Design, synthesis, and biological evaluation of (hydroxyphenyl)naphthalene and -quinoline derivatives: Potent and selective nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) for the treatment of estrogen-dependent diseases

Article abstract of DOI:10.1021/jm701447v

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of the weak estrogen estrone (E1) to the highly potent estradiol (E2). This reaction takes place in the target cell where the estrogenic effect is exerted via the estrogen receptor (ER). Estrogens, especially E2, are known to stimulate the proliferation of hormone-dependent diseases. 17β-HSD1 is overexpressed in many breast tumors. Thus, it is an attractive target for the treatment of these diseases. Ligand- and structure-based drug design led to the discovery of novel, selective, and potent inhibitors of 17β-HSD1. Phenyl-substituted bicyclic moieties were synthesized as mimics of the steroidal substrate. Computational methods were used to obtain insight into their interactions with the protein. Compound 5 turned out to be a highly potent inhibitor of 17β-HSD1 showing good selectivity (17β-HSD2, ERα and β), medium cell permeation, reasonable metabolic stability (rat hepatic microsomes), and little inhibition of hepatic CYP enzymes.

Full text of DOI:10.1021/jm701447v

2158
J. Med. Chem. 2008, 51, 2158–2169
Design, Synthesis, and Biological Evaluation of (Hydroxyphenyl)naphthalene and -quinoline
Derivatives: Potent and Selective Nonsteroidal Inhibitors of 17ꢀ-Hydroxysteroid Dehydrogenase
Type 1 (17ꢀ-HSD1) for the Treatment of Estrogen-Dependent Diseases
Martin Frotscher,† Erika Ziegler,† Sandrine Marchais-Oberwinkler,† Patricia Kruchten,† Alexander Neugebauer,†
Ludivine Fetzer,† Christiane Scherer,† Ursula Müller-Vieira,‡ Josef Messinger,§ Hubert Thole,§ and Rolf W. Hartmann*,†
8.2 Pharmaceutical and Medicinal Chemistry, Saarland UniVersity, P.O. Box 151150, D-66041 Saarbrücken, Germany, Pharmacelsus CRO,
Science Park 2, D-66123 Saarbrücken, Germany, and SolVay Pharmaceuticals, Hans-Böckler-Allee 20, D-30173 HannoVer, Germany
ReceiVed NoVember 16, 2007
Human 17ꢀ-hydroxysteroid dehydrogenase type 1 (17ꢀ-HSD1) catalyzes the reduction of the weak estrogen
estrone (E1) to the highly potent estradiol (E2). This reaction takes place in the target cell where the estrogenic
effect is exerted via the estrogen receptor (ER). Estrogens, especially E2, are known to stimulate the
proliferation of hormone-dependent diseases. 17ꢀ-HSD1 is overexpressed in many breast tumors. Thus, it
is an attractive target for the treatment of these diseases. Ligand- and structure-based drug design led to the
discovery of novel, selective, and potent inhibitors of 17ꢀ-HSD1. Phenyl-substituted bicyclic moieties were
synthesized as mimics of the steroidal substrate. Computational methods were used to obtain insight into
their interactions with the protein. Compound 5 turned out to be a highly potent inhibitor of 17ꢀ-HSD1
showing good selectivity (17ꢀ-HSD2, ERR and ꢀ), medium cell permeation, reasonable metabolic stability
(rat hepatic microsomes), and little inhibition of hepatic CYP enzymes.
Chart 1. Interconversion of Estrone (E1) to Estradiol (E2)
Introduction
Estrogens are a family of steroid hormones that play a central
role in the growth, development, and maintenance of a diverse
range of tissues. They are also known, however, to be involved
in hormone-dependent diseases. For instance, they stimulate the
estrogen receptor (ER)a-mediated proliferation of breast cancer
and endometrial cells leading to a progression of cancer and to
endometriosis.1,2 Antagonizing estrogenic effects by selective
estrogen receptor modulators (SERMs) or decreasing estrogen
levels by aromatase inhibitors are established treatments for
these diseases.3,4 However, these therapies have some limita-
tions: both strategies affect sites where estrogen is required for
normal function. SERMs are also known to induce carcinoma
in other tissues like endometrium.3 As estrogenic effects are
predominantly caused by the most potent estrogen, estradiol
(E2), a novel and promising therapeutic approach could be the
inhibition of the last step of E2 biosynthesis which is catalyzed
by certain 17ꢀ-hydroxysteroid dehydrogenases. In general, this
class of dehydrogenases modulates potencies of steroidal sex
hormones.
enzymes, which intracellularly activate circulating E1 (estrone)
to E2 (Chart 1). In pathologically altered tissues, e.g. breast
tumors, it is known that 17ꢀ-HSD1 is often overexpressed
leading to elevated local E2 levels.7 Therefore, 17ꢀ-HSD1 is
being discussed as an attractive target for the treatment and even
the prevention8 of estrogen-dependent diseases such as breast
cancer and endometriosis. The advantage of this novel thera-
peutic approach should be a reduction of side effects compared
to established treatments.
17ꢀ-HSD1 contains 327 amino acids and exists as a
homodimer with a subunit mass of 34.9 kDa.9 The enzymatic
reaction is coupled to the consumption of the cosubstrate
NAD(P)H.10 In primates, 17ꢀ-HSD1 is mainly expressed in
placenta, ovarian granulosa cells, and to a lesser extent in
endometrium, prostate, and adipose tissue, but not in adrenals
or testes.11,12 This tissue-specific expression pattern makes
17ꢀ-HSD1 an attractive drug target in women’s health
diseases. Currently, 16 crystal structures of the enzyme are
known.13–21 Only a few inhibitors of 17ꢀ-HSD1 have been
reported, most of them having a steroidal structure.22–34 No
drug candidate has been described so far. As a biological
counterpart, 17ꢀ-hydroxysteroid dehydrogenase type 2 (17ꢀ-
HSD2) catalyzes the oxidation of E2 to E1 (Chart 1), thus
deactivating E2 and protecting the cell from excessively high
concentrations of active estrogen. This enzyme should not
be affected by potential inhibitors of 17ꢀ-HSD1. The aim of
our work described in the following is the design, synthesis,
Fourteen members of this enzyme family are known; 11 of
them regulate the concentrations of active androgens and
estrogens in a tissue-specific manner in humans.5 This modula-
tion of hormone activities by the target cells themselves is
known as intracrinology.6 17ꢀ-Hydroxysteroid dehydrogenase
type 1 (17ꢀ-HSD1) is the most important of these 17ꢀ-HSD
* To whom correspondence should be addressed. Tel: +49 681 302 3424.
Fax: +49 681 302 4386. E-mail: rwh@mx.uni-saarland.de, http://www.
pharmmedchem.de.
†
Saarland University.
Pharmacelsus CRO.
Solvay Pharmaceuticals.
‡
§
a Abbreviations: 17ꢀ-HSD1, 17ꢀ-hydroxysteroid dehydrogenase type 1;
17ꢀ-HSD2, 17ꢀ-hydroxysteroid dehydrogenase type 2; E1, estrone; E2,
estradiol; ER, estrogen receptor; CYP, cytochrome P450; PDB-ID, protein
data bank identification code; Papp, apparent permeability coefficient; RBA,
relative binding affinity; RMSd, root-mean-square distance; SAR: struc-
ture–activity relationship; DME, dimethoxyethane.
10.1021/jm701447v CCC: $40.75
2008 American Chemical Society
Published on Web 03/07/2008

Synthesis of (Hydroxyphenyl)naphthalene and -quinoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2159
Figure 1. Schematic presentation of the active site of 17ꢀ-HSD1 containing E2. Turquoise labels denote polar amino acids and orange labels stand
for lipophilic amino acids. Hydrogen bonds are marked in yellow.
and biological evaluation of potent and selective nonsteroidal
inhibitors of 17ꢀ-HSD1.
dole seemed to be appropriate scaffolds as they can adopt a
nearly flat conformation similar to the steroid (Chart 2). The
synthesis of the heterocyclic compounds seemed to be promising
in order to examine whether the heteroatom could establish
additional interactions with the polar amino acids Tyr218 and/
or Ser222, which are located close to C6 of the steroidal B-ring
(Figure 1). For instance, the distance between the Tyr218 oxygen
and C6 of the steroid is 3.8 Å (PDB-ID: 1FDT).
Design of Inhibitors
17ꢀ-HSD1 Substrate Binding Site. Although 16 crystal-
lographic structures of binary and ternary complexes of 17ꢀ-
HSD1 have been published,13–21 there is no structure of the
enzyme with the substrate E1 available. Therefore, the ternary
complex PDB-ID: 1FDT containing E2 and NADP+ was used
to study the three-dimensional architecture of the enzyme. The
substrate binding site is a hydrophobic tunnel with polar areas
at each end formed by His221/Glu282 on one side and Ser142/
Tyr155 (along with Asn114 and Lys159 members of the
catalytic tetrad) on the other side. These amino acids establish
hydrogen bonds to the two hydroxy groups of E2 OH(3) and
OH(17), respectively. Interestingly, there are two additional
polar amino acids (Tyr218, Ser222) in this region of hydro-
phobic amino acids, having no counterpart in the steroid for
interaction. Figure 1 shows a schematic presentation of the active
site and the binding mode of E2.
In front of the active site a flexible entry loop (amino acids
188-201) is located which is not properly resolved in the crystal
structures. This entry loop can adopt two distinct conformations:
the loop can close the entrance to either the substrate binding
site or the cofactor binding pocket.35
Design of Steroidomimetics. In order to reduce the risk of
undesired side effects caused by interaction with steroid
receptors and to have straightforward access to structural
diversity, we focused on the design of nonsteroidal inhibitors.
The steroidomimetics should contain two polar groups mimick-
ing the functional groups in the 3- (A-ring) and the 17-positions
(D-ring). The distance between them should be approximately
10–13 Å corresponding to the geometry in the steroid. Phe-
nyltetralone, phenylnaphthalene, phenylquinoline, and phenylin-
Chemistry
The synthesis of the compounds is depicted in Schemes 1–6.
Scheme 1 shows the synthesis of compounds 1–3 from
6-hydroxy-1-tetralone via triflation, Suzuki coupling, and aro-
matization as described in the literature.36
Different quinoline and naphthalene derivatives were coupled
to the aromatic/heteroaromatic moiety via the Suzuki reaction
using two different methods. Compounds 4a–6a, 7–9, 10a, and
20a-24a were prepared following method A [Pd(PPh3)4,
sodium carbonate in toluene/water 1/1].37 Compounds 12–19
were obtained by parallel synthesis using microwave-assisted
reactions according to method B [Pd(OAc)2, potassium carbon-
ate in DME/water/ethanol 7/3/2] (Schemes 2 and 4).
Quinoline intermediates 23a and 24b were prepared by
reaction of 3-methoxyaniline with 2-bromomalonaldehyde or
2-(4-methoxyphenyl)malondialdehyde under acidic conditions
(Scheme 3).
All synthesized compounds containing a methoxy substituent
were demethylated using aluminum chloride (method C) for
compounds 21–24 or boron tribromide (method D) for com-
pounds 3–6, 11, and 20 (Schemes 2 and 4).
The 2-phenylquinoline derivative 25 was obtained by reaction
of 6-methoxyquinoline-N-oxide with freshly prepared 3-meth-
oxybenzenemagnesium bromide and subsequent ether cleavage
using boron tribromide (method D, Scheme 5).

2160 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Chart 2. Designed Compounds
Frotscher et al.
Scheme 1. Synthesis of Compounds 1–3a
to compound 26a, which was demethylated using boron
tribromide (Scheme 6).
Biological Results
Inhibition of Human 17ꢀ-HSD1. As source of enzyme, both
recombinant as well as placental enzyme were used. The amount
of labeled E2 formed from tritiated substrate in the presence of
cofactor and inhibitor was determined by HPLC. The percent
inhibition values of all hydroxy compounds are shown in Tables
1 and 2, and the IC50 values of selected compounds are shown
in Table 3. The hybride inhibitor (EM-1745) described by Poirier
et al.19 was used as reference compound and gave similar values
as described (IC50 ) 52 nM). Compounds showing less than
10% inhibition at 1 µM were considered to be inactive. All
molecules with methoxy groups showed no activity (data not
shown).
Phenylnaphthalenes. The tetralone 1 turned out to have weak
inhibitory activity. Compounds 2 and 3 bearing a hydroxy
substituent at the 1-position of the naphthalene and a 3′-
hydroxyphenyl or 4′-hydroxyphenyl substituent also showed
weak (2) to moderate (3) inhibition (Table 1). An interesting
structure–activity relationship (SAR) can be observed after
shifting the hydroxy group of the naphthalene from the 1 to the
2 position. Three isomers were prepared bearing the second OH
group in position 2′ (4), 3′ (5), or 4′ (6) of the phenyl moiety.
a Reagents and conditions: (a) trifluoromethanesulfonic anhydride,
CH2Cl2, 0°C, 30 min; (b) 3-hydroxy- or 4-methoxybenzeneboronic acid,
aq Na2CO3, Pd(PPh3)4, DME, reflux, 4 h; (c) Pd/C, p-cymene, reflux, 6–24
h; (d) BBr3, CH2Cl2. -78 °C, overnight.
Synthesis of the indole 26 started with commercially available
4-methoxyaniline and 2-bromo-1-(3-methoxyphenyl)ethanone
following a procedure described by von Angerer et al.38 leading

Synthesis of (Hydroxyphenyl)naphthalene and -quinoline DeriVatiVes
Scheme 2. Synthesis of Compounds 4–20a
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2161
a Reagents and conditions: (a) Method A: aq Na2CO3, Pd(PPh3)4, toluene or DME, 80 °C, overnight; Method B: K2CO3, Kirschning catalyst, DME/
water/ethanol 7/3/2, microwave irradiation 150 °C, 100 W, 300 s; (b) BBr3, CH2Cl2, -78 °C, overnight; (c) Pd/C, H2, dry THF, rt, overnight.
Scheme 3. Synthesis of Compounds 23a–24ba
hydroxyphenyl substituent of the most active compound 5. All
synthesized compounds 10–20 of this series show no or only
moderate inhibition. The most active compound is the benzoic
acid derivative 12, displaying 76% inhibition at a concentration
of 1 µM. Compound 20, a 3′-hydroxypyridyl-substituted
naphthol, showed a moderate activity (58% at 1 µM), indicating
a Reagents and conditions: (a) ethanol, rt, 18 h and then acetic acid,
100 °C, 10 days or EtOH, concd HCl, 80 °C, 3 days.
that a nitrogen atom in this area of the active site is not well
tolerated.
Phenylindoles and Phenylquinolines. As discussed above,
insertion of a hydrogen-bond acceptor or donor into the nonpolar
phenylnaphthalene scaffold might lead to additional interactions
with Tyr218 and/or Ser222, thus increasing inhibitory activity
for 17ꢀ-HSD1. In order to explore this hypothesis, hydroxy-
substituted quinoline and indole derivatives 21–26 were syn-
thesized and evaluated for their 17ꢀ-HSD1 inhibitory activity
(Table 2).
Compounds 4 and 6 show no affinity to 17ꢀ-HSD1, whereas
the isomer with the phenolic OH-group in the meta position
(compound 5) is a highly potent inhibitor of the enzyme.
Interestingly, this compound has recently been described to be
a weak inhibitor of tyrosinase.39
The question whether both hydroxy substituents are necessary
for strong inhibition was examined. Starting from the structure
of the highly active 5 with hydroxy groups in obviously
favorable positions, two compounds 8 and 9 were synthesized,
each bearing only one hydroxy substituent. Compound 9 (3′-
OH on the phenyl moiety) shows a moderate activity, whereas
8 (2-OH on the naphthalene moiety) is devoid of activity.
Heterocycles and phenyl moieties bearing substituents other
than OH, capable of acting as hydrogen-bond donors and/or
acceptors, were introduced as potential alternatives for the 3′-
In the class of the quinolines 21–25, SARs are similar to those
observed in the naphthalene series: monohydroxylated phe-
nylquinolines 21 and 22 show little or no inhibition. The 3′-
hydroxyphenyl-substituted quinolin-7-ol 24 is a moderate
inhibitor (57% inhibition at 1 µM), in contrast to its 4′-
hydroxyphenyl-substituted isomer 23, a weak ER ꢀ ligand40
which is inactive. This again reflects the importance of the
substitution pattern at the hydroxyphenyl moiety. On the other

2162 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Frotscher et al.
Scheme 4. Synthesis of Compounds 21–24
a
a Reagents and conditions: (a) aq Na2CO3, Pd(PPh3)4, toluene or DME, 80 °C, 4 h-3 d; (b) AlCl3, toluene, 90 °C, 2 h.
Scheme 5. Synthesis of Compound 25a
Table 1. Inhibition of Human 17ꢀ-HSD1 by Naphthalene Compounds
1–20
a Reagents and conditions: (a) dry toluene, 90 °C, 2 h; (b) BBr3 CH2Cl2,
-78 °C, 3 h.
Scheme 6. Synthesis of Compound 26a
inhibition of 17ꢀ-HSD1a (%)
compd R1
R2
n
V
Z
100 nM
1 µM
1
n.i.
n.i.
n.i.
n.i.
91
n.i.
16
n.i.
26
23
23
55
n.i.
94
n.i.
62
n.i.
61
n.i.
n.i.
76
n.i.
19
2
3
4
5
6
7
8
9
1-OH 3′-OH
1-OH 4′-OH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
1 CH CH
a Reagents and conditions: (a) N,N-dimethylaniline, 180 °C, 2 h; (b) BBr3,
CH2Cl2, -78 °C, overnight.
2-OH 2′-OH
2-OH 3′-OH
2-OH 4′-OH
3-OH 3′-OH
2-OH H
hand variations of the position of the nitrogen atom within the
scaffold only have little effect on inhibitory potency: both 20
and 25 show moderate inhibition values similar to that of 24.
Indole 26, bearing a hydroxyphenyl substituent at the five-
membered ring, shows no activity.
H
3′-OH
10 2-OH 3′-NO2
11 2-OH 3′-NH2
12 2-OH 3′-COOH
13 2-OH 4′-COOH
n.i.
n.i.
36
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
30
14 2-OH 3′-NHCOCH3 1 CH CH
The most active members 24 and 25 in this series of
heterocyclic compounds are analogues of the most active
naphthalene 5. The introduction of a nitrogen atom in the
scaffold, however, resulted in a reduction of activity (at
100 nM: 24% inhibition for 24 compared to 91% for 5). This
not only indicates that no additional bond is established between
the nitrogen atom of the scaffold and the side chains of Tyr218
and Ser222 but it also shows that the polarity brought into the
scaffold is not well tolerated by the enzyme.
15 2-OH 3′-CH2OH
16 2-OH 4′-CH2OH
17 2-OH H
18 2-OH H
19 2-OH H
1 CH CH
1 CH CH
0 CH S
0 S CH
1 N CH
1 N CH
n.i.
13
n.i.
n.i.
32
20 2-OH 3′-OH
58
a Recombinant 17ꢀ-HSD1, substrate [3H]-E1 (30 nM), NADPH (1 mM)
procedure A; mean value of two determinations, deviation <20%, n.i. )
no inhibition (inhibition <10%).
For selected compounds, IC50 values were determined. As
shown in Table 3, compound 5 with an IC50 value of 116 nM
is the most potent inhibitor identified in this series. Moving the
OH group on the naphthalene from the 2 position to the 1
position results in a loss of activity (3: IC50: 2.4 µM). Insertion
of a nitrogen in the phenyl ring also reduces activity by a factor
of 10 (20).
Selectivity. As 17ꢀ-HSD2 deactivates E2 by oxidation to E1,
which is beneficial for the treatment of estrogen-dependent
diseases, this enzyme should not be affected by inhibitors of
the type 1 enzyme. Furthermore, inhibitors of 17ꢀ-HSD1 should
have low or no affinity at all for the estrogen receptors R and
ꢀ (ER R and ꢀ), since binding to these receptors could counteract
the therapeutic concept of 17ꢀ-HSD1 inhibition. Compounds
3, 5, 9, and 20sa selection of the most potent inhibitors of 17ꢀ-
HSD1 found in this studyswere tested for their activities toward
17ꢀ-HSD2 and binding affinities to ER R and ꢀ.
Briefly, the inhibition assay for 17ꢀ-HSD2 was performed
by HPLC measurement of the amount of labeled E1 formed
from [3H]-E2 in the presence of NAD+ and inhibitor. IC50 values
were determined and selectivity factors calculated (Table 3).
While compound 3 shows selectivity for 17ꢀ-HSD2, the other
compounds exhibit selectivity for 17ꢀ-HSD1, compound 5 being
the most selective (factor 48).
For the ER assays recombinant human protein was used and
a competition assay applying [3H]-E2 and hydroxyapatite was
run. All tested compounds show very little to moderate affinity
to the ERs, respectively. The most promising compound so far,

Synthesis of (Hydroxyphenyl)naphthalene and -quinoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2163
Table 2. Inhibition of Human 17ꢀ-HSD1 by Quinoline and Indole
Compounds 21–26
Table 4. Metabolic Stability of Compound 5 Using Rat Liver
Microsomes
a,b
a,c
compd
t1/2 (min)
Clint (µL/min/mg protein)
5
12.6
40.8
6.8
366.9
113.3
679.6
diazepam
diphenhydramine
a Mean value of three determinations. b t1/2: half-life. c Clint: intrinsic body
clearance.
molecular interactions with the protein. There are two possibili-
ties for our steroidomimetics to superimpose with E2 resulting
in similar RMSd values (Figure 2). Each of the two hydroxy
groups of compound 5 can mimic the 3-OH or the 17-OH of
E2.
inhibition of 17ꢀ-HSD1a (%)
compd
n
X
Y
R1
R2
100 nM
1 µM
21
22
23
24
25
26
1
1
1
1
1
0
N
N
N
N
CH
CH
CH
CH
H
H
3′-OH
4′-OH
n.i.
n.i.
n.i.
24
18
n.i.
n.i.
57
CH OH 4′-OH
CH OH 3′-OH
N
N
In this context, it is striking that taking out one hydroxy
function of compound 5, leading to two monohydroxylated
compounds, results in an active (9) and an inactive compound
(8). Obviously, the hydroxy group on the phenyl ring is more
important for binding than the one on the naphthyl moiety. This
finding favors binding mode B (the hydroxyphenyl group
superimposes with the steroidal A ring), as it can be hypoth-
esized that the hydroxy group mimicking the steroidal phenolic
3-OH is more important for binding than the hydroxy group
mimicking the 17-oxygen function: the enzymatic product E2
(17ꢀ-OH) binds less tightly than the substrate E1 (17-CO) in
the binding site.
OH 3′-OH
OH 3′-OH
14
63
n.i.
n.i.
a Recombinant 17ꢀ-HSD1, substrate [3H]-E1 (30 nM), NADPH (1 mM)
procedure A; mean value of two determinations, deviation <20%, n.i. )
no inhibition (inhibition <10%).
5, presents low affinities to the ERR (RBA: 0.2%) and the ERꢀ
(RBA: 0.8%, Table 3).
Further Biological Evaluation of Compound 5. Compound
5 was investigated for permeation of Caco-2 cells. These cells
exhibit morphological and physiological properties of the human
small intestine41 and are generally accepted to be an appropriate
model for the prediction of peroral absorption. Depending on
the Papp data obtained, compounds can be classified as low (Papp
(× 10-6 cm/s) < 1), medium (1 < Papp < 10), or highly
permeable (Papp >10). Compound 5 shows medium cell
permeation (Papp) 8.9 ( 0.45 × 10-6 cm/s, n ) 3).
Compound 5 was tested for metabolic stability. It was
incubated with rat liver microsomes. Samples were taken at
defined time points and the remaining percentage of parent
compound was determined by LC-MS/MS. Half-life and
intrinsic clearance were evaluated and compared to the two
reference compounds diazepam and diphenhydramine. The short
half-life t1/2 ) 12 min and the relatively high intrinsic clearance
(367 µL/min/mg protein) indicate a moderate metabolic stability
for compound 5 (Table 4).
Compound 5 was also investigated for possible drug-drug
interaction using six human hepatic enzymes: CYP1A2, 2B6,
2C9, 2C19, 2D6, and 3A4. The latter two enzymes are very
crucial for two reasons: they are responsible for approximately
75% of drug metabolism and a genetic polymorphism is
described. IC50 values of compound 5 and control inhibitors
were determined for each enzyme. None of the enzymes was
strongly inhibited by compound 5. This result indicates a low
risk of CYP-inhibition induced drug-drug interaction
(Table 5).
The surprising result that the introduction of a nitrogen in
compound 5 does not increase activity further can be explained
by the fact that the nitrogen cannot interact with the amino acids
Tyr218 and/or Ser222 and supports our hypothesis that com-
pound 5 binds according to mode B and not to mode A. For a
better understanding, we docked the quinoline compound 24 in
the active site according to binding mode B using the docking
program Gold 3.0 (flexible ligand, rigid protein) and determined
the distances to the two polar amino acids Tyr218 and Ser222.
The distance OTyr218-Nquinoline is 4.58 Å (Figure 3). The distance
OSer222-Nquinoline is 5.88 Å (not shown) but can adopt 3.50 Å
due to the conformational flexibility of the CH2OH group
(Figure 3). At this latter distance a hydrogen bond interaction
should be possible between the OSer222-Nquinoline and should
increase activity.
To obtain further insight, a molecular dynamics simulation
of a binary complex of 17ꢀ-HSD1 in the conformation shown
in Figure 3 and compound 24 was performed in order to identify
the preferred position of the CH2OH group of Ser222. The
system was simulated for 500 ps. After 25 ps, the CH2OH group
rotated back to the initial position found in the X-ray structure.
This conformation remained stable until the end of the simula-
tion. The formation of an additional hydrogen bond interaction
with quinoline 24 could thus be excluded. This means that the
quinoline nitrogen has no partner for interaction. The nitrogen
consequently being located in a hydrophobic surrounding should
Molecular Modeling. After having obtained interesting
biological data for the synthesized compounds, theoretical
studies were performed to develop greater insight into their
Table 3. IC50 Values and Selectivity Data for Selected Compounds
17ꢀ-HSD1
17ꢀ-HSD2
selectivity
compd
a,b
a,c
IC50 (nM)
IC50 (nM)
factord
ERR RBAe (%)
ERꢀ RBAe (%)
3
5
9
2425
116
2257
1232
302
5641
4007
0.1
48
1.8
5
0.2
5
0.8
0.01 < RBA < 0.1
0.01 < RBA < 0.1
0.01 < RBA < 0.1
1
20
>10000
>8
a Mean value of three determinations, standard deviation <20% except 20: 24% for 17ꢀ-HSD1. b Human placenta 17ꢀ-HSD1, substrate [3H]-E1 (500
nM), NADH (500 µM) procedure B. c Human placenta 17ꢀ-HSD2, substrate [3H]-E2 (500 nM), NAD+ (1500 µM). d IC50 (17ꢀ-HSD2)/IC50 (17ꢀ-HSD1).
e RBA (relative binding affinity) estradiol: 100%.

2164 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Frotscher et al.
Table 5. Inhibition of Selected Hepatic CYP Enzymes by Compound 5 and Control Inhibitors
IC50 (µM) ( SDa
CYP1A2
CYP2B6
CYP2C9
CYP2C19
CYP2D6
CYP3A4
5
3.99 ( 0.25
furafylline
3.04 ( 0.08
13.40 ( 0.45
tranylcypromine
6.96 ( 0.025
1.05 ( 0.02
sulfenazole
0.250 ( 0.027
7.54 ( 0.51
tranylcypromine
3.04 ( 0.17
32.56 ( 0.72
1.58 ( 0.04
ketoconazole
0.054 ( 0.001
control inhibitor
quinidine
0.011 ( 0.001
a Mean value of three determinations.
Figure 2. Principal ways of superimposition of 5 (green) and E2 (red). RMSd values: 0.36 Å for A (left) and 0.46 Å for B (right).
of 17ꢀ-HSD1 to be applied clinically this is a prerequisite. There
is no debate that the application of an estrogen receptor agonist
is detrimental for treating estrogen-dependent diseases. A 17ꢀ-
HSD1 inhibitor with additional antagonistic activity is also not
welcome for our concept of local estrogen deprivation as such
a compound would exert systemic effects.
The sharp SAR mentioned above suggests a rather rigid
architecture of the substrate binding site of 17ꢀ-HSD1. At least
as far as the amino acid residues interacting with the hydroxy
groups (i.e., His221/Glu282 and Ser142/Tyr155) are concerned,
conformational flexibility seems to be marginal. This assumption
is in agreement with the fact that the geometry of the steroid
binding site is highly conserved in the different X-ray structures
available in the protein data banksindependent of the presence
or absence of ligands.
The rigid structure of the binding site could also be an
explanation for our finding that phenyl naphthalenes very likely
bind in one of the two possible binding modes (Figure 2, mode
B). This result is important for further structural optimizations
in this class of compounds.
Figure 3. Possible hydrogen bond interactions of the 3-phenylquinoline
compound 24 and the polar amino acids Tyr218 and Ser222 within
the active site of 17ꢀ-HSD1. The CH2OH group of Ser222 is rotated
manually toward the nitrogen atom of 24 resulting in a feasible
hydrogen bond. Distances are expressed in Å.
In the present study, compound 5 was identified as a highly
active inhibitor of 17ꢀ-HSD1 showing good selectivity toward
17ꢀ-HSD2, ER R and ER ꢀ. Furthermore it displays a medium
Caco-2 permeability, a reasonable metabolic stability and a low
inhibition of the most important hepatic CYP enzymes. This
compound will be used as a first lead in the further drug design
process.
decrease the inhibitory activity of compound 24 strongly
(compared to compound 5) as was seen in the inhibition test.
Discussion and Conclusion
It is striking in this study that the structure–activity relation-
ships encountered in the investigated class of compounds are
very sharp: the ortho OH compound 4 and the para OH
compound 6 show no affinity for 17ꢀ-HSD1, whereas the isomer
with the OH group in the meta position of the phenyl ring (5)
is a highly potent inhibitor of the enzyme. Moving the OH group
at the naphthalene moiety of 5 to the 1- or 3-position
(compounds 2 and 7) results in a dramatic loss of activity, again
indicating the pivotal role of the hydroxy group positions on
the inhibitory activity. This is all the more remarkable because
hydroxyphenylnaphthols designed as ligands for the structurally
related estrogen receptors do not show this clear-cut SAR.36 In
this context there is an even more interesting aspect: the
substitution pattern for optimal 17ꢀ-HSD1 inhibition and high
affinity for ER are different. Compound 3 is a strong ligand for
the estrogen receptors36 having low 17ꢀ-HSD1 inhibitory
activity whereas compound 5 displaying a strong inhibition of
17ꢀ-HSD1 shows only low affinity for the ERs. For an inhibitor
Experimental Section
Chemical Methods. IR spectra were measured neat on a Bruker
Vector 33FT-infrared spectrometer.
1H NMR spectra were recorded on a Bruker AM500 (500 MHz)
instrument at 300 K in CDCl3, CD3OD, DMSO-d6, or acetone-d6.
Chemical shifts are reported in δ values (ppm); the hydrogenated
residues of deuterated solvent were used as internal standard
(CDCl3: δ ) 7.26 ppm in 1H NMR and δ ) 77 ppm in 13C NMR,
CD3OD: δ ) 3.35 ppm in 1H NMR and δ ) 49.3 ppm in 13C
NMR, DMSO-d6: δ ) 2.58 ppm in 1H NMR and δ ) 39.7 ppm in
1
13C NMR, acetone-d6: δ ) 2.05 ppm in H NMR and δ ) 29.8
ppm in 13C NMR). Signals are described as s, d, t, dd, m, and b
for singlet, doublet, triplet, double–doublet, multiplet, and broad,
respectively. All coupling constants (J) are given in Hz.
Mass spectra (ESI and APCI) were measured on a TSQ Quantum
instrument unless otherwise mentioned.

Synthesis of (Hydroxyphenyl)naphthalene and -quinoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2165
Chemical names follow IUPAC nomenclature. Starting materials
were purchased from Aldrich, Acros, Lancaster, or Fluka and were
used without purification.
Reactions under microwave irradiation were performed using an
Emrys Optimizer Workstation apparatus from CMS.
Column chromatography was performed using silica gel (70–200
µm), and the reaction progress was determined by TLC analyses
on ALUGRAM SIL G/UV254 (Macherey-Nagel).
aqueous solution of sodium carbonate (2 equiv), and tetrakis(triph-
enylphosphine) palladium(0) (0.1 equiv) in toluene or DME was
stirred at 80 °C under nitrogen. The reaction mixture was cooled
to room temperature, and water was added. The mixture was
extracted with dichloromethane, washed with brine, dried over
MgSO4, and concentrated to dryness. The product was purified by
column chromatography.
Method B. General Procedure for Parallel Synthesis of
Compounds 12–19. Aryl halide (1 equiv), boronic acid (2 equiv),
K2CO3 (3 equiv), and Pd(OAc)2 were suspended in 5 mL of DME/
water/ethanol 7/3/2. The reaction mixture was exposed to micro-
wave irradation 300 s, 100 W, 150 °C. After reaching room
temperature, the reaction mixture was filtered, concentrated, and
purified by preparative HPLC (Waters fraction lynx autopurification
system, Varian Inertsil C18-column 50 × 21 mm, particle size 3
µm, gradient elution, solvents: A: acetonitrile, formic acid (0.01%),
B: water, formic acid (0.01%). Flow rate: 25 mL/min. Gradient:
0–100% A within 13 min (linear), then 100% A for 2 min). Purity
data are given in the Supporting Information.
2-Methoxy-6-(2-methoxyphenyl)naphthalene (4a). The title
compound was prepared by reaction of 2-bromo-6-methoxynaph-
thalene (300 mg, 1.26 mmol, 1 equiv) with 2-methoxybenzene
boronic acid (192 mg, 1.26 mmol, 1 equiv) for 18 h according to
method A (quantitative yield). The product was pure enough to be
used directly for the subsequent ether cleavage without purification.
6-Phenyl-2-naphthol (8). The title compound was prepared by
reaction of 6-bromo-2-naphthol (500 mg, 2.24 mmol) with benzene
boronic acid for 22 h according to method A. The product was
purified by column chromatography using hexane/ethylacetate 8/2
as eluent: yield 429 mg (87%); C16H12O; MW 220.
2-Methoxy-6-(3-nitrophenyl)-naphthalene (10a). The title com-
pound was prepared by reaction of 3-bromonitrobenzene (1.00 g,
4.95 mmol) with 6-methoxynaphthaleneboronic acid for 20 h
according to method A. The product was purified by column
chromatography using hexane as eluent: yield 557 mg (40%);
C18H17NO3; MW 295.
The following compounds were prepared according to previously
described procedures: 5-oxo-5,6,7,8-tetrahydronaphthalen-2-yl
trifluoromethanesulfonate (1a),36 6-(3-hydroxyphenyl)-3,4-dihy-
dronaphthalen-1(2H)-one (1),36 6-(3-hydroxyphenyl)-1-naphthol
(2),36
6-(4-methoxyphenyl)-3,4-dihydronaphthalen-1(2H)-one
(3b),36 6-(4-hydroxyphenyl)-1-naphthol (3),36 6-(2-hydroxyphenyl)-
2-naphthol (4),36 2-methoxy-6-(3-methoxyphenyl)naphthalene
(5a),36 6-(3-hydroxyphenyl)-2-naphthol (5),36 2-methoxy-6-(4-
methoxyphenyl)naphthalene (6a),36 6-(4-hydroxyphenyl)-2-naphthol
(6),367-(3-hydroxyphenyl)-2-naphthol (7),36 3-(2-naphthyl)phenol
(9),40 4-(quinolin-3-yl)phenol (22),40 6-methoxy-2-(3-methoxyphe-
nyl)quinoline 25a,43 and 2-(3-hydroxyphenyl)quinolin-6-ol (25).43
6-(4-Methoxyphenyl)-1-naphthol (3a). A mixture of 6-(4-
methoxyphenyl)-3,4-dihydronaphthalen-1(2H)-one (510 mg, 2.02
mmol, 1 equiv) in p-cymene (7 mL) and palladium on charcoal
(528 mg) was heated to reflux for 24 h, cooled, and filtered through
Celite. The filtrate was washed twice with 1 M NaOH and twice
with water. The aqueous layer was extracted several times with
ethyl acetate. The organic layers were combined, dried over MgSO4,
and concentrated under reduced pressure. The crude product was
purified by column chromatography (dichloromethane/hexane 5/5
and then 7/3) to give 69 mg of the expected product (14% yield):
C17H14O2; MW 250; MS (ESI) 251 (M + H)+.
7-Methoxy-3-(4-methoxyphenyl)quinoline (23a).39 To a
solution 2-(4-methoxyphenyl)malondialdehyde (797 mg, 4.48
mmol) in EtOH (20 mL) and 3-methoxyaniline (551 mg, 4.48
mmol, 1.00 equiv) was added concentrated hydrochloric acid
(37%; 10 mL). The reaction was heated at 100 °C for 3 days.
After the solution was cooled to room temperature, water was
added, and the mixture was extracted with dichloromethane. The
organic layer was basified, washed with brine, and dried over
MgSO4. After evaporation of the solvent and column chroma-
tography (hexane/ethyl acetate 8/2), the pure product was
obtained: yield 221 mg (19%); C17H15NO2; MW 265; MS (ESI)
266 (M + H), 251, 223, 208, 180, 152.
3-(6-Methoxy-2-naphthyl)phenylamine (11a). A mixture of
2-methoxy-6-(3-nitrophenyl)naphthalene (200 mg, 0.71 mmol, 1
equiv) and Pd/C (5%; 50 mg) in dry THF (100 mL) was stirred
under hydrogen overnight. After filtration through Celite and
evaporation of the solvent, the crude product was purified by
preparative chromatography to afford 36 mg of the desired product
(20% yield): C17H15NO; MW 249.
3-Bromo-7-methoxyquinoline (24b). To a solution of bromo-
malonaldehyde (1.80 g, 12.3 mmol) in 30 mL of ethanol was added
3-methoxyaniline (1.25 mL, 11.2 mmol, 0.911 equiv). The mixture
was stirred at room temperature overnight. Acetic acid (20 mL)
was added, and the reaction mixture was heated to 100 °C for 10
days. After the solution was cooled to room temperature, the solvent
was evaporated, and the residue was partitioned between water and
ethyl acetate. The organic layer was basified, washed with brine,
and dried over MgSO4. After evaporation of the solvent under
reduced pressure, the product was purified by column chromatog-
raphy using hexane/ethyl acetate 8/2 as eluent: yield 700 mg (20%);
C10H8BrNO; MW 238.
5-Methoxy-2-(3-methoxyphenyl)-1H-indole (26a). To a boiling
mixture of 4-methoxyaniline (2.46 g, 19.9 mmol, 6.44 equiv) and
N,N-dimethylaniline (3.5 mL) was added dropwise 2-bromo-1-(3-
methoxyphenyl)ethanone (0.70 g, 3.1 mmol) in solution in ethyl
acetate (12 mL). After completion of the addition, the mixture was
kept at 180 °C for 2 h. The reaction mixture was cooled to room
temperature, and a dark solid was formed. Ethyl acetate was added
along with 2 M HCl. The aqueous layer was extracted with ethyl
acetate several times. The combined organic layers were washed
with brine and dried over MgSO4, filtered, and evaporated.
Purification by column chromatography (dichloromethane/hexane
7/3) afforded 760 mg of 26a (15%): C16H15NO2; MW 253; MS
(ESI) 254 (M + H)+.
3-(6-Hydroxy-2-naphthyl)benzoic acid (12). The title com-
pound was prepared by reaction of 6-bromo-2-naphthol (44.6 mg,
0.2 mmol, 1 equiv) with 3-carboxyphenylboronic acid (66.4 mg,
0.4 mmol, 2 equiv) for 5 min according to method B: yield 27.8
mg (53%); C17H12O3; MW 264; MS 265 (M + H)+.
4-(6-Hydroxy-2-naphthyl)benzoic acid (13). The title com-
pound was prepared by reaction of 6-bromo-2-naphthol (44.6 mg,
0.2 mmol, 1 equiv) with 4-carboxyphenylboronic acid (66.4 mg,
0.4 mmol, 2 equiv) for 10 min according to method B: yield 21.1
mg (40%); C17H12O3; MW 264; MS (ESI) 265 (M + H)+.
N-[3-(6-Hydroxy-2-naphthyl)phenyl]acetamide (14). The title
compound was prepared by reaction of 6-bromo-2-naphthol (44.6
mg, 0.2 mmol, 1 equiv) with 3-acetanilideboronic acid (71.6 mg,
0.4 mmol, 2 equiv) for 5 min according to method B: yield 22.4
mg (40%); C18H15NO2; MW 277; MS (ESI) 278 (M + H)+.
6-[3-(Hydroxymethyl]phenyl]-2-naphthol (15). The title com-
pound was prepared by reaction of 6-bromo-2-naphthol (44.6 mg,
0.2 mmol, 1 equiv) with 3-(hydroxymethyl)phenylboronic acid (60.8
mg, 0.4 mmol, 2 equiv) for 5 min according to method B: yield
24.6 mg (49%); C17H14O2; MW 250; MS (ESI) 251 (M + H)+.
6-[4-(Hydroxymethyl)phenyl]-2-naphthol (16). The title com-
pound was prepared by reaction of 6-bromo-2-naphthol (44.6 mg,
0.2 mmol, 1 equiv) with 4-(hydroxymethyl)benzeneboronic acid
(60.8 mg, 0.4 mmol, 2 equiv) for 5 min according to method B:
yield 21.1 mg (40%); C17H14O2; MW 250; MS (ESI) 251 (M +
H)+.
Suzuki Coupling. Method A. General Procedure for Syn-
thesis of Compounds 4a, 8, 10a, 20a, 21a, 22a, and 24a. A
mixture of aryl bromide (1 equiv), boronic acid (1 equiv), 2%
6-(3-Thienyl)-2-naphthol (17). The title compound was prepared
by reaction of 6-bromo-2-naphthol (44.6 mg, 0.2 mmol, 1 equiv)

2166 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Frotscher et al.
with 3-thiopheneboronic acid (51.2 mg, 0.4 mmol, 2 equiv) for 10
min according to method B: yield 45.1 mg (99%); C14H10S; MW
226; MS (ESI) 227 (M + H)+.
6-(2-Thienyl)-2-naphthol (18). The title compound was prepared
by reaction of 6-bromo-2-naphthol (44.6 mg, 0.2 mmol, 1 equiv)
with 2-thiopheneboronic acid (51.2 mg, 0.4 mmol, 2 equiv) for 10
min according to method B: yield 43.7 mg (97%); C14H10S; MW
226; MS (ESI) 227 (M + H)+.
6-Pyridin-3-yl-2-naphthol (19). The title compound was pre-
pared by reaction of 6-bromo-2-naphthol (44.6 mg, 0.2 mmol, 1
equiv) with 3-pyridineboronic acid (49.2 mg, 0.4 mmol, 2 equiv)
for 5 min according to method B: yield 17.6 mg (40%); C15H11NO;
MW 221; MS (ESI) 222 (M + H)+.
3-Methoxy-5-(6-methoxy-2-naphthyl)pyridine (20a). The title
compound was prepared by reaction of 6-methoxynaphthalenebo-
ronic acid (258 mg, 1.28 mmol) with 3-bromo-5-methoxypyridine
for 24 h according to method A. The product was purified by
column chromatography using hexane/ethyl acetate 2/1: yield 237
mg (84%); C17H15NO2; MW 265.
3-(3-Methoxyphenyl)quinoline (21a). The title compound was
prepared by reaction of 3-bromoquinoline (200 mg, 0.96 mmol)
with 3-methoxybenzene boronic acid for 19 h according to method
A. The product was purified by column chromatography using
hexane/ethyl acetate 75/25: yield 147 mg (70%); C16H13NO; MW
235; MS (ESI) 236 (M + H)+, 221, 193, 167, 139.
3-(4-Methoxyphenyl)quinoline (22a). The title compound was
prepared by reaction of 3-bromoquinoline (500 mg, 2.40 mmol)
with 4-methoxybenzene boronic acid according to method A during
18 h. The product was purified by column chromatography using
hexane/ethyl acetate 9/1: yield 504 mg (89%) of a white powder;
C16H13NO; MW 235; MS(ESI) 236 (M + H)+, 221, 193, 192, 167,
165, 154.
7-Methoxy-3-(3-methoxyphenyl)quinoline (24a). The title com-
pound was prepared by reaction of 3-bromo-7-methoxyquinoline
(255 mg, 1.07 mmol) with 3-methoxybenzeneboronic acid for 4.5 h
according to method A. The product was purified by column
chromatography using hexane/ethyl acetate 9/1: yield 215 mg
(76%); C17H15NO2; MW 265; MS (ESI) 266 (M + H)+.
Ether cleavage. Method C. General Procedure. To a solution
of methoxy derivative in dry toluene was added aluminum
trichloride under N2. The reaction mixture was heated at 90 °C for
2 h and cooled to room temperature. The reaction was quenched
by the addition of 2% Na2CO3 and extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
MgSO4. and evaporated to dryness.
Method D. General Procedure. To a solution of methoxy
derivative in dry dichloromethane (cooled to –78 °C) was added
boron tribromide (1 M solution in cyclohexane) under N2. The
reaction mixture was stirred at -78 °C for 1 h, at room temperature
for 1 h, and then allowed to warm to room temperature. The reaction
was quenched by the addition of 2% Na2CO3 and extracted with
dichloromethane. The combined organic layers were washed with
brine, dried over MgSO4, filtered, and evaporated.
by column chromatography (dichloromethane/methanol 95/5) af-
forded 13.1 mg (7%) of 20: C15H11NO2; MW 237; MS (ESI) 238
(M + H)+.
3-(Quinolin-3-yl)phenol (21). The title compound was prepared
by reaction of 3-(3-methoxyphenyl)quinoline (101 mg, 0.429 mmol)
with aluminum trichloride (343 mg, 2.58 mmol, 6.00 equiv)
according to method C. Purification by preparative thin layer
chromatography (silica, 1 mm thickness, dichloromethane/methanol
95/5) afforded 81 mg of the desired product (85%): C15H11NO;
MW 221; MS (ESI) 222 (M + H)+.
3-(4-Hydroxyphenyl)quinolin-7-ol (23).39 The title compound
was prepared by reaction of 7-methoxy-3-(4-methoxyphenyl)quino-
line (96 mg, 0.37 mmol, 1 equiv) with aluminum chloride (392
mg, 2.95 mmol, 6.00 equiv) according to method C. Purification
by column chromatography (dichloromethane/methanol 96/4) af-
forded 55 mg of the desired product (63%): C15H11NO2; MW 237;
MS (ESI) 238 (M + H)+.
3-(3-Hydroxyphenyl)quinolin-7-ol (24). The title compound
was prepared by reaction of 7-methoxy-3-(3-methoxyphenyl)quino-
line (108 mg, 0.407 mmol) with aluminum chloride (433 mg, 3.26
mmol, 8.00 equiv) according to method C. Purification by column
chromatography (dichloromethane/methanol 95/5) afforded 73 mg
of the desired product (76%): C15H11NO2; MW 237; MS (ESI) 238
(M + H)+.
2-(3-Hydroxyphenyl)-1H-indol-5-ol (26). The title compound
was prepared by reacting 5-methoxy-2-(3-methoxyphenyl)-1H-
indole (89 mg, 0.35 mmol) with boron tribromide (6.0 equiv)
according to method D. Purification by preparative thin-layer
chromatography (hexane/ethyl acetate 4/6) afforded 60 mg (76%)
of compound 26: C14H11NO2; MW 225; MS (ESI) 226 (M + H)+.
Biological Methods. [2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were
purchased from Perkin-Elmer, Boston. Quickszint Flow 302 scin-
tillator fluid was bought from Zinsser Analytic, Frankfurt. Other
chemicals were purchased from Sigma, Roth, or Merck.
1. Inhibition of 17ꢀ-HSD1. The synthesized compounds were
tested for their ability to inhibit 17ꢀ-HSD1 according to procedure
A (percentage of inhibition). For selected compounds, IC50 values
were determined according to procedure B. Procedures A and B
differ in enzyme source and substrate concentration. The two
procedures have been compared and give similar results.
Procedure A: Percentage of Inhibition Determination.
Enzyme Preparation. Recombinant baculovirus was produced by
the “Bac to Bac Expression System” (Invitrogen). Recombinant
bacmid was transfected to Sf9 insect cells using “Cellfectin
Reagent” (Invitrogen). Sixty hours later, cells were harvested; the
microsomal fraction was isolated as described by Puranen.44
Aliquots containing 17ꢀ-HSD1 were stored frozen until determi-
nation of enzymatic activity.
Assay. Recombinant protein (0.1 µg/mL) was incubated in 20
mM KH2PO4 pH 7.4 with 30 nM [3H]-estrone and 1 mM NADPH
for 30 min at room temperature, in the presence of potential
inhibitors at concentrations of 1 µM or 100 nM. Inhibitor stock
solutions were prepared in DMSO. Final concentration of DMSO
was adjusted to 1% in all samples. The enzyme reaction was stopped
by addition of 10% trichloroacetic acid (final concentration).
Samples were centrifuged in a microtiter plate at 4000 rpm for 10
min. Supernatants were applied to reverse phase HPLC on a Waters
Symmetry C18 column, equipped with a Waters Sentry Guard
column. Isocratic HPLC runs were performed at room temperature
at a flow rate of 1 mL/min of acetonitrile/water (48:52) as mobile
phase. Radioactivity of the eluate was monitored by a Packard Flow
Scintillation Analyzer. Total radioactivities for estrone and estradiol
were determined in each sample, and percent conversion of estrone
to estradiol was calculated.
6-(3-Nitrophenyl)-2-naphthol (10). The title compound was
prepared by reaction of 2-methoxy-6-(3-nitrophenyl)naphthalene
(200 mg, 0.72 mmol) with boron tribromide (5.6 equiv) according
to method D. Purification by column chromatography (hexane/ethyl
acetate 9/1) afforded 89.5 mg of the desired product (47%):
C16H11NO3; MW 265; MS (ESI) 264 (M - H)-.
6-(3-Aminophenyl)-2-naphthol (11). The title compound was
prepared by reaction of 3-(6-methoxynaphthalene-2-yl)phenylamine
(87.9 mg, 0.35 mmol, 1 equiv) with boron tribromide (5 equiv)
according to method D. Purification by column chromatography
(hexane/ethyl acetate 3/1) afforded 12 mg of the desired product
(14%): C16H13NO; MW 235; MS (ESI) 236 (M + H)+.
5-(6-Hydroxy-2-naphthyl)pyridin-3-ol (20). The title compound
was prepared by reaction of 3-methoxy-5-(6-methoxy-2-naphth-
yl)pyridine (200 mg, 0.754 mmol) with aluminum chloride (804
mg, 6.03 mmol, 8.00 equiv) according to method C. Purification
Procedure B: IC50 Value Determination. Enzyme Pre-
paration. 17ꢀ-HSD1 and 17ꢀ-HSD2 were partially purified from
human placenta according to the previously described procedures.19,45
Fresh human placenta was homogenized and fractionally centrifuged
at 1000g, 10000g, and 150000g. The pellet fraction contained the
microsomal 17ꢀ-HSD2, while 17ꢀ-HSD1 was obtained after
precipitation with ammonium sulfate from the cytosolic fraction.

Synthesis of (Hydroxyphenyl)naphthalene and -quinoline DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2167
Assay. Enzymatic activities were determined as described45–47
with minor modifications. Briefly, the enzyme preparation was
incubated with NADH (500 µM) in the presence of potential
inhibitors at 37 °C in a phosphate buffer supplemented with 20%
glycerol and EDTA 1 mM. 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 [3H]-
E1 (final concentration: 500 nM). After 30 min, the enzymatic
reaction was stopped with HgCl2, the steroids were extracted with
ether and the solvent evaporated. The steroids were dissolved in
acetonitrile. E1 and E2 were separated using an acetonitrile/water
mixture (45/55) as the mobile phase on a C18 reversed-phase
chromatography column (Nucleodur C18 Gravity, 3 µm, Macherey-
Nagel, Düren) connected to a HPLC-system (Agilent 1100 Series,
Agilent Technologies, Waldbronn). Detection and quantification of
the steroids were performed using a radioflow detector (Berthold
Technologies, Bad Wildbad). The conversion rate was calculated.
Each value is given as a mean value of at least three independent
experiments. The subsequent IC50 values were determined.
2. Inhibition of 17ꢀ-HSD2. The 17ꢀ-HSD2 inhibition assay
was performed as previously described for 17ꢀ-HSD1 according
to procedure B, using [3H]-E2 as substrate (final concentration: 500
nM) and NAD+ [1500 µM] as cofactor.
3. ER Affinity. The binding affinity of selected compounds to
the ER R and ER ꢀ was determined according to Zimmermann et
al.48 The human recombinant protein was used. Briefly, 0.25 pmol
of ERR or ERꢀ, respectively, were incubated with [3H]-E2 (10 nM)
and inhibitor for 1 h at room temperature. Inhibitor dilutions were
made in DMSO (5% final concentration). Nonspecific-binding was
determined with diethylstilbestrol (10 µM). After incubation,
ligand–receptor complexes were selectively bound to hydroxyapatite
(5 g/60 mL TE-buffer). The formed complex was washed and
resuspended in ethanol. For radiodetection, scintillator cocktail
(Quickszint 212, Zinsser Analytic, Frankfurt, Germany) was added
and samples were measured in a liquid scintillation counter (Rack
Beta Primo 1209, Wallac, Turku, Finland). For determination of
the relative binding affinity (RBA), the inhibitor concentration
required to displace 50% of the bound [3H]-E2 was determined.
RBA values were calculated according to following equation: RBA
(%) ) IC50(estradiol)/IC50(compound)100. The RBA value for E2
was arbitrarily set at 100%.
4. Caco-2 Transport Experiments. Caco-2 cell culture and
transport experiments were performed according to Yee49 with small
modifications. Cell culture time was reduced from 21 to 10 days
by increasing seeding density from 6.3 × 104 to 1.65 × 105 cells
per well. Four reference compounds (atenolol, testosterone, keto-
profene, erythromycin) were used in each assay for validation of
the transport properties of the Caco-2 cells. The compounds were
applied to the cells as mixtures (cassette dosing) to increase the
throughput of the cell permeability tests. The initial concentration
of the compounds in the donor compartment was 50 µM (for each
compound in buffer, containing either 1% ethanol or DMSO).
Samples were taken from the acceptor side after 0, 60, 120, and
180 min and from the donor side after 0 and 180 min. Each
experiment was run in triplicate. The integrity of the monolayers
was checked by measuring the transepithelial electrical resistance
(TEER) before the transport experiments and by measuring lucifer
yellow permeability after each assay. All samples of the Caco-2
transport experiments were analyzed by LC/MS/MS after dilution
with buffer of the opposite transwell chamber (1:1, containing 2%
acetic acid). The apparent permeability coefficients (Papp) were
calculated using equation Papp ) (dQ/dtAc0), where dQ/dt is the
appearance rate of mass in the acceptor compartment, A the surface
area of the transwell membrane, and c0 the initial concentration in
the donor compartment.
protein in phosphate buffer 100 mM pH 7.4 and 90 µL of NADP-
regenerating system (NADP+ 1 mM, glucose-6-phosphate 5 mM,
glucose-6-phosphate dehydrogenase 5 U/mL, MgCl2 5 mM).
The reaction was initiated by the addition of test compound to
the preincubated microsomes/buffer mix at 37 °C. The samples were
removed from the incubations after 0, 15, 30, and 60 min and
processed for acetonitrile precipitation. The samples were analyzed
by LC-MS/MS. Two control groups were run in parallel: positive
controls (PC; n ) 1) using 7-ethoxycoumarin as reference
compound to prove the quality of the microsomal enzymatic activity
and negative controls (NC; n ) 1), using boiled microsomes (boiling
water bath, 25 min) without regenerating system to ensure that the
potential apparent loss of parent compound in the assay incubation
is due to metabolism. The amount of compound in the samples
was expressed in percentage of remaining compound compared to
time point zero (100%). These percentages were plotted against
the corresponding time points and the half-life time was derived
by a standard fit of the data.
Intrinsic clearance (Clint) estimates were determined using the
rate of parent disappearance. The slope (-k) of the linear regression
from log [test compound] versus time plot was determined as well
as the elimination rate constant: k ) ln 2/t1/2. The equation
expressing the microsomal Clint can be derived: Clint ) kVfu (µL/
min/mg protein), where fu is the unbound fraction. V gives a term
for the volume of the incubation expressed in micro liters per mg
protein. As fu is not known for the tested compound, the calculation
was performed with fu ) 1 (V ) incubation volume (µL)/
microsomal protein (mg) ) 6667).
6. Inhibition of human hepatic CYPs. The commercially
available P450 inhibition kits from BD Gentest (Heidelberg,
Germany) were used according to the instructions of the manufac-
turer. Compound 5 was tested for inhibition of the following
enzymes: CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4. Inhibitory
potencies were determined as IC50 values.
Molecular modeling. X-ray structures of 17ꢀ-HSD1 were
obtained from the Protein Databank (PDB, www.pdb.org; 0). Water
molecules, E2 and sulfate ions were removed from the PDB file
and missing protein atoms were added.
Close contacts were fixed (Arg37) and correct atom types were
set. Finally hydrogen atoms and neutral end groups were added.
Superimpositions of E2 (from PDB ID: 1FDT) and inhibitors of
17ꢀ-HSD1 were performed by Schrödinger Maestro/Macromodel.50
Docking of inhibitors into the substrate binding site was
performed by the automated docking program GOLD 3.0.51
The molecular dynamics simulation was performed by the
AMBER 8 suite of programs.52 The simulation system was
minimized (5000 steps of steepest descent followed by 10000 steps
of conjugate gradient) and equilibrated at 300 K (20000 steps, step
size 1 fs). Finally, 500 ps of molecular dynamics simulation (step
size 1.0 fs, 100000 steps) at constant temperature (300 K) was
performed. Temperature was regulated by coupling to an external
bath (Berendsen’s method)53 using a bath coupling constant of 1.0
ps during equilibration and 1.5 ps during production. The general-
ized Born model54 was used to account for solvation effects.
Acknowledgment. We thank Ms. Anja Palusczak and Ms.
Beate Geiger for their help in performing the in Vitro tests (17ꢀ-
HSD1 procedure B, 17ꢀ-HSD2, ERR and ERꢀ). We are grateful
to Hormos Medical Corp. (Turku, Finland) for performing the
inhibition test on 17ꢀ-HSD1 (procedure A) and to Dr. Donald
Poirier (Laval University, Quebec, Canada) for providing the
hybride inhibitor EM-1745. We thank Dr. B. Husen (Solvay
Pharmaceuticals, Hannover, Germany) for discussions in the
field of estrogen-dependent diseases. We thank the Fonds der
chemischen Industrie for financial support. P.K. and C.S. are
grateful to the European Postgraduate School 532 (DFG) for a
scholarship.
5. Metabolic Stability Assay. The assay was performed with
liver microsomes from male Sprague–Dawley rats (BD GentestTM
,
Heidelberg, Germany). Stock solutions (10 mM in acetonitrile) were
diluted to give working solutions in 20% acetonitrile. The incubation
solutions consisted of a microsomal suspension of 0.33 mg/mL of
Supporting Information Available: Spectroscopic data (1H
NMR, 13C NMR, IR) and purity data of compounds 2, 3b, 3-5,

2168 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
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