Bicyclic Substituted Hydroxyphenylmethanone Type Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type1 (17β-HSD1): The Role of the Bicyclic Moiety

Article abstract of DOI:10.1002/cmdc.201000457

An attractive target that has still to be explored for the treatment of estrogen-dependent diseases, such as breast cancer and endometriosis, is the enzyme responsible for the last step in the biosynthesis of estradiol (E2): 17β-hydroxysteroid dehydrogena

Full text of DOI:10.1002/cmdc.201000457

MED
DOI: 10.1002/cmdc.201000457
Bicyclic Substituted Hydroxyphenylmethanone Type
Inhibitors of 17b-Hydroxysteroid Dehydrogenase Type 1
(17b-HSD1): The Role of the Bicyclic Moiety
Alexander Oster, Tobias Klein, Claudia Henn, Ruth Werth, Sandrine Marchais-Oberwinkler,
Martin Frotscher, and Rolf W. Hartmann*[a]
An attractive target that has still to be explored for the treat-
ment of estrogen-dependent diseases, such as breast cancer
and endometriosis, is the enzyme responsible for the last step
in the biosynthesis of estradiol (E2): 17b-hydroxysteroid dehy-
drogenase type 1 (17b-HSD1). It catalyzes the reduction of the
weakly active estrone (E1) into E2, which is the most potent es-
trogen in humans. Inhibition of 17b-HSD1 lowers intracellular
E2 concentrations and thus presents a therapeutic target for
estrogen-dependent pathologies. Recently, we reported a new
class of highly active and selective 17b-HSD1 inhibitors: bicy-
clic substituted hydroxyphenylmethanones. Here, further struc-
tural variations on the bicyclic moiety are described, especially
focusing on the exchange of its hydroxy function. Twenty-nine
novel inhibitors were synthesized and evaluated for 17b-HSD1
inhibition in a cell-free and cellular assay, for selectivity toward
17bHSD2 and estrogen receptors (ER) alpha and beta, as well
as for metabolic stability. The best compound exhibited IC50
values of 12 nm (cell-free assay) and 78 nm (cellular assay),
high selectivity for 17b-HSD1, and reasonable metabolic stabili-
ty. A molecular docking study provided insight into the pro-
tein–ligand interactions of this compound with 17b-HSD1.
Introduction
17b-Hydroxysteroid dehydrogenases (17b-HSDs) are responsi-
ble for the oxido-reduction reactions of steroid hormones at
the 17 position, especially for estrogens and androgens, and
use subtype-specifically NADPH (in the case of 17b-HSD1) or
NAD+ (17b-HSD2) as co-factors. Although these are reversible
reactions, under in vivo conditions the catalysis is unidirection-
al due to the fact that NADP+ is abundant in its reduced form,
whereas NAD+ is mainly present in its oxidized form.[1] 17b-
HSDs catalyze the conversion of hormones with high receptor
affinity to the corresponding low affinity forms and vice versa.
They are expressed in the gonads and show characteristic ex-
pression patterns in peripheral tissues.[2] Therefore 17b-HSDs
play a pivotal role in the local regulation of hormone levels.[2]
In addition to their individual tissue and subcellular distribu-
tions, they also differ in their substrate specificities.[3,4]
Figure 1. Intervention of aromatase and 17b-HSD1 inhibitors in estrogen
synthesis.
17b-HSD1 is the most extensively characterized 17b-HSD
subtype. It catalyzes the last step in the biosynthesis of the
highly active estrogen 17b-estradiol (E2)—the reduction of the
weakly active estrone (E1). (Figure 1)
sults in a strong reduction of estrogen levels in the whole
body leading to unwanted side effects. Therefore, intracellular
regulation of estrogen levels in the diseased tissues by 17b-
HSD1 is a promising therapeutic approach for the treatment of
It is well known that E2, the natural ligand of the estrogen
receptors a and b (ERa and ERb), has a high impact on the de-
velopment and progression of estrogen-dependent diseases,
such as breast cancer, endometriosis, and endometrial hyper-
plasia.[5–7] Reduction of estrogen levels by interference with E2
biosynthesis has been used as a treatment of ER-positive post-
menopausal breast cancer for almost thirty years. Up to now,
aromatase inhibitors (AIs) are the first-line therapeutics for
these patients.[8,9] In the past few years, AIs have been inten-
sively investigated.[10–15] However, inhibition of E2 synthesis by
AIs does not affect the last but only the penultimate step. It re-
[a] A. Oster, T. Klein, C. Henn, R. Werth, Dr. S. Marchais-Oberwinkler,
Dr. M. Frotscher, Prof. Dr. R. W. Hartmann
Pharmaceutical and Medicinal Chemistry, Saarland University
& the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS)
Campus C2 3, P.O. Box 151150, 66123 Saarbrꢀcken (Germany)
Fax: (+49)681-302-70308
E-mail: rwh@mx.uni-saarland.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000457.
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Inhibitors of 17b-Hydroxysteroid Dehydrogenase Type 1
estrogen-dependent diseases. An analogous concept has al-
ready been proved successful for the treatment of androgen-
dependent diseases, such as benign prostatic hyperplasia and
alopecia, by using 5a-reductase inhibitors.[16–19] Proof-of-con-
cept for the treatment of breast cancer was performed in dif-
ferent mouse models.[20–22] However, no 17b-HSD1 inhibitor has
reached clinical trials so far. Furthermore, inhibition of 17b-
HSD1 could have an additional antitumor effect, because it is
also involved in the metabolism of retinoic acid,[23,24] a natural
compound known to have tumor inhibiting activity.[25] As the
therapeutic efficacy of 17b-HSD1 inhibitors should not be
counteracted, selectivity towards 17b-hydroxysteroid dehydro-
genase type 2 (17b-HSD2) is required. This enzyme catalyzes
the reverse reaction—oxidation of E2 into E1. Additionally, for
reducing the risk of intrinsic estrogenic effects, affinity of the
inhibitors to the ERs should be avoided.
duced. Synthesis, biological evaluation and structure–activity
relationships (SAR) of new highly potent and selective 17b-
HSD1 inhibitors are described. Furthermore, the probable bind-
ing mode of the most potent compound was investigated by
molecular docking studies to explain the SAR obtained.
Design
Binding mode investigations of three potent compounds of
this class (B1–B3) revealed a steroid-like binding mode with
the benzoyl moiety occupying an apolar subpocket, previously
described to have an impact on ligand binding.[44,45] The
phenyl ring adjacent to the thiophene was found to mimic the
steroidal A ring binding in the C-terminal region of the protein.
It is known that there is space for additional substituents in
this region since several highly active steroidal inhibitors have
extensions in position 2.[46] The most potent inhibitors of these
derivatives bear an additional phenyl ring connected by a
C2 linker.[46] Thus, the expected steroid-like binding mode of
B1–B3 prompted us to further investigate this C-terminal
region by exchanging the phenyl hydroxy group.
In recent years, the structural architecture of 17b-HSD1 has
been investigated and numerous crystallographic data have
been determined[24] and assigned to the different steps of the
catalytic cycle.[26] Several, mostly steroidal, compounds have al-
ready been published as 17b-HSD1 inhibitors,[24,27–30] using this
information. With regards to nonsteroidal inhibitors, there are
three compound classes with IC50 values in the low nanomolar
range described: thiophenepyrimidinones,[31,32] hydroxyphenyl-
naphthols,[33–36] and bis(hydroxyphenyl)heterocycles.[37–41] Re-
cently, we reported bicyclic substituted hydroxyphenylmetha-
nones as a new class of 17b-HSD1 inhibitors (Figure 2a).[42]
Chemistry
The synthesis of compounds 1–29 is depicted in Scheme 1.
The methoxylated intermediate 1b was synthesized via Frie-
del–Crafts acylation of 2-bromothiophene with 3-methoxyben-
zoyl chloride using aluminium chloride. The methoxy function
was cleaved according to method B (BBr3, CH2Cl2, ꢀ788C!RT,
Figure 2. Bicyclic substituted hydroxyphenylmethanones as inhibitors of
17b-HSD1.
These inhibitors also show a very promising biological profile.
As the hydroxy function on the benzoyl part is more important
for inhibitory activity (Figure 2b), replacement of the phenyl
OH group was regarded as an appropriate modification to
gain further interactions and to avoid possible phase II metab-
olism in this position.[43]
Scheme 1. Synthesis of compounds 1–29. Reagents and conditions: a) AlCl3,
anhyd CH2Cl2, 08C, 0.5 h then RT, 1 h for compound 1b; b) Cs2CO3,
Pd(PPh3)4, DME/EtOH/water (1:1:1), microwave conditions (150 W, 15 bar,
1508C, 15 min) for compound 2a; c) Cs2CO3, Pd(PPh3)4, DME/water (1:1),
reflux, 4 h for compounds 20a–22a and 29 (Method A2) and 2 h for com-
pounds 1, 3–19 and 23–28 (Method A1); d) Method B: BBr3, CH2Cl2, ꢀ788C,
18 h for compounds 1a, 2 and 20–22.
Herein, we report the replacement of the hydroxy group of
the phenyl ring adjacent to the thiophene in bicyclic substitut-
ed hydroxyphenyl methanones. In order to evaluate whether it
is possible to achieve additional interactions in the C-terminal
region of the target enzyme, various substituents were intro-
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477

R. W. Hartmann et al.
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18 h)[47] to yield compound 1a in quantitative yields. Starting
from these brominated key intermediates 1a and 1b and the
appropriate commercially available boronic acids, Suzuki cross-
coupling reactions[48] were carried out using two different
methods. Compound 2a was prepared in a one-pot synthesis
by means of microwave-assisted Suzuki reaction (Cs2CO3, dime-
thoxyethane (DME)/EtOH/water (1:1:1), Pd(PPh3)4, MW (150 W,
1508C, 15 bar 15 min)).[38] All other compounds were prepared
following method A. Using DME/water (1:1) as solvent, caesium
carbonate and tetrakis(triphenylphosphine)palladium as cata-
lyst, the reaction mixtures were stirred under reflux for 2 h
(method A1; 1, 3–19 and 23–28) or 4 h (method A2; 20a–22a
and 29). Demethylations of 2a, sulfonamide derivatives 21 and
22, and nitrile 20 were performed using method B (BBr3,
CH2Cl2, ꢀ788C!RT, 18 h).
Table 1. Inhibition of human 17b-HSD1 and 17b-HSD2 by compounds 1–
29.
Compd
X
IC50 [nm][a]
17b-HSD1[b] 17b-HSD2[c]
SF[d]
B1
B2
B3
1
2
3
4
5
6
7
2-hydroxy
3-hydroxy
4-hydroxy
H
2-methoxy
3-methoxy
4-methoxy
95
22
33
155
86
95
18
109
478
128
196
342
507
1036
44%[e]
26%[e]
915
195
502
586
797
1065
768
702
789
1197
1034
1003
354
563
559
169
253
319
299
173
283
410
0.2
5
14
0.8
2
4
2
2
Biology
220
510
Activity: Inhibition of human 17b-HSD1
3,4-dimethoxy
3,4,5-trimethoxy
2,3,4-trimethoxy
2-ethoxy
3-ethoxy
4-ethoxy
44%[e]
Human placenta was used as enzyme source and partially puri-
fied following a described procedure.[49] Tritiated E1 was incu-
bated with 17b-HSD1, cofactor and inhibitor. The separation of
substrate and product was performed by HPLC. The inhibition
values of compounds 1–29 are shown in Table 1.
54%[e]
515
8
9
2
2
6
94
78
498
62
35
47
60
31
54
40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1
H
13
30
16
12
26
22
26
28
2
4
4
14
0.8
6
Compound 1, lacking the OH function on the phenyl ring,
showed an inhibitory activity, which is slightly weaker than the
activities of the hydroxylated compounds B1–B3. As com-
pound 2 (ortho-methoxy) and 3 (meta-methoxy) revealed re-
markable IC50 values around 90 nm, it became apparent that a
hydrogen-bond donor is beneficial (B1–B3)[42] but not necessa-
rily needed in this region for inhibitory potency. The ortho- and
meta-ethoxy compounds 9 and 10, respectively, exhibited simi-
lar 17b-HSD1 inhibition as their corresponding methoxy deriva-
tives 2 and 3 indicating that there is some space for further
substituents. The good inhibitory activity of 12 (IC50 =62 nm)
indicates the presence of additional space, which was further
investigated by the introduction of substituents on each posi-
tion of the benzyloxy moiety (13–19).
2-methoxy
3-methoxy
4-methoxy
3,5-dimethoxy
2-chloro
3-chloro
4-chloro
3-nitrile
3-methanesulfonamide
N-methylmethanesulfonamide
3-(4-methylbenzenesulfonamide)
2-naphthalene
5-(2,3-dihydrobenzofurane)
5-(1,3-benzodioxol)
5-indole
36
225
150
125
12
300
55
66
5
1
2
4
151
192
107
6-indole
6-indazole
While the introduction of a carbonitrile in the meta position
adjacent to the thiophene (20, IC50 =225 nm) or methanesulfo-
namide group (21, IC50 =150 nm; 22, IC50 =125 nm) did not
induce significant changes in enzyme inhibition compared to
the unsubstituted compound 1 (IC50 =155 nm), the methylben-
zenesulfonamide 23 was clearly more potent and exhibited
the highest 17b-HSD1 inhibitory activity (IC50 =12 nm) in this
study, similar to those obtained in the previous study B1–
B3.[42]
Compared to compound 1 (IC50 =155 nm), the naphthalene-
substituted derivative 24 showed a slight drop in activity
(IC50 =300 nm). Introduction of a dihydrobenzofuran moiety
(25), which represents a rigidification of the ethoxy substituent
of compound 11 (IC50 =498 nm), led to increased inhibition
values (IC50 =55 nm). Obviously, rigidified systems are more ap-
propriate for inhibitory activity in this position of the molecule
indicating a steric hindrance for larger substituents in para po-
sition. This observation was confirmed by comparing com-
[a] Mean value of three determinations, standard deviation less then
26%; [b] Human placenta, cytosolic fraction, substrate E1 (500 nm), cofac-
tor NADH (500 mm); [c] Human placenta, microsomal fraction, substrate
E2 (500 nm), cofactor NAD+ (1500 mm); [d] Selectivity factor (SF): IC50
(17b-HSD2)/IC50 (17b-HSD1); [e] Inhibition at 1 mm (inhibitor concentra-
tion).
pounds 6 and 26; by replacing the two methoxy functions (6)
with an acetale (26), the weak inhibition (44% at 1 mm) could
be increased to an IC50 value of 66 nm.
Selectivity: Inhibition of human 17b-HSD2 and affinities for
ERa and ERb
17b-HSD2 acts as a biological counterpart of the type 1
enzyme by catalyzing the reverse reaction. Therefore, inhibi-
tion of this enzyme must be avoided. In an assay similar to the
17b-HSD1 test, human placental microsomes were incubated
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Inhibitors of 17b-Hydroxysteroid Dehydrogenase Type 1
with tritiated E2 in the presence of NAD+ and inhibitor to eval-
uate inhibition of 17b-HSD2. Labeled product was quantified
after HPLC separation. Inhibition values of all compounds are
given in Table 1. In vitro selectivity toward 17b-HSD2 is ex-
pressed as a selectivity factor (SF), describing the ratio of the
concentrations required to inhibit the isoenzymes by 50% (IC50
17b-HSD2/IC50 17bHSD1).
Table 2. Inhibition of 17b-HSD1 in cell-free and cellular assay by selected
compounds.
SF values of methoxy and ethoxy compounds 1–2, 4–9 and
11 do not exceed 2. Higher SF values were observed for com-
pounds bearing substituents in the meta position. However, SF
values up to 50, as achieved in the previous study,[42] were not
obtained.
Compd
R
IC50 [nm][a]
cell-free assay[b]
cellular assay[c]
154
10
12
78
62
Interestingly, enlargement of the ether resulted in a slight in-
crease in selectivity: compound 3 (SF=4) versus 10 (SF=6).
Remarkably, the benzyloxy derivative 12 showed a 13-fold
higher inhibition of 17b-HSD1 compared to the type 2 enzyme
indicating that the space within this area of the latter enzyme
might be more restricted than in 17b-HSD1. Accordingly, intro-
duction of substituents into the benzyloxy moiety might lead
to further enhancement of selectivity. While this hypothesis
was confirmed by the meta-methoxy (13), dimethoxy (16) and
the chloro derivatives (17–19), the monomethoxy compounds
(14–15) showed different results. Compounds 14 (meta-me-
thoxy, SF=16) and 15 (para-methoxy, SF=12) revealed similar
selectivity toward 17b-HSD2 as the nonsubstituted benzyloxy
compound 12 (SF=13).
53%[d]
13
14
15
35
47
60
441
565
526
16
17
31
54
40%[d]
34%[d]
Considering the sulfonamides 21–23, it is striking that the
introduction of a phenyl ring also increases selectivity (for 21
and 22, SF=4 versus 23, SF=14). These results attract increas-
ing notice to a phenyl ring within this area of the protein with
regard to its influence on activity and selectivity.
18
19
40
36
49%[d]
47%[d]
As estrogens function via activation of ERs, the affinity of
17b-HSD1 inhibitors toward ERa and ERb should be as low as
possible in order to avoid stimulatory effects. Since E1 shows
tenfold lower affinity toward ERa[50] and 50-fold lower affinity
toward ERb[50] compared with E2 (relative binding affinity
(RBA)=100% for E2), inhibitors having an RBA value below
0.1% were considered to possess too little affinity to exert es-
trogenic effects. As E1 is the only active estrogen present
during 17b-HSD1 inhibition, inhibitors with much lower recep-
tor affinity than E1 are negligible for receptor activation. All
compounds having a SF value higher than 10 were evaluated
in this competition assay. None of them showed higher RBA
values than 0.1% (data not shown) confirming our design con-
cept focusing on nonsteroidal inhibitors.
23
25
12
55
78
357
[a] Mean value of three determinations, standard deviation less then
24%; [b] Human placenta, cytosolic fraction, substrate E1 (500 nm), cofac-
tor NADH (500 mm); [c] T47D cells, substrate E1 (50 nm); [d] inhibition at
1 mm (inhibitor concentration).
ethoxy derivative 10, without an additional phenyl ring, exhib-
ited a three- to fourfold higher intracellular inhibitory activity
(IC50 =154 nm) than compounds 13–15.
Methylbenzenesulfonamide 23 turned out to be the most
potent 17b-HSD1 inhibitor of this study both in the cell-free
(IC50 =12 nm) and in the cellular assay (IC50 =78 nm).
In order to find an appropriate species for demonstrating in
vivo efficacy in a disease-oriented animal model, compounds
1, 13 and 23 were tested in a cell-free assay using the cytosolic
fraction of mouse liver preparation and under conditions simi-
lar to our standard cell-free assay. However, no mentionable in-
hibition of 17b-HSD1 or 17b-HSD2 could be detected (data not
shown). Therefore, the search for an optimal species pertaining
Further biological evaluation
Using T47D cells expressing both 17b-HSD1 and 17b-HSD2, the
intracellular potencies of selected compounds were evaluated
(Table 2). Regarding the benzyloxy-substituted compounds 12–
19, only the monomethoxy-substituted benzyloxy derivatives
13–15 showed moderate inhibition of E2 formation (IC50 =526,
441 and 565 nm, respectively). However, the dimethoxy (16)
and chloro substituents (17–19) have a negative impact on cel-
lular activity, which might be explained by insufficient cell pen-
etration or increased intracellular metabolism. Interestingly, the
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479

R. W. Hartmann et al.
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to in vivo proof-of-concept for this class of compounds is on-
going.
oxygen atom and Tyr155. The second hydrogen bond of the
ketone with Ser142, which was proposed for B1–B3, was not
predicted by modeling studies to be present for 23. The meth-
ylbenzenesulfonamide substituent is predicted to interact with
two amino acids, which are located in the C-terminal region.
His221 is suggested to form a hydrogen bond with one sulfo-
namide oxygen atom, while Phe259 might be involved in p–p
interactions with the benzene moiety.
Furthermore, metabolic stability of the most active com-
pound 23 (IC50 =12 nm) and the most selective compound 13
(SF=30) of this study were evaluated using human liver micro-
somes (Table 3). Compounds 13 and 23 showed intrinsic clear-
Table 3. Metabolic stability of compounds 13, 23 and control com-
pounds using human liver microsomes.
Compd
CLint[a]
[mLminꢀ1 mgꢀ1 protein]
t =
1
2
Discussion and Conclusions
[min]
From our previous work in this class of compounds,[42] it was
demonstrated that variation of the OH position on the phenyl
ring do not result in marked differences in the inhibitory activi-
ty of evaluated compounds B1–B3. In this paper, we have re-
ported the replacement of the hydroxy group on the phenyl
ring in the class of the bicyclic substituted hydroxyphenyl
methanones. Various substituents were introduced in order to
evaluate whether additional interactions in the C-terminal
region are beneficial for inhibitory activity. The biological data
show that 17b-HSD1 inhibition can also be achieved without
the OH functionality on the phenyl ring. Considering the
design concept of the previous study, this hydroxy group was
expected to form hydrogen bonds with His221 and Glu282 in
the C-terminal region of the steroid binding pocket. Obviously,
the hydrogen-bond donor property is not necessary for strong
inhibitory activity with regard to the methoxy derivatives.
Glu282, which is expected to accept a hydrogen bond from
this OH group, does not seem to be involved in inhibitor bind-
ing at all. In general, its relevance for ligand binding is dis-
cussed controversially. While site-directed mutagenesis studies
indicated no significant interaction between Glu282 and the 3-
OH group of the substrate,[51] hydrogen bonding between
both was found in several independent crystal structures.[52,53]
The hydrogen-bond acceptor property of the phenol might
play a more important role since compound 1, without hy-
droxy functionality on the phenyl ring, showed slightly re-
duced 17b-HSD1 inhibition. However, the potential hydrogen-
bond donor His221 is already known as an important factor
for substrate interaction.[54] Although this amino acid is de-
scribed to be implicated in enzyme stabilization[44] by interact-
ing with Glu282, it appears to be also involved in the binding
of our inhibitors.
dextromethorphan
verapamil
13
23
10.7
105
81.2
109
129
13.2
17.1
12.8
[a] Intrinsic metabolic clearance (CLint); test concentration=3 mm.
ances (CLint) of 81.2 and 109 mLminꢀ1 mgꢀ1 protein, respec-
tively, and therefore can be considered to be in the high clear-
ance category. Regarding the reference drugs, dextromethor-
phan and verapamil, the commonly used calcium channel
blocker verapamil revealed a very similar CLint of 105 mLminꢀ1
mgꢀ1 protein defining a high but not too high liability to meta-
bolic degradation.
Molecular Modeling
A docking study was performed in order to gain deeper insight
into the molecular interactions between the most potent in-
hibitor 23 and 17b-HSD1 and to understand the SAR. In
Figure 3, the hypothetical binding mode of compound 23 is
shown, indicating hydrogen-bonding interactions between the
OH functionality and Asn152, as well as between the keto
Regarding the mono-methoxy and ethoxy derivatives (2–5
and 9–11, respectively), it becomes apparent that the para po-
sition seems to be less favorable than meta and ortho. More-
over, di- and trimethoxy derivatives (6–8) showed a strong de-
crease in activity as well, indicating that the space around this
phenyl ring is sterically restricted. This finding is in accordance
with our docking results. According to the orientation of the
phenyl ring, the disubstitution of 6 compels the methoxy
groups to adopt a conformation leading to steric clashes with
either Tyr218 or Phe259. Indeed, taking into account that the
1,3-benzodioxol compound 26 showed a much higher affinity
than the corresponding dimethoxy compound 6, it can be
concluded that the binding pocket is very restricted.
Figure 3. Hypothetical binding mode of compound 23 (cyan) obtained by
docking into 17b-HSD1 (PDB: 1FDT). Co-factor NADPH (orange), interacting
residues (green), and cartoon-rendered tertiary structure (grey) of the active
site are shown. Hydrogen bonds are indicated (a). The figure was gener-
ated with the Molecular Operating Environment (MOE, version: 2009.10;
Chemical Computing Group Inc., Montreal, Canada).
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Inhibitors of 17b-Hydroxysteroid Dehydrogenase Type 1
1H NMR and 13C NMR spectra were measured on a Bruker AM 500
spectrometer (500 MHz) at 300 K. Chemical shifts (d) are reported
in parts per million (ppm), with the residual solvent as the internal
standard (CDCl3: d=7.24 ppm (1H NMR), d=77 ppm (13C NMR);
CD3COCD3: d=2.05 ppm (1H NMR), d=30.8 ppm (13C NMR)). Sig-
nals are described as singlet (s), doublet (d), triplet (t), doublet of
doublets (dd), doublet of doublets of doublets (ddd), multiplet (m),
doublet of triplets (dt), triplet of doublets (td) and quadruplet (q).
All coupling constants (J) are given in hertz (Hz). Mass spectra
were recorded on a TSQ Quantum (Thermo Finnigan) instrument
using electrospray ionization (ESI).
Concerning flexible substituents, 17b-HSD1 inhibitory activi-
ty as well as selectivity toward the type 2 enzyme revealed the
meta position to be the most favorable. Starting with com-
pound 3, it is striking that an enlargement of the meta sub-
stituents resulted in increased selectivity (SF values: 3 <10 <
12 <13/16–19). Interestingly, the IC50 values for 17b-HSD1 in-
hibition of all benzyloxy derivatives are in the same range and
similar to that of the ethoxy compound 10. Therefore, al-
though influencing the selectivity toward 17b-HSD2, the
phenyl ring of the benzyloxy substituent does not seem to
specifically interact with 17b-HSD1. Further investigation is still
warranted as to whether the phenyl ring is pointing out of the
17b-HSD1 active site.
Tested compounds possessed >98% chemical purity as measured
by HPLC. The methods for HPLC analysis and a table of data for all
tested compounds are provided in the Supporting Information.
Sulfonamide 23 turned out to be the most potent inhibitor
of this study. Compared to its methanesulfonamide analogue
21, the introduction of the phenyl led to 12-fold higher 17b-
HSD1 inhibition. The docking study revealed the benzene ring
of 23 to be implicated in a p–p interaction with Phe259. Com-
paring B2 (meta-OH) and 23, both compounds interact with
Asn152. The relevance of this amino acid for ligand binding
was already confirmed by a co-crystal structure of a highly
potent, steroidal 17b-HSD1 inhibitor.[44] B2 and 23 showed sim-
ilar IC50 values although the hydrogen-bonding interaction of
23 with Ser142 was not present in the observed binding
mode. The p–p interactions found for compound 23 might be
able to compensate the loss of the hydrogen bond between
the ketone and Ser142.
General procedure for Suzuki coupling (Method A): A mixture of
2-bromothiophene derivative (1 equiv), boronic acid derivative
(1.2 equiv), Cs2CO3 (4 equiv) and Pd(PPh3)4 (0.01 equiv) in an
oxygen-free dimethoxyethane (DME)/water (1:1) solution was re-
fluxed under N2 for 2 h (method A1) or 4 h (method A2). The reac-
tion mixture was cooled to RT. The aqueous layer was extracted
with EtOAc. The combined organic layers were washed with brine,
dried over MgSO4, filtered and concentrated to dryness. The prod-
uct was purified by column chromatography (CC), CC followed by
preparative TLC, or CC followed by preparative HPLC.
General procedure for ether cleavage (Method B): A solution of
methoxybenzene derivative (1 equiv) in dry CH2Cl2 at ꢀ788C (dry
ice/acetone bath) was treated dropwise with a solution of BBr3 in
CH2Cl2 (1m, 3 equiv per methoxy function). The reaction mixture
was stirred for 20 h at RT under N2. Water was added to quench
the reaction, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with brine, dried over
Na2SO4, filtered and concentrated to dryness. The product was pu-
rified by CC, CC followed by preparative HPLC, or recrystallization.
In summary, we have reported on the further developments
in the class of bicyclic substituted hydroxyphenylmethanones.
The high inhibitory activities of inhibitors bearing large sub-
stituents like benzylsulfonamide or benzyloxy revealed that
these groups take advantage of the additional space in the
C-terminal region. Although comparable inhibitory potencies
were achieved, they have to be considered critically in terms
of ligand efficiency. However, further bio-isosteric replacements
of the phenolic hydroxy group might lead to compounds com-
bining high potency with good selectivity and intracellular ac-
tivity. The most promising compound of this study, methylben-
zenesulfonamide 23, shows strong inhibitory activity in both
cellular and cell-free assays, as well as no affinity toward estro-
gen receptors a and b. It might be a candidate for further lead
optimization, especially regarding its selectivity and cellular
activity.
General procedure for purification using preparative HPLC:
Compounds were purified via an Agilent Technologies Series 1200
preparative HPLC using a RP C18 Nucleodur 100–5 column (30ꢁ
100 mm/50 mm from Macherey–Nagel GmbH) as the stationary
phase with a linear gradient (solvents: CH3CN/H2O) starting from
20%!100% CH3CN over 36 min.
(5-Bromo-2-thienyl)(3-methoxyphenyl)methanone (1b): A mix-
ture of 2-bromothiophene (145 mg, 0.89 mmol), 3-methoxybenzoyl
chloride (152 mg, 0.89 mmol) and AlCl3 (119 mg, 0.89 mmol) in
anhyd CH2Cl2 was stirred at 08C for 0.5 h. The reaction mixture was
warmed to RT and stirred for 1 h. HCl (1m) was used to quench the
reaction. The aqueous layer was extracted with EtOAc. The com-
bined organic layers were washed with brine, dried over MgSO4, fil-
tered and concentrated to dryness. Purification by CC (hexane/
EtOAc 97:3) gave the product as a white powder (77%, 204 mg):
1H NMR (CD3COCD3): d=7.55 (d, J=4.1 Hz, 1H), 7.47 (t, J=7.9 Hz,
1H), 7.42 (td, J=1.5, 7.9 Hz, 1H), 7.35–7.34 (m, 1H), 7.33 (d, J=
4.1 Hz, 1H), 7.22 (ddd, J=0.9, 2.5, 8.2 Hz, 1H), 3.88 ppm (s, 3H);
13C NMR (CD3COCD3): d=187.9, 161.8, 140.5, 137.4, 133.9, 131.6,
124.0, 123.1, 120.5, 115.5, 56.9 ppm.
Experimental Section
Chemistry
Chemical names follow IUPAC nomenclature. Starting materials
were purchased from Aldrich, Acros, Lancaster, Combi Blocks,
Merck or Fluka and were used without purification. Column chro-
matography (CC) was performed on silica gel (70–200 mm). Prepa-
rative thin layer chromatography (TLC) was performed on 1 mm
SIL G-100 UV254 silica-coated glass plates (Macherey–Nagel). Reac-
tions were monitored using Alugram SIL G UV254 TLC plates (Ma-
cherey–Nagel). All microwave irradiation experiments were carried
out in a CEM-Discover monomode microwave apparatus.
(5-Bromo-2-thienyl)(3-hydroxyphenyl)methanone (1a): The title
compound was prepared by reaction of 1b (650 mg, 2.19 mmol)
and BBr3 (6.57 mmol) according to method B. The product was ob-
tained as a white powder (quantitative yield, 620 mg) and used in
1
the next step without further purification: H NMR (CD3COCD3): d=
8.76 (s, 1H), 7.54 (d, J=4.1 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.34–
7.32 (m, 2H), 7.30 (t, J=2.1 Hz, 1H), 7.13 ppm (ddd, J=1.3, 2.5,
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481

R. W. Hartmann et al.
MED
8.2 Hz, 1H); 13C NMR (CD3COCD3): d=187.9, 159.5, 147.3, 140.5,
137.3, 133.8, 131.7, 123.8, 122.1, 121.6, 117.3 ppm.
4.1 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.35 (dt, J=1.3, 7.8 Hz, 1H), 7.34
(t, J=2.1 Hz, 1H), 7.12 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 7.02 (d, J=
8.8 Hz, 2H), 3.85 ppm (s, 3H); 13C NMR (CD3COCD3): d=188.6,
162.7, 159.4, 154.7, 143.2, 141.5, 138.0, 131.5, 129.5, 127.8, 125.1,
122.1, 121.1, 117.3, 116.5, 56.8 ppm; MS (ESI): m/z: 311 [M+H]+.
(3-Hydroxyphenyl)(5-phenyl-2-thienyl)methanone (1): The title
compound was prepared by reaction of 1a (150 mg, 0.53 mmol),
benzene boronic acid (78 mg, 0.64 mmol), Cs2CO3 (691 mg,
2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) according to method A1.
Purification by CC (hexane/EtOAc 8:2) gave the product as a beige
(3-Hydroxyphenyl)[5-(6-methoxypyridin-3-yl)-2-thienyl]metha-
none (5): The title compound was prepared by reaction of 1a
(150 mg, 0.53 mmol), 6-methoxypyridine-3-boronic acid (98 mg,
0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg,
5 mmol) according to method A1. Purification by CC (hexane/EtOAc
85:15) gave the product as a beige powder (63%, 104 mg):
1H NMR (CD3COCD3): d=8.86 (s, 1H), 8.60 (dd, J=0.6, 2.4 Hz, 1H),
8.06 (dd, J=2.5, 8.5 Hz, 1H), 7.71 (d, J=4.1 Hz, 1H), 7.55 (d, J=
4.1 Hz, 1H), 7.40 (t, J=7.8 Hz, 1H), 7.36 (dt, J=1.3, 7.6 Hz, 1H),
7.33–7.32 (m, 1H), 7.12 (ddd, J=1.3, 2.5, 7.8 Hz, 1H), 6.88 (dd, J=
0.6, 8.5 Hz, 1H), 3.94 ppm (s, 3H); 13C NMR (CD3COCD3): d=188.7,
166.4, 159.5, 151.0, 146.4, 141.3, 138.6, 137.9, 131.6, 126.1, 122.1,
121.3, 117.3, 113.1, 55.0 ppm; MS (ESI): m/z: 312 [M+H]+.
1
powder (28%, 42 mg): H NMR (CD3COCD3): d=8.81 (s, 1H), 7.81–
7.80 (m, 2H), 7.72 (d, J=4.1 Hz, 1H), 7.60 (d, J=4.1 Hz, 1H), 7.49 (t,
J=7.9 Hz, 2H), 7.44–7.39 (m, 2H), 7.37 (dt, J=1.5, 7.6 Hz, 1H),
7.34–7.33 (m, 1H), 7.13 ppm (ddd, J=1.5, 2.5, 7.9 Hz, 1H); 13C NMR
(CD3COCD3): d=188.8, 159.4, 154.3, 144.2, 141.3, 137.9, 135.2,
131.6, 131.2, 131.1, 128.1, 126.3, 122.1, 121.3, 117.3 ppm; MS (ESI):
m/z: 281 [M+H]+.
(3-Methoxyphenyl)[5-(2-methoxyphenyl)-2-thienyl]methanone
(2a): A mixture of 1b (200 mg, 0.67 mmol), 2-methoxybenzene
boronic acid (122 mg, 0.80 mmol), Cs2CO3 (873 mg, 2.68 mmol) and
Pd(PPh3)4 (8 mg, 7 mmol) was suspended in an oxygen-free DME/
EtOH/H2O (1:1:1) solution (7.5 mL). The reaction mixture was ex-
posed to microwave irradiation (15 min, 150 W, 1508C, 15 bar).
After reaching RT, water was added and the aqueous layer was ex-
tracted with EtOAc. The combined organic layers were washed
with brine, dried over MgSO4, filtered and concentrated to dryness.
Purification by CC (hexane/EtOAc 9:1) gave the product as a
yellow powder (67%, 145 mg): 1H NMR (CD3COCD3): d=7.85 (dd,
J=1.9, 7.9 Hz, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.49–7.44 (m, 2H), 7.42–
7.38 (m, 2H), 7.21–7.18 (m, 2H), 7.07 (dt, J=0.9, 7.9 Hz, 1H), 4.01
(s, 3H), 3.89 ppm (s, 3H); 13C NMR (CD3COCD3): d=189.1, 161.7,
158.1, 149.9, 144.1, 141.7, 136.5, 132.2, 131.5, 130.2, 127.8, 123.8,
123.1, 123.0, 119.9, 115.5, 114.1, 57.1, 56.8 ppm.
[5-(3,4-Dimethoxyphenyl)-2-thienyl](3-hydroxyphenyl)metha-
none (6): The title compound was prepared by reaction of 1a
(150 mg, 0.53 mmol), 3,4-dimethoxybenzene boronic acid (117 mg,
0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg,
5 mmol) according to method A1. Purification by CC (hexane/EtOAc
1
8:2) gave the product as a yellow powder (73%, 132 mg): H NMR
(CD3COCD3): d=8.78 (s, 1H), 7.68 (d, J=4.1 Hz, 1H), 7.51 (d, J=
4.1 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.38–7.34 (m, 3H), 7.32–7.31 (m,
1H), 7.12 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 7.06–7.04 (m, 1H), 3.92 (s,
3H), 3.87 ppm (s, 3H); 13C NMR (CD3COCD3): d=189.7, 159.4, 152.6,
151.8, 141.5, 138.0, 131.6, 128.1, 125.4, 122.0, 121.1, 121.0, 117.3,
114.1, 111.7, 57.3, 57.2 ppm; MS (ESI): m/z: 341 [M+H]+.
(3-Hydroxyphenyl)[5-(2-methoxyphenyl)-2-thienyl]methanone
(2): The title compound was obtained by reaction of 2a (145 mg,
0.45 mmol) and BBr3 (2.70 mmol) according to method B. Purifica-
tion by CC (hexane/EtOAc 8:2) followed by preparative HPLC gave
(3-Hydroxyphenyl)[5-(3,4,5-trimethoxyphenyl)-2-thienyl]metha-
none (7): The title compound was prepared by reaction of 1a
(150 mg, 0.53 mmol), 3,4,5-trimethoxybenzene boronic acid
(136 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4
(6 mg, 5 mmol) according to method A1. Purification by CC
(hexane/EtOAc 8:2) gave the product as a yellow powder (71%,
140 mg): 1H NMR (CD3COCD3): d=8.76 (s, 1H), 7.68 (d, J=4.1 Hz,
1H), 7.56 (d, J=4.1 Hz, 1H), 7.40 (t, J=7.8 Hz, 1H), 7.35 (dt, J=1.3,
7.6 Hz, 1H), 7.33–7.32 (m, 1H), 7.12 (ddd, J=1.3, 2.5, 7.9 Hz, 1H),
7.07 (s, 2H), 3.93 (s, 6H), 3.78 ppm (s, 3H); 13C NMR (CD3COCD3):
d=188.7, 159.4, 156.0, 154.8, 143.8, 141.4, 137.8, 131.6, 130.7,
126.2, 122.1, 121.2, 117.3, 105.9, 61.7, 57.7 ppm; MS (ESI): m/z: 371
[M+H]+.
1
the product as a yellow oil (55%, 76 mg): H NMR (CD3COCD3): d=
7.83 (dd, J=1.6, 7.9 Hz, 1H), 7.69 (d, J=4.1 Hz, 1H), 7.67 (d, J=
4.1 Hz, 1H), 7.41–7.35 (m, 4H), 7.18 (d, J=7.9 Hz, 1H), 7.14–7.12
(m, 1H), 7.07–7.04 (m, 1H), 4.00 ppm (s, 3H); 13C NMR (CD3COCD3):
d=189.2, 159.3, 158.1, 149.7, 144.1, 141.7, 136.4, 132.1, 131.5,
130.1, 127.7, 123.8, 122.9, 122.1, 121.0, 117.3, 114.0, 57.1 ppm; MS
(ESI): m/z: 311 [M+H]+.
(3-Hydroxyphenyl)[5-(3-methoxyphenyl)-2-thienyl]methanone
(3): The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 3-methoxybenzene boronic acid (97 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 9:1) gave the
product as a beige powder (94%, 154 mg): 1H NMR (CD3COCD3):
d=7.71 (d, J=4.1 Hz, 1H), 7.60 (d, J=4.1 Hz, 1H), 7.42–7.35 (m,
4H), 7.34 (t, J=1.9 Hz, 1H), 7.32 (t, J=1.9 Hz, 1H), 7.13 (ddd, J=
1.3, 2.2, 7.6 Hz, 1H), 7.00 (ddd, J=1.3, 2.2, 7.6 Hz, 1H), 3.89 ppm (s,
3H); 13C NMR (CD3COCD3): d=188.8, 162.4, 159.5, 154.2, 144.3,
141.3, 137.8, 136.5, 132.3, 131.6, 126.6, 122.1, 121.3, 120.5, 117.3,
116.8, 113.4, 56.8 ppm; MS (ESI): m/z: 311 [M+H]+.
(3-Hydroxyphenyl)[5-(2,3,4-trimethoxyphenyl)-2-thienyl]metha-
none (8): The title compound was prepared by reaction of 1a
(150 mg, 0.53 mmol), 2,3,4-trimethoxybenzene boronic acid
(136 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4
(6 mg, 5 mmol) according to method A1. Purification by CC
(hexane/EtOAc 8:2) gave the product as a yellow powder (62%,
122 mg): 1H NMR (CD3COCD3): d=8.80 (s, 1H), 7.66 (d, J=4.1 Hz,
1H), 7.59–7.56 (m, 2H) 7.39 (t, J=7.8 Hz, 1H), 7.35 (dt, J=1.3,
7.6 Hz, 1H), 7.33–7.32 (m, 1H), 7.11 (ddd, J=1.3, 2.5, 7.9 Hz, 1H),
6.92 (d, J=9.1 Hz, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 3.87 ppm (s, 3H);
13C NMR (CD3COCD3): d=189.1, 159.4, 156.6, 152.9, 149.7, 143.7,
141.8, 136.6, 131.5, 126.7, 124.4, 122.0, 121.7, 121.0, 117.3, 110.4,
62.1, 62.0, 57.5 ppm; MS (ESI): m/z: 371 [M+H]+.
(3-Hydroxyphenyl)[5-(4-methoxyphenyl)-2-thienyl]methanone
(4): The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 4-methoxybenzene boronic acid (97 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 8:2) followed
by preparative TLC (hexane/EtOAc 7:3) gave the product as a
yellow powder (96%, 158 mg): 1H NMR (CD3COCD3): d=8.72 (s,
1H), 7.72 (d, J=8.8 Hz, 2H), 7.66 (d, J=4.1 Hz, 1H), 7.45 (d, J=
5-(2-Ethoxyphenyl)-2-thienyl](3-hydroxyphenyl)methanone (9):
The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 2-ethoxybenzene boronic acid (106 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 85:15) gave
482
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ChemMedChem 2011, 6, 476 – 487

Inhibitors of 17b-Hydroxysteroid Dehydrogenase Type 1
the product as a yellow oil (88%, 151 mg): 1H NMR (CD3COCD3):
d=7.87 (dd, J=1.5, 7.9 Hz, 1H), 7.74 (d, J=4.1 Hz, 1H), 7.69 (d, J=
4.1 Hz, 1H), 7.41–7.35 (m, 3H), 7.34 (t, J=2.1 Hz, 1H), 7.18–7.16 (m,
1H), 7.12 (ddd, J=1.3, 2.5, 7.6 Hz, 1H), 7.06 (td, J=1.3, 7.6 Hz, 1H),
4.28 (q, J=6.9 Hz, 2H), 1.55 ppm (t, J=6.9 Hz, 3H); 13C NMR
(CD3COCD3): d=189.2, 157.3, 144.0, 141.8, 138.4, 136.3, 132.1,
131.5, 130.1, 127.6, 123.8, 122.8, 122.1, 121.0, 117.3, 114.8, 113.0,
66.4, 16.1 ppm; MS (ESI): m/z: 325 [M+H]+.
121.3, 120.6, 117.6, 117.3, 114.3, 112.6, 66.7, 57.0 ppm; MS (ESI):
m/z: 417 [M+H]+.
(3-Hydroxyphenyl)(5-{3-[(3-methoxybenzyl)oxy]phenyl}-2-thien-
yl)methanone (14): The title compound was prepared by reaction
of 1a (150 mg, 0.53 mmol), 3-[(3-methoxybenzyl)oxy]benzene bor-
onic acid (165 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and
Pd(PPh3)4 (6 mg, 5 mmol) according to method A1. Purification by
CC (hexane/EtOAc 8:2) gave the product as a beige powder (74%,
163 mg): 1H NMR (CD3COCD3): d=8.75 (s, 1H), 7.71 (d, J=4.1 Hz,
1H), 7.60 (d, J=4.1 Hz, 1H), 7.44–7.36 (m, 5H), 7.34–7.30 (m, 2H),
7.13 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 7.10–7.08 (m, 3H), 6.91–6.89 (m,
1H), 5.21 (s, 2H), 3.81 ppm (s, 3H); 13C NMR (CD3COCD3): d=188.8,
162.0, 161.4, 159.5, 154.1, 141.3, 140.7, 137.8, 136.5, 132.3, 131.6,
131.4, 126.6, 122.1, 121.6, 121.3, 120.7, 117.7, 117.3, 115.2, 115.0,
114.4, 71.6, 56.6 ppm; MS (ESI): m/z: 417 [M+H]+.
[5-(3-Ethoxyphenyl)-2-thienyl](3-hydroxyphenyl)methanone (10):
The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 3-ethoxybenzene boronic acid (106 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 8:2) followed
by preparative TLC (hexane/EtOAc 7:3) gave the product as a
1
beige powder (91%, 157 mg): H NMR (CD3COCD3): d=8.80 (s, 1H),
7.69 (d, J=4.1 Hz, 1H), 7.56 (d, J=4.1 Hz, 1H), 7.41–7.34 (m, 4H),
7.32 (dt, J=1.3, 7.6 Hz, 1H), 7.28 (t, J=2.1 Hz, 1H), 7.13 (ddd, J=
1.3, 2.5, 7.6 Hz, 1H), 6.96 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 4.11 (q, J=
6.9 Hz, 2H), 1.38 ppm (t, J=6.9 Hz, 3H); 13C NMR (CD3COCD3): d=
188.7, 161.6, 159.4, 154.2, 144.1, 141.3, 137.7, 136.4, 132.2, 131.6,
126.4, 122.1, 121.2, 120.3, 117.3, 117.2, 113.9, 65.3, 16.1 ppm; MS
(ESI): m/z: 325 [M+H]+.
(3-Hydroxyphenyl)(5-{3-[(4-methoxybenzyl)oxy]phenyl}-2-thien-
yl)methanone (15): The title compound was prepared by reaction
of 1a (150 mg, 0.53 mmol), 3-[(4-methoxybenzyl)oxy]benzene bor-
onic acid (165 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and
Pd(PPh3)4 (6 mg, 5 mmol) according to method A1. Purification by
CC (hexane/EtOAc 8:2) gave the product as a beige powder (59%,
131 mg): 1H NMR (CD3COCD3): d=8.83 (s, 1H), 7.71 (d, J=4.1 Hz,
1H), 7.60 (d, J=4.1 Hz, 1H), 7.44 (d, J=8.5 Hz, 2H), 7.41–7.36 (m,
5H), 7.34–7.33 (m, 1H), 7.13 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 7.07
(ddd, J=1.3, 2.5, 7.9 Hz, 1H), 6.96 (d, J=8.5 Hz, 2H), 5.14 (s, 2H),
3.80 ppm (s, 3H); 13C NMR (CD3COCD3): d=186.9, 181.7, 161.6,
161.5, 159.4, 144.3, 141.3, 137.8, 136.5, 132.3, 131.6, 131.3, 131.0,
126.5, 122.1, 121.3, 120.6, 117.8, 117.3, 115.8, 114.4, 71.5, 56.6 ppm;
MS (ESI): m/z: 417 [M+H]+.
[5-(4-Ethoxyphenyl)-2-thienyl](3-hydroxyphenyl)methanone (11):
The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 4-ethoxybenzene boronic acid (106 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 8:2) gave the
product as a beige powder (71%, 122 mg): 1H NMR (CD3COCD3):
d=8.76 (s, 1H), 7.72 (d, J=8.8 Hz, 2H), 7.67 (d, J=4.1 Hz, 1H), 7.45
(d, J=4.1 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.35 (dt, J=1.3, 7.8 Hz,
1H), 7.33–7.32 (m, 1H), 7.11 (ddd, J=0.9, 2.5, 7.8 Hz, 1H), 7.01 (d,
J=8.8 Hz, 2H), 4.11 (q, J=6.9 Hz, 2H), 1.38 ppm (t, J=6.9 Hz, 3H);
13C NMR (CD3COCD3): d=188.6, 162.0, 159.4, 154.8, 143.1, 141.5,
138.1, 131.5, 129.5, 127.6, 125.0, 122.0, 121.1, 117.3, 117.0, 65.3,
16.0 ppm; MS (ESI): m/z: 325 [M+H]+.
(3-Hydroxyphenyl)(5-{3-[(3,5-dimethoxybenzyl)oxy]phenyl}-2-thi-
enyl)methanone (16): The title compound was prepared by reac-
tion of 1a (150 mg, 0.53 mmol), 3-[(3,5-dimethoxybenzyl)oxy]ben-
zene boronic acid (184 mg, 0.64 mmol), Cs2CO3 (691 mg,
2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) according to method A1.
Purification by CC (hexane/EtOAc 8:2) followed by preparative
1
HPLC gave the product as a beige powder (47%, 112 mg): H NMR
(CD3COCD3): d=7.71 (d, J=4.1 Hz, 1H), 7.59 (d, J=4.1 Hz, 1H),
7.42–7.35 (m, 5H), 7.33–7.32 (m, 1H), 7.12 (ddd, J=1.3, 2.5, 7.9 Hz,
1H), 7.09–7.06 (m, 1H), 6.68 (d, J=2.2 Hz, 2H), 6.44 (t, J=2.2 Hz,
1H), 5.17 (s, 2H), 3.79 ppm (s, 6H); 13C NMR (CD3COCD3): d=188.8,
163.1, 161.4, 159.5, 154.1, 144.3, 141.5, 141.3, 137.8, 136.5, 132.3,
131.6, 126.6, 122.0, 121.3, 120.7, 117.7, 117.3, 114.4, 107.2, 101.4,
71.6, 56.7 ppm; MS (ESI): m/z: 447 [M+H]+.
{5-[3-(Benzyloxy)phenyl]-2-thienyl}(3-hydroxyphenyl)methanone
(12): The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 3-(benzyloxy)benzene boronic acid (146 mg,
0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg,
5 mmol) according to method A1. Purification by CC (hexane/EtOAc
1
8:2) gave the product as a beige powder (71%, 146 mg): H NMR
(CD3COCD3): d=8.76 (s, 1H), 7.70 (d, J=4.1 Hz, 1H), 7.58 (d, J=
4.1 Hz, 1H), 7.52–7.51 (m, 2H), 7.43–7.32 (m, 9H), 7.13 (ddd, J=1.3,
2.5, 7.6 Hz, 1H), 7.08 (ddd, J=1.3, 2.5, 7.6 Hz, 1H), 5.22 ppm (s,
2H); 13C NMR (CD3COCD3): d=188.7, 161.4, 159.4, 154.1, 144.2,
141.3, 139.1, 137.8, 136.5, 132.3, 131.6, 130.3, 129.7, 129.5, 126.5,
122.1, 121.3, 120.7, 117.7, 117.3, 114.4, 71.7 ppm; MS (ESI): m/z: 387
[M+H]+.
(3-Hydroxyphenyl)(5-{3-[(2-chlorobenzyl)oxy]phenyl}-2-thienyl)-
methanone (17): The title compound was prepared by reaction of
1a (150 mg, 0.53 mmol), 3-[(2-chlorobenzyl)oxy]benzene boronic
acid (168 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and
Pd(PPh3)4 (6 mg, 5 mmol) according to method A1. Purification by
CC (hexane/EtOAc 8:2) gave the product as a beige powder (51%,
113 mg): 1H NMR (CD3COCD3): d=8.78 (s, 1H), 7.71 (d, J=4.1 Hz,
1H), 7.68–7.66 (m, 1H), 7.61 (d, J=4.1 Hz, 1H), 7.50–7.48 (m, 1H),
7.46–7.45 (m, 1H), 7.43–7.36 (m, 6H), 7.34–7.33 (m, 1H), 7.14–7.10
(m, 2H), 5.31 ppm (s, 2H); 13C NMR (CD3COCD3): d=188.8, 161.2,
159.4, 154.0, 144.3, 141.3, 137.8, 136.6, 136.6, 134.8, 132.4, 131.7,
131.6, 131.5, 131.3, 129.2, 126.7, 122.1, 121.3, 121.0, 117.6, 117.3,
114.4, 69.1 ppm; MS (ESI): m/z: 421 [M+H]+.
(3-Hydroxyphenyl)(5-{3-[(2-methoxybenzyl)oxy]phenyl}-2-thien-
yl)methanone (13): The title compound was prepared by reaction
of 1a (150 mg, 0.53 mmol), 3-[(2-methoxybenzyl)oxy]benzene bor-
onic acid (165 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and
Pd(PPh3)4 (6 mg, 5 mmol) according to method A1. Purification by
CC (hexane/EtOAc 7:3) gave the product as a beige powder (64%,
142 mg): 1H NMR (CD3COCD3): d=8.74 (s, 1H), 7.71 (d, J=4.1 Hz,
1H), 7.60 (d, J=4.1 Hz, 1H), 7.48 (m, 1H), 7.42–7.36 (m, 5H), 7.34–
7.31 (m, 2H), 7.13 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 7.08–7.04 (m, 2H),
6.97 (td, J=1.3, 7.6 Hz, 1H), 5.21 (s, 2H), 3.90 ppm (s, 3H); 13C NMR
(CD3COCD3): d=188.8, 161.6, 159.4, 159.2, 154.2, 144.2, 141.3,
137.8, 136.5, 132.3, 131.6, 131.2, 130.9, 127.0, 126.5, 122.3, 122.1,
(3-Hydroxyphenyl)(5-{3-[(3-chlorobenzyl)oxy]phenyl}-2-thienyl)-
methanone (18): The title compound was prepared by reaction of
1a (150 mg, 0.53 mmol), 3-[(3-chlorobenzyl)oxy]benzene boronic
acid (168 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and
Pd(PPh3)4 (6 mg, 5 mmol) according to method A1. Purification by
ChemMedChem 2011, 6, 476 – 487
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
483

R. W. Hartmann et al.
MED
CC (hexane/EtOAc 8:2) gave the product as a beige powder (55%,
122 mg): 1H NMR (CD3COCD3): d=8.80 (s, 1H), 7.70 (d, J=4.1 Hz,
1H), 7.58 (d, J=4.1 Hz, 1H), 7.57–7.56 (m, 1H), 7.48–7.46 (m, 1H),
7.43–7.34 (m, 8H), 7.13 (ddd, J=1.3, 2.5, 7.9 Hz, 1H), 7.10–7.08 (m,
1H), 5.24 ppm (s, 2H); 13C NMR (CD3COCD3): d=188.8, 161.1, 159.4,
154.0, 144.3, 141.7, 141.3, 137.7, 136.5, 135.8, 132.4, 132.1, 131.6,
129.7, 129.3, 127.8, 126.6, 122.1, 121.3, 120.9, 117.6, 117.3, 114.4,
70.7 ppm; MS (ESI): m/z: 421 [M+H]+.
ness to give the product as a beige powder (87%, 168 mg):
1H NMR (CD3COCD3): d=8.79 (s, 1H), 8.76 (s, 1H), 7.76 (t, J=1.9 Hz,
1H), 7.73 (d, J=4.1 Hz, 1H), 7.60–7.57 (m, 2H), 7.48 (t, J=7.9 Hz,
1H), 7.42–7.36 (m, 3H), 7.34 (t, J=1.9 Hz, 1H), 7.13 (ddd, J=1.2,
2.5, 7.6 Hz, 1H), 3.07 ppm (s, 3H); 13C NMR (CD3COCD3): d=188.8,
159.5, 153.5, 144.5, 141.5, 137.8, 132.3, 131.7, 126.7, 122.4, 122.1,
121.3, 119.3, 117.3, 40.7 ppm; MS (ESI): m/z: 374 [M+H]+.
N-{3-[5-(3-Methoxyphenyl)-2-thienyl]benzyl}methanesulfon-
amide (22a): The title compound was prepared by reaction of 1b
(200 mg, 0.67 mmol), 3-{[(methylsulfonyl)amino]methyl}benzene
boronic acid (183 mg, 0.80 mmol), Cs2CO3 (1.32 g, 4.04 mmol) and
Pd(PPh3)4 (12 mg, 10 mmol) according to method A2. Purification
by CC (hexane/EtOAc 9:1) gave the product as a beige powder
(94%, 253 mg): 1H NMR (CD3COCD3): d=7.87–7.86 (m, 1H), 7.75–
7.72 (m, 1H), 7.61 (d, J=4.1 Hz, 1H), 7.51–7.45 (m, 4H), 7.39–7.38
(m, 1H), 7.22 (ddd, J=1.6, 2.5, 7.6 Hz, 1H), 7.42 (s, 1H), 7.41 (s, 1H),
3.90 (s, 3H), 2.92 ppm (s, 3H); 13C NMR (CD3COCD3): d=188.7,
161.8, 154.2, 144.2, 141.7, 138.0, 135.4, 131.6, 131.4, 130.6, 127.5,
127.1, 126.6, 123.1, 120.1, 115.5, 56.9, 48.3, 41.6 ppm.
(3-Hydroxyphenyl)(5-{3-[(4-chlorobenzyl)oxy]phenyl}-2-thienyl)-
methanone (19): The title compound was prepared by reaction of
1a (150 mg, 0.53 mmol), 3-[(4-chlorobenzyl)oxy]benzene boronic
acid (168 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and
Pd(PPh3)4 (6 mg, 5 mmol) according to method A1. Purification by
CC (hexane/EtOAc 8:2) gave the product as a beige powder (63%,
141 mg): 1H NMR (CD3COCD3): d=8.76 (s, 1H), 7.71 (d, J=4.1 Hz,
1H), 7.60 (d, J=4.1 Hz, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.45–7.38 (m,
6H), 7.37 (dt, J=1.3, 7.6 Hz, 1H), 7.34–7.33 (m, 1H), 7.13 (ddd, J=
1.3, 2.5, 7.9 Hz, 1H), 7.09–7.08 (m, 1H), 5.24 ppm (s, 2H); 13C NMR
(CD3COCD3): d=188.8, 161.2, 159.4, 154.0, 144.3, 141.3, 138.2,
137.8, 136.5, 135.1, 132.4, 131.6, 131.2, 130.4, 126.6, 122.1, 121.3,
120.9, 117.7, 117.3, 114.4, 70.9 ppm; MS (ESI): m/z: 421 [M+H]+.
N-{3-[5-(3-Hydroxybenzoyl)-2-thienyl]benzyl}methanesulfon-
amide (22): The title compound was prepared by reaction of 22a
(253 mg, 0.63 mmol) and BBr3 (1.89 mmol) according to method B.
The crude mixture was washed with MeOH, and the suspension
was filtered and the precipitated product was evaporated to dry-
3-[5-(3-Methoxybenzoyl)-2-thienyl]benzonitrile (20a): The title
compound was prepared by reaction of 1b (300 mg, 1.01 mmol),
3-cyanobenzene boronic acid (178 mg, 1.21 mmol), Cs2CO3 (1.32 g,
4.04 mmol) and Pd(PPh3)4 (12 mg, 10 mmol) according to meth-
od A2. Purification by CC (hexane/EtOAc 9:1) gave the product as a
beige powder (88%, 282 mg): 1H NMR (CDCl3): d=7.95 (t, J=
1.6 Hz, 1H), 7.90–7.88 (m, 1H), 7.67–7.64 (m, 2H), 7.55 (t, J=7.9 Hz,
1H), 7.47 (dt, J=1.3, 7.6 Hz, 1H), 7.42 (t, J=7.9 Hz, 1H), 7.40–7.39
(m, 2H), 7.15 (ddd, J=1.3, 2.8, 8.2 Hz, 1H), 3.88 ppm (s, 3H);
13C NMR (CDCl3): d=187.5, 159.7, 149.7, 139.0, 135.7, 134.7, 132.1,
130.5, 130.1, 129.6, 129.5, 125.0, 121.7, 118.8, 118.2, 113.8, 113.6,
55.5 ppm.
1
ness to give a beige powder (81%, 197 mg): H NMR (CD3COCD3):
d=8.76 (s, 1H), 7.86 (s, 1H), 7.75–7.72 (m, 2H), 7.62 (d, J=4.0 Hz,
4H), 7.50–7.48 (m, 2H), 7.41 (t, J=7.8 Hz, 1H), 7.37 (dt, J=1.4,
7.6 Hz, 1H), 7.34–7.33 (m, 1H), 7.13 (ddd, J=1.2, 2.4, 7.6 Hz, 1H),
6.61 (s, 1H), 4.41 (s, 1H), 4.40 (s, 1H), 2.92 ppm (s, 3H); 13C NMR
(CD3COCD3): d=188.8, 159.5, 154.0, 144.3, 141.7, 141.3, 137.9,
135.4, 131.6, 131.4, 130.5, 127.5, 127.1, 126.5, 122.1, 121.3, 117.3,
48.3, 41.6 ppm; MS (ESI): m/z: 388 [M+H]+.
3-[5-(3-Hydroxybenzoyl)-2-thienyl]benzonitrile (20): The title
compound was prepared by reaction of 20a (282 mg, 0.88 mmol)
and BBr3 (2.64 mmol) according to method B. Purification by recrys-
tallization (EtOH/CH3CN) gave the product as a beige powder
(66%, 176 mg): 1H NMR (CD3COCD3): d=8.78 (s, 1H), 8.23 (t, J=
1.6 Hz, 1H), 8.12 (ddd, J=0.9, 1.9, 7.9 Hz, 1H), 7.83–7.81 (m, 1H),
7.78–7.76 (m, 2H), 7.72 (td, J=0.7, 7.9 Hz, 1H), 7.41 (t, J=7.6 Hz,
1H), 7.40–7.38 (m, 1H), 7.36–7.35 (m, 1H), 7.14 ppm (ddd, J=1.6,
2.5, 7.6 Hz, 1H); 13C NMR (CD3COCD3): d=188.8, 151.3, 145.5, 141.0,
140.7, 137.8, 136.5, 134.2, 132.4, 131.7, 131.3, 127.9, 122.2, 121.5,
117.3, 115.4, 110.3 ppm; MS (ESI): m/z: 306 [M+H]+.
N-{3-[5-(3-Hydroxybenzoyl)-2-thienyl]phenyl}-4-methylbenzene-
sulfonamide (23): The title compound was prepared by reaction
of 1a (150 mg, 0.53 mmol), 3-{[(4-methylphenyl)sulfonyl]amino}-
benzene boronic acid (239 mg, 0.64 mmol), Cs2CO3 (691 mg,
2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) according to method A1.
The crude mixture was washed with MeOH. The suspension was fil-
tered and the precipitated product was evaporated to dryness to
1
give a beige powder (53%, 126 mg): H NMR (CD3COCD3): d=7.75
(d, J=8.2 Hz, 2H), 7.70 (d, J=4.1 Hz, 1H), 7.62 (t, J=1.8 Hz, 1H),
7.50–7.48 (m, 2H), 7.40 (t, J=7.6 Hz, 1H), 7.38–7.32 (m, 5H), 7.27
(ddd, J=0.9, 2.2, 8.2 Hz, 1H), 7.13 (ddd, J=1.3, 2.5, 7.9 Hz, 1H),
2.35 ppm (s, 3H); 13C NMR (CD3COCD3): d=188.7, 159.5, 153.4,
145.8, 144.5, 141.2, 141.0, 138.9, 137.8, 136.1, 132.1, 131.6, 131.6,
129.1, 126.6, 123.8, 122.7, 122.1, 121.3, 119.6, 117.3, 22.4 ppm; MS
(ESI): m/z: 450 [M+H]+.
N-{3-[5-(3-Methoxybenzoyl)-2-thienyl]phenyl}methanesulfon-
amide (21a): The title compound was prepared by reaction of 1b
(300 mg, 1.01 mmol), 3-[(methylsulfonyl)amino]benzene boronic
acid (178 mg, 1.21 mmol), Cs2CO3 (1.32 g, 4.04 mmol) and Pd(PPh3)4
(12 mg, 10 mmol) according to method A2. The product was ob-
tained as a beige powder (96%, 376 mg) and used in the next step
without further purification: 1H NMR (CD3COCD3): d=7.76 (t, J=
1.9 Hz, 1H), 7.70 (d, J=4.1 Hz, 1H), 7.55–7.52 (m, 2H), 7.48–7.43
(m, 3H), 7.41 (ddd, J=0.9, 1.9, 7.9 Hz, 1H), 7.38–7.37 (m, 1H), 7.21–
7.19 (m, 1H), 3.87 (s, 3H), 3.07 ppm (s, 3H); 13C NMR (CD3COCD3):
d=188.6, 161.6, 153.6, 144.3, 141.4, 141.1, 137.9, 136.2, 131.5,
126.7, 123.7, 123.1, 122.4, 120.1, 119.3, 115.5, 59.7, 40.7 ppm.
(3-Hydroxyphenyl)[5-(2-naphthyl)-2-thienyl]methanone (24): The
title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 2-naphthaleneboronic acid (110 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 8:2) gave the
product as a yellow powder (57%, 100 mg): 1H NMR (CD3COCD3):
d=8.86 (s, 1H), 8.36 (s, 1H), 8.03–8.00 (m, 2H), 7.95–7.91 (m, 2H),
7.76 (d, J=4.1 Hz, 1H), 7.74 (d, J=4.1 Hz, 1H), 7.59–7.54 (m, 2H),
7.42 (t, J=7.6 Hz, 1H), 7.39 (dt, J=1.5, 7.6 Hz, 1H), 7.37–7.36 (m,
1H), 7.15–7.13 ppm (m, 1H); 13C NMR (CD3COCD3): d=188.8, 159.5,
154.3, 144.4, 141.3, 138.0, 135.6, 131.6, 130.9, 130.3, 129.7, 128.9,
128.8, 127.1, 126.7, 125.8, 122.1, 121.3, 117.3 ppm; MS (ESI): m/z:
331 [M+H]+.
N-{3-[5-(3-Hydroxybenzoyl)-2-thienyl]phenyl}methanesulfon-
amide (21): The title compound was prepared by reaction of 21a
(200 mg, 0.52 mmol) and BBr3 (1.56 mmol) according to method B.
The crude mixture was washed with MeOH, and the suspension
was filtered and the precipitated product was evaporated to dry-
484
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 476 – 487

Inhibitors of 17b-Hydroxysteroid Dehydrogenase Type 1
[5-(2,3-Dihydro-1-benzofuran-5-yl)-2-thienyl](3-hydroxyphenyl)-
methanone (25): The title compound was prepared by reaction of
1a (150 mg, 0.53 mmol), 2,3-dihydro-1-benzofuran-5-boronic acid
(105 mg, 0.64 mmol), Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4
(6 mg, 5 mmol) according to method A1. Purification by CC
(hexane/EtOAc 7:3) gave the product as a yellow powder (65%,
(d, J=4.1 Hz, 1H), 7.66–7.63 (m, 2H), 7.38 (t, J=7.9 Hz, 1H), 7.28
(dt, J=1.3, 7.9 Hz, 1H), 7.22–7.21 (m, 1H), 7.06 ppm (ddd, J=0.9,
2.5, 7.9 Hz, 1H); 13C NMR (CD3SOCD3): d=186.6, 179.5, 157.4, 153.2,
140.5, 139.9, 138.7, 136.7, 134.4, 129.7, 125.1, 124.7, 124.2, 123.3,
119.5, 118.4, 115.1, 111.1 ppm; MS (ESI): m/z: 321 [M+H]+.
1
111 mg): H NMR (CD3COCD3): d=7.67–7.66 (m, 2H), 7.57–7.55 (m,
1H), 7.44 (d, J=4.1 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.35 (dt, J=1.3,
7.6 Hz, 1H), 7.32–7.31 (m, 1H), 7.11 (ddd, J=1.3, 2.5, 7.9 Hz, 1H),
6.82 (d, J=8.5 Hz, 1H), 4.63 (t, J=8.8 Hz, 2H), 3.29 ppm (t, J=
8.8 Hz, 2H); 13C NMR (CD3COCD3): d=188.6, 159.4, 141.6, 138.1,
131.5, 130.9, 128.4, 127.8, 125.1, 124.8, 122.0, 121.0, 117.2, 111.5,
73.6, 73.5 ppm; MS (ESI): m/z: 323 [M+H]+.
Biology
[2,4,6,7-3H]E2 and [2,4,6,7-3H]E1 were bought from Perkin–Elmer
(Boston, USA). Quickszint Flow 302 scintillator fluid was bought
from Zinsser Analytic (Frankfurt, Germany). 17b-HSD1 and 17b-
HSD2 were obtained from human placenta according to previously
described procedures.[49] Fresh human placenta was homogenized
and cytosolic fraction and microsomes were separated by centrifu-
gation. For the partial purification of 17b-HSD1, the cytosolic frac-
tion was precipitated with (NH4)2SO4. 17b-HSD2 was obtained from
the microsomal fraction.
[5-(1,3-Benzodioxol-5-yl)-2-thienyl](3-hydroxyphenyl)methanone
(26): The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 1,3-benzodioxole-5-boronic acid (106 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A1. Purification by CC (hexane/EtOAc 8:2) gave the
product as a yellow powder (70%, 120 mg): 1H NMR (CD3COCD3):
d=8.76 (s, 1H), 7.66 (d, J=4.1 Hz, 1H), 7.46 (d, J=4.1 Hz, 1H), 7.39
(t, J=7.8 Hz, 1H), 7.35 (dt, J=1.3, 7.6 Hz, 1H), 7.33–7.32 (m, 1H),
7.30 (ddd, J=0.6, 1.9, 8.2 Hz, 1H), 7.28–7.27 (m, 1H), 7.12 (ddd, J=
1.3, 2.5, 7.9 Hz, 1H), 6.93 (d, J=7.9 Hz, 1H), 6.08 ppm (s, 2H);
13C NMR (CD3COCD3): d=188.6, 159.4, 154.4, 150.6, 143.4, 141.4,
137.9, 131.6, 129.4, 125.6, 122.4, 122.1, 121.2, 117.3, 110.7, 108.2,
103.7 ppm; MS (ESI): m/z: 325 [M+H]+.
For the use of human placenta, informed consent was obtained
from the parturient.
Inhibition of 17b-HSD1: Inhibitory activities were evaluated by an
established method with minor modifications.[49] Briefly, the
enzyme preparation was incubated with NADH (500 mm) in the
presence of potential inhibitors at 378C in a phosphate buffer
(50 mm) supplemented with 20% glycerol and EDTA (1 mm). Inhibi-
tor stock solutions were prepared in dimethyl sulfoxide (DMSO).
The final concentration of DMSO was adjusted to 1% in all sam-
ples. The enzymatic reaction was started by addition of a mixture
of unlabeled- and [2,4,6,7-3H]E1 (final concentration: 500 nm,
0.15 mCi). After 10 min, the reaction was stopped by the addition of
HgCl2, and the mixture was extracted with Et2O. After evaporation,
the steroids were dissolved in CH3CN. E1 and E2 were separated
using CH3CN/water (45:55) as the mobile phase on a C18 reverse-
phase chromatography column (Nucleodur C18 gravity, 3 mm; Ma-
cherey–Nagel, Dꢂren, Germany) connected to a HPLC-system (Agi-
lent 1100 series, Agilent Technologies, Waldbronn, Germany). De-
tection and quantification of the steroids were performed using a
radioflow detector (Berthold Technologies, Bad Wildbad, Germany).
The conversion rate was calculated after analysis of the resulting
chromatograms according to Equation (1). Each value was calculat-
ed from at least three independent experiments.
(3-Hydroxyphenyl)[5-(1H-indol-5-yl)-2-thienyl]methanone
(27):
The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), indole-5-boronic acid (103 mg, 0.64 mmol), Cs2CO3
(691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) according to
method A1. Purification by CC (hexane/EtOAc 7:3) gave the prod-
uct as a yellow powder (47%, 80 mg): 1H NMR (CD3COCD3): d=
10.46 (s, 1H), 8.70 (s, 1H), 7.90–7.89 (m, 1H), 7.69 (d, J=4.1 Hz,
1H), 7.67 (d, J=8.2 Hz, 1H), 7.53 (d, J=4.1 Hz, 1H), 7.49 (dd, J=
1.5, 8.2 Hz, 1H), 7.46–7.45 (m, 1H), 7.40 (t, J=7.6 Hz, 1H), 7.36 (dt,
J=1.5, 7.6 Hz, 1H), 7.34–7.33 (m, 1H), 7.12 (ddd, J=1.3, 2.5, 7.9 Hz,
1H), 6.54–6.53 ppm (m, 1H); 13C NMR (CD3COCD3): d=186.4, 159.4,
138.1, 137.5, 131.5, 129.0, 125.1, 122.9, 122.0, 121.0, 119.8, 117.3,
111.1, 103.9 ppm; MS (ESI): m/z: 320 [M+H]+.
(3-Hydroxyphenyl)[5-(1H-indol-6-yl)-2-thienyl]methanone
(28):
The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), indole-6-boronic acid (103 mg, 0.64 mmol), Cs2CO3
(691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) according to
method A1. Purification by CC (hexane/EtOAc 7:3) gave the prod-
uct as a yellow powder (49%, 83 mg): 1H NMR (CD3COCD3): d=
10.48 (s, 1H), 8.77 (s, 1H), 7.90–7.89 (m, 1H), 7.69 (d, J=4.1 Hz,
1H), 7.66 (d, J=8.2 Hz, 1H), 7.53 (d, J=4.1 Hz, 1H), 7.49 (dd, J=
1.5, 8.2 Hz, 1H), 7.45–7.44 (m, 1H), 7.40 (td, J=0.6, 7.6 Hz, 1H),
7.36 (dt, J=1.5, 7.6 Hz, 1H), 7.34–7.33 (m, 1H), 7.12 (ddd, J=1.3,
2.5, 7.9 Hz, 1H), 6.54–6.53 ppm (m, 1H); 13C NMR (CD3COCD3): d=
188.6, 159.4, 156.9, 142.9, 141.6, 138.5, 138.1, 131.5, 131.2, 129.0,
128.5, 125.1, 122.9, 122.0, 121.0, 119.8, 117.3, 111.1, 103.8 ppm; MS
(ESI): m/z: 320 [M+H]+.
%E2
ð1Þ
%conversion ¼
ꢁ 100
%E2 þ %E1
Inhibition of 17b-HSD2: The 17b-HSD2 inhibition assay was per-
formed similarly to the 17b-HSD1 procedure. The microsomal frac-
tion was incubated with NAD+ (1500 mm), test compound and a
mixture of unlabeled E2 and [2,4,6,7-3H]E2 (final concentration:
500 nm, 0.11 mCi) for 20 min at 378C. Further treatment of the sam-
ples and HPLC separation was carried out as mentioned above.
The conversion rate was calculated after analysis of the resulting
chromatograms according to Equation (2).
%E1
ð2Þ
%conversion ¼
ꢁ 100
%E1 þ %E2
(3-Hydroxyphenyl)[5-(2H-indazol-5-yl)-2-thienyl]methanone (29):
The title compound was prepared by reaction of 1a (150 mg,
0.53 mmol), 2H-indazole-5-boronic acid (104 mg, 0.64 mmol),
Cs2CO3 (691 mg, 2.12 mmol) and Pd(PPh3)4 (6 mg, 5 mmol) accord-
ing to method A2 refluxing the mixture for 14 h instead of 4 h. Pu-
rification by CC (hexane/EtOAc 6:4) gave the product as a yellow
ER affinity: The binding affinity of selected compounds to the ERa
and ERb was determined according to Zimmermann et al.[55] Briefly,
ERa or ERb (0.25 pmol) were incubated with [2,4,6,7-3H]E2 (10 nm)
and test compound for 1 h at room temperature. The potential in-
hibitors were dissolved in DMSO (5% final concentration). Evalua-
tion of nonspecific binding was performed with diethylstilbestrol
1
powder (75%, 127 mg): H NMR (CD3SOCD3): d=13.27 (s, 1H), 9.87
(s, 1H), 8.24 (s, 1H), 8.17 (s, 1H), 7.79 (dd, J=1.5, 8.5 Hz, 1H), 7.71
ChemMedChem 2011, 6, 476 – 487
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
485

R. W. Hartmann et al.
MED
(10 mm). After incubation, ligand–receptor complexes were selec-
tively bound to hydroxyapatite (5 g per 60 mL TE buffer). The com-
plex formed was separated, washed and resuspended in EtOH. For
radiodetection, scintillator cocktail (Quickszint 212, Zinsser Analytic,
Frankfurt, Germany) was added and samples were measured in a
liquid scintillation counter (Rack Beta Primo 1209, Wallac, Turku,
Finland). For determination of the relative binding affinity (RBA), in-
hibitor and E2 concentrations required to displace 50% of the re-
ceptor-bound labeled E2 were determined. RBA values were calcu-
lated according to Equation (3). The RBA value for E2 was set at
100%.
leted, rotatable bonds were defined, and AM1-BCC charges kept.
For the protein, nonpolar hydrogen atoms were deleted and
charges were added to the structure. The docking area has been
defined by a box, centered on the steroid binding site. Grid points
of 90ꢁ90ꢁ90 with 0.250 ꢃ spacing were calculated around the
docking area for all the ligand atom types using AutoGrid 4.2. For
each inhibitor, 50 separate docking calculations were performed.
Each docking calculation consisted of 25ꢁ105 energy evaluations
using the Lamarckian genetic algorithm local search (GALS)
method. Each docking run was performed with a population size
of 250. A mutation rate of 0.02 and a crossover rate of 0.8 were
used to generate new docking trials for subsequent generations.
The docking results from each of the 50 calculations were clus-
tered on the basis of a root mean square deviation (RMSD) of 2.0 ꢃ
between the Cartesian coordinates of the ligand atoms and were
ranked on the basis of the free binding energy.
IC50ðE2Þ
ð3Þ
RBA½%ꢂ ¼
ꢁ 100
IC50ðcompoundÞ
Inhibition of 17b-HSD1 in T47D cells: A stock culture of T47D cells
was grown in RPMI 1640 medium supplemented with 10% fetal
calf serum (FCS), l-glutamine (2 mm), penicillin (100 IUmLꢀ1), strep-
tomycin (100 mgmLꢀ1), insulin zinc salt (10 mgmLꢀ1) and sodium
pyruvate (1 mm) at 378C under 5% CO2 humidified atmosphere.
The cells were seeded into a 24-well plate at 1ꢁ106 cells per well
in Dulbecco’s modified Eagle’s medium (DMEM) medium with FCS,
l-glutamine and the antibiotics added in the same concentrations
as mentioned above. After 24 h, the medium was changed for
fresh serum-free DMEM and a solution of test compound in DMSO
was added. The final concentration of DMSO was adjusted to 1%
in all samples. After a preincubation of 30 min at 378C with 5%
CO2, the incubation was started by addition of a mixture of unla-
beled E1 and [2,4,6,7-3H]E1 (final concentration : 50 nm, 0.15 mCi).
After 0.5 h incubation, the enzymatic reaction was stopped by re-
moving of the supernatant medium. The steroids were extracted
with Et2O. Further treatment of the samples was carried out as
mentioned for the 17b-HSD1 assay.
Acknowledgement
We are grateful to the Deutsche Forschungsgemeinschaft
(HA1315/8–1) for financial support of this work. We thank Dr.
Jçrg Haupenthal for coordinating the biological tests and Jan-
nine Ludwig for her help in performing the in vitro tests (17b-
HSD1, 17b-HSD2, cellular inhibition assay).
Keywords: 17b-HSD1
inhibitors · structure–activity relationships
· breast cancer · endometriosis ·
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Published online on February 17, 2011
ChemMedChem 2011, 6, 476 – 487
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
487