Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a selective 17β-HSD1 inhibitor with sub-nanomolar IC50for a proof-of-principle study

Article abstract of DOI:10.1016/j.ejmech.2016.11.004

Current endocrine therapeutics for the estrogen-dependent disease endometriosis often lead to considerable side-effects as they act by reducing estrogen action systemically. A more recent approach takes advantage of the fact that the weak estrogen estrone (E1) which is abundant in the plasma, is activated in the target cell to the highly estrogenic estradiol (E2) by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1). 17β-HSD1 is overexpressed in endometriosis and thus a promising target for the treatment of this disease, with the prospect of less target-associated side-effects. Potent inhibitors from the class of bicyclic substituted hydroxyphenylmethanones with sulfonamide moiety recently described by us suffered from high molecular weight and low selectivity over 17βHSD2, the physiological adversary of 17β-HSD1. We describe the structural optimizations leading to the discovery of (5-(3,5-dichloro-4-methoxyphenyl)thiophen-2-yl)(2,6-difluoro-3-hydroxyphenyl)methanone 20, which displayed a sub-nanomolar IC₅₀towards 17β-HSD1 as well as high selectivity over the type 2 enzyme, the estrogen receptors α and β and a range of hepatic CYP enzymes. The compound did neither show cellular toxicity, nor PXR activation nor mutagenicity in the AMES II assay. Additional favourable pharmacokinetic properties (rat) make 20 a suitable candidate for proof-of-principle studies using xenotransplanted immunodeficient rats.

Full text of DOI:10.1016/j.ejmech.2016.11.004

European Journal of Medicinal Chemistry xxx (2016) 1e14
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Treatment of estrogen-dependent diseases: Design, synthesis and
profiling of a selective 17 -HSD1 inhibitor with sub-nanomolar IC50
b
for a proof-of-principle study
,
Ahmed S. Abdelsamie a b, Chris J. van Koppen c, Emmanuel Bey c, Mohamed Salah a,
d
d
e
e
€
Carsten Borger , Lorenz Siebenbürger , Matthias W. Laschke , Michael D. Menger ,
Martin Frotscher a
,
*
a Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, D-66123 Saarbrücken, Germany
b Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, 12622 Cairo, Egypt
c ElexoPharm GmbH, Campus A12, D-66123 Saarbrücken, Germany
d PharmBioTec GmbH, Science Park 1, D-66123 Saarbrücken, Germany
e Institute for Clinical and Experimental Surgery, Saarland University, D-66421, Homburg/Saar, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Current endocrine therapeutics for the estrogen-dependent disease endometriosis often lead to
considerable side-effects as they act by reducing estrogen action systemically. A more recent approach
takes advantage of the fact that the weak estrogen estrone (E1) which is abundant in the plasma, is
Received 22 September 2016
Received in revised form
2 November 2016
Accepted 3 November 2016
Available online xxx
activated in the target cell to the highly estrogenic estradiol (E2) by 17
b-hydroxysteroid dehydrogenase
type 1 (17 -HSD1). 17 -HSD1 is overexpressed in endometriosis and thus a promising target for the
b
b
treatment of this disease, with the prospect of less target-associated side-effects. Potent inhibitors from
the class of bicyclic substituted hydroxyphenylmethanones with sulfonamide moiety recently described
Keywords:
by us suffered from high molecular weight and low selectivity over 17
of 17 -HSD1. We describe the structural optimizations leading to the discovery of (5-(3,5-dichloro-4-
methoxyphenyl)thiophen-2-yl)(2,6-difluoro-3-hydroxyphenyl)methanone 20, which displayed a sub-
nanomolar IC50 towards 17 -HSD1 as well as high selectivity over the type 2 enzyme, the estrogen re-
ceptors and and a range of hepatic CYP enzymes. The compound did neither show cellular toxicity,
bHSD2, the physiological adversary
17b-Hydroxysteroid dehydrogenase type 1
Enzyme inhibitor
Steroidogenic enzymes
Estrogen-dependent disease
Intracrinology
b
b
a
b
nor PXR activation nor mutagenicity in the AMES II assay. Additional favourable pharmacokinetic
properties (rat) make 20 a suitable candidate for proof-of-principle studies using xenotransplanted
immunodeficient rats.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
17b-Hydroxysteroid-dehydrogenase type 1 (17b-HSD1) is a
member of the NADPH/NADþ-dependent oxidoreductases. It ca-
talyses the activation of estrone (E1) to the most potent estrogen
estradiol (E2; Fig. 1) within the target cell. Besides its beneficial
physiological effects, E2 is also known to play crucial roles in the
development of estrogen-dependent diseases (EDD). Thus, endo-
metriosis [1], breast cancer [2,3], ovarian tumor [4] and other EDD
are typically attended by locally increased E2/E1-ratios and high
Abbreviations: (h)17
b
-HSD1, (human) 17
b-hydroxysteroid dehydrogenase type
1; (h)17 -HSD2, (human) 17
b
b-hydroxysteroid dehydrogenase type 2; ADME, ab-
sorption, distribution, metabolism, and excretion; BSHs, bicyclic substituted
hydroxyphenylmethanones; CC, column chromatography; DBPO, dibenzoyl
peroxide; DCM, dichloromethane; DME, dimethoxyethane; E1, estrone; E2, 17b-
estradiol; EDD, estrogen-dependent disease; ER, estrogen receptor; GnRH, gonad-
otropin-releasing hormone; HPLC, high performance liquid chromatography; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAD(H), nicotin-
amide adenine dinucleotide; NADP(H), nicotinamide adenine dinucleotide phos-
phate; NBS, N-bromosuccinimide; PXR, pregnane X receptor; RBA, relative binding
affinity; SEM, standard error of the mean; SERM, selective estrogen receptor
levels of 17
b
-HSD1 mRNA in the diseased tissue. Therefore, inhi-
bition of 17
b
-HSD1 is considered to be a valuable treatment option
modulator; SF, selectivity factor over 17b-HSD2; TLC, thin layer chromatography.
* Corresponding author.
for EDD: The tissue-selective expression of 17
b-HSD1 and its
intracrine mode of action [5] offer the prospect of a therapy which
E-mail address: m.frotscher@mx.uni-saarland.de (M. Frotscher).
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

2
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
Fig. 1. Interconversion of E1 and E2.
is associated with less side-effects compared to established (but
unsatisfactory) treatments with GnRH-analogues, [6,7], aromatase-
inhibitors [8e13], anti-estrogens [14] and selective estrogen-
receptor modulators (SERMs) [14]. The validity of this concept is
selectivity.
2. Design
supported by the observation that a 17
decrease of E2-levels in endometriotic specimens [15]. In addition,
17 -HSD1 inhibitors were shown to reduce the E1-stimulated tu-
b-HSD1 inhibitor led to a
As a starting point, structural modifications of lead compound A
b
(Chart 1) were carried out focussing on the substitution patterns of
rings A and C (compounds 1e25, Chart 1), taking into account the
following previous SAR data:
mor cell growth in vitro and in animal models, suggesting the
suitability of this target for the treatment of breast cancer [16e18].
17b
-HSD1 inhibitors should be selective over 17
b-HSD2, the phys-
iological adversary of 17
b
-HSD1 which inactivates E2 by oxidation
ꢀ The possibilities for variations of ring A-substitution are very
to E1 (Fig. 1). Moreover, they should not bind to estrogen receptors
in order to keep systemic interference with estrogenic pathways to
a minimum.
restricted: An OH-group in position 3 of ring A is important for
inhibitory activity towards 17b-HSD1 and should be retained. In
addition, the target enzyme only tolerates small additional
substituents on ring A.
ꢀ Much more flexibility exists concerning the substitution pattern
of ring C: here, even bulky substituents should be tolerated, and
the OH-group can be omitted e albeit its presence can be ex-
A number of 17
many of them with a steroidal scaffold [19e25]. Our group reported
on several classes of non-steroidal 17 -HSD1 inhibitors, [26e30],
b-HSD1 inhibitors are described in the literature,
b
among them the bicyclic substituted hydroxyphenylmethanones
(BSHs) which displayed very strong inhibition of the target protein
[26,29]. Previous studies in this compound class revealed that in-
hibition of the target enzyme strongly depends on the substitution
pattern of the benzoyl ring (Fig. 2, ring A) [26,29]. Here, already
minor structural modifications were found to induce dramatic
changes in activity. Thus, bulky substituents led to a loss of activity
whereas the introduction of fluorine atoms resulted in considerably
more active compounds [26,29]. In contrast, it was rather selec-
pected to lead to increased selectivity over 17b-HSD2.
First, based upon a simplified analogue of lead A (compound 1,
Chart 1), the synthesis of a couple of compounds bearing small
substituents, especially fluorine, on ring A was envisaged in order
to identify a beneficial substitution pattern (compounds 2e5)
which should be maintained in the further design process. Subse-
quent structural modifications aimed at the optimization of ring C
and its substitution pattern. To this purpose, electron-donating and
ewithdrawing groups of different sizes were introduced. In addi-
tion, the effect of a replacement of benzene with pyridine was
evaluated (compounds 6e25).
Moreover, compounds 26 and 27 as non-fluorinated analogues
of 20 and 21 were synthesized to re-evaluate the effect of the
fluorine atoms on ring A on activity and selectivity. Finally, the
phenyl ring C of the most interesting compounds 20 and 21 was
decorated with heterocyclic moieties in order to increase solubility
(compounds 28e32, Chart 2).
tivity (towards 17b-HSD2) than inhibitory potency which was
influenced by substituents at the phenyl ring (ring C) [26].
The majority of these SAR data was derived from inhibitors
bearing a bulky aromatic sulfonamide moiety. There was evidence,
however, that - in terms of biological activities towards human 17b-
HSD1 and 2 - the sulfonamides do not have substantial advantages
compared to compounds devoid of the sulfonamide group (Fig. 2)
[29]. Consequently, the design and synthesis of novel potential
inhibitors lacking this group was aimed at, thus lowering molecular
weight while preserving or increasing inhibitory potency and
Fig. 2. Comparison of compound A with sulfonamide moiety and compound B, lacking this group.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
3
Chart 1. Design of synthesized compounds 1e25.
3. Chemistry
Friedel-Crafts conditions (method A). Subsequent ether cleavage of
intermediates I and IV (method B) yielded VI and VII, respectively,
in a quantitative yield (Scheme 1).
The key intermediates IeV were synthesized in good yield from
the appropriate benzoyl chlorides and 2-bromothiophene using
For the preparation of compounds 1e7 the key intermediates
Chart 2. Design of synthesized compounds 26e32.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

4
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
O
Cl
a
b
Br
Br
Br
O
O
S
R1
R
R2
S
+
S
R2
R2
R1
R1
HO
R3
R
R3
I(R = OCH3, R1 = R2 = R3 = H)
VI(R1 = R2 = H)
VII(R1 = R2 = F)
II(R = OCOCH3, R1 = CH3, R2 = R3 = H)
III(R = OCH3, R1 = F, R2 = R3 = H)
IV(R = OCH2CH3, R1 = R2 = F)
V(R = OCH3, R1 = R2 = R3 = F)
Scheme 1. Synthesis of intermediates I-VII. a) method A, AlCl3, anhydrous CH2Cl2, 0 ꢁC, 0.5 h and then rt, 3 h. b) method B, BBr3, CH2Cl2, -78 ꢁC to rt, overnight.
IeV were submitted to a Suzuki reaction (method C1 or C2) with
the appropriate boronic acid to give 1a-7a. The latter were sub-
mitted to ether cleavage with BBr3 in dichloromethane to afford the
final compounds (1, 3e7) or hydrolysis using 10% NaOH in ethanol
to give compound 2, respectively (Schemes 2 and 3).
4. Biological results and discussion
4.1. Inhibition of human 17
HSD2, ER , and ER
b-HSD1 and selectivity towards 17b-
a
b
The compounds 8e21 and 24e27 were obtained from the in-
termediates VI or VII by standard methods (either Suzuki reaction
(method C1) alone or followed by ether cleavage (method B))
(Scheme 4).
The synthesis of compound 23 was accomplished as shown in
Scheme 5: Bromination of 2-chloro-6-methylphenol with NBS in
acetic acid selectively gave the ring-brominated compound 22c,
which underwent methylation upon treatment with methyl iodide.
The resulting compound 22b was transformed to the boronic acid
ester 22a. Finally, Suzuki cross coupling reaction of the latter with
VII yielded compound 22, which was submitted to ether cleavage
with BBr3, resulting in 23.
The boronic acid ester 22a was also used as starting material for
the syntheses leading to compounds 28e30 (Scheme 5). By using
NBS and DBPO in CCl4, 22a was converted to the brominated in-
termediate 28b. Nucleophilic substitution of the bromine atom
with 1-piperazine-1-ylethanone yielded 28a. The latter was reac-
ted with VII in a Suzuki reaction (method C1) to afford compound
28, which gave access to 29 (via ether cleavage) and 30 (via amide
hydrolysis under acidic conditions).
The azide intermediate 31c was prepared by reaction of 28b
with NaN3 in DMF (Scheme 6). The subsequent Suzuki cross-
coupling reaction with VII yielded 31b, which was submitted to a
cycloaddition reaction with acetic acid vinyl ester [31] to give the
1,2,3-triazole substituted compound. Interestingly, the latter was
exclusively isolated as acetic acid ester 31a. Saponification with
2M-NaOH gave the phenol 31, which was transformed to the diol 32
by reaction with BBr3 (method B).
Human placental enzymes were used for both 17
and were obtained according to described methods [32e34]. In the
17 -HSD1 assay, incubations were run with cytosolic fractions,
tritiated E1, cofactor and inhibitor. The separation of substrate and
product was accomplished by HPLC. The 17 -HSD2 assay was
b-HSD assays
b
b
performed similarly using tritiated E2 as substrate and the micro-
somal fraction. Activities are given as IC50-values (Tables 1e4).
As expected, 17b-HSD1 inhibition of the synthesized com-
pounds was strongly dependent on the substitution pattern of the
benzoyl moiety (ring A, see Chart 1): Whereas compound 1 showed
moderate activity (IC50 ¼ 105 nM, Table 1), the presence of a methyl
group was detrimental for inhibitory potency (compound 2). A very
strong inhibition of the target enzyme, however, could be achieved
by the introduction of one or more fluorine atoms (compounds
3e5). The most interesting properties in terms of activity towards
17b-HSD1 and selectivity over 17b-HSD2 was detected for the 2,6-
difluorinated compound 4. Therefore, this substitution pattern of
the benzoyl moiety was maintained in the subsequent optimization
of ring C.
The replacement of the phenyl ring C with pyridine (compounds
6 and 7) or omission of the fluorine substituents (8) led to a more or
less pronounced drop in activity, compared to compound 4
(Table 2). For the sulfonamides 9e11 this effect was also observed.
The introduction of a nitrile group proved to be beneficial for ac-
tivity (compounds 12 and 13), but led to low selectivity over h17b-
HSD2.
Starting from 13, the nitrile group was omitted or replaced by
chlorine while maintaining an oxygen-function of ring C, in order to
F
a
b or c
Br
F
O
S
O
O
S
S
F
R2
F
R2
R2
R1
HO
R1
R1
R
R
R3
R3
R3
I(R = OCH3, R1 = R2 = R3 = H)
II(R = OCOCH3, R1 = CH3, R2 = R3 = H) 2a(R = OCOCH3, R1 = CH3, R2 = R3 = H)
III(R = OCH3, R1 = F, R2 = R3 = H)
IV(R = OCH2CH3, R1 = R2 = F)
V(R = OCH3, R1 = R2 = R3 = F)
1a(R = OCH3, R1 = R2 = R3 = H)
1(R1 = R2 = R3 = H)
2(R1 = CH3, R2 = R3 = H)
3(R1 = F, R2 = R3 = H)
4(R1 = R2 = F)
3a(R = OCH3, R1 = F, R2 = R3 = H)
4a(R = OCH2CH3, R1 = R2 = F)
5a(R = OCH3, R1 = R2 = R3 = F)
5(R1 = R2 = R3 = F)
Scheme 2. Synthesis of compounds 1e5. a) method C1, 2,4-difluorophenylboronic acid, Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, 4 h. b) method B, BBr3, CH2Cl2, -78 ꢁC to rt,
overnight. c) 10% NaOH, ethanol, reflux, 2 h.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17b-HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004

A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
5
R1
R2
R1
R2
Br
a
b
O
S
F
O
O
S
F
S
F
F
F
F
O
O
HO
6a(R1 = N, R2 = CH)
7a(R1 = CH, R2 = N)
6(R1 = N, R2 = CH)
7(R1 = CH, R2 = N)
IV
Scheme 3. Synthesis of compounds 6 and 7. a) method C2, 3- pyridinylboronic acid pinacol ester or 4-pyridinylboronic acid for 6a and 7a, respectively, Na2CO3, Pd(PPh3)4, toluene/
ethanol (1:1), reflux, overnight. b) method B, BBr3, CH2Cl2, -78 ꢁC to rt, overnight.
enhance selectivity (compounds 14, 15, 17e19). Compound 16 lacks
this oxygen function and was synthesized for comparison reasons.
An OH-group in 4-position of ring C proved to be beneficial (see e.g.
The determined estrogen receptor affinities are given in Table 5.
The data suggest that fluorination of the benzoyl moiety increases
ER-affinity (cf. compound 20 vs. 26). Increasing the number or size
of substituents adjacent to the oxygen-function of the phenyl ring
(ring C) decreases ER-affinity (cf. compound 18 vs. 11, 13, 20, 21, 24,
and 25).
compound 15: IC50
¼
3
nM, SF
¼
24 and compound 18:
IC50 ¼ 0.5 nM, SF ¼ 40) whereas the removal of the OH-group
(compound 16) or shifting its position from para to ortho (com-
pound 19), led to a decrease or a complete loss of selectivity,
respectively (Table 2). From this optimization step, compound 18
emerged as the most active and selective inhibitor.
Compounds 20 and 25, which were highly active towards the
target enzyme and selective over 17
affinities towards ER and , in particular in view of their sub-
nanomolar activity towards 17 -HSD1.
b-HSD2, displayed very low
a
b
It was striking that compounds bearing an additional substitu-
ent on ring C in ortho-position to the oxygen-functionality (hydroxy
or methoxy) stand out in terms activity and selectivity (compounds
13, 17 and 18, Table 2). This prompted us to investigate compounds
with two ortho-substituents (compounds 20e25, Table 3). In fact,
this structural modification resulted in highly active compounds,
b
We next profiled the most interesting compound 20 in several
relevant toxicity assays. First, compound 20 did not affect cell
viability up to a concentration of 5.6 mM, as measured in the MTT
cytotoxicity assay in HEK293 cells after an incubation period of
66 h, yielding a safety margin of 11,160-fold (i.e., IC20 in the MTT
generally displaying sub-nanomolar IC50-values for h17
b
-HSD1
assay divided by the IC50 value in the human 17b-HSD1 assay).
inhibition. Interestingly, compounds bearing chloro- and methyl-
substituents showed similarly strong inhibition of the target
enzyme. In terms of selectivity towards the type 2 enzyme it is
noteworthy that generally the phenols were superior compared to
their methoxy-analogs. An exception was compound 20 which was
not only one of the most active but also the most selective com-
Secondly, for determination of the mutagenic potential of com-
pound 20, an AMES II test was performed using TA98 (frameshift
mutation) or TAMix (base-pair substitution, TA7001-TA7006)
strains of Salmonella typhimurium in the presence or absence of rat
liver S9 fraction. Compound 20 did not show mutagenic potential
up to the highest tested concentration of 100 mM in the absence or
pound of its class (IC50(h17
b
-HSD1) ¼ 0.5 nM; SF ¼ 82). To get an
presence of rat liver S9 fraction. We next investigated whether
compound 20 has the potential of CYP3A4 induction. For this,
compound 20 was tested for activation of the pregnane X receptor
(PXR), which is responsible for the induction of CYP3A4, and other
members of the CYP3 subfamily of hepatic CYP450 enzymes,
respectively. Compound 20 tested at a concentration of 3.16 mM did
not stimulate the PXR receptor (effect < 5% activation, data not
shown). Compound 20 was also profiled for inhibition of several
evidence for the importance of fluorine atoms on the benzoyl part
on the inhibitory activity issue of this compound class, compounds
26 and 27 were synthesized. Comparison with the inhibition data of
compound 20 and its analog non-fluorinated compound 26 high-
lights again the beneficial impact of the fluorine atoms at the
benzoyl moiety. The same dependence of activity on benzoyl
fluorination can be seen when comparing compounds 21 and 27
(Table 3).
hepatic CYP450 enzymes at a compound concentration of 1
mM,
The dichlorinated inhibitor 20 showed favourable properties
concerning activity and selectivity, but exhibited high lipophilicity
(clogD (pH ¼ 7.4): 4.9, as determined using the ACD-Labs Percepta
software). In order to render the compound more hydrophilic, one
of the chlorine atoms was replaced by heterocyclic substituents
(compounds 28e32, Table 4). The synthesized compounds showed
greatly reduced lipophilicities (clogD range (pH ¼ 7.4): 1.7 (30) e
3.3 (31)) and were highly active towards the target enzyme: apart
which is 2000-fold higher than the IC50 value for 17 -HSD1 inhi-
b
bition. Compound 20 only moderately inhibited CYP1A2, 2B6, 2C9,
2C19, 2D6, and 3A4 by 11%, 4%, 0%, 27%, 0% and 36%, respectively.
We next investigated whether our best current compound 20 can
be administered to rodents to allow proof-of-principle testing of a
potent 17
this, we subcutaneously administered compound 20 at a dose of
150 mol/kg body weight once-a-day for four consecutive days,
b-HSD1 inhibitor in a rodent model of endometriosis. For
m
from the dihydroxy compound 32, all compounds displayed IC50
-
during which we determined plasma levels of 20, and measured
also the plasma level of compound 20 four days after the last
dosing. As shown in Fig. 3, compound 20 subcutaneously admin-
istered as suspension in 0.5% gelatin/5% mannitol in water led to a
gradual increase in the plasma concentration from 24 nM measured
at day 2, 42 nM at day 4, and to 72 nM at day 8, 4 days after the last
dosing at day 4. These in vivo plasma concentrations are 10-fold
values in the one-digit nanomolar range (Table 4). Selectivity over
the type 2 enzyme, however, was in most cases less favourable than
for compounds 20e25.
The affinities of selected compounds to the recombinant human
estrogen receptors
a and b were determined by incubation of
tritiated E2 with the respective receptor and compound in 1000-
fold excess, based on E2 concentration. Receptor affinities are
expressed as the percentage of E2 replaced by a compound. ER-
binding of 50% is equal to a relative binding affinity (RBA) of 0.1%
of that of E2 [35].
higher than the concentrations required in vitro in the 17
assay to block 17 -HSD1by 80e100% (data not shown).
b-HSD1
b
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

6
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
R4
R4
R2
Br
R3
R3
O
S
O
O
S
F
S
F
b
a
R2
R1
HO
R1
R2
R1
F
F
HO
HO
13, 15, 18, 19, 21, 25
VI(R1 = R2 = H)
VII(R1 = R2 = F)
8-12, 14, 16, 17, 20, 24
a
Cl
Cl
Cl
b
OH
O
O
O
S
S
Cl
HO
HO
27
26
Compd R1
R2
H
R3
R4
H
H
H
Comd R1
R2
R3
R4
H
H
H
8
H
H
H
H
16
17
18
H
H
H
Cl
Cl
Cl
H
9
SO2NH2
H
H
OCH3
OH
10
SO2NH2
O
O
N
11
H
OCH3
H
19
OH Cl
H
H
S
O
12
H
CN
H
20
H
Cl
OCH3
Cl
O
OH
13
14
H
H
CN
H
H
H
21
24
H
H
Cl
OH
Cl
OCH3
CH3
OCH3
CH3
15
H
H
OH
H
25
H
CH3
OH
CH3
Scheme 4. Synthesis of compounds 8e21 and 24e27. a) method C1, corresponding boronic acid, Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, 4 h. b) method B, BBr3, CH2Cl2, -78 ꢁC to
rt, overnight.
5. Conclusions
To our knowledge, the 17b-HSD1 inhibitors identified in this
study are the most active ones ever described for this enzyme. The
most interesting compounds were 20, 21, and 25 as their high
potency towards the target protein was accompanied by good se-
lectivities over the human type 2 enzyme and the estrogen re-
The aim of the present study was the design of potent and se-
lective, low molecular weight inhibitors of human 17b-HSD1, which
can be used to conduct a proof of principle study in an animal
disease model for EDD. Starting point were sulfonamide-
substituted bicyclic substituted hydroxyphenylmethanones previ-
ously described by us. Structural modifications focused on the
substitution patterns of the benzoyl- and the phenyl-moieties.
ceptors
a and b.
We profiled compound 20 in several ADME and toxicity assays.
Compound 20 did not activate PXR at the tested concentration of
3.16 mM and did not show mutagenicity in the AMES II assay up to
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
7
Scheme 5. Synthesis of compounds 22e23, and 28e30. a) NBS, AcOH, rt, overnight. b) CH3I, K2CO3, DMF, rt, overnight. c) bispinacolato diborane, Pd(dppf)Cl2, KOAc/DMSO, 2 h. d)
method C1, VII, Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, overnight. e) method B, BBr3, CH2Cl2, -78 ꢁC to rt, overnight. f) NBS, DBPO, CCl4, reflux, 2 h. g) NEt3, 1-piperazin-1-yl-
ethanone. h) HCl 3 M, reflux, 3 h.
the highest tested concentration of 100
not show cellular toxicity up to a concentration of 5.6
measured after 66 h of incubation in the MTT cell viability assay.
Given the very potency of compound 20 towards 17 -HSD1 (IC50 of
m
M. Compound 20 also did
6. Experimental section
mM, as
6.1. Chemical methods
b
0.5 nM), it is therefore to be expected that no cytotoxic effects will
be observed in therapeutic doses. In addition, compound 20
showed only moderate CYP450 enzyme inhibition at the relatively
Chemical names follow IUPAC nomenclature. Starting materials
were purchased from Aldrich, Acros, Lancaster, Maybridge, Combi
Blocks, Merck, or Fluka and were used without purification.
Column chromatography (CC) was performed on silica gel
high concentration of 1 mM. Immunocompromised rodents such as
athymic nude and severe-compromised immunodeficient (SCID)
mice and rats are frequently utilized to determine in vivo efficacy of
preclinical drug candidates towards implanted human cells,
including breast cancer cells and human endometriotic tissue
samples. The very promising pharmacokinetic profile of compound
20 in rat after repetitive subcutaneous administration will allow for
the first time proof-of-principle studies in rodent models of breast
(70e200 mm), reaction progress was monitored by thin layer
chromatography (TLC) on Alugram SIL G UV254 (Macherey-Nagel).
All microwave irradiation experiments were carried out in a
CEM-Discover monomode microwave apparatus.
1H NMR and 13C NMR spectra were measured on a Bruker
AM500 spectrometer (500 MHz) at 300 K. Chemical shifts are re-
ported in
d (parts per million: ppm), by reference to the hydroge-
cancer and endometriosis, in which inhibition of 17
considered to be therapeutically highly beneficial.
b
-HSD1 is
nated residues of deuteriated solvent as internal standard (CDCl3:
d
¼ 7.24 ppm (1H NMR) and
d
¼ 77 ppm (13C NMR), CD3OD:
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

8
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
Scheme 6. Synthesis of compounds 31 and 32. a) DMF, rt, overnight. b) method C1, VII, Cs2CO3, Pd(PPh3)4, DME/water (1:1), reflux, overnight. c) CH3COOCH]CH2, microwave,
120 ꢁC, 10 h.d) 2 M NaOH, THF, rt, 2 h. e) method B, BBr3, CH2Cl2, -78 ꢁC to rt, overnight.
Table 1
Optimization of A-ring-substitution: Activities of compounds 1e5 towards h17b-HSD1 and 2.
Cmpd
Ring A
IC50 [nM]a
SFd
Cmpd
Ring A
IC50 [nM]a
h17 -HSD Type
2c
SFd
h17
1b
b
-HSD Type
2c
b
1b
1
3
5
110
140
1.3
1.1
0.2
2
4
3400
300
0.1
2.5
47
55
3.5
9.0
7.2
1.4
a
Mean value of three determinations, standard deviation less than 15%.
Human placenta, cytosolic fraction, substrate [3H]-E1, 500 nM, cofactor NADH, 500
b
c
m
M.
Human placenta, microsomal fraction, substrate [3H]-E2, 500 nM, cofactor NADþ, 1500
m
M.
d
SF (selectivity factor): IC50(17b-HSD2)/IC50(17b-HSD1).
d
d
d
¼ 3.35 ppm (1H NMR) and
¼ 2.05 ppm (1H NMR) and
¼ 2.50 ppm (1H NMR) and
d
d
d
¼ 49.3 ppm (13C NMR), CD3COCD3:
¼ 29.9 ppm (13C NMR), CD3SOCD3
¼ 39.5 ppm (13C NMR)). Signals are
The following compounds were prepared according to previ-
ously described procedures: (5-bromo-thiophen-2-yl)(3-
methoxyphenyl)methanone (I), [26], (5-bromo-thiophen-2-yl)(3-
hydroxy-phenyl)methanone (IV), [29], (5-bromo-thiophen-2-
yl)(3-hydroxy-phenyl)methanone (VI), [26], (5-Bromo-thiophen-
2-yl)-(2,6-difluoro-3-hydroxy-phenyl)-methanone (VII), [29], 4-
bromo-2-chloro-6-methyl-phenol (22c), [36], 5-bromo-1-chloro-
2-methoxy-3-methyl-benzene (22b) [37].
The Supplementary Data section reports the synthesis of
compounds II, V, 1a-5a, 7a, 3e7, 9e29, 31b and 32. For each
general synthetic procedure, one representative example is given
below.
described as s, d, t, dd, ddd, m, dt, q, sep, br. for singlet, doublet,
triplet, doublet of doublets, doublet of doublets of doublets,
multiplet, doublet of triplets, quadruplet, septet, and broad,
respectively. All coupling constants (J) are given in hertz (Hz).
Mass spectra (ESI) were recorded on a TSQ Quantum (Thermo
Finnigan) instrument.
Tested compounds are >95% chemical purity as measured by
HPLC. The methods for HPLC analysis and a table of data for all
tested compounds are provided in the supporting information.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
9
Table 2
Optimization of C-ring-substitution: Activities of compounds 6e19 towards h17
b-HSD1 and 2.
Cmpd
Ring C
IC50 [nM]a
h17 -HSD Type
1b 2c
SFd
Cmpd
Ring C
IC50 [nM]a
SFd
b
h17
1b
b-HSD Type
2c
6
8
27
47
1.7
3.2
7
9
150
39
0.7
1.6
11
35
55
86
10
12
150
8.0
85
19
0.6
2.4
11
13
130
3.3
180
1.4
8.8
29
14
16
18
38
10
2.1
8.3
15
17
3
71
11
24
1.2
1.3
8.5
18
0.5
20
40
19
27
29
1
a-d
See Table 1.
6.1.1. General procedure for Friedel-Crafts acylation. Method A.
(IeV)
(1.2 equiv), cesium carbonate (4 equiv) and tetrakis(-
triphenylphosphine) palladium (0.05 equiv) was suspended in an
oxygen-free DME/water (1:1) solution and refluxed under nitrogen
atmosphere. The reaction mixture was cooled to room tempera-
ture. 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.
An ice-cooled mixture of monosubstituted thiophene derivative
(1.5 equiv), arylcarbonyl chloride (1 equiv), and aluminumtri-
chloride (1 equiv) in anhydrous dichloromethane was warmed to
room temperature and stirred for 2e4 h 1 M HCl was used to
quench the reaction. The aqueous layer was extracted with ethyl
acetate. The combined organic layers were washed with brine,
dried over magnesium sulfate, filtered, and concentrated to dry-
ness. The product was purified by CC.
6.1.3.2. Method C2. (6a and 7a). A mixture of arylbromide (1
equiv), boronic acid derivative (1.2 equiv), Na2CO3 (2 equiv) and
tetrakis(triphenylphosphine) palladium (0.05 equiv) was sus-
pended in an oxygen-free toluene/ethanol (1:1) solution was
refluxed overnight under nitrogen atmosphere. The aqueous layer
was extracted with ethyl acetate. The combined organic layers were
washed with brine, dried over magnesium sulfate, filtered and
concentrated to dryness. The product was purified by CC.
6.1.2. General procedure for ether cleavage. Method B. (VI, VII,
1,3e7, 13, 15, 18, 19, 21, 23, 25, 27, 29, and 32)
To a solution of methoxybenzene derivative (1 equiv) in anhy-
drous dichloromethane at ꢂ78 ꢁC (dry ice/acetone bath), boron
tribromide in dichloromethane (1 M, 5 equiv per methoxy func-
tion) was added dropwise. The reaction mixture was stirred over-
night at room temperature under nitrogen atmosphere. Water was
added to quench the reaction, and the aqueous layer was extracted
with ethyl acetate. The combined organic layers were washed with
brine, dried over magnesium sulfate, filtered, and concentrated to
dryness. The product was purified by CC.
6.1.4. (5-Bromo-thiophen-2-yl)-(2-fluoro-3-methoxy-phenyl)-
methanone (III)
The title compound was prepared by reaction of 2-
bromothiophene
(1297
mg,
7.95
mmol),
2-fluoro-4-
methoxybenzoyl chloride (1000 mg, 5.30 mmol) and aluminum
chloride (707 mg, 5.30 mmol) according to method A. The product
was purified by CC (hexane/ethyl acetate 96:4); yield: 57%
6.1.3. General procedure for Suzuki coupling
6.1.3.1. Method C1. (1a-5a, 8e12, 14, 16, 17, 20, 22, 24, 26, 28, and
31b). A mixture of arylbromide (1 equiv), boronic acid derivative
(1000 mg). 1H NMR (500 MHz, acetone-d6)
d
7.32 (d, J ¼ 7.4 Hz, 1H),
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

10
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
Table 3
Optimization of C-ring-substitution: Activities of compounds 20e27 towards h17b-HSD1 and 2.
Cmpd
20
Ring C
IC50 [nM]a
SFd
82
21
Cmpd
Ring C
IC50 [nM]a
SFd
16
30
h17
1b
b-HSD Type
h17
1b
b-HSD Type
2c
2c
0.5
0.3
41
21
23
2.4
0.2
39
22
7.2
6.5
24
26
0.9
6.0
18
20
42
25
27
0.4
73
7.0
18
250
540
7.4
a-d
See Table 1.
Table 4
Introduction of hydrophilic moieties: Activities of compounds 28e32 towards h17
b-HSD1 and 2.
Cmpd
Ring D
IC50 [nM]a
h17 -HSD Type
1b 2c
SFd
Cmpd
Ring D
IC50 [nM]a
h17 -HSD Type
1b 2c
SFd
b
b
28
30
32
1.4
4.7
18
21
26
26
15
6
29
31
2.1
11
5
4
2.1
9
1
a-d
See Table 1.
7.33 (ddd, J ¼ 7.3, 4.9, 1.4 Hz, 1H), 7.12 (d, J ¼ 7.4 Hz, 1H), 7.09 (t,
J ¼ 7.4 Hz, 1H), 6.92e6.87 (m, 1H), 3.82 (s, 3H); 13C NMR (125 MHz,
6.1.5. (5-(2,4-Difluoro-phenyl)-thiophen-2-yl)-(3-hydroxy-
phenyl)-methanone (1)
The title compound was prepared by reaction of (5-(2,4-
difluoro-phenyl)-thiophen-2-yl)-(3-methoxy-phenyl)-methanone
(1a) (417 mg, 1.26 mmol) and boron tribromide (3.8 mmol)
according to method B. The product was purified by CC
acetone-d6)
d
184.05, 151.12 (d, J ¼ 246.0 Hz), 149.54, 143.29, 136.87,
128.24 (dd, J ¼ 9.9, 4.4 Hz), 126.67 (d, J ¼ 13.0 Hz), 122.18 (d,
J ¼ 4.3 Hz), 117.46 (d, J ¼ 3.0 Hz), 116.52 (d, J ¼ 1.3 Hz), 115.94 (dd,
J ¼ 12.8, 4.1 Hz), 54.21; MS (ESI): 316.41 (MþH)þ.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
11
Table 5
Binding affinities of selected compounds for the estrogen receptors
6.1.7. (3-Ethoxy-2,6-difluoro-phenyl)-(5-pyridin-4-yl-thiophen-2-
yl)-methanone (6a)
The title compound was prepared by reaction of (5-bromo-thi-
ophen-2-yl)-(3-ethoxy-2,6-difluoro-phenyl)-methanone (III)
a
and b.
Cmpd.
% Bindinga
Cmpd.
% Bindinga
ERab
ERbc
ERab
ERbc
(300 mg, 0.86 mmol), pyridine-3-boronic acid pinacol ester
(355 mg, 1.73 mmol), sodium carbonate (2.5 mL, 2 M) and tetra-
kis(triphenylphosphine) palladium (5 mmol) according to method
C2. The product was purified by CC (dichloromethane/methanol
99.5:0.5); yield: 91% (450 mg). The product was used in the next
step without any characterization.
11
13
18
20
0
4
6
55
8
21
24
25
26
37
51
17
8
11
69
22
0
44
73
27
a
Mean value of three determinations, standard deviation less than 15%.
b
c
c(ER
c(ER
a
b
) ¼ 1 nM, c(E2) ¼ 3 nM, c(test compound) ¼ 3
m
M.
m
) ¼ 4 nM, c(E2) ¼ 10 nM, c(test compound) ¼ 10
M.
6.1.8. (5-(3-Chloro-phenyl)-thiophen-2-yl)-(2,6-difluoro-3-
hydroxy-phenyl)-methanone (8)
The title compound was prepared by reaction of (5-bromo-thi-
100
75
ophen-2-yl)-(2,6-difluoro-3-hydroxy-phenyl)-methanone
(VII)
(430 mg, 1.35 mmol), 3-chlorophenylboronic acid (253 mg,
1.62 mmol), cesium carbonate (1756 mg, 5.39 mmol) and tetra-
kis(triphenylphosphine) palladium (5
C1. The product was purified by CC (hexane/ethyl acetate 90:10);
yield: 74% (350 mg). 1H NMR (500 MHz, acetone-d6)
9.02 (s, 1H,
mmol) according to method
50
d
OH), 7.84 (tt, J ¼ 2.4, 1.2 Hz, 1H), 7.78e7.76 (m, 1H), 7.69 (d,
J ¼ 4.1 Hz, 1H), 7.66 (dt, J ¼ 4.1, 0.9 Hz, 1H), 7.52 (td, J ¼ 7.9, 0.5 Hz,
1H), 7.47 (ddd, J ¼ 8.0, 2.0, 1.1 Hz, 1H), 7.23e7.21 (m, 1H), 7.06e7.04
25
s.c.dosing
(m, 1H); 13C NMR (125 MHz, acetone-d6)
d 180.77, 153.31, 152.56
(dd, J ¼ 240.6, 5.8 Hz), 148.44 (dd, J ¼ 245.9, 7.7 Hz), 143.92, 142.68
(dd, J ¼ 12.9, 3.2 Hz), 138.12, 135.80, 135.69, 131.92, 130.20, 126.96,
126.90, 125.81, 120.42 (dd, J ¼ 9.1, 3.9 Hz), 117.97 (dd, J ¼ 23.9,
19.7 Hz), 112.47 (d, J ¼ 22.8 Hz); MS (ESI): 351.63 (MþH)þ.
0
0
24 48 72 96 120 144 168 192
Time (h)
Fig. 3. Pharmacokinetic profile of compound 20 in Sprague-Dawley rats following
repetitive subcutaneous administration. Compound 20 was subcutaneously adminis-
6.1.9. 2-(3-Chloro-4-methoxy-5-methyl-phenyl)-4,4,5,5-
tered once-a-day at a dose of 150 mmol/kg body weight in 0.5% gelatin/5% mannitol in
water to three female Sprague-Dawley rats. Plasma samples were taken at 24 h, 48 h,
tetramethyl-(1,3,2)dioxaborolane (22a)
5-Bromo-1-chloro-2-methoxy-3-methylbenzene (22b) (5,00 g,
20,2 mmol, 1,00 equiv), bis(pinacolato)diboron (8,09 g, 31,8 mmol,
1,50 equiv), potassium acetate (5,95 g, 60,6 mmol, 3,00 equiv) and
1,10-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride
(739 mg, 1,01 mmol, 0,05 equiv) were dissolved under N2 in 40 ml
dry DMSO and the mixture was stirred at 80 ꢁC for 2 h. The reaction
was quenched with water, diluted with diethyl ether and filtered
over celite. The phases were separated and the aqueous layer was
extracted two times with diethyl ether. The combined organic
layers were washed three times with water; one time with brine,
dried over MgSO4, filtered and concentrated under reduces pres-
sure. The crude product was purified by CC (hexane/ethyl acetate
85:15); yield: 88% (4.71 g). 1H NMR (500 MHz, acetone-d6)
72 h and 168 h. Arrows indicate the administration of compound 20 during the first
four days, immediately after plasma sampling. Data shown are the mean
three animals.
SEM of
(dichloromethane/methanol 99.75:0.25); yield: 41% (330 mg). 1H
NMR (500 MHz, acetone-d6)
d
8.77 (s, 1H), 7.94 (td, J ¼ 8.8, 6.3 Hz,
1H), 7.75 (dd, J ¼ 4.0, 1.1 Hz, 1H), 7.63 (dd, J ¼ 4.0, 1.0 Hz, 1H),
7.43e7.40 (m, 1H), 7.39e7.37 (m, 1H), 7.35 (ddd, J ¼ 2.5, 1.5, 0.5 Hz,
1H), 7.27e7.21 (m, 1H), 7.20e7.15 (m, 1H), 7.14 (ddd, J ¼ 7.7, 2.6,
1.5 Hz, 1H); 13C NMR (125 MHz, acetone-d6)
d 187.84, 163.90 (dd,
J ¼ 250.2,12.1 Hz),160.35 (dd, J ¼ 254.0,13.3 Hz),158.45,144.41 (dd,
J ¼ 91.1, 3.7 Hz), 140.16, 136.06, 131.44 (dd, J ¼ 9.9, 4.4 Hz), 130.63,
128.25e127.77 (m), 121.15, 120.39, 118.70 (dd, J ¼ 12.9, 4.1 Hz),
116.32, 113.36 (dd, J ¼ 21.8, 3.6 Hz), 105.70 (t, J ¼ 26.4 Hz); MS (ESI):
317.63 (MþH)þ.
d
7.54e7.56 (m, 1H), 7.48e7.50 (m, 1H), 3.82 (s, 3H), 2.31 (t,
J ¼ 0.6 Hz, 3H), 1.33 (s, 12H).
6.1.10. 2-(3-Bromomethyl-5-chloro-4-methoxy-phenyl)-4,4,5,5-
tetramethyl-(1,3,2)dioxaborolane (28b)
2-(3-Chloro-4-methoxy-5-methylphenyl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (22a) (200 mg, 0,71 mmol, 1,00 equiv) and N-
bromosuccinimide (113 mg, 0,63 mmol, 0,90 equiv) were dissolved
under N2 in 17 ml CCl4, followed by a catalytic amount of dibenzoyl
peroxide. The mixture was stirred under reflux for 1 h. The reaction
was quenched with water and extracted three times with
dichloromethane. The combined organic layers were washed two
times with water, one time with brine, dried over Na2SO4, filtered
and concentrated under reduced pressure. The crude product was
purified by CC (hexane/ethyl acetate 85:15); yield: 70% (180 mg). 1H
6.1.6. (5-(2,4-Difluoro-phenyl)-thiophen-2-yl)-(3-hydroxy-2-
methyl-phenyl)-methanone (2)
Compound 2a (430 mg, 1.16 mmol) in ethanol (5 ml) was
refluxed in 10% NaOH (15 mL) for 2 h on a water bath. The reaction
mixture was cooled, diluted with water and neutralized with acetic
acid. The product was purified by CC (dichloromethane/methanol
99.5:0.5); yield: 58% (220 mg). 1H NMR (500 MHz, acetone-d6)
d
9.51 (s, 1H, OH), 8.83 (dd, J ¼ 15.1, 8.5 Hz, 1H), 8.49 (d, J ¼ 3.4 Hz,
1H), 8.37 (d, J ¼ 3.4 Hz, 1H), 8.24e8.02 (m, 3H), 7.95 (d, J ¼ 7.9 Hz,
1H), 7.89 (d, J ¼ 7.3 Hz, 1H), 3.11 (s, 3H); 13C NMR (125 MHz,
NMR (500 MHz, acetone-d6)
J ¼ 1.5 Hz, 1H), 4.71 (s, 2H), 4.00 (s, 3H), 1.33e1.35 (m, 12H).
d
7.75 (d, J ¼ 1.5 Hz, 1H), 7.68 (d,
acetone-d6)
d
189.56, 163.02 (dd, J ¼ 251.3, 12.8 Hz), 159.45 (dd,
J ¼ 253.4, 12.4 Hz), 155.85, 144.44 (dd, J ¼ 10.1, 4.1 Hz), 140.21,
135.70, 130.55 (dd, J ¼ 9.9, 4.4 Hz), 127.34, 127.28, 126.11, 122.33,
118.97, 117.80 (dd, J ¼ 12.8, 4.0 Hz), 116.62, 112.45 (dd, J ¼ 21.8,
3.6 Hz), 104.79 (t, J ¼ 26.4 Hz), 11.99; MS (ESI): 331.04 (MþH)þ.
6.1.11. 1-(4-(3-Chloro-2-methoxy-5-(4,4,5,5-tetramethyl-(1,3,2)
dioxaborolan-2-yl)-benzyl)-piperazin-1-yl)-ethanone (28a)
2-(3-(Bromomethyl)-5-chloro-4-methoxyphenyl)-4,4,5,5-
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

12
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
tetramethyl-1,3,2-dioxaborolane (28b) (180 mg, 0,50 mmo, 1,00
equiv) was dissolved under N2 in ml dry THF and 1-
6.1.15. (5-(3-Chloro-4-methoxy-5-(1,2,3)triazol-1-ylmethyl-
phenyl)-thiophen-2-yl)-(2,6-difluoro-3-hydroxy-phenyl)-
methanone (31)
1
acetylpiperazine was added. The mixture was stirred for 1 h un-
der reflux. The reaction was quenched with water and extracted
three times with ethyl acetate. The combined organic layers were
washed two times with water, one time with brine, dried over
MgSO4, filtered and concentrated under reduced pressure. The
crude product was purified by CC (ethyl acetate/ethanol 8:2), to
give the desired product as yellow solid; yield: 70% (198 mg). 1H
Acetic
acid
3-(5-(3-chloro-4-methoxy-5-(1,2,3)triazol-1-
ylmethyl-phenyl)-thiophene-2-carbonyl)-2,4-difluoro-phenylester
(31a) (110 mg, 0.24 mmol, 1.00 equiv) was dissolved under N2 in a
degased mixture of 5 ml THF and 600 ml of 2 M NaOH. The mixture
was stirred for 2 h at room temperature. The reaction was acidified
to pH 6 with 1 M HCl and extracted three times with ethyl acetate.
The combined organic layers were washed two times with water,
one time with brine, dried over MgSO4, filtered and concentrated
under reduced pressure to give the desired product; yield: 95%
NMR (500 MHz, acetone-d6)
d
7.69 (d, J ¼ 1.5 Hz, 1H), 7.65 (d,
J ¼ 1.5 Hz, 1H), 3.90 (s, 3H), 3.57 (s, 3H), 2.77 (t, J ¼ 5.2 Hz, 2H), 2.70
(t, J ¼ 5.2 Hz, 2H), 2.47 (t, J ¼ 5.2 Hz, 2H), 2.42 (t, J ¼ 5.2 Hz, 2H), 2.00
(s, 3H), 1.34 (s, 12H).
(90 mg). 1H NMR (500 MHz, acetone-d6)
d 9.05 (br. s, 1H), 8.11 (d,
J ¼ 0.9 Hz,1H), 7.91 (d, J ¼ 2.2 Hz,1H), 7.70 (d, J ¼ 0.9 Hz,1H), 7.67 (d,
J ¼ 2.2 Hz,1H), 7.65e7.62 (m,1H), 7.61e7.58 (m,1H), 7.21 (td, J ¼ 8.9,
5.5 Hz, 1H), 7.03 (td, J ¼ 8.8, 1.9 Hz, 1H), 5.78 (s, 2H), 3.89 (s, 3H); MS
(ESI): 462.15 (MþH)þ.
6.1.12. (5-(3-Chloro-4-methoxy-5-piperazin-1-ylmethyl-phenyl)-
thiophen-2-yl)-(2,6-difluoro-3-hydroxy-phenyl)-methanone (30)
1-(4-(3-Chloro-5-(5-(2,6-difluoro-3-hydroxybenzoyl)-thio-
phen-2-yl)-2-methoxybenzyl)piperazin-1-yl)ethanone
(28)
6.2. Biological methods
(150 mg, 0.29 mmol, 1.00 equiv) was dissolved in 20 ml 3 M
aqueous HCl and heated to 80 ꢁC for 3 h. The reaction was washed
two times with ethyl acetate, the aqueous layer was basified to pH
10 with 2 M NaOH and washed two times with ethyl acetate. The
aqueous layer was neutralized with 2 M HCl and extracted three
times with ethyl acetate. The combined organic layers were washed
one time with water, one time with brine, dried over MgSO4,
filtered and concentrated under reduced pressure to give the
desired pure product in 40% yield as pale yellow solid. 1H NMR
[2,4,6,7-3H]-E1 and [2,4,6,7-3H]-E2 were bought from Perkin-
Elmer, Boston. Quickszint Flow 302 scintillator fluid was bought
from Zinsser Analytic, Frankfurt.
6.2.1. Preparation of human 17
b
-HSD1 and 17b-HSD2
Human 17 -HSD1 and 17
b
b-HSD2 were obtained from human
placenta according to previously described procedures [32]. Fresh
human placenta was homogenized, and cytosolic fraction and mi-
crosomes were separated by fractional centrifugation. For the
(500 MHz, acetone-d6)
d 7.83e7.79 (m, 2H), 7.67e7.60 (m, 2H), 7.19
(m, 1H), 7.00 (td, J ¼ 9.0, 1.9 Hz, 1H), 3.92 (s, 3H), 3.59 (s, 2H),
partial purification of 17b-HSD1, the cytosolic fraction was precip-
2.89e2.81 (m, 4H), 2.53e2.45 (m, 4H); 13C NMR (125 MHz, acetone-
itated with ammonium sulfate. 17
microsomal fraction.
b-HSD2 was obtained from the
d6) d 180.8, 156.8, 153.3, 151.5, 149.4, 147.5, 143.6, 142.9, 142.7, 138.2,
135.4, 130.7, 129.6, 128.4, 127.9, 126.7, 120.5, 118.1, 112.3, 112.2, 62.0,
56.9, 50.4, 44.3; MS (ESI): 479.22 (MþH)þ.
6.2.2. Inhibition of human 17b-HSD1
Inhibitory activities were evaluated by an established method
with minor modifications [32]. Briefly, the enzyme preparation was
6.1.13. 2-(3-Azidomethyl-5-chloro-4-methoxy-phenyl)-4,4,5,5-
tetramethyl-(1,3,2)dioxaborolane (31c)
incubated with NADPH (500 mM) in the presence of potential in-
hibitors 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. The 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 concentra-
2-(3-(Bromomethyl)-5-chloro-4-methoxyphenyl)-4,4,5,5-tetra-
methyl-1,3,2-dioxaborolane (28b) (800 mg, 2.19 mmol, 1.00 equiv)
was dissolved under N2 in 8 ml dry DMF and sodium azide (143 mg,
2.19 mmol, 1.00 equiv) was added. The mixture was stirred at room
temperature overnight. The mixture was quenched with water and
extracted three times with ethyl acetate. The combined organic
layers were washed two times with water, one time with brine,
dried over MgSO4, filtered and concentrated under reduced pres-
sure. The crude product was purified by CC (hexane/ethyl acetate
tion: 500 nM, 0.15 mCi). After 10 min, the incubation was stopped
with HgCl2 and the mixture was extracted with diethylether. After
evaporation, the steroids were dissolved in acetonitrile. E1 and E2
were separated using acetonitrile/water (45:55) as mobile phase in
a C18 reverse phase chromatography column (Nucleodur C18 125/
3100-5, Macherey-Nagel) connected to a HPLC-system (Agilent
1200 Series, Agilent Technologies). Detection and quantification of
the steroids were performed using a radioflow detector (Ramona,
raytest). The conversion rate was calculated after analysis of the
resulting chromatograms according to the following equation:
8:2); yield: 71% (503 mg). 1H NMR (500 MHz, acetone-d6)
d 7.72 (d,
J ¼ 1.6 Hz, 1H), 7.69 (d, J ¼ 1.6 Hz, 1H), 4.55 (s, 2H), 3.93 (s, 3H), 1.34
(s, 12H).
6.1.14. Acetic acid 3-(5-(3-chloro-4-methoxy-5-(1,2,3)triazol-1-
ylmethyl-phenyl)-thiophene-2-carbonyl)-2,4-difluoro-phenylester
(31a)
%conversion ¼ [%E2/(%E2 þ %E1)] ꢃ 100
(5-(3-(Azidomethyl)-5-chloro-4-methoxyphenyl)-thiophen-2-
yl)(2,6-difluoro-3-hydroxyphenyl)methanone (31b) (50 mg,
Each value was calculated from at least three independent
experiments.
0.11 mmol, 1.00 equiv), was dissolved in 106 ml vinyl acetate and the
reaction was heated at 120 ꢁC under microwave for 10 h. The re-
action was concentrated under reduced pressure and purified by CC
using ethyl acetate as eluent; yield: 27% (15 mg). 1H NMR (500 MHz,
6.2.3. Inhibition of human 17
The h17 -HSD2 inhibition assay was performed similarly to the
h17 -HSD1 procedure. The microsomal fraction was incubated with
NADþ [1500
M], test compound and a mixture of unlabeled- and
b-HSD2
b
b
DMSO-d6)
d
8.11 (d, J ¼ 0.9 Hz, 1H), 7.92 (d, J ¼ 2.5 Hz, 1H), 7.70 (d,
m
J ¼ 0.9 Hz, 1H), 7.68e7.66 (m, 1H), 7.64e7.61 (m, 2H), 7.53 (td,
J ¼ 8.9, 5.5 Hz, 1H), 7.26 (td, J ¼ 8.8, 1.9 Hz, 1H), 5.78 (s, 2H), 3.89 (s,
3H), 2.34 (s, 3H); MS (ESI): 504.12 (MþH)þ.
[2,4,6,7-3H]-E2 (final concentration: 500 nM, 0.11
mCi) for 20 min at
37 ꢁC. Further treatment of the samples and HPLC separation was
carried out as mentioned above.
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
13
The conversion rate was calculated after analysis of the resulting
chromatograms according to the following equation:
typhimurium in the presence or absence of rat liver S9 fraction ac-
cording to the manufacturer's instructions.
%conversion ¼ [%E1/(%E1 þ %E2)]ꢃ100
6.2.7. Hepatic CYP450 inhibition and PXR assays
The inhibition of hepatic CYP enzymes CYP1A2, CYP2B6,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4 by 1 mM of compound 20
was determined in microsomes of baculovirus-infected insect cells
expressing the recombinant human enzyme according to the
manufacturer's instruction (BD Gentest/Corning). Agonist activity
of compound 20 on the pregnane X receptor (PXR) was performed
at CEREP (now Eurofins) in a cofactor recruitment cell-free assay.
6.2.4. ER affinity
The binding affinity of selected compounds to ERa and ERb was
determined according to the recommendations of the US Envi-
ronmental Protection Agency (EPA) by their Endocrine Disruptor
Screening Program (EDSP) using recombinant human proteins.
Briefly, 1 nM of ER
a
and 4 nM of ER
b
, respectively, were incubated
6.2.8. Rat pharmacokinetics
with [3H]-E2 (3 nM for ER
a
and 10 nM for ER ) and test compound
b
All animal procedures were performed in accordance with the
Guide for the Care and Use of Laboratory Animals. Experiments
were conducted on female Sprague-Dawley rats (body weight
255e274 g) purchased from Charles River Laboratory (Sulzfeld,
Germany). After an acclimatization period of 1 week, compound 20
for 16e20 h at 4 ꢁC.
The inhibitors were dissolved in DMSO (5% final concentration).
Evaluation of non-specific [3H]-E2 binding was performed with
unlabeled E2 at concentrations 100-fold of [3H]-E2 (300 nM for ER
a
and 1000 nM for ER ). After incubation, ligand-receptor complexes
b
was subcutaneously administered at a dose of 150 mmol/kg (66 mg/
were selectively bound to hydroxyapatite (83.5 g/l in TE-buffer).
The bound complex was washed three times and resuspended in
ethanol. For radiodetection, scintillator cocktail (Quickszint 212,
Zinsser Analytic, Frankfurt) was added and samples were measured
in a liquid scintillation counter (1450 LSC & Luminescence Counter,
Perkin Elmer).
kg) body weight (n ¼ 3) per animal. Suspensions of compound 20 in
0.5% porcine gelatine/5% mannitol (w/w) in demineralized water
were prepared every day following 10 min in an ultrasonic bath,
30 min before administration. Before application of suspension
(4 mL/kg body weight) at 0 h, 24 h, 48 h and 72 h, rats were
anesthetized with 2% isoflurane. At 24 h, 48 h, 72 h, 96 h and 168 h,
From these results the percentage of [3H]-E2 displacement by
the compounds and unlabeled E2 was calculated. The plot of %
displacement versus unlabeled E2 concentration resulted in
sigmoidal binding curves. The E2 concentrations to displace 50% of
the receptor bound [3H]-E2 were determined in each experiment
and E2 IC50 values were used as reference. The E2 IC50 values
blood samples of 50
renewed subcutaneous administration of compound 20) and
ml were taken from the tail vein (always before
collected in 0.2 ml Eppendorf tubes containing 5
ml of 106 mM
sodium citrate buffer. After centrifugation at 5000 rpm at 4 ꢁC, the
plasma samples were first frozen at ꢂ20 ꢁC and within 24 h, stored
at ꢂ80 ꢁC. For bioanalysis, plasma samples were thawed and 10
mL
accepted were 3 20% nM for ER
a
and 10 20% nM for ER
b.
of plasma were added to 50 mL acetonitrile containing diphenhy-
Compounds were tested at concentrations of 1000 ∙ IC50 (E2).
Compounds with less than 50% displacement of [3H]-E2 at a con-
centration of 1000 ∙ IC50 (E2) were classified as RBA <0.1%.
dramine (750 nM) as internal standard. Samples and calibration
standards (in rat plasma) were centrifuged at 15,000 rpm for
5 min at 4 ꢁC. The solutions were transferred to fresh vials for HPLC-
MS/MS analysis (Accucore RP-MS, TSQ Quantum triple quadrupole
6.2.5. MTT cell viability assay
mass spectrometer, APCI interface). After injection of 10 mL (per-
Evidence of cell viability by the MTT assay is based on the
reduction of the yellow, water-soluble dye 3-(4,5-dimethyl-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) to a blue-violet, water
insoluble formazane. HEK293 cells (2 ꢃ 105 cells per well) were
seeded in 24-well flat-bottom plates. Culturing of cells, incubations
and absorbance measurements were performed as described pre-
viously [38] with minor modifications. 3 h after seeding the cells in
Dulbecco's minimal essential medium with 5% fetal calf serum and
formed in duplicate), data were analyzed based on the ratio of the
peak areas of compound 20 and internal standard. Detection limit
of compound 20 in plasma was 10 nM.
Author contributions
A.S.A., E.B., M.S. and M.F. are responsible for design, synthesis
and chemical characterization of the compounds. C.J.v.K., M.S., C.B.,
L.S., M.F., M.W.L. and M.D.M. are responsible for the biological as-
says. A.S.A., C.J.v.K. and M.F. wrote the manuscript.
penicillin G (100 units/ml)/streptomycin (100
tion was started by the addition of compound 20 at 10, 5, 2.5, 1.25
and 0.625 M in a total volume of 1 ml with a final DMSO con-
centration of 1%. After 66 h, 50 l of a 5 mg/ml MTT in phosphate-
mg/ml), the incuba-
m
m
Notes
buffered saline, pH 7.4 (PBS) was added to the medium, and incu-
bated in a CO2 incubator at 37 ꢁC. After 30 min, medium was
The authors declare no competing financial interest.
removed and 250 ml of DMSO containing 10% SDS and 0.5% acetic
acid were added to dissolve the cells and MTT crystals. Absorbance
of formazane was measured at 570 nm in an Omega plate reader
spectrometer. The decrease in fluorescence at 570 nm after incu-
bation in the presence of compound 20 compared with the fluo-
rescence measured in the presence of the vehicle control alone (1%
DMSO) was determined, followed by the calculation of the IC20
value using GraphpadPrism 3. Arbitrarily, below IC20 cell viability is
not considered to be affected.
Acknowledgments
We are grateful to the Egyptian Ministry of Higher Education
and Scientific Research (MoHESR) and the Deutscher Akademischer
Austauschdienst (DAAD) for financial support of this work (A/09/
92319). Thanks are due to Professor Rolf W. Hartmann for valuable
discussions. We thank Dr. Claudia Scheuer and Julia Parakenings for
their technical assistance in performing the pharmacokinetic pro-
file experiments.
6.2.6. Mutagenicity testing
Mutagenic potential of compound 20 was evaluated at 100, 33,
Appendix A. Supplementary data
11, 3.7, 1.2 and 0.4
mM using Xenometrix AMES II mutagenicity
assay kit with TA98 and TA7001-TA7006 strains of Salmonella
Supplementary data related to this article can be found at http://
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b

14
A.S. Abdelsamie et al. / European Journal of Medicinal Chemistry xxx (2016) 1e14
(2008) 1931e1940.
dx.doi.org/10.1016/j.ejmech.2016.11.004.
[19] D. Poirier, Advances in development of inhibitors of 17
b hydroxysteroid de-
hydrogenases, Anticancer Agents Med. Chem. 9 (2009) 642e660.
[20] S. Starcevic, P. Brozic, S. Turk, J. Cesar, T.L. Rizner, S. Gobec, Synthesis and
biological evaluation of (6- and 7-phenyl) coumarin derivatives as selective
References
ꢀ
ꢁ
ꢀ ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
nonsteroidal inhibitors of 17
b
-hydroxysteroid dehydrogenase type 1, J. Med.
[1] T. Smuc, M.R. Pucelj, J. Sinkovec, B. Husen, H. Thole, T. Lanisnik Rizner,
Expression analysis of the genes involved in estradiol and progesterone action
in human ovarian endometriosis, Gynecol. Endocrinol. 23 (2007) 105e111.
[2] Y. Miyoshi, A. Ando, E. Shiba, T. Taguchi, Y. Tamaki, S. Noguchi, Involvement of
Chem. 54 (2011) 248e261.
[21] D. Poirier, Inhibitors of 17
b-hydroxysteroid dehydrogenases, Curr. Med.
Chem. 10 (2003) 453e477.
[22] D. Poirier, 17b-Hydroxysteroid dehydrogenase inhibitors: a patent review,
up-regulation of 17b-hydroxysteroid dehydrogenase type 1 in maintenance of
Expert Opin. Ther. Pat. 20 (2010) 1123e1145.
intratumoral high estradiol levels in postmenopausal breast cancers, Int. J.
[23] J.M. Day, H.J. Tutill, A. Purohit, M.J. Reed, Design and validation of specific
Cancer 94 (2001) 685e689.
inhibitors of 17b-hydroxysteroid dehydrogenases for therapeutic application
[3] A. Jansson, 17b-hydroxysteroid dehydrogenase enzymes and breast cancer,
in breast and prostate cancer, and in endometriosis, Endocr. Relat. Cancer 15
J. Steroid Biochem. Mol. Biol. 114 (2009) 64e67.
(2008) 665e692.
[4] C.H. Blomquist, M. Bonenfant, D.M. McGinley, Z. Posalaky, D.J. Lakatua, S. Tuli-
[24] J.M. Day, H.J. Tutill, A. Purohit, 17
Minerva Endocrinol. 35 (2010) 87e108.
ꢀ ꢀ
b-hydroxysteroid dehydrogenase inhibitors,
Puri, D.G. Bealka, Y. Tremblay, Androgenic and estrogenic 17b-hydroxysteroid
dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors:
evidence for the type 1, 2 and 5 isoforms, J. Steroid Biochem. Mol. Biol. 81
(2002) 343e351.
ꢀ
ꢀ
[25] P. Brozic, T. Lanisnik Rizner, S. Gobec, Inhibitors of 17b-hydroxysteroid de-
hydrogenase type 1, Curr. Med. Chem. 15 (2008) 137e150.
[26] A. Oster, S. Hinsberger, R. Werth, S. Marchais-Oberwinkler, M. Frotscher,
R.W. Hartmann, Bicyclic substituted hydroxyphenylmethanones as novel in-
[5] T.M. Penning, Molecular endocrinology of hydroxysteroid dehydrogenases,
Endocr. Rev. 18 (1997) 281e305.
hibitors of 17b-hydroxysteroid dehydrogenase type 1 (17b -HSD1) for the
treatment of estrogen-dependent diseases, J. Med. Chem. 53 (2010)
8176e8186.
[6] W.R. Miller, J.M. Bartlett, P. Canney, M. Verrill, Hormonal therapy for post-
menopausal breast cancer: the science of sequencing, Breast Cancer Res. Treat.
103 (2007) 149e160.
[27] A. Spadaro, M. Frotscher, R.W. Hartmann, Optimization of hydrox-
[7] N.J. Bush, Advances in hormonal therapy for breast cancer, Semin. Oncol. Nurs.
23 (2007) 46e54.
ybenzothiazoles as novel potent and selective inhibitors of 17b-HSD1, J. Med.
Chem. 55 (2012) 2469e2473.
[8] A. Cavalli, A. Bisi, C. Bertucci, C. Rosini, A. Paluszcak, S. Gobbi, E. Giorgio,
A. Rampa, F. Belluti, L. Piazzi, P. Valenti, R.W. Hartmann, M. Recanatini,
[28] A.S. Abdelsamie, E. Bey, N. Hanke, M. Empting, R.W. Hartmann, M. Frotscher,
Inhibition of 17 -HSD1: SAR of bicyclic substituted hydrox-
b
Enantioselective nonsteroidal aromatase inhibitors identified through
a
yphenylmethanones and discovery of new potent inhibitors with thioether
linker, Eur. J. Med. Chem. 82 (2014) 394e406.
multidisciplinary medicinal chemistry approach, J. Med. Chem. 48 (2005)
7282e7289.
[29] A.S. Abdelsamie, E. Bey, E.M. Gargano, C.J. van Koppen, M. Empting,
M. Frotscher, Towards the evaluation in an animal disease model: fluorinated
[9] M. Le Borgne, P. Marchand, M. Duflos, B. Delevoye-Seiller, S. Piessard-Robert,
G. Le Baut, R.W. Hartmann, M. Palzer, Synthesis and in vitro evaluation of 3-
(1-azolylmethyl)-1H-indoles and 3-(1-azolyl-1-phenylmethyl)-1H-indoles as
inhibitors of P450 arom, Arch. Pharm. Weinh. 330 (1997) 141e145.
[10] C. Jacobs, M. Frotscher, G. Dannhardt, R.W. Hartmann, 1-imidazolyl(alkyl)-
substituted di- and tetrahydroquinolines and analogues: syntheses and
evaluation of dual inhibitors of thromboxane A(2) synthase and aromatase,
J. Med. Chem. 43 (2000) 1841e1851.
17b-HSD1 inhibitors showing strong activity towards both the human and the
rat enzyme, Eur. J. Med. Chem. 103 (2015) 56e68.
€
[30] S. Marchais-Oberwinkler, C. Henn, G. Moller, T. Klein, M. Negri, A. Oster,
A. Spadaro, R. Werth, M. Wetzel, K. Xu, M. Frotscher, R.W. Hartmann,
J. Adamski, 17b-Hydroxysteroid dehydrogenases (17beta-HSDs) as thera-
peutic targets: protein structures, functions, and recent progress in inhibitor
development, J. Steroid Biochem. Mol. Biol. 125 (2011) 66e82.
[11] M. Le Borgne, P. Marchand, B. Delevoye-Seiller, J.M. Robert, G. Le Baut,
R.W. Hartmann, M. Palzer, New selective nonsteroidal aromatase inhibitors:
synthesis and inhibitory activity of 2,3 or 5-(alpha-azolylbenzyl)-1H-indoles,
Bioorg Med. Chem. Lett. 9 (1999) 333e336.
[31] S.G. Hansen, H.H. Jensen, Microwave irradiation as an effective means of
synthesizing unsubstituted N-linked 1,2,3-triazoles from vinyl acetate and
azides, Synlett 20 (2009) 3275e3276.
[32] P. Kruchten, R. Werth, S. Marchais-Oberwinkler, M. Frotscher, R.W. Hartmann,
Development of a biological screening system for the evaluation of highly
ꢁ ꢁ
[12] M.P. Leze, M. Le Borgne, P. Pinson, A. Palusczak, M. Duflos, G. Le Baut,
R.W. Hartmann, Synthesis and biological evaluation of 5-[(aryl)(1H-imidazol-
1-yl)methyl]-1H-indoles: potent and selective aromatase inhibitors, Bioorg
Med. Chem. Lett. 16 (2006) 1134e1137.
active and selective 17b-HSD1-inhibitors as potential therapeutic agents, Mol.
Cell Endocrinol. 301 (2009) 154e157.
[33] W. Qiu, R.L. Campbell, A. Gangloff, P. Dupuis, R.P. Boivin, M.R. Tremblay,
[13] R. Maltais, A. Trottier, A. Delhomme, X. Barbeau, P. Lagüe, D. Poirier, Identi-
D. Poirier, S.X. Lin, A concerted, rational design of type 1 17b-hydroxysteroid
dehydrogenase inhibitors: estradiol-adenosine hybrids with high affinity,
FASEB J. 16 (2002) 1829e1831.
fication of fused 16beta,17beta-oxazinone-estradiol derivatives as
family of non-estrogenic 17 -hydroxysteroid dehydrogenase type
hibitors, Eur. J. Med. Chem. 93 (2015) 470e480.
a
new
in-
b
1
[34] K.M. Sam, S. Auger, V. Luu-The, D. Poirier, Steroidal spiro-gamma-lactones
[14] V. Adamo, M. Iorfida, E. Montalto, V. Festa, C. Garipoli, A. Scimone, M. Zanghi,
N. Caristi, Overview and new strategies in metastatic breast cancer (MBC) for
treatment of tamoxifen-resistant patients, Ann. Oncol. 18 (Suppl 6) (2007)
vi53e57.
that inhibit 17 b-hydroxysteroid dehydrogenase activity in human placental
microsomes, J. Med. Chem. 38 (1995) 4518e4528.
[35] J. Moreno-Farre, P. Workman, F.I. Raynaud, Analysis of potential drug-drug
interactions for anticancer agents in human liver microsomes by high
throughput liquid chromatography/mass spectrometry assay, Recent Adv.
Res. Updat. 7 (2006) 207e224.
[15] B. Delvoux, T. D'Hooghe, C. Kyama, P. Koskimies, R.J. Hermans,
G.A. Dunselman, A. Romano, Inhibition of type 1 17
b-hydroxysteroid dehy-
drogenase impairs the synthesis of 17 -estradiol in endometriosis lesions,
b
[36] D.J. Kertesz, C. Brotherton-Pleiss, M. Yang, Z. Wang, X. Lin, Z. Qiu,
D.R. Hirschfeld, S. Gleason, T. Mirzadegan, P.W. Dunten, S.F. Harris,
A.G. Villasenor, J.Q. Hang, G.M. Heilek, K. Klumpp, Discovery of piperidin-4-yl-
aminopyrimidines as HIV-1 reverse transcriptase inhibitors. N-benzyl de-
rivatives with broad potency against resistant mutant viruses, Bioorg Med.
Chem. Lett. 20 (2010) 4215e4218.
J. Clin. Endocrinol. Metab. 99 (2014) 276e284.
[16] B. Husen, K. Huhtinen, M. Poutanen, L. Kangas, J. Messinger, H. Thole, Evalu-
ation of inhibitors for 17b-hydroxysteroid dehydrogenase type 1 in vivo in
immunodeficient mice inoculated with MCF-7 cells stably expressing the
recombinant human enzyme, Mol. Cell Endocrinol. 248 (2006) 109e113.
[17] B. Husen, K. Huhtinen, T. Saloniemi, J. Messinger, H.H. Thole, M. Poutanen,
[37] M. Iwao, H. Takehara, S. Furukawa, M. Watanabe, A regiospecific synthesis of
carbazoles via consecutive palladium-catalyzed cross-coupling and aryne-
mediated cyclization, Heterocycles 36 (1993) 1483e1488.
Human hydroxysteroid (17-b) dehydrogenase 1 expression enhances estro-
gen sensitivity of MCF-7 breast cancer cell xenografts, Endocrinology 147
(2006) 5333e5339.
[38] J. Haupenthal, C. Baehr, S. Zeuzem, A. Piiper, RNAse A-like enzymes in serum
inhibit the anti-neoplastic activity of siRNA targeting polo-like kinase 1, Int. J.
Cancer 121 (2007) 206e210.
[18] J.M. Day, P.A. Foster, H.J. Tutill, M.F. Parsons, S.P. Newman, S.K. Chander,
G.M. Allan, H.R. Lawrence, N. Vicker, B.V. Potter, M.J. Reed, A. Purohit, 17b-
hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for
endocrine therapy of hormone-dependent breast cancer, Int. J. Cancer 122
Please cite this article in press as: A.S. Abdelsamie, et al., Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a
selective 17 -HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.004
b