A Novel, High-Affinity, Fluorescent Progesterone Receptor Antagonist

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Bioconjugate Chem. 2004, 15, 359−365

359

A Novel, High-Affinity, Fluorescent Progesterone Receptor Antagonist. Synthesis and in Vitro Studies Claudia Ho¨dl,† Wolfgang S. L. Strauss,‡ Reinhard Sailer,‡ Christoph Seger,§ Rudolf Steiner,‡ Ernst Haslinger,† and H. Wolfgang Schramm*,† Institute for Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens-University, Universita¨tsplatz 1, A-8010 Graz, Austria, Institut fu¨r Lasertechnologien in der Medizin und Messtechnik an der Universita¨t Ulm, Helmholtzstrasse 12, D-89081 Ulm, Germany, and Institute of Pharmacy, Department of Pharmacognosy, Leopold-Franzens-University, Innrain 52, A-6020 Innsbruck, Austria. Received September 16, 2003; Revised Manuscript Received February 3, 2004

The present paper describes the chemical synthesis and in vitro characterization of a novel, highaffinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptornegative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by nearinfrared optical imaging.

INTRODUCTION

The progesterone receptor (PR)1 is a ligand-activated transcription factor that belongs to the nuclear receptor superfamily, which includes the steroid hormone receptors as well as thyroid, vitamin D, and retinoic acid receptors. It contains conserved functional domains, including the N-terminus, a centrally located DNA binding domain (DBD), and the C-terminal ligand binding domain (LBD). The human PR exists as two isoforms (A and B) in most PR-expressing cells, which arise from a single gene located in the region 11q22-q23 of chromosome 11. The isoform A (PRA) is a 164 amino acid N-terminally truncated variant of the isoform B (PRB). Although both isoforms have similar DNA and ligand binding properties, they have distinct functional properties. Current knowledge of the ligand-receptor interaction at the molecular level mainly relies on the crystal structure of the LBD of the PR complex formed after binding of progesterone (1) as well as on results of sitedirected mutagenesis experiments (2) and classical molecular dynamics simulations (3). According to the physi* Corresponding author: phone +43/316/380-538; fax+43/316/ 380-9846; e-mail: [email protected]. † Karl-Franzens-University. ‡ Institut fu ¨ r Lasertechnologien in der Medizin und Messtechnik an der Universita¨t Ulm. § Leopold-Franzens-University. 1 Abbreviations: CH, cyclohexane; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; MeCN, acetonitrile; MeOH, methanol; MMTV, mouse mammary tumor virus; NIR, near-infrared; PBS, phosphate-buffered saline; PR, progesterone receptor; THF, tetrahydrofuran.

ological role of its natural ligand, i.e., establishment and maintenance of pregnancy, high PR levels are found in the female reproductive system. Current development of drugs interacting with the PR comprise (1) selective PR antagonists (4, 5) expected to be useful for the treatment of chronic conditions, e.g., endometriosis or leiomyomas, and (2) radioligands (6-8) to enable determination of PR levels in vivo for assessing the hormone responsiveness of breast cancer without tissue biopsy. During the past decade, optical imaging using nearinfrared (NIR) light has emerged as a new diagnostic modality, in particular for breast tissue (9). Since contrast and image resolution is limited by multiple scattering of the photons, reliable tissue characterization based on intrinsic absorption and scattering inhomogeneities is often difficult, in particular with respect to distinguish between benign and malignant lesions (10, 11). Similar to other clinical imaging modalities, it is likely that sensitivity and specificity of optical imaging will be improved by contrast agents. Considering photophysical properties, cyanine dyes are one of the most promising classes of compounds suitable as contrast agents for NIR optical imaging (12, 13). Recently, indocyanine green has been successfully applied to detect breast cancer probing tumor vascularization and vessel permeability (14). In addition to tumor targeting by such unspecific mechanisms, contrast agents which specifically target receptors overexpressed in tumor cells (15-18), or which are activated by tumor-associated enzymes (19-23), have recently been developed, thus enabling molecular imaging. PR status of breast cancer was found to be an independent predictive factor for benefit from adjuvant

10.1021/bc034169o CCC: $27.50 © 2004 American Chemical Society Published on Web 02/26/2004

360 Bioconjugate Chem., Vol. 15, No. 2, 2004

endocrine therapy. Patients with steroid hormone receptor-positive tumors, expressing estrogen receptor as well as PR, have the most favorable prognosis (24). Therefore, reliable and noninvasive determination of PR levels in breast cancer, preferentially with high sensitivity and without harmful radiation, i.e., by NIR optical imaging, seem to be highly desirable. However, no appropriate PRbinding contrast agent is known, so far. In the present paper, synthesis and in vitro studies of a novel, highaffinity, fluorescent PR antagonist is reported, which might serve as lead compound for the development of PRbinding contrast agents for NIR optical imaging. EXPERIMENTAL PROCEDURES

Materials and Chemical Methods. (11β,17β)-17Hydroxy-11-[4-(dimethylamino)phenyl]-17-(1-propynyl)estra-4,9-dien-3-one (1) and fluorescein 5-isothiocyanate (FITC, isomer I) were obtained from Sigma-Aldrich. All other reagents for syntheses were purchased from either Sigma-Aldrich or Merck and were used without further purification. Reagent-grade solvents were obtained from Merck and were purified and dried using standard methods. Solvents of analytical and spectroscopic grade and deuterated NMR solvents were purchased from Merck and Uetikon, respectively. UV/vis spectra were recorded on a Shimadzu UV-160A spectrometer. Absorption maxima λmax are given in nm. Fluorescence spectra were measured using a Kontron Instruments spectrofluorometer. Excitation and emission maxima λmax are given in nm. IR spectra were recorded on a Perkin-Elmer FTIR 2000 spectrometer. Absorption maxima νmax are given in cm-1 and are referred to as s (strong), m (medium), w (weak), and br (broad). EI-mass spectra were measured with a Finnigan MAT 212 or Varian MAT 312 spectrometer at 70 eV ionizing voltage. Intensities are given in % of the base peak. NMR spectra were recorded at 300 K on a Varian Unity Inova 400 NMR spectrometer equipped with a tunable broad band probe. Inverse detected gradient selected 2D experiments (gCOSY, HSQC, HMBC) were performed using standard pulse sequence programs. HMBC experiments were optimized for a long range-coupling constant of 8 Hz. Spectra were referenced internally using tetramethylsilane. Chemical shifts δ are given in ppm. Coupling constants (J values) are expressed in Hz, and multiplicities are referred to as s (singlet), d (dublet), t (triplet), q (quartet), m (multiplet), and br (broad). TLC analyses were performed on Merck F254 silica gel plates. Spots were visualized either by UV absorbance at 254 nm or fluorescence emission excited at 366 nm. Column chromatography was carried out on Merck silica gel 60, 70-230 mesh, using EtOAc:cyclohexane (CH) (3:2) as eluent. Preparative RP-HPLC was performed using a Labomatic HD-200 pump equipped with a Labocord-700 UV/vis detector. (11β,17β)-17-Hydroxy-11-[4-(methylamino)phenyl]17-(1-propynyl)estra-4,9-dien-3-one (2). (11β,17β)-17Hydroxy-11-[4-(dimethylamino)phenyl]-17-(1-propynyl)estra-4,9-dien-3-one (1) (500 mg, 1.164 mmol) was dissolved in a 1:1 mixture of MeOH and THF (7.0 mL), and freshly calcined CaO (555 mg, 9.897 mmol) was added. I2 (739 mg, 2.912 mmol) dissolved in the same solvent (1.5 mL) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. After CaO removal, it was diluted with 10% Na2S2O3 and extracted with CH2Cl2. The combined organic layers were washed with water and brine and dried over Na2SO4. The solvent was evaporated after filtration, and the crude product was purified by column chromatography. Fractions containing

Ho¨dl et al.

the steroidal compounds 1 and 2 were further purified by HPLC (ProntoSIL 120-5-C18H, 5 µm, 200 × 20 mm, MeCN:H2O ) 84:16, 23 mL/min, λ ) 254 nm) to afford compound 2 as a white solid (135 mg, 28%) after lyophilization, tR ) 3.4 min. Educt 1 was recovered (130 mg, 26%), tR ) 4.3 min. TLC (EtOAc:CH ) 3:2): Rf ) 0.5; TLC (CH2Cl2:MeOH ) 9:1): Rf ) 0.71. UV/vis (MeOH): 209.0, 250.0, 303.5; UV/VIS (MeCN): 254.0, 295.5. IR (KBr): 3387 (m (br), N-H, O-H); 2929 (s, C-H); 2870 (m, C-H); 2250 (w, CtC); 1654 (s (br), Cd O), 1615 (s, CdC). MS (m/z): 415 (M+, 100); 397 (7); 331 (11); 266 (71); 213 (23); 120 (47); 107 (76); 91 (54); calculated for C28H33NO2, 415. 1H NMR (CDCl3): 0.50 (s, 3H, H-18); 1.40 (m, 1H, H-16); 1.42 (m, 1H, H-7); 1.68 (m, 2H, H-14, H-16); 1.84 (s, 3H, H-21); 1.89 (td, J ) 11.4, J ) 3.2, 1H, H-15); 1.98 (m, 1H, H-7); 2.16 (m, 1H, H-15); 2.21 (m, 1H, H-12); 2.29 (m, 4H, H-1, 2x(H-2), H-12); 2.42 (dd, J ) 10.4, J ) 0.8, 1H, H-8); 2.52 (m, 2H, H-6); 2.73 (m, 1H, H-1); 2.86 (s, 3H, NCH3); 4.31 (d, J ) 6.4, 1H, H-11); 5.52 (s, 1H, H-4); 6.61 (m, 2H, H-3′, H-5′); 6.97 (m, 2H, H-2′, H-6′). 13C NMR (CDCl3): 3.7 (q, C-21); 13.6 (q, C-18); 23.2 (t, C-16); 25.6 (t, C-1); 27.2 (t, C-7); 31.0 (t, C-6); 36.7 (t, C-2); 38.8 (t, C-12, C-15); 39.0 (d, C-8); 39.4 (d, C-11); 40.5 (q, NCH3); 46.7 (s, C-13); 49.7 (d, C-14); 79.9 (s, C-17); 82.0 (s, C-20/C-19); 82.4 (s, C-19/ C-20); 112.7 (d, C-3′, C-5′); 122.5 (d, C-4); 127.3 (d, C-2′, C-6′); 128.8 (s, C-10); 132.0 (s, C-1′); 146.8 (s, C-9); 148.4 (s, C-4′); 156.9 (s, C-5); 199.5 (s, C-3). (11β,17β)-17-Hydroxy-11-(4-[(6-hydroxyhexyl)methylamino]phenyl)-17-(1-propynyl)estra-4,9-dien-3-one (3). Compound 2 (200 mg, 0.481 mmol) and freshly distilled 6-bromohexanol (500 mg, 2.760 mmol) were dissolved in anhydrous DMF (4.0 mL) and stirred in the presence of dry K2CO3 (80 mg, 0.579 mmol) at 65 °C for 18 h. The solvent was removed in vacuo, and the residue was purified by column chromatography, yielding 134 mg (54%) of compound 3 as a white solid. For biological studies 25 mg of compound 3 were additionally purified by HPLC (YMC-Pack ODS-A (C18), 10 µm, 250 × 20 mm, MeCN:H2O ) 2:3, 12 mL/min, λ ) 264 nm), tR ) 9.3 min. After lyophilization 13.5 mg (54%) were recovered. TLC (EtOAc:CH ) 3:2): Rf ) 0.3; TLC (CH2Cl2: MeOH ) 9:1): Rf ) 0.87. UV/vis (MeOH): 208.5, 263.5, 303.5. IR (KBr): 3421 (m, O-H); 2934 (s, C-H); 2861 (m, C-H); 2250 (w, CtC); 1653 (s, CdO), 1611 (s, CdC). MS (m/z): 515 (M+, 100), 475 (12), 428 (83), 388 (17), 366 (12), 120 (18); calculated for C34H45NO3, 515. 1H NMR (CDCl3): 0.53 (s, 3H, H-18); 1.34 (m, 1H, H-16); 1.42 (m, 1H, H-7); 1.46 (m, 6H, H-3′′, H-4′′, H-5′′); 1.55 (m, 2H, H-2′′); 1.70 (m, 2H, H-14, H-16); 1.87 (s, 3H, H-21); 1.88 (td, J ) 12.0, J ) 3.2, 1H, H-15); 2.00 (m, 1H, H-7); 2.24 (m, 3H, 2x(H-12), H-15); 2.39 (m, 4H, H-1, 2x(H-2), H-8); 2.55 (m, 2H, H-6); 2.75 (m, 1H, H-1); 2.86 (s, 3H, NCH3); 3.24 (m, 2H, H-1′′); 3.61 (br, 2H, H-6′′); 4.32 (d, J ) 6.8, 1H, H-11); 5.73 (s, 1H, H-4); 6.57 (m, 2H, H-3′, H-5′); 6.97 (m, 2H, H-2′, H-6′). 13C NMR (CDCl3): 3.8 (q, C-21); 13.7 (q, C-18); 23.3 (t, C-16); 25.6 (t, C-2′′); 25.8 (t, C-1); 26.7 (t, C-3′′/C-4′′); 26.9 (t, C-4′′/C-3′′); 27.4 (t, C-7); 31.1 (t, C-6); 32.7 (t, C-5′′); 36.9 (t, C-2); 38.2 (q, NCH3); 38.8 (t, C-12/C-15); 38.9 (t, C-15/C-12); 39.1 (d, C-8); 39.5 (d, C-11); 46.8 (s, C-13); 49.8 (d, C-14); 52.9 (t, C-1′′); 62.8 (t, C-6′′); 80.2 (s, C-17); 82.3 (s, C-19/C-20); 82.5 (s, C-20/ C-19); 112.2 (d, C-3′, C-5′); 123.6 (d, C-4); 127.6 (d, C-2′, C-6′); 129.0 (s, C-10); 131.3 (s, C-1′); 146.9 (s, C-9); 147.3 (s, C-4′); 157.0 (s, C-5); 199.7 (s, C-3). O-(6-[N-Methyl-N-(4-[(11β,17β)-17-hydroxy-3-oxo17-(1-propynyl)estra-4,9-dien-11-yl]phenyl)amino]hexyl)-N-(3′,6′-dihydroxy-3-oxospiro[isobenzofuran1(3H),9′-[9′H]xanthen]-5-yl)-carbamothioate (4). Com-

Fluorescent Progesterone Receptor Antagonist

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Scheme 1: Synthesis of Conjugate 4 and Its Precursors 2, 3a

a

(a) CaO/I2/MeOH/THF; (b) BrCH2(CH2)4CH2OH/DMF; (c) FITC (isomer I)/DMF.

pound 3 (120 mg, 0.234 mmol) and fluorescein 5-isothiocyanate (136 mg, 0.349 mmol) were dissolved in anhydrous DMF (5.0 mL) and stirred at 70 °C for 5 h. It was diluted with ice/H2O to give a brownish precipitate. The crude product was extracted with EtOAc, washed with H2O, and dried over Na2SO4. The solvent was evaporated after filtration, and the residue was purified by column chromatography. HPLC purification (YMC-Pack ODS-A (C18), 10 µm, 250 × 20 mm, MeOH: H2O ) 4:1, 12 mL/ min, λ ) 220 nm) was applied to afford conjugate 4 as a yellow solid (18.5 mg, 8.6%) after lyophilization, tR ) 5.3 min. TLC (CHCl3:MeOH ) 4:1): Rf ) 0.63. UV/vis (MeOH): 220.5, 284.0, 454.5, 480.5. IR (KBr): 3430 (s, O-H); 2934 (m, C-H); 2861 (m, C-H); 1737 (w, CdO, lactone); 1636 (s, CdO), 1612 (s, CdC). MS (m/z): 905 (M+, 13), 887 (14), 597 (5), 408 (19), 390 (90), 374 (76); 348 (100); calculated for C55H56N2O8S, 905. 1H NMR (acetone-d6): 0.56 (s, 3H, H-18); 1.36 (m, 1H, H-16); 1.41 (m, 3H, H-7, 2x(H-3′′/H-4′′)); 1.51 (m, 2H, H-4′′/H-3′′); 1.58 (m, 2H, H-2′′); 1.68 (m, 1H, H-16); 1.74 (m, 1H, H-14); 1.77 (m, 2H, H-5′′); 1.82 (s, 3H, H-21); 1.89 (m, 1H, H-15); 2.04 (m, 1H, H-7); 2.11 (m, 1H, H-15); 2.20 (m, 1H, H-1); 2.28 (m, 1H, H-12); 2.29 (m, 2H, H-2); 2.37 (m, 1H, H-12); 2.49 (m, 1H, H-8); 2.61 (m, 2H, H-6); 2.78 (m, 1H, H-1); 2.87 (s, 3H, NCH3); 3.31 (t, J ) 6.8, 2H, H-1′′); 4.08 (m, 2H, H-6′′); 4.36 (d, J ) 6.8, 1H, H-11); 4.55 (s, 1H, OH17); 5.63 (s, 1H, H-4); 6.70 (m, 6H, H-3′, H-5′, H-1′′′′, H-2′′′′, H-7′′′′, H-8′′′′); 6.75 (d, J ) 2.0, 2H, H-4′′′′, H-5′′′′); 7.02 (m, 2H, H-2′, H-6′); 7.23 (m, 1H, H-6′′′); 9.00 (s (br), 2H, OH-3′′′′, OH-6′′′′); 10.40 (s (br), 1H, NH). 13C NMR (acetone-d6): 3.4 (q, C-21); 14.3 (q, C-18); 23.9 (t, C-16); 26.5 (t, C-1); 27.2 (t, C-3′′/C-4′′); 27.3 (t, C-2′′); 27.4 (t, C-4′′/C-3′′); 28.5 (t, C-7); 29.1 (t, C-5′′); 31.6 (t, C-6); 37.5 (t, C-2); 38.3 (q, NCH3); 39.7 (t, C-12/C-15); 39.8 (t, C-15/ C-12); 40.1 (d, C-8); 40.3 (d, C-11); 47.7 (s, C-13); 50.7 (d, C-14); 53.1 (t, C-1′′); 60.5 (t, C-6′′); 80.1 (s, C-17); 81.4 (s, C-19/C-20/C-9′′′′); 83.7 (s, C-20/C-9′′′′/C-19); 84.2 (s, C-9′′′′, C-19, C-20); 103.3 (d, C-4′′′′, C-5′′′′); 111.5 (s, C-1′′′′a,

C-8′′′′a); 113.1 (d, C-3′, C-5′/C-2′′′′, C-7′′′′); 113.3 (d, C-2′′′′, C-7′′′′/C-3′, C-5′); 117.3 (d, C-3′′′); 123.0 (d, C-4); 125.2 (d, C-6′′′); 128. 5 (d, C-2′, C-6′); 129.6 (s, C-10); 130.1 (d, C-1′′′′, C-8′′′′); 132.7 (s, C-1′); 140.8 (s, C-2′′′); 147.7 (s, C-4′); 148.2 (s, C-9); 149.7 (s, C-4′′′); 153.3 (s, C-4′′′′a, C-5′′′′a); 157.1 (s, C-5); 160.3 (s, C-3′′′′, C-6′′′′); 169.0 (s, C-7′′′); 189.4 (s, CS); 198.6 (s, C-3). Cell Culture and Media. T47-D cells (breast cancer, human, steroid hormone receptor-positive) were obtained from American Type Culture Collection (ATCC number HTB-133 (25)) and OV2774 cells (ovarian cancer, human, steroid hormone receptor-negative) were kindly provided by Prof. D. G. Kieback, Department for Obstetrics and Gynecology, Maastricht University Medical Center, The Netherlands. T47-D-Cl24 cells stably expressing luciferase under control of hormone responsive mouse mammary tumor virus (MMTV) promoter (26) were generated. Vectors ∆Ma-Luc (generous gift of Prof. R. Schu¨le, Department of Obstetrics and Gynecology, University Hospital Freiburg, Germany) and pcDNA6/HisA (invitrogen) were cotransfected into T47-D cells using Rotifect (Roth) as recommended by the supplier. Stable transfectants were selected with blasticidin (2.5 µg/mL), and individual colonies were picked and cultured separately. Clones were screened for luciferase expression after incubation with progesterone (10-6 mol/L). Clone 24 was chosen considering level and dynamic range of luciferase expression after progesterone incubation (10-11 to 10-6 mol/L). T47-D and T47-D-Cl24 cells were maintained in RPMI 1640 medium (invitrogen) supplemented with 10% fetal calf serum (FCS, Biochrom) and antibiotics (penicillin (100.000 U/L), streptomycin (100.000 µg/ L), invitrogen) and cultured in a humidified atmosphere containing 5% CO2 at 37 °C. Twenty-four hours prior to the experiments cells were seeded at a density of 100 cells per mm2 in 96-well plates (Nunc) or on microscope slides (Marienfeld) and were cultivated in RPMI 1640 medium

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(without phenol red) supplemented with 5% dextran charcoal stripped FCS (DCS) and antibiotics at 5% CO2 and 37 °C. Incubation media were made up by adding an appropriate volume of the stock solutions of compounds 1-4 and progesterone (prepared in dimethyl sulfoxide (DMSO) and stored at -20 °C) to RPMI 1640 medium (without phenol red, plus 5% DCS and all other supplements). In each case, the final DMSO concentration in the incubation medium was 0.1% (v/v). Incubations were performed after washing the cultures with phosphate-buffered saline (PBS). Alkaline Phosphatase Assay. T47-D cells were coincubated with increasing concentrations of either compound 1 (reference antagonist) or test compounds 2, 3, or 4 (10-11 to 10-7 mol/L) and progesterone (10-9 mol/L). The alkaline phosphatase assay was performed as reported previously (27, 28) with some minor modifications. Briefly, after 48 h incubation cells were washed with 0.9% saline and stored at -80 °C for at least 30 min.. After thawing cells were incubated with 50 µL p-nitrophenyl phosphate (5 mM, Fluka) dissolved in aqueous diethanolamine (1 M, supplemented with 0.5 mM magnesium chloride and 20 µM zinc sulfate, and adjusted to pH 9.8) for 2-5 h in the dark at room temperature. Absorbance of p-nitrophenolate was measured in a Lucy 1 multimode plate reader (anthos labtec instruments) at 405 nm (vs 690 nm as reference). For each compound, at least four independent experiments (with six data points each) were carried out. After background subtraction (absorbance of cells without hormonal treatment), absorbance was normalized to the absorbance resulting from the progesterone incubation. Median (central tendency) ( median absolute deviation (variability) were calculated. IC50 values were determined from dose-response curves and are given in nmol/L. Transactivation Assay. T47-D-Cl24 cells were coincubated with increasing concentrations of compound 1 (reference antagonist) or test compounds 2, 3, or 4 (10-12 to 10-8 mol/l) and progesterone (10-9 mol/L). After 24 h incubation, luciferase activities were measured by the Reporter Gene Luciferase Assay, Constant Light Signal (Roche Diagnostics) as recommended by the supplier in the Lucy 1 reader. For each compound, four independent experiments (with six data points each) were carried out using white 96-well plates. Luciferase activity is given as normalized response value relative to the luciferase activity produced by progesterone. Median (central tendency) ( median absolute deviation (variability) were calculated. IC50 values were determined from doseresponse curves and are given in nmol/L. Fluorescence Microscopy. T47-D and OV2774 cells were incubated with either compound 4 or fluorescein (Riedel-de Haen) for 5-7 h at 37 °C. Incubation concentrations were varied between 10-8 mol/L and 10-6 mol/ L. After incubation, cells were washed twice with PBS. Fluorescence images were obtained with an upright Axioplan I microscope (Zeiss) equipped with a highly sensitive CCD camera (ST138 (Princeton Instruments) equipped with a thermoelectrically cooled CCD-512EFT chip (EEV)) at 40× magnification (oil immersion NA 1.3). Fluorescence was excited in the blue spectral region (band-pass filter: 450-490 nm) and detected above 520 nm (dichroic mirror: 510 nm, long-pass filter: 520 nm). For both compounds, an identical exposure time of 1 s was applied to minimize photobleaching. Image acquisition was performed using WinView 32 (Princeton Instruments). Images were stored as TIFF files, and contrast and brightness were adjusted manually (Adobe Photoshop 6.0, Adobe Systems, Inc.).

Ho¨dl et al.

Figure 1. Fluorescence excitation and emission spectra of conjugate 4 (solid lines) and fluorescein (dotted lines) in PBS (supplemented with 10% FCS). Excitation spectra were recorded at the emission maxima (515 nm (fluorescein) and 524 nm (conjugate 4)), and emission spectra were recorded at the excitation maxima of each compound (488 and 498 nm) at a concentration of 10-6 mol/L. Table 1. Antiprogestagenic Activity of Conjugate 4, Intermediates 2, 3, and Parent Compound 1 alkaline phosphatase assaya transactivation assayb compound IC50 valuesc 1 2 3 4

0.045 0.170 0.063 1.5

rel potencyd IC50 valuesc rel potencyd 1 0.26 0.71 0.03

0.021 0.073 0.071 0.550

1 0.29 0.30 0.04

a Inhibition of progesterone-induced (10-9 mol/L) alkaline phosphatase activity in T47-D cells. b Inhibition of progesteroneinduced (10-9 mol/L) transcriptional activity in T47-D-Cl24 cells. c IC values are were determined from dose-response curves and 50 are given in nmol/L. d Relative potency is defined as the ratio of IC50 (1) to IC50 (x) with x ) 2, 3, 4.

RESULTS

Chemistry. Synthesis of the conjugate 4 was carried out starting from the antiprogestin 1, mifepristone (RU486), which was converted to intermediate 2 by treating with calcium oxide and iodine in methanol/ tetrahydrofuran (29). Alkylation of the methylamino group of 2 with 6-bromohexanol in anhydrous dimethylformamide in the presence of potassium carbonate resulted in intermediate 3, which was reacted with FITC in dry dimethylformamide (30), yielding compound 4 as yellow crystals. The synthetic pathway of conjugate 4 is depicted in Scheme 1. The colorless, nonfluorescent lactone form of conjugate 4 was prevalent in the aprotic NMR solvent, whereas an aqueous environment as used in biological studies caused spontaneous conversion into its quinoid, fluorescent form. Fluorescence excitation and emission spectra of fluorescein and conjugate 4 are quite similar (Figure 1), although a bathochromic shift of about 10 nm was observed for excitation and emission maxima (488 nm vs 498 nm and 515 nm vs 524 nm). Antiprogestagenic Activity. Antagonistic activity of conjugate 4 and intermediates 2 and 3 was determined in cell culture using the alkaline phosphatase assay in T47-D cells and a transactivation assay in T47-D-Cl24 cells stably transfected with the luciferase gene linked to the hormone responsive MMTV promoter. Inhibition of progesterone-induced increase of either alkaline phosphatase or luciferase activity was found for all compounds. IC50 values determined from dose-response curves and the relative potencies (defined as the ratio of the IC50 value of the reference antagonist 1 to the IC50 value of the individual test compound) are summarized in Table 1. Antiprogestagenic activity of the intermedi-

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Figure 2. Fluorescence images (40× magnification) of conjuagte 4 in steroid hormone receptor-positive T47-D breast cancer cells at incubation concentrations of 10-8 mol/L (A) and 10-7 mol/L (B) as well as in steroid hormone receptor-negative OV2774 ovarian cancer cells (C, 10-7 mol/L), and fluorescence image of fluorescein in T47-D cells (D, 10-7 mol/L). Incubations were carried out 5-7 h prior to microscopy at 37 °C.

ates 2 and 3 were 3- to 4-fold lower than that of the reference antagonist 1. Conjugate 4 exhibited considerable antiprogestagenic activity with IC50 values in the nanomolar range, although the relative potency of conjugate 4 was approximately 30-fold lower as compared with the parent compound 1. Fluorescence Microscopy. Subcellular distribution of conjugate 4 was examined by fluorescence microscopy. For steroid hormone receptor-positive T47-D cells, fluorescence of conjugate 4 was almost confined to the cell nuclei (without staining the nucleoli) at the lowest incubation concentration of 10-8 mol/L. Predominantly diffuse fluorescence arising from the cytoplasm was barely above background (intrinsic cell fluorescence) (Figure 2a). When incubation concentration was increased to 10-7 mol/L or 10-6 mol/L, fluorescence was distributed more homogeneously within the whole cell, and cell nuclei were stained prominently only in selected cases (Figure 2b). In contrast, fluorescence of conjugate 4 could not be detected in the cell nuclei of steroid hormone receptor-negative OV2774 cells independently of the incubation concentration. Cells exhibited a weak fluorescence with sometimes a distinct pattern within the cytoplasm at an incubation concentration of 10-7 mol/L (Figure 2c), whereas fluorescence intensity was close to intrinsic cell fluorescence at 10-8 mol/L. Control experiments performed with fluorescein in T47-D cells revealed diffuse fluorescence within the cytoplasm together with fluorescent granules in the perinuclear region, which is exemplified for an incubation concentration of 10-7 mol/L in Figure 2d. DISCUSSION

Conjugate 4 was obtained in a three-step synthesis. After demethylation of mifepristone to intermediate 2

(step 1), the methylamino group was alkylated with 6-bromohexanol, resulting in intermediate 3 (step 2), which was reacted with FITC, yielding the desired conjugate 4 (step 3). Oxidative demethylation of 4-substituted N,N-dimethylanilines (31) has previously been applied to obtain intermediate 2 and a related antiprogestin (29). Alternatively, intermediate 2 could also be obtained by reacting 1 with iodobenzene diacetat (32). The other two steps were performed following standard procedures. Structures of all compounds were confirmed by NMR spectroscopy, and clean spectra were obtained in all cases. Although the protons at positions 3′′′ and 5′′′ as well as the carbon atoms 1′′′ and 5′′′ of conjugate 4 could not be resolved at the applied magnetic field strength, the NMR spectra clearly confirm the prevalence of the lactone form of conjugate 4 in the aprotic solvent. Several protolytic forms of fluorescein are known. The neutral species can be obtained as a solid as a colorless lactone, yellow zwitterion or red quinoid (33). Since covalent attachment of fluorescein does not perturb the protolytic transitions of the chromophore (34), the dianion form of conjugate 4 is the predominant fluorescent species in an aqueous environment at near neutral pH values (33, 34). To demonstrate ligand-receptor interaction of conjugate 4 and intermediates 2 und 3 in cell culture, antiprogestagenic activity of all compounds was determined using two independent assays, which was compared with that of the parent compound 1, mifepristone, as reference antagonist. Compounds 1-3 exhibited antiprogestagenic activity in the same order of magnitude. Even after attachment of the bulky fluorescein moiety to intermediate 3, considerable antiprogestagenic acitivity was maintained. Although the interactions of conjugate 4 and the intermediates 2 and 3 with other steroid hormone recep-

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tors were not evaluated, it is likely that all three compounds display a similar receptor interaction profile as the parent compound 1, e.g., considerable antiglucocorticoid activity (35). Since functional activity is roughly in agreement with receptor binding affinity (RBA) (36), considerable affinity of conjugate 4 to PR is supposed. Specific binding of the conjugate 4 to PR is strongly supported by fluorescence microscopy. Nuclear localization was observed solely in steroid hormone receptorpositive T47-D cells, whereas nuclei of steroid hormone receptor-negative OV2774 cells remained unstained. Moreover, coincubation of conjugate 4 with a 10-fold excess of the antiprogestin 1 prevented nuclear staining (R. Sailer et al., unpublished data). Fluorescence microscopy of conjugate 4 is concordant to findings obtained recently with chimeras of PR and green fluorescent protein (GFP). Binding of either agonist (17,21-dimethyl19-nor-pregn-4,9-diene-3,20-dione, promegestone, a synthetic progestin) or antagonist (compound 1) to either GFP-PRA or GFP-PRB chimeras caused import of the receptors into the nucleus, but excluding the nucleoli (37). The different fluorescence intensity of conjugate 4 in the nuclei of different cells demonstrates the heterogeneity in PR expression, which varies during the cell cycle (38, 39). Accumulation of conjugate 4 within the cytoplasm at higher incubation concentration is most likely to result from unspecific accumulation or binding to other proteins after saturation of the specific PR binding sites. So far, efforts to develop fluorescent ligands with high affinity for PR resulted in the synthesis of a nitrobenzoxadiazole derivative of a 11β-substituted 19-nor-steroid ((11β,17β)-17-hydroxy-11-(4-[(7-nitro-4-benzofurazanyl)amino-methyl]phenyl]-17-(1-propynyl)estra-4,9-dien-3one (40)). This compound exhibits high affinities for PR as well as glucocorticoid receptor (40, 41) and high fluorescence quantum yield in hydrophobic solvents (41). Although fluorescence was maintained after unspecific binding to proteins, the ligand became nonfluorescent after specific binding to PR (41). Since ligand-receptor interaction is not severely impaired after attachment of either the nitrobenzoxadiazole fluorophore or fluorescein to a 11β-substituted 19-nor-steroid, the distance between the fluorophore and the steroid moiety might be important to obtain a fluorescent PR complex. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. However, it should be mentioned that fluorescence polarization might be used to determine ligand-steroid hormone receptor interaction (Fluormone, Invitrogen; PCT International Application (32), Boehringer Ingelheim) in homogeneous systems. But it remains to be elucidated, whether the ligands used in these assays are also fluorescent in viable cells. To summarize, conjugate 4 is a novel, high-affinity, fluorescent PR antagonist, which might serve as a lead compound for the development of PR-binding contrast agents for NIR optical imaging. It is anticipated that fluorescein can be replaced by an appropriate NIR dye (12, 13) without further compromising ligand-PR interaction. However, further development of a contrast agent for PR imaging should consider (1) receptor binding profile and (2) tissue distribution. Ligand specificity is likely to be increased, when the conjugate is assembled with a highly selective antiprogestin (4, 5, 42, 43), thus minimizing binding to other steroid hormone receptors. Minimizing the uptake of the conjugate into nontarget tissues is more challenging as outlined for several receptor-specific small-molecule radiopharmaceuticals (44).

Ho¨dl et al. ACKNOWLEDGMENT

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