Bicyclic substituted hydroxyphenylmethanones as novel inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) for the treatment of estrogen-dependent diseases

Article abstract of DOI:10.1021/jm101073q

Estradiol (E2), the most important estrogen in humans, is involved in the initiation and progression of estrogen-dependent diseases such as breast cancer and endometriosis. Its local production in the target cell is regulated by 17β-hydroxysteroid dehydro

Full text of DOI:10.1021/jm101073q

8176 J. Med. Chem. 2010, 53, 8176–8186
DOI: 10.1021/jm101073q
Bicyclic Substituted Hydroxyphenylmethanones as Novel Inhibitors of 17β-Hydroxysteroid
Dehydrogenase Type 1 (17β-HSD1) for the Treatment of Estrogen-Dependent Diseases†
Alexander Oster, Stefan Hinsberger, Ruth Werth, Sandrine Marchais-Oberwinkler, Martin Frotscher, and Rolf W. Hartmann*
Pharmaceutical and Medicinal Chemistry, Saarland University, and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Campus C23, D-66123 Saarbru€cken, Germany
Received August 18, 2010
Estradiol (E2), the most important estrogen in humans, is involved in the initiation and progression of
estrogen-dependent diseases such as breast cancer and endometriosis. Its local production in the target cell
is regulated by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), which catalyzes E2-formation by
reduction of the weak estrogen estrone (E1). Because the enzyme is expressed in the diseased tissues, inhi-
bition of 17β-HSD1 is considered as a promising therapy for the treatment of estrogen-dependent diseases.
For the development of novel inhibitors, a structure- and ligand-based design strategy was applied, resulting
in bicyclic substituted hydroxyphenylmethanones. In vitro testing revealed high inhibitory potencies toward
human placental 17β-HSD1. Compounds were further evaluated with regard to selectivity (17β-HSD2,
estrogen receptors ERR and ERβ), intracellular activity (T47D cells), and metabolic stability. The most
promising compounds, 14 and 15, showedIC50 values in the low nanomolar range in the cell-free and cellular
assays (8-27 nM), more than 30-fold selectivity toward 17β-HSD2 and no affinity toward the ERs. The data
obtained make these inhibitors interesting candidates for further preclinical evaluation.
Introduction
Both therapeutic approaches show disadvantages by their
rather radical reduction of systemic estrogen concentration.
A softer therapy seems to be the inhibition of 17β-hydroxy-
steroid dehydrogenase type 1 (17β-HSD1). This enzyme reg-
ulates the ratio of the weak active estrogen E1 and its potent
metabolite E2 by catalyzing the reduction of E1 to E2 using
NAD(P)H as a cofactor (Chart 1).
Breast cancer and endometriosis are estrogen-dependent
diseases, whichshowa strong global prevalence. Breast cancer
is one of the two leading causes of cancer death in women.
Although some therapeutic approaches are already available,
the five-year survival rate for breast cancer patients only
remains around 80% (www.cancer.org). The Endometriosis
Foundation of America has pointed out that endometriosis
is one of the top three causes of female infertility (www.
endofound.org). However, until recently, there has been no
oral medication available to cure this disease.
Interestingly, mRNA of 17β-HSD1, which is also used as a
prognostic marker, is often overexpressed in breast cancer
tissues10,11 and endometriotic lesions.12 Considering the fact
that therapeutic approaches regulating intracellular hormone
concentrations have already been used successfully in andro-
gen dependent diseases such as benign prostatic hyperplasia
and alopecia,13-16 17β-HSD1 appeared to be a promising
target for the local reduction of E2 and accordingly for the
treatment of breast cancer and endometriosis. In contrast to
the applied therapies, a basal estrogenic activity will be
maintained because the E1 level should not be affected. Thus,
side effects caused by a total absence of estrogenic activity
could be avoided. The tissue specific influence of 17β-HSD1
inhibitors seems especially suitable for the therapy of post-
menopausal women, in which the local production (e.g., in
breast or endometrium) represents the major source of E2.
As it has recently been suggested, 17β-HSD1 is also involved
in the metabolism of retinoic acid,17,18 a natural compound
which is known to have antitumor activity.19 Thus, inhibition
of 17β-HSD1 should have an additional beneficial effect.
Efficacy of 17β-HSD1 inhibitors has been shown by several
groups in different animal models. Husen et al.20,21 and Day
et al.22 used xenograft models with MCF7 breast cancer cells
stably expressing recombinant human 17β-HSD1 or the breast
cancer cell line T47D, respectively, in order to demonstrate in
vivo efficacy of their inhibitors by decreasing tumor growth. In
The local production of estradiol (E2a) plays a pivotal role
for the initiation and development of both diseases.1,2 Thus, the
reduction of tissue levels of active estrogens by inhibiting their
synthetic pathway moves steadily to the forefront. Two ther-
apeutic approaches have mainly been investigated within the
past years. Aromatase inhibitors3-8 (to reduce the transforma-
tion of androgens into estrogens) and sulfatase inhibitors9 (to
reduce the conversion of estrone sulfate (E1S) into estrone
(E1)) intervene in the penultimate step of E2 biosynthesis.
†For the sake of clarity, IUPAC nomenclature is not strictly followed
except for the experimental part where the correct IUPAC names are given.
*To whom correspondence should be addressed. Phone: þ49 681 302
70300. Fax: þ49 681 302 70308. E-mail: rwh@mx.uni-saarland.de.
Website: http://www.pharmmedchem.de.
a Abbreviations: 17β-HSD1, 17β-hydroxysteroid dehydrogenase type
1; 17β-HSD2, 17β-hydroxysteroid dehydrogenase type 2; E1, estrone; E2,
17β-estradiol; E1S, estrone sulfate; ER, estrogen receptor; NADP(H),
nicotinamide adenine dinucleotide phosphate; NAD(H), nicotinamide
adenine dinucleotide; IBX, 2-iodoxybenzoic acid; SAR, structure-activity
relationship; RBA, relative binding affinity; SF, selectivity factor; HPLC,
high performance liquid chromatography; CC, column chromatography;
TLC, thin layer chromatography; IUPAC, international union of pure and
applied chemistry.
r
pubs.acs.org/jmc
Published on Web 10/26/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8177
transgenic mice, it was proven that 17β-HSD1 inhibitors are
able to reduce the conversion of E1 into E2.23 With regards to
endometriosis, two in vivo models have been established.24,25
Using nude mice implanted with endometrial tissue from
human donors, the expression of steroid hormone receptors
and steroid converting enzymes, as well as the proliferation
of the ectopic tissue, can be analyzed.24 In a second model,
endometriosis is induced in marmoset monkeys, either by
endometrial reflux or invasively by laparotomy.25 Despite
encouraging in vivo data, no 17β-HSD1 inhibitor has entered
clinical evaluation yet. Thus, the applied inhibitors do not seem
to be appropriate candidates for clinical application.
The therapeutic concept requires selectivity of potent in-
hibitors toward 17β-hydroxysteroid dehydrogenase type 2
(17β-HSD2) because it catalyzes the reverse reaction, the
oxidation of E2 into E1. Furthermore, to reduce the risk of
intrinsic estrogen effects, affinity toward both estrogen recep-
tors R and β (ERR and ERβ) should be avoided, even if the
pathophysiological role of ERβ is not completely clarified.
The selective inhibition of 17β-HSD1 was the goal of several
groups investigating inhibitors over the past years.18,26-30
Their drug design efforts were facilitated by the existence of
several X-ray structures of human 17β-HSD1 and will be
further alleviated by the recently published first insights in
17β-HSD1 enzyme kinetics and ligand binding by dynamic
motion investigations.31 Although running the higher risk of
unwanted side effects, most of these groups focused on the
development of compounds bearing a steroidal scaffold. Re-
garding nonsteroidal inhibitors, aside from phytoestrogens27,32
and thiophenepyrimidinones,33,34 there are two additional
compound classes showing inhibitory activity in the low nano-
molar range, namely the (hydroxyphenyl)naphthols35-39 and
the bis(hydroxyphenyl)heterocycles.40-45 Recently, we also
reported on heterocyclic substituted biphenylols,46 which dem-
onstrate notable inhibitory activity. Molecular modeling stud-
ies indicated that linear and angulate molecules out of this
class unfold their activity via interaction with the catalytic cen-
ter and a rather lipophilic subpocket located below the cata-
lytic site, respectively.46
crystal structure 1EQU (PDB-ID) indicated that their hetero-
cyclic part interacts with different areas of the active site.46
Designed using E1 as a template, compound A shows the same
interaction pattern as the natural substrate. In detail: the meta-
OH-phenyl ring imitates the steroidal A-ring and the methoxy-
pyridine interacts as hydrogen-bond acceptor with Ser142 and
Tyr155, which together with Asn114 and Lys159 constitute the
catalytic tetrade. While the OH-phenyl ring of B also mimics
the steroidal A-ring, its chlorothiophene part points in the
direction of a mainly apolar subpocket consisting of Leu95,
Leu96, Asn152, Tyr155 and Phe192.
Our strategy focuses on the design of inhibitors, which fit
into a three-point-pharmacophore model derived from the
different interactions found for A and B (Chart 3). A closer
analysis of the amino acids in these regions by computational
docking studies provides ideas for substituents as appropriate
interacting partners as well as for the structural architecture of
the compounds.
To mimic the interactions established by the steroidal A-ring
of the substrate with the amino acids His221 and Glu282, a
hydroxy-substituted phenyl moiety will be one important
component of our inhibitors. The second part should interact
with the catalytic tetrade. Inhibitors with two H-bond accep-
tor groups located in this region are described to inhibit 17β-
HSD1.46 One functional group with two hydrogen bond
acceptor properties could eventually be more appropriate to
target the corresponding amino acids. A keto-group, as in the
natural substrate E1, should be the functionality of choice.
Moreover, it disrupts the linearity present in A, thus allowing
for binding with a third interaction region, which is consti-
tuted by a mainly apolar subpocket located beneath the cata-
lytic center. In this subpocket, Asn152 is the only hydrophilic
amino acid. This amino acid was already exploited for active
compound design: recently, Mazumdar et al.47 reported on
the crystal structure of the steroidal inhibitor E2B (Chart 2),
whose meta-amidobenzyl substituent was found to interact
with Asn152. Consequently, a second OH-phenyl moiety was
Chart 3. Design Concept and Pharmacophore Model
In the following, we will report on the design, synthesis, and
biological evaluation of a novel class of 17β-HSD1 inhibitors,
namely bicyclic substituted hydroxyphenylmethanones.
Design
Molecular docking studies of the most potent heterocyclic
substituted biphenylol inhibitors A and B (Chart 2) using the
Chart 1. Interconversion of Estrone (E1) and Estradiol (E2)
Chart 2. Most Potent 17β-HSD1 Inhibitors of Heterocyclic Substituted Biphenylol Compound Class (Compounds A and B) and E2B

8178 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Oster et al.
introducedasa further component of the inhibitor structurein
order to fit in this apolar subpocket and interact with Asn152
(Chart 4).
benzoyl chlorides, resulting in compounds 1a-3a and 6a-10a.
Subsequently, treatment with boron tribromide led to the cor-
responding dihydroxylated final compounds 1-3 and 6-10.
The brominated key intermediate 4b was synthesized via
Friedel-Crafts acylation of 2-bromothiophene with 3-methoxy-
benzoyl chloride. Using 4b, Suzuki cross coupling reactions48
with the appropriate commercially available boronic acids were
carried out via two different methods (B for compounds
14a-16a and C41 for compounds 4a, 5a, 11a-13a, and 18a,
The design concept resulted in compounds 1-5, which were
selected as the starting point for this study. Thiophene was
chosen as the connecting group between the hydroxyphenyl
and the keto-group in order to fit in the rather lipophilic sub-
strate binding site (known as hydrophobic tunnel) and to fit
the distance between the various interacting partners in the
protein. After identifying 1 and 4 as most promising, small
substituents were introduced into these compounds (6-20).
Finally, the substitution pattern of the thiophene was changed
(21) and it was replaced by thiazole (22-24).
49
Scheme 1). The methoxy functions were cleaved with BBr3
yielding compounds 4, 5, 11-16, and 18.
Ether cleavage of the key intermediate 4b gave 17a in
quantitative yield. Cross coupling of 17a with the appropriate
hydroxylatedbenzeneboronicacidsresultedinthe dihydroxy-
lated compounds 17 and 19.
Chemistry
3-Ethyl-4-methoxybenzeneboronic acid was synthesized using
the method of Allan et al.50 and was used for Suzuki reaction in
order to yield the monomethoxy derivative 20a. The latter was
heated with pyridinium hydrochloride51 to yield compound 20.
The preparation of compounds 21-24 (Scheme 2) started
with a Grignard reaction using 3-methoxyphenylmagnesium
bromide and the appropriate brominated aromatic carbalde-
hydes (method F). The OH-groups of compounds 21c-23c
were subsequently oxidized to the keto-functions with 2-
iodoxybenzoic acid (IBX) (method G). Suzuki cross coupling
reactions using the 4-methoxy-3-methylbenzeneboronic acid
and the brominated methanones 21b-23b led to compounds
21a-23a. Demethylation with BBr3 (method D) yielded the
final compounds 21 and 23, whereas pyridinium hydrochlo-
ride (method E) was used for the preparation of 22.
For the preparation of 24, Suzuki coupling was performed
using the hydroxylated intermediate 24c because its oxidation
failed. Subsequently, 24b was oxidized by means of IBX in
anhydrous THF. Reaction of 24a with pyridinium hydro-
chloride led to 24.
The synthesis of the thiophene derivatives 1-20 is depicted
in Scheme 1. The first step in the preparation of compounds
1-3 and 6-10 was the coupling of 2-bromothiophene with
3-methoxy- and 4-methoxybenzeneboronic acid, respectively,
thus leading to intermediates 1b and 6b. A Friedel-Crafts
acylation (method A) was then performed with the appropriate
Chart 4. Schematic Representation of the Inhibitor Template
Binding to the Amino Acids of the Active Site (Possible Hydro-
gen Bond Interactions Are Depicted in Dashed Lines)
Biological Results
Activity: Inhibition of Human 17β-HSD1. Placental en-
zyme was isolated following a described procedure38 and
Scheme 1. Synthesis of Compounds 1-20a
a Reagents and conditions: (a) method A, AlCl3, anhydrous CH2Cl2, 0 °C, 0.5 h and then rt, 1 h, for compounds 1a-3a, 4b, 6a-10a; (b) method B,
Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, 2 h, for compounds 14a-16a, 17, 19, 20a; (c) method C, Cs2CO3, Pd(PPh3)4, DME/EtOH/water (1:1:1),
microwave conditions (150 W, 15 bar, 150 °C, 15 min), for compounds 4a, 5a, 11a-13a, 18a; (d) method D, BBr3, CH2Cl2, -78 °C, 20 h, for compounds 1-16,
17a, 18; (e) method E, pyridinium hydrochloride, 220 °C, 18 h, for compound 20; (f) Na2CO3, Pd(PPh3)4, THF/water (1:1), reflux, 18 h, for compound 1b.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8179
Scheme 2. Synthesis of Compounds 21-24a
a Reagents and conditions: (a) method F, anhydrous THF, 80 °C, 3 h, for compounds 21c-24c; (b) method G, 2-iodoxybenzoic acid, anhydrous
THF, 0 °C, 10 min and 60 °C, 18 h, for compounds 21b-23b, 24a; (c) method B, Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, 2 h, for compounds
21a-23a; (d) method D, BBr3, CH2Cl2, -78 °C, 20 h, for compounds 21, 23; (e) method E, pyridinium hydrochloride, 220 °C, 18 h, for compounds 22,
24; (f) Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, 4 h, for compound 24b
incubated with tritiated E1, cofactor, and inhibitor. After
HPLC separation of substrate and product, the amount of
labeled E2 formed was quantified. The inhibition values of
compounds 1-24 are presented in Tables 1 and 2.
Compounds 1-5 with their keto and two OH functions
were designed to interact with three distinct areas of the active
site. Among these inhibitors, the meta-meta substituted 1
showed the best IC50 value (22 nM), demonstrating that in this
class of compounds, the meta-OH substituted phenyl moiety
is also very relevant for 17β-HSD1 inhibition as observed with
the bis(hydroxyphenyl)heterocycles.41 Displacement of the
hydroxy group on the benzoyl ring led to a decrease in activity
(2 and 3). The position of the OH group at the phenyl ring has
replacement of the CH3 for a hydroxy (18: IC50 = 86 nM) or a
methoxy (19: IC50 = 108 nM). Interestingly, the ethyl deriv-
ative 20 once again revealed a high biological potency (IC50 =
20 nM), thus indicating that an oxygen in this position impinges
on enzyme affinity.
Originally chosen as a suitable lipophilic component to
fit the mainly apolar substrate binding site, we replaced the
2,5-disubstituted thiophene with 2,4-disubstituted thiophene
and thiazole (21-24) in order to investigate the relevance of
the middle ring on inhibitory activity. In the structurally re-
lated class of bis(hydroxyphenyl)heterocycles, a strong cor-
relation between the nature of the heterocycle and the affinity
toward 17β-HSD1 was already shown.41 As 14 is one of the
most active inhibitorsinthisseries, itssubstitutionpattern was
maintained for this study. The biological results of com-
pounds 21-24 are shown in Table 2.
With regard to 2,4-disubstituted 21, the decrease of activ-
ity (IC50=199 nM) underlines the importance of the sulfur
position. Exchanging thiophene with thiazole led to com-
pounds 22-24. Compound 22, which bears the sulfur of the
thiazole in the same position as 14, the thiophene-sulfur, still
demonstrated a very good activity (IC50=35 nM). Interest-
ingly, the potency of 23 and 24 strongly decreased, thereby
confirming that the position of the sulfur plays a pivotal role
for 17β-HSD1 inhibition.
Selectivity: Inhibition of Human 17β-HSD2 and Affinities
to ERr and ERβ. Inhibition of 17β-HSD type 2 must be
avoided, as it acts as a biological counterpart by catalyz-
ing the reverse reaction: oxidation of E2 to E1. Compared
with the type 1 enzyme, inhibition of 17β-HSD2 was deter-
mined in a similar assay using placental microsomes. These
were incubated with tritiated E2 in the presence of NADþ
and the inhibitor. The separation and quantification of the
labeled product was performed by HPLC using radiodetec-
tion. IC50 values and the percentage of inhibition, respec-
tively, are shown in Tables 1 and 2. In vitro selectivity toward
17β-HSD2 is also expressed as selectivity factor (SF) describ-
ing the ratio of the concentration required to inhibit the
activity of the isoenzymes by 50% (IC50 17β-HSD2/IC50
17β-HSD1).
a smaller impact on inhibitory activity: para-OH 4 (IC50
=
33 nM) was equipotent to meta-OH 1, the potency of ortho-OH 5
was only slightly reduced (IC50=95 nM). The best substitu-
tion patterns identified in 1 (meta-meta) and 4 (meta-para)
were sustained in the design of the further compounds. To
examine the influence of additional small substituents in para-
position of the benzoyl group, compounds 6-10 were synthe-
sized. Introduction of a methyl substituent decreased the inhib-
itory activity in both series strongly (6 and 8). An exchange of
methyl by fluorine did not affect inhibition (7 and 9).
Introduction of methyl in para-position of the phenyl ring
led to a 10-fold decrease in activity (11), whereas meta-CH3,
para-OH 14 turned out to be a very potent compound (IC50 =
8 nM). Fluorinated compounds with different substitution
patterns showed a similarly high inhibition of 17β-HSD1
(IC50s: 12, 30 nM; 13, 6 nM; 15, 19 nM). It is remarkable that
the substitution with fluorine, in contrast to methyl, does
not decrease the high inhibitory potency of the parent com-
pounds 1 and 4, regardless of their position at the benzoyl
or phenyl ring. To provide more insight into the role of differ-
ent substituents, 14 and 15 were used as starting points for
further structural modifications. To combine the character-
istics of methyl and fluorine, a CF3 group (16), as well as
chlorine (17) as its bioisosteric equivalent, was introduced.
Both inhibitors confirmed the expected high inhibitory activ-
ity with IC50 values of 14 and 5 nM, respectively. In contrast to
this finding, a slight decrease in activity was caused by the

8180 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Oster et al.
Table 1. Inhibition of Human 17β-HSD1 and 17β-HSD2 by Compounds 1-20
IC50 [nM]a
compd
R1
R2
17β-HSD1b
17β-HSD2c
SFd
1
2
3
4
5
3-OH
4-OH
2-OH
3-OH
3-OH
3-OH
3-OH
3-OH
4-OH
2-OH
22
368
945
33
109
376
567
478
18
5
1
0.6
14
0.2
95
1
22
64%e
18
109
31%e
49
5
8
CH3
F
9
3
10
11
12
13
NO2
594
207
30
240
465
57
0.4
2
4-CH3
4-F
2-F
2
6
19
3
4
33
52%e
21
478
13%e
69
14
6
CH3
F
7
3
48
31
6
14
15
16
17
18
19
20
CH3
F
8
19
382
588
95
CF3
Cl
16
5
86
246
590
793
786
49
7
OH
OCH3
CH2CH3
108
20
7
39
a Mean value of three determinations, standard deviation less than 23%. b Human placenta, cytosolic fraction, substrate E1, 500 nM, cofactor
NADH, 500 μM. c Human placenta, microsomal fraction, substrate E2, 500 nM, cofactor NADþ, 1500 μM. d Selectivity factor: IC50 (17β-HSD2)/IC50
(17β-HSD1). e Inhibition at 1 μM (inhibitor concentration).
Table 2. Inhibition of Human 17β-HSD1 and 17β-HSD2 by Com-
pounds 14, 21-24
turned out to be potent inhibitors of 17β-HSD2 (SF = 0.6
and SF=0.2, respectively). Introduction of further substit-
uents on the benzoyl ring did not strongly influence the SF,
with the exception of compound 10, whose nitro group is also
responsible for the reverse selectivity in favor of 17β-HSD2
(SF=0.4). Concerning the different phenyl ring modified
compounds, insertion of CH3 (14, SF=48) and F (15, SF =31)
in the meta position (ortho to OH) increased selectivity com-
pared to the unsubstituted compound 4 (SF = 14). While
other positions had less of a high impact (11-13), this meta
position seems to be a key positionfor 17β-HSD1activity and
selectivity toward 17β-HSD2. Chlorine (17) and ethyl (20)
showed comparable high selectivities (SFs=49 for 17 and 39
for 20). The fact that chlorine and trifluoromethyl substitu-
ents have comparable electronic influences on a phenyl ring
implies that other parameters are responsible for the differ-
ence in selectivity between 17β-HSD1 and 2.
Regarding the influence of the different middle rings on
selectivity toward 17β-HSD2, it is striking that when com-
pared with 14, the isomeric thiophene 21 showed a strong
reduction of selectivity. Only thiazole 22 showed a compara-
tively high SF of 14. Surprisingly, thiazoles 23 and 24 turned
out to be more suitable inhibitors for 17β-HSD2, thereby
demonstrating that the nature of the middle ring has a major
impact on activity and selectivity.
a Mean value of three determinations, standard deviation less than
14%. b Human placenta, cytosolic fraction, substrate E1, 500 nM,
cofactor NADH, 500 μM. c Human placenta, microsomal fraction,
substrate E2, 500 nM, cofactor NADþ, 1500 μM. d selectivity factor:
IC50 (17β-HSD2)/IC50 (17β-HSD1). e Inhibition at 1 μM (inhibitor
concentration).
Regarding compounds 1-5, it became apparent that just
the modification of the OH-substitution pattern influences
the selectivity toward the type 2 enzyme. While compound 4
showed the best SF (14) out of these five, compounds 3 and 5,
which bear one of their OH functions in the ortho position,
To keep the risk of unwanted side effects as low as possible,
intrinsic estrogenic effects should be avoided. Therefore, it is a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8181
Table 3. Inhibition of 17β-HSD1 in Cell-Free and Cellular Assay by Selected Compounds
a Mean values of three determinations, standard deviation less than 23%. b Human placental, cytosolic fraction, substrate E1, 500 nM, cofactor
NADH, 500 μM. c T47D cells, substrate E1, 50 nM.
prerequisite for 17β-HSD1 inhibitors to show no or low affinity
for the ERs. Relative binding affinities (RBA) of compounds,
which showed a selectivity factor higher than 10, were deter-
mined using recombinant human protein in a competitive
binding assay applying tritium labeled E2 and hydroxyapatite.
Because RBA of E2 is equated with 100%, compounds showing
a RBA value below 0.1% are considered to have neglectable
receptor affinity. Within this study, none of the evaluated inhib-
itors crossed this value, neither for ERR nor ERβ.
Further Biological Evaluations. Besides the cell-free assay,
T47D, a breast cancer cell line reflecting the conditions in many
breast tumors by expressing subtypes 1 and 2 of 17β-HSD, was
used to determine the intracellular potency of compounds hav-
ing a SF higher than 10 (Table 3). While inhibitors 4, 14, and 15
confirmed their strong activity in T47D-cells with IC50 values in
the low nanomolar range (between 17 and 30 nM), compounds
17 and 22 showed inhibitory activities in the 100 nM range.
Compared to the cell-free assay, only chloro-(17) and ethyl-
derivative (20) demonstrated a remarkable reduction of in-
hibitory activity in T47D cells.
Additionally, metabolic stability of compounds 14, 17,
and 22 was evaluated using human liver microsomes. The
corresponding data of the selected compounds and the ref-
erence compounds dextromethorphan and verapamil are pre-
sented in Table 4. All three inhibitors can be classified in the
medium clearance category according to their half-life times
around 100 min and thus are considered to have a reasonable
metabolic stability. None of the small variations among these
three inhibitors caused considerable differences in liability of
the compounds to metabolic degradation.
Discussion and Conclusions
The results of molecular modeling studies in the class of
heterocyclic substituted biphenylols46 provided the basis for a
successful design strategy using a new three-point-pharmaco-
phore model. The determined biological results place emphasis
on the validity of this structure- and ligand-based design
approach, resulting in compound 1 as a new lead for further
optimization. Interestingly, the position of a hydroxy function
at the benzoyl ring has more influence on inhibitory activity
than the one at the phenyl ring. This suggests that either the
expected hydrogen-bond interactions of the latter with His221
and Glu282 are less important for 17β-HSD1 inhibition or
that the protein is flexible enough, at least in this part of the
enzyme, to allow equal interaction with each derivative 1, 4,
and 5. In contrast to previous studies,43 an exchange of this
OH-phenyl moiety might also lead to highly active 17β-HSD1
inhibitors.
The introduction of further substituents, such as methyl and
fluorine in different positions on the benzoyl and phenyl ring,
resulted in interesting modulations of biological activities.
Compounds 6 and 8, bearing the methyl substituent in the para
position of the benzoyl ring, showed a strong decrease of inhib-
itory activity, once again emphasizing the sharp structure-
activity relationships within the rather apolar subpocket. There
is plausibly a sterical hindrance caused by the CH3 group, thus
leading to a shifted position of the molecule that is unable to
establish suitable hydrogen bond interactions. Taking into
account that the geometrical radius of fluorine is smaller
than methyl, the fluorine substituted hydroxyphenyl-part of

8182 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Oster et al.
Table 4. Metabolic Stability of Compounds 14, 17, and 22 and Ref-
erence Compounds Using Human Liver Microsomes
of 17β-HSD2 available, these results cannot be interpreted on
a sound basis.
The most important advantage of this compound class is its
high cellular inhibitory activity. While most of the described
intracellularly active 17β-HSD1 inhibitors were tested using
low substrate concentrations (2-30 nM),33,50 the new com-
pound class revealed IC50 values in the low nanomolar range
in spite of using a much higher substrate concentration of
50 nM. Evaluated in our assay, the most potent inhibitors of
the bis(hydroxyphenyl)heterocycles showed cellular inhibi-
tory activities around 300 nM (IC50).43 Therefore, the keto
function seems to have a positive influence on cell permeation,
protein binding, and/or intracellular metabolism. Only com-
pounds 17 and 20 showed intracellularly a reduction of inhib-
itory activity compared to the cell-free assay. Increased lipo-
philicity might be responsible for this finding.
Concerning the metabolic stability study, the tested inhibi-
tors 14, 17, and 22 revealed a medium intrinsic clearance
confirming a reasonable metabolic profile. The fact that all
three inhibitors showed comparable profiles indicates that
neither the exchange thiophene-thiazole nor a variation of the
additional substituent next to the hydroxy function can give
rise to some significant metabolic liability.
In this paper, we described the structure-based design ap-
proach, synthesis, and biological evaluations of bicyclic sub-
stituted hydroxyphenylmethanones as new highly potent and
selective inhibitors of 17β-HSD1. The applied design concept is
based on the newly built pharmacophore model, which resulted
in compound 1 as the starting point of the study. The position
of the hydroxy functions, the influence of further small sub-
stituents and an alternative central ring were investigated.
Structural optimization of this lead led to the development of
14, 15, 17, and 22, all of which show inhibitory activities in the
very low nanomolar range in cell-free and cellular assays,
remarkable selectivity toward 17β-HSD2, no affinity on both
ERs, and very good metabolic stability (proven in case of 14, 17,
and 22) tested on human liver microsomes. Hence, they repre-
sent promising 17β-HSD1 inhibitors, which fulfill all prerequi-
sites for consideration as clinical candidates. Nevertheless,
further optimizations appear possible, especially with regards
to the phenyl ring and will therefore be investigated in a future
study.
compd
dextrometorphana
CLint [μL/min/mg protein]
t1/2 [min]
10.7
105
129
13.2
100
108
81.4
verapamila
14a
13.8
12.9
17.0
17b
22a
a Test concentration = 3 μM. b Test concentration = 0.5 μM.
compounds 7 and 9 obviously still fits well into the subpocket,
as can be seen by their high inhibitory activities.
Regarding the hydroxyphenyl, the introduction of methyl
and fluorine in ortho- to the para-OH substituent (14 and 15)
improved inhibitory activity and even selectivity toward 17β-
HSD2. It is striking that derivatives with more polar substit-
uents (OH in compound 18 and OCH3 in compound 19) in
this position could not exert such a high inhibitory activity,
and especially their selectivity factor decreased. The fact that
the Cl and CH2CH3 compounds 17 and 20 have similar inhib-
itory properties comparable to those of 14 and 15, led to the
assumption that the amino acids surrounding this region of the
17β-HSD1 binding site are predominantly lipophilic. Only
compound 16, which bears a CF3 group, demonstrated a low
selectivity factor because of its strong inhibition of 17β-HSD2.
The ethyl derivative 20, which has a marginally lower SF than
14, revealed a slightly reduced 17β-HSD2 inhibition, indicat-
ing that an enlargement of this lipophilic substituent might
lead to lower inhibition of the type 2 enzyme.
Concerning selectivity toward ERR and ERβ, each inhibi-
tor out of this new compound class showed neglectable affi-
nity to the ERs, which confirms our strategy of developing
nonsteroidal inhibitors. Despite the fact that the keto function
and one hydroxy group of the bicyclic substituted hydroxy-
phenylmethanones have to be considered as mimicks of the
oxygen functions of E1, obviously, the absence of the sterane
core and the additional phenolic ring are the main responsible
factors that we succeeded in obtaining compounds without
ER affinity.
While the presence of the keto linker is the only structural
difference between this new class of 17β-HSD1 inhibitors and
the previously described bis(hydroxyphenyl)heterocycles,40-43
several structure-activity relationships are in accordance.
For example, the influence of the (hetero)aromatic middle
ring is comparable. The exchange of the 2,5-disubstituted
thiophene with 2,4-disubstituted thiophene 21 or different
thiazoles 22-24 revealed that the position of the sulfur plays a
critical role for enzyme inhibition. However, only compounds
in which the sulfur is located between the two substituents show
inhibition values in the low nanomolar range. Because it was
shown that 2,4-thiophenes are also able to exhibit strong 17β-
HSD1 inhibition,41 the molecular electrostatic potential of the
inhibitors can be discussed as a possible reason for the differ-
ences in inhibitory activity.41 With regards to thiazoles, the
corresponding compounds in the class of bis(hydroxyphenyl)-
heterocycles also revealed similar results. In both studies,
there is a rather radical loss of inhibitory activity for all
thiazole derivatives with nitrogen located between the two
substituents, thus pointing out that this substitution pattern
of thiazoles is detrimental for 17β-HSD1 inhibition. Interest-
ingly, while compound 22 resembles the best inhibitor 14 in
activityand selectivity, compounds 23 and 24 inhibited signifi-
cantly stronger 17β-HSD2 than the type 1 enzyme. As there is
neither an X-ray structure nor an applicable homology model
Experimental Section
Chemical Methods. Chemical names follow IUPAC nomen-
clature. Starting materials were purchased from Aldrich, Acros,
Lancaster, Maybridge, Combi Blocks, Merck, or Fluka and
were used without purification.
Column chromatography (CC) was performed on silica gel
(70-200 μm), 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).
All microwave irradiation experiments were carried out in a
CEM-Discover monomode microwave apparatus.
1H NMR and 13C NMR spectra were measured on a Bruker
AM500 spectrometer (500 MHz) at 300 K. Chemical shifts are
reported in δ (parts per million, ppm), by reference to the hydro-
genated residues of deuteriated solvent as internal standard
(CDCl3, δ=7.24 ppm (1H NMR) and δ=77 ppm (13C NMR);
CD3OD, δ=5.84ppm(1HNMR) andδ= 49.3 ppm (13CNMR);
CD3COCD3, δ = 2.05 ppm (1H NMR) and δ = 30.8 ppm
(13C NMR); CD3SOCD3, δ = 2.50 ppm (1H NMR) and δ =
39.5 ppm (13C NMR)). Signals are described as s, d, t, dd, ddd, m,
dt, td, and q for singlet, doublet, triplet, doublet of doublets, doublet

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8183
of doublets of doublets, multiplet, doublet of triplets, triplet of
doublets, and quadruplet, respectively. All coupling constants
(J) are given in hertz (Hz).
Mass spectra (ESI) were recorded on a TSQ Quantum (Thermo
Finnigan) instrument.
Tested compounds have >95% chemical purity as measured
by HPLC. The methods for HPLC analysis and a table of data
for all tested compounds are provided in the Supporting In-
formation.
brine, dried over magnesium sulfate, filtered, and concentrated
to dryness. The product was purified by CC.
General Procedure for Oxidation. Method G. A mixture of
aliphatic alcohol derivative (1 equiv) and 2-iodoxybenzoic acid
(2 equiv) in anhydrous THF was stirred at 0 °C. After 10 min, the
reaction mixture was stirred and heated to 60 °C for 18 h. After
cooling to room temperature, saturated sodium thiosulfate solu-
tion was added to quench the reaction and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were
washed succesively with 1 M NaOH and brine, dried over mag-
nesium sulfate, filtered, and concentrated to dryness. The prod-
uct was purified by CC.
General Procedure for Purification Using Preparative HPLC.
All declared compounds were purified via an Agilent Technol-
ogies series 1200-preparative HPLC using a RP C18 Nucleodur
100-5 column (30 mm ꢀ 100 mm/50 μM from Macherey Nagel
GmbH) as stationary phase with a linear gradient run (solvents:
acetonitrile, water) starting from 20% acetonitrile up to 100% in
36 min.
(3-Hydroxyphenyl)[5-(3-hydroxyphenyl)-2-thienyl]methanone
(1). The title compound was prepared by reaction of (3-methoxy-
phenyl)[5-(3-methoxyphenyl)-2-thienyl]methanone (1a) (138 mg,
0.43 mmol) and boron tribromide (2.58 mmol) according to
method D. The product was purified by CC (hexane/ethyl acetate
8:2) followed by preparative HPLC; yield: 75% (95 mg). 1H
NMR (CD3OD): 7.64 (d, J = 4.1 Hz, 1H), 7.42 (d, J = 4.1 Hz,
1H), 7.33 (t, J = 7.9 Hz, 1H), 7.27 (dt, J = 1.3 Hz andJ = 7.6 Hz,
1H), 7.24-7.17 (m, 3H), 7.12 (t, J = 2.1 Hz, 1H), 7.03 (ddd, J =
0.9 Hz and J = 2.5 Hz and J = 7.9 Hz, 1H), 6.80 (ddd, J = 0.9 Hz
and J = 2.5 Hz and J = 7.9 Hz, 1H). 13C NMR (CD3OD):
189.90, 159.40, 159.00, 155.05, 140.60, 139.10, 137.90, 131.45,
130.80, 125.40, 121.30, 120.70, 118.60, 117.50, 116.55, 114.00.
(3-Hydroxyphenyl)[5-(4-hydroxyphenyl)-2-thienyl]methanone
(4). The title compound was prepared by reaction of (3-metho-
xyphenyl)[5-(4-methoxyphenyl)-2-thienyl]methanone (4a) (178 mg,
0.55 mmol) and boron tribromide (3.30 mmol) according
to method D. The product was purified by CC (hexane/ethyl
acetate 8:2) followed by preparative TLC (hexane/ethyl acetate
1:1); yield: 51% (83 mg). 1H NMR (CD3COCD3): 8.79 (s, 1H),
8.72 (s, 1H), 7.67-7.64 (m, 3H), 7.42 (d, J = 4.1 Hz, 1H), 7.37
(t, J = 7.9 Hz, 1H), 7.35 (td, J = 1.3 Hz and J = 7.6 Hz,
1H), 7.33-7.32 (m, 1H), 7.11 (ddd, J = 0.9 Hz and J = 2.5 Hz
and J = 7.9 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H). 13C NMR
(CD3COCD3): 188.60, 160.60, 159.35, 155.20, 142.80, 141.50,
138.05, 131.50, 129.60, 126.80, 124.70, 122.00, 121.05, 117.95,
117.25.
The following compounds were prepared according to previously
described procedures: 4-fluoro-3-methoxybenzoyl chloride,52
4-nitro-3-methoxybenzoyl chloride,52 3-ethyl-4-methoxyben-
zeneboronic acid (20b),50 2-(4-methoxyphenyl)thiophene (6b).53
General Procedure for Friedel-Crafts Acylation. Method A.
A mixture of monosubstituted thiophene derivative (1 equiv),
arylcarbonyl chloride (1 equiv), and aluminumtrichloride (1 equiv)
in anhydrous dichloromethane was stirred at 0 °C for 0.5 h. The
reaction mixturewas warmedtoroom temperatureandstirredfor
1 h. 1 M HCl was used to quench the reaction. The aqueous layer
was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered,
and concentrated to dryness. The product was purified by CC.
General Procedures for Suzuki Coupling. Method B. A mix-
ture of arylbromide (1 equiv), boronic acid derivative (1.2 equiv),
cesium carbonate (4 equiv), and tetrakis(triphenylphosphine)
palladium (0.01 equiv) in an oxygen free DME/water (1:1) solu-
tion was refluxed under nitrogen atmosphere for 2 h. The reac-
tion mixture was cooled to room temperature. The aqueous layer
was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered,
and concentrated to dryness. The product was purified by CC
(except for 17 (CC followed by preparative HPLC) and 19 (CC
followed by preparative TLC)).
Method C. A mixture of arylbromide (1 equiv), boronic acid
derivative (1.2 equiv), cesium carbonate (4 equiv), and tetrakis-
(triphenylphosphine) palladium (0.01 equiv) was suspended in
an oxygen free DME/EtOH/water (1:1:1) solution. The reaction
mixture was exposed to microwave irradiation (15 min, 150 W,
150 °C, 15 bar). After reaching room temperature, water was
added and the aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
magnesium sulfate, filtered, and concentrated to dryness. The
product was purified by CC.
General Procedures for Ether Cleavage. 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 eq per methoxy function)
was added dropwise. The reaction mixture was stirred for 20 h at
room temperature under nitrogen atmosphere. 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, filtered, and concentrated to dryness.
The product was purified by CC, CC followed by preparative TLC,
or CC followed by preparative HPLC, respectively.
Method E. A mixture of methoxybenzene derivative (1 equiv)
and pyridinium hydrochloride (37 equiv per methoxy function)
was heated to 220 °C for 18 h. After cooling to room tempera-
ture, water, 1 M HCl and ethyl acetate were added. The aqueous
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 followed by preparative TLC or preparative HPLC,
respectively.
[5-(4-Hydroxy-3-methylphenyl)-2-thienyl](3-hydroxyphenyl)-
methanone (14). The title compound was prepared by reaction
of [5-(4-methoxy-3-methylphenyl)-2-thienyl](3-methoxyphenyl)-
methanone (14a) (180 mg, 0.53 mmol) and boron tribromide
(3.18 mmol) according to method D. The product was purified by
1
CC (hexane/ethyl acetate 7:3); yield: 90% (148 mg). H NMR
(CD3COCD3): 8.68 (s, 1H), 8.66 (s, 1H), 7.65 (d, J = 4.1 Hz, 1H),
7.57-7.56 (m, 1H), 7.47 (dd, J = 2.2 Hz andJ = 8.2 Hz, 1H), 7.41
(d, J = 4.1 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.35 (dt, J = 1.3 Hz
and J = 7.6 Hz, 1H), 7.32-7.31 (m, 1H), 7.11 (ddd, J = 1.3 Hz
and J= 2.5Hz andJ = 7.9 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 2.27
(s, 3H). 13C NMR (CD3COCD3): 188.55, 159.35, 158.65, 155.50,
142.65, 141.60, 138.05, 131.50, 130.70, 127.25, 126.90, 126.75,
124.55, 122.00, 121.00, 117.25, 117.20, 17.10.
[5-(3-Fluoro-4-hydroxyphenyl)-2-thienyl](3-hydroxyphenyl)-
methanone (15). The title compound was prepared by reaction
of [5-(3-fluoro-4-methoxyphenyl)-2-thienyl](3-methoxyphenyl)-
methanone (15a) (200 mg, 0.58 mmol) and boron tribromide
(3.48 mmol) according to method D. The product was purified by
CC (hexane/ethyl acetate 7:3) followed by preparative TLC (hexane/
ethyl acetate 6:4); yield: 90% (164 mg). 1H NMR (CD3COCD3):
9.09 (s, 1H), 8.74 (s, 1H), 7.66 (d, J = 4.1 Hz, 1H), 7.56 (dd, J =
2.2 Hz and J = 12.3 Hz, 1H), 7.47-7.44 (m, 2H), 7.39 (t, J = 7.8
Hz, 1H), 7.35 (dt, J = 1.5 Hz and J = 7.6 Hz, 1H), 7.34-7.32
General Procedure for Grignard Reaction. Method F. To a
solution of carbaldehyde derivative (1 equiv) in anhydrous THF,
3-methoxyphenylmagnesium bromide (1.0 M in THF/toluene,
2.2 equiv) was added dropwise. The reaction mixture was stirred
for 3 h at 80 °C under nitrogen atmosphere. Brine was added to
quench the reaction, and the aqueous layer was extracted with
ethyl acetate. The combined organic layers were washed with

8184 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Oster et al.
(m, 1H), 7.13 (ddd, J = 1.3 Hz and J = 2.5 Hz and J = 7.8 Hz,
1H), 7.09 (t, J = 8.8 Hz, 1H). 13C NMR (CD3COCD3): 188.65,
159.35, 154.45, 153.45, 147.80, 143.50, 141.30, 137.95, 131.55,
127.50, 125.65, 124.70, 122.05, 121.15, 120.35, 117.25, 115.60.
[5-(3-Chloro-4-hydroxyphenyl)-2-thienyl](3-hydroxyphenyl)-
methanone (17). The title compound was prepared by reaction
of (5-bromo-2-thienyl)(3-hydroxyphenyl)methanone (17a) (150 mg,
0.53 mmol), 3-chloro-4-hydroxybenzene boronic acid (110 mg,
0.64 mmol), cesium carbonate (691 mg, 2.12 mmol), and tetrakis-
(triphenylphosphine) palladium (6 mg, 5 μmol) according to
method B. The product was purified by CC (hexane/ethyl acetate
8:2) followed by preparative HPLC; yield: 42% (73 mg). 1H
NMR (CD3COCD3): 7.79 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 4.1
Hz, 1H), 7.60 (dd, J = 2.2 Hz and J = 8.5 Hz, 1H), 7.51 (d, J =
4.1 Hz, 1H), 7.39 (td, J = 0.6 Hz and J = 7.8 Hz, 1H), 7.35 (dt,
J = 1.5 Hz and J = 7.8 Hz, 1H), 7.33-7.32 (m, 1H), 7.13-7.11
(m, 2H). 13C NMR(CD3COCD3): 188.60, 159.40, 156.05, 153.20,
143.55, 141.35, 137.95, 131.55, 129.40, 128.10, 128.00, 125.65,
123.10, 122.00, 121.15, 119.30, 117.25.
[5-(3-Ethyl-4-hydroxyphenyl)-2-thienyl](3-hydroxyphenyl)-
methanone (20). The title compound was prepared by reaction of
[5-(3-ethyl-4-methoxyphenyl)-2-thienyl](3-hydroxyphenyl)metha-
none (20a) (116 mg, 0.34 mmol) and pyridinium hydrochloride
(1.45 g, 12.58 mmol) according to method E. The product was
purified by CC (hexane/ethyl acetate 6:4) followed by prepara-
tive TLC (hexane/ethyl acetate 6:4); yield: 47% (52 mg). 1H
NMR (CD3COCD3): 8.72 (s, 1H), 8.68 (s, 1H), 7.66 (d, J=4.1
Hz, 1H), 7.57 (d, J =2.5 Hz, 1H), 7.47 (d, J =2.5 Hz and J= 8.2
Hz, 1H), 7.42 (d, J =4.1 Hz, 1H), 7.39 (t, J =7.9 Hz, 1H), 7.34
(dt, J =1.5 Hz and J =7.6 Hz, 1H), 7.32-7.31 (m, 1H), 7.11
(ddd, J =1.3 Hz and J =2.5 Hz and J =7.9 Hz, 1H), 6.93 (d,
J =8.2 Hz, 1H), 2.70 (q, J =7.6 Hz, 2H), 1.24 (t, J =7.6 Hz,
3H). 13C NMR (CD3COCD3): 187.60, 158.35, 157.30, 154.65,
149.55, 141.65, 140.60, 137.05, 132.35, 130.50, 128.25, 125.90,
123.60, 121.00, 120.00, 116.50, 116.25, 23.90, 14.50.
E1 and E2 were separated using acetonitrile/water (45:55) as
mobile phase in a C18 reverse phase chromatography column
€
(Nucleodur C18 Gravity, 3 μm, Macherey-Nagel, Duren) con-
nected to a HPLC-system (Agilent 1100 series, Agilent Tech-
nologies, Waldbronn). Detection and quantification of the
steroids were performed using a radioflow detector (Berthold
Technologies, Bad Wildbad). The conversion rate was calcu-
lated after analysis of the resulting chromatograms according
to the following equation:
%E2
%conversion ¼
ꢀ 100
%E2 þ %E1
Each value was calculated from at least three independent
experiments.
2. Inhibition of 17β-HSD2. The 17β-HSD2 inhibition assay
was performed similarly to the 17β-HSD1 procedure. The
microsomal fraction was incubated with NADþ [1500 μM], test
compound, and a mixture of unlabeled- and [2,4,6,7-3H]-E2
(final concentration: 500 nM, 0.11 μCi) 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
resulting chromatograms according to the following equation:
%E1
%conversion ¼
ꢀ 100
%E1 þ %E2
3. ER Affinity. The binding affinity of selected compounds to
the ERR and ERβ was determined according to Zimmermann
et al.54 Briefly, 0.25 pmol of ERR or ERβ, respectively, were
incubated with [2,4,6,7-3H]-E2 (10 nM) and test compound
for 1 h at room temperature. The potential inhibitors were
dissolved in DMSO (5% final concentration). Evaluation of
nonspecific-binding was performed with diethylstilbestrol
(10 μM). After incubation, ligand-receptor complexes were
selectively bound to hydroxyapatite (5 g/60 mL TE-buffer). The
complex formed was separated, washed, and resuspended in
ethanol. For radiodetection, scintillator cocktail (Quickszint
212, Zinsser Analytic, Frankfurt) was added and samples were
measured in a liquid scintillation counter (Rack Beta Primo
1209, Wallac, Turku, Finland). For determination of the rela-
tive binding affinity (RBA), inhibitor and E2 concentra-
tions required to displace 50% of the receptor bound labeled
E2 were determined. RBA values were calculated according to
the following equation:
[2-(4-Hydroxy-3-methylphenyl)-1,3-thiazol-5-yl](3-hydroxy-
phenyl)methanone (22). The title compound was prepared by reac-
tion of [2-(4-methoxy-3-methylphenyl)-1,3-thiazol-5-yl](3-meth-
oxyphenyl)methanone (22a) (65 mg, 0.19 mmol) and pyridinium
hydrochloride (1.63 g, 14.06 mmol) according to method E. The
product was purified by CC (hexane/ethyl acetate 8:2) followed by
preparative TLC (hexane/ethyl acetate 6:4); yield: 46% (27 mg).
1H NMR (CD3COCD3): 8.26 (s, 1H), 7.87-7.86 (m, 1H), 7.78
(dd, J = 2.2 Hz and J = 8.5 Hz, 1H), 7.43-7.40 (m, 2H), 7.38-
7.37 (m, 1H), 7.16-7.14 (m, 1H), 6.97 (d, J = 8.2 Hz, 1H), 2.29
(s, 3H). 13C NMR (CD3COCD3): 188.25, 176.30, 161.20, 159.65,
151.65, 141.25, 131.75, 131.60, 128.20, 127.40, 126.35, 121.90,
121.65, 117.20, 17.15.
IC50ðE2Þ
RBA½%ꢁ ¼
ꢀ 100
IC50ðcompoundÞ
Biological Methods. [2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were
bought from Perkin-Elmer, Boston. Quickszint Flow 302 scin-
tillator fluid was bought from Zinsser Analytic, Frankfurt.
17β-HSD1 and 17β-HSD2 were obtained from human pla-
centa according to previously described procedures.38 Fresh
human placenta was homogenized, and cytosolic fraction and
microsomes were separated by centrifugation. For the partial
purification of 17β-HSD1, the cytosolic fraction was precipi-
tated with ammonium sulfate. 17β-HSD2 was obtained from the
microsomal fraction.
1. Inhibition of 17β-HSD1. Inhibitory activities were evalu-
ated by an established method with minor modifications.38
Briefly, the enzyme preparation was incubated with NADH
(500 μM) in the presence of potential inhibitors at 37 °C in a
phosphate buffer pH 7.4 (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 addition of a mixture of unlabeled- and [2,4,6,7-3H]-E1 (final
concentration: 500 nM, 0.15 μCi). After 10 min, the incubation was
stopped with HgCl2 and the mixture was extracted with dieth-
ylether. After evaporation, the steroids were dissolved in acetonitrile.
The RBA value for E2 was arbitrarily set at 100%.
4. Inhibition of 17β-HSD1 in T47D Cells. A stock culture of
T47D cells was grown in RPMI 1640 medium supplemented with
10% FCS, L-glutamine (2 mM), penicillin (100 IU/mL), strepto-
mycin (100 μg/mL), insulin-zinc-salt (10 μg/mL), and sodium
pyruvate (1 mM) at 37 °C under 5% CO2 humidified atmosphere.
The cells were seeded into a 24-well plate at 1 ꢀ 106 cells/well in
DMEM medium with FCS, and L-glutamine and the antibiotics
added in the same concentrations as mentioned above. After 24 h,
the medium was changed for fresh serum free DMEM and a solu-
tion of test compound in DMSO was added. Final concentration
of DMSO was adjusted to 1% in all samples. After a preincu-
bation of 30 min at 37 °C with 5% CO2, the incubation was started
by addition of a mixture of unlabeled- and [2,4,6,7-3H]-E1 (final
concentration: 50 nM, 0.15 μCi). After 0.5 h incubation, the
enzymatic reaction was stopped by removing of the supernatant
medium. The steroids were extracted with diethylether. Further
treatment of the samples was carried out as mentioned for the
17β-HSD1 assay.
5. Metabolic Stability. Human liver microsomes (final protein
concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4, and
test compound (final substrate concentration = 3 μM; final

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8185
DMSO concentration = 0.25%) were preincubated at 37 °C
prior to the addition of NADPH (final concentration=1 mM)
to initiate the reaction. Dextromethorphan and verapamil were
used as references. All incubations were performed singularly for
each test compound. Each compound was incubated for 0, 5, 15,
30, and 45 min (control: 45 min). The reactions were stopped by
the addition of 50 μL of methanol containing internal standard at
the appropriate time points. The incubation plates were centri-
fuged at 2000 rpm for 20 min at 4 °C to precipitate the protein.
The sample supernatants were combined in cassettes of up to four
compounds and analyzed using LC-MS/MS.
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Acknowledgment. We are grateful to the Deutsche For-
schungsgemeinschaft (HA1315/8-1) for financial support of
this work. We thank Jannine Ludwig for her help in perform-
ing the in vitro tests.
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Supporting Information Available: Experimental and spectro-
scopic data of all compounds as well as HPLC purity determi-
nation of all tested compounds. This material is available free of
charge via the Internet at http://pubs.acs.org
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