Benzopyrans are selective estrogen receptor β agonists with novel activity in models of benign prostatic hyperplasia

Article abstract of DOI:10.1021/jm060491j

Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor β (ERβ) in vitro assays. X-ray crystal structures of SERBA-1 in ERα and β help explain observed β-selectivity of this

Full text of DOI:10.1021/jm060491j

J. Med. Chem. 2006, 49, 6155-6157
6155
Scheme 1a
Benzopyrans Are Selective Estrogen Receptor â
Agonists with Novel Activity in Models of Benign
Prostatic Hyperplasia
Bryan H. Norman,* Jeffrey A. Dodge,
Timothy I. Richardson, Peter S. Borromeo,
Charles W. Lugar, Scott A. Jones, Keyue Chen, Yong Wang,
Gregory L. Durst, Robert J. Barr,
Chahrzad Montrose-Rafizadeh, Harold E. Osborne,
Robert M. Amos, Sherry Guo, Amechand Boodhoo, and
Venkatesh Krishnan
DiscoVery Chemistry Research, Bone and Inflammation Research,
Lead Optimization Biology, Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, Indiana 46285
ReceiVed April 26, 2006
a Reaction conditions: (a) diisopropylethylamine, Tf2O (63%); (b) i.
t-BuLi, ZnCl2; ii. 2, Pd(PPh3)4 (55%); (c) H2, 60 psi Pd/C (75%); (d)
Me(OMe)NMgCl (90%); (e) 4-methoxymethoxyphenyllithium (97%); (f)
i. TsOH, MeOH; ii. NaBH3CN, HCl (45%); (g) chiral chromatography.
Abstract: Benzopyran selective estrogen receptor beta agonist-1
(SERBA-1) shows potent, selective binding and agonist function in
estrogen receptor â (ERâ) in vitro assays. X-ray crystal structures of
SERBA-1 in ERR and â help explain observed â-selectivity of this
ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate
in CD-1 mice (ERâ effect), while having no effect on gonadal hormone
levels (ERR effect) at 10× the efficacious dose, consistent with in vitro
properties of this molecule.
Table 1. In Vitro Properties of SERBA-1 and SERBA-2
SERBA-1
SERBA-2
binding Ki ERR (nM)
binding Ki ERâ (nM)
binding selectivity
2.68 ((0.21)
0.19 ((0.01)
14-fold
14.5 ((6.4)
1.54 ((0.45)
9-fold
Estrogen receptors (ERsa) are part of a superfamily of nuclear
hormone receptors (NHRs) that behave as ligand-activated
transcription factors and operate as part of a complex signaling
network.1 The importance of tissue distribution, coactivator/
corepressor activity, and agonist/antagonist/modulator function
of many NHRs has become an area of current scientific interest
and study.2 Furthermore, with the recent demonstration that
selective ER modulators (SERMs) can have beneficial effects
in important diseases such as osteoporosis and breast cancer,2,3
the desire to better understand NHR ligands has increased.
Ligands which bind to the ER have been studied for many
decades, and it was generally assumed that these ligands bound
to a single ER (now called ERR). In 1996, a second ER subtype
was reported (ERâ).4 Thus, there has been much attention given
toward improving our understanding of these receptors, their
tissue distribution and biological functions.5 While biological
tools, such as ERR and â knockout animals, have provided some
clarity to the physiological roles of both ERs,6 we and others
have focused on the pharmacological effects of subtype specific
ligands.7-9 Toward that end, we report on the characterization
of the selective ERâ agonist, selective estrogen receptor beta
agonist-1 (SERBA-1).
PC3/ERR-ERE EC50 (nM)
% rel. efficacy (R agonist)
PC3/ERâ-ERE EC50 (nM)
% rel. efficacy (â agonist)
FS
19.4 ((1.13)
32.5 ((18.2)
94 ((1.7)
85 ((14)
0.66 ((0.04)
101 ((1.3)
32-folda
3.61 ((1.51)
100 ((10)
11-folda
a Functional selectivity (FS) calculation was weighted using relative
efficacy (RE), that is, FS ) (R EC50)‚(â RE)/(â EC50)‚(R RE).
amide 5, which was used to prepare the aryl ketone 6, via
reaction with the appropriate aryllithium reagent. Deprotection
and cyclization of 6 was accomplished in one pot, via treatment
with toluenesulfonic acid, followed by the addition of sodium
cyanoborohydride under acidic conditions. Careful monitoring
of pH (<4, bromocresol green) favored the all-cis stereochem-
istry, as shown. Chiral chromatography (Chiralpak AD, 80%
heptane-2-propanol) separated the enantiomers to allow for the
isolation of SERBA-1 and SERBA-2, with absolute stereochem-
istries as drawn.12
The in vitro properties of SERBA-1 and SERBA-2 are shown
3
in Table 1. The binding Kis were generated using H-estradiol
and recombinant, full-length, human ERs in a competitive
binding assay. The more potent enantiomer, SERBA-1, showed
potent binding affinity for both ERs, but was 14-fold selective
for the â isoform. The functional activity and selectivity was
measured using a transcription assay in the cotransfected human
prostate cancer PC3/ER (R or â)-ERE cell line. SERBA-1 was
potent (EC50 ) 0.66 nM) and selective (32-fold) toward ERâ
and showed full agonist function in both ERR and ERâ assays
(>90% relative efficacy). We felt that, based on the in vitro
properties of SERBA-1, this molecule may have unique proper-
ties in rodent models for BPH.
Recent studies by Katzenellenbogen et al.,13 Wyeth,14 and
Schering15 have shown the utility of X-ray crystallography,
molecular modeling, and structure-based drug design (SBDD)
in the discovery of ER subtype selective ligands. Although the
ligand binding pockets of these two proteins are very similar
The benzopyran SERBA-1 was prepared as part of an SAR
effort to identify potent and selective agonists of ERâ. The
synthesis, which is outlined in Scheme 1, begins with a
palladium-mediated Negishi cross coupling reaction between
the bis-protected hydroquinone 110 and the cyclopentyl vinyl
triflate 211 to give cyclopentene 3. Hydrogenation of the olefin
allowed us to set the critical cis-relative stereochemistry in the
cyclopentane 4. The methyl ester was converted to the Weinreb
* To whom correspondence should be addressed. Tel.: 317-277-2235.
Fax: norman@lilly.com.
a Abbreviations: ER, estrogen receptor; BPH, benign prostatic hyper-
plasia; NHR, nuclear hormone receptor; SERM, selective estrogen receptor
modulator; SERBA, selective estrogen receptor beta agonist; SBDD,
structure based drug design; SV, seminal vesicle; T, testosterone; DHT,
dihydrotestosterone; DES, diethylstilbestrol.
10.1021/jm060491j CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/02/2006

6156 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Letters
Figure 1. Surface diagram of the X-ray structure of SERBA-1
complexed to ERR (1A) and ERâ (1B).
(only two residue changes), significant binding selectivities have
been obtained using SBDD tools. In general, the approach has
followed a path of utilizing the conservative residue differences
between ERR and ERâ (M421(R) f I373(â) and L384(R) f
M336(â)) by targeting molecular design features that can
specifically exploit these changes. Inherent in this approach is
the fact that most molecules bind similarly in the ERR and ERâ
pockets.
We have recently solved SERBA-1/ERR and ERâ X-ray
cocrystal structures (Figure 1) and report significant differences
in the manner in which these molecules bind within the binding
pockets. The phenols in SERBA-1 bind in a predictable manner
in both structures. That is, one phenol (D ring phenol) is bound
within the glutamic acid-arginine network and the A ring phenol
is bound to the appropriate histidine. However, SERBA-1
displays a different orientation in these structures, which
corresponds to a (ca. 180°) rotation on its bisphenol axis.
Additionally, the A ring phenol, while bound to histidine in
both structures, locates to different sides of the imidazole
functionality for this interaction. This finding is unique and helps
explain the observed selectivity of SERBA-1. It also provides
a unique opportunity for SBDD that is not wholly tied to
exploiting the differences between two conservative residue
changes. We intend to report on our SBDD approach to
improved selectivities in due course.
We have recently developed a mouse model for the purpose
of evaluating ERâ agonist effects on mouse prostate while, at
the same time, measuring ERR-driven risks. It is known that
nonselective ER agonists can have indirect effects on prostate
involution by lowering serum gonadal hormone levels16,17 via
an ERR pathway. These R effects are also displayed in
regression of the testes and seminal vesicle (SV). We felt that
a potent SERBA would demonstrate involution of the prostate
(â effect) at doses that showed no R-related effects on testes
and SV. Additionally, we felt that there should be no effect on
circulating levels of the androgens testosterone (T) and dihy-
drotestosterone (DHT) at these doses. Our mouse prostate model
evaluates each of these endpoints after 7 days of oral dosing,
using intact vehicle, castrate, and finasteride controls.
The mouse efficacy data for SERBA-1 are shown in Figure
2. It should be noted that this molecule produces the desired
effect on prostate weight in a dose-response manner, while
having no effect on testes and SV weights in this dose range.
Additionally, SERBA-1 had no effect on T and DHT levels, at
up to 10× the minimum efficacy dose (0.1 mg/kg). It should
be pointed out that the nonselective ER agonist diethylstilbestrol
(DES), showed significant regression of prostate, testes, and
SV, while also lowering T and DHT (data not shown).18 There
were no doses where the DES effects were displayed only on
prostate tissue. Finally, the BPH drug, finasteride, a 5R-reductase
inhibitor, is known to exert its prostate regression effects by
inhibiting the conversion of T to DHT.19 Finasteride demon-
Figure 2. Effect of SERBA-1 on the prostate wet weight in CD-1
mice, measured after 7 days of oral daily doses. Prostate weights (PW)
were normalized to body weight (BW) and the quotient PW/BW (as a
percentage of intact control) is reported. Castrate and finasteride controls
are also shown. *Statistically significant (P < 0.05).
strated prostate involution in our mouse model, albeit at a
significantly higher dose (30 mg/kg).
In conclusion, we have shown that a potent and selective ERâ
agonist, such as SERBA-1, has unique properties in in vitro
studies and in rodent prostate models. We feel that these data
suggest an opportunity for this class of compound to show
efficacy in human BPH. Additionally, we have demonstrated
the value of SBDD in this benzopyran series and are optimistic
that these tools will allow for significant selectivity improve-
ments, which will be disclosed in subsequent papers.
Acknowledgment. We thank Gregory Stephenson for as-
sistance in the assignments of the absolute stereochemistry of
the enantiomers 7a and 7b.
Supporting Information Available: Atomic coordinates for
ERR and ERâ complexes with SERBA-1 have been deposited in
the PDB, with accession codes 2I0J and 2I0G, respectively.
Experimental details of the synthesis and characterization of
compounds 3-7, in vitro and in vivo assays, and X-ray crystal-
lographic procedures and statistics. This material is available free
of charge via the Internet at http://pubs.acs.org.
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