Novel and potent 17β-hydroxysteroid dehydrogenase type 1 inhibitors

Article abstract of DOI:10.1021/jm049045r

Structure-based drug design using the crystal structure of human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) led to the discovery of novel, selective, and the most potent inhibitors of 17β-HSD1 reported to date. Compounds 1 and 2 contain a side cha

Full text of DOI:10.1021/jm049045r

J. Med. Chem. 2005, 48, 2759-2762
2759
Novel and Potent 17â-Hydroxysteroid
Dehydrogenase Type 1 Inhibitors
Harshani R. Lawrence,† Nigel Vicker,†
Gillian M. Allan,† Andrew Smith,† Mary F. Mahon,†,§
Helena J. Tutill,‡ Atul Purohit,‡ Michael J. Reed,‡
and Barry V. L. Potter*,†
Medicinal Chemistry, Department of Pharmacy and
Pharmacology and Sterix Ltd., University of Bath,
Claverton Down, Bath, BA2 7AY, U.K., Endocrinology and
Metabolic Medicine and Sterix Ltd., Faculty of Medicine,
Imperial College London, St. Mary’s Hospital, London,
W2 1NY, U.K., and Department of Chemistry, University of
Bath, Claverton Down, Bath, BA2 7AY, U.K.
Figure 1. Estrone template modification for design of new
inhibitors of 17â-HSD1.
information to design potential druglike inhibitors of
17â-HSD1. It has been suggested that the steroid and
the NADPH binding sites can be independently tar-
geted.10 17â-HSD1 is a 327 amino acid residue protein
with a subunit mass of 35 kDa and exists as a ho-
modimer. Several groups have reported selective inhibi-
tors for 17â-HSD1,11 many of which share common
structural features such as a phenolic ring and hydro-
phobic scaffolds that interact with the hydrophobic
amino acid residues in the active site that recognize the
steroid substrate.
Poirier et al. have reported estrone-based steroidal
inhibitors of 17â-HSD1, with E2 derivatives bearing a
short side chain at C17R and C16R positions of the
estrone skeleton.12 In an alternative approach, this
group recently reported hybrid inhibitors of 17â-HSD1,
the most potent being EM1745 with an IC50 of 52 nM,
which had the estradiol template linked via an eight-
methylene spacer to the adenosine moiety.13 The crystal
structure of 17â-HSD1 and EM1745 was resolved to 1.6
Å 14 and shows the substrate and cofactor binding sites
occupied in the enzyme in a fashion similar to estradiol
in the crystal structure of the ternary complex 17â-
HSD1-E2-NADP+. This work highlights the size of the
binding site and its accessibility to large flexible mol-
ecules.
As part of an ongoing program, we targeted the
discovery of small-molecule inhibitors of 17â-HSD1 by
incorporating a functionalized side chain at the 16
position of estrone (Figure 1). Structure-based drug
design and parallel synthesis were used to optimize the
interactions with the active site residues and to probe
possible binding with the cofactor. From the therapeutic
perspective of estrogen-dependent breast cancer, inhibi-
tors of 17â-HSD1 should not display estrogenic effects.
Attempts to generate dual action inhibitors that show
antiestrogenic effects and inhibit 17â-HSD1 have been
reported.15 In our approach functionality is incorporated
onto the steroid scaffold aimed at reducing estrogenicity.
Here, we report the design, synthesis, and in vitro
biological properties of compounds that represent a
selective, potent, and novel class of 17â-HSD1 inhibitor
(Table 1).
Received November 25, 2004
Abstract: Structure-based drug design using the crystal
structure of human 17â-hydroxysteroid dehydrogenase type
1 (17â-HSD1) led to the discovery of novel, selective, and the
most potent inhibitors of 17â-HSD1 reported to date. Com-
pounds 1 and 2 contain a side chain with an m-pyridylmethyl-
amide functionality extended from the 16â position of a steroid
scaffold. A mode of binding is proposed for these inhibitors,
and 2 is a steroid-based 17â-HSD1 inhibitor with the potential
for further development.
Breast cancer is the most common cancer diagnosed
among women, with an estimated global incidence in
2002 of ∼1 150 000.1 Approximately 60% of these are
hormone-responsive breast cancer with circulating es-
trogens playing a vital role in promoting the growth and
development of such tumors, and in this regard 17â-
estradiol (E2) is crucial for tumor growth and develop-
ment.2 Elevated 17â-hydroxysteroid dehydrogenase type
1 (17â-HSD1) activity has been found in many human
tissues such as placenta, liver, ovary, endometrium and
in breast cancers.3 In human breast tissue, the principal
activity of 17â-HSD1 is to convert weakly active estrone
(E1) to active 17â-estradiol (E2), using nicotinamide
adenine dinucleotide (NADH) or its phosphate deriva-
tive (NADPH) as the cofactor.4,7 Occupation and activa-
tion of the estrogen receptor by estradiol are crucial for
the stimulation of growth and development in hormone-
dependent breast cancer.5,6 Because E2 enhances breast
cell proliferation in tumors,7 suppression of the activity
of 17â-HSD1 constitutes a way of reducing tumor
estrogen levels and promoting tumor regression. There-
fore, 17â-HSD1 inhibition is an attractive target for the
design of potential antitumor drugs, but in vivo efficacy
of an agent against this target has never been demon-
strated.
Detailed knowledge of the biochemistry and molecular
biology of 17â-HSD1 has grown rapidly in the recent
years.8 Crystallographic structural information on 17â-
HSD1 with the enzyme in its native form, in complex
with estradiol and NADP (PBD code 1FDT), with
estradiol alone9 and with equilin and NADP10 provides
In the design of selective and potent inhibitors of 17â-
HSD1, several structural requirements were taken into
account (Figure 1). To obtain an inhibitor capable of
exhibiting interactions with the substrate and the
cofactor binding sites of 17â-HSD1, we designed mol-
* To whom correspondence should be addressed. Phone: +44 1225
386639. Fax: +44 1225 386114. E-mail: B.V.L.Potter@Bath.ac.uk.
† Department of Pharmacy and Pharmacology, University of Bath.
§ Department of Chemistry, University of Bath.
‡ Imperial College London.
10.1021/jm049045r CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/25/2005

2760 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Letters
Table 1. Inhibition of 17â-HSD1 and 17â-HSD2 in T47D and
MDA-MB-231 Cell Lines, Respectivelya,22,23
a Mean of at least two measurements typically ( 5% variation.
b At 10 µM. The asterisk (/) indicates point of attachment.
Figure 2. Compound 2 (green) docked into 1FDT with
cofactor present (pink) and substrate removed.
ecules containing moieties with the ability to interact
at each site with a linker of suitable length. The moieties
selected to act as scaffolds and to bind to the substrate
binding site were estrone and 2-ethylestrone; these were
to be substituted at the 16 position with a distal
carboxylic acid group that could be reacted with amines.
The estrone skeleton is present in many inhibitors, and
its binding mode is well demonstrated by X-ray crystal-
lographic studies. As regards the spacer, we reasoned
that a side chain bearing an amide function might
favorably interact with amino acid residues in the active
site of the enzyme while inclusion of an amide also aids
the ease of synthesis for rapid optimization. Therefore,
our strategy to incorporate carboxylamide and methyl-
ene carboxylamide linkers at the 16 position adds a
point of diversity into the estrone pharmacophore, thus
facilitating the probing of key interactions in the
substrate binding site of the enzyme (Figure 1). The
library design included different linkers and a diverse
set of commercially available R2 substituents. Full
details of this work will be published elsewhere. The
most promising side chain was a methylene carboxyl-
amide moiety at the 16 position of the estrone (Figure
1), and molecular modeling studies guided the potential
directions for synthetic lead optimization. To reduce the
estrogenic activity, we focused on compounds with
2-ethyl and 2-methoxy groups on the A ring that are
reported to display very low estrogenic activity,16,17 and
this strategy has previously been used successfully by
our group to lower estrogenicity.17
The phenolic group on the A ring of estradiol under-
goes bifurcated hydrogen bonding with His221 and
Glu282 residues of the protein, and we found that the
compounds with a hydroxyl group on the A ring showed
high inhibition of 17â-HSD1. The amino acid residues
in the catalytic region of the active site of 17â-HSD1
(Tyr 155, Ser 142, Glu 192) were targeted via hydrogen
bonding with the 17-keto group on the estrone deriva-
tives demonstrating high inhibition of 17â-HSD1. The
R2 substituents were chosen from commercially avail-
able amines that incorporated motifs of diverse char-
acteristics.
Figure 3. Crystal data and structure refinement of the
hydrochloride salt of the major diastereoisomer of the 3-O-
benzyl derivative of 1. The asymmetric unit also contains one
molecule of water. Ellipsoids are represented at the 30%
probability level.
docking program GOLD.18 The top-ranked conforma-
tions of 1 and 2 gave high fitness scores of 74.0 and 81.1,
respectively. The steroid backbones of 1 and 2 are
essentially overlaid when docked into 1FDT, forming
the same hydrophobic interactions with the exception
of the ethyl group in the 2 position of 2, which is capable
of forming an extra hydrophobic interaction with Leu262
and Phe259. The main loci of common hydrophobic
interactions involving the steroid backbone of 1 and 2
are with Leu149, Val225, Phe226, Phe259; these hy-
drophobic interactions are also observed in the crystal
structures reported wherein the steroid nucleus is
cocrystallized.8-10
Figure 2 depicts 2 docked into 1FDT with key
hydrophobic interactions with the steroid backbone and
the phenolic oxygen at the 3 position and the carbonyl
oxygen at the 17 position. In these potent inhibitors it
was proposed that functionality in the 16â side chain
(see Figure 3) might interact with the nicotinamide
portion of the cofactor.
In 1FDT the nicotinamide carbonyl and the amide
nitrogen form hydrogen bonds to the NH of Val188 and
the oxygen of Thr140, respectively. The docked confor-
mation of 2 shows that the carbonyl oxygen of the amide
in the side chain is 3.16 Å from the closest nicotinamide
NH. This observation indicates that there may be an
interaction between the nicotinamide amide moiety and
the amide carbonyl of the 16â side chain. In addition, a
16â side chain with sp3 substitution may hinder the Pro-
(S) hydrogen being transferred to the 17 carbonyl 2.21
Å away because one of the hydrogen atoms in the
methylene linker attached to the 16 position is only 2.63
Å from the Pro(S) hydrogen and the methine hydrogen
at the 16 position is 1.81 Å away. The pyridyl nitrogen
of the 16â side chain is 3.16 Å from a phosphate oxygen
Compounds 1 and 2 were identified as the most po-
tent and selective inhibitors of 17â-HSD1 from our
compound library with IC50 values of 37 and 27 nM,
respectively. To understand how these compounds bind
in accordance with our strategy, 1 and 2 were docked
into 1FDT with estradiol removed using the flexible

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2761
Scheme 1. Synthetic Routes to Phenol Libraries 7 and 10 and 2-Methoxyestrone Analogue 3a
a Reagents and conditions: (i) BnBr, K2CO3, DMF, 90-97%; (ii) (a) -10 °C, LDA, THF; (b) ethyl bromoacetate, -60 °C, 78-99%; (iii)
H2, Pd-C, EtOH, 94%; (iv) NaOH, MeOH/THF/H2O (1:1:0.5), 80-90 °C, 90%; (v) EDCI, DMAP, Et3N, 3-aminomethylpyridine (2 equiv),
DCM, 48-70%; (vi) H2, Pd-C, THF/MeOH (1:1), 97%; (vii) NH2SO2Cl, DMA, 98%; (viii) Cl-trityl-Cl resin, DIPEA, DCM, (loading of
estrone is 0.86 mmol/g); (ix) 4 M NaOH, THF, room temp, 2 h; (x) 3-aminomethylpyridine, PyBroP, HOBt, DIPEA, DCM, 4 days; (xi)
piperazine, THF, reflux, 10-45%; (xii) (a) -10 °C, LDA, THF; (b) ethyl bromoacetate, -60 °C, 95%; (xiii) H2, Pd-C, THF/MeOH (1:1),
65%; (xiv) 4 M NaOH, THF, room temp, 2 h, 90%; (xv) PyBroP, HOBt, DIPEA, DCM, 50%.
and could form an interaction similar to that of Ile14
with an alternative phosphate oxygen at the same
phosphorus center. These additional interactions of 1
and 2 with the cofactor may in part explain the high
potency of these novel inhibitors.
Finally, the amides 9 were reacted with piperazine
to give the deprotected phenol library 10 with good
yields and high purities. Phenol libraries 7 and 10 were
purified using SiO2 chromatography and characterized
by spectroscopic methods (1H NMR, HPLC, LRMS,
HRMS) to provide compounds with average purities of
>90%. The synthesis of 3 is also outlined in Scheme 1,
where 2-methoxybenzylestrone16 11 was reacted with
ethyl bromoacetate via enolization to give 12. The
intermediate 12 was sequentially reacted with Pd-C/
H2 and NaOH to give the acid 13 in good yield. The final
compound 3 was obtained in good yield as a mixture of
diastereomers, using standard coupling conditions, i.e.,
PyBroP, HOBt, DIPEA, DCM. Focused libraries of
amides displaying a variety of electronic and steric
properties were generated to identify potential synthetic
leads.
The synthetic strategies for 17â-HSD1 inhibitors are
shown in Scheme 1. The target compounds described
here were prepared as part of a larger program to
develop selective leads for 17â-HSD1. As a result, we
utilized several different methods to obtain compounds.
Here, we highlight the synthesis of the most potent
compounds. The syntheses of the estrone derivatives
with substitution at the 16 position were achieved using
solid- and solution-phase parallel synthesis (Scheme 1).
Estrone and 2-ethylestrone (R1 ) H and Et, respectively)
were O-benzylated at the 3 position and then alkylated
at the 16 position via enolization to give benzyl protected
ethyl ester 5.19 The intermediate 5 was sequentially
reacted with Pd-C/H2 and NaOH to obtain the inter-
mediate 6 in high yield. Acid 6 was subsequently reacted
using parallel synthesis with commercially available
amines using EDCI, DMAP, and Et3N in DCM to obtain
a focused library of amides 7 in good yields.
Additionally, as part of our combinatorial approach,
a procedure reported by Poirier et al. was employed to
produce the phenol library 10.20 This allowed rapid
access to large numbers of diverse compounds. The
2-ethylestrone derivative 5 was debenzylated using Pd-
C/H2 to release the hydroxyl group, and the product
reacted with sulfamoyl chloride to give the 3-O-sulfa-
mate in excellent yield. This was attached to Cl-trityl-
Cl resin to give the intermediate 8 with 0.86 mmol/g
loading, using the standard conditions of DIPEA in
DCM. The resin-bound sulfamate ester was then hy-
drolyzed and coupled with a series of amines using
PyBroP and HOBt to provide resin-bound amides 9.
1
The major diastereomers (approximately 75% by H
NMR) of 1 and 2 were separated by SiO2 chromatogra-
phy and have a â orientation at the 16 position. The
major diastereomers of 1 and 2 with >99% diastereo-
meric purity were used for biological studies. The X-ray
crystal structure (Figure 3) of the major diastereomer
of the 3-O-benzyl derivative of 1 confirmed that the side
chain has a â orientation.21
The in vitro inhibitory activities of the 1-3 were
evaluated against types 1 and 2 17â-HSDs using cell-
based assays (Table 1). For this, T47D or MDA-MB-231
cells were used to assess the inhibitory potency of
compounds in cells with 17â-HSD1 and 17â-HSD2
activities because T47-D and MDA-MB-231 cells have
previously been shown to possess predominantly reduc-
tive and oxidative 17â-HSD activity, respectively.22,23
Of those reported to date for 17â-HSD1, 1 and 2 were
the most potent and selective inhibitors discovered by
our approach.

2762 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
Letters
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The inhibitory activities shown by 1-3 suggest that
the m-pyridyl unit is beneficial to the activity and
selectivity in these compounds, and this supports our
proposed novel mode of binding for these compounds.
Small hydrophobic groups are tolerated at the steroid
2 position. Hence, 2 has a potency similar to that of 1
and may possess added hydrophobic interactions in the
active site. Indeed, in addition to its potent activity and
selectivity, 2 has built-in structural features proven to
lower estrogenicity. Compound 3 containing a methoxy
group at the 2 position is 10-fold less active than 2.
Because the methoxy group is less hydrophobic than an
ethyl group and has some directional electronics, the
hydrophobic interactions with Leu262 and Phe259 may
be less favorable. In addition, the H-bonding interac-
tions in the phenolic region may be lowered.
In conclusion, structure-based drug design using the
crystal structure of human 17â-HSD1 has led to the
discovery of novel potent inhibitors of 17â-HSD1. Com-
pounds 1 and 2, with IC50 values of 37 and 27 nM,
respectively, and containing a side chain with an
m-pyridylmethylamide functionality extended from the
16â position of a steroid scaffold, were identified from
libraries using solid- and solution-phase parallel syn-
thesis. A novel mode of binding is proposed for these
new inhibitors. Moreover, 2 is a steroid-based 17â-HSD1
inhibitor with the potential for further development.
Acknowledgment. This work was supported by
Sterix Ltd. as a member of the Ipsen Group. We thank
Ms. A. C. Smith for technical assistance.
Supporting Information Available: Spectroscopic data
for 1-3, examples of R2 substituents from commercially
available amines used in the library construction as depicted
in Scheme 1, details of biological assays, and Figure 2 enlarged.
This material is available free of charge via the Internet at
http://pubs.acs.org. Crystallographic data for the 3-O-benzyl
derivative of 1 have been deposited with the Cambridge
Crystallographic Data Centre as CCDC 253309. Copies of the
data can be obtained free of charge on application to CCDC,
12 Union Road, Cambridge CB2 1EZ, U.K. [fax(+44) 1223
336033, e-mail: deposit@ccdc.cam.ac.uk].
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C
33H39ClN2O4, M ) 563.11, λ ) 0.710 73 Å,
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c ) 26.723(3) Å, â ) 92.047(4)°, U ) 1445.0(2) Å3, Z ) 2, Dc )
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)
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JM049045R