Lead optimization of 17β-HSD1 inhibitors of the (hydroxyphenyl) naphthol sulfonamide type for the treatment of endometriosis

Article abstract of DOI:10.1021/jm201735j

The reduction of estrone to estradiol, the most potent estrogen in human, is catalyzed by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). A promising approach for the treatment of estrogen-dependent diseases is the reduction of intracellular estradiol formation by inhibition of 17β-HSD1. For the species-specific optimization of the (hydroxyphenyl)naphthols, a combinatorial approach was applied and enhanced by a focused synthesis that resulted in the aromatic-substituted (hydroxyphenyl)naphthol sulfonamides. Rigidification of 12 led to the 4-indolylsulfonamide 30, which is a highly active and selective human 17β-HSD1 inhibitor, as well as a highly potent and selective inhibitor of 17β-HSD1 from Callithrix jacchus. It shows no affinity to the estrogen receptors I?± and β and good intracellular activity (T47D). Thus, compound 30 shows good properties for further ADMET studies and might be a candidate for the in vivo proof of concept in C. jacchus.

Full text of DOI:10.1021/jm201735j

Article
pubs.acs.org/jmc
Lead Optimization of 17β-HSD1 Inhibitors of the
(Hydroxyphenyl)naphthol Sulfonamide Type for the Treatment of
Endometriosis
Claudia Henn,†,‡ Almuth Einspanier,§ Sandrine Marchais-Oberwinkler,† Martin Frotscher,†
and Rolf W. Hartmann*,†,‡
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2 3, D-66041 Saarbrucken, Germany
̈
‡Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Center for Infection Research (HZI), Campus C2 3,
66123 Saarbrucken, Germany
̈
§Faculty of Veterinary Medicine, Institute of Physiological Chemistry, An den Tierkliniken 1, 04103 Leipzig, Germany
S
* Supporting Information
ABSTRACT: The reduction of estrone to estradiol, the most
potent estrogen in human, is catalyzed by 17β-hydroxysteroid
dehydrogenase type 1 (17β-HSD1). A promising approach for
the treatment of estrogen-dependent diseases is the reduction
of intracellular estradiol formation by inhibition of 17β-HSD1.
For the species-specific optimization of the (hydroxyphenyl)-
naphthols, a combinatorial approach was applied and
enhanced by a focused synthesis that resulted in the
aromatic-substituted (hydroxyphenyl)naphthol sulfonamides.
Rigidification of 12 led to the 4-indolylsulfonamide 30, which
is a highly active and selective human 17β-HSD1 inhibitor, as well as a highly potent and selective inhibitor of 17β-HSD1 from
Callithrix jacchus. It shows no affinity to the estrogen receptors α and β and good intracellular activity (T47D). Thus, compound
30 shows good properties for further ADMET studies and might be a candidate for the in vivo proof of concept in C. jacchus.
Notably, mRNA of 17β-HSD1, which is often used as a
prognostic marker, has been found highly expressed in breast
cancer11 and endometriotic tissue.12 Therefore, the develop-
ment of 17β-HSD1 inhibitors is an attractive approach to
reduce the intracellular E2 levels and to treat EDDs, particularly
since such an intracrine concept has already been proven to be
successful for the treatment of the androgen-dependent
diseases BPH (benign prostatic hyperplasia) and alopecia by
using 5α-reductase inhibitors.13,14
INTRODUCTION
■
Estradiol (E2), the most important estrogen in females, is
responsible for the development and differentiation of
estrogen-sensitive tissues, for example, breast and endometrial
tissues. It is mainly produced in the granulosa cells of the
ovaries. In a first step, estrone (E1) is formed by aromatization
of androstenedione, followed by the local conversion of E1 to
E2 by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1,
Figure 1). Besides its physiological effects, E2 is involved in the
To decrease undesirable pharmacological effects, the 17β-
HSD1 inhibitors should be selective toward 17β-hydroxysteroid
dehydrogenase type 2 (17β-HSD2), which catalyzes the
deactivation of E2 to E1 (Figure 1) and is downregulated in
malignant breast cancer cells. Specific inhibition of 17β-HSD2
was recently published as a novel approach to prevent
osteoporosis.15−19 Furthermore, potential inhibitors should
not show any affinity to estrogen receptors α and β (ERα and
β) to reduce the risk of estrogenic side effects.
Some 17β-HSD1 inhibitors are already described. Most of
them are based on a steroidal skeleton.20−22 In the last couple
of years, selective nonsteroidal inhibitors were published by
others23−26 and our group. Our work is based on a long time
experience in steroid mimetics, which led to the development
Figure 1. Interconversion of E1 to E2.
development and progression of estrogen-dependent diseases
(EDDs) like breast cancer,1,2 ovarian tumor,3 endometriosis,4,5
endometrial hyperplasia,6 and uterine leiomyoma.7 The
conventional treatment for these diseases is therapy with
antiestrogens,8 selective estrogen receptor modulators
(SERMs), or aromatase inhibitors9,10 to block the synthesis
of estrogens on the systemic level.
Received: December 23, 2011
Published: March 1, 2012
© 2012 American Chemical Society
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of selective inhibitors of CYP19,27,28 CYP17,29−31
CYP11B2,32−34 and CYP11B135,36 and resulted in three classes
of nonsteroidal 17β-HSD1 inhibitors: the bis(hydroxyphenyl)-
heterocycles,37−42 the hydroxyphenylmethanones,43−45 and the
(hydroxyphenyl)naphthols.46−48 Recently, we reported on the
optimization of the latter class that led to the identification of a
highly active and selective sulfonamide substituted derivative A
with no affinity to ERα and ERβ (Figure 2).49
find compounds for in vivo experiments. Because of the high
phylogenetic similarity of monkeys and humans, models of the
former species are preferable. An appropriate animal should be
Callithrix jacchus (common marmoset), as there is an
endometriosis model described.54 The high overall sequence
identity of 80% between human and C. jacchus 17β-HSD1
(cj17β-HSD1) increases up to 87%, if only the steroidal binding
site is considered, with five major amino acid variations
observed in common marmoset: A191P, E194Q, S222N,
V225I, and E282N.55 This high identity makes the animal
model very appropriate for in vivo evaluation of designed
inhibitors. Two different forms of endometriosis can be
induced: developing and established endometriosis. Further-
more, the pathogenesis of endometriosis and the relationship
between local and central estrogen metabolism have been
studied, which is important for validation of the intracrine
concept of 17β-HSD1 inhibitors.
After having established an assay to screen our in-house
library for compounds showing good inhibition of cj17β-HSD1,
the lead compound in the class of the sulfonamide substituted
hydroxyphenylnaphthols (Figure 2), however, showed only
very low inhibitory potency (16% inhibition at 50 nM) and no
selectivity toward C. jacchus 17β-hydroxysteroid dehydrogenase
type 2 (cj17β-HSD2) (23% inhibition at 50nM).55 Therefore, it
is our aim to increase the potency toward cj17β-HSD1 without
decreasing the activity toward human 17β-hydroxysteroid
dehydrogenase type 1 (h17β-HSD1) in this class of compounds
to identify a potential candidate for the in vivo proof of concept
in the common marmoset.
Figure 2. Recently published inhibitor.
As this compound showed good intracellular activity and
favorable pharmacokinetics, it should be the starting point for
the establishment of an in vivo proof of concept in a disease-
oriented animal model. Several animal models for breast cancer
and endometriosis using different species are available.50−54 For
an in vivo experiment with rodents, there are either rodent
enzyme active inhibitors necessary, or experiments with
xenograft models or transgenic mice have to be performed.
Previous studies had revealed that representative inhibitors of
the aforementioned classes only show low inhibition of E2
formation in the rat and in the mouse.42,43,45 Therefore, we
focus on a species in which 17β-HSD1 shows high sequence
identity to the human enzyme to increase the probability to
In this study, we report on the optimization of the
hydroxyphenylnaphthols at the sulfonamide core. In a previous
study, it was found that the exchange of aliphatic sulfonamides
by aromatic sulfonamides led to an increase in activity toward
Figure 3. Library compounds.
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a
Scheme 1. Synthesis of Compound 1b
a
Reagents and conditions: (a) NBS, reflux, 2 h. (b) Na2CO3, Pd(PhP3)4, toluene/H2O/MeOH (3:2:2). (c) Cs2CO3, Pd(PhP3)4, dimethoxyethane/
EtOH/H2O (1:1:1), 15 min microwave irradiation (150 °C, 1.5 bar).
h17β-HSD1.45 Therefore, we designed and synthesized a library
(parallel synthesis) of aromatic-substituted sulfonamides
(Figure 3), and the compounds were evaluated for inhibition
of h17β-HSD1 in a cell-free assay. The most promising
compounds were resynthesized in a larger amount and were
further evaluated biologically. To optimize and to derive a
meaningful SAR (structure−activity relationship), substituents
in the phenyl moieties were varied regarding their electronic
and lipophilic properties (Table 1).
Scheme 2. Synthesis of Library Compounds 1−11, 14, 15,
a
and 17
CHEMISTRY
■
The available 2-bromo-6-methoxynaphthalene was brominated
with N-bromosuccinimide followed by Suzuki cross-coupling
with 3-(methoxyphenyl) boronic acid, sodium carbonate, and
Pd(PhP3)4 in toluene/H2O/MeOH (3:2:2) to obtain com-
pound 1c. In a second Suzuki cross-coupling with 3-
(aminophenyl) boronic acid, cesium carbonate, and Pd(Ph3P)4
in dimethoxyethane/EtOH/H2O (1:1:1), compound 1b was
prepared (Scheme 1).
The library synthesis was started using 3-(2-methoxy-6-(3-
methoxyphenyl)naphthalen-1-yl)aniline (1b), and an ether
cleavage according to method C was performed yielding the
demethylated compound 1a. The diversity was introduced with
different commercially available sulfonyl chlorides (1−11, 14,
15, 17, method A, Scheme 2).
a
Reagents and conditions: (a) BBr3, CH2Cl2, −78 to rt, 18 h, method
C. (b) PS-morpholine, PS-DMAP, THF, 48 h, rt, method A.
The important intermediate 12b was synthesized in four
steps. After bromination of β-naphthol in glacial acetic acid, the
β-hydroxy function was protected with benzylbromide (12d).
Via two successive Suzuki cross-couplings with 3-(benzox-
yphenyl)- and 3-(aminophenyl) boronic acid 12b was obtained
(Scheme 3).
The synthesis of the protected sulfonamides 8a−29a and the
deprotection yielding 8−29 are shown in Scheme 4. The
sulfonamide coupling with compound 1b or compound 12b
and the commercially available sulfonyl chlorides was
performed according to method B (DMAP, pyridine, rt, 6
days). In a last step, compounds 8a, 10a, 11a, 15a, 17a, and
18a were demethylated with boron tribromide (method C;
BBr3, CH2Cl2, −78 °C to rt, 18 h, Scheme 4). Compound 9a,
12a−14a, 16a, and 19a−29a were debenzylated using the same
reagent according to method D (BBr3, CH2Cl2, −25 °C for 1 h,
rt for 2 h, Scheme 4).
For the synthesis of the rigidified sulfonamides 30 and 31,
the 4-indolylphenylsulfonamide and the 6-indolylphenylsulfo-
namide residues were introduced to compound 12c by a Suzuki
coupling with the corresponding indoyl boronic acids. A
tetrabutyl ammonium chloride (TBAC)-catalyzed sulfonamide
coupling resulted in compounds 30a and 31a. Cleavage of the
benzyloxy moieties with boron tribromide (method D)
afforded 30 and 31 (Scheme 5).
BIOLOGICAL RESULTS
■
Inhibition of 17β-HSD1 and Selectivity toward 17β-
HSD2 in Human and C. jacchus. The assays were performed
with placental enzymes from human and C. jacchus (cj). For the
17β-HSD1 assay, the cytosolic fraction, tritiated E1, cofactor,
and inhibitor were used. The 17β-HSD2 assay was performed
similarly using the microsomal fraction and tritiated E2.
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a
Scheme 3. Synthesis of Compound 12b
a
Reagents and conditions: (a) Br2, acetic acid, reflux, 3 h. (b) Benzylbromide, K2CO3, EtOH, reflux, 15 h. (c) Cs2CO3, Pd(Ph3P)4, THF/H2O (1:1),
reflux, 4 h. (d) Cs2CO3, Pd(Ph3P)4, DME/EtOH/H2O (1:1:1), 15 min microwave irradiation (150 °C, 1.5 bar).
a
Scheme 4. Synthesis of Compound 8−29
a
Reagents and conditions: (a) DMAP, pyridine, rt, 6 days, method B. (b) BBr3, CH2Cl2, −78 °C to rt, 18 h, method C. (c) BBr3, CH2Cl2, −25 °C
for 1 h, rt for 2 h, method D.
In a first step, a synthetic library was designed to introduce
aromaticity into the sulfonamide core (Figure 3). Biological
evaluation of these compounds revealed that several are highly
potent h17β-HSD1 inhibitiors. Compounds that exhibited
more than 30% inhibition of h17β-HSD1 at 10 nM were
resynthesized, and IC50 values and selectivity factors (SFs)
toward human 17β-hydroxysteroid dehydrogenase type 2
(h17β-HSD2) as well as percentage inhibition values against
cj17β-HSD1 and 17β-HSD2 were determined (8−11, 14, 15,
and 17). Furthermore, to elaborate a meaningful SAR,
substituents differing in their electronic and lipophilic proper-
ties were introduced in the aromatic moiety, and the
corresponding compounds were also tested against the
human and cj enzymes (12, 13, 16, and 18−31; Table 1)
All compounds showed IC50 values toward h17β-HSD1 in
the low nanomolar range (between 12 and 92 nM) as well as
selectivity toward h17β-HSD2 (SF between 2 and 71). The
only exception is the dinitro-substituted sulfonamide 16 with
30-fold reduced activity toward h17β-HSD1. Interestingly, 16
turned out to be a moderate h17β-HSD2 inhibitor. By
introduction of meta- and para-substituents into the phenyl
substituent of 12, selectivity toward h17β-HSD2 could be
further improved up to a SF of 71 for compound 23.
To explore whether rigidification leads to an improvement of
affinity toward the target, as it has been shown in previous
studies,56 and to get more insights into the binding site, two
rigidified conformers were synthesized. The rigidification of 12
resulted in two compounds with different conformations in the
sulfonamide moiety, the indoles 30 and 31. Interestingly, one
conformation remained highly active toward h17β-HSD1 (30),
while the other conformer lost activity (31).
The activity toward cj17β-HSD1 and also the selectivity
toward cj17β-HSD2 could be slightly optimized for all
compounds. Remarkably, the rigidified 12 (compound 30)
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a
Scheme 5. Synthesis of Compounds 30 and 31
a
Reagents and conditions: (a) Cs2CO3, Pd(Ph3P)4, DME/EtOH/H2O (1:1:1), 15 min microwave irradiation (150 °C, 1.5 bar). (b) DCM, TBAC,
NaOH, 3 h, 10 °C. (c) BBr3, CH2Cl2, −25 °C for 1 h, rt for 2 h, method D.
turned out to be a highly active and selective inhibitor of cj17β-
HSD1 (79% inhibiton of cj17β-HSD1 and 43% inhibition of
cj17β-HSD2 at 50 nM).
values for selected compounds (Table 2). All tested
compounds showed IC50 values around or below 1000 nM.
Compound 17 exhibiting an IC50 value of 157 nM turned out
to be the most active one of this series.
To further characterize the inhibitors regarding their
biophysical properties, tPSA values were calculated using the
commercial ChemDrawUltra 12.0 program and are shown in
Table 3. Except for 16 (190.25 Å, Table 3), all compounds
showed tPSA values that are in an adequate range for oral
bioavailabity58 (Supporting Information, Table 4).
A selection of the compounds (reference A, 12, 17, 23, and
29) was also tested using the cytosolic and microsomal fraction
of mouse liver homogenate, which are described to be
responsible for the E1 and E2 conversion, respectively,57 to
examine whether the aromatic sulfonamides show activity in
this species. In the cytosolic fraction, E1 conversion was
observed, but the compounds did not show inhibitory activity.
In the microsomal fraction, E2 oxidation was monitored, and
the compounds exhibited moderate inhibiton of E1 formation
(Supporting Information, Table 2).
Selectivity toward ERs α and β. Because of the similarity
of the compounds to the estrogen scaffold, it cannot be
excluded that the phenyl naphthalene sulfonamides show
affinity toward the ERs α and β. Agonistic as well as
antagonistic effects are not compatible with the therapeutic
concept for 17β-HSD1 inhibitors. Agonistic properties would
stimulate cell proliferation and thus counteract the effect of
these inhibitors. Antagonistic qualities are likely to lead to
unwanted pharmacological effects and could reduce the
advantages of the new therapeutic intracrine concept as
compared to antiestrogens or SERMs. The compounds were
tested in an assay using the recombinant human ERs in
competition with [3H]-E2. The affinity of E2 was set to 100%.
A relative binding affinity (RBA) of 0.1% means that the
compound shows a 1000-fold weaker affinity to the receptor
than E2 and is considered as satisfactory. All compounds,
including reference A, showed RBA values lower than 0.1%
toward ERα and ERβ and were classified as very low affinity
ligands (Supporting Information, Table 3).
DISCUSSION AND CONCLUSION
■
The identification of a highly active h17β-HSD1 inhibitor (A)
with properties suitable for further preclinical studies provided
the starting point for the optimization concerning activitiy
toward cj17β-HSD1. Starting with a parallel synthesis approach,
several highly potent h17β-HSD1 inhibitors were discovered.
With the 4-indolyl-substituted hydroxyphenyl naphthalene
sulfonamide 30, a highly potent h- and cj17β-HSD1 inhibitor
was identified, which might be an appropriate candidate for in
vivo proof of concept.
The introduction of aromatic moieties differing in their
electronic and lipophilic effects into the sulfonamide core is
well tolerated by h17βHSD1 but did not resulted in higher
activities. This finding cannot exclude additional beneficial
interactions in the binding site initiated by the introduced
substituents. However, they are obviously compensated by
adverse effects, like losing a hydrogen bond from one of the
two essential hydroxy groups46 or by stripping the hydrate
sheath for diffusion into the binding pocket
The strongly reduced activity of compound 16 could be
explained by the high electron-withdrawing effects of the two
bulky nitro groups, disrupting potential π−π interactions with
the protein. A crystal structure will be necessary to shed light
on the real binding mode of the synthesized compounds.
Further Biological Evalution. To get insights into cellular
activity, the mammary tumor T47D cell line, which expresses
h17β-HSD1 and h17β-HSD2, was used to determine IC50
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Table 1. IC50 Values toward h17β-HSD1, Selectivity Factor toward h17β-HSD2, and Inhibition of cj17β-HSD1 and cj17β-HSD2
for Compounds 8−31
a
b
Mean values of three determinations, standard deviation less than 18%. Human placenta, cytosolic fraction, substrate [3H]-E1 + E1 (500 nM),
c
d
cofactor NADH (500 μM). Human placenta, microsomal fraction, substrate [3H]-E2 + E2 (500 nM), cofactor NAD+ (1500 μM). IC50 (17β-
e
f
HSD2)/IC50 (17β-HSD1). C. jacchus placenta, cytosolic fraction, substrate [3H]-E1 + E1 (500 nM), cofactor NADH (500 μM). C. jacchus
h
g
placenta, microsomal fraction, substrate [3H]-E2 + E2 (500 nM), cofactor NAD+ (1500 μM). n.i., no inhibition, i.e., inhibition ≤10%. Aliphatic
sulfonamide inhibitor A49 is used as a reference.
Rigidification is a common strategy in drug design to
minimize entropic costs of binding and thus optimize the
interaction. In this study, rigidification of the sulfonamide core
led to the discovery of the highly active h- and cj17β-HSD1
inhibitor 30. The structure of this compound obviously is very
close to the biologically active conformation of 12. Contrarily,
for the other conformation, rigidified in compound 31, a
significantly reduced inhibition of h17β-HSD1 was found. This
conformation is obviously not able to form sufficiently strong
interactions in the active site of h17β-HSD1.
Interestingly, the rigidification of 12 leading to 30 has a
strong influence on inhibitory activity for the common
marmoset enzyme as compared to the human. The discovery
of compound 30 as the biologically active conformation of this
class of inhibitors is of great importance and can be used for
further structural optimization.
Through the introduction of aromatic substituents, the
activity toward cj17β-HSD1 could be increased significantly,
and also, selectivity toward cj17β-HSD2 could be achieved for
this class of compounds. H-bond acceptors in the para-position
of the phenyl moiety seem to be beneficial for selectivity in C.
jacchus. Considering the fact that the expression of 17β-HSD2
is downregulated in EDD tissues, it is nevertheless important to
obtain selectivity toward this isozyme to avoid systemic
enhancement of E2 levels and the side effects associated with it.
The differences in inhibitory activities between h- and cj17β-
HSD1 in this compound class could be indications for different
binding modes for the two species. However, an in-depth
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Table 2. IC50 Values in the T47D Cell Assay for Selected
Compounds
EXPERIMENTAL SECTION
■
Chemistry. Chemical names follow IUPAC nomenclature. Starting
materials were purchased from commercial suppliers and were used
without further purification. Column flash chromatography was
performed on silica gel (70−200 μm), preparative thin-layer
chromatography (PTLC) was performed on 1 mm SIL-G-100 UV254
glass plates (Macherey-Nagel), and reaction progress was monitored
by TLC on TLC Silica Gel 60 F254 (Merck). NMR spectra were
measured on a Bruker AM500 spectrometer (500 MHz) at 300 K.
Chemical shifts are reported in δ (parts per million: ppm), TMS was
used as an internal reference, and hydrogenated residues of deuteriated
solvent were used as an internal standard [CDCl3: δ = 7.24 ppm (1H
NMR) and δ = 77 ppm (13C NMR); CD3COCD3: δ = 2.05 ppm (1H
NMR) and δ = 29.8 ppm (13C NMR)]. Signals are described as s, d, t,
dd, m, dt, and ddd for singlet, doublet, triplet, doublet of doublets,
multiplet, doublet of triplets, and doublet of doublet of doublets,
respectively. All coupling constants (J) are given in Hertz (Hz). The
purity of final products (≥95%) was confirmed by analytical high-
performance liquid chromatography (HPLC). HPLC/MS was
performed on a MSQ electrospray mass spectrometer (Thermo
Fisher). The system was operated by the standard software Xcalibur. A
RP C18 NUCLEODUR 100-5 (125 mm × 3 mm) column was used as
the stationary phase with water/acetonitrile mixtures as eluents. Mass
spectra (ESI) were recorded on a TSQ Quantum (Thermofischer)
instrument. Melting points were measured using melting point
apparatus SMP3 (Stuart Scientific). The apparatus is uncorrected.
General Procedure for Sulfonamide Library (1−11, 14, 15, and
17): Method A. A solution of sulfonylchloride (1.2 equiv) in 2 mL of
THF was added to a solution of 1-(3-aminophenyl)-6-(3-
hydroxyphenyl)naphthalene-2-ol 1a (1.0 equiv) and PS-morpholine
in 2 mL of THF (55 mg). After it was stirred for 15 h at rt, a catalytic
amount of PS-DMAP was added, and the mixture was stirred another
15 h at rt. Catalytic amounts of PS-Tris(2-aminoethyl)-amine and PS-
isocyanate were added to the mixture of compounds 15 and 17. After
filtration and evaporation, the compounds were purified by preparative
HPLC (Varian Inertsil C18 50 mm × 21 mm) (H2O/CH3CN + 0.1%
TFA) (100:0) → (0:100). The remaining compounds were stirred for
15 h at 60 °C and afterward finished with the same procedure as 15
and 17. After filtration and evaporation, the compounds were purified
by combi-flash chromatography (hexane/ethylacetate (2:1 → 1:1).
General Procedure for the Synthesis of the Protected Phenyl-
naphthalenesulfonamides: Method B.60 3-(2-methoxy-6-(3-
methoxyphenyl)naphthalene-1-yl)aniline (1b) or 3-(2-benzyloxy)-6-
(3-benzyloxy)phenyl)naphthalene-1-yl)aniline (12b) (1 equiv) was
dissolved in pyridine abs. and was spiked with the accordant sulfonyl
chloride (1.5 equiv) and DMAP as a catalyst. The reaction mixture was
stirred for 6 days at rt. The reaction was quenched by adding 10 mL of
2 N HCl and extracted with ethyl acetate. The organic layers were
washed with saturated NaHCO3 and brine, dried over MgSO4, and
evaporated under vacuum. The product was purified by flash
chromatography to give the title compounds.
cell assay
a
a
a
IC50 (nM)
IC50 (nM)
IC50 (nM)
b
b
b
compd
17βHSD1
compd
17βHSD1
compd
17βHSD1
A
71
646
498
316
196
462
396
753
17
18
19
20
21
22
23
24
157
402
555
429
462
637
701
1009
25
26
27
28
29
30
525
440
8
10
11
12
13
14
15
633
240
246
1100
a
Mean values of three determinations, standard deviation less than
b
25%. T47D cells, substrate [3H]-E1 + E1 (50 nM).
Table 3. Biophysical Characterization of A, 16, and 30
a
a
compd
tPSA (Å)
MW (g/mol)
clog P
A
86.63
190.25
77.84
405
557
491
3.91
6.08
6.59
16
30
a
Calculated values with ChemDrawUltra 12.0.
investigation of the SAR for further elucidation is hampered by
a somewhat blurred relationship.
All aromatic sulfonamides showed a decrease in cellular
activity in T47D cells, especially the indolyl derivative 30 (IC50
= 1100 nM), which could be explained by the higher molecular
weight and the increased clog P value as compared to the
reference A (491 vs 405 g/mol and clog P 6.59 vs 3.91,
respectively; Supporting Information, Table 4). Intracelluar
metabolism might also play a role for the decreased cellular
activity.
Another important issue in drug development is oral
bioavailability. Molecular weight and lipophilicity are not
sufficient for prediction of oral bioavailability. The polar surface
area and the number of free rotatable bonds are more
appropriate parameters59 and should be considered for the
development of potential drugs. The polar surface area of 77.84
Å2 for compound 30 is in an excellent range (86.63 Å2 for
reference A), and also, the more rigid structure is promising.
For mimetics of the steroidal scaffold, selectivity toward the
ERs is very important. To avoid estrogenic side effects, neither
agonistic nor antagonistic activities can be tolerated. With RBA
values below 0.1% for ERα and β, all herein reported
compounds are not expected to exert an ER-mediated effect.
In this paper, we described a species-specific optimization of
hydroxyphenyl sulfonamide naphthols starting with a combi-
natorial library approach followed by optimization of
discovered hits. The goal of the present study was the
identification of cj17β-HSD1 inhibitors. Thus, it was successful,
as we discovered the rigidified sulfonamide 30, which is highly
potent toward h17β-HSD1 as well as toward cj17β-HSD1 and
shows selectivity toward 17β-HSD2 of both species and the
ERs. This h- and cj17β-HSD1 inhibitor seems to be an
appropriate candidate for the proof of concept in the
endometriosis common marmoset model. Nevertheless, before
performing an in vivo experiment, further parameters like
metabolic stability and pharmacokinetic properties will be
investigated.
General Procedure for Ether Cleavage: Method C.61,62 A 3-(2-
methoxy)-6-(3-methoxy)phenyl)naphthalene-1-yl)sulfonamide deriva-
tive (1 equiv) was dissolved in dichloromethane abs. and was cooled to
−78 °C (acetone/dry ice). Boron tribromide (1 M, 3.5 equiv per
methoxy group) was added dropwise under stirring. The reaction
mixture was stirred for 18 h from −78 °C to rt. The reaction was
quenched by adding 10 mL of water and extracted with ethyl acetate.
The organic layers were washed with saturated NaHCO3 and brine,
dried over MgSO4, and evaporated under reduced pressure. The
product was purified by flash chromatography to give the title
compounds.
General Procedure for Ether Cleavage: Method D.61,62 A 3-(2-
benzyloxy)-6-(3-benzyloxy)phenyl)naphthalene-1-yl)sulfonamide de-
rivative (1 equiv) was dissolved in dichloromethane abs. and was
cooled to −25 °C. Boron tribromide (1 M, 10 equiv per benzyloxy
group) was added dropwise under stirring. The reaction mixture was
stirred for 1 h at −25 °C and then for 3 h at rt. The reaction was
quenched by adding 10 mL of water and extracted with ethyl acetate.
The organic layers were washed with saturated NaHCO3 and brine,
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dried over MgSO4, and evaporated under reduced pressure. The
product was purified by flash chromatography to give the title
compounds.
5-Chloro-N-(3-(2-hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)-
phenyl)thiophene-2-sulfonamide (1). The title compound was
prepared by reaction of 1-(3-aminophenyl)-6-(3-hydroxyphenyl)-
naphthalen-2-ol (1a) and 5-chlorothiophene-2-sulfonyl chloride
according to method A; yield, 35% (8.8 mg). MS (ESI): 508 (M +
H)+.
4,5-Dibromo-N-(3-(2-hydroxy-6-(3-hydroxyphenyl)naphthalen-
1-yl)phenyl)thiophene-2-sulfonamide (2). The title compound was
prepared by reaction of 1-(3-aminophenyl)-6-(3-hydroxyphenyl)-
naphthalen-2-ol (1a) and 4,5-dibromothiophene-2-sulfonyl chloride
according to method A; yield, 21% (6.6 mg). MS (ESI): 629 (M +
H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-
thiophene-2-sulfonamide (11). The title compound was synthesized
by reaction N-(3-(2-methoxy-6(3-methoxyphenyl)naphthalen-1-yl)
phenyl) thiophene-2-sulfonamide (11a) (50 mg, 0.1 mmol) and
boron tribromide (0.75 mmol) according to method C. The product
was purified by flash chromatography hexane/ethylacetete (1:1); yield,
34% (16 mg). MS (ESI): 473 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-
benzenesulfonamide (12). The title compound was synthesized by
reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)naphthalen-
1-yl)phenyl)-3-methylbenzene-sulfonamide (12a) (215 mg, 0.33
mmol) and boron tribromide (3.32 mmol) according to method D.
The product was purified by preparative HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (35:65) → (0:100) in 42 min]; yield,
23%. MS (ESI): 468 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-2-
nitrobenzene-sulfonamide (13). The title compound was synthesized
by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-2-nitrobenzenesulfonamide (13a) (190 mg,
0.27 mmol) and boron tribromide (2.70 mmol) according to method
D. The product was purified by preparative HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (35:65) → (0:100) in 42 min]; yield, 9%
(12 mg). MS (ESI): 530 (M + H2O)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-3-
nitrobenzene-sulfonamide (14). The title compound was synthesized
by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-3-nitrobenzene-sulfonamide (14a) (150 mg,
0.22 mmol) and boron tribromide (2.20 mmol) according to method
D. The product was purified by preparative HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (35:65) → (0:100) in 42 min]; yield,
21% (24 mg). MS (ESI): 513 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
nitrobenzene-sulfonamide (15). The title compound was synthesized
by reaction N-(3-(2-methoxy-6(3-methoxyphenyl)naphthalen-1-yl)
phenyl)4-nitrobenzene-sulfonamide (15a) (150 mg, 0.28 mmol) and
boron tribromide (2.8 mmol) according to method C. The product
was purified by flash chromatography hexane/ethylacetete (1:1); yield,
43% (62 mg). MS (ESI): 512 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-
2,4-dinitrobenzene-sulfonamide (16). The title compound was
synthesized by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)phenyl)-2,4-dinitro-benzenesulfonamide
(16a) (150 mg, 0.26 mmol) and boron tribromide (2.60 mmol)
according to method D. The product was purified by flash
chromatography hexane/ethyl acetate (5:1); yield, 21% (24 mg).
MS (ESI): 558 (M + H)+.
2-Cyano-N-(3-(2-hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)-
phenyl)benzene-sulfonamide (17). The title compound was synthe-
sized by reaction of 2-cyano-N-(3-(2-methoxy-6(3-methoxyphenyl)-
naphthalen-1-yl) phenyl)benzene sulfonamide (17a) (226 mg, 0.16
mmol) and boron tribromide (1.12 mmol) according to method C.
The product was purified by flash chromatography hexane/ethyl
acetate (15:1 → 5:1); yield, 23% (38 mg). MS (ESI): 493 (M + H)+ .
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-3-
cyanobenzene-sulfonamide (18). The title compound was synthe-
sized by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-3-cyanobenzene-sulfonamide (18a) (200 mg,
0.33 mmol) and boron tribromide (3.30 mmol) according to method
D. The product was purified by flash chromatography hexane/ethyl
acetate (10:1 → 6:1); yield, 49% (72 mg). MS (ESI): 495 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
cyanobenzene-sulfonamide (19). The title compound was synthe-
sized by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-4-cyanobenzene-sulfonamide (19a) (170 mg,
0.25 mmol) and boron tribromide (2.50 mmol) according to method
D. The product was purified by preparative TLC hexane/ethyl acetate
(2:1); yield, 50% (66 mg). MS (ESI): 493 (M + H)+.
Methyl 5-(N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-
yl)phenyl)sulfamoyl)-4-methoxythiophene-3-carboxylate (3). The
title compound was prepared by reaction of 1-(3-aminophenyl)-6-
(3-hydroxyphenyl)naphthalen-2-ol (1a) and methyl 5-(chlorosulfon-
yl)-4-methoxythiophene-3-carboxylate according to method A; yield,
19% (5.4 mg). MS (ESI): 562 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-5-
(2-(methylthio)pyrimidin-4-yl)thiophene-2-sulfonamide (4). The
title compound was prepared by reaction of 1-(3-aminophenyl)-6-
(3-hydroxyphenyl)naphthalen-2-ol (1a) and 5-(2-(methylthio)-
pyrimidin-4-yl)thiophene-2-sulfonyl chloride according to method A;
yield, 22% (6.7 mg). MS (ESI): 598 (M + H)+ .
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-
benzo[d]thiazole-6-sulfonamide (5). The title compound was
prepared by reaction of 1-(3-aminophenyl)-6-(3-hydroxyphenyl)-
naphthalen-2-ol (1a) and benzo[d]thiazole-6-sulfonyl chloride accord-
ing to method A; yield, 42% (11 mg). MS (ESI): 525 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-2-
nitro-4-(trifluoromethyl)benzenesulfonamide (6). The title com-
pound was prepared by reaction of 1-(3-aminophenyl)-6-(3-
hydroxyphenyl)naphthalen-2-ol (1a) and 2-nitro-4-(trifluoromethyl)-
benzene-1-sulfonyl chloride according to method A; yield, 18% (5.1
mg). MS (ESI): 581 (M + H)+.
5-Chloro-N-(3-(2-hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)-
phenyl)-6aH-pyrrolo[3,2-d]thiazole-6-sulfonamide (7). The title
compound was prepared by reaction of 1-(3-aminophenyl)-6-(3-
hydroxyphenyl)naphthalen-2-ol (1a) and 5-chloro-6aH-pyrrolo[3,2-
d]thiazole-6-sulfonyl chloride according to method A; yield, 24% (6.6
mg). MS (ESI): 548 (M + H)+. Compounds 8−11, 14, 15, and 17
were synthesized in the parallel synthetic approach according to
method A and afterward resynthesized in a bigger scale according to
descriptions below.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
bromo-2,5-fluorobenzene-sulfonamide (8). The title compound was
synthesized by reaction of 4-bromo-2,5-difluoro-N-(3-(2-methoxy-6(3-
methoxyphenyl)naphthalen-1-yl) phenyl) benzenesulfonamide (8a)
(70 mg, 0.15 mmol) and boron tribromide (1.05 mmol) according to
method C. The product was purified by preparative HPLC (Agilent
PrepC18 (H2O/CH3CN + 0.1% TFA) (20:80) → (0:100) in 35 min;
yield, 63% (52 mg). MS (ESI): 583 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
bromo-2-trifluoro-methoxybenzenesulfonamide (9). The title com-
pound was synthesized by reaction of N-(3-(2-(benzyloxy)-6-(3-
(benzyloxy)phenyl)naphthalen-1-yl)phenyl)-4-bromo-2-(trifluoro-
methoxy)benzenesulfonamide (9a) (280 mg, 0.34 mmol) and boron
tribromide (3.40 mmol) according to method D. The product was
purified by flash chromatography hexane/ethylacetete (5:3); yield,
84% (180 mg). MS (ESI): 631 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-
quinolin-6-sulfonamide (10). The title compound was synthesized by
reaction N-(3-(2-methoxy-6(3-methoxyphenyl)naphthalen-1-yl)
phenyl)quinoline-6-sulfonamide (10a) (61 mg, 0.12 mmol) and
boron tribromide (0.9 mmol) according to method D. The product
was purified by flash chromatography hexane/ethylacetete (1:1); yield,
63% (30 mg). MS (ESI): 519 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-2-
fluorobenzene-sulfonamide (20). The title compound was synthe-
sized by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-2-fluorobenzene-sulfonamide (20a) (190 mg,
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0.29 mmol) and boron tribromide (2.90 mmol) according to method
D. The product was purified by preparative TLC hexane/ethyl acetate
(3:2); yield, 71% (100 mg). MS (ESI): 508 (M + Na)+.
PrepC18 (H2O/CH3CN + 0.1% TFA) (35:65) → (0:100) in 38 min];
yield, 25% (60 mg). MS (ESI): 474 (M + H)+.
6-(3-Hydroxyphenyl)-1-(1-(phenylsulfonyl)-1H-indol-4-yl)-
naphthalen-2-ol (30). The title compound was synthesized by
reaction of 4-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)naphthalen-1-
yl)-1-(phenylsulfonyl)-1H-indole (30a) (70 mg, 0.14 mmol) and
boron tribromide (1.40 mmol) according to method D. The product
was purified by HPLC [Agilent PrepC18 (H2O/CH3CN + 0.1% TFA)
(75:25) → (0:100) in 55 min]; yield, 10% (5.00 mg). MS (ESI): 493
(M + H)+.
5-(2-(Benzyloxy)-6-(3-(benzyloxy)phenyl)naphthalen-1-yl)-1-
(phenylsulfonyl)-1H-indole (31a) and 1-[1-(Benzensulfonyl)indol-6-
yl]-6-(3-hydroxyphenyl)naphthalen-2-ol (31). Powdered sodium
hydroxide (5 mg, 0.11 mmol) and a catalytic amount of tetra-butyl
ammonium chloride were stirred for 10 min in dichloromethane at 0
°C under nitrogen atmosphere. (6-(2-(Benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)-1H-indole (31b) (20 mg, 0.038 mmol) was
added without further purfication and stirred for a while. A solution of
benzenesulfonyl chloride (7 mg, 0.04 mmol) in dichloromethane was
added dropwise during a period of 15 min. The solution was stirred for
3 h under 10 °C. The product was extracted with ethyl acetate, and the
combined organic layers were dried over MgSO4 and evaporated
under vacuum. The product was used without further purification for
the ether cleavage according to method D (3.60 mmol boron
tribromide were used). The product was purified by HPLC [Agilent
PrepC18 (H2O/CH3CN + 0.1% TFA) (75:25) → (0:100) in 40 min];
yield, 11% (2.3 mg). MS (ESI): 493 ([M + H])+.
Biophysical Characterization. The molecular weight, clog P, and
tPSA values of all compounds were calculated from CambridgeSoft
Chem & Bio Draw using the ChemDrawUltra 12.0 program.
Biological Methods. [2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were
purchased from Perkin-Elmer (Boston). Quickszint Flow 302
scintillator fluid was bought from Zinsser Analytic (Frankfurt).
T47D cells were obtained from ECACC (Salisbury). FCS was
purchased from Sigma (Taufkirchen). Cell culture media and dextran-
coated charcoal-stripped FCS (DCC-FCS) were ordered from
CCPRO (Oberdorla). Other chemicals were purchased from Sigma,
Roth, or Merck.
Enzyme Preparation (17βHSD1 and 17βHSD2) of Placenta
from Human and C. jacchus. 17β-HSD1 and 17β-HSD2 were
obtained from human placenta and C. jacchus placenta according to
previously described procedures.63,64 Fresh human placenta and frozen
C. jacchus placenta were homogenized and separated by partial
centrifugation. The pellet fraction contained the microsomal 17β-
HSD2, while 17β-HSD1 was obtained after precipitation with
ammonium sulfate from the cytosolic fraction. Aliquots of 17β-
HSD1 and 17β-HSD2 were stored frozen at −80 °C.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-3-
fluorobenzene-sulfonamide (21). The title compound was synthe-
sized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-3-fluorobenzene-sulfonamide (21a) (150
mg, 0.23 mmol) and boron tribromide (2.30 mmol) according to
method D. The product was purified by preparative TLC hexane/ethyl
acetate (2:1); yield, 13% (15 mg). MS (ESI): 508 (M + Na)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
fluorobenzene-sulfonamide (22). The title compound was synthe-
sized by reaction of N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-4-fluorobenzene-sulfonamide (22a) (250 mg,
0.38 mmol) and boron tribromide (3.80 mmol) according to method
D. The product was purified by flash chromatography hexane/ethyl
acetate (5:3); yield, 44% (80 mg). MS (ESI): 486 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-3-
hydroxybenzene-sulfonamide (23). The title compound was
synthesized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)phenyl)-3-methoxybenzene-sulfonamide
(23a) (100 mg, 0.15 mmol) and boron tribromide (2.10 mmol)
according to method D. The product was purified by flash
chromatography hexane/ethyl acetate (5:3); yield, 72% (52 mg).
MS (ESI): 484 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
hydroxybenzene-sulfonamide (24). The title compound was
synthesized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)phenyl)-4-methoxybenzene-sulfonamide
(24a) (150 mg, 0.22 mmol) and boron tribromide (2.20 mmol)
according to method D. The product was purified by flash
chromatography hexane/ethyl acetate (2:1 → 1:1); yield, 10% (11
mg). MS (ESI): 484 (M + H)+.
N-(3-(2-(Hydroxy)-6-(3-(hydroxy)phenyl)naphthalen-1-yl)-
phenyl)-2-methylbenzene-sulfonamide (25). The title compound
was synthesized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)phenyl)-2-methylbenzene-sulfonamide (25a)
(40 mg, 0.08 mmol) and boron tribromide (0.8 mmol) according to
method D. The product was purified by HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (60:40) → (0:100) in 42 min]; yield,
10% (11 mg). MS (ESI): 482 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-3-
methylbenzene-sulfonamide (26). The title compound was synthe-
sized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-3-methylbenzene-sulfonamide (26a) (150
mg, 0.23 mmol) and boron tribromide (2.30 mmol) according to
method D. The product was purified by HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (35:65) → (0:100) in 42 min]; yield,
14% (15 mg). MS (ESI): 499 (M + H2O)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-4-
methylbenzene-sulfonamide (27). The title compound was synthe-
sized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)phenyl)-
naphthalen-1-yl)phenyl)-4-methylbenzene-sulfonamide (27a) (213
mg, 0.32 mmol) and boron tribromide (3.20 mmol) according to
method D. The product was purified by HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (35:65) → (0:100) in 35 min]; yield,
45% (70 mg). MS (ESI): 499 (M + H2O)+.
N-(4-(N-(3-(2-(Hydroxy)-6-(3-(hydroxy)phenyl)naphthalen-1-yl)-
phenyl)sulfamoyl)-phenyl)-acetamide (28). The title compound was
synthesized by reaction N-(4-(N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)phenyl)sulfamoyl)-phenyl)-acetamide (28a)
(299 mg, 0.40 mmol) and boron tribromide (4.00 mmol) according
to method C. The product was purified by HPLC [Agilent PrepC18
(H2O/CH3CN + 0.1% TFA) (55:45) → (0:100) in 42 min]; yield,
37% (70 mg). MS (ESI): 525 (M + H)+.
N-(3-(2-Hydroxy-6-(3-hydroxyphenyl)naphthalen-1-yl)phenyl)-
2,2,2-trifluoro-ethanesulfonamide (29). The title compound was
synthesized by reaction N-(3-(2-(benzyloxy)-6-(3-(benzyloxy)-
phenyl)naphthalen-1-yl)phenyl)-2,2,2-trifluoro-ethanesulfonamide
(29a) (300 mg, 0.5 mmol) and boron tribromide (5.00 mmol)
according to method D. The product was purified by HPLC [Agilent
Inhibition of 17β-HSD1 in Cell-Free Assay. Inhibitory activities
toward human and C. jacchus enzymes were evaluated by a well-
established method with minor modifications.65−67 Briefly, the enzyme
preparation was incubated with NADH (500 μM) in the presence of
potential inhibitors at 37 °C in a phosphate buffer (50 mM)
supplemented with 20% glycerol and EDTA. Ten millimolar inhibitor
stock solutions were prepared in DMSO. The final concentration of
DMSO was adjusted to 1% in all samples. The enzymatic reaction was
started by addition of a mixture of unlabeled- and [3H]-E1 (final
concentration, 500 nM; 0.15 μCi). After 10 min, the incubation was
stopped with HgCl2 (10 mM), and the mixture was extracted with
diethylether. After evaporation, the steroids were dissolved in
acetonitrile. E1 and E2 were separated using acetonitrile/water
(45:55) as the mobile phase in a C18 rp chromatography column
(Nucleodur C18 Gravity, 3 μm, Macherey-Nagel, Duren) connected
̈
to a HPLC system (Agilent 1200 Series, Agilent Technologies,
Waldbronn). Detection and quantification of the steroids were
performed using a radioflow detector (Ramona Star, Raytest,
Straubenhardt). The conversion rate was calculated according to
following equation:
% E2
% E2 + % E1
% conversion =
× 100
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Each value was calculated from at least three independent experiments.
Inhibition of 17β-HSD2. The 17β-HSD2 inhibition assays were
performed similarly to the 17β-HSD1 procedure. The microsomal
fraction was incubated with NAD+ (1500 μM), test compound, and a
mixture of unlabeled- and [3H]-E2 (final concentration, 500 nM; 0.14
μCi) for 20 min at 37 °C (the reaction was stopped with 1 mM
HgCl2). Further treatment of the samples and HPLC separation were
carried out as mentioned above.
ER Affinity. The binding affinity of selected compounds to the ERα
and ERβ was determined according to Zimmermann et al.68 Briefly,
0.25 pmol of ERα or ERβ, respectively, was incubated with
[2,4,6,7-3H]-E2 (10 nM) and test compound for 1 h at rt. The
potential inhibitors were dissolved in DMSO (5% final concentration).
Nonspecific binding was performed with diethylstilbestrol (10 μM).
After incubation, ligand−receptor complexes were selectively bound to
hydroxyapatite (5 g/60 mL TE buffer). The formed complex was
separated, washed, and resuspended in ethanol. For radiodetection,
scintillator cocktail (Quickszint 212, Zinsser Analytic, Frankfurt) was
added, and samples were measured in a liquid scintillation counter
(Wallac Micro Beta TriLux, Perkin-Elmer). For determination of the
RBA, inhibitor and E2 concentrations required to displace 50% of the
receptor bound labeled E2 were determined. RBA values were
calculated according to the following equation:
ABBREVIATIONS USED
■
h17β-HSD1, human 17β-hydroxysteroid dehydrogenase type 1;
cj17β-HSD1, Callithrix jacchus 17β-hydroxysteroid dehydrogen-
ase type 1; h17β-HSD2, human 17β-hydroxysteroid dehydro-
genase type 2; cj17β-HSD2, Callithrix jacchus 17β-hydroxyste-
roid dehydrogenase type 2; BPH, benign prostatic hyperplasia;
E1, estrone; E2, 17β-estradiol; NADP(H), nicotinamide
adenine dinucleotide phosphate; EDD, estrogen-dependent
disease; ER, estrogen receptor; SERM, selective estrogen
receptor modulator; SAR, structure−activity relationship;
equiv, equivalent; HPLC, high-performance liquid chromatog-
raphy; RBA, relative binding affinity; rt, room temperature; SF,
selectivity factor
REFERENCES
■
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IC (E2)
50
RBA (%) =
× 100
IC (compound)
50
The RBA value for E2 was arbitrarily set at 100%.
Inhibition of 17β-HSD1 in Cellular Assay Using T47-D. A stock
culture of T47D cells was grown in RPMI 1640 medium
supplemented with 10% FCS, L-glutamine (2 mM), penicillin (100
IU/mL), streptomycin (100 μg/mL), insulin-zinc-salt (10 μg/mL),
and sodium pyruvate (1 mM) at 37 °C under 5% CO2 humidified
atmosphere.
̌
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The cells were seeded into a 24-well plate at 5 × 105 cells/well in
DMEM medium with FCS and L-glutamine, and the antibiotics were
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 37 °C with 5% CO2, the incubation was started by addition of a
mixture of unlabeled- and [2,4,6,7-3H]-E1 (final concentration, 50 nM;
0.15 μCi). After 30 min of incubation, the enzymatic reaction was
stopped by removal of the supernatant medium. The steroids were
extracted into diethylether. Further treatment of the samples was
carried out as mentioned for the 17β-HSD1 assay.
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ASSOCIATED CONTENT
* Supporting Information
Synthesis and characterization of intermediates as well as
HPLC purity determination for final compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
(8) Adamo, V.; Iorfida, M.; Montalto, E.; Festa, V.; Garipoli, C.;
Scimone, A.; Zanghi, M.; Caristi, N. Overview and new strategies in
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AUTHOR INFORMATION
Corresponding Author
*Tel: +49 681 302 70300. Fax: +49 681 302 70314. E-mail:
rwh@mx.uni-saarland.de or rolf.hartmann@helmholtz-hzi.de.
■
(10) Bush, N. J. Advances in hormonal therapy for breast cancer.
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dehydrogenases involved in local oestrogen synthesis have prognostic
Notes
significance in breast cancer. Br. J. Cancer 2005, 92 (3), 547−552.
̌
̌
The authors declare no competing financial interest.
For the sake of clarity, IUPAC nomenclature is not strictly
followed except for the experimental part, where the correct
IUPAC names are given.
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ACKNOWLEDGMENTS
■
We thank Benjamin Kirsch for his help in performing the
synthesis. We thank Jeannine Jung, Martina Jankowski, and
Janine Ludwig for their help in performing the in vitro tests.
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