Bioconjugate Chem. 1994, 5, 182-193
Ia2
ARTICLES Progestin Radiopharmaceuticals Labeled with Technetium and Rhenium: Synthesis, Binding Affinity, and in Vivo Distribution of a New Progestin N~S2-MetalConjugate James P. O’Nei1,tJ Kathryn E. Carlson,? Carolyn J. Anderson,$ Michael J. Welch,g and John A. Katzenellenbogen’vt Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, and Division of Radiation Sciences, Washington University Medical School, 600 South Kingshighway, St. Louis, Missouri 63110. Received December 27, 1993’
We have prepared and evaluated three metal conjugates of a progestin-monoamine-monoamide (MAMA’) bisthiol chelate system. These conjugates of rhenium and technetium-99 and -99m, are structural analogs of the bisamino-bisthiol (BAT) conjugates we have described recently, but the MAMA’ chelate, being more polar than the BAT system, gives a conjugate that is much less lipophilic, having an octanolwater partition coefficient that is nearly 80-fold lower. In competitive binding assays, the Re- and ggTc-MAMA’-progestin conjugates bind to the progesterone receptor with affinities greater than that of progesterone itself, and in a direct binding assay, the equilibrium dissociation constant (&) of the 99mTc-MAMA’conjugate was 0.97 nM. As is typical for llp-substituted progestins, these conjugates also have substantial binding affinity for glucocorticoid receptors. In tissue distribution studies in immature female rats, the pr~gestin-~~~Tc-MAMA’ conjugates show selective uptake for principal target tissue (such as uterus) over that of blood and nontarget tissue (such as muscle); these uptake ratios reach maximum levels of 5 and 4, respectively. Uptake by fat, liver, and kidney is quite high; however, only the uptake in uterus is displaceable upon coinjection of the selective progestin ORG2058. Metabolism studies show that the radioactivity in the uterus is essentially unmetabolized out to 4 h, while liver activity is completely due to metabolites. Other tissues show an intermediate fraction of unmetabolized conjugates that decreases with time. The in vivo behavior of the progestin-WmTcMAMA’ conjugate is similar to that of the labeled BAT conjugate: its uptake selectivity is somewhat greater than that of the BAT conjugate, but its target tissue uptake is lower. Factors that may be responsible for limiting the target tissue uptake properties of these conjugates are their moderate affinity for progesterone receptor, their substantial binding to glucorticoid receptors, and their large overall molecular size.
INTRODUCTION Because of its wide availability, convenient half-life, and appropriate y energy, technetium-99m is frequently the radionuclide of choice in the development of diagnostic imaging agents (1). Nevertheless, there are certain situations in which it is difficult to utilize technetium-99m: being a metal, it requires a chelate system to form stable complexes (1, 2) and other systems of organometallic bonding, while intriguing in structure, are difficult to prepare at the no-carrier-added level, as is required for receptor-based imaging agents (3). Since the technetium complexes are large, generally having molecular weights in excess of 250, their incorporation into small ligands for receptors, such as steroid hormones and neurotransmitters, provides a formidable challenge: (1) metal complex substituents of this size can severely compromise the binding affinity of these receptor ligands, and (2) the bulk of these conjugates can alter the physicochemical properties of receptor ligands to such an extent that their receptorUniversity of Illinois. Current address: Center for Functional Imaging, Lawrence Berkeley Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720. +
t
Washington University Medical School.
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Abstract publishedin Aduance ACS Abstracts, April 1,1994. 1043-1802/94/2905-0182$04.50/0
mediated biodistribution is limited by permeability barriers or overwhelmed by nonspecific uptake. In a recent study in which we examined the receptor binding affinity of four progestin-metal conjugates, we reported the preparation of a conjugate I between a progestin related to the Roussel-Uclaf antiprogestin RU486 IV and a bisamine-bisthiol (BAT)-technetium complex that retained high affinity for the progesterone receptor (4). While we could demonstrate nanomolar receptor binding of this conjugate in in vitro assays, its nonspecific binding was high and its tissue distribution in vivo showed high uptake by nontarget tissues (5). We postulated that the poor distribution properties of I might be largely due to the very lipophilic character of the bisamine-bisthiol (BAT) chelate system that was used. To lower lipophilicity, we considered other more polar metal chelate systems, and we have recently described the preparation of a smaller and considerably less lipophilic monoamine-monoamide-bisthiol-metal chelate system I1 (MAMA’) that forms stable, neutral complexes with rhenium and technetium (6). The MAMA‘-metal chelate system is a structural isomer of the well known MAMA system (7, 8). In this present report, we describe the preparation of a conjugate I11 of the RU486-related progestin with this 0 1994 American Chemical Society
Progestin NpSp-Metal Labeled Conjugates
Chart 1 CH3
Bioconjugate Chem., Vol. 5, No. 3, 1994
183
3
I BAT ProgestinOxo Metal Complexes
RU486 IV
II
MAMA’ Oxo Metal Complexes
ProgesteroneV
R5020 VI
new less lipophilic chelate system (9). The synthesis of the MAMA’-metal conjugates with rhenium and technetium-99 and -99m and an investigation of the progesterone receptor binding affinity, the measurement of lipophilicity, and the in vivo tissue distribution of these conjugates are presented. This new progesterone-metal conjugate represents the first metal-labeled steroid that retains nanomolar binding affinity for its receptor and has typical steroid-like lipophilicity; the in vivo tissue distribution characteristics of the wmTc-MAMA’ conjugate I11 are improved and show a higher component of receptormediated uptake than do the more lipophilic BAT congeners I.
111 MAMA Progestin Oxo Metal Complexes ( M = Re, Tc)
OR02058 VI1 X = OH X=F FENP Vlll
Products International Corp., Mt. Prospect, IL); 1,4-bis(5-phenyloxazol-2-y1)benzene(POPOP), 1-octanol (Aldrich Chemical Co., Milwaukee,WI); protosol (DuPont New England Nuclear, Boston, MA). All in vitro assays were done in the following buffer: 0.01 M Tris, 0.0015M EDTA, 0.02 % NaN3,20 mM Na molybdate, 20% glycerol, pH 7.4 at room temperature. Cytosols. Cytosols were prepared and stored as previouslyreported: ratPgR (IO),ratliver GuR (11),and human PR from T4,D tissue culture cells (12,13). Relative Binding Affinity (RBA). Assays were performed as previouslyreported (14).Severalconcentrations of unlabeled competitor or buffer, together with 10 nM tritiated tracer, were incubated with cytosol at 0 OC for MATERIALS AND METHODS 18-24 h. Unbound ligand was removed with charcoaldextran. Competitor solutions were prepared in 1:lDMF/ Biological Procedures. Materials. Radioligandswere buffer to ensure solubility. Progesterone receptor assays obtained from the following sources: [17a-methyPH]utilized either rat uterine cytosol from 3-day estrogenpromegestone (R5020), 86 Ci/mmol, and [6-meth~l-~HIprimed immature rats or cytosol from T4,D tissue culture llfl,l7fl-dihydroxy-6-methyl-l7a-( 1-propyny1)androsta(- 1.5nM receptor plus 1pM hydrocortisone to block cells 1,4,6-trien-3-one (RU283621, 77 Ci/mmol (DuPont New any glucocorticoid receptor) and [3HlR5020as the tracer. England Nuclear, Boston, MA). i3H1RU38486, 38 Ci/ Glucocorticoid receptor assays utilized liver cytosol from mmol, was a gift from Roussel-Uclaf, Romainville, France, 3-day adrenalectomized adult male rats ( w 1nM type I1 Unlabeled ligands: promegestone and RU28362 (DuPont sites) with 13H]RU28362 as the tracer. New England Nuclear, Boston, MA); 16a-ethyl-21-hyRadioactivity was determined in a Nuclear Chicago droxy-19-norpregn-4-ene-3,20-dione (ORG2058)(Organon Isocap 300 scintillation counter with adjustable windows, Corp., Oss, The Netherlands); RU486 (RU38486;RousselC from the tritium set to exclude 95% of the ~ T counts Uclaf, Romainville, France); hydrocortisone (Sigma Chemichannel. The 5% spill was subtracted from the tritium cal Co., St. Louis, MO). Other chemicals were obtained counts. from the following sources: activated charcoal, Trizma Log PDeterminations. The log Pvalues were estimated base, 3-(N-morpholino)propanesulfonic acid (MOPS), as previously reported (15) from the log k’, values n-decylamine (Sigma Chemical Co., St. Louis, MO); determined by HPLC chromatography, following the dextran grade C (Schwarz/Mann, Orangeburg, NY); direcommendations of Minick (161,using an HPLC equipped methylformamide (DMF) (Fisher Scientific,Fairlawn, NJ); with an autosampler and Spectra System software (Ther(ethy1enedinitrilo)tetraaceticacid tetrasodium salt (EDTA) mo Separation Products, Warrenville, IL). and sodium azide (Eastman Organic Chemicals,Rochester, NY); sodium molybdate (Mallinckrodt Inc., St. Louis, Direct Binding Assays. Rat uterine cytosol containing MO); Triton X-114 (Central Solvents and Chemicals Co., 1.5 nM progesterone receptor, preincubated with 1pM Bedford Park, IL); 2,5-diphenyloxazole (PPO) (Research hydrocortisone, was incubated with various concentrations
-
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Bioconjugate Chem., Vol. 5, No. 3, 1994
of [3Hl(R5020 or RU486) or 9 9 m T(MAMA' ~ or BAT) progestin, in the absence or presence of a 100-fold excess of unlabeled R5020 (R5020 is used to block the progesterone receptor). Incubations were at 0 "C for 3-5 h. Unbound ligand was removed by charcoal-dextran. For conjugate ~ 7 was diluted with all in vitro assays, the g g m T 99Tc conjugate 6 to give a specific activity of -86 Ci/ mmol. The charcoal-dextran slurry used to remove unbound ligand was prepared as previously reported (14)and used in a ratio of 1 part charcoal-dextran slurry per 10 parts cytosol solution at 0 "C. Data were plotted according to the method of Scatchard (17). Radioactivity was determined in a Nuclear Chicago Isocap 300 scintillation counter with adjustable windows, set to exclude 95% of the 99Tc counts from the tritium channel. The 5% spill was subtracted from the tritium counts. Animal Uptake Methods. Immature female SpragueDawley rats (25 days old, -50 g except where noted) were injected (IV, lateral tail vein), under methoxyflurane (2,2dichloro-1,l-difluoroethylmethyl ether) anesthesia, with ca. 25 pCi of 99mTc conjugate 7 or 5 pCi of f3H1RU486in a 4/ 1,physiological saline/ethanol solution. To ascertain whether the uptake was mediated by a high affinity, limited capacity system, in one set of animals 18 pg of unlabeled ORG2058 (RBA = 170% relative to R5020) was coinjected with the radiopharmaceutical. Animals were sacrificed by decapitation at the times indicated, and samples of tissue and blood were weighed. Radioactivity in organ and standard samples was determined with a Beckman Gamma 8000 automatic well-type y counter (Beckman Instruments, Fullerton, CA) (5,18) for 9gmTc,while 3H radioactivity was determined following digestion using a previously reported protocol (19). Metabolism Studies. The radiolabeled progestin 7was extracted from blood or tissue samples at various time points with ethanol (15,20).Blood samples (100 pL) were obtained by cardiac puncture, while tissue samples were dissected from various organs directly after sacrifice. The blood or tissue samples were weighed and their radioactivity measured; the samples were then diluted with EtOH (500 pL) and homogenized. After centrifugation (5 min, 2500 g), the pellet and supernatant were separated and counted. A 100-pLaliquot of the supernatant was analyzed by TLC (5% iPrOH/CH2Clz) with comparison to an authentic sample of radiolabeled progestin 7. No attempt was made to identify the specific metabolites. Chemical Procedures. Materials. Solvents and reagents were purchased from various commercial sources, Aldrich, Mallinckrodt, Sigma, Fisher, Baker, Eastman, or Alfa, and were used as received, unless otherwise noted. NassmTc04in saline solution was eluted from a 99Mo/ 99mT generator ~ purchased from DuPont or Mallinckrodt. I99Tc1(n-Bu)4NTcOC14 was prepared as previously described (5 ) from [99Tc] (n-Bu)4Tc04 obtained from Alan Davison (MIT). 99mTc-glucoheptonate kits (Glucoscan kits) were purchased from Du Pont, N. Billerica, MA. General. Analytical thin layer chromatography (TLC) was performed using 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 refers to the method of Still et al. (21).Short silica plugs used Woelm silica gel (0.032-0.064 mm) or Merck silica gel (0.040-0.063 mm). High-performance liquid chromatography (HPLC) was performed isocratically with a preparative Si02 column (Whatman Partisil M-9,O.g cm X 50 cm). The eluate was monitored by UV absorbance at 254 nm and a sodium
O'Neil et al.
iodide scintillation flow detector, where appropriate. Proton magnetic resonance (IH NMR) spectra at 400 MHz are reported downfield from a tetramethylsilane internal standard (6 scale). Both low- and high-resolution fast atom bombardment (FAB) mass spectra were obtained employing a dithiothreitol matrix. 1l p - [a-[N-[ [2 [( Triphenylmethyl)thio]ethyl]aminolcarbonyl]methyll -N[2-[(triphenylmethyl)thiolethyllamino] -p-tolyl]- 17a-propynyl-17p-hydroxy-4,9estradien-3-one (MAMA'-TrrProgestin Conjugate,3). To a stirring solution of llp-[a-[(methylsulfonyl)oxylp-tolyl]-l7c~-propynyl-l7~-hydroxy-4,9-estradien-3-one ( 1) (4)(115.2 mg, 0.23 mmol) in CHzClz (3 mL) in a Reactivial" was added a solution of N-[[[2-[(triphenylmethyl)thiolethyl]aminolacetyll-S-(triphenylmethyl)-2-aminoethanethio1 (MAMA'-Trz) (2) (5) (332 mg, 0.49 mmol) in CHzC12 (1mL). The vial was flushed with nitrogen, sealed with a septum and cap, and placed in a 70 "C oil bath for 2 h. The crude reaction mixture was subjected to silica gel flash chromatography (2:l EtOAc/hexanes) to provide the thiolprotected progestin-ligand conjugate 3 (156.5 mg, 62%) as a white foam: 'H-NMR (CDCl3, 400 MHz) 6 7.55 (t, l H , J = 5.7 Hz), 7.43-7.35 (m, 12 H, ArH), 7.25-7.13 (m, 20H, ArH), 7.02 (d, 2H, J = 9 Hz), 5.75 (s, l H , 4-CH), 4.33 (d, l H , 7.1 Hz, lla-CH), 3.40 (q, 2H, J = 13.4 Hz, ArCHzN), 3.10-2.89 (m, 2H), 2.86 (d, 2H, J = 4.2 Hz), 2.70 (dt, l H , J = 14.6, 5.1 Hz), 2.56-2.18 (m, 14H), 2.20-1.84 (m, 3H), 1.91 (s, 3H, C=CCH3), 1.78-1.64 (m, 3H), 1.50-1.18 (m, 2H), 0.46 (s,3H, l8-CH3); MS (LRFABMS) mlz (relative intensity) 1080 (281, 1079 (51), 1078 (611, 835 (131, 680 (141, 679 (261, 591 (271, 399 (26); HRFAB calcd for C ~ ~ H ~ Z N Z(M O ~+S1) Z 1077.5063, found 1077.5076. 1l p - [a-[N-[[[(2-Mercaptoethyl)amino]carbonyl]methyll -N- (2-mercaptoethyl)aminol-p-tolyl]-17a-propyny1-17p-hydroxy-4,9-estradien-3-one(llp-MAMA'Progestin Conjugate,4). To a solution of MAMA'-Trzprogestin conjugate (3) (60 mg, 0.56 mmol) in ethyl acetate (1mL) and ethyl alcohol (1mL) was added a solution of Hg(0Ac)z (44.4 mg, 0.139 mmol) in ethyl alcohol (2 mL). The mixture was heated to reflux for 10 min and then cooled to room temperature. Gaseous HzS was bubbled through the solution until a dark black precipitate had formed, at which time the reaction vessel was capped and the solution allowed to stir for 5 min. The resulting heterogeneous mixture was filtered through Celite to remove the HgS residue, and the filtrate was concentrated to dryness in vacuo. Silica gel flash chromatography of the resulting yellow oil (EtOAc followed by 1:9 EtOH/ EtOAc) provided, after concentration in vacuo, free bisthiol conjugate 4 (12.2 mg, 37%) as a colorless oil. A standard solution of this compound was also prepared in nitrogendegassed EtOH at a concentration of 1mg/mL for use in the 9 9 m Tradiochemical ~ labeling experiments. [ll p- [a-[N- [[[(2-Mercaptoethyl)amino]carbonyl] methyl]-N-(2-mercaptoethyl)amino]-p-tolyl]-l7a-propyny1-17/3-hydroxy-4,9-estradien-3-onato(4-)]oxorhenium(V) (MAMA'-Re(V)-Oxo-ProgestinConjugate,5). To a solution of MAMA'-Trz-progestin conjugate (3) (150 mg, 0.139 mmol) in EtOAc (4 mL) was added a solution of Hg(0Ac)z (11mg, 0.034 mmol) in EtOH (3 mL). The mixture was heated to reflux for 20 min and then cooled to room temperature. Gaseous HzSwas bubbled through the solution until a black precipitate had formed, at which time the reaction vessel was capped and the solution allowed to stir for 5 min. The resulting heterogeneous mixture was filtered through Celite to remove the HgS residue, and the filtrate was concentrated to dryness in vacuo to provide the bisthiol4 as a yellow oil. To the free
Progestin N&-Metal
Labeled Conjugates
ligand 4, diluted with MeOH (5 mL), was added NaOAc (1N in MeOH, 2 mL) followed by (PPh3)2Re(O)Cl3(128 mg, 0.153 mmol). The reaction mixture was heated to 90 "C in a closed Reactivial" for 2 h and then cooled to room temperature. The resulting mixture was diluted with EtOAc (15 mL) and filtered through Celite. The purple solution was washed with water and the organic portion dried over MgS04, filtered, and concentrated in vacuo. Silica gel flash chromatography of the resulting oil (gradient elution from 2:l EtOAc/hexanes to 3:l EtOAc/ hexanes) provided 5 (33 mg, 0.067 mmol) as a purple solid. The two diastereomers about the tertiary amine of the ligand could be separated on the analytical scale by normalphase HPLC (Whatman Si-5,4.6 mm X 25 cm, 50% (1:20 iPrOH-CHzC12) 50 % hexanes). lH-NMR (CDCl3, 400 MHz) 6 7.70-7.31 (m, 4H, ArH), 5.80 (s, l H , 4-CH), 5.06 (d, lH, J = 14.3 Hz), 4.96 (dd, lH, J = 16.2,2 Hz, ArCHz), 4.63 (d, l H , J = 14.3 Hz), 4.58 (dd, l H , J = 11.2,6.6 Hz), 4.10 (d, l H , J =5.9 Hz, lla-CH), 3.76 (d, l H , J = 16.2 Hz, ArCHz), 3.69 (tt, l H , J = 12.9, 2.7 Hz), 3.21 (m, 2H), 3.03 (ddd, l H , J = 12.0,9.5,3.2 Hz), 2.88 (dt, l H , J = 13.4,4.4 Hz), 2.78 (dq, l H , J = 14.9, 4.9 Hz), 2.61 (m, 2H), 2.502.18 (m, 6H), 2.12-1.15 (m, 10 H), 1.90 (s,3H, C=CCH3), 0.45 (s,1.5H, one diastereomer 18-CH3),0.44 (s,1.5H, other diastereomer 18-CH3); MS (LRFABMS) mlz (relative intensity) 794 (15) (M + 2), 793 (35) (M + 11,791 (22) (M 1, la5Re),615 (15), 613 (33), 581 (18), 463 (28), 461 (42), 459 (22), 309 (1001, 279 (100); HRFAB calcd for C34H42N z 0 4 S ~ ~ ~(M ~ R+e1) 793.2144, found 793.2130. [ l 10- [a- [N- [[[2-Mercaptoethyl)amino]carbonyl]methyl] -N- (2-mercaptoethy1)aminol-p-tolyl] -17a-propynyl-l7~-hydroxy-4,9-estradien-3-onato(4-)]oxo~Tc]technetium( V) (MAMA'-PgTc] Tc(V)-Oxo-Progestin Conjugate, 6). Deprotection of the ligand system was performed in a manner similar to that described in the preparation of rhenium conjugate 5. Thus, bisthiol4 was prepared from protected compound 3 (44.6 mg, 0.41 mmol) and Hg(0Ac)z (26 mg, 0.081 mmol). To the crude ligand in MeOH (2 mL) was added solid (n-Bu)4NTcOC4 (15 mg, 0.030 mmol) and NaOAc (0.400 mL, 1N in MeOH). The mixture was allowed to react at room temperature for 20 min before the soluble product was isolated by filtration through Celite with EtOAc and washed with water. The organic extract was dried with MgS04 and concentrated by solvent evaporation under a stream of nitrogen. Silica gel flash chromatography (2:l Et0Ac:hexanes) provided 6 (16.6 mg, 78.3%) as a yellow solid. 'H-NMR (CDCl3, 400 MHz) 6 7.39-7.30 (m, 4H, ArH), 5.81 (s l H , 4-CH), 5.07 (d, l H , J = 14.2 Hz), 4.95 (dd, lH, J = 16.1, 1.5 Hz, ArCHZ), 4.63 (d, lH, J = 14.2 Hz), 4.60 (dd, l H , J = 10.7, 4.2 Hz), 4.48 (bd, lH, J = 8 Hz, lla-CH), 3.77 (d, l H , J = 16.1 Hz,ArCHz), 3.70 (tt,l H , J =13.2,3.2Hz), 3.23 (m, 2H), 3.04 (ddd, lH, J = 12.7, 10.3, 2.9 Hz), 2.90 (dt, IH, J =13.7,4.4Hz),2.79 ( d q , l H , J = 14.9,4.9Hz),2.62 (bm, 2H), 2.55-2.20 (m, 6H), 2.06 (dt, lH, J = 12.9, 4.41, 1.95 (m, lH), 1.91 (s,3H, CECCH~),1.81 (8, lH), 1.74 (m, 2H), 1.65-1.18 (m, 5H), 0.46 (s, 1.5H, one diastereomer l8-CH3), 0.45 (s, 1.5H, other diastereomer l8-CH3); MS (LRFABMS) mlz (relative intensity) 706 (5) (M + 2), 705 (13) (M + l),613 (7), 463 (5), 461 (8),406 (5), 399 (71,391 (13); HRFAB calcd for C34H42Nz04Sz99Tc (M + 1) 705.1615, found 705.1632. [ l l p - [a- [N- [[[2-Mercaptoethyl)amino]carbonyl]methyl] -N- (2-mercaptoethy1)aminol-p- tolyl]- 17a-propyny1-17/3-hydroxy-4,9-estradien-3-onato(4-)]oxoPgmTc] t echne t iu m ( V) (MAMA'-PgmTc]Tc( V)-OxoProgestin Conjugate, 7). Water (5 mL) was added to a Glucoscan kit (200 mg glucoheptonate, 0.06 mg SnCly
+
Bioconjugate Chem., Vol. 5, No. 3, 1994
185
2Hz0), swirled, and allowed to stand for 10 min at room temperature. Na9gmTc(VII)04(17.9 mCi) in saline (300 pL) was added to 500 pL of the previously prepared glucoheptonate solution, swirled, and allowed to stand for 15 min at room temperature. A solution of 110-MAMA'progestin (4) (40 pg, 0.068 pmol) in EtOH (800 pL) was added to the Tc-glucoheptonate solution and stirred for 20 min at room temperature. The reaction mixture was extracted with CHzC12 (3 X 1.5 mL), and the combined organic layers were dried over MgSO4 and filtered. The solvents were removed in vacuo, and the residue was redissolved in CHzClz and applied to a short (2 cm) silica pipet column. The labeled progestin was eluted from the column with CHzClz (2 mL) followed by 5% iPrOH/CHzClp (2 mL) and the solvent removed in vacuo. The residue was redissolved in 500 p L of 1/1hexane/ ( 5 % iPrOH/CH2Clz) and purified by normal-phase HPLC (Whatman M9, partisil5,9.4 mm X 50 cm), eluting with 160% (1:20iPrOHCHzC12)/40%)hexanes] toprovide5.47mCi (31%)of l l p MAMA'-ghTc-progestin (7) (tR = 16.8 min) in a ligandfree form. This material (7) coeluted with the 99Tcanalog (6) by both normal phase and reversed-phase (HzOacetonitrile gradient on C-18 silica gel) HPLC. RESULTS
Chemical Synthesis. The synthesis of compounds 3-7 is shown in Scheme 1and is described below. Each of the metal conjugates 5-7 was prepared from the same NZSZ (MAMA')-progestin conjugate (4). The progestin conjugate 4 was prepared by N-alkylation of ligand 2 (4)with the methanesulfonate (mesylate)of the previously reported RU486 analog 1 (4),followed by removal of the triphenylmethane (trityl) sulfur-protecting groups. The metal conjugates 5 and 6, prepared at macroscopic levels, were characterized by standard analytical techniques, while the 99mT~ conjugate 7, which could only be prepared at the tracer level, was identified by ita coelution upon HPLC analysis with the previously characterized 99Tc counterpart 6. As was discussed in previous publications (4,5), the insertion of a metal-oxo core into an NzSz ligand-progestin system can result in four diastereomeric products. These are denoted as the syn and anti diastereomeric pairs, where syn and anti are defined by the orientation of the N-progestin substituent relative to the metal-oxo bond. The syn diastereomeric pair is typically formed in far higher yield than the anti pair (4, 22); in fact, in these systems, the anti diastereomers could not be detected. All three metal-progestin conjugates 5-7 could be isolated in ligand-free form by normal-phase HPLC, since the metal conjugates elute far ahead of the free ligand 4. S-Trityl-MAMA'-Progestin (3). The conjugate 3 was prepared by N-alkylation of the MAMA' ligand system (5)with the benzylic 0-mesylate of the RU486 analog used under basic conditions provided in our previous work (4), by triethylamine. Extensive column chromatography was required to obtain analytically pure material, as the trityl protecting groups have a tendency to hydrolyze, thereby causing extensive streaking during chromatography. MAMA'-Progestin (4). S-deprotection of MAMA'progestin 3 was accomplished by treatment with Hg(0Ac)Z to displace the trityl protecting groups and form the steroidal mercury salt, followed by protonation with hydrogen sulfide (23)to provide free ligand 4. As the MAMA'-progestin (4) is prone to disulfide formation under ambient conditions, this compound was used directly as described in the Chemical Procedures for the macroscopic preparations (conjugates 5 and 6) or was prepared
186
O'Nell et al.
Bloconjugate Chem., Vol. 5, No. 3, 1994
Scheme 1 0.
O
h
/NH
HN\
'STr
TrS
I
2
L
Et3N, CICH2CH2CI 63 Yo
1
-0
3
NaOAc, MeOH 48 %
'
SH SH
4
( ~ - B u ) ~([gSrcITcOCI,) N
NaOAc,MeOH 78 Yo
Na ([99mT~]T~04), saline Glucoscan kit
*
7 (M = ""'Tc)
31 Yo Table 1. Binding Affinity of the Progestin-Metal Complexes and Related Ligands for the Progesterone Receptor (PR) and Glucocorticoid Receptor (GR) and Their Log P Values RBAa ( % ) PR (rat)b GR (rat)c PR (human)d log P compd (R5020 = 100) (dexamethasone = 100) (R5020 = 100) (from kb) 13f1 1.08 f 0.46 9f3 3.87 progesterone (V) 170 f 5 319 f 67 24 f 7 4.69 RU486 (IV) 198 f 39 1.98 f 0.42 87 f 3 4.21 ORG2058 (VII) 100 2.81 f 1.60 100 4.34 R5020 (VI) 697 f 88e 1.42 f 0.46 360 f 18 4.41 FENP (VIII) 8f3 6.41 BAT-BgTc complex I (syn pair) 25 f 1 56 f 21 25 f 5f 31 f 8f BAT-Re complex I 5f3 6.45 35 f 8 100 f 15 16f3 5.25 MAMA-g9Tc complex 6 (syn pair) 117 f 2 7.9 f 0.1 4.90 MAMA-Re complex 5 (syn pair) 45 f 8 syn A 28 f 8 113 f 46 not assayed not assayed syn B 52 f 26 101 f 65 not assayed not assayed a
The receptor bindingaffinity is determined by a competitive radiometricbinding assay. Values are the average of two or more determinations
f range (n= 2) or SD (n2 3) and are expressedon a percent scale relative tothe affinityof the tritium-labeledtracer (14). Cytosolpreparations were from estrogen-primedimmature rat uterus, with [3HlR5020as tracer (10). Cytosol preparations were from saline perfused liver of 3-day adrenalectomized mature male rats with [sH]RU28362as tracer (11). Cytosol preparations were from human T47D breast cancer cells grown as previously described (13).e Data from ref 24. f Data from ref 4.
as a standard solution in degassed ethanol and stored at -20 "C for use in the tracer-level preparation of conjugate 7. MAMA'-Re-Oxo Conjugate 5. The Re0 core was inserted into the MAMA'-progestin (4) through ligand exchange with (PhSP)zRe(O)Clain basic (NaOAc) methanol. The product was purified by flash column chromatography and further examined by normal-phase HPLC. Only the syn diastereomeric pair could be detected. The two syn diastereoisomers could be partially separated by HPLC, but only at long elution times which provided broad peaks. Thus, the two syn diastereomers could be isolated for in vitro binding assays, but not on a time or quantity scale that would have been practical for in vivo biological studies. MAMA'-99Tc-Oxo Conjugate 6. The TcO core was inserted into the MAMA'-progestin (4) through ligand exchange with an equimolar amount of (n-Bu)rN([99TclTcOCl4) in basic (NaOAc) methanol. The syn diastereomeric pair was isolated by flash column chromatography
and further purified by normal-phase HPLC. No attempt was made to separate the two syn diastereomers, as their separation in the case of the rhenium conjugate 5 had proved to be a tedious task; also, previous work (4,5) had shown that the ratio of in vitro binding affinities for the separate isomers is essentially equivalent for the Re and Tc compounds, and in our hands, the PR binding affinity of the two syn diastereomers of 5 differed by less than a factor of 2 (see below and Table 1). MAMA'99mTc-Oxo Conjugate 7. Technetium-99mprogestin 7 was prepared by a chelate exchange reaction with preformed mTc-glucoheptonate and the MAMA'progestin (4) and was purified by normal-phase HPLC. Only the syn diastereomeric pair was detected, and these compounds were not separated (cf. discussion above for MAMA'-Re-progestin 5). In order to maximize the exchange process, reaction conditions were adjusted to provide a much more dilute reaction in both aqueous medium (i.e., dilution of the Glucoscan kit), as well as organic medium (Le., EtOH equal in volume to the aqueous
*/
Progestin N2S2-Metal Labeled Conjugates
phase). Preparations were studied over a range of pH values (pH 4-11), and maximum incorporation was seen under slightly acidic conditions (pH 5), although for convenience the WTc-glucoheptonate obtained in kit form (pH 7) was found to provide satisfactory yields. Although the conjugate 7 was not formed as readily as the correspondingBAT--Tc compounds previously reported (5), the MAMA’ conjugate appeared to be more stable to aqueous conditions and to concentration in vacuo than were the BAT conjugates. The sample of conjugate 7 used for the in vivo and in vitro studies had a radiochemical purity in excess of 95 % , as determined by reversed-phase analytical HPLC. The specific activity of the preparation was estimated to be ca. 100 Ci/mmol, based on the history of the 99Mo/99mTc generator which was used (5). In vitro binding experiments were conducted with material that was diluted with the 99Tc-progestin 6 to a specific activity of 86 Ci/mmol. In Vitro Studies. Relative Binding Affinityof Metall Progestin Conjugates 111for the Progesterone Receptor (PR) and Glucocorticoid Receptor (GR). The relative binding affinities (RBA) of the MAMA’-Re and -WTc conjugates 5 and 6 as well as previously prepared BATRe a n d - q c conjugates ( 4 3 )and some related compounds (cf. Scheme 1) to the progesterone receptor and the glucocorticoid receptor are shown in Table 1. The RBAs are determined by a competitive radiometric binding assay. Even though they are radioactive, the 99Tcconjugates can be assayed in this competitive binding assay with a tritiumlabeled tracer, using standard dual label scintillation counting methods (5);the specific activity (SA) of wTc is low (0.0017Ci/mmol) compared to the SAof tritiated tracer ligands (77-86 Ci/mmol), so the counts from the 99Tc conjugates are appreciable only at the highest concentrations. The RBA value of the 99Tcconjugate 6 for the progesterone receptor is similar to that of the analogous unlabeled Re conjugate 5. This was observed previously with the BAT conjugates I (4) and is expected, since the Re and Tc complexes are similar in size and electron density distribution (25, 26). Both the MAMA’-Re and -99Tc conjugates assayed as syn pairs have rat PR binding affinities that are ca. three times greater than that of progesterone itself (35-4576 vs 13%). When the syn diastereomers of the Re conjugate 5 are separated, one was found to have a binding affinity approximately two times greater than the other. The PR binding affinities of the MAMA’ conjugates 5 and 6 for rat PR appear to be equivalent or marginally higher than those of the corresponding BAT conjugates I. Many progestins and antiprogestins have markedly different binding affinities to the PR from different species (27-29). Almost uniformly, the compounds presented in Table 1 show lower affinity to the human PR than to rat PR (relative to R5020). The studies presented in this paper use the rat as the animal model, but the ultimate goal is to design compounds for use in humans. Therefore, binding affinity measurements in both human and rat are important. In addition to PR, immature rat uterus contains substantial levels of glucocorticoid receptor (GR) and small levels of mineralocorticoid receptor (72% and 5 % ,relative to progesterone receptor = 100%) (K. E. Carlson, unpublished). All in vitro binding studies with PR included 1 pM of hydrocortisone to block these corticosteroid receptors. Since no attempt was made to block them in the in vivo tissue distribution experiments, this heterologousbinding to other receptors may influence the results
Bioconjugate Chem., Vol. 5, No. 3, 1994 2.0 /- ?99
187
B.
5ee c Ific
-
Blocked
0.0 (VI)
/Total
L / B l o c k e d
0.0 0
10
20
0
10
20
Free (nM) Figure 1. Binding curves for mTc-MAMA’ (A), mTc-BAT (B), [3HlR5020 (C), and [aH]RU486 (D)in rat uterine cytosol. Shown is the total binding,the nonspecific binding (NS) (receptor blocked by lOOX excess unlabeled R5020),and specific binding (difference between total and NS), after 3 h incubation at 0 O C . For comparison,the mTc-BAT conjugate is included, using data adapted from ref 5.
of the in vivo experiments. As shown in Table 1, the 99TcBAT conjugates I have GR affinities comparable to their affinities for PR, while the Re- and 99Tc-MAMA’ conjugates 5 and 6 have GR affinities nearly three times higher than PR affinities. The GR affinities of these metal conjugates are 10times greater than those of the selective progesterone receptor ligands ORG2058 and R5020 but lower than that of the parent ligand, RU486. Lipophilicity Estimates (Log P ) . Octanol/water partition coefficients ( P ) ,as a measure of lipophilicity, have been shown to correlate well with the nonspecific binding of steroids (30). The chromatographic method of Minick (161, to measure log k’,, has been found to accurately reproduce octanol/water partition values measured with the shake flask method. The regression line relating the chromatographically determined log k’, values of the standards to their measured log P values shows very high statistical reliability (15)and was extrapolated to the high log k’, values of the progestin conjugates, although no standards existed in this range. As shown in Table 1,the MAMA’ conjugates have lower log P values than those of the corresponding BAT-substituted conjugates previously studied ( 4 ) and are in fact approximately 80 times less lipophilic. Unlike the BAT compounds, which show ca. 100-foldhigher lipophilicities than those of simple steroidal progestins, the MAMA’-metal conjugates have log Pvalues approaching those of the unsubstituted steroids themselves (V-VIII). Direct Binding Assays. The direct assay of progesterone receptor binding with the tritium-labeled ligands R5020 (VI) and RU486 (IV) as well as 99mTc-MAMA’-progestin conjugate 7 and the previously prepared 99mTc-BAT conjugate I is shown in Figure 1. All four ligands show high affinity (& 0.46-2.74 nM)),saturable binding to PR. The extent of nonspecific binding in this assay is evident from the slope of the “nonspecific” binding curve. The lipophilic BAT conjugate I shows the highest nonspecific binding, as was expected, since nonspecific binding typically correlates with lipophilicity. The nonspecific binding of the MAMA‘ conjugate is somewhat less, being more comparable to that of R5020.
-
188
Bloconjugate chef??., V O ~5, . No. 3, 1994 I \
O’Neil et ai. S
W
I
1
E
U \
U
C
3 0
m
0.0
0.5
1.o
1.5
Specific Bound (nM) Figure 2. Data shown in Figure 1converted to Scatchard plots, showing Kd values for RU486 = 0.409nM, R5020 = 0.458 nm, mT~-MAMA’ = 0.965 nM, and mTc-BAT = 2.136 nM.
When the binding data are expressed as Scatchard plots (Figure 21, it is clear that the metal conjugates bind to nearly the same number of sites as do R5020 and RU486, consistent with their binding only to PR (glucocorticoid receptor was blocked with hydrocortisone). The small differences observed in site concentration for the MAMA’ conjugate could be due to uncertainties in the estimated specific activities of the radioactive metals. The & for I3H1R5020 in these assays is 0.46 nM, while the Kd for 99mTc-MAMA’conjugate 6 is 0.97 nM. The ratio of the dissociation constants for the metal conjugate vs R5020 (Kd(RS020)/Kd(conjug*~)) is analogous to an RBA value. For the gemT~ conjugate, this ratio is 47 5% , very close to the RBA value of 35% f 8 (Table 1). The concordance Of Kd ratios with RBA values for metal conjugates is particularly significant, as it verifies that the RBA values obtained in the competitive binding assay (with 99Tcand unlabeled Re) are those of the intact metal conjugates and not the decomplexed steroid. In Vivo Tissue Distribution of 9 9 m TConjugate ~ 7. Tissue uptake studies were performed in immature female Sprague-Dawley rats, 21-25 days old, that were primed with estradiol to increase their uterine titer of PR ( 5 , 1 0 ) . The rats were injected IV (tail vein) with the radiopharmaceutical, and the uptake in various tissues was determined at the times indicated in Tables 2 and 3. Some rats were coinjected with a large dose (18pg) of ORG2058 (RBA = 198% relative to R5020) to block PR sites. ORG2058 is one of the most selective ligands for PR (cf. Table 1); it should not block other receptors (glucocorticoid, mineralocorticoid) present in the rat tissues (11, 29). The tissue uptake data are presented as percent of injected dose per gram tissue ( % ID/g) in Tables 2 and 3. Some of these data are shown in Figures 3-6. The principal target organ for these radiolabeled progestins is the uterus, and there is substantial uterine uptake for the MAMA’-99mTc conjugate (Table 2 and Figure 3). There is also high uptake in the nontarget organs (liver and kidney) involved in the metabolism and excretion of steroids. However, binding to GR may also account for some of the uptake into these organs, as they are rich in this receptor (29, 31-33). Fat also shows high uptake, suggesting that the nonspecific uptake may still be dominated by the lipophilicity of these metal conjugates. Nevertheless, the uptake in the uterus is higher than that in a nontarget tissue such as muscle, and the uterus to muscle ratio after 6 and 18 h is 4.1 and 2.9, respectively (Figure 4); uterus to blood ratios peak earlier, at 3 h, at even higher ratios. When PR is blocked in vivo by coinjection of a large dose of ORG2058 (see Table 3 and Figure 5), only the uterus shows a significant decrease in uptake (55%,60%,
and 71% at 1,3, and 6 h, respectively), suggesting that the uterine uptake is PR-mediated. In this blocking experiment, the statistical significance of the changes in the various tissues was calculated by student’s T-test (34),n = 5; only the change in uterine activity is statistically significant at the 99% confidence level (0.01 > p > 0.001) (cf. Figure 5). Thus, it appears that we are observing PRmediated uptake of conjugate 7 in the uterus, as ORG2058 blocks PR, but is unlikely to block GR. The uterine uptake of the conjugate which cannot be blocked by ORG2058, however, might be due to its binding to GR, since the conjugate has substantial affinity for this receptor. In order to make a critical evaluation of the chemical structure components affecting the animal uptake, we also conducted uptake studies using r3H1RU486,which is the parent ligand for both the BAT and MAMA’ conjugates. The biodistribution of I3H1RU486is presented in Table 4; its comparison with the two gemTcsystems at 3 h is shown in Figure 6. The efficiency of ovarian and uterine uptake of r3H1RU486 was 2.7-4.3 times as great as that of the 99mTc-MAMA‘ conjugate at 3 h @I < 0.05). However, its uptake specificity (percent of uptake that can be blocked) is only marginally greater: 86% vs 60% at 3 h, respectively. Both of these properties are likely to be related to the binding affinity of the ligand to PR: RU486 has a 4.8 times greater affinity than the Tc-MAMA‘ complex. The 99mTc-MAMA’is retained longer in the target tissues than is RU486: at 6 h, 35% of the initial 99mT MAMA’ ~ uptake remains in the uterus, while only 18% of the RU486 uptake remains. A slow clearance from the target tissues is a desirable characteristic for ligands designed for imaging (30). Perhaps the best overall comparison among the three compounds can be seen at the 3 h time point shown in Figure 6. Here, the higher level of uterine uptake byRU486 and the improveduterus to muscle uptake ratio are evident. Liver and fat uptake of RU486, however, are still high and comparable to those of the two conjugates, suggesting that the progestin portion of the conjugate is mainly responsible for this uptake, not the metal chelate. A comparison between the two 99mT~ conjugates (MAMA‘ 7 and BAT I) at 3 h is also seen in Figure 6; the uterine uptake by the BAT conjugate is greater, as is its uterine to blood ratio, but relative to muscle and fat, uterine uptake is the same as for the MAMA’ complex. The fraction of uterine activity displaced by ORG2058 is somewhat greater for the MAMA‘ 7 (70% at 6 h) compared to BAT I (63% at 6 h), but this difference is not statistically significant. It is notable that all three of the PR ligands show uterine uptake that is lower and less selective than 21-fluoro-16aethyl-19-norprogesterone (FENP),where 1h % ID/g levels are as follows: uterus, 6.43; muscle, 0.42; blood, 0.25; fat, 3.56; and liver, 3.34 (24). Metabolism Studies. The in vivo stability of the 99mTc-MAMA’-progestinconjugate 7 was investigated in immature rats. At various times after injection, blood and tissues were extracted with ethanol and the soluble activity analyzed by thin-layer chromatography to determine the percent activity that is unmetabolized. Correction is made for the extraction efficiency of the 9f”TcMAMA’-progestin conjugate 7, and the results are shown graphically in Figure 7. As has been noted with other receptor-binding radiopharmaceuticals (15,19),the activity in the target tissue remains largely the administered compound, even out to 4 h, as receptor sequesters the tracer and prevents its metabolism. In contrast, even at the earliest time (1h), liver activity is almost exclusivelydue to metabolites, while
Bioconjugafe Chem., Vol. 5, No. 3, 1994
Progestin N2S2-Metal Labeled Conjugates
189
Table 2. Biodistribution Data for 11&MAMA'-99mTc-Progestin 7 at 1,3, 6, and 18 h (%
%ID/g f SD (n = 5)
%ID/g f SD (n = 10)
a
tissue blood lung liver kidney muscle fat uterus ovaries
lh 0.245 f 0.027 0.655 f 0.078 4.906 f 1.190 1.963 f 0.259 0.586 f 0.088 1.094 f 0.525 0.753 f 0.217 0.674 f 0.181
3h
6h
18h ~-
0.155 f 0.039 0.300 f 0.046 3.528 f 0.883 1.830 f 0.194 0.226 f 0.070 0.817 f 0.270 0.591 f 0.082 9.535 f 0.213
0.109 f 0.021 0.166 f 0.032 2.797 f 0.640 1.751 f 0.475 0.066 f 0.019 0.203 f 0.104 0.266 f 0.049 0.161 f 0.063
0.073 f 0.017 0.056 f 0.010 1.140 f 0.267 1.108 f 0.399 0.016 f 0.007 0.036 f 0.030 0.046 f 0.014 0.041 f 0.013
uterus/muscle uterus/ blood
1.282 f 0.364 3.166 f 0.736
2.680 f 0.669 5.006 f 1.111
4.115 f 0.808 2.529 f 0.384
2.875 f 0.532 0.630 f 0.241
% ID/g: percent injected dose/gram tissue. I
Table 3. Biodistribution Data for ll~-MAMA'-99mT~-Progestin 7 at 1,3, and 6 h (% ID/g)a
tissue blood lung liver kidney muscle fat uterus ovaries
%ID/gf SD (n = 10) 1h (block) 3 h (block) 6 h (block) 0.145 f 0.031 0.098 f 0.019 0.070 f 0.006 0.479 f 0.145 0.209 f 0.058 0.097 f 0.016 3.509 f 0.806 3.021 f 0.547 2.161 f 0.260 1.453 f 0.305 1.362 f 0.289 1.394 f 0.352 0.450 f 0.116 0.214 f 0.140 0.040 f 0.012 0.747 f 0.173 0.438 f 0.074 0.125 f 0.046 0.339 f 0.091 0.234 f 0.019 0.076 f 0.018 0.461 f 0.126 0.210 f 0.028 0.077 f 0.018
uterus/muscle uterus/blood
0.753 f 0.280 2.338 f 0.802
a
~~
1.093 f 0.721 2.388 f 0.502
I
I
U
5.0
1.900 f 0.726 1.086 f 0.274
The rats used in these experiments were each coinjected with 18
T
4.0
-
3.0
-
2.0
-
1.0
-
0.0
'
'
l'hour
'
I
0
4.0
I
ute rus/muscle
I
I
I
I
I
3
6
9
12
15
18
I
Figure 4. Time course of the change in the ratio ( S D ) of target (uterus) to nontarget (blood or muscle) tissue uptake for mTcprogestin 7.Data are taken from Table 2 and are decay corrected.
3hour
2
I
Hours
6 hour
W
I
uterus/blood
I
pg of ORG2058 (block). % ID/g: percent injected dose/gram tissue.
1
-
I
I
3.0
-
6 hour
'T
'
18 hour
E cd &J
T
1.o
n
n
8
0.0
uterus blood
2.0
\
fat
1.o
muscle liver kidney
Figure 3. Biodistribution of mTc-progestin 7 in immature estrogen-primedfemale rats presented as percent injected dose/ gram (ASD) of tissue at 1,3,6, and 18 h following injection. Data are taken from Table 2 and are decay corrected.
in the kidney and blood, the percent that is unmetabolized declines rather slowly from an initial value of 50%. This time course and pattern of metabolism is similar to that of the BAT conjugates (4).
0.0
uterus blood
fat
muscle liver kidney
Figure 5. Uptake of mTc-progestin 7 a t 6 h when unblocked or when the PR has been blocked by an excess of Org 2058, presented as percent injected dose/gram tissue (MD). Data are taken from Tables 2 and 3.
vivo was not sufficiently selective for target tissues to be useful for imaging human breast tumors based on their content of steroid receptors (4, 5 ) . DISCUSSION Because the BAT-metal conjugate system is very hydrophobic, we suspected that the high lipophilicity of In this paper, we describe the synthesis and evaluation this complex, which has an octanol-water partition coefof a new conjugate between a progestin with high affinity ficient nearly 100-fold higher than that of typical for the progesterone receptor (PR) and a new metal complex (MAMA'). This conjugate represents an extenprogestins, might be the principal reason for its reduced sion of our recent work in which we described the uptake selectivity. The progestin-MAMA'-metal conpreparation of the first receptor ligand labeled with jugate described here is our first attempt to effect a technetium-99m that retains nanomolar receptor binding substantial reduction in the lipophilicity of these progestin affinity (4,5). While this first system, a p r o g e ~ t i n - ~ ~ ~ T c - metal conjugate systems. Relative to the BAT chelate, the MAMA' system has four fewer methyl groups and has BAT conjugate, displayed high receptor affinity, its a carbonyl function in place of one methylene group. nonspecific binding was high and its tissue distribution in
190
Bioconjugate Chem., Vol. 5, No. 3, 1994
6.0
E
5.0
bol
4.0
2
1
MAMA‘
\
n U t?
3.0 2.0 1.o 0.0
uterus blood
fat
muscle liver
Figure 6. Comparison of the biodistribution at 3 h of -TcMAMA’7, ~Tc-BAT I (taken from ref 5), and [3H]RU486(IV). Data presented as percent injected dose/gram tissue (MD).
In a recent publication, we have described the preparation of the basic MAMA’-rhenium and -technetium chelates, and we have presented a detailed spectroscopic and structural analysis of both the syn and anti diastereomers of a simple N-benzyl derivative (6). This MAMA’ system has many features similar to those of other conjugates of the well-studied N2S2 class (7, 8). The preparation of the corresponding progestin-MAMA’ conjugate proceeded in an analogous fashion to the preparation of the BAT conjugate and presented no particular difficulties, and the rhenium and technetium-99, and -99m conjugates could be readily prepared and purified. With the MAMA’ conjugates, the syn diastereomers predominated over the anti to an even greater extent than with the BAT conjugates, so that only the syn isomers could be isolated and characterized. The progestin-MAMA’-metal conjugates 5 and 6 have affinities for PR that are three to four times greater than the natural ligand (progesterone) for this receptor, as was also true for the progestin-BAT-metal conjugates I. With both of these systems, it is remarkable that a steroid conjugated to a metal complex of nearly equal size can retain such high affinity for receptor. It should be noted, however, that the affinity of these conjugates is lower than that of the synthetic ligands R5020, RU486, and ORG2058, a fact that may contribute to the moderate level of their receptor-mediated tissue distribution. Both in the BAT and the MAMA’ series, there is a significant reduction in affinity for human PR vs rat PR, relative to the tracer R5020. This is also true for progesterone and the synthetic ligands ORG2058 and RU486. Such species-dependent differencesin PR ligand binding affinity and selectivity is well known (27-29),but the reason is unclear. The cDNA-deduced amino acid sequences for the rat and human PR ligand binding domains are 88-96% similar (35,36);on the other hand, it is known that even a single amino acid change in PR can result in a profound alteration in ligand binding specificity (37). There is considerable sequence homology between the hormone binding domains of PR and GR (38),and these two receptors (together with the mineralocorticoid and androgen receptors) are considered to be within the same evolutionary family (39). This sequence/structural similarity is reflected in the tendency of ligands for these receptors to exhibit considerable “receptor cross-talknor “heterologous binding” (40, 41). In fact, the synthetic progestins R5020 and ORG2058 are useful as selective tracers for PR in in vitro binding assays because of their
O’Neil et al.
high selectivity for PR vs GR, but this characteristic is not shared by most ligands with l l p substituents, such as RU486 (28, 42). Both the BAT- and MAMA’-metalprogestin conjugates also demonstrate considerable binding affinity for the GR. This too may be a factor compromising the selectivity of their in vivo distribution. One of our principal intents in developing the MAMA’progestin conjugates was to create a system that would be similar to the BAT conjugates, but would have lower lipophilicity. We hoped that substitution of the MAMA’ for the BAT-metal chelate would accomplish this without compromising the high PR binding affinity of the BAT conjugates. This was, in fact, borne out by experiment, where as expected, the MAMA’ conjugates have octanolwater partition coefficients nearly 100-fold lower than those of the BAT systems, now being within the range that is typical for high affinity ligands for PR. This was achieved without a decrease in affinity for PR. The in vivo tissue distribution of the p r o g e ~ t i n - ~ ~ ~ T c MAMA’ conjugate was quite similar to that of the corresponding BAT complex. Displaceable (receptormediated) uptake was only observed in the principal target tissue the uterus, and activity in this tissue was greater than in blood or muscle, but fat uptake and activity in liver and kidney was very high. The 80-fold lower lipophilicity of the MAMA’ conjugate is not reflected in a dramatic way in its uptake behavior relative to the BAT complex: the uptake of the MAMA’ conjugate into fat is only half that of the BAT complex,but the uterine uptake is also reduced by a similar fraction. It is instructive to make a comparison of the tissue distribution of the BAT-and MAMA’-progestin conjugates with that of RU486 and other labeled progestins that we have studied earlier ( 1 9 , 2 4 , 4 3 ) . As a class, the progestins tend to have lower target tissue uptake than, for example, the estrogens, and their uptake by fat and tissues involved in metabolism and clearance (liver and kidney) is relatively high. Nevertheless, the BAT-and MAMA’-metal conjugates have target tissue uptake efficiency and selectivity at the low end of all the compounds we have studied. The role that lipophilicity plays in uptake behavior as evidenced with the BAT and MAMA’ conjugates does not seem to be a dominant one. The possibility that elevated liver uptake is due to their binding by GR in this tissue is unlikely, since metabolism studies on the two conjugates have shown that most of the activity in the liver is due to metabolites. Also, RU486, which has very high affinity for GR, does not show liver uptake that is very much greater than that of other PR tracers such as ORG2058, R5020, and FENP, which all have very low GR affinity. In fact, in other studies we have found that even tracers with very high GR binding affinity fail to show substantial receptor-mediated uptake by GR target tissues (44),indicating that the GR system itself may be poorly suited for selective ligand accumulation. Two other factors that may account for the uptake behavior of the metal conjugates are receptor binding affinity and overall molecular size. Even though the BAT and MAMA’ conjugates have nanomolar binding affinity for PR, exceeding that of progesterone several fold, their affinity for PR may still be too low to result in desirable distribution behavior. The affinity of progesterone itself is insufficient for target tissue uptake and retention, and it may be that affinities approaching those of FENP, ORG2058, and R5020 may be required. In fact, most progestins of moderate affinity for PR that we have studied have relatively poor target tissue uptake (5, 43, 45).
Bioconjugate Chem., Vol. 5, No. 3, 1994 191
Progestin N2S2-Metal Labeled Conjugates
Table 4. Biodistribution Data for 3H-RU486 at 1,3, and 6 h (% ID/gP % ID/g f SD (n = 3)
tissue blood lung liver spleen muscle fat uterus ovaries
1 2 3 4
5 6 7 8
lh
3h
6h
3 h (block)
0.550 f 0.080 0.980 f 0.360 5.240 f 0.960 0.820 f 0.160 0.940 f 0.200 4.000 f 1.600 3.030 f 1.670 2.940 f 0.680
0.410 f 0.140 0.440 f 0.170 3.020 f 0.890 0.350 f 0.130 0.350 f 0.100 2.270 f 1.250 2.570 f 1.300 1.450 f 0.600
0.390 f 0.130 0.240 f 0.040 1.140 f 0.040 0.210 f 0.060 0.190 f 0.010 1.220 f 0.200 0.570 f 0.160 0.520 f 0.030
0.230 f 0.100 0.190 f 0.030 1.130 f 0.230 0.170 f 0.030 0.200 f 0.070 1.570 f 0.250 0.360 f 0.110 0.540 f 0.140
3.000 f 0.857 1.800 f 0.836 uterus/muscle 3.223 f 1.904 7.534 f 4.377 1.462 f 0.637 1.565 f 0.832 uterus/blood 5.509 f 0.582 6.268 f 3.825 a One set of animals at 3 h was coinjected with 18 pg of ORG2058 (block). % ID/g: percent injected dose/gram tissue. 9 10
I
1
I
I
I
tivity that is also intermediate between that of the steroids and the metal conjugates, suggests that the size of the tracer may be important. In this study, we have determined that lipophilicity and heterologous binding to other receptors may be less important factors in tissue uptake than previouslythought and the issues of overall molecular size and PR binding affinity more important. Our future investigations on technetium-labeled ligands for steroid receptors will address these considerations. ACKNOWLEDGMENT
0
60
120
180
240
Minutes Figure 7. Metabolism of mTc progestin 7 in uterus and nontarget tissues, blood, kidney, and liver.
We are grateful for support of this research through grants from the Department of Energy (DE FG02 86ER60401to J.A.K. and DE FG02 84ER60218to M.J.W.). J.P.O. was partially supported through an NIH training fellowship (5T32CA09067). NMR spectra at 400 MHz and mass spectra were obtained on instruments supported by the National Science Foundation (CHE 90-01438EQ) and by the National Institutes of Health (GM27029, respectively). We wish to thank Donald Seielstad for insightful conversation regardingsteroid receptor structure and Drs. James DiZio and Henry VanBrocklin for helpful discussions and assistance in the preparation of steroidal precursors. We also wish to thank Pam Roque and Henry Lee for their competent aid with the in vivo experiments and especially Tammy Pajeau-Stinson for undertaking the biodistribution study of [3H]RU486. LITERATURE CITED
Figure 8. Size comparison of FENP (upper left), RU486 (upper right), -Tc-BAT I (lower left), and mTc-MAMA’ 7 (lower right) using space-filling representations (46).
Finally, the issue of molecular size may be involved: The BAT and MAMA’ conjugates as ligands for PR are nearly twice the size of a typical steroid, such as FENP, as shown in Figure 8. Differences in their transport and tissue permeability may compromise their target tissue uptake in ways that may not be apparent from a study of only steroid-sized ligands. The fact that RU486, which is a PR ligand midway in size between R5020 and the metal complex, has a target tissue uptake efficiency and selec-
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Katzenellenbogen, J. A. (1993) Fluorine-18-labeledprogestin ketals: synthesis and target tissue uptake selectivity of potential imaging agents for receptor-positive breast tumors. J. Med. Chem. 36, 1120-1127. (46) Space filling models were constructed in the SYBYL 6.0 molecular modeling package (TRIPOS Assoc., St. Louis, MO) on a Silicon Graphics Indigo Elan computer system. Atomic coordinates for FENP and RU486 as well as the BAT-metal complex were obtained from the Cambridge Structural Database (Allen, F. H., Davies, J. E., Galloy, J. J., Johnson, O., Kennard, O., Macrae, C. F., Mitchell, E. M., Mitchell, G. F., Smith, J. M., and Watson, D. G. (1991) The development of versions 3 and 4 of the CambridgeStructural Database System. J. Chem. Znf. Comp. Sci. 31, 187-204). The progestin NzSz conjugates were constructed by merging the complex coordinates with those of RU486, replacing the dimethyl amino functionality. Atomic coordinates for the MAMA' structure were obtained directly from our previously published crystal structure (6).
