Synthesis and characterization of iodinated tetrahydroquinolines targeting the G protein-coupled estrogen receptor GPR30

Article abstract of DOI:10.1021/jm9011802

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ERR/β and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta-[c]quinolines exhibited IC₅₀ values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with ¹²⁵I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9011802

1004 J. Med. Chem. 2010, 53, 1004–1014
DOI: 10.1021/jm9011802
Synthesis and Characterization of Iodinated Tetrahydroquinolines Targeting the G Protein-Coupled
Estrogen Receptor GPR30
Chinnasamy Ramesh,† Tapan K. Nayak,‡, Ritwik Burai,† Megan K. Dennis,‡ Helen J. Hathaway,‡,§ Larry A. Sklar,§,^
Eric R. Prossnitz,‡,§ and Jeffrey B. Arterburn*,†,§
†Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003, ‡Department of Cell Biology and
Physiology, School of Medicine, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, §UNM Cancer Center,
University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, College of Pharmacy, University of New Mexico Health
Science Center, Albuquerque, New Mexico 87131, and ^Department of Pathology, School of Medicine, University of New Mexico Health Science
Center, Albuquerque, New Mexico 87131
Received August 7, 2009
A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential
targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and
specificity of binding to GPR30 versus the classical estrogen receptors ERR/β and functional responses
associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta-
[c]quinolines exhibited IC50 values lower than 20 nM in competitive binding studies with GPR30-
expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and
blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of
selected compounds were radiolabeled with 125I using the iodogen method. In vivo biodistribution
studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human
endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal,
and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally
related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the
radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity
of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and
suggests that further optimization of this parameter may lead to improved targeting characteristics.
Introduction
ovarian, and endometrial cancers indicate roles for both
ERR/β and GPR30 intumoregenesis and suggestthe potential
for clinical diagnostic and prognostic applications based on
receptor expression.9,10 The development of drugs that are
capable of differentiating the pharmacology of classical estro-
gen receptors, which have different tissue distribution profiles
and distinct patterns of gene regulation, by selectively mod-
ulating the activity of the individual receptor subtypes ERR/β
is widely recognized as an important strategy for obtaining
improved therapeutics.11,12 Unraveling the pharmacological
profiles and specificities of these three estrogen receptors will
contribute toward understanding the interrelated physiologi-
cal roles of each receptor and facilitate the development of the
next generation of receptor-specific drugs.
Estrogen is a critical hormone that regulates a multitude of
biological processes. The nuclear estrogen hormone receptors
(ERR and ERβ)a are best characterized for their regulation of
gene expression and consequently are important targets in
many disease states that include cancer, skeletal, neurological,
and immunological conditions. New evidence of estrogen’s
role in nongenomic signal transduction pathways has ex-
panded the classical paradigm of hormone function and
suggests corresponding significance for mammalian biology.1,2
The discovery of the G protein-coupled estrogen receptor
GPR30 (IUPHAR designation: GPER), a seven transmem-
brane GPCR, has introduced an entirely new class of receptor
to the milieu of nongenomic and genomic estrogen-mediated
signaling.3-6 Significant overlap exists between the cellular
and physiological aspects of GPR30 function and that of
the classical estrogen receptors,7 as well as in their ligand
specificity and pharmacological profiles.8 Studies with breast,
We combined virtual and biomolecular screening to
identify the first GPR30-selective agonist 1-[4-(6-bromo-
benzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta-
[c]quinolin-8-yl]ethanone (G-1, 1, Figure 1).13 This compound
has found application as a molecular probe for in vitro and in
vivo characterization of GPR30-mediated effects.14-25
A
*To whom correspondence should be addressed. Phone: (575) 646-
2738. Fax: 575 646-2649. E-mail: jarterbu@nmsu.edu.
focused effort including synthetic chemistry and virtual and
biomolecular screening through the New Mexico Molecular
Libraries Screening Center recently provided the complemen-
tary GPR30-selective antagonist 4-(6-bromo-benzo[1,3]dioxol-
5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline (G-15, 2,
Figure 1).26 This compound is capable of blocking cellular
a Abbreviations: PI3K, phosphoinositide 3-kinase; PIP3, phosphati-
dylinositol (3,4,5)-trisphosphate; RFP, red fluorescent protein; PH,
pleckstrin homology domain; ERR, estrogen receptor subtype R; ERβ,
estrogen receptor subtype β; SPECT/CT, single photon emission com-
puted tomography with integrated X-ray computerized tomography
scanner; ID, injected dose.
r
pubs.acs.org/jmc
Published on Web 12/30/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1005
Figure 1. Structures of 17β-estradiol (E2), GPR30-selective
agonist (G-1), and antagonist (G15).
activation by estrogen in cells expressing GPR30 but has no
effect on estrogen-stimulated intracellular calcium mobilization
or nuclear accumulation of PIP3 induced through ERR or ERβ.
The intriguing pair of compounds, which share the tetrahydro-
3H-cyclopenta[c]quinoline scaffold, have the potential to
further investigations of fundamental questions regarding
GPR30 physiology, including assessment of potential clinical
roles for this receptor in disease progression and therapeutic
response.
There remain significant opportunities for further delineat-
ing the individual biological roles of these estrogen receptors
through the application of radiolabeled GPR30-selective
ligands. The commercial availability of [3H]-17β-estradiol
has facilitated the characterization of receptor distribution
and ligand binding of the classical estrogen receptors using
cellular extracts, cell culture, and in vivo models. The devel-
opment of estrogen receptor ligands radiolabeled with posi-
tron- or γ-emitting halogen isotopes for PET and SPECT
imaging applications, as well as potential therapeutic applica-
tions based on estrogen receptor targeting, has been inten-
sively studied over the past 30 years.27-31 Clinical oncologists
have successfully used [18F]FES for staging and visualizing
primary and metastatic carcinomas.32,33 The quantification of
ERR and ERβ levels affords predictive value for determining
outcomes of hormone therapy in breast cancer.34,35 The
development of radiolabeled 17R-iodovinylestradiols has pro-
gressed to clinical assessment of 123I-labeled 11β-methoxyio-
dovinylestradiol for estrogen receptor imaging in breast
cancer.36 Radiolabeled analogues incorporating Auger-emit-
ting isotopes 125I and 123I have potential as therapeutic agents
for estrogen receptor expressing tumors.30,37 The high specific
activity and sensitivity of detection possible using the γ-emit-
ting isotope 125I offer practical advantages for receptor binding
studies in the laboratory and allow efficient determination of
receptor content in tissues and convenient detection and
quantification of images. The development of GPR30-selective
radiotracers would have significant value for characterizing
receptor binding properties and investigations of imaging
applications based on targeting this receptor. The desired
performance characteristics of these agents should include
high selectivity for GPR30, low affinity for ERR/β, effective
receptor-mediated uptake, and retention accompanied by
rapid clearance from nontarget tissue and organs in order to
provide images that directly correspond to GPR30 expression
levels with minimum background detection levels.
Figure 2. Structure of direct and pendent iodo-substituted tetra-
hydro-3H-cyclopenta[c]quinoline derivatives.
antagonists. The corresponding tributylstannane derivatives
were radiolabeled with 125I and used to determine GPR30
receptor binding affinity in cell culture and to evaluate
biodistribution and in vivo imaging in a mouse tumor model.
Results and Discussion
Synthetic Chemistry. We designed a focused series of
iodinated analogues using structure-activity considerations
obtained during our development of the GPR30-selective
agonist and antagonist compounds 1 and 2 (Figure 1).13,26
The compounds illustrated in Figure 2 possess either direct
halogenation of the aryltetrahydro-3H-cyclopenta[c]quinoline
scaffold (3-6) or conjugation of an iodinated appendage
(7-9). The iodinated analogue 3 of 1 was a promising candi-
date because of the relatively minor increase in steric volume
resulting from iodide substitution. The positions 6-8 of the
tetrahydrocyclopenta[c]quinoline scaffold were also identified
as possible sites for introducing halogen substitutions. Series
4-6 have iodo substitutions at positions 6-8 of the 1,2,3,
4-tetrahydroquinoline. The other derivatives (7, 8) contain
iodophenyl appendages connected at the C8 position with a
flexible N-ethylcarboxamide or urea linkages, respectively.
Hydrazone 9 was prepared directly from 1.
The tetrahydro-3H-cyclopenta[c]quinoline scaffold is
synthetically accessible using the three-component Povarov
cyclization.38 We evaluated a series of reaction conditions
employing a variety of protic and Lewis acid catalysts in
order to optimize reaction rate, yield, and diastereoselectiv-
ity for the construction of the compounds in this series. Our
optimized procedure was based on the application of lantha-
nide salts as catalysts for the Povarov cyclization,39 and we
employed Sc(OTf)3 in acetonitrile, which generally provided
rapid reaction times, high product yield, and diastereoselec-
tive formation of the syn- or endo-products (Scheme 1). This
approach provided a direct route for the preparation of the
desired iodides 3-6. In several cases the endo diastereomer
ratio was further enriched by crystallization. All compounds
used in biological studies were evaluated as racemic mixtures
consisting of >95% endo diastereomer.
Herein, we report the synthesis of a series of iodo-substi-
tuted quinoline derivatives 3-9 as selective ligands and
potential targeted imaging agents for GPR30 that meet some
of the performance characteristics outlined above. These
compounds were evaluated against a panel of functional
and competitive ligand binding assays using GPR30 and
ERR/β transfected COS7 as well as Hec50 and SKBr3 cells,
which endogenously express only GPR30, to evaluate recep-
tor selectivity and potential cross-reactivity. Selected deriva-
tives 8 and 9 were demonstrated to be GPR30-selective

1006 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Ramesh et al.
The synthesis of 4 from 1-(4-amino-3-iodophenyl)ethanone,
6-bromopiperonal, and cyclopentadiene was accomplished in
moderate yield as illustrated in Scheme 1. This derivative
possesses an iodide substituent at the 6-position of the tetra-
hydro-3H-cyclopenta[c]quinoline system and retains the
ketone group of 1. Monitoring the reaction revealed that both
the intermediate imine formation and subsequent cyclization
were slow in comparison with the other aniline substrates,
attributed to increased steric hindrance of the o-iodide, and the
yield was not improved by attempted cyclization of the pre-
formed imine. The related iodinated compounds 5 and 6
substituted at the 7- and 8-positions were prepared analogously
from the corresponding m- or p-iodoanilines, respectively. The
derivative 10, possessing a pendant aminoethyl functional
t
group, was prepared using Boc-protected aminophenethyla-
mine45 and was isolated in 95% yield after deprotection with
TFA/CH2Cl2. The amine 10 was coupled with p-iodobenzoic
acid to provide benzamide 7 as shown in Scheme 2. The related
urea 8 was prepared by coupling 10 with m-iodophenyl
isocyanate (Scheme 2).
The synthesis of 9 required 5-iodopyridin-2-ylhydrazine,40
which was prepared by substitution of commercially avail-
able 2-chloro-5-iodopyridine with hydrazine hydrate in pyr-
idine as shown in Scheme 3. The desired hydrazone 9 was
obtained by melting the hydrazine with 1 at 170-180 ꢀC,
followed by preparative reverse phase chromatography.
Assessment of the lipophilicity and solubility was an
additional consideration for selecting possible imaging
agents. The log Po/w values for the selected iodides 3-9 were
calculated and tabulated in Table 1. All seven iodinated
compounds exhibited increased lipophilicity relative to 1.
These log Po/w values paralleled the qualitative experimental
observations of the solubility of the compounds. Compound
7 was poorly soluble in alcohols (MeOH and EtOH), halo-
genated organic solvents (CH2Cl2, CHCl3), and acetonitrile
but was soluble in DMSO with heating. The urea-linked
derivative 8 exhibited a greater range of solubility, including
alcoholic, halogenated solvents, acetonitrile, and DMF. The
hydrazone 9 was soluble in a variety of protic and aprotic
solvents.
Scheme 1a
a Reagents: Sc(OTf)3, CH3CN, 23 ꢀC, 2-5 h.
Scheme 2a
With the iodinated compounds 3-9 in hand, we proceeded
to synthesize the tributyltin-substituted analogues in pre-
paration for radiolabeling experiments.41 The accessibility of
the corresponding stannane and efficiency of subsequent
iodine exchange combined with analysis of receptor binding
and activation properties (vida infra) were factors considered
for the selection of compounds that were advanced for
radiolabeling experiments and in vivo imaging studies.
Compound 3 is a close structural analogue of 1; however,
attempts to prepare the corresponding stannane 12 directly
by palladium-catalyzed exchange of 1 or 3 with hexabutyl-
distannane were unsuccessful (Scheme 4). The benzylic
amine functionality is susceptible to cyclopalladation and
likely results in catalyst degradation.42 An alternative route
a Reagents: (a) PyBOP, iPrNEt2, DMF; (b) Et3N, DMF.
Scheme 3a
a Reagents: (a) H2NNH2, H2O, pyridine, 120 ꢀC, 5 h; (b) 1, 170-
180 ꢀC melt.
Table 1. Binding and Functional Characterization of GPR30-Targeted Compounds
GPR30
binding,
GPR30
agonism
(calcium)b
GPR30
antagonism
(calcium)c
ERR
agonism
(PI3K)d
ERR
antagonism
(PI3K)e
GPR30
agonism
(PI3K)d
GPR30
antagonism
(PI3K)e
ERR
ERβ
compd CLogP bindinga bindinga IC50 (nM)
E2
1
3.37
4.55
4.94
5.82
6.00
6.00
6.71
7.00
6.35
þþþ
-
-
-
-
-
-
-
þ/-
þþþ
-
-
3-6
7
þþþ
þþþ
þþþ
-
-
-
þþþ
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
þþþ
þþþ
þþþ
þ/-
þ/-
-
-
-
-
3
ND
ND
ND
1.7
þ/-
4
þ/-
þ
þ/-
5
-
þ
þ/-
þ/-
þ/-
þþ
þ
-
6
þ/-
þ/-
-
þ
-
7
16.2
8.4
þ
þþ
þ/-
þþþ
þ
8
-
-
9
-
1.7
-
-
a
þ/- denotes that 10 μM compound blocks ∼25% of 10 nM E2-Alexa binding. - denotes <10% inhibition of E2 binding. b þþþ denotes that
calcium mobilization in response to 10 μM compound is 80-100% of reference compound (200 nM E2). þ denotes that calcium mobilization in response
c
to 10 μM compound is 20-40% of reference compound. þþ denotes that 10 μM compound blocks 80-100% of 200 nM E2-induced calcium
mobilization. þ denotes that 10 μM compound blocks 50-60% of E2-induced calcium mobilization. þ/- denotes that 10 μM compound blocks less
than 50% of E2-induced calcium mobilization. d þþþ denotes activation of PI3K similar by 10 μM compound similar to activation induced by 10nM
e
reference compound (E2). þþ and þ/- denote decreased activation of PI3K (qualitative assay). þþþ denotes complete antagonism of 10 nM
E2-induced PI3K activation by 10 μM compound. þ and þ/- denote lesser degrees of antagonism of 10 nM E2-mediated PI3K activation by 10 μM
compound.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1007
Scheme 5a
Scheme 4a
a Reagents: (a) (SnBu3)2, Pd(PPh3)4, dioxane, 100 ꢀC, 16 h; (b)
Sc(OTf)3, CH3CN, 6-bromopiperonal.
Scheme 6a
a Reagents: (a) (SnBu3)2, Pd(PPh3)4, dioxane, 100 ꢀC; (b) Sc(OTf)3,
CH3CN, cyclopentadiene, 4-aminoacetophenone; (c) 4-aminoacetophe-
none, 170-180 ꢀC melt.
to 12 was attempted using the SnBu3-substituted piperonal
11, prepared from 6-bromopiperonal by Pd-catalyzed stan-
nylation; however, the cyclization of 11 was unsuccessful
using either our optimized procedure or various other protic
and Lewis acid catalysts. Attempts to form the intermediate
imine 14 under a variety of forcing conditions were also
unsuccessful, resulting in unreacted 11 and aminoacetophe-
none. The failure to form imine 14 contrasts with the facile
formation of the brominated analogue 13 and can be attrib-
uted to the increased steric demands of the stannyl group of
11 in comparison with 6-bromopiperonal. Attempted stan-
nylation of brominated imine 13 to form 14 was also
unsuccessful.
a Reagents: (a) (SnBu3)2, Pd(PPh3)4, dioxane, 100 ꢀC, 15 h; (b)
Sc(OTf)3, CH3CN, 6-bromopiperonal, 2 h.
Scheme 7a
While the direct Pd-catalyzed tributylstannylation of iodides
4-9 appears feasible, in practice we were unable to effect
this transformation because of the competing reactivity of
the 6-bromobenzo[1,3]dioxol-5-yl discussed previously. The
attempted stannylation of 6 to obtain 15 was unsuccessful;
therefore, an alternative stepwise approach was investigated as
shown in Scheme 5. The Pd-catalyzed exchange of 4-iodoani-
line with (SnBu3)2 to produce 16 required extended reaction
times and heating and proceeded in low yield (∼10%) accom-
panied by unreacted starting material under these conditions,
presumably due to increased electron density from the p-amine
substituent. The attempted cyclization of 16 under a variety of
conditions resulted in poor yields of 15, and significant
amounts of the proto-dehalogenated material were observed.
Preliminary initial iodination experiments of 15 with iodogen
demonstrated poor incorporation of iodide and proteo-
destannylation as the primary competing reaction. The poor
preparative yield of tributylstannane 15 and the subsequent
inefficient iodination of this derivative were recognized as
significant disadvantages for a putative imaging agent. In con-
trast, the three-component cyclization of the m-tributylstanny-
laniline 17 proceeded efficiently to provide 18 (Scheme 6).
This result confirms the positional dependence of substitution
on the aniline ring that was problematic for para-substituted
analogue 16.
a Reagents: (a) 3-iodophenyl isocyanate, CH2Cl2; (b) (SnBu3)2, Pd-
(PPh3)4, dioxane, 100 ꢀC; (c) Sc(OTf)3, CH3CN, 6-bromopiperonal.
the urea 19 (Scheme 7). Palladium catalyzed exchange with
bis(tributyltin) gave the tributyltin derivative 20. Scandium-
(III) catalyzed cyclization of 20 with 6-bromopiperonal and
cyclopentadiene gave the desired product 21 in good yield.
Stannylation of 2-chloro-5-iodopyridine provided an effi-
cient route for the preparation of 2-chloro-5-(tributylstannyl)-
pyridine. This compound was previously synthesized by halogen-
metal exchange with n-butyllithium followed by stannylation
with tributylstannyl chloride.43 Nucleophilic substitution of
chloride with hydrazine gave the desired 2-hydrazinyl-5-(tri-
butylstannyl)pyridine (Scheme 8). Hydrazone formation with 1
occurred upon melting, and the resulting product 22 was
purified by preparative reverse phase chromatography.
Receptor Binding and Functional Properties. The iodinated
compounds 3-9 were evaluated in cell-based binding and
functional assays to determine the potency and type of
signaling response (agonism or antagonism) associated with
The primary aliphatic amine of 4-(2-aminoethyl)aniline
reacted preferentially with m-iodophenyl isocyanate to give

1008 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Ramesh et al.
Scheme 8a
their lack of binding to ERR, compounds 3-8 were unable to
induce either activation of PI3K or inhibit estrogen-induced
PI3K activation in ERR transfected COS7 cells. Compound
9, which exhibited very weak binding to ERR, was also
unable to influence PI3K activity in ERR transfected COS7
cells. Additionally, none of compounds 3-9 were able
to activate PI3K in COS7 cells lacking estrogen receptor
expression. Thus, none of the seven iodinated derivatives
show any functional activity mediated through ERR or
any ability to activate PI3K via other mechanisms. Com-
pound 3 showed strong GPR30-dependent activation
of PI3K as expected for an isostructural analogue of 1.
A regioisomeric dependence of iodide substitution on
GPR30-mediated PI3K activation was observed for com-
pounds 4-7. Compounds 4 and 7 exhibited partial agonist
properties on PI3K, acting as agonists when applied to cells
in the absence of estrogen but also demonstrating the ability
to inhibit the full activation of PI3K by estrogen. Compound
5 was a weaker agonist of PI3K than 3, and compound 6 had
no functional effect. Compounds 8 and 9 were able to block
the estrogen-induced PI3K activation via GPR30 and thus
appear to function as antagonists of GPR30 function in this
context.
Because rapid signaling via GPR30 results in increased
intracellular calcium levels,5 we investigated whether com-
pounds 3-9 had similar agonist and antagonist properties in
a cell-based calcium assay using SKBr3 cells, which endo-
genously express only GPR30. In these cells, stimulation
with GPR30 agonists, including estrogen and 1, elicits a
rapid increase in intracellular calcium levels that can be easily
monitored using the cell-permeant fluorescent calcium in-
dicator Indo-1. Consistent with the PI3K activation profiles,
compounds 8 and 9 blocked estrogen-induced calcium mo-
bilization and thus functioned as antagonists of estrogen-
mediated GPR30 function. Additionally, compound 4 ap-
pears to weakly antagonize estrogen-mediated calcium mo-
bilization. Compounds 3 and 5-7 can all be characterized as
partial agonists in the context of this assay, as all compounds
mobilize calcium when applied alone and all four com-
pounds display some degree of inhibition of estrogen-
mediated calcium mobilization. However, as in the PI3K
assay, compound 3 is a potent agonist of calcium mobiliza-
tion similar to 1.
Some variability in compound profiles exists between the
two GPR30 functional assays (i.e., compound 6 was inactive
in the PI3K assay but a partial agonist of GPR30 in the
calcium assay and compound 3 is a potent agonist in
the PI3K assay and a partial, though still potent, agonist in
the calcium assay). This is potentially due to the different
cellular systems used in the assays and also may be related to
assay sensitivity and the differing time courses of the assays.
The PI3K assay employs ectopically expressed receptors in
cells which may lack required cofactors for full compound
activity; thus, some compounds may elicit weaker or no
responses in this system than in a system in which cells with
endogenous receptor are used, such as the calcium assay used
to profile the compounds. Additionally, the PI3K assay is a
phenotypic assay in which many fields of cells must be
analyzed and an average response determined relative to
control stimulated cells, whereas the calcium assay is a more
quantitative assay in which smaller changes in signaling are
more likely to be observed. Importantly, compounds 8 and 9
displayed consistent activity as antagonists of GPR30 func-
tion in both functional assays.
a Reagents: (a) (SnBu3)2, Pd(PPh3)4, dioxane, 100 ꢀC; (b) H2NNH2,
H2O, pyridine, 120 ꢀC, 24 h ; (c) 1, 170-180 ꢀC melt.
ligand-induced GPR30- and ERR/β-mediated signaling
(summarized in Table 1 with numerical binding data pro-
vided in the Supporting Information). These compounds
were profiled using transfected COS7 cells expressing
GPR30, ERR, or ERβ, in addition to Hec50 and SKBr3
cells, which endogenously express only GPR30. Functional
characterization of the compounds using either the extent (or
inhibition of E2-mediated increase) in intracellular calcium
or the activation of PI3K as measured by production of PIP3
in the nucleus is as previously described.5,13,26 The specificity
of the response in these assays was demonstrated in control
COS7 cells that do not endogenously express GPR30 or
ERR/β. The comparison of the observed ligand-induced
responses across cells expressing individual receptors in
comparison to estrogen-induced responses, in addition to
comparison of differential binding to receptors, provides a
profile of the selectivity/cross-reactivity of the compounds.
We have previously shown the specificity of binding of the
GPR30-selective ligand 1 to GPR30 versus the classical
estrogen receptors ERR/β using cell-based competitive bind-
ing assays.13 In order to characterize the selectivity of
iodinated compounds 3-9, the same cell based assay em-
ploying transfected COS7 cells was used to evaluate binding
of these compounds to the classical estrogen receptors. These
assays confirmed that compounds 3, 5, and 8, like 1, do not
exhibit binding to either of the classical estrogen receptors
ERR/β. Compounds 4, 6, and 7 exhibited minimal binding to
ERβ at high concentrations (blocking less than 35% of E2-
Alexa633 binding when present at 10 μM). Hydrazone 9 was
the only iodinated derivative to show any detectable binding
to ERR, which was also minimal. These low levels of
competition at 10 μM are compared to an IC50 for estrogen
in the same assay of approximately 0.4 nM.13
Cell-based functional assays for compounds 3-9 were
carried out in parallel to binding studies in order to assess
the functional characteristics of this series of compounds.
Stimulation of cells expressing ERR or GPR30 results in the
receptor-dependent activation of PI3K which can be visua-
lized using the pleckstrin homology (PH)-domain of Akt
fused to RFP (PH-RFP), which serves as a reporter of PIP3
localization. Receptor activation leads to the translocation
of this reporter from a cytoplasmic or plasma membrane-
associated localization to the nucleus.5,13 We evaluated the
ability of compounds 3-9 alone to induce PI3K activation or
the ability of these compounds to block estrogen-stimulated
PI3K activation to determine the agonist and antagonist
properties of each compound in transfected COS7 cells
expressing ERR or GPR30; additionally COS7 cells trans-
fected with PH-RFP reporter alone were treated with all
compounds to confirm that the results in receptor-trans-
fected cells were receptor-dependent. As expected because of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1009
Table 2. In Vivo Biodistribution (% ID/g) of GPR30-Targeted Radioiodinated Tracer
1 h PI
2 h PI
2 h PI (block)a
organ
heart
blood
8*
9*
8*
9*
8*
9*
2.03 ( 0.35
3.18 ( 0.45
2.51 ( 0.14
6.31 ( 0.81
2.65 ( 0.96
26.12 ( 5.53
9.56 ( 4.90
4.51 ( 1.75
6.76 ( 2.71
2.15 ( 0.14
0.76 ( 0.03
1.14 ( 0.56
1.42 ( 0.25
1.35 ( 0.26
0.14 ( 0.02
1.72 ( 0.86
0.99 ( 0.03
5.23 ( 0.52
14.29 ( 1.25
7.97 ( 1.04
7.01 ( 1.46
5.49 ( 0.31
8.04 ( 1.83
9.86 ( 0.86
24.04 ( 7.39
7.05 ( 0.42
6.24 ( 1.04
1.86 ( 0.26
1.36 ( 0.08
5.33 ( 0.64
5.01 ( 0.63
0.52 ( 0.27
7.03 ( 1.01
4.41 ( 0.25
1.36 ( 0.09
3.14 ( 0.40
2.63 ( 0.51
5.88 ( 0.18
2.08 ( 0.21
8.14 ( 0.87
3.83 ( 0.87
5.42 ( 2.38
1.90 ( 0.13
1.70 ( 0.13
0.48 ( 0.09
0.52 ( 0.05
2.83 ( 0.51
2.04 ( 0.10
0.10 ( 0.04
0.43 ( 0.10
1.08 ( 0.15
3.52 ( 0.09
13.02 ( 2.42
7.32 ( 1.75
4.83 ( 0.42
5.50 ( 1.35
5.40 ( 0.50
9.76 ( 2.04
36.35 ( 4.24
5.52 ( 0.52
5.44 ( 0.40
2.72 ( 0.25
1.30 ( 0.14
3.55 ( 0.33
3.33 ( 0.26
0.41 ( 0.10
4.00 ( 1.10
3.47 ( 0.17
1.50 ( 0.36
2.23 ( 0.43
1.77 ( 1.19
6.74 ( 0.60
2.22 ( 0.52
4.99 ( 0.85
2.86 ( 1.00
3.84 ( 1.72
3.60 ( 0.62
0.99 ( 0.11
0.69 ( 0.14
0.70 ( 0.07
0.67 ( 0.23
0.63 ( 0.15
0.08 ( 0.07
0.26 ( 0.07
0.56 ( 0.08
3.72 ( 1.19
9.01 ( 0.56
5.21 ( 1.30
5.20 ( 1.94
5.94 ( 1.30
9.11 ( 2.48
7.82 ( 4.19
29.99 ( 3.34
5.30 ( 1.76
3.25 ( 0.52
2.03 ( 0.75
1.42 ( 0.58
1.67 ( 0.10
2.23 ( 0.61
0.64 ( 0.15
5.60 ( 1.80
1.98 ( 0.50
lung
liver
spleen
large intestine
small intestine
stomach
kidney
adrenalb
bone
muscle
uterusb
mammaryb
brain
urinary bladder
tumorb
a Receptor blocking studies performed by co-injecting 5 μg of 1 with the radiotracer. b Uptake values at 2 h PI are significantly different (p < 0.05)
from the values at 2 h PI when the radiotracer was co-injected with 5 μg of 1 for blocking the receptor.
125I Radiolabeling and GPR30 Binding. The iodides 8 and 9
were selected for radiolabeling based on the results of the
binding and functional assays combined with considerations
of their chemical properties and synthetic accessibility of the
tributylstannylated precursors. Although compound 3 ex-
hibited a favorable GPR30-selective binding and functional
profile, efforts to prepare the tributylstannyl precursor 12
were unsuccessful. The tributylstannyl precursor, N-{2-[4-(6-
bromo-benzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclo-
penta[c]quinolin-8-yl]ethyl}-4-tributylstannylbenzamide for
7 was synthesized but not selected for 125I labeling because of
the poor solubility characteristics. Radioiodinated 8* and 9*
were prepared using the iodogen method from tributylstan-
nyl precursors 21 and 22, respectively. A 1:1 stoichiometry of
125I/:tributylstannyl precursor was used for radiolabeling
and greatly simplified the purification process by avoiding
the need to separate excess precursor. Typical radiochemical
yields ranged from 40% to 55%. Over 99.9% unincorpo-
rated 125I and unlabeled ligands were successfully separated
from the radioiodinated 8* and 9* using solid phase
extraction as determined by thin layer chromatography.
The experimental log Po/w values of 7.0 ( 0.3 and 6.0 (
0.2 for 8* and 9* determined by the shake-tube method were
similar to the corresponding calculated values of 7.00
and 6.35 and followed the same relative ordering of lipophi-
licity 8 > 9.
radioiodinated 8* and 9*. Both the radiotracers 8* and 9*
were rapidly metabolized and eliminated via the hepatobili-
ary system (gall bladder uptake of over 30% ID/g). Radio-
tracer 8* was rapidly cleared from the intestines, kidney, and
urinary bladder (Table 2).
However, increased uptake and retention was observed in
the uterus, adrenal and mammary glands, and tumor, in-
dicative of receptor-mediated uptake. Blocking studies also
revealed GPR30-mediated uptake of 8* in tumor, adrenal,
and reproductive organs, sites of known or expected GPR30
expression. Radiotracer 9* demonstrated a significantly
different pharmacokinetic profile than radiotracer 8*
(Table 2). The blood uptake values of 9* were nearly 3-fold
that of 8* at 1 h PI (14.3% ID/g vs 3.2% ID/g), indicative of
high plasma-protein binding. However, blocking studies also
revealed GPR30-mediated uptake of 9* in tumor, adrenal,
and reproductive organs. In spite of receptor-mediated
uptake, the tumor was not visualized by a whole body 80 s/
projections SPECT/CT studies due to poor targeting char-
acteristics of both the radiotracers (Supporting Information,
S10). Quantitative SPECT/CT studies revealed further dis-
parities between 8* and 9*. The thyroid uptake of 9* was
almost 7 times greater than that of 8* (4.1% ID vs 0.6% ID).
Whereas the gall bladder (with bile content) uptake of 9* was
nearly 3 times lower than that of 8* (1.8% ID vs 5.7% ID).
For 8*, the organ with the largest uptake was intestine (over
70% ID), whereas for 9* it was the stomach (over 15% ID).
The imaging studies suggest that 8* is less susceptible to in
vivo dehalogenation compared to 9* but was also more
susceptible to metabolism because of increased gall bladder
activity. The moderate increase in kidney uptake of com-
pound 8* upon coadministration of excess 1 could be due
changes in metabolism or excretion kinetics in the presence
of large doses of blocking agent. The high stomach uptake of
9* could presumably be associated with the presence of free
iodide, which would also correspond with the high thyroid
levels observed. The blocking studies using the structurally
related agonist 1 verified the critical involvement of GPR30-
mediated uptake of the radiotracers; however, the corres-
ponding displacement by E2 was not characterized. This
represents a limitation of the present study, since endogen-
ous estrogens constitute the dominant competing ligands in
Competition binding studies were performed on adherent
monolayers of GPR30-expressing human endometrial
Hec50 cancer cells. The IC50 values of 6, 7, 8, and 9 were
1.7, 16.2, 8.4, and 1.7 nM, respectively (Table 1). This
binding assay was verified by evaluating the receptor binding
affinity for the GPR30 agonist 1 (IC50 ≈ 7 nM), which
compares similarly to the value obtained using a competitive
binding assay with a fluorescent estrogen ligand and recom-
binant GPR30 (IC50 ≈ 11 nM),13 and reported values for
17β-estradiol (IC50 ≈ 3-6 nM).5,6 The recently described
GPR30 antagonist 2 exhibited slightly weaker binding
(IC50 ≈ 20 nM) relative to 1.26
In Vivo Studies. In vivo biodistribution studies were
performed on female ovariectomized athymic (NCr) nu/nu
mice bearing GPR30-expressing human endometrial
Hec50 tumors by intravenously injecting trace amounts of

1010 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Ramesh et al.
intact (not ovariectomized) animals and, although challen-
ging to address experimentally, should be incorporated into
the evaluation of next generation agents that exhibit im-
proved targeting characteristics.
water-methanol mobile phase. NMR spectra were acquired
at ambient temperatures (18 ( 2 ꢀC) unless otherwise noted.
Reactions were monitored by thin-layer chromatography on
˚
silica gel (60 A pore size, 5-17 μm) polyester backed sheets that
were visualized under a UV lamp, iodine vapor, phosphomo-
1
lybdic acid, or anisaldehyde. The H NMR spectra in CDCl3
were referenced to TMS unless otherwise noted. The 13C {1H}
NMR spectra were recorded at 75 or 100 MHz and referenced
relative to the 13C {1H} peaks of the solvent. Spectra are
reported as follows: shift (ppm), multiplicity, coupling constants
(Hz), and number of protons. Melting points are uncorrected.
The purity of all compounds used in biological studies was
determined to be >95% by analytical HPLC equipped with
photodiode array (PDA) and ESI-MS detection. The com-
pounds (1 mg/mL CH3CN, 20 μL) were injected into a Waters
Symmetry C18 5 μm, 3.0 mm ꢀ 150 mm column and eluted as
specified. Reported binding and functional characterization
values are based on a minimum of three measurements.
General Procedure for Sc(OTf)3 Mediated Cyclization. A
catalytic amount of Sc(OTf)3 (10 mol %) in anhydrous acetonitrile
(0.5 mL) was added to a mixture of 6-bromopiperonal (1 equiv),
aniline derivative (1 equiv), and cyclopentadiene (5 equiv) in
acetonitrile (4 mL). The reaction mixture was stirred at ambient
temperature (∼23 ꢀC) for 2-5 h with monitoring of the product
formation by thin layer chromatography using ethyl acetate/
hexanes as eluent. The volatiles were removed in vacuo. The crude
product was purified as specified.
1-[4-(6-Iodobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclo-
penta[c]quinolin-8-yl]ethanone (3). According to the general proce-
dure, 6-iodopiperonal (0.055 g, 0.2 mmol), p-aminoacetophenone
(0.027 g, 0.2 mmol), cyclopentadiene (0.066 g, 1.0 mmol), and
Sc(OTf)3 (0.010 g, 0.5 mmol) were combined with a reaction time
of 2 h. The volatiles were removed in vacuo. The residue was
purified by preparative silica gel column chromatography using
ethyl acetate/hexanes (10:90) to provide 3 (0.085 g, 92%) as a
colorless solid. Mp 106-109 ꢀC; IR (KBr, cm-1) 3320, 2880, 1665,
1575, 1242; 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 1.9 Hz,
1H), 7.62 (dd, J = 8.4, 1.9 Hz, 1H), 7.28 (s, 1H), 7.07(s, 1H), 6.61
(d, J = 8.4 Hz, 1H), 6.01 (d, J = 1.1 Hz, 1H), 5.99 (d, J = 1.1 Hz,
1H), 5.96-5.94 (m, 1H), 5.68-5.66 (m, 1H), 4.82 (d, J = 3.1 Hz,
1H), 4.14 (d, J = 8.8 Hz, 1H), 4.06 (bs, 1H), 3.19-3.13 (m, 1H),
2.56-2.44 (m, 4H), 1.83-1.79 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 196.5, 149.8, 148.6, 147.8, 136.3, 133.7, 130.4, 130.0,
128.6, 127.6, 125.1, 118.9, 115.2, 107.9, 101.7, 60.8, 45.3, 42.2, 31.2,
26.0. HPLC-MS: Elution with 60-90% CH3CN (gradient 1.5%
min-1) in H2O exhibited a single peak at 12.38 min. ESI-MS m/z
[ESþ] calcd for C21H18INO3 [M þ H]þ 460.03; found 460.09.
1-(4-(6-Bromobenzo[d][1,3]dioxol-5-yl)-6-iodo-3a,4,5,9b-tetr-
ahydro-3H-cyclopenta[c]quinolin-8-yl)ethanone (4). According
to the general procedure 6-bromopiperonal (0.115 g, 0.5 mmol),
1-(4-amino-3-iodophenyl)ethanone (0.140 g, 0.53 mmol),
cyclopentadiene (0.165 g, 2.5 mmol), and Sc(OTf)3 (0.0246 g,
0.5 mmol) were combined with a reaction time of 3 h. The
volatiles were removed in vacuo. The residue was purified by
preparative silica gel column chromatography using ethyl
acetate/hexanes (08:92) to provide 4 (0.151 g, 56%) as a white
solid. Mp 110-114 ꢀC; IR (KBr, cm-1) 3447, 2920, 1670, 1588,
1476, 1242; 1H NMR (300 MHz, CDCl3) δ 8.11 (d, J = 2.0 Hz,
1H), 7.65 (d, J = 2.0 Hz, 1H), 7.14 (s, 1H), 7.06 (s, 1H), 6.03 (d,
J = 1.3 Hz, 1H), 6.02 (d, J = 1.3 Hz, 1H), 5.92-5.88 (m, 1H),
5.68-5.66 (m, 1H), 4.99 (d, J = 3.5 Hz, 1H), 4.53 (bs, 1 H), 4.14
(d, J = 8.2 Hz, 1H), 3.25-3.19(m, 1H), 2.52-2.42 (m, 4H),
1.87-1.77 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 195.1, 148.9,
147.7, 147.6, 137.3, 133.4, 132.9, 130.7, 129.6, 129.6, 125.6,
113.0, 107.4, 101.8, 85.4, 77.2, 56.6, 45.9, 42.3, 31.4, 25.9.
HPLC-MS: Elution with 60-90% CH3CN in H2O (gradient
1.5% min-1) exhibited a single peak at 17.40 min. HRMS: calcd
for C21H17BrINO3 [M þ H]þ 537.9515; found 537.9525.
4-(6-Bromobenzo[1,3]dioxol-5-yl)-7-iodo-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinoline (5). According to the general procedure
Conclusions
In this study, we developed a series of iodinated compounds
that exhibit high affinity and selectivity toward GPR30 versus
the classical estrogen receptors. The synthetic compounds
were characterized using cell-based competitive binding and
functional assays to identify binding affinity, specificity, and
agonist or antagonist response associated with receptor-
mediated signaling pathways. Two compounds incorporating
either conjugated phenylurea or hydrazone linkages, respec-
tively, were selected for radiolabeling with 125I to provide 8*
and 9*. These compounds functioned as antagonists of
GPR30-mediated calcium mobilization and were used for
competitive ligand binding assays in cell culture. Comparative
in vivo studies were used to evaluate the biodistribution,
pharmacokinetics, and the potential for GPR30-targeted
tumor imaging in an animal model. Although both radio-
tracers 8* and 9* exhibited receptor-mediated uptake in
tumor, adrenal, and reproductive organs, neither was effective
for tumor imaging due to poor targeting characteristics, high
background, and issues with plasma protein binding and
rapid metabolism. The striking differences in the in vivo
characteristics of 8* and 9* reflect the structural, physico-
chemical, and metabolic differences associated with the iodi-
nated appendages used. Dehalogenation, high plasma-protein
binding, and uptake in the stomach were problematic for the
m-iodophenylurea 9*. The iodopyridin-2-ylhydrazone 8* was
less susceptible to dehalogenation but exhibited increased
metabolism and high uptake in the intestine. Both compound
classes were significantly more lipophilic than either estradiol
or the GPR30-selective probes 1 and 2, which likely contri-
butes toward the observed nontarget tissue uptake. The in
vivo data from 8* and 9* provide valuable insight for the
design of the next generation of GPR30-targeted radiotracers.
The iodide 3 exhibited favorable characteristics including
reduced lipophilicity and high selectivity as a GPR30 agonist;
however, the inaccessibility of the tributylstannyl precursor
through the synthetic routes investigated prevented further
evaluation of the radioiodinated compound. It may be possi-
ble to incorporate the less sterically demanding trimethylstan-
nyl group, which could serve as a precursor for 125I labeling to
yield this promising agent.44 We have previously described
99mTc-labeled 17β-estradiol derivatives that incorporate
pyridin-2-yl-hydrazine chelates for SPECT imaging applica-
tions.45,46 These studies suggest that incorporating this
99m
Tc-imaging modality into the tetrahydrocyclopenta[c]-
quinoline scaffold could result in GPR30-selective agents with
decreased lipophilicity, increased chemical and metabolic
stability, reduced plasma-protein binding, and overall im-
provements in targeting characteristics for in vivo imaging
applications.
Experimental Section
General Methods. Reagents and solvents were obtained from
commercial sources and used without further purification.
Chromatographic separations were performed using medium
pressure flash chromatography and ethyl acetate/hexanes or
methanol/dichloromethane as eluent. Reverse phase chroma-
tography employed C-18 columns and water-acetonitrile or

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1011
6-bromopiperonal (0.229 g, 1 mmol), m-iodoaniline (0.219 g, 1
mmol), cyclopentadiene (0.330 g, 5 mmol), and Sc(OTf)3 (0.0492 g,
0.1 mmol) were combined with a reaction time of 2 h. The volatiles
were removed in vacuo. The residue was purified by preparative
silica gel column chromatography using ethyl acetate/hexanes
(10:90) to provide 5 (0.354 g, 72%) as a white solid. Mp
chromatography using ethyl acetate/hexanes (25:75) to provide
8 (0.09 g, 67%) as a white solid. Mp 210-213 ꢀC; IR (KBr,
cm-1) 3341, 2870, 1636, 1580, 1474; 1H NMR (300 MHz,
DMSO-d6) δ 8.60 (s, 1H), 7.98-7.97(m, 1H), 7.25-7.20 (m,
3H), 7.14 (s, 1H), 7.01-6.96 (m, 1H), 6.85 (s, 1H), 6.76 (dd, J =
8.2, 1.76 Hz, 1H), 6.65 (d J = 8.1, 1H), 6.11-6.05 (m, 3H),
5.87-5.83 (m, 1H), 5.58-5.53(m, 1H), 5.45 (bs, 1H), 4.63 (d, J
= 2.8 Hz, 1H), 3.97 (d, J = 8.8 Hz, 1H), 3.28-3.22 (m, 2H),
3.05-2.95 (m, 1H), 2.62-2.54 (m, 2H), 2.45-2.40 (m, 1H),
1.69-1.60 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 154.7,
147.0, 146.9, 144.3, 142.0, 134.6, 134.3, 130.58, 129.4, 129.3,
128.6, 126.1, 125.6, 124.9, 116.7, 116.1, 112.3, 112.1, 108.5,
101.8, 94.7, 56.0, 45.5, 41.8, 40.8, 40.3, 35.0, 31.1. HPLC-MS:
Elution with 63-93% CH3CN in H2O containing 0.01% formic
acid (gradient 1% min-1) exhibited a two peaks at tR = 26.40
min (exo) and 27.88 min (endo). HRMS: calcd for C28H25BrI-
N3O3 [M þ H]þ 658.0202; found 658.0193.
1
195-198 ꢀC; IR (KBr, cm-1) 3432, 2881, 1573, 1496, 1217; H
NMR (300 MHz, CDCl3) δ 7.01 (s, 1H), 7.05 (dd, J = 8.0, 2.0 Hz,
1H), 7.03 (s, 1H), 6.97 (d, J = 2.0 Hz, 1H), 6.77 (d, J = 8.0 Hz,
1H), 6.00 (d, J = 1.5 Hz, 1H), 5.98 (d, J = 1.5 Hz, 1H), 5.83-5.79
(m, 1H), 5.69-5.64 (m, 1H), 4.87 (d, J = 3.3 Hz, 1H), 4.03 (d, J =
9 Hz, 1H), 3.55 (bs, 1H), 3.21-3.09 (m, 1H), 2.58-2.49 (m, 1H),
1.84-1.75 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 147.5, 147.3,
146.8, 133.9, 133.4, 130.6, 130.5, 128.1, 125.7, 124.4, 113.0, 112.9,
107.8, 101.7, 101.7, 90.9, 56.3, 45.6, 40.9, 31.3. HPLC-MS:
Elution with 73:27 CH3CN/H2O containing 0.01% formic acid
exhibited a single peak at tR = 22.38 min. HRMS: calcd for
C19H15BrINO2 [M þ H]þ 495.9409; found 495.9412.
N-{1-[4-(6-Bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinolin-8-yl]ethylidene}-N0-(5-iodopyridin-2-yl)-
hydrazine (9). The (5-iodopyridin-2-yl)hydrazine (0.047 g, 0.2
mmol) was heated with 1 (0.082 g, 0.2 mmol) at 170-180 ꢀC for
5 min in vacuo (2 mmHg). The crude hydrazone was purified by
reverse phase column chromatography using acetonitrile/water
(75:25) to provide 9 (0.101 g, 85%) as a pale-yellow solid. Mp
4-(6-Bromobenzo[1,3]dioxol-5-yl)-8-iodo-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinoline (6). According to the general proce-
dure, 6-bromopiperonal (0.460 g, 2 mmol), p-iodoaniline (0.438 g,
2 mmol), cyclopentadiene (0.66 g, 10 mmol), and Sc(OTf)3
(0.098 g, 0.2 mmol, 10 mol %) were combined with a reaction
time of 2 h. The product 6 (0.890 g, 90%) was isolated by filtration
as a white solid. Mp 175-178 ꢀC; IR (KBr, cm-1) 3435, 2887,
1588, 1481, 1239; 1H NMR (300 MHz, CDCl3) δ 7.33 (d, J = 2.0
Hz, 1H), 7.23 (dd, J = 8.4, 2.0 Hz, 1H), 7.11 (s, 1H), 7.02 (s, 1H),
6.38 (d, J = 8.4 Hz, 1H), 5.99 (d, J = 1.2 Hz, 1H), 5.98 (d, J = 1.2
Hz, 1H), 5.84-5.80 (m, 1H), 5.68-5.66 (m, 1H), 4.85 (d, J = 3.3
Hz, 1H), 4.05 (d, J = 9.0 Hz, 1H), 3.5 (bs, 1H), 3.20-3.09 (m, 1H),
2.59-2.48 (m, 1H), 1.84-1.74 (m, 1H); 13C NMR (75 MHz,
CDCl3) δ 147.5, 147.3, 145.0, 137.5, 134.3, 134.0, 133.4, 130.7,
128.8, 118.5, 113.0, 112.9, 107.9, 101.7, 80.5, 56.4, 45.7, 41.9, 31.3.
HPLC-MS: Elution with 73:27 CH3CN/H2O containing 0.01%
formic acid exhibited a single peak at tR = 22.10 min. HRMS:
calcd for C19H15BrINO2 [M þ H]þ 495.9409; found 495.9407.
N-{2-[4-(6-Bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinolin-8-yl]ethyl}-4-iodobenzamide (7). The
amine 10 (0.082 g, 0.2 mmol) and 4-iodobenzoic acid (0.049 g,
0.2 mmol) were combined in dry DMF (1 mL). Disopropylethyl-
amine (0.025g, 0.2 mmol) was added, followed by the addition of
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate (0.103 g, 0.2 mmol) and allowed to stir at ambient
temperature for 20 h. The precipitate formed was filtered and
washed with water and dried in vacuo to provide 10 (0.100 g,
78%) as a white solid. Mp 217-220 ꢀC; IR (KBr, cm-1) 3338,
1631, 1481, 1244, 1038; 1H NMR (300 MHz, DMSO-d6) δ 8.58
(t, J = 5.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz,
2H), 7.24 (s, 1H), 7.14(s, 1H), 6.84 (m, 1H), 6.77 (dd, J = 8.1, 1.7
Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 6.09 (s, 1H), 6.07 (s, 1H),
5.82-5.77 (m, 1H), 5.58-5.55 (m, 1H), 5.47 (bs, 1H), 4.62 (d,
J = 3.0 Hz, 1H), 3.96 (d, J = 8.9 Hz, 1H), 3.34-3.38 (m, 2H),
3.04-2.96 (m, 1H), 2,67 (t, J = 7.3 Hz, 2H), 2.45-2.38 (m, 1H),
1.68-1.60 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 165.3,
147.0, 146.9, 144.3, 137.5, 137.0, 134.3, 134.0, 131.0, 129.4,
129.1, 128.7, 126.1, 124.8, 116.1, 112.3, 112.1, 108.5, 101.8,
98.5, 56.0, 45.54, 41.8, 41.3, 34.3, 31.1. HPLC-MS: Elution
with 60-90% CH3CN in H2O (gradient 1.5% min-1) exhibited
a single peak at 15.98 min. HRMS: calcd for C28H24BrIN2O3
[M þ H]þ 664.9913; found 664.9916.
1
128-131 ꢀC. IR (KBr, cm-1) 3351, 2919, 1611, 1578, 1493; H
NMR (300 MHz, CDCl3) δ 8.26 (d, J = 1.8 Hz, 1H), 7.98 (s, 1H),
7.79 (dd, J = 8.8, 2.0 Hz, 1H), 7.43-7.40 (m, 2H), 7.24-7.18 (m,
1H), 7.13 (s, 1H), 7.02 (s, 1H), 6.61 (d, J = 8.2 Hz, 1H), 5.98 (d,
J = 1.3 Hz, 1H), 5.97 (d, J = 1.3 Hz, 1H) 5.93-5.899 (m, 1H),
5.68-5.63 (m, 1H), 4.89 (d, J = 3.1 Hz, 1H), 4.13(d, J = 8.8 Hz,
1H), 3.69 (s, 1H), 3.23-3.16 (m, 1H), 2.60-2.51 (m, 1H), 2.16 (s,
3 H), 1.84-1.76 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 156.2,
153.0, 147.4, 147.2, 146.0, 145.5, 144.5, 134.1, 133.9, 130.3, 129.7,
126.5, 125.6, 124.1, 115.9, 113.0, 112.8, 109.8, 107.9, 101.7, 79.2,
56.5, 45.9, 42.1, 31.3, 12.3. HPLC-MS: Elution with 73:27
CH3CN/H2O containing 0.01% formic acid exhibited two peaks
at tR = 17.18 min (exo) and 22.33 min (endo). HRMS: calcd for
C26H22BrIN4O2 [M þ H]þ 629.0049; found 628.9939.
1-[2-(4-Aminophenyl)ethyl]-3-(3-iodophenyl)urea (19). A solu-
tion of 3-iodophenyl isocyanate (0.490 g, 2.0 mmol) in dry
dichloromethane (10 mL) was added to 4-aminophenethyla-
mine (0.272 g, 2.0 mmol) in an ice bath and subsequently allowed
to stir for 2 h at ambient temperature. The white solid precipitate
was separated by centrifugation and dried in vacuo to provide 19
(0.703 g, 92%) as a colorless solid. Mp 161-163 ꢀC; IR
(KBr, cm-1) 3312, 1628, 1577, 1516, 1472; 1H NMR (300 MHz,
DMSO-d6) δ 8.60 (s, 1H), 7.96 (t, J = 1.76 Hz, 1H), 7.24-7.18 (m,
2H), 6.98 (t, J = 8.0 Hz, 1H), 6.86 (d, J = 8.3 Hz, 2H), 6.50 (d, J =
8.3 Hz, 2H), 6.09-6.05 (m, 1H), 4.84 (s, 2H), 3.25-3.18 (m, 2H),
2,54 (t, J = 7.19 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) δ
154.7, 147.7, 142.0, 130.6, 129.3, 128.9, 126.1, 125.5, 116.7, 114.0,
94.7.
1-[2-(4-Aminophenyl)ethyl]-3-(3-tributylstannanylphenyl)urea
(20). A mixture of 1-[2-(4-aminophenyl)ethyl]-3-(3-iodophe-
nyl)urea (0.381 g, 1 mmol), bistributyltin (1.16 g, 2 mmol), and
tetrakis(triphenylphosphine)palladium (0.115 g, 0.10 mmol) was
refluxed in dry dioxane at 100 ꢀC for 3 h under argon. The
volatiles were removed in vacuo. The residue was purified by
preparative silica gel column chromatography using ethyl acet-
ate/hexanes (40:60) to provide 20 (0.331 g, 61%) as a colorless
liquid. IR (KBr, cm-1) 3410, 2920, 2912, 1632, 1238; 1H NMR
(300 MHz, CDCl3) δ 7.30 (d, J = 1.4 Hz, 1H), 7.23-7.20 (m, 2H),
7.14-7.12(m, 1H), 6.92 (d, J = 8.3 Hz, 2H), 6.80 (s, 1H), 6.56 (d,
J = 8.3 Hz, 2H), 5.13 (t, J = 5.7 Hz, 1H), 3.48 (bs, 2H), 3.37 (q,
J = 6.0 Hz, 2H), 2.65 (t, J = 7.0 Hz, 2H), 1.56-1.46 (m, 6H),
1.36-1.24 (m, 6H). 1.06-1.00 (m, 6H), 0.86 (t, J = 7.4 Hz, 9H);
13C NMR (75 MHz, CDCl3) δ 156.0, 144.6, 143.1, 138.1, 131.6,
129.5, 128.8, 128.5, 128.4, 120.7, 115.3, 41.6, 35.3, 29.1, 27.3, 13.6,
9.5. HPLC-MS: Elution with 60-90% CH3CN in H2O
(gradient 1% min-1) exhibited a single peak at tR = 24.60 min.
1-{2-[4-(6-Bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinolin-8-yl]ethyl}-3-(3-iodophenyl)urea (8). A
solution of 3-iodophenyl isocyanate (0.10 g, 0.4 mmol) in dry
DMF (1 mL) was added to the amine 10 (0.082 g, 0.2 mmol) and
Et3N (0.02 g, 0.2 mmol) in dry DMF (1 mL) in an ice bath and
allowed to stir overnight at ambient temperature. The reaction
mixture washed with water (15 mL), and the product was
extracted with methylene chloride (30 mL). The organic layer
was dried over anhydrous sodium sulfate and evaporated under
reduced pressure. The residue was purified by silica gel column

1012 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Ramesh et al.
ESI-MS m/z (ESþ) calcd for C27H43N3OSn [M þ Na]þ 568.23;
found 568.09.
using a reverse phase C18 Sep-pak column (Waters, Milford,
MA). Unreacted 125I and unlabeled ligand were eluted in
distilled water (weak solvent), and ethanol was used to extract
radioiodinated 8* and 9*. To assess the iodination efficiency,
thin-layer chromatography (TLC) was performed using silica
gel strips (Gelman Sciences, Inc., Ann Arbor, MI). Ethanol and
0.9% (w/v) sodium chloride solutions were used as organic and
aqueous mobile phases, respectively. The developed strips were
scanned using a thin-layer-chromatography-imaging scanner
(AR-2000, BioScan, Inc., Washington, DC). Ethanol was eva-
porated using nitrogen gas to obtain concentrated solutions of
radioiodinated 8* and 9*. The log Po/w values were experimen-
tally determined using the shake-tube method as previously
described.45 For biological experiments, the concentrated etha-
nol solution was reconstituted in aqueous solutions containing
0.1% albumin, 0.1% Tween-20, and 10% ethanol.
(1-{2-[4-(6-Bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinolin-8-yl]ethyl}-3-(3-tributylstannylphenyl)-
urea) (21). According to the general procedure, 6-bromopiperonal
(0.034 g, 0.147 mmol), 20 (0.080 g, 0.147 mmol), cyclopentadiene
(0.048 g, 0.735 mmol), and Sc(OTf)3 (0.007 g, 0.014 mmol) were
combined with a reaction time of 8 h. The volatiles were removed
in vacuo. The residue was purified by preparative silica gel column
chromatography using ethyl acetate/hexanes (35:65) to provide 21
(0.090 g, 75%) as a white solid. Mp 125-127 ꢀC; IR (KBr, cm-1):
3336, 2955, 2924, 1635, 1564, 1241; 1H NMR(300MHz, CDCl3) δ
7.25-7.16 (m, 6H), 7.02 (s, 1H), 6.68(s, 1H), 6.80 (dd, J = 7.7, 1.4
Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.22 (bs, 1H), 5.99 (d, J = 1.3
Hz, 1H), 5.98 (d, J = 1.3 Hz, 1H), 5.82-5.79 (m, 1H), 5.66-5.60
(m, 1H), 4.84 (d, J = 2.8 Hz, 1H), 4.78 (bs, 1H), 4.05 (d, J = 8.2
Hz, 1H), 3.47 (q, J = 5.8 Hz, 2H), 3.21-3.10 (m, 1H), 2.71 (t, J =
6.9 Hz, 2H), 2.60-2.51 (m, 1H), 1.83-1.74 (m, 1H), 1.57-1.47
(m, 6H), 1.37-1.25 (m, 6H), 1.06-1.01 (m, 6H), 0.87 (t, J = 7.4
Hz, 9H); 13C NMR (75 MHz, CDCl3) δ 155.9, 147.4, 147.2, 143.6,
143.3, 138.0, 134.4, 133.9, 131.9, 130.2, 129.2, 128.87, 128.85,
128.5, 126.6, 126.3, 121.0, 116.3, 113.0, 112.8, 108.0, 101.7, 56.7,
45.9, 42.0, 41.7, 35.4, 31.2, 29.0, 27.3, 13.6, 9.5. HPLC-MS:
Elution with 93:7 CH3CN/H2O containing 0.01% formic acid
exhibited a single peak at tR = 22.67 min. ESI-MS m/z [ESþ]
calcd for C40H52BrN3O3Sn [M þ H]þ 822.22; found 822.20.
N-{1-[4-(6-Bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-
3H-cyclopenta[c]quinolin-8-yl]ethylidene}-N0-(5-tributylstannanylpyr-
idin-2-yl)hydrazine (22). A mixture of 2-chloro-5-tributylstannylpyr-
idine (0.401 g, 1 mmol) and hydrazine hydrate (3 mL) was heated
at 120 ꢀC in pyridine (5 mL) for 24 h. The volatiles were removed
by rotary evaporation. Sodium hydroxide (1 N, 25 mL) was added to
the residue, and the product was extracted with dichloromethane
(3 ꢀ 15 mL) and dried over anhydrous Na2SO4. The volatiles were
removed in vacuo to provide (5-tributylstannanylpyridin-2-yl)-
hydrazine. The hydrazine was heated with 1 (0.144 g, 0.35 mmol)
at 170-180 ꢀC for 5 min in vacuo (2 mmHg). The crude hydrazone
was purified by reverse phase column chromatography using metha-
nol/water (90:10) to provide 22 (0.180 g, 65%) as a pale-yellow solid.
Mp 114-116 ꢀC; IR (KBr, cm-1) 3351, 2919, 1611, 1578, 1493; 1H
NMR (300 MHz, CDCl3) δ 8.09-8.08 (m, 1H), 7.94 (bs, 1H), 7.65
(dd, J= 8.2, 1.6 Hz, 1H), 7.48 (d, J= 1.6 Hz, 1H), 7.44 (dd, J=8.3,
2.0 Hz, 1H), 7.37 (dd, J = 8.2, 1.0 Hz, 1H), 7.16 (s, 1H), 7.03(s, 1H),
6.62 (d, J = 8.3 Hz, 1H), 5.99 (d, J = 1.4 Hz, 1H), 5.97 (d, J = 1.4
Hz, 1H), 5.94-5.91 (m, 1H), 5.70-5.64 (m, 1H), 4.90 (d, J=3.0Hz,
1H), 4.15 (d, J = 8.6 Hz, 1H), 3.67 (bs, 1H), 3.25-3.15 (m, 1H),
2.62-2.53 (m, 1H), 2.22 (s, 3H), 1.85 (m, 1H), 1.59-1.49 (m, 6H),
1.41-1.25 (m, 6H), 1.08-1.03 (m, 6H), 0.89 (t, J = 7.2 Hz, 9H); 13C
NMR (75 MHz, CDCl3) δ 157.1, 153.7, 147.5, 147.2, 145.8, 145.7,
143.3, 134.2, 133.9, 130.3, 130.1, 126.4, 125.7, 125.4, 124.1, 115.8,
113.0, 112.8, 108.0, 107.9, 101.7, 56.6, 45.9, 42.1, 31.3, 29.0, 27.3, 13.6,
12.2, 9.5. HPLC-MS: Elution with 73:27 CH3CN/H2O containing
0.01% formic acid exhibited two peaks at tR = 8.40 min (exo) and
11.08 min (endo). ESI-MS m/z [ESþ] calcd for C38H49BrN4O2Sn
[M þ H]þ 793.21; found 793.23.
Intracellular Calcium Mobilization. SKBr3 cells (1 ꢀ 107/mL)
wereincubatedinHBSS containing 3 μM Indo1 a.m. (Invitrogen)
and 0.05% pluronic acid F-127 for 1 h at room temperature. Cells
were then washed twice with HBSS, incubated at room tempera-
ture for 20 min, washed again with HBSS, resuspended in HBSS
at a density of 108cells/mL, and kept on ice until assay, performed
at a density of 2 ꢀ 106 cells/mL. Ca2þ mobilization was deter-
mined ratiometrically using λex of 340 nm and λem of 400/490 nm
at 37 ꢀC in a spectrofluorometer (QM-2000-2, Photon Technol-
ogy International) equipped with a magnetic stirrer. The relative
490 nm/400 nm ratio was plotted as a function of time. All
experiments were performed a minimum of three times.
PI3K Activation. The PIP3 binding domain of Akt fused to
mRFP1 (PH-RFP) was used to localize cellular PIP3. COS7
cells (cotransfected with GPR30-GFP or ERR-GFP and PH-
RFP) or SKBr3 (transfected with PH-RFP) cells were plated on
coverslips, and serum was starved for 24 h followed by stimula-
tion with ligands as indicated. The cells were fixed with 2% PFA
in PBS, washed, mounted in Vectashield containing DAPI
(Vector Labs), and analyzed by confocal microscopy using a
Zeiss LSM510 confocal fluorescent microscope. All experiments
were performed a minimum of three times.
Ligand Binding Assays. Binding assays for ERR and ERβ
were performed as previously described.5 Briefly, COS7 cells
were transiently transfected with either ERR-GFP or ERβ-
GFP. Following serum starvation for 24 h, cells (∼5 ꢀ 104)
were incubated with compounds 3-9 for 10 min in a final
volume of 10 μL prior to addition of 10 μL of 20 nM E2-
Alexa633 in saponin-based permeabilization buffer. Following
5 min at room temperature, cells were washed once with 1 mL of
PBS/2%BSA, resuspended in 200 μL, and analyzed on a FACS
Calibur flow cytometer (BD Biosciences).
For GPR30 binding, 1-{2-[4-(6-bromobenzo[1,3]dioxol-5-
yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]ethyl}-
3-(3-iodo(125)-phenyl)urea (8*), was used. Briefly, Hec50 cells
were cultured in phenol red free DMEM/F-12 containing 10%
charcoal-stripped FBS, plated in 24-well tissue culture plates,
and grown to 80% confluence. Wells were rinsed with PBS, and
cells were incubated with competitor (6, 7, 8, 9) for 30 min prior
to addition of approximately 0.5-1 μCi of radioligand. Wells
were incubated at 37 ꢀC for 1 h and rinsed with PBS, and
radioactivity was collected by ethanol extraction and counted
in a Wallac Wizard 1480 γ counter (Perkin-Elmer, Gaithers-
burg, MD). All binding experiments were performed a mini-
mum of three times.
Radioiodination. The 125I-labeled ligands 8* and 9* were
prepared by oxidative radioiododestannylation of the corre-
sponding tributylstannyl derivatives 21 and 22 using iodogen-
coated reaction tubes (Thermo Fisher Scientific, Rockford, IL).
The tributylstannyl precursor was dissolved in ethanol (5 μL)
and allowed to stand at room temperature for 30 min with
occasional gentle stirring. A 100 μL solution of 10 mM Tris
(pH 7) was added to an iodogen-coated tube, followed by
addition of sodium 125I (∼5 mCi, catalog no. NEZ033L005MC,
Perkin-Elmer, Waltham, MA), followed by incubation at room
temperature for 2-5 min. The tributylstannyl precursor (1 equiv
in 5 μL) was added to the iodogen tube and incubated for 5 min.
The reaction mixture was transferred to a new iodogen-coated
tube and allowed to stand for an additional 1 h with occasional
gentle stirring. The product was purified by solid phase extraction
Cell Culture. ERR/β-negative and GPR30-negative COS7
cells and ERR/β-negative and GPR30-expressing human endo-
metrial carcinoma Hec50 cells were cultured in DMEM med-
ium, fetal bovine serum (10%), 100 units/mL penicillin, and 100
μg/mL streptomycin. ERR/β-negative and GPR30-expressing
SKBr3 cells were cultured in RPMI-1640 medium, fetal bovine
serum (10%), 100 units/mL penicillin, and 100 μg/mL strepto-
mycin. Cells were grown as a monolayer at 37 ꢀC in a humidified
atmosphere of 5% CO2 and 95% air.

Article
Animal Studies. All animal experiments were performed in
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1013
the epidermal growth factor receptor through release of HB-EGF.
Mol. Endocrinol. 2000, 14, 1649–1660.
accordance with the NIH Guide for the Care and Use of
Laboratory Animals and were approved by the UNM Institu-
tional Animal Care and Use Committee. A human endometrial
carcinoma xenograft tumor model was developed by injecting
4 million Hec50 cells subcutaneously in 8-week-old female
ovariectomized athymic (NCr) nu/nu mice (NCI-Frederick,
Frederick, MD). After 4-6 weeks, tumors ranging from 0.7 to
0.9 cm in diameter were observed. Once the tumors were formed,
biodistribution and SPECT/CT studies were performed. Hec50
tumor-bearing mice were injected intravenously (tail vein) with
individual radioiodinated 8* and 9*. To determine GPR30
receptor specificity, in a different set of experiments 5 μg of 1
was co-injected with the radiotracer. At the desired time points,
animals were sacrificed by CO2 euthanasia and organs were
carefully removed and isolated to determine the biodistribution
characteristics of the radiotracer. The organ samples were
weighed, and the corresponding radioactivity was measured
using an automated γ counter after verifying the counting
efficiency with standards. The percent injected dose per gram
of tissue (% ID/g) was calculated by comparison with standards
representing the injected dose per animal.
NanoSPECT/CT imaging studies were performed using a
multipinhole NanoSPECT/CT small animal imager (Bioscan
Inc., Washington, DC). Whole body imaging studies were
carried out using 1.5-1.7% isoflurane on a temperature con-
trolled bed after injecting 2.5 MBq of radioiodinated derivatives
in tumor-bearing mice.
Statistical Analysis. All numerical data were expressed as the
mean of the values ( the standard error of mean (SEM).
Graphpad Prism, version 4 (San Diego, CA), was used for
statistical analysis, and a P value less than 0.05 was considered
statistically significant.
(5) Revankar, C. M.; Cimino, D. F.; Sklar, L. A.; Arterburn, J. B.;
Prossnitz, E. R. A transmembrane intracellular estrogen receptor
mediates rapid cell signaling. Science 2005, 307, 1625–1630.
(6) Thomas, P.; Pang, Y.; Filardo, E. J.; Dong, J. Identity of an
estrogen membrane receptor coupled to a G-protein in human
breast cancer cells. Endocrinology 2005, 146, 624–632.
(7) Prossnitz, E. R.; Arterburn, J. B.; Smith, O. H.; Oprea, T. I.; Sklar,
L. A.; Hathaway, H. J. Estrogen signaling through the transmem-
brane G protein-coupled receptor GPR30. Annu. Rev. Physiol.
2008, 70, 165–190.
(8) Prossnitz, E. R.; Sklar, L. A.; Oprea, T. I.; Arterburn, J. B. GPR30:
a novel therapeutic target in estrogen-related disease. Trends
Pharmacol. Sci. 2008, 29, 116–123.
(9) Filardo, E. J.; Graeber, C. T.; Quinn, J. A.; Resnick, M. B.; Giri,
D.; DeLellis, R. A.; Steinhoff, M. M.; Sabo, E. Distribution of
GPR30, a seven membrane-spanning estrogen receptor, in primary
breast cancer and its association with clinicopathologic determi-
nants of tumor progression. Clin. Cancer Res. 2006, 12, 6359–6366.
(10) Smith, H. O.; Leslie, K. K.; Singh, M.; Qualls, C. R.; Revankar, C.
M.; Joste, N. E.; Prossnitz, E. R. GPR30: a novel indicator of poor
survival for endometrial carcinoma. Am. J. Obstet. Gynecol. 2007,
196, 386.e1–386.e11.
(11) Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Estrogen recep-
tor transcription and transactivation: estrogen receptor alpha and
estrogen receptor beta: regulation by selective estrogen receptor
modulators and importance in breast cancer. Breast Cancer Res.
2000, 2, 335–344.
(12) Ariazi, E. A.; Ariazi, J. L.; Cordera, F.; Jordan, V. C. Estrogen
receptors as therapeutic targets in breast cancer. Curr. Top. Med.
Chem. 2006, 6, 181–202.
(13) Bologa, C. G.; Revankar, C. M.; Young, S. M.; Edwards, B. S.;
Arterburn, J. B.; Kiselyov, A. S.; Parker, M. A.; Tkachenko, S. E.;
Savchuck, N. P.; Sklar, L. A.; Oprea, T. I.; Prossnitz, E. R. Virtual
and biomolecular screening converge on a selective agonist for
GPR30. Nat. Chem. Biol. 2006, 2, 207–212.
(14) Albanito, L.; Madeo, A.; Lappano, R.; Vivacqua, A.; Rago, V.;
Carpino, A.; Oprea, T. I.; Prossnitz, E. R.; Musti, A. M.; Ando, S.;
Maggiolini, M. G protein-coupled receptor 30 (GPR30) mediates
gene expression changes and growth response to 17β-estradiol and
selective GPR30 ligand G-1 in ovarian cancer cells. Cancer Res.
2007, 67, 1859–1866.
(15) Brailoiu, E.; Dun, S. L.; Brailoiu, G. C.; Mizuo, K.; Sklar, L. A.;
Oprea, T. I.; Prossnitz, E. R.; Dun, N. J. Distribution and
characterization of estrogen receptor G protein-coupled receptor
30 in the rat central nervous system. J. Endocrinol. 2007, 193, 311–
321.
(16) Pang, Y.; Dong, J.; Thomas, T. Estrogen signaling characteristics
of Atlantic Croaker G protein-coupled receptor 30 (GPR30) and
evidence it is involved in maintenance of oocyte meiotic arrest.
Endocrinology 2008, 149, 3410–3426.
(17) Albanito, L.; Sisci, D.; Aquila, S.; Brunelli, E.; Vivacqua, A.;
Madeo, A.; Lappano, R.; Pandey, D. P.; Picard, D.; Mauro, L.;
Ando, S.; Maggiolini, M. Epidermal growth factor induces G
protein-coupled receptor 30 expression in estrogen receptor-
negative breast cancer cells. Endocrinology 2008, 149, 3799–3808.
(18) Teng, J.; Wang, Z.-Y.; Prossnitz, E. R.; Bjorling, D. E. The G
protein-coupled receptor GPR30 inhibits human urothelial cell
proliferation. Endocrinology 2008, 149, 4024–4034.
(19) Sirianni, R.; Chimento, A.; Ruggiero, C.; Luca, A. D.; Lappano,
R.; Ando, S.; Maggiolini, M.; Pezzi, V. The novel estrogen recep-
tor, G protein-coupled receptor 30, mediates the proliferative
effects induced by 17β-estradiol on mouse spermatogonial GC-1
cell line. Endocrinology 2008, 149, 5043–5051.
(20) Wang, C.; Dehghani, B.; Magrisso, I. J.; Rick, E. R.; Bonhomme,
E.; Cody, D. B.; Elenich, L. A.; Subramanian, S.; Murphy, S. J.;
Kelly, M. J.; Rosenbaum, J. S.; Vandenbark, A. A.; Offner, H.
GPR30 contributes to estrogen-induced thymic atrophy. Mol.
Endocrinol. 2008, 22, 636–648.
(21) Madak-Erdogana, Z.; Kieser, K. J.; Kim, S. H.; Komm, B.;
Katzenellenbogen, J. A.; Katzenellenbogen, B. S. Nuclear and
extranuclear pathway inputs in the regulation of global gene
expression by estrogen receptors. Mol. Endocrinol. 2008, 22,
2116–2127.
(22) Alyea, R. A.; Laurence, S. E.; Kim, S. H.; Katzenallenbogen, B. S.;
Katzenallenbogen, J. A.; Watson, C. S. The roles of membrane
estrogen receptor subtypes in modulating dopamine transporters in
PC-12 cells. J. Neurochem. 2008, 106, 1525–1533.
Acknowledgment. This work was supported by NIH
Grants R01 CA127731 (J.B.A., E.R.P.), CA116662 (E.R.P.),
and U54MH074425 and U54MH084690 (L.A.S.), the Uni-
versity of New Mexico Cancer Research and Treatment Center
(NIH Grant P30 CA118100), the New Mexico Cowboys for
Cancer Research Foundation (J.B.A.), Oxnard Foundation
(E.R.P.), and the Stranahan Foundation (E.R.P.). SPECT/CT
images in this paper were generated in the Keck-UNM Small
Animal Imaging resource established with funding from the
W. M. Keck Foundation (L.A.S.). Images in this paper were
generated in the University of New Mexico Cancer Center
Fluorescence Microscopy Facility, supported as detailed on
the Web page http://hsc.unm.edu/crtc/microscopy/Facility.
html. Flow cytometry data were generated in the Flow Cyto-
metry Shared Resource Center supported by the University of
New Mexico Health Sciences Center and the University of
New Mexico Cancer Center.
Supporting Information Available: Additional synthetic pro-
cedures and characterization data; HPLC-MS data for 8, 9, 21,
22; and SPECT/CT images of 8* and 9*. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Edwards, D. P. Regulation of signal transduction pathways
by estrogen and progesterone. Annu. Rev. Physiol. 2005, 67, 335–
376.
(2) Lange, C. A.; Gioeli, D.; Hammes, S. R.; Marker, P. C. Integration
of rapid signaling events with steroid hormone receptor action in
breast and prostate cancer. Annu. Rev. Physiol. 2007, 69, 171–199.
(3) http://www.iuphar-db.org/GPCR/ReceptorDisplayForward?re-
ceptorID=3055.
(4) Filardo, E. J.; Quinn, J. A.; Bland, K. I; Frackelton, A. R., Jr.
Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-
coupled receptor homolog, GPR30, and occurs via trans-activation of
(23) Kamanga-Sollo, E.; White, M. E.; Chung, K. Y.; Johnson, B. J.;
Dayton, W. R. Potential role of G-protein-coupled receptor 30
(GPR30) in estradiol-17β-stimulated IGF-I mRNA expression in

1014 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Ramesh et al.
bovine satellite cell cultures. Domest. Anim. Endocrinol. 2008, 35,
254–262.
(24) Otto, C.; Rohde-Schulz, B.; Schwarz, G.; Fuchs, I.; Klewer, M.;
Brittain, D.; Langer, G.; Bader, B.; Prelle, K.; Nubbemeyer, R.;
Fritzemeier, K.-H. G protein-coupled receptor 30 localizes to the
endoplasmic reticulum and is not activated by estradiol. Endocri-
nology 2008, 149, 4846–4856.
(25) Dun, S. L.; Brailoiu, G. C.; Gao, X.; Brailoiu, E.; Arterburn, J. B.;
Prossnitz, E. R.; Oprea, T. I.; Dun, N. J. Expression of estrogen
receptor GPR30 in the rat spinal cord, autonomic and sensory
ganglia. J. Neurosci. Res. 2009, 87, 1610–1619.
Schubert, E. K.; Dunnwald, L. K.; Krohn, K. A.; Mankoff, D. A.
Quantitative fluoroestradiol positron emission tomography ima-
ging predicts response to endocrine treatment in breast cancer.
J. Clin. Oncol. 2006, 24, 2793–2799.
(36) Rijks, L. J. M.; Boer, G. J.; Endert, E.; de Bruin, K.; Janssen, A. G.
M.; van Royen, E. A. The Z-isomer of 11β-methoxy-17R-[123I]-
iodovinylestradiol is a promising radioligand for estrogen receptor
imaging in human breast cancer. Nucl. Med. Biol. 1997, 24, 65–75.
(37) Fischer, T.; Schomacker, K.; Schicha, H. Diethylstilbestrol (DES)
labeled with Auger emitters: potential radiopharmaceutical for
therapy of estrogen receptor-positive tumors and their metastases?
Int. J. Radiat. Biol. 2008, 84, 1112–1122.
(38) Kouznetsov, V. V. Recent synthetic developments in a powerful
imino Diels-Alder reaction (Povarov reaction): application to the
synthesis of N-polyheterocycles and related alkaloids. Tetrahedron
2009, 6, 2721–2750.
(26) Dennis, M. K.; Burai, R.; Ramesh, C.; Petrie, W. K.; Alcon, S. N.;
Nayak, T. K.; Bologa, C. G.; Leitao, A.; Brailoiu, E.; Deliu, E.;
Dun, N. J.; Sklar, L. A.; Hathaway, H. J.; Arterburn, J. B.; Oprea,
T. I.; Prossnitz, E. R. In vivo effects of a GPR30 antagonist. Nat.
Chem. Biol. 2009, 5, 421–427.
(27) Hochberg, R. B.; Rosner, W. Interaction of 16 R-[125I]iodo-estra-
diol with estrogen receptor and other steroid-binding proteins.
Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 328–332.
(39) Kobayashi, S.; Sugiura, M.; Kitagawa, H.; Lam, W. W.-L. Rare-
earth metal triflates in organic synthesis. Chem. Rev. 2002, 102,
2227–2302.
(28) Katzenellenbogen, J. A.; Carlson, K. E.; Heiman, D. F.; Goswami,
R. Receptor-binding radiopharmaceuticals for imaging breast
tumors: estrogen-receptor interactions and selectivity of tissue
uptake of halogenated estrogen analogs. J. Nucl. Med. 1980, 21,
550–558.
(40) Moran, D. B.; Morton, G. O.; Albright, J. D. Synthesis of
(pyridinyl)-1,2,4-triazolo[4,3-a]pyridines. J. Heterocycl. Chem.
1986, 23, 1071–1077.
(41) Hanson, R. N.; Napolitano, E.; Fiaschi, R. Synthesis and evalua-
tion of 11β-substituted 21-choloro/iodo-(17R,20E/Z)-19-norpreg-
na-1,3,5(10),20-tetraene-3,17β-diols: high affinity ligands for the
estrogen receptor. J. Med. Chem. 1998, 41, 4686–4692.
(42) Arterburn, J. B.; Rao, K. B.; Perry, M. C. Novel 17R-ethynyles-
tradiol derivatives: Sonogashira couplings using unprotected phe-
nylhydrazines. Tetrahedron Lett. 2000, 41, 839–842.
(43) Sirisoma, N. S.; Johnson, C. R. R-Iodocycloalkenones: synthesis of
(()- epibatidine. Tetrahedron Lett. 1998, 39, 2059–2062.
(44) Pimlott, S.; Stevenson, L.; Wyper, D.; Sutherland, A. Rapid and
efficient radiosynthesis of [123I]I-PK11195, a single photon emis-
sion computed tomography tracer for peripheral benzodiazepine
receptors. Nucl. Med. Biol. 2008, 35, 537–542.
(45) Ramesh, C.; Bryant, B.; Nayak, T.; Revankar, C. M.; Anderson,
T.; Carlson, K. E.; Katzenellenbogen, J. A.; Sklar, L. A.;
Norenberg, J. P.; Prossnitz, E. R.; Arterburn, J. B. Linkage effects
on binding affinity and activation of GPR30 and estrogen
receptors ERR/β with tridentate pyridin-2-yl hydrazine tricarbo-
nyl-Re/99mTc(I) chelates. J. Am. Chem. Soc. 2006, 128, 14476–
14477.
(29) Cummins, C. H. Radiolabeled steroidal estrogens in cancer re-
search. Steroids 1993, 58, 245–259.
(30) Hanson, R. N. Synthesis of Auger electron-emitting radiopharma-
ceuticals. Curr. Pharm. Des. 2000, 6, 1457–1468.
(31) Van de Wiele, C.; De Vos, F.; Slegers, G.; Van Belle, S.; Dierckx, R.
A. Radiolabeled estradiol derivatives in breast cancer: a review.
Eur. J. Nucl. Med. 2000, 27, 1421–1433.
(32) McGuire, A. H.; Dehdashti, F.; Siegel, B. A.; Lyss, A. P.; Brodack,
J. W.; Mathias, C. J.; Mintun, M. A.; Katzenellenbogen, J. A.;
Welch, M. J. Positron tomographic assessment of 16 R-[18F] fluoro-
17β-estradiol uptake in metastatic breast carcinoma. J. Nucl. Med.
1991, 32, 1526–1531.
(33) Yoshida, Y.; Kurokawa, T.; Sawamura, Y.; Shinagawa, A.;
Okazawa, H.; Fujibayashi, Y.; Kotsuji, F. The positron emission
tomography with F18 17β-estradiol has the potential to benefit
diagnosis and treatment of endometrial cancer. Gynecol. Oncol.
2007, 104, 764–766.
(34) Yoo, J.; Dence, C. S.; Sharp, T. L.; Katzenellenbogen, J. A.; Welch,
M. J. Synthesis of an estrogen receptor β-selective radioligand:
5-[18F]fluoro-(2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentanenitrile
and comparison of in vivo distribution with 16R-[18F]fluoro-17β-
estradiol. J. Med. Chem. 2005, 48, 6366–6378.
(46) Nayak, T. K.; Hathaway, H. J.; Ramesh, C.; Arterburn, J. B.; Dai,
D.; Sklar, L. A.; Norenberg, J. P.; Prossnitz, E. R. Preclinical
development of a neutral, estrogen receptor-targeted, tridentate
99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative for imaging
of breast and endometrial cancers. J. Nucl. Med. 2008, 49, 978–
986.
(35) Linden, H. M.; Stekhova, S. A.; Link, J. M.; Gralow, J. R.;
Livingston, R. B.; Ellis, G. K.; Petra, P. H.; Peterson, L. M.;