.50 Hz, 2H), 6.81 (s, 1H), 6.72–6.74 (m, 3H); 13
C
NMR (CD3OD): 160.50, 131.85, 122.10 (2C), 117.05, 116.80, 116.00,
113.30 (2C), 102.15; IR: 3500, 2935, 1620, 790 cmꢁ1; MS (ESI): 253
(M+H)+; Anal. Calcd C15H12N2O2. C, 71.42; H, 4.79; N, 11.10. Found: C,
71.38; H, 4.71; N, 11.25.
7.1.10.2. 3-[5-(4-Hydroxyphenyl)-1,3-oxazol-2-yl]phenol (18). The
title compound was prepared by reaction of 5-(4-methoxyphenyl)-2-
(3-methoxyphenyl)-1,3-oxazole (18i) (100 mg, 0.35 mmol) according
to the procedure reported above. The product was purified by prepara-
tive TLC (hexane/ethyl acetate 5:5); yield: 65% (59 mg); yellow pow-
der; 1H NMR (CD3COCD3): 8.80 (s, 1H, OH), 8.75 (s, 1H, OH), 7.69 (d,
J = 8.20 Hz, 2H), 7.60 (m, 2H), 7.46 (s, 1H, H-oxazole), 7.35 (t,
J = 8.20 Hz, 1H), 6.98–6.95 (m, 3H); 13C NMR (CD3COCD3): 160.90,
158.95, 158.75, 152.55, 130.95, 129.80, 126.80, 122.50, 120.65,
118.25 (2C), 118.15, 116.85, 115.40, 113.55; IR: 3480, 1602, 1510,
852 cmꢁ1; MS (ESI): 254 (M+H)+; Anal. Calcd C15H11NO3: C, 71.14; H,
4.38; N, 5.53. Found: C, 70.92; H, 4.35; N, 5.60.
AUCðE2Þ
%conversion ¼ AUCðE2ÞþAUCðE1Þ ꢂ 100. Each value was calculated from
at least three-independent experiments.
7.2.2. Inhibition of 17b-HSD2
7.1.10.3. 3-[4-(4-Hydroxyphenyl)-1,3-oxazol-2-yl]phenol (19). The
title compound was prepared by reaction of 4-(4-methoxy-
phenyl)-2-(3-methoxyphenyl)-1,3-oxazole (19i) (100 mg, 0.36 mmol)
according to the procedure reported above. The product was puri-
fied by preparative TLC (hexane/ethyl acetate 5:5); yield: 82%
(74 mg); yellow powder; 1H NMR (CD3COCD3): 8.27 (s, 1H, H-oxa-
zole), 7.93 (d, J = 8.50 Hz, 2H), 7.37 (s, 1H), 7.33 (d, J = 7.60 Hz, 1H),
7.20 (t, J = 7.60 Hz, 1H), 6.97 (d, J = 8.50 Hz, 2H), 6.79 (m, 1H); 13C
NMR (CD3COCD3): 161.80, 159.70, 157.75, 141.55, 133.70,
132.90, 129.70, 128.05, 119.25, 116.70, 115.75, 114.90, 112.35;
IR: 3300, 1595, 1259, 804 cmꢁ1; MS (ESI): 252 (MꢁH)ꢁ; Anal. Calcd
C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.00; H, 4.48; N,
5.58.
The 17b-HSD2 inhibition assay was performed similarly to the
17b-HSD1 procedure. The microsomal fraction was incubated with
NAD+ [1500 lM], 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 cal-
culated after analysis of the resulting chromatograms according to
AUCðE1Þ
following equation: %conversion ¼ AUCðE1ÞþAUCðE2Þ ꢂ 100.
7.2.3. ER affinity
The binding affinity of select compounds to the ERa and ERb
was determined according to Zimmermann et al.76 Briefly,
0.25 pmol of ERa or ERb, respectively, was incubated with
[2,4,6,7-3H]-E2 (10 nM) and test compound for 1 h at room tem-
perature. The potential inhibitors were dissolved in DMSO (5% fi-
nal concentration). Non-specific-binding was performed with
diethylstilbestrol (10 lM). After incubation, ligand–receptor com-
plexes 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
(Quickszint 212, Zinsser Analytic, Frankfurt) was added and sam-
ples 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 deter-
7.1.11. 4,40-(1,3-Oxazol-2,5-diyl)diphenol (17)
The title compound was prepared by reaction of 2,5-bis(4-
methoxyphenyl)-oxazole (17i) (260 mg, 0.90 mmol, 1 equiv) and
pyridinium hydrochloride (2.90 g, 25.7 mmol, 37 equiv) heated to
220 °C for 18 h. After cooling to rt, water (10 mL) and ethyl acetate
(20 mL) were added. The aqueous layer was washed with ethyl ace-
tate. The combined organic layers were dried over sodium sulfate,
filtered, evaporated under reduced pressure and purified by pre-
parative TLC (hexane/ethyl acetate 5/5); yield: 82% (186 mg), yellow
powder; mp: 163 °C; 1H NMR (CD3OD): 7.89 (d, J = 7.80 Hz, 2H), 7.60
(d, J = 8.80 Hz, 2H), 7.32 (s, 1H), 6.91–6.86 (m, 4H); 13C NMR
(CD3OD): 162.35, 161.30, 159.35, 152.78, 132.80, 129.00 (2C),
126.80 (2C), 125.80 (2C), 116.90 (2C); IR: 3387, 1611, 1506, 1170,
834 cmꢁ1; MS (ESI): 254 (M+H)+; Anal. Calcd C15H11NO3: C, 71.14;
H, 4.38; N, 5.53. Found: C, 71.02; H, 4.18; N, 5.63.
mined. RBA values wereðEc2Þalculated according to the following
IC50
equation: RBA½%ꢃ ¼
ðcompoundÞ ꢂ 100. The RBA value for E2 was
IC50
arbitrarily set at 100%.
7.2.4. T-47D cell assays
7.2. Biological methods
7.2.4.1. Evaluation of the estrogenic activity. Phenol red-free
medium was supplemented with sodium bicarbonate (2 g/L),
streptomycin (100 lg/mL), insulin zinc salt (10 lg/mL), sodium
[2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were bought from Perkin El-
mer, Boston. Quickszint Flow 302 scintillator fluid was bought
from Zinsser Analytic, Frankfurt.
pyruvate (1 mM),
L-glutamine (2 mM), penicillin (100 U/mL) and
DCC-FCS 5% (vol/vol). RPMI 1640 (without phenol red) was used

6434
E. Bey et al. / Bioorg. Med. Chem. 16 (2008) 6423–6435
for the experiments. Cells (7500 cells/96-well plate) were grown
for 48 h in phenol red-free medium. Compound 18 was added at
a final concentration of 100 nM. Inhibitors and E2 were diluted in
ethanol (final ethanol concentration was adjusted to 1%). As a po-
sitive control E2 was added at a final concentration of 0.1 nM. Eth-
anol was used as negative control. Medium was changed every 2–3
days and supplemented with the respective additives. After 8 days
of incubation, the cell viability was evaluated measuring the
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra-
zoliumbromide (MTT). The cleavage of MTT to a blue formazan
by mitochondrial succinat-dehydrogenase was quantified
spectrophotometrically at 590 nm as described by Denizot and
Lang77 with minor modifications. The control proliferation
was arbitrarily set at 1 and the stimulation induced by the inhibi-
tor was calculated according to following equation:
%stimulation ¼ ½proliferationðcompound-inducedÞꢁ1ꢃ ꢂ 100%. Each value is
end groups were added. Close contacts were fixed (Arg37) and cor-
rect atom types were set.
Docking of inhibitors into the substrate-binding site was per-
formed by the automated docking program GOLD 3.0.79
Acknowledgments
We are grateful to the Deutsche Forschungsgemeinschaft
(HA1315/8-1) for financial support of this work. We thank Dr.
Christiane Scherer for performing the CaCo-2 cell test, Kerstin Esch,
Anja Palusczak and Beate Geiger for their help in performing the
in vitro tests (17b-HSD1, 17b-HSD2, ERa and ERb) and Matthias
Negri for discussions about molecular modelling. Patricia Kruchten
is grateful to the European Postgraduate School 532 (DFG) for a
scholarship.
calculated as
experiments.
a
m½proelaifenratiovnaðEl2u-ienducoefdÞꢁa1ꢃt least three independent
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culated using equation Papp ¼
, where dQ is the appearance rate
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