Progestin-rhenium complexes: metal-labeled steroids with high

Mark D. Bartholomä , Anika S. Louie , John F. Valliant and Jon Zubieta ..... Frank Wüst , Kathryn E Carlson , John A Katzenellenbogen , Hartmut Spie...
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Bloconlugte Chem. 1991, 2, 353-366

353

Progestin-Rhenium Complexes: Metal-Labeled Steroids with High Receptor Binding Affinity, Potential Receptor-Directed Agents for Diagnostic Imaging or Therapy James P. DiZio,? Rita Fiaschi,? Alan Davison,* Alun G. Jones,# and John A. Katzenellenbogen'*t Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801, Department of Chemistry, Room 6-435, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Radiology, Harvard Medical School, 50 Binney Street, Boston, Massachusetts 02115. Received July 1, 1991 In order to investigate the possibility of developing diagnostic imaging agents for steroid-positivetumors that are labeled with the readily available radionuclide technetium-99m, we prepared four conjugate systems in which a progestin is linked to a metal chelate system. Three of these are bis-amino bis-thiol (BAT or N2S2) systems and are linked through carbon-21 of progesterone or the 17a- or llg-position of a nortestosterone type progestin. The fourth, an amino-amido-thiol-alcohol chelate (NzOS)system, is linked at the 16a,l7a-positionsof a dihydroprogesterone. As a model for technetium-labeled complexes, all four chelate systems were converted to their oxo-rhenium complexes. Of the four possible diastereomers in the 16a,l7a-system,only one was isolated, while of the four possible diastereomers in the other systems, a syn pair and an anti pair (linker methylene vs rhenium-oxo, relative to the NzS2 plane) were separated in the l7a-substituted series, a syn pair was isolated in the 21-substituted series, and a syn pair and the two individual anti diastereomers were separated in the 116-substituted series. In competitive radiometric receptor binding assays, the 21-, 17a-, and 16a,l7a-linked systems had low affinity for the progesterone receptor (less than 0.3% that of promegestone (R5020) or 2% that of progesterone). By contrast, the two anti diastereomers of the 116-linked system had affinities that were 10% and 44% that of R5020 (or 64 % and 283 5% that of progesterone) and the syn pair had an affinity 25% that of R5020 (or 161% that of progesterone). The latter finding indicates that it is possible to prepare metal-labeled steroids that retain high affinity for steroid receptors. These and related systems, when complexed with radioactive metals, may be useful in vivo as receptor-directed agents for diagnostic imaging or therapy of steroid receptor-positive tumors.

INTRODUCTION In the development of in vivo diagnostic imaging agents for receptor systems such as steroid receptors, a major consideration is that the radiolabeled hormone analogue maintains high binding affinity for the receptor (1). Thus, the logical choices for labeling have been radionuclides such as carbon-11, fluorine-18, and isotopes of bromine and iodine, as these single-atom systems present a minimum of increased steric bulk when substituted on the ligand (I). While many receptor ligands labeled with the aforementioned radionuclides have been prepared, their use on a routine basis is constrained by the relative difficulty in producing or obtaining these radionuclides. By contrast, diagnostic imaging agents based on technetium-99m have the advantage of radionuclide availability through the molybdenum-99/technetium-99mgenerator system (2). So, it is no surprise that almost all routine in vivo radioimaging is based on technetium-99m. The availability advantage of this radiotracer, however, is compromised by the nature of the labeling system, for attachment of a metal ion to an organic molecule requires the construction of a chelating system. Various neutral chelating systems have been used, the most common being the bis-amino bis-thiol (BAT or N2S2) systems (3) and amide analogues ( 4 ) thereof, the propylene amine oximes

* Address correspondence to John A. Katzenellenbogen, Department of Chemistry, Box 37 Roger Adams Laboratory, University of Illinois, 1209 W. California St., Urbana, Illinois 61801.

+ University of

Illinois. Massachusetts Institute of Technology. 1 Harvard Medical School. 1043-1802/91/2902-0353$02.50/0

(HMPAO) (5), and the boronate-capped tris-dioximes (BATO) (6). These three and other technetium chelate systems are large, having molecular weights of 350 or greater, comparable to those of receptor ligands themselves, and they are bulky. Thus, they appear unsuitable for labeling receptor ligands where steric complementarity is a prime consideration. In our development of diagnostic imaging agents for steroid-receptor-positive tumors of the breast, we and others have prepared estrogens labeled with radionuclides of fluorine (3, bromine (8),and iodine (9) and measured their binding affinity toward the estrogen receptor. We have found that there are sites on the hormone analogue where even a minimal increase in steric bulk reduces binding affinity markedly, whereas other sites are quite tolerant of groups as bulky as bromine and iodine and may even show increased binding affinity (I,10). In more recent work on radiolabeled progestins for breast tumor imaging, we were struck by the fact that some synthetic progestins bearing very large substituents retain either good affinity for the progesterone receptor or have high biological activity. A number of these are shown in Chart I. The 21-(phenylse1eno)progestin(11)has an affinity for progesterone receptor that is only somewhat lower than that of progesterone, and 17a- (12) and 16a,l7a-substituted (13) systems are known to be potent progestins. Finally, certain derivatives of the antiprogestin RU 38486 (RU486, mefipristone) have high affinities for receptor (14).

Emboldened by these observations, we set out to prepare a series of progestins in which a metal-chelating system of the N2S2 type (and in one case a NzSO system in which 0 1991 American Chemical Society

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No. 5, 1991

Chart I

17a-substituted RBA = 138"

21-substituted FiBA = 59"

1lpsubstituted

RBA = 690"

one thiol is replaced by alcohol) is attached to the steroidal skeleton through the 21-, 17a-, 16a,17a-, or llp-site. In order to avoid the use of radioactive metals at this preliminary stage, we prepared the oxo-rhenium rather than the oxo-technetium complexes of these systems, and determined their binding affinities toward the progesterone receptor. In all cases, introduction of the oxo-rhenium chelate system lowers the receptor binding affinity of these systems, except the 118-substituted one; one diastereomer in this system has an affinity much greater than that of progesterone. These findings demonstrate that it is possible to prepare metal-chelate conjugates of receptor-binding hormone analogues that retain high binding affinity for their receptor. Such agents may have potential use in vivo as receptor-directed agents for diagnosis or therapy. EXPERIMENTAL PROCEDURES General. Solvents and reagents were purchased from the following commercial sources: Aldrich, Mallinckrodt, Sigma, Fisher, Baker, Eastman, or Alfa. Tetrahydrofuran (THF) was distilled from sodium/ benzophenone ketyl immediately prior to use. Other solvents were used as received, unless otherwise noted. Analytical thin layer chromatography (TLC) was performed with Merck silica gel F-254 glass-backed plates. Visualization was achieved by phosphomolybdic acid (PMA) or anisaldehyde spray reagents, iodine, or UV illumination. Flash chromatography was performed according to Still (15), using Woelm silica gel (0.032-0.064 mm) and Merck silica gel (0.04H.063 mm). Highperformance liquid chromatography (HPLC) was performed isocratically on a Spectra-Physics Model 8700 or a Varian 5060 liquid chromatograph with an analytical 5-pm Si02 column (4.6 mm X 30 cm, Varian Si-5 Micro Pak) or a preparative Si02 column (WhatmanPartisil M-9, 0.9 cm X 50 cm). The UV absorbance of the eluent was monitored at 254 nm. Melting points were determined on either a Thomas Hoover, Fisher Johns, or Hacker melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded on Perkin-Elmer 1320 spectrophotometer. The data are given in inverse centimeters with only diagnostic bands reported. Proton magnetic resonance

16a, 17a-substituted RBA = 12913

progesterone R B A - 100

('H NMR) spectra were obtained on a Varian XL-200 (200 MHz), a General Electric QE-300 (300 MHz), or a Nicolet NT-360 (360 MHz) spectrometer; chemical shifts are reported downfield of a tetramethylsilane internal standard (6 scale). Low-resolution electron-impact mass spectra were obtained on a Finnigan MAT CH-5 spectrometer. High-resolution, electron-impact, exact mass determinations were obtained on a Varian MAT 731 spectrometer. Both low- and high-resolution fast atom bombardment (FAB) mass spectra were obtained on a VG instrument (ZAB HF), employing a dithiothreitol matrix. Elemental analyses were performed by the Microanalytical Service of the University of Illinois. A standard procedure for product isolation was utilized that involved quenching of the reaction mixture in an aqueous solution, followed by a thorough extraction with an organic solvent, washing of the extracts, drying of the organic extract over an anhydrous salt, filtration, and evaporation of solvent under reduced pressure. This is indicated by the phrase "product isolation" followed by a list of the components in parentheses. The chemical nomenclature for norgestrel is 13@-ethyl-17a-ethynyl-l7j3hydroxy-4-gonen-3-one. Compounds 1 (I6),2 (I7),BAT I (I 7), C2 (18),C3 (IB),C4 (19), and D3 (20)were prepared according to literature procedures. BAT I1 Chelator. N,iV'-Bis[2-methyl-2-[(triphenylmethyl)thio]propyl]ethylenediamine (BAT11). BAT I (7.06 g, 29.9 mmol) was dissolved in 70 mL of trifluoroacetic acid. Triphenylmethanol(l5.8 g, 60.6 mmol) was added and the solution was allowed to stir for 30 min. The solvent was evaporated over 18 h under a stream of nitrogen. The thick brown oil was diluted with 200 mL of Et20 and the resulting white precipitate was collected and washed with 50 mL of EtzO. The white solid was partitioned between 100 mL of EtOAc and 50 mL of 1N NaOH. Product isolation (EtOAc, MgS04) and purification (flash chromatography, 10/1 EtlO/MeOH) gave 7.1 g (33%)of BAT I1 as a white foam. The product runs as a streak with a median Rf = 0.30 on silica TLC: 'H NMR (CDC13) 6 7.62 (d, J = 7.7,12 H, o-ArH), 7.13-7.26 (m, 18 H, m,p-ArH), 2.38 (s,4 H, NCHzCHzN), 1.75 (s,4 H, NCH&(CH&), 1.03 (s,12 H, CH3; MS (LRFAB) m / z (relative intensity) 721 (M+ + H, 181,479 (5), 243 (100);

Progestin-Rhenium Complexes

HRFAB calcd for CMHSZNZSZ (+H) 721.3650, found 721.3641. Anal. Calcd for CMHSZNZSZ: C, 79.95; H, 7.27, N, 3.89. Found: C, 79.80; H, 7.45, N, 3.73. Progesterone C-21 Series (A). 214 [(Trifluoromethyl)sulfonyl]oxy]progesterone (Al). Trifluoromethanesulfonic anhydride (0.30mL, 1.8 mmol) was added dropwise to a stirring 5 mL, -78 “C CHZC12 solution containing 0.5 g (1.5 mmol) of deoxycorticosterone and 0.23 mL (2.0 mmol) of 2,6-lutidine. After 30 min, the orange reaction mixture was allowed to warm to room temperature whereupon the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (1/1 EtOAc/hexane) to give 0.55 g (79%)of A1 as a white foam. The product is unstable and cannot be stored, therefore it was carried on to the next step immediately: ‘H NMR (CDC13,200 MHz) 6 5.84 (s, 1 H, 4-CH),4.97 (AB q, 2 H, A6 = 0.046, J = 16.71 Hz, CHzOTf), 4.20 (5, br, 1 H, 17-CH), 1.20 (s, 3 H, 18-CH3), 0.73 ( 8 , 3 H, 19-CH3); MS (LRFAB) m/z (relative intensity) 463 (M+ + H, 20); HRFAB calcd for CzzH2gOgSF3 (+H) 463.1758, fc ind 463.1766. 21-[N-[2-1N-[2’-Methyl-2’-[ (triphenylmethy1)thiolpropyl]amino]ethyl]-N-[ 2’-methyl-2’-[(triphenylmethyl)thio]propyl]amino]progesterone (21-BATI1 progesterone, A2). Steroid A1 (150 mg, 0.32 mmol) dissolved in 1 mL of CHzClz was added dropwise to a 3 mL, -78 OC solution of BAT I1 (500 mg, 0.69 mmol) in CHZC12. After the addition, the reaction mixture was allowed to warm to room temperature and was stirred for 2 h. Product isolation (HzO and 1.5 equiv of 1 N NaOH, CHzClZ, MgS04) resulted in a crude yellow oil that was purified through the use of two separate silica gel flash columns. The first column was eluted with 1/20 MeOH/ EtOAc; this separated the desired product from BAT I1 and most of the side products. The second column was eluted with 1/20 MeOH/CHzClZ; this separated the desired product from the deoxycorticosterone side product. The product runs as a streak with a median Rf = 0.30 on silica TLC (1/20 MeOH/CHzClz). The isolated yield after purification was 0.24 g (72%)of A2 as a white foam: ‘H NMR (CDC13, 300 MHz) 6 7.57-7.62 (m, 12 H, o-ArH), 7.13-7.26 (m, 18 H, m,p-ArH), 5.74 ( 8 , 1 H, 4-CH), 3.47 (AB q, 2 H, A6 = 0.057, J = 18.79 Hz, CHzN), 1.17 (s, 3 H, 18-CH3),0.92 ( ~ , H, 6 C(CH3)2),0.89 ( ~ , H, 6 C(CH&), 0.62 (s,3H, 19-CH3);MS (LRFAB)m/z (relativeintensity) 1033 (M+ + H, 23), 859 (3), 791 (7), 547 (13), 244 (100); HRFAB calcd for C~~H&NZSZ (+H) 1033.5739, found 1033.5746. Norgestrel 17a Series (B). A5(6)- and A5(lo)-3,3(Ethy1enedioxy)-138-ethyl-17a-ethynylgonen-178-01 (Bl). A catalytic amount of p-TsOH (6 mg, 0.03 mmol) was added to a solution containing ethylene glycol (0.054 mL, 0.97 mmol), benzene (25 mL), and norgestrel (100 mg, 0.32 mmol). The mixture was heated to reflux with azeotropic distillation for 4 h and then allowed to cool to room temperature. Product isolation (aqueous saturated NaHC03, EtOAc, MgSO4) and purification (flash chromatography, 1/4 EtOAc/hexane) gave 99 mg (87 5%) of B1 as a 1/2 mixture of As(6)/A6(10)double bond isomers (glass): IH NMR (CDC13) 6 2.59 (s, 1 H, C W H ) , 3.92-4.02 (m, 4 H, OCHzCHzO), 5.44-5.49 (m, 0.33 H, 6-CH); MS (LREI, 70 eV) m/z (relative intensity) 356 (M+, 3), 294 (l),273 (l),99 (loo),86 (9), 55 (8).Anal. Calcd for C23H3203: C, 77.49; H, 9.05. Found: C, 77.23; H, 9.30. As@)-and AS(’O)-3,3-( Ethy1enedioxy)-138-ethyl-17a(3’-hydroxypropynyl)gonen-17~-ol (B2). n-BuLi in hexane (1.6 M, 0.058 mL, 0.093 mmol) was added to a solution of steroid B1 (15 mg, 0.042 mmol) in 0.5 mL of

Bloconlugete Chem., Vol. 2, No. 5, 1991 355

THF at -78 “C. After stirring for 10 min, the dianion mixture was then added to a heterogeneous slurry of 2.5 mg of paraformaldehyde in 0.5 mL of THF a t -78 OC. The reaction was warmed to room temperature and stirred for 30 min. Product isolation (cold aqueous saturated NH4C1, CHzClz, MgS04) and purification (flash chromatography, 3/4 EtOAc/hexane) gave 12 mg (74%)of B2 as a 1/2 mixture of As(6)/A5(10)double bond isomers (glass): lH NMR (CDCl3) 6 3.95-4.01 (m, 4 H, OCHzCHzO), 4.33 ( ~ , 2H, CHzOH), 5.46 (d, J = 5.13 Hz, 0.33 H, 6-CH); MS (LREI, 70 eV) m/z (relative intensity) 386 (M+, 3), 368 (l),330 (2), 99 (loo), 86 (19), 55 (4). Anal. Calcd for C24H~04: C, 74.57; H, 8.87. Found: C, 74.34; H, 9.10. A5(6)- and A5(lo)-3,3-( Ethy1enedioxy)-138-ethyl-17a[3’-[(methylsulfonyl)oxy]propynyl]gonen- 178-01 (B3). A solution of containing 5 mL of THF (at room temperature), steroid B2 (12 mg, 0.031 mmol), and Et3N (0.022mL, 0.16 mmol) was treated with 4.8pL (0.061mmol) mesyl chloride and allowed to stir for 1 h. Product isolation (HzO, EtOAc, MgS04) and purification (flash chromatography, 3/4 EtOAc/hexane) gave 12.2 mg (85%)of €33 as a 1/2 mixture of As@)/A5(lo)double bond isomers (glass). The product is slightly unstable on silica gel: lH NMR (CDC13) 6 3.12 (s,3H, OSOZCH~), 3.94-4.00 (m, 4 H, OCHZCHzO),4.93 (s,2 H, CHzOMs),5.45-5.47 (m,0.33 H, 6-CH); MS (LREI, 70 eV) m/z (relative intensity) 464 (M+, 2), 446 (2), 373 (4),99 (loo),79 (25), 43 (27). Anal. Calcd for CZSH&~S: C, 64.63; H, 7.81; S, 6.90. Found: C, 64.78; H, 7.90; S, 6.78. A5@)-and A5(lo)-3,3-(Et hylenedioxy)-13@-ethyl17a[3-[N-[2’4N-[ 2”-methyl-2”-[(triphenylmethyl)t hiolpropyl]amino]ethyl]-N-[2’’-methyl-2”-[(triphenylmethyl)thio]propyl]amino]propynyl]gonen- 178-01 (17a-BAT I1 norgestrel, B4). Steroid B3 (1.7 g, 3.66 mmol) and BAT I1 (5.27 g, 7.31 mmol) were dissolved in 50 mL of o-dichlorobenzene and the mixture was then heated to 90 “C and stirred for 2 h. While still at 90 OC, the solvent was partially evaporated (blown off) under a stream of nitrogen for 0.5 h. The reaction mixture was then stirred for 15 h at room temperature while the rest of the solvent was evaporated under a nitrogen stream, resulting in a thick brown oil. Product isolation (0.5 N NaOH, CHzClz, MgSO4) and purification (flash chromatography, 3/4 EtOAc/hexane, fully purified after two separate columns) gave product B4 as a 1/2 mixture of A5@)/A5(lo)double bond isomers (2 g, 51 % , glass). The product runs as a streak with a median Rf= 0.35 on silica TLC: 1H NMR (CDCl3) 6 7.58-7.65 (m, 12 H, o-ArH), 7.14-7.26(m,18H,m,p-ArH),5.43-5.48(m,0.33H,6-CH), 3.90-4.00 (m, 4 H, OCHzCHzO), 3.31-3.29 ( 8 , 2 H, CECCHZN, 2/1 ratio), 2.47-2.54 (m, 2 H, NCHzCHzN), 2.32-2.37 (m, 2 H, NCHzCHzN), 0.97 (s, 6 H, C(CHs)z), 0.91 (s, 6 H, C(CH3)z); MS (LRFAB) m/z (relative intensity) 1089 (M+ + H, 28), 847 (6), 603 (22), 529 (lo), 244 (100);HRFAB calcd for C,ZH~O~NZSZ (+H) 1089.6002, found 1089.5980. Anal. Calcd for C ~ Z H ~ O ~ N C, Z S79.45; Z: H, 7.69; N, 2.57. Found: C, 79.43; H, 7.95; N, 2.37. Estradiene 118 Series (C). 3,3-(Ethylenedioxy)-5(10),9(ll)-estradien-l78-01(C5). Ethylene glycol (6mL, 96.7 mmol) and concentrated HzS04 (6 drops) were added to a suspension of estradienone C4 (2.05 g, 7.53 mmol) in 60 mL of benzene. The mixture was slowly distilled over 1.5h so that about 20 mL of benzene was lost. The reaction was then cooled to room temperature. Product isolation (solid NaHC03 and HzO, EtzO, MgSO4) and purification (flash chromatography, 1/1 EtOAc/hexane) gave 2.3 g (96%)Of C5 as a glass: lH NMR (CDCl3) 6 5.55-5.57 (m, 1 H, ll-CH), 3.98 (8, 4 H, OCHzCHzO), 3.76 (t, 1 H, J =

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8.62 Hz, 17-CH),0.73 (s,3 H, 18-CH3);MS (LREI, 70 eV) m / z (relative intensity) 316 (M+, loo), 288 (62), 243 (841, 197 (31), 86 (69), 43 (92). Anal. Calcd for C20H2803: C, 75.91; H, 8.92. Found: C, 76.05; H, 9.01. 3,3-(Ethylenedioxy)-(5a,lOa)-and -(5@,10B)-epoxy9(1l)-estren-178-01(C6andC7). SteroidC5 (0.60g,0.190 mmol) was dissolved in 2 mL of CHzClz and cooled to 10 "C. Pyridine (1pL, 0.012 mmol), CF3COCF3 (3 pL, 0.017 mmol), and a 30% solution of HzOz (29 pL, 0.284 mmol) were added, respectively. The reaction was allowed to warm to room temperature and was vigorously stirred for 8 h. Product isolation (10% sodium thiosulfate, CHzClz, CuSO4, HZO, MgS04) gave a clear foam. The crude ratio of a/@-epoxideformation was 4.15/1 (by NMR). The a-isomer was partially purified (flash chromatography, 1/1 EtOAc/hexane), giving 0.45 g of a 6/1 cy/@-epoxide mixture as a glass. The a-epoxide has an Rf= 0.23 whereas the 0-epoxide has an Rf = 0.26 on silica TLC (1/1EtOAc/ hexane). The 6/1 mixture of isomers was used in the next reaction sequence. Analytically pure a-epoxide C6 and @-epoxideC7 were obtained by taking select fractions from the flash column: 1H NMR of C6 (CDC13) 6 6.02-6.04 (m, 1H, 11-CH), 3.87-3.97 (m, 4 H, OCH2CH20), 3.75 (t, br, 1H, J = 8.44 Hz, 17-CH), 2.48 (ddd, 1 H, J = 2.54, 4.58, 14.58 Hz, 8-CH), 0.75 (s, 3 H, l8-CH3); lH NMR of C7 (CDCl3) 6 5.84-5.86 (m, 1 H, 11-CH), 3.87-3.96 (m, 4 H, OCH~CHZO), 3.74 (dt, 1H, J = 8.15,4.56 Hz, 17-CH),0.73 (s, 3 H, 18-CH3). Anal. Calcd for (6/1 a/@-epoxide mixture) CzoHzs04: C, 72.26; H, 8.49. Found: C, 72.15; H, 8.75. e-[(2-Tetrahydropyranyl)oxy]-p-bromotoluene ( T l ) . Dihydropyran (1.22 mL, 13.4 mmol) was added to a 5 "C solution of 4-bromobenzyl alcohol (0.5 g, 2.7 mmol) dissolved in 7 mL of THF. A catalytic amount (20 mg) of p-toluenesulfonic acid was added and the reaction mixture was allowed to warm to room temperature and stirred for 3 h. Product isolation (solid NaHC03 and Hz0, EtzO, MgS04) and purification (flash chromatography, 1/6 EtOAc/hexane) gave 0.7 g (97%) of T1 as a liquid: 'H NMR (CDC13,300 MHz) 6 7.36 (AB q, 4 H, A6 = 0.22, J = 8.43 Hz, Ar-H), 4.69 (t, br, 1H, J = 3.38, OCHO), 4.60 (AB q, 2 H, A6 = 0.28, J = 12.28 Hz, ArCHzO), 3.90 (ddd, 1H, J = 3.21, 8.24, 11.31 Hz, OCHHCHz), 3.51-3.58 (m, 1 H, OCHHCHz), 1.5-1.9 (m, 6 H, OCHZCHZCHZCHZ). Anal. Calcd for C12H1502Br: C, 53.15; H, 5.58; Br, 29.47. Found: C, 53.16; H, 5.59; Br, 29.50. 3,3-(Ethylenedioxy )-5a-hydroxy-1 lB-[a-[(2'-tetrahydropyranyl)oxy]-p-tolyl]-9(lO)-estren-l7B-ol(C8). Aryl bromide T1 (0.647 g, 2.39 mmol), diluted with 3 mL of THF, was added dropwise to a heterogeneous solution of magnesium turnings (61mg, 2.5 mmol) in 3 mL of THF. The solution was then heated to reflux for 3 h and then cooled to 5 "C whereupon CUI (10 mg, 0.0525 mmol) was added. After stirring for 15 min, a 6/1 mixture of C6/C7 (0.1g, 0.301 mmol), dissolved in 3 mL of THF, was slowly added dropwise to the reaction mixture and the solution was stirred for 1h a t 5 "C. Product isolation (coldaqueous saturated NH4C1, CHzC12, MgS04) and purification (flash chromatography, 4/1 EtOAc/hexane) gave 0.139 g (88%) of C8 as a mixture of THP diastereomers (glass): 'H NMR (CDC13, 300 MHz) 6 7.22 (AB q , 4 H, A6 = 0.05, J = 8.24 Hz, Ar-H), 4.71 (t, br, 1 H, J = 3.40, OCHO), 4.60 (AB q, 2 H, A6 = 0.30, J = 11.93 Hz, ArCH20, doubling of some signals due to diastereomeric mixture), 4.36 (s, 1 H, 5-COH), 4.25 (d, br, 1 H, J = 7.01 Hz, 11-CH), 3.93-4.04 (m, 4 H, OCHZCHZO),3.87-3.93 (m, 1 H, OCHZCHZCHZ), 3.61 (t, br, 1 H, J = 7.93 Hz, 17-CH), 3.50-3.57 (m, 1 H,

DiZio et al.

OCHzCHzCHz), 0.35 ( 8 , 3 H, l8-CH3). Anal. Calcd for C32H4406: C, 73.25; H, 8.45. Found C, 73.30; H, 8.46. 3,3-(Ethylenedioxy)-5a-hydroxy11&[ a-[(%'-tetrahydropyranyl)oxy]-ptolyl]-9 (10)-estren- 17-one (C9). (nPr)lNRuOd (65 mg, 0.19 mmol) was added to a solution containing steroid C8 (1.08 g, 2.06 mmol), N-methylmorpholine N-oxide (362 mg, 3.09 mmol), and 15 mL of CHzClz. The reaction mixture slowly turned black as it was stirred for 10 min. After quenching with 7 mL of a 10% sodium thiosulfate solution and the further addition of 10 mL of CHzClz, the entire mixture was filtered through Celite. The organic layer of the filtrate was separated, washed with water, dried over MgSO4, and evaporated under reduced pressure. Product purification (flash chromatography, 3/ 1EtOAc/hexane) gave 1.04 g (98%) of C9 as a mixture of THP diastereomers (glass): 'H NMR (CDC13,300 MHz) 6 7.23 (AB q, 4 H, A6 = 0.048, J = 7.68 Hz, Ar-H), 4.71 (t, br, 1H, J = 3.42 Hz, OCHO), 4.60 (AB q, 2 H, A6 = 0.31, J = 12.00 Hz, ArCH20, doubling of some signals due to diastereomeric mixture), 4.40 (8, 1 H, 5-COH), 4.32 (d, br, 1 H, J = 7.14 Hz, 11-CH), 3.95-4.05 (m, 4 H, OCHZCHZO), 3.88-3.94 (m, 1H, OCHZCHZCHZ), 3.50-3.59 (m, 1 H, OCHzCHzCHz), 0.49 (s, 3 H, l8-CH3). Anal. Calcd for C32H4206: C, 73.53; H, 8.10. Found: C, 73.34; H, 8.19. 3,3-(Ethylenedioxy)-5a-hydroxy-1 1@-[a-[(%'-tetrahydropyrany1)oxy]-ptolyll- l7a-propynyl-9(lO)-estren178-01 (C10). Steroid C9 (1.2 g, 2.30 mmol), dissolved in 10 mL of THF, was added dropwise over 10 min to a room temperature heterogeneous solution of propynyllithium (0.54 g, 11.84 mmol) in 50 mL of THF. The reaction mixture was stirred for 1 h and concentrated to half its volume. Product isolation (Hz0, EtzO, MgS04) and purification (flash chromatography, 2/ 1EtOAc/hexane) gave 1.1g (85%) of C10 as a glass: 'H NMR (CDCls, 300 MHz) 6 7.21 (AB q, 4 H, A6 = 0.049, J = 8.14 Hz, Ar-H), 4.71 (t, br, 1 H, J = 3.20 Hz, OCHO), 4.60 (AB q, 2 H, A6 = 0.30, J = 11.98 Hz, ArCHz0, doubling of some signals due to diastereomeric mixture), 4.32 (d, br, 1H, J = 7.30 Hz, 11-CH), 3.95-4.02 (m, 4 H, OCHzCHzO), 3.88-3.94 3.52-3.56 (m, 1 H, OCHZCHZ(m, 1 H, OCHZCH~CHZ), CHZ),1.89 (s, 3 H, C=CCH3), 0.44 (s,3 H, 18-CH3). Anal. Calcd for C35H4606: C, 74.70; H, 8.25. Found: C, 74.51; H, 8.46. 1 la-(a-Hydroxy-p-tolyl)-170-propynyl- 17B-hydroxy-4,9-estradien-3-one (Cll). Steroid ClO (195 mg) was dissolved in 4 mL of 7/3 acetic acid/water and the solution was heated at 50 "C for 1.5 h and then cooled to room temperature. Product isolation (aqueous saturated NaHC03, CHzClZ, MgS04) and purification (flash chromatography, 2 / 1 EtOAc/hexane) gave 100 mg (70%) of C11 as a glass: 'H NMR (CDC13,300 MHz) 6 7.22 (AB q, 4 H, Ab = 0.070, J = 8.26 Hz, Ar-H), 5.78 (s, 1H, 4-CH), 4.66 (d, 2 H, J = 5.71 Hz, ArCHzOH), 4.43 (d, br, 1 H, J = 6.20 Hz, II-CH), 1.90 (s, 3 H,C=CCH3), 0.50 (9, 3 H, l8-CH3); MS (LREI, 70 eV) m/z (relative intensity) 416 (M+, 54), 398 (30), 334 (45), 235 (70), 225 (541, 91 (100); HREI calcd for CzsH3203 416.2352, found 416.2356. 1 l@-[a-[(Methylsulfonyl)oxy]-p-tolyl]-l7a-propynyl-l7~-hydroxy-4,9-estradien-3-one (C12). Methanesulfonyl chloride (7.44 pL, 0.096 mmol) was slowly added to a stirring mixture containing steroid C l l ( 2 0 mg, 0.048 mmol), 13.4 pL of Et3N (0.096 mmol), and 1mL of THF at room temperature. The reaction mixture was stirred for 0.5 h. Product isolation (HzO, EtzO, MgSO4) and purification (flash chromatography, 2 / 1 EtOAc/hexane) gave 21 mg (88%)of C12 as a glass. The product has a half-life of 1day when subjected to ambient conditions

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tolyl]- 17a-propynyl- 178-hydroxy-4,9-estradien-3but is moderately stable if stored a t -20 “C: ‘H NMR one (118-BAT I estradiene, C14). Crude C14 was (CDCl3,300MHz) 6 7.29 (AB q , 4 H,A6 = 0.106, J = 8.10 synthesized by the AgNO3reaction described above. This Hz, Ar-H), 5.79 (s, 1H, 4-CH), 5.21 (d, 2 H, J = 5.71 Hz, material was used for the Re-insertion reaction: MS (LRArCHzOMs), 4.45 (d, br, 1 H, J = 7.07 Hz, 11-CH), 2.88 FAB) m/z (relative intensity) 635 (M+ + H, 25), 460 (5), (s, 3 H, OS02CH3), 1.90 (8, 3 H, CWH3), 0.47 (9, 3 H, 279 (8); HRFAB calcd for C38H&N& (+H) 635.3705, l8-CH3); MS (LRFAB) m / z (relative intensity) 495 (M+ 635.3722. + H,271,461 (25), 399 (32); HRFAB calcd for C ~ ~ H ~ ~ O S Sfound I (+HI: 495.2205, found 495.2215. Pure C14, suitable for RBA determinations, was obtained by the Hg(0Ac)z method described to form D8 1lb-[a-[N-[2-[N-[2’-Methyl-2’-[ (triphenylmethy1)from D7 (65% yield): ‘H NMR for C14 (CDC13,300MHz) t hio]propyl]amino]et hyll-N- [2’-met hyl-2’- [ ( t r i 6 7.21 (AB q, 4 H, A6 = 0.161, J = 8.04 Hz, Ar-H), 5.78 (8, phenylmethyl)thio]propyl]amino]-ptolyll- l7a-pro1H, 4-CH), 4.43 (d, br, 1H, J = 6.75 Hz, 11-CH), 3.71 (8, pynyl-l78-hydroxy-4,9-estradien-3-one (1 W B A T I1 2 H, ArCH2N), 3.08 ( 8 , br, 3 H, SH and NH), 1.90 (s,3 H, estradiene,C13). A solution of BAT I1 (1.5 g, 2.12 mmol) CZCCH~),1.347 ( ~ , H, 6 C(CH&), 1.333 ( ~ , H, 3 C(CH3)and steroid C12 (0.5g, 1.01mmol) in lOmL of l,2-dichloro3 18-CH3). (CH3)),1.325 (s,3 H, C(CH3)(CH3)),0.50 ( ~ , H, ethane was stirred at 80 “C for 2 h. While still at 80 “C, the solvent was evaporated under a stream of nitrogen. General Re0 Insertion Reaction. Exemplified by The resulting thick brown-orange oil was cooled to room Re0 insertion into A3 to form A4, the following method temperature. Product isolation (1 N NaOH, CH2C12, is completely general and was used to form all the steroidMgSOr) and purification (flash chromatography, 1/1 BAT Re chelates. EtOAc/ hexane, required two or more separate columns) Steroid A3 (102 mg, 0.19 mmol) and oxotrichlorobisgave C13 (0.71 g, 63%) as a glass. The product runs as (triphenylphosphine)rhenium(V) (155 mg, 0.19 mmol) a streak with a median Rf = 0.30 on silica TLC and follows were dissolved in 1.90 mL (1.90 mmol) of 1N methanolic directly behind a prominent yellow band: lH NMR NaOAc. The reaction mixture was heated at reflux for 45 (CDCl3) 6 7.02-7.61 (m, 34 H, Ar-H), 5.74 ( 8 , 1H, 4-CH), min and then allowed to cool to room temperature. 4.45 (d, br, 1 H, J = 6.73 Hz, 11-CH), 3.43 (s, br, 2 H, Product isolation (H20, EtOAc, MgS04) gave a green oil. ArCH2N), 1.90 (s,3 H, CECCH~),0.95 (s,6 H, C(CH3)2), [2 1-[N-[24N-( 2’-Methyl-2’-mercaptopropyl)amino]0.94 ( ~ ,H, 6 C(CH3)2),0.44 (s,3 H, 18-CH3);MS (LRFAB) ethyl]-N-(2’-met hyl-2’-mercaptopropyl)amino]prom/z (relative intensity) 1119 (M+ + H, 40), 877 (8), 633 gesteronato(4-)]oxorhenium( V) (21-N& Re Chelate, (15), 559 (17), 244 (100). HRFAB calcd for C~-&&N~SZ A4). Pure product (diastereomeric mixture of A4syn1,2) (+HI 1119.5896, found 1119.5879. was obtained as a glass (29%) after two separate silica General S-Deprotection Reaction. Exemplified by flash columns (1/1EtOAc/hexane). The syn (purple) dithe deprotection of C13 to form C14, the following method astereomeric pair were the only desired products isolated. is completely general and was used to deprotect all of the The diastereomers were inseparable by HPLC: lH NMR steroid-BAT chelators. (CDC13,300 MHz) 6 5.74 ( 8 , l H, 4-CH), 4.78/4.77 (AB 9, A homogeneous mixture containing AgN03 (58.5 mg, 2 H, A6 0.133/0.206, J = 19.29/19.22 Hz, COCH2N, 1/1 0.345 mmol), pyridine (30 pL, 0.37 mmol), and 1 mL of diastereomeric ratio), 4.05-4.18 (m, 1 H, NCHHCH2N), EtOH was added to a 65 “C solution of steroid C13 (193 3.91-3.98 (m, 1H, NCHHCHzN), 3.90 (d, 1 H, J = 10.97 mg, 0.172 mmol) dissolved in 2 mL of 2 / 1 EtOAc/EtOH. Hz, amide-NCHHC(CHa)z,diastereomers displaced by 5.9 The reaction mixture was stirred a t 65 “C for 45 min, Hz), 3.65 (d, 1 H, J = 13.35 Hz, amine-NCHHC(CHdz), reduced to one-third of its volume, and then cooled to 3.61 (d, 1H, J = 10.97 Hz, amide-NCHHC(CH3)2),3.22room temperature. With stirring, 20 mL of Et20 was 3.33 (m, 1 H, NCHHCHzN), 2.89-2.98 (m, 1 H, NCHadded, causing the disilver salt to precipitate. The HCHzN), 2.63 (d, 1 H, J = 13.35 Hz, amine-NCHHCprecipitate was collected by filtration over Celite. As the (CH3)2),1.91 ( ~ , H, 3 C(CH3)(CH3)),1.70 ( ~ , H, 3 C(CH3)precipitate could not be separated from the Celite pad, (CHa), diastereomers displaced by 6.7 Hz), 1.58 ( 8 , 3 H, the inseparable mixture of Celite and precipitate was dried. C(CH&CH3), diastereomers displaced by 19.9 Hz), 1.47 The dried Celite/precipitate mixture was stirred for 30 (s,3 H, C(CH&CH3)), 1.19 (s,3 H, 18-CH3,diastereomers min in a solution of 15 mL of acetone and 0.6 mL of 6 N displaced by 2.4 Hz), 0.77 (s, 3 H, 19-CH3, diastereomers HC1. Product isolation (aqueous saturated NaHC03, displaced by 2.4 Hz); IR (solution,CHCl3) 1708(20-ketone), EtOAc, MgSO4) gave 133 mg of C14 as a crude off-white, 1664 (3-ketone), 938 cm-’ (Re=O); MS (LRFAB) m/z amorphous solid. The crude product was used in this form (relative intensity) 749 (M+ + H (Re = 187), loo), 732 (31, for the next reaction sequence. A3 and B5 were produced 549 (81,419 (121,279(11);HRFAB calcd for C31H4903N2S2in an analogous manner and afforded similar yields of Re (+H) 749.2821, found 749.2822. crude product. [ 138-Ethyl-178-hydroxy-l7a-[34N-[2-[N-(t’-methyl2l-[N-[ 24 N-( 2‘-Methyl-2‘-mercaptopropyl)amino]- 2’-mercaptopropyl)amino]ethyl]-N-(2’-methyl-2’ethyl]-N-(2’-methyl-2’-mercaptopropyl)amino]pro- mercaptopropyl)amino]propynyl]-4-gonen-3-onatogesterone (21-BAT I progesterone, A3): MS (LRFAB) (4-)]oxorhenium(V) (17a-NzS2Re Chelate, B6). Pure m/z (relative intensity) 549 (M+ H, loo), 531 (20), 515 product (B6) was obtained as a glass (40%) after two (181, 461 (10); HRFAB calcd for C ~ ~ H S ~ O Z(H) N ~ S ~separate silica flash columns using 1/1EtOAc/hexane as 549.3548, found 549.3551. the eluent in the first column and 3/2/1 EtzO/CH2Clz/ hexane as the eluont in the second column. Syn (BGsynl, 138-Ethyl- 1 7 4 3 4N-[2’-[ N-( t”-methyl-2”-mercapB6syn2) and anti (BGantil, B6anti2) diastereomericpairs topropy1)aminolet hyll-N- (2”-met hyl-%”-memaptowere obtained in a 4/1 (syn/anti) ratio. The diastereopropyl)amino]propynyl]- 178-hydroxy-4-gonen-3mers in each diastereomeric pair were inseparable by one (17a-BAT I norgestrel, B5): MS (LRFAB) m / z (relative intensity) 561 (M+ + H, loo), 527 (a), 325 (2); HPLC. HRFAB calcd for C32H6202N2S2 (+H) 561.3548, found Analysis for syn diastereomer mixture B6syn1,2: ‘H 561.3542. NMR (CDC13,300 MHz) 6 5.85 (8, 1H, 4-CH),4.652/4.655 118-[~-[N-[2-[N-(2’-Methyl-2’-mercaptopropyl)ami- (AB q, 2 H, A6 = 0.091/0.118, J = 17.67/17.80 Hz, CeCCHzN, 1/1diastereomeric mixture), 4.21 (ddd, 1H, nolethyll-N-(%’-methyl-2’-mercaptopropyl)amino]-p

+

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= 0.399, J = 11.00 Hz, ArCH2N), 3.03 (d, 1 H, J = 13.08 Hz, amide-NCHHC(CHs)z),2.13 (8, 3 H, C(CHs)(CHs)), 1.94 (9, 3 H, C(CH3)(CH,)), 1.91 ( 8 , 3 H, CWCHs), 1.69 (s,3 H, C(CHd(CHs)), 1-43(8,3 H, c(CH3)(CH3)),0.46 (8, 3 H, l8-CH3); IR (solution, CHCl3) 1655 (3-ketone), 953 cm-l (Re=O); MS (LRFAB) m/z (relative intensity) 835 1-90( ~ , H, 3 C(CH3)CH3), 1.74 (9, 3 H, C(CH3)CH3), 1.62 (M+ + H (Re = 187), 19), 435 (121, 399 (51,279 (8);HR1-03(t, ( ~ , H, 3 C(CH3)(CH3)),1.46 (s,3 H, C(CH~)(CHB)), FAB calcd for C38H5103N2S2Re (+HI 835.2977, found 3 H, J = 7.33 Hz, 18-CHzCH3);IR (solution, CHC13) 3420 835.2953. (br, OH), 1663 (3-ketone), 938 cm-l (Re=O); MS (LRAnalysis for anti diastereomer C15anti2: 'H NMR FAB) m/z (relative intensity) 761 (M+ + H (Re = 187), (CDC13,300 MHz) 6 7.25 (AB q, 4 H, A6 = 0.045, J = 8.24 33), 433 (ll),279 (37);HRFAB calcd for C32H4903N2S2Re Hz, Ar-H), 5.80 (s, 1 H, 4-CH), 4.45 (d, br, 1 H, J = 7.03 (+H) 761.2815, found 761.2806. Hz, 11-CH), 4.09 (ddd, 1 H, J = 5.98, 12.54,