First Dual Inhibitors of Steroid Sulfatase (STS) and 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1): Designed Multiple Ligands as Novel Potential Therapeutics for Estrogen-Dependent Diseases

Article abstract of DOI:10.1021/acs.jmedchem.7b00062

STS and 17β-HSD1 are attractive targets for the treatment of estrogen-dependent diseases like endometriosis and breast cancer. The simultaneous inhibition of both enzymes appears more promising than blockage of either protein alone. We describe a designed

Full text of DOI:10.1021/acs.jmedchem.7b00062

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Brief Article
First Dual Inhibitors of Steroid Sulfatase (STS) and 17#-Hydroxysteroid
Dehydrogenase Type 1 (17#-HSD1): Designed Multiple Ligands as
Novel Potential Therapeutics for Estrogen-Dependent Diseases
Mohamed Salah, Ahmed Saad Abdelsamie, and Martin Frotscher
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00062 • Publication Date (Web): 13 Apr 2017
Downloaded from http://pubs.acs.org on April 15, 2017
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First Dual Inhibitors of Steroid Sulfatase (STS) and 17βꢀ
Hydroxysteroid Dehydrogenase Type 1 (17βꢀHSD1 : Designed
)
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Multiple Ligands as Novel Potential Therapeutics for Estroꢀ
genꢀDependent Diseases
Mohamed Salah§, Ahmed S. Abdelsamie§#, and Martin Frotscher§*
§ Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, D-66123 Saarbrücken, Germany
# Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, 12622 Cairo, Egypt
KEYWORDS: steroidogenic enzyme inhibition; steroid sulfatase; 17β-hydroxysteroid dehydrogenase type 1; estrogen-
dependent disease; designed multiple ligand.
ABSTRACT: STS and 17β-HSD1 are attractive targets for the treatment of estrogen-dependent diseases like endometriosis
and breast cancer. The simultaneous inhibition of both enzymes appears more promising than blockage of either protein
alone. We describe a designed multiple target approach resulting in highly potent dual inhibitors. The most interesting
compound 9 showed nanomolar IC50s for both proteins, membrane permeability and no interference with estrogen re-
ceptors. It efficiently reversed E1S- and E1-induced T47D cell proliferation.
INTRODUCTION
Estrogens exert proliferative and antiapoptotic effects and
are involved in the etiology of estrogen-dependent diseas-
es (EDD) such as endometriosis and a high percentage of
breast cancers. Therapeutic interventions comprise endo-
crine treatment with GnRH analogs, selective estrogen
receptor modulators (SERMs) or aromatase inhibitors.
Figure 1. The sulfatase pathway of local estrogen biosyn-
thesis
These options, however, do not prevent relapses and
often lead to severe side effects. Thus, there is considera-
ble unmet medical need for novel treatments, and the
exploration of novel biological targets is required.
The latter is reduced to 17β-estradiol (E2), the most po-
tent estrogen in humans, predominantly by the action of
17β-HSD1 (Figure 1).3 This route of estrogen biosynthesis
has been termed “sulfatase pathway”.4 It also includes the
STS catalyzed transformation of androstenediol sulfate to
the estrogenic androstenediol, whose proliferative effect
on estrogen-sensitive cells is known.5 17β-HSD2 catalyzes
the reverse reaction, i.e. the inactivation of E2 by oxida-
tion to E1, and is the physiological adversary of the type 1
enzyme. Both STS and 17β-HSD1 are overexpressed in
endometriotic lesions,6,7 and there is strong evidence that
elevated local E2 levels are mainly due to estrogen activa-
tion via the sulfatase pathway whereas local E2 formation
from androgen precursors (“aromatase pathway”) is of
considerably less relevance.8 The sulfatase pathway also
plays a crucial role in estrogen-dependent breast cancer,
and STS expression is an important prognostic factor in
this disease.9,10 Strikingly, the tumor tissue of breast can-
cer patients who were treated with aromatase inhibitors
showed increased expression of both STS and 17β-HSD1.11
Thus, STS and 17β-HSD1 are key enzymes for local estro-
gen activation in EDD. In conclusion, their inhibition is a
promising approach for therapeutic intervention. The
validity of this concept is supported by the observation
that a 17β-HSD1 inhibitor led to a decrease of E2-levels in
Intriguingly, the progression of EDD is in many cases
strongly coupled to the local estrogen biosynthesis, i.e.
the formation of active estrogen within the diseased tis-
sue itself. Normally, the activities of enzymes involved in
local estrogen activation (steroid sulfatase (STS), aroma-
tase, 17β-HSD1) and de-activation (17β-HSD2, sulfotrans-
ferase) are well balanced. In case of EDD, a mismatch
between activation and de-activation results in elevated
local estrogen levels, leading to increased cell prolifera-
tion and reduced apoptosis. This mismatch is caused by
aberrant expression of the involved enzymes in situ.1,2
Therefore, the selective inhibition of local estrogen bio-
synthesis is a promising therapeutic approach, with the
prospect of fewer side effects compared to existing thera-
pies. In this context, STS and 17β-HSD1 play major roles as
they catalyze the final steps in estrogen biosynthesis with-
in the target cell (intracrinology): STS converts the inac-
tive estrone-3-sulfate (E1S), the main transport and stor-
age form of estrogens, to the weakly estrogenic estrone
(E1).
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endometriotic specimens.12 In transgenic mice, 17β-HSD1
inhibitors from different compound classes, described in
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5
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8
inhibitors reversed estrogen-induced endometrial hyper-
plasia.13 17β-HSD1 inhibitors were shown to reduce the E1-
stimulated tumor cell growth in vitro and in vivo, suggest-
ing the suitability of this target for the treatment of breast
cancer.14,15 In a mouse model, STS inhibition significantly
reduced the growth of endometriotic lesions, while plas-
ma E2 levels remained unchanged.16
a series of publications and summarized in a recent re-
view article.20 Selected DASIs showed favourable proper-
ties in vitro and in vivo.20,26 However, significant reduction
of plasma E2 levels was reported indicating a not exclu-
sively local mode of action.25
In this report we describe for the first time the rational
design, synthesis and in vitro profilation of non-steroidal
dual STS/17β-HSD1 inhibitors (DSHIs) as potential drugs
for the treatment of EDD, with the prospect of local ac-
tion.
Selective inhibition of either enzyme, however, bears
intrinsic drawbacks: Selective 17β-HSD1 inhibition does
not prevent the formation of the estrogenic agents E1 and
androstenediol. Selective STS inhibition, on the other
hand, does not block E2 formation from E1, which is pro-
duced from testosterone via the aromatase pathway. The
latter should not be very distinct but could be of rele-
vance for the progression of EDD.
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RESULTS AND DISCUSSION
While STS inhibitors differ considerably regarding molec-
ular structure, most of them bear an unsubstituted aryl
sulfamate group (exemplified by cmpd 14, figure 2) as a
common feature which serves as the main pharmaco-
phore for target inhibition. This structural motif was
adopted for the design of dual inhibitors and transferred
to an appropriate position of a highly potent inhibitor of
17β-HSD1.
Consequently, the idea of simultaneous inhibition of both
STS and 17β-HSD1 arises as a novel and attractive treat-
ment approach. This aim could be achieved by admin-
istration of two single inhibitors, each selective for one of
the two targets (multi component therapy). Different
patient-specific rates of biotransformation, however, may
result in complex PK/PD-correlations, leading to difficult
predictability of pharmacological effects. Another draw-
back is the risk of drug-drug-interactions.
Scheme 1. Synthesis of compounds 1-12
OH
a
b,c
Br
Br
O
S
F
S
S
R
O
F
1a
3b-12b
An intriguing concept is the inhibition of both targets
with a single drug whose structure is rationally derived
for this dual mode of action (designed multiple ligand,
DML).17 Preferably, 17β-HSD2 should not be inhibited.
O
b
d
O
S
OH
NH2
O
S
F
S
O
R
F
1b, 2b
3c-12c
O
S
O
d,c
a,c
O
NH2
4
O
S
F
O
2 R 3
F
1-12
HO
Position
Position
Cpd
R
Cpd
R
-OSO2NH2
-OSO2NH2
1
2
3
4
5
6
H
H
4
2
3
3
4
4
7
8
2-Me
3-F
4
4
4
4
4
4
H
9
3-Cl
2-F
2-F
2-Cl
10
11
12
3-Me
2,3-diF
Figure 2. Structure of compounds 13, 14 and general
structure of dual STS/17β-HSD1 inhibitors (DSHIs). R: e.g.
Cl, F, CH3.
2-F ,3-Cl
Reagents
and
conditions:
a)
2,6-difluoro-3-
For 17β-HSD1 and STS, a number of steroidal and non-
steroidal inhibitors have been described.18–22 Examples are
compounds 13 (17β-HSD1 inhibitor,)23 and 14 (STX-64)
which was the first STS inhibitor to enter clinical trials
(figure 2).24 Interestingly, DML approaches have success-
fully been applied for STS inhibitors, leading to the dis-
covery of compounds combining STS inhibition with
estrogen receptor modulation or inhibition of aromatase
(dual aromatase sulfatase inhibitors, DASIs).20 First de-
scribed by Woo et al.,25 the DASI concept has been thor-
oughly investigated and led to the discovery of many dual
methoxybenzoyl chloride, anhydrous AlCl3, anhydrous
o
CH2Cl2, 0 C, 0.5h and then room temperature, 3h; b) corre-
sponding phenyl boronic acid, Cs2CO3, Pd(PPh3)4,
DME/water (1:1), 110 °C, 4h; c) BBr3, CH2Cl2, -78oC to room
o
temperature, overnight; d) DMA, sulfamoyl chloride, 0 C,
and then room temperature, overnight.
We reported on the discovery of bicyclic substituted hy-
droxyphenylmethanones (BSHs) such as compound 13
23
(figure 2) as highly active 17β-HSD1 inhibitors.
Their
scaffold consists of a phenyl- and a benzoyl-moiety which
2
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group (28% and 22% of parent compound remaining after
are linked by a thiophene ring. Extensive SAR revealed
1
2
3
4
5
6
7
8
that their 17β-HSD1 inhibitory activity is maintained even
if bulky substituents are attached to the phenyl moiety
(Figure 2, compound 13, dotted box). This characteristic
triggered the implementation of the envisaged design
strategy by attaching a sulfamate group essential for STS
inhibition to the phenyl group of the BSHs. A general
structure of the potential DSHIs based on the above men-
tioned strategy is shown in Figure 2. The potential dual
inhibitors 1-12 were synthesized using standard methods
according to scheme 1.
30 min, resp.) whereas the other compounds proved to be
stable under these conditions. The different stabilities can
be correlated to the pKa values of the formed phenolic
OH groups which are significantly lower in case of 11 and
12 compared to the other compounds (see Supporting
Information, Table S2), making their phenolates better
leaving groups in substitution reactions. The loss of the
sulfamate “warhead” is in agreement with the comparably
low STS inhibition by 11 and 12. The fact that both com-
pounds showed strong inhibition of 17β-HSD1 is not in
conflict with their instability because the sulfamate group
is not relevant for 17β-HSD1 inhibition.
9
10
11
12
13
14
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Inhibition of human STS, 17β-HSD1 and 17β-HSD2 in
cell-free assays
Table 1: Inhibitory activities of compounds 1-14 to-
wards hSTS, h17β-HSD1 and 2 in cell-free assays
STS inhibition was determined by incubation of human
placental STS with E1S and inhibitor. E1 formation was
quantified by ELISA (see Supporting Information). Inhibi-
tion of 17β-HSD1 and 17β-HSD2 was evaluated using the
respective radiolabeled steroid (E1 or E2) and human
placental 17β-HSD1 (cytosolic fraction) or 17β-HSD2 (mi-
crosomal fraction). The radiolabeled estrogens were sepa-
rated and quantified using HPLC with scintillation detec-
tion (see Supporting Information). Inhibitory activities
are expressed as IC50 values (Table 1). 13 and 14 were used
as reference compounds.
IC50 [nM]a
Cpd
R1
R2
SFe
h17β-
h17β-
hSTSb
Compounds 1-3 were highly active towards 17β-HSD1, but
did not show inhibition of STS. Introduction of a fluorine
atom to compound 3 led to the dual inhibitor 4, whose
inhibition of STS, however, was clearly less pronounced
than that of 17β-HSD1. Shifting the sulfamate group from
position 3 to position 4 resulted in the highly active com-
pound 5 which equipotently inhibited STS and 17β-HSD1.
The STS inhibitory potency of compound 5 matched that
of the reference compound 14, one of the most potent STS
inhibitors. The fluorine atom of 5 could be replaced with
chlorine, leading to compound 6 with practically identical
inhibitory properties, whereas a methyl group slightly
decreased potency (compound 7). Compounds 5 and 6
only showed marginal selectivity over 17β-HSD2. Reloca-
tion of the substituents F, Cl and methyl from position 2
to position 3 (compounds 8-10) on the one hand led to a
decrease of STS inhibition. On the other hand, in case of
the halogenated inhibitors 8 and 9 this modification fur-
ther increased activity towards 17β-HSD1 and selectivity
over 17β-HSD2. This finding prompted us to synthesize
compounds 11 and 12, bearing halogen atoms in both
position 2 and 3, thus possibly combining strong STS
inhibition (halogen in position 3) with strong 17β-HSD1
inhibition and selectivity over 17β-HSD2 (halogen in posi-
tion 2).
HSD1c
HSD2d
1
2
H
-
H
-
ni
ni
3.4
22.5
2.2
7.1
28.0
4.1
8.2
0.2
14.2
2.5
3
H
F
-
ni
31.3
17.4
22.2
19.1
40.0
13.7
36.1
31.2
23.2
18.4
71.3
ni
4
-
123.4
19.5
19.4
35.0
81.8
143.1
220.8
105.5
244.5
ni
5
H
H
H
F
F
12.2
10.2
43.5
2.5
1.8
6
Cl
Me
H
H
H
F
1.9
7
0.9
5.5
8
9
Cl
Me
F
1.1
33.0
1.0
10
11
12
13
14
32.4
2.7
8.6
13.2
23.1
NA
Cl
-
F
1.4
-
3.1
-
-
15.1
ni
a
Mean value of at least two independent experiments each
b
conducted in duplicates , standard deviation less than 15%;
Human placenta, microsomal fraction, substrate E1S [300
nM]; c Human placenta, cytosolic fraction, substrate [3H]-E1 +
d
E1 [500 nM], cofactor NADH [0.5 mM]; Human placenta,
microsomal fraction, substrate [3H]-E2 + E2 [500 nM], cofac-
In fact, a strong 17β-HSD1 inhibition was achieved, com-
parable to that of compounds 8 and 9. In addition, 11 and
12 showed selectivity over 17β-HSD2. There was, however,
no improvement concerning STS inhibition. Investigation
of compound stability in buffer (20 mM Tris-HCl, pH =
7.2, 37 °C) revealed fast cleavage of the sulfamate moiety
in case of 11 and 12, under formation of the phenolic OH-
e
tor NAD+ [1.5 mM]; SF (selectivity factor): IC50(17β-HSD2) /
IC50(17β-HSD1); ni: no inhibition (< 10% inhibition at 1 µM);
NA: not applicable.
Intracellular inhibition of human STS and 17β-HSD1; Irre-
versible inhibition of human STS
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The estrogen-dependent human breast cancer cell line Table 2: Inhibitory activities of compounds 5, 6, 8, 9,
T47D expresses both STS and 17β-HSD1.27 Intact cells
were incubated with compounds 5, 6, 8 and 9, respective-
ly, and the corresponding radiolabeled substrate, E1S or
E1. After incubation, the steroids were separated and
quantified using HPLC with radio-detection. Table 2
shows the IC50 values of the cellular inhibition assays.
13 and 14 towards hSTS and h17β-HSD1 in cellular
assays
1
2
3
4
5
6
7
8
R2
R
1 O
S
O
NH2
O
O
S
F
F
All compounds displayed strong inhibition of the target
enzymes with IC50 values in the nanomolar range, indicat-
ing good cell penetration. The fact that the compounds
were able to strongly inhibit STS in spite of the long incu-
bation time of 24h suggested an irreversible mode of STS
inhibition, as described for other sulfamate-containing
STS inhibitors, e.g. for 14.20
5 6, 8 9
,
,
HO
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
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46
47
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IC50 [nM]a
hSTSb
Cpd
R1
R2
h17β-
hSTSb
HSD1c
irreversible
For further support of this irreversible mode of action,
T47D cells were pretreated with compounds 5, 6, 8, 9 and
the reference 14, respectively. After removal of the com-
pounds by extensive washing, STS activity was evaluated
by incubation with E1S. In all cases, conversion to E1 was
strongly inhibited with IC50 values very similar to those
obtained in the “regular” assay for cellular STS inhibition
(Table 2). In contrast, 17β-HSD1 activity was fully restored
after incubation with 9 (Table S3), suggesting that the
persisting STS inhibition is a result of irreversible inhibi-
tion rather than of compound retention.
5
6
H
H
F
F
Cl
H
H
-
2.1
3.4
5.1
42.1
60.9
20.0
22.2
7.9
3.1
3.9
5.7
16.4
ni
8
9
Cl
-
15.6
ni
13
14
-
-
1.9
ni
2.1
a
Mean value of at least two independent experiments each
conducted in triplicates using intact T47D cells, standard
deviation less than 15%; Substrate [3H]-E1S + E1S [5 nM];
b
b
Estrogen stimulation of T47D cell proliferation
Substrate [3H]-E1 + E1 [50 nM]; ni: no inhibition (< 5% inhibi-
tion at 1 µM).
The proliferation of T47D cells in response to estrogen
treatment was evaluated by adding E1S, E1 or E2 to the
culture medium at concentrations ranging from 0.1 nM to
500 nM and evaluation of cell viability after 7 days of
incubation (Figure 3). Stimulation of proliferation was
similar for E1S, E1 and E2.
It was initially observed at an estrogen concentration of
10 nM and reached a maximum at 250 nM. In the follow-
ing experiments, 100 nM of estrogen (either 50 nM E1S
and 50 nM E1 –reflecting that both may be present in vivo
- or 100 nM E2) was used.
Effect of DSHIs on estrogen stimulated cell proliferation
Compound 9 was applied to E1S/E1-stimulated T47D cells
at concentrations of 100, 200 and 400 nM, approximately
corresponding to 5, 10 and 20 times its IC50 value of cellu-
lar 17β-HSD1 inhibition. The reference compounds 13 and
14 were applied in concentrations of 50, 100 and 200 nM,
approximately corresponding to the same multiples of
their IC50 values, in case of 14 that of the cell-free assay.
The dual inhibitor 9 was able to decrease the proliferative
effect of E1S/E1 stimulation dose-dependently, reaching
control levels when applied in a concentration of 400 nM
(Figure 4). Similar results were obtained for compounds 5,
6 and 8 (Figure S1). In contrast, the selective references 13
and 14 did not decrease the stimulatory estrogen effect
below 150% and 200% of the control, respectively.
Figure 3. Concentration dependent stimulation of T47D
cell proliferation. Black bars: E1S-stimulation, striped: E1-
stimulation, gray: E2-stimulation. The control (no estro-
gen-stimulation) was arbitrarily set to 100%. Cells were
incubated with the respective additives for 7 days without
passage. Medium was changed every 2–3 days.
medium after feeding the cells with E1S and E1, depending
on the presence of the different types of inhibitors. This
point was investigated by incubating the cells with radio-
labeled E1S and E1 in the presence or absence of inhibi-
tors, and quantification of estrogen levels after 48 h using
HPLC with radio-detection (Table 3). In the absence of
inhibitor, almost complete conversion of both E1S and E1
to E2 was observed, whereas in the presence of the dual
inhibitor 9 no significant conversion of E1S or E1 occurred.
In the presence of 14 no conversion of E1S occurred but all
The stronger anti-proliferative effects of the dual inhibi-
tors in comparison to the selective ones were assumed to
be attributed to the differences in E2 and E1 levels in the
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firmed using estrogen-independent cells: Compound 9
the E1 was transformed into E2. In case of 13, E1S was
1
2
3
4
5
6
7
8
almost completely consumed and 88.4% E1 were found,
besides a minor amount of E2 (7.9%). These results are in
agreement with the residual proliferation induction found
in case of the selective inhibitors. In addition, they ex-
plain the more pronounced residual cell proliferation
after application of 14 compared to 13 (200% vs. 150%), as
in the first case the amount of the strongly estrogenic E2
was high (49.2%) whereas in the latter case the less estro-
genic E1 was the predominant estrogen.
did not affect the viability of HEK293 cells, even in the
highest applied concentration of 1 µM (see supporting
information). Moreover, no influence of 9 on the prolifer-
ation of E2-stimulated cells could be detected, indicating
that the compound did not deploy its anti-proliferative
effect by estrogen receptor antagonism. These data clearly
demonstrate that the effect of compound 9 on E1S/E1-
stimulated cells is caused by inhibition of STS and 17β-
HSD1, which results in the blockage of E2 formation.
9
10
11
12
13
14
15
16
17
18
19
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CONCLUSIONS
A designed multiple target approach was successfully
applied, leading to the discovery of potent dual inhibitors
of STS and 17β-HSD1. Inhibitor design was facilitated by
combining structural elements necessary for strong 17β-
HSD1 blockage, identified using in-house SAR infor-
mation, with a sulfamate function which is the major
pharmacophore for STS inhibition. Twelve potential dual
inhibitors were synthesized, all of which proved to be
highly active against 17β-HSD1. Activity towards STS re-
quired an additional substituent at the aromatic moiety
bearing the sulfamate group. An electron-withdrawing
substituent is preferred; strong electron-withdrawing
effects, however, impaired STS inhibition by reducing the
chemical stability of the sulfamate function.Compound 9
turned out to be the most interesting dual inhibitor. In
cellular assays it showed well-balanced activity against
both target proteins, with IC50 values of about 20 nM and
an irreversible mode of action towards STS. Moreover, it
displayed the highest selectivity over 17β-HSD2. At 400
nM it efficiently reversed the E1S and E1 stimulated prolif-
eration of T47D cells, showing neither cytotoxicity nor
estrogen receptor interference.
Figure 4. Concentration dependent inhibition of E1S- and E1-
stimulated cell growth for compounds 9, 13 and 14 on T47D
cells. Cells were grown in phenol red-free RPMI 1640 medi-
um supplemented with 5% stripped FCS. Control represents
vehicle treated cells. EM (estrogenic medium) represents E1S
[50 nM] and E1 [50 nM] treated cells. 9 was tested at 100, 200
and 400 nM, resp. 13 and 14 were tested at 50, 100 and 200
nM, resp. Cells were incubated with the respective additives
for 7 days without passage. Medium was changed every 2–3
days. Vehicle: ethanol. ** p < 0.01. *** p < 0.001.
Table 3: Percentages of estrogens upon incubating
T47D cells with E1S and E1 (50 nM each) for 48 hours,
in the presence of vehicle, 9, 13 and 14
a
%
Cpd
E1S
E1
E2
Vehicle b
9 c
3.9
50
3.5
50
0.9
48.9
88.4
0.8
95.2
1.1
13 d
14 e
7.9
Figure 5. Effect of compound 9 on estrogen-stimulated cell
proliferation of T47D. Cells were grown in phenol red-free
RPMI 1640 medium supplemented with 5% stripped FCS.
Proliferation was stimulated with E1S and E1 at a concentra-
tion of 50 nM each or E2 (100 nM). 9 (400 nM) was added in
the presence or absence of estrogens. Cells were incubated
with the respective additives for 7 days without passage.
Medium was changed every 2–3 days. Vehicle: ethanol. *** p
< 0.001.
49.2
a
Mean value of at least two independent experiments each
b
conducted in triplicates, standard deviation less than 10%;
vehicle: ethanol; cmpd 9 [400 nM]; cmpd 13 [200 nM];
cmpd 14 [200 nM].
c
d
e
As depicted in Figure 5, compound 9 has no effect on
non-stimulated cells at a concentration of 400 nM, which
was the highest concentration in which 9 was applied in
the anti-proliferation assay. Thus, the dual inhibitor 9 did
not exert estrogenic or cytotoxic effects at this concentra-
tion. The lack of unspecific cytotoxicity was also con-
In summary, compound 9 is the first rationally derived
dual inhibitor of STS and 17β-HSD1. It may serve as a lead
for the development of novel therapeutics for EDD.
5
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Journal of Medicinal Chemistry
Page 6 of 7
3 equiv) in anhydrous dichloromethane (10 ml). The
EXPERIMENTAL SECTION
1
2
3
4
5
6
7
8
product was purified by column chromatography (di-
chloromethane/methanol 98.5:1.5) to give 60.00 mg (0.13
mmol/ 27%) of the analytically pure compound.
C17H10ClF2NO5S2; MW 445; mp: 172-173; 1H NMR (500
MHz, (CD3)2CO) δ 9.00 (s, 1H), 7.97 (d, J = 2.3 Hz, 1H),
7.80 (dd, J = 8.6, 2.3 Hz, 1H), 7.67 (d, J = 4.1 Hz, 1H), 7.64 –
7.62 (m, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.47 (s, 2H), 7.21 –
7.14 (m, 1H), 7.03 – 6.96 (m, 1H); 13C NMR (126 MHz,
(CD3)2CO) δ 180.76, 152.52 (dd, J = 240.6, 5.9 Hz), 152.31,
148.41 (dd, J = 246.0, 7.6 Hz), 148.06, 144.15, 142.67 (dd, J =
12.8, 3.2 Hz), 138.18, 133.45, 129.05, 129.03, 127.25, 127.04,
125.62, 120.44 (dd, J = 9.1, 3.9 Hz), 117.89 (dd, J = 23.9, 19.6
Hz), 112.45 (dd, J = 22.8, 3.9 Hz); MS (ESI): 446.12 (M+H)+.
The purity of all tested compounds was ≥ 95%, as evaluat-
ed by LC/MS. Purchased chemicals were reagent grade
and used without purification (supporting information).
Compound 9a was prepared according to method B by
the reaction of 2-bromothiophene (0.82 g, 5 mmol, 1
equiv) and (3-chloro-4-methoxyphenyl)boronic acid (1.11
g, 6.00 mmol, 1.2 equiv) in the presence of caesium car-
bonate (6.50 g, 20.00 mmol, 4 equiv) and tetrakis-
(triphenylphosphine) palladium (0.29 g, 0.25 mmol,
0.05 equiv) in DME/water 1:1 (50 ml). The product was
purified by column chromatography (cyclohex-
ane/dichloromethane 7:1) to give 1.01 g (4.50 mmol/ 90%)
of the analytically pure compound. C11H9ClOS; MW 224;
1H NMR (300 MHz, (CD3)2SO) δ 7.72 (d, J = 2.3 Hz, 1H),
7.57 (dd, J = 8.6, 2.3 Hz, 1H), 7.50 (dd, J = 5.1, 1.2 Hz, 1H),
7.47 (dd, J = 3.6, 1.2 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.11
(dd, J = 5.1, 3.6 Hz, 1H), 3.88 (s, 3H); MS (ESI): 225.07
(M+H)+.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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53
54
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58
59
60
ASSOCIATED CONTENT
Supporting Information. Chemistry: Chemical methods;
General synthetic methods (A, B, C, D, and E); Detailed syn-
thesis procedures and compound characterization; Purity
data; Molecular formula strings. Biology: Chemicals; Detailed
biological assays; Statistical method. This material is availa-
ble free of charge via the Internet at http://pubs.acs.org.
Compound 9b was prepared according to Method C by
the reaction of 9a (0.90 g, 4.00 mmol, 1 equiv) and boron
tribromide (1 M) in dichloromethane (12.00 ml, 12.00
mmol, 3 equiv) in anhydrous dichloromethane (20 ml).
The product was purified by column chromatography
(cyclohexane/dichloromethane 4:1) to give 0.65 g (3.08
mmol/ 77%) of the analytically pure compound.
C10H7ClOS; MW 210; 1H NMR (300 MHz, (CD3)2SO) δ 10.36
(s, 1H), 7.62 (d, J = 2.3 Hz, 1H), 7.46 (dd, J = 5.1, 1.2 Hz, 1H),
7.44 – 7.35 (m, 2H), 7.09 (dd, J = 5.1, 3.6 Hz, 1H), 7.00 (d, J
= 8.4 Hz, 1H); MS (ESI): 211.03 (M+H)+.
AUTHOR INFORMATION
Corresponding Author
*E-mail address: m.frotscher@mx.uni-saarland.de (M.
Frotscher), Tel.: +49-681 302 70330
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
Compound 9c was prepared according to method D by
the reaction of 9b (0.63 g, 3.00 mmol, 1 equiv) and sul-
famoyl chloride (1.73 g, 15.00 mmol, 5 equiv) in DMA (20
ml). The product was purified by column chromatography
(cyclohexane/ethylacetate 2:1) to give 0.45 g (1.56 mmol/
52%) of the analytically pure compound. C10H8ClNO3S2;
Funding Sources
We are grateful to the Deutsche Forschungsgemeinschaft for
financial support (FR 3002/1-1) of this work.
ACKNOWLEDGMENT
Thanks are due to C. Hoffmann and J. Ludwig for support in
synthesis and biological evaluation.
Dedicated to Professor Rolf W. Hartmann on the occasion of
his 65th birthday.
1
MW 289; H NMR (300 MHz, (CD3)2SO) δ 8.29 (s, 2H),
7.88 (d, J = 2.2 Hz, 1H), 7.69 (dd, J = 8.6, 2.3 Hz, 1H), 7.64 –
7.59 (m, 2H), 7.52 (d, J = 8.6 Hz, 1H), 7.16 (dd, J = 4.9, 3.9
Hz, 1H); MS (ESI): 290.01 (M+H)+.
Compound 9d was prepared according to method A by
the reaction of 2,6-difluoro-3-methoxybenzoyl chloride
(0.41 g, 2.00 mmol, 1 equiv) and 9c (0.87 g, 3.00 mmol, 1.5
equiv) in the presence of anhydrous aluminum chloride
(0.53 g, 4.00 mmol, 2 equiv) in anhydrous dichloro-
methane (10 ml). The product was purified by column
chromatography (cyclohexane/ethylacetate 1:1) to give
0.59 g (1.30 mmol/ 65%) of the analytically pure com-
ABBREVIATIONS
BSHs, bicyclic substituted hydroxyphenylmethanones;
DSHIs, dual STS/17β-HSD1 inhibitors; E2, 17β-estradiol; EDD,
estrogen-dependent diseases; E1, estrone; E1S, estrone-3-
sulfate; 17β-HSD1 or 2, 17β-hydroxysteroid dehydrogenase
type1 or 2; STS, steroid sulfatase.
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pound. C18H12ClF2NO5S2; MW 459; H NMR (300 MHz,
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