Substituted 6-phenyl-2-naphthols. Potent and selective nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1): Design, synthesis, biological evaluation, and pharmacokinetics

Article abstract of DOI:10.1021/jm800367k

17β-Estradiol (E2) is implicated in the genesis and the development of estrogen-dependent diseases. Its concentration is mainly regulated by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), which catalyzes the reduction of the weak estrogen estrone (E1) to the highly potent E2. This enzyme is thus an important target for the treatment of hormone-dependent diseases. Thirty-seven novel substituted 6-phenyl-2-naphthols were synthesized and evaluated for 17β-HSD1 inhibition, selectivity toward 17β-HSD2 and the estrogen receptors (ERs) α and β, and pharmacokinetic properties. SAR studies revealed that the compounds most likely bind according to binding mode B to the active site, i.e., the 6-phenyl moiety mimicking the steroidal A-ring. While substitution at the phenyl ring decreased activity, introduction of substituents at the naphthol moiety led to highly active compounds, especially in position 1. The 1-phenyl compound 32 showed a very high inhibitory activity for 17β-HSD1 (IC₅₀ = 20 nM) and good selectivity (17β-HSD2 and ERs) and pharmacokinetic properties after peroral application.

Full text of DOI:10.1021/jm800367k

J. Med. Chem. 2008, 51, 4685–4698
4685
Substituted 6-Phenyl-2-naphthols. Potent and Selective Nonsteroidal Inhibitors of
17ꢀ-Hydroxysteroid Dehydrogenase Type 1 (17ꢀ-HSD1): Design, Synthesis, Biological
Evaluation, and Pharmacokinetics
Sandrine Marchais-Oberwinkler,† Patricia Kruchten,† Martin Frotscher,† Erika Ziegler,† Alexander Neugebauer,†
Umadevi Bhoga,† Emmanuel Bey,† Ursula Mu¨ller-Vieira,‡ Josef Messinger,§ Hubert Thole,§ and Rolf W. Hartmann*,†
8.2 Pharmaceutical and Medicinal Chemistry, Saarland UniVersity, P.O. Box 15 11 50, D-66041 Saarbru¨cken, Germany, Pharmacelsus CRO,
Science Park 2, D-66123 Saarbru¨cken, Germany, and SolVay Pharmaceuticals Research Laboratories, Hans-Bo¨ckler-Allee 20,
D-30173 HannoVer, Germany
ReceiVed April 1, 2008
17ꢀ-Estradiol (E2) is implicated in the genesis and the development of estrogen-dependent diseases. Its
concentration is mainly regulated by 17ꢀ-hydroxysteroid dehydrogenase type 1 (17ꢀ-HSD1), which catalyzes
the reduction of the weak estrogen estrone (E1) to the highly potent E2. This enzyme is thus an important
target for the treatment of hormone-dependent diseases. Thirty-seven novel substituted 6-phenyl-2-naphthols
were synthesized and evaluated for 17ꢀ-HSD1 inhibition, selectivity toward 17ꢀ-HSD2 and the estrogen
receptors (ERs) R and ꢀ, and pharmacokinetic properties. SAR studies revealed that the compounds most
likely bind according to binding mode B to the active site, i.e., the 6-phenyl moiety mimicking the steroidal
A-ring. While substitution at the phenyl ring decreased activity, introduction of substituents at the naphthol
moiety led to highly active compounds, especially in position 1. The 1-phenyl compound 32 showed a very
high inhibitory activity for 17ꢀ-HSD1 (IC50 ) 20 nM) and good selectivity (17ꢀ-HSD2 and ERs) and
pharmacokinetic properties after peroral application.
Chart 1. Interconversion of Estrone (E1) to Estradiol (E2)
Introduction
It is well recognized that estrogens play a central role in
female physiology. 17ꢀ-Estradiol (E2a), the endogenous ligand
of the estrogen receptors, however, is also known to be involved
in the development of estrogen-dependent diseases, inducing
cell proliferation in breast cancer1 and playing a critical role in
the development and growth of endometriosis.2
Until today, two different strategies have been developed for
the treatment of hormone-dependent breast cancer:3,4 (1) reduc-
tion of the estrogen biosynthesis either by aromatase inhibitors,
which prevent the transformation of androgens into estrogens,
or by using GnRH analogues, which inhibit ovarian estrogen
formation; (2) blockage of estrogen action at the receptor level
via selective estrogen receptor modulators (SERMs).
Another approach to reduce the estrogen action could be
achieved by inhibition of an enzyme catalyzing the last step in
E2 biosynthesis. Three different 17ꢀ-hydroxysteroid dehydro-
genase subtypes (1, 7, and 12) are principally able to catalyze
the reduction of estrone (E1) to E2. The primary role of 17ꢀ-
HSD7 and 17ꢀ-HSD12 is supposed to be in the cholesterol
synthesis5 and regulation of the lipid biosynthesis,6 respectively.
Moreover, Day et al.7 showed that 17ꢀ-HSD12, although highly
expressed in breast cancer cell lines, is inefficient in E2
formation. A pronounced reduction of the estrogen action should
be obtained by inhibition of 17ꢀ-HSD1.
17ꢀ-HSD1 (EC1.1.1.62) transforms the weak estrogen E1 into
the most potent estrogen E2 (Chart 1). 17ꢀ-HSD1 is a cytosolic
enzyme, which was crystallized with different steroidal ligands.8–16
The X-ray structures provide information about the active site
of the enzyme; it is an elongated hydrophobic tunnel with two
polar ends (His221, Glu282 on one side and Ser142, Tyr155
on the other side; the last two amino acids are involved in the
catalytic tetrad). Interestingly, another two polar amino acids
(Ser222 and Tyr218) are also located in this narrow cavity close
to the B/C ring of the steroid but do not interact directly with
it.
* To whom correspondence should be addressed. Phone: +49 681 302
3424. Fax: +49 681 302 4386. E-mail: rwh@mx.uni-saarland.de. Web site:
http://www.pharmmedchem.de.
†
Saarland University.
Pharmacelsus CRO.
Solvay Pharmaceuticals Research Laboratories.
‡
17ꢀ-HSD1 is often overexpressed in breast cancer cells17–20
resulting in high intracellular E2 concentrations. Inhibition of
17ꢀ-HSD1 will prevent the transformation of E1 into E2 and
thus will reduce the intracrine21 effect of E2. In contrast to
aromatase inhibition the E1 level will not be affected and a
basal estrogenic activity will be maintained. 17ꢀ-HSD1 inhibi-
tors therefore might be softer therapeutics for the treatment of
estrogen-dependent diseases; i.e., they should lead to fewer side
effects. 17ꢀ-HSD1 is therefore an attractive target for the design
of new drugs for breast cancer and endometriosis.
§
a Abbreviations: 17ꢀ-HSD1,17ꢀ-hydroxysteroid dehydrogenase type 1;
17ꢀ-HSD2, 17ꢀ-hydroxysteroid dehydrogenase type 2; E1, estrone; E2, 17ꢀ-
estradiol; ER, estrogen receptor; SERM, selective estrogen receptor modula-
tor; SAR, structure-activity relationship; PDB ID, Protein Data Bank
identification code; NADP(H), nicotinamide adenine dinucleotide phosphate;
RBA, relative binding affinity; DME, dimethoxyethane; DMAP, 4-dim-
ethylaminopyridine; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;
HOBt, 1-hydroxybenzotriazole; m-CPBA, m-chloroperoxybenzoic acid;
DMS, dimethyl sulfate. For the sake of clarity, IUPAC nomenclature is
not strictly followed except for the experimental part where the correct
IUPAC names are given.
10.1021/jm800367k CCC: $40.75
2008 American Chemical Society
Published on Web 07/17/2008

4686 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Marchais-Oberwinkler et al.
Recently, Laplante et al.22 showed that potent estradiol type
inhibitors were able to reduce the E1 induced T-47D cell
proliferation by 62% in vitro. Husen et al.23,24 and Day et al.7
have described mouse models to evaluate the efficacy of 17ꢀ-
HSD1 inhibitors in vivo. Both groups have used ovariectomized
immunodeficient mice and implanted MCF-7 cells stably
transfected with human 17ꢀ-HSD123,24 or T-47D cells,7 respec-
tively. The E1 induced tumor growth could be reduced by 17ꢀ-
HSD1 inhibitors.
Chart 2. Synthesized Compounds
Regarding the therapeutic concept, it is important that
inhibitors of 17ꢀ-HSD1 are selective toward 17ꢀ-HSD2 (which
inactivates E2 to E1, thus acting as an adversary to the type 1
enzyme) and toward the estrogen receptors R and ꢀ; i.e., they
should have no or little affinity to these proteins.
Over the past years, several groups have reported about 17ꢀ-
HSD1 inhibitors, most of them having a steroidal structure.22,25,26
The first nonsteroidal inhibitors of 17ꢀ-HSD1, the thiophenepy-
rimidinones, were published by Messinger et al.27 However,
most of the nonsteroidal 17ꢀ-HSD1 inhibitors described so far
do not appear to be druglike. Recently, on the basis of our
experience in the design of steroid mimicking compounds,28–30
we reported on the discovery of (hydroxyphenyl)naphthalenes
as potent and selective inhibitors of 17ꢀ-HSD1.31 The most
promising compound of this series of steroidomimetics was the
6-(3-hydroxyphenyl)-2-naphthol 1 (Chart 2).
Compound 1 can be considered as a scaffold for structure
optimization. Introduction of additional substituents leading to
new interactions with amino acids from the active site might
increase potency and selectivity of this class of inhibitors. In
this report, the rational design of novel, selective inhibitors of
17ꢀ-HSD1 (based on the analysis of possible binding modes
of the scaffold in the substrate binding site of the enzyme) will
be presented. Subsequently, synthesis and biological evaluation
of the derived substituted 6-phenyl-2-naphthols (Chart 2) will
be described.
Design
There are two modes for the binding of compound 1 into the
substrate binding site (Figure 1). Binding mode A is character-
ized by interactions of the phenyl-OH with Ser142 and Tyr155
from the catalytic tetrad while the naphthol-OH interacts with
His221 and Glu282. Binding mode B is characterized by the
formation of hydrogen bonds between the phenyl-OH and
His221, Glu282, as well as between the naphthol-OH and
Ser142, Tyr155. Docking of compound 1 into the active site of
17ꢀ-HSD1 (PDB ID 1FDT with the amino acids of the flexible
loop in the B conformation), after having removed E2, shows
that the space available for additional substituents on the
lipophilic core structure is different in the two binding modes.
This is visualized in Figure 1 with colored arrows: green arrows
indicate that there is space available for large substituents. In
orange, positions are shown with limited space (small substit-
uents), and in red, positions are marked where no space is
available for substitution.
In case compound 1 binds according to mode A, space is
available around position 4′ of the phenyl ring and position 1
of the naphthalene moiety to introduce a substituent; a com-
pound substituted at these positions should have some activity.
In contrast, introducing a substituent in position 5′, 6′, 3, 4, or
7 should result in an inactive compound. Accordingly, in case
compound 1 binds in binding mode B, a substituent in position
2′ and 1 should be tolerated and lead to some activity while
substitution in position 4′ and 6′ should result in inactive
compounds. These considerations are only valid when the amino
acids of the flexible loop (188-201) are in the B conformation,
while there is more space available for substitution in the A
conformation.
Compounds substituted at positions 4′ and 5′ of the phenyl
ring and positions 1, 3, 4, and 8 of the naphthalene moiety were
synthesized to get more insight in the binding mode of this class
of compounds and to find out about the best position to gain
activity.
Chemistry
Substitution at the Phenyl Ring. The synthesis of com-
pounds 2-18 substituted in positions 4′ and 5′ of the phenyl
ring is depicted in Schemes 1–4. Scheme 1 shows the synthesis
of compounds 2 and 3 substituted in position 4′ of the phenyl
ring with an amide moiety. These compounds were obtained
from Suzuki coupling32 using 4-bromo-2-methoxyaniline 2c and
6-methoxy-2-naphthalene boronic acid 2d as reactants. The

Substituted 6-Phenyl-2-naphthols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4687
Scheme 2. Synthesis of Compounds 4-6a
a Reagents and conditions: (a) Pd(PPh3)4, aqueous Na2CO3, DME, 80°C,
12-26 h; (b) BBr3, CH2Cl2, -78 °C, 4 h; (c) KMnO4, pyridine/H2O (2:5),
75 °C, 65 h; (d) acrylic acid, PPh3, Pd(OAc)2, NEt3, xylene, 100 °C, 11 h.
Scheme 3. Synthesis of Compounds 7-12a
Figure 1. Two possible binding modes for compound 1. For each
binding mode A and B, investigation of the space available around 1
was done for introduction of substituents. Green arrows indicate enough
space for large substituents. Orange arrows indicate space for smaller
substituents, and red arrows indicate no space for any substituents.
Dotted lines represent distances (Å).
Scheme 1. Synthesis of Compounds 2 and 3a
a Reagents and conditions: (a) RNH2, EDCI, HOBt, CH2Cl2, 0 °C to
room temp, overnight; (b) BBr3, CH2Cl2, -78 °C, 4 h; (c) Pd(OH)2, H2,
THF, room temp, 20 h.
according to the pathway described in Scheme 3. These
compounds were obtained after Suzuki coupling, acylation in
presence of EDCI and HOBt34 and ether cleavage (boron
tribromide). The intermediates 7b and 7c were prepared by
oxidation of the methyl derivatives 4a and 4b, respectively, with
potassium permanganate35 (Scheme 2). The acrylic acid 9c, the
precursor of 9b, was prepared from the corresponding bromo
derivative 5c via Heck reaction36 (Scheme 2). Subsequent
catalytic hydrogenation of the double bond (9a and 10a) was
performed using Pearlman’s catalyst.37
a Reagents and conditions: (a) Pd(PPh3)4, aqueous Na2CO3, toluene, 80
°C, overnight; (b) RCOCl, CH2Cl2, DMAP, room temp, overnight; (c) BBr3,
CH2Cl2, -78 °C, overnight.
Compounds 13-16 with a nitro instead of a hydroxy moiety
in position 3′ of the phenyl ring and an amide function in
position 5′ were also synthesized. The preparation of these
compounds 13-16 is described in Scheme 4 and is similar to
the route leading to (3′-hydroxyphenyl)naphthalenes 7 and 8
mentioned above. Reduction of the nitro groups, either by
hydrogenation in presence of palladium on charcoal as catalyst38
or by action of tin with hydrochloric acid,39 afforded amines
17 and 18.
Substitution at the Naphthalene Ring. The synthesis of
compounds 19-38 substituted in positions 1, 3, 4, and 8 of the
naphthalene moiety is depicted in Schemes 5–9.
Substituents introduced in position 1 include carboxylic acid
amides, bromine, phenyl, and sulfones. Synthesis of the amides
aniline moiety was N-acylated with two different acid chlorides.
Subsequent ether cleavage with boron tribromide33 led to 2 and
3.
Compounds 4-6 bearing alkyl or aromatic substituents in
position 5′ of the phenyl ring were synthesized according to
the route described in Scheme 2. Suzuki coupling between the
bromo derivatives (4b, 5c) and the appropriate boronic acid (one
Suzuki reaction for 4a and two successive Suzuki couplings
for 5a and 6a) followed by a demethylation step using boron
tribromide gave compounds 4-6.
Compounds 7 and 8 with an amide moiety directly linked to
position 5′ of the phenyl ring and compounds 9-12 bearing
the amide function linked via a C2 spacer were synthesized

4688 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Scheme 4. Synthesis of Compounds 13-18a
Marchais-Oberwinkler et al.
Scheme 6. Synthesis of Compounds 33 and 34a
a Reagents and conditions: (a) 3-methoxybenzeneboronic acid, Pd(PPh3)4,
toluene, aqueous Na2CO3, 80 °C, overnight; (b) BBr3, CH2Cl2, -78 °C to
room temp, overnight; (c) benzeneboronic acid, Pd(PPh3)4, toluene, aqueous
Na2CO3, 80 °C, 23 h.
a Reagents and conditions: (a) Pd(PPh3)4, aqueous Na2CO3, DME, 80
°C, overnight; (b) RNH2, EDCI, HOBt, CH2Cl2, 0 °C to room temp, 48 h;
(c) BBr3, CH2Cl2, -78 °C, 4 h; (d) H2, Pd/C, EtOH, 0 °C to room temp,
overnight; (e) Sn, HCl, THF, 50 °C, 1 h.
Scheme 7. Synthesis of Compounds 31 and 32a
Scheme 5. Synthesis of Compounds 19-30a
a Reagents and conditions: (a) DMF, reflux, 11 h; (b) m-CPBA, CH2Cl2,
0 °C to room temp, 12 h; (c) BBr3, CH2Cl2, -78 °C to room temp, overnight.
Scheme 8. Synthesis of Compounds 35 and 36a
a Reagents and conditions: (a) Br2, acetic acid, reflux, 2 h; (b) Sn, HCl,
acetic acid, reflux, 3 h; (c) DMS, K2CO3, acetone, reflux, 3 h; (d)
3-methoxybenzeneboronic acid, Pd(PPh3)4, toluene, aqueous Na2CO3, 80
°C, overnight; (e) LiOH, THF/H2O (1:1), reflux, 90 min; (f) RNH2, EDCI,
HOBt, CH2Cl2, 0 °C to room temp, overnight; (g) BBr3, CH2Cl2, -78 °C
to room temp, overnight.
a Reagents and conditions: (a) TiCl4, dichloromethyl methyl ether,
CH2Cl2, 0 °C to room temp, overnight; (b) 3-methoxybenzeneboronic acid,
Pd(PPh3)4, toluene, aqueous Na2CO3, 80 °C, 24 h; (c) H2NSO3H, NaOClO,
H2O/acetone (1:2), 0 °C, 30 min; (d) (i) SOCl2, room temp, 30 min; (ii)
RNH2, DME or CH2Cl2, DMAP, room temp, overnight; (e) RNH2, EDCI,
HOBt, CH2Cl2, room temp, overnight; (f) BBr3, CH2Cl2, -78 °C to room
temp, overnight; (g) triethylphosphonoacetate, NaH, dry DME, room temp,
1 h; (h) LiOH, THF/H2O (3:1), reflux, overnight; (i) Pd(OH)2, H2, ethanol/
THF (2:1), room temp, overnight.
conversion to the amides 19a-27a, the ether functions were
deprotected to give the desired compounds 19-27. In another
synthetic route, the aldehyde 19c was subjected to the
Horner-Wadsworth-Emmons conditions42 to introduce an
acrylic ester moiety (compound 28c). Hydrolysis of the ester,
amide formation, and ether cleavage afforded compounds 28
and 29. Reduction of the double bond by hydrogenation using
Pearlman’s catalyst led to 30.
19-30 is shown in Scheme 5. Formylation at position 1 of the
naphthalene moiety, performed according to the procedure
described by Royer and Buisson,40 followed by Suzuki cross-
coupling, led to the key intermediate, aldehyde 19c. It was
oxidized41 into the corresponding carboxylic acid 19b. After

Substituted 6-Phenyl-2-naphthols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4689
Scheme 9. Synthesis of Compounds 37 and 38a
Table 1. Inhibition of Human 17ꢀ-HSD1 and 17ꢀ-HSD2 by
Compounds 19-30
a Reagents and conditions: (a) pyridinium hydrochloride; 190 °C, 2 h;
(b) NaOMe, CuBr, reflux, 3 h; (c) Tf2O, pyridine, CH2Cl2, 0 °C; (d)
3-methoxybenzeneboronic acid, Pd(PPh3)4, DME, aqueous Na2CO3, 80 °C,
overnight.
The synthesis of the 1-phenylnaphthalene 32 is depicted in
Scheme 6. Regioselective Suzuki reaction of the 1,6-dibro-
monaphthalene 31b led to the 1-bromo-6-(3′-methoxyphenyl)-
naphthalene 31a. Subsequent ether cleavage led to the 1-bro-
monaphthol 31, which in turn was submitted to a second Suzuki
coupling to give compound 32.
The synthesis of the 1-sulfonylnaphthalenes 33 and 34 was
performed according to the route described in Scheme 7 from
the key intermediate 31a. Aromatic nucleophilic substitution
of the 1-bromonaphthalene 31a by sodium benzenethiolate or
4-methylbenzenethiolate43 led to the thioethers 33b and 34b.
Hydrolysis of the methoxy groups and oxidation with m-CPBA44
gave the corresponding sulfones 33 and 34.
Introduction of amide moieties in position 3 of the 2-naphthol
core was carried out as depicted in Scheme 8. The intermediate
35c was synthesized using the route described by Murphy et
al.45 It was submitted to Suzuki coupling, amide formation, and
ether cleavage to give the final compounds 35 and 36.
The synthesis of compounds substituted with a cyano moiety
at position 4 (37) or 8 (38) of the 2-naphthol system started
from the common intermediate 37c (Scheme 9). The latter was
obtained using the route described by Mewshaw et al.46 Starting
from 37c the 4-cyanonaphthalene 37 was prepared in a five-
step pathway according to the literature.46 Suzuki coupling
between 37c and 3-methoxybenzeneboronic acid followed by
a subsequent ether cleavage with pyridinium hydrochloride
afforded the 8-cyanonaphthalene 38.
a Recombinant human 17ꢀ-HSD1, substrate [3H]E1 [30 nM], NADPH
[1 mM], procedure A, mean value of two determinations. b Human placental
17ꢀ-HSD1, substrate [3H]E1 [500 nM], NADH [500 µM], procedure B,
mean value of three determinations, relative standard deviation of 10% for
27. c Human placental 17ꢀ-HSD2, substrate [3H]E2 [500 nM], NAD+ [1500
µM], procedure D, mean value of three determinations, relative standard
deviation of 38% for 19 and 27% for 20. d Recombinant human 17ꢀ-HSD2
[3H]E2 [30 nM], NAD+ [1 mM], procedure C, mean value of two
determinations. n.d. ) not determined, n.i. ) no inhibition (inhibition
<10%).
Biological Results
Inhibition of Human 17ꢀ-HSD1. As source of enzyme, both
recombinant and human placental enzymes were used. The
incubations were run with tritiated E1, cofactor, and inhibitor
and led to comparable results using both enzymes. The
separation of substrate and product was performed by HPLC.
The percent inhibition values of all hydroxy compounds are
shown in Tables 1 and 2 except for compounds 2 and 3 and
5-18, which are inactive and are not reported. The IC50 values
of selected compounds are shown in Table 3. 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).
the enzyme. A lack of space in this region of the active site
might be responsible for the loss of activity compared to
compound 1. In case the compounds bind according to binding
mode A, the flexible loop might reduce the space in the area of
the catalytic tetrad. It could be expected that the loop also adopts
other conformations; space should then be present in the region
close to positions 4′ and 5′ of the phenyl ring. The high diversity
of substituents introduced at these positions, leading to inactive
compounds, shows either that the loop remains located in front
of the substrate binding site or that these compounds do not
bind according to binding mode A.
The unsubstituted compound 1 identified as lead in the
Compounds 19-38 are substituted in positions 1, 3, 4, and 8
of the naphthalene ring. Introduction of a substituent having
the ability to be a hydrogen-bond acceptor or donor might enable
compounds to additionally interact with Tyr218 and/or Ser222,
the two polar amino acids present in the active site, thus
increasing 17ꢀ-HSD1 inhibitory activity. A compound binding
according to binding mode A, substituted in position 1 of the
naphthalene, might be able to establish hydrogen bond interac-
previous work31 was used as reference compound.
Compounds substituted at positions 4′ and 5′ of the phenyl
rings of 2-18 were all inactive except compound 4, with a
methyl group in position 5′ which presented a medium activity
(42% and 14% inhibition at 1 µM and 100 nM, respectively).
Substituents at position 4′ of the phenyl ring as well as at
position 5′ with the exception of methyl are not tolerated by

4690 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Marchais-Oberwinkler et al.
Table 2. Inhibition of Human 17ꢀ-HSD1 and 17ꢀ-HSD2 by
Compounds 31-38
position 8 of the naphthalene ring (38) reduced activity, while
the same substituent in position 4 (37) was very well tolerated
(Table 2).
For selected compounds IC50 values were determined (Table
3). Compounds 31 and 32 with an IC50 value of 40 and 20 nM,
respectively, were the most potent inhibitors identified in this
series (3-4 times more potent than the reference compound
1).
Selectivity. To assess the selectivity of the most interesting
compounds, inhibition of 17ꢀ-HSD2 and affinity to the estrogen
receptors R and ꢀ (ER R and ꢀ) were determined.
Since 17ꢀ-HSD2 catalyzes the oxidative transformation of
E2 into E1, thus “inactivating” E2, inhibition of this enzyme is
counterproductive for the treatment of estrogen-dependent
diseases. Briefly, human placental microsomes as source of 17ꢀ-
HSD2 were incubated with [3H]E2 in the presence of NAD+
and inhibitor. The amount of labeled E1 formed from substrate
was determined after HPLC separation. Inhibition of 17ꢀ-HSD2
was measured for compounds showing more than 75% inhibition
of 17ꢀ-HSD1 at 1 µM. Compounds substituted at the phenyl
ring have weak to no inhibitory activity toward 17ꢀ-HSD2
(<20%; data not shown). Compounds substituted at the
naphthalene ring are also weak to moderate inhibitors of 17ꢀ-
HSD2 (Tables 1 and 2). By use of the IC50 values, selectivity
factors ((IC50 HSD2)/(IC50 HSD1)) were calculated (Table 3).
It turned out that compounds 31 and 32 show reasonable
selectivity.
Furthermore, inhibitors of 17ꢀ-HSD1 should have low or no
affinity for ER R and ꢀ, since binding to these receptors could
counteract the therapeutic concept of 17ꢀ-HSD1 inhibition. For
the most interesting compounds, binding affinities were deter-
mined. The ER assays were performed using recombinant
human protein, [3H]E2, and inhibitor. Separation of bound and
free E2 was carried out using hydroxyapatite. Compounds 31
and 32 show very little affinity to the ERs (Table 3).
Since the ER binding affinity experiments do not explore the
intrinsic activity at the receptor, compounds 1 and 32 were
further investigated. By use of the estrogen dependent mammary
tumor cell line T-47D, cell proliferation was monitored after
incubation with the test compounds. E2 was used as positive
control. At an E2 concentration of 0.1 nM, a strong stimulation
of cell proliferation was observed after 8 days of incubation.
Administered in the same concentration, compounds 1 and 32
did not show any stimulatory effect. The compounds had to be
applied in a much higher concentration (100 nM, 1000-fold
excess compared to E2) to see a weak stimulation (41% and
39%, respectively, of the E2 effect).
Pharmacokinetic Evaluation of Compounds 1 and 32. To
get an idea about the in vivo behavior of compounds 1 and 32,
their pharmacokinetics were determined in the rat. After peroral
administration of 1 and 32 to male rats (10 mg/kg) (n ) 4) in
a cassette dosing approach, plasma samples were collected over
24 h and concentrations determined by LC-MS/MS. The
pharmacokinetic parameters of 1 and 32 are presented in Table
4. Each compound first showed a continuous increase in plasma
concentration over time for 6 h for 1 and for 4 h for 32 (Figure
2). Maximal plasma concentration (Cmax obs) was higher for 1
(2226 ng/mL) than for 32 (860 ng/mL); the time of maximal
plasma concentration (tmax obs) was measured 6 and 4 h after
administration for 1 and 32, respectively. Subsequently, plasma
concentrations decrease again. Plasma levels were very low 24 h
after administration. The mean profile of the plasma concentra-
tion of 1 and 32 is depicted in Figure 2. These results indicate
that both compounds exhibit a good pharmacokinetic profile in
a Recombinant human 17ꢀ-HSD1, substrate [3H]E1 [30 nM], NADPH
[1 mM], procedure A, mean value of two determinations. b Human placental
17ꢀ-HSD1, substrate [3H]E1 [500 nM], NADH [500 µM], procedure B,
mean value of three determinations. c Human placental 17ꢀ-HSD2, substrate
[3H]E2 [500 nM], NAD+ [1500 µM], procedure D, mean value of three
determinations. n.d. ) not determined. n.i. ) no inhibition (inhibition
<10%).
tions with these amino acids. In order to explore this hypothesis,
a number of compounds substituted with an amide in position
1 (19-30) were synthesized and evaluated for their 17ꢀ-HSD1
inhibitory activities (Table 1). The amide moiety was linked
directly to the position 1 (19-27) or linked via a C2 spacer
(28-30) (unsaturated or saturated). Amides 19-30 show a
moderate activity (between 45% and 80% inhibition at 1 µM)
except for the pyridine 25 and the pyrimidine 26, which are
inactive. Introduction of the spacer between the naphthalene
and the amide function does not enhance the activity. The
sulfones 33 and 34 also turned out to be moderately active (33%
and 75% inhibition at 1 µM, respectively) (Table 2). Introduction
of an amide or of a sulfone led to a significant drop in activity
compared to compound 1. There is some space available in this
position, but the nature of the substituent does not seem to be
appropriate. It is unlikely that hydrogen bond interactions are
formed with the two targeted amino acids Ser222 and Tyr218.
A bromine and a phenyl ring were also inserted in position
1 of the naphthalene. The corresponding compounds 31 and 32
turned out to be at least as active as the reference compound 1
(Table 2). This shows that there is space available for an
aromatic moiety in this region of the active site. The enhanced
activity of compound 32 might be indicative of π-π stacking
interactions.
Substitution in position 3 of the naphthalene was also
investigated. The amides 35 and 36, however, exhibited only
little and moderate activity. Introduction of a cyano function in

Substituted 6-Phenyl-2-naphthols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4691
Table 3. IC50 Values, Selectivity Factors, and Binding Affinities for the Estrogen Receptors R and ꢀ for Selected Compounds
compd
17ꢀ-HSD1 IC50 [nM]a,b
17ꢀ-HSD2 IC50 [nM]a,c
selectivity factorsd
ERR RBA (%)e
0.2
0.01 < RBA < 0.1
0.01 < RBA < 0.1
ERꢀ RBA (%)e
0.8
0.01 < RBA < 0.1
0.01 < RBA < 0.1
1
31
32
116
40
20
5641
639
540
48
16
27
a Mean value of three determinations. b Human placental 17ꢀ-HSD1, substrate [3H]E1 [500 nM], NADH [500 µM], procedure B. c Human placental
17ꢀ-HSD2, substrate [3H]E2 [500 nM], NAD+ [1500 µM], procedure D. d IC50(17ꢀ-HSD2)/IC50(17ꢀ-HSD1). e RBA: relative binding affinity, estradiol:
100%.
Table 4. Pharmacokinetic Parameters of Compounds 1 and 32 in Rats after Oral Application
parametersa
compd
dose [mg/kg]
Cmax obs [ng/mL]
Cz [ng/mL]
tmax obs [h]
tz [h]
t1/2z [h]
AUC0-tz [ng·h/mL]
AUC0-∞ [ng·h/mL]
1
32
10
10
2226
860
159
115
6
4
24
24
4
6
29 694
11 701
30 698
12 669
a Cmax obs: maximal measured concentration. Cz: last analytical quantifiable concentration. tmax obs: time to reach the maximum measured concentration. tz:
time of the last sample that has an analytical quantifiable concentration. t1/2z: half-life of the terminal slope of a concentration-time curve, AUC0-tz: area
under the concentration-time curve up to the time tz of the last sample. AUC0-∞: area under the concentration-time curve extrapolated to infinity.
lishes hydrogen bond interactions with His221 and Glu282, and
the naphthalene-OH with Ser142 and Tyr155 as described for
compound 1.31 Additionally, the phenyl-OH is stabilized via
π-π interaction with Tyr218 in a parallel-displaced geometry
(distance between the two ring centers, 5.09 Å). The phenyl-
naphthalene backbone is stabilized by hydrophobic interactions
(van der Waals). The phenyl ring in position 1 of the
naphthalene seems to be involved in hydrophobic interactions
with the nicotinamide part of the cofactor. The distance of the
ring centers (4.72 Å) and the closest contact distance between
a carbon of the phenyl ring of 32 and of the nicotinamide (3.63
Å) are in a good range, as it is described for π-π interactions
by McGaughey et al.47 Furthermore, this 1-phenyl ring could
additionally establish π-π interactions with Phe226 and with
Tyr155 (both T-shape interaction). The distances between the
ring centers are 4.23 and 5.42 Å, respectively. From the docking
pose, it also becomes apparent that a small hydrophobic pocket
is located close to position 4 of the naphthalene. The finding
that the 4-cyano compound 37 is active is in agreement with
the observation that there is space available for a small
substituent.
From the protein structure, steric hindrance and electrostatic
repulsion might be the reason for the fact that amide groups in
position 3 of the naphthalene (35 and 36) decrease activity of
the parent compound 1. However, in the case of the phenylamide
36, the aromatic moiety might be able to reach the hydrophobic
area close to position 4, stabilizing the molecule and explaining
a regaining in activity compared to the methylamide 35.
Figure 2. Mean profile ((SEM) of plasma levels (ng/mL) in rat versus
time after oral application (10 mg/kg) of compounds 1 and 32 in cassette
dosing.
the rat and that they might be good candidates for further
experiments in disease-oriented models.
Molecular Modeling
The biological results show that introduction of substituents
in position 4′ or 5′ of the phenyl ring is detrimental for activity
with the exception of a small group in 5′ (medium activity).
Substitution at the naphthalene ring is well tolerated in positions
1 and 4, while a decrease in activity is observed in positions 3
and 8. A correlation between these experimental data and the
space available around each position of compound 1 (deduced
from the docking poses presented in Figure 1) can be established
in the case of binding mode B, which appears to be the most
favorite binding mode of this series of substituted (3′-hydroxy-
phenyl)naphthalenes.
The most potent inhibitor described in this report, the
1-phenylnaphthol 32, was docked in the enzyme (PDB ID
1FDT) to better understand the favorable interactions achieved
by 32 in the active site. The obtained pose is shown in Figure
3. It is located in the substrate binding pocket according to
binding mode B. It has a flat geometry, like compound 1, only
the 1-phenyl moiety is turned about 60° away from the plane
of the naphthalene.
Discussion and Conclusion
The biological results obtained confirm the hypothesis31 that
substituted (3′-hydroxyphenyl)naphthalenes bind in the active
site according to binding mode B; i.e., the phenyl-OH moiety
of the compounds mimic the A-ring of E2. In this region, there
is no or very little (5′-methylphenyl, compound 4) space for a
substituent. On the other hand, space is available around the
naphthalene scaffold in positions 3 and 8 and especially 1 and
4 (region mimicking the C/D ring of E2). These results are in
agreement with the activity of substituted steroidal inhibitors
of 17ꢀ-HSD1 reported in the literature; the most active
compounds are substituted with large groups only at the D
ring48,49 (position 15 or 16) and a few at the B ring50 (position
6 or 7). Only a small substituent like ethyl or methoxy51 is
tolerated in position 2 of the steroid (A-ring), but activity is
often reduced. These findings are very important for drug design:
the space available for substituents on the scaffold of our
compounds is very limited (in the region corresponding to the
Compound 32 seems to be stabilized in the active site by
hydrogen bond and hydrophobic interactions (van der Waals
and arene-arene interactions). The phenyl-OH moiety estab-

4692 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Marchais-Oberwinkler et al.
Figure 3. Structure of the 17ꢀ-HSD1 binding pocket (green amino acids) with the docked compound 32 (yellow) following binding mode B in the
presence of the cofactor. Hydrogen bonding interactions and π-π stacking interactions are marked by violet lines and blue lines, respectively. All
distances are expressed in Å. The figure was generated with Pymol (http://www.pymol.org).
A-ring of E2), while an empty cavity is present in the
neighborhood of the catalytic tetrad (around the D-ring of E2).
Furthermore, our results indicate that position 1 of the
naphthalene ring is appropriate for the introduction of large
substituents and that aromatic groups are better tolerated than
amides or sulfones. The latter substituents were introduced with
the aim of establishing hydrogen bonds with Ser222 and Tyr218,
which might have been adequate partners in case of binding
mode A. The fact that all compounds 19-30 show a decrease
in activity is certainly in disfavor of binding mode A, as in this
mode hydrogen bond interactions should have been possible.
The low activity of these compounds, however, also demon-
strates that these groups are not able to interact with the
nicotinamide moiety of the cofactor (binding mode B).
It remains to be elucidated whether it is possible to establish
interactions with Ser222 and Tyr218 to improve activity and
selectivity. It is striking that these two polar amino acids, which
do not interact with the steroid, are present in the active site.
However, they might be involved in the stabilization of the three-
dimensional structure of the protein and thus might not be free
to establish another interaction.
In binding mode B, the phenyl moiety of the most potent
inhibitor 32 is located in the catalytic region where space is
available. It might undergo π-π stacking interactions with the
nicotinamide part of the cofactor (parallel-displaced geometry).
Other π-π interactions might also occur with the aromatic
amino acids Phe226 and Tyr155. It remains unclear why the
1-bromo compound 31 is more active than 1. It might be due
to the electronic effect of the bromine. However, hydrophobic
interactions of the bromine with the cofactor cannot be excluded.
Concerning the selectivity of the highly active 17ꢀ-HSD1
inhibitors, selected compounds were tested for inhibition of 17ꢀ-
HSD2 and affinity to ER R and ꢀ. Compounds 31 and 32
showed a high selectivity for 17ꢀ-HSD2, exhibiting selectivity
factors of 16 and 27, respectively. They also showed very little
affinity to ER R and ꢀ (between 0.01% and 0.1% that of E2).
This result is in accordance with the data reported by Mewshaw
et al.,46 who found that the introduction of a phenyl group into
the 1 position of 6-(4-hydroxyphenyl)-2-naphthol reduced ER
affinity strongly. This shows that there is no or little space in
the ERs to introduce a bulky substituent at the 1 position.46 In
contrast to substitution in the 1 position, a gain in ER affinity
was described for compounds substituted at the 4 position with
a cyano group.46 Therefore, position 1, and not position 4, is
appropriate to achieve selective 17ꢀ-HSD1 inhibition. The
affinity of 32 to the ERs corresponds to a very small agonistic
effect. Tested in 1000-fold higher concentration than E2 using
the ER-positive T-47D mammary tumor cell line, it showed 39%
of the stimulatory effect of E2.
In this report, we described the synthesis of substituted (3′-
hydroxyphenyl)-2-naphthols as inhibitors of 17ꢀ-HSD1 and the
evaluation of their biological properties. SAR studies using the
17ꢀ-HSD1 inhibition data revealed that the compounds most
likely bind according to binding mode B into the active site. A
new, potent, and selective 17ꢀ-HSD1 inhibitor, compound 32,
was discovered. It contains a phenyl group at the 1 position of
the naphthalene that may interact with binding partners in the
catalytic region. It is highly selective toward 17ꢀ-HSD2 and
the ER R and ꢀ and shows a good pharmacokinetic profile after
peroral application. It could be used in an in vivo disease-
oriented model to further validate the concept that 17ꢀ-HSD1
might be a promising target for the treatment of estrogen-
dependent diseases.
Experimental Section
Chemical Methods. IR spectra were measured neat on a Bruker
Vector 33 FT 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 deuteriated 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, q, dd, ddd, m,
b for singlet, doublet, triplet, quadruplet, doublet of doublet, doublet
of doublet of 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 (ThermoFisher).
Chemical names follow IUPAC nomenclature.
Starting materials (compounds 2d, 5c, 13c, 19e) were purchased
from Aldrich, Acros, Lancaster, or Fluka and were used without
purification. No attempts were made to optimize yields.
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). Preparative chro-
matography was performed on glass plate SIL G-100/UV254 (TLC,
silica, 1 mm thick) from Macherey-Nagel.
The following compounds were prepared according to previously
described procedures: 4-bromo-2-methoxyaniline 2c,52 1-bromo-
3-methoxy-5-methylbenzene 4b,53 1,6-dibromo-2-methoxynaph-
thalene 31b,54 methyl 7-bromo-3-methoxy-2-naphthoate 35c,45
8-cyano-6-methoxy-2-naphthyl trifluoromethanesulfonate 37b,46 and
3-bromo-7-methoxy-1-naphthonitrile 37c.46

Substituted 6-Phenyl-2-naphthols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4693
General Procedure for Suzuki Coupling. Method A. A mixture
of aryl bromide (1 equiv), boronic acid (1 equiv), 2% aqueous
solution of sodium carbonate (2 equiv), and tetrakis(triphenylphos-
phine)palladium(0) (0.1 equiv) in toluene or DME was stirred at
80 °C under nitrogen for 4-26 h. The reaction mixture was cooled
to room temperature, quenched by the addition of 2% HCl, and
extracted with dichloromethane. The organic layer was washed with
brine, dried over MgSO4, and concentrated to dryness. The product
was purified by chromatography.
2-Hydroxy-6-(3-hydroxyphenyl)-N-methyl-1-naphthamide (19).
The title compound was prepared by reaction of 2-methoxy-6-(3-
methoxyphenyl)-N-methyl-1-naphthamide 19a (250 mg, 0.78 mmol,
1 equiv) with boron tribromide (3.9 mmol, 5 equiv) according to
method F. Purification by column chromatography (dichloromethane/
methanol, 98:2) afforded the desired product in 95% yield (217
1
mg). C18H15NO3, MW 293. MS (ESI): 292 (M - H)-. H NMR
(CD3OD): δ 8.01 (d, J ) 1.9 Hz, 1H), 7.90 (dd, J ) 2.5 Hz, J )
8.8 Hz, 2H), 7.76 (dd, J ) 1.9 Hz, J ) 8.8 Hz, 1H), 7.31 (t, J )
7.9 Hz, 1H), 7.22-7.21 (m, 1H), 7.19 (d, J ) 8.8 Hz, 1H),
7.17-7.16 (m, 1H), 6.82 (ddd, J ) 0.6 Hz, J ) 2.2 Hz, J ) 7.9
Hz, 1H), 3.06 (s, 3H).
General Procedures for Amide Bond Formation. Method
B. A mixture of carboxylic acid (1 equiv) and amino derivative (1
equiv) dissolved in dichloromethane was added dropwise to a
solution of EDCI (1 equiv) and HOBt (1 equiv) in dichloromethane
at 0 °C. The reaction mixture was stirred at room temperature
overnight. After evaporation of the solvent, the residue was
dissolved in ethyl acetate, washed with saturated sodium carbonate
solution and brine, dried over MgSO4, filtered, and concentrated.
The product was purified by chromatography.
Method C. A mixture of carboxylic acid (1 equiv), EDCI (1
equiv), HOBt (1 equiv), and triethylamine (1 equiv) in dichlo-
romethane cooled at 0 °C was added dropwise to a solution of the
amino derivative (1 equiv) in dichloromethane. The reaction mixture
was refluxed for 1.5 h and quenched by addition of aqueous HCl
(0.1 M). The organic layer was separated, washed with sodium
carbonate and brine, dried over MgSO4, filtered, and concentrated.
The crude product was purified by chromatography.
Method D. Thionyl chloride (4 equiv) was added to the
carboxylic acid (1 equiv) under nitrogen and stirred in the presence
of a drop of DMF at room temperature for 30 min. After evaporation
of the thionyl chloride, the acid chloride (1 equiv) was dissolved
in dry THF or dichloromethane and added to the corresponding
amine (1 equiv) in solution in CH2Cl2 together with triethylamine
(1.2 equiv) and a catalytic amount of DMAP. During the addition,
the temperature was kept at 0 °C. The reaction mixture was refluxed
overnight, quenched by the addition of water, and extracted with
ethyl acetate. The organic phase was dried over MgSO4 and
concentrated to dryness. The desired amide was purified by
chromatography.
General Procedures for Ether Cleavage. Method E. To a
solution of methoxy derivative (1 equiv) in toluene was added
aluminum chloride (3-5 equiv per methoxy function) at room
temperature under N2. The reaction mixture was heated at 90 °C
for 2 h and then allowed to cool to room temperature. The reaction
was quenched by the addition of 2% Na2CO3. After extraction with
ethyl acetate, the combined organic layers were washed with brine
and dried over MgSO4. After evaporation of the solvent, the crude
product was purified by chromatography.
Method F. To a solution of methoxy derivative (1 equiv) in
dichloromethane cooled at -78 °C under N2 was slowly added
boron tribromide (1 M solution in dichloromethane, 3-5 equiv per
methoxy function). The reaction mixture was stirred at -78 °C 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 and dried over MgSO4. After evaporation of the solvent the
product was purified by chromatography.
Method G. The methoxy derivative (1 equiv) and pyridinium
hydrochloride (12 equiv) were heated at 220 °C for 3 h. The reaction
mixture was cooled at room temperature, and 1 N HCl (4 mL) was
added. The resulting precipitate was collected and dissolved in a
small amount of ethyl acetate. The organic layer was washed with
water and dried over Na2SO4, and the solvent was evaporated in
vacuo.
2-Hydroxy-6-(3-hydroxyphenyl)-N-phenyl-1-naphthamide (20).
The title compound was prepared by reaction of 2-methoxy-6-(3-
methoxyphenyl)-N-phenyl-1-naphthamide 20a (217 mg, 0.57 mmol,
1 equiv) with boron tribromide (3.42 mmol, 6 equiv) according to
method F. Purification by column chromatography (dichloromethane/
methanol, 95:5) afforded the desired product in 23% yield (45 mg).
C23H17NO3, MW 355. MS (ESI): 356 (M + H)+. 1H NMR
(CD3OD): δ 7.99 (d, J ) 1.2 Hz, 1H), 7.93 (d, J ) 8.8 Hz, 1H),
7.88 (d, J ) 8.8 Hz, 1H), 7.77 (d, J ) 7.6 Hz, 2H), 7.74 (dd, J )
1.8 Hz, J ) 8.8 Hz, 1H), 7.38 (t, J ) 7.9 Hz, 2H), 7.27-7.26 (m,
1H), 7.21 (d, J ) 8.8 Hz, 1H), 7.17-7.16 (m, 1H), 7.15-7.14 (m,
2H), 6.78 (ddd, J ) 0.9 Hz, J ) 2.4 Hz, J ) 7.9 Hz, 1H).
2-Hydroxy-N,6-bis(3-hydroxyphenyl)-1-naphthamide (21). The
title compound was prepared by reaction of 2-methoxy-N,6-bis(3-
methoxyphenyl)-1-naphthamide 21a (150 mg, 0.36 mmol, 1 equiv)
with boron tribromide (2.9 mmol, 8 equiv) according to method F.
Purification by column chromatography (dichloromethane/methanol,
93:7) afforded the desired product in 50% yield (66 mg).
C23H17NO4, MW 371. MS (ESI): 372 (M + H)+. 1H NMR
(CD3OD): δ 8.00 (d, J ) 1.6 Hz, 1H), 7.92 (d, J ) 8.5 Hz, 1H),
7.88 (d, J ) 9.1 Hz, 1H), 7.75 (dd, J ) 1.9 Hz, J ) 8.8 Hz, 1H),
7.44 (t, J ) 1.9 Hz, 1H), 7.27 (t, J ) 7.9 Hz, 1H), 7.18-7.17 (m,
5H), 6.78-6.77 (m, 1H), 6.61-6.60 (m, 1H).
6-(3-Hydroxyphenyl)-1-(piperidin-1-ylcarbonyl)-2-naphthol
(22). The title compound was prepared by reaction of 2-hydroxy-
6-(3-hydroxyphenyl)-1-naphthoic acid (160 mg, 0.57 mmol, 1
equiv) with piperidine (113 mL, 97 mg, 1.14 mmol, 2 equiv)
according to method D. After purification of the crude product by
column chromatography (dichloromethane/methanol, 95:5) com-
pound 22 was obtained in 8% yield (16 mg). C22H21NO3, MW 347.
1H NMR (CD3OD): δ 8.04 (d, J ) 1.9 Hz, 1H), 7.90 (d, J ) 8.8
Hz, 1H), 7.77 (dd, J ) 1.9 Hz, J ) 8.8 Hz, 1H), 7.66 (d, J ) 8.8
Hz, 1H), 7.30 (t, J ) 7.9 Hz, 1H), 7.23-7.21 (m, 1H), 7.20 (d, J
) 8.8 Hz, 1H), 7.17-7.16 (m, 1H), 6.82 (ddd, J ) 0.9 Hz, J ) 2.5
Hz, J ) 8.2 Hz, 1H), 3.98-3.97 (m, 1H), 3.87-3.83 (m, 1H),
3.69-3.67 (m, 1H), 3.31-3.29 (m, 1H), 1.81-1.79 (m, 3H),
1.75-1.36 (m, 3H).
6-(3-Hydroxyphenyl)-1-(morpholin-4-ylcarbonyl))-2-naph-
thol (23). The title compound was prepared by reaction of 4-[2-
methoxy-(6-(3-methoxyphenyl)-1-naphthyl)]-1-morpholine 23a (195
mg, 0.52 mmol, 1 equiv) with boron tribromide (2.6 mmol, 5 equiv)
according to method F. Purification by column chromatography
(dichloromethane/methanol, 95:5) afforded the compound 23 in 93%
yield (170 mg). C21H19NO4, MW 349. MS (ESI): 348 (M - H)-.
1H NMR (CD3OD): δ 8.04 (d, J ) 1.6 Hz, 1H), 7.92 (d, J ) 8.8
Hz, 1H), 7.79 (dd, J ) 1.9 Hz, J ) 8.8 Hz, 1H), 7.70 (d, J ) 8.8
Hz, 1H), 7.33-7.30 (m, 1H), 7.23-7.21 (m, 1H), 7.21 (d, J ) 8.8
Hz, 1H), 7.18-7.17 (m, 1H), 6.83-6.81 (m, 1H), 3.97-3.96 (m,
2H), 3.88-3.87 (m, 2H), 3.71-3.70 (m, 1H), 3.59-3.57 (m, 1H),
3.41-3.36 (m, 1H), 3.31-3-30 (m, 1H).
6-(3-Hydroxyphenyl)-1-(piperazin-1-ylcarbonyl))-2-naph-
thol (24). The title compound was prepared by reaction of tert-
butyl 4-(2-methoxy-6-(3-methoxyphenyl)-1-naphthoyl)piperazine-
1-carboxylate 24a (200 mg, 0.42 mmol, 1 equiv) with boron
tribromide (2.52 mmol, 6 equiv) according to method F. The desired
compound was obtained in 60% yield (81 mg). C21H20N2O3, MW
General Procedure for Reduction of the Double Bond.
Method H. A suspension of the olefinic compound (1 equiv) and
a catalytic amount of Pd(OH)2 in a mixture ethanol/THF (2:1) was
stirred at room temperature for 20 h under hydrogen atmosphere.
After completion of the reaction, the crude was filtered and
concentrated.
1
348. MS (ESI): 349 (M + H)+. H NMR (CD3OD): δ 8.05 (d, J
) 1.3 Hz, 1H), 7.93 (d, J ) 9.1 Hz, 1H), 7.79 (dd, J ) 1.6 Hz, J
) 8.5 Hz, 1H), 7.69 (d, J ) 8.8 Hz, 1H), 7.31 (t, J ) 7.9 Hz, 1H),

4694 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Marchais-Oberwinkler et al.
7.23 (d, J ) 8.8 Hz, 1H), 7.21-7.20 (m, 1H), 7.17-7.16 (m, 1H),
6.84-6.82 (m, 1H), 4.38-4.35 (m, 1H), 4.08-4.05 (m, 1H), 3.68
(m, 1H), 3.59-3.58 (m, 1H), 3.48-3.47 (m, 1H), 3.42-3.41 (m,
1H), 3.40-3.31 (m, 1H), 3.20-3.19 (m 1H).
quantitative yield (40 mg). C20H19NO3, MW 321. MS (ESI): 322
1
(M + H)+. H NMR (CD3OD): δ 8.04 (d, J ) 8.8 Hz, 1H), 7.98
(d, J ) 2.2 Hz, 1H), 7.76 (dd, J ) 1.9 Hz, J ) 8.8 Hz, 1H), 7.72
(d, J ) 8.8 Hz, 1H), 7.31 (t, J ) 7.91 Hz, 1H), 7.23-7.22 (m,
1H), 7.18-7.17 (m, 1H), 7.16 (d, J ) 8.8 Hz, 1H), 6.80 (ddd, J )
0.9 Hz, J ) 2.5 Hz, J ) 7.9 Hz, 1H), 3.41-3.38 (m, 2H), 2.74 (s,
3H), 2.60-2.57 (m, 2H).
2-Hydroxy-6-(3-hydroxyphenyl)-N-pyridin-2-yl-1-naphtha-
mide (25). The title compound was prepared by reaction of
2-methoxy-6-(3-methoxyphenyl)-N-(2-pyridyl)-1-naphthamide 25a
(33 mg, 0.086 mmol, 1 equiv) with boron tribromide (0.52 mmol,
6 equiv) according to method F. Purification by column chroma-
tography (dichloromethane/methanol, 99:1) afforded the desired
compound in 36% yield (11 mg). C22H16N2O3, MW 356. MS (ESI):
357 (M + H)+. 1H NMR (acetone-d6): δ 9.79 (bs, 1H), 8.48 (d, J
) 6.3 Hz, 1H), 8.29-8.28 (m, 1H), 8.25 (d, J ) 5.0 Hz, 1H),
8.09-8.08 (m, 1H), 8.00 (d, J ) 8.8 Hz, 1H), 7.89-7.88 (m, 1H),
7.82 (dd, J ) 1.8 Hz, 6.9 Hz, 1H), 7.33-7.32 (m, 4H), 7.14-7.13
(m, 1H), 6.86-6.85 (m, 1H).
1-Bromo-6-(3-hydroxyphenyl)-2-naphthol (31). The title com-
pound was prepared by reaction of 1-bromo-2-methoxy-6-(3-
methoxyphenyl)naphthalene 31a (500 mg, 1.46 mmol, 1 equiv) with
boron tribromide (7.3 mmol, 5 equiv) according to method F. It
was obtained in quantitative yield (460 mg). C16H11BrO2, MW 315.
1
MS (ESI): 313-315 (M - H)-. H NMR (CD3OD): δ 8.19 (d, J
) 8.8 Hz, 1H), 8.02 (d, J ) 1.9 Hz, 1H), 7.83 (dd, J ) 0.9 Hz, J
) 8.5 Hz, 1H), 7.83 (d, J ) 8.8 Hz, 1H), 7.32 (t, J ) 7.9 Hz, 1H),
7.24 (d, J ) 8.8 Hz, 1H), 7.25-7.23 (m, 1H), 7.19-7.18 (m, 1H),
6.83 (ddd, J ) 1.3 Hz, J ) 2.5 Hz, J ) 8.2 Hz, 1H).
2-Hydroxy-6-(3-hydroxyphenyl)-N-(pyrimidin-2-yl)-1-naph-
thamide (26). The title compound was prepared by reaction of
2-methoxy-6-(3-methoxyphenyl)-N-(pyrimidin-2-yl)-1-naphtha-
mide 26a (117 mg, 0.30 mmol, 1 equiv) with boron tribromide
(2.12 mmol, 7 equiv) according to method F. Purification by column
chromatography (dichloromethane/methanol, 98:2) afforded the
desired compound in 96% yield (99 mg). C21H15N3O3, MW 357.
MS (ESI): 358 (M + H)+. 1H NMR (acetone-d6): δ 9.90 (bs, 1H),
9.60 (s, 1H), 8.59 (d, J ) 5.8 Hz, 2H), 8.40 (s, 1H), 8.13 (d, J )
5.2 Hz, 1H), 8.05-8.04 (m, 1H), 7.95 (d, J ) 9.1 Hz, 1H), 7.75
(dd, J ) 1.8 Hz, J ) 6.9 Hz, 1H), 7.28 (t, J ) 8.1 Hz, 1H),
7.20-7.19 (m, 3H), 7.11 (t, J ) 5.0 Hz, 1H), 6.80-6.79 (m, 1H).
2-Hydroxy-6-(3-hydroxyphenyl)-N-(5-methyl-1,3,4-thiadiazol-
2-yl)-1-naphthamide (27). The title compound was prepared by
reaction of 2-methoxy-6-(3-methoxyphenyl)-N-(5-methyl-1,3,4-
thiadiazol-2-yl)-1-naphthamide 27a (620 mg, 1.53 mmol, 1 equiv)
with boron tribromide (12.24 mmol, 8 equiv) according to method
F. Purification by column chromatography (dichloromethane/
methanol, 98:2) afforded compound 27 in 20% yield (115 mg).
C20H15N3O3S, MW 377. MS (ESI): 378 (M + H)+. 1H NMR
(acetone-d6): δ 12.30 (bs, 1H), 10.03 (s, 1H), 9.07 (s, 1H), 7.66 (s,
1H), 7.55 (d, J ) 9.1 Hz, 1H), 7.31 (d, J ) 8.8 Hz, 1H), 7.23 (d,
J ) 7.8 Hz, 1H), 6.84 (t, J ) 7.8 Hz, 2H), 6.74 (d, J ) 8.1 Hz,
1H), 6.68-6.67 (m, 1H), 6.33-6.32 (m, 1H), 2.22 (s, 3H).
(2E)-3-[2-Hydroxy-6-(3-hydroxyphenyl)-1-naphthyl)]-N-me-
thylacrylamide (28). The title compound was prepared by reaction
of (2E)-3-[2-methoxy-6-(3-methoxyphenyl)-1-naphthyl)]-N-methy-
lacrylamide 28a (127 mg, 0.37 mmol, 1 equiv) with boron
tribromide (2.31 mmol, 8 equiv) according to method F. Purification
by column chromatography (dichloromethane/methanol, 95:5) af-
forded the desired product in 60% yield (71 mg). C20H17NO3, MW
6-(3-Hydroxyphenyl)-1-phenyl-2-naphthol (32). The title com-
pound was prepared by reaction of 1-bromo-6-(3-hydroxyphenyl)-
2-naphthol 31 (50 mg, 0.16 mmol, 1 equiv) with benzeneboronic
acid (19.4 mg, 0.16 mmol, 1 equiv) according to method A. The
crude product was purified by column chromatography (hexane/
ethyl acetate, 7:3) to give 32 in 30% yield (15 mg). C22H16O2, MW
1
312. MS (ESI): 311 (M - H)-. H NMR (CD3OD): δ 8.01 (d, J
) 1.9 Hz, 1H), 7.84 (d, J ) 8.8 Hz, 1H), 7.58 (dd, J ) 1.9 Hz, J
) 8.8 Hz, 1H), 7.56-7.53 (m, 2H), 7.48-7.45 (m, 2H), 7.42-7.40
(m, 2H), 7.30-7.27 (m, 1H), 7.26 (d, J ) 8.8 Hz, 1H), 7.21-7.19
(m, 1H), 7.17 (t, J ) 1.9 Hz, 1H), 6.80 (ddd, J ) 0.9 Hz, J ) 2.2
Hz, J ) 7.9 Hz, 1H).
6-(3-Hydroxyphenyl)-1-(phenylsulfonyl)-2-naphthol (33). To
a solution of 6-(3-hydroxyphenyl)-1-(phenylsulfanyl)-2-naphthol
33a (71 mg, 0.21 mmol, 1 equiv) in anhydrous dichloromethane
(10 mL) at 0 °C was added m-CPBA (192 mg, 0.82 mmol, 3.9
equiv) in CH2Cl2 (10 mL). The reaction mixture was stirred at 0
°C for 1 h and at room temperature for 12 h. The mixture was
poured into ice-water, and the crude was extracted with ethyl
acetate. The combined organic layers were washed with NaHCO3
and brine and dried over Na2SO4. After purification of the crude
product by column chromatography compound 33 was obtained in
50% yield (38 mg). C22H16O4S, MW 376. MS (ESI): 375(M -
1
H)-. H NMR (acetone-d6): δ 8.49 (d, J ) 9.1 Hz, 1H), 8.23 (d,
J ) 8.8 Hz, 1H), 8.11 (s, 1H), 8.06 (d, J ) 7.9 Hz, 1H), 7.82 (dd,
J ) 1.8 Hz, J ) 8.8 Hz, 1H), 7.70-7.62 (m, 3H), 7.27-7.26 (m,
2H), 7.16-7.15 (m, 2H), 6.85 (dt, J ) 1.8 Hz, J ) 8.8 Hz, 1H).
6-(3-Hydroxyphenyl)-1-(4-methylphenyl)sulfonyl)-2-naph-
thol (34). To a solution of 2-methoxy-6-(3-methoxyphenyl)-1-[(4-
methylphenyl)sulfanyl]naphthalene 34b (385 mg, 1.03 mmol, 1
equiv) in anhydrous dichloromethane (10 mL) at 0 °C was added
m-CPBA (1.42 g, 8.24 mmol, 8.2 equiv) in CH2Cl2 (10 mL). The
reaction mixture was stirred at 0 °C for 1 h and at room temperature
for 12 h, poured into ice-water, and extracted with ethyl acetate.
The combined organic layers were washed with NaHCO3 and brine
and dried over Na2SO4. A monodemethylation occurred during the
oxidation step. The second methoxy group of the product (105 mg,
0.26 mmol, 1 equiv) was then removed by reaction with boron
tribromide (2.1 mmol, 8 equiv) according to method F. The product
was obtained in 79% yield (80 mg). C23H18O4S, MW 390. MS
1
319. MS (ESI): 320 (M + H)+. H NMR (CD3OD): δ 8.29 (d, J
) 15.8 Hz, 1H), 8.27 (d, J ) 9.1 Hz, 1H), 7.99 (d, J ) 1.9 Hz,
1H), 7.81 (d, J ) 9.1 Hz, 1H), 7.79 (dd, J ) 1.9 Hz, J ) 8.8 Hz,
1H), 7.31 (t, J ) 7.91 Hz, 1H), 7.24-7.21 (m, 1H), 7.21 (d, J )
8.8 Hz, 1H), 7.20-7.19 (m, 1H), 7.11 (d, J ) 15.8 Hz, 1H), 6.82
(ddd, J ) 0.9 Hz, J ) 2.5 Hz, J ) 7.9 Hz, 1H), 2.93 (s, 3H).
(2E)-3-[2-Hydroxy-6-(3-hydroxyphenyl)-1-naphthyl)]-N-phe-
nylacrylamide (29). The title compound was prepared by reaction
of (2E)-3-[2-methoxy-6-(3-methoxyphenyl)-1-naphthyl)]-N-pheny-
lacrylamide 29a (70 mg, 0.17 mmol, 1 equiv) with boron tribromide
(1.36 mmol, 8 equiv) according to method F. Purification by column
chromatography (dichloromethane/methanol, 95:5) afforded the
product in 13% yield (8 mg). C25H19NO3, MW 381. MS (ESI):
382 (M + H)+. 1H NMR (CD3OD): δ 8.47 (d, J ) 15.8 Hz, 1H),
8.34 (d, J ) 8.8 Hz, 1H), 8.03 (d, J ) 1.9 Hz, 1H), 7.86 (d, J )
9.1 Hz, 1H), 7.83 (dd, J ) 1.9 Hz, J ) 8.8 Hz, 1H), 7.76 (d, J )
7.6 Hz, 1H), 7.40-7.36 (m, 3H), 7.34-7.31 (m, 1H), 7.26-7.24
(m, 1H), 7.24 (d, J ) 9.1 Hz, 1H), 7.21-7.20 (m, 1H), 7.17-7.14
(m, 1H), 6.83 (ddd, J ) 0.9 Hz, J ) 2.5 Hz, J ) 7.9 Hz, 1H).
3-[2-Hydroxy-6-(3-hydroxyphenyl)-1-naphthyl)]-N-methyl-
propanamide (30). The title compound was prepared by hydro-
genation of (2E)-3-[2-hydroxy-6-(3-hydroxyphenyl)-1-naphthyl)]-
N-methylacrylamide 28 (40 mg, 0.13 mmol, 1 equiv) with Pd(OH)2
according to method H. The compound 30 was obtained in
1
(ESI): 389 (M - H)-. H NMR (acetone-d6): δ 8.49 (d, J ) 8.8
Hz, 1H), 8.22 (d, J ) 8.8 Hz, 1H), 8.12 (d, J ) 2.1 Hz, 1H), 7.95
(d, J ) 8.5 Hz, 1H), 7.82 (dd, J ) 2.1 Hz, J ) 8.3 Hz, 1H), 7.43
(d, J ) 8.8 Hz, 2H), 7.31-7.26 (m, 2H), 7.19-7.18 (m, 2H),
6.87-6.86 (m, 1H).
3-Hydroxy-7-(3-hydroxyphenyl)-N-methyl-2-naphthamide (35).
The title compound was prepared by reaction of 3-methoxy-7-(3-
methoxyphenyl)-N-methyl-2-naphthamide 35a (93 mg, 0.29 mol,
1 equiv) with boron tribromide (5.5 mmol, 19 equiv) according to
method F. The product 35 was obtained in a quantitative yield (87
1
mg). C18H15NO3, MW 293. MS (ESI): 294 (M + H)+. H NMR
(CD3OD): δ 8.43 (s, 1H), 8.03 (s, 1H), 7.75 (s, 2H), 7.29 (t, J )
7.9 Hz, 1H), 7.26 (s, 1H), 7.19 (d, J ) 7.6 Hz, 1H), 7.15 (s, 1H),
6.80 (dd, J ) 2.2 Hz, J ) 7.9, 1H), 3.02 (s, 3H).

Substituted 6-Phenyl-2-naphthols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4695
3-Hydroxy-7-(3-hydroxyphenyl)-N-phenyl-2-naphthamide (36).
The title compound was prepared by reaction of 3-methoxy-7-(3-
methoxyphenyl)-N-phenyl-2-naphthamide 36a (86 mg, 0.22 mol,
1 equiv) with boron tribromide (4.45 mmol, 20 equiv) according
to method F. After purification of the crude product by column
chromatography (hexane/ethyl acetate, 6:4) compound 36 was
obtained in 50% yield (40 mg). C23H17NO3, MW 355. MS (APCI):
355 (M)+. 1H NMR (DMSO-d6): δ 11.54 (bs, 1H), 10.14 (bs, 1H),
8.73 (s, 1H), 8.47 (bs, 1H), 8.08 (s, 1H), 7.84-7.81 (m, 4H), 7.42
(dd, J ) 0.9 Hz, J ) 8.5 Hz, 2H), 7.36 (s, 1H), 7.31 (t, J ) 8.2
Hz, 1 H), 7.23-7.18 (m, 3H), 6.88-6.86 (m, 1H).
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 acetonitrile/water (48:52) as running
solvent. Radioactivity of the eluate was monitored by a Packard
flow scintillation analyzer. Total radioactivities for E1 and E2 were
determined in each sample. The conversion rate was calculated
according to the following formula: % conversion ) 100[(cpm E2
in sample with inhibitor)/(cpm E1 in sample with inhibitor + cpm
E2 in sample with inhibitor)]/[(cpm E2 in sample without inhibitor)/
(cpm E1 in sample without inhibitor + cpm E2 in sample without
inhibitor)]. Percentage of inhibition was calculated according to
the following equation: % inhibition ) 100 - % conversion. Each
value was calculated from two independent experiments.
Procedure B Using Human Placental Enzyme. Assay. Inhibi-
tory activities were evaluated by an established method with minor
modifications.57–59 Briefly, the enzyme preparation was incubated
with NADH (500 µM) in the presence of potential inhibitors at 37
°C in a phosphate buffer (50 mM) supplemented with 20% 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 and [3H]E1 (final concentration: 500 nM, 0.15
µCi). After 10 min, 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 chroma-
tography column (Nucleodur C18 Gravity, 3 µm, Macherey-Nagel,
Du¨ren) 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
according to following equation: % conversion ) % E2/(% E2 +
% E1) × 100. Each value was calculated from at least three
independent experiments.
3. Inhibition of 17ꢀ-HSD2. The synthesized compounds were
tested for their ability to inhibit 17ꢀ-HSD2 according to procedure
C (recombinant human enzyme) or D (human placental enzyme).
For select compounds, IC50 values were determined according to
procedure D (human placental enzyme). Procedures C and D differ
from enzyme source and substrate concentration. The two proce-
dures have been compared and give similar results.
Procedure C Using Recombinant Human Enzyme. The 17ꢀ-
HSD2 inhibition assay was performed as previously described for
17ꢀ-HSD1 according to procedure A from the recombinant human
protein, using [3H]E2 as substrate (30 nM) and NAD+ (1 mM) as
cofactor.
Procedure D Using Human Placental Enzyme. The 17ꢀ-HSD2
inhibition assay was performed similarly to the 17ꢀ-HSD1 proce-
dure. The microsomal fraction was incubated with NAD+ (1500
µM), test compound, and a mixture of unlabeled and [3H]E2 (final
concentration: 500 nM, 0.11 µCi) for 20 min at 37 °C. Further
treatment of the samples and HPLC separation was carried out as
mentioned above for 17ꢀ-HSD1.
3-Hydroxy-7-(3-hydroxyphenyl)-1-naphthonitrile (37). The
title compound was prepared by reaction of 3-methoxy-7-(3-
methoxyphenyl)-1-naphthonitrile 37a (100 mg, 0.35 mmol, 1 equiv)
with pyridinium hydrochloride (492 mg, 4.3 mmol, 12 equiv) according
to method G. The desired compound was obtained in 64% yield (58
1
mg). C17H11NO2, MW 261. MS (ESI): 260 (M - H)-. H NMR
(CD3OD): δ 8.20 (s, 1H), 7.89 (d, J ) 8.5 Hz, 1H), 7.85 (dd, J ) 1.9
Hz, J ) 6.9 Hz, 1H), 7.61 (d, J ) 2.2 Hz, 1H), 7.50 (d, J ) 2.5 Hz,
1H), 7.35 (dd, J ) 2.2 Hz, J ) 7.6 Hz, 1H), 7.24-7.23 (m, 1H), 7.20
(dd, J ) 2.2 Hz, J ) 1.9 Hz, 1H), 6.87-6.86 (m, 1H).
7-Hydroxy-3-(3-hydroxyphenyl)-1-naphthonitrile (38). The
title compound was prepared by reaction of 7-methoxy-3-(3-
methoxyphenyl)-1-naphthonitrile 38a (110 mg, 0.39 mmol, 1 equiv)
with pyridinium hydrochloride (547 mg, 4.76 mmol, 12.2 equiv)
according to method G. The compound was obtained in 62% yield
(63 mg). C17H11NO2, MW 261. MS (ESI): 260 (M - H)-. 1H NMR
(CD3OD): δ 8.30 (bs, 1H), 8.18 (d, J ) 1.9 Hz, 1H), 7.98 (d, J )
8.8 Hz, 1H), 7.47-7.46 (m, 1H), 7.36-7.33 (m, 1H), 7.28 (dd, J
) 2.2 Hz, J ) 8.8 Hz, 1H), 7.24-7.22 (m, 1H), 7.17-7.16 (m,
1H), 6.87-6.85 (m, 1H).
Biological Methods. [2,4,6,7-3H]E1 and [2,4,6,7-3H]E2 were
purchased from Perkin-Elmer, Boston, MA. Quickszint Flow 302
scintillator fluid was bought from Zinsser Analytic, Frankfurt,
Germany. T-47D cells (passage 9) were obtained from ECACC,
Salisbury, U.K. FCS was purchased from Sigma, Taufkirchen,
Germany. Cell culture media and dextran coated charcoal stripped
FCS (DCC-FCS) were bought from CCPRO, Oberdorla, Germany.
Other chemicals were purchased from Sigma, Roth, or Merck.
1. 17ꢀ-HSD1 and 17ꢀ-HSD2 Enzyme Preparation. Recom-
binant Human Enzyme (Procedures A and C). 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.55 Aliquots containing 17ꢀ-HSD1 or 17ꢀ-HSD2 were
stored frozen until determination of enzymatic activity.
Human Enzyme Enriched from Placental Tissue (Procedures
B and D). 17ꢀ-HSD1 and 17ꢀ-HSD2 were obtained from human
placenta according to previously described procedures.11,56 Fresh
human placenta was homogenized, and the enzymes were separated
by subcellular fractionation (centrifugation method). For the
purification of 17ꢀ-HSD1, the cytosolic fraction was precipitated
with ammonium sulfate. 17ꢀ-HSD2 was obtained from the mi-
crosomal fraction. Aliquots containing 17ꢀ-HSD1 or 17ꢀ-HSD2
were stored frozen.
2. Inhibition of 17ꢀ-HSD1. The synthesized compounds were
tested for their ability to inhibit 17ꢀ-HSD1 according to procedure
A (recombinant human enzyme) or B (human placental enzyme).
For select compounds, IC50 values were determined according to
procedure B (human placental enzyme). Procedures A and B differ
from enzyme source and substrate concentration. The two proce-
dures have been compared and give similar results.
Procedure A Using Recombinant Human Enzyme. Assay.
Recombinant human 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).
4. ER Affinity
The binding affinity of select compounds to the ERR and
ERꢀ was determined according to Zimmermann et al.60 using
recombinant human proteins. Briefly, 0.25 pmol of ERR or ERꢀ,
respectively, was incubated with [3H]E2 (10 nM) and test
compound for 1 h at room temperature. The potential inhibitor
was dissolved in DMSO (5% final concentration). Nonspecific
binding was performed with diethylstilbestrol (10 µM). After
incubation, ligand-receptor complexes were selectively bound
to hydroxyapatite (5 g/60 mL TE buffer). The formed complex
was separated, washed, and resuspended in ethanol. For radio-
detection, scintillator cocktail (Quickszint 212, Zinsser Analytic,
Frankfurt) was added and samples were measured in a liquid

4696 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Marchais-Oberwinkler et al.
scintillation counter (Rack Beta Primo 1209, Wallac, Turku).
For determination of the relative binding affinity (RBA),
inhibitor and E2 concentrations required to displace 50% of the
receptor bound labeled E2 were determined. RBA values were
calculated according to the following equation: RBA[%] )
IC50(E2)/IC50(compound) × 100. The RBA value for E2 was
arbitrarily set at 100%.
This test was performed according to the Laws on Animal Care
and Use and had been approved by the local Animal Care
Committee.
Molecular Modeling. All molecular modeling studies were
performed on an Intel(R) P4 CPU, 3.00 GHz, running Linux
Suse 9.3. X-ray structure of 17ꢀ-HSD1 (PDB ID 1FDT,
www.pdb.org using the amino acids 188-201 in the B
conformation) was prepared using the BIOPOLYMER module
of SYBYL version 7.0 (Sybyl, Tripos Inc., St. Louis, MO).
Water molecules, E2, and sulfate ions were stripped from the
PDB file, and missing protein atoms were added and correct
atom types set. Finally hydrogen atoms and neutral end groups
were added. All basic and acidic residues were considered
protonated and deprotonated, respectively. Furthermore, the
crystal structure was minimized for 200 steps with the steepest
descent minimizer as implemented in SYBYL with the backbone
atoms kept at fixed positions in order to fix close contacts
(Arg37).
Docking of selected inhibitors, built with SYBYL and energy-
minimized in MMFF94s force field as implemented in Sybyl,
into the substrate binding site was performed by the docking
program GOLD version 3.0.1.62 Since the GOLD docking
program allows flexible docking of ligands, no conformational
search was employed to the ligand structures. Ligands were
docked in 50 independent genetic algorithm (GA) runs. Active-
site origin was set at the center of the steroid binding site, while
the radius was set equal to 13 Å. The automatic active-site
detection was switched on. Further, the CHEMSCORE fitness
function was used and genetic algorithm default parameters were
set as suggested by the GOLD authors.
5. Proliferation Assay
Cell Culture. Stock culture of T-47D cells (ecacc, U.K.) was
maintained in RPMI-1640 supplemented with sodium bicarbon-
ate (2 g/L), streptomycin (100 µg/mL), insuline zinc salt (10
µg/mL), sodium pyruvate (1 mM), penicillin (100 U/mL), and
FCS 10% (vol/vol). Cells were cultured at 37 °C under 5% CO2
humidified atmosphere. Medium was changed every 2-3 days,
and cells were subcultured every 4-5 days.
Evaluation of Estrogenic Effects on the Estrogen
Dependent Human Breast Cancer Cell Line T-47D. Phenol
red-free medium was supplemented with sodium bicarbonate
(2 g/L), streptomycin (100 µg/mL), insuline zinc salt (10 µg/
mL), sodium pyruvate (1 mM), L-glutamine (2 mM), penicillin
(100 U/mL), and DCC-FCS 5% (vol/vol). RPMI 1640 (without
phenol red) was used for the experiments. Cells (7500 cells/
96-well plate) were grown for 48 h in phenol red-free medium.
The compounds tested were added at a final concentration of
100 nM. Inhibitors and E2 were diluted in ethanol (final ethanol
concentration was adjusted to 1%). As a positive control E2
was added at a final concentration of 0.1 nM. Ethanol was used
as negative control. Medium was changed every 2-3 days and
supplemented with the respective additive. After 8 days of
incubation, the cell viability was evaluated measuring the
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
liumbromide (MTT). The cleavage of MTT to a blue formazan
by mitochondrial succinate-dehydrogenase was quantified spec-
trophotometrically at 590 nm as described by Denizot and
Lang61 with minor modifications. The control proliferation was
arbitrarily set at 1, and the stimulation induced by the inhibitor
was calculated according to following equation: % stimulation)
[proliferation(compound - induced) - 1]/[proliferation(E2 -
induced) - 1] × 100%. Each value is calculated as a mean
value of at least three independent experiments.
The quality of the docked poses was evaluated on the basis
of the scoring function, which gives a good measure to
discriminate between the found binding modes, and mainly on
visual inspection of the putative binding modes of the ligands.
Acknowledgment. We thank Anja Palusczak and Beate
Geiger for their help in performing the in vitro tests (17ꢀ-HSD1
procedure B, 17ꢀ-HSD2 procedure D, ERR, and ERꢀ). We are
grateful to Hormos Medical Corp. (Finland) for performing the
inhibition test on 17ꢀ-HSD1 (procedure A) and 17ꢀ-HSD2
(procedure C). We thank Matthias Negri for useful discussion
concerning the modeling section and for his help with Figure
3. P.K. is grateful to the European Postgraduate School 532
(DFG) for a scholarship.
6. Evaluation of Plasma Concentrations of Compounds 1
and 32 after Peroral Application to Adult Male Rats in
Cassette Dosing
Supporting Information Available: Additional experimental
details on the synthesis and spectroscopic data of 1-38 and purity
data of compounds 2, 3, 5, 7, 8, 10, 11, 19-24, 26, 28-32, 35, 37,
38. This material is available free of charge via the Internet at http://
pubs.acs.org.
Four adult male Wistar rats (Janvier, France) were used.
Animals were housed in a temperature-controlled room (20-22
°C) and maintained in a 12 h light/12 h dark cycle. Food and
water were available ad libitum. Rats were anesthetized with a
ketamine (135 mg/kg)/xylazine (10 mg/kg) mixture and can-
nulated with silicone tubing via the right jugular vein. Prior to
the first blood sampling, animals were connected to a counter-
balanced system and tubing to perform blood sampling in the
freely moving rat. Compounds 1 and32, dissolved in labrasol/
water (1:1) as vehicle, were administered perorally at doses of
10 mg/kg in a cassette dosing approach. Each application group
consisted of four rats. At time 0, compounds 1 and 32 were
applied and blood samples (200 µL) were taken at 1, 2, 3, 4, 6,
8, 10, and 24 h postdose, collected in heparinized tubes, and
stored on ice. Plasma was harvested and kept at -20 °C until
being assayed. HPLC-MS/MS analysis and quantification of the
samples were carried out on a Surveyor HPLC system coupled
with a TSQ Quantum (ThermoFisher) triple quadrupole mass
spectrometer equipped with an electrospray ion interface (ESI).
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