17β-hydroxysteroid dehydrogenase type 2 inhibition: Discovery of selective and metabolically stable compounds inhibiting both the human enzyme and its murine ortholog

Article abstract of DOI:10.1371/journal.pone.0134754

Design and synthesis of a new class of inhibitors for the treatment of osteoporosis and its comparative h17β-HSD2 and m17β-HSD2 SAR study are described. 17a is the first compound to show strong inhibition of both h17β-HSD2 and m17β-HSD2, intracellular activity, metabolic stability, selectivity toward h17β-HSD1, m17β-HSD1 and estrogen receptors α and β as well as appropriate physicochemical properties for oral bioavailability. These properties make it eligible for pre-clinical animal studies, prior to human studies. Copyright:

Full text of DOI:10.1371/journal.pone.0134754

RESEARCH ARTICLE
17β-Hydroxysteroid Dehydrogenase Type 2
Inhibition: Discovery of Selective and
Metabolically Stable Compounds Inhibiting
Both the Human Enzyme and Its Murine
Ortholog
Emanuele M. Gargano1, Giuseppe Allegretta2, Enrico Perspicace1, Angelo Carotti3,
Chris Van Koppen4, Martin Frotscher1, Sandrine Marchais-Oberwinkler1,2*, Rolf
W. Hartmann1,2
*
1 Pharmaceutical and Medicinal Chemistry, Saarland University, Saarbrücken, Germany, 2 Helmholtz
Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany, 3 Dipartimento Farmaco-Chimico,
Università degli Studi di Bari, Bari, Italy, 4 ElexoPharm GmbH, Saarbrücken, Germany
* rolf.hartmann@helmoltz-hzi.de (RWH); s.marchais@mx.uni-saarland.de (SMO)
OPEN ACCESS
Citation: Gargano EM, Allegretta G, Perspicace E,
Carotti A, Van Koppen C, Frotscher M, et al. (2015)
17β-Hydroxysteroid Dehydrogenase Type 2
Abstract
Design and synthesis of a new class of inhibitors for the treatment of osteoporosis and its
comparative h17β-HSD2 and m17β-HSD2 SAR study are described. 17a is the first com-
pound to show strong inhibition of both h17β-HSD2 and m17β-HSD2, intracellular activity,
metabolic stability, selectivity toward h17β-HSD1, m17β-HSD1 and estrogen receptors α
and β as well as appropriate physicochemical properties for oral bioavailability. These prop-
erties make it eligible for pre-clinical animal studies, prior to human studies.
Inhibition: Discovery of Selective and Metabolically
Stable Compounds Inhibiting Both the Human
Enzyme and Its Murine Ortholog. PLoS ONE 10(7):
e0134754. doi:10.1371/journal.pone.0134754
Editor: Alessio Lodola, University of Parma, ITALY
Received: June 10, 2015
Accepted: July 13, 2015
Published: July 31, 2015
Copyright: © 2015 Gargano et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Osteoporosis is a common, age-related disease, characterized by a systemic impairment of
bone mass and microarchitecture, increasing bone fragility and risk of fractures [1]. It has been
shown that the drop in 17β-estradiol (E2) and testosterone (T) levels, occurring with ageing, is
the main factor driving the onset and progression of this disease [2]. 17β-Hydroxysteroid dehy-
drogenase type 2 (17β-HSD2) catalyzes the conversion of the highly active E2 and T into the
weakly potent 17-ketosteroids estrone (E1) and Δ4-androstene-3,17-dione (Δ4-AD), respec-
tively [3]. It is expressed in osteoblastic cells [4], therefore its inhibition can lead to the desired
increase of E2 and T levels in the bone tissue and may thus be a novel approach for the treat-
ment of osteoporosis.
Some steroidal [5–7] and non steroidal [8, 9] 17β-HSD2 inhibitors are already described. In
our group we also developed and reported about several classes of non-steroidal 17β-HSD2
inhibitors [10–14], with a strong inhibition of human 17β-hydroxysteroid dehydrogenase type
2 (h17β-HSD2) and a good selectivity toward h17β-HSD1. Since h17β-HSD1 is the biological
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors acknowledge the Deutsche
Forschungsgemeinschaft (DFG) for financial support
(Grants HA1315/12-1). The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript. Elexopharm
GmbH had no financial interest in this collaboration.
Competing Interests: The authors have declared
that no competing interests exist. Chris Van Koppen
is an employee of Elexopharm GmbH. He was in
PLOS ONE | DOI:10.1371/journal.pone.0134754 July 31, 2015
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
charge of the biological testing of the compounds.
This work was based on a scientific collaboration
between Elexopharm GmbH, the Saarland University
and the Helmholtz Institute for Pharmaceutical
Research Saarland. This does not alter the authors'
adherence to PLOS ONE policies on sharing data
and materials related to this study.
counterpart of h17β-HSD2, catalyzing the opposite conversion, selectivity toward this enzyme
is an important feature to take into consideration. Potent and selective h17β-HSD1 inhibitors
have also been described for the treatment of estrogen-dependent diseases [15, 16].
Given that the most commonly used animal model for osteoporosis studies are established
in rodents [17, 18], we aimed at the development of new inhibitors displaying a good inhibition
of mouse 17β-hydroxysteroid dehydrogenase type 2 (m17β-HSD2) and a reasonable selectivity
toward m17β-HSD1. In order to have a compound suitable for animal testing and following
human studies, the designed inhibitors should also display h17β-HSD2 inhibitory activity and
selectivity toward h17β-HSD1. Other characteristics to be implemented are preferably low
affinity to the estrogen receptors (ERs) α and β in order to maximize the E2 local effect and to
minimize systemic side effects as well as metabolic stability.
Since the 3D-structure for both human and mouse 17β-HSD2 is up to date not available a
ligand based approach was chosen for the design of new inhibitors. A set of h17β-HSD2 inhibi-
tors was selected and tested for m17β-HSD2 inhibitory activity in order to get insight in the
SAR and tracking the lead for the rational design of new inhibitors.
Results and Discussion
h17β-HSD2, h17β-HSD1, m17β-HSD2 and m17β-HSD1 cell-free assays were performed simi-
larly, by incubating enzyme, tritiated substrate, cofactor and inhibitor, according to described
procedures [19–22].
As starting point 25 previously described h17β-HSD2 inhibitors (Fig 1), belonging to the
2,5-thiophene amide, 1,3-phenyl amide and 1,4-phenyl amide class [12–14], were tested for
m17β-HSD2 inhibition, in order to elaborate a comparative SAR and to develop an optimiza-
tion strategy.
IC50 or percent of inhibition values for both h17β-HSD2 and m17β-HSD2 are given
(Table 1, compounds 1a-2g, 3a, 4a-4f) to facilitate comparison.
In the 2,5-thiophene amide class (compounds 1a-2g), h17β-HSD2 inhibitors [13, 14], a
broad range of inhibitory activities was detected, depending on the substitution pattern; the
most active compounds show IC50 values around 60 nM (Table 1). Conversely, the inhibitory
activity towards m17β-HSD2 was only marginally affected by these changes (inhibitory activity
around 30% at 1 μM). Only compound 2a shows a more pronounced m17β-HSD2 inhibition
combined with good inhibition of the human enzyme (65% at 1 μM and IC50 = 61 nM). Unfor-
tunately, compound 2a turned out to be metabolically very unstable, with a half-life of only 4
minutes in the human liver S9 fraction [13]. In a further screening, all the tested 2,5-thiophene
amide displayed a high metabolic instability [13].
It is striking that neither the nature of substituents on ring A and C or their substitution pat-
tern does appear to exert an effect on m17β-HSD2 inhibition, whereas it is decisive for the
h17β-HSD2 one. This result suggests that the inhibitors in this class are likely to have different
binding modes in the two enzyme isoforms.
Fig 1. Previously described h17β-HSD2 inhibitors, tested for m17β-HSD2 inhibition.
doi:10.1371/journal.pone.0134754.g001
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
Table 1. Inhibition of Human and Mouse 17β-HSD2 by 2,5-Thiophene Amide, 1,3-Phenyl Amide and
1,4-Phenyl Amide Derivatives in a Cell-Free Assay.
IC50 (nM)a or % inh. at 1 μMa, d
Cmpd
R1
R2
h17β-HSD2b
m17β-HSD2c
1a
1b
1c
1d
1e
1f
3-OMe
3-Me
3-OMe
3-Me
68
29%
42%
30%
19%
n.i.
52
3-Me
3-OMe
3-OMe
3-OH
58
2-OMe
3-OH
490
33%
410
170
510
48%
62
2-OH
3-OH
18%
40%
n.i.
1g
1h
1i
3-N(Me)2
3-F
3-OMe
3-OMe
3-OMe
3-OMe
3-Me
4-CN
n.i.
1j
2-F, 3-OMe
2-F, 3-OMe
2-F, 3-OMe
3-OMe
3-OH
26%
45%
65%
16%
26%
35%
45%
37%
n.i.
1k
2a
2b
2c
2d
2e
2f
62
3-OH
61
3-OMe
3-OH
370
390
330
160
330
n.i.
4-OH
3-OH
3-Me
3-OH
3-F
3-OH
2g
3a
4a
4b
4c
4d
4e
4f
4-CN
3-OH
3-Me
3-Me
1100
520
1200
35%
11%
640
480
50%
25%
11%
n.i.
3-OMe
4-OMe
3-OH
3-OMe
3-OMe
3-OH
3-OMe
3-OH
3-OMe
3-OH
n.i.
29%
22%
4-OH
3-OH
aMean value of at least two determinations, standard deviation less than 20%.
bHuman placental microsomal fraction, substrate E2 [500 nM], cofactor NAD+ [1500 μM].
cMouse liver microsomal fraction, substrate E2 [500 nM], cofactor NAD+ [1500 μM].
dn.i.: no inhibition, i.e., inhibition ꢀ10%.
doi:10.1371/journal.pone.0134754.t001
Exchange of the central thiophene by a 1,3-disubstituted phenyl led to compounds 4a-4c for
n = 0 and 4d-4f for n = 1, with weak inhibitory activity towards both the human and the mouse
enzyme (Table 1).
In contrast, compound 3a (Table 1), bearing a 1,4-disubstituted phenyl moiety as central
ring, shows moderate inhibition of both h17β-HSD2 and m17β-HSD2 and also revealed excep-
tional metabolic stability in the human liver S9 fraction, with a half-life time>120 minutes
[13]. It was therefore taken as starting point for the design of a small library of inhibitors where
the substitution pattern and the physicochemical nature of substituents on the A and C rings
was varied (Fig 2). A larger number of derivatives, bearing substituents with different physico-
chemical properties on the A ring were prepared, according to their chemical accessibilities.
Compounds 25a and 25 were also synthesized to investigate the effect of the methylene linker
between the amide function and the C ring.
The synthesis of the 1,4-phenyl derivatives 5a-23a, 25a, 7 and 25, depicted in Fig 3, was
accomplished following a two- or three-step reaction pathway.
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
Fig 2. Chemical Structures of the Designed Compounds.
doi:10.1371/journal.pone.0134754.g002
First, amidation was carried out by reaction of the commercially available 4-bromobenzoyl
chloride 5d with substituted anilines 5c or with the 1-(3-methoxyphenyl)-N-methylmethana-
mine 25c using standard conditions (method A: triethylamine, dichloromethane, from 0C to
room temperature, overnight) affording the brominated intermediates 5b, 6b and 11b in
almost quantitative yields. Subsequently, Suzuki coupling (Method B) using tetrakis(triphenyl-
phosphine)palladium and cesium carbonate in a mixture of DME/EtOH/H2O (1:1:1,3 mL) as
solvent and microwave irradiation (150°C, 150 W for 20 minutes), provided the biphenyl
derivatives 5a-21a and 25a, and the 1,3-benzodioxole derivatives 22a and 23a in good yields.
Compounds 7a and 25a were submitted to ether cleavage using boron trifluoride-dimethyl sul-
fide complex, yielding the hydroxylated molecules 7 and 25.
The synthesis of the retroamide 24a, displayed in Fig 4, follows a two-step procedure.
First the commercially available 3-methoxybenzoyl chloride 24c was reacted with 4-bromo-
N-methyl aniline 24d according to method A and afforded the brominated intermediate 24b
with 70% yield. Subsequently, Suzuki coupling following method B afforded compound 24a in
68% yield.
Compounds 5a-25a, 7 and 25 were tested for h17β-HSD2, m17β-HSD2 and h17β-HSD1
inhibition (Table 2, results expressed as percentage of inhibition or IC50 value).
Compounds 18a and 24a were not tested for inhibition of m17β-HSD2, due to their low
inhibitory activity towards the human enzyme. Among the different synthesized 1,4-biphenyl
Fig 3. Synthesis of 1,4-Phenyl Derivatives 5a-23a, 25a, 7, 25. Reagents and conditions: (i) Et3N, CH2Cl2,
room temperature, overnight; (ii) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave irradiation (150°C,
150W, 20 min); (iii) BF3ÁSMe2, CH2Cl2, room temperature, overnight.
doi:10.1371/journal.pone.0134754.g003
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
Fig 4. Synthesis of 1,4-Phenyl Retroamide Derivative 24a. Reagents and conditions: (i) Et3N, CH2Cl2,
room temperature, overnight; (ii) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave irradiation (150°C,
150W, 20 min).
doi:10.1371/journal.pone.0134754.g004
amides without methylene linker (n = 0, Table 2, compounds 5a-21a), the best h17β-HSD2
inhibitory activity and selectivity toward h17β-HSD1 was achieved for compounds 6a, 7a and
Table 2. Inhibition of h17β-HSD2, m17β-HSD2 and h17β-HSD1 by Biphenyl Amide Derivatives with Different Substitution Patterns on the A and C
Rings in Cell-Free System.
IC50 (nM)a or % inh. at 1 μMa, c
IC50 (nM)a or % inh. at 1 μMa, c
m17β-HSD2f,g
Cmpd
R1
R2
h17β-HSD2b
h17β-HSD1c
s. f.d,f
3a
3-Me
3-Me
1100
44%
260
11500
n.i.
10
50%
56%
260
5a
3-OMe
3-Me
3-Me
n.d.
25
6a
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
4-OMe
4-OMe
3-OH
6400
6400
22%
n.i.
7a
3-OMe
-H
330
20
290
8a
51%
50%
710
n.d.
n.d.
38
48%
33%
66%
58%
62%
42%
57%
73%
67%
52%
140
9a
2-OMe
4-OMe
3-OMe
4-OMe
3-OH
10a
11a
12a
7
27000
n.i.
27%
19%
36%
1000
950
n.d.
n.d.
n.d.
8
n.i.
15%
8000
12600
5000
85500
13300
n.i.
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
25
3-F
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-Me
3-Cl
13
3-N(Me)2
3-OMe,4-OMe
3-Me,4-Me
3-F,4-F
2-F, 3-OMe
2-F, 3-Me
2-F, 3-Me
-
650
8
520
164
44
300
31%
64%
460
n.d.
n.d.
24
n.d.
35%
11300
3710
n.i.
67%
48%
56%
56%
70%
n.d.
330
11
3-OMe
3-Me
51%
560
n.d.
20
-
10900
n.i.
-
-
11%
310
n.d.
283
118
2-F, 3Me
2-F, 3-Me
3-OMe
3-OH
87000
31000
43%
190
260
aMean value of at least three determinations, standard deviation less than 20% except for 11a (hHSD2): 26%, 7(hHSD1): 25%,.
bHuman placental, microsomal fraction, substrate E2 [500 nM], cofactor NAD+ [1500 μM].
cHuman placental, cytosolic fraction, substrate E1 [500 nM], cofactor NADH [500 μM].
ds.f. (selectivity factor) = IC50(17β-HSD1)/IC50(17β-HSD2).
en.i.: no inhibition (inhibition of <10%).
fn.d.: not determined.
gMouse liver microsomal fraction, substrate E2 [500 nM], cofactor NAD+ [1500 μM].
doi:10.1371/journal.pone.0134754.t002
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
17a (Table 2, IC50 values between 260 and 330nM, s.f. between 20 and 44), showing that a
3-OMe-group on ring C in combination with either a 3-OMe- or a 3-Me-group on ring A leads
to a maximum in potency and selectivity in this series of compounds.
The presence of a methyl group in 4-position of ring A is tolerated by the h17β-HSD2 (com-
pound 17a, IC50 = 310nM) and increases the selectivity toward h17β-HSD1 (17a: s.f. 44; 6a: s.f.
25). In contrast, compound 16a bearing a 3,4-dimethoxy substituted A ring displays a slight
decrease in h17β-HSD2 inhibitory activity if compared to the corresponding compound 7a
with only one methoxy group on that ring. The rigidification of the two methoxy substituents
by the synthesis of a 1,3-benzodioxole ring (compounds 22a and 23a) could not overcome the
drop in potency. Compounds 6a, 7a and 17a displayed the strongest m17β-HSD2 inhibitory
activity (IC50 = 260, 290 and 140 nM, respectively).
In general, the introduction of methyl- or methoxy-groups, especially in the 3-positions of
rings A and B, had a positive impact on inhibitory activity, which is similar for both 17β-HSD2
isoforms (Table 2). Therefore, inhibitors belonging to the 1,4-phenyl class are likely to bind in
a conserved area common to both enzyme, in contrast to the 2,5-thiophene amide class. As
17β-HSD2 belongs to the SDR superfamily, characterized by the conserved Rossmann fold and
catalytic triad [23], it is possible that these inhibitors bind in or very close to these regions. Fur-
thermore, since compounds 6a and 17a lack of the two oxygen functions to mimic the E2 inter-
actions with the enzyme, they are likely to bind to the active site in an alternative mode,
significantly influenced by the methyl substitution patterns.
Compounds with a methylene linker 25a and 25 (Table 2), showed h17β-HSD2 inhibitory
activity in the same order of magnitude as the corresponding derivative 20a lacking the methy-
lene group, but displayed improved selectivities over h17β-HSD1. In contrast to what was
observed for human 17β-HSD2, compounds 25a and 25 showed a significant difference in
terms of m17β-HSD2 inhibition, indicating that the hydroxy group of compound 25 might
function as H-bond donor in the interaction with the enzyme. A similar behavior was observed
in the 2,5-thiophene amide class with regard to the h17β-HSD2 inhibitory activity[13]: all the
tested 2,5-thiophene amides with a methylene linker achieved the highest h17β-HSD2 inhibi-
tion when substituted with an hydroxy group on the C ring. It might be therefore speculated,
that the addition of the methylene linker can influence the binding mode of the inhibitors in
both classes.
The 1,4-phenyl amides are likely to be competitive inhibitors, as derivatives with a similar
structure were found to inhibit the enzyme following this mode of action.
The most potent m17β-HSD2 inhibitors 6a-8a, 10a, 14a, 15a, 17a, 19a, 21a, 23a and 25
were also tested for their m17β-HSD1 inhibitory activity. None of the tested compounds
showed any inhibition of m17β-HSD1 at a concentration of 1 μM, indicating a significant selec-
tivity toward this enzyme.
The inhibitory activity of compounds 6a, 17a and 25 was also evaluated in the human mam-
mary cell line MDA-MB-231 containing endogenous 17β-HSD2 (Table 3). The compounds
Table 3. Human 17β-HSD2 Cellular Inhibition of Compounds 6a, 17a and 25.
Cmpd
% inhb HSD2a at 250 nM
% inhb HSD2a at 1250 nM
6a
60%
66%
67%
87%
90%
88%
17a
25
aMDA-MB-231 cell line, substrate E2 [200 nM].
bMean value of two determinations, standard deviation less than 15%.
doi:10.1371/journal.pone.0134754.t003
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
were tested at 250 nM and 1250 nM, representing approximately the IC50 observed in the cell-
free assay, and its 5-fold value. As displayed in Table 3, all three compounds showed an inhibi-
tion between 60% and 67%at the lower concentration and approximately 90% inhibition at the
higher concentration, indicating that the inhibitors can permeate the membrane and are able
to inhibit the enzyme in a concentration dependent manner.
Compounds 6a, 7a, 17a, 21a, 25a and 25 were tested for their affinity toward the ERs α and
β according to described methods [24] (assay details are available in the Materials and Meth-
ods). Even when applied in a 1000-fold excess relative to E2, no inhibitor was able to displace
more than 20% of the steroid from the corresponding receptor, indicating a very low binding
affinity to the ERs.
The metabolic stabilities of the most active compounds 6a, 17a and 25 were evaluated using
human liver microsomes (S9 fraction). In addition, compounds 5a, 14a, 20a and 25a were also
tested in order to investigate whether structure modifications might exert an effect on meta-
bolic stability (Table 4, assay details are available in Materials and Methods). All compounds,
except 25, revealed a very high stability, which was not influenced by the nature of the substitu-
ents. Compound 25, exhibiting a short half-life, bears a hydroxy group, potentially susceptible
to phase ΙΙ metabolism. Interestingly, all the tested inhibitors from the 1,4-phenyl amide class,
for n = 0, demonstrated high metabolic stability, which seemingly constitutes a positive feature
of the whole class. As species differences for the metabolism of drugs that are not or only partly
metabolized are usually small [25], sufficient metabolic stability in species other than human
can be anticipated.
Conclusion
The aim of this work was the design of a compound, which should be suitable for application
in both an animal model of osteoporosis and in humans. We report here the discovery of
compound 17a, which is the first to show an appropriate profile for this purpose, with strong
inhibition of both human and mouse 17β-HSD2 and selectivity toward the respective type 1
enzymes and the ERs. It also displayed good cellular inhibitory activity, high metabolic stability
and good physicochemical parameters (MW = 345 and cLogP = 4.75) predictor for good oral
bio-availability [26]. A comparative SAR study for h17β-HSD2 and m17β-HSD2 is also
described for the 1,4-phenyl and the 2,5-thiophene classes of inhibitors, revealing that only
Table 4. Half-life in Human Liver Microsomes S9 Fraction of Representative Compounds 3a, 5a, 6a,
14a, 17a, 20a, 25a and 25.
Cmpd
R1
R2
Inhibitorc t1/2(min)a,b
3a
3-Me
3-Me
>120
116
106
82
5a
3-OMe
3-Me
6a
3-Me
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OH
14a
17a
20a
25a
25
3-Cl
3-Me,4-Me
2-F, 3-Me
2-F, 3-Me
2-F, 3-Me
107
104
103
6
aMean of at least two determinations, standard deviation less than 25%.
b1 mg/ml pooled mammalian liver S9 fraction (BD Gentest), 2 mM NADPH regenerating system, 1 mM
UDPGA and 0.1 mM PAPS at 37°C for 0, 5, 15 and 60 minutes.
cInhibitor tested at a final concentration of 1 μM.
doi:10.1371/journal.pone.0134754.t004
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
compounds belonging to the first series similarly inhibit the two enzymes, probably through a
similar binding mode. The species specific characterization of the thiophene and the phenyl
derivatives pointed out the superiority of the latter class of inhibitors, which is able to equally
inhibit the two isoenzymes and additionally displays a high metabolic stability. In vivo assays
in a mouse osteoporosis model will be carried out soon and the results reported in due course
in a specialized journal dealing with bone diseases.
Materials and Methods
Chemical Methods
Chemical names follow IUPAC nomenclature. Starting materials were purchased from
Aldrich, Acros, Combi-Blocks or Fluorochem and were used without purification. Column
chromatography was performed on silica gel (70–200 μm) and reaction progress was moni-
tored by TLC on Alugram SIL G/UV254 (Macherey-Nagel). Visualization was accomplished
with UV light.1H NMR and 13C NMR spectra were measured on a Bruker AM500 spectrome-
ter (at 500 MHz and 125 MHz, respectively) at 300 K and on Bruker Fourier 300 (at 300 MHz
and 75 MHz, respectively) at 300K. Chemical shifts are reported in δ (parts per million: ppm),
by reference to the hydrogenated residues of deuteriated solvent as internal standard: 2.05 ppm
(1H NMR) and 29.8 and 206.3 ppm (13C NMR) for CD3COCD3, 7.26 ppm (1H NMR) and
77.0 ppm (13C NMR) for CDCl3. Signals are described as br (broad), s (singlet), d (doublet),
t (triplet), dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets)
and m (multiplet). All coupling constants (J) are given in Hertz (Hz).
Melting points (mp) were measured in open capillaries on a Stuart Scientific SMP3 appara-
tus and are uncorrected.
Mass spectrometry was performed on a TSQ Quantum (ThermoFisher, Dreieich, Ger-
many). The triple quadrupole mass spectrometer was equipped with an electrospray interface
(ESI). The Surveyor-LC-system consisted of a pump, an auto sampler, and a PDA detector.
The system was operated by the standard software Xcalibur. A RP C18 NUCLEODUR 100–5
(3 mm) column (Macherey-Nagel GmbH, Dühren, Germany) was used as stationary phase. All
solvents were HPLC grade. In a gradient run (acetonitrile/water) the percentage of acetonitrile
(containing 0.1% trifluoroacetic acid) was increased from an initial concentration of 0% at 0
min to 100% at 15 min and kept at 100% for 5 min. The injection volume was 15 μL and flow
rate was set to 800 μL/min. MS analysis was carried out at a needle voltage of 3000 V and a cap-
illary temperature of 350°C. Mass spectra were acquired in positive mode from 100 to 1000 m/
z and UV spectra were recorded at the wave length of 254 nm and in some cases at 360 nm.
All microwave irradiation experiments were carried out in a 507 CEM-Discover microwave
apparatus.
All tested compounds exhibited ꢁ 95% chemical purity as measured by LC/MS.
The following compounds were prepared according to previously described procedures:
4-bromo-N-methyl-N-(m-tolyl)benzamide 7a[13].
Method A, general procedure for amide formation. To a solution of bromoben-
zoylchloride (2 mmol) was added the corresponding N-methylaniline (2 mmol) followed by
Et3N (2 mmol) in CH2Cl2 (10 mL) at 0°C. After a few minutes, the ice bath was removed and
the reaction mixture was warmed up to room temperature and stirred at room temperature
overnight. The reaction mixture was extracted twice with CH2Cl2 (2 × 15 mL). The organic
layer was dried over MgSO4, filtered and the solution was concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography using hexanes and EtOAc
as eluent or by trituration in a mixture of diethyl ether / petroleum ether to afford the desired
compound.
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
Method B, general procedure for Suzuki-Miyaura coupling. In a sealed tube the previ-
ously prepared bromo-N-heteroarylcarboxamide derivative (1 eq.) was introduced, followed by
the corresponding boronic acid (1.5 eq.), cesium carbonate (3 eq.), tetrakis(triphenylpho-
sphine)palladium (0.02 eq.) and a mixture of DME/EtOH/H2O (1:1:1, v:v:v, 3 mL) as solvent.
The reactor was flushed with N2 and submitted to microwave irradiation (150°C, 150 W) for
20 minutes. After cooling to room temperature, a mixture of EtOAc/H2O (1:1, v:v, 2 mL) was
added to stop the reaction. The aqueous layer was extracted with EtOAc (3 × 10 mL). The
organic layer was washed once with brine and once with water, dried over MgSO4, filtered and
the solution was concentrated under reduced pressure. The residue was purified by column
chromatography using hexanes and EtOAc as eluent to afford the desired compound.
Detailed synthesis procedures of the compounds
4-Bromo-N-(4-methoxyphenyl)-N-methylbenzamide (11b). The title compound was
prepared by reaction of 4-bromobenzoyl chloride (5c) (1272 mg, 5.8mmol) and 4-methoxy-N-
methylaniline (11d) (400 mg, 2.9mmol) according to method A. The residue was purified by
silica gel column chromatography (hexanes/EtOAc80:20) to afford the desired product as yel-
low solid (932 mg, 99%). C15H14BrNO2; MW 320; mp: 61–62°C; MS (ESI) 320, 322 [M]+; IR
(cm-1) 1635, 2840, 2850, 2917;1H NMR (C2D6CO, 500 MHz) δ (ppm) 3.36 (s, 3H), 3.74 (s,
3H), 6.82 (d, J = 9.0 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0
Hz, 2H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 38.5, 55.7, 115.2, 123.7, 129.3, 131.4, 131.6,
137.1, 138.6, 159.1, 169.5.
4-Bromo-N-(3-methoxyphenyl)-N-methylbenzamide (6b). The title compound was pre-
pared by reaction of 4-bromobenzoyl chloride(5c) (1272 mg, 5.8mmol) and 3-methoxy-N-
methylaniline (6d) (400 mg, 2.9mmol) according to method A. The residue was purified by sil-
ica gel column chromatography (hexanes/EtOAc80:20) to afford the desired product as yellow
solid (935 mg, quantitative). C15H14BrNO2; MW 320; mp: 98–100°C; MS (ESI) 320, 322 [M]+;
IR (cm-1) 1638, 2840, 2945, 3012;1H NMR (C2D6CO, 500 MHz) δ (ppm) 3.40 (s, 3H), 3.71 (s,
3H), 6.70–6.72 (m, 1H), 6.74–6.78 (m, 2), 7.17 (t,J = 8.0 Hz, 1H), 7.26–7.28 (m, 2H), 7.39–7.41
(m, 2H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 38.2, 55.7, 113.2, 113.8, 120.2, 123.9, 130.7,
131.3, 131.6, 137.0, 147.0, 161.2, 169.4.
4-Bromo-N-methyl-N-(m-tolyl)benzamide (5b). The title compound was prepared by
reaction of 4-bromobenzoyl chloride(5c) (724 mg, 3.3mmol) and N-methyl-m-toluidine (5d)
(200 mg, 1.6mmol) according to method A. The residue was purified by silica gel column chro-
matography (hexanes/EtOAc80:20) to afford the desired product as white solid (500 mg, 99%).
C15H14BrNO; MW 304; mp: 90–92C; MS (ESI) 304, 306 [M]+; IR (cm-1) 1586, 1638, 2853,
2923, 3058;1H NMR (C2D6CO, 300 MHz) δ (ppm) 2.24 (s, 3H), 3.39 (s, 3H), 6.90–6.93 (d,
J = 8.0Hz, 1H), 6.99–7.05 (m, 2H), 7.14 (t, J = 8.0 Hz, 1H), 7.23–7.26 (m, 2H), 7.37–7.40 (m,
2H); 13C NMR (C2D6CO, 75 MHz) δ (ppm) 21.2, 38.4, 123.9, 125.2, 128.1, 128.5, 129.8, 131.4,
131.6, 137.0, 140.0, 145.8, 169.4.
4-Bromo-N-(3-methoxybenzyl)-N-methylbenzamide (25b). The title compound was
prepared by reaction of 4-bromobenzoyl chloride (5c) (579 mg, 2.6mmol) and 3-methoxy-N-
methylbenzylamine (25d) (200 mg, 1.3mmol) according to method A. The residue was purified
by silica gel column chromatography (hexanes/EtOAc80:20) to afford the desired product as
yellow oil(430 mg, 97%).C16H16BrNO2; MW 334; MS (ESI) 334, 336 [M]+; IR (cm-1) 1630,
2834, 2923;1H NMR (C2D6CO, 500 MHz) δ(ppm) 2.91 (s, 3H), 3.79 (s, 3H), 4.52–4.70 (m,
2H), 6.76–6.80 (m, 1H), 6.86 (dd, J = 8.8 Hz, 5Hz, 1H), 6.95 (s, 1H), 7.28 (t, J = 8.0 Hz, 1H),
7.44 (d, J = 7.6 Hz, 2H), 7.62 (s, 2H); 13C NMR (C2D6CO, 125 MHz) δ(ppm) 37.2, 51.0, 55.5,
113.3, 113.6, 114.4, 119.7, 121.0, 123.9, 129.9, 130.5, 132.3, 137.0, 161.1.
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
N-(4-bromophenyl)-3-methoxy-N-methylbenzamide (24b). The title compound was
prepared by reaction of 3-methoxybenzoyl chloride (24c) (336 mg, 2.0mmol) and 4-bromo-N-
methylaniline(24d) (367 mg, 2.0mmol) according to method A. The residue was purified by sil-
ica gel column chromatography (hexanes/EtOAc80:20) to afford the desired product as white
solid (440 mg, 70%). C15H14BrNO2; MW 320; mp: 97–100°C; MS (ESI) 320, 322 [M]+; IR (cm-
1) 1634, 2834, 2929, 3009;1H NMR (C2D6CO, 300 MHz) δ(ppm) 3.41 (s, 3H), 3.69 (s, 3H),
6.84–6.88 (m, 3H), 7.12–7.17 (m, 3H), 7.44–7.47 (m, 2H); 13C NMR (C2D6CO, 75 MHz) δ
(ppm) 38.3, 55.6, 114.8, 116.3, 120.1, 121.7, 129.8, 130.0, 133.1, 138.8, 145.7, 160.1, 170.3.
N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (8a). The title com-
pound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenzamide (6b)
(80 mg, 0.25mmol), phenylboronic acid (40 mg, 0.33mmol), cesium carbonate (244 mg,
0.75mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg) in DME/EtOH/H2O
(1/1/1/, 3 mL) according to method B. The residue was purified by silica gel column chroma-
tography (hexanes/EtOAc70:30) to afford the desired product as yellow solid (77 mg, 97%).
C21H19NO2; MW 317; mp: 102–104°C; MS (ESI) 318 [M+H]+; IR (cm-1) 1588, 1636, 2831,
2917, 2960, 3040;1H NMR (C2D6CO, 300 MHz) δ (ppm) 3.44 (s, 3H), 3.70 (s, 3H), 6.72–6.77
(m, 2H), 6.80 (t, J = 2 Hz, 1H), 7.17 (t, J = 8 Hz, 1H), 7.33–3.37 (s, 1H), 7.41–7.46 (m, 4H),
7.51–7.54 (m, 2H), 7.60–7.63 (m, 2H); 13C NMR (C2D6CO, 75 MHz) δ (ppm) 38.4, 55.7, 113.0,
113.8, 120.1, 126.9, 127.7, 128.6, 129.8, 130.1, 130.6, 136.7, 140.8, 142.6, 147.4, 161.2, 170.2.
2'-Methoxy-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (9a). The
title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenza-
mide (6b) (112 mg, 0.35mmol), 2-methoxyphenylboronic acid (70 mg, 0.46mmol), cesium car-
bonate (342 mg, 1.05mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 8 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc70:30) to afford the desired product as colorless oil (113 mg, 93%). C22H21NO3;
MW 347; MS (ESI) 348 [M+H]+; IR (cm-1) 1597, 1640, 2837, 2939;1H NMR (C2D6CO, 500
MHz) δ (ppm) 3.44 (s, 3H), 3.69 (s, 3H), 3.77 (s, 3H), 6.73–6.78 (m, 3H), 6.99 (dt, J = 1.0, 7.3
Hz, 1H), 7.07 (dd, J = 1.0, 8.3 Hz, 1H), 7.18 (dt, J = 1.0, 7.5 Hz, 1H), 7.24 (dd, J = 2.0, 7.5 Hz,
1H), 7.30–7.34 (m, 1H), 7.35–7.39 (m, 4H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 38.4, 55.6,
55.9, 112.5, 113.0, 113.7, 120.0, 121.7, 129.1, 129.5, 130.0, 130.4, 130.6, 131.3, 136.0, 140.7,
147.4, 157.5, 161.1, 170.4.
3'-Methoxy-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (7a). The
title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenza-
mide (6b) (81 mg, 0.25mmol), 3-methoxyphenylboronic acid (46 mg, 0.3mmol), cesium car-
bonate (247 mg, 0.75mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc70:30) to afford the desired product as white solid (74 mg, 85%). C22H21NO3; MW
347; mp: 131–134°C; MS (ESI) 348 [M+H]+; IR (cm-1) 1582, 1635, 2837, 2941, 2963;1H NMR
(C2D6CO, 500 MHz) δ(ppm) 3.43 (s, 3H), 3.70 (s, 3H), 3.84 (s, 3H), 6.73–6.76 (m, 2H), 6.80–
6.81 (m, 1H), 6.92(ddd, J = 1.0, 2.0, 8.0 Hz, 1H), 7.14–7.19 (m, 3H), 7.32–7.35 (m, 1H), 7.41–
7.42 (m, 2H), 7.51–7.53 (m, 2H); 13C NMR (C2D6CO, 125 MHz) δ(ppm) 38.4, 55.6, 55.7,
113.0, 113.1, 113.8, 114.3, 120.0, 120.1, 126.9, 130.0, 130.6, 130.8, 136.8, 142.2, 142.5, 147.37,
147.38, 161.2, 170.2.
3'-Hydroxy-N-(3-hydroxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (7). To a
solution of 3'-methoxy-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (7a)
(100 mg, 0.29mmol) in CH2Cl2 (10 mL) was added trifluorobromine dimethyl sulphide com-
plex BF3.SMe2 (0.36 mL, 3.46 mM) at room temperature. The reaction mixture was stirred
overnight. The reaction was quenched with MeOH (10 mL). The solvent was removed under
reduced pressure at 25°C. Water was added to dissolve inorganic salts and the precipitate was
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
filtered off and triturated with diethyl ether to afford the desired product as white solid (80 mg,
87%). C20H17NO3; MW 319; mp: 168–171°C; MS (ESI) 320[M+H]+; IR (cm-1) 1589, 3191;1H
NMR (C2D6CO, 300 MHz) δ(ppm) 3.42 (s, 3H), 6.64–6.68 (m, 3H), 6.82–6.86 (m, 1H), 7.07–
7.12 (m, 3H), 7.26 (t, J = 8.0 Hz, 1H), 7.39–7.44 (m, 2H), 7.47–7.49 (m, 2H), 8.42 (s, 1H), 8.48
(s, 1H); 13C NMR (C2D6CO, 75 MHz) δ(ppm) 38.4, 114.4, 114.6, 115.0, 115.6, 119.0, 119.1,
126.8, 130.1, 130.7, 130.8, 136.6, 142.3, 142.7, 147.4, 158.8, 158.9, 170.1.
4'-Methoxy-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (10a). The
title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenza-
mide (6b) (123 mg, 0.38mmol), 4-methoxyphenylboronic acid (75 mg, 0.49mmol), cesium car-
bonate (371 mg, 1.14mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 9 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc80:20) to afford the desired product as white solid (70 mg, 53%). C22H21NO3; MW
347; mp: 128–130°C; MS (ESI) 348 [M+H]+; IR (cm-1) 1601, 1625, 2840, 2935, 2963;1H NMR
(C2D6CO, 500 MHz) δ (ppm) 3.43 (s, 3H), 3.70 (s, 3H), 3.82(s, 3H), 6.72–6.75 (m, 2H), 6.78–
6.79 (m, 1H), 6.98 (d, J = 2.3 Hz, 1H), 7.00 (d, J = 2.3 Hz, 1H), 7.15–7.18 (m, 1H), 7.37–7.40
(m, 2H), 7.46–7.48 (m, 2H), 7.55–7.58 (m,2H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 38.4,
55.6, 55.7, 112.9, 113.8, 115.2, 120.1, 126.3, 128.8, 130.1, 130.6, 130.1, 135.9, 142.4, 147.5, 160.7,
161.2, 170.3.
3'-Fluoro-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (13a). The
title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenza-
mide (6b) (100 mg,0.31mmol), 3-fluorophenylboronic acid (57 mg, 0.41mmol), cesium car-
bonate (303 mg, 0.93mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 7 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc70:30) to afford the desired product as yellow solid (91 mg, 89%). C21H18FNO2;
MW 335; mp: 97–100°C; MS (ESI) 336 [M+H]+; IR (cm-1) 1587, 1637, 2834, 2923, 2960;1H
NMR (C2D6CO, 300 MHz) δ (ppm) 3.44 (s, 3H), 3.71 (s, 3H), 6.73–6.77 (m, 2H), 6.80–6.81
(m, 1H), 7.09–7.21 (m, 2H), 7.36–7.50 (m, 5H), 7.55–7.57 (m, 2H); 13C NMR (C2D6CO, 75
MHz) δ (ppm) 38.4, 55.8, 113.1, 113.9, 114.3, 114.6, 115.2, 115.4, 120.2, 123.7, 127.1, 130.2,
130.8, 131.6, 131.8, 137.4, 141.2, 143.3, 143.4, 147.4, 161.3, 162.6, 165.8, 170.1.
3'-Chloro-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (14a).
The title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methyl-
benzamide (6b) (80 mg, 0.25mmol), 3-chlorophenylboronic acid (52 mg, 0.33mmol), cesium
carbonate (244 mg, 0.75mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc70:30) to afford the desired product as colorless oil (47 mg, 53%). C21H18ClNO2;
MW 352; MS (ESI) 352, 354 [M]+; IR (cm-1) 1594, 1639, 2837, 2938;1H NMR (C2D6CO, 500
MHz) δ (ppm) 3.43 (s, 3H), 3.70 (s, 3H), 6.73–6.76 (m, 2H), 6.80–6.81(m, 1H), 7.17 (t,J = 8 Hz,
1H), 7.38–7.40 (m, 1H), 7.43–7.47 (m, 3H), 7.54–7.59 (m, 3H), 7.63–7.64 (m, 1H); 13C NMR
(C2D6CO, 125 MHz) δ (ppm) 38.3, 55.7, 113.1, 113.8, 120.2, 126.3, 127.0, 127.6, 128.5, 130.1,
130.7, 131.4, 135.3, 137.4, 141.0, 142.9, 147.3, 161.2, 170.0.
N-(3-methoxyphenyl)-N,3'-dimethyl-[1,1'-biphenyl]-4-carboxamide (6a). The title
compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenzamide
(6b) (77 mg, 0.24mmol), 3-methylphenylboronic acid (42 mg, 0.31mmol), cesium carbonate
(235 mg, 0.72mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg) according to
method B. The residue was purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford the desired product as colorless oil (56 mg, 71%). C22H21NO2; MW 331; MS
(ESI) 332 [M+H]+; IR (cm-1) 1586, 1640, 2840, 2929;1H NMR (C2D6CO, 300 MHz) δ(ppm)
3.38 (s, 3H), 3.44 (s, 3H), 3.70 (s, 3H), 6.74–6.76 (m, 2H), 6.79–6.80 (m, 1H), 7.15–7.20 (m,
2H), 7.31 (t,J = 8 Hz, 1H), 7.39–7.43 (m, 4H), 7.49–7.52 (m, 2H); 13C NMR (C2D6CO, 75
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
MHz) δ(ppm) 21.5, 38.4, 55.7, 113.0, 113.9, 120.2, 124.9, 126.9, 128.5, 129.4, 129.8, 130.1,
130.7, 136.6, 137.4, 140.9, 142.9, 147.5, 161.3, 170.3.
3'-(Dimethylamino)-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide
(15a). The title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-
methylbenzamide (6b) (80 mg, 0.25mmol), 3-(dimethylamino)phenylboronic acid (54 mg,
0.33mmol), cesium carbonate (244 mg, 0.75mmol) and tetrakistriphenylphosphine palladium
(0.02 eq., 6 mg) according to method B. The residue was purified by silica gel column chroma-
tography (hexanes/EtOAc70:30) to afford the desired product as yellow oil (49 mg, 54%).
C23H24N2O2; MW 360; MS (ESI) 361 [M+H]+; IR (cm-1) 1597, 1639, 2797, 2834, 2939;1H
NMR (C2D6CO, 300 MHz) δ (ppm) 2.97 (s, 6H), 3.43 (s, 3H), 3.70 (s, 3H), 6.72–6.76 (m, 3H),
6.79 –-6.80 (m, 1H), 6.86–6.93 (m, 2H), 7.17 (t,J = 8.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.38–
7.41 (m, 2H), 7.49–7.52 (m, 2H); 13C NMR (C2D6CO, 75 MHz) δ (ppm) 38.5, 40.8, 55.7, 111.8,
113.0, 113.9, 116.0, 120.2, 127.0, 130.0, 130.4, 130.7, 136.5, 141.6, 143.9, 147.5, 152.2, 161.3,
170.4.
N-(3-methoxyphenyl)-N,3',4'-trimethyl-[1,1'-biphenyl]-4-carboxamide (17a). The title
compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenzamide
(6b) (100 mg, 0.31mmol), 3,4-dimethylphenylboronic acid (62 mg, 0.41mmol), cesium carbon-
ate (303 mg, 0.93mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 7 mg) according
to method B. The residue was purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford the desired product as yellow oil (106 mg, 99%). C23H23NO2; MW 345; MS
(ESI) 346[M+H]+; IR (cm-1) 1599, 1640, 2840, 2914, 2942;1H NMR (C2D6CO, 500 MHz) δ
(ppm) 2.26 (s, 3H), 2.29 (s, 3H), 3.43 (s, 3H), 3.70 (s, 3H), 6.73–6.75 (m, 2H), 6.79 –-6.80 (m,
1H), 7.15–7.19 (m, 2H), 7.32 (dd, J = 2.0Hz, 8.0Hz, 1H), 7.38–7.40 (m, 3H), 7.47–7.49 (m, 2H);
13C NMR (C2D6CO, 125 MHz) δ(ppm) 19.4, 19.8, 38.4, 55.6, 112.9, 113.8, 120.1, 125.0, 126.5,
128.8, 130.0, 130.6, 131.0, 136.2, 137.0, 137.8, 138.3, 142.8, 147.4, 161.2, 170.3.
3',4'-Difluoro-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (18a).
The title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methyl-
benzamide (6b) (100 mg, 0.31mmol), 3,4-difluorophenylboronic acid (65 mg, 0.41mmol),
cesium carbonate (303 mg, 0.93mmol) and tetrakistriphenylphosphine palladium (0.02 eq.,
7 mg) according to method B. The residue was purified by silica gel column chromatography
(hexanes/EtOAc70:30) to afford the desired product as yellow oil (97 mg, 88%). C21H17F2NO2;
MW 353; MS (ESI) 354[M+H]+; IR (cm-1) 1599, 1639, 2837, 2948;1H NMR (C2D6CO, 500
MHz) δ (ppm) 3.43 (s, 3H), 3.70 (s, 3H), 6.73–6.76 (m, 2H), 6.80 –-6.81 (m, 1H), 7.17 (t, J =
8.0 Hz, 1H), 7.35–7.41 (m, 1H), 7.42–7.44 (m, 2H), 7.45–7.48 (m, 1H), 7.52–7.54 (m, 2H), 7.56
(dd, J = 2.0 Hz, 8.0 Hz, 1H), 7.60 (dd, J = 2.0 Hz, 8.0 Hz, 1H); 13C NMR (C2D6CO, 125 MHz)
δ (ppm) 38.3, 55.7, 113.0, 113.8, 116.6, 116.7, 118.5, 118.7, 120.2, 124.3, 124.4, 126.9, 130.1,
130.7, 137.3, 140.3, 147.3, 161.2, 170.0.
3',4'-Dimethoxy-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide
(16a). The title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-
methylbenzamide (6b) (100 mg, 0.31mmol), 3,4-dimethoxyphenylboronic acid (75 mg,
0.41mmol), cesium carbonate (303 mg, 0.93mmol) and tetrakistriphenylphosphine palladium
(0.02 eq., 7 mg) according to method B. The residue was purified by silica gel column chroma-
tography (hexanes/EtOAc70:30) to afford the desired product as white solid (83 mg, 71%).
C23H23NO4; MW 377; mp: 153–156°C; MS (ESI) 378[M+H]+; IR (cm-1) 1604, 1635, 2843,
2938, 2969, 3003, 3068;1H NMR (C2D6CO, 300 MHz) δ(ppm) 3.51 (s, 3H), 3.69 (s, 3H), 3.90
(s, 3H), 3.92 (s, 3H), 6.62–6.72 (m, 3H), 6.89–6.91 (d, J = 8.0 Hz, 1H), 7.04–7.17 (m, 3H), 7.38
(s, 4H); 13C NMR (C2D6CO, 75 MHz) δ(ppm) 38.6, 55.6, 56.1, 110.4, 111.6, 112.2, 113.0, 119.4,
119.6, 126.1, 129.4, 130.0, 133.2, 134.4, 142.2, 146.3, 149.2, 149.3, 160.2, 170.5.
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
2'-Fluoro-3'-methoxy-N-(3-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide
(19a). The title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-
methylbenzamide (6b) (80 mg, 0.25mmol), 2-fluoro-3-methoxyphenylboronic acid (56 mg,
0.33mmol), cesium carbonate (244 mg, 0.75mmol) and tetrakistriphenylphosphine palladium
(0.02 eq., 6 mg) according to method B. The residue was purified by silica gel column chroma-
tography (hexanes/EtOAc 90:10) to afford the desired product as brown solid (43 mg, 47%).
C22H20FNO3; MW 365; mp: 125–127°C; MS (ESI) 366[M+H]+; IR (cm-1) 1592, 1644, 2837,
2948, 2975, 3009;1H NMR (C2D6CO, 300 MHz) δ(ppm) 3.44 (s, 3H), 3.70 (s, 3H), 3.90 (s, 3H),
6.72–6.81 (m, 3H), 6.95–7.00 (m, 1H), 7.10–7.20 (m, 3H), 7.39 –-7.45 (m, 4H); 13C NMR
(C2D6CO, 75 MHz) δ(ppm) 38.3, 55.7, 56.6, 113.1, 113.8, 113.9, 120.1, 122.4, 125.2, 125.3,
129.0, 129.1, 129.6, 130.6, 137.1, 137.5, 147.3, 161.2, 170.1.
2'-Fluoro-N-(3-methoxyphenyl)-N,3'-dimethyl-[1,1'-biphenyl]-4-carboxamide (20a).
The title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methyl-
benzamide (6b) (72 mg, 0.22mmol), 2-fluoro-3-methylphenylboronic acid (45 mg, 0.29mmol),
cesium carbonate (215 mg, 0.66mmol) and tetrakistriphenylphosphine palladium (0.02 eq.,
5 mg) according to method B. The residue was purified by silica gel column chromatography
(hexanes/EtOAc70:30) to afford the desired product as yellow solid (70 mg, 91%). C22H20
FNO2; MW 349; mp: 91–94°C; MS (ESI) 350 [M+H]+; IR (cm-1) 1599, 1635, 2840, 2920, 2963,
3055;1H NMR (C2D6CO, 500 MHz) δ (ppm) 2.29 (d, J = 2.5 Hz, 3H), 3.44 (s, 3H), 3.70 (s, 3H),
6.73–6.77 (m, 2H), 6.79–6.80 (m, 1H), 7.13 (t, J = 8.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.25 (t,
J = 8.0 Hz, 1H), 7.39–7.44 (m, 4H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 14.6, 14.7, 38.3,
55.7, 113.1, 113.8, 120.1, 125.0, 125.1, 126.2, 126.3, 128.6, 128.7, 129.0, 129.07, 129.09, 129.6,
130.6, 131.9, 132.0, 137.0, 137.9, 147.3, 157.9, 159.9, 170.1.
4-(Benzo[d][1,3]dioxol-5-yl)-N-(3-methoxyphenyl)-N-methylbenzamide (22a). The
title compound was prepared by reaction of 4-bromo-N-(3-methoxyphenyl)-N-methylbenza-
mide (6b) (80 mg, 0.25mmol), benzo[d][1,3]dioxol-5-ylboronic acid (55 mg, 0.33mmol),
cesium carbonate (244 mg, 0.75mmol) and tetrakistriphenylphosphine palladium (0.02 eq.,
6 mg) according to method B. The residue was purified by silica gel column chromatography
(hexanes/EtOAc70:30) to afford the desired product as yellow solid (68 mg, 76%). C22H19NO4;
MW 361; mp: 139–141°C; MS (ESI) 362 [M+H]+; IR (cm-1) 1592, 1644, 2837, 2948, 2975,
3009;1H NMR (C2D6CO, 500 MHz) δ(ppm) 3.43 (s, 3H), 3.70 (s, 3H), 6.02 (s, 2H), 6.72–6.73
(m, 1H), 6.74–6.75 (m, 1H), 6.79 –-6.80 (m, 1H), 6.89–6.90 (m, 1H), 7.10–7.12 (m, 2H), 7.17
(t, J = 8.0 Hz, 1H), 7.37–7.39 (m, 2H), 7.44–7.46 (m, 2H); 13C NMR (C2D6CO, 125 MHz)
δ(ppm) 38.4, 55.7, 102.3, 108.0, 109.4, 112.9, 113.8, 120.1, 121.4, 126.5, 130.0, 130.6, 135.0,
136.2, 142.4, 147.4, 148.5, 149.3 161.2, 170.2.
3'-Methoxy-N-(4-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (11a). The
title compound was prepared by reaction of 4-bromo-N-(4-methoxyphenyl)-N-methylbenza-
mide (11b) (100 mg, 0.31mmol), 3-methoxyphenylboronic acid (61 mg, 0.40mmol), cesium
carbonate (303 mg, 0.93mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 7 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc70:30) to afford the desired product as colorlessoil (106 mg, 98%). C22H21NO3;
MW 347; MS (ESI) 348 [M+H]+; IR (cm-1) 1635, 2840, 2935;1H NMR (C2D6CO, 300 MHz)
δ (ppm) 3.40 (s, 3H), 3.75 (s, 3H), 3.85 (s, 3H), 6.82–6.86 (m, 2H), 6.92 (ddd, J = 1.0 Hz, 2.6
Hz, 8.3 Hz, 1H), 7.12–7.19 (m, 4H), 7.32–7.40 (m, 3H), 7.50–7.53 (m, 2H); 13C NMR
(C2D6CO, 75 MHz) δ (ppm) 38.7, 55.6, 55.7, 113.1, 114.3, 115.2, 120.0, 126.9, 129.2, 130.1,
130.8, 136.9, 139.0, 142.2, 142.3, 159.0, 161.2, 170.2.
4'-Methoxy-N-(4-methoxyphenyl)-N-methyl-[1,1'-biphenyl]-4-carboxamide (12a). The
title compound was prepared by reaction of 4-bromo-N-(4-methoxyphenyl)-N-methylbenza-
mide (11b) (78 mg, 0.24mmol), 4-methoxyphenylboronic acid (48 mg, 0.32mmol), cesium
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
carbonate (235 mg, 0.72mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg)
according to method B. The residue was purified by silica gel column chromatography (hex-
anes/EtOAc 90:10) to afford the desired product as yellow solid (73 mg, 88%). C22H21NO3;
MW 347; mp: 129–132°C; MS (ESI) 348 [M+H]+; IR (cm-1) 1635, 2840, 2932, 2957;1H NMR
(C2D6CO, 300 MHz) δ(ppm) 3.39 (s, 3H), 3.74 (s, 3H), 3.83 (s, 3H), 6.82–6.82 (m, 2H), 6.98–
7.01 (m, 2H), 7.11–7.14 (m, 2H), 7.34–7.37 (m, 2H), 7.44–7.47 (m, 2H), 7.54–7.57 (m, 2H);
13C NMR (C2D6CO, 75 MHz) δ(ppm) 38.7, 55.6, 55.7, 115.2, 126.2, 128.8, 129.2, 130.2, 133.0,
135.9, 139.1, 142.1, 159.0, 160.6, 170.3.
3'-Methoxy-N-methyl-N-(m-tolyl)-[1,1'-biphenyl]-4-carboxamide (5a). The title com-
pound was prepared by reaction of 4-bromo-N-(3-methylphenyl)-N-methylbenzamide (5b)
(123 mg, 0.40mmol), 3-methoxyphenylboronic acid (79 mg, 0.52mmol), cesium carbonate
(393 mg, 1.20mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 9 mg) according to
method B. The residue was purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford the desired product as yellow oil (101 mg, 77%). C22H21NO2; MW 331; MS
(ESI) 332 [M+H]+; IR (cm-1) 1582, 1601, 1639, 2834, 2935, 3034;1H NMR (C2D6CO, 300
MHz) δ(ppm) 2.24 (s, 3H), 3.42 (s, 3H), 3.84 (s, 3H), 6.90–7.00 (m, 3H), 7.08 (s, 1H), 7.11–7.18
(m, 3H), 7.33 (t, J = 8.0 Hz, 1H), 7.38–7.41 (m, 2H), 7.49–7.52 (m, 2H); 13C NMR (C2D6CO,
75 MHz) δ(ppm) 21.2, 38.5, 55.6, 113.1, 114.3, 120.0, 125.1, 126.9, 127.9, 128.4, 129.8, 130.1,
130.8, 136.7, 139.9, 142.3, 142.5, 146.2, 161.2, 170.2.
2'-Fluoro-N,3'-dimethyl-N-(m-tolyl)-[1,1'-biphenyl]-4-carboxamide (21a). The title
compound was prepared by reaction of 4-bromo-N-(3-methylphenyl)-N-methylbenzamide
(5b) (93 mg, 0.31mmol), 2-fluoro-3-methylphenylboronic acid (62 mg, 0.40mmol), cesium
carbonate (303 mg, 0.93mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 7 mg)
according to method B. The residue was purified by silica gel column chromatography
(hexanes/EtOAc70:30) to afford the desired product as white solid (103 mg, quantitative).
C22H20FNO; MW 333; mp: 73–76°C; MS (ESI) 334 [M+H]+; IR (cm-1) 1604, 1634, 2853,
2920;1H NMR (C2D6CO, 500 MHz) δ (ppm) 2.24 (s, 3H), 2.28 (d, J = 2.4 Hz, 3H), 3.43 (s, 3H),
6.94–6.97 (m, 1H), 6.98–7.01 (m, 1H), 7.07 (s, 1H), 7.11–7.16 (m, 2H), 7.23 –-7.26 (m, 2H),
7.38–7.41 (m, 4H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 14.6, 14.7, 21.2, 38.5, 125.05,
125.09, 125.12, 126.2, 126.3, 128.5, 128.6, 128.7, 128.99, 129.01, 129.07, 129.09, 129.6, 129.8,
131.9, 132.0, 136.9, 137.8, 139.9, 146.1, 157.9, 159.9, 170.1.
4-(Benzo[d][1,3]dioxol-5-yl)-N-methyl-N-(m-tolyl)benzamide (22a). The title com-
pound was prepared by reaction of 4-bromo-N-(3-methylphenyl)-N-methylbenzamide (5b)
(79 mg, 0.26mmol), benzo[d][1,3]dioxol-5-ylboronic acid (56 mg, 0.34mmol), cesium carbon-
ate (254 mg, 0.78mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg) according
to method B. The residue was purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford the desired product as yellow solid (44 mg, 49%). C22H19NO3; MW 345; mp:
111–113°C; MS (ESI) 346[M+H]+; IR (cm-1) 1602, 1634, 2788, 2911, 2966;1H NMR (C2D6CO,
500 MHz) δ (ppm) 2.24 (s, 3H), 3.42 (s, 3H), 6.02 (s, 2H), 6.89 (d, J = 8.0 Hz, 1H), 6.94 (d,
J = 8.0 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 7.07–7.11 (m, 3H), 7.14 (t, J = 8.0 Hz, 1H), 7.35–7.37
(m, 2H), 7.42–7.44 (m, 2H); 13C NMR (C2D6CO, 125 MHz) δ (ppm) 21.2, 38.5, 102.3, 107.9,
109.4, 121.4, 125.1, 126.5, 127.9, 128.4, 129.8, 130.2, 135.1, 136.2, 139.9, 142.3, 146.3, 148.5,
149.3, 170.2.
2'-Fluoro-N-(3-methoxybenzyl)-N,3'-dimethyl-[1,1'-biphenyl]-4-carboxamide (25a).
The title compound was prepared by reaction of 4-bromo-N-(3-methoxybenzyl)-N-methyl-
benzamide (25b) (200 mg, 0.60mmol), 2-fluoro-3-methylphenylboronic acid (120 mg,
0.78mmol), cesium carbonate (587 mg, 1.80mmol) and tetrakistriphenylphosphine palladium
(0.02 eq., 14 mg) according to method B. The residue was purified by silica gel column chroma-
tography (hexanes/EtOAc70:30) to afford the desired product as yellow oil (200 mg, 87%).
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
C23H22FNO2; MW 363; MS (ESI) 364 [M+H]+; IR (cm-1) 1630, 2834, 2923, 3034;1H NMR
(C2D6CO, 500 MHz) δ(ppm) 2.32 (s, 3H), 2.97 (s, 3H), 3.81 (s, 3H), 4.59–4.73 (m, 2H), 6.80–
7.00 (m, 3H), 7.17 (t,J = 8 Hz, 1H), 7.27–7.32 (m, 2H), 7.33–7.36 (m, 1H), 7.57–7.59 (m, 2H),
7.62 (s, 2H); 13C NMR (C2D6CO, 125 MHz) δ(ppm) 14.6, 14.7, 37.3, 51.0, 55.5, 113.6, 114.4,
121.0, 125.1, 125.2, 126.2, 126.3, 128.1, 128.7, 128.8, 129.1, 129.2, 129.7, 129.8, 130.5, 132.0,
132.1, 137.1, 138.0, 158.0, 159.9, 161.1.
2'-Fluoro-N-(3-hydroxybenzyl)-N,3'-dimethyl-[1,1'-biphenyl]-4-carboxamide (25). To
a solution of 2'-fluoro-N-(3-methoxybenzyl)-N,3'-dimethyl-[1,1'-biphenyl]-4-carboxamide
(25a) (100 mg, 0.28mmol) in CH2Cl2 (10 mL) was added trifluorobromine dimethyl sulphide
complex BF3.SMe2 (0.18 mL, 1.68mM) at room temperature. The reaction mixture was stirred
overnight. The reaction was quenched with MeOH (10 mL). The solvent was removed under
reduced pressure at 25°C. Water was added to dissolve inorganic salts and the precipitated was
filtered off and triturated with diethyl ether to afford the desired product as yellow solid (66
mg, 67%). C22H20FNO2; MW 349; mp: 123–126°C; MS (ESI) 350 [M+H]+; IR (cm-1) 1600,
2917, 3181;1H NMR (C2D6CO, 500 MHz) δ (ppm) 2.32 (s, 3H), 2.96 (s, 3H), 4.55–4.69 (m,
2H), 6.72–6.90 (m, 3H), 7.16–7.22 (m, 2H), 7.29 (t, J = 8.0 Hz, 1H), 7.33 –-7.36 (m, 1H), 7.57–
7.58 (m, 2H), 7.62 (s, 2H); 13C NMR (C2D6CO, 75 MHz) δ (ppm) 14.6, 14.7, 115.2, 125.1,
125.2, 126.2, 126.4, 128.0, 128.7, 128.9, 129.17, 129.21, 129.7, 129.8, 130.6, 132.0, 132.1, 137.0,
138.0, 157.3, 158.6, 160.6
3-Methoxy-N-(3'-methoxy-[1,1'-biphenyl]-4-yl)-N-methylbenzamide (24a). The title
compound was prepared by reaction of N-(4-bromophenyl)-3-methoxy-N-methylbenzamide
(24b) (80 mg, 0.25mmol), 3-methoxyphenylboronic acid (50 mg, 0.33mmol), cesium carbonate
(244 mg, 0.75mmol) and tetrakistriphenylphosphine palladium (0.02 eq., 6 mg) according to
method B. The residue was purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford the desired product as yellow oil (59 mg, 68%). C22H21NO3; MW 347; MS
(ESI) 348 [M+H]+; IR (cm-1) 1640, 2840, 2936;1H NMR (C2D6CO, 300 MHz) δ(ppm) 3.45 (s,
3H), 3.66 (s, 3H), 3.85 (s, 3H), 6.80 –-6.84 (m, 1H), 6.90–6.94 (m, 3H), 7.09–7.19 (m, 3H),
7.24–7.27 (m, 2H), 7.34 (t, J = 8.0 Hz, 1H), 7.57–7.60 (m, 2H); 13C NMR (C2D6CO, 75 MHz)
δ(ppm) 38.4, 55.6, 55.7, 113.2, 114.0, 114.9, 116.4, 120.0, 121.9, 128.3, 128.4, 129.7, 130.9,
139.0, 139.6, 142.3, 145.7, 160.1, 161.3, 170.3.
logP Determination
The logP values were calculated from ACD/Labs Percepta 2012 Release program. The loga-
rithm of partition constant P (log P) was calculated using the “GALAS” method (Global
Adjusted Locally According to Similarity). The program predicts clogP by comparing the mol-
ecule with structurally similar molecules where experimental data are known.
Biological methods
[2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were purchased from Perkin-Elmer, Boston. Quickszint
Flow 302 scintillator fluid was bought from Zinsser Analytic, Frankfurt. Other chemicals were
purchased from Sigma, Roth or Merck.
Ethics statement. Anonymized placental samples were obtained from Saarbrücken-Dud-
weiler Hospital’s Department of Gynecology. No author involved in this study has received
information about the patients. The microsomal fraction of the mouse enzyme (m17β-HSD2)
was obtained from mouse livers, which were bought from Pharmacelsus GmbH (Saarbrücken,
Germany).
h17β-HSD1 and h17β-HSD2 enzyme preparation. Cytosolic (h17β-HSD1) and micro-
somal (h17β-HSD2) fractions were obtained from human placenta according to previously
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
described procedures [20, 27]. Fresh tissue was homogenized and the enzymes were separated
from the mitochondria, cell membrane. Nucleus and other rests by fractional centrifugation at
1000 g, 10.000 g and 150.000 g. The pellet fraction containing the microsomal h17β-HSD2 was
used for the determination of h17β-HSD2 inhibition, while h17β-HSD1 was obtained after pre-
cipitation with ammonium sulfate from the cytosolic fraction for use of testing of h17β-HSD1
inhibition. Aliquots containing h17β-HSD1 or h17β-HSD2 were stored frozen.
m17β-HSD2 enzyme preparation and inhibition. The microsomal fraction (m17β-
HSD2) was obtained from mouse liver as described for h17β-HSD2. Inhibitory activities were
evaluated by a method identical to the one described for the human enzyme.
m17β-HSD1 enzyme preparation. Recombinant m17β-HSD1 enzyme was produced by
transfection of HEK 293 cells with a m17β-HSD1 expression plasmid (coding sequence of
NM_010475 in pCMV6Entry vector, OriGene Technologies, Inc.) according to a described
procedure[28]. 48 hours after transfection cells were homogenized by sonication (3 x 10 s) in a
buffer containing saccharose (40 mMTris, 250 mM saccharose, 5 mM EDTA, 7 mM DTT, 1
mM PMSF, pH 7,5). Cell lysate was centrifuged (1000 g, 15 min, 4°C) and 20% glycerol was
added to the supernatant before aliquots were frozen and stored at -70°C.
Inhibition of h17β-HSD2 and m17β-HSD2 in cell-free assay. Inhibitory activities were
evaluated following an established method with minor modifications[29–31]. Briefly, the
enzyme preparation was incubated with NAD+ [1500 μM] in the presence of potential inhibi-
tors at 37C in a phosphate buffer (50 mM) supplemented with 20% of glycerol and EDTA
1mM. Inhibitor stock solutions were prepared in DMSO. Final concentration of DMSO was
adjusted to 1% in all samples. The enzymatic reaction was started by addition of a mixture of
unlabelled- and [3H]-E2 (final concentration: 500 nM, 0.11 μCi). After 20 min, the incubation
was stopped with HgCl2 and the mixture was extracted with ether. After evaporation, the ste-
roids were dissolved in acetonitrile/water (45:55). E1 and E2 were separated using acetonitrile/
water (45:55) as mobile phase in a C18 RP chromatography column (Nucleodur C18, 3μm,
Macherey-Nagel, Düren) connected to a HPLC-system (Agilent 1100 Series, Agilent Technolo-
gies, Waldbronn). Detection and quantification of the steroids were performed using a radio-
flow detector (Berthold Technologies, Bad Wildbad). The conversion rate was calculated
according to the following equation: %conversion = (%E1/(%E1+%E2))×100. Each value was
calculated from at least two independent experiments.
Inhibition of h17β-HSD1 and m17β-HSD1 in cell-free assay. The 17β-HSD1 inhibition
assay was performed similarly to the h17β-HSD2 test. The human cytosolic enzyme was incu-
bated with NADH [500 μM] while the mouse recombinant enzyme was reacted with NADPH
[500 μM].Test compound and a mixture of unlabelled- and [3H]-E1 (final concentration: 500
nM, 0.15 μCi) were added and mixed for 10 min at 37°C. Further treatment of the samples and
HPLC separation was carried out as mentioned above for h17β-HSD2.
Inhibition of h17β-HSD2 in a cellular assay. Cellular h17β-HSD2 inhibitory activity is
measured using the breast cancer cell-line MDA-MB-231[32] (17β-HSD1 activity negligible).
[3H]-E2 (200 nM) is taken as substrate and is incubated with the inhibitor for 6 h at 37°C.
After ether extraction, substrate and product are separated by HPLC and detected with a radio-
flow detector. Potency is evaluated as percentage of inhibition (inhibitor concentrations used:
1250 nM and 250 nM).
Estrogen receptor affinity in a cell-free assay. The binding affinity of selected compounds
to ERα and ERβ was determined according to the recommendations of the US Environmental
Protection Agency (EPA) by their Endocrine Disruptor Screening Program (EDSP)[24] using
recombinant human proteins. Briefly, 1 nM of ERα and 4 nM of ERβ, respectively, were incu-
bated with [3H]-E2 (3 nM for ERα and 10 nM for ERβ) and test compound for 16–20 h at 4°C.
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Selective and Stable Inhibiotors of Human and Mouse 17β-HSD2
The potential inhibitors were dissolved in DMSO (5% final concentration). Evaluation of
non-specific-binding was performed with unlabeled E2 at concentrations 100-fold of [3H]-E2
(300 nM for ERα and 1000 nM for ERβ). After incubation, ligand-receptor complexes were
selectively bound to hydroxyapatite (83.5 g/LinTE-buffer). The bound complex was washed
three times and resuspended in ethanol. For radiodetection, scintillator cocktail (Quickszint
212, Zinsser Analytic, Frankfurt) was added and samples were measured in a liquid scintilla-
tion counter (1450 LSC & Luminescence Counter, Perkin Elmer).
From these results the percentage of [3H]-E2 displacement by the compounds was calcu-
lated. The plot of % displacement versus compound concentration resulted in sigmoidal bind-
ing curves. The compound concentrations necessary to displace 50% of the receptor bound
[3H]-E2 were determined. Unlabeled E2 IC50 values were determined in each experiment and
used as reference. The E2 IC50 determined were 3 20% nM for ERα and 10 20% nM for ERβ.
Relative Binding Affinity was determined by applying the following equation: RBA[%] =
(IC50(E2)/IC50(compound)) Á 100[24]. This results in a RBA value of 100% for E2.
After the assay was established and validated, a modification was made to increase through-
put. Compounds were tested at concentrations of 1000 times the IC50(E2). Compounds with
less than 50% displacement of [3H]-E2 at a concentration of 1000 times IC50(E2) were classi-
fied as RBA <0.1%.
Metabolic Stability in a cell-free assay. Compounds 7a, 8a, 16a, 19a, 22a, 27a and 27
were tested according to established method[33–35]For evaluation of phase I and II metabolic
stability 1 μM compound was incubated with 1 mg/ml pooled mammalian liver S9 fraction
(BD Gentest), 2 mM NADPH regenerating system, 1 mM UDPGA and 0.1 mM PAPS at 37°C
for 0, 5, 15 and 60 minutes at a final volume of 100 μL. The incubation was stopped by precipi-
tation of S9 enzymes with 2 volumes of cold acetonitrile containing internal standard. Concen-
tration of the remaining test compound at the different time points was analyzed by LC-MS/
MS and used to determine half-life (t1/2).
Acknowledgments
We thank Josef Zapp for the NMR measurements, Larissa Müller, Isabella Mang and Martina
Jankowski for the biological tests.
Author Contributions
Conceived and designed the experiments: EMG SMO. Performed the experiments: EMG GA
EP CVK. Analyzed the data: EMG SMO. Contributed reagents/materials/analysis tools: EMG
EP. Wrote the paper: EMG AC SMO MF RWH.
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