17β-HSD2 inhibitors for the treatment of osteoporosis: Identification of a promising scaffold

Article abstract of DOI:10.1016/j.bmc.2010.12.013

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyses the conversion of active 17β-hydroxysteroids into the less active 17-ketosteroids thereby controlling the availability of biologically active estrogens (E2) and androgens (T) in the tissues. The skeletal disease osteoporosis occurs mainly in post-menopausal women and in elderly men when the levels of estrogens and androgens, respectively, decrease. Since 17β-HSD2 is present in osteoblasts, inhibition of this enzyme may provide a new and promising approach to prevent the onset of osteoporosis, keeping a certain level in estrogens and androgens in bone cells of ageing people. Hydroxynaphthyl, hydroxyphenyl and hydroxymethylphenyl-substituted moieties were synthesised as mimetics of the steroidal substrate. Compound 8 has been identified as promising scaffold for 17β-HSD2 inhibitors displaying high activity and good selectivity toward 17β-HSD1, ERα and ERβ.

Full text of DOI:10.1016/j.bmc.2010.12.013

Bioorganic & Medicinal Chemistry 19 (2011) 807–815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
17b-HSD2 inhibitors for the treatment of osteoporosis: Identification
of a promising scaffold
Marie Wetzel a, Sandrine Marchais-Oberwinkler a, Rolf W. Hartmann a,b,
⇑
a Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, D-66123 Saarbrücken, Germany
b Helmholtz Institute for Pharmaceutical Sciences Saarland (HIPS), Saarland University, Campus C23, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
17b-Hydroxysteroid dehydrogenase type 2 (17b-HSD2) catalyses the conversion of active 17b-hydroxy-
steroids into the less active 17-ketosteroids thereby controlling the availability of biologically active
estrogens (E2) and androgens (T) in the tissues. The skeletal disease osteoporosis occurs mainly in
post-menopausal women and in elderly men when the levels of estrogens and androgens, respectively,
decrease. Since 17b-HSD2 is present in osteoblasts, inhibition of this enzyme may provide a new and
promising approach to prevent the onset of osteoporosis, keeping a certain level in estrogens and andro-
gens in bone cells of ageing people. Hydroxynaphthyl, hydroxyphenyl and hydroxymethylphenyl-substi-
tuted moieties were synthesised as mimetics of the steroidal substrate. Compound 8 has been identified
as promising scaffold for 17b-HSD2 inhibitors displaying high activity and good selectivity toward 17b-
Received 28 September 2010
Revised 29 November 2010
Accepted 3 December 2010
Available online 9 December 2010
Keywords:
17b-HSD2
Osteoporosis
Steroidomimetics
Non steroidal inhibitors
Substituted hydroxynaphthyl
Hydroxyphenyl
HSD1, ERa and ERb.
Ó 2010 Elsevier Ltd. All rights reserved.
Hydroxymethylphenyl
1. Introduction
elderly men results in a disproportion between bone loss and bone
formation and often leads to osteoporosis.
Osteoblasts (OB) and osteoclasts (OC), two bone cells, are
responsible for bone formation and bone resorption, respectively.
In bone physiology of healthy individuals, bone mass is maintained
by a well regulated balance between the activity of OBs and OCs.
Osteoporosis1 is a skeletal disease, characterised by a reduced
bone mineral density (BMD), increased bone fragility and predis-
position to fractures. The most common osteoporotic fractures oc-
cur at the hip, spine and wrist, leading to pain and amplifying
mortality. In post-menopausal women and elderly men, there is
an acceleration of bone loss, because of higher activity of the OCs
compared to the one of OBs. As androgens and estrogens appear
to be the most important sex steroids2 involved in osteoclastic
resorption and osteoblastic formation, a decrease in estrogens in
post-menopausal women and in both androgens and estrogens in
Antiresorptive agents, including bisphosphonates and selective
estrogen receptor modulators (SERM) are often used to treat oste-
oporosis. Bisphosphonates (like alendronate)3 are currently the
most potent agents for the treatment of osteoporosis. However
they lead to reduction of only 50% of fracture risks in post-meno-
pausal women3,4 and elderly men.5 The SERM raloxifene,6 is also
efficient to treat osteoporosis but is associated with an increased
risk of venous thromboembolism.7
OH
O
17β-HSD2, NAD+
17β-HSD1, NADPH
HO
HO
E2
E1
O
OH
Abbreviations: 17b-HSD1, 17b-hydroxysteroid dehydrogenase type 1; 17b-
HSD2, 17b-hydroxysteroid dehydrogenase type 2; E1, estrone; E2, 17b-estradiol;
ER, estrogen receptor; RBA, relative binding affinity; SF, selectivity factor; T,
testosterone; OPG, osteoprotegerin; NAD(P)H, nicotinamide adenine dinucleotide
phosphate; A-dione, androstenedione; ALP, alkaline phosphatase; OB, osteoblast;
OC, osteoclast; BMD, bone mineral density; SERM, selective estrogen receptor
modulator.
17β-HSD2, NAD+
O
17β-HSD3, NADPH
O
T
A-dione
⇑
Corresponding author. Tel.: +49 681 302 70300; fax: +49 681 302 70308.
E-mail address: rwh@mx.uni-saarland.de (R.W. Hartmann).
URL: http://www.pharmmedchem.de (R.W. Hartmann).
Chart 1. Interconversion of 17b-estradiol (E2) to estrone (E1) by 17b-HSD1 and 17b-
HSD2 and of testosterone (T) to androstenedione (A-dione) by 17b-HSD2 and 17b-HSD3.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.013

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M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
As all therapies used nowadays for the treatment of osteoporo-
sis have limitations and none of them offers a complete cure, there
is a need to develop other efficient drugs for this disease.
androgen levels can easily be obtained either systematically by
the use of aromatase12 and CYP1713,14 inhibitors or locally by
inhibitors of 17b-HSD115–19 or 5 -reductase,20–22 an intracellular
a
There is evidence that androgens and estrogens play a crucial
role in the development of osteoporosis.8 Although they belong
to the antiresorptive agents, estrogens act on the OBs, regulating
the ability of the OC precursors to differentiate to OCs (osteoclasto-
genesis). Estrogens can also enhance OB proliferation9 as well as
osteoprotegerin (OPG) gene expression10 and alkaline phosphatase
(ALP) activity.10 While OB proliferation and ALP increase11 are ben-
eficial for bone formation, OPG via osteoclastogenesis inhibition re-
duces bone resorption. Therefore a drug, which could increase the
estrogen levels in the OBs should result in a local increase of OB
cell proliferation, OPG and ALP production, and should have bene-
ficial effect on osteoporosis. While a decrease in estrogen and
increase of both sex hormones should also be feasible.
High levels of E2 and T should be obtained in bone cells only in
order to avoid development of breast and prostate cancer. This goal
could be achieved by inhibiting 17b-hydroxysteroid dehydroge-
nase type 2 (17b-HSD2, Chart 1) in the target tissues.
17b-HSD2 catalyses the conversion of active 17b-hydroxysteroid
estrogen E2 and androgen T into their less active 17-ketosteroids
using the cofactor NAD+. 17b-HSD2 is a trans-membrane protein23
and its 3D-structure is unknown. It is mainly expressed in placenta,
liver, small intestine, endometrium and osteoblastic cells.24 As 17b-
HSD2 is present in bone cells24,25 and is responsible for the deacti-
vation of active estrogens and androgens, inhibitors of 17b-HSD2
could maintain a certain level in active sex steroids in bone tissue,
and therefore could protect against bone loss and structure deteri-
oration. Therefore 17b-HSD2 is a new attractive target for the treat-
ment of osteoporosis.
Only very few inhibitors of 17b-HSD2 (Chart 2) have been de-
scribed until now.26–29 The steroidal C-18 spirolactone (Chart 2)
was the first 17b-HSD2 inhibitor identified.26–28 Within the non-
steroidal ones, some flavonoids like 3-hydroxyflavone have been
described in the literature as 17b-HSD2 inhibitors.30 However,
regarding 17b-HSD1 they were not very selective. The pyrrolidi-
none A is until now the most potent inhibitor described in the lit-
erature. It was evaluated in vivo in a monkey osteoporosis model
by Bagi et al.31 In spite of the small effects observed at the highest
dose and a strong variability when administrated orally, certainly
O
OH
O
N
O
S
F
HO
C-18 spirolactone
62-66% at 1µM
Pyrrolidinone
IC50 = 10 nM
A
Chart 2. Inhibitors of 17b-HSD2.
Table 1
Inhibition of human 17b-HSD2 and 17b-HSD1 by compounds 1–25
4´
3´
R
1
R
3
R
2
2
1
OH
B, 1-14, 19-25
15-18
Compd
R1
R2
R3
Inhibition of 17b-HSD2a [%] at 1
lM
Inhibition of 17b-HSD1b [%] at 1
lM
B
1
2
3
4
5
6
7
2-OH
2-OH
2-OH
2-OH
H
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
—
—
—
—
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
1-OH
30-OH
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
20
n.i.
n.d.
17
12
20
32
20
74
31
n.i.
13
12
n.i.
16
26
n.i.
n.i.
13
57
75
22
13
43
64
n.i.
100
n.i.c
n.i.c
44
20-OH
40-OH
H
30-OH
61c
n.i.
23c
28
H
30-OH
40-OCH3
40-OH
8
9
20
14
n.i.
15
16
40-CH2OH
40-F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
40-NO2
40-CN
40-COCH3
40-COOH
—
16
n.i.
n.i.
n.i.
n.i.
n.i.
12
52
19
n.i.
n.i.
12
4-Pyridyl
5-Pyrimidyl
4-OCH3-3-pyridyl
3-Furanyl
—
—
—
—
—
—
—
—
—
—
40-SO2NHCH3
40-NHSO2CH3
30-NHSO2CH3
40-CONHCH3
30-CONHCH3
30-NHCOCH3
30-NHCOPh
n.i.
a
Human placenta, microsomal fraction, substrate [3H]-E2 + E2 [500 nM], NAD+ [1.5 mM], mean value of three determinations, relative standard deviation
<10%.
b
Human placenta, cytosolic fraction, substrate [3H]-E1 + E1 [500 nM], NADH [0.5 mM], mean value of three determinations, relative standard deviation <10%.
Recombinant human 17b-HSD1, substrate [3H]-E1 + E1 [30 nM], cofactor NADPH [1 mM], mean value of two determinations, relative standard deviation
c
<20%, 15n.d.: not determined, n.i.: no inhibition (inhibition <10%).

M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
809
Table 2
Inhibition of human 17b-HSD2 and 17b-HSD1 by compounds 26–44
4 R2
N
3
R3
n
OR1
26-44
Compd
n
R1
Position
R2
R3
Inhibition of 17b-HSD2a [%]
Inhibition of 17b-HSD1b [%]
substitution
at 1
lM
at 1 lM
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
H
CH3
H
3
3
3
3
3
3
3
4
4
3
3
3
3
3
3
3
3
4
4
H
H
COCH3
COPh
COPh
COPh(30-OMe)
COPh(40-OMe)
COCH2-Ph(30-OMe)
COCH2-Ph(40-OMe)
SO2CH3
SO2Ph(30-CH3)
COCH3
COPh
n.i.
n.i.
n.i.
15
14
12
n.i.
n.i.
12
n.i.
n.i.
46
13
20
n.i.
39
10
n.i.
14
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
16
n.i.
n.i.
n.i.
25
n.i.
n.i.
CH3
CH3
CH3
CH3
CH3
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
H
COPh
COPh(30-OMe)
COPh(40-OMe)
COCH2-Ph(30-OMe)
COCH2-Ph(30-OH)
COCH2-Ph(40-OMe)
SO2CH3
H
H
SO2Ph(30-CH3)
a
Human placenta, microsomal fraction, substrate [3H]-E2 + E2 [500 nM], NAD+ [1.5 mM], mean value of three determinations, relative standard deviation <10%.
b
Human placenta, cytosolic fraction, substrate [3H]-E1 + E1 [500 nM], NADH [0.5 mM], mean value of three determinations, relative standard deviation <10%, n.i.: no
inhibition (inhibition <10%).
R1
OTf
OTf
d
c
Br
O
OH
O
5e R1: OCH3
5d
5b
5a
e or f
a
b
e
R1: OH
5c R1: OTf
4
R
R
3
OH
R: 4-pyridyl
OH
15
14
R: 4-COOH
16 R: 5-pyrimidyl
17 R: 4-OCH3-3-pyridyl
18
5 R: H
19 R: 4-SO2NHCH3
20 R: 4-NHSO2CH3
7
R: 4-OCH3
8 R: 4-OH
R: 3-furanyl
21
9
R: 3-NHSO2CH3
R: 4-CH2OH
10
22 R: 4-CONHCH3
23
R: 4-F
11 R: 4-NO2
12
R: 3-CONHCH3
24 R: 3-NHCOCH3
R: 4-CN
13 R: 4-COCH3
Scheme 1. Synthesis of compounds 5 and 7–24. Reagents and conditions: (a) AlCl3, toluene, reflux, 2 h; (b) (CF3CO)2O, pyridine, 0 °C, 30 min; (c) Br2, CCl4, diethylether, 0 °C,
4 h; (d) LiBr, Li2CO3, DMF, reflux, overnight; (e) R-boronic acid, Pd(PPh3)4, Na2CO3, DME/EtOH 1:1, 85 °C, 2 h for 5 and 7–18, Method A; (f) R-boronic acid, Pd(PPh3)4, Na2CO3,
DME/water 2:1, microwave irradiation (150 °C, 150 W, 25 min) for 19–24, Method B.
due to an inappropriate pharmacokinetic profile, this study vali-
dates 17b-HSD2 as potential target for the treatment of osteoporo-
sis. Therefore there is a need for new potent inhibitors with good
pharmacokinetic properties for further in vivo experiments.
In this report, the design of novel and selective inhibitors de-
rived from substituted 1-hydroxynaphthyl, 4-hydroxymethylphe-
nyl and 4-hydroxyphenyl as well as their synthesis and biological
evaluation regarding inhibition of 17b-HSD2 and 17b-HSD1 will
be presented (Tables 1 and 2).
2. Design of inhibitors
As flavonoids are known to interact with several pharmacolog-
ical targets, we refrained from using these structures as a starting
point. A similar structure, 6-(3-hydroxyphenyl)-2-naphthol (com-
pound B) was recently identified by Frotscher et al.15 as a highly
potent 17b-HSD1 inhibitor. In spite of the low sequence identity32
and homology32 (23% and 45%, respectively), the active site of
17b-HSD1 and 17b-HSD2 is considered to be very similar as the

810
M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
H
H
O
Cl
O
N
B(OH)2
O
N
R
NH2
a
b
+
B(OH)2
25 R: 5-hydroxy-2-naphthyl
27 R: 4-hydroxymethylphenyl
36 R: 4-hydroxyphenyl
25a
Scheme 2. Synthesis of compounds 25, 27 and 36. Reagents and conditions: (a) Et3N, CH2Cl2, room temperature, 1 h, Method C; (b) R-Br or R-OTf, Pd(PPh3)4, Na2CO3, DME/
EtOH 1:1, 85 °C, 2 h, Method A.
substrate of one enzyme is the product of the catalytic reaction of
the other enzyme (Chart 1). Taking advantage of our experience
developing 17b-HSD1 inhibitors, the hydroxyphenylnaphthol scaf-
fold of compound B was considered as promising starting point for
the identification of new inhibitors of 17b-HSD2. To improve the
activity of compound B in favour of 17b-HSD2 inhibition, the
optimal position of the hydroxy groups was investigated first. In
the second step, one hydroxy group was replaced by different
substituents using the best scaffold. Subsequently, the naphthalene
core was exchanged either by hydroxyphenyl or by hydroxymeth-
ylphenyl moiety.
3. Chemistry
Compounds 1–4 and 6 were synthesised following the proce-
dure described by Frotscher et al.15 The synthesis of 5 started from
the commercially available 6-methoxy-1-tetralone 5e, which is
converted to the hydroxy analogue 5d by an ether cleavage using
aluminium trichloride33 and subsequently triflated34 to 5c. Bro-
mination,35 followed by dehydrobromination35 gave the desired
1-naphthol 5a in very good yield. Different substituted phenyl (5,
7–14, 19–24) or heteroaromatic rings (15–18) were introduced
into the 6-position of the naphthalene core via Suzuki coupling36
reaction (Scheme 1, Method A or B).
Synthesis of 25, 27 and 36 occurred in a two steps reaction:
amidation (Method C) between benzoyl chloride and 3-amino-
phenylboronic acid37 afforded the intermediate 25a and a subse-
quent Suzuki coupling (Scheme 2) with the appropriate boronic
acid led to the target compounds.
4
Br
R
3
a
n
OH
Starting from 4-hydroxymethylbromophenyl or 4-bromophenol,
n
HO
26 R: 3-NHCOCH3, n=1
33 R: 4-NHSO2CH3, n=1
compounds 26, 33, 35 and 43 were obtained via
a cross-
coupling reaction under microwave assisted conditions (Scheme 3,
Method B).
The synthesis of compounds 28–32 and 37–42 started from the
3-bromo-N-methylaniline which was coupled to different benzoyl
chlorides following method C (Scheme 4) to form the amides
28a–32a. Subsequent Suzuki cross couplings with the appropriate
35 R: 3-NHCOCH3, n=0
43 R: 4-NHSO2CH3, n=0
Scheme 3. Synthesis of compounds 26, 33, 35 and 43. Reagents and conditions:
R-boronic acid, Pd(PPh3)4, Na2CO3, DME/water 2:1, microwave irradiation (150 °C,
150 W, 15 bar), 25 min, Method B.
O
Cl
H
N
Br
N
3
n
a
R1
+
R1
O
4
Br
28a R1: H, n= 0
29a R1: 3-OCH3, n= 0
30a R1: 4-OCH3, n= 0
31a R1: 3-OCH3, n= 1
32a R1: 4-OCH3, n= 1
B(OH)2
B(OH)2
b
b
OH
OR2
HO
OR
2
N
4
3
n
N
R1
43R1
n
O
O
28 R1: H, n= 0
37 R1: H, R2: H, n= 0
29 R1: 3-OCH3, n= 0
30 R1: 4-OCH3, n= 0
31 R1: 3-OCH3, n= 1
32 R1: 4-OCH3, n= 1
38 R1: 3-OCH3, R2: CH3, n= 0
39 R1: 4-OCH3, R2: CH3, n= 0
40 R1: 3-OCH3, R2: CH3, n= 1
c
41 R1: 3-OH, R2: H, n=1
42 R1: 4-OCH3, R2: CH3, n= 1
Scheme 4. Synthesis of compounds 28–32 and 37–42. Reagents and conditions: (a) Et3N, CH2Cl2, room temperature, 1 h, Method C; (b) Pd(PPh3)4, Na2CO3, DME/water 2:1,
microwave irradiation (150 °C, 150 W, 15 bar, 25 min), Method B; (c) BBr3, CH2Cl2, ꢀ78 °C to room temperature, overnight.

M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
811
boronic acids afforded 28–32, 37–40 and 42. Compound 40 was
further demethylated38 with boron tribromide to give compound
41 in high yield.
was accomplished by HPLC. The 17b-HSD1 assay was performed
similarly using tritiated E1 as substrate. The percent inhibition val-
ues of compounds 1–44 are shown in Tables 1 and 2, and the IC50
values of selected compounds are reported in Table 3. Compounds
The synthesis37 of the sulfonamide derivative 34a was carried
out by reaction of 4-bromoaniline with 3-methylphenylsulfonyl-
chloride (Scheme 5). Subsequent Suzuki coupling with the corre-
sponding boronic acids afforded sulfonamides 34 and 44.
showing less than 10% inhibition tested at a concentration of 1 lM
were considered to be inactive. The 6-(3-hydroxyphenyl)-2-naph-
thol B identified in a previous work15 was used as internal refer-
ence (20% 17b-HSD2 inhibition vs 100% 17b-HSD1 inhibition at
1
lM).
4. Biological results
To identify the best scaffold for a high 17b-HSD2 inhibition, the
questions were examined whether two hydroxy substituents are
necessary for inhibition and which positions on the 6-phenylnaph-
thalene moiety were optimal. Compounds bearing only one hydro-
xy group, either on the naphthalene (compounds 3 and 5) or on the
phenyl (compound 4) core turned out to have a weak 17b-HSD2
inhibitory activity (Table 1).
Compounds with two OH groups can be separated in two differ-
ent groups depending on the position of the hydroxy moiety on the
naphthalene ring: 2-naphthols (B, 1–2) and 1-naphthols (6–25). 2-
Naphthols are not appropriate as scaffold for 17b-HSD2 inhibitors
as all compounds turned out to be inactive.
4.1. Inhibition of human 17b-HSD2 and selectivity toward 17b-
HSD1, ERa and b
17b-HSD2 and 17b-HSD1 inhibitory activities of the synthesised
compounds were evaluated in cell-free assays. As 17b-HSD1
catalyses the reduction of E1 to E2, it should not be affected by
17b-HSD2 inhibitors. Moreover, inhibitors of 17b-HSD2 should
have no affinity for the estrogen receptors ER
pected that E2 effects are ER mediated.
a and b, as it is ex-
Human placental enzymes were used as source of enzymes for
both assays and were obtained according to described methods.39–
41
Concerning the 1-naphthols, compounds 6 and 7 substituted
with a meta-hydroxy (6) or a para-methoxy (7) group on the
In the 17b-HSD2 assay, incubations were run with tritiated E2,
cofactor and inhibitor.41 The separation of substrate and product
O
O
Cl
S
S
S
O
O
NH2
Br
HN
HN
O
O
a
b
+
Br
34a
R
34 R: CH2OH
44
R: OH
Scheme 5. Synthesis of compounds 34 and 44. Reagents and conditions: (a) pyridine, room temperature, overnight; (b) R-phenylboronic acid, Pd(PPh3)4, Na2CO3, DME/water
2:1, microwave irradiation 150 °C, 150 W, 15 bar, 25 min, Method B.
Table 3
IC50 values, selectivity factor and binding affinities for the estrogen receptors (ER)
a
and b for selected compounds
Compd
Cell-free assay
ER
a
RBAd (%)
ERb RBAd (%)
a
b
17b-HSD2 IC50 [nM]
17b-HSD1 IC50 [nM]
Selectivity factorc
B
8
20
24
5641
302
600
696
116
2425
614
0.02
8
1
0.2
5
n.d.
n.d.
0.8
5
n.d.
n.d.
8516
12
a
b
c
Human placenta, microsomal fraction, substrate [3H]-E2 + E2 [500 nM], cofactor NAD+ [1.5 mM], mean value of three determinations, relative standard deviation <10%.
Human placenta, cytosolic fraction, substrate [3H]-E1 + E1 [500 nM], cofactor NADH [0.5 mM], mean value of three determinations, relative standard deviation <20%.
IC50 (17b-HSD1)/IC50 (17b-HSD2).
d
RBA: relative binding affinity, E2: 100%; n.d.: not determined.
OH
OH
8A
8B
8B
8A
BA
BA BB
BB
HO
HO
B
8
Chart 3. Superimposition of compounds B and 8.

812
M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
phenyl moiety displayed only very low activity. However, a para-
hydroxy substituent (8) improved activity (74% inhibition at
thol 24 64% inhibition at 1
compared to the 1-naphthol 20 75% inhibition at 1
l
M) and sulfonamide 43 (to be
lM) were also
1
lM) indicating that (1) the para-position is more appropriate
inactive. These results emphasise the importance of the aromatic
than the meta-position and (2) H-bond interactions are likely to oc-
cur with the enzyme.
ring 8A of the 1-naphthol, which certainly stabilise the molecule
in the active site via p–p-interactions. However, introduction of a
Looking at these results, it appears that two hydroxy groups are
crucial for the activity and the distance between them must be
around 11 Å as observed for E2 and 8. The structure of inactive B
is very similar to the active 8: they both consist of a hydroxylated
naphthol and a hydroxylated phenyl, and the distance between the
two OH groups are identical. These compounds differ only in their
hydroxy substitution pattern which seems to play a very important
role for 17b-HSD2 or 17b-HSD1 inhibition. In order to better
understand these results, 8 and B were superimposed using their
hydroxy groups as fixed points and energetically minimized with
Hyper Chem Pro 6.0. (Chart 3). It can be observed that the central
aromatic rings of B and 8 occupy a different space in the enzyme. It
is likely that the presence of at least one amino acid of the active
site of 17b-HSD2 around ring BB is responsible for the selectivity
observed for 8 in favour of 17b-HSD2.
methyl on the nitrogen of the phenyl amide 37 or of the benzyla-
mide 41 led to a regain of activity (46% and 39% inhibition at
1 lM for 37 and 41, respectively). All methoxy derivatives (38–
40 and 42) were devoid of activity.
In order to evaluate the selectivity profile of these compounds,
all synthesised molecules were tested for their 17b-HSD1 inhibi-
tion (Tables 1 and 2). For the most potent inhibitors identified in
this report, IC50 data for 17b-HSD2 and 17b-HSD1 were determined
as well as their binding affinity to ER
a and b (Table 3). The most
interesting compound identified in this study is the 4-hydroxy-
phenyl-1-naphthol 8, with an IC50 of 302 nM for 17b-HSD2 inhibi-
tion. A certain selectivity toward 17b-HSD1 has been achieved
(selectivity factor: 8). Taking into account the structural similarity
between E2 and compound 8, it was important to evaluate the
affinity of 8 to the ERs, which is expressed as relative binding affin-
ity.42,43 The RBA measured represents the ligand affinity to ER, rel-
ative to that of E2 which is arbitrarily set up at 100%. With a RBA
The dihydroxylated compound
8 can be considered as a
promising core structure. In order to improve its activity and to
limit the number of hydroxy functions (often responsible for a fast
metabolism as OH groups are substrates for phase II metabolism),
either the hydroxy group of the phenyl moiety was replaced by dif-
ferent substituents (9–14, 19–25) or the 4-hydroxyphenyl was ex-
changed by different heteroaromatic rings (15–18).
value of 5% for ER
a and b, compound 8, however, has a similar
affinity as E1 which is weakly estrogenic.44
5. Discussion and conclusion
Introduction of a methylene linker between the phenyl and the
hydroxy group (9) led to a strong decrease in activity, possibly due
to the flexibility of the OH group and a too long distance between
the two hydroxy groups.
Exchange of the hydroxy moiety by a withdrawing group like
fluoro (10), nitro (11), cyano (12), acetyl (13) or carboxylic (14)
led to inactive compounds, indicating that only electron donating
groups are tolerated by the enzyme in this region or that a H-bond
donor group is necessary for activity. A series of sulfonamides, re-
versed sulfonamides and amides in positions 3 or 4 were intro-
duced as replacement of the hydroxy function. Amides were
better tolerated in 3-position and sulfonamides in 4-position
The goal of this study was the identification of a new non-ste-
roidal scaffold for 17b-HSD2 inhibition. This was a difficult task,
considering the structural similarities between 17b-HSD2 and
17b-HSD1 in their active sites. Taking advantage of our experience
developing 17b-HSD1 inhibitors in the class of hydroxyphenyl-
naphthols,15,16 compound 8 was identified as potent and selective
17b-HSD2 inhibitor while compound B showed a high inhibitory
activity for 17b-HSD1 only. Compounds B and 8 share several
structural similarities, they differ only in their hydroxy substitu-
tion pattern: in B, one OH is in 2-position of the naphthalene and
the other is in meta on the phenyl while in 8, the naphthalene is
hydroxylated in 1-position and the phenyl in para (Chart 3).
Because of the different hydroxy substitution pattern not only
activity for 17b-HSD2 could be gained but also selectivity toward
17b-HSD1 could be reached. A minor structural change in the
ligand can completely reverse the activity and selectivity profile.
This indicates that the small differences between the active sites
of these two enzymes are very important and can lead to a radical
ligand discrimination. Further studies about selectivity regarding
other 17b-HSDs and SDR enzymes will be evaluated in the future.
Selectivity toward the ER which is an important issue could be
gained by introduction of large substituents into this scaffold.
As neither a 3D-structure nor a good homology model of 17b-
HSD2 exists, the information on the active site architecture is
limited and identification of amino acids which could be important
for interaction with the potential inhibitors is not possible. There-
fore in this study, drug design was focused on a ligand-based ap-
proach using the substrate E2 and other ligands like B (17b-HSD1
inhibitor). In case of 17b-HSD1, several X-ray structures of the en-
zyme with or without ligand are available.45 In a previous work we
mentioned that the hydroxyphenyl moiety of B certainly mimics
the steroidal A-ring.15 The superimposition between the OH moie-
ties of B (17b-HSD1 inhibitor) and of 8 (17b-HSD2 inhibitor)
(Chart 3) also indicates that the hydroxyphenyl moiety of B most
likely does not overlap with the one of 8, thus the OH-phenyl of
8 would mimic the steroidal D-ring, interacting with the catalytic
triad. It can be expected that in this area of the enzyme, close to
the catalytic triad, there is more free space available than close
(64% 17b-HSD2 inhibition at 1 lM for amide 24; 57% and 75% for
sulfonamide 19 and reverse sulfonamide 20). These results point
out the importance of the H-bond donor group directly attached
to the phenyl ring: good inhibition data were obtained in case of
the amide (in 3-position 24) or sulfonamide (in 4-position 20)
which are linked to the ring by the NH and therefore able to mimic
the OH group of 8. The inactivity of 25 is probably due to a steric
clash between the large phenylamide group and amino acids from
the active site.
Various heteroaromatic rings were also introduced in 6-position
of the naphthalene (compounds 15–18). None of them turned out
to be active, proving that the hydroxy substituent on the phenyl of
8 can not be replaced by a pure H-bond acceptor group, a H-donor
in this position is essential for the activity.
In order to simplify the rather lipophilic scaffold of the naphtha-
lene, the 1-naphthol moiety was replaced either by 4-hydroxy-
methylphenyl or by 4-hydroxyphenyl groups. The exchange of
the 1-naphthol for a 4-hydroxymethylphenyl was detrimental for
the activity: the amide 26 (to be compared to the 1-naphthol 24
64% inhibition at 1
lM) and sulfonamide 33 (to be compared to
the 1-naphthol 20 75% inhibition at 1
lM) turned out to be com-
pletely inactive (Table 2). In order to gain activity in this class of
compounds, different substituted amides (27–32) and sulfonamide
(34) were introduced but also turned out to be either weakly
active or inactive. Elimination of the methylene linker (compounds
35–44) leading to 4-hydroxyphenyl derivatives was not appropri-
ate to regain activity: the amide 35 (to be compared to the 1-naph-

M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
813
to the A-ring for a large substituent. This results is indeed in accor-
dance with the biological data obtained for 20 and 24, where the
Chemical names follow IUPAC nomenclature. Starting materials
were used as obtained from Aldrich, Acros and Combi-blocks with-
out further purification. No attempts were made to optimise yields.
The following compounds were prepared according to pre-
viously described procedures: 6-(2-hydroxyphenyl)-2-naphthol 1,34
6-(4-hydroxyphenyl)-2-naphthol 2,34 6-phenyl-2-naphthol 3,15
3-(2-naphthyl)phenol 4,46 6-(3-hydroxyphenyl)-1-naphthol 6,34
6-(4-hydroxyphenyl)-1-naphthol 8,34 5-oxo-5,6,7,8-tetrahydro-2-
naphthyl trifluoromethanesulfonate 5c,34 6-hydroxy-1,2,3,4-tetra-
hydronapthalen-1-one 5d,33 3-benzamidophenylboronic acid 25a.47
hydroxy moiety (8, 74% inhibition at 1
ger methylsulfonamide (20, 75% inhibition at 1
(24, 64% inhibition at 1 M) without loss in activity. This finding
lM) is replaced by the lar-
lM) or acetamide
l
will be further validated by substitution of OH-naphthalene or by
introduction of substituent close to this hydroxy moiety in 8.
Attempts to simplify the scaffold of compound 8 failed: one hy-
droxy group is not enough to stabilise the compound in the active
site. The aromatic ring 8A of the naphthalene, mimicking the ste-
roidal A-ring seems to be necessary for inhibitory activity, it might
interact with the enzyme via
p–p
-interactions.
6.1.1. General procedures for Suzuki coupling
Exchange of the hydroxy group on the phenyl moiety by elec-
tron withdrawing groups or exchange of the hydroxyphenyl by
heteroaromatic rings led to a complete loss in activity indicating
the crucial role of the hydrogen bond donating properties of the
OH-phenyl moiety to stabilise the compound in the active site.
The structure–activity relationship which can be deduced from
the biological data gives very useful informations to map the un-
known enzyme active site of 17b-HSD2.
In this report, a new scaffold for 17b-HSD2 inhibitors was iden-
tified: the 4-hydroxyphenyl-1-naphthol 8, with a good inhibitory
activity (IC50 = 302 nM), a selectivity factor of 8 toward 17b-
HSD1 and only weak estrogen receptor binding affinity. As
hydroxynaphthyl and hydroxyphenyl compounds have been
associated with the formation of reactive intermediates and
DNA-adducts, the safety of compound 8 will be further evaluated
and attempts to improve the biological profile (activity, selectivity)
will be performed.
6.1.1.1. Method A. To a mixture of arylbromide (1 equiv) and tet-
rakis(triphenylphosphine) palladium (0) (0.02 equiv) in DME was
added a 2 M aqueous solution of sodium carbonate (2 equiv). The
mixture was purged with N2 and stirred at room temperature for
5 min. Subsequently a solution of boronic acid (1.2 equiv) in EtOH
was added. The mixture was heated to 90 °C for 2 h. The reaction
mixture was cooled to room temperature, quenched with water
and extracted with ethyl acetate. The organic layer was washed
with brine, dried over sodium sulfate and concentrated to dryness
under reduced pressure. The product was purified by column
chromatography.
6.1.1.2. Method B. A mixture of arylbromide (1 equiv), boronic
acid (1.2 equiv), sodium carbonate (2 equiv), and tetrakis(triphen-
ylphosphine) palladium (0) (0.02 equiv) was suspended in an oxy-
gen-free DME/water (2:1) solution. The reaction mixture was
exposed to microwave irradiation (25 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 sodium sulfate
and concentrated to dryness under reduced pressure. The product
was purified by column chromatography.
6. Experimental section
6.1. Chemical methods
1H NMR and 13C NMR spectra were recorded on a Bruker DRX-
500 instrument in CDCl3, CD3OD and acetone-d6. Chemicals shifts
are reported in d values (ppm), the hydrogenated residues of deu-
terated solvent were used as internal standard (CDCl3:
d = 7.26 ppm in 1H NMR and d = 77 ppm in 13C NMR, CD3OD:
d = 3.35 ppm in 1H NMR and d = 49.3 ppm in 13C NMR, acetone-
d6: d = 2.05 ppm in 1H NMR and d = 29.8 ppm and 206.3 ppm in
13C NMR). Signals are described as br, s, d, t, dd, ddd, dt, qt and
m for broad, singlet, doublet, triplet, doublet of doublets, doublet
of doublet of doublets, doublet of triplets, quintuplet and multiplet,
respectively. All coupling constants (J) are given in Hertz.
IR spectra were measured neat on a Bruker Vector 33FT-infrared
spectrometer. Melting points were measured on Stuart Scientific
apparatue SMP3.
Mass spectra (ESI) were measured on a TSQ Quantum (Thermo
Electron Corporation) instrument.
All microwave irradiation experiments were carried out in a
CEM-Discover microwave apparatus.
Flash chromatography was performed using Silica Gel 40 (35/
40–63/70 lM) with hexane/ethyl acetate mixtures as eluents.
6.1.2. General procedure for amide bound formation
6.1.2.1. Method C. N-Methylaniline derivative (1 equiv) was
mixed with Et3N (2 equiv) in dichloromethane. The solution was
cooled at 0 °C. The acyl chloride (1.2 equiv) was slowly added
and the mixture stirred at room temperature for 1 h. The reaction
mixture was extracted with ethyl acetate. The organic layer was
washed with water, dried over sodium sulfate and concentrated
to dryness under reduced pressure. The product was purified by
column chromatography.
6.1.3. Detailed synthesis procedure for the most active
compounds
6.1.3.1.
4-(5-Hydroxy-2-naphthyl)-N-methylbenzenesulfona-
mide (19). The title compound was prepared by reaction
of 5-hydroxy-2-naphthyl trifluoromethanesulfonate 5a (100 mg,
0.34 mmol, 1 equiv) with 4-[(methylamino)sulfonyl]phenylbo-
ronic acid (88 mg, 0.41 mmol, 1.2 equiv) according to method B.
The product was purified by column chromatography (gradient
hexane/ethyl acetate 1:0 to 3:1) to afford the analytically pure
compound in 90% yield (84 mg) as a yellow powder. C17H15NO3S;
MW 313; mp: 204–207 °C; 1H NMR (acetone-d6): d 9.10 (s, 1H),
8.36 (d, J = 8.6 Hz, 1H), 8.21 (d, J = 1.8 Hz, 1H), 8.04–8.01 (m, 2H),
7.99–7.95 (m, 2H), 7.85–7.84 (m, 1H), 7.51 (d, J = 8.4 Hz, 1H),
7.39–7.35 (m, 1H), 6.97 (dd, J = 1.0 Hz, J = 7.6 Hz, 1H), 6.36 (qt,
J = 5.1 Hz, 1H), 2.64 (d, J = 5.1 Hz, 3H); 13C NMR (acetone-d6): d
154.0, 145.7, 139.6, 137.7, 136.1, 133.2, 129.9, 128.6, 127.9,
127.8, 126.9, 125.4, 124.6, 124.1, 120.6, 109.7; IR: 3259, 2971,
1378 cmꢀ1; LC/MS m/z: 314 (M+H)+.
Reaction progress was monitored by thin-layer chromatography
analyses on Alugram SIL G/UV254 (Macherey-Nagel). Visualisation
was accomplished with UV light. Purifications with preparative
HPLC were carried out on a Agilent 1200 series HPLC system from
Agilent Technologies, using a RP C18 Nucleodur 100-5 column
(30 ꢁ 100 mm/50
lm—from Macherey-Nagel GmbH) as stationary
phase with acetonitrile/water as solvent in a gradient from 20:80
to 100:0.
Tested compounds show P95% chemical purity as measured by
HPLC. The methods for HPLC analysis and a table of data for all
tested compounds are provided in the Supplementary data.
6.1.3.2. N-[4-(5-Hydroxy-2-naphthyl)phenyl]methanesulfona-
mide (20). The title compound was prepared by reaction of

814
M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
5-hydroxy-2-naphthyl trifluoromethanesulfonate 5a (100 mg,
0.34 mmol, 1 equiv) with 4-[(methylsulfonyl)amino]phenylbo-
ronic acid (88 mg, 0.41 mmol, 1.2 equiv) according to method B.
The product was purified by column chromatography (hexane/
ethyl acetate 3:1) to give the analytically pure compound in 57%
yield (61 mg) as an orange powder. C17H15NO3S; MW 313; mp:
194–195 °C; 1H NMR (acetone-d6): d 9.33 (s, 1H), 8.92 (s, 1H),
8.53 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 1.6 Hz, 1H), 8.05–8.01 (m, 2H),
8.00 (dd, J = 1.6 Hz, J = 8.8 Hz, 1H), 7.72–7.69 (m, 2H), 7.67
(d, J = 8.8 Hz, 1H), 7.57–7.55 (m, 1H), 7.16–7.13 (m, 1H), 3.27
(s, 3H); 13C NMR (acetone-d6): d 154.1, 138.9, 138.6, 137.8, 136.3,
131.6, 128.9, 127.7, 125.7, 125.0, 124.6, 123.8, 121.5, 120.9,
120.3, 109.1, 39.5; IR: 3384, 2934, 1692, 1519, 1322 cmꢀ1; LC/MS
m/z: 314 (M+H)+.
0.083 mmol, 1 equiv) in anhydrous dichloromethane cooled at
ꢀ78 °C under nitrogen was slowly added boron tribromide
(0.83 mL, 0.83 mmol, 10 equiv). The reaction mixture was stirred
at ꢀ78 °C for 1 h and allowed to warm up to room temperature
overnight. The reaction was quenched by addition of water and ex-
tracted with ethyl acetate. The combined organic layers were
washed with brine and dried over sodium sulfate evaporated to
dryness under reduced pressure and purified by column chroma-
tography (dichloromethane/methanol 20:1) to give the analytically
pure compound in 72% yield (20 mg) as a yellow oil. C21H19NO3;
MW 333; 1H NMR (acetone-d6): d 8.51 (s, 1H), 8.16 (s, 1H), 7.58
(d, J = 7.3 Hz, 1H), 7.49–7.45 (m, 3H), 7.42 (t, J = 1.8 Hz, 1H), 7.19
(ddd, J = 0.9 Hz, J = 2.0 Hz, J = 7.9 Hz, 1H), 7.05–7.00 (m, 1H),
6.94–6.90 (m, 2H), 6.68–6.64 (m, 2H), 6.50 (s, 1H), 3.43 (s, 2H),
3.27 (s, 3H); 13C NMR (acetone-d6): d 158.4, 158.2, 150.9, 145.8,
130.8, 129.9, 129.0, 126.3, 121.0, 116.9, 116.6, 114.2, 41.4; LC/MS
m/z: 334 (M+H)+.
6.1.3.3. 3-(5-Hydroxy-2-naphthyl)-N-methylbenzamide (23).
The title compound was prepared by reaction of 5-hydroxy-2-
naphthyl trifluoromethanesulfonate 5a (100 mg, 0.34 mmol,
1 equiv) with 3-(N-methylaminocarbonyl)phenylboronic acid
(74 mg, 0.41 mmol, 1.2 equiv) according to method B. The analyti-
cally pure product was obtained after column chromatography
(gradient dichloromethane/methanol 1:0 to 20:1) in 54% yield
(51 mg) as a brown oil. C18H15NO2; MW 277; 1H NMR (acetone-
d6): d 9.14 (s, 1H), 8.34 (d, J = 8.6 Hz, 1H), 8.30 (t, J = 1.9 Hz, 1H),
8.14 (d, J = 1.9 Hz, 1H), 7.95–7.93 (m, 1H), 7.92–7.90 (m, 1H),
7.88 (s, 1H), 7.82 (dd, J = 1.9 Hz, J = 8.6 Hz, 1H), 7.60–7.57 (m,
1H), 7.48 (d, J = 8.2 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 6.95 (dd,
J = 0.9 Hz, J = 7.6 Hz, 1H), 2.95 (d, J = 4.7 Hz, 3H); 13C NMR (ace-
tone-d6): d 154.2, 141.9, 132.8, 132.7, 130.5, 129.8, 129.5, 129.4,
127.8, 127.0, 126.6, 126.3, 124.7, 123.9, 120.3, 109.4, 26.7; IR:
3166, 1574, 1272 cmꢀ1; LC/MS m/z: 278 (M+H)+.
6.2. Biological assays
[2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were bought from Perkin–
Elmer, Boston. Quickszint Flow 302 scintillator fluid was bought
from Zinsser Analytic, Frankfurt. 17b-HSD2 and 17b-HSD1 were
obtained from human placenta according to previously described
procedures.39–41,48 Fresh human placenta was homogenised and
centrifuged. The pellet fraction contains the microsomal 17b-
HSD2, while 17b-HSD1 was obtained after precipitation with
ammonium sulfate from the cytosolic fraction.
6.2.1. Inhibition of 17b-HSD2
Inhibitory activities were evaluated by a well established meth-
od with minor modifications.39 Briefly, the enzyme preparation
6.1.3.4. N-[3-(5-Hydroxy-2-naphthyl)phenyl]acetamide (24).
The title compound was prepared by reaction of 5-hydroxy-2-
naphthyl trifluoromethanesulfonate 5a (100 mg, 0.34 mmol,
1 equiv) with 3-acetamidophenylboronic acid (74 mg, 0.41 mmol,
1.2 equiv) according to method B. The analytically pure product
was obtained after column chromatography (gradient dichloro-
methane/methanol 1:0 to 20:1) in 42% yield (40 mg) as a yellow
powder. C18H15NO2; MW 277; mp: 105–106 °C; 1H NMR (ace-
tone-d6): d 9.24 (s, 1H), 9.04 (s, 1H), 8.32 (d, J = 8.6 Hz, 1H), 8.10–
8.08 (m, 1H), 8.05 (d, J = 1.6 Hz, 1H), 7.75 (dd, J = 1.8 Hz,
J = 8.8 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.49–7.45 (m, 2H), 7.42 (t,
J = 7.9 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 6.93 (dd, J = 1.0 Hz,
J = 7.4 Hz, 1H), 2.13 (s, 3H); 13C NMR (acetone-d6): d 154.0, 147.6,
136.3, 130.1, 127.7, 126.0, 124.8, 123.7, 122.8, 120.3, 119.0,
118.7, 109.2, 24.3; IR: 3208, 3082, 2944, 1670 cmꢀ1; LC/MS m/z:
278 (M+H)+.
was incubated with NAD+ [1500
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. 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]-E2 (final
concentration: 500 nM, 0.11 lCi). After 20 min at 37 °C, the incu-
bation was stopped with HgCl2 and the mixture was extracted with
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 rp chromatography column (Nucleodur C18
Gravity, 3 lm, Macherey-Nagel, Düren) connected to a HPLC-sys-
tem (Agilent 1100 Series, Agilent Technologies, Waldbronn). Detec-
tion and quantification of the steroids were performed using a
radioflow detector (Berthold Technologies, Bad Wildbad). The con-
version rate was calculated according to following equation: %con-
version = (%E1/(%E1 + %E2) ꢁ 100). Each value was calculated from
at least three independent experiments.
6.1.3.5.
N-(40-Hydroxybiphenyl-3-yl)-N-methylbenzamide
(37). The title compound was prepared by reaction of N-(3-
bromophenyl)-N-methylbenzamide 28a (100 mg, 0.34 mmol,
1 equiv) with 4-hydroxyphenylboronic acid (56 mg, 0.41 mmol,
1.2 equiv) according to method B. The analytically pure product
was obtained after column chromatography (hexane/ethyl acetate
6:4) in 19% yield (20 mg) as a beige powder. C20H17NO2; MW 303;
mp: 175–176 °C; 1H NMR (acetone-d6): d 8.51 (s, 1H), 7.34–7.31
(m, 3H), 7.30–7.29 (m, 2H), 7.28 (s, 1H), 7.26 (t, J = 8.0 Hz, 1H),
7.23–7.20 (m, 1H), 7.19–7.15 (m, 2H), 3.44 (s, 3H); 13C NMR (ace-
tone-d6): d 170.7, 158.4, 146.6, 142.7, 137.9, 132.1, 130.2, 130.1,
129.4, 128.8, 128.5, 126.1, 125.6, 124.9, 116.6, 38.2; IR: 3380,
3080, 1696, 1598, 1379, 1257 cmꢀ1; LC/MS m/z: 304 (M+H)+.
6.2.2. Inhibition of 17b-HSD1
The 17b-HSD1 inhibition assay was performed similarly to the
17b-HSD2 procedure. The cytosolic fraction was incubated with
NADH [500
l
M], test compound and a mixture of unlabelled-
and [2,4,6,7-3H]-E1 (final concentration: 500 nM, 0.15
l
Ci) for
10 min. Further treatment of the samples and HPLC separation
was carried out as mentioned above.
6.2.3. ER affinity
The binding affinity of selected compounds to the ER
a and ERb
was determined according to Zimmermann et al.49 using recombi-
nant human proteins. Briefly, 0.25 pmoles of ER
a or ERb, respec-
6.1.3.6.
N-(40-Hydroxybiphenyl-3-yl)-2-(3-hydroxyphenyl)-N-
tively, were incubated with [3H]-E2 (10 nM) and test compound
for 1 h at room temperature. The potential inhibitors were dis-
solved in DMSO (5% final concentration). Non-specific-binding
methylacetamide (41). To a solution of N-(40-methoxybiphe-
nyl-3-yl)-2-(3-methoxyphenyl)-N-methylacetamide 40 (30 mg,

M. Wetzel et al. / Bioorg. Med. Chem. 19 (2011) 807–815
815
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was performed with diethylstilbestrol (10 lM). After incubation, li-
gand–receptor complexes were selectively bound to hydroxyapa-
tite (5 g/60 mL TE-buffer). The formed complex was separated,
washed and resuspended in ethanol. For radiodetection, scintilla-
tor cocktail (Quickszint 212, Zinsser Analytic, Frankfurt) was added
and samples were measured in a liquid scintillation counter (Rack
Beta Primo 1209, Wallac, Turku). From these results the percentage
of [3H]-E2 displacement by the compounds was calculated. The
plot of % displacement versus compound concentration resulted
in sigmoidal binding curves. The compound concentration to dis-
place 50% of the receptor bound [3H]-E2 were determined. Unla-
belled E2 was used as a reference. For determination of the
relative binding affinity42 the ratio was calculated according to
IC50ðE2Þ
the following equation: RBA% ¼
ðcompoundÞ ꢁ 100.50 This results
IC50
in an RBA value of 100% for E2. After the assay was established
and validated a modification was made to increase throughput.
Compounds were tested at concentrations of 1000 ꢁ IC50(E2) and
10,000 ꢁ IC50(E2). Results were reported as RBA ranges. Com-
pounds with less than 50% displacement of [3H]-E2 at a concentra-
tion of 10,000 ꢁ IC50(E2) were classified as RBA <0.01%, compounds
that displace more than 50% at 10,000 ꢁ IC50(E2) but less than 50%
at 1000 ꢁ IC50(E2) were classified as 0.01% < RBA < 0.1%.
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Acknowledgements
We are grateful to the Deutsche Forschungsgemeinschaft for
financial support (HA1315/8-1). We thank Jannine Ludwig, Jeannine
Jung and Jörg Haupenthal for their help in performing the enzyme
inhibition tests (17b-HSD2, 17b-HSD1) and the ER tests.
ˇ
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Supplementary data
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Supplementary data (chemical synthesis and characterisation of
all compounds as well as HPLC purity determination) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.12.013.
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