Introduction of an electron withdrawing group on the hydroxyphenylnaphthol scaffold improves the potency of 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) inhibitors

Article abstract of DOI:10.1021/jm2008453

Estrogen deficiency in postmenopausal women or elderly men is often associated with the skeletal disease osteoporosis. The supplementation of estradiol (E2) in osteoporotic patients is known to prevent bone fracture but cannot be administered because of a

Full text of DOI:10.1021/jm2008453

Article
pubs.acs.org/jmc
Introduction of an Electron Withdrawing Group on the
Hydroxyphenylnaphthol Scaffold Improves the Potency of
17β-Hydroxysteroid Dehydrogenase Type 2 (17β-HSD2) Inhibitors
Marie Wetzel,† Sandrine Marchais-Oberwinkler,† Enrico Perspicace,† Gabriele Moller,§ Jerzy Adamski,§,∥
̈
and Rolf W. Hartmann*,†,‡
‡
†Pharmaceutical and Medicinal Chemistry, Saarland University, and Helmholtz Institute for Pharmaceutical Sciences Saarland
(HIPS), Campus C2 , D-66041 Saarbrucken, Germany
̈
3
§Genome Analysis Center, Institute of Experimental Genetic, Helmholtz Zentrum Munchen, 85764 Neuherberg, Germany
̈
∥Lehrstuhl fur Experimentelle Genetik, Technische Universitat Munchen, 85350 Freising-Weihenstephan, Germany
̈
̈
̈
S
* Supporting Information
ABSTRACT: Estrogen deficiency in postmenopausal women or elderly men is often associated
with the skeletal disease osteoporosis. The supplementation of estradiol (E2) in osteoporotic
patients is known to prevent bone fracture but cannot be administered because of adverse effect.
As 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) oxidizes E2 to its inactive form
estrone (E1) and has been found in osteoblastic cells, it is an attractive target for the treatment
of osteoporosis. Twenty-one novel, naphthalene-derived compounds have been synthesized and
evaluated for their 17β-HSD2 inhibition and their selectivity toward 17β-HSD1 and the
estrogen receptors (ERs) α and β. Compound 19 turned out to be the most potent and
selective inhibitor of 17β-HSD2 in cell-free assays and had a very good cellular activity in MDA-
MB-231 cells, expressing naturally 17β-HSD2. It also showed marked inhibition of the E1-
formation by the rat and mouse orthologous enzymes and strong inhibition of monkey 17β-
HSD2. It is thus an appropriate candidate to be further evaluated in a disease-oriented model.
endometrium and to a lesser extend also in kidney, pancreas,
INTRODUCTION
■
colon, uterus, breast, and prostate. It was also found in osteo-
17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2)
catalyzes the conversion of the biologically active sex steroids
estradiol (E2) and testosterone (T) into their inactive forms
using NAD+ as cofactor (Chart 1). It is a transmembrane
blastic cells.1−4
In bone physiology, osteoblastic cells5 are responsible for
bone formation and mineralization and osteoclastic cells (OCs)
for bone resorption. The subtle remodeling balance maintained
between the OB and OC activities is crucial for bone stability.
Osteoporosis6 is a systemic skeletal disease characterized by
low bone mass and deterioration of bone tissue. Symptoms of
this disease include bone fragility6 and increase in fractures,6
often at the hips, spine, and wrist. Osteoporosis occurs
especially in postmenopausal women and elderly men, when
the levels in active sex steroids (E2 and T)7 drop.
Chart 1. Interconversion of Estradiol (E2) to Estrone (E1)
by 17β-HSD2, 17β-HSD4, and 17β-HSD1 and of Testo-
sterone (T) to Androstenedione (A-dione) by 17β-HSD2,
17β-HSD4, 17β-HSD3, and 17β-HSD5
Only few drugs are efficient for the treatment of
osteoporosis. Among them, the antiresorptive agent bisphos-
phonate alendronate8 is the most potent drug but the risk of
fractures in postmenopausal women8−10 and elderly men11 is
only reduced by 50%. Estrogens stop bone loss in osteoporotic
patients but are no longer used because of several nonskeletal
effects such as an increased risk of deep vein thrombosis,
pulmonary embolism, and breast cancer.12 Furthermore, low
dose of synthetic estrogens, like ethinylestradiol, has no practical
protein,1 and no crystal structure is available yet. This enzyme
is abundantly expressed in placenta, liver, small intestine, and
Received: June 28, 2011
Published: October 6, 2011
© 2011 American Chemical Society
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Journal of Medicinal Chemistry
Article
application. Raloxifene,13 a selective estrogen receptor modu-
lator (SERM) is frequently used to treat osteoporosis. Draw-
backs of this therapy are the increased risks of thromboembolism,14
hot flushes,15 and leg cramps.15 Therefore, there is a need to
develop novel drugs that are more efficient and selective for
osteoporosis.
Table 1. Inhibition of Human 17β-HSD2 and 17β-HSD1 by
Compounds 1−21
Estrogens are known to play a key role in bone physiology.
They act directly on OBs, which regulate the ability of the OC
precursors to differentiate to OCs (osteoclastogenesis). The
mode of action of estrogens on bone mass is not yet well
understood. 17β-HSD2, which is expressed in OBs, can be
considered as a molecular switch, as it is involved in the
regulation of the level of active E2 and T in the target cell.
Therefore, a local intracellular enhancement of E2 and T in
bones should be feasible. This strategy has already been
successfully applied for several enzymes like aromatase,16−18
CYP17,19−22 17β-HSD1,23−35 and 5α-reductase.36−40
Among the few 17β-HSD2 inhibitors described in the litera-
ture41−50 there is only one class of nonsteroidal compounds:46−48
the cis-pyrrolidinones. By application of a nonsteroidal inhibitor
from this class in a monkey osteoporosis model, 17β-HSD2 has
been validated as a target for the treatment of osteoporosis by
Bagi et al.51 Treatment with this compound led to a slight
decrease in bone resorption and increase in bone formation
resulting in maintenance of bone balance and bone strength.50
As only small effects were observed, there is a need to develop
new potent 17β-HSD2 inhibitors with better in vivo efficacy.
Additionally, these compounds should show a high selectivity
toward 17β-HSD1, the isoenzyme catalyzing the reverse reac-
tion, the reduction of estrone (E1) to E2.
inhibition of
17β-HSD2 (%) 17β-HSD1
inhibition of
a
bd
,
c
compd
spiro-δ-
R1
R2
at 1 μM
(%) at 1 μM log P
68
ni
lactone
A
H
H
H
74
20
25
65
79
37
11
49
36
69
84
32
87
51
78
26
83
69
84
44
98
74
45
20
28
20
25
48
15
10
45
24
31
47
41
36
66
62
73
74
73
52
69
60
43
ni
3.93
B
CH3
1
CH3 2′-CH3
4.68
4.41
4.41
4.06
3.54
4.06
3.80
3.54
5.60
4.35
4.09
4.35
4.09
4.51
4.24
4.24
4.85
5.11
4.85
4.49
3.96
2
H
H
2′-CH3
3′-CH3
3
4
CH3 2′-OCH3
2′-OH
CH3 3′-OCH3
5
H
6
7
H
H
H
3′-OCH3
3′-OH
8
9
3′-phenyl
10
11
12
13
14
15
16
17
18
19
20
21
CH3 2′-F
2′-F
CH3 3′-F
3′-F
CH3 2′,5′-diF
H
H
In a previous work,52 focusing on the design of new nonsteroidal
17β-HSD2 inhibitors, 6-(4′-hydroxyphenyl)-1-naphthol (com-
pound A, Table 1) has been identified as a promising scaffold
with an IC50 of 302 nM for 17β-HSD2 and a selectivity factor (SF)
of 8 toward 17β-HSD1. This hit needed to be improved regarding
activity and selectivity. According to our hypothesis,52 the OH
substituted benzene ring of the naphthalene moiety in A mimics
the A-ring of E2 and the OH-phenyl the D-ring, leading to a close
binding of the OH-phenyl to the catalytic triad and the cofactor. As
the substrate of one enzyme (17β-HSD2 in this case) is the
product of the catalytic reaction of the other enzyme (17β-HSD1
in this case), it can be concluded that their active sites must be very
similar too. Therefore, with analyzing 17β-HSD1 from which in
contrast to 17β-HSD2 the crystal structure is known, it becomes
apparent that there is some space available around the OH-phenyl
for substituents.
In this work, first the hypothesis concerning the binding
mode of hydroxyphenyl naphthol derivatives to 17β-HSD2 was
validated. Afterward, the available space for the inhibitors in the
active site of the enzyme was explored by synthesis of several
derivatives where the size of substituents at positions 2′ and 3′
of the hydroxyphenyl ring was varied. With the expectation that
electronic effects play an important role, electron donating
(EDG) and electron withdrawing (EWG) groups were also
introduced into the OH-phenyl moiety.
H
H
H
2′,5′-diF
2′,6′-diF
2′-CF3
CH3 3′-CF3
H
H
H
3′-CF3
3′-Cl
3′-CN
a
Human placenta, microsomal fraction, substrate [3H]E2 + E2 [500
nM], NAD+ [1500 μM], mean value of three determinations, relative
b
standard deviation of <10%. Human placenta, cytosolic fraction,
substrate [3H]E1 + E1 [500 nM], NADH [500 μM], mean value of
c
three determinations, relative standard deviation of <10%. Calculated
log P data. ni: no inhibition (inhibition of <10%).
d
(method A or B). Ether cleavage was performed using boron
tribromide for compounds 2, 7, and 13 (method C); boron
trifluoride dimethylsulfide complex for compounds 3, 5, and 15
(method D); and pyridinium hydrochloride for compounds 11
and 19 (method E).
Compound 9 bearing an additional phenyl ring on the
hydroxyphenyl moiety was synthesized according to the route
described in Scheme 2. Compound 1a was first methylated
using methyl iodide,54 and subsequently the triflate group was
exchanged by a boronic ester group (intermediate 9c) with
bis(neopentylglycolato)diboron and a crown ether under basic
conditions according to the method of Xu et al.55 In parallel,
compound 9b was synthesized by a Suzuki coupling with
4-bromo-2-iodo-1-methoxybenzene 9e and phenylboronic acid.
Subsequent Suzuki coupling (method B) between compounds
9c and 9b led to compound 9a. Demethylation using boron
tribromide (method C) afforded compound 9.
CHEMISTRY
■
Synthesis of 1−8 and 10−20 (Scheme 1) started from the
commercially available 6-methoxy-1-tetralone which was
triflated and aromatized in 1a in very good yield according to
the method described by us.52 The cross-coupling reaction53 of
the triflate 1a with the appropriate methoxyphenylboronic acid
led to compounds 1, 3a, 4, 6, 8, 10, 12, 14, 16−18, and 20
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Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Compounds 1−8 and 10−20
a
Reagents and conditions: (a) boronic acid, Pd(PPh3)4, aq Na2CO3, DME/EtOH, 90 °C, 2 h for 1, 3a, 4, 6, 12, and 17, method A; (b) boronic acid,
Pd(PPh3)4, Cs2CO3, DME/water, microwave irradiation (150 °C, 150 W, 25 min) for 8, 10, 14, 16, 18, and 20, method B; (c) BBr3, CH2Cl2, −78
°C to room temp, overnight, for 2, 7, and 13, method C; (d) BF3·SMe2, CH2Cl2, room temp, overnight, for 3, 5, and 15, method D; (e) pyridinium
hydrochloride, 220 °C, 3 h for 11 and 19, method E.
a
Scheme 2. Synthesis of Compounds 9 and 21
a
Reagents and conditions: (a) MeI, K2CO3, 18-crown-6, acetone, reflux, overnight; (b) bis(neopentylglycolato)diboron, PdCl2(dppf), dppf, KOAc,
1,4-dioxane, 85 °C, 4 h; (c) PhI(OAc)2, I2, EtOAc, 60 °C, dark conditions, 4 h; (d) boronic acid, Pd(PPh3)4, Cs2CO3, DME/water, microwave
irradiation (150 °C, 150 W, 25 min), method B; (e) BBr3, CH2Cl2, −78 °C to room temp, overnight, method C; (f) boronic acid, Pd(PPh3)4, aq
Na2CO3, DME/EtOH, 90 °C, 2 h, method A; (g) BF3·SMe2, CH2Cl2, room temp, overnight, method D.
7549
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Article
The synthesis of 21 was carried out by palladium-catalyzed
Suzuki coupling reaction using the intermediate 9c (method A)
and the commercially available 5-bromo-2-hydroxybenzonitrile,
followed by ether cleavage with boron trifluoride dimethyl-
sulfide complex (method D, Scheme 2).
small substituent in the 2′- or 3′-position, but the methyl is certainly
not able to achieve any specific interactions, as no gain in activity is
observed compared to the parent hydroxyphenylnaphthol A.
Exchange of the methyl group for a hydrophilic hydroxy sub-
stituent was detrimental for activity in the 2′-position (5, inactive)
but tolerated in the 3′-position (8, similar activity as the
unsubstituted A). It can therefore be hypothesized that there is
no polar amino acid in the active site close to the 2′-position of
the inhibitor. Furthermore, the 3′-OH group of compound 8
did not show specific interactions with the enzyme, as no
increase in activity was observed compared to A. It is striking that
an exchange of hydroxy (8) by methoxy (7) in the 3′-position
decreased the inhibitory activity strongly.
BIOLOGICAL CHARACTERIZATION
■
Inhibition of Human 17β-HSD2. 17β-HSD2 inhibitory
activity of the synthesized compounds was first evaluated in a
cell-free assay. Human placental microsomal enzyme was used
as source. The incubation was performed with tritiated E2,
cofactor, and inhibitor. The separation of substrate and product
was accomplished by HPLC with a subsequent quantifica-
tion by peak integration.56 The percent inhibition values of
compounds 1−21 are shown in Table 1, and the IC50 values of
the most active compounds are reported in Table 2.
A phenyl ring was also introduced into the 3′-position (9).
This compound displayed a slight increase in activity (84%
inhibition at 1 μM for 9 vs 74% inhibition at 1 μM for A),
confirming our previous hypothesis that there is space available
around the hydroxyphenyl core.
Table 2. IC50, Selectivity Factor, and Binding Affinities for
In order to evaluate the influence of electron withdrawing
groups (EWGs) or electron donating groups (EDGs) on the
17β-HSD2 inhibitory activity, compounds 10−21 bearing
EWGs were synthesized to be compared to compounds with
EDGs like methyl (3) or phenyl (9) groups. All methoxy
derivatives (10, 12, 14, and 18) showed lower activity than the
corresponding hydroxy compounds (11, 13, 15, and 19). Most
of the hydroxy compounds exhibited identical or better potency
than the parent compound A except 21. Compound 11,
substituted with a fluoro group in the 2′- position, turned out to
be highly active (87% inhibition at 1 μM, IC50 = 132 nM).
Disubstituted fluorine compounds (2′,5′-diF 15 and 2′,6′-diF
16) resulted in highly active inhibitors but were slightly weaker
than the monofluoro 11 (IC50 of 238 nM for 15 and 132 nM
for 11). Compounds 17 (R2 = 2′-CF3) and 19 (R2 = 3′-CF3)
showed high (17) and very high (19) activity (84% and 98%
inhibition at 1 μM, respectively), resulting in IC50 values of
19 nM for 19 and 156 nM for 17. However, exchange of the
trifluoromethyl group for bioisosteric chloro (20) or cyano
(21) groups was detrimental for the activity (74% and 45%
inhibition for 20 and 21, respectively) when compared with
inhibitory efficiency of 19 (98% at 1 μM).
the Estrogen Receptors α and β for Selected Compounds
cell-free assay
de
,
de
,
17β-HSD2 17β-HSD1 selectivity ERα RBA
be ce
ERβ RBA
a
,
,
compd IC50 (nM) IC50 (nM) factor
(%)
(%)
spiro-δ-
lactone
34
nd
nd
nd
nd
A
302
275
261
132
292
238
156
19
2425
1748
919
8
5
5
3
6
<0.1
<0.1
<0.1
0.1−1.0
<0.1
9
4
11
13
15
17
19
20
2245
713
17
2
<0.1
0.1−1.0
0.1−1.0
1.0−10
<0.1
0.1−1.0
0.1−1.0
1.0−10
0.1−1.0
0.1−1.0
309
1
926
6
611
32
307
1130
4
0.1−1.0
a
Human placenta, microsomal fraction, substrate [3H]E2 + E2 [500
nM], cofactor NAD+ [1500 μM], mean value of three determinations,
b
relative standard deviation of <10%. Human placenta, cytosolic
fraction, substrate [3H]E1 + E1 [500 nM], cofactor NADH [500 μM],
mean value of three determinations, relative standard deviation of
c
d
<20%. IC50(17β-HSD1)/IC50 (17β-HSD2). RBA: relative binding
e
affinity. E2: 100%. nd: not determined.
Selectivity and Cellular Activity. 17β-HSD1, as the main
enzyme implicated in the reduction of estrone (E1) to E2 in sex
steroid metabolism,57 should not be affected by inhibitors of
17β-HSD2. Furthermore, inhibitors of 17β-HSD2 should have
none or little affinity for the estrogen receptors α and β (ERα
and ERβ), as most of the E2 effects are ER mediated. There-
fore, all compounds were tested for selectivity toward 17β-
HSD1 and a selection of the most potent inhibitors for binding
affinity to ERα and β.
The assay for 17β-HSD1 inhibition was performed in a
similar way as for the 17β-HSD2. Human placental cytosolic
17β-HSD1 was incubated with tritiated E1, cofactor, and
inhibitor. The amount of labeled E2 formed was determined
after HPLC separation. For the most potent compounds, IC50
values were determined and selectivity factors (SFs) calculated
(SF = IC50(17β-HSD1)/IC50(17β-HSD2), Table 2). All com-
pounds except 15 showed selectivity toward 17β-HSD1. The
most selective one, with a selectivity factor of 32, was com-
pound 19 (R2 = 3′-CF3), which was also the most potent one
exhibiting an IC50 of 19 nM. The 2′-F compound 11 also
showed a high SF of 17 toward 17β-HSD1.
Compounds showing less than 10% inhibition at 1 μM were
considered to be inactive. Compound A, identified in a previous
work,52 was used as internal reference (74% 17β-HSD2 inhibi-
tion vs 20% 17β-HSD1 inhibition at 1 μM), and a spiro-δ-lactone
(compound 10 published by Poirier et al.43) was taken as external
control (68% at 1 μM in our test; 62−66% at 1 μM in their test43).
For selected substituents (R2 = 2′-CH3, 2′-OCH3, 3′-OCH3,
2′-F, 3′-F, 2′,5′-diF, 3′-CF3), both hydroxyphenyl 2, 5, 8, 11, 13,
15, 19 and corresponding methoxyphenyl derivatives 1, 4, 6,
10, 12, 14, 18 were synthesized and tested for 17β-HSD2
inhibitory activity. As observed for the unsubstituted compound
A, the hydroxylated molecules (2, 8, 11, 13, 15, and 19) were
always more potent than the respective methoxylated analogues
except for the compound pair 4 (R2 = 2′-OCH3) and 5. For
compounds 3, 9, 16, 17, 20, and 21, the hydroxylated
derivatives only were therefore tested.
Introduction of a lipophilic group like methyl (compounds 2 and
3) led to good 17β-HSD2 inhibitory activity (65% inhibi-
tion for 2 and 79% for 3 when tested at 1 μM), which was at the
level of unsubstituted A (74% inhibition at 1 μM). This indicates that
there is space around the hydroxyphenyl moiety for introduction of a
For the determination of ER binding affinity, a competition
assay was used, with tritiated E2. Receptor bound and free E2
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Journal of Medicinal Chemistry
Article
were separated by means of hydroxyapatite. Compounds 3, 9,
11, and 19 showed a relative binding affinity (RBA) of less than
0.1% to ERα, compared to the affinity of E2, which is arbitrarily
set to 100%. The indicated compounds are therefore classified
as low affinity ligands of the ERα.
Table 3. Inhibition of Rat, Mouse, and Monkey E1 and E2-
Formation by Compound 19
inhibition of E1-
inhibition of E2-
ak
,
bk
,
formation (%)
formation (%)
compd 19
1 μM
100 nM
1 μM
100 nM
The log P values were calculated using the ChemDrawPro,
version 11.0, program in order to get insight into the lipo-
philicity of this class of compounds and are shown in Table 1.
The values for all compounds except 9 and 18 are lower than 5,
fulfilling the criteria of Lipinski’s rule of five. It therefore can be
expected that they are able to easily permeate cell membranes.
The most interesting compounds 11, 17, and 19 with IC50
values below 200 nM were further investigated in a cellular assay,
using the human breast cancer cell line MDA-MB-231,58 which
endogenously expresses 17β-HSD2. Cells were incubated with
tritiated E2 and inhibitor for 3.5 h. Separation was performed by
HPLC similar as in the cell-free inhibition assays. Compounds
17 and 19 showed very similar inhibitory activity in MDA-MB-
231 cells as in the cell-free assay, i.e., IC50 of 171 nM for 17 and
31 nM for 19 in cellular assay vs 156 and 19 nM, respectively, in
the cell-free assay. Interestingly, compound 11 showed an IC50
of 1227 nM in the cellular assay compared to 132 nM in the cell-
free assay, indicating that this compound is less appropriate to
permeate the cell membrane or is metabolically unstable.
Further in Vitro Assays. In order to refine the profile of
the most potent compound 19, it was further tested for
selectivity on other enzymes, namely, 17β-HSD types 4 and 5.
17β-HSD4 catalyzes the same reaction as 17β-HSD2 (Chart 1),57
and 17β-HSD5 (beside 17β-HSD3) is responsible for the
reduction of the androgen A-dione to T.57 Both enzymes
should not be inhibited by 17β-HSD2 inhibitors. To investigate
this important issue, experiments were performed with E. coli
bacteria expressing human 17β-HSD4 and 17β-HSD5.56,59
These cells were incubated with cofactor, inhibitor, and tritiated
E2 in the case of 17β-HSD4 and with A-dione in the case of
17β-HSD5. After RP-HPLC separation of substrate and
product, the amount of labeled E1 or T formed was quantified.
Compound 19 was found to be selective toward 17β-HSD4 and
17β-HSD5 (40% 17β-HSD4 inhibition and 21% 17β-HSD5
inhibition at 1 μM).
According to the potency of compound 19 in cell-free and
cellular assays and its selectivity toward 17β-HSD1, 17β-HSD4,
17β-HSD5, and ERs, it can be considered as potential candidate
for further development. Before evaluation in an animal
model of the efficacy of this new 17β-HSD2 inhibitor 19, it is
reasonable to determine its profile on orthologous enzymes from
different species. Compound 19 was therefore tested for inhibition
of 17β-HSD2/E1-formation (activity) and 17β-HSD1/E2-for-
mation (selectivity) using rat, mouse, and monkey enzymes.
In rat and mouse assays, liver enzymes were used, while in
monkey assay, placental enzymes were employed. Compound 19
showed good inhibition of the rat and mouse 17β-HSD2 enzymes
and very good inhibition on the monkey enzyme. In this case, the
efficacy was in the same range as for the human enzyme (Table 3).
In addition, selectivity toward 17β-HSD1/E2-formation in
monkey seems to be similar to that observed in human.
c
e
g
i
c
d
d
human
rat
98
77
72
99
87
60
52
26
75
27
f
nd
nd
nd
nd
h
j
mouse
i
j
monkey
75
55
a
Substrate [3H]E2 + E2 [500 nM], NAD+ [1500 μM], mean value of
b
three determinations, relative standard deviation of <10%. Substrate
[3H]E1 + E1 [500 nM], NADH [500 μM], mean value of three
c
determinations, relative standard deviation of <10%. Human placenta,
d
e
microsomal fraction. Human placenta, cytosolic fraction. Rat liver,
f
g
microsomal fraction. Rat liver, cytosolic fraction. Mouse liver,
h
i
microsomal fraction. Mouse liver, cytosolic fraction. Monkey
placenta, microsomal fraction. Monkey placenta, cytosolic fraction.
j
k
nd: not determined.
moiety by a sulfonamide did not affect the activity toward 17β-
HSD2 (74% vs 75% inhibition at 1 μM, respectively), indicating
that some space is available around the phenyl core. We
hypothesized therefore that the phenyl ring mimics the D-ring
of E2 and thus that introduction of additional substituents on
this phenyl moiety would be tolerated and would allow an
increase of the 17β-HSD2 inhibitory activity. In this paper, we
aimed at verifying whether this hypothesis is correct and
whether introduction of various substituents on the phenyl ring
could improve this activity.
The biological activity of the phenyl-substituted compound 9
confirms that the compound can indeed bind in the active site
according to the previous hypothesis (the hydroxynaphthalene
mimics the A-ring and the hydroxyphenyl the D-ring). The
additional phenyl might be accommodated between cofactor
and steroid where space is available.
The electronic effects of the substituents on the 17β-HSD2
inhibitory activity were further elucidated. No matter where
they were introduced, EDGs (Me, OH, Ph) in either the 2′- or
3′-position did not lead to significant differences in activity
except for the hydroxy group where position 3′ was better
(compounds 5 and 8). None of the synthesized compounds
(2, 3, 8, and 9) reached a better activity compared to reference
A, indicating that these substituents are tolerated (there is space
available) but not able to establish specific interactions with
amino acid residues. The position of the substituent on the
phenyl ring will induce either a pronounced rotation of the
phenyl toward the naphthalene (position 2′, ortho effect) or a
weak rotation (position 3′). In both cases, only the 4′-hydroxy
substituent stays at the same position and anchors the molecule
into its binding site. Our finding that there is no difference in
activity between 2′- and 3′-substituted compounds indicates
that the phenyl ring is not able to establish π−π stacking
interactions with amino acid side chains of the protein.
When the substituent was an EWG (F, CF3, Cl, CN), the
highest potency was observed for compound 19 (R2 = 3′-CF3)
in which the additional group was located at the 3′-position.
Exchange of the trifluoromethyl group by the bioisosteric
groups like chloro (20) and cyano (21) decreased inhibition. In
the case of the CN group, the different activities might be due
to the fact that CN is hydrophilic whereas CF3 is lipophilic. The
linear shape of CN giving steric hindrance might also explain
the moderate activity of 21.
DISCUSSION AND CONCLUSION
■
Compound A, identified by us in a previous study52 represents
a promising scaffold as 17β-HSD2 inhibitor: substituted by two
hydroxy groups (polar), the naphthalene and phenyl cores
(hydrophobic) mimick the substrate E2. We have previously
shown52 that an exchange of the hydroxy group on the phenyl
7551
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As shown in many other cases5 and also observed for
compound 11, the substitution with F results in an increase in
inhibitory activity. However, the introduction of a second
fluorine as in 15 or 16 does not further enhance activity,
indicating that the effects are not additive.
that 17β-HSD2 inhibitors are able to inhibit osteoclastogenesis
and induce bone formation.
EXPERIMENTAL SECTION
■
Chemical Methods. 1H NMR and 13C NMR spectra were
recorded on a Bruker DRX-500 instrument in CDCl3 and acetone-d6.
Chemicals shifts are reported in δ values (ppm). The hydrogenated
residues of deuteriated solvent were used as internal standard (CDCl3,
δ = 7.26 ppm in 1H NMR and δ = 77.0 ppm in 13C NMR; acetone-d6,
In our previous study,52 it was observed that the hydroxy
group bound to the phenyl moiety was important for activity
(74% at 1 μM for A with OH vs 20% inhibition for B with
OMe). This is confirmed in this study, as all methoxy
compounds displayed lower 17β-HSD2 inhibitory activity
than their corresponding hydroxy analogues. Contrary to our
compounds, the other described nonsteroidal 17β-HSD2
inhibitors,46−48 the cis-pyrrolidinones, do not need any hydro-
philic group to be active, allowing us to presume that they
might have a different binding mode compared to ours.
As other 17β-HSD enzymes of the short-chain dehydrogen-
ase/reductase (SDR) and the aldo−keto reductases (AKR)
family participate in steroid interconversions,60 the develop-
ment of enzyme-specific inhibitors is a challenge. We therefore
tested the compounds on other 17β-HSDs, like 17β-HSD types
1, 2, 4, and 5. Selectivity toward 17β-HSD1 was low with nearly
all of the tested hydroxyphenylnaphthol derivatives; however,
the most potent 17β-HSD2 inhibitors 11 and 19 showed the
highest selectivity toward 17β-HSD1, exhibiting promising
selectivity factors (SFs) of 17 and 32, respectively. Although
some inhibition of 17β-HSD4 and -5 was observed with the
best inhibitor compound 19, the substance can be assumed to
be selective toward both enzymes. We were surprised by the
fact that compound 19 showed some minor inhibition of 17β-
HSD4. In our previous projects we have not seen any effect on
this particular enzyme with steroidal or nonsteroidal inhib-
itors.56,61 Nevertheless, the observed inhibition of compound
19 is fairly low and the impact on the ubiquitously expressed
17β-HSD4 contributing to the peroxisomal β-oxidation of fatty
acids62 is most probably negligible.
1
δ = 2.05 ppm in H NMR and δ = 30.8 ppm and 206.3 ppm in 13C
NMR). Signals are described as s, br, d, t, dd, dt, qt, and m for singlet,
broad, doublet, triplet, doublet of doublets, doublet of triplets, quin-
tuplet, and multiplet, respectively. All coupling constants (J) are given
in Hertz. IR spectra were measured neat on a Bruker Vector 33FT
infrared spectrometer. Melting points (mp) were determined in open
capillaries on a Stuart Scientific SMP3 apparatus and are uncorrected.
Mass spectra (GC/MS) were measured on a GCD series G1800A
(Hewlett-Packard) instrument with an Optima-5-MS (0.25 μM, 30 m)
column (Macherey Nagel). All microwave irradiation experiments
were carried out in a CEM-Discover monomode microwave apparatus.
Flash chromatography was performed on silica gel 40 (35/40 to
63/70 μM) with hexane/ethyl acetate mixtures as eluents and the
reaction progress was determined by thin-layer chromatography
analyses on Alugram SIL G/UV254 (Macherey-Nagel). Visualization
was accomplished with UV light.
Tested compounds are ≥95% chemical purity as measured by LC/
MS. Data for all tested compounds are provided in the Supporting
Information. The Surveyor LC system consisted of a pump, an
autosampler, and a PDA detector. Mass spectrometry was performed
on a MSQ electrospray mass spectrometer (ThermoFisher, Dreieich,
Germany). The system was operated by the standard software
Xcalibur. A RP-C18 NUCLEODUR 100-5 (125 mm × 3 mm) column
(Macherey-Nagel GmbH, Duhren, Germany) was used as stationary
̈
phase. All solvents were HPLC grade. In a gradient run the percentage
of acetonitrile (containing 0.1% trifluoroacetic acid) in 0.1%
trifluoroacetic acid was increased from an initial concentration of 0%
at 0 min to 100% at 15 min and kept at 100% for 5 min. The injection
volume was 15 μL, and flow rate was set to 800 μL/min. MS analysis
was carried out at a spray voltage of 3800 V and a capillary
temperature of 350 °C and a source CID of 10 V. Spectra were
acquired in positive mode from 100 to 1000 m/z at 254 nm for the UV
trace.
Compounds 11 and 19 also displayed very little binding
affinity to ERα, which is responsible for cell proliferation. Only
compound 19 showed a very good cellular activity (IC50
=
31 nM in MDA-MB-231 cells). Thus, it is able to permeate the
cell membrane and is not quickly metabolized.
Starting materials (different boronic acids and compounds 1e, 9f,
and 21b) were purchased from Aldrich, Alfa Aeser, Acros, and Combi-
Blocks and were used without further purification. No attempts were
made to optimize yields.
The following compounds were prepared according to previously
described procedures: 5-hydroxy-2-naphthyl trifluoromethanesulfonate
1a,52 4-bromo-2-iodo-1-methoxybenzene 9e.27
An osteoblastic cell-line expressing 17β-HSD2 and the ERs
like SV-HFO should be promising to prove our concept that
17β-HSD2 inhibition is able to induce bone formation and
inhibit osteoclastogenesis. For further in vivo experiments,
species differences have to be taken into consideration,63,64 and
compound 19 should inhibit the enzyme of the chosen species.
In this pursuit, 19 was tested on rat, mouse, and monkey
enzymes and revealed a high (72−77% inhibition at 1 μM in
mouse and rat enzymes) to very high (99% inhibition at 1 μM
in monkey enzyme) inhibitory activity for 17β-HSD2/E1-
formation. Showing a better activity and selectivity in marmoset
(Callithrix jacchus) enzymes, this species should be appropriate
for proving our concept.
In the present study, we describe the synthesis of substituted
6-phenyl-1-naphthols as inhibitors of 17β-HSD2 and the
evaluation of their biological activities. A new highly potent
inhibitor of 17β-HSD2 has been discovered (compound 19)
with very good cellular activity and good selectivity toward 17β-
HSD1, 17β-HSD4, 17β-HSD5, and ERs. Compound 19 is also
able to inhibit E1-formation in rat, mouse, and monkey. Further
biological analyses still need to be investigated to get
information on the efficacy of the compound to demonstrate
General Procedures for Suzuki Coupling. Method A. To a
mixture of aryl bromide (1 equiv) and tetrakis(triphenylphosphine)-
palladium(0) (0.02 equiv) in DME was added a 2 N aqueous solution
of sodium carbonate (2 equiv), and the mixture (oxygen free) was
purged with N2. The resultant solution was stirred at room tem-
perature for 5 min, and a solution of boronic acid (1.3 equiv) in EtOH
was added. The mixture was heated to 90 °C and stirred for 2 h. The
reaction mixture was cooled to room temperature, quenched by water,
and extracted with ethyl acetate. The organic layer was washed with
brine, dried over sodium sulfate, filtered, and concentrated to dryness.
The product was purified by column chromatography.
Method B. A mixture of aryl bromide (1 equiv), boronic acid
(1 equiv), cesium carbonate (3 equiv), and tetrakis(triphenylphosphine)-
palladium(0) (0.02 equiv) was suspended in an oxygen-free DME/
water (2:1) solution. The reaction mixture was exposed to microwave
irradiation (25 min, 150 W, 150 °C, 15 bar). After the mixture reached
room temperature, water was added and the aqueous layer was
extracted with ethyl acetate. The combined organic layers were washed
with brine, dried over sodium sulfate, filtered, and concentrated to
dryness. The product was purified by column chromatography.
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General Procedures for Ether Cleavage. Method C. To a
solution of methoxy derivative (1 equiv) in dry dichloromethane
cooled at −78 °C under nitrogen was slowly added boron tribromide
(1 M solution in dichloromethane, 5−10 equiv per methoxy function).
The reaction mixture was stirred at −78 °C for 1 h and then allowed
to warm to room temperature overnight. The reaction was quenched
by water, and the aqueous layer was extracted with ethyl acetate. The
combined organic layers were washed with brine and dried over
sodium sulfate, evaporated to dryness under reduced pressure, and
purified by column chromatography.
Method D. To a solution of methoxy derivative (1 equiv) in dry
dichloromethane, boron trifluoride−dimethylsulfide complex (75
equiv per methoxy function) was added dropwise at room temper-
ature. The reaction mixture was stirred at room temperature overnight.
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 sodium sulfate, evaporated to dryness under
reduced pressure, and purified by column chromatography.
Method E. To pyridinium hydrochloride (100 equiv) at 190 °C
was added methoxy derivative (1 equiv), and the solution was stirred
for 3 h. The reaction mixture was cooled to room temperature and was
stirred with 1 N HCl. The mixture was dissolved in ethyl acetate, and
the combined organic layers were washed with water, dried over
sodium sulfate, filtered, evaporated to dryness under reduced pressure,
and purified by column chromatography.
6-(4-Hydroxy-3-methylphenyl)-1-naphthol (3). The title com-
pound was prepared by reaction of 6-(4-methoxy-3-methylphenyl)-1-
naphthol 3a (20 mg, 0.08 mmol, 1 equiv) with boron trifluoride−
dimethylsulfide complex (6.00 mmol, 75 equiv) according to method
D. The product was purified by column chromatography (hexane/
ethyl acetate, 1:1) to give 12 mg (60%) of the analytically pure
compound as a brown solid. C17H14O2; MW 250; mp 164−166 °C;
1H NMR (acetone-d6) δ 8.90 (s, br, 1H), 8.26 (s, br, 1H), 8.23 (d, J ≈
8.5 Hz, 1H), 7.96 (d, J ≈ 1.9 Hz, 1H), 7.69 (dd, J ≈ 2.0 Hz, J ≈ 8.5
Hz, 1H), 7.53 (d, J ≈ 1.9 Hz, 1H), 7.43 (dd, J ≈ 2.0 Hz, J ≈ 8.5 Hz,
1H), 7.40−7.37 (m, 1H), 7.28−7.24 (m, 1H), 6.91 (d, J ≈ 8.5 Hz,
1H), 6.84 (dd, J ≈ 0.8 Hz, J ≈ 7.4 Hz, 1H), 2.27 (s, 3H); 13C NMR
(acetone-d6) δ 156.1, 154.0, 139.6, 136.4, 133.0, 130.4, 127.4, 126.4,
125.6, 124.9, 124.7, 123.5, 120.2, 118.2, 116.0, 108.7, 16.3; IR 3337,
2952, 1506, 1274 cm−1; GC/MS m/z 250 (M)+.
6-(3-Fluoro-4-hydroxyphenyl)-1-naphthol (13). The title
compound was prepared by reaction of 6-(3-fluoro-4-methoxyphenyl)-
1-naphthol 12 (20 mg, 0.08 mmol, 1 equiv) with boron tribromide
(0.80 mmol, 10 equiv) according to method C. The product was
purified by column chromatography (hexane/ethyl acetate, 1:1) to
give 8 mg (40%) of the analytically pure compound as a yellow oil.
1
C16H11O2F; MW 254; H NMR (acetone-d6) δ 8.99 (s, br, 1H), 8.74
(s, br, 1H), 8.29 (d, J ≈ 8.8 Hz, 1H), 8.05 (d, J ≈ 2.0 Hz, 1H), 7.74
(dd, J ≈ 2.1 Hz, J ≈ 8.7 Hz, 1H), 7.57 (dd, J ≈ 2.1 Hz, J ≈ 8.7 Hz,
1H), 7.50−7.47 (m, 1H), 7.44 (d, J ≈ 8.3 Hz, 1H), 7.34−7.30
(m, 1H), 7.15−7.11 (m, 1H), 6.91 (dd, J ≈ 0.9 Hz, J ≈ 7.6 Hz, 1H);
13C NMR (acetone-d6) δ 154.2, 136.3, 127.6, 125.4, 124.4, 124.0,
123.7, 120.3, 119.1, 115.5, 109.0; IR 3247, 1692, 1524, 1277 cm−1
;
GC/MS m/z 254 (M)+.
6-(2, 5-Difluoro-4-hydroxyphenyl)-1-naphthol (15). The title
compound was prepared by reaction of 6-(2,5-difluoro-4-methox-
yphenyl)-1-naphthol 14 (40 mg, 0.14 mmol, 1 equiv) with boron
trifluoride−dimethylsulfide complex (10.5 mmol, 75 equiv) according
to method D. The product was purified by column chromatography
(hexane/ethyl acetate, 8:2) to afford 20 mg (50%) of the analytically
pure product as a beige solid. C16H10F2O2; MW 272; mp 178−181 °C;
1H NMR (acetone-d6) δ 9.22 (s, br, 1H), 9.05 (s, br, 1H), 8.29 (d, J ≈
8.8 Hz, 1H), 7.99−7.97 (m, 1H), 7.63 (dt, J ≈ 1.9 Hz, J ≈ 8.8 Hz,
1H), 7.44 (d, J ≈ 8.4 Hz, 1H), 7.42−7.39 (m, 1H), 7.36−7.32
(m, 1H), 6.96−6.90 (m, 2H); 13C NMR (acetone-d6) δ 162.4, 159.6,
154.4, 153.2, 135.2, 134.5, 126.6, 125.9, 125.1, 123.5, 122.0, 120.8,
117.9, 117.5, 108.9, 104.6; IR 3427, 1641, 1522, 1268 cm−1; GC/MS
m/z 272 (M)+.
6-[4-Hydroxy-2-(trifluoromethyl)phenyl]-1-naphthol (17).
The title compound was prepared by reaction of 5-hydroxy-2-naphthyl
trifluoromethanesulfonate 1a (100 mg, 0.34 mmol, 1 equiv) with
4-hydroxy-2-trifluoromethylphenylboronic acid (98 mg, 0.44 mmol,
1.3 equiv) according to method A. The product was purified by
column chromatography (hexane/ethyl acetate, 8:2) to give 70 mg
(68%) of the analytically pure compound as a brown solid.
C17H11F3O2; MW 304; mp 140−142 °C; 1H NMR (acetone-d6)
δ 9.03 (s, br, 1H), 9.01 (s, br, 1H), 8.25 (d, J ≈ 8.8 Hz, 1H), 7.73
(s, 1H), 7.42−7.39 (m, 2H), 7.36−7.31 (m, 2H), 7.28 (d, J ≈ 2.5 Hz,
1H), 7.18 (dd, J ≈ 2.5 Hz, J ≈ 8.6 Hz, 1H), 6.96−6.93 (m, 1H); 13C
NMR (acetone-d6) δ 157.9, 154.3, 138.8, 135.4, 134.9, 128.7, 127.8,
127.2, 125.0, 122.6, 120.4, 119.4, 113.7, 109.5, 60.5; IR 3361, 1691,
1322 cm−1; GC/MS m/z 304 (M)+.
6-[4-Hydroxy-3-(trifluoromethyl)phenyl]-1-naphthol (19).
The title compound was prepared by reaction of 6-[4-methoxy-3-
(trifluoromethyl)phenyl]-1-naphthol 18 (100 mg, 0.15 mmol, 1 equiv)
with pyridinium hydrochloride (15 mmol, 100 equiv) according to
method E. The product was purified by column chromatography
(hexane/ethyl acetate, 7:3) to afford 40 mg (42%) of the analytically
pure product as a brown solid. C17H11F3O2; MW 304; mp 158−160
°C; 1H NMR (acetone-d6) δ 8.28 (d, J ≈ 8.5 Hz, 1H), 8.04 (d, J ≈ 2.0
Hz, 1H), 7.91 (d, J ≈ 2.0 Hz, 1H), 7.85 (dd, J ≈ 2.0 Hz, J ≈ 8.5 Hz,
1H), 7.71 (dd, J ≈ 2.0 Hz, J ≈ 8.5 Hz, 1H), 7.42 (d, J ≈ 8.5 Hz, 1H),
7.29 (m, 1H), 7.22−7.19 (m, 1H), 6.89 (dd, J ≈ 0.9 Hz, J ≈ 7.3 Hz,
1H); 13C NMR (acetone-d6) δ 156.3, 154.1, 138.0, 136.3, 133.0, 127.7,
125.5, 124.9, 124.4, 123.9, 120.2, 118.6, 109.1; IR 3229, 3066, 2986,
1619, 1263 cm−1; GC/MS m/z 304 (M)+.
5-(5-Hydroxy-2-naphthyl)biphenyl-2-ol (9). The title compound
was prepared by reaction of 2-methoxy-5-(5-methoxy-2-naphthyl)-
biphenyl 9a (75 mg, 0.22 mmol, 1 equiv) with boron tribromide (2.2
mmol, 10 equiv) according to method C. The product was purified by
column chromatography (hexane/ethyl acetate, 8:2), then by
preparative HPLC (gradient acetonitrile/water from 20:80 to 100:0)
to give 10 mg (15%) of the analytically pure compound as an orange
oil. C22H16O2; MW 312; 1H NMR (acetone-d6) δ 8.16 (d, J ≈ 8.5 Hz,
1H), 7.96 (d, J ≈ 2.0 Hz, 1H), 7.67 (dd, J ≈ 2.0 Hz, J ≈ 8.5 Hz, 1H),
7.61 (d, J ≈ 2.0 Hz, 1H), 7.59−7.55 (m, 2H), 7.51 (dd, J ≈ 2.0 Hz,
J ≈ 8.5 Hz, 1H), 7.32−7.28 (m, 3H), 7.22−7.16 (m, 2H), 7.00
(d, J ≈ 8.5 Hz, 1H), 6.76 (d, J ≈ 7.6 Hz, 1H); 13C NMR (acetone-d6)
δ 154.8, 153.9, 139.7, 139.2, 136.4, 133.7, 130.3, 128.9, 128.1, 127.7,
127.5, 125.2, 124.7, 124.6, 123.6, 120.2, 117.5, 108.7; GC/MS m/z
312 (M)+.
6-(2-Fluoro-4-hydroxyphenyl)-1-naphthol (11). The title
compound was prepared by reaction of 6-(2-fluoro-4-methoxyphenyl)-
1-naphthol 10 (100 mg, 0.09 mmol, 1 equiv) with pyridinium
hydrochloride (9 mmol, 100 equiv) according to method E. The
product was purified by column chromatography (hexane/ethyl
acetate, 7:3) to afford 48 mg (54%) of the analytically pure product
6-(3-Chloro-4-hydroxyphenyl)-1-naphthol (20). The title
compound was prepared by reaction of 5-hydroxy-2-naphthyl trifluoro-
methanesulfonate 1a (200 mg, 0.68 mmol, 1 equiv) and 3-chloro-
4-hydroxyphenylboronic acid (153 mg, 0.89 mmol, 1.3 equiv) accord-
ing to method B. The product was purified by column chromatog-
raphy (hexane/ethyl acetate, 7:3) to give 117 mg (64%) of the
analytically pure compound as a beige solid. C16H11ClO2; MW 270;
1
as a beige solid. C16H11FO2; MW 254; mp 145−146 °C; H NMR
(acetone-d6) δ 9.15 (s, br, 1H), 9.07 (s, br, 1H), 8.28 (d, J ≈ 8.5 Hz,
1H), 7.83 (d, J ≈ 8.5 Hz, 2H), 7.62 (dt, J ≈ 1.8 Hz, J ≈ 8.5 Hz, 1H),
7.50−7.46 (m, 1H), 7.42 (d, J ≈ 8.5 Hz, 1H), 7.32−7.31 (m, 1H), 6.92
(dd, J ≈ 1.0 Hz, J ≈ 7.3 Hz, 1H), 6.84−6.81 (m, 1H); 13C NMR
(acetone-d6) δ 162.7, 154.0, 148.1, 136.0, 132.4, 130.1, 127.8, 127.5,
126.5, 123.1, 120.2, 115.1, 115.0, 112.9, 109.1, 104.2; IR 3225, 1623,
1214 cm−1; GC/MS m/z 254 (M)+.
1
mp 112−114 °C; H NMR (acetone-d6) δ 9.10 (s, br, 2H), 8.31 (d,
J ≈ 8.5 Hz, 1H), 8.07 (d, J ≈ 2.0 Hz, 1H), 7.80 (d, J ≈ 2.0 Hz, 1H),
7.75 (dd, J ≈ 2.0 Hz, J ≈ 8.5 Hz, 1H), 7.64 (dd, J ≈ 2.0 Hz, J ≈ 8.5 Hz,
1H), 7.47 (d, J ≈ 8.4 Hz, 1H), 7.36−7.32 (m, 1H), 7.19 (d, J ≈ 8.4 Hz,
1H), 6.94 (dd, J ≈ 1.0 Hz, J ≈ 7.4 Hz, 1H); 13C NMR (acetone-d6) δ
154.0, 153.5, 137.9, 136.2, 134.5, 129.2, 127.7, 127.6, 125.4, 125.3,
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124.4, 123.8, 121.7, 120.2, 118.1, 109.0; IR 3330, 1599, 1283, 1187
cm−1; GC/MS m/z 270−272 (M)+.
[1,2,6,7-3H]A-dione at 21 nM. Further treatment of the samples and
HPLC separation were carried out as mentioned above.56
Biological Assays. [2,4,6,7-3H]E2 and [2,4,6,7-3H]E1 were
bought from Perkin-Elmer, Boston, MA. Quickszint Flow 302 scintil-
lator fluid was bought from Zinsser Analytic, Frankfurt, Germany.
Cytosolic (17β-HSD1) and microsomal (17β-HSD2) fractions were
obtained from human and Callithrix jacchus placenta according to
previously described procedures29,65,66 and from rat and mouse liver
tissues.67 Fresh tissue was homogenized and centrifuged. The pellet
fraction contains the microsomal 17β-HSD2 and was used for the
determination of E1-formation, while 17β-HSD1 was obtained after
precipitation with ammonium sulfate from the cytosolic fraction for
use of testing of E2-formation.
Inhibition of 17β-HSD2 in a Cellular Assay. Cellular 17β-
HSD2 activity is measured using the breast cancer cell-line MDA-MB-
23158 (17β-HSD1 activity negligible). [3H]E2 (200 nM) is taken as
substrate and is incubated with the inhibitor for 3.5 h at 37 °C. After
ether extraction, substrate and product are separated by HPLC and
detected with a radioflow detector. Potency is evaluated as percentage
of inhibition (inhibitor concentration of 1 μM) and as IC50.
ER Affinity.29 The binding affinity of selected compounds to the
ERα and ERβ was determined according to Zimmermann et al.73
using recombinant human proteins. Briefly, 0.25 pM ERα or ERβ was
incubated with [3H]E2 (10 nM) and test compound for 1 h at
room temperature. The potential inhibitors were dissolved in DMSO
(5% final concentration). Evaluation of 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, Germany) was added
and the samples were measured in a liquid scintillation counter (Rack
Beta Primo 1209, Wallac, Turku, Finland). From these results the
percentage of [3H]E2 displacement by the compounds was calculated.
The plot of percent displacement versus compound concentration
resulted in sigmoidal binding curves. The compound concentration to
displace 50% of the receptor bound [3H]E2 was determined.
Unlabeled E2 was used as a reference. For determination of the
relative binding affinity (RBA) the ratio was calculated according to
the following equation:74
Human 17β-HSD4 and 17β-HSD5 were cloned into the modified
pGEX-2T vector.56 For the multidomain enzyme 17β-HSD4, only the
steroid converting SDR domain was subcloned.56
Inhibition of 17β-HSD2/E1-Formation. Inhibitory activities
were evaluated by a well established method with minor
modifications.68−70 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) 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 addition of a mixture of unlabeled
E2 and [2,4,6,7-3H]E2 (final concentration of 500 nM, 0.11 μCi).
After 20 min at 37 °C, the incubation was stopped with HgCl2 and the
mixture was extracted with ether. After evaporation, the steroids were
dissolved in acetonitrile. E1 and E2 were separated using acetonitrile/
water (45:55) as mobile phase in a C18 RP chromatography column
(Nucleodur C18 Gravity, 3 μm, Macherey-Nagel, Duren, Germany)
̈
connected to a HPLC system (Agilent 1100 series, Agilent
Technologies, Waldbronn, Germany). Detection and quantification
of the steroids were performed using a radioflow detector (Berthold
Technologies, Bad Wildbad, Germany). The conversion rate was
calculated according to following equation:
This results in an RBA of 100% for E2. After the assay was established
and validated, a modification was made to increase throughput.
Compounds were tested at concentrations of 1000 IC50(E2) and
10000 IC50(E2). Results were reported as RBA ranges. Compounds
with less than 50% displacement of [3H]E2 at a concentration of
10000 IC50 (E2) were classified with RBA < 0.01%. Compounds that
displace more than 50% at 10000 IC50 (E2) but less than 50% at 1000
IC50(E2) were classified with 0.01% < RBA < 0.1%.
Each value was calculated from at least three independent experiments.
Inhibition of 17β-HSD1/E2-Formation. The 17β-HSD1 inhib-
ition assay was performed similarly to the 17β-HSD2 procedure. The
cytosolic fraction was incubated with NADH [500 μM], test
compound, and a mixture of unlabeled E1 and [2,4,6,7-3H]E1 (final
concentration of 500 nM, 0.15 μCi) for 10 min. Further treatment of
the samples and HPLC separation were carried out as mentioned
above.
Inhibition of 17β-HSD4. Inhibitory activity was assessed as
originally described71,72 with minor modifications.56 Briefly, bacteria
containing recombinant 17β-HSD4 were resuspended in PBS and
enzymatic assay was performed at pH 7.7. The enzyme preparation
was incubated with NAD+ [7.5 mM]. Inhibitor (dissolved in DMSO)
was added in a final concentration of 1 μM. Final concentration of
DMSO was adjusted to 1% in all samples. The enzymatic reaction was
started with the addition of [6,7-3H]E2 at 21 nM. The incubation at
37 °C was stopped with 0.21 M ascorbic acid in methanol/acetic acid
(99:1) after the time needed to convert approximately 30% of the
substrate in a control assay without inhibitor. Steroids were extracted
from the assay mixture by SPE using Strata C18-E columns
(Phenomenex), eluted with methanol, and separated by RP-HPLC
(column, Luna, 5 μm C18(2), 150 mm; Phenomenex) at a flow rate
of 1 mL/min acetonitrile/water (43:57). Radioactivity was detected
by scintillation counting after mixing with ReadyFlowIII (Beckman).
Conversion was calculated from integration of substrate and
product peaks. Assay was run in triplicate of three independent
experiments.
log P Determination. The log P values were calculated from
CambridgeSoft Chem & Bio Draw, version 11.0, using the
ChemDrawPro, version 11.0, program.
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical synthesis, characterization of all compounds, and
HPLC purity determination. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 681 302 70300. Fax: +49 681 302 70308. E-mail:
rwh@mx.uni-saarland.de.
ACKNOWLEDGMENTS
■
We thank Jannine Ludwig, Martina Jankowski, Jeannine Jung,
and Dr. Jorg Haupenthal for their help in performing the
̈
enzyme and cellular inhibition tests (17β-HSD1, 17β-HSD2)
and the ER tests. We acknowledge Prof. A. Einspanier for the
gift of monkey placentas. We are grateful to the Deutsche
Forschungsgemeinschaft for financial support (Grants
HA1315/12-1 and AD 127/10-1).
Inhibition of 17β-HSD5. The 17β-HSD5 inhibition assay was
performed similarly to the 17β-HSD4 procedure. The recombinant
enzyme was incubated with NADPH [6 mM], inhibitor [1 μM], and
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(12) Delmas, P. D. Treatment of postmenopausal osteoporosis.
Lancet 2002, 359 (9322), 2018−2026.
ABBREVIATIONS USED
■
17β-HSD1, 17β-hydroxysteroid dehydrogenase type 1; 17β-
HSD2, 17β-hydroxysteroid dehydrogenase type 2; A-dione, 4-
androstene-3,17-dione; AKR, aldo−keto redutase; APCI,
atmospheric pressure chemical ionization; DME, dimethoxy-
ethane; DMSO, dimethylsulfoxide; E1, estrone; E2, 17β-
estradiol; EDG, electron donating group; equiv, equivalent;
EDTA, ethylenediaminetetraacetic acid; ER, estrogen receptor;
ESI, electronspray interface; EtOH, ethanol; EWG, electron
withdrawing group; HPLC, high pressure liquid chromatog-
raphy; NAD(P)(H), nicotinamide adenine dinucleotide
(phosphate); OB, osteoblast; OC, osteoclast; PBS, phosphate
buffered saline; RBA, relative binding affinity; RP, reversed
phase; SAR, structure−activity relationship; SDR, short chain
dehydrogenase/reductase; SERM, selective estrogen receptor
modulator; SF, selectivity factor; T, testosterone
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̈
Eckert, S.; Ensrud, K. E.; Avioli, L. V.; Lips, P.; Cummings, S. R.
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ADDITIONAL NOTE
■
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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