Integrated “3+1” Oxorhenium(V) Complexes as Estrogen Mimics

Bioconjugate Chem. 1999, 10, 119−129

119

Integrated “3+1” Oxorhenium(V) Complexes as Estrogen Mimics Marc B. Skaddan, and John A. Katzenellenbogen* Department of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, Illinois 61801. Received August 19, 1998; Revised Manuscript Received October 7, 1998

The diagnosis and staging of breast cancer could be improved by the development of imaging radiopharmaceuticals that provide a noninvasive determination of the estrogen receptor (ER) status of tumor cells. Toward this goal, we have synthesized a number of integrated “3+1” oxorhenium(V) complexes designed to mimic estradiol and a class of nonsteroidal estrogens, the tetrahydrochrysenes (THC). The monodentate component of the estradiol mimic is a p-hydroxyphenethyl thiol ligand with ethyl substituents at the benzylic and homobenzylic positions. Model complexes of this ligand were easily made, but steric hindrance of the secondary thiol prevented the formation of the complex with the disubstituted ligand. The three “3+1” oxorhenium(V) complexes prepared to mimic the THC class mimics represent the first pyridinedithiol rhenium complexes of their kind to be made. These complexes are quite stable to air and moisture. The target tridentate ligand was prepared from chelidamic acid, and the VT NMR of the rhenium complex displays interesting fluxional behavior. The binding affinities of these complexes for the estrogen receptor are low, and their lipophilicities are rather high. Nevertheless, our findings provide a further refinement of our understanding of ligand structurebinding affinity correlations for the estrogen receptor.

INTRODUCTION

Metastatic breast cancer is the most prevalent form of cancer of women in the United States and is the number one killer of women ages 40-55 (1). Recent figures show that a woman’s lifetime risk of developing breast cancer is 1 in 8 (2). Early detection is imperative for breast cancer survival, as the 5 year survival rate for localized breast cancer is 96% (3). However, if the cancer has spread regionally, the survival rate drops to 75%, and distant metastases further reduce the survival rate to 20% (3). Clearly, better diagnostic techniques for early detection would increase survival. Breast cancers that retain estrogen receptor (ER)1 can usually be treated effectively by endocrine therapy, such as the use of the antiestrogen tamoxifen. In fact, about 60% of ER+ patients respond to endocrine therapy, whereas the response of ER- patients is much lower (4). However, only 70% of breast cancers are ER+; so, it is important to be able to determine the ER status of each tumor. Current methods used to determine the ER status of breast tumor cells are invasive and do not fully account for cell heterogeneity. One way to eliminate the invasiveness and minimize the heterogeneity problems of current tumor ER assays would be to image breast tumors based on their content of ER, using positronemission tomography (PET) or single photon emission computed tomography (SPECT). These techniques utilize the emission characteristics of certain radionuclides attached to radiopharmaceuticals that have appropriate distribution properties to image tissues. Considerable * To whom correspondence should be addressed. Phone: (217) 333-6310. Fax: (217) 333-7325. E-mail: [email protected]. 1 Abbreviations: ER, estrogen receptor; DHP, dihydropyran; MEM, methoxyethoxymethyl; MOPS, 3-morpholinopropanesulfonic acid; PET, positron emission tomography; RBA, receptor-binding affinity; SPECT, single photon emission computerized tomography; THC, tetrahydrochrysene.

advances have been made in the development of steroids labeled with either fluorine-18 for PET imaging (5-16) or iodine-123 for SPECT imaging (5, 17-21) of ER+ breast tumors. However, the radionuclide of choice for breast tumor imaging would be Tc-99m, due to its convenient 6 h half-life and its wide availability. We have been interested in the development of Tc-99mcontaining steroid mimics for use as imaging agents for receptor-positive breast tumors. In the “conjugated” or “pendant” design, a Tc-99m containing moiety is tethered to an existing steroid, such as progesterone or estradiol (1) (22-29). Such complexes are generally stable, but their large overall size can greatly affect their physical properties and their affinity for steroid receptors (22, 29). Smaller complexes are produced by the alternative “integrated” approach, in which part of the steroid backbone itself is replaced by the requisite Tc-99m chelate (30-32). One way to incorporate Tc-99m into such integrated complexes is via the “3+1” design, in which a tridentate and monodentate ligand surround an oxometal core (33). The advantage of such a design is the wellestablished stability of many “3+1” complexes themselves (27-29, 33-35), compared with that of the related 2+2 complexes. In addition, appropriately designed “3+1” complexes lack syn and anti stereoisomers that are often produced in the tetradentate oxorhenium and technetium complexes. We report here the synthesis of several “3+1” oxorhenium complexes that are in the series of the estradiol mimic 2 and the tetrahydrochrysene (THC) mimic 4 (Figure 1). The syntheses involve rhenium as the surrogate metal for Tc-99m (36). As is illustrated in Figure 1, estradiol mimic 2 would contain a tridentate aliphatic ligand (where the heteroatom X could be a sulfur, oxygen, or alkyl nitrogen), intended to mimic the D-ring of estradiol. The monodentate ligand is aromatic, with a 2 carbon “spacer” separating the phenyl ring from the thiol, and is intended to mimic the A, B, and C rings of estradiol. The THC mimic 4 involves thiophenol as the

10.1021/bc980094q CCC: $18.00 © 1999 American Chemical Society Published on Web 12/11/1998

120 Bioconjugate Chem., Vol. 10, No. 1, 1999

Figure 1. Estrogens and their mimics.

monodentate ligand and a unique pyridinedithiol as the tridentate ligand. In this case, both ligands are equally involved in mimicking the rings of THC. EXPERIMENTAL SECTION

General. All reagents and solvents were obtained from Aldrich, Acros, Alfa Aesar, or Fisher. All reactions were performed under a nitrogen atmosphere unless otherwise indicated. THF was distilled from sodium/benzophenone immediately prior to use. Methylene chloride was distilled from CaH2 prior to use. Triethylamine was dried by distillation from CaH2 and stored over KOH. Dimethylformamide was distilled from and stored over 4 Å molecular sieves. MeOH was distilled from Mg/I2 and stored over 3 Å molecular sieves. Hexanes were distilled from calcium sulfate (Drierite) before use in flash chromatography. Reaction progress and flash chromatography was monitored by analytical thin-layer chromatography. Silica gel used in flash chromatography was 32-63 µm. Radial chromatography (Chromatotron, Harrison Research, model 7924T) was performed using a circular glass plate with a 2 mm depth of silica gel 60 PF254 (with gypsum). TLC plates were visualized using short-wave UV light (254 nm), potassium permanganate, or phosphomolybdic acid. 1H and 13C NMR were obtained at 400 or 500 MHz and are reported in parts per million relative to incomplete deuteration signals of CDCl3, CD3OD (CD3 signal used), DMSO-d6 or acetone-d6. Mass spectra were obtained with a VG Instruments 70-VSE (EI, CI) or a VG Instruments ZAB-SE (FAB) mass spectrometer. Melting points are uncorrected. Elemental analyses were performed by the Microanalytical Service Laboratory of the University of Illinois. 4-Methoxyphenethyl Thioacetate (8a). MsCl (770 µL, 9.94 mmol) was added dropwise to a solution of 4-methoxyphenethyl alcohol (1.01 g, 6.63 mmol) and Et3N (1.40 mL, 9.94 mmol) in THF (40 mL) at 0 °C. A white ppt of Et3N hydrochloride formed immediately. The reaction was stirred for 1 h, at which time potassium thioacetate (2.27 g, 19.9 mmol) in DMF (10 mL) was added, resulting in an orange solution which turned red after several h. After 6 h, the reaction was stopped and concentrated, then dissolved in CH2Cl2 (60 mL). This was washed with saturated LiCl twice, followed by a wash with brine and then water (30 mL). The brine and water washes were separately back-extracted with CH2Cl2 (30 mL). The organics were then combined, dried over Na2SO4, filtered, and concentrated to a dark oil. Flash

Skaddan and Katzenellenbogen

chromatography (60% CH2Cl2/PhH) and radial chromatography (30% EtOAc/Hex), followed by Kugelrohr distillation (125 °C, 0.9 mmHg) provided 8a as a yellow oil (1.06 g, 76%). 1H NMR (CDCl3, 400 MHz): δ 2.33 (s, 3H, -SCOCH3), 2.95 [ABq, JAB ) 7.3 Hz, ∆υ ) 119 Hz, 2H, Ar(CH2)2SCOCH3], 2.95 [ABq, JAB ) 6.3 Hz, ∆υ ) 102 Hz, 2H, Ar(CH2)2SCOCH3], 3.79 (s, 3H, ArOCH3), 6.84 (d, J ) 8.8 Hz, 2, ArH), 7.14 (d, J ) 8.9 Hz, 2H, ArH). 13C NMR (CDCl , 100 MHz): δ 30.6, 30.7, 34.8, 55.2, 3 113.8, 129.5, 132.0, 158.2, 195.7. MS (EI, 70 eV): m/z 210 (M+, 5), 134 (100), 121 (64). HRMS calcd for C11H14O2S: 210.0715. Found: 210.0715. 4-Methoxyphenethyl Thiol (9a). A solution of 1 M HCl in MeOH/dioxane was prepared by diluting commercially available 4 M HCl/dioxane with freshly distilled, degassed MeOH. Following Okuno’s procedure (37), 4 mL of this solution was added to 8a (200 mg, 0.951 mmol) and the reaction was refluxed at 80 °C under Ar for 3 h. The reaction was then concentrated under high vacuum overnight, providing an orange oil which was Kugelrohr distilled (65 °C, 0.2 mmHg) to provide 9a as a pungent-smelling yellow oil (70.1 mg, 44%). 1H NMR (CDCl3, 400 MHz): δ 1.37 (t, J ) 7.8 Hz, 1H, -SH), 2.75 (m, 2H, ArCH2CH2SH), 2.87 (t, J ) 7.3 Hz, 2H, ArCH2CH2SH), 3.80 (s, 3H, ArOCH3), 6.85 (d, J ) 8.7 Hz, 2H, ArH), 7.11 (d, J ) 8.7 Hz, 2H, ArH). MS (CI, CH4): m/z 168 (M+, 22), 135 (100), 61 (23). HRMS calcd for C9H13OS: 169.0686. Found: 169.0687. (4-Methoxyphenethylthiolato)(3-thiapentane-1,5dithiolato)oxorhenium(V) (5a). A solution of bis(2mercaptoethyl)sulfide (35.5 µL, 0.272 mmol) and 9a (45.8 mg, 0.272 mmol) in CH3CN (1 mL) was added dropwise to a solution of [BnEt3N]ReOCl4 (38) (146 mg, 0.272 mmol) in MeOH (1 mL) at 0 °C, followed by Et3N (114 µL, 0.816 mmol). A brown solution immediately formed. After stirring for 1 h, the crude mixture was concentrated and purified by flash chromatography (CH2Cl2) to produce 5a as a foamy red solid (78.7 mg, 55%). IR (NaCl): 959 cm-1 (RedO); mp 51-53 °C (dec). 1H NMR (CDCl3, 400 MHz): δ 1.95 [ddd, J ) 14.6, 10.1, 4.7 Hz, 2H, -S(CH2)2S-], 3.07-3.17 [m, 4H, -S(CH2)2Ar-], 3.80 (s, 3H, ArOCH3), 3.91 [ddd, J ) 10.2, 4.1, 1.5 Hz, 2H, -S(CH2)2S-], 4.00-4.06 [m, 2H, -S(CH2)2S-], 4.29 [ddt, J ) 13.4, 4.9, 1.3 Hz, 2H, -S(CH2)2S-], 6.85 (d, J ) 8.6 Hz, 2H, ArH), 7.24 (d, J ) 8.6 Hz, 2H, ArH). MS (CI, CH4): m/z 523 (M + H+ for 187Re, 3), 521 (M + H+ for 185 Re, 2), 227 (19), 195 (51), 135 (100), 121 (20). HRMS calcd for C13H20O2S4187Re: 520.9876. Found: 520.9893. 4-Hydroxyphenethyl Thioacetate (8b). Diisopropyl azodicarboxylate (4.27 mL, 21.7 mmol) was added dropwise to a solution of PPh3 (5.70 g, 21.7 mmol) in THF (75 mL) at 0 °C. A white precipitate of the ylide formed, and the heterogeneous solution was stirred in an ice bath for 30 min. A solution of the 4-hydroxyphenethyl alcohol (1.00 g, 7.24 mmol) in THF was added slowly to the ylide, followed by thiolacetic acid (1.55 mL, 21.7 mmol). The reaction was stirred for 1 h at 0 °C, then room-temperature overnight, changing from a white to green to a golden yellow color during this time. The reaction was then concentrated and purified by flash chromatography twice (2.5% MeOH/CH2Cl2 and 2.5% acetone/CH2Cl2), yielding 8b as a yellow oil (756 mg, 53%). 1H NMR (CDCl3, 400 MHz): δ 2.34 (s, 3H, -SCOCH3), 2.95 [ABq, JAB ) 7.2 Hz, ∆υ ) 127 Hz, 2H, Ar(CH2)2SCOCH3], 2.95 (ABq, JAB ) 6.1 Hz, ∆υ ) 110 Hz, 2H, Ar(CH2)2SCOCH3), 6.07 (s, 1, ArOH), 6.80 (d, J ) 8.3 Hz, 2H, ArH), 7.07 (d, J ) 8.1 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz): δ 30.6, 30.8, 34.7, 115.3, 129.6, 131.8, 154.3, 197.3. MS (EI,

Oxorhenium Complexes Estrogen Mimics

70 eV): m/z 197 (M + H, 44), 120 (100), 107 (66). HRMS calcd for C10H12O2S: 196.0558. Found: 196.0558. 4-Hydroxyphenethyl Thiol (9b). Ten milliliters of 1 M HCl in MeOH/dioxane (vide supra) was added to 8b (466 mg, 2.37 mmol), and the reaction was refluxed at 80 °C under Ar for 3 h. Following evaporation of the solution under high vacuum, Kugelrohr distillation (75 °C, 0.1 mmHg) provided 9b as a near-colorless oil (338 mg, 92%). 1H NMR (CDCl3, 400 MHz): δ 1.37 (t, J ) 7.7 Hz, 1H, SH), 2.75 [m, 2H, Ar(CH2)2SH], 2.85 [m, 2H, Ar(CH2)2SH], 4.70 (s, 1H, ArOH), 6.78 (d, J ) 8.4 Hz, 2H, ArH), 7.07 (d, J ) 8.1 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz) δ 26.3, 39.2, 115.3, 129.8, 132.0, 153.9. MS (EI, 70 eV): m/z 154 (M+, 27), 120 (81), 107 (100). Anal. Calcd for C8H10OS: C, 62.30; H, 6.54. Found: C, 62.28; H, 6.59. (4-Hydroxyphenethylthiolato)(3-thiapentane-1,5dithiolato)oxorhenium(V) (5b). A solution of bis(2mercaptoethyl)sulfide (39.3 µL, 0.301 mmol) and 9b (46.4 mg, 0.301 mmol) in CH3CN (0.8 mL) was added dropwise to a solution of [BnEt3N]ReOCl4 (161 mg, 0.301 mmol) in MeOH (1 mL) at 0 °C under Ar, followed by Et3N (1 drop). A red precipitate immediately formed. After stirring for 2 h at 0 °C, the crude mixture was concentrated and purified by radial chromatography (1% acetone/ CH2Cl2) to produce a red residue. This was dissolved in CH2Cl2, and ether was slowly added over the top of the CH2Cl2 layer. Crystals immediately formed. After 24 h, the mixture was filtered to give 5b as red crystals (37.4 mg, 24%). IR (KBr): 955 cm-1 (RedO); mp 153-155 °C (dec). 1H NMR (CD2Cl2, 400 MHz): δ 1.97 [ddd, J ) 14.5, 10.4, 5.0 Hz, 2H, -S(CH2)2S-], 3.02 [m, 2H, Ar(CH2)2S], 3.10 [ddd, J ) 14.4, 13.2, 4.1 Hz, 2, -S(CH2)2S-], 3.95 [m, 2H, Ar(CH2)2S-], 4.29 [ddt, J ) 13.4, 4.8, 1.2 Hz, 2H, -S(CH2)2S-], 5.19 (s, 1H, ArOH), 6.77 (d, J ) 8.5 Hz, 2H, ArH), 7.17 (d, J ) 8.3 Hz, 2H, ArH). 13C NMR (CD2Cl2, 100 MHz): δ 38.8, 39.1, 44.1, 47.0, 115.3, 129.9, 132.9, 155.4. MS (FAB): m/z 509 (M+ for 187Re, 16), 507 (M+ for 185Re, 10), 307 (74), 289 (48), 154(100), 136 (94). HRMS Calcd for C12H18O2S4187Re: 508.9747. Found: 508.9741. 2-(4-Hydroxyphenyl)butanoic Acid (12). Following the procedure by Aaron (39), sodium (1.58 g, 69.2 mmol) was added to a solution of naphthalene (8.87 g, 69.2 mmol) in THF (60 mL) and was stirred for 4 h with occasional sonication, during which time it turned the characteristic deep blue color. This was added to a solution of 4-methoxyphenylacetic acid (5.00 g, 30.1 mmol) in THF (70 mL) dropwise, which was then stirred for several h. The reaction was then cooled in an ice bath, and to it, EtBr (2.56 mL, 34.3 mmol) was added dropwise. The ice bath was removed, and the reaction was stirred overnight. It was then quenched with water (20 mL), concentrated, dissolved in ether, and extracted with 1 M Na2CO3 (3 × 25 mL). The extracts were washed with ether, then acidified and extracted with ether. The ether extracts were dried with Na2SO4, concentrated, and distilled (131 °C, 0.5 mmHg) to provide 11 as a yellow oil (5.28 g), which was used in the next step without further purification. The yellow oil was refluxed with HBr (15 mL) and acetic acid (15 mL) for 16 h. It was neutralized with saturated Na2CO3, extracted into CH2Cl2, washed with saturated Na2CO3, and acidified with HCl. The aqueous washes were back-extracted with CH2Cl2. The organics were combined, dried with Na2SO4, and purified by flash chromatography (5% MeOH/CH2Cl2 to 10% MeOH/ CH2Cl2) to give 12 as a slightly yellow solid (715 mg, 13% two-step yield): mp 125.5-129 °C. 1H NMR (CD2Cl2/ CD3OD, 500 MHz): δ 0.87 (t, J ) 7.4 Hz, 3H, -CH3),

Bioconjugate Chem., Vol. 10, No. 1, 1999 121

1.71 (dquin, J ) 13.7, 7.4 Hz, 1H, -CH2Me), 2.02 (dquin, J ) 13.4, 7.5 Hz, 1H, -CH2Me), 3.36 (t, J ) 7.6 Hz, 1H, CH), 6.75 (d, J ) 8.7 Hz, 2H, ArH), 7.13 (d, J ) 8.4 Hz, 2H, ArH). 13C NMR (CD3OD, 100 MHz): δ 12.5, 27.8, 54.0, 116.2, 129.9, 131.7, 157.5, 178.5. MS (CI, CH4): m/z 181 (M + H+, 49), 163 (15), 149 (11), 135 (100), 121 (8), 107 (14). HRMS Calcd for C10H13O3: 181.0865. Found: 181.0870. 2-(4-Hydroxyphenyl)butanol (13). A solution of 12 (180 mg, 0.924 mmol) in THF (10 mL) was added to a solution of LAH (4.33 mg, 4.7 mmol) in THF (1 mL), and the reaction was stirred overnight. The reaction was quenched with saturated potassium sodium tartrate, and the gummy aluminum salts from the reaction were filtered through Celite. Concentration, followed by radial chromatography (2.5-10% MeOH/CH2Cl2) provided 13 as an off-white solid (34.9 mg, 23%). 1H NMR (CDCl3/ CD3OD, 500 MHz): δ 0.80 (t, J ) 7.4 Hz, 3H, -CH3), 1.49 [ddq, J ) 13.6, 9.5, 7.4 Hz, 1H, -CH(CH2OH)CH2CH3], 1.68 [dqd, J ) 13.5, 7.4, 5.4 Hz, 1H, -CH(CH2OH)CH2CH3], 2.61 [ddt, J ) 9.5, 8.4, 5.4 Hz, 1H, -CH(CH2OH)Et], 3.66 [dd, J ) 10.8, 8.4 Hz, 1H, -CH(CH2OH)Et], 3.75 [dd, J ) 10.8, 5.5 hz, 1H, -CH(CH2OH)Et], 6.75 (d, J ) 8.7 Hz, 2H, ArH), 7.02 (d, J ) 8.6 hz, 2H, ArH). 13C NMR (CD2Cl2, CD3OD, 100 MHz): δ 12.0, 25.4, 49.9, 67.5, 115.6, 129.4, 133.9, 155.5. MS (CI, CH4): m/z 167 (M + H+, 27), 149 (33), 135 (100), 107 (97). HRMS Calcd for C10H15O2: 167.1072. Found: 167.1078. 2-(4-Hydroxyphenyl)butyl Thioacetate (14). DIAD (83.0 µL, 0.420 mmol) was added dropwise to a solution of PPh3 (110 mg, 0.420 mmol) in THF (5 mL) at 0 °C. Following formation of the white ylide precipitate, the solution was stirred for 30 min, at which time a solution of 13 (34.9 mg, 0.210 mmol) in THF was added dropwise, followed by thiolacetic acid (1.54 mL, 21.6 mmol). The reaction was stirred for 1 h at 0 °C, then overnight at room temperature. The reaction was then concentrated, and purification by radial chromatography (1% acetone/ CH2Cl2) provided 14 as a slightly yellow oil (33.3 mg, 71%). 1H NMR (CDCl3, 400 MHz): δ 0.78 (t, J ) 7.4 Hz, 3H, -CH(CH2SCOCH3)CH2CH3], 1.56 (ddq, J ) 13.5, 9.6, 7.4 Hz, 1H, -CH(CH2SCOCH3)CH2CH3], 1.78 (dqd, J ) 13.5, 7.4, 5.0 Hz, 1H, -CH(CH2SCOCH3)CH2CH3], 2.29 (s, 3H, -SCOCH3), 2.58 [dddd, J ) 9.6, 8.4, 6.6, 5.0 Hz, 1H, -CH(CH2SCOCH3)Et], 3.04 (dd, J ) 13.3, 8.5 Hz, 1H, -CH(CH2SCOCH3)Et], 3.21 [dd, J ) 13.3, 6.6 Hz, 1H, -CH(CH2SCOCH3)Et], 5.56 (s, 1H, -OH), 6.79 (d, J ) 8.5 Hz, 2H, ArH), 7.01 (d, J ) 8.5 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz): δ 12.0, 28.5, 30.6, 35.7, 46.4, 115.2, 128.7, 135.2, 154.3, 196.8. MS (CI, CH4): m/z 225 (M + H+, 7), 149 (100), 135 (23), 131 (36), 107 (48). HRMS calcd for C12H17O2S: 225.0949. Found: 225.0950. 2-(4-Hydroxyphenyl)butanethiol (15). Five milliliters of 1 M HCl in MeOH/dioxane (vide supra) was added to 14 (18.5 mg, 0.0825 mmol), and the reaction was refluxed at 80 °C under Ar for 3 h. Following evaporation of the solution under high vacuum, Kugelrohr distillation (65 °C, 0.1 mmHg) provided 15 as a near-colorless oil which crystallized in the collection flask to a white solid (17.2 mg, 100%). mp 64-65.5 °C. 1H NMR (CDCl3, 400 MHz): δ 0.81 (t, J ) 7.3 Hz, 3, -CH3), 1.19 (ddd, J ) 8.6, 7.5, 0.6 Hz, 1H, -SH), 1.57 (m, 1H -CH2CH3), 1.82 (m, 1H, -CH2CH3), 2.56 (m, 1H, CH), 2.74 (m, 2H, -CH2SH), 4.73 (s, 1H, OH), 6.80 (d, J ) 8.3 Hz, 2H, Ar3H), 7.04 (d, J ) 8.5 Hz, 2, Ar-2H). 13C NMR (CDCl3, 100 MHz): δ 12.1, 28.3, 31.0, 50.3, 115.3, 129.0, 135.3, 154.1. MS (CI, CH4): m/z 183 (M + H+, 36), 149 (56), 135 (52), 107 (100), 89 (18). Anal. Calcd for C10H14OS: C, 66.65; H, 7.83. Found: C, 66.69; H, 8.08.

122 Bioconjugate Chem., Vol. 10, No. 1, 1999

[2-(4-Hydroxyphenyl)butanethiolato](3-thiapentane-1,5-dithialato)oxorhenium(V) (6). A solution of 15 (19.3 mg, 0.106 mmol) and bis(2-mercaptoethyl)sulfide (13.0 µL, 0.106 mmol) in CH3CN (0.6 mL) was added dropwise to a solution of [BnEt3N]ReOCl4 (56.8 mg, 0.106 mmol) in MeOH (1 mL). A reddish solid immediately precipitated from solution. To this was added Et3N (1 drop), and the reaction was stirred at 0 °C for 1 h. The reaction was concentrated, then purified by radial chromatography (1% acetone/CH2Cl2) to give 6 as a red powder (17 mg, 31%). IR (KBr): 932 cm-1 (RedO); mp 188-189 °C, dec. 1H NMR (DMSO-d6, 500 MHz): δ 0.71 (t, J ) 7.4 Hz, 3H, -CH3), 1.54 (m, 1H, -CH2CH3), 2.21 [m, 2H, -S(CH2)2S-], 2.76 (m, 1H, CH), 3.01 [m, 2H, -S(CH2)2S-], 3.76 (dd, J ) 12.8, 7.1 Hz, 1H, -CHCH2S), 3.86 (dd, J ) 13.0, 7.5 Hz, 1H, -CHCH2S-), 4.03 [m, 2H, -S(CH2)2S-], 4.27 [dt, J ) 12.5, 6.1 Hz, 2H, -S(CH2)2S-], 6.67 (d, J ) 8.4 Hz, 2H, Ar-3H), 7.00 (d, J ) 8.4 Hz, 2H, Ar-2H), 9.11 (s, 1H, OH). 13C NMR (d6DMSO, 125 MHz): δ 12.1, 27.8, 43.0, 43.1, 45.5, 49.2, 115.0, 128.4, 134.7, 155.6. MS (FAB): m/z 537 (M, 187Re isotope, 3). HRMS calcd for C14H22O2S4187Re: 537.0060. Found: 537.0054. 1-(p-Anisyl)-butan-2-one (17). Following the synthesis according to Myers (40), NaOMe (13.7 g, 253 mmol) was added in portions over a period of 3 h to a solution of distilled p-anisaldehyde (31.0 g, 228 mmol) and ethyl R-bromobutyrate (44.5 g, 228 mmol) with cooling in an ice bath. After stirring for 2 days, the crude reaction was diluted with water, acidified with acetic acid, and extracted with ether. The organics were dried over Na2SO4, filtered, and concentrated to a yellow oil. The oil was Kugelrohr distilled, and after a forerun of anisaldehyde was collected (∼10 g, 90 °C, 0.1 mmHg), the epoxides were collected (110-115 °C, 0.1 mmHg) to give 16 as a slightly yellow oil (39.4 g, 69%, 100% corrected). A smaller portion was further purified by radial chromatography (20% EtOAc/Hex) and redistilled to give analytically pure material. MS (CI, CH4): m/z 251 (M + H+, 12), 237 (16), 177 (100), 149 (16), 121 (10). Anal. Calcd for C14H18O4: C, 67.18; H, 7.25. Found: C, 67.10; H, 7.04. A solution of NaOH (6.92 g, 173 mmol) in EtOH (110 mL) was added to the epoxide and refluxed 3 h, at which time it was cooled and concentrated. It was then diluted with water (35 mL) and washed with ether (75 mL). This was followed by acidification with 6 M HCl and extraction with ether. The ether was then washed with water (2 × 50 mL) and water (50 mL) with a trace amount of NaHCO3. The organics were dried over Na2SO4, filtered, and concentrated. The concentrate was then heated on an oil bath at 90-100 °C for 4 h, then 130 °C for 1 h. After cooling, the red oil was dissolved in ether (100 mL) and washed sequentially with 1 M NaOH (50 mL), water (2 × 40 mL), and brine (40 mL). The organics were dried over Na2SO4, and concentrated to an orange oil. Kugelrohr distillation (90 °C, 0.1 mmHg) provided 17 as a slightly yellow oil (21.2 g, 76%). 1H NMR (CDCl3, 400 MHz): δ 1.01 (t, J ) 7.3 Hz, 3H, -CH2CH3), 2.45 (q, J ) 7.3 Hz, 2H, -CH2CH3), 3.61 (s, 2H, ArCH2COEt), 3.78 (s, 3H, -OCH3), 6.85 (d, J ) 8.8 Hz, ArH), 7.11 (d, J ) 8.8 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz) δ 7.7, 34.9, 48.7, 55.1, 114.0, 126.4, 130.2, 158.5, 209.2. MS (EI, 70 eV): m/z 178 (M+, 23), 121 (100), 91 (10), 78 (23), 57 (12). Anal. Calcd for C11H14O2: C, 74.13; H, 7.92. Found: C, 74.20; H, 7.99. 4-(4-Methoxyphenyl)-3-hexanone (18). Following the procedure by Myers (40), EtI (28.1 mL, 351 mmol) was added dropwise to a slurry of 17 (20.9 g, 117 mmol) and NaOMe (12.6 g, 234 mmol) in an ice bath. The

Skaddan and Katzenellenbogen

solution was then refluxed, adding excess EtI when needed to keep the reaction stirring. After 1 h, the reaction was cooled, concentrated, and diluted with 10 mL of water. This was then extracted with ether (30 mL). The ether layer was washed with 10% NaS2O3 (10 mL), water (10 mL), and brine (10 mL). The ether layer was then dried with Na2SO4, filtered, and concentrated to a yellow oil. Kugelrohr distillation (81 °C, 0.1 mmHg) provided 18 as a slightly yellow oil (21.4 g, 89%). A small portion was purified by radial chromatography (20% EtOAc/hexane) and redistilled to give analytically pure product. 1H NMR (CDCl3, 400 MHz): δ 0.80 (t, J ) 7.4 Hz,3H,-CHCH2CH3),0.94(t,J)7.3Hz,3H,-COCH2CH3), 1.66 (dquin, J ) 13.8, 7.5 Hz, 1H, -CHCH2CH3), 2.01 (dquin, J ) 13.7, 7.3 Hz, 1H, -CHCH2CH3), 3.46 [t, J ) 7.5 Hz, 1H, Ar-CH(Et)COEt], 2.37 (ABqq, JAB ) 7.3 Hz, ∆υ ) 16.7 Hz, J ) 7.3 Hz, 2H, -COCH2CH3), 3.77 (s, 3H, ArOCH3), 6.84 (d, J ) 8.8 Hz, 2H, ArH), 7.11 (d, J ) 8.7 Hz, 2H, ArH). 13C NMR δ 7.8, 12.0, 25.2, 34.8, 55.1, 59.5, 114.0, 129.1, 131.1, 158.6, 211.4. MS (EI, 70 eV): m/z 206 (M+, 14), 149 (100), 121 (44), 91 (13). Anal. Calcd for C13H18O2: C, 75.69; H, 8.79. Found: C, 75.88; H, 8.94. 4-(4-Hydroxyphenyl)-3-hexanone (19). HBr (15 mL) and acetic acid (15 mL) were added to 18, and the mixture was refluxed for 5 h and then cooled. The reaction was diluted with water (40 mL) and extracted with ether (3 × 40 mL). The ether layer was washed with saturated NaHCO3 until neutral to pH paper. The ether was then washed with 10% Na2S2O3 (30 mL) and water (40 mL). The phenol was extracted with 1 M NaOH (2 × 40 mL), acidified with HCl, and extracted with ether (3 × 35 mL). The ether layer was washed with water (30 mL), brine (40 mL), then dried with Na2SO4, filtered, and concentrated to provide 19 as a brown solid (5.73 g, 88%): mp 64-65.5 °C. 1H NMR (CDCl3, 400 MHz): δ 0.80 (t, J ) 7.3 Hz, 3H, -CHCH2CH3), 0.96 (t, J ) 7.3 Hz, 3H, -COCH2CH3), 1.69 (dquin, J ) 13.7, 7.6 Hz, 1H, -CHCH2CH3), 2.01 (dquin, J ) 13.7, 7.3 Hz, 1H, -CHCH2CH3), 2.42 (ABqq, JAB ) 7.4 Hz, ∆υ ) 18.4 Hz, J ) 7.3 Hz, 2H, -COCH2CH3), 3.50 [t, J ) 7.5 Hz, 1H, ArCH(Et)COEt], 6.89 (d, J ) 8.7 Hz, 2H, ArH), 6.96 (s, 1H, ArOH), 7.05 (d, J ) 8.5 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz): δ 7.9, 12.0, 25.1, 35.0, 59.6, 115.7, 129.3, 130.4, 155.2, 213.7. MS (EI, 70 eV): m/z 192 (M+, 16), 135 (100), 107 (55). Anal. Calcd for C12H16O2: C, 74.97; H, 8.39. Found: C, 75.12; H, 8.40. 4-[4-(2-Methoxyethoxymethoxy)phenyl]-3-hexanone (20). To a solution of NaH (1.25 g, 31.2 mmol) and THF (20 mL), cooled to 0 °C, was added 19 (4.00 g, 20.8 mmol) slowly. After stirring for 30 min, MEMCl (7.25 mL, 41.6 mmol) was added slowly. This was stirred for 6 h at room temperature, then quenched with MeOH with cooling and concentrated. The residue was dissolved in 110 mL of ether, then washed with 15% NaOH (2 × 40 mL), water (40 mL), and brine (45 mL). The ether layer was dried over Na2SO4 and concentrated to a yellow oil. Flash chromatography (10% acetone/CH2Cl2) and radial chromatography (2.5% acetone/CH2Cl2) provided 20 as a slightly yellow oil (4.91 g, 84%). 1H NMR (CDCl3, 400 MHz): δ 0.79 (t, J ) 7.4 Hz, 3H, -CHCH2CH3), 0.93 (t, J ) 7.3 Hz, 3H, -COCH2CH3), 1.65 (dquin, J ) 13.8, 7.5 Hz, 1H, -CHCH2CH3), 2.00 (dquin, J ) 13.8, 7.3 Hz, 1H, -CHCH2CH3), 2.36 (ABqq, JAB ) 7.3 Hz, ∆υ ) 16.1 Hz, J ) 7.3 Hz, 2H, -COCH2CH3), 3.35 [s, 3H, CH3O(CH2)2OCH2OAr], 3.46 [t, J ) 7.4 Hz, 1H, ArCH(Et)COEt], 3.54 [m, 2H, CH3O(CH2)2OCH2OAr], 3.80 [m, 2H, CH3O(CH2)2OCH2OAr], 5.23 [s, 2H, CH3O(CH2)2OCH2OAr], 6.97 (d, J ) 8.9 Hz, 2H, ArH), 7.10 (d, J ) 8.5 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz): δ 7.8, 12.0, 25.3, 34.9, 58.9,

Oxorhenium Complexes Estrogen Mimics

59.5, 67.5, 71.5, 93.4, 116.4, 129.1, 132.5, 156.3, 211.4. MS (EI, 70 eV): m/z 280 (M+, 8), 223 (38), 149 (4), 135 (9), 107 (10), 89 (100), 59 (88), 43 (33). Anal. Calcd for C16H24O4: C, 68.54; H, 8.63. Found: C, 68.30; H, 8.90. 4-[4-(2-Methoxyethoxymethoxy)phenyl]-3-hexanol (21). A solution of 20 (2.48 g, 8.85 mmol) in THF (10 mL) was added to a slurry of LAH (839 mg, 22.1 mmol) in THF (15 mL) at 0 °C. The reaction was warmed to room temperature and stirred for 1.5 h. It was quenched sequentially with 840 µL of water, 840 µL of 15% NaOH, and 2.5 mL of water. The white slurry was then passed through Celite and concentrated. Flash chromatography (10% acetone/CH2Cl2) provided 21 as a colorless oil (2.24 g, 90%). 1H NMR (CDCl3, 400 MHz, major diastereomer): δ 0.75 (t, J ) 7.4 Hz, 3H, -CH3), 0.95 (t, J ) 7.4 Hz, 3H, -CH3), 1.27 (d, J ) 5.0 Hz, 1H, OH), 1.29 [ddq, J ) 13.9, 8.2, 7.3 Hz, 1H, -CH(OH)CH2CH3], 1.55 [dqd, J ) 13.9, 7.5, 3.9 Hz, 1H, -CH(OH)CH2CH3], 1.63 [ddq, J ) 13.6, 10.3, 7.3 Hz, 1H, -CH(CH2CH3)Ar], 1.75 [dqd, J ) 13.6, 7.4, 4.9 Hz, 1H, -CH(CH2CH3)Ar], 2.43 [dt, J ) 10.4, 5.2 Hz, 1H, -CH(Et)Ar], 3.36 [s, 3H, CH3O(CH2)2CH2OAr], 3.56 [m, 2H, CH3O(CH2)2CH2OAr], 3.61 [m, 1H, -CH(OH)Et], 3.82 [m, 2H, CH3O(CH2)2CH2OAr], 5.25 [s, 2H, CH3O(CH2)2CH2OAr], 7.00 (d, J ) 8.8 Hz, 2H, ArH), 7.11 (d, J ) 8.6 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz, major diastereomer): δ 10.1, 12.2, 25.0, 27.8, 52.6, 58.9, 67.5, 71.6, 76.0, 93.5, 116.1, 129.8, 134.4, 155.9. MS (EI, 70 eV): m/z 282 (M+, 1), 223 (30), 148 (56), 136 (36), 107 (19), 89 (100), 59 (92). HRMS Calcd for C16H26O4: 282.1831. Found: 282.1841. 4-[4-(2-Methoxyethoxymethoxy)phenyl]-3-thioacetyl-hexane (22). Triethylamine (1.66 mL, 11.9 mmol) and MsCl (0.921 mL, 11.9 mmol) were added to a solution of 21 (2.24 g, 7.93 mmol) in THF (15 mL) and cooled to 0 °C in an ice bath. The reaction was stirred for 30 min at 0 °C, then for 1 h at room-temperature. Reaction was stopped and diluted with 20 mL of water. Following concentration, the aqueous layer was extracted with ether (1 × 40 mL, 1 × 15 mL). The organics were combined and washed with water (15 mL), dried with Na2SO4, and concentrated to a yellow oil (2.72 g, 91%). This was used in the next step without further purification. The mesylate (361 mg, 0.960 mmol) was combined with tetrabutylammonium thioacetate [760 mg, 2.39 mmol, prepared according to Logusch (41)] in DMF (2 mL). This was heated at 50 °C overnight. The crude mixture was diluted with ether, washed with saturated LiCl, water, and brine, then dried over Na2SO4, filtered, and concentrated to a dark oil. This was purified by radial chromatography (0.5% acetone/CH2Cl2) to provide the thioacetate 22 as a yellow-orange oil (121 mg, 37%). 1H NMR (CDCl3, 400 MHz, major diastereomer): δ 0.68 (t, J ) 7.4 Hz, 3H, CH3), 0.84 (t, J ) 7.3 Hz, 3H, CH3), 1.24 [m, 1H, -CH(SCOCH3)CH2CH3], 1.56 [m, 2H, -CH(SCOCH3)CH2CH3], 1.93 [dqd, J ) 13.6, 7.4, 3.8 Hz, 1H, -CH(CH2CH3)Ar], 2.34, (s, 3H, -SCOCH3), 2.52 [ddd, J ) 11.1, 8.6, 3.8 Hz, 1H, -CH(CH2CH3)Ar], 3.37 [s, 3H, CH3O(CH2)2CH2OAr], 3.56 [m, 2H, CH3O(CH2)2CH2OAr], 3.68 [td, J ) 8.9, 3.5 Hz, 1H, -CH(Et)Ar], 5.25 [s, 2H, CH3O(CH2)2CH2OAr], 6.97 (d, J ) 8.6 Hz, 2H, ArH), 7.08 (d, J ) 8.6 Hz, 2H, ArH). 13C NMR (CDCl3, 100 MHz, major diastereomer): δ 11.0, 12.2, 25.4, 26.4, 30.8, 50.8, 51.6, 59.0, 57.6, 71.6, 93.6, 115.9, 129.4, 135.6, 155.8, 195.9. MS (EI, 70 eV): m/z 340 (M+, 1), 264 (40), 223 (51), 135 (21), 107 (15), 89 (100), 59 (75). HRMS calcd for C18H28O4S: 340.1708. Found: 340.1709.

Bioconjugate Chem., Vol. 10, No. 1, 1999 123

3-Mercapto-4-(4-hydroxyphenyl)hexane (23). Ten milliliters of 1 M HCl in MeOH/dioxane was added to 22 (121 mg, 0.350 mmol) and the reaction was refluxed at 80 °C under Ar for 3 h. Evaporation of the solvent under high vacuum provided a dark oil. Purification by radial chromatography (0.5% acetone/CH2Cl2) gave the thiol 23 as a yellow-orange oil (34.1 mg, 46%). 1H NMR (CDCl3, 400 MHz, major diastereomer): δ 0.73 (t, J ) 7.3 Hz, 3H, CH3), 0.95 (t, J ) 7.3 Hz, 3H, CH3), 1.26 [m, 1H, -CH(SH)CH2CH3], 1.31 (d, J ) 7.2 Hz, 1H, -SH), 1.63 [m, 2H, ArCH(CH2CH3)CH(SH)CH2CH3], 2.03 [dqd, J ) 13.5, 7.4, 3.8 Hz, 1H, -CH(CH2CH3)Ar], 2.50 [ddd, J ) 11.0, 7.4, 3.5 Hz, 1H, -CH(Et)Ar], 2.87 [dtd, J ) 9.2, 7.3, 3.5 Hz, 1H, -CH(SH)Et], 5.09 (s, 1H, Ar-OH), 6.78 (d, J ) 8.3 Hz, 2H, ArH), 7.02 (d, J ) 8.5 Hz, 2H, ArH). MS (CI, CH4): m/z 211 (M + H+, 40), 177 (100), 135 (72), 121 (14), 107 (80). HRMS Calcd for C12H19OS: 211.1157. Found: 211.1161. 2,6-Bis(thiacetomethyl)pyridine (25). DIAD (4.25 mL, 21.6 mmol) was added dropwise to a solution of PPh3 (5.67 g, 21.6 mmol) in THF (100 mL) at 0 °C. Following formation of the white ylide precipitate, the solution was stirred for 30 min, at which time a solution of 2,6pyridinedimethanol (1.00 g, 7.19 mmol) in THF/DMF was added dropwise, followed by thiolacetic acid (1.54 mL, 21.6 mmol). The reaction was stirred for 1 h at 0 °C, then overnight at room temperature. A green slurry developed immediately upon addition of the thiolacetic acid, which eventually changed to a yellow color. The reaction was then concentrated and purified by flash chromatography (5% MeOH/CH2Cl2) and radial chromatography (CH2Cl2 to 5% MeOH/CH2Cl2). Recrystallization from EtOAc/ hexanes 3 times provided 25 as yellow crystals (980 mg, 53%): mp 46.5-47.5 °C. 1H NMR (CDCl3, 400 MHz): δ 2.36 (s, 6H, -SCOCH3), 4.22 (s, 4H, ArCH2SCOCH3), 7.21 (d, J ) 7.7 Hz, 2H, ArH), 7.56 (t, J ) 7.7 Hz, 1H, ArH). 13C NMR (CDCl , 100 MHz): δ 30.2, 35.0, 121.6, 137.4, 3 157.0, 194.9. MS (CI, CH4): m/z 256 (M + H+, 100), 214 (55). Anal. Calcd for C11H13NO2S2: C, 51.74; H, 5.13; N, 5.49; S, 25.11. Found: C, 51.64; H, 5.19; N, 5.42; S, 25.07. 2,6-Dithiomethylpyridine Hydrochloride (26). Four milliliters of 1 M HCl in MeOH/dioxane (vide supra) was added to 25 (200 mg, 0.783 mmol) and the reaction was refluxed at 80 °C under Ar for 3 h. Evaporation under high vacuum provided 26 as a slightly yellow solid (144 mg, 89%). This solid was used in the next reaction without further purification. 1H NMR (CD3OD, 400 MHz) δ 4.09 (s, 4H, ArCH2SH), 7.93 (d, J ) 8.0 Hz, 2H, ArH), 8.48 (t, J ) 8.0 Hz, 1H, ArH). MS (CI, CH4): m/z 172 (M + H+, 100), 138 (39). (4-Hydroxythiophenolato)(2,6-dithiomethylpyridinato)oxorhenium(V) (4b). A solution of 26 (28.0 mg, 0.172 mmol) and 4-hydroxythiophenol (24.1 mg, 0.172 mmol) in CH3CN (0.6 mL) and MeOH (0.4 mL) was added to a solution of [BnEt3N]ReOCl4 in MeOH (1 mL) under an Ar atmosphere. A brownish solution formed immediately, which intensified when Et3N (95.9 µL, 0.688 mmol) was added. The reaction was stirred for 1 h, concentrated, and purified by radial chromatography (1% acetone/CH2Cl2) to give 4b as a red solid (47.2 mg, 55%). IR (KBr): 962 cm-1 (RedO); mp 108-112 °C (dec). Normal-phase HPLC [tR ) 8.6 min, 50% hexane/50% (5% IPA/CH2Cl2), UV detection] showed only one visible peak. 1H NMR (acetone-d , 400 MHz): δ 4.8-5.6 (bm, 5H, 6 -SCH2pyr, ArOH), 6.89 (d, J ) 8.7 Hz, 2H, -SArH), 7.53 (d, J ) 8.7 Hz, 2H, -SArH), 7.70-7.88 (bd, 2H, pyrH), 7.97 (t, J ) 7.7 Hz, 1H, pyr-4H). MS (EI, 70 eV): m/z 497 (M+, 100), 372 (80). HRMS Calcd for C13H12NO2S3187Re: 496.9588. Found: 496.9581.

124 Bioconjugate Chem., Vol. 10, No. 1, 1999

(4-Methoxythiophenolato)(2,6-dithiomethylpyridinato)oxorhenium(V) (4c). A solution of 26 (20 mg, 0.0927 mmol) and 4-methoxythiophenol (13.0 mg, 0.0927 mmol) in a 1:1 mixture of MeOH/CH3CN (1 mL) was added dropwise to a solution of [BnEt3N]ReOCl4 in MeOH (1.5 mL). This was followed by the dropwise addition of Et3N (52 µL, 0.371 mmol), and the reaction was stirred for 1 h, then warmed to 40 °C for 15 min. Concentration, followed by radial chromatography (CH2Cl2) provided 4c as a dark red solid (18.5 mg, 39%). IR (KBr): 964 cm-1 (RedO); mp 103 °C (dec). 1H NMR (CD2Cl2, 500 MHz): δ 3.85 (s, 3H, Ar-OCH3), 4.8-5.6 (m, 4H, pyr-CH2S-), 6.96 (d, J ) 8.8 Hz, -SArH), 7.59 (d, J ) 8.9 Hz, 2H, -SArH), 7.78 (bd, 2H, pyrH), 7.97 (t, J ) 7.7 Hz, 1H, pyrH). MS (FAB): m/z 511 (M + H+ for 187Re, 8), 509 (M + H+ for 185Re, 4), 141 (100). HRMS Calcd for C14H14NO2S3187Re: 510.9744. Found: 510.9741. Dimethyl Chelidamate (28). Using the procedure by Bradshaw (42), chelidamic acid (6.00 g, 32.8 mmol), MeOH (80 mL), and concentrated H2SO4 (1.2 mL) were combined and refluxed for 21 h. The reaction was cooled, neutralized with saturated Na2CO3, and reacidified with concentrated HCl. This was diluted with water (80 mL) and extracted with CH2Cl2 (1 × 100 mL, 2 × 50 mL). The organics were combined, dried with Na2SO4, filtered, and concentrated to a yellow solid. Two recrystallizations from MeOH gave 28 as a light yellow solid (3.51 g, 51%): mp 168.5-169.5 °C [lit. (42) 169-169.5 °C]. 1H NMR (CDCl3, 400 MHz): δ 3.96 (s, 6H, ArCO2CH3), 7.80 (bs, 2H, pyrH), 9.8-10.2 (bs, 0.4H, pyrOH), 11.4-11.9 (bs, 0.4H, pyrOH). MS (EI, 70 eV): m/z 211 (M+, 100), 183 (71), 167 (15), 153 (96), 121 (52), 92 (64), 79 (8), 68 (14), 59 (31). Anal. Calcd for C9H9NO5: C, 51.19; H, 4.30; N, 6.63. Found: C, 51.16; H, 4.24; N, 6.63. Dimethyl 4-[(Tetrahydro-2-pyranyl)oxy]-2,6-pyridinedicarboxylate (29). To a solution of 28 (3.11 g, 14.7 mmol) in CH2Cl2 (44 mL) was added 3,4-dihydro2H-pyran (6.72 mL, 73.6 mmol) and pyridinium ptoluenesulfonate (377 mg, 1.50 mmol). The reaction was stirred overnight at room temperature. The crude reaction mixture was washed with brine (2 × 15 mL) and water (20 mL), dried over Na2SO4, filtered, concentrated, and recrystallized from EtOAc/hexane to give 29 as white crystals (2.97 g, 68%): mp 119.5-121.5 °C [lit. (42) 120121.5 °C]. 1H NMR (CDCl3, 400 MHz): δ 1.5-1.78 (m, 3H, THP), 1.82-2.04 (m, 3, THP), 3.61-3.80 (m, 2H, THP), 3.98 (s, 6H, pyrCO2CH3), 5.66 (t, J ) 2.75 Hz, 1H, THP), 7.92 (s, 2H, pyrH). 13C NMR (CDCl3, 100 MHz): δ 17.9, 24.7, 29.5, 53.1, 62.0, 96.3, 116.0, 149.6, 165.06, 165.12. MS (CI, CH4): m/z 296 (M + H+, 5), 240 (13), 212 (100), 85 (26), 57 (11). Anal. Calcd for C14H17NO6: C, 56.94; H, 5.80; N, 4.74. Found: C, 56.80; H, 6.99; N, 4.73. 4-(Tetrahydro-2H-2-pyranyloxy)-2,6-bis(hydroxymethyl)pyridine (30). Following the procedure used by Scrimin (43), NaBH4 (1.06 g, 28.1 mmol) was added to a solution of 29 in EtOH (90 mL). Crushed CaCl2 (3.11 g, 28.1 mmol) was then added slowly to the reaction, producing a heterogeneous pink color. After stirring overnight, the reaction was diluted with water (100 mL), and the EtOH was removed by evaporation in vacuo. The residue was added to brine (100 mL), and extracted with CHCl3 (4 × 200 mL). This was evaporated to a light green oil, which solidified upon standing under vacuum for several days, giving a crude mass of 1.53 g (69%). Recrystallization from EtOAc provides 30 as off-white crystals (1.14 g, 51%): mp 115-116 °C. 1H NMR (CD3OD, 500 MHz): δ 1.5-1.75 (m, 2H, THP), 1.8-2.0 (m, 3H, THP), 2.70 (bs, 2H, pyrCH2OH), 3.58 (dtd, J ) 11.3, 4.1,

Skaddan and Katzenellenbogen

1.5 Hz, 1H, THP), 3.77 (ddd, J ) 11.4, 10.1, 3.2 Hz, 1H, THP), 4.61 (s, 4H, pyrCH2OH), 5.55 (t, J ) 3.2 Hz, 1H, THP), 6.89 (s, 2H, pyrH). MS (CI, CH4): m/z 240 (M + H+, 13), 222 (10), 184 (25), 156 (100), 85 (35). Anal. Calcd for C12H17NO4: C, 60.24; H, 7.16; N, 5.85. Found: C, 59.93; H, 7.13; N, 5.85. 4-(Tetrahydro-2H-2-pyranyloxy)-2,6-bis(thioacetylmethyl)pyridine (31). Diisopropyl azodicarboxylate (2.30 mL, 11.5 mmol) was added slowly to a solution of PPh3 (3.01 g, 11.5 mmol) in THF (75 mL) at 0 °C. Upon formation of the white ylide precipitate., the reaction was stirred for 30 min at 0 °C. A solution of 30 (1.08 g, 4.50 mmol) in THF/DMF was added to the reaction, followed by thiolacetic acid (820 µL, 11.5 mmol). The reaction was stirred for 1 h at 0 °C, then overnight at room temperature. The reaction was concentrated, redissolved in CH2Cl2 (60 mL), and washed with saturated LiCl (2 × 30 mL) and brine (1 × 30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. After three flash chromatography purifications (5% MeOH/CH2Cl2), the thioacetate 31 was isolated as a yellow oil (1.15 g, 72%). 1H NMR (CDCl3, 400 MHz): δ 1.24 (m, 2H, THP), 1.52-2.0 (m, 6H, THP), 2.34 (s, 6H, -SCOCH3), 3.60 (dtd, J ) 11.1, 4.0, 1.5 Hz, 1H, THP), 3.76 (m, 1H, THP), 4.14 (ABq, JAB ) 13.8 Hz, ∆υ ) 13.8 Hz, 4H, ArCH2SCOMe), 6.86 (s, 2H, pyrH). 13C NMR (CDCl3, 100 MHz): δ 18.2, 24.8, 29.7, 30.2, 35.4, 61.9, 95.7, 109.6, 158.4, 164.4, 194.9. MS (CI, CH4): m/z 356 (M + H+, 5), 300 (15), 272 (81), 247 (26), 205 (55), 161 (100), 147 (32), 119 (89), 85 (34), 57 (16). HRMS Calcd for C16H22NO4S2: 356.0997. Found: 356.0997. [4-Hydroxy-2,6-bis(thiomethyl)pyridinato][4-hydroxythiophenolato]oxorhenium(V) (4a). A solution of 1 M HCl in MeOH/dioxane (1 mL) was added to 31 (50 mg, 0.141 mmol) and refluxed at 80 °C for 3 h under Ar. Concentration under high vacuum provided the dithiol HCl salt 32 as an extremely hygroscopic off-white solid, which darkened with time at room temperature. A solution of the thiol (25.2 mg, 0.113 mmol) and p-hydroxythiophenol (15.8 mg, 0.113 mmol) in 1:1 MeOH/ CH3CN (1 mL) was added to a solution of [BnEt3N]ReOCl4 in MeOH (1 mL) at 0 °C under Ar. This was followed by the dropwise addition of Et3N (16 µL, 0.113 mmol), resulting in a dark solution. The reaction was warmed to room temperature for 45 min. Concentration, followed by radial chromatography (5-10% MeOH/CH2Cl2), provided a red, sticky oil, which when slowly evaporated from CH2Cl2 provided 4a as red solid (22.5 mg, 39%). IR (NaCl): 965 cm-1 (RedO); mp ∼100 °C (dec). 1H NMR (DMSO-d6, 400 MHz, 95 °C): δ 4.98 (ABq, JAB ) 17.6 Hz, ∆υ ) 195 Hz, 4H, pyrCH2S-), 6.76 (d, J ) 8.5 Hz, 2H, -SArH), 7.25 (s, 2H, pyrH), 7.28 (d, J ) 8.5 Hz, 2H, -SArH). 13C NMR (CD3OD/CDCl3, 100 MHz): δ 51.2, 106.7, 134.8, 138.0, 155.8, 168.8. MS (FAB): m/z 514 (M+ for 187Re, 3), 512 (M+ for 185Re, 2), 461 (100). HRMS calcd for C13H13NO3S3187Re: 513.9615. Found: 513.9613. Measurement of the Octanol/Water Partition Coefficient. The log Po/w values were estimated from log k′w values determined by reversed-phase HPLC following the method outlined by Minick (44). A Chromegabond C8 (5 mm, 60 Å, ES Industries) 15 cm × 4.6 mm column served as the stationary phase. The organic mobile phase was methanol containing 0.25% (v/v) 1-octanol, and the aqueous phase consisted of octanol-saturated water containing 0.25% (v/v) M MOPS (3-morpholinopropanesulfonic acid, Sigma) buffer and 0.15% (v/v) n-decylamine, adjusted to pH 7.4. The flow rate was 1 mL/min.

Oxorhenium Complexes Estrogen Mimics Scheme 1

Estrogen Receptor Binding Affinity. Receptorbinding affinity (RBA) values were determined by a competitive radiometric binding assay, using tritiated estradiol as the tracer and lamb uterus cytosol as the source of receptor, according to a previously established method (45). RESULTS AND DISCUSSION

Syntheses. Synthesis of Complexes That Mimic Estradiol (5ab, 6, and 2). Before the preparation of the precursor monodentate ligand for 2, we first synthesized some model compounds which were intended to not only help us determine the route by which to introduce the thiol to monodentate ligands but also serve as a “reference” to compare relative-binding affinities to 2. These complexes included one with a simple phenethyl thiol monodentate ligand (5a,b) and one with an ethyl group at the benzylic position of the monodentate ligand (6). The synthesis of the first model systems 5a and 5b follows the straightforward route outlined in Scheme 1. Starting with the parent phenethyl alcohol (7a, 7b), either Mitsunobu or mesylation-displacement with potassium thioacetate work well in generating the thioacetates 8a and 8b, respectively. Treatment of the thioacetate with anhydrous HCl in methanol/dioxane generates the free thiols 9a and 9b cleanly. Attempts to free the thiol under alkaline conditions (e.g., K2CO3 in MeOH/H2O and LAH) only resulted in formation of the disulfide, even when strictly anaerobic conditions were observed. Complexation of these thiols with the commercially available bis(2-mercaptoethyl) sulfide and a readily available rhenium source, Bu4N[ReOCl4] (38), furnished the desired complexes 5a and 5b in modest yields. The synthesis of the monoethyl model system 6 is outlined in Scheme 2 and is analogous to the syntheses of 5a and 5b. Carboxylic acid 11 is prepared from Scheme 2

Bioconjugate Chem., Vol. 10, No. 1, 1999 125

p-methoxyphenylacetic acid (11), using a known literature procedure (39), and is used without purification in the deprotection step to afford phenol 12 in low yield. Reduction with LAH generates alcohol 13 in low yield because of difficulties in the workup. A Mitsunobu reaction on the alcohol provides the thioacetate 14 in good yield. The thiol 15 is then released in a manner analogous to 9a and 9b, and the complex is obtained without complication. The attempted synthesis of desired complex 2 is described in Scheme 3. The skeleton for the monodentate ligand was generated by first reacting p-anisaldehyde and ethyl a-bromobutyrate in a Darzens reaction to afford the glycidic ester 16 in good yield (40). Saponification, followed by thermal decarboxylation produced ketone 17 in good yield (40). Ethylation of the more stable benzylic enolate with EtI as the solvent afforded the branched ketone 18 in excellent yield. Deprotection with HBr and AcOH produced the free phenol 19, again in excellent yield (40). After reprotection with the MEM group, the protected ketone 20 was reduced by LAH reduction to generate the alcohol 21 as a 5:1 mixture of diastereomers by NMR (presumably 3R*,4S*:3R*,4R* according to Felkin-Ahn rules). We did not separate the diastereomers at this point because we chose to determine the relativebinding affinities of the diastereomeric mixture of rhenium complexes, not knowing which would be of higher affinity. If appreciable binding were observed, we then would have made efforts to separate the two diastereomers. Mesylation of alcohol 21, followed by displacement with thioacetate, generated the thioacetate 22 in low yield. A competing elimination reaction to produce the trisubstituted olefin and the difficulty of an SN2 attack of the thioacetate on the highly hindered secondary carbon are thought to be responsible for the low yield. Typical Mitsunobu conditions to generate the thioacetate failed for this reaction. In addition, attempts to form the thiol via BuLi-assisted reductive fragmentation of a thioketal (derived from ketone 20) failed (46), and reduction of the Lawesson’s Reagent-produced thioketone (also derived from ketone 20) was also unsuccessful. The thioacetate 22 was subjected to the same acidic conditions as in Schemes 1 and 2 to generate the thiol 23 in modest yield; the yield was again limited by acid-catalyzed formation of the olefin. However, in contrast to thiols 9ab or 15, when the thiol was subjected to the standard complexation conditions, complex 2 failed to form. Efforts to generate the complex by changing the temperature, order of reagent addition, and rhenium source also failed. It is believed that the difficulty in producing this complex is again due to the highly hindered nature of the secondary thiol used in the reaction, especially considering the fact that complexation of thiol 15, which lacks just one of the

126 Bioconjugate Chem., Vol. 10, No. 1, 1999

Skaddan and Katzenellenbogen

Scheme 3

Scheme 4

ethyl groups, results in clean formation of complex 6 (Scheme 2). Synthesis of Complexes That Mimic a Tetrahydrochrysene Estrogen (4a-c). Complexes 4a-c were designed to mimic a tetrahydrochrysene (THC) estrogen (cf. Figure 1). The reasoning behind including complexes with and without the p-hydroxy functionality on the tridentate ligand was to compare the effect this would have on the relative-binding affinity. One would expect the binding to be appreciably higher in 4a where both hydroxyls are present, as this best represents the THC that the complex is trying to mimic. Alternatively, when one removes the p-hydroxy group from the tridentate ligand (as in 4b and 4c) or protects the hydroxy on the monodentate ligand (4b), one should see a dramatic drop in binding affinity compared to 4a. We synthesized the tridentate pyridine dithiol ligand for 4b and 4c, which does not contain the p-hydroxy group, by starting with commercially available 2,6pyridinedimethanol (24, Scheme 4). The Mitsunobu reaction was then used to transform the diol 24 into the dithioacetate 25 after recrystallization. Thioacetate 25 was then treated with anhydrous hydrochloric acid under argon to afford the dithiol 26 as the HCl salt in good yield. Subjecting the crude dithiol 26 to standard complexation conditions with either p-methoxythiophenol or p-hydroxythiophenol afforded the red complexes 4b and 4c, respectively.

A concern that we had prior to the successful formation of these complexes was that the planar aromatic pyridine would inhibit bonding of the tridentate ring to the oxorhenium core, because of the rigidity it enforces on the framework of the tridentate ligand. However, all complexation reactions involving this type of tridentate ligand went smoothly, and all of the complexes produced were stable to air and moisture for extended periods of time. To our knowledge, this is the first example of a benzylic dithiopyridine complexed to an oxorhenium center. The synthesis of complex 4a requires the use of a starting material which will eventually give a tridentate ligand having a p-hydroxy functionality. To this end, we started with commercially available chelidamic acid (27, Scheme 5). Esterification provides the diester 28, which, upon protection of the phenol with DHP, gives the fully protected diester 29 (42). Although LAH was not effective in reducing the diesters, a mixture of NaBH4 and CaCl2 produced diol 30 in good yield (43). The thioacetates were then introduced by using the standard Mitsunobu conditions to give the dithioacetate 31. Removal of the thioacetates under acidic conditions also conveniently removed the THP group, giving the desired tridentate dithiol 32 as the very hygroscopic HCl salt, in quantitative yield. The crude 32 was then subjected to the standard complexation conditions to give the novel rhenium complex 4a in modest yield. Variable Temperature NMR Studies on the Fluxional 3+1 Complex 4a. An X-ray crystal structure of a complex similar to 4b (where there is no para hydroxyl on the pyridine ring) shows that the arenethiolate ligand is bent toward one side of the molecule (47). Therefore, 4a is asymmetric, and VT NMR studies on 4a seem to confirm this observation (Figure 2). At -60 °C, the rotation of the arenethiolate ligand is slow on the NMR time scale, and the magnetically inequivalent benzylic and meta protons of the tridentate ligand appear as two sharp sets of AB quartets and two singlets, respectively. As the temperature is elevated, these peaks start to coalesce as the arenethiolate ligand flips back and forth more rapidly. This is evident at 16 °C where these signals are significantly broadened. At 95 °C, the rotation of the arenethiolate ligand is fast on the NMR time scale, and one observes a symmetric time-averaged structure. This is evident by the fact that the two singlets at -60 °C have now nicely coalesced into one singlet at 95 °C. Similarly,

Oxorhenium Complexes Estrogen Mimics

Bioconjugate Chem., Vol. 10, No. 1, 1999 127

Scheme 5

Figure 2. VT NMR of 4a [A (CD3OD, -60 °C); B (CD3OD, 16 °C); C (DMSO-d6, 95 °C)]. Table 1. Relative-Binding Affinity (RBA) and Log PO/w Values of Rhenium Complexes complex

RBA (0 °C)

log Po/w

1 5a 5b 6 4a 4b 4c

100 4b). However, the significance of this trend at very low affinities is uncertain. The lipophilicities of the THC mimics were determined by measuring their log Po/w values according to the method of Minick (44). The values of 4b and 4c (4.14 and

3.46, respectively) are rather similar to that of estradiol, while 4a, which one would expect to be even more polar than 4c because of the addition of a hydroxyl group, has a remarkably high log Po/w value of 5.35 by this method, even though normal-phase TLC and solubility in polar organic solvents show the opposite trend. The unique and not well-understood properties of this novel rhenium complex structure, especially its interactions with the reversed-phase HPLC column used to determine the log Po/w in this experiment, might contribute to this unexpected result. It is interesting to note, however, that Kung and co-workers reported a similar finding in comparing the log Po/w values of N2S2 oxotechnetium complexes of the diamine vs amine-amide type (48). The replacement of two hydrogens with an oxo substituent in this case resulted in an 8-fold increase in the log Po/w value. CONCLUSIONS

We have described the synthesis of a number of novel “3+1” oxorhenium(V) complexes (4a-c, 5ab, and 6) for use as estrogen mimics and have determined their binding affinites for the estrogen receptor (ER). We were also able to synthesize the monodentate ligand 23 needed to prepare complex 2 (the best mimic of estradiol); however, the hindered nature of the thiol in 23 prevented this ligand from complexing with rhenium to form the desired complex. The relative-binding affinity (RBA) values of the estradiol model complexes (5a, 5b, and 6) are low (the highest value is 0.018% vs 100% for estradiol) and indicate that these complexes are not, in fact, good mimics of estradiol. The series of pyridinedithiol complexes 4a-c as mimics for THC have also been synthesized. These pyridinedithiol oxorhenium complexes are the first of their kind. The complexes are quite stable to air and moisture, and VT NMR studies reveal the fluxional nature of the arenethiolate ligand in these complexes. These complexes also have poor binding affinity to the ER. The lipophilicity of complexes 4b and 4c is fairly close to that of estradiol, while complex 4a shows a log Po/w value much higher than expected. Even though molecular modeling suggests that the distance between the phenolic hydroxyl groups in complex 4a is actually comparable to that of diethylstilbestrol compounds (49), the location of the polar and bulky square pyramidal oxorhenium core toward the center of a normally lipophilic region of steroidal and nonsteroidal estrogens might account for the low binding affinity observed for the THC class mimics (30). The low binding affinity of all complexes described here is probably due to the poor geometric and electronic match between the complexes and the native ligands. Future work on these integrated complexes will involve

128 Bioconjugate Chem., Vol. 10, No. 1, 1999 Chart 1

the use of a chelate system whose geometry (e.g., square planar) and arrangement of polar moieties is more akin to that of the native ligands. ACKNOWLEDGMENT

We are grateful for the support of this research through grants from the Department of Energy (DE FG02 86ER60401) and the National Institutes of Health (PHS 5 R01 CA25836). The assistance of Ms. Kathy E. Carlson in the receptor-binding experiments is appreciated. Helpful discussions regarding molecular modeling, log Po/w measurements, and X-ray crystal structures from Frank Wu¨st are also deeply appreciated. NMR experiments were performed in the Varian Oxford Instrument Center for Excellence NMR Laboratory (VOICE NMR Lab), in part funded by grants from the National Institutes of Health (PHS 1 S10 RR1044-01), the National Science Foundation (NSF CHE 96-0152), and the Keck Foundation. Mass spectral data were acquired on spectrometers purchased with funds in part from the Division of Research Resources, National Institutes of Health (RR 01575 and RR 04648), the National Science Foundation (PCM 8121494), and the National Institute of General Medical Sciences (GM27029). LITERATURE CITED (1) Harris, J. R., Lipman, M. E., Veronesi, U., and Willett, W. (1992) Breast cancer. N. Engl. J. Med. 327, 319. (2) Feuer, E. J., Wun, L. M., Boring, C. C., Flanders, W. D., Timmel, M. J., and Tong, T. (1993) The lifetime risk of developing breast cancer. J. Natl. Cancer Inst. 85, 892. (3) American Cancer Society (1996) Cancer Facts and Figures. (4) McGuire, W. L. (1975) Estrogen Receptors in Human Breast Cancer. Raven Press, New York. (5) Cummins, C. H. (1993) Radiolabeled steroidal estrogens in cancer research. Steroids 58, 245. (6) VanBrocklin, H. F., Pomper, M. G., Carlson, K. E., Welch, M. J., and Katzenellenbogen, J. A. (1992) Preparation and evaluation of 17-ethynyl-substituted 16R-[18F]fluoroestradiols: Selective receptor-based PET imaging agents. Int. J. Rad. Appl. Instrum. B 19, 363-374. (7) VanBrocklin, H. F., Carlson, K. E., Katzenellenbogen, J. A., and Welch, M. J. (1993) 16β-([18F]Fluoro)estrogens: Systematic investigation of a new series of fluorine-18-labeled estrogens as potential imaging agents for estrogen-receptorpositive breast tumors. J. Med. Chem. 36, 1619-1629. (8) VanBrocklin, H. F., Rocque, P. A., Lee, H. V., Carlson, K. E., Katzenellenbogen, J. A., and Welch, M. J. (1993) 16β[18F]Fluoromoxestrol: a potent, metabolically stable positron emission tomography imaging agent for estrogen receptor positive human breast tumors. Life Sci. 53, 811-819. (9) VanBrocklin, H. F., Liu, A., Welch, M. J., O’Neil, J. P., and Katzenellenbogen, J. A. (1994) The synthesis of 7R-methylsubstituted estrogens labeled with fluorine-18: Potential breast tumor imaging agents. Steroids 59, 34-45. (10) Kochanny, M. J., VanBrocklin, H. F., Kym, P. R., Carlson, K. E., O’Neil, J. P., Bonasera, T. A., Welch, M. J., and Katzenellenbogen, J. A. (1993) Fluorine-18-labeled progestin ketals: Synthesis and target tissue uptake selectivity of potential imaging agents for receptor-positive breast tumors. J. Med. Chem. 36, 1120-1127. (11) Brandes, S. J., and Katzenellenbogen, J. A. (1987) Fluorinated androgens and progestins: molecular probes for an-

Skaddan and Katzenellenbogen drogen and progesterone receptors with potential use in positron emission tomography. Mol. Pharmacol. 32, 391-403. (12) Buckman, B. O., Bonasera, T. A., Kirschbaum, K. S., Welch, M. J., and Katzenellenbogen, J. A. (1995) Fluorine-18-labeled progestin 16R,17R-dioxolanes: Development of high-affinity ligands for the progesterone receptor with high in vivo target site selectivity. J. Med. Chem. 38, 328-337. (13) Pomper, M. G., Katzenellenbogen, J. A., Welch, M. J., Brodack, J. W., and Mathias, C. J. (1988) 21-[18F]Fluoro-16Rethyl-19-norprogesterone: Synthesis and target tissue selective uptake of a progestin receptor based radiotracer for positron emission tomography. J. Med. Chem. 31, 1360-1363. (14) Pomper, M. G., Pinney, K. G., Carlson, K. E., van Brocklin, H., Mathias, C. J., Welch, M. J., and Katzenellenbogen, J. A. (1990) Target tissue uptake selectivity of three fluorinesubstituted progestins: Potential imaging agents for receptorpositive breast tumors. Int. J. Rad. Appl. Instrum. B 17, 309319. (15) Verhagen, A., Luurtsema, G., Pesser, J. W., de Groot, T. J., Wouda, S., Oosterhuis, J. W., and Vaalburg, W. (1991) Preclinical evaluation of a positron emitting progestin ([18F]fluoro-16R-methyl-19-norprogsterone) for imaging progesterone receptor positive tumours with positron emission tomography. Cancer Lett. 59, 125-132. (16) Verhagen, A., Elsinga, P. H., de Groot, T. J., Paans, A. M. J., deGoeij, C. C. J., Sluyser, M., and Vaalburg, W. (1991) A fluorine-18 labeled progestin as an imaging agent for prostestin receptor positive tumors with positron emission tomography. Cancer Res. 51, 1930-1933. (17) Hochberg, R. B., Hoyte, R. M., and Rosner, W. (1985) E-17R-(2-[125I]iodovinyl)-19-nortestosterone: The synthesis of a gamma emitting ligand for the progesterone receptor. Endocrinology 117, 2550-2552. (18) Hochberg, R. B., MacLusky, N. J., Chambers, J., Eisenfeld, A. J., Naftolin, F., and Schwartz, P. E. (1985) Concentration of [16R-125I]iodoestradiol in human ovarian tumors in vivo and correlation with estrogen receptor content. Steroids 46, 775-778. (19) Hoyte, R. M., Rosner, W., Johnson, I. S., Zielinski, J., and Hochberg, R. B. (1985) Synthesis and evaluation of potential radioligands for the progesterone receptor. J. Med. Chem. 28, 1695-1699. (20) Lamb, D. J., Bullock, D. W., Hoyte, R. M., and Hochberg, R. B. (1988) D9-[16R-125I]iodo-19-nortestosterone: a gammaemitting photoaffinity label for the progesterone receptor. Endocrinology 122, 1923-1932. (21) Salman, M., Stotter, P. L., and Chamness, G. C. (1989) 125I-Ligand for progesterone receptor: 17R-(6′-iodohex-1′ynyl)-19-nortestosterone. J. Steroid Biochem. 33, 25-31. (22) DiZio, J. P., Fiaschi, R., Davison, A., Jones, A. G., and Katzenellenbogen, J. A. (1991) Progestin-rhenium complexes: Metal-labeled steroids with high receptor binding affinity, potential receptor-directed agents for diagnostic imaging or therapy. Bioconjugate Chem. 2, 353. (23) DiZio, J. P., Anderson, C. J., Davison, A., Ehrhardt, G. J., Carlson, K. E., and Welch, M. J. (1992) Technetium- and rhenium-labeled progestins: Synthesis, receptor binding and in vivo distribution of an 11β-substituted progestin labeled with technetium-99 and rhenium-186. J. Nucl. Med. 33, 558. (24) O’Neil, J. P., Carlson, K. E., Anderson, C. J., Welch, M. J., and Katzenellenbogen, J. A. (1994) Progestin radiopharmaceuticals labeled with technetium and rhenium: Synthesis, binding affinity, and in vivo distribution of a new progestin N2S2-metal conjugate. Bioconjugate Chem. 5, 182. (25) Top, S., Vessieres, A., and Jaouen, G. (1994) Synthetic strategy for organometallic complexes of rhenium with exceptionally high affinity for the oestradiol receptor; their potential use as imaging and therapeutic agents. J. Chem. Soc., Chem. Commun. 453. (26) Top, S., El Hafa, H., Vessieres, A., Quivy, J., Vaissermann, J., Hughes, D. W., McGlinchey, M. J., Mornon, J. P., Thoreau, E., and Jaouen, G. (1995) Rhenium carbonyl complexes of β-estradiol derivatives with high affinity for the estradiol receptor: An approach to selective organometallic radiopharmaceuticals. J. Am. Chem. Soc. 117, 8372.

Oxorhenium Complexes Estrogen Mimics (27) Wu¨st, F., Spies, H., and Johannsen, B. (1996) Synthesis of "3+1" mixed-ligand oxorhenium(V) complexes containing modified 3,17β-estradiol. Bioorg. Med. Chem. Lett. 6, 2729. (28) Wu¨st, F., Spies, H., and Johannsen, B. (1997) Synthesis of 17R-substituted mercaptoalkynyl derivatives of 3,17βestradiol. Tetrahedron Lett. 38, 2931. (29) Wu¨st, F. R., Carlson, K. E., Katzenellenbogen, J. A., Spies, H., and Johannsen, B. (1999) Synthesis and binding affinities of new 17R-substituted estradiol-rhenium mixed-ligand and thioether-carbonyl complexes. Steroids (in press). (30) Hom, R. K., and Katzenellenbogen, J. A. (1997) Synthesis of a tetradentate oxorhenium(V) complex mimic of a steroidal estrogen. J. Org. Chem. 62, 6290. (31) Sugano, Y., and Katzenellenbogen, J. A. (1996) Synthesis of tetradentate bisamino-bisthiol complexes of oxorhenium(V) as structural mimics of steroids. Bioorg. Med. Chem. Lett. 6, 361. (32) Chi, D. Y., O’Neil, J. P., Anderson, C. J., Welch, M. J., and Katzenellenbogen, J. A. (1994) Homodimeric and heterodimeric bis(aminothiol) oxometal complexes with rhenium(V) and technetium(V). Control of heterodimeric complex formation and an approach to metal complexes that mimic steroid hormones. J. Med. Chem. 37, 928. (33) Spies, H., Fietz, T., Glaser, M., Pietzsch, H. J., and Johannsen, B. (1995) The “n+1” concept in the synthesis strategy of novel rhenium and technetium tracers (M. Nicolini, G. Bandoli, and U. Mazzi, Eds.)Vol. 4, pp 243-246, SGEditoriali, Padova, Italy. (34) Mastrostamatis, S. G., Papadopoulas, M. S., Pirmettis, I. C., Paschali, E., Varvarigou, A. D., Stassinopolou, C. I., Raptapoulou, C. P., Terzis, A., and Chiotellis, E. (1994) Tridentate ligands containing the SNS donor atom set as a novel backbone for the development of technetium brainimaging agents. J. Med. Chem. 37, 3212. (35) Pirmettis, J. C., Papadopoulas, M. S., Mastrostamatis, S. G., Raptopoulou, C. P., Terzis, A., and Chiotellis, E. (1996) Synthesis and characterization of oxotechnetium(V) mixedligand complexes containing a tridentate N-substituted bis(2-mercaptoethyl)amine and a monodentate thiol. Inorg. Chem. 35, 1685. (36) Niccolini, M., Bandoli, G., and Mazzi, U. (1995) Technetium and Rhenium in Chemistry and Nuclear Medicine 4, SGEditoriali, Padova, Italy. (37) Okuno, H., Uoto, K., Tomohiro, T., and Youinou, M. T. (1990) Synthesis of tetranuclear iron-sulphur protein analogues with tetrathiol ligands attached to macrocycles which provide intramolecular hydrophobic domains. J. Chem. Soc., Dalton Trans. 3375. (38) Spies, H., Fietz, T., Pietzsch, H. J., Johannsen, B., Leibnitz, P., Reck, G., Scheller, D., and Klosterman, K. (1995) Neutral

Bioconjugate Chem., Vol. 10, No. 1, 1999 129 oxorhenium(V) complexes with tridentate dithiolates and monodentate alkane- or arene-thiolate coligands. J. Chem. Soc., Dalton Trans. 2277. (39) Aaron, C., Dull, D., Schmiegel, J. L., Jaeger, D., Ohashi, Y., and Mosher, H. S. (1967) The resolution and configuration of R-substituted phenylacetic acids. J. Org. Chem. 32, 2797. (40) Myers, T. C., Pratt, R. J., Morgan, R. L., O’Donnell, J., and Jensen, E. V. (1955) Hormone analogues. I. Preparation of intermediate ketones for the synthesis of stilbestrol analogues. J. Am. Chem. Soc. 77, 5655. (41) Logusch, E. W. (1988) A simple preparation of S-alkyl homocysteine derivatives: S-phosphonomethyl homocysteines as inhibitors of glutamine synthetase. Tetrahedron Lett. 29, 6055. (42) Bradshaw, J. S., Colter, M. L., Nakatsuji, Y., Spencer, N. O., Brown, M. F., Izatt, R. M., Arena, G., Tse, P.-K., Wilson, B. E., Lamb, J. D., and Dalley, N. K. (1985) Proton-ionizable crown compounds. 2. Synthesis, complexation properties, and structural studies of macrocyclic polyether-diester ligands containing a 4-hydroxypyridine subcyclic unit. J. Org. Chem. 50, 4865. (43) Scrimin, P., Tecilla, P., Tonellato, U., and Vendrame, T. (1989) Aggregate structure and ligand location strongly influence Cu2+ binding ability of cationic metallosurfactants. J. Org. Chem. 54, 5988. (44) Minick, D. J., Frenz, J. H., Patrick, M. A., and Brent, D. A. (1988) A comprehensive method for determining hydrorophobicity constants by reverse-phase high-performance liquid chromatography. J. Med. Chem. 31, 1923. (45) Katzenellenbogen, J. A., Johnson, H. J., Jr., and Myers, H. N. (1973) Photoaffinity labels for estrogen binding proteins of rat uterus. Biochemistry. 12, 4085. (46) Wilson, S. R., Caldera, P., and Jester, M. A. (1982) Synthesis of sulfides and mercaptans from thioketals. J. Org. Chem. 47, 3319. (47) The crystal structure of this complex was kindly provided by Frank Wu¨st at the Forschungszentrum Rossendorf e.V., Dresden, Germany. (48) Meegalla, S. K., Plossl, K., Kung, M. P., Chumpradit, S., Stevenson, D. A., Kushner, S. A., McElgin, W. T., Mozley, P. D., and Kung, H. F. (1997) Synthesis and characterization of Tc-99m labeled tropanes as dopamine transporter imaging agents. J. Med. Chem. 40, 9-17. (49) Wu¨st, F. R. (1998) Forschungszentrum Rossendorf e.V., Dresden, Germany (personal communication).

BC980094Q