Estrogen receptor imaging agents

Abstract

Compounds useful for molecular imaging of cells expressing estrogen receptors are provided. Also provided are intermediates for making the compounds and methods of making the compounds using a modular convergent strategy. Further, methods of making the intermediates are described, as well as methods of diagnosing disease in a subject by using the compounds as imaging agents.

Claims

1. A compound according to formula (I) ##STR00029## wherein n is a number in the range of 1-10; m is a number in the range of 4-10; R.sup.1 is H or a C.sub.1-3 alkyl group; and R.sup.2 is either absent, or selected from the group consisting of H and a C.sub.1-3 alkyl group.

2. A compound according to formula (II) ##STR00030## wherein m is a number in the range of 4-10; and R.sup.1 is H or a C.sub.1-3 alkyl group; and R.sup.2 is either absent, or selected from the group consisting of H and a C.sub.1-3 alkyl group.

3. A compound according to formula (III) ##STR00031## wherein m is a number in the range of 4-10; R.sup.1 is H or a C.sub.1-3 alkyl group; and R.sup.2 is either absent, or selected from the group consisting of H and a C, 1-3 alkyl group.

4. A process for the preparation of a compound according to formula (I) comprising the step of reacting a compound according to formula (II) with a compound according to formula (IV) using a Huisgen [3+2] cycloaddition reaction, ##STR00032## wherein n is a number in the range of 1-10; m is a number in the range of 4-10; R.sup.1 is H or a C.sub.1-3 alkyl group; and R.sup.2 is either absent, or selected from the group consisting of H and a C.sub.1-3 alkyl group, and wherein x is a number in the range of 1-10.

5. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically effective carrier or diluent.

6. A method of diagnosing a disease characterized by overexpression of estrogen receptors in a subject, the method comprising detecting a cell of the subject expressing estrogen receptors by contacting the compound according to claim 1 with the cell, imaging the cell with positron emission tomography (PET) or single photon emission computed tomography (SPECT), and determining from the imaging whether estrogen receptors are overexpressed in the cell compared to a normal cell, wherein the subject is diagnosed as having the disease if overexpression of estrogen receptors is detected in the cell, wherein the disease is breast cancer or endometrial cancer.

7. A method of determining effectiveness of an anticancer treatment of a subject with breast cancer or endometrial cancer, the method comprising providing an anti-cancer treatment to the subject followed by detecting cells of the subject expressing estrogen receptors by contacting the compound according to claim 1 with the cells, imaging the cells by positron emission tomography (PET) or single photon emission computed tomography (SPECT), and determining from the imaging whether estrogen receptors are overexpressed in the cells compared to normal cells, wherein the treatment is considered effective if overexpression of estrogen receptors is decreased in the cells compared to before the treatment.

8. A process for the preparation of a compound according to formula (XVIII) ##STR00033## comprising the steps of transforming estra-5(10),9(11)-diene-3,17-dione 3-ethylenedioxy ketal (1) into 11-(4-Hydroxy-phenyl)-estra-4,9-diene-3,17-dione (3) by reacting (1) initially with a peroxide reagent to give an epoxide, treating the product obtained with the Grignard reagent derived from trimethylsilyloxyphenyl bromide to obtain compound (3), alkylating (3) with dimethylaminoethyl chloride to obtain compound (4), aromatizing (4) with acetyl bromide and acetic anhydride to obtain compound (5), reducing and saponifying (5) to obtain compound (6), demethylating (6) with alpha chloroethyl chloroformate to obtain compound (7), and reacting (7) with reagent (8a), ##STR00034## ##STR00035## thereby obtaining the compound according to formula (XVIII).

9. A process for the preparation of a compound according to formula (XIX) ##STR00036## comprising the steps of transforming estra-5(10),9(11)-diene-3,17-dione 3-ethylenedioxy ketal (1) into 11-(4-Hydroxy-phenyl)-estra-4,9-diene-3,17-dione (3), by reacting (1) initially with a peroxide reagent to give an epoxide, treating the product obtained with the Grignard reagent derived from trimethylsilyloxyphenyl bromide to obtain compound (3), alkylating (3) with dimethylaminoethyl chloride to obtain compound (4), aromatizing (4) with acetyl bromide and acetic anhydride to obtain compound (5), reducing and saponifying (5) to obtain compound (6), demethylating (6) with alpha chloroethyl chloroformate to obtain compound (7), and reacting (7) with reagent (8b) ##STR00037## ##STR00038## thereby obtaining the compound according to formula (XIX).

10. A process for the preparation of a compound according to formula (XX) ##STR00039## comprising the steps of: preparing 2-(4-bromophenoxy)-N,N-dimethylethanamine (1) by reacting 4-bromophenol with 2-dimethylaminoethyl chloride, converting (1) to (4-(2-(dimethylamino)ethoxy)phenyl)magnesium bromide (2) by treating with reagents comprising Mg, and iodine, and reacting (2) with 3,3-Ethylenedioxy-5(10)--epoxy-estr-9-ene-17-one (3) in the presence of CuI and acetic acid ##STR00040## thereby obtaining the compound according to formula (XX).

11. A kit comprising a compound according to Formula (II) ##STR00041## wherein m is a number in the range of 4-10; and R.sup.1 is H or a C.sub.1-3 alkyl group; and R.sup.2 is either absent, or selected from the group consisting of H and a C.sub.1-3 alkyl group, a labeling moiety, and instructions for reacting the compound and the labeling moiety to provide a compound according to claim 1, wherein the labeling moiety is a compound of formula (IV) ##STR00042## wherein, n is a number in the range of 1-10.

12. A kit comprising a compound according to Formula (III) ##STR00043## wherein m is a number in the range of 4-10; R.sup.1 is H or a C.sub.1-3 alkyl group; and R.sup.2 is either absent, or selected from the group consisting of H and a C.sub.1-3 alkyl group, a labeling moiety, and instructions for reacting the compound and the labeling moiety to provide a compound according to claim 1, wherein the labeling moiety is a compound of formula (XXI) ##STR00044## wherein n is a number in the range of 1-10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the structures of a set of .sup.18F-radiolabeled estradiols previously described (Bennink, R. J. et al., 2001; Pomper, M. G. et al., 1990; Kiesewetter, D. O. et al., 1984; VanBrocklin, H. F. et al., 1993; VanBrocklin, H. F., 1994; Seimbille, Y. et al., 2002).

(2) FIG. 2 is a diagram of a synthetic scheme for alkynyl and azido 11-antiestrogen precursors.

(3) FIG. 3 is a comparison of NMR spectra and thin layer chromatograms associated with the syntheses of the intermediate 4 (FIG. 2) using a first approach (lower panel) and using a second (alternate) approach (upper panel).

(4) FIG. 4 is a diagram of a scheme for convergent synthesis of .sup.18F-radiolabeling of 11-substituted antiestrogen.

(5) FIG. 5 is a diagram of a scheme for synthesis of .sup.18F radiolabeled 11 substituted estradiol showing variation in click chemistry conditions.

(6) FIG. 6 is a diagram showing eight different click reactions (right) performed, and radiochromatographic purifications (left) for the synthesis of .sup.18F radiolabeled 11 substituted estradiol.

(7) FIG. 7A shows preparative radio HPLC (high performance liquid chromatography) purification of the .sup.18F radiolabeled 11 substituted estradiol product, under different chromatographic conditions. Peaks corresponding to the product and reactants are indicated by arrows. FIG. 7B shows radio HPLC traces confirming the assignment of peaks in the purification shown in FIG. 7A.

(8) FIG. 8 is a diagram showing attachment of different imaging modalities to the alkynyl 11-antiestrogen precursor of FIG. 2 using click chemistry.

(9) FIG. 9 is a diagram showing synthesis of a lipophilic .sup.18F radiolabeled 11 antiestrogen in addition to an amphiphilic .sup.18F radiolabeled 11 antiestrogen.

(10) FIG. 10 is a proton NMR spectrum of demethylated material.

(11) FIG. 11 is a proton NMR spectrum of material with propargyl tetraethylene glycol linker.

(12) FIG. 12 is a carbon-13 NMR spectrum of material with propargyl tetraethylene glycol linker.

(13) FIG. 13 shows an LC-MS scan showing retention times of starting material, tetraethylene glycolated product, and N,N-dimethyl standard.

(14) FIG. 14 is a high resolution mass spectrum of propargylated tetraethylene glycolated material.

(15) FIG. 15 shows high resolution mass spectra of N-demethylated material.

(16) FIG. 16 shows an HPLC trace indicating the purity of propargyl tetraethylene glycolated material.

(17) FIG. 17 shows an HPLC trace indicating the purity of propargyl tetraethylene glycolated material using a different solvent system than in FIG. 16 or FIG. 18.

(18) FIG. 18 shows an HPLC trace indicating the purity of propargyl tetraethylene glycolated material using a different solvent system than in FIG. 16 or FIG. 17.

(19) FIG. 19 is a proton NMR spectrum of the depicted N-fluoropropyl derivative.

(20) FIG. 20 shows an expanded proton NMR spectral region for the N-fluoropropyl derivative.

(21) FIG. 21 is an expanded proton NMR spectral region for the N-fluoropropyl derivative.

(22) FIG. 22 is a carbon-13 NMR spectrum for the N-fluoropropyl derivative.

(23) FIG. 23 shows LC-MS traces for the N-fluoropropyl derivative.

(24) FIG. 24 shows a high resolution mass spectrum for the N-fluoropropyl derivative.

(25) FIG. 25 is a proton NMR spectrum of the depicted N-demethylated antiestrogen.

(26) FIG. 26 is a diagram showing the variation in the conditions for synthesizing .sup.18F-estradiol substituted at the 11-position.

(27) FIGS. 27A and 27B show mass spectra of the compounds depicted above the respective spectra. FIG. 27C is an HPLC analysis of the progress of reaction, and shows peaks corresponding to the reactant .sup.18F-PEG.sub.3-N.sub.3, and the products .sup.18F-11-FES and .sup.19F-11-FES. FIG. 27D shows a preparative HPLC purification of the product obtained as a result of the click reaction between the reactants .sup.18F-PEG.sub.3-N.sub.3 and HCC-11-ES, the alkynyl 11-antiestrogen precursor. Upper trace shows UV absorption and lower trace shows radioactivity.

(28) FIG. 28 shows optimization of conditions of the click reaction for the synthesis of .sup.18F radiolabeled 11 antiestrogen using the reactants .sup.18F-PEG.sub.3-N.sub.3 and the alkynyl and 11-antiestrogen precursor of FIG. 2.

(29) FIG. 29A is a graph of the pharmacokinetics of .sup.18F radiolabeled 11 antiestrogen (.sup.18F-11-FES) showing half-life measurements of the compound in the blood. FIG. 29B is a table of different formulations used in the pharmacokinetics measurements shown in FIG. 29A. FIG. 29C is a graph of the biodistribution of .sup.18F-11-FES. FIG. 29D is a comparison of the biodistribution of .sup.18F-16-FES (left bar of each pair, Kiesewetter (1984)) and .sup.18F-11-FES of the present invention (right bar of each pair).

(30) FIG. 30A is a graph of the biodistribution of .sup.18F-11-FES in a breast cancer model of a xenograft of MCF7 cells in nu/nu mice. FIG. 30B is a graph of comparison of the biodistribution of .sup.18F-11-FES in wild type mice (left bar of each pair) and mice having the MCF7 xenograft (right bar of each pair). FIG. 30C shows a summary of the data shown in FIG. 30B.

(31) FIG. 31 is an image of a 2 hour dynamic PET scan of a C57B1/6 mouse administered with .sup.18F-11-FES. The image shows that within 3 minutes post injection the majority of activity is in the liver (2.sup.nd image from left), from which it quickly moves to the intestines (3.sup.rd image from left). At approximately 15 minutes post injection (far right image), a significant amount of activity has accumulated in the bladder.

DETAILED DESCRIPTION OF THE INVENTION

(32) Described herein are estrogen receptor (ER) targeted, 11-aryl estradiol-based molecular imaging (MI) agents having high specific activity, high specific receptor binding affinity, and metabolic and clearance characteristics that render them useful as imaging agents for mammalian subject, including human subjects.

(33) Previous structure activity relationship (SAR) studies of steroidal estrogens have indicated that small substituents at the 11-position conferred agonist activity, usually with an increase of binding affinity (Dao et al., 2012, and Hanson, R. N.; Hua, E. et al., 2012, and references therein). Alkyl or heteroalkyl groups beyond 3-4 atoms in length have been shown to impart ER antagonist properties, often without a significant loss of binding affinity (Hanson, R. N.; Hua, E. et al., 2012, and references therein). Analysis of the properties of anti-estrogen-doxorubicin bioconjugates has shown that the 11-aryl estradiol-based antiestrogens possessed several key features as potential candidates for MI for (ER+)-breast cancer (Dao et al., 2012). The 11-aryl estradiol-based antiestrogens (AEs) retained high ER affinity, expressed ER antagonist properties, showed selectivity for ER-expressing cancer cells, and a linker between the steroid and doxorubicin improved pharmaceutical properties and allowed convergent attachment of different terminal groups.

(34) The 11-aryl group of the EAs of the present invention occupies a secondary pocket of the ER that extends to the surface. In the antagonist conformation, the 4-position of the aryl group accesses the solvent-exposed space, thereby permitting further conjugation with the imaging moieties of the present invention without interference with receptor binding specificity.

(35) The present invention employs a convergent strategy that allows the imaging tag component (imaging moiety), and the 11-aryl estradiol-based antiestrogen component having a complementary linker at the 4-position of the aryl group, to be prepared separately and then linked with an orthogonal ligation. Further, the preparation of labeled 11- substituted estradiols described herein is modular. The imaging moiety and the 11- substituted estradiol precursor are independently made with high yields and purity.

(36) As a first step toward synthesis of 11 substituted estradiol-imaging conjugates having a radiohalogenated moiety, a high affinity alkynyl 11-phenyl substituted estradiol component (9a) was prepared. Separately, an exemplary radio imaging label component, fluorinated triethylene azide (8b), was prepared. The compound, alkynyl 11-phenyl substituted estradiol (9a), was prepared by two methods, both of which are shown in FIG. 2. The precursor was synthesized from estra-5(10),9(11)-diene-3,17-dione 3-ethylenedioxy ketal (1) and transformed into the key intermediate 11-(4-Hydroxy-phenyl)-estra-4,9-diene-3,17-dione (3). An alkylation step was used to prepare the precursor 11-[4-(2-dimethylamino-ethoxy)-phenyl]-estra-4,9-diene-3,17-dione (4).

(37) Initially the dimethylaminoethoxy group was introduced after the 11 phenyl group to synthesize the intermediate (4). In an alternate approach the side chain was introduced prior to the Grignard reaction. In this route, 4-bromophenol was alkylated with dimethyl chloroethylamine to give 4-bromo-(2-dimethylaminoethoxy) benzene. The Grignard reaction was carried out as the next step to produce the intermediate (4). The alternate approach offered a number of advantages: the synthesis of the steroidal portion was shortened by one step; the yield of the product was higher, and the separation/isolation process was easier (FIG. 3). Further, the 4-bromo phenol starting material is significantly less expensive than the 1-bromo-(4-trimethysilyloxy) benzene, and reaction using the alternate approach was more reproducible than reaction according the initial scheme. Starting from the intermediate (4), the alkynyl antiestrogen was obtained as illustrated in the synthetic scheme shown in FIG. 2. The molecular imaging probe, such as an azido fluorinated ligand, can be attached using the click chemistry of azide-alkyne Husigen cycloaddition employing copper or ruthenium as a catalyst (FIG. 4, and Hanson, R. N. et al., 2012; Dao, K.-L. et al., 2012; Hanson, R. N.; Hua, E. et al., 2012; Adam, H. J. et al., 2012; Dao, K.-L. et al., 2013; Haddad, T. et al., 2012; Hanson, R. N.; Hua, E.; Adam, H. J. et al., 2012; Hanson, R. N.; Kirss, R. et al., 2012; Hanson, R. N.; McCaskill, E. et al., 2012; Olmsted, S. L. et al., 2012).

(38) The preparation of the fluorinated component proceeded in good overall yield. The synthesis began with the -chloro triethylene glycol derivative which underwent displacement with sodium azide to give (10) (FIG. 4). Tosylation gave intermediate (11), which was treated with tetra-n-butylammonium fluoride (TBAF) to give the fluorinated precursor (12). Conversion to the final product 15 was achieved in good yields using classical click conditions. All of the intermediates and final products were characterized by IR, NMR, and LC-MS. The convergent modular approach proved to be a successful strategy for assembling the target anti-estrogen (AE).

(39) To synthesize 11-AE radiolabeled with .sup.18F as shown in the structure in FIG. 5, a variety of different click conditions were investigated to determine the best method to convert the alkynated AE precursor into the radiolabeled click products. Because of the short half-life of .sup.18F radionuclide, it is necessary that the conversion method be fast and proceed in high yield. The Table in FIG. 6 summarizes the click reactions that were carried out with a pre-automated synthesizer (Center for Systems Biology, Mass General Hospital, Boston, Mass.). As the results in the radioHPLC trace (FIG. 5) indicate, the in situ click reaction in conditions 4, 7, and 8 gave the best radiochemical yields for the conversion of .sup.18F-PEG-N.sub.3 to the .sup.18F-AE radioligand. However, method 4 involved an additional ligand, bathophenantrolinedisulfonic acid disodium salt (BPDS) in a mixture of DMSO/H.sub.2O (50:50), potentially resulting in more impurities requiring HPLC purification. Although, the radiochemical yield for method 7 was lower, the reaction conditions are much simpler, and only one catalyst copper (I) tetrakis(acetonitrile)copper(I) hexafluorophosphate was used. In reaction 8, 40 L of 80 mM BPDS was added to the scale up reaction to ensure that adequate amount of Cu.sup.+1 was formed, and was available for catalyzing throughout the click reaction. Indeed, this method gave 95% conversion and produced isolated yields ranging from 9.73 mCi to 9.12 mCi. All the reactions in FIG. 5 used microwave synthesis conditions (temp 60 C., power 30 watts, and 5 minutes), except method 7 in which a 10 min irradiation time was used.

(40) Use of MeCN:H.sub.2O (50:50) with 0.1% TFA as the solvent for HPLC purification resulted in elution of the radiolabeled antiestrogen product at 5 minutes, followed closely by .sup.18F-PEG-N.sub.3 at about 6 minutes. Changing the eluting solvent to MeCN:H.sub.2O (37.5:62.5) with 0.1% TFA resulted in improved separation, with the .sup.18F-radiolabled 11-substituted antiestrogen eluting at about 10 min, .sup.18F-PEG-N.sub.3 eluting at about 7.5 min, and the alkyne precursor eluting at 22 minutes (UV HPLC trace).

(41) Results of preperative radioHPLC of the .sup.18F labeling click product and radioHPLC analysis of the separated compounds demonstrated that 11-AE described herein (9a; FIG. 4) can be radiolabeled with .sup.18F radionuclide using click chemistry with the azido-PEG-.sup.18F ligand (12). The method of synthesis gave a sustainable high activity conversion, and the HPLC purification scheme indicated that the labeling compound is 100% pure (FIG. 7) and is suitable for in vivo imaging.

(42) In addition to the .sup.18F oligoethylene glycol azide (12) used to prepare the ER-targeted radioimaging probe .sup.18F 11-AE (FIG. 5), a series of reagents that can be used for other imaging modalities (FIG. 8), were coupled (clicked) to the 11-AE intermediate described herein (9a) or to other targeting groups. For example, a convergent approach of making a prosthetic metallated .sup.99mTc/Re chelate that can be directly clicked on the alkynyl (9a) or the azido (9b) 11- substituted estradiol (FIG. 2) is described (FIG. 8). The synthesis of the final click product depends on optimal conditions, especially the Cu(I) catalyst ligands and solubility of the reagents in the reaction solvents. The Re(CO).sub.3-11AE was synthesized and characterized.

(43) It is important to note that the click chemistry depicted in FIG. 8 is entirely reversible. That is, any of the labeling moieties depicted can terminate in either an azido group and be reacted with a non-labeled 11-AE precursor terminating in an alkynyl group, or any of the labeling moieties can terminate in an alkynyl group and be reacted with a non-labeled 11-AE precursor terminating in an azido group. The invention contemplates each such possible combination, and the corresponding methods of synthesizing an ER imaging agent using each and every such combination.

(44) 11-aryl estradiol-based antiestrogens labeled with Raman active moieties as MI agents are also described herein. Surface-enhanced Raman scattering (SERS) spectroscopy in combination with optical microscopy yields a novel noninvasive and label-free method to assess and image cellular processes based on their biochemical changes (Yigit, M. V. et al., 2011; Qian, X. et al., 2008). The advantage of using Raman spectroscopy over conventional optical imaging method is that it affords minimal sample preparation, high sensitivity to small intracellular fluctuations, as well as high spatial resolution. Raman spectra from within the cell reflect the biochemical composition found within the laser focal volume of approximately 0.3-1.3 m.sup.3 in size, which is determined by the diffraction limit of the applied laser light. Using the spectral parameters of a cell's components, it is possible to image cellular organelles such as the nucleus, chromatin, mitochondria, and lipid bodies at the resolution of conventional microscopy without the use of external labels or dyes (Yigit, M. V. et al., 2011; Chernenko, T. et al., 2009; Kneipp, J. et al., 2009).

(45) In general, the technique is based on identification of molecular vibrations that are characteristic of distinct functional group in a molecule. The functional groups used as the Raman active moiety for coupling to 11-aryl substituted estradiol herein are deteuruated triphenyphosphine (dTPP) and cyano benzyl derivatives (FIG. 8). Vibrational spectra are obtained by illuminating the specimen either with infrared radiation or laser light in the visible range of the spectrum. This method of obtaining vibrational spectra is based upon the inelastic scattering of photons and is known as the Raman effects (Alhasan, M. K. et al., 2012; Pysz, M. A. et al., 2010; Chernenko, T. et al., 2009). The dTPP and azido cyano benzyl labels (FIG. 8) were coupled to the alkynyl and azido 11-substituted estradiol described herein (FIG. 2) using click conditions to form in vitro Raman imaging probes for ER hormone dependent breast cancer cells. The probes were characterized and results obtained indicate that the Raman labels are nontoxic, stable, do not suffer from photobleaching, and possess a distinctive and specific Raman signature at near 2100 cm.sup.1. In the local optical field of the labels SERS also provides sensitive information about the immediate molecular environment of the label, e.g. dTPP in the cell, thereby allowing imaging the native cellular constituents and organelles.

(46) Described herein also is an exemplary traditional fluorescent probe, fluorescein isothiocyanate (FITC), modified with complementary oligo ethoxy glycol (OEG) linker (FIG. 8), which is subsequently attached to 11AE (9a FIG. 2) to form a fluorescent probe for ER imaging. Such probe is useful in the measurement of cellular binding uptake and/or cellular internalization of the designed ligands by ER.

EXAMPLES

Example 1. Modification of Steroid precursor(11R,13S)-11-(4-(2-(dimethylamino)ethoxy)phenyl)-13-methyl-7,8,11,12,13,14,15,16-octahydro-1H-cyclopenta[a]phenanthrene-3,17(2H,6H)-dione (intermediate 4; FIG. 2)

Synthesis of 2-(4-bromophenoxy)-N,N-dimethylethanamine

(47) ##STR00023##

(48) To a solution of 4-bromophenol (6.8 g, 40 mmol) in DCM (150 mL) CsCO.sub.3 (19.5 g, 100 mmol) was added, and mixture was allowed to stir at room temperature for about 1 hour. Next, 2-dimethylaminoethyl chloride hydrochloride (5.6 g, 40 mmol) was added to the mixture. The reaction was stirred at reflux for approximately 24 hours. The mixture was then filtered and the filtrate was concentrated to yield a clear oily crude material. The crude material was purified using silica gel chromatography (100% DCM then 1-10% MeOH in DCM) to obtain pure 2-(4-bromophenoxy)-N,N-dimethylethanamine (4 g, 16 mmol, 42% yield). .sup.1H NMR (500 MHz, CHLOROFORM-d) 7.33 (d, J=8.79 Hz, 2H), 6.78 (d, J=8.79 Hz, 2H), 3.99 (t, J=5.37 Hz, 2H), 2.64-2.88 (m, 2H), 2.31 (br. s., 6H). .sup.13C NMR (100 MHz, CHLOROFORM-d) 158.1, 132.3, 116.6, 66.4, 58.3, 46.1.

Synthesis of (4-(2-(dimethylamino)ethoxy)phenyl)magnesium bromide

(49) ##STR00024##

(50) Glassware for the synthesis was rinsed with DCM and acetone, and dried in an oven overnight at 113 C. The apparatus for the reaction was assembled and flame dried with a propane torch. Magnesium turnings (3 g, 125 mmol) were placed into a three-necked flask and kept overnight in an oven heated to 113 C. After it was cooled, the apparatus was placed under argon atmosphere. Distilled anhydrous THF (50 mL) was then added to the three-necked flask followed by the addition of a small granule of iodine which immediately turned the color of the contents of the flask to orange. The mixture was allowed to stir for roughly 12 minutes followed by the addition of 2-(4-bromophenoxy)-N,N-dimethylethanamine (1 ml dissolved in 30 mL of THF) in 5 mL aliquots every 20 minutes until half of the 2-(4-bromophenoxy)-N,N-dimethylethanamine (4 g, 16 mmol) was added to the mixture. The mixture was heated to about 65 C. Next, the remainder of the 2-(4-bromophenoxy)-N,N-dimethylethanamine was added in 0.5 mL aliquots every 20 minutes. As a result, the contents first became greenish yellow, and then went to lighter yellow and finally became greenish smoky grey. The reaction mixture was heated for another hour and then cooled to room temperature.

Synthesis of (11R,13S)-11-(4-(2-(dimethylamino)ethoxy)phenyl)-13-methyl-7,8,11,12,13,14,15,16-octahydro-1H-cyclopenta[a]phenanthrene-3,17(2H,6H)-dione

(51) ##STR00025##

(52) 3,3-Ethylenedioxy-5(10)--epoxy-estr-9-ene-17-one (2.014 g, 6.1 mmol) was dissolved in anhydrous THF (15 mL) under argon. Copper (I) iodide (0.160 g, 0.840 mmol) was added to the solution at 10 C. and stirred for 15 min Freshly prepared Grignard reagent, (4-(2-(dimethylamino)ethoxy)phenyl)magnesium bromide, was added dropwise in 5.0 mL aliquots. The reaction was gradually warmed to ambient temperature and stirring was continued for 16 hours. The reaction was quenched by the addition of ammonium chloride (0.8 g, 15 mmol) in 35 mL of water and 35 mL of EtOAc at 10 C. The organic layer was washed with water (235 mL). The organic solvent was removed under reduced pressure and the resulting residue was dissolved in a mixture of acetic acid (14 mL) and water (6 mL). The resultant mixture was warmed at 50-60 C. for 1.5 hours, after which it was diluted with ethyl acetate (20 mL). The solution was neutralized by the addition of saturated aqueous sodium bicarbonate. The organic layer was separated, washed with brine solution, dried over magnesium sulfate and evaporated to dryness to give a crude, yellow oil. Purification using silica gel column chromatography (70:30 hexane/ethyl acetate) afforded the desired product (2.00 g, 76%) as yellow solid: .sup.1H NMR (CDCl.sub.3, 400 MHz): 0.53 (3H, s), 4.38 (1H, d, J=6.9), 5.78 (1H, s), 6.71 (2H, d), 6.97 (2H, d). .sup.13C NMR (CDCl.sub.3, 100 MHz): 14.17, 21.76, 25.99, 27.06, 30.68, 35.02, 36.87, 38.14, 38.34, 39.71, 47.53, 50.77, 115.53, 122.82, 128.30, 129.83, 135.52, 145.62, 155.59, 155.99, 197.54, 217.44; m.p. 248 C.

Example 2. Pharmacokinetics and Biodistribution Analysis of 18F-11-FES

(53) Pharmacokinetics measurements were carried out after administration of .sup.18F-11-FES to C57 BL/6 mice (FIGS. 29 A and B). A number of co-solvents, dimethyl sulfoxide (DMSO, 5%), 4% 1:1 Solutol:dimethyl acetamide (DMA), and ethanol (8%) were tested. A 36% reduction in terminal concentration of .sup.18F-11-FES was found when ethanol was used as a co-solvent. Biodistribution measurements showed that other than kidney, liver, blood and feces, which are associated with excretion, the highest concentration of .sup.18F-11-FES was found in the uterus (an ER expressing organ) (FIG. 29C). A comparison of the biodistribution of .sup.18F-11-FES and .sup.18F-16-FES (Kiesewetter, J. Nucl. Med., 1984, vol. 25, 1212) is shown in FIG. 29 D.

Example 3. Biodistribution of 18F-11-FES in an Animal Model of Breast Cancer

(54) Biodistribution of .sup.18F-11-FES was also measured in Nu/Nu mice implanted with a xenograft of MCF7 breast cancer cells. ER expressing tumors and uterus were each observed to accumulate substantial amounts of .sup.18F-11-FES, which was higher than in any other tissue/material not associated with excretion, with the exception of bone (FIGS. 30 A and C). Similar results were obtained when the biodistribution of .sup.18F-11-FES in the tissues of Nu/Nu mice implanted with breast cancer xenograft was compared with that in the tissues of wild type C57BL/6 mice (FIGS. 30 B and C).

Example 4. PET Scan of 18F-11-FES in C57BL/6 Mouse

(55) A PET scan was carried out on a C57BL/6 mouse administered with .sup.18F-11-FES. A 2 hour dynamic PET scan of the mouse is shown in FIG. 31. Within 3 minutes post injection the majority of activity was found to be localized in the liver (second panel from the left), form which the label was observed to quickly localize to the intestines (third panel from the left). A significant amount of activity was observed to accumulate in the bladder approximately 15 minutes post injection (right panel).

Example 5. F-11-AE with Optimized N-Linker

(56) An ER-specific MI compound should have high uptake and retention within target tissues (e.g., uterus and tumor), lower concentration in blood and nontarget tissue, and increased renal clearance. The single factor most likely to influence those parameters is the basicity/hydrophobicity of the nitrogen. Unlabeled N-modified F-11- intermediates shown below (right) are candidate molecules that have modification of the linker to yield reduced values of c Log P.

(57) ##STR00026##

(58) Substitution on the N-group includes secondary to quaternary and carbonyl groups on either side (amide). Synthesis of these unlabeled F-11-AE derivatives is illustrated below.

(59) ##STR00027## ##STR00028##
Subsequent ligation to fluorotriethylene glycol azide provides the final compounds as the unlabeled derivatives. The final compounds are all more hydrophilic than the F-11-AE described herein by 0.5-2.5 log units. Analysis of the binding, cell penetration and metabolic properties is carried out to indicate the influence various substituents on the nitrogen has on biological properties. Based upon the results, one or more compounds are selected for evaluation as their [.sup.18F]-analogs.

(60) The relative binding affinity (RBA) of the compounds is determined using a competitive binding assay with the full-length recombinant ER- and ER- from PanVera (Madison, Wis.). Because many breast cancer cells express both subtypes, the binding affinity of the alkynyl precursor and final F-11-AE is determined for both subtypes. F-11-AE described herein and E2 are used as controls.

(61) Efficacy studies for the synthesized F-11-AE compounds are performed using a cell-based inhibition ER trans-activation assay. The assay uses heLA, HepG and MCF-7 cells transfected with plasmids encoding ER (or empty vector) using lipofectamine-2000 (Invitrogen). The cells are treated with increasing concentrations of the compounds or controls, followed by harvesting at 20-24 h. Evaluation for luciferase activity using the Luciferase Assay Kit (Promega) provides data from which the efficacy (IC.sub.50) is determined Compounds having c Log P values in the 1.5-4.5 range are selected for labeling and use as imaging agents.

(62) In vitro metabolic evaluation of representative 18F-11-AE is performed as follows. In vitro metabolism measurements utilizes the rat hepatocyte culture, as previously described (Jonson et al., 1999). Rodents do not have steroid hormone binding globulin (SHBG) or Human serum albumin (HAS) and conditions are adjusted to account for this. The compounds of interest are labeled using click chemistry (FIG. 2) and added (100 L, 1.0 mCi) to the cells (3.9 mL). After a brief swirling to initiate the process, an aliquot (0.1 mL) is removed, transferred to a 0.5 mL centrifuge tube containing ethanol (0.1 mL). The TO sample is vortexed, sonicated, and centrifuged to separate supernatant from the cell pellet. Additional aliquots are removed and processed in a similar fashion at predetermined time intervals. The supernatant (20 L) is spotted on TLC plates and developed with an appropriate solvent system. Air dried plates are scanned using an automated radio-TLC scanner and the metabolites/parent compound radiotracer peaks are integrated to determine metabolic disposition. The effect of SHBG on metabolism is determined by repeating the previous experiments but adding human SHBG (80 g, 1 mg/mL in buffer) to the isolated hepatocytes prior to the addition of the 18F-11-AE derivative. The control is the hepatocyte culture with just addition of the buffer solution. The effects of HSA, for which the antiestrogen may have affinity, are examined similarly.

(63) The in vivo bioistribution, pharmacokinetic and metabolic stability measurements of the [18F]-F-11-AE synthesized in this Example are performed as follows. The labeled compounds are evaluated initially in normal female mice to determine uterine uptake (% ID/g) and selectivity (T/NT). Imaging also provides data regarding non-target disposition and major clearance (hepatic vs renal) pathways. Each compound is compared to .sup.18F-FES. Uptake of high doses of estradiol demonstrates specific ER binding in target tissues. Based upon the preliminary studies, the compounds with the best uterine uptake values, highest selectivity (uterus to nontarget tissue) and clearance pathway (renal>hepatic) compared to FES is evaluated in implanted breast tumor models. Tissue distribution, selectivity and clearance are determined Procedures for both normal and tumor bearing animal studies are well established. The results are analyzed to evaluate the effect of RBA, c Log P, and other effects of the substituents on each of the biological parameters associated with effective imaging. The compound(s) with the best properties are evaluated more extensively for their in vivo metabolism.

(64) Metabolism in vivo of the [18F]-F-11-AE is evaluated using normal female rats. Groups of rats are sacrificed at 30, 60 and 120 minutes post administration of the radiotracer. Blood is separated to give protein bound and free components and each is evaluated by radio-TLC to determine the number and type of radiolabeled species. Liver and bladder are examined to determine the number and type of labeled compounds present in both at each time point. The values are compared to results found with FES. In rats, FES is rapidly metabolized to more polar species. By 60 minutes after injection, less than 15% of circulating radioactivity is due to [18F]-FES; the remainder is metabolites. It has been reported that injection of blood from rats obtained 2 hours after injection into different rats showed that the metabolites did not accumulate in ER-rich tissues (Mathias, C. J. et al., 1987). The extent to which the 11-substituent modifies the metabolism of the radiotracer is determined. The structural modifications is expected to lead to an uptake in the target tissue, comparable to FES (FIG. 4). Reduction of the c Log P values is expected to reduce hepatic uptake and clearance with faster elimination via renal excretion. This produces lower blood levels and less colonic background. Faster blood clearance lowers retention in nontarget tissues, resulting in improved T/NT values. Reduced in vivo metabolism also leads to better pharmacokinetic properties.

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