Fluorine-substituted corticosteroids: synthesis and evaluation as potential receptor-based imaging agents for positron emission tomography of the brain.

NW/. Med. Biol.

Vol. 19,

No. 4, pp. 461-480,

1992

ht. J. Radiat.Appl. Instrum. Part B

Copyright

0

0883-2897/92 $5.00 + 0.00 1992 Pergamon Press Ltd

Printed in Great Britain. All rights rescrwd

Fluorine-substituted Corticosteroids: Synthesis and Evaluation as Potential Receptor-based Imaging Agents for Positron Emission Tomography of the Brain MARTIN

G. POMPER’, MONICA J. KOCHANNY’, ANDREA M. THIEME’, KATHRYN E. CARLSON’, HENRY F. VANBROCKLIN’, CARLA J. MATHIAS*, MICHAEL J. WELCH’ and JOHN A. KATZENELLENBOGEN’*

‘Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, IL 61801 and 2Division of Radiation Sciences, The Edward Mallinckrodt Institute of Radiology, Washington University Medical School, 510 South Kingshighway, St Louis, MO 63110, U.S.A. (Received 4 October 1991)

We have prepared eight fluorine-substituted corticosteroids representing ligands selective for Type I and Type II corticosteroid receptor subtypes as potential imaging agents for corticosteroid receptor-containing regions of the brain. Receptor binding affinity assays show that fluorine substitution for hydroxyl or hydrogen in these steroids generally results in some reduction in affinity, with the result that the absolute affinity of these fluorine-substituted ligands for receptor is less than that typical for steroid hormones that show receptor-based, target selective uptake in viva. Five of these compounds were prepared in fluorine-18 labeled form by a simple sulfonate ester displacement reaction, and their tissue distribution was studied in the adrenalectomized rat. There is no selective accumulation nor selective retention of the Type I selective corticosteroids (‘*F-RU 26752, 21-[‘8F]fluoroprogesterone, 21-[“Flfluoro-1 la-hydroxyprogesterone) in either the brain, or other target tissues (pituitary, kidney, liver). The Type II selective corticosteroids (‘*F-RU 28362, ‘*F-triamcinolone acetonide) show uptake into the hippocampus which can be partially blocked by a competing ligand; in target tissues outside the brain, the blocking is more complete. All of the ‘*F-labeled compounds show considerable defluorination, evident as high bone activity levels. These results, coupled with earlier findings in the literature, suggest that radiolabeled corticosteroid receptor ligands with both greater metabolic stability and higher receptor binding affinity and selectivity are needed for imaging corticosteroid receptors in the hippocampus.

Introduction Corticosteroids possess diverse physiological actions including maintenance of mineral balance and me*All correspondence should be addressed to: Dr John A. Katzenellenbogen, 461 Roger Adams Laboratory, Box 37, 1209 West California Street, University of Illinois at Urbana-Champaign, Urbana, IL 61801, U.S.A. Abbreviations used: deoxycorticosterone for 21-hydroxypregn-4-ene-3,20-dione; corticosterone for 1lb,21-dihydroxypregn-4-ene-3,20-dione; cortisol for 11/?,17a,21-trihydroxypregn-4-ene-3,20-dione; aldosterone for 1lb,21dihydroxy-3,20-dioxopregn-4-en-18-al; dexamethasone for 9a-fluoro-ll/?,l7a,2l-trihydroxy-l6a-methylpregna1,4-diene-3,20-dione; triamcinolone acetonide for 9c(fluoro-ll/?,2l-dihydroxy-l7a,17a-[l-methylethylidenebis (oxy)]pregna-1,4-diene-3,20-dione; RU 26752 for 3’-(3oxo-7a-propyl-l7B-hydroxyandrost-4-ene-l7a-yl)-propionic acid lactone; RU 28362 for 1lg,l7fi-dihydroxy-6methyl-l7a-pregna-l,4,6-trien-20-yn-3-one; RU 26988 for 1lB.l7B-dihvdroxv-17a-preana-l,4,6-trien-2O-yn-3.. . one; 6a-methylprednisblone ‘for-1 1/?,17a,21-trihydroxy6a-methylpregna-1,4-diene-3,20-dione.

diation of the stress response (Sapolsky et al., 1986). Their production is controlled via the hypothalame-pituitary-adrenal axis, a classic closed loop endocrine system. Within the central nervous system, corticosteroid receptors are present at highest concentrations within hippocampal neurons, but may also be found within lateral septum and parts of the amygdala and cortex. Three types of high-affinity corticosteroid receptors have been identified: Type I (mineralocorticoid-preferring, MR), Type I (corticosterone-preferring, CR) and Type II (glucocorticoidpreferring, GR); the difference between MR and CR is not structural but functional (Funder et al., 1988). Type I receptors are more localized to the hippocampus than Type II receptors, which, although in highest concentration in the hippocampus, are more diffusely distributed throughout the brain. Functionally, Type I receptors have been suggested to be involved in control of salt appetite (MR), while Type II receptors mediate negative

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MARTING. POMPERet al.

feedback inhibition of the adrenocortical axis when circulating glucocorticoid concentrations are in the stress range; both types may provide an index of brain aging (De Kloet et al., 1986). An intriguing series of reports concerning stressinduced hippocampal damage suggested a possible role for excessive corticosteroid production in the pathogenesis of Alzheimer’s dementia (Sapolsky et al., 1986; Sapolsky and McEwen, 1988). In that disease, the hippocampus, an area of the brain intimately involved in learning and memory, degenerates with a consequent reduction in corticosteroid receptor number. The clinical diagnosis of Alzheimer’s dementia is one of exclusion, and is incorrect lO-30% of the time; definitive diagnosis is possible only at biopsy or necropsy. Much needed is a non-invasive, early diagnostic test that would eliminate other forms of dementia which, unlike Alzheimer’s dementia, are potentially treatable. Diagnostic imaging for Alzheimer’s dementia via positron emission tomography (PET) and single photon emission computerized tomography (SPECT) has recently become a focus of attention (Holman et al., 1985; Johnson et al., 1987; Jagust et al., 1987; DeKosky and Bass, 1985). However, the majority of the studies to date have been performed with the intention of exploiting the well documented alterations

in regional cerebral blood flow and glucose metabolism. An image of a selective corticosteroid hormone receptor deficit might represent a more precise physiological assessment of the Alzheimer’s dimentia patient. As certain receptor deficits are known to occur early in the course of Alzheimer’s dementia (DeKosky and Bass, 1985) corticosteroid receptor imaging may provide an early marker for the disease. In this study, we have prepared as potential imaging agents for the hippocampal receptors, fluorinesubstituted analogs of both synthetic ligands (those selective for Type I sites [RU 26752 (2) (Coirini et al., 1985)] and Type II sites [RU 28362 (3) and 26988 (4) (Coirini et al., 1985), dexamethasone (14) triamcinolone acetonide (13)] and natural ligands [deoxycorticosterone (7), corticosterone (8) and cortisol(9)]) (Fig. 1). We have determined the binding affinity of these compounds for Type I and II corticosteroid receptors and have selected five of them to prepare in i8F-labeled form for tissue distribution studies in adrenalectomized male rats. Methods Chemical synthesis

Melting points are uncorrected. Visualization of TLC was achieved with short wave U.V. light and/or

R

X

RU 26752 (1) 3’-Fhroro-RU26752 (2) :

X

R

RU 28362 (3) H RU 26988 (4) 3’-Fhroro-RU 28362 (5) : J’-Fh,toro-RU 26988 (6) F

f&

X

0

X

A& Y

&s H

0

IZ

’

X

deo~ycortIwsterone (7) coo=ytgne (CORP. 8) 21-fluoroIxogestefone (10) 2’-fl;;Zyd$Z;y21-fluoro-11~,17adIhydxyprogesterone (12)

Y

X

2

Y

z

-0

:: &I ! OH OH OH ;

&

::

F

OH OH

uifmcinolone acetoni& (TCA. 13)

dcxamclhasone @Ex,

OH 14)

K

OH O;I” CH?

21-fluoro-TC!A (15) 2l-fluoro-DEX

(16)

Fig. 1. Structures of corticosteroids and fluorine-substituted analogues.

F

OH CHs

Fluorine-substituted corticosteroids phosphomolybdic acid spray. Flash chromatography (Still et al., 1978) was performed using Woelm 32-63 pm silica gel. A standard method for product isolation was used: it involved an aqueous quench and organic extraction, drying of the extract and removal of the solvent under vacuum; the components used are given in parentheses following the phrase “product isolation”. r9F NMR spectra were obtained at 338.76 MHz and are reported in ppm upfield from internal CFCI, (4 scale). Elemental analyses are within f 0.4% of theory. High-performance liquid chromatography (HPLC) was performed isocratically on a 5 pm analytical silica gel column (4.6 mm x 30 cm, Varian Si-5 Micro Pak), a 10 pm preparative silica gel column (9 mm x 50 cm, Whatman Partisil M-9) or a Cu column (10 mm x 50 cm, Whatman Partisil M-9, ODS-2). HPLC eluent was monitored via U.V. absorbance (254nm); for radiochemical purification, HPLC eluent was also monitored with a NaI(TI) radioactivity detector. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl; all other solvents were distilled from CaH,. The BF,-S(CH3), complex was prepared by saturating (CH,)*S with BFr gas. Dimethylformamide (DMF) was freshly distilled and stored over sieves under argon. All reactions were carried out under a nitrogen atmosphere unless otherwise indicated. Chemicals were obtained from commercial sources and were used as received, unless otherwise noted. RU 27987, RU 26988 and RU 26752 were gifts of Roussel UCLAF of Paris, France. RU 28362 and [6-methyl-‘H]RU 28362 were purchased from DuPont-NEN. 6ar-Methylprednisolone was purchased from Steraloids Inc. The following compounds were prepared according to published procedures: deoxycorticosterone 21-methanesulfonate (35) (Simons ez al., 1980) cortisol 21-methanesulfonate (38) (Simons and Thompson, 1981) dexamethasone 21-methanesulfonate (41) (Simons et al., 1980). 3’(3-Oxo- 17/I-hydroxy-4,6androstadiene- 17a-yl)propionic acid y -1actone (canrenone, 18). Potassium canrenoate 17 (5 g, 12.6mmol) was suspended in THF (100 mL). Dropwise addition of 3 N HCl (50 mL) afforded a solution which was allowed to stir for 60min at room temperature. Product isolation (CHCI, ,saturated NaHCO, , brine, Na, Sod) gave a yellow oil that was recrystallized (EtOAc) to afford yellow needles (8.42mmo1, 67%): m.p. 165°C (Lit, 149-151°C); ‘H NMR (200 MHz, CDCl,) 6 1.02 (s, 3 H, 18-CH,), 1.12 (s, 3 H, 19-CH,), 5.66 (s, 1 H, 4-H), 6.10 (m, 2 H, 6-H, 7-H); i.r. (KBr) 1765 (C=O, lactone), 1650 (ketone), 1615 cm-‘; MS (EI), 340 (M+, 100) 627 (99) 136 (68) 107 (80), 91 (57), 55 (41). Anal. calcd for Cr2H2r03: C, 77.60; H, 8.31. Found: C, 77.87; H, 8.27. 3-t -Butoxy - 1 -chloropropane (20) (Alexakis et al., 1988). 3-Chloro-1-propanol 19 (3.0 mL, 36 mmol)

was stirred with CH,Cl, (130 mL) and concentrated HZSO, (192 pL, 3.6 mmol). An excess of isobutylene was bubbled through the reaction mixture at room

463

temperature. After 7 h, product isolation (water, halfsaturated NaHCO,, brine, Na,S04) and Kugelrohr distillation provided a clear oil (3.67 g, 68%): b.p. 140°C (5mm); ‘H NMR (300MHz, CDCl,) 6 1.20 [s, 9 H, O(CH,),], 1.96 (quintet, 2 H, CH,CH,CH,), 3.49 [t, 2H, CZf,O(CH,),], 3.65 (t, 2H, CH,Cl); i.r. (neat) 2950) [C-H, C(CH,),], 1090 [CH,-0-C(CH,),] 793, 760cm-I; MS (EI) 135 (M+-CHj, 100) 87 (5), 77 (16), 57 (loo), 49 (7), 40 (100). Anal. calcd for C,H,,OCl: C, 55.80; H, 10.06. Found: C, 55.68; H, 10.10. 3’[3-Oxo- 7a-(3”-t-butoxypropyl)- 17/Landrosta-4ene - 17-yljwopionic acid lactone (22a) and 3’-[3-0x0 7B-(3”-t-butoxypropyl)- 17fl-androsta-4-ene- 17-yl]propionic acid lactone (22b). Magnesium turnings

(96 mg, 4.0 mmol) were heated to dryness under a stream of argon. After cooling to room temperature, THF (1 mL) and a crystal of iodine were added followed by 1,2-dibromoethane (10 p L). Alkoxy chloride 20 was added, with heating, over 15 min. The reaction mixture was allowed to reflux for 1 h after the end of addition, at which time an aliquot of Grignard reagent 21 was titrated according to the l,lO-phenanthroline method (Watson and Eastham, 1964): 1.16 M. Under a blanket of argon, LiCl (5 mg, 0.12 mmol) and CuCl (7.8 mg, 79 pmol) were added to a mixture of ether-THF (1: 1 v/v, 1.72 mL). Canrenone 18 (100 mg, 0.29 mmol) was added and the mixture was cooled to c. -30°C in a CO,-CHCI,-CCl, bath. A THF solution of Grignard reagent 21 (c. 0.68 N, 603 pL, 0.41 mmol) was added dropwise and the reaction mixture was allowed to stir at -30°C for 30 min, at which time 5 N HCl (588 JoL) was added. The mixture was stirred for 5 min as it was allowed to approach room temperature. Product isolation (2 N NHICl, 0.5 N NH,Cl, water, Na,SO,) and flash chromatography (15% benzene-ether) provided 22a and 22b in a ratio of 2.1: 1, respectively. 22a: 71.8mg, 54%, white foam; ‘H NMR (200 MHz, CD(&) 6 0.97 (s, 3 H, 18-CH,), 1.16 [s, 9H, C(CH,),], 1.23 (s, 3H, 19-CH,), 3.30 [t, 2H, J = 5.2 Hz, CH,OC(CH,),], 5.75 (s, 1 H, 4-H); i.r. (KBr) 2950 (C-H, C(CH,),], 1772 (c---O, lactone), 1670 (ketone), 1195, 117Ocm-I; MS (EI) 456 (M+, 1.5), 400 (41), 383 (11), 341 (100) 57 (90). Anal. (exact mass HREIMS) calcd for &H,O,, 456.3239. Found, 456.3249. 22b: 34.5 mg, 26%, clear oil; ‘H NMR (200 MHz, CDCl,) 6 0.97 (s, 3 H, 18-CH,), 1.16 [s, 9 H, C(CH,),], 1.25 (s, 3 H, 19-CH,), 3.30 (t, 2 H, J = 5.2 Hz, CH,OC(CH,),], 5.71 (s, 1 H, 4-H); i.r. (CHCI,) 1760 (c---O, lactone), 1675 (ketone), 1658, 1210, 780cm’; MS (EI) 456 (M+, 2), 400 (28), 383 (3), 341 (85), 57 (100). Anal. (exact mass HREIMS) calcd for CZ9H.,.,04, 456.3239. Found, 456.3259. 3’-[3-0x0- 7a-(3”-hydroxypropyl)-17/Chydroxyandrosta-4-ene-17-yl&ropionic

acid

lactone

(23).

Canrenone derivative 22a (346 mg, 0.46 mmol) was dissolved in dioxane (1.4 mL), followed by addition

464


of concentrated HCl (2mL). The dark brown solution was stirred at room temperature for 90min. Product isolation (water, EtOAc, saturated aqueous NaHCO,, brine, Na, SO,) and flash chromatography (EtOAc) provided a white foam (231 mg, 76%); ‘H NMR (300 MHz, CDCI,) 6 0.99 (s, 3 H, 18-CH,), 1.23 (s, 3 H, 19-CH,), 3.63 (t, 2 H, J = 6.1 Hz CH,OH), 5.74 (s, 1 H, 4-H); i.r. (KBr) 3440 (OH), 1770 (C==O, lactone), 1662 (C=O, ketone), 1450, 1192, 1171 cm-‘; MS (EI) 400 (M+, 33) 341 (loo), 145 (11) 105 (18) 74 (19) 55 (19). Anal. (exact mass, HREIMS) calcd for CzsH,,04, 400.2614. Found, 400.2634. 3’-[3-0x0- 7u-[3”-[(methanesuIfonyl)oxy]propyl]178 -hydroxy -androsta -4-ene - 17-yl]propionic acid lactone (24). Keto lactone 23 (46.1 mg, 0.115 mmol)

was dissolved in THF and the solution cooled to 0°C. Et,N (39 pL, 0.28 mmol) was added followed by a solution of mesyl chloride (2OpL, 0.25 mmol) in THF (150 pL). After stirring for 45 min, product isolation (water, EtOAc, Naz S04) and flash chromatography (80% EtOAc-hexane) gave a white foam (44.1 mg, 80%); ‘H NMR (200 MHz, CDCl,) 6 0.99 (s, 3 H, 18-CH,), 1.22 (s, 3 H, 19-CH,), 3.00 (s, 3 H, SOzCH,), 4.20 (t, 2 H, J = 5.2 Hz, CH,OSO>CH,), 5.72 (s, 1 H, 4-H); MS (EI) 478 (M+, 16) 341 (23) 96 (84) 79 (100) 48 (40) 34 (46). Anal. (exact mass, HREIMS) calcd for C26H3806S, 478.2382. Found, 478.2386. 3’-[3-0x0- 7u-[3”-[[[(2,2,2- trtJ¶uoroethyl)sulfonyl]oxy]propyl]]-178-hydroxy-androsta-I-ene-17-yl]propionic acid lactone (25). Ketol lactone 23 (150 mg,

0.37 mmol) was dissolved in CH2Cl, and the solution cooled to -15°C (CO,-CH,OH). Et,N (68 pL, 0.48 mmol) was added followed by tresyl chloride (52 pL, 0.47 mmol). After stirring for 15 min, the reaction mixture was diluted with cold CHIC& (1 mL), product isolation (ice-water, cold 10% HCl, saturated aqueous NaHCO,, brine, Na*SO,) and flash chromatography (70% EtOAc-hexane) gave a white foam (192 mg, 94%); ‘H NMR (300 MHz, CDCI,) 6 0.99 (s, 3 H, 18-CH,), 1.22 (s, 3 H, 19CH,), 3.90 (q, 2 H, J = 8.7Hz, SOzCH,CF,), 4.34 (m, 2H, CH,0S02CH,F,), 5.72 (s, 1 H, 4-H); i.r. (KBr) 2950, 1770 (C=O, lactone), 1658 (0, ketone), 1380 (S=O), 1270, 1180, 1135cm-‘; MS (FAB) 547 (M+ +H, 100) 531 (50) 309 (15) 155 (64) 135 (40) 119 (100). Anal. (exact mass, HRFABMS) calcd for C,,H,,SO,F, (M + H), 546.2341. Found, 546.2335. 3’ - [3 - 0x0 - 7a - (3” -Jluoropropyl) - I 78 - hydroxy androsta-4-ene- 17-yl] propionic acid lactone (3’-FRU 26752, 2). Tresylate 25 (29.1 mg, 53 pmol) was dissolved in THF (1.1 mL). n -Bu,NF (1 M in THF,

213 pL, 0.21 mmol) was added and the reaction mixture allowed to stir at 60°C. After 25 min, the mixture was passed through a 2 cm plug of neutral alumina, concentrated in vacua, and the residue subjected to flash chromatography (40% EtOAchexane) to yield a clear oil (10.4 mg, 49%) which

solidified upon standing. m.p. 184185°C; ’ H NMR (300 MHz, CDCl,) S 1.00 (s, 3 H, 18-CH3), 1.23 (s, 3 H, 19-CH,), 4.44 (dm, 2 H, Jur= 47 Hz, CH*F), 5.73 (s, 1 H, 4-H); 19F NMR (338 MHz CDCl,) r$ -219.20 (ttd, Jur= 48, 28, 4 Hz, CH,F); i.r. (KBr) 2940, 1770 (C==O, lactone), 1662cm-’ (c--O, ketone); MS (EI) 402 (M+, 85) 291 (32) 279 (33) 123 (59) 119 (34). Anal. (exact mass, HREIMS) calcd for CzsH3, FO,, 402.2507. Found, 402.2577. 6a-Methyl-II/?-hydroxy-androsta-1,4-diene-3,17dione (27). 6a -Methylprednisolone 26 (1.5 g,

4.00 mmol) was dissolved in 50% aqueous acetic acid (144 mL). Sodium bismuthate (6.9 g, 24.8 mmol) was added and the mixture was stirred for 5 h at room temperature. Product isolation (CHCI,, saturated aqueous NaHCO,, Na,SO,) followed by flash chromatography (60% EtOAc-hexane) gave a white solid (1.20 g, 96%): m.p. 247°C; ‘H NMR (300 MHz, D,SO-d6) 6 1.04 (s, 3 H, 18-CH,), 1.04 (d, 3 H, J = 5.9 Hz, 6u-CH,), 1.38 (s, 3 H, 19-CH,), 4.22 (m, 1 H, lla-H), 4.78 (d, J = 3.1 Hz, -OH), 5.79 (d, 1 H, J = 1 Hz, 4-H), 6.12 (dd, 1 H, J = 10, 1.2 Hz, 2-H), 7.27 (d, 1 H, J = 10 Hz, 1-H); i.r. (KBr) 3440 (OH), 1735, 1658 (C=O, 17-ketone), 1620, 1600, 1412 cm-‘; MS (EI) 314 (M+, 3) 136 (100) 121 (17) 91 (9). Anal. calcd for CZOH2503:C, 76.40; H, 8.33. Found: C, 76.27; H, 8.25. 6u-Methyl- 1 I/?-hydroxy-androsta- 1,4,6-triene3,17-dione (28). Dione 27 (2.70 g, 8.59 mmol) was

dissolved in 2-pentanol (145 mL). Chloranil (tetrachloro-1,4-benzoquinone) (12.71 g, 51.7 mmol) was added followed by approx. 4g of CaCO, and the mixture was heated at reflux for 5 h. The mixture was allowed to cool to room temperature, filtered, and concentrated in vacua. The reddish-orange residue was redissolved in c. 200 mL of CHCI,, and product isolation (H,O, 5% aqueous NaOH, H,O, brine, Na,SO,), followed by flash chromatography (50% EtOAc-hexane) gave a yellow solid (1.83 g, 68%): m.p. 198-200°C; ‘H NMR (300 MHz, CDCI,) 6 1.23 (s, 3 H, 18-CH,), 1.43 (s, 3 H, 19CH,), 1.92 (s, 3 H, 6-CH,), 4.51 (m, 1 H, 1lu-H), 5.97 (s, 1 H, 4-H), 6.15 (s, 1 H, 7-H), 6.32 (dd, 1 H, J = 9.8, 1.2 Hz, 2-H), 7.31 (d, 1 H, J = 9.8 Hz, 1-H); i.r. (KBr) 3410 (OH), 1730 (d), 1658 (c---O, 17-ketone), 1615 (C==O, 3-ketone), 1409 cm-‘; MS (EI) 312 (M+, 66) 294 (59) 279 (43) 185 (loo), 159 (41). Anal. calcd for C,,H,,O,: C, 76.89; H, 7.74. Found: C, 76.55; H, 7.87. I7u-[3’- Hydroxypropynyl)]- 1 It?, 17/?-dihydroxy- 6methyl-androsta-1,4,6-triene-3-one (29). 2-Propynyl tetrahydropyranyl ether (475 pL, 3.52 mmol) in pentane (18 mL) was cooled to 0°C. n-BuLi (2.27 mL, 3.52 mmol) was added and a white precipitate formed. Dione 28 (500 mg, 1.60 mmol) in THF (10 mL, freshly distilled) was added to the flask and the mixture was allowed to stir for 5 h. Product isolation (saturated aqueous NH,Cl, EtOAc, H,O, brine, Na,SO,), followed by flash chromatography (60% EtOAc-hexane) gave the 17a-propargyl tetrahydropyranyl ether as a yellow oil (230 mg, 5 1%).

Fluorine-substituted

’ H NMR (300 MHz, CDCI,) 6 1.20 (s, 3 H, 18-CH,), 1.41 (s, 3 H, 19-CH,), 1.91 (s, 3 H, 6-CH,), 3.47-3.79 (m, 2 H, -OHC2 of THP), 4.26 (d, 2 H, J = 4.6, C=CCH,O), 4.50 (bs, 1 H, llu-H), 4.73 (d, 1 H, J = 1.6 Hz, -OCHO), 5.87 (s, 1 H, 4-H), 6.13 (s, 1 H, 7-H), 6.31 (dd, 1 H, J = 10, 1.2 Hz, 2-H), 7.31 (d, 1 H, J = lOHz, 1-H); i.r. (KBr) 3430 (OH), 2920, 2875, 1650 (C==O, ketone), 1600, 1020cm-‘; MS (FAB) 453 (M+ + H, loo), 369 (35) 309 (33), 185 (68), 155 (100) 135 (87) 119 (100). Anal. (exact mass, HRFABMS) calcd for &H,,O, (M +H), 453.2641. Found, 453.2641. The tetrahydropyranyl ether (63.1 mg, 0.139 mmol) was dissolved in THF (1 mL, freshly distilled). Water (3 mL) was added followed by HOAc (3 mL). The reaction mixture was stirred at 50°C for 4 h. After cooling to room temperature and dilution with water (5 mL), product isolation (saturated aqueous NaHCO,, EtOAc, Na,SO,), followed by flash chromatography (EtOAc) gave a white solid (38.3 mg, 75%): m.p. 130-140°C; ‘H NMR (300MHz, CD,CN) 6 1.11 (s, 3 H, 18-CH,), 1.38 (s, 3 H, 19-CH,), 1.89 (d, 3H, J= 1.6Hz, 6-CHr), 4.10 (s, 2 H, C=CCH,O), 4.39 (d, 1 H, J = 2.1 Hz, lla-H), 5.96 (s, 1 H, 4-H), 6.01 (s, 1 H, 7-H), 6.20 (dd, 1 H, J = 9.9, 1.6 Hz, 2-H), 7.34 (d, 1 H, J = 9.9 Hz, 1-H); i.r. (KBr) 3400, 2980-2870, 1645 (0, ketone), 1600, 1035 cm-‘; MS (EI) 368 (M+, 8), 317 (17), 185 (47), 159 (100) 135 (43) 85 (36). Anal. (exact mass, HREIMS) calcd for CZ3H,,O,, 368.1988. Found, 368.1980. I7u-[3’-Methanesulfonyloxypropynyl)]-11/?,17/3dihydroxy-6-methyl-androsta1,4,6-triene-3-one (30). Trio1 29 (42.7 mg, 0.12 mmol) was dissolved in

THF (1.3 mL, freshly distilled), and the solution was cooled to 0°C. Et,N (3 1 pL, 0.22 mmol) was added, followed by a solution of mesyl chloride in THF (0.21 mmol, 10% v/v). After stirring at 0°C for 2 h, an additional 8 PL (57 pmol) of triethylamine was added followed by 4 /IL (52 pmol) of mesyl chloride. After 30 min, product isolation (ice-water, EtOAc, brine, Na,SO,) and flash chromatography (80% EtOAc-hexane) afforded a brownish oil (32.1 mg, 62%); ‘H NMR (300 MHz, CDCI,) d 1.22 (s, 3 H, 18-CH,), 1.41 (s, 3 H, 19-CH3), 1.91 (s, 3 H, 6-CHJ), 3.06 (s, 3 H, SO,CH,), 4.51 (bs, 1 H, 11x-H), 4.84 (s, 2 H, C=CCH20), 5.86 (s, 1 H, 4-H), 6.14 (s, 1 H, 7-H), 6.31 (dd, 1 H, J = 10.2, 1.3 Hz, 2-H), 7.31 (d, 1 H, J = 10.2 Hz, 1-H); MS (FAB) 447 (M+ + H, loo), 309 (90), 195 (40) 155 (100) 135 (loo), 119 (100). Anal. (exact mass, HRFABMS) calcd for C,HjOSO, (M + H), 447.1823. Found, 447.1832. 17u- [3’ - Fluoropropynyl)]- llfi,l7j - dihydroxy - 6methyl-androsta-1,4,6-triene-3-one

(3*-F-28362,

5).

Mesylate 30 (12.6mg, 28.2mmol) was dissolved in THF (0.540 mL, freshly distilled). n-Bu,NF (1 M in THF, 102 pL, 0.102 mmol) was added and the reaction mixture was heated at 60°C for 20 min. After cooling to room temperature, the reaction mixture was passed through a 3 cm plug of neutral alumina.

corticosteroids

465

Concentration in vacua was followed by flash chromatography (40% EtOAc-hexane) to produce a light yellow oil (10.4mg, 28.1 mmol, 100%); ‘H NMR (300MHz, CDCl,) 6 1.22 (s, 3 H, 18-CH,), 1.41 (s, 3 H, 19-CH,), 1.91 (s, 3 H, 6-CH,), 4.52 (d, 1 H, J = 2.7 Hz, lla-H), 4.96 (d, 2 H, JHIF= 47.5 Hz, C=CCH,F), 5.87 (s, 1 H, 4-H), 6.14 (s, 1 H, 7-H), 6.31 (dd, 1 H, J= 10.2, 1.8Hz, 2-H), 7.32 (d, 1 H, J = 10.2Hz, 1-H); “F NMR (338 MHz, CDCl,) 4-214.62 (t, JHF= 47.8 Hz, CH,F); MS (EI) 370 (M+, 7), 252 (lo), 237 (17), 203 (46) 185 (29) 159 (100) 135 (44), 91 (24), 41 (27). Anal. (exact mass, HREIMS) calcd for Cz3H2,03F, 370.1944. Found, 370.1951. 1 lb-Hydroxy -4-androsten -3,17-dione (31). Cortisol 9 (2.00 g, 5.52 mmol) was dissolved in 200 mL of 50% aqueous acetic acid. Sodium bismuthate (9.55 g, 34.1 mmol) was added, and the heterogeneous mixture stirred at room temperature for 4 h. At this time, sodium hydroxide (2 N, 150 mL) was added to bring the pH of the mixture to approx. 5. Product isolation (CHCI,, saturated sodium bicarbonate to neutrality, Nar SO,) gave 1.30 g (78%) of the product dione 31 as a white powder. Recrystallization from EtOAc afforded white needles, m.p. 195-196°C; ‘H NMR (300 MHz, CDCI,) 6 1.16 (s, 3 H, 18-CH,), 1.46 (s, 3 H, 19-CH,), 4.46 (t, 1 H, J = 2 Hz, 1la-H), 5.69 (s, 1 H, 4-H); i.r. (CHCl,) 3615 (OH), 1734 (C=O, C-17), 1663 (C==O, C-3); MS (EI) 302 (M+, 88), 163 (loo), 123 (67). Anal. calcd for CgH,,Oj, C, 75.46; H, 8.67. Found, C, 75.32; H, 8.73. Ilb-Hydroxy-1,4,6-androstatrien-3,17-dione

(32).

Dione 31 (253 mg, 0.84 mmol), chloranil (1.23 g, 5.01 mmol) and CaCO, (625 mg) were stirred together in 25 mL of 2-pentanol. The mixture was refluxed for 6 h. At this time, the mixture was filtered, rinsing with CHCl,, and the solvent removed under reduced pressure. The residue was redissolved in chloroform and washed with three portions each of water, 5% NaOH, water. Product isolation (Na,SO,) and flash chromatography (50% EtOAc-hexane) yielded 188mg (76%) of dione 32 as an off-white foam, m.p. 186188°C. ‘H NMR (360 MHz, CDCl,) 6 1.24 (s, 3 H, 18-CH,), 1.46 (s, 3 H, 19-CH,), 4.54 (t, 1 H, J = 3 Hz, 1la-H), 5.99 (s, 1 H, 4-H), 6.18 (dd, 1 H, J = 10,2 Hz, 6-H), 6.28-6.33 (m, 2 H, 2-H, 7-H), 7.28 (d, 1 H, J = 9 Hz, 1-H); MS (EI) 298 (M+, 72), 280 (63) 185 (62), 184 (78), 171 (100) 159 (61), 134 (61), 121 (78), 115 (76). Anal. (exact mass HREIMS) calcd for C,gH,,O,, 298.1569. Found, 298.1577. 1 I/?, 17fl-Dihydroxy- 17a-(3’-hydroxypropynyl)1,4,6-androstatrien-3-one (33). A solution of propar-

gyl tetrahydropyranoyl ether (203 p L, 1.5 1 mmol) in 8.5 mL of pentane was cooled to 0°C under N,. n-BuLi (973 pL, 1.51 mmol) was added, resulting in the formation of a white precipitate. THF (1.2 mL) was added dropwise to dissolve the precipitate. A solution of dione 32 (150 mg, 0.50 mmol) in 3 mL of THF was added to the anion solution. The reaction was stirred at 0°C for 8 h, then allowed to warm

466


slowly to room temperature and stirred overnight. Product isolation (saturated NHICI, EtOAc) and flash chromatography (50% EtOAc-hexane) gave 44 mg (20%) of the 17a-propargyl tetrahydropyranyl ether as a yellow oil. ‘H NMR (360 MHz, CDCl,) 6 1.21 (s, 3 H, 18-CH,), 1.45 (s, 3H, 19-CH,), 3.46-3.80 (m, 2H, -OCH, of THP), 4.26 (s, 2H, C=CCH,O), 4.53 (bs, 1 H, lla-H), 4.73 (t, 1 H, J = 3 Hz, -OCHO), 5.97 (s, 1 H, 4-H), 6.08 (dd, 1 H, J = 10, 1 Hz, 6-H), 6.25 (dd, 1 H, J= 10, 2.5 Hz, 7-H), 6.29 (dd, 1 H, J = 10, 2 Hz, 2-H), 7.30 (d, 1 H, J = 10 Hz, 1-H); MS (FAB) 439 (M+ + l), 279, 223, 205, 177, 157, 137, 121. Anal. (exact mass HRFABMS) calcd for (&HJ505 (M + l), 439.2484. Found, 439.2480. The tetrahydropyranyl ether (44 mg, 0.10 mmol) was dissolved in 335pL of tetrahydrofuran. H,O (1 mL) was added, followed by glacial acetic acid (1 mL). The reaction was stirred at 50°C for 2.5 h. The solution was then added to 20mL H,O, and saturated NaHCO, added to neutrality. Product isolation (EtOAc, Na,SO,) and flash chromatography (80% EtOAc-hexane) yielded 35 mg (99%) of the alcohol 33 as a pale yellow foam, m.p. 150-152°C. ‘H NMR (360 MHz, acetone-d,) 6 1.,22 (s, 3 H, 18-CH,), 1.47 (s, 3 H, 19-CH,), 4.15 (s, 2H, C=CCH,O), 4.50 (bs, 1 H, lla-H), 5.87 (s, 1 H, 4-H), 6.17 )bd, 2 H, J = 10 Hz, 2-H, 6-H), 6.30 (dd, 1 H, J = 10, 2.5 Hz, 7-H), 7.38 (d, 1 H, J = 10 Hz, 1-H); MS (FAB) 355 (M+ + l), 279, 223, 195, 171. Anal. (exact mass HRFABMS) calcd for CzzH2,04 (M + l), 355.1909. Found, 355.1909. I lb, 17j - Dihydroxy - 17~ - (3’ - methanesulfonyloxy propynyl)-1,4,6-androstatrien-3-one (34). Trio1 33 (20 mg, 0.056 mmol) and Et,N (13 FL, 0.093 mmol) were dissolved in 750 PL of THF and cooled to 0°C. Mesyl chloride (7 y L, 0.090 mmol) was added as a solution in 120 PL of THF over 5 min. The reaction was stirred at 0°C for 30 min, then warmed to room temperature and stirred for 3 h. At this time the reaction was again cooled to O”C, and Et,N (4 pL, 0.029 mmol) and a solution of mesyl chloride (2 p L, 0.026 mmol) in 40 FL of THF were added. Stirring was continued at 0°C for 15 min, then at room temperature for 1 h. The mixture was filtered and product isolation (H,O, EtOAc, Na,SO,) and flash chromatography (80% EtOAc-hexane) yielded 20 mg (81%) of the mesylate 34 as a yellow oil. ‘H NMR (360 MHz, acetone-d,) 6 1.25 (s, 3 H, 18-CH,), 1.47 (s, 3 H, 19-CH,), 3.14 (s, 3 H, -SO,CH,), 4.52 (bs, 1 H, 11&-H), 4.93 (s, 2H, C=CCH,O), 5.88 (s, 1 H, 4-H), 6.16 (bd, 2 H, J = 10 Hz, 2-H, 6-H), 6.31 (dd, 1 H, J = 10, 2.5Hz, 7-H), 7.37 (d, 1 H, J = 10 Hz, 1-H); MS (FAB) 433 (M+ + l), 279, 195, 171. lib,1 7b-Dihydroxy-I7a-(3’~fluoropropynyl)-1,4,6androstatrien-3-one (6). Mesylate 34 (8 mg,

0.018 mmol) was dissolved in 250 PL of THF. nBu,NF (1 M in THF, 9 1 pL, 0.091 mmol) was added, and the reaction heated at 60°C for 15 min. Flash

chromatography (70% EtOAc-hexane) gave 5 mg (78%) of the fluoride 6 as a colorless oil. ‘H NMR (360MHz, CDC&) S 1.23 (s, 3 H, 18-CH3), 1.45 (s, 3 H, 19-CH1), 4.54 (bs, 1 H, 1la-H), 4.96 (d, 2 H, JHF = 48 Hz, C=CCH,F), 5.97 (s, 1 H, 4-H), 6.08 (dd, 1 H, J= 10, 1.5Hz, 6-H), 6.26 (dd, 1 H, J= 10, 2.5 Hz, 7-H), 6.30 (dd, 1 H, J = 10, 1.5 Hz, 2-H), 7.30 (d, 1 H, J = lOHz, 1-H); MS (FAB) 357 (M+ + l), 339, 279, 202, 186, 171. Anal. (exact mass HRFABMS) calcd for C,,H,,F03 (M + l), 357.1866. Found, 357.1862. Deoxycorticosterone ZI-triJlate (36). Deoxycorticosterone 7 (50 mg, 0.15 mmol) was dissolved in 1 mL of CH2Clz, and the solution cooled to -78°C under N,. To the cold solution was added triflic anhydride (39 pL, 0.23 mmol), followed by 2,6-lutidine (27.5 pL, 0.24 mmol). The mixture was stirred at - 78°C for 1.5 h, at which time the reaction was diluted with 4 mL of cold (- 78°C) CH,Cl,, then quenched with 2 mL of -78’C methanol. Removal of the solvent under reduced pressure, followed by flash chromatography (50% EtOAc-hexane) yielded 51 mg (73%) of the triflate 36 as a pale orange foam, m.p. 48-51°C (decomp.); ‘H NMR (300 MHz, CDCl,) S 0.73 (s, 3 H, 18-CH,), 1.19 (s, 3 H, 19CH,), 4.90 (AB quartet, centered at 6 = 4.90, Av = 15 Hz, J = 16 Hz, 2H, 21-H), 5.77 (s, 1 H, 4-H); MS (FAB) 463 (M + l), 331, 279, 195. 21 -Fluoroprogesterone (10). Triflate 36 (20 mg, 0.043 mmol) was dissolved in 800 PL of THF, nBu,NF (1 M in THF, 44 ~1L, 0.044 mmol) was added, and the solution stirred at room temperature for 10 min. Removal of the solvent under reduced pressure, followed by flash chromatography (50% EtOAc-hexane) yielded 13 mg (90%) of the fluoride 10 as a white solid, m.p. 19&191”C [lit. (Spitznagle and Marino, 1977) 140-143”C]; ‘H NMR (300 MHz, CDCl,) 6 0.71 (s, 3 H, 18-CH,), 1.19 (s, 3 H, 19CH,), 4.77 (AB quartet, centered at 6 = 4.77, Av = 20 Hz, JHH = 16 Hz, with additional doublet splitting due to JHF = 48 Hz, 2 H, 21-H), 5.74 (s, 1 H, 4-H); 19F NMR (CDCl,) 4 -225.87 (dt, JHF= 45, 3 Hz); MS (EI) 332 (M+, 58), 290 (44), 124 (100). Anal. (exact mass HREIMS) calcd for Cz,HZ9F02, 332.2152. Found, 332.2146. Corticosterone 21-mesylate (37). Corticosterone 8 (30 mg, 0.087mmol) and Et,N (19 pL, 0.14mmol) were dissolved in 1 mL of THF and cooled to O’C. Mesyl chloride (11 pL, 0.14 mmol) in 8OyL of THF was added dropwise over 5 min. The reaction was stirred at 0°C for 25 min, then warmed to room temperature for 1 h, and filtered. Product isolation (H20, EtOAc, Na2S0,) and flash chromatography (50% EtOAc-hexane) yielded 34mg (92%) of the mesylate 37 as a white solid, m.p. 148°C; ‘H NMR (200 MHz, CDCl,) 6 0.95 (s, 3 H, 18-CH,), 1.44 (s. 3 H, 19-CH,), 3.24 (s, 3 H, -S02CHI), 4.43 (bs, 1 H, 11&-H), 4.78 (AB quartet, centered at 6 = 4.78, Av = 25 Hz, J = 18 Hz, 2H, 21-H), 5.68 (s, 1 H, 4-H); MS (FAB) 425 (M+ + l), 331, 279, 223, 195.

Fluorine-substituted corticosteroids 21 -Fluoro - 1 l/3 -hydroxyprogesterone (11). Mesylate 37 (17.5 mg, 0.041 mmol) was dissolved in 750 PL of o-dichlorobenzene. n-Bu,NF (1 M in THF, 41 pL, 0.041 mmol) was added, and the reaction stirred for 5 min at 100°C. Flash chromatography (40% EtOAc-hexane) yielded 5 mg (34%) of the fluoride 11 as a white solid, m.p. 196198°C; ‘H NMR (360 MHz, CDCI,) 6 4.42 (t, 1 H, J = 3 Hz, 1la-H), 4.77 (AB quartet, centered at 6 = 4.77, Av = 22 Hz, JHH= 16 Hz, with additional doublet splitting due to JHF= 48 Hz, 2 H, 21-H), 5.69 (s, 1 H, 4-H); 19F NMR (CDCI,) 4 -225.98 (tt, JHF= 48, 1.5 Hz); MS (EI) 348 (M+, 27), 330 (28) 163 (loo), 124 (go), 123 (69), 91 (61) 79 (56). Anal. (exact mass HREIMS) calcd for Cz,Hr9F03, 348.2101. Found, 348.2107. 21-Fluoro-ll/I,178-dihydroxyprogesterone

(12).

Mesylate 38 (100 mg, 0.23 mmol) was dissolved in 10 mL o-dichlorobenzene with heating at 96°C. nBu,NF (1 M in THF, 227 pL, 0.23 mmol) was added and the mixture stirred at 96°C for 15 min. The products were isolated by flash chromatography (40% EtOAc-hexane) followed by preparative layer chromatography (4% 2-propano1-63% methylene chloride-33% hexane, 4 developments). The fluoride 12 (23 mg, 28%) and the oxetanone 39 (15 mg, 20%) were isolated as off-white solids. Fluoride 12: m.p. 21 l-213°C; ‘H NMR (360 MHz, acetone-d,) 6 0.94 (s, 3 H, 18-CH,), 1.49 (s, 3 H, 19 CH,), 4.49 (t, 1 H, J = 3 Hz, lla-H), 5.25 (AB quartet, centered at 6 = 5.25, Av = 115 Hz, JHH= 17 Hz, with additional doublet splitting due to JHF= 48 Hz, 2 H, 21-H), 5.57 (s, 1 H, 4-H); 19F NMR (acetone-d,) 4 -232.38 (t, J,,r = 45 Hz); MS (EI) 364 (M+, loo), 346 (34) 331 (28), 242 (56), 227 (73). Anal. (exact mass HREIMS) calcd for Cz,H29F04, 364.2050. Found, 364.2055. Oxetanone 39: m.p. 226.5-228°C; ‘H NMR (300 MHz, acetone-d,) S 1.11 (s, 3 H, l8-CH,), 1.48 (s, 3 H, 19-CH,), 4.52 (quintet, 1 H, J = 3 Hz, 1la-H), 4.96 (AB quartet, centered at 6 = 4.96, Av = 34 Hz, J = 15 Hz, 2 H, 21-H), 5.58 (s, 1 H, 4-H); i.r. (KBr) 3410, 2965, 1805, 1652, 1620, 952cm-‘; MS (EI) 344 (M+, 35) 326 (12), 302 (100) 163 (73), 136 (67), 123 (70) 91 (57), 79 (53). Anal. (exact mass HREIMS) calcd for CZlH,,04, 344.1988. Found, 344.1992. Triamcinolone acetonide fonate (40). Triamcinolone

21 -tr@oromethanesul-

acetonide 13 (52mg, 0.12 mmol) was dissolved in 8 mL of CH,Cl,, and cooled to -78°C. Triflic anhydride (60 pL, 0.36mmol) was added, followed by 2,6-lutidine (42.5 pL, 0.36mmol), and the reaction mixture stirred at -78’C for 30 min. The reaction was quenched at - 78°C with 4 mL of methanol, then filtered through SiO,-Al,O,, rinsing with EtOAc. Flash chromatography (40% EtOAc-hexane, then 35% EtOAc-hexane) gave 51 mg (75%) of triflate 40 as a pale yellow solid, m.p. 192-194°C; ‘H NMR (360 MHz, acetone-d,) 6 0.99 (s, 3 H, 18-CH,), 1.22 (s, 3 H, /I-acetonide CH,), 1.42 (s, 3 H, a-acetonide

467

CH,), 1.61 (s, 3 H, 19CH,), 4.42 (bd, 1 H, J,,=9Hz, lla-H), 5.01 (d, lH, J=4Hz, 16/?-H), 5.54 (AB quartet, centered at 6 = 5.54, Av = 173 Hz, J = 18 Hz, 2 H, 21-H), 6.03 (s, 1 H, 4-H), 6.20 (dd, 1 H, J = 10, 2Hz, 2-H), 7.28 (d, 1 H, J = 10 Hz, 1-H); i.r. (CHCl,) 1740 (C=O, C-20), 1667 (C=O, C-3), 1425 (S=O), 1223 (s-_=o); MS (FAB) 567 (M + l), 529, 309, 195, 155, 135, 119. Anal. calcd for C,,H,F,O,S: C, 53.00; S, 5.66. Found: C, 53.00; H, 5.40; F, 13.27; S, 5.76. 21 -Fluoro-21 -&oxytriamcinolone

acetonide

(15).

Triflate 40 (21 mg, 0.037 mmol) was dissolved in 1 mL of THF, and the solution cooled to 0°C. n-Bu,NF (1 M in THF, 75 pL, 0.075 mmol) was added and the reaction stirred at 0°C for 15min. The mixture was filtered through SiO,, rinsing with EtOAc. Flash chromatography (50% EtOAc-hexane) gave 2 mg (12%) of the fluoride 15 as a white solid; ’ H NMR (360 MHz, CDCI,) 6 0.94 (s, 3 H, 18-CH,), 1.20 (s, 3 H, /I-acetonide CH,), 1.42 (s, 3 H, a-acetonide CHJ), 1.54 (s, 3 H, 19-CH,), 4.44 (bd, 1 H, JHF= 6 Hz, 1 la-H), 5.05 (d, 1 H, J = 5 Hz, 168-H) 5.13 (AB quartet, centered at 6 = 5.13, Av = 131 Hz, J,,” = 17 Hz, with additional doublet splitting due to J,,=48Hz, 2H, 21-H), 6.14(s, 1 H, 4-H), 6.35(dd, 1 H, J= 10, 2Hz, 2-H), 7.16 (d, 1 H, J = lOHz, 1-H); i.r. (CHCI,) 1734 (C=O, C-20), 1667 (C==O, C-3), 1055 (C-F); MS (FAB) 437 (M+ + l), 309, 275, 195, 155, 135, 119. Anal. (exact mass HRFABMS) calcd for C,H,,F,O, (M + l), 437.2140. Found, 437.2140. 21 -Fluoro -21 -deoxydexamethasone (16). Mesylate 41 (20 mg, 0.042 mmol) was stirred as a suspension in 3 mL of o-dichlorobenzene. n-Bu,NF (1 M in THF, 85 pL, 0.085 mmol) was added, at which point the mixture became homogeneous. The reaction was stirred at 75°C for 15 min. The products were isolated by flash chromatography (70% EtOAchexane) followed by preparative layer chromatography (4% 2-propanol-63% methylene chloride-33% hexane). The fluoride 16 (6.2 mg, 37%) and the oxetanone 43 (8.4mg, 53%) were obtained as white solids. Fluoride 16: m.p. 239-241°C; ‘H NMR (360 MHz, CD,CN) 6 0.85 (d, 3 H, J = 7 Hz, 16a-CH,), 0.98 (s, 3 H, 18-CH,), 1.52 (s, 3 H, 19-CH,), 4.25 (bd, 1 H, JHI.= 6 Hz, 1la-H), 5.20 (AB quartet, centered at 6 = 5.20, Av = 98 Hz, JHH= 17 Hz, with additional doublet splitting due to JHF= 48 Hz, 2 H, 21-H), 6.01 (bs, 1 H, 4-H), 6.22 (dd, 1 H, J = 10, 2 Hz, 2-H), 7.24 (d, 1 H, J = 10 Hz, 1-H); MS (FAB) 395 (M+ + l), 357, 279, 237, 195. Oxetanone 43: m.p. 242-243°C; ‘H NMR (360 MHz, CD,CN) 6 1.06 (d, 3 H, J = 7 Hz, 16aCHJ), 1.15 (s, 3 H, 18-CHr), 1.52 (s, 3 H, 19-CH3), 4.30 (bd, 1 H, JHF= 7 Hz, 1la-H), 4.92 (AB quartet, centered at 6 = 4.92, Av = 8 Hz, J = 15 Hz, 2 H, 21H), 6.01 (bs, 1 H, 4-H), 6.22 (dd, 1 H, J = 10, 2 Hz, 2-H), 7.24 (d, 1 H, J = 10 Hz, 1-H); MS (FAB) 375 (M+ + l), 337, 279, 223, 195.

468

MARTING. POMPER et al.

General procedure for synthesis of “F-labeled corticosteroids. Fluorine-18 was prepared from [tBO]H20 as previously described (Kilboum et al., 1985). The

aqueous activity was added to a 1 M n-Bu,NOH solution in a Vacutaine#. Water was removed by azeotropic distillation with CH,CN (0.2-l .5 mL) at 110°C under a stream of nitrogen. The dried activity was resolubilized in 200 pL of freshly distilled THF and transferred to a glass vial containing the steroid precursor. After the indicated reaction time, the reaction mixture was allowed to come to room temperature, then diluted with EtOAc (1.5 mL) and passed through a 50 mg plug of SiO,. The SiO, was rinsed with an additional 1.5 mL of EtOAc. Removal of the solvent under a stream of nitrogen left a residue which was redissolved 1,2-dichloroethane (normal phase) or CH,CN (reversed phase) prior to injection onto the HPLC. 3’-[3-0x0- 7u-(3’-[“Fd4uoropropyl)- 17jLhydroxyandrosta-4-ene-17-yl)propionic acid lactone (3’[‘*F]F-RU 26752, 2). The reaction employed 1.5 mg

(3.1 pmol) of mesylate 17 and 2.7 pL of n-Bu,NOH. Activity (200 mCi) was transferred in THF (200 p L). Reaction was at 70°C for 20min. Purification via HPLC [Whatman M-9, 75% hexane-25% (5% iPrOH-CH,Cl,), 5 mL/min] gave 13.5 mCi, 14% (1427%); tR = 35 min; sp.act. = 2456 Ci/mmol. 1 7a-[3’-[‘8F]-Fluoropropynyl]- 11 p, 17p-dihydroxy6-methyl-androsta-1,4,6-triene-jr-one (3’~[‘*F]F-RU 28362, 5). The reaction employed 1.45 mg

(3.25 pmol) of mesylate 24 and 3 pL of n-Bu,NOH in the resolubilization. Activity (192 mCi) was transferred to substrate in THF (200mL). Reaction was at 70°C for 20min. Purification via HPLC [Whatman M-9, 90% (5% i-PrOH-CH,CI,-10% hexane), 5 mL/min], gave 5.34 mCi, 4.6% (3.419%) t, = 17 mitt; sp.act. = 432 Ci/mmol 21-[‘*F]Fluoro-4-pregnene-3,20-dione ([‘*F]-10). The reaction employed 1.7 mg (4.2 pmol) of deoxycorticosterone-2 1-mesylate (35) and 3.5 p L of n Bu,NOH in the resolubilization. Activity (227 mCi) was transferred to the substrate in THF (200 p L). Reaction was at 70°C for 20 min. Purification via HPLC [Whatman M-9, 20% (5% i-PrOHCH,Cl,)-80% hexane, 5 mL/min] gave 28.5 mCi, 20% 4.5-20%) t, = 16 min; sp.act. = 1088 Ci/mmol. 21-[‘*F]Fluorodione ([‘*F]-12).

1 lfl-hydroxy-4-pregnene-3,20-

The reaction employed 1.4 mg (3.5 pmol) of mesylate 37 and 2.8 pL on n-Bu,NOH in the resolubilization. Activity (209 mCi) was transferred to the substrate in THF (2OOpL). Reaction was at 70°C for 20min. Purification via HPLC [Whatman M-9, 57% (5% i-PrOH-CH,C1,)43% hexane, 5 mL/min] gave 31.4mCi, 23% (1 l-23%), t, = 18 min; sp.act. = 1618 Ci/mmol. 21[‘8F]-9c(,21-dijuoro-ll~-hydroxy-16u,17a-[lmethylethylidene-bis(oxy)]pregnal,Cdiene-3,20dione ([‘*F]-25). The reaction employed 1.5 mg

(2.65 pmol) of triflate 40 and 3.0 pL of n-Bu,NOH in the resolubilization. Activity (162 mCi) was trans-

ferred to the substrate in THF (200 pL). Reaction was at 0°C for 20min. Purification was via HPLC. Reversed phase HPLC [Whatman M-9, ODS-2, 50% CH, CN-50% H, 0, 5 mL/min] gave 2.3 mCi (1.4%), tR = 15 min. The solvents were removed azeotropitally under reduced pressure. Normal phase HPLC [Whatman M-9, 55% (5% i-PrOH-CH,Cl,w5% hexane, 5 mL/min] of the residue gave 1.2 mCi (0.75%) tR = 17 min; sp.act. = 41 Ci/mmol. 21[‘8F]-Ar,21-difluoroll~,l7u-dihydroxy16amethylpregna-1,4-diene-3,20-dione ([‘*F]-16). A typical reaction employed 1.5 mg (2.9 pmol) of dexamethasone-21-triflate 42 and 2.8 pL of n-Bu,NOH in the resolubilization. Activity (3.8 mCi) was transferred to the substrate in 2OOpL of THF. Reaction was at 25°C for 20min. Purification via HPLC [Whatman M-9, 60% (5% i-PrOH-CH,Cl,+lO% hexane, 5 mL/min] gave 1.1 mCi (28%) t, = 26 min. Biological procedures

Radioligands were obtained from the following sources: [1,2,6,7-‘Hlaldosterone, 81 Ci/mmol (Amersham Corp., Arlington Heights, Ill.); and [6-methyl3H] 11/I,17fl-dihydroxy-6-methoxy- 17cr-(1-propynyl)androsta-1,4,6-trien-3-one (RU 28362), 77 Ci/mmol (DuPont-New England Nuclear, Boston, Mass.); unlabeled ligands: RU28362 (DuPont-New England Nuclear, Boston, Mass.); corticosterone and aldosterone (Sigma Chemical Co., St Louis, MO.). The following compounds were obtained from the sources indicated: dextran, grade C (Schwarz-Mann, Orangeburg, N.Y.); 2-mercaptoethanol, (ethylene-dinitrilo)tetraacetic acid, tetrasodium salt (EDTA) (Eastman Organic Chemicals, Rochester, N.Y.); Triton X-l 14 (Chem Central-Indianapolis, Indianapolis, Ind.); sodium azide, 1,4-bis(5-phenyloxazol-2-yl)benzene (Popop) (Aldrich Chemical Co., Milwaukee, Wis.); sodium molybdate (Mallinckrodt Inc., St Louis, MO.); glycerin and N,N-dimethyl formamide (DMF) (Fisher Scientific, Fair Lawn, N.J.); and 2,5-diphenyloxazole (PPO) (Research Products International Corp., Elk Grove Village, Ill.). Both intact and 3-day adrenalectomized (ADX), 300 g male rats were obtained from Sasco Inc., St Louis, MO. The adrenalectomized animals were maintained with physiological saline in place of regular drinking water. Preparations of cytosol. Cytosols for glucocorticoid receptor (Type II sites, liver) and mineralocorticoid receptor (Type I sites, kidney) were prepared from the tissues of 300 g male Long-Evans rats which had been adrenalectomized 3 days previously. The tissues were homogenized in GU buffer (0.01 M Tri-HC14.0015 M EDTA-O.02% sodium azide20 mM Na molybdate-0.012 M thioglycerol, 10% glycerol, pH 7.4 at 25”C), homogenized at 1 g tissue/ 1.5-1.7 mL buffer, centrifuged for 1 h at 140,OOOg and the supernatant stored in liquid nitrogen. For assays, the kidney cytosol was diluted 1: 2 with buffer (_ 12 mg/mL) and the liver cytosol was diluted 1: 10 with buffer (-6mg/mL) (Pinney et al., 1990).


Relative binding ajinity (RBA). Assays were a modification of that previously reported for the estrogen receptor (Katzenellenbogen et al., 1974). Cytosol was incubated with buffer or several concentrations of unlabeled competitor together with 10 nM ‘Htracer at 0°C for 18-24 h. The unlabeled competitor was prepared in 1:l dimethylformamide (DMFt buffer to ensure solubihty. Glucocorticoid receptor assays utilized liver cytosol (- 1 nM Type II sites) and [3H]RU 28362 as the tracer. Mineralocorticoid assays were performed with kidney cytosol (- 0.2 nM Type I sites plus 10m6M RU 28362 to block Type II sites) and [3H]aldosterone as the tracer. The charcoaldextran slurry used to remove unbound ligand was prepared as previously reported (Katzenellenbogen et al., 1973) and was generally used at 1 part to 10 parts of cytosol solution, at 0°C. In vivo uptake studies. Purified “F ligands were dissolved in 10% ethanol-saline or in 40% DMScsaline and 70 PCi were injected (iv. femoral vein) under ether anesthesia into 3-day adrenalectomized mature male rats. Tissue distribution was determined at various times post injection (see Tables 2-6). The animals were sacrificed, tissues were extracted, weighed and counted as previously described (Katzenellenbogen et al., 1982). The accumulation of “F into bone was calculated as %ID/g and also as %ID/organ using 7.68 gm of bone/100 g total body weight in a mature rat. In experiments to show blocking, 3 mg of unlabeled corticosterone was coinjetted with the “F ligand into ADX rats (Tables 2, 4, 5 and 6 “blocked”), except for “F RU 28362, 5, which was blocked with 700 pg of unlabeled RU 28362 (Table 3 “blocked”). Additionally, to show natural “blocking”, one set of animals was left intact (Table 3 “intact”). The receptors in these animals would be occupied by the endogenous hgands. Specific activities. Effective specific activity was determined from a decayed sample by a competitive binding assay as previously described (Senderoff et al., 1982; Kiesewetter et al., 1984).

Results Chemical synthesis of jluorocorticosteroids

The anticipated ease of introduction of a fluorine substituent at the C-3’-position of the 7a-propyl moiety made 2 a logical synthetic target as a fluorinesubstituted analog of the Type I selective hgand RU 26752 (1). The synthesis of fluoropropyl corticosteroid 2 is depicted in Scheme 1. Transformations up to and including the synthesis of lactone 23 represent modifications of published procedures (Nedelec et al., 1982). Acid caused cychzation of the potassium salt of hydroxy acid 17 to canrenone 18. 3-t-Butoxy-lpropyl-magnesium chloride 21 was synthesized from the corresponding alkoxy chloride 20, that was in turn prepared from 3-chloro-1-propanol (19) and isobutylene. Copper-catalyzed 1,6-conjugate addition of 21 to canrenone 18 provided a 2.1: 1 ratio of

corticosteroids

469

22a: 22b; the preponderance of 22a, resulting from bottom side attack at C-7 is consistent with both steric and stereoelectronic effects (Marshall and Roebke, 1966). The desired canrenone derivative 22a was deprotected under acidic conditions and the resulting lactone 23 was treated with either methanesulfonyl chloride (mesyl chloride, R = CH,) or 2,2,2-trichloride (tresyl chloride, fluoroethanesulfonyl R = CF, CH,) to give the corresponding methanesulfonate (mesylate) 24 or 2,2,2-trifluoroethanesulfonate (tresylate) 25. 3’-Fluoro-RU 26752 (2) was generated by rapid fluoride displacement of the tresylate precursor 25 in tetrahydrofuran at 60°C. The mesylate precursor 24 was used only for radiochemical synthesis (vide infra). Selective Type II receptor ligands include RU 26988 (4) and RU 28362 (3). The latter was developed in part to improve upon kinetic features of RU 26988, which has been shown to dissociate rapidly from receptor (Coirini et al., 1985). The synthesis of 3’fluoro-RU 28362 (5) depicted in Scheme 2 differs from that previously reported (Teutsch et al., 1981) in that the starting material is commercially available 6a-methylprednisolone 26. This approach involves synthesis of the 17a-(3-fluoro-1-propynyl) derivative from the mesylate precursor, a method successfully employed in the synthesis of 17a-fluoropropynyl nortestosterone (Brandes and Katzenellenbogen, 1987). Oxidative cleavage of the C-21 side chain of 26 (Heller et al., 1962) to give the dienedione 27 was followed by chloranil desaturation (Agnello and Laubach, 1960). Reaction of trienedione 28 with an excess of 3-hthio-2-propynyl tetrahydropyranyl ether afforded the 17a-substituted-17/?-alcohol which, after deprotection of the propynyl ether, gave trio1 29, the 3’-hydroxy analogue of RU 28362. Treatment with excess mesyl chloride and triethylamine in tetrahydrofuran provided the mono mesylate 30 selectively (Brandes and Katzenellenbogen, 1987; Simons et al., 1980; Simons and Thompson, 1981). Conversion of 30 to 3’-fluoro-RU 28362 (5) was achieved by treatment with tetrabutylammonium fluoride. Synthesis of 3’-fluoro-RU 26988 (6) from commercially available cortisol (9) (Scheme 2) was carried out in an analogous manner to that described for compound 5. Fluorinated analogs (10-12) of the naturally occurring corticosteroids deoxycorticosterone (7) corticosterone (8) and cortisol (9) were sought as potentially selective ligands for the Type I receptor. Literature precedent suggested the C-21 position as a synthetically accessible site for I*F labeling (Spitznagle et al., 1981; Feliu and Rottenberg, 1987). The synthesis of the fluorinated analogues 10-12 from the 21-hydroxy precursors is outlined in Scheme 3. Syntheses of mesylates 35 and 37 were adapted from the procedure reported for preparation of cortisol 21-mesylate 38 (Simons and Thompson, 1981). Thus, treatment of the 21-hydroxy steroids in tetrahydrofuran with triethylamine and mesyl chloride gave selectively the

470


Cd CIwOH

C”,

, cat. H2S04

‘CH,

-

ClMOr-Bu

22a

22b

3N HCI *

0 22a

23 0

0

n-Bu4NF (4996) F

24 R = CHS (80%) 25 R = CF$H, (94%)

2

Scheme

21-mesylates 35, 37 and 38 in high yield. Alternatively, treatment of 7 with triflic anhydride and 2,6-lutidine in methylene chloride at -78°C gave the 21-triflate 36. 21-Fluoroprogesterone (10) was prepared by reaction of 36 with excess tetrabutylammonium fluoride in tetrahydrofuran; the mesylate 35 was used only for radiochemical synthesis (vi& infu). Mesylates 37 and 38 were converted to the 21-fluoro compounds 11 and 12 by treatment with tetrabutylammonium fluoride in o-dichlorobenzene at

1

100°C. In the case of cortisol 21-mesylate 38, a competing reaction was intramolecular displacement of the mesylate by the 17cl-oxygen to form the C-17 Spiro oxetan-3’-one 39. This reaction has been noted previously upon treatment of mesylate 38 with potassium tert -butoxide or anhydrous potassium fluoride (Pons and Simons, 1981). Among synthetic corticosteroids, triamcinolone acetonide (TCA, 13) and dexamethasone (DEX, 14) show high affinity and selectivity for the Type II

Fluorine-substituted corticosteroids

o&

NaBiQ

c

oa

:

t 26 R=CH,,

471

27 R = CH,, A1 96%

A~

9 R=H

31 R=H

JP HO

chloranll, CaC03

0

0

-

0

78%

1) LiCIcCH@THP

00

t

2) THF, HzO, HOAc

R 28 R= CH, 32 R=H

29 R=CH3 33 R=H

68% 76%

38% 20%

30 R= CH3 62% 34 R=H 81%

n-Bu4NF

5 R=CH, 6 R=H

99% 78% Scheme 2

receptor. The 21-fluoro analogues 15 and 16 were thus prepared as outlined in Scheme 4. Reaction of triamcinolone acetonide with triflic anhydride and 2,6-lutidine provided the 21-triflate 40. Treatment of 40 with tetrabutylammonium fluoride in tetrahydrofuran at 0°C provided 21-deoxy-21-fluorotriamcinolone acetonide 15 in low yield, along with several highly polar by-products. Similarly, reaction of dexamethasone with either mesyl chloride in pyridine (Simons et al., 1980) or triflic anhydride and 2,6-lutidine gave the corresponding mesylate 41 or triflate 42. The mesylate was converted to 2 1-deoxy-2 1-fluorodexamethasone 16

upon treatment with tetrabutylammonium fluoride in o-dichlorobenzene at 75°C. Again, intramolecular displacement of the mesylate to form oxetanone 43 was a competing reaction. The triflate precursor was used only for radiochemical synthesis (uide infra). Corticosteroid

receptor binding affinity

Binding to Type I corticosteroid receptor utilized kidney cytosol preparations from adrenalectomized rats, a source rich in these receptors, in which Type II sites were blocked with micromolar concentrations of the Type II specific ligand RU 28362 in unlabeled form, using either [‘H]RU 26752 or [3H]aldosterone

MARTING. POMPER et al.

412

c@ OH

0

X

IIY

CH,SO&l

0

’

7 f

(CFT&&O Y

x

Y

X

iH

ii OH

:“6“H ii OH H

OH

:3

o&

n-Bu,$F

10 ::

OH

OH

R

CH3 2 CH:

90% 73% 92% 72%

+ o#

x

Y

H OH OH

H H OH

39

20%

90% 92% 80%

Scheme 3

14

X=OH.

Y=CH3

40

x, Y =

41 42

X=OH. X = OH,

R = CF, Y=R=CH3 Y = CH,, R = CF,

n-BuJW

15 16

x, Y =

-0

-0 >( X=OH. Y =CH3

___

12% 37%

Scheme 4

43

53%

75% 87% 15%

Fluorine-substituted corticosteroids as the radiotracer; binding data are given relative to aldosterone. Binding to Type II corticosteroid receptors was assayed using liver cytosol from adrenalectomized rats, with [3H]RU 28362 as tracer; binding data are expressed relative to dexamethasone. The RBA values in Table 1 are consistent with those found in the literature on related compounds (Ojasoo and Raynaud, 1978; Moguilewsky and Raynaud, 1980; De Kloet et al., 1984) and reflect the greater general affinity of the natural corticosteroids for the Type I rather than the Type II sites. Fluorine substitution for hydrogen, as in the Roussel compounds [RU 26752 vs 2, RU 28362 vs 5 and RU 26988 (Teutsch ef al., 1981; Gomez-Sanchez and Gomez-Sanchez, 1983) vs 6], lowers binding affinity toward the intended receptor type by a factor of 2-3, whereas fluorine for hydroxyl substitution can either maintain (deoxyCORT vs 10) or decrease (CORT, cortisol, TCA and DEX vs 11,12,15 and 16) binding affinity. Although the binding affinities of the fluorine-substituted corticosteroids were high, the absolute affinity (&) of corticosteroids for their receptors is lower than that of ligands for other steroid hormone systems (see Discussion). Radiochemical synthesis of fluorocorticosteroids

Radiochemical synthesis of [‘“F]-2 and [‘“F]-5 each proceeded by [‘8F]fluoride ion displacement of a mesylate precursor. [“F]Fluoride ion was generated by proton bombardment of an oxygen-18 enriched water target, as previously described (Kilbourn et al., 1985). For the synthesis of [‘“F]-2 and [‘*F]-5, resolubilized [‘*F]fluoride ion (n-Bu,N” F) in tetrahydrofuran was transferred to a glass vial containing the appropriate precursor, and the reaction mixture Table

I. Corticosteroid

was heated at 70°C for 20 min. Radiolabeled material was isolated after purification by normal phase HPLC. In the case of [‘“F]-2, radiochemical yields varied between 14 and 27% (decay corrected), while an effective specific activity (Senderoff et al., 1982) of 2456 Ci/mmol was attained; for [‘*F&5, the yield was only 5% (decay corrected), with an effective specific activity of 432 Ci/mmol. A similar low yield was reported earlier in a related system (Pomper et al., 1987). Alterations in reaction conditions and resolubilizing techniques [Kryptofix@/potassium carbonate (Coenen et al., 198611did not raise the yield. Radiochemical purities for [‘*F]-2 and [‘*F]-5 were each above 95%. The [18F]fluorocorticosteroids [‘8F]-10 and [“F]-11 were synthesized from the mesylate precursors in the same manner as described above. For compound [‘8F]-lO, radiochemical yields ranged from 5 to 22% with an effective specific activity of 1088 Ci/mmol, while for [‘*F]-11 the yield ranged from 12 to 23% with an effective specific activity of 1618 Ci/mmol. Radiochemical synthesis of [“F]-15 proceeded from the triflate precursor. The reaction was carried out in tetrahydrofuran at 0°C. Radiochemical yield was only 0.6%, with an effective specific activity of 41 Ci/mmol. The low yield reflects problems in the purification of the radiolabeled product. Two successive HPLC purifications (reversed phase followed by normal phase) were required to separate the product from coeluting mass. Approximately half of the incorporated activity was lost upon concentration of the eluent from the first HPLC purification, suggesting decomposition of the product. Radiochemical synthesis of [‘*F]-16 from the mesylate precursor resulted in only 0.5% incorporation of receptor

binding Relative

Comoound

413

affinities binding

affinity (RBA)

Type 1 (rat kidney cytosol) (+ 10m6 M RU 28362)

Type 11 (rat liver cytosol)

IO0 (0.5 nM)* 9.3

0.61 100 (3.8 nM)*

RefiWtW

Aldosterone Dexamethasone 14 NlltWal Deoxycorticosterone 7 Corticosterone 8 Cortisol 9 Synrhetic RU 26752 1 RU 28362 3 RU 26988 4 Triamcinolone acetonide 13 Fluorinated 3’-Fluoro-RU 26752 2 3’-Fluoro-RU 28362 5 3’-Fluoro-RU 26988 6 21-Fluoroprogesterone 10 21-Fluoro-I lb-hydroxyprogesterone 11 21-Fluoro-I lj?,17a-dihydroxyprogesterone 12 2l-Fluoro-2l-deoxytriamcinolone acetonide 15 21-Fluoro-21-deoxvdexamethasone 16 *Equilibrium dissocation constant for the radiotracer too low to measure. tLiterature value (Teutsch et al., 1981). ILiterature value, rat kidney cytosol (Gomez-Sanchez

-

II3 75 42 146 0.007 o.st 7.6

15 8.4 1.5 192 290$ 1029

65 -

0.2 59 13 0.77 4.0 15 174

0.045 188 41 28 I.2 3.8 is given in parentheses;

and Gomez-Sanchez,

-,

indicates

1983).

a value

474


the radiolabel. This is presumably due to competing intermolecular displacement to form oxetanone 43. Radiochemical synthesis was thus carried out using the triflate precursor in tetrahydrofuran at room temperature. Radiochemical yield was 34% (decay corrected) with an effective specific activity of 27 Ci/mmol. Biodistribution of radiolabeled corticosteroids [18F]-2, 5, 10,11 and 15

The purified radiotracers were reconstituted in the indicated vehicles, and 70 PCi portions were injected (i.v., femoral vein) into mature male Sprague-Dawley rats. Except where noted, rats were adrenalectomized 3 days prior to injection to reduce the levels of endogenous corticosteroid which would compete with injected radiolabeled material, and might cause anomalously low uptake values. To ascertain whether uptake was mediated by binding to receptor, one set of animals was given a saturating dose of unlabeled ligand (corticosterone or RU 28362) together with the radiotracer (values indicated as “blocked”). 3’-[“F]Fluoro-RU 26752. Purified 3’-[‘*FlfluoroRU 26752 ([‘*F]-2) was injected using a 40% DMS&saline vehicle. This vehicle was chosen because the relatively large amount of ethanol which would have been required to dissolve the radiotracer (> 50% aqueous ethanol) has caused central nervous system depressant effects in other in uiuo studies (Feliu and Rottenberg, 1987). Tissue distribution of radioactivity was determined at 2 and 30 min (Table 2). Blocking employed 3 mg of corticosterone per animal administered together with radiotracer [‘*F]-2. The data in Table 2 show relatively little uptake into the brain, with somewhat higher pituitary uptake, but a comparison of blocked and unblocked values at 30min indicates that this uptake is not receptor-mediated. The considerable uptake into

Table 3. Biodistribution Tissue

of 3’-[‘sF]fluoro-RU

30 min (ADX)§

28362 ([“F]-5)

Table

2. Biodistribution of 3’-[‘*F]fluoro-RU 26752 ([‘*F]-2) in adrenalectomized, mature male rats (n = 4).

% ID/g? Blood Liver Kidney Muscle Fat Cortex Hippocampus Pituitary Rest of brain Total brain

0.384 3.72 I.25 0.124 0.253 0.342 0.373 0.844 0.349 0.351 0.142

% ID /organ t Blood Liver Kidney Ml&c Fat Rest of brain Total brain Bone

f 0.014 f 0.066 f 0.036 + 0.005 f 0.056 f 0.005 li: 0.005 i 0.023 f 0.008 f 0.008 f 0.19

0.265 0.715 0.503 0.121 0.513 0.075 0.084 0. I80 0.074 0.075 2.49

f f * + + k k k + f f

0.013 0.100 0.059 0.014 0.061 0.028 0.029 0.030 0.023 0.023 0.17

3.25 k 0.28 4.52 f 0.75 0.434 f 0.039 3.01 + 0.24 7.91 k 1.61 0.108 f 0.014 0.117+0.017 66.17: 5.53

3.875 5.90 0.448 3.55 14.77 0.123 0.138 56.47

k k f f f f it f

0.221 1.26 0.076 0.44 1.76 0.037 0.040 3.22

0.219 0.504 0.473 0.102 0.271 0.060 0.068 0.144 0.063 0.064 2.87

i: 0.054 5 0.36 + 0.14 + 0.012 f0.171 f 0.042 * 0.051 + 0.058 + 0.026 + 0.028 f 0.01 I

5.48 + 0.62 27.69 f 2.83 l.l96?0.148 3.54 f 0.26 7.01 f 4.47 0.599 * 0.043 0.651 + 0.045 3.17 7 0.35

‘All values are mean k SD. tlD is injected dose. iBlocked: coinjection with 3 mg corticosterone.

fat attests to the lipophilicity of 2, and the very high values for bone reflect significant in uivo defluorination. 3’-[‘8F]Fluoro-RU 28362. Purified 3’-[‘*F]fluoroRU 28362 [“F]-5 was reconstituted in 10% ethanol-saline and injected into adrenalectomized and intact mature male rats. Tissue distribution was determined at 0.5 and 3 h (Table 3). Blocking employed 7OOpg of unlabeled RU 28362 (3) given concurrently with the radiolabeled material. The data in Table 3 at 30 min indicate little uptake in brain or hippocampus (tissue levels below blood levels), and no evidence of receptor-mediated distribution (compare blocked and unblocked values). Since nearly

in adrenalectomized

30 min (ADX) blocked1

30 min (intact)

(ADX) and intact, mature male rates (n = 4)’ 3h blockedt

(2:X)

% ID/gt Blood Liver Kidney Cortex Hippocampus Pituitary Rest of brain Total brain Bone

0.380 2.482 1.238 0.173 0.152 1.070 0.165 0.168 0.802

+ 0.028 i 0.401 + 0.107 f 0.039 f 0.028 f0.188 k 0.031 + 0.031 + 0.042

0.380 + 0.020 2.140~0.195 1.126f0.150 0.187~0.041 0. I64 * 0.034 0.802 + 0.166 0.173 k 0.034 0.176 f 0.034 0.677 f 0.074

0.406 i 0.019 1.877 f 0.197 1.064 f 0.025 0.131 +0.012 0.177~0.011 0.822 + 0.125 0.122 * 0.011 0.125 k 0.010 0.956 k 0.144

0.455 k 0.109 f 0.1 IO f 3.488 k

0.283 0.056 0.057 0.801

0.1 I6 i 0.030 0.544 i 0.163 0.336 f 0. I I I 0.058 + 0.01211 0.054 f 0.018’1 0.277?0.119 0.054 f 0.019 0.055 f 0.018 4.064 + 0.400

% ID /organ t Blood Kidney Rest of brain Total brain Bone

5.286 1.050 0.295 0.322 17.41

+ 0.275 i: 0.090 & 0.057 f 0.063 f 1.49

5.450 f 0.378 1.027f0.197 0.302 f 0.071 0.329 ? 0.076 15.15 f 2.16

5.172 0.841 0.186 0.203 18.90

2.401 0.427 0.202 0.205 79.94

0.433 0.132 0.102 0.104 22.84

1.615 0.282 0.092 0.098 88.35

‘All values are mean f SD. tID is injected dose. tBlocked: coinjected with 7OOpg unlabeled gn =5. ‘In = 3. /In = I.

RIJ 28362,

f f f f f

30 min blockedi

30 min

2 min

Tissue

0.408 0.080 0.029 0.028 2.63

0.168 f 0.044 0.792 + 0.256 0.508 + 0. I92

f f f k +

+ k f f +

0.414 0.095 0.036 0.035 II.25

3h (intact) 0.069 f 0.021 0.249 f 0.081 0.147 f 0.059 0.037lI 0.03211 0.049 * 0.052 0.023 & 0.008 0.023 i 0.007 5.336 f 0.418 0.868 + 0.246 0.112i_0.045 0.036 f 0.010 0.037 f 0.012 103.94 * 7.73

Fluorine-substituted corticosteroids 90% of the activity was located in bone by 3 h, the significance of the uptake data into other organs at this later time is questionable. Again, pituitary activity levels are higher than brain. Both liver and pituitary show partial blocking in the intact animals but less or none in those administered exogenous unlabeled steroid. High activity levels in bone indicate complete metabolic defluorination. 21 -[“F]Fhoroprogesterone. Biodistribution of 21[‘*Flfluoroprogesterone ([“F]-10) employed a 10% ethanol-saline vehicle. Blocking utilized 3 mg of corticosterone per animal administered interperitoneally, 30 min prior to injection of the radiotracer. Tissue distribution data are given in Table 4. The data show brain activity equal to blood, with no selective uptake by the pituitary or hippocampus. Blood and tissue activity levels drop from 15 min to 2 h. Comparison of the 2 h blocked and unblocked values indicate that the activity which is present is not necessarily receptor-bound. Activity levels in bone indicate considerable metabolic defluorination. 21 -[“F]Fluoro - 1 l/I -hydroxyprogesterone. Purified 21-[‘8F]fluoro-l l/I-hydroxyprogesterone ([“F]-11) was injected using a 10% ethanol-saline vehicle, and blocking utilized 3 mg of corticosterone. Tissue distribution was determined at 2min, 30 min and 2 h (Table 5). At 2min, target tissue uptake (hippocampus, cortex, pituitary) exceeds that of the blood. However, 30 min and 2 h values indicate that most of the activity has been “washed out”; that which remains appears not to be due to receptor-mediated uptake. Metabolic defluorination is comparable to that seen with compound [‘8F]-10.

Table 5. Biodistribution

Tissue % ID/g’, Blood Liver Kidney MWclc Fat Cortex Hippocampus Pituitary Rest of brain Total brain Thymus Bone

415

Table 4. Biodistribution of 21-[‘*F]Ruoroprogestcrone ([‘*F]-10) in adrenalectomized, mature male rats In = 4)’ Tissue

2h

15min 0.226 I .56 0.543 0.143 0.623 0.208 0.380 0.261 0.318 0.148 0.380

f + + f f + + f f + ?

0.033 0.27 0.114 0.034 0.572 0.082 0.160 0.082 0.108 0.044 0.067

0.030 0.103 0.056 0.023 0.127 0.023 0.024 0.026 0.026 0.021 I .27

* + f f f f f + k + ;

0.007 0.025 0.016 0.009 0.058 0.005 0.006 0.006 0.008 0.005 0.30

0.023 f 0.006 0.108 k 0.009 0.039 * 0.007 0.016 f 0.010 0.135~0.119 0.019 + 0.006 0.017 i 0.006 0.032 & 0.014 0.021 f 0.008 0.018 + 0.003 1.18 + 0.14

% ID/organ $ Blood Liver Kidney Muscle Fat Rest of brain Thymus Bone

3.50 II.27 0.443 4.37 18.61 0.558 3.52 9.34

f f f + + k i +

0.27 0.57 0.069 0.71 16.42 0.177 0.80 2.67

0.431 0.665 0.042 0.656 3.60 0.048 0.478 28.49

k f i f f f f f

0.092 0.165 0.011 0.238 1.61 0.015 0.100 5.90

0.380 0.802 0.036 0.505 4. I8 0.041 0.464 30.54

21 -[“F]Fluoro -21 -deoxytriamcinolone

30 min blocked7

2 min

30 min

0.559 f 0.056 2.495 f 0.573 4.136+_0.542 0.186 + 0.095 0.430 f 0.162 0.893 f 0.178 I.101 * 0.210 1.27 f 0.23 0.920 f 0.155 0.925 f 0.156

0.310 f 0.029 2.584 + 0.243 1.06~0.10 0.173 + 0.007 0.324 + 0.039 0.127~0.011 0.132 f 0.018 0.276 k 0.010 0.133 *0.012 0.133 +0.012 0.024 f 0.004 0.905 + 0.137

0.277 2.44 0.879 0.165 0.281 0.146 0.156 0.239 0.163 0.162 0.018 0.652

4.344 f 0.413 19.86 + 1.64 0.951 f 0.068 4.85kO.15 8.96 f 0.97 0.231 f 0.010 0.245 f 0.010 0.650 f 0.174 19.80 + 3.10

4.20 f 21.25 f 0.800 k 4.99 + 8.40* 0.277 + 0.296 + 0.436 f 15.40 +

8.09 f 0.78 19.22 f 4.67 3.625 k 0.561 5.400 + 3.760 12.26 + 4.54 I .52 f 0.30 1.642kO.311 4.346 + 0.677

*All values are mean + SD. TBlocked: coinjected with 3 mg corticosterone. In = 2. @I = 3. 1lID is injected dose.

0.062 0.089 0.006 0.283 3.42 0.018 0.075 4.97

acetonide.

Biodistribution of 21-[‘8F]fluoro-21-deoxytriamcinolone acetonide ([‘“F]-15) employed a 40% DMS&saline vehicle. Corticosterone (3 mg per animal) was used as the blocking agent. The data in Table 6 indicate little uptake or retention of activity in brain target tissues (hippocampus, cortex). The pituitary showed significant uptake, which appeared at 1 h to be at least partially receptor-mediated. The low brain uptake, being less than blood at 15 min,

f 0.046 k 0.24 f 0.166 f 0.025 ?r.0.048 f 0.025 f 0.029 k 0.026 + 0.030 f 0.030 f 0.005 f 0.120

2h blocked

2 h: 0.046 0.341 0.096 0.022 0.016 0.013 0.013 0.025 0.012

f * k * f f f + i -

(ADX) and

0.003 0.174 0.007 0.001 0.001 0.001 0.001 0.002 0.001

0.030 0.334 0.072 0.014 0.015 0.01 I 0.009 0.017 0.010

? f f * * * i f i -

0.007 0.070 0.022 0.004 0.005 0.003 0.002 0.006 0.002

0.835 k 0.340

0.843 f 0. I28

0.785 f 3.52* 0.102 f 0.731 * 0.537 + 0.023 f

0.468 + 0.083 2.86 + 0.32 0.055 f 0.009 0.460 f 0.106 0.479*0.114 0.016 i 0.002

% ID /organ 11 Blood Liver Kidney Muscle Fat Rest of brain Total brain Thymus Bone

+ f f k + ? + f

*All values are mean f SD. tn =3. SID is injected dose. §Blocked: preinjected with 3 mg corticosterone

of 21-[‘8F]fluoro-I Ifi-hydroxyprogesterone ([‘*F]-11) in adrenalectomized intact. mature male rats (n = 4).

0.193 * 0.033

2 h blockedt§

% IDlgf Blood Liver Kidney Muscle Fat Cortex Hippocampus Pituitary Rest of brain Thymus Bone

0.53 2.89 0.183 0.55 1.11 0.045 0.052 0.103 2.37

0.124 1.17 0.018 0.043 0.013 0.000

21.75 + 7.02

20.86 + I .75

476

MARTIN G. POMPER et al.

Table 6. Biodistribution of 21-[‘8F]fluoro-21-deoxytriamcinolone acetonide C118F1-15) in adrenalectomized mature male rats Cn = 4)’ 15min

Tissue

Ih

Ih

blockedt

% ID/g1 Blood Cortex Hippocampus Pituitary Rest of brain Bone

0.289 f 0.121 + 0.112 + 0.953 + 0.1 IO + 0.408 :

0.053 0.024 0.034 0.089 0.022 0.018

% ID /organ t Blood Rest of brain Bone

3.973 * 0.415 0.187 t 0.037 8.670 + 1.594

0.019 0.066 0.010 0.080 0.009 0.226

0.233 f 0.034 0.049 + 0.002 0.057 + 0.020 0.332 k 0.081 0.045 + 0.006 1.373Io.171

2.975 f 0.227 0.104 & 0.017 31.926 + 4.827

3.337 + 0.709 0.078 f 0.010 30.496 + 5.605

0.195 0.504 0.066 0.601 0.058 1.430

f i: * F + i

*All values are mean f SD. TBlocked: coinjection with 3 mg corticosterone. IID is injected dose.

suggests that the passage of this compound across the blood-brain barrier is limited. Activity levels in bone indicate significant metabolic defluorination. Bone activity as an index of metabolic defuorination

The time course of radioactivity uptake in bone as a percent of the injected dose per organ is shown in Fig. 2 for the five “F-labeled compounds. Because bone is known to have a high avidity for fluoride ion (Fiserova-Bergerova, 1973; Wallace-Durbin, 1954), this uptake is assumed to be due to free fluoride ion generated by metabolic scission of the carbonfluorine bond (see Discussion). Compound [‘*F]-2, which bears fluorine at a primary but unactivated position, gives the most rapid bone uptake. The remaining four compounds show similar initial rates of bone activity increase, and of those studied beyond 1 h, the uptake of [‘8F]-10 and [‘8F]-ll level off below 30% ID/organ. Compared to the other three compounds, these two are cleared from the blood quite quickly (cf. blood levels at different times, Tables 2-6), so the cessation of their bone uptake may simply reflect the completion of this

looI

-0

1

HOURS

2

3

AFTER

INJECTION

4

Fig. 2. Time-course of the uptake of radioactivity in bone following iniection of the ‘*F-labeled corticosteroids in mature adrenalectomized male rats. Data are plotted from the percent injected dose for bone taken from Tables 26 Uptake into bone indicates metabolic defluorination of the ligands.

clearance. [‘*F]-5, which bears fluorine at a primary but activated (propargylic) position, shows progressive, and extensive bone uptake. Clearance of this synthetic corticosteroid is relatively slow (cf. Table 3). Although [18F]-15 was not studied beyond 1 h, its bone activity at this time is considerable. As clearance of this synthetic corticosteroid is slow (cf. Table 6), continued metabolic defluorination at later times would be expected.

Discussion There is great appeal in the possibility of utilizing fluorine-18 substituted corticosteroids as PET imaging agents for corticosteroid receptors in the hippocampus, in order to have a biochemical marker for cognitive neurodegenerative disorders including Alzheimer’s disease (Sapolsky and McEwen, 1988). The challenges involved in developing such an imaging agent are well recognized: required is a ‘8F-labeled corticosteroid with (a) high affinity for the desired receptor (so that target site uptake will be selective and be retained compared to non-target regions), (b) low heterologous and non-receptor binding (so that non-target uptake will be modest) and (c) good metabolic stability with clean clearance (so that levels of recirculating metabolites are low) (Katzenellenbogen et al., 1982). The synthesis and biodistribution of several 18Flabeled corticosteroids has been previously described: 21-[‘8F]fluoropregnenolone proved metabolically labile. undergoing defluorination (Spitznagle et al., 1981), while 21-[‘8F]fluoroprednisone showed little target tissue (brain) uptake because of significant metabolic reduction of the C-20 carbonyl group with subsequent biliary excretion (Feliu and Rottenberg, 1987). The authors of the second study concluded that fluorocorticosteroids which could obviate C-20 reduction would be most promising as imaging agents; however, the use of intact, rather than adrenalectomized, rats might have obscured their data since receptor uptake of tracer levels of an exogenously administered radiocorticosteroid could conceivably be blocked by endogenous adrenocortical steroids (Rhees et al., 1975). Among the most selective and highest affinity Type I and Type II corticosteroid receptor ligands are three products of the Roussel Co. (Coirini et al., 1985), RU 26752 (l), RU 28362 (3) and RU 26988 (4) (Fig. 1); the first compound is selective for Type I receptors, while the latter two bind predominantly to Type II sites. Those compounds possess no C-20 carbonyl and thus would not undergo reduction. We prepared fluorine-substituted derivatives (2, 5 and 6) of these compounds by fluoride ion displacement of suitable leaving groups. We also prepared the fluorine-substituted analogues (l&12, 15 and 16) of several other naturally occurring (deoxycorticosterone, corticosterone and cortisol) and synthetic (dexamethasone and triamcinolone acetonide) corticosteroids because


of their potential receptor selectivity (Type I for l&12, Type II for 15 and 16) and high affinity, as well as their synthetic accessibility. The binding affinity of these fluorosteroids for Type I and Type II corticosteroid receptors was determined in standard competitive radiometric binding assays. The substitution of fluorine for a hydroxy group causes only a modest change in the relative binding affinities vs the parent ligand (increases: 10 vs 7; 16 vs 14; decreases: 11 vs 8, 12 vs 9, 15 vs 13). The substitution of fluorine for hydrogen causes a modest decrease in binding affinity of the Type I selective ligand (2 vs l), but a greater decrease in the Type II selective ligands (5 and 6 vs 3 and 4). Thus, these changes in affinity range from an increase to 166% of the parent ligand to a decrease to 4% of the parent ligand. We have seen similar effects of fluorine-substitution on sex steroidal hormones (Kiesewetter et al., 1984) but in certain cases, fluorine substitution can cause a much more pronounced increase in binding affinity (Pomper et al., 1988). While some of these compounds have high relative binding affinities, a significant fact is that the corticosteroids as a class have lower receptor binding affinity than do the estrogens, progestins and androgens. Aldosterone is considered to bind to Type I and Type II rat receptors with Kd values of 2.8-3.5 and 33-47 nM, respectively (Gomez-Sanchez and Gomez-Sanchez, 1983; Sheppard and Funder, 1987). However, in vitro, when the Type II sites are blocked with an excess of either RU 26988 or RU 28362 (both pure Type II binders), aldosterone binds to Type I receptors with an enhanced affinity, Kd of 040.5 nM (cf. Table 1) (Gomez-Sanchez and Gomez-Sanchez, 1983; Sheppard and Funder, 1987). While such in vitro competition assays may give a true estimate of aldosterone’s relative affinity for Type I receptors, they do not accurately reflect the receptor milieu in the living animal-in vivo, aldosterone would bind to the two receptor types with affinities of -3 and - 40 nM, respectively. Dexamethasone has a Kd of 4-8 nM for the Type II receptor (Manz et al., 1982). Comparatively, estrogens, progestins and androgens have much higher in vitro as well as in vivo affinities for their receptors (E2, 0.1 nM for rat uterine estrogen receptors; R5020, 0.41 nM for rat uterine Pg receptor; R1881, 0.6 nM for rat prostate androgen receptor) (Jensen and Jacobson, 1962; McGuire et al., 1977; Bonne and Raynaud, 1975). Thus, a corticosteroid having the same relative binding affinity as an estrogen will have 30-80 times lower absolute affinity, in vivo. On the basis of compound class and binding affinity, we selected five compounds for preparation in “F-labeled form and in vivo tissue distribution studies in adrenalectomized rats. Although a somewhat different set of time points was taken and a somewhat different set of tissues was examined in each case, the tissue distribution was followed as a function of time, and at one or two times the

corticosteroids

411

effectiveness of a blocking dose of exogenous or endogenous (intact animals) unlabeled corticosteroid was determined. In no case is there evidence of selective uptake by the intended brain target, the hippocampus, either in terms of elevated tissue activity levels or prolonged retention of activity over non-target sites. Only one Type II ligand ([‘*F]-15) shows a small drop in hippocampus: blood ratios upon receptor blocking, while the others show no evidence of blocking: the ratio of [‘*F]-15 at 1 h drops from 0.34 in adrenalectomized animals to 0.24 in intact ones, but the change is not statistically significant (0.1 < P < 0.5). The pituitary, a known target site for corticosteroids located outside the blood-brain barrier, shows selective uptake only with the same two Type II ligands. In the case of [18F]-5 and [“F]-15, this uptake appears partially blocked by competing exogenous unlabeled ligands, but only the change with [I8F]-15 is statistically significant ([“‘F]-5: 30 min (0.1 < P < 0.5);3 h (0.1 < P < 0.5);[‘*F]-15 1 h (0.001 < P < 0.01)). The fact that the pituitary shows selective accumulation of these ligands relative to blood levels (pit/blood g 3) while the hippocampus and other brain regions do not accumulate the “‘Fligand over blood levels (hippo/blood g 0.3-0.4) suggests that the blood-brain barrier may play a significant role in limiting brain tissue levels of both ‘*F-RU 28362 and “F-TA. Uptake into classic target tissues, kidney for Type I and liver for Type II, shows little blocking by an injected unlabeled competitor. Since both of these tissues are involved in detoxification and removal of steroids from the body, the radioactivity accumulated in them is mostly in the form of metabolites and thus may not reflect the distribution of the ‘*F-hgand itself. Unlike the sex steroid receptor systems that we have studied previously (Kiesewetter et al., 1984; Pomper et al., 1988) the corticosteroid selective tissues in the brain (hippocampus) do not retain exogenous ligand for very long (t,,* of [3H]corticosterone in hippocampal tissue is - 1 h) (McEwen et al., 1970). We see a similar time course of ligand retention for two of the synthetic IsFligands ([‘“F]-5, [18F]-15) and a more rapid loss of the rest. A factor that may contribute substantially to the ineffectiveness of these five fluorine-substituted corticosteroids as receptor selective ligands in vivo is their metabolic instability. The development of uptake contrast between target and non-target sites in vivo depends upon the selective clearance of non-specifically bound ligand from non-target tissues, when clearance is based on a metabolic process that releases radioactive components that recirculate and enter tissues, then the contrast potential inherent in the compound can become compromised. In all cases with these compounds, bone activity at the longest time points studied (0.5-3 h) is high (Fig. 2); since free fluoride ion is efficiently and selectively accumulated

478

MARTING. POMPEKet al.

by bone (Fiserova-Bergerova, 1973; Wallace-Durbin, 1954), this indicates that the carbon-fluorine bond is being cleaved during metabolism. While fluorine substitution is generally associated with stability (Smith, 1973) many fluorine-substituted compounds are, in fact, readily metabolized (Pochpasky ef al., 1990; Marcotte and Robinson, 1982). The process does not involve direct C-F bond scission, but hydroxylation of an CI C-H bond to produce an unstable fluorohydrin, a process in which the fluorine-substituent may actually be activating (Pochapsky et al., 1990). The relative metabolic lability of fluorine in the five fluorine-substituted steroids follows expectations for this type of reaction; but probably reflects, as well, the clearance rate of these steroids. [‘*F]-5, the analog of RU 28362, which has fluorine at a vulnerable propargylic position, gives 88% of the injected activity in bone at 3 h. The two 21-fluoro substituted steroids that are analogs of natural corticosteroids ([‘*F]-10 and [‘sF]-11) show rapid but limited defluorination lability. This is consistent with C-21 as a known site of hydroxylation and the rapid clearance of these compounds. Most surprising, however, was the propensity of compound [“F]-2 towards metabolic defluorination. Here the fluorine is situated at an unactivated primary position, yet 56% of the injected activity is deposited in the bone at 30min. There are other examples of metabolic lability of primary carbon-fluorine bonds (Pochasky et al., 1990). While our lack of success in observing selective uptake of these five corticosteroids in brain target sites in uivo may derive, in part, from their metabolic lability, it is more likely that contrast is compromised by a combination of the low corticosteroid receptor density in the hippocampus, the relatively low receptor binding affinity of these ligands and their high non-specific binding. There are a number of studies on selective binding and uptake of corticosteroids in the hippocampus that address these issues. The presence of Type I and Type II corticosteroid receptors in the hippocampus can be demonstrated easily by in vitro binding assays on cytosol preparations (Coirini et al., 1985; De Kloet et al., 1984; Sutanto et al., 1988). In such studies, receptor titers averaged over the whole hippocampus are 140-200 fmol/mg for Type II. This is less than ER or PgR in the uterus (1000 and 800 fmol/mg, respectively) (Carlson and Katzenellenbogen, 1990) but similar to AR in rat prostate (120 fmol/mg). However, tissue autoradiographic studies show that the receptor distribution within the hippocampus is non-uniform, with much higher densities of receptor localized in certain subregions (McEwen et al., 1970; Sutanto et al., 1988; Sarrieau et al., 1988; Coutart et al., 1987; Reul and De Kloet, 1986; Sarrieau et al., 1984) and other regions having few or no receptors. Additionally, there is evidence that excess corticosterone failed to block uptake of radioactive ligand into certain subportions of the hippocampus

(McEwen et al., 1970; Coutart et al., 1987; Sarrieau et al., 1984), suggesting non-specific uptake. Both of these methods (cytosol binding and autoradiography) however, involve adsorption or washing processes that selectively remove free and non-specifically bound ligand, which increases specific to non-specific contrast. The situation with brain localization and corticosteroids in uivo is quite different. Here, contrast between hippocampal uptake vs other brain regions is rather poor when total tissue activity is assayed (c. 2: l), whereas selective accumulation (c. 10: 1) is evident when the nuclear fractions, enriched in corticosteroid receptors, are compared (McEwen et al., 1969; De Nicola et al., 1981). This suggests that non-specific uptake of corticosteroids in the brain is high enough that the limited capacity, receptormediated component is largely obscured at the whole tissue level. Finally, in those cases where the time course of brain activity is followed after injection of a radiolabeled corticosteroid, a rapid decline of activity is observed in all regions, with the hippocampus to blood ratio being c. 2: 1 (McEwen et al., 1969); the ratio of other brain regions to blood is c. 0.5: 1 (McEwen et al., 1969, 1970). Again, however, selective and prolonged retention is observed in the nuclear fraction from hippocampus (McEwen et al., 1970). Where such in viuo uptake studies have been done with the pituitary, investigations have observed higher and more selective uptake that can be effectively displaced; displacement in the hippocampus is less pronounced (De Nicola et al., 1981). While it should be possible to label corticosteroids with radionuclides that are metabolically stable, the problems in achieving contrast between target and non-target regions that derive from low receptor titer and its limited distribution remain. It is clear that ligands with both greater metabolic stability and higher receptor binding affinity and selectivity will be required. Note added in proof Recently, DaSilva et al. [DaSilva J. N., Crouzel C., Stulzaft O., Khalili-Varasteh M. and Hantraye P. (1992) Synthesis, tissue distribution in rats and PET studies in baboon brain of no-carrier-added [‘sFjRU 52461: in uivo evaluation as a brain glucocorticoid receptor radioligand. Nucl. Med. Biol. 19, 167-1731 reported the synthesis of [‘sF]RU 52461 (which is the same compound as [‘*F]-5 in this paper). They also describe tissue distribution in rats, as well as imaging in baboons. As do we, they find competable uptake only in the pituitary, with no evidence of receptor-mediated uptake elsewhere in the brain. The authors conclude that this compound is unsuitable as an imaging agent for brain glucocorticoid receptors due to its failure to sufficiently penetrate the blood-brain barrier. Acknowledgements-We are grateful for support of this research through a grant from the American Health Assistance Foundation (to J.A.K.) and from the Department of Energy (DOE DE FG-02 84ER60218 to M.J.W.). We received helpful comments from Drs Edward Roy and

Fluorine-substituted Robert Sapolsky. High-field NMR spectra and high-resolution mass spectra were obtained on instruments supported by grants from the National Institutes of Health to the University of Illinois (RR02299 and GM27029, respectively). We thank Dr G. Teutsh and Roussel Uclaf for samples of RU 26752, 26988 and 28362.

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The fast method of Cu-porphyrin complex synthesis for potential use in positron emission tomography imaging.

Radiolabeled Phosphonium Salts as Mitochondrial Voltage Sensors for Positron Emission Tomography Myocardial Imaging Agents.

Synthesis and evaluation of 18F-labeled mitiglinide derivatives as positron emission tomography tracers for β-cell imaging.

Positron emission tomography radiotracers for imaging hypoxia.

Synthesis of fluorinated analogues of sphingosine-1-phosphate antagonists as potential radiotracers for molecular imaging using positron emission tomography.

Design, synthesis, radiolabeling and in vivo evaluation of potential positron emission tomography (PET) radioligands for brain imaging of the 5-HT₇ receptor.

Stable confinement of positron emission tomography and magnetic resonance agents within carbon nanotubes for bimodal imaging.

Positron emission tomography imaging of atherosclerosis.

Positron emission tomography imaging of coronary atherosclerosis.

Asymmetric Synthesis of Spirocyclic 2-Benzopyrans for Positron Emission Tomography of σ1 Receptors in the Brain.

Cyclotrons and positron emission tomography radiopharmaceuticals for clinical imaging.

Radiochemical synthesis of 2'-[18F]-labelled and 3'-[18F]-labelled nucleosides for positron emission tomography imaging.

Positron emission tomography imaging in sarcoidosis.

computed tomography imaging and rheumatoid arthritis.

Positron emission tomography-guided stereotactic brain biopsy.

Synthesis and evaluation of fluorinated fingolimod (FTY720) analogues for sphingosine-1-phosphate receptor molecular imaging by positron emission tomography.

Design, radiosynthesis, and evaluation of radiotracers for positron emission tomography imaging of stearoyl-CoA desaturase-1.

Positron emission tomography-magnetic resonance imaging in the evaluation of brain tumors: current status and future prospects.

A Novel Potential Positron Emission Tomography Imaging Agent for Vesicular Monoamine Transporter Type 2.

magnetic resonance imaging of the breast.

The methodology of TSPO imaging with positron emission tomography.

magnetic resonance imaging.

Synthesis and preliminary in vivo evaluation of well-dispersed biomimetic nanocrystalline apatites labeled with positron emission tomographic imaging agents.

¹⁸F-fluorodopa positron-emission tomography: an emerging imaging modality for patients with brain metastases.