Highly Potent 17β-HSD2 Inhibitors with a Promising Pharmacokinetic Profile for Targeted Osteoporosis Therapy

Article abstract of DOI:10.1021/acs.jmedchem.8b01373

Intracellular elevation of E2 levels in bone by inhibition of 17β hydroxysteroid dehydrogenase type 2 (17β-HSD2) without affecting systemic E2 levels is an attractive approach for a targeted therapy against osteoporosis, a disease which is characterized by loss of bone mineral density. Previously identified inhibitor A shows high potency on human and mouse 17β-HSD2, but poor pharmacokinetic properties when applied perorally in mice. A combinatorial chemistry approach was utilized to synthesize truncated derivatives of A, leading to highly potent compounds with activities in the low nanomolar to picomolar range. Compound 33, comparable to A in terms of inhibitor potency against both human and mouse enzymes, displays high in vitro metabolic stability in human and mouse liver S9 fraction as well as low toxicity and moderate hepatic CYP inhibition. Thus, compound 33 showed a highly improved peroral pharmacokinetic profile in comparison to A, making 33 a promising candidate for further development.

Full text of DOI:10.1021/acs.jmedchem.8b01373

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Highly Potent 17β-HSD2 Inhibitors with a Promising
Pharmacokinetic Profile for Targeted Osteoporosis Therapy
Lorenz Siebenbuerger,† Victor Hernandez-Olmos,‡ Ahmed S. Abdelsamie,§,∥ Martin Frotscher,∥
Chris J. van Koppen,⊥ Sandrine Marchais-Oberwinkler,# Claudia Scheuer,¶ Matthias W. Laschke,¶
Michael D. Menger,¶ Carsten Boerger,† and Rolf W. Hartmann*,∥,∇
†PharmBioTec GmbH, Science Park 1, 66123 Saarbru
̈
cken, Germany
‡Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology TMP,
Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany
§Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, 12622 Cairo, Egypt
∥Department of Pharmacy, Saarland University, Campus C2.3, 66123 Saarbru
̈
cken, Germany
⊥Elexopharm GmbH, Campus A1, 66123 Saarbru
̈
cken, Germany
#Institute for Pharmaceutical Chemistry, Philipps University Marburg, 35032 Marburg, Germany
¶Institute for Clinical and Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany
∇Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1,
66123 Saarbrucken, Germany
̈
S
* Supporting Information
ABSTRACT: Intracellular elevation of E2 levels in bone by inhibition of 17β hydroxysteroid dehydrogenase type 2 (17β-
HSD2) without affecting systemic E2 levels is an attractive approach for a targeted therapy against osteoporosis, a disease which
is characterized by loss of bone mineral density. Previously identified inhibitor A shows high potency on human and mouse 17β-
HSD2, but poor pharmacokinetic properties when applied perorally in mice. A combinatorial chemistry approach was utilized to
synthesize truncated derivatives of A, leading to highly potent compounds with activities in the low nanomolar to picomolar
range. Compound 33, comparable to A in terms of inhibitor potency against both human and mouse enzymes, displays high in
vitro metabolic stability in human and mouse liver S9 fraction as well as low toxicity and moderate hepatic CYP inhibition.
Thus, compound 33 showed a highly improved peroral pharmacokinetic profile in comparison to A, making 33 a promising
candidate for further development.
The resulting oxidized steroid hormones are much less active
on the estrogen receptors α and β (ERα and ERβ) and the
androgen receptor than their reduced forms. Systemic
administration of E2 leads to prevention of osteoporosis, but
is accompanied by serious side effects such as an increased risk
of venous emboli and breast cancer. As 17β-HSD2 is expressed
in bone OBs,4 inhibition of this enzyme leads to increased E2
and T levels inside OBs, making 17β-HSD2 inhibition a
promising approach for a targeted therapy. Bagi et al. validated
the beneficial effect of 17β-HSD2 inhibitors on bone quality in
an ovariectomized monkey model.5 However, only a minor
effect was seen on bone mass, which might be due to the poor
pharmacokinetic (PK) profile of the administered compound
INTRODUCTION
■
Healthy bone is primarily maintained by two different cell
types, osteoblasts (OBs) and osteoclasts (OCs). OBs are
responsible for the formation of new bone matrix and OCs for
its degradation. An imbalance in the activities of these cell
types in favor of matrix degradation, as it is often the case in
elderly people, leads to osteoporosis. Osteoporosis is
characterized by decreased bone mineral density and impaired
bone microarchitecture, resulting in an increased risk of bone
fractures.1 Osteoporosis is directly associated with a decrease
of the plasma levels of the steroid hormone 17β-estradiol (E2),
as it occurs in women after menopause.2 Furthermore, a
decrease of testosterone (T) has been linked to osteoporosis in
elderly men.3 17β-Hydroxysteroid dehydrogenase type 2 (17β-
HSD2) catalyzes the NAD+-dependent oxidation of E2 into
estrone (E1) and of T into 4-androstene-3,17-dione (A-dione).
Received: September 3, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Chart 1. Reported Nonsteroidal 17β-HSD2 Inhibitors25,28
or its moderate potency. Therefore, the development of novel
potent inhibitors with improved in vivo efficacy is highly
desirable. Both steroidal6−8 and nonsteroidal9−18 inhibitors of
17β-HSD2 have been described in the literature. Different
scaffolds of 17β-HSD2 inhibitors have been identified and
optimized,11−20 resulting in compounds with strong 17β-
HSD2 inhibition and high selectivity toward 17β-hydroxyste-
roid dehydrogenase type 1 (17β-HSD1). 17β-HSD1 on the
other hand is an attractive target for the treatment of
endometriosis and breast cancer.19−24 Very recently, lead
compound A, a highly active 17β-HSD2 inhibitor (Chart 1,
human IC50 = 2 nM, mouse IC50 = 25 nM, human liver S9 t1/2
= 60 min, mouse liver S9 t1/2 = 46 min), was administered
subcutaneously to mice in a proof-of-principle model for bone
fracture healing.25 Strong efficacy of the compound was
observed by histological and biomechanical testing because of
the large formed callus and bone formation at the site of the
fracture compared to vehicle controls. These findings underline
the beneficial effect of 17β-HSD2 inhibitors on the formation
of new bone substance. However, compound A is not suitable
for osteoporosis therapy because of its poor PK properties after
oral administration (Figure 1), which is regarded a prerequisite
for long-term therapy of osteoporosis. It is important that
newly designed inhibitors should also inhibit the mouse 17β-
HSD2 ortholog because this species is appropriate for the
evaluation of compounds in an osteoporosis model.26 As there
is no 17β-HSD1 detectable in OBs,4 no selectivity toward 17β-
HSD1 is required to increase E2 levels inside the bone. In fact,
nonselective compounds might be favorable because in
contrast to nonselective inhibitors, selective inhibitors will
increase E2 levels in tissues where both 17β-HSD2 and 17β-
HSD1 are present. In the breast tissue,27 for example, both
reductive and oxidative activities of 17β-HSDs are equally
present and inhibition of the oxidative activity by 17β-HSD2
inhibitors could therefore lead to increased E2 concentrations
inside the breast, which leads to an increased risk for breast
cancer. In addition to sufficient inhibitory potency on the
target, compounds should show good in vitro and in vivo
ADME-Tox properties such as metabolic stability, aqueous
solubility, as well as low cytotoxicity and CYP inhibition.
Plasma concentrations should be in the range of the IC90 in
order to show efficacy in a rodent proof-of-principle model.
Furthermore, the compound should be suitable for oral
applications because this route of administration is the most
adequate for long-term osteoporosis therapy. In this study, we
describe the design, synthesis, and biological assessment of
novel 17β-HSD2 inhibitors. The obtained compounds show
the highest in vitro inhibition values of both human and mouse
17β-HSD2 described so far for osteoporosis therapy.
Furthermore, the compounds show good metabolic stability
in human and mouse liver S9 fraction, thus toward phase 1 and
phase 2 metabolizing enzymes. Finally, high plasma concen-
trations of the compounds are reached for a considerable
length of time in a mouse PK study after oral administration.
DESIGN
■
Starting point of this study was the sulfonamide compound A
(Chart 1), which has recently been shown to be a suitable
candidate for a bone fracture healing proof-of-principle study,
showing high potency on the human and murine 17β-HSD2,
as well as moderate aqueous solubility and metabolic
stability.25 Subcutaneous application of compound A in high
dose (50 mg/kg) to mice is necessary to achieve sufficiently
high plasma concentrations to inhibit mouse 17β-HSD2 for at
least 24 h after a single dose. However, compound A is not
suitable for peroral applications, as A in a single dose of 50
mg/kg is cleared entirely from the plasma after 6 h (Figure 1).
Compounds B and C, designed as 17β-HSD1 inhibitors,28
show that the sulfonamide moiety is not required to achieve
high potency on both human 17β-HSD2 and 17β-HSD1 when
compared to A. Compound B shows high potency on human
17β-HSD2 and moderate potency on the murine 17β-HSD
enzyme, as well as moderate selectivity toward 17β-HSD1.
Compound C shows moderate activity on the human 17β-
HSD2 and an increased selectivity over 17β-HSD1 compared
to B, making B and C promising optimized hit compounds for
Figure 1. Mean profile ( ) S.E.M. of plasma concentration [nM] in
C57Bl/6 mice vs time after oral application of compounds A, 2, 5, 33,
and 34 in single dosing experiments (n = 3). Dashed line represents
the in vitro 17β-HSD2 IC90 value for 33 in mouse.
B
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Chart 2. Designed and Synthesized 17β-HSD2 Inhibitors
further optimization. Furthermore, they showed that a p-
methoxy or a hydroxy group, flanked by one or two m-chloro
or methyl substituents on the phenyl ring, leads to an increased
activity on both human 17β-HSDs (results not shown).28
Newly designed compounds should show similar or improved
potency on both human and mouse 17β-HSD2, as well as
improved solubility and metabolic stability compared to
reference compound A. A ligand-based approach using
combinatorial chemistry methods was employed in order to
synthesize new compounds. In a first step, we investigated
whether the structure−activity relationship (SAR) obtained
from the 17β-HSD1 inhibitor class28 could be transferred to
the tri-F- and methyl-scaffold of B and C, respectively. These
hit compounds already show high to moderate potency on
17β-HSD2 as well as selectivity over 17β-HSD1 and serve as
ideal templates for further SAR studies. Therefore, a set of
mono- and disubstituted p-methoxy and p-hydroxy derivatives
combined with the benzoyl moieties of B and C have been
synthesized (Chart 2, compounds 1−11) and tested for their
activity on human and murine 17β-HSD1 and 17β-HSD2. The
newly designed compounds show similar or increased
inhibitory potency on human 17β-HSD2 compared to the
reference compounds A, B, and C. Regarding activity on
mouse 17β-HSD2, an increased potency compared to B and C
was achieved, but not compared to compound A. Because of
the close structural similarity of compounds 1−11 of the first
library, a second, structurally more diverse library (12−39,
Chart 2) was designed and synthesized in order to gain deeper
insight into the SAR of the compounds concerning both
human and mouse 17β-HSD2 inhibition. In this library, phenyl
rings bearing different hydrophilic electron-donating substitu-
ents and different heterocyclic moieties were combined with
the tri-F benzoyl thiophene motif of B. Compounds of both
libraries that showed best inhibitory activities on human and
mouse 17β-HSD2 were tested for metabolic stability in human
and murine liver S9 fraction. On the basis of the testing results,
a SAR regarding potency and selectivity on both human and
murine 17β-HSD2 as well as metabolic stability was obtained.
Selected compounds were further evaluated for other ADME-
Tox parameters. Finally, the compounds with the highest
metabolic stability were tested in a mouse PK study.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of compounds 1−39 was
achieved following a two- to four-step reaction sequence
(Scheme 1) with derivatization occurring mostly at the last two
C
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Compounds 1−39
a
Reagents and conditions: (a) method A, AlCl3, anhydrous CH2Cl2, 0 °C, 0.5 h and then rt, 3 h. (b) Method B, BBr3, CH2Cl2, −93 °C to rt,
overnight. (c) Method C1, corresponding boronic acid for 1−37, Cs2CO3, Pd(PPh3)4, toluene/DME/water (0.7:0.9:2), 85 °C, 16 h. Method C2
for 38, Na2CO3 (2 M), Pd(PPh3)4, toluene/ethanol (1:1), reflux, overnight.
steps. The tri-F and methyl thiophene scaffolds of B and C can
be easily prepared as their 2-bromothiophene intermediates.
Mild Suzuki coupling conditions (method C1) can be applied
to introduce a large number of aromatic rings to the thiophene
core, which makes the reaction scheme ideal for combinatorial
chemistry on a robotic synthesis platform. Starting from the
commercially available substituted benzoyl chlorides 1a and
7a, a Friedel−Crafts acylation using a standard protocol
(method A) was performed on 2-bromothiophene to obtain 1b
and 7b or on 2-methylthiophene to obtain 39b, respectively.
Ether cleavage of 1b and 39b with BBr3 under anhydrous
conditions yielded intermediate 1c and final compound 39
(method B). The bromo compound 1c was then converted to
final compounds 1−4, 12−14, and 18−38 in a Suzuki−
Miyaura cross-coupling reaction with the corresponding
boronic acids using tetrakis(triphenylphosphine) palladium(0)
as a catalyst with cesium carbonate in oxygen-free toluene/1,2-
dimethoxyethane (DME)/water at 85 °C for 16 h (method
C1).28 Compound 38 was obtained using a similar Suzuki
coupling procedure with toluene/ethanol as a solvent and
sodium carbonate as a base (method C2).28 In the case of
compounds 22, 23, 25, 33, and 38, the corresponding boronic
acid pinacol esters 22a, 23a, 25a, 33a, and 38a were
synthesized by Miyaura borylation reaction29 before the Suzuki
coupling was performed (see the Supporting Information).
Suzuki coupling of 7b with the corresponding boronic acids
yielded the final hydroxy compounds 7−9. The dihydroxy
derivatives 5, 6, 10, 11, and 15−17 were obtained by the
cleavage of the respective methylethers with BBr3 in dichloro-
methane (DCM) at low temperature (method B).
increased inhibition values compared to their 2-methyl
analogues (7−11, 20−46% inhibition at 100 nM) by a factor
of approximately 100 or higher, whereas in general, the di-m-
Cl/Me, p-OH/OMe motif (1−6, IC50 = 1.3−11 nM) does not
lead to an increase in potency compared to A (IC50 = 2 nM)
and B (IC50 = 1.4 nM). Because of the low potency of 7−11,
only compounds 1−6 were tested for their inhibition of the
murine enzymes m17β-HSD2 and m17β-HSD1 at concen-
trations of 50 and 1000 nM, respectively. All compounds show
decreased activity on the murine 17β-HSD1 compared to
m17β-HSD2. IC50 values were determined for compounds
showing the highest inhibition on m17β-HSD2. In general, all
tested compounds show strongly decreased activity on the
murine enzymes compared to the human ones. The best two
compounds are two and four times less active on the m17β-
HSD2 with IC50 values of 58 nM for 5 and 89 nM for 2,
respectively, compared to compound A (IC50 = 25 nM).
This discrepancy between the inhibition values on human
and mouse 17β-HSDs led to a second design step to establish a
SAR on both human and murine 17β-HSDs. To this end, a set
of isosteres of the hydroxy group, different cyclic moieties, and
a methyl group were introduced to the tri-F scaffold, resulting
in compounds 12−39. In general, the compounds of this series
showed increased or similar activity on the human 17β-HSD2
when compared to sulfonamide compound A, with IC50 values
in the picomolar to lower nanomolar range. Selectivity factors
of up to 24 were reached within these compounds.
Comparing the monosubstituted methoxy derivatives 12−14
(IC50 = 1.1−2.3 nM) with the hydroxy derivatives 15−17
(IC50 = 0.32−1.1 nM), the former show slightly decreased
activity on human 17β-HSD2. A possible explanation is the
lack of an H-bond donor in the case of the methoxy
compounds. 15−17 represents the most active 17β-HSD2
inhibitors described with the p-hydroxy 15 (IC50 = 0.32 nM)
as the most potent one with a sixfold increase in activity
compared to A (IC50 = 2 nM). The increased or similar
potency of the plain para OMe or OH substituted compounds
Inhibition of 17β-HSD2 and 17β-HSD1. All final
compounds have been tested for their inhibitory activities on
both human 17β-HSD2 and 17β-HSD1. The results are
presented in Table 1.
The inhibition values of compounds 1−11 are similar to
those already obtained from compounds B and C. Compounds
bearing the 2,4,5-tri-F motif (1−6, IC50 = 1.3−11 nM) show
D
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
E
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
12 (IC50 = 2.3 nM) and 15 (IC50 = 0.32 nM) compared to the
p-OH/OMe-m-Me/Cl compounds 1−6 (IC50 = 1.3−11 nM)
shows that for the tri-F compound class, the meta substituent
does not lead to an enhanced potency on human 17β-HSD2,
as observed in the SAR of the closely related di-F class.28
Substitution of the hydroxy or methoxy oxygen by an amino
function as seen in compounds 19−21 (IC50 = 4−9.2 nM)
leads to decreased potency on the human 17β-HSD2
compared to their oxygen analogues 12−17 (IC50 = 0.32−
2.3 nM). A combination of both, an amino function with an
OMe or OH group (22, IC50 = 7.1 nM, 23, IC50 = 3.4 nM),
does not restore the activity as seen for 12 (IC50 = 2.3 nM)
and 15 (IC50 = 0.32 nM). Introduction of F or Me on the ring
to the most active aniline 20 (IC50 = 5.4 nM) is tolerated well
by the enzyme and leads to equipotent or slightly less potent
compounds 24−28 (IC50 = 2.2−10 nM). Cyclization to the
corresponding indole compounds 33−35 (IC50 = 1.1−3.8 nM)
results in comparable to slightly increased potency compared
to the aniline derivatives 18 (IC50 = 4.4 nM) and 19 (IC50
9.2 nM). The plain phenyl−substituted compound 32 (IC50
=
=
0.85 nM, SF 8) shows one of the highest inhibition values for
17β-HSD2 with moderate 17β-HSD1 activity, indicating that
no additional substituent is required for this class to achieve
high potency on the human 17β-HSD2 as well as moderate
selectivity. Different heterocyclic moieties 33−38 (IC50 = 1.1−
6.3 nM) are also tolerated by the enzyme, but lead to a
decreased potency compared to 32 (IC50 = 0.85 nM), except
the indole derivatives 33 (IC50 = 1.1 nM) and 34 (IC50 = 1.3
nM), which are equipotent on the target enzyme. The highly
decreased potency of the truncated molecule 39 (56% at 100
nM for human 17β-HSD2 vs 13% at 100 nM for 17β-HSD1)
compared to 32 (IC50 = 0.85 nM) not only illustrates the
necessity of a bulkier group on the right side of the molecule
connected to the thiophene to achieve high potency on human
17β-HSD2 but also points out that the tri-F-benzoyl thiophene
moiety alone is already 17β-HSD2 selective. These findings
demonstrate clearly that a wide range of different substituted
phenyl moieties is tolerated by human 17β-HSD2 within this
compound class and no bigger sulfonamide tail, as seen in A, is
needed for high potency.
The compounds of the second library 12−37 were tested for
their inhibition of the murine 17β-HSD2 at 50 nM and HSD1
at 1 μM. IC50 values were obtained for compounds showing
more than 50% inhibition at 50 nM. The compounds show
strongly decreased potency compared to their human ortholog
as well as selectivity over murine 17β-HSD1, as observed for
the first library (compounds 1−11) before. However, some
properties observed in the SAR of 12−38 on the human 17β-
HSD2 can also be seen on the murine enzyme. The amino
derivatives 19−21 (19, 22%, 20, 22%, and 21, 19% inhibition
of murine 17β-HSD2) show decreased activity compared to
their hydroxy analogues (15, 47%, 16, 34%, and 17, 38%
inhibition of murine 17β-HSD2). On contrary to the findings
on the human enzyme, addition of methyl or fluorine on the
phenyl ring (24, 48%, 25, 35%, 26, 37%, and 28, 35%
inhibition of murine 17β-HSD2) leads to an increase in activity
compared to unsubstituted m-aniline 20 (22% inhibition of
murine 17β-HSD2). Furthermore, the unsubstituted phenyl
(32, 27% inhibition of murine 17β-HSD2) is only weakly
active, as are most of the heterocyclic compounds 36−38 as
well. An exception are the bulkier indole derivatives 33−35
(33, 63%, 34, 69%, 35, 43% inhibition of murine 17β-HSD2)
which show the same trend in activity regarding their
F
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
substitution pattern as observed for the human enzyme.
Especially, compounds 33 (m17β-HSD2 IC50 = 34 nM) and
34 (m17β-HSD2 IC50 = 34 nM) show the highest inhibition
values on the mouse enzyme obtained in this study, being
equipotent to reference compound A (m17β-HSD2 IC50 = 25
nM).
IN VIVO EVALUATION OF PLASMA
CONCENTRATION IN MICE
■
Osteoporosis is a disease affecting mostly elderly persons, and
therapy often occurs over decades. For a drug that is to be
administered frequently, the oral route is preferred. Thus, the
two compounds showing the highest in vitro potency and
metabolic stability, 2 and 5 of the first and 33 and 34 of the
second library, were tested in a PK study in C57Bl/6 mice. 2
and 5 were applied at a single dose of 50 mg/kg, 33 and 34 at a
three times lower dose of 16.7 mg/kg. The obtained
concentration curves are depicted in Figure 1. The obtained
PK parameters are listed in Table 3. All compounds show
FURTHER IN VITRO BIOLOGICAL EVALUATION
■
Cytotoxicity. In order to identify suitable candidates for
applications in vivo, it was necessary for the administered
compounds to be nontoxic. Therefore, the most active
compounds toward potency on mouse 17β-HSD2 (2, 5, 33,
and 34) were evaluated in a cellular toxicity MTT assay over
66 hours using HEK293 cells. All compounds showed safety
factors (LC20/IC50 human 17β-HSD2) >1000 and, thus, are
regarded as nontoxic.
a b
,
Table 3. PK Parameters of Selected Compounds
dose
[mg/kg]
Cmax
[nM]
Tmax
[h]
C24h
[nM]
AUC0−∞
[ng·h/mL]
Cmpd
Aqueous Solubility and Metabolic Stability. In order
to achieve a sufficient plasma concentration for a sufficient
period of time following peroral administration of the
compound, aqueous solubility and metabolic stability are two
important factors for good oral absorption and low clearance of
the drug. Solubility of the compounds in aqueous solution and
in vitro metabolic half-life using human and mouse liver S9
fraction have been determined. The results are depicted in
Table 2. Both compounds 2 and 5 show increased solubility
A
2
5
33
34
50
50
50
16.7
16.7
252
167
269
1211
390
2
6
8
2
2
ND
ND
ND
46
434
609
1428
5503
1293
53
a
b
Data are mean values. Abbreviations: Cmax, highest plasma
concentration of a drug after administration; Tmax, time to reach
max; C24h, plasma concentration at 24 h timepoint, AUC, area under
the concentration−time curve. ND, nondetectable (below lowest level
of detection); methods were validated, CV was below 20%, in rare
cases higher; GraphPadPrism 6 was used for AUC calculation.
C
Table 2. Aqueous Solubility and Metabolic Stability of
Selected Compounds
ad
,
bd
,
cd
,
aq solubility
t1/2 human S9
t1/2 mouse S9
bigger area-under-the-curve values (AUC) than A. Compound
33 had the highest value with a 13-fold increase in AUC
compared to A. 33 also shows the highest plasma
concentrations with a Cmax of 1211 nM. Furthermore, both
indole derivatives 33 and 34 can still be detected after 24 h (46
and 53 nM, respectively), enabling accumulation of the drug
after multiple dosing, each once-a-day. The most likely
desirable inhibitor concentration should be around the IC90,
which is assumed as 10 times the IC50, based on the in vitro
inhibition curves of 33. During the course of the day, only 33 is
likely to reach this concentration for 19 h (dotted line, 300 nM
for 33), which makes 33 a suitable candidate for an in vivo
proof-of-principle study.
Cmpd
(μM)
(min)
(min)
A
2
5
33
34
50−100
>200
>200
50−100
50−100
60
19
32
51
34
46
70
18
≫60
49
e
e
e
a
Solubility of compound in phosphate-buffered saline, pH 7.2,
b
containing 2% DMSO. Pooled human liver S9 fraction (1 mg/
mL), 2 mM NADPH regenerating system, 1 mM UDPGA, 0.1 mM
PAPS, 1 μM test compound at 37 °C for 0, 15, and 60 min. Pooled
mouse liver S9 fraction (1 mg/mL), 2 mM NADPH regenerating
c
system, 1 mM UDPGA, 0.1 mM PAPS, 1 μM test compound at 37 °C
d
for 0, 15, and 60 min. Mean value of two determinations, SD < 15%.
e
Inhibition of Human Hepatic CYP Enzymes. To analyze
the potential risk of drug−drug interaction, inhibition of 33
toward human hepatic CYP enzymes CYP2C19, CYP2D6, and
CYP3A4 was evaluated. Compound 33 was tested at 1.2 μM
and showed no or moderate inhibition (22, 0, and 41%).
Single value.
(>200 μM) compared to A (50−100 μM), whereas the
solubility of the indole derivatives 33 and 34 (50−100 μM) is
in the same range as seen for reference A (50−100 μM).
Comparing metabolic stability in human liver S9 fraction, the
CONCLUSIONS
■
17β-HSD2 has been shown to be a suitable target for the
treatment of osteoporosis.5 Originating from the closely related
field of 17β-HSD1 inhibitors,28 the potent 17β-HSD2 inhibitor
A was developed and successfully tested in a bone fracture
model in mice when applied subcutaneously.25 However, this
compound shows an unfavorable oral PK profile administered
in mice and therefore is not a suitable candidate to be
evaluated in an osteoporosis proof-of-principle model. The aim
of this work was to develop a low molecular weight compound
with similar potency on both human and mouse 17β-HSD2
and with increased aqueous solubility and metabolic stability
with regards to A to increase plasma concentrations. In a first
design step, different phenol-derived moieties were synthe-
sized. Among these, 2 and 5 showed decreased potency on the
most newly synthesized compounds (2, t1/2 = 19 min, 5, t1/2
=
32 min, and 34, t1/2 = 34 min) show two to four times
decreased half-life times compared to A (t1/2 = 60 min). Only
the half-life time of compound 33 (51 min) is similar to that of
A (t1/2 = 60 min). Having a look at the stability in murine liver
S9 fraction, most of the tested compounds (2, t1/2 = 70 min,
33, t1/2 ≫ 60 min, 34, 49 min) are more or equally stable than
A (t1/2 = 46 min), except 5 (t1/2 = 18 min), which is three
times less stable than A. It is noted that 33 shows the highest
metabolic stability both in human and mouse liver S9 fraction.
G
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and sep for singlet, broad singlet, doublet, triplet, doublet of doublets,
doublet of doublets of doublets, multiplet, doublet of triplets,
quadruplet, and septet, respectively. All coupling constants (J) are
given in hertz (Hz). Melting points have been recorded on a SMP40
apparatus (Stuart-Equipment, Staffordshire, United Kingdom.) Mass
spectra (ESI) have been recorded on the system mentioned above.
Tested compounds were recrystallized from acetonitrile/water +0.1%
formic acid and are of >95% chemical purity as measured by HPLC
unless otherwise stated. MS/MS measurements were performed on a
TSQ Quantum Acess Max (ThermoFisher, Dreieich, Germany)
coupled to an Acella UHPLC system. An electrospray interface (ESI)
was used as an ion source. The Acella-LC-system consisted of a pump
and an auto sampler. The system was operated by the standard
software Xcalibur. All solvents were HPLC grade. All newly
synthetized compounds passed an in silico PAINS filter.
General Procedure for Friedel−Crafts Acylation (Method A). An
ice-cooled mixture of monosubstituted thiophene derivate (1 or 1.5
equiv), arylcarbonyl chloride (1 equiv), and aluminumtrichloride (1
equiv) in anhydrous dichloromethane was warmed to room
temperature and stirred for 2−4 h. HCl (1 M) was used to quench
the reaction. The aqueous layer was extracted with ethyl acetate. The
combined organic layers were washed with brine, dried over
magnesium sulfate, filtered, and concentrated to dryness. The product
was purified by CC.
General Procedure for Ether Cleavage (Method B). To a solution
of methoxybenzene derivative (1 equiv) in anhydrous dichloro-
methane at −78 °C (dry ice/acetone bath), boron tribromide in
dichloromethane (1 M, 3 equiv per methoxy function) was added
dropwise. The reaction mixture was stirred overnight at room
temperature under nitrogen atmosphere. Water was added to quench
the reaction, and the aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
magnesium sulfate, filtered, and concentrated to dryness. The product
was purified by CC.
General Procedure for Suzuki Coupling (Method C1). A mixture
of arylbromide (1 equiv), boronic acid derivative (1.1 equiv), cesium
carbonate (3.5 equiv), and tetrakis(triphenylphosphine) palladium
(0.035 equiv) was suspended in an oxygen-free toluene/DME/water
(0.7:0.9:2) solution and heated under argon atmosphere to 85 °C for
16 h. The reaction mixture was cooled to room temperature. Water
was added, and the aqueous layer was extracted with ethyl acetate
three times. The combined organic layers were dried over magnesium
sulfate, filtered, and concentrated to dryness. The product was
purified by preparative HPLC.
mouse 17β-HSD2, but increased solubility and metabolic
stability compared to A. The decreased potency on the mouse
enzyme led to the synthesis of a second library, introducing
different hydrophilic substituents as well as different aromatic
moieties to gain deeper insights into the SAR of the mouse
17β-HSD2, resulting in compounds 12−39. In this series,
compound 15 is the most potent 17β-HSD2 inhibitor (IC50
=
320 pmolar) currently described in the literature for
osteoporosis therapy and the most selective of its compound
class (22-fold). The highest murine 17β-HSD2 inhibitory
activities are shown by compounds 29, 33, and 34 which are
equipotent to compound A. These findings clearly demon-
strate that the sulfonamide moiety of A does not significantly
contribute to the binding to the enzyme of this compound
class and can therefore be omitted. Taking into account
principles for the design of orally bioavailable compounds,30,31
the decreased molecular weight below 500 should facilitate
permeability via passive diffusion of the compounds through
the gastro-intestinal barrier and hence increase absorption of
the drug. The compounds show comparable or increased
aqueous solubility and metabolic stability with 33 showing
better profile than A. The best two compounds of each library,
2 and 5 of the first and 33 and 34 of the second library, were
tested in a mouse oral PK study. All tested compounds showed
an increase in AUC compared to A, with 33 showing a 13-fold
increase. Furthermore, 33 is able to maintain a pharmacolog-
ically relevant plasma concentration for much longer than A,
lasting for at least 8 hours after a single dose. In summary, 33
was identified as a highly potent inhibitor of human and mouse
17β-HSD2 with a superior pharmacological profile and is
therefore a promising candidate for an in vivo proof-of-
principle study in a mouse osteoporosis model.
EXPERIMENTAL SECTION
■
Chemical Methods. Chemical names follow IUPAC nomencla-
ture. Starting materials were purchased from Aldrich, Acros,
Lancaster, Maybridge, Combi Blocks, Merk, or Fluka and were used
without purification.
Column chromatography (CC) was performed on silica gel (70−
200 μm), and reaction progress was monitored by thin-layer
chromatography on Alugram SIL G UV254 (Macherey-Nagel).
All Suzuki couplings have been conducted according to method C1
on a combinatorial chemistry synthesis platform (Chemspeed Isynth,
Method C2. A mixture of arylbromide (1 equiv), boronic acid
derivative (1.2 equiv), sodium carbonate (2 equiv), and tetrakis-
(triphenylphosphine) palladium (0.01 equiv) was suspended in an
oxygen-free toluene/ethanol (1:1) solution and was refluxed for 20 h
under nitrogen atmosphere. The aqueous layer was extracted with
ethyl acetate. The combined organic layers were washed with brine,
dried over magnesium sulfate, filtered, and concentrated to dryness.
The product was purified by CC.
Chemspeed AG, Fullinsdorf, Switzerland). In the case of preparative
̈
high-performance liquid chromatography (HPLC) purification, the
compounds were purified using a setup produced by Waters
Corporation containing a 2767 Sample Manager, a 2545 binary
gradient pump, a 2998 PDA detector, and a 3100 electron spray mass
spectrometer. The system has been used for a part of the analytical
analysis as well as the preparative separation. In the latter case after
separation, the solvent flow has been split using a flow splitter and a
515 HPLC pump for makeup flow. Water containing 0.1% formic acid
and acetonitrile containing 0.1% formic acid were used as solvents for
the analysis and separation. A Waters X-Bridge column (C18, 150 ×
4.6 mm, 5 μM) has been used with a flow of 1 mL/min for the
analysis, and a Waters X-Bridge column (C18, 150 × 19 mm, 5 μM)
has been used with a flow of 20 mL/min for the separation. 1H NMR
spectra were measured on a Bruker AM500 spectrometer (500 MHz)
at 300 K or on a Bruker Fourier 300 (300 MHz) at 295.5 K. Chemical
shifts are reported in δ (parts per million: ppm), by reference to the
hydrogenated residues of the deuterated solvent as an internal
standard [CDCl3: δ = 7.24 ppm (1H NMR) and δ = 77 ppm (13C
NMR), CD3OD: δ = 3.35 ppm (1H NMR) and δ = 49.3 ppm (13C
NMR), CD3COCD3: δ = 2.05 ppm (1H NMR) and δ = 29.9 ppm
(13C NMR), CD3SOCD3 δ = 2.50 ppm (1H NMR) and δ = 39.5 ppm
(13C NMR)]. Signals are described as s, br s, d, t, dd, ddd, m, dt, q,
Biological Methods. [2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were
obtained from PerkinElmer, Boston. A Quickszint Flow 302
scintillator fluid was purchased from Zinsser Analytic, Frankfurt.
Other chemicals were bought from Sigma, Roth, Merck, or Santa
Cruz Biotechnology.
Ethics Statement. Placental samples were obtained anonymously
from Saarbrucken-Dudweiler Hospital’s Department of Gynecology.
̈
None of the authors involved in this study has received any
information about the patients. The microsomal fraction of the mouse
enzyme (m17β-HSD2) was obtained from mouse livers, which were
purchased from Pharmacelsus GmbH (Saarbru
animal experiments were approved by the local governmental animal
protection committee (Landesamt fur Verbraucherschutz, Abteilung
̈
cken, Germany). All
̈
̈
̈
C Lebensmittel-und Veterinarwesen, Saarbrucken, Germany) and
were conducted in accordance with the European legislation on
protection of animals (Guide line 2016/63/EU) and the National
Institutes of Health Guidelines for the Care and Use of Laboratory
H
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Animals (http://oacu.od.nih.gov/regs/index.htm. Eighth Edition;
2011).
acid was added to dissolve the cells and MTT crystals. Then,
fluorescence was measured in a BioTek Synergy 2 plate reader. The
decrease in fluorescence (at 570 nm) in the presence of the test
compound compared to the fluorescence in the presence of the
vehicle control (1% DMSO) was determined followed by the
calculation of IC20 values using GraphpadPrism 6 curve fitting. In
each experiment, a reference compound was included.
Aqueous Solubility Determination. Aqueous solubility was
evaluated as previously described.36 Final concentrations of 5, 15,
50, 100, and 200 μM of A, 2, 5, 33, and 34 in an aqueous solution
containing 2% DMSO were prepared, and the solution clarity and
potential compound precipitation were determined by eye after 1 and
24 h at room temperature. In each experiment, a reference compound
was included.
Metabolic Stability in a Cell-Free Assay. Compounds were tested
according to an established method.37 For evaluation of phases I and
II metabolic stability, 1 μM compound was incubated with 1 mg/mL
pooled mammalian (human or mouse) liver S9 fraction (BD
Gentest), 2 mM NADPH regenerating system, 1 mM UDPGA, and
0.1 mM PAPS at 37 °C for 0, 5, 15, and 60 min at a final volume of
100 μL. The incubation was stopped by precipitation of S9 enzymes
with 2 volumes of ice-cold acetonitrile containing diphenhydramine as
an internal standard. Concentration of the remaining test compound
at different time points was analyzed by LC-MS/MS and used to
determine half-life (t1/2). In each experiment, a reference compound
was included.
Inhibition of Human Hepatic CYP Enzymes. Inhibition of hepatic
CYP enzymes CYP2B6, CYP2C19, and CYP3A4 by the test
compound (1.2 μM) was measured in baculosomes expressing the
recombinant human enzymes according to the manufacturer’s
instruction (Life Technologies). In each experiment, a reference
compound was included.
PK Experiments. Experiments were conducted on female C57BL/6
mice (body weight 20−25 g). Compounds were orally administered
at a dose of 50 mg/kg (n = 3) or 16.7 mg/kg (n = 3) per animal. The
administered suspensions of solid inhibitors were prepared by mixing
them with 0.5% gelatin and 5% mannitol (w/w) in water. Blood
samples were collected in citrate tubes from the tail vein at 2, 4, 8, and
24 h. Obtained blood samples (50 μL blood + 5 μL citrate buffer)
were centrifuged at 3000 rpm. Subsequently, 10 μL of mouse plasma
was added to 50 μL of acetonitrile containing diphenhydramine as an
internal standard, and obtained samples were stored at −20 °C until
HPLC-MS/MS analysis. Samples and calibration standards were
centrifuged at 15 000 rpm for 5 min at 4 °C. The solution was
transferred into fresh vials for HPLC-MS/MS analysis (Accucore RP-
MS, TSQ Quantum triple quadrupole mass spectrometer, electrospray
interface). After injection of 10 μL, data were analyzed based on the
ratio of the peak areas of analyte and internal standard.
Preparation of h17β-HSD1 and h17β-HSD2 Enzymes. Cytosolic
(h17β-HSD1) and microsomal (h17β-HSD2) fractions were obtained
from human placenta similar to previously described procedures.23,32
The placenta was homogenized, and the enzymes were separated by
fractional centrifugation at 1000g, 10 000g, and 150 000g. The pellet
containing the microsomal h17β-HSD2 fraction was used for the
determination of h17β-HSD2 inhibition, whereas h17β-HSD1 was
obtained by ammonium sulfate precipitation from the cytosolic
fraction. Aliquots of the enzyme were stored at −78 °C.
m17β-HSD2 Enzyme Preparation and Inhibition. The micro-
somal fraction (m17β-HSD2) was obtained from mouse liver similar
to the procedure described for h17β-HSD2. Inhibitory activities were
evaluated by a method identical to the one described for the human
enzyme.
m17β-HSD1 Enzyme Preparation. Recombinant m17β-HSD1
enzyme was produced by transfection of HEK293 cells with an
17β-HSD1 expression plasmid (coding sequence of NM_010475 in
pCMV6Entry vector, OriGene Technologies, Inc.) according to a
described procedure.33 Transfected cells were homogenized after 48 h
by sonication (3 × 10 s) in a 40 mM Tris buffer containing 250 mM
saccharose, 5 mM ethylenediaminetetraacetic acid (EDTA), 7 mM
DTT, 1 mM phenylmethylsulfonyl fluoride, pH 7.5. Cell lysate was
centrifuged (1000g, 15 min, 4 °C), and 20% glycerol was added to the
supernatant before aliquots were frozen and stored at −70 °C.
Inhibition of h17β-HSD2 and m17β-HSD2 in Cell-free Assay.
Inhibitory activities were evaluated following an established method
with minor modifications.34 Briefly, the enzyme preparation was
incubated with NAD+ (1500 μM) in the presence of potential
inhibitors at 37 °C in a phosphate buffer (50 mM), pH 7.4
supplemented with 20% of glycerol and 1 mM EDTA. Inhibitor stock
solutions were prepared in DMSO. Final concentration of DMSO was
adjusted to 1% in all samples. The enzymatic reaction was started by
the addition of a mixture of unlabeled- and [3H]-E2 (final
concentration: 500 nM, 0.11 μCi). After 20 min, the incubation
was stopped with 100 mM HgCl2 and the mixture was extracted with
ether. After evaporation, the steroids were dissolved in acetonitrile/
water (45:55). E1 and E2 were separated using acetonitrile/water
(45:55) as a mobile phase in a C18 RP chromatography column
connected to a HPLC system (Agilent 1100 Series, Agilent
Technologies, Waldbronn). Detection and quantification of the
steroids were performed using a radio-flow detector (Berthold
Technologies, Bad Wildbad). The conversion rate was calculated
according to the following equation: % conversion = (% E1/(% E1 +
% E2)) × 100. Each value was calculated from at least two
independent experiments. In each experiment, a reference compound
was included.
Inhibition of h17β-HSD1 and m17β-HSD1 in Cell-Free Assay.
The 17β-HSD1 inhibition assay was performed similarly to the h17β-
HSD2 test. The human enzyme was incubated with NADH (500
μM), whereas the mouse recombinant enzyme was incubated with
NADPH (500 μM). Test compound and a mixture of nonlabeled-
and [3H]-E1 (final concentration: 500 nM, 0.15 μCi) were added and
mixed for 10 min at 37 °C. Further treatment of the samples and
HPLC separation was carried out as-mentioned above for h17β-
HSD2. In each experiment, a reference compound was included.
MTT Cytotoxicity Assay. In general, the assay was performed
similar to a protocol described in the literature.35 HEK293 cells (2 ×
105 cells per well, 1 mL per well) were seeded in 96-well flat-bottom
plates. Culturing of cells, incubations, and OD measurements were
performed as described previously with minor modifications. In short,
4 h after seeding the cells, the incubation was started by the addition
of compounds (100, 50, 25, 12.5, and 6.25 μM) in a final DMSO
concentration of 1% or vehicle (DMSO) in quadruplicates. After 66 h,
50 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) solution in phosphate-buffered saline
is added to the medium and cells were then incubated for 30 min at
37 °C in a CO2 incubator. After this, the medium was removed by
suction and 250 μL of DMSO containing 10% SDS and 0.5% acetic
(5-(4-Methoxy-3,5-dimethylphenyl)thiophen-2-yl)(2,4,5-tri-
fluoro-3-hydroxyphenyl)methanone (1). The title compound was
prepared by reaction of (5-bromothiophen-2-yl) (2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (4-methoxy-
3,5-dimethylphenyl)boronic acid (54.0 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.5 mg, 9.95 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 73% (81.5 mg); mp
1
162−164 °C; H NMR (300 MHz, CDCl3, δ): 7.49 (dd, J = 3.9, 1.7
Hz, 1H), 7.34 (s, 2H), 7.24−7.27 (m, 1H), 6.99 (td, J = 8.7, 6.1 Hz,
1H), 3.76 (s, 3H), 2.38 (s, 6H); MS (ESI): 393.2 (M + H)+; UV λ
(nm) = 250, 355; RT HPLC: 13.48 min (13 min 10−95% MeCN in
water with 0.1% formic acid); purity >99%.
(5-(3,5-Dichloro-4-methoxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-
3-hydroxyphenyl)methanone (2). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (3,5-
dichloro-4-methoxyphenyl)boronic acid (66.3 mg, 0.300 mmol),
cesium carbonate (326 mg, 1.00 mmol), and tetrakis-
(triphenylphosphine)palladium (11.5 mg, 9.95 μmol) according to
method C1. The product was purified by preparative HPLC; yield:
1
59% (73.2 mg); mp 156−158 °C; H NMR (300 MHz, acetone-d6,
I
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
δ): 7.86 (s, 2H), 7.71−7.69 (m, 2H), 7.13 (ddd, J = 10.0, 8.1, 5.7 Hz,
1H), 3.95 (s, 3H); MS (ESI): 431.0 (M − H)−; UV λ (nm) = 340;
RT f HPLC: 14.14 min (13 min 10−95% MeCN in water with 0.1%
formic acid); purity >99%.
the reaction of (3-(5-bromothiophene-2-carbonyl)-2-methylphenyl
acetate (7b) (84.8 mg, 0.250 mmol), (3,5-dichloro-4-
methoxyphenyl))boronic acid (66.3 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.5 mg, 9.95 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 61% (59.9 mg); mp
(5-(3-Chloro-4-hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (3). The title compound was prepared by
the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (3-chloro-
4-hydroxyphenyl)boronic acid (51.7 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.5 mg, 9.95 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 40% (43.4 mg); mp
1
209−211 °C; H NMR (300 MHz, acetone-d6, δ): 8.60 (br s, 1H),
7.84 (s, 2H), 7.64 (d, J = 3.9 Hz, 1H), 7.45 (d, J = 4.2 Hz, 1H), 7.17
(t, J = 7.5 Hz, 1H), 7.04 (dd, J = 8.1, 1.2 Hz, 1H), 6.98 (dd, J = 7.5,
1.2 Hz, 1H), 3.94 (s, 3H), 2.19 (s, 3H); MS (ESI): 393.12 (M + H)+;
UV λ (nm) = 334; Rf HPLC: 14.42 min (13 min 10−95% MeCN in
water with 0.1% formic acid); purity 98%.
1
107−110 °C; H NMR (300 MHz, acetone-d6, δ): 9.61 (br s, 1H),
(5-(3-Chloro-4-hydroxyphenyl)thiophen-2-yl)(3-hydroxy-2-
methylphenyl)methanone (9). The title compound was prepared by
the reaction of (3-(5-bromothiophene-2-carbonyl)-2-methylphenyl
acetate (7b) (84.8 mg, 0.250 mmol), (3-chloro-4-hydroxyphenyl))-
boronic acid (51.7 mg, 0.300 mmol), cesium carbonate (326 mg, 1.00
mmol), and tetrakis(triphenylphosphine)palladium (11.5 mg, 9.95
μmol) according to method C1. The product was purified by
preparative HPLC; yield: 3.5% (3.00 mg); mp 106−108 °C; 1H NMR
(300 MHz, acetone-d6, δ): 7.79 (d, J = 2.1 Hz, 1H), 7.60 (dd, J = 8.4,
2.1 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 7.39 (d, J = 4.2 Hz, 1H), 7.18−
7.11 (m, 2H), 7.03 (dd, J = 7.8, 0.9 Hz, 1H), 6.95 (dd, J = 7.5, 0.9 Hz,
1H), 2.18 (s, 3H).; MS (ESI): 345.11 (M + H)+; UV λ (nm) = 233,
355; Rf HPLC: 10.90 min (13 min 10−95% MeCN in water with
0.1% formic acid); purity 99%.
7.82 (d, J = 2.2 Hz, 1H), 7.68−7.59 (m, 2H), 7.53 (d, J = 4.1 Hz,
1H), 7.13 (d, J = 8.6 Hz, 1H), 7.18−7.04 (m, 1H), 7.13 (d, J = 8.6
Hz, 1H); MS (ESI): 385.1 (M + H)+; UV λ (nm) = 250, 361; Rf
HPLC: 11.04 min (13 min 10−95% MeCN in water with 0.1% formic
acid); purity 95%.
(5-(3-Fluoro-4-hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (4). The title compound was prepared by
the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (3-fluoro-
4-hydroxyphenyl)boronic acid (46.8 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.5 mg, 9.95 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 29% (30.8 mg); mp
1
164−166 °C; H NMR (300 MHz, acetone-d6, δ): 9.55 (br s, 1H),
(3-Hydroxy-2-methylphenyl)(5-(4-hydroxy-3,5-dimethylphenyl)-
thiophen-2-yl)methanone (10). The title compound was prepared by
the reaction of (3-hydroxy-2-methylphenyl)(5-(4-methoxy-3,5-
dimethylphenyl)thiophen-2-yl)methanone (7) (16 mg, 0.045
mmol), and boron tribromide (0.14 mmol, 3.0 equiv) according to
method B. The product was purified by preparative HPLC; yield 66%:
7.65 (dd, J = 4.1, 1.5 Hz, 1H), 7.60 (dd, J = 11.9, 2.2 Hz, 1H), 7.52−
7.48 (m, 1H), 7.51 (d, J = 4.1 Hz, 1H), 7.14−7.06 (m, 1H), 7.11 (t, J
= 8.8 Hz, 1H); MS (ESI-): 367.1 (M − H)−; UV λ (nm) = 363; Rf
HPLC: 10.97 min (13 min 10−95% MeCN in water with 0.1% formic
acid); purity 98%.
1
(10.0 mg); mp 180−183 °C; H NMR (300 MHz, methanol-d4, δ):
(5-(4-Hydroxy-3,5-dimethylphenyl)thiophen-2-yl)(2,4,5-trifluoro-
3-hydroxyphenyl)methanone (5). The title compound was prepared
by the reaction of (5-(4-methoxy-3,5-dimethylphenyl)thiophen-2-
yl)(2,4,5-trifluoro-3-hydroxyphenyl)methanone (1) (51 mg, 0.13
mmol), and boron tribromide (0.39 mmol, 3.0 equiv) according to
method B. The product was purified by CC; yield: 98% (51 mg); mp
7.25−7.39 (m, 4 H), 7.08−7.17 (m, 1 H), 6.88 (d, J = 7.6 Hz, 1 H),
6.93 (d, J = 7.9 Hz, 1 H), 2.26 (s, 6 H), 2.16 (s, 3 H); MS (ESI):
339.3 (M + H)+; UV λ (nm) = 363; Rf HPLC: 10.98 min (13 min
10−95% MeCN in water with 0.1% formic acid); purity 99%.
(5-(3,5-Dichloro-4-hydroxyphenyl)thiophen-2-yl)(3-hydroxy-2-
methylphenyl)methanone (11). The title compound was prepared
by the reaction of (5-(3,5-dichloro-4-methoxyphenyl)thiophen-2-
yl)(3-hydroxy-2-methylphenyl)methanone (8) (34.0 mg, 0.086
mmol) and boron tribromide (0.23 mmol, 3.0 equiv) according to
method B. The product was purified by preparative HPLC; yield 92%:
1
198−200 °C; H NMR (300 MHz, CDCl3, δ): 7.48 (dd, J = 4.1, 2.0
Hz, 1H), 7.32 (br s, 2H), 7.22 (d, J = 4.1 Hz, 1H), 6.98 (ddd, J = 9.6,
8.1, 5.8 Hz, 1H), 4.90 (s, 1H), 2.30 (s, 6H); MS (ESI): 379.10 (M +
H)+; UV λ (nm) = 377; Rf HPLC: 11.28 min (13 min 10−95%
MeCN in water with 0.1% formic acid); purity >99%.
1
(30.0 mg). H NMR (300 MHz, methanol-d4, δ): 7.66 (s, 2 H),
(5-(3,5-Dichloro-4-hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-
3-hydroxyphenyl)methanone (6). The title compound was prepared
by the reaction of (5-(3,5-dichloro-4-methoxyphenyl)thiophen-2-
yl)(2,4,5-trifluoro-3-hydroxyphenyl)methanone (2) (48 mg, 0.11
mmol), and boron tribromide (0.34 mmol, 3.0 equiv) according to
method B. The product was purified by CC; yield: quantitative (84
mg); mp 212−214 °C; 1H NMR (300 MHz, acetone-d6, δ): 9.92 (s, 1
H); 9.30 (s, 1 H); 7.81 (s, 2 H); 7.68 (dd, J = 1.8, 4.1 Hz, 1 H);
7.65−7.61 (m, 1 H); 7.13 (ddd, J = 5.6, 8.1, 10.0 Hz, 1 H); MS (ESI):
419.20 (M + H)+; UV λ (nm) = 355; Rf HPLC: 11.54 min (13 min
10−95% MeCN in water with 0.1% formic acid). Purity 98%.
(3-Hydroxy-2-methylphenyl)(5-(4-methoxy-3,5-dimethylphenyl)-
thiophen-2-yl)methanone (7). The title compound was prepared by
the reaction of (3-(5-bromothiophene-2-carbonyl)-2-methylphenyl
acetate (7b) (84.8 mg, 0.250 mmol), 4-methoxy-3,5-dimethylphenyl)-
boronic acid (54.0 mg, 0.300 mmol), cesium carbonate (326 mg, 1.00
mmol), and tetrakis(triphenylphosphine)palladium (11.5 mg, 9.95
μmol) according to method C1. The product was purified by
preparative HPLC; yield: 26% (23.0 mg); mp 194−196 °C; 1H NMR
(300 MHz, acetone-d6, δ): 8.57 (br s, 1H), 7.47 (s, 2H), 7.44 (d, J =
3.9 Hz, 1H), 7.39 (d, J = 3.9 Hz, 1H), 7.16 (t, J = 7.8 Hz, 1H), 7.02
(dd, J = 8.1, 1.2 Hz, 1H), 6.95 (dd, J = 7.5, 1.2 Hz, 1H), 3.75 (s, 3H),
2.32 (s, 6H), 2.18 (s, 3H); MS (ESI): 353.21 (M + H)+; UV λ (nm)
= 236, 348; Rf HPLC: 13.83 min (13 min 10−95% MeCN in water
with 0.1% formic acid); purity >99%.
7.32−7.43 (m, 2 H), 7.06−7.19 (m, 1 H), 6.82−6.98 (m, 2 H), 2.15
(s, 3 H); MS (ESI): 365.2 (M + H)+; UV λ (nm) = 347, 246 (sh); Rf
HPLC: 11.29 min (13 min 10−95% MeCN in water with 0.1% formic
acid); purity >99%.
(5-(4-Methoxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (12). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (7b) (94.8 mg, 0.281 mmol), 4-
methoxyphenylboronic acid (45.6 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)-
palladium (11.6 mg, 10.0 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 40% (40.7 mg); mp
1
150−152 °C; H NMR (300 MHz, acetone-d6, δ): 7.81−7.70 (m,
2H), 7.64 (dd, J = 4.1, 1.8 Hz, 1H), 7.48 (d, J = 4.1 Hz, 1H), 7.17−
6.99 (m, 3H), 3.87 (s, 3H); MS (ESI): 365.2 (M + H)+. UV λ (nm) =
363; Rf HPLC: 12.14 min (13 min 10−95% MeCN in water with
0.1% formic acid); purity 98%.
(5-(3-Methoxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (13). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 3-
methoxyphenylboronic acid (45.6 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.6 mg, 10.0 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 43% (44.0 mg); mp
(5-(3,5-Dichloro-4-methoxyphenyl)thiophen-2-yl)(3-hydroxy-2-
methylphenyl)methanone (8). The title compound was prepared by
1
161−163 °C; H NMR (300 MHz, MeOH-d4, δ): 7.81−7.48 (m,
J
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 7.46−7.26 (m, 3H), 7.09−6.95 (m, 2H), 3.89 (s, 3H); MS
(ESI): 365.2 (M + H)+; UV λ (nm) = 345; RT HPLC = 12.30 min
(13 min 10−95% MeCN in water with 0.1% formic acid); purity
>98%.
3-methoxyphenyl)methanone (19a) (300 mg, 0.82 mmol) and boron
tribromide (4.17 mmol) according to method B. The product was
purified by CC (dichloromethane/methanol 99:1); yield: 31% (90
1
mg); H NMR (500 MHz, acetone-d6, δ) 9.79 (s, 1H, OH), 7.63
(5-(2-Methoxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (14). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 2-
methoxyphenylboronic acid (45.6 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)-
palladium (11.6 mg, 10.0 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 68% (70.0 mg); mp
(ddd, J = 41.7, 4.1, 1.8 Hz, 1H), 7.57−7.52 (m, 2H), 7.36 (d, J = 4.1
Hz, 1H), 7.07 (ddd, J = 10.0, 8.2, 5.6 Hz, 1H), 6.83−6.74 (m, 3H),
4.94 (s, 2H); UV λ (nm) = 399, 260 (sh); RT HPLC = 10.02 (13 min
10−95% MeCN in water with 0.1% formic acid); purity 99%.
(5-(3-Aminophenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (20). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (3-
aminophenyl)boronic acid (41.1 mg, 0.300 mmol), cesium carbonate
(326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)palladium
(11.5 mg, 9.95 μmol) according to method C1. The product was
purified by preparative HPLC; yield: 24% (25.1 mg); mp 182−184
1
167−169 °C; H NMR (300 MHz, acetone-d6, δ): 7.87 (dd, J = 7.8,
1.7 Hz, 1H), 7.71 (d, J = 4.2 Hz, 1H), 7.63 (dd, J = 4.2, 1.8 Hz, 1H),
7.43 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.24−7.19 (m, 1H), 7.15−7.05
(m, 2H), 4.03 (s, 3H); MS (ESI): 365.2 (M + H)+; UV λ (nm) =
359; RT HPLC = 12.16 min (13 min 10−95% MeCN in water with
0.1% formic acid); purity 97%.
1
°C; H NMR (300 MHz, MeOH-d4, δ): 7.57−7.53 (m, 1H), 7.44−
7.41 (m, 1H), 7.20−7.14 (m, 1H), 7.09−6.93 (m, 3H), 6.78−6.74
(m, 1H); MS (ESI): 350.2 (M + H)+; UV λ (nm) = 343; RT HPLC =
10.19 min (13 min 10−95% MeCN in water with 0.1% formic acid);
purity 96%.
(5-(4-Hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (15). The title compound was prepared
by the reaction of (5-(4-methoxyphenyl)thiophen-2-yl)(2,4,5-tri-
fluoro-3-hydroxyphenyl)methanone (12) (20.0 mg, 54.9 μmol) and
boron tribromide (0.165 mmol, 3.0 equiv) according to method B.
The product was purified by preparative HPLC; yield: 18% (3.50
(5-(2-Aminophenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (21). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), (2-
aminophenyl)boronic acid (41.1 mg, 0.300 mmol), cesium carbonate
(326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)palladium
(11.6 mg, 10.0 μmol) according to method C1. The product was
purified by preparative HPLC; yield: 53% (52.1 mg); mp 216−218
1
mg); mp 145−148 °C; H NMR (300 MHz, acetone-d6, δ): 7.70−
7.63 (m, 2H), 7.61 (dd, J = 4.1, 1.9 Hz, 1H), 7.43 (d, J = 4.1 Hz, 1H),
7.05−6.91 (m, 3H); MS (ESI): 351.2 (M + H)+; UV λ (nm) = 365;
RT HPLC = 10.15 min (13 min 10−95% MeCN in water with 0.1%
formic acid); purity 96%.
1
°C; H NMR (300 MHz, MeOH-d4, δ): 7.64 (dd, J = 3.8, 1.7 Hz,
(5-(3-Hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (16). The title compound was prepared
by the reaction of (5-(3-methoxyphenyl)thiophen-2-yl)(2,4,5-tri-
fluoro-3-hydroxyphenyl)methanone (13) (26.2 mg, 71.9 μmol) and
boron tribromide (0.216 mmol, 3.0 equiv) according to method B.
The product was purified by preparative HPLC; yield: 83% (21.0
mg); mp 215−218 °C; 1H NMR (500 MHz, acetone-d6, δ): 7.66 (dd,
J = 3.9, 1.7 Hz, 1H), 7.54 (d, J = 4.1 Hz, 1H), 7.33−7.26 (m, 2H),
7.25−7.23 (m, 1H), 7.11 (ddd, J = 10.0, 8.1, 5.5 Hz, 1H), 6.95−6.91
(m, 1H); MS (ESI): 351.3 (M + H)+; UV λ (nm) = 264 (sh), 348;
RT HPLC = 10.22 min (C18, 13 min 10−95% MeCN in water with
0.1% formic acid); purity 98%.
1H), 7.45−7.38 (m, 1H), 7.33 (dd, J = 7.7, 1.5 Hz, 1H), 7.26−7.14
(m, 1H), 7.11−6.95 (m, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.84−6.71 (m,
1H); MS (ESI): 350.2 (M + H)+; UV λ (nm) = 281, 316, 377; Rf =
10.98 min (13 min 10−95% MeCN in water with 0.1% formic acid);
purity >99%.
(5-(3-Amino-4-methoxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (22). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 2-
methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (22a)
(74.7 mg, 0.300 mmol), cesium carbonate (326 mg, 1.00 mmol), and
tetrakis(triphenylphosphine)palladium (11.6 mg, 10.0 μmol) accord-
ing to method C1. The product was purified by preparative HPLC;
yield: 31% (33.3 mg); mp 181−183 °C; 1H NMR (300 MHz,
MeOH-d4, δ): 7.61−7.52 (m, 1H), 7.29 (d, J = 3.9 Hz, 1H), 7.22−
7.10 (m, 1H), 7.05−6.86 (m, 1H), 6.78 (d, J = 8.1 Hz, 1H), 4.61 (br
s, 2H), 3.94 (s, 3H); MS (ESI): 380.3 (M + H)+; UV λ (nm) = 285
(sh), 380; RT HPLC = 10.54 min (13 min 10−95% MeCN in water
with 0.1% formic acid); purity >99%.
(5-(3-Amino-4-hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (23). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole (23a) (73.5 mg,
0.300 mmol), cesium carbonate (326 mg, 1.00 mmol), and
tetrakis(triphenylphosphine)palladium (11.6 mg, 10.0 μmol) accord-
ing to method C1. The product was purified by preparative HPLC;
yield: 35% (35.7 mg). 1H NMR (300 MHz, MeOH-d4, δ): 7.58−7.51
(m, 1H), 7.33 (d, J = 3.9 Hz, 1H), 7.17 (d, J = 1.5 Hz, 1H), 7.04 (dd,
J = 8.0, 2.1 Hz, 1H), 7.01−6.92 (m, 1H), 6.78 (d, J = 8.1 Hz, 1H);
MS (ESI): 366.2 (M + H)+; UV λ (nm) = 285 (sh), 365; RT HPLC
= 8.32 min (13 min 10−95% MeCN in water with 0.1% formic acid);
purity >99%.
(5-(2-Hydroxyphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (17). The title compound was prepared
by the reaction of (5-(2-methoxyphenyl)thiophen-2-yl)(2,4,5-tri-
fluoro-3-hydroxyphenyl)methanone (14) (34.3 mg, 94.1 μmol) and
boron tribromide (0.282 mmol, 3.0 equiv) according to method B.
The product was purified by preparative HPLC; yield: 45% (14.7
1
mg). H NMR (300 MHz, acetone-d6, δ): 7.82 (dd, J = 7.9, 1.5 Hz,
1H), 7.75 (d, J = 4.2 Hz, 1H), 7.64 (dd, J = 4.1, 1.6 Hz, 1H), 7.31−
7.21 (m, 1H), 7.15−7.03 (m, 2H), 6.98 (t, J = 7.5 Hz, 1H); MS
(ESI): 351.2 (M + H)+; UV λ (nm) = 265 (sh), 290 (sh), 363; RT
HPLC = 10.56 min (13 min 10−95% MeCN in water with 0.1%
formic acid); purity >99%.
(5-(3-(Hydroxymethyl)phenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (18). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (3-
(hydroxymethyl)phenyl)boronic acid (45.6 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.5 mg, 9.95 μmol) according to method C1. The
product was purified by preparative HPLC; yield: 24% (25.1 mg); mp
170−172 °C; 1H NMR (300 MHz, MeOH-d4, δ): 7.75 (s, 1H), 7.65
(d, J = 6.7 Hz, 1H), 7.58 (dd, J = 4.0, 1.8 Hz, 1H), 7.51 (d, J = 4.1 Hz,
1H), 7.47−7.37 (m, 2H), 6.97 (ddd, J = 9.7, 8.1, 5.7 Hz, 1H), 4.67 (s,
2H); MS (APCI−): 364.1 (M − H)−; UV λ (nm) = 345; Rf HPLC:
10.61 min (13 min 10−95% MeCN in water with 0.1% formic acid);
purity 99%.
(5-(3-Amino-4-methylphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (24). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 3-amino-
4-methylphenylboronic acid (45.3 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.6 mg, 10.0 μmol) according to method C1. The
(5-(4-Aminophenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (19). The title compound was prepared
by the reaction of (5-(4-aminophenyl)thiophen-2-yl)(2,4,5-trifluoro-
K
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
product was purified by preparative HPLC; yield: 15% (15.7 mg); mp
208−210 °C; 1H NMR (300 MHz, MeOH-d4, δ): 7.61 (d, J = 2.2 Hz,
1H), 7.14 (d, J = 3.9 Hz, 1H), 7.11−6.97 (m, 2H), 6.86 (d, J = 7.8
Hz, 1H), 6.80 (d, J = 7.5 Hz, 1H), 2.22 (s, 3H); MS (ESI): 364.2 (M
+ H)+; UV λ (nm) = 323; RT HPLC = 12.23 min (13 min 10−95%
MeCN in water with 0.1% formic acid); purity >99%.
(5-(3-Amino-2-methylphenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (25). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 2-methyl-
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (25a) (69.9
mg, 0.300 mmol), cesium carbonate (326 mg, 1.00 mmol), and
tetrakis(triphenylphosphine)palladium (11.6 mg, 10.0 μmol) accord-
ing to method C1. The product was purified by preparative HPLC;
yield: 7% (7.60 mg); mp 201−203 °C; 1H NMR (300 MHz, MeOH-
d4, δ): 7.58 (dd, J = 3.9, 1.8 Hz, 1H), 7.43 (d, J = 4.0 Hz, 1H), 7.19−
6.88 (m, 4H), 2.22 (s, 3H); MS (ESI): 364.2 (M + H)+; UV λ (nm)
= 280 (sh), 353; RT HPLC = 10.97 min (13 min 10−95% MeCN in
water with 0.1% formic acid); purity 97%.
onic acid (53.7 mg, 0.300 mmol), cesium carbonate (319 mg, 0.979
mmol), and tetrakis(triphenylphosphine)palladium (11.3 mg, 9.78
μmol) according to method C1. The product was purified by
preparative HPLC; yield: 12% (12.9 mg); mp 264 °C (decom-
position); 1H NMR (300 MHz, DMSO-d6 δ): 10.16 (s, 1H) 7.76 (d, J
= 8.9 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.64 (dd, J = 4.1, 1.5 Hz,
1H), 7.61−7.56 (m, 1H), 7.19−7.07 (m, 1H), 2.07 (s, 3H); MS
(ESI): 392.2 (M + H)+; UV λ (nm) = 257 (sh), 363; RT HPLC: 9.91
min (13 min 10−95% MeCN in water with 0.1% formic acid); purity
86%.
3-(5-(2,4,5-Trifluoro-3-hydroxybenzoyl)thiophen-2-yl)benzamide
(31). The title compound was prepared by the reaction of (5-
bromothiophen-2-yl)-(2,4,5-trifluoro-3-hydroxyphenyl)methanone
(1b) (96.0 mg, 0.285 mmol), (3-carbamoylphenyl)boronic acid (49.5
mg, 0.300 mmol), cesium carbonate (326 mg, 1.00 mmol), and
tetrakis(triphenylphosphine)palladium (11.5 mg, 9.95 μmol) accord-
ing to method C1. The product was purified by preparative HPLC
giving the formiate of the title compound; yield: 21% (25.7 mg); mp
1
260 °C (decomposition); H NMR (300 MHz, DMSO-d6 δ): 11.35
(br s, 1H), 8.28 (t, J = 1.8 Hz, 1H), 8.16 (br s, 1H), 8.00−7.92 (m,
2H), 7.77 (d, J = 4.3 Hz, 1H), 7.73 (dd, J = 4.3, 1.7 Hz, 1H), 7.66−
7.50 (m, 3H), 7.28−7.20 (m, 1H); MS (ESI−): 376.1 (M − H)−; UV
λ (nm) = 341; Rf HPLC: 9.14 min (13 min 10−95% MeCN in water
with 0.1% formic acid); purity >99%.
(5-(3-Amino-4-fluorophenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (26). The title compound was prepared
by the reaction of (5-(3-amino-4-fluorophenyl)thiophen-2-yl)(2,4,5-
trifluoro-3-methoxyphenyl)methanone (26a) (150 mg, 0.53 mmol)
and boron tribromide (1.97 mmol) according to method B. The
product was purified by CC (dichloromethane/methanol 99.5:0.5);
yield: 31% (45 mg). 1H NMR (500 MHz, acetone-d6, δ) 9.79 (s, 1H,
OH), 7.64 (dd, J = 4.1, 1.8 Hz, 1H), 7.44 (d, J = 4.0 Hz, 1H), 7.31−
7.27 (m, 1H), 7.14−7.02 (m, 3H), 5.32 (s, 2H); MS (ESI): 368.3 (M
+ H)+; UV λ (nm) = 276 (sh), 348; RT HPLC = 10.91 min (13 min
10−95% MeCN in water with 0.1% formic acid); purity 95%.
(5-(3-Amino-4,5-difluorophenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (27). The title compound was prepared
by the reaction of (5-(3-amino-4,5-difluorophenyl)thiophen-2-yl)-
(2,4,5-trifluoro-3-methoxyphenyl)methanone (27a) (100 mg, 0.25
mmol) and boron tribromide (1.25 mmol) according to method B.
The product was purified by CC (dichloromethane/methanol
(5-Phenylthiophen-2-yl)(2,4,5-trifluoro-3-hydroxyphenyl)-
methanone (32). The title compound was prepared by the reaction
of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-hydroxyphenyl)-
methanone (1b) (94.8 mg, 0.281 mmol), phenylboronic acid (36.6
mg, 0.300 mmol), cesium carbonate (326 mg, 1.00 mmol), and
tetrakis(triphenylphosphine)palladium (11.6 mg, 10.0 μmol) accord-
ing to method C1. The product was purified by preparative HPLC;
yield: 51% (47.9 mg); mp 214−216 °C; 1H NMR (300 MHz,
acetone-d6, δ): 9.91 (br s, 1H), 7.87−7.77 (m, 2H), 7.69 (dd, J = 4.1,
1.8 Hz, 1H), 7.61 (d, J = 4.1 Hz, 1H), 7.55−7.39 (m, 3H), 7.12 (ddd,
J = 10.0, 8.1, 5.7 Hz, 1H); MS (ESI): 335.2 (M + H)+; UV λ (nm) =
336; RT HPLC = 12.23 min (13 min 10−95% MeCN in water with
0.1% formic acid); purity >99%.
1
99.5:0.5); yield: 46% (45 mg). H NMR (500 MHz, acetone-d6 δ)
9.77 (s, 1H, OH), 7.67 (dd, J = 4.1, 1.8 Hz, 1H), 7.52 (d, J = 4.1 Hz,
1H), 7.16−7.09 (m, 2H), 6.98 (ddd, J = 11.1, 6.6, 2.2 Hz, 1H), 5.39
(s, 2H); MS (ESI): 386.2 (M + H)+; UV λ (nm) = 270 (sh), 343; RT
HPLC = 11.29 min (13 min 10−95% MeCN in water with 0.1%
formic acid); purity 98%.
(5-(3-Amino-4,6-difluorophenyl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (28). The title compound was prepared
by the reaction of (5-(3-amino-4-fluorophenyl)thiophen-2-yl)(2,4,5-
trifluoro-3-methoxyphenyl)methanone (28a) (150 mg, 0.53 mmol)
and boron tribromide (1.97 mmol) according to method B. The
product was purified by CC (dichloromethane/methanol 99.5:0.5);
yield: 31% (45 mg). 1H NMR (500 MHz, acetone-d6 δ) 9.76 (s, 1H,
OH), 7.69 (ddd, J = 4.1, 1.8, 1.0 Hz, 1H), 7.52 (dd, J = 4.1, 1.0 Hz,
1H), 7.35−7.07 (m, 3H), 5.29 (s, 2H); MS (ESI): 386.3 (M + H)+;
UV λ (nm) = 283 (sh), 341; RT HPLC = 11.29 min (13 min 10−
95% MeCN in water with 0.1% formic acid); purity 90%.
(5-(1H-Indol-4-yl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (33). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (33a) (72.9 mg,
0.300 mmol), cesium carbonate (319 mg, 0.979 mmol), and
tetrakis(triphenylphosphine)palladium (11.3 mg, 9.78 μmol) accord-
ing to method C1. The product was purified by preparative; yield:
1
95% (99.6 mg); mp 204−208 °C; H NMR (300 MHz, acetone-d6,
δ): 10.67 (br s, 1H), 7.73 (dd, J = 4.0, 1.8 Hz, 1H), 7.67 (d, J = 4.1
Hz, 2H), 7.60−7.52 (m, 2H), 7.48 (dd, J = 7.4, 0.9 Hz, 1H), 7.28−
7.20 (m, 1H), 7.15 (ddd, J = 10.0, 8.1, 5.6 Hz, 1H), 6.96 (ddd, J = 3.2,
2.0, 1.0 Hz, 1H); MS (ESI): 374.3 (M + H)+; UV λ (nm) = 276. 374;
RT HPLC = 10.22 min (13 min 10−95% MeCN in water with 0.1%
formic acid); purity >99%.
(5-(1H-Indol-5-yl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (34). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1g) (94.8 mg, 0.281 mmol), (1H-indol-
5-yl)boronic acid (48.3 mg, 0.300 mmol), cesium carbonate (326 mg,
1.00 mmol), and tetrakis(triphenylphosphine)palladium (11.6 mg,
10.0 μmol) according to method C1. The product was purified by
preparative HPLC; yield: 49% (51.6 mg). 1H NMR (500 MHz,
acetone-d6, δ): 8.09 (dd, J = 1.9, 0.6 Hz, 1H), 7.65 (dd, J = 4.1, 1.9
Hz, 1H), 7.60−7.51 (m, 3H), 7.47−7.41 (m, 1H), 7.11 (ddd, J =
10.1, 8.2, 5.7 Hz, 1H), 6.63−6.57 (m, 1H); MS (ESI): 374.3 (M +
H)+; UV λ (nm) = 265 (sh), 380; Rf = 11.27 min (13 min 10−95%
MeCN in water with 0.1% formic acid); purity >99%.
N-(3-(5-(2,4,5-Trifluoro-3-hydroxybenzoyl)thiophen-2-yl)-
phenyl)acetamide (29). The title compound was prepared by the
reaction of (5-bromothiophen-2-yl) (2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 3-
acetylamidophenylboronic acid (53.7 mg, 0.300 mmol), cesium
carbonate (326 mg, 1.00 mmol), and tetrakis(triphenylphosphine)
palladium (11.6 mg, 10.0 μmol) according to method C1. The
1
product was purified by preparative HPLC; yield: 8% (8.72 mg); H
NMR (300 MHz, acetone-d6, δ): 9.31 (br s, 1H), 8.21−8.14 (m, 1H),
7.72−7.60 (m, 2H), 7.60−7.34 (m, 3H), 7.12 (ddd, J = 10.0, 8.1, 5.6
Hz, 1H), 3.31 (s, 3H); MS (ESI): 392.3 (M + H)+; UV λ (nm) = 255
(sh), 346; RT HPLC = 12.16 min (13 min 10−95% MeCN in water
with 0.1% formic acid); purity >99%.
N-(4-(5-(2,4,5-Trifluoro-3-hydroxybenzoyl)thiophen-2-yl)-
phenyl)acetamide (30). The title compound was prepared by the
reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-hydroxyphenyl)-
methanone (1b) (94.8 mg, 0.281 mmol), 4-acetylamidophenylbor-
(5-(1H-Indol-6-yl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (35). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (96.0 mg, 0.285 mmol), (1H-indol-
6-yl)boronic acid (48.3 mg, 0.300 mmol), cesium carbonate (326 mg,
L
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.00 mmol), and tetrakis(triphenylphosphine)palladium (11.5 mg,
9.95 μmol) according to method C1. The product was purified by
preparative HPLC; yield: 81% (85.7 mg); 1H NMR (300 MHz,
acetone-d6, δ): 10.50 (br s, 1H), 7.91 (m, 1H), 7.67 (d, J = 8.4 Hz,
1H), 7.64 (dd, J = 4.1, 1.9 Hz, 1H), 7.53 (d, J = 4.1 Hz, 1H), 7.51−
7.46 (m, 2H), 7.13−7.05 (m, 1H), 6.56−6.52 (m, 1H); MS (ESI-):
372.1 (M − H)−; UV λ (nm) = 271, 389; Rf HPLC: 11.81 min (13
min 10−95% MeCN in water with 0.1% formic acid); purity 96%.
(5-(1H-Benzo[d]imidazole-5-yl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (36). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 1H-
benzimidazole-5-boronic acid pinacol ester (73.2 mg, 0.300 mmol),
cesium carbonate (320 mg, 0.984 mmol), and tetrakis-
(triphenylphosphine)palladium (40.4 mg, 35.0 μmol) according to
method C1. The product was purified by preparative HPLC; yield:
AUTHOR INFORMATION
Corresponding Author
*E-mail: rolf.hartmann@helmholtz-hzi.de. Phone: +(49) 681
98806 2000.
■
ORCID
Martin Frotscher: 0000-0003-1777-8890
Chris J. van Koppen: 0000-0003-2799-6728
Sandrine Marchais-Oberwinkler: 0000-0001-9941-7233
Rolf W. Hartmann: 0000-0002-5871-5231
Notes
The authors declare the following competing financial
interest(s): L.S., V.H.-O., A.S.A., M.F., C.J.v.K., S.M.-O.,
C.B., and R.W.H. are inventors of a patent application,
which covers the respective compound class. ElexoPharm
GmbH is the owner of this patent application. R.W.H. is CEO
of ElexoPharm GmbH.
1
7% (7.60 mg). H NMR (300 MHz, DMSO-d6 δ): 12.58 (br s, 1H),
8.32 (s, 1H), 8.04 (br s, 1H), 7.70−7.64 (m, 4H), 7.19−7.11 (m,
1H); MS (ESI): 375.2 (M + H)+; UV λ (nm) = 347; RT HPLC =
7.02 min (C18, 13 min 10−95% MeCN in water with 0.1% formic
acid); purity >99%.
ACKNOWLEDGMENTS
The authors thank Nadja Weber, Christopher Hornes, Birgit
■
(5-(Pyridin-3-yl)thiophen-2-yl)(2,4,5-trifluoro-3-hydroxyphenyl)-
methanone (37). The title compound was prepared by the reaction
of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-hydroxyphenyl)-
methanone (1b) (94.8 mg, 0.281 mmol), pyridine-3-boronic acid
(36.9 mg, 0.300 mmol), cesium carbonate (326 mg, 1.00 mmol), and
tetrakis(triphenylphosphine)palladium (11.3 mg, 9.78 μmol) accord-
ing to method C1. The product was purified by preparative HPLC;
yield: 15% (14.2 mg); mp 133−135 °C; 1H NMR (500 MHz,
DMSO-d6 δ): 9.05 (d, J = 2.0 Hz, 1H), 8.63 (dd, J = 4.8, 1.4 Hz, 1H),
8.22 (dt, J = 8.0, 1.9 Hz, 1H), 7.81 (d, J = 4.1 Hz, 1H), 7.74 (dd, J =
4.1, 1.4 Hz, 1H), 7.67−7.48 (m, 1H), 7.24 (ddd, J = 10.0, 8.2, 5.7 Hz,
1H); MS (ESI): 336.3 (M + H)+; UV λ (nm) = 331; RT HPLC =
9.23 min (13 min 10−95% MeCN in water with 0.1% formic acid);
purity 92%.
̈
Wiegand, Isabella Mang, and Jannine Ludwig for their help
with the in vitro tests, Julia Parakenings for her help with the
animal experiments, and Jennifer Pham and Anke Kaiser for
their help with synthesizing the compounds. Furthermore, the
authors thank Dr. Emmanuel Bey for his help within the
project. The research project has been financially supported by
the federal ministry for education and research under the sign
031A467. The authors are responsible for the content of the
publication.
ABBREVIATIONS
■
(5-(Quinolin-7-yl)thiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (38). The title compound was prepared
by the reaction of (5-bromothiophen-2-yl)(2,4,5-trifluoro-3-
hydroxyphenyl)methanone (1b) (94.8 mg, 0.281 mmol), 7-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (38a) (76.5 mg, 0.300
mmol), sodium carbonate (74.2 mg, 0.700 mmol), and tetrakis-
(triphenylphosphine)palladium (3.24 mg, 2.80 μmol) according to
method C2. The product was purified by preparative HPLC; yield:
OBs, osteoblasts; OCs, osteoclasts; E2, estradiol; T, testoster-
one; 17β-HSD2, 17β-hydroxysteroid dehydrogenase type 2;
E1, estrone; A-dione, androstenedione; 17β-HSD1, 17β-
hydroxysteroid dehydrogenase type 1; PK, pharmacokinetic;
SAR, structure−activity relationship; MTT, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide; DCM, dichloro-
methane
1
5% (5.9 mg). H NMR (300 MHz, DMSO-d6 δ): 8.97 (dd, J = 4.2,
1.7 Hz, 1H), 8.46−8.38 (m, 2H), 8.20−8.02 (m, 2H), 7.94 (d, J = 4.0
Hz, 1H), 7.74 (dd, J = 4.1, 1.5 Hz, 1H), 7.58 (dd, J = 8.1, 4.2 Hz,
1H), 7.13 (d, J = 6.2 Hz, 1H); MS (ESI): 386.2 (M + H)+; UV λ
(nm) = 355; RT HPLC = 10.45 min (13 min 10−95% MeCN in
water with 0.1% formic acid); purity 84%.
REFERENCES
■
(1) Compston, J. E. Sex Steroids and Bone. Physiol. Rev. 2001, 81,
419−447.
(2) Riggs, B. L.; Khosla, S.; Melton, L. J. A Unitary Model for
Involutional Osteoporosis: Estrogen Deficiency Causes Both Type I
and Type II Osteoporosis in Postmenopausal Women and
Contributes to Bone Loss in Aging Men. J. Bone Miner. Res. 1998,
13, 763−773.
(3) Chin, K.-Y.; Ima-Nirwana, S. Sex Steroids and Bone Health
Status in Men. Int. J. Endocrinol. 2012, 2012, 208719.
(4) Dong, Y.; Qiu, Q. Q.; Debear, J.; Lathrop, W. F.; Bertolini, D. R.;
Tamburini, P. P. 17Beta-Hydroxysteroid Dehydrogenases in Human
Bone Cells. J. Bone Miner. Res. 2009, 13, 1539−1546.
(5) Bagi, C. M.; Wood, J.; Wilkie, D.; Dixon, B. Effect of 17β-
Hydroxysteroid Dehydrogenase Type 2 Inhibitor on Bone Strength in
Ovariectomized Cynomolgus Monkeys. J. Musculoskeletal Neuronal
Interact. 2008, 8, 267−280.
(5-Methylthiophen-2-yl)(2,4,5-trifluoro-3-hydroxyphenyl)-
methanone (39). The title compound was prepared by the reaction
of (5-methylthiophen-2-yl)(2,4,5-trifluoro-3-methoxyphenyl)-
methanone (39a) (121 mg, 0.423 mmol) and boron tribromide
(1.38 mmol, 3.3 equiv) according to method B. The product was
1
purified by preparative HPLC; yield: 55% (63.1 mg). H NMR (300
MHz, MeOH-d4, δ) 7.47−7.43 (m, 1H), 7.02−6.90 (m, 2H), 2.62 (s,
3H); MS (ESI): 273.2 (M + H)+; UV λ (nm) = 260 (sh), 309; RT
HPLC = 10.33 min (13 min 10−95% MeCN in water with 0.1%
formic acid); purity 95%.
ASSOCIATED CONTENT
■
(6) Bydal, P.; Auger, S.; Poirier, D. Inhibition of Type 2 17beta-
Hydroxysteroid Dehydrogenase by Estradiol Derivatives Bearing a
Lactone on the D-Ring: Structure-Activity Relationships. Steroids
2004, 69, 325−342.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b01373.
̈
(7) Tremblay, M. R.; Luu-The, V.; Leblanc, G.; Noel, P.; Breton, E.;
Labrie, F.; Poirier, D. Spironolactone-Related Inhibitors of Type II
17β-Hydroxysteroid Dehydrogenase: Chemical Synthesis, Receptor
Binding Affinities, and Proliferative/Antiproliferative Activities.
Bioorg. Med. Chem. 1999, 7, 1013−1023.
Molecular formula strings of all final compounds (CSV)
Synthetic procedure for intermediates 1b, 1c, 19a, 22a,
23a, 25a, 26a, 27a, 28a, 33a, 38a, and 39b (PDF)
M
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(8) Poirier, D.; Bydal, P.; Tremblay, M. R.; Sam, K.-M.; Luu-The, V.
Inhibitors of Type II 17β-Hydroxysteroid Dehydrogenase. Mol. Cell.
Endocrinol. 2001, 171, 119−128.
(9) Vuorinen, A.; Engeli, R.; Meyer, A.; Bachmann, F.; Griesser, U.
J.; Schuster, D.; Odermatt, A. Ligand-Based Pharmacophore
Modeling and Virtual Screening for the Discovery of Novel 17β-
Hydroxysteroid Dehydrogenase 2 Inhibitors. J. Med. Chem. 2014, 57,
5995−6007.
(10) Wood, J.; Bagi, C. M.; Akuche, C.; Bacchiocchi, A.; Baryza, J.;
Blue, M.-L.; Brennan, C.; Campbell, A.-M.; Choi, S.; Cook, J. H.; et al.
4,5-Disubstituted Cis-Pyrrolidinones as Inhibitors of Type II 17β-
Hydroxysteroid Dehydrogenase. Part 3. Identification of Lead
Candidate. Bioorg. Med. Chem. Lett. 2006, 16, 4965−4968.
(11) Perspicace, E.; Cozzoli, L.; Gargano, E. M.; Hanke, N.; Carotti,
A.; Hartmann, R. W.; Marchais-Oberwinkler, S. Novel, Potent and
Selective 17β-Hydroxysteroid Dehydrogenase Type 2 Inhibitors as
Potential Therapeutics for Osteoporosis with Dual Human and
Mouse Activities. Eur. J. Med. Chem. 2014, 83, 317−337.
(12) Perspicace, E.; Giorgio, A.; Carotti, A.; Marchais-Oberwinkler,
S.; Hartmann, R. W. Novel N-Methylsulfonamide and Retro-N-
Methylsulfonamide Derivatives as 17β-Hydroxysteroid Dehydrogen-
ase Type 2 (17β-HSD2) Inhibitors with Good ADME-Related
Physicochemical Parameters. Eur. J. Med. Chem. 2013, 69, 201−215.
(13) Marchais-Oberwinkler, S.; Xu, K.; Wetzel, M.; Perspicace, E.;
Hydroxyphenylnaphthol Steroidomimetics as Potent and Selective
17β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors for the
Treatment of Estrogen-Dependent Diseases. J. Med. Chem. 2011,
54, 534−547.
(24) Oster, A.; Klein, T.; Werth, R.; Kruchten, P.; Bey, E.; Negri, M.;
Marchais-Oberwinkler, S.; Frotscher, M.; Hartmann, R. W. Novel
Estrone Mimetics with High 17β-HSD1 Inhibitory Activity. Bioorg.
Med. Chem. 2010, 18, 3494−3505.
(25) van Koppen, C.; Abdelsamie, A.; Herath, S.; Biskupek, Y.;
̈
Boerger, C.; Siebenburger, L.; Salah, M.; Scheuer, C.; Marchais-
Oberwinkler, S.; Frotscher, M.; Pohlemann, T.; Menger, M. D.;
Hartmann, R. W.; Laschke, M. W.; van Koppen, C. J. Targeted
Endocrine Therapy: Design, Synthesis and Proof-of-Principle of 17ß-
Hydroxysteroid Dehydrogenase Type 2 Inhibitors in Bone Fracture
Healing. J. Med. Chem. 2018, submitted.
(26) Watanabe, K.; Hishiya, A. Mouse Models of Senile
Osteoporosis. Mol. Aspects Med. 2005, 26, 221−231.
́
̈
(27) Martel, C.; Rheaume, E.; Takahashi, M.; Trudel, C.; Couet, J.;
Luu-The, V.; Simard, J.; Labrie, F. Distribution of 17ß-Hydroxysteroid
Dehydrogenase Gene Expression and Activity in Rat and Human
Tissues. J. Steroid Biochem. Mol. Biol. 1992, 41, 597−603.
̈
(28) Abdelsamie, A. S.; van Koppen, C. J.; Bey, E.; Salah, M.; Borger,
C.; Siebenburger, L.; Laschke, M. W.; Menger, M. D.; Frotscher, M.
̈
Treatment of Estrogen-Dependent Diseases: Design, Synthesis and
Profiling of a Selective 17β-HSD1 Inhibitor with Sub-Nanomolar
IC50 for a Proof-of-Principle Study. Eur. J. Med. Chem. 2017, 127,
944−957.
(29) Ishiyama, T.; Murata, M.; Miyaura, N. Palladium(0)-Catalyzed
Cross-Coupling Reaction of Alkoxydiboron with Haloarenes: A Direct
Procedure for Arylboronic Esters. J. Org. Chem. 1995, 60, 7508−7510.
(30) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and Computational Approaches to Estimate Solubility
and Permeability in Drug Discovery and Development Settings1PII of
Original Article: S0169-409X(96)00423-1. The Article Was Originally
Published in Advanced Drug Delivery Reviews 23 (1997) 3. Adv.
Drug Delivery Rev. 2001, 46, 3−26.
(31) Navia, M. A.; Chaturvedi, P. R. Design Principles for Orally
Bioavailable Drugs. Drug Discovery Today; Elsevier Current Trends,
May 1, 1996; pp 179−189.
(32) Zhu, D.-W.; Lee, X.; Breton, R.; Ghosh, D.; Pangborn, W.;
Duax, W. L.; Lin, S.-X. Crystallization and Preliminary X-Ray
Diffraction Analysis of the Complex of Human Placental 17β-
Hydroxysteroid Dehydrogenase with NADP+. J. Mol. Biol. 1993, 234,
242−244.
(33) Sam, K. M.; Boivin, R. P.; Tremblay, M. R.; Auger, S.; Poirier,
D. C16 and C17 Derivatives of Estradiol as Inhibitors of 17 Beta-
Hydroxysteroid Dehydrogenase Type 1: Chemical Synthesis and
Structure-Activity Relationships. Drug Des. Discovery 1998, 15, 157−
180.
(34) Kruchten, P.; Werth, R.; Marchais-Oberwinkler, S.; Frotscher,
M.; Hartmann, R. W. Development of a Biological Screening System
for the Evaluation of Highly Active and Selective 17β-HSD1-
Inhibitors as Potential Therapeutic Agents. Mol. Cell. Endocrinol.
2009, 301, 154−157.
(35) Emmerich, J.; van Koppen, C. J.; Burkhart, J. L.; Hu, Q.;
̈
Siebenburger, L.; Boerger, C.; Scheuer, C.; Laschke, M. W.; Menger,
M. D.; Hartmann, R. W. Lead Optimization Generates CYP11B1
Inhibitors of Pyridylmethyl Isoxazole Type with Improved Pharmaco-
logical Profile for the Treatment of Cushing’s Disease. J. Med. Chem.
2017, 60, 5086−5098.
(36) Lu, C.; Kirsch, B.; Maurer, C. K.; de Jong, J. C.; Braunshausen,
A.; Steinbach, A.; Hartmann, R. W. Optimization of Anti-Virulence
PqsR Antagonists Regarding Aqueous Solubility and Biological
Properties Resulting in New Insights in Structure−activity Relation-
ships. Eur. J. Med. Chem. 2014, 79, 173−183.
(37) Gargano, E. M.; Perspicace, E.; Hanke, N.; Carotti, A.;
Marchais-Oberwinkler, S.; Hartmann, R. W. Metabolic Stability
Optimization and Metabolite Identification of 2,5-Thiophene Amide
̈
Negri, M.; Meyer, A.; Odermatt, A.; Moller, G.; Adamski, J.;
Hartmann, R. W. Structural Optimization of 2,5-Thiophene Amides
as Highly Potent and Selective 17β-Hydroxysteroid Dehydrogenase
Type 2 Inhibitors for the Treatment of Osteoporosis. J. Med. Chem.
2013, 56, 167−181.
(14) Al-Soud, Y. A.; Marchais-Oberwinkler, S.; Frotscher, M.;
Hartmann, R. W. Synthesis and Biological Evaluation of Phenyl
Substituted 1H-1,2,4-Triazoles as Non-Steroidal Inhibitors of 17β-
Hydroxysteroid Dehydrogenase Type 2. Arch. Pharm. 2012, 345,
610−621.
(15) Wetzel, M.; Gargano, E. M.; Hinsberger, S.; Marchais-
Oberwinkler, S.; Hartmann, R. W. Discovery of a New Class of
Bicyclic Substituted Hydroxyphenylmethanones as 17β-Hydroxyste-
roid Dehydrogenase Type 2 (17β-HSD2) Inhibitors for the
Treatment of Osteoporosis. Eur. J. Med. Chem. 2012, 47, 1−17.
̈
(16) Wetzel, M.; Marchais-Oberwinkler, S.; Perspicace, E.; Moller,
G.; Adamski, J.; Hartmann, R. W. Introduction of an Electron
Withdrawing Group on the Hydroxyphenylnaphthol Scaffold
Improves the Potency of 17β-Hydroxysteroid Dehydrogenase Type
2 (17β-HSD2) Inhibitors. J. Med. Chem. 2011, 54, 7547−7557.
(17) Wetzel, M.; Marchais-Oberwinkler, S.; Hartmann, R. W. 17β-
HSD2 Inhibitors for the Treatment of Osteoporosis: Identification of
a Promising Scaffold. Bioorg. Med. Chem. 2011, 19, 807−815.
(18) Xu, K.; Wetzel, M.; Hartmann, R. W.; Marchais-Oberwinkler, S.
Synthesis and Biological Evaluation of Spiro-δ-Lactones as Inhibitors
of 17β-Hydroxysteroid Dehydrogenase Type 2 (17β-HSD2). Lett.
Drug Des. Discovery 2011, 8, 406−421.
(19) Miralinaghi, P.; Schmitt, C.; Hartmann, R. W.; Frotscher, M.;
Engel, M. 6-Hydroxybenzothiophene Ketones: Potent Inhibitors of
17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1) Owing to
Favorable Molecule Geometry and Conformational Preorganization.
ChemMedChem 2014, 9, 2294−2308.
(20) Spadaro, A.; Negri, M.; Marchais-Oberwinkler, S.; Bey, E.;
Frotscher, M. Hydroxybenzothiazoles as New Nonsteroidal Inhibitors
of 17b-Hydroxysteroid Dehydrogenase Type 1 (17b- HSD1). PLoS
One 2012, 7, No. e29252.
(21) Henn, C.; Einspanier, A.; Marchais-Oberwinkler, S.; Frotscher,
M.; Hartmann, R. W. Lead Optimization of 17β-HSD1 Inhibitors of
the (Hydroxyphenyl)Naphthol Sulfonamide Type for the Treatment
of Endometriosis. J. Med. Chem. 2012, 55, 3307−3318.
(22) Spadaro, A.; Frotscher, M.; Hartmann, R. W. Optimization of
Hydroxybenzothiazoles as Novel Potent and Selective Inhibitors of
17β-HSD1. J. Med. Chem. 2012, 55, 2469−2473.
(23) Marchais-Oberwinkler, S.; Wetzel, M.; Ziegler, E.; Kruchten, P.;
Werth, R.; Henn, C.; Hartmann, R. W.; Frotscher, M. New Drug-like
N
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
17β-Hydroxysteroid Dehydrogenase Type 2 Inhibitors. Eur. J. Med.
Chem. 2014, 87, 203−219.
O
DOI: 10.1021/acs.jmedchem.8b01373
J. Med. Chem. XXXX, XXX, XXX−XXX