Design, synthesis, biological evaluation and pharmacokinetics of bis(hydroxyphenyl) substituted azoles, thiophenes, benzenes, and aza-benzenes as potent and selective nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1)

Article abstract of DOI:10.1021/jm8006917

17β-Estradiol (E2), the most potent female sex hormone, stimulates the growth of mammary tumors and endometriosis via activation of the estrogen receptor α (ERα). 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1), which is responsible for the catalytic r

Full text of DOI:10.1021/jm8006917

J. Med. Chem. 2008, 51, 6725–6739
6725
Design, Synthesis, Biological Evaluation and Pharmacokinetics of Bis(hydroxyphenyl)
substituted Azoles, Thiophenes, Benzenes, and Aza-Benzenes as Potent and Selective
Nonsteroidal Inhibitors of 17ꢀ-Hydroxysteroid Dehydrogenase Type 1 (17ꢀ-HSD1)
,†
Emmanuel Bey,† Sandrine Marchais-Oberwinkler,† Ruth Werth,† Matthias Negri, Yaseen A. Al-Soud,§,† Patricia Kruchten,†
Alexander Oster,† Martin Frotscher,† Barbara Birk,‡ and Rolf W. Hartmann*,†
Pharmaceutical and Medicinal Chemistry, Saarland UniVersity, PO Box 15 11 50, D-66041, Saarbru¨cken, Germany, Pharmacelsus CRO,
Science Park 2, D-66123 Saarbru¨cken, Germany
ReceiVed June 6, 2008
17ꢀ-Estradiol (E2), the most potent female sex hormone, stimulates the growth of mammary tumors and
endometriosis via activation of the estrogen receptor R (ERR). 17ꢀ-Hydroxysteroid dehydrogenase type 1 (17ꢀ-
HSD1), which is responsible for the catalytic reduction of the weakly active estrogen estrone (E1) into E2, is
therefore discussed as a novel drug target. Recently, we have discovered a 2,5-bis(hydroxyphenyl) oxazole to be
a potent inhibitor of 17ꢀ-HSD1. In this paper, further structural optimizations were performed: 39 bis(hydrox-
yphenyl) azoles, thiophenes, benzenes, and aza-benzenes were synthesized and their biological properties were
evaluated. The most promising compounds of this study show enhanced IC50 values in the low nanomolar range,
a high selectivity toward 17ꢀ-HSD2, a low binding affinity to ERR, a good metabolic stability in rat liver
microsomes, and a reasonable pharmacokinetic profile after peroral application. Calculation of the molecular
electrostatic potentials revealed a correlation between 17ꢀ-HSD1 inhibition and the electron density distribution.
Introduction
most important of which is 17ꢀ-HSD1. The primary function
of 17ꢀ-HSD7 and 17ꢀ-HSD12 is supposed to be in the
cholesterol synthesis6 and in the regulation of the lipid biosyn-
thesis,7 respectively. Moreover, Day et al.8 showed that 17ꢀ-
HSD12, although highly expressed in breast cancer cell lines,
is inefficient in E2 formation.
Estrogens, the most potent one being 17ꢀ-estradiol (E2a), act
as female sex hormones and are predominantly produced before
menopause by the ovaries. They unfold their activity by
stimulation of the estrogen receptors (ERs) R and ꢀ. Besides
their physiological effects, they are, however, also involved in
the initiation and progression of estrogen-dependent diseases
like mammary tumor1 and endometriosis.2 Presently, the three
main endocrine therapies for the treatment of breast cancer are:3,4
inhibition of estrogen biosynthesis by aromatase inhibitors or
GnRH agonists or antagonists and interference with the estrogen
action at the receptor level by selective estrogen receptor
modulators (SERMs) or pure antiestrogens.5 Besides specific
disadvantages of each therapeutic concept, all of these strategies
have in common that they reduce estrogen levels systemically,
leading to the corresponding side effects.
17ꢀ-HSD1 is NAD(P)H-dependent and intracellularly con-
verts the weak estrogen E1 into the strong estrogen E2. As it is
often overexpressed in breast cancer cells9-12 and endometrio-
sis,13 17ꢀ-HSD1 is regarded as a promising novel target for
the treatment of estrogen-dependent diseases. Appropriate
inhibitors of this enzyme should exhibit fewer side effects
compared to the current treatments, as they should selectively
reduce the concentration of active E2 in the diseased tissues.14
As a biological counterpart, 17ꢀ-hydroxysteroid dehydroge-
nase type 2 (17ꢀ-HSD2) catalyzes the deactivation of E2 into
E1. It protects the cell from excessively high concentrations of
active estrogens15 and should therefore not be affected by
inhibitors of 17ꢀ-HSD1. In addition, 17ꢀ-HSD1 inhibitors
should not show affinity to the ERs to avoid intrinsic estrogenic
effects.
A softer approach could be inhibition of the enzyme involved
in the last step of the E2 biosynthesis: 17ꢀ-hydroxysteroid
dehydrogenase (17ꢀ-HSD), which is able to convert estrone (E1)
into E2. There are three subtypes (1, 7, and 12) described, the
17ꢀ-HSD1 was crystallized with different steroidal ligands.16-24
The published X-ray structures provide insight into the active
site, a narrow hydrophobic tunnel with polar contacts at each
end. On one side His221/Glu282 are located, on the other
Ser142/Tyr155 (two members of the catalytic tetrad).25 Surpris-
ingly, close to the hydrophobic B/C region of the steroid, two
polar amino acids, Tyr218 and Ser222, can be found that do
not interact with E2.
* To whom correspondence should be addressed. Phone: +49 681 302
70302. Fax: +49 681 302 70308. E-mail: rwh@mx.uni-saarland.de. Website:
http://www.pharmmedchem.de.
†
Pharmaceutical and Medicinal Chemistry, Saarland University.
Pharmacelsus CRO.
Home address: Department of Chemistry, College of Science, Univer-
‡
§
sity of Al al-Bayt, Al-Mafraq, Jordan
Home address: Department of Pharmaceutical Sciences, University of
Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
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; NADP(H), nicotinamide adenine dinucleotide phosphate; NAD(H),
nicotinamide adenine dinucleotide; RBA, relative binding affinity; ESP,
electrostatic potential; Papp, apparent permeability coefficient; SAR,
structure-activity relationship; CC, column chromatography; MEP, mo-
lecular electrostatic potential; AUC, area under the curve; semi-QMAR,
semiquantitative MEP-activity relationship. For the sake of clarity, IUPAC
nomenclature is not strictly followed except for the Experimental Section,
where the correct IUPAC names are given.
Several steroidal and nonsteroidal inhibitors of 17ꢀ-HSD1
have been described. The first report on steroidal compounds
by Penning26 was as early as 1996. In the past few years, several
articles of other groups followed.27 Regarding the nonsteroidal
inhibitors, only four compound classes have been described so
far, all of them very recently: thienopyrimidinones A,28,29
biphenyl ethanones B,30 and from our group, 6-(hydroxyphenyl)
naphthols C31,32 and bis(hydroxyphenyl) azoles D33 (Chart 1).
10.1021/jm8006917 CCC: $40.75
2008 American Chemical Society
Published on Web 10/15/2008

6726 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Chart 1. Nonsteroidal 17ꢀ-HSD1 Inhibitors
Bey et al.
pounds 31ii, 32ii, 19i-28i, 31i, and 32i were synthesized using
method B (sodium carbonate, THF/water (1:1), Pd(PPh3)4,
reflux, 20 h). During the first cross-coupling reaction leading
to the mono(methoxyphenyl) substituted derivatives 1ii-8ii,
18ii-27ii, 31ii, and 32ii, no disubstituted compounds were
obtained, indicating that the (methoxyphenyl)bromo azoles and
thiophenes are less reactive than their dibromo aryl precursors.
The bromo intermediate 18ii was treated with n-BuLi in
anhydrous THF and subsequently hydrolyzed with water to yield
the monomethoxylated thiophene 18i. The methoxy groups of
1i-8i, 18i-28i, 31i, and 32i were cleaved with boron tribromide
(method E: BBr3, CH2Cl2, -78 °C to rt, 18 h, Scheme 1).
The 1,3,4-thiadiazole 9 was prepared in a three-step synthetic
pathway based on the method described by Gierczyk and
Zalas.36 The 3-methoxybenzoyl chloride was treated with
hydrazine monohydrate to give the resulting 3-methoxy-N′-(3-
methoxybenzoyl)benzohydrazide, which was cyclized into the
corresponding thiadiazole using Lawesson reagent in DME
under microwave assisted conditions. In a last step, the methoxy
substituents were cleaved with boron tribromide (method E:
BBr3, CH2Cl2, -78 °C to rt, 18 h).
Chart 2. Title Compounds
The synthesis of the 1,2,4-thiadiazoles 10 and 11 is shown
in Scheme 2. The commercially available 4-methoxybenzonitrile
and 3-hydroxybenzonitrile were converted into the thioamide
intermediates 10ii and 11i, respectively, using aqueous am-
monium sulfide under microwave assisted reaction.37 These
thioamides were submitted to strong acidic conditions, resulting
in a mixture of the thiadiazoles 10i and 11, which were separated
by column chromatography. The bis(methoxyphenyl) compound
could not be isolated. Compound 10i was demethylated with
boron tribromide (method E: BBr3, CH2Cl2, -78 °C to rt, 18 h,
Scheme 2).
The most promising compound in the latter series was the
2,5-bis(hydroxyphenyl) oxazole E, with an IC50 of 310 nM and
a selectivity factor of 56 against 17ꢀ-HSD2. In general, it was
discovered that the inhibitory activity of these compounds
depends on the existence of hydroxy rather than methoxy groups
on the phenyl substituents and on the OH substitution pattern,
meta-para and para-meta substituted compounds being more
active than para-para substituted ones. Furthermore, it became
apparent that inhibition also depends on the nature of the
heterocycle. Hydrogen bond donor functions turned out to be
inappropriate, whereas in several compounds, H-bond acceptor
atoms were favorable. This finding led to the hypothesis that
active compounds are capable of interacting with Tyr218 or
Ser222 via H-bonds.33
To enhance activity and selectivity and to get a better insight
into the interaction of these compounds with the active site of
17ꢀ-HSD1, the significance of the OH groups will be further
evaluated and other five-membered heterocycles, especially
sulfur containing ones, will be investigated. Furthermore, it will
be evaluated whether six-membered rings are also appropriate
to connect the two hydroxyphenyl moieties.
In the following, we describe the synthesis of 39 bis(hydrox-
yphenyl) azoles, thiophenes, benzenes, and aza-benzenes (Chart
2) as well as the determination of their 17ꢀ-HSD1 inhibitory
activity and selectivity toward 17ꢀ-HSD2, ERR, and ERꢀ.
Furthermore, cell permeability using CaCo-2 cells, metabolic
stability in rat liver microsomes, inhibition of the most important
hepatic CYP enzymes, and pharmacokinetic properties in the
rat of selected compounds were determined. For a better
understanding of the SARs, molecular electrostatic potentials
(MEPs) were calculated.
The synthesis of compounds 12-15 is presented in Scheme
3. The dimethoxylated-1,2,4-triazoles 12i-15i were synthesized
by reaction of the N-acylimidates38 12ii-15ii with methylhy-
drazine (method D: MeNHNH2, CH2Cl2, 30-40 °C, 4 h). The
methoxy groups of compounds 12i-15i were cleaved with
borontrifluoride dimethyl sulfide complex35 (method F:
BF3 ·SMe2, CH2Cl2, rt, 20 h, Scheme 3).
Compound 16 was prepared according to Sharpless39 using
3-azidophenol and 3-hydroxyphenyl acetylene.
Compounds 17, 29, 30, 37, and 38 were obtained, under
microwave assisted conditions in a one-pot synthesis (method
C: DME/EtOH/water (1:1:1), Cs2CO3, Pd(PPh3)4, MW (150 W,
150 °C, 15 bar, 15 min)) with benzene boronic acid for 17,
hydroxylated benzene boronic acid for 29, 30, 37, and 38 and
the corresponding dibrominated heterocycle.
The synthesis of compounds 33-36 is depicted in Scheme
4. Starting from the commercially available dibrominated
benzene and methoxylated benzene boronic acids, compounds
33-36 were prepared via two successive Suzuki reactions
following the conditions of method A. In the first reaction, only
the monosubstituted compounds 33ii and 35ii were obtained
due to the fact that the (methoxyphenyl)bromobenzenes are less
reactive than the dibromobenzenes. Consequently, longer reac-
tion times were required for the second cross-coupling reaction
(20 versus 4 h). The methoxy groups of compounds 33i-36i
were cleaved using boron tribromide (method E: BBr3, CH2Cl2,
-78 °C to rt, 18 h, Scheme 4). 1,2,4,5-Tetrazine 39 was
synthesized following the procedure described by Guither et
al.40 Briefly, 3-hydroxybenzonitrile was refluxed with hydrazine
Chemistry
Starting from the commercially available dibrominated het-
erocycles and methoxylated benzene boronic acids, compounds
1-8, 19-28, 31, and 32 were prepared via two successive
Suzuki reactions34 followed by a demethylation step using
borontribromide35 as reagent. The Suzuki cross-coupling was
carried out using three different methods. Intermediates 1ii-8ii,
18ii-27ii, and 1i-8i were prepared following method A (aq
sodium carbonate, toluene, Pd(PPh3)4, reflux, 4 h) and com-

Nonsteroidal Inhibitors of 17ꢀ-HSD1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6727
Scheme 1. Synthesis of Compounds 1-8, 18-28, 31, and 32a
a Reagents and conditions: (a) For compounds 1ii-8ii, 18ii-27ii, and 1i-8i: method A: aq Na2CO3, toluene, Pd(PPh3)4, reflux, 4 h; for compounds 31ii,
31i, 32i, 32ii, and 19i-28i: method B: Na2CO3, THF/water (1:1), Pd(PPh3)4, reflux, 20 h; (b) (1) n-BuLi, THF dry, -78 °C, 15 min, (2) water. (c) Method
E: BBr3, CH2Cl2, -78 °C to rt, 18 h.
Scheme 2. Synthesis of Compounds 10 and 11a
a Reagents and conditions: (a) conc HCl, 38 °C, 8 h; (b) method E: BBr3, CH2Cl2, -78 °C to rt, 18 h.
Scheme 3. Synthesis of Compounds 12-15a
a Reagents and conditions: (a) CH2Cl2, NEt3, 30-40 °C, 6 h; (b) method D: MeNHNH2, CH2Cl2, 30-40 °C, 4 h; (c) method F: BF3 ·SMe2, CH2Cl2, rt,
20 h.
monohydrate and sulfur. Treatment with sodium nitrite led to
Compounds showing less than 10% inhibition at 1 µM were
cyclization resulting in 3,3′-(1,2,4,5-tetrazine-3,6-diyl)diphenol 39.
considered to be inactive.
The inhibition values of the test compounds are shown in
Table 1. It becomes apparent that 11 compounds are more active
than the previously described oxazole E33 (IC50 ) 310 nM).
All methoxy compounds (data not shown) and para-para
dihydroxylated derivatives are inactive except phenylene 34,
which is a weak inhibitor (IC50 > 1000 nM). The shift of one
Biological Results
Activity: Inhibition of Human 17ꢀ-HSD1. Placental enzyme
was partially purified following a described procedure.41 Tri-
tiated E1 was incubated with 17ꢀ-HSD1, cofactor, and inhibitor.
The amount of labeled E2 formed was quantified by HPLC.

6728 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bey et al.
Table 1. Inhibition of Human 17ꢀ-HSD1 and 17ꢀ-HSD2 by Compounds 1-39, O-O Distances, and Phenyl-het-phenyl Angles

Nonsteroidal Inhibitors of 17ꢀ-HSD1
Table 1. Continued
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6729
a O-O distance between the hydroxy substituents, for E2 d ) 11.0 Å. b Angle between the two phenyl moieties in deg. c Mean values of three determinations,
standard deviation less than 12% except 3: 18% for 17ꢀ-HSD1. d Human placental, cytosolic fraction, substrate [3H]-E1 + E1 [500 nM], cofactor NADH
[500 µM]. e Human placental, microsomal fraction, substrate [3H]-E2 + E2 [500 nM], cofactor NAD+ [1500 µM]. f IC50 (17ꢀ-HSD2)/IC50 (17ꢀ-HSD1); ni:
no inhibition; nt: not tested.
hydroxy substituent from the para into the meta position leads
to highly active compounds except for thiazoles 1, 7 (IC50 values
> 1000 nM) and 5 (IC50> 5000 nM), which have a weak
inhibitory activity and triazoles 12 and 14, which are inactive.
Moving the second OH substituent also in the meta position
(meta-meta derivatives) results in potent compounds except
for triazole 16, which is a weak inhibitor (IC50 > 5000 nM),
and selenophene 30 and triazole 15, which are inactive.
The exchange of the para-OH group of the highly active
thiophene 22 (IC50 ) 69 nM) with hydrogen reduces activity
(compound 19, IC50 ) 342 nM). More dramatically, the
replacement of the meta-OH function of compound 22 with

6730 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Scheme 4. Synthesis of Compounds 33-36a
Bey et al.
a Reagents and conditions: (a) method A: Na2CO3 10% in water, toluene, Pd(PPh3)4, reflux, 4 h for 33ii and 35ii and 20 h for 33i–36i; (b) method E:
BBr3, CH2Cl2, -78 °C to rt, 18 h.
hydrogen results in the inactive compound 20. Similarly, the
exchange of the meta-hydroxyphenyl moiety as well as the two
hydroxy groups of 22 with hydrogens leads to the inactive
compounds 17 and 18. This exemplifies the importance of the
existence of two OH substituents and their positions at the
phenyl moieties with at least one being in the meta position.
The synthesized thiazoles 1-8 show lower activities than the
thiophene analogues 17-28 except compound 3 (IC50 ) 50 nM),
which exhibits similar inhibition to 22 (IC50 ) 69 nM) and 27
(IC50 ) 77 nM). The introduction of a second nitrogen
(compounds 9-11) in the heterocyclic scaffold of these potent
inhibitors does not strongly reduce activity (9, IC50 ) 336 nM;
11, IC50 ) 169 nM). For meta-meta disubstituted compounds,
the introduction of nitrogen in compound 4 decreases activity
slightly (9, IC50 ) 336 vs 243 nM), whereas introduction in
compound 8 increases activity (11, IC50 ) 169 vs 455 nM), as
it is observed for the meta-para disubstituted compound 7 (10,
IC50 ) 413 vs > 1000 nM).
Moving the hydroxyphenyl moiety of the highly active
thiophene 22 (IC50 ) 69 nM) from position 5 to the 3 position
decreases activity dramatically (32, IC50 > 1000 nM). Exchange
of the sulfur atom of the potent thiophene 23 (IC50 ) 173 nM)
with selenium results in the inactive compound 30.
In the case of the six-membered rings, benzene and some of
its aza analogues were investigated. The 1,3-bis(hydroxyphenyl)
substituted phenylenes were not active, whereas the 1,4-
substituted compounds showed some activity, the most active
benzene compound being the meta-meta derivative 35 (IC50
) 173 nM). The introduction of one or more nitrogens into the
central benzene ring of 35 (compounds 37-39) results in an
increase (pyridine 37, IC50 ) 101 nM), no change (tetrazine
39, IC50 ) 201 nM), and a decrease of inhibitory activity
(pyrazine 38, IC50 ) 1000 nM).
Table 2. Binding Affinities for the Human Estrogen Receptors R and ꢀ
of Selected Compounds
RBAa(%)
compound
ERRb
ERꢀb
3
<0.01
0.01< RBA< 0.1
1.5
0.1 < RBA < 1
0.1 < RBA < 1
0.01< RBA < 0.1
<0.01
22
25
27
35
37
0.1< RBA< 1
0.01< RBA< 0.1
0.01< RBA< 0.1
<0.001
0.01 < RBA< 0.1
a RBA (relative binding affinity), E2: 100%, mean values of three
determinations, standard deviations less than 10%; b Human recombinant
protein, incubation with 10 nM [3H]-E2 and inhibitor for 1 h.
compounds 37 and 39, which show high selectivity factors of
34 and 35, respectively. The meta-para-disubstituted inhibitors
mostly show higher selectivity, with compound 3 exhibiting the
highest selectivity factor of 80.
A further prerequisite for 17ꢀ-HSD1 inhibitors to be used as
potential drugs is that these compounds do not show affinity
for ERR and ERꢀ or only a marginal one because binding to
these receptors could counteract the therapeutic efficacy. The
binding affinities of selected compounds were determined using
recombinant human protein in a competition assay applying
[3H]-E2 and hydroxyapatite (Table 2). All tested compounds
show very marginal or marginal affinity to the ERs.
Further Biological Evaluations. The intrinsic estrogenic
activity of a representative compound of each class was
determined using the ER-positive mammary tumor T-47D cell
line. No agonistic, i.e., no stimulatory effect, was observed after
application of the inhibitors even at a concentration 1000-fold
higher than E2 (data not shown).
A selection of active and selective compounds was investigated
for permeation of CaCo-2 cells. These cells exhibit morphological
and physiological properties of the human small intestine42 and
are a generally accepted 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)). Thiazole 3 shows medium
cell permeation, while thiophenes 22, 25 and phenylene 35 exhibit
high cell permeability (Table 3).
Furthermore, in Table 1, the angles between the two phenyl
moieties are presented as a structural parameter. No correlation
to the activities of the corresponding compounds can be
observed.
Selectivity: Inhibition of Human 17ꢀ-HSD2 and Affini-
ties for ERr and ERꢀ. Because 17ꢀ-HSD2 catalyzes the
inactivation of E2 to E1, inhibitory activity toward this enzyme
must be avoided. The 17ꢀ-HSD2 inhibition was determined
using an assay similar to the 17ꢀ-HSD1 test. Placental mi-
crosomes were incubated with tritiated E2 in the presence of
NAD+ and inhibitor. Separation and quantification of labeled
product (E1) was performed by HPLC using radio detection. A
selection of the most potent 17ꢀ-HSD1 inhibitors was tested
for inhibition of 17ꢀ-HSD2. IC50 values and selectivity factors
(IC50 17ꢀ-HSD2/IC50 17ꢀ-HSD1) are presented in Table 1.
It is striking that most meta-meta bis(hydroxyphenyl)
substituted inhibitors present only poor selectivity except for
A representative compound of the thiazole, thiophene, and
phenylene class (compounds 4, 25, and 35) was evaluated for
their phase 1 metabolic stability using 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 (Table 4). All tested compounds show longer
half-lives than the antihistaminic drug diphenhydramine (values
in the range between 12.6 and 22.7 min vs 6.8 min).

Nonsteroidal Inhibitors of 17ꢀ-HSD1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6731
Table 3. CaCo-2 Cell Permeation of Highly Active 17ꢀ-HSD1
tion was considered and the molecular electrostatic potentials
(MEPs) of selected compounds were determined. The geometry
of the compounds had been fully optimized in the gas phase at
the B3LYP/6-311++G (d,p) level of density functional theory
(DFT). MEPs were plotted for every compound on its electron
density with GaussView 3.09. The electrostatic potential
distribution of the charge density is presented by a color code
ranging from -3.1 × 10-2 to 4.5 × 10-2 Hartree (Figures 1
and 2 and Supporting Information). For better comparison, the
MEPs of different compounds were divided into three regions
corresponding to each aromatic system.
Inhibitors
compound
Papp (× 10-6cm/s)a,b
classification
3
22
25
35
7.8
22.0
14.4
12.5
0.1
medium
high
high
high
low
atenolol
testosterone
ketoprofene
9.4
25.7
medium
high
a Permeability of reference compounds similar to the values described
b
(atenolol,71 testosterone,67 ketoprofene72). Papp: apparent permeability
coefficient, mean values of three determinations, standard deviations less
than 10%.
In Figure 1, the MEPS of five-membered heterocyclic
compounds are arranged based on their increasing inhibitory
potency. While the positions of the hydroxyphenyl moieties are
fixed, the nature of the heterocycle is varied. It becomes apparent
that the heterocycle influences the ESP distribution of the whole
molecule. To rationalize these MEPs, the ESP distribution ranges
(C1), the mean values of the distribution ranges (C2), and the ∆
of ESP (C3) were analyzed; negative ESP values (red/orange/
yellow) in region I and II and less negative to almost neutral
ESP values (green/yellow) in region III are obviously an
important factor for high inhibitory potency. Trying to establish
a semiquantitative MEP-activity relationship (“semi-QMAR”)
optimal ESP ranges for areas I, II, and III for potent inhibition
were identified (hydrogens and the OH groups were not
considered): for region I ESP from -1.7 to -1.2 × 10-2, for
region II -1.6 to -0.9 × 10-2, and for region III -1.2 to -0.5
× 10-2 Hartree. Similarly, the optimal ∆ values of ESP for
each region were determined: 0.5, 0.7, and 0.7 Hartree,
respectively. Both the shift of a certain ESP distribution range
on the scale and the change of the ∆ value result in a decrease
of inhibitory activity. The combination of these two criteria is
substantiated with +, -, and 0 in Figure 1D, indicating
favorable, unfavorable, and neutral impact on activity, respectively.
The 2,5-disubstituted imidazloe33 exhibits a polarization
between top and bottom sides of the molecule: the vertex side
(NH) has positive ESP values, while on the opposite side
(N-C), negative to neutral values are predominant. This
polarization (∆) 7.2 × 10-2 Hartree) results in an inactive
compound, indicating that the ESP of the central heterocycle is
crucial for 17ꢀ-HSD1 inhibitory activity.
Table 4. Half-Lives and Intrinsic Clearances of Compounds 4, 25, and
35 in Rat Liver Microsomesa
b
compound
half-life (min)
CLint (µL /min/mg protein)
4
12.6
18.6
22.7
40.8
6.8
367.2
248.5
203.4
113.3
679.6
25
35
diazepamc
diphenhydraminec
a 0.33 mg/mL protein, NADP+-regenerating system, [inhibitor]: 1 µM,
incubation at 37 °C, samples taken at 0, 15, 30, and 60 min, determination
of parent compound by MS. b CLint: intrinsic body clearance. c Values of
reference compounds similar to described values.
Table 5. Pharmacokinetic Parameters of Compound 3 and 39 in Male
Rats after Oral Application of 10 mg/kg
compound
parameters
3
39
Cmax obs (ng/mL)a
Cz (ng/mL)b
tmax obs (h)c
tz (h)d
7.8
6.6
8.0
10.0
1.5
99.2
106.0
54.0
3.0
10.0
1.2
t1/2z (h)e
AUC0-tz (ng/mL)f
1204.0
a Cmax obs: maximal measured concentration. b Cz: last analytical quantifi-
c
able concentration. tmax obs: time to reach the maximum measured
concentration. d tz: time of the last sample which has an analytically
e
quantifiable concentration. t1/2z: half-life of the terminal slope of a
concentration time curve. f AUC0-tz: area under the concentration time curve
up to the time tz of the last sample.
The same compounds (4, 25, and 35) were further investigated
for inhibition of the six most important human hepatic enzymes:
CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4. All compounds show
very low inhibition of CYP1A2, 2B6, 2C19, and 2D6 (IC50> 4
µM). In the case of CYP2C9 and CYP3A4 (4: 2.1 and 0.8; 25:
0.8 and 1.9; 35: 1.9 and 2.1 µM, respectively), inhibition was
higher but still clearly below the IC50 values of 17ꢀ-HSD1
inhibition. These results indicate a low risk of drug-drug
interaction caused by CYP inhibition.
The pharmacokinetic profiles of the most active and selective
azole compound 3 and one of the six-membered ring compounds
(39) were determined in rats after oral administration in a
cassette. The most potent six-membered ring compound 37 was
not chosen, as it was unstable in buffer over 24 h. Each group
consisted of four male rats, and the compounds were adminis-
tered in doses of 10 mg/kg. Plasma samples were collected over
24 h, and plasma concentrations were determined by HPLC-
MS/MS. The pharmacokinetic parameters are presented in Table
5. Maximal plasma concentration (Cmax) and AUC value are
much higher for compound 39compared to compound 3.
A good example for the change in activity is well demon-
strated by the ESP distribution in region III for thiazoles 1 and
3 (Figure 1C1, C2). The dramatic loss of activity of compound
1 (IC50 > 1000 nM) in comparison to 3 (IC50 ) 50 nM) is due
to a nonoptimal electron density distribution.
The MEP maps of the six-membered ring compounds are
depicted in Figure 2. A similar charge density distribution was
observed except for the only planar compound of this series,
tetrazine 33, which showed no polarization between top and
bottom side of the molecule. It remains to be clarified whether
this is the reason for the reasonable activity of compound 33.
Discussion and Conclusion
The present paper shows that the 2,5-disubstituted oxazole
from a previous study33 could be optimized. The most active
and selective thiazole 3 shows an IC50 value of 50 nM and a
selectivity factor of 80 (compound E, IC50) 310 nM, selectivity
factor: 56).
The biological results obtained confirm similar findings
described in our previous article:33 the OH substitution pattern
of our compounds is decisive for inhibitory activity. Comparison
of the monohydroxylated thiophenes 19 and 20 (meta- and
para-, respectively) shows that the meta-hydroxy group is crucial
Computational Chemistry
To obtain an insight into which physicochemical parameter
might influence biological activity, the charge density distribu-

6732 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bey et al.
Figure 1. (A) Structure and 17ꢀ-HSD1 inhibitory activity of F,33 E,33 and compounds 1, 3, and 17 ordered by increasing activity. (B) MEP maps,
dorsal and ventral view. (C1) ESP distribution range (Hartree; × 10-2). (C2) mean value of distribution range (Hartree; × 10-2). (C3) ∆ of ESP.
(D) “semi-QMAR”. MEP surfaces were plotted with GaussView 3.09.
Figure 2. (A) Structure and 17ꢀ-HSD1 inhibitory activity of compounds 29 and 31-33 ordered by increasing activity. (B) MEP maps, dorsal and
ventral view. (C1) ESP distribution range (Hartree; × 10-2). (C2) Mean value of distribution range (Hartree; × 10-2). (C3) ∆ of ESP. (D) “semi-
QMAR”. MEP surfaces were plotted with GaussView 3.09.

Nonsteroidal Inhibitors of 17ꢀ-HSD1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6733
for activity. The inactivity of compound 18 indicates that the
phenyl moiety of the meta-hydroxyphenyl thiophene 19 is also
important for potency. The replacement of the meta-hydroxy
group of 19 with hydrogen, leading to the inactive compound
17, demonstrates the importance of the hydrogen bond interac-
tion for activity.
HSD2 dramatically (IC50 17ꢀ-HSD2/IC50 17ꢀ-HSD1, 28 vs 80).
In general, the most potent compounds exhibit a really low
affinity for the ERR and ERꢀ and show no stimulation of cell
proliferation (agonistic effect) in the ER-positive T-47D cell
line. It is worth mentioning that 3, in spite of its good CaCo-2
permeability, shows a really low bioavailability compared to
39. Glucuronidation and/or sulfatation might be responsible for
the low plasma levels of the parent compound.
In the present report, we described the synthesis of bis(hy-
droxyphenyl) azoles, thiophenes, benzenes, and aza-benzenes
and the evaluation of their biological properties. The most
promising compounds of this study, 3, 22, and 39, show a high
selectivity toward 17ꢀ-HSD2, a low binding affinity to the ERR,
a high CaCo-2 permeability, and a reasonable pharmacokinetic
profile after peroral application. These new compounds should
be useful tools to further investigate in vivo 17ꢀ-HSD1 as a
target for the treatment of estrogen-dependent diseases.
As observed for the previously described bis(hydroxyphenyl)
derivatives,33 the distance between the two oxygens obviously
has to be close to the value observed for the substrate (d ) 11
Å). The para-para disubstituted compounds show distances
longer than 12.5 Å. They are all inactive. Concerning the
meta-para and meta-meta disubstituted compounds, which
have O-O distances between 8.5 and 12.8 Å, medium to high
inhibitory activities are observed for most compounds. They
could be able to establish hydrogen bond interactions with
His221/Glu282 and Ser142/Tyr155. However, the inactivity of
thiazoles 1, 5, and 7 and triazoles 12, 14-16, which are all
meta-para and meta-meta disubstituted, respectively, indicates
that this distance is not the only decisive criterion for activity.
The heterocycle also influences the inhibitory potencies of the
compounds.
In the five-membered ring series, the potency of 1,2,3-
triazoles described previously33 led us to further investigate this
class of compounds. The inactivity of compounds 12-15 shows
that either the nitrogen distribution in the ring or the methyl
group are not tolerated by the enzyme. The replacement of the
N-Me moiety of 14 with sulfur (compound 10), leading to a
fairly potent compound, indicates that S in this position has a
positive influence. To further investigate the role of the nitrogen
in the ring, thiazoles 1-8 and thiophenes 21-28, 31, and 32
were investigated. The fact that thiazole 3 and thiophene 22
exhibit almost identical potencies shows that the nitrogen does
not contribute to binding, i.e., that there is obviously no
hydrogen bond interaction.
Experimental Section
Chemical Methods. Chemical names follow IUPAC nomen-
clature. Starting materials were purchased from Aldrich, Acros,
Lancaster, Roth, Merck, or Fluka and were used without purification.
Column chromatography (CC) was performed on silica gel
(70-200 µm) coated with silica, preparative thin layer chroma-
tography (TLC) on 1 mm SIL G-100 UV254 glass plates (Macherey-
Nagel), and reaction progress was monitored by TLC on Alugram
SIL G UV254 (Macherey-Nagel).
Melting points were measured on a Mettler FP1 melting point
apparatus and are uncorrected.
IR spectra were recorded on a Bruker Vector 33 spectrometer
(neat sample).
1H NMR and 13C NMR spectra were measured on a Bruker AM500
spectrometer (500 MHz) at 300 K. Chemical shifts are reported in δ
(parts per million: ppm), by reference to the hydrogenated residues of
deuteriated solvent as internal standard (CDCl3: δ ) 7.24 ppm (1H
NMR) and δ ) 77 ppm (13C NMR); CD3OD: δ ) 3.35 ppm (1H
NMR) and δ ) 49.3 ppm (13C NMR); CD3COCD3: δ ) 2.05 ppm
(1H NMR) and δ ) 29.9 ppm (13C NMR); CD3SOCD3: δ ) 2.50
ppm (1H NMR) and δ ) 39.5 ppm (13C NMR). Signals are described
as s, d, t, dd, m, and dt for singlet, doublet, triplet, doublet of doublets,
multiplet, and doublet of triplets, respectively. All coupling constants
(J) are given in Hertz (Hz).
In this report, we could also show that six-membered rings
are appropriate for the design of highly active 17ꢀ-HSD1
inhibitors. Comparison of the almost equipotent phenylene 35,
pyridine 37, and tetrazine 39 confirms the hypothesis that
nitrogens are tolerated in the ring but do not contribute to a
specific interaction.
Mass spectra (ESI) were recorded on a TSQ Quantum (Ther-
mofischer) instrument. Elemental analyses were performed at the
Department of Instrumental Analysis and Bioanalysis, Saarland
University.
The role of the angle between the two hydroxyphenyl moieties
was also investigated in order to find out whether there is a
correlation between this parameter and the inhibitory potency.
The broad range of angles calculated (128° for 11 to 180° for
37, two highly active compounds) could not be correlated with
high or low inhibitory potency, indicating that the enzyme
presents some flexibility for ligand binding. Interestingly, for
smaller angles between the two hydroxyphenyl groups, as in
case of compounds 10 and 11 (N atom at the vertex), the
meta-meta substitution results in a higher activity, while for
larger angles, in presence of a sulfur atom at the vertex of the
five-membered heterocycle (2,5-disubstituted thiazoles and 2,5-
disubstituted thiophenes), the optimal substitution pattern is
meta-para/para-meta. This phenomenon will be further
investigated.
Our finding that there exists a correlation between the MEPs
and the biological activities of the compounds (semi-QMAR)
might be exploited for further structure optimization, underlining
the relevance of this descriptor for biological activity. Further-
more, the MEPs could be used to investigate how these
inhibitors approach and bind to the enzyme as it is described
for genistein and the estrogen receptor.43
5-Bromo-2-(4-methoxyphenyl)-1,3-thiazole (1ii),44 4-methox-
ythiobenzamide (10ii),45 3-bromo-2-(4-methoxyphenyl)thiophene
(31ii),46 3-bromo-4′-methoxybiphenyl (33ii),47 4′-bromo-3-meth-
oxybiphenyl (35ii),48 2-(4-methoxyphenyl)-5-(3-methoxyphenyl)-
1,3-thiazole (1i),49 2,5-bis(4-methoxyphenyl)-1,3-thiazole (2i),50
2,4-bis(4-methoxyphenyl)-1,3-thiazole (6i),35 3-hydroxythiobenza-
mide (11i),16 2,5-bis(4-methoxyphenyl)thiophene (21i),51 2,4-bis(4-
methoxyphenyl)thiophene (26i),52 2,3-bis(4-methoxyphenyl)th-
iophene (31i),53 3,4′′-dimethoxy-1,1′:3′,1′′-terphenyl (33i),54 4,4′′-
dimethoxy-1,1′:3′,1′′-terphenyl (34i),54 4,4′-(1,3-thiazole-2,5-
diyl)diphenol (2),55 4,4′-(1,3-thiazole-2,4-diyl)diphenol (6),35 3,3′-
(1,2,4-thiadiazole-3,5-diyl)diphenol (11),56 3,3′-(1H-1,2,3-triazole-
1,4-diyl)diphenol (16),57 2,5-diphenylthiophene (17),58 4,4′-thiene-
2,5-diyldiphenol (21),51 4,4′-thiene-2,4-diyldiphenol (26),55 1,1′:
3′,1′′-terphenyl-3,4′′-diol (33),59 1,1′:3′,1′′-terphenyl-4,4′′-diol
(34),60 1,1′:4′,1′′-terphenyl-3,3′′-diol (35),59 1,1′:4′,1′′-terphenyl-
3,4′′-diol (36),59 3,3′-pyrazine-2,5-diyldiphenol (38),61 and 3,3′-
(1,2,4,5-tetrazine-3,6-diyl)diphenol (39)40 were prepared following
described procedures.
General Procedure for Suzuki Coupling. Method A. A mixture
of arylbromide (1 equiv), methoxybenzene boronic acid (1 equiv),
sodium carbonate (2 equiv), and tetrakis(triphenylphosphine) pal-
ladium (0.005 equiv) in an oxygen free toluene/water (1:1) solution
Interestingly, the exchange of a CH in the thiophene 22 by a
N leading to the thiazole 3 increases selectivity toward 17ꢀ-

6734 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bey et al.
was stirred at 100 °C for 4 h under nitrogen. The reaction mixture
was cooled to rt. The aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
magnesium sulfate, filtered, and concentrated to dryness. The
product was purified by CC.
mg, 4.6 µmol) according to method A. The product was purified
by CC (hexane/ethyl acetate 9:1); yield: 58% (160 mg).
3-[5-(4-Hydroxyphenyl)-1,3-thiazol-2-yl]phenol (3). The title
compound was prepared by reaction of 2-(3-methoxyphenyl)-5-(4-
methoxyphenyl)-1,3-thiazole (3i) (40 mg, 0.13 mmol) and boron
tribromide (0.81 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 80%
(50 mg); MS (ESI): 270 (M + H)+; Anal. (C15H11NO2S) C, H, N.
2,5-Bis(3-methoxyphenyl)-1,3-thiazole (4i). The title compound
was prepared by reaction of 5-bromo-2-(3-methoxyphenyl)-1,3-
thiazole (3ii) (250 mg, 0.93 mmol), 3-methoxybenzeneboronic acid
(170 mg, 1.11 mmol), sodium carbonate (197 mg, 1.86 mmol), and
tetrakis(triphenylphosphine) palladium (5.4 mg, 4.6 µmol) according
to method A. The product was purified by CC (hexane/ethyl acetate
9:1); yield: 40% (111 mg).
3,3′-(1,3-Thiazole-2,5-diyl)diphenol (4). The title compound was
prepared by reaction of 2,5-bis(3-methoxyphenyl)-1,3-thiazole (4i)
(100 mg, 0.36 mmol) and boron tribromide (2.02 mmol) according
to method E. The product was purified by preparative TLC (hexane/
ethyl acetate 5:5); yield: 85% (82 mg); MS (ESI): 270 (M + H)+;
Anal. (C15H11NO2S) C, H, N.
Method B. A mixture of arylbromide (1 equiv), methoxybenzene
boronic acid (1.2 equiv), sodium carbonate (2 equiv), and tetraki-
s(triphenylphosphine) palladium (0.005 equiv) in an oxygen free
tetrahydrofurane/water (1:1) solution was stirred at 100 °C for 20 h
under nitrogen. The reaction mixture was cooled to rt. The aqueous
layer was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered,
and concentrated to dryness. The product was purified by CC.
Method C. A mixture of aryl dibromide (1 equiv), methoxy-
benzene boronic acid (2.4 equiv), cesium carbonate (4 equiv), and
tetrakis(triphenylphosphine) palladium (0.001 equiv) was suspended
in an oxygen-free DME/EtOH/water (1:1:1) solution. The reaction
mixture was exposed to microwave irradiation (15 min, 150 W,
150 °C, 15 bar). After reaching rt, water was added and the aqueous
layer was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over magnesium sulfate, filtered,
and concentrated to dryness. The product was purified by prepara-
tive TLC.
General Procedure for Synthesis of 1,2,4-Triazoles. Method D.
A solution of acyl chloride (1 equiv) in dichloromethane was added
dropwise to a mixture of ethyl imino ester (1 equiv) and dry
triethylamine (1 equiv) in 20 mL dichloromethane and heated to
30-40 °C for 6 h. After cooling to rt, the mixture was poured into
3% NaHCO3 solution (25 mL). The layers were separated and the
organic layer was washed with water, dried over sodium sulfate,
and evaporated to dryness under reduced pressure. The resulting
N-acylimino esters 12ii-15ii were heated to 30-40 °C with methyl
hydrazine (2 equiv) in CH2Cl2 for 4 h. The solvent was removed
under reduced pressure and the 1,2,4-triazoles 12i-15i were
crystallized from CH2Cl2/ Et2O.
General Procedure for Ether Cleavage. Method E. To a
solution of methoxybenzene derivative (1 equiv) in dry dichlo-
romethane at -78 °C (dry ice/acetone bath), boron tribromide in
dichloromethane (1 M, 3 equiv per methoxy function) was added
dropwise. The reaction mixture was stirred for 20 h at rt under
nitrogen. Water was added to quench the reaction, and the aqueous
layer was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over sodium sulfate, evaporated to
dryness under reduced pressure, and purified by preparative TLC.
Method F. To a solution of bis(methoxyphenyl) derivative (1
equiv) in dry dichloromethane, borontrifluoride dimethyl sulfide
complex (75 equiv) was added dropwise at rt. The reaction mixture
was stirred for 20 h. Water was added to quench the reaction, and
the aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over sodium sulfate,
evaporated to dryness under reduced pressure, and purified by
preparative TLC.
3-[2-(4-Hydroxyphenyl)-1,3-thiazol-5-yl]phenol (1). The title
compound was prepared by reaction of 5-(3-methoxyphenyl)-2-(4-
methoxyphenyl)-1,3-thiazole (1i) (40 mg, 0.13 mmol) and boron
tribromide (0.81 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 77%
(28 mg); MS (ESI): 270 (M + H)+; Anal. (C15H11NO2S) C, H, N.
5-Bromo-2-(3-methoxyphenyl)-1,3-thiazole (3ii). The title com-
pound was prepared by reaction of 2,5-dibromo-1,3-thiazole (500
mg, 2.06 mmol), 3-methoxybenzeneboronic acid (376 mg, 2.47
mmol), sodium carbonate (437 mg, 4.12 mmol), and tetrakis(triph-
enylphosphine) palladium (11 mg, 10 µmol) according to method
A. The product was purified by CC (dichloromethane/methanol 95:
5); yield: 50% (278 mg).
4-Bromo-2-(4-methoxyphenyl)-1,3-thiazole (5ii). The title com-
pound was prepared by reaction of 2,4-dibromo-1,3-thiazole (500
mg, 2.06 mmol), 4-methoxybenzeneboronic acid (376 mg, 2.47
mmol), sodium carbonate (437 mg, 4.12 mmol), and tetrakis(triph-
enylphosphine) palladium (11 mg, 10 µmol) according to method
A. The product was purified by CC (hexane/ethyl acetate 9:1); yield:
55% (305 mg).
2-(4-Methoxyphenyl)-4-(3-methoxyphenyl)-1,3-thiazole (5i). The
title compound was prepared by reaction of 4-bromo-2-(4-meth-
oxyphenyl)-1,3-thiazole (5ii) (250 mg, 0.93 mmol), 3-methoxy-
benzeneboronic acid (170 mg, 1.11 mmol), sodium carbonate (197
mg, 1.86 mmol), and tetrakis(triphenylphosphine) palladium (5.4
mg, 4.6 µmol) according to method A. The product was purified
by CC (hexane/ethyl acetate 9:1); yield: 52% (143 mg).
3-[2-(4-Hydroxyphenyl)-1,3-thiazol-4-yl]phenol (5). The title
compound was prepared by reaction of 2-(4-methoxyphenyl)-4-(3-
methoxyphenyl)-1,3-thiazole (5i) (70 mg, 0.24 mmol) and boron
tribromide (1.44 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 78%
(50 mg); MS (ESI): 268 (M - H)-; Anal. (C15H11NO2S) C, H, N.
4-Bromo-2-(3-methoxyphenyl)-1,3-thiazole (7ii). The title com-
pound was prepared by reaction of 2,4-dibromo-1,3-thiazole (500
mg, 2.06 mmol), 3-methoxybenzeneboronic acid (376 mg, 2.47
mmol), sodium carbonate (437 mg, 4.12 mmol), and tetrakis(triph-
enylphosphine) palladium (11 mg, 10 µmol) according to method
A. The product was purified by CC (hexane/ethyl acetate 9:1); yield:
50% (270 mg).
2-(3-Methoxyphenyl)-4-(4-methoxyphenyl)-1,3-thiazole (7i). The
title compound was prepared by reaction of 4-bromo-2-(3-meth-
oxyphenyl)-1,3-thiazole (7ii) (250 mg, 0.93 mmol), 4-methoxy-
benzeneboronic acid (170 mg, 1.11 mmol), sodium carbonate (197
mg, 1.86 mmol), and tetrakis(triphenylphosphine) palladium (5.4
mg, 4.6 µmol) according to method A. The product was purified
by CC (hexane/ethyl acetate 9:1); yield: 79% (218 mg).
3-[4-(4-Hydroxyphenyl)-1,3-thiazol-2-yl]phenol (7). The title
compound was prepared by reaction of 2-(3-methoxyphenyl)-4-(4-
methoxyphenyl)-1,3-thiazole (7i) (70 mg, 0.24 mmol) and boron
tribromide (1.44 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 80%
(52 mg); MS (ESI): 268 (M - H)-; Anal. (C15H11NO2S) C, H, N.
2,4-Bis(3-methoxyphenyl)-1,3-thiazole (8i). The title compound
was prepared by reaction of 4-bromo-2-(3-methoxyphenyl)-1,3-
thiazole (7ii) (250 mg, 0.93 mmol), 3-methoxybenzeneboronic acid
(170 mg, 1.11 mmol), sodium carbonate (197 mg, 1.86 mmol), and
tetrakis(triphenylphosphine) palladium (5.4 mg, 4.6 µmol) according
to method A. The product was purified by CC (hexane/ethyl acetate
9:1); yield: 18% (50 mg).
2-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-1,3-thiazole (3i). The
title compound was prepared by reaction of 5-bromo-2-(3-meth-
oxyphenyl)-1,3-thiazole (3ii) (250 mg, 0.93 mmol), 4-methoxy-
benzeneboronic acid (170 mg, 1.11 mmol), sodium carbonate (197
mg, 1.86 mmol), and tetrakis(triphenylphosphine) palladium (5.4
3,3′-(1,3-Thiazol-2,4-diyl)diphenol (8). The title compound was
prepared by reaction of 2,4-bis(3-methoxyphenyl)-1,3-thiazole (8i)
(70 mg, 0.24 mmol) and boron tribromide (1.44 mmol) according

Nonsteroidal Inhibitors of 17ꢀ-HSD1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6735
to method E. The product was purified by preparative TLC (hexane/
ethyl acetate 5:5); yield: 78% (50 mg); MS (ESI): 268 (M - H)-;
Anal. (C15H11NO2S) C, H, N.
3,3′-(1,2,4-Thiadiazol-2,5-diyl)diphenol (9). A solution of 3-hy-
droxythiobenzamide (100 mg, 0.17 mmol, 2 equiv.) in DMSO (10
mL) was stirred for 5 h at rt with 3 mL of concentrated chlorhydric
acid. The crude mixture was poured into water and the resulting
precipitate was filtered, washed with water, and dried overnight in
a desiccator; yield: 92% (41 mg); MS (ESI): 269 (M - H)-. Anal.
(C14H10N2O2S) C, H, N.
3-[3-(4-Methoxyphenyl)-1,2,4-thiadiazol-5-yl]phenol (10i). A so-
lution of 4-methoxythiobenzamide (10ii) (318 mg, 1.90 mmol, 1
equiv.) in DMSO was heated for 8 h at 38 °C with 3-hydrox-
ythiobenzamide (11i) (290 mg, 1.90 mmol, 1 equiv.) and concen-
trated hydrochloric acid (193 µL, 1.90 mmol, 1 equiv.). After
cooling to rt, the crude mixture was poured into water and the
resulting precipitate was collected by filtration. The mixture of 10i
and 11 was separated by CC (hexane/ethyl acetate 8:2); yield: 30%
(70 mg) for 10i and 42% (215 mg) for 11.
3-[3-(4-Hydroxyphenyl)-1,2,4-thiadiazol-5-yl]phenol (10). The
title compound was prepared by reaction of 3-[3-(4-methoxyphe-
nyl)-[1,2,4]-thiadiazol-5-yl]-phenol (10i) (150 mg, 0.53 mmol) and
boron tribromide (1.59 mmol) according to method E. The product
was purified by preparative TLC (hexane/ethyl acetate 5:5); yield:
91% (130 mg); MS (ESI): 271 (M + H)+; Anal. (C14H10N2O2S)
C, H, N.
3-[5-(3-Hydroxyphenyl)-1-methyl-1H-1,2,4-triazol-3-yl]phenol (15).
The title compound was prepared by reaction of 3,5-bis(3-methox-
yphenyl)-1-methyl-1H-1,2,4-triazole (15i) (100 mg, 0.37 mmol) and
borontrifluoride dimethyl sulfide complex (27.7 mmol) according to
method F. The product was purified by preparative TLC (ethyl acetate);
yield: 64% (58 mg); MS (ESI): 268 (M + H)+.
2-Bromo-5-(3-methoxyphenyl)thiophene (18ii). The title com-
pound was prepared by reaction of 2,5-dibromothiophene (465 µL,
4.13 mmol), 3-methoxybenzeneboronic acid (753 mg, 4.95 mmol),
sodium carbonate (876 mg, 8.26 mmol), and tetrakis(triphenylphos-
phine) palladium (24 mg, 20 µmol) according to method A. The
product was purified by CC (hexane/ethyl acetate 9:1); yield: 41%
(445 mg).
2-(3-Methoxyphenyl)thiophene (18i). To a solution of 2-bromo-
5-(3-methoxyphenyl)thiophene (18ii) (100 mg, 0.37 mmol, 1 equiv.)
in dry THF cooled to -78 °C for 5 min, n-BuLi (1.6 M in hexane,
0.28 mL, 0.44 mmol, 1.2 equiv.) was added dropwise and stirred
for 15 min at -78 °C. The crude mixture was carefully hydrolyzed
by addition of water (10 mL) and layers were separated. The
aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over magnesium
sulfate, and evaporated to dryness under reduced pressure; yield:
98% (69 mg).
3-(2-Thienyl)phenol (18). The title compound was prepared by
reaction of 2-(3-methoxyphenyl)thiophene (18i) (80 mg, 0.42 mmol)
and boron tribromide (1.26 mmol) according to method E. The
product was purified by preparative TLC (hexane/ethyl acetate 7:3);
yield: 85% (63 mg); MS (ESI): 177 (M + H)+; Anal. (C10H8OS)
C, H, N.
2-(3-Methoxyphenyl)-5-phenylthiophene (19i). The title com-
pound was prepared by reaction of 2-bromo-5-(3-methoxyphe-
nyl)thiophene (18ii) (400 mg, 1.52 mmol), benzeneboronic acid
(223 mg, 1.82 mmol), sodium carbonate (322 mg, 3.04 mmol), and
tetrakis(triphenylphosphine) palladium (8.8 mg, 7.6 µmol) according
to method B. The product was purified by CC (hexane/ethyl acetate
7:3); yield: 70% (283 mg).
3-(5-Phenyl-2-thienyl)phenol (19). The title compound was
prepared by reaction of 2-(3-methoxyphenyl)-5-phenylthiophene
(19i) (100 mg, 0.37 mmol) and boron tribromide (2.22 mmol)
according to method E. The product was purified by preparative
TLC (hexane/ethyl acetate 5:5); yield: 81% (75 mg); MS (ESI):
253 (M + H)+; Anal. (C16H12OS) C, H, N.
2-Bromo-5-(4-methoxyphenyl)thiophene (20ii). The title com-
pound was prepared by reaction of 2,5-dibromothiophene (V ) 465
µL, 4.13 mmol), 4-methoxybenzeneboronic acid (753 mg, 4.95
mmol), sodium carbonate (876 mg, 8.26 mmol), and tetrakis(triph-
enylphosphine) palladium (24 mg, 20 µmol) according to method
A. The product was purified by CC (hexane/ethyl acetate 9:1); yield:
75% (815 mg).
2-(4-Methoxyphenyl)-5-phenylthiophene (20i). The title com-
pound was prepared by reaction of 2-bromo-5-(4-methoxyphe-
nyl)thiophene (20ii) (400 mg, 1.52 mmol), benzeneboronic acid
(223 mg, 1.82 mmol), sodium carbonate (322 mg, 3.04 mmol), and
tetrakis(triphenylphosphine) palladium (8.8 mg, 7.6 µmol) according
to method B. The product was purified by CC (hexane/ethyl acetate
7:3); yield: 75% (303 mg).
3-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-1-methyl-1H-1,2,4-
triazole (12i). The title compound was prepared from para-anisoyl
chloride (170 mg, 1.0 mmol), ethyl 3-methoxybenzimidate (179
mg, 1.0 mmol), and methyl hydrazine (92 mg, 2.0 mmol) according
to method D; yield: 85% (250 mg); mp 108-110 °C (CH2Cl2/
Et2O); MS (ESI): 296 (M + H)+.
3-[5-(4-Hydroxyphenyl)-1-methyl-1H-1,2,4-triazol-3-yl]phenol (12).
The title compound was prepared by reaction of 3-(3-methoxyphe-
nyl)-5-(4-methoxyphenyl)-1-methyl-1H-1,2,4-triazole (12i) (100
mg, 0.37 mmol) and borontrifluoride dimethyl sulfide complex
(27.75 mmol) according to method F. The product was purified by
preparative TLC (ethyl acetate); yield: 46% (42 mg); MS (ESI):
268 (M + H)+.
3,5-Bis(4-methoxyphenyl)-1-methyl-1H-1,2,4-triazole (13i). The
title compound was prepared from para-anisoyl chloride (170 mg,
1.0 mmol), ethyl 4-methoxybenzimidate (179 mg, 1.0 mmol), and
methyl hydrazine (92 mg, 2.0 mmol) according to method D; yield:
78% (230 mg); mp 141-143 °C (CH2Cl2/ Et2O); MS (ESI): 296
(M + H)+.
4-[5-(4-Hydroxyphenyl)-1-methyl-1H-1,2,4-triazol-3-yl]phenol (13).
The title compound was prepared by reaction of 3,5-bis(4-methox-
yphenyl)-1-methyl-1H-1,2,4-triazole (13i) (100 mg, 0.37 mmol) and
borontrifluoride dimethyl sulfide complex (27.7 mmol) according to
method F. The product was purified by preparative TLC (ethyl acetate);
yield: 63% (57 mg); MS (ESI): 268 (M + H)+.
3-(4-Methoxyphenyl)-5-(3-methoxyphenyl)-1-methyl-1H-1,2,4-
triazole (14i). The title compound was prepared from meta-anisoyl
chloride (170 mg, 1.0 mmol), ethyl 4-methoxybenzimidate (179
mg, 1.0 mmol), and methyl hydrazine (92 mg, 2.0 mmol) according
to method D; yield: 77% (227 mg); mp 114-116 °C (CH2Cl2/
Et2O); MS (ESI): 296 (M + H)+.
4-[5-(3-Hydroxyphenyl)-1-methyl-1H-1,2,4-triazol-3-yl]phenol (14).
The title compound was prepared by reaction of 3-(4-methoxyphe-
nyl)-5-(3-methoxyphenyl)-1-methyl-1H-1,2,4-triazole (14i) (100
mg, 0.37 mmol) and borontrifluoride dimethyl sulfide complex (27.7
mmol) according to method F. The product was purified by
preparative TLC (ethyl acetate); yield: 53% (48 mg); MS (ESI):
268 (M + H)+.
3,5-Bis(3-methoxyphenyl)-1-methyl-1H-1,2,4-triazole (15i). The
title compound was prepared from meta-anisoyl chloride (170 mg,
1.0 mmol), ethyl 3-methoxybenzimidate (179 mg, 1.0 mmol), and
methyl hydrazine (92 mg, 2.0 mmol) according to method D; yield:
67% (198 mg); mp 67-69 °C (CH2Cl2/ Et2O); MS (ESI): 296 (M
+ H)+.
4-(5-Phenyl-2-thienyl)phenol (20). The title compound was
prepared by reaction of 2-(4-methoxyphenyl)-5-phenylthiophene
(20i) (100 mg, 0.37 mmol) and boron tribromide (2.22 mmol)
according to method E. The product was purified by preparative
TLC (dichloromethane/methanol 99:1); yield: 95% (89 mg); MS
(ESI): 253 (M + H)+; Anal. (C16H12OS) C, H, N.
2-(3-Methoxyphenyl)-5-(4-methoxyphenyl)thiophene (22i). The
title compound was prepared by reaction of 2-bromo-5-(3-meth-
oxyphenyl)thiophene (18ii) (150 mg, 0.57 mmol), 4-methoxyben-
zeneboronic acid (223 mg, 0.68 mmol), sodium carbonate (120 mg,
1.14 mmol), and tetrakis(triphenylphosphine) palladium (3.2 mg,
2.8 µmol) according to method B. The product was purified by
CC (hexane/ethyl acetate 9:1); yield: 75% (126 mg).
3-[5-(4-Hydroxyphenyl)-2-thienyl]phenol (22). The title com-
pound was prepared by reaction of 2-(3-methoxyphenyl)-5-(4-

6736 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bey et al.
methoxyphenyl)thiophene (22i) (150 mg, 0.51 mmol) and boron
tribromide (3.06 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 93%
(127 mg); MS (ESI): 269 (M + H)+; Anal. (C16H12O2S) C, H, N.
2,5-Bis(3-methoxyphenyl)thiophene (23i). The title compound
was prepared by reaction of 2-bromo-5-(3-methoxyphenyl)thiophene
(18ii) (150 mg, 0.57 mmol), 3-methoxybenzeneboronic acid (223
mg, 0.68 mmol), sodium carbonate (120 mg, 1.14 mmol), and
tetrakis(triphenylphosphine) palladium (3.2 mg, 2.8 µmol) according
to method B. The product was purified by CC (hexane/ethyl acetate
9:1); yield: 78% (132 mg).
3,3′-Thiene-2,5-diyldiphenol (23). The title compound was pre-
pared by reaction of 2,5-bis(3-methoxyphenyl)thiophene (23i) (150
mg, 0.51 mmol) and boron tribromide (3.06 mmol) according to
method E. The product was purified by preparative TLC (hexane/
ethyl acetate 5:5); yield: 95% (130 mg); MS (ESI): 269 (M + H)+;
Anal. (C16H12O2S) C, H, N.
4-Bromo-2-(3-methoxyphenyl)thiophene (24ii). The title com-
pound was prepared by reaction of 2,4-dibromothiophene (1.00 g,
4.13 mmol), 3-methoxybenzeneboronic acid (753 mg, 4.95 mmol),
sodium carbonate (876 mg, 8.26 mmol), and tetrakis(triphenylphos-
phine) palladium (24 mg, 20 µmol) according to method A. The
product was purified by CC (hexane/ethyl acetate 9:1); yield: 72%
(782 mg).
2-(3-Methoxyphenyl)-4-phenylthiophene (24i). The title com-
pound was prepared by reaction of 4-bromo-2-(3-methoxyphe-
nyl)thiophene (24ii) (250 mg, 0.93 mmol), benzeneboronic acid
(137 mg, 1.12 mmol), sodium carbonate (197 mg, 1.86 mmol), and
tetrakis(triphenylphosphine) palladium (6 mg, 4.64 µmol) according
to method B. The product was purified by CC (petroleum ether/
ethyl acetate 7:3); yield: 91% (225 mg).
3-(4-Phenyl-2-thienyl)phenol (24). The title compound was
prepared by reaction of 2-(3-methoxyphenyl)-4-phenylthiophene
(24i) (225 mg, 0.85 mmol) and boron tribromide (3.6 mmol)
according to method E. The product was purified by preparative
TLC (dichloromethane/methanol 99:1); yield: 69% (147 mg); MS
(ESI): 253 (M + H)+.
4-Bromo-2-(4-methoxyphenyl)thiophene (25ii). The title com-
pound was prepared by reaction of 2,4-dibromothiophene (1.00 g,
4.13 mmol), 4-methoxybenzeneboronic acid (753 mg, 4.95 mmol),
sodium carbonate (876 mg, 8.26 mmol), and tetrakis(triphenylphos-
phine) palladium (24 mg, 20 µmol) according to method A. The
product was purified by CC (hexane/ethyl acetate 9:1); yield: 78%
(847 mg).
2-(4-Methoxyphenyl)-4-(3-methoxyphenyl)thiophene (25i). The
title compound was prepared by reaction of 4-bromo-2-(4-meth-
oxyphenyl)thiophene (25ii) (150 mg, 0.57 mmol), 3-methoxyben-
zeneboronic acid (223 mg, 0.68 mmol), sodium carbonate (120 mg,
1.14 mmol), and tetrakis(triphenylphosphine) palladium (3.2 mg,
2.8 µmol) according to method B. The product was purified by
CC (hexane/ethyl acetate 9:1); yield: 70% (118 mg).
3-[5-(4-Hydroxyphenyl)-3-thienyl]phenol (25). The title com-
pound was prepared by reaction of 2-(4-methoxyphenyl)-4-(3-
methoxyphenyl)thiophene (25i) (150 mg, 0.51 mmol) and boron
tribromide (3.06 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 80%
(109 mg); MS (ESI): 267 (M - H)-; Anal. (C16H12O2S) C, H, N.
2-(3-Methoxyphenyl)-4-(4-methoxyphenyl)thiophene (27i). The
title compound was prepared by reaction of 4-bromo-2-(3-meth-
oxyphenyl)thiophene (24ii) (150 mg, 0.57 mmol), 4-methoxyben-
zeneboronic acid (223 mg, 0.68 mmol), sodium carbonate (120 mg,
1.14 mmol), and tetrakis(triphenylphosphine) palladium (3.2 mg,
2.8 µmol) according to method B. The product was purified by
CC (hexane/ethyl acetate 9:1); yield: 70% (118 mg).
2,4-Bis(3-methoxyphenyl)thiophene (28i). The title compound
was prepared by reaction of 4-bromo-2-(3-methoxyphenyl)thiophene
(24ii) (150 mg, 0.57 mmol), 3-methoxybenzeneboronic acid (223
mg, 0.68 mmol), sodium carbonate (120 mg, 1.14 mmol), and
tetrakis(triphenylphosphine) palladium (3.2 mg, 2.8 µmol) according
to method B. The product was purified by CC (hexane/ethyl acetate
9:1); yield: 72% (121 mg).
3,3′-Thiene-2,4-diyldiphenol (28). The title compound was pre-
pared by reaction of 2,4-bis(3-methoxyphenyl)thiophene (28i) (150
mg, 0.51 mmol) and boron tribromide (3.06 mmol) according to
method E. The product was purified by preparative TLC (hexane/
ethyl acetate 5:5); yield: 88% (120 mg); MS (ESI): 267 (M - H)-;
Anal. (C16H12O2S) C, H, N.
4,4′-(Seleniene-2,5-diyl)diphenol (29). The title compound was
prepared by reaction of 2,5-dibromoselenophene (150 mg, 0.52
mmol), 4-hydroxybenzeneboronic acid (172 mg, 1.25 mmol),
cesium carbonate (679 mg, 2.08 mmol), and tetrakis(triphenylphos-
phine) palladium (6.0 mg, 5.2 µmol) according to method C. The
product was purified by preparative TLC (hexane/ethyl acetate 6:4);
yield: 49% (81 mg); MS (ESI): 317 (M + H)+; Anal. (C16H12O2Se)
C, H, N.
3,3′-(Seleniene-2,5-diyl)diphenol (30). The title compound was
prepared by reaction of 2,5-dibromoselenophene (150 mg, 0.52
mmol), 3-hydroxybenzeneboronic acid (172 mg, 1.25 mmol),
cesium carbonate (679 mg, 2.08 mmol), and tetrakis(triphenylphos-
phine) palladium (6.0 mg, 5.2 µmol) according to method C. The
product was purified by preparative TLC (hexane/ethyl acetate 6:4);
yield: 42% (69 mg); MS (ESI): 317 (M + H)+; Anal. (C16H12O2Se)
C, H, N.
3-Bromo-2-(4-methoxyphenyl)thiophene (31ii). The title com-
pound was prepared by reaction of 2,3-dibromothiophene (234 µL,
2.1 mmol), 4-methoxybenzeneboronic acid (383 mg, 2.52 mmol),
sodium carbonate (403 mg, 4.2 mmol), and tetrakis(triphenylphos-
phine) palladium (12 mg, 10 µmol) according to method B. The
product was purified by CC (hexane); yield: 70% (387 mg).
4,4′-(Thiene-2,3-diyl)diphenol (31). The title compound was
prepared by reaction of 2,3-bis(4-methoxyphenyl)thiophene (31i)
(150 mg, 0.51 mmol) and boron tribromide (3.06 mmol) according
to method E. The product was purified by preparative TLC (hexane/
ethyl acetate 5:5); yield: 70% (95 mg); MS (ESI): 269 (M + H)+;
Anal. (C16H12O2S) C, H, N.
3-Bromo-2-(3-methoxyphenyl)thiophene (32ii). The title com-
pound was prepared by reaction of 2,3-dibromothiophene (234 µL,
2.1 mmol), 3-methoxybenzeneboronic acid (383 mg, 2.52 mmol),
sodium carbonate (403 mg, 4.2 mmol), and tetrakis(triphenylphos-
phine) palladium (12 mg, 10 µmol) according to method B. The
product was purified by CC (hexane); yield: 58% (320 mg).
2-(3-Methoxyphenyl)-3-(4-methoxyphenyl)-thiophene (32i). The
title compound was prepared by reaction of 3-bromo-2-(3-meth-
oxyphenyl)thiophene (32ii) (150 mg, 0.57 mmol), 4-methoxyben-
zeneboronic acid (223 mg, 0.68 mmol), sodium carbonate (120 mg,
1.14 mmol), and tetrakis(triphenylphosphine) palladium (3.2 mg,
2.8 µmol) according to method B. The product was purified by
CC (hexane/ethyl acetate 7:3); yield 40% (68 mg).
3-[3-(4-Hydroxyphenyl)-2-thienyl]phenol (32). The title com-
pound was prepared by reaction of 2-(3-methoxyphenyl)-3-(4-
methoxyphenyl)thiophene (32i) (150 mg, 0.51 mmol) and boron
tribromide (3.06 mmol) according to method E. The product was
purified by preparative TLC (hexane/ethyl acetate 5:5); yield: 56%
(77 mg); MS (ESI): 269 (M + H)+; Anal. (C16H12O2S) C, H, N.
3,3′′-Dimethoxy-1,1′:4′,1′′-terphenyl (35i). The title compound
was prepared by reaction of 4′-bromo-3-methoxybiphenyl (35ii)
(500 mg, 1.90 mmol), 3-methoxybenzeneboronic acid (346 mg, 2.28
mmol), sodium carbonate (403 mg, 3.80 mmol), and tetrakis(triph-
enylphosphine) palladium (11 mg, 9.5 µmol) according to method
A (heating the mixture 20 h instead of 4 h). The product was
purified by CC (hexane/ethyl acetate 95:5); yield: 14% (77 mg).
3,4′′-Dimethoxy-1,1′:4′,1′′-terphenyl (36i). The title compound
was prepared by reaction of 4′-bromo-3-methoxybiphenyl (35ii)
(500 mg, 1.90 mmol), 4-methoxybenzeneboronic acid (346 mg, 2.28
mmol), sodium carbonate (403 mg, 3.80 mmol), and tetrakis(triph-
3-[4-(4-Hydroxyphenyl)-2-thienyl]phenol (27). The title com-
pound was prepared by reaction of 2-(3-methoxyphenyl)-4-(4-
methoxyphenyl)thiophene (27i) (150 mg, 0.51 mmol) and boron
tribromide solution (3.06 mmol) according to method E. The
product was purified by preparative TLC (hexane/ethyl acetate 5:5);
yield: 85% (116 mg); MS (ESI): 267 (M - H)-; Anal. (C16H12O2S)
C, H, N.

Nonsteroidal Inhibitors of 17ꢀ-HSD1
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6737
enylphosphine) palladium (11 mg, 9.5 µmol) according to method
A (heating the mixture 20 h instead of 4 h). The product was
purified by CC (hexane/ethyl acetate 95:5); yield: 90% (496 mg).
3,3′-Pyridine-2,5-diyldiphenol (37). The title compound was
prepared by reaction of 2,5-dibromo pyridine (150 mg, 0.63 mmol),
3-hydroxybenzeneboronic acid (231 mg, 1.52 mmol), cesium
carbonate (821 mg, 2.52 mmol), and tetrakis(triphenylphosphine)
palladium (7.3 mg, 6.3 µmol) according to method C. The product
was purified by preparative TLC (dichloromethane/methanol 98:
2); yield: 67% (111 mg); MS (ESI): 262 (M - H)-; Anal.
(C17H13NO2) C, H, N.
was used for the experiments. Cells were grown for 48 h in phenol
red-free medium. Compounds 4, 10, 22, 25, 35, and 36 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
two to three days and supplemented with the respective additive.
After eight days of incubation, the cell viability was evaluated
measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphen-
yl-tetrazoliumbromide (MTT). The cleavage of MTT to a blue
formazan by mitochondrial succinate-dehydrogenase was quantified
spectrophotometrically at 590 nm as described by Denizot and
Lang66 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 ) ((pro-
liferation(compound-induced) - 1)/(proliferation(E2-induced) - 1))
× 100%. Each value is calculated as a mean value of at least three
independent experiments
5. Caco-2 Transport Experiments. Caco-2 cell culture and
transport experiments were performed according to Yee67 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. The
compounds were applied to the cells as a mixture (cassette dosing)
to increase the throughput. The initial concentration of the
compounds in the donor compartment was 50 µM (0.2 M MES,
pH: 6.5, 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 perme-
ability 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.
6. Metabolic Stability Assay. The assay was performed with
liver microsomes from male Sprague-Dawley rats (BD Gentest,
USA). 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 protein in
phosphate buffer 100 mM pH 7.4 and 90 µL 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 (final
concentration 1 µM) 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-ethoxycou-
marin 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 percent-
ages were plotted against the corresponding time points and the
half-life time was derived by a standard fit of the data.
Biological Methods. [2,4,6,7-3H]-E2 and [2,4,6,7-3H]-E1 were
bought from Perkin-Elmer, Boston. Quickszint Flow 302 scintillator
fluid was bought from Zinsser Analytic, Frankfurt.
17ꢀ-HSD1 and 17ꢀ-HSD2 were obtained from human placenta
according to previously described procedures.22,62 Fresh human
placenta was homogenized and centrifuged. The pellet fraction
contains the microsomal 17ꢀ-HSD2, while 17ꢀ-HSD1 was obtained
after precipitation with ammonium sulfate from the cytosolic
fraction.
1. Inhibition of 17ꢀ-HSD1. Inhibitory activities were evaluated
by a well established method with minor modifications.41,63,64
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 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 a mixture of
unlabeled- and [2,4,6,7-3H]-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 chromatog-
raphy 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.
2. Inhibition of 17ꢀ-HSD2. The 17ꢀ-HSD2 inhibition assay
was performed similarly to the 17ꢀ-HSD1 procedure. The microso-
mal fraction was incubated with NAD+ [1500 µM], test compound,
and a mixture of unlabeled- and [2,4,6,7-3H]-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.
3. ER Affinity. The binding affinity of select compounds to the
ERR and ERꢀ was determined according to Zimmermann et al.65
Briefly, 0.25 pmol of ERR or ERꢀ, respectively, were incubated
with [2,4,6,7-3H]-E2 (10 nM) and test compound for 1 h at rt. The
potential inhibitors were dissolved in DMSO (5% final concentra-
tion). Nonspecific binding was performed with diethylstilbestrol
(10 µM). After incubation, ligand-receptor complexes were
selectively bound to hydroxyapatite (5 g/ 60 mL TE-buffer). The
formed complex was separated, washed, and resuspended in ethanol.
For radiodetection, scintillator cocktail (Quickszint 212, Zinsser
Analytic, Frankfurt) was added and samples were measured in a
liquid scintillation counter (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%.
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 µL/mg protein. As fu
4. Evaluation of the Estrogenic Activity Using T-47D Cells.
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% (v/v). RPMI 1640 (without phenol red)

6738 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Bey et al.
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7. 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. Compounds 4, 25, and 35 were tested for inhibition of the
following enzymes: CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4.
Inhibitory potencies were determined as IC50 values.
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8. In Vivo Pharmacokinetics. Male Wistar rats weighing
300-330 g (Janvier France) 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. They were anesthetized
with a ketamine (135 mg/kg)/xylyzine (10 mg/kg) mixture and
cannulated with silicone tubing via the right jugular vein and attached
to the skull with dental cement.68 Prior to the first blood sampling,
animals were connected to a counterbalanced system and tubing to
perform blood sampling in the freely moving rat.
Compounds 3 and 39 were applied orally in a cassette dosing in
4 rats at the dose of 10 mg/kg body weight by using a feeding
needle. The compounds were dissolved in a mixture labrasol/water
(1:1) and given at a volume of 5 mL/kg. Blood samples (0.2 mL)
were taken at 0, 1, 2, 3, 4, 6, 8, 10, and 24 h postdose and collected
in heparinized tubes. They were centrifuged at 3000 g for 10 min,
and plasma was harvested and kept at -20 °C until analyzed.
HPLC-MS/MS analysis and quantification of the samples was
carried out on a Surveyor HPLC-system coupled with a TSQ
Quantum (Thermofischer) triple quadrupole mass spectrometer
equipped with an electrospray interface (ESI).
ˇ
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Hartree) and blue for positive ones (4.5 × 10-2 Hartree).70
Acknowledgment. We are grateful to the Deutsche Fors-
chungsgemeinschaft (HA1315/8-1) for financial support. We
thank Dr. Christiane Scherer for performing the CaCo-2 cell
test, Anja Palusczak and Beate Geiger for their help in
performing the enzyme inhibition tests (17ꢀ-HSD1, 17ꢀ-HSD2,
ERR, and ERꢀ), and Dr. Ursula Mu¨ller-Viera, Pharmacelsus
CRO, Saarbru¨cken, Germany, for the stability tests with rat liver
microsomes. Patricia Kruchten is grateful to the European
Postgraduate School 532 (DFG) for a scholarship. Dr. Yaseen
A. Al-Soud is grateful to the Alexander von Humboldt Founda-
tion (AvH) for a fellowship.
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Supporting Information Available: Spectroscopic data of all
compounds (1H NMR, 13C NMR, IR), purity data of final
compounds and MEP maps of compounds 7, 10, 14, 27, and G.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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