Structure-based design, synthesis and in vitro characterization of potent 17β-hydroxysteroid dehydrogenase type 1 inhibitors based on 2-substitutions of estrone and D-homo-estrone

Article abstract of DOI:10.1016/j.bmcl.2009.09.113

In search for specific drugs against steroid-dependent cancers we have developed a novel set of potent inhibitors of steroidogenic human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD 1). The X-ray structure of 17β-HSD 1 in complex with estradiol served

Full text of DOI:10.1016/j.bmcl.2009.09.113

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6740–6744
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design, synthesis and in vitro characterization of potent
17b-hydroxysteroid dehydrogenase type 1 inhibitors based on 2-substitutions
of estrone and D-homo-estrone
Gabriele Möller a, Dominga Deluca a, Christian Gege b, , Andrea Rosinus a, Dorota Kowalik a, Olaf Peters b,à
,
c,–
Peter Droescher b,à, Walter Elger c,§, Jerzy Adamski a, , Alexander Hillisch
*
a Helmholtz Zentrum München, Institute of Experimental Genetics, Genome Analysis Center, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany
b Jenapharm GmbH & Co. KG, Medicinal Chemistry, Otto-Schott-Str. 15, 07745 Jena, Germany
c EnTec GmbH, Adolf-Reichwein-Str. 20, 07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2009
Revised 27 September 2009
Accepted 29 September 2009
Available online 3 October 2009
In search for specific drugs against steroid-dependent cancers we have developed a novel set of potent
inhibitors of steroidogenic human 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD 1). The X-ray
structure of 17b-HSD 1 in complex with estradiol served as basis for the design of the inhibitors. 2-Substi-
tuted estrone and D-homo-estrone derivatives were synthesized and tested for 17b-HSD 1 inhibition. The
best 17b-HSD 1 inhibitor, 2-phenethyl-D-homo-estrone, revealed an IC50 of 15 nM in vitro. The inhibitory
potency of compounds is comparable or better to that of previously described inhibitors. An interaction
within the cofactor binding site is not necessary to obtain this high binding affinity for substances
developed.
Keywords:
Steroid metabolism
Cancer
Ó 2009 Elsevier Ltd. All rights reserved.
Inhibitors
Molecular docking
Structure-based drug design
Human diseases can be treated by manipulation of selective
targets contributing to pathogenesis. Several such targets are de-
fined as enzymes including 17b-hydroxysteroid dehydrogenases
(17b-HSDs).1,2 The latter control the biological potency of steroid
hormones by redox reactions at position 17 of the steroid scaf-
fold.3–5 The 17b-HSDs belong to the short-chain dehydrogenase/
reductase superfamily (SDR)6 except for 17b-HSD 5 which is an
aldo-ketoreductase.7
Observations on the prognostic value of 17b-HSDs in breast
cancer,8–10 prostate cancer,11 and in endometriosis12–14 have
boosted research on these enzymes. Approaches against breast
and prostate cancers involve the development of new safe and
17b-HSD-specific drugs.15–18 Several strategies include 17b-HSD
1 as a target and drug design has been facilitated by the known
crystal structure of the enzyme.19 An effective inhibitor of conver-
sion of estrone to estradiol by 17b-HSD 1 should deplete active
hormone from the signal transduction pathway.
Among naturally available substances the phytoestrogens are
the most potent inhibitors of 17b-HSDs20,21 but unfortunately they
are often non-specific.22 Therefore, distinct highly specific 17b-
HSD 1 inhibitors have been developed.14,23 Strategies for that in-
cluded modifications of the steroid scaffold at positions 6, 16,
and/or 1716,24–28 and the substitution of hydroxyl moieties with
sulfamates29,30 or fluorine.31 Even hybrid inhibitors based on estra-
diol derivatized with adenosine were reported.17,32,33 Non-steroi-
dal inhibitors of 17b-HSD 1 have as well been identified.34
However, none of the published inhibitors has progressed to clini-
cal trials and further research is necessary.
In this work we present the development of an inhibitor for
17b-HSD 1 for treatment of estradiol-dependent diseases as de-
scribed in our patents.35,36 We show the design, synthesis, and
in vitro characterization of several 2-substituted derivatives of es-
trone and D-homo-estrone. The main difference from previous re-
ports is the substitution of estrone and D-homo-estrone in position
2 at the aromatic A-ring. The X-ray structure of 17b-HSD 1 shows
an unoccupied lipophilic subpocket there and modifications at C2
were only marginally pursued so far.16,37 In addition, these 2-
substituted inhibitors should show reduced intrinsic estrogenici-
* Corresponding author. Tel: +49 89 3187 3155; fax: +49 89 3187 3225.
E-mail address: adamski@helmholtz-muenchen.de (J. Adamski).
Present address: X2-Pharma GmbH, Im Neuenheimer Feld 584, 69120 Heidelberg,
Germany.
à
Present address: Bayer Schering Pharma AG, Müllerstraße 178, 13353 Berlin,
Germany.
§
Present address: Schorlemerallee 12B, 14195 Berlin, Dahlem, Germany.
Present address: Bayer Schering Pharma AG, Apratherweg 18a, 42096 Wuppertal,
–
Germany.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.113

G. Möller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744
6741
ty,37 since these do not fit well into the ER
a
binding site,27 and
O
O
should posses altered properties with respect to phase II metabo-
lism, due to shielding of the phenolic 3-hydroxy group.27
H
H
H
a
I
Compounds 1 (estrone, E1) and 6 (Table 1) are commercially
available while the synthesis of compounds 2–438, 537, 7–839 has
been published in the literature. The synthesis of the other deriva-
tives was performed as described by us in full in our patents.35,36
In brief, incorporation of the 2-pentanoyl moiety into 3-methoxyes-
tra-1,3,5(10)-trien-17-one was accomplished under Friedel–Crafts
conditions with valeroyl chloride and aluminium trichloride at
5 °C to give the 2-acylated intermediate.40 Cleavage of the 3-methyl
ether with hydrobromide in acetic acid under reflux afforded prod-
uct 9. Derivative 10 was synthesized from 3-acetoxy-2-iodo-estra-
1,3,5(10)-trien-17-one38 via a Sonogashira coupling with phenyl-
acetylene and subsequent deacetylation. Hydrogenation of the acet-
ylated 2-phenethynyl intermediate with palladium on charcoal
furnished the 2-phenethyl-substituted derivative 11 (Scheme 1).
Modification of the steroidal core towards the 14,15-dehydro
derivative 12 was accessible by application of Saegusa’s general
H
H
H
H
H
H
AcO
AcO
b, c
O
HO
11
Scheme 1. Reagents and conditions: (a) PhC„CH, Pd(OAc)2, PPh3, CuI, NEt3/THF
(3:2), rt, 89%; (b) H2 (1 bar), Pd/C, EtOAc, rt, 3 h; (c) NaOMe, MeOH/THF, rt, 89% for
both steps.
procedure to compound 6.41 The 14
a,15a-cyclopropa[a]estra-
The D-homo moiety (Table 2) was incorporated via a modified
Tiffenau–Demjanow ring expansion, starting from the 17-keto
derivatives.46,47
1,3,5(10),8-tetraene core42 was substituted in the 2-position by
ortho-lithiation similar as previously described43 to give product
13. Incorporation of the 6-oxo moiety towards compound 14 was
accomplished with chromium trioxide in acetic acid as described
with similar derivatives previously.44
For example, reaction of 1 with trimethylsulfonium iodide and
potassium tert-butylate afforded the oxirane, which was opened
with sodium azide and then cyclized with trimethylsilyliodide to
afford the D-homo derivative 18 in high overall yield (Scheme 3).
Starting with compound 6, product 22 was obtained on the
same route. After acetylation of compound 18, incorporation of
the halogen moiety in 2-position was accomplished via an ortho-
thallation as described previously,38 albeit in lower yield (Scheme
4). Subsequent deprotection furnished the derivatives 19, 20, and
21, respectively. The 2-allylated product 23 was obtained via the
route described for the corresponding estrone derivative (Scheme
3).39 Starting material 18 was reacted with allyl bromide and ce-
sium carbonate as base in N,N-dimethylformamide to give the 3-
O-allyl intermediate, which was rearranged in refluxing diethylan-
iline to afford a mixture of the 2-allyl and 4-allyl isomer (ratio
ꢀ1:2). Separation by HPLC furnished the desired 2-allyl substituted
product 23. Derivative 24 was synthesized similar as described
Halogenation of 18-homo-1,3,5(10)-trien-17-one was executed
as described for derivative 2 to give compound 15.38 Fluorination
was accomplished by silylation of ketone 2 and reaction of the
resulting enol ether with N-fluoropyridinium triflate (NFPT) to give
compound 16 (Scheme 2).45 Sodium methoxide catalyzed epimer-
ization45 of derivative 16 in methanol afforded an equimolar mix-
ture, wherein the 16b-fluoro derivative 17 was separated by HPLC
in 33% yield.
Table 1
Inhibition of 17b-HSD 1 by 2-substituted estrone derivatives
R18
O
O
O
O
H
R16
H
above for derivative 11 (Scheme 4). a-Fluorination of ketone 19
R2
O
with AccufluorÒ NFTh was conducted as previously described48
and furnished a diastereomeric mixture, which was separated by
HPLC to give pure compounds 25 and 26.
H
H
H
HO
HO
12
14
Inhibition of catalytic activity of human 17b-HSD 1 towards E1
was assessed as originally described by us and a detailed descrip-
tion for that is given in the Supplementary data section.31,49
The X-ray structure of 17b-HSD 1 in complex with estradiol (E2)
and NADP+ (PDB entry code 1FDT)19 was used for the docking
H
O
O
H
O
H
HO
HO
13
O
O
Compd
R2
R16
R18
% Inhibition @ 2
lM
IC50 (nM)
1 (E1)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
H
H
H
H
H
H
H
H
H
H
H
H
—
—
—
H
H
H
H
H
H
H
H
H
H
H
H
—
—
—
Me
H
H
90
95
95
96
86
70
94
91
85
90
97
84
97
80
86
99
92
109
140
233
148
89
207
109
545
218
56
H
H
H
F
F
Cl
Br
NC
Et
MeO
Allyl
n-Propyl
CH3(CH2)3CO
Ph-C„C
Phenethyl
—
—
—
Cl
Cl
Cl
Cl
Cl
a, b
H
H
H
H
HO
HO
2
16
c
O
Cl
47
633
193
392
121
101
35
H
H
HO
17
a
-F
Scheme 2. Reagents and conditions: (a) TMSOTf, NEt3, toluene, reflux; (b) NFPT,
CH2Cl2, rt, 1 N HCl; (c) NaOMe, MeOH, rt, 11 d, 33%.
b-F

6742
G. Möller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744
FLEXX parameters, as implemented in the 6.7 release of SYBYL, were
used. Substrate binding was analyzed using the program MOLCAD
Table 2
Inhibition of 17b-HSD 1 by 2-substituted D-homo-derivatives of estrone
in SYBYL
.
O
R17
The crystal structure of 17b-HSD 1 in complex with E2 was pre-
viously applied in molecular dynamics simulations and ligand–
protein docking studies of non-steroidal52–54 as well as steroidal
derivatives.16,28,30,55 In the latter studies mainly the region in the
16 and 17 position of the steroid was investigated, which points to-
wards the NADP+ cofactor. In our study, we focused on position 2 of
estrone and D-homo-estrone. From the surface of the substrate-
binding pocket in the 17b-HSD 1 X-ray structures it is clearly vis-
ible that the position 2 of E2 is located in a lipophilic environment
consisting of the side-chains of Val143, Met147, Phe259, Leu262
and Met279. In this region the substrate binding pocket forms a
lipophilic tunnel to the exterior of the protein. The existence of
such a lipophilic tunnel inspired us to synthesize steroidal com-
pounds substituted with lipophilic residues (such as halogen or al-
kyl) in position 2. In addition, fluorine substituents were
H
R2
H
H
HO
Compd
R2
R17
% Inhibition @ 2
lM
IC50 (nM)
18
19
20
21
22
23
24
25
26
H
Cl
Br
I
MeO
Allyl
H
H
H
H
H
H
H
91
100
100
100
92
30
77
73
123
85
32
15
87
126
97
95
86
Phenethyl
Cl
Cl
a
-F
b-F
100
introduced in position 16a and b to stabilize the 17 carbonyl group
of estrone from metabolic attack. The five-membered D-ring was
also enlarged to a six-membered (D-homo) ring.
O
O
Several 2-substituted estrone derivatives were tested for their
ability to inhibit recombinant 17b-HSD 1 activity (Table 1). Struc-
ture–activity relationships focused mainly on the role (size and
polarity) of substitutions at position 2 of the steroidal A-ring and
some modifications of the B- and D-ring (Fig. 1).
H
H
H
a, b, c
H
H
H
H
H
HO
HO
HO
1
18
d, e
Based on the observed IC50-values all compounds in this series
are submicromolar to nanomolar inhibitors of 17b-HSD 1. To facil-
itate normalized comparison we analyzed the natural substrate es-
trone (E1) which shows under the same conditions an IC50 of
109 nM. Compared to halogen/pseudohalogen substituents such
as chlorine, bromine and carbonitrile (compounds 2–4) in 2-posi-
tion do not lead to increased inhibition. The same is true for the
18-homo estrone derivative 15 and the 2-methoxy-derivatives
(compounds 6, 12–14). We had made similar observations for
17-fluorine-substituted estrogens in a previous study.31 It is pro-
posed that an intramolecular hydrogen bond between the 3-hydro-
xyl group and the oxygen atom of the 2-methoxy substituent of the
steroid reduces binding to His221 and Glu282 of 17b-HSD 1 and
positions the methoxy group in the plane of the steroidal A-ring.
This might lead to repulsive interactions with Phe259. This intra-
molecular hydrogen bond and the respective conformation of the
methoxy group is observed in a number of small molecule X-ray
structures bearing the O-methoxy-phenyl ring system.56
O
H
23
Scheme 3. Reagents and conditions: (a) MeSI (2 equiv), KOtBu (2.2 equiv), DMSO,
rt, 80%; (b) NaN3 (1.3 equiv), DMF, 60 °C, 57%; (c) NaI, Me3SiCl, CH3CN, rt, 75%; (d)
AllBr, Cs2CO3, DMF, 60 °C, 3 h, 89%; (e) PhNEt2, reflux, 8 h, 34% (and 4-allyl isomer).
O
O
H
H
H
a, b
I
H
H
H
H
H
H
Modification of compound 6 with an additional carbonyl group
in position 6 (compound 14) or double bond in position 14–15
(compound 12) leads to diminished inhibition while introduction
HO
AcO
18
c, d
O
of a 8–9 double bond together with a 14a,15a-cyclopropyl substi-
tuent does not alter the IC50 in comparison with compound 6. The
docking studies suggest that the 6-oxo group in compound 14
could lead to repulsive interactions with Ser222 and Tyr218.
Slightly increased inhibition was observed with the 2-ethyl sub-
stituent (compound 5 with IC50 = 89 nM) while 2-allyl, propyl,
and pentanoyl resulted in equally potent or weaker inhibitors.
Especially the terminal CH3-group of the 2-ethyl substituent is
proposed to fill a subpocket formed by Val143, Met147, Leu149,
Phe259, and Leu262 of 17b-HSD 1 while the other substituents
are longer and interact with Leu262, which is more exposed to
the solvent. A significant increase in inhibitory activity compared
to E1 was observed for the 2-phenethynyl and 2-phenethyl sub-
stituents (compounds 10, IC50 = 56 nM and 11, IC50 = 47 nM).
These longer substituents are proposed to form lipophilic contacts
with Leu262 and Phe259, which might explain the increased
inhibition.
HO
24
Scheme 4. Reagents and conditions: (a) Ac2O, pyridine, cat. DMAP, rt, quant.; (b)
Tl(OCOCF3)3, TFA, 10 °C, then KI, 40%; (c) PhC„CH, Pd(OAc)2, PPh3, CuI, NEt3/THF
(3:1), rt, 80%; (d) H2 (1 bar), Pd/C, EtOAc/THF, rt; NaOMe, MeOH/CH2Cl2, rt, 65%.
experiments to support structure–activity relationships (SAR)
studies and design of inhibitors. The 3D-structures of the steroidal
compounds were generated within SYBYL 6.7 (Tripos Inc. SYBYL, 2000)
and energy minimized using the MMFF94 force field.50 The FLEXX
program version 1.851 interfaced with SYBYL was used to perform
the docking experiments to 17b-HSD 1. Glu282 was treated as
charged residue, whereas His221 was kept uncharged. All default
The most potent inhibitor in this series was obtained by an
additional substitution of compound 2 (2-chloro-estrone) in posi-

G. Möller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744
6743
Figure 1. Molecular modelling. Lipophilic surface areas are colored brownish, green corresponds to neutral and blue to hydrophilic parts of the protein surface. Leu149 and
Phe259 are not depicted since they are positioned far below and above the steroid plane. (Top) Substrate binding pocket of 17b-HSD 1 in complex with estradiol and NADP+
according to PDB data 1FDT. (Bottom) Modelled complex of 2-phenethyl-D-homo-estrone 24 with 17b-HSD 1 and NADP+.
tion 16 with fluorine. 2-chloro-16b-fluorine-estrone (compound
17) is significantly more potent (IC50 = 35 nM) than the 16 -isomer
(IC50 = 101 nM). An electrostatic repulsive interaction between the
androgens on binding to 17b-HSD 1. The lead compound D-
homo-estrone 18, having a six-membered A and D-ring (Table 2),
revealed an IC50 of 30 nM which is even better than that obtained
with the most potent compound 17 from the series based on 2-
substituted E1. This is surprising, because the SAR around the 2-
position between the two lead series (estrone and D-homo-es-
trone) is completely parallel (Tables 1 and 2). Chlorine, bromine,
and methoxy modifications lead to weaker inhibitors. Allyl is neu-
tral in comparison to compound 18, whereas 2-phenethyl in-
creases inhibition and represents the most potent compound in
this study (compound 24, IC50 = 15 nM). This suggests a very sim-
ilar binding mode of the two series which is also supported by our
docking experiments. In contrast to the estrone series (compounds
16 and 17), fluorine substitution in the position vicinal to the car-
bonyl moiety (position 17) leads to weaker inhibitors than plain D-
homo-estrone 18. This effect cannot be explained in detail with the
applied docking procedures.
a
partially negatively charged 16a-fluorine substituent and the
amide carbonyl oxygen of NADP+ (distance ꢁ3 Å) might explain
this difference of the two stereoisomers.
The structure–activity study was extended to compounds with
another steroid-like scaffold, the D-homo-estrone (Table 2), in
which the five-membered D-ring of estrone is expanded to an ali-
phatic 6-ring. This modification was purely driven by the favour-
able synthetic accessibility of these compounds. It has been
shown that androstanedione, androstenedione, and testosterone
bind to 17b-HSD 1 in a reverse binding mode in comparison to
E2.57,58 When bound substrate conformations are compared, the
six-membered A ring of the androgens overlays approximately
with the five-membered D-ring of E2. This observation allows for
the assumption that D-homo-estrogens probably behave like

6744
G. Möller et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6740–6744
18. Frotscher, M.; Ziegler, E.; Marchais-Oberwinkler, S.; Kruchten, P.; Neugebauer,
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In conclusion, novel and potent inhibitors of 17b-HSD 1 were
identified applying structure based design, chemical synthesis
and biochemical testing of the recombinant enzyme in an iterative
manner. The inhibitors were derived from estrone, the natural sub-
strate of 17b-HSD 1. The X-ray structure of 17b-HSD 1 guided the
design of the compounds. The most potent compound is character-
ized by an IC50 in the low nanomolar range, sevenfold better than
that of estrone. With respect to inhibitory potency, the compounds
are comparable to previously described inhibitors such as non-ste-
roidal compounds or compounds that are linked to cofactors in
positions 16 and 17.16,26,34e However, the increase in potency is ob-
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Acknowledgment
35. Hillisch, A.; Regenhardt, W.; Gege, C.; Peters, O.; Bothe, U.; Adamski, J.; Möller,
G.; Rosinus, A.; Elger, W.; Schneider, B. Patent WO06/003013, 2006.
36. Gege, C.; Regenhardt, W.; Peters, O.; Hillisch, A.; Adamski, J.; Möller, G.; Deluca,
D.; Elger, W.; Schneider, B. Patent WO06/003012, 2006.
This project was supported by BMBF-Project ‘Bioinstrumente
Jena 0312255’.
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Supplementary data
Supplementary data associated with this article can be found in
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59. The binding affinity of the compounds to human ER
determined by in vitro competition experiments using [3H]-17b-estradiol
([3H]-E2) as ligand (5 nM) and unlabeled E2 as reference. hER
was
produced in SF9 insect cells using baculovirus expression vector.
a (hERa) was
a
a
Experiments were performed in quadruplicates. The IC50 of E2 for hER
a
was determined as 1.25 0.7 Â 10À8 M.