Synthesis and Characterization of Tricarbonyl-Re/Tc(I) Chelate Probes Targeting the G Protein-Coupled Estrogen Receptor GPER/GPR30

The discovery of the G protein-coupled estrogen receptor GPER (also GPR30) and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPER-mediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer.


Introduction
Estrogens are involved in a diverse array of physiological responses. The genomic roles of the nuclear estrogen receptors ERa and ERb have been characterized in greatest detail. ERa, and to some extent ERb, are important drug targets because of their roles in development, reproduction, skeletal physiology and the nervous, cardiovascular, and immune systems. Estrogen also rapidly triggers a variety of secondary messenger (non-genomic) signaling events that contribute to complex physiological, morphological and behavioral effects. Estrogen-responsiveness constitutes a major determinant of therapy selection and prognosis in breast cancer with both genomic and non-genomic pathways regulating tumor biology [1][2][3][4]. The recent identification of the involvement of a G protein-coupled estrogen receptor GPR30 (IUPHAR designation: GPER) in tumor signaling pathways and studies demonstrating the prognostic value of assessing GPER expression suggest GPER may serve as a potentially important biomarker and therapeutic target in cancer [5][6][7][8][9].
We have developed the first GPER-selective agonist G-1, a tetrahydro-3H-cyclopenta[c]quinoline, and the structurally related antagonists G15 and G36, all of which exhibit high affinity for GPER and high selectivity for GPER over ERa/b (Figure 1) [10][11][12]. Structure activity studies have identified the important role of the 6-bromo-benzo [1,3]dioxolane substituent, and the presence of an ethanone hydrogen-bond acceptor group at the C8-position was associated with agonism. These functional probes have been used to distinguish GPER-mediated effects in a wide variety of in vitro and in vivo studies, particularly in systems expressing multiple estrogen receptors, generating high confidence in their use as leads in drug discovery programs [13]. Based on the demonstrated receptor selectivity of this scaffold, we initiated a program focusing on the development of novel targeted imaging agents for the in vivo characterization of GPER expression in normal and disease model systems.
We recently described the generation of the first radiolabeled agents based on the GPER-targeting tetrahydro-3H-cyclopenta[c]quinoline ( = ''G'') scaffold of G-1/G15, incorporating the cemitting isotopes 125 I, and 111 In (Figure 2) [14,15]. While the C8-iodo compound 1 exhibited promising GPER-targeting characteristics, radiolabeling of the stannane precursor resulted in poor yields and competing deiodination due to the strong electrondonating effect of nitrogen and prevented practical application of this agent. The pendant hydrazone 2 and urea 3 derivatives underwent 125 I-radiolabeling and were effective competitive ligands for GPER binding, but showed poor tumor targeting characteristics using in vivo xenograft model studies. The relatively high background and non-target tissue uptake was attributed to the lipophilicity of the pendant groups and complications due to rapid metabolism. In complementary studies, we constructed a series of acyclic and macrocyclic polyamino-polycarboxylate ligands and evaluated the resulting 111/113 In(III) chelates to determine the effect of ionic charge on GPER targeting in vitro. The neutral DOTA conjugate 4 was capable of activating rapid GPER-mediated signaling pathways while charged complexes were inactive. Rapid initiation of GPER-mediated signaling by neutral, but not charged, synthetic estrogen probes has also been demonstrated [16]. These results are consistent with previous studies demonstrating the predominant intracellular localization for GPER in the endoplasmic reticulum of most cell types, with only minor amounts detectable at the plasma membrane [17]. Chelate 4 exhibited receptor-mediated uptake in the uterus, mammary glands, adrenal glands and tumor with the tumor being clearly visualized, although high uptake in the intestines, hepatobiliary excretion and the formation of polar metabolites limit the usefulness of this agent. These studies have identified the need for alternative radiolabeling modalities to improve intracellular targeting properties and obtain enhanced in vivo performance. Improvements in small animal imaging scanners for single photon emission computed tomography (SPECT) have increased the resolution and sensitivity of detection affording numerous advantages for preclinical studies and opportunities for employing the favorable nuclear decay properties of the important diagnostic radionuclide 99m Tc (T 1/2 = 6 h, 140 keV c-radiation) that is widely available through 99 Mo/ 99m Tc generators [18]. Whereas all isotopes of technetium are radioactive, the third-row congener rhenium ( 185 Re and 187 Re) serves as an effective non-radioactive chemical surrogate for technetium, yet is also available as the medically useful radioisotopes: 186/188 Re. The organometallic M(CO) 3 + core (M = 99m Tc, Re) is conveniently generated in aqueous media and offers favorable characteristics that are advantageous for the design of small molecule imaging agents, such as a small steric profile and promiscuous chelation chemistry with a variety of bidentate and tridentate ligands to yield kinetically stable complexes [19].
In this study, we describe the synthesis of a promising new class of GPER-targeted tricarbonyl-Re(I) chelates 5-10 ( Figure 3), and profile their targeting properties, activity and selectivity using cellbased functional assays for receptor-mediated signaling. Within the series of Re-chelates evaluated, the connecting linkage and chelate structure were found to contribute significantly to the physicochemical and biological properties, effectively determining the functional role of the compounds as agonists or antagonists of GPER-mediated signaling. Efficient radiolabeling with [ 99m Tc(CO) 3 (H 2 O) 3 ] + provides a promising new class of GPERtargeted SPECT imaging agents.

Synthesis of chelate ligands
We designed a series of six new GPER-targeted organorhenium probes (5-10) using the tetrahydro-3H-cyclopenta[c]quinoline scaffold shared by G-1 and G15. We have previously described an efficient, diastereoselective synthetic route for G-1 [20]. Three different chelating heterocyclic aminocarboxylate ligands with demonstrated capacity for the formation of neutral tricarbonylrhenium(I) complexes were selected; namely pyridin-2-yl-hydrazinylethanoic acid (5,(7)(8)(9) [21,22], pyridin-2-yl-methylaminoethanoic acid (6) [23,24], and 1,2,3-triazol-4-yl-methylaminoethanoic acid (10) [25,26]. The C8 position of the cyclopenta[c]quinoline scaffold was selected for attachment of the chelating ligands, based on our previous structure-activity studies demonstrating the plasticity of this site for derivatization. The proximal functionality and length of the connecting linkages between targeting moiety and chelate groups were varied within this series to evaluate the combined structural effects on the interaction of the complexes with GPER.  The t Boc-protected 5-bromopyridin-2-yl-hydrazinylethanoate 11 was converted to the 5-ethynyl derivative 12 using a palladium-catalyzed Sonogashira coupling with trimethylsilylacetylene, followed by silver(I)triflate-mediated removal of the silyl group ( Figure 4). A second Sonogashira coupling of alkyne 12 with the C8-iodo compound 1 gave the corresponding ethyne-linked synthetic intermediate. Removal of the t Boc-and t butyl-ester groups with trifluoroacetic acid was accompanied by regioselective hydrolysis of the alkyne to the ethanone group [27]. The pyridin-2-yl-hydrazinylethanoic acid ligand 13 was purified by silica gel chromatography.
Radical initiated bromination of 5-bromo-2-methylpyridine 14 with N-bromosuccinimide, followed by N-alkylation with t Bocglycine-t butyl ester gave the picoline amine derivative 15 ( Figure 5). A step-wise sequence of Pd-catalyzed ethynylation with trimethylsilylacetylene, removal of TMS-group, Sonogashira coupling of the resulting ethyne with the C8-iodo compound 1, and finally deprotection of the t Boc-and t butyl-ester groups occurred with concomitant hydrolysis of the alkyne in TFA to afford the ethanone-linked pyridin-2-yl-methylaminoethanoic acid ligand 16.
Facile palladium-catalyzed hydrodebromination of the bromobenzodioxole group complicated direct attempts to prepare the ethane-bridged chelate ligand 18 by catalytic hydrogenation of the ethyne-linked synthetic intermediate produced in Figure 4. Therefore, we designed an alternative approach to 18 using the protected aniline derivative 17 as a constituent of the threecomponent aza-Diels Alder (Povarov) reaction ( Figure 6). The palladium-catalyzed C-C coupling of 4-ethynylaniline with protected 5-bromopyridin-2yl-hydrazinylethanoic derivative 11, followed by catalytic hydrogenation of the resulting alkyne using 10% Pd/C gave the aniline derivative 17. The cyclization of 17 with 6-bromopiperonal and cyclopentadiene was catalyzed by KHSO 4 to provide the tetrahydro-3H-cyclopenta[c]quinoline derivative 18 in moderate yield. The endo-diastereoselectivity achieved with this catalyst was relatively low (3.3:1); however, attempts to improve this using the scandium catalyst Sc(OTf) 3 were unsuccessful due to competing chelation by the hydrazine-2yl pyridine. Removal of the t butyl-protecting groups using TFA, gave the ethane-linked pyridin-2-yl-hydrazinylethanoic acid ligand 18.

Complexation with [Re(CO) 3 (H 2 O) 3 ] +
All of the aminocarboxylate conjugates evaluated were effective for complexation of Re(CO) 3 (H 2 O) 3 in aqueous ethanol as shown in Table 1. The chelation occurred at ambient temperature; however, reduced reaction times and increased yields of the complexes were achieved at higher temperatures. Evaluation of the stability of the triazol-4-yl-methylamine, picolinamine and pyridin-2yl-hydrazine ligands in aqueous ethanol buffered with NaHCO 3 at 90uC showed no observable decomposition after 1 h. The different solubilities of the ligands were considered when selecting the EtOH/H 2 O solvent mixture. The picoline-based chelate 6-Re was more hydrophobic than the pyridylhydrazine complex 5-Re, and while this may be expected from the isosteric replacement of ''NH'' with ''CH 2 '' groups, this substitution resulted in large differences in chromatographic mobility using reverse phase HPLC where the retention times were 20.63 min and 16.32 min respectively. The complexation of triazol-4-ylmethylamine ligand 25 at room temperature was relatively slow. Increasing the reaction temperature to 65uC for 1 h gave complex 10-Re in high yield (92%).

Ligand Interactions with GPER and ERa/b Receptors
The series of tricarbonylorganorhenium(I) complexes 5-Re : 10-Re was evaluated using established functional assays for GPER   and profiled for potential cross-reactivity via the nuclear estrogen receptor ERa ( Table 2). The capacity of the complexes to elicit rapid mobilization of intracellular calcium was evaluated in SKBr3 breast cancer cells, which are ERa-and ERb-negative but express GPER. The pyridylhydrazine and picolinamine complexes 5 and 6 were both potent agonists of GPER-mediated calcium mobilization. These complexes possess conserved ethanone groups in the connecting linkage, analogous to the parent agonist ligand G-1, further supporting structure-activity models that indicate a role for the hydrogen bond accepting properties of the ethonanone group in agonism of this ligand. In sharp contrast, complexes 7-Re, 8-Re and 10-Re were inactive as agonists at concentrations up to 30 mM. The assay was then modified to assess possible antagonism by examining the capacity of the complexes to block Ca 2+ mobilization by 17b-estradiol. The triazole-linked conjugate 9-Re was identified as an antagonist of estrogen activation, while complexes 7-Re, 8-Re and 10-Re had no effect on this response.
The stimulation of GPER as well as ERa and ERb by the native hormone 17b-estradiol activates PI3K and results in the nuclear accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), as shown by the nuclear translocation of an Akt PH domain fluorescent protein fusion protein reporter (PH-RFP). To determine whether these complexes activated the PI3K pathway, we employed COS7 cells transiently transfected with either GPER-GFP or ERa-GFP together with PH-RFP that serves as a reporter of PIP3 production. The complexes 5-Re and 6-Re were again found to be agonists of GPER-mediated PI3K activation, confirming their activity profile. Switching the assay protocol to detect antagonism of PI3K activation by 17b-estradiol, the complex 9-Re was similarly identified as an antagonist. None of the complexes were able to activate PI3K through ERa, demonstrating that the conjugation of linked chelate groups did not alter the selectivity profile to allow interaction with the nuclear receptor, consistent with the observation that there was no significant binding to ERa or ERb at concentrations up to 10 mM, where 17b-estradiol yields a K i of 0.3-0.5 nM (data not shown).
The activity profile in receptor-mediated signaling obtained from functional assays reveals the importance of structural effects associated with the linkage to the heterocyclic aminocarboxylate ligand at the C8 position of the tetrahydroquinoline scaffold. The pyridylhydrazine and picolinamine complexes 5-Re and 6-Re possess ethanone linkages that are analogous to the methyl ketone group of G-1, and were similarly found to be potent agonists of GPER signaling in both the calcium and PI3K assays. In contrast, the triazole-linked complex 9-Re antagonized GPER-mediated signaling in both of these functional assays. The 1,2,3-triazole linkage is capable of functioning as a hydrogen-bond acceptor; however, the increased steric constraints and rigid planar ring structure in 9-Re may prevent the required conformational alignment in the receptor-bound complex. The ethane-linked complex 7-Re was inactive towards initiating or blocking GPERmediated signaling. The conformational mobility of the flexible ethane linkage would produce a relatively large rotational steric volume and unfavorable entropic contribution that may impede the process of ligand-binding, accompanied by reduced affinity due to the hydrophobicity and absence of a H-bond accepting group in this linkage. The direct connection of heterocyclic chelates to the quinoline scaffold in compounds 8-Re and 10-Re contributes a relatively large steric volume in this region which precludes interactions with GPER. The extension of the linkage through a planar triazole group yielded antagonist complex 9, and emphasizes the importance of the linkage structure on receptor targeting properties.

Radiolabeling with [ 99m Tc(CO) 3 (H 2 O) 3 ] +
The tricarbonyl approach was used to prepare the 99m Tcradiolabeled complexes 5-8. The [ 99m Tc(CO) 3 3 ] + into the pyridin-2-yl hydrazine 5-Tc and picolylamine 6-Tc complexes. A reduction to 85% incorporation was observed for the directly linked pyridin-2yl hydrazine 8-Tc complex. The radiolabeled complexes were conveniently purified by reverse-phase solid phase extraction, efficiently removing excess ligand and inorganics. In order to assess a more rapid radiosynthetic method for improved specific activity, the [ 99m Tc(CO) 3 (H 2 O) 3 ] + intermediate was mixed with the ligands and heated at 80uC for 30 min. Under these complexation conditions, degradation products were evident by HPLC analysis and the radiochemical purity was less than 70%. All 99m Tc-labeled complexes demonstrated good stability (.95%) in mouse plasma and PBS buffer after incubation at 37uC for 24 h. The complexes were stable in the presence of biologically relevant chelating ligands, exhibiting less than 10% transchelation upon incubation with 1 mM cysteine solution or 1 mM histidine solution at 37uC for 24 h. The log P (o/w) values (mean 6 SEM, n = 4) of the ethanone-linked pyridin-2-yl hydrazine complex 5-Tc (4.660.3) was lower than that for the picolinamine complex 6-Tc (5.060.1), revealing the contribution of the hydrazine group to the hydrophilicity of the complex. Increased log P (o/w) values were also observed for the ethane-linked 7-Tc (4.960.1) and directly connected pyridin-2-yl hydrazine complex 8-Tc (5.560.1).
Excessive lipophilicity was previously identified as a major limitation resulting in non-target tissue uptake for the urea-and hydrazone-linked I-125 radiolabeled analogs 2 and 3 with log P (o/ w) values of 7.00 and 6.35 respectively [13]. The tricarbonyl-Tc(I) chelate structure in 5-Tc therefore represents a significant improvement in log P (o/w) values compared to previous generations of GPER-targeted imaging agents.

Conclusions
We have identified a new class of organometallic probes that selectively targets GPER in cellular assays. The tridentate heterocyclic aminocarboxylate ligands formed stable complexes from the cationic precursor [Re(CO) 3 (H 2 O) 3 ] + in aqueous ethanol. These conditions were successfully adapted for efficient 99m Tcradiolabeling. The ethanone-linked complexes 5-Re and 6-Re yielded selective activation of GPER-mediated signaling pathways comparable to the parent ligand G-1, while the triazole-linked complex 9-Re was an antagonist capable of blocking GPER signaling. These results demonstrate that structural features of the linkage and chelate were critical determinants of receptormediated signaling through GPER. We anticipate that continuing in vivo SPECT imaging studies of GPER expression with the promising new probe complex 5-Tc using will reveal important new insights on the role of GPER in normal and disease states.

Materials and Methods
Reagents and solvents were purchased from commercial sources and used without further purification. Preparative chromatography was performed using Sorbent technologies prepacked silica gel columns under medium pressure with ethyl acetate/hexanes (EtOAc/Hexanes) or methanol/dichloromethane (MeOH/ CH 2 Cl 2 ) as eluent. Reactions were followed by thin-layer chromatography (TLC) on silica gel (60 Å pore size, 5-17 mm) polyester backed sheets that were visualized under a UV lamp, iodine vapor, phosphomolybdic acid, or anisaldehyde. 1 H NMR spectra were acquired at 300 MHz or 400 MHz, and 13 C NMR were acquired at 75 MHz or 100 MHz spectrometers at ambient temperatures (1862uC) unless otherwise noted. The 1 H NMR spectra in CDCl 3 were referenced to TMS unless otherwise noted. The 13 C {1H} NMR spectra were recorded at 75 or 100 MHz and referenced relative to the 13 C {1H} peaks of the solvent. Spectra are reported as (ppm), (multiplicity, coupling constants (Hz), and number of protons). Tri and Py are abbreviations of triazole and pyridine, respectively. Analytical HPLC was obtained using a Waters 2695 HPLC with Waters 2996 Photodiode Array (PDA) and Micromass ZQ ESI-MS detection (cone voltage 62 V, Capillary Voltage 3 kV). The compound (1 mg/mL CH 3 CN, 20 mL) was injected into Waters SymmetryH C 18 5 mm 3.06150 mm column eluted with CH 3 CN/H 2 O as specified. High resolution mass spectra were obtained from the University of California at Riverside.

General Procedure A; Deprotection of t Boc-ligands
Trifluoroacetic acid (0.8 mL) was added to the protected ligand (0.1 mmol) in dichloromethane (1 mL) and allowed to stir at ambient temperature for 1 h. The reaction mixture was diluted with dichloromethane (5 mL) and excess trifluoroacetic acid was neutralized with saturated NaHCO 3 , the organic layer was separated, dried over anhydrous Na 2 SO 4 , evaporated and concentrated in vacuo, and purified as described to isolate the ligand.

G-CH 2 CH 2 -5-pyridyl-2-NHNHCH 2 CO 2 H (18)
Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH 2 Cl 2 (10: 90) to isolate the product 18 (0.081g, 60%) as a colorless solid consisting of a mixture of syn: anti (3.3: 1) determined by NMR; 1  G-5-pyridyl-2-NHNHCH 2 CO 2 H (20) Following general procedure A, the residue was purified by silica gel column chromatography using MeOH/CH 2 Cl 2 (15: 85) to isolate the product 20 (0.048 g, 90%) as a colorless solid consisting of a mixture of syn: anti (3: 1) determined by NMR; 1  G-triazole-CH 2 NHCH 2 CO 2 H (25) Following general procedure A, the residue was purified by addition of water, producing a solid that was filtered and purified by silica gel column chromatography using MeOH/CH 2 Cl 2 (20: 70) to isolate the product 25 (0.039 g, 75%) as a white solid. 1 3 (1.1 eq) and NaHCO 3 (1.0 eq) in water was added to the ligand in EtOH and allowed to stir at rt for 3-6 h. Volatiles were removed, water was added, the resulting precipitate was filtered and washed with water. The residue was purified as described to isolate the rhenium complex.

Intracellular calcium mobilization
SKBr3 cells (1610 7 /mL) were incubated in HBSS containing 3 mM Indo1-AM (Invitrogen) and 0.05% pluronic acid F-127 for 1 h at RT. Cells were then washed twice with HBSS, incubated at RT for 20 min, washed again with HBSS, resuspended in HBSS at a density of 10 8 cells/mL and kept on ice until assay, performed at a density of 2610 6 cells/mL. Ca ++ mobilization was determined ratiometrically using l ex 340 nm and l em 400/490 nm at 37uC in a spectrofluorometer (QM-2000-2, Photon Technology International) equipped with a magnetic stirrer.

PI3K activation
The PIP3 binding domain of Akt fused to mRFP1 (PH-RFP) was used to localize cellular PIP3. COS7 cells co-transfected with GPER-GFP or ERa-GFP and PH-RFP were plated on coverslips and serum starved for 24 h followed by stimulation 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.

Receptor binding
Binding assays for ERa and ERb were performed as previously described [30]. Briefly, COS7 cells were transiently transfected with ERa-GFP or ERb-GFP. Following serum starvation for 24 h, cells (,5610 4 ) were incubated with Re-labeled derivatives for 10 min in a final volume of 10 mL prior to addition of 10 mL of 20 nM E2-Alexa633 in saponin-based permeabilization buffer. Following 5 min at RT, cells were washed once with 1 mL PBS/ 2%BSA, resuspended in 200 mL and analyzed on a FACS Calibur flow cytometer (BD Biosciences).
99m Tc-radiolabeling The carbonyl complex [ 99m Tc(CO) 3 (H 2 O) 3 ]+ was prepared by adding 3.7 GBq of freshly eluated Na-99m TcO 4 to the IsolinkH kit (Tyco healthcare, Mallinckrodt, St. Louis, MO, USA) as previously described [20], The alkaline [ 99m Tc(CO) 3 (H 2 O) 3 ] + mixture was then neutralized to pH 7 with acetic acid. The ligands were dissolved in ethanol and 10 mg of each of the derivatives was added to the prepared [ 99m Tc(CO) 3 (H 2 O) 3 ] + mixture. The reaction mixture was stirred for 2 h at room temperature. The inorganic impurities from IsolinkH kit, aqua ions of 99m Tc (if any) and excess ligand were separated using solid phase extraction (SPE) technique. SPE was performed using C-18 SepPak Plus cartridges (Waters, Milford, MA USA). The impurities and excess of ligand were eluted with 4 fractions of 0.5 mL of the weak solvent (50% ethanol in water). Elution of the final 99m Tc-labeled complexes was performed with 4 fractions of 0.5 mL strong solvent (100% ethanol). HPLC was performed to assess radiochemical purity and specific activity. Ten mL of the final product was added to 200 mL of HPLC grade ethanol (JT Baker, Phillipsburg, NJ, USA). To asses radiochemical purity and specific activity, 10 mL of the diluted quality control sample was injected on a reverse-phase C-18 column (JT Baker, Phillipsburg, NJ, USA) using HPLC grade ethanol and HPLC grade water as previously described [22]. Stability, transchelation and partition coefficient studies were performed as previously described [22].